从虎杖中提取分离大黄素和白藜芦醇工艺研究
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摘要
虎杖系蓼科蓼属植物虎杖的根茎和根。白藜芦醇是植物受外界刺激而产生的一类低分子量、具有生物活性的植物抗毒素。虎杖中的白藜芦醇含量是迄今研究植物中最高的。综合提取分离虎杖中两种甚至两种以上的有效成分,未见研究报道。本文选择虎杖作为研究对象,探索虎杖中大黄素和白藜芦醇的综合提取分离,为生产企业提供有价值的参考数据。
建立了紫外分光光度法(UV法)、薄层扫描法(TLC法)以及高效液相色谱法(HPLC法)分析检测白藜芦醇和大黄素的方法。以HPLC法精度最高,主要可用于最终产品含量测定,而TLC法、UV法可用于工业生产的定性检查或检测,且简便、快速。
对不同采收期虎杖的白藜芦醇、大黄素含量进行测定,结果表明:白藜芦醇、大黄素在8、9月份的含量最高,可达0.162%、0.648%。综合考虑虎杖采收期应在8、9月较为合理。
对虎杖中白藜芦醇提取进行了单因素试验和正交试验,综合考虑确定白藜芦醇和大黄素最佳提取工艺为:用10倍量的75%乙醇热回流提取3次,每次提取120min,此法优于水热回流法,与超声提取法相近。进行了虎杖中白藜芦醇和大黄素综合提取放大实验,结果表明:从虎杖中综合提取白藜芦醇和大黄素采用乙醇热回流提取法,提取液经浓缩、萃取、水解、脱色、重结晶等方法可有效地从虎杖中分离出高纯度白藜芦醇和大黄素,其中白藜芦醇(98.04%)转化率为64.76%,大黄素(98.11%)转化率为32.08%。
Polygonum cuspidatum is the root and rootstock of Polygonum cuspidatum Sieb.et Zucc belonged to Polygonaaceae. Resveratrol is a kind of small molecular weight and having biologic active phytoalexin produced by plants when they react against the stimulus of enviroment. The resveratrol content in Polygonum cuspidatum is the highest among all studied plant species. It has not been reported that two or more effective component are comperehensively extrated and separated from Polygonum cuspidatum. Adopting Polygonum cuspidatum as the research materials, we studied the method of comprehensive extraction and separation of resveratrol and emodin from Polygonum cuspidatum, and offered valuable reference data for the corporation.
The procedures to analyze resveratrol and emodin with UV, TLC and HPLC methods were set up. The HPLC analysis method can be used for detemining final product because of its highest preciseness. The TLC and UV analysis methods can be used for analyzing half-finished product in producting process because of its convenience and quickness.
The content of resveratrol and emodin in Polygonum cuspidatum of diffent picking period is determined, and is the highest in August and September which achieve 0.162% and 0.648%. August and September is reasonable picking period of Polygonum cuspidatum.
Based on the results of single factor experiments and orthogonal experiments, the best extracting procedures is extracting them with 10 times amount of 75% ethanol at 60℃for 2 hours and repeating the process three times. This method is better than heat return extraction of water, and near to ultrasonic extraction. Experiments of comprehensive extraction and separation of resveratrol and emodin from Polygonum cuspidatum were enlarged. After concentrating, extracting, hydrolyzing, decolorizing and crystalling, resveratrol and emodin can be effectively extracted and separated from Polygonum cuspidatum. The extracting ratio of resveratrol (98.04%) is 64.76%. The extracting ratio of emodin (98.11%) is 32.08%.
