复方板蓝根颗粒化学成分的分离分析及活性评价
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摘要
复方板蓝根颗粒为《中华人民共和国卫生部药品标准》所收载的中药成方制剂,由板蓝根和大青叶两种药材组成,具有清热解毒、凉血之功效,用于瘟病发热、出斑、风热感冒、咽喉肿烂、流行性乙型脑炎、肝炎、腮腺炎等。
本论文主要包括以下几个方面的研究:
1.采用系统溶剂提取或萃取的方法,将复方板蓝根颗粒及其单味药逐级分离,得到了样品B1、B2、B3、B4、D1、D2、D3、D4、FF1、FF2、FF3、FF4和FF共13个化学部位。
2.利用高效液相色谱与质谱联用技术,以及多种化学鉴别方法,对各粗提物的化学成分进行了分离分析,确定了复方板蓝根颗粒及其单味药的主要化学成分。
3.利用体外实验研究的方法,对复方板蓝根颗粒及其单味药各粗提物进行了活性评价。结果表明,复方板蓝根颗粒及其单味药中糖类、氨基酸类以及香豆素类化合物为复方板蓝根颗粒及其单味药免疫增强作用的主要有效成分。
这一研究结果为该方质量控制标准的制定和药效物质基础的研究打下了基础。
The chemical components in traditional Chinese medicine (TCM) are responsible for their therapeutic effects and are very important in the modernization of TCM. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques have been proved to be powerful tool in the analysis of TCM due to the high efficiency, sensitivity, speed, resolution and low level of sample consumption. Especially with the development of high performance liquid chromatography couple to mass spectrometry (HPLC-MS), it has provided direct, rapid and effective method for the authentication of known substances, structural identification of unknown substances and the selection of bioactive components in TCM.
In this paper we separated different chemical composition by systemic solvent step by step extraction from compound Indigowoad Root granule and its single herbal medicine. Then we obtained the thirteen samples B1, B2, B3, B4, D1, D2, D3, D4, FF1, FF2, FF3, FF4 and FF.
The analytical method of compound Indigowoad Root granule and its single herbal medicines using HPLC and MS had been preliminarily established. We also analyzed the chemical composition of all samples by HPLC-MS and other chemical identification techniques. So the mainly chemical components of compound Indigowoad Root granule and its single herbal medicine had been identified. The results showed that the mainly chemical components in the samples B1, D1 and FF1 are adenosine, uridine and alkaloids such as indigo, indirubin, hydroxy indirubin and tryptanthrin B. The mainly ones in the samples B2, D2 and FF2 are alkaloids such as 2,4(1H,3H)-quinazolinedione and 2,5-dihydroxy-indole and purine pyrimidine compounds such as guanine, adenine and uracil. The mainly ones in the samples B3, D3 and FF3 are epigoitrin and L-proline. And the mainly ones in the samples B4, D4 and FF4 are carbohydrates, amino acids and coumarin category.
Then we studied on the biological activity of compound Indigowoad Root granule and its single herbal medicines by the evaluation in vitro. Through the impact of the compound Indigowoad Root granule and its single herbal medicines extracts on the proliferation of the isolated mouse spleen lymphocyte and the activity of the peritoneal macrophage, we found the effect of every extracts on the immune function of the isolated mouse. The results showed that every extracts can certainly stimulated the proliferation of spleen lymphocyte and activity of peritoneal macrophages. And the samples B3, B4, D4, FF3 and FF4 presented much stronger effect on the proliferation of spleen lymphocyte.
The studies on the chemical composition and biological activity of compound Indigowoad Root granule and its single herbal medicine showed that the effective components of immunity enhancement are mainly existed in the larger polarity part, which are the carbohydrates, amino acids and coumarin category. The results were useful for the quality control of the compound Indigowoad Root granule and the bioactive material basis of this prescription.
本论文主要包括以下几个方面的研究:
1.采用系统溶剂提取或萃取的方法,将复方板蓝根颗粒及其单味药逐级分离,得到了样品B1、B2、B3、B4、D1、D2、D3、D4、FF1、FF2、FF3、FF4和FF共13个化学部位。
2.利用高效液相色谱与质谱联用技术,以及多种化学鉴别方法,对各粗提物的化学成分进行了分离分析,确定了复方板蓝根颗粒及其单味药的主要化学成分。
3.利用体外实验研究的方法,对复方板蓝根颗粒及其单味药各粗提物进行了活性评价。结果表明,复方板蓝根颗粒及其单味药中糖类、氨基酸类以及香豆素类化合物为复方板蓝根颗粒及其单味药免疫增强作用的主要有效成分。
这一研究结果为该方质量控制标准的制定和药效物质基础的研究打下了基础。
The chemical components in traditional Chinese medicine (TCM) are responsible for their therapeutic effects and are very important in the modernization of TCM. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques have been proved to be powerful tool in the analysis of TCM due to the high efficiency, sensitivity, speed, resolution and low level of sample consumption. Especially with the development of high performance liquid chromatography couple to mass spectrometry (HPLC-MS), it has provided direct, rapid and effective method for the authentication of known substances, structural identification of unknown substances and the selection of bioactive components in TCM.
