The Investigation Of The Anti-osteoporotic And Estrogenic Activities Of Phenolic Glycosides In Curculigo Orchioides

Curculigo orchioides is the dry rhizome of Curculigo orchioides Gaertn., one of the traditional Chinese medicine. Curculigo orchioides are considered to be pungent flavor, hot-natured and poisonous in traditional Chinese medicine. They have the effects of kidney-tonifying and yang-boosting, maintaining healthy energy, strengthening the muscles and bone, expelling wind and rheumatism, etc. People usually use Curculigo orchioides for immunoregulation, anti-osteoporotic, protecting the liver and cardiovascular system. Curculigo orchioides are used in many of traditional Chinese prescriptions for anti-osteoporotic, so cause great interest and argument. The main constituents are phenol and phenolic glycoside, cycloartane-type triterpene saponin, lignan, flavone, etc. In previous we used OVX rat model to investigate the antiosteoporotic activity of the ethanol extract of Curculigo orchioides, and used the osteoblast and osteoclast to investigate the antiosteoporotic activity of phenolic glycosides in vitro. The main contents of our study are as follows:The method of determination orcinol glucoside, curculigoside and curculigine A in Curculigo orchioides by HPLC was established. The contents of orcinol glucoside, curculigoside and curculigine A were used as parameters, and they were determined by HPLC. Study of the concentration of ethanol, extraction time, times of extraction and solvent volum on the impact of extraction efficiency. The extraction conditions of phenolic glycosides in Curculigo orchioides were optimized by orthogonal design. The optimal extraction process was refluxed three times with 70% ethanol (10 times) for 2h each time.Curculigo orchioides were refluxed with optimal extraction process. Then the solvent was evaporated under vacuum and dissolved into distilled water to give 1g/ml (crude medicine/volume) sample solution. The solution was instilled into the prepared D101 macroporous resin, till the sample was absolutely adhered to the resin. After that the resin was washed with distilled water and ethanol solutions of different concentrations (water, 10%,20%,30%,40%,50%,60%) successively. Resin elution was detected by HPLC to determine the time of replacing the eluent. Orcinol glucoside was found in the 10% ethanol solution and Curculigoside and curculigine A in the 30% ethanol solution.The desorption solution was collected and concentrated to dryness under vacuum. Then the dryness was dissolved into distilled water and purified taking the same approach. Finally, purity of Orcinol glucoside was 39%, Curculigoside A and curculigine A were 46%and 4%, respectively.10 compounds were isolated and identified from 10% and 30% ethanol solution, by D101 macroporous resin column, gel column, ODS reversed phase column, MCI column, preparative liquid.8 of them are phenolic compounds, others are furan compounds. We selected the 3 months old ovariectomized rat model to investigate the anti-osteoporosis effect of Curculigo orchioides and its phenolic glycosides. It was found that uterines of female rats after ovariectomy were atrophy, indicated that menopausal status has been caused. Serum alkaline phosphatase activity, U-Ca/Cr value increased; femoral bone mineral density and the activity of serum antioxidant enzymes decreased, while MDA content was increased. Curculigo orchioides and different doses of benzoic acid esters of phenolic glycosides could improve bone density in ovariectomized rats, and in the higher and highest dose groups, it was significantly different from the OVX rats (P <0.01). Orcinol glucosid had no significant effect on increasing bone density in ovariectomized rats. This indicates that the effective fraction of anti-osteoporosis is benzoic acid esters of phenolic glycosides, such as Curculigoside, etc. These compounds decrease bone loss by inhibiting bone resorption. Metabolomics and serum antioxidant enzyme activity analysis showed that the phenolic glycosides from Curculigo orchioides by increasing the antioxidant capacity to improve bone metabolism and reduce bone loss.We investigated the estrogenic effects of the ethanol extract of Curculigo orchioides, including each alcohol elution part through D101 macroporous resin by analysising the body weight and uterus growth of 17 days old rats. The results indicate that ethanol extract of Curculigo orchioides and each elution part have no significant estrogenic effect. Then we used of estrogen receptor-positive cell model (MCF-7) investigated the effect of phenolic compounds:Curculigoside, curculigine A,2,6 - dimethoxy benzoic acid, Orcinol glucoside, Orcinol gentiobioside. We found estradiol (E2) promoted cell proliferation significantly, while the tested drugs had no significant effect on cell proliferation, We concluded that the phenolic compounds had no estrogen-like effects.Ovariectomized rats were administrated orcinol phenolic glycosides and benzoic acid esters of phenolic glycosides for 12 weeks, the tissue morphology of estrogen receptor-positive organs breast, vagina and uterus did not change significantly compared to OVX rats, but the expression of progesterone receptor, estrogen receptorαandβdecreased. These results suggest that orcinol phenolic glycosides and benzoic acid esters of phenolic glycosides had anti-estrogenic effect.In summary, we obtained the method of preparation phenolic glycosides from Curculigo orchioides, isolated and identificated the major phenolic glycosides, clarified the material basis of its anti-osteoporosis and anti-estrogenic effects. By anti-osteoporosis and estrogen-like activity analysis, the phenolic glycosides from Curculigo orchioides reduce bone loss by increasing the antioxidant capacity and anti-estrogenic effect. The disadvantage is that the sudy in anti-estrogenic effect of phenolic glycosides from Curculigo orchioides is still not comprehensive enough. In-depth study, we should reveal the characteristics of its anti-estrogenic, and define its beneficial or detrimental effect on human body.