Special Article. Soybean Foods and Their Benefits: Potential Mechanisms of Action. Adetayo O. Omoni, MSc, and Rotimi E. Aluko, PhD

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1 Special Article August 2005: Soybean Foods and Their Benefits: Potential Mechanisms of Action Adetayo O. Omoni, MSc, and Rotimi E. Aluko, PhD Isoflavones have been proposed to be the active component responsible for the beneficial effects of soybean foods, and appear to work in conjunction with the proteins to protect against cancer, cardiovascular disease, and osteoporosis. Most of the research activities on the benefits of soybean foods have focused on the role these isoflavones play in disease prevention or treatment; however, there is also some evidence that the benefits are attributable to certain peptides or protein fractions from soybeans. This review will focus on some of the potential mechanisms whereby soybeans exert their protective effects against heart disease, cancer, and osteoporosis. Key words: soybean, isoflavones, peptides, cardiovascular disease, cancer 2005 International Life Sciences Institute doi: /nr.2005.aug INTRODUCTION Soybean foods have generated a lot of interest recently as a result of evidence that populations consuming large amounts of soybeans have a lower risk of some chronic diseases, most notably heart disease and cancer. 1-3 Soybean (Glycine max) is an ancient legume that is traditionally used to prepare both fermented and nonfermented foods, and is a staple among Asian populations. Soybeans are extremely versatile and can be made into a variety of foods. Asians consume an average of 20 to 80 g of traditional soy foods daily, the most common of which are tofu, miso, and tempeh. 4,5 Americans consume much less soy, only about 1 to 3 g daily, 5,6 and this is mostly in processed forms such as soy drinks, breakfast cereals, energy bars, and soy burgers. Ms. Omoni and Dr. Aluko are with the Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada. Address for correspondence: Dr. Rotimi E. Aluko, Department of Human Nutritional Sciences, University of Manitoba, H515 Duff Roblin, Winnipeg, Manitoba, Canada, R3T 2N2; Phone: ; Fax: ; alukor@cc.umanitoba.ca. The consumption of soy foods may reduce the risk of cardiovascular disease and cancer, and this effect is seen particularly among Asian populations, in whom consumption of soy foods is high, compared with Western populations, who eat smaller amounts of soy. 1-3 In addition to cancer and heart disease, data suggest that soy may also reduce the risk of osteoporosis and help to alleviate menopausal symptoms, 3,7,8 both of which are major health concerns for women. The aim of this review is to discuss current knowledge relating to the possible mechanisms by which some of the bioactive components in soybeans protect against cardiovascular diseases, cancer, and osteoporosis. BIOACTIVE COMPONENTS OF SOY AND SOY FOODS Soy foods contain an array of biologically active compounds called phytochemicals that may confer important health benefits. 9,10 These include: saponins, phytates, protease inhibitors, phenolic acids, and lecithin, all known for their anti-cancer potential; 4,6 phytosterols, which are recognized for their cholesterol-lowering effects; isoflavones, which are known for several health benefits 11 ; and omega-3 fatty acids, which are well recognized for their cardioprotective effects. Of all the bioactive components of soybeans, isoflavones have attracted the most attention. 4,12 Soy Isoflavones Soybeans, specifically the isolated soy proteins, are considered a rich source of isoflavones. 1,7,13 A 25 g portion of soy protein contains approximately 50 mg of isoflavones, although the amounts vary depending on the variety of bean and the growing conditions. 8 Asian populations consume as much as 80 mg of total isoflavones daily, while in the United States, consumption is usually not more than 5 mg/d. 14,15 Isoflavones exist in soybean and unfermented soy foods mostly as glycosides (genistin, diadzin, and, to a lesser extent, glycetin). In fermented soy foods, isoflavones are in the aglycone form (genistein, daidzein, and glycetein). 16 Genistein is 272 Nutrition Reviews, Vol. 63, No. 8

