Demonstration of Anti-hemolytic

Property of Angelica keiskei (Ashitaba)

Leaf Ethanolic Extract In Vitro
Katherine Mae G. Acejo, Rae Erica A. Beltran, Vien Athena Meg M. Galicia,
Maiden M. Garduce, R-Jay J. Manapat, Raphaela Angela L. San Andres
Gamaliel De Vera, RMT, MSMT

UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY
DEPARTMENT OF MEDICAL TECHNOLOGY
Abstract
Angelica keiskei, or more commonly known as Ashitaba, which originated from Japan and is
extensively used in China and parts of Southeast Asia is commonly used because of its
biologic functions such as anti-lipidemic, anti-carcinogenic, anti-thrombotic, anti-bacterial,
anti-hyperglycemic and anti-hemolytic. In this study, the researchers tested the plant for the
phytochemicals that may be attributed to its anti-hemolytic effect. Then they used Ashitaba
leaves extract with ethanol as the solvent and tested its anti-hemolytic property on 5% red
cell suspension. After inducing hemolysis through the addition of hydrogen peroxide, the
researchers compared the anti-hemolytic property of the Ashitaba Leaf Ethanolic Extract
(ALEE) from the control, Butylated-hyroxytoluene (BHT) in terms of percent inhibition
computed from the absorbance measured through spectrophotometry. Furthermore, 250
ug/ml, 500 ug/ml and 750 ug/ml of the extract were also used to determine which
concentration has the highest antihemolytic property. Results have shown that ALEE has a
higher anti-hemolytic activity than the standard used. Among the three concentrations of the
extract, 750 ug/mL yielded the highest anti-hemolytic property followed by 500 ug/mL. The
concentration with the lowest anti-hemolytic property is 250 ug/mL. This is due to the
flavonoids, phenols and tannins, which are present in the extract used.
INTRODUCTION are due to inhalation of polluted air or
chemicals, cigarette smoking, taking drugs
Erythrocytic damage caused by
and living an unhealthy lifestyle. However,
free radicals such as reactive oxygen
incidence of oxidative stress in
species (ROS) containing oxygen
erythrocytes can be altered by substances
molecules formed by the incomplete one-
called anti-oxidants.
electron reduction of oxygen, results in
hemolysis which releases hemoglobin Angelica keiskei, commonly known
content in the plasma [1]. Other diseases as Ashitaba, is a native plant from Japan.
associated with oxidative stress include Ashitaba has been used in Japan and China
heart and blood vessel disorders, chronic as a medicinal herb due to its power of
fatigue, arthritis, and hemolytic anemia. supplying vital nutrients and biologic
The risks of manifesting these conditions functions such as anti-carcinogenic, anti-
bacterial, anti-hyperglycemic, anti-
thrombotic, and anti-hypertensive effects.
It contains thirteen minerals, eleven
vitamins, chlorophyll, enzymes, carotene,
saponins, protein, plant fibers, glycosides,
coumarins, germanium, and a unique and
rare class of flavonoids called chalcones.
Ashitaba contains two physiologically
active flavonoid chalcones namely,
xanthoangelol and 4-hydroxyderricin that
are potent anti-oxidants [2]. Exogenous
consumption of these anti-oxidants from
plant, animal, and mineral sources have
proved beneficial to human health and is
effective to reduce incidence of free
radical-induced diseases.
Human erythrocytes are reliable
models of oxidative stress in clinical
laboratories. RBCs act as transporters of
oxygen and carbon dioxide to the lungs
and tissues. These cells are said to be
radical scavengers as they contain anti-
oxidant systems not only for the cell itself,
but also in organs and tissues of the
human body [3].
MATERIALS AND METHOD
The study was carried out using
cohort design which wanted to find out if
Angelica keiskei have an anti-hemolytic
property, through observation of different
treatments induced in red blood cells. The
independent variable was the type of anti-
hemolytic substance used while the
dependent variable was the amount of
hemolysis produced. The study was
conducted within the premises of the
University of Santo Tomas. Venous blood
collected from the students of the UST
Medical Technology Program was used in
the study.
Inclusion criteria include healthy
volunteers, 18-40 years of age, male or
female with normal CBC results while the
exclusion criteria include children or young
adults below 18 years old and adults who
are 40 years old and above. Healthy adults,
18-40 years of age, with history of
hematologic disorders and who had any
parasitic or bacterial infection in their
blood were not accepted for this study.
A minimum of seven samples per group
was used in the study to achieve at least
80% power of the statistical test, at 5%
level of significance. This sample size was
based on the result of Antonio et al which
stated that the mean absorbance of
methanolic extract of ashitaba was 0.224
(SD = 0.008) and ascorbic acid was 0.213
(SD = 0.005). G*Power version 3.1 was used
in the calculation. Furthermore, replicates
of the 7 samples, which were selected for
this study, were allocated for each of the 3
treatment groups or concentrations and 3
trials for each of those treatment groups.
Venous blood was collected from
twelve healthy adult volunteers who gave
explicit informed oral and written consent
for the investigation. A nondisclosure
agreement, signed by the researchers, was
given to each of the participants to
guarantee confidentiality. An initial
questionnaire was answered by each
participant and a CBC was done to verify
the integrity of their blood sample.
Among the twelve participants who
underwent a complete blood count on their
blood samples, the samples of the top seven
participants with normal hemoglobin,
hematocrit and RBC counts proceeded to
the experiment proper. Normal levels of
these three parameters are important to
assure that the RBCs of the participants are
within normal limits. Too high or too low
