ORIGINAL COMMUNICATION

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1 (2004) 58, & 2004 Nature Publishing Group All rights reserved /04 $ ORIGINAL COMMUNICATION A biochanin-enriched isoflavone from red clover lowers LDL cholesterol in men P Nestel 1 *, M Cehun 1, A Chronopoulos 1, L DaSilva 2, H Teede 2 and B McGrath 2 1 Baker Medical Research Institute Wynn Domain, Monash University, Melbourne, Australia; 2 Department of Vascular Medicine, Monash University, Melbourne, Australia Objective: To determine whether the two major isoflavones in red clover differ in their effect on low-density lipoprotein cholesterol (LDL-C). Design: A randomised, placebo-controlled, double-blind trial; two parallel groups taking one of the two isoflavones within which treatment and placebo were administered in a crossover design. Setting: Free-living volunteers. Subjects: A total of 46 middle-aged men and 34 postmenopausal women. Intervention: Two mixtures of red clover isoflavones enriched in either biochanin (n ¼ 40) or formononetin (n ¼ 40) were compared. Placebo and active treatment (40 mg/day) were administered for 6 weeks each in a crossover design within the two parallel groups. Main outcome measures: Plasma lipids were measured twice at the end of each period. Results: Baseline LDL-C concentrations did not differ significantly between men (n ¼ 46) and women (n ¼ 34), nor between those randomised to biochanin or formononetin. Interaction between time and treatments, biochanin, formononetin and corresponding placebos (two-way ANOVA) on LDL-C showed a significant effect of biochanin treatment alone. The biochanin effect was confined to men; median LDL-C was 3.61 ( ) mmol/l with biochanin and 3.99 ( ) mmol/l with the corresponding placebo (RM ANOVA with Dunnett s adjustment Po0.05). The difference between placebo and biochanin effects on LDL-C was 9.5%. No other lipid was affected and women failed to respond significantly to treatment. Conclusion: Isolated isoflavones from red clover enriched in biochanin (genistein precursor) but not in formononetin (daidzein precursor), lowered LDL-C in men. This may partly explain the previous failure to demonstrate cholesterol-lowering effects with mixed isoflavones studied predominantly in women. Sponsorship: Novogen Ltd, North Ryde NSW, Australia, provided partial support including provision of tablets and outside monitoring. (2004) 58, doi: /sj.ejcn Keywords: isoflavones; red clover; biochanin; formononetin; LDL cholesterol Introduction Since the publication of the meta-analysis of the lipidlowering effect of soy protein (Anderson et al, 1995), the precise constituent in soy responsible for reducing the low-density lipoprotein cholesterol (LDL-C) concentration remains uncertain. The isoflavone content is likely to contribute strongly, since isoflavone-depleted soy protein appears less effective (Gardner et al, 2001; Wangen et al, 2001) and one large study has shown a dose-related effect *Correspondence: P Nestel, Baker Medical Research Institute, PO Box 6492, St Kilda Rd Central, Melbourne 8008, Australia. paul.neslet@baker.edu.au Received 5 March 2003; revised 23 April 2003; accepted 16 May 2003 when increasing amounts of isoflavones within soy protein were compared (Crouse et al, 1999). However, trials of purified isoflavones have to date been disappointing (Nestel et al, 1997, 1999; Hodgson et al, 1998; Howes et al, 2000; Simons et al, 2000). Further, there is large variability in response to soy as shown in the meta-analysis, since about half of the 31 studies analysed by Anderson et al (1995) showed only minimal LDL-C lowering for reasons that have not been resolved. Among recent well-designed and executed trials, the reduction in non-high-density lipoprotein cholesterol (HDL-C) has been as low as 2.6% (Teixeira et al, 2000) or that of LDL-C no greater than with the milk protein-based control diets (Teede et al, 2001; Meinertz et al, 2002). As is seen with other cholesterol-lowering nutritional

2 404 interventions, the best responses to soy protein were shown by individuals with the highest initial LDL-C levels (Crouse et al, 1999; Vigna et al, 2000). On the other hand, two recent studies that compared two soy protein products with very different isoflavone contents reported no specific LDL-C lowering attributable to isoflavones (Jenkins et al, 2002; Lichtenstein et al, 2002). Studies with intact soy protein have been conducted in both men and women with no apparent gender difference (Crouse et al, 1999). Adding to these variables is the large interindividual variation in isoflavone absorption (Tsunoda et al, 2002). A puzzling result is the apparent absence of a response to purified isoflavones when taken in amounts that show a benefit within soy protein. Alternative