2 the most abundant isoflavone in soybean 17 and is proposed to be the most biologically active. 6 Soy isoflavones exert both estrogenic and anti-estrogenic effects, depending on the tissue in which they are acting. 12,16,18 They are structurally and functionally similar to 17 estradiol, the most potent mammalian estrogen, and are thus called phytoestrogens. 19 They also have non-hormonal effects, including signal transduction and antioxidant activity. 19 Isoflavones have been proposed to be the active component responsible for the beneficial effects of soy foods. 8,20 They appear to work in conjunction with soy protein to exert anti-carcinogenic, anti-atherogenic, and anti-osteoporotic effects, 7,21 and most of the research on the benefits of soy foods have focused on the role that they play in disease prevention or treatment. 19,22 There is also some evidence that these benefits are attributable to certain peptides or protein fractions from soybeans. Biotransformation of Isoflavones in the Intestine After ingestion, soy isoflavones are biotransformed in the intestinal tract, a process that is highly dependent on intestinal bacterial metabolism. 9,16,19,23,24 The glycosides diadzin and genistin cannot be absorbed intact into the peripheral circulation of healthy adults; they have to be changed to the aglycones genistein and diadzein via 16, 23,25 the action of intestinal -glucosidases (Figure 1), and can be further metabolized into both estrogenic and anti-estrogenic metabolites. 16 The extent to which isoflavone glycosides are bioavailable is therefore dependent on gut microflora. POTENTIAL MECHANISMS OF ACTION Soy Isoflavones and Cardiovascular Disease Of all the acclaimed benefits of soy foods, perhaps the most conclusive one is its protective effects against cardiovascular disease, which is one of the leading causes of death worldwide, 26,27 with elevated levels of plasma low-density lipoproteins (LDL) and triglycerides Figure 1. Biotransformation of soy isoflavones in the intestine. presenting a higher risk. 26 By contrast, high-density lipoproteins (HDL) are beneficial. 28 Postmenopausal women are at greater risk for cardiovascular disease because natural or surgical menopause is associated with elevated levels of circulating total and LDL cholesterol. 29 The reduction in blood total and LDL cholesterol concentrations with the consumption of products containing soy protein has been shown repeatedly in humans and several animal models (Table 1) However, the exact component responsible for this action has yet to be clearly defined. Isoflavones have been proposed to be the active ingredient responsible for the hypocholesterolemic effects of soy. 14, 33 A previous study suggested that isoflavone-rich soy protein is considerably more effective than isoflavone-depleted soy protein, though this finding is controversial. 7 The cholesterol-lowering effect is one of several proposed mechanisms by which soy reduces the risk of heart disease. 34 In a study to evaluate the effects of isoflavone-rich and isoflavone-depleted soy protein on plasma lipid concentrations in postmenopausal, moderately hypercholesterolemic women, isoflavone-rich soy protein lowered total and LDL cholesterol more than soy protein depleted of isoflavones, though no significant differences were observed in HDL cholesterol or triacylglycerol concentrations. This difference in total and LDL cholesterol lowering between the two soy protein supplements suggests an effect attributable to the isoflavone-containing fraction. 34 Similar results were observed among 18 postmenopausal women with normal and moderately elevated cholesterol levels in a randomized crossover trial to assess the hypocholesterolemic effects of soy isoflavones. Subjects were fed isolated soy protein beverage containing one of three isoflavone levels, 7.1 mg/d isoflavone (control), 65 mg/d isoflavone (low isoflavone), and 132 mg/d isoflavone (high isoflavone). The high-isoflavone diet lowered plasma LDL cholesterol by 6.5%, though there were no significant changes in total or HDL cholesterol, triacylglycerol, apolipoprotein (Apo) A I, ApoB, lipoprotein(a), or LDL peak particle diameter. Though small, a decrease of LDL cholesterol of this magnitude could be associated with a 16% reduction in cardiovascular disease risk. 26 A number of explanations have been put forward for this cholesterol-lowering effect of soy. It has been suggested that soy reduces blood cholesterol levels by reducing cholesterol and bile acid absorption from the gastrointestinal tract 35 and increasing bile acid excretion. 36 When this happens, hepatic cholesterol metabolism shifts to provide cholesterol for enhanced bile acid synthesis, and cholesterol biosynthesis and LDL receptor activity increase. The result is an increased removal of cholesterol from the blood via the LDL receptor, thereby Nutrition Reviews, Vol. 63, No

