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 Chapter 7

Isoflavones: Vegetable Sources, Biological Activity, and

Isoflavones: Vegetable
Analytical Methods for Sources, Biological Activity, and
Their Assessment
Analytical Methods for Their Assessment

Daniela-Saveta Popa and Marius Emil Rusu

Daniela-Saveta Popa and Marius Emil Rusu
Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

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Abstract
Phytoestrogens are natural compounds found in various plant species and they have the
ability to bind to the estrogenic receptors, exerting agonist and/or antagonist effects. The
main classes of phytoestrogens are isoflavones, lignans, and coumestranes. Isoflavones
are plant bioactive nonsteroidal polyphenolic metabolites with antioxidant properties.
They have a very close structure with 17β-estradiol and possess estrogenic/antiestrogenic
effects. The main dietary source of isoflavones is soy (Glycine max L.). Other legumes,
such as red clover (Trifolium pratense L.), alfalfa (Medicago sativa L.), and Genista species,
have important content in isoflavones, showing nutritional or phytotherapeutic inter-
est. In plants, isoflavones can be found mainly as non-active glycosides which are con-
verted after ingestion, in the corresponding aglycones (e.g., genistein, daidzein) that have
pharmacological activity. Many studies have demonstrated the benefits of dietary iso-
flavones in menopause and multiple chronic pathologies, including cardiovascular dis-
eases, osteoporosis, and hormonal cancers. Dietary intake of isoflavones is widespread,
mainly due to the consumption of soybean products. Analytical methods applied for the
quantification of isoflavones allow both assessment of dietary intake of isoflavones and
highlighting natural sources with phytotherapeutic potential. Health benefits of isofla-
vones justify the interest for this class of functional food; therefore, further clinical and
epidemiological studies are required.

Keywords: nutraceuticals, phytoestrogens, isoflavones, vegetables, analysis

1. Introduction

Phytoestrogens are natural nonsteroidal compounds able to bind to estrogenic receptors and
have both estrogenic and antiestrogenic activities. They are widespread in the plant kingdom
being considered ubiquitous. The main classes of phytoestrogens are isoflavones, coumes-
tans, and lignans.

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134 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Isoflavones are plant-derived secondary metabolites with a polyphenolic structure and antiox-
idant properties [1]. They pertain to the flavonoid class and are found mostly in plants belong-
ing to Fabaceae family. Soy (Glycine max L.) is the major natural source of isoflavones, and
the benefits associated with a soy diet occur mostly because of these phytochemicals. Other
natural sources of isoflavones are red clover (Trifolium pratense L.), alfalfa (Medicago sativa L.),
and species of the genus Genista. All of these plants present phytotherapeutic and nutraceuti-
cal significance, and their by-products, herbal teas, and food supplements are often used.
Several epidemiological studies have demonstrated the benefits of dietary isoflavones in
menopause and multiple chronic pathologies, including cardiovascular diseases, osteoporo-
sis, and hormonal cancers. The main mechanisms of action of isoflavones, their benefits to
human health, and the factors involved in the modulation of their bioactivity are shown in
this chapter. Moreover, the analytical methods used for their quantification in plant and food
samples are introduced. These are very important methods to evaluate the human exposure
to isoflavones and also to assess the optimum intake for human well-being.

2. Characteristics of isoflavones

2.1. Chemistry and metabolism of isoflavones

Isoflavones (IFs) are yellow pigments derived from 3-phenyl-benzopyrone (3-phenyl-chro-
mone) structure. They are found in plants mostly as biologically inactive glycosides: 7-O-β-
D-glycosides, 6″-O-acetyl-7-O-β-D-glucosides, and 6″-O-malonyl-7-O-β-D-glycosides [1, 2].
After ingestion, glycosides are not bioavailable to be absorbed through enterocytes [3]. They
are hydrolyzed into bioactive aglycones by both intestinal mucosa and bacterial β-glucosidases
from the gut microbiota. Only these forms are absorbed into systemic circulation directly or
after subsequent metabolism in the bowel by intestinal bacteria [3]. Soybeans incorporate pre-
dominantly genistin, daidzin, and glycitin as inactive glycosides, which are hydrolyzed into
their corresponding biologically active aglycones: genistein, daidzein, and glycitein. Other
isoflavones observed in legumes are ononin and sissotrin, with their aglycones, formonone-
tin, and biochanin A, respectively (Figure 1).

The absorption of aglycones is fast and efficient. Plasmatic isoflavone levels increase up to
micromolar-level values after the consumption of soy-based foods, compared to the nanomo-
lar (≤40 nm) levels found in diets without soy [4]. First pharmacokinetic study on isolated and
purified isoflavones was performed, when a single dose of 50 mg of aglycone or the equiva-
lent dose of β-glycoside, respectively, was given to healthy adult volunteers. The plasmatic
peak values (Cmax) were 341 ± 74 ng/mL for genistein and 194 ± 30.6 ng/mL for daidzein. The
times when the values reached the peaks were 5.2 and 6.6 hours (tmax) in the case of direct
aglycones ingestion and 9.3 and 9.0 h in the case of the ingestion of β-glycosides, genistin, and
daidzin, due to the time required for their hydrolysation. The bioavailability of genistein and
daidzein (based on the area under the curve in plasma concentration versus time graph) was
higher after consumption of β-glycosides [5].

Formononetin and biochanin A can be transformed to daidzein and genistein, respectively,

through 4′-O-demethylation by the gut microflora or in the liver [6]. Aglycones can be fur-
ther metabolized through several steps: reduction, deoxygenation, hydroxylation, and C-ring
 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 135
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Figure 1. Chemical structure of main isoflavones.

cleavage. Daidzein forms S-(−)equol and O-desmethylangolensin (O-DMA) via dihydro-

daidzein (Figure 2). Similarly, genistein is metabolized first as dihydrogenistein and then
as 5′-hydroxy-equol and p-ethyl phenol (Figure 2). Another possible minor pathway is the
hydroxylation of isoflavone rings at different positions, catalyzed by hepatic cytochrome
P450 isoenzymes [2]. Metabolites with phenolic or polyphenolic structures are conjugated
to O-glucuronides and sulfate esters during and after absorption through the gut barrier and
more intense in the liver. The conjugated metabolites are urinary or biliary excreted and have
enterohepatic circulation [4, 7].

Gut microbiota play a very important role in the isoflavone metabolism. The positive effects of a
soy-rich diet derive from the existence of microorganisms in the gut capable of intense metabo-
lization of isoflavones. It is the so-called equol producer phenotype, responsible for metaboliz-
ing daidzein to equol and identified through the equol/daidzein ratio in the 24-hour urine. Asian
people (Japanese, Korean, or Chinese) and Western adult vegetarians are 50–60% equol produc-
ers, but equol producers are only 25–30% in Western population. This phenotype is rather stable
and cannot be modulated through prebiotic or probiotic nutritional interventions [8]. Otherwise,
there are differences between human and animal metabolism, and therefore in vivo results are
not relevant to humans [9]. All tested animals had equol in urine after the ingestion of soy or
clover [8]. Notably in rodents, equol constitutes 70–90% from the serum isoflavones, compared
to humans where only 30% of the daidzein absorbed is metabolized as equol [4].

