京大戟化学成分的研究
|
摘要
京大戟为大戟科大戟属植物大戟(Euphorbia pekinensis Rupr)的干燥根。具有泻水逐饮,消肿散结功效,用于水肿胀满、痰饮、瘰疠、痈疽肿毒等症。临床上常用于胸膜炎积水,晚期血吸虫病腹水,肝硬化腹水及精神分裂症等。
本实验利用乙醇提取,经硅胶柱层析分得7个化合物,并利用理化性质以及UV、MS、NMR等波谱数据分析,鉴定了它们的结构,分别是3,3′-二甲氧基鞣花酸-4′-O-β-D-吡喃木糖苷(3,3′-di-O-methyl ellagic acid-4′-O-β-D-xylopyranoside)、3,3′-二甲氧基鞣花酸-4′-O-β-D-吡喃葡萄糖苷(3,3′-di-O-methyl ellagic acid-4′-O-β-D-glucopyranoside)、3,3′-二甲氧基鞣花酸(3,3′-di-O-methyl ellagicacid)、阿魏酸二十八酯(octacosyl ferulate)、大戟二烯醇(euphol)、β-谷甾醇(β-sitosterol)和胡萝卜苷(daucosterol)。这些化合物除了β-谷甾醇之外均为首次从京大戟中分离得到。
本实验还建立了京大戟中大戟二烯醇的HPLC测定方法。方法学研究表明,该方法具有准确、灵敏、便捷等特点。
本实验深入研究了京大戟中主要的化学成分,为完善京大戟质量标准以及为京大戟的开发利用提供了新的科学依据。
“Jing Daji”, the root of Euphorbia pekinensis Rupr,is widely used for treatment of edema,removing the full bilge, pleurisy and schizophrenia etc. So far, there are many researches in chemical constituents and pharmacological function of the root of Euphorbia pekinensis Rupr., but there isn’t report about the index of content determination in it’s literatures. For the sake of the better use of this herb medicine, this paper describes the extraction, isolation and identification. Moreover, the determination method has been established for content of euphol in the root of Euphorbia pekinensis Rupr in this paper. A series of tests proved that the determination method is accurate, reliable and reproducible to determine euphol in the root of Euphorbia pekinensis Rupr.
Extraction and Separation
Dried roots (5kg) were extracted by heat reflux in 90% Ethanol (solvent volume were 30, 25 and 20 L , respectively ) for three times (2,1.5 and 1h, respectively), the ethanol was evaporated in vacuum and concentrated to cream. The cream was subjected to silica gel column chromatography eluted with ethyl acetate– methanol– water (5:1:0.5) to afford compounds A (300mg),B (50mg),C (50mg),D (90mg) E(350mg),F(80mg) and G(60mg).
Compounds A, B, C, D, E, F and G were identified respectively by UV, IR,MS, and 1DNMR as 3,3′-di-O-methyl-ellagic acid-4′-O-β-D-xylopyranoside , 3,3′-di-O–methyl-ellagic acid-4-O-β-D-glucopyranoside, 3, 3′-di-O-methyl-ellagic acid, Octacosyl ferulate, euphol,β-sitosterol and daucosterol.
The content determination of euphol in root of the Euphorbia pekinensis Rupr was studied in the paper. We present a more sensitive and accurate quanti-fication method by HPLC for determining the major chemical constituent-euph- ol in root of the Euphorbia pekinensis Rupr.
HPLC was performed on Agilent 1200 HPLC instrument and a Eclipse SDB-C18 column(250 mm×4.6 mm,5μm)at 25℃with the rate of 1.0 ml/min,using the mixture of acetonitrile, methanol and water as mobile phase (49:49:2).
Conclusions:
ⅰThe linear range of euphol is 0.1μg～5.0μg. y=846.00x+17.00 r=0.9999
ⅱEuphol solution was injected into HPLC for six times, the RSD of precision tests is 0.44 % (n=6).
ⅲThe repeatability tests for six samples showed RSD of euphol was 1.75%, (n=6) respectively.
ⅳThe S/N was 3.1 after the concentration of euphol solution had been diluted to 0.0005 mg/ml and had been determined by HPLC, So the detectability is 0.01μg (10ng)。
ⅴThe test of stability showed its RSD was 0.64%, so the euphol solution is stable in ten hours.
