Artigos Fito

91
Ginger (Zingiber officinale Rosc.) is one of the

important and most widely used spices. India
is one of the largest ginger producing country
in the World. The productivity of ginger
remains low in India due to constraints like
diseases and improper management. Ginger is
generally a heavy feeder and responds well to
fertilizer application. Nowdays micronutrient
deficiency is a common problem in ginger
growing soils and application of micronutrient
especially zinc (Zn) improves growth, yield and
quality of this crop (Parthasarathy et al. 2010).
Srinivasan et al. (2004) reported that Zn
deficiency existed in 49% of soil samples collected
from various ginger-growing regions of India
and reported significant increase in yield due
to Zn application.
Ginger is valued for its aroma and flavor and
it has medicinal properties, which in turn is
determined by the composition of its steam
volatile oil. This is comprised mainly of
sesquiterpene hydrocarbons, monoterpene
hydrocarbons and oxygenated monoterpenes.
The monoterpene constituents are believed to
be the most important contributors to the
Influence of zinc on yield and quality profile of ginger (Zingiber officinale Rosc.)
S Hamza, N K Leela, V Srinivasan, C R Nileena & R Dinesh
Division of Crop Production and Post Harvest Technology,
Indian Institute of Spices Research, Kozhikode-673 012, Kerala, India.
E-mail: hamza@spices.res.in
Received 19 January 2012; Revised 09 May 2012; Accepted 23 July 2012
Abstract
Field experiments were conducted for three consecutive years (200809. 200910, 201011) in a Zn
deficient soil to study the variation in quality and oil composition of ginger due to incorporation
of Zn in the fertilizer schedule. The treatments consisted of recommended package of practice
(POP) without zinc, POP + 5 kg Zn ha
-1
, POP + 10 kg Zn ha
-1
as zinc sulphate. The results
showed that application of Zn increased the fresh yield of ginger from 7.72 to 9.57 kg 3m
-2
indicating
an increase of 23%. Zinc application also increased the oil, oleoresin, -sesquiphellandrene,
farnesene, camphene and Z-citral contents of ginger oil. Contrarily, Zn application decreased
zingiberene, -pinene, -curcumene and 1, 8 cineol contents of ginger oil. The quality components
such as fiber content varied from 3.05% to 3.43%, oil content from 1.33% to 1.73%, oleoresin from
3.35% to 5.41%, zingiberene from 13.1% to 21.8%, - pinene from 0.67% to 2.23%,
-sesquiphellandrene from 5.92% to 10.20%, farnesene from 5.58% to 11.1%, camphene from
3.06% to 5.36%, Z-citral from 3.73% to 6.54%, -curcumene from 5.32% to 7.70%, 1,8 cineol from
2.70% to 5.29%, -phellandrene from 1.87% to 4.18% and citral contents from 5.03% to 6.54%.
Keywords: ginger, ginger quality, zinc deficiency, zinc nutrition
Journal of Spices and Aromatic Crops
Vol. 22 (1) : 9194 (2013) Indian Society for Spices
www.indianspicesociety.in/josac/index.php/josac
92
aroma of ginger and they tend to be relatively
more abundant in the fresh (green) rhizome
than in the dried ginger (Zacharia 2008).
Oxygenated sesquiterpenes are relatively minor
constituents of the volatile oil but appear to be
significant contributors to its flavor properties.
Zn plays a key role in several critical cellular
functions, such as protein metabolism, gene
expression, structural and functional integrity
of bio membranes, photosynthate, C
metabolism, and Indole 3 acetic-acid (IAA)
metabolism. So Zn deficiency may alter several
physiological processes in plants. Keeping these
points in view, experiments were conducted
with the objective to study the quality and oil
composition of ginger in relation to Zn
fertilization.
The field experiment was conducted for three
years (200811) at Indian Institute of Spices
Research Experimental Farm, Peruvannamuzhi
and in farmers plot at Wayanad District, Kerala.
Ginger variety IISR Varada was planted in
raised beds of size 3 m 3 m. The soil of
experimental field was Ustic Humitropept deficient
in Zn (available Zn 0.56 mg kg
-1
). The crop was
manured as per the POP recommendation
(KAU 2009). The treatments consisting of the
recommended POP without Zn, POP+ 5 kg Zn
ha
-1
as ZnSO
4
, POP+ 10 kg zinc ha
-1
as ZnSO
4
were imposed at 45 DAP. The experimental
design was RBD with seven replications. The
crop was harvested after maturity and yield data
was recorded. The rhizome samples were taken
treatment wise peeled, dried and oil, fiber and
oleoresin contents were estimated as per
standard procedures (ASTA 1997).
Zn fertilization increased the yield to the tune
of 23.0% (Table 1). Similar yield increase was
earlier reported by Srinivasan et al. (2009) in
ginger for zinc application. The quality
parameters such as oil, oleoresin,
-sesquiphellandrene, farnesene, camphene,
-citral, -curcumene and that of zingibeiene,
-pinene and 1-8 cineol contents decreased
significantly due to Zn fertilization (Table 2).
Whereas, Zn did not influence -phellandrene
and citral and fibre contents. Sadanandan &
Hamza (1998) reported the increase in oil and
Hamza et al.
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93
oleoresin contents of ginger due to Zn
fertilization.
The potential of ginger in culinary, non-
culinary and medicinal fields is based on the
chemistry of volatile oil and non-volatile
pungent principles. The oil yield is about
2%3% and the oil consists of 64% sesquiterpene
hydrocarbons, 6% carbonyl compounds, 5%
alcohols, 2% monoterpene hydrocarbons and
1% esters. The main compounds are zingiberene
(29.5%) and sesquiphellandrene (18.4%)
(Zachariah 2008). The composition of these
constituents varies based on maturity, genotype
and agroclimatic conditions (Zachariah et al.
1999; Gaston & Cyril 2005).
It is concluded that application of Zn was
beneficial in increasing yield of ginger by 23%
also it increased the oil, oleoresin content,
-sesquiphellandrene, farnesene, camphene and
z-citral content of ginger oil. Zn fertilization
also found to decrease zingiberene, -pinene,
-curcumene and 1, 8 cineol content of ginger
oil. It was found that fiber content varied from
3.05 to 4.40%, oil content from 1.33% to 1.73%,
oleoresin from 3.35% to 5.41%, zingiberene
from 13.1% to 21.8%, - pinene from 0.67% to
2.23%, sesquiphellandrene from 5.92% to
10.20%, farnasene from 5.58% to 11.1%,
camphene from 3.06 to 5.36%, z-citral from
3.73% to 6.54%, -curcumene from 5.32% to
7.70%, 1,8 cineol from 2.70% to 5.29%, -
phellandrene from 1.87% to 4.18% and citral
from 5.03% to 6.54%.
References
ASTA 1997 Official Analytical Methods. 4
th
Edn,
American Spice Trade Association,
Washington DC.
Gaston V & Cyril P 2005 Chemistry of Ginger.
pp. 87160. In: Ravindran P N & Nirmal
Babu K (Eds. ) Ginger: The Genus
Zingiber, Medicinal and Aromatic
Plants-Industrial Profiles CRC PRESS
Boca Raton London, New York
Washington, D.C.
KAU 2009, Package of practices, Kerala
Agricultural University, Thrissur, 2009.
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Zinc effect on ginger
94
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Hamza S 2010 Integrated Nutrient
Management in Major Spices. Indian J.
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Sadanandan A K & Hamza S 1998 Effect of
organic farming on nutrient uptake
yield and quality of ginger (Zingiber
officinale). pp. 8994. In: Proc. National
Seminar on Water and Nutrient
Management for Sustainable Production
and Quality of Spices Indian Society for
Spices, Calicut.
Srinivasan V, Hamza S & Dinesh R 2009 Critical
limits of zinc in soil and plant for
increased productivity of ginger
(Zingiber officinale Rosc.). J. Indian Society
of Soil Sci. 57(2): 191195.
Srinivasan V, Hamza S, Krishnamurthy K S &
Thankamani C K 2004 Threshold level
of soil zinc for optimum production of
ginger (Zingiber officinale R), J. Spices
Arom. Crops 13 (1): 5557.
Zacharia T J 2008 Ginger. (pp: 7093) In:
Parthasarathy V A, Chempakam B &
John Zachariah T (Eds.), Chemistry of
Spices, CAB International, UK.
Zachariah T J, Sasikumar B & Nirmal Babu K
1999 Variation for quality components
in ginger and turmeric and their
interaction with environments. (pp.
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Hamza et al.
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 201329, 10 pages
http://dx.doi.org/10.1155/2013/201329
Research Article
Prescription Pattern of Chinese Herbal Products for
Diabetes Mellitus in Taiwan: A Population-Based Study
Chung-Yu Huang,
1,2
Yueh-Ting Tsai,
1
Jung-Nien Lai,
1,3
and Feng-Lin Hsu
4
1
Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No. 155, Section 2, Linong Road,
Taipei 112, Taiwan
2
Department of Traditional Medicine, Wan Fang Hospital, Taipei Medical University, Taipei 11696, Taiwan
3
Department of Chinese Medicine, Taipei City Hospital, Yangming Branch, Taipei 111, Taiwan
4
Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, No. 250, Wuxing Street, Taipei 11031, Taiwan
Correspondence should be addressed to Jung-Nien Lai; kareny@ms10.hinet.net
Received 23 December 2012; Revised 2 April 2013; Accepted 17 April 2013
Academic Editor: Srinivas Nammi
Copyright 2013 Chung-Yu Huang et al. Tis is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Background. Traditional Chinese medicine (TCM), when given as a therapy for symptom relief, has gained widespread popularity
among diabetic patients. Te aim of this study is to analyze the utilization of TCM among type 2 diabetic patients in Taiwan.
Methods. Te use of TCM for type 2 diabetic patients were evaluated using a randomly sampled cohort of 1,000,000 benefciaries
recruited from the National Health Insurance Research Database. Results. Overall, 77.9% (n = 31,289) of type 2 diabetic patients
utilized TCM and 13.9% (n = 4,351) of them used TCM for the treatment of type 2 diabetes. Among the top ten most frequently
prescribed herbal formulae, four remedies, Zhi-Bo-Di-Huang-Wan, Qi-Ju-Di-Huang-Wan, Ji-Sheng-Shen-Qi-Wan and Ba-Wei-Di-
Huang-Wan are derivative formulae of Liu-Wei-Di-Huang-Wan. In other words, Liu-Wei-Di-Huang-Wan and its derivatives were
found to be the most common herbal formulae prescribed by TCM doctors for the treatment of diabetes in Taiwan. Conclusion.
Although some evidence does support the use TCMto treat diabetes, the results fromthe current study may have been confounded
by placebo efect, which emphasize the need for well conducted, double-blind, randomized, placebo-controlled studies in order to
further evaluate the efcacy of Liu-Wei-Di-Huang-Wan on patients with type 2 diabetes.
1. Introduction
Type 2 diabetes is becoming a pandemic disorder, and the
related alarming increase in the prevalence of both microvas-
cular and macrovascular disease has raised signifcant con-
cerns [17]. Importantly, clinically signifcant morbidity is
present at diagnosis, but the development of diabetes-related
microvascular and macrovascular diseases may occur much
earlier and well before diagnosis. Due to a lack of appro-
priate care for preclinical diabetes, diabetes expenditures
have grown dramatically annually due to increased medical
care required by patients with diabetes-related complications.
Even worse, although many drugs improve glycemic control,
they do not necessarily provide real-world benefts. Previ-
ous reports have indicated that combination therapy with
metformin and glyburide and the use of thiazolidinediones
increase the risk of a composite end point involving car-
diovascular events and mortality [8, 9]. In addition, some
diabetes medication unfortunately results in a number of
common side efects such as nausea or upset stomach; these
unwanted conditions drive patients to seek alternative advice
[10]. Terefore, despite recent advances in intensive glycemic
control, diabetes mellitus continues to be an important public
health concern because it causes substantial morbidity and
mortality as well as long-term complications [11, 12]. Not
surprisingly, alternative therapies have become increasingly
popular and are quickly approaching conventional therapy in
their frequency of use as a treatment for diabetes and/or dia-
betes-related complications [1315].
Previous studies of traditional Chinese medicines have
found that tianhuafen (Radix Trichosanthis) [16], gegen (Ra-
dix Puerariae) [17], maimendong (Radix Ophiopogonis) [18],
2 Evidence-Based Complementary and Alternative Medicine
rougui (Cortex Cinnamomi) [19, 20], huangbai (Cortex Phel-
lodendri) [21] and aconitum (Aconitum carmichaeli) [22]
may have antidiabetic activity, and dihuang (Radix Rehman-
niae) [23], shanzhuyu (Fructus Corni) [24], renshen (Radix
Ginseng) [25], gancao (Radix Glycyrrhizae Praeparata) [26],
and bitter orange (Aurantii Fructus) [27] have been suggest-
ed to increase insulin secretion. Unfortunately, evidence ob-
tained in human studies is limited regarding patterns of use
of classical traditional Chinese medicine (TCM) in relation
to type 2 diabetes, which seem to be an area in which com-
plementary and alternative medicines have recently grown in
popularity. Furthermore, TCMs now seems to be marketed
without established efcacy or safety in many Western coun-
tries [28, 29]. Owing to the previously and to a lack of knowl-
edge about the prescription profle of TCMs, researchers and
conventional doctors have found it difcult to explore the
potential mechanisms of TCMtherapies targeting type 2 dia-
betes. Furthermore, it also has proved difcult to assess the
cost efectiveness of using TCM therapy and to observe the
interaction between Chinese herbs and conventional diabetes
drugs.
TCM, which includes acupuncture, traumatology manip-
ulative therapies, and Chinese herbal products, has been an
important part of health care in Taiwan for hundreds of years
and is fully reimbursed under the current National Health
Insurance (NHI) system. Previous studies using the NHI
research database have reported that female [30] and individ-
uals aged in their 30s [31] are more likely to use TCM among
the general population in Taiwan. Chinese herbal remedies
are the most commonTCMmodality usedby this population,
followed by acupuncture and traumatology manipulative
therapies. Te unique approach to TCM diagnosis involves
gathering clinical symptoms and signs, and then a treatment
principle is put forward in accordance with the aforemen-
tioned diagnostic process. In this situation, researchers in
Taiwan have found that symptoms, signs, and ill-defned con-
ditions are one of the most common reasons for TCM visits
across various diferent patient populations [3234]. Accord-
ingly, the claims that database, part of the Taiwan National
Health Insurance Research Database, provides are a platform
for understanding the utilization of TCM therapies by li-
censed TCM doctors [30, 31, 35]. Te aim of our study is to
analyze a random sample from this comprehensive database
and to determine the TCMutilization patterns of newly diag-
nosed type 2 diabetes patients in Taiwan. Te results of this
study should provide valuable information that will enable
physicians to respond to patient use of TCM in an informed
way, which will in turn strengthen further the patient-phy-
sician relationship when treating diabetes and diabetes-relat-
ed complications.
2. Materials and Methods
2.1. Data Resources. Tis study was designed as a population-
based study analyzing a sample of one million subjects select-
ed at randomfromthe 22 million benefciaries of the Nation-
al Health Insurance scheme of Taiwan and aimed to deter-
mine the prevalence of using prescribed Chinese herbal pre-
scriptions (CHP) among diabetes patients between January 1,
All type 2 diabetes patients,
Exclusion of prevalent cases of
Exclusion of 505 patients aged
type 2 diabetes before the end
A random sample of one million individuals from NHIRD
= 1,000,000
(1) having at least three outpatient visits with diabetes diagnosis
within 1 year, = 52,772
(2) having one or more hospital admission with diabetes
diagnosis, = 8,556
= 51,868
type 2 diabetes patients,
Newly diagnosed case of
= 40,668
type 2 diabetes patients in adults,
Newly diagnosed case of
= 40,163
Total = 53,294
TCM nonusers,
= 8,874
TCM users,
= 31,289
Exclusion of 949 cases with
type 1 diabetes and 477
patients with missing data on
gender and age
of 1997, = 11,200
<20 years
Figure 1: Flow recruitment chart of subjects from the one million
random samples obtained from the National Health Insurance
Research Database (NHIRD), 1997 to 2008, in Taiwan.
1998 and December 31, 2008. All data were obtained from
the National Health Insurance Research Database (NHIRD),
which includes all the reimbursement data of the NHI with
the identifcation numbers of all individuals being encrypted
and transformed; this database is maintained by the National
Health Research Institutes of Taiwan [36]. Te NHIRD data-
base contains patients gender and date of birth, all records of
clinical visits and hospitalization, prescribed drugs and dos-
ages, including CHP, and three major diagnoses coded in
the International Classifcation of Diseases, Ninth Revision,
and Clinical Modifcation (ICD-9-CM) formats [37].
2.2. Study Subjects. Te selection of study subjects from the
random sample of one million individuals was performed
as follows (Figure 1). First, we included all patients that (1)
had at least three outpatient visits with a diabetes diagnosis
within 1 year ( = 52,772) or (2) having one or more hospital
admission with diabetes diagnosis ( = 8,556) [38]. A total
of 53,294 subjects were obtained. Second, we excluded all
patients with type 1 diabetes ( = 949) or with missing infor-
mation on gender and age ( = 477). Tird, cases of diabetes
Evidence-Based Complementary and Alternative Medicine 3
( = 11, 200) that had been diagnosed before the end of 1997
were also excluded to ensure that all the subjects included
were newly diagnosed with type 2 diabetes in the time period
19982008. Fourth, subjects under 20 years of age ( = 505)
were also excluded to limit the study sample to adults. Finally,
40,163 study subjects were included in the study cohort.
2.3. Study Variables. To determine the key independent vari-
ables for utilization of TCM among diabetes patients, we se-
lected a series of demographic factors based onprevious stud-
ies [3, 3841]. Te subjects were categorized into four groups
according to age: 2039, 4059, 6079, and 80 years. Taiwan
was divided into seven geographic regions: Taipei city, Kaoh-
siung city, Northern region, Central region, Southern region,
Eastern region and Outlying islands. We split the monthly
wage of individuals into four levels: newTaiwandollars (NT$)
0, 119,999, 20,00039,999, and 40,000. We also searched
the NHIRDdatabase for clinical complications and treatment
records related to diabetes as independent variables. Te
complications associated with diabetes included nephropa-
thy (ICD-9 code, 581.81, 583.81, 585.1585.9), retinopathy
(362.01362.07, 365.44, 366.41, 369.00369.9), neuropathy
(353.5, 536.3, 354.0355.9, 713.5, 337.1, 357.2), peripheral cir-
culatory disorders (250.70), and other specifed manifesta-
tions (250.80) [42]. Te reimbursement database contains all
details related to the prescription of conventional medicines
for treating diabetes mellitus. Ten, for the fnal analysis, we
categorized the types of preparations into the following cate-
gories: sulfonylureas, biguanides, -glucosidase, meglitinide,
thiazolidinediones, guar gum, and insulin.
2.4. Statistical Analysis. Data analysis consisted of descriptive
statistics, including the prescription rates of TCMusers strat-
ifed by patients demographic characteristics, indications for
the prescription of TCM, and the most frequently prescribed
herbal formulae used when treating diabetes. Primary indi-
cations were classifed according to their ICD-9 code. Te
diagnoses were coded according to the ICD-9 and grouped
into a series of distinct broad disease categories. Te potential
efects of Chinese herbs contained in the ten most commonly
prescribed CHPs were grouped according to previous in vivo
and in vitro studies and are summarized in Table 4 [1627].
Multiple logistic regression was conducted to evaluate the
factors that correlated with TCM use. A signifcance level of
= 0.05 was selected. Te statistical sofware package SAS
9.13 was used for data management and analysis.
3. Results
Te database of outpatient claims contained information
on 40,163 patients with type 2 diabetes from 1998 to 2008.
Among them, 31,289 (77.9%) patients used TCM outpatient
services at least once. Most TCM users (91.2%) also received
diabetes treatment. Among all TCM users, 13.9% ( = 4,351)
used TCM for the treatment of type 2 diabetes. Details of
the demographic distribution of TCM users and nonusers
are presented in Table 1. Te mean age of TCM nonusers
was slightly higher than that of TCM users. Tere were more
TCM users than TCM nonusers with an income level of NT$
20,00039,999 or residing in Central Taiwan.
Te adjusted odds ratios (aORs) and 95% confdence
intervals (95% CIs) obtained by multiple logistic regression
are also presented in Table 1. Compared with the age group
4059 years (aOR = 1.00), those aged 2039 years were more
likely to be TCMusers. Tere was also a signifcant diference
between TCM users and nonusers with there being more of
the former in the income group of NT$20,00039,999. Afer
adjusting for other factors, patients with more type 2 diabetes
chronic complications (one complication: OR = 1.10, 95%
CI: 1.041.16; two complications: OR = 1.28, 95% CI: 1.19
1.38; more than three complications: OR = 1.25, 95% CI: 1.13
1.38) were more likely to seek TCM treatment than those
with no chronic complication (aOR = 1.00). Tere was no
signifcant diference in the diabetes treatment modalities
received (monotherapy (aOR=1.00) or combinationtherapy)
between TCMusers and TCMnonusers, except among those
who took more than fve types of antidiabetic drugs (OR
= 1.13, 95% CI: 1.011.26) for the control of blood sugar or
HbA1c.
Among the diabetes patients visiting TCM doctors,
3,627,622 (92.6%) visits involved the prescription of TCM,
while the rest were prescribed acupuncture and traumatol-
ogy manipulative therapies. Analysis of the major disease
categories for all TCM visits made by 31,289 TCM users
are summarized in Table 2. Te fndings show that symp-
toms, signs, and ill-defned conditions were the most com-
mon reason for using Chinese herbal prescriptions (CHP)
(16.8%, = 608,535), followed by endocrine, nutritional
and metabolic diseases, and immunity disorders (12.1%,
= 439,612), and diseases of digestive system (11.5%, =
417,611). Details of the most frequently prescribed CHP for
treating type 2 diabetes by TCM doctors are provided in
Table 3. Liu-Wei-Di-Huang-Wan (Rehmannia six pill) was
the most frequently prescribed CHP, followed by Bai-Hu-
Jia-Ren-Shen-Tang (white tiger plus ginseng combination),
Zhi-Bo-Di-Huang-Wan(zhibai Rehmannia six pill), Qi-Ju-Di-
Huang-Wan (chichu Rehmannia pill), Yu-Quan-Wan (jade
spring pill), Ji-Sheng-Shen-Qi-Wan (economic health shenqi
pill), Xue-Fu-Zhu-Yu-Tang (persica and achyranthes com-
bination), Ba-Wei-Di-Huang-Wan (eight-favour Rehmannia
pill), Bai-Hu-Tang (white tiger combination), and Gan-Lu-
Yin (sweet combination drink). Among the top ten most
frequently prescribed herbal formulae, Zhi-Bo-Di-Huang-
Wan, Qi-Ju-Di-Huang-Wan, Ji-Sheng-Shen-Qi-Wan, and Ba-
Wei-Di-Huang-Wan are four derivative formulae of Liu-Wei-
Di-Huang-Wan, which all contain Rhizoma Rehmanniae Pre-
parata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alis-
matis, Cortex MoutanRadicis, and Poria. Inother words, Liu-
Wei-Di-Huang-Wan and its various derivatives are the most
common herbal formulae prescribed by TCM doctors for
the treatment of diabetes in Taiwan. Te ten most frequently
prescribed CHPs include Chinese herbs that have been his-
torically used to lower serum glucose. Te potential efects
of these Chinese herbs when used to treat type 2 diabetes
are summarized in Table 4 and include increasing insulin
secretion, enhancing glucose uptake by adipose and muscle
Table 1: Demographic characteristics and results of multiple logistic regressions showing the adjusted odds ratio (aOR) and 95% CI
(confdence interval) for diabetes from 1998 to 2008 in Taiwan.
Characteristic All patients TCM
a
nonusers (%) TCM users (%) aOR
b
(95% CI
c
)
Number of cases 40,163 8,874 31,289
Gender
Male 20,971 (52.2) 5,718 (64.4) 15,253 (48.7) 1.00
Female 19,192 (47.8) 3,156 (35.6) 16,036 (51.3) 1.98 (1.882.08)
Age at diagnosis (years)
Mean SD 56.7 12.4 58.3 12.9 56.3 12.2
2039 3,175 (7.9) 609 (6.9) 2,566 (8.2) 1.11 (1.011.22)
4059 20,523 (51.1) 4,184 (47.1) 16,339 (52.2) 1.00
6079 15,244 (38.0) 3,653 (41.2) 11,591 (37.0) 0.79 (0.750.83)
80 1,221 (3.0) 428 (4.8) 793 (2.5) 0.46 (0.410.52)
Insured salaries (NT$
d
/month)
0 9,981 (24.9) 2,207 (24.9) 7,774 (24.9) 1.00
119,999 21,256 (52.9) 4,801 (54.1) 16,455 (52.6) 1.01 (0.951.08)
20,00039,999 5,414 (13.5) 982 (11.1) 4,432 (14.2) 1.33 (1.211.46)
40,000 3,512 (8.7) 884 (9.9) 2,628 (8.4) 1.02 (0.921.12)
Insured region
Taipei city 6,975 (17.4) 1,746 (19.7) 5,229 (16.7) 1.00
Kaohsiung city 2,802 (7.0) 606 (6.8) 2,196 (7.0) 1.21 (1.091.35)
Northern Taiwan 11,316 (28.2) 2,652 (29.9) 8,664 (27.7) 1.10 (1.021.18)
Central Taiwan 7,172 (17.8) 1,070 (12.1) 6,102 (19.5) 1.92 (1.762.09)
Southern Taiwan 10,447 (26.0) 2,436 (27.4) 8,011 (25.6) 1.12 (1.041.20)
Eastern Taiwan 1,130 (2.8) 288 (3.3) 842 (2.7) 0.97 (0.831.12)
Outlying islands 319 (0.8) 75 (0.8) 244 (0.8) 1.11 (0.851.45)
Number of diabetic complications
0 17,913 (44.6) 4,213 (47.5) 13,700 (43.8) 1.00
1 12,833 (32.0) 2,823 (31.8) 10,010 (32.0) 1.10 (1.041.16)
Nephropathy 2,990 (7.4) 724 (8.1) 2,266 (7.2)
Retinopathy 2,764 (6.9) 674 (7.6) 2,090 (6.7)
Neuropathy 5,339 (13.3) 993 (11.2) 4,346 (13.9)
Peripheral circulatory disorders (PCD) 844 (2.1) 213 (2.4) 631 (2.0)
Other specifed manifestations 896 (2.2) 219 (2.5) 677 (2.2)
2 6,296 (15.7) 1,226 (13.8) 5,070 (16.2) 1.28 (1.191.38)
Nephropathy + retinopathy 1,039 (2.6) 241 (2.7) 798 (2.5)
Nephropathy + neuropathy 1,588 (4.0) 300 (3.4) 1,288 (4.1)
Nephropathy + PCD 256 (0.6) 64 (0.7) 192 (0.6)
Nephropathy + others 273 (0.7) 61 (0.7) 212 (0.7)
Retinopathy + neuropathy 1,579 (3.9) 250 (2.8) 1,329 (4.3)
Retinopathy + PCD 234 (0.6) 45 (0.5) 189 (0.6)
Retinopathy + others 206 (0.5) 46 (0.5) 160 (0.5)
Neuropathy + PCD 582 (1.4) 107 (1.2) 475 (1.5)
Neuropathy l + others 429 (1.1) 81 (0.9) 348 (1.1)
PCD + others 110 (0.3) 31 (0.4) 79 (0.3)
3 3,121 (7.7) 612 (6.9) 2,509 (8.0) 1.25 (1.131.38)
Table 1: Continued.
Characteristic All patients TCM
a
nonusers (%) TCM users (%) aOR
b
(95% CI
c
)
Number of medical treatments for diabetes
None 3,370 (8.4) 627 (7.1) 2,743 (8.8)
Monotherapy
e
6,048 (15.1) 1,443 (16.3) 4,605 (14.7) 1.00
Two-drug combination
f
13,609 (33.9) 3,127 (35.2) 10,482 (33.5) 1.01 (0.941.09)
Tree-drug combination 8,675 (21.6) 1,937 (21.8) 6,738 (21.5) 1.00 (0.931.09)
Four-drug combination 5,026 (12.5) 1,093 (12.3) 3,933 (12.6) 1.01 (0.921.10)
Over fve-drug combination 3,435 (8.5) 647 (7.3) 2,788 (8.9) 1.13 (1.011.26)
a
TCM refers to traditional Chinese medicine;
b
OR refer to odds ratio;
c
CI refers to confdence interval;
d
NT$ refers to new Taiwan dollars, of which US$ 1 =
NT$30 approximately.
e
Monotherapy is the use of a single antidiabetic drug (sulfonylureas, biguanides, -glucosidase, meglitinide, thiazolidinediones, guar gum, or insulin).
f
combination therapy is the use of more than one antidiabetic drug.
tissues, inhibiting glucose absorption from intestine, inhibit-
ing glucose production from hepatocytes, and decreasing in-
sulin resistance or enhancing insulin sensitivity.
4. Discussion
Te prevalence of type 2 diabetes in Taiwan over the 11 years
inthe study was 4.0%, whichis inline withthe estimates given
by previous surveys [1, 43]. Worthy of note, the utilization of
TCMamong adults with type 2 diabetes in Taiwan during the
study period was 77.9%, which appears to be high compared
with previous fndings [14, 15]. TCM is a unique traditional
therapy approach for various ailments that has been used in
Taiwan for over hundreds of years, and this long period of use
may contribute signifcantly to the high prevalence of TCM
usage among type 2 diabetic subjects. In addition, it should
be noted that TCM treatment is covered by the NHI system.
Terefore, unsurprisingly, the prevalence of CHP for treating
type 2 diabetes among adults is comparatively higher in Tai-
wan than in other countries [15, 44]. Te present study in-
cludes all patients who were newly diagnosed with type 2
diabetes by qualifed conventional doctors between 1998 and
2008 from a random sample of one million subjects among
the insured general population; importantly the rate of in-
sured individuals has been consistently above 96%since 1997,
and therefore we can rule out the possibility of selection bias.
Te present results show that, although 91% of type 2
diabetic patients in Taiwan have received antidiabetic treat-
ment, over half of them still have sufered from one or
more diabetes complications during the 11-year follow-up.
Nephropathy and neuropathy were the two most common
diabetes complications. One possibility is that type 2 diabetes
has a long asymptomatic preclinical phase that is likely to go
undetected [4549], and the injurious efects of asympto-
matic hyperglycemia, therefore, have resulted in a high inci-
dence of microvascular and macrovascular complications [4,
42]. Te present study found that patients with type 2 diabetes
who developed more than one site of involvement re-diabetes
complications were more likely to seek advice from a TCM
doctor. However, regardless of their experience in receiving
more than one type of antidiabetic drug with the aim of
improving their poor control of serumblood sugar, the choice
of any of the major medical options available to patients with
type 2 diabetes was not associated with the use of TCM.
Hence, we suggest that when TCM is used to treat type 2 dia-
betes in Taiwan, this is generally an adjunct to diabetes treat-
ment rather than a replacement for it.
Te present fndings show that, among diabetes patients,
females and those aged 2039 years were more likely to be
TCM users than males and other age groups as shown in
Table 1. As showninTable 2, symptoms, signs, andill-defned
conditions were the most common reasons for using CHP
(16.8%, = 608, 535), followed by endocrine, nutritional
and metabolic diseases, and immunity disorders (12.1%,
= 439,612) and diseases of digestive system (11.5%, =
417,611). Further analysis found that TCM doctors tended
to use Chinese herbal remedies targeting diabetes as well as
gastrointestinal disorders that might be the uncomfortable
side efects of diabetes drugs. Although previous studies have
demonstrated that acupuncture might be related to an alter-
native therapy for treating hyperglycemia and diabetes com-
plications, the present study indicated that acupuncture in
Taiwan is used by this study population mainly for diseases
of the musculoskeletal system and connective tissue.
Liu-Wei-Di-Huang-Wan was the most frequently pre-
scribed formula for treating type 2 diabetes in Taiwan dur-
ing the study period, as shown in Table 3. Liu-Wei-Di-Huang-
Wan is among the most highly regarded ancient Chinese
herbal formulae and was frst documented in the classical
Chinese text Xiao Er Yao Zheng Zhi Jue (Key to Terapeutics
of Childrens Diseases) circa 1119 A.D. In the classical lit-
erature, Liu-Wei-Di-Huang-Wan is said to nourish yin and to
invigorate the kidney, which might indicate it as a poten-
tially efcacious therapy for reducing hyperglycemia and
relieving neuropathic andnephropathic complications india-
betes mellitus [5053]. Among the top ten most frequently
prescribed formulae for treating type 2 diabetes, Zhi-Bo-Di-
Huang-Wan, Qi-Ju-Di-Huang-Wan, Ji-Sheng-Shen-Qi-Wan,
and Ba-Wei-Di-Huang-Wan, which are all derivatives of Liu-
Wei-Di-Huang-Wan, are prescribed to alleviate various com-
mon symptoms of type 2 diabetes, namely, unusual thirst,
blurred vision, frequent urination, and cold feeling in the
Table 2: Frequency distribution of traditional Chinese medicine (TCM) visits by major disease categories (according to 9th ICD codes)
among diabetes patients from 1998 to 2008 in Taiwan.
Major disease category ICD-9-CM codes
No. of visits (no. of patients)
Chinese herbal remedies
Acupuncture or
manipulative therapies
Total of TCM
Infectious and parasitic diseases 001139 21,083 (761) 69 (13) 21,152 (772)
Neoplasms 140239 33,132 (366) 613 (20) 33,745 (381)
Endocrine, nutritional and
metabolic diseases, and
immunity disorders
240279 439,612 (5,565) 1,995 (127) 441,607 (5,620)
Diabetes 250 377,621 (4,328) 1,505 (65) 379,126 (4,350)
Others 61,991 (1,703) 490 (62) 62,481 (1,742)
Mental disorders 290319 24,834 (858) 536 (28) 25,370 (876)
Diseases of nervous system and
sense organs
320389 104,033 (3,962) 4,593 (802) 108,626 (4,513)
Diseases of circulatory system 390459 180,821 (3,647) 5,857 (444) 186,678 (3,892)
Diseases of respiratory system 460519 377,262 (10,505) 1,423 (126) 378,685 (10,537)
Diseases of digestive system 520579 417,611 (9,387) 1,332 (129) 418,943 (9,432)
Diseases of genitourinary system 580629 171,262 (4,145) 1,294 (63) 172,556 (4,170)
Diseases of skin and
subcutaneous tissue
680709 61,387 (2,784) 280 (41) 61,667 (2,810)
Diseases of musculoskeletal
system and connective tissue
710739 318,920 (8,808) 82,936 (12,682) 401,856 (16,932)
Symptoms, signs, and ill-defned
conditions
780799 608,535 (14,216) 3,839 (458) 612,374 (14,345)
Injury and poisoning 800999 17,994 (1,389) 90,542 (14,141) 108,536 (14,625)
Supplementary classifcation
+
V01V82, 115 (9) 0 (0) 115 (9)
E800E999 0 (0) 0 (0) 0 (0)
Others
851,021 (15,034) 96,311 (11,164) 947,332 (18,954)

Total 3,627,622 (27,135) 291,620 (22,891) 3,919,242 (31,289)
Others include ICD-9-CM codes 280289, 630677, 740759, 760779 and missing/error data;
+
Supplementary classifcation of factors infuencing health
status and contact with health service, external causes of injury and poisoning.
Table 3: Ten most common herbal formulae prescribed by TCM doctors for the treatment of type 2 diabetes among 31,289 patients from
1998 to 2008 in Taiwan.
Herbal formulae English name
Number of
person-days
= 775,447 (%)
Average daily dose (g)
Average duration for
prescription (days)
Liu-Wei-Di-Huang-Wan Rehmannia six pill 62,249 (8.0) 8.0 47.9
Bai-Hu-Jia-Ren-Shen-Tang
White tiger plus ginseng
combination
42,676 (5.5) 7.5 47.4
Zhi-Bo-Di-Huang-Wan Zhibai Rehmannia six pill 37,918 (4.9) 5.7 45.4
Qi-Ju-Di-Huang-Wan Chichu Rehmannia pill 37,796 (4.9) 5.7 64.4
Yu-Quan-Wan Jade spring pill 35,878 (4.6) 5.6 54.8
Ji-Sheng-Shen-Qi-Wan
Economic health shenqi
pill, life-saving renal Chi
pill
27,347 (3.5) 7.1 43.6
Xue-Fu-Zhu-Yu-Tang
Persica and achyranthes
combination
22,708 (2.9) 5.3 45.9
Ba-Wei-Di-Huang-Wan
Eight-favour Rehmannia
pill
19,247 (2.5) 9.1 41.5
Bai-Hu-Tang White tiger combination 18,801 (2.4) 7.0 42.1
Gan-Lu-Yin Sweet combination drink 18,502 (2.4) 5.1 38.1
Table 4: Potential efects of herbs present in the ten most common herbal formulae prescribed by TCM doctors for treating type 2 diabetes.
Herbal formulae Number of herbs Ingredient herbs
Liu-Wei-Di-Huang-Wan 6
Rhizoma Rehmanniae Praeparata
A,B,D,E
, Fructus Corni
A,D
, Rhizoma Dioscoreae
B,E
,
Rhizoma Alismatis
B
, Cortex Moutan Radicis, Poria
B,E
.
Bai-Hu-Jia-Ren-Shen-Tang 5
Gypsum Fibrosum, Rhizoma Anemarrhenae
E
, Radix Glycyrrhizae Praeparata
A
,
Semen Oryzae Sativae, Radix Ginseng
A,B,C,D
.
Zhi-Bo-Di-Huang-Wan 8
Rhizoma Anemarrhenae
E
, Cortex Phellodendri, Rhizoma Rehmanniae
Praeparata
A,B,D,E
, Fructus Corni
A,D
, Rhizoma Dioscoreae
B,E
, Rhizoma Alismatis
B
,
Cortex Moutan Radicis, Poria
B,E
.
Qi-Ju-Di-Huang-Wan 8
Flos Chrysanthemi, Fructus Lycii
B,E
, Rhizoma Rehmanniae Praeparata
A,B,D,E
,
Fructus Corni
A,D
, Rhizoma Dioscoreae, Rhizoma Alismatis
B
, Cortex Moutan
Radicis, Poria
B,E
.
Yu-Quan-Wan 9
Radix Trichosanthis, Radix Puerariae
A,B
, Radix Ophiopogonis
A,C,D
, Radix Ginseng,
Poria
B,E
, Radix Astragali
B,E
, Radix Glycyrrhizae Praeparata, Fructus Mume, Radix
Astragali Praeparata.
Ji-Sheng-Shen-Qi-Wan 10
Semen Plantaginis, Radix Achyranthis Bidentatae, Ramulus Cinnamomi, Radix
Aconiti, Rhizoma Rehmanniae Praeparata, Fructus Corni, Rhizoma Dioscoreae
B,E
,
Rhizoma Alismatis
B
, Cortex Moutan Radicis, Poria
B,E
.
Xue-Fu-Zhu-Yu-Tang 11
Chinese Angelia Root, Rhizoma Rehmanniae Praeparata, Peach Kernel, Safower,
Bitter Orange
A
, Red Peony Root, Bupleurum Root, Glycyrrhiza, Platycodon Root,
Chuanxiong Rhizome, Cyathula Root.
Ba-Wei-Di-Huang-Wan 8
Ramulus Cinnamomi
E
, Radix Aconiti
B
, Rhizoma Rehmanniae Praeparata, Fructus
Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan Radicis, Poria
B,E
.
Bai-Hu-Tang 4
Gypsum Fibrosum, Rhizoma Anemarrhenae, Radix Glycyrrhizae Praeparata,
Semen Oryzae Sativae.
Gan-Lu-Yin 10
Rhizoma Rehmanniae, Radix Ophiopogonis
A,C,D
, Radix Glycyrrhizae Praeparata,
Herba Dendrobii, Radix Asparagi, Eriobotryae Folium, Bitter Orange
A
, Scutellariae
radix, Wormwood Herb, Rhizoma Rehmanniae Praeparata.
A
Increase in insulin secretion,
B
enhancement of glucose uptake by adipose and muscle tissues,
C
inhibition of glucose absorption by the intestine,
D
inhibition
of glucose production by hepatocytes, and
E
decrease in insulin resistance or enhancement of insulin sensitivity.
limbs, respectively. Other frequently prescribed formulae are
associated with severe dysphoric thirst and lassitude (Bai-
Hu-Jia-Ren-Shen-Tang or white tiger plus ginseng decoction,
Bai-Hu-Tang or white tiger decoction, Yu-Quan-Wan or jade
spring combination, and Gan-Lu-Yin or sweet dewdecoction
or sweet combination drink) and with peripheral neuropa-
thy due to blood stasis (Xue-Fu-Zhu-Yu-Tang or Persica
and Carthamus Combination). Although, previous in vitro
studies have found that some Chinese herbs are able to
decrease serum levels of glucose, glycosylated proteins, and
hemoglobin A1C, possibly by blocking intestinal absorption
and/or inhibiting hepatic glucose-6-phosphatase [50, 54],
there have not yet been any clinical trials that have demon-
strated the efcacy and safety of Liu-Wei-Di-Huang-Wan and
its derivatives when treating diabetes type 2. In general, TCM
doctors treated diabetes patients complaints according to
the syndrome diferentiation theory rather than by making
a specifc diagnosis; this is based on holistic consideration
of diabetes patients who are sufering from symptoms and
complications at various sites. In this context and in line
with previous results [51], the present study found that TCM
doctors in Taiwan prescribed herbal therapies mainly to opti-
mize the bodys ability to function normally, rather than as a
cure for diabetes. Moreover, despite inadequate data on the
clinical safety and efcacy of CHP when treating diabetic
patients, a large number of patients use them. Tus, based on
the present trend in TCM utilization, herbal remedies for
treating diabetes and/or diabetic complications will continue
to be used. Although we respect the patients choice of med-
ical care, we recommend that TCM practitioners and physi-
cians should carefully monitor patient blood glucose levels
and the potential side efects of CHP when they are being
used alongside or in lieu of diabetes drugs. Further studies
are warranted to assess the formulae generally used by TCM
doctors in this study in order to determine whether they
are really useful as add-on treatments for patients receiving
antidiabetic treatment.
Te present study has three limitations. First, this study
did not include Chinese herbal remedies or decoctions that
were purchased directly from TCM herbal pharmacies, nor
did we include health foods containing herbs. Tus, the fre-
quency of CHP utilization might have been underestimated.
However, because the NHI systemcovers TCMprescriptions,
which generally cost less than the herbs sold in Taiwans
markets, the likelihood that subjects purchased a lot of other
herbs outside the NHI database is not high. Second, we are
unable to drawany conclusionabout the relationship of blood
glucose and haemoglobin A1C levels with respect to TCM
utilization owing to the lack of actual clinical data. Tird,
this was a retrospective study and thus does not include a
randomized placebo group. Tus, great caution is necessary
when interpreting the results of the most commonly pre-
scribed Chinese formulae obtained in the present study due
to the possibility of a placebo efect.
5. Conclusions
Our results suggest that, based on the coexistence of both
conventional and traditional Chinese medical treatments, of
most the diabetes patients consume herbal therapies with
the intention of relieving their diabetes-related symptoms,
rather than because they have rejected standard diabetes
treatments. Liu-Wei-Di-Huang-Wan and its derivatives are
the most frequently prescribed formulae by TCM doctors
in Taiwan for diabetes patients. Having recognized the use
of TCM, exploring any potential interactions and adverse
efects, and integrating both technologies into a holistic treat-
ment system may be benefcial to the overall health, presence
of comorbidities, and quality of life, of patients with type 2
diabetes. It is worth noting that although some evidence does
support the use of TCMto treat diabetes, the results fromthe
current study may have been confounded by the placebo
efect. Tis emphasizes the need for well-conducted, double-
blind, randomized, placebo-control studies to further evalu-
ate efcacy when Liu-Wei-Di-Huang-Wan is given to patients
with type 2 diabetes.
Acknowledgments
Tis research was conducted at the Institute of Traditional
Medicine at the School of Medicine, National Yang-Ming
University, Taipei. Te authors would like to express sincere
gratitude for the partial support provided for this project in
the form of Grants from the Department of Health, Taipei
City Government (38), the Committee on Chinese Medicine
and Pharmacy (CCMP100-RD-033), and the National Sci-
ence Council (NSC 99-2320-B-010-011-MY2), Taiwan.
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Digestion 2011;83(suppl 1):16
DOI: 10.1159/000323397
Flavor Perception in Human Infants:
Development and Functional Significance
GaryK.Beauchamp JulieA.Mennella
Monell Chemical Senses Center, Philadelphia, Pa. , USA
such as carrot and garlic, occurs through flavorings in breast
milk, infant formula and early foods. These early experiences
mold long-term food and flavor preferences which can im-
pact upon later health. Copyright 2011 S. Karger AG, Basel
Flavor Perception
The flavor of a food or beverage is defined here as the
perceptual combination of three anatomically distinct
sensory systems, namely smell (cranial nerve 1), oral
chemical somatosensory stimulation (cranial nerve 5)
and taste (cranial nerves 7, 9, and 10). Thus we do not con-
sider visual or auditory stimuli as contributing to
flavor, although these sensory systems are certainly in-
volved in food identification, choice and appreciation.
A remarkable aspect of flavor perception is that, al-
though the sensory systems that underlie it are complete-
ly anatomically separate, the sensory impression is uni-
tary. That is, we do not typically analyze the flavor of a
carbonate beverage, for example, into its sweetness, the
degree to which it tingles, and its fruitiness; instead we
perceive a carbonated beverage as a single impression.
How the brain accomplishes this melding of the senses
into a single percept is the focus of much current research
using various methods but particularly, in humans, func-
tional magnetic resonance imaging.
Key Words
Taste Smell Flavor Infant Child Food Imprinting
Abstract
Background: Foods people consume impact on their health
in many ways. In particular, excess intake of salty, sweet and
fatty foods and inadequate intake of fruits and vegetables
have been related to many diseases including diabetes, hy-
pertension, cardiovascular disease and some cancers. The
flavor of a food determines its acceptability and modulates
intake. It is thus critical to understand the factors that influ-
ence flavor preferences in humans. Aim: To outline several
of the important factors that shape flavor preferences in hu-
mans. Methods: We review a series of studies, mainly from
our laboratories, on the important role of early experiences
with flavors on subsequent flavor preference and food in-
take. Results and Conclusions: Some taste preferences and
aversions (e.g. liking for sweet, salty and umami; disliking for
bitter) are innately organized, although early experiences
can modify their expression. In utero events may impact on
later taste and flavor preferences and modulate intake of nu-
trients. Both before and after birth, humans are exposed to
a bewildering variety of flavors that influence subsequent
liking and choice. Fetuses are exposed to flavors in amniotic
fluid modulating preferences later in life and flavor learning
continues after birth. Experience with flavors that are bitter,
sour or have umami characteristics, as well as volatile flavors
Published online: March 10, 2011
Gary K. Beauchamp
Monell Chemical Senses Center
3500 Market Street, Philadelphia, PA 19104 (USA)
Tel. +1 267 519 4710, Fax +1 215 898 2084
E-Mail beauchamp @ monell.org
2011 S. Karger AG, Basel