2.2. Isoflavone content in different sources

Isoflavones can be found in legumes [10–12], nuts, and some fruits, such as currants and rai-
sins [13], coffee [14], and cereals [15], but the most important dietary sources are soybeans and
136 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Figure 2. Metabolic pathways of daidzein and genistein.

their by-products [10, 12]. The content of isoflavones in several plants and foods is presented
in Tables 1 and 2. Soy can be ingested as textured soy protein, as soy milk or drink, added to
many fortified foods (e.g., energized bars, cereals, baby formula), or consumed as fermented
soybean products, such as miso, natto, and tempeh (Table 3) [12]. Also, many food supple-
ments containing soy isoflavones are on the market [16].
Isoflavone content in plants can vary greatly (up to threefold) for the same variety by growth
conditions, geographical areas, years, biotic stress factors (e.g., pests), and abiotic stress fac-
tors, such as temperature, nutritional status, or drought [4]. Dietary culture has an especially
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Food description Daidzein Genistein Glycitein Total IFs

Soybeans, green, mature 61.70 60.07 7.07 128.83
seeds, raw

Soybeans, mature seeds, 62.07 (27.77–78.86) 80.99 (39.78–89.32) 14.99 (9.01–22.37) 154.53 (85.68–178.81)
raw (the mean values from
Australia, Brazil, China,
Europe, Japan, Korea,
Taiwan, the USA)

Soybeans, mature seeds, 30.76 31.26 3.75 65.11

cooked, boiled

Beans, common, raw 0.29 0.30 0.00 0.59

(Phaseolus vulgaris)

Beans, adzuki, mature 0.36 0.23 0.00 0.59

seeds, raw

Beans, pinto, mature seeds, 0.01 0.17 - 0.18

raw

Black bean, sauce 5.96 4.04 0.53 10.26

Chickpeas, mature seeds, 0.21 0.06 0.18 0.38

raw

Chickpeas, mature seeds, 0.00 0.02 - 0.02

cooked, boiled

Peas, green, split, mature 0.32 0.11 0.00 0.44

seeds, raw

Table 1. Isoflavone content in selected legumes (mg/100 g, edible portion—the mean value derived from multiple
experiments) [12].

Food description Coumestrol Formononetin Biochanin A

Egg, whole, raw, fresh 0.00 0.05 0.05

Alfalfa seeds, sprouted, raw 1.60 1.43 0.04

Clover sprouts, raw 14.08 3.15 0.59

Red clover 1322.00 833.00 -

Soybeans, mature seeds, raw 0.02 8.46 0.00

Soybeans, mature seeds, sprouted, 0.34 0.03 0.00

raw

Lima beans, large, mature seeds, raw 0.14 0.32 0.27

Lima beans, large, mature seeds, 0.00 0.01 0.00

boiled

Chickpeas, mature seeds, raw 0.01 0.12 1.54

Chickpeas, mature seeds, canned 0.00 0.00 -

Table 2. Coumestrol, Formononetin, and Biochanin A in selected foods (mg/100 g, edible portion—the mean value
derived from multiple experiments) [12].
138 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Food description Daidzein Genistein Glycitein Total IFs

Miso 16.43 23.24 3.00 41.45

Natto 33.22 37.66 10.55 82.29

Tempeh 22.66 36.15 3.82 60.61

Tofu, raw, regular, 8.56 12.99 1.98 22.73

prepared with calcium
sulfate

Soybeans, green, raw 20.34 22.57 7.57 48.95

(includes edamame)

Soybeans, green, 7.41 7.06 4.60 17.92

cooked, boiled,
drained, without salt
(includes edamame)

Soybeans, mature 12.86 18.77 2.88 34.39

seeds, sprouted, raw

Instant beverage, 40.07 62.18 10.90 109.51

soy, powder, not
reconstituted

Soy cheese, unspecified 5.79 11.14 - 25.72

Soy drink 2.75 5.10 - 7.85

Soy flour (textured) 67.69 89.42 20.02 172.55

Soy meal, defatted, raw 80.77 114.71 16.12 209.58

Soy protein drink 27.98 42.91 10.76 81.65

Soy protein isolate 30.81 57.28 8.54 91.05

Soy yogurt 13.77 16.59 2.80 33.17

Table 3. Isoflavone content in soy foods (mg/100 g, edible portion—the mean value derived from multiple
experiments) [12].

big influence on isoflavone content in the diet. Asian and vegetarian diets provide 20–50 mg
isoflavones/day, in some cases reaching 100 mg/day, while the Western diet contributes only
0.2–1.5 mg isoflavones/day [2]. Based on recent report of European Food Safety Authority
(EFSA), in Europe the dietary isoflavone intake is usually under 1 mg/day, despite an increase
in the soy food consumption [17]. The differences between the types of diets refer to the
amount of isoflavone in foods, as well as the type of food consumed. In the Western diet,
solid processed soy products (such as tofu) and soymilk dominate the diet, and they contain
both glycosides (genistin and daidzin which are stable during processing) and aglycones.
In the Asian diet, most soy products are obtained through fermentation and have higher
amounts of aglycones [3]. Miso, fermented soybean paste (Japan); doenjang, fermented soy-
bean paste (Korea); douchi, fermented soybeans (China); and tempeh, fermented soybean
cake (Indonesia) are staple foods in some Asian countries. Simultaneously, health benefit pro-
biotics are formed in these foods during the fermentation processes [18].
 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 139
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Besides soy, other plants in the Fabaceae family have a high content of isoflavones: spe-
cies of clover, mainly red clover (Trifolium pratense L.), alfalfa (Medicago sativa L.), and hop
clover (Medicago lupulina L.), form important part of animal feed. These plants are used in
phytotherapy, as medicinal teas or as food supplements. Red clover (Trifolium pratense L.)
incorporates mainly genistein, daidzein and formononetin, and their respective β-glycosides
[19–22]. It also contains important quantities of coumestrol, a phytoestrogen part of coumes-
tan class [19, 20], and antioxidant compounds [23, 24]. Data from scientific literature show
that red clover extracts can be used as replacement for conventional hormonal therapy in
menopause or hormone-dependent diseases [25]. Alfalfa (Medicago sativa L.) contains isofla-
vones (genistein, daidzein, formononetin, biochanin A) in addition to other phytoestrogens
(coumestrol) and many nutrients. It is used in phytotherapy for its antianemic, antihemor-
rhagic, and remineralization properties [26] and for its hypocholesterolemic, antimicrobial,
hypolipidemic, antioxidant, antiulcer, neuroprotective, and estrogenic properties [27]. Species
of Genista (G. tinctoria L., G. sagittalis L.) contain essentially genistin and genistein [19, 20, 28,
29]. They are known for their hypoglycemic [30], anti-inflammatory, antiulcer, spasmolytic,
antioxidant, and estrogenic properties [31]. Among these plants, Genista tinctoria L. show anti-
oxidant and antitoxic activities [32, 33], protective effect against ultraviolet (UV) radiation,
and in vitro melanoma cell proliferation [31].

2.3. Mechanism of estrogen-like action of isoflavones

According to the xenohormesis theory, plants synthesize phytochemicals to withstand and
adapt under stress. Indeed, isoflavone biosynthesis depends on the environmental conditions
in which the plant grows and is stimulated by stress. The stress-induced plant compounds
have the ability to upregulate stress adaptive pathways in animals and humans. In the body,
the biological effects of isoflavones are exercised by modulating pathways mediated by estro-
gen receptors (ERs) or various key enzymes involved in cellular signaling or metabolism and
antioxidant potential [4].