In this paper we separated different chemical composition by systemic solvent step by step extraction from compound Indigowoad Root granule and its single herbal medicine. Then we obtained the thirteen samples B1, B2, B3, B4, D1, D2, D3, D4, FF1, FF2, FF3, FF4 and FF.
The analytical method of compound Indigowoad Root granule and its single herbal medicines using HPLC and MS had been preliminarily established. We also analyzed the chemical composition of all samples by HPLC-MS and other chemical identification techniques. So the mainly chemical components of compound Indigowoad Root granule and its single herbal medicine had been identified. The results showed that the mainly chemical components in the samples B1, D1 and FF1 are adenosine, uridine and alkaloids such as indigo, indirubin, hydroxy indirubin and tryptanthrin B. The mainly ones in the samples B2, D2 and FF2 are alkaloids such as 2,4(1H,3H)-quinazolinedione and 2,5-dihydroxy-indole and purine pyrimidine compounds such as guanine, adenine and uracil. The mainly ones in the samples B3, D3 and FF3 are epigoitrin and L-proline. And the mainly ones in the samples B4, D4 and FF4 are carbohydrates, amino acids and coumarin category.
Then we studied on the biological activity of compound Indigowoad Root granule and its single herbal medicines by the evaluation in vitro. Through the impact of the compound Indigowoad Root granule and its single herbal medicines extracts on the proliferation of the isolated mouse spleen lymphocyte and the activity of the peritoneal macrophage, we found the effect of every extracts on the immune function of the isolated mouse. The results showed that every extracts can certainly stimulated the proliferation of spleen lymphocyte and activity of peritoneal macrophages. And the samples B3, B4, D4, FF3 and FF4 presented much stronger effect on the proliferation of spleen lymphocyte.
The studies on the chemical composition and biological activity of compound Indigowoad Root granule and its single herbal medicine showed that the effective components of immunity enhancement are mainly existed in the larger polarity part, which are the carbohydrates, amino acids and coumarin category. The results were useful for the quality control of the compound Indigowoad Root granule and the bioactive material basis of this prescription.
引文
[1] 中华人民共和国卫生部药典委员会编,中华人民共和国卫生部药品标准,第十二册
[2] 张贵君主编,中药鉴定学 [M]. 北京:科学出版社,2002
[3] 刘云海,秦国伟,丁水平,等. 板蓝根化学成分的研究(Ⅲ) [J]. 中草药,2002,33(2):97～99
[4] 刘云海,秦国伟,丁水平,等. 板蓝根化学成分的研究(Ⅰ) [J]. 中草药,2001,32(12):1057～1060
[5] 刘海利,吴立军,李华,等. 板蓝根的化学成分研究 [J]. 沈阳药科大学学报,2002,19(2):93～95
[6] 丁水平,刘云海,李敬,等. 板蓝根化学成分研究(Ⅱ) [J]. 医药导报,2001,20(8):475～476
[7] 李彬,陈万生,郑水庆,等. 四倍体板蓝根中的两个新生物碱 [J]. 药学学报,2000,35(7):508～510
