Red Clover Isoflavones Enriched with Formononetin Lower Serum

ORIGINAL ARTICLE Red clover isoﬂavones enriched with formononetin lower serum LDL cholesterol—a randomized, double-blind, placebo-controlled study

PB Clifton-Bligh1,2, M-L Nery1, RJ Clifton-Bligh1,2, S Visvalingam3, GR Fulcher1,2, K Byth4 and R Baber2,3

BACKGROUND: Although postmenopausal combined hormone replacement therapy reduces the risk of hip fracture, long-term use may be associated with an increased risk of breast cancer, and in women more than 10 years after menopause it is associated with an increased risk of cardiovascular disease. Isoflavones, because of preferential binding to estrogen receptor beta, may retain the beneficial effects on bone but lessen the adverse effects on the breast. OBJECTIVE: The objective of this study was to study the effects of an isoflavone obtained from red clover (Rimostil) on bone mineral density, and on low-density lipoprotein (LDL) cholesterol. DESIGN: In a double-blind, randomized, placebo-controlled trial, 50 mg of Rimostil was given to women who were menopausal for at least 1 year. Bone mineral density of the spine, femoral neck and forearm and serum LDL cholesterol were measured at baseline and at 6-month intervals. The duration of follow-up was 2 years. RESULTS: There was no beneficial effect of Rimostil on bone density at any site. There was a 12% fall in serum LDL cholesterol in the Rimostil-treated arm, which was significantly greater than the 2% drop seen in the control arm (P = 0.005). European Journal of Clinical Nutrition (2015) 69, 134–142; doi:10.1038/ejcn.2014.207; published online 5 November 2014

INTRODUCTION In a previous study using red clover extract enriched with After the onset of menopause, there is a rise in the serum formononetin, we found that the forearm bone mineral density cholesterol and an increased risk of cardiovascular disease.1 (BMD) increased and the serum low-density lipoprotein (LDL) fi Menopausal women also experience frequent troublesome cholesterol was signi cantly decreased in a group of postmeno- 8 fl episodic hot flushes. The most effective therapy to diminish both pausal women. This study had several aws: the absence of an the frequency and severity of hot flushes is exogenous estrogen. appropriate placebo group, the unexpected large increases in BMD in the forearm in a 6-month period and the absence of a Estrogen therapy, when given early in menopause, is also 2,3 dose–response relationship between the fall in the serum LDL associated with a reduction in cardiovascular events, but when cholesterol and increasing amounts of red clover extract. given more than 10 years after the onset of menopause it is With this in mind, we have undertaken a larger double-blind, associated with an increased risk of cardiovascular events, 4,5 placebo-controlled study over a 2-year period, in which BMD was including an increased risk of stroke. In contrast, the risk of measured in the lumbar spine, femoral neck and forearm together hip fracture is reduced significantly. In addition, the use of with measures of serum LDL cholesterol concentrations. combined estrogen and progestin for more than 5 years is Thus, the primary aim of this study was to evaluate the efficacy associated with an increased risk of breast cancer.4 Estradiol of Isoflavones (Formononetin enriched, P-081, Rimostil, Sydney, interacts with both the estrogen receptor α (ERα), produced in the NSW, Australia) on vertebral body, femoral neck and forearm BMD breast and the endometrium, and the estrogen receptor β (ERβ), and on serum LDL cholesterol, as measured by the within-patient predominantly in trabecular bone.6 The above considerations changes from baseline at 24 months. Secondary end points were have led to a search for substances that interact preferentially with changes in HDL cholesterol, ApoA, ApoB, Lp(a), triglycerides, the estrogen receptors in bone but to a lesser extent with the serum 25OH-vitamin D, serum calcium and urinary deoxypyridino- estrogen receptors in the breast and endometrium. In this respect, line (DPYR). The hemoglobin and serum alanine aminotransferase the isoflavones produced in soy and red clover have seemed (ALT) were measured to assess potential adverse effects of the promising despite the fact that they have much less affinity for the active treatment. ERβ than estradiol itself,6 although s-equol, a metabolite of fi β daidzein produced in the intestine, has an af nity for ER as high PARTICIPANTS AND METHODS as estradiol itself but a low affinity for ERα.7 Soy protein extracts contain mostly genistein and daidzein, whereas red clover extracts Participants were recruited by newspaper advertisements between the years 2000 and 2006 in the local geographical community. To be included contain more biochanin A and formononetin (the precursors for in the study, the women had to be at least 1 year postmenopausal, have a genistein and daidzein, respectively). serum follicle stimulating hormone level of 430 U/ml, have a serum