3. The estrogenic/antiestrogenic effects

Isoflavones produce both estrogenic and antiestrogenic effects through several ways. Due to
their structure similar to that of 17β-estradiol, they have the ability to bind to the nuclear ERs,
but their affinity for these receptors is rather weak. Only genistein shows stronger affinity for
ERβ to which it binds preferentially. Its relative affinity (0.87) is closer to that of the reference
hormone, 17β-estradiol. Daidzein affinity for these receptors is 0.005, but equol, its metabolite,
has a 5.7 times stronger affinity, thus increasing its estrogenic potential. The affinity for ERα
decreases as follows: genistein > equol > daidzein, with the values of 0.04, 0.005, and 0.001,
respectively. The affinities of other isoflavones are less than 0.0001 [2, 4].

Isoflavones induce agonist/antagonist effects depending on the level of the endogenous estro-
gen. For people with high levels of estrogen, (women premenopause, especially in the fol-
licular phase of the menstrual cycle), the isoflavones bind to the estrogen receptors. Because
of their weak estrogen potency, isoflavones exert an antagonist effect. They block the action of
140 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

endogenous estrogens on their receptors. In case of low concentration of endogenous estro-

gens (women in menopause, after ovariectomy, or males), the estrogenic action of isoflavones
becomes evident, showing additive agonist effect [34]. This is the reason why isoflavones can
be used as a long-term complementary or alternative hormone therapy [35].
Isoflavones and their active metabolites can bind to the membrane ERs and induce rapid
non-genomic effects by which they modulate cellular metabolism. Thus, they can change the
protein kinase and lipid kinase cell signaling pathways [1]. It is believed that the activation
of these signaling pathways by isoflavones causes some beneficial effects, in particular in the
tissues that are not specific targets for the estrogens. At the circulatory system, the isoflavones
induce vasodilation by increasing the production of nitric oxide (NO) after the activation of
the endothelial NO− synthase. At the central nervous system, they improve the cognitive func-
tion by affecting cell membrane permeability and altering the neuronal excitability. In the
skeletal system, the isoflavones inhibit the tyrosine kinase causing changes in the alkaline
phosphatase activity. On the other hand, they induce the apoptosis of the osteoclasts, sup-
press the formation of osteoclasts [34], and prevent the bone demineralization [35].
Also, isoflavones influence the activity of some of the enzymes involved in the metabolism
of the sex steroid hormones. In this way they inhibit 5α-reductase (the enzyme responsible
for the conversion of testosterone to 5α-dihydrotestosterone) and aromatase (involved in the
conversion of testosterone to estradiol) in low concentrations, but they increase the aromatase
activity at high concentrations. Isoflavones have an affinity for sex hormone-binding globulin
(SHBG) and they induce its expression. Therefore, they affect the free-steroid hormone level
in the systemic circulation. But these outcomes depend on many factors, including species,
gender, and the hormonal status [35].

Xenoestrogens can modulate the enzyme activity of aromatase. Thus, they induce alterations
in the metabolism of fats and carbohydrates through effects on ERα. The decrease of endog-
enous estrogen levels on ERα, aromatase inhibition or the existence of mutations affecting
the enzyme activity has been correlated with visceral obesity or truncate, hyperlipidemia,
glucose intolerance and insulin resistance, low physical activity, and reduced energy expen-
diture. Isoflavones compensate for the deficit of estrogens and have the ability to prevent the
associated negative effects. Asian diets, rich in isoflavones, are correlated with low incidence
of obesity and metabolic syndrome, favorable plasma profile, and a reduced body mass index
in postmenopausal women [4].

4. Health benefits of isoflavones

4.1. Isoflavones and their effects on diseases

Numerous epidemiological and clinical studies have demonstrated the protective role of
dietary isoflavones against development of specific menopause symptoms [36–38] and sev-
eral chronic diseases, including cardiovascular diseases [39, 40], osteoporosis [38], cognitive
impairment [37], and hormone-dependent cancers [41–43]. Based on human health benefits of
soy diet, the Food and Drug Administration (FDA) approved the use of the following health
 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 141
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claim on the labels: “25 grams of soy protein a day, as part of a low in saturated fat and cho-
lesterol, may reduce the risk of heart disease” [44].

Isoflavones, as all polyphenols, have a strong antioxidant activity. They can neutralize free
radicals and prevent the lipid peroxydation by stopping the chain reactions. Also, isoflavones
induce the antioxidant enzymes (glutathione peroxidase, catalase, and superoxide dismutase)
and inhibit the expression of some enzymes, such as xanthine oxidase [1]. The antioxidant
protective action of isoflavones from soy or plant extracts, such as Trifolium pratense L. or
Genista tinctoria L., was proven in clinical studies [45, 46], as well as in animal models [32, 47].

4.2. Anticarcinogenic activity of isoflavones

The anticarcinogenic potential of isoflavones is based on multiple actions: binding to estro-
gen receptors (ERs), changing of cell signaling pathways, and inhibition of the key enzymes
involved in the metabolism of sex hormones. Also, the anticarcinogenic potential of iso-
flavones has positive effects through independent mechanisms which do not involve ERs,
such as antioxidant activity, reduction in the bioactivation of carcinogens, and stimulation of
detoxification [2, 48].

Anticarcinogenic activity of genistein has been assessed more thoroughly among isofla-
vones. Genistein initiates apoptosis, alters cell proliferation and angiogenesis, and inhibits
metastasis in many types of cancer cells [49]. It is a tyrosine kinase inhibitor. Therefore, in
breast cancer cells, it slows down tumorigenesis; in the circulatory system, it prevents tumor
vascularization; in the nervous system, it induces neuroprotective effects. In addition, genis-
tein affects tumorigenesis by inhibiting DNA topoisomerases I and II [50], alteration of epi-
genetic regulations (both histone methylation and DNA methylation), and activating tumor
suppressor genes [51]. As a polyphenol, genistein has antioxidant [1] and anti-inflammatory
potential [52]. Another possible action pathway for genistein is the competitive inhibition of
estrone metabolism through cytochrome P450 isoenzymes by altering the 2-hydroxy-estrone
(2-OH-E1)/16α-hydroxy-estrone (16α-OH-E1) ratio, as noticed in vitro [53]. While 2-OH-E1 is a
weak estrogen, 16α-OH-E1 has an important role in carcinogenesis, showing a strong estrogen
effect and genotoxic properties [54]. 16α-OH-E1 covalently binds to the estrogenic receptors
and thus stimulates cell proliferation [55]. The ratio 2-OH-E1/16α-OH-E1 has been proposed
and studied as a biomarker of breast cancer risk [55–59], but now its significance is controver-
sial. In high concentrations, genistein decreases the hydroxylation of estrone in position 2 in
favor of hydroxylation in position 16α [55]. Other studies show that genistein has no muta-
genic or clastogenic activity in vivo. But in high concentration of genistein, it has clastogenic
potential in vitro, explained by the topoisomerase inhibitory effect, which is known to cause
chromosome damage above a certain threshold dose [60].
Anti-proliferative effects of high concentrations of genistein were demonstrated in all breast
cancer cells, both ER positive and ER negative. However, there are several studies showing
that genistein shows both anti-proliferative and proliferative effects, depending on the con-
centration, type of tumor, level of endogenous estrogens present in the tissue, or development
stage. At low physiological concentrations, genistein stimulates tumorigenesis and cancels the
142 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

effects of tamoxifen in ER-positive breast cancer cells [50]. Similar dual effects were observed
in the case of tamoxifen and other selective estrogen receptor modulators (SERMs) [16].