[8] Deng KM,Wu XY,Yang GJ,et al. Alkaloids from Isatis indigotica [J]. Chinese Chemical Letters,1997,8(3):237～238
[9] 李玲,梁华清,廖时萱,等. 马蓝的化学成分研究 [J]. 药学学报,1993, 28 (3):238～240
[10] Elliott MC,et al. Phytochemistry,1970,9:1629
[11] Huang QS,et al. Planta Medica,1981,42(3):308
[12] Wu XY,Qin GW. New Alkaloids from Isatis indigotica [J]. Terahedron,1997,53(39):13323～13328
[13] 徐国钧主编,生药学 [M]. 北京:人民卫生出版社,1995
[14] 郑汉臣主编,药用植物学与生药学 [M]. 北京:人民卫生出版社,2003
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[16] 匡海学主编,中药化学,[M]. 北京:中国中医药出版社,2003
[17] 肖姗姗,金郁,孙毓庆. 板蓝根化学成分、药理及质量控制研究进展[J]. 沈阳药科大学学报,2003,20(6):455～459
[18] 胡兴昌,程佳蔚,刘士庄,等. 板蓝根凝集素效价与抑制感冒病毒作用关系的实验研究 [J]. 上海中医药大学学报,2001,15(3):238～240
[19] 李闻文,施凯. 板蓝根、喜树果抗 HFRSV 和 HSV-2 作用的实验研究 [J]. 湖南医科大学学报,1994,19(4):309～311
[20] 孙广莲,胡志力,孟红,等. MTT法检测板蓝根抗巨细胞毒效应 [J].山东中医药大学学报,2000,24(2):137～138
[21] 蒋锡源,杨珍珠,胡志军,等. 50 种治疗肝炎中草药与制剂体外抑制Hbs-Ag 活性的比较 [J]. 现代应用药学,1992,9(5):208～211
[22] 李玲,董同义,李修禄,等. 大青叶类药材及其制剂质量控制的研究[J]. 药学学报,1994,29(2):128～131
[23] Yamada Harudi. Antiviral compositions containing new glycoprotein from Isatis tinctoria [P]. JP:11 60,599 [99 60,599],1999-03-02
[24] 侯琦,程桂芳,张成义,等. 4种刺激剂及抗炎药对HL-60细胞生成IL-8的影响 [J]. 药学学报,2000,35(3):173～176
[25] 许益民,陆平成,王永珍,等. 板蓝根多糖促进免疫功能的实验研究 [J]. 中西医结合杂志,1991,11(6):357～359
[26] 梁永红,侯华新,黎丹戎,等. 板蓝根二酮B体外抗癌活性研究 [J]. 中草药,2000,31(7):531～533
[27] 郑剑玲,王美惠,杨秀珍,等. 大青叶和板蓝根提取物的抑菌作用研究 [J]. 中国微生态学杂志,2003,15(1):18～19
[28] 孙立新,宁黎丽,毕开顺,等. 板蓝根和大青叶质量的化学模式识别研究 [J]. 中药材,2000,23(10):609～613
[29] 张连同,邱世翠,吕俊华,等. 大青叶体外抑菌作用研究 [J]. 时珍国医国药,2002,23(5):283～284
[30] 刘盛,陈万生,乔传卓,等. 不同种质板蓝根和大青叶的抗甲型流感病毒作用 [J]. 第二军医大学学报,2000,21(3):204～206
[31] Nai K M,Chung Y L,Xiao Y W,et al. Inhibtion of RANTES expression by in influenza virus-infected humanbronchial epithelial cells [J]. BiochemPharm,2004,67:167～174
[32] 刘云海,李清香,石玉梅. 大青叶抗内毒素实验 [J]. 中药材,1994,17(6):36～38
[33] 张汉明,张戈,乔传卓. 板蓝根和大青叶不同部位的靛蓝、靛玉红含量测定及其部分成分的抗内毒素作用比较 [J]. 药学实践杂志,2000, 18(5):347
[34] 张淑杰,马立人,顾定伟,等. 大青叶水煎剂调节小鼠免疫细胞分泌IL-2、TNF-α的体外研究 [J]. 陕西中医,2003,23(8):757～759
[35] Tatsuya I,Takanori O,Keizo K,et al. Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression [M]. J Ethnopharm,2000,72:141～150
[36] Barbara C S,Mirza M H,Barbara H K,et al. Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin [J]. BiochemPharm,2003,66:2313～2321
[37] 田颂九,胡昌勤,马双成,等编著,色谱在药物分析中的应用 [M]. 北京:化学工业出版社,2006
[38] 陈耀祖,陈绍媛主编,中药现代化研究的化学法导论 [M].北京:科技出版社,2003
[39] 孙立新,唐虹,尹萍,等. RP-HPLC法测定板蓝根、大青叶中靛蓝、靛玉红的含量 [J]. 沈阳药科大学学报. 2000,17(3):191
[40] 董娟娥,龚明贵,粱宗锁. 板蓝根、大青叶中靛蓝和靛玉红的测定方法比较 [J]. 西北农林科技大学学报,2007,35(2):215～219
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[44] 黄芳,熊雅婷,徐丽华,等. 板蓝根不同提取物中抗病毒成分表告依春在大鼠体内的药代动力学 [J]. 中国药科大学学报,2006,37(6): 519～522
