手参块茎化学成分及2-异丁基苹果酸葡萄糖氧基苄酯定量分析方法研究
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摘要
手参(Gymnadenia conopsea(L)R．Br)为兰科手参属(Gymnadenia)珍稀药用植物。作为我国传统中药，手参具有较高的临床药用价值，但目前存在着药材质量不一、野生资源枯竭的现象。近年来对手参药用部位即块茎的化学成分研究报道较多，而对手参药材质量标准研究仅一篇文献报道即手参块茎指纹图谱研究。为了更好地实现对手参药用资源进行质量控制，以及为人工栽培、扩大药源等研究工作提供参考，我们对手参块茎进行了化学成分研究，并利用分离获得的已知的活性化合物为对照品进行了定量分析方法研究。
采用多种色谱技术进行分离纯化，从手参块茎的乙醇提取物中分离获得20个化合物，并运用理化性质和各种波谱技术鉴定了19个化合物，分别为：十六烷酸甘油三酯(1)、十八烷酸甘油三酯(2)、十八烷酸(3)、β-谷甾醇(4)、胡萝卜苷(5)、对-羟基苯甲醛(6)、丁二酸(7)、对-羟基苄基二硫醚(8)、4，4′-对羟基苄基亚砜(9)、腺嘌呤核苷(10)、coelovirinsB(11)、gymnadesideⅠ(12)、dactylorhin E(13)、militarine(14)、loroglossin(15)、dactylorhin B(16)、dactylorhin A(17)、coelovirins F(18)、coelovirins G(19)。其中1个为新的天然产物，8个为首次从本属植物分得。
在对该植物化学成分研究基础上，首次以5个2-异丁基苹果酸葡萄糖氧基苄酯(包括已知的4个活性化合物)为对照品，利用HPLC法进行了定量分析方法研究，从而为制定手参药材质量标准奠定了基础。并运用所建立的定量分析方法对5种不同产地的手参进行了比较，以及分析了其他7种兰科植物，对扩大手参药材资源及其化学成分研究提供了一定的参考价值。
以上述工作为基础，对手参块茎化学成分提取及分离方法，以及2-异丁基苹果酸葡萄糖氧基苄酯类化合物的波谱学规律等进行了探讨。此外，本文综述了手参属植物的化学成分、药理活性和临床应用的最新研究进展，以及2-异丁基苹果酸葡萄糖氧基苄酯类化合物在植物中的分布，这些均为本属植物的深入研究提供了参考。
Gymnadenia conopsea ( L ) R. Br ( family orchidaceae ) is a rare traditional Chinese medicinal herb with highly clinical values. There have been more studies on chemical constituents of the tubers of G. conopsea in recent years, but only one literature has been reported on the research of quality standard, which is chemical fingerprint analysis of the tubers of G. conopsea. In order to evaluate the quality of this medicinal plant, and provide references for interspecies identification and cultivation, a systematic photochemical study was carded out at first, and then a high performance liquid chromatogramphy (HPLC) method was developed for the quantitative determination of the known active compounds in the tubers of G. conopsea.
Twenty compounds were isolated from the ethanol extracts of the tubers of G. conopsea by various column chromatographies, nineteen of which were determined on the basis of physical properties and spectroscopic analysis as follows: hexadecanoic triglyceride (1), octadecylic triglyceride (2), stearic acid (3),β-sitosterol(4 ), daucosterol( 5 ), p-hydroxybenzaldehyde( 6 ), succinic acid( 7 ), di-(p-hydroxybenzyl) disulfide (8), 4,4′-dihydroxybenzyl sulfoxide (9), adenosine ( 10 ), coelovirins B ( 11 ), gynmadesideⅠ( 12), dactylorhin E ( 13), militarine ( 14), loroglossin ( 15), dactylorhin B ( 16), dactylorhin A ( 17), coelovirins F ( 18 ) and coelovirins G (19) . Compound 8 was obtained from nature products for the first time, eight compounds were obtained from the plants of Gymnadenia R.Br. for the first time.
Five glucosyloxybenzyl 2-isobutylmalates, four of which were the known active compounds, were isolated from the tubers of G. conopsea and selected as the marker compounds, and a HPLC method was firstly developed for simultaneous quantitative determination of these five compounds in the tubers of G. conopsea. The assay had been successfully applied to determinate the contents of these compounds in G. conopsea and other seven plants of orchidaceae collected from different regions of China.
Based on the above researches, several issues have been discussed covering the extraction and isolation methods of the chemical constituents of the tubers of G. conopsea, and the spectroscopic features of glucosyloxybenzyl 2-isobutylmalates, et al. Furthermore, this dissertation reviewed the advances on the study of chemical constituents, pharmacological activities and clinical applications of the plants of Gymnadenia R.Br., and summarized distribution of glucosyloxybenzyl 2-isobutylmalates in the plants of Orchidaceae. All these findings are helpful for further research on the plants of Gymnadenia R.Br.
