Transformation of Daidzein to Equol and Its Bioactivity - ACS

Chapter 8

Transformation of Daidzein to Equol and Its Bioactivity 1

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Mariko Uehara , Yoshiko Ishimi , Shin-ichi Katsumata , and Kazuharu Suzuki Downloaded by FUDAN UNIV on March 30, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch008

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Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan Project for Bio-index, National Institute of Health and Nutrition, Tokyo 162-8636, Japan 2

The structures of soybean isoflavones are similar to that of estrogen and have received attention as alternatives to hormone replacement therapy. Daidzein that is a major isoflavone found in soybean is metabolized to equol by intestinal microflora, and the metabolite exhibits a stronger estrogenic activity than daidzein. Recent studies suggest that the clinical effectiveness of isoflavones might be due to their ability to produce equol in the intestine. Although typical laboratory animal species consistently produce high levels of equol, only 30-50% of the human population can produce equol. Fructooligosaccharides, indigestible sugars, increased the bioavailability of daidzein and enhanced the transformation of daidzein to equol by stimulating intestinal microflora. In particular, equol may have indirect and direct effects on bone loss in rodents, osteoporotic models, and osteopenia. Possible bioactivity of equol based on its estrogenic properties with regard to bone metabolism and taking into consideration the status of intestinal microflora is discussed.

Several plant constituents have estrogen-like physiological effects. These constituents are called phytoestrogens (1, 2). Isoflavones (genistein and daidzein) in soybeans and lignans (enterolactone and enterodiol) that are formed in the intestine from their precursors in various grains, seeds, fruits, some vegetables, and tea are commonly known as phytoestrogens (3, 4). Dietary © 2008 American Chemical Society

Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by FUDAN UNIV on March 30, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch008

82 phytoestrogens may prevent sex hormone-related diseases, such as breast cancer and prostate cancer, through mechanisms that have not yet been adequately documented (3, 5, 6). On the other hand, they may also prevent postmenopausal osteoporosis through an estrogenic-like effect. For instance, the intake of soy products, genistein, daidzein, or other phytoestrogens has been found to prevent postovariectomized bone loss in rats and mice (7, 8), and post- or perimenopausal women (9-11). Almost all phytoestrogens, such as genistein and daidzein, in food exist as glycosides; thus, to facilitate the intestinal absorption and the physiological effect of these glycosides, it is necessary to hydrolyze the glycosidic bonds (1-3). These bonds are hydrolyzed by the glucosidases of intestinal bacteria such as lactobacilli, bacteroides, and bifidobacteria (12, 13). Recently it has been determined that the metabolites of isoflavones are more important than their precursors (14). The soybean isoflavone daidzein is metabolized to equol in the gastrointestinal tract by gut microflora (14, 15). Equol possesses a stronger affinity for estrogen receptors than daidzein (16) and has the strongest transcriptional activity among soybean isoflavonoids in yeast 2-hybrid systems (17). Furthermore, equol is a chiral molecule, which can exist as the enantiomers #(+)-equol and S(-)-equol. Setchell et al. (18) established S-equol as the exclusive product of human gut microfloral synthesis from daidzein and also showed that both enantiomers were bioavailable. Thus, equol is a biologically active metabolite of daidzein. Recent studies suggest that the clinical effectiveness of isoflavones might be due to their ability to produce equol in the intestine (14). Maximal responses to isoflavone intake are observed in individuals who are effective producers of equol. Although typical laboratory animal species consistently produce high levels of equol, only 30-50% of the population can produce equol (14, 15, 19). It is important to clarify the key factors involved in the transformation of daidzein into equol and the bioactivity of equol.

Equol Has a Direct Beneficial Effect on Bone Loss Due to Ovariectomy To determine a direct effect of equol on bone loss, eight-week-old female mice were assigned to the following five groups: sham-operation (sham), ovariectomy (OVX), OVX + 0.1 mg/day equol (0.1 Eq), OVX + 0.5 mg/day equol (0.5 Eq), and OVX + 0.03 mg/day 17(3-estradiol (E ) (20). Equol and E were subcutaneously administered by using a mini-osmotic pump. Four weeks after intervention, uterine weight was reduced by OVX and restored by E administration. In contrast, equol at doses used in this study did not affect 2

