【作者】 陈自泓；

【导师】 黄可儿；

【作者基本信息】 广州中医药大学 ， 中药学， 2019， 硕士

【摘要】 目的:优选黄芪多糖半仿生提取法的最佳工艺,分析半仿生提取醇沉工艺和水提醇沉工艺所得的黄芪多糖,在多糖均一性、分子量以及对胃粘膜损伤保护作用的差异,并初步探讨其作用机制。方法:以黄芪总多糖提取率、提取收率及总皂苷提取收率为考察指标,采用正交实验优化半仿生提取最佳工艺,并与传统水提工艺进行比较。采用Sevag去蛋白、DEAE-52纤维素柱层析对传统水提醇沉法与半仿生提取醇沉法处理得到黄芪多糖,采用高效凝胶渗透色谱法(GPC)检测两种工艺所得黄芪多糖的均一性及分子量的差异。比较两种提取工艺得到的黄芪多糖对乙醇及吲哚美辛诱导大鼠胃粘膜损伤的保护作用。研究半仿生提取醇沉工艺获得的黄芪多糖对乙醇及吲哚美辛诱导大鼠胃粘膜损伤的保护作用,并从胃粘膜组织中凋亡信号通路Bcl-2、Bax、Caspase-3蛋白表达探讨治疗作用机制。结果:1、正交实验优化黄芪多糖半仿生提取法工艺:以黄芪总多糖提取率(p/%)、总皂苷提取率(P/%)、提取收率(P/%)作为指标;①在模拟胃液提取条件下,贡献度加权分析结果显示,贡献度大小由大到小依次是提取温度(28.9)>料液比(12.8)>提取时间(1.5)。其中料液比(P<0.05)和提取温度(P<0.01)起主要作用,均具有统计学差异(P<0.05),提取时间作用次之,无统计学差异(P>0.05)。②在模拟肠液提取条件下,贡献度加权分析结果显示,加权后贡献度大小由大到小依次是料液比(49.6)>提取温度(12.8)>提取时间(4.0)。其中料液比(P<0.01)和提取温度(P<0.05)起主要作用,均具有统计学差异(P<0.05),提取时间作用次之,无统计学差异(P>0.05);实验结果:结合经济效益,半仿生提取法优选工艺为模拟胃液为料液比1:30,提取温度100℃,提取时间1h;模拟肠液为料液比1:30,提取温度100℃,提取时间1 h。2、两种工艺得到的黄芪多糖在均一性及分子量的比较:两种提取工艺得到的黄芪多糖均为非均一多糖,两者之间在分子量上存在差异,半仿生提取法的分子量分布约在3万-670万之间,且其低分子量段含量稍高于高分子量段多糖,而传统水提法的分子量分布约为5万-1670万,其高分子量段含量远高于其他分子量段多糖。提示半仿生提取获得的多糖在分子量方面与传统水提法获得的多糖是有明显的区别。3、两种工艺得到的黄芪多糖对乙醇及吲哚美辛诱导的大鼠胃粘膜损伤保护作用观察:①乙醇诱导大鼠胃粘膜损伤保护实验结果显示,与模型组比较,半仿生提取醇沉黄芪多糖组大鼠的胃粘膜损伤评分明显降低均有统计学差异(P<0.05)。与水提取醇沉黄芪多糖组比较,半仿生提取醇沉黄芪多糖组的大鼠胃粘膜损伤分值明显降低均有统计学差异(P<0.05)。②吲哚美辛诱导大鼠胃粘膜损伤实验结果显示,与模型组比较,水提醇沉黄芪多糖组、半仿生提取醇沉黄芪多糖组大鼠的胃粘膜损伤评分显著性降低,具有统计学差异(P<0.05)。与水提醇沉黄芪多糖组比较,半仿生提取醇沉黄芪多糖组的大鼠胃粘膜损伤分值降低无统计学差异(P>0.05)。实验结果提示,半仿生提取工艺获得的黄芪多糖对乙醇及吲哚美辛诱导的大鼠胃粘膜损伤均有保护作用,且优于传统水提工艺的黄芪多糖,4、半仿生提取醇沉黄芪多糖对乙醇及吲哚美辛诱导大鼠胃粘膜损伤的形态学影响:①在乙醇诱导大鼠胃粘膜损伤的保护实验中,半仿生提取醇沉黄芪多糖高、低剂组均能显著降低胃粘膜的损伤分数,均具有统计学差异(P<0.05),高剂量组效果明显优于低剂量组。②在吲哚美辛诱导大鼠胃粘膜损伤的保护实验中,半仿生提取醇沉黄芪多糖高剂量组大鼠的胃粘膜损伤评分显著降低,具有统计学差异(P<0.05),低剂量组无明显保护作用。实验结果提示,半仿生提取醇沉黄芪多糖对乙醇及吲哚美辛诱导的胃粘膜损伤具有明显的保护作用,且有剂量关系。5、半仿生提取醇沉黄芪多糖对乙醇诱导大鼠胃粘膜损伤保护作用的机制研究:通过免疫组化检测抑凋亡蛋白Bcl-2和促凋亡蛋白Bax和Caspase-3的表达情况,与正常对照组比较,模型组大鼠胃粘膜组织中抑凋亡蛋白Bcl-2的表达降低有统计学差异(P<0.05),促凋亡蛋白Bax和Caspase-3的表达增强有统计学差异(P<0.01)。说明模型的抑凋亡蛋白Bcl-2的表达降低,促凋亡蛋白Bax和Caspase-3的表达升高。与模型对照组比较,半仿生提取醇沉黄芪多糖高、低剂量对胃粘膜组织中抑凋亡蛋白Bcl-2的表达增强有统计学差异(P<0.01),促凋亡蛋白Bax和Caspase-3的表达降低有统计学差异(P<0.05和P<0.01)。结果提示,半仿生提取醇沉黄芪多糖对乙醇诱导大鼠胃粘膜损伤的保护作用是通过凋亡信号途径,即升高抑凋亡蛋白Bcl-2的表达,降低促凋亡蛋白Bax和Caspase-3的表达,发挥对胃粘膜损伤的保护作用。结论:黄芪多糖半仿生提取的优选工艺条件为模拟胃液为料液比1:30,提取温度100℃,提取时间1 h;模拟肠液为料液比1:30,提取温度100℃,提取时间1 h;优选的半仿生提取法工艺在黄芪总多糖、提取收率、保护乙醇及吲哚美辛诱导的大鼠胃粘膜损伤作用上均优于传统水提工艺;两种工艺得到的黄芪多糖为非均一多糖,分子量上存在差异。从形态学观察发现,半仿生提取醇沉黄芪多糖能预防乙醇及吲哚美辛诱导的大鼠胃粘膜损伤,且有剂量关系。其对乙醇诱导胃粘膜损伤的保护作用机制是通过凋亡途径,即升高抑凋亡蛋白Bcl-2的表达,降低促凋亡蛋白Bax和Caspase-3的表达,而发挥对胃粘膜损伤的保护作用。更多还原

【Abstract】 Objective:This study was aiming at optimizing the best semi-bionic extraction process of Astragalus polysaccharide and analysing the differences in the Astragalus polysaccharide homogeneity,molecular weight and the gastroprotective effects between the semi-bionic extraction alcohol precipitation process and water extraction alcohol precipitation process.Furthermore,the mechanism of gastric mucosal protection of Astragalus polysaccharide was discussed preliminaryly.MethodsTaking the extraction yield and extraction rate of polysaccharide and saponins of Astragalus as indicators,orthogonal design experiments were projected to optimize the semi-bionic extraction process which would be compared with traditional extraction process further.The crude Astragalus polysaccharide obtained from the traditional extraction process and the semi-bionic extraction process were purified by Sevag deproteination method and DEAE-52 cellulose column chromatography.The differences in the Astragalus polysaccharide homogeneity,molecular weight between the two different extraction processes were detected by high performance gel permeation chromatography(HPGPC).Additionally,the gastroprotective effects of Astragalus polysaccharides extracted by two