The effect of processing and formulation on the

bioavailability of isoflavones from red clover

Dorthe Mller Srensen

Master of Science Thesis, May 2015
Title: The effect of processing and formulation on the bioavailability of isoflavones from red clover

Danish title: Effekten af forarbejdning og formulering p biotilgngeligheden af isoflavoner fra

rdklver

Project period:
4th February 2014 4th May 2015
3rd and 4th semester MSc

Author: Dorthe Mller Srensen

Student number: 201210044

Supervisor: Per Bendix Jeppesen

Co-supervisor: Max Norman Tandrup Lambert

M.Sc. Molecular nutrition and food technology

Aarhus University Department of food science
Preface

This master thesis has been carried out in the 3rd and 4th semester of the MSc programme in Molecular
Nutrition and Food Technology, Aarhus University, Denmark. The project was conducted under
supervision of Associate Professor PhD, Per Bendix Jeppesen with the help from PhD stud., M.Sc.
Max Norman Tandrup Lambert.

The project is presented as a literature review and a scientific article based on experimental work
carried out at the Diabetes Research Laboratory, the Department of Endocrinology, MEA, Aarhus
University Hospital. The project is part of a larger research project on osteoporosis in menopausal
women formulated by Associate Professor PhD, Per Bendix Jeppesen.

The review article titled Bioavailability and physiological effects of isoflavones a review will be
submitted to the journal Journal of natural products.

Due to problems with the highly sensitive and advanced analytical equipment necessary for analyses
of plasma samples, it has only been possible to analyse a minor portion of the plasma samples from
the clinical study within the timeframe of this project. The scientific article titled Bioavailability of
isoflavones from fermented and unfermented red clover formulations is therefore based on results
from approximately 10% of the samples. The remaining samples will be analysed and results
incorporated in the article at a later time.

Acknowledgements

I would like to thank my supervisor Associate Professor PhD, Per Bendix Jeppesen for supervision
during the project period, and PhD stud, M.Sc. Max Norman Tandrup Lambert for practical help
during the study, sharing of theoretical knowledge and proof reading.

A big thank you goes to laboratory technicians Lene Truds and Eva Mlgrd Jensen and M.Sc
Catrine Thybo for all their practical assistance and huge support during the study. I wish to thank
PhD stud. Joan Bach Nielsen and PhD stud. Thomas Nordstrm Kjr for their assistance with blood
sampling during the study, as well as the Biochemical Department at Aarhus University Hospital
THG. A thank you also goes to the participants for making the study possible and being good sports.

Id like to thank Lars Jrgensen. DBlab for analysis of red clover formulations, Hans-Christian
Vollstedt and Henrik Jrgensen, Natur-Drogeriet A/S for production of capsules and tablets and
Xavier Frett, Rime Bahij El-Houri and Flemming Nielsen, SDU for analysis of plasma samples.

Moreover, I want to thank Michael Mohr Jensen, Herrens Mark, for producing and providing red
clover extract as well as funding for the project.
 Bioavailability and physiological effects of isoflavones a review
Author name: Dorthe Mller Srensen
Address: Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Tage-
Hansens Gade 2, DK-8000 Aarhus C, Denmark
E-mail: dorthe_ms@yahoo.dk

Abstract

Isoflavones are phytoestrogens found in plants that resemble estrogen in chemical structure, and bind
to estrogen receptors with a preference for estrogen receptor (ER). They can act as estrogen
agonists or antagonists and they may exert tissue specific effects. Soy and red clover (RC) are rich
sources of isoflavones, which has led to the prospect of soy and RC being alternatives to conventional
hormone replacement therapy (HRT). Following ingestion the isoflavones are metabolised by
enzymes, absorbed, conjugated in the liver, circulated in plasma and finally excreted in urine.
Bioavailability is important for the biological activity of isoflavones and many factors affect
isoflavone absorption including isoflavone source and dose, food matrix and individual metabolism.
Accumulating evidence suggests that isoflavones may potentially confer health benefits to many
diseases including menopausal symptoms, osteoporosis, hormone-related cancers and cardiovascular
diseases (CVD). These results are consistent with epidemiological evidence showing lower
incidences of cancer and CVD in populations consuming diets high in soy. Although a few concerns
have been raised about soy intake being harmful, especially to breast cancer patients, the intake of
isoflavones is generally evaluated as safe. The aim of this review is to summarize data on
bioavailability and health effects of isoflavones.

1 Introduction

The menopause results in a reduction in production of endogenous estrogen, and a hypoestrogenic

state that can cause menopausal symptoms. Symptoms include mood and behavioural changes, hot
flushes, sleep disturbance, vaginal dryness and more. Lack of estrogen production is also associated
with an increased risk of suffering from diseases like osteoporosis and cardiovascular disease (CVD).
The risk of cardiovascular morbidity and mortality is lower in premenopausal women compared to
men of the same age, but the risk increases as women reach menopause [109]. Due to an increased
rate of bone loss during menopause, postmenopausal women have an increased risk of developing
osteoporosis in the long run [3]. Currently hormone replacement treatment (HRT) is available,
however usage is associated with side effects of which most concerning is an increased risk of cancer.
Alternatives like phytoestrogens from plants are becoming increasingly popular as they are thought
to proffer the same beneficial effects of HRT without proffering side effects.

1
Asians have lower incidences of CVD, prostate and breast cancer compared to western populations,
and Asian women experience fewer menopausal symptoms [54,151]. Hendrich et al. have shown that
the plasma level of daidzein was higher in Japanese women than in Asian women who had immigrated
to Hawaii, and the incidence of breast cancer was lower for Japanese women [38]. Asian migrants
who maintain their usual diet do not have increased risk of coronary heart disease and hormone related
cancer types, compared to migrants who change to a western style diet [54]. This suggests that dietary
and environmental factors have an impact apart from racial differences, and that a higher intake of
phytoestrogens from soy products results in a lower risk of developing diseases associated with
estrogen deficiency [47,101,134,151].

There is growing evidence that isoflavones have beneficial effects on menopausal symptoms and
hormone-dependent disease, but inconsistencies are present in this field. This paper introduces
chemistry, metabolism and bioavailability of isoflavones, and summarizes results from studies on the
physiological effects of isoflavones (incl. osteoporosis, cancer, diabetes, menopausal symptoms and
CVD).

2 Estrogen and HRT

Estrogens are the main sex hormone in females, and are C18 steroids derived from androgens, which
are synthesized from cholesterol. Several types of estrogen exist, and 17 -estradiol (E2) is the most
potent form followed by estrone (E1) and estriol (E3) [36]. The latter two require conversion to E2
locally in order to attain full estrogenic activity [83]. E2 is primarily formed in the granulosa cells of
the ovaries, and E1 and E3 are mainly formed from E2 in the liver [36]. Aromatase enzymes catalyse
the final step in estrogen formation and are mostly expressed in ovarian granulosa cells in
premenopausal women [36,83]. In postmenopausal women the main sites of aromatase expression
are extragonadal sites like adipose tissue and skin [83].

Estrogen action is mostly mediated through estrogen receptors (ERs) which function as ligand-
activated transcription factors [83]. Binding of a ligand to the receptors results in conformational
changes, which leads to changes in the rate of transcription of estrogen-regulated genes. In the human
body, there are two different types of ERs; ER and ER which are different in structure and
biological function, and ligands have different binding affinities for the two receptors [36,80,83].
Endogenous estrogen binds mainly to ER [140]. The two receptors are expressed differently in
different tissues, where ER is found mainly in breast and ovarian tissue and ER is found in brain,
bone and bladder tissues [36,140].

Apart from these two ERs that are both found inside the cells, recent research has found G-protein
coupled receptors in cell membranes, which are activated by estrogen. The mechanisms and
involvement of these receptors in physiological processes are still being researched [70].

2
In breast tissue, estrogen stimulates growth of the ductal epithelium and connective tissue. Estrogen
however can impact negatively on this tissue, as it stimulates the growth of breast cancer cells [36].
Estrogens affect the cardiovascular system because they are natural vasoprotective agents. They
increase the formation and release of nitric oxide (NO) and prostacyclin in endothelial cells as well
as reduce vascular smooth-muscle tone. Controversy exists about whether estrogen treatment can
prevent atherosclerosis in postmenopausal women as epidemiological and intervention studies show
contradicting results [36]. Estrogen also affects bones as osteoclasts and osteoblasts contain ERs. In
osteoblasts, estrogen stimulates the secretion of the anabolic growth factor IGF-1, and inhibits the
secretion of cytokines, IL-1, TNF and IL-6 that are involved in bone resorption. Estrogen also inhibits
the function of osteoclasts by stimulating the synthesis and secretion of osteoprotegrin (OPG).
Estrogen is thereby involved in reducing bone resorption and maintaining bone health [36,86].

When women reach the perimenopausal stage, ovarian production of E2 decreases, which results in
decreasing plasma levels of E2. The E2 which is present is formed in extragonadal tissues and the
amounts that are synthesized increase with body weight [36]. Estrogen deficiency in postmenopausal
women is partly responsible for decreases in bone mass and increased risk of fracture over time, as
estrogen deficiency leads to increased bone resorption and decreased formation of new bone tissue.
Lack of activation of the ERs in bone tissue, leads to a decline in secretion of the growth factor IGF-
1 and OPG, stimulating bone remodelling and inhibiting osteoclast function respectively [36,88,140].

HRT is a treatment used for menopausal women to relieve menopausal symptoms and/or decrease
the risk of hormone-dependant diseases. The treatment includes estrogen sometimes combined with
a progestin, which is a synthetic progesterone that ought to counteract the proliferative action of
estrogen in the endometrium [22]. HRT is associated with an increased risk of breast cancer in
postmenopausal women, and the risk increases with duration of use [11,24]. The Womens Health
Initiative (WHI) study, a large randomized controlled study, found that HRT use was associated with
a 26% increased risk of developing breast cancer [102]. A recent meta-analysis of 52 epidemiological
studies revealed that current or recent users of HRT had a 37% increased risk of developing ovarian
cancer [23]. There are contradicting results regarding the effect of combining estrogen with progestin.
It is unclear whether progestin exerts proliferative or antiproliferative effect on the human breast, as
study results depend on the structure of progestin and the chosen tissue [107]. However, there are
several large observational studies that indicate a further increase in cancer risk when using progestin,
including The Nurses Health study reporting a yearly increase in cancer risk of 3.3% when using
estrogen alone compared to 9% for estrogen plus a progestin [22]. In The Million Women study
including 828,923 postmenopausal women they found a relative risk (RR) of 1.66 for developing
breast cancer in current users of HRT compared with never users. The risk was greater for users of
estrogen-progestin preparations than for estrogen-only preparations (RR: 2.0 and 1.3 respectively)
[11].

Estrogen has been shown to have vasculoprotective effects including beneficial effects on the lipid
profile and NO production. In The Nurses Health study the risk of coronary events was reduced by
45% in users of HRT [35]. There is however also evidence of the opposite including the WHI study

3
which was ended prematurely because of an increased risk of coronary events [102]. These
contradictions could be attributable to biases in observational studies, but there might also be a timing
effect where the influence of HRT on the risk of CVD is associated with the timing of initiation of
HRT (age and time since menopause) [41].

Selective estrogen receptor modulators (SERM) are nonsteroidal compounds that mimic the effect of
estrogen in some tissues, but have an antagonistic effect in other tissues [36]. This type of
pharmaceuticals is used for treatment of diseases related to hypoestrogenic states including
osteoporosis, CVD and cancer [9,90]. The combination of agonistic and antagonistic effects makes
SERMs useful for treatment of symptoms of postmenopausal women. The estrogenic effect on bones
and brain is desired to maintain bone strength and brain function, and agonistic effects on breast and
endometrial tissue are avoided [36].

Isoflavones are considered to be natural SERMs, even though their mechanism of action differs from
the synthetically manufactured SERMs [9]. They bind to ERs and are tissue specific in that they have
preferential and stronger binding to ER compared to ER [57]. The different isoflavones have
varying binding affinities to ERs [90,108]. Their preferred binding to ER results in a protective
effect against breast cancer as it inhibits mammary cell growth, which is an advantage compared to
HRT [90].

3 Isoflavone sources and structures

Secondary metabolites are organic compounds found in plants. Unlike the primary metabolites, they
function as part of a plants defence system. The secondary metabolites are generally divided into
three main groups; terpenes, phenolics and nitrogen-containing compounds [13,127]. The phenolic
group contains flavonoids that are a type of phytoestrogen, which are nonsteroidal compounds that
have structural similarities to estrogens (17--estradiol) and can exert estrogenic effects in the body
by binding to ERs. Flavonoids are the main type of phytoestrogens found in the western diet, and are
a compelling group of compounds from a nutritional and health perspective. Isoflavones which are
found in soy and red clover (RC) are part of this group of compounds [112,130,134].

Soy and RC (Trifolium pratense L.), both from the Leguminosae family, are abundant sources of
isoflavones [148]. Soy has been part of the traditional Asian diet for centuries, and it has been known
since 1931 that soybeans contain high amounts of isoflavones [1]. Soy is to date the most studied
source of phytoestrogens. RC is used in agriculture as it improves soil quality by fixing nitrogen. In
the 1940s the estrogenic effects of RC isoflavones were first documented, where a decrease in fertility
of grazing sheep on RC fields was found; later experiments have shown similar effects in cattle [9].

The main isoflavones found in soy are genistein, daidzein and glycitein [72]. In RC the main
isoflavones are formononetin and biochanin A, which is a unique feature of RC. Genistein and

4
daidzein are the demethylated forms of biochanin A and formononetin respectively, and are found in
smaller amounts in RC (Figure 1) [148].

Biochanin A Formononetin

Genistein Daidzein

ODMA Equol

Figure 1: Chemical structure of main isoflavones in soy and red clover and daidzein metabolites.

Isoflavones can exist as aglycones or in a conjugated form as glycosides. In soy and RC the
isoflavones are found in a complex mixture of aglycones and the glycoside conjugates -, malonyl-
and acetyl glycosides [15,91]. The malonyl- and acetyl glycosides are less stable than the -glycosides
and readily convert into -glycosides upon exposure to heat or other processing steps [112]. The -
glycosides are predominant in soy, whereas in RC the isoflavones are mainly found as malonyl
glycosides [148,156].

The concentration and composition of the isoflavones in plants depends on factors like plant cultivar,
environmental conditions and industrial processing [112]. The pattern of conjugation of the
isoflavones affects the ease with which the glycosides are hydrolysed or degraded by bacteria and
thereby the bioavailability of the isoflavones [53]. Processing of isoflavone containing foods can alter
the total content of isoflavones as well as the ratio between glycosides and aglycones. Generally
processing increases hydrolysis and thereby the content of aglycones, and in fermentation processes
the glucose moiety is removed and the level of aglycones is increased [74,91]. The aglycones are heat
stable, which means that it is mainly the conjugation pattern that is affected by heating processes and
not the total isoflavone content [112].

5
4 Absorption and metabolism of isoflavones

The gut microflora of an individual influences the bioavailability of isoflavones from the diet. The
composition of the gut microflora varies between individuals, and is established early in life (age 0-
3 years). It is considered to be relatively stable but dietary changes can affect the composition and
activity of colonic microbiota [110].

The glycoside form, in which the isoflavones are mainly found in their natural state, is not highly
bioavailable for humans, and a conversion is needed before the isoflavones are absorbed [111,115].
The reason that active transport isnt possible is thought to be due to their high hydrophilicity and
molecular weight [47]. The first step of isoflavone metabolism is the hydrolysis of glycosides into
their aglycone form. This hydrolysis is highly efficient and is carried out by glycoside hydrolases
along the entire length of the intestinal tract [15,91,101,114]. The intestinal bacteria are also
responsible for the demethylation of methylated forms of isoflavones, and less than 5% remain intact
[9,111]. The glycoside hydrolases are either endogenously present in the small intestine, eg. lactase
phlorizin hydrolase (LPH), or from bacteria colonizing the small and large intestine (-glucosidases)
[85,101].

Figure 2: Gut microfloral metabolism and distribution of isoflavones.

Aglycones are absorbed directly or further metabolised into other metabolites [15]. It is believed that
the direct absorption of aglycones takes place by non-ionic passive diffusion in the jejunum (Figure

6
2) [89,111]. When ingesting isoflavones there is an increase in plasma concentrations of isoflavones
1-2 hours after ingestion. This reflects direct absorption of the aglycones and is more pronounced
when ingesting aglycone rich formulations [47,53,89]. When ingesting glycoside-rich formulations
the main peak in most cases appears after 5-8 hours which is due to the hydrolytic cleavage and
absorption taking place in the lower part of the small intestine and in the colon, as well as some
enterohepatic recycling of conjugates [85,89]. In these cases there might also be a small peak early
after ingestion, which is due to some of the hydrolytic cleavage and absorption taking place in the
small intestine, and direct absorption of the minor proportion of aglycones present in the formulation
[89].

In the second step of isoflavone metabolism the aglycones are either absorbed by the liver to undergo
first pass hepatic conjugation, or metabolised further in the large intestine [134]. After absorption in
the liver the aglycones are either released in their active form to the blood stream, or undergo
conjugation; mostly into glucuronides and a small proportion as sulfates [115,134]. Genistein and
daidzein both have two conjugation sites and can therefore be found as mono- or di- glucuronides or
sulfates, as well as mixed conjugates with one sulphated and one glucuronidated site [119].
Isoflavones are found in blood either in the aglycone form or as conjugates, and generally low
concentrations of aglycones are found in blood as the first pass enterohepatic conjugation is efficient
[111,153]. The conjugation is carried out by UDP-glucuronyltransferase isozymes, and they have
both tissue and substrate specificity [115]. A study on ewes revealed that in tissues the isoflavones
are mainly found as glucuronides, and that there are large variations in concentrations between tissues
where the highest concentrations were found in kidney and liver [137].

The formed glucuronides enter the systemic circulation and are transported to target cells and tissues
where they exert their biological activity. Some of the isoflavones are excreted in the bile, and
intestinal glucuronidases can hydrolyse them making them available for re-absorption and further
enterohepatic recycling or for metabolism. The conjugation helps retain the isoflavones in the body
within the enterohepatic circulation, and prolongs their pharmacological activity [111,134]. The
extent of biological activity of the conjugated isoflavones is at present not fully clarified [153].
Studies have shown that the conjugates might have biological activity through direct actions or by
serving as immediate sources of aglycones in target tissues [119]. In an animal study using quercetin
it was shown that quercetin aglycones are found in tissues but not in plasma where it is mainly found
as glucuronides. -glucuronidase activity was also found in lung, liver, kidney and muscle tissue and
this suggests that the active aglycones are formed at target tissues [12]. The same mechanisms might
apply for isoflavones. The glucuronides are water-soluble and are eventually excreted in the urine
[151]. It has been shown that the conjugate patterns in urine and plasma are different, which suggests
that further metabolism takes place during renal clearance. Double conjugated metabolites are the
main conjugates found in plasma, whereas in urine the monoglucuronides predominate. This suggests
that the highly polar double conjugates cannot readily undergo renal excretion, and deglucuronidation
and desulfation of the metabolites takes place before excretion [43,119].

7
The ratio of the different isoflavones is different in bile and urine. Daidzein is found in higher
concentrations than genistein in urine, and a study of daidzein and genistein metabolites in blood and
urine after ingestion of soy flour showed that the recovery in urine was higher for daidzein than for
genistein, even though the composition ratio of genistein:daidzein was 1.7. This indicates that
genistein and its conjugated metabolites are excreted into the bile rather than urine to a larger extent
than daidzein, and undergo more enterohepatic circulation [43]. Genistein is more susceptible to
degradation by intestinal microflora compared to daidzein due to its 5-OH group, and less genistein
may therefore be available for absorption resulting in lower urinary recovery [14,149]. Unknown
metabolites of genistein may be present in urine, and therefore an underestimation of the excreted
amounts of genistein takes place [43,53]. Plasma genistein concentrations are consistently higher
than daidzein concentrations, which can be explained by the excretion into the bile mentioned before,
but also by the fact that daidzein is more extensively distributed in tissues in the body [111].
Furthermore, plasma daidzein has a faster clearance rate than genistein, and is therefore found in
plasma for a shorter time [103,144].

