Open access peer-reviewed chapter

New Insights Regarding the Potential Health Benefits of Isoflavones

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Corina Danciu, Suciu Oana, Diana Simona Antal, Florina Ardelean, Aimée Rodica Chiş, Codruţa Şoica, Florina Andrica, Cristina Dehelean and Vlaicu Brigitha

Submitted: 03 February 2017 Reviewed: 15 February 2017 Published: 23 August 2017

DOI: 10.5772/67896

From the Edited Volume

Flavonoids - From Biosynthesis to Human Health

Edited by Goncalo C. Justino

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Isoflavones are a class of plant secondary metabolites, with an estrogen‐like structure presenting a plethora of biological activities. The chapter discusses important facts about this class of phytoestrogens, from biosynthesis to the latest research about their health benefits. The following major points discussed are: biosynthesis, regulation, isolation, metabolism and bioavailability, isoflavones in diet and intake, and new insights regarding the therapeutic effect including cancer chemoprevention. The chapter ends with a mini review of own research of the anti‐inflammatory and chemopreventive activity of isoflavonoid genistein alone and incorporated in modern pharmaceutical formulations. The chapter updates the interested researchers in the field with the latest progress regarding potential health benefits of isoflavones.


  • isoflavones
  • biosynthesis
  • regulation
  • isolation
  • metabolism
  • bioavailability
  • therapeutic effect

1. Introduction

Isoflavonoids, a class of secondary metabolites including over 1000 structures [1], are polyphenolic derivatives of 1,2‐diphenylpropane, as opposed to the larger group of flavonoids having a 1,3‐diphenylpropane skeleton. They encompass several subgroups, the most prominent being the isoflavones, the rotenoids, the pterocarpans and the coumestans (Figure 1). The multiplicity of isoflavonoid structures is accounted by different oxidation levels of the backbone, the presence of additional heterocyclic rings and the diversity of substituents. These substances occur mostly in free forms as aglycones (Figure 2); glycosides are formed with glucose, rhamnose or apiose as the sugar moiety and are mainly O‐glycosides.

Figure 1.

Flavonoid and isoflavonoid backbones. 1: Flavones, 2: Isoflavones, 3: Rotenoids, 4: Pterocarpans and 5: Coumestans.

Figure 2.

Main isoflavone aglycones: 1: Genistein, 2: Daidzein, 3: Glycitein, 4: Biochanin A and 5: Formononetin.

Isoflavonoids are a major biochemical marker of the Fabaceae, especially of the subfamily Faboideae [2]. As much as 95% of the isoflavonoid aglycones and about 90% of isoflavones were reported in this family [3]. In recent years, the distribution of isoflavonoids has reliably been proven in a variety of nonlegumes, including bryophytes, gymnosperms and angiosperms. In flowering plants, they occur in over 50 families, and their distribution pattern is unrelated with the degree of phylogenetic closeness. Such families include among monocots such as the Iridaceae, Liliaceae, Asphodelaceae, Poaceae, Zingiberaceae and Cyperaceae as well as a large number of dicots such as Asteraceae, Apiaceae, Malvaceae, Rosaceae, Rutaceae or Solanaceae [1, 4]. Among plant organs, isoflavonoids are mainly present in underground parts, wood and bark as compared to flowers and leaves [1]. In planta, they act as anti‐microbial compounds synthesized in response to the attack of pathogens, representing the first identified type of phytoalexins. Isoflavonoids may either be preexistent in plant tissues before microbial attacks or be produced only upon exposure to infection or environmental stressors. The prominent distribution of these secondary metabolites in legumes is related to their physiological role in nodulation. The isoflavonoids present in root exudates are associated with the attraction of symbiotic Rhizobium bacteria and promotion of their growth within root nodules [5], leading to an improved nitrogen uptake.


2. Biosynthesis and regulation

The biosynthesis of isoflavonoids occurs as a branch of the phenylpropanoid pathway, involved in the biologic obtainment of all flavonoids [6]. The departing point is represented by the amino acid phenylalanine, sequentially converted to cinnamate, p‐coumarate and p‐coumaroyl‐CoA by the relevant enzymes of each step (phenylalanine ammonia‐lyase, cinnamic acid‐4‐hydroxylase, and 4‐coumarate‐CoA ligase, respectively). Subsequently, chalcone synthase is involved in the creation of the 15‐carbon flavonoid backbone from p‐coumaroyl‐CoA; several derivatives may be produced via different branch pathways. The crucial enzyme for isoflavone biosynthesis is isoflavone synthase, a cytochrome P450 monooxygenase, which catalyzes the migration of the aryl moiety and transforms flavanones to isoflavones [7]. The reaction involves keto‐enol tautomerism of flavanones and epoxidation, followed by dehydration [8].

Relevant metabolic pathways for the biosynthesis of the major isoflavonoids genistein (precursor naringenin) and daidzein (precursor liquiritigenin) have been well‐studied in soybean (Glycine max (L.) Merr.). Their understanding opened possibilities of metabolic engineering, leading to the development of soybean lines with an increased content of isoflavones in comparison to wild‐type seeds. On the other hand, the health benefits of isoflavones prompted strategies to induce the synthesis of these compounds by non‐legume plants (broccoli and tomatoes). This approach could be achieved in plants with an active phenylpropanoid pathway [9]. In fact, the flavanone naringenin is not only the substrate of isoflavone synthase, but as well that of flavanone‐3‐hydroxylase, involved in the biosynthesis of flavonols, anthocyanins and condensed tannins. The introduction of the isoflavone synthase gene in plants that do not express this enzyme was able to trigger genistein production in corn [7] and the model cruciferous Arabidopsis thaliana [10]. The disadvantage was represented by low isoflavone content, related to the competition between the flavonoid and the isoflavone pathways. Blocking the alternative flavonoid/anthocyanin branch of the phenylpropanoid pathway while upregulating the synthesis of isoflavones by introducing foreign transcription factors yielded very favorable results. Yu et al. could increase up to fourfold the level of isoflavones in soy by introducing the transcription factors C1 and R from corn in soybean (involved in the regulation of the phenylpropanoid pathway genes), and by co‐suppressing flavanone‐3‐hydroxylase, hence diverting the whole substrate to the isoflavonoid pathway [11]. An additional regulation of isoflavone biosynthesis involves the enzymes catalyzing the glycosylation of aglycones, as the majority of isoflavonoids accumulate in conjugated form. In soybean, the malonylglycosides are predominant, followed by glucosides. These conjugates are obtained upon catalysis by malonyl transferase and glycosyl transferase, respectively, in specific positions. The engineered obtainment of isoflavonoids in nonlegumes is crucially related to the presence of isoflavonoid‐specific malonyl transferases and glycosyl transferases [12]. In soy, the content and composition of isoflavonoids are subjected to polygenic regulation and highly variable in response to drought, temperature, fertilization, carbon dioxide content and genetic factors [13]. The level of isoflavonoids is higher in wild‐growing populations than in cultivated soybean; this situation is thought to be a consequence of domestication [14].


3. Isolation

The isoflavonoid aglycones such as genistein and daidzein are compounds with a low polarity and hence practically insoluble in water. The polarity is lowered by methylation, as in formononetin and biochanin A. After glycosylation, the water solubility increases; glucosides have a higher solubility in water than their malonylated and acetylated derivatives. The glycosidic bond may be hydrolyzed under acidic or basic conditions [15]. Early extraction of isoflavones was performed by refluxing alcohol, but had the disadvantage of converting malonyl‐ and acetyl‐glucosides into glucosides and aglycones [16]. A mixture of methanol 80% was as well proposed [17]. An optimized extraction method was developed by Griffith and Collison, using acetonitrile/water, without the addition of an acid [18]. Acetonitrile is considered to yield higher extraction ratios than solvents such as acetone, ethanol and methanol, during the analysis of 12 main soy isoflavones from foods; the organic solvent (53%) is mixed with water [19]. The preparative isolation of isoflavones could be achieved by high‐speed countercurrent chromatography (HSCCC). In one setting of HSCCC, acid‐free solvents were employed; the isolation of malonylglucosides was performed with the aid of a solvent tert‐butyl‐methyl ether/n‐butanol/acetonitrile/water in a ratio of 1/3/1/5 [20]. Monoglucosylated and acetylated isoflavones were obtained more recently by HSCCC after a cleaning‐up step on Amberlite XAD‐7 material [21].

Quantification of isoflavones is usually performed by HPLC‐DAD, using reversed‐phase columns and eluents containing 95% acetonitrile with 0.1% trifluoroacetic acid. Validated methods with good peak resolution are available [18]. Detection may be performed at 262 nm. For the quantification in biologic samples (urine, saliva and blood), HPLC‐MS/MS spectrometry is employed after solid‐phase extraction (SPE) of isoflavones; the glycosides are hydrolyzed enzymatically prior to SPE [22].


4. Bioavailability and metabolism

Bioavailability of isoflavones is based on data from absorbtion, metabolism, distribution and excretion studies. After the intake of pure compounds, isoflavone‐rich extracts or foods containing high levels of isoflavones, the parent compound and their metabolite can be found in plasma and urine of human volunteers. Following ingestion, soy isoflavones attain maximal plasma concentration within 4–8 h and then eliminate from the body through the bile and kidneys with an elimination half‐life that is on average 8 h [23, 24]. Aglycones are well absorbed due to their low water solubility and small molecular weight [25]. After ingestion, isoflavone glycosides are hydrolyzed by intestinal glucosidases, which partially release the aglycones daidzein, genistein and glycitein [24]. These may be absorbed or converted to a number of metabolites including equol and p‐ethyl phenol [24].

It was proved that intestinal microflora plays an important role in the metabolism and bioavailability of isoflavones [26]. It is considered that about 50% of Asians and 25% of non‐Asians host the intestinal bacteria that convert daidzein into the isoflavonoid equol [27]. Variation in individual metabolism of phytoestrogens due to differences in gut microflora might influence the serum concentration of phytoestrogens. It was found that the capacity to produce equol is higher among Japanese and Korean men than among American men [28]. After an intake of 50 mg isoflavone, the urinary excretion was 42% for daidzein and 16% for genistein [29]. Fermented soy products, or supplements in which the soy extract has been hydrolyzed, contain mostly the aglycone forms of isoflavones; however, following ingestion, the plasma profile of isoflavone metabolites is the same, no matter the form ingested [30]. Free aglycones, released after hydrolysis, are absorbed by passive diffusion across the intestin [31].