采用多种色谱技术进行分离纯化，从手参块茎的乙醇提取物中分离获得20个化合物，并运用理化性质和各种波谱技术鉴定了19个化合物，分别为：十六烷酸甘油三酯(1)、十八烷酸甘油三酯(2)、十八烷酸(3)、β-谷甾醇(4)、胡萝卜苷(5)、对-羟基苯甲醛(6)、丁二酸(7)、对-羟基苄基二硫醚(8)、4，4′-对羟基苄基亚砜(9)、腺嘌呤核苷(10)、coelovirinsB(11)、gymnadesideⅠ(12)、dactylorhin E(13)、militarine(14)、loroglossin(15)、dactylorhin B(16)、dactylorhin A(17)、coelovirins F(18)、coelovirins G(19)。其中1个为新的天然产物，8个为首次从本属植物分得。
在对该植物化学成分研究基础上，首次以5个2-异丁基苹果酸葡萄糖氧基苄酯(包括已知的4个活性化合物)为对照品，利用HPLC法进行了定量分析方法研究，从而为制定手参药材质量标准奠定了基础。并运用所建立的定量分析方法对5种不同产地的手参进行了比较，以及分析了其他7种兰科植物，对扩大手参药材资源及其化学成分研究提供了一定的参考价值。
以上述工作为基础，对手参块茎化学成分提取及分离方法，以及2-异丁基苹果酸葡萄糖氧基苄酯类化合物的波谱学规律等进行了探讨。此外，本文综述了手参属植物的化学成分、药理活性和临床应用的最新研究进展，以及2-异丁基苹果酸葡萄糖氧基苄酯类化合物在植物中的分布，这些均为本属植物的深入研究提供了参考。
Gymnadenia conopsea ( L ) R. Br ( family orchidaceae ) is a rare traditional Chinese medicinal herb with highly clinical values. There have been more studies on chemical constituents of the tubers of G. conopsea in recent years, but only one literature has been reported on the research of quality standard, which is chemical fingerprint analysis of the tubers of G. conopsea. In order to evaluate the quality of this medicinal plant, and provide references for interspecies identification and cultivation, a systematic photochemical study was carded out at first, and then a high performance liquid chromatogramphy (HPLC) method was developed for the quantitative determination of the known active compounds in the tubers of G. conopsea.
Twenty compounds were isolated from the ethanol extracts of the tubers of G. conopsea by various column chromatographies, nineteen of which were determined on the basis of physical properties and spectroscopic analysis as follows: hexadecanoic triglyceride (1), octadecylic triglyceride (2), stearic acid (3),β-sitosterol(4 ), daucosterol( 5 ), p-hydroxybenzaldehyde( 6 ), succinic acid( 7 ), di-(p-hydroxybenzyl) disulfide (8), 4,4′-dihydroxybenzyl sulfoxide (9), adenosine ( 10 ), coelovirins B ( 11 ), gynmadesideⅠ( 12), dactylorhin E ( 13), militarine ( 14), loroglossin ( 15), dactylorhin B ( 16), dactylorhin A ( 17), coelovirins F ( 18 ) and coelovirins G (19) . Compound 8 was obtained from nature products for the first time, eight compounds were obtained from the plants of Gymnadenia R.Br. for the first time.
Five glucosyloxybenzyl 2-isobutylmalates, four of which were the known active compounds, were isolated from the tubers of G. conopsea and selected as the marker compounds, and a HPLC method was firstly developed for simultaneous quantitative determination of these five compounds in the tubers of G. conopsea. The assay had been successfully applied to determinate the contents of these compounds in G. conopsea and other seven plants of orchidaceae collected from different regions of China.
Based on the above researches, several issues have been discussed covering the extraction and isolation methods of the chemical constituents of the tubers of G. conopsea, and the spectroscopic features of glucosyloxybenzyl 2-isobutylmalates, et al. Furthermore, this dissertation reviewed the advances on the study of chemical constituents, pharmacological activities and clinical applications of the plants of Gymnadenia R.Br., and summarized distribution of glucosyloxybenzyl 2-isobutylmalates in the plants of Orchidaceae. All these findings are helpful for further research on the plants of Gymnadenia R.Br.
引文
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[11] Gustafsson S, Joegren G P. Genetic diversity in the rare orchid, Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea [J]. Conservation Genetics, 2002, 3 (3): 225-234.
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[1] Morikawa T, Xie H H, Matsuda H, et al. Glucosyloxybenzyl 2-isobutylmalates from the tubers of Gymnadenia conopsea [J]. Journal of Natural Products, 2006, 69 (6): 881-886.
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[1] 刘美凤，韩芸，邢东明，等．竹叶兰化学成分研究[J]．中国中药杂志，2004，29(2)：147-149．
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[10] Huang S Y, Li G Q, Shi J G, et al. Chemical constituents of the rhizomes of Coeloglossum viride var. bracteatum [J]. Journal of Asian Natural Products Research, 2004, 6 (1): 49-61.
[11] Morikawa T, Xie H H, Matsuda H, et al. Glucosyloxybenzyl 2-isobutylmalates from the tubers of Gymnadenia conopsea [J]. Journal of Natural Products, 2006, 69 (6): 881-886.