Accessible online at:
www.karger.com/dig
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Digestion 2011;83(suppl 1):16 2
Importance of Flavor Perception
It is generally believed that the senses of vision and
hearing are the most important, at least for humans, be-
cause it is so devastating to lose them. In contrast, the
chemical senses of flavor are considered of secondary im-
portance: their loss, while unpleasant, is not as serious.
This view is debatable on two grounds. First, most people
do not realize how difficult it would be to live without any
flavor perception because it is extremely rare to lose all
three sensory systems simultaneously. Indeed, it is even
quite rare to lose the sense of taste (ability to detect sweet,
sour, salty, bitter and umami), but when it does occur, for
example following radiation therapy for head and neck
cancer, eating and nutrition is often severely compro-
mised [1] . Second, while loss of the flavor senses may not
be as disruptive as loss of vision and hearing, the behav-
iors that they mediate, particularly food choice and in-
take, are among the most important ones in terms of the
health problems found in developed and developing so-
cieties. That is, a full understanding of flavor perception
is central to preventing such diseases as hypertension,
obesity, heart disease, diabetes and some cancers. All of
these diseases are strongly impacted by food choices
which in turn are determined, in part, by how much we
like (or dislike) the flavor of the food. Rather than being
secondary senses, the chemical senses of flavor take a pri-
mary role when it comes to major issues of human health.
Expanding Our Conception of Flavor and Taste
Flavor is by definition a perceptual quality elicited by
molecules associated with foods and beverages. During
the past two decades, major advances have been made in
our understanding of how these flavor molecules are rec-
ognized at the intersection between the mucus mem-
branes of the oral and nasal cavities and the neural pro-
cesses that send flavor messages to the brain. We now
know much about olfactory receptor structure and func-
tion [2] , the receptors for pungent chemicals such as cap-
saicin, carbonation and the cooling of menthol [3] , and
taste receptor structure and function.
We focus here on one of these sensory systems, the
sense of taste. For hundreds of years, investigators have
speculated on the nature of the receptors for taste com-
pounds, but it has not been until the last 10 years or so
that definitive studies of their molecular nature have
been published [4] . We now know that sweet stimuli are
primarily or exclusively recognized by a dimer consisting
of two closely related 7-transmembrane receptors (T1R3
and T1R2) that together form a venus flytrap structure
atop taste receptor cells of taste buds on the tongue and
the palate. Umami taste (the taste characteristic of the
sodium salt of glutamic acid MSG in humans and
apparently a more general amino acid receptor in many
other species) is also recognized by a dimer (T1R3 and
T1R1), but other glutamate receptors have also been im-
plicated. Bitter taste is recognized by a family of about 25
7-transmembrane receptors (the T2Rs), although we are
still unsure as to the sensitivities of each receptor to spe-
cific bitter compounds as well as whether there may be
other mechanisms of detecting bitter compounds. Still
something of a puzzle is the nature of sour and salty re-
ceptors. It is thought that sour taste is mediated by a
channel protein and a number of candidate receptors
have been suggested. Salt taste has long been thought to
be detected by an epithelial sodium channel (an ENaC),
and recently this has been strongly supported by studies
demonstrating in mouse models that if the ENaC is dis-
rupted, salt taste response is altered [5, 6] . However, these
mice with the disrupted ENaC retain substantial respon-
siveness to sodium and other salts demonstrating that
there is/are additional mechanisms for detecting salt that
remain to be defined. The taste system may also be re-
sponsive to other classes of compounds (e.g. calcium
salts, fatty acids), but less is known about the underlying
mechanisms for detecting these nutrients [4] .
The identification of taste receptors for sweet, umami
and bitter compounds about a decade ago set off an ex-
plosion of studies to characterize them, to explore the re-
lationships between structure and function, to investi-
gate their variation within and between species and to
evaluate their functional characteristics. It also set in
place a search to determine whether they might be ex-
pressed in tissues outside the mouth. Surprisingly or
perhaps in hindsight not so surprising so-called taste
receptors were identified in the gut, the pancreas and the
brain [7, 8] .
Understanding the functional significance of these re-
ceptors in these non-oral organs is now an extremely ac-
tive area of research and it is expected that they may play
a significant role in nutrient recognition and regulation
[7, 8] . Had investigators first identified these receptors in
the gut, they may well have been labeled gut chemosen-
sory receptors. In this case there would have been sur-
prise to discover that they are also found in the mouth!
One can now consider the ingestive and digestive tube,
from the mouth through the stomach and intestine, to be
a single super organ. This super organ first recognizes
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Flavor Perception in Infants:
and discriminates among nutrients consciously by oral
and nasal receptors when decisions to accept or reject are
made. Next, once nutrients pass the mouth and enter the
gut, these same receptors, as well as others, no longer sig-
nal the presence of nutrients consciously but instead re-
spond by producing local hormonal signals or by sending
messages through the vagus nerve to the unconscious
parts of the brain that are important for nutrient recogni-
tion and utilization. These conscious and unconscious
signals are orchestrated so that food can be efficiently
recognized and processed and that non-foods (e.g. bitter
toxic compounds) are avoided or detoxified.
Origin of Flavor Preferences
Flavor perception and preferences guide food selec-
tion, but what determines flavor preferences? We have
been investigating this question in a program of research
that has focused on the interacting role of genetic and ex-
periential influences in the human fetus, the infant and
the child. In this section, we briefly describe some of our
findings. This work has been more thoroughly reviewed
recently elsewhere [9] from which this summary is taken.
Liking for taste stimuli is generally strongly influ-
enced by inborn (innate) factors [ 10 ] . For example, foods
that are sweet are innately preferred by most or all herbi-
vores and omnivores, presumably since sweetness re-
flects the presence of caloric sugars in plants. Infants and
children have higher preferences than adults and these
preferences can be modified by experience [for review,
see 11 ] . For umami, an innate component to liking is also
likely [ 11 ] . Bitter taste signals the presence of potentially
toxic compounds and hence substances that are bitter are
generally disliked and avoided [ 12 ] . That many of the bit-
ter foods that people like most (e.g. coffee, tea, beer) have
pharmacological properties helps explain their desirabil-
ity and the fact that people need to learn to like them.
Nevertheless, with experience even bitter vegetables can
come to be liked [ 13 , 14 ] , although whether the bitterness
of these is actually liked or whether the other flavor char-
acteristics are sufficient to overcome the dislike of bitter-
ness is not known.
A sensitivity to and preference for salty tasting sub-
stances (almost the only one being NaCl) also appears to
have an innate component that develops at around 4
months of age. By 2 years of age, childrens preferences for
salty foods are even greater than they are for adults [ 1 , 15 ] .
Along with proposed innate factors, prenatal develop-
mental events modify the infants and childs preferences
for salty tastes. Severe maternal emesis can have an en-
during influence on response of offspring to salty taste
[1618] . Similarly, we have shown [19] that several behav-
ioral measures related to salty taste preference are in-
versely related to birth weight over the first 4 years of life.
Recent unpublished studies from our laboratories suggest
that early experiences may profoundly modify the degree
of salt liking in children. If verified, these observations
are significant since it is generally accepted that excess
salt intake can lead to or exacerbate hypertension. One
set of factors predisposing children and adults to high salt
intake may be the heightened preferences that are caused
by in utero and early postnatal experiences.
Flavor Learning: Prenatal Life
Preferences for flavor compounds detected by the
sense of smell are generally more highly influenced than
taste preferences by learning with learning early in life,
even in utero, being particularly salient [ 10 , 20 ] . The sen-
sory environment in which fetuses live, the amniotic sac,
changes as a function of the food choices of the mother
since these foods flavor amniotic fluid [ 21 ] . Experiences
with such flavors lead to heightened preferences for these
flavors shortly at birth [ 22 , 23 ] and at weaning [ 24 ] . In an
experimental study, infants whose mothers were ran-
domly assigned to drink carrot juice during the last tri-
mester of pregnancy enjoyed carrot-flavored cereals more
than infants whose mothers did not drink carrot juice or
eat carrots [ 24 ] . Thus, like other mammals, prenatal ex-
periences with food flavors transmitted from the moth-
ers diet to amniotic fluid lead to greater acceptance and
enjoyment of these foods during weaning.
Postnatal Flavor Learning via Infant Formula
Flavor learning continues after birth as a consequence
of exposure to commercial infant formulas and/or to
breast milk. Studies of formula feeding allow us to pre-
cisely control sensory experiences of the infant, and
through this research we have demonstrated there is a
sensitive learning period in the first several months of life
during which unpalatable flavors (to those not familiar
with them) can be rendered palatable.
This body of research has randomized infants to feed
either standard commercial cow milk formulas (CMF) or
extensively protein hydrolysate formulas (PHF) since
these formulas differ enormously in flavor. In particular,
the flavor of PHF is extremely unpleasant to those unfa-
miliar with them, having a bitter and sour taste, and a
nauseating smell and aftertaste, perhaps because many
amino acids taste sour and bitter [ 25 ] . Infants less than
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34 months of age who have never been exposed to hydro-
lyzed casein (HC) formulas readily accept them and ap-
pear not to dislike them as determined both by their will-
ingness to consume them and by the absence of charac-
teristic facial responses of rejection when being fed them
[ 26 , 27 ] . In marked contrast, infants over 56 months of
age with no experience with HC formulas strongly dislike
and reject them [ 26 , 27 ] . However, infants tested at about
this same age who have never been exposed to HC formu-
las during the first few months of life not only do not reject
them but even appear to relish them [ 28 ] .
A clinical trial to characterize the timing and duration
of this sensitive period [ 28 ] demonstrated that 3 months
of PHF exposure led to a similar acceptance as 1 month
of exposure. Although these infants were more accepting
than infants with no exposure, they were less accepting
than infants with 7 months of exposure, thereby provid-
ing strong evidence for a dosing effect. The infants age
when flavor experiences began was also significant.
Among infants exposed to PHF for 1 month, those who
first fed PHF at 3.5 months rejected PHF relative to CMF
on its first presentation more than infants exposed at
younger ages, which provides evidence for a timing effect
with younger infants being more willing to sample.
Although we have identified one sensitive period for
the acceptance of PHF, one cannot assume there is only
one sensitive period in flavor programming, any more
than there is only one sensitive period for auditory learn-
ing [ 29 ] or visual learning [ 30 ] . The model system that we
identified allows us to explore the ability to change be-
havior based on experiences during only one period. Like
other senses, windows of plasticity in flavor learning may
occur at different developmental periods. Moreover, they
surely do not shut abruptly, and significant plasticity is
retained as the child ages. Thus, learning of new flavor
preferences can occur across the life span. What we argue
here is that there is greater plasticity and more permanent
effects of early compared with later flavor experiences.
But what exactly is learned during this early sensitive
period? In one study we exploited the fact that although
all brands of PHF share common flavor attributes and are
all perceived as unpleasant by unexposed older children
and adults, they each have unique flavor characteristics
[ 25 ] . Infants were found to prefer the flavor of the brand
of PHF on which they had been fed over an unfamiliar
brand. In other words, flavor acceptance is quite specific
to the flavors experienced. This suggests that during PHF
exposure infants form a relatively precise flavor image
which, when later matched, enhances intake and elicits
pleasure.
Among infants tested prior to being introduced to ta-
ble foods, experiences with flavors in formula impact on
preferences for the taste qualities in food. Infants feeding
PHF ate larger amounts of infant cereals that tasted sa-
vory, sour and bitter, and ate them at a faster rate than
those fed milk-based formulas [14]. That they liked the
savory- and bitter-tasting cereals is indicated by the few-
er facial expressions of distaste displayed during feeding.
When compared to CMF, and presumably breast milk
[ 31 , 32 ] , PHF formulas have more pronounced savory, bit-
ter and sour tastes and stronger odors [ 25 ] . Thus, infants
who regularly feed PHF have more experiences early in
life during sensitive periods with these taste and flavor
qualities which impacts on later food choice.
The effects of formula-based flavor experiences are
long-lived. Four- to 5-year-old children who were fed
PHF during infancy exhibited more positive responses to
foods and beverages characterized by sensory attributes
associated with them (e.g. sour taste, aroma, chicken,
broccoli) when compared with same-aged children with-
out such experience [ 33 , 34 ] . These results are consistent
with studies on children and adults with phenylketonuria
(PKU). The dietary regimen to treat PKU consists of an
unpalatable (to unexposed individuals) HC formula with
phenylalanine removed. When given a choice, children
and adolescents with PKU preferred their bad-tasting
formula to that of the new formulation that was more pal-
atable to naive children and adults [ 35 ] . In other words,
the characteristic flavor of the formula experienced in
early life is imprinted and remains as a preference for a
considerable time.
Postnatal Flavor Learning via Human Milk
We believe that the principles revealed in studies with
controlled exposures to infant formulas apply directly to
the natural breastfeeding situation. Human milk con-
tains not only nutrients such as carbohydrates, proteins,
vitamins and minerals, but also flavors which are derived
from the foods, spices and beverages ingested or inhaled
(e.g. tobacco) by the mother [ 20, 24, 3639 ] . In this regard,
the natural feeding experience of the breast-fed infants is
characterized by a great variability of flavor experiences
that depend directly on the dietary choices made by the
mother. For example, when nursing mothers consume
garlic, vanilla or many other flavors, their milk becomes
recognizably flavored and these flavors are detected by
the nursing infant. Thus breast-fed infants experience
flavor compounds from the foods and beverages that the
mother has chosen to consume, and these experiences
influence the infants subsequent liking and acceptance
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Flavor Perception in Infants:
of these flavors in foods as has been reported for other
mammals [ 40 42 ] . Breast milk may thus serve as a
bridge between the in utero experiences with flavors in
amniotic fluid to those in solid foods at weaning and be-
yond. Breastfeeding (unlike formula feeding) provides
the infant with the potential for a rich source of varying
flavor experiences. Since the sweetness and textural
properties of human milk, such as viscosity and mouth
coating, also vary both between and within mothers [ 31,
32 ] , breastfeeding provides an even richer variation in
oral sensory stimulation.
How does this early sensory experience with flavors in
breast milk impact on food choice? The study referred to
earlier [ 24 ] on carrot flavor exposure in utero also includ-
ed a group of breastfeeding infants whose mothers con-
sumed carrot juice during the first 3 months of the in-
fants life. The exposure to flavors in breast milk en-
hanced the infants responses to carrot flavor when they
were tested at weaning to approximately the same degree
as the in utero experience did. This demonstrates that
there is redundancy in early flavor learning and that it
extends from before birth into early infancy at least.
In a related study, breast-fed infants were more accept-
ing of peaches than formula-fed infants, as determined
by intake, rate of consumption and facial expressions.
This enhanced acceptance of fruit was likely due to more
exposure to fruit flavors since their mothers ate more
fruits during lactation [ 13 ] . We suggest that breast-fed in-
fants learn to prefer flavors associated with fruits and
vegetables by experiencing these flavors in mothers milk.
This highlights the importance of a varied diet for both
pregnant and lactating women. This variety of flavor ex-
periences during early ontogeny may be the reason that,
compared to formula-fed infants, breast-fed infants are
less picky [ 43 ] and are more willing to try new foods [ 44 ] .
Conscious efforts to increase vegetable and fruit con-
sumption among pregnant and lactating women could be
a potent strategy to enhance fruit and vegetable con-
sumption of their children.
In summary, the flavor world of infants, particularly
those that are breast-fed, is complex. Early experiences
with flavor compounds carried in amniotic fluid and in
breast milk modify later flavor and food preferences.
These experiences interact with genetic differences in fla-
vor perception [ 45, 46 ] and together genes and experience
play a central role in establishing food likes and dislikes
thereby impacting on the health and wellness of the in-
fant, the child and the adult. An appreciation of the role
of early experiences during sensitive periods in flavor
learning and a greater understanding of the different
means by which infants communicate their liking of
tastes and flavors, will aid in our development of evi-
dence-based strategies to facilitate healthy eating by ev-
eryone.
Acknowledgement
Preparation of the manuscript was supported in part by NIH
Grant HD37119.
Disclosure Statement
No conflicts of interest exist.

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http://dx.doi.org/10.1155/2013/343594
Review Article
Treating Type 2 Diabetes Mellitus with Traditional Chinese and
Indian Medicinal Herbs
Zhijun Wang,
1
Jeffrey Wang,
1,2
and Patrick Chan
3
1
Center for Advancement of Drug Research and Evaluation, College of Pharmacy, Western University of Health Sciences,
309 E. Second Street, Pomona, CA 91766, USA
2
Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, 309 E. Second Street,
Pomona, CA 91766, USA
3
Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences,
309 E. Second Street, Pomona, CA 91766, USA
Correspondence should be addressed to Jefrey Wang; jwang@westernu.edu and Patrick Chan; chanp@westernu.edu
Received 1 February 2013; Accepted 1 April 2013
Academic Editor: Weena Jiratchariyakul
Copyright 2013 Zhijun Wang et al. Tis is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Type II diabetes mellitus (T2DM) is a fast-growing epidemic afecting people globally. Furthermore, multiple complications and
comorbidities are associated with T2DM. Lifestyle modifcations along with pharmacotherapy and patient education are the
mainstay of therapy for patients aficted with T2DM. Western medications are frequently associated with severe adverse drug
reactions and high costs of treatment. Herbal medications have long been used in the treatment and prevention of T2DM in
both traditional Chinese medicine (TCM) and traditional Indian medicine (TIM). Tis review examines in vivo, in vitro, and
clinical evidence supporting the use of various herbs used in TCM and TIM. Te problems, challenges, and opportunities for the
incorporation of herbal frequently used in TCM and TIM into Western therapy are presented and discussed.
1. Introduction
Type 2 diabetes mellitus (T2DM) is a chronic illness due to
endocrine dysfunction. Uncontrolled, diabetes is associated
with various acute and chronic comorbidities. T2DM is
a rapidly growing health concern in both developed and
developing nations. T2DM accounts for over 90% of cases
globally [1, 2]. According to the World Health Organization
(WHO), in 2011, approximately 364 million people globally
sufer from diabetes (DM), with projections that DM-related
deaths will double from2005 to 2030 [3]. In 2004, 3.4 million
people died directly from the consequences of high blood
glucose. Te prevalence of DM worldwide was calculated as
2.8% in 2000. Tis is expected to increase to 4.4% by 2030
[4]. Te growing concern is the epidemic growth in obesity
and increase in the elderly population, which will continue
to increase the prevalence of DM. Another study, using data
from 91 countries, estimates that the prevalence can be as
high as 7.7%(439 million adults) by 2030 [2]. Other estimates
include a 70% increase in DM in developing countries and
20% increase in developed nations.
In the United States, T2DM is quickly becoming an
epidemic. Te Center for Disease Control (CDC) estimates
that in the United States alone, 25.8 million Americans, or
8.3% of the population, sufer from DM, with 7 millions
currently undiagnosed [5]. DMis higher, 26.9%, inthe elderly
(65 years or older). But it is also rapidly becoming a disease
observed in younger patients with almost 2 millions over the
age of 20 being newly diagnosed with DM in 2010. More
alarmingly, 35%of adults over the age of 20 and 50%of elderly
had prediabetes. Tis equates to 79 million people in the
US. DM is the primary cause of renal failure, non-traumatic
lower-limb amputations, and newly diagnosed retinopathy.
DM is the 7th leading cause of mortality of Americans.
1.1. Current Pharmacological Agents in the Treatment of
T2DM. T2DM is a chronic disease that afects millions of
people globally and is associated with multiple comorbidities
and complications. DM education, prevention, and care are
complex and should be designed to be patient specifc.
Physicians, nurses (and nurse practitioners), pharmacists,
and dieticians are ofen recruited as a balanced health-care
teaminmanaging a patients diabetes. Te AmericanDiabetes
Association (ADA) promotes diabetes self-management edu-
cation, a process in which the patient is equipped with
the knowledge and skills to provide self-care, manage crisis
(severe hyperglycemia and hypoglycemia), and make lifestyle
changes [6, 7].
Primary non-pharmacological interventions include ap-
propriate diet and exercise. Diet should be balanced and
aimed to reduce weight. At least thirty minutes of moderate to
intense exercise canimprove T2DMandweight management.
Intense lifestyle modifcations (LSMs) are the mainstay of all
treatment modalities and should be encouraged in both pop-
ulations who are at risk for developing diabetes and patients
who are sufering from diabetes. For patients requiring phar-
macological interventions of T2DM, metformin, a biguanide,
is frst-line treatment for most patients who are unable
to achieve their glycemic goals with LSM. Used for years,
metformin increases glucose uptake by the skeletal mus-
cles [8], inhibits hepatic gluconeogenesis [9], and increases
insulin sensitivity [10] (summarized in Table 1). Not only is
metforminthe frst-line recommendationfor the treatment of
T2DM, but there is also evidence of metformin being a useful
agent in preventing T2DM in high-risk populations [11].
Sulfonylureas are a commonly used second-line class
of antidiabetic drugs which increases insulin secretion by
binding to K
ATP
(potassium) channels of the -islet cells
in the pancreas [12]. Second-generation sulfonylureas are
largely used due to their potency, fewer drug interactions,
and less severe adverse reactions [6]. Insulin, which has long
been considered last-line therapy in the treatment of T2DM
and is the primary treatment of Type 1 Diabetes Mellitus
(T1DM, insulin-dependent DM), is now a viable addition
to metformin as a second-line agent in lieu of sulfonylureas
[6, 7]. Insulin is efective in reducing blood glucose and
HbA
1C
. Insulin regimens are patient-specifc and can involve
various combinations.
Other less validated classes of medications that can
be added to metformin include the thiazolidinediones
(TZDs) and GLP-1 agonists (glucagon-like peptide-1). TZDs
(pioglitazone, rosiglitazone) act by modulating peroxisome
proliferator-activated receptor (PPAR), a nuclear receptor
involved in the regulation of glucose and lipid metabolism.
Activation of PPAR leads to increased insulin sensitivity
primarily in adipose tissue but has also shown to have an
efect on skeletal muscle and liver [13]. In the United States,
TZDs carry a black box warning of increased cardiovascular
events due to a trial that demonstrated an increased risk
of myocardial events with rosiglitazone [14]. GLP-agonists
(exenatide, liraglutide) are peptides derived from naturally
occurring incretinhormones produced inthe small intestines
afer meals [15]. It binds to GLP-1 receptors in the pancreas to
stimulate insulin secretion and suppress glucagon secretion.
Te meglitinides class (repaglinide, nateglinide) has a
similar mechanism to sulfonylureas [16] but binds to a
diferent site from sulfonylureas on the K
ATP
channels of
the -islet cells in the pancreas, also stimulating insulin
release. Tere is a reduced risk of hypoglycemia with the
meglitinides. -Glucosidase inhibitors (acarbose, miglitol)
work primarily in the gut by inhibiting -glucosidase
enzymes on the intestinal brush border. -Glucosidase is a
key enzyme for breaking down carbohydrates such as starch,
dextrin, and disaccharides for absorption [17]. -Glucosidase
inhibitors may also stimulate GLP-1 secretion. Dipeptidyl
peptidase-4 (DPP-4) is a protease enzyme responsible
for the inactivation of hormones GLP-1 and GIP (gastric
inhibitory peptide) [18]. Inhibition of DPP-4 by inhibitors
(sitagliptin, saxagliptin, linagliptin) increases endogenous
levels of GLP-1. Endogenous amylin and its analogues
(pramlintide) bind to amylin receptors in the brain [19].
Endogenous amylin is secreted along with insulin from
pancreatic -islet cells. Pramlintide delays gastric emptying,
reducing postprandial glucose levels [20].
1.2. Pharmacological Prevention of T2DM. Te prevention of
T2DM in patients primarily focuses on education, diet, and
exercise. While the use of pharmacological approaches for
prevention is not routinely practiced, the ADA recommends
that health-care practitioners consider the use of metformin
in patients who are at high risk for developing diabetes. In the
Diabetes Prevention Program(DPP) trial, metformin 850 mg
twice daily was given to female patients considered at risk for
developing DM[11]. One groupwas administeredmetformin,
another group underwent intensive LSM, the last group
was given placebo medication. Afer a four-year study, the
incidence of DM was decreased by 58% ( < 0.001) with the
LSM group and 31% ( < 0.001) with the metformin-treated
population when compared to placebo. As such, metformin
is the only current medication that has been advocated to be
used in the prevention of diabetes in high-risk populations
such as those with a history of gestational diabetes, morbidly
obese, and those with progressive hyperglycemia [6, 21].
Te Troglitazone in Prevention of Diabetes (TRIPOD)
study demonstrates preservation of pancreatic -islet cell
function [22]. TZD (troglitazone) was administered in high-
risk Hispanic women as identifed with the development of
gestational diabetes within the previous four years. In women
receiving 400 mg troglitazone for 30 months, the cumulative
incidence of diabetes was reduced signifcantly in treated
women (5.4%) compared to placebo (12.1%; < 0.01).
Troglitazone was discontinued in the USA in 1998 due to
potential liver damage associated with the drug.
Over 1300 patients with impaired glucose tolerance in a
multi-center study were selected for the STOP-NIDDM trial
and given either acarbose three times daily or placebo [23].
Afer treatment for an average of 3.3 years, 17%of the patients
in the acarbose-treated group developed diabetes compared
to 26% in the placebo group ( = 0.001).
Native Asian Indians with impaired glucose tolerance
(IGT) enrolled inthe IndianDiabetes PreventionProgramme
(IDPP-1) study received placebo, LSM, metformin, or LSM
plus metformin [24]. Patients were followed for three years,
and the cumulative 3-year incidences of diabetes were 39.3%
with LSM(relative risk reduction [RRR] = 28.5%, = 0.018),
40.5% with metformin (RRR = 26.5%, = 0.029), and 39.5%
with LSM plus metformin (RRR = 28.2%, = 0.22). Results
demonstrated that LSM or metformin alone can signifcantly
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GLUT2
ADP ATP
Apoptosis
Glucokinase
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TCM/TIM
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-glucosidase, -amylase, aldose reductase
1
Non-carbohydrates
Figure 1: Mechanisms of antidiabetic efect of TCM and TIM herbs. (1) Reduced carbohydrate absorption, such as inhibition of -
glucosidase, -amylase, and aldose reductase, (2) increased glucose uptake in muscle and adipose tissues, (3) activation of PPAR, (4)
increased insulin sensitivity/upregulation of receptor expression, (5) exertion of antioxidant efects and decreasing -cell apoptosis, (6)
stimulation of -cell insulin secretion, (7) inhibition of hepatic gluconeogenesis/glycogenolysis, and (8) prevention of endogenous incretins
from degradation/suppression of glucagon. Te numbered mechanisms of actions correspond to Table 1.
lower the incidence of diabetes, but the combination of LSM
and metformin did not display any added beneft.
Te IDPP-2 study recruited native Asian Indians with
IGTand received LSMplus placebo or LSMplus pioglitazone.
Followup three years later did not show improvements or
reduction in the development of T2DM [25]. Te cumulative
risk was 29.8% in the pioglitazone group and 31.6% in the
placebo group.
In the DREAM trial (Diabetes Reduction Assessment
with Ramipril and Rosiglitazone Medication), rosiglitazone
was administered in hopes of preventing T2DM [26] in
patients with IGT or impaired fasting glucose (IFG). Patients
were followed for a median of 3 years. Te incidence of DMin
the rosiglitazone treatment group was 10.6% and 25% in the
placebo group ( < 0.0001). Te risk of T2DM or death was
reducedby 60%inpatients who have a highrisk of developing
T2DM. Heart failure, which is a concern of rosiglitazone, was
0.5% in the rosiglitazone arm compared to 0.1% ( = 0.01)
in the placebo arm.
Te NAVIGATOR (Nateglinide and Valsartan in Im-
paired Glucose Tolerance Outcomes Research) study group
randomized patients with IGT to receive nateglinide or
placebo with a median followup of 5 years [27]. Te cu-
mulative incidence of diabetes was nonsignifcant in the
nateglinide group (36%) compared to the placebo group
(34%; = 0.05).
Te efects of low-dose combination of metformin and
rosiglitazone were examined in patients with IGT in the
CANOE (Canadian Normoglycemia Outcomes Evaluation)
trial [28]. Te median followup was 3.9 years and demon-
strated that this combination was efective in reducing the
incidence of developing DM in the treatment group (14%)
compared to the placebo group (39%; < 0.0001), with
a relative risk reduction of 66%. A signifcant reduction in
insulin sensitivity in the placebo group (1.24) compared to
the treatment group (0.39; = 0.0006) was also observed.
Orlistat, a gastrointestinal lipase inhibitor used in the
treatment of obesity, was used in the XENDOS (Xenical
in the Prevention of Diabetes in Obese Subjects) trial [29].
Patients were recruited onthe basis of BMI (body mass index)
>30 kg/m
2
, which is classifed as obese. Approximately 21%
of the patients exhibited IGT in both the orlistat treatment
group and the placebo group. Te results of the four-year
study showed the cumulative incidence of diabetes to be 6.2%
in the orlistat-treatment group and 9.0%in the placebo group
(37.3% risk reduction; = 0.0032).
1.3. Traditional Chinese Medicine (TCM) and Traditional
Indian Medicine (TIM) for Treatment and Prevention of DM.
Although there are currently a number of efective Western
T2DM medications available for treatment, management of
T2DMusing medications withfewer side efects at lower costs
is still a big challenge. Tese medications frequently have
side efects, such as weight gain, bone loss, and increased
risk of cardiovascular events [30]. Tese side efects could
become more prevalent due to continuous use. Furthermore,
treatment is very costly as well, since T2DM is a chronic
disease and long-term medications are necessary. Herbal
medications can be a good alternative to replace or at least
supplement to Western medications [3134]. Te Indian
and Chinese cultures have had several thousand years of
history and experience in the prevention and treatment of
T2DM with herbal medicine. As later discussed, several
herbal medications have beenprovento be clinically efective.
Because herbal medicines are usually derived from natural
plants, they are considered to be relatively safe and have fewer
side efects compared to the conventional drugs.
Herbal medications treating T2DM can target multiple
mechanisms including enhancement of insulin sensitivity,
stimulation of insulin secretion, or reduction of carbohydrate
absorption [31]. Unlike Western medicine which usually
contains a single active ingredient aiming for a specifc
mechanism, herbal concoctions may contain various active
ingredients targeting multiple mechanisms. Herbal medicine
is based on the holistic theory, which puts an emphasis
on the integrated body. Western drugs are typically more
potent than herbal medicine in lowering blood glucose levels.
However, herbal supplements have shown to be able to treat
diabetic complications [35]. Tus herbal medicine can also be
used as supplementation or in combination with the Western
medicine to improve better therapeutic outcomes.
In the Chinese and Indian cultures, traditional medicine
has long been the foundation in the treatment and prevention
of many diseases. Approximately 800 plants have been iden-
tifed in the treatment or prevention of T2DM. Many formu-
lations are present as a single herbal extract or in a complex
formula. Over 400 extracts have shown to be efective in vitro
or in vivo [32]. Te pharmacological mechanisms of the herbs
can be classifed as (1) decreasing carbohydrate absorption,
(2) improving insulin sensitivity, (3) increasing peripheral
glucose uptake, (4) stimulating insulin secretion, (5) poten-
tiating endogenous incretins, (6) exerting antioxidant efects
and decreasing cell apoptosis, and (7) increasing the glyco-
genesis or inhibiting hepatic glycogenolysis (Figure 1) [31, 32,
36]. Since many formulations contain multiple extracts and
compounds, each herbal preparation may contain multiple
mechanisms. In the following sections, we summarize the
Chinese and English literature and list the most efective
TCM and TIM herbal preparations under these identifable
mechanisms. Most of the studies have been conducted using
in vitro systems and diabetic animals. However, there has
been an increase in randomized placebo-controlled clinical
trials testing the efectiveness of various TCM and TIM in
both healthy and T2DM patients (Table 2).
2. Commonly Used TCM/TIM for T2DM
2.1. Herbs in Both TCM and TIM
2.1.1. Gymnema sylvestre Schult (syn. Periploca sylvestris Retz).
Gymnema sylvestre Schult, belonging to genus Gymnema and
family of Apocynaceae, grows in the tropical forests of south-
ern and central India, southern China, Vietnam, Australia,
and African countries. Te leaves of G. sylvestre have been
used for treatment of diabetes, hypercholesterolemia, joint
pain, and snake bites in India and China [37, 38]. Te leaf
extract of the G. sylvestre has also been marketed as herbal
supplements for diabetic patients [39]. Te major chemical
components are gymnemic acids I-VII, triterpenoid saponins
(gymnemosides A-F and gymnemoside W1-2), conduritol A,
and dihydroxy gymnemic triacetate.
Te major bioactive constituents are gymnemic acids,
a group of oleanane-type triterpenoid saponins includ-
ing gymnemic acids I-VII, gymnema saponins and their
derivatives such as deacylgymnemic acid (DAGA) which
is the 3-O-glucuronide of gymnemagenin (3,16,21,22,23,28-
hexahydroxy-olean-12-ene) [38].
In alloxan-induced diabetic mice, body weight as well as
pancreas and liver weight were increased by oral adminis-
tration of the leaf or callus extract of G. sylvestre at a dose
of 200 mg/kg [39]. Te efect of the extracts was similar
to 4 unit/kg of insulin. Hepatic glycogen levels were also
increased (2.15 to 2.47 mg/g versus 1.35 mg/g for the extracts
and control, respectively), which in turn could stimulate the
secretion of insulin. In streptozotocin (STZ)-induced DM
rats, the hexane, acetone, and methanol extracts decreased
plasma glucose levels. Te acetone extract was found to
be most potent. Oral administration of 600 mg/kg of the
acetone extract for 45 days decreased the glucose level
from 443 to 114 mg/L. Dihydroxy gymnemic triacetate was
identifed to be the major active component; at 520 mg/kg,
it showed signifcant efects on lowering blood glucose level
by increasing plasma insulin levels. G. sylvestre extracts
were shown to be able to regenerate pancreatic cells and
increase circulating insulin level by stimulating its secretion
[40].
In one small clinical study, the fasting blood glucose
(FBG) and HbA
1C
levels were improved in T2DM patients
afer receiving 200 mg of ethanolic extract of G. sylvestre
either daily or their usual treatment for 18 to 20 months
[41]. In a second clinical trial, the subjects showed reduced
polyphagia, fatigue, blood glucose (fasting and postprandial),
and HbA
1C
in comparison to the control group following an
oral dose of 500 mg of herbal extract for a period of 3 months
[42]. In an uncontrolled trial involving 65 patients with
T1DMand T2DM, the FBGand HbA
1C
levels were decreased
11% and 0.6%, respectively, afer oral dose of 800 mg daily of
G. sylvestre extract [41].
2.1.2. Momordica charantia. Momordica charantia, a tendril-
bearing vine belonging to the Cucurbitaceae family (also
known as bitter melon/gourd, karela, or balsam pear), is a
popular plant used for the treatment of diabetes in China,
SouthAmerica, India, the Caribbean, andEast Africa [32, 43].
In M. charantia seeds, the major components have been
identifed to be eleostearic acid and stearic acid, which
account for approximately 45% of total weight. Several
glycosides, such as charantin and vicine, were isolated from
the M. charantia stem and fruit. Other components include
polypeptide-p, lipids, triterpenoids, and alkaloids [43].
Te methanol extract of M. charantia exhibited hypo-
glycemic efects in diabetic male ddY mice at a dose of
400 mg/kg. M. charantia can also suppress glucose tolerance
and postprandial hyperglycaemia in rats [43] by inhibiting
Table 2: Recently completed and current clinical trials of herbs.
Herb Trial name Status Sponsors Clinical trial no.
Gymnema sylvestre Schult.
Double Blind Randomized
Trial to Compare Gurmar
(Gymnema sylvestre) with
Metformin in Type 2 Diabetes
Status
currently
unknown
(1) Postgraduate Institute of Medical
Education and Research
(2) Indian Council of Medical
Research
(3) International Clinical
Epidemiology Network (INCLEN)
TRUST
NCT00396851
Momordica charantia
Te Efect of Metamin 3D on
the Lipid and Glucose in
Subjects with Metabolic
Syndrome
Completed
2009
Taichung Veterans General
Hospital, Taiwan
NCT01120873
Folium mori
(1-deoxynojirimycin extract)
Efect of Mulberry Leaf
Extract on Blood Glucose
Completed
2011
(1) Ewha Womans University
(2) Bundang CHA Medical Center
(3) Ministry of Knowledge
Economy, Korea
NCT01385865
Trigonella foenum-graecum L.
Efect of Fenugreek on Blood
Sugar and Insulin in Diabetic
Humans
Completed
2008
(1) Pennington Biomedical
Research Center
(2) Louisiana State University
Health Sciences Center in New
Orleans
NCT00597350
Rhizoma coptidis
Trial of Diferent Dosages Ge
Gen Qin Lian Decoction in
the Treatment of Type 2
Diabetes
Currently
recruiting
patients
Guanganmen Hospital of China
Academy of Chinese Medical
Sciences
NCT01219803
Rhizoma coptidis
(berberine extract)
Efcacy and Safety of
Berberine in the Treatment of
Diabetes with Dyslipidemia
Completed
2006
Shanghai Jiao Tong University
School of Medicine, China
NCT00462046
Terapeutic Efects of
Berberine in Patients with
Type 2 Diabetes
Completed
2004
(1) Shanghai Jiao Tong University
School of Medicine
(2) National Institutes of Health
(NIH)
NCT00425009
Ginkgo biloba
Ginkgo Biloba Extract and the
Insulin Resistance Syndrome
Completed
2005
National Center for
Complementary and Alternative
Medicine (NCCAM)
NCT00032474
Radix ginseng Mey
(ginsenosides extract)
A Clinical Trial of Ginseng in
Diabetes
Completed
2008
Washington University School of
Medicine
NCT00781534
Data retrieved from the U.S. National Institutes of Health (http://clinicaltrials.gov/).
the absorption of carbohydrates from the gastrointestinal
tract.
Leung et al. reviewed clinical trials examining the
hypoglycemic efects with M. charantia in T2DM patients.
However, contradictory clinical outcomes were observed
among these trials, probably due to poor methodological
design without baseline characterizations along with non-
standardized extraction method [43]. Nevertheless, the M.
Charantia juice fromthe fresh fruit showed glucose-lowering
efects in T2DMpatients [44, 45], but not the extract fromthe
dried fruit [46].
Te extract of M. charantia using ethyl acetate was
able to activate peroxisome proliferator-activated receptors
(PPAR and ) and upregulate the expression of the acyl CoA
oxidase gene in H4IIEC3 hepatoma cells. Te suppression of
peroxidation and apoptosis resulted in improvements in -
cell function and enhanced insulin excretion. In adipocytes,
the momordicosides from M. charantia stimulated glucose
transporter-4 (GLUT4) translocation to the cell membrane
and increased the activity of adenosine monophosphate-
activated protein kinase (AMPK), which could enhance glu-
cose uptake from the blood. Animal studies showed that the
extract could also enhance insulin sensitivity and lipolysis. In
STZ rats, gluconeogenesis was inhibited by M. charantia via
downregulation of hepatic glucose-6-phosphatase (G6P) and
fructose-1,6-bisphosphatase activities [47].
2.1.3. Morus alba L. Te mulberry tree (Morus alba L.) grows
widely in Asian countries, and various substituents of its
leaves, Foliummori, have been applied clinically in TCM[48]
as hypoglycemic, hypotensive, and diuretic agents. Folium
mori have been traditionally used to treat hyperglycemia.
Te main bioactive components are favonoids, alkaloids (1-
deoxynojirimycin), and polysaccharides [33].
In T2DM mice (high sucrose-fed KK-Ay mice), Folium
mori extract reduced insulin resistance following 8-week
treatment. Both FBG levels and urinary glucose levels were
signifcantly lowered in mice fed with a diet supplemented
with Folium mori extract in a dose-dependent manner
[49]. In Goto-Kakizaki rats, a spontaneous nonobese animal
model for T2DM, the Folium mori extract demonstrated
reduced postprandial blood glucose levels [50]. In human
subjects, it showed that a food-grade mulberry powder
enriched 1-deoxynojirimycin suppressed postprandial blood
glucose serge [50, 51].
In vitro cell studies showed that in adipocytes, Folium
mori extract increased glucose uptake and thus enhanced the
translocation of GLUT-4 with concentrations ranging from 5
to 45 mcg/mL [52, 53]. In db/db mice, the extract ameliorated
adipocytokines in white adipose tissue possibly due to the
inhibition of oxidative stress [52]. One of the alkaloids, 1-
deoxynorimycin, is also a potent inhibitor of -glucosidase
[54].
2.1.4. Trigonella foenum-graecum L. Te fenugreek is an
annual plant in the family Fabaceae. Te fenugreek seed was
a traditional remedy used by ancient Egyptians and spread
to Asian countries such as China and India [55]. Fenugreek
seeds are a rich source of the polysaccharide galactomannan
and also contain saponins such as diosgenin, yamogenin,
gitogenin, tigogenin, and neotigogens. Other active con-
stituents include mucilage, volatile oils, and alkaloids [56, 57].
Te hypoglycemic efects of T. foenum-graecum in rats
were frstly reported in 1974 [58]. Soon aferwards, the amino
acid 2S,3R,4S, 4-hydroxyisoleucine, purifed from fenugreek
seeds, showed insulinotropic efects which increased periph-
eral glucose uptake in vitro [5961]. Te activities of hepatic
enzymes hexokinase, glucokinase, G6P, and fructose-1,6-
bisphosphatase were reduced in DMrats [62, 63]. Plasma glu-
cose levels decreased afer receiving the T. foenum-graecum
extract in both non-DM patients and DM patients [64, 65].
Insulin levels were signifcantly higher in the fenugreek
treatment group incomparisonto the placebo treatment [66].
A meta-analysis of T. foenum-graecum showed that the herb
may reduce HbA
1C
by 1.13% ( = 0.03) [67].
2.2. Other Herbs in TCM
2.2.1. Radix rehmanniae . Radix rehmanniae is the root of
Rehmannia glutinosa Libosch, under the family of Scro-
phulariaceae or Gesneriaceae. It has been widely used for
treatment of diseases relating to blood, immune, endocrine,
nervous, and cardiovascular systems.
Te bioactive components of Radix rehmanniae include
catalpol, rehmannioside A, B, C, and D, phenethyl alcohol
derivatives such as leucosceptoside A and purpureaside C,
monocyclic sesquiterpenes as well as their glycosides [68].
Radix Rehmanniae showed hypoglycemic activity in nor-
mal and STZ-induced DM mice. In Chinese medicine, it is
usually prepared in combination with other herbs such as
Radix ginseng, Radix scutellariae [69], and Radix astragali
[70]. Tese combinations stimulated insulin secretion and
-cell proliferation through insulin receptor substrate 2
induction. It also showed improvements in diabetic foot ulcer
healing in rats through the processes of tissue regeneration,
angiogenesis, and infammation control [70]. Te postulated
mechanisms of action are stimulation of insulin secretion,
regulation of glucose metabolism in DM rats, and reduction
of hepatic glycogen content of non-DM mice [31, 71].
2.2.2. Stephania tetrandra Moore. Stephania tetrandra Moore
is an herbaceous perennial vine of the Menispermaceae
family, which is a fundamental herb used in TCM for
the reduction of swelling and also providing an analgesic
efect. Te root of S. tetrandra has demonstrated to have
anti-infammatory, anti-allergic and hypotensive efects in
experimental animal studies [72].
Te major components are alkaloids, including tetran-
drine, fangchinoline, bisbenzylisoquinoline, protoberber-
ine, morphinane, and phenanthrene [73]. At 0.33 mg/kg,
fangchinoline signifcantly decreased blood glucose and
increased blood insulin in STZ-mice by potentiating insulin
release [31]. In another study, formononetin, one of the
active components in Radix astragali, potentiated the efect
of S. tetrandra on lowering the blood glucose level and
increasing the blood insulin level, although no direct anti-
hyperglycemic efect of formononetin was observed [72]. Te
postulated antidiabetic mechanism of S. tetrandra extract is
the stimulationof insulinrelease inpancreatic -cells [72, 74].
2.2.3. Rhizoma coptidis. Rhizoma coptidis is the rhizome of
Coptis chinensis Franch that belongs to the Ranunculaceae
family, recorded as Coptidis Rhizoma (CR) in the Chinese
Pharmacopeia with the Chinese name of Huang Lian. It has
been widely used to clear heat, dry dampness, and eliminate
toxins from the body. It is also a commonly used herb
in various formulas against intestinal infections, diarrhea,
infammation, hypertension, and hypoglycemia.
Te most well-known components of Rhizoma coptidis
are isoquinoline alkaloid and berberine [75] which has
variety of biological activities such as tumor reduction, anti-
microbial, anti-Alzheimers disease, anti-hyperglycemic, anti-
infammatory, and anti-malarial [76]. Te berberine com-
pounds of Rhizoma coptidis have been studied for its anti-
hyperglycemic efects. Te other alkaloids include palmatine,
jateorrhizine, epiberberine, and coptisine.
Both the extract and pure berberine signifcantly
decreased blood glucose and serum cholesterol levels in
high fat diet-fed mice at the dose of 200 mg/kg by gavage.
In alloxan-induced diabetic mice, berberine showed an
anti-hyperglycemic efect and also blunted blood glucose
increase induced by intraperitoneal glucose or adrenaline
administration in normal mice. Te activity of berberine was
similar to sulfonylureas or biguanides [31, 77].
Te anti-hyperglycemic efects of berberine could be due
to the improvement of insulin sensitivity by activating the
AMPK pathway or inducing insulin receptor expression.
Berberine could also improve fatty acid oxidation via acti-
vation of AMPK and acetyl-CoA carboxylase. Furthermore,
six quaternary protoberberine-type alkaloids of berberine
inhibited aldose reductase activity in vitro with an IC
50
less
than 200 M [77, 78]. However, no evidence from in vivo
studies is available to verify this mechanism.
2.2.4. Radix astragali. Radix astragali, Chinese name of
Huang Qi, is the dried root of perennial herbs Astragalus
membranaceus (Fisch.) Bunge and Astragalus mongholicus
(Fabaceae) Bunge of the Leguminosae family and grows
in northern China. Te major active compounds in Radix
astragali are isofavones and isofavonoids (formononetin,
calycosin, and ononin), saponins (astragaloside IV, astra-
galoside II, astragaloside I and acetylastragaloside), and
astragalus polysaccharides [79].
Radix astragali possesses a broad spectrumof efects such
as immunostimulation, hepatoprotection, diuresis, analgesia,
expectorant, and sedation. In traditional Chinese medicinal
theory, the herb is capable of consolidating the exterior of
the body and can alleviate heat in the muscles by ascending
positive qi [70, 80].
Afer treating DMSprague-Dawley rats with Radix astra-
gali decoction (500 mg/kg IP daily) for two months, improve-
ments in insulin sensitivity and attenuation of fatty liver
development were observed. However, blood glucose levels,
-cell function, and glucose tolerance were not substantially
improved. Radix astragali polysaccharides reduced hyper-
glycemia and led to indirect preservation of -cell function
and mass via immunomodulatory efects in T1DM mice. In
addition, its polysaccharides restored glucose homeostasis
in T2DM mice/rats by increasing insulin sensitization. For-
mononetin, calycosin and ononin might exert a synergistic
hypoglycemic efect withfangchinoline inSTZ-diabetic mice,
most likely by increasing insulin release [70, 80].
2.2.5. Eriobotrya japonica Lindl. Te loquat Eriobotrya japon-
ica Lindl., a fruit tree in the family Rosaceae, is indigenous
to central and south China. Te dried leaves of E. japonica,
also called Folium eriobotryae, have been used for treat-
ment of chronic bronchitis, cough, and diabetes. Te active
compounds in E. japonica are identifed to be triterpenes,
sesquiterpenes, favonoids, megastigmane glycosides and
polyphenolic compounds including ursolic acid, oleanolic
acid, cinchonain Ib, procyanidin B-2, chlorogenic acid, and
epicatechin [8183].
In an in vitro study using insulin receptor substrate-1
cells, the aqueous extract and the cinchonain Ib (one of the
components in E. japonica) enhanced insulin secretion in
a dose-dependent manner [84]. In vivo studies showed that
the aqueous extract of E. japonica could transiently reduce
blood glucose levels [84]. Te 70% ethanol extract exerted
a signifcant hypoglycemic efect on alloxan-diabetic mice
following oral doses of 15, 30, and 60 g/kg (crude drug).
Te total sesquiterpenes were found to signifcantly lower
blood glucose levels in both normal and alloxan-diabetic
mice [85]. Shih et al. found that the extract, with major
components of tormentic acid, maslinic acid, corosolic acid,
oleanolic acid, and ursolic acid, could ameliorate high fat
induced hyperglycemia, hyperleptinemia, hyperinsulinemia
and hypertriglyceridemia [86]. Another study found that
the co-fermentation of Folium eriobotryae and green tea leaf
reduced the blood glucose level by 23.8% within 30 min in
maltose-loaded SD rats at a dose of 50 mg/kg, although this
efect was not observed in the sucrose- and glucose-loaded
rats [87].
2.2.6. Ginkgo biloba. Te Ginkgo biloba tree, native to China,
dates back to the prehistoric ages of 250300 million years
and is frequently called a living fossil [88]. Today, the biloba
tree can live more than 1,000 years [89]. In the United States,
G. biloba is one of the most frequently used over-the-counter
(OTC) herbal supplements [90]. Extract from its leaves
contains ginkgo favonoid glycosides, terpene lactones, and
ginkgolic acids. Many human clinical trials have examined
possible ginkgo uses in cerebrovascular disease, tinnitus,
sexual dysfunction, intermittent claudication, migraine pro-
phylaxis, and alleviating symptoms of the common cold
[15, 9194]. However, the most common use of ginkgo is
the prevention and treatment of Alzheimers disease and
dementia [95100].
Te administration of the ginkgo extract, EGb 761, in
rats with DM increased glucose uptake into hepatic and
muscle tissues [101] and decreased atherogenesis, a common
comorbidity of DM [102]. In vitro assays determined the
possible anti-diabetic efect of ginkgo to be through the
inhibition of -glucosidase and amylase activities [103].
Clinical investigations of the anti-diabetic properties of
ginkgo in humans have produced mixed results. Healthy
human subjects showed no reduction in blood glucose levels
with an accompanying signifcant increase in plasma insulin
levels [104]. Te randomized double-blinded clinical study
involving non-DM, pre-T2DM, and T2DM patients showed
that ginkgo did not increase insulin sensitivity nor reduced
blood glucose levels [105, 106]. However, in T2DM patients,
ingestion of G. biloba extract showed increased clearance of
insulin, resulting in a reduction plasma insulin levels and
elevated blood glucose. Gingko may improve endothelial
function in T2DM patients with early stages of nephropathy,
but without afecting blood glucose levels [107]. While popu-
lar for its many possible indications, gingko appears to have
limited anti-diabetic properties to warrant its use in diabetes.
2.2.7. Radix ginseng. Radix ginseng is native in the north-
ern hemisphere, most notably, in eastern Asia (northern
China, Korea, and eastern Siberia) and northern America.
Subsequently, ginseng is ofen named from its originAsian
ginseng, American ginseng, Chinese ginseng, to name a few.
More than 700 compounds have been identifed in ginseng,
with the most active components being identifed as the
ginsenosides (Rb1, Re, Rd), polysaccharides, peptides, and
polyacetylenic alcohols [108]. Te geographical origin of
ginseng, in combination with the extraction and processing
method, produces variable anti-diabetic results [32, 108, 109].
Te anti-diabetic activity of Ridix ginseng has been
explored in both animal and human studies. Hypoglycemic
activity is greater in lipophilic extracts than aqueous extracts.
In DM rats, Korean ginseng (0.11.0 g/mL) stimulated the
release of insulin from isolated pancreatic islets. American
ginseng (100 mg/kg) produced lowered levels of serum glu-
cose and HbA
1C
inDMrats [110]. Vuksanand colleagues have
conducted a number of human clinical trials demonstrating
that ginseng reduced postprandial blood glucose, fasting
blood glucose, and HbA
1C
levels [111115]. Similar results
were presented at American Diabetes Association Annual
Meeting in 2003 [116].
Pharmacologically, ginseng has antioxidant properties.
It also reduces -cell apoptosis by upregulating adipocytic
PPAR- protein expression [117]. Ginseng impairs glucose
absorption by decreasing glucosidase activity [118]. It may
also increase insulin sensitivity in peripheral tissues [32]. One
of the active components, ginsenoside Rb1, can enhance glu-
cose transport by inducing the diferentiation of adipocytes
via upregulating the expression of PPAR- and C/EBP-
[119]. In addition, ginsenoside Rb1 can increase GLUT-4
activity leading to increased uptake of glucose from blood by
adipocytes [120].
2.2.8. Fructus schisandrae. Fructus schisandrae (also known
as fve-favor berry in China), the fruit of a deciduous
woody vine native to forests of northern China, is tradi-
tionally used as a tonic or sedative agent. It has been used
in TCM to astringe the lungs and nourish the kidneys. It
was reported that Fructus schisandrae can enhance hepatic
glycogen accumulation and decrease hepatic triglycerides. It
has also been used in various TCM formulas, such as the
modifed Ok-Chun-San and modifed Huang-Lian-Jie-Du-
Tang, to treat diabetes [121]. Te major chemical components
include lignans such as schizandrins (schizandrin A) and
gomisins (gomisin A, J, N, and angeloylgomisin H), and
polysaccharides [72],
In vitro, gomisin J, gomisin N and schizandrin A
increased basal glucose uptake in HepG2 cells [122]. Several
other schizandrins were found to be able to prevent -cell
apoptosis and decrease insulin resistance [123]. In an in vitro
study using 3T3-L1 adipocytes, several fractions of ethanol
extract showed the stimulation efect of PPAR-. Among
these fractions, FS-60, a subfraction from the 70% ethanol
extract, was identifed to be most potent with the major com-
ponents of schizandrinA, gomisinA, and angeloylgomisinH.
In an in vivo study using pancreatectomized DM rats, FS-60
lowered serumglucose levels during the OGTT similar to the
level of the fasting stage. During hyperglycemic clamp, FS-60
increased the frst phase insulin secretion in diabetic animals
[121].
Te major mechanisms are hypothesized to be the stimu-
lation of insulin secretion and increased insulin sensitivity by
ameliorating insulin resistance via increased PPAR- activity.
Fructus schisandrae can also improve glucose homeostasis in
DM mice by inhibiting aldose reductase [121].
2.2.9. Pueraria lobata (Gegen). Gegen is the dried root
of Pueraria lobata (Willd.) Ohwi, a semiwoody, perennial
and leguminous vine native to South east Asia, and also
known as yegen, kudzu root, and kudzu vine root [124,
125]. For more than 2000 years, gegen has been used as an
herbal medicine for the treatment of fever, acute dysentery,
diarrhea, DM, and cardiovascular diseases [126, 127]. Over
seventy compounds have been identifed in gegen, with
isofavonoids (puerarin) and triterpenoids being the major
constituents.
In vitro studies showed that puerarin contained in P.
lobata can enhance the glucose uptake in a dose-dependent
manner performed in high glucose-treated preadipocytes
[128]. Puerarin also promoted insulin-induced preadipocyte
diferentiation and upregulated mRNAexpression of PPAR,
which can regulate glucose homeostasis, adipocyte difer-
entiation, and lipid metabolism [129]. In China, a clini-
cal trial was conducted in DM patients using the Gegen
Qin Lian decoction which showed a dose-dependent efect
on reducing HbA
1C
and FBG [130]. Possible mechanisms
of action from in vitro and in vivo studies include -
glucosidase inhibition, increased expression and activity of
PPAR-, upregulation of GLUT-4 mRNA, increased plasma
endorphins, and preservation of pancreatic islets [131
133].
2.2.10. Cornus ofcinalis Sieb. et Zucc. Cornus ofcinalis
Zucc., native to China, Japan, and Korea, is a common herbal
medicine of the family of Cornaceae. Fructus corni is the
dried ripe sarcocarp of C. ofcinalis Sieb. et Zucc. Cornaceae,
which has been widely prescribed as a tonic agent in Chinese
medicinal formula and possess activities of improving the
function of the liver and kidney [134]. Te major active
components are iridoid glycosides, morroniside, loganin,
mevaloside, loganic acid, ursolic acid and oleanolic acid, 5-
hydroxymethyl-2-furfural, and 7-O-galloyl-D-sedoheptulose
[135].
Te ethanol extract of Fructus corni induced the expres-
sion of GLUT-4 by stimulating the proliferation of pancreatic
islets, resulting in increased insulin secretion [136]. One of
the active components, ursolic acid, was found to be an
inhibitor of protein tyrosine phosphatase (PTP) 1B, which
sensitizes the efects of insulin[137]. Te Fructus corni extract
decreased blood sugar in STZ mice and reduced renal oxida-
tive stress and glycation products in STZ-induced diabetic
rats. Te underlying mechanisms include the inhibition of
glucosidase, reduction of gene expression for hepatic gluco-
neogenesis, protection of -cells against toxic challenges, and
enhancement of insulin secretion.
2.3. Other Herbs in TIM
2.3.1. Barringtonia racemosa. Barringtonia racemosa is an
evergreenmangrove tree that grows inBangladesh, Sri Lanka,
and the west coast of India, with the bark and leaves used
for snake bites, rat poisoning, boils, and gastric ulcers. Te
extracts fromdiferent parts have various biological activities
including anti-cancer, analgesic, anti-DM, anti-bacterial, and
anti-fungal activities. Its seeds are aromatic and useful incolic
and ophthalmic disorders [138].
Several diterpenoids and triterpenoids have been iden-
tifed in B. racemosa extract and a pentacyclic triterpenoid,
bartogenic acid, is the major active component [138, 139].
Te hexane, ethanol and methanol extracts as well as
the pure compound of bartogenic acid inhibited intestinal
-glucosidase activity at concentrations ranging from 0.02
0.2 g/mL in an in vitro enzymatic study. In an in vivo rat
study, the methanol extract was found to suppress the rise of
blood glucose level afer receiving maltose [140].
2.3.2. Syzygium cumini (L.) Skeels. Syzygium cumini (L.)
Skeels, frequently referred to as Skeels, is a tropical tree
native to India, China, and Indonesia. Skeels is also known
as Eugenia jambolana, Jamun, Jambu, Black Plum, or Black
Berry and has been frequently used to treat DMin India [140]
and Brazil [141]. Studies using DM rats showed reductions
in blood glucose, post prandial glucose, cholesterol, and
free fatty acid [142, 143]. Pharmacologically, the extracts
of S. cumini have shown -glucosidase inhibitory activi-
ties [144, 145]. Hepatic enzymatic activities of glucokinase
and phosphofructokinase, hepatic enzymes which play a
role in glucose metabolism, were signifcantly reduced in
DM animals [145, 146]. Adenosine deaminase activity was
inhibited in Skeels-treated erythrocytes procured from both
DM and non-DM patients. However, clinical trials have not
produced favorable results. Two double-blind, randomized
trials involving non-DM and DM patients did not support
the use of Skeels in DM [147, 148]. In patients with DM
consumed tea prepared from S. cumini leaves, FBG levels
were not reduced signifcantly.
2.3.3. Tinospora cordifolia. Tinospora cordifolia, also called
Guduchi of the Menispermaceae family, is a succulent
climbing shrub, indigenous to the tropical areas of India,
Myanmar, and Sri Lanka. Te aqueous stem extract is
used for curing gastrointestinal pain [149]. T. cordifolia
also has anti-spasmodic, anti-pyretic, anti-allergic, anti-
infammatory, immunmodulatory, and anti-leprosy activities
[150]. Te bioactive ingredients are alkaloids (palmatine,
jatrorrhizine and magnoforine), diterpenoid lactones, gly-
cosides, steroids, sesquiterpenoid, phenolics, aliphatic com-
pounds, and polysaccharides.
Te anti-DMactivity of T. cordifolia has been investigated
in DM mice. Te aqueous and alcoholic extract of the
plant can improve glucose tolerance in DM rats. Grover and
coworkers found that T. cordifolia ameliorated diabetic neu-
ropathy at a dose of 400 mg/kg [151]. Te 70%ethanol extract
signifcantly decreased blood glucose levels and attenuated
the rate of bloodglucose elevationafer 2 g/kg glucose loading
following an oral dose of 100 or 200 mg/kg of T. cordifolia
for 14 days [152]. Te extract was also found to be able to
prevent diabetic retinopathy in STZ diabetic rats at a dose of
250 mg/kg [153].
Te anti-DM efect is related to the amelioration of
oxidative stress by reducing the production of thiobarbi-
turic acid-reactive substances. Te extract can increase the
expression of thioredoxin and glutaredoxin. T. cordifolia
extract can also adjust alter carbohydrate metabolism and
reduce gluconeogenesis via inhibiting G6P and fructose 1,6-
diphosphatase [154]. Other possible mechanisms examined
are the enhancement of the insulin release and inhibition of
-glucosidase [155].
2.3.4. Ocimum basilicum. Ocimum basilicum, with the com-
mon name of basil, or sweet basil, is a culinary herb of
the family Lamiaceae (mints), which is sometimes known
as Saint Josephs Wort. Basil was originally from India and
widely used in Southern Asian.
Basil is a potent anti-septic and preservative agent and
also demonstrates slight sedative efects, regulation of diges-
tion, and diuresis. Clinically, it has been used to treat
headache, cough, upper respiratory tract infection, and kid-
ney dysfunction. Laboratory studies have foundthat basil also
has activities in lowering blood sugar, stimulating nervous
system, and protection from radiation [156, 157].
Te major components of basil consist of apigenin,
linalool, and ursolic acid, which previously demonstrated
anti-viral activity [158]. Basil improved lipid metabolism
in hypercholesterolemic rats [159]. In an in vitro cell line
study using human macrophages, the ethanol extract of
basil reduced cholesterol synthesis [160]. Te aqueous extract
of O. basilicum can inhibit rat intestinal sucrase, maltase,
and porcine pancreatic -amylase activities which may have
positive efect for treatment of DM [161]. In a clinical trial
in DM patients in India, the basil leaf extract decreased
the fasting blood glucose by 21.0 mg/dL, and postprandial
blood glucose fell by 15.8 mg/dL. Te results suggest that
O. basilicum may be used as a dietary therapy in mild to
moderate T2DM [162].
2.3.5. Berberis aristata. Berberis aristata (also known as
Zarshik, Daruharidra) of the family Berberidaceae is an
Ayurvedic herb which has been used since ancient times in
South Asia as an herbal tonic agent to improve hepatic and
cardiac functions [163, 164].
Te main constituents of the root have been identifed
as berberine, berbamine and palmatine [165]. Te extract of
B. aristata (root) has a strong potential to regulate glucose
homeostasis by decreasing gluconeogenesis and oxidative
stress. In DM rats, the extract increased the glucokinase
and G6P dehydrogenase activities but decreased G6P activ-
ity [165]. In patients with sub-optimal glycemic control,
HbA
1C
, basal insulin, insulinresistance, total and low-density
lipoprotein cholesterol, and triglycerides were signifcantly
reduced afer 90-day treatment with combination of B.
aristata extract and Silybum marianum extract [166].
3. Problems, Challenges, and Opportunities
Tere are two entirely diferent approaches in the future
research on TCM/TIM. Te sharp shooter approach is
to select a particular plant with a specifc activity or a
biological target. By using bioactivity-guided isolation and
structural elucidation, one can discover a new chemical
entity for a specifc disease target. Many Western drugs, such
as chemotherapeutic agent paclitaxel, were discovered this
way, while some others such as metformin were developed
upon further structural modifcation. Obviously, this is a
validated approach for drug discovery and development for
the treatment of human diseases. On the other hand, instead
of targeting a specifc receptor or mechanism, one can select
proper combinations of herbs or ingredients, and optimize
the outcome of treatment by diferent combinations and dose
regimens. Tis approach is termed the shotgun approach.
While it is not necessary to identify the exact active ingre-
dient(s), it is still necessary to have good quality control of
the preparations to ensure reproducibility. Certain chemical
markers in the preparations can be selected as markers to
standardize the raw materials and processing procedures.
Once a reproducible preparationis obtained and its biological
activity is established, it can be used to treat certain given
disease in the general patient population. Because the extract
contains multiple components which may interact with
multiple disease targets, it might be advantageous to a single
chemical entity, especially in the area of disease prevention.
However, pharmaceutical scientists are facing unique
challenges in developing herbal products as anti-DM agents.
(1) Patentability. Because herbal medicines derive from
natural plants, their active components cannot be
patented as novel materials. It is also difcult to patent
their usage since much information is already in the
public domain. But it is possible to patent a unique
combination and/or the extraction process.
(2) Product standardization. Tis should be achieved
via proper control on raw material, extract pro-
cess and fnal formulation. Without efective quality
control, consistency of the herbal product may be
compromised. Improved methods for quality control
of herbal products, such as bioactivity-guided phar-
macokinetic methods and genomic fngerprinting
techniques are promising.
(3) Placebo-controlled, randomized clinical trials. TCM/
TIM physicians philosophy to individualize formula
for diferent patients has signifcantly hindered the
systematic scientifc investigation according to West-
ern medicine standards. In comparison to their West-
erncounterparts, the anti-DMefcacies of TCM/TIM
herbs have not been well studied using randomized,
double-blinded clinical trials, although many animal
studies have been carried out. However, the imple-
mentation of placebo-controlled, randomized clinical
trials is a prerequisite for the evidence-based practice
of using TCM/TIM preparations for DM prevention.
(4) Toxicity and herb-drug interaction. TCM herbs are
generally thought to be relatively safe and with milder
side efects. However, their activities are usually not
as potent as Western medications. Tus high doses
(sometimes as high as 10 g per day) are usually
required to achieve optimal therapeutic efcacy. In
addition, the toxicity of herbal products cannot be
ignored. Most common complaints of herbal supple-
ments ingestion are gastrointestinal related, including
stomach upset, diarrhea, constipation, nausea, and
vomiting. In addition, more serious adverse efects
may also occur. For example, ginseng abuse syndrome
is a result of chronic ingestion of excessive amounts
of ginseng. Tis is characterized by hypertension and
CNS stimulation, insomnia, and nervousness. Tere
is also an increased awareness of herb-drug inter-
action in pharmacokinetics and pharmacodynamics.
When used in combination with established anti-DM
medications, herbal supplementation may predispose
patients at an increased risk of hypoglycemia. For
example, Ginkgo biloba extract interacts with selective
serotonin reuptake inhibitors used in the treatment of
depression, resulting in the serotonin syndrome, and
with thiazide diuretics resulting in decreased efcacy.
It is encouraging to note that two drugs based on plant
extract have been approved by the FDA for the treatment
of human diseases. Veregen (Polyphenon E) Ointment is the
frst prescription botanical drug approved by FDA in 2006.
It is an extract of green tea as a prescription drug for the
topical (external) treatment of genital warts caused by the
humanpapilloma virus (HPV). More recently, FDAs approval
of crofelemer (Fulyzaq) signals the frst time an orally
administered botanical has received drug approval from the
Administration. Crofelemer derived from the latex of the
South American sangre de drago tree (dragons blood, Croton
lechleri) is the frst drug to be approved in the United States
to treat HIV-associated diarrhea. With experience gained
through the developmental and regulatory processes comes
high hope that many TCM/TIM-based anti-DM products
will be available for the general population.
4. Conclusions
It is evident that many TCM/TIM herbs possess anti-DM
activities by interacting with various proven drug targets
where Western drugs interact. Because of their empirically
known oral efcacy and safety profles, nutritional supple-
ment status, multiple components for multiple drug targets,
low cost, and easy access, TCM/TIM herbs such as ginseng,
mulberry, and Radix coptidis are excellent candidates for
long-term use for the prevention and treatment of T2DM.
During the development stage, product standardization,
quality control and assurance, placebo-controlled and ran-
domized clinical trials are essential components that need to
be perfected in order to translate their potential into a reality
that millions of people could beneft upon.
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Cellular Longevity
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EDITORS CORNER EDITORS CORNER
www.landesbioscience.com Oxidative Medicine and Celluar Longevity 227
Derived from classical Chinese philosophy and with frequent applications in Chinese medicine, yin yang has grown into an interest-
ing concept that may have a number of applications. The term is usually used to depict the existence of polarizing or opposing forces in
nature. Yet, the concept of yin yang can be much more involved, especially in biological systems, and pertain to events that not only are
intimately connected, but also have signicant repercussions upon each other. Interestingly, one does not need to search far in biology and
medicine to learn of the concept of yin yang. For example, nicotinamide, which is the amide form of vitamin B3 (niacin), is a robust
cytoprotectant that can scavenge reactive oxygen species during oxidative stress and increase survival of both neuronal and vascular cells
during multiple injury paradigms that include anoxia, excitotoxicity, ethanol toxicity, and elevated glucose. Furthermore, nicotinamide also
may limit inammatory injury, protect against trauma, and improve cognition under some conditions. However, there is another side to
nicotinamide that is relevant to the concept of yin yang and involves opposing forces with sirtuins. It appears that reduced concentrations
of nicotinamide may be necessary to increase cell longevity to permit activity of sirtuins. Elevated concentrations of nicotinamide block
the activity of sirtuins, agents that have been tied to increases in longevity and cellular protection such as during metabolic disorders with
diabetes mellitus. Therefore, in relation to the biology of cell survival and cell longevity, a complex relationship exists between nicotinamide
and sirtuins that require a careful balance to maximize cell survival and extend cell longevity.
In this issue of Oxidative Medicine and Cellular Longevity, we present a unique group of papers that bring home the role of yin yang
for our readers. In the review paper by Bouayed and Bohn, the authors take direct aim at the concept of yin yang in bringing to light the
sometimes unrecognized and unexpected effects of exogenous antioxidants. Exogenous dietary antioxidants, such as vitamin C, vitamin E,
carotenoids, and polyphenols, are necessary for the proper functioning of the endogenous cellular antioxidant system, but under certain
conditions exogenous agents also may yield pro-oxidant behavior. As a result, the authors outline the intimate connections between oxidant
and antioxidant pathways and the need to better align these systems. In the next review by Attia, the author describes the challenges of drug
development and unexpected consequences that may arise following clinical approval and use of drug treatments as a result of unexpected
opposing forces of nature. Attia provides striking insight to adverse drug reactions, especially those that are idiosyncratic in nature, and how
knowledge and modulation of reactive metabolic pathways that may ensue with drug activation may prevent unintended adverse conse-
quences during future clinical drug deployment. Sayed-Ahmed et al. extend this concept of nely controlling opposing cellular pathways
in their original work using a model of hepatocellular carcinoma. They show that initiation of hepatocellular carcinoma results in the gen-
eration of reactive oxygen species and the concomitant reduction of antioxidant pathways, but that specic and directed strategies such as
with thymoquinone that can oppose specic oxidant stress pathways can prevent the generation of hepatocellular carcinoma. Remarkably,
opposing cellular pathways also involve metabolic pathways in individuals that may not have extensive disease as shown by the unique
clinical study in non-diabetic males by Tahara et al. The authors show for the rst time that insulin resistance promotes elevated serum
levels of advanced glycation end products yet is inversely correlated with testosterone resulting in low testosterone levels in diabetic men.
Interestingly, application of exogenous therapeutic strategies may not always upset the homeostasis of intrinsic cellular pathways through
the development of reactive metabolic pathways but rather through the elimination of essential molecules. For example, colleagues of
Fatani et al. illustrate for us that deciency in carnitine, an essential cofactor for -oxidation of long-chain fatty acids in the mycocardium,
occurs during Fanconi Syndrome and can result in ifosfamide-induced cardiotoxicity requiring the supplementation of carnitine to prevent
this disorder. Gautam et al. introduce an additional level of scrutiny for the concept of yin yang to inform us with their novel clinical
study of lymphocytes in individuals of progressive ages that external factors such as aging should also be recognized in scenarios that can
alter the balance between the opposing forces of oxidant generation and antioxidant scavenging in biological pathways. In our nal article
for this issue, Lee et al. identify a unique relationship between neurons and astrocytes that allow neurons to transfer -synuclein to astro-
cytes and trigger the production of inammatory factors that may injure neurons. Yet, this observation represents only one side of the coin
and in the true spirit of the concept of yin yang, we learn that some factors released from -synuclein-stimulated astrocytes can impart
protection upon neurons. Our papers in this issue of Oxidative Medicine and Cellular Longevity bring to the forefront the concept of yin
yang in complex biological systems. Although the origins of this concept may have originated with the advent of medical care in ancient
civilizations, it is no less relevant then than it is now to fully understand and develop effective therapeutic strategies for modern medicine.
Yin yang
A balancing act for oxidative stress
Kenneth Maiese
Department of Neurology and Neurosciences; University of Medicine & Dentistry of New Jersey;
New Jersey Medical School; Newark, NJ USA
Oxidative Medicine and Cellular Longevity 3:4, 227-227; July/August 2010; 2010 Landes Bioscience
Correspondence to: Kenneth Maiese; Email: maieseke@umdnj.edu
Submitted: 08/16/10; Accepted: 08/16/10
Previously published online: www.landesbioscience.com/journals/oximed/article/13340
DOI: 10.4161/oxim.3.4.13340
Cellular Longevity
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in Medicine
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AIDS
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http://dx.doi.org/10.1155/2013/456747
Research Article
An Integrative Platform of TCM Network Pharmacology and
Its Application on a Herbal Formula, Qing-Luo-Yin
Bo Zhang,
1,2
Xu Wang,
1
and Shao Li
1
1
Bioinformatics Division and Center for Synthetic and Systems Biology, TNLIST/Department of Automation, Tsinghua University,
Room 1-107, FIT Building, Beijing 100084, China
2
Joint Computational Center of Drug Discovery, Tianjin International Joint Academy of Biotechnology & Medicine,
Tianjin 300457, China
Correspondence should be addressed to Shao Li; shaoli@mail.tsinghua.edu.cn
Received 6 January 2013; Accepted 4 February 2013
Academic Editor: Aiping Lu
Copyright 2013 Bo Zhang et al. Tis is anopenaccess article distributedunder the Creative Commons AttributionLicense, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Te scientifc understanding of traditional Chinese medicine (TCM) has been hindered by the lack of methods that can explore
the complex nature and combinatorial rules of herbal formulae. On the assumption that herbal ingredients mainly target a
molecular network to adjust the imbalance of human body, here we present a-self-developed TCM network pharmacology
platform for discovering herbal formulae in a systematic manner. Tis platform integrates a set of network-based methods that
we established previously to catch the network regulation mechanism and to identify active ingredients as well as synergistic
combinations for a given herbal formula. We then provided a case study on an antirheumatoid arthritis (RA) formula, Qing-Luo-
Yin (QLY), to demonstrate the usability of the platform. We revealed the target network of QLY against RA-related key processes
including angiogenesis, infammatory response, and immune response, based on which we not only predicted active and synergistic
ingredients fromQLY but also interpreted the combinatorial rule of this formula. Tese fndings are either verifed by the literature
evidence or have the potential to guide further experiments. Terefore, such a network pharmacology strategy and platform is
expected to make the systematical study of herbal formulae achievable and to make the TCM drug discovery predictable.
1. Introduction
Traditional Chinese medicine (TCM) is a whole medical sys-
tem deriving from thousands of years of clinical application
that has evolved independently from or parallel to allopathic
conventional medicine and has been considered as one of the
main items of the complementary or alternative medical sys-
tem [1, 2]. Te treatments of TCM formulate the therapeutic
use of herbs using the combinatorial principle of Sovereign-
Minister-Assistant-Envoy (Jun-Chen-Zuo-Shi in Chinese) on
the basis of a patients syndrome (ZHENG in Chinese) and
attempt to regain the balance state of life and body functions
[3]. However, unlike modern drugs developed by targeting
a specifc protein, understandings of the molecular basis of
traditional herbal formulae are still very limited, posing a
serious challenge for the modernizationof TCM[4]. With the
recent advent of high-throughput technologies, experimental
analyses of the active ingredients screening and the mech-
anisms of action of herbal formulae have become increas-
ingly various. Investigators ofen examine a herbal formula
from diferent facets by combining chemical or metabolic
fngerprint [5, 6], pharmacodynamic and pharmacokinetic
technology [7, 8], and genomic, proteomic or metabolomics
analyses [911]. However, herbal formulae with numerous
chemical compounds are too complex to be examined solely
by conventional experimental approaches. Moreover, a herbal
formula contains hundreds of chemical compounds and its
therapeutic efects are mainly produced by complex interac-
tions among ingredients [12]. Current experimental methods
are restricted to tap into the deeper well and comprehensively
elucidate the molecular mechanisms of TCM. Te dearth
of modern methods in TCM study and deconvolution of
complexity of TCM urgently requires new strategies and
appropriate approaches.
As the beginning of TCM network pharmacology, we
proposed the possible relationship between TCMand molec-
ular networks in 1999 [13] and established a network-based
herbal formulae research framework illustrated by a network-
based case study on Cold/Hot herbal formulae and Hot/Cold
syndromes in 2007 [14, 15]. Shortly afer, the age of network
pharmacology has clearly begun [16, 17], we believe net-
work pharmacology approaches, focused on examining the
network connectivity and dynamics as components of drug
targets and designing the optimal therapeutic strategies, can
reveal the underlying complex relationships between a herbal
formula and the whole body. Tus, we further explored the
new subject of TCM network pharmacology by updating the
research paradigm from current one target, one drug to
network target, multicomponent therapeutics, which refers
to the comprehensive analysis for therapeutic efects of herbal
formulae on the basis of the identifcation of the network
target underlying a given disease or TCM syndrome as well
as the target network of a given herbal formula [15, 1820].
To date, accumulating evidence suggests that the network
pharmacology analysis is a powerful way to study the molec-
ular mechanisms that are responsible for combinational
efects of herbal formula [18, 2025]. For example, Sun et al.
presented a network analysis to explore the mechanism of
anti-Alzheimer herbal ingredients by evaluating the distance
between the herbal targets and Alzheimer-related proteins
in the protein interaction network [21]. Wang et al. used a
systems biology model integrating oral bioavailability and
drug-likeness screening, target identifcation, and network
methods to analyze the synergistic mechanism of four herbs
in combined treatment of cardiovascular disease [22].
In this work, to better recognize the active ingredients
in herbal formula and uncover the combinational rules
of ingredients, we integrate our previous methods into a
TCM network pharmacology platform to illustrate network
connections between multiple targets of ingredients in herbal
formula and multiple genes of a specifc disease. Te methods
we created for TCM network pharmacology in the past years
include network-based disease gene prediction, drug target
prediction, drug-gene-disease comodule association, herb
network analysis, and synergistic drug combination screen-
ing [20, 2531]. Te good performance of these methods
had been demonstrated in discovery of bioactive compounds
and elucidation of action mechanism for herbal formulae
[23, 32].
We further apply this integrative platform to unveil
the molecular mechanisms of antirheumatoid arthritis (RA)
formula named Qing-Luo-Yin (Q-L-Y), including four herbs:
Ku-Shen (Sophora favescens), Qing-Feng-Teng (Sinomenium
acutum), Huang-Bai (Phellodendron chinensis) and Bi-Xie
(Dioscorea collettii) [33]. Here, we revealed the target net-
work of QLY against RA-related key processes including
angiogenesis, infammatory response, and immune response
and report that the four herbs may produce interactions for
enhancing efciency and reducing toxicity through acting in
concert on the target network closely associated with RA. Te
Jun herb, Ku-Shen, treats the main causes of RA, for example,
infammatory response, immune response, and angiogenesis.
Te Chen herb, Qing-Feng-Teng, serves to augment the anti-
infammatory and antiangiogenesis efects of Jun. Te Zuo-
Shi herbs, Huang-Bai and Bi-Xie, are used to modulate the
therapeutic efects of Jun-Chen herbs and to counteract the
side efects of Ku-Shen possibly by targeting some of-target
genes (i.e., PTGS1). Moreover, we found that the synergism
among major ingredients from Ku-Shen and Qing-Feng-
Teng may derive from the feedback loop and compensatory
mechanisms (i.e., TNF-, IL1B-, and VEGFA induced NF-
B pathways). We also identifed several ingredient groups
such as Saponins and Alkaloids that act as active components
in QLY using the cluster analysis of their target profles.
Te above fndings are either verifed by the literature
evidence or have the potential to guide further experiments.
Hopefully, our platform is eventually extensible to other
herbal formulae, which provides a reliable and practical
strategy to identify active herbal ingredients and potential
synergistic pairs, to reveal the mechanisms of herbal formulae
and to facilitate TCM drug discovery and modernization as
well.
2.1. Inputs of the Integrative Platform of TCM Network
Pharmacology. To address the challenges in the study of
the molecular basis and combinatorial principle of herbal
formulae, we developed a network-based integrative strategy
to provide a unifed framework as a platform for TCM
network pharmacology (Figure 1). Tis platform contains
two diferent types of entities as inputs: herbal ingredients
with known chemical structure, disease-specifc genes, and
targets of drug treating this disease. Previously, we built a
HerbBioMap database to collect the chemical ingredients in
621 herbs [34]. For QLY, we select all available ingredients
for each of four herbs in this formula from HerbBioMap and
the available literature on the four herbs [3437]. We also
conducted chemical analysis to identify and determine the
major ingredients in QLY, for example, Matrine, Kurarinone,
Sinomenine, Berberine, and Diosgenin [38, 39]. Finally, afer
excluding the repeated ingredients, a total of 235 ingredients
with 112 in Ku-Shen, 49 in Qing-Feng-Teng, 54 in Huang-Bai,
and 20 in Bi-Xie were collected in this study. Te structure,
canonical name, and CID number of these ingredients were
obtained from PubChem [40]. For RA, known RA-related
genes were retrieved from the OMIM Morbid Map [41].
Putative RA genes were predicted by our CIPHER method
[27]. Te known targets of RA drugs were obtained from
DrugBank database [42]. For RA disease network construc-
tion, these genes and gene products are treated as seeds
to obtain their partner genes in the context of the human
protein-protein interaction network (HPRD, Release 7) [43].
2.2. Predicting Target Profles for Each Ingredients in QLY.
Comprehensively determining compound-target interaction
profles and mapping these on signaling and metabolic
pathways will become increasingly necessary for elucidating
the mechanisms of action of drugs [44]. In silico prediction
Herb
combinations
Compounds
DMIM
Target profle
drugCIPHER
Disease genes
LMMA
Network target
analysis
TCM drug discovery
Optimization of
therapeutic strategies
comCIPHER
NADA
NIMS
CIPHER
CSPN
Herbal formula
Omics data
Disease
(TCM syndrome)
Terapeutic mechanism
of herbal formula
Disease molecular
network
Figure 1: Aschematic map of the integrative TCMnetwork pharmacology platformthat is based on our network target theory and combined
some of our self-developed methods including CIPHER, drugCIPHER, comCIPHER, DMIM, NIMS, NADA, LMMA, and CSPN.
of target profles of small molecular compounds especially
is a critical step for the study of TCM network pharma-
cology. Recently, we developed a regression model called
drugCIPHER that can predict the links between drugs and
target proteins by combining the drug chemical similarity
and protein-protein interaction information in a heteroge-
neous network that correlates chemical, pharmacological,
and genomic spaces [26]. In accordance with the rationale
of like attracts like, this method is based on the hypothesis
that drugs with similar chemical structures or therapeutic
efects tend to bind to functionally related or modularized
target proteins in the molecular network. Tus, drugCIPHER
can infer the target profle for a given herbal ingredient
with known structure by integrating and making full use
of all available FDA-approved drug structures, drug-target
interactions, and human protein-protein interactions. Te
prediction principle of drugCIPHER is also featured in the
TCM holism thinking.
In this study, we used the drugCIPHER-CS step in
our drugCIPHER method to predict the target profle for
each herbal ingredient from QLY. Te drugCIPHER-CS
score refers to the likelihood of ingredient-target interaction
calculated from the correlation between the query ingredi-
ents structure similarity vector in the drug space and the
target-related genes closeness vector in the target space. Te
resulting proteins with high likelihoods are considered as
potential targets of the herbal ingredient. We selected the top
100 proteins with high precision rate as a target profle for
each ingredient [26]. We then assembled the target profles
of all available ingredients in every of four herbs and resulted
in an integrative target profles of QLY.
2.3. Principal Component Analysis (PCA). To classify the
herbal ingredients from QLY using the predicted target
profles, PCA was performed to reduce the dimensionality of
multivariate data into a multidimensional space, allowing for
clear visualization of the variation between diferent herbal
ingredients. In this step, we use PCAto reduce the dimension
of the target profle of each ingredient in QLY while most
of the variance is preserved by linearly transforming the
variables into a smaller number, say , of variables that we
denote by
1
, . . . ,
=1
, = 1, . . . , ,
(1)
where = [(1), . . . , ()]
denotes the target profle of each

