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Isopropanolic Cimicifuga racemosa is favorable on bone markers but neutral on an osteoblastic cell line

2008; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2008.03.042

1556-5653

Miguel Ángel García‐Pérez, Begoña Pineda, Carlos Hermenegildo, Juan J. Tarı́n, Antonio Cano,

Bone health and osteoporosis research

Postmenopausal women treated with an isopropanolic extract of Cimicifuga racemosa underwent a decrease in the urinary concentration of N-telopeptides, a marker of bone resorption, and an increase in alkaline phosphatase, a marker of bone formation, at the third month of therapy. Serum from treated women did not modify the activity of alkaline phosphatase or the expression of three genes, runt-related transcription factor-2 (Runx-2), alkaline phosphatase, and osteocalcin, when added to the MC3T3-E1 osteoblastic cell line. Postmenopausal women treated with an isopropanolic extract of Cimicifuga racemosa underwent a decrease in the urinary concentration of N-telopeptides, a marker of bone resorption, and an increase in alkaline phosphatase, a marker of bone formation, at the third month of therapy. Serum from treated women did not modify the activity of alkaline phosphatase or the expression of three genes, runt-related transcription factor-2 (Runx-2), alkaline phosphatase, and osteocalcin, when added to the MC3T3-E1 osteoblastic cell line. There is no equivalent alternative to hormone therapy (HT) in the control of menopause symptoms. However, many symptomatic women do not take HT due to either reservations about potential harmful effects of hormones or poor tolerance of the side effects (1Nelson H.D. Humphrey L.L. Nygren P. Teutsch S.M. Allan J.D. Postmenopausal hormone replacement therapy: scientific review.JAMA. 2002; 288: 872-881Crossref PubMed Scopus (897) Google Scholar). A series of "natural" alternatives has been proposed; among them, Cimicifuga racemosa (CR), or black cohosh, has received attention.The efficacy of CR in the correction of menopausal symptoms is a matter of debate (2Mahady G.B. Black cohosh (Actaea/Cimicifuga racemosa): review of the clinical data for safety and efficacy in menopausal symptoms.Treat Endocrinol. 2005; 4: 177-184Crossref PubMed Scopus (57) Google Scholar). Differences in the composition of the extracts of CR might explain some of the discrepancies. Some investigators have claimed that the CR extracts may have some bone-sparing effects. There is direct density data in rodents (3Seidlova-Wuttke D. Jarry H. Pitzel L. Wuttke W. Effects of estradiol-17β, testosterone and a black cohosh preparation on bone and prostate in orchidectomized rats.Maturitas. 2005; 51: 177-186Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and information on changes in bone metabolic parameters in humans or human cell lines (4Wuttke W. Gorkow C. Seidlova-Wuttke D. Effects of black cohosh (Cimicifuga racemosa) on bone turnover, vaginal mucosa, and various blood parameters in postmenopausal women: a double-blind, placebo-controlled, and conjugated estrogens-controlled study.Menopause. 2006; 13: 185-196Crossref PubMed Scopus (72) Google Scholar, 5Viereck V. Grundker C. Friess S.C. Frosch K.H. Raddatz D. Schoppet M. et al.Isopropanolic extract of black cohosh stimulates osteoprotegerin production by human osteoblasts.J Bone Miner Res. 2005; 20: 2036-2043Crossref PubMed Scopus (38) Google Scholar). The protective effects of CR have been described in osteoblasts, with increased production of osteoprotegerin (5Viereck V. Grundker C. Friess S.C. Frosch K.H. Raddatz D. Schoppet M. et al.Isopropanolic extract of black cohosh stimulates osteoprotegerin production by human osteoblasts.J Bone Miner Res. 2005; 20: 2036-2043Crossref PubMed Scopus (38) Google Scholar), and in osteoclasts, whose differentiation was inhibited (6Qiu S.X. Dan C. Ding L.S. Peng S. Chen S.N. Farnsworth N.R. et al.A triterpene glycoside from black cohosh that inhibits osteoclastogenesis by modulating RANKL and TNFα signaling pathways.Chem Biol. 2007; 14: 860-869Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar).Our study of the bone effects of an isopropanolic extract of CR (iCR) had two goals. First, we checked whether women taking the iCR preparation exhibited any changes in a panel of bone biochemical markers. Second, we