葡萄籽中原花青素提取研究
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摘要
本文对葡萄籽中原花青素的提取和纯化工艺进行了研究，提出了一条较为简单、合理的工艺路线，包括提取、柱层析、溶剂沉淀三个主要步骤。
在提取阶段，以提取率和提取物中原花青素含量为指标通过对提取时间、提取温度、提取溶剂、溶剂用量及料液比五个方面的考察，确定较优的提取工艺条件：溶剂为60％乙醇、温度为30℃、分三次提取、每次30min、料液比为1∶3(m／v)，得到的粗提物中原花青素含量为57.2％，收率为92.7％。
在柱层析步骤中，通过层析法筛选了九种大孔吸附树脂，并用聚酰胺层析作对照，确定较为合适的柱层析填料为X-5大孔吸附树脂。实验结果表明：原花青素在X-5吸附树脂中的吸附行为符合Freundlich等温吸附方程，吸附量随溶液浓度的增加而增大，吸附达到平衡的时间随溶液浓度的增加而增加，当平衡浓度为180mg／ml时，吸附平衡时间约为3h。当以C_0=64.5mg／ml的溶液以0.5BV／h流量通过吸附床时，动态吸附量153.2mg／g树脂；而C_0=42.3mg／ml时，树脂的动态吸附容量为141.8mg／g，为据此可推算工程应用中的最大进样量。用40％乙醇洗脱吸附在树脂上的原花青素，洗脱率为86.1％，原花青素含量高于80％。
层析后的产物经过乙酸乙酯溶解，4倍体积的石油醚沉淀后，原花青素含量上升到97.7％，收率为99.1％。
从提取到分离纯化的全过程，收率为79.1％，产物中原花青素含量为97.7％。该工艺技术具有重要的生产应用价值。
By studying conditions of extracting and purifying proanthocyanidins from grape seeds, the separation process of proanthocyanidins from grape seeds was established in this paper, which was simple and reasonable. The process is composed of three parts: extraction, column chromatography, and precipitation.
In extraction process, five extraction conditions were studied: temperature, extracting time, concentration of solvent, amount of solvent and ratio of solvent to grape seed. With the extraction yield of proanthocyanidins and the selectivity of extraction as optimal condition, the optimal extraction conditions are as followed: solvent 60% ethanol, temperature 30℃, solvent volume to grape seeds weight is 3:1, extracting for 3times,and the time is 30min respectively. Extraction yield of proanthocyanidins was about 92.7%, the content of proanthocyanidins in product is 57.2%.
After studying nine macro porous adsorption resins with column chromatography, comparing with polyamide chromatography, it was revealed that X-5 resin chromatography was proper in purifying proanthocyanidins. The results of experiments show that the adsorption isotherm can be correlated with Freundlich isotherm equation. When the equilibrium concentration is 180mg/ml, the adsorption equilibrium time is about 3h. When the flow rate was 0.5BV/h, and the Co was 64.5mg/ml, the static adsorption capacity is 153.2mg/g resin; if Co=42.3mg/ml, the static adsorption capacity is 141.8mg/g, by which the maximal loading capacity in industrial adsorption column can be estimated. Desorption can be achieved by 40% ethanol. Desorption ratio is 86.1%. The purity of Proanthocyanidins is more than 80%.
After one deposition, the purity of proanthocyanidins is 97.7%, and the yield is 99.1%, the deposition solvent was petroleum ether, the proanthocyanidins was dissolved in acetic ether, and the ratio is 4:1.
In the end, the 97.7% content of proanthocyanidins in product can be got, and the yield of proanthocyanidins is 79.1% by the process. The value of technology is important in industrial production.
在提取阶段，以提取率和提取物中原花青素含量为指标通过对提取时间、提取温度、提取溶剂、溶剂用量及料液比五个方面的考察，确定较优的提取工艺条件：溶剂为60％乙醇、温度为30℃、分三次提取、每次30min、料液比为1∶3(m／v)，得到的粗提物中原花青素含量为57.2％，收率为92.7％。
在柱层析步骤中，通过层析法筛选了九种大孔吸附树脂，并用聚酰胺层析作对照，确定较为合适的柱层析填料为X-5大孔吸附树脂。实验结果表明：原花青素在X-5吸附树脂中的吸附行为符合Freundlich等温吸附方程，吸附量随溶液浓度的增加而增大，吸附达到平衡的时间随溶液浓度的增加而增加，当平衡浓度为180mg／ml时，吸附平衡时间约为3h。当以C_0=64.5mg／ml的溶液以0.5BV／h流量通过吸附床时，动态吸附量153.2mg／g树脂；而C_0=42.3mg／ml时，树脂的动态吸附容量为141.8mg／g，为据此可推算工程应用中的最大进样量。用40％乙醇洗脱吸附在树脂上的原花青素，洗脱率为86.1％，原花青素含量高于80％。
层析后的产物经过乙酸乙酯溶解，4倍体积的石油醚沉淀后，原花青素含量上升到97.7％，收率为99.1％。
从提取到分离纯化的全过程，收率为79.1％，产物中原花青素含量为97.7％。该工艺技术具有重要的生产应用价值。
By studying conditions of extracting and purifying proanthocyanidins from grape seeds, the separation process of proanthocyanidins from grape seeds was established in this paper, which was simple and reasonable. The process is composed of three parts: extraction, column chromatography, and precipitation.
