【作者】 徐露；

【导师】 董志；

【作者基本信息】 重庆医科大学 ， 药理学， 2008， 硕士

【摘要】 目的:通过略加改进的传统提取方法,从传统补益药当归[Angelica Sinensis(Oliv.)Diels]中,提取分离出当归多糖(Angelica Sinensis polysacchride ASP),用改良的H2SO4-苯酚法测量ASP含量。再用不同浓度的ASP作用于D-半乳糖所至的亚急性衰老小鼠模型。通过观察小鼠体重、行为学、脑组织与脾脏切片、脾脏指数、脑指数的变化,及测量小鼠血清和脑组织中SOD、MDA、GSH-PX含量和小鼠脑细胞凋亡数,来确定ASP是否具有抗衰老作用及探寻其可能的作用机制,从而为更深入的研究ASP药理作用和研发新型的抗衰老药物提供理论指导和实验依据。方法:①当归多糖的提取和含量测定。本研究在传统提取方法的基础上略进行改进,先用乙醇(Ethanol,EtOH)沉淀ASP,再过滤,80oC水提取,加入饱和氢氧化钙(Calcium hydroxide,C a(OH)2)调节PH到10,过滤,再用乙酸调节滤液PH到4～5,离心过滤,脱色后,减压浓缩滤液为原来1/3。浓缩液中加入乙醇,静置,滤过,沉淀物用无水乙醇、丙酮和乙醚洗涤三次,50oC烘干得到ASP粗当归多糖。采用改良的H2SO4-苯酚法测量ASP含量,测定波长为490nm;②当归多糖对D-半乳糖致衰老小鼠的抗衰老作用。90只昆明小鼠随机分为6组,一组为空白组,另5组每日皮下注射10%D-半乳糖0.25ml/20g,连续40d,其中一组为衰老组,一组为香菇多糖阳性对照组,其余三组为高中低剂量的当归多糖组,实验的第一天开始,每日灌胃香菇多糖和不同剂量的当归多糖进行干预。Morris水迷宫实验检测小鼠的学习记忆功能,羟胺法测定小鼠血清和脑组织SOD含量,TBA法测定血清和脑组织MDA含量,采用二硫代二硝基苯甲酸法(DTNB)测谷胱甘肽过氧化物酶(GSH-Px)活性。脾脏和脑组织切片HE染色观察脑细胞形态学变化,TUNEL染色法测定小鼠脑细胞凋亡指数,记录各小鼠脑指数和脾脏指数以及体重变化。结果:①提取的当归多糖的含量结果。用改良的H2SO4-苯酚法测量ASP含量,检测波长为490nm,测得含量为71%;②ASP对D-半乳糖所致亚急性衰老模型小鼠的抗衰老作用检测结果。与模型组比较,通过Morris水迷宫实验和跳台实验,ASP组衰老小鼠学习记忆力较高(p<0.01);通过羟胺法测得ASP组小鼠血清和脑组织SOD活力明显强于模型组(p<0.05,p<0.01);TBA法检测出ASP组小鼠血清和脑组织MDA含量明显减少(p<0.05,p<0.01);二硫代二硝基苯甲酸法(DTNB)测出ASP组血清和脑组织GSH-PX含量得到增加( p<0.05,p<0.01 ); ASP组的脾脏指数和脑指数也明显提高(p<0.05,p<0.01),体重也有了增加;脑平片HE染色观察到ASP组脑细胞变性和坏死情况得到改善;脾脏HE染色观察到ASP组小鼠脾脏淋巴细胞增多;TUNEL染色法测出ASP组脑细胞凋亡指数降低(p<0.05,p<0.01)。结论:在传统提取当归多糖的基础上略加改动得到的ASP粗多糖含量比传统方法得到的当归粗多糖更高,因此对实验结果的影响更加微小。改良的H2SO4-苯酚法测量出的ASP含量也使ASP用量更加精确。抗衰老实验结果表明ASP对D-半乳糖所致亚急性衰老小鼠有一定的抗衰老作用:ASP能提高衰老动物的学习记忆力、增强衰老小鼠体内SOD、GSH-PX活力、减少MDA含量、脾脏指数、脑指数和体重得到提高、脑组织细胞变性坏死情况得到改善、提高小鼠免疫力、降低脑细胞凋亡数。研究提示:当归多糖对于预防和治疗老年病可能具有较好的潜在的开发和应用前景。更多还原

【Abstract】 Objective: We have used a method that is different from traditional method to extracted ,isolated and gained an ASP from neutral angelica sinensis. ASP content was determined by the modified method of sulphuric acid-phenol. Different concentration ASP acted on subacute senile mouse model induced by D-galactose. We observed weight、behavior、brain and spleen section、spleen index、brain index and We measured SOD、MDA and GSH-PX content in the serum and brain tissue and cerebral cell apoptosis number to determine whether ASP has anti-aging effects and find its possible mechanism for further study of ASP pharmacology effects. The experimental study my be provide the new theory and experiment instruction to develop new anti-aging drug.Methods:①Extraction separation of ASP and content determining: We have used a method that is different from traditional method. We used ethanol to precipitate ASP, filtering, extracting in 80OC. We added saturable Ca(OH)2 solution to adjust PH to 10 and acetic acid to adjust PH to4-5. Solution was centrifugal filtered. After decoloring, filter liquor was decompress concentrated to 1/3 original volume. Alcohol was added into concentrate liquid, standing, filtering. Anhydrous ethanol,acetone,ether washed precipitate three times respectively. ASP was obtained when it was dried in 50 oC. ASP content was determined by the modified method of sulphuric acid-phenol;②Effect of ASP on anti-aging in D-galactose induced rats. Sixty mice were divided randomly into 6 groups (normal saline,D-galactose,lentinan,low,middle and high does of ASP groups). The D-galactose group,lentinan group and ASP groups were injected with 10% D-galactose 0.25ml/20g for 40 days. The ASP groups and lentinan group were given various dosage of ASP and lentinan 100mg/kg respectively by intragastric administration from the first day. We undergone diving platform test and Morris maze test to determine the study and memory ability . We observed the change on spleen index、brain index and weight. SOD activities,MDA content and GSH-PX activities were measured respectively in blood serum and brain tissue. The morphous of brain and spleen cells were observed with the method of HE and the apoptotic index of brain cells was determined with the method of TUNEL.Results:①ASP content results. ASP content was determined by the modified method of sulphuric acid-phenol in the 490nm wavelength. ASP content is 71.0%.②Effect of ASP on anti-aging in D-galactose induced aged rats. Comparing with model group, ASP could improve the study and memory ability of mice、increase the activities of SOD and GSH-PX and decrease MDA content in blood serum and brain tissue,markedly counteract the atrophy of the spleen,improve brain cell degeneration and decrease apoptotic index,and increase the number of lymphocyte.Conclution: The method that is different from traditional method to extracted ,isolated and gained an ASP is better than traditional method. We gained ASP in higher content with modified method and it will occur less effects on this experiment. ASP content was determined by the modified method of sulphuric acid-phenol accurately. This experiment suggests that ASP having good anti—aging action in mice. ASP may be developed as a drug that could prevent and treat senile disease.更多还原