金钗石斛的化学成分与生物活性研究

【作者】 张雪；

【导师】 姚新生； 王乃利；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2005， 博士

【摘要】 金钗石斛（Dendrobium nobile Lindl．）为兰科石斛属多年生附生草本植物，其入药始载于《神农本草经》，被列为上品，具有清热养阴、生津益胃、润肺止咳等功效，是药用范围较广的名贵中药，享有“仙草”之美誉。本文综合运用硅胶柱层析、ODS柱层析、Sephadex LH-20柱层析以及反相HPLC等多种色谱学分离手段，对金钗石斛60％乙醇提取物的乙酸乙酯萃取物的化学成分进行了系统研究，从中分离得到化合物53个。通过理化性质和现代波谱学手段(UV、IR、MS、¹H-NMR、¹³C-NMR和2D-NMR)鉴定了它们的结构，分别为：dendronobilin A（1）^*，dendronobilin B（2）^*，dendronobilin C（3）^*，dendronobilin D（4）^*，dendronobilin E（5）^*，dendronobilin F（6）^*，dendronobilin G（7）^*，dendronobilin H（8）^*，dendronobilin I（9）^*，10β，13，14-trihydroxyalloaromadendrane （10），10，12-dihydroxypicrotoxane （11），6α，10，12-trihydroxypicrotoxane （12），dendrobiumane A（13），bullatantirol （14），dendrodensiflorol （15），dendrobane A（16），nobilin A（17）^*，nobilin B（18）^*，nobilin C（19）^*，nobilin D（20）^*，nobilin E（21）^*，crepidatin（22），鼓槌联苄（chrysotobibenzyl）（23），dendrobin A（24），鼓槌石斛素（chrysotoxine）（25），石斛酚（moscatilin）（26），gigantol（27），流苏菲（fimbriatone）（28），毛兰菲（confusarin）（29），flavanthrinin （30），2，5-二羟基-4，9-二甲氧基菲（2，5-dihydroxy-4，9-dimethoxyphenanthrene）（31），3，7-二羟基-2，4-二甲氧基菲（3，7-dihydroxy-2，4-dimethoxyphenanthrene）（32），nobilone （33）^*，dendroflorin （34），丁香脂素（syringaresinol）（35），松脂素（pinoresinol）（36），5’-甲氧基-（-）-松脂素（medioresinol）（37），lirioresinol-A（38），2-甲基蒽醌（2-methylanthraquinone）（39），nobilate（40）^*，丁香酸（syringicacid）（41），2-羟基苯丙醇（2-hydroxyphenylpropanol）（42），香草醛（vanillin）（43），罗布麻宁（apocynin）（44），松柏醛（coniferyl aldehyde）（45），丁香醛（syringaldehyde）（46），丁香乙酮（syringylethanone）（47），对羟基苯甲醛（p-hydroxybenzaldehyde）（48），3-羟基-4-甲氧基苯乙醇（3-hydroxy-4-methoxyphenylethanol）（49），α-羟基丁香丙酮（α-hydroxysyringylethanone）（50），二氢松柏醇（dihydroxyconiferyl alcohol）（51），对羟基苯甲酸（p-hydroxybenzoic acid）（52），对羟基苯丙酸（p-hydroxyphenylpropionic acid）（53）。53个化合物中包括倍半萜类化合物16个，联苄类化合物11个，菲类化合物5个，芴酮类化合物2个，木脂素类化合物4个，蒽醌类化合物1个，酚酸类化合物14个。其中化合物1-9，17-21，33，40为新化合物，化合物14，30-31，38-39，41-42，44-53为首次从该属植物中分离得到，化合物13，15，22，23，25，28，29，32，34-37，43为首次从该种植物中分离得到。采用DPPH自由基清除法（DPPH法）对金钗石斛中分离得到的各单体化合物清除自由基的能力进行了考察。结果表明，金钗石斛中分离得到的53个化合物中，有24个化合物显示不同程度的清除DPPH自由基的活性。菲类、联苄类和木脂素类化合物多显示很好的活性，芴酮类和酚酸类化合物中部分结构活性较好。其中绝大部分化合物的活性都强于目前食品工业中常用的合成抗氧化剂BHT，有9个化合物显示出比Vc更强或相当的活性。同时采用氧自由基清除能力法（ORAC法）对金钗石斛中分离得到的绝大部分单体化合物清除自由基的能力进行了考察。结果表明，进行ORAC活性测试的49个化合物中，有30个化合物显示不同程度的氧自由基清除活性，其中绝大部分的活性都强于V_C。菲类、联苄类、木脂素类和芴酮类化合物普遍显示较好的活性，酚酸类化合物中部分结构活性较强。采用Griess法考察了金钗石斛中分离得到的绝大部分单体化合物抑制大鼠巨噬细胞NO释放的活性。结果表明，进行测试的46个化合物中，有12个化合物显示不同程度的抑制活性，其中5个化合物显示出比阳性药resveratrol更强或相当的活性。菲类、联苄类和芴酮类化合物多具有不同程度的活性，木脂素类和酚酸类化合物中只有个别结构具有较弱的活性。采用ConA刺激小鼠脾细胞增殖的模型，考察了金钗石斛中分离得到的大部分单体化合物对T淋巴细胞增殖的影响。结果初步表明，进行测试的37个化合物中，有10个化合物显示不同程度的抑制T淋巴细胞增殖的活性，其中化合物29（毛兰菲）显示很强的抑制作用。本实验结果同时表明糖基部分是倍半萜糖苷类化合物重要的活性基团，并首次发现石斛中的菲类、联苄类、酚酸类等芳香化合物具有该方面的药理作用。对金钗石斛中分离得到的部分化合物还进行了“对KCl诱导的豚鼠心室肌细胞胞浆[Ca²⁺]_i升高的影响”的测试，结果初步表明化合物11，22，25，41，45对KCl诱导的心室肌细胞[Ca²⁺]_i升高有抑制作用，提示这些化合物可能对高血压、心绞痛、心律失常、心肌梗死等与钙通道关系密切的疾病有一定的预防和治疗作用。本文在化学和生物活性研究的基础上，证实了金钗石斛抗氧化活性的作用物质基础为菲类、联苄类、芴酮类、木脂素类和酚酸类等富含酚羟基的化合物，初步表明了抑制NO释放活性的作用物质基础为菲类、联苄类和芴酮类化合物，同时发现菲类和联苄类化合物还具有免疫抑制作用，由此表明中药石斛中大量存在的上述化合物可通过清除体内过剩自由基、抑制NO过度释放和抑制过高的免疫反应等多方面的调节作用而对多种疾病起到预防和治疗作用。在DPPH法和ORAC法活性测试的基础上，对两种方法活性结果与化合物结构的相关性进行了探讨。对于DPPH法，化合物结构中酚羟基的数目和位置、羟基邻位甲氧基的数目和侧链取代基的性质都会对活性产生重要的影响。而ORAC法中活性和结构之间的相关性并不明显。更多还原