compounds in soy carry fewer experimental credentials and have been reviewed recently (Erdman, 2000). Only one of the trials with pure isoflavones included men (Hodgson et al, 1998), although it has been recognized that women and men may respond differently to other dietary interventions such as changing the intakes of saturated fatty acids and cholesterol. Clifton and Nestel (1992) in a study of 26 men and 25 women matched for age, LDL-C and BMI, found that men responded to dietary saturated fat and cholesterol with a greater rise in LDL-C, whereas women showed greater increments in HDL-C. Cobb et al (1992) also found higher LDL-C/HDL-C responses among men than women as the saturation of the dietary fat was altered. The primary objective of the present study was therefore to evaluate the changes in LDL-C with supplements of the two main individual isoflavones (biochanin and formononetin) present in red clover. This was studied in 46 men and 34 women with preparations that comprised predominantly biochanin (B; the precursor of genistein) or predominantly formononetin (F; the precursor of daidzein) in a randomised, double-blind, placebo-controlled trial. Methods Study population Middle-aged men and women were recruited by advertisement. The women had been postmenopausal for 41 y and shown to have FSH values above 40 mmol/l. In all, 82 subjects were recruited, but two withdrew prior to randomisation. Men (n ¼ 46) and women (n ¼ 34) were of similar mean age, but the men were heavier (means and standard deviations): men 80.5 (11.0) kg and women 65.6 (11.9) kg (Po0.001); respective BMI values for men and women were 25.9 (2.7) and 24.5 (3.7) kg/m 2 (Po0.02) (Table 1). The 80 subjects were randomised to either B or F (80 mg daily in two tablets) irrespective of sex; the two treatment groups did not differ in average age or average body mass. Their baseline lipid values are shown in Table 2. Inclusion criteria were: age y; postmenopausal status for women; plasma total cholesterol mmol/l; plasma triacylglycerolo3.5 mmol/l. Exclusion criteria were: smoking, alcohol intake 44 standard drinks daily, BMI434 kg/ Table 1 Subjects Characteristics of subjects by gender and by treatment Number Age (y) Body mass (kg) BMI (kg/m 2 ) Men (7) 80.5 (11.1)* 25.9 (2.7)** Women (6) 65.6 (12) 24.5 (3.7) Formononetin (7) 73.6 (11.5) Formononetin placebo (11.1) Biochanin (6) 75.1 (15.6) Biochanin placebo (15.7) n Significantly different between men and women (Po0.001); ** (Po0.02). Differences in body weight between the two treated groups and their placebo periods were not significant. Values are means (s.d.) Table 2 Baseline lipids measured after run-in, prerandomisation phase Men Women Lipid (mmol/l) Biochanin Formononetin Biochanin Formononetin Total cholesterol 5.57 (0.85) 5.56 (1.05) 5.40 (1.05) 6.17 (0.87) LDL-C 3.73 (0.85) 3.67 (0.95) 3.37 (0.94) 3.85 (1.05) HDL-C 1.21 (0.3) 1.30 (0.3) 1.62 (0.46) 1.78 (0.44) Triacylglycerol 1.42 (0.93) 1.29 (0.56) 0.90 (0.48) 1.18 (0.47) Values are means (s.d.), n=80. m 2, medication for lowering lipids or blood pressure, diabetes mellitus, a past or present history of cancer or chronic bowel disease, allergy to soy, unusual dietary habits and unwillingness to discontinue supplements of any kind or adhere to a predetermined diet. For women, hormone replacement therapy within the last 2 months and absence of a recent normal mammogram (42 y) were additional exclusions. The project was approved by the Human Ethics Committee of the Alfred Group of Hospitals and signed consent was obtained from the volunteers. Experimental design The 80 subjects were allocated by random numbers to receive either B or F with equal numbers receiving B and F. Each isoflavone was compared against placebo pills of similar colour and consistency. Among the 46 men, 25 received B and 21 F; among the 34 women, 15 received B and 19 F. Each study began with a 2-week run-in, during which subjects became acquainted with procedures such as recording daily the taking of placebo pills and completing 3-day food frequency questionnaires (simplified by the Anti- Cancer Council of Victoria, Australia, that produced and analysed the food records). The questionnaires were completed during the final 3 days of the run-in and the final intervention periods, respectively. Volunteers had been