[12] Kizu H, Kaneko E, Tomimori T. Studies on nepalese crude drugs. ⅩⅩⅥ. chemical constituents ofpaneh aunle, the roots ofDactylorhiza hatagirea D. Don [J]. Chem Pharm Bull, 1999, 47(11): 1618- 1625.
[1] Cai M, Zhou Y, Gesang S L et al. Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC-DAD-MS [J]. Journal of Chromatography B, 2006, 844: 301-307.
[1] Morikawa T, Xie H H, Matsuda H, et al. Glucosyloxybenzyl 2-isobutylmalates from the tubers of Gymnadenia conopsea [J]. Journal of Natural Products, 2006, 69 (6): 881-886.
[2] Huang S Y, Li G Q, Shi J G, et al. Chemical constituents of the rhizomes of Coeloglossum viride var. bracteatum [J]. Journal of Asian Natural Products Research, 2004, 6 (1): 49-61.
[3] Matsuda H, Morikawa T, Xie H H, et al. Antiallergic phenathrenes and stilbene from the tubers of Gymnadenia conopsea [J]. Planta Medica, 2004, 70 (9): 847-855.
[4] Hye S Y, Mi K P. Isolation of 4,4′-dihydroxybenzyl sulfoxide from Gastrodia elata [J].Arch Pharm Res, 1997,20(1):91-92.
[5] Cai M, Zhou Y, Gesang S L et al. Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC- DAD-MS [J]. Journal of Chromatography B, 2006, 844: 301-307.
[6] Delauney P. New researches relating to the presence of loroglossin in indigenous orchids [J] Compt. Rend, 1923, 176: 598-600.
[2] 李帅，王栋，匡海学等．手掌参的化学成分研究[J]．中草药，2001，32(1)：18．
[3] 扬畅杰，徐明，于风琴．朝鲜族药爱给荪的鉴别与独特应用[J]．中国民族民间医药杂志，1999，39：217-218．
[4] 雒成林，刘峰林，金建平．藏医与中医对手参的应用探讨[J]．甘肃中医学院学报，2001，18(4)：43-44．
[5] Van Waes J, Debergh P. In vitro germination of some Westem European orchids [J]. Physiol Plant, 1986, 67 (2): 253-61.
[6] 金洪，王俊杰，张众．手掌参的组织培养研究[J]．内蒙古农牧学院学报，1995, 16(2): 74-77.
[7] Uphoff W. Identification of European orchids by determination of the anthocyanin concentration during development of the blossoms[J]. Experientia, 1982, 38 (7): 778-80.
[8] 谢静，张浩，俞森，等．藏药手参原植物的生药鉴定[J]．华西药学杂志，2005，20(3)：197-199．
[9] Gustafsson S, Thoren P. Microsatellite loci in Gymnadenia conopsea, the fragrant orchid[J]. Molecular Ecology Notes, 2001, 1 (1/2): 81-82.
[10] Campbell V, Rowe G, Beebee T, et al. Solation and characterization ofmicrosatellite primers for the fragrant orchid Gymnadenia conopsea (L.) R.Brown.(Orchidaceae) [J]. Molecular Conservation Genetics, 2002, 3 (2): 209-210.
[11] Gustafsson S, Joegren G P. Genetic diversity in the rare orchid, Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea [J]. Conservation Genetics, 2002, 3 (3): 225-234.
[12] Gustafsson S, Loenn M. Genetic differentiation and habitat preference of flowering-time variants within Gymnadenia conopsea[J]. Heredity, 2003, 91 (3), 84-292.
[13] Matsuda H, Morikawa T, Xie H H, et al. Antiallergic phenathrenes and stilbene from the tubers of Gymnadenia conopsea [J]. Planta Medica, 2004, 70 (9): 847-855.
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[1] Cai M, Zhou Y, Gesang S L et al. Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC-DAD-MS [J]. Journal of Chromatography B, 2006, 844: 301-307.
[1] Morikawa T, Xie H H, Matsuda H, et al. Glucosyloxybenzyl 2-isobutylmalates from the tubers of Gymnadenia conopsea [J]. Journal of Natural Products, 2006, 69 (6): 881-886.
[2] Huang S Y, Li G Q, Shi J G, et al. Chemical constituents of the rhizomes of Coeloglossum viride var. bracteatum [J]. Journal of Asian Natural Products Research, 2004, 6 (1): 49-61.
[3] Matsuda H, Morikawa T, Xie H H, et al. Antiallergic phenathrenes and stilbene from the tubers of Gymnadenia conopsea [J]. Planta Medica, 2004, 70 (9): 847-855.
[4] Hye S Y, Mi K P. Isolation of 4,4′-dihydroxybenzyl sulfoxide from Gastrodia elata [J].Arch Pharm Res, 1997,20(1):91-92.
[5] Cai M, Zhou Y, Gesang S L et al. Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC- DAD-MS [J]. Journal of Chromatography B, 2006, 844: 301-307.
[6] Delauney P. New researches relating to the presence of loroglossin in indigenous orchids [J] Compt. Rend, 1923, 176: 598-600.