In fermented soybean products (e.g., natto, miso, tempeh), aglycons can suffer changes under
the effect of enzymes produced by the microorganisms involved in the fermentation process.
Thus, ortho-hydroxygenistein (6-OHG, 8-OHG, 3′-OHG) and ortho-hydroxydaidzein (6-OHD,
8-OHD, 3′-OHD) were identified. These compounds are not synthesized by the plants. The
hydroxylation reaction that occurs in the ortho position gives molecules a high antioxidant
potential and a free radical scavenging activity. Moreover, several of their abilities have been
proven: to suppress cell proliferation and to inhibit tyrosinase (anti-melanogenesis proper-
ties) and antimutagenic, anti-inflammatory, and hepatoprotective properties [18].

Equol has a higher estrogenic potential than daidzein, its precursor, and a preferential affin-
ity for ERβ, as it has already been stated. This detail is of high interest for its beneficial effect
in the treatment of prostate cancer, since both isomers, S-(−)equol and R-(+)equol, can bind
in vivo dihydrotestosterone without having an affinity for the androgen receptor. Therefore,
equol prevents the endogenous hormone to exert its stimulating effect on prostate growth. In
addition, equol possesses the highest antioxidant capacity of all isoflavones tested. It causes
blood vessel relaxation and modifies the inflammatory response in activated macrophages
and has beneficial effects in cardiovascular and inflammatory diseases [52].

4.3. Effects of isoflavones on hormone-dependent cancers

Clinical studies show contradictory results of the efficacy of isoflavones in the treatment of
breast cancer. The effects depend on a number of factors such as age, gender, hormonal status,
type of isoflavones consumed (soy proteins or isolated isoflavones), dose, diet (type of food),
and extent of consumption [2].

A recent meta-analysis of 35 studies shows that soy isoflavones lower the risk of breast cancer
in both premenopausal and post-menopausal women. The effect is more evident in Asian
women than in those living in Western countries, probably due to differences in quality (tra-
ditionally fermented foods) and quantity of the isoflavone products ingested [41]. In Asian
women, a diet rich in soy food lowers breast cancer risk with 30% [61]. A higher prevalence
of equol-producer phenotype in Asian population can be an essential factor. Equol-producer
phenotype is associated with a substantial reduction in the risk of breast cancer. Several spe-
cific biomarkers are favorable modified, such as sex hormone-binding globulin (SHBG) and
steroid hormone levels in plasma, a higher urinary 2-hydroxy-estrone/16α-hydroxy-estrone
ratio, and a lower mammographic breast density [2]. However, because several studies have
provided mixed or contradictory results, the general recommendation for patients diagnosed
with estrogen-dependent breast cancer is to avoid consuming high quantities of products con-
taining isoflavone. Indeed, isoflavones are selective estrogen receptor modulators (SERMs),
and their effects would depend on multiple factors.

Another meta-analysis of five cohort studies that included more than 11,000 female patients
diagnosed with breast cancer focused on the post-diagnostic relationship between consump-
tion of soy foods and mortality or cancer recurrence. The study concluded that the ingestion
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of soy foods reduced mortality and recurrence in all types of breast cancer, especially in the
ER-negative, ER-positive/PR-positive, and postmenopausal patients [42]. In women diag-
nosed with breast cancer under tamoxifen treatment, the consumption of plants containing
isoflavones did not alter plasma levels of the drug and its metabolites [62]. Moreover, a recent
study shows that a moderate intake of soy isoflavones (5–10 g soy protein/day) would have
an optimal effect on tamoxifen treatment on these patients [63].

In some studies [64], excessive consumption of soy was associated with a negative impact on
male fertility and reproductive hormones and the disruption of the thyroid gland function. In
other studies these effects were inconsistent [65].

Isoflavones can modulate the toxicity of other xenoestrogens, but the interactions are complex
and difficult to predict relying only on in vitro steroid receptor affinities [66]. In these kinds of
interactions, multiple mechanisms are involved, both estrogen and non-estrogen type, such
as oxidative stress [32, 47, 53]. European Food Safety Authority (EFSA) has recently conducted
a systematic study of published medical literature, focusing on the correlation between the
intake of soy isoflavones and the induced effects on the breast (mammographic density, pro-
liferative marker Ki67 expression), uterus (endometrial thickness, histopathology changes),
and thyroid (the thyroid hormone). Results showed that the intake of 35–150 mg isoflavones/
day does not affect these organs in peri- and postmenopausal women [17]. Isoflavones have
demonstrated prostate cancer efficacy in several studies: in vitro, on prostate cancer cell lines,
in vivo, and in numerous clinical trials [43, 67, 68]. Conclusion of a recent meta-analysis sug-
gests that phytoestrogen intake, mostly genistein and daidzein, can be correlated with a
decreased risk of prostate cancer [69].

5. Recent advances in analytical methods of isoflavones

5.1. Isolation of isoflavones in foods and vegetable materials

In recent years, due to the health benefits provided by isoflavones, higher attention has been
paid to the analytical methods that allow identification and quantification of isoflavones from
different types of samples: (a) food, for dietary intake assessing [15, 70]; (b) food supplements,
for standardization of nutraceuticals [5, 71]; (c) vegetable products, for phytotherapeutic eval-
uation [19, 20, 28]; and (d) human biological samples (plasma, urine) [5]. These analytical
methods are commonly used for isoflavone bioavailability assessing and in pharmacokinetic
or pharmacological studies.

Isoflavones are solubilized from food or vegetable material by refluxing or maceration, shak-
ing, and stirring [72]. The isolation of isoflavones from the mixture can be achieved either by
conventional methods, liquid-liquid extraction [11, 15, 19] or Soxhlet, or by modern ones—
supercritical fluid extraction, ultrasound-assisted extraction [19, 71], pressurized fluid extrac-
tion, microwave-assisted extraction, and solid-phase extraction [5, 73] (Table 4).

The methods used to isolate isoflavones from food are selected function of the nature of
the food, the type of the isoflavones analyzed (the total of aglycones or aglycones and
144 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

Analytes Sample Extraction Detection Run time (min) LOQ References

method
3 IFs Soy dry extract Sonication/ HPLC-DAD 20 40–100 ng/mL [71]
Steam bath

3 IFs, Cou* 10 plant species UAE** ULPC-PDA 4 1.97–4.08 ng/mL [19]

12 IFs Soybean seeds Maceration HPLC-UV 60 NA# [70]

12 IFs Soybeans, soy Maceration HPLC-DAD 30 <600 nmol/L [72]

products

3 IFs Coffee Refluxing HPLC-DAD 35 13.7–25.0 ng/mL [14]

17 IFs Soymilk Refluxing LC-ESI(+)-MS/ NA# NA# [74]

MS

7 IFs, Cou* 2 plant extracts Refluxing or LC-ESI(−)-MS/ 18 40 ng/mL [20]

Maceration MS

5 IFs, Cou* 7 plant extracts Maceration or ULPC- 5.5 5–10.78 ng/mL [28]
percolation ESI(+)-MS/
MS

5 IFs Legumes SPE C18 UHLPC- 18 0.1–1 ng/mL [73]

ESI(+)-MS/
MS

5 IFs Coffee SPE C18 HPLC-ESI-MS/ 18 0.05–1 ng/mL [75]

MS
* Cou, coumestrol.
** UAE, ultrasound-assisted extraction.
# NA, not available.

