月腺大戟化学成分的研究
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摘要
月腺大戟(Euphorbia ebracteolata Hayata),又名狼毒,为大戟科大戟属植物,以根入药。月腺大戟始载于《神农本草经》,其味辛、性平,有大毒,具有逐水去痰和破积杀虫的功效。可治水肿腹胀、淋巴结核、慢性气管炎等症。临床多用于癌症和结核病的治疗。
本实验针对月腺大戟的化学成分及其主要成分的含量测定做了较为系统的研究。
本实验采用85%乙醇回流提取,柱层析分离等手段从月腺大戟根中分离得到了7种单体,经理化性质及波谱数据的分析鉴定了化学结构。它们分别是β-香树脂醇乙酸酯(β-amyrin acetate ), 24-亚甲基环阿尔廷醇( 24-methylene cycloartanol),3, 3′-二乙酰基-4,4′-二甲氧基-2,2′,6,6′-四羟基二苯甲烷(3,3′-diacetyl-4,4′-dimethoxy-2,2′,6,6′-tetrahydroxydiphenylmethane),岩大戟内酯B(jolkinolide B),狼毒乙素(2,4-dihydroxy-6-methoxy-3-methy-1-acetoph- enone),月腺大戟甲素(ebractedatanolide A)和β-谷甾醇(β-sitosterol)。
本实验还建立了月腺大戟中两种三萜类成分β-香树脂醇乙酸酯和24-亚甲基环阿尔廷醇的HPLC含量测定方法。方法学实验证明,该含量测定方法具有便捷、准确、灵敏、选择性高等特点。
本研究结果为月腺大戟质量标准的建立及月腺大戟的开发利用提供了新的科学依据。
Euphorbia ebracteolata Hayata, as one of the original plant of traditional Chinese medicine Langdu, is mainly distributed in Anhui, Shandong, Henan, Hubei, and Jiangsu provinces etc. in China. The plant is used for the treatment insecticide, abdominal distension, lymphnodes and chronic bronchitis etc.. Also, it can be used to treat of pulmonary ruberculosis and chronic tracheitis in clinical.
According to the literature, tannins, flavonoids, hypnones, terpenoids, sterols and other biological active compounds were found in the Euphorbia ebracteolata Hayata. In order to find a kind of triterpenoids chemical constituent as a representational compound with a high content in Euphorbia ebracteolata Hayata, we studied chemical constituent in the root of Euphorbia ebracteolata Hayata and determined the content of the bioactivity compound in it.
The extraction and isolation were accomplished with heat reflux and column chromatographical method, seven compounds were obtained and identified by IR, 1H NMR and 13C NMR spectra analysis asβ-amyrin acetate,24-methylene cycl- oanol,3,3′-diacetyl-4,4′-dimethoxy-2,2′,6,6′-tetrahydrxydiphenylmethane, jolkinolide B, 2,4-dihydroxy-6-methoxy-3-methy-1-acetophenone, ebractedatanolide A andβ-sitosterol.
Among the seven compounds mentioned above,β-amyrin acetate and 24- methylene cycloartanol, not only are in relative high content, but also have important bioactivity. Therefore, the method of quantitative analysis has been established for determining the contents of the two compounds in Euphorbia ebracteolata Hayata with HPLC.A series of methodical experiments proved that this method has the advantages of accuracy, precision, sensitivity, convenience etc. Extraction and Separation
Dried leaves (3kg) were extracted by heat reflux in ethanol (85%) (30L, 24L, 18L respectively) for three times (2h, 1.5h, 1h respectively). The extraction was obtained after ethanol had been evaporated.
The extraction was subjected to silica gel column chromatography using petroleum etherethyl acetate as mobile phase. Seven partitions were obtained as A, B, C, D , E ,F, G and H parts.
E, F, G and H were subjected to silica gel and ODS colum chromatography repeatedly. Monomer compounds Y2 (200mg), Y3 (90mg), Y4 (120mg), Y5 (90mg), Y6(20mg) and Y7(50mg) were obtained. The part B was recrystallized by petroleum etherethyl acetate to gain monomer compound Y1 (120mg).
Compounds Y1, Y2, Y3, Y4, Y5, Y6 and Y7 were identified by IR, 1H NMR, 13C NMR etc. asβ-amyrin acetate,24-methylene cycloartanol,3,3′-diacetyl-4,4′- dimethoxy-2,2′,6,6′-tetrahydroxydiphenylmethane, jolkinolide B, 2,4-dihyd- roxy-6-methoxy-3-methy-1-acetophenone, ebractedatanolide A andβ-sitosterol resp- ectively.