The extensive metabolization of the aglycones is evident in the extremely low content of the free form in body fluids, of less than 1% in human plasma and urine [32]. The metabolization of genistein has intensively been studied and thoroughly reviewed [25]. Following the faith of most xenobiotics, it undergoes detoxification, being conjugated to glucuronides and sulfates. Among them, monoglucuronides are present in the highest proportion (62.5%), followed by diglucuronides, sulfoglucuronides, disulfates and monosulfates in human urine after dietary intake of soy products [33]. The percentage of sulfates was reported to be slightly higher in blood (20%) than in urine (13%) [32]. In humans, the plasma level of genistein (all forms) is situated in the micromolar range, while the level of free genistein is in the higher nanomolar range. The rate of metabolization after oral ingestion is less than half an hour, with conjugations occurring in the intestine, the liver but as well in the kidneys, heart and lungs. The tissue distribution of genistein is highest in the gastro‐intestinal tract and liver, consistent with its enterohepatic recycling [34]. While the differences between individuals are very high, the oral bioavailability of genistein is low due to extensive metabolization and high expression level of efflux transporters (such as breast cancer resistance protein (BCRP)) [25].


5. Dietary intake and sources of isoflavones

As already mentioned above, isoflavones are flavonoid compounds that are biogenetically produced by plants belonging mainly to the Fabaceae family. Main sources of dietary isoflavones are soybeans (Glycine max) [35, 36] and red clover (Trifolium pratense) [37]. Other plants with a high content of isoflavones are: mung beans (Vigna radiata) [38], kudzu (Pueraria lobata), lupine (Lupinus spp.), fava bean (Vicia faba), psoralea (Psoralea corylifolia) [39], poinciana (Caesalpinia pulcherrima) [40] and alfalfa (Medicago sativa) [41].

Soybeans are widely employed for the preparation of food and dietary supplements. They contain both isoflavone aglycones: genistein, daidzein, glycitein, and glucosides: 7‐O‐glucosides: genistin, daidzin, glycitin, and three 6″‐O‐acetyl glucosides: 6″‐O‐acetyl‐genistin, 6″‐O‐acetyldaidzin, and 6″‐O‐acetyl‐glycitin, and three 6″‐O‐malonyl‐glucosides: 6″‐O‐malonyl‐genistin, 6″‐O‐malonyldaidzin, and 6″‐O‐malonylglycitin (Figure 3) [42, 43]. The highest percentage of soy isoflavones in soybeans is represented by genistein glucosides, approximately 50%. Daidzein glucosides are about 40% and glycitein glucosides 5–10% [15]. More exactly, the concentration of daidzein and daidzin in soy extract is 10.4 and, respectively, 244.5 mg/g soy extract corresponding to 1.7 and 39.6%. Concerning the concentration of genistein and genistin, it is 5.3 mg/g soy extract and, respectively, 319.6 mg/g soy extract representing 0.9 and 51.8% [44].

Figure 3.

Structure of main isoflavone glycosides.

Comparing the isoflavones from soybeans with those extracted from red clover, it is remarkable that there are four main isoflavones (daidzein, genistein, formononetin and biochanin A) from red clover and only three in soybeans [15].

In China, the first reference to soybeans dates from 2853 BC [45]. In contrast, Western cultures have adopted these products only lately. A variety of soy foods is currently available throughout the world, produced with modern processing techniques or using the traditional methods. They can be classified into fermented (tofu, okara, yuba, fresh greed soybeans, whole dry soybeans, soy nuts, whole‐fat soy flour, soy sprouts, soymilk and soymilk products) and nonfermented (tempeh, natto, miso, fermented tofu and soymilk products and soy sauces) products [46]. Epidemiological studies among the Japanese population suggest that fermented soybean products have better effects than nonfermented soy products, probably because of a higher bioavailability of isoflavone aglycones [47].

During the processing of raw soybeans, the composition of isoflavones is altered. The loss of isoflavones in the water used to soak raw soybeans, whey and the okara, was 4, 18 and 31%, respectively [42]. The isoflavone loss during coagulation in tofu processing was 44% and during alkaline extraction in soy protein isolate production was 53% [48]. The recoveries of isoflavones in tofu and in soy beverage comparing to their initial concentration in raw soybeans were found to be 36 and 54%, respectively [42].

Several reviews are available on the content of isoflavones in soy foods, including Japanese foods [49], foods used in the US [50, 51] and Europe [52]. A comparison of the most frequently used foods containing soy is performed in Table 1.

CountryJapanese foodsFood in the USFood in Europe
μg/g wet foodμg/gμg/g
Soybean (raw)1006.31437.7613.3863.3580840
Soybean (boiled)135.8472.574.170.6150320
Soybean sprout49.679.35067
Mung bean sprout3.
Okara—bean curd residue33.
Tofu (momen type)166.2269.2
Tofu (silken type)130.1206.491.584.2
Tofu (packed type)168.6280.7
Tofu (baked type)166.8291.2102.6104.3
Tofu (dried type)168.2556.7
Deep fried tofu138.0184.3
Deep fried tofu (thick type)148.7257.4
Deep fried tofu (thin type)84.2179.1
Tempeh—fermented soybean from Indonesia525.91326.2226.6361.5
Navy beans (haricot)0.1374.080.1300.110

Table 1.

Concentrations of daidzein and genistein in Japanese foods [49], food in the US, [50, 51] and food in EU [52].

Soy foods, such as tofu and tempeh, are extremely rich in isoflavones compared to other type of foods [53]. Raw soybeans contain the highest level of genistein and daidzein in the Japanese foods. The soybeans in US have a higher amount of genistein (863.3 μg/g) and daidzein (613.3 μg/g) than in Europe, which have 840 μg/g genistein and 580 μg/g daidzein [51, 52]. The richest sources of isoflavones are tofu, tempeh and miso in Japanese, European and American foods. Small variation from one region to another may occur. The isoflavone level in vegetables, fruits and other types of food are extremely low, sometimes in traces or not detectable. Among these, the navy beans have a higher level of genistein 4.08 μg/g.

One gram of soy protein in soybeans and traditional soy foods contain about 3.5 mg of isoflavones. One serving of a traditional soy food (100 g of tofu or 250 mL soymilk) provides about 25 mg isoflavones. In more refined products, it is possible that 80–90% of the isoflavone content to be lost during processing [27].

5.1. Soy foods in Asia

In Asia, soybeans are used in producing traditional foods such as tofu, soymilk and fermented products, while in Western nations, soybeans are used in the form of refined soy protein ingredients that are further used in food processing [45]. In Japan, the most popular soy food is tofu, served at all meals and in dessert products. Fermented foods such as natto and miso were very popular among Japanese and today are also largely consumed. Japanese adults consume approximately 6–11 g of soy protein and 25–50 mg of isoflavones (expressed as aglycone equivalents) per day. The results were higher than in Hong Kong and Singapore [54]. Concerning genistein and daidzein, the annual report of the national nutrition survey in 1997 in Japan shows that the dietary intake of isoflavones daidzein and genistein was 64.6 and 111.6 μmol/day/capita (16.4 and 30.1 mg/day/capita). The isoflavones intake was mostly attributable to tofu, natto and miso [49].

The mean plasma concentrations of total isoflavones are estimated to be 492.7 nM for genistein and 282.5 nM for daidzein in Japanese men, and 33.2 nM for genistein and 17.9 nM for daidzein in British men [55].

In China, not only tofu, yuba, soymilk and many regional specialities are served, but also soy powder mixes are becoming popular [45]. Isoflavone intake differs very much from a region to another [54]. In Taiwan, the meat substitutes from soy (chicken‐like and fish‐like products) are highly appreciated and, in Indonesia, tempeh is the most popular soy food [45].

5.2. Soy foods in Europe

Meat and dairy substitutes are the most popular soyfoods in Europe [45]. Traditional soyfoods rich in isoflavones (tofu, tempeh and miso) are rarely eaten in the UK, but soya dairy alternatives (milk, cheese and yogurts) are more commonly eaten. Some commercial products (bread, biscuits and breakfast cereals) contain soy ingredients as food additives which contribute to isoflavone intake [56].

Data from the 1998 UK Total Diet Study shows that daily intake of isoflavone aglycones (daidzein, genistein and glycitein) is approximately 3 mg/day [57].

In a group of 9 omnivores and a group of 10 vegetarians, mean isoflavone intake was measured after a 7‐day food diary; mean daily isoflavone intake in the omnivorous and vegetarian groups was 1.2 and 7.4 mg, respectively. Main isoflavones food sources for the omnivorous group were soya yogurts, wholemeal bread and rolls, and for the vegetarian group were soymilk (plain), meat‐substitute foods with soy protein isolate, beans, raisins and wholemeal bread and rolls [58].

5.3. Soy foods in the US

The consumption of soy food in the US resembles with the one in Europe. Popular soy foods in the US are tofu, soy sauce, soymilk, miso and tempeh. There can be found some new, adapted soy foods such as tempeh burgers, veggie burgers, tofu hot dogs, tofu ice cream, soymilk yogurt, soymilk cheeses, soy flour pancake mix and myriad [46]. Isoflavone intake is less than 3 mg/day in the US [27].

The Study of Women’s Health Across the Nation demonstrated that median intake of daidzein and genistein by white subjects, African American subjects, and Japanese subjects in the US were 6.2 and 3.9 μg/day, 2.7 and 1.7 μg/day, and 4676 and 7151 μg/day, respectively. Women were aged between 42 and 52 years [59].

5.4. Soy foods in Africa

In certain African countries, soy foods gained acceptance due to the high protein level and nutrition quality. In other countries, it is used because of the food aid. In South Africa, modern soy foods are used [45]. The intake of soybeans in South Africa (0.64 g/day) is comparable with the soybeans intake in Germany (0.64 g/day).

As it can be observed in Table 2, the soybeans intake in Asian countries is higher than in the Western countries. For example, in 2001, in Taiwan, Japan and China, per capita consumption was approximately 19.15, 7.73 and 7.31 kg/year, respectively. In contrast, in the US, Germany and South Africa, per capita consumption was 0.33, 0.24 and 0.23 kg/year, respectively. Concerning isoflavone intake among Japanese adults, it is ranged from about 30–50 mg/day but is less than 3 mg/day in the US and Europe [27]. In Asian countries, the mean isoflavone consumption is 25–50 mg/day, whereas in Western countries, 1–2 mg/day is typical [60]. However, in the last decade, in Western countries, production and intake of soy foods have increased due to its important health benefits (relief of menopausal symptoms, improvement in bone health and reduced risk of certain types of cancers) [61].

South Africa0.230.64
World average2.396.54

Table 2.

Annual per capita consumption of soybeans (2001).

Source: Ref. [45].


6. Isoflavones: new insights regarding the therapeutic effect

An increased consumption of soy products has been remarqued in the last decade of the twentieth century and was associated with the awareness concerning the health benefits of these products, besides their role as a source of proteins [62]. The therapeutic activities of soy isoflavones were also evaluated in numerous studies for their estrogenic [63], lipid‐lowering [64], anti‐diabetic [65], anti‐inflammatory [66], cardioprotective [67, 68] or anticancer effects [69]. In vitro and in vivo studies were performed in order to evaluate the therapeutic potential and the mechanisms of action of these compounds [70, 71].

