细柱五加叶及其同属植物活性成分研究

【作者】 倪娜；

【导师】 刘向前；

【作者基本信息】 中南大学 ， 制药工程， 2008， 硕士

【摘要】 五加科(Araliaceae)五加属(Acanthopanax Miq.)植物在全世界约有37种,主要分布于亚洲的中国、韩国和日本,其根皮为民间常见中药,俗称“五加皮”,具有滋补、抗风湿、抗疲劳、活血化淤等功效,临床主要用于治疗风湿痹痛、腰膝酸软等病症。我国的五加属植物资源丰富,有26种18变种,《中国药典》收载的五加皮为细柱五加(Acanthopanax gracilistylus W.W.Smith)根皮。韩国已发现五加属植物11种3变种3变型,《韩国药典》收载的五加皮为无梗五加(Acanthopanaxsessiliflorum Seemann)根皮。由于五加属植物丰富的资源以及在医药领域上的广泛应用,为了寻找更多新的活性成分,扩大其药用价值,本论文对细柱五加叶及其同属植物中的活性成分(三萜、二萜及挥发油)进行了系统研究。五加属植物中的三萜化合物大多具有生物活性,为了筛选抗HMGB1药物,对湖南省长沙地区细柱五加叶中的三萜成分进行研究。将其热甲醇提取物经Dimon HP-20P柱层析分离,其中100%和80%MeOH部分再经硅胶柱层析、Sephadex LH-20柱层析、TLC及重结晶等方法分离得到6个化合物。化合物1～5采用EI-MS、HR-MS、1D-NMR、2D-NMR、IR以及与已知化合物的文献数据比较,鉴定为羽扇豆烷型三萜化合物acankoreanogenin A、acankoreoside B和acankoreoside A。其中化合物1为acankoreanogenin A,化合物2为acankoreanogenin A,化合物3为acankoreoside B(首次从该植物中得到),化合物4为acankoreanogenin A,化合物5为acankoreoside A。化合物6经GC-MS定性分析,主要为邻苯二甲酸二丁酯等酯类成分。对分离产物acankoreanogenin A和acankoreoside A进行抗HMGB1活性研究,结果显示二者均有一定的抗炎活性,acankoreanogenin A对HMGB1有较好的抑制效果,有望成为一种新型抗HMGB1药物,此发现为该植物的深入开发利用提供了有利依据。研究表明五加皮中的二萜化合物是其有效成分之一,被证实具有较好的抗炎功能。对细柱五加根皮的乙醚提取物进行多次硅胶柱层析及制备薄层层析,从中分离得到两个抗炎二萜,经鉴定化合物7为五加酸(acanthoic acid),化合物8为贝壳烯酸(kaurenoic acid)。首次利用反相HPLC建立了五加酸和贝壳烯酸的定量分析方法,确定了最佳色谱条件:检测波长为207nm,柱温为30℃,流速为1.0ml/min,流动相为乙腈-水(0～60min乙腈10%→100%,60～70min乙腈100%)。经方法学验证,此方法简便快捷,准确性高,适用于抗炎二萜成分的分离定量。同时对8种中韩五加属植物根皮中的二萜成分五加酸和贝壳烯酸进行定量分析,为制定一种五加属植物新的质量控制标准提供实验依据。五加属植物中的挥发油多具有抗菌消炎、祛痰止咳之功效,也是主要活性成分之一。采用水蒸气蒸馏法提取8种中韩五加属植物挥发油,然后用气相色谱-质谱-计算机联用技术对其化学成分进行定性及定量分析。同时比较分析8种五加属植物根皮挥发油的共有成分,多含有α-蒎烯、β-蒎烯、匙叶桉油烯醇和正-十六烷酸等,为五加属植物进一步开发利用提供研究依据。本论文的研究结果对抗HMGB1药物筛选具有重要的意义,为中草药活性成分阐明、植物化学分类学和新药筛选提供重要的理论依据,对我国天然植物资源的开发利用以及中草药现代化研究具有一定的参考价值。更多还原