【Abstract】 The traditional Chinese medicine "Shi Hu", derived from the dried or fresh stems of several Dendrobium species （Orchidaceae）, are widely used as a Yin tonic to nourish the stomach and promote the production of body fluid. Of which, Dendrobium nobile Lindl. is one of the most famous one and has been recorded in the Chinese Pharmacopoeia （2005 Edition） as one of the original materials of "Shi Hu".In our systematical study on the chemical constituents of Dendrobium nobile, 53 compounds were obtained from the 60% ethanol extract of the stems of this plant through various chromatographic techniques. Their structures were elucidated on the basis of physico-chemical property and spectroscopic analysis. The 53 compounds from Dendrobium nobile are dendronobilin A （1）^*, dendronobilin B （2）^*, dendronobilin C （3）^*, dendronobilin D （4）^*, dendronobilin E （5）^*, dendronobilin F （6）^*, dendronobilin G （7）^*, dendronobilin H （8）^*, dendronobilin I （9）^*, 10β,13,14-trihydroxyalloaromadendrane （10）, 10,12-dihydroxypicrotoxane （11）, 6α,10,12-trihydroxypicrotoxane （12）, dendrobiumane A （13）, bullatantirol （14）, dendrodensiflorol （15）, dendrobane A （16）, nobilin A （17）^*, nobilin B （18）^*, nobilin C （19）^*, nobilin D （20）^*, nobilin E （21）^*, crepidatin （22）, chrysotobibenzyl) （23）, dendrobin A （24）, chrysotoxine （25）, moscatilin （26）, gigantol （27）, fimbriatone （28）, confusarin （29）, flavanthrinin （30）, 2,5-dihydroxy-4,9-dimethoxyphenanthrene （31）, 3,7-dihydroxy-2,4-dimethoxyphenanthrene （32）, nobilone （33）^*, dendroflorin （34）, syringaresinol （35）, pinoresinol （36）, medioresinol （37）, lirioresinol-A （38）, 2-methylanthraquinone （39）, nobilate （40）^*, syringic acid （41）, 2-hydroxyphenylpropanol （42）, vanillin （43）, apocynin （44）, coniferyl aldehyde （45）, syringaldehyde （46）, syringylethanone （47）, p-hydroxybenzaldehyde （48）, 3-hydroxy-4-methoxyphenylethanol （49）,α-hydroxysyringylethanone （50）, dihydroxyconiferyl alcohol （51）, p-hydroxybenzoic acid （52）, p-hydroxyphenylpropionic acid （53）, respectively. Compounds 1-9, 17-21, 33, 40 are new compounds, compounds 14, 30-31, 38-39, 41-42, 44-53 were isolated for the first time from the Dendrobium genus and compounds 13, 15, 22, 23, 25, 28, 29,32,34-37,43 were isolated from this plant for the first time.All the compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl （DPPH） free radical scavenging method. The results showed that 24 compounds displayed scavenging activity on different levels. Most of the phenanthrenes, bibenzyls and lignans showed good activity and some of fluorenones and phenolic acids were active in this assay. Almost all the active compounds possessed higher scavenging activity than BHT, which is commonly used in food industry as a synthetical antioxidant. 