Isoflavones are known as phytoestrogens due to their ability to bind the estrogen receptors and to exhibit estrogenic‐like properties. Dietary supplements containing soy isoflavones are used to alleviate menopause disturbances as an alternative to hormone therapy [72]. Isoflavones attracted the attention of researchers after the observation of fertility problems in sheep grazing on a clover type rich in isoflavones [62]. The decreased risk of breast, prostate and colon cancer in Asian countries was associated with a higher intake of soy products in these population compared to western countries [70].

The possible benefits of isoflavones thus generated several studies to establish their therapeutic properties. Isoflavones exhibit both estrogenic and anti‐estrogenic activity, binding to both α and β subtypes of estrogen receptor (ER) [73]. It was stated that isoflavones act as estrogen antagonists or agonists depending on estrogen concentration. Therefore, they are estrogen antagonists in a high estrogen environment, but when the estrogen quantity is reduced, they act as agonists [74]. The affinity for the estrogen receptors is different; genistein presents a 20–30 times higher affinity for ERβ than for ERα, while daidzein presents a weak affinity for both receptors, still higher for ERβ [70, 75]. Administration of 80 mg/day red clover isoflavones (containing genistein, daidzein, formononetin and biochanin A) for 90 days reduced hot flushes and night sweats, important vasomotor symptoms commonly found in postmenopausal women [76]. Low doses of isoflavones (25 mg/day containing 51.8% genistein, 43.3% daidzein and 4.9% glycitein) can reduce depression and insomnia in postmenopausal women [72].

Soy isoflavones present beneficial effects on lipid metabolism. The lipid‐lowering activity of isoflavones has been observed in studies performed in animals. Soy isoflavones reduced the levels of triglycerides (TG) and low‐density lipoprotein (LDL) in obese rats, and also exhibited benefits on obesity. The mechanisms involved are the suppression of mechanistic target of rapamycin complex 1 (mTORC1) activity that determines a reduction of AKT phosphorylation [77]. A decrease of total cholesterol, LDL cholesterol levels and an improvement of ApoA1/ApoB ratio were noticed after the administration of 435 mg isoflavones/day for 2 months in women with type 2 diabetes [64].

The benefits of isoflavones in glucose and lipid metabolism have been previously reported, but the mechanisms of action are not yet fully understood [78]. Potential benefits in obesity were also observed after intake of isoflavones. Dietary modifications in early stages of this condition are important to prevent cardiovascular and metabolic disorders [79]. Some of the mechanisms involved in the anti‐diabetic properties of isoflavone genistein are the enhancing of β‐cell proliferation and the regulation of insulin secretion [65]. Isoflavones intake might reduce the risk of type 2 diabetes [53]. However, the beneficial role of isoflavones in this disease was not supported by other studies [78, 80]. A decreased risk of type 2 diabetes was noticed for women with high concentrations of genistein in plasma, but this correlation was not observed in men [81]. After the administration of a diet supplemented with 0.02% (w/w) genistein for 8 weeks (10–12 mg genistein/day) in Zucker diabetic fatty (ZDF) rats, there were no beneficial effects on glucose homeostasis or on skeletal muscle oxidative stress [78]. The conflicting results regarding the benefits of isoflavones in diabetes led to the assumption that other soy compounds might be responsible for this activity [65].

Genistein not only reduces weight gain in female obese ob/ob mice after the administration of 600 mg/kg diet for 4 weeks, but also promotes oxidative stress in the vasculature and inflammation in the perivascular adipose tissue [82]. Daidzin and glycitin (0.06% in diet) decrease blood glucose, insulin and HbA1c levels in mice with obesity and diabetes induced by a high‐fat diet [79].

The effects of isoflavones on components of the metabolic syndrome were also evaluated. An extract from the roots of Pueraria lobata, administered 0.2% in the diet of female stroke‐prone spontaneously hypertensive rats (SP‐SHR) improved blood glucose levels, decreased plasma total cholesterol levels and reduced blood pressure, thus indicating beneficial effects on risk factors that led to the development of metabolic syndrome. The major isoflavones in the extract were puerarin (25.3%), daidzin (7.1%) and daidzein (0.8%) [83].

Even though the effects on obesity and diabetes were not sustained by other studies, an increase in bone mass was noticed in obese mice treated with 600 mg genistein/kg for 4 weeks [84]. The bone protective properties were also observed for other compounds. Formononetin, an isoflavone found mainly in the roots of Astragalus membranaceus and Astragalus mongholicus, improved the mechanical properties of the bones and exerted beneficial effects in rats with osteoporosis induced by ovariectomy after the administration of 10 mg/kg formononetin for 4 weeks [85]. The results of a meta‐analysis investigating trials that evaluated the effects of isoflavone in osteoporosis, revealed that the effects of these compounds depend on dose and duration of administration. An increase in mineral density of the bone by 54% and a reduction in the fracture risk were noticed in women included in these studies [86]. A clinical trial regarding the effects of soy isoflavones on bone loss and menopausal symptoms did not reveal benefits in this condition. Women included in the study received tablets containing 200 mg soy isoflavones (genistein and daidzein)/day for 2 years [87].

The cardiovascular diseases are more frequent in women after menopause, due to the modifications in the production of estrogen [88]. Compounds with estrogenic properties, such as the isoflavones found in soy, were evaluated for their potentially cardioprotective activity [89, 90]. The administration of 80 mg/day isoflavones extracted from soybeans for 12 weeks in patients with primary or recurrent ischemic stroke determined a decrease in high sensitivity, C‐reactive protein and improved vascular endothelial function [68]. A meta‐analysis evaluating the results of nine trials concluded that isoflavone supplementation improves endothelial function in postmenopausal women that presents low flow‐mediated dilatation (FMD) levels, but not in ones with high baseline FMD levels [89]. Nevertheless, the benefits of isoflavones regarding the protective effects in ischemic stroke are questionable. Yu et al. associated the high intake of soy isoflavones (53.6 mg isoflavones/day) with an increase in the risk of ischemic stroke in women [91].

The protective effect against the inflammatory vascular disease of isoflavones is due to their anti‐inflammatory activity. The inhibition of monocyte adhesion to endothelial cells that involves the activation of PPARγ is related to this effect [66]. The anti‐inflammatory properties are due to inhibition of interleukin‐6 (IL‐6), interleukin‐12 (IL‐12), interleukin‐1β (IL‐1β), and tumor necrosis factor‐α (TNF‐α) production, NF‐κB regulation and their antioxidant activity [92]. Pro‐oxidant effects at high doses were also noticed for these compounds. The supplementation with 640 mg/kg daidzein in pigs revealed antioxidant properties in muscle, but were accompanied by pro‐oxidant effects in fat and liver tissues [93].

Administration of soy isoflavones (daidzein, genistein and glycetin) in capsules, 20 mg twice daily in female patients improved irritable bowel disease and association with vitamin D can determine a synergistic effect [94]. The estrogen‐like effects of isoflavones genistein and daidzein seem to be involved in their beneficial effects on sleep status. A high intake of isoflavones was related to an optimal sleep duration and an increased quality of sleep in Japanese adults [95].

Age‐related skin modifications in women emerging with a decline in estrogen production can be reduced by isoflavones. Genistein improves skin appearance and is used to reduce wrinkles and skin dryness in cosmetic preparations. It increases the skin resistance and contributes to skin reparation [96].

Anti‐bacterial effects were also reported for isoflavones. For instance, biochanin A, a methylated isoflavone from red clover, inhibits the growth of Chlamydia trachomatis and Chlamydia pneumoniae [97]. Isoflavones act as anti‐viral agents against several types of viruses including herpes simplex virus and human immunodeficiency virus (HIV). Genistein, a tyrosine kinase inhibitor, is one of the compounds most studied for these properties and revealed positive effects in inhibiting HIV‐1 infection, especially when applied in entry and early post‐entry stages [98].


7. Isoflavonoids: new insights regarding cancer chemoprevention

It is very well known that dietary components are, nowadays, considered important therapeutic agents used to prevent various chronic disorders, especially cancer, cardiovascular pathologies and inflammation processes [99]. For example, according to the statistics, the incidence of prostate cancer or breast cancer in Asian countries has been lower than in Western countries, mainly because of their high consumption of soy products [100]. Furthermore, the incidence of breast cancer in Asian women who had immigrated to Western countries proved to be similar to that of Western women [101]. In addition, soy isoflavones, with the main representative genistein, proved to be promising phytotherapeutic drugs with chemopreventive effects in chronic disorders caused by exposure to solar UVB radiations, including nonmelanoma skin cancer [102, 103].

Genistein has been extensively studied in different types of cancers cells and cancer animal models [104]. Unfortunately, the low oral bioavailability of genistein [105], due to its high lipophilicity and its extensive metabolism by the phase II enzymes [106], has limited its use in clinical trials [107].

The data regarding the chemopreventive effects of daidzein are limited, more studies being focused on the curative effect of daidzein in cancer and not in its prophylactic properties [108].

Previous in vitro evidences have shown that genistein may act not only as an agonist, but also as an antagonist on cancer cells depending on both its concentration and the type of cancer cells on which was tested [109]. In this regard, genistein because of its biphasic effect may be involved not only in preventing, but also in promoting cancer [110].

The in vitro chemopreventive effect of genistein might be related to its involvement in epigenetic regulations of gene expression, having a direct effect on histone modifications and DNA methylation [111]. Genistein is also a strong inhibitor of the tyrosine kinase [112] and topoisomerase activities [113]. The in vitro apoptotic effect of genistein may be related to the inactivation of NF‐kB and Akt signaling pathways [114], although its specific mechanisms of inducing apoptosis have not being fully understood [115]. According to a previous in vitro study on pancreatic cancer cells, genistein suppressed the ovarian cancer cell growth and migration through the inhibition of mRNA [116]. The chemopreventive properties of genistein have also been proved on MCF‐7 breast cancer cells, in which genistein has inhibited the cell proliferation by inactivating the IGF‐1R‐PI3 K/Akt pathway and decreasing the Bcl‐2/Bax mRNA and protein expressions [115]. Another mechanism responsible for the chemopreventive effects of genistein might consist in the suppression of the microsomal CYP1a1 gene expression in Hepa‐1c1c7 liver cancer cells [117].

The association between genistein and daidzein has proved antiproliferative effects on human colon adenocarcinoma grade II cell line (HT‐29) [118], the anticancer effect of daidzein being related to copper‐dependent pathway and redox cycle [108].

