【作者】 施树云；

【导师】 赵昱； 李校堃；

【作者基本信息】 浙江大学 ， 药物分析学， 2007， 博士

【摘要】 本论文对三种民间的药用菊科植物:橐吾属植物黑紫橐吾（Ligulariaatroviolacea（Franch.）Hand.-Mazz.）、蒲公英属植物蒙古蒲公英（Taraxacummongolicum Hand.-Mazz.）和六棱菊属植物臭灵丹[Laggera pterodonta（DC.）Benth]进行了研究,全文有以下四个部分组成。第一章黑紫橐吾的化学成分及部分化合物的抗乙型肝炎病毒活性研究黑紫橐吾（Ligularia atroviolacea）主要分布于我国云南省西北部。2005年日本学者对我国的Ligularia atroviolacea进行了初步的植物化学成分研究,探明艾里莫芬烷型倍半萜为该物种的代表性次生代谢产物。但迄今,尚未见有文献对该植物的化学成分进行详细的研究报道。因此我们选择产自我国云南省丽江县的黑紫橐吾进行了系统的植物化学成分研究并对其主要组分以及新化合物进行了初步的抗乙型肝炎病毒活性测试。本论文采用常规的柱层析、制备薄层层析、重结晶等分离手段对植物中的提取物进行了分离纯化,并利用现代波谱方法(¹H-NMR,¹³C-NMR,¹H-¹H COSY,NOESY,HMBC,HSQC,ESI-MS,HR-MS,UV,IR,X-ray等)进行结构鉴定,确定了化学结构。主要的实验结果如下:一、从黑紫橐吾（Ligularia atroviolacea）中共分离得到32个单体化合物,鉴定了24个化合物,其中化合物（Ⅰ-2～Ⅰ-10）为新化合物,化合物（Ⅰ-11）为首次以天然产物的形式分得,其它化合物均为首次从该植物中分离得到:1.8β-氢-艾里莫芬烷-3,7（11）-二烯-12,8α;15,6α-二内酯（Ⅰ-1）;2.8β-羟基-艾里莫芬烷-3,7（11）-二烯-12,8α;15,6α-二内酯（Ⅰ-2）;3.8β-甲氧基-艾里莫芬烷-3,7（11）-二烯-12,8α;15,6α-二内酯（Ⅰ-3）;4.艾里莫芬烷-3,7（11）,8-三烯-12,8α;15,6α-二内酯（Ⅰ-4）;5.3β-当归酰基-艾里莫芬烷-7（11）-烯-12,8β-内酯-15-酸（Ⅰ-5）;6.1α-氯-6β-异丁酰基-9-酮-10β-羟基-呋喃雅槛蓝烷（Ⅰ-6）;7.10β-氢-8-酮-艾里莫芬烷-3（4）,6（7）-二烯-12,15-二酸（Ⅰ-7）;8.10α-氢-8-酮-艾里莫芬烷-3（4）,6（7）-二烯-12,15-二酸（Ⅰ-8）;9.8β-[艾里莫芬烷-3′,7′（11′）-二烯-12′,8′α;5′,6′α-二内酯]-艾里莫芬烷-二烯-12,8α;15,6α-3,7（11）-二内酯（Ⅰ-9）;10.黑紫橐吾A（Ⅰ-10）;11.呋喃雅槛蓝烷-3-烯-15,6α-内酯（Ⅰ-11）;12.呋喃雅槛蓝烷-15β,6α-内酯（Ⅰ-12）;13.1α-羟基-6β-异丁酰基-9-酮-10β-氢-呋喃雅槛蓝烷（Ⅰ-13）;14.1α-羟基-6β-异丁酰基-9-酮-10α-氢-呋喃雅槛蓝烷（Ⅰ-14）;15.1α,10β-二羟基-6β-当归酰基-9-酮-呋喃雅槛蓝烷（Ⅰ-15）;16.8β-羟基-艾里莫芬烷-7（11）-烯-12,8α;15,6α-二内酯（Ⅰ-16）;17.3β-当归酰基-8β-氢-艾里莫芬烷-7（11）-烯-12,8α;14β,6α-二内酯（Ⅰ-17）;18.3β-（2′-甲基丁酰基）-8β-氢-艾里莫芬烷-7（11）-烯-12,8α;14β,6α-二内酯（Ⅰ-18）;19.β-谷甾醇（Ⅰ-19）;20.豆甾醇（Ⅰ-20）;21.豆甾-4-烯-3β-醇（Ⅰ-21）;22.胡萝卜苷（Ⅰ-22）;23.6-O-乙酰氧基-α-D-吡喃葡萄糖（Ⅰ-23）;24.正二十六烷酸（Ⅰ-24）.以上共计艾里莫芬烷类倍半萜18个,甾醇类化合物4个,糖类衍生物1个,长链脂肪烃1个。通过X-ray单晶衍射技术确定了化合物（Ⅰ-1）和（Ⅰ-6）的结构。二、黑紫橐吾的主要次生代谢产物为艾里莫芬烷类倍半萜。三、根据抗乙型肝炎病毒活性实验,化合物（Ⅰ-1）、（Ⅰ-2）、（Ⅰ-3）和（Ⅰ-4）能显著抑制HBsAg的表达。第二章蒲公英的化学成分及抗病毒活性研究蒲公英（Herba Taraxaci）为我国常用中药材,种类繁多,资源丰富。在国内以蒙古蒲公英（简称蒲公英）（Taraxacum mongolicum）为主流品。具有清热解毒、消肿散结和利胆利尿等功效。国外对药用蒲公英（Taraxacum officinale）的化学成分研究比较全面,主要含倍半萜、三萜和黄酮类成分,药理活性报道较少,而未见蒙古蒲公英（Taraxacum mongolicum）的化学成分研究报道。国内对蒲公英属植物的化学成分研究报道甚少,多引用Taraxacum officinale的化学成分。为了进一步阐明蒲公英的药效物质基础,本研究对蒙古蒲公英进行了系统的植物化学研究。从中分离得到44个单体化合物,鉴定了其中的40个化合物的结构,其中4个为新化合物（Ⅱ-14～Ⅱ-15和Ⅱ-32～Ⅱ-33）,16个为首次从本属植物中分离得到的化合物（Ⅱ-1,Ⅱ-3,Ⅱ-6～Ⅱ-13,Ⅱ-16,Ⅱ-20～Ⅱ-22,Ⅱ-31和Ⅱ-34）,13个化合物（Ⅱ-18,Ⅱ-23～Ⅱ-30和Ⅱ-35～Ⅱ-38）为首次从该植物中分得,且实验中首次从该属植物中分离鉴定出木脂素类化合物。