ingredient in QLY and
is transforming weights with the

property of the orthogonality and unit norm:
= 1, ,
= 0, =.
(2)
2.4. Network Target Analysis. To better elucidate the holistic
therapeutic efects of QLY, we attempted to fgure out the
target network and mechanism of action of QLY by our
platform. First, genes or proteins involved in RA were
compiled by combining RA-causing genes from OMIM and
CIPHER prediction [27, 41] and the target proteins of anti-
RA drugs from DrugBank [42]. Second, RA-related genes or
proteins were used as seeds to fsh their partner interacting
proteins in the HPRD [43]. Te searching of such partner
proteins resulted in an expanded network as the RA-specifc
network. Tird, the candidate targets of chemical compounds
in each herb predicted by drugCIPHER were mapped into
the RA-specifc network and were used as a new query to
identify the target network of each herb, respectively. Fourth,
in order to understand the possible biological functions of
eachherb, the functional distributionof these target networks
was further examined. Finally, the combinational rationale
of QLY was interpreted according to the detailed analysis of
comodule associations and enriched biological functions.
2.5. Biological Function Enrichment Analysis. For biological
functional analysis of QLY, we use the functional enrichment
tool of the DAVID database to analyze the enriched GO
(Gene Ontology) terms for the assembled target-related
proteins of QLY with a false discovery rate less than 0.05
by the Fisher exact test [45, 46]. We only selected the GO
functional terms with value less than 0.05 afer Benjaminis
correction.
3. Results
3.1. A Self-Developed Platform of TCM Network Pharmacol-
ogy. Te concept of network target that we proposed [15,
1820] is the core of the integrative platformof TCMnetwork
pharmacology, by which we hypothesize that the relationship
between a herbal formula and a disease or TCM syndrome
can be transferred into a network context. Te key modules
in a disease-specifc molecular network are considered as the
therapeutic target of a given herbal formula. Tus, we can
disclose the action mechanisms and the active ingredients
as well as their combinations in a herbal formula from the
network target viewpoint. Tis platform fully integrates our
developed methods, in which the good performance has
been validated, respectively [2532], for understanding the
therapeutic mechanismof herbal formula fromthe following
three aspects (Figure 1).
(1) Network target construction: the construction of a
disease-specifc network as the therapeutic target,
such as the prioritization of candidate genes for
a given disease (CIPHER) [27], and construction
of disease-specifc networks in the molecular level
(LMMA) [28] or in the pathway-pathway interaction
level (CSPN) [29] by combined knowledge and high-
throughput omics data.
(2) Target prediction and herbal pair extraction: the
prediction of target profles of herbal ingredients
(drugCIPHER) [26, 47] as well as the extraction of
common herbal pairs from herbal formulae treating
specifc diseases (DMIM) [25].
(3) Comodule analysis based on the network target: such
as drug-gene-disease comodule analysis between
drug (ingredient) targets from herbal formula and
disease-specifc network target (comCIPHER) [31]
and network target-based computational screening
of active ingredients (NADA) [30] and synergistic
therapeutic combinations (NIMS) [20, 48].
Diferent from conventional herbal formulae research
strategies that fnd active ingredients based on a certain
disease, our network target strategy [2532] can lead to more
discoveries of active ingredients against various diseases in a
network level by capturing each ingredients target profle in
a genome-wide scale. Tus, our platform has two signifcant
characteristics: discovery-oriented and generally applicable,
especially in predicting active ingredients, synergistic ingre-
dient combinations as well as active ingredient groups from
a given formula, and providing the comprehensive molecular
mechanisms of the formula. Here, we examine the Qing-Luo-
Yin as a case study in the following sections.
3.2. Clustering Active Ingredients in QLY Based on the Target
Profles. QLY, which derives from a Xinan medical family
[49], is an efective formula in the treatment of arthritis and
a typical antiangiogenic herbal formula in TCM [50]. Tis
formula is composed of Ku-Shen (Sophora favescens), Qing-
Feng-Teng (Sinomenium acutum), Huang-Bai (Cortex Phel-
lodendri Chinensis), and Bi-Xie (Dioscorea tokoro Makino)
(Figure 2(a)) [51]. Our previous studies have revealed the
antiangiogenic and anti-infammatory efects [33] and the
network regulation actions of QLY [14].
To further understand the molecular details about how
QLY can be administrated on RA, we used our platform to
predict the target profles of each ingredient in QLY and
utilizedPCAto visually assess the distinctionof target profles
of eachherb anddetermine whether herbal ingredients canbe
grouped. Te analysis of dimensionality of all target profles
showed that two frst components could account for >60%
of the variance present in all targets contained within the
target profles. To see whether the variation retained in
the two components contains relevant information about
the mechanism of QLY, each ingredient is projected onto
the two components as illustrated in Figure 2(b). Te PCA
analysis showed that four herbs cannot be separated into
four independent clusters only according to the target profles
(Figure 2(b)), suggesting that the features of target profles of
the ingredients from diferent herbs are overlapped. Further,
to determine whether the herbal ingredients with the similar
chemical properties can be clustered together, the results
showed that most herbal ingredients in QLY can be roughly
divided into three groups, which exactly were mapped to
three types of chemical components, namely, saponins, gly-
cosides, and alkaloids (Figure 2(c)). Figure 2(c) showed that
the herbal ingredients ineachwell-separatedcluster may have
similar mechanisms of actionandcanafect diferent stages or
pathological processes of RA in the form of active ingredient
groups. Together, these fndings indicate that predicted target
profles can be used to identify active ingredient groups
leading to similar efects in a herbal formula.
3.3. Predicting Active and Synergistic Ingredients from QLY.
To examine what ingredients can produce synergistic efects
on key pathological processes involving RA, angiogenesis,
infammatory, and immune response and what are the
synergistic mechanisms among them, we took advantage
of the previous conclusion that synergism may arise from
modulations of compensatory actions or feedback loops in
the network [20, 52] to estimate the interaction between
Ku-Shen Qing-Feng-Teng
Huang-Bai Bi-Xie
(a)
0 2 4 6
2
0
2
4
P
r
o
j
e
c
t
i
o
n

o
n
t
o

P
C
2
2
Projection onto PC1
KS
QFT
HB
BX
(b)
Matrine
Sinomenine
Kurarinone
Diosgenin
Berberine
2
0
2
4
2 0 2 4
P
r
o
j
e
c
t
i
o
n