Among the soy isoflavones, GLY has been the most potent activator of extracellular signal‐regulated kinase (ERK1/2), exhibiting a significant antiproliferative effect on RWPE‐1 nontumorigenic prostate epithelial cells [119]. Thus, according to another study on the same type of cells, GLY has also shown to decrease the expression of cytokeratin 18 and prostate‐specific antigen (PSA) [120].

Furthermore, in vivo data have shown that oral consumption of genistein during the early prepubertal period decreased the susceptibility of developing breast cancer later in life [121]. Genistein, as a phytoestrogen, may interact with the estrogen receptors. In this regard, a previous study on HER2 overexpressing mice has shown that genistein mimicked the estradiol effects, in the presence of estrogen receptor alpha [112], while in postmenopausal women, in the absence of estradiol, genistein directly reduced the anticancer activity of cisplatin, a cytostatic drug commonly used in breast cancer [122]. For instance, according to Tonetti et al. in vitro and in vivo studies, the concomitant administration of tamoxifen with genistein or daidzein might not be safe because this association has produced bigger size tumors than tamoxifen alone [123].

Soy isoflavones exhibited in vivo protective effects against skin chronic disorders including cancer by reducing pro‐inflammatory cytokines and oxidative stress and through activation of NF‐kB [124]. For example, genistein has suppressed UV‐induced skin carcinogenesis in mice through its moderate inhibitory effect on ornithine decarboxylase activity [125]. Moreover, 7,3′,4′‐trihydroxyisoflavone, a major metabolite of daidzein, has reduced UVB‐induced skin cancer in mice through inhibition of cyclooxygenase‐2 (COX‐2) expression by suppressing NF‐kB transcription activity [103]. In this regard, genistein loaded‐PLA nanocapsules indicated to be a promising formulation with chemopreventive effects against skin cancer in porcine ear skin not only by increasing the penetration of genistein in skin deeper layers, but also by limiting its degradation in time [107].

According to Ghaemi et al. study on human papillomavirus (HPV) associated‐cervical cancer in mice, genistein has also indicated immunomodulatory effects through increment of interferon‐gamma (IFN‐gamma) level, lymphocyte proliferation and lactate dehydrogenase (LDH) release [126].

Consequently, the isoflavones from soy products may be considered promising alternative therapies to prevent various types of cancer, more experimental and clinical studies being necessary for establishing the safe dose that can be used especially in patients susceptible to hormone‐dependent tumors.


8. Anti‐inflammatory and chemopreventive activity of isoflavonoid genistein alone and incorporated in modern pharmaceutical formulations

From the main isoflavones reported above, we have studied genistein. One of the most important lines in our research group on this topic involves the analysis of the chemopreventive effect of the phytoestrogen genistein against malignant melanoma. The isoflavonoid genistein (4′,5,7‐trihydroxyisoflavone) is the aglycone of heteroside genistin. It is the most studied compound from the class of isoflavones together with daidzein, glycitein, formononetin, equol and biochanin A. It is the major active compound from soy seeds, Glycine max (L.) Merr., family Fabaceae [127129]. Regarding the biological activities, recent papers report that: genistein induces apoptosis, inhibits cell proliferation, modulates cell cycle progression on different cancerous cell lines, inhibits angiogenesis, suppress lymphocyte activation and proliferation, stabilizes mast cells and presents mild anti‐inflammatory properties [130, 131]. The phytoestrogen also inhibits the production of reactive oxygen species (ROS) which is directly correlated with DNA modification and tissue damage. Production of ROS, especially by activated cells of the immune system, has been postulated to play an important role in carcinogenesis, particularly in tumor promotion [132, 133].

In a recent complex study employing, the B164A5 and B16F10 murine melanoma cell lines, we have shown testing a wide range of concentrations (150, 100, 50, 30, 15, 5 and 1 µM) that this phytocompound is an active antiproliferative and pro‐apoptotic agent on this two cell lines. MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay has shown an IC50 of 41.1 µM genistein for B164A5 cells and 61.4 µM genistein for B16F10 cells. The carboxyfluorescein diacetate succinimidyl ester (CFSE) assay has shown that after 24 h of incubation, proliferation of B16 cells was decreased when treated with 30 μM genistein. Genistein at 100 µM was able to cause G2/M arrest in the cell cycle of these murine melanoma cell lines [134]. DAPI staining was performed in order to detect first signs of apoptosis. When B16 cells were incubated with 100 μM genistein, this phenomenon could be detected and translated by a reduction of the cell number and increase in nuclear fragmentation compared to the control group [134]. Western blot analysis was furtherer conduced, for four important proteins involved in the process of apoptosis, namely caspase 3, poly(ADP‐ribose) polymerase (PARP), Bax and Bcl‐2. Incubation with 100 μM genistein conduced for both B16 cell lines to cleaved caspase‐3 as well as cleaved PARP as responsible for the mechanism of apoptotic events. In another study using all concentrations previously tested (150, 100, 50, 30, 15, 5 and 1 µM genistein) and after a period of incubation of 72 h, we have shown that the phytoestrogen does not induce caspase‐2 activation in vitro on B16 melanoma cell lines [135]. In order to get the full picture about apoptosis, namely to detect early and late apoptotic cells, annexin V‐FITC/7AAD staining was performed by fluorescence cytometry in parallel for the B16 melanoma cells as well as for the bone marrow‐derived dendritic cells (BMDCs). At its highest concentration, genistein induced slightly more apoptotic events in the B16 cells than in the BMDCs [134].The aim of the above‐mentioned study was to find a drug that “kills” the cancerous cells and stimulate the immune activity. On this purpose, the potential immune stimulatory activity of genistein was tested by measuring the anti‐tumorigenic cytokine IL‐12p70 released by murine primary BMDCs. The phytoestrogen, at the concentration of 5 μM decreased the level of IL‐12p70 of the LPS‐stimulated DCs. These findings were in line with the observation that also the IL‐12p35 mRNA levels were downregulated. T cell activity was further screened by analyzing the concentration of IFN‐γ and IL‐2 cytokine in the supernatant of spleen cells of OT I mice expressing the ovalbumin‐specific transgenic T cell receptors. Results have shown that genistein had no effect on the concentration of IFN‐γ and IL‐2 cytokine [134].

Besides the chemopreventive activity for murine melanoma, our research group have investigated genistein alone and incorporated in randomly methylated β‐cyclodextrin (RAMEB), hydroxypropyl‐β‐cyclodextrin (HPBCD) and hydroxypropyl‐γ‐cyclodextrin (HPGCD) in a molar ratio 1:1 for a range of biological activities. This approach was chosen in order to increase the water solubility of this lipophilic compound. CDs are cyclo‐oligosaccharides presenting a hydrophilic outside and hydrophobic inner side with the ability to form host‐guest inclusion complexes with an increased number of chemical structures [136]. Firstly, quantum chemical calculations were performed analyzing the behavior in gas phase, in water and in dimethyl sulfoxide, the solvent used for the solubilization of active agents for all the mentioned assays. Additionally, it was proofed that incorporation of genistein in the above‐mentioned CDs took place by a series of consecrated techniques such as phase solubility studies, differential scanning calorimetry (DSC), X‐ray diffraction and scanning electron microscopy (SEM) assays [136]. Genistein and its inclusion complexes were studied in vitro on four types of cancer cells lines such as HeLa (cervical adenocarcinoma), MCF‐7 (breast adenocarcinoma), A2780 (human ovarian carcinoma) and A431 (skin epidermoid carcinoma) cell lines using the following concentrations: 1, 3, 10, 30, 60 and 90 μM and a period of incubation of 72 h. A2780 human ovarian carcinoma cell line proofed to be the most sensitive to genistein, followed by HeLa. Proliferation was not significantly affected for the other two cell lines. After incorporation in the above‐mentioned CDs, changes in the antiproliferative action occurred with respect to the tested cell line. Cervical adenocarcinoma HeLa cell line was more sensitive for all three inclusion complexes when compared to pure genistein. The same behavior was found for A2780 cell line, except for the complex with RAMEB. Complexation with RAMEB conduced to an increased IC50 also for MCF‐7 and A431 cell lines. For this two cell lines, complexation of genistein with HPBCD seemed to be the best option [136]. In the same study, genistein and its CD complexes were analyzed by the agar disk‐diffusion method and the dilution method against several bacterial strains: Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Pseudomonas aeruginosa and Staphylococcus aureus. Tested compounds at the concentration of 10 mM presented antibacterial activity only for B. subtilis [137]. The last line of this study was drawn toward tests for antiangiogenic effects employing the chorioallantoic membrane of the chicken embryo. Pure genistein presented antiangiogenic effects and the HPBCD complex showed superior activity. Also for the other two complexes, namely HPGCD and RAMEB could be described an antiangiogenic effect but a decreased one as evaluated by applying the 0–5 score [136].

Another attempt to increase the bioavailability of this lipophilic phytoestrogen was directed toward the synthesis and analysis of a genistein ester derivative with myristic acid and complexed with beta cyclodextrin. The successful synthesis of the new compound as well as the successful inclusion in beta cyclodextrin was determined using consecrated assays such as TLC analysis, HPLC analysis, FTIR spectroscopy, MS spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Samples were tested in vitro, using the MTT proliferation assay on three human cell lines: HeLa—cervix adenocarcinoma, A2780—ovary carcinoma and A431—skin epidermoid carcinoma. Results have shown that, after a period of incubation of 72 h at the concentrations of 10 and 30 µM, respectively, genistein is an active agent on HeLa (cervix adenocarcinoma) and A2780 (ovary carcinoma) cell lines. The new formulations did not decrease the viability of the cancerous cells. This behavior may be explained by the increased stability of the complex within the in vitro environment [138].

A new modern formulation explored by the pharmaceutical industry, but not only, is the polyurethane microstructures (PM). What determined us to focus on these compounds? Depending on the structure of the particle, such approach can offer: the possibility of amending the lipo‐ or water‐solubility of inclusion structures, protection from external agents such as UV radiation, strong acidic or alkaline environments, drug delivery toward a specific receptor or retard activity of the biologically active compound due to the use of transport vehicles with low speed of degradation [139]. Based on these hypotheses, we have synthesized PM with a yield of encapsulation of 68.3% genistein (w/w). The formulation was tested in vitro using the MTT proliferation assay on three human breast cancer cell lines MCF7, MDA‐MB‐231 and T47D‐human breast adenocarcinoma cell lines. Tests were performed also for the antimicrobial and antifungal activity against the following strains: S. aureus, E. coli, P. aeruginosa, Salmonella enteritidis, B. subtilis, Bacillus cereus and Candida albicans employing the dilution method. Results made us to conclude that the PM are a bad in vitro carrier partner for genistein [137].