HPLC determination of triterpenoids (Y1 and Y2)
In this paper we presented a more sensitive quantification method by HPLC for determining the main triterpenoids.
HPLC was performed on a Agilent 1100 HPLC instrument with a Agilent HC-C8 column (150 mm×4.6 mm,5μm) with the mixture of CH3CN-H2O(85:15) solution as mobile phase and the flow rate is 1.0mL·min-1 at 25℃. Results
ⅰThe linear range ofβ-amyrin acetate was 0.1μg～5.0μg. Y=7.511×103X+10.00 r=0.9998 The linear range of 24-methylene cycloartanol was 0.1μg～5.0μg. Y=13.403×103X+21.81 r=0.9999.
ⅱThe solution of compound Y1 and Y2 were injected into HPLC for six times, the result of precision tests was RSD<1%.
ⅲThe reprodurity tests for six samples showed RSD<2%(n=6).
ⅳWhen the concentration of compound Y1 and Y2 solution was descreased to 0.0005 mg·mL-1 and determined by HPLC, S/N≥3, so the detectabilities were 1.0ng.
ⅴThe solution of compound Y1 and Y2 were stable in tweleve hours because of it’s RSD<1%.
ⅵThe average recoveries were 99.27%(compound Y1) and 96.92% (compound Y2) respectively.
ⅶThe samples of Euphorbia ebracteolata Hayata have been determined fromdifferent place, the results showed that the average contens of compound Y1 and Y2 were 0.049% and 0.173%(from Anguo in Hebei), 0.062% and 0.266%(from southward), 0.000% and 0.138%(from northward).
The results of this paper can offer a new index of content determination for establishment of quality standard of Euphorbia ebracteolata Hayata, also, can offer science foundation for the development of new traditional medicine from this plant.
本实验针对月腺大戟的化学成分及其主要成分的含量测定做了较为系统的研究。
本实验采用85%乙醇回流提取,柱层析分离等手段从月腺大戟根中分离得到了7种单体,经理化性质及波谱数据的分析鉴定了化学结构。它们分别是β-香树脂醇乙酸酯(β-amyrin acetate ), 24-亚甲基环阿尔廷醇( 24-methylene cycloartanol),3, 3′-二乙酰基-4,4′-二甲氧基-2,2′,6,6′-四羟基二苯甲烷(3,3′-diacetyl-4,4′-dimethoxy-2,2′,6,6′-tetrahydroxydiphenylmethane),岩大戟内酯B(jolkinolide B),狼毒乙素(2,4-dihydroxy-6-methoxy-3-methy-1-acetoph- enone),月腺大戟甲素(ebractedatanolide A)和β-谷甾醇(β-sitosterol)。
本实验还建立了月腺大戟中两种三萜类成分β-香树脂醇乙酸酯和24-亚甲基环阿尔廷醇的HPLC含量测定方法。方法学实验证明,该含量测定方法具有便捷、准确、灵敏、选择性高等特点。
本研究结果为月腺大戟质量标准的建立及月腺大戟的开发利用提供了新的科学依据。
Euphorbia ebracteolata Hayata, as one of the original plant of traditional Chinese medicine Langdu, is mainly distributed in Anhui, Shandong, Henan, Hubei, and Jiangsu provinces etc. in China. The plant is used for the treatment insecticide, abdominal distension, lymphnodes and chronic bronchitis etc.. Also, it can be used to treat of pulmonary ruberculosis and chronic tracheitis in clinical.
According to the literature, tannins, flavonoids, hypnones, terpenoids, sterols and other biological active compounds were found in the Euphorbia ebracteolata Hayata. In order to find a kind of triterpenoids chemical constituent as a representational compound with a high content in Euphorbia ebracteolata Hayata, we studied chemical constituent in the root of Euphorbia ebracteolata Hayata and determined the content of the bioactivity compound in it.
The extraction and isolation were accomplished with heat reflux and column chromatographical method, seven compounds were obtained and identified by IR, 1H NMR and 13C NMR spectra analysis asβ-amyrin acetate,24-methylene cycl- oanol,3,3′-diacetyl-4,4′-dimethoxy-2,2′,6,6′-tetrahydrxydiphenylmethane, jolkinolide B, 2,4-dihydroxy-6-methoxy-3-methy-1-acetophenone, ebractedatanolide A andβ-sitosterol.