In extraction process, five extraction conditions were studied: temperature, extracting time, concentration of solvent, amount of solvent and ratio of solvent to grape seed. With the extraction yield of proanthocyanidins and the selectivity of extraction as optimal condition, the optimal extraction conditions are as followed: solvent 60% ethanol, temperature 30℃, solvent volume to grape seeds weight is 3:1, extracting for 3times,and the time is 30min respectively. Extraction yield of proanthocyanidins was about 92.7%, the content of proanthocyanidins in product is 57.2%.
After studying nine macro porous adsorption resins with column chromatography, comparing with polyamide chromatography, it was revealed that X-5 resin chromatography was proper in purifying proanthocyanidins. The results of experiments show that the adsorption isotherm can be correlated with Freundlich isotherm equation. When the equilibrium concentration is 180mg/ml, the adsorption equilibrium time is about 3h. When the flow rate was 0.5BV/h, and the Co was 64.5mg/ml, the static adsorption capacity is 153.2mg/g resin; if Co=42.3mg/ml, the static adsorption capacity is 141.8mg/g, by which the maximal loading capacity in industrial adsorption column can be estimated. Desorption can be achieved by 40% ethanol. Desorption ratio is 86.1%. The purity of Proanthocyanidins is more than 80%.
After one deposition, the purity of proanthocyanidins is 97.7%, and the yield is 99.1%, the deposition solvent was petroleum ether, the proanthocyanidins was dissolved in acetic ether, and the ratio is 4:1.
In the end, the 97.7% content of proanthocyanidins in product can be got, and the yield of proanthocyanidins is 79.1% by the process. The value of technology is important in industrial production.
引文
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[2]孔庆山，刘崇怀，潘兴，刘三军。国内外鲜食葡萄发展现状、趋势、问题于对策[J]．中国农业信息快讯，2002，3：3-6。
[3]许申鸿，杭瑚，郝晓丽．，葡萄籽化学成分分析及其抗氧化性质的研究[J]．食品工业科技，2000，21(2)：18-20．
[4]Yinrong Lu, L. Yeap Foo. The polyphenol of grape Pomace[J]. Food chemistry, 1999,65:1-8.
[5]石碧，狄莹．植物多酚[M]．北京，科学出版社，2000：10，137．
[6]国植，徐莉．原花青素：具有广阔发展前景的植物药[J]．国外医药-植物药分册，1996，11(5)，196-204．
[7]Corinne Prieur,Ja(?)ques Rigaud, Veronique Cheynier, et al. Ol(?)eric and polymeric procyanidins from grape seed.[J] Phytochemistry. 1994,36(3): 781-784.
[8]L. Yeap Foo., Yinrong LU, Herbert Wong. Biphenyl-linked biflavanoids from grape pomace[J]. Phytochemistry, 1998,47(6): 1137-1140.
[9]Victor A P. DE Freitas, Yves Glories, Guy Bourgeois, et al. Characterization of oligomeric and polymeric procyanidins from grape seed by liquid secondary ion mass spectrometry[J]. Phytochemistry. 1998,49(5) 1435-1441.
[10]Jorge M. Ricardo Da Silva. et al. Procyanidin dimers and trimers from grape seeds[J]. Phytochemistry, 1991,30(4): 1259-1264.
[11]Celestino Santos-Buelga. Augustin Scalbert. Proanthocyanidins and tannin-like compounds-nature, occurrence, dietary intake and effects on nutrition and health[J]. J Sci Food Agric. 2000, 80:1094-1117.
[12]孙达旺．植物单宁化学[M]．北京：中国林业出版社，1992：130-142．
[13]韩菊，魏福祥，瞿丽萍，等．葡萄籽中低聚原花色素的性能研究[J]．食品科学．2003，24(2)：36-38．
[14]Andrew K. Smith, et al. Effect of viscosity on the bitterness and astringency of grape seed tannin[M]. Food quality and preference, 1996,7:161-166.
[15]赵超英，姚小曼．葡萄籽提取物原花青素的营养保健功能(综述)[J]．中国食品卫生杂志，2000．12(6)：38-41．
[16]高军涛，Huiru Tang，候京武，等，葡萄籽中多酚类物质对氧自由基清除作用的ESR研究[J]．波谱学杂志．1999，16(5)：409-415．
[17] C. Negro, L. Tommasi, A. Miceli. Phenolic compounds and antioxidant activity from red grape marc extracts [J]. Bioresource technology. 2003, 87:41-44.
[18] 林亲录，施兆鹏，秦丹．葡萄籽中的天然抗氧化剂及其保健功能[J]．食品与发酵工业．2002，28(4)：75-78
[19] 郑光耀．葡萄籽原花青素提取物的生理活性、药理作用及其应用[J]．林产化工通讯．2000，34(1)：28-33．
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