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广西六堡茶化学成分分离、活性分析及茶褐素提取工艺研究IV广西师范学院硕士学位论文I摘要六堡茶是中国黑茶之一，具有悠久的生产历史，多种生物活性。针对六堡茶的研究主要是加工工艺，化学成分的研究较少。本课题对广西六堡茶的有效化学成分进行分析，并对茶褐素进行提取工艺优化。主要结果如下：1.活性分析：六堡茶经95%乙醇浸提后，用石油醚、乙酸乙酯、正丁醇依次萃取，各萃取相蒸干，4个极性部分的浸膏分别进行抗氧化、抗菌和抗肿瘤实验。抗氧化实验中分别选取DPPH自由基、羟基自由基、超氧阴离子自由基通过测定吸光度值判断各极性部分的抗氧化能力，通过数据分析，乙酸乙酯相和正丁醇相具有较好的抗氧化能力，特别是对DPPH自由基的清除率值较高；通过最小抑菌浓度法(MIC)选取革兰氏阴性的铜绿假单胞菌、大肠杆菌以及革兰氏阳性的金黄色葡萄球菌、白色念珠菌四种菌种对经过水提萃取后的4个极性部分和95%乙醇提取萃取后的4个极性部分进行抗菌活性实验，对大肠杆菌的抑菌效果优于对其它细菌的抑制作用；浸膏分别对胃癌细胞MGC-7901、人宫颈癌细胞Hela、肝癌细胞7404进行毒性测定，通过数据分析，依据IC50值判断活性效果，与预期差别较大，对这三种癌细胞的抑制作用较小。2.化学成分分析：六堡茶提取物经萃取后，石油醚相和乙酸乙酯相通过GC-MS分析挥发性成分，并对石油醚相、乙酸乙酯相和正丁醇相用柱层析进行分离鉴定。化学成分分离得到9个化合物，已鉴定出5个化合物，分别是棕榈酸、咖啡因、没食子酸、没食子酸甲酯、α-菠甾醇。3.工艺提取纯化：采用纤维素酶辅助法提取六堡茶中的茶褐素，分别对酶解温度(A)、酶解用量(B)、pH值(C)、酶解时间(D)四个影响因素通过单因素和正交实验对比分析，得到了茶褐素提取的最佳工艺条件，产率达到24.50%。用大孔树脂对茶褐素进行富集纯化，首先选取D900、DM-301、ADS-17、D101、S-8五种不同型号的大孔吸附树脂通过静态吸附，比较吸附率与解析率的综合效果，确定了D101作为纯化茶褐素的大孔吸附树脂。其吸附率是43.65%，解析率是67.09%。确定了茶褐素溶液浓度、洗脱剂、洗脱速度、pH值对纯化的影响。选用葡聚糖凝胶柱层析的方法对茶褐素进行分级分离，丙酮-水做洗脱剂，按照色谱带的不同得到了6个级分，分别通过紫外和红外光谱观察，数据分析表明茶褐素的组成与结构复杂。关键词：六堡茶；活性分析；化学成分；提取；纯化；分级广西六堡茶化学成分分离、活性分析及茶褐素提取工艺研究II广西师范学院硕士学位论文IIIAbstractLiubaoTeabelongstooneoffamousblackteainChina,whichhasalonghistoryofproductionandavarietyofbiologicalactivity.Processingtechnologyistheprimaryresearch,whilestudiesonchemicalcompositionislimited.Inthispaper,theactivechemicalcompositionofLiubaoTeaandthepurificationprocessofTheabrownsextractionwerestudied.Themainexperimentscontentsareasfollows:1.ActivityAnalysisPetroleumether,ethylacetateandn-butanolwereusedtoextractthe95%ethanolextractedliquidfromLiubaoTea,thenthefourpolarityextractedwerestudiedontheantioxidant,antibacteri-alandanticancerexperiments.DPPHradical,hydroxylradicalandsuperoxideanionradicalweretextedintheantioxidantexperiments.TheantioxidantactivitywasdeterminedbyUVabsorbancevalue,withtheresultsdatas,ethylacetateandn-butanolpHasehaveexcellentantioxidantcapacity,especiallyclearancevalueofDPPHradicalisthebest.Gram-negativePseudomonasaeruginosa,EscherichiacoliandGram-positiveStapHylococcusaureus,CandidaalbicanswereselectedtodetecttheantibacterialactivityaboutthefourpHasewiththeminimumbacterialconcentration.TheinhibitioneffectonEscherichiacoliisbetterthanotherpHase.Thenhumangastriccancercells(MGC-7901),humancervicalcancercells(Hela),Humanlivercancercells(7404)wereselectedtodeterminetheanticanceractivityaboutthefourpHase.BymeasuringIC50values,whichshowgreatlydifferencewithwhatwereexpected,thefourphasehavesmallinhibitioneffecttothethreecells.2.ChemicalcomponentsAnalysisThePetroleumetherandethylacetatepHaseweretestedbyGC-MStodeterminethevolatilecomponents.ThenPetroleumether,ethylacetateandn-butanolwereseparatedtoanalysisthecompounds.WiththegelcolumnchromatograpHy,ninecompoundswereobtained,fivecompoundswereidentifiedbyNMR,IR,MS.Thefivecompoundswerepalmiticacid,Gallicacid,Gallicin,caffenine,α-spinasterol.3.ProcessofextractionandPurificationTheabrowninewasextractedwithcelluloseauxiliarymethod,theeffectsoftheenzymedose,enzymatichydrolysistemperature,enzymatichydrolysistimeandthepHofextractionwereinvestigatedbyorthogonaltest.Thebestextractionratewithcellulosewas24.503%.MacroporousresinwasselectedtopurifyTheabrownines,absorptionanddesorptionratioofD900,DM-301,ADS-17,S-8,D101toTBswereanalyzedwithstaticadsorption.D101wasselectedtopurifyTBs.Theabsorptionratiois43.65%anddesorptionratiois67.09%.The广西六堡茶化学成分分离、活性分析及茶褐素提取工艺研究IVconcentrationoftheTBs,theeluent,theelutionrate,pHvalueweredeterminedintheexperiment.Acetone-waterastheelution,sepHadexasthepackingofthegelcolumnchromatograpHytodofractionationexperimentonTBs,sixfractionswereobtainedaccordingtothedifferenceofthepigmentsribbon.EveryfractionhassimilaritywiththeanalyzedofthedatasofUV-VISangIR.DatasshowthecompositionandthestructureoftheTBswerecomplexity.Keyword:LiubaoTea;activeanalysis;chemicalcomponents;extract;purification;fraction广西师范学院硕士学位论文V目录摘要......................................................................................................................................................IAbstract............................................................................................................................................III目录.....................................................................................................................................................V第一章绪论.......................................................................................................................................11.1六堡茶研究进展...................................................................................................................11.2黑茶中化学成分分析研究进展...........................................................................................21.2.1生物碱类.......................................................................................................................................21.2.2茶多酚类.......................................................................................................................................31.2.3氨基酸类.............................................................