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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 purify flavonoids extracts from persimmon (FEP) by using aqueous two-phase system of polyethylene glycol 600 (PEG 600) - (NH 4) 2 SO 4. Methods FEP yield was used as an index to investigate the effects of PEG 600, PEG (NH4) 2SO4, temperature, MgCl2 dosage and pH on FEP extraction rate in PEG 600- (NH4) 2SO4 aqueous two- , And the Box-Behnken center combination method was used to design a 3-factor-3 response surface methodology. Design-Expert 8.05 software was used to analyze the response surface. KM mice were used as experimental subjects. Hemostasis, blood coagulation and acute anti-inflammatory effects of FEP were detected by tail-cut method, capillary method and xylene-induced inflammation method, and compared with notoginseng powder and aspirin respectively. Results The optimum conditions of purification were as follows: PEG 600 mass fraction 12%, (NH4) 2SO4 28%, pH 8, final yield 95.97%. FEP can significantly shorten the bleeding time and clotting time in mice, and have a significant inhibitory effect on the ear swelling induced by xylene in mice. Conclusions The aqueous two-phase system formed by this method is simple and convenient to operate with high efficiency and mildness and effectively maintains biological activity. It is considered that FEP may be added as an active ingredient to daily necessities or drugs for oral administration.