o
n
t
o

P
C
2
Projection onto PC1
Saponins
Glycosides
Alkaloids
Matrine
Sinome mmmmmm nine S
Kurarinone ar
Diosgenin
Ber BBBBBBBBBBBBBB berine eri
(c)
Figure 2: (a) Four herbs Ku-Shen (Sophora favescens), Qing-Feng-Teng (Sinomenium acutum), Huang-Bai (Cortex Phellodendri Chinensis),
and Bi-Xie (Dioscorea tokoro Makino) in QLY. (b) and (c) Principal component analysis of the target profles for each herbal ingredient in
QLY. Each dot represents one herbal ingredient plotted against its target profle. Ingredients are color coded according to the four herbs (b)
and the three types of the chemicals (c) in QLY. Samples were distributed by their similarity in target profles using dimensionality reduction.
Te diferent chemical types cluster in terms of target profles can be separated from each other. Te four herb clusters in terms of target
profles are partially intermix with each other.
ingredients. Adapting such criteria, we derived an ingredient-
ingredient interaction network in terms of target proteins
of each ingredient (Figure 3). We identifed six potentially
synergistic pairs between main ingredients from Ku-Shen
and other herbs, including Matrine and Sinomenine, Matrine
and Kurarinone, Matrine and Berberine, Kurarinone and
Sinomenine, Kurarinone and Berberine, and Kurarinone and
Diosgenin. Tese prediction results can be supported by the
literature evidence. For example, Matrine and Sinomenine
were evaluated as a synergistic combination by endothelial
cell proliferation assay in previous studies [20] and were
confrmed from the mechanism here. As shown in Figure 3,
the prediction showed that Matrine can bind to the IL1R1,
which suppresses infammatory and immune response by
IKBKB
(Huang-Bai) Berberine
TNF
MAPK14
MAPK1
PTK2
RAF1
AKT1
CASP9
AKT2 IL1B
IL1R1
VEGFA
KDR
PIK3R1
RELB
RELA
IKBKG
TNFRSF1A
Diosgenin (Bi-Xie)
TNF pathway
VEGF pathway
IL1B pathway
Feedback loop
Sinomenine
(Qing-Feng-Teng)
SRC
TRAF2
NFKB1
NFKB2
NFKBIA
NFKBIA
Kurarinone (Ku-Shen)
Synergistic interaction
Matrine (Ku-Shen)
Figure 3: Putative therapeutic mechanism for selected major active
ingredients in QLY and potential synergistic pairs. Te upper
network is the ingredient synergy network (including Kurarinone
and Matrine inKu-Shen, Sinomenine inQing-Feng-Teng, Berberine
in Huang-Bai, and Diosgenin in Bi-Xie), and the lower network
is RA-specifc molecular network which was constructed manually
based on the RA-related pathways and the potential targets of the
major ingredients. Herbal ingredients and RA-related genes were
represented as triangle and circle, respectively. TNF-, IL1B-, and
VEGF-induced pathways were highlighted in color line as indicated.
NFKB1, NFKB2, RELA, and RELBcan formheterogeneous complex
as an important transcription factor in the development of RA. Te
blue line linking two ingredients indicated synergy with the width
of the line correlating with the number of synergistic mechanisms:
the wider line represented feedback and compensatory mechanisms
and the narrow line represented feedback or compensation.
blocking the NF-B pathway activated by IL1B. Sinomenine
can suppress angiogenesis and infammation by inhibiting
NFKB1 and SRC. Sinomenine may complement Matrine-
induced inactivation of NF-B to reduce its induction of
angiogenesis, infammation, andimmune response. Inpartic-
ular, two ingredients fromKu-Shen, Kurarinone and Matrine,
may lead to potent synergistic interaction with each other,
thus refecting interactions from diferent ingredients in the
same herb. Because some studies indicated the relationship
between NF-B and oxidative stress in rheumatoid arthritis
[53], we inferred that Kurarinone as an antioxidant [54] is
likely to inhibit NF-B activation in terms of predicted target
profles. Kurarinone can inhibit AKT1 and PTK2, which is
downstream of IL1B. Tis may sensitize the efect of Matrine
via modulation of NF-B and AKT1. In addition, four other
synergistic pairs were also identifed by network-based syner-
gism hypothesis. For example, Matrine and Berberine act on
diferent targets (IL1R1 and KDR) of two cross-talk pathways
(IL1B and VEGFA pathway) that regulate the SRC activity.
Kurarinone and Sinomenine act on diferent targets (AKT1
and SRC) of the same pathway (AKT1-SRC-PTK2 pathway)
that regulates the NFKB1. Kurarinone and Berberine act on
diferent targets (NFKB1 and KDR) of two related pathways
(VEGF and NF-B pathway) that regulate diferent targets
(SRC and NFKB1). Kurarinone and Diosgenin act on the
same type of target (NFKB1 and NFKB2) in the feedback loop
(NFKB1-NFKB2-RELA-RELB complex) and diferent targets
(PTK2 and RAF1) of two related pathways.
3.4. Target Networks and Combinatorial Rules of QLY. Dif-
ferent from the conventional trial-and-error drug studies,
our network-based strategy tries to make the TCM drug
discovery predictable and to make the systematical study of
combinatorial rules in herbal formulae achievable. As shown
in Figure 4, the network target analysis of 235 ingredients in
QLYherbs indicated the detailed mechanisms of herb combi-
nations with increased efcacy and decreased toxicity in RA
therapy. Ku-Shen, as Jun herbs in QLY, acts on the principle
RA pathological processes, such as infammation, immune
response, and angiogenesis. Qing-Feng-Teng as a Chen herb
and Huang-Bai and Bi-Xie as Zuo-Shi herbs seemto augment
or modulate the therapeutic efects of Jun herb through
targeting RA-relatedgenes including NFkB1, HTR3A, CASP1,
and PPARG. Interestingly, these results of network target
analysis demonstrate an unexpected mechanism of QLY for
RA therapies, in which Aryl hydrocarbon receptor (AHR)
contributing to the pathogenesis of RA [55] is regulated by
multiple ingredients in QLY (e.g., Kuraridin, Sophorafa-
vanone and Xanthohumol in Ku-Shen, Salicylaldehyde and
Allantoin in Qing-Feng-Teng, -Elemene in Huang-Bai, and
Piperitol in Bi-Xie). Besides, the pharmacological activities
of Ku-Shen, Qing-Feng-Teng, and Bi-Xie are associated with
targeting the NF-B pathway. Tese results demonstrated
the synergistic efects among the four herbs of QLY. Our
predictions also include the mechanisms of decreasedtoxicity
of Zuo and Shi herb in QLY. For instance, Xanthohumol in
Jun herb may cause adverse drug reactions through afecting
of-target genes such as PTGS1, resulting in gastrointestinal
haemorrhage, haematuria, and abdominal pain [56]. In the
target network, we found that cis-limonene oxide phello-
chinin A and ferulic acid in Huang-Bai may neutralize the
adverse efects of Ku-Shen through modulating PTGS1.
In addition, by examining the functional distribution of
the potential targets of QLY, we found that the signifcantly
enriched GO terms QLY acted include the key processes
in the development of RA, such as infammatory response,
regulation of cytokine production, regulation of angiogen-
esis, and leukocyte activation (Table 1). Terefore, by mod-
ulating these pathological processes, QLY may promote the
recovery of network balance from a disease state to a normal
state. Together, these results reveal not only the target net-
work of QLY against RA-related angiogenesis, infammatory
response, immune response, and NF-B activity but also the
Jun-Chen-Zuo-Shi principle of QLY from the connections
of functional modules in the network target.
4. Discussion
Many common diseases such as cancer and rheumatoid
arthritis as well as cardiovascular diseases are complex bio-
logical systems caused by multiple molecular abnormalities
Reducing toxicity
Infammatory
response
Immune
response
Angiogenesis
Adverse
reaction
Enhancing efciency
Reducing toxicity
Efect of Ku-Shen
Efect of Qing-Feng-Teng
Efect of Huang-Bai
Efect of Bi-Xie
Enhancing efciency
Enhancing efciency
cascade
IKK-NF-B
Rheumatoid arthritis
Figure 4: Target network and functional enrichment of the four herbs from QLY. A target network of each herb was uniquely identifed
by mapping the possible targets of each herb into RA-specifc molecular network. Te functions for the target networks were obtained by
the functional enrichment tool (DAVID). Tese enriched biological functions are associated with RA. Targeting PTGS1 may lead to some
adverse efects. Genes labeled in red color denote RA genes collected from OMIM, genes labeled in purple color denote the targets of the
FDA-approved drugs for treating RA, and genes labeled in blue color represent RA genes predicted by CIPHER.
Table 1: Enriched RA-related GO terms in the Qing-Luo-Ying target network.
Function category GO term ID GO terms
P value
(Benjaminis correction)
Angiogenesis
GO:0009611 Response to wounding 1.30 29
GO:0045765 Regulation of angiogenesis 1.62 04
GO:0010594 Regulation of endothelial cell migration 0.002
Infammatory response GO:0006954 Infammatory response 1.23 13
Immune response
GO:0001817 Regulation of cytokine production 1.42 04
GO:0006955 Immune response 0.002
GO:0045321 Leukocyte activation 1.17 05
GO:0001816 Cytokine production 0.004
GO:0051249 Regulation of lymphocyte activation 0.001
GO:0006952 Defense response 2.88 12
GO:0042981 Regulation of apoptosis 2.32 17
NF-B activity GO:0051092 Positive regulation of NF-B transcription factor activity 0.037
[57, 58]. During the therapy, many drugs that modulate a
single target might not always yield the desirable outcome
even if they completely interdict the functions of their direct
targets [59, 60]. From a network perspective, the entity
that needs to be targeted and modulated must shif from
single proteins to entire disease molecular networks [61,
62]. Te efcacy of such therapies can be explained by the
fact that drugs targeting diferent proteins in the disease
network or pathway could trigger a synergistic response, and
their combinations can eliminate compensatory reactions
and feedback controls, thereby overcoming the robustness of
diseases [63, 64]. Tese perspectives illuminate that the level
of complexity of the proposed therapies should be increased.
Interestingly, the properties of TCM herbal formula are con-
sistent with the coming network-based therapeutic strategies.
However, currently it is hard to unveil the complex systems
embedded in the TCM repertoire, especially the interactions
between the complex biological systems of human body and
the complex chemical systems of herbal formulae.
To provide a novel route for the systematic studies of
herbal formulae, here we report a self-developed integra-
tive platform of TCM network pharmacology (Figure 1) to
acquire a better understanding of the underlying mechanisms
and combinatorial rules of herbal formula. To the best of
our knowledge, this is the frst self-developed TCM network
pharmacology platformfor studying herbal formulae [47, 48,
65]. Indeed, the performance of all methods in the platform
have been properly tested, respectively [2532], and some
key methods have been recognized as one of the leading
approaches in network biology and network pharmacology
[66, 67]. For instance, CIPHER achieves a high-precision
accuracy in disease gene prediction that outperforms the
state-of-art methods [27], drugCIPHER takes the lead in
the genome-wide drug target prediction [26], and NIMS
is regarded as a novel method in network pharmacology
[67]. Tese methods can help solve challenging problems in
studying chemical and biological basis of herbal formula. For
example, drugCIPHER provides a new way to identify target
profles of most ingredients in herbal formulae [26, 47].
In this work, by QLY as a case study, we demonstrate
that this platform is efective on identifying bioactive ingre-
dients, synergistic ingredient pairs, and ingredient groups
(Figures 2 and 3) and elaborating the combinational rules
of QLY (Figure 4), which tentatively validated by statistical
approaches as well as literature. For example, the previous
experimental studies have shown the anti-infammatory
actions of Matrine, the antiangiogenic efect and anti-IL1B
expression of Sinomenine, the immune-regulatory efect of
Berberine [6871], and synergistic efects between Matrine
and Sinomenine [20], which proved the outputs of our
platform. Although the target networks of QLY need to be
further experimentally determined, this platform is useful
for uncovering the systematic-level mechanisms that are not
easily detectable in experimental studies. For instance, our
analysis revealed that not only Ku-Shen and three other herbs
may synergize by targeting diferent biological processes (e.g.,
angiogenesis, infammatory, and immune response), but also
Huang-Bai may antagonize the adverse reaction of Ku-Shen
through some of-target genes (e.g., PTGS1) that deserve
further experimental testing.
Te aim of herbal formulae treatment is to adjust an
imbalance state of disease-specifc network, which refers to
the network interaction and node activity or expression in a
given disease context deviating from health status (Figure 5).
Recent studies of cancer therapy have shown that disease-
specifc networks are dynamic and can change with time and
space in order to adapt to diferent interventions, resulting
in compensatory efects and drug resistance [72, 73]. For a
given disease-specifc molecular network, the combination of
interventions can best restore the disease network to a desired
normal state. Tus, our platform can be used to reveal the
behavior of network balance regulation featured by herbal
formulae. Our results suggest that various ingredients in QLY
may weakly target diferent proteins within the RAmolecular
network, shut down the whole pathological process by net-
work interaction or biochemical synergism, then maintain a
delicate balance of the human body, and fnally activate its
own capability of disease resistance. In this study, we clarifed
that the synergistic efects among six main ingredients in
QLY are caused by acting on the compensatory pathway
and feedback loop in the TNF/IL1B/VEGF-induced NF-B
pathways involved in RA and the synergistic mechanism
of QLY is partially associated with the modulation of NF-
B imbalanced network (Figures 3 and 4). Recently, the
combination intervention of NF-B system has provided the
evidence for the efciency of network balance regulation
in cancer therapy. For instance, NF-B-blocking therapies
against tumors with constitutive or chemotherapy-induced
NF-B activation represent one of the few examples where
inhibition of NF-B network serves as a homeostatic switch
for enhancing genotoxic damage but promotes the secretion
of protumorigenic factor, IL-1 [74, 75]. In this case, NF-
B inhibition combined with anti-IL-1 therapy will rebalance
the adverse efects of perturbed NF-B network [76]. Indeed,
the adjustment of dysregulated NF-B network implicated
in other diseases can help to understand the efects of QLY
on RA. QLY is most likely to modulate angiogenesis within
infammation or tumor environment owing to its modulation
of the NF-B network. It is noted that the Jun-Chen herb (Ku-
Shenand Qing-Feng-Teng) inQLYhas shownthe therapeutic
benefts on tumor development [70, 77]. Recently we also
identifed and experimentally verifed a novel angiogenesis
inhibitor, vitexicarpin, from a herb paired with Huang-Bai
in QLY [32]. We believe that integrative adjustment of the
imbalanced network is expected to be one of the trends of
future drug discovery, especially discovery of combinatory
drugs from herbal formulae.
In addition, we can also capture the formula-syndrome
relationship from a network target viewpoint. Te treatment
strategy of herbal formula is characterized by guiding the
combination of herbs in the light of the imbalance state of
the human body, such as TCM Cold and Hot syndromes. We
investigated the imbalanced molecular network associated
with Cold syndrome and Hot syndrome in the context of
the neuroendocrine-immune system and identifed several
key Hot syndrome-related molecules, such as IL1B, TNF, and
VEGF [14]. Our previous results demonstrated that QLY can
suppress angiogenesis and infammation in collagen induced
arthritis rats [33]. Te present work also demonstrates at
the molecular level that QLY as a Cold-natured formula
is likely to modulate these network hub molecules of Hot
syndrome (IL1B, TNF, and VEGF), aiming to expel the
pathogenetic hot for curing Hot syndrome-related RA and
exert the antiangiogenesis, anti-infammation, and immune-
regulatory actions (Figure 4 and Table 1). All together, these
fndings evidenced that the mechanism of QLY can be
interpreted by its actions on a therapeutic network and its
adjustment of the network imbalance state.
As illustrated by Qing-Luo-Yin, we demonstrate that
the implementation of our TCM network pharmacology
Mutations or amplifcations
Aberrant and continuous activation
Loss of negative feedback control
Normal state
Herbal formula
Herbs
Disease/TCM syndrome state
Figure 5: Regulation of network imbalance as an important therapeutic principle of herbal formula. Mutation/amplifcation and aberrant
signal transduction cause the multiple changes of the normal network structure, leading to the imbalance of health state. Combinations of
herbs used in herbal formula (substantially certain chemical compounds) can weakly target diferent proteins within the disease-specifc
network so as to restore the imbalanced disease state.
platform can not only recover the known knowledge but also
provide new fndings that deserve further experimental vali-
dations for discovering the active ingredients and therapeutic
mechanism of herbal formulae. Terefore, this sustainable
development platform coupling with the rich experience of
TCM is hopeful of shifing the paradigm for conquering
complex diseases from the conventional one target, one
drug to the network target, multicomponent therapeutics,
ofering bright prospects and solid supports for translating
TCM from experience-based to evidence-based medicine
and accelerating TCM drug discovery as well.
Conflict of Interests
Te authors declare that they have no confict of interests.
Acknowledgments
Tis work is supported by the National Natural Science Foun-
dation of China (nos. 81225025, 60934004, and 91229201), the
CATCMproject, and the SATCMkey discipline of TCMBi-
Bing in Yijishan Hospital of Wannan Medical College.
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of
http://dx.doi.org/10.1155/2013/697893
Review Article
Observational Studies on Evaluating the Safety and
Adverse Effects of Traditional Chinese Medicine
Jung-Nein Lai,
1,2
Jin-Ling Tang,
3,4
and Jung-Der Wang
5
1
Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei City 112, Taiwan
2
Department of Chinese Medicine, Taipei City Hospital, Yangming Branch, Taipei City 111, Taiwan
3
Center for Evidence Based Medicine, Peking University Health Science Centre, Peking University, Beijing 100871, China
4
Division of Epidemiology, School of Public Health and Primary Care, Te Chinese University of Hong Kong, Hong Kong
5
Department of Public Health, College of Medicine, National Cheng Kung University, Tainan City 701, Taiwan
Correspondence should be addressed to Jung-Der Wang; jdwang@ntu.edu.tw
Received 7 April 2013; Revised 23 June 2013; Accepted 10 August 2013
Academic Editor: Lixing Lao
Copyright 2013 Jung-Nein Lai et al. Tis is an open access article distributed under the Creative Commons Attribution License,
Background. Tis study aims to share our experiences when carrying out observational studies of traditional Chinese medicine
(TCM). Methods. We have proactively monitored the safety profles of Duhuo Jisheng Tang (DJT), Suan Zao Ren Tang (SZRT),
and TMN-1. A list of adverse events (AEs), complete blood counts, and liver and kidney function tests were obtained from the
participants during their scheduled hospital visits. Retrospective observational studies were conducted based on the reimbursement
database of the National Health Insurance system, Taiwan, to explore the relationship between the use of TCM that have been
adulterated by aristolochic acid and the risk fromboth nephrotoxins and carcinogens. Results. Atotal of 221, 287, and 203 AEs were
detected afer SZRT, DJT, and TMN-1 had been taken, respectively. Dizziness, headache, stomach ache, and diarrhea were judged
to be probably related to SZRT treatment. Retrospective observational studies found an association between the consumption of
aristolochic acid-containing Chinese formulae such as Mu Tong and an increased risk of CKD, ESRD, and urinary tract cancer.
Conclusion. Prospective and retrospective observational studies seem to have specifc advantages when investigating the safety and
adverse efects of TCM therapies, as well as possibly other alternative/complementary therapies.
1. Introduction
Traditional Chinese medicine (TCM), a long and widely used
form of medical care in ethnic Chinese communities and
nearby regions, has recently been adopted by other ethnic
groups worldwide [15]. Most indications and contraindica-
tions of TCM therapies currently in the market are solely
based on documentation found in ancient books [6] asso-
ciated with a traditional belief that these herbs and/or their
combinations are usually safe. Randomized controlled trials
(RCTs) are recommended to evaluate the intended efects of
TCMtherapies because these are the most convincing design
for controlling bias and potential confounding factors during
comparative studies of clinical interventions [710]. While
modern medicine has generally developed from physiology
and biochemistry and the mechanisms of action of such
drugs are understood at cellular and molecular levels, the
therapeutic principles of TCM usually take a holistic view
involving activating systems, improving system connection,
and enhancing human disease resistance [11]. TCM doctors
usually prescribe herbs tailored to the patients symptoms/
signs and constitution, which ofen requires some modif-
cation of the ancient Chinese formulae. As a result, herbal
formulae that consist of modifed prescriptions of herbs
continue to appear and are also prescribed without any
systematic evaluation [1]. Tus there remains a big question
in TCM research as to how to evaluate TCM prescriptions;
this is because they are highly individualized and difer from
patient to patient. Tus, one possibility is the evaluation
of many diferent herbal formulae during the same trial or
another possibility is to adopt an approach that difers from
that used when evaluating purifed-compound medicines.
Without prior pilot studies to generate safety and efcacy
data, it is not surprising that very few TCM therapies have
undergone high-quality clinical trials and have been proven
to efectively treat diseases or symptoms [9, 12, 13]. Further-
more, because there are considerable variations in the quality
of TCM trials, negative results from RCTs cannot be used
as sufcient evidence for the absence of efcacy, especially
when these TCM remedies have already been on the market
or have been approved by regulatory agencies. Ephedrine
and artemisinin are currently used in western medicine for
treating asthma and malaria, respectively, and have a long
history of use as Chinese herbal remedies. Tese examples
indicate that there the clinical efects of TCM therapies show
a positive association between the active ingredients used in
modern therapies [14, 15] and the traditional use of the plants
from which they are derived. Tus it should be recognized
that, for some, the efects of traditional Chinese medicines
when treating symptoms are as efective as conventional
westernmedicines, but this also gives themthe same potential
to have harmful side efects [16]. Furthermore, adulteration,
inappropriate formulation, and a lack of understanding of
Chinese herbs and their interactions have led to adverse reac-
tions that are sometimes life threatening or lethal. Tus, the
safety concerns normally applied to conventional medicines
equally apply to traditional Chinese medicines.
Unlike efcacy data, safety information obtained even
fromcase series [17] has direct and immediate applications to
patient care. If a treatment is proved to be misused or involved
in oversight during the course of clinical care, then stopping
their use immediately will protect lives and save resources.
Aristolochic acid nephropathy (AA nephropathy), which
has been observed in patients taking Mu Tong and Fangchi
(traditional Chinese formulae that have been adulterated
by aristolochic acid), is a good example. If the causation
between a side efect and a TCMtherapy can be corroborated,
updating contraindication of the treatment will beneft future
TCMusers. As Chinese herbal medicines are used in humans
all the time, continuous surveillance of patient safety while
taking TCM treatments, which can be likened to postmarket
surveillance, should be a convenient and powerful way to
detect any potential harm caused by a TCM therapy. In
this paper, we share our experiences conducting prospective
observational clinical studies that use active surveillance of
the safety profles of various TCM therapies (Figure 1, lef
column). In addition, a retrospective observational study
was also conducted involving the passive surveillance of the
safety profle of Chinese formulae that are known to have
been adulterated with Aristolochia spp. Tese approaches we
believe, when used to assess the safety of Chinese herbal
therapies, may have wider applications.
2.1. Study Design 1: Active Surveillance of Safety Profles. An
efective way of detecting TCM safety hazards is through
active surveillance of a well-defned group of people who
are taking the medication, who are then followed using the
guidelines of GCP (good clinical practice) [18]. First we
organized at the beginning of study design an expert focus
group composed of practicing TCM doctors to determine if
they had noticed any adverse events (AEs) associated with
the treatment of subjects with a specifc health condition.
Ten, these reported potential AEs were included in our list
for surveillance with the AEs most frequently reported for
conventional medicine by the National Reporting System;
these consisted of abdominal fullness, diarrhea, vomiting,
nausea, urticaria, itching, purpura, jaundice, skin vesicles
(or local reddish swelling), edema, hypotension, bradycardia,
dyspnea, fever, muscle cramps, and sleepiness. In addition,
all subjects were required to take objective laboratory tests in
order to detect any abnormal fndings with respect to liver
function, kidney function and blood counts, both at baseline
and afer TCM therapy. During the active safety surveillance,
an AE was defned as any medical complaint except the
symptoms/syndrome under study for efcacy. Any abnormal
change in laboratory values that was judged by the inves-
tigator or the study nurse to be clinically signifcant was
also included. Te aforementioned potential AEs were listed
on the case report form and regularly screened for at every
clinic visit. When the subject flled-in the questionnaire, she
or he was required to recall if any of the listed AEs or
any symptoms/syndrome under study had occurred between
her/his last visit and the current one. Ten, both the clinical
investigator and the study nurse evaluated the severity and
causality of each AE. If either one of themsuspected the AEto
be related to the TCM therapy, a further causality assessment
was conducted for every AE to determine if an adverse drug
reaction was involved [19, 20].
We proactively monitored the safety profle of two tra-
ditional formulae (Duhuo Jisheng Tang and Suan Zao Ren
Tang) [21, 22] together with a new invented formula named
TMN- 1 [23]. Tese formulae have already been published
in evidence-based journals as examples that can be used
to demonstrate our strategy of promoting evidence-based
medicine as part of TCM.
2.1.1. Prospective Observational Study 1: Short-Term Safety
Profling of the Traditional Chinese Formula, Namely, Suan
Zao Ren Tang. Suan Zao Ren Tang (SZRT) has a long history
of use as part of the traditional Chinese pharmacopoeia and
was frst documented in the classical Chinese text Jin Gui
Yao Lue (Essential Prescriptions from the Golden Cabinet)
circa 210 A.D. by Zhong-Jing Zhang [24]. In the classical
literature, SZRT is said to nourish the blood and calm the
nerves eventually bring about a tranquillizing sensation and
reduce sleep disturbance. TCM doctors rely on the indica-
tions and contraindications mentioned in the thousand-year-
old classical Chinese medicine literature when prescribing
this TCM therapy. We carry out active safety surveillance for
four weeks in order to outline the safety profle of SZRT [22].
Profling of a Traditional Chinese Formula Containing Xixin,
Namely, Duhuo Jisheng Tang. Xixin(Radix et Rhizoma Asari),
also known as Saishin in Japan or Sesin in Korea, is widely
used in many parts of Asia despite the fact that it contains
some minute amounts of aristolochic acid (AA) [2527].
Although, since 2004, a total of 393 Chinese herbal products
containing Xixin have been reimbursed under the National
Health Insurance (NHI) in Taiwan [28, 29] where the
Prospective observational studies
Potential efectiveness
Randomized controlled trials
Approval for clinical application
In vitro and animal studies of mechanisms of action
New drug development
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Retrospective observational studies
(analysis of national databases or established cohorts)
(or phase II trials)
Postmarketing surveillance actively (phase IV trials)
Update evidence-based guideline
Informed TCM doctors to prescribe with more precaution
minimize their use in daily practice
Adverse events
Formulas with potential toxicity
drug-herb interaction
Frequently prescribed traditional Chinese formulas
Formulas with potential toxicity
New invented formulas
Identifcation of relevant substances
Figure 1: Surveillance of safety and adverse efects of traditional Chinese medicine.
regulations stipulate that AA must be undetectable in the
fnal herbal products [30], the efects of Xixins plant-derived
nephrotoxins and carcinogens on TCM consumers need
to be monitored proactively. Duhuo Jisheng Tang (DJT), a
traditional Chinese formula described by the ancient Chinese
physician Sun Simiao in 652 AD for the treatment of lower
back and knee pain [31] was prescribed to 725,549 patients
between 1996 and 2004 in Taiwan. DJT has been proposed
to be the cause of AA-related nephropathy in a case report
[32]. Terefore, a study using active safety surveillance system
was conducted to determine whether DJT use among patients
with osteoarthritis (OA) causes acute nephrotoxicity.
Profling of a New Invented Chinese Formula, Namely, TMN-1.
A new formulation called TMN-1, aimed at providing relief
to climacteric women with hot fushes, has been created by a
TCMexpert who has hadmore than20 years of practice expe-
rience in Taiwan. Te product contains a fxed ratio of the
three commercially available traditional Chinese medicines,
Jia Wey Shiau Yau San (JWSYS), Zhi Bo Di Huang Wan
(ZBDHW), and Xiang Sha Liu Jun Zi Tang (XSLJT). All three
preparations have traditionally been individually prescribed
and are well documented in ancient Chinese medicinal
texts (e.g., JWSYS in Prescriptions of the Bureau of Taiping
Peoples Welfare Pharmacy; ZBDHW in Key to Terapeutics
of Childrens Diseases; and XSLJY in Collected Exegesis of
Recipes) [6]. Tis type of prescriptionpatternhas beenneither
recommended in the ancient Chinese pharmacopoeias or
textbooks nor is it taught as part of the TCM academic
program. Hence, there have been concerns regarding herb-
herb and/or formula-formula interactions and these cannot
be overlooked; as a result, further studies on the safety of such
mixed formulae are warranted.
2.2. Study Design 2: Analysis of National Databases and/or
Established Cohorts
2.2.1. Retrospective Observational Study Targeting the Long-
Term Safety Profles of Traditional Chinese Formulae Tat
Have Been Adulterated with Aristolochic Acid. Te datasets
for studying the safety of either conventional medicines or
TCM therapies can be obtained from any established large
health insurance system or from other established cohorts
[2]. Many examples are conducted on the reimburse-
ment database established and managed by the National
Health Research Institutes (NHRI) for the National Health
Insurance (NHI) of Taiwan [3336]. Out of a total population
of 23,400,826 people enrolled within the NHI in Taiwan
in 2002, information can be obtained on a random sample
of 200,000 or 1,000,000 individuals covering the period of
January 1, 1997, to December 31, 2004. We conducted retro-
spective observational studies based on the reimbursement
database of the National Health Insurance to explore the
relationship between the use of traditional Chinese formulae
that have been adulterated by aristolochic acid in terms of
nephrotoxic and carcinogenic risk.
3. Results
3.1. Prospective Observational Study 1. In all, 61 (91%) of
the initial 67 women aged between 45 and 55 years who
formed the intention to treat (ITT) group completed the
SZRT study without any major protocol violation. However,
the study raised notable safety issues. A total of 221 AEs were
detected/reported during the study period. Te most ofen
reported AEs were abdominal distention, diarrhea, cough,
headache, and dizziness with the incidence rates of 4.2, 3.6,
3.0, 1.8, and 1.8 per 1000 person-days, respectively, and 1.4,
1.2, 1.0, 0.6, and 0.6 per 1000 person-sachets, respectively
(Table 1). Five events were judged to be probably related to the
treatment. Tese consisted of three single events of dizziness/
headache/stomach ache and two events of diarrhea during 4-
week therapy period. Tree participants withdrew from the
study due to the AEs. Intolerable side efects resulting in
withdrawal included three events of stomach ache, diarrhea,
and dizziness. We further carried out periodic evaluation of
any unexpected symptoms during the treatment period and
found that gastrointestinal discomfort occurred two days
afer SZRT consumption. We were unable to rule out the
possibility that consuming SZRT might cause a deteriora-
tion in some preexisting gastrointestinal and/or neurological
symptoms during climacteric period.
3.2. Prospective Observational Study 2. Amongst the 71 par-
ticipants with OA knee, a total of 287 AEs were detected/
reported during the study period and were coded according
to the Coding Symbols for Tesaurus of Adverse Reaction
Terms. Te most ofen reported AEs were rashes, abdominal
fullness, coughs, somnolence, muscle cramps, and diarrhea
with the incidence rates of 14.5, 12.9, 12.4, 11.9, 10.3, and 10.3
per 1000 person-days, respectively, and 7.5, 6.9, 6.6, 6.3, 5.5,
and 5.5 per 1000 person-sachets, respectively (Table 1). Tese
AEs were tolerable and did not have any signifcant efects
on the subjects daily activities. None of the subjects showed
any abnormalities with respect to urinalysis, creatinine, blood
urea nitrogen, urinary N-acetyl-glucosaminidase or retinal
binding protein [37].
We also conducted an ITT analysis and found that, afer
four weeks of treatment, 44 of the 68 patients reported no
change in the symptom of faccidity, nine reported improve-
ments, whereas ffeen reported deterioration. Tirty-eight
patients reported no change in the symptom of aversion
to cold afer four weeks of treatment; fourteen reported
improvements, while 16 reported deterioration. Contrary to
the hypotheses proposed in an ancient text on traditional
Chinese medicine, Qianjin Yaofang, there seemed to be no
consistent improvements with regard to these two symptoms
afer 4 weeks of treatment.
3.3. Prospective Observational Study 3. When we carried
out another clinical observation to monitor a new invented
formula, TMN-1, it was found that fve out of 203 adverse
events were judged to be probably related to the treatment;
these included three single events of nausea, abdominal pain,
and abdominal fullness and two events of diarrhea over
the 12-week therapy period (Table 1) [18]. Further analyses
showed that TMN-1 treatment resulted in an inferior beneft
among postmenopausal women, compared to the beneft
obtained among women during the perimenopausal period
[23].
In addition, during the study period, there was an
isolated occurrence of SAE-acute thrombocytopenia [38]. A
52-year-old woman, who was aficted with perimenopausal
symptoms, including hot fushes, sweating and irregular
menstrual cycle, had been taking TMN-1 three times a day
without any complaints; however, on the 55th day she dis-
covered gingival bleeding. Te investigator at the study
site found that the woman had numerous petechiae and
ecchymoses over her bilateral shoulders, forearms, palms,
and thighs. Her complete blood count showed WBC
3,700/mm
3
, RBC 4,150,000/mm
3
, Hemoglobin 12.6 mg/dL,
and platelets 2,000/mm
3
. Her platelet count on the 28th
day afer medication had been 254,000/mm
3
, and there-
fore a diagnosis of acute thrombocytopenia was made. She
immediately received 12 units of platelet transfusion. Since
her platelet count had returned to 99,000/mm
3
by the follow-
ing day, she was subsequently discharged afer consultation
with a haematologist. As a result of a detailed enquiry into
the SAE patients history, we found that there had been con-
comitant uses of conventional medicines (acetaminophen,
mefenamic, and chlorpheniramine) and other folk herbs
(Moringa oleifera tea) before the occurrence of this SAE.
Following our emergency management, the patient is now
fully aware that she may be susceptible to acetaminophen,
mefenamic, or chlorpheniramine and must be careful to
avoid these medications for the rest of her life.
3.4. Retrospective Cohort Study. A retrospective follow-up
study was conducted using a systematic random sample
(200,000 people) from the National Health Insurance reim-
bursement database over the period 19972002. Te inci-
dence rates of chronic kidney disease (CKD) and end-stage
renal disease (ESRD) were calculated for the whole sample
and for those individuals who had ever used TCM product
suspected to contain AA. A total of 2,343 new CKD patients
and 25,843 newESRDpatients 199,843 persons were included
in the fnal analysis, with an average incidence rate of
1964/106 person-years for CKDand 279/106 person-years for
ESRD. Afer controlling for other risk factors, including age,
hypertension, and diabetes, consumption of 60 g of Mu Tong
or Fangchi as part of a herbal supplement was associated with
an increased risk of developing both chronic kidney disease
and kidney failure [39, 40].
Using the same database, we also conducteda population-
based case-control study in Taiwan to examine the asso-
ciation between prescribed Chinese herbal products that
contain aristolochic acid and urinary tract cancer. A total
of 4,594 patients newly diagnosed with urinary tract cancer
(from January 1, 2001, to December 31, 2002) and a random
sample of 174,701 control subjects from the entire insured
population (from January 1, 1997, to December 31, 2002)
were enrolled. Afer adjustment for age, sex, residence in a
township where black foot disease was endemic, and history
of chronic urinary tract infection, having been prescribed,
more than 60 g of Mu Tong and an estimated consumption
of more than 150 mg of aristolochic acid were independently
associated with an increased risk of urinary tract cancer in
the multivariable analyses (Mu Tong: at 61100 g, OR = 1.6,
95% CI = 1.3 to 2.1, and at >200 g, OR = 2.1, 95% CI = 1.3 to
3.4; aristolochic acid: at 151250 mg, OR = 1.4, 95% CI = 1.1
to 1.8, and at >500 mg, OR = 2.0, 95% CI = 1.4 to 2.9). A
statistically signifcant linear dose-response relationship was
observed between the prescribed dose of Mu Tong or the
estimated cumulative dose of aristolochic acid and the risk
of urinary tract cancer, as shown in Table 2 [41]. However,
we did not fnd the association between Xixin consumption
Table 1: Active surveillance system for herbs safety: top 20 adverse events detected by the panel of investigators and study nurses during the
study period.
Adverse events
Risk
Suan Zao Ren Tang Duhuo Jisheng Tang TMN-1

/10
3
person-days
a
/10
3
person-sachets
a
/10
3
person-days
b
/10
3
person-sachets
b
/10
3
person-days
c
/10
3
person-sachets
c
Body as a whole
Abdominal pain 1.8 0.6 8.3 4.4 1.8 0.6
Abdominal fullness 4.2 1.4 12.9 6.9 1.7 0.6
Dizziness 1.8 0.6
Chest pain 0.6 0.2 3.1 1.7 0.3 0.1
Fever 0.6 0.2 1.0 0.2 0.2 0.1
Somnolence 11.9 6.3
Musculoskeletal system
Muscle cramps 10.3 5.5 0.1 0.1
Headache 1.8 0.6 4.6 2.5
Respiratory system
Cough 3.0 1.0 12.4 6.6 2.6 0.9
Rhinitis 1.8 0.6 8.3 4.4 1.9 0.7
Pharyngitis 1.8 0.6 8.3 4.4 2.5 0.9
Digestive system
Diarrhea 3.6 1.2 10.3 5.5 1.6 0.6
Nausea 1.2 0.4 3.1 1.7 0.8 0.3
Vomit 0.6 0.2 0.5 0.3 0.3 0.1
Oral ulcer 1.5 0.8 0.2 0.1
Stomachache 0.6 0.2 0.5 0.2
Skin and appendages
Urticaria 0.5 0.3 0.1 0.0
Pruritus 0.6 0.2 1.6 0.6
Skin discolor 0.5 0.3 0.3 0.1
Rash 0.6 0.2 14.5 7.7 0.8 0.3
Number of cases was used as the numerator for calculation of risk.

a
Te denominators for the calculation of the risks were 1,691 person-days and 5,122 person-sachets.
b
Te denominators for the calculation of the risks were 1,936 person-days and 3,633 person-sachets.
c
Te denominators for calculation of risks were 10,133 person-days and 28,744 person-sachets.
and an increased risk of CKD, ESRD, and urinary tract cancer
[3941].
4. Discussion
Although randomized control trials (RCT) have been recom-
mended as the gold standard to prove the efcacy of TCM
therapies, the intensive requirements in terms of resources
and time preclude, this approach having widespread imple-
mentation [7, 42]. Te inclusion and exclusion criteria
imposed on subject recruitment during RCT further limit its
generalizability. While most ancient books of TCM stipulate
the combined use of many herbs under a specifc herbal
formulae, TCM doctors usually modify or add on pre-
scriptions for patients because of syndrome diferentiation
[1]; thus, it is extremely difcult, if not impossible, for the
prescribed mixtures of herbs to meet the strict requirements
of chemistry and quality control of products associated with
RCT. Issues may become even more complicated if, as the
results of a poorly designed RCT trial, the results contradict
the traditional wisdom of practitioners. Tus, there is a
tremendous opportunity for observational studies to provide
evidence in the area of TCM, beginning with safety profles
and the testing of old indications followed by the proposing of
new hypotheses that will allowthe initiation of efcacy trials.
Our establishment of an active safety system has shown
that the old indications in terms of traditional Chinese
formulas need to be regularly updated according to the data
generated from evidence-based studies. Although this type
of study is observational in nature and may not be suitable
for making strong inferences, it provides an opportunity to
corroborate previous claim of indications. In fact, the results
of the DJS study demonstrated that the previously proposed
indication of DJT with respect to faccidity of the lower back
Table 2: Number of new cases (), adjusted hazards ratios (HR), and 95% confdence intervals (CI) estimated from multivariate Cox
regression model for chronic kidney disease and adjusted odds ratios (ORs) with 95% confdence intervals (CIs) for new occurrence of
kidney failure and urinary tract cancer from multivariable logistic regression models on a random sample of the National Health Insurance
database followed from 19972002.
(a)
Cox regression model for chronic kidney disease
a
Study Chinese herb No. of cases HR 95% CI
Mu-Tong
0 g 1,979 1.0
130 g 248 1.0 0.81.1
3160 g 63 1.3 1.031.8
61100 g 27 1.4 0.962.1
101200 g 22 1.7 1.12.6
>200 g 4 0.7 0.31.9
Fangchi
0 g 1,875 1.0
130 g 389 1.0 0.91.2
3160 g 42 1.3 0.981.9
61100 g 18 1.8 1.12.8
101200 g 10 1.4 0.82.7
>200 g 9 2.2 1.14.2
(b)
Multiple logistic regression model for kidney failure
b
Study Chinese herb No. of cases/controls Adjusted OR 95% CI
Mu-Tong
0 g 22,188/157,939 1.0
130 g 2,542/20,122 1.12 0.861.47
3160 g 492/3,729 1.16 0.831.62
61100 g 226/1,569 1.47 1.012.14
101200 g 209/1,054 2.14 1.473.11
>200 g 186/438 5.82 3.898.71
Fangchi
0 g 21,985/157,543 1.0
130 g 3,145/24,868 0.68 0.580.78
3160 g 362/1,528 1.14 0.911.44
61100 g 169/492 1.60 1.202.14
101200 g 116/295 1.62 1.172.23
>200 g 66/125 1.94 1.292.92
(c)
Multiple logistic regression model for urinary tract cancer
c
Study Chinese herb
No. of
cases/controls
Adjusted
OR
95% CI
Mu-Tong
0 g 3,987/149,464 1.0
160 g 489/22,354 1.0 0.91.2
61100 g 50/1,485 1.6 1.32.1
101200 g 46/1003 2.0 1.42.7
>200 g 22/395 2.1 1.33.4
Fangchi
0 g 3,927/150,456 1.0
160 g 623/23,456 0.9 0.81.0
(c) Continued.
Multiple logistic regression model for urinary tract cancer
c
Study Chinese herb
No. of
cases/controls
Adjusted
OR
95% CI
61100 g 15/427 0.7 0.41.2
>100 g 29/362 1.3 0.92.0
Estimated cumulative dose of aristolochic acid, in mg
0 3,274/121,820 1.0
1150 1151/48,869 1.0 0.961.1
151250 69/2,032 1.4 1.11.8
251500 64/1,403 1.6 1.22.1
>500 36/577 2.0 1.42.9
a
Hazards ratios (HR) and 95%confdence intervals (CI) obtained fromCox proportional hazards regressionmodels withall variables (sex, age, hypertension,
diabetes, cumulative dosage of nonsteroidal antiinfammatory drugs, Mu Xiang, Mu-Tong, and Fangchi) ftted simultaneously.
b
Multivariable odds ratios were adjusted for potential confounders (sex, age, hypertension, diabetes, chronic hepatitis, chronic urinary tract infection,
chronic neuralgia, musculoskeletal disease, cumulative dosage of nonsteroidal antiinfammatory drugs, Mu Xiang, Mu-Tong, and Fangchi) ftted simulta-
neously.
c
Multivariable odds ratios were adjusted for potential confounders (sex, age, hypertension, diabetes, residence in township where black foot disease was
endemic, chronic urinary tract infection, Mu Xiang, Mu-Tong, and Fangchi) ftted simultaneously.
and knee and aversion to cold, as written on Qianjin Yaofang
(Invaluable Prescriptions for Ready Reference), seemed to be
wrong. Although the probable ADRs are tolerable and did not
have any signifcant efects on the subjects daily activities, we
recommend that regulatory agencies need to require pharma-
ceutical companies to informtheir consumers and healthcare
professionals of any of this type of fndings, which are based
on these scientifc evidence, before their DJT products enter
the market. Te above empirical experiences have shown that
active safety surveillance systemis feasible for the detectionof
adverse efects andthe validationof oldindications of TCMas
long as close cooperation among investigators, study nurses,
and patients is able to be established. Similarly, we believe that
TCMdoctors nowwill be alert to the possibility that prescrib-
ing SZRT may cause severe gastrointestinal adverse efects or
dizziness based on our observational results. As safety and/or
adverse event evidence accumulates, these should infuence
the prescription behavior of TCM doctors. Traditional use,
which refers to documentary evidence that related to the
recorder use of a traditional Chinese herbs/formula has been
used over three or more generations of recorded use for
a specifc health related or medicinal purpose, is one type
of information which that can be used to support claims
on TCM. Te present fndings are another type of evidence
indicating the urgent need for changes of the labels of popular
SZRT products in order to refect these new concerns. We
therefore believe that there should be updating of the old
contraindication of SZRT based on the relative strengths of
the evidence-based claims and their likely impact on con-
sumers. Furthermore, this type of active safety systemenables
us to exclude without doubt the possibility that TMN-1 alone
was the cause of a serious case of thrombocytopenia. Hence,
the aforementioned observational studies are able to provide
more and better evidence for rational TCM use and in the
process improve patient care regardless of whether they have
a positive or a negative result.
Of all analytical studies, prospective cohort studies best
meet the criteria to clearly demonstrate an appropriate tem-
poral sequence between treatment and outcome. No acute
renal tubular damage was observed afer receiving four weeks
of DJS (possibly a total of 1.342.01 g of AAI) during the
study period [43]. Possibly, as compared to the reported
case of the AA-related nephropathy that involved a patient
ingesting 400 g of DJT for over four months, the ingestion
levels of AA in our observational study were very low.
However, it is worth noting in this context that this type of
study design may not always be feasible due to its resources
requirements in terms of a large sample size that will allow
the detection of a low incidence as well as the need for a long
periodic followup in order to assess correctly aristolochic
acid exposure. Terefore, we must learn from the case of AA
nephropathy that was frst detected by a Belgian physician
[17] rather than from countries with a long history of taking
Chinese herbs, such as China, Japan, Korea, or Taiwan.
Although traditional Chinese therapies have been used for
many thousands of years, the data generated from our obser-
vational studies show that they cannot be guaranteed to
be totally safe [3941, 44, 45]. Furthermore, the herbology
theory of traditional Chinese medicine, in which diferent
herbs are mixed together to counteract their toxicity, may not
always be correct. Tus, a retrospective observational study
should be useful when exploring serious adverse efects, such
as chronic renal failure and/or urothelial cancer associated
with exposure to aristolochic acid containing Chinese for-
mulae. As the sensitivity of such studies might be reduced
by limited sample sizes and misclassifcation errors resulting
from consumption of herbs outside of prescriptions, future
long-term active surveillance of Xixin (potential containing
aristolochic acid) consumers or users is still needed so that
a careful watch on the safety of current regulatory policy is
maintained.
In conclusion, prospective or retrospective observational
studies have specifc advantages when investigating safety
of and adverse efects of TCM therapies and possibly
other alternative/complementary therapies. Te usefulness
of such studies to prove efcacy is limited because bias in
patient selection may occur. Te evidence we obtain from
such studies, however, can provide highly useful data on the
safety profle of daily TCM practice, which will also serve as
prior information when designing randomized control trials.
In general, it is ethically and fnancially more feasible to carry
out observational studies to generate data and/or information
on the adverse efects of existing TCM therapies that to
tackle a risk/beneft evaluation [4648]. Moreover, the more
information we obtain about the safety and adverse events
of TCMtherapies and alternative/complementary medicines,
the greater is the likelihood that physicians trained in con-
ventional medicine will be encouraged to use such medicines
rationally. Tis will possibly help to bridge the gaps between
these two felds [49].
Acknowledgments
Tis research was conducted at the Institute of Traditional
Medicine at the School of Medicine, National Yang-Ming
University, Taipei. Te authors would like to express sin-
cere gratitude for the partial support provided for this
project in the form of grants from the Committee on Chi-
nese Medicine and Pharmacy (CCMP100-RD-033) and the
National Research Institutes of Chinese Medicine (NRICM-
9903).
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MEDIATORS
INFLAMMATION
of
http://dx.doi.org/10.1155/2013/739716
Review Article
Chinese Herbal Medicine and Depression:
The Research Evidence
Lee Butler
1
and Karen Pilkington
2
1
Department of Complementary Medicine, School of Life Sciences, University of Westminster, London W1W 6UW, UK
2
Department of Human and Health Sciences, School of Life Sciences, University of Westminster, 115 New Cavendish Street,
London W1W 6UW, UK
Correspondence should be addressed to Karen Pilkington; k.pilkington@westminster.ac.uk
Received 14 August 2012; Revised 5 November 2012; Accepted 11 December 2012
Academic Editor: Cynthia R. Long
Copyright 2013 L. Butler and K. Pilkington. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Background. Alternative approaches for managing depression are oen sought and herbal mixtures are widely used in China. e
aimof this paper was to provide an overall picture of the current evidence by analysing published systematic reviews and presenting
a supplementary systematic review of trials in Western databases. Methods. Searches were conducted using AMED, Cochrane
Library, EMBASE, MEDLINE/PubMed, PsycINFO, and trial registers. Results were screened and selected trials were evaluated by
two reviewers working independently. Systematic reviews were identied and assessed using key criteria. Results. Five systematic
reviews were located addressing the Chinese literature, adjunctive use of Chinese herbs, and the formulae Chaihu-Shugan-San,
Xiao Yao San, and Free and Easy Wanderer Plus. e supplementary review located 8 trials, 3 of which were not included in
previous reviews. Positive results were reported: no signicant dierences from medication, greater eect than medication or
placebo, reduced adverse event rates when combined or compared with antidepressants. However, limitations in methodology and
reporting were revealed. Conclusions. Despite promising results, particularly for Xiao Yao Sanandits modications, the eectiveness
of Chinese herbal medicine in depression could not be fully substantiated based on current evidence. Further well-designed, well-
reported trials that reect practice may be worth pursuing.
1. Introduction
Knowledge about the use of complementary therapies by
people with mental illness is increasing. Previous studies
have demonstrated that psychiatric disorders are common
among those seeking to use complementary and alternative
medicine (CAM) and that anxiety and depression are among
the most common reasons for people to seek care from com-
plementary practitioners [14]. Over 50% of respondents
self-diagnosed with anxiety attacks or severe depression
reported using complementary and alternative therapies to
treat their condition [2]. A recent survey in England also
found that presence of anxiety or depression was an indepen-
dent predictor of complementary therapy use [5]. People with
mental illness appear to derive a broad range of benets from
complementary practices and these include enhancement
of social, spiritual, general, and self-functioning [6]. One
survey of women with depression revealed active seeking
for treatments based on natural approaches and for those
that match the individuals own values and beliefs [7]. People
with depression may also be seeking to avoid adverse eects
associated with conventional medication. However, CAM
use has been found to be common even in those taking
prescription drugs [5].
Kessler et al. [2] found that use of herbs or diet supple-
ments was particularly prevalent in those with psychiatric
problems. Use of CAM in the USA increased by 2% between
2002 and 2007 [8] with natural products reported to be the
most frequently used therapies. Similar increases have been
observed in Australia [9] while use in the UK continues to
be substantial [5]. Users were found to be more likely to have
received mental health and primary care treatment and to be
dissatised with their overall healthcare than nonusers [10].
Chinese herbal medicines were not specically mentioned,
but several of the herbs listed are included in Chinese herbal
formulae. e UK survey also revealed frequent use of herbs
but limited reported use specically of Chinese herbs [5]. In
contrast, 7% of participants in a national survey in Australia
reported using Chinese herbal medicine, of whom32.9%had
visited a Chinese herbal medicine practitioner [11].
It is dicult to fully assess the extent of use of Chinese
herbs in depression outside China. Chinese herbs may be
less likely to be reported in surveys as participants may be
unable to name the specic herbs that they have received.
Additionally, in contrast to Western herbs, access to Chi-
nese herbs is generally via consultation with a practitioner
rather than purchase over the counter which limits their
use somewhat. Patterns of use may also vary according to
ethnic group and be higher in those who consider Chinese
medicine to be their primary medical system. For example,
use of herbs was shown to be greater in Chinese and Japanese
women in midlife than in Whites and African Americans
[12]. Considerable promotion of the benets of Chinese
herbs is also found on the internet suggesting that use may
be more widespread than is apparent from surveys.
Numerous systematic reviews and research papers have
been published on the eects of Western herbs such as
St Johns wort (Hypericum perforatum) in the treatment of
depression (e.g., [1315]). Chinese herbs appear to have
received less attention although there has been research
interest in one specic formula, iao ao San. Systematic
reviews of the evidence on Chinese herbs in depression have
been published previously but none has addressed all RCTs
of Chinese herbs in the Western literature.
2. Aim
e aim of this paper was to assess the evidence on Chinese
herbal medicine treatments for depression based on previous
systematic reviews plus a supplementary systematic review
of trials in the Western literature. A secondary aim was to
explore the proportion of studies on this topic that can be
found in Western databases.
3.1. Searches. Randomised controlled trials (RCTs) of the
treatment of depression with Chinese herbal medicine
were located by searching AMED, EMBASE, MEDLINE,
PsycINFO, PubMed, Cochrane Field for Complementary
Medicine website, and the Clinical Trials Register of the
Cochrane Collaboration Depression, Anxiety & Neurosis
Group (CCDANTR). Only trials published in English were
included at this stage. In the second stage, more compre-
hensive searches were conducted and trials in all languages
were included. e above databases plus British Nursing
Index (BNI) were searched from inception to July 2010 using
the Ovid interface. e search strategy was as follows: exp
depression/ OR exp depressive disorder/ OR exp depressive
disorders/ OR exp major depression/ OR depressed.ti,ab. OR
depression.ti,ab. OR depressive.ti,ab. OR dysthymi
.ti,ab. AND
exp drugs, chinese herbal/ OR exp Medicine, Chinese Tradi-
tional/ OR exp Medicine, Oriental Traditional/ OR exp chinese
drug/ OR exp chinese medicine/ OR exp chinese herb/ OR (exp
medicinal herbs/ AND chinese.mp.) OR (chinese adj3 herb
)
OR (chinese.ti,ab. AND herb
.ti,ab.). Cochrane CENTRAL

was searched and a search was conducted of the Cur-
rent Controlled Trials metaregister (http://www.controlled-
trials.com/mrct/) to identify possible unpublished trials. e
most recent search for trials was carried out in July 2011.
Systematic reviews were identied from the above
searches plus searches of the Cochrane Register of Systematic
Reviews and the Database of Abstracts of Reviews of Eects
(DARE). Searches for systematic reviews continued up to July
2012.
Search results were screened for trials by two reviewers
working independently using the following inclusion criteria.
(i) Participants: diagnosed with clinical depression using
conventional or traditional Chinese criteria. Trials
in which participants with dierent diagnoses were
treated (i.e., depression or bipolar disorder) were
included if outcomes were reported separately for
patients with depression.
(ii) Interventions: any intervention using Chinese herbal
formulae (combinations of herbs) or single Chinese
herbs.
(iii) Comparison interventions: included placebo, no
treatment, or any active treatment.
(iv) Outcomes: improvement in depression (partial or
complete alleviation of symptoms), as measured by
validated outcome measure, clinical assessment of
improvement, and participants subjective experi-
ences where reported.
Trials in which Chinese herbs were combined with
other complementary therapies (i.e., Chinese herbs and
acupuncture) were excluded. Studies on participants with
depression associated with other conditions, for example, a
medical/physical condition, menopause, premenstrual syn-
drome, or bipolar disorder were identied initially. owever,
as the herbal mixtures used may have had eects other than
on depression, the main data analysis focused on those in
depression not associated with another condition. Selections
were compared and dierences resolved by discussion.
Systematic reviews were identied by one researcher (KP)
using the following inclusion criteria: reviews of Chinese
herbal medicine in depression reported to be systematic and
including methods for searching and selection of studies.
3.2. Data Extraction and Assessment of Methodological Qual-
ity. Key criteria were used for systematic reviews. ese
included focus, date of searches, sources, inclusion criteria,
extraction and appraisal methods, number of trials included,
method of synthesis of results, and conclusions. A data
extraction sheet was designed to capture key data from trials
and allow an accurate comparison of the studies selected.
Translations were obtained for studies published in Chinese.
Data collected included details of study design, participants,
diagnostic criteria, interventions, outcomes measures, and
results for the primary outcome measure.
RCTs were evaluated using two rating scales: the Jadad
criteria, use of which results in a score of between 0 and 5
based on 3 aspects of the methods: randomisation, blinding,
and extent of followup [16]. is scale places great emphasis
on blinding, which may not be feasible in trials involving
mixtures of Chinese herbs. erefore, a second more com-
prehensive scale was deemed necessary. e checklist pro-
posed by Downs and Black [17] was considered appropriate.
Previous reviews had found that there was good correlation
with Jadad [16] andhadindependently validatedits accuracy
[18, 19]. Downs and Black [17] use a checklist of 27 questions
which are intended to give broader overview of the methods
used in RCTs and non-RCTs. e nal question relating to
power was adapted so that trial either scored 0 if power was
not discussed or 1 if a power calculation was conducted and
sucient numbers recruited. e total possible score was,
therefore, 28. Trials scoring 3 or more on the Jadad scale were
considered to be at low risk of bias [16]. e approach taken
by Malcomson et al. [20] was to consider trials scoring 50%
or more on the Downs and Black scale as of good quality.
In this paper, total scores for both scales and key risk of bias
measures are reported.
4. Results
4.1. Previously Published Systematic Reviews. Six reports of
5 systematic reviews were identied [2126]. Each of these
focused on a specic aspect: trials in the Chinese literature
[21], trials of the specic herbal formulae Xiao Yao San
[22, 23], Free and Easy Wanderer Plus (a modied version of
Xiao Yao San) [24], and Chaihu-Shugan-San [25], and trials
of Chinese herbs as adjunctive treatment [26].
e rst of these systematic reviews included 18 tri-
als found by searching Chinese databases [21]. e trials
involved a total of 1,260 patients and included trials in
depression associated with various conditions such as cancer,
stroke, and the menopause. e authors concluded that there
was no evidence to support a benecial eect in depression.
e ndings are dicult to interpret and the validity of
the conclusions is unclear. Trials of combined treatment
(acupuncture plus herbs) were included as were those in
which treatment appeared to be aimed at the medical condi-
tion rather than depression. Assessment of quality involved
assigning Jadad scores but these were not reported. A meta-
analysis was conducted but the results of trials comparing
Chinese herbs against placebo and those comparing Chinese
herbs against active treatment appear to have been combined
rather than reported separately.
A second systematic review focused specically on Xiao
Yao San (also known as Free and Easy Wanderer Powder
or Rambling Powder) [22, 23]. is formula together with
a modied version, Free and Easy Wanderer Plus (Jia wei
Xiao Yao San or augmented Xiao Yao San) are two of
the more frequently used formulae in Chinese medicine
[27]. Two similar reviews have been published: the rst,
published in Chinese, included 32 trials (2,253 patients)
and the second, published in English including 26 trials
(1,837 patients) [22, 23]. e following comments relate to
the second publication. e authors concluded that Xiao
Yao San combined with antidepressants was more eective
than antidepressants alone. Adverse eects were reported
for antidepressants but not in relation to Xiao Yao San. e
majority of trials were found to be of poor methodological
quality. e conclusions also need to be interpreted in the
context of potential publication bias as revealed by a funnel
plot which demonstrated signicant asymmetry. ere was
also considerable variation between the formulae used in the
trials: only 8 trials used the traditional formula for Xiao Yao
San, the remaining trials using a modication of the original
formula. e total number of herbs used in each trial varied
between 7 and 17.
e third review which was also published in 2011
addressed trials of Free and Easy Wanderer Plus [24].
Searches of Chinese and Western databases were carried out
and 14 trials selected, all conducted in China and 9 of which
included participants with major depression. Only trials
scoring at least 3 on the Jadad scale were included, which
had resulted in a further 50 trials being excluded from the
meta-analysis. e authors concluded that the herbal formula
may be eective in depression, may enhance conventional
antidepressants, and may have a better safety prole than
standard antidepressants.
In the rst of the reviews published in 2012, English and
Chinese databases were searched for RCTs in which Chi-
nese herbs were used in combination with antidepressants
[26]. Methods appeared rigorous and results presented as
weighted mean dierence (WMD) based on Hamilton rating
scale for depression (HAM-D) scores. Seven RCTs involving
576 participants were identied. Meta-analysis indicated
that integrated traditional and Western medicine based on
syndrome dierentiation produced a greater reduction in
mean HAM-D scores than Western medicine alone (WMD
2.39 95% CI 2.96, 1.83). No serious adverse eects were
reported for combined treatment, but all trials were evaluated
as being at risk of bias or the risk of bias was unclear.
e h systematic review was also published in 2012
[25]. A range of Western and Chinese databases were
searched for trials of the formula, Chaihu-Shugan-San in
depression, and papers were selected by two researchers.
Ten RCTs involving 835 subjects were included. Meta-
analyses based on 6 RCTs indicated that Chaihu-Shugan-
San in combination with various selective serotonin reuptake
inhibitors (SSRIs) was more eective that antidepressant
drugs alone based on HAM-D scores (WMD = 3.56; 95%
CI 5.09 to 2.03). Based on two trials, Chaihu-Shugan-San
as monotherapy was also more eective than antidepressants
in improving depressive symptoms (WMD = 3.09; 95% CI
5.13 to 1.06). No serious adverse events were reported. As
with previous reviews, all studies were judged to be of poor
methodological quality and at risk of bias.
No systematic reviews were found in which Western
databases were searched for trials of all Chinese herbs or
herbal formulae in depression. In the systematic reviews
described above, either only Chinese databases were searched
or the review only included trials of one specic formula. A
systematic review was conducted to address this gap (sup-
plementary systematic review) and the results are described
below.
4.2. Supplementary Systematic Review. A total of 1600 cita-
tions were retrieved (151 from the initial search; 1676 from
the comprehensive searches of which 1449 remained aer
removing duplicates). No unpublished trials were identied
from the trials register. Forty-ve potentially relevant cita-
tions were selected from these. Trials were identied of Chi-
nese herbs in the treatment of depression, in perimenopausal
depression, poststroke depression and depression associated
with other medical and psychiatric conditions, and in the
prevention of postnatal depression. For depression as the
primary diagnosis, no nonrandomised controlled trials were
located but two uncontrolled studies were located: one in
which 40 cases of melancholia were treated with a formula
called modied Wen Dan Tang [28] and the other in which
20 patients with prolonged partial remitted major depressive
disorder associated with fatigue or loss of energy were treated
with Japanese formulae comparable to the two Chinese
formulae: Liu Wei Di Huang Wan and Bai Wei Di Huang Wan
[29]. e RCTs [3038] are discussed inthe following section,
and Figure 1 shows a summary of the selection process and
the excluded studies [28, 29, 3972].
4.2.1. Summary of Trials Located. A total of nine reports
of controlled trials of Chinese herbs in depression were
identied [3038] (see Table 1 for a summary of the trials).
Two reports appeared to be of dierent outcomes from
one trial [31, 32]. In all cases, the trials were described as
RCTs. Participants had been diagnosed with depression by
various means: using the Diagnostic and Statistical Manual
of Mental Disorders (DSM-IV) criteria, the Chinese Clas-
sication of Mental Disorders (CCMD-3) criteria, CCMD-
3 plus the International Classication of Diseases (ICD-10),
and Chinese medicine diagnostic methods. In 7 trials, the
inclusion criteria included a minimum score on the HAM-D
scale. e minimum scores required were generally between
17 and 20 reecting mild-to-moderate depression. However,
the mean scores were higher than this in some trials (and
over 30 in two trials), suggesting that more severe cases of
depression were included. Two trials were placebo-controlled
[34, 38] and one was a double-placebo-controlled trial [31,
32]. Two trials compared a Chinese herbal formula with
the antidepressants: uoxetine and maprotiline, respectively
[30, 33]. In two trials, Chinese herbs combined with an
antidepressant were compared against antidepressant alone
[35, 36]. In both cases, the Chinese herbs were combined
with a tricyclic antidepressant but in Yang et al.s study [35],
the comparison was against an SSRI. e nal trial was a 3-
arm trial comparing two dierent Chinese herbal formulae
combined with uoxetine against uoxetine alone [37].
4.2.2. Setting, Size, and Duration. All trials had been con-
ducted in China and patients appeared to have been recruited
from a variety of sources. e authors were based at hospitals
or medical colleges and one could assume that this is where
the trials were conducted but this was not explicitly stated in
all cases. e number of subjects recruited ranged from 60 to
164 (total 756) and the treatment duration ranged between
30 days and 12 weeks. None of the trials conducted a later
followup to ascertain whether changes were maintained or
whether remission was only temporary.
4.2.3. Chinese Herbs Investigated. All trials involvedthe use of
Chinese herbal formulae rather than a single herb. A variety
of dierent Chinese herbal formulae were investigated. Of
the herbs included in the formulae, only one, Hypericum
(St Johns wort) has documented anti-depressive activity
[73]. is herb was included in the formula used in one
trial. According to the same reference source, four of the
herbs used have potential sedative or anxiolytic properties.
Preliminary evidence has also been reported in studies from
China on relevant eects of several herbs included Xiao Yao
San [24].
4.2.4. Outcome Measures. e eects of the herbal formulae
were assessed in 7 of the trials by comparing HAM-D
scores before and aer treatment. Rating scales used in
addition to HAM-D included the Self-Rating Anxiety Scale
(SAS), Self-Rating Depression Scale (SDS), Montgomery-
Asberg Depression Rating Scale (MADRS), Clinical Global
Impression (CGI), TCM syndrome and symptom dierenti-
ation (TCM-SSD), and Treatment Emergent Symptom Scale
(TESS). In one trial, response was only measured by clinical
assessment of symptom improvement or resolution [30].
4.2.5. Reporting of Adverse Events. All but two trials reported
types of adverse events by intervention. Zhang et al. [38]
and Sun et al. [34] found there to be no statistical dierence
between incidence of adverse events in the Chinese herb
group and the placebo group. A signicant dierence was
reported between treatment with a Chinese herbal mixture
with or without an antidepressant compared with the antide-
pressant alone, with the herbal mixture apparently causing
reduced adverse event rates [3133, 36, 37]. In the other
studies, a formal statistical comparison was not carried out
and adverse events simply reported by group.
4.2.6. Overall Quality of Methods and Reporting. All studies
were described as randomised but several did not describe
how randomisation had been carried out. e Jadad assess-
ment also revealed that an eective process for blinding (or
masking) treatment was only described in three trials. From
the assessment based on the Downs and Black checklist,
the following items were found to be reported in few, if
any, studies: how recruitment to the study was carried out,
whether intervention and control groups were wellmatched
at baseline, which statistical tests were used, whether compli-
ance was reliable, and whether the study had sucient power.
e mean Jadad score was 2.4 (out of 5) and 3 trials scored
more than 3. e mean adapted Downs and Blacks score was
17 (out of 28). Only two trials scored more than 20 (both of
which also scored 4 on the Jadad scale). e scores on each
of the aspects addressed by the Downs and Blacks checklist
varied from 5 to 11 (out of 11) for reporting, 0 to 1 (out of
3) for external validity, 1 to 6 (out of 7) for internal validity
(bias), and3 to 6 (out of 6) for internal validity (confounding).
No trial discussed power or provided a rationale for the
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Records identied through from
initial database searches
Records identied through
comprehensive searches
Records screened
Records excluded
Articles assessed for
eligibility
Studies included in
qualitative synthesis
9 reports
Based on title/abstract:
2 bipolar disorder [39, 40]
1 dysthymia [41]
6 menopausal depression [4348]
1 postoperative depression [42]
8 poststroke depression [4956]
2 premenstrual syndrome [57, 58]
1 prevention of postnatal depression [59]
4 combination treatment [6063]
1 efect of educational intervention [64]
2 no control group [37, 38]
1 animal study [65]
Based on full text:
1 bipolar disorder [66]
2 combination treatment [67, 68]
4 not a clinical trial [6972]
( = 151)
( = 1676)
( = 1600)
( = 1600)
( = 1555)
( = 45)
( = 8)
Records afer duplicates removed
[2836]
( = 36) Articles excluded
F 1: Flowchart showing selection process.
number of participants recruited. Based on the risk of bias
assessment, trials were either at risk of bias or the overall risk
of bias was unclear.
4.2.7. Pooling of Results. e herbal formulae and control
treatments used, trial duration, and trial design all diered to
such an extent that it was not possible to pool data to present
any meaningful statistics. Where changes in depression
scores were measured, clinically signicant reductions were
reported within groups with active treatment, either Chinese
herbs or antidepressants. However, between-group compar-
isons suggested that Chinese herbs were more eective than
antidepressants [30], were comparable to antidepressants
[3133], did not increase eectiveness but reduced adverse
eects or relapse rates when used as additive therapy with
antidepressants [3537], or were more eective than placebo
[34, 38].
4.3. Comparison with the Other Systematic Reviews. Only
three trials were locatedthat hadnot beenincludedinthe pre-
vious reviews [30, 34, 36]. Two of these assessed the formula,
Shugan Jieyu and the third, modied Xiao Yao San. Two trials
were included in the previous reviewfocusing on the Chinese
literature [33, 37], three were included in the systematic
review of Xiao Yao San formula [31, 32, 35, 37] and two were
in the Free and Easy Wanderer Plus review [31, 38]. None of
the trials were included in the reviews of Chaihu-Shugan-San
or Chinese herb/antidepressant combination treatment. A
comparison of the systematic reviews is presented in Table 2.
In terms of methodology and reporting, the current review
found similar issues to the previous systematic reviews.
ese included lack of reporting of whether groups were
matched on baseline characteristics, allocation concealment
or blinding of assessors. Little or no detail was provided
on dropout and intention-to-treat analysis. ese address
issues related to internal validity. As described above, the
current review also revealed issues aecting external validity
in that the process of recruitment of participants and the
specic location of the trial were not reported in most
trials.
An evaluation of the results of the meta-analyses and
the strength and quality of the supporting evidence was
conducted. e results are presented in Table 3. is demon-
strates that the overall evidence was generally of low quality
and even moderate evidence was compromised by aspects
such as the heterogeneity of the interventions and diagnoses.
5. Discussion
is paper provides an overall picture of the current evi-
dence base for Chinese herbal medicine in the treatment of
depression. Five published systematic reviews were located.
A supplementary systematic review to address a gap in the
coverage located eight trials, of which three had not been
included in previous systematic reviews.
Positive results were reported almost universally. ese
included one or more of the following: greater anti-depressive
eects than placebo, equivalent eects to antidepressants,
less problems with adverse eects than antidepressants, or
reduction of the adverse eects caused by antidepressants
whenusedincombination. Intrials comparing Chinese herbs
against antidepressants, lack of power calculations means
that it is unclear whether a lack of dierence between the
Chinese herbal formula and the antidepressant is due to a
true dierence or simply a trial that was underpowered to
detect a dierence. f the CTs found, ve scored less than
3 on the Jadad scale which suggests bias may have been
introduced. In several cases, the lowscore was due to blinding
being impossible but lack of information on withdrawals and
dropouts was also a problem. is nding correlates with
previous reviews, which also reported low scores on Jadad or
unclear or high risk of bias for many of the included trials. In
fact, based on risk of bias assessments, all trials were either at
risk of bias or the extent of possible bias was unclear.
All 8 trials were conducted in China and the methods
used in most of the trials were similar. Diagnosis was
using conventional diagnostic frameworks and the response
measured using the HAM-D instrument. Patients appear to
have been recruited via hospitals in most cases but the exact
process of recruiting patients was not reported in any trial.
us, it is dicult to assess whether the patients selected
were representative of the population from which they were
selected. Similarly, trials were reported to be randomised
but it is dicult to judge whether allocation was eectively
concealed. Infact, the limited reporting precludes anaccurate
assessment of the methods and, therefore, the reliability of the
results. Again, this is a similar nding to those of the other
systematic reviews in this area.
Herbs were used in combination and at least 6 dierent
herbs appear to have been included in many of the formulae.
Several of the herbs have sedative or anxiolytic potential
activity which may be benecial in depressed patients [39].
e remaining herbs have a range of uses and actions and
it becomes more apparent why a formula such as Xiao Yao
San with or without modications might be used widely for a
range of conditions. It is also possible that using these herbs in
combination may produce eects that would not be achieved
with each herb alone.
Chinese herbal mixtures are supplied as the dried plant
parts, pills or capsules, or inpowder form[27]. Dierent parts
of the plant are used and preparation of the dose may entail
processes such as decoction which involves boiling. e eect
of these processes on the activities of the component herbs
is dicult to predict. In terms of trial design, blinding or
masking of the patient is, in many cases, impossible. Even if
pills or capsules are prepared, unblinding may take place due
to the smell of the herbs. is obviously may introduce some
bias on the part of the patient which is particularly relevant
in depression where the main outcome measure is based on
self-report. Blinding of assessors may limit the extent of bias
somewhat but was not reported in the trials included in this
paper, nor have other reviewers found consistent reporting of
blinding.
Two systematic reviews have focused on the formula Xiao
Yao San or a modication of this [23, 24]. In practice, this
formula forms the basis for an array of modied formulae.
From the Chinese medicine perspective, these are all based
on the core formula, with additional herbs added to address
specic problems. ey are referred to as modied Xiao
Yao San or Jia wei Xiao Yao San (san means powder while
wan means pill). us, two preparations may have a similar
name but contain dierent herbs [27]. is causes potential
problems in interpreting the results of trials using these
formulae. It also causes problems in practice in the reporting
of adverse events and checking for interactions. e formulae
used are considered safe by TCM practitioners based upon
experiential evidence but there is obviously a potential for
interactions and adverse eects. Some insight into the more
frequent adverse eects is revealed by the results of these
trials but the small size of the trials means that less common
adverse eects may not have been encountered.
e issue of diagnostic frameworks is also worth consid-
ering when assessing the relevance of these trials to practice.
Chinese medicine recognises patterns of signs and symptoms
and diagnoses that do not t with a Western framework [27].
People who might be diagnosed in Western medicine with
depression may receive dierent Chinese medical diagnoses
T
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T 3: Summary of results and supporting evidence (based on meta-analyses).
Outcome Intervention Control Result (95% CI) Evidence (participants) Quality
(comments)
HAM-D
score
Chinese herbs + ADs ADs alone WMD 2.39 [2.96, 1.83] 7 RCTs (576)
Low (trials unclear/high risk of
bias, varied herbs)
WMD 3.56 [5.09, 2.03] 6 RCTs (506)
Low (trials low quality,
heterogeneity)
WMD 0.51 [0.71, 0.31] 14 RCTs (921)
bias, heterogeneity)
OR
1.75 [1.26, 2.44] 8 RCTs (648)

Low/moderate (varied
diagnoses)
Chinese herbs ADs WMD 3.09 [5.13, 1.06] 2 RCTs (164)
Low (trials high risk of bias,
heterogeneity)
WMD 0.43 [2.14, 2.99] 3 RCTs (NR)
bias, publication bias)
OR
1.09 [0.60, 1.98] 4 RCTs (250)

Low/moderate (varied
diagnoses)
TESS score
Chinese herbs Placebo OR
9.40 [5.57, 15.89] 3 RCTs (321) Low (heterogeneity)

Chinese herbs/ADs ADs alone WMD 2.51 [3.18, 1.84] 4 RCTs (263) Low (heterogeneity)
Chinese herbs ADs WMD 1.86 [2.57, 1.15] 1 RCT (60)
Very low (single trial, high risk of
bias)
TESS: Treatment emergent symptoms and side eects; NR: not reported;

Overall quality of the evidence was assessed based on reported quality/potential bias
in RCTs, heterogeneity, publication bias, consistency of interventions and diagnoses.

