公丁香和母丁香化学成分的高速逆流色谱分析
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摘要
桃金娘科(Myrtaceae)蒲桃属(Syzygium)植物丁香(Eugenia caryophylla Thunb.)是《中国药典》2005年版收录的中草药,其中公丁香是植物丁香药用未开放的干燥花蕾,母丁香是植物丁香的成熟果实,丁香油具有良好的抗菌消炎作用。公丁香和母丁香的主要活性成分是植物丁香不同生长时期的次生代谢产物,为阐明公丁香和母丁香药理作用差异的物质基础,本文利用高速逆流色谱法分析比较公丁香和母丁香非挥发油成分的同时,并利用高速逆流色谱和高效液相色谱技术对母丁香的化学成分进行了系统研究。
本论文由三部分组成,主要包括以下几方面的研究内容:
首先,综述了公丁香和母丁香化学成分、药理作用、分离提取方法的研究进展以及高速逆流色谱的应用现状与发展趋势。
其次,将公丁香和母丁香95%乙醇回流提取液经逐级萃取、大孔吸附树脂进一步净化,得到公丁香和母丁香的粗提物。利用高速逆流色谱法分离公丁香与母丁香粗提物,并对其主要化学成分进行比较分析。采用溶剂体系正己烷-乙酸乙酯-甲醇-水(5:8:6:13,v/v/v/v)和(5:8:9:10,v/v/v/v)分布洗脱,公丁香和母丁香以主机转速880 r/min,流速1.2 mL/min,单次进样量70 mg各分离得到9个洗脱组分,两张高速逆流色谱分离图谱的重叠率为66.67%,其中包含6个共有峰,并且各共有峰HSCCC保留时间的相对标准偏差RSD的平均值为2.260%,与国家标准关于方法学考察资料技术参数对RSD≤3%的要求相符合,可以判定公丁香和母丁香化学成分内在质量即存在稳定性和均一性,也具有差异性,说明高速逆流色谱法比较公丁香和母丁香化学成分的可行性,为构建公丁香和母丁香高速逆流色谱指纹图谱奠定基础。同时本实验以上述条件成功地从公丁香和母丁香粗提物中分离得到槲皮素、鞣花酸、5,7-二甲氧基-2-甲基色原酮、5,7-二甲氧基-2,6-二甲基色原酮、3,5,3’4’-四羟基-7-甲氧基黄酮五种非挥发油成分,通过标准品对照以及1~H-NMR、(13)~C-NMR、MS等波谱方法对各化合物的结构进行鉴定。其中化合物5,7-二甲氧基-2,6-二甲基色原酮的结构是首次通过核磁数据进行表征。
最后,在第二部分实验的基础上,利用高效液相色谱技术分离纯化母丁香强极性和弱极性粗提物,得到没食子酸、5,7,3′,4′,5′-五甲氧基黄酮、母丁香酚三种高纯度化合物,通过标准品对照以及1~H-NMR、(13)~C-NMR、MS等波谱方法对各化合物的结构进行鉴定。其中化合物5,7,3′,4′,5′-五甲氧基黄酮在母丁香中尚未发现。
Eugenia caryophylla Thunb., a commonly used herb, is documented in the Traditional Chineses Medicine (TCM) collection edition 2005. Flos Caryophyllate is the dry flower buds of Eugenia caryophylla Thunb., while Fructus Caryophylli is the mature fruits of Eugenia caryophylla Thunb.. It has been clearly known that effective components of these herbs are their secondary metabolites. The present paper comparatively analyzed chemical components of Flos Caryophyllata and Fructus Caryophylli by high-speed counter-current chromatography technique.
This dissertation consists of three sections:
The first part is the introduction section. In this section, an extensive review including chemical components, pharmaceutical effect, purification methods of Flos Caryophyllate and Fructus Caryophylli was included. A brief review about the latest progress of HSCCC was also included in this section.
