Box-Behnken设计-响应面法优化番石榴叶总黄酮提取工艺及总黄酮对糖尿病小鼠糖耐量的影响研究

摘要

目的:优化番石榴叶总黄酮提取工艺,考察番石榴叶总黄酮对糖尿病模型小鼠糖耐量的影响.方法:在单因素试验的基础上,以提取时间、料液比、乙醇用量为自变量,金丝桃苷、槲皮素-3-O-β-D-吡喃阿拉伯糖苷、槲皮素-3-O-α-L-呋喃阿拉伯糖苷和浸出物得率的综合评分为因变量,采用Box-Behnken设计-响应面法优化番石榴叶总黄酮回流提取工艺并进行验证试验.取60只小鼠随机分为空白组、模型组、二甲双胍组(170 mg/kg)和番石榴叶总黄酮低、中、高剂量组(23.41、46.81、93.62 mg/kg,以总黄酮含量计),每组10只.除空白组小鼠外,其余各组小鼠复制糖尿病模型,成模后灌胃给药,每天1次,连续21 d,末次给药6h后,灌胃葡萄糖2.0 g/kg,于给予葡萄糖后0、0.5、1.0、2.0 h测定血糖值,计算血糖变化曲线下面积(AUC).结果:番石榴叶总黄酮最优工艺为提取2次,每次1.0h,料液比1∶17,乙醇体积分数56％,验证试验中金丝桃苷、槲皮素-3-O-β-D-吡喃阿拉伯糖苷、槲皮素-3-O-α-L-呋喃阿拉伯糖苷含量分别为2.57、3.38、2.26 mg/g(RSD＜2％,n=3),浸膏得率为25.71％ (RSD＜ 1.19％,n=3),综合评分平均值为96.41 (RSD=1.34％,n=3),与预测值99.98的相对误差为3.57％.与空白组比较,各组小鼠AUC显著升高;与模型组比较,二甲双胍组和番石榴叶总黄酮中、高剂量组AUC显著降低,差异均有统计学意义(P＜0.05或P＜0.01).结论:优化的提取工艺稳定可行、质量可控;番石榴叶总黄酮可促进糖尿病小鼠血糖恢复正常.%OBJECTIVE:To optimize the extraction technology of total flavonoids from Psidium guajava leaves and investigate the effects of total flavonoids from P guajava leaves on glucose tolerance in diabetic model mice.METHODS:Based on single factor test,using extraction time,solid-liquid ratio and the amount of ethanol as independent variables,comprehensive scores of the yield of hyperin,quercetin-3-O-β-D-arabinopyranoside,quercetin-3-O-α-L-furanarabino side ethyl acetate and extract as dependent variable,Box-Behnken design-response surface method was used to optimize the reflux extraction process of total flavonoids from P guajava leaves,and validation test was also conducted.Sixty mice were randomly divided into blank group,model group,metformin group (170 mg/kg),P.guajava leaves total flavonoids low-dose,medium-dose and high-dose groups (23.41,46.81,93.62 mg/kg,by the content of total flavonoids),with 10 mice in each group.Except for blank group,diabetic model was induced in those groups,and then model mice were given relevant medicine intragastrically once a day for consecutive 21 d,6 h after last medication given glucose 2.0 g/kg intragastrically.The levels of blood glucose were determined 0,0.5,1.0,2.0 h after intragastric administration of glucose,and the area under the curve (AUC) of blood glucose was calculated.RESULTS:The optimal technology of P guajava leaves total flavonoids was as follows as extracting twice,lasting for 1.0 h each time,solid-liquid ratio of 1 ∶ 17,ethanol volume fraction of 56％.In validation test,the contents of hyperin,quercetin-3-O-β-D-arabinopyranoside and quercetin-3-O-α-L-furanarabino side ethyl acetate were 2.57,3.38,2.26 mg/g(RSD＜2％,n=3);the yield of extract was 25.71％ (RSD=1.19％,n=3);average comprehensive score was 96.41 (RSD=l.34％,n=3);relative error was 3.57％ with the predicted value of 99.98.Compared with blank group,AUC of mice were increased significantly in each group;compared with model group,AUC of metformin group,P.guajava leaves total flavonoids medium-dose and high-dose groups were all decreased significantly,with statistical significance (P＜0.05 or P＜0.01).CONCLUSIONS:The optimal extraction technology is stable,feasible and controllable in quality.Total flavonoids from P.guajava leaves can promote the recovery to normal level of blood glucose.