Among the seven compounds mentioned above,β-amyrin acetate and 24- methylene cycloartanol, not only are in relative high content, but also have important bioactivity. Therefore, the method of quantitative analysis has been established for determining the contents of the two compounds in Euphorbia ebracteolata Hayata with HPLC.A series of methodical experiments proved that this method has the advantages of accuracy, precision, sensitivity, convenience etc. Extraction and Separation
Dried leaves (3kg) were extracted by heat reflux in ethanol (85%) (30L, 24L, 18L respectively) for three times (2h, 1.5h, 1h respectively). The extraction was obtained after ethanol had been evaporated.
The extraction was subjected to silica gel column chromatography using petroleum etherethyl acetate as mobile phase. Seven partitions were obtained as A, B, C, D , E ,F, G and H parts.
E, F, G and H were subjected to silica gel and ODS colum chromatography repeatedly. Monomer compounds Y2 (200mg), Y3 (90mg), Y4 (120mg), Y5 (90mg), Y6(20mg) and Y7(50mg) were obtained. The part B was recrystallized by petroleum etherethyl acetate to gain monomer compound Y1 (120mg).
Compounds Y1, Y2, Y3, Y4, Y5, Y6 and Y7 were identified by IR, 1H NMR, 13C NMR etc. asβ-amyrin acetate,24-methylene cycloartanol,3,3′-diacetyl-4,4′- dimethoxy-2,2′,6,6′-tetrahydroxydiphenylmethane, jolkinolide B, 2,4-dihyd- roxy-6-methoxy-3-methy-1-acetophenone, ebractedatanolide A andβ-sitosterol resp- ectively.
HPLC determination of triterpenoids (Y1 and Y2)
In this paper we presented a more sensitive quantification method by HPLC for determining the main triterpenoids.
HPLC was performed on a Agilent 1100 HPLC instrument with a Agilent HC-C8 column (150 mm×4.6 mm,5μm) with the mixture of CH3CN-H2O(85:15) solution as mobile phase and the flow rate is 1.0mL·min-1 at 25℃. Results
ⅰThe linear range ofβ-amyrin acetate was 0.1μg～5.0μg. Y=7.511×103X+10.00 r=0.9998 The linear range of 24-methylene cycloartanol was 0.1μg～5.0μg. Y=13.403×103X+21.81 r=0.9999.
ⅱThe solution of compound Y1 and Y2 were injected into HPLC for six times, the result of precision tests was RSD<1%.
ⅲThe reprodurity tests for six samples showed RSD<2%(n=6).
ⅳWhen the concentration of compound Y1 and Y2 solution was descreased to 0.0005 mg·mL-1 and determined by HPLC, S/N≥3, so the detectabilities were 1.0ng.
ⅴThe solution of compound Y1 and Y2 were stable in tweleve hours because of it’s RSD<1%.
ⅵThe average recoveries were 99.27%(compound Y1) and 96.92% (compound Y2) respectively.
ⅶThe samples of Euphorbia ebracteolata Hayata have been determined fromdifferent place, the results showed that the average contens of compound Y1 and Y2 were 0.049% and 0.173%(from Anguo in Hebei), 0.062% and 0.266%(from southward), 0.000% and 0.138%(from northward).
The results of this paper can offer a new index of content determination for establishment of quality standard of Euphorbia ebracteolata Hayata, also, can offer science foundation for the development of new traditional medicine from this plant.
引文
[1]江苏新医学院.中药大词典:下册[M].上海:上海人民出版社,1977.
[2]赵奎君.狼毒类生药原植物调查及分类鉴定研究[J].中国中药杂志,1995,20(1):5.
[3] Ahan,Beung Tae.Pharmacognostic study on Euphorbia ebracteolata.(II).On the chemical study of the tannins and related compounds[J]. Saengyak Hakhoechi,1992,23(4):211.
[4] Ahan,Beung Tae. Phenolic compounds from Euphorbia ebracteolata.[J]. Saengyak Hakhoechi,1996,27(2):136.
[5] Lee,Sang Cheol. Pharmacognostic study on Euphorbia ebracteolata.(I). Flavonoid constituents[J].Saengyak Hakhoechi,1992,23 (3):126.
[6]张涵庆.月腺大戟根中有效成分的研究[J].植物学报,1987,29(4):429.
[7]张涵庆.月腺大戟根中有效成分乙素和丙素的结构研究[J].植物资源与环境,1992,1(3):6.
[8]王文祥.月腺大戟根中的乙酰间苯三酚类衍生物[J].药学学报,1999,34 (7):514.