The second section focuses on Comparative analysis of Flos Caryophyllata and Fructus Caryophylli by high-speed counter-current chromatography. The herbs were first extracted by 95% ethanol continous reflux. The crude extracts were further extracted by petroleum ether and ethyl acetate sequentially, followed by macroporous resin fractionation. The fractions were then separated by HSCCC in a stepwise elution technology respectively. The two-phase solvent system composed of hexane: ethyl acetate: methanol: water (5:8:6:13, v/v/v/v, A solvent system) and hexane: ethyl acetate: methanol: water (5:8:9:10, v/v/v/v, B solvent system) that were optimized and determined according to their physical parameters. The separation was initially done by filling the column with upper phase of A solvent system as the stationary phase followed by elution with lower phase of A solvent system for 210 min. Afterwards it eluted with lower phase of B solvent system for another 340 min. Rotation speed of the chromatography was 880 r/min and the flow rate maintains 1.2 mL/min. The sample volume was 70 mg. Nine peaks were identified among which 6 peaks were consistent from both samples. The average of relative standard deviation (RSD) from the 6 peaks’HSCCC retention times was 2.260%, which is much lower than the national standared criteria 3%. Overlay ratios between two HSCCC chromatograms were around 66.67%, which manifest efficiency of HSCCC method in the test. In addition, five compounds including ellagic acid, 5,7-dimethoxy-2-methyl-chromen-4-one, 5,7-dimethoxy- 2,6-dimethyl- chromen -4-one, 3,5,3’4’-tetrahydroxy-7-methoxyflavone and quercetinpurified from the two herbs, among which quercetin, ellagic acid and 3,5,3’4’-tetrahydroxy-7-methoxyflavone are known compounds isolated from Eugenia caryophylla Thunb.. To be mentioned here, 5,7-dimethoxy- 2,6-dimethyl-chromen-4-one is a compound that firstly identified by NMR information so far.
Based on the second section, compounds from Fructus Caryophylli was systematically isolated by using HPLC method. Three compounds including gallic acid, bancroftione and recrystallized 5,7,3’,4’,5’-pentamethoxyflavanone were purified. Among which 5,7,3’,4’,5’-pentamethoxyflavanone and was firstly identified from Fructus Caryophylli.
本论文由三部分组成,主要包括以下几方面的研究内容:
首先,综述了公丁香和母丁香化学成分、药理作用、分离提取方法的研究进展以及高速逆流色谱的应用现状与发展趋势。
其次,将公丁香和母丁香95%乙醇回流提取液经逐级萃取、大孔吸附树脂进一步净化,得到公丁香和母丁香的粗提物。利用高速逆流色谱法分离公丁香与母丁香粗提物,并对其主要化学成分进行比较分析。采用溶剂体系正己烷-乙酸乙酯-甲醇-水(5:8:6:13,v/v/v/v)和(5:8:9:10,v/v/v/v)分布洗脱,公丁香和母丁香以主机转速880 r/min,流速1.2 mL/min,单次进样量70 mg各分离得到9个洗脱组分,两张高速逆流色谱分离图谱的重叠率为66.67%,其中包含6个共有峰,并且各共有峰HSCCC保留时间的相对标准偏差RSD的平均值为2.260%,与国家标准关于方法学考察资料技术参数对RSD≤3%的要求相符合,可以判定公丁香和母丁香化学成分内在质量即存在稳定性和均一性,也具有差异性,说明高速逆流色谱法比较公丁香和母丁香化学成分的可行性,为构建公丁香和母丁香高速逆流色谱指纹图谱奠定基础。同时本实验以上述条件成功地从公丁香和母丁香粗提物中分离得到槲皮素、鞣花酸、5,7-二甲氧基-2-甲基色原酮、5,7-二甲氧基-2,6-二甲基色原酮、3,5,3’4’-四羟基-7-甲氧基黄酮五种非挥发油成分,通过标准品对照以及1~H-NMR、(13)~C-NMR、MS等波谱方法对各化合物的结构进行鉴定。其中化合物5,7-二甲氧基-2,6-二甲基色原酮的结构是首次通过核磁数据进行表征。
最后,在第二部分实验的基础上,利用高效液相色谱技术分离纯化母丁香强极性和弱极性粗提物,得到没食子酸、5,7,3′,4′,5′-五甲氧基黄酮、母丁香酚三种高纯度化合物,通过标准品对照以及1~H-NMR、(13)~C-NMR、MS等波谱方法对各化合物的结构进行鉴定。其中化合物5,7,3′,4′,5′-五甲氧基黄酮在母丁香中尚未发现。
Eugenia caryophylla Thunb., a commonly used herb, is documented in the Traditional Chineses Medicine (TCM) collection edition 2005. Flos Caryophyllate is the dry flower buds of Eugenia caryophylla Thunb., while Fructus Caryophylli is the mature fruits of Eugenia caryophylla Thunb.. It has been clearly known that effective components of these herbs are their secondary metabolites. The present paper comparatively analyzed chemical components of Flos Caryophyllata and Fructus Caryophylli by high-speed counter-current chromatography technique.