In vivo assays were also performed in order to test the chemopreventive effects of genistein against murine melanoma. During the research in our group, we have observed in a murine model of melanoma, obtained by subcutaneous injection of 0.1 ml of 1*105 B164A5 cells/mouse that, genistein after a period of 15 days at a dose of 15 mg/kg, body weight decreased tumor volume and weigh with about 30% and reduced distance tumors. Noninvasive measurements using the Multiprobe Adapter System (MPA5) from Courage‐Khazaka, Germany, Mexameter® MX 18 showed that genistein reduced the quantity of melanin and the degree of erythema directly correlated with the number of days of treatment [140]. Being very well known, the link between inflammation and cancer, we have analyzed the effect of genistein in an animal model of ear inflammation alone and after incorporation in hydroxypropyl‐beta‐cyclodextrin (HPBCD) and randomly‐methylated‐beta‐cyclodextrin (RAMEB). Cyclodextrins (CDs) are well known agents used to increase the hydro solubility of active lipophilic agents [139]. The study concluded that the phytoestrogen, at a dose of 2 mg can be reconsidered as an active anti‐inflammatory natural compound on C57BL/6 J animal model of inflammation. Additionally, complexation of genistein with the above‐mentioned CDs was done and led to a stronger anti‐inflammatory effect [141]. In a recent study, in order to attempt to increase the bioavailability of this phytoestrogen, we have adopted a new strategy that combines two elements: the formulation and the modality of administration. The formulation was lamellar lyotropic liquid crystal in which genistein was incorporated at the concentration 3% and the formulation was applied local, with or without electroporation (EP), using the Mezoforte Duo Mez 120905‐D device on C57BL6J. Results have shown that tumors appeared later when electroporation was applied. During the 21 days of the experiment, genistein incorporated in the new modern formulation, applied topically classic decreased the tumor volume, the degree of erythema and amount of melanin for mice bearing B16 murine melanoma tumors. When the formulation was applied by electroporation, the prognosis was even better. However, the new approach had no effect in terms of serum concentrations of the protein S100B and serum neuron‐specific enolase (NSE), or the tissue expression of the platelet‐derived growth factor receptor β (PDGFRβ) antibody [142]. Also, soy total extract incorporated in the new modern lamellar lyotropic liquid crystal formulation was tested in vitro on the B164A5 mouse melanoma cell line for its pro‐apoptotic potential, employing two consecrated assays: 4’,6‐diamidino‐2‐phenylindole (DAPI) and annexin‐FITC‐7AAD double staining. 200 μg/ml of soy extract, respectively 200 μg/ml of soy extract incorporated into the lamellar lyotropic liquid crystalline formulation were incubated for 72 h together with this murine melanoma cell line. Results have shown that soy extract has pro‐apoptotic properties and incorporation in the new formulation does not affect in a negative manner this effect thus being a suitable excipient for in vivo studies [143]. In a recent study, diffusion and penetration of genistein, respectively, genistein incorporated in lamellar lyotropic liquid crystalline formulation through different membranes (a synthetic membrane in vitro, chick chorioallantoic membrane (CAM) ex ovo, and excised human epidermis ex vivo) were also investigated by conventional treatment without EP, and also with the mediation of EP by the help of a Franz diffusion cell system. In vivo ATR‐FTIR and ex vivo Raman spectroscopy were applied in order to analyze the effect on mice skin [144]. Results have shown that the new formulation is a suitable carrier for the lipophilic genistein. The formulation with the active agent penetrated the skin, but when electroporation was applied, the transdermal drug transport was more rapid and effective. This observation was validated by both ATR‐FTIR and Raman spectroscopy [144].

The research of our group on the phytoestrogen genistein points toward the clear conclusion that this phytocompound is an active chemopreventive agent against malignant melanoma both in vitro and in vivo. A series of attempts were made in order to increase the bioavailability of this lipophilic compound. We cannot say that we have found the optimal formulation, but we have managed to improve results compared to pure substance. Further studies will be conducted on this matter.


