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题名: 植物环肽酶环化,环二肽后合成,新环肽及其它植物化学成分
作者: 贾爱群
学位类别: 博士
答辩日期: 2004
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 周俊
学位专业: 植物学
中文摘要: 本论文主要由三部分组成。第一部分共二章,研究了太子参环肽B (Heterophyllin B)的酶环化反应及传统中药三七(Panax notoginseng(Burk.)F.H.chen)中三个环二肽的后合成。ostharvest synthesis);第二部分共三章,研究了两种石竹科植物——丽江蚤缀(A renaria oreophila Hook.)以及漆姑草(Saginajaponica Ohwi)中新化合物的结构解析,并通过对三种北极石竹科植物StellariacrossiPes、cerastiuni arcticum和C regelii中五种环肽的初步解析,进一步证明了环肽作为石竹科特征成分之一的观点,初步佐证了北极和青藏高原区域是石竹科两大分布中心之一,附带通过GC-MS分析了该三种北极石竹科的挥发油成分,佐证了卷耳属和繁缕属有较近的亲缘关系:论文的第三部分综述了近年来(1994-2003)非石竹科植物中环肽的分布及其活性。利用多种植物生理技术(包括细胞破碎实验、分子排阻层析、超滤、透析、离子交换层析以及冷冻干燥等)对太子参环肽B进行了酶环化反应研究,利用对照实验,并结合植化手段,证明了三七中三个环二肽是后合成产物;利用植化的各种材料和方法(包括常压、低压硅胶柱层析,D101大孔树脂,MCI-gel CHP20P,Sephadex LH-20,Rp-8及RP-18,制备HPLC,GC-MS等),从以上五种石竹科植物中共分离鉴定了47个化合物,其中发现了18个新化合物,化合物类型涉及植物环肽、植物蜕皮甾酮(C27)及其苷、C27甾体皂苷、黄酮及其苷、香豆素、肉桂酸等。另外在检测(分离环肽的基础上,我们通过GC-MS对北极的3种石竹科植物进行了挥发油成分鉴定,共指认了其中的54个挥发油成分。一.植物环肽的两个合成问题1.太子参环肽B的酶环化反应自1991年我们组从太子参中发现太子参环肽以来,历经10多年共分离鉴定植物环肽100余个,其中多为石竹科环肽,不仅建立了完善的植物环肽检测分离和鉴定方法,而且为环肽作为石竹科的特征成分提供了大量的资料积累。但植物环肽的生源问题一直困扰我们,为此我们提出了3种假说,我们遵循从简单入手(abitio)的原则设计了生物合成路线。并通过对太子参环肽B的酶环化实验,初步证明了该环肽是在该植物体内环化酶的作用下,由相应的链肽转化而来的。该实验的成功也同时验证了甘氨酸和苯丙氨酸之间是该类植物环肤进行环化的位点的统计结果;同时该实验也是迄今为止国内外第一个植物环肽的酶环化合成。2.三七中三个环二肽的后合成,植物的后合成(postharvest synthesis)是指某种植物在采摘后干燥过程(postharvest treatments)中产生了某些化学成分,这些成分是活体内原来没有的或原来有而在后来发生量的增减变化。1968年德国的Kucera M.,staryE和Pitra J.发现毛花洋地黄(Digitalis lanataEhth.)毛花苷的后合成现象并提出了该概念。36年之后,我们对三七中的环二肽进行了后合成实验,结果发现其中的3个环二肽是后合成产物。二.五种石竹科植物的化学成分1.三种北极石竹科植物的环肽成分及其化学分类和分布意义.2002年10月,中国科学院北极考察队从挪威的Svalbard(74-81°N,10-35°E)带回3种各样均小于100克的石竹科植物全草stellaria crassiPes,cerastium arcticum和C regelii,本论文对该3种植物的化学成分尤其是环肽成分进行了研究,通过现代分析、分离手段(HPLC,AAA,FAB+-MS等)共分离纯化了6个新植物环肽,并初步鉴定了其中5个。在此基础上我们试图为石竹科环肽的化学分类及石竹科植物的分布和演化提供佐证。另外在检测分离北极石竹科植物环肽的基础上,我们通过GC.Ms对北极的3种石竹科植物进行了挥发油成分鉴定,共指认了其中的54个挥发油成分。”分别从£crassiPes,c arcticum和C.regelii的甲醇提取物中分离得到6个新环肽1-6,分别命名为arcticuminA(1)、arcticuminB(2)、arcticuminC(3)·regelinA(4)、erassiPinB(5)和crasscrassipinA,其中1-5的化学结构得到了初步解析。北极石竹科环肽的发现佐证了石竹科第二分布中心的存在以及环肽是石竹科的特征成分。并通过对该3种植物的挥发油成分分析,佐证了卷耳属和繁缕属有较近的亲缘关系。2.两种石竹科植物的化学成分(1).丽江蚤缀的化学成分作为石竹科环肽和其它化学成分研究的继续,我们从丽江蚤缀(A renaria oreoPhila Hook)全草的乙醇提取物中共分离鉴定了16个化合物(丽江蚤缀环肽A-H(6-13),E-对-甲氧基肉桂酸(14),Z-对-甲氧基肉桂酸甲酯(15),Z-对-甲氧基肉桂酸(16),E-对-羟基肉桂酸甲酯(17),柚皮素(Naringenin)(18),胡萝卜昔(19),β-谷甾醇(20),5,7,4'-三羟基-3',5'-二甲氧基黄酮(21)),其中8个新植物环肽(丽江蚤缀环肽A-H(6-13))。在分离过程中初步检测显示有微量生物碱的存在。(2).漆姑草的化学成分我们从漆姑草(Sagina jaPonica ohwi.)