Effects of extracts from Trifolium medium L. and Trifolium pratense L. on development of estrogen deficiency-induced osteoporosis in rats

U Cegieła1, J Folwarczna1, M Pytlik1, G Zgórka2. 1Department of Pharmacology, Medical University of Silesia, Katowice, 41-200 Sosnowiec, Poland, 2Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 20-093 Lublin, Poland

Estrogen deficiency contributes to development of osteoporosis in postmenopausal women. There is an interest in using phytoestrogens to alleviate menopausal symptoms. Phytoestrogens are plant derived substances with estrogenic activity, which, binding to estrogen receptors, may both compete with estradiol, decreasing its effects, and activate the estrogen receptors. They are sometimes considered natural selective estrogen receptor modulators (SERMs). Red clover (Trifolium pratense L.) is a well-known source of phytoestrogens – isoflavones; it has been already reported to favorably affect the skeletal system in experimental conditions. Trifolium medium L. is a much richer isoflavone source than T. pratense. The aim of the present study was to examine the effects of standardized extracts, obtained from aerial parts of T. medium and T. pratense, on development of estrogen deficiency-induced osteoporosis in rats.

The lyophilized ethanol-water extracts from T. medium and T. pratense were administered orally to 3-month-old bilaterally ovariectomized (OVX) Wistar rats at doses corresponding to 10 and 20 mg/kg of isoflavones daily for 4 weeks. The mean isoflavone (daidzein + genistein + formononetin + biochanin A) content for the T. medium and T. pratense extracts was 7.2% and 2.8% dry weight, respectively. The results were compared to those obtained in two control groups: OVX rats and sham-operated rats. Moreover, OVX rats receiving estradiol (0.2 mg/kg p.o. daily) served as a positive control. The ovariectomy or sham-operation were performed, under ketamine-xylazine anaesthesia, 7-8 days before the start of drug administration. The number of rats was 10 per group. The rats were fed a soy-free diet ad libitum. Serum bone resorption (RatLaps) and formation (osteocalcin) markers, bone mass, mass of bone mineral, histomorphometric parameters and mechanical properties of tibial metaphysis and femoral diaphysis (in three-point bending tests) and femoral neck (in a compression test) were studied. One-way ANOVA followed by Duncan’s post-hoc test was used to evaluate the significance of the results.

In OVX control rats, the mechanical properties of tibial metaphysis (cancellous bone) and the femoral neck were strongly worsened in comparison with sham-operated control rats, and those of femoral diaphysis (compact bone) were unaffected. Histomorphometric parameters indicated increased bone resorption. The bone turnover markers were increased, and bone mineral mass/bone mass ratios were decreased. Estradiol counteracted the worsening of the tibial strength, the changes in bone histomorphometric parameters and increases in bone turnover markers. Only T. pratense extract increased the strength of the tibial metaphysis (this effect was weaker than that of estradiol). Both Trifolium extracts counteracted the changes in some bone histomorphometric parameters. Both extracts did not affect bone turnover markers, but they significantly increased the strength of the femoral diaphysis. Moreover, calcium and phosphorus content in the bone mineral markedly increased. The extracts did not affect the uterus mass.

In conclusion, the effects of T. medium and T. pratense extracts differed from those exerted by estradiol, indicating more complex mechanism of action. It is possible that other than isoflavones constituents of the extracts, like sugars or phenolic acids, contributed to their skeletal effect.

Acknowledgements: This scientific project was supported by the Polish Ministry of Science and Higher Education (Grant No N N405 617638).