杨梅果实酚类物质提取物降糖活性及其相关机理研究

发布时间：2018-09-13 07:56

【摘要】：本论文以杨梅(Myrica rubra Sieb et. Zucc)果实为材料,研究了17个品种果肉的总酚、总黄酮含量和抗氧化活性,并对其主要酚类物质进行了定性和定量；利用人肝癌HepG2细胞模型,评价了不同品种杨梅果实酚类物质提取物体外促进细胞葡萄糖消耗的活性,筛选出具有降糖潜力的杨梅品种,分析了发挥降糖活性的可能的主效成分；利用糖尿病小鼠模型,评价了‘荸荠’杨梅果实酚类物质提取物的体内降糖活性；进一步利用体外和体内模型,研究了‘荸荠’杨梅果实降糖活性相关作用机制。主要结果如下：1)不同品种杨梅果实总酚、总黄酮含量以及抗氧化活性差异显著,其中‘荸荠’、‘炭梅’、‘慈荠’、‘晚稻’等深色果肉品种总酚、总黄酮含量较高,而‘粉红’、‘水晶’等浅色果肉品种酚类物质含量较低。利用高效液相色谱(HPLC)和液相色谱-质谱联用(LC-MS)技术,在‘荸荠’果实中鉴定了12种酚类物质,包括5种花色苷类物质,即飞燕草色素己糖苷、矢车菊素-3-O-半乳糖苷、矢车菊素-3-O-葡萄糖苷、天竺葵色素-3-O-葡萄糖苷、芍药色素-3-O-葡萄糖苷和7种黄酮醇糖苷类物质,即杨梅素-3-O-鼠李糖苷、杨梅素脱氧己糖苷没食子酸酯、槲皮素-3-O-半乳糖苷、槲皮素-3-O-葡萄糖苷、槲皮素-3-O-鼠李糖苷、山奈酚-3-O-半乳糖苷和山奈酚-3-O-葡萄糖苷。酚类物质组成和含量在不同品种间差异显著。不同品种杨梅果实抗氧化活性与总酚、总黄酮含量呈显著正相关关系。2)利用人肝癌HepG2细胞模型,评价了9个杨梅品种果实酚类物质提取物和5种主要酚类化合物单体促进细胞葡萄糖消耗的活性。结果表明,不同品种杨梅果实酚类物质提取物促进细胞葡萄糖消耗的活性差异显著,‘炭梅’、‘荸荠’、‘慈荠’等品种提取物促进细胞葡萄糖消耗活性较强,这与其较高的总酚、总黄酮含量及DPPH·自由基清除能力呈显著正相关关系。进一步研究表明,矢车菊素-3-O-葡萄糖苷和3种槲皮素糖苷单体均表现出显著促进细胞葡萄糖消耗活性,而杨梅素-3-O-葡萄糖苷该活性不显著。相关性分析结果表明,不同品种杨梅果实提取物的葡萄糖消耗活性与其矢车菊素-3-O-葡萄糖苷含量(R2=0.922)和3种槲皮素糖苷单体总含量(R2=0.847)呈显著正相关性。因此,矢车菊素-3-O-葡萄糖苷和槲皮素糖苷单体可能是杨梅果实提取物促进HepG2细胞葡萄糖消耗的主效组分。3)利用KK-Ay糖尿病小鼠模型,研究了‘荸荠’果实酚类物质提取物(200mg/kg BW)的体内降糖活性。结果表明,‘荸荠’果实提取物饲喂糖尿病小鼠5周能够显著降低小鼠的空腹血糖水平,提高其口服糖耐量和胰岛素敏感性,显著降低血清胰岛素、胰高血糖素和瘦素浓度等糖代谢相关指标。此外,‘荸荠’果实提取物还能显著降低糖尿病小鼠的血清总胆固醇、甘油三酯、低密度脂蛋白、谷丙转氨酶和IL-1p水平,显著降低小鼠肝脏重量、肝脏脂质含量,缓解糖尿病小鼠肝脂肪变性等症状。4)对‘荸荠’果实酚类物质提取物的降糖作用机制进行了初步探索。体外HepG2细胞实验结果表明,0.4-10 μg/ml的‘荸荠’果实提取物能够显著抑制细胞糖异生作用、增强细胞糖酵解能力；这可能与其激活AMPK蛋白磷酸化、抑制下游糖异生相关PGC-1α、 PEPCK和G6Pase基因表达有关。KK-Ay小鼠实验结果表明,‘荸荠’果实提取物同样能够显著激活AMPK蛋白磷酸化、抑制小鼠肝脏PGC-1α和PEPCK基因表达。此外,‘荸荠’果实提取物显著下调小鼠肝脏组织中脂质代谢相关PAP、ACAT和CIDEA等基因和炎症因子TNFα、IL-6和MCP-1等基因表达水平。以上结果表明,‘荸荠’果实提取物降糖活性可能与其激活AMPK蛋白磷酸化,抑制肝糖异生,促进肝糖酵解,改善肝脏糖脂代谢,缓解肝脏炎症有关。本研究结果可为进一步挖掘不同杨梅资源降糖活性及相关作用机制提供基础,也可为从果品营养保健角度指导杨梅产业育种、引导人群尤其是糖尿病患者的果品消费等提供理论依据。
[Abstract]:In this paper, Myrica rubra Sieb et. Zucc fruits were used as materials to study the total phenols, total flavonoids and antioxidant activities in the flesh of 17 varieties of Myrica rubra fruits, and the main phenolic substances were qualitatively and quantitatively analyzed. The phenolic substances extracted from different Myrica fruits of Myrica rubra Sieb et. Zucc were used to evaluate the cell-promoting grapes in vitro by using HepG2 cell model of human liver cancer. The hypoglycemic activity of phenolic extracts from Myrica rubra fruit was evaluated by diabetic mice model, and the hypoglycemic potential of Myrica rubra fruit was studied by in vitro and in vivo models. The main results were as follows: 1) There were significant differences in total phenols, total flavonoids and antioxidant activities among different varieties of Myrica rubra fruits. Among them, dark flesh varieties such as `water chestnut', `charcoal plum', `ciqu', `late rice', etc. contained higher total flavonoids, while light flesh varieties such as `pink', `crystal'contained higher total phenols. 12 phenolic compounds, including 5 anthocyanins, including delphinidin hexoside, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, geranidin-3-O-glucoside, peonidin-3-O-glucoside, were identified by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). - Glucosides and seven flavonol glycosides, namely myricetin-3-O-rhamnoside, myricetin deoxyhexoside gallate, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-galactoside and kaempferol-3-O-glucoside. Phenolic compounds and contents in different varieties The antioxidant activities of different Myrica rubra fruits were positively correlated with total phenols and total flavonoids. 