1Department of Endocrinology, Royal North Shore Hospital, St Leonards, NSW, Australia; 2Northern Clinical School, University of Sydney, St Leonards, NSW, Australia; 3Menopause Clinic, Royal North Shore Hospital, St Leonards, NSW, Australia and 4NHMRC Clinical Trials Centre, University of Sydney, St Leonards, NSW, Australia. Correspondence: Professor P Clifton-Bligh, Department of Endocrinology, Royal North Shore Hospital, St Leonards, NSW 2065, Australia. E-mail: [email protected] Received 21 May 2014; revised 24 July 2014; accepted 16 August 2014; published online 5 November 2014 Red clover isoflavones, LDL cholesterol, Bone mineral density PB Clifton-Bligh et al 135 25OH-vitamin D level440 nmol/l and a BMD T-score 4 − 2.0 in the spine Randomization was performed in blocks of 6 in a 1:1 design. The study and hip. Nine participants whose serum 25OH-vitamin D levels were participants and all of the study staff members were blinded to the subsequently found to be less than 40 nmol/l were inadvertently treatment. randomized and continued in the trial. The mean 25OH-vitamin D levels The study was approved by the Royal North Shore Hospital Human at baseline and at 6, 12, 18 and 24 months were not significantly different Research Ethics Committee and conducted according to ICH-GCP guide- between active treatment and placebo groups. The mean age of lines. Each study participant provided written informed consent. participants was 54 ± 4 (s.d.) years. Participants were excluded if hormone replacement therapy had been taken within 2 months of screening into the study, if there was a history of Sample size calculation clinical vertebral fracture or other minimal trauma fracture or if they had The five primary outcome variables were the within-patient change from any intercurrent medical condition, such as celiac disease, primary baseline at 24 months for BMD of the spine, femoral neck, proximal and hyperparathyroidism, renal impairment or thyroid overactivity, that would distal radius and for serum LDL cholesterol. Differences between the active be likely to confound the study outcome. Participants were also excluded if and control arms were tested at the 1.0% significance level for each of they had previously taken bisphosphonates, strontium, fluoride or these outcomes in order to maintain an overall 5% significance level for raloxifene or if they were a vegetarian consuming more than 10 gm of the primary outcomes. Previous experience in our department suggested legume daily. None of the participants took any of these bone-preserving that the standard deviation of the within-patient percentage change from 10 medications during the study, and participants were instructed not to take baseline BMD at any of the four sites would not exceed 3.4%. A sample soy or red clover products during the 2-year study period and to keep a size of 65 per arm ensures 80% power to detect a difference of at least 2% food diary so that food intake could be assessed. between the mean within-patient percentage change from baseline A total of 183 participants were initially screened for the study; 30 observed in the active and control arms at 24 months. A sample size of 71 participants were excluded because of failure to meet the study’s per arm was recommended to allow for up to 10% discontinuation. inclusion/exclusion criteria and 6 participants withdrew consent before randomization. In all, 147 participants entered into the study and were Statistical analyses randomized: 75 participants in the active treatment group and 72 participants in the control group. Throughout the trial, the data were collected and collated by an A full medical examination was performed on each participant at entry independent manager. Novogen (Sydney, NSW, Australia), who supplied into the study and annually throughout the 2-year period. BMD the study medication, had no role in the collection or analysis of the study measurements were performed every 6 months on the AP spine (L2–L4) data or in the submission of the manuscript for publication. The analysis and femoral neck using the Norland XR36 densitometer (Fort Atkinson, WI, was carried out by an independent professional statistician, and all authors USA), and the proximal and distal radius using the Norland pDEXA scanner vouch for the integrity of the data. (Fort Atkinson, WI, USA). Both scanners were calibrated and an external The statistical software packages S-PLUS Version 8 (TIBCO Software Inc., phantom was used to monitor quality control on a daily basis. From 2002, Palo Alto, CA, USA) and SPSS version 21 (IBM Corporation, Armonk, NY, USA) because of the involvement of the site in an international clinical trial, an were used to analyze the data by the intention-to-treat principle. Two-tailed tests were used throughout. Each of the five primary outcome variables was additional external Hologic phantom (Bedford, MA, USA) was also fi incorporated into the Norland XR36 daily quality control regimen and tested at a signi cance level of 1% in order to maintain an overall 5% level. fi – Exploratory analyses were used to test each of the secondary outcomes at the monitored by Synarc (Portland, OR, USA) (coef cient of variation 0.26 fi – 0.55%). The monthly average measurements of the Hologic phantom 5% signi cance level. Two-sample t tests (or Mann Whitney tests for the between 2002 and 2007 varied between 99.6 and 100.4% of the highly skewed Lp(a) and ALT variables) were used to test for imbalances designated value. A thoracolumbar spine X-ray was performed at entry between the treatment arms at baseline (see Table 1). 11 fi into, and at the conclusion of, the study. Linear mixed-effects (LME) models were tted to the spine, femoral Fasting blood samples were collected every 6 months for the neck, proximal radius and distal radius BMD measurements, to the serum measurement of serum total cholesterol, high-density lipoprotein (HDL) LDL, HDL and total cholesterol, to the triglycerides, ApoA, ApoB, serum cholesterol, LDL cholesterol, ApoA, ApoB, triglycerides, Lp(a), serum calcium, serum 25OH-vitamin D, urine DPYR, hemoglobin and log- calcium and serum 25OH-vitamin D. Biochemical safety measures to transformed Lp(a) and ALT measurements and to the Greene score, systolic assess the safety of the active medication included serum ALT and and diastolic blood pressure measurements. Treatment (active or placebo), fi hemoglobin. A fasting 2-hour second void morning urine sample was also time and their two-way interaction were considered as xed effects. The fi collected every 6 months for the measurement of urine DPYR. patient identi er and time were considered as random effects. Time was fi A trans-vaginal ultrasound to measure endometrial thickness was considered as a factor with ve levels (baseline 0, 6, 12, 18 and 24 months). performed at entry into, and at the conclusion of, the study. In the LME models used to assess linear trends within the patient over time, – Study participants were also asked to monitor their menopausal time was considered as a continuous covariate. Normal Q Q plots were used fi symptoms throughout the study using a Greene Climacteric Scale9 diary; to assess whether the residuals from the tted models showed any obvious each of the 21 items in the Greene Climacteric Scale were graded 0–3in departure from the underlying assumption of normality. fi severity so that the maximum score possible was 63; and they were also The LME models included treatment and time as a ve-level factor. The encouraged to write down any adverse events or the use of concomitant two-way interaction between these terms provided effect estimates which fi medications in the diary. are summarized in Table 2. For all outcome measures, a signi cant Laboratory Assays: The serum cholesterol, HDL cholesterol, LDL interaction between the effects of treatment and time was interpreted as cholesterol, triglycerides, ApoA and ApoB were measured using the Roche evidence of a difference between the within patient changes observed modular assay system (Roche Diagnostics, Basel, Switzerland). The Lp(a) over time in both the control and active arms of the study. was measured initially with a Biopool ELISA assay (Umea, Sweden) and Many exploratory analyses of secondary outcome measures were fi subsequently with a Mercodia (Upsala, Sweden) ELISA assay (CSLS). Serum performed and false positive results may have occurred. Any such ndings fi 250H-vitamin D was measured between 2000 and 2005 using the Nichols should be treated with caution and con rmed in further independent Advantage (San Clemente, CA, USA) assay (inter-assay variation 10–14%) studies. and from the 22 July 2005 using the Diasorin Liaison (Saluggia, Italy) assay. The assays were cross-correlated (Diasorin Liaison = 1.07 Nichols+18, r = 0.794). The Nichols serum 25OD-vitamin D values were transformed RESULTS into Diasorin Liaison equivalents using this equation. Urinary DPYR was The patient characteristics at baseline by treatment arm are listed measured using the Siemens (Erlangen, Germany) Immulite assay with an in Table 1. With the exception of triglycerides (control group lower inter-assay variation of 9–15%. than active), there were no significant differences between Each study participant was asked to take two tablets of the study drug treatment arms at baseline. daily: this was either P-081 (marketed since as Rimostil), with each tablet containing 25 mg of isoflavone predominantly formononetin and biochanin, A total of 20 participants in the active arm withdrew from the or placebo, which was identical in shape, size and color. Both groups of study before study completion. Of these, 15 dropped out in the women were given supplemental calcium (CalSup) at a dose of 1000 mg first 6 months. In all, 30 participants in the control arm withdrew per day. Compliance with the study medication was assessed by pill counts from the study, and of these 19 dropped out in the first 6 months. at each study visit. The most common reason for withdrawal from the study was