4.1 Isoflavone metabolites

Generally there is a low recovery of isoflavones in urine, and the faecal excretion is also low,
indicating that a large percentage of dietary isoflavones are metabolised beyond deglycosylation
[112,134]. In a study by Watanabe et al. recoveries of daidzein and genistein in urine and faeces were
55% and 20% respectively after ingestion of baked soybean powder [144]. Because of large inter-
individual variations in gut microflora, great variation in the amount of metabolites are found in
different individuals, and the diet has an influence on the degree of metabolism of isoflavones
[134,151].

The formed metabolites differ in structure and estrogenic effect from the parent molecule. Equol (7-
hydroxy-3-[4-hydroxyphenyl]-chroman) and o-desmethylangolensin (ODMA) are the main
metabolites of daidzein, and dihydrodaidzein and 4-hydroxyequol are also formed. ODMA is weakly
estrogenic (appr. one third of daidzein affinity to ER) and equol has a higher estrogenic effect than
daidzein (twofold higher affinity to ER). Fewer metabolites of genistein are known but one example
is p-ethyl phenol, which is hormonally inert [38,108,134,151].

Equol is the most abundant and potent metabolite of daidzein and is structurally similar to estradiol
[140]. The two enantiomeric forms, S- and R-equol, show different binding affinities to ERs [151].
Human intestinal bacteria show enantiomeric specificity in their production of equol, and only
produce S-equol [117,151]. The ability to produce equol is affected by race due to differences in gut
microflora. Approximately 30% of the population in America and Europe are able to produce equol,
whereas in Asia the proportion is 50% [89,91,111,140]. The conversion of daidzein into equol might
be affected by the habitual diet. It has been shown in some studies that a high carbohydrate, fibre and
polyunsaturated fat intake increases the rate of conversion, and there is a higher proportion of equol
producers among vegetarians than non-vegetarians [89,104,111,118]. Other studies have correlated

8
high equol production with high fat and meat intake, and some have shown that fibres have adverse
effect on isoflavone absorption. Yet other studies fail to show an effect of fibre and isoflavone intake
on the ability to produce equol [60]. The contradictory results may be due to variations in age, diet
and intestinal flora of the subjects [103]. Pre- and probiotics might also have an influence on
production of equol because of their effect on gut microflora [130]. There is a time delay of 6-8 hours
after ingestion before equol appears in plasma, which suggests that the metabolism takes place in the
colon [89,111]. A study revealed a higher plasma concentration of equol after ingestion of glycosides
compared to aglycones, as they are present in the colon and have a longer transit time [156]. A study
of the effect of food matrix showed a higher urinary equol excretion when ingesting tempeh, which
is a fermented solid food matrix, compared to soy milk and textured vegetable protein (TVP). A solid
food matrix might protect daidzein from degradation until it reaches the large intestine, where it is
metabolised to equol [28].

The isoflavones biochanin A and formononetin are demethylated, to a great extent, in vivo into
daidzein and genistein respectively [44,72]. This either happens by microbial demethylation or by
oxidative demethylation catalysed by cytochrome P450 enzymes [72]. Therefore, a proportion of the
biochanin A and formononetin ingested is found as daidzein and genistein in plasma, and generally,
the concentrations of biochanin A and formononetin are lower than daidzein and genistein [44,111].

5 Bioavailability

5.1 Aglycones vs. glycosides

Studies have shown that isoflavones from fermented soy products are more available to humans than
unfermented products, which is thought to be due to the formation of aglycones from glycosides in
the fermentation process [15,51]. In a study by Okabe et al. a higher maximal concentration (60%)
and area under the plasma concentration-time curve (AUC) (20%) were observed for the aglycone-
rich soybean powder compared with the glycoside rich form [89]. Cassidy et al. compared
pharmacokinetic parameters of daidzein and genistein after ingestion of tempeh (rich in aglycones)
and TVP (rich in glycosides) and found higher maximal concentrations (10-114 %) and AUCs (20-
120 %) after tempeh intake [15]. A study on three different soymilks (untreated, fermented and
enzymatically treated) showed that soy aglycones were absorbed faster and in higher amounts than
their glucosides [51]. Izumi et al. showed higher concentrations of isoflavones in plasma after both
single-bolus dosage and long term intake of aglycone- or glycoside-rich supplements based on
soybean extract [47].

Xu et al. however couldnt show differences in bioavailability when subjects were fed TVP, tempeh,
tofu and cooked soybeans, even though the four foods contained different amounts of aglycones and
glycosides [150]. Richelle et al. conducted a study where a prehydrolysis process was carried out,
and the bioavailability of the isoflavones was studied. The absorption patterns were very similar for

9
the aglycone and glycoside soy drink ingested, and there was no delay observed in the absorption of
glycosides [101]. These results indicate that although the deglycosylation step is essential for
absorption, it might not be rate-limiting, even though this has been suggested in other studies
[85,101]. These include studies by Setchell et al. and Izumi et al. who have shown that the time to
reach maximum plasma concentration is longer for glycosides than for aglycones [47,111].

Even though a higher bioavailability of aglycones is expected, a few studies have shown the opposite
including studies by Rufer et al. and Setchell et al. Both studies used pure isoflavones, which might
show different behaviour compared with food matrices, as interactions between the mixtures of
isoflavones are not found as in soy products. Furthermore, a small number of subjects were included
in the studies decreasing their power [105,111].

The higher bioavailability of glycosides is unexpected as the aglycones can be directly absorbed
without hydrolysis, and the hydrophilic properties and larger molecular weight of glycosides is
thought to result in poorer absorption in the gut [47]. It is known that apolar compounds are more
easily absorbed over the cellular membrane, and a study of isoflavone uptake in Caco-2 cells showed
that daidzein and genistein aglycones were taken up more easily into the cells, and the glucosides
were not transported through the cell monolayer [79].

The stability of aglycones and glycosides may also affect their degree of absorption. Differences in
their chemical structure result in glycosides being more stable and water soluble, and are therefore to
a large degree delivered to the -glucosidases in the large intestine and absorbed. The aglycones are
less stable against bacterial degradation and if allowed to reach the large intestine may, to a larger
extent be degraded into known or unknown metabolites prior to absorption. The sugar moiety of the
glycoside might protect against this form of degradation. [85,105]. Inter-individual differences in
intestinal conditions leads to variation in the degree of degradation of isoflavones and this contributes
to the disparities seen in bioavailability.

Other factors that might contribute to the contradicting results are differences in study design, food
matrix and composition of formulations. There is no consensus on linearity between dosage and
bioavailability, and it is therefore difficult to compare studies using different doses [105]. Some
studies are cross-over studies whereas others are not, and the inter-individual variations therefore
affect results to different extents, and might play a more important role in bioavailability than the
molecular form of isoflavones. Aglycones and glycosides bind differently to food components eg.
proteins, and bioavailability is influenced by whether the isoflavones are administered in a food
matrix or as pure compounds [98]. These differences make it difficult to determine the exact effect
of the attached sugar moiety, and suggests that isoflavone metabolism is more complex than it
appears.

Active transport of isoflavone glycosides has not been proven, and a study has denied the transport
of flavonoids by the sodium dependent glucose transporter (SGLT1), which has been shown to
transport quercetin glycosides in other studies [56]. It has been shown that attachment of a glucose

10
group enhances the bioavailability of quercetin compared to aglycones supporting the existence of a
glucose transporter [42]. Even though this has not been shown for isoflavones similar mechanisms
might exist for their absorption and future research might lead to the discovery of yet unknown
transporters. If these are present, they would be expressed differently in individuals, and this could
partly explain why there are differences between the studies. However, no isoflavone or quercetin
glycosides are found in human plasma so hydrolysis must take place before transport to the liver
[34,105].

It has been shown that aglycones are absorbed faster and in greater amounts than glycosides, which
is due to their hydrophobic character and lower molecular weight. A hydrolytic step is not needed
prior to their absorption and they are absorbed in the upper part of the gastro intestinal tract. The
contradictions in the field suggest that it is a challenge to simply compare aglycones and glycosides
as many other factors affect absorption and bioavailability including food matrix, isoflavone source,
intestinal bacteria and background diet.

5.2 Effect of matrix

The composition of isoflavones in supplements or foods varies depending on the origin of the
isoflavones and processing methods. The absorption of compounds is affected by the solubility of a
substance in the intestine, and therefore it is expected that isoflavones from a liquid matrix are
absorbed faster than isoflavones from a solid food matrix. This was shown in a study where faster
absorption rates and earlier peak serum concentrations were seen for soy milk compared with TVP
[15]. de Pascual-Teresa et al. studied the absorption of isoflavones after ingestion of cookies,
chocolate bars or juice, and the maximum concentrations in serum were the same for all three
matrices. The observed differences were a faster absorption of genistein following consumption of
the juice compared to cookies and chocolate bars, as well as a lower total urinary recovery of genistein
following ingestion of juice. The differences in the results between the groups didnt reach statistical
significance, but the data suggested that absorption of isoflavones may be affected by the food matrix
[91]. In a study looking at urinary recovery of isoflavones after ingestion of different soy foods, no
differences between soy milk, tempeh and TVP were seen in total urinary isoflavone excretion.
However, when looking at the individual isoflavones and subject groups food effects could be found
[28]. Franke et al. also studies urinary excretion of isoflavones after ingestion of four different soy
foods, and absorption patterns were different for the studied isoflavones. In accordance with some
previous studies, they found higher bioavailability for soymilk, but for the protein powder drink the
bioavailability was lower than the solid foods. Differences were also found between the two solid
foods, indicating that not only the consistency of the food matrix but also the composition (eg. fat and
fibre content) influences bioavailability [29].

Many supplements containing isoflavones from various sources are available, but it is unknown
whether they have the same clinical effect as isoflavones from phytoestrogen-rich foods.
Furthermore, it is not known whether possible differences in effects are caused by differences in

11
bioavailability, metabolism or other factors [111]. Studies comparing bioavailability of isoflavones
from supplements and food matrices are limited, and show different results. Gardner et al. showed
that the bioavailability of isoflavones was higher for soy foods than isoflavone supplements. Even
though the dosage of isoflavones was lower when provided via soy foods, a higher serum
concentration of genistein and daidzein were seen when ingesting soy foods compared to tablets. The
exact isoflavone content in the soy foods was not chemically determined but estimated from
databases, and the isoflavone form was not known and could be very different from the 98% glucoside
content in the supplements [30]. Vergne et al. compared bioavailability of daidzein and genistein after
ingestion of a soy supplement and soy based cheese, and found a higher bioavailability for
supplements. The total isoflavone intake was similar for the two formulations, but there were
differences in aglycone content (highest in cheese) and the ratio between daidzein and genistein [139].
Differences in aglycone content and ratios between isoflavones could have a great influence on the
results. Another factor which could influence bioavailability in these cases is the excretion of bile.
This is induced when ingesting protein and lipids, and has been shown in an vitro study to potentially
increase bioavailability [142]. As the isoflavones are found in a more pure form in supplements and
are not bound to other components as in a food matrix, a higher bioavailability from supplements
could be expected. However, synergistic effects between isoflavones and other components may be
present in food matrices affecting bioavailability of isoflavones in yet unknown ways.

It is not possible to draw clear conclusions on the differences in bioavailability between supplements
and isoflavone rich foods, and lacking standardisation of biological products is a further challenge as
large variations in isoflavone content are seen in commercial preparations, and large discrepancies
between declared and actual content have been found [111,139].

5.3 Intestinal microflora

Endogenous glucosidases in the small intestine have been shown to be responsible for hydrolysis of
isoflavones in germ-free rats [14]. This suggests that hydrolysis of glycosides is to a certain extent
carried out by mammalian glucosidases, but glucosidases produced by gut microflora are also an
important source and the gut microflora thereby has an influence on the bioavailability of isoflavones
[104,134]. Enterococci display high -glucosidase activity followed by Lactobacillus, Bacteroides,
and Bifidobacterium. These microorganisms are present in high numbers in the small and large
intestine [134]. Gut microflora are also responsible for metabolism and degradation of isoflavones
and the extent of this is reflected in isoflavone recovery in faeces. Xu et al. showed that urinary
recovery of isoflavones was twice as high in high faecal excretors compared to low excretors [149].
The bacteria in the high excretors are not as effective as in the low excretors and the isoflavones are
therefore more available for absorption, and the intestinal microflora thereby has an impact on
isoflavone bioavailability. There are large inter-individual differences in the absorption and excretion
of isoflavones, which might be due to the influence of differences in gut microflora populations as
well as past exposure to isoflavones and glucosidase production [53,101,156].

12
Probiotics are able to increase populations of bacteria that are beneficial to host health, and have been
shown to alter enzyme activity and contribute to conversion of glycosidic isoflavones into aglycones
in soy products [84]. It is expected that the bioavailability of isoflavones would be increased by
ingestion of probiotics as it increases bacterial populations in the gastrointestinal tract and enzyme
activity [61]. In a study by Cohen et al. there was a 40% reduction in urinary excretion of isoflavones
after probiotic administration indicating increased circulating levels of isoflavones [21]. Nettleton et
al. and Larkin et al. however failed to show increased plasma levels of phytoestrogens after
administration of probiotics, even though the gut microflora was significantly altered [61,84]. It
cannot be excluded that different bacterial strains, higher levels of probiotic bacteria or longer-term
intake of probiotics might show different results. Amount of isoflavone intake may also affect results,
as hydrolysis may not be a rate-limiting step at low doses.

Gut transit time (GTT) may also have an effect on the bioavailability of isoflavones, as it affects the
degree of metabolism. The longer the isoflavones are in the gut, the more time there is for
microorganisms to degrade them and a smaller amount is available for absorption [38]. Zheng et al.
also showed a higher absorption of isoflavones in women with a rapid GTT and a low isoflavone
degradation phenotype [155].

5.4 Effect of diet

Fibres might have an influence on the absorption of isoflavones, and a study by Piazza showed an
increase in isoflavone absorption upon ingestion of inulin. This is thought to be due to a stimulation
of bacterial growth and thereby increased hydrolysis of glycosides into aglycones [93,140]. Tew et
al. however showed that the total urinary excretion of genistein was reduced with increased fibre
content in the diet. They suggest that the insoluble fibres bind to the isoflavones and prevent their
absorption throughout the gut. Furthermore, the insoluble fibres bind water and thereby cause bulking
of the luminal content of the intestine, and a dilution of the gut microflora responsible for the
hydrolysis of the isoflavones prior to absorption [131]. Other dietary compounds such as protein and
fat may also affect the absorption of isoflavones. A short-term study failed to show differences in
bioavailability even though the protein and fat content varied in the diets. This may be because the
study was too short, hence a longer term study would be better suited to show whether diet has an
effect on bioavailability through effects on gut microflora [150].

Several studies have shown that a daily intake of isoflavones results in reduced bioavailability, and
the explanation for this is not known. It may be due to the substrate concentration exceeding the
hydrolytic capacity, rate-limiting uptake of aglycones or the isoflavones inducing their own
metabolism [8,69,115]. Others however fail to show an effect of habitual intake. Wiseman et al.
demonstrated that a habitual intake of soy isoflavones did not significantly affect concentrations of
isoflavones and their metabolites in plasma, urine and faeces. Apart from an increase in -glucosidase
activity in the subjects consuming high soy diets, there doesnt seem to be an effect of habitual soy
intake on bioavailability [145]. In a study by Lampe et al. there was no difference in isoflavone

13
excretion between short- (3 days) and long-term (1 month) intake groups of premenopausal women
[60].

5.5 Conclusion on bioavailability

Bioavailability has been shown to depend on many factors. The form isoflavones are in upon
ingestion affect bioavailability where most studies have shown that the aglycones are more
bioavailable than the glycosides as the hydrolysis step in the intestines is not needed [47,89]. No clear
conclusions can be drawn regarding the effect of food matrix on bioavailability, but there is a
tendency that it is higher for liquids compared to solid foods [16].

Gut microflora has a big impact on bioavailability and large inter-individual differences are seen.
Even though clear results have not been found, the composition of habitual diets (fibre, fat, protein)
is suggested to affect bioavailability through influences on gut microflora, and the extent of daily
intake of isoflavones might also have an impact.

Many clinical trials have been carried out studying bioavailability of isoflavones, but large differences
in the study designs and participant number make it difficult to draw clear conclusions. Differences
in gut microflora, GTT and degradation phenotype may be a large contributor to differences in
absorption and blunt the effects of other studied factors (matrix, aglycone/glycoside etc.).

6 Safety

The use of isoflavones as an alternative to HRT is becoming increasingly popular. When using
isoflavone supplements there is potential of ingesting large amounts of isoflavones per day; much
larger than doses obtained by isoflavone containing foods. This makes it important to assess the safety
of ingesting large amounts of isoflavones daily.

In a 2-year study including healthy menopausal women, Steinberg et al. evaluated the safety of
supplements derived from soy hypocotyl containing mainly daidzein and glycitein in doses of 80 or
120 mg/day. They evaluated clinical blood chemistry measures (including complete blood count,
thyroid and liver function, estradiol and lipid profiles), adverse events and did a physical examination
(including blood pressure, endometrial thickness and mammogram). No statistical differences were
found in blood chemistry markers except for urea after 2 years intervention, which remained within
the reference range, and no differences were observed in the physical examinations. Two adverse
events were reported, but the incidences were not different from those predicted from statistical
probabilities, and their overall conclusion was that 2 year intake of isoflavones from soy hypocotyl
is safe [125]. A study by Nahas also found use of soy isoflavones for 10 months in postmenopausal
women safe as no serious adverse events were reported and clinical outcomes including endometrial
thickness, mammography and hormonal profiles did not change as a result of soy extract supplement

14
intake [81]. Powles et al. carried out a safety study of isoflavones from RC and found that they were
safe and well tolerated by women with a family history of breast cancer. After 40 mg daily intake
during 3 years no significant differences in breast density and endometrial thickness were seen
between the isoflavone and placebo group [96]. Several more studies of varying size (19-138
subjects), isoflavone dose (36-92 mg/day) and duration (6-36 months) have also evaluated isoflavone
intake as safe based on mammographic breast density, endometrial thickness and reports of adverse
events [7,71,92,106].

There are also studies that report of serious adverse effects after long-term intake of isoflavones.
Unfer et al. conducted a large 5-year study in healthy postmenopausal women where they observed
endometrial hyperplasia in 3.8 % of the subjects in the intervention group compared to 0 % in the
placebo group. No cases of endometrial hyperplasia were observed in the intervention group halfway
through the study (after 30 months) and no cases of endometrial carcinoma were observed during the
study period. It should be noted that the daily isoflavone dose was 150 mg which is about triple the
daily intake of Asian populations [136]. In a smaller study by Wolff et al. 32 postmenopausal were
given 80 mg/day of RC isoflavones during six months, and at the end of the study endometrial
alterations were seen in three women. It should be noted that this study has a small study population
and there was no placebo group for comparison [146].