3 advised to maintain their entry patterns of food intake and the macronutrients that may affect LDL-C as shown in Table 3 demonstrated no significant differences during the trial period. The average difference in body mass between a treatment period and its corresponding placebo period of o0.8 kg (Table 1) was not significant. The subjects were allocated randomly to receive placebo or active treatment first, for a period of 6 weeks. This was followed by a 1-week washout (placebo) before the crossover to the alternate 6-week period. The total duration of the study was 15 weeks. Blood was collected into EDTA-containing tubes twice after each 6-week period, mostly on consecutive days and never more than 3 days apart. Subjects had fasted overnight for not less than 10 h. The two isoflavone preparations were obtained from Novogen Ltd (North Ryde, NSW, Australia). The mixtures were concentrated ethanolic extracts from red clover separated into two fractions. B comprised biochanin:formononetin in a ratio of approximately 3.5:1 with 4% genistein and o1% daidzein. F comprised formononetin:biochanin in a ratio of 4.9:1 with o1% genistein and daidzein. Urinary excretion was measured over a 24 h period at the end of the placebo and isoflavone periods. Laboratory procedures Plasma cholesterol and triacylglycerol concentrations and HDL-C were measured in a specialised laboratory using standard enzyme-based methods and measurement in a Cobas-Bio automated analyser (Roche, Basel, Switzerland). LDL-C was calculated. Urinary isoflavones were measured as described by Tsunoda et al (2002). Briefly, aliquots of urine were incubated for Table 3 study Macronutrient intakes at the end of run-in period and end of Daily intake End run-in period End of study Energy (kj) 7953 (2994) 7458 (2454) Fat (g) 73.1 (33.1) 67.0 (28.9) (%en) Saturated fatty acids (g) 27.8 (12.9) 25.7 (11.6) (%en) Monounsaturated FA (g) 25.4 (12.0) 23.7 (10.9) (%en) Polyunsaturated FA (g) 13.1 (7.8) 11.3 (6.2) (%en) Protein (g) 88.9 (32.4) 83.8 (27.5) (%en) Carbohydrate (g) 223 (87) 212 (75) (%en) Dietary fibre (g) 26.1 (10.0) 24.1 (8.7) Cholesterol (mg) 257 (117) 244 (100) None of the differences between run-in period and end of study was significant. Alcohol consumption was o4% energy. Calculated from 3-day food frequency data. Values are means (s.d.), n= h at 371C with glucuronidase and the free isoflavones were eluted with 3 ml ethanol from a C-18 solid-phase extraction column (Waters, Sydney, Australia), and separated by HPLC. The HPLC system consisted of a C-18 stationaryphase column and a gradient acetonitrile/water mobile phase. The limit of detection of the assay for each isoflavone was 5 ng/ml and the inter-assay CV was o15%. Data analysis Responses to isoflavones vs. placebo were analysed initially for the 46 men and the 34 women for within-subject interactions and time or order effects. The analyses included both isoflavones and their respective placebo periods. Interaction between time and treatment was tested by twoway ANOVA. Since this indicated an effect of biochanin treatment on LDL-C, further analyses were carried out separately for men and women. Comparisons were analysed by RM ANOVA with appropriate adjustments for multiple comparisons. The distribution of LDL-C was not normal among the men and Friedman repeated-measures analysis of variance on ranks was substituted and adjusted by Dunnett s all pairwise multiple comparison procedure. In men, the differences between each treatment and its placebo were compared for B vs. F by one-way ANOVA adjusted by Tukey Test. Results Table 4 shows the trial data for plasma lipids in men and women, respectively. The distribution of LDL-C among men was not normal and hence both mean (standard deviation) and medians (25 75% range) are given. ANOVA showed that there was no effect of order of therapy on any lipid variable. Following 6 weeks on placebo, total plasma cholesterol and LDL-C values did not differ significantly between men and women. HDL-C concentration was significantly greater among the women (Po0.01, t- test). Plasma triacylglcerol tended to be higher among men, but not significantly so. Analysis of differences according to isoflavone randomisation, especially for LDL-C, showed no significant differences during placebo phases, although the two comparisons were marginally different. Men randomised to B tended to show higher initial LDL-C levels than women (P ¼ 0.061, t-test). Among men, LDL-C levels did not differ significantly between those drawn to B or F; among the women, those drawn to F had marginally higher initial LDL- C levels than those drawn to B (P ¼ 0.067, t-test). Effect of treatment on LDL-C was significant for B treatment (P ¼ 0.026; two-way ANOVA). The results were further analysed among men and women by type of treatment. Among men, the respective median LDL-C concentrations for placebo and B were 3.99 and 3.61 mmol/l (Friedman RM ANOVA on ranks: P ¼ 0.026; with Dunnett s adjustment Po0.05). Median LDL-C tended to be lower with F than with placebo (3.62 vs mmol/l), but 405