© 2015 Macmillan Publishers Limited European Journal of Clinical Nutrition (2015) 134 – 142 Red clover isoflavones, LDL cholesterol, Bone mineral density PB Clifton-Bligh et al 136 10 participants, significant wedging in the thoracic spine in 3 Table 1. Patient characteristics (mean and s.d.) by treatment group at participants and significant wedging in the third lumbar vertebra baseline in 1 participant. Ninety participants had further X-rays at Month Variable Baseline values 24. Thoracic vertebral compression not previously present at the baseline X-ray was reported in five participants: two in the active Placebo Active P-valuea arm and three in the control arm. No incidence of a clinical vertebral fracture was reported in either arm. Mean s.d. Mean s.d. There was no significant evidence of any difference between treatments in the within-patient changes observed over time for Age 54.1 3.7 54.6 4.0 0.433 BMI 25.3 4.5 26.3 4.1 0.161 the primary outcomes of BMD of the spine, femoral neck, distal Systolic BP 126.5 18.3 126.7 18.0 0.948 radius or proximal radius (all P-values for the time by treatment Diastolic BP 76.1 9.0 75.3 9.1 0.627 interactions 40.25) (Table 2). Across both the active and control arms, there was a statistically significant decrease over time in the BMD BMD of the spine, femoral neck, distal radius and proximal radius Spine 1.04 0.14 1.02 0.12 0.357 (test for linear within patient trend P = 0.008, Po0.001, Po0.001 Femoral neck 0.90 0.11 0.86 0.11 0.060 and Po0.001, respectively). Proximal radius 0.76 0.07 0.77 0.08 0.944 Five participants, four in the active arm and one in the control Distal radius 0.33 0.05 0.32 0.05 0.188 arm, commenced statin therapy during the study. The lipid Lipids parameters of these participants were excluded from all lipid Total cholesterol 5.80 0.88 5.91 1.05 0.505 analyses presented in Table 2 (the lipid results were virtually Triglycerides 1.11 0.63 1.33 0.60 0.041 identical when the five participants were included). There was HDL cholesterol 1.82 0.49 1.67 0.35 0.052 significant evidence of a difference between treatments in the LDL cholesterol 3.43 0.86 3.68 0.94 0.125 within-patient changes observed over time for the primary ApoA 1.75 0.25 1.69 0.21 0.155 outcome of serum LDL cholesterol (P = 0.005 for time by treatment ApoB 0.99 0.24 1.05 0.25 0.172 interaction) and for serum total cholesterol (P = 0.015). In Lp (a) 207.00 252.00 271.00 294.00 0.139 particular, a significant reduction was seen at each time point for the active arm, and this reduction was significantly greater Other Serum calcium 2.33 0.10 2.35 0.11 0.128 than any change observed in the control arm at all time points Serum ALT 23.4 10.9 22.8 13.1 0.544 except 12 months for both serum LDL cholesterol and for serum Serum 25OH VitD 100.0 28.7 97.5 32.6 0.623 total cholesterol (Figure 2). DPYR 8.9 3.4 8.4 2.7 0.406 There was no significant evidence of any difference between Hemoglobin 134.5 8.0 133.9 7.6 0.643 treatments in the within-patient changes observed over time for Greene score 11.0 8.0 8.9 7.3 0.166 urinary DPYR or serum 25OH-vitamin D (Table 2). Across both the fi Abbreviations: BMI, body mass index; BMD, bone mineral density; BP, blood active and control arms, there was a statistically signi cant pressure; LDL, low-density lipoprotein; HDL, high-density lipoprotein; reduction in serum 25OH-vitamin D over time (test for linear DPYR, deoxypyridinoline. Units used: Age, years; BMD, g/cms2; total within-patient trend Po0.001). There was no significant evidence cholesterol, triglycerides, HDL and LDL, mmol/l; ApoA and ApoB, g/l; Lp (a), of any difference between treatments in the within-patient mg/l; serum calcium, mmol/l; serum ALP, U/l; serum 25OH-vitamin D, nmol/l; changes observed over time for serum calcium, serum ALT (as a DPYR (urine deoxypyridinoline), nmol/mmol creatinine; Hemoglobin, g/l. marker of liver function) and hemoglobin, nor was there any a two-sample t test (or Mann–Whitney test for the skewed lipoprotein significant overall linear within-patient trend with time in any of (a) and ALT). these variables (data not shown). There was no significant evidence of any difference between worsening of menopausal symptoms. Study participants were not treatments in the within-patient changes observed over time for told to expect any change in the frequency of hot flushes during serum HDL cholesterol or for serum triglycerides (Table 2). Linear the study period. A total of 55 participants in the active arm and trend analyses showed a significant overall within-patient increase fi 42 in the control arm completed the 2-year study as per protocol. in HDL cholesterol and a signi cant overall within-patient Figure 1. decrease in serum triglycerides with time, neither of which fi o If patients dropped out of the study in the first few weeks, they differed signi cantly by treatment (P 0.001 and P = 0.012, respectively, for linear within-patient trend with time across active were excluded from the pill count analysis. Not all the patients fi were diligent in returning their study medications at the end of and control arms). There was no signi cant evidence of any each 6-month period. Where data were available, compliance was difference between treatments in the within-patient changes observed over time for serum ApoA, ApoB or serum Lp(a) (time by almost identical in the active arm (88% ± 15%) and in the control treatment interaction P = 0.744, 0.056 and 0.089, respectively). arm (89 ± 13%). In all, 21 of those receiving active treatment and Coagulation factors were measured in 15 participants in the 29 of those receiving placebo did not record any pill count. fi fl active arm and 11 in the placebo arm. There was no signi cant Urinary iso avones were not measured in this study. difference between the two groups for the prothrombin time, In all, 110 participants had a pelvic ultrasound at the time of APTT, serum fibrinogen and factor VIII (data not shown). o entry into the study. Endometrial thickness was normal ( 6 mm) There was no significant evidence of a difference between in all except one participant, where it was noted to be 9 mm. treatments in the within-patient changes observed over time for ’ A curette was performed by the participant s gynecologist; the Greene climacteric scores (time by treatment interaction endometrial histology was found to be normal and the participant P = 0.855). The average body mass index of the control group at continued in the study. Fifty-eight participants had a further pelvic the completion of the study was 25.3 ± 4.1 (s.d.) compared with ultrasound at Month 24. One patient in the placebo arm had an 26.3 ± 4.2 (s.d.) in the active group. (P = 0.150). Significant within- endometrial thickness of 11 mm; a repeat ultrasound was patient decreases from baseline to month 24 were observed for performed a few months later and was normal. systolic and diastolic BP in both the active and control arms A total of 142 participants had an X-ray of their thoracolumbar (Po0.05 in all instances). However, the decreases were compar- spine at the baseline visit. Vertebral wedging was reported in 14 able in both arms (P = 0.703 for systolic and P = 0.540 for diastolic participants: minor wedging in the thoracic spine was reported in BP) (Table 2).

