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Lipids and cardiovascular/metabolic health

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

Abstract

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).

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References

  1. Kannel WB, Hjortland MC, McNamara PM, Gordon T . Menopause and risk of cardiovascular disease: the Framingham study. Ann Intern Med 1976; 85: 447–452.

    Article  CAS  Google Scholar 

  2. Hodis HN, Mack WJ . A ‘window of opportunity’: the reduction of coronary heart disease and total mortality with menopausal therapies is age and time-dependent. Brain Res 2011; 1379: 244–252.

    Article  CAS  Google Scholar 

  3. Rossouw JE, Prentice RL, Manson JE, Wu L, Barad D, Barnabei VM et al. Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA 2007; 297: 1465–1477.

    Article  CAS  Google Scholar 

  4. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML et al. Writing group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women. JAMA 2002; 288: 312–333.

    Google Scholar 

  5. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women, Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA 1998; 280: 605–613.

    Article  CAS  Google Scholar 

  6. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, Vandersaag PT et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998; 139: 4252–4263.

    Article  CAS  Google Scholar 

  7. Setchell KD, Clerici C, Lephart ED, Cole SJ, Heenan C, Castellanni D et al. S-Equol, a potent ligand for estrogen receptor β is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am J Clin Nutr 2005; 81: 1072–1079.

    Article  CAS  Google Scholar 

  8. Clifton-Bligh PB, Baber RJ, Fulcher GR, Nery ML, Moreton T . The effect of isoflavones extracted from red clover (Rimostil®) on lipid and bone metabolism. Menopause 2001; 8: 259–265.

    Article  CAS  Google Scholar 

  9. Greene JG . Constructing a standard climacteric scale. Maturitas 1998; 29: 25–31.

    Article  CAS  Google Scholar 

  10. Cooper L, Clifton-Bligh PB, Nery ML, Figtree G, Twigg S, Hibbert E et al. Vitamin D supplementation and bone mineral density in early postmenopausal women. Am J Clin Nutr 2003; 77: 1324–1328.

    Article  CAS  Google Scholar 

  11. Fitzmaurice GM, Laird NM, Ware JM . Applied Longitudinal Analysis 2004.

  12. Hall JM, McDonnell DP . The estrogen receptor beta-isoform (ER beta) of the human estrogen receptor modulates ER alpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 1999; 140: 5566–5578.

    Article  CAS  Google Scholar 

  13. Kousteni S, Almeida M, Han L, Bellido T, Jilka R, Manolagas SC . Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor. Mol Cell Biol 2007; 27: 1516–1530.

    Article  CAS  Google Scholar 

  14. Galea GL, Meakin LB, Sugiyama T, Zebda N, Sunters A, Taipaleenmaki H et al. Estrogen receptor α mediates proliferation of osteoblastic cells stimulated by estrogen and mechanical strain, but their acute down-regulation of the Wnt antagonist Sost is mediated by estrogen receptor β. J Biol Chem 2013; 288: 9035–9048.

    Article  CAS  Google Scholar 

  15. Morito K, Aomori T, Hirose T, Kinjo K, Hasegawa J, Ogawa S et al. Interaction of phytoestrogens with estrogen receptors alpha and beta (11). Biol Pharm Bull 2002; 25: 48–52.

    Article  CAS  Google Scholar 

  16. Gautam AK, Bhargavan B, Tyagi AM, Srivastava K, Yadav DK, Kumar M et al. Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats. J Nutr Biochem 2011; 22: 318–327.

    Article  CAS  Google Scholar 

  17. Ming L-G, Chen K-M, Xian CJ . Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodelling. J Cell Physiol 2013; 228: 513–521.

    Article  CAS  Google Scholar 

  18. Karieb S, Fox SW . Phytoestrogens directly inhibit TNFα-induced bone resorption in RAW264.7 cells by suppressing c-fos-induced NFATc1 expression. J Cell Biochem 2011; 112: 476–487.

