紫心甘薯多糖的提取与生物活性研究
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摘要
活性多糖分布广泛,主要来源于植物、动物、微生物等。多年来的药理研究表明多糖作为免疫调节剂,在抗肿瘤、抗病毒、抗感染、抗氧化、降血糖血脂等各个方面均有广泛的作用,而且安全低毒性,是现代医学与食品领域关注的研究热点之一。
本文以浙江省临安市太阳镇紫心甘薯基地的“渝紫263”为研究对象,利用分离纯化技术、化学结构分析和动物模型实验等多种手段,研究了紫心甘薯多糖(PPSP)的提取工艺、分离纯化过程中的条件控制与实验优化,并对其体内降血糖调血脂作用以及体内外抗氧化活性进行了深入研究,主要结论如下:
(1)研究了PPSP的分离提取工艺的最佳条件。在热水浸提过程中影响PPSP含量的一些单因素实验基础上,设计了三因素正交试验,获得最佳工艺条件为料液比1:15、提取温度70℃、提取2h;通过对醇沉过程中PPSP水溶液的浓缩比和乙醇用量的条件优化,并综合考虑多糖含量和乙醇用量,获得其最佳醇沉条件为浓缩比1:2、3倍体积乙醇沉淀。
(2)通过柱层析分离获得了四个多糖组分。通过DEAE-纤维素柱层析结合不同浓度和种类的缓冲液洗脱,对PPSP进行分离和分部收集。控制洗脱条件依次为:蒸馏水、0.05 mol/LNaHCO_3缓冲液洗脱、50 mmol/L PBS溶液、0-2 mol/LNaCl以及0.5 mol/LNaOH+1.5 mol/LNaCl洗脱;采用苯酚-硫酸法检测多糖在OD_(490)的吸收峰,绘制洗脱曲线;收集单峰对应管,经透析除盐,真空干燥得到四个多糖组分PPSPⅠ、PPSPⅡ、PPSPⅢ和PPSPⅣ;并对PPSP的两个组分PPSPⅡ、PPSPⅢ分别进行了初步的结构分析。
(3)开展PPSP的降血糖调血脂的体内活性研究,以链脲佐菌素(STZ)糖尿病大鼠为模型,测定了PPSP溶液灌胃对糖尿病大鼠血糖、糖化血清蛋白(GSP)、肝糖原、胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)的影响。结果显示,PPSP可显著提高肝糖原合成能力,降低糖尿病大鼠血糖,调节血脂及血清中GSP、TC、TG的含量。表明PPSP具有一定的降血糖调血脂作用,分析作用机制可能与调节肝糖原代谢有关。
(4)检测到PPSP具有较强的体内外抗氧化活性。采用还原力、羟基自由基和DPPH·自由基反应体系,检测不同剂量PPSP的体外抗氧化活性。建立链脲佐菌素诱导糖尿病大鼠模型,测定并比较多糖组与模型组的肝脏和胰腺组织内的丙二醛(MDA)含量、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)和总抗氧化(T-AOC)活性。结果显示大鼠肝脏和胰腺组织内的MDA含量均有明显降低,SOD、GSH和T-AOC活性都有显著升高;肝脏切片结果显示中剂量多糖能有效减少糖尿病所致的肝脏细胞损伤。
Polysaccharides are widely distributed in the world, mainly obtained from plants, animals, microorganisms and so on. By previous pharmacological studies, they have been reported as an immune modulator to play a wide range of functional roles in anti-tumor, anti-virus, anti-infective, anti-oxidation and the regulation for blood glucose and lipids. On the basis of their low toxicity, more and more concerns have recently been paid to the correlative research with bioactive polysaccharides in the modern medicine and food industry field.
In this study, we chose" Yu purple sweet potato 263" as our research objective, which harvested from Sun Town in Lin'an City of Zhejiang province. By use of biochemical purification, chemical structure analysis and animal model experiments and other means, we developed the studies of purple sweet potato from polysaccharide (PPSP) in its extraction, isolation and purification process, and experimental optimization of the conditions, and its role in regulating blood lipid and glucose in vivo, as well as antioxidant activity in vitro and in vivo. In summary, the main conclusions are drawn as follows:
(1) The optimum extraction conditions of PPSP were studied by designing orthogonal experiment, and the following optimum conditions were known as: the ratio of the sample weight to the solvent volume is 1:15; the temperature is 70℃; 2 hours for extraction. In addition, the polysaccharide yield was shown to be influenced by two factors in the process of ethanol precipitation, the ideal conditions for precipitation were as follow: the ratio of sample weight to volume of concentrated solution is 1:2; the final concentration of ethanol is 85% and 3 times.
