具有降血糖活性的茶多糖组分分离纯化与结构鉴定
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摘要
国内外已有大量报道证实茶多糖具有显著的降血糖功能,但有关其降血糖方式和结构的报道还不多。资料显示水溶性茶多糖具有显著的降血糖功能,因此为了得到具有较高生理活性的茶多糖,本论文采用水提法提取茶多糖,然后从茶多糖对自由基的清除作用、对相关糖代谢酶活性的影响以及对葡萄糖往3T3-L1细胞转运的促进作用三个方面,体外研究茶多糖的降血糖方式,以此为依据,依次采用离子交换法和凝胶柱层析法从茶多糖中分离具有降血糖活性的组分,并对其一级结构进行深入研究和探讨。
为提高茶多糖含量测定的精确度,在茶多糖提取以前,首先对传统蒽酮-硫酸法测定茶多糖的条件作了改进。结果显示,测定茶多糖含量应以半乳糖为标准单糖,675 nm比色。确定了茶多糖的测定条件以后,在不破坏茶多糖生理活性的温度范围内优化了茶多糖的提取工艺,得出水提法提取茶多糖的最佳工艺条件为:浸提时间90 min,浸提温度70℃,料水比1 g:10 mL,浸提3次。所得提取率为3.20 %,高于资料报道的2.97 %的最高提取率。所提茶多糖经一系列纯化处理,纯度达到了89 %。
接着从对羟基自由基和超氧自由基的清除作用、对小肠α-葡萄糖苷酶和肝脏糖代谢酶活性的影响、以及对葡萄糖往3T3-L1细胞转运的促进作用三个方面,体外研究茶多糖的降血糖方式。结果发现茶多糖对羟基自由基和超氧自由基没有明显的清除作用,对葡萄糖往3T3-L1细胞转运也没有明显的促进作用,对小肠α-葡糖苷酶活性的抑制作用也较低,但可显著增强肝脏葡萄糖激酶和己糖激酶活性。浓度为1 mg/mL的茶多糖可使葡萄糖激酶和己糖激酶相对活性分别提高82.97 %和99.57 %;10 mg/mL的茶多糖可使它们分别提高152.09 %和156.10 %。
采用DEAE-Sepharose CL-6B离子交换色谱柱分离具有增强肝脏葡萄糖激酶和己糖激酶活性的多糖组分,共分出5个组分:FA、FB、FC、FD、FE,其中FA和FC均能显著增强己糖激酶和葡萄糖激酶活性。茶多糖浓度为1 mg/mL时,FA对己糖激酶和葡萄糖激酶相对活性的提高率分别为102.92 %和60.31 %;FC对己糖激酶和葡萄糖激酶相对活性的提高率分别为121.17 %和137.81 %。进一步用Sephadex G75分离FA和FC,得FA-1、FA-2、FC-1和FC-2。FA-1和FC-1可显著提高己糖激酶和葡萄糖激酶的相对活性,提高率分别为106.42 %和72.92 %以及142.22 %和140.34 %(茶多糖浓度为1 mg/mL)。FA-2仅能显著增加己糖激酶的活性(P<0.05),不能增强葡萄糖激酶活性,FC-2对己糖激酶和葡萄糖激酶活性都没有增强作用。进一步用Sephadex G150柱纯化FA-1和FC-1,Sephadex G25柱纯化FA-2,得单一组分。
根据Sephadex G25对多糖的分离范围可得出FA-2的相对分子量较小;红外光谱和紫
Tea polysaccharides (TPS) have been documented good hypoglycemic action at home and abroad, nevertheless, few reports were involved in its structure and anti-hyperglycemia manner. Since water-soluble TPS displayed significant anti-hyperglycemic activity, TPS were extracted with water from coarse green tea leaves. Then its anti-hyperglycemic action was investigated from following aspects: scavenging of free radicals, effect on enzymes involved in carbohydrate metabolism and 3T3-L1 adipocytes. The anti-hyperglycemic components were fractionated using ion-exchange and gel columns and their structures were characterized with varieties of chemical and physical methods.
First, Anthrone-Sulfuric acid Method, which was usually used to detect polysaccharide, was improved and found that using galactose as standard sugar and colorimetric analysis at 675 nm was better than that using glucose and at 620 nm. For increasing the extraction rate of TPS, the extraction parameters were optimized. Results showed that the optimum conditions for extraction of TPS were heating at 70℃for 90 min with a 1:10 ratio of tea to water, repeating three times. Under this condition, the extraction rate was 3.20 %, higher than the most extraction rate of 2.97 % in previous reports. The extracted TPS was purified with Sevag method, polyamide column chromatography and Sephadex G 50 column chromatography and obtained TPS1-1 with a 89 % purity.
Then from following aspects to detect the anti-hyperglycemic effect of TPS: scavenging of free radicals, effect on enzymes involved in carbohydrate metabolism and 3T3-L1 adipocytes. Results exhibited that TPS can not stimulate the transportation of glucose into 3T3-L1 cells and scavenge free radicals in vitro, however, can increase significantly the activities of glucokinase and hexokinase in vitro, though have no obvious effect on alvine glycosidase. 1 mg/mL of TPS elevated glucokinase and hexokinase 82.97 % and 99.57 %, respectively. 10 mg/mL elevated 152.09 % and 156.10 %, respectively.