ⅵThe average recovery of euphol was 100.4%( RSD=0.99% ), ( n=6)。
The results of this paper can provide a new index of content determination for establishment of quality standards of the root of Euphorbia pekinensis Rupr. and also can offer science foundation for the development and utilization of this medicinal plant.
本实验利用乙醇提取,经硅胶柱层析分得7个化合物,并利用理化性质以及UV、MS、NMR等波谱数据分析,鉴定了它们的结构,分别是3,3′-二甲氧基鞣花酸-4′-O-β-D-吡喃木糖苷(3,3′-di-O-methyl ellagic acid-4′-O-β-D-xylopyranoside)、3,3′-二甲氧基鞣花酸-4′-O-β-D-吡喃葡萄糖苷(3,3′-di-O-methyl ellagic acid-4′-O-β-D-glucopyranoside)、3,3′-二甲氧基鞣花酸(3,3′-di-O-methyl ellagicacid)、阿魏酸二十八酯(octacosyl ferulate)、大戟二烯醇(euphol)、β-谷甾醇(β-sitosterol)和胡萝卜苷(daucosterol)。这些化合物除了β-谷甾醇之外均为首次从京大戟中分离得到。
本实验还建立了京大戟中大戟二烯醇的HPLC测定方法。方法学研究表明,该方法具有准确、灵敏、便捷等特点。
本实验深入研究了京大戟中主要的化学成分,为完善京大戟质量标准以及为京大戟的开发利用提供了新的科学依据。
“Jing Daji”, the root of Euphorbia pekinensis Rupr,is widely used for treatment of edema,removing the full bilge, pleurisy and schizophrenia etc. So far, there are many researches in chemical constituents and pharmacological function of the root of Euphorbia pekinensis Rupr., but there isn’t report about the index of content determination in it’s literatures. For the sake of the better use of this herb medicine, this paper describes the extraction, isolation and identification. Moreover, the determination method has been established for content of euphol in the root of Euphorbia pekinensis Rupr in this paper. A series of tests proved that the determination method is accurate, reliable and reproducible to determine euphol in the root of Euphorbia pekinensis Rupr.
Extraction and Separation
Dried roots (5kg) were extracted by heat reflux in 90% Ethanol (solvent volume were 30, 25 and 20 L , respectively ) for three times (2,1.5 and 1h, respectively), the ethanol was evaporated in vacuum and concentrated to cream. The cream was subjected to silica gel column chromatography eluted with ethyl acetate– methanol– water (5:1:0.5) to afford compounds A (300mg),B (50mg),C (50mg),D (90mg) E(350mg),F(80mg) and G(60mg).
Compounds A, B, C, D, E, F and G were identified respectively by UV, IR,MS, and 1DNMR as 3,3′-di-O-methyl-ellagic acid-4′-O-β-D-xylopyranoside , 3,3′-di-O–methyl-ellagic acid-4-O-β-D-glucopyranoside, 3, 3′-di-O-methyl-ellagic acid, Octacosyl ferulate, euphol,β-sitosterol and daucosterol.
The content determination of euphol in root of the Euphorbia pekinensis Rupr was studied in the paper. We present a more sensitive and accurate quanti-fication method by HPLC for determining the major chemical constituent-euph- ol in root of the Euphorbia pekinensis Rupr.
HPLC was performed on Agilent 1200 HPLC instrument and a Eclipse SDB-C18 column(250 mm×4.6 mm,5μm)at 25℃with the rate of 1.0 ml/min,using the mixture of acetonitrile, methanol and water as mobile phase (49:49:2).
Conclusions:
ⅰThe linear range of euphol is 0.1μg～5.0μg. y=846.00x+17.00 r=0.9999
ⅱEuphol solution was injected into HPLC for six times, the RSD of precision tests is 0.44 % (n=6).
ⅲThe repeatability tests for six samples showed RSD of euphol was 1.75%, (n=6) respectively.
ⅳThe S/N was 3.1 after the concentration of euphol solution had been diluted to 0.0005 mg/ml and had been determined by HPLC, So the detectability is 0.01μg (10ng)。
ⅴThe test of stability showed its RSD was 0.64%, so the euphol solution is stable in ten hours.
ⅵThe average recovery of euphol was 100.4%( RSD=0.99% ), ( n=6)。
The results of this paper can provide a new index of content determination for establishment of quality standards of the root of Euphorbia pekinensis Rupr. and also can offer science foundation for the development and utilization of this medicinal plant.
