土鳖虫醇提物制备方法及其制备物对MC3T3-E1成骨细胞促增殖作用活性评价

Study on the Preparation Process of Ethanol Extracts from Eupolyphaga Sinensis and the Evaluation by MC3T3-E1 Osteoblast Proliferation Activity

目的：研究土鳖虫最佳醇提物制备工艺。方法：使用浸渍法和匀浆法用50%乙醇以浸提时间（4 h-4 d）、温度（4-60℃）、料液比（4-12倍）和是否搅拌为因素,以提取物总获得率和蛋白获得率为指标提取土鳖虫;使用正交试验法优化浸渍法和匀浆法的提取条件;使用MTT法体外评价两种制备方法所得土鳖虫醇提物对MC3T3-E1成骨细胞促增殖作用的影响以探索土鳖虫最佳醇提物的制备工艺。结果：搅拌因素对两种方法制得提取物的获得率均影响甚微;在不搅拌的状态下浸渍法制得的提取物和总蛋白获得率随温度升高而增加,料液比对匀浆法物质获得率影响较大;正交试验优选浸渍法最佳提取方案为以10倍量料液比在60℃条件下静置4 d,其总获得率为11%;匀浆法最佳方案为：以8倍量料液比在50℃条件下静置12 h,其总获得率为10.2%;匀浆法和浸渍法提取土鳖虫的总获得率无统计学差异,但匀浆法制得的提取物对MC3T3-E1成骨细胞的促增殖作用更明显,当用药浓度均为0.2 mg/m L时,匀浆法和浸渍法制备物的促细胞增殖率分别达到171.59%和48.45%。结论：优化后的两种方法制备物获得率差异不显著,但匀浆法所得提取物对MC3T3-E1成骨细胞的促增殖作用优于浸渍法。

Objective：The present study was undertaken to investigate the preparation methods of the alcohol extracts of Eupolyphaga sinensis. Methods：The Eupolyphaga sinensis extracts were separately prepared by the soak or homogenate methods with 50% ethanol and the effects of different treatments：temperature（4~60 ℃）,time（4 h^4 d）,solid-liquid ratio（4~12 times）and agitation on the total yield and the total protein yield were measured. The extracting conditions were optimized through orthogonal test based on single factor. Effects of ethanol extracts of Eupolyphaga sinensis on proliferation of MC3 T3-E1 osteoblasts were assayed by MTT methods. Results：Whether stirring did not affect the amount of substance extracted by the two methods. With increasing the treating temperature,the total yield and the total protein yield increased which displayed in two methods. The ratio of solid to liquid had great influence on yield of the homogenate method. The optimized extraction process was decided by orthogonal experiment which was at 60 ℃,10 times of solid-liquid ratio,4 d,nonagitation for the soak method and at 50 ℃,8 times of solid-liquid ratio,12 h and non-agitation for the homogenate method. The maximum total yield by the soak or the homogenate method were 11% and 10.2%,respectively,which was not statistically different. MTT assay showed that most of the samples promoted the MC3 T3-E1 osteoblasts cells proliferation when the concentration was lower than 0.5 mg/m L. The promotion ability of the samples prepared by homogenate methods was stronger than by the soak methods. When the concentration of the sample was 0.2 mg/m L,the cell proliferation rate of the homogenate and soaking method reached to 171.59% and 48.45%,respectively. Conclusion：The amount of the total yields didn＇t show different between the two methods but the ability to promote cells proliferation of the samples extracted by the homogenate method was better than by the soak method.