Table 4. HPLC and UPLC methods applied for analysis of isoflavones in different samples.

glycosides), and the instrumental method used for identification and quantification. Several
examples are presented below.

Liggins et al. isolated isoflavones from cereals and derivatives after a prior sonication in a polar
solvent (methanol/water 4:1, v/v), in order to break apart the cellular material, followed by fil-
tration and evaporation of the solvent under nitrogen. In order to determine the total aglycones,
glycosides were hydrolyzed in an acid medium (0.1 M acetate buffer, pH 5) by overnight incu-
bation at 37 °C in the presence of cellulase (enzyme used for hydrolytic removal of the hydroly-
sis resulted carbohydrates). Aglycones were extracted into ethyl acetate and were derivatized
and analyzed using GC-MS [15]. Otieno et al. analyzed isoflavones from fermented and unfer-
mented soy milk. For the solubilization of analytes, the freeze-dried sample was refluxed in
methanol for 1 hour and filtered, and after adding the internal standard, the solvent has been
evaporated to dryness under nitrogen. The residue has been suspended into a buffer (10 mm
ammonium acetate containing 0.1% trifluoroacetic acid) and centrifuged, and the supernatant
was filtered and analyzed using high-performance liquid chromatography (HPLC) [74].
Extraction and analysis of isoflavones in soybeans can be realized through maceration of the
powdered beans with 70% ethanol at room temperature, for 24 hours under constant stirring.
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After centrifugation and filtering, the supernatant is analyzed directly by HPLC [70]. Also,
analysis of isoflavones contained in food supplements requires a simple preparation of the
samples: fine powdering of tablets, refluxing in 80% methanol for 1 hour, filtering, and injec-
tion into the HPLC system [5].

Hydroalcoholic extracts or tinctures can be prepared from either fresh or dry and pulverized
vegetable materials. The hydroalcoholic extracts can be made in 70% ethanol or methanol, by
refluxing and filtration; by cold maceration, pressing, and filtration [20]; by percolation [28];
or using modern methods, such as ultrasound-assisted extraction in 50% ethanol [19]. The
extracts can be analyzed directly by LC-MS/MS, after an adequate dilution [20], or they can be
subjected to an acid hydrolysis [19] in order to release aglycones. Further, the aglycones can
be assessed directly or after liquid-liquid extraction, for a concentration of the analytes [19].

In biological samples (e.g., plasma and human urine) isoflavones can be found in different
forms: as aglycones (active metabolites), aglycone derivatives (with or without bioactivity),
or conjugated metabolites (β-glucuronides and sulfate esters). Isoflavone analysis can focus
on individual quantification of aglycones and their metabolites or quantification of aglycones
after the hydrolysis of conjugated forms. Hydrolysis of conjugated metabolites is achieved
by incubation at 37 °C with a mixture of β-glucuronidase/sulfatase in the presence of a buffer
(0.5 M acetate) at pH 4.5 for several hours or overnight. Isolation of free forms and/or of those
freed after hydrolysis can be done by liquid-liquid extraction or solid-phase extraction [5].

5.2. Quantification of isoflavones in foods and vegetable materials

For isoflavone identification, the following chromatographic methods are used: gas chroma-
tography coupled with mass spectrometry (GC-MS) [5, 15], high-performance liquid chro-
matography (HPLC) with UV detector (photodiode array, PDA) [28, 70, 71], fluorescence
detector (FLD), electrochemical detector (ECD) or mass spectrometer detector (MS) [20, 74,
75], and, less often, capillary electrophoresis (CE).

Quantification of isoflavones and their derivatives can be achieved in two ways: (a) by deter-
mining the free aglycons after a prior acid hydrolysis [19, 70, 72], alkaline hydrolysis [72], or
enzymatic hydrolysis [72] of the glycosides in the sample and (b) by simultaneously analyz-
ing the glycosides and aglycones present in the sample [20, 28]. GC-MS methods are used
less lately, because they require an additional step of isoflavone derivatization to the volatile
compounds [5, 15]. This additional step increases both the time and the cost of the analysis
and represents a potential source of error [28].
Generally, HPLC-UV is not sensitive enough (Table 4) for the quantification of small levels
of isoflavones from plant extracts [19] or human plasma [5]. This method often requires a
hydrolysis step to transform glycosides into aglycones followed by the quantification of total
aglycones from the sample [71].

In order to correctly identify new isoflavones or isoflavone derivatives present in the samples
analyzed, liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass
spectrometry (LC-MS/MS) are the preferred methods (Table 4), due to the advantages: speed,
selectivity, sensitivity, and robustness. In addition, mass spectrometry detection allows sure
146 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

determination of the compounds based on molecular weight and ion charge. For the quanti-
fication of isoflavones, the pseudo-molecular ions or the ionic fragments resulted after frag-
mentation are monitored. In LC-MS/MS analysis, compound identification can be achieved
even if their separation is not complete, and it is an advantage [74]. A shorter analysis can
be realized by ultra-performance liquid chromatography (UPLC) [19, 28]. This method uses
columns with very small size of the packing particles (1.7 μm) and consequently performs
separations with superior resolution in a shorter time and a lower consumption of the mobile
phase.

The isoflavones have polyphenolic structure and can easily lose a proton to form negative
pseudo-molecular ions [M-H]− [20]. However, they can also be detected after ionization in
positive mode to [M + H]+ [74]. Isoflavones are polar compounds and they form ions in solu-
tion. For these type of compounds, electro-spray ionization (ESI) is the most commonly used
source to obtain analytical ions. Atmospheric pressure chemical ionization (APCI) is the
source preferred for non-polar analytes that ionize in the gas phase. The isoflavones often give
poor response in this ionization source [28]. The fragmentation patterns of isoflavone glyco-
sides (malonyl-glycosides, acetyl-glycosides, glycosides, aglycones) follow a similar trend.
However each compound has a unique fragmentation pattern that allows their accurate iden-
tification (Table 5) [74].

Isoflavone [M + H]+ Transitions [M − H]− Tranzitions

Daidzein 255 [28, 74] →199 [28] 253 [20, 73, 75] →208, 132 [73, 75]

Formononetin 269 [28] →197 [28] 267 [20, 75] →252, 223 [75]

Genistein 271 [28, 73, 74] →153 [28] 269 [20, 73, 75] →159, 133 [73, 75]

Biochanin A 283 [73, 75] →268, 239 [73, 75]

Glycitein 285 [74] →270, 257, 229, 196, 283 [20]

166 [74]

Daidzin 417 [28, 73, 74] →255 [28, 73, 74], 199 415 [20] →253 [20]
[73]

Ononin 429 [20] →267 [20]

Genistin 433 [28, 74, 75] →271 [28, 73–75], 91 431 [20] →268, 269 [20]
[73, 75]

Glycitin 447 [74] →428, 285 [74]

Ac-daidzin 459 [74] →441, 255 [74]

Ac-genistin 475 [74] →431, 271 [74]

Ac-glycitin 489 [74] →471, 285 [74]

Mal-daidzin 503 [74] →485, 255 [74]

Mal-genistin 519 [74] →501, 271 [74]

Table 5. Ions (m/z) and transitions monitored for isoflavone quantification.

