三種太白山藥用植物的活性成分研究.pdf

1、對(duì)傳統(tǒng)藥用植物和民間中草藥的化學(xué)成分研究能夠發(fā)現(xiàn)一些有藥理活性的先導(dǎo)化合物,是發(fā)現(xiàn)創(chuàng)新藥物的重要途徑之一,并為進(jìn)一步開發(fā)利用中草藥資源提供科學(xué)依據(jù)。秦嶺太白山是我國(guó)有名的“天然藥庫(kù)”,擁有豐富的植物資源,其中具有藥用價(jià)值的植物數(shù)量眾多。本文在前期研究的基礎(chǔ)上,在國(guó)家自然科學(xué)基金和陜西省科技統(tǒng)籌創(chuàng)新工程計(jì)劃項(xiàng)目的資助下,對(duì)太白山產(chǎn)的三種藥用植物太白銀蓮花(Anemone taipaiensis)、小花草玉梅(Anemonerivulari

2、svar.flore-minore)和頂花板凳果(Pachysandra terminalis)進(jìn)行了成分及藥理活性研究。運(yùn)用硅膠柱色譜、Sephadex LH-20柱色譜、反相ODS柱色譜及半制備HPLC等分離手段研究了這三種藥用植物的化學(xué)成分,運(yùn)用波譜技術(shù)(NMR、MS等)、化學(xué)分析、單晶衍射等手段對(duì)化合物進(jìn)行結(jié)構(gòu)鑒定,共分離鑒定了54個(gè)化合物,包含16個(gè)新化合物。對(duì)得到的皂苷類成分采用MTT試驗(yàn)、流式細(xì)胞術(shù)和免疫印跡法做了抗腫瘤活

3、性的初步研究;對(duì)得到的甾體生物堿類成分進(jìn)行了3種標(biāo)準(zhǔn)菌和4種耐藥菌的抗菌活性篩選。具體研究?jī)?nèi)容和結(jié)果如下:
　　一、太白銀蓮花的化學(xué)成分研究對(duì)太白銀蓮花根莖70％乙醇提取物正丁醇萃取部位的大極性片段進(jìn)行了分離,共得到10個(gè)齊墩果烷型三萜皂苷,其中包括4個(gè)新化合物。分別為:3b-O-{a-L-rhamno-pyranosyl-(1→2)-a-L-arabinopyranosyl} siaresinolic acid28-O-{a-L

4、-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-(

5、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-glucopyr

6、anosyl(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-

7、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)

8、-[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-copyranos

9、yl-(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

10、-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-ara

11、binopyranosyl}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-

12、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-

13、D-glucopyranoside}(T-10),其中化合物T-1,T-4,T-9和T-10為新化合物,這些皂苷都屬于齊墩果烷型,基于三種苷元類型:oleanolic acid,hederagenin和siaresinolic acid。
　　二、小花草玉梅的化學(xué)成分研究對(duì)小花草玉梅根莖70%乙醇提取物的正丁醇萃取部位進(jìn)行了分離,共得到37個(gè)化合物,其中35種為齊墩果烷型三萜皂苷,共包含9個(gè)新化合物。分別為:β-谷甾醇(X-1),

14、3b-O-L-arabinopyranosyl oleanolic acid28-O-β-D-glucopyranoside(X-2),β-胡蘿卜苷(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

15、 acid(X-6),HN-saponin F(X-7),sapindoside B(X-8),白頭翁皂苷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-arabino

16、pyranosyl}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),白頭翁皂苷C(X-16),紅毛七皂苷 D(X-17),3b-O-{b-D-ribopyranosyl-(1→3)-a-L-rhamnopyranos

17、yl-(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-rha

18、mnopyranosyl-(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-glucopyran

19、oside}(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

20、-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-glu

21、copyranoside}(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→

22、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-glu

23、copyranosyl-(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-glucopyrano

24、syl-(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}

25、(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-3

26、4),刺楸皂苷 B(X-35),sieboldianoside A(X-36)和hederacolchiside E(X-37),除X-1和X-3為甾體類化合物,X-24為羽扇豆烷型三萜皂苷,其余都為齊墩果烷型三萜皂苷,化合物X-4,X-18,X-22,X-24,X-25,X-28,X-29,X-33和X-34為新化合物,這些化合物基于五種苷元:oleanolic acid,hederagenin,gypsogenin,21α-hydr