4 406 Table 4 Plasma lipid data for all treatments in men and women Lipid (mmol/l) Biochanin Placebo Formononetin Placebo Cholesterol Men 5.39 (0.75) 5.54 (0.75) 5.52 (1.27) 5.66 (1.23) Women 5.25 (1.01) 5.27 (1.06) 6.10 (0.94) 6.21 (1.11) LDL-C Men Mean 3.64 (0.93) 3.82 (0.84) 3.65 (1.09) 3.68 (1.09) Median 3.61 ( ) 3.99 a ( ) 3.62 ( ) 3.87 ( ) Women Mean 3.24 (0.86) 3.26 (0.95) 3.83 (0.92) 3.92 (0.98) HDL-C Men 1.18 (0.28) 1.21 (0.29) 1.28 (0.28) 1.35 (0.31) Women 1.64 (0.38) 1.59 (0.33) 1.79 (0.53) 1.80 (0.49) Triacylglycerol Men 1.36 (0.71) 1.21 (0.47) 1.29 (0.71) 1.37 (0.71) Women 0.92 (0.41) 0.90 (0.49) 1.09 (0.43) 1.18 (0.49) a RM ANOVA with Dunnetts adjustment for multiple comparisons, Po0.05; comparison of median LDL-C concentrations at the end of biochanin and placebo periods. n=25 men biochanin; 21 men formononetin; 15 women biochanin; 19 women formononetin. Values are means (s.d.) except for medians (25 75%) for LDL-C in men. the difference was not significant. The difference between the LDL-C medians for B and placebo in men was 0.38 mmol/l (9.5%). The difference between the two isoflavone effects was tested directly by comparing the mean differences in LDL-C between each isoflavone and its placebo. Among the 25 men receiving B, the mean difference for LDL-C was 0.18 mmol/l and the corresponding value among the 21 men receiving F was 0.03 mmol/l. The difference for LDL-C between mean (placebo minus B) vs. mean (placebo minus F) was significant (Po0.05; one-way ANOVA adjusted by Tukey Test for all pairwise multiple comparisons). Isoflavones recovered in urine showed as expected that most, but by no means all, the primary isoflavones, formononetin and biochanin were converted into daidzein and genistein, respectively (Tsunoda et al, 2002). Further, the total excreted by individuals consuming B was less than that excreted with the consumption of F, as noted by others (Busby et al, 2002). Of particular interest was whether a minority of individuals who excreted substantial amounts of equol, a metabolite of daidzein and hence of formononetin, showed a different LDL-C response than nonequol excretors, despite formononetin itself having no effect. In total, 15 subjects were found to excrete substantial amounts of equol as defined by Rowland et al (2000). Eight had been randomised to F and seven to B: the mean (s.d.) LDL-C concentrations were 3.39 (0.74) and 3.29 (0.65) mmol/l with placebo and isoflavone treatments, respectively. This difference was not significant, nor was the difference in LDL-C significant between equol excretors taking F or B, although the numbers are small. Total excretion of recovered isoflavones was 18.9 (23.6) mg/day, indicative of the large variability among individuals. Discussion The results support the possibility that individual isoflavones affect LDL-C differentially. Only in men was LDL-C lowered significantly with a mixture in which biochanin predominated (B). Two issues arise therefore: the reasons for the different gender response and for the greater benefit from biochanin, the precursor of genistein. The present findings of a differential response between men and women and between the responses to biochanin and formononetin may help explain the failure by ourselves (Nestel et al, 1997, 1999) and others (Hodgson et al, 1998; Howes et al, 2000; Simons et al, 2000) to demonstrate an effect on plasma LDL-C with isolated isoflavones. In each of the previous studies, the isoflavones contained a mixture of B and F that would have diluted any effect attributable to B alone. The present findings are consistent with other evidence for a differential effect of dietary interventions on LDL-C between women and men. Gender differences were noted by Savolainen et al (1991), who found that men had a significantly greater rise in LDL-C than women in response to a high dietary fat intervention. Clifton and Nestel (1992) had shown that women responded to an increase in saturated fat and cholesterol with a rise in HDL-C, whereas older men, especially those with higher baseline LDL-C levels, showed substantial increases in LDL-C. Cobb et al (1992) also reported a gender difference in lipoprotein

5 responses to changes in the saturation of fat. Among women, HDL-C underwent the greater change so that the LDL- C:HDL-C ratio in men remained higher than in women. However, Geil et al (1995) found that both men and women responded similarly to a fat-reduced diet. In this context, the failure of isolated isoflavones, from both soy and red clover, to lower LDL-C might be viewed as being partly due to the predominance of women in these studies. Clearly, however, the effects of isoflavones differ from those of dietary total fat, fatty acid type and cholesterol. Changes in the consumption of the latter commonly give rise to changes in HDL-C as well as to LDL- C and under appropriate circumstances, also in triacylglycerol. With the exception of the large study