 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 147
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6. Conclusion

Dietary intake of isoflavones is widespread, mainly due to the high consumption of soybean
products. Health benefits of isoflavones justify the interest for this class of bioactive com-
pounds, but the controversial outcomes of some clinical and epidemiological studies require
further investigations. In the context of these researches, the analytical methods applied for
assessment of isoflavones are very valuable. They allow for the evaluation of dietary intake of
isoflavones, equating the health benefits and the circumstances in which they are exerted, and
highlight the natural sources of isoflavones with phytotherapeutic potential.

Author details

Daniela-Saveta Popa1* and Marius Emil Rusu2

*Address all correspondence to: [email protected]

1 Department of Toxicology, Faculty of Pharmacy, “Iuliu Haţieganu” University of Medicine
and Pharmacy Cluj-Napoca, Cluj-Napoca, Romania
2 Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, “Iuliu
Haţieganu” University of Medicine and Pharmacy Cluj-Napoca, Cluj-Napoca, Romania

References

[1] Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients. 2010;2(12):1231–

1246. Doi: 10.3390/nu2121231

[2] Bolca S. Bioavailability of soy-derived isoflavones and human breast cancer. In:
Watson RR, Preedy VR, Zibadi S, editors. Polyphenols in Human Health and Disease.
Amsterdam: Elsevier; 2014. pp. 1241–1256. Doi: 10.1016/B978-0-12-398456-2.00094-3

[3] Setchell KD, Brown NM, Zimmer-Nechemias L, Brashear WT, Wolfe BE, Kirschner
AS, Heubi JE. Evidence for lack of absorption of soy isoflavone glycosides in humans,
supporting the crucial role of intestinal metabolism for bioavailability. Am J Clin Nutr.
2002;76(2):447–453.

[4] Cederroth CR, Nef S. Soy, phytoestrogens and metabolism: a review. Mol Cell
Endocrinol. 2009;304(1–2):30–42. Doi: 10.1016/j.mce.2009.02.027

[5] Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner
AS, Cassidy A, Heubi JE. Bioavailability of pure isoflavones in healthy humans and anal-
ysis of commercial soy isoflavone supplements. J Nutr. 2001;131(4 Suppl):1362S–1375S.
[6] Tolleson WH, Doerge DR, Churchwell MI, Marques MM, Roberts DW. Metabolism of
biochanin A and formononetin by human liver microsomes in vitro. J Agric Food Chem.
2002;50(17):4783–4790.
148 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

[7] Gaya P, Medina M, Sánchez-Jiménez A, Landete JM. Phytoestrogen metabolism by adult

human gut microbiota. Molecules. 2016;21(8),1034:1-17. Doi: 10.3390/molecules21081034

[8] Setchell KD, Clerici C. Equol: history, chemistry, and formation. J Nutr.
2010;140(7):1355S–1362S. Doi: 10.3945/jn.109.119776

[9] Douglas CC, Johnson SA, Arjmandi BH. Soy and its isoflavones: the truth behind the
science in breast cancer. Anticancer Agents Med Chem. 2013;13(8):1178–1187. Doi:
10.2174/18715206113139990320

[10] Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and
genistein contents of vegetables. Br J Nutr. 2000;84(5):717–725.

[11] Megías C, Cortés-Giraldo I, Alaiz M, Vioque J, Girón-Calle J. Isoflavones in chickpea

(Cicer arietinum) protein concentrates. J Funct Foods. 2016;21:186–192. Doi: 10.1016/j.
jff.2015.12.012
[12] USDA Database for the Isoflavone Content of Selected Foods Release 2.1. 2015. https://
www.ars.usda.gov/ARSUserFiles/80400525/Data/isoflav/Isoflav_R2-1.pdf.
[13] Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and
genistein content of fruits and nuts. J Nutr Biochem. 2000;11(6):326–331.
[14] Alves RC, Almeida IMC, Casal S, Oliveira MBPP. Method development and validation
for isoflavones quantification in coffee. Food Chem. 2010;122:914–919. Doi: 10.1016/j.
foodchem.2010.03.061

[15] Liggins J, Mulligan A, Runswick S, Bingham SA. Daidzein and genistein content of cere-
als. Eur J Clin Nutr. 2002;56(10):961–966.

[16] Patisaul HB, Jefferson W. The pros and cons of phytoestrogens. Front Neuroendocrinol.
2010;31(4):400–419. Doi: 10.1016/j.yfrne.2010.03.003

[17] EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food).
Scientific opinion on the risk assessment for peri- and post-menopausal women tak-
ing food supplements containing isolated isoflavones. EFSA J 2015;13(10):4246. Doi:
10.2903/j.efsa.2015.4246

[18] Chang TS. Isolation, bioactivity, and production of ortho-hydroxydaidzein and ortho-
hydroxygenistein. Int J Mol Sci. 2014 Apr 3;15(4):5699–5716. Doi: 10.3390/ijms15045699

[19] Kiss B, Popa DS, Hanganu D, Pop A, Loghin F. Ultra-performance liquid chromatogra-
phy method for the quantification of some phytoestrogens in plant material. Rev Roum
Chim. 2010;55(8):459–465.

[20] Vlase L, Popa DS, Tero-Vescan A, Olah N. New liquid chromatography: mass spec-
trometry assay for natural phytoestrogens from vegetable extracts. Acta Chromatogr.
2011;23:509–520.
[21] Hanganu D, Vlase L, Olah N. LC/MS analysis of isoflavones from fabaceae species
extracts. Farmacia, 2010;58(2):177–183.
 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 149
http://dx.doi.org/10.5772/66531

[22] Valls J, Millán S, Martí MP, Borràs E, Arola L. Advanced separation methods of food
anthocyanins, isoflavones and flavanols. J Chromatogr A. 2009;1216(43):7143–7172. Doi:
10.1016/j.chroma.2009.07.030

[23] Kicel A, Wolbiś M. Phenolic content and DPPH radical scavenging activity of the flowers
and leaves of Trifolium repens. Nat Prod Commun. 2013;8(1):99–102.

[24] Mu H, Bai YH, Wang ST, Zhu ZM, Zhang YW. Research on antioxidant effects and estro-
genic effect of formononetin from Trifolium pratense (red clover). Phytomedicine. 2009
Apr;16(4):314–319. Doi: 10.1016/j.phymed.2008.07.005
[25] Beck V, Rohr U, Jungbauer A. Phytoestrogens derived from red clover: an alternative
to estrogen replacement therapy? J Steroid Biochem Mol Biol. 2005;94(5):499–518. Doi:
10.1016/j.jsbmb.2004.12.038

[26] Grigorescu E, Ciulei I, Stanescu U. Index fitoterapeutic. Bucuresti: Editura Medicala;

1986. p. 252.

[27] Bora KS, Sharma A. Phytochemical and pharmacological potential of Medicago sativa: a
review. Pharm Biol. 2011;49(2):211–220. Doi: 10.3109/13880209.2010.504732

[28] Kiss B, Popa DS, Păltinean R, Loghin F. A high-throughput UPLC-MS/MS for the simul-
taneous analysis of six phytoestrogens from Genista tinctoria extracts. J Liq Chromatogr
Relat Technol. 2012;35(19):2735–2752.

