茶多酚提取新工艺研究
摘要
本文简要介绍了茶多酚(Tea-polyphenols，TP)的组成、结构、性质、应用及其提取理论，研究了茶多酚新的提取工艺：(1)确定了非水溶剂浸提TP的工艺条件，并对其在水溶液中的抗氧化性进行了初步探讨。HPLC及紫外光谱分析表明：乙醇可以抑制茶多酚，尤其是儿茶素的氧化；采用40％(v／v)乙醇，在40℃浸提三次，每次10min，茶多酚的浸提率为32.1wt%(以干茶叶计)，其中EGCG、EGC、C、EC、ECG分别为7.5％、4.1％、0.1％、0.6％、2.2wt％，茶多酚和儿茶素的浸提率较采用GB8313-87浸提方法分别提高29.4wt％和16.1wt％。(2)研究了利用聚酰胺提纯茶多酚的工艺条件：常温下，聚酰胺对茶多酚的饱和吸附量为73.4mg／g(树脂)，吸附-脱附实验确定分离TP的最佳吸附量为50mg／g(干树脂)。实验采用120×30mm聚酰胺色谱柱，先用120ml、5vol％的乙醇溶液在流速为1.0ml／min。的条件下洗脱咖啡因，再用285ml、70vol％的乙醇复合洗脱液在流速为1.5ml／min的条件下分离色素与茶多酚，TP的回收率平均为59.4wt％，咖啡因的脱除率为96.3wt％。整个提取过程的茶多酚总得率平均为19.2wt％(以1g干茶叶计)，液相分析各成分的组成为EGCG：33.3％；EGC：18.8％；ECG：8.8％；EC：2.6％；C：0.5％；GA：1.0wt%；儿茶素总含量为65.1wt％，咖啡因含量仅为1.04wt％。(3)探讨了流动相组成、酸的种类、流速、pH及进样量对高效液相色谱(HPLC)分析茶多酚的影响。确定分析的最佳条件为：温度t=30℃，检测波长λ=280nm，C_(18)反相柱(250×4.6mm,5μ)，流动相为甲醇：水：乙酸：25：74.5：0.5(v／v)，进样量v=6μl，流速μ=1.0ml／min等度洗脱。此时，实验的分析时间为45min，分离度为1.46，回归方程的相关系数为0.996，回收率为96-98％。
On basis of the composition, structure, properties and application of Tea poly- phenols(TP) and its extracting theory , the technological process of TP extraction was investigated. (1) Technological process of extraction for TP with organic solvent was investigated; the anti-oxidation of TP was elementarily discussed also. Experimental results by HPLC and UV Spectra show: oxidation of TP in water especially catechins can be inhibited by ethanol aqueous, the extraction ratio of TP is 32.1wt% when extracting with 40vol% ethanol aqueous, three times at 40 癈, lOmin each time, EGCG, EGC, C, EC and ECG is 7.5%, 4.1%, 0.1%, 0.6% and 2.2wt% respectively. The yields of TP and catechins increases by 29.4% and 16.1wt% compared with extracts by GB8313-87. (2) Technological process of purification was completed by chromatographic column packed with 30-40 mesh polyamide. Employing 120 X 30mm column, the saturated adsorption amount determined by dynamic experiments of TP is 73.4mg/g (dry resin) , the optimum adsorption amount by adsorption-desorption experiment for TP separation is 50mg/g(dry resin); At this condition, purification was carried out first by 120ml, 5vol% ethanol aqueous at the flow rate 1 .Oml/min to elute caffeine, then pigment by 285ml 70vol% rated ethanol aqueous at 1.Oml/min. The recovery ratio of TP in the process of purification is 59.4wt% on average, and desorbing ratio of caffeine 96.3wt%. The total yields of TP is 19.2wt%( Ig dry tea leaves), the contents of catechins analyzed by HPLC in the product is 65.1wt%, in which EGCG33.3%, EGC 18.8%, ECG8.8%, EC2.6%, CO.5% and GA1.0wt% respectively, especially, the content of caffeine is as low as 1.04wt%. (3) Effects of mobile phase, acid, flow rate, pH, and injection volume on analysis of TP by High-performance Liquid Chromatograph were discussed as well. The contents of catechins of TP were determined by 250X4.6mm reversed phase column packed with 5um Cig modified silica, the analysis condition is as follows: mobile phase is aqueous rated by ethanol: water: acetic acid =25:74.5:0.5(v/v) , flow rate 1.Oml/min, temperature 30"C, injection volume 6ul, UV detector at 280nm, isocratical elution. According to these conditions, the experimental separating time is 45min; the resolution 1.46, the value of relative co-efficiency of the regression equation is 0.996 and recovery ratio 96-98wt%.