3 Table 1. Cholesterol-lowering Effects of Soy Products Reference Population (n) Soy Product Fed, Dose, Duration Outcome Human Studies Goodman-Gruen and Kritz-Silverstein, Postmenopausal women (n 208) Gardner et al., Postmenopausal, moderately hypercholesterolemic women (n 94) Wangen et al., Normocholesterolemic and mildly hypercholesterolemic women (n 18) Merz-Demlow et al., Normocholesterolemic premenopausal women (n 13) Daily soy isoflavone intake assessed for one year with questionnaire; average intake of genistein was mg/d, range mg/d Soy protein with trace amounts of isoflavones (Soy ) or 80 mg aglycone isoflavones (Soy ) per day for 12 weeks Daily consumption of soy protein isolate (SPI) beverage powders providing an average of (control), (low isoflavone), or (high isoflavone) mg isoflavones per day for three 93-day periods in a randomized crossover design SPI consumed as a beverage powder with three levels of soy isoflavones: (control), (low), and (high) mg per day for three menstrual cycles each in a randomized crossover design Women with moderate and high genistein consumption had significantly improved HDL cholesterol Total and LDL cholesterol concentrations decreased more in the Soy group than in the Soy group; no significant differences in HDL cholesterol and triacylglycerol concentrations between the groups High-isoflavone diet reduced plasma LDL cholesterol by 6.5% compared with control; low- and high-isoflavone diet reduced LDL:HDL cholesterol by 8.5% and 7.7%, respectively; isoflavone consumption did not significantly affect plasma concentrations of total or HDL cholesterol, triacylglycerol, ApoA I, ApoB, or lipoprotein (a) or the LDL peak particle diameter High-isoflavone diet lowered LDL cholesterol by 7.6% to 10%, total:hdl cholesterol by 10.2%, and LDL:HDL cholesterol by 13.8% Mitchell and Collins, Healthy men (n 10) Soy milk (1 L/d) for 4 weeks No significant effect of soy supplementation on plasma cholesterol, triglyceride levels, or HDL:LDL cholesterol ratios compared with controls Potter et al., Hypercholesterolemic, postmenopausal women (n 66) Wong et al., Normocholesterolemic (n 13) and hypercholesterolemic (n 13) men Soy protein (40 mg/d) plus moderate and higher concentrations of isoflavones (1.39 and 2.25 mg isoflavone/g protein, respectively) for 6 months Soy protein incorporated into the National Cholesterol Educational Program Step I diet (constituting 75% total protein content) fed for a total of 10 weeks in a randomized crossover design (average daily soy protein consumption approximately 50 g) Non-HDL cholesterol was reduced in both groups fed moderate and higher concentrations of isoflavone compared with controls; HDL cholesterol increased in the two groups; LDL receptor mrna was reduced in the two groups compared with controls Plasma concentration of LDL cholesterol and LDL:HDL cholesterol in both normocholesterolemic and hypercholesterolemic men were significantly lower (approximately 6% and 11%, respectively) when soy protein diet was consumed 274 Nutrition Reviews, Vol. 63, No. 8

4 Table 1. (Cont d) Cholesterol-lowering Effects of Soy Products Reference Population (n) Soy Product Fed, Dose, Duration Outcome Nestel et al., Menopausal and perimenopausal women (n 21) Soy isoflavone tablets (80 mg/d) for 5 to 10 weeks No effect on plasma lipids (plasma cholesterol, triglyceride and HDL cholesterol); arterial compliance improved by 26% Animal Studies Adams et al., Atherosclerosis-susceptible mice, male and ovariectomized female ApoE-null mice, and LDLreceptor-null ApoB transgenic mice (n 416) Demonty et al., Male Sprague-Dawley rats (n 36) Fukui et al., Male Sprague-Dawley rats (n 30) Lucas et al., Ovariectomized female golden Syrian hamsters (n 48) Tsai and Huang, Male golden Syrian hamsters (n 24) One of five experimental diets: soy plus isoflavones, -conglycinin (7S globulin, a major soy storage protein), conglycinin-devoid soy protein, glycinin (11S globulin, a major soy storage protein), or W008 (a soy peptide fraction) for 4 months SPI and casein with similar isoflavone content (1.82 mg/g protein) for 21 days 20% SPI; 20% isoflavone-free SPI (IF SPI) for 2 weeks Ethanol extracted isoflavone-depleted SPI (ID SPI) (240 g/kg of diet) for 70 days SPI containing 1.24 mg genistein and 0.61 mg diadzein/g, and ethanol-extracted SPI (E SPI) low in isoflavones (0.21 mg genistein and 0.06 mg diadzein/g) fed for 10 weeks Reduced atherosclerosis observed in all mice fed the conglycinin diets compared with mice fed the soy plus isoflavones diet; extent of atherosclerosis reduced in ovariectomized female mice fed all soy proteincontaining diets compared with controls Plasma total triglycerides decreased by 26% in rats fed soy protein and casein plus isoflavones compared with control Plasma total cholesterol concentrations of rats fed SPI and IF-SPI were comparable and significantly lower than that of rats fed control diet; higher fecal steroid excretion in the SPI group ID-SPI lowered serum triglyceride concentrations compared to controls, no significant differences in serum HDL concentrations, liver total lipids and liver total cholesterol SPI group exhibited significantly lower serum total cholesterol concentration compared with the E SPI group; both SPI and E SPI groups had lower LDL cholesterol, LDL ApoB, and LDL:HDL cholesterol compared with the controls Nutrition Reviews, Vol. 63, No