European Journal of Clinical Nutrition (2015) 134 – 142 © 2015 Macmillan Publishers Limited 05McilnPbihr iie uoenJunlo lnclNtiin(05 134 (2015) Nutrition Clinical of Journal European Limited Publishers Macmillan 2015 ©

Table 2. Estimated mean within-patient change from baseline and associated 95% CI from the linear mixed-effects model involving time, treatment and their interaction

Variable Treatment Change from baseline at 6mths Change from baseline at 12mths Change from baseline at 18mths Change from baseline at 24mths Time by Treatment interaction P-valuea Mean (95%CI) P-valueb Mean (95%CI) P-valueb Mean (95%CI) P-valueb Mean (95%CI) P-valueb

BMD Spine Placebo 0.005 (−0.007 to 0.017) 0.353 − 0.007 (−0.023 to 0.009) 0.236 − 0.006 (−0.023 to 0.011) 0.791 − 0.019 (−0.037 to − 0.0002) 0.523 0.554 Active 0.013 (0.001 to 0.024) 0.006 (−0.009 to 0.022) − 0.003 (−0.019 to 0.013) − 0.010 (−0.028 to 0.007) Femoral neck Placebo 0.008 (−0.003 to 0.019) 0.321 − 0.010 (−0.023 to 0.004) 0.288 − 0.011 (−0.024 to 0.001) 0.602 − 0.016 (−0.030 to − 0.003) 0.754 0.265 Active 0.0004 (−0.010 to 0.011) 0.000 (−0.012 to 0.013) − 0.007 (−0.018 to 0.004) − 0.013 (−0.025 to − 0.001) Proximal radius Placebo − 0.006 (−0.017 to 0.005) 0.404 − 0.006 (−0.015 to 0.003) 0.782 − 0.017 (−0.025 to − 0.009) 0.960 − 0.017 (−0.025 to − 0.009) 0.983 0.942 Active − 0.012 (−0.022 to − 0.002) − 0.008 (−0.016 to 0.0003) − 0.017 (−0.024 to − 0.010) − 0.017 (−0.024 to − 0.009) Distal radius Placebo − 0.003 (−0.008 to 0.002) 0.151 − 0.005 (−0.010 to −0.001) 0.289 − 0.010 (−0.016 to −0.004) 0.105 − 0.010 (−0.016 to − 0.004) 0.414 0.468 Active 0.002 (−0.003 to 0.007) − 0.002 (−0.006 to 0.002) − 0.004 (−0.009 to 0.002) − 0.007 (−0.012 to − 0.001)