【Abstract】 There are about 37 kinds of Acanthopanax Miq.plants of Araliaceae in the world,which mainly being distributed in China,Korea and Japan of Asia.The root bark,commonly called "Cortex Acanthopanacis",is a common traditional Chinese drug,which have efficiencies of invigoration, antirheumatism,antifatigue and stimulate circulation to end stasis.It is used for treat the rheumatism pain,waist and knee aching etc.A mass of Acanthopanax Miq.plants grow in our country,about 26 kinds and 18 variety,and the root bark of Acanthopanax gracilistylus W.W.Smith is recorded in Chinese Pharmacopeia.There are 11 kinds,3 variety and 3 form in Korea,and the root bark of Acanthopanax sessiliflorum Seemann is recorded in Korea Pharmacopeia.Just for the further development and utilization of these plants and search more active ingredients,we research on the active ingredients including triterpene,diterpene and voltatile oil from the leaves of Acanthopanax gracilistylus W.W.Smith and other Acanthopanax Miq.plants.Triterpene from Acanthopanax Miq.plants has much bioactivity. To develop new anti-HMGB1 drugs,we research on the triterpene components of Acanthopanax gracilistylus W.W.Smith leaves collected from Changsha in Hunan.The hot methanol extract was primarily separated by Diaion HP-20P CC.Then the 100%and 80%portions were orderly separated by Silica gel CC,Sephadex LH-20 CC,TLC, recrystallization,and finally isolated six components.Multiple spectrum techniques,like EI-MS、HR-MS、1D-NMR、2D-NMR、IR were used for qualitative analysis of compound 1～5.After compared with the data of known compounds,three lupane triterpene components were identified. There were acankoreanogenin A（compound 1,2 and 4）,acankoreoside B （compound 3）and acankoreoside A（compound 5）,which compound 3 was isolated from this plant for first time.Compound 6 were the mixture of ester by GC-MS analysis.Anti-HMGB1 activity test was made,the results showed acankoreanogenin A and acankoreoside A both have some antiphlogistic activity,and acankoreanogenin A has a good catastaltic effect to HMGB1.Maybe it will be a new anti-HMGB1 drug in future. The conclusion of study would provide gist for the further development and utilization of Acanthopanax gracilistylus W.W.Smith.The research showed diterpene,one of the active ingredients from Cortex Acanthopanacis,has antiphlogistic activity.The mothed of Silica gel CC and TLC was repeatedly used to isolate the ether extract of root bark of Acanthopanax gracilistylus W.W.Smith,and two components were obtained.By identified,compound 7 was acanthoic acid and compound 8 was kaurenoic acid.RP-HPLC method was established for quantitative analysis of two diterpenes for first time.The best condition of HPLC:wavelength is 207nm,temp is 30℃,flow rate is 1ml/min, mobile phase is CH₃CN-H₂O（0～60min CH₃CN 10%→100%,60～70min CH₃CN 100%）.Through the methodology test,the method was simple and fast to use,and could gain good accuracy.The diterpene compounds from 8 kinds of Acanthopanax Miq.plants were quantitative analysed by this RP-HPLC method.The results would surely aid to construct the new standard for the identification and quality control of Acanthopanax Miq. plants.Most of voltatile oil from Acanthopanax Miq.plants has antiseptic, antiphlogistic,apophlegmatic and antitussive activity.The method of water-steam distillation was used to extract the essential oils from 8 kinds of Acanthopanax Miq.plants,and the chemical components were identified by GC-MS-DS techniques.Compared with these essential oils, and the common compounds wereα-pinene,β-pinene,spathulenol and n-hexadecanoic acid.The results would provide investigative basis for further development of Acanthopanax Miq.plants.The results of this paper has important effect to choose anti-HMGB1 drug.And it will enrich natural products database,and provide important theoretical foundation for identification of active ingredients,systematics of phytochemistry and new drug screening.So it will has referred value on developing and exploitaging our country’s natural products resource.更多还原