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83 uterine atrophy in OVX mice. Bone mineral density (BMD) of the whole body measured by a PIXImus densitometer was reduced in OVX mice, while it was maintained by the administration of 0.5 mg/day equol as well as E . The BMD of the femur and lumbar spine was also reduced by OVX, and treatment with 0.5 mg/day equol prevented bone loss in dual-energy X-ray absorptiometry (DXA). In particular, the BMD of the proximal femur was the same as that of the proximal femurs of the mice in the sham group. E prevented OVX-induced bone loss from all regions. These results suggest that equol inhibits bone loss apparently without estrogenic activity in the reproductive organs of OVX mice. 2


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Antibiotics Decrease Equol Production from Daidzein The efficacy of daidzein in inhibiting bone loss was examined. The effect of antibiotics on equol production and femoral BMD was determined in OVX mice that were administered daidzein (aglycone). Eight-week-old female mice were assigned to the following seven diet groups: sham, OVX, OVX + 0.3% kanamycin (KN), OVX + 0.1% daidzein (Dz), OVX + 0.04% KN + Dz (0.04 KN), OVX + 0.08% K N + Dz (0.08 KN), and OVX + 0.3% K N + Dz (0.3 KN). Four weeks after treatment, kanamycin decreased the equol concentration in the serum (Table I). Uterine weight was reduced by OVX and the reduced weight was unchanged by kanamycin or daidzein administration. Proximal and distal femur BMD was reduced in OVX mice, while it was maintained by the administration of 0.1% daidzein (Table I). However, kanamycin destroyed the preventive effect of daidzein on bone loss induced by OVX. These results suggest that it might be important to promote or activate intestinal microflora, which produce equol, to obtain the maximal effects of isoflavones on the prevention of bone loss in an estrogen-deficient status.

The Prebiotics Fructooligosaccharides Modify Isoflavone Metabolism Fructooligosaccharides (FOS)—a mixture of indigestible and fermentable sugars—stimulate the growth of bifidobacteria in the intestine (21-24). It was postulated that dietary FOS may affect the bioavailability of isoflavone glycoside conjugates and therefore improve their absorption in the intestine. The kinetics of isoflavones in rats fed a 5% FOS-supplemented diet or a control diet were examined by measuring genistein and daidzein concentrations in blood collected from three different veins and by measuring urinary excretion at 24-



ND

+KN0.3

b

113.2 ± 22.2

92.4 ± 9.5

+Dz+KN0.08

+Dz+KN0.3

30.3 ± 0 . 5 9 a,b 31.2 ± 0 . 4 1 a 31.7 ± 0 . 5 4 a 30.5 ± 0 . 6 1 a,b 30.4 ± 1.00 a,b 28.8 ± 0 . 5 b

39.5 ± 0.75 a 36.9 ± 0.76 b 37.3 ± 1.05 b 39.8 ± 1.06 a 35.8 ± 0.64 b 35.1 ± 0.97 b 36.6 ± 0.59 b

Proximal

2

B M D (mg/cm ) Middle 31.9 ± 0 . 4 6 a

b,c 35.2 ± 0.45

35.3 ± 0.69 b,c 33.7 ± 0.69 c

36.7 ± 0.67 a 35.3 ± 0.53 b,c

b,c

a

34.1 ± 1.05

38.6 ± 0.41

Distal

Values are Means ± S E M , n=6. sham: sham operated, O V X : ovariectomized, +KN0.3: OVX+0.3%kanamycin, +Dz: OVX+0.1% daidzein, OVX+Dz+0.04%kanamycin, +Dz+KN0.08: OVX+Dz+0.08%kanamycin, +Dz+KN0.3: OVX+Dz+0.3%ka Means with different letters differ, P < 0.05.

b

b

111.7 ± 50.2

a

+Dz+KN0.04

3530.1 ± 790.0

ND

OVX

+Dz

ND

Sham

Serum equol (nmol/L)

Table I. Serum Equol Concentration and Femoral Bone Mineral Density (BMD)