Lipids Total cholesterol Placebo − 0.151 (−0.245 to 0.043) 0.018 − 0.265 (−0.487 to − 0.043) 0.741 − 0.128 (−0.245 to 0.090) 0.033 − 0.113 (−0.207 to 0.081) 0.07 0.015 Active − 0.477 (−0.663 to − 0.291) − 0.316 (−0.527 to − 0.105) − 0.457 (−0.666 to − 0.248) − 0.361 (−0.546 to − 0.176) Trigycerides Placebo 0.039 (−0.099 to 0.176) 0.905 − 0.046 (−0.179 to 0.088) 0.375 − 0.019 (−0.181 to 0.144) 0.957 − 0.056 (−0.167 to 0.055) 0.137 0.560 Active 0.027 (−0.103 to 0.157) − 0.130 (−0.258 to − 0.001) − 0.025 (−0.182 to 0.132) − 0.173 (−0.279 to − 0.066) HDL Placebo − 0.061 (−0.137 to 0.016) 0.485 − 0.042 (−0.113 to 0.030) 0.277 0.015 (−0.068 to 0.099) 0.592 0.049 (−0.033 to 0.132) 0.521 0.141 Active − 0.098 (−0.172 to − 0.025) 0.013 (−0.055 to 0.081) − 0.016 (−0.096 to 0.064) 0.087 (0.008 to 0.165) LDL Placebo − 0.088 (−0.282 to 0.107) 0.008 − 0.137 (−0.352 to 0.078) 0.386 − 0.121 (−0.294 to 0.099) 0.031 − 0.067 (−0.244 to 0.111) 0.004 0.005 Active − 0.456 (−0.643 to − 0.270) − 0.268 (−0.471 to − 0.006) − 0.455 (−0.663 to − 0.274) − 0.432 (−0.600 to − 0.263) Apo A Placebo − 0.074 (−0.132 to − 0.016) 0.579 − 0.084 (−0.142 to − 0.026) 0.911 − 0.049 (−0.108 to 0.010) 0.369 − 0.018 (−0.069 to 0.033) 0.346 0.744 e lvriso clover Red Active − 0.097 (−0.153 to − 0.040) − 0.079 (−0.134 to − 0.024) − 0.086 (−0.143 to − 0.029) − 0.052 (−0.101 to − 0.003) Clifton-Bligh PB Apo B Placebo 0.018 (−0.017 to 0.062) 0.013 − 0.059 (−0.111 to −0.006) 0.484 − 0.016 (−0.064 to 0.032) 0.056 − 0.032 (−0.080 to 0.016) 0.066 0.056 Active − 0.061 (−0.104 to − 0.018) − 0.085 (−0.134 to − 0.035) − 0.081 (−0.127 to − 0.035) − 0.095 (−0.141 to − 0.049) log(Lipo (a)) Placebo 0.032 (−0.067 to 0.131) 0.091 0.079 (−0.028 to 0.186) 0.185 − 0.009 (−0.103 to 0.116) 0.019 0.153 (0.009 to 0.298) 0.029 0.089 Active − 0.086 (−0.182 to 0.009) − 0.022 (−0.126 to 0.082) − 0.214 (−0.333 to − 0.105) − 0.074 (−0.218 to 0.007)