It is difficult to determine the most appropriate isoflavone dose and study duration for safety studies.
In some of the previously mentioned studies the dose of isoflavones is higher than recommended
values, which might induce cellular changes that would not have occurred at lower doses [136]. Soy
foods and isoflavone preparations have different compositions of isoflavones, and the results from
safety evaluations in one study cannot necessarily be used to establish safety profiles of other
isoflavone preparations. Despite differences in study designs, most studies suggest minimal health
risks with isoflavones supplementation and epidemiological data support the claim that isoflavone
intake is safe as a lower rate of endometrial cancer is seen in Asian women compared to Western
societies [10,125]. Isoflavones modify ERs in different ways depending on the structure of the
receptor, which means that they can exert agonistic, partial agonistic and antagonistic effects. Because
individuals have different distributions of ERs, they can also have different sensibilities towards the
isoflavones, and this can affect the results of safety evaluations [136].

7 Physiological effects of isoflavones

7.1 Menopause

Isoflavones have estrogenic effect because they have structural similarities to estrogen and similar
molecular weights. Estrogenic effects are reached when a steady-state plasma concentration of
isoflavones of 50-800 ng/ml is reached, which corresponds to concentrations found in Asian
populations [9]. The 4 hydroxyl group in the isoflavone structure is the binding site for ERs, and

15
therefore the structures of the different isoflavones have an impact on their estrogenic effect [111].
Morito et al. showed that methylation of the hydroxyl group reduced the affinity of the isoflavones
towards ER, as formononetin and biochanin A bound less strongly to ER and compared to
genistein [75]. There is also a difference in the binding affinities of genistein and daidzein. Genistein
has a much higher binding affinity to ER than daidzein, which is due to a decrease in polarity of the
ester functionality caused by interactions between the proximal hydroxyl group and the carbonyl
group [80].

The effect of isoflavones on ERs are similar to SERMs, and they show agonistic or antagonistic effect
depending on the concentration of endogenous estrogen and the type of ER [115,151]. In case of a
high level of endogenous estrogen, phytoestrogens bind to ERs and reduce the effect of endogenous
estrogen, and thereby induce antagonistic effects. On the contrary, if there are low levels of
endogenous estrogen in the body, phytoestrogens act as stand-ins for estrogen and exert agonistic
effects [66,80].

It has been shown in several studies that equol producers have greater benefits of isoflavone
containing diets compared to non-equol producers [113,118]. This is assumed to be because S-equol
is more estrogenic compared to daidzein and other metabolites of daidzein, as it has similar binding
affinities to ER and ER as genistein, which is the most potent soy isoflavone [130,151]. The
binding affinity of S-equol to ER is relatively poor, which is also the case for the ability of R-equol
to bind to both types of ERs [117]. It is unique for S-equol that it possesses estrogenic effects as well
as antiandrogen action, which might be the reason why a greater effect of isoflavone diets is seen in
equol producers [118]. Equol binds ER receptors with 20% the affinity of 17-estradiol, but is
excreted in amounts 10 to 1000 times greater than endogenous estrogen depending on the diet
[46,117]. A larger proportion of equol is found in serum in its free form compared to daidzein and
estradiol [151]. Only 50% of equol is protein bound, compared to 95% of endogenous estrogen. Only
the unbound compounds can bind to ERs, and this difference means that the biological potency of
equol is enhanced compared to estrogen [117]. Furthermore, there are differences in the renal
clearance of the different isoflavones and metabolites, and equol remains in plasma for a longer time
than genistein and daidzein which further enhances its biological effect [118,156].

The estrogenic effect of isoflavones is thought to help relieve menopausal symptoms including hot
flushes. In a study by Lipovac et al. administration of 80 mg/d of RC isoflavone aglycones resulted
in a 74% reduction in menopausal symptoms (hot flush/night sweat frequency and Kupperman index)
in postmenopausal Austrian women [67]. A 78% reduction in menopausal symptoms was seen in
postmenopausal Ecuadorian women who received RC supplements for 90 days. A placebo effect was
seen in this study, as a 23% reduction in Kupperman index was seen after administration of placebo
[40]. Many of the studies of menopausal symptoms have outcome measures based on subjective
evaluations, including the two mentioned studies. In one study sweat secretion over 24 h was
measured in menopausal women suffering from menopausal symptoms. A mean reduction of 15% in
hot flush frequency and 32% in intensity of hot flushes was found after daily intake of RC during 12
weeks [59].

16
There are also many examples of studies that either fail to show an effect or show similar effects in
intervention and placebo groups. In a study by del Giorno et al. similar reductions in menopausal
symptoms were seen in groups receiving 40 mg isoflavones and placebo [32]. Tice et al. investigated
the effect of two RC supplements providing 82 mg and 57 mg isoflavones on the frequency of hot
flushes in postmenopausal women. They found similar reductions of around 37% in the intervention
and placebo groups [133]. These results might be a reflection of the placebo effect where expectations
influence perception, which becomes very apparent when subjective outcome measures are used.

Isoflavones are thought to help relieve menopausal symptoms through their estrogenic effects, and
many studies have investigated the effects of phytoestrogens on menopausal symptoms with varying
results. There are many factors contributing to the contradicting results in these studies, and
heterogeneity between study designs (isoflavone preparations, outcome measures etc.) is a major one.
The lack of clear results might also partly be explained by the differences in ability to produce equol.
Equol producers might have the most gain of isoflavones in relieving hot flushes, and there has been
made no distinction between producers and non-producers in most studies [114].

7.2 Osteoporosis

When women reach menopause the lack of estrogen production results in an imbalance in bone
metabolism where the rate of bone resorption exceeds the rate of bone formation. This leads to a
decrease in bone mineral density (BMD) and an increased risk of fractures and development of
osteoporosis. Phytoestrogens might be able to improve the imbalance due to their estrogenic effects.
In vitro studies and studies on rodents have shown that isoflavones reduce bone resorption and
therefore potentially could have antiosteoporotic effect [114]. ERs have been found in osteoblast and
osteoclast cells, and it has been shown that the action of daidzein is mediated through ERs in these
cells, resulting in stimulation of osteoblasts and inhibition of osteoclasts [31]. Occhiuto treated
ovariectomized rats with RC isoflavones and showed a reduction in the number of osteoclasts. The
treated rats had increased bone mineral content and femoral density as a result of reduced bone
resorption [88].

The antiosteoporotic effect has also been investigated in several human studies, but contradicting
results exist. Epidemiological and cross-sectional studies have shown a reduction in fractures and
increases in BMD with increasing intake of soy products [73,152]. Some intervention studies have
shown an improvement in BMD after administration of phytoestrogens, while others have failed to
show a beneficial effect of isoflavones on BMD. Atkinson et al. investigated the effect of 1 year daily
supplementation of RC isoflavones (appr. 40 mg/d) on bone density in women. Isoflavone
supplementation resulted in a smaller decrease in spine bone mineral content (BMC) and BMD
compared with placebo; percentage changes in BMD were - 1.08 % and -1.86 % for treatment and
placebo groups respectively. A similar pattern was seen for the hip but differences were not
significant, probably due to the hip consisting primarily of cortical bone which has a slower bone

17
turnover [6]. Examples of studies that fail to show a positive effect of isoflavones on bone include a
study by Levis et al.where postmenopausal women received 200 mg/d isoflavones from soy during 2
years. No significant differences were found in spinal (-2.0% vs. -2.3%), femoral neck (-2.2% vs. -
2.1%) and total hip (-1.4 % vs. -1.2%) BMD between the isoflavone and placebo group [64]. Tai et
al. also failed to show differences in rates of bone loss between 2 year daily intake of soy isoflavones
(300 mg/d) or placebo, evaluated from lumbar spine and femoral neck BMD values as well as bone
turnover markers [126]. The reason for conflicting results in the intervention studies might be
differences in study designs including dosage, product form and study duration. Especially the source
of isoflavones may have a significant influence on the antiosteoporotic effects as intervention studies
using RC generally show more positive effects than soy, which may be due to the unique composition
and higher content of isoflavones in RC. Furthermore, it might be important in such studies to make
a discrepancy between equol producers and non-producers as the former may have a more
pronounced effect of isoflavone treatment on bone state [31,114].

Formononetin, which is found in high concentrations in RC, has been found to have an increased
potency on osteoblast function compared to daidzein, and exerts its action by activating the p38
mitogen-activated protein kinase dependent pathway. Gautam et al. have shown that formononetin
does not exert its action through ERs in the same way as daidzein, and is purely osteogenic [31].
Tyagi et al. showed that formononetin restored bone in osteopenic rats by enhancing bone formation
[135]. Due to a high degree of demethylation in the gut, the concentration of formononetin in plasma
is however lower than daidzein and genistein, and the effect on bones is mostly from the two latter
isoflavones and the metabolite equol [111].

Through their estrogenic effect, isoflavones are thought to have a beneficial impact on bone health in
postmenopausal women with an imbalance in bone metabolism. Isoflavones have been shown to
inhibit osteoclasts and stimulate osteoblasts in in vitro and animal studies. Soy isoflavones are the
most extensively studied and results are inconsistent, but the few studies on RC show very promising
results, which is most likely due to its high content of formononetin [6,17,52,59].

7.3 Cancer

Four Asian retrospective epidemiological studies have shown that when ingesting isoflavones early
in life they exert a protective effect towards breast cancer. Korde et al. investigated correlations
between soy intake and risk of breast cancer in Asian American women, and found an inverse
relationship which was strongest for childhood soy intake (RR: 0.40), but was also seen for adolescent
and adult soy intake. Lee et al. found a protective effect of soy protein and isoflavone intake against
premenopausal breast cancer in Chinese women. A high soy food intake during adolescence was
associated with a 43% reduced risk of premenopausal breast cancer [55,63,121,147]. Thanos et al.
found similar results among non-Asians (Canadian study). They found a 20% reduction in breast
cancer risk for a high isoflavone intake during adolescence [132]. These findings are further

18
supported by animal studies. Lamartiniere et al. found a reduction in chemically induced mammary
cancer in rats after prepubertal exposure to genistein [58].

Studies have shown that daidzein and especially genistein have anticarcinogenic effect because they
inhibit the proliferation of cancer cells. The exact mechanism behind is unknown but is thought not
to be exclusively hormonal and include several properties like activation of ERs, inhibition of growth
promoting steroid hormones, antioxidative and antiangiogenic properties [47,112,140]. Pino et al.
have shown that isoflavone intake is associated with an increase in sex hormone-binding globulin
(SHBG) in postmenopausal women, especially in those women having a low level of SHBG at
baseline [94]. An increase in SHBG decreases the free fraction of estradiol in plasma and thereby its
biological activity, and this can partly explain the anticarcinogenic properties of isoflavones [1].

The anticarcinogenic effects of isoflavones are different in equol-producers and non equol-producers,
as there is an enhanced expression of estrogen-responsive genes in equol-producers [151].
Furthermore, equol-producers might have a more favourable hormonal profile. A study by Duncan et
al. showed that equol-producers had lower plasma levels of sex hormones and higher levels of SHBG,
which is a hormonal pattern, associated with a lower cancer risk [26]. The bacteria involved in
converting daidzein to equol, also reduces the enterohepatic recycling of reproductive hormones
[151].

However, concern has been raised about isoflavones being harmful to breast cancer patients. Several
studies have evaluated the effect of isoflavones on breast-cell proliferation, and an increase in cell
proliferation was not found in response to isoflavone intake by Hargreaves et al and Cheng et al.
[18,37]. Even though these are small and short-term studies, the results are positive as opposed to the
increased cell proliferation seen with HRT [25,78]. Epidemiological studies of this issue have also
been done, and an analysis of three cohort studies carried out by Nechuta et al. showed that isoflavone
intake by cancer survivors was associated with at reduced risk of breast cancer recurrence and
mortality [82].

Epidemiological studies suggest that ingestion of isoflavones early in life has a protective effect
against cancer later in life, and there are animal studies that support these findings. The exact
mechanisms behind potential beneficial effects are hard to determine because of many pathways
being involved in cancerous diseases and isoflavones potentially acting in many different ways
(hormonal, antioxidative etc.) It is difficult to make definite conclusions on whether phytoestrogens
have an anti-carcinogenic effect. Even though there is much evidence to support this hypothesis, the
nature of the disease makes it difficult to investigate the effects, as there is potential for many biases.
Hence, a greater number of larger scale and longer-term studies are needed.

19
7.4 Diabetes

The prevalence of diabetes and metabolic syndrome is rapidly increasing worldwide. Isoflavones
could potentially have a beneficial effect on these conditions by affecting lipid metabolism and
glucose homeostasis.

Several animal studies have shown promising effects of isoflavones on plasma lipid profiles and
glucose levels. In a study with Zucker diabetic fatty rats lower plasma concentrations of total
cholesterol, triglycerides (TG) and free fatty acids (FFA) were seen after administration of isoflavone
rich soy protein for ten weeks. Furthermore, lower levels of fasting blood glucose were detected in
the soy-fed group throughout the treatment period [27]. Nordentoft et al. carried out a study with
KKAy mice and found a 38% reduction in TG and 31% reduction in total cholesterol after 9 weeks
feeding with isoflavone rich soy protein. The soy protein was also shown to have antihyperglycemic
and antihyperinsulinemic properties as a 64% reduction in plasma glucose and a 85% reduction in
insulin was seen [87]. This shows that the animals have become more sensitive towards the action of
insulin, which is positive in the treatment of diabetes.

Many trials have been conducted to investigate the effect of soy proteins on plasma lipid profiles in
humans [5,99,129]. A meta-analysis of 38 studies has shown that soy proteins reduce serum
concentrations of total cholesterol (9%), LDL cholesterol (13%) and TG (11%) in humans. The effect
is most pronounced in hypercholesterolemic subjects [5]. Another meta-analysis of 11 studies
compared cholesterol lowering effects of isoflavone enriched and depleted soy protein. Enriched soy
protein significantly reduced serum total (1.77%) and LDL (3.58%) cholesterol compared to depleted
soy protein when ingesting 100 mg isoflavones per day [129]. These studies have created the
foundation for the health claim from The Food and Drug Administration (FDA), that 25 grams of soy
protein a day may reduce the risk of heart disease. In an intervention study by Hermansen et al.
reductions in LDL cholesterol, LDL/HDL cholesterol ratio and TG levels of 10%, 12% and 22%
respectively were found in type 2 diabetic subjects supplemented with isoflavone-rich soy protein for
6 weeks. No effect of soy protein was seen on glucose and insulin responses [39]. Studies using
isoflavone sources other than soy protein have also shown lipid lowering effects. A 2 year study
including postmenopausal women who were administered 50 mg/d isoflavones as RC supplements
showed a 12% reduction in serum LDL cholesterol [20]. Clerici et al. conducted a study with soy
germ-enriched pasta which is rich in isoflavone aglycones. Intake of this pasta was compared with
conventional pasta in hypercholesterolemic adults and showed reductions in total and LDL
cholesterol of 7.3% and 8.6% respectively, and the effect was more pronounced in equol producers
[19]. It is known that estrogen has an impact on lipid profiles, and the positive effects of isoflavones
might be due to their estrogenic properties.

There are however also studies that fail to show an effect of isoflavones on the lipid profile.
Administration of a RC supplement to postmenopausal women with moderately elevated plasma
cholesterol levels didnt significantly alter plasma levels of total, LDL and HDL cholesterol as well
as TGs [45]. Lichtenstein et al. showed that isoflavones added to soy and animal protein diets did not

20
have an effect on plasma lipid levels in moderately hypercholesterolemic subject [65]. As the subjects
in these studies only had moderately elevated levels, the possible effects of the intervention may be
difficult to detect.

Glucose metabolism impairment is an important feature of the metabolic syndrome and the effects of
isoflavones on glucose metabolism is another way in which isoflavones might affect the risk of
developing type 2 diabetes. Zhang et al. found in a meta-analysis that soy isoflavone supplementation
reduced body weight, blood glucose and insulin in postmenopausal non-Asian women. The effect on
body weight and insulin levels was most pronounced in women with normal body weight (BMI < 30)
and the best effect on blood glucose is obtained with long-term and lower dosage of isoflavones [154].
In a one year study by Squadrito et al. the effect of pure genistein was investigated in postmenopausal
women with metabolic syndrome. They found a mean difference change in HOMA-IR of -1.4 and a
significant reduction in blood glucose and insulin levels after 12 months intervention. Beneficial
effects on lipid profiles and blood pressure were also found in this study. The study showed that
genistein treatment significantly improved features of the metabolic syndrome, and after the
intervention only 30% of the women in the genistein group met the criteria of metabolic syndrome
[124].

However, contradictions exist regarding the effect of phytoestrogens on glucose metabolism. A meta-
analysis of 10 intervention studies showed that daily ingestion of isoflavone mixtures did not reduce
serum levels of glucose in non-Asian women [100]. Gopert et al. failed to find an effect of soy protein
isolate on markers of glycaemic control after 8 weeks intervention in type 2 diabetics who did not
manage their disease with medication. Fasting and postprandial glucose and insulin levels, HbA1C
and HOMA-IR were not reduced after the intervention compared to intake of milk protein [33]. The
contradictory results regarding glucose homeostasis may be caused by differences in baseline values
before intervention and study duration, as shorter studies generally fail to show effect.

Other dietary compounds having a more pronounced effect on lipid metabolism than isoflavones
might contribute to the contradictory results. It is not clear whether the positive effects on lipid
profiles and glucose homeostasis seen in studies with soy protein are due to isoflavones or other
components of soy (proteins, fibers, saponins etc.) or a combination. In a meta-analysis it was shown
that depleted soy protein significantly lowered LDL and total cholesterol and increased HDL
cholesterol compared with animal protein, which suggests a beneficial effect of the proteins in soy
[129]. There is however also evidence that fibre and isoflavones in the soy have lipid lowering
properties [39]. All this suggests that several compounds in soy have beneficial effect on plasma lipid
profiles and there might even be synergistic effects [95,128]. Other factors potentially contributing to
contradictions are differences in the ability of the subjects to produce equol, differences in plasma
cholesterol states of subjects before intervention and the limited sample sizes in some of the studies,
which might affect the ability to detect significant differences.

The exact mechanisms behind the effects of isoflavones on lipid and glucose metabolism are not clear
and remain controversial. One way of action is thought to be through activation of peroxisome

21
proliferator activated receptors (PPARs). PPAR is involved in numerous processes in the body
including lipid metabolism, glucose homeostasis, insulin sensitivity and inflammation. Isoflavones
may act as selective PPAR modulators with an agonistic effect similar to endogenous ligands [143].
Mueller et al. showed in an in vitro study that isoflavones from RC, and especially their metabolites,
are potent PPAR binders and activators. Their maximal PPAR activities range from 15-32% of the
maximal activity of rosiglitazone, which is a synthetic PPAR activator used for treatment of type 2
diabetes but with negative effects on lipid profiles and weight [76].

Isoflavones have also been shown to act directly on pancreatic -cells. In vitro studies carried out by
Liu et al. showed that genistein increased glucose stimulated insulin secretion by increasing adenylate
cyclase activity and thereby elevating intracellular cAMP levels. This leads to activation of the
cAMP/PKA cascade and increased insulin secretion [68]. Genistein is a potent inhibitor of tyrosine-
specific protein kinases, and this effect may partially explain the effect genistein has on insulin
secretion as tyrosine kinases have been shown to be involved in the regulation of islet -cell function
[4,123].

Many other mechanisms have been suggested including inhibition of -glucosidase and glucose-6-
phospahatase and reduction of intestinal glucose uptake [2,62,138].

Evidence suggests that soy protein has beneficial effects on the conditions associated with the
metabolic syndrome including decreases in LDL and total cholesterol as well as blood glucose. These
effects probably stem from several compounds in soy, and isoflavones might be a contributor, as
effects have also been shown with RC supplements and pasta rich in isoflavone aglycones. A clear
conclusion can however not be drawn as there are also studies failing to show beneficial effects of
isoflavones on plasma lipid profiles and glucose levels, and this is most likely due to large between-
study heterogeneity and the beneficial effects on the metabolic syndrome stemming from many
compounds in the diet.

