三种太白山药用植物的活性成分研究

摘要

对传统药用植物和民间中草药的化学成分研究能够发现一些有药理活性的先导化合物,是发现创新药物的重要途径之一,并为进一步开发利用中草药资源提供科学依据。秦岭太白山是我国有名的“天然药库”,拥有丰富的植物资源,其中具有药用价值的植物数量众多。本文在前期研究的基础上,在国家自然科学基金和陕西省科技统筹创新工程计划项目的资助下,对太白山产的三种药用植物太白银莲花(Anemone taipaiensis)、小花草玉梅(Anemonerivularisvar.flore-minore)和顶花板凳果(Pachysandra terminalis)进行了成分及药理活性研究。运用硅胶柱色谱、Sephadex LH-20柱色谱、反相ODS柱色谱及半制备HPLC等分离手段研究了这三种药用植物的化学成分,运用波谱技术(NMR、MS等)、化学分析、单晶衍射等手段对化合物进行结构鉴定,共分离鉴定了54个化合物,包含16个新化合物。对得到的皂苷类成分采用MTT试验、流式细胞术和免疫印迹法做了抗肿瘤活性的初步研究;对得到的甾体生物碱类成分进行了3种标准菌和4种耐药菌的抗菌活性筛选。具体研究内容和结果如下:
　　一、太白银莲花的化学成分研究对太白银莲花根茎70％乙醇提取物正丁醇萃取部位的大极性片段进行了分离,共得到10个齐墩果烷型三萜皂苷,其中包括4个新化合物。分别为:3b-O-{a-L-rhamno-pyranosyl-(1→2)-a-L-arabinopyranosyl} siaresinolic acid28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(T-1),3b-O-{a-L-rhamno-pyranosyl-(1→2)-[glucopyranosyl(1→4)]-a-L-arabinopyranosyl} siaresinolic acid28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(T-2),Hederagenin28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glu-copyranoside}(T-3),3b-O-{β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl(1→4)-[a-L-rhamnopyranosyl-(1→2)]-a-L-arabinopyranosyl}siaresinolic acid28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(T-4),3b-O-{b-D-glucopyranosyl-(1→4)-b-D-glucopyranosyl-(1→4)-[a-L-rhamnopyranosyl-(1→2)]-a-L-arabinopyranosyl}hederagenin28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-gluco-pyranosyl-(1→6)-b-D-glucopyranoside}(T-5),3b-O-{b-D-xylopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl} hederage-nin28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyrano-side}(T-6),3b-O-{b-D-xylopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-[b-D-glu-copyranosyl-(1→4)-b-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl}hederagenin28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(T-7),3b-O-{a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl} oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-8),3b-O-{b-D-xylopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl}hederagenin28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glu-copyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-gluco-pyranoside}(T-9),3b-O-{b-D-xylopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl}oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-rha-mnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-10),其中化合物T-1,T-4,T-9和T-10为新化合物,这些皂苷都属于齐墩果烷型,基于三种苷元类型:oleanolic acid,hederagenin和siaresinolic acid。
　　二、小花草玉梅的化学成分研究对小花草玉梅根茎70%乙醇提取物的正丁醇萃取部位进行了分离,共得到37个化合物,其中35种为齐墩果烷型三萜皂苷,共包含9个新化合物。分别为:β-谷甾醇(X-1),3b-O-L-arabinopyranosyl oleanolic acid28-O-β-D-glucopyranoside(X-2),β-胡萝卜苷(X-3),3β-O-{β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl}hederagenin(X-4),刺楸皂苷A(X-5),3b-O-{β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl}oleanolic acid(X-6),HN-saponin F(X-7),sapindoside B(X-8),白头翁皂苷D(X-9),sapindoside B(X-10),pulsatilloside D(X-11),prosapogenin CP4(X-12),3b-O-{b-D-xylopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl}oleanolic acid(X-13),3b-O-{β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl}oleanolicacid28-O-β-D-glucopyranoside(X-14),cussonside B(X-15),白头翁皂苷C(X-16),红毛七皂苷 D(X-17),3b-O-{b-D-ribopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl}hederagenin28-O-β-D-glucopyranoside(X-18),3b-O-{β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl}hederagenin28-O-β-D-glucopyran-oside(X-19),3b-O-a-L-arabinopyranosyl