【作者】 石燕玲；

【导师】 何慧；

【作者基本信息】 华中农业大学 ， 食品科学， 2008， 硕士

【摘要】 目前国内外关于灵芝多糖及灵芝三萜类化合物的提取纯化与药理作用研究比较透彻,然而对灵芝肽（GLP）的研究却鲜有涉及;本室前期工作已对灵芝活性肽进行了初步分离,并证实其具有很好的抗氧化活性及对CCl₄致肝损伤有很好的辅助保护作用。为了深入研究灵芝肽的保肝活性及其构效关系,本文对灵芝原料进行了选择,考察了8种树脂对灵芝肽的脱盐效果;通过体外试验、动物实验,探究了灵芝肽与灵芝多糖、灵芝三萜类化合物的协同抗氧化、保肝作用;分别采用了D-氨基半乳糖（D-GalN）、乙醇及卡介苗/脂多糖（BCG+LPS）三种急性肝损伤小鼠模型,进一步研究了灵芝肽的保肝作用;采用Sephadex G-15葡聚糖凝胶层析法,考察了灵芝肽分子量分布;借助于RP-HPLC、HPLC-MS/MS等手段解析了一种灵芝肽结构,初步探讨了灵芝肽的结构与其活性的关系。1.灵芝原料的选择、灵芝肽的提取与脱盐（1）比较了灵芝子实体粉、两种灵芝菌丝体粉、赤芝子实体菌盖及菌柄水提液的粗蛋白含量和抑制羟基自由基活性,结果显示:灵芝子实体粉与灵芝菌丝体粉1的粗蛋白质含量及对羟基自由基的抑制率基本相当,其粗蛋白质含量虽不及灵芝菌丝体粉2,但与灵芝菌丝体粉2比较,其对羟基自由基的抑制率的差异相对较小;由于灵芝菌丝体粉1已商品化,经综合考虑,选择了灵芝菌丝体粉1作为本实验的原料。（2）灵芝水提液超滤后经Cu²⁺-Sephadex G-25配位色谱法分离后得到两个肽峰,是两组复合肽。（3）比较S-8、XAD-16、DA201-C、NAK-Ⅱ、D4020、AB-8六种大孔树脂的静态吸附率、脱盐率及静态解吸附效果,结果显示:S-8树脂对灵芝肽的吸附率及在70%乙醇中的解吸附率均最大,但是DA201-C树脂的脱盐率最高,且其吸附与解吸附效果也接近于S-8树脂,因此选择DA201-C树脂进行了动态吸附的正交试验,确定了最佳的吸附条件为:上样流速为1.6 mL/min、pH值为5.5、上样浓度为2.0mg/mL;比较经大孔树脂（S-8树脂和DA201-C树脂）和阴、阳离子交换树脂处理的两种脱盐方法,结果显示:大孔树脂的脱盐效果优于阴、阳离子交换树脂;而大孔树脂DA201-C与S-8相比较,虽然后者的氮回收率稍高,但其·OH抑制活性和脱盐率均不如前者。经综合考虑,宜选用大孔树脂DA201-C作为灵芝肽脱盐的柱材料。2.灵芝肽对三种肝损伤模型的保肝作用（1）与D-GalN肝损伤模型组小鼠比较,当灌胃中（120mg/kg·bw）、高剂量（180mg/kg·bw）GLP时,能显著（p＜0.05）或极显著（p＜0.01）地降低小鼠血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）活性,同时也能显著地抑制肝匀浆中谷胱甘肽（GSH）含量、超氧化物歧化酶（SOD）活力的下降及丙二醛（MDA）含量的升高（p＜0.01）;生化指标测定及病理学观察均显示出明显的剂量-效应关系,高剂量给肽组小鼠肝细胞病变明显好转或恢复。（2）急性酒精性肝损伤模型的生化指标变化明显,而肝组织病理学变化不明显。模型组的ALT、AST、TG、GSH、MDA、SOD等指标与对照组相比,均发生了显著变化,说明造模是成功的。与乙醇肝损伤模型组小鼠比较,当灌胃低（60mg/kg·bw）、中（120mg/kg·bw）、高剂量（180mg/kg·bw）GLP时,均能极显著地降低血清AST/ALT的活性和TG的含量（p＜0.01）,可极显著地拮抗小鼠肝匀浆中GSH含量与SOD活力的下降与MDA的产生（p＜0.01）,并极显著地抑制肝、脾指数的增大（p＜0.01）;肝组织病理学观察显示:高剂量给肽组小鼠肝细胞与正常组小鼠肝细胞基本相同。中剂量组的效果与阳性对照药物硫普罗宁组（50mg/kg·bw）相当,其中尤以高剂量灵芝肽的保肝作用明显。（3）与BCG+LPS致肝损伤模型组小鼠比较,当灌胃中（120mg/kg·bw）、高剂量（180 mg/kg·bw）GLP时,能显著（p＜0.05）或极显著（p＜0.01）降低小鼠血清中ALT、AST活性以及肝、脾指数,同时也能显著地抑制肝匀浆中GSH含量、GSH-Px/SOD活力的下降（p＜0.01）及MDA与NO含量的升高;生化指标测定及病理学观察均显示出明显的剂量-效应关系,高剂量组的各项实验结果均趋近于正常组,某些生化指标甚至好于正常组。总之,灵芝肽灌胃剂量为180 mg/kg·bw时,对D-氨基半乳糖（D-GalN）、乙醇及卡介苗加脂多糖（BCG+LPS）三种急性肝损伤小鼠模型均有很好的保护作用。3.灵芝肽、多糖、三萜类化合物及其协同抗氧化、保肝作用研究（1）当浓度为7.5mg/mL GLP与浓度为25mg/mL的灵芝多糖（GLPS）组合时,其抑制羟基自由基效果比优于单组分的作用效果,但尚未达到协同增效效果。（2）当GLP与GLPS组合时,无论有无自由基诱导剂,组合组抑制小鼠体外肝匀浆中MDA生成的效果均优于单组分组;当浓度为12.5mg/mL的GLPS与三种剂量的GLP组合时,均呈现出了协同增效效果。（3）在D-GalN肝损伤模型实验中,GLP（150mg/kg·bw）、GLPS（800mg/kg·bw）、灵芝三萜类化合物（GLT）（100mg/kg·bw）及三者组合,均能极显著降低小鼠血清中ALT、AST活性,同时也能极显著地抑制肝匀浆中SOD/GSH-Px活性、GSH含量的下降及MDA含量的升高（p＜0.01）;但生化指标除GSH外,三者组合组均未显示出明显的协同增效作用;但三者组合组小鼠肝细胞病变明显好转或恢复,效果好于单组分组。4.灵芝肽分子量分布及灵芝肽结构初探GLP经葡聚糖凝胶G-15层析分离,出现2个吸收峰,峰1组分的分子量范围在5398.8-1148.9Da之间;峰2组分的分子量范围在1148.9-52.0Da之间,峰2主要洗脱物的分子量范围为530.0-244.5Da;采用HPLC、MS、MS/MS对与HPLC图中主峰Ⅰ对应的,M/Z为365.1的质谱峰进行结构解析,该化合物是氨基酸序列为Ser-Asp-Gly-Ser四肽。更多还原