1.洋艾素（Ⅱ-1）;2.槲皮素（Ⅱ-2）;3.槲皮素-3′,4′,7-三甲醚（Ⅱ-3）;4.木犀草素（Ⅱ-4）;5.木犀草素-7-O-β-D-葡萄糖苷（Ⅱ-5）;6.木犀草素-7-O-β-D-半乳糖苷（Ⅱ-6）;7.芫花素（Ⅱ-7）;8.Isoetin（Ⅱ-8）;9.3′,5,7-三羟基-4′-甲氧基-二氢黄酮（Ⅱ-9）;10.芫花素-4′-O-β-D-芦丁糖苷（Ⅱ-10）;11.橙皮苷（Ⅱ-11）;12.槲皮素-7-O-[β-D-吡喃葡萄糖基（1→6）-β-D-吡喃葡萄糖苷]（Ⅱ-12）;13.槲皮素-3,7-O-β-D-二吡喃葡萄糖苷（Ⅱ-13）;14.Isoetin-7-O-β-D-glucopyranosyl-2′-O-α-L-arabinopyranoside（Ⅱ-14）;15.Isoetin-7-D-β-D-glucopyranosyl-2′-O-α-D-glucopyranoside（Ⅱ-15）;16.Isoetin-7-O-β-D-glucopyranosyl-2′-O-β-D-xyloypyranoside（Ⅱ-16）;17.咖啡酸（Ⅱ-17）;18.阿魏酸（Ⅱ-18）;19.绿原酸（Ⅱ-19）;20.3,5-O-双咖啡酰基奎尼酸（Ⅱ-20）;21.3,4-O-双咖啡酰基奎尼酸（Ⅱ-21）;22.4,5-O-双咖啡酰基奎尼酸（Ⅱ-22）;23.1-羟甲基-5-羟基-苯-2-O-β-D-吡喃葡萄糖苷（Ⅱ-23）;24.对羟基苯甲酸（Ⅱ-24）;25.对香豆酸（Ⅱ-25）;26.3,5-二羟基苯甲酸（Ⅱ-26）;27.没食子酸（Ⅱ-27）;28.没食子酸甲酯（Ⅱ-28）;29.丁香酸（Ⅱ-29）;30.七叶内酯（Ⅱ-30）;31.Refescidrine（Ⅱ-31）;32.蒙古蒲公英A（Ⅱ-32）;33.蒙古蒲公英B（Ⅱ-33）;34.Isodonsesquitin A（Ⅱ-34）;35.蒲公英甾醇乙酸酯（Ⅱ-35）;36.伪蒲公英甾醇乙酸酯（Ⅱ-36）;37.羽扇豆醇乙酸酯（Ⅱ-37）;38.正十六烷酸（Ⅱ-38）.另外,从石油醚部位分得大量的豆甾醇和β-谷甾醇（Ⅱ-39和Ⅱ-40）。以上可以看出,蒲公英的次生代谢产物主要是黄酮类和酚酸类化合物。根据抗病毒活性实验结果表明:酚酸中的异绿原酸类化合物体外对单纯疱疹病毒和甲型人流感病毒均有较强的抑制作用。第三章臭灵丹中异绿原酸的色谱制备研究臭灵丹（Laggera pterodonta）具有显著的抗菌消炎之功效,在我国云南具有丰富的植物资源,极具开发价值。经研究,热水提醇沉法制备的臭灵丹提取物主要成分为异绿原酸类化合物。且从第二章可以看出,异绿原酸类化合物具有较强的抗病毒活性。由于三个主要的异绿原酸类化合物结构相似,难于分离,为了进一步研究的需要,我们研究了它们的色谱分离制备行为。本文立足于药效实际,从臭灵丹原料出发,采用高效液相色谱分离纯化出可用于新药开发的三个活性化合物,走出了一条对臭灵丹进行精深加工和综合开发的新工艺路线。主要内容如下:1.对臭灵丹的活性成分进行分析方法研究。提出了臭灵丹中三种主要异绿原酸类化合物的高效液相色谱（RP-HPLC）的同时分析方法,适用于原材料及产品的质量控制,快速、准确。2.运用高效液相色谱制备技术,实现了多酚类混合物中三种主要活性成分3,4-O-双咖啡酰基奎尼酸,3,5-O-双咖啡酰基奎尼酸和4,5-O-双咖啡酰基奎尼酸的同步分离,提出了一条在制备流程中同时得到三种高纯产品的新工艺路线,实验中对色谱填料、流动相组成、流速、进样量等工艺参数进行了优化,得出制备型液相色谱分离纯化异绿原酸的最佳色谱条件如下:制备柱尺寸:300×100 mm;C₁₈固定相（粒径10μm）;流动相:MeOH:H₂O:HAc=27:73:0.1（v/v/v）;流速:45mL/min;检测波长:254nm;柱温:25℃;进样体积:20 mL;进样量:3.6 g。在此色谱条件下,三个目标化合物均能得到很好的分离,产品纯度≥95%。其中4,5-O-双咖啡酰基奎尼酸单体产品填补了国内空白。论文的第四章为综述。更多还原

【Abstract】 This dissertation dealt with the chemistry of the natural resources of three folk medical plants,Ligularia atroviolacea（Franch.）Hand.-Mazz.（Asteraceae）, Taraxacum mongolicum Hand.-Mazz.（Asteraceae）and Laggera pterodonta（DC.） Benth.