    Article  CAS  Google Scholar 

  19. Fanti P, Monier-Faugere MC, Genz Z, Schmidt J, Morris PE, Cohen D et al. The phytoestrogen genistein reduces bone loss in short-term ovariectomized rats. Osteoporosis Int 1998; 8: 274–281.

    Article  CAS  Google Scholar 

  20. Chang KL, Hu Y-C, Hsieh B-S, Cheng H-L, Hsu H-W, Huang L-W et al. Combined effect of soy isoflavones and vitamin D3 on bone loss in ovariectomized rats. Nutrition 2013; 29: 250–257.

    Article  CAS  Google Scholar 

  21. Su S-J, Yeh Y-T, Shyu H-W . The preventive effect of biochanin A on bone loss in ovariectomized rats: involvement in regulation of growth and activity of osteoblasts and osteoclasts. Evidence-based complementary and alternative medicine, published online 2013; 16: 594857.

    Google Scholar 

  22. Tyagi AM, Srivastava K, Singh AK, Kumar A, Changkija B, Pandey R et al. Formononetin reverses established osteopenia in adult ovariectomized rats. Menopause 2012; 19: 856–863.

    Article  Google Scholar 

  23. Weaver CM, Martin BR, Jackson GS, McCabe GP, Nolan JR, McCabe LD et al. Antiresorptive effects of phytoestrogen supplements compared with estradiol or risedronate in postmenopausal women using 41Ca methodology. J Clin Endocrinol Metab 2009; 94: 3798–3805.

    Article  CAS  Google Scholar 

  24. Potter SM, Baum JA, Teng H, Stillman R, Shay NF, Erdman JW Jr . Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr 1998; 68: 1375S–1379S.

    Article  CAS  Google Scholar 

  25. Brink E, Coxam V, Robins S, Wahala K, Cassidy A . Branca F on behalf of the PHYTOS investigators. Long-term consumption of isoflavone-enriched foods does not affect bone mineral density, bone metabolism, or hormonal status in early postmenopausal women: a randomized, double-blind, placebo-controlled study. Am J Clin Nutr 2008; 87: 761–770.

    Article  CAS  Google Scholar 

  26. Tai TY, Tsai KS, Tu ST, Wu JS, Chang CI, Chen CL et al. The effect of soy isoflavone on bone mineral density in postmenopausal Taiwanese women with bone loss: a 2-year randomized, double-blind, placebo-controlled study. Osteoporosis Int 2012; 23: 1571–1580.

    Article  CAS  Google Scholar 

  27. Levis S, Strickman-Stein N, Ganjei-Azar P, Xu P, Doerge DR, Krischer J . Soy isoflavones in the prevention of menopausal bone loss and menopausal symptoms: a randomized double-blind trial. Arch Intern Med 2011; 171: 1363–1369.

    Article  CAS  Google Scholar 

  28. Kenny AM, Mangano KM, Abourizk RH, Bruno RS, Anamani DE, Kleppinger A et al. Soy proteins and isoflavones affect bone mineral density in older women: a randomized controlled trial. Am J Clin Nutr 2009; 90: 234–242.

    Article  CAS  Google Scholar 

  29. Marini H, Minutoli L, Polito F, Bitto A, Altavilla D, Atteritano M et al. Effects of phytoestrogen genistein on bone metabolism in osteopenic postmenopausal women: a randomized trial. Ann Intern Med 2007; 146: 839–847.

    Article  Google Scholar 

  30. D’Anna R, Cannata ML, Marini H, Atteritano M, Cancellieri F, Corrado F et al. Effects of the phytoestrogen genistein on hot flushes, endometrium and vaginal epithelium in post-menopausal women: a 2-year randomized, double-blind, placebo-controlled study. Menopause 2009; 16: 301–306.