9 compounds showed activity equivalent to or higher than Vc.At the same time, oxygen radical absorbance capacity method was used to evaluate most of the compounds isolated from Dendrobium nobile. Among the 49 tested compounds, 30 compounds displayed oxygen radical scavenging activity on different levels, most of which showed stronger activity than Vc. The phenanthrenes, bibenzyls, lignans and fluorenones generally possessed good activity and some of phenolic acids were active in this assay.Most of the compounds were assayed against NO production on LPS and IFN-γactivated murine macrophage-like cell line RAW 264.7 by Griess method. Among the 46 tested compounds, 12 compounds displayed inhibitory effects on different levels, 5 of which showed activity equivalent to or higher than positive control resveratrol. Some of lignans and phenolic acids showed week activity and most of the phenanthrenes, bibenzyls and fluorenones were active in this assay.The immunomodulatory activity of most compounds were evaluated. Among the 37 tested compounds, 10 compounds were found to inhibit the proliferation of T lymphocytes of mice induced by Con A, in which compound 29 （confusarin） showed inhibitory activity significantly. The results also showed that glycons of sesquiterpene glycosides were important immunomodulatory activity. And this is the first report of aromatic compounds, such as phenanthrenes, bibenzyls and phenolic acids, with immunosuppressive activity from Dendrobium genus.Some of the compounds on [Ca²⁺]_ⅰof Guinea pig’s cardiomyocytes increase induced by KCl were also evaluated. The results showed that compounds 11, 22, 25, 41, 45 could inhibit the increase of [Ca²⁺]_ⅰmobilized by KCl. It suggested that these compounds could have certain prophylactic and therapeutic effects on such diseases as hypertension, angina pectoris, arrhythmia, myocardical infarction, which have close relationship with calcium channel.On the basis of chemical and bioactive study, this paper confirmed that the antioxidant components from Dendrobium nobile were phenanthrenes, bibenzyls, fluorenones, lignans and phenolic acids, which possessed phenolic hydroxyl groups in their structures, primarily showed that active compounds against NO production on LPS and IFN-γactivated murine macrophage RAW 264.7 were phenanthrenes, bibenzyls and fluorenones, and found new immunosuppressive components, phenanthrenes and bibenzyls. Our study also indicated the above-mentioned compounds in TCM "Shi Hu" could have certain prophylactic and therapeutic effects on many diseases through various modulatory effects, such as scavenging the redundant radicals in the body, inhibiting NO excessive production and inhibiting exorbitant immunoreaction.On the basis of DPPH assay and ORAC assay, the structure-activity relationship was discussed. The number and position of phenolic hydroxyl groups, the number of methoxyl groups substituted in ortho position to the hydroxyl group and the character of side chain in the structure seemed to play an important role in the activity in DPPH assay. But the relativity wasn’t obvious between activity and structure in ORAC assay.更多还原