鹿瓜多肽体外活性测定方法的探讨研究

Research on the method for determining the in vitro activity of Cervus and Cucumis polypeptide

目的:建立鹿瓜多肽体外活性测定方法。方法:根据鹿瓜多肽药理作用机制,选用敏感骨细胞株作为试验对象,CCK-8比色法探讨鹿瓜多肽对骨细胞株的增殖促进作用。同时对试验条件,包括培养基、细胞接种密度、药物作用时间、胎牛血清(FBS)浓度等进行探讨,建立鹿瓜多肽体外活性测定方法。对该方法线性、重复性进行验证,同时用该方法对2个厂家分别生产的鹿瓜多肽注射液和注射用鹿瓜多肽样品进行活性测定。结果:鹿瓜多肽对大鼠骨肉瘤细胞株(UMR106细胞株)具有增殖促进作用。选用不含谷氨酰胺,含1%FBS的DMEM培养液进行试验,细胞接种密度为2×10~4个·m L^(-1),药物作用细胞72 h,药物质量浓度在0.5~4 mg·m L^(-1)范围内,鹿瓜多肽对UMR106细胞株具有增殖促进作用,刺激指数可达2.97。对该方法进行方法学验证:鹿瓜多肽质量浓度在0.5~4 mg·m L^(-1)范围内,以药物浓度为横坐标,吸收度值为纵坐标进行线性拟合,相关系数(R^2)分别为0.971和0.968,均大于0.95,线性良好;对2个厂家生产的鹿瓜多肽注射液和注射用鹿瓜多肽样品重复测定3次,刺激指数平均值分别为2.72和1.86,RSD分别为9.6%和3.9%。结论:该方法可用于鹿瓜多肽体外活性测定。

Objective：To establish the method to determine the in vitro activity of Cervus and Cucumis polypeptide.Methods：According to the pharmacological mechanism of Cervus and Cucumis polypeptide,the different sensitive bone cell strains were chosen,and CCK-8 method was applied to evaluate the proliferation activity of the Cervus and Cucumis polypeptide on bone cell strains.Experiment conditions,including media,cell density,drug incubation time and concentration of fatal bovine serum（FBS）concentration,etc.,were explored.Then,the method for the detection of in vitro activity of Cervus and Cucumis polypeptide was established.The linearity and reproducibility of the method were validated.This method was applied for the determination of the activities of the Cervus and Cucumis polypeptide samples from 2 different manufacturers.Results：Cervus and Cucumis polypeptide enhanced the proliferation of rat osteosarcoma cell strain（UMR106 cell strain）.DMEM medium without glutamine but containing 1%FBS was chosen.Cell density was 2.0×10~4 m L^（-1） and the drug incubation time was 72 hours.The concentration of drug was between 0.5 mg·m L^（-1） and 4 mg·m L^（-1）.The Cervus and Cucumis polypeptide could promote the proliferation of UMR-106 cells,and the stimulation index could reach 2.97.As for the validation,the drug concentration was plotted on the abscissa and the absorbance value was plotted on the ordinate.With concentration of Cervus and Cucumis polypeptide between 0.5 mg·m L^（-1） and 4 mg·m L^（-1）,the linearity of the method was good,correlation coefficients R^2 were 0.971 and 0.968,respectively（both more than 0.95）.The activities of Cervus and Cucumis polypeptide samples from two different manufacturers were evaluated 3 times,and the average stimulation indexes were 2.72 and 1.86 with RSD 9.6%and 3.9%,respectively.Conclusion：The method is suitable for determining the in vitro activity of Cervus and Cucumis polypeptide.