建立了紫外分光光度法(UV法)、薄层扫描法(TLC法)以及高效液相色谱法(HPLC法)分析检测白藜芦醇和大黄素的方法。以HPLC法精度最高,主要可用于最终产品含量测定,而TLC法、UV法可用于工业生产的定性检查或检测,且简便、快速。
对不同采收期虎杖的白藜芦醇、大黄素含量进行测定,结果表明:白藜芦醇、大黄素在8、9月份的含量最高,可达0.162%、0.648%。综合考虑虎杖采收期应在8、9月较为合理。
对虎杖中白藜芦醇提取进行了单因素试验和正交试验,综合考虑确定白藜芦醇和大黄素最佳提取工艺为:用10倍量的75%乙醇热回流提取3次,每次提取120min,此法优于水热回流法,与超声提取法相近。进行了虎杖中白藜芦醇和大黄素综合提取放大实验,结果表明:从虎杖中综合提取白藜芦醇和大黄素采用乙醇热回流提取法,提取液经浓缩、萃取、水解、脱色、重结晶等方法可有效地从虎杖中分离出高纯度白藜芦醇和大黄素,其中白藜芦醇(98.04%)转化率为64.76%,大黄素(98.11%)转化率为32.08%。
Polygonum cuspidatum is the root and rootstock of Polygonum cuspidatum Sieb.et Zucc belonged to Polygonaaceae. Resveratrol is a kind of small molecular weight and having biologic active phytoalexin produced by plants when they react against the stimulus of enviroment. The resveratrol content in Polygonum cuspidatum is the highest among all studied plant species. It has not been reported that two or more effective component are comperehensively extrated and separated from Polygonum cuspidatum. Adopting Polygonum cuspidatum as the research materials, we studied the method of comprehensive extraction and separation of resveratrol and emodin from Polygonum cuspidatum, and offered valuable reference data for the corporation.
The procedures to analyze resveratrol and emodin with UV, TLC and HPLC methods were set up. The HPLC analysis method can be used for detemining final product because of its highest preciseness. The TLC and UV analysis methods can be used for analyzing half-finished product in producting process because of its convenience and quickness.
The content of resveratrol and emodin in Polygonum cuspidatum of diffent picking period is determined, and is the highest in August and September which achieve 0.162% and 0.648%. August and September is reasonable picking period of Polygonum cuspidatum.
Based on the results of single factor experiments and orthogonal experiments, the best extracting procedures is extracting them with 10 times amount of 75% ethanol at 60℃for 2 hours and repeating the process three times. This method is better than heat return extraction of water, and near to ultrasonic extraction. Experiments of comprehensive extraction and separation of resveratrol and emodin from Polygonum cuspidatum were enlarged. After concentrating, extracting, hydrolyzing, decolorizing and crystalling, resveratrol and emodin can be effectively extracted and separated from Polygonum cuspidatum. The extracting ratio of resveratrol (98.04%) is 64.76%. The extracting ratio of emodin (98.11%) is 32.08%.
引文
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[3] 张喜云,虎杖的化学成分、药理作用及提取分离,天津药学,1999,11(3):13～14
[4] 牛培勤,郭传勇,白藜芦醇药理作用的研究进展,医药导报,2006,25(6):524～525
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[7] Goel RK, Das Gupta G, Ram SN et al. Antiulcerogenic and anti-in-flammatory effects of emodin, solated from rhamnus triquerta wall. India J Exp Bid,1991,29 (3):230~232
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[9] Huang S S, Yeh SF, Hong CY, Effect of anthraquinone derivatives on lipid peroxidation in rat heart mitochondria: Structure-activity relationship, J Nat Prod, 1995, 58 (9): 1365~1371