different extraction processes were compared using indomethacin-induced or enthanol-induced gastric mucosal injury model in rats respectively..What’s more,the expression of Bcl-2,Bax and Caspase-3 in gastric mucosa tissues were investigated to explore the underlying gastroprotective mechanism of semi-bionic extracted Astragalus polysaccharides.wasResults1.Results of orthogonal experiments taking the total polysaccharide extraction rate(p/%),total saponin extraction rate(p/%)and extraction yield(p/%)as indicators showed that under simulated gastric juice conditions,the weighted contribution to the semi-bionic extraction process from large to small was the extraction temperature(28.9)>material-liquid ratio(12.8)>extraction time(1.5),of which,the material-liquid ratio(P<0.05)and extraction temperature(P<0.01)played a major role and had statistical differences(P<0.05),however,the extraction time was not so obvious.(P>0.05).Under intestinal juice situation,The weighted contribution from large to small was the material-liquid ratio(49.6)>extraction temperature(12.8)>extraction time(4.0).Among these 3 factors,the material-liquid ratio(P<0.01)and extraction temperature(P<0.05)were the major factors and had statistical differences(P(0.05),the extraction time was secondly with no statistical difference(P>0.05).From the perspective of economic benefits,the optimized process condition for the semi-bionic extraction is 1:30 of solid-liquid ratio,100℃ of extraction temperature and 1 h of extraction time no matter under the simulated gastric juice condition or the intestinal juice condition.2.Compare two kinds of extraction processes of Astragalus polysaccharides in terms of the homogeneity and the molecular weight,we found that the Astragalus polysaccharides obtained from two kinds of extraction processes are heterogeneous polysaccharide and the molecular weight of them are more different.The molecular weight distribution of the semi-bionic extracted Astragalus polysaccharides was about 30 thousand to 6.7 million,and the content of polysaccharides in low molecular weight segment was slightly higher than that in high molecular weight segment.Nevertheless,the molecular weight distribution of the Astragalus polysaccharides extracted by traditional water extraction method was about 50 thousand to 16.7 million,and the content of polysaccharides in high molecular weight segment was far higher than that in other molecular weight segment.Results illustrated above indicated that Astragalus polysaccharide obtained from semi-bionic extraction was significantly different from that obtained from the traditional extraction in term of molecular weight.3.The results of protective effects of Astragalus polysaccharide obtained from two different extraction processes on two kinds of gastric mucosal injury models in rats are depicted below:① Results of protective effects on ethanol-induced gastric mucosal lesion showed that the gastric mucosal injury scores of rats administrated with semi-bionic extracted Astragalus polysaccharide markedly decreased when compared with the model group(P<0.05)and the goup(P<0.05)which treated with water extraction alcohol precipitated Astragalus polysaccharide.