【作者】 宋鲁彬；

【导师】 黄建安； 刘仲华；

【作者基本信息】 湖南农业大学 ， 茶学， 2008， 博士

【摘要】 黑茶是我国特种茶之一,具有较长时间渥堆发酵的加工工艺,在长时间的渥堆过程中,微生物参与茶叶内物质的转化,产生特殊色香味品质,同时,由于微生物的参与,黑茶产生了新的不同于普通茶叶的药理功能及保健功能。与其他种类的茶叶相比,对黑茶的研究较少。虽然近年来对黑茶研究逐渐增多,但黑茶产生功效的作用机理大多仍不明确,这严重阻碍了黑茶进一步发展及开发。因此,本研究以茯砖茶、花砖茶、青砖茶、黑砖茶、六堡茶、普洱茶为材料,以沱茶、米砖茶为对比材料,以高通量药物筛选为手段对黑茶的抗代谢综合症、高胆固醇血症、抗炎、抗消化道肿瘤活性进行了系统的药理功能分析及评价;采用高速逆流色谱对黑茶的物质成分进行了分离研究以及采用高效薄层色谱对黑茶脂溶性色素以及茯砖茶金花脂溶性荧光物质进行了分析研究。1.采用柱层析硅胶作为支持剂的部位分离法从茶叶的乙醇回流提取液中得到石油醚洗脱组分、氯仿洗脱组分、乙酸乙酯洗脱组分、乙醇洗脱组分;以热水回流提取方式得到水提取成分。高效薄层检测发现石油醚洗脱组分主要是类胡萝卜素物质;氯仿洗脱组分主要是色素类物质及茶碱;且黑茶的石油醚洗脱组分以及氯仿洗脱组分在366nm紫外光下有强荧光。高效液相色谱检测分析后发现黑茶中没食子酸的含量较高;在所有的黑茶中,普洱茶中没食子酸的含量最高;而红茶类的米砖茶中没食子酸的含量也较高。同时发现黑茶中茶碱的含量较高,花砖茶中茶碱的含量则是其他黑茶的4～10倍。2.选择PPARd、PPARγ、FXR激活、FXR抑制、LXR共5种核受体模型评价黑茶组分的抗高脂血症、抗动脉粥样硬化、抗肥胖等代谢综合症的生理活性,结果表明:(1)茯砖茶对PPARγ受体模型以及PPARδ核受体模型具有较好的激活作用,对LXR激活模型、FXR激活模型的激活作用都较强,有望能够从茯砖茶中发现抗脂减肥、治疗动脉粥样硬化、以及治疗糖尿病等抗代谢综合症的药物;普洱茶对这几种模型的作用结果显示普洱茶可能具有良好的对糖及脂类代谢的双向调节作用。(2)六堡茶对FXR受体抑制模型表现出较好的活性;普洱茶中的中高极性对FXR受体抑制模型的活性也较好;而青砖茶中低极性成分对FXR受体抑制模型具较好的活性。因此,六堡茶、普洱茶及青砖茶具有较好的FXR抑制剂的筛选前景。同时,黑茶的高极性物质对FXR受体抑制模型也具较好的活性。(3)沱茶乙酸乙酯洗脱组分具有增加FXR受体抑制敏感性的作用,这一作用可能是由儿茶素表现的。(4)沱茶的水提取成分对两种PPARs核受体模型几乎没有激活作用,而黑茶的水提取成分对于PPARs模型有较好的激活作用。这说明黑茶经过微生物发,酵之后,能够产生较多的易溶于水的抗脂减肥的生理活性成分。因此,直接饮用黑茶能够降脂减肥、防止动脉粥样硬化。3.选择TNF、IL-1分泌细胞模型评价黑茶组分炕炎活性,黑茶组分对炎症分泌因子模型没有明显的抑制活性。4.选择SGC-7901、HCT-8细胞株为模型评价黑茶组分对消化道肿瘤的抑制作用,结果显示黑茶对消化道肿瘤生长的抑制效果较好。黑茶的乙酸乙酯洗脱组分对消化道肿瘤的抑制作用较强。因此可使用乙酸乙酯洗脱的方式从黑茶中提取具有消化道肿瘤抑制作用的药物前提物。黑茶中对对消化道肿瘤细胞HCT-8起抑制作用的物质的极性要比对SGC-7901起抑制作用的物质的极性高。5.金花对所选择的5种核受体模型都具有良好活性作用,实验结果表明金花中对核受体有作用的活性成分的极性跨度较大。对消化道肿瘤的抑制实验结果表明,金花同时具有很强的对消化道肿瘤的抑制作用。且低极性的金花氯仿洗脱组分的抑制活性要比中极性的金花乙酸乙酯洗脱组分的抑制活性更强,与黑茶中的物质相比,金花中起抑制作用的物质更倾向于低极性。对金花中脂溶性荧光物质进行检测时也发现其中有多种生理活性物质。对金花的生理功能评价的结果显示,金花是很值得进一步开发的抗代谢综合症、抗高脂血症、调节糖代谢的药物前体物,同时也是抗消化道肿瘤药物的前体物。6.使用HSCCC分离黑茶中的物质成分。利用乙酸乙酯-水为溶剂系统,乙酸乙酯为流动相,可以一次性的将黑茶中将的茶碱、咖啡碱、没食子酸与儿茶素分离;茶碱、咖啡碱、没食子酸的纯度不低于99%。出峰顺序依次是ECG、EGCG、EC和DL-C、没食子酸、咖啡碱、茶碱。对黑茶水提取成分的分离中使用双向洗脱法,可缩短HSCCC分离时间,并且使出峰峰型较为尖锐。7.在可见光下,色素在硅胶薄层板经石油醚:丙酮=7:3的展开剂展开后,清晰分离为9条色素带,按R_f值从大到小依次是β-胡萝卜素(β-Car,0.87,橙黄色)、未知色素(0.74,橙黄色)、脱镁叶绿素酸脂a(Poa,0.65,灰绿色)、脱镁叶绿素a(Pya,0.53,褐灰绿色)、脱镁叶绿素酸脂b(Pob,0.49,绿黄色)、脱镁叶绿素b及叶绿素酸脂a的混合色斑(Pyb/Cda,0.43,蓝绿色或黄绿色)、叶绿素酸脂b(Cdb,0.37,褐黄色)、叶绿素b及叶黄素(Chlb/Xan,0.35,绿黄色或土黄色)、新黄质(Neo,0.21,土黄色)。并且,在366 nm紫外光下,黑茶在R_f=0.70处有明显的荧光斑。从黑茶中分离出的色素的光谱吸收特征与文献上色素的光谱吸收特征差异较大:且不同黑茶中各色素的比例以及绝对含量上都有差异。8.以脂溶性色素的含量为集群依据,采用SPSS软件对茶样进行聚类分析,集群分析树形图将两种绿茶(沱茶、炒青绿茶)归为一群,六堡茶单独成群,黑茶及米砖茶归为一类。其归类结果反映了不同茶类本身的色泽特征及茶叶相互之间的相似程度。9.以HPTLC分析金花中的脂溶性荧光物质的光谱吸收特征,发现R_f=0.27处的条带在241 nm、288 nm、366 nm处有较强的紫外吸收,它的最大吸收峰出现在366 nm处;而R_f=0.58处的条带在241nm、290 nm处有较强的紫外吸收,它的最大吸收峰在290 nm处。同时以制备性薄层硅胶板对脂溶性荧光物质进行分离,以GS-MS联用分析,从金花中检测到下列物质:docosane、Hentriacontanc、Bis(2-ethylhexyl)phthalate、A-Neooleana-3(5),12-diene、Olean-12-ene、4,4,6a,6b,8a,11,11,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3-one、a-Neooleana-3,12-diene、10,13-Dimethyl-17-(1,4,5-trimethyl-hex-2-enyl)-1,2,9,10,11,12,13,15,16,17-decahydrocyclopenta[a]phenanthren-3-one、Squalene、Friedelin。这些物质都是首次从金花中发现。更多还原