【Abstract】 There are many research reports about polysacchrides and triterpene from Ganoderma lucidum（GLPS and GLT） but Ganoderma lucidum peptides（GLP） at present.Previously we have reported that GLP has been separated and it possess potent antioxidative capacity and hepatoprotective effects against CCl₄-induced liver injury in mice.In order to research the hepatoprotective effects and relationship between its structure and effect,material was selected and desalination was done,and the hepatoprotective effects of GLP were studied against D-galactosamine（D-GalN） /alcohol/Bacille-Calmette-Gu’erin and lipopolysaccharide（BCG+LPS） induced liver damage in mice.The hepatoprotective effects and antioxidative capacities of GLP, GLPS and GLT were explored.GLP were separated by Sephadex G-15 and the distribution profiles of GLP were studied.The amino acid sequence of a peptide was analyzed by RP-HPLC and HPLC-MS/MS.1.Selection of material and extraction/desalination of GLP（1） The content of crude protein and ratio of inhibitory activities on -OH of water-solution from five material of Ganoderma lucidum（GL） were determined.The result showed that the content of crude protein and the ratio of inhibitory activities on·OH of GL fruit body and GL mycelium one（GLM1） were equivalent.The ratio of inhibitory activities on·OH of GLM1 was close to GLM2,though the content of crude protein of GLM1 were inferior to GLM2.Finally,GLM1 was selected to be material in the experiment,because it is a product by industry.（2） Two fractions-peptides were separated by Cu²⁺-Sephadex G-25 ligand chromatogram method.（3） Desalination of GLP by six kinds of macroporous adsorption resins was investigated.The result revealed that desorption rate and desalination rate of S-8 were both relatively highest with 70%ethanol as desorpting agent,but the best resin to desalt GLP was DA201-C,and absorption rate and desorption rate of it was approached to S-8.According to the static adsorption,the DA201-C resin was chosen to do the dynamic adsorption tests,and the optimum adsorbing condition was 1.6 mL/min of flowing velocity,5.5 of pH and 2.0mg/mL of concentration.Compared with Cation and Anion exchange resin treatment and S-8 resin,DA201-C resin treatment was the best desalt method,the ratio of inhibitory activities on·OH of GLP and desalting rate of which were highest except that its nitrogen recovery ratio was not better than S-8.2.The hepatoprotective effects of GLP against D-GalN/alcohol/BCG+LPS induced liver damage in mice（1） Compared to mice of D-GalN-induced hepatic damage,pretreatment of mice with GLP at doses of 120 mg/kg-bw and 180mg/kg·bw were manifested by a significant decrease in the activities of marker enzymes（AST,ALT） in serum and MDA level in liver（p＜0.01）,and by a significant increase in SOD activity and GSH level in liver （p＜0.01）.The biochemical results were supplemented by histopathological examination of liver sections.Specially,at the dose of 180mg/kg·bw GLP,the biochemical parameters and the liver histopathological characters were close to normal mice.