7.5 Cardiovascular disease

The metabolic syndrome is associated with an increased risk of coronary heart disease and
cardiovascular mortality, and increasing age and menopause is associated with an increased risk of
suffering from CVD [20,39]. Estrogen deficiency leads to unfavourable changes in lipid metabolism
and an increase in serum cholesterol levels, which are risk factors for developing CVD. Apart from
evidence for the beneficial effects of isoflavones on plasma lipid profiles described in section 7.4,
there are other ways in which isoflavones affect the risk of CVD.

Endothelial NO has a vasodilator function and is involved in the regulation of blood flow and vascular
tone. NO has also been shown to have anti-inflammatory and antiatherogenic effects, and decreased
NO synthesis is partly responsible for the initiation of atherosclerosis [122,151]. Endothelial nitric
oxide synthase (eNOS) is activated by estrogen, and it is of interest whether phytoestrogens can

22
activate eNOS in postmenopausal women with reduced estrogen production. Administration of RC
isoflavones to human endothelial cells showed an increase in NO release and activation of eNOS.
This is a genomic effect where an upregulation of eNOS expression is seen, similar to that induced
by estrogen. The effect was enhanced when the isoflavones were provided together with menopausal
concentrations of estradiol [122]. Joy et al. showed that equol is a potent activator of NO production
in human endothelial cells, and documented that equol induces relaxation of aortic rings [49].
Inducible nitric oxide synthase (iNOS) is another form of NOS predominantly found in macrophages,
and overproduction of NO by iNOS is associated with development of atherosclerosis. Studies have
shown that daidzein, genistein and equol inhibit NO production and expression of iNOS, which may
be part of the explanation for their protective effects [50,120].

Atherosclerosis is an inflammatory process that is associated with increased expression of certain

genes including cytokines, growth factors and acute-phase proteins [9]. Isoflavones have been shown
to inhibit the activation of transcription factor NF-B that plays an important role in the expression
of various genes related to inflammation [77]. C-reactive protein (CRP) is a marker of inflammation
that is often elevated in patients with atherosclerosis. Serum concentrations of CRP were reduced by
42% in a study with soy germ pasta rich in isoflavone aglycones [19]. Isoflavones inhibit cell
adhesion, alter growth factor activity and inhibit cell proliferation which are all factors involved in
atherosclerotic lesions and plaque formation. These effects are believed to be mediated through
inhibition of tyrosine kinases [4,97]. Oxidative modifications of LDL is also an important mechanism
in atherosclerosis, and the antioxidant properties of isoflavones may reduce lipid oxidation, by acting
as free radical scavengers through hydrogen/electron donation [151]. The number and position of the
hydroxyl groups have an impact on the antioxidative effect of isoflavones, and some metabolites are
more antioxidative than the parent molecules, eg. equol is a more effective antioxidant than daidzein
and genistein [151]. Being an equol producer might therefore lead to a greater reduction of the risk
of developing CVD.

Activation of PPARs is associated with an improved lipid profile as it increases HDL cholesterol,
decreases circulating TG levels and shifts LDL cholesterol particle size to a less atherogenic form.
PPAR activation may also exert antiatherosclerotic actions through other mechanisms including
reducing oxidative stress, accumulation of advanced glycation end products and inflammation [48].
Phytoestrogens have been shown to be PPAR agonists, and may exert their antiatherosclerotic effect
through this activation.

Apart from potential effects on plasma lipid profiles, isoflavones have also been shown to increase
production of NO by eNOS (which has a vasodilator function), as well as inhibit NO production by
iNOS (which is associated with development of atherosclerosis). Isoflavones are shown to reduce the
expression of genes associated with inflammatory processes in atherosclerosis and prevent cell
adhesion and proliferation through inhibition of tyrosine kinases. Their antioxidative effects limit
lipid oxidation. All these factors contribute to the capability of isoflavones to lower the risk of CVD.

23
8 Conclusion

Research on the physiological effects of isoflavones has provided evidence of potential ameliorating
health effects in several diseases including menopausal symptoms, osteoporosis, cancer and type 2
diabetes. They exert their beneficial effects through estrogenic activities and antioxidant properties
as well as activation of PPAR pathways. Soy and RC are rich sources of isoflavones. The fact that
HRT increases the risk of breast cancer has further augmented the popularity of isoflavones in use as
a safer alternative treatment.

Isoflavones are largely metabolised in humans, and some of the metabolites have higher biological
activity compared to parent molecules; for example equol, a metabolite of daidzein. Bioavailability
is important to consider when studying the physiological effects of isoflavones. Studies have shown
that the degree of absorption depends on many factors including food matrix, isoflavone form and
background diet.

There are generally many contradictory results in studies of bioavailability and effects of isoflavones.
The reason for this may be, in part, the source and treatment of isoflavones preparations. The presence
of synergistic effects between isoflavones and other components in food has not been elucidated and
synergistic components could be removed during extraction processes, moreover isoflavones may
also become inactivated during processing [6]. Another challenge is the poor standardisation and
uncheck claims of contents of biological products, which means that there are great variations
between commercial preparations. Furthermore, large inter-individual differences are observed as a
consequence of variations in intestinal microflora or genetics, leading to differences in absorption
and metabolism of isoflavones. A study of polymorphisms in genes has shown that there is an
association between polymorphisms in certain genes and conjugation patterns of isoflavones. These
polymorphisms affect the activity of enzymes and transporters and therefore influence metabolism
and bioavailability of isoflavones, and through this their biological activity [141].

There are ways in which bioavailability could be enhanced and potentially increase the effects of
isoflavones. Addition of probiotics might have a beneficial effect through influences on gut
microflora even though research has not shown a direct correlation between ingestion of probiotics
and increased bioavailability. Microencapsulation is a technique used to stabilise food additives and
pharmaceutical products, which might represent another useful method to increase the effects of
isoflavones by enhancing residence time and obtaining more constant plasma concentrations [116].

Even though many studies have been carried out on bioavailability and physiological effects of
isoflavones, it is still important to execute large, well-constructed human studies of longer duration,
with focus on the specific composition of the isoflavone products. Most studies have used soy
isoflavones and it is of interest to study the effects of RC as it is one of the richest sources of
isoflavones with a unique isoflavone composition, and has shown promising effects on relieving
menopausal symptoms and decreasing bone demineralisation [59]. It might also be valuable to take

24
account of genotype when investigating the bioavailability and potential beneficial effects of
isoflavones.

Conflict of interests

The authors declare no conflicts of interests.

References
1. Adlercreutz, C.H., Goldin, B.R., Gorbach, S.L., et al.Soybean phytoestrogen intake and cancer risk.
The Journal of nutrition 125, 3 Suppl (1995), 757S770S.

2. Ae Park, S., Choi, M.S., Cho, S.Y., et al.Genistein and daidzein modulate hepatic glucose and lipid
regulating enzyme activities in C57BL/KsJ-db/db mice. Life Sciences 79, 12 (2006), 12071213.

3. Ahlborg, H.G., Johnell, O., Nilsson, B.E., Jeppsson, S., Rannevik, G., and Karlsson, M.K.Bone loss in
relation to menopause: a prospective study during 16 years. Bone 28, 3 (2001), 32731.

4. Akiyama, T., Ishida, J., Nakagawa, S., et al.Genistein, a specific inhibitor of tyrosine-specific protein
kinases. Journal of Biological Chemistry 262, 12 (1987), 55925595.

5. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E.Meta-analysis of the effects of soy protein
intake on serum lipids. The New England journal of medicine 333, (1995), 276282.

6. Atkinson, C., Compston, J.E., Day, N.E., Dowsett, M., and Bingham, S. a.The effects of phytoestrogen
isoflavones on bone density in women: A double-blind, randomized, placebo-controlled trial 1-3.
American Journal of Clinical Nutrition 79, 3 (2004), 326333.

7. Balk, J.L., Whiteside, D. a., Naus, G., DeFerrari, E., and Roberts, J.M.A pilot study of the effects of
phytoestrogen supplementation on postmenopausal endometrium. Journal of the Society for
Gynecologic Investigation 9, 4 (2002), 238242.

8. Barnes, S., Sfakianos, J., Coward, L., and Kirk, M.Soy isoflavonoids and cancer prevention. Underlying
biochemical and pharmacological issues. Dietary Phytochemicals in Cancer Prevention and
Treatment 401, (1996), 87100.

9. Beck, V., Rohr, U., and Jungbauer, a.Phytoestrogens derived from red clover: an alternative to
estrogen replacement therapy? The Journal of steroid biochemistry and molecular biology 94, 5
(2005), 499518.

10. Bedell, S., Nachtigall, M., and Naftolin, F.The pros and cons of plant estrogens for menopause.
Journal of Steroid Biochemistry and Molecular Biology 139, (2014), 225236.

11. Beral, V.Breast cancer and hormone-replacement therapy in the Million Women Study. Lancet 362,
9382 (2003), 419427.

25
12. Bieger, J., Cermak, R., Blank, R., et al.Tissue distribution of quercetin in pigs after long-term dietary
supplementation. The Journal of nutrition 138, 8 (2008), 14171420.

13. Bourgaud, F., Gravot, A., Milesi, S., and Gontier, E.Production of plant secondary metabolites: A
historical perspective. Plant Science 161, 2001, 839851.
http://linkinghub.elsevier.com/retrieve/pii/S0168945201004903.

14. Bowey, E., Adlercreutz, H., and Rowland, I.Metabolism of isoflavones and lignans by the gut
microflora: A study in germ-free and human flora associated rats. Food and Chemical Toxicology 41,
5 (2003), 631636.

15. Cassidy, A., Brown, J.E., Hawdon, A., et al.Factors affecting the bioavailability of soy isoflavones in
humans after ingestion of physiologically relevant levels from different soy foods. The Journal of
nutrition 136, 1 (2006), 4551.

16. Cassidy, A.Potential risks and benefits of phytoestrogen-rich diets. International journal for vitamin
and nutrition research. Internationale Zeitschrift fr Vitamin- und Ernhrungsforschung. Journal
international de vitaminologie et de nutrition 73, 2 (2003), 1206.

17. Cegiea, U., Folwarczna, J., Pytlik, M., and Zgrka, G.Effects of Extracts from Trifolium medium L. and
Trifolium pratense L. on Development of Estrogen Deficiency-Induced Osteoporosis in Rats.
Evidence-based complementary and alternative medicine: eCAM 2012, 921684 (2012), 111.

18. Cheng, G., Wilczek, B., Warner, M., Gustafsson, J.-A., and Landgren, B.-M.Isoflavone treatment for
acute menopausal symptoms. Menopause (New York, N.Y.) 14, 3 (2007), 468473.

19. Clerici, C., Setchell, K.D.R., Battezzati, P.M., et al.Pasta naturally enriched with isoflavone aglycons
from soy germ reduces serum lipids and improves markers of cardiovascular risk. The Journal of
nutrition 137, 10 (2007), 22702278.

20. Clifton-Bligh, P.B., Nery, M.-L., Clifton-Bligh, R.J., et al.Red clover isoflavones enriched with
formononetin lower serum LDL cholesterola randomized, double-blind, placebo-controlled study.
European Journal of Clinical Nutrition 69, 1 (2014), 134142.

21. Cohen, L. a., Crespin, J.S., Wolper, C., et al.Soy isoflavone intake and estrogen excretion patterns in
young women: Effect of probiotic administration. In Vivo 21, 3 (2007), 507512.

22. Colditz, G.A., Hankinson, S.E., Hunter, D.J., et al.The use of estrogens and progestins and the risk of
breast cancer in postmenopausal women. The New England journal of medicine 332, 24 (1995),
158993.

23. Collaborative Group on Epidemiological Studies of Ovarian Cancer.Menopausal hormone use and
ovarian cancer risk: individual participant meta-analysis of 52 epidemiological studies. The Lancet
6736, 14 (2015), 18.

24. Collaborative Group on Hormonal Factors in Breast Cancer.Breast cancer and hormone replacement
therapy: collaborative reanalysis of data from 51 epidemiological studies of 52,705 women with
breast cancer and 108,411 women without breast cancer. Collaborative Group on Hormonal Factors
in Breast Cancer. Lancet 350, 9084 (1997), 10471059.

26
25. Conner, P., Sderqvist, G., Skoog, L., et al.Breast cell proliferation in postmenopausal women during
HRT evaluated through fine needle aspiration cytology. Breast Cancer Research and Treatment 78,
(2003), 159165.

26. Duncan, A.M., Merz-Demlow, B.E., Xu, X., Phipps, W.R., and Kurzer, M.S.Premenopausal equol
excretors show plasma hormone profiles associated with lowered risk of breast cancer. Cancer
Epidemiology Biomarkers and Prevention 9, June (2000), 581586.

27. Dyrskog, S.E.U., Jeppesen, P.B., Colombo, M., Abudula, R., and Hermansen, K.Preventive effects of a
soy-based diet supplemented with stevioside on the development of the metabolic syndrome and
type 2 diabetes in Zucker diabetic fatty rats. Metabolism: clinical and experimental 54, 9 (2005),
11811188.

28. Faughnan, M.S., Hawdon, A., Ah-Singh, E., Brown, J., Millward, D.J., and Cassidy, A.Urinary
isoflavone kinetics: the effect of age, gender, food matrix and chemical composition. The British
journal of nutrition 91, 4 (2004), 56774.

29. Franke, A. a, Ashburn, L. a, Kakazu, K., Suzuki, S., Wilkens, L.R., and Halm, B.M.Apparent
bioavailability of isoflavones after intake of liquid and solid soya foods. The British journal of
nutrition 102, 8 (2009), 12031210.

30. Gardner, C.D., Chatterjee, L.M., and Franke, A. a.Effects of isoflavone supplements vs. soy foods on
blood concentrations of genistein and daidzein in adults. The Journal of nutritional biochemistry 20,
3 (2009), 22734.

31. Gautam, A.K., Bhargavan, B., Tyagi, A.M., et al.Differential effects of formononetin and cladrin on
osteoblast function, peak bone mass achievement and bioavailability in rats. Journal of Nutritional
Biochemistry 22, (2011), 318327.

32. Del Giorno, C., da Fonseca, A.M., Bagnoli, V.R., de Assis, J.S., Soares, J.M., and Baracat, E.C.Effects of
Trifolium pratense on the climacteric and sexual symptoms in postmenopause women. Revista da
Associacao Medica Brasileira 56, 5 (2010), 558562.

33. Gobert, C.P., Pipe, E. a, Capes, S.E., Darlington, G. a, Lampe, J.W., and Duncan, A.M.Soya protein
does not affect glycaemic control in adults with type 2 diabetes. The British journal of nutrition 103,
3 (2010), 412421.

34. Graefe, E.U., Wittig, J., Mueller, S., et al.Pharmacokinetics and bioavailability of quercetin glycosides
in humans. Journal of clinical pharmacology 41, 5 (2001), 492499.

35. Grodstein, F., Manson, J.E., Colditz, G.A., Willett, W.C., Speizer, F.E., and Stampfer, M.J.A
prospective, observational study of postmenopausal hormone therapy and primary prevention of
cardiovascular disease. Annals of Internal Medicine 133, 12 (2000), 933941.

36. Gruber, C.J., Tschugguel, W., Schneeberger, C., and Huber, J.C.Production and actions of estrogens.
The New England journal of medicine 346, 5 (2002), 34052.

27
37. Hargreaves, D.F., Potten, C.S., Harding, C., et al.Two-week dietary soy supplementation has an
estrogenic effect on normal premenopausal breast. Journal of Clinical Endocrinology and
Metabolism 84, 11 (1999), 40174024.

38. Hendrich, S.Bioavailability of isoflavones. Journal of chromatography. B, Analytical technologies in

the biomedical and life sciences 777, 1-2 (2002), 20310.

39. Hermansen, K., Sndergaard, M., Hie, L., Carstensen, M., and Brock, B.Beneficial effects of a soy-
based dietary supplement on lipid levels and cardiovascular risk markers in type 2 diabetic subjects.
2001.

40. Hidalgo, L. a, Chedraui, P. a, Morocho, N., Ross, S., and San Miguel, G.The effect of red clover
isoflavones on menopausal symptoms, lipids and vaginal cytology in menopausal women: a
randomized, double-blind, placebo-controlled study. Gynecological endocrinology: the official
journal of the International Society of Gynecological Endocrinology 21, 5 (2005), 257264.

41. Hodis, H.N. and Mack, W.J.Hormone replacement therapy and the association with coronary heart
disease and overall mortality: Clinical application of the timing hypothesis. Journal of Steroid
Biochemistry and Molecular Biology 142, (2014), 6875.

42. Hollman, P.C.H., De Vries, J.H.M., Van Leeuwen, S.D., Mengelers, M.J.B., and Katan, M.B.Absorption
of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. American Journal of
Clinical Nutrition 62, 6 (1995), 12761282.

43. Hosoda, K., Furuta, T., and Ishii, K.Metabolism and disposition of isoflavone conjugated metabolites
in humans after ingestion of kinako. Drug metabolism and disposition: the biological fate of
chemicals 39, 9 (2011), 17627.

44. Howes, J., Waring, M., Huang, L., and Howes, L.G.Long-term pharmacokinetics of an extract of
isoflavones from red clover (Trifolium pratense). Journal of alternative and complementary medicine
(New York, N.Y.) 8, 2 (2002), 13542.

45. Howes, J.B., Sullivan, D., Lai, N., et al.The effects of dietary supplementation with isoflavones from
red clover on the lipoprotein profiles of post menopausal women with mild to moderate
hypercholesterolaemia. Atherosclerosis 152, (2000), 143147.

46. Hutchins, A.M., Slavin, J.L., and Lampe, J.W.Urinary isoflavonoid phytoestrogen and lignan excretion
after consumption of fermented and unfermented soy products. Journal of the American Dietetic
Association 95, 5 (1995), 54551.

47. Izumi, T., Piskula, M.K., Osawa, S., et al.Soy isoflavone aglycones are absorbed faster and in higher
amounts than their glucosides in humans. The Journal of nutrition 130, 7 (2000), 16959.

48. Jandeleit-Dahm, K. a M., Calkin, A., Tikellis, C., and Thomas, M.Direct antiatherosclerotic effects of
PPAR agonists. Current opinion in lipidology 20, (2009), 2429.

49. Joy, S., Siow, R.C.M., Rowlands, D.J., et al.The isoflavone equol mediates rapid vascular relaxation:
Ca 2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt

28
 phosphorylation in human endothelial cells. Journal of Biological Chemistry 281, 37 (2006), 27335
27345.

50. Kang, J.S., Yoon, Y.D., Han, M.H., et al.Equol inhibits nitric oxide production and inducible nitric oxide
synthase gene expression through down-regulating the activation of Akt. International
immunopharmacology 7, (2007), 491499.

51. Kano, M., Takayanagi, T., Harada, K., Sawada, S., and Ishikawa, F.Bioavailability of isoflavones after
ingestion of soy beverages in healthy adults. The Journal of nutrition 136, 9 (2006), 22912296.

52. Kawakita, S., Marotta, F., Naito, Y., et al.Effect of an isoflavones-containing red clover preparation
and alkaline supplementation on bone metabolism in ovariectomized rats. Clinical interventions in
aging 4, (2009), 91100.

53. King, R. a and Bursill, D.B.Plasma and urinary kinetics of the isoflavones daidzein and genistein after
a single soy meal in humans. The American journal of clinical nutrition 67, 5 (1998), 86772.

54. Knight, D.C. and Eden, J.A.A review of the clinical effects of phytoestrogens. Obstetrics and
gynecology 87, 5 Pt 2 (1996), 897904.

55. Korde, L. a., Wu, A.H., Fears, T., et al.Childhood soy intake and breast cancer risk in Asian American
women. Cancer Epidemiology Biomarkers and Prevention 18, April (2009), 10501059.