levels of these parameters may affect the
absorbance of the red cell suspensions.
 The chemicals and reagents that were
used in this study such as distilled water
(dH2O), Phosphate buffer solution (PBS),
100 mmol hydrogen peroxide (H202), and
normal saline solution (NSS) were all
purchased from Bambang, Sampaloc,
Manila. Butylated hydroxytoluene (BHT)
was purchased from Belman Laboratories.
The equipment and materials that
were utilized in this study were the
following: spectrophotometer, vortex,
centrifuge, glass stirring rods, Pasteur
pipettes, micropipettes, beakers, test
tubes, test tube racks, parafilm, graduated
cylinders, and cuvettes which were
provided by the University of Santo
Tomas.
The Ashitaba plant was acquired from
the formerly known Manila Seedling Bank
now auctioned by the Quezon City
Government located at Quezon Memorial
Circle Garden. The plant was then
identified and verified by the National
Museum, Botany Division, Manila,
Philippines.
The leaves of the Ashitaba were
removed from the plant and were washed,
dried and cut into smaller pieces. The
small pieces of Ashitaba leaves were
brought to the University of Santo Tomas
Research Center for the Natural and
Applied Sciences (UST-RCNAS) where
they were grinded and the pulverized. The
leaves were weighed using an analytical
balance. Extraction was done using a
percolator for 48hrs with 180mL of
ethanol as the solvent. The percolate was
collected and filtered using a rotary
evaporator at 40°C until a viscous
consistency was obtained. The
concentrated extract was further dried at
35°C, kept in an amber colored glass at 0 to
8°C, and used for analysis. The extract was
tested for the presence of tannins,
chalcones, phenols and flavonoids by a
biochemist in the UST Research Center for
the Natural and Applied Sciences (UST-
RCNAS).
The extraction procedure done by the
UST RCNAS used 260 mL of ethanol. The
total weight of the powdered Ashitaba
leaves was 42.55 grams. The weight of the
native extract obtained after the
extraction procedure was 3.5 grams.
Therefore, the native extract ratio was
12:1. This means that about 8%
extractable matter was obtained from the
powdered Ashitaba leaves in the
extraction procedure used.
The extract was diluted into three
different concentrations (250 μg/mL, 500
μg/mL, 750 μg/mL). To make 250 μg/mL,
250 μg of ALEE was diluted into 1 ml of
NSS. For 500 μg/ml, 500 μg of ALEE was
mixed with 1 ml NSS and lastly, to make
750 μg/ml, 1 ml NSS was used to dilute
750 μg of the extract. The same diluent
was also used to prepare the different
concentrations of BHT.
Collect venous blood Add 1.9mL of Normal
Saline Solution to produce
5% Red Cell Suspension
Centrifuge at 3,400
rpm for 3 minutes
Aspirate the supernatant
from the last washing
Pipette 2mL of blood into
a Wasserman Test Tube
Repeat the process
until the 3rd washing
Add Normal Saline
Solution until the tube
Gently invert the tube
is half-filled
Re-suspended the cells
Gently invert the tube
with half-full volume of
Normal Saline Solution
Centrifuge the solution
for 1 minute at 3,400 Decant the Supernatant
rpm
Figure 1. Preparation of 5% Red cell suspension
Venous blood was collected using EDTA as
the anticoagulant and was centrifuged at
3400 rpm for 3 minutes. First, 2 ml of
blood was pipetted into the Wassermann
test tube using a Pasteur pipette then the
tube was half-filled with normal saline
solution. Second, the test tube was
covered with parafilm and the solution
was mixed by gentle inversion. Third, the
solution was centrifuged for 1 minute at
3,400 rpm then the parafilm was removed
and the supernatant was aspirated using a
Pasteur pipette or by decanting the
supernatant as quickly as possible to avoid
disturbance in cell button. Then the cells
were re-suspended with another half-full
volume of NSS then were covered with
parafilm and mixed by gentle inversion.
The process was done in the first washing
until the 3rd washing/last washing. Lastly,
after aspirating the supernatant from the
last washing, 1.9 mL of normal saline
solution was added to produce 5% red cell
suspension.
The required amount of NSS to be
added to the packed cell volume was
calculated as follows:
Total volume desired =
Packed Cell Volume x 100
Desired % conc.
Volume of NSS to be added = Total
Volume - Packed Cell Volume
The 5% red cell suspension was divided
into three treatments namely Standard,
Sample, and Control (the red blood cells
used in the experiment were one day old).
The Sample was made up of 100 μL of 5%
RCS and 50 μL of ALEE at different
concentrations (250, 500 and 750 μg/mL in
NSS) while the Standard was made up of
100 μL of 5% RCS and 50 μL of BHT at
different concentrations (250, 500, 750
μg/mL). The control was made up of 100 μL
of 5% RCS. These three treatments were
subjected to incubation at room
temperature for five minutes. Then 200 μL
of 100 mmol hydrogen peroxide was added
to the three treatments.
SAMPLE STANDARD CONTROL
100 μL of 5% 100 μL of 5% 100 μL of 5%
RCS + 50μL of RCS + 50 μL of RCS
ALEE BHT
Incubate at room temperature for 5 minutes
Add 200 μL of 100 mM H2O2
Incubate at 37oC for 1 hour
Dilute with 3 mL PBS
Centrifuge at 2000 rpm for 10 minutes
Measure absorbance of supernatant at 540nm
Figure 2. Procedure to test inhibitory effects on
H2O2 induced hemolysis
The tubes were incubated at 37 degrees
Celsius for an hour, diluted with 3mL
phosphate buffer solution and then
centrifuged at 2000 rpm at 10 minutes. The
absorbance of the three treatments was
measured at 540nm using a Genesys 10 UV
spectrophotometer. A graph was used to
compare the absorbance of the three
treatments.
In order to determine the effect of the
extract and the standard, the inhibitory
activity of each of their concentration was
expressed as % Inhibition of Hemolysis and chosen. These seven participants were #03,
computed using the absorbance as follows: #05 #07, #09, #10, #11, and #12.

