【作者】 王珂；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2011， 硕士

【摘要】 桑叶(mulberry leaf)为桑树(Morus alba L.)的叶,黄酮是其中主要的生物活性成分,为深度利用资源,本文对黄酮的提取分离技术进行了工艺研究,并后研究了桑叶黄酮对高尿酸血症模型的降尿酸的作用。研究通过高压液相色谱法对桑叶黄酮类化合物进行成分分析,结果表明,其主要成分为芦丁,杨梅素,槲皮素,山奈素等。采用正交试验法对提取桑叶黄酮的工艺进行优化,结果表明,最佳的浸提条件为：在80℃下70%乙醇回流提取2次,每次1.5h,料液比为1：20(w/v)。为得到纯度较高的桑叶总黄酮,采用大孔树脂吸附法进行黄酮纯化,以桑叶总黄酮的得率和纯度为指标,比较了五种大孔树脂的吸附性能,结果显示HPD826大孔树脂对桑叶总黄酮吸附性能最好；进一步采用正交试验法对HPD826大孔树脂纯化桑叶黄酮的工艺进行优化,所获最佳条件组合为：取浓度为4mg/ml左右的桑叶提取液上样,上样体积为1.5树脂体积数(BV),上样速率控制在2BV/h左右,之后用3 BV,60%的乙醇,以1 BV/h的速率洗脱,纯化后的桑叶黄酮得率为1.95%,纯度55.41%。药理学研究表明：桑叶黄酮具有调节α-葡萄糖苷酶、降血糖、抗肿瘤等药理活性。而且黄酮类化合物对高尿酸血症有效。所以选择体外实验探讨桑叶黄酮(MLF)对大鼠肝脏匀浆液中黄嘌呤氧化酶(XO)活性的影响,结果显示：MLF剂量依赖性地抑制大鼠肝脏匀浆液XO活性,在100.00 mg/L抑制率与别嘌醇(AP)无显著差异,提示MLF可能具有降尿酸活性。进一步研究其对正常小鼠血清UA含量和肝脏匀浆液XO活性的影响,以50、100、200mg/kg*d-1剂量MLF灌胃正常小鼠14d,测定相关指标。结果显示：MLF三种剂量与对照相比对正常小鼠体内血清UA含量和肝脏匀浆XO活性均无显著性影响,对小鼠体重和脏器系数无显著影响。采用氧嗪酸钾诱导的高尿酸血症小鼠模型评价了MLF的降尿酸活性,以50、100、200mg/kg*d-1剂量MLF灌胃模型小鼠14d,检测相关指标,结果显示：200mg/kg bw MLF可极显著(P<0.01)降低模型小鼠血清尿酸含量,下降16.67%；并能显著(P<0.05)抑制肝脏XO活性,抑制率达6.05%。而50mg/kg bw MLF对肝脏XO活性的无明显影响(P>0.05),但能极显著降低模型小鼠血清尿酸含量(P<0.01)。长期高尿酸血症导致肾损害和痛风,肾损害亦可致尿酸排泄障碍。为进一步探讨桑叶黄酮对病理状态下的尿酸代谢及相关肾损伤的影响,本实验继续研究了MLF对腺嘌呤联合高糖饲料诱导的大鼠高尿酸血症、肾损伤的防治作用。在腺嘌呤联合高糖饲料诱导的高尿酸病理状态下,分别给予200、100、50mg/kg* d-1的MLF,以别嘌醇为阳性对照药物。结果显示：MLF中、高剂量组在给药1w后,血清UA水平分别降低17.83%和21.17%；2w后降低21.09%和27.15%。3w后MLF中剂量组血清尿酸接近于正常大鼠水平,XO活力比HC组显著降低25.01%。200mg/kg*d-1 MLF可使血清TG,FFA水平降低41.95%,42.05%,同时MLF可减小腺嘌呤造模引发的肝脏、心脏系数的异常。降低高尿酸病理状态下肌酐和尿素氮含量,并通过电镜观察,MLF对腺嘌呤所引起肾脏损伤有一定的保护作用。综合以上结果：推断MLF具有明显的降尿酸活性,并对高尿酸病理状态下的肾脏损伤有一定的保护作用；MLF可能通过抑制XO和调节脂质紊乱来调节血清UA水平。更多还原

【Abstract】 Leaves from Morus alba L.is a kind of traditional Chinese medicine. Mulberry leaf flavonoids are the mainly active ingredient in mulberry leaves. The technique of extraction and separation of active ingredient has been investigated in this paper. Thenceforth, the hypouricemic activity of the mulberry leaves flavonoids was explored.Mulberry leaf flavonoids were analysed with high performance liquid chromatography. Rrutin, myricetin, quercetin, kaempferol are the main ingredients in it. Factors of temperature, extraction time, ethanol concentration,volume of solvent and extraction frequency were evaluated for their effects on extraction process by orthogonal designed experiments. The results showed that the optimum conditions were 80℃,2 times,1.5 hours,70% ethanol-herb ratio of volume to mass was 20:1 respectively.In order to get purer mulberry leaf flavonoids, macroporous resin was adopted. The adsorption ability of macroporous resin was evaluated by yield and purity of the flavonoids. The kind of HPD826 was the best of the five macroporous resin. The optimum process conditions were 3 BV 60% ethanol as eluting solvent, adding sample rate in about 2 