淫羊藿醇提工艺及醇提物对软骨细胞保护作用的研究

选取淫羊藿所含主要活性成分朝藿定A、朝藿定B、朝藿定C、淫羊藿苷、宝藿苷Ⅰ的转移率为评价指标,采用均匀设计-综合评分法考察乙醇用量、乙醇浓度、提取时间等因素的影响,通过多元线性逐步回归优选条件参数,经验证试验确定淫羊藿醇提工艺条件。并根据该工艺条件制备淫羊藿醇提物,干预白细胞介素-1β(IL-1β)诱导的软骨细胞损伤模型,采用Annexin V-FITC/PI染色流式细胞术检测软骨细胞的凋亡率,分析淫羊藿醇提物对软骨细胞损伤模型的影响。优化的淫羊藿醇提工艺条件为:取淫羊藿粗粉,加入18倍量70%乙醇溶液,回流提取2次,每次提取60 min,在该工艺条件下朝藿定A、朝藿定B、朝藿定C、淫羊藿苷、宝藿苷Ⅰ的提取率分别达到94.21%,94.76%,93.85%,96.17%,96.85%,淫羊藿醇提工艺合理、可行,重现性好。所制得的淫羊藿醇提物能显著降低IL-1β诱导软骨细胞损伤模型的早期凋亡率、晚期凋亡和坏死率、总凋亡率(P<0.05或P<0.01),提示淫羊藿醇提物对IL-1β诱导的软骨细胞凋亡具有一定抑制作用,可为深入研究其对骨关节炎的防治作用奠定基础。

Ethanol extraction technology of Epimedii Folium and protective effect of ethanol extract on chondrocyte

Uniform design-comprehensive scoring method was used to investigate the effects of ethanol dosage,ethanol concentration and extraction time,based on the evaluation index from transfer rates of epimedin A,epimedin B,epimedin C,icariin and baohuosideⅠ,which are the main active components in Epimedii Folium.Furthermore,the optimum conditions for the ethanol extraction process were determined by multiple linear stepwise regression and empirical test.Then,the ethanol extract of Epimedii Folium prepared according to the optimized technological conditions was used to intervene the injury model of chondrocyte induced by interleukin-1 beta(IL-1β).Annexin V-FITC/PI staining flow cytometry was used to detect the apoptotic rate of chondrocyte and analyze the effect of ethanol extract of Epimedii Folium on chondrocyte injury model.The optimum conditions of ethanol extraction were as follows.Crude powder of Epimedii Folium was added with 18 times of 70%ethanol solution,and extracted for twice in the refluxing process,for 60 minutes each time.Under the conditions,the extraction rates of the above five active components were 94.21%,94.76%,93.85%,96.17%and 96.85%,respectively.The optimized ethanol extraction process of Epimedii Folium was reasonable,feasible and reproducible.This ethanol extract could significantly reduce the early apoptotic rate,late apoptotic and necrotic rate,total apoptotic rate(P<0.05 or P<0.01)of chondrocyte injury model induced by IL-1β,suggesting that the ethanol extract of Epimedii Folium can inhibit the apoptosis of chondrocytes induced by IL-1βto a certain extent,which lays a foundation for further study on its prevention and treatment of osteoarthritis.