5 reducing blood cholesterol levels (particularly the LDL fraction). There is evidence that soy protein increases fecal excretion of bile acids. In rats fed isoflavone-rich and isoflavone-depleted soy protein, higher fecal steroid excretion was observed in the isoflavone-rich group, though both groups had lowered total cholesterol compared with the control group. The authors concluded that the cholesterol-lowering effects of soy may be attributed to the protein content, with the isoflavones and other minor constituents playing a role. 11 Soy isoflavones are also believed to reduce the risk of heart disease by reducing the susceptibility of LDL to oxidation via its antioxidant action. 16,37 The effects of a soy protein diet enriched with isoflavones and one depleted of isoflavones on LDL resistance to oxidation were compared among 24 healthy subjects in a randomized crossover design. Plasma concentrations of 8-epiprostaglandin F2, a biomarker of lipid oxidation, were significantly lower after the high-isoflavone diet (21.2 mg diadzein, 34.8 mg genistein), showing increased resistance of LDL to Cu 2 -induced oxidation. This antioxidant action may be significant with regard to risk of atherosclerosis and cardiovascular disease in general. 16 Similar results were observed in male golden Syrian hamsters. 10 The resistance of LDL to Cu 2 - induced oxidation was greater in hamsters fed isoflavone-rich soy protein than in those fed isoflavonedepleted protein, as assessed by the lower concentrations of thiobarbituric acid reactive substances, another biomarker of lipid oxidation, and the longer lag time required for the formation of conjugated dienes. The isoflavone-enriched group also had significantly higher serum total antioxidant capacity, particularly -tocopherol content, showing sparing effects on -tocopherol contents in both serum and liver. These findings suggest that the intake of soy isoflavones enhanced the resistance of LDL to oxidation, contributing to antioxidant defense, and reduced the consumption of -tocopherol in both the serum and liver of hamsters. 10 Improvement of arterial compliance is another possible mechanism by which soy isoflavones protect against heart disease. 38 Arterial compliance (arterial elasticity) is an important cardiovascular disease risk factor that diminishes with age and menopause. 39 The effect of soy isoflavones (80 mg daily) on arterial compliance was tested in menopausal and perimenopausal women over 5- to 10-week periods in a placebo-controlled crossover trial. 40 Though there was no effect on plasma lipids, systemic arterial compliance improved by 26% compared with placebo to about the same extent as that achieved with conventional hormone replacement therapy (HRT). The fact that plasma lipids were not changed suggests that other constituents of soybean (apart from its isoflavones) may be responsible for lipid lowering. 40 Soy Isoflavones and Cancer The consumption of high levels of soy foods has been shown to be associated with a reduced risk of several types of cancer, including breast, endometrial, and prostate cancers, particularly among Asian populations. 5,6,15,41,42 This is in contrast to Western populations, where there is a higher incidence of these cancers. A number of in vivo studies are supportive of a protective role for soy foods in cancer and some studies have shown that soy isoflavones are responsible for these protective effects. 15 This protective effect appears to be strongest for breast cancer. Administration of soy isoflavones early in life enhances the early maturation and differentiation of the mammary gland of rats, suggesting that exposure in early life may be important. 3,7,17 Table 2 shows a summary of previous studies on the effects of soy products on cancer and associated DNA damage. Several possible mechanisms have been identified for the anticarcinogenic activity of soy isoflavones. One of the mechanisms by which soy isoflavones are believed to exert their anticarcinogenic effects is via their antioxidant properties, 14 which result in a decrease in lipid peroxidation 16 and oxidative DNA damage, 14 both important risk factors for carcinogenesis. These antioxidant properties are suggested to be related to the chemical structure of soy isoflavones. Consumption of soy containing naturally occurring amounts of isoflavones reduced lipid peroxidation in vivo among 24 healthy subjects, as evidenced by lower levels of plasma concentrations of 8-epiprostaglandin F2, a biomarker of in vivo lipid peroxidation. 