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[48] 马莉,冯少华,唐建元,等.不同制备工艺板蓝根多糖对小鼠脾淋巴细胞增殖作用的影响 [J]. 中国药房,2007,18(3):169～171
[2] 张贵君主编,中药鉴定学 [M]. 北京:科学出版社,2002
[3] 刘云海,秦国伟,丁水平,等. 板蓝根化学成分的研究(Ⅲ) [J]. 中草药,2002,33(2):97～99
[4] 刘云海,秦国伟,丁水平,等. 板蓝根化学成分的研究(Ⅰ) [J]. 中草药,2001,32(12):1057～1060
[5] 刘海利,吴立军,李华,等. 板蓝根的化学成分研究 [J]. 沈阳药科大学学报,2002,19(2):93～95
[6] 丁水平,刘云海,李敬,等. 板蓝根化学成分研究(Ⅱ) [J]. 医药导报,2001,20(8):475～476
[7] 李彬,陈万生,郑水庆,等. 四倍体板蓝根中的两个新生物碱 [J]. 药学学报,2000,35(7):508～510
[8] Deng KM,Wu XY,Yang GJ,et al. Alkaloids from Isatis indigotica [J]. Chinese Chemical Letters,1997,8(3):237～238
[9] 李玲,梁华清,廖时萱,等. 马蓝的化学成分研究 [J]. 药学学报,1993, 28 (3):238～240
[10] Elliott MC,et al. Phytochemistry,1970,9:1629
[11] Huang QS,et al. Planta Medica,1981,42(3):308
[12] Wu XY,Qin GW. New Alkaloids from Isatis indigotica [J]. Terahedron,1997,53(39):13323～13328
[13] 徐国钧主编,生药学 [M]. 北京:人民卫生出版社,1995
[14] 郑汉臣主编,药用植物学与生药学 [M]. 北京:人民卫生出版社,2003
[15] 阴健,郭力弓主编,中药现代研究与临床应用,[M]. 北京:学苑出版社,1995
[16] 匡海学主编,中药化学,[M]. 北京:中国中医药出版社,2003
[17] 肖姗姗,金郁,孙毓庆. 板蓝根化学成分、药理及质量控制研究进展[J]. 沈阳药科大学学报,2003,20(6):455～459
[18] 胡兴昌,程佳蔚,刘士庄,等. 板蓝根凝集素效价与抑制感冒病毒作用关系的实验研究 [J]. 上海中医药大学学报,2001,15(3):238～240
[19] 李闻文,施凯. 板蓝根、喜树果抗 HFRSV 和 HSV-2 作用的实验研究 [J]. 湖南医科大学学报,1994,19(4):309～311
[20] 孙广莲,胡志力,孟红,等. MTT法检测板蓝根抗巨细胞毒效应 [J].山东中医药大学学报,2000,24(2):137～138
[21] 蒋锡源,杨珍珠,胡志军,等. 50 种治疗肝炎中草药与制剂体外抑制Hbs-Ag 活性的比较 [J]. 现代应用药学,1992,9(5):208～211
[22] 李玲,董同义,李修禄,等. 大青叶类药材及其制剂质量控制的研究[J]. 药学学报,1994,29(2):128～131
[23] Yamada Harudi. Antiviral compositions containing new glycoprotein from Isatis tinctoria [P]. JP:11 60,599 [99 60,599],1999-03-02
[24] 侯琦,程桂芳,张成义,等. 4种刺激剂及抗炎药对HL-60细胞生成IL-8的影响 [J]. 药学学报,2000,35(3):173～176
[25] 许益民,陆平成,王永珍,等. 板蓝根多糖促进免疫功能的实验研究 [J]. 中西医结合杂志,1991,11(6):357～359
[26] 梁永红,侯华新,黎丹戎,等. 板蓝根二酮B体外抗癌活性研究 [J]. 中草药,2000,31(7):531～533
[27] 郑剑玲,王美惠,杨秀珍,等. 大青叶和板蓝根提取物的抑菌作用研究 [J]. 中国微生态学杂志,2003,15(1):18～19
[28] 孙立新,宁黎丽,毕开顺,等. 板蓝根和大青叶质量的化学模式识别研究 [J]. 中药材,2000,23(10):609～613
[29] 张连同,邱世翠,吕俊华,等. 大青叶体外抑菌作用研究 [J]. 时珍国医国药,2002,23(5):283～284
[30] 刘盛,陈万生,乔传卓,等. 不同种质板蓝根和大青叶的抗甲型流感病毒作用 [J]. 第二军医大学学报,2000,21(3):204～206
[31] Nai K M,Chung Y L,Xiao Y W,et al. Inhibtion of RANTES expression by in influenza virus-infected humanbronchial epithelial cells [J]. BiochemPharm,2004,67:167～174
[32] 刘云海,李清香,石玉梅. 大青叶抗内毒素实验 [J]. 中药材,1994,17(6):36～38
[33] 张汉明,张戈,乔传卓. 板蓝根和大青叶不同部位的靛蓝、靛玉红含量测定及其部分成分的抗内毒素作用比较 [J]. 药学实践杂志,2000, 18(5):347
[34] 张淑杰,马立人,顾定伟,等. 大青叶水煎剂调节小鼠免疫细胞分泌IL-2、TNF-α的体外研究 [J]. 陕西中医,2003,23(8):757～759
[35] Tatsuya I,Takanori O,Keizo K,et al. Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression [M]. J Ethnopharm,2000,72:141～150
[36] Barbara C S,Mirza M H,Barbara H K,et al. Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin [J]. BiochemPharm,2003,66:2313～2321
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