柿子黄酮类化合物的双水相系统纯化及止血凝血抗炎作用研究

Purification of Flavonoids Extracts from Persimmon by Aqueous Two-phase System and Study on Its Hemostatic, Coagulant and Anti-inflammatory Effects

目的采用聚乙二醇600(PEG 600)-(NH4)2SO4的双水相体系纯化柿子黄酮(flavonoids extracts from persimmon,FEP)并考察其止血凝血抗炎作用。方法以FEP得率为指标,采用单因素试验设计,探讨PEG 600-(NH4)2SO4双水相体系中的PEG 600质量分数、(NH4)2SO4质量分数、温度、MgCl2用量、pH对FEP提取率的影响,并采用Box-Behnken中心组合方法设计3因素3水平的响应面法试验,选择Design-Expert 8.05软件进行响应面分析。以KM小鼠为试验对象,采用剪尾法、毛细管法、二甲苯致炎法对FEP进行止血、凝血、急性抗炎作用的初步研究,并分别与三七粉和阿司匹林进行比较。结果最佳纯化条件:PEG 600质量分数为12%,(NH4)2SO4质量分数为28%,pH值为8,最终得率为95.97%。FEP可使小鼠的出血时间和凝血时间明显缩短,对二甲苯所致的小鼠耳肿胀具有较显著的抑制作用。结论本法所形成的双水相体系,操作简便,高效温和并有效保持生物活性;可以考虑将FEP作为活性成分添加入口腔用生活用品或药品。

OBJECTIVE To explore hemostatic, coagulant and anti-inflammatory effect and the extraction mechanism of the separation-purification of the flavonoids extracts from persimmon（FEP） in aqueous two-phase systems（ATPS） of PEG 600 and （NH4）2SO4. METHODS The yield rate of flavonoids from persimmon was taken as an index, to optimize the conditions such as the concentration of PEG 600, the concentration of （NH4）2SO4, temperature, amounts of MgCl2 and pH were studied by single factor experiments and response surface methodology（RSM） with a three-factor[the concentration of PEG 600, the concentration of （NH4）2SO4, pH] and three-level experiment design were developed by Box-Behnken central composite design method. Furthermore, hemostatic, coagulant and anti-inflammatory effect of FEP were studied by breaking tail method, capillary tubes method and the ear swelling method induced by dimethylbenzene in KM mice and results were compared to Panax notoginseng powder and aspirin. RESULTS The result showed that the optimum condition was as follows： 12% PEG 600, 28% （NH4）2SO4, pH 8 and the yield rate of flavonoids reached 95.57%. The FEP could shorten bleeding time（BT）, coagulation time and restrain ear swelling induced by dimethylbenzene. CONCLUSION The purification method for flavonoid by ATPS is easy, efficient, and moderate which can keep the bioactivity. The FEP is a potential product added in oral daily necessities and oral drugs as active ingredient.