16 In another study to evaluate the effects of soy isoflavone supplementation on markers of oxidative stress in men and women, soy supplementation was given in the form of Novasoy tablets containing 50 mg of isoflavones daily for 3 weeks. There was a significant decrease in the levels of 5-hydroxymethyl-2- deoxyuridine, a biomarker of oxidative DNA damage; though no significant decrease in lipid peroxidation was observed. The results from this study suggest that dietary supplementation with soy isoflavones can decrease levels of oxidative DNA damage, which is associated with the process of carcinogenesis. 14 A similar decrease in oxidative DNA damage was also observed among men consuming 1Lofsoymilk daily for 4 weeks. 43 Another mechanism by which soy isoflavones may protect against cancer is through the induction of phase II enzymes, 44 which is associated with cancer chemopreventive potential at both the initiation and promotion phases. 44 Induced levels of the phase II enzymes glutathione-s-transferase (GST) and quinone reductase (QR) 276 Nutrition Reviews, Vol. 63, No. 8

6 Table 2. Effect of Soy Products on Cancer and Oxidative DNA Damage Reference Population Soy Product Fed, Dose, Duration Outcome Human Studies Djuric et al., Men (n 6) and women (n 6) Wiseman et al., 24 healthy subjects (19 women and 5 men) Mitchell & Collins, Healthy men (n 10) Novasoy tablets containing 50 mg isoflavones; women took 1 tablet daily and men took 2 tablets daily for 3 weeks One of two experimental diets over two 17-day periods in a randomized crossover design: low-soy isoflavone diet (21.2 mg diadzein, 34.8 mg genistein) or high-soy isoflavone diet (0.9 mg diadzein, 1.0 mg genistein) Soy isoflavone supplementation decreased levels of oxidative DNA damage in men and women, evidenced by decreased levels of 5-hydroxymethyl-2-deoxyuridine, a biomarker of oxidative DNA damage (47% and 61% decrease in women and men, respectively) High-isoflavone diet significantly reduced plasma concentrations of F 2 -isoprostane-8-epiprostaglandin F 2, a biomarker of in vivo lipid peroxidation Soy milk (1 L/d) for 4 weeks Decrease in oxidative DNA damage by approximately 67% Animal Studies Constantinou et al., Female Sprague Dawley rats (n 119) One of five experimental diets for 120 days: genistein (200 mg/kg diet); diadzein (200 mg/kg diet); genistein plus diadzein at 100 mg/kg diet each; 16% soy protein isolate plus isoflavones (SPI ); 16% isoflavone-depleted SPI (SPI ) Cohen et al., Rats (n 150) 20% intact soy protein (SP), 10% SP, 20% isoflavone-depleted SP (IDSP), 10% IDSP for 18 weeks Applet and Reicks, Female Sprague Dawley rats (n 86) Soy containing three levels of isoflavones (0.03, 0.4, 0.81 mg/g diet; low, middle and high level of isoflavones, respectively) for 13 weeks SPI was the most effective diet, significantly reducing mammary tumor multiplicity by 50%; diadzein and SPI diet also significantly reduced tumor multiplicity by 32% compared with controls; no significant reduction in mammary tumor incidence in any diet No significant differences in mammary tumor incidence, latency, multiplicity, or volume compared with controls Rats fed high-isoflavone diet had lower mammary tumor incidence (40%) compared with controls (71.4%); rats fed middle and low isoflavone diet had tumor incidence of 53.3 and 57.1%, respectively Nutrition Reviews, Vol. 63, No

7 have been proposed as suitable biomarkers for identifying compounds likely to inhibit carcinogenesis. 45 QR and GST are enzymes that help the body get rid of the toxic products of oxidative metabolism of aromatic hydrocarbons. 44 There is evidence to support the mechanism of soy isoflavones as antioxidants and as phase II enzyme inducers. Soy isoflavones increased antioxidant and phase II enzyme activity, especially QR, GST, and UDP-glucuronosyltransferase, in various tissues of rats fed a high-isoflavone diet for 2 weeks. 45 Similar results were found in a study by Constantinou et al. 44 comparing the anti-tumor effects of isoflavone-enriched soy protein isolate and isoflavonedepleted soy protein isolate with those of its isoflavones, genistein and diadzein. Although the diadzein and isoflavone-enriched soy groups showed significantly reduced tumor multiplicity compared with the controls, both soy groups (but not the genistein or diadzein groups) upregulated transcriptional expression of the antioxidant enzymes QR and GST. These results suggest that the mode of preventive action of soy protein isolate is distinct from that of the main isoflavones, and that isoflavone-depleted soy may be more beneficial than isoflavone-enriched soy in preventing mammary tumors in these experimental animals. 