Other fl vns D hlseo,Bn iea density mineral Bone cholesterol, LDL avones, Serum calcium Placebo 0.005 (−0.024 to 0.034) 0.013 − 0.002 (−0.035 to 0.030) 0.193 − 0.005 (−0.034 to 0.023) 0.705 − 0.006 (−0.037 to 0.026) 0.220 0.010 al et Active − 0.046 (−0.073 to − 0.020) − 0.032 (−0.063 to − 0.001) 0.002 (−0.025 to 0.029) − 0.033 (−0.062 to − 0.003) log(ALT) Placebo 0.035 (−0.069 to 0.139) 0.188 − 0.010 (−0.096 to 0.076) 0.753 0.048 (−0.072 to 0.168) 0.262 − 0.041 (−0.130 to 0.048) 0.404 0.627 Active − 0.061 (−0.159 to 0.037) − 0.029 (−0.110 to 0.052) − 0.047 (−0.160 to 0.066) − 0.093 (−0.177 to −0.009) Serum 25OH VitD Placebo − 3.913 (−10.293 to 2.467) 0.461 − 12.660 (−19.912 to − 5.408) 0.131 − 11.655 (−20.381 to − 2.929) 0.051 − 17.970 (−24.929 to − 11.012) 0.452 0.338 Active − 0.686 (−6.679 to 5.307) − 5.172 (−11.888 to 1.544) 0.059 (−8.096 to 8.214) − 14.383 (−20.886 to − 7.879) DPYR Placebo − 1.889 (−2.854 to − 0.924) 0.252 − 2.140 (−3.047 to − 1.333) 0.021 − 0.687 (−1.919 to 0.546) 0.802 − 1.266 (−2.126 to − 0.406) 0.87 0.051 Active − 1.106 (−2.031 to − 0.180) − 0.664 (−1.420 to 0.192) − 0.901 (−2.039 to 0.236) − 1.167 (−1.983 to − 0.350) Hemoglobin Placebo − 1.957 (−3.397 to − 0.517) 0.103 − 2.024 (−3.658 to − 0.389) 0.188 − 1.105 (−3.121 to 0.911) 0.689 − 2.549 (−4.216 to − 1.381) 0.021 0.061 Active − 0.309 (−1.666 to 1.048) − 0.527 (−2.037 to 0.984) − 0.543 (−2.422 to 1.337) 0.153 (−1.406 to 1.711) Greene score Placebo NA 0.099 (−1.945 to 2.142) 0.399 0.610 (−1.497 to 2.717) 0.567 0.782 (−1.548 to 3.112) 0.660 0.855 Active NA 1.313 (−0.627 to 3.253) 1.459 (−0.534 to 3.453) 1.501 (−0.694 to 3.696) Systolic BP Placebo NA NA NA − 6.1 (−10.7 to − 1.6) 0.703 0.703 Active NA NA NA − 5.0 (−8.7 to − 1.4) Diastolic BP Placebo NA NA NA − 4.1 (−6.5 to − 1.7) 0.540 0.540 Active NA NA NA − 3.0 (−5.6 to −0.4) Abbreviations: BMD, bone mineral density; BP, blood pressure; CI, confidence interval; DPYR, deoxypyridinoline; HDL, high-density lipoprotein; LDL, low-density lipoprotein. atest of difference between active and placebo within-patient response to treatment over time. btest of difference between active and placebo within-patient change from baseline at specified time. – 142 137 Red clover isoflavones, LDL cholesterol, Bone mineral density PB Clifton-Bligh et al 138

Enrolled Excluded n=183 n=36 Failure to meet inclusion criteria

Randomized: Yes Intention-to-treat:Yes

Allocation n=147

Placebo Active Treatment n=72 n=75

6-month 6-month follow-up follow-up

Lost to follow up: n=16 Lost to follow-up: n=15 Analyzed: n=56 Analyzed: n=60

12-month 12-month follow-up follow-up

Lost to follow-up: n=9 Lost to follow-up: n=4 Analyzed: n=47 Analyzed: n=56

18-month 18-month follow-up follow-up

Lost to follow-up: n=4 Lost to follow-up: n=1 Analyzed: n=43 Analyzed: n=55

24-month 24-month follow-up follow-up

Lost to follow-up: n=1 Lost to follow-up: n=0 Analyzed: n=42 Analyzed: n=55

Figure 1. Flowchart of study participants and subject participation.

Adverse events abnormal LFTs of unknown cause, which recovered sponta- Several serious adverse events occurred during the study: in the neously, fractured L4 and fractured ribs (all n = 1). active arm, one participant withdrew 3 weeks after randomization Sinusitis, upper respiratory tract infection, urine tract infection, with a diagnosis of colon carcinoma, and another participant in muscle and joint pain were common in both treatment arms. this arm lost 22 kg in weight, and despite full investigation as to Several participants were troubled by constipation. other probable causes, may have been due to voluntary excessive There was no instance of breast cancer in either treatment arm, caloric restriction. Other reported adverse events in the active arm and although participants were encouraged to maintain regular include biopsy-confirmed benign thyroid nodules (n = 2), bowel government-funded 2-yearly mammograms, systematic mammo- obstruction (n = 2, one small bowel and one large bowel) and type grams were not carried out during the study. 2 diabetes, duodenal ulcer, gastric ulcer and celiac disease (all n = 1). In the control arm, one participant suffered a subarachnoid hemorrhage after a fall from which she recovered. Another patient DISCUSSION died of a ruptured berry aneurysm. Other reported adverse events The findings of the studies showing an increased risk of breast in the placebo arm include superficial thrombophlebitis (n = 2), cancer and cardiovascular disease in postmenopausal women biopsy-confirmed benign thyroid nodules (n = 2), deep-vein receiving estrogen and progesterone therapy have led to the thrombosis, diverticulitis, gastric ulcer, biliary colic, cholecystitis, search for alternative agents, which might retain the benefits of