56. Kottra, G. and Daniel, H.Flavonoid glycosides are not transported by the human Na+/glucose
transporter when expressed in Xenopus laevis oocytes, but effectively inhibit electrogenic glucose
uptake. The Journal of pharmacology and experimental therapeutics 322, 2 (2007), 829835.

57. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., et al.Interaction of estrogenic chemicals and
phytoestrogens with estrogen receptor . Endocrinology 139, 10 (1998), 42524263.

58. Lamartiniere, C.A., Cotroneo, M.S., Fritz, W.A., Wang, J., Mentor-Marcel, R., and Elgavish,
A.Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate.
The Journal of nutrition 132, 3 (2002), 552S558S.

59. Lambert, M. and Jeppesen, P.The effects of bioavailable red clover isoflavones for menopausal
symptoms and associated diseases. Planta Medica 80, 16 (2014).

60. Lampe, J.W., Skor, H.E., Li, S., Whl, K., Howald, W.N., and Chen, C.Wheat bran and soy protein
feeding do not alter urinary excretion of the isoflavan equol in premenopausal women. The Journal
of nutrition 131, November 2000 (2001), 740744.

61. Larkin, T. a., Price, W.E., and Astheimer, L.B.Increased probiotic yogurt or resistant starch intake
does not affect isoflavone bioavailability in subjects consuming a high soy diet. Nutrition 23, 10
(2007), 709718.

62. Lee, D.S. and Lee, S.H.Genistein, a soy isoflavone, is a potent alpha-glucosidase inhibitor. FEBS
letters 501, 1 (2001), 8486.

29
63. Lee, S.-A., Shu, X.-O., Li, H., et al.Adolescent and adult soy food intake and breast cancer risk: results
from the Shanghai Womens Health Study. The American journal of clinical nutrition 89, 4 (2009),
19201926.

64. Levis, S., Strickman-Stein, N., Ganjei-Azar, P., Xu, P., Doerge, D.R., and Krischer, J.Soy isoflavones in
the prevention of menopausal bone loss and menopausal symptoms: a randomized, double-blind
trial. Archives of internal medicine 171, 15 (2011), 13639.

65. Lichtenstein, A.H., Jalbert, S.M., Adlercreutz, H., et al.Lipoprotein response to diets high in soy or
animal protein with and without isoflavones in moderately hypercholesterolemic subjects.
Arteriosclerosis, Thrombosis, and Vascular Biology 22, (2002), 18521858.

66. Liew, L.The Natural Estrogen Diet & Recipe Book: Healthy Recipes for Perimenopause. Hunter House
Publishers, 2003.

67. Lipovac, M., Chedraui, P., Gruenhut, C., et al.The effect of red clover isoflavone supplementation
over vasomotor and menopausal symptoms in postmenopausal women. Gynecological
Endocrinology 28, 3 (2012), 203207.

68. Liu, D., Zhen, W., Yang, Z., Carter, J.D., Si, H., and Reynolds, K. a.Genistein acutely stimulates insulin
secretion in pancreatic beta-cells through a cAMP-dependent protein kinase pathway. Diabetes 55,
4 (2006), 10431050.

69. Lu, L.J. and Anderson, K.E.Sex and long-term soy diets affect the metabolism and excretion of soy
isoflavones in humans. The American journal of clinical nutrition 68, 6 Suppl (1998), 1500S1504S.

70. Maggiolini, M. and Picard, D.The unfolding stories of GPR30, a new membrane-bound estrogen
receptor. The Journal of endocrinology 204, 2 (2010), 10514.

71. Marini, H., Bitto, A., Altavilla, D., et al.Breast safety and efficacy of genistein aglycone for
postmenopausal bone loss: a follow-up study. The Journal of clinical endocrinology and metabolism
93, 12 (2008), 47874796.

72. Maul, R. and Kulling, S.E.Absorption of red clover isoflavones in human subjects: results from a pilot
study. The British journal of nutrition 103, 11 (2010), 156972.

73. Mei, J., Yeung, S.S., and Kung, a W.High dietary phytoestrogen intake is associated with higher bone
mineral density in postmenopausal but not premenopausal women. The Journal of clinical
endocrinology and metabolism 86, March (2001), 52175221.

74. Messina, M.Soy foods, isoflavones, and the health of postmenopausal women. The American journal
of clinical nutrition 100, Supplement 1 (2014), 423S430S.

75. Morito, K., Aomori, T., Hirose, T., et al.Interaction of phytoestrogens with estrogen receptors alpha
and beta (II). Biological & pharmaceutical bulletin 25, 1 (2002), 4852.

76. Mueller, M. and Jungbauer, A.Red clover extract: a putative source for simultaneous treatment of
menopausal disorders and the metabolic syndrome. Menopause (New York, N.Y.) 15, 6 (2008),
11201131.

30
77. Muraoka, K., Shimizu, K., Sun, X., et al.Flavonoids exert diverse inhibitory effects on the activation of
NF-B. Transplantation Proceedings 34, 02 (2002), 13351340.

78. Murkes, D., Conner, P., Leifland, K., et al.Effects of percutaneous estradiol-oral progesterone versus
oral conjugated equine estrogens-medroxyprogesterone acetate on breast cell proliferation and bcl-
2 protein in healthy women. Fertility and Sterility 95, 3 (2011), 11881191.

79. Murota, K., Shimizu, S., Miyamoto, S., et al.Unique uptake and transport of isoflavone aglycones by
human intestinal caco-2 cells: comparison of isoflavonoids and flavonoids. The Journal of nutrition
132, 7 (2002), 19561961.

80. Muthyala, R.S., Ju, Y.H., Sheng, S., et al.Equol, a natural estrogenic metabolite from soy isoflavones:
convenient preparation and resolution of R- and S-equols and their differing binding and biological
activity through estrogen receptors alpha and beta. Bioorganic & medicinal chemistry 12, 6 (2004),
155967.

81. Nahas, E. a P., Nahas-Neto, J., Orsatti, F.L., Carvalho, E.P., Oliveira, M.L.C.S., and Dias, R.Efficacy and
safety of a soy isoflavone extract in postmenopausal women: a randomized, double-blind, and
placebo-controlled study. Maturitas 58, 3 (2007), 249258.

82. Nechuta, S.J., Caan, B.J., Chen, W.Y., et al.Soy food intake after diagnosis of breast cancer and
survival: an in-depth analysis of combined evidence from cohort studies of US and Chinese women.
The American journal of clinical nutrition 96, (2012), 12332.

83. Nelson, L.R. and Bulun, S.E.Estrogen production and action. Journal of the American Academy of
Dermatology 45, 3 Suppl (2001), S11624.

84. Nettleton, J. a, Greany, K. a, Thomas, W., Wangen, K.E., Adlercreutz, H., and Kurzer, M.S.Plasma
phytoestrogens are not altered by probiotic consumption in postmenopausal women with and
without a history of breast cancer. The Journal of nutrition 134, 8 (2004), 19982003.

85. Nielsen, I.L.F. and Williamson, G.Review of the factors affecting bioavailability of soy isoflavones in
humans. Nutrition and cancer 57, 1 (2007), 110.

86. Nilsson, S., Mkel, S., Treuter, E., et al.Mechanisms of estrogen action. Physiological reviews 81, 4
(2001), 153565.

87. Nordentoft, I. and Jeppesen, P.Increased insulin sensitivity and changes in the expression profile of
key insulin regulatory genes and beta cell transcription factors in diabetic KKAy-mice after feeding.
Journal of Agricultural and Food Chemistry, October 1999 (2008), 43774385.

88. Occhiuto, F., Pasquale, R. De, Guglielmo, G., et al.Effects of phytoestrogenic isoflavones from red
clover (Trifolium pratense L.) on experimental osteoporosis. Phytotherapy research: PTR 21, 2
(2007), 1304.

89. Okabe, Y., Shimazu, T., and Tanimoto, H.Higher bioavailability of isoflavones after a single ingestion
of aglycone-rich fermented soybeans compared with glucoside-rich non-fermented soybeans in
Japanese postmenopausal women. Journal of the science of food and agriculture 91, 4 (2011), 658
63.

31
90. Oseni, T., Patel, R., Pyle, J., and Jordan, V.C.Selective estrogen receptor modulators and
phytoestrogens. Planta medica 74, 13 (2008), 165665.

91. De Pascual-Teresa, S., Hallund, J., Talbot, D., et al.Absorption of isoflavones in humans: effects of
food matrix and processing. The Journal of nutritional biochemistry 17, 4 (2006), 25764.

92. Penotti, M., Fabio, E., Modena, A.B., Rinaldi, M., Omodei, U., and Vigan, P.Effect of soy-derived
isoflavones on hot flushes, endometrial thickness, and the pulsatility index of the uterine and
cerebral arteries. Fertility and Sterility 79, 5 (2003), 11121117.

93. Piazza, C., Privitera, M.G., Melilli, B., et al.Influence of inulin on plasma isoflavone concentrations in
healthy postmenopausal women. American Journal of Clinical Nutrition 86, (2007), 775780.

94. Pino, A.M., Valladares, L.E., Palma, M. a., Mancilla, A.M., Yez, M., and Albala, C.Dietary isoflavones
affect sex hormone-binding globulin levels in postmenopausal women. Journal of Clinical
Endocrinology and Metabolism 85, 8 (2000), 27972800.

95. Potter, S.M.Soy protein and cardiovascular disease: The impact of bioactive components in soy.
Nutrition Reviews 56, 8 (1998), 231235.

96. Powles, T.J., Howell, A., Evans, D.G., et al.Red clover isoflavones are safe and well tolerated in
women with a family history of breast cancer. Menopause international 14, 1 (2008), 612.

97. Raines, E.W. and Ross, R.Biology of atherosclerotic plaque formation: possible role of growth factors
in lesion development and the potential impact of soy. The Journal of nutrition 125, (1995), 624S
630S.

98. Rawel, H.M., Rohn, S., and Kroll, J.Influence of a sugar moiety (rhamnosylglucoside) at 3-O position
on the reactivity of quercetin with whey proteins. International Journal of Biological
Macromolecules 32, 3-5 (2003), 109120.

99. Reynolds, K., Chin, A., Lees, K. a, Nguyen, A., Bujnowski, D., and He, J.A meta-analysis of the effect of
soy protein supplementation on serum lipids. The American journal of cardiology 98, 5 (2006), 633
640.

100. Ricci, E., Cipriani, S., Chiaffarino, F., Malvezzi, M., and Parazzini, F.Effects of soy isoflavones and
genistein on glucose metabolism in perimenopausal and postmenopausal non-Asian women: a
meta-analysis of randomized controlled trials. Menopause (New York, N.Y.) 17, 5 (2010), 10801086.

101. Richelle, M., Pridmore-Merten, S., Bodenstab, S., Enslen, M., and Offord, E.A.Hydrolysis of isoflavone
glycosides to aglycones by beta-glycosidase does not alter plasma and urine isoflavone
pharmacokinetics in postmenopausal women. The Journal of nutrition 132, 9 (2002), 258792.

102. Rossouw, J.E., Anderson, G.L., Prentice, R.L., et al.Risks and benefits of estrogen plus progestin in
healthy postmenopausal women: principal results From the Womens Health Initiative randomized
controlled trial. 2002.

103. Rowland, I., Faughnan, M., Hoey, L., Whl, K., Williamson, G., and Cassidy, A.Bioavailability of
phyto-oestrogens. The British journal of nutrition 89 Suppl 1, (2003), S4558.

32
104. Rowland, I.R., Wiseman, H., Sanders, T.A., Adlercreutz, H., and Bowey, E.A.Interindividual variation
in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the
gut microflora. Nutrition and cancer 36, 1 (2000), 2732.

105. Rfer, C.E., Bub, A., Mseneder, J., Winterhalter, P., Strtz, M., and Kulling, S.E.Pharmacokinetics of
the soybean isoflavone daidzein in its aglycone and glucoside form: A randomized, double-blind,
crossover study. American Journal of Clinical Nutrition 87, 5 (2008), 13141323.

106. Sammartino, a, Di Carlo, C., Mandato, V.D., Bifulco, G., Di Stefano, M., and Nappi, C.Effects of
genistein on the endometrium: ultrasonographic evaluation. Gynecological endocrinology: the
official journal of the International Society of Gynecological Endocrinology 17, 1 (2003), 4549.

107. Santen, R.J., Pinkerton, J., McCartney, C., and Petroni, G.R.Risk of breast cancer with progestins in
combination with estrogen as hormone replacement therapy. The Journal of clinical endocrinology
and metabolism 86, 1 (2001), 1623.

108. Schmitt, E., Dekant, W., and Stopper, H.Assaying the estrogenicity of phytoestrogens in cells of
different estrogen sensitive tissues. Toxicology in Vitro 15, 4-5 (2001), 433439.

109. Van der Schouw, Y.T., van der Graaf, Y., Steyerberg, E.W., Eijkemans, J.C., and Banga, J.D.Age at
menopause as a risk factor for cardiovascular mortality. Lancet 347, 9003 (1996), 7148.

110. Scott, K.P., Gratz, S.W., Sheridan, P.O., Flint, H.J., and Duncan, S.H.The influence of diet on the gut
microbiota. Pharmacological research: the official journal of the Italian Pharmacological Society 69,
(2013), 5260.

111. Setchell, K.D., Brown, N.M., Desai, P., et al.Bioavailability of pure isoflavones in healthy humans and
analysis of commercial soy isoflavone supplements. The Journal of nutrition 131, 4 Suppl (2001),
1362S75S.

112. Setchell, K.D.Phytoestrogens: the biochemistry, physiology, and implications for human health of
soy isoflavones. The American journal of clinical nutrition 68, 6 Suppl (1998), 1333S1346S.

113. Setchell, K.D.R., Brown, N.M., Desai, P.B., et al.Bioavailability, disposition, and dose-response effects
of soy isoflavones when consumed by healthy women at physiologically typical dietary intakes. The
Journal of nutrition 133, 4 (2003), 102735.

114. Setchell, K.D.R., Brown, N.M., and Lydeking-Olsen, E.The clinical importance of the metabolite
equol-a clue to the effectiveness of soy and its isoflavones. The Journal of nutrition 132, 12 (2002),
357784.

115. Setchell, K.D.R., Brown, N.M., Zimmer-Nechemias, L., et al.Evidence for lack of absorption of soy
isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for
bioavailability. The American journal of clinical nutrition 76, 2 (2002), 44753.

116. Setchell, K.D.R., Brzezinski, A., Brown, N.M., et al.Pharmacokinetics of a slow-release formulation of
soybean isoflavones in healthy postmenopausal women. Journal of Agricultural and Food Chemistry
53, 6 (2005), 19381944.

33
117. Setchell, K.D.R., Clerici, C., Lephart, E.D., et al.S-equol, a potent ligand for estrogen receptor beta, is
the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal
bacterial flora. The American journal of clinical nutrition 81, 5 (2005), 10729.

118. Setchell, K.D.R. and Cole, S.J.Method of defining equol-producer status and its frequency among
vegetarians. The Journal of nutrition 136, 8 (2006), 218893.

119. Shelnutt, S.R., Cimino, C.O., Wiggins, P. a, Ronis, M.J.J., and Badger, T.M.Pharmacokinetics of the
glucuronide and sulfate conjugates of genistein and daidzein in men and women after consumption
of a soy beverage. The American journal of clinical nutrition 76, 3 (2002), 58894.

120. Sheu, F., Lai, H.H., and Yen, G.C.Suppression effect of soy isoflavones on nitric oxide production in
RAW 264.7 macrophages. Journal of agricultural and food chemistry 49, (2001), 17671772.

121. Shu, X.O., Jin, F., Dai, Q., et al.Soyfood intake during adolescence and subsequent risk of breast
cancer among Chinese women. Cancer epidemiology, biomarkers & prevention: a publication of the
American Association for Cancer Research, cosponsored by the American Society of Preventive
Oncology 10, May (2001), 483488.

122. Simoncini, T., Fornari, L., Mannella, P., et al.Activation of nitric oxide synthesis in human endothelial
cells by red clover extracts. Menopause (New York, N.Y.) 12, 1 (2004), 6977.

123. Sorenson, R.L., Brelje, T.C., and Roth, C.Effect of tyrosine kinase inhibitors on islets of Langerhans:
evidence for tyrosine kinases in the regulation of insulin secretion. Endocrinology 134, 4 (1994),
19751978.

124. Squadrito, F., Marini, H., Bitto, A., et al.Genistein in the metabolic syndrome: results of a
randomized clinical trial. The Journal of clinical endocrinology and metabolism 98, 8 (2013), 3366
74.

125. Steinberg, F.M., Murray, M.J., Lewis, R.D., et al.Clinical outcomes of a 2-y soy isoflavone
supplementation in menopausal women. American Journal of Clinical Nutrition 93, 2 (2011), 356
367.

126. Tai, T.Y., Tsai, K.S., Tu, S.T., et al.The effect of soy isoflavone on bone mineral density in
postmenopausal Taiwanese women with bone loss: a 2-year randomized double-blind placebo-
controlled study. Osteoporosis international: a journal established as result of cooperation between
the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA 23,
5 (2012), 157180.

127. Taiz, L. and Zeiger, E.Plant physiology. Sinauer Associates, Inc., Publishers, 2006.

128. Taku, K., Melby, M.K., Takebayashi, J., et al.Effect of soy isoflavone extract supplements on bone
mineral density in menopausal women: meta-analysis of randomized controlled trials. Asia Pacific
journal of clinical nutrition 19, 1 (2010), 3342.

129. Taku, K., Umegaki, K., Sato, Y., Taki, Y., Endoh, K., and Watanabe, S.Soy isoflavones lower serum
total and LDL cholesterol in humans: A meta-analysis of 11 randomized controlled trials. American
Journal of Clinical Nutrition 85, 4 (2007), 11481156.

34
130. Tamura, M.Effects of intestinal flora on the metabolism and absorption of isoflavones. JAPAN
AGRICULTURAL RESEARCH QUARTERLY 40, May 2005 (2006), 4550.

131. Tew, B.Y., Xu, X., Wang, H.J., Murphy, P.A., and Hendrich, S.A diet high in wheat fiber decreases the
bioavailability of soybean isoflavones in a single meal fed to women. The Journal of nutrition 126, 4
(1996), 8717.

132. Thanos, J., Cotterchio, M., Boucher, B. a., Kreiger, N., and Thompson, L.U.Adolescent dietary
phytoestrogen intake and breast cancer risk (Canada). Cancer Causes and Control 17, 10 (2006),
12531261.

133. Tice, J. a, Ettinger, B., Ensrud, K., Wallace, R., Blackwell, T., and Cummings, S.R.Phytoestrogen
supplements for the treatment of hot flashes: the Isoflavone Clover Extract (ICE) Study: a randomized
controlled trial. 2003.

134. Turner, N.J., Thomson, B.M., and Shaw, I.C.Bioactive isoflavones in functional foods: the importance
of gut microflora on bioavailability. Nutrition reviews 61, 6 Pt 1 (2003), 20413.

135. Tyagi, A.M., Srivastava, K., Singh, A.K., et al.Formononetin reverses established osteopenia in adult
ovariectomized rats. Menopause: The Journal of The North American Menopause Society 19, 8
(2012), 856863.

136. Unfer, V., Casini, M.L., Costabile, L., Mignosa, M., Gerli, S., and Di Renzo, G.C.Endometrial effects of
long-term treatment with phytoestrogens: A randomized, double-blind, placebo-controlled study.
Fertility and Sterility 82, 1 (2004), 145148.

137. Urpi-Sarda, M., Morand, C., Besson, C., et al.Tissue distribution of isoflavones in ewes after
consumption of red clover silage. Archives of Biochemistry and Biophysics 476, 2 (2008), 205210.