44 However, another study evaluating the effects of intact and isoflavone-depleted soy protein on N- nitroso-n-methylurea (NMU)-induced rat mammary tumorigenesis showed no effect on tumor incidence, latency, multiplicity, or volume. The results from this study do not support the hypothesis that the isoflavone components of soy protein, or the soy protein itself, inhibit chemically induced mammary tumor development. 6 As with animal experiments, studies in human populations relating soy consumption to reduced risk of breast cancer have produced conflicting results, and a few have shown stimulatory effects. Petrakis et al. 46 reported that prolonged consumption of soy protein isolate by a group of healthy premenopausal US women increased hyperplastic epithelial cells in duct fluid aspirates, showing a stimulatory effect on the breast. Soy Isoflavones and Osteoporosis The ovarian hormone deficiency associated with menopause results in an increased rate of bone turnover, which causes an imbalance between bone resorption and bone formation, thereby accelerating bone loss that leads to osteoporosis. 25,47 HRT is an effective treatment in reducing the rate of bone loss and risk of fracture, though not very popular among postmenopausal women because of the undesirable side effects and long-term risks of breast and endometrial cancer associated with prolonged use. 18,25,47,48 Soy isoflavones have been shown to alleviate osteoporosis without the side effects of HRT. 12 For this reason, soy isoflavones are called selective estrogen receptor modulators (SERMs), and may be a possible alternative to HRT. 7,12 Soy isoflavones have been suggested to alleviate osteoporosis by inhibiting bone resorption and stimulating bone formation. Evidence that soy isoflavones reduce bone resorption has been demonstrated by a number of studies. Picherit et al. 49 investigated the ability of longterm daily intake of soy isoflavones in reversing osteopenia in the adult ovariectomized rat, which was used as a model of postmenopausal women. Soy isoflavones reduced the urinary excretion of deoxypyridinoline, a specific biomarker of bone resorption. 49 Similar results were also observed among postmenopausal women, in whom soy protein intake was associated with significantly lower urinary deoxypyridinoline excretion. 50,51 Soy isoflavones appear to stimulate osteoblastic activity through the promotion of insulin-like growth factor-i (IGF-I) production. 52 It is well recognized that IGF-I enhances osteoblastic activity in humans. 25 IGF-I is a protein involved in the bone formation process, and therefore an increase in IGF-I is indicative of increased bone formation. In a study by Arjmandi et al. 52 using a rat model of osteopenia, soy increased the gene expression of IGF-I, as indicated by higher femoral mrna levels. Incorporation of soy protein with normal isoflavone content (2.3 mg/g protein) had a greater effect on femoral IGF-I mrna than the isoflavone-depleted soy protein-based diet (approximately 0.1 mg/g protein). This finding indicates that isoflavones may have a role in enhancing the synthesis of IGF-I at the bone level. There is additional evidence that soy promotes bone formation. Increased levels of bone alkaline phosphatase, a biomarker of bone formation, was observed among perimenopausal women fed an isoflavone-rich soy protein diet. 18 In the same study, lumbar spine bone mineral density (BMD) and bone mineral content (BMC) were assessed at baseline and at the end of the treatment. There was a significant difference in BMD (5.6%) and BMC (10.1%) between the isoflavone-rich and control groups, and the authors concluded that soy isoflavones attenuated bone loss from the lumbar spine in estrogendeficient perimenopausal women. 18 Similar results were also observed among hypercholesterolemic, postmenopausal women assessed for total and regional BMC and BMD before and after administration of 40 mg/d of soy protein containing moderate and higher concentrations of isoflavones (1.39 and 2.25 mg isoflavone/g of protein, respectively). The high-isoflavone diet significantly increased both BMC and BMD in the lumbar spine but not elsewhere. 30 Increased BMD and mechanical bone strength and intestinal calcium absorption were also observed in ovariectomized, osteoporotic rats fed soy milk. 53 Soy is also a rich source of calcium, and this may 278 Nutrition Reviews, Vol. 63, No. 8