This dissertation consists of three sections:
The first part is the introduction section. In this section, an extensive review including chemical components, pharmaceutical effect, purification methods of Flos Caryophyllate and Fructus Caryophylli was included. A brief review about the latest progress of HSCCC was also included in this section.
The second section focuses on Comparative analysis of Flos Caryophyllata and Fructus Caryophylli by high-speed counter-current chromatography. The herbs were first extracted by 95% ethanol continous reflux. The crude extracts were further extracted by petroleum ether and ethyl acetate sequentially, followed by macroporous resin fractionation. The fractions were then separated by HSCCC in a stepwise elution technology respectively. The two-phase solvent system composed of hexane: ethyl acetate: methanol: water (5:8:6:13, v/v/v/v, A solvent system) and hexane: ethyl acetate: methanol: water (5:8:9:10, v/v/v/v, B solvent system) that were optimized and determined according to their physical parameters. The separation was initially done by filling the column with upper phase of A solvent system as the stationary phase followed by elution with lower phase of A solvent system for 210 min. Afterwards it eluted with lower phase of B solvent system for another 340 min. Rotation speed of the chromatography was 880 r/min and the flow rate maintains 1.2 mL/min. The sample volume was 70 mg. Nine peaks were identified among which 6 peaks were consistent from both samples. The average of relative standard deviation (RSD) from the 6 peaks’HSCCC retention times was 2.260%, which is much lower than the national standared criteria 3%. Overlay ratios between two HSCCC chromatograms were around 66.67%, which manifest efficiency of HSCCC method in the test. In addition, five compounds including ellagic acid, 5,7-dimethoxy-2-methyl-chromen-4-one, 5,7-dimethoxy- 2,6-dimethyl- chromen -4-one, 3,5,3’4’-tetrahydroxy-7-methoxyflavone and quercetinpurified from the two herbs, among which quercetin, ellagic acid and 3,5,3’4’-tetrahydroxy-7-methoxyflavone are known compounds isolated from Eugenia caryophylla Thunb.. To be mentioned here, 5,7-dimethoxy- 2,6-dimethyl-chromen-4-one is a compound that firstly identified by NMR information so far.
Based on the second section, compounds from Fructus Caryophylli was systematically isolated by using HPLC method. Three compounds including gallic acid, bancroftione and recrystallized 5,7,3’,4’,5’-pentamethoxyflavanone were purified. Among which 5,7,3’,4’,5’-pentamethoxyflavanone and was firstly identified from Fructus Caryophylli.
引文
[1]刘军红,廖国玲.中药指纹图谱的研究综述.农业科学研究, 2010, 28(4): 383-387.
[2]谢培山.中药色谱指纹图谱鉴别的概念、属性、技术与应用.中国中药杂志, 2001, 26(10): 653-655.
[3]高荫榆,魏强,范青生,等.高速逆流色谱分离提取天然产物技术研究进展.食品科学, 2008, 29(2): 461-465.
[4]顾铭,张贵峰,苏志国,等.高速逆流色谱技术在丹参指纹图谱中的应用.中国药品标准, 2005, 6(6): 7-12.
[5]韩群鑫,黄寿山,陈杰.丁香应用技术的研究进展.江西植保, 2006, 29(1): 24-26.
[6]周丽光.关东丁香化学成分研究[D].长春:吉林大学, 2008.
[7]李新娥.丁香及其混伪品的鉴别.传统医药, 2007, 16(18): 50.
[8]崔国静,石思佳,宋桂英.丁香的鉴别.传统中医药, 2010:48.
[9]但春.丁香和升麻的化学成分研究[D].成都:中国科学院成都生物研究所, 2006.
[10]刘洪宇,朱姝,小松かつ子,等.丁香水溶性化学成分的研究.中药材, 2008, 31(7): 998-1000.
[11]董文宁,邱琴,刘成琴,等.超临界CO2流体萃取法提取母丁香挥发油化学成分的研究. World Health Digest, 2007, 4(7): 125-128.