（Asteraceae）.The thesis is composed of four chapters.ChapterⅠPhytochemieal Investigation and Evaluation of Anti-HBV Activity of Ligularia atroviolaceaLigularia atroviolacea was distributed in the northwestern Yunnan province, China.Previous investigation of the same plant collected from Yunnan Province led to the isolation of eremophilane derivatives.These interesting findings have prompted us to a phytochemical examination of Ligularia atroviolacea grown in Yunnan.The compounds were isolated by means of silica gel column chromatography,preparative thin layer chromatography and recrystallization,etc. The structures of these compounds were elucidated by spectroscopic experiments (¹H-NMR,¹³C-NMR,¹H-¹H COSY,NOESY,HMBC,HSQC,ESI-MS,HR-MS, UV,IR,X-ray,etc)and chemical methods.24 compounds have been isolated and elucidated from Ligularia atroviolacea,and nine of them are new compounds （Ⅰ-2～Ⅰ-10）.1.8β-H-eremophil-3,7（11）-dien-12,8α;15,6α-diolide（Ⅰ-1）;2.8β-Hydroxy-eremophil-3,7（11）-dien-12,8α;15,6α-diolide（Ⅰ-2）;3.8β-Methoxy-eremophil-3,7（11）-dien-12,8α;15,6α-diolide（Ⅰ-3）;4.Eremophil-3,7（11）,8-trien-12,8;15,6α-diolide（Ⅰ-4）;5.3β-Angeloyloxy-eremophil-7（11）-en-12,8β-olide-15-oic acid（Ⅰ-5）;6.1α-Chloro-6β-isobutyroxy-9-oxo-10β-hydroxy-furanoeremophilane（Ⅰ-6）;7.10β-H-8-oxo-eremophil-3（4）,6（7）-dien-12,15-dioic acid（Ⅰ-7）;8.10α-H-8-oxo-eremophil-3（4）,6（7）-dien-12,15-dioic acid（Ⅰ-8）;9.8β-[Eremophil-3’,7’（11’）-dien-12’,8’α;5’,6’α-diolide]-eremophil-3,7（11）-dien-12,8α;15,6α-diolide（Ⅰ-9）; 10.Ligulatrovine A（Ⅰ-10）;11.Furanoeremophil-3-en-15,6α-olide（Ⅰ-11）;12.Furanoeremophil-15β,6α-olide（Ⅰ-12）;13.1α-Hydroxy-6β-isobutyroxy-9-oxo-10β-H-furanoeremophilane（Ⅰ-13）;14.1α-Hydroxy-6β-isobutyroxy-9-oxo-10α-H-furanoeremophilane（Ⅰ-14）;15.1α,10β-Dihydroxy-6β-angeloyloxy-9-oxo-furanoeremophilane（Ⅰ-15）;16.8β-Hydroxy-eremophil-7（11）-en-12,8α;15,6α-diolide（Ⅰ-16）;17.3β-Angeloyloxy-8β-H-eremophil-7（11）-ene-12,8α;14β,6α-diolide（Ⅰ-17）;18.3β-（2’-methylbutanoyloxy）-8β-H-eremophil-7（11）-ene-12,8α;14β,6α-diolide（Ⅰ-18）;19.β-Sitosterol（Ⅰ-19）;20.Stigmasterol（Ⅰ-20）;21.3β,Δ⁴-Stigmasten-3-ol（Ⅰ-21）;22.Daucosterol（Ⅰ-22）;23.6-O-Acetyl-α-D-glucopyranoside（Ⅰ-23）;24.Cerotic acid（Ⅰ-24）.The structure of compounds（Ⅰ-1）and（Ⅰ-6）were confirmed by X-ray diffraction analysis.Compound（Ⅰ-11）was first obtained as natural products.All other compounds were first reported from this plant.The antiviral research on selected constituents from Ligularia