(2) Four components were separated from PPSP with a single peak after eluted by DEAE-cellulose column chromatography under a combination of different concentrations and choosing with different kinds of elution buffers as follow: water, 0.05mol/L NaHCO_3 buffer,50mmol/L PBS buffer, 0-2mol/LNaCl and 0.5mol/LNaOH +1.5mol/LNaCl buffer and subsequently collected as PPSPⅠ, PPSPⅡ, PPSPⅢand PPSPⅣafter further dialysis desalting and desiccated under vacuum. The preliminary structural analysis was further made respectively for PPSPⅡand PPSPⅢ.
(3) The effects of PPSP on the metabolism of glucose and lipids in diabetic rat induced by streptozotocin (STZ) were studied in vivo as well as underlined mechanism. By observing the serum glucose, glycosylated serum protein (GSP), hepatic glycogen, total cholesterol (TC), triglyceride(TG) and high density lipoprotein cholesterol (HDL-C), the results indicate that PPSP could significantly increase the synthesis of hepatic glycogen, while it might decrease the content of serum glucose, GSP, TC and TG, which suggests that PPSP can significantly attenuate hyper -lipidemia and hyperglycemia in diabetic rat, probably due to the regulatory function of PPSP on the metabolism of hepatic glucogen and antioxidation.
(4) PPSP was shown its significant antioxidant effect in vitro and in vivo. By establishing the reaction system of hydroxyl radical, DPPH free radical and reducing power, we detected antioxidant activity of PPSP with different doses in vitro. The contents of MDA, the activities of SOD, GSH and T-AOC in liver and pancreas were respectively measured in oxidative diabetes mellitus rat model caused by streptozotocin. In addition, we found that the contents of MDA were decreased and the activities of SOD, GSH and T-AOC were increased in both liver and pancreas Moreover, liver rescue was observed in the liver tissue obtained from STZ-inducing diabetic rat by middle dose PPSP extract taken.
本文以浙江省临安市太阳镇紫心甘薯基地的“渝紫263”为研究对象,利用分离纯化技术、化学结构分析和动物模型实验等多种手段,研究了紫心甘薯多糖(PPSP)的提取工艺、分离纯化过程中的条件控制与实验优化,并对其体内降血糖调血脂作用以及体内外抗氧化活性进行了深入研究,主要结论如下:
(1)研究了PPSP的分离提取工艺的最佳条件。在热水浸提过程中影响PPSP含量的一些单因素实验基础上,设计了三因素正交试验,获得最佳工艺条件为料液比1:15、提取温度70℃、提取2h;通过对醇沉过程中PPSP水溶液的浓缩比和乙醇用量的条件优化,并综合考虑多糖含量和乙醇用量,获得其最佳醇沉条件为浓缩比1:2、3倍体积乙醇沉淀。
(2)通过柱层析分离获得了四个多糖组分。通过DEAE-纤维素柱层析结合不同浓度和种类的缓冲液洗脱,对PPSP进行分离和分部收集。控制洗脱条件依次为:蒸馏水、0.05 mol/LNaHCO_3缓冲液洗脱、50 mmol/L PBS溶液、0-2 mol/LNaCl以及0.5 mol/LNaOH+1.5 mol/LNaCl洗脱;采用苯酚-硫酸法检测多糖在OD_(490)的吸收峰,绘制洗脱曲线;收集单峰对应管,经透析除盐,真空干燥得到四个多糖组分PPSPⅠ、PPSPⅡ、PPSPⅢ和PPSPⅣ;并对PPSP的两个组分PPSPⅡ、PPSPⅢ分别进行了初步的结构分析。
(3)开展PPSP的降血糖调血脂的体内活性研究,以链脲佐菌素(STZ)糖尿病大鼠为模型,测定了PPSP溶液灌胃对糖尿病大鼠血糖、糖化血清蛋白(GSP)、肝糖原、胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)的影响。结果显示,PPSP可显著提高肝糖原合成能力,降低糖尿病大鼠血糖,调节血脂及血清中GSP、TC、TG的含量。表明PPSP具有一定的降血糖调血脂作用,分析作用机制可能与调节肝糖原代谢有关。
(4)检测到PPSP具有较强的体内外抗氧化活性。采用还原力、羟基自由基和DPPH·自由基反应体系,检测不同剂量PPSP的体外抗氧化活性。建立链脲佐菌素诱导糖尿病大鼠模型,测定并比较多糖组与模型组的肝脏和胰腺组织内的丙二醛(MDA)含量、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)和总抗氧化(T-AOC)活性。结果显示大鼠肝脏和胰腺组织内的MDA含量均有明显降低,SOD、GSH和T-AOC活性都有显著升高;肝脏切片结果显示中剂量多糖能有效减少糖尿病所致的肝脏细胞损伤。
Polysaccharides are widely distributed in the world, mainly obtained from plants, animals, microorganisms and so on. By previous pharmacological studies, they have been reported as an immune modulator to play a wide range of functional roles in anti-tumor, anti-virus, anti-infective, anti-oxidation and the regulation for blood glucose and lipids. On the basis of their low toxicity, more and more concerns have recently been paid to the correlative research with bioactive polysaccharides in the modern medicine and food industry field.