全草的乙醇提取物中共分离鉴定了26个化合物(漆姑草环肽A-D(22-25),蜕皮甾酮A(26),E-对甲氧基肉桂酸甲酯(27),Z-对-羟基肉桂酸甲酯(28),伞形花内酯(29),7-甲氧基香豆素(30),6-羟基香豆素(31),6-甲氧基香豆素(32),5,7-二羟基香豆素(33),5,7-二甲氧基香豆素(34),7-O-β-D-葡萄叶,南糖苷-6-C-木糖吡喃糖苷-5,4'-二羟基黄酮(35),2″-O-β-D-木糖吡喃糖苷-8-葡萄吡喃糖苷-5,4’-二羟基黄酮(36),,(25R)-螺甾-5-烯-1β,3β-二醇1-O-{O-α-L-鼠李吡喃糖苷-(1→2)-O-[β-D-木糖吡喃糖苷-(1→3)]-β-D-岩藻吡喃糖苷}(OphiopogoninD)(37),(25R)-ruscogenin 1-O-[2-O-(乙酰基)α-L-鼠李吡喃糖苷-(1→2)][β-D-木糖吡喃糖苷-(1→3)〕-β-D-岩藻吡喃糖苷(38),(25R)ruscogenin1-O-[3-O-(乙酰基)-α-L鼠李吡喃糖苷-(1→2)][β-D-木糖吡喃糖苷-(1→3)〕-β-D-岩藻吡喃糖苷(39),5,7’-三羟基-6-六甲氧基黄酮(e naxinll)(40),5,7,2’-三羟基-8-六甲氧基黄酮(Scutevulin)(41),5,7-二羟基-8,2’-二甲氧基黄酮(42),5,7,3’,4’-四羟基-6-甲氧基黄酮(43),蜕皮甾酮(44),Shidasterone(45),3,16p,29-三羟基-4β-醛基-12-烯-28-酸齐墩果烷(46),正十九烷-10-醇(47),其中4个新植物环肽(22-25),1个新蜕皮甾酮(26)。运用本组建立的环肽检测方法——茚三酮原位反应TLC法分离跟踪到3个脑苷脂类化合物,对该3个化合物的鉴定工作有待继续。论文的最后对1994年以来非石竹科植物的环肽成分进行了综述,包括环肽的名称、分子式、分子量、旋光、熔点、生物活性、来源及参考文献等方面。
英文摘要: The thesis is mainly composed of three parts. The first part including two chapters is about detailed description of the enzymatic cyclization of Heterophyllin B (I, HB), and the postharvest synthesis of cyclodipeptides II-IV from Panax notoginseng. The second part involving three chapters deals with chemical constituents of two Caryophyllaceae plants named Arenaria oreophila Hook, and Sagina japonica Ohwi from Yunnan Province, and three Caryophyllaceae plants named Stellaria crassipes、Cerastium arcticum and C. regelii from Acric Regions. The last part reviews the recent progress (1994-2003) of cyclopeptides from plants of non-Caryophyllaceae families. As the first enzymatic cyclization of higher plant cyclopeptides in the world, we isolated the crude enzymes which cyclized to produce HB successfully. By controlling experiment and the traditional phytochemical methods, it was proved that the three cyclodipeptides II-IV from Panax notoginseng were postharvest synthesis products. Systematic phytochemical investigations on above Caryophyllaceae species led to the isolation and elucidation of 47 compounds, in which 18 compounds are new ones. The elucidated structures covered cyclopeptides, ecdysterones (C27) derivatives, C27 steroidal saponins, flavonoids, cumarins, and aromatic derivatives etc. Additional, 54 constituents were identified from three Arctic Region Caryophyllaceae plants by GC-MS. 1. Two questions on the synthesis of cyclopeptides 1.1. Enzymatic cyclization of Heterophyllin B (HB)Since 1991, over 100 cyclopeptides were isolated from plants by our group, especially from Caryophyllaceae. Based on these researches, we established the methods of detecting, isolating and elucidating of plant cyclopeptides, and further more, we think that the cyclopetides are the characteristic components of Caryophyllaceae. But the biogenesis of the cyclopeptides has puzzled us for these years. To answer the question, we