[12]赵晨曦,梁逸曾.公丁香与母丁香挥发油化学成分的GC/MS研究.现代中药研究与实践, 2004, 18: 92-95.
[13]陈星灿,刘定安,宫锡坤,等.中药抗菌作用研究.中医药学报, 1998, 1: 36-37.
[14]宋义忠,孔秋莲,孟宪君,等.丁香提取物对花椰菜保鲜效果的研究.上海蔬菜, 2003, 2: 40-41.
[15]沈琦,胡晋红,徐莲英.不同来源丁香油、高良姜油对氟尿嘧啶透皮吸收的影响.药学服务与研究, 2001, 1(1): 30.
[16]沈琦,胡晋红,徐莲英..肉桂等3种挥发油对苯甲酸透皮吸收的影响.中国医院药学杂志, 2001, 21(4): 197-199.
[17]沈琦,蔡贞贞,徐莲英.中药丁香促进5-氟脲嘧啶透皮吸收的作用研究.中草药, 1999, 30(8): 601-602.
[18]张明发,陈光娟,沈雅琴,等.丁香温经止痛和温中止泻药理研究.中药材, 1992, 15(4): 33-36.
[19]许青媛,陈春梅,张小莉,等,丁香及其主要成分的抗凝作用.中药药理与临床, 1990, 6(6): 31-32.
[20]张军锋,张树军.丁香属植物的化学成分及其药理作用的研究进展.海南大学学报自然科学版. 2007, 25(2): 200-205.
[21]李锦绣.丁香现代药理研究进展.实用中医药杂志. 2002, 18(6).
[22]王桂清,姬兰柱,张洪,等.公、母丁香乙醇提取物离体抑菌活性比较.农药, 2006, 45(12): 852-855.
[23]俞忠明,章建民.丁香超临界流体萃取工艺的研究.浙江中医杂志, 2006, 41(12): 724-725.
[24]付振喜,王建清,金政伟,等.定向挥发油的提取工艺及化学成分分析.安徽农业科学, 2010, 38(11): 5628-5630.
[25]韩志峰,沈洁,郭立伟,等.微滤法用于丁香挥发油含有水体的油水分离研究.中国中药杂志, 2011, 36(1): 41-44.
[26]郭辉.高速逆流色谱技术在药物和天然产物分离中的应用. Strait Pharmaceutical Journal, 2007,19(3): 95-97.
[27]赵莹.高速逆流色谱在天然产物分离中的应用.食品与药品. 2007, 9(5): 24-27.
[28]但春,蔡敏,王瑞,等.公丁香极性成分的HPLC-MS/MS分析.分析测试学报. 2005, 24:113-114.
[29]周丽光,冯雪松,黄开毅,等.关东丁香化学成分研究.中药材. 2008, 31(5): 679-681.
[30]魏征骥,卢丹,刘金平,等.紫丁香化学成分的研究.长春中医学院学报. 2004, 20(1): 33-34.
[31]叶龙忠,汪敏燕,陈秀,等.高速逆流色谱研究进展.化工生产与技术, 2003, 10(4): 27-29.
[32]时新刚,陈志伟,刘东武,等.高速逆流色谱应用研究进展.生命科学仪器, 2009, 8(12): 3-6.
[33]戴德舜,王义明,罗国安.高速逆流色谱研究进展.分析化学, 2001, 29(5): 586-587.
[34]刘迪,陈雪峰,宋晓宇.高速逆流色谱技术应用现状.食品与机械, 2006, 22(2): 98-100.
[35]张天佑.让逆流色谱技术为中药现代化服务.世界科学技术-中药现代化, 2001, 3(6): 17-19.
[36]徐宝财,张桂菊,王关兴,等.高速逆流色谱技术及其在药物分离分析中的应用.药物分析杂志, 2006, 26(10): 1516-1520.
[37] Gu Ming, Ouyang Fan, Su Zhiguo. Comparison of high-speed counter-current chromatography and high-performance liquid chromatography on fingerprinting of Chinese traditional medicine. J. Chromatography A, 2004, 1022: 139-144.
[38]高蕾,杨光丽,陈俐娟,等.高速逆流色谱快速分离四种甘草黄酮及其结构鉴定.四川化工. 2007, 10(2): 34-38.