【Abstract】 Dark teas are special tea in China.All Dark teas have post-fermentation processing and there are many microorganisms participated in the process.Therefore unique flavor and function to health had been produced.The study of Dark tea is still weakly and obstructed the further development of Dark tea.Therefore,Puer-tea,Liubao-tea,Fuzhuan-tea,Huazhuan-tea,Qingzhuan-tea,and Heizhuan-tea has been taken as study materials,and Tuo-tea,Mizhuan-tea has been taken as contrast materials,HTS has been used to evaluate their physiological function of them,HSCCC has been used to separate compounds of Dark Tea,HPTLC has been used to studies their liposoluble pigment and liposoluble fluorescence components.1.Two methods had been used to withdraw the composition material from tea.At the first method 95%ethanol was used to withdraw the composition material from tea and at second method 95℃water was used.Silica-gel has been used as holder for the ethanol liquor and solvent used to wish it,Petroleum ether Elution & Chloroform elution & Ethyl-acetate elution & Ethanol elution had been got.Aqueous extraction had been got by the second method.HPTLC analysis shows that Petroleum ether Elution compound composed with carotenoids and the Chloroform elution composed with pigment and alkaloids.Dark tea Aether-petrolei elution and the Chloroform elution can give fluorescence on 366nm light.HPLC analysis shows that the content of Gallic acid in Dark-balck tea is higher than in Tuo tea.And the content of Gallic acid in Pu-er tea is higher that in other Dark-black.Mi-zhuan tea also has high content of Gallic axid.And HPLC analysis also shows that Dark-black tea has high content of Theophylline,and the content of Theophylline in Hua-zhuan tea is 4 to 10 times higher than that in other Dark tea.2.To evaluate the faction of Dark tea,PPARδmodel,PPARγ,model,FXR activate model and FXR restrain model,LXR model had been taken on the study of Dark tea anti-activity on metabolizing Syndrome such as hyperlipoidemia, atherosclerosis,fatness & etc.And following conclusions had been got:(1) Fu-zhuan tea have been found being low pole.and have strong power on PPARd model,PPARγmodel,LXR activate model & LXR model.Therefore it promised potential therapy on hyperlipoidemia,atherosclerosis,fatness & diabetes mellitus would be found.Pu-er tea has good regulation of glyco-metabolism and lipid metabolism.(2) Liu-bao tea have good power on FXR restraint model,The mid-high components of Pu-er tea have good power on FXR restraint model,The mid-low components of Qing-zhuan have good power on FXR restraint model too.Therefore Liu-bao tea,Pu-er tea and Qing-zhuan teahas high value of being FXR inhibitor.And high pole of Dark tea have good power on FXR restraint model.(3) Tuo tea Ethyl-acetate elution can reinforce sensitivity of FXR restrain and it titles Tuo tea good foreground on medicine.(4) Tu0 tea Aqueous extraction has no power on PPARs model but Dark tea Aqueous extraction have.It is a hint that Dark tea can produce more water-soluble activity.components on anti-fatness,anti-hyperlipoidemia,anti-atherosclerosis than Tuo tea.And it hints that drinking Dark tea can prevent hyperlipoidemia, atherosclerosis and fatness.3 TNF,IL-1 model had been taken to evaluate the faction of Dark tea on anti-inflame,and the results show that Dark tea have no activity to TNF & IL-1.4 SGC-7901 cells,and HCT-8 