  1. 1. Reynaud J, Guilet D, Terreux R, Lussignol M, Walchshofer N. Isoflavonoids in non‐leguminous families: an update. Nat Prod Rep. 2005;22(4):504-15
  2. 2. Veitch NC. Isoflavonoids of the leguminosae. Nat Prod Rep. 2013;30(7):988-1027. DOI: 10.1039/c3np70024k
  3. 3. Hegnauer R, Grayer‐Barkmeijer RJ. Relevance of seed polysaccharides and flavonoids for the classification of the leguminosae: a chemotaxonomic approach. Phytochemistry. 1993;34:3-16
  4. 4. Mackova Z, Koblovska R, Lapcik O. Distribution of isoflavonoids in non‐leguminous taxa – an update. Phytochemistry. 2006;67:849-55
  5. 5. Dakora FD. Defining new roles for plant and rhizobial molecules in sole and mixed plant cultures involving symbiotic legumes. New Phytologist. 2003;158(1):39-49
  6. 6. Subramanian S, Stacey G, Yu O. Distinct, crucial roles of flavonoids during legume nodulation. Trends Plant Sci. 2007;12(7):282-5. DOI: 10.1016/j.tplants.2007.06.006
  7. 7. Jung W, Yu O, Lau SM, O’Keefe DP, Odell J, Fader G, McGonigle B. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nat Biotechnol. 2000;18(2):208-12; erratum Jung W, Yu O, Lau S‐MC, O’Keefe DP, Odell J, Fader G, McGonigle B. Nat Biotechnol. 2000;18:559
  8. 8. Hagmann M, Grisebach H. Enzymatic rearrangement of flavanone to isoflavone. FEBS Letters. 1984;175(2):199-202
  9. 9. Dhaubhadel S. Regulation of isoflavonoid biosynthesis in Soybean Seeds, Soybean‐Biochemistry, Chemistry and Physiology, Prof. Tzi‐Bun Ng (Ed.), 2011; ISBN: 978‐953‐307‐219‐7, InTech, Available from:‐biochemistry‐chemistry‐and‐physiology/regulation‐ofisoflavonoid‐biosynthesis‐in‐soybean‐seeds
  10. 10. Yu O, Jung W, Shi J, Croes RA, Fader GM, McGonigle B, Odell JT. Production of the isoflavones genistein and daidzein in non‐legume dicot and monocot tissues. Plant Physiology. 2000;124(2):781-93
  11. 11. Yu O, Shi J, Hession AO, Maxwell AA, McGonigle B, Odell JT. Metabolic engineering to increase isoflavone biosynthesis in soybean seeds. Phytochemistry. 2003;63(7):753-63
  12. 12. Liu CJ, Blount JW, Steele CL, Dixon RA. Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. Proc Nat Acad Sci USA. 2002;99(22):14578-83
  13. 13. Dastmalchi M, Dhaubhadel S. Soybean seed isoflavonoids: biosynthesis and regulation. Phytochemicals – biosynthesis, function and application. Recent Adv Phytochem, 2014;44:1-21
  14. 14. Li M‐W, Muñoz Nacira B., Wong C‐F, Wong F‐L, Wong K‐S, Wong JW‐H, Xinpeng Q, Li K‐P, Ng M‐S, Lam H‐M. QTLs regulating the contents of antioxidants, phenolics, and flavonoids in soybean seeds share a common genomic region. Front Plant Sci. 2016;7:854. DOI: 10.3389/fpls.2016.00854
  15. 15. Albulescu M, Popovici M. Isoflavones‐biochemistry, pharmacology and therapeutic use. Rev Roum Chim. 2007;52(6):537-50
  16. 16. Nguyenle T, Wang E, Cheung AP. An investigation on the extraction and concentration of isoflavones in soy‐based products. J Pharm Biomed Anal. 1995;14(1-2):221-32
  17. 17. Coward L, Smith M, Kirk M, Barnes S. Chemical modification of isoflavones in soyfoods during cooking and processing. Am J Clin Nutr. 1998;68(6):1486S–91
  18. 18. Griffith AP, Collison MW. Improved methods for the extraction and analysis of isoflavones from soy‐containing foods and nutritional supplements by reversed‐phase high‐performance liquid chromatography and liquid chromatography – mass spectrometry. J Chromatography A. 2001;913:397-413
  19. 19. Murphy P, Barua K, Hauck CC. Solvent extraction selection in the determination of isoflavones in soy foods. J Chromatogr B. 2002;777(1-2):129-38
  20. 20. Degenhardt A, Winterhalter P. Isolation and purification of isoflavones from soy flour by high‐speed countercurrent chromatography. Eur Food Res Technol. 2001;213(4):277-80
  21. 21. Stürtz M, Lander V, Schmid W, Winterhalter P. Preparative isolation of isoflavones from soy and red clover. Mol Nutr Food Res. 2006;50(4-5):356-61
  22. 22. Cao Y, Calafat AM, Doerge DR, Umbach D, Bernbaum JC, Twaddle NC, Ye X, Rogan WJ. Isoflavones in urine, saliva, and blood of infants: data from a pilot study on the estrogenic activity of soy formula. J Expo Sci Environ Epidemiol. 2009;19(2):223-34. DOI: 10.1038/jes.2008.44
  23. 23. 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 analysis of commercial soy isoflavone supplements. J Nutr. 2001;131:1362-75
  24. 24. Cassidy A. Factors affecting the bioavailability of soy isoflavones in humans. J AOAC Int. 2006;89:1182-8
  25. 25. Yang Z, Kulkarni K, Zhu W, Hu M. Bioavailability and pharmacokinetics of genistein: mechanistic studies on its ADME. Anticancer Agents Med Chem. 2012;12(10):1264-80
  26. 26. Setchell KD, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroidal estrogens of dietary origin: possible roles in hormone‐dependent disease. Am J Clin Nutr. 1984;40:569-78
  27. 27. Messina M. Soy and health update: evaluation of the clinical and epidemiologic literature. Nutrients. 2016;8:754. DOI: 10.3390/nu8120754
  28. 28. 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
  29. 29. Manach C, Williamson G, Morand C, Scalbert A, Rémésy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr. 2005;81:230-42
  30. 30. Setchell KD, Brown NM, Desai PB, Zimmer‐Nechimias L, Wolfe B, Jakate AS, Creutzinger V, Heubi JE. Bioavailability, disposition, and dose‐response effects of soy isoflavones when consumed by healthy women at physiologically typical dietary intakes. J Nutr. 2003;133:1027-35
  31. 31. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr. 2000;130:2073-208
  32. 32. Gu L, House SE, Prior RL, Fang N, Ronis MJ, Clarkson TB, Wilson ME, Badger TM. Metabolic phenotype of isoflavones differ among female rats, pigs, monkeys, and women. J Nutr. 2006;136(5):1215-21
  33. 33. Adlercreutz H, van der Wildt, Kinzel J, Attalla H, Wähälä K, Mäkelä T, Hase T, Fotsis T. Lignan and isoflavonoid conjugates in human urine. J Steroid Biochem Mol Biol. 1995;52(1):97-103
  34. 34. Coldham NG, Sauer MJ. Pharmacokinetics of [(14)C] genistein in the rat: gender‐related differences, potential mechanisms of biological action, and implications for human health. Toxicol Appl Pharmacol. 2000;164(2):206-15
  35. 35. Messina MJ. Legumes and soybeans: overview of their nutritional profiles and health effects. Am J Clin Nutr. 1999;70:439-50
  36. 36. Liu ZM, Ho SC, Chen YM, Ho S, To K, Tomlinson B, Woo J. Whole soy, but not purified daidzein, had a favorable effect on improvement of cardiovascular risks: a 6‐month randomized, double-blind, and placebo‐controlled trial in equol‐producing postmenopausal women. Mol Nutr Food Res. 2014;58:709-17. DOI: 10.1038/ejcn.2015.24
  37. 37. Pakalapati G, Li L, Gretz N, Koch E, Wink M. Influence of red clover (Trifolium pratense) isoflavones on gene and protein expression profiles in liver of ovariectomized rats. Phytomedicine. 2009;16:845-55. DOI: 10.1016/j.phymed.2009.03.003
  38. 38. Zaheer K, Akhtar MH. An updated review of dietary isoflavones: nutrition, processing, bioavailability and impacts on human health. Crit Rev Food Sci Nutr. 2017;57(6):1280-1293. DOI: 10.1080/10408398.2014.989958
  39. 39. Kaufman PB, Duke JA, Brielmann H, Boik J, Hoyt JE. A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: implications for human nutrition and health. J Altern Complement Med. 1997;3:7-12. DOI: 10.1089/acm.1997.3.7
  40. 40. Kumar MP, Sankeshi V, Naik RR, Thirupathi P, Das B, Raju TN. The inhibitory effect of Isoflavones isolated from Caesalpinia pulcherrima on aldose reductase in STZ induced diabetic rats. Chem Biol Interact. 2015;237:18-24. DOI: 10.1016/j.cbi.2015.05.010
  41. 41. Soto‐Zarazúa MG, Rodrigues F, Pimentel FB, Bah MM, Oliveira MB. The isoflavone content of two new alfalfa‐derived products for instant beverage preparation. Food Funct. 2016;7:364-71. DOI: 10.1039/c5fo01115a
  42. 42. Jackson CJ, Dini JP, Lavandier C, Rupasinghe HP, Faulkner H, Poysa V, Buzzell D, DeGrandis S. Effects of processing on the content and composition of isoflavones during manufacturing of soy beverage and tofu. Process Biochem. 2002;37:1117-23. DOI: 10.1016/S0032‐9592(01)00323‐5
  43. 43. Zhang J, Ge Y, Han F, Li B, Yan S, Sun J, Wang L. Isoflavone content of soybean cultivars from maturity group 0 to VI grown in northern and southern China. J Am Oil Chem Soc. 2014;91:1019-28. DOI: 10.1007/s11746‐014‐2440‐3
  44. 44. Soukup ST, Helppi J, Müller DR, Zierau O, Watzl B, Vollmer G, Diel P, Bub A, Kulling SE. Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross‐species and sex comparison. Arch Toxicol. 2016;90:1335-47. DOI: 10.1007/s00204‐016‐1663‐5
  45. 45. Golbitz P, Jordan J. Soyfoods: Market and Products. Soy Applications in Food. Taylor & Francis; Boca Raton, Florida, USA. 2006. pp. 2-20
  46. 46. Golbitz P. Traditional soyfoods: processing and products. J Nutr. 1995;125:570-2
  47. 47. Nagino T, Mitsuyoshi KA, Masuoka N, Chiaki KA, Michitoshi AN, Miyazaki K, Kamachi K, Isozaki M, Suzuki C, Kasuga C, Tanaka A. Intake of a fermented soymilk beverage containing moderate levels of isoflavone aglycones enhances bioavailability of isoflavones in healthy premenopausal Japanese women: a double‐blind, placebo‐controlled, single‐dose, crossover trial. Biosci Microbiota Food Health. 2016;35:9-17. DOI: 10.12938/bmfh.2015‐011
  48. 48. Wang HJ, Murphy PA. Mass balance study of isoflavones during soybean processing. J. Agric Food Chem. 1996;44:2377-83. DOI: 10.1021/jf950535p
  49. 49. Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, Kinae N. Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J Nutr. 2000;130:2243-50
  50. 50. de Kleijn MJ, van der Schouw YT, Wilson PW, Adlercreutz H, Mazur W, Grobbee DE, Jacques PF. Intake of dietary phytoestrogens is low in postmenopausal women in the United States: the Framingham study1-4. J Nutr. 2001;131:1826-32
  51. 51. Bhagwat S, Haytowitz DB, Holden JM. USDA Database for the Isoflavone Content of Selected Foods Release 2.0. Maryland: US Department of Agriculture. 2008;15
  52. 52. Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein contents of vegetables. Br J Nutr. 2000;84:717-25. DOI: 10.1017/S0007114500002075
  53. 53. Ding M, Pan A, Manson JE, Willett WC, Malik V, Rosner B, Giovannucci E, Hu FB, Sun Q. Consumption of soy foods and isoflavones and risk of type 2 diabetes: a pooled analysis of three US cohorts. Eur J Clin Nutr. 2016;70:1381-7. DOI: 10.1038/ejcn.2016.117
  54. 54. Messina M, Nagata C, Wu AH. Estimated Asian adult soy protein and isoflavone intakes. Nutr Cancer. 2006;55:1-2. DOI: 10.1207/s15327914nc5501_1
  55. 55. Masilamani M, Wei J, Sampson HA. Regulation of the immune response by soybean isoflavones. Immunol Res. 2012;54:95-110. DOI: 10.1007/s12026‐012‐8331‐5