[10] 展玉涛,卫新革,陈颖伟,等,大黄素对肝纤维化大鼠血清透明质酸的影响,中西医结合肝病杂志,1999,9(4):27
[11] 刘冠贤,叶任高,谭志明,等,大黄素延缓狼疮性肾炎肾间质纤维化作用的研究. 中国实验临床免疫学杂志, 1999,11(3):24～7
[12] 吴建新,徐家裕,袁耀宗,大黄素与善得定对重症胰腺炎胰缺血的影响及机制,中国中西医结合杂志,1997,17(6):356～9
[13] 靳珠华,马德录,林秀珍,等,大黄素对豚鼠离体肠管作用的影响,中国中西医结合杂志, 1994,14(7):429～31
[14] 姜晓峰,甄永苏,大黄素逆转肿瘤细胞多药抗药性的作用,药学学报,1999,34(3):164～167
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[17] Bertelli AAE, Giovannini L, Giannessi D,et al,Antiplatelet activity of synthetic and natural resveratrol in red wine,Int J Tissue Reac,1995,17:1~3
[18] Arichi H, imura Y, Okuda H, et a1, effects of stilbene compounda of the roots of Polygonum cuspidatum Sieb,et Zucc, on lipid metabolism,Chem Pharm Bul1, 982, 0:1766~1770
[19] Kimura Y, Okuda H, Arichi S, Effects of stilbenes on arachidonate metabolism in leukocytes, Biochim. Biophys,Acta,1985,843:275～278
[20] Kimura Y,Okuda H,Kubo M,Effects of stilbenes isolates from medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes, Ethnopharmacol,1995,45:131~139
[21] Chung M I,Teng C M,Cheng K L,et a1,An antiplatelet principie of Veratrum formosanum, Planta Med,1992,58:274~276
[22] Pace-Asciak C R, Hahn S, Diamandis E P, et a1, The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: Implications for protection against coronary heart disease, Clin Chim Acta, 1995, 235:207~219
[23] Jang M, Cai L, Udeani GO, et al, Cancer chemopreventive aclivity of resveratrol, a natural product derived from grapes, Science, 1997: 275
[24] 傅喆暾，张蓉，黄宝康，等，天然植物中活性成分白藜芦醇的作用研究进展，现代中药研究与实践,2005,19(2):62～64
[25] 陈莉娜,臧威进,唐玉海,白藜芦醇心血管保护作用研究进展,生理科学进展,2003,34(3):272～274
[26] 王瑾,杜江,邱德文,天然活性成分白藜芦醇抗氧化作用的研究进展,时珍国医国药, 2005,16(2):155～156
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[28] 吕秋军,温利青,张敏,等,白藜芦醇的辐射预防及其分子机制的研究,中华放射医学与防护杂志,2004,24(1):21～22
[29] 金顺姬,段链,黄梅,等,白藜芦醇和抗坏血酸对预防非典型肺炎方剂Ⅰ和Ⅵ所致小鼠外周血液淋巴细胞DNA损伤的保护作用,中草药,2003,34(12):114～117
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[31] 程存归,李丹婷,陈秉初。微波辅助提取虎杖大黄素的研究。林产化学与工业。2005,25(3):97～99页
[32] 姜波,虎杖中大黄素的提取分离研究,中医药学报,2004,32(5):40～41
[33] 曾里,袁佩,虎杖蒽醌提取分离方法的优化研究,中国药业,2003,12(9):45～46
[34] 张喜云,虎杖的化学成分、药理作用与提取分离,天津药学,1999,11(3):13～14
[35] Siemann, E.H., Creasy, et a1, Concentration of the phytoalexin resveratrol in wine. Am. J. Enol.Vitic, 1992, 43(1): 49~52
[36] 熊英,邵云东,胡光祥,等,不同工艺的虎杖提取物中白藜芦醇甙含量的比较,天津药学,2000,12(4):79
[37] 刘树兴,程丽英,陈志亮,等,白藜芦醇提取工艺的研究,食品与发酵工业,2004,(12):153～156
[38] 周锦珂,李金华,葛发欢,超临界CO2萃取虎杖中白藜芦醇的工艺研究,中药材,2004,27(9):675～676
[39] 瞿卫林,陈晓祥,赵伯涛,大孔吸附树脂分离虎杖中白藜芦醇的研究,中国野生植物资源,2005, 24(6):60～64
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[2] 国家药典委员会编,中华人民共和国药典(2005年版一部),北京:化学工业出版社,2005:145～146
[3] 张喜云,虎杖的化学成分、药理作用及提取分离,天津药学,1999,11(3):13～14
[4] 牛培勤,郭传勇,白藜芦醇药理作用的研究进展,医药导报,2006,25(6):524～525
[5] 焦东海,蒋小维,阮宜吾,等,全国首届大黄学术研讨会文献概述,中医杂志,1988:29(11):66～68
[6] Kumar A, Dhawans S, Aggarwal BB.Emodin inhibits TNF-induced NF-kappa B activation, IkappaB degradation, and expression of cell surface adhesion protein in human vadcular endothlelical cells. Oncogene, 1998, 17(7):913~918