For purification of the anti-hyperglycemic components, DEAE-Sepharose CL 6B was used to separate the components, which can stimulate the activities of hexokinase and glucokinase. Five components were collected and two, FA and FC, showed obvious activity. FA and FC were further isolated by Sephadex G75, and FA-1, FA-2, FC-1 and FC-2 were collected. Among the four components, FA-1 and FC-1 were detected significant stimulating activity in glucokinase and hexokinase. 1 mg/mL of FA-1 increased the activities of hexokinase and glucokinase 106.42 % and 72.92 %, respectively. 1 mg/mL of FC-1 increased the activities of
为提高茶多糖含量测定的精确度,在茶多糖提取以前,首先对传统蒽酮-硫酸法测定茶多糖的条件作了改进。结果显示,测定茶多糖含量应以半乳糖为标准单糖,675 nm比色。确定了茶多糖的测定条件以后,在不破坏茶多糖生理活性的温度范围内优化了茶多糖的提取工艺,得出水提法提取茶多糖的最佳工艺条件为:浸提时间90 min,浸提温度70℃,料水比1 g:10 mL,浸提3次。所得提取率为3.20 %,高于资料报道的2.97 %的最高提取率。所提茶多糖经一系列纯化处理,纯度达到了89 %。
接着从对羟基自由基和超氧自由基的清除作用、对小肠α-葡萄糖苷酶和肝脏糖代谢酶活性的影响、以及对葡萄糖往3T3-L1细胞转运的促进作用三个方面,体外研究茶多糖的降血糖方式。结果发现茶多糖对羟基自由基和超氧自由基没有明显的清除作用,对葡萄糖往3T3-L1细胞转运也没有明显的促进作用,对小肠α-葡糖苷酶活性的抑制作用也较低,但可显著增强肝脏葡萄糖激酶和己糖激酶活性。浓度为1 mg/mL的茶多糖可使葡萄糖激酶和己糖激酶相对活性分别提高82.97 %和99.57 %;10 mg/mL的茶多糖可使它们分别提高152.09 %和156.10 %。
采用DEAE-Sepharose CL-6B离子交换色谱柱分离具有增强肝脏葡萄糖激酶和己糖激酶活性的多糖组分,共分出5个组分:FA、FB、FC、FD、FE,其中FA和FC均能显著增强己糖激酶和葡萄糖激酶活性。茶多糖浓度为1 mg/mL时,FA对己糖激酶和葡萄糖激酶相对活性的提高率分别为102.92 %和60.31 %;FC对己糖激酶和葡萄糖激酶相对活性的提高率分别为121.17 %和137.81 %。进一步用Sephadex G75分离FA和FC,得FA-1、FA-2、FC-1和FC-2。FA-1和FC-1可显著提高己糖激酶和葡萄糖激酶的相对活性,提高率分别为106.42 %和72.92 %以及142.22 %和140.34 %(茶多糖浓度为1 mg/mL)。FA-2仅能显著增加己糖激酶的活性(P<0.05),不能增强葡萄糖激酶活性,FC-2对己糖激酶和葡萄糖激酶活性都没有增强作用。进一步用Sephadex G150柱纯化FA-1和FC-1,Sephadex G25柱纯化FA-2,得单一组分。
根据Sephadex G25对多糖的分离范围可得出FA-2的相对分子量较小;红外光谱和紫
Tea polysaccharides (TPS) have been documented good hypoglycemic action at home and abroad, nevertheless, few reports were involved in its structure and anti-hyperglycemia manner. Since water-soluble TPS displayed significant anti-hyperglycemic activity, TPS were extracted with water from coarse green tea leaves. Then its anti-hyperglycemic action was investigated from following aspects: scavenging of free radicals, effect on enzymes involved in carbohydrate metabolism and 3T3-L1 adipocytes. The anti-hyperglycemic components were fractionated using ion-exchange and gel columns and their structures were characterized with varieties of chemical and physical methods.
First, Anthrone-Sulfuric acid Method, which was usually used to detect polysaccharide, was improved and found that using galactose as standard sugar and colorimetric analysis at 675 nm was better than that using glucose and at 620 nm. For increasing the extraction rate of TPS, the extraction parameters were optimized. Results showed that the optimum conditions for extraction of TPS were heating at 70℃for 90 min with a 1:10 ratio of tea to water, repeating three times. Under this condition, the extraction rate was 3.20 %, higher than the most extraction rate of 2.97 % in previous reports. The extracted TPS was purified with Sevag method, polyamide column chromatography and Sephadex G 50 column chromatography and obtained TPS1-1 with a 89 % purity.
Then from following aspects to detect the anti-hyperglycemic effect of TPS: scavenging of free radicals, effect on enzymes involved in carbohydrate metabolism and 3T3-L1 adipocytes. Results exhibited that TPS can not stimulate the transportation of glucose into 3T3-L1 cells and scavenge free radicals in vitro, however, can increase significantly the activities of glucokinase and hexokinase in vitro, though have no obvious effect on alvine glycosidase. 1 mg/mL of TPS elevated glucokinase and hexokinase 82.97 % and 99.57 %, respectively. 10 mg/mL elevated 152.09 % and 156.10 %, respectively.
For purification of the anti-hyperglycemic components, DEAE-Sepharose CL 6B was used to separate the components, which can stimulate the activities of hexokinase and glucokinase. Five components were collected and two, FA and FC, showed obvious activity. FA and FC were further isolated by Sephadex G75, and FA-1, FA-2, FC-1 and FC-2 were collected. Among the four components, FA-1 and FC-1 were detected significant stimulating activity in glucokinase and hexokinase. 1 mg/mL of FA-1 increased the activities of hexokinase and glucokinase 106.42 % and 72.92 %, respectively. 1 mg/mL of FC-1 increased the activities of
引文
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