cells had been taken to evaluate anticancer of Dark tea and the results show that all Dark-black tea have strong power on it.Dark tea Ethyl-acetate elution have strong restrain power on alimentary canal cancer.So it hints that eluting methods can be used on obtaining inhibitor of alimentary canal cancer from Dark tea.The inhibitor to HCT-8 cells in Dark-black tea has high pole than that to SGC-7901 cells.5.Jin-hua has a good physical activity on 5 NR models,The results of study show that the pole of Jin-hua activity components is broadly.The study on Jin-hua repressive power on alimentary canal cancer shows that Jin-hua has good antineoplastic effect.And the power in Chloroform elution is more stronger than in Ethyl-acetate elution.Furthermore there many function materials have been found in Jin-hua liposoluble Fluorescence Components.The study results show that Jin-hua is a precursor of medicine with anti-activity on metabolizing Syndrome and antineoplastic effect.6.A preparative high-speed counter-current chromatography(HSCCC) method for isolation and purification of Gallic acid,Theine,Theophylline from Ethyl acetate Elution was successfully established by using ethyl acetate-water as the two-phase solvent system.The lower phase was used as the stationary phase of HSCCC.The mobile phase used in HSCCC was the upper phase.With this solvent system,not only theine,Theophylline,Gallic acid be isolation,but also Catechin be separated.And with same solvent system,bidirectional elution mode has been used on separating Dark tea Aqueous extraction and the mode can short time of HSCCC analysis.7.In visible light,HPTLC analysis of Dark tea liposoluble pigment shows that 9 bands had been separated by aether petrolei-acetone(7/3,V/V) on Silica-gel plate. from high R_f to lower R_f lined asβ-Car(0.87),known pigment(0.74),Poa(0.65),Pya (0.53),Pob(0.49),Pyb/Cda(0.35),Neo(0.21).The bands of Dark tea have fluorescence on Rf=0.70.The light absorb character of Dark tea pigment are different from the light absorb character of pigment that said on article,and the ration or content in Dark tea are different from each other.8.According to pigment content,the cluster analysis of Dark tea by SPSS shows that the two green tea had been classed in one group,Liu-bao tea separated as a group and Dark-black tea and Mi-zhuan tea classed in one group.The result is satisfactory to the real.9.HPT1C analysis of Jin-hua liposoluble fluorescence components and found that the band on R_f=0.27 has three major light absorb on 241nm,288nm and 366nm, and the maximal is on 366nm.The band on R_f=0.58 has two major light absorb on 241nm and 190 nm,and the maximal is on 290nm.Using preparation TLC isolate liposoluble fluorescence and GS-MS analysis shows that the follows material in it: Docosane,Hentriacontane,Bis(2-ethylhexyl)phthalate、A-Neooleana-3(5),12-diene、Olean-12-ene、a-Neooleana-3,12-diene、4,4,6a,6b,8a,11,11,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b -octadeeahydro-2H-picen-3-one, 10,13-Dimethyl-17-(1,4,5-trimethyl-hex-2-enyl)-1,2,9,10,11,12,13,15,16,17-decahydr ocyclopenta[a]phenanthren-3-one、Squalene、Friedelin.更多还原