引文
[1]马立军.京大戟、红大戟、草大戟的鉴别.基层中药杂志[J],2000.14(5).
[2]李同琴,郭秋红,田质芬.大戟科植物药用历史沿革及价值的探讨.中医药学刊[J],2003,21(5).
[3]中国科学院中国植物志编辑委员会.中国植物志第44卷第3分册[M].北京:科学出版社,1997.
[4]Metcalf C.R.,Chalk L. Anatomy of the D icotyledons[M].Oxford:Clarendon Press,1950.
[5]孟娜,周守标,蒋继宏.五种大戟属植物nrDNA的ITS序列分析及其叶的比较解剖学研究[J].广西植物,2006,26(1):18.
[6]帅彦平,贾忠建.我国大戟二萜酯及其生理活性研究新进展[J].高等学校化学学报,1997,18(7):1107-1112.
[7]潘勤,闵知大.甘遂中巨大戟萜醇型二萜酯类化学成分的研究[J].中草药, 2003,(6).
[8]石建功,贾忠建,杨立.甘青大戟二萜化合物研究[J].高等学校化学学报, 1994,(6).
[9]李玉林,索有瑞.藏药大果大戟中的巨大戟烷型二萜酯类成分[J].中草药, 2005,(12).
[10]潘莉.唐古特瑞香和大果大戟的化学成分研究[D].中国科学院研究生院(成都生物研究所),2006.
[11]王文祥,丁杏苞.月腺大戟中二萜化学成分的研究[J].药学学报, 1998,(2).
[12]张峻,杨成金,吴大刚.小狼毒的二萜化学成分研究(Ⅰ)[J].中草药, 1998,(2).
[13]李娟,阮汉利;张悦.湖北大戟根中的环阿尔廷型三萜[J].天然产物研究与开发,2007,06
[14]石心红,王宇行,孔令义.准噶尔大戟根中黄酮类成分的研究[J].中国药学杂志,2006,41(20):1538-1540.
[15]裴月湖,韩冰,冯宝民.狼毒大戟化学成分的研究[J].中草药, 2002,(7).
[16]张维库,杨国恩,李茜,等.对叶大戟化学成分的研究[J].中国中药杂志, 2006,(20).
[17]郭增军,佐建峰,卜筱茜.九牛造化学成分研究[J].中药材, 2007,(4).
[18]孔令义,闵知大.大戟根化学成分的研究[J].药学学报, 1996,(7).
[19]郑维发.甘遂醇提物中4种二萜类化合物的体内抗病毒活性研究[J].中草药,2004,35(1):65-68.
[20]王立岩.甘遂的化学成分及其生物活性的研究[D].沈阳药科大学2003级研究生论文集,2003.
[21]刘春安,彭明.抗癌中草药大辞典[M].武汉:湖北科学技术出版社,1994:169.
[22]蔡鹰,陆瑜,梁秉文.泽漆根体内抗肿瘤作用研究[J].中药材, 1999, 22(11): 579-581.
[23]曹仁烈.中药水浸剂在试管内抗皮肤真菌的观察[J].中华皮肤科杂志,1957(4):286.
[24]宋友文,谢珍,肯洪灿.脚癣狐臭酊剂抗皮肤病真菌及安全性研究[J].中国实验方剂学杂志,1997,3(2):13-15.
[25]陈志宝,赵岩,邓旭明.狼毒大戟的化学成分、药理作用及临床应用[J].黑龙江八一农垦大学学报,2001,13(3):52-57.
[26]尚溪瀛,文成英,刘丽波.大戟注射液对L615白血病小鼠体内药物实验及DNA含量的检测[J].中医药报,2000,2:76.
[27]Evans F J,Taylor S E. Pro-inflammatory,tumour-promotingand anti-tumour diterpenes of the plant families Euphorbiaceaeand Thymelaeaceae[J].Fortschr Chem Org Naturst,1983,44:1-99.
[28]左风,译.大戟提取物的抗炎作用[J].国外医学·中医中药分册,1998,20(3):39.
[29]陈希琛.甘遂引产成分的初步研究[J].药学通报,1982,17(6):43.
[30]南京神经精神病防治院.京大戟治疗精神病的初步研究[J].江苏医药,1977年第10期.
[31]周志明,徐长桂.龙虎草治疗肝硬变腹水的临床观察[J].中国医刊,1960-01-31
[32]王潇.十枣汤临床研究近况[J].中国中医急症,1999,8(4):182-184.