2) The activities of phenolic extracts from 9 Myrica rubra fruits and 5 main phenolic compounds monomers in promoting cell glucose consumption were evaluated by HepG2 cell model. The phenolic extracts had significant difference in promoting glucose consumption. The extracts of'Prunus mume','Chinese water chestnut','Ciqiao'and other varieties had strong activity in promoting glucose consumption, which was positively correlated with their higher total phenols, total flavonoids content and DPPH free radical scavenging capacity. The results of correlation analysis showed that the glucose consumption activity of myricetin-3-O-glucoside extracts from different Myrica rubra fruits was related to the content of cyanidin-3-O-glucoside (R2=0.922) and three quercetin glycosides (R2=0.922). Therefore, cyanidin-3-O-glucoside and quercetin-glucoside monomers may be the main components of Myrica rubra fruit extract to promote glucose consumption in HepG2 cells. 3. The hypoglycemic activity of phenolic substances extract (200mg/kg BW) from'water chestnut'fruit was studied in KK-Ay diabetic mice. The results showed that the extract of the fruit of the water chestnut significantly decreased the fasting blood glucose level, increased the oral glucose tolerance and insulin sensitivity in diabetic mice, and significantly reduced the serum insulin, glucagon and leptin levels of glucose metabolism related indicators. In addition, the extract of the fruit of the water chestnut also significantly reduced the glucose level in diabetic mice. The levels of serum total cholesterol, triglyceride, low density lipoprotein, alanine aminotransferase and interleukin-1p in mice with urinary disease were significantly reduced liver weight, liver lipid content, alleviating liver steatosis and other symptoms in diabetic mice. The results showed that 0.4-10 ug/ml extract of `water chestnut'fruit could significantly inhibit cell gluconeogenesis and enhance cell glycolysis, which may be related to activating AMPK protein phosphorylation and inhibiting the expression of downstream gluconeogenesis-related PGC-1a, PEPCK and G6Pase genes. In addition, the extract of `water chestnut'fruits significantly decreased the expression levels of genes related to lipid metabolism, such as PAP, ACAT and CIDA, and inflammatory factors TNFalpha, IL-6 and MCP-1 in liver tissues of mice. Hypoglycemic activity may be related to activating phosphorylation of AMPK protein, inhibiting hepatic gluconeogenesis, promoting hepatic glycolysis, improving liver glycolipid metabolism and alleviating liver inflammation. It provides a theoretical basis for guiding the population, especially the consumption of fruits for diabetics.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S667.6

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