（2） The mice’index markedly changed except liver histopathological characters with alcohol treatment.Mice intoxicated with alcohol alone developed extremely evident hepatocellular damage from an extremely significant change in activities of AST, ALT and SOD and content of TG,GSH and MDA,when compared with normal control group.Compared to mice of alcohol-induced hepatic damage,pretreatment of mice with GLP at doses of 60 mg/kg·bw,120 mg/kg·bw and 180mg/kg·bw were manifested by a significant decrease in the activities of marker enzymes（AST,ALT） and content of TG in serum and MDA level in liver（p＜0.01）,and by a significant increase in activity of SOD and GSH level in liver（p＜0.01）.The elevated liver index and spleen index was markedly reduced by GLP treatment（P＜0.01）.At the dose of 180mg/kg·bw GLP,the liver histopathological characters were close to normal mice. At the dose of 120 mg/kg·bw GLP,the effect was equivalent to mice pretreated with 50 mg/kg·bw tiopronin.（3） Compared to mice of BCG+LPS-induced hepatic damage,pretreatment of mice with GLP at doses of 120 mg/kg·bw and 180 mg/kg·bw were manifested by a significant decrease in the activities of marker enzymes（AST,ALT） in serum and liver index and spleen index（p＜0.05,p＜0.01）,and by a significant increase in activities of SOD and GSH level and a significant decrease in content of MDA and NO in liver（p＜0.01）.The biochemical results were supplemented by histopathological examination of liver sections.Specially,at the dose of 180 mg/kg·bw GLP,the biochemical parameters and the liver histopathological characters were close to normal mice,and some were better than normal mice.In conclusion,pretreatment of mice with GLP at doses of 180 mg/kg·bw afforded a significant protection in the alleviation of D-GalN/Alcohol/BCG+LPS-induced hepatocellular injury.3.Study on antioxidant activity of GLP and GLPS and hepatoprotective effects of GLP,GLPS and GLT（1） The ratio of inhibitory activities on·OH of GLP（7.5mg/mL）+GLPS（25mg/mL） was higher than the same concentration GLP or GLPS,but was not remarkable.（2） The inhibitory effect on liver MDA of GLP+GLPS was excelled to the same concentration GLP or GLPS in vitro.It showed prominent effect when GLPS （12.5mg/mL） was compounded with GLP at each concentration.（3） Compared to mice of D-GalN-induced hepatic damage,pretreatment of mice with GLP（150mg/kg·bw）,GLPS（800mg/kg·bw）,GLT（100mg/kg·bw） and combination of them were manifested by a significant decrease in the activities of marker enzymes （AST,ALT） in serum and MDA level in liver（p＜0.01）,and by a significant increase in activity of SOD/GSH-Px and GSH level in liver（p＜0.01）.The change of every index was not evident except GSH in mice treated with combination of GLP,GLPS and GLT.The liver histopathological characters of mice treated with combination of GLP,GLPS and GLT markedly got better and recovered,better than the same concentration GLP or GLPS or GLT. 4.Study on distribution profiles and structure of GLPGLP were separated by Sephadex G-15 column and finally divided into two peaks, one distributed between 5398.8 to 1148.9Da in molecular weight,and the other distributed between 1148.9 to 52.0Da,the main of which was between 530.0 to 244.5Da.By means of HPLC-MS,MS/MS,the amino acid sequence of a peptide, with M/Z 365.1 from peak I in HPLC,was determined,which was Ser-Asp-Gly-Ser.更多还原