[39]彭爱一,曲学伟,李慧,等.高速逆流色谱分离纯化九里香中的黄酮类化合物.色谱, 2010, 28(4): 383-387.
[40]王玮,王琳.黄酮类化合物的研究进展.沈阳医学院学报, 2002, 4(2): 115-116.
[41]黄华艺,查锡良.黄酮类化合物抗肿瘤作用研究进展.中国新药与临床杂志. 2002, 21 (7): 428-433.
[42]王凤美,陈军辉,李磊,等.高速逆流色谱法分离制备丹酚酸B.天然产物的研究与应用, 2006, l8(1): 100-104.
[43]曹学丽.高速逆流色谱分离技术及应用.化学工业出版社. 2005, 1: 214.
[44] Yan JZ, Chen G, Tong SQ, et al. Preparative isolation and purification of germacrone and curdione from the essential oil of the rhizomes of Curcuma wenyujin by high-speed counter-current chromatography. J. Chromatogr A, 2005, 1070(1-2): 207-210.
[45] Liu RM, Feng L, Sun AL, et al. Preparative isolation and purification of coumarins from Cnidiummonnieri(L.) Cusson by high-speed counter-current chromatography. J. Chromatogr A, 2004, 1055(1-2): 71-76.
[46]宁敏等.高速逆流色谱分离弥勒牙菜中的生物总碱.化学研究与应用, 2002, 14(4): 479-4801.
[47]王春燕,蔡定国,柳正良,等.芦荟甙和芦荟大黄素的高速逆流色谱法分离纯化.中国医药工业杂志, 2001, 32(4): 145-147.
[48]夏兴,戈梅,陈代杰,等.利用高速逆流色谱从真菌HCCB00106转化液分离转化产物.中国抗生素杂志, 2007, 32(3): S3-S4.
[49]顾铭,苏志国.高速逆流色谱用于天然产物分离和指纹图谱构建.生物加工过程, 2003, 1(2): 59-62.
[50] Lee YW, Pack TW, Voyksner RD, et al. J. Liq. Chromatogr, 1990, 13: 2389.
[51] Lee YW, Voyksner TW, Pack TW, et al. Anal Chem, 1990, 62: 241.
[52] Kong ZR, Rinehart RL, Milberg RM, et al. J. Liq. Chrom. & Rcl Technol, 1998, 21(1%2): 65.
[53] Yao Shun, Liu Renmin, Huang Xuefeng, et al. Preparative isolation and purification of chemical constituents from the root of Adenophora tetraphlla by high-speed counter-current chromatography with evaporative light scattering detection. J. Chromatogr A. 2007, 1139: 254-262.
[54] Zhou Tingting, Zhu Zhenyu, Wang Chen, et al. On-line purity monitoring in high-speed counter-current chromatography: Application of HSCCC-HPLC-DAD for the preparation of 5-HMF, neomangiferin and mangiferin from Anemarrhena asphodeloides Bunge. Journal of Pharmaceutical and Biomedical Analysis, 2007, 44: 96-100.
[55]曹学丽.高速逆流色谱技术在药物研究开发中的应用.世界科学技术-中医药现代化, 2007, 9(1): 54-58.
[56] Oka H, Ito Y. Separation of lac dye components using high-speed counter-current chromatography. Counter-current Chromatography, 2000(4): 24-29.
[57]方东升,谢阳,陈勇,等.高速逆流色谱法分离纯化环孢菌素.中国抗生素杂志, 2005, 30(1): 48-51.
[58]郅文波,邓秋云,宋江楠,等.高速逆流色谱双水相体系分离蛋白质.色谱, 2005, 23(1): 12-17.
[59]汪正范.色谱定性与定量:色谱指纹图谱与色谱指纹谱.第二版.北京:化学工业出版社, 2007: 108-118.
[60]冯启余,曹玉华,龚立冬.中药指纹图谱共有峰的自动识别.分析科学学报, 2009, 25(1): 41-46.
[61]顾铭,欧阳藩,苏志国.高速逆流色谱法分离纯化丹参并尝试制订中药指纹图谱.生物工程学报, 2003, 19(6): 740-744.
[62]杨秀伟,郭庆梅.蓝桉果实化学成分的研究.中国中药杂志, 2007, 32(6): 496-500.