BV/h, eluting rate in about 1 BV/h. The yield of Mulberry leaf flavonoids by this process was 1.95%,and the purity was 55.41%.Pharmacology study showed that mulberry leaf flavonoids could adjust the activity of a-glucosidase, lower blood glucose, antitumor, etc. Results indicated that MLF inhibit rat liver XO activity on dose dependent. Normal mice were administered orally with MLF at doses of 50,100,150 mg./kg*d-1.The results showed that mulberry leaf flavonoids had no significant effect on uric acid levels and liver XO activity in normal mice.Experimental animal model of hyperuricemia was induced by uricase inhibitor potassium oxonate. Potassium oxonate-induced hyperuricemic mice were administered orally with MLF at doses of 50,100,150 mg/kg*d-1, while distilled water and allopurinol at 50 mg/kg*d-1 were given to the hyperuricemic control group and positive control group. The results showed that oral administration of 200 mg/kg*d-1 MLF to hyperuricemic mice exhibited significant reduction in the blood uric acid levels 16.67% and in XO activity 6.05%. The oral administration of 50 mg./kg*d-1 MLF also caused remarkable reduction in the blood uric acid levels, but without remarkable reduction in XO activity.Long-term hyperuicemia caused kidney damage and gout, renal damage cause uric acid excretion obstacles. We studied the hypouricemic effect of MLF by using model of adenine-reduced hyperuricemia in rats. In this test, Adenine-induced hyperuricemic mice were administered orally with MLF at doses of 50,100,150 mg/kg*d-1, while distilled water and allopurinol at 50 mg/kg*d-1 were given to the hyperuricemic control group and positive control group. The results showed that oral administration of 100 mg/kg*d-1 MLF to hyperuricemic mice exhibited significant reduction in the blood uric acid levels at different time periods:17.83% at 7th d, 21.17% at 14th d, and oral administration of 200 mg/kg*d-1 MLF caused remarkable reduction in the blood uric acid levels at different time periods:21.09% at 7th d, 27.15% at 14th d. Oral administration of 200 mg/kg*d-1 MLF also caused remarkable reduction in the blood triglyceride levels 41.95% and in the blood free fatty acid levels 42.05% at 21th d. Transmission electron microscopy (TEM) observation results showed that MLF protected kidney cells of rats which treated by adenine.All the results mentioned above showed that MLF could decrease the levels of blood uric acid. It also protect kidney cell and prevent it from further damage, improve the metabolism of triglyceride and adjust body weight in hyperuricemic mice. It was concluded that MLF had good activity on adenine-reduced hyperuricemic rats, but its molecular mechanism need further study.更多还原