used an osteoblastic cell line to explore whether serum from treated women was capable of stimulating the cellular function as well as the expression of three capital genes for bone formation: runt-related transcription factor-2 (Runx-2), alkaline phosphatase (ALP), and osteocalcin.We conducted a 3-month prospective trial on a group of women with surgical or natural menopause, defined as amenorrhea ≥1 year and follicle-stimulating hormone (FSH) levels >40 IU/L. Forty-five women were treated with 40 mg/day of iCR (Remifemin; Schaper & Brümmer, Ringelheim, Germany), and 37 women participated as untreated controls. Caucasian origin and low level of menopausal symptoms were additional conditions for eligibility. In addition to the information obtained from their medical history, the women were considered healthy according to a basic as well as a complete gynecologic evaluation and a routine blood analysis. All were nonsmokers, nondrinkers, and generally sedentary. Exclusion criteria were immobility, recent bone fractures ( 40 IU/L. Forty-five women were treated with 40 mg/day of iCR (Remifemin; Schaper & Brümmer, Ringelheim, Germany), and 37 women participated as untreated controls. Caucasian origin and low level of menopausal symptoms were additional conditions for eligibility. In addition to the information obtained from their medical history, the women were considered healthy according to a basic as well as a complete gynecologic evaluation and a routine blood analysis. All were nonsmokers, nondrinkers, and generally sedentary. Exclusion criteria were immobility, recent bone fractures (<1 year ago), or use of medication known to influence calcium metabolism. Compliance was checked with the use of diary cards. Our investigation conformed to the principles outlined in the Declaration of Helsinki, was approved by the institutional review board at our center, and written informed consent was obtained from all patients. At the beginning of the study and at the completion of the third month of the follow-up period, blood and urine samples were obtained (08:00 to 10:00 am) after an overnight fast. Serum and urine were immediately frozen at −80°C until assay. Serum was analyzed for routine biochemical parameters, including ALP and a complete lipid profile with an autoanalyzer (Olympus AV 5200; Olympus, Tokyo, Japan). Also in serum, intact parathyroid hormone (pg/mL) was measured by immunoradiometry (IRMA), and follicle-stimulating hormone (FSH), estradiol, and testosterone were measured by specific immunoassays as previously described elsewhere (7García-Pérez M.A. Moreno-Mercer J. Tarín J.J. Cano A. Similar efficacy of low and standard doses of transdermal estradiol in controlling bone turnover in postmenopausal women.Gynecol Endocrinol. 2006; 22: 179-184Crossref PubMed Scopus (18) Google Scholar, 8Oviedo P.J. Hermenegildo C. Tarin J.J. Cano A. Therapeutic dosages of raloxifene do not modify myeloperoxidase and F2α-isoprostane levels in postmenopausal women.Fertil Steril. 2005; 84: 1789-1792Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). Urine was analyzed for creatinine and total calcium with the use of the autoanalyzer Olympus AV 600 (Olympus). The urinary concentration of N-telopeptide of type I collagen (NTx) was measured by an enzyme-linked immunoabsorbent assay (ELISA), as previously described elsewhere (7García-Pérez M.A. Moreno-Mercer J. Tarín J.J. Cano A. Similar efficacy of low and standard doses of transdermal estradiol in controlling bone turnover in postmenopausal women.Gynecol Endocrinol. 2006; 22: 179-184Crossref PubMed Scopus (18) Google Scholar). Measurements of bone mineral density (BMD, g/cm2) were performed at the lumbar spine from L2 to L4 (LS BMD) and at the nondominant proximal femoral neck (FN BMD) with the use of a dual-energy x-ray absorptiometry (DEXA) system (Norland XR-36; Norland Medical Systems Inc., Fort Atkinson, WI). The mouse cell line MC3T3-E1 was used to clarify any effect of iCR extract on osteoblastic function. To that end, MC3T3-E1 cells were grown according to previously published conditions (9Matsuzaki E. Takahashi-Yanaga F. Miwa Y. Hirata M. Watanabe Y. Sato N. et al.Differentiation-inducing factor-1 alters canonical Wnt signaling and suppresses alkaline phosphatase expression in osteoblast-like cell lines.J Bone Miner Res. 2006; 21: 