oleanolic acid28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(X-20),刺五加苷A1(X-21),3β-O-{β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl}oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-22),sieb-oldianoside B(X-23),3β-O-α-L-arabinopyranosyl betulinic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-24),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl}oleanolic acid28-O-β-D-glucop-yranoside(X-25),3b-O-{b-D-glucopyranosyl-(1→2)a-L-arabinopyranosyl}oleanolic acid28-O-{b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(X-26),3β-O-α-L-arabinop-yranosyl gypsogenin28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-27),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl}gypsogenin28-O-β-D-gluco-pyranoside(X-28),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl}gypsogenin28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-29),cauloside F(X-30),hederasaponin B(X-31),3b-O-{b-D-ribopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl}hederagenin28-O-{a-L-rhamnopyranosyl-(1→4)-b-D-glucopyranosyl-(1→6)-b-D-glucopyranoside}(X-32),3b-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl}21α-hydroxy oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-33),3-O-{β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyran-osyl-(1→2)]-α-L-arabinopyranosyl}21α-hydroxy oleanolic acid28-O-{α-L-rhamno-pyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-34),刺楸皂苷 B(X-35),sieboldianoside A(X-36)和hederacolchiside E(X-37),除X-1和X-3为甾体类化合物,X-24为羽扇豆烷型三萜皂苷,其余都为齐墩果烷型三萜皂苷,化合物X-4,X-18,X-22,X-24,X-25,X-28,X-29,X-33和X-34为新化合物,这些化合物基于五种苷元:oleanolic acid,hederagenin,gypsogenin,21α-hydroxy oleanolic acid和betulinic acid,其中后两种苷元为首次从银莲花属植物种分离得到。
　　三、顶花板凳果的化学成分研究顶花板凳果全草用90%乙醇回流提取,浸膏先后经过酸、碱处理,后用氯仿萃取得总碱部位,对总碱部位的分离得到7个生物碱类化合物。分别为:1α-6α,10β-dihydroxy-2,3,7,7,10α-pentamethyl-2-azabicyclo-[4.3.1]decan-9-one(D-1),pachysamine A(D-2),20α-dimethylamino-3β-senecioylamino-16β-hydroxy-pregn-5-ene(D-3),20α-dimethylamino-3β-senecioylamino-pregn-5-ene(D-4),Epipachysamine E(D-5),Epipachysamine B(D-6)和20α-dimethylamino-pregnane-3β,4α-diol(D-7),除D-1外其余6个均为板凳果属特征的富贵草型生物碱,D-1,D-3,D-4为新化合物。
　　四、化合物的活性研究采用MTT法筛选了20个皂苷类化合物X-4～X-23对5种人源型肿瘤细胞的细胞毒性并初步归纳了齐墩果烷型皂苷抗肿瘤的构效关系,结果表明:苷元C-3位连有Rha(1→2)Ara-片段的单糖链齐墩果酸皂苷具有较高活性,其中活性最强的为X-12,对U87MG细胞的IC50为8.21mM。采用流式细胞术AnnexinV/PI双染检测发现X-12作用于U87MG细胞时呈时间依赖性地诱导其发生凋亡,在24 h和72 h均能显著增加凋亡细胞比例;免疫印迹法结果显示X-12作用于U87MG细胞时参与诱导Survivin的表达下调、Bcl-2/Bax表达比率的降低、Caspase3和Caspase9的裂解激活,这可能是X-12诱导U87MG细胞发生凋亡的分子机制。
　　选用S.aureusATCC29213和E.coliATCC25922两种标准菌株测试了富贵草型甾体生物碱类化合物D-2～D-7的最低抑菌浓度(MIC),结果显示D-2～D-7对ATCC29213有抑菌活性,其中D-3的MIC最低，为32 μg/mL；对D-3的进一步测试表明其对另一种标准菌(S.aureus ATCC 14990)和四种耐药菌(MRSA、MRSE、VRSAMu50和LAC)有抑菌活性，其中对MRSA、MRSE和LAC的MIC均为25 μg/mL，明显优于阳性对照头孢他啶(MIC = 64～128 μg/mL)；生长曲线和杀菌曲线的测试结果表明，在耐药菌株(LAC和MRSE)对头孢他啶完全耐药的情况下，D-3表现出对耐药菌株的全程生长抑制作用，同时在浓度为50 μg/mL时就表现出良好的杀菌活性，相比较传统抗生素有明显优势。
　　课题创新性及研究意义：
　　1.对分布于太白山的3 种药用植物的化学成分进行了系统研究，鉴定了 45 个三萜皂苷类成分、7 个生物碱成分和2 个甾体成分。
　　2.发现了 16 个新化合物，其中以 21α-hydroxy oleanolic acid和betulinic acid 为苷元的皂苷系首次在银莲花属植物中发现，新生物碱 D-1 亦为板凳果属植物中首次发现的结构类型。
　　3.通过体外抗肿瘤、抗菌活性筛选，发现一批具有显著抗肿瘤、抗菌活性的皂苷和生物碱成分。
　　4.本课题为开发这三种太白山药用植物资源提供了科学依据，为进一步研究它们的活性物质积累了资料，也为开发抗肿瘤、抗菌新药提供了有价值的先导化合物。

目录

封面

声明

目录

缩略语表

中文摘要

英文摘要

前言

文献回顾

正文

小结

参考文献

附录

个人简历及研究成果

致谢