Study On Traditional Chinese Medicines And Their Active Constituents Promoting Osteoblastic Bone Formation

Osteoblasts are the important cells in bone formation and, therefore, agents stimulating osteoblastic bone formation would have potential activity of antiosteoporosis. In order to screen and exploit novel and effective medicines to prevent and cure osteoporosis, osteoblast-like UMR1O6 cells were employed as a screening model of anti-osteoporosis medicines for the first time. The cell proliferation and differentiation were measured as index of stimulating osteoblastic bone formation. Cell proliferation was assayed by M~F method with fluoride sodium as positive control. And cell differentiation was assayed by measuring the cellular alkaline phosphatase activity with dexaniethasone as positive control. The influence of ethanol and dimethyl sulfoxide on cell proliferation and differentiation assay was also investigated.Some 搆idney-tonifying?traditional Chinese medicines were selected on the basis of the theory of traditional Chinese medicines and examined for the activity of stimulating osteoblastic bone formation in vitro. After co-culturing the crude extracts of 15 搆idney-tonifying?traditional Chinese medicines with UMIR1 06 cells, 9 were found to have significant promoting effect on osteoblast proliferation. Fifteen traditional Chinese medicines other than 搆idney-tonifying?and 53 plants not yet for medicinal use were also studied, 4 from the former and 2 from the later were found to stimulate the cell proliferation significantly. And 6 from 12 traditional Chinese medicines provided promoting effect on the cell differentiation. Four traditional Chinese medicines showed activities of stimulating both osteoblastic proliferation and differentiation, they are Psoralea coryl~folia L., Viscum coloratum (Kom.) Nakai, Epimedium sagittatum Maxim and Cyathula officinalis Kuan.The effect of various polar fractions from Psoralea corylzfolia L. ethanol extract on cell proliferation and differentiation were further examined and its ethyl acetate fraction were found to be active fraction. Also the effect of its ethyl acetate fraction on ovariectimized rats, as a pharmacological model of osteoporosis, was studied and biphosphate served as positive control. Compared with OVX, the ethyl acetate fraction from Psoralea coryl~folia L. significantly increased bone mineral density and bone calcium content of rats.In order to isolate the active compounds in ethyl acetate fraction of Psoralea coryl~folia L., activity-guided isolation was performed along with chromatographic3techniques. Four active compounds were isolated from ethyl acetate fraction and identified by their NMR spectral data and physical-chemical properties. They are corylin and bavachin, which showed stimulating effect on osteoblastic proliferation and differentiation, isopsoralen and psoralen exhibited activity of promoting cell differentiation in some degree.There was no report, up to dale, on the determination of flavonoids in Psoralea cory4folia L.. In this study, an HPLC method for simultaneous determination of corylin and bavachin was set up. HIPLC analysis was carried out on an ODS column(25mmX 4.6mm). The mobile phase was methanol and 2OmmoIIl ammonium acetate buffer, pH 4.0 (67: 33). The flow rate was 0.8 mllmin and the wavelength of UV detector was set at 240 nra. The sample was prepared by first refluxing raw materials for 6 h and then extract 3 times with ethyl acetate. And the RSD of bavachin and corylin was 3.1% and 3.6%, respectively; the recovery was 94.9% and 96.2%. The contents of bavachin and corylin in Psoralea coiyljfolia L. from 8 regions were determined.In addition, the effect of some flavonoid compounds on osteoblast-like cells was also studied. Icariin, the main constituent of Epimedium sagittatum Maxim, and its metabolite icariside II showed similar activity in cell proliferation. The total flavonoids from J?scum coloratum (Kom.) Nakai could stimulate cell proliferation and significantly promote cellular ALP activity. Daidzein and its derivatives exhibited no significant activity in cell...