Odds ratios were based on a decrease of at least 50%in HAM-D scores.
depending on the overall pattern. Virtually all the trials have
used standardised Western-based diagnostic frameworks.
e advantage is more ready interpretation in a Western
context but the primary problem is whether the trials reect
usual Chinese medicine practice. Related to this is the fact
that virtually all the research has to date been conducted in
China so it is dicult to translate the results into a Western
healthcare context.
None of the trials reviewed here conducted a followup
at a later date to ascertain duration of antidepressant eects
aer the herbal treatment. e longest duration was 12 weeks.
Without this information it is impossible to establish whether
the herbal medicine had a temporary eect or whether its
eects were long lasting or to be able to calculate relapse
rates. National guidelines on the management of depression
[74] state that drugs administered for depression should be
continued for 6 months aer the last episode to avoid relapse.
If the same is true of herbal formulas, then a long-term
trial would be necessary to test long-term benets of herbal
medication.
Analysis of the overall strength and quality of the
evidence from the various systematic reviews revealed a
number of aspects which compromise rm conclusions on
the eectiveness of Chinese herbal formulae in the treatment
of depression.
5.1. Limitations of is Review. Only systematic reviews
indexed in Western databases were included. However, the
Chinese literature was addressed to some extent in that each
of these systematic reviews involved searches of Chinese
databases. In the supplementary review, all except one of
the trials included were originally published in Chinese.
erefore, the data presented and quality assessments were
based on translations rather than the original reports.
6. Conclusions
Overall, this paper has provided an insight into the research
that has been conducted on Chinese herbs in depression.
e intention of this paper was to assess the eectiveness of
Chinese herbal medicine treatments for depression. Positive
results were reported in all the trials identied and in all
but one of the systematic reviews. ese results included
no signicant dierences when compared with medication,
greater eect than medication or placebo, and reduction in
adverse event rates when used as additive therapy. However,
because of limitations in the strength and quality of the
evidence, it has not proved possible to either fully substantiate
or disprove claims of eectiveness of Chinese herbal medicine
in the management of depression. Limitations in reporting
and in methodology, dierent control interventions, and use
of varied formulae precluded any reliable conclusions. In
addition, virtually all trials located were for 12 weeks or less
so that it is unclear whether reported eects were sustained
in the longer term. Adverse eects were reported but trials
were generally small and the preparations used varied so
that further evidence on safety would be required for rm
conclusions.
It is clear that a large number of trials of Chinese herbs
in depression have been conducted. Based on the ndings of
the supplementary review, only a small proportion of these
are currently listed in Western databases. Similarly, in the
case of the systematic reviews, it is also possible that further
systematic reviews are available in the Chinese literature.
However, research to change or support practice in Western
contexts needs to be accessible, thus supporting the rationale
for focusing onevidence foundinthe Westernliterature while
drawing attention to the wider literature on this topic.
Conclusions of potentially benecial eects, particularly
for the formula Xiao Yao San and modied versions of this
formula, need to be interpreted in the light of limitations
related to reporting and methods but also because of the
variation in the combinations of herbs used.
Nevertheless, this overview of the evidence indicates that
well-designed trials in a Western context may be worth
pursuing. ese will require decisions such as which frame-
work is used to diagnose depression, whether any exibility
is allowed in the prescribing of Chinese herbs and the
most appropriate comparison intervention. It is possible
that pragmatic trials comparing overall care via dierent
systems will be most informative. Reporting does need to
be addressed because it aects how an RCT is judged,
particularly when scoring systems such as Jadad are used to
assess quality. Assessment of preference and expectations will
also be important in interpreting the results where disguising
the treatment is not possible.
onct of nterests
K. Pilkington has no conict of interests to declare. L. Butler
has a part-time private practice which includes Chinese
herbal medicine. Neither author has a direct nancial rela-
tionship with the website mentioned in the paper.
Acknowledgments
e authors thank Yin-fan Lee and Si Jie Liu, who kindly
helped with the translation of papers and Professor Volker
Scheid who was consulted for clarication and conrmation
of specic points.
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MEDIATORS
INFLAMMATION
of
http://dx.doi.org/10.1155/2013/914563
Research Article
Dried Ginger (Zingiber officinalis) Inhibits Inflammation in
a Lipopolysaccharide-Induced Mouse Model
You Yeon Choi,
1
Mi Hye Kim,
1
Jongki Hong,
2
Sung-Hoon Kim,
3
and Woong Mo Yang
1
1
Department of Prescriptionology, College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee University,
Seoul 130-701, Republic of Korea
2
College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
3
Cancer Preventive Material Development Research Center, College of Oriental Medicine, Kyung Hee University,
Seoul 130-701, Republic of Korea
Correspondence should be addressed to Woong Mo Yang; wmyang@khu.ac.kr
Received 21 January 2013; Revised 5 April 2013; Accepted 3 June 2013
Academic Editor: Y. Ohta
Copyright 2013 You Yeon Choi et al. Tis is an open access article distributed under the Creative Commons Attribution License,
Objectives. Ginger rhizomes have a long history of human use, especially with regards to their anti-infammatory properties.
However, the mechanisms by which ginger acts on lipopolysaccharide-(LPS-)induced infammation have not yet been identifed.
We investigated the anti-infammatory efects of dried Zingiber ofcinalis (DZO) on LPS-induced hepatic injury. Methods. ICR
mice were given a DZO water extract (100, 1000 mg/kg) orally for three consecutive days. On the third day, they were administered
by LPS intraperitoneally. To investigate the anti-infammatory efects of DZO, histological, cytokine expression, and protein
factor analyses were performed. Results. Oral administration of DZO signifcantly reduced pathological changes in the liver and
proinfammatory cytokines including interferon-(IFN-) and interleukin-(IL-)6 in the serum. In addition, DZO inhibited LPS-
induced NF-B activation by preventing degradation of the IB-, as well as the phosphorylation of ERK1/2, SAPK/JNK, and p38
MAPKs. Tese were associated with a decrease in the expression of inducible nitric oxide synthase (iNOS) and cyclooxyenase-2
(COX-2). Conclusions. Our data provide evidence for the hepatoprotective mechanisms of DZO as an anti-infammatory efect.
Furthermore, use of DZO to treat could provide therapeutic benefts in clinical settings.
1. Introduction
Te liver is well known to play an important role in the
initiation of multiorgan failure, the most lethal complication
in the infammatory response [1]. In particular, severe liver
injury results from the massive death of liver cells leading
to severe impairment of liver function [2]. Microbes and
their virulence factors enter the hepatic circulation where
they frst activate Kupfer cells to produce proinfammatory
mediators, including necrosis factor-(TNF-), interleukin,
(IL-)1, IL-6 and eicosanoids [3]. Tese mediators cause not
only microbial killing but also structural and functional liver
damage concerning mainly the parenchymal cells [4].
Lipopolysaccharide (LPS), the major constituent of the
outer cell wall of Gram-negative bacteria, has been widely
used to examine the mechanisms of infammation that
produce typical hepatic necrosis followed by fulminant
hepatic failure [5]. It has been reported that under LPS
stimulation, Kupfer cells release pro-infammatory cytokines
[6]. In addition, activation of LPS-induced nuclear factor-
kappa B (NF-B) mediates the mitogen-activated pro-
tein kinases (MAPKs) and subsequently regulates cycloox-
ygenase-(COX-)2 expressions, as well as inducible nitric
oxide synthases (iNOS) expressions [7]. Furthermore, COX-2
and iNOS expression have been proven to contribute to
infammatory disease [8]. Terefore, these cytokines repre-
sent an ideal target for neutralization of LPS [9]. Currently,
long-term use of anti-infammatory drugs is associated with
side efects such as fever, facial fushing, and aching muscles.
Terefore, using a natural product to treat infammatory
diseases may be more efective and have fewer side efects
[10].
Ginger rhizome (Zingiber ofcinale Roscoe, Zingiber-
aceae) has long been used in the world as a popular spice
food as well as a medicinal herb because of its high con-
tent of antioxidants and anti-infammatory properties [11,
12]. Ginger rhizome comprises various kinds of chemicals
including 6- and 8-series of gingerols and shogaols, among
which gingerol is the major ingredient representing a vari-
ety of bioactivities including antitumor promotional and
antiproliferative [13]. Studies by Nonn et al. have shown that
6-gingerol inhibited the TNF-, and IL-1-induced increase
in the p38-dependent NF-B activation and expression of
pro-infammatory genes of IL-6 and IL-8 in normal prostatic
epithelial cells [14]. Extensive studies in recent years have
displayed that 6-gingerol of ginger metabolites inhibits COX-
2 expression by blocking the activation of p38 MAP kinase
and NF-B in phorbol ester-stimulated mouse skin [15]. 6-
Shogaol suppressed LPS-induced up-expression of iNOS and
COX-2 in murine macrophages [16]. Tese efects may be due
to the pharmacological activity of biologically active com-
pounds involved in reducing infammation [17]; however,
the anti-infammatory mechanisms of DZO on LPS-induced
liver damage are not yet fully understood.
Tis study addressed the question of whether DZO has
a hepatoprotective efect in LPS-induced infammations. We
also attempted to understand the mechanism by which DZO
can reduce LPS-induced infammation as well as hepatic
failure.
2.1. Chemicals and Regents. LPS (Escherichia coli 0111:B4)
was purchased from Sigma-Aldrich (BD Bioscience, USA).
Mouse IFN-, IL-6, and the TMB substrate reagent ELISA
kit were purchased fromBDBioscience (San Jose, CA, USA).
RIPA bufer and protease inhibitor cocktails were obtained
from Roche (Indianapolis, IN, USA). Dual-color protein
standards, protein assays, Tween-20, acrylamide, ammonium
persulfate, skim milk, enhanced chemiluminescence (ECL)
detection reagent, and PVDF membranes were purchase
fromBio-Rad Laboratories (Hercules, USA). Rabbit antibeta-
actin, iNOS, COX-2, NF-B, IB-, phosphor-IB-, and
anti-rabbit alkaline phosphatase-conjugated secondary anti-
body were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA, USA). Anti-ERK1/2, phospho-ERK1/2, anti-
SAPK/JNK, phospho-SAPK/JNK, anti-p38, and phospho-
p38 MAPK were purchased from Cell Signaling Technology
Inc. (Beverly, MA, USA).
2.2. Preparation of DZO. Dried Zingiber ofcinalis (DZO)
was purchased from Omni Herb Inc. (Andong-si, Gyeong-
buk, Republic of Korea). DZO extract was prepared by
decocting 250 g dried herb with 5 L boiling distilled water
for 1 h 30 min. Te fltrate was concentrated under reduced
pressure and lyophilized. Afer fltration, an aqueous solution
of the extract was concentrated in a rotary evaporator,
freeze dried for 3 days, and stored at 4
C until used. Te
yield of DZO was approximately 10.45% w/w (dried weight
26.12 g). A voucher specimen (DZO001) was deposited at
our laboratory. Before each experiment, DZO extract was
dissolved in distilled water and vortexed for 2 min at room
temperature.
2.3. HPLC Analysis of Standards to DZO. HPLC analyses
were carried out with an Agilent Series 1100 HPLC system
(Palo Alto, CA, USA) consisting of a quaternary delivery
system, an autosampler, and a diode array detector (DAD).
Te chromatographic separation analysis was carried out on
a Shiseido UG 120 C18 (250 4.6 mm, i.d., 5 m) column.
Te mobile phases consisted of solvent A (acetonitrile, ACN)
and solvent B (water). Te standard materials used for the
quantitative analysis of DZO were gingerol and shogaol. A
gradient program was performed: 012 min (4050% A);
1224 min (5070% A); 2430 min (70100% A); 3040 min
(100% A), back to the initial conditions for equilibration. UV
detection wavelength was set at 220 nm. Te fow rate and
injection volume were set at 1 mL/min and 10 L, respectively,
at room temperature (25
C).
2.4. Animal Treatment and Induction of Infammation.
Female ICR mice (7 weeks old, weighing 2830 g) were
obtained from Japan SLC Inc. (Hamamatsu, Japan). Te
mice were kept in sterilized cages ( = 5) under standard
conditions (12 hlight and12 hdark), at 24 2
Cwitha relative
humidity of 4080%. Tey were fed a laboratory diet, water
was provided ad libitum, and they were housed for 7 days
before experimentation. Tey were arbitrarily divided into
four groups: normal (control group; no treatment), LPS (a
negative control group; treated with LPS 35 mg/kg), LPS +
DZO100 (treated with LPS and DZO100 mg/kg), and LPS +
DZO1000 mg/kg (treated with LPS and DZO1000). Afer the
7-day adaptation period, mice in groups 3 and 4 were orally
administered by DZO in distilled water for three consecutive
days, while groups 1 and 2 received an equivalent volume of
water as the control. On day 3, 1 h afer DZO administration,
all animals in groups 2, 3, and 4 were intraperitoneally
injected with LPS dissolved in distilled water. Blood from
suborbital and liver samples was collected 6 h afer the LPS
challenge. All experiments were conducted according to the
guidelines of the Committee on Care and Use of Laboratory
Animals of the Kyung Hee University. (KHUASP(SE)-12-
020).
2.5. Histological Assays. Liver tissue was fxed in 10%bufered
formaldehyde, embedded in parafn for 24 h, and serially
sectioned to a thickness of 5 m. Sections were stained with
hematoxylin and eosin (H&E) and examined for general
morphology. Pathological changes were evaluated under a
light microscope using the Leica Application Suite (LAS;
Leica Microsystems, Bufalo Grove, IL, USA). Digital images
were taken at a magnifcation of 100 and 400.
2.6. Determination of Cytokine Levels (IFN- and IL-6).
Serum samples were obtained from centrifuged blood
(14000 g, 30 min) and stored at 80
C until needed. IL-6

and IFN- concentrations were measured using a mouse
TNF-, IL-4, IL-6, and IFN- ELISA kit (BD Bioscience, San
Jose, CA, USA), according to the manufacturers instructions.
Te optical density of each well was read on an ELISAReader
(Molecular Devices, Downingtown, PA, USA) using 450 nm
and 570 nm flters.
2.7. Preparations of Protein Extracts. Liver tissue was frozen
in liquid nitrogen and then homogenized. For the cytoplas-
mic extracts, 100 mg frozen liver tissue homogenate ( = 5 in
each group) was incubated on ice for 15 min in cytoplasmic
bufer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM DTT, 0.15% Nonidet P-40, 50 mM -
glycerophosphate, 10 mM NaF, and 5 mM Na
3
VO
4
) and the
protease inhibitor cocktail. Te resulting homogenate was
centrifuged at 12000 g for 30 min at 4
Cand the supernatant

was carefully removed without disturbing the pellet. Te
supernatant was used to detect activated IB- and phospo-
IB-. To extract the nuclear proteins, nuclear bufer (20 mM
HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1 mMDTT, 0.50%Nonidet P-40, 50 mM-glycerophosphate,
10 mM NaF, and 5 mM Na
3
VO
4
, containing the protease
inhibitor cocktail) was added to the pellet and then incubated
on ice for 15 min. Tis was centrifuged at 12000 g for 15 min
at 4
C to determine the NF-B. Levels of iNOS, COX-

2, and MAPKs (ERK1/2, SAPK/JNK, p38) were confrmed
using whole protein extracts. Liver tissue ( = 5 in each
group) was homogenized on ice for 15 min in RIPA bufer
(50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 150 mM NaCl) and the protease inhibitor
cocktail. Te homogenate was incubated on ice for 15 min
afer vortexing, and this process was repeated four times.
Finally, the homogenate was centrifuged at 10,000 g for
30 minat 4
C, andthe supernatant was analyzedfor the whole

protein extracts.
2.8. Determination of NF-B, I-B, MAP Kinase (ERK1/2,
SAPK/JNK, p38), COX-2, and iNOS. Samples (40 g) of
protein from each liver homogenate (nuclear, cytoplasmic,
and whole fraction) were loaded onto 15% polyacrylamide
gels for electrophoresis. Ten, the proteins were transferred to
polyvinylidene fuoride (PVDF) membranes and incubated
at room temperature for 60 min in TBS bufer containing
0.1% Tween (TBS-T) and 5% dried skim milk to block
nonspecifc binding. Te membrane was incubated overnight
with one of the primary antibodies (antibodies; -actin,
NF-B, phospho-IB-, ERK1/2, phospho-ERK, SAPK/JNK,
phospho-SAPK/JNK, p38, phospho-p38, iNOS, and COX-2;
dilution 1 : 1000 in TBS-T). Anti-rabbit alkaline phosphatase-
conjugated secondary antibody (dilution 1 : 2000 in TBS-T)
was used to detect the target proteins. Afer incubation for 2 h
at room temperature, blots were detected using an enhanced
chemiluminescence (ECL) detection reagent. -Actin was
used as an internal loading control. Te relative band density
was calculated with respect to the -actin blot developed
under similar conditions using a computerized densitometry
system.
2.9. Statistical Analysis. All values are expressed as means
SD. Diferences between the mean values of normally dis-
tributed data were assessed by one-way ANOVA (Dunnetts
test) and Students test. Statistical signifcance was accepted
at < 0.05.
3. Results
3.1. Phytochemical Analyses of Standard Materials to DZO
Extract. HPLC was used for detection of DZO constituent
researchwithgingerol andshogaol as standardmaterials. Two
peaks on DZOwere synchronized with gingerol and shogaol,
which are components of Zingiber ofcinalis (Figure 1).
3.2. DZO Attenuates Infammatory Responses in LPS-Induced
Hepatic Failure. Liver architecture and structure were exam-
ined for fve specimens taken fromeach of the four treatment
groups (normal, LPS, DZO100, and DZO1000). Histological
analysis indicated normal liver architecture and structure
in the normal control group (Figure 2(a)). However, LPS-
induced livers showed broad hemorrhagic necrosis and
extensive areas of portal infammation (Figure 2(b)). Tese
pathological changes were less severe in DZO treatment
groups (Figures 2(c) and 2(d)).
3.3. Efects of DZO on Serum Levels of IFN- and IL-
6 in LPS-Induced Infammation. Te regulatory efects of
DZO (100, 1000 mg/kg) on the systemic secretion of circu-
lating cytokines, such as IFN- and IL-6, were examined
by ELISA. Serum levels of IFN- and IL-6 signifcantly
increased 6 h afer LPS challenge compared to the normal
group. In contrast, serum levels of IFN- and IL-6 in DZO
(100, 1000 mg/kg) treatment groups were signifcantly lower
(Figure 3).
3.4. DZO Reduces the Expression of NF-B and IB- in
LPS-Induced Infammation. To understand the mechanisms
of DZO inhibition of the pro-infammatory cytokines, we
investigated whether DZO inhibits both the degradation
of IB- and the nuclear translocation of NF-B. DZO
(100, 1000 mg/kg) signifcantly suppressed the LPS-induced
degradationof IB-(Figure 4(a)) as well as the translocation
of NF-B (Figure 4). It also inhibited LPS-induced phospho-
rylation of IB- in the cytoplasm.
3.5. DZO Reduces the Expression of MAP Kinase (ERK1/2,
SAPK/JNK, and p38) in LPS-Induced Infammation. Te
efects of DZO on LPS-induced phosphorylation of ERK1/2,
SAPK/JNK, and p38 MAPKs were examined by western blot.
DZO markedly reduced LPS-induced phosphorylation of all
compounds, whereas the nonphosphorylated forms used as
a control remained the same (Figure 5). Tus, DZO might
suppress the production of LPS-induced pro-infammatory
mediators by inhibiting phosphorylation of the signaling
proteins.
3.6. DZO Inhibits the Expression of iNOS and COX-2 in LPS-
Induced Infammation. Te infammatory factors iNOS and
COX-2 are correlated with LPS stimulation. We tested the
anti-infammatory efects of DZOon LPS-induced iNOS and
COX-2 expression, by western blot. Both factors increased
(min)
0 5 10 15 20 25 30 35
m
A
U
0
50
100
150
200
250
(2) Shogaol
(1) Gingerol
(a)
(min)
0 5 10 15 20 25 30 35
m
A
U
0
20
40
60
80
100
120
(2) Shogaol
(1) Gingerol
(b)
Figure 1: HPLC chromatogram of standard materials (a) and DZO (b). Two external standards were used for HPLC analysis.
(a) (b) (c) (d)
Figure 2: Efects of two doses of DZO on histopathological changes in liver tissues of mice treated with LPS. (a) Normal group: liver tissue
structure showed no pathological abnormalities. (b) LPS group: liver tissue structure showed apparent broad hemorrhagic necrosis. (c) LPS +
DZO group (100 mg/kg): liver tissue structure showed minimal hepatocellular necrosis. (d) LPS + DZO group (1000 mg/kg): liver tissue
structure was similar to normal group. Typical images were chosen from each experimental group (100 in the upper panel and 400 in the
lower panel).
signifcantly in the LPS-induced groups, but DZO inhibited
their expression in the treatment groups (100, 1000 mg/kg)
(Figure 6).
4. Discussion
We demonstrated that pretreatment with DZO signifcantly
reduced the infammatory response of LPS-induced infam-
mation. LPS induced liver-related infammation in a mouse
model of viral hepatitis that closely resembles human viral
hepatitis [18]. A previous study reported that LPS-induced
histological changes showed lymphocyte and neutrophil
infltrationincreasing in the central and portal areas [19]. Our
histological analysis showed the inhibitory efects of DZO
on necrotic hepatocytes and tissue damage with excessive
production of infammatory cytokines in the LPS-induced
liver and serum. In particular, DZO1000 group largely
attenuated LPS-induced broad hemorrhagic necrosis of liver
structure similar to normal group. Tese results indicate that
DZO is efective for controlling the response of LPS-induced
liver damage.
Several studies have indicated that the infammatory res-
ponse to LPS challenge is associated with the release of pro-
infammatory cytokines such as IFN- and IL-6 [20]. In
addition, mediators generated by LPS stimulation assist the
innate immune response, but their overproduction results in
acute infammation that can cause tissue injury and organ
failure [21]. Here, we confrmed that DZOinhibits the expres-
sion of LPS-induced IFN- and IL-6, which are signifcantly
elevated in LPS-induced infammation. Tese results indicate
200000
150000
100000
50000
0
###
I
F
N
-
(
p
g
/
m
L
)
DZO
LPS (35 mg/kg)

+ + +
100 1000
(a)
150000
100000
50000
0
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
I
L
-
6

(
p
g
/
m
L
)
(b)
Figure 3: Efect of two doses of DZO on IFN- (a) and IL-6 (b) levels in the serum of mice treated with LPS. Te results are presented as the
mean SEM ( = 5). Te serum levels of IFN- and IL-6 in DZO-treated group (100 or 1000 mg/kg) were signifcantly attenuated.
#
Indicates
signifcance for the diference between normal control group and LPS group (
< 0.001).
Indicates signifcant diference fromLPS group

(
< 0.001,

< 0.01).
500
400
300
200
100
0
P-IB
Normal LPS
LPS +
DZO100
LPS +
DZO1000
Nf-B
-Actin
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
N
F
-
B
/
-
a
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l

(
%

o
f

c
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t
r
o
l
)
250
200
150
100
50
0
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
P
-
I
B
/
-
a
c
t
i
n

l
e
v
e
l

(
%

o
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o
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)
(a) (b)
Figure 4: Efects of two doses of DZO on the activation of NF-B (a) and IB- (b) in the liver of mice treated with LPS. DZO inhibited
the degradation of IB- and NF-B nuclear translocation. Similar results were obtained in three independent experiments, and the results
obtained from one of three representative experiments are shown.
#
Indicates signifcance for the diference between normal control group
and LPS group (
< 0.001).

Indicates signifcant diference from LPS group (
< 0.001).
ERK
p-ERK
500
0
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
Normal LPS
LPS +
DZO100
LPS +
DZO1000
1500
1000
P
-
E
R
K
1
/
2
/
E
R
K
1
/
2

l
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(
%

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o
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)
(a)
JNK
p-JNK
400
600
200
0
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
Normal LPS
LPS +
DZO100
LPS +
DZO1000
P
-
J
N
K
/
J
N
K

l
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(
%

o
f

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)
(b)
p38
P-p38
500
400
300
200
100
0
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
Normal LPS
LPS +
DZO100
LPS +
DZO1000
P
-
p
3
8
/
p
3
8

l
e
v
e
l

(
%

o
f

c
o
n
t
r
o
l
)
(c)
Figure 5: Efects of two doses of DZO on the phosphorylation of MAPKs (ERK1/2 (a), SAPK/JNK (b), and p38 MAPKs (c)) in the liver of
mice treated with LPS. DZO remarkably attenuated LPS-induced phosphorylation of ERK1/2, SAPK/JNK, and p38 MAPKs. Similar results
were obtained in three independent experiments, and the results obtained fromone of three representative experiments are shown.
#
Indicates
signifcance for the diference between normal control group and LPS group (
< 0.001).
Indicates signifcant diference fromLPS group

(
< 0.01).
COX-2
iNOS
-Actin
500
400
300
200
100
0
Normal LPS
LPS +
DZO100
LPS +
DZO1000
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
C
O
X
-
2
/
-
a
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t
i
n

l
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(
%

o
f

c
o
n
t
r
o
l
)
500
400
300
200
100
0
800
700
600
###
DZO
LPS (35 mg/kg)

+ + +
100 1000
i
N
O
S
/
-
a
c
t
i
n

l
e
v
e
l

(
%

o
f

c
o
n
t
r
o
l
)
(a) (b)
Figure 6: Efects of two doses of DZO on the expression of COX-2 (a) and iNOS (b) in the liver of mice treated with LPS. Te expression
of COX-2 and iNOS was increased signifcantly afer exposure to LPS for 6 h, and this efect was blocked by pretreatment with DZO at a
dose of 100 or 1000 mg/kg.
#
Indicates signifcance for the diference between normal control group and LPS group (
< 0.001).

Indicates
signifcant diference from LPS group (
< 0.001,

< 0.01,

< 0.05).
that DZO might suppress the infammatory response via
the inhibition of infammatory cytokines supporting our
histological analysis.
Te transcription regulator NF-B plays a pivotal role
in activating subsequent signaling pathways, especially the
regulation of pro-infammatory molecules [22]. Also, activa-
tion of LPS-induced NF-B causes phosphorylation of IB-
kinase (IKK), leading to degradation of IB- and translo-
cation of NF-B into the nucleus [23]. Tese studies may
provide a target to specifcally downregulate the expression
of NF-B with inhibition of IB- degradation. In this study,
DZO inhibited LPS-induced NF-B transcription activity as
well as IB- protein expression in the liver compared to
negative control group.
Many studies have reported that MAPKs mediate the
activation of the transcription factor NF-B [24]. To explore
the mechanisms of NF-B inactivation by DZO, the efects
of DZO on LPS-induced phosphorylation of the Erk1/2,
SAPK/JNK, and p38 MAP kinases were examined. All of the
kinases were overexpressed afer exposure to LPS, but that
expression was reduced afer DZO exposure. Tis suggests
that the hepatoprotective activity of DZO is due to NF-B
inhibition via the inhibition of LPS-induced phosphorylation
of MAPKs.
Furthermore, NF-B activation mediates the expression
of rapid-response genes, including pro-infammatory medi-
ators such as iNOS and COX-2 [25]. Te present study
confrmed that LPS stimulates iNOS and COX-2, which
was associated with overexpression of NF-B, whereas orally
administrated DZO greatly reduces the expression of iNOS
and COX-2 and hence the expression of NF-B. It is likely
that the anti-infammatory activity of DZO contributes to
the reduced expression of iNOS and COX-2 in LPS-induced
liver injury. Also, proinfammation cytokines, IL-6 and TNF-
are upregulated by the expression of COX-2 and iNOS
[26]. Taken together, these data suggest that DZO inhibits
the expression of iNOS and COX-2 through inactivation
of NF-B by reducing IB- phosphorylation. We assume
that the hepatoprotective efects of DZO may be due to
the anti-infammatory compounds such as gingerols and
shogaols. Tese results are helpful in understanding the anti-
infammations properties of DZO.
5. Conclusion
We found that DZO inhibits LPS-induced infammation via
regulation of NF-B and MAP kinases. DZO signifcantly
inhibits the production of IFN- and IL-6 and suppresses
NF-B by degradation of IB-. Tese activities appear to
be mediated via downregulation of the ERK1/2, SAPK/JNK,
and p38 MAP kinases signaling pathways and suppression
of iNOS and COX-2. Our data provide evidence for a
mechanism by which DZO acts as an anti-infammatory
agent. Strategic use of DZOin treating infammatory diseases
could provide therapeutic benefts for future clinical use.
Conflict of Interests
Te authors clearly declare that they have no conficts of
interests at all.
Acknowledgments
Tis work was supported by a Grant from the Kyung Hee
University in 2012 (KHU-20121731) and the National Re-
search Foundation of Korea (NRF) Grant funded by the
Korea government (MEST) (no. 2012-0005755).
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[11] H. Kikuzaki and N. Nakatani, Antioxidant efects of some
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[12] R. C. Lantz, G. J. Chen, M. Sarihan, A. M. S olyom, S. D. Jolad,
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MEDIATORS
INFLAMMATION
of
Glutamate: from discovery as a food avor to role as a basic
taste (umami)
13
Kenzo Kurihara
ABSTRACT
In 1908 Kikunae Ikeda identied the unique taste component of
konbu (kelp) as the salt of glutamic acid and coined the term umami
to describe this taste. After Ikedas discovery, other umami taste
substances, such as inosinate and guanylate, were identied. Over
the past several decades, the properties of these umami substances
have been characterized. Recently, umami has been shown to be the
fth basic taste, in addition to sweet, sour, salty, and bitter. Am J
Clin Nutr 2009;90(suppl):719S22S.
INTRODUCTION
Kikunae Ikeda, a professor of physical chemistry at the
University of Tokyo interested in the unique taste of kelp (konbu)
and meat, suspected that there was an unknown, unidentied
taste component in these foods. Accordingly, in 1907 he began
a project to attempt to identify this taste component in dried
konbu. Within a year, he had discovered the sodium salt of
glutamic acid to be the taste component and termed it umami.
Glutamic acid itself had already been described chemically in
1866 by Ritthausen (1). Subsequently, Fischer (2) reported that
glutamic acid at rst tasted sour and then developed a peculiar
insipid taste. Fischer thus did not notice that glutamate had
a distinctive taste. The uniqueness of the taste of sodium glu-
tamate and of other umami substances (eg, inosinate, guanylate)
would not become widely accepted until the end of the 20th
century.
IKEDAS DISCOVERY THAT GLUTAMATE IS AN UMAMI
COMPONENT IN KONBU
Ikeda was born in Kyoto in 1864 and matriculated at the
University of Tokyo in 1885; by 1896 he had already achieved the
rank of associate professor of physical chemistry (3). In 1899 he
was invited to study physical chemistry in the laboratory of
Wilhelm Ostwald in Leipzig, Germany. Ostwald was one of the
founders of classical physical chemistry and received the Nobel
Prize in Chemistry in 1909 for his work. Soon after returning
from abroad, Ikeda was promoted to professor of physical
chemistry at the University of Tokyo (1901).
Ikeda recognized that there were 4 dened taste qualities,
namely sweet, sour, bitter, and salty. In addition, however, he
considered that there might be another taste quality, which was
quite distinct from the 4 known tastes and dominant in foods such
as kelp and possibly meat. He coined the term umami to name
this putative taste. In 1907 he began a project to identify
chemically the umami component in kelp. His focus on kelp
derived partly from the fact that, in Japan, large quantities of
kelp (Laminaria japonica), termed konbu, are harvested and
consumed as food. He thus had a bountiful supply of starting
material. His use of kelp also derived in part from his familiarity
with the taste of konbu. In Kyoto, where Ikeda was born, konbu
was used in a variety of foods; he thus had been exposed to this
taste from childhood.
To identify the umami component in konbu, Ikeda began with
dried konbu, because the proteins are denatured during drying
and thus are not extracted in water. He began his water extraction
by using 12 kg of dried konbu. Most of the mannitol and NaCl in
the liquid was removed by crystallization. The umami taste-
imparting material remained in solution. Preliminary tests in-
dicated that the umami material was the salt of an organic acid,
and subsequent steps were focused on isolating the organic acid.
Ikeda made various salts of the organic acid, but he could not
obtain a precipitate, because they were all highly soluble in water.
Finally, by using lead nitrite, he produced a salt of the organic
acid that would precipitate. On cooling, the resinous precipitate
was pulverized and treated with hydrogen sulde in the presence
of water and barium carbonate. This procedure converted the lead
salt of the organic acid into a barium salt, which was soluble in
water in the presence of chloride; the lead ion precipitated as lead
sulde. Subsequent addition of silver sulfate (dissolved in a large
volume of hot water) caused the complete removal of barium
chloride from the organic acid, and the barium was then removed
by precipitation with sulfuric acid. This nal solution was
concentrated and left to crystallize. This procedure of crystal-
lization led to removal of silver ion because it did not precipitate.
About 30 g of the organic acid was obtained. Because konbu in
water has the umami taste and is at neutral pH, the organic acid
must exist as a salt form in water. Ikeda therefore prepared
a solution of the isolated organic acid, adjusted the pH to neu-
trality, and conrmed that this solution elicited a strong umami
taste.
Molecular weight and elementary analyses of the organic acid
crystals revealed the molecular formula C
5
H
9
NO
4
. Ikeda rec-
1
From Aomori University, Aomori, Japan.
2
Presented at the 100th Anniversary Symposium of Umami Discovery:
The Roles of Glutamate in Taste, Gastrointestinal Function, Metabolism, and
Physiology, held in Tokyo, Japan, 1013 September 2008.
3
Address correspondence to K Kurihara, Aomori University 2-3-1,
Kobata, Aomori 030-0943, Japan. E-mail: kurihara@aomori-u.ac.jp.
First published online July 29, 2009; doi: 10.3945/ajcn.2009.27462D.
Am J Clin Nutr 2009;90(suppl):719S22S. Printed in USA. 2009 American Society for Nutrition 719S

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ognized the compound to be glutamic acid. The structure of
glutamic acid had already been described by Ritthausen in 1866
(1) and by Fischer (2), who subsequently reported that glutamic
acid had at rst a sour and then a peculiar, insipid taste. Fischer
thus did not notice that glutamate produces a unique taste. The
reason is that it is the salt form of glutamate, not the acid, that
elicits the umami taste. In this regard, it is noteworthy that the
pH of most foods approximates neutrality; at neutral pH, glu-
tamate is present almost exclusively as a salt. Fischer had tasted
the acid. Although Ikeda had isolated the acid, he prepared and
tasted it as a salt. In fact, various soluble salts (eg, Na, K, or Ca
salt) of glutamate elicit umami taste.
Ikeda completed his work in 1908, only a year after he had
begun. Soon thereafter, together with Saburosuke Suzuki, he
commercialized his discovery.
THE CONTENT OF UMAMI SUBSTANCES IN FOOD
Ikeda was also interested in identifying an umami component
in bonito akes (sh akes). Ikedas protege Shintaro Kodama
undertook this project and in 1913 identied 5#-inosinate (salt of
inosine-5#-monophosphate) as the umami taste in bonito (4).
Several decades later, 5#-guanylate was also shown to elicit an
umami taste (5) and was found to be the main umami compo-
nent in shiitake mushrooms.
The concentrations of these umami substances (glutamate, 5#-
inosinate, 5#-guanylate) were subsequently measured in a vari-
ety of foods. Free glutamate is found in both animal and plant
foodstuffs. Notable examples include konbu, green tea, seaweed,
tomato, potato, Chinese cabbage, soybean, Parmesan cheese,
sardines, prawns, and clams. Free glutamate is found in high
concentrations in ripe tomatoes (140 mg/100 g) and provides the
characteristic umami taste of this vegetable. Much higher con-
centrations of free glutamate are found in Parmesan cheese, an
animal product with a strong umami taste (1200 mg/100 g) (7).
Konbu dashi, a Japanese soup and cooking stock of pure
umami taste, contains 20 mg/100 ml glutamate (Figure 1, left;
note the presence of aspartate as well, another umami substance
of lesser intensity). Figure 1 (right) also shows that breast milk
contains free glutamate at a concentration similar to that present
in konbu dashi. Humans thus become familiar with the taste of
glutamate and umami very early in life at concentrations found
in some foods.
Inosinate is found only in animal food products, including
dried sardines, bonito akes, horse, mackerel, tuna, pork, beef,
and chicken, typically in the 100300 mg/100 g range (8). Fresh
sh often contains little free inosinate and thus no umami taste;
aging for even a few hours produces a rise in inosinate con-
centrations (as cells degrade), and the emergence of a charac-
teristic umami taste. Guanylate occurs only in foods of plant
origin, notably mushrooms (eg, dried shiitake mushrooms and
matsutake and enokitake mushrooms, among others), typically in
concentrations ranging between 10 and 150 mg/100 g (9).
SYNERGISM BETWEEN GLUTAMATE
AND NUCLEOTIDES
Konbu dashi alone does not elicit a strong umami taste. Avery
strong umami taste can be achieved by adding bonito akes or
dried sardines, which contain inosinate. That is, the mixing of
glutamate and a nucleotide (inosinate) greatly enhances the taste
of umami. A similar effect is achieved elsewhere, such as in
China, Europe, and the United States, by cooking beef or chicken
(which contains inosinate) with vegetables containing free glu-
tamate and/or the addition of cheese (eg, Parmesan cheese).
This synergism was originally identied by A Kuninaka and
systematically examined by Yamaguchi (9), but extent of the
synergism varies among the species of animals. For example, it is
observed in rats and mice, although it is rather weak in com-
parison with humans. And, in rats and mice, sensory synergism
with nucleotides is also observed with amino acids other than
glutamate (10). On the other hand, the dog shows synergism only
between glutamate and the nucleotides (11). As discussed in
detail in other articles in this supplement, the synergism also
appears at the receptor level: candidate umami receptors in mice
show glutamate-nucleotide synergism in their activation. In
mouse receptor studies in vitro, this synergism also occurs
FIGURE 1. Free amino acid concentrations in konbu dashi (left) and breast milk (right). Breast milk data are from reference 6.
FIGURE 2. Mean (6SEM) results of at least 3 nerve preparations that
show enhancement of canine chorda tympani nerve response to glycine by
NaCl. The relative response (ordinate) represents the ratio of the response to
100 mmol/L glycine in the presence of NaCl (at the concentrations indicated
on the abscissa) divided by the response in the absence of NaCl. Reproduced
with permission from reference 15.
720S KURIHARA
between nucleotides and many amino acids (12), whereas in
studies of human receptors, synergism occurs only between
glutamate and the nucleotides (13).
TASTE COMPONENTS OF FOODS
Although foods typically contain many taste components, the
identifying tastes of some are produced by a small number of
taste components. By using the omission test in human taste
experiments, Konosu et al (14) studied the essential taste com-
ponents of a variety of foods. They found, for example, that the
taste of crabmeat could be duplicated by a solution containing
certain amino acids (glycine, alanine, arginine), umami sub-
stances (glutamate and inosinate), and salts (NaCl and K
2
HPO
4
).
If the umami substances were deleted from the solution, the taste
of crab was lost. The charactertistic tastes of other sea foods
were found to depend on the presence of different amino acids,
such as glycine (in high concentrations), to elicit the taste of
scallops and methionine to reproduce the taste of sea urchin.
Both NaCl and K
2
HPO
4
are needed to produce crabmeat taste.
But, although K
2
HPO
4
makes a minor contribution to the taste
(its elimination does not markedly change the crabmeat-like
taste), the role of NaCl is critical. The removal of NaCl from the
solution almost completely eliminates the taste of crab. As
shown in Figure 2, the response of taste nerves to glycine is
greatly enhanced by the presence of NaCl (15), with maximal
enhancement occurring at 100 mmol/L NaCl (0.58%). Similar
effects were obtained in psychometric studies in humans (16). It
should be noted that 100 mmol/L NaCl elicits only a weak salty
taste (physiologic saline, 0.9% NaCl, is also not very salty).
UMAMI AS A BASIC TASTE
The taste of umami is very familiar to the Japanese, who have
long used pure umami solutions such as konbu dashi in their
cooking. In contrast, the perception of a specic umami taste has
not emerged in Western cultures, most likely because pure,
umami-tasting ingredients have not existed or been used in
Western cooking until recently. In 1982 a group of Japanese
investigators founded the Umami Research Association, a col-
laboration among scientists specializing in physiology, molecular
biology, nutrition, and food chemistry. Over the past 25 y, the
association has held a number of international symposia on
umami, together with scientists from Europe and the United
States (Table 1).
Discussion of the key issues surrounding the establishment of
umami as a basic taste began at the rst symposium. Psycho-
physical investigators put forth the idea that umami substances
did not produce a unique taste but instead potentiated one or more
of the then 4 basic tastes. However, the studies of Yamaguchi (17)
later showed convincingly that such was not the case and that
umami was a taste phenomenon independent of the 4 basic
tastes. Electrophysiologists argued that the response of taste -
bers to the sodium salt of glutamate resulted from the sodium
ion, not from glutamate (umami) (18). But Kumazawa and
Kurihara (11) showed a marked synergism between MSG and
the nucleotides in the canine chorda tympani nerve and further
showed that the large response to MSG and guanylate could not
be inhibited by amiloride, an inhibitor of the response to NaCl
(Figure 3). Such ndings show that the large synergistic re-
sponse could not be explained by an effect of the sodium ion.
Ninomiya and Funakoshi (20) also reported that single glosso-
pharyngeal nerve units exist that are sensitive only to umami
substances in the mouse. And Bayliss and Rolls (21) reported
the existence of single bers that respond most sensitively to
MSG in the taste cortex of macaques. Such ndings together
provided a solid, physiologic basis for postulating the existence
of a unique umami receptor, which subsequently led to a search
for, and, through the use of molecular techniques, the recent
identication of candidate umami receptors that reect the
properties of umami taste in vivo (12, 13, 22). Taken together,
TABLE 1
International symposia on umami
Year Occasion Location
1982 Umami Research Association founded Japan
1985 First International Symposium on Umami Hawaii
1990 Second International Symposium on Glutamate Sicily
1993 Umami session, ninth International Symposiumon Olfaction
and Taste
Sapporo, Japan
1997 Umami session, 12th International Symposium on Olfaction
and Taste
San Diego, CA
1998 International Symposium on Glutamate Bergamo, Italy
2004 Umami session, 14th International Symposium on Olfaction
and Taste
Kyoto, Japan
FIGURE 3. Mean (6SEM) results of the effect of amiloride on the
response of the canine chorda tympani nerve to umami substances
[monosodium glutamate (MSG) + guanosine-5#-monophosphate (GMP):
100 mmol/L + 0.5 mmol/L] and NaCl (100 mmol/L); n = 34/group. The
relative response (ordinate) represents the ratio of the response to the test
agent(s) in the presence of amiloride divided by the response in the absence
of amiloride. Reproduced with permission from reference 19.
GLUTAMATE: A FOOD FLAVOR AND A BASIC TASTE (UMAMI) 721S
the results of the past quarter century of umami research have
established umami as the fth basic taste, thus afrming the
suspicions that led Ikeda to begin his studies of umami a century
ago. (Other articles in this supplement to the Journal include
references 2351.)
The authors travel expenses to participate in the symposium and an hon-
orarium were paid by the International Glutamate Technical Committee, the
sponsor of the symposium, a nongovernmental organization funded by indus-
trial producers and users of glutamate in food.
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722S KURIHARA
Chinese Medicine, 2010, 1, 84-90
doi:10.4236/cm.2010.13016 Published Online December 2010 (http://www.SciRP.org/journal/cm)
Copyright 2010 SciRes. CM
The Foundation of Traditional Chinese Medicine
Patrick Kim Cheng Low, Sik-Liong Ang
Universiti Brunei Darussalam Brunei Darussalam, Brunei
Email: patrick_low2003@yahoo.com, angsikliong@gmail.com
Received August 31, 2010; revised September 17, 2010; accepted September 30, 2010
Abstract

In this paper, the authors examine and interpret the concept of Traditional Chinese Medicine (TCM) whom
the Chinese believes and practices for so many centuries. The authors also explain the ancient Chinese con-
cepts of Chi and Tao (Yin and Yang) which are the foundation of the TCM. This paper also seeks to discuss
the ways of attaining a healthy body with the clarity of mind as well as to demonstrate the benefits of such
healthy lifestyle.

Keywords: Traditional Chinese Medicine (TCM), Chinese Concepts of Tao (Yin and Yang) and Chi,
Meditation, The Yellow Emperors Canons of Internal Medicine, The Tao of Revitalization
1. Introduction

Medicine-the science of healing the sick has been prac-
ticed for thousands of years. Even the earliest human had
used plants and herbs as medicines and had tried simple
surgical procedures, such as putting splint on broken
bones. Over the last two hundred years, we have made
huge progress in the clinical and surgical procedures in
medicine [1]. Chinese medicine which is also considered
as an alternative medicine is gradually being accepted
and is practiced even in the Western world.

1.1. The Papers Aim & Objectives

In this paper, the aim and objectives are to illustrate the
concept of Traditional Chinese Medicine (TCM) which
is of paramount importance for one to achieve a healthy
lifestyle. The authors interpret and explain that the an-
cient Chinese concepts of Chi and Tao (Yin and Yang)
are the foundation of the TCM. The paper also seeks to
discuss the ways of attaining a healthy body with the
clarity of mind as well as to demonstrate the benefits of
such healthy lifestyle.