[29] Tero-Vescan A, Imre S, Vari CE, Osan A, Dogaru MT, Csedo C. Determination of
some isoflavonoids and flavonoids from Genista tinctoria L. by HPLC-UV. Farmacia,
2009;57(1):120–127.
[30] Rauter AP, Martins A, Lopes R, Ferreira J, Serralheiro LM, Araújo ME, Borges C,
Justino J, Silva FV, Goulart M, Thomas-Oates J, Rodrigues JA, Edwards E, Noronha
JP, Pinto R, Mota-Filipe H. Bioactivity studies and chemical profile of the antidia-
betic plant Genista tenera. J Ethnopharmacol. 2009;122(2):384–393. Doi: 10.1016/j.
jep.2008.10.011

[31] Rigano D, Cardile V, Formisano C, Maldini MT, Piacente S, Bevelacqua Y, Russo A,

Senatore F. Genista sessilifolia DC. and Genista tinctoria L. inhibit UV light and nitric
oxide-induced DNA damage and human melanoma cell growth. Chem Biol Interact.
2009;180(2):211–219. Doi: 10.1016/j.cbi.2009.02.010

[32] Popa DS, Bolfă P, Kiss B, Vlase L, Păltinean R, Pop A, Cătoi C, Crişan G, Loghin F.
Influence of Genista Tinctoria L. or methylparaben on subchronic toxicity of bisphenol A
in rats. Biomed Environ Sci. 2014;27(2):85–96. Doi: 10.3967/bes2014.021

[33] Hanganu D, Olah NK, Benedec D, Mocan A, Crisan G, Vlase L, Popica I, Oniga I.
Comparative polyphenolic content and antioxidant activities of Genista tinctoria L. and
Genistella sagittalis (L.) Gams (Fabaceae). Pak J Pharm Sci. 2016;29(1 Suppl):301–307.
[34] Hwang CS, Kwak HS, Lim HJ, Lee SH, Kang YS, Choe TB, Hur HG, Han KO. Isoflavone
metabolites and their in vitro dual functions: they can act as an estrogenic agonist or
150 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

antagonist depending on the estrogen concentration. J Steroid Biochem Mol Biol.

2006;101(4–5):246–253. Doi: 10.1016/j.jsbmb.2006.06.020
[35] Pilšáková L, Riečanský I, Jagla F. The physiological actions of isoflavone phytoestrogens.
Physiol Res. 2010;59(5):651–664.

[36] Franco OH, Chowdhury R, Troup J, Voortman T, Kunutsor S, Kavousi M, Oliver-

Williams C, Muka T. Use of plant-based therapies and menopausal symptoms: a
systematic review and meta-analysis. JAMA. 2016;315(23):2554–2563. Doi: 10.1001/
jama.2016.8012

[37] Cheng PF, Chen JJ, Zhou XY, Ren YF, Huang W, Zhou JJ, Xie P. Do soy isoflavones
improve cognitive function in postmenopausal women?. Meta Anal Menopause.
2015;22(2):198–206. Doi: 10.1097/GME.0000000000000290

[38] Pawlowski JW, Martin BR, McCabe GP, McCabe L, Jackson GS, Peacock M, Barnes S,
Weaver CM. Impact of equol-producing capacity and soy-isoflavone profiles of supple-
ments on bone calcium retention inpostmenopausal women: a randomized crossover
trial. Am J Clin Nutr. 2015;102(3):695–703. Doi: 10.3945/ajcn.114.093906

[39] Cavallini DC, Manzoni MS, Bedani R, Roselino MN, Celiberto LS, Vendramini RC, de
Valdez G, Abdalla DS, Pinto RA, Rosetto D, Valentini SR, Rossi EA. Probiotic soy prod-
uct supplemented with isoflavones improves the lipid profile of moderately hyper-
cholesterolemic men: a randomized controlled trial. Nutrients. 2016;8(1),52:1–18. Doi:
10.3390/nu8010052

[40] Wong JM, Kendall CW, Marchie A, Liu Z, Vidgen E, Holmes C, Jackson CJ, Josse RG,
Pencharz PB, Rao AV, Vuksan V, Singer W, Jenkins DJ. Equol status and blood lipid pro-
file in hyperlipidemia after consumption of diets containing soy foods. Am J Clin Nutr.
2012;95(3):564–571. Doi: 10.3945/ajcn.111.017418

[41] Chen M, Rao Y, Zheng Y, Wei S, Li Y, Guo T, Yin P. Association between soy isofla-
vone intake and breast cancer risk for pre- and post-menopausal women: a meta-
analysis of epidemiological studies. PLoS One. 2014;9(2):e89288. Doi: 10.1371/journal.
pone.0089288

[42] Chi F, Wu R, Zeng YC, Xing R, Liu Y, Xu ZG. Post-diagnosis soy food intake and
breast cancer survival: a meta-analysis of cohort studies. Asian Pac J Cancer Prev.
2013;14(4):2407–2412. Doi: http://dx.doi.org/10.7314/APJCP.2013.14.4.2407.

[43] Zhang Q, Feng H, Qluwakemi B, Wang J, Yao S, Cheng G, Xu H, Qiu H, Zhu L, Yuan M.
Phytoestrogens and risk of prostate cancer: an updated meta-analysis of epidemiologic
studies. Int J Food Sci Nutr. 2016;9:1–15. Doi: 10.1080/09637486.2016.1216525

[44] US Food and Drug Administration. Guidance for Industry: A Food Labeling Guide (11.
Appendix C: Health Claims). 2013. http://www.fda.gov/Food/GuidanceRegulation/
GuidanceDocumentsRegulatoryInformation/LabelingNutrition/ucm064919.htm.

[45] Azadbakht L, Kimiagar M, Mehrabi Y, Esmaillzadeh A, Hu FB, Willett WC. Dietary

soya intake alters plasma antioxidant status and lipid peroxidation in postmenopausal
 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 151
http://dx.doi.org/10.5772/66531

women with the metabolic syndrome. Br J Nutr. 2007;98(4):807–813. Doi: 10.1017/

S0007114507746871

[46] Cha YS, Park Y, Lee M, Chae SW, Park K, Kim Y, Lee HS. Doenjang, a Korean fermented
soy food, exerts antiobesity and antioxidative activities in overweight subjects with the
PPAR-γ2 C1431T polymorphism: 12-week, double-blind randomized clinical trial. J Med
Food. 2014;17(1):119–127. Doi: 10.1089/jmf.2013.2877

[47] Popa DS, Hanganu D, Vlase L, Kiss B, Loghin F, Crişan G. Protective effect of
Trifolium pratense extract on oxidative stress induced by bisphenol A in rats. Farmacia.
2014:62(2):341–349.
[48] Lockwood B. Nutraceuticals. A Guide for Healthcare Professionals. Second edition,
London: Pharmaceutical Press; 2007. pp. 192–200.

[49] Spagnuolo C, Russo GL, Orhan IE, Habtemariam S, Daglia M, Sureda A, Nabavi
SF, Devi KP, Loizzo MR, Tundis R, Nabavi SM. Genistein and cancer: current sta-
tus, challenges, and future directions. Adv Nutr. 2015;6(4):408–419. Doi: 10.3945/
an.114.008052

[50] Kwon Y. Effect of soy isoflavones on the growth of human breast tumors: findings from
preclinical studies. Food Sci Nutr. 2014;2(6):613–622. Doi: 10.1002/fsn3.142.