[9] Itokawa Hideji. Constituents of Euphorbia ebracteolata[J]. Chemical and Pharmaceutical Bulletin,1970,18(6):1276.
[10]王文祥.月腺大戟中二萜化学成分的研究[J].药学学报,1998,33(2):12.
[11] Xu Zhihong. Casbane diterpenoids from Euphorbia ebracteolata. Chemical studies of Lang Du,a traditional Chinese medicine[J]. Phytochemistry,1998,49 (1):149.
[12] Xu Zhihong. An isopimarane diterpene from Euphorbia ebracteolata Hayata[J]. Journal of Asian Natural Products Research,2000,2(4):257.
[13]董云发.月腺大戟中乙酰间苯三酚衍生物[J].植物资源与环境,1992,1(2):1.
[14]王文祥.月腺大戟的化学成分研究[J].中草药,1999,30(1)1:1.
[15]孙晓飞.月腺大戟化学成分研究[J].中国中药杂志,1999,24(4):226.
[16]江苏新医学院.中药大词典:下册[M].上海:上海人民出版社,1977.
[17]曹春林.中药制剂汇编[M].北京:人民出版社,1983.
[18]刘寿山.中药研究文献摘要[M].北京:科学出版杜,1959.
[19]陈学文,姬志勤,方丽萍,等.月腺大戟抑菌活性成分的研究[J].江苏农业科学,2006,3:84-85.
[20] Liu W K. Biochemical Pharmacology,2002,63(5):951.
[21]杜娟,徐瑞军,崔日希,等.月腺大戟水提物诱导P388白血病细胞的凋亡[J].中国医院药学杂志,2007,27 (4):454-458.
[22]刘玉玺,孙晓飞,王明正,等.狼毒大戟碱性提取液的抗惊厥作用[J].中国中西医结合杂志,1994,14(5):282.
[23]郭晓庄主编.有毒中草药大辞典[M].天津:天津科技翻译出版社,1992,第一版:446-451.
[24]杨宝印,杨正裕,高国栋,等.瑞香狼毒对小鼠移植肿瘤的抑制作用[J].中药通报, 1986,11(1):58-59.
[25]杨正裕,徐培生,杨宝印,等.瑞香狼毒对小鼠移植性Lewis肺癌生长的抑制作用[J].中药通报, 1988,13(4):245.
[26]单颖,张越,傅德才.瑞香狼毒制剂成分分析[J].中国中药杂志, 1991,16(4):228.
[27] Feng WJ. Studies on antitumor active compounds of stellera chamaejasme L. and their mechanism of action[J]. Wakan Iyaku Gakkaishi, 1991,8(2):96.
[28]魏春雁.瑞香狼毒化学成分与生物活性研究的回顾与展望[J].中国药学杂志, 2001,36(9):577-580.
[29]中医辞典编辑委员会.简明中医辞典[M].北京:人民卫生出版社,1982: 253.
[30]董曼军,陈俊生,徐依民,等.狼毒注射液治疗银屑病的临床研究[J].中华皮肤科杂志,1992,25(1):58.
[31]徐晨耕,谈光新,施旭东,等.月腺大戟治疗传染性肺结核的临床研究[J].中国防痨杂志,2001,23 (增刊):226-227.
[32]乔春峰,韩金斌,贺震旦,等.RP-HPLC法测定狼毒中岩大戟内酯A和B的含量[J].药物分析杂志,2006,26(9): 1204-1206.
[33]赵奎君,徐国钧,金蓉鸾,等.HPLC法测定月腺大戟根中狼毒甲素及狼毒乙素的含量[J].中草药.1995,26(2):66-67.
[34]浮光苗,余伯阳,朱丹妮.月腺大戟化学成分的研究[J].中国药科大学学报,2003,34(4):377-379.
[35]孙晓飞,王淑萍,郑泽荣.月腺大戟化学成分研究[J].中国中药杂志,1999,24(4):226-227.
[36]董云发,丁云梅.月腺大戟根中乙酰基间苯三酚衍生物[J].植物资源与环境,1992,1(2):1-3.
[37]王文祥,丁杏苞.月腺大戟根中的乙酰间苯三酚类衍生物[J].药学学报,1999,34(7):514-517.
[38]刘桂芳,付玉琴,杨志强,等.狼毒大戟抗癌活性成分二萜内酯的分离鉴定[J].中药通报,1988,13(5):35-36.
[39]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,1998,33(2):128-131.
[2]赵奎君.狼毒类生药原植物调查及分类鉴定研究[J].中国中药杂志,1995,20(1):5.