atroviolacea was also investigated.The results indicated that compounds（Ⅰ-1）,（Ⅰ-2）,（Ⅰ-3）and（Ⅰ-4）had significant inhibitory effects on the expression of HBsAg secretion from HepG2.2.15 cells.ChapterⅡPhytochemieal Investigation and Evaluation of Antiviral Activity of Taraxacum MongolicumHerba Taraxaci,which has a long history as a Traditional Chinese Medicine, has different species as well as rich resources in China.Among which,the most popular species is Taraxacum mongolicum.Its roots,stems and leaves have long been used as folk medicine in China,passed with efficacy of inhibiting bacteria, anti-inflammation,resisting endotoxin,strengthening stomach,normalizing the secretion and discharge of bile,improving lactation,inhibiting tumors and immunological regulation.Previous phytochemical constituents of this genus were concentrated on Taraxacum officinale.In order to find out the active components of Taraxacum mongolicum,which was investigated and 40 compounds were isolated and elucidated,and four of them are new compounds（Ⅱ-14～Ⅱ-15 andⅡ-32～Ⅱ-33）.The structures of known and new compounds were mainly elucidated by spectral data and shown to be:1.Artemitin（Ⅱ-1）;2.Quercetin（Ⅱ-2）;3.Quercetin-3’,4’,7-trimethyl ether（Ⅱ-3）;4.Luteolin（Ⅱ-4）;5.Luteolin-7-O-β-D-glucopyranoside（Ⅱ-5）;6.Luteolin-7-O-β-D-galactopyranoside（Ⅱ-6）;7.Genkwanin（Ⅱ-7）;8.Isoetin（Ⅱ-8）;9.3’,5,7-Trihydroxy-4’-methoxyflavanone（Ⅱ-9）;10.Genkwanin-4’-O-β-D-lutinoside（Ⅱ-10）;11.Hesperidin（Ⅱ-11）;12.Quercetin-7-O-[β-D-glucopyranosyl（1→6）-β-D-glucopyranoside]（Ⅱ-12）;13.Quercetin-3,7-O-β-D-diglucopyranoside（Ⅱ-13）;14.Isoetin-7-O-β-D-glucopyranosyl-2’-O-α-L-arabinopyranoside（Ⅱ-14）;15.Isoetin-7-O-β-D-glucopyranosyl-2’-O-α-D-glucopyranoside（Ⅱ-15）;16.Isoetin-7-O-β-D-glucopyranosyl-2’-O-β-D-xyloypyranoside（Ⅱ-16）;17.Caffeic acid（Ⅱ-17）;18.Furulic acid（Ⅱ-18）;19.3-O-Caffeoylquinic acid（Ⅱ-19）;20.3,5-Di-O-caffeoylquinic acid（Ⅱ-20）;21.3,4-Di-O-caffeoylquinic acid（Ⅱ-21）;22.4,5-Di-O-caffeoylquinic acid（Ⅱ-22）;23.1-Hydroxymethyl-5-hydroxy-phenyl-2-O-β-D-glucopyranoside（Ⅱ-23）; 24.p-Hydroxybenzoic acid（Ⅱ-24）;25.p-Coumaric acid（Ⅱ-25）;26.3,5-Dihydroxybenzoic acid（Ⅱ-26）;27.Gallic acid（Ⅱ-27）;28.Gallicin（Ⅱ-28）;29.Syringic acid（Ⅱ-29）;30.Esculetin（Ⅱ-30）;31.Rufescidride（Ⅱ-31）;32.Mongolicumin A（Ⅱ-32）;33.Mongolicumin B（Ⅱ-33）;34.Isodonsesquitin A（Ⅱ-34）;35.Taraxasteryl acetate（Ⅱ-35）;36.