138. Vedavanam, K., Srijayanta, S., OReilly, J., Raman, A., and Wiseman, H.Antioxidant action and
potential antidiabetic properties of an isoflavonoid-containing soyabean phytochemical extract
(SPE). Phytotherapy Research 13, 7 (1999), 601608.

139. Vergne, S., Bennetau-Pelissero, C., Lamothe, V., et al.Higher bioavailability of isoflavones after a
single ingestion of a soya-based supplement than a soya-based food in young healthy males. The
British journal of nutrition 99, 2 (2008), 333344.

140. Vitale, D.C., Piazza, C., Melilli, B., Drago, F., and Salomone, S.Isoflavones: estrogenic activity,
biological effect and bioavailability. European journal of drug metabolism and pharmacokinetics 38,
1 (2013), 1525.

141. Wakeling, L. a. and Ford, D.Polymorphisms in genes involved in the metabolism and transport of soy
isoflavones affect the urinary metabolite profile in premenopausal women following consumption of
a commercial soy supplement as a single bolus dose. Molecular Nutrition and Food Research 56, 12
(2012), 17941802.

142. Walsh, K.R., Zhang, Y.C., Vodovotz, Y., Schwartz, S.J., and Failla, M.L.Stability and bioaccessibility of
isoflavones from soy bread during in vitro digestion. Journal of agricultural and food chemistry 51,
16 (2003), 46039.

35
143. Wang, L., Waltenberger, B., Pferschy-Wenzig, E.-M., et al.Natural product agonists of peroxisome
proliferator-activated receptor gamma (PPAR): a review. Biochemical pharmacology 92, 1 (2014),
7389.

144. Watanabe, S., Yamaguchi, M., Sobue, T., et al.Pharmacokinetics of soybean isoflavones in plasma,
urine and feces of men after ingestion of 60 g baked soybean powder (kinako). The Journal of
nutrition 128, 10 (1998), 17105.

145. Wiseman, H., Casey, K., Bowey, E. a., et al.Influence of 10 wk of soy consumption on plasma
concentrations and excretion of isoflavonoids and on gut microflora metabolism in healthy adults.
American Journal of Clinical Nutrition 80, 8 (2004), 692699.

146. Wolff, L.P.G., Martins, M.R., Bedone, A.J., and Monteiro, I.M.U.Endometrial evaluation in
menopausal women after six months of isoflavones. Revista da Associacao Medica Brasileira 52, 6
(2006), 419423.

147. Wu, A.H., Wan, P., Hankin, J., Tseng, C., Yu, M.C., and Pike, M.C.Adolescent and adult soy intake and
risk of breast cancer in Asian-Americans. Carcinogenesis 23, 9 (2002), 14911496.

148. Wu, Q., Wang, M., and Simon, J.E.Determination of isoflavones in red clover and related species by
high-performance liquid chromatography combined with ultraviolet and mass spectrometric
detection. Journal of chromatography. A 1016, 2 (2003), 195209.

149. Xu, X., Harris, K.S., Wang, H.J., Murphy, P. a, and Hendrich, S.Bioavailability of soybean isoflavones
depends upon gut microflora in women. The Journal of nutrition 125, 9 (1995), 23072315.

150. Xu, X., Wang, H.J., Murphy, P.A., and Hendrich, S.Neither background diet nor type of soy food
affects short-term isoflavone bioavailability in women. The Journal of nutrition 130, 4 (2000), 798
801.

151. Yuan, J.-P., Wang, J.-H., and Liu, X.Metabolism of dietary soy isoflavones to equol by human
intestinal microflora--implications for health. Molecular nutrition & food research 51, 7 (2007), 765
81.

152. Zhang, X., Shu, X.-O., Li, H., et al.Prospective cohort study of soy food consumption and risk of bone
fracture among postmenopausal women. Archives of internal medicine 165, (2005), 18901895.

153. Zhang, Y., Hendrich, S., and Murphy, P.A.Glucuronides are the main isoflavone metabolites in
women. The Journal of nutrition 133, 2 (2003), 399404.

154. Zhang, Y.B., Chen, W.H., Guo, J.J., et al.Soy isoflavone supplementation could reduce body weight
and improve glucose metabolism in non-Asian postmenopausal women-A meta-analysis. Nutrition
29, 1 (2013), 814.

155. Zheng, Y., Lee, S.-O., Verbruggen, M. a, Murphy, P. a, and Hendrich, S.The apparent absorptions of
isoflavone glucosides and aglucons are similar in women and are increased by rapid gut transit time
and low fecal isoflavone degradation. The Journal of nutrition 134, 10 (2004), 25342539.

36
156. Zubik, L. and Meydani, M.Bioavailability of soybean isoflavones from aglycone and glucoside forms
in American women. The American journal of clinical nutrition 77, 6 (2003), 145965.

37
 Bioavailability of isoflavones from fermented and unfermented red
clover formulations

Authors: Dorthe Mller Srensen

Address: Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Tage-
Hansens Gade 2, DK-8000 Aarhus C, Denmark

Abstract

Background
Red clover is a rich source of isoflavones, which have been shown to have beneficial physiological
effects. Bioavailability is a necessity for isoflavones to exert their biological activity and the purpose
of this study was to determine the bioavailability of isoflavones from fermented and unfermented red
clover formulations.

Method
Fifteen healthy women aged 19-39 years participated in a crossover trial and were each given five
red clover formulations (fermented liquid extract, fermented freeze-dried extract in capsules, tablets
and yoghurt and unfermented capsules) separated by a wash-out period of minimum 5 days. The red
clover formulations were ingested immediately prior to a standardised breakfast meal, and blood
samples were collected at baseline and at 2, 4, 6, 8, 12, 24 and 48 hours after administration. These
were analysed for isoflavone content, thereafter the bioavailability was evaluated from calculated
incremental area under the concentration-time curves (iAUC).

Results
Daidzein represented the highest concentrations in plasma followed by genistein and formononetin.
Peak plasma concentrations of total isoflavones were seen after 6 hours for fermented liquid extract
and unfermented capsule (0.24 and 0.23 mol/L respectively). The fermented capsule showed a
tendency for higher area under the curve for total isoflavones than unfermented capsule (iAUC0-24h:
1.21 0.28 vs. 0.99 0.12 mol24h/L, P = 0.36). For daidzein the bioavailability was significantly
higher in the fermented liquid extract compared to the unfermented capsule (iAUC0-24h: 0.62 0.07
vs. 0.27 0.05 mol24h/L, P < 0.05). There was a tendency for the same picture when looking at
formononetin, but differences were not statistically significant. For genistein the bioavailability was
surprisingly significantly higher in unfermented capsule compared to the fermented liquid extract
(iAUC0-24h: 0.61 0.07 vs. 0.33 0.05 mol24h/L, P < 0.05) and the fermented capsule (iAUC0-24h:
0.61 0.07 vs. 0.35 0.07 mol24h/L, P < 0.05). This might be due to differences in isoflavone
composition in the formulations. There were no differences in area under the curve between the
fermented liquid extract and capsule when looking at the individual and total isoflavone curves.

1
Conclusion
There is a tendency for a higher bioavailability of total isoflavones in the fermented red clover
formulations compared to unfermented. No differences in bioavailability were observed between
liquid and solid formulation of fermented red clover. Results are based on data from a small
percentage of the total number of collected plasma samples, due to challenges with analytical
equipment. A comparison of all five included formulations based on an adequate number of time
points will be possible when all samples have been analysed.

Abbreviations
RC red clover
CVD cardiovascular disease
AUC area under concentration-time curve
iAUC incremental area under concentration-time curve
BMI body mass index
TVP textured vegetable protein
lex liquid extract
fcap fermented capsule
ufcap unfermented capsule

1 Introduction

Isoflavones are phytoestrogen components present in the human diet, of which soy and red clover
(RC) (Trifolium pratense L.) are abundant sources. RC is native to Europe and Western Asia, and is
used to improve soil fertility by nitrogen fixation. It is also used in traditional medicine to treat
conditions such as burns, wounds, fever, coughs and asthma (Booth et al., 2006).

A lower incidence of cardiovascular diseases (CVDs), cancer and menopausal symptoms is widely
reported in Asian women compared to western populations (Yuan, Wang, & Liu, 2007). The Asian
diet is rich in soy, and evidence suggests that isoflavones may contribute health beneficial effects
(Jerome-Morais, Diamond, & Wright, 2011). Studies on cell cultures, animals and humans have
shown that isoflavones exert estrogenic, antioxidant, anti-carcinogenic, antiosteoporotic and
antiatherogenic effects (Beck, Rohr, & Jungbauer, 2005).

The biological effects of isoflavones depend on the plasma levels of the compounds, which are highly
influenced by their bioavailability. Isoflavones exist as aglycones and glycosides, and in plants
isoflavones are mainly found as glycosides which are not readily absorbed due to their hydrophilicity
and high molecular weight (Izumi et al., 2000). Post-ingestion aglycones readily penetrate the
enterocyte by passive diffusion and are absorbed in the liver, whereas isoflavone glycosides require
hydrolysis by glucosidases in the intestine. Glucosidases are mainly produced by intestinal bacteria,
where gut microflora have a considerable impact on bioavailability and are thought responsible for
the large inter-individual variations (Richelle, Pridmore-Merten, Bodenstab, Enslen, & Offord, 2002;

2
K. D. Setchell et al., 2001). The absorbed aglycones are either released into the bloodstream in a free
active form or undergo conjugation. The first pass enterohepatic conjugation is efficient and
isoflavones in plasma are mainly conjugated, either with glucuronic acid or, to a lesser extent, with
sulphate (Turner, Thomson, & Shaw, 2003). Urinary and faecal recovery of isoflavones is low,
showing that a large proportion of the isoflavones are metabolised. The metabolites are mainly formed
in the large intestine and differ from parent molecules in their pharmacological activity (Vitale,
Piazza, Melilli, Drago, & Salomone, 2013).

Due to the hydrolytic step required for absorption of isoflavone glycosides, the bioavailability of
aglycones is expected to be higher. Isoflavone supplements containing aglycones (30 mg) or
glycosides (50 mg) given to healthy men and women showed that aglycones were absorbed faster and
in greater amounts (Izumi et al., 2000). A similar result was obtained using a food matrix where
isoflavones from fermented soy resulted in a higher bioavailability compared to unfermented soy
(Okabe, Shimazu, & Tanimoto, 2011). However, there is also evidence indicating no differences in
glycoside and aglycone bioavailability. In one study where 16 women were given tablets with soy
isoflavones as aglycone (32 mg) or glycoside (52 mg), only slight differences were seen in maximum
concentration and area under the plasma concentration-time curve (AUC). Overall the bioavailability
did not differ between the two (Zubik & Meydani, 2003).

These contradictions in results are more likely to be the consequence of heterogeneous study designs
(i.e. differences in the ethnicity of subjects, sources and dose of isoflavones, food matrix, duration of
study, number of subjects and habitual diet) which confound outcomes when assessing the effect of
isoflavone form (aglycone vs. glycoside). Furthermore, large inter-individual differences in the
absorption and metabolism of isoflavones by gut microflora present a further challenge to interpreting
results.

Bioavailability strongly influences the biological effects of isoflavones, and no clear conclusions can
be drawn regarding the impact of a glycoside moiety on bioavailability. As RC extract has been shown
to exert beneficial effects on menopausal symptoms and bone health, it is of pharmacological and
scientific interest to find the form with the highest bioavailability of isoflavones. It is also of interest
to study the bioavailability of isoflavones from fermented RC in different matrices, due to practical
issues with the liquid RC extract (unappealing taste, reduced durability, challenges during travels).

With this in mind, the present study aims to test thoroughly the bioavailability of five standardised
RC formulations considering diet, food matrix and molecular form. RC formulations were
administered in a crossover human study and the pharmacokinetic profiles of the isoflavone
formulations were followed for 48 h to investigate the effect of fermentation and matrix on
bioavailability.

3
2 Materials and methods

2.1 Red clover formulations

Five formulations of RC were tested; fermented liquid extract, fermented capsules, fermented tablets,
yoghurt with fermented RC and unfermented capsules.

Herrens Mark (Nr. Aaby, Denmark) provided the RC preparations used for the fermented
formulations. The production process includes harvesting of the RC plants followed by heating in
stainless steel drums. The mass is then filtered and transferred to cooling tanks where lactid acid
bacteria is added and the extract is fermented for six months. During the fermentation the pH falls,
and the pH of the end product is approximately 4, which acts to preserve the extract. This novel
patented fermentation process for RC, gives an extract with a high proportion of aglycone isoflavones.
The RC extract was freeze-dried prior to usage in the fermented capsules, tablets and yoghurt
formulation.

The fermented capsules and tablets were produced in collaboration with Natur-Drogeriet A/S
(Hrning, Denmark). The mixture in the capsules consisted of freeze-dried RC extract, silicon
dioxide, magnesium stearate and potato starch. The freeze-dried extract was ground in a blender, and
all ingredients were sieved before being mixed manually. The mixture was encapsulated in gelatine
capsules using an automated filling machine. The fermented tablets consisted of freeze-dried RC
extract, magnesium carbonate and magnesium stearate. The freeze-dried extract was ground in a
blender, and all ingredients were sieved before being mixed in an automated mixer and compressed
to tablets. The yoghurt formulation was prepared by the project team as a mixture of skyr (Levevis,
Dansk Supermarked A/S, Hjbjerg, Denmark), freeze dried RC extract, blueberries (Vores, Dansk
Supermarked A/S, Hjbjerg, Denmark), stevia powder (steviol-glycosides, Steviafarma Industrial
S/A, Maring, Brazil) and almond essence (Dr. Oetker, Glostrup, Denmark). The unfermented
capsules are a commercial RC supplement containing 400 mg pulverised RC per capsule (Jordens
Frugter, Aarhus, Denmark).

2.2 HPLC analysis of isoflavones in red clover formulations

Quantification of isoflavones in the RC formulations was performed by DB lab (DB Lab A/S, Odense,
Denmark) utilising high performance liquid chromatography (HPLC) with UV detection. Analysis
was carried out pre- and post-acidic hydrolysis to determine aglycone content.

The samples were prepared for analysis in the following ways. 10 g RC extract was mixed with 50
mL methanol (VWR chemical, Sborg, Denmark) and filtered through a 0.45 m nylon filter (Q-
Max, Frisenette, Knebel, Denmark). Acidic hydrolysis of RC extract was carried out by mixing 20
mL RC extract, 8.3 mL 37% hydrochloric acid (Merck KGaA, Darmstadt, Germany) and 71.7 mL

4
ethanol (Merck KGaA, Darmstadt, Germany). The mixture was heated to 80 C for 5 hours. 400 mg
of freeze-dried RC extract was mixed with appr. 80 mL ethanol and treated in ultrasonic bath for 40
minutes. The mixture was topped with ethanol until 100 mL and filtered through a 0.45 m nylon
filter. Acidic hydrolysis of freeze-dried RC extract was carried out by mixing 400 mg freeze-dried
RC extract with 60 mL ethanol and 20 mL 5M hydrochloric acid. The mixture was heated to 80 C
for 5 hours, then topped with ethanol until 100 mL and filtered through a 0.45 m nylon filter. The
powder from the unfermented capsules was treated in the same way as the freeze-dried RC extract.

The HPLC system was a Summit 4 (Thermo Scientific, San Jose, CA) consisting of a P680 LPG
pump, ASI 100T autosampler, TCC-100 column oven and PDA-100 detector. It was equipped with
a Polaris 3 C18-A column (250 x 3 mm, 3 m particle size, Agilent technologies, Glostrup, Denmark).
The mobile phase consisted of 10% 0,1% formic acid (Sigma-Aldrich, Brndby, Denmark) in a
mixture of water (A) and acetonitrile (B) (VWR chemical, Sborg, Denmark) as follows; 0 to 57 min
18% B, 57 to 62 min 90% B, 62 to 70 min 18% B. The flow rate was 0,4 mL/min and the injection
volume was 10 L. The isoflavones were quantified by UV detection at 260 nm.

2.3 Subjects and study design

The study protocol was approved by the Local research ethics committee in region Central Jutland
(Denmark) and the trial registered at ClinicalTrials.gov (NCT02264223). Written informed consent
was obtained from all participants.

A power calculation using a least significant difference of 8% and values of AUC from a study by
Okabe as a guideline showed that 13 participants were necessary. In order to consider the usual
percentage dropout, 15 participants were recruited.(Okabe et al., 2011)

A randomized crossover study of 15 healthy women aged 19-39 years was performed. Exclusion
criteria included current hormone treatment, pregnancy, lactation, use of medications that affect
uptake (except contraceptives), drug or alcohol abuse, cardiovascular, psychiatric, neurological or
renal disorders and acute diseases. During the screening process health status was evaluated by
measuring blood pressure and markers of liver and kidney function.

The participants received all five formulations and are used as their own controls. The participants
received RC formulations in random order, and each intervention was separated by wash-out periods
of a minimum of 5 days. The participants followed an isoflavone-free diet one week before trial start
and during the whole duration of the trial.

After an overnight fast, the RC formulations were provided immediately prior to breakfast.
Participants received standardized meals on the intervention day and the following day, which were
prepared by members of the project team. On the intervention day lunch was given at noon after blood
was drawn at 4 h and dinner was given at 18:00 approximately 10 h after RC administration. The

5
nutritional composition of the standardised diet followed the nutritional recommendations for a
healthy diet and the mean energy content was 9075 kJ/day. The percentage distribution of
macronutrients was 17,5 % protein, 52,5 % carbohydrates and 30,5 % fat (for details on the diet see
table A.1 in Appendix A). Diet diaries for the day before each intervention and the two intervention
days were filled out. Blood samples were collected before consumption (baseline) of the RC and 2,
4, 6, 8, 12, 24 and 48 h after intake. EDTA plasma was centrifuged (3000 x g, 10 min, 20 C) and
stored at -80 C until further analysis.

To obtain the 40 mg isoflavones aglycone equivalents required in the study, the participants were
administered 61.7 mL RC extract, 2.86 g freeze-dried RC extract or fifteen 500 mg unfermented
capsules.

2.4 Extraction of isoflavones in plasma

Plasma samples were pretreated with -glucuronidase from Helix Pomatia (type HP-2, > 100,000
U/mL, Sigma-Aldrich, Brndby, Denmark). 1 mL plasma was incubated with 120 L -
glucuronidase and 1010 L sodium acetate buffer (0.945 g sodium acetate powder (VWR chemical,
Sborg, Denmark), 172.5 L acetic acid (glacial) (Merck KGaA, Darmstadt, Germany), 50 mL pure
water) for 1 hour at 45 C. The plasma samples were extracted by mixing the hydrolysed plasma
mixture with 3 mL acetonitrile (HPLC purity > 98%, Sigma-Aldrich, Brndby, Denmark). The
mixture was vortexed for 30 s every 5 min for 3 times and centrifuged at 190 x g at 20 C for 10 min.
The supernatant was collected, filtered through a 0.22 m filter (Q-Max, Frisenette, Knebel,
Denmark) and evaporated to dryness at 34 C in a speed vacuum centrifuge (Scan Speed 40, Scanvac,
Lynge, Denmark). The isoflavones were reconstituted in 400 L of mobile phase (0.1% formic acid
(HPLC purity > 98%, Sigma-Aldrich, Brndby, Denmark) in 60% acetonitrile and 40% water) and
50 L ethanol (HPLC purity > 98%, Sigma-Aldrich, Brndby, Denmark).