1307-1316Crossref PubMed Scopus (36) Google Scholar); when at 90% of confluence, serum from women was added to the medium. After 12 days of culture, the medium was removed, and ALP activity and protein content were measured in the supernatant from sonicated cells. To further confirm that we were using fully mature osteoblasts, a parallel series of experiments was performed in which MC3T3-E1 cells were treated with β-glycerophosphate (10 mM) and ascorbic acid (50 μg/mL), two osteoblastic differentiation inductors. The activity of ALP was assayed in triplicate by a commercial kit (THERMO Electron Corporation, Louisville, KY) following the manufacturer's instructions. Also in MC3T3-E1 cells, we used real-time polymerase chain reaction (RT-PCR) to quantify the expression of three genes related with osteoblastic differentiation and function, Runx-2, ALP, and osteocalcin. The extraction of RNA as well as the reverse transcription procedure was performed as previously described (10Garcia-Perez M.A. Del V.R. Noguera I. Hermenegildo C. Pineda B. Martinez-Romero A. Cano A. Estrogen receptor agonists and immune system in ovariectomized mice.Int J Immunopathol Pharmacol. 2006; 19: 807-819Crossref PubMed Scopus (21) Google Scholar). The primers used for the PCR reaction were glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense 5′-ACC ACA GTC CAT GCC ATC AC-3′ and antisense 5′-TCC ACC ACC CTG TTG CTG TA-3′; Runx-2 sense 5′-CCG CAC GAC AAC CGC ACC AT-3′ and antisense 5′-CGC TCC GGC CCA CAA ATC TC-3′; ALP sense 5′-GGT GAA CGG GAA AAT GTC TC-3′ and antisense 5′-CTG GAC CTC TCC CTT GAG TG-3′; and osteocalcin sense 5′-CTC TCC TAC AAG AAC GGC AC-3′ and antisense 5′-TCA CTA GCC AGA AAT CGG TGG-3′. Amplified PCR products were separated on 2% agarose gel and stained with ethidium bromide for visualization. The intensity of ethidium bromide–stained bands was quantified using the histogram function in Adobe PhotoShop version 7.0 (Adobe Systems Inc., San Jose, CA) and was normalized with the glyceraldehyde phosphate dehydrogenase (GAPDH) housekeeping gene. The statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL) v 14.0 for Windows. Kolmogorov-Smirnov one-sample test was used to check whether variables were normally distributed. Fixed-effects (models with only fixed effects and the residual term) designs of analysis of variance (ANOVA) and Student's t-test were applied for comparisons of means of data. In addition to parallel comparisons between groups, effects on each participant were compared with her own baseline values. P≤.05 was considered statistically significant. The treatment was well tolerated, and no patient withdrew from the study due to adverse reactions. Basic clinical data including age (55.5 ± 5.9 years in the treated group and 55.3 ± 5.1 years in the control), years since menopause (10.4 ± 5.3 vs. 8.5 ± 4.5, respectively), and body mass index (26.8 ± 3.8 vs. 27.4 ± 4.2 kg/m2, respectively) were comparable in both groups. Values for LS BMD and FN BMD were within the normal range for the age, and were similar in both groups. There were no differences between the groups for routine biochemical parameters, lipids or hormones (estradiol, testosterone, FSH, and parathyroid hormone) at each time-point of the study, or when values at 3 months were compared with baseline within each group. The whole profile of the effects of iCR on bone is presented in Figure 1. Women treated with iCR exhibited statistically significant differences for NTx, which was decreased, and ALP, which was increased (panel A). No difference between groups was detected, however, for data obtained with the use of MC3T3-E1 cells. Moreover, ALP remained unchanged when cells were treated with serum alone or with serum plus β-glycerophosphate and ascorbic acid (panel B). Consistent with this data, there were no statistically significant changes in any of the three studied genes (Runx-2, ALP, or osteocalcin) when compared with the GAPDH constitutive gene (panel C). Our data confirm that the use of an iCR extract was well tolerated and did not alter analytical parameters. This neutrality included the lipid profile, in contrast to the recognized changes induced by oral estrogens. Moreover, the treatment was not associated with changes in the reproductive hormones FSH, testosterone, or estradiol. The bone changes