φ-Taraxasteryl acetate（Ⅱ-36）;37.Lupenol acetate（Ⅱ-37）;38.Palmitic acid（Ⅱ-38）;39.Stigmasterol（Ⅱ-39）;40.β-Sitosterol（Ⅱ-40）.It can be seen from the above results,the flavonoids and phenolic acids are the main metabolism of Taraxacum mongolicum.The anti-viral research of constituents from Taraxacum mongolicum was also carried out.Isochlorogenic acids display significant antiviral activity.ChapterⅢStudies on Behavior of Chromatographic Separation of Isochlorogenic Acids from Laggera pterodontaLaggera pterodonta is mainly distributed in Africa and southwestern of Asia, especially in Yunnan Province of China.It has anti-flammatory and antibacterial properties.The isochlorogenic acids were the main compounds of Laggera pterodonta.For advanced investigation,the behavior of chromatographic separation of isochlorogenic acids from Laggera pterodonta was studied.In this dissertation,modern technologies such as preparative high performance liquid chromatography and macroporous were employed to purify the three bioactive compounds.Main contents are as follows:1.Flexible analytical methods of effective components in Laggera pterodonta were established in the dissertation.Simultaneous and quick determination methods of three main isochlorogenic acids by HPLC were presented.2.3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid and 4,5-O-di-caffeoylquinic acid were prepared by preparative high performance liquid chromatography method.A new method was presented that three target compounds could be purified in one step.For 300×100 mm chromatography column,the suitable chromatography conditions were obtained to prepare 3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid in one step,such as the solid phase,composition of mobile phase and flow rate.The chromatography conditions were as follows:C₁₈ （grain size 10μm）as stationary phase;mobile phase was MeOH/H₂O/HAc[27:73: 0.1（v/v/v）],the rate of flow was 45 mL/min and sample capacity was 3.6 g.Under the chromatography stations,the purities of three isochlorogenic acids were above 95%.ChapterⅣreviewed the development of eremophilane derivatives.更多还原