**5. Dietary intake and sources of isoflavones**

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].

The isoflavonoid aglycones such as genistein and daidzein are compounds with a low polar‐ ity 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

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 quanti‐ fication 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

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 kid‐ neys 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 glyco‐ sides are hydrolyzed by intestinal glucosidases, which partially release the aglycones daidzein,

**3. Isolation**

260 Flavonoids - From Biosynthesis to Human Health

prior to SPE [22].

cleaning‐up step on Amberlite XAD‐7 material [21].

**4. Bioavailability and metabolism**

As already mentioned above, isoflavones are flavonoid compounds that are biogenetically produced by plants belonging mainly to the Fabaceae family. Main sources of dietary iso‐ flavones 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*‐glu‐ cosides: 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*‐malo‐

nyl‐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].

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 cul‐ tures 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 prod‐ ucts) 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].


**Figure 3.** Structure of main isoflavone glycosides.

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].

nyl‐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

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

In China, the first reference to soybeans dates from 2853 BC [45]. In contrast, Western cul‐ tures 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 prod‐ ucts) 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

and 51.8% [44].

red clover and only three in soybeans [15].

262 Flavonoids - From Biosynthesis to Human Health

**Figure 3.** Structure of main isoflavone glycosides.

because of a higher bioavailability of isoflavone aglycones [47].

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**.


**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**

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

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

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

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)

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

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].

are highly appreciated and, in Indonesia, tempeh is the most popular soy food [45].

content to be lost during processing [27].

264 Flavonoids - From Biosynthesis to Human Health

attributable to tofu, natto and miso [49].

for daidzein in British men [55].

**5.2. Soy foods in Europe**

isoflavone intake [56].

**5.1. Soy foods in Asia**

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 daid‐ zein 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 con‐ sumption 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,


**Table 2.** Annual per capita consumption of soybeans (2001).

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 symp‐ toms, improvement in bone health and reduced risk of certain types of cancers) [61].