    Article  Google Scholar 

  31. Kreijkamp-Kaspers S, Kok L, Grobbee DE, de Haan EH, Aleman A, Lampe JW et al. Effect of soy protein containing isoflavones on cognitive function, bone mineral density, and plasma lipids in postmenopausal women: a randomized controlled trial. JAMA 2004; 292: 65–74.

    Article  CAS  Google Scholar 

  32. Alekel DL, Van Loan MD, Koehler KL, Hanson LN, Stewart JW, Hanson KB et al. The soy isoflavones for reducing bone loss (SIRBL) study: a 3-year randomized controlled trial in postmenopausal women. Am J Clin Nutr 2010; 91: 218–230.

    Article  CAS  Google Scholar 

  33. Chilibeck PD, Vatanparast H, Pierson R, Case A, Olatunbosun O, Whiting SJ et al. Effect of exercise training combined with isoflavone supplementation on bone and lipids in postmenopausal women: a randomized clinical trial. J Bone Miner Res 2013; 28: 780–793.

    Article  CAS  Google Scholar 

  34. Wong WW, Lewis RD, Steinberg FM, Murray MJ, Cramer MA, Amato P et al. Soy isoflavone supplementation and bone mineral density in menopausal women: a 2-year multicenter clinical trial. Am J Clin Nutr 2009; 90: 1433–1439.

    Article  CAS  Google Scholar 

  35. Atkinson C, Compston JE, Day NE, Dowsett M, Bingham SA . The effects of phytoestrogen isoflavones on bone density in women: a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr 2004; 79: 326–333.

    Article  CAS  Google Scholar 

  36. Zhang X, Shu X-O, Li H, Yang E, Li Q, Gao Y-T et al. Prospective cohort study of soy food consumption and risk of bone fracture among postmenopausal women. Arch Intern Med 2005; 165: 1890–1895.

    Article  Google Scholar 

  37. Setchell KD, Lydeking-Olsen E . Dietary phytoestrogens and their effect on bone, evidence from in vitro and in vivo, human observational, and dietary intervention studies. Am J Clin Nutr 2003; 78: 593S–609S.

    Article  CAS  Google Scholar 

  38. Steinberg FM, Murray MJ, Lewis RD, Cramer MA, Amato P, Young RL et al. Clinical outcomes of 2y soy isoflavone supplementation in menopausal women. Am J Clin Nutr 2011; 93: 356–367.

    Article  CAS  Google Scholar 

  39. Wu J, Oka J, Tabata I, Higuchi M, Toda T, Fuku N et al. Effects of isoflavone and exercise on BMD and fat mass in postmenopausal Japanese women: a 1-year randomized controlled trial. J Bone Miner Res 2006; 21: 780–789.

    Article  CAS  Google Scholar 

  40. Morabito N, Crisafulli A, Vergara C, Gaudio A, Lasco A, Frisina N et al. Effects of genistein and hormone- replacement therapy on bone loss in early postmenopausal women: a randomized double-blind, placebo-controlled study. J Bone Miner Res 2002; 17: 1904–1912.

    Article  CAS  Google Scholar 

  41. Ye Y-B, Wang Z-L, Zhuo S-Y, Lu W, Liao H-F, Verbruggen M et al. Soy germ isoflavones improve menopausal symptoms but have no effect on blood lipids in early postmenopausal Chinese women: a randomized placebo-controlled trial. Menopause 2012; 19: 791–798.

    Article  Google Scholar 

  42. Dalais FS, Ebeling PR, Kotsopoulos D, McGrath BP, Teede HJ . The effects of soy protein containing isoflavones on lipids and idices of bone resorption in postmenopausal women. Clin Endocrinol (Oxf) 2003; 58: 704–709.

    Article  CAS  Google Scholar 

  43. Taku K, Umegaki K, Sato Y, Taki Y, Endoh K, Watanabe S . Soy isoflavones lower serum total and LDL cholesterol in humans: a meta-analysis of 11 randomized controlled trials. Am J Clin Nutr 2007; 85: 1148–1156.

