Elucidation of the action mechanism of erxian decoction, a Chinese medicinal formula for menopause: frompharmacological approach to analytical approach

Abstract

 As the aging in reproductive system proceeds, females will eventually enter the period of menopause, during which a series of physiological changes occurs. The decline of estrogen level during menopausal transition is thought to associate with various menopausal symptoms. Although hormone replacement therapy can be adopted to deal with the estrogen-deficient state, side effects such as cancer risk cannot be overlooked.

Alternatively, Erxian Decoction (EXD), a Chinese medicine formula for treating menopausal symptoms has been used clinically for more than 60 years without adverse effects reported. Some pharmacological properties of EXD have been reported in previous research, which are thought to be contributed by its multiple bioactive components. Thus in the present study, the pharmacological properties of EXD have been further evaluated. The drug compatibility of Traditional Chinese Medicine (TCM) formula, EXD, was also demonstrated. At last, a novel approach for identification of bioactive components from Chinese medicine formula was introduced using EXD as study model. To evaluate the multiple pharmacological properties of EXD, proteins involved in steroidogenesis in ovaries of aged female rats were measured by immunoblotting analysis. On top of that, serum lipid profiles and the related proteins were determined by colorimetric assay and immunoblotting analysis respectively. Also, anti-osteoporotic properties and drug compatibility of EXD were evaluated by in vitro methods such as proliferation assay, osteoclast differentiation assay, ELISA assay or immunoblotting analysis. Lastly, a novel approach for identification of bioactive components in relation to the subsequent bioactivity from traditional Chinese medicinal formula was introduced using HPLC profiles. From the results, it was demonstrated that EXD can modulate steroidogenesis in aged female rat model at least through up-regulation of ovarian aromatase, protein kinase B and estrogen receptor beta at protein level. Besides, EXD also exerts antihyperlipidemic effects in aged female rats as reflected from the decreased serum total cholesterol and LDL-cholesterol levels via regulation of HMG CoA reductase and LDL-receptor, the key proteins for cholesterol synthesis and LDL-cholesterol clearance. In vitro study has also demonstrated the anti-osteoporotic properties of EXD through stimulation of osteoblast proliferation and inhibition of proliferation and differentiation of osteoclast precursor cells. The later was proved to be mediated by down-regulation of NFATc1 proteins, a key protein for osteoclastogenesis. The roles of the drugs categories according to the drug compatibility of traditional Chinese medicine contributing to the optimal anti-osteoporotic properties of EXD were also elucidated. Since the diverse pharmacological properties of a Chinese medicinal formula are often the results of the effects of complex bioactive constituents in the extract, yet identification of the bioactive components has been a tedious task. Thus in the last part of the study, a novel approach for identification of bioactive component from Chinese medicinal formula has been developed. By comparing the HPLC profiles of EXD extracted by different decoction method in relation to their pharmacological properties, six bioactive chemicals were successfully identified which may contribute to the stimulatory effect of EXD on ovarian aromatase and hepatic catalase expression.