85 and 48-h after a single intragastric administration of isoflavone glycoside conjugates (lOOmg mixture of daidzin, genistin and glycitin/kg body weight (BW)). The genistein concentration in the portal blood increased rapidly, reaching a peak in both the FOS-fed and control groups at 1 h after administration. The concentrations in the central venous blood were approximately half of those in the portal blood. In the FOS-fed group, both genistein and daidzein remained detectable in the tail venous blood after 24 to 48 h. The urinary excretion of both isoflavones at 24- and 48-h after administration was significantly higher in the FOS-fed group than in the control group. In a similar study, the equol concentration started to increase in the central venous blood at 12 h after the administration of isoflavone glycoside conjugates (lOOmg/kg BW) with FOS feeding and was significantly higher in the FOS-fed group than in the control group at 48 and 72 h (Table II). Thus, FOS modified the absorption and enterohepatic recirculation of isoflavones and enhanced equol production from daidzein. Furthermore, in sham-operated and OVX mice, FOS increased the activity of P-glucosidase in cecum contents to hydrolyze the glycosidic bonds of isoflavone conjugates and stimulated the transformation of daidzein to equol in the administered isoflavone conjugates. This combination of isoflavones and FOS feeding inhibited bone loss induced by OVX. The other synergistic effects of dietary isoflavones and FOS supplementation on postgastrectomy (GX) osteopenia were also determined. Decreases in femoral trabecular BMD and bone breaking force by GX were inhibited in rats fed FOS with and without isoflavone diets, whereas supplementation with only isoflavone did not prevent bone loss in gastrectomized rats. Notably, a combination of isoflavones and FOS diet enhanced the production of equol from daidzein in gastrectomized rats.

Changes in Intestinal Microbiota on Administration of FOS and Isoflavones Determined by Terminal Restriction Fragment Length Polymorphism Analysis To determine changes in intestinal microbiota produced by the administration of FOS and isoflavones, terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes was used (25). Fecal samples were collected between 0 and 24 h, 24 and 48 h, and 48 and 72 h after a single intragastric administration of isoflavone conjugates. Clone libraries of 16S rDNA were constructed and the predominant terminal restriction fragments were identified by comparing T-RFLP patterns in the fecal community with those of corresponding 16S rDNA clones. Sequence analysis indicated that despite isoflavone administration, FOS-fed rats were colonized mainly by members of Bifidobacterium and Lactobacillus. The results of real-



198.9 ± 55.4

Control

FOS

214.8 ± 41.5

3h 144.7 ± 17.5 1048.5 ± 648.5

371.4 ± 67.2

2361.0 ± 966.5

Serum equol (nmol/L) 12h 24h 277.8 ± 75.5 1185.3 ± 280.8

6h 282.2 ± 59.1

* Significantly differentfromthe control at a particular time point, P < 0.05.

Values are Means ± SEM, n=5-7.

lh 271.8 ± 49.6

72h 507.6 ± 0.4 2172.6 ± 494.0 * 1211.0 ± 1.1 *

48h 861.5 ± 81.1

Table II. Time Course of Changes of Equol Concentrations in Central Venous Serum in Rats Fed the Control Diet of the 5% Fructooligosaccharide (FOS) Diet after a Single Dose of Isoflavone Glycoside Conjugates



87 time polymerase chain reaction (PCR) indicated that Lactobacillus was classified as L. murinus or L. animalis. Bifidobacterium is one of species, which can produce equol. However, identifying equol-producing bacteria is complicated. It is known that equol is produced from daidzein through its intermediate metabolite dihydrodaidzein (26). Only one lactic acid bacterium (Lactococcus garvieae) has yet been identified that can produce equol directly from daidzein (27). From a human fecal sample, Decroos et al have isolated a stable mixed microbial culture comprising four species (Lactobacillus mucosae, Enterococcus faecium, Finegoldia magna, and Veillonella sp) that is capable of transforming daidzein into equol and have examined the influence of some environmental conditions in the colon on equol production (28). They suggested that hydrogen in particular but also propionate and butyrate influenced equol production in a positive manner because hydrogen gas probably acts as an electron donor in the biotransformation, and short chain fatty acids are related to the production of hydrogen gas (29). FOS increase hydrogen gas, propionate, and butyrate in vivo, but in an in vitro study, FOS inhibited equol production. Further studies should be conducted to clarify this discrepancy between the results of the in vivo and in vitro studies.

Conclusions It is important to promote or activate intestinal microbiota for the transformation of daidzein to equol. FOS might be candidate ingredients for enhancing equol production, but further studies should be conducted to clarify the discrepancy between the results of in vivo and in vitro studies. Furthermore, in a human study involving French postmenopausal women, FOS did not increase urinary equol production (28). However, racial differences might exist with regard to isoflavone metabolism. Further human studies involving Asian subjects are required.

Acknowledgment We would like to thank Dr. Herman Adlercreutz, Dr. Atsutane Ohta and Dr. Kazuki Kanazawa for constructive discussions and valuable suggestions about phytoestrogens and FOS. Dr. Hidetoshi Kubota, Mr. Hiroya Endo and Ms. Yuko Baba from Meiji Seika Kaisha Ltd. are also acknowledged for the technical support of T-RFLP.


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Transformation of Daidzein to Equol and Its Bioactivity - ACS

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