②Results of protective effects on indomethacin-induced gastric mucosal lesion showed that there was no significant difference between the group treated with semi-bionic extracted Astragalus polysaccharide and the group treated with water extraction alcohol precipitated Astragalus polysaccharide in the term of the gastric mucosal injury scores(P>0.05),but scores of both groups were notably lower(P<0.05)than that in the model group.These results unequivocally indicated that semi-bionic extracted Astragalus polysaccharide could effectively prevent the gastric mucosal lesion no matter the lesion induced by ethanol or indomethacin,and the efficacy was better than that of the water extraction alcohol precipitated Astragalus polysaccharide.4.The influence of semi-bionic extracted Astragalus polysaccharide to the ethanol-induced gastric mucosal lesion and the indomethacin-induced gastric mucosal lesion revealed that treatment with low dose or high dose of semi-bionic extracted Astragalus polysaccharide could also significantly reduce the ethanol-induced gastric mucosal injury scores(P<0.05),especially for the high dose group.In addition,in the indomethacin-induced gastric mucosal injury model,scores were reduced obviously in the high dose group(P<0.05),but were not significantly different between the low dose group and the model group(P>0.05).The experimental results proved that the semi-bionic extracted Astragalus polysaccharide had obvious protective effects on gastric mucosal injury no matter induced by ethanol or indomethacin,and presented dosage dependence relations.5.The underlying mechanism of the gastroprotective effect of semi-bionic extracted Astragalus polysaccharide on ethanol-induced gastric mucosal lesion was researched by detecting the expression of anti-apoptotic protein(Bcl-2)and pro-apoptotic protein(Bax,Caspase-3)using immunohistochemical detection technique.In comparison with the normal control group,the expression of Bcl-2 of the gastric mucosa was significantly decreased(P<0.05),and the expression of Bax and Caspase-3 was significantly increased in the model group(P<0.01).Nevertheless,the decrease of Bcl-2 and the increase of Bax and Caspase-3 were markedly reversed by the application of low dose and high dose semi-bionic extracted Astragalus polysaccharide when compared with that in the model group.(P<0.05 or P<0.01),.Taken together,theseresults suggested that the semi-bionic extracted Astragalus polysaccharide was able to protect the gastric mucosal lesion induced by ethanol through the adjustion of apoptotic signaling pathway such as up-regulate anti-apoptotic protein Bcl-2 expression and down-regulate pro-apoptotic protein Bax and Csspase-3 expression.ConclusionBased on the results of the orthogonal tests and the pharmacodynamics experiments,we found that the optimized process condition for the semi-bionic extraction is 1:30 of solid-liquid ratio,100℃ of extraction temperature and 1 h of extraction time no matter under the simulated gastric juice condition or the intestinal juice condition,and this semi-bionic extraction process is superior to the traditional water extraction process in terms of yield and extraction rate of total Astragalus polysaccharide,the gastroprotective efficacy on ethanol or indomethacin-induced gastric mucosal lesion.The Astragalus polysaccharides obtained from two kinds of extraction processes are heterogeneous polysaccharide with different relative molecular weight.Moreover,the semi-bionic extracted Astragalus polysaccharide can effectively prevent the gastric mucosal lesion induced by ethanol or indomethacin,and present dosage dependence relations.Additionally,the underlying mechanism of rats’gastroprotective effect of semi-bionic extracted Astragalus polysaccharides on ethanol-induced gastric mucosal lesion is through the adjustion of apoptotic signaling pathway such as up-regulate anti-apoptotic protein Bcl-2 expression and down-regulate pro-apoptotic protein Bax and Csspase-3 expression..更多还原