    Article  CAS  Google Scholar 

  44. Howes JB, Sullivan D, Lai N, Nestel P, Pomeroy S, West L et al. The effects of dietary supplementation with isoflavones from red clover on the lipoprotein profiles of postmenopausal women with mild to moderate hypercholesterolaemia. Atherosclerosis 2000; 152: 143–147.

    Article  CAS  Google Scholar 

  45. Nestel PJ, Pomeroy S, Kay S, Komesaroff P, Behrsing J, Cameron JD et al. Isoflavones from red clover improve arterial compliance but not plasma lipids in menopausal women. J Clin Endocrinol Metab 1999; 84: 895–898.

    CAS  PubMed  Google Scholar 

  46. Nestel P, Cehun M, Chronopoulos A, DaSilva L, Teede H, McGrath B . A biochanin-enriched isoflavone from red clover lowers LDL cholesterol in men. Eur J Clin Nutr 2004; 58: 403–408.

    Article  CAS  Google Scholar 

  47. Schult TM, Ensrud KE, Blackwell T, Ettinger B, Wallace R, Tice JA . Effect of isoflavones on lipids and bone turnover markers in menopausal women. Maturitas 2004; 48: 209–218.

    Article  Google Scholar 

  48. Atkinson C, Oosthuizen W, Scollen S, Loktionov A, Day NE, Bingham SA . Modest protective effects of isoflavones from red clover-derived dietary supplement on cardiovascular disease risk factors in perimenopausal women, and evidence of an interation with apoE genotype in 49-65 year old women. J Nutr 2004; 134: 1759–1764.

    Article  CAS  Google Scholar 

  49. Hidalgo LA, Chedraui PA, Morocho N, Ross S, San Miguel G . The effect of red clover isoflavones on menopausal symptoms, lipids and vaginal cytology in menopausal women: a randomized, double-blind, placebo-controlled trial. Gynecol Endocrinol 2005; 21: 257–264.

    Article  CAS  Google Scholar 

  50. Gould AL, Davies GM, Alemao E, Yin DD, Cook JR . Cholesterol reduction yields clinical benefits: meta-analysis including recent trials. Clin Ther 2007; 29: 778–794.

    Article  CAS  Google Scholar 

  51. Bush TL, Barrett-Connor E, Cowan LD, Criqui M, Wallace RB, Suchindran CM et al. Cardiovascular mortality and non-contraceptive use of estrogen in women: results from the lipid research clinics program follow-up study. Circulation 1987; 75: 1102–1109.

    Article  CAS  Google Scholar 

  52. Mihaylova B, Emberson J, Blackwell L, Keech A, Simes J, Barnes EH et al. The effects of lowering LDL cholesterol with statin therapy in people with low risk of vascular disease: meta-analysis of individual data from 27 randomized trials. Lancet 2012; 380: 581–590.

    Article  CAS  Google Scholar 

  53. Probstfield JL, Rifkind BM . The lipid research clinics coronary primary prevention trial: design, results, and implications. Eur J Clin Pharmacol 1991; 40: S69–S75.

    Article  Google Scholar 

  54. Zhang X, Shu XO, Gao YT, Yang G, Li Q, Li H et al. Soy food consumption is associated with lower risk of coronary heart disease in Chinese women. J Nutr 2003; 133: 2874–2878.

    Article  CAS  Google Scholar 

  55. Anthony MS, Clarkson TB, Bullock BC, Wagner JD . Soy protein versus soy phytoestrogens in the prevention of diet-induced coronary artery atherosclerosis of male cynomolgus monkeys. Arterioscler Thromb Vasc Biol 1997; 17: 2524–2531.

    Article  CAS  Google Scholar 

  56. Sockt ET, Chapados NA, Lavoie JM . LDL receptor and PCSK9 transcripts are decreased in liver of ovariectomized rats: Effects of exercise training. Horm Metab Res 2014; 46: 550–555.