designed the enzymatic cyclization method of HB obeying the ab initio rule. The success of enzymatic cyclization experiment of HB proved that there is/are enzyme(s) in the body of Pseudostellaria heterophylla, and under the catalyzed of the enzyme(s), HB (I) could be cyclized from the corresponding linear peptide. The conclusion also proved that the segments which consist of NH-Phe-Gly-CO residue could be cyclized into cyclopeptides. This is the first enzymatic cyclization of higher plant cyclopeptides in the world. 1.2.. The postharvest synthesis of three cyclodipeptides in Panax notoginseng The postharvest synthesis is an interesting phenomenon in the plants, it referred to some components have been produced or increased the mass of the components during the wilting pereoid. In 1968, Kucera M., Stary R and Pitra J. first found this phenomenon in Digitalis lanata Ehrh. and give the definition. During the investing of the components of Panax notoginseng, we found that the three cyclodipeptides II-IV named cyclo(Tyr-Leu) (TI), cyclo(Ile-Ala) (111), cyclo(Leu-He) (IV) are postharvest synthesis products during the wilting periods. 2. Chemical constituents of five Caryophyllacea species 2.1. Cyclopeptides from three Arctic Caryophyllaceae plants, chemotaxonomy and distribution significances of Caryophyllaceae cyclopeptides Three arctic Caryophyllaceae species named Stellaria crassipes, Cerastium arcticum and C. regelii were collected from Svalbard (74-81° N, 10-35° E) (<100g), 59 compounds were isolated and identited by modern analytical amd isolated methds, such as HPLC, FAB+-MS, GC-MS and so on, among these compounds, there are 6 new cyclopeptides named arcticumin A (1)> arcticumin B (2^ arcticumin C (3)n regelin A (4)-, crassipin B (5) and crassipin A, and 1-5 were identified cyclopeptides primarily. Based on these data, it is proposed that cyclopeptides are characteristic components of Caryophyllaceae and Arctic region is one part of the distribution center of Caryophyllaceae. By analysis of the constituents of volatile oils from three Caryophyllaceae species in Arctic Region, it is further proved that that Stellaria and Cerastium are two generas that are close relationship. 2.2. Chemical constituents from two Caryophyllaceae species 2.2.1. Chemical constituents from Arenaria oreophila Hook. As a continued study of Caryophyllaceae cyclopeptides and also a systematic research on other constituents, two Caryophyllaceae species Arenaria oreophila Hook and Sagina japonica Ohwi were investigated. From the former species, 16 compounds were isolated and elucidated as arenariphilin AC 6), arenariphilin B(7), arenariphilin C(8), arenariphilin D(9), arenariphilin E(10), arenariphilin F( 11), arenariphilin G (12), arenariphilin H (13), p-E-methoxycinnamic acid (14), 3-(4-methoxyphenyl)-2-Z-propenoic acid methyl ester (15), p-Z-methoxycinnamic acid (16), p-E-hedroxycinnamic acid methyl ester (17), Naringenin (18), daucosterol(19). 