2.5 LC-MS analysis of isoflavones in plasma

The plasma samples were analysed for daidzein, genistein, formononetin and biochanin A content by
LC-MS. The LC-MS consisted of a TSQ Vantage triple quadrupole mass spectrometer (Thermo
Scientific, San Jose, CA) and an HPLC system with an Accella 1250 pump and a CTC PAL
autosampler (Thermo Scientific, San Jose, CA). The Mass spectrometer was fitted with an ESI source
and run in SRM mode with a collision gas pressure of 1.5 mTorr and with the following tune settings.
The capillary and vaporizer temperatures were 200 C and 350 C respectively, the sheath gas and
ion sweep gas pressures were 40 and 0 respectively, the auxiliary gas flow was 20 L/min and the
spray voltage was 3000 V in negative mode. The parent ions, product ions and collision energies for
each compound are given in Table 1.
.

6
 Table 1: Parent ions, product ions and collision energies on MS.
Parent ion Product ion Collision energy
Daidzein 253 224 28
Genistein 269 133 33
Formononetin 267 252 22
Biochanin A 283 239 34

The HPLC column was a Kinetex 2.6 m, C18, 100 , 150 x 4.6 mm (Phenomenex, Vrlse,
Denmark). The column was kept at a constant temperature of 30 C and the injection volume of
sample was 10 l. Elution was performed with a flow of 200 L/min with ultrapure water with 0.1%
formic acid (HPLC purity > 98%, Sigma-Aldrich, Brndby, Denmark) (solvent A) and acetonitrile
(HPLC purity > 98%, Sigma-Aldrich, Brndby, Denmark) with 0.1% formic acid (solvent B) with
the following gradient programme; 0 to 1 min isocratic 15% solvent B, 1 to 20 min linear gradient
from 15 to 25% solvent B, 20 to 25 min isocratic 25% solvent B, 25 to 40 min linear gradient from
25 to 60% solvent, 40 to 55 min linear gradient from 60 to 99% solvent B, 55 to 60 min isocratic 99%
solvent B, 60 to 62 min linear gradient from 99 to 15% solvent B and 62 to 72 min isocratic elution
with 15% solvent B.

2.6 Statistical analyses

Means and standard deviations were calculated for demographic data and diet registrations. Statistical
differences between the macronutrient compositions of the diet for the five interventions were
analysed by one-way ANOVA with a Bonferroni post-test. Differences were considered significant
when p < 0.05.

The bioavailability was evaluated from the incremental area under the concentration-time curve
(iAUC), which was calculated using the trapezoidal rule using the 0 hour plasma concentration as
baseline value. Results are given as means SEM. Statistical differences between the bioavailability
of formulations were evaluated with a paired students t test. Differences were considered significant
when p < 0,05.

iAUC calculation and statistical analyses were carried out using GraphPad Prism version 4.03 for
Windows.

3 Results

3.1 Isoflavones in red clover formulations

The concentrations of isoflavones in the RC formulations were determined pre- and post-acidic
hydrolysis, and the results for the hydrolysed samples for the four main isoflavones are shown in

7
Table 2. The values were used to calculate the dosage of the formulations to obtain 40 mg total
isoflavones, and the intake of the individual isoflavones per dose are also given in the table. The
isoflavones glycitein and ononin were found in minor amounts.

Table 2: Isoflavone concentrations in RC after acidic hydrolysis (aglycone equivalents) and intake of isoflavones at
dosage of 40 mg total isoflavones.
Fermented extract Freeze-dried fermented Unfermented capsules
extract (mg/kg)
Conc. Intake per Conc. Intake per Conc. Intake per
(mg/L) dose (mg) (mg/kg) dose (mg) (mg/kg) dose (mg)
Daidzein 31 1.9 164 0.5 393 2.9
Genistein 54 3.3 1286 3.7 1166 8.7
Formononetin 342 21.1 7842 22.4 1446 10.8
Biochanin A 221 13.7 4690 13.4 2360 17.6
Total 648 40 13982 40 5365 40

The distribution of isoflavones in the unfermented capsule is very different from the fermented
formulations. The percentage content of formononetin is considerably lower in the unfermented
capsule (27 % vs. 54 %), and the content of the three other isoflavones are higher (22 % vs. 8 % for
genistein, 7 % vs. 3 % for daidzein, 44 % vs. 34% for biochanin A). It is therefore necessary to look
at the iAUCs for all four isoflavones when comparing the bioavailability of different formulations.

The fermentation process changes the ratio between aglycones and glycosides, and the aglycone
content in the fermented RC formulations is approximately twice as high as the unfermented RC
(Table 3).

Table 3: Dose of RC formulations to obtain 40 mg isoflavone aglycone equivalents and distribution of isoflavone forms.
Formulation Dose Aglycone (%) Glycoside (%) D+F/G+B
ratio
Fermented capsule 6 pcs. 71 29 1.34
Unfermented capsule 15 pcs. 35 65 0.52
Liquid extract 61.7 mL 76 24 1.36
Yoghurt 2.86 g 71 29 1.34
Fermented tablet 4 pcs. 71 29 1.34
Ratio of daidzein and formononetin over genistein and biochanin A.
Amount of freeze dried RC extract mixed into yoghurt formulation.

3.2 Demographic data and diet

Fifteen healthy women were recruited and completed the five interventions. The mean age of the
participants was 25 6 years. Data from pre-study screening are given in Table 4.

8
Table 4: Data from prestudy screening; age, body mass index (BMI), systolic blood pressure (Sys BP), diastolic blood
pressure (Dia BP), ALAT, ASAT, creatinine (Crea). (means SD, n=15)
Age BMI Sys BP Dia BP ALAT ASAT Crea
(years) (kg/m2) (mm Hg) (mm Hg) (U/l) (U/l) (mol/l)
25 6 23 3 115 12 70 7 16 5 20 5 66 6

The diets on the two days of intervention were standardized and macronutrient compositions of the
diets during the five interventions are shown in Table 5. These were assessed through diet diaries and
no statistical differences were observed between intakes of the five formulations.

Table 5: Macronutrient composition of diets (mean of the two intervention days) (means SD, n=15).
Energy Protein Fat Carbohydrate Fibre
(kJ/d) (g/d) (g/d) (g/d) (g/d)
Fermented 8981 1052 89 10 70 12 285 50 30 3
capsule
Unfermented 8463 1062 84 12 67 8 274 47 29 4
capsule
Liquid extract 8885 1048 88 8 69 11 287 42 31 3

Yoghurt 8823 861 89 7 68 13 283 39 30 4

Fermented 8582 791 89 7 66 9 277 37 29 3

tablet

P value > 0.05 > 0.05 > 0.05 > 0.05 > 0.05

3.3 Plasma concentrations of isoflavones

Due to challenges with the highly sensitive analytical equipment, it has only been possible to
determine the isoflavone content in approximately 10% of the collected plasma samples within the
given time frame of the project. We have chosen to analyse samples taken at 0, 2, 6 and 24 hours for
five participants and three formulations (liquid extract (lex), fermented capsule (fcap) and
unfermented capsule (ufcap)).

Figure 1 shows plasma concentration curves for total isoflavones (sum of daidzein, genistein,
formononetin and biochanin A) for the three formulations.

9
 Total isoflavones

Plasma concentration (mol/L)

0.275
lex
0.250 fcap
ufcap
0.225

0.200

0.175

0.150
0.000
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

Figure 1: Concentration-time curves for total isoflavones (sum of daidzein, genistein, formononetin and biochanin A)
for liquid extract (lex), fermented capsule (fcap) and unfermented capsule (ufcap). Data are shown as means SEM.

Before ingestion the total isoflavone concentrations are 0.15 - 0.18 mol/L for the three formulations.
Peak concentrations of total isoflavones were reached after 6 hours for liquid extract and unfermented
capsule, and reached maximum concentrations of 0.24 and 0.23 mol/L respectively. There was no
clear peak concentration for the fermented capsule. Twenty-four hours after the ingestion of RC the
total isoflavone plasma concentration fell, but did not reach equivalence to baseline value. Similar
patterns are seen for the individual isoflavones (daidzein, genistein, formononetin) with peak plasma
concentrations being reached after 6 hours (data not shown). The relationship between formulation
and maximum concentrations are however different for the individual isoflavones which is reflected
in the iAUCs shown in Table 6. When looking at daidzein and formononetin the highest iAUC is
obtained in the fermented formulations, whereas for genistein the highest iAUC is seen for the
unfermented capsule.

Table 6: Values for iAUC0-24h for plasma isoflavones after ingestion of RC formulations. Total isoflavones were calculated
as the sum of daidzein, genistein, formononetin and biochanin A. (means SEM, n=5)
iAUC0-24h (mol24h/L)
Isoflavone Liquid extract Fermented capsule Unfermented capsule
Total 1.04 0.15 1.21 0.28 0.99 0.12
Daidzein 0.62 0.07 0.58 0.12 0.27 0.05a
Genistein 0.33 0.05 0.35 0.07 0.61 0.07b
Formononetin 0.09 0.03 0.07 0.02 0.06 0.01
Biochanin A - - -
Values for biochanin A are not shown as the plasma concentration changes are very low and it is not possible to
calculate the area under the concentration curve.
a
Significantly different from liquid extract (p < 0.05).
b
Significantly different from liquid extract and fermented capsule (p < 0.005).

The mean values of iAUC for total isoflavones for the three formulations are shown in Figure 2. There
is a tendency that total bioavailability of isoflavones is higher for the fermented capsules compared

10
with the unfermented, however no statistical differences can be seen between the total isoflavone
iAUCs for the three formulations.

Figure 2: iAUC 0-24h for liquid extract (lex), fermented capsule (fcap) and unfermented capsule (ufcap) for total
isoflavones. Paired t-test shows no significant differences. Data are shown as means SEM.

When looking at daidzein and genistein separately statistical differences were obtained (Figure 3).
For daidzein, iAUC was significantly higher for the liquid extract compared to unfermented capsules.
For genistein, iAUC for the unfermented capsules was significantly higher compared to the two
fermented formulations.

Figure 3: iAUC 0-24h for liquid extract (lex), fermented capsule (fcap) and unfermented capsule (ufcap) for daidzein
and genistein. * P < 0.05, ** P < 0.005. Data are shown as means SEM.

There were large variations in changes in plasma concentrations between the subjects, especially in
the initial increase after 2 hours.

11
4 Discussion

Enhancing the bioavailability of isoflavones increases their biological activity. Physiologically

relevant plasma concentrations are needed to exert health beneficial effects. Hence, it is important to
take bioavailability into account when investigating potential health effects of isoflavones.
Bioavailability of isoflavones depends on many factors including isoflavone source and form, food
matrix and habitual diet. Many studies have been carried out to investigate the impact and magnitude
of these factors on bioavailability. Studies have mainly shown that aglycones have higher
bioavailability than glycosides, which is due to their more lipophilic character and lower molecular
weight (Izumi et al., 2000; Okabe et al., 2011). A few studies have failed to show differences between
the two forms or even find that glycosides are more bioavailable (K. D. Setchell et al., 2001; Zubik
& Meydani, 2003). Studies on the effect of food matrix have demonstrated that there is tendency for
higher bioavailability for liquids compared to solids (Cassidy et al., 2006). Studies on bioavailability
are difficult to compare due to heterogeneity between study designs (study duration, isoflavone
source, dose etc.) and extensive differences in inter-individual metabolism. Variations in extraction
and analytical methods used further contribute to disparities apparent in plasma concentrations
between studies.

We found that all four isoflavones of interest (daidzein, genistein, formononetin and biochanin A)
could be detected in the plasma samples, and that daidzein represented the highest concentrations
followed by genistein and formononetin. Biochanin A was detected in negligible amounts.
Approximately the same pattern was observed in a study by Maul et al. where a RC supplement with
similar composition as ours was ingested. They found daidzein in highest concentrations followed by
formononetin and genistein (Maul & Kulling, 2010). It is however contradictory to studies using soy,
where plasma concentrations of genistein have generally been higher than daidzein despite similar
intakes, but this may be due to differences in isoflavone composition in the source (Izumi et al., 2000;
Watanabe et al., 1998). The iAUC for total isoflavones was 1.04 0.15, 1.21 0.28 and 0.99 0.12
mol24h/L for liquid extract, fermented capsule and unfermented capsule respectively. There is no
statistical significant difference between the values for the three formulations but there is a tendency
for a higher bioavailability for the fermented formulations.

Looking at the isoflavones separately different patterns emerge. In the case of daidzein the largest
plasma concentrations and iAUC is seen for the liquid extract, which is also expected as the
isoflavones are mainly found in aglycone form and they are in a liquid matrix. The iAUC is
significantly higher in the extract compared to the unfermented capsules. For formononetin there was
a tendency for higher iAUC for the fermented formulations compared to the unfermented formulation,
but differences did not reach statistical significance. However, when looking at genistein, the
unfermented capsule shows a significantly higher iAUC than the two fermented formulations. This
is unexpected due to isoflavones found mainly as glycosides. There is however, a difference in the
ratios of daidzein and formononetin over genistein and biochanin A in the fermented and unfermented
formulations (Table 3). The ratio for the fermented formulations is on average 1.35 whereas the ratio
is 0.52 for the unfermented capsule. This should be taken into consideration and the most reliable

12
picture regarding bioavailability in general is obtained when looking at total isoflavone
concentrations. There are no significant differences in bioavailability between the fermented liquid
extract and fermented capsules either when looking at total or the individual isoflavones.

When investigating the effect of isoflavone form and matrix on bioavailability differences in
isoflavone source can be a challenge. In this study the isoflavones in the fermented liquid, tablet,
capsule and yoghurt formulation stem from the same source, this is advantageous as it strengthens
the ability to study the effect matrix on bioavailability. There are slight differences in the percentage
distribution of the four studied isoflavones between the liquid extract and the freeze-dried extract but
these are considered to negligibly influence the results. The cause for these slight differences stems
from the different batches of RC used for the liquid and freeze-dried extract. As the isoflavone content
depends on environmental conditions and varies between harvests, it is natural that variations in
content and distribution are seen. However, a clear difference is apparent when comparing the
distributions for the fermented and unfermented formulations. The content of daidzein and its
methylated form formononetin is 1.3 times higher in the fermented formulations than in the
unfermented formulation, whereas the concentration of genistein and its methylated form biochanin
A is almost doubled in the unfermented formulation compared to the fermented formulations.

It is unknown why these differences are seen, especially for formononetin, which is expected to
represent the highest concentrations in RC preparations. During the analyses of the formulations, the
hydrolysed samples were not analysed for remaining glycosides and it is unknown whether a full
hydrolysis was achieved. If this is not the case, the deviation will be most pronounced for the
unfermented formulation containing the highest concentration of glycosides. Another factor
contributing to the lower isoflavone concentrations found in the unfermented capsules may be the
presence of fibre material from the RC plant, as this formulation consists solely of pulverised RC. In
contrast, the fermented RC is a more pure formulation devoid of plant material. These differences in
isoflavone distribution may affect bioavailability and confound results, and are probably the main
reason why different patterns are seen when considering daidzein and genistein separately. It could
have been an advantage to test an unfermented formulation from the same RC source as the fermented
formulation; unfortunately, this was not possible in the study. The variations in isoflavone distribution
seen in the fermented and unfermented RC formulation represents the general pattern of the market
for supplements, where a wide inter- and intra- product variation in the composition is often present
and little standardisation takes place (K. D. Setchell et al., 2001). As isoflavones are shown to have
different biological activities, it is of utmost importance to consider the isoflavone source when
studying physiological effects.

The low concentrations of isoflavones detected in the unfermented capsules used required fifteen
capsules to be taken to obtain the equivalent dose of 40 mg total isoflavones (the chosen dose
resembles average daily intakes of Asian populations (Messina, Nagata, & Wu, 2006)). Fifteen is an
unrealistic amount to take on a daily basis to relieve menopausal symptoms or prevent bone
demineralisation. Hence development of fermented RC formulations with higher and standardised
isoflavone concentrations would provide a clearly preferable solution.

13
We initially attempted to use quadropole time-of-flight mass spectrometer (QTOF-MS) to quantify
isoflavones (aglycones and metabolites) in plasma, as this has not been done in previous studies and
would give information on isoflavone metabolism. However, we did not manage to measure and
quantify the isoflavones with this method. Due to these and other challenges with analytical
equipment and time constraints during the analysis stage of the trial, it was not possible to analyse all
the collected plasma samples. Therefore, the concentration time curves are only based on four
measurements. This increases the risk of skewness of data curves leading to less reliable data and
increased false negative/false positive outcomes. The prioritised time points have been chosen by
looking at results from other bioavailability studies, and consideration must be given the missing
measurements, particularly when determining the maximal concentration point. In other studies the
concentration time data shows that there is a steady decline between 8 and 24 hours post intake, this
implies that our graph closely resembles reality. In the early hours after ingestion our graphs may be
less reliable and representative, especially for the fermented formulation, as maximum concentrations
are expected to be reached at an earlier time. This has been shown in a study with soy powder where
maximum concentration was reached 1-2 hours after ingestion for the aglycone-rich powder whereas
for the glycoside-rich formulation maximum concentration was obtained after 4-5 hours (Okabe et
al., 2011). Our results show peak concentrations at 6 hours for all formulations. Remaining samples
(five for each intervention) will be analysed later, and when values at 4 and 8 hours are available,
differences in time to reach maximum concentration may be observed. There are large variations in
the absorption patterns between the subjects, and some of the graphs have shapes that are difficult to
explain. This may be due to the few points on the curve; once data from all time points are available,
a clearer picture of the absorption and excretion patterns will be obtainable.

The isoflavone concentrations in our samples are lower than expected. In a study by Maul et al.
subjects ingested 38.8 mg RC isoflavones as capsules and plasma samples were taken after 6.5 hours
(Maul & Kulling, 2010). The dose is close to the dose in the present study, and even though the
isoflavone composition was different, the daidzein and formononetin content was similar. Despite of
this, plasma concentrations of these two isoflavones were considerably higher in the female
participants in their study (0.297 M and 0.082 M for daidzein and formononetin respectively)
compared to our values for the 6 hour samples (0.097 M and 0.048 M for daidzein and
formononetin respectively). de Pascual-Teresa measured isoflavone uptake after ingestion of 50 mg
soy isoflavones incorporated in food matrices. Maximum plasma concentrations were 0.4 0.6 M
which are approximately five times higher than the concentrations measured in this study (de Pascual-
Teresa et al., 2006). The low plasma concentrations we measured are unexpected as the isoflavone
dose was similar in the studies and the participants were healthy and of European origin. The average
isoflavone intake in European countries is 1.4 mg/day, so when an isoflavone dose approximately
thirty times higher was administered, we expected higher plasma isoflavone concentrations than twice
the baseline value (Zamora-Ros et al., 2012).

A possible explanation could be that the extraction method requires optimisation and only a small
percentage of the isoflavones are extracted from the plasma. Several methods have been used for

14
sample preparation when analysing isoflavones in plasma, and this can contribute to observed
differences between studies. In the current study, the extraction method used was based on
experiences with isoflavone analyses and similar to methods in other studies. Time did however not
allow for validation of the method, and the method might not be optimal for this purpose, as the use
of other extraction solvents and an internal standard might be appropriate. Furthermore, the hydrolysis
time was based on results from a study by Taylor et al. showing that daidzein and genistein were
hydrolysed after 40 minutes, but a longer hydrolysis time may be necessary to reach higher
concentrations of the aglycones (Taylor, Grace, & Bingham, 2005). This is especially important when
using a triple quadropole MS where you specify the molecules of interest. With incomplete hydrolysis
there is potential for missed intact glucuronides and sulphated conjugates. Due to their hydrophobic
nature, aglycones may bind to plastic surfaces they come in contact with during sample preparation,
which can lead to significant losses of isoflavones (Griffith & Collison, 2001). It may therefore be
more appropriate to use glass utensils where possible, especially for the steps where the sample is in
contact with the surface for a longer time. As the utensils used are seldom mentioned in literature,
focus has not been put on this during our sample preparations, and time did not allow for recovery
studies, which could have clarified whether significant losses took place. The low plasma
concentrations make the results more vulnerable to variations in measurements, and several factors
in our sample preparation may contribute to these low concentrations. However, as it is a cross-over
study and all samples were treated in the same way, it is estimated that even though the extraction
method does not extract all the isoflavones, comparison of the formulations are still possible as they
have been exposed to the same magnitude of error.