% Inhibition of Hemolysis = Table 2. Percent Inhibition of the different

concentrations of Sample and Standard
[(Acontrol – Asample)/ Acontrol] X 100
% Inhibition of Hemolysis
The % Inhibition of Hemolysis of the ASHITABA BHT
different concentrations of ALEE and BHT
was the unit of comparison used for each 250 500 750 250 500 750

Participant
concentration. μg/ μg/m μg/m μg/m μg/m μg/m
mL L L L L L
RESULTS AND DISCUSSION
03 6.67 16.67 20.0 8.89 12.22 17.22

Pytochemical Testing 05 50.26 57.18 58.46 48.21 52.82 56.15

Table 1. Results of Phytochemical Testing 07 7.36 42.86 46.32 3.46 5.19 5.63

Phytochemicals Phytochemicals 09 8.59 45.31 55.08 4.29 13.28 17.19

Tested Present
10 7.64 31.27 44.36 18.18 20.36 23.27
Flavonoids Positive (+)
11 8.78 29.75 49.75 6.83 14.63 49.26

Tannins and Phenols Positive (+) 12 15.92 34.07 43.70 16.67 16.67 29.25

Alkaloids Negative (-) Comparison of Percent Inhibition of

ALEE and BHT

Bate-Smith and Metcalf method was used
for the determination of flavonoids and it
resulted in a strong violet coloration that
indicates the presence of flavonoids in the
extract.
In the overall antihemolytic property of
ALEE and BHT (average of the means of 250
μg/mL, 500 μg/mL, 750 μg/mL), ALEE is
more effective in inhibiting hemolysis than
BHT in the test performed.

Ferric chloride test was used in

determining the presence of tannins and
phenols and the test resulted positive by
the presence of bluish black color.
Percent Inhibition

40
Mayer’s test was used for the 30
determination of alkaloids and it resulted 20
negative due to the absence of precipitate. 10
Percent Inhibition of the Three 0
Treatments ALEE BHT