8 be a possible explanation for the observed increased in intestinal calcium absorption. From these studies, it is evident that soy foods play a role in attenuating bone loss (Table 3); however, the exact osteoprotective mechanism of soy isoflavones remains unclear and requires further elucidation. Soy Proteins and Peptides in Cardiovascular Disease and Cancer Some evidence exists to support the role of certain isoflavone-free soy protein and peptide fractions in cardiovascular disease and cancer. Sirtori et al. 54 have shown that 7S globulin, a major storage protein in soybean, reduced plasma cholesterol concentration by 35% in rats. Similar results were observed by Lovati et al. 55 in an in vitro study evaluating LDL receptor activity in Hep G2 cells (a human hepatoma cell line) exposed either to small natural peptides produced by enzymatic digestion of Croksoy R 70 (a commercial isoflavone-poor soy concentrate) or to synthetic peptides corresponding to specific sequences of the complete 7S globulin (Table 4). The findings from this study demonstrated that 7S globulins stimulate the expression of LDL receptors and the degradation of LDL in the cultured hepatocytes. The data also support the hypothesis that there are bioactive peptides produced from the digestion of soy protein that are absorbed from the small intestine and have beneficial effects on lipoprotein metabolism and cardiovascular health by up-regulating LDL-receptor activity in liver cells. Similar degradation of LDL by an alcohol-extracted 7S globulin was also shown, which demonstrates that the active molecules are the proteins and not the alcohol-soluble isoflavones. 55 The authors concluded that the isoflavone-free protein component is likely to be responsible for this biochemical effect of soy. To further demonstrate this effect in vivo, Adams et al. 56 assessed the effects of dietary -conglycinin, a major soy storage protein, and other soy peptide fractions on the development of atherosclerosis in atherosclerosis-susceptible mice. Male and ovariectomized female ApoE-null mice and LDL-receptor-null ApoB transgenic mice were randomly assigned to one of six treatment groups differing only in their source of dietary protein: 1) casein/lactalbumin, 2) isoflavone-containing soy protein isolate, 3) -conglycinin, 4) glycinin (11S globulin, another major soy storage protein), 5) -conglycinin-devoid soy protein, or 6) W008 (a peptide fraction produced by hydrolysis and precipitation of soy protein isolate). After 4 months of feeding, the aortic atherosclerosis (cholesteryl ester content) and plasma lipoprotein cholesterol concentrations of the mice were quantified. Results showed that the extent of atherosclerosis was reduced in ovariectomized female mice fed all soy protein-containing diets compared with mice fed casein/lactalbumin-based diets. Furthermore, compared with mice fed isoflavone-containing soy protein isolate, atherosclerosis was reduced only in mice fed the -conglycinin-containing diet. These reductions were 39% and 67% in male and ovariectomized female ApoE-null mice, respectively, and 66% in the male LDL-receptor-null mice. These observed effects were unrelated to variations in isoflavone content of the protein source and only minimally related to plasma lipoprotein cholesterol concentrations. The authors thus concluded that a diet rich in -conglycinin has atheroprotective effects that greatly exceed those of isoflavone-containing soy protein isolate. Interestingly, however, their results show that these effects do not depend on LDL receptors or influence plasma lipoproteins. Evidence also shows that dietary soy peptides and soy protein isolates (without isoflavones) have antioxidative properties. Takenaka et al. 57 demonstrated that isoflavone-free soy protein isolate and soy peptide reduced paraquat-induced oxidative stress in rats. Soy protein may also have cancer-protective effects. Azuma et al. 58 and Kanamoto et al. 59 demonstrated that feeding an insoluble, high-molecular weight protein fraction (HMF) prepared from a proteinase-treated soybean protein isolate suppressed colon and liver tumorigenesis induced by azoxymethane and dietary deoxycholate in experimental animals. Bile acid is known to play a critical role in liver and colon tumorigenesis. The authors proposed that HMF exerted these protective effects in colon and liver tumorigenesis by interfering with the enterohepatic circulation of bile acids, thus inhibiting resorption of bile acids in the intestine and increasing fecal bile acid excretion. Peptides found in the feces of HMF-fed animals were found to be rich in hydrophobic amino acids, so the authors suggested that the HMF protein forms non-dissociable complexes with bile acids