[7] Goel RK, Das Gupta G, Ram SN et al. Antiulcerogenic and anti-in-flammatory effects of emodin, solated from rhamnus triquerta wall. India J Exp Bid,1991,29 (3):230~232
[8] 路铭,陈琼华,大黄的生化研究,中国药科大学学报,1989,20(4):223～226
[9] Huang S S, Yeh SF, Hong CY, Effect of anthraquinone derivatives on lipid peroxidation in rat heart mitochondria: Structure-activity relationship, J Nat Prod, 1995, 58 (9): 1365~1371
[10] 展玉涛,卫新革,陈颖伟,等,大黄素对肝纤维化大鼠血清透明质酸的影响,中西医结合肝病杂志,1999,9(4):27
[11] 刘冠贤,叶任高,谭志明,等,大黄素延缓狼疮性肾炎肾间质纤维化作用的研究. 中国实验临床免疫学杂志, 1999,11(3):24～7
[12] 吴建新,徐家裕,袁耀宗,大黄素与善得定对重症胰腺炎胰缺血的影响及机制,中国中西医结合杂志,1997,17(6):356～9
[13] 靳珠华,马德录,林秀珍,等,大黄素对豚鼠离体肠管作用的影响,中国中西医结合杂志, 1994,14(7):429～31
[14] 姜晓峰,甄永苏,大黄素逆转肿瘤细胞多药抗药性的作用,药学学报,1999,34(3):164～167
[15] 吴建新,徐家裕,袁耀宗,大黄素与善得定对重症胰腺炎胰缺血的影响及机制,中国中西医结合杂志,1997,17(6):356～359
[16] Fremont L, Belguendouz L, Delpal S, Antioxidant activity of resveratrol andalcohol-free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids,1ife Sci, 1999, 4: l1
[17] Bertelli AAE, Giovannini L, Giannessi D,et al,Antiplatelet activity of synthetic and natural resveratrol in red wine,Int J Tissue Reac,1995,17:1~3
[18] Arichi H, imura Y, Okuda H, et a1, effects of stilbene compounda of the roots of Polygonum cuspidatum Sieb,et Zucc, on lipid metabolism,Chem Pharm Bul1, 982, 0:1766~1770
[19] Kimura Y, Okuda H, Arichi S, Effects of stilbenes on arachidonate metabolism in leukocytes, Biochim. Biophys,Acta,1985,843:275～278
[20] Kimura Y,Okuda H,Kubo M,Effects of stilbenes isolates from medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes, Ethnopharmacol,1995,45:131~139
[21] Chung M I,Teng C M,Cheng K L,et a1,An antiplatelet principie of Veratrum formosanum, Planta Med,1992,58:274~276
[22] Pace-Asciak C R, Hahn S, Diamandis E P, et a1, The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: Implications for protection against coronary heart disease, Clin Chim Acta, 1995, 235:207~219
[23] Jang M, Cai L, Udeani GO, et al, Cancer chemopreventive aclivity of resveratrol, a natural product derived from grapes, Science, 1997: 275
[24] 傅喆暾，张蓉，黄宝康，等，天然植物中活性成分白藜芦醇的作用研究进展，现代中药研究与实践,2005,19(2):62～64
[25] 陈莉娜,臧威进,唐玉海,白藜芦醇心血管保护作用研究进展,生理科学进展,2003,34(3):272～274
[26] 王瑾,杜江,邱德文,天然活性成分白藜芦醇抗氧化作用的研究进展,时珍国医国药, 2005,16(2):155～156
[27] 周建军,虎杖中二苯乙烯类化合物药理作用研究进展,西北药学,2000,15(2):86
[28] 吕秋军,温利青,张敏,等,白藜芦醇的辐射预防及其分子机制的研究,中华放射医学与防护杂志,2004,24(1):21～22
[29] 金顺姬,段链,黄梅,等,白藜芦醇和抗坏血酸对预防非典型肺炎方剂Ⅰ和Ⅵ所致小鼠外周血液淋巴细胞DNA损伤的保护作用,中草药,2003,34(12):114～117
[30] 杨秀贤,王昕,吴寿金,从虎杖中提取大黄素的生产工艺,中草药,1996,27(2):87
[31] 程存归,李丹婷,陈秉初。微波辅助提取虎杖大黄素的研究。林产化学与工业。2005,25(3):97～99页
[32] 姜波,虎杖中大黄素的提取分离研究,中医药学报,2004,32(5):40～41
[33] 曾里,袁佩,虎杖蒽醌提取分离方法的优化研究,中国药业,2003,12(9):45～46
[34] 张喜云,虎杖的化学成分、药理作用与提取分离,天津药学,1999,11(3):13～14
[35] Siemann, E.H., Creasy, et a1, Concentration of the phytoalexin resveratrol in wine. Am. J. Enol.Vitic, 1992, 43(1): 49~52
[36] 熊英,邵云东,胡光祥,等,不同工艺的虎杖提取物中白藜芦醇甙含量的比较,天津药学,2000,12(4):79
[37] 刘树兴,程丽英,陈志亮,等,白藜芦醇提取工艺的研究,食品与发酵工业,2004,(12):153～156
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