1.2. What is Chinese Medicine?

Chinese Medicine is also called Traditional Chinese
Medicine (TCM). It includes a range of traditional medi-
cine practices originating in China. TCM practices in-
clude treatments such as Chinese herbal medicine,
acupuncture, dietary therapy, and also both the Tui Na and
Shiatsu massages. Chi Kung (Qigong) and Tai Chi
movements (Taijiquan) are also closely associated with
TCM. TCM has been practiced by the Chinese for thou-
sands of years and is rooted in meticulous observation of
how nature, the cosmos, and the human body are inter-
acting. Major theories include; Tao (Yin and Yang), Chi,
the Five Phases (Wu Xing), the human body Meridian/
hannel system and Zang Fu organ theory.

1.3. What is Tao? What is Yin and Yang

Lao Tzu, was one of the earliest philosopher in the Chi-
nese history, who describes the marvel of Tao as an
evolving force that operates throughout the universe. Tao
is the first cause of the universe. Lao Tzu said that Tao is
the way and he emphasized this in the first verse of his
Tao Teh Ching [2] that:

(, )

Translated as:

The Tao that can be said is not the everlasting Tao.
If a name can be named, it is not the everlasting name.
That which has no name is the origin of heaven and earth;
That which has a name is the mother of all things.

(Lao Tzus Tao Teh Ching, Verse.1) [3]
P. K. C. Low ET AL.

85
Therefore Tao is always without a name and that it is
the origin of heaven and earth. Tao can also be said to be
the Absolute that it can be said to be the movement
and a stillness without a beginning, Yin and Yang (also
known as Tai Chi) are things that can be said to be with-
out a beginning (Cleary 2003).
The Tai Chi (Ultimate Principle of Existence) in-
volved The two dynamic powers (the white space
represents the Yang and the black space represents the
Yin) exists in equilibrium and from which a coordinated
and vigorous force is produced. [4]
This classic symbol for Yin and Yang appears like a
pair of fish swimming in a circle around each other; the
tail of one is formed from the head of the other. Here, we
can see that Yin-Yang are born out of each other and are
transformed into each other. Each of the Yin-Yang con-
tains the seed of the other; there is a tiny seed circle of
dark Yin contained in the white part of Yang, as there is a
seed circle of white Yang contained in the darkness of
Yin. Tao is the force, which flows through all lives. Each
person is to nurture the breathing or what is also known
as the integral life force (Chi or Qi) that has been
given to him/her. Unlike Western thinking, time is not
linear but cyclical. And overall, each and every Taoism
believers goal is to align him(her)self, by having a bal-
ance (the perfect sense of balance is embodied in the idea
of Yin-Yang) or being harmonious with the Tao. In the
universe, there should always be a balance of nature.
Ying (female) and Yang (male) are always at work, and
there should be a good balance between them; and hence
the avoidance of extremes. This is indeed what the con-
cept of Traditional Chinese Medicine (TCM), which is
anchored in Taoism roots, is based on. The fact that one
who knows how to maintain good health, one would al-
ways carry out ones daily life in accordance to nature.
Thus, one would need to follow the principle of Yin and
Yang and keeps in conformity with the art of predicting
the consequences of what would happen based on the
interaction of Yin and Yang. By doing so, one would be-
able to modulate or transform ones life in harmony with
nature. By way of recuperating the essence and the vital
energy, one would then master and practice the way of

Figure 1. Tai Chi diagram.
maintaining harmony as well as good health. For in-
stance, if ones behavior in daily life is kept in regular
patterns including ones food and drink intake, its fixed
amount, as well as ones daily activities where one
would not overwork ones body. And if, one is also
based on Taoism practices, it is taken that one should be
aware and, in fact, be sensitive of the balance of Yin and
Yang in nature. One can also take it as axiomatic that in
the morning after a good night sleep, one is often re-
freshed and energized. In the morning when the Yang
(Chi or energy) is at its high and at its abundance, one
becomes naturally active, thereby, as one uses ones en-
ergy, one gets tired. One needs to balance the Yang with
Yin (taking rest or naps) during the day so that one
maintains ones energy level (This is very true when one
gets older). When the evening comes, the Yin (Chi) be-
comes abundant or overwhelming; one then has to con-
serve the Yang energy as well in balance to be ready for
rest. If the Yang energy is used in a way that it is in an
extreme manner, ones whole body is not in balance and
one becomes totally exhausted so much so that one may
not even rest well. One, thus, needs to follow the way of
nature and be in balance. Also, in the same way, one
cannot pull the seedling to assist its growth. Thus, the
Chinese saying: to pull seedlings to help them grow,
meaning to work hard in a self-defeating way or going
against nature for quick results; and this is foolish. The
Western equivalent here is that of penny wise and
pound foolish. And in fact, in everything, nature must
take its course. And by understanding the balance of Yin
and Yang in nature with regards to the daily living, one
could live a healthier life in body and in spirit.

1.4. Chi and the Balance of Yin and Yang

Western Medicine is different from the TCM because the
TCM has a concept of Chi as a form of energy. It is be-
lieved that this energy exists in all things (living and
non-living) including air, water, food and sunlight. Chi is
said to be the unseen vital force that nourishes ones
body and sustains ones life. It is also believed that an
individual is born with an original amount of Chi at the
beginning of ones life and as one grows and lives, one
acquires Chi from eating and drinking, from breathing
the surrounding air and also from living in ones envi-
ronment. An individual would become ill or dies if ones
Chi in the body is imbalanced or exhausted.
When one studies the principle of the Life Force; the
Chi and the Tao (Yin and Yang); one would understand
how this Life Force manifests in nature. Through
self-cultivation, one basically enriches ones Chi for op-
timum health and longevity. This happens when onesub-
scribes to this Life Force from nature that flows freely
into ones mind and body. However, this requires one to

86 P. K. C. Low ET AL.
live freely from desires, worries and emotions. To live
freely, one has to detach from the worldly possessions.
For the instance, money is to be spent, there is to-ing and
fro-ing, thus a going and a coming of it; and there is a
non-attachment to the money or the material things for
better flow. Furthermore, one is required to discipline
oneself by having a proper diet, sleep and exercise so
that one would not disturb and interrupt with the move-
ment of Life Force which may cause the Chi to dissipate
in ones body. This dissipation of Chi would result one to
fall into sickness, disease, physical and mental sufferings.
It is the Taoists belief that the practice of Chi Kung, Tai
Chi movements and meditation helps one to harmonize
ones Life force with ones environment and nature.
Stationary and Moving: One would like to ask this
question: Why, for clarity of the mind, do both the Chi
and the balance of stationary (Yin) and moving (Yang)
are so important?
First of all, take note of it: When one is asleep, ones
body is heavy and resting (stationary) and not moving. In
fact, the body takes time to recreate, rejuvenate its
strength or regenerate energies for the body, the store-
house of energy (Chi).
Doctors normally advise one to have enough sleep
(usually 6-8 hours) and younger children require more
sleep than adult (some more than 12 hours) for growth and
tissue repairs. When one is short of sleep, ones mind is
not clear. One may feel groggy; in fact, one normally feels
tired and one cannot think straight or properly since the
lack of sleep affects ones focus and concentration. If one
sleeps too much, (one feels heavy) one also feels drowsy
and cannot think effectively as well.
When one is awake, ones body feels lighter, and one
can move from one place and another, by spending on its
kinetic energy. On one hand, too many activities will
make ones body and mind feel tired and on the other
hand, too few activities will also make the body and the
mind feel bored.
So it is meant that there should be an optimum point
when sleep (potential energy) and being awakened (ki-
netic energy) are balanced for the body and the mind to
be effective and active.
As everything is centered on the nature, Taoism en-
courages man to take the path of nature because the path
which nature itself would follow is not for human inter-
ference or interventions. Water flows downwards and
that it is the natural flow or spontaneously natural. Forc-
ing ones way against nature, going against the grain,
forcing nature to bend to ones will is not good as it
harms oneself. But by relaxing and allow nature to go its
way, everything will fall into place. It is wu wei er wu bu
wei-by doing nothing, everything is done.
2. Chi Kung (Qigong) for Good Health

The practice of Chi Kung (Qigong) is to regulate and con-
trol the Chi within the body. Chi Kung practice involves
the manipulation and balance of the Chi within the practi-
tioners body. According to Taoism, the regulation of Chi
is carried out through the three interconnected components:
the Mind, the Body and the Spirit. For the Taoist, the
training of the mind and the body is through meditation,
contemplation and physical exercises. It sometimes also
includes the ingestion of Chinese herbs to regulate ones
Chi within the body. The development of Traditional
Chinese Medicine has added more detailed to the Chi
within the human body. In this system, Chi travels through
the body along twelve main meridian channels and nu-
merous smaller branches and tributaries. These main me-
ridians also correspond to twelve main organs: the lung,
large intestines, stomach, spleen, heart, small intestine,
urinary bladder, kidney, liver, gallbladder, pericardium,
and the triple warmer, which represents the entire torso
region. The amount and flow of Chi is affected by ones
emotional state which is ultimately related to the Mind, the
Body and the Spirit. To put it simply, most Chi Kung
practitioners use this concept of the proper Chi flow
through those meridians as a basic premise.

2.1. Tai Chi for Good Health

Maintaining the balance of the Yin-Yang equilibrium is
also the concept of Tai Chi and can be illustrated in prac-
tice by the Tao practitioners in the Tai Chi movements.
Today, Tai Chi becomes a form of relaxation or de-
stressing exercise which is sometimes known as the
Moving Meditation. This is developed by a Taoist
monk during the thirteenth century in ancient China. The
principle of Tai Chi movement is based on the philoso-
phy of Lao Tzu (Tao Teh Ching) and the Chinese medi-
cine (TCM). In practicing Tai Chi, one performs certain
prescribed movements and one must constantly maintain
an upright and naturally balance posture (central equilib-
rium) during the process. Hence, movements which are
too extreme resulting in the out of balance body posture
are avoided in this exercise. By doing so, it is said that
Tai Chi would help to de-stress ones body. Practicing
Tai Chi regularly is said to be beneficial because the re-
laxation exercise also promotes steady breathing, regu-
lates blood circulation, and relieves tension by doing
gentle movement of the body. Meditation in motion is
also believed to help in refreshing and replenishing ones
energy to its normal level.

2.2. Meditation for Good Health

The tradition of resting in silence of an undistracted or
clear mind is thousands of years old. It is practiced by
P. K. C. Low ET AL.

87
seekers in all of the worlds spiritual traditions, as well
as in hospitals and even corporate boardrooms. Medita-
tion has been used in the East, but only became popular
in the West in the 1960 s, after the British pop group,
the Beatles, visited India. Scientists now agree that
meditation could raise the levels of important chemicals
in our bodies that help to fight off infections [5]. A very
simple meditation technique that one can adopt is to find
a stable and comfortable posture so that one can become
aware of ones body in the present moment. To do so,
one can sit on a chair with ones feet flat on the floor; or
sit cross legged on a meditation cushion. What matters
is that one has a sense of stability, comfort and ease. The
next step is to bring ones awareness into the present
moment and one becomes aware of ones environment
and the sounds around oneself. Take a few deep breaths
and relax and later on, one can then simply become
aware of how the breath breathes itself in its own
rhythms. This simple meditation exercise will help one
to be fully aware of the present moment and minimize
ones mind from wandering off to other thoughts or
things. The art of the meditation is to see the wandering
of the mind and to acknowledge it in the moment, and
then to return to ones breathing. In some ways, medita-
tion is a remembering or self-remembering and it is a
process of waking up, of being with the breath or the
body, and then to return to ones usual way when one is
not in the meditating mode. As we know that thinking,
planning, remembering and worrying often take up a lot
of times in our living, and it is very important that we
should understand the condition of our body so that we
can perform in an optimum way; it is with constant
practice of meditation and live more fully with lesser
thinking and worrying that we would be able to have a
healthy body and a healthy mind. [6]
In ancient China, scholars were very good in following
the nature, waking up when the rooster crowed. They sit
calmly meditating when the sun was rising; it was felt
that this gave them energy for the day. Meditative
awareness really reduces tension and heals (re-charges)
ones body; Meditation quiets or calm ones minds and
gently opens ones heart; it steadies ones spirit. [6] After
reading and writing for a long time, the scholars walked
into the open air admiring nature. When it is time for
them to sleep, they got rid of any worries by telling
themselves not to ponder on them; they cleared their
mind of any concerns. Such ways, attitude or conscious-
ness enabled them to live or experienced less stress, if
not, no stress at all.

2.3. TCM Based on Yellow Emperors Canons
of Internal Medicines

The Yellow Emperor said, Since ancient times, it is
considered that the existence of men has depended on the
interactions of Yin and Yang energies (Chi) in various
proportions and in many ways, and that human life is
based on Yin-Yang principles. As all things on earth and
in the universe communicate through the Yin-Yang prin-
ciples, therefore, human being can be considered as a
small universe because the human body has everything
that the universe has; all follows the Yin and Yang prin-
ciples [7].
Extending this concept further, the Emperor remarked,
Besides these Yin and Yang energies are the foundation
of life, the survival of men also depends on the five ele-
ments on earth (metal, wood, water, fire and earth), it is
the so called, Life depend on five. The five elements
further correspond to the three Yins (cold, dryness and
wetness) and the three Yangs (wind, fire and summer
heat). It is so called, Energies depend on the three. If
one cannot balance these elements and the Yin-Yang en-
ergies, and one violates the principles of preserving
health frequently, ones health will be hurt by the imbal-
ance of the Yin and Yang energies or the negative factors
and would contract disease For instance, based on the
concept that the human energy is connected with that of
the universe and that during the times when there is no
strong wind or heavy rain storm, the human body will be
fresh and cool in a calm environment. If one can keep
ones spirit quiet by refraining from the emotions of over
joy or excessive anger, one would have a peaceful mind
as clear as a blue sky. By this time, ones Yang energy is
substantial to guard the body against any negative factors
and one would attain a healthy body. Similarly, if one
has the ability of adapting oneself to the sequence and
variations of any environment such as the four seasons,
one would be able to preserve ones health in a good way.
Conversely, when one is in extreme anger, the Yang en-
ergy and the blood would rush upward to the head. If the
blood stagnates in the chest due to constant anger and
emotional upset; the blood circulation and the vital en-
ergy would be obstructed. This would result in an un-
healthy body and often one would contract sickness such
as high blood pressure or a heart attack.
As one can clearly see, Yin and Yang are always in
constant dynamic motion maintained by a continuous
adjustment of the relative levels of Yin and Yang. When
either Yin or Yang are out of balance, they naturally af-
fect each other and change their proportions to achieve a
new balance. And there are four possible ways in which
a Yin-Yang imbalance can occur as illustrated in the dia-
gram below:
1) Preponderance or Dominance of Yin
When Yin is excessive, it induces the decrease of Yang
and it also means that the Yin consumes Yang.
2) Preponderance of Yang
When Yang is excessive, it induces the decrease of Yin
and it also means that Yang consumes Yin.

88 P. K. C. Low ET AL.
3) Weaknesses of Yin
When Yin is weak, Yang will be seen in apparent ex-
cess. This apparent excess is only in relation to the defi-
cient quality of Yin.
4) Weaknesses of Yang
When Yang is weak, Yin will be seen in apparent ex-
cess. Similarly, this apparent excess is only in relation to
the deficient quality of Yang.
Therefore, in these illustrations, it is very critical to be
able to see the differences between the two states: the
preponderance of Yin and the weakness of Yang. This is
because on one state it is the truly excess Yin and on the
other state it is the weakness of Yang that Yin is seen as
apparent excess. Similarly, this differentiation applies
between the preponderance of Yang and the weakness of
Yin. [8]
It can be said that the theory of Yin and Yang is fun-
damental in Traditional Chinese Medicine (TCM) and
every physiological process and every symptom or sign
of a human body can be analyzed in the light of the
Yin-Yang theory. In other words, TCM sees illness as an
imbalance in the patients whole system. It tries to get to
the underlying root cause of a health problem. The aim is
to heal the persons mind, body and spirit rather than just
his or her sore throat or stomach ache. [11] Ultimately,

Figure 2. Preponderance of Yin and Yang.

Figure 3. Weaknesses of Yin and Yang.
every treatment modality is aimed at 1) improving Yang,
2) improving Yin, 3) reducing excess Yang and reducing
excess Yin, and understanding the application of the the-
ory of Yin-Yang theory is of great importance.

2.4. The Tao of Revitalization-The Chinese Sys-
tem of Self-Healing

To maintain, nourish, revitalize and prolong our lives, it
is said that we should spend our lives simply to fulfill
two basic needs namely:
1) Ingestion (eating and drinking)
2) Motion
a) Mind Movement (Thinking)
b) Body Movement (Breathing and internal
organs and external limbs movement.
These two basic conditions if not fully or properly sat-
isfied will affect ones life. For instance, if a person is
not given sufficient nutrients through eating and drinking,
his/her body will not be healthy and his/her life will
shorten. Similarly, if one does not exercise mentally or
physically, ones body will weaken. It was with these
considerations that the ancient Taoists created the Tao of
Revitalization which is the method of thinking, breathing,
and exercising for one to maintain good health. [9] It is
believed that a human body is a perfectly well-balanced,
self-regenerative and self-protecting organism and that
the body itself can respond to natural healing treatments
instead of drug intakes. The Tao of revitalization is a
series of mental and physical movements, almost effort-
less internal exercises that can energize the body's own
life-force to repel fatigue, illness and disease and prevent
them from reoccurring. Amongst all the internal exer-
cises recommended for good health, a very simple series
of physical and mental exercises that one can adopt are
the five animal exercises which are related to the five
elements.
Nowadays, we are living in a world full of stress for
the reason that everything we do has to be fast in an ur-
gent manner to meet daily targets and in most ways, we
are living and working in a competitive, rush, rush world.
Every day, most of us have little time to exercise. To
spend less yet quality time to relieve or reduce stress in
an efficient way, the authors here recommend these sim-
ple exercises based on the Traditional Chinese Medicine
(TCM) which had been practiced by the ancient Taoists a
long time ago; thus, although they are old, they indeed
bear modern relevance.

2.4.1. The Five Animal Exercises
Taoists designed the five exercises after five animals
whose movements were proven effective for the healing of
human beings and they were the dragon (fire), bear (earth),
eagle (metal), monkey (water) and tiger (wood). By imi-
P. K. C. Low ET AL.

89
tating their characteristic movements human beings can
alleviate the imbalanced functioning of their organs.

2.4.2. The Dragon Exercise (Fire Element)
The purpose of the Dragon Exercise is to instill the
characteristics of the dragon into the mind and body of
the practitioner. This exercise affects the mind by help-
ing to overcome feelings of depression, anger, hostility,
and all the anxieties brought on by being overwhelmed
by adverse circumstances, for the dragon, flying through
the heavens, is above all mundane concern.
One begins the exercise by standing still. Then, one
takes a few deep breaths while imagining as vividly as
possible that one is a dragon with glowing eyes, open
mouth with fangs, glistening emerald scales, curling tail,
paws splayed showing long claws. Then, raising one
foot, assume the pose and character of a dragon. While
imagining that ones hands are claws, hold one arm up
with claws down and hold the other arm down with
claws up. As this is not a formalized pose, a certain
degree of freedom of expression is allowed within the
confines of the image. Hold the pose as long as one
holds the image without straining. One repeats as many
times as one comfortably can. The most important as-
pect of this and all the other exercises is the union of
the body and the mind. If the image fades or the mind
wanders during the pose, one stops and begins again.
No benefit will be obtained unless the body and the
mind are in union.
Since the dragon represents the fire element, the physi-
cal effect of its exercise is to bring equilibrium to the heart,
blood vessels, and absorption in the small intestines.

2.4.3. The Bear Exercise (Earth Element)
The power and strength of the bear becomes evident
when it stands and walks on its hind legs. In this position,
the most prominent physical feature of the bear also be-
comes obvious-its stomach, which protrudes outward and
prevents the bear from walking straight.
One begins this exercise by standing still. One takes a
few deep breaths while visualizing oneself as a bear.
Then with legs stiff, stomach pushed out, arms sloping
out in front, walk slowly forward. As one does this, one
will feel the movement of ones abdomen and the stimu-
lation of the area of the spleen-pancreas. One continues
walking this way as long as the image remains fixed in
ones mind. One repeats as many times as is convenient.
The bear is associated with the earth element, and so this
exercise affects the enzyme production of the spleen-
pancreas and the functioning of the stomach muscle. This
exercise is therefore recommended for bad digestion,
hyper-and hypoglycemia, and diabetes.

2.4.4. The Eagle Exercise (Metal Element)
To the ancient Taoists the flying eagle represented the
spirit because of its god-like qualities - silence, serenity,
and invisibility. The eagle is also an accomplished hunter.
It soars effortlessly to great heights, and its sharp eyes
are alert to all details of the landscape below. The eagle
manifests its attributes of intelligence, alertness, and ease
when it hunts.
One begins the Eagle Exercise by standing still. One
takes a few deep breaths while imagining oneself as an
eagle. When the visualization is complete, one begins to
walk slowly with ones arms held out to the side in a
slant, or with ones hands gently clasped behind oneself.
As one walk, imagine one is an eagle, effortlessly float-
ing through the blue sky, untouchable, divine. Ones
body should be very relaxed, but ones mind and eyes
should be very alert, noticing everything without focusing
on anyone thing in particular. One continues the exercise
as long as the mind does not wander. If it does, one stops
and begins again. Though this exercise can be performed
anytime, anywhere, it is especially effective if done out-
doors, after the evening meal.
The eagle is associated with the metal element, so the
Eagle Exercise stimulates the lungs, skin, and the large
intestine.

2.4.5. The Monkey Exercise (Water Element)
To the ancient Taoists the monkey epitomized boundless
activity, curiosity, and free will. The monkey is con-
stantly active, whether on the ground, swinging in the
trees, or leaping playfully about, uninhibited by any cul-
tural conventions.
One begins by standing or sitting. One takes a few deep

Table 1. Shows the relationship between the exercises and the organs.
Elements Fire Earth Metal Water Wood
Exercise Dragon Bear Eagle Monkey Tiger
Organs
Heart Small Intestine
Triple Heater ( Endo-
crine) Heart Constrictor
(Blood Vessels)
Spleen-pancreas
Stomach Muscle
Lungs Large Intes-
tine Skin
Kidneys
Bladder
Bones
Liver Gallbladder
Nerves

P. K. C. Low ET AL.

90
breaths while imagining oneself as a monkey. When the
visualization is complete, one kicks off ones shoes,
throws off ones clothes, and begins to act like a little
monkey. One sits on the floor, crouches in a chair, leaps
about, bounces up and down, hangs upside down or by
one arm, whatever is physically possible to do without
strain or exertion. This exercise is completely free- style;
all the movements and actions should act out impulses
and whims as they occur to one. Monkeys also rub and
scratch themselves a great deal. One may do this also,
especially in the area of the kidneys. As the embodiment
of free will, the monkey inspires an exercise that is
free-style in the broadest sense. This exercise is best done
in private as the presence of others might be inhibiting.
The monkey is associated with the water element, so
the Monkey Exercise stimulates the functions of the kid-
neys and bladder. This exercise is recommended for
those feeling confined or restricted by circumstances in
which there is a lack of freedom. To the Taoist will
power resides in the kidneys. The Monkey Exercise is
also recommended for any problems involving the kid-
neys, bladder, and urinary tract.

2.4.6. The Tiger Exercise (Wood Element)
The tiger demonstrates its power in its ability to capture
something by leaping over it and mauling it. The tiger
pose is an imitation of this leaping over movement.
One begins by standing still. One takes a few deep
breaths while imagining oneself as a tiger. When the
visualization is complete, bend ones knees slightly and
rise up on ones toes while reaching up and out until
ones arms are straight. Keep the claws down, as if one
has reached over and out to grab something. One main-
tains this position as long as one can hold the image
without straining the body. One repeats as many times as
is comfortable.
Since tiger represents the wood element, this exercise
is recommended for healing and detoxifying the liver, to
sooth inflamed nerves, to balance gallbladder functions,
and to detoxify the brain and body cells.

3. Conclusion

The ancient concepts of Chi and Tao (Yin and Yang) are
the foundation of Traditional Chinese Medicine (TCM)
and accordingly, disease or sickness is caused by a dis-
ruptive flow of energy or the imbalance of the Yin and
Yang energies around our human bodies. Hence, TCM
provides a holistic treatment meaning the whole person
is being treated his or her body, mind and spirit. It is
believed that mind-body systems such as Meditation, Chi
Kung (Qigong) and Tai Chi exercises could send the
mind into an altered state to harness its healing power.
The Tao of revitalization aims to energize, train and
strengthen the internal organs so that they may become
strong and healthy. The exercises also make the circula-
tory system run smoothly without pressuring the heart in
speeding up the heart rate and that all the exercises are
carried out slowly according to ones ability.

4. References

[1] A. Rooney, Medicine, Stem Cells, Genes and Super-
beams, Harcourt Education, UK, 2006.
[2] M.-J . Cheng, Lao-Tzu, My Words Are Very Easy to
Understand: Lectures on the Tao Teh Ching, North At-
lantic Books, Richmond, California, 1981.
[3] Lao-tzu, Tao Te Ching, translated by S. Mitchell, 2010
http://acc6.its.brooklyn.cuny.edu/~phalsall/texts/taote-v3.
html
[4] P. K. C. Low and S.-L. Ang Taoism and Corporate So-
cial Responsibility, S. O. Idowu, Ed., Encyclopaedia of
Corporate Social Responsibility, Springer.(in press)
[5] C. Wallerstein, Need to Know Alternative Medicine,
Harcourt Education, UK, 2003.
[6] J . Kornfield, Meditation for Beginners, Bantam Books,
UK, 2004.
[7] L. S. Wu and Q. Wu, Yellow Emperors Canon of Internal
Medicine, Original Note Wang Bing (Tang Dynasty),
China Science & Technology Press, Beijing, 1997.
[8] G. Maciocia, The Foundation of Chinese Medicine: A
Comprehensive Text for Acupuncturists and Herbalist,
Churchill Livingston, London, 1989.
[9] S. Chang, The Chinese System of Self- Healing, Tao
Publishing, USA, 1989.

Title
Understanding traditional Chinese medicine from a
systems theory perspective
Author(s) Tsui, WK
Citation 2013, p. 1-20
Issue Date 2013
URL http://hdl.handle.net/10722/183666
Rights Creative Commons: Attribution 3.0 Hong Kong License

Understanding Traditional Chinese Medicine from a Systems Theory Perspective

Wai Kin Tsui

Department of Electrical & Electronic Engineering
University of Hong Kong

J une 2013

Abstract: The theory of the five elements, the yin-yang theory, the theory of qi and the
meridian theory are the core components of traditional Chinese medicine. This paper
attempts to integrate all of these theories under the framework of systems theory: the
flow of the dynamical system is interpreted as the flow of qi; the structure of the
dynamical system is understood as the structure of inter-promotion and inter-restraint
of the five subsystems; the state space of the dynamical system is decomposed into
two invariant sets which are similar to yin qi and yang qi; and the information system
resembles that which controls the circulation of qi and regulates yin-yang. The result
of integrating systems theory with traditional Chinese medicine is a more modern
interpretation and understanding of traditional Chinese medicine.

Keywords: Systems theory; Dynamical system; Eigenvalue; Basic theories of
traditional Chinese medicine.

Introduction
According to traditional Chinese medicine (TCM), the human body is an indivisible
whole. Through the interconnection of qi, blood, body fluid secretion, nerves,
meridians and the various zang-fu organs, this indivisible whole forms an extremely
complex system. According to systems theory, entities are composed of multiple
functional subsystems which interconnect and interact with each other to form whole
organic systems, each with a specific purpose. This paper attempts to use systems
theory to describe the physiology of the human body (Xutian et al., 2009; Dong and
Dai 2003; Wang et al., 2005) and to establish a scientific foundation for the basic
theories of TCM (Chen and Xu, 2003).

The Five-Element Theory
The human body is a dynamical system that can be described by a set of nonlinear
differential equations (Wagner, 1999):
( ) x f x x = , (1)
where f is a sufficiently smooth function and is an open subset of
n
R .
( ) ( ) ( )
1
, ,
n
x x t x t = is the state vector in . The state vector
0
x is called the
equilibrium point of the system if ( )
0
0 f x = .
TCM uses five basic substances, their abstract features and the relationships between
them to describe the organic structure of the human body. Each organ and tissue is
attributed to one of the five subsystems, as represented by the five zang organs (the
heart, liver, spleen, lungs and kidneys). These five abstract functional subsystems,
which are different from the anatomical organs, have characteristics similar to those
of the five basic material elements, i.e., wood, fire, earth, metal and water.
Practitioners of Chinese medicine believe that deficiency or excess in the material
make-up of a zang-fu organ affects the function of that organ, which in turn promotes
or restrains its material metabolism. According to the five-element theory, each
abstract functional subsystem promotes and/or restrains the functions of other abstract
functional subsystems: wood promotes fire, fire promotes earth, earth promotes metal,
metal promotes water and water promotes wood; meanwhile, water restrains fire, fire
restrains metal, metal restrains wood, wood restrains earth and earth restrains water,
thereby forming the five-element structure of the human body (Wiseman and Ellis,
1993) (Fig. 1).

Figure 1: A causal diagram representing the interconnection and interaction of the five
abstract functional subsystems of the human body.

First, let us consider the systems of inter-promotion and inter-restraint operating
within the five subsystems. For simplicity, let us suppose we have a system described
by five variables which represent five abstract functional subsystems (Zhang et al.,
2011; Zhuang et al., 2003). At the equilibrium point
0
x , the linearized system can be
written as

dy
Ay
dt
= ,
where A is the J acobian matrix evaluated at
0
x , ( )
0
A Df x = . The linear system has
the following structure:

1 1 1 1
2 2 2 2
3 3 3 3
4 4 4 4
5 5 5 5
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
y y
y y
d
y y
dt
y y
y y

,
where
k
and
k
are positive parameters, 1,2, ,5 k = .
For simplicity, we will only consider the system of inter-promotion operating within
the five subsystems. All values of 0
k
= and the system has the following structure:

1 1 1
2 2 2
3 3 3
4 4 4
5 5 5
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
y y
y y
d
y y
dt
y y
y y

=

. (2)
The matrix A has eigenvalues
2 5
, 2, 1,0,1, 2
k i
k
e k
= = , where
( )
15
1 2 3 4 5
= , ( ) ( ) Re 0 1,0,1
k
k > = and ( ) ( ) Re 0 2,2
k
k < = .
The general solution of the linearized system (2) can be written as

( ) ( ) ( )
( ) ( )
1 1
1 2 1 3 1
1
2 2
4 2 5 2
cos 2 5 sin 2 5
cos 4 5 sin 4 5 ,
k
i
k
i
t t
t
y t C e C e t k C e t k
t t
C e t k C e t k

= + +

+ +

where 1, 2, ,5 k = , ( )
1
cos 2 5 = , ( )
2
cos 4 5 = , ( )
1
sin 2 5 = , and
( )
2
sin 4 5 = .
The concept of qi is fundamental to TCM. Qi can be understood as the flow of energy
through the human body (Low and Ang, 2010). All life activities result from
movements and changes in qi. The flow of the dynamical system of the human body
is the flow of qi. Qi can be categorized as either yin qi or yang qi. In this paper, we
use mathematical operations to define yin qi and yang qi.

Yin-Yang Theory
The ancient Chinese concept of yin-yang describes the interrelationships between two
apparently opposite sets of things or phenomena in the universe. Things and
phenomena that are warm, active, excited, hyperactive and/or exhibiting an ascending
motion are categorised as yang. Cold, passive and inhibited things or phenomena, and
those showing a declining or descending motion, fall under the category of yin.

Invariant Manifolds
An equilibrium point
0
x is said to be hyperbolic if the J acobian matrix ( )
0
A Df x =
has no eigenvalues with zero real parts. If the equilibrium point is hyperbolic, the
solution of the linearized system can be written as a direct sum of the stable
eigenspace
s
E and the unstable eigenspace
u
E . The stable eigenspace
s
E is the
space spanned by the eigenvectors whose corresponding eigenvalues have negative
real parts, and the unstable eigenspace
u
E is the space spanned by the eigenvectors
whose corresponding eigenvalues have positive real parts.
We first analyze the local behavior of the dynamical system (1) near a hyperbolic
equilibrium point. If the J acobian matrix at the equilibrium point
0
x has exactly k
eigenvalues with positive real parts,
0
x is called a type-k equilibrium point. There
exists a neighborhood of
0
x and local stable and unstable manifolds
( ) ( )
{ } 0 0
:lim
s
loc t
t
W x x x x
=
and ( ) ( )
{ } 0 0
: lim
u
loc t
t
W x x x x
= ,
where ( )
t
x is the flow of the nonlinear system of (1). ( )
0
s
loc
W x and ( )
0
u
loc
W x are
two invariant sets which have the same dimensions as those of the eigenspaces
s
E
and
u
E

(Perko, 1996) .

Energy Functions
If
0
x is a type-k equilibrium point, there exists a nonsingular matrix P such that the
matrix A can be diagonalized to form the block diagonal matrix

1
0
0
A
P AP
A
+

=

,
where
k k
A R

+
has eigenvalues with positive real parts and
( ) ( ) n k n k
A R

has
eigenvalues with negative real parts. Let ( )
1 2
, y y y = , where
1
k
y R and
( )
2
n k
y R

.
The state vector
1
y P x
= satisfies the differential equation

( )
( )
1 1 1
2 2 2
0
0
A f y y y
d
A f y y y dt
+

= +

.
We define an energy function ( ) V y . If
0
x is a type-k equilibrium point, there exist
two positive definite matrices
1
k k
B R

,
( ) ( )
2
n k n k
B R

, and the energy function
( )
1 1 1 2 2 2
T T
V y y B y y B y = + .
The function ( ) V x is a nonnegative function of the nonlinear system (1).
If ( )
0
and 0
s
loc
x W x x , ( ) 0 V x <
; if ( )
0
and 0
u
loc
x W x x , ( ) 0 V x >
. The
energy function decreases in time along the orbit with state space in ( )
0
s
loc
W x and
increases in time along the orbit with state space in ( )
0
u
loc
W x .

Yin Yang of the Human Body
Consider the nonlinear dynamical system (1). The stable manifold ( )
0
s
loc
W x denotes
the set of points that flow forwards in time to the equilibrium point, and the unstable
manifold ( )
0
u
loc
W x denotes the set of points that flow backwards in time to the
equilibrium point. We take the concept of the stable manifold to define yin qi, and that
of the unstable manifold to define yang qi. These two invariant manifolds are used to
study the qualitative behavior of our bodies. Qi can be defined as vital energy or
life force.
Under normal conditions, the human body should maintain homeostasis. The local
stable and unstable manifolds represent two opposite and complementary flows of
energy: energy release and energy absorption. According to TCM, yin and yang
should remain in a state of dynamic balance (Seki et al., 2005). Diseases arise when
their balance is destroyed. The stable and unstable manifolds represent two aspects of
motion: contraction and expansion (Porkert, 1974). When these two aspects no longer
counterbalance each other, the result is a surfeit of yin or yang.
Evaporation and condensation are processes of change in the state of matter. The
human metabolism can be divided into two main categories: catabolism and
anabolism. Together, evaporation, condensation, catabolism and anabolism constitute
the processes of energy release and absorption (Adams, 2007; Goldberg et al., 1976;
Ni, 1995). Under normal conditions, the human body should maintain a dynamic
balance in its metabolism and energy flow. Anabolism uses energy to construct cells
and living tissues, while catabolism breaks down organic matter to release energy for
physiological activities. The organs promote material metabolism and produce the
substances necessary to maintain the bodys physical form, e.g., amino acids, proteins
and enzymes. These fundamental substances are also the material bases for the bodys
activities. Chinese medicine holds that the bodys physical forms pertain to yin, and
its activities or functions pertain to yang (Porkert, 1974).
The internal organs show different qualities associated with yin and yang. The five
zang organs are solid, and their main activities are the synthesis and storage of
essential substances and qi. The six fu organs (gallbladder, stomach, large intestine,
small intestine, bladder and triple burner) are hollow organs, chiefly responsible for
transforming, transporting, digesting and excreting bodily fluids. Chinese medicine
holds that the zang organs pertain to yin and the fu organs pertain to yang (Ni, 1995) .

Theory of Qi
In the context of TCM, system openness describes the interaction between the human
body and its external environment that takes place in the exchange of materials,
energy and information. As an open system, the human body must constantly
exchange substances, energy and information with nature to maintain its metabolism.
People obtain nutrition from nature through breathing and eating, which supply
materials and energy for consumption. Through a series of digestive processes,
nutrients are converted into the substances necessary for the human body to sustain its
life activities.
According to TCM, qi is both the essential substance of the human body and the vital
energy which drives the circulation of essential substances among the five subsystems.
This flow of energy is derived also from the essential substance for various
physiological processes. Qi is the flow of energy that is gained or released during
circulation.
Qi transformation refers to the various processes by which materials are generated
and transformed in the movement of qi (Maciocia, 1989). For example, food and
water are transformed into qi, blood and body fluids, which are in turn converted into
the essential substances of the five zang organs. This entails the metabolism and
transformation of substances, as well as the process of energy conversion. The energy
required for the conversion process is provided by yin qi and yang qi.
The movement of qi is known as qi activity, which can be divided into four basic
patterns: ascending, descending, exiting and entering. Energy conversion occurs
during the collision and interaction of yin qi and yang qi. The kinetic energy of yang
qi is reduced, resulting in downward movements, while the kinetic energy of yin qi
increases, resulting in upward movements. For this reason, it is also called yin
ascending and yang descending (Kaptchuk, 1991).
In the context of the human body, qi is the flow of energy gained or released during qi
transformation and qi activity. The processes involved in qi transformation and qi
activity must take place in a coordinated manner to maintain a dynamic balance of
energy. In other words, the coordination and orderly interaction of yin-yang in the
human body is important to sustain normal life activities.

Balance between Yin and Yang
Metabolism, homeostasis and adaptation are central to the functioning of all living
systems. Human life is affected by the structure of the body, eating habits and changes
in the external environment. The body transforms food and air into qi and blood, and
then converts qi and blood into substances necessary to maintain metabolism. The
movement of qi is dependent on energy transformations which drive the flow of qi
and blood through the whole body.
The external environment changes constantly. During the exchange of materials,
energy and information between the body and its surroundings, the body must
maintain a dynamic balance in its physical form and kinetic energy. Yin and yang play
important roles in qi transformation and qi activity. The human body must maintain a
dynamic balance between yin and yang. According to TCM, maintaining this balance
ensures that our bodies are healthy and disease-free(Ni, 1995).

Rhythmic Changes
Rhythm of the Five Zang-Organs
Human beings have a very close relationship with nature. The human body must adapt
to changes in the external environment. The Earths rotation and orbit produce,
respectively, a twenty-four-hour cycle of day and night, and a four-season cycle.
These cyclical changes directly affect the human bodys biological clock. In TCM,
each of the five zang organs has a corresponding season, with periods of heightened
activity and, conversely, rest.
The five elements also represent five phases or five activities in the cycle of the
seasons. According to TCM, the five zang organs of the human body liver, heart,
spleen, lungs and kidney correspond to spring, summer, long summer, autumn and
winter, respectively (Kendall, 2002). Due to their cyclical structure of inter-promotion
and inter-restraint, the five zang organs of the human body show a regular annual
rhythm correlating with the four seasons of a year. There are also mathematical
grounds for this phenomenon from the mathematical model. According to the
solutions of system (2),
1
2
sin
5
k
t

+

and
1
2
cos
5
k
t

+

1, ,5 k = have the
same phase shift, which equals one fifth of the period 2; and
2
4
sin
5
k
t

+

and
2
4
cos
5
k
t

+

1, ,5 k = have the same phase shift, which equals one fifth of the
period 4.

Rhythm of Yin and Yang
The waxing and waning of yin and yang describe the alternating changes in yin and
yang, wherein one progresses as the other declines, and vice versa. Yin and yang are
never static. In nature, yin and yang change periodically on a daily and yearly basis.
Correspondingly, the yin-yang balance in the human body changes according to
natural circadian rhythms (Ramsey et al., 2007; Moore-Ede, 1986; Habbal and
Al-J abri, 2009).
The human body is an open system. As materials and energy are exchanged with the
external environment, the human body must maintain a dynamic internal balance of
matter and energy. One of the most important functions of qi is to provide sufficient
power to mobilize both qi and blood to sustain the metabolic process.
During the day, the bodys physiological functions are more active. Greater energy
consumption is required to supply the demands of physical and intellectual labor
during the daytime. The catabolic process, which releases energy from the breakdown
of large molecules, is one illustration of the decline of yin as yang increases. However,
yin and yang must work in a complementary fashion. Consuming excessive amounts
of basic substances leads to a loss of qi, which is essential to the functioning of the
five zang organs. At night, the bodys physiological functions are comparatively
suppressed. During this time, the human body expends more energy on anabolism,
which constructs molecules from smaller units. This process is an illustration of the
decline of yang as yin increases. In the human body, yin and yang exhibit a regular
alternating cycle corresponding to the natural rhythm of day and night.

The Orderliness of the System of the Human Body
TCM emphasizes the correspondence between nature and humankind. In other words,
the human body and nature are understood to exhibit similar rhythmical changes. The
five-element system in the human body changes in a regular fashion according to the
earths rotation and orbit. Similarly, yin and yang undergo orderly changes in the
human body which correlate with changes in the external environment: yin waxes as
yang wanes, and vice versa.
As an open system, the human body must maintain a constant exchange of substances,
energy and information with its natural environment. To operate effectively, the body
must also have an information system to coordinate the interaction of the body with
its external environment, as well as the interaction between the bodys five abstract
functional subsystems and their various organs and tissues. By this means, an orderly
and self-organizing system is constructed to maintain the proper functioning of the
human body.
In the context of TCM, governing the exterior to infer the interior refers to the
process by which the operation of the zang-fu organs and the mechanisms of qi and
blood, yin and yang can be deduced from the external conditions of the body
(Kaptchuk, 1991), because the information available on the surface of the body is
understood to reflect physiological and pathological changes inside the body.
The bio-holographic law (Schjelderup, 1992) refers to a certain relationship of
correspondence between the component and the whole. The health of qi and blood is
reflected in local parts of the body. When a disease occurs inside our organs, related
information can be obtained from the eyes, ears, nose, tongue, pulse, acupuncture
points, etc. The site of disease can thus be inferred. There is a close relationship
between acupuncture points and the zang-fu organs (Vickers and Zollman, 1999).
Acupuncture points are the input and output terminals of messages relating to the
interior of the human body (Gao and Gao, 2008). When an acupuncture point is
stimulated, a signal is transmitted to its corresponding zang-fu organ(s). By this
means, we can obtain information about the health of particular organs, and adjust
their functioning. The signal also spreads to other acupuncture points.
Practitioners of traditional Chinese medicine believe that the meridians are
responsible for the circulation of qi and blood, thereby regulating yin and yang
(Wiseman and Ellis, 1993). The flow of qi and blood in the human body are observed
to follow the direction of the meridian lines. The operation of qi and blood follows
some of the basic principles of yin and yang. Their sequential operation should meet
the energy needs of zang-fu organs and tissues. At the same time, their proper
functioning ensures that yin and yang remain balanced within the human body despite
changes in the external environment.

Synergetics
The theory of synergetics was proposed by H. Haken in 1969 (Haken, 1997). Each
system under study is composed of many subsystems. Synergetics is the study of the
interaction and collaboration of these various subsystems. At a fundamental level, it
addresses the concepts of stability and instability, control parameters, order
parameters and the enslavement principle. The behavior of an open system is affected
by aspects of the external environment (control parameters). Driven by its order
parameters, the system moves from a disorderly to an orderly state, forming a new
structure and functionality and ultimately evolving into a self-organizing system.

The Processes of Evolution
Nature is not static. Living systems are constantly evolving. The ancient Chinese
philosopher Lao Tzu said: All things connote the yin and yang. The yin and yang
keep acting upon each other. And thus things keep changing and unifying themselves
(Lao, 1990). Yin and yang represent two aspects of motion: contraction and expansion.
When a system is in a state of balance between yin and yang, it is able to maintain
homeostasis. When homeostasis is lost, due to the failure of yin to restrict yang or
excessively rapid changes in the external environment, the system becomes unstable,
which damages its components and functioning (Arthur, 1990; Crespi, 2004). As a
result, the structure of the system is no longer conducive to its development. The
system moves from one equilibrium point to another equilibrium point and thus enters
a process of evolution by which its structure is created anew

(Corning, 1995). Figure 2
shows the system transition from one equilibrium point to another equilibrium point.
The yin-yang of the system also changes from yin into yang and yang into yin (also
called mutual transformation between yin and yang).

Figure 2.
1 2 3
, and x x x are equilibrium points. The system moves from one
equilibrium point to another equilibrium point through the mutual transformation
between yin and yang.

Order Parameter Principle
To establish a structure for a system is to establish relationships of mutual
coordination and cooperation between the subsystems. The order parameters
determine the behaviour of each subsystem, and thus the behavior of the whole
system.
The human body is composed of five abstract functional
subsystems, { }
1 2 5
, , , S S S S = , where S is the system of the human body, and
, 1,2, ,5
k
S k = refers to each of the five abstract functional subsystems.
Relationships of inter-promotion and inter-restraint operate between these five
subsystems. Each abstract functional subsystem is composed of its corresponding
zang-fu organs and tissues:

{ }
,1 ,2 ,
, , , , 1,2, ,5
k
k k k k n
S S S S k = = ,
where
,
, 1,2, ,
k j k
S j n = is the sub-subsystem of the k-th abstract functional
subsystem. The system of the human body is the result of long-term evolution. In the
course of evolution, the human body continuously improves its information systems.
Controlled by order parameters, the body regulates resource allocation and the
structure of each subsystem, including the zang-fu organs and tissues, so that the
human body becomes a self-organizing system.
Order parameters play a role in coordinating internal operations. TCM places a great
emphasis on the coordinated and orderly behavior of qi and blood. In physiological
terms, the five abstract functional subsystems are linked by interrelationships of
promotion and restraint. However, the zang-fu organs and tissues of the human body
are incapable of operating independently. All organs must be linked together as a
whole before they can carry out their proper functions. The qi of the human body is
also the qi of the five abstract functional subsystems, which are highly coordinated
and cooperate closely. The distribution and excretion of body fluids depend on the
movement of qi, which should circulate smoothly and in an orderly manner. When qi
and blood flow too fast, the human body is over-stimulated, and behaves in an
uncontrolled fashion; when supplies of qi and blood are inadequate, the body
collapses. The circulation of qi and blood must, therefore, be coordinated and proceed
in an orderly fashion. An intermediary is required to provide the information
necessary to coordinate their operation.
During the exchange of energy and matter between the human body and the external
environment, the body must maintain a dynamic balance in both its kinetic energy and
its physical form. The five abstract functional subsystems of the human body must be
synchronized with natures rhythms. Again, the body requires information to set its
biological clock so that various organs can conduct their activities in an orderly
manner

(Albegov, 2010).
Order parameters also play a role in coordinating the bodys yin and yang. The human
body carries out different physiological activities at different times. According to
TCM, its yin and yang should be synchronized with the yin and yang of nature. The
zang-fu organs exhibit different yin and yang properties, and require different
amounts of energy to operate. The zang-fu organs also need information to regulate
their operation and thereby ensure that yin and yang remain coordinated and orderly.
This in turn enables qi to flow smoothly and maintain a harmonious balance within
the body.
The spontaneous harmonization of yin and yang refers to the yin and yang of the
human body: under normal conditions, these two aspects maintain homeostasis
automatically; and in a pathological state, these two aspects have the ability to restore
their balance (Wang, 2012). Yin and yang must work complementarily, via mutual
interaction and restriction, to achieve a state of harmonious balance. The spontaneous
harmonization of yin and yang means that in the long-term evolutionary process, the
human body continuously optimizes its system structure and its information systems
to ensure that yin and yang maintain the best conditions for homeostasis and
self-recovery.

Conclusion and Perspectives
TCM can be characterized as holistic, addressing the essence of human life. TCM has
a unique theory and methodology. After thousands of years of evolution, TCM has
gradually formed a unique theoretical system. Systems theory also emphasizes the
holistic: linkages between internal elements of the system, and between the system
and its external environment. This paper uses systems theory to interpret the basic
theories of TCM.
TCM is based on the theory of the five elements, the yin-yang theory, the theory of qi
and the theory of the meridians. Each of these theories has its own specialty, but
integration under a single framework provides a more modern interpretation that
facilitates scientific understanding. We show in this paper that systems theory can
explain the structure of the human body through the theory of the five elements;
physiological activities and pathological changes through the theories of qi and
yin-yang; and information and control mechanisms through the theory of the
meridians.
With the rapid development of science and technology, Chinese medicine should
make use of modern science and technology to promote the modernization of
technology as well as its theoretical system. The basic theory of TCM comes from the
long-term observation and summarization of all experiences and practices. TCM
offers a thorough explanation of the physiological and pathological changes of the
human body in various capacities, and has very important scientific implications
nowadays. Systems theory makes the integration of TCM and Western medicine a
reality.