[51] Casati L, Sendra R, Sibilia V, Celotti F. Endocrine disrupters: the new players able to
affect the epigenome. Front Cell Dev Biol. 2015;3:37. Doi: 10.3389/fcell.2015.00037

[52] Setchell KD, Clerici C. Equol: pharmacokinetics and biological actions. J Nutr.
2010;140(7):1363S–1368S. Doi: 10.3945/jn.109.119784

[53] Gheldiu AM, Popa DS, Loghin F, Vlase L. Oxidative metabolism of estrone modified by
genistein and bisphenol A in rat liver microsomes. Biomed Environ Sci. 2015;28(11):834–
838. Doi: 10.3967/bes2015.116

[54] Lee AJ, Mills LH, Kosh JW, Conney AH, Zhu BT. NADPH-dependent metabolism of
estrone by human liver microsomes. J Pharmacol Exp Ther. 2002;300(3):838–849.

[55] Eliassen AH, Missmer SA, Tworoger SS, Hankinson SE. Circulating 2-hydroxy- and
16alpha-hydroxy estrone levels and risk of breast cancer among postmenopausal
women. Cancer Epidemiol Biomarkers Prev. 2008;17(8):2029–2035. Doi: 10.1158/1055-
9965.EPI-08-0262

[56] Ziegler RG, Fuhrman BJ, Moore SC, Matthews CE. Epidemiologic studies of estro-
gen metabolism and breast cancer. Steroids. 2015;99(Pt A):67-75. Doi: 10.1016/j.
steroids.2015.02.015

[57] Morimoto Y, Conroy SM, Pagano IS, Isaki M, Franke AA, Nordt FJ, Maskarinec G. Urinary
estrogen metabolites during a randomized soy trial. Nutr Cancer. 2012;64(2):307–314.
Doi: 10.1080/01635581.2012.648819

[58] Atkinson C, Skor HE, Dawn Fitzgibbons E, Scholes D, Chen C, Wähälä K, Schwartz
SM, Lampe JW. Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-
152 Superfood and Functional Food - The Development of Superfoods and Their Roles as Medicine

hydroxyestrone concentrations: an observational study of young to middle-aged

women. J Steroid Biochem Mol Biol. 2003;86(1):71–77.

[59] Lu LJ, Cree M, Josyula S, Nagamani M, Grady JJ, Anderson KE. Increased urinary excre-
tion of 2-hydroxyestrone but not 16alpha-hydroxyestrone in premenopausal women
during a soya diet containing isoflavones. Cancer Res. 2000;60(5):1299–1305.
[60] McClain MR, Wolz E, Davidovich A, Bausch J. Genetic toxicity studies with genistein.
Food Chem Toxicol. 2006;44(1):42–55.
[61] Messina M. Impact of soy foods on the development of breast cancer and the prog-
nosis of breast cancer patients. Forsch Komplementmed. 2016;23(2):75–80. Doi:
10.1159/000444735

[62] Hu YC, Wu CT, Lai JN, Tsai YT. Detection of a negative correlation between prescrip-
tion of Chinese herbal products containing coumestrol, genistein or daidzein and risk
of subsequent endometrial cancer among tamoxifen-treated female breast cancer sur-
vivors in Taiwan between 1998 and 2008: a population-based study. J Ethnopharmacol.
2015;169:356–362. Doi: 10.1016/j.jep.2015.04.028

[63] Braakhuis AJ, Campion P, Bishop KS. Reducing breast cancer recurrence: the role of
dietary polyphenolics. Nutrients. 2016;8(9),547:1-15. Doi: 10.3390/nu8090547

[64] Chavarro JE, Toth TL, Sadio SM, Hauser R. Soy food and isoflavone intake in rela-
tion to semen quality parameters among men from an infertility clinic. Hum Reprod.
2008;23(11):2584–2590. Doi: 10.1093/humrep/den243
[65] D’Adamo CR, Sahin A. Soy foods and supplementation: a review of commonly per-
ceived health benefits and risks. Altern Ther Health Med. 2014;20(Suppl 1):39–51.
[66] You L, Sar M, Bartolucci EJ, McIntyre BS, Sriperumbudur R. Modulation of mammary
gland development in prepubertal male rats exposed to genistein and methoxychlor.
Toxicol Sci. 2002;66(2):216–225. Doi: 10.1093/toxsci/66.2.216

[67] Hwang YW, Kim SY, Jee SH, Kim YN, Nam CM. Soy food consumption and risk of pros-
tate cancer: a meta-analysis of observational studies. Nutr Cancer. 2009;61(5):598–606.
Doi: 10.1080/01635580902825639
[68] van Die MD, Bone KM, Williams SG, Pirotta MV. Soy and soy isoflavones in prostate
cancer: a systematic review and meta-analysis of randomized controlled trials. BJU Int.
2014;113(5b):E119–E130. Doi: 10.1111/bju.12435

[69] He J, Wang S, Zhou M, Yu W, Zhang Y, He X. Phytoestrogens and risk of prostate cancer:

a meta-analysis of observational studies. World J Surg Oncol 2015;13:231. Doi: 10.1186/
s12957-015-0648-9

[70] Sun J, Sun B, Han F, Yan S, Yang H, Kikuchi A. Rapid HPLC method for determination
of 12 isoflavone components in soybean seeds. Agric Sci China. 2011;10(1):70–77. Doi:
10.1016/S1671-2927(09)60291-1
 Isoflavones: Vegetable Sources, Biological Activity, and Analytical Methods for Their Assessment 153
http://dx.doi.org/10.5772/66531

[71] César Ida C, Braga FC, Soares CD, Nunan Ede A, Pianetti GA, Condessa FA, Barbosa
TA, Campos LM. Development and validation of a RP-HPLC method for quantification
of isoflavone aglycones in hydrolyzed soydry extracts. J Chromatogr B Analyt Technol
Biomed Life Sci. 2006;836(1–2):74–78. Doi: 10.1016/j.jchromb.2006.03.030
[72] Shao S, Duncan AM, Yang R, Marcone MF, Rajcan I, Tsao R. Systematic evaluation of
pre-HPLC sample processing methods on total and individual isoflavones in soybeans
and soy products. Food Res Int. 2011;44:2425–2434. Doi: 10.1016/j.foodres.2010.12.041

[73] Vila-Donat P, Caprioli G, Maggi F, Ricciutelli M, Torregiani E, Vittori S, Sagratini G.

Effective clean-up and ultra high-performance liquid chromatography–tandem mass
spectrometry for isoflavone determination in legumes. Food Chem. 2015;174:487–494.
Doi: 10.1016/j.foodchem.2014.11.047
[74] Otieno DO, Rose H, Shah NP. Profiling and quantification of isoflavones in soymilk
from soy protein isolate using extracted ion chromatography and positive ion fragmen-
tation techniques. Food Chem. 2007;105:1642–1651. Doi: 10.1016/j.foodchem.2007.04.036

[75] Caprioli G, Navarini L, Cortese M, Ricciutelli M, Torregiani E, Vittori S, Sagratini G.

Quantification of isoflavones in coffee by using solid phase extraction (SPE) and high-
performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). J
Mass Spectrom. 2016;51(9):698–703. Doi: 10.1002/jms.3802