【作者】 余琼；

【导师】 阮征； 姚彦斌；

【作者基本信息】 南昌大学 ， 农业硕士（专业学位）， 2023， 硕士

【摘要】 随着饮食习惯和生活习惯的改变,非酒精性脂肪肝病(Nonalcoholic fatty liver disease,NAFLD)的发病率和患病率逐年上升,给患者带来极大的痛苦和经济负担。NAFLD的发病机制目前尚未完全阐明,且无有效的治疗性药物。2019年卫健委公布黄芪为药食两用的药材,黄芪活性成分包括黄芪多糖、皂苷、异黄酮等,黄芪活性成分具有减轻胰岛素抵抗、调节糖脂代谢、抑制炎症反应等多种生物活性,来发挥对NAFLD肝损伤的保护作用。但目前黄芪有效活性成分防治NAFLD的作用机制尚不完全清楚。因此本论文利用网络药理学、分子对接、代谢组学的技术,从多层次、多角度系统探究黄芪活性成分干预NAFLD小鼠肝损伤的机制。最后,结合实际应用,开发了一款黄芪护肝保健饮料。本文主要结论如下:1.运用网络药理学方法预测黄芪化合物活性成分,潜在靶点及分子机制。构建黄芪化合物与NAFLD的“成分-靶点-通路”的互作网络,包括药物相似性的评价、口服生物利用度的预测、蛋白质-蛋白质相互作用(PPI)网络构建和分析基因本体(GO)术语和通路注释。最终确定了黄芪化合物的5种活性成分调控NAFLD相关的5个关键靶点与15条主要信号通路,GO生物富集分析结果显示黄芪化合物干预NAFLD主要涉及调控氧化还原反应、胰岛素的调节、炎症反应以及脂肪细胞的分解等。利用分子对接模拟方法进一步说明了配体的活性位点和结合程度,分子对接结果显示黄芪化合物山奈酚、染料木素、大豆苷元均能与NAFLD靶点INS、PPARG、AKT1结合,且都有较好的结合效果,均有潜在防治NAFLD作用。2.以高脂高糖诱导的非酒精性脂肪肝为模型,用染料木素(Genistein,GEN)、大豆苷元(Daidzein,DAI)两种黄芪异黄酮进行干预。结果表明,两种黄芪异黄酮显著降低小鼠体重、血清TG、TC、肝功能因子AST与ALT活性、肝脏炎症水平、血清LPS水平。这表明两种黄芪异黄酮均能改善高脂高糖膳食引起的肝脏脂代谢紊乱和炎症反应,均对NAFLD小鼠的肝损伤具有保护作用。使用HPLC-Q-TOF/MS技术对GEN和DAI抗NAFLD的作用机制进行初步研究。四组小鼠肝脏共筛选并鉴定71个肝脏显著性差异代谢物,涉及的代谢通路主要有磷脂代谢、花生四烯酸代谢、三羧酸循环等,初步阐明了两种黄芪异黄酮抗NAFLD对内在作用机理,为后续研究奠定了基础。3.通过响应面优化实验得到了黄芪护肝饮料的最佳工艺配比,黄芪基液的添加量为13.10%、糖的添加量5.90%、柠檬酸的添加量为0.05%、黄原胶的添加量为0.14%、牛磺酸0.03%、维生素C 0.02%。采用恒温加速实验,对该饮料保质期进行了预测,其保质期可达12个月,保质期内饮料感官、理化及微生物指标都在国家标准范围。得到一款色泽自然、澄清透明、口感纯正、功效成分稳定的护肝保健饮料。更多还原

【Abstract】 With the change of eating habits and living habits,the incidence and prevalence of Nonalcoholic fatty liver disease(NAFLD)increase year by year,which brings great pain and economic burden to patients.The pathogenesis of NAFLD has not been fully elucidated,and there are no effective therapeutic drugs.In 2019,the Commission of Health announced that Astragalus membranaceus was a medicinal material for both medicine and food.The active ingredients of Astragalus membranaceus include Astragalus polysaccharide,saponins,isoflavones,etc.The active ingredients of Astragalus membranaceus have various biological activities such as alleviating insulin resistance,regulating glucose and lipid metabolism,inhibiting inflammatory response,etc.,so as to play a protective role against NAFLD liver injury.However,the mechanism of effective active ingredients of Astragalus against NAFLD is still unclear.Therefore,in this paper,based on the overall research idea of network pharmacology,molecular docking and metabolomics,the possible mechanism of Astragalus membranaceus active ingredient’s intervention in liver injury of NAFLD mice was systematically elaborated from multiple levels and perspectives.Finally,combined with the practical application,developed a astragalus liver protection drink.The main conclusions of this paper are as follows:1.Network pharmacological methods were used to predict the active constituents,potential targets and molecular mechanisms of Astragalus compounds.Construct a compose-target-pathway interaction network between Astragalus compounds and NAFLD,including evaluation of drug similarity,prediction of oral bioavailability,construction of protein-protein interaction(PPI)network,and analysis of gene ontology(GO)terminology and pathway annotation.Finally,five key targets and 15 major signaling pathways related to the regulation of NAFLD by five active components of Astragalus compounds were determined.GO bioenrichment analysis results showed that the intervention of Astragalus compounds in NAFLD mainly involved the regulation of REDOX reaction,insulin regulation,inflammatory response and adipose cell decomposition.Molecular docking simulation method was used to further elucidate the active sites and binding degree of the ligands.The results of molecular docking showed that the astragalus compounds kaverol,genistein and daidzein could all bind to NAFLD targets INS,PPARG and AKT1,and all had good binding effect,and all had potential control effect on NAFLD.2.Non-alcoholic fatty liver induced by high fat and high sugar was taken as the model,and two kinds of astragalus isoflavones(Genistein,GEN)and(Daidzein,DAI)were used for intervention.The results showed that the two kinds of astragalus isoflavones significantly decreased the body weight,serum TG,TC,activity of liver function factors AST and ALT,liver inflammation level and serum LPS level in mice.These results indicated that both kinds of astragalus isoflavones could improve liver lipid metabolism disorder and inflammatory response caused by high fat and high sugar diet,and both had protective effects on liver injury in NAFLD mice.HPLC-QTOF/MS technique was used to study the mechanism of action of GEN and DAI against NAFLD.A total of 71 metabolites with significant differences in liver were screened and identified in the liver of mice in the four groups.The metabolic pathways involved mainly included phospholipid metabolism,arachidonic acid metabolism,tricarboxylic acid cycle,etc.The intrinsic mechanism of anti-NAFLD of two kinds of astragalus isoflavones was preliminarily clarified,which laid a foundation for subsequent studies.3.The optimal process ratio of Astragalus liver protection drink was obtained through response surface optimization experiment.The addition amount of Astragalus base solution was 13.10%,sugar was 5.90%,citric acid was 0.05%,xanthan gum was0.14%,taurine was 0.03% and vitamin C 0.02%.The shelf life of the beverage was predicted by constant temperature acceleration experiment.The shelf life of the beverage was up to 12 months,and the sensory,physical and chemical and microbial indexes of the beverage were within the range of national standards.Get a natural color,clear and transparent,pure taste,functional ingredients stable liver health drink.更多还原