  56. 56. Mulligan AA, Welch AA, McTaggart AA, Bhaniani A, Bingham SA. Intakes and sources of soya foods and isoflavones in a UK population cohort study (EPIC‐Norfolk). Eur J Clin Nutr. 2007;61:248-54. DOI: 10.1038/sj.ejcn.1602509
  57. 57. Clarke DB, Lloyd AS. Dietary exposure estimates of isoflavones from the 1998 UK Total Diet Study. Food Addi Contam. 2004;21:305-16. DOI: 10.1080/02652030410001668781
  58. 58. Ritchie MR, Cummings JH, Morton MS, Steel CM, Bolton‐Smith C, Riches AC. A newly constructed and validated isoflavone database for the assessment of total genistein and daidzein intake. Br J Nutr. 2006;95:204-13. DOI: 10.1079/BJN20051603
  59. 59. Greendale GA, FitzGerald G, Huang MH, Sternfeld B, Gold E, Seeman T, Sherman S, Sowers M. Dietary soy isoflavones and bone mineral density: results from the study of women’s health across the nation. Am J Epidemiol. 2002;155:746-54. DOI: 10.1093/aje/155.8.746
  60. 60. Reverri EJ, LaSalle CD, Franke AA, Steinberg FM. Soy provides modest benefits on endothelial function without affecting inflammatory biomarkers in adults at cardiometabolic risk. Mol Nutr Food Res. 2015;59:323-33. DOI: 10.1002/mnfr.201400270
  61. 61. Sathyapalan T, Rigby AS, Bhasin S, Thatcher NJ, Kilpatrick ES, Atkin SL. Effect of soy in men with type 2 diabetes mellitus and subclinical hypogonadism – a randomized controlled study. J Clin Endocrinol Metab. 2017 Feb 1;102(2):425-433. DOI: 10.1210/jc.2016-2875
  62. 62. Wang Q, Ge X, Tian X, Zhang Y, Zhang J, Zhang P. Soy isoflavone: the multipurpose phytochemical (review). Biomed Rep. 2013;1(5):697-701
  63. 63. Vitale DC, Piazza C, Melilli B, Drago F, Salomone S. Isoflavones: estrogenic activity, biological effect and bioavailability. Eur J Drug Metab Pharmacokinet. 2013;38(1):15-25
  64. 64. Chi XX, Zhang T, Zhang DJ, Yu W, Wang QY, Zhen JL. Effects of isoflavones on lipid and apolipoprotein levels in patients with type 2 diabetes in Heilongjiang Province in China. J Clin Biochem Nutr. 2016;59(2):134-8
  65. 65. Gilbert ER, Liu D. Anti‐diabetic functions of soy isoflavone genistein: mechanisms underlying its effects on pancreatic β‐cell function. Food Funct. 2013;4(2):200-12. DOI: 10.1039/c2fo30199g
  66. 66. Chacko BK, Chandler RT, D’Alessandro TL, Mundhekar A, Khoo NK, Botting N, Barnes S, Patel RP. Anti‐inflammatory effects of isoflavones are dependent on flow and human endothelial cell PPARgamma. J Nutr. 2007;137(2):351-6
  67. 67. Gil‐Izquierdo A, Penalvo JL, Gil JI, Medina S, Horcajada MN, Lafay S, Silberberg M, Llorach R, Zafrilla P, Garcia‐Mora P, Ferreres F. Soy isoflavones and cardiovascular disease epidemiological, clinical and –omics perspectives. Curr Pharm Biotechnol. 2012; 13(5):624-31
  68. 68. Chan YH, Lau KK, Yiu KH, Li SW, Chan HT, Fong DY, Tam S, Lau CP, Tse HF. Reduction of C‐reactive protein with isoflavone supplement reverses endothelial dysfunction in patients with ischaemic stroke. Eur Heart J. 2008;29(22):2800-7. DOI: 10.1093/eurheartj/ehn409
  69. 69. Zhang YF, Kang HB, Li BL, Zhang RM. Positive effects of soy isoflavone food on survival of breast cancer patients in China. Asian Pac J Cancer Prev. 2012;13(2):479-82
  70. 70. Pilšáková L, Riečanský I, Jagla F. The physiological actions of isoflavone phytoestrogens. Physiol Res. 2010;59(5):651-64
  71. 71. Kalaiselvan V, Kalaivani M, Vijayakumar A, Sureshkumar K, Venkateskumar K. Current knowledge and future direction of research on soy isoflavones as a therapeutic agents. Pharmacogn Rev. 2010;4(8):111-7. DOI: 10.4103/0973‐7847.70900
  72. 72. Hirose A, Terauchi M, Akiyoshi M, Owa Y, Kato K, Kubota T. Low‐dose isoflavone aglycone alleviates psychological symptoms of menopause in Japanese women: a randomized, double‐blind, placebo‐controlled study. Arch Gynecol Obstet. 2016;293(3):609-15. DOI: 10.1007/s00404‐015‐3849‐0
  73. 73. Möller FJ, Diel P, Zierau O, Hertrampf T, Maass J, Vollmer G. Long‐term dietary isoflavone exposure enhances estrogen sensitivity of rat uterine responsiveness mediated through estrogen receptor alpha. Toxicol Lett. 2010;196(3):142-53. DOI: 10.1016/j.toxlet.2010.03.1117
  74. 74. 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 antagonist depending on the estrogen concentration. J Steroid Biochem Mol Biol. 2006;101(4-5):246-53
  75. 75. Gencel VB, Benjamin MM, Bahou SN, Khalil RA. Vascular effects of phytoestrogens and alternative menopausal hormone therapy in cardiovascular disease. Mini Rev Med Chem. 2012;12(2):149-74
  76. 76. Lipovac M, Chedraui P, Gruenhut C, Gocan A, Kurz C, Neuber B, Imhof M. The effect of red clover isoflavone supplementation over vasomotor and menopausal symptoms in postmenopausal women. Gynecol Endocrinol. 2012;28(3):203-7. DOI: 10.3109/09513590.2011.593671
  77. 77. Huang C, Pang D, Luo Q, Chen X, Gao Q, Shi L, Liu W, Zou Y, Li L, Chen Z. Soy isoflavones regulate lipid metabolism through an AKT/mTORC1 pathway in diet‐induced obesity (DIO) male rats. Molecules. 2016;21(5). pii: E586. DOI: 10.3390/molecules21050586
  78. 78. van Bree BW, Lenaers E, Nabben M, Briedé JJ, Jörgensen JA, Schaart G, Schrauwen P, Hoeks J, Hesselink MK. A genistein‐enriched diet neither improves skeletal muscle oxidative capacity nor prevents the transition towards advanced insulin resistance in ZDF rats. Sci Rep. 2016;6:22854. DOI: 10.1038/srep22854
  79. 79. Zang Y, Igarashi K, Yu C. Anti‐obese and anti‐diabetic effects of a mixture of daidzin and glycitin on C57BL/6J mice fed with a high‐fat diet. Biosci Biotechnol Biochem. 2015;79(1):117-23.DOI: 10.1080/09168451.2014.955453
  80. 80. Nanri A, Mizoue T, Takahashi Y, Kirii K, Inoue M, Noda M, Tsugane S. Soy product and isoflavone intakes are associated with a lower risk of type 2 diabetes in overweight Japanese women. J Nutr. 2010;140(3):580-6. DOI: 10.3945/jn.109.116020
  81. 81. Ko KP, Kim CS, Ahn Y, Park SJ, Kim YJ, Park JK, Lim YK, Yoo KY, Kim SS. Plasma isoflavone concentration is associated with decreased risk of type 2 diabetes in Korean women but not men: results from the Korean genome and epidemiology study. Diabetologia. 2015;58(4):726-35. DOI: 10.1007/s00125‐014‐3463‐x
  82. 82. Simperova A, Al‐Nakkash L, Faust JJ, Sweazea KL. Genistein supplementation prevents weight gain but promotes oxidative stress and inflammation in the vasculature of female obese ob/ob mice. Nutr Res. 2016;36(8):789-97. DOI: 10.1016/j.nutres.2016.03.011
  83. 83. Peng N, Prasain JK, Dai Y, Moore R, Arabshahi A, Barnes S, Carlson S, Wyss JM. Chronic dietary kudzu isoflavones improve components of metabolic syndrome in stroke‐prone spontaneously hypertensive rats. J Agric Food Chem. 2009;57(16):7268-73. DOI: 10.1021/jf901169y
  84. 84. Michelin RM, Al‐Nakkash L, Broderick TL, Plochocki JH. Genistein treatment increases bone mass in obese, hyperglycemic mice. Diabetes Metab Syndr Obes. 2016;9:63-70. DOI: 10.2147/DMSO.S97600
  85. 85. Kaczmarczyk‐Sedlak I, Wojnar W, Zych M, Ozimina‐Kamińska E, Taranowicz J, Siwek A. Effect of formononetin on mechanical properties and chemical composition of bones in rats with ovariectomy‐induced osteoporosis. Evid Based Complement Alternat Med. 2013;2013:457052. DOI: 10.1155/2013/457052
  86. 86. Wei P, Liu M, Chen Y, Chen DC. Systematic review of soy isoflavone supplements on osteoporosis in women. Asian Pac J Trop Med. 2012;5(3):243-8. DOI: 10.1016/S1995‐7645(12)60033‐9
  87. 87. Levis S, Strickman‐Stein N, Ganjei‐Azar P, Xu P, Doerge DR, Krischer J. Soy isoflavones in the prevention of menopausal bone loss and menopausal symptoms: a randomized, double‐blind trial. Arch Intern Med. 2011;171(15):1363-9. DOI: 10.1001/archinternmed.2011.330
  88. 88. Yang XP, Reckelhoff JF. Estrogen, hormonal replacement therapy and cardiovascular disease. Curr Opin Nephrol Hypertens. 2011;20(2):133-8. DOI: 10.1097/MNH.0b013e3283431921
  89. 89. Li SH, Liu XX, Bai YY, Wang XJ, Sun K, Chen JZ, Hui RT. Effect of oral isoflavone supplementation on vascular endothelial function in postmenopausal women: a meta‐analysis of randomized placebo‐controlled trials. Am J Clin Nutr. 2010;91(2):480-6. DOI: 10.3945/ajcn.2009.28203
  90. 90. Goodman‐Gruen D, Kritz‐Silverstein D. Usual dietary isoflavone intake is associated with cardiovascular disease risk factors in postmenopausal women. J Nutr. 2001;131(4):1202-6
  91. 91. Yu D, Shu XO, Li H, Yang G, Cai Q, Xiang YB, Ji BT, Franke AA, Gao YT, Zheng W, Zhang X. Dietary isoflavones, urinary isoflavonoids, and risk of ischemic stroke in women. Am J Clin Nutr. 2015;102(3):680-6. DOI: 10.3945/ajcn.115.111591
  92. 92. Yu J, Bi X, Yu B, Chen D. Isoflavones: anti‐inflammatory benefit and possible caveats. Nutrients. 2016;8(6). pii: E361. DOI: 10.3390/nu8060361
  93. 93. Chen W, Ma X, Lin Y, Xiong Y, Zheng C, Hu Y, Yu D, Jiang Z. Dietary supplementation with a high dose of daidzein enhances the antioxidant capacity in swine muscle but experts pro‐oxidant function in liver and fat tissues. J Anim Sci Biotechnol. 2016;7:43. DOI: 10.1186/s40104‐016‐0102‐z
  94. 94. Jalili M, Hekmatdoost A, Vahedi H, Poustchi H, Khademi B, Saadi M, Zemestani M, Janani L. Co‐administration of soy isoflavones and Vitamin D in management of irritable bowel disease. PLoS One. 2016;11(8):e0158545. DOI: 10.1371/journal.pone.0158545
  95. 95. Cui Y, Niu K, Huang C, Momma H, Guan L, Kobayashi Y, Guo H, Chujo M, Otomo A, Nagatomi R. Relationship between daily isoflavone intake and sleep in Japanese adults: a cross‐sectional study. Nutr J. 2015;14:127. DOI: 10.1186/s12937‐015‐0117‐x
  96. 96. Polito F, Marini H, Bitto A, Irrera N, Vaccaro M, Adamo EB, Micali A, Squadrito F, Minutoli L, Altavilla D. Genistein aglycone, a soy‐derived isoflavone, improves skin changes induced by ovariectomy in rats. Br J Pharmacol. 2012;165(4):994-1005. DOI: 10.1111/j.1476‐5381.2011.01619.x
  97. 97. Hanski L, Genina N, Uvell H, Malinovskaja K, Gylfe Å, Laaksonen T, Kolakovic R, Mäkilä E, Salonen J, Hirvonen J, Elofsson M, Sandler N, Vuorela PM. Inhibitory activity of the isoflavone biochanin A on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation. PLoS One. 2014;9(12):e115115. DOI: 10.1371/journal.pone.0115115
  98. 98. Stantchev TS, Markovic I, Telford WG, Clouse KA, Broder CC. The tyrosine kinase inhibitor genistein blocks HIV‐1 infection in primary human macrophages. Virus Res. 2007;123(2):178-89
  99. 99. Nabavi SF, Daglia M, Tundis R, Loizzo MR, Sobarzo‐Sanchez E, Orhan IE, et al. Genistein: a boon for mitigating ischemic stroke. Curr Top Med Chem. 2015;15(17): 1714-21
  100. 100. Dong X, Xu W, Sikes RA, Wu C. Combination of low dose of genistein and daidzein has synergistic preventive effects on isogenic human prostate cancer cells when compared with individual soy isoflavone. Food Chem. 2013;141(3):1923-33. DOI: 10.1016/j.foodchem.2013.04.109