    Article  Google Scholar 

  57. Hodis HN, Mack WJ, Kono N, Azen SP, Shoupe D, Hwang-Levine J et al. Isoflavone soy protein supplementation and atherosclerosis progression in healthy postmenopausal women: a randomized controlled trial. Stroke 2011; 42: 3168–3175.

    Article  CAS  Google Scholar 

  58. Teede HJ, McGrath BP, De Silva L, Cehun M, Fassoulakis A, Nestel PJ . Isoflavones reduce arterial stiffness. A placebo-controlled study in men and postmenopausal women. Arterioscler Thomb Vasc Biol 2003; 23: 1066–1071.

    Article  CAS  Google Scholar 

  59. Van Popele NM, Grobbee DE, Bots ML, Asmar R, Topouchian J, Reneman RS et al. Association between arterial stiffness and atherosclerosis: The Rotterdam study. Stroke 2001; 32: 454–460.

    Article  CAS  Google Scholar 

  60. Vlachopoulos C, Aznaouridis K, Stefanadis C . Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol 2010; 55: 1318–1327.

    Article  Google Scholar 

  61. Howes JB, Tran D, Brillante D, Howes LG . Effects of dietary supplementation with isoflavones from red clover on ambulatory blood pressure and endothelial function in postmenopausal type 2 diabetes. Diabetes Obes Metab 2003; 5: 325–332.

    Article  CAS  Google Scholar 

  62. Nestel P, Fujii A, Zhang L . An isoflavone metabolite reduces arterial stiffness and blood pressure in overweight men and postmenopausal women. Atherosclerosis 2007; 192: 184–189.

    Article  CAS  Google Scholar 

  63. Sun T, Wang J, Huang L-H, Cao Y-X . Antihypertensive effect of formononetin through regulating the expressions of eNOS, 5-HT 2A/1B receptors and α1-adrenoreceptors in spontaneously rat arteries. Eur J Pharmacol 2013; 699: 241–249.

    Article  CAS  Google Scholar 

  64. Van de Weijer PH, Barentsen R . Isoflavones from red clover (Promensil) significantly reduce menopausal hot flush symptoms compared with placebo. Maturitas 2002; 42: 187–193.

    Article  CAS  Google Scholar 

  65. Tice JA, Ettinger B, Ensrud K, Wallace R, Blackwell T, Cummings SR . Phytoestrogen supplements for the treatment of hot flashes: the isoflavones clover extract (ICE) study: a randomized controlled trial. JAMA 2003; 290: 207–214.

    Article  CAS  Google Scholar 

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Acknowledgements

Study medication used in the trial was supplied by Novogen Ltd. The cost of the biochemical testing was met by Novogen Ltd.

Disclaimer

A partial professional salary was paid to MLN, the study coordinator who recruited and supervised all study participants. None of the other authors received any payment from Novogen Ltd. None of the authors or members of their families hold any stock in Novogen Ltd. This double-blind, placebo-controlled study was not registered with the clinical trials registry because the study commenced before the requirement was introduced to register all clinical trials.

Author contributions

PBC-B, GRF and RB developed the overall research plan and designed the protocol. MLN was responsible for participant recruitment and for the progress of each participant through the study protocol to the time of completion or withdrawal. PBC-B, RJC-B, SV, GRF and RB were responsible for ongoing clinical assessment of each participant throughout the trial. PBC-B and KB made major contributions to the writing of the manuscript and take responsibility for the validity of the data. KB performed statistical analyses of the data. SV died during the course of the study. All other authors have read and approved the final manuscript.

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Correspondence to P B Clifton-Bligh.

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Clifton-Bligh, P., Nery, ML., Clifton-Bligh, R. et al. Red clover isoflavones enriched with formononetin lower serum LDL cholesterol—a randomized, double-blind, placebo-controlled study. Eur J Clin Nutr 69, 134–142 (2015). https://doi.org/10.1038/ejcn.2014.207

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