0 -sitosterol(20), 5, 7, 4' -trihydroxy-3', 5' -dimethoxyl flavone (21)。 Compounds 1-8 are new cyclopeptides. In this species, we also discovered primarily minor alkaloids, but couldn't obtain them due to their low content. 2.2.1. Chemical constituents from Sagina japonica Ohwi S. japonica is a folk medicine used for anti-inflammation. From the ethanol extracts of whole plants 26 compounds were isolated and elucidated as japonicin A (22), japonicin B (23), japonicin C(24), japonicin D (25), japonicone A (25), 3-(4-methoxyphenyl)-2-E-propenoic acid methyl ester(27), β-Z-hedroxycinnamicacid methyl ester (28) , umbelliferone (29), 7-methoxycoumarin (30), 6-methoxycoumarin (31), 6-methoxycoumarin (32), 5, 7- β-hydroxycoumarin (33), 5,7-β-methoxycoumarin (34), 7-0-β -D-Glucopyranosyloxy-isomollupentin(35) , 2"-0-β -D-Xylopyranosyl-8-Galactopyranosyloxy-4',5,7-trihydroxyflavone (36), (25-R-)Ophiopogonin D, (25R)-spirost-5-ene-1 β,3 β -diol[(25R)-ruscogenin]l-O-{O-α-L-rhamnopyranosyl-(l-"2)-O-[β -D-xyloptranosyl-(l→3)]-β-D-fucopyranoside} (37), (25 R)-ruscogenin l-0-[2-0-(acetyl)- α -L-rhamnopyranosyl-(l→2)][β-D-xylopyranosyl-(l→3)]-β-D-fucopyranoside (38), (25 R)-ruscogenin l-O-[3-0-(acetyl)-α-L- rhamnopyranosyl-(l→2)][ β-D-xylopyranosyl-(l→3)]-β-D-fucopyranoside (39), Tenaxin II (5,7,2'-trihydroxy-6-methoxyflavone) (40), Scutevulin (5,7,2'-trihydroxy-8-methoxyflavone) (41), 5,7-dihydroxy-8,2'-dimethoxy flavone (42), 5,7,3',4'-tetrahydroxy-6-methoxyflavone (43), Ecdysterone (44), Shidasterone (45), 3, 16β , 29-trihydroxy-olean-4β-ald-12-ene-28-oic acid (46), 10-hydroxy-n-ninteenane (47)。Among these compounds, compounds 22-26 are new ones (four new cyclopeptides and one new ecdysterone). The end part of the thesis reviewed the recent progress (1994-2003) of cyclopeptides which isolated from plants of non-Caryophyllacea in aspects of compound name, molecular weight, molecular formula, optical rotation, melting point, bioactivity, source, and reference.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/708
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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植物环肽酶环化,环二肽后合成,新环肽及其它植物化学成分.贾爱群[d].中国科学院昆明植物研究所,2004.20-25
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