in the intestine through hydrophobic binding, which are then excreted into feces. HMF may therefore be used as a functional food to prevent the tumor-promoting activity of bile acids on the liver and colon. CONCLUSIONS AND FUTURE DIRECTIONS The consumption of soy foods has been proven to protect against heart disease, some forms of cancer, and osteoporosis. However, there is still no conclusive evidence to date showing whether the protective effects of soy are derived solely from isoflavones, from soy protein itself, or from a combination. Research findings are inconsistent; some evidence supports a role for the isoflavones contained in soybeans and many soy foods, while other evidence supports certain peptides or peptide fractions derived from soy. In addition to this, soy foods also contain other bioac- Nutrition Reviews, Vol. 63, No

9 Table 3. Effect of Soy Products on Bone Health Reference Population Soy Product Fed, Dose, Duration Outcome Human Studies Horiuchi et al., Postmenopausal Japanese women (n 85) Alekel et al., Perimenopausal women (n 69) Potter et al., Hypercholesterolemic, postmenopausal women (n 66) Animal Studies Picherit et al., Adult ovariectomized rats (n 36) Arjmandi et al., Ovariectomized rats with established bone loss (n 36) Daily soy protein intake assessed over a period of three consecutive days using a questionnaire (mean intake 12.6 g/d) Soy protein intake significantly correlated with higher bone mineral density (BMD) and lower urinary excretion of deoxypyridinoline SPI with isoflavones (80.4 mg/d) for 24 weeks Soy isoflavones attenuated bone loss from the lumbar Soy protein (40 mg/d) plus moderate and higher concentrations of isoflavones (1.39 and 2.25 mg isoflavone/g protein, respectively) for 6 months Soybean isoflavone fed as powdered soy isoflavone concentrate; daily intake of 0, 20, 40 and 80 mg/kg body weight for 84 days Soy protein with normal isoflavone content (2.3 mg/g protein) and reduced isoflavone content (approximately 0.1 mg/g protein) for 65 days spine: BMD and bone mineral content (BMC) increased by 5.6% and 10.1%, respectively, compared with controls; increased levels of serum bone-specific alkaline phosphatase, a biomarker of bone formation High-isoflavone diet significantly increased both BMC and BMD in the lumbar spine but not elsewhere Daily soybean isoflavone consumption decreased bone turnover as evidenced by decreased urinary excretion of deoxypyridinoline, a specific bone marker of bone resorption Increased expression of femoral insulin-like growth factor-i mrna levels, an indicator of increased bone formation 280 Nutrition Reviews, Vol. 63, No. 8

10 Table 4. Effect of Croksoy R 70, its Enzyme Digests and Subfractions of Different Molecular Weight on LDL Receptor Activity in Hep G2 Cells* Uptake Degradation mg 125 I-LDL/g cell protein Lipoprotein-deficient Serum (LPDS) Croksoy R 70, g/l Croksoy R 70 enzyme digested, g/l Fractions from Croksoy R 70 enzyme digested, g/l MW 3000 Da MW 3000 Da MW 1000 Da * Values are means SEM, n 3. P 0.05 compared with LPDS P compared with LPDS P 0.05 compared with Croksoy R 70 at the same concentration Reproduced from Lovati et al. 55 with copyright permission of the American Society for Nutritional Sciences. tive components that could exert beneficial effects: the cholesterol-lowering effects of soluble fiber and phytosterols; the anti-carcinogenic potential of saponins, phytic acid, Bowman-Birk inhibitor, and lecithin; and the cardioprotective effects of omega-3 fatty acids. Furthermore, these components may have synergistic effects. It is evident that there is still a lot to be learned about soy foods. A question that remains to be answered conclusively is what component of soy foods is responsible for its protective effects against heart disease, cancer, and osteoporosis. This gap in knowledge could form the basis for more research on the benefits of soy, and further elucidation of its mode of action. Other areas worthy of research are the possible variations in effects of the form of soy foods consumed (fermented, nonfermented) on different tissues and the possible synergistic effects of the many bioactive components of soy. Furthermore, tissue-specific metabolism and biotransformation of soy isoflavones, and the effects and mechanisms of action of its different metabolites in vivo, will go a long way in contributing to the understanding of the mechanisms of action of soy isoflavones. ACKNOWLEDGEMENT The authors thank the Natural Sciences and Engineering Research Council of Canada for financial support provided to the research program of Dr. Aluko. Nutrition Reviews, Vol. 63, No

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