On basis of the composition, structure, properties and application of Tea poly- phenols(TP) and its extracting theory , the technological process of TP extraction was investigated. (1) Technological process of extraction for TP with organic solvent was investigated; the anti-oxidation of TP was elementarily discussed also. Experimental results by HPLC and UV Spectra show: oxidation of TP in water especially catechins can be inhibited by ethanol aqueous, the extraction ratio of TP is 32.1wt% when extracting with 40vol% ethanol aqueous, three times at 40 癈, lOmin each time, EGCG, EGC, C, EC and ECG is 7.5%, 4.1%, 0.1%, 0.6% and 2.2wt% respectively. The yields of TP and catechins increases by 29.4% and 16.1wt% compared with extracts by GB8313-87. (2) Technological process of purification was completed by chromatographic column packed with 30-40 mesh polyamide. Employing 120 X 30mm column, the saturated adsorption amount determined by dynamic experiments of TP is 73.4mg/g (dry resin) , the optimum adsorption amount by adsorption-desorption experiment for TP separation is 50mg/g(dry resin); At this condition, purification was carried out first by 120ml, 5vol% ethanol aqueous at the flow rate 1 .Oml/min to elute caffeine, then pigment by 285ml 70vol% rated ethanol aqueous at 1.Oml/min. The recovery ratio of TP in the process of purification is 59.4wt% on average, and desorbing ratio of caffeine 96.3wt%. The total yields of TP is 19.2wt%( Ig dry tea leaves), the contents of catechins analyzed by HPLC in the product is 65.1wt%, in which EGCG33.3%, EGC 18.8%, ECG8.8%, EC2.6%, CO.5% and GA1.0wt% respectively, especially, the content of caffeine is as low as 1.04wt%. (3) Effects of mobile phase, acid, flow rate, pH, and injection volume on analysis of TP by High-performance Liquid Chromatograph were discussed as well. The contents of catechins of TP were determined by 250X4.6mm reversed phase column packed with 5um Cig modified silica, the analysis condition is as follows: mobile phase is aqueous rated by ethanol: water: acetic acid =25:74.5:0.5(v/v) , flow rate 1.Oml/min, temperature 30"C, injection volume 6ul, UV detector at 280nm, isocratical elution. According to these conditions, the experimental separating time is 45min; the resolution 1.46, the value of relative co-efficiency of the regression equation is 0.996 and recovery ratio 96-98wt%.
引文
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74 GB8313-87 茶多酚含量的测定
2. Okabe, Sachiko, et al. Mechanistic aspects of green tea as a cancer preventive: effect of components on human stomach cancer cell lines. Jpn. J. Cancer Res.,1999, 90(7), 733-739.
3. Kondo, K, et al. Scavenging mechanisms of (-)-epigallocatechin gallate and (-)-epicatechin gallate on peroxyl radicals and formation of superoxide during the inhibitory action. Free Radical Biol. Med.,1999, 27(7/8), 855-863.
3．刘静波等，茶多酚对动物血清血脂和载脂蛋白水平的影响和抗氧化作用。茶叶科学，2000，20(1)：67—70
5 沈生荣等，铁存在下儿茶素的抗自由基效应。茶叶科学，1997，17：119-123
6 Yatsu, Mitsuo, et al. Cosmetics containing tea catechins. Jpn. Kokai Tokkyo Koho JP 11180850 A2 6 Jul 1999
7 Wang, H. et al, Epimerisation of catechins in green tea infusions. Food Chem., 2000, 70(3), 337-344.
8 Mabe, Katsuhiro et al, In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob. Agents Chemother. 1999, 43(7), 1788-1791.
9 陈玉琼等，罐装绿茶水浸提条件的研究，茶叶科学[J]，2000；20(1)：119-123
10 Chang,-CMJ et al. Separation of catechins from green tea using carbon dioxide extraction. Food-Chem. Jan 2000:68(1):109-113
11 孙智达等，高酯型儿茶素茶多酚制备方法的研究，华中农业大学学报，1995，14(5)，496-523
12 W.E.Bronner et al. Journal of chromatography, A. 805(1998)137-142
13 Roberts,E.A.H&Myers.M.J. (1959a) J. Sci. Fd & Agr.,10.176
14 Roberts,E.A.H&Myers.M.J.(1959b) J. Sci. Fd & Agr.,10.172
15 滝野庆则等 (1964)Agr.Biol.Chem.,28.64
16 萧伟强等，茶儿茶素体外氧化产物分析，茶叶科学，1999，19(2)：145-149
17 陶荣达．茶多酚的制备和应用研究进展．化学世界，1997，(2)：64—67
18 Sitech HF, Chanpkl. Inhibitory effects of Phenolics, teas and saliva on the formation of mutageric nitrosation products of salted fish .Int. J.Cancer, 1982,30:714—719.
19 中国农业科学院茶叶研究所．茶树生理及茶叶生化实验手册．北京：农业出版社，1983：53—55．
20 陈为钧．万圣勤．茶叶中多酚类物质的研究进展．天然产物研究开发，1994，6(12)：75—80．
21 赵保路等．茶多酚机理研究．中国药理学报，1991，12(5)：20—22．
22 陈瑞锋，李名君等．茶叶天然抗氧化剂的提取和应用．见：中国农业科学院茶叶研究所．茶叶科学论文集．上海：上海科学技术出版社，1991：157—169．
23 杨贤强等．茶多酚抑癌变机理研究．中国药理学报，1994，5(4)：350—353．
24 沈生荣．儿茶素抑癌变作用的研究．茶叶科学，1993，13(2)：141—146
25 杨秀芳，杨贤强，程书均等．茶多酚抑癌变作用进展．茶叶，1998，24(1)，36—38
26 陈宗懋．ECG和EGCG抗癌变研究．茶叶文摘，1988，2(6)：1—6．
27 沈生荣．茶多酚复合体及L—EGCG清除超氧阴离子自由基特性研究．浙江农业大学学报，1992，18(4)：11—16
28 Hibasami H, Achiwa Y, Fujikawa T, et al. Induction of programmed cell death (apoptosis) in human lymphoid leukemia cells by catechin compounds. Anticancer res. 1996.16:1943—1946.
29 陈上春．茶多酚提取工艺的研究．中国农业化学会志(台湾)，1989，27(1)：82—88．
30 王颖．从茶叶废料中提取茶多酚和咖啡碱．广西化工，1995，24(2)：42—46．
31 葛宜掌，金红．茶多酚的离子沉淀提取法．应用化学，1995，12(2)：107—109．
32 葛宜掌，金红．可溶金属盐离子提取茶多酚的最佳工艺与参数．精细化工，1995，12(6)：3—6
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74 GB8313-87 茶多酚含量的测定