[33]虞觐冠.十枣汤临床运用体会[J].安徽中医学院学报,1998,17(1):29-30.
[34]孙颂三.巴豆霜对泻下和免疫功能的影响[J].中草药,1993,24(5):251.
[35]吴世德.狼毒抗癌作用研究[J].进展与综述,2000,29(10):30-31.
[36]杨王君,王世岭.千金子提取液对大鼠肺成纤维细胞增值的影响及细胞毒性作用[J].中国临床康复,2005,9(27):l01-103.
[37]王新,张宗友.巴豆提取物诱导小鼠小肠组织中蛋白质差异表达的初步研究[J].胃肠病学和肝病学杂志,2000,9(2):103.
[38]邓毅.甘草甘遂配伍对小鼠血清GPT、GOT、LDH影响的实验研究[J].中医研究,2007,20(3):15-16.
[39]孙志刚.口服狼毒蛋煎剂对大鼠慢性毒性实验研究[J].中医药学刊,2001, 19(6):620-621.
[40]向丽华.24味有毒中药长期毒性实验对大鼠脏器指数的影响[J].中国中医基础医学杂志,2006,12(1):35-371.
[41]黄文权.甘草、甘遂伍用对大鼠心肝肾功能及形态的影响[J].第二军医大学学报,2001,23(12):1439-1441.
[42]杨志军.甘遂与甘草配伍对小鼠肝脏组织中MDA、GSH2PX影响的实验研究[J].中医研究,2006,19(7):15-16.
[43]赵玫.致癌剂DEN、促癌剂巴豆油对大鼠肝α1抑制因子3基因表达的影响[J].生物化学杂志,l992,8(6):730.
[44]李啸红,李娟,张艳.巴豆对小鼠骨髓及胚胎肝细胞微核率的影响[J].中国优生与遗传杂志,2002,l0(3):43-47.
[45]漆淑华,吴大刚,罗晓东.桂林乌桕中的香豆素和鞣花酸类化合物[J].天然产物研究与开发,2004,16(4):297.
[46]浮光苗.月腺大戟化学成分的研究[J]中国药科大学学报, 2003,(04).
[47]潘勤.甘遂的化学成分研究[D].中国药科大学,2003.
[2]李同琴,郭秋红,田质芬.大戟科植物药用历史沿革及价值的探讨.中医药学刊[J],2003,21(5).
[3]中国科学院中国植物志编辑委员会.中国植物志第44卷第3分册[M].北京:科学出版社,1997.
[4]Metcalf C.R.,Chalk L. Anatomy of the D icotyledons[M].Oxford:Clarendon Press,1950.
[5]孟娜,周守标,蒋继宏.五种大戟属植物nrDNA的ITS序列分析及其叶的比较解剖学研究[J].广西植物,2006,26(1):18.
[6]帅彦平,贾忠建.我国大戟二萜酯及其生理活性研究新进展[J].高等学校化学学报,1997,18(7):1107-1112.
[7]潘勤,闵知大.甘遂中巨大戟萜醇型二萜酯类化学成分的研究[J].中草药, 2003,(6).
[8]石建功,贾忠建,杨立.甘青大戟二萜化合物研究[J].高等学校化学学报, 1994,(6).
[9]李玉林,索有瑞.藏药大果大戟中的巨大戟烷型二萜酯类成分[J].中草药, 2005,(12).
[10]潘莉.唐古特瑞香和大果大戟的化学成分研究[D].中国科学院研究生院(成都生物研究所),2006.
[11]王文祥,丁杏苞.月腺大戟中二萜化学成分的研究[J].药学学报, 1998,(2).
[12]张峻,杨成金,吴大刚.小狼毒的二萜化学成分研究(Ⅰ)[J].中草药, 1998,(2).
[13]李娟,阮汉利;张悦.湖北大戟根中的环阿尔廷型三萜[J].天然产物研究与开发,2007,06
[14]石心红,王宇行,孔令义.准噶尔大戟根中黄酮类成分的研究[J].中国药学杂志,2006,41(20):1538-1540.
[15]裴月湖,韩冰,冯宝民.狼毒大戟化学成分的研究[J].中草药, 2002,(7).
[16]张维库,杨国恩,李茜,等.对叶大戟化学成分的研究[J].中国中药杂志, 2006,(20).
[17]郭增军,佐建峰,卜筱茜.九牛造化学成分研究[J].中药材, 2007,(4).