[63] Taylor, D.R., Warner, J.A. and Wright, J.A. (1977), J. Chem. Soc., Perkin Trans. 1, 397–405.
[64] H.Schmid.. Uber die Inhaltstoffe von Eugenia caryphyllata(L.)ThunbgⅢ. Isolierung und Konstitution des Eugenitins. Volumen XXXII,FasciculusⅢ, 1949,105: 813-820.
[65]张荣劲,杨义芳.高速逆流色谱分离天然产物的溶剂体系选择.中草药, 2008, 39(2): 298-303.
[66]姚舜,柳仁民,黄雪峰,等.高速逆流色谱在天然产物分离中的方法学研究.中国天然药物, 2008, 6(1): 13-19.
[67] Yoichiro Ito. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. Journal of Chromatography A, 2005, 1065: 145-168.
[68]赵德玉,毕桂宏.中药指纹图谱的不同方法特点及应用.中华中医药学刊, 2007, 25(3): 601-603.
[69]刘东,鞠建华,杨峻山.狭叶崖爬藤化学成分的研究.中草药, 2003, 34(1): 4-6.
[70] Shu-Juan Liu, Zong-Tao Lin, Hong Wang,et al.Quantification ofeugenol and bancroftione in Caryophylli Fructus using high-performance liquid chromatography. Journal of Chinese Pharmaceutical Sciences, 2010, 19: 459-463.
[71]常连举,张宗和,黄嘉玲,等.没食子酸的制备与应用综述.生物质化学工程, 2010, 44(4): 48-52.
[2]谢培山.中药色谱指纹图谱鉴别的概念、属性、技术与应用.中国中药杂志, 2001, 26(10): 653-655.
[3]高荫榆,魏强,范青生,等.高速逆流色谱分离提取天然产物技术研究进展.食品科学, 2008, 29(2): 461-465.
[4]顾铭,张贵峰,苏志国,等.高速逆流色谱技术在丹参指纹图谱中的应用.中国药品标准, 2005, 6(6): 7-12.
[5]韩群鑫,黄寿山,陈杰.丁香应用技术的研究进展.江西植保, 2006, 29(1): 24-26.
[6]周丽光.关东丁香化学成分研究[D].长春:吉林大学, 2008.
[7]李新娥.丁香及其混伪品的鉴别.传统医药, 2007, 16(18): 50.
[8]崔国静,石思佳,宋桂英.丁香的鉴别.传统中医药, 2010:48.
[9]但春.丁香和升麻的化学成分研究[D].成都:中国科学院成都生物研究所, 2006.
[10]刘洪宇,朱姝,小松かつ子,等.丁香水溶性化学成分的研究.中药材, 2008, 31(7): 998-1000.
[11]董文宁,邱琴,刘成琴,等.超临界CO2流体萃取法提取母丁香挥发油化学成分的研究. World Health Digest, 2007, 4(7): 125-128.
[12]赵晨曦,梁逸曾.公丁香与母丁香挥发油化学成分的GC/MS研究.现代中药研究与实践, 2004, 18: 92-95.
[13]陈星灿,刘定安,宫锡坤,等.中药抗菌作用研究.中医药学报, 1998, 1: 36-37.
[14]宋义忠,孔秋莲,孟宪君,等.丁香提取物对花椰菜保鲜效果的研究.上海蔬菜, 2003, 2: 40-41.
[15]沈琦,胡晋红,徐莲英.不同来源丁香油、高良姜油对氟尿嘧啶透皮吸收的影响.药学服务与研究, 2001, 1(1): 30.
[16]沈琦,胡晋红,徐莲英..肉桂等3种挥发油对苯甲酸透皮吸收的影响.中国医院药学杂志, 2001, 21(4): 197-199.
[17]沈琦,蔡贞贞,徐莲英.中药丁香促进5-氟脲嘧啶透皮吸收的作用研究.中草药, 1999, 30(8): 601-602.
[18]张明发,陈光娟,沈雅琴,等.丁香温经止痛和温中止泻药理研究.中药材, 1992, 15(4): 33-36.
[19]许青媛,陈春梅,张小莉,等,丁香及其主要成分的抗凝作用.中药药理与临床, 1990, 6(6): 31-32.
[20]张军锋,张树军.丁香属植物的化学成分及其药理作用的研究进展.海南大学学报自然科学版. 2007, 25(2): 200-205.