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Review article
Garlic: Health benets and actions
Chia-Wen Tsai
a
, Haw-Wen Chen
a
, Le-Yen Sheen
b
, Chong-Kuei Lii
a,
*
a
Department of Nutrition, China Medical University, Taichung, Taiwan
b
Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan
a r t i c l e i n f o
Article history:
Received 24 October 2011
Received in revised form
21 November 2011
Accepted 11 December 2011
Available online 31 January 2012
Keywords:
cancer
cardiovascular disease
drug metabolism
foodedrug interaction
garlic
a b s t r a c t
Recent years have seen an increasing emphasis on foods and food components in disease
prevention. Garlic (Allium sativum L.), one of the best-researched herbal remedies, holds
a unique position in history, traditionally employed to treat infection, colds, diabetes, heart
disease, and a host of other disorders. Clinically, it has been evaluated for lowering blood
pressure, cholesterol, and glucose concentration, as well as for the prevention of arterio-
sclerosis and cancer. Epidemiologically, garlic consumption inversely correlates with the
risk of oral, stomach, esophageal, colon, and prostate cancers. In addition, the biological
activities of garlic, including antibacterial, antithrombotic, antioxidant, immunomodula-
tory, and antidiabetic actions and modulation of drug metabolism, have been extensively
investigated. Here, we briey summarize the recent ndings on garlic and its sulfur-
containing compounds in preventing cardiovascular diseases and cancer, along with its
modulation of drug-metabolizing enzymes and membrane transporter activities. Finally,
garlic safety and drug interaction are discussed.
Copyright 2012, China Medical University. Published by Elsevier Taiwan LLC. All rights
reserved.
1. Chemicals and bioactive components of
garlic
The unique avor and health-promoting functions of garlic
are generally attributed to its rich content of sulfur-containing
compounds, i.e., alliin, g-glutamylcysteine, and their deriva-
tives. Processing a fresh and intact garlic bulb by crushing,
grinding, or cutting induces the release of the vacuolar
enzyme alliinase, which very quickly catalyzes alliin to allicin
[1,2]. Allicin is, however, a very unstable compound, soon
rearranged and transformed into numerous lipid-soluble
sulfur-containing byproducts, mostly diallyl disulde (DADS)
but also diallyl sulde (DAS), diallyl trisulde (DATS),
allylmethyl trisulde, and diallyl tetrasulde [1]. These
compounds emit strong odors and are kept in garlic oil. Under
appropriate conditions, allicin can be transformed into other
lipid-soluble products such as ajoene and vinyldithiin. Ajoene
is identied as a principal product in garlic extract prepared by
using ether as a solvent [3].
In contrast to the processes stated above, alternative
pathways occur incase of different means of garlic storage. An
aging process caused by immersing intact or sliced raw garlic
in alcohol or vinegar for several months results in sulfur-
containing compounds in this aged product dramatically
different from those found in garlic oil. This aging process
is supposed to cause considerable loss of allicin. Meanwhile,
with the action of g-glutamyltranspeptidase, the other
sulfur-containing precursor g-glutamylcysteine is trans-
formed into water-soluble S-allylcysteine (SAC) and subse-
quent metabolites, including S-allylmercaptocysteine (SAMC)
* Corresponding author. Department of Nutrition, China Medical University, Taichung, Taiwan.
E-mail address: cklii@mail.cmu.edu.tw (C.-K. Lii).
Available online at www.sciencedirect.com
www. bi omed- onl i ne. com
B i o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9
2211-8020/$ e see front matter Copyright 2012, China Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
doi:10.1016/j.biomed.2011.12.002
and S-methylcysteine. Unlike the oily sulfur compounds,
these water-soluble compounds are odorless but have a more
delicate and less characteristic avor [4].
In addition to sulfur-containing compounds as stated
above, garlic is also rich in trace elements. In raw garlic, the
amounts of zinc, manganese, copper, selenium, and iodine in
100 g fresh weight of garlic are 556.1, 446.9, 143.3, 5.5 and
2.5 mg, respectively [5]. The protein content of raw garlic
ranges from 2.6% to 3.0%, depending on the variety of garlic.
The average content of free amino acids is 2.13%. Concentra-
tions of dietary ber and total tocopherols in raw garlic are
2310 and 103.1 mg/100 g fresh weight, respectively. Ascorbic
and total polyphenols levels are 73.6 and 1.9 mg in 100 g dry
weight [6]. Over 70 fatty acids have been determined, with
linoleic (46e53%), palmitic (20e23%), oleic (4e13%), and a-
linolenic (3e7%) acids being most abundant, accounting for
80% of the total lipids [7].
2. Garlic preparations and supplements
Because of the complex chemistry of garlic, variations in
processing methods can yield quite different preparations.
Raw garlic homogenate, the major preparation of garlic, is the
most common form of garlic consumed, and allicin the main
compound present in fresh raw garlic homogenate. There are
currently many garlic supplements on the market, garlic oil,
powder, and aged extract being the most popular.
Garlic oil is mostly obtained by steam distillation, with
a yield around 2.5e3.0 g/kg fresh garlic. In garlic oil, DAS,
DADS, and DATS, differing in their number of sulfur atoms,
and allylmethyl sulde are the four most abundant volatile
allyl suldes [8]. Garlic power is generated from garlic cloves
that have beendehydrated and pulverized into powder. Due to
deactivation of alliinase by heat during dehydration, the
major active constituents of garlic powder are alliin and
a small amount of oil-soluble sulfur compounds.
To overcome the strong and irritant odor and the possible
side effects of raw garlic and garlic oil, including growth
retardation and destruction of gut microora, an aging
process has been applied to garlic. Aged garlic is prepared by
soaking whole or sliced garlic cloves in alcohol or vinegar
solution for 6e20 months, which removes the several irritant
sulfur-containing compounds and also stabilizes some
unstable compounds such as allicin [4,9]. The water-soluble
compounds SAC and SAMC are the most abundant sulfur-
containing components, and trace amounts of oil-soluble
allyl suldes exist in aged garlic. In contrast to odoriferous
garlic oil and rawgarlic, garlic powder and odorless aged garlic
product are currently the most popular garlic supplements on
the market.
3. Garlic and cardiovascular disorders
Cardiovascular disease is a common human chronic disease,
and it is the leading cause of morbidity and mortality in the
USA [10]. The etiology of cardiovascular disorders is multi-
factorial, with, for example, hypercholesterolemia, hyper-
tension, diabetes mellitus, heredity, hyperhomocysteinemia,
increase in oxidative damage, and smoking as well-
demonstrated risk factors [11].
Due to the accompanying inammation in the plaque,
cardiovascular disorders are regarded as chronic
inammation-related diseases [11]. The increased production
and release of inammatory mediators, such as reactive
oxygen species (ROSs), tumor necrosis factor alpha (TNF-a),
interleukin 6 (IL-6), arachidonic acid metabolites, and nitric
oxide is noted in the atherosclerotic lesion [12]. This results in
a greater expression of adhesion molecules, including P-
selectin, E-selectin, vascular cell adhesion molecule (VCAM),
intercellular adhesion molecule (ICAM), and monocyte
chemotactic protein-1 on the cell surfaces of monocytes,
leukocytes and vascular endothelial cells, which accelerates
the adherence of monocytes and leukocytes to the vascular
endothelium and their subsequent transmigration into the
subendothelial space.
Within the intima, activated macrophages release ROSs,
scavenge oxidized low-density lipoprotein (oxLDL), become
foam cells, and lead to the development of the fatty streak in
the early stage of atherosclerosis [13,14]. This explains why
phytochemicals with anti-chronic inammation, hypolipi-
demic, and antioxidative properties are thought to be capable
of decreasing the incidence of atherosclerosis.
Garlic has been regarded as a potent antiatherogenic food
[15]. Its lowering of blood cholesterol is believed largely due to
a reduction in LDL-cholesterol [16,17], which may be due to
inhibition of hepatic hydroxymethylglutaryl-CoA reductase
activity by alliin and allicin [18]. Over the past decade, several
intervention studies and systemic meta-analytic reviews have
investigated the effectiveness and properties of garlic in pre-
venting cardiovascular disease (Table 1).
A double-blind placebo-controlled randomized study
including 51 patients with coronary conditions indicated that
12 months treatment with 300 mg/d garlic powder signi-
cantly decreased the total cholesterol and LDL-cholesterol
levels [19]. A reduction of 32.9 and 27.3 mg/dL in LDL-
cholesterol resulting from the garlic was observed in men
and women, respectively.
A similar reduction in total cholesterol and LDL-
cholesterol, along with an increase in high-density lipopro-
tein-cholesterol, were also reported in hypercholesterolemic
adults who were administered 10 g/d garlic extract for 4
months, 5 g/d raw garlic for 6 weeks, or 600 mg/d garlic
powder for 12 weeks [20e22]. Oily macerate of garlic (1620 mg/
day for 30 days) was found to signicantly lower the levels of
total cholesterol, LDL-cholesterol, and triacylglycerides in 70
hypertensive adults [23]. However, Burggraaf and colleagues
reported that 12 weeks of 2.1 g/d garlic powder administration
did not change the lipid proles in overweight subjects with
normal blood lipid levels [24].
A meta-analysis including 29 trials recently revealed that
garlic supplementation markedly reduced blood total choles-
terol levels (e0.19 mmol/L; 95% CI e0.33 to e0.06 mmol/L)
and triacylglyceride levels (e0.11 mmol/L; 95% CI e0.19
to e0.06 mmol/L), but exhibited no signicant effect on LDL- or
high-density lipoprotein-cholesterol [25]. A similar reduction
in total blood cholesterol and triglycerides has been reported
in systemic reviews [26,27]. Inconsistent clinical evidence
warrants more study before reaching convincing conclusions.
Bi o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 18
Garlic is reported to prevent cardiovascular disease by
multiple effects, one of which is inhibition of platelet aggre-
gation. A single intravenous dose of aqueous extracts of garlic
(10e100 mg/kg) dose-dependently inhibited blood throm-
boxane B
2
concentration in rabbits [28]. Maximuminhibition of
thromboxane B
2
occurred 0.5 hours after injection and lasted
until 6 hours afterwards. In another study, oral administration
of aqueous extract of fresh garlic inhibited cyclooxygenase
(COX) activity in rabbit platelets, resulting in a suppression of
thromboxane formation and blood aggregation [29]. Similarly,
in eight males (aged 40e50 years), the consumption of one
crushed clove of garlic daily for 16 weeks resulted in an 80%
reduction in serum thromboxane B
2
levels [30].
Allicin and allicin-derived thiosulnates are recognized as
major compounds responsible for the antithrombotic activity
of garlic [31]. Besides an inhibition of COX activity, other
possible mechanisms for garlics inhibition of platelet aggre-
gation include suppression of intraplatelet Ca
2
mobilization,
an increase in cyclic AMP and cyclic GMP levels, an increase in
platelet-derived nitric oxide production, and a reduction in
platelet binding to brinogen [32].
Garlics protection against cardiovascular diseases has
been partly attributed to its potent anti-inammatory activity
[33]. The ethyl acetate-soluble fraction of garlic is proven to be
effective in inhibiting nuclear factor kB (NF-kB) activation, as
well as expression of COX-2 and inducible nitric oxide syn-
thase in IL-3-dependent murine pro-B-cells Ba/F3 through the
Toll-like receptor-dependent pathway [34]. Thiacremonon,
a novel organosulfur compound of garlic, inhibits 12-O-tetra-
decanoylphorbol-13-acetate-induced ear edema in ICR mice,
and carrageenan- and Mycobacterium butyricum-induced
inammatory and arthritic responses in the paws of Sprague-
Dawley rats [35]. Garlic oil reportedly suppresses 1-chloro-2,
4-dinitrobenzene-induced contact hypersensitivity as deter-
mined by ear swelling [36].
After 30 days of administration of 600 mg/kg garlic
powder, an increase in interferon-g and a decrease in IL-4 in
phytohemagglutinin-activated splenocytes were noted,
suggestingthat garlic treatment mayfavor a T-helper type2 cell
or humoral immune response [37]. 1,2-Vinyldithiin was
recently reported to signicantly suppress IL-6 and monocyte
chemoattractant protein-1 secretion by macrophage-secreted
factors stimulated human preadipocytes isolated from the
subcutaneous adipose tissue of nonobese young women [38].
DAS has been reported to prevent COX-2 upregulation and
prostaglandin E
2
(PGE
2
) secretion in primary human synovial
broblasts and articular chondrocytes induced by IL-1b and
monosodium urate crystal, and ameliorates crystal-induced
synovitis, potentially throughthe NF-kBsignaling pathway [39].
The presence of proinammatory cytokines initiates
numerous physiological changes in vessel walls, such as
enhanced adhesion of leukocytes to the endothelium.
A recent in vitro study indicated that the chloroform extract of
aged black garlic attenuated TNF-a-induced VCAM-1 expres-
sion via an NF-kB-dependent pathway in human umbilical
vein endothelial cells (HUVEC), hence decreasing the adhe-
siveness of monocytes on endothelial cells [40]. In primary
human coronary artery endothelial cells, aqueous extract of
garlic (0.25e4.0 mg/mL) dose-dependently curbs ICAM-1 and
VCAM-1 expression induced by IL-1a [41]. When stimulated by
oxLDL, DADS and DATS suppress VCAM-1 and E-selectin
expression in HUVECand the subsequent adhesionof HL-60 to
endothelial cells [42].
Taken together, although solid clinical evidence that
garlics effect in protecting blood vessels can be attributed to
its anti-inammatory properties is lacking, the potent anti-
inammatory action of garlic and its sulfur-containing
compounds obtained from in vitro and animal studies
supports the potential value of garlic in preventing
atherogenesis.
Evidence indicates that garlic also acts to maintain
vascular tone and cardiac function. Experiments on labora-
tory animals and investigations of humans has proved that
diets supplemented with garlic can restore endothelial func-
tions. Allicin is believed to be the active component of
raw garlic protecting coronary endothelial function and
Table 1 e Effect of garlic supplementation on human cardiovascular disorders.
Preparation Subjects/dose Effect Reference
Garlic powder 51 patients with CVD, 300 mg/d, 12 mo Y total cholesterol and LDL-C [19]
42 mildly hypercholesterolemic men,
600 mg/d, 12 wk
Y total cholesterol and LDL-C [22]
[ HDL-C
90 normolipidemic and overweight
adults, 2.1 g/d, 12 wk
No changes in blood total cholesterol,
LDL-C, and TG
[24]
Raw garlic 30 hypercholesterolemic adults, 5 g/d, 42 d Y total cholesterol and TG [21]
[ HDL-C
Garlic extract 23 hypercholesterolemic adults (13 with
hypertension), approximately 10 g/d, 4 mo
Y total cholesterol, LDL-C, and TG [165]
[ HDL
Y SBP and DBP
Aged garlic extract 11 healthy adults, methionine- induced
hyperhomocysteinemia, 4 mL/d, 6 wk
[ NO and endothelium-derived
hyperpolarizing factor
[166]
65 patients with intermediate CVD risk,
250 mg/d co-administered with B vitamins
(B
12
, B
6
, and folic acid) and L-arginine, 12 mo
Y total cholesterol, LDL-C, and homocysteine [140]
[ HDL
Garlic oil 20 hypertensive patients, 250 mg/d, 2 mo Y SBP, DBP, and oxLDL [13]
Oil-macerated garlic 70 hypertensive adults, 1620 mg/d, 30 d Y total cholesterol, LDL-C, and TG [23]
CVD cardiovascular disease; DBP diastolic blood pressure; HDL-C high-density lipoprotein-cholesterol; LDL-C low-density lipoprotein-
cholesterol; NO nitric oxide; oxLDL oxidized LDL; SBP systolic blood pressure; TG triglycerides.
B i o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 19
vasoreactivity in pulmonary hypertensive rats [43]. Enhance-
ment of nitric oxide synthase activity and greater nitric oxide
production partly explained this hypotensive action.
Our recent work demonstrated that DADS and DATS
protect the activity and protein expression of endothelial nitric
oxide synthase in response to an oxLDL insult to the endo-
thelial cells [44]; this is partly attributable to the mediation of
phosphatidylinositol 3-kinase/protein kinase B signaling and
prevention of eNOS degradation caused by DADS and DATS
[44]. SAC supplementation reduces the incidence of stroke in
stroke-prone spontaneously hypertensive rats [45], and lowers
mortality and infarct size in a rat model of acute myocardial
infarction induced by coronary artery ligation [46].
In an animal experiment inducing diabetes by streptozo-
tocin, rats were orally administered 0e100 mg/kg/d garlic oil
for consecutive 16 days; streptozotocin-induced cardiac
contractile dysfunction and apoptosis were markedly
improved by the garlic oil [47]. In a hypercholesterolemic
animal experiment, rats were fed a 1.0% garlic- and 0.5%
turmeric-supplemented diet for 10 weeks. Enhanced vaso-
relaxation in the aortic ring response to adenosine, acetyl-
choline, and isoproterenol, along with attenuation of the
contractile response to 5-hydroxytryptamine, was seen in
animals given the garlic- and turmeric-supplemented diet,
thus lowering their blood pressure [48].
In a randomized, placebo-controlled, cross-over design
involving 15 patients with angiographically proven coronary
artery disease, brachial artery ow-mediated endothelium-
dependent dilation was improved by aged garlic extract [49].
Similarly to aged garlic extract, garlic oil in a dose of 250 mg/d
for 2 months demonstrably improved both systolic and dia-
stolic blood pressure in 20 hypertensive patients [13].
4. Garlic and cancer
The past fewdecades have seenmany epidemiological studies
on the correlation between garlic consumption and incidence
of cancer, from which an inverse relationship has emerged.
Setiawan et al observed a negative doseeresponse relation-
ship between the monthly intake of garlic and the risk of
stomach cancer in Shanghai and Qingdao, China [50]. A recent
study found an odds ratios among individuals with a high
versus a low intake of garlic and onions that correlated with
a starkly reduced risk of colorectal adenoma [51]. In persons
who consume a high proportion of garlic, a decreased
susceptibility to stomach and colon cancers has also been
reported [52].
Based on the US Food and Drug Administrations evidence-
based review system for scientically evaluating the risk of
diverse types of cancer, 19 human studies revealed garlics
antitumorigenic potential in stomach, colon, rectal, breast,
lung, and endometrial cancers. Very limited evidence
supports a relation between garlic consumption and reduced
risk of colon, prostate, esophageal, larynx, oral, ovary, or renal
cell cancer [53].
Several human intervention studies have plotted garlics
anticarcinogenic traits. A preliminary double-blind, random-
ized clinical trial using high-dose aged garlic extract (2.4 mL/d)
as the active treatment and low-dose aged garlic extract
(0.16 mL/d) as the control was performed involving 51 patients
with colorectal adenomas/precancerous lesions of the large
bowel [54]. After 12 months of treatment, 37 patients (19 in the
active and 18 in control group) completed the study, the size
and number of colon adenomas in the high-dose group being
signicantly lower ( p 0.04).
An earlier double-blind intervention study of 5033 subjects
(2526 in the intervention and 2507 in the control group) was
performed in China. A dose of 200 mg/d DATS in combination
with 100 mg/d selenium was taken by the intervention group
each month for 3 years. The results showed that DATS offered
protectionagainst gastric cancer for males [55]. Inthis study, it
is interesting to note that no such protection occurred in
females.
Numerous animal model studies found in the literature
were carried out using either garlic extract or individual garlic-
derived compounds. The development of aatoxin B1- or
diethylnitrosamine-induced liver cancer in rats was limited
by fresh garlic [56] and garlic oil [57]; the latter also protected
against ferric nitrilotriacetate-induced kidney cancer growth
in rats [58]. DADS suppressed 7,12-dimethylbenzo[a]anthra-
cene (DMBA)-induced rat mammary tumor [59]. DAS and
DATS protected against DMBA-, phorbol ester-, and benzo[a]
pyrene-induced skin tumorigenesis in mice [60e63]; DATS
also inhibited the growth of PC-3 human prostate cancer
xenografts in male nude mice [64]. Similarly, ajoene signi-
cantly inhibited B16/BL6 melanoma growth and metastasis to
the lung in C57BL/L mice [65]. Aside from oil-soluble organo-
sulfur compounds, water-soluble SAC inhibited the growth
and malignant progression of highly metastatic human
nonsmall-cell lung carcinoma in nude mice [66].
Although the precise mechanism of garlics anticancer
efcacy is still not clear, molecular actionsuch as regulationof
cell proliferation, increase in tumor apoptosis, blocking of the
cell cycle, inhibition of carcinogen activation, increase in
phase II drug-metabolizing enzymes, enhanced antioxidation
capacity, change in proteasome-dependent protein degrada-
tion, and modulation of immune response have been
proposed and extensively probed in recent years (Table 2).
In many cancer cells, garlic organosulfur compounds
display potential for suppressing the growthof cancer cells and
producing cell cycle arrest. DAS increases the accumulation of
sub-G1 DNA and the concomitant accumulation of cells in the
G2/M phase in a dose-dependent manner in human anaplastic
thyroid carcinoma cells [67], as well as in human colon cancer
cells [68]. DAS, DADS, and DATS further exhibit differential
effects in terms of lowering cyclin-dependent kinase-Cdk7 and
Table 2 eMechanisms underlying the anti-cancer actions
of garlic.
1. Induces apoptosis/arrests the cell cycle
2. Blocks invasion/metastasis
3. Suppresses cell proliferation
4. Inhibits activation of carcinogen
5. Enhances antioxidation
6. Decreases histone deacetylase activity
7. Interrupts tubulin polymerization
8. Changes proteasome activity
Bi o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 20
raising cyclin B1 protein levels in J5 human liver tumor cells,
thus arresting the cells in the G2/M phase [69]. Among those
lipid-soluble allyl suldes, which differ in their number of
sulfur atoms, DATS revealed a better growth inhibition of
human melanoma A375 cells and skin basal cell carcinoma
cells than was seen with DADS and DAS [70]. The induction of
apoptosis and cell cycle arrest by garlic allyl suldes have also
beenreported indifferent types of cancer cell, e.g., human lung
adenocarcinoma [71], glioblastoma [72], prostate cancer [73,74],
neuroblastoma [75], gastric cancer [76], bladder cancer [77],
colon cancer [78], and mammary cancer [79].
Garlic organosulfur compounds resulting in cell cycle
arrest and apoptosis can be linked to the modulation of
several key elements in cellular signal transduction. It has
been demonstrated that DATS-induced apoptosis of PC3
human prostate cancer cells involves c-Jun N-terminal kinase
(JNK) and extracellular-signal-regulated kinase-mediated
phosphorylation of Bcl-2 [80]. Inactivation of the Akt signaling
pathway also likely plays a role in DATS-induced mitochon-
drial translocation of Bad and caspase-mediated apoptosis in
PC3 and DU145 human prostate cancer cells [81]. Likewise, the
DATS arrest of DU145 cells in G2/M phase is effected by
hyperphosphorylation of Cdc25C [82] and delayed cdk1
translocation into the nucleus [83], as well as by oxidative
modication of beta-tubulin in human colon cancer cells,
which impedes the polymerization of tubulin [84].
A similar interruption of tubulin polymerization has been
reported by treating SW480 and NIH3T3 broblasts with
SAMC; this subsequently arrests the cells in mitosis and trig-
gers the JNK1 and caspase-3 signaling pathways, leading to
apoptosis [85]. In B16F-10 melanoma cells, DADS-induced
apoptosis is attributed to the mitochondrion-dependent
pathway by upregulating p53 and caspase-3 while down-
regulating NF-kB-mediated Bcl-2 activation [86]. Recently,
both the extrinsic and intrinsic death pathways have been
shown to be involved in allicin induction of apoptosis in
gastric SGC-7901 cancer cells [87].
Garlic organosulfur compounds may also act epigenetically
and exert anticarcinogenic activity. Histone acetylation
notably increases in colonocytes isolated from DADS-treated
rats and also in erythroleukemia cells from SAMC-treated
mice, suggesting that histone deacetylase is the target of
garlic allyl compounds [88, 89]. In addition to DADS, other
garlic organosulfur compounds have been tested; allyl
mercaptan, a metabolite of DADS, has been shownto exert the
most potent inhibitory effect on histone acetylase in assays
with HeLa nuclear extracts, lysates from human colon cancer
cells, or puried human histone deacetylase-8 [78,90]. Allyl
mercaptan inhibition of histone deacetylase activity results
in increasing histone acetylation and Sp3 transcription factor
binding to the p21WAF1 gene promoter region, elevating p21
expression and producing cell cycle arrest in HT29 colon
cancer cells [90]. Enzyme kinetics assays further reveal an
inhibition of allyl mercatpan on histone deacetylase via
a competitive mechanism (Ki 24 mM) [90].
Evidence indicates that tumor invasion and metastasis are
suppressed in the presence of garlic and its organosulfur
compounds. DATS administration retards the growth of PC-3
human prostate cancer xenograft cells in athymic mice [64],
and prevents progression to invasive carcinoma and lung
metastasis in transgenic adenocarcinoma of mouse prostate
(TRAMP) cells [91]. In in vitro experiments, the DADS-
suppressed invasion of human prostate cancer LNCaP cells
was attributed to an inhibition of matrix metalloproteinase-2
(MMP-2) and MMP-9 activity and to a tightening of the tight
junctions [92].
Garlics suppression of tumor invasion may also be attrib-
uted to its action on E-cadherin expression. SAC and SAMC
restoration of E-cadherin expression suppresses the prolifer-
ation and invasion of prostate cancer cells [93]. This increase
in E-cadherin expression and inhibition of cell proliferation
are also noted in oral squamous cancer CAL 27 cells in the
presence of SAC [94]. The invasive activities of SW480 and
SW620 colorectal cancer cells are inhibited by aged garlic
extract, whereas aged garlic extract has no effect on the
invasion of HT29 cells, suggesting that the anti-invasive
action of aged garlic extract is cancer cell-dependent [95]. In
the presence of ajoene, human leukemia HL60 cells were
arrested in the G2/M phase; both trypsin- and chymotrypsin-
like proteasome catalytic activities were inhibited [96].
Taken together, most animal and cell studies suggest that
garlic is a potent chemopreventive agent for several types of
cancer, acting by inhibiting cell proliferation, arresting the cell
cycle, inducing cell apoptosis, and blocking invasion and
metastasis.
5. Garlic and the detoxication system
The cancer-chemopreventive effect of garlic organosulfur
compounds is believed to be associated with the modulation
of carcinogen metabolism, including effects on phase I and II
detoxication enzymes. Phase I enzymes, mainly cytochrome
P450 (CYP), detoxify a variety of endogenous and exogenous
chemicals and activate many carcinogens [97]. Phase II
enzymes catalyze the conjugation of phase I metabolites to
various water-soluble molecules, such as glutathione (GSH),
glucuronic acid, or sulfate, accelerating the metabolite
excretion rate. The efcacy of DAS, DADS, and DATS in the
transcriptional regulation of phase I and II detoxication
enzyme expression positively correlates with the suppression
of aatoxin B1- and benzo[a]pyrene-induced liver and forest-
omach neoplastic formation in mice and rats [98,99].
Decreased 7,12-dimethylbenzo[a] anthracene-induced
DNA adduct formation in rat mammary tissue by DADS [59],
protection against benzo[a]pyrene-induced skin tumorigen-
esis and micronucleated reticulocyte formation in mice by
DAS [63], and the suppression of aatoxin B1-induced DNA
breaks by allicin, DAS, DADS, and SAC in HepG2 cells [100] can
also be explained by their effectiveness in modulating
metabolism of carcinogens.
Among the CYP isozymes, a decrease in CYP2E1 activity
and protein levels has been reported in rats fed a diet con-
taining 5%garlic powder [101]. This downregulation of CYP2E1
by garlic suppresses the formation in rats of hepatic pre-
neoplasia induced by diethylnitrosamine [102]. The formation
of lycidamide, an active metabolite of acrylamide, in rat liver
tissues falls because of the inhibition of CYP2E1 by DAS [103].
In addition to DAS, a reduction in the activity and expression
of CYP2E1 results from garlic oil, DADS, and allyl methyl
B i o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 21
sulde [104,105]. In contrast to downregulation of CYP2E1, the
activities of isozymes CYP1A1, CYP1A2, CYB2B1, and CYP3A2,
as well as their protein and mRNA levels, are upregulated by
garlic organsosulfur compounds. Dosing rats with 200 mg/kg
DAS and allyl methyl sulde raises CYP1A1, CYP1A2, and
CYP3A2 protein levels in a time-dependent manner, a rise
being noted 24 hours after treatment [105]. A dose-dependent
increase in rat liver CYP1A1, CYP2B1, and CYP3A1 activities
and gene transcription is also caused by garlic oil (30e200 mg/
kg body weight), probably from the combined effect of the
three major allyl suldes, DAS, DADS, and DATS, in the garlic
oil [104,106,107].
Besides acting at the stage of gene transcription, the
constituents of garlic may bind to CYP and change its enzyme
activity. Using human liver microsomes, the activity of
CYP2C9, CYP2C19, CYP3A4, CYP3A5, and CYP3A7, but not
CYP2D6, is inhibited by incubation with garlic oil or extracts of
fresh garlic, garlic powder, or aged garlic [108]. In the case of
CYP2E1, diallyl sulfone and diallyl sulfoxide, metabolites of
DAS, act as suicide substrates [109]; this inhibited CYP2E1
activity explains partly the action of DAS in attenuating
acetaminophen-, carbon tetrachloride- and ischemic-
reperfusion-induced toxicity in rat livers [110,111].
Phase II detoxication enzymes are known to play a key
role in accelerating the excretion rate of numerous xenobi-
otics. Induction of phase II enzymes such as glutathione
S-transferase (GST), epoxide hydrolase (EH), UDP-glucuronyl
transferase (UGT), sulfotransferase, and NAD(P)H quinone
oxidoreductase 1 (NQO1) is considered to be a crucial mech-
anism protecting organisms against chemical insults. It is
thus reasonable to speculate that the induction potency of
phytocompounds on phase II enzymes is associated with their
efcacy in chemoprevention [112,113].
GST is among the most important phase II enzymes, its
vital role in cancer prevention being supported by the nding
that the incidence of 7,12-dimethylbenzanthracene-induced
skin cancer was signicantly elevated in the pi form of GST
(GSTP)-null mice [114]. The increase in GST activity caused by
garlic organosulfur compounds, including allyl methyl trisul-
de, allyl methyl disulde, DATS and DAS, strongly correlates
with the inhibition of benzo[a]pyrene-induced forestomach
neoplasia [115]. The effect of DAS, DADS, and DATS on the
transcriptional regulation of GST enzyme expression is also
positively correlated with their suppression of aatoxin B1-
and benzo[a]pyrene-induced liver and forestomach neoplastic
formation [98,99].The decrease in 7,12-dimethylbenz[a]
anthraxcene-induced hamster buccal pouch carcinogenesis
caused by SAC is also accompanied by enhanced GST activity
and an increased GSH level [116].
Upregulation of phase II detoxication enzyme gene tran-
scription involves a series of signaling pathways and tran-
scriptional factors. Among these, the pivotal role of nuclear
factor E2-related factor 2 (Nrf2) is well documented [112,117].
Activation and binding of Nrf2 to the promoter antioxidant
response element/electrophile response element increases
the transcription of GST, NQO1, UGT, and sulfotransferase.
After treatment with garlic organosulfur compounds, Nrf2
nuclear translocation is increased and NQO1 expression is
upregulated in HepG2 cells and in mice [118,119]. Increased
hepatic NQO1 and GST activity helps to attenuate carbon
tetrachloride-induced liver injury in rats orally dosed with
500 mmol/kg DATS for 5 consecutive days [120].
Dozens of organosulfur compounds have been identied in
garlic products, and these appear to vary in their biological
activity. It is interesting to ask what chemical characteristic of
these garlic-derived compounds determines their potency to
modulate drug metabolism. Evidence from structur-
eefunction relationship studies indicates that the number of
both allyl groups and sulfur atoms in each organosulfur
compound is a determining factor in the transcription of
phase I and II enzymes.
With phase II detoxication enzymes, the number of sulfur
atoms and allyl groups correlates positively with their potency
to enhance gene transcription. DATS displays the best
induction of NQO1, follows by DADS; DAS has only a minor
effect [118]. Compared with DATS, DADS at a 10-fold higher
dose (100 mmol/kg) increased the expression of GST and NQO1
in rat liver, whereas DAS did not [121]. Similar ndings
(DATS > DADS > DAS) have been reported for the induction of
GSTP in rat liver [107,122]. An increase in UGT activity is also
noted in HepG2 cells treated with DAS, dipropyl sulde (DPS),
and DADS; the effective concentration of DAS and DPS (50 mM)
is much higher than that of DADS (2.5 mM) [123]. Feeding rats
a diet containing 5% garlic powders markedly raises hepatic
UGT activity in an alliin content-dependent manner [101].
A comprehensive study to examine the effect of the allyl
suldes DAS, DADS, DPS, and dipropyl disulde (DPDS) on the
hepatic, renal, intestinal, and pulmonary phase II enzymes
GST, EH, UGT, and NQO, was performed by Guyonett et al
[124]. After orally dosing Wistar rats with 1 mmol/kg of each of
the compounds for 4 consecutive days, DADS exerted the
greatest inducibility of all phase II detoxication enzymes,
with pulmonary EH activity unchanged. In addition, induction
of NQO activity was seen only in DADS-treated animals. The
increases in GST and EH activity caused by DAS, DPS, and
DPDS were only noted in liver. Later, the increase in hepatic
GST and NQO1 expression and activity by treatment with
allyl-containing compounds was demonstrably greater than
that by propyl-containing ones: i.e., DADS >DPDS and DATS >
dipropyl trisulde [118,125,126]. These ndings suggest that
garlic alk(en)yl suldes have different potencies for inducing
phase II enzymes, and that such induction is tissue-specic.
As for phase I enzymes, garlic components with an allyl
side chain are better at inducing most CYP isozyme expres-
sions than are propyl- or methyl-containing ones. However,
the effect of sulfur atom number on CYP expression differs
from that seen in their action on phase II enzymes: garlic
compounds with a higher number of sulfur atoms displayed
lower inducibility [105,107,115,122,126]. This discrepancy
suggests that the regulatory mechanismof garlic organosulfur
compounds on phase II and CYP isozymes is different, and the
precise active mechanism warrants further study.
6. Garlic and antioxidation
Oxidative stress is a state wherein the balance between radi-
cals generated and the free radical- or oxidant-scavenging
capacity of the endogenous antioxidant system is disrupted.
Oxidative stress is documented as being involved in the
Bi o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 22
pathogenesis of chronic diseases, including cardiovascular
disorders and cancer. Hence, compounds with antioxidant
properties may be used to prevent oxidative stress-mediated
diseases [127].
Numerous studies have demonstrated garlic and its orga-
nosulfur compounds to be potent antioxidants by displaying
radical-scavengingactivity andmodulating cellular antioxidant
enzyme activity. Aged garlic extract and SAC have been shown
toscavengeROSs, protect endothelial cells frominjurybyoxLDL
[128], and defend PC12 neuron cells from damage by hydrogen
peroxide [129]. Garlic extract has been proven to be as effective
as N-acetylcysteine in lessening ROS formation and GSH
depletion induced by acetaminophen in rat primary hepato-
cytes [130]. Garlic pretreatment with 1 g/kg for 5 weeks reduces
iron-catalyzed lipid peroxidation by lowering malondialdehyde
levels in rat liver and colon, along with enhancing the status of
theantioxidants [131]. Likewise, garlicreduces iron-inducedcell
proliferation and autophagy and protects mitochondrial
membranes by lowering iron storage in the liver [131]. Garlic oil
is effective in reducing tributyltin-induced oxidative damage in
mice and human amniotic cells [132], as well as decreasing
sodium nitrite-induced neurotoxicity in rats [133].
The aforementioned garlic protection against oxidant-
induced damage can be attributed to an increase in the
activities of superoxide dismutase, GSH reductase, g-gluta-
mate cysteine ligase, and GST, and also in GSH production
[133e135]. Activated Nrf2 demonstrably plays a key role in
garlic enhancement of both antioxidant defense capability
and drug metabolism enzymes, as described above [134].
The antioxidant properties of garlic have been ascertained
in animal models of disease. In the fructose-induced meta-
bolic syndrome model in rats, aqueous garlic extract attenu-
ates oxidative stress and prevents vascular remodeling by
suppressing NAD(P)H-oxidase [136]. In db/db mice with type 2
diabetes, the consumption of 5%freeze-dried aged black garlic
for 7 weeks signicantly raised superoxide dismutase, cata-
lase, and glutathione peroxidase activity and lessened lipid
peroxidation in the liver [137]. In rats with streptozotocin
induced-diabetes, garlic oil helps to normalize impaired
antioxidant status [138]. Less neuron damage accompanied by
increased levels of synaptophysin and presynaptic SNAP25
(synaptosomal-associated protein of 25 kDa, a member of the
soluble N-ethylmaleimide-sensitive factor attachment
protein receptors, which play a key role in presynaptic vesicle
fusion and exocytosis), have been seen in Alzheimers APP
transgenic (Tg) mice treated with a diet containing 2% aged
garlic extract and its active component SAC (20 mg/kg diet)
[129]. SAC also reduces lipid peroxidation and superoxide
radical production, and elevates Cu-Zn-superoxide dismutase
activity in 1-methyl-4-phenylpyridinium-induced parkin-
sonism in mice [139].
In recent years, several human intervention studies have
examined the antioxidant potency of garlic in humans. Two
months of garlic oil (250 mg/d) supplementation greatly
reduced oxLDL and 8-iso-prostaglandin F
2
alpha levels,
accompanied by a signicant decline in both systolic and
diastolic blood pressure, in hypertensive patients [13].
A similar fall in oxLDL production has been reported by dosing
70 hypertensive adults with 1620 mg/d oily macerate of garlic
for 30 d [23]. In double-blind placebo-controlled study, plasma
oxLDL levels sharply fell in those administered 200 mg/d aged
garlic extract combined with multi-micronutrients (folic acid,
vitamins B
6
and B
12
, and L-arginine) for 1 year, compared with
controls [140]. Taken together, these results suggest that garlic
has potent antioxidant activity in delaying the onset and
development of cardiovascular disease, cancer, diabetes, and
neurodegenerative diseases caused by an imbalance between
free radical production and antioxidant defense.
7. Garlic and drug interaction
As stated above, garlic denitely modulates drug-metabolizing
enzyme activity and membrane transporter levels in the liver,
lung, kidney, and intestinal tissues. This raises some possi-
bility that garlic supplementation could cause interactions
between food and drugs and change the therapeutic efcacy of
any drugs administered. To resolve this question, in vitro and
in vivo experiments have multiplied in recent years. Increased
toxicity of the human immunodeciency virus protease
inhibitor ritonavir has beenreportedinpatients withAIDS who
were co-administered garlic [141]. This can be explained, at
least in part, by its inhibition of the excretion of ritonavir:
allicin, for example, has been reported to inhibit the p-glyco-
protein-mediated efux of ritonavir in Caco-2 cells [142].
However, examining the permeability of rat jejunum and
the Caco-2 cell monolayer has shown that aged garlic extract
raises saquinavir and darunavir efux [143]. A higher efux of
darunavir after addition of aged garlic extract has recently
been noted in rat liver slices and isolated hepatocytes,
whereas the efux of saquinavir decreases [144]. The authors
propose the competitive binding at the same binding sites and
a positive cooperative effect with distinct binding places is
likely to be responsible for garlics effect in altering the efux
of saquinavir and darunavir, respectively [144]. Greater
multidrug resistance-associated protein 2 expression is also
reported in kidney brush-border membranes with DADS, but
not with SAC [145].
For in vivo models, the pharmacokinetics of the diuretic
drug hydrochlorothiazide in rats has been calculated
following 3 weeks administration of garlic homogenate. The
results show that garlic homogenate increases the bioavail-
ability and half-life of hydrochlorothiazide while decreasing
its clearance [146]. The diuretic effect of hydrochlorothiazide
is concomitantly increased by garlic homogenate. Enhance-
ment of the antihypertensive and cardioprotective efcacy of
captopril in rats by garlic homogenate and SAC was also re-
ported in a later work [147].
In our laboratory, the effect of garlic oil on the pharmaco-
kinetics of atorvastatin has recently been determined. Rats
were orally administered 50 mg/kg of garlic oil for 5 consec-
utive days, and then a single dose of atorvastatin (10 mg/kg)
was given. The rise of p-glycoprotein levels in liver and 3A1/2
activity in both intestinal and liver tissue appear to be nega-
tively correlated to the area under the curve (AUC) of plasma
concentration of atorvastatin and its metabolite 2-OH-ator-
vastatin (unpublished data).
It would also be intriguing to learn whether and how garlic
supplementation interacted with drugs in humans and
changed their therapeutic efcacy. To date, limited research
B i o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 23
has been carried out (Table 3). In a clinical trial involving 10
healthy subjects, 600 mg garlic extract was given daily for 21
days, the results indicating that garlic extract increased intes-
tinal p-glycoprotein expression and decreased the AUC of
plasma concentration of saquinavir [148]. Our study evaluated
the pharmacokinetics of two hypocholesterolemic drugs,
simvastatin and pravastatin, whose AUCs were not changed.
Due to its antithrombotic activity, garlic ranks among the
most widely used herbal medicines, typically ingested by
people receiving warfarin [149]. Changes in the pharmacoki-
netics of warfarin as a result of garlic have been determined in
a clinical trial involving 12 healthy male volunteers. The
results showed that the plasma concentrationetime prole of
warfarin and platelet aggregation unaltered when warfarin
was co-administered with garlic (2 g/d) for 2 weeks [150].
An inuence of garlic on the pharmacokinetics of doce-
taxel was also rated in 10 women with metastatic breast
cancer [151], treated with 30 mg/m
2
docetaxel given weekly for
3 of 4 weeks. Three days after the initial dose of docetaxel,
patients received 600 mg of garlic twice daily for 12 consecu-
tive days. The results indicated that the clearance of docetaxel
and additional pharmacokinetic parameters including peak
concentration, AUC, and half-life were not affected.
Although garlic had no signicant effect on the pharma-
cokinetics of docetaxel, these authors found that patients
with the CYP3A5*3C/*3C genotype had a lower mean AUCratio
than those with the CYP3A5*1A/*1A genotype [151]. This
nding suggests that genetic background is a determining
factor in the outcome of garlic and drug interaction. Under-
standing the genotype of each individual tested may help in
evaluating whether garlic interacts with the particular drug
and changes its therapeutic efcacy.
Although the results remain inconsistent and contradic-
tory, the possibility that garlic will affect the therapeutic
efcacy of certain drugs cannot be excluded based on its
potency in terms of modulating drug-metabolizing enzymes
and the activity and expression of membrane transporters..
More well-designed studies are warranted to clarify whether
garlic affects the metabolism of drugs and alters their
pharmacokinetics.
8. Safety of garlic
Consumed for hundreds of years, garlic is regarded as a safe
food. However, in addition to the possible interaction with
drugs cited above, several health risks have been reported to
be associated with the excess consumption of garlic, or with
contact with garlic it in the workplace. In particular, gastro-
intestinal tract injury and allergic reactions caused by garlic
attract concern. Increased exfoliation of the gastric surface
epithelial cells in healthy subjects has been reported after the
intragastric infusionof a single dose of rawgarlic of over 0.75 g
[152]. By injecting 0.5 mL of raw garlic juice into the ligated
duodenum of rats, injury to the duodenal mucosal lining fol-
lowed 2 hours after exposure, with severe damage including
ulcers and bleeding occurring after 24 hours [153].
Damage to the stomach and intestine may account for the
decrease in body weight seen after rats were given aqueous
extracts of garlic (300 or 600 mg/kg/d for 21 days) and garlic oil
(200 mg/kg, three times aweekfor 6 weeks) [36,154]. Inachronic
toxicity test, however, nodifferences inbody weight gainandin
urinary, hematological, serological, and histological examina-
tions were observed in Wistar rats given garlic extract at doses
of 2 g/kg ve times a week for 6 months [155]. These inconsis-
tencies require more careful experimental designs to clarify
whether garlic displays an adverse effect on gastrointestinal
tract and growth; for instance, differences in garlic species,
garlic preparations, and the dosage tested merit consideration.
Over recent decades, the allergenic potential of garlic has
become well recognized. Cases of allergic reactions e e.g.,
contact dermatitis, asthma, urticaria, pemphigus, and
anaphylaxis e have been reported in association with garlic
use [156]. Allergic contact dermatitis in response to garlic was
initially reported in 1950; to date, most cases have appeared in
chefs and housewives in frequent contact with garlic
[157e161]. Among Ferna ndez-Vozmediano et al.s 13 curry
chefs, four tested DADS-positive, all showing dermatitis of the
nondominant hand, with hyperkeratosis and ssuring of the
thumb, index, and middle nger [162]. Allergy of the hands in
the case of a 58-year-old male taking garlic to treat his
hyperlipidemia also related to his use of garlic tablets [163].
Based on such evidence, garlic is classied as a type I allergen
[159], the allergens being identied as DADS, allylpropyl
disulde, allylmercaptan, and allicin [164].
9. Conclusions
Past decades have seen myriad studies, especially in vitro and
in animal models, addressing the protective effect of garlic
against cardiovascular disease and cancer. This protection
can arise from its diverse biological activities: enhanced
antioxidant defense, lowering of blood lipids, inhibition of
blood aggregation, enhancement of cancer cell cycle arrest/
apoptosis, inhibition of invasion and/or metastasis, and
Table 3 e Garlic and drug interactions.
Preparation Subjects/dose Effect Reference
Garlic extract 12 healthy males, 2 g/d
(3.71 mg allicin/tablet), 2 wk
No changes in the pharmacokinetics
of warfarin
[150]
Garlic extract 10 women with breast cancer,
600 mg/d (3.6 mg allicin/tablet), 17 d
No changes in the pharmacokinetics
of docetaxel
[151]
Garlic extract 10 healthy males, 600 mg/d
(12 mg g-glutamylcysteine/4.8 mg alliin), 21 d
[ duodenal p-glycoprotein level [148]
No change in CYP 3A4 expression
No changes in bioavailability of simvastatin,
pravastatin, and saquinavir
Bi o Me d i c i ne 2 ( 2 0 1 2 ) 1 7 e2 9 24
modulation of drug metabolism and/or the immune response.
However, the results observed in human clinical and inter-
vention studies have been inconsistent. The risk of garlice
drug interactions is attracting increasing interest, especially
in the elderly and in those with chronic diseases. Further
experiments are warranted to understand the actual health
benets and impact of garlic.
Acknowledgments
This research was supported by China Medical University,
Grant No. CMU97-134 and CMU98-CT-21.
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May 2013, Vol.11, No.3
175
Journal of Integrative Medicine
www.jcimjournal.com/jim

