Isolation And Identification Of Flavonoids From Epimedium Koreanum Nakai And The Mechanisms Of Their Anti-osteoporosis Activity

As a traditional Chinese herb medicine, Epimedium have been paid much attention and became a research focus around the world for its abundant resources, various pharmacological activities and doubtless clinical effects. In order to developeand utilize Epimedium better, a series studies have been carried out:(1) By using model of primary mouse bone marrow stromal cells (MSCs) and methods of MTT and ALP activity assay, the antiosteoporotic fractions were screened out;(2) Six compounds were separated from the antiosteoporotic fraction by column chromatography coupled with silica gel and Sephadex LH-20, and identified by spectral analysis (ESI-MS, ¹HNMR and ¹³CNMR) respectively;(3) By using model of primary mouse bone marrow stromal cells (MSCs) methods of MTT, ALP activity assay, alizarin red stain and assay, red O stain and assay, we studied the effects of active fraction, icariin which is the main component of active fraction and its metabolic product named baohuosideâ… on osteogenic and adipogenic differentiation of MSCs;(4) By using model of primary mouse osteoblasts (OBs) and methods of MTT, red O stain and assay, we studied the effects of active fraction, icariin and baohuoside I on adipocytic trans-differentiation of OBs;(5) Using the methods of western blot and real-time PCR, we studied the mechanisms (targets or pathways) that may affect osteogenic and adipogenic differentiation of MSCs;(6) MTT assay and DPPH radical scavenging assay were used to observe the effects of six flavonoids from Epimedium koreanum Nakai on proliferation of preosteoclast RAW264.7 and their antioxidative and antitumor abilities.The results showed that:(1) On cellular level, the fraction of ethyl acetate and water extract could promote osteogenic differentiation of MSCs;(2) Six flavonoids were isolated from the ethyl acetate extract of the aerial part. Their structures were identified as icariin (â… ), baohuosideâ…¡(â…¡), epimedokoreanin B (â…¢), baohuoside I (â…£) hyperoside (â…¤) and luteolin (â…¥);(3) On cellular level, the fraction of ethyl acetate could promote osteogenic differentiation and showed no inhibition on adipogenic differentiation of MSCs. Among certain concentrations, icariin and its metabolic product baohuoside I could promote osteogenic differentiation and inhibited adipogenic differentiation of MSCs. The effects were associated with chemical structure, concentrations and times. The effects were the best when icariin were in the concentrations of 10^-9and 10^-8mol/L on 14d.(4) On cellular level, among certain concentrations the fraction of ethyl acetate, icariin and baohuoside I could inhibit adipocytic trans-differentiation of osteoblasts. The effects were the best when icariin were in the concentrations of 10^-9and 10^-8mol/L on 6d.(5) ERαis one of the possible targets of icariin that affect osteogenic and adipogenic differentiation of MSCs.The results of (1)-(5) suggests that icariin, as the active ingredient of Epimedium, can regulate a dual differentiational process of bone marrow stromal cells into the osteogenic and adipogenic lineages, and trans-differentiational process of primary osteoblasts into the adipocyte lineages, causing a lineage shift toward osteoblasts. So we deduced that the protective effects of icariin on bone may be mediated by increasing number of osteoblasts to directly increase bone formation and decreasing adipocytic cell formation from marrow stromal cells, which may promote osteoblast proliferation, differentiation and mineralization, and secreting less cytokines which may inhibit osteoclast formation and activation. ERαis one of the possible targets of icariin.(6) Other activities screenings showed that the effects of flavonoids on the proliferation of RAW264.7 are bi-directional, depending on concentrations and species, and Epimedium may cure osteoporosis by inhibiting the proliferation and differentiation of preosteoclast; The antitumor activity of Epimedium may be partially related to the antioxidantive activity of flavonoids.