[21]李锦绣.丁香现代药理研究进展.实用中医药杂志. 2002, 18(6).
[22]王桂清,姬兰柱,张洪,等.公、母丁香乙醇提取物离体抑菌活性比较.农药, 2006, 45(12): 852-855.
[23]俞忠明,章建民.丁香超临界流体萃取工艺的研究.浙江中医杂志, 2006, 41(12): 724-725.
[24]付振喜,王建清,金政伟,等.定向挥发油的提取工艺及化学成分分析.安徽农业科学, 2010, 38(11): 5628-5630.
[25]韩志峰,沈洁,郭立伟,等.微滤法用于丁香挥发油含有水体的油水分离研究.中国中药杂志, 2011, 36(1): 41-44.
[26]郭辉.高速逆流色谱技术在药物和天然产物分离中的应用. Strait Pharmaceutical Journal, 2007,19(3): 95-97.
[27]赵莹.高速逆流色谱在天然产物分离中的应用.食品与药品. 2007, 9(5): 24-27.
[28]但春,蔡敏,王瑞,等.公丁香极性成分的HPLC-MS/MS分析.分析测试学报. 2005, 24:113-114.
[29]周丽光,冯雪松,黄开毅,等.关东丁香化学成分研究.中药材. 2008, 31(5): 679-681.
[30]魏征骥,卢丹,刘金平,等.紫丁香化学成分的研究.长春中医学院学报. 2004, 20(1): 33-34.
[31]叶龙忠,汪敏燕,陈秀,等.高速逆流色谱研究进展.化工生产与技术, 2003, 10(4): 27-29.
[32]时新刚,陈志伟,刘东武,等.高速逆流色谱应用研究进展.生命科学仪器, 2009, 8(12): 3-6.
[33]戴德舜,王义明,罗国安.高速逆流色谱研究进展.分析化学, 2001, 29(5): 586-587.
[34]刘迪,陈雪峰,宋晓宇.高速逆流色谱技术应用现状.食品与机械, 2006, 22(2): 98-100.
[35]张天佑.让逆流色谱技术为中药现代化服务.世界科学技术-中药现代化, 2001, 3(6): 17-19.
[36]徐宝财,张桂菊,王关兴,等.高速逆流色谱技术及其在药物分离分析中的应用.药物分析杂志, 2006, 26(10): 1516-1520.
[37] Gu Ming, Ouyang Fan, Su Zhiguo. Comparison of high-speed counter-current chromatography and high-performance liquid chromatography on fingerprinting of Chinese traditional medicine. J. Chromatography A, 2004, 1022: 139-144.
[38]高蕾,杨光丽,陈俐娟,等.高速逆流色谱快速分离四种甘草黄酮及其结构鉴定.四川化工. 2007, 10(2): 34-38.
[39]彭爱一,曲学伟,李慧,等.高速逆流色谱分离纯化九里香中的黄酮类化合物.色谱, 2010, 28(4): 383-387.
[40]王玮,王琳.黄酮类化合物的研究进展.沈阳医学院学报, 2002, 4(2): 115-116.
[41]黄华艺,查锡良.黄酮类化合物抗肿瘤作用研究进展.中国新药与临床杂志. 2002, 21 (7): 428-433.
[42]王凤美,陈军辉,李磊,等.高速逆流色谱法分离制备丹酚酸B.天然产物的研究与应用, 2006, l8(1): 100-104.
[43]曹学丽.高速逆流色谱分离技术及应用.化学工业出版社. 2005, 1: 214.
[44] Yan JZ, Chen G, Tong SQ, et al. Preparative isolation and purification of germacrone and curdione from the essential oil of the rhizomes of Curcuma wenyujin by high-speed counter-current chromatography. J. Chromatogr A, 2005, 1070(1-2): 207-210.
[45] Liu RM, Feng L, Sun AL, et al. Preparative isolation and purification of coumarins from Cnidiummonnieri(L.) Cusson by high-speed counter-current chromatography. J. Chromatogr A, 2004, 1055(1-2): 71-76.
[46]宁敏等.高速逆流色谱分离弥勒牙菜中的生物总碱.化学研究与应用, 2002, 14(4): 479-4801.