European Journal of Clinical Nutrition (2015) 134 – 142 © 2015 Macmillan Publishers Limited Red clover isoflavones, LDL cholesterol, Bone mineral density PB Clifton-Bligh et al 139 Another potential benefit of isoflavones might be to lower serum LDL cholesterol. The lipid-lowering effects of isoflavones have been variable in postmenopausal women, summarized in Table 4. In most studies using soy isoflavones, there was no change in serum LDL cholesterol,25,31,38–41 whereas a significant fall occurred in two studies.33,42 A meta-analysis of studies using soy isoflavones43 showed a significant, albeit small, reduction in serum LDL of 0.13 mmol/l (Po0.0001). With red clover isoflavones, there was no change in serum LDL cholesterol in five studies44–48 and a significant fall of 11.6% in one study.49 In our study using red clover isoflavones enriched with formononetin at 50 mg/day and conducted over a 24-month period, there was a significant fall in the serum LDL cholesterol, which was first Figure 2. LDL cholesterol measurements by time in the active observed at 6 months and maintained at 24 months. The serum treatment group (closed bars) and the placebo group (open bars). LDL cholesterol in the treated arm fell by 12% from 3.68 mmol/l to Using a linear mixed-effects model, there was significant evidence of 3.25 mmol/l, a drop of 0.43 (95% CI -0.26–0.60) versus a a difference between treatments in the within-patient change nonsignificant decline in the control arm of 0.07 ((95% CI -0.11- observed over time (P=0.005). 0.24). Gould,50 in a large population study, showed that for every estrogen therapy, with a concomitant reduction in the risk of 1 mmol/l reduction in serum LDL cholesterol there was a 15.6% adverse effects. reduction in all-cause mortality and a 28% reduction in coronary Estrogen exerts its effects by binding to ERα and ERβ6,12 and heart disease mortality. Bush et al.51 followed up women aged 40- also by influencing the function of cell kinases through interaction 49 years for 8.5 years and there was a significant reduction in with the estrogen receptor.13 ERβ is found predominantly in bone, death from cardiovascular disease in estrogen users (RR = 0.34), whereas ERα is significantly expressed in the breast and and the reduction in mortality was related to a rise in the HDL endometrium but also significantly in bone. It is ERβ and not cholesterol. When statins were used to reduce serum LDL ERα that mediates the reduction in sclerostin with changes in cholesterol, for a 1 mmol/l reduction in serum LDL cholesterol, estradiol and mechanical loading.14 the risk ratio for mortality was 0.79 irrespective of the baseline Isoflavones have estrogen-like activity and bind preferentially to serum cholesterol and the presence or absence of previous ERβ but with a much lower affinity than estradiol.6 Soy and red vascular disease.52 In a Lipid Research Clinics’ coronary primary clover extracts contain isoflavones; soy isoflavones consist prevention trial in men, a 12.6% reduction in serum LDL predominantly of genistein and daidzein, whereas red clover cholesterol was associated with a 19% reduction in myocardial isoflavones consist predominantly of formononetin and biochanin. infarction.53 In a Shanghai cohort of middle- and older-aged Biochanin is converted into genistein and formononetin into women, the risk of coronary heart disease fell by 75% in those daidzein, and it is not certain whether formononetin and consuming a high-soy protein diet,54 although this benefit cannot biochanin have any intrinsic biologic activity before conversion. be attributed to the impact of isoflavones specifically. Isoflavones Genistein has 20–30 times the binding affinity for ERβ compared fed to cynomolgus monkeys was associated with a significant with ERα. The binding affinity of biochanin to estrogen receptors is reduction in the extent of atherosclerosis.55 10 000 times less than estradiol.6 Formononetin stimulated ERβ- The exact mechanism whereby isoflavones may lower serum dependent beta galactosidase production to a greater extent than LDL cholesterol is unclear. In ovariectomized rats deprived of via ERα.15 However, formononetin stimulated osteoblast differ- estradiol, hepatic expression of LDL receptor and PcsK9 is reduced entiation via the MAP kinase pathway and did not activate the and restored by estradiol supplementation.56 osteoblast ER.16 Genistein also enhanced osteoblast differentiation Soy isoflavones at 91 mg/day for 2.7 years reduced the rate of by the p38 MAP kinase pathway17 and reduced the RANKL/OPG progression of atherosclerosis, as measured by a change in carotid mRNA expression ratio in human osteoblasts.18 artery intima-media thickness in postmenopausal women within 5 Early studies with soy isoflavones in ovariectomized rats years of the onset of menopause.57 Promensil and formononetin showed the prevention of trabecular and cortical bone loss.19,20 improved systemic arterial compliance.45,58 Aortic stiffness Biochanin maintained femoral BMD and formononetin restored measured as pulse-wave velocity is strongly associated with femoral and tibial trabecular architecture21,22 in ovariectomized atheroclerosis59 and with cardiovascular mortality,59,60 and thus rats. Red clover isoflavones, however, did not reduce bone cardiovascular benefits of isoflavone therapy may occur indepen- resorption from bone prelabeled with 41Ca.23 Treatment of dently of any change in serum LDL cholesterol. postmenopausal women with soy isoflavones and red clover We did find a significant lowering of blood pressure over the 24- isoflavones have produced variable results. A summary of the month period of our study, but there was no significant difference effect of isoflavones on BMD is shown in Table 3.24–35 Some between the active treatment and control groups. Red clover studies showed a preservation of particularly hip BMD,29–33 isoflavones enriched with formononetin61 and a metabolite of especially with genistein.29–31 Red clover isoflavones enriched formononetin62 reduced systolic and diastolic blood pressure in with biochanin were associated with preservation of spinal BMD.35 postmenopausal women and formononetin had an antihyperten- In our present study using red clover isoflavones enriched with sive effect in spontaneously hypertensive male rats.63 formononetin, there was no impact on the rate of loss of BMD The isoflavone genistein, 54 mg/day, significantly reduced the over a 24-month period at the spine, hip and forearm, which frequency of hot flushes in postmenopausal women.30,31 The red proceeded at a rate expected in the postmenopausal state. Zhang clover extract, Promensil, reduced the frequency of hot flushes.64 et al.36 showed in a large Shanghai population study that high Rimostil, 82 mg/day, given over a 12-week period had no impact dietary soy protein intake was associated with a significant on the frequency of hot flushes,65 and in our study, Rimostil, reduction in fracture rate. A typical soy diet consists of 15–50 mg/ 50 mg/day, also had no significant effect on menopausal day of isoflavones.37 Many of the studies using soy isoflavones symptoms evaluated by the Green Climacteric Scale. involved much higher doses of isoflavones rich in genistein and There are several possible limitations to the interpretation of the daidzein. It is possible that other factors in soy extract apart from results. The number of participants recruited may not have been isoflavones may have a role in reducing incident fractures. large enough to allow the ascertainment of significant differences