27、oxy oleanolic acid和betulinic acid,其中后兩種苷元為首次從銀蓮花屬植物種分離得到。
　　三、頂花板凳果的化學(xué)成分研究頂花板凳果全草用90%乙醇回流提取,浸膏先后經(jīng)過(guò)酸、堿處理,后用氯仿萃取得總堿部位,對(duì)總堿部位的分離得到7個(gè)生物堿類化合物。分別為:1α-6α,10β-dihydroxy-2,3,7,7,10α-pentamethyl-2-azabicyclo-[4.3.1]decan-9-one(D

28、-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-di

29、ol(D-7),除D-1外其余6個(gè)均為板凳果屬特征的富貴草型生物堿,D-1,D-3,D-4為新化合物。
　　四、化合物的活性研究采用MTT法篩選了20個(gè)皂苷類化合物X-4～X-23對(duì)5種人源型腫瘤細(xì)胞的細(xì)胞毒性并初步歸納了齊墩果烷型皂苷抗腫瘤的構(gòu)效關(guān)系,結(jié)果表明:苷元C-3位連有Rha(1→2)Ara-片段的單糖鏈齊墩果酸皂苷具有較高活性,其中活性最強(qiáng)的為X-12,對(duì)U87MG細(xì)胞的IC50為8.21mM。采用流式細(xì)胞術(shù)Annex

30、inV/PI雙染檢測(cè)發(fā)現(xiàn)X-12作用于U87MG細(xì)胞時(shí)呈時(shí)間依賴性地誘導(dǎo)其發(fā)生凋亡,在24 h和72 h均能顯著增加凋亡細(xì)胞比例;免疫印跡法結(jié)果顯示X-12作用于U87MG細(xì)胞時(shí)參與誘導(dǎo)Survivin的表達(dá)下調(diào)、Bcl-2/Bax表達(dá)比率的降低、Caspase3和Caspase9的裂解激活,這可能是X-12誘導(dǎo)U87MG細(xì)胞發(fā)生凋亡的分子機(jī)制。
　　選用S.aureusATCC29213和E.coliATCC25922兩種標(biāo)準(zhǔn)菌

31、株測(cè)試了富貴草型甾體生物堿類化合物D-2～D-7的最低抑菌濃度(MIC),結(jié)果顯示D-2～D-7對(duì)ATCC29213有抑菌活性,其中D-3的MIC最低，為32 μg/mL；對(duì)D-3的進(jìn)一步測(cè)試表明其對(duì)另一種標(biāo)準(zhǔn)菌(S.aureus ATCC 14990)和四種耐藥菌(MRSA、MRSE、VRSAMu50和LAC)有抑菌活性，其中對(duì)MRSA、MRSE和LAC的MIC均為25 μg/mL，明顯優(yōu)于陽(yáng)性對(duì)照頭孢他啶(MIC = 64～128

32、μg/mL)；生長(zhǎng)曲線和殺菌曲線的測(cè)試結(jié)果表明，在耐藥菌株(LAC和MRSE)對(duì)頭孢他啶完全耐藥的情況下，D-3表現(xiàn)出對(duì)耐藥菌株的全程生長(zhǎng)抑制作用，同時(shí)在濃度為50 μg/mL時(shí)就表現(xiàn)出良好的殺菌活性，相比較傳統(tǒng)抗生素有明顯優(yōu)勢(shì)。
　　課題創(chuàng)新性及研究意義：
　　1.對(duì)分布于太白山的3 種藥用植物的化學(xué)成分進(jìn)行了系統(tǒng)研究，鑒定了 45 個(gè)三萜皂苷類成分、7 個(gè)生物堿成分和2 個(gè)甾體成分。
　　2.發(fā)現(xiàn)了 16 個(gè)新化合

33、物，其中以 21α-hydroxy oleanolic acid和betulinic acid 為苷元的皂苷系首次在銀蓮花屬植物中發(fā)現(xiàn)，新生物堿 D-1 亦為板凳果屬植物中首次發(fā)現(xiàn)的結(jié)構(gòu)類型。
　　3.通過(guò)體外抗腫瘤、抗菌活性篩選，發(fā)現(xiàn)一批具有顯著抗腫瘤、抗菌活性的皂苷和生物堿成分。
　　4.本課題為開發(fā)這三種太白山藥用植物資源提供了科學(xué)依據(jù)，為進(jìn)一步研究它們的活性物質(zhì)積累了資料，也為開發(fā)抗腫瘤、抗菌新藥提供了有價(jià)值的先導(dǎo)化