  101. 101. Yu D, Shin HS, Lee YS, Lee D, Kim S, Lee YC. Genistein attenuates cancer stem cell characteristics in gastric cancer through the downregulation of Gli1. Oncol Rep. 2014;31(2):673-8. DOI: 10.3892/or.2013.2893
  102. 102. Brownlow B, Nagaraj VJ, Nayel A, Joshi M, Elbayoumi T. Development and in vitro evaluation of vitamin E‐enriched nanoemulsion vehicles loaded with genistein for chemoprevention against UVB‐induced skin damage. J Pharm Sci. 2015;104(10):3510-23. DOI: 10.1002/jps.24547
  103. 103. Lee DE, Lee KW, Byun S, Jung SK, Song N, Lim SH, et al. 7,3′,4′‐Trihydroxyisoflavone, a metabolite of the soy isoflavone daidzein, suppresses ultraviolet B‐induced skin cancer by targeting Cot and MKK4. J Biol Chem. 2011;286(16):14246-56. DOI: 10.1074/jbc.M110.147348
  104. 104. Ahn JC, Biswas R, Chung PS. Combination with genistein enhances the efficacy of photodynamic therapy against human anaplastic thyroid cancer cells. Lasers Surg Med. 2012;44(10):840-9. DOI: 10.1002/lsm.22095
  105. 105. Xiong P, Wang R, Zhang X, DeLa Torre E, Leon F, Zhang Q, et al. Design, synthesis, and evaluation of genistein analogues as anti‐cancer agents. Anticancer Agents Med Chem. 2015;15(9):1197-203
  106. 106. Wiegand H, Wagner AE, Boesch‐Saadatmandi C, Kruse HP, Kulling S, Rimbach G. Effect of dietary genistein on phase II and antioxidant enzymes in rat liver. Cancer Genomics Proteomics. 2009;6(2):85-92
  107. 107. Zampieri AL, Ferreira FS, Resende EC, Gaeti MP, Diniz DG, Taveira SF, et al. Biodegradable polymeric nanocapsules based on poly(DL‐lactide) for genistein topical delivery: obtention, characterization and skin permeation studies. J Biomed Nanotechnol. 2013;9(3):527-34
  108. 108. Ullah MF, Ahmad A, Bhat SH, Khan HY, Zubair H, Sarkar FH, et al. Simulating hypoxia‐induced acidic environment in cancer cells facilitates mobilization and redox‐cycling of genomic copper by daidzein leading to pro‐oxidant cell death: implications for the sensitization of resistant hypoxic cancer cells to therapeutic challenges. Biometals. 2016;29(2):299-310. DOI: 10.1007/s10534‐016‐9916‐6
  109. 109. Kim SH, Kim CW, Jeon SY, Go RE, Hwang KA, Choi KC. Chemopreventive and chemotherapeutic effects of genistein, a soy isoflavone, upon cancer development and progression in preclinical animal models. Lab Anim Res. 2014;30(4):143-50. DOI: 10.5625/lar.2014.30.4.143
  110. 110. Liu Y, Santillo MF, Flynn TJ, Ferguson MS. Sex hormone modulation of both induction and inhibition of CYP1A by genistein in HepG2/C3A cells. In Vitro Cell Dev Biol Anim. 2015;51(4):426-31. DOI: 10.1007/s11626‐014‐9848‐9
  111. 111. Zhang Y, Chen H. Genistein, an epigenome modifier during cancer prevention. Epigenetics. 2011;6(7):888-91
  112. 112. Sakla MS, Shenouda NS, Ansell PJ, Macdonald RS, Lubahn DB. Genistein affects HER2 protein concentration, activation, and promoter regulation in BT‐474 human breast cancer cells. Endocrine. 2007;32(1):69-78. DOI: 10.1007/s12020‐007‐9006‐1
  113. 113. Zhang Z, Wang CZ, Du GJ, Qi LW, Calway T, He TC, et al. Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53‐dependent pathway in human colon cancer cells. Int J Oncol. 2013;43(1):289-96. DOI: 10.3892/ijo.2013.1946
  114. 114. Li HQ, Luo Y, Qiao CH. The mechanisms of anticancer agents by genistein and synthetic derivatives of isoflavone. Mini Rev Med Chem. 2012;12(4):350-62
  115. 115. Chen J, Duan Y, Zhang X, Ye Y, Ge B, Chen J. Genistein induces apoptosis by the inactivation of the IGF‐1R/p‐Akt signaling pathway in MCF‐7 human breast cancer cells. Food Funct. 2015;6(3):995-1000. DOI: 10.1039/c4fo01141d
  116. 116. Xu L, Xiang J, Shen J, Zou X, Zhai S, Yin Y, et al. Oncogenic MicroRNA‐27a is a target for genistein in ovarian cancer cells. Anticancer Agents Med Chem. 2013;13(7):1126-32
  117. 117. Froyen EB, Steinberg FM. Genistein decreases basal hepatic cytochrome P450 1A1 protein expression and activity in Swiss Webster mice. Nutr Res. 2016;36(5):430-9. DOI: 10.1016/j.nutres.2016.01.001
  118. 118. Lepri SR, Zanelatto LC, da Silva PB, Sartori D, Ribeiro LR, Mantovani MS. Effects of genistein and daidzein on cell proliferation kinetics in HT29 colon cancer cells: the expression of CTNNBIP1 (beta‐catenin), APC (adenomatous polyposis coli) and BIRC5 (survivin). Hum Cell. 2014;27(2):78-84. DOI: 10.1007/s13577‐012‐0051‐6
  119. 119. Clubbs EA, Bomser JA. Glycitein activates extracellular signal‐regulated kinase via vascular endothelial growth factor receptor signaling in nontumorigenic (RWPE‐1) prostate epithelial cells. J Nutr Biochem. 2007;18(8):525-32. DOI: 10.1016/j.jnutbio.2006.09.005
  120. 120. Clubbs EA, Bomser JA. Basal cell induced differentiation of noncancerous prostate epithelial cells (RWPE‐1) by glycitein. Nutr Cancer. 2009;61(3):390-6. DOI: 10.1080/01635580802582728
  121. 121. Wang J, Jenkins S, Lamartiniere CA. Cell proliferation and apoptosis in rat mammary glands following combinational exposure to bisphenol A and genistein. BMC Cancer. 2014;14:379. DOI: 10.1186/1471‐2407‐14‐379
  122. 122. Hu XJ, Xie MY, Kluxen FM, Diel P. Genistein modulates the anti‐tumor activity of cisplatin in MCF‐7 breast and HT‐29 colon cancer cells. Arch Toxicol. 2014;88(3):625-35. DOI: 10.1007/s00204‐013‐1184‐4
  123. 123. Tonetti DA, Zhang Y, Zhao H, Lim SB, Constantinou AI. The effect of the phytoestrogens genistein, daidzein, and equol on the growth of tamoxifen‐resistant T47D/PKC alpha. Nutr Cancer. 2007;58(2):222-9. DOI: 10.1080/01635580701328545
  124. 124. Khan AQ, Khan R, Rehman MU, Lateef A, Tahir M, Ali F, et al. Soy isoflavones (daidzein & genistein) inhibit 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced cutaneous inflammation via modulation of COX‐2 and NF‐kappaB in Swiss albino mice. Toxicology. 2012;302(2-3):266-74. DOI: 10.1016/j.tox.2012.08.008
  125. 125. Rahman Mazumder MA, Hongsprabhas P. Genistein as antioxidant and antibrowning agents in in vivo and in vitro: a review. Biomed Pharmacother. 2016;82:379-92. DOI: 10.1016/j.biopha.2016.05.023
  126. 126. Ghaemi A, Soleimanjahi H, Razeghi S, Gorji A, Tabaraei A, Moradi A, et al. Genistein induces a protective immunomodulatory effect in a mouse model of cervical cancer. Iran J Immunol. 2012;9(2):119-27. DOI: IJIv9i2A5
  127. 127. Jiang W, Hu M. Mutual interactions between flavonoids and enzymatic and transporter elements responsible for flavonoid disposition via phase II metabolic pathways. RSC Adv. 2012;2(21):7948-63. DOI: 10.1039/C2RA01369J
  128. 128. Poluzzi E, Piccinni C, Raschi E, Rampa A, Recanatini M, Ponti FD. Phytoestrogens in postmenopause: the state of the art from a chemical, pharmacological and regulatory perspective. Curr Med Chem. 2014;21(4):417-36. DOI: 10.2174/09298673113206660297
  129. 129. Taylor CK, Levy RM, Elliott JC, Burnett BP. The effect of genistein aglycone on cancer and cancer risk: a review of in vitro, preclinical, and clinical studies. Nutr Rev. 2009;67(7):398-415. DOI: 10.1111/j.1753‐4887.2009.00213.x
  130. 130. Liao CY, Lee CC, Tsai C, Hsueh CW, Wang CC, Chen IH, et al. Novel Investigations of flavonoids as chemopreventive agents for hepatocellular carcinoma. BioMed Research International. 2015;2015:e840542. DOI: org/10.1155/2015/840542
  131. 131. Polkowski K, Mazurek AP. Biological properties of genistein. A review of in vitro and in vivo data. Acta Pol Pharm. Mar-Apr 2000;57(2):135-55
  132. 132. Peterson G. Evaluation of the biochemical targets of genistein in tumor cells. J Nutr. 1995;125(3 Suppl):784S–9
  133. 133. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140(6):883-99. DOI: 10.1016/j.cell.2010.01.025
  134. 134. Pfarr K, Danciu C, Arlt O, Neske C, Dehelean C, Pfeilschifter JM, et al. Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin. PLoS One. 2015;10(3):e0118802. DOI: 10.1371/journal.pone.0118802. eCollection 2015
  135. 135. Danciu C, Caraba A, Bojin F, Soica C, Simu G, Ciurlea S,Peev C, Citu IM, Panzaru I. Genistein does not induce caspase 2 activation in vitro on B16 melanoma cell lines. Farmacia. 2014;62(4):753-60
  136. 136. Danciu C, Soica C, Oltean M, Avram S, Borcan F, Csanyi E, et al. Genistein in 1:1 inclusion complexes with ramified cyclodextrins: theoretical, physicochemical and biological evaluation. Int J Mol Sci. 2014;15(2):1962-82. DOI: 10.3390/ijms15021962
  137. 137. Danciu C, Borcan F, Soica C, Zupko I, Csányi E, Ambrus R, et al. Polyurethane microstructures‐‐a good or bad in vitro partner for the isoflavone genistein? Nat Prod Commun. 2015;10(6):951-4
  138. 138. Danciu C, Soica C, Dehelean C, Zupko I, Csanyi E, Pinzaru I, et al. Preliminary in vitro evaluation of genistein chemopreventive capacity as a result of esterification and cyclodextrin encapsulation. Anal Cell Pathol. 2015;2015(2015):e262930. DOI: org/10.1155/2015/262930
  139. 139. Loftsson T, Brewster ME. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J Pharm Sci. 1996;85(10):1017-25
  140. 140. Danciu C, Borcan F, Bojin F, Zupko I, Dehelean C. Effect of the isoflavone genistein on tumor size, metastasis potential and melanization in a B16 mouse model of murine melanoma. Nat Prod Commun. 2013;8(3):343-6
  141. 141. Danciu C, Soica C, Csanyi E, Ambrus R, Feflea S, Peev C, et al. Changes in the anti‐inflammatory activity of soy isoflavonoid genistein versus genistein incorporated in two types of cyclodextrin derivatives. Chem Cent J. 2012;6(1):58. DOI: 10.1186/1752‐153X‐6‐58
  142. 142. Danciu C, Berkó S, Varju G, Balázs B, Kemény L, Németh IB, et al. The effect of electroporation of a lyotropic liquid crystal genistein‐based formulation in the recovery of murine melanoma lesions. Int J Mol Sci. 2015;16(7):15425-41. DOI: 10.3390/ijms160715425
  143. 143. Danciu C, Biris M, Boglárka B, Csanyi E, Pavel IZ, Pop G, Soica C, Ceuta L, Nita L, Morgovan C,Stoian D.Pro‐apoptotic effect of soy total extract incorporated in lyotropic liquid crystals formulation. Revista de Chimie. 2015;66(7):1038-41
  144. 144. Balázs B, Sipos P, Danciu C, Avram S, Soica C, Dehelean C, Varju G, Eros E, Szucs MB, Berkó S, and Csányi E. ATR‐FTIR and Raman spectroscopic investigation of the electroporation‐mediated transdermal delivery of a nanocarrier system containing an antitumor drug. Biomed Opt Express. 2016;7(1):67-78. DOI: 10.1364/BOE.7.000067

Written By

Corina Danciu, Suciu Oana, Diana Simona Antal, Florina Ardelean, Aimée Rodica Chiş, Codruţa Şoica, Florina Andrica, Cristina Dehelean and Vlaicu Brigitha

Submitted: 03 February 2017 Reviewed: 15 February 2017 Published: 23 August 2017