Research Article
Effects of Chinese herbal medicine Yiqi Huaju
Qingli Formula in metabolic syndrome patients
with microalbuminuria: a randomized
placebo-controlled trial
Tian-zhan Wang
1
, Yu Chen
2,3
, Yan-ming He
2
, Xiao-dong Fu
1
, Yi Wang
2
, Yan-qiu Xu
2
, Hong-
jie Yang
2
, Hong-li Xue
2
, Yi Liu
1
, Xiao-tao Feng
4
, Teng Zhang
2,3
, Wen-jian Wang
1,3
1. Institute of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China
2. Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of
Traditional Chinese Medicine, Shanghai 200437, China
3. Clinical Research Institute of Integrative Medicine, Shanghai University of Traditional Chinese Medicine,
Shanghai 200437, China
4. Guangxi Scientifc Experimental Center of Traditional Chinese Medicine, Guangxi University of Chinese
Medicine, Nanning 530001, Guangxi Zhuang Autonomous Region, China
BACKGROUND: Microalbuminuria (MAU) is a key component of metabolic syndrome (MetS)
and is an early sign of diabetic nephropathy as well. Although routine Western medicine
treatments are given to MetS patients to control high blood pressure, hyperglycemia and
dyslipidemia, some patients still experience progressive renal lesions and it is necessary to
modify and improve the treatment strategy for MetS patients.
OBJECTIVE: To investigate the effcacy of Yiqi Huaju Qingli Herb Formula, a compound traditional
Chinese herbal medicine, in MetS patients with MAU when it is combined with routine Western
medicine treatment.
DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Sixty patients with MetS were
randomized into the Chinese herbal formula group (CHF, Yiqi Huaju Qingli formula treatment in
combination with Western medicine) and control group (placebo in combination with Western
medicine). All treatments were administered for 12 weeks.
MAIN OUTCOME MEASURES: Urinary microalbumin (MA), urinary albumin-to-creatinine ratio
(UACR), 24-hour total urine protein (24-hTP), body mass index (BMI), waist circumference
(WC), waist-to-hip ratio (WHR), fasting plasma glucose (FPG), 2-hour postprandial plasma
glucose (2-hPPG), glycosylated hemoglobin (HbA1c), homeostasis model assessment for
insulin resistance (HOMA-IR), blood lipid profle and blood pressure were observed.
RESULTS: Compared with the control group, CHF treatment significantly decreased BMI
(P<0.05), WC (P<0.01) and WHR (P<0.01). Both groups had significant decreases in FPG,
2-hPPG, HbA1c, HOMA-IR, MA, and UACR, with CHF treatment showing better effects on these
parameters compared with the control treatment (P<0.05). Both treatments signifcantly reduced
the levels of total cholesterol, low-density lipoprotein cholesterol and triacylglycerol (TAG), and a
greater reduction in TAG was observed with CHF treatment (P<0.05). The level of high-density
lipoprotein cholesterol did not change in the control group after treatment (P>0.05), whereas it
signifcantly increased with CHF treatment (P<0.01). Compared with before the treatment, signifcant
decreases in systolic blood pressure, diastolic blood pressure and mean arterial blood pressure
were observed in both groups (P<0.01). However, there was no signifcant difference between
the two groups (P>0.05).
CONCLUSION: Combined treatment of Yiqi Huaju Qingli Formula and Western medicine signifcantly
alleviated MAU, which may correlate with the improvement of insulin sensitivity and glucose and
lipid metabolism.
TRIAL REGISTRATION IDENTIFIER: This trial was registered in the Chinese Clinical Trial
Registry with the identifer ChiCTR-TRC-11001633.
May 2013, Vol.11, No.3
176
1 Introduction
Microalbuminuria (MAU) is one of the criteria for the
diagnosis of metabolic syndrome (MetS), as established
by the World Health Organization in 1998
[1]
. MAU is
one of the most common microvascular complications of
diabetic nephropathy (DN), and ultimately develops into
end-stage renal disease
[2]
. Besides the fact that diabetes is
closely associated with MAU, hypertension and obesity
can also impair renal function and result in MAU. Thus,
MAU is not only one of the important components of
MetS, but also an independent risk factor of cardiovascular
disease
[3]
. With the control of hypertension, hyperglycemia
and dyslipidemia, some patients still exhibit MAU and
progressive renal lesion, which probably result from multiple
risk factors involved in patients with MetS. As shown in
our previous study, Yiqi Huaju Formula treatment of the
MetS subjects without MAU was effective in improving
central obesity and fatty liver disease
[4,5]
. Traditional Chinese
medicine (TCM) theory on the development of MAU
regards it as evil heat that damages the kidneys, and
blood stasis that blocks the collateral pathway. We added
some heat-eliminating herbs to Yiqi Huaju Formula to
compose a new Yiqi Huaju Qingli Formula
[6]
. The previous
experiments showed that Yiqi Huaju Qingli Formula
improved insulin sensitivity in rats with type 2 diabetes
mellitus (T2DM) and reduced MAU
[7]
. In this study, we
further investigated the effects on MAU subjects coupled
with MetS when we added Yiqi Huaju Qingli Formula to
routine treatment.
2 Materials and methods
2.1 Subjects
2.1.1 Inclusion criteria
(1) Patients from 18 to 65 years old. (2) Subjects with
abdominal obesity (waist circumference (WC) > 90 cm for
men and WC > 85 cm for women), triacylglycerol (TAG)
1.70 mmol/L (150 mg/dL), high-density lipoprotein cholesterol
(HDL-C) < 1.04 mmol/L (40 mg/dL), elevated blood pressure
(systolic blood pressure (SBP) 130 mmHg, diastolic
blood pressure (DBP) 80 mmHg or previously diagnosed
hypertension), elevated fasting blood glucose (fasting
plasma glucose (FPB) 6.1 mmol (110 mg/dL), and 2-hour
postprandial plasma glucose (2-hPPG) 7.8 mmol (140 mg/
dL) or previously diagnosed diabetes). Presence of at least
three of the aforementioned factors (in addition to diabetes)
was suffcient to establish a clinical diagnosis of MetS. MetS
was defned according to the diagnostic criteria for metabolic
syndrome by the Joint Committee for Developing Chinese
Guidelines on Prevention and Treatment of Dyslipidemia in
Adults
[8]
. (3) On different days, two continuous tests of urinary
albumin-to-creatinine ratio (UACR) within 30 to 300 mg/g.
(4) Informed consent form was signed.
2.1.2 Exclusion criteria
Subjects were excluded from this study if they had any
of the following conditions: (1) type 1 diabetes; (2) SBP >
180 mmHg or DBP > 110 mmHg; (3) severe cardiovascular,
cerebrovascular diseases or chronic liver diseases; (4) UACR >
300 mg/g, or 24-hour total urine protein (24-hTP) > 0.5 g, or
serum creatinine > 176 mmol/L; (5) pregnant or lactating
women; (6) mental disorders; (7) cancer.
2.1.3 Participants
Sixty participants were recruited from the Department
of Endocrinology at Yueyang Hospital of Integrated Tradi-
tional Chinese and Western Medicine, Shanghai University
of Traditional Chinese Medicine, and the Department of
Integrative Medicine at Huashan Hospital, Fudan University
between December 2011 and June 2012. The participants
were randomly divided into Chinese herb formula
group (CHF group; n = 30) and control group (n = 30)
according to the random number table. The study protocol
was approved by the Ethics Committee of Yueyang Hospital
of Integrated Traditional Chinese and Western Medicine,
Shanghai University of Traditional Chinese Medicine, and
was registered in Chinese Clinical Trail Registry (Trial
registration identifer: ChiCTR-TRC-11001633).
2.2 Treatment
All of the participants were educated for diet control
KEYWORDS: metabolic syndrome X; microalbuminuria; insulin resistance; drugs, Chinese
herbal; randomized controlled trials
DOI: 10.3736/jintegrmed2013032
Wang TZ, Chen Y, He YM, Fu XD, Wang Y, Xu YQ, Yang HJ, Xue HL, Liu Y, Feng XT, Zhang T, Wang WJ.
Effects of Chinese herbal medicine Yiqi Huaju Qingli Formula in metabolic syndrome patients with microalbumin-
uria: a randomized placebo-controlled trial. J Integr Med. 2013; 11(3): 175-183.
Received March 18, 2013; accepted April 15, 2013.
Open-access article copyright 2013 Tian-zhan Wang et al.
Correspondence: Teng Zhang, MD, Professor; Tel: +86-21-61561782-6159; E-mail: zhangteng501@hotmail.
com. Wen-jian Wang, MD, Professor; Tel: +86-21-52888220; E-mail: wj6518@163.com
May 2013, Vol.11, No.3
177
and proper exercise as a basic treatment. Routine Western
medicine treatment was maintained for all the participants
with or without the addition of CHF.
2.2.1 CHF group
The participants from the CHF group received the
routine Western medication and an additional Chinese
herb formula, which was composed of Huangqi (Radix
Astragali), Huanglian (Rhizoma Coptidis), Puhuang
(Pollen Typhae), Zexie (Artemisiae Rhizoma Alismatis),
Ludouyi (Testa Vignae Radiatae), Liuyuexue (Serissa
Japonica), and Fuzi (Radix Aconiti Lateralis Preparata).
The extract of the indicated herbs in the powder form was
produced by the Department of Pharmacy of Yueyang
Hospital of Integrated Traditional Chinese and Western
Medicine, Shanghai University of Traditional Chinese
Medicine (No. 110502). The extract powder was placed
into packs, each of which contained the equivalent of
23.5 g crude herbs.
2.2.2 Control group
Participants in the control group received placebo instead
of CHF in addition to their routine Western medicine. The
placebo (No. 110602) was formulated with 5% dosage
of CHF and was similar to CHF in both taste and color.
The placebo and CHF packaging was also identical. The
participants were blinded to their treatments of CHF or
placebo (one bag per dose, twice a day) for 12 weeks.
2.3 Clinical and biochemical measurements
2.3.1 Demographic characteristics and blood pressure
At the beginning and end of the study, SBP, DBP and
mean artery blood pressure (MABP) were recorded;
body height, WC, and hip circumference were measured,
and then body mass index (BMI) and waist-to-hip ratio
(WHR) were calculated.
2.3.2 Blood sample collection and analyses
Venous blood samples were collected following an overnight
12-h fast for the analyses of FPG, fasting plasma insulin
(FPI), glycosylated hemoglobin (HbA1c) and lipid profle
including total cholesterol (TC), TAG, HDL, and low-
density lipoprotein cholesterol (LDL-C). Insulin resistance
was evaluated by homeostasis model assessment for insulin
resistance (HOMA-IR) (FPI (U/mL) FPG (mmol/L)/22.5).
Venous blood samples were collected after a 75-g oral glucose
tolerance test for the measurement of 2-hPPG.
FPG, 2-hPPG, TAG and TC were determined using
enzymatic methods with kits from Shanghai Jingyuan
Company. HDL-C was tested using enzymatic methods
with kits from Japan Jishui Company. LDL-C was tested
by elimination method and kits were purchased from Japan
Jishui Company. Aforementioned indexes were analyzed
by the automatic biochemistry analyzer (Hitachi 7600).
HbA1c was analyzed by high-performance liquid chromatog-
raphy with kits from Sysmex Corporation. Plasma insulin
was analyzed by the fully automated chemilluminescent
immunoassay analyzer. (CENTAUR XP, Simens, Germany).
2.3.3 Urine sample collection and analyses
First morning urine was collected for the detection of
urinary microalbumin (MA), urine creatinine, and UACR.
Twenty-four-hour urine was gathered for the analyses of
24-hTP. Kehua Biomed Company provided the kit for
urine creatinine fest and Randox Company provided the
kit for 24-h urinary albumin analysis. MA was analyzed
using immunoturbidimetry with brain natriuretic peptide-
specifc protein detection machine (Simens, Germany).
2.4 Safety assessment
Routine blood test, routine urine test, liver function test,
kidney function test, electrolytes and electrocardiogram
were performed before and after both treatments.
2.5 Statistical methods
Statistical analysis was performed using SPSS (SPSS,
Chicago, IL, USA; version 16). Data were presented as
mean standard deviation if normally distributed and
median (interquartile range) if distributions were skewed.
Independent-samples t tests were used to compare normally
distributed continuous variables; otherwise, Mann-Whitney
test was applied. Within-group comparison between baseline
and follow-up was assessed using paired t test or Wilcoxon
signed rank sum test. Fisher exact test was used for
between-group comparison and McNemar test for within-
group comparison of categorical variables. P value less
than 0.05 was considered statistically signifcant.
3 Results
3.1 General information of patients
Thirty patients (17 male and 13 female) were enrolled in
the CHF group with the average age of (53.17 7.82) years.
Another 30 patients (18 male and 12 female) were included in
the control group with the average age of (53.33 8.51) years.
All the patients met the diabetes diagnosis criteria. Twenty-
nine patients in the CHF group and 28 patients in the
control group were diagnosed with hypertension prior
to the study. All the patients, regardless of whether they
had hypertension, were treated with angiotensin receptor
blockers due to their positive MAU. Some of them received
additional calcium channel blocker treatment for better
control of their blood pressure. The baseline data, including
the application of the hypoglycemics and antihypertensives,
were comparable, and there was no statistical difference
between the CHF group and the control group (P>0.05)
(Table 1). See Figure 1 for fowchart of the recruitment process.
3.2 Changes in BMI, WC and WHR
As shown in Table 2, there were no signifcant changes
in terms of BMI, WC and WHR in the control group after
treatment (P>0.05). In contrast, significant decreases in
BMI, WC and WHR were observed in the CHF group
after treatment (P<0.01). Significantly decreased BMI
May 2013, Vol.11, No.3
178
3.4 Changes in TC, TAG, LDL-C and HDL-C
As shown in Table 4, both the CHF and control treatments
resulted in signifcant reduction of the levels of TC, TAG
and LDL-C, respectively (P<0.01). A more significant
reduction of TG was observed after CHF treatment compared
with that after control treatment (P<0.05). The level of
HDL-C was not altered in the control group after treatment
(P>0.05), whereas it was signifcantly increased by CHF
treatment (P<0.01), although there was no significant
difference of HDL-C between the CHF group and the
control group after treatment (P>0.05).
3.5 Changes in MA, UACR and 24-hTP
As shown in Table 5, both CHF and control treatment
significantly reduced the levels of MA and UACR
(P<0.01, P<0.05). The reductions of MA and UACR in
the CHF group were much greater than those in the control
group (P<0.05). The level of 24-hTP was not altered by
the control treatment (P>0.05), whereas it was signifcantly
decreased by the CHF treatment (P<0.01). There was a
signifcant difference of 24-hTP between the CHF group
and the control group after treatment (P<0.05).
3.6 Changes in SBP, DBP and MABP
As shown in Table 6, signifcant decreases in SBP, DBP
and MABP were observed in patients from both CHF
and control groups as compared with before treatment
(P<0.01). However, there was no significant difference
between the two groups (P>0.05).
3.7 Safety evaluation of CHF treatment
Two patients undergoing the CHF treatment had displayed
mild diarrhea and gastrointestinal discomfort, but completed
the treatment after these symptoms were alleviated. No
other adverse reactions were observed. There were no
abnormal manifestations regarding blood biochemistry,
Table 1 Baseline demographic characteristics of the two groups
Item CHF (n=30) Control (n=30)
Age (mean standard deviation,
years)
53.177.82 53.338.51
Gender (male/female) 17/13 18/12
Diabetes (cases) 30 30
Diabetes duration (mean
standard deviation, years)
11.035.92 11.736.09
Anti-diabetic treatment
Metformin (cases) 21 20
Sulfonylurea (cases) 12 11
Alpha glucosidase inhibitors
(cases)
22 21
Hypertension (cases) 29 28
Antihypertensive treatments
Angiotensin receptor blockers
(cases)
30 30
Calcium channel blockers
(cases)
24 23
Lipid-lowering treatment
Statins (cases) 16 15
Fibrates (cases) 2 3
Figure 1 Flowchart of the patients in the trial
(P<0.05), WC (P<0.01) and WHR (P<0.01) were also
noted in the CHF group as compared to the control group.
3.3 Changes in FPG, 2-hPPG, HbA1c and HOMA-IR
As shown in Table 3, significant reductions in FPG,
2-hPPG, HbA1c and HOMA-IR were recorded in both
CHF and control groups after treatment (P<0.01, P<0.05).
When these parameters were compared between the CHF
and control groups, the CHF group showed statistically
greater effects in reducing the levels of FPG, 2-hPPG,
HbA1c and HOMA-IR after treatment (P<0.05).
May 2013, Vol.11, No.3
179
Table 2 BMI, WC, and WHR before and after treatment
(Mean standard deviation)
Group n
BMI (kg/m
2
) WC (cm) WHR
Before treatment After treatment Before treatment After treatment Before treatment After treatment
CHF 30 26.812.11 23.342.43
**
94.716.98 87.308.60
**
0.950.06 0.870.05
**
Control 30 26.432.57 26.812.75 94.4511.20 94.6710.87 0.950.06 0.940.05
**
P<0.01, vs before treatment;
P<0.05,
P<0.01, vs control group. BMI: body mass index; WC: waist circumference; WHR: waist-
to-hip ratio.
Table 3 FPG, 2-hPPG, HbA1c and HOMA-IR before and after treatment
Group n
FPG (mmol/L) 2-hPPG (mmol/L) HbA1c (%) HOMA-IR
Before
treatment
After
treatment
Before
treatment
After
treatment
Before
treatment
After
treatment
Before
treatment
After
treatment
CHF 30 8.202.72 6.201.31
**
12.023.79 8.482.56
**
7.981.49 7.021.26
**
4.401.24 2.401.92
**
Control 30 8.112.46 7.021.41
**
13.433.74 10.203.24
**
8.101.38 7.621.36
*
4.381.83 3.551.88
*
*
P<0.05,
**
P<0.05,

vs control group. FPG: fasting plasma glucose; 2-hPPG: 2-hour postprandial plasma
glucose; HbA1c: glycosylated hemoglobin; HOMA-IR: homeostasis model assessment for insulin resistance.
Table 4 TC, TAG, LDL-C and HDL-C before and after treatment
(Mean standard deviation, mmol/L)
Group n
TC TAG LDL-C HDL-C
Before
treatment
After
treatment
Before
treatment
After
treatment
Before
treatment
After
treatment
Before
treatment
After
treatment
CHF 30 4.690.78 4.220.77
**
2.210.51 1.520.58
**
3.040.85 2.510.71
**
1.070.28 1.190.30
**
Control 30 4.650.54 4.220.66
**
2.180.51 1.850.44
**
3.010.76 2.560.52
**
1.070.32 1.130.21
**
P<0.05, vs control group. TC: total cholesterol; TAG: triacylgycerol; LDL-C: low-density lipoprotein
cholesterol; HDL-C: high-density lipoprotein cholesterol.
Table 5 MA, UACR and 24-hTP before and after treatment
Group n
MA (mg/L) UACR (mg/g) 24-hTP (g per 24 h)
CHF 30 101.5875.39 47.1127.85
**
120.5475.75 63.3239.86
**
0.140.04 0.070.03
**
Control 30 98.5066.92 78.8760.65
*
121.4588.72 101.8374.56
*
0.130.06 0.110.07
*
P<0.05,
**
P<0.05, vs control group. MA: urinary microalbumin; UACR: urinary albumin-to-creatinine

ratio; 24-hTP: 24-hour total urine protein.
Table 6 SBP, DBP and MABP before and after treatment
(Mean standard deviation, mmHg)
Group n
SBP DBP MABP
CHF 30 139.238.66 125.935.32
**
83.537.39 79.035.66
**
101.436.10 95.304.81
**
Control 30 137.0011.15 127.976.91
**
82.937.60 79.775.26
**
101.737.19 96.774.97
**
*
P<0.05,
**
P<0.01, vs before treatment. SBP: systolic blood pressure; DBP: diastolic blood pressure; MABP: mean artery blood pressure.
May 2013, Vol.11, No.3
180
urine biochemistry, liver function, kidney function,
electrolytes and electrocardiogram after both CHF and
control treatments.
4 Discussion
The incidence of MetS, which is characterized by abdominal
obesity, hyperglycemia, dyslipidemia and elevated blood
pressure, is increasingly on the rise. With it, the risks of
cardiovascular diseases and diabetes increase, and the
development and progression of renal damage resulting
from the concomitant multiple risk factors increase as
well. MAU, an important component of MetS, is a sensitive
sign not only of early stages of DN, but also of extensive
vascular dysfunction. Renal function in patients with MetS
is more vulnerable, as compared to that of the patients
with diabetes or hypertension alone, due to the MetS
patients multiple risk factors. A previous study showed
that the number of patients with MAU was 14% greater in
diabetic patients coupled with MetS, as compared to that
in diabetic patients without MetS. Increasing numbers of
MetS components corresponded with increased prevalence
of MAU
[9]
. Now MAU is considered one of the strongest
predictors for cardiovascular diseases. In the Copenhagen
Prospective study
[10]
, the prevalence of coronary heart
disease with albumin excretion rate (AER) > 5 mg/min
was 11% and the mortality was 28%, while the prevalence of
coronary heart disease with AER < 5 mg/min was 5% and
the mortality was 13% (P<0.001). Obviously, AER above
5 mg/min was a strong risk factor for coronary heart disease
and cardiovascular death. Therefore, early intervention on
MAU is critical to the prevention, or delaying the onset,
of renal lesions in MetS, and to the prevention of cardio-
vascular disease as well.
The patients in this study had been receiving routine
treatments to control high blood pressure, hyperglycemia,
and dyslipidemia, and their blood pressure, glucose and
lipid levels were controlled to a certain extent. However,
they still developed MAU. This indicates that there were
some unidentifed or uncontrolled factors resulting in the
renal lesion besides the known and controlled factors in
MetS patients. These unknown factors caused the patients
to experience residual risk such as MAU. This indicates
the need for improving clinical treatment strategies to
enhance the therapeutic effcacy for MAU.
Insulin resistance, one of the essential mechanisms of
MetS, was previously considered to have some indirect
impact on the membrane permeability of renal glomeruli
through hyperglycemia, dyslipidemia and elevated blood
pressure; the direct impact of insulin resistance on renal
damage was thought to be low. In fact, MAU is closely
associated with insulin resistance
[11,12]
. Chan et al
[13]
found
that among patients with T2DM, insulin resistance was
more severe in those with MAU compared with those without
MAU. Moreover, insulin resistance was positively correlated
to the urinary albumin excretion rate (r=0.25, P<0.002).
In non-diabetic patients, MAU was positively correlated
with insulin resistance as well
[14]
. In a prospective study,
insulin resistance and the number of MetS components
were demonstrated as critical factors that impact the
prevalence of chronic renal lesions even after adjustment
of hypertension and diabetes
[15]
. These fndings can help
us to understand why there were 5% to 10% patients with
MAU among the newly diagnosed diabetics
[16-18]
. Patients
with MAU prior to diabetes may have experienced long-term
insulin resistance and hyperinsulinemia. Insulin resistance
and hyperinsulinemia lead to glomerular hyperfiltration,
hyperperfusion, hypertension, proinflammatory and pro-
thrombotic states, which can activate the renin-angiotensin
system, and damage renal function. Moreover, abdominal
obesity, the anthropometric symbol of insulin resistance, is
also an independent risk factor of MAU
[19]
. Thus, increasing
insulin sensitivity may be an important step in reducing
renal lesions in MetS. However, currently there is no
safe or ideal insulin-sensitizing agent. The typical insulin
sensitizer rosiglitazone may increase cardiovascular risks
(myocardial infarction, stroke, congestive heart failure and
death) and has been restricted in its clinical usage
[20]
. In
contrast, TCM offers new possibilities in safely improving
insulin sensitivity.
According to TCM principles, the pathogenesis of insulin
resistance involves spleen-defciency resulting in obstruction
of transformation. This illustrates the pathological condition
under which the absorbed nutrients (glucose, lipids and
others) are unable to be converted into the energy required
for normal function, which is promoted by spleen qi. This
abnormality results in the accumulation of glucose and
lipids, subsequently causing elevated levels of lipids,
glucose, blood pressure and adipocytic infiltration of internal
organs. These elevated parameters are transformed into
pathological factors resulting in target organ injuries,
abdominal obesity, hyperglycemia, hypertension and
dyslipidemia as well. Yiqi Huaju Formula was designed
under the guidance of this principle to prevent and treat MetS
component diseases
[21]
. The previous study demonstrated
that the formula significantly increased insulin sensitivity,
improved abnormal body fat distribution, hyperglycemia,
and dyslipidemia, inhibited inflammatory cytokines, and
effectively promoted fibrinolysis
[4,5]
. In order to address
MetS coupled with MAU, in which endothelial function and
renal local microcirculation are affected by insulin resistance,
we added some heat-eliminating herbs and kidney-tonifying
herbs to the Yiqi Huaju Formula to compose a new herb
formula Yiqi Huaju Qingli Formula for the treatment of
MetS coupled with MAU. As to Yiqi Huaju Qingli, Yiqi
means improving insulin resistance and promoting the
May 2013, Vol.11, No.3
181
transformation of nutrients to energy such as adenosine
triphosphate. Huaju Qingli means rectifying the abnormal
body fat distribution, hyperglycemia, and dyslipidemia
caused by insulin resistance and regulating levels of
adipokines, infammatory cytokines, prothrombotic factors,
and oxidative stress molecules, thus improving endothelial
function and local renal microcirculation and alleviating
MAU
[22]
.
In this study, MetS patients coupled with MAU were
treated with CHF or placebo respectively for 12 weeks.
After treatment, patients treated with placebo in addition
to hypoglycemic and antihypertensive medications in
the control group had significant reductions in MA and
UACR, but no statistical changes in 24-hTP. These results
indicate that the medications are effective for MetS
coupled with MAU, but they do not satisfactorily address
the entire picture. In contrast, applying Yiqi Huaju Qingli
Formula in addition to hypoglycemic and antihypertensive
medications in the CHF group resulted in more signifcant
reductions in MA and UACR, and a signifcant reduction
in 24-hTP. And there were signifcant differences between
the two groups after treatment. These results demonstrate
clear improvement gained when CHF treatment is added
to the routine Western medications in the treatment of
MetS patients couple with MAU.
The routine treatment resulted in reductions in FPG,
2-hPPG, HbA1c, TC, TAG and LDL-C in the control group,
but the reductions were much greater with the addition of
Yiqi Huaju Qingli Formula in the CHF group, and there
were significant differences between the two groups after
treatment. Additionally, the level of HDL-C in the CHF
group increased signifcantly, but remained nearly unchanged
in the control group. These fndings showed that, when treating
glucose and lipid profiles in MetS patients with MAU,
combining CHF and Western pharmaceutical medications
is superior to the routine Western medicine treatment
alone.
Hypertension in both groups was well controlled before
the study initiated because of the application of antihy-
pertensives. However, SBP, DBP and MBP in both groups
were further significantly decreased after the 12-week
treatment, and there was no statistical difference between
the two groups at the end of the study. These results suggest
that the decrease in blood pressure was involved in the
improvement of renal function in the patients; however,
blood pressure changes may not be the main mechanism
of the Chinese herb formula in the improvement of MAU.
HOMA-IR was signifcantly decreased in both groups,
though more reduction was seen in the CHF group, which
suggests that CHF has a superior effect in improving
insulin sensitivity. The effect of the CHF treatment in
increasing insulin sensitivity was supported by the changes
in anthropometric parameters. Significantly decreased
BMI, WC and WHR were noted in the CHF group after
treatment, while there were no signifcant changes in the
control group. Obesity, and especially central obesity, is
a hallmark of insulin resistance
[19]
. Our previous study
investigated the effects of the major components of the
formula and found that astragalus polysaccharides, berberine,
and pollen typhae favonoids increased insulin sensitivity
through phosphatidylinositide 3-kinases or -arrestin-2
signal transduction and enhanced the consumption of glucose
in adipocytes
[23-27]
. We also observed that Yiqi Huaju Qingli
Formula increased insulin sensitivity and decreased MAU
in diabetic rats, and the results were consistent with the
clinical outcomes in the present study
[28]
.
It is very important to control hyperglycemia and hyperten-
sion for the treatment of MAU subjects coupled with diabetes
or with MetS. All the patients enrolled in this study were
treated with hypoglycemic and antihypertensive medica-
tions before the study initiated; however, they still had
positive MAU, which suggested that the routine treatment
did not address all aspects of MetS and MAU diseases.
When we added Yiqi Huaju Qingli Formula to the routine
medications for the subjects in the CHF group, the therapeutic
effect for MAU was improved more greatly. The most
significant differences between the CHF group and the
control group were the dramatic increased insulin sensitivity
and signifcant improvement on anthropometric parameters,
which represent abnormal body fat distribution in the CHF
group. Much more signifcant reductions in plasma glucose
and the lipid profle were also observed. All the results suggest
that the insulin-sensitizing effects of CHF can explain the
main difference between the two groups.
5 Acknowledgements
We thank Wei-hua Chen for providing technical assistance.
We acknowledge all the physicians, nurses and assistant
nurses of the hospital services in which the intervention
took place.
This work was supported by Ministry of Education
211 Project, Fudan University; Project of Innovation of
Shanghai Municipal Committee of Science and Technology
(No. 08dj1400600); National Natural Science Foundation
of China (No. 81001574); Leading Medical Projects at
Science and Technology Commission of Shanghai Municipality
(No. 12401905100); Three-year Projects to Promote Traditional
Chinese Medicine, Shanghai (No. ZYSNXD-CC-ZDYJ050);
Project of Shanghai Cerebrated TCM Doctor Workshop
(No. ZYSNXD-CC-MZY034); Shanghai Association of
Chinese Integrative Medicine (No. zxyQ-1245); The Fok
Ying-Tong Education Foundation for Young Teacher of
University (No. 114036); The Program for Professor of
Special Appointment (Eastern Scholar) at Shanghai Institutions
of Higher Learning; Program for Pu Jiang Scholar at Science
May 2013, Vol.11, No.3
182
and Technology Commission of Shanghai Municipality;
Key Disciplines of Clinical Integrative Chinese and
Western Medicine at State Administration of Traditional
Chinese Medicine of the Peoples Republic of China;
the Foundation of Leading Academic Discipline Project of
Shanghai Municipal Education Commission (No. J50307).
6 Competing interests
The authors declare that they have no competing interests.
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Nutrients 2011, 3, 694-711; doi:10.3390/nu3060694

nutrients
ISSN 2072-6643
www.mdpi.com/journal/nutrients
Review
Reducing Sodium in Foods: The Effect on Flavor
Djin Gie Liem, Fatemeh Miremadi and Russell S. J. Keast *
School of Exercise and Nutrition Sciences, Centre for Physical Activity and Nutrition Research,
Sensory Science Group, Deakin University, Melbourne, 3125, VIC, Australia;
E-Mails: gie.liem@deakin.edu.au (D.G.L.); fmir@deakin.edu.au (F.M.)
* Author to whom correspondence should be addressed; E-Mail: russell.keast@deakin.edu.au;
Tel.: +61-3-9244-6944; Fax: +61-3-9244-6017.
Received: 5 May 2011; in revised form: 31 May 2011 / Accepted: 10 June 2011 /
Published: 20 June 2011

Abstract: Sodium is an essential micronutrient and, via salt taste, appetitive. High
consumption of sodium is, however, related to negative health effects such as hypertension,
cardiovascular diseases and stroke. In industrialized countries, about 75% of sodium in the
diet comes from manufactured foods and foods eaten away from home. Reducing sodium
in processed foods will be, however, challenging due to sodiums specific functionality in
terms of flavor and associated palatability of foods (i.e., increase of saltiness, reduction of
bitterness, enhancement of sweetness and other congruent flavors). The current review
discusses the sensory role of sodium in food, determinants of salt taste perception and a
variety of strategies, such as sodium replacers (i.e., potassium salts) and gradual reduction
of sodium, to decrease sodium in processed foods while maintaining palatability.
Keywords: salt taste; flavor; sensory

1. Introduction
Sodium chloride (NaCl) is the prototypical stimulus for salty taste [1]. Sodium improves the
sensory properties of foods, by increasing saltiness, decreasing bitterness, and increasing sweetness
and other congruent flavor effects [2]. Factors which determine an individuals liking and acceptance
of salty foods are poorly understood, however it is thought that environmental factors such as the level
of sodium in foods and habitual diet play a significant role [3,4]. While sodium is essential for normal
human functioning, current sodium intakes far exceed recommendations for good health [5]. Excessive
OPEN ACCESS
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sodium intake is associated with an increase in blood pressure, which is a major cause of
cardiovascular diseases. It has been estimated that 62% of stroke and 49% of coronary heart disease is
caused by high blood pressure [6]. Excess sodium consumption has also been associated with
numerous other negative health effects, including gastric cancer [7], decreased bone mineral
density [8] and possibly obesity [9].
A report by Asaria et al. [10] calculated that a modest 15% reduction in population sodium intake
could prevent 8.5 million cardiovascular-related deaths worldwide over 10 years. A meta analysis
prepared by the WHO concludes that there is strong evidence for the cost effectiveness of national
sodium reduction strategies [10,11]. For example, cardiovascular diseases are the most expensive
health issue accounting for 11% of total health expenditure around the world [12]. The average sodium
reduction strategy is expected to cost only 0.3% of the current expenditure on hypertension control
program in conjunction with other cardiovascular-associated costs worldwide [10]. Lowering sodium
intake is beneficial for hypertensive and normotensive people although it affects hypertensive people
to a greater degree.
Despite the negative health consequences and associated health care costs of high sodium
consumption, humans consume well above the recommended levels in most developed nations,
making sodium reduction a priority for public health [11,13]. For this reason, a range of strategies to
reduce sodium in different foods have been applied. However, success is often limited as sodium
reduction has adverse affects on taste quality and flavor perception [14]. The objective of this paper is
to (1) review the role sodium plays in the flavor of food, (2) to assess strategies to reduce sodium in
processed foods.
2. Physiological Role of Sodium in the Body, Recommended Sodium Intake, and Sodium
Consumption Trends
Within the body, sodium regulates extracellular volume, maintains acid-base balance, neural
transmission, renal function, cardiac output and myocytic contraction [1]. World Health Organization
(WHO) recommendations indicate that, in order to prevent chronic diseases, an adult upper daily limit
intake of sodium should be less than 87 mmol Na/day (<5 g NaCl/day) [15]. The average US sodium
intake is estimated to be 140160 mmol Na/day (8.29.4 g NaCl/day) [13], United Kingdom
161 mmol Na/day (9.4 g NaCl/day) [16] and Asian countries higher than 206 mmol Na/day
(12.0 g NaCl/day) [15]. These studies illustrate consumption levels well in excess of sodium required
for optimum health. In westernized countries, approximately 75% of sodium in the diet comes from
processed foods, and foods eaten away from home [15,17]. The processing of foods often involves
adding sodium to foods for a variety of flavor or processing reasons. For example chick peas, sweet
corn and peas, which have naturally very low sodium content, have 10 to 100 times more sodium
post-processing (Table 1) [15].
It is believed that the relatively high sodium intake of almost all societies today became common
beginning between 500010,000 years ago [18,19]. It is generally thought that food preservation was
the main driver of high sodium consumption in the early days [20]. This early use of sodium is likely
to be the origin of the current high consumption of sodium. Interestingly, sodium consumption did not
change dramatically over time. For example, in 300 B.C. the average daily sodium intake in certain
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parts of China was approximately 3000 mg Na/day (7.6 g NaCl/day) for women and 5000 mg Na/day
(12.7 g NaCl/day) for men [21]. In his book Neptunes gift: a history of common salt,
Multhauf estimated that the average salt consumption in 1850 in Britain and France was about
40005000 mg Na/day (10.212.7 g NaCl/day) [20], which is rather similar to the current intake of
sodium [22]. In 2010, Bernstein and Willett [23] analyzed 38 studies, published between 1957 and
2003, which investigated sodium consumption, and found that individuals consistently consumed
about 3700 mg Na/day (9.4 g NaCl/day) throughout this period. Despite the absence of any great
increase in sodium consumption in the past centuries, salt intake has remained too high as people
transitioned from preserved foods to modern processed foods [23].
Table 1. Comparison of the sodium content of some of the natural and processed foods [15].
Food item Description Sodium content (mg/100 g)
Beef Topside, roast, lean and fat
Corned beef, canned
48
950
Bran Bran, wheat
Bran Flakes
28
1000
Cheese Hard, average
Processed
620
1320
Chick-peas Dried, boiled in unsalted water
Canned, re-heated, drained
5
220
Potato Raw, boiled in unsalted water
9
250
Peas Raw, boiled in unsalted water
Trace
250
Potato
chips
Homemade, fried in blended oil
Oven chips, frozen, baked
12
53
Salmon Raw, steamed
Canned
Smoked
110
570
1880
Sweet corn On-the-cob, whole, boiled in unsalted water
Kernels, canned, re-heated, drained
1
270
Tuna Raw
Canned in oil, drained
Canned in brine, drained
47
290
320
Cereals and cereal products such as bread, breakfast cereals, biscuits and cakes, contribute
about 3050% of the estimated total intake of sodium in UK and US (see Figure 1). In Asian countries,
such as Japan, a large proportion of dietary sodium comes from sodium added in cooking
(i.e., soy sauce) [15,24]. Fast foods also contribute a significant amount to the daily sodium intake,
e.g., one large slice of pizza alone contributes 1000 mg sodium or 43% of upper daily limit intake of
sodium (2300 mg Na/day; 5.8 g NaCl/day) [25].

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Figure 1. Percentage contribution of food types to average daily intake of sodium [15,24].

3. Flavor Perception
Flavor is a unitary percept, however what we perceive as an unitary whole is a combination of
inputs from independent sensory systems: taste, smell and chemical irritation (part of the sense of
touch) [14]. For example, when we consume an orange flavored soft-drink, the sense of taste is activated
by non-volatile sugars and acids, the sense of smell by volatile aromas added to characterize the
soft-drink (orange), and the chemical irritation by carbon dioxide. We do not perceive sweetness
independently of orange aroma or carbon dioxide tingle; we perceive the sensations simultaneously as
an orange flavored soft-drink. This central integration of taste, smell and chemical irritation ensures
that there is ample opportunity for interactions between the senses. Removing or reducing one
component of flavor, for example sweet taste can have effects beyond simply loss of sweetness; it can
influence the entire flavor profile [2].
4. Taste Perception
The sense of taste presumably evolved to detect both toxins and nutrients in foods. To perform this
task the taste system is subserved by five taste qualities: sweet, elicited by sugars indicating
carbohydrates in foods; umami, elicited by glutamic acid and other amino acids indicating protein in
foods; sour, elicited by protons indicating acidic foods; bitter indicating toxic foods; and salt, elicited
by sodium content of foods [26].
Taste receptor cells, which mediate the sense of taste, are located throughout the oral cavity.
Most taste receptor cells are components of taste buds, which are clustered on three types of papillae
(i.e., fungiform, foliate, and circumvallate) located on the tongue [27]. Papillae contain several
hundred taste buds, each of which is composed of 50 to 150 taste receptor cells. The taste receptors at
the apical end of the taste receptor cells are exposed to the internal environment in the oral cavity.
When food or drink enters the mouth, chemicals from those foods may activate taste receptors. The
chemical signal is converted to an electrical signal and sent via the seventh, ninth and tenth cranial
afferent nerve fibers to the gustatory processing regions of the brain [2,26].
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Perceived taste is characterized by four separate attributesquality, intensity, temporal and spatial
patterns [2]. Taste quality is the most important defining feature of taste sensation and is defined as a
descriptive noun given to categorize sensations that taste compounds elicit: sweet, sour, salty, bitter
and umami. Perceived intensity is related to the strength of the taste sensation and, when plotted
against tasted concentrations, creates a psychophysical function. Temporal pattern is used to describe
the time course of the taste perception [2,14], and spatial topography relates to location and localizability
of taste sensation. If we use sodium as an example, the taste quality elicited is saltiness, the intensity of
saltiness varies with the concentration of sodium in a food, and the time course of saltiness varies from
when you put the salty food in your mouth through to mastication, swallowing and aftertaste. The
location of saltiness originates throughout the entire oral cavity where taste receptor cells are located.
It is important to note that there is no such thing as a tongue map, saltiness can be perceived across the
tongue, rather than by specific areas on the tongue as wrongly suggested by the tongue map [27].
Salt Taste Perception
The evolutionary importance of sodium is illustrated in the fact that one taste quality is devoted to
identifying sodium in foods. Lithium also has a pure salt taste, but due to toxicity will not be an
approved food ingredient, however other minerals such as potassium may also have a salty component
to their taste [28].
The exclusivity of sodium as a stimulus for salty taste can be explained by its unique sodium-specific
transduction mechanism involving epithelial sodium channels (ENaCs) on the taste receptor cells [29].
There are two ENaCs subtypes, one specific for sodium, which is activated at low sodium
concentrations and is believed responsible for the appetitive nature of salt taste. The second ENaCs is
permeable to multiple cations, activated at higher sodium concentrations, and is believed responsible
for the aversive nature of cations. The seminal research revealing putative salt taste mechanisms was
based on a mouse model, but there is little reason to believe the mechanisms would differ in
humans [2,26].
Salt taste perception begins when sodium activates ENaCs on taste receptors and an afferent signal
is sent to gustatory processing regions of the brain. At low sodium concentrations, the afferent signal
may be too weak and not able to produce a noticeable difference from a similar solution without
sodium. As the concentration of sodium increases the afferent signal strength will increase and reach a
level where an individual will be able to discriminate a sodium solution from water, but remain unable
to identify the taste quality. This is known as the detection threshold and is often used as a measure of
individual sensitivity to sodium. Actual identification of saltiness occurs when the concentration of
sodium is high enough not only to activate the taste receptors, but also produce electrical impulses,
which can be carried via sensory neurons to the brain where they are decoded and after which the taste
quality can be identified. This is known as the recognition threshold. The sodium concentrations above
the recognition threshold are in the range of perceived saltiness, which is termed suprathreshold
(Figure 2) [2,26]. The concentration of sodium required to elicit saltiness will vary considerably between
food matrices, e.g., it is easier to detect 50 mM NaCl in an aqueous solution than in a bread matrix.

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Figure 2. Scale diagram of taste perception as concentration increases (adopted from [26]).

5. Taste Interactions
The perceived intensity of a single compound in an aqueous solution increases as its concentration
increases. However, in everyday life we mostly consume mixtures, rather than singular taste stimuli,
notable exceptions being sucrose (table sugar) and NaCl (sodium chloride or table salt). The perceived
intensity of mixtures may be additive or non-additive, which result in suppression or enhancement
outcomes. Suppression takes place when the total intensity of a mixture, existing of two or more
suprathreshold stimuli of the same modality, is less than the sum its components (e.g., 1 + 1 < 2) [14].
Enhancement is the opposite of suppression (the intensity of a mixture is greater than the sum of its
components [14].
5.1. Levels of Taste Interactions
Interactions between taste compounds such as mixture suppression and enhancement can occur at
three different levels including chemical interactions, oral physiological interactions and central
cognitive interactions. Chemical interactions may occur in the food matrix, for example gluten in bread
may bind sodium, making it unavailable for taste reception [14].
Oral physiological interactions involve one compound interfering with taste receptor cells or taste
transduction mechanisms associated with a second compound. These are classified as peripheral
interactions, as they occur at the epithelial/cellular level. An example of oral peripheral interaction is
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the effect sodium salts have on bitterness, where sodium interferes with the bitter taste transduction
prior to the taste signal being sent to processing regions of the brain [2,30].
To demonstrate this peripheral effect, Kroeze and Bartoshuk [31] used a split-tongue technique in
which sodium salt and a bitter stimulus were simultaneously applied to separate parts of the tongue, or
were applied as a mixture to the same part of the tongue. The intensity of bitterness was reduced more
when the stimuli were applied to the tongue in a mixture together, compared to when salt and bitter
taste stimuli were applied to separate parts of the tongue. This illustrated that sodium interferes with
the bitter taste transduction, although the mode of action in the periphery is unknown.
Cognitive interactions refer to central processing of taste stimuli after afferent signals are sent to
taste processing regions of the brain. Mixture suppression, which takes place after stimuli were applied
to separate parts of the tongue, is an example of cognitive interaction [31,32].
5.2. Taste Interactions Involving Saltiness
When two compounds with different taste qualities are mixed, a number of interactions may occur,
including non-monotonic (both enhancement and suppression) and asymmetrical intensity shifts [2,30].
In food matrices, sodium salts may influence other taste qualities independent of intensity/concentration.
For example, it has been suggested that saltiness is suppressed at high concentrations of of NaCl and
KCL, but enhanced at low concentrations of NaCl and KCl saltiness [2]. Salt and sour taste mixtures
symmetrically affect each others intensity with enhancement at low intensities/concentrations and
suppression or no effect at high intensities/concentrations [33,34]. Bitterness is suppressed by
sodium at all intensities/concentrations, while salt taste is less affected by bitterness. Sodium
enhances sweetness at low intensities/concentrations, has variable effects through the moderate
intensity/concentration range, and is suppressive or has no effect on sweetness at higher
intensity/concentration. Sweetness suppresses salty taste at moderate intensities [2]. Figure 3 shows a
schematic overview of binary interactions of taste qualities at different levels of concentrations. These
schematic reviews are just indications of what happens to taste qualities when two are mixed.
Variations, depending on the food matrix, may occur.
Figure 3. Schematic review of binary interactions of taste qualities at different levels of
intensity/concentration, Adapted with permission from Elsevier [2].

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Figure 3. Cont.

Interactions between tastes get more complex when three taste qualities interact, or when more
complex food matrices are involved [32]. Breslin et al. [35] investigated the interaction between
sodium (salt), sucrose (sweet) and urea (bitter) in a mixture. They found that perceived bitterness is
suppressed when sodium is added to a bitter-sweet mixture. Due to the decreased perceived bitterness,
perceived sweetness increased. The latter was a result of the bitterness being less able to reduce
perceived sweetness. This interaction takes place at a cognitive level. These findings are in line with
Gillette et al. [36], who suggested that addition of NaCl to three soups decreased bitterness and
increased sweetness. Similarly, Fuke and Konosu [37] reported that addition of umami tasting sodium
salts of 5-ribonucleotides reduced bitterness and increased sweetness in an artificial prawn extract.
Pangborn, who has been recognized as one of the most influential researchers in the area of taste
interaction [3840], performed a series of experiments in the early 1960s investigating sucrose, citric
acid and NaCl taste interrelationships. Several different food matrixes were used, e.g., pear nectar [41]
tomato juice [42] and lima bean puree [43]. The results from the food matrix mirrored those in simple
aqueous media [38,39,44], and from other studies [35,36].
Overall, reduction of sodium from food will have multiple flavor effects, as shown in Figure 4. The
primary effect will be loss of saltiness. There may also be an increase of bitterness, due to the effect of
sodium as an effective bitterness inhibitor; removing sodium will cause bitterness to be released from
suppression. An increase in bitterness will result in mixture suppression, thereby decreasing
sweetness [35]. In terms of taste liking, reducing sodium may decrease appetitive salt and sweet taste,
and increase aversive bitter taste. A reduction in appetitive salty and sweet taste, may also result in a
decreased perception of appetitive aromas associated with those tastes [14,35]. Overall, reducing sodium
in foods not only reduces perceived saltiness, but is also associated with a wide range of complex taste
interactions, which may negatively impact the liking of foods. Without sufficient knowledge about
taste interactions the search for sodium replacers and other strategies to decrease sodium in industry
sourced foods will be challenging.

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Figure 4. Influence of sodium reduction on flavor. The height of the boxes making up total
flavor reflects intensity of the named attribute in standard or reduced sodium
food [14,35].

6. Factors Affecting Sensitivity and Preference for Salt Taste in Foods
Supermarket shelves contain very few low sodium processed foods, which makes it difficult for
consumers to follow a low sodium diet [45]. Individuals continue to consume large amounts of sodium
and liking is preferentially directed to sodium-rich foods despite knowledge of the negative health
effects [6,46,47]. Little is known about the factors which affect an individuals salt taste sensitivity,
liking and selection of salty foods, and whether there is an association between oral sodium sensitivity
and liking of salty foods.
6.1. Genetic Factors
To our knowledge only two studies have tested the association between heritability and salt taste
sensitivity as assessed by recognition threshold [48,49]. They concluded that salt taste sensitivity had
no major heritable components and environmental factors may play a greater role in salt taste
sensitivity. No measures of salt preference were included in these studies.

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6.2. Environmental Factors
Environmental factors appear to have a larger influence on salt taste preference, than genetic
factors [3]. Infants and childrens liking for salt in specific foods is thought to be influenced by
consumption of salty foods commonly available after 6-months of age [4], when infants are able to
detect and preferentially ingest NaCl solutions compared to water. This developmental change is
thought to be a result of experience, and salt taste preference is said to be learned rather than
innate [4]. Further evidence of the role of environmental factors in salt taste preferences are based on
observations that exposure to sodium determines salt taste preferences [50]. Subjects on a sodium
reduced diet experienced increased saltiness in foods and a decreased liking for higher concentrations
of sodium chloride in foods [51,52]. Some researchers suggest a link between sodium intake and both
salt taste sensitivity and preference [51,52], which is likely caused by the sensory perception of
salt [53]. It is also believed that changes in salt taste preference were due to sensory experience of salt
taste rather than actual amount of sodium consumed.
The evidence, however, is not conclusive, and other research has shown no link between salt taste
sensitivity and consumption or liking of salty food [54], or between consumption of reduced sodium
diet and increasing preference for salty foods [55,56]. However, these studies have generally involved
a dramatic sodium reduction, e.g., a 50% reduction over a short period of time. Bertino et al. [51]
hypothesized a biphasic response to sodium reduction which is characterized by an initial phase of
increased attraction to salt taste followed by a decreased preference for salt which would explain the
discrepancies in results.
7. Methods of Sodium Reduction
7.1. Sodium Restricted Diet
A simplistic view on sodium reduction is to just tell consumers they eat too much sodium and
expect them to change their eating behavior. Community-based intervention trials, however, have
demonstrated that only 2040% of participants were able to reduce their sodium intake below the
recommended upper limit of 100 mmol Na/day (5.8 mg NaCl/day) despite intense counseling [45].
Due to the need for counseling, this intervention is not feasible at a population level.
Sodium reduced diets are difficult to maintain as they often require a change in dietary behavior
for example, actively choosing low salt foods of which there is very limited choice on supermarket
shelves [57]. It is widely accepted that sodium reduction could be more effectively achieved
by reducing the sodium content of processed foods and by applying multisensory principles,
e.g., enhancing aromas, to optimize the flavor characteristics of the foods, rather than by just giving
dietary advice alone [1,57,58].
7.2. The United Kingdom StrategySodium Reduction by Stealth
The UK approach is based around stealth reduction methods which refers to a gradual reduction of
salt in processed foods that is unnoticeable to consumers [59]. Grigis et al. [60] gradually reduced the
sodium content of white bread by 25% over a period of 6 weeks and consumers generally could not
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notice a difference in flavor. For food industry this would mean that they can meet sodium reduction
targets by gradually decreasing sodium in their product over the course of a couple of years, without
losing consumers. This strategy has been successful with the sodium content of many processed foods
in supermarkets being reduced by 2030% in the past 3 years. These results are expected to be
duplicated when revised targets are set for a further 1020% reduction to meet the UKs daily intake
target of 6 g/day by 2012 [61]. The reduction by stealth approach has the benefit of not requiring any
behavior change from consumers, which is traditionally difficult to achieve. This approach has resulted
in the UK population intake of NaCl reducing by approximately 1 g/day [24,57].
7.3. Use of Salt Substitute
An ideal situation would be the replacement of sodium with a compound that elicits a similar pure
saltiness when consumed. However, such replacements appear improbable due to the specificity of
sodium to the ENaC responsible for salt taste. Sodium chloride replacers such as potassium chloride,
calcium chloride and magnesium sulfate have been used to replace or enhance salt taste in a number of
food products [28]. While these compounds do contribute a certain salty taste quality, they may also
provide undesirable after tastes such as bitter, metallic and astringent tastes, which has limited their
current use in food manufacturing [62,63]. These other non-sodium cations are believed to activate a
second type of salt taste ENaC, which is non-specific and believed responsible for the off-tastes and
flavors. So while the use of other cations is appealing, in particular potassium due to the added health
benefit of increasing dietary potassium, the concentration which can be used in processed food will be
limited [63]. Potentially, the intensity of perceivable bitter taste and associated off flavors can be
decreased by bitter blockers, or sweeteners such as sucrose and thaumatin, which is an intensely
sweet tasting protein [64].
Furthermore, food grade acids have been shown effective in enhancing saltiness of sodium. Little
and Brinner [65] observed the effects of modifying NaCl and citric acid content of tomato soup on
both taste preference and saltiness [65]. Saltiness intensity increased with increasing citric acid
concentration. Taste preferences followed a hyperbolic function with an initial increase to a point of
maximum taste preference (between 0.150.3% citric acid) and then dropping quite rapidly thereafter.
Maximum taste preference was reached at both moderate NaCl (0.6%) and acid (0.3%) concentrations.
It is also interesting to note that a low NaCl soup with citric acid could match or exceed the liking
of a high NaCl soup [65]. A similar study investigated the effects of taste mixtures of NaCl and a
combination of acetic and lactic acid in both simple solutions and bread [66]. As in the soup study,
increasing concentrations of acid caused increased perceived saltiness at all salt variations in bread
samples. Highest pleasantness rating was reached at a moderate acid (0.6%) and highest NaCl (1.2%)
concentration in bread. The maximum taste preference of both studies involved only moderate acid
concentrations. This suggests the use of acids to enhance salty taste is limited to lower concentrations,
because sour notes may dominate and affect hedonic responses at higher concentrations. However,
there may be some food matrices in which the use of acids would not be successful.

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7.4. Other Approaches to Sodium Reduction
Due to the influence of sodium reduction on the total sensory profile of foods, the addition of
flavor, by means of herbs and spices, to sodium reduced product might be a viable solution [67].
Monosodium glutamate, which is responsible for the umami flavor, has been suggested as good flavor
enhancer in low NaCl products, without substantially increasing the total sodium content of the
product [59]. Kremer and colleagues replaced NaCl in a variety of foods, with different levels of
naturally brewed soy sauce and measured the total NaCl and food acceptance [68]. They suggested
that salad dressings (50% NaCl reduced), stir-fried pork (17% NaCl reduced) and soups (29% NaCl
reduced), were still acceptable when NaCl was substantially decreased and substituted with soy
sauce [68]. The study by Manabe [69] suggested that 17% of NaCl in egg custard could be decreased
by the addition of dried bonito (fish) [69].
8. Concerns for Food Industry for Sodium Reduction
Sodium chloride is a widely used food additive due to its low cost, its ability to increase liking of
foods via flavor modification, and other functional abilities in a food matrix [24,57]. Sodium chloride
is required in food processing for some technological functions such as dough development in bread,
and water binding and preservation in meats. However it is also believed that the sodium content of
many food products exceeds technological requirements and primarily acts to enhance sensory
effects [70]. A survey of UK food manufacturers revealed the most commonly cited function of
sodium chloride in a range of food products was to impart flavor and the most commonly cited
constraint to sodium reduction was palatability and consumer preference [71]. Given that sodium
reduction will likely decrease preference for foods, there will be considerable pressure from food
manufacturers to maintain current sodium levels in processed foods.
8.1. Loss of Palatability and Consumer Acceptance
Taste is one of the most important factors in food choice. Humans and animals have a liking for salt
taste [27]. Therefore, when a large reduction in sodium content occurs there is a decline in consumer
acceptance [72,73]. Small incremental decreases in sodium have shown to be effective, as consumers
may not be able to detect gradual reductions in sodium up to a point [60]. However, with a continual
decline of sodium from products it is inevitable that a point will be reached where a difference in
flavor profile will be detectable by consumers and the food will be less liked. A recent study by
Lucas et al. [54] showed that a significant sodium reduction (i.e., >50%) of a meal component (hash
brown) could occur with only a minor decrease in liking [54]. Therefore, large sodium reductions in
foods are possible, but ideally the salt taste elicited by sodium needs to be replaced so that the
consumer acceptance is not negatively affected [1].
8.2. Texture and Other Quality Characteristics
Reducing sodium chloride level may affect texture and other quality characteristics including,
moisture levels, fat content, pH, starter cultures, various additives, and processing conditions [1]. For
example, sodium chloride is able to bind proteins and fats and hold water. Therefore, meat batters with
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low sodium need to have a sodium replacer, which not only replaces salt taste, but also needs to
compensate for the other functions, which are lost when sodium is decreased [74].
Sodium chloride also limits the growth of yeast and enables the gluten structure in bread to develop.
The reduction of sodium chloride in bread may therefore result in an increase in the growth of yeast
and an undeveloped gluten structure, which has a negative effect on the texture of bread [75].
Furthermore, reduction of sodium chloride in cheese may affect starter culture activity. This negative
effect of sodium reduction, however, did not prevent food industry in the U.S. to market Cheddar
cheeses with different levels of sodium chloride [76].
8.3. Preservation and Microbial Safety
Sodium reduces water activity in foods and is therefore able to limit the growth of pathogens and
spoilage organism in a variety of food system [67]. For example, in processed meats and cheeses
sodium chloride limits the growth and production of toxin by Clostridium Botulinum [77]. Similarly,
sodium diacetate and sodium lactate limit the growth of Listeria monocytogenes and lactic acid
bacteria in ready-to eat meats, which makes it safe for human consumption [78]. Sodium reduction
may therefore pose a risk for unwanted bacteria growth and shorten shelf life. When sodium is reduced
extra care needs to be taken in terms of cooking, packaging and storage temperatures. Furthermore,
other preservatives may need to be added and all newly developed low sodium products need to be
tested on microbiological safety and shelf life [67].
8.4. Other Functions of Sodium
In addition to safety risks and processing challenges involved in producing low sodium foods, there
is also an economic consideration. Sodium chloride is relatively cheap and any substitute used will
increase the cost of the product. Furthermore, in order to find suitable salt replacers, bitter blockers and
or flavor enhancers substantial effort and money needs to go into research, development and consumer
testing. The food industry will need to take all functions of sodium in foods into account when
addressing sodium reduction.
9. Conclusion
Saltiness is an important sensory attribute of many foods, and sodium chloride contributes more
than just saltiness to the characteristic flavor of many food types. Whilst ensuring adequate dietary
sodium intake is vital to health, our intake of excessive amounts of sodium has been linked to
development of hypertension and subsequent pathologies [79]. Changing the dietary sodium content of
a population that has adapted to a high sodium diet will not be easy, and will entail a number of
strategies [24]. Part of the reason previous attempts to reduce sodium in processed foods were not
successful was due to loss of palatability of foods [73]. One strategy to reduce sodium is to replace
sodium with potassium salts, and while potassium chloride elicits weak saltiness, at higher
concentrations it also elicits metallic and bitter taste limiting its utility in food. The stealth approach
of gradual sodium reduction in processed foods [60], thereby modifying consumers salt taste
experience over time is recognized as arguably the best current strategy to reduce sodium in foods.
Nutrients 2011, 3

707
Initiatives carried out in several countries will provide much-needed data about whether this approach
is successful in reducing sodium intake and blood pressure at the population level.
There are potential barriers such as the technological function of sodium in foods, and decreased
consumer liking, that could be put forward by the food industry to stop sodium reduction. However,
mandating sodium reduction in certain food categories would urge food industry to come up with
creative solutions, which will enable sodium reduction without compromising on food safety and
consumer acceptance. There is no doubt that sodium reduction in foods will be difficult, but equally
there is no doubt that reducing the level of sodium in foods is essential for population health.
Acknowledgments
The authors are funded by the Central Research Grants Scheme (CRGS), Deakin University,
Melbourne, Australia. There is no conflict of interest to disclose. All authors contributed to the
preparation of the manuscript and agreed the final version.
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