Table 3. Effects of isoﬂavones on bone

Isoﬂavone type Dose (mg/d) Duration in weeks Skeletal effects

Soy protein 40 24 BMD increased24 Soy isoflavones 110 52 BMD loss unchanged25 Genistein, Daidzein 300 104 BMD loss unchanged26 Soy isoflavones 200 104 BMD loss unchanged27 Soy isoflavones 105 52 BMD unchanged28 Genistein 54 104 BMD increased at femoral neck29,30 Genistein 93 104 BMD loss at femoral neck slightly reduced31 Soy isoflavones 120 156 BMD loss at femoral neck reduced32 Soy isoflavones 165 104 BMD loss of total hip reduced33 Soy isoflavones 120 104 Whole-body BMD loss slightly reduced34 Red clover isoflavones enriched with biochanin 43 52 BMD spine preserved35 Red clover isoflavones enriched with formononetin 50 104 BMD spine, hip and forearm not preserved (current study) Abbreviation: BMD, bone mineral density.

Table 4. Effects of Isoﬂavones on LDL Cholesterol

Isoﬂavone type Dose (mg/d) Duration in weeks LDL cholesterol effects

Soy isoflavones 110 52 No change25 Soy isoflavones (soy protein mostly genistein) 93 52 No change31 Soy isoflavones 165 104 6% reduction33 Soy isoflavones 80, 120 104 No change38 Soy isoflavones 75 52 No change39 Soy isoflavones (soy protein) 40 12 0.60 mm/l reductionl42 69 Genistein 54 52 No change40 Soy germ isoflavones (50–60% daidzein) 84, 126 12,24 No change41 Red clover isoflavones 43, 86 10 No change44 Red clover isoflavones biochanin enriched 40 10 No change45 80 Red clover isoflavones enriched with formononetin 40 6 No change46 Red clover isoflavones 80 13 11.6% reduction49 Red clover isoflavones enriched with formononetin 57.2 12 No change47 Red clover enriched with biochanin 43 52 No change48 Red clover isoflavones formononetin enriched 50 104 12% reduction (current study) Abbreviation: LDL, low-density lipoprotein.

in BMD between the two treatment groups. However, the effect CONFLICT OF INTEREST sizes measured for within-person changes would suggest that The authors declare no conflict of interest. even if much larger numbers were studied a significant benefitof Rimostil on BMD would not have been seen. The larger number ACKNOWLEDGEMENTS of dropouts from the inactive treatment group versus the active treatment group may have skewed results in an Study medication used in the trial was supplied by Novogen Ltd. The cost of the unpredictable way. biochemical testing was met by Novogen Ltd. In summary, the present study has shown that, compared with placebo, 50 mg/day Rimostil significantly lowers the primary DISCLAIMER outcome variable of serum LDL cholesterol over 24 months. The A partial professional salary was paid to MLN, the study improvement is evident by 6 months. Our previous study8 also fi coordinator who recruited and supervised all study participants. showed a signi cant reduction in serum LDL cholesterol with None of the other authors received any payment from Novogen Rimostil. In contrast to our previous study, the use of Rimostil Ltd. None of the authors or members of their families hold any fi compared with placebo did not signi cantly alter the loss of BMD stock in Novogen Ltd. This double-blind, placebo-controlled study in the forearm, or in the lumbar spine and femoral neck. It is was not registered with the clinical trials registry because the fl β possible that the binding of the iso avones in Rimostil to the ER study commenced before the requirement was introduced to was too weak to prevent loss of BMD. There was no adverse register all clinical trials. effect on endometrial thickness. The constellation of adverse effects in patients taking Rimostil was no different from those seen in the control group. It is uncertain whether the fall in the AUTHOR CONTRIBUTIONS serum LDL cholesterol might lead to a change in the risk of PBC-B, GRF and RB developed the overall research plan and cardiovascular disease, or whether the observed fall in the serum designed the protocol. MLN was responsible for participant LDL cholesterol with Rimostil would augment any fall in the recruitment and for the progress of each participant through serum LDL cholesterol brought about by other agents such as the study protocol to the time of completion or withdrawal. PBC-B, statins. RJC-B, SV, GRF and RB were responsible for ongoing clinical

European Journal of Clinical Nutrition (2015) 134 – 142 © 2015 Macmillan Publishers Limited Red clover isoflavones, LDL cholesterol, Bone mineral density PB Clifton-Bligh et al 141 assessment of each participant throughout the trial. PBC-B and KB 22 Tyagi AM, Srivastava K, Singh AK, Kumar A, Changkija B, Pandey R et al. made major contributions to the writing of the manuscript and Formononetin reverses established osteopenia in adult ovariectomized rats. take responsibility for the validity of the data. KB performed Menopause 2012; 19:856–863. statistical analyses of the data. SV died during the course of the 23 Weaver CM, Martin BR, Jackson GS, McCabe GP, Nolan JR, McCabe LD et al. study. All other authors have read and approved the final Antiresorptive effects of phytoestrogen supplements compared with estradiol or manuscript. risedronate in postmenopausal women using 41Ca methodology. 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