[3] Ahan,Beung Tae.Pharmacognostic study on Euphorbia ebracteolata.(II).On the chemical study of the tannins and related compounds[J]. Saengyak Hakhoechi,1992,23(4):211.
[4] Ahan,Beung Tae. Phenolic compounds from Euphorbia ebracteolata.[J]. Saengyak Hakhoechi,1996,27(2):136.
[5] Lee,Sang Cheol. Pharmacognostic study on Euphorbia ebracteolata.(I). Flavonoid constituents[J].Saengyak Hakhoechi,1992,23 (3):126.
[6]张涵庆.月腺大戟根中有效成分的研究[J].植物学报,1987,29(4):429.
[7]张涵庆.月腺大戟根中有效成分乙素和丙素的结构研究[J].植物资源与环境,1992,1(3):6.
[8]王文祥.月腺大戟根中的乙酰间苯三酚类衍生物[J].药学学报,1999,34 (7):514.
[9] Itokawa Hideji. Constituents of Euphorbia ebracteolata[J]. Chemical and Pharmaceutical Bulletin,1970,18(6):1276.
[10]王文祥.月腺大戟中二萜化学成分的研究[J].药学学报,1998,33(2):12.
[11] Xu Zhihong. Casbane diterpenoids from Euphorbia ebracteolata. Chemical studies of Lang Du,a traditional Chinese medicine[J]. Phytochemistry,1998,49 (1):149.
[12] Xu Zhihong. An isopimarane diterpene from Euphorbia ebracteolata Hayata[J]. Journal of Asian Natural Products Research,2000,2(4):257.
[13]董云发.月腺大戟中乙酰间苯三酚衍生物[J].植物资源与环境,1992,1(2):1.
[14]王文祥.月腺大戟的化学成分研究[J].中草药,1999,30(1)1:1.
[15]孙晓飞.月腺大戟化学成分研究[J].中国中药杂志,1999,24(4):226.
[16]江苏新医学院.中药大词典:下册[M].上海:上海人民出版社,1977.
[17]曹春林.中药制剂汇编[M].北京:人民出版社,1983.
[18]刘寿山.中药研究文献摘要[M].北京:科学出版杜,1959.
[19]陈学文,姬志勤,方丽萍,等.月腺大戟抑菌活性成分的研究[J].江苏农业科学,2006,3:84-85.
[20] Liu W K. Biochemical Pharmacology,2002,63(5):951.
[21]杜娟,徐瑞军,崔日希,等.月腺大戟水提物诱导P388白血病细胞的凋亡[J].中国医院药学杂志,2007,27 (4):454-458.
[22]刘玉玺,孙晓飞,王明正,等.狼毒大戟碱性提取液的抗惊厥作用[J].中国中西医结合杂志,1994,14(5):282.
[23]郭晓庄主编.有毒中草药大辞典[M].天津:天津科技翻译出版社,1992,第一版:446-451.
[24]杨宝印,杨正裕,高国栋,等.瑞香狼毒对小鼠移植肿瘤的抑制作用[J].中药通报, 1986,11(1):58-59.
[25]杨正裕,徐培生,杨宝印,等.瑞香狼毒对小鼠移植性Lewis肺癌生长的抑制作用[J].中药通报, 1988,13(4):245.
[26]单颖,张越,傅德才.瑞香狼毒制剂成分分析[J].中国中药杂志, 1991,16(4):228.
[27] Feng WJ. Studies on antitumor active compounds of stellera chamaejasme L. and their mechanism of action[J]. Wakan Iyaku Gakkaishi, 1991,8(2):96.
[28]魏春雁.瑞香狼毒化学成分与生物活性研究的回顾与展望[J].中国药学杂志, 2001,36(9):577-580.
[29]中医辞典编辑委员会.简明中医辞典[M].北京:人民卫生出版社,1982: 253.
[30]董曼军,陈俊生,徐依民,等.狼毒注射液治疗银屑病的临床研究[J].中华皮肤科杂志,1992,25(1):58.
[31]徐晨耕,谈光新,施旭东,等.月腺大戟治疗传染性肺结核的临床研究[J].中国防痨杂志,2001,23 (增刊):226-227.
[32]乔春峰,韩金斌,贺震旦,等.RP-HPLC法测定狼毒中岩大戟内酯A和B的含量[J].药物分析杂志,2006,26(9): 1204-1206.
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[34]浮光苗,余伯阳,朱丹妮.月腺大戟化学成分的研究[J].中国药科大学学报,2003,34(4):377-379.
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