The participants were asked to avoid isoflavone containing foods in their diet one week prior to the
study and for the whole duration of the study. We expected the baseline concentrations to be close to
zero, and were surprised to find concentrations, which were high compared to the measurements at
later time points and values found in other studies. The mean baseline values for daidzein and
genistein were 0.07 and 0.04 M respectively, and are at least ten times higher than values found for
the Danish participants in the European Prospective Investigation into Cancer and Nutrition (EPIC).
They found mean plasma concentrations of daidzein and genistein of 0.004 M (Peeters et al., 2007).
The reason for this difference is unknown but variations caused by the sample preparation method
mentioned previously might contribute. Consequently, the iAUCs have been used taking baseline
values into account.

Isoflavones in liquid formulations might be more bioavailable as they are already in solution and the
solubility affects the rate of absorption. One study has shown higher AUC and maximal
concentrations of isoflavones when ingesting soy milk compared to solid textured vegetable protein
(TVP) (Cassidy et al., 2006). However, other studies have failed to show a clear effect as differences
between absorption of isoflavones from soy milk and TVP were only observed for genistein and only
in women (Faughnan et al., 2004). Other factors than food matrix may affect the bioavailability
including isoflavone composition as this will be different in soy milk and TVP even though they are
both from soy. In this study the liquid and solid (capsule) formulations have approximately the same
isoflavone composition and no significant differences in bioavailability were seen. This evaluation is

15
however based on a limited number of analysed samples and low plasma concentrations, and patterns
may change when all results are available. It would be beneficial if bioavailability is preserved after
processing the extract to a capsule, as capsules would solve a number of issues regarding palatability,
storage and logistical challenges of the extract.

Studies have shown that the bioavailability of isoflavones is higher for aglycones than for glycosides
as the initial hydrolysis step is not needed. Okabe et al. found an increase of 60% and 20% of the
maximal plasma concentration and the AUC respectively, when ingesting an aglycone-rich soybean
formulation compared with a formulation rich in glycosides (Okabe et al., 2011). One method of
converting glycosides into aglycones is by fermentation with lactic acid bacteria. This process
doubled the amount of aglycones in the fermented RC formulations in contrast to the unfermented
formulation in this study (Table 2). We failed to show the expected difference between fermented
and unfermented formulations in terms of total isoflavone uptake. There is a tendency for a higher
bioavailability of the fermented formulations when looking at Figure 2, however the differences are
small and were non-significant. As the results are at present only based on five participants, the
statistical foundation is weak and it is expected that the patterns will become more clear and robust
when samples from all 15 participants and all data points have been analysed.

The isoflavone content in RC is unique with a high content of formononetin and biochanin A
compared to soy. This has an influence on its biological effects as formononetin has been shown to
have an increased potency on osteoblast function compared to daidzein (Gautam et al., 2011).
However, a great conversion of formononetin and biochanin A into their demethylated forms daidzein
and genistein takes place, and lower concentrations of formononetin and biochanin A are often found
in plasma compared to daidzein and genistein even when administered in higher doses. Few studies,
with a limited number of participants, have looked at plasma concentrations of formononetin and
biochanin A after red clover intake. They found that substantial demethylation takes place, and even
though the intakes of the methylated isoflavones are 10 - 40 times higher than the demethylated
isoflavones, the ratio of methylated over demethylated isoflavone ranges from 0.2 - 0.5 in plasma
(Howes et al., 2000; Maul & Kulling, 2010; K. D. Setchell et al., 2001). We expected to see increases
in concentrations of formononetin and biochanin A based on the findings of these studies and due to
the high contents in the RC formulations. Surprisingly we found no increases in biochanin A
concentrations and the increases for formononetin are relatively minor. Issues with the sample
preparation, as mentioned earlier, might explain this. Despite the low concentrations, an evaluation
of the iAUC for formononetin shows a tendency for a higher bioavailability for the fermented
formulations, which could be a reflection of the higher concentration of formononetin in these
formulations.

Equol is a metabolite of daidzein that has higher estrogenic activity than daidzein and therefore
potentially exerts greater biological effects than its parent molecule. Equol is produced in the large
intestine by gut microbiota but not all people are able to produce equol. Appr. 30% of the American
and European population have this ability whereas in Asia the proportion is appr. 50% (Okabe et al.,
2011). Some of the participants in this study may be equol producers, which would lead to lower

16
plasma concentrations of daidzein. Plasma concentrations of equol were not measured in this study,
but would have given a fuller picture of the total amount of isoflavones being absorbed. However,
the main purpose of this study is to compare the bioavailability of different formulations. As it is a
cross-over study the subjects act as their own controls and it can be assumed that the subjects capable
of equol production produce consistently across all five formulations.

Diet may have an effect on isoflavone pharmacokinetics, as it affects gut microflora and thereby
metabolism and absorption. In this study standardised diets were given to the participants during the
interventions, and no differences in energy or macronutrient intake were seen between the five
interventions (Table 5). The diet on the day before the intervention was not standardised but registered
in diet diaries. Large variations were seen between the participants and between the interventions; as
such it might have been advantageous to extend the standardised diets to start prior to the intervention
day. Registration of diet the day prior to ingestion of a formulation reflects habitual diets of the
participants, this may have a greater influence on bioavailability than the post ingestion diet given
during the study. These large differences in habitual diet may partly explain the large inter-individual
differences in bioavailability. Gut transition time and genetics also have an impact on absorption and
metabolism of isoflavones and contribute to inter-individual variations, but as this is a cross-over
study the influence of this variation is reduced.

The standardised diet followed nutritional recommendations for a healthy lifestyle and was therefore
higher in dietary fibres than the diets registered on the days prior to formulation intake, and the
habitual diets of a great proportion of the Danish population. It has been shown that dietary fibre has
an impact on bioavailability, some studies have shown increased bioavailability after addition of
inulin while others show reduced absorption with increasing fibre content in the diet (Piazza et al.,
2007; Tew, Xu, Wang, Murphy, & Hendrich, 1996). The direct effect of fibres in the diet on
bioavailability is therefore unclear, and it is uncertain whether it is a long term effect or whether the
fibre content in the diet has a more immediate impact when ingested simultaneously with isoflavones.
It is a possibility that the relatively high fibre content in the standardised diet contributes to the low
isoflavone plasma concentrations seen in this study.

Bioavailability of isoflavones may be influenced by the context in which the isoflavones are ingested,
especially in the case of isoflavones taken as supplements. An in vitro study by Walsh et al. showed
that concentrations of bile affected the bioavailability of isoflavones, it is proposed that isoflavones
in a food matrix or eaten together with foods containing fat and protein have higher bioavailability
than isoflavones from supplements taken without a meal (Walsh, Zhang, Vodovotz, Schwartz, &
Failla, 2003). In the current study the RC formulations were provided together with a breakfast meal,
which should even out variations caused by differences in bile secretion.

Lactic acid bacteria are used as probiotics as they influence gut microflora and they may therefore
affect bioavailability of isoflavones. The studies in this field show mixed effects of probiotics on
bioavailability, which might primarily be due to differences in dosage levels and duration of intake
(Cohen et al., 2007; Larkin, Price, & Astheimer, 2007; Nettleton et al., 2004). In order for probiotics

17
to have a beneficial impact on gut microflora long-term intake is probably necessary. Lactic acid
bacteria are used in the fermentation process for four RC preparations used in the present study. The
bacteria found in the freeze-dried extract are in lower concentrations due to losses during processing.
The presence of lactic acid bacteria in these RC formulations might contribute to increased absorption
of isoflavones after long-term intake.

In the current study, young healthy women were chosen as subjects in order to reduce variations in
health status. However, the main target group for RC products is menopausal and postmenopausal
women experiencing hot flashes and bone demineralisation. It is unknown whether the absorption
patterns would have been different for these women. A study of the effect of age on urinary excretion
of isoflavones failed to show a difference between pre- and postmenopausal women (Faughnan et al.,
2004). Another study on plasma also failed to show a significant effect of age and menopausal stage
on the bioavailability of isoflavones (K. D. R. Setchell et al., 2003). It is therefore assumable that the
absorption patterns observed in this study will be similar in postmenopausal women.

Because bioavailability is important, methods of increasing absorption are of interest. Apart from the
potential factors mentioned previously (fermentation, probiotics) microencapsulation may be a useful
method. Microencapsulation is used in the food and pharmaceutical industry and could increase
bioavailability of isoflavones by increasing residence time and obtaining more constant plasma
concentrations (K. D. R. Setchell et al., 2005). Future studies of the effects of this method as well as
large long-term studies of the influence of isoflavone form, matrix, dose etc. on bioavailability will
be useful in determining the optimal dosage and form of isoflavone intake to obtain desired
physiological effects.

We attempted to investigate the bioavailability of isoflavones from fermented and unfermented RC

formulations (five in total) by analysing blood samples from fifteen healthy young women recruited
for this cross-over study. Due to considerable challenges with analytical equipment, it was only
possible to analyse approximately 10 % of all samples within the time frame of this project, and
interpretations are based on this reduced number of samples. The results suggest a tendency for total
isoflavone bioavailability being higher for fermented RC (liquid extract and capsules) compared to
unfermented capsules, even though the results did not reach statistical significance. When looking at
daidzein there was a significantly higher bioavailability for the fermented extract compared to the
unfermented capsule. The total isoflavone bioavailability was almost equal in the fermented liquid
extract and capsules, indicating that processing does not reduce isoflavone bioavailability. Stronger
conclusions can be drawn when data from all samples are available.

18
Conflict of interest
The authors declare that they have no conflicts of interests.

Acknowledgements

The author wishes to thank Michael Mohr Jensen (Herrens Mark) for providing RC extract and Hans-
Christian Vollstedt (Naturdrogeriet) for production of the fermented capsules and tablets. Also a
thank you to Lars Jrgensen (DBLab), Xavier Frett, Flemming Nielsen and Rime Bahij El-Houri
(University of Southern Denmark) for analytical assistance, and to the women who participated in
the study.

References

Beck, V., Rohr, U., & Jungbauer, a. (2005). Phytoestrogens derived from red clover: an alternative
to estrogen replacement therapy? The Journal of Steroid Biochemistry and Molecular Biology,
94, 499518.

Booth, N. L., Piersen, C. E., Banuvar, S., Geller, S. E., Shulman, L. P., & Farnsworth, N. R. (2006).
Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a
literature review. Menopause (New York, N.Y.), 13, 251264.

Cassidy, A., Brown, J. E., Hawdon, A., Faughnan, M. S., King, L. J., Millward, J., Setchell, K.
D. R. (2006). Factors affecting the bioavailability of soy isoflavones in humans after ingestion
of physiologically relevant levels from different soy foods. The Journal of Nutrition, 136, 45
51.

Cohen, L. a., Crespin, J. S., Wolper, C., Zang, E. a., Pittman, B., Zhao, Z., & Holt, P. R. (2007).
Soy isoflavone intake and estrogen excretion patterns in young women: Effect of probiotic
administration. In Vivo, 21, 507512.

De Pascual-Teresa, S., Hallund, J., Talbot, D., Schroot, J., Williams, C. M., Bugel, S., & Cassidy,
A. (2006). Absorption of isoflavones in humans: effects of food matrix and processing. The
Journal of Nutritional Biochemistry, 17, 25764.

Faughnan, M. S., Hawdon, A., Ah-Singh, E., Brown, J., Millward, D. J., & Cassidy, A. (2004).
Urinary isoflavone kinetics: the effect of age, gender, food matrix and chemical composition.
The British Journal of Nutrition, 91, 56774.

Gautam, A. K., Bhargavan, B., Tyagi, A. M., Srivastava, K., Yadav, D. K., Kumar, M., Singh,
D. (2011). Differential effects of formononetin and cladrin on osteoblast function, peak bone
mass achievement and bioavailability in rats. Journal of Nutritional Biochemistry, 22, 318
327.

Griffith, a P., & Collison, M. W. (2001). Improved methods for the extraction and analysis of
isoflavones from soy-containing foods and nutritional supplements by reversed-phase high-

19
 performance liquid chromatography and liquid chromatography-mass spectrometry. Journal of
Chromatography. A, 913, 397413.

Howes, J. B., Sullivan, D., Lai, N., Nestel, P., Pomeroy, S., West, L., Howes, L. G. (2000). The
effects of dietary supplementation with isoflavones from red clover on the lipoprotein profiles
of post menopausal women with mild to moderate hypercholesterolaemia. Atherosclerosis,
152, 143147.

Izumi, T., Piskula, M. K., Osawa, S., Obata, A., Tobe, K., Saito, M., Kikuchi, M. (2000). Soy
isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in
humans. The Journal of Nutrition, 130, 16959.

Jerome-Morais, A., Diamond, A. M., & Wright, M. E. (2011). Dietary supplements and human
health: for better or for worse? Molecular Nutrition & Food Research, 55, 122135.

Larkin, T. a., Price, W. E., & Astheimer, L. B. (2007). Increased probiotic yogurt or resistant starch
intake does not affect isoflavone bioavailability in subjects consuming a high soy diet.
Nutrition, 23, 709718.

Maul, R., & Kulling, S. E. (2010). Absorption of red clover isoflavones in human subjects: results
from a pilot study. The British Journal of Nutrition, 103, 156972.

Messina, M., Nagata, C., & Wu, A. H. (2006). Estimated Asian adult soy protein and isoflavone
intakes. Nutrition and Cancer, 55, 112.

Nettleton, J. a, Greany, K. a, Thomas, W., Wangen, K. E., Adlercreutz, H., & Kurzer, M. S. (2004).
Plasma phytoestrogens are not altered by probiotic consumption in postmenopausal women
with and without a history of breast cancer. The Journal of Nutrition, 134, 19982003.

Okabe, Y., Shimazu, T., & Tanimoto, H. (2011). Higher bioavailability of isoflavones after a single
ingestion of aglycone-rich fermented soybeans compared with glucoside-rich non-fermented
soybeans in Japanese postmenopausal women. Journal of the Science of Food and Agriculture,
91, 65863.

Peeters, P. H. M., Slimani, N., van der Schouw, Y. T., Grace, P. B., Navarro, C., Tjonneland, A.,
Bingham, S. a. (2007). Variations in plasma phytoestrogen concentrations in European adults.
The Journal of Nutrition, 137, 12941300.

Piazza, C., Privitera, M. G., Melilli, B., Incognito, T., Marano, M. R., Leggio, G. M., Drago, F.
(2007). Influence of inulin on plasma isoflavone concentrations in healthy postmenopausal
women. American Journal of Clinical Nutrition, 86, 775780.

Richelle, M., Pridmore-Merten, S., Bodenstab, S., Enslen, M., & Offord, E. A. (2002). Hydrolysis
of isoflavone glycosides to aglycones by beta-glycosidase does not alter plasma and urine
isoflavone pharmacokinetics in postmenopausal women. The Journal of Nutrition, 132, 2587
92.

20
Setchell, K. D., Brown, N. M., Desai, P., Zimmer-Nechemias, L., Wolfe, B. E., Brashear, W. T.,
Heubi, J. E. (2001). Bioavailability of pure isoflavones in healthy humans and analysis of
commercial soy isoflavone supplements. The Journal of Nutrition, 131, 1362S75S.

Setchell, K. D. R., Brown, N. M., Desai, P. B., Zimmer-Nechimias, L., Wolfe, B., Jakate, A. S.,
Heubi, J. E. (2003). Bioavailability, disposition, and dose-response effects of soy isoflavones
when consumed by healthy women at physiologically typical dietary intakes. The Journal of
Nutrition, 133, 102735.

Setchell, K. D. R., Brzezinski, A., Brown, N. M., Desai, P. B., Melhem, M., Meredith, T., Blatt,
Y. (2005). Pharmacokinetics of a slow-release formulation of soybean isoflavones in healthy
postmenopausal women. Journal of Agricultural and Food Chemistry, 53, 19381944.

Taylor, J. I., Grace, P. B., & Bingham, S. a. (2005). Optimization of conditions for the enzymatic
hydrolysis of phytoestrogen conjugates in urine and plasma. Analytical Biochemistry, 341,
220229.

Tew, B. Y., Xu, X., Wang, H. J., Murphy, P. A., & Hendrich, S. (1996). A diet high in wheat fiber
decreases the bioavailability of soybean isoflavones in a single meal fed to women. The
Journal of Nutrition, 126, 8717.

Turner, N. J., Thomson, B. M., & Shaw, I. C. (2003). Bioactive isoflavones in functional foods: the
importance of gut microflora on bioavailability. Nutrition Reviews, 61, 20413.

Vitale, D. C., Piazza, C., Melilli, B., Drago, F., & Salomone, S. (2013). Isoflavones: estrogenic
activity, biological effect and bioavailability. European Journal of Drug Metabolism and
Pharmacokinetics, 38, 1525.

Walsh, K. R., Zhang, Y. C., Vodovotz, Y., Schwartz, S. J., & Failla, M. L. (2003). Stability and
bioaccessibility of isoflavones from soy bread during in vitro digestion. Journal of
Agricultural and Food Chemistry, 51, 46039.

Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y., Adlercreutz, H.
(1998). Pharmacokinetics of soybean isoflavones in plasma, urine and feces of men after
ingestion of 60 g baked soybean powder (kinako). The Journal of Nutrition, 128, 17105.

Yuan, J.-P., Wang, J.-H., & Liu, X. (2007). Metabolism of dietary soy isoflavones to equol by
human intestinal microflora--implications for health. Molecular Nutrition & Food Research,
51, 76581.

Zamora-Ros, R., Knaze, V., Lujan-Barrosso, L., Kuhnle, G., Mulligan, A., Touillaud, M.,
Gonzales, C. a. (2012). Dietary intakes and food sources of phytoestrogens in the European
Prospective Investigation into Cancer and Nutrition (EPIC) 24-hour dietary recall cohort.
European Journal of Clinical Nutrition, 66, 932941.

Zubik, L., & Meydani, M. (2003). Bioavailability of soybean isoflavones from aglycone and
glucoside forms in American women. The American Journal of Clinical Nutrition, 77, 1459
65.

21
Appendix A

Table A.1: Standardised diet during intervention

Day Meal Menu
1 Breakfast 1 bread roll, 2 slices of cheese, butter, marmalade, orange- or
apple juice, 1 apple, coffee or tea
1 Lunch Sandwich with chicken, tomato, cucumber, lettuce and
mayonnaise
1 Dinner Pasta with leeks, ham, cheese, cream and eggs
Mixed salad (carrot, pointed cabbage, chinese cabbage, corn,
tomatoes)
Salad dressing
1 Snacks 2 carrots, 1 bread roll, butter, 1 banana, 1 pear, 1 muesli bar with
chocolate
2 Breakfast 1 bread roll, 2 slices of cheese, butter, marmalade, orange- or
apple juice, 1 apple, coffee or tea
2 Lunch 2 pita breads with ham, tuna, mixed salad (carrot, pointed cabbage,
chinese cabbage, corn, tomatoes) and salad dressing
2 Dinner Lasagne with chicken, tomato, onion, garlic, carrots, courgettes
and mushrooms
Mixed salad (carrot, pointed cabbage, chinese cabbage, corn,
tomatoes)
Salad dressing
2 Snacks 2 carrots, 1 bread roll, butter, 1 banana, 1 pear, 1 muesli bar with
nuts
3 Breakfast 1 bread roll, 2 slices of cheese, butter, marmalade, orange- or
apple juice, 1 apple, coffee or tea

22