[47]王春燕,蔡定国,柳正良,等.芦荟甙和芦荟大黄素的高速逆流色谱法分离纯化.中国医药工业杂志, 2001, 32(4): 145-147.
[48]夏兴,戈梅,陈代杰,等.利用高速逆流色谱从真菌HCCB00106转化液分离转化产物.中国抗生素杂志, 2007, 32(3): S3-S4.
[49]顾铭,苏志国.高速逆流色谱用于天然产物分离和指纹图谱构建.生物加工过程, 2003, 1(2): 59-62.
[50] Lee YW, Pack TW, Voyksner RD, et al. J. Liq. Chromatogr, 1990, 13: 2389.
[51] Lee YW, Voyksner TW, Pack TW, et al. Anal Chem, 1990, 62: 241.
[52] Kong ZR, Rinehart RL, Milberg RM, et al. J. Liq. Chrom. & Rcl Technol, 1998, 21(1%2): 65.
[53] Yao Shun, Liu Renmin, Huang Xuefeng, et al. Preparative isolation and purification of chemical constituents from the root of Adenophora tetraphlla by high-speed counter-current chromatography with evaporative light scattering detection. J. Chromatogr A. 2007, 1139: 254-262.
[54] Zhou Tingting, Zhu Zhenyu, Wang Chen, et al. On-line purity monitoring in high-speed counter-current chromatography: Application of HSCCC-HPLC-DAD for the preparation of 5-HMF, neomangiferin and mangiferin from Anemarrhena asphodeloides Bunge. Journal of Pharmaceutical and Biomedical Analysis, 2007, 44: 96-100.
[55]曹学丽.高速逆流色谱技术在药物研究开发中的应用.世界科学技术-中医药现代化, 2007, 9(1): 54-58.
[56] Oka H, Ito Y. Separation of lac dye components using high-speed counter-current chromatography. Counter-current Chromatography, 2000(4): 24-29.
[57]方东升,谢阳,陈勇,等.高速逆流色谱法分离纯化环孢菌素.中国抗生素杂志, 2005, 30(1): 48-51.
[58]郅文波,邓秋云,宋江楠,等.高速逆流色谱双水相体系分离蛋白质.色谱, 2005, 23(1): 12-17.
[59]汪正范.色谱定性与定量:色谱指纹图谱与色谱指纹谱.第二版.北京:化学工业出版社, 2007: 108-118.
[60]冯启余,曹玉华,龚立冬.中药指纹图谱共有峰的自动识别.分析科学学报, 2009, 25(1): 41-46.
[61]顾铭,欧阳藩,苏志国.高速逆流色谱法分离纯化丹参并尝试制订中药指纹图谱.生物工程学报, 2003, 19(6): 740-744.
[62]杨秀伟,郭庆梅.蓝桉果实化学成分的研究.中国中药杂志, 2007, 32(6): 496-500.
[63] Taylor, D.R., Warner, J.A. and Wright, J.A. (1977), J. Chem. Soc., Perkin Trans. 1, 397–405.
[64] H.Schmid.. Uber die Inhaltstoffe von Eugenia caryphyllata(L.)ThunbgⅢ. Isolierung und Konstitution des Eugenitins. Volumen XXXII,FasciculusⅢ, 1949,105: 813-820.
[65]张荣劲,杨义芳.高速逆流色谱分离天然产物的溶剂体系选择.中草药, 2008, 39(2): 298-303.
[66]姚舜,柳仁民,黄雪峰,等.高速逆流色谱在天然产物分离中的方法学研究.中国天然药物, 2008, 6(1): 13-19.
[67] Yoichiro Ito. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. Journal of Chromatography A, 2005, 1065: 145-168.
[68]赵德玉,毕桂宏.中药指纹图谱的不同方法特点及应用.中华中医药学刊, 2007, 25(3): 601-603.
[69]刘东,鞠建华,杨峻山.狭叶崖爬藤化学成分的研究.中草药, 2003, 34(1): 4-6.
[70] Shu-Juan Liu, Zong-Tao Lin, Hong Wang,et al.Quantification ofeugenol and bancroftione in Caryophylli Fructus using high-performance liquid chromatography. Journal of Chinese Pharmaceutical Sciences, 2010, 19: 459-463.
[71]常连举,张宗和,黄嘉玲,等.没食子酸的制备与应用综述.生物质化学工程, 2010, 44(4): 48-52.