[18]孔令义,闵知大.大戟根化学成分的研究[J].药学学报, 1996,(7).
[19]郑维发.甘遂醇提物中4种二萜类化合物的体内抗病毒活性研究[J].中草药,2004,35(1):65-68.
[20]王立岩.甘遂的化学成分及其生物活性的研究[D].沈阳药科大学2003级研究生论文集,2003.
[21]刘春安,彭明.抗癌中草药大辞典[M].武汉:湖北科学技术出版社,1994:169.
[22]蔡鹰,陆瑜,梁秉文.泽漆根体内抗肿瘤作用研究[J].中药材, 1999, 22(11): 579-581.
[23]曹仁烈.中药水浸剂在试管内抗皮肤真菌的观察[J].中华皮肤科杂志,1957(4):286.
[24]宋友文,谢珍,肯洪灿.脚癣狐臭酊剂抗皮肤病真菌及安全性研究[J].中国实验方剂学杂志,1997,3(2):13-15.
[25]陈志宝,赵岩,邓旭明.狼毒大戟的化学成分、药理作用及临床应用[J].黑龙江八一农垦大学学报,2001,13(3):52-57.
[26]尚溪瀛,文成英,刘丽波.大戟注射液对L615白血病小鼠体内药物实验及DNA含量的检测[J].中医药报,2000,2:76.
[27]Evans F J,Taylor S E. Pro-inflammatory,tumour-promotingand anti-tumour diterpenes of the plant families Euphorbiaceaeand Thymelaeaceae[J].Fortschr Chem Org Naturst,1983,44:1-99.
[28]左风,译.大戟提取物的抗炎作用[J].国外医学·中医中药分册,1998,20(3):39.
[29]陈希琛.甘遂引产成分的初步研究[J].药学通报,1982,17(6):43.
[30]南京神经精神病防治院.京大戟治疗精神病的初步研究[J].江苏医药,1977年第10期.
[31]周志明,徐长桂.龙虎草治疗肝硬变腹水的临床观察[J].中国医刊,1960-01-31
[32]王潇.十枣汤临床研究近况[J].中国中医急症,1999,8(4):182-184.
[33]虞觐冠.十枣汤临床运用体会[J].安徽中医学院学报,1998,17(1):29-30.
[34]孙颂三.巴豆霜对泻下和免疫功能的影响[J].中草药,1993,24(5):251.
[35]吴世德.狼毒抗癌作用研究[J].进展与综述,2000,29(10):30-31.
[36]杨王君,王世岭.千金子提取液对大鼠肺成纤维细胞增值的影响及细胞毒性作用[J].中国临床康复,2005,9(27):l01-103.
[37]王新,张宗友.巴豆提取物诱导小鼠小肠组织中蛋白质差异表达的初步研究[J].胃肠病学和肝病学杂志,2000,9(2):103.
[38]邓毅.甘草甘遂配伍对小鼠血清GPT、GOT、LDH影响的实验研究[J].中医研究,2007,20(3):15-16.
[39]孙志刚.口服狼毒蛋煎剂对大鼠慢性毒性实验研究[J].中医药学刊,2001, 19(6):620-621.
[40]向丽华.24味有毒中药长期毒性实验对大鼠脏器指数的影响[J].中国中医基础医学杂志,2006,12(1):35-371.
[41]黄文权.甘草、甘遂伍用对大鼠心肝肾功能及形态的影响[J].第二军医大学学报,2001,23(12):1439-1441.
[42]杨志军.甘遂与甘草配伍对小鼠肝脏组织中MDA、GSH2PX影响的实验研究[J].中医研究,2006,19(7):15-16.
[43]赵玫.致癌剂DEN、促癌剂巴豆油对大鼠肝α1抑制因子3基因表达的影响[J].生物化学杂志,l992,8(6):730.
[44]李啸红,李娟,张艳.巴豆对小鼠骨髓及胚胎肝细胞微核率的影响[J].中国优生与遗传杂志,2002,l0(3):43-47.
[45]漆淑华,吴大刚,罗晓东.桂林乌桕中的香豆素和鞣花酸类化合物[J].天然产物研究与开发,2004,16(4):297.
[46]浮光苗.月腺大戟化学成分的研究[J]中国药科大学学报, 2003,(04).
[47]潘勤.甘遂的化学成分研究[D].中国药科大学,2003.