consisted of a dual protective effect, with reduction in the levels of NTx, an indicator of bone resorption, and increase in ALP, an indicator of bone formation. The mechanism used by the iCR extract to limit bone remodeling is unknown at present. Nevertheless, a recent experimental study has shed some light by showing that 25-acetylcimigenol xylopyranoside, a triterpenoid glycoside isolated from CR, potently blocks the osteoclastogenesis induced by either receptor activator of nuclear factor κB ligand (RANKL) or tumor-necrosis factor-α (6Qiu S.X. Dan C. Ding L.S. Peng S. Chen S.N. Farnsworth N.R. et al.A triterpene glycoside from black cohosh that inhibits osteoclastogenesis by modulating RANKL and TNFα signaling pathways.Chem Biol. 2007; 14: 860-869Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Further information has been provided by another study in which an iCR extract increased the production of osteoprotegerin by cultured osteoblasts (5Viereck V. Grundker C. Friess S.C. Frosch K.H. Raddatz D. Schoppet M. et al.Isopropanolic extract of black cohosh stimulates osteoprotegerin production by human osteoblasts.J Bone Miner Res. 2005; 20: 2036-2043Crossref PubMed Scopus (38) Google Scholar). The counterbalancing effects of osteoprotegerin on osteoclastogenesis might influence the reduction in the bone-remodeling activity observed in our study. Experiments with MC3T3-E1 cells, an osteoblastic cell line, were designed to further clarify the mechanism used by the iCR extract. For this purpose, we used serum from women, a model whose advantage resides in that it most faithfully reproduces conditions similar to the physiologic environment (11Mikkola T. Ranta V. Orpana A. Viinikka L. Ylikorkala O. Hormone replacement therapy modifies the capacity of plasma and serum to regulate prostacyclin and endothelin-1 production in human vascular endothelial cells.Fertil Steril. 1996; 66: 389-393Abstract Full Text PDF PubMed Google Scholar, 12Oviedo P.J. Hermenegildo C. Tarin J.J. Cano A. Raloxifene promotes prostacyclin release in human endothelial cells through a mechanism that involves cyclooxygenase-1 and -2.Fertil Steril. 2005; 83: 1822-1829Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In contrast to the changes in bone markers, there was no change in ALP activity. The use of RT-PCR to explore changes in the expression of genes involved in osteoblast differentiation and function gave a negative result as well. There is no clear explanation for the discrepancy with the bone markers data in women. Potential hypotheses might include the murine origin of the cell line or the increased local concentrations of iCR metabolites on bone in vivo. There has been some debate on whether some CR actions might be mediated by a hypothetical agonism on estrogen receptors (ER), but experiments exploring the affinity of an ethanolic extract concluded in a lack of affinity for either ERα or ERβ. We have not performed specific experiments to discern whether there is any binding affinity of our isopropanolic extract on ER, but the poor modulation ability on bone metabolism as well as the lack of effect on the levels of FSH reduces support to this hypothesis. Similar findings have been reported by other investigators (13Liske E. Hanggi W. Henneicke-von Zepelin H.H. Boblitz N. Wustenberg P. Rahlfs V.W. Physiological investigation of a unique extract of black cohosh (Cimicifugae racemosae rhizoma): a 6-month clinical study demonstrates no systemic estrogenic effect.J Womens Health Gend Based Med. 2002; 11: 163-174Crossref PubMed Scopus (174) Google Scholar). We detected biochemical changes consistent with a small protective effect of iCR against the increased bone loss that occurs after menopause. Experiments with a murine osteoblastic cell line did not confirm any effect on osteoblasts. We cannot discard that the outcome might be different with another CR preparation because the bands obtained with thin-layer chromatography do not overlap for the most common ethanolic and isopropanolic extracts (14Zierau O. Bodinet C. Kolba S. Wulf M. Vollmer G. Antiestrogenic activities of Cimicifuga racemosa extracts.J Steroid Biochem Mol Biol. 2002; 80: 125-130Crossref PubMed Scopus (95) Google Scholar). The authors thank Rosa Aliaga, B.Sc. and Elvira Calap, B.Sc., for their excellent technical assistance.