In this study, we chose" Yu purple sweet potato 263" as our research objective, which harvested from Sun Town in Lin'an City of Zhejiang province. By use of biochemical purification, chemical structure analysis and animal model experiments and other means, we developed the studies of purple sweet potato from polysaccharide (PPSP) in its extraction, isolation and purification process, and experimental optimization of the conditions, and its role in regulating blood lipid and glucose in vivo, as well as antioxidant activity in vitro and in vivo. In summary, the main conclusions are drawn as follows:
(1) The optimum extraction conditions of PPSP were studied by designing orthogonal experiment, and the following optimum conditions were known as: the ratio of the sample weight to the solvent volume is 1:15; the temperature is 70℃; 2 hours for extraction. In addition, the polysaccharide yield was shown to be influenced by two factors in the process of ethanol precipitation, the ideal conditions for precipitation were as follow: the ratio of sample weight to volume of concentrated solution is 1:2; the final concentration of ethanol is 85% and 3 times.
(2) Four components were separated from PPSP with a single peak after eluted by DEAE-cellulose column chromatography under a combination of different concentrations and choosing with different kinds of elution buffers as follow: water, 0.05mol/L NaHCO_3 buffer,50mmol/L PBS buffer, 0-2mol/LNaCl and 0.5mol/LNaOH +1.5mol/LNaCl buffer and subsequently collected as PPSPⅠ, PPSPⅡ, PPSPⅢand PPSPⅣafter further dialysis desalting and desiccated under vacuum. The preliminary structural analysis was further made respectively for PPSPⅡand PPSPⅢ.
(3) The effects of PPSP on the metabolism of glucose and lipids in diabetic rat induced by streptozotocin (STZ) were studied in vivo as well as underlined mechanism. By observing the serum glucose, glycosylated serum protein (GSP), hepatic glycogen, total cholesterol (TC), triglyceride(TG) and high density lipoprotein cholesterol (HDL-C), the results indicate that PPSP could significantly increase the synthesis of hepatic glycogen, while it might decrease the content of serum glucose, GSP, TC and TG, which suggests that PPSP can significantly attenuate hyper -lipidemia and hyperglycemia in diabetic rat, probably due to the regulatory function of PPSP on the metabolism of hepatic glucogen and antioxidation.
(4) PPSP was shown its significant antioxidant effect in vitro and in vivo. By establishing the reaction system of hydroxyl radical, DPPH free radical and reducing power, we detected antioxidant activity of PPSP with different doses in vitro. The contents of MDA, the activities of SOD, GSH and T-AOC in liver and pancreas were respectively measured in oxidative diabetes mellitus rat model caused by streptozotocin. In addition, we found that the contents of MDA were decreased and the activities of SOD, GSH and T-AOC were increased in both liver and pancreas Moreover, liver rescue was observed in the liver tissue obtained from STZ-inducing diabetic rat by middle dose PPSP extract taken.
引文
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