by Howes et al (2000), isolated red clover isoflavones have not lowered triacylglycerol significantly. In that study, a significant inverse relationship was observed between triacylglycerol levels and the excretion of genistein and isoflavone metabolites, suggesting that triacylglycerol fell in those women who absorbed isoflavones best. It should, however, be noted that studies of soy protein have also not been uniformly effective in changing plasma lipid levels. Many of the trials included in the large metaanalysis of soy protein (Anderson et al, 1995) failed to lower LDL-C. Other recent trials have also not found differences between soy protein isolates and control proteins, mostly casein (Gardner et al, 2001; Teede et al, 2001). By contrast, Crouse et al (1999) showed a clear soy isoflavone-related effect on LDL-C. Despite the large number of subjects in that study, 156 women and men, there was no clear gender isoflavone interaction, since LDL-C lowering was of marginal significance in the postmenopausal women (P ¼ 0.07) and in the men; premenopausal women showed no change. The basal LDL-C concentration appeared to be the best predictor of the isoflavone-mediated reduction in LDL-C, much as has been observed with other dietary interventions. To what extent baseline LDL-C might have been a factor in the present study is uncertain, but we believe it to be minor for the following reasons. LDL-C concentrations did not differ significantly between the men and women during the placebo phase. Among those randomised to B, men had slightly higher LDL-C levels than women but not significantly so. Among men, in whom alone LDL-C concentrations were lowered with B, the placebo LDL-C levels were not significantly different between those treated with B and F. On the other hand, women randomised to receive F tended to show, at the end of the placebo phase, higher LDL-C levels: 3.92 (0.98) mmol/l than those randomised to B: 3.26 (0.95) mmol/l; P ¼ 0.067; yet there was no difference in LDL- C lowering. The differential responses to B and F might have been due to the fact that genistein, which is derived from biochanin shows substantially greater affinity for the oestrogen receptor than does daidzein, which is derived from formononetin (Setchell, 1998). The precise mechanism for the cholesterolreducing effect of isoflavones is not known. Indeed, the controversy over the need to have isoflavones retained within soy protein is not resolved. However, there is a suggestion that isoflavones, like oestrogens, may upregulate the LDL receptors (Lovati et al, 1987; Baum et al, 1998) and thereby accelerate clearance of LDL-C from the circulation. If that requires interaction with oestrogen receptors in liver and other tissues, then biochanin/genistein might be expected to exert a greater effect than formononetin/ daidzein. Urinary excretion of isoflavones is a very approximate indication of absorption. The recovery during the intervention with F of 18.9 mg/day or just under 25% of the ingested amount is similar to amounts that we have reported (Nestel et al, 1997; Tsunoda et al, 2002). Nonurinary routes of excretion and failure to measure minor metabolites (only equol and O-desmethylangloensin were measured) contribute to underestimation. Out of 80 subjects, 15 excreted large amounts of equol and the others mostly none. It is generally accepted that equol excretors are in the minority (Rowland et al, 2000). The interest in equol derives partly from the fact that equol binds to oestrogen receptors with greater affinity than its parent compound formononetin and equally with that of genistein (Morito et al, 2001). In summary, LDL-C was lowered significantly with a biochanin-rich isoflavone mixture isolated from red clover, but this was seen only in men and not in women. It is a novel finding that requires confirmation. A formononetinenriched isoflavone mixture was ineffective in both sexes. Neither HDL-C nor triacylglycerol concentrations were affected by isoflavone supplementation. Acknowledgements Novogen Ltd (North Ryde, NSW, Australia) provided the isoflavone supplements and placebo tablets and arranged an outside monitor to supervise the trial. All investigators contributed to the design and interpretation of the experiment. References Anderson JW, Johnstone BM & Cook-Newell MB (1995): Metaanalysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333, Baum JA, Teng H, Erdman Jr JW, Weigel RM, Klein BP, Persky VW, Freels S, Surya P, Bakhit RM, Ramos E, Shay NF & Potter SM (1998): Long-term intake of soy protein improves blood lipid profiles and increases mononuclear cell low-density-lipoprotein receptor messenger RNA in hypercholesterolemic, postmenopausal women. Am. J. Clin. 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