Among the twelve participants that Antihemolytic Agent

underwent a complete blood count on their Figure 3. Mean Percent Inhibition of ALEE
blood samples, the top 7 participants with and BHT from all the participants in all
CBC results within the normal range were concentrations
Table 3. Percent Inhibition of ALEE and BHT
from all the participants in all concentrations
Antihemolytic Mean Std. 95% Confidence
Agent Error Interval
Lower Upper
Bound Bound
ALEE (sample) 32.38 4.614 21.092 43.670
BHT (standard) 20.66 5.99 5.978 35.335
Comparison of Percent Inhibition of Each
Concentration of ALEE and BHT (250
μg/mL, 500 μg/mL, 750 μg/mL)
The 750 μg/ml and 500 μg/ml of the
anti-hemolytic agents yield higher
percent inhibition than 250 μg/mL. This
is observable on all the concentrations of
both the sample (ALEE) and the standard
(BHT).
Using the IBM SPSS Statistics Version
20 program for statistical analysis, the
concentration of ALEE in the samples has
a significant effect in its antihemolytic
activity.
Table 4. Percent Inhibition of the three
concentrations (250 μg/mL, 500 μg/mL and
750 μg/mL) of ALEE and BHT from all
accepted participants
The mean % inhibition of each
concentration of ALEE are significantly
different: 750μg/ml is significantly
different (p<0.001) 500 μg/ml while 500
μg/ml is significantly different (p<0.001)
250 μg/ml. Between concentrations A and
B of ALEE, B has a significantly greater
mean % inhibition because based on the
table, it has a higher mean % inhibition
(p=0.007). Meanwhile, concentration C
has a significantly greater % inhibition
than B because based on the table, C has a
higher mean % inhibition than B
(p=0.040).
50
Percent Inhibition
40
30
20
10
0
250 500 750
Concentration (μg/ml)
ALEE BHT
Figure 4. Mean Percent Inhibition of the
three concentrations (250 μg/mL, 500 μg/mL
and 750 μg/mL) of ALEE and BHT from all
accepted participants
The Ashitaba plant obtained is
positive for tannins, phenols and
flavonoids but tested negative for
alkaloids. The high percent inhibition of
ALEE (compared with BHT) is attributed
to the presence of sufficient amounts of
flavonoids that are able to scavenge and
chelate the hydrogen peroxide induced in
the samples. It is also attributed to the
phenolic compounds that are important
because of the conjugated ring structures
and hydroxyl groups which have the
potential to stabilize free radicals involved
in oxidative processes through
hydrogenation or complexing with
oxidizing species. Hagerman et al. have
reported that the high molecular weight
phenolics (tannins) have abilities to
quench free radicals and their
effectiveness depends on their molecular
weight, the number of aromatic rings and
nature of hydroxyl groups. Free radical
scavenging activity of the ALEE might be
due to the presence of these high
molecular weight phenolics such as
catechin and rutin derivatives. Recent
investigations have shown that many
flavonoids and related polyphenols
contribute significantly to the anti-oxidant
activity of medicinal plants. These
flavonoids and phenols are not present in
BHT.
The low percent inhibition of ALEE
(w/ the use of 250 μg/mL of the extract) is
attributed to the insufficient amount of
these phytochemicals due to the small
amount of extract used. It is therefore
concluded by the researchers that the
efficacy of the anti-hemolytic property of
Ashitaba also depends on the
concentration of the extract used.
CONCLUSION
Based on the results obtained, Angelica
keiskei leaf ethanolic extract (ALEE) has an
anti-hemolytic property. The Ashitaba
plant tested positive for tannins,
flavonoids, and phenols but was negative
for alkaloids. Generally, ALEE yielded
more anti-hemolytic activity than that of
the standard (BHT) in the test performed.
The concentration of the extract that
yielded the highest percent inhibition of
hemolysis on 5% red cell suspension was
750 μg/mL followed by 500 μg/mL, while
250 μg/mL yielded the lowest percent
inhibition of hemolysis.
RECOMMENDATIONS
The researchers recommend further
studies on the anti-hemolytic property of
Ashitaba in vivo so that its effect inside the
body can also be tested. The same study to
be done on a larger sample size for the
consistency of the results. It may also be
done for the comparison of the effects on
healthy and unhealthy participants to
assess the efficacy of the extract. Further
studies on antihemolytic activity of the
other parts of the Ashitaba plant like stems
and roots. These may also contain other
phytochemicals which are not present in
the leaves. Perform the same experiment
using aged versus young red cells with the
extract to determine if it has the ability to
extend the shelf-life of red blood cells. Use
of other possible standards aside from
BHT. In a study conducted by Antonio et
al., ascorbic acid was the standard of
choice against their extract. Lastly, use
other methods to test the anti-hemolytic
property of the Ashitaba plant such as
automated analyzers and assays.
REFERENCES
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(2009). Protective Effect of Ascorbic Acid
Against Oxidative Damage Induced by
Hydrogen Peroxide in Cultured Human
Peripheral Blood Lymphocytes. Indian Journal
of Clinical Biochemistry, 24(3). 294-300
[2] Maronpot R. (2015). Toxicological
Assessment of Ashitaba Chalcone. Food
and Chemical Toxicology.
[3] Pandey, K. B. & Rizvi, S. I. (2011).
Biomarkers of Oxidative Stress in Red Blood
Cells. Biomed Pap Med Fac Univ Palacky
Olomouc Czech Repub. DOI
10.5507/bp.2011.027.