绣线菊二萜生物碱生物合成的研究及滑桃树种壳的化学成分分析
其他题名Studies on Biosynthesis of Diterpene alkaloids of Spiraea japonica and Chemical Constituents of Seed Endothelium of Trewia nudiflora
赵沛基
学位类型硕士
导师沈月毛
2004
学位授予单位中国科学院昆明植物研究所
学位授予地点中国科学院昆明植物研究所
学位专业植物学
关键词绣线菊 二萜生物碱 饲喂 生物合成 串联质谱 滑桃树 种壳 新木脂素 抗菌活性
摘要本论文建立了绣线菊二萜生物碱的LC-ESI-MS定性和定量检测方法。根据电喷雾二级串联质谱的撞击能量差异可以区别绣线菊二萜生物碱atisine和hetisine的骨架。在优化质谱条件下,通过分析9个绣线菊二萜生物碱的质谱裂解途径,发现hetisine型二萜生物碱的二级质谱裂解能比atisine型二萜生物碱的二级质谱裂解能高因此可以根据电喷雾二级串联质谱的撞击能量差别,简便快速地鉴定粉花绣线菊不同变种中二萜生物碱的结构类型;用Xtalre C 18柱分离绣线菊二萜生物碱,流动相为甲醇一水[95:5(水中含0.1%三乙胺)],使用电喷雾离子阱质谱检测,建立了粉花绣线菊中二萜生物碱的LC-ESI-MS定量检测方法;在粉花绣线菊椭圆叶变种(SPiraeajoponica vara ovalolia)无菌苗的悬浮培养系中,检测到了多个atisine型二萜生物碱。在200mg/L的剂量下,几丁质可以诱导无菌苗悬浮培养系中spiramineA/B的产量高达150mg/L(是空白对照的8倍)。饲喂双标记的丝氨酸(2-13C,15N-serine),通过LC-ESI-MS和多级质谱检测,在产物中发现含有同位素标记的绣线菊二萜生物碱(21-13C,15N-spiramineA/B和21-13c,15N-spiraxninec。),21-13C,15N-spiramineA/B的含量约为spiralnine A/B的1/20,表明绣线菊二萜生物碱的氮源为丝氨酸,首次证明atisine型二萜生物碱的合成途径是从二萜到二萜生物碱(Fig.1)。从滑桃树(Trewta nudiflora)种壳的乙醇提取物中分离到H个化合物,它们的结构分别鉴定为:9'-甲基-americano1A(1)、9'-乙基-isoalnericanolA(2)、9'-乙基-americanofA(3)、9'-丁基-americano1A(4)、2,3-二氢-3,6,7-三羟基-1-苯丙吡喃-4-酮(5)、americanin(6)、isoamericanin(7)、α,2,,4,4'-tetrahydroxydidrochalaone(8)、isoliquiritigenin(9)、3,4-二羟基桂皮醛(10)和α-谷甾醇(11),其中化合物1-5是新化合物(Fig.2)。制备了化合物6的两个乙酰化产物3,4-二乙酰基-amerioanin (6a)和3,4,9-三乙酰基-americanin(6b)。以羧节青霉素钠盐、硫酸链霉素和利福平为阳性对照,检测了十二个化合物(1、2、3、4、5、6、7、8、9和10以及6a和6b)(结构式见第二章图l,p26)的抗细菌活性:化合物4、6和9对革兰氏阳性菌金黄色葡萄球菌(Staphylococcus aureus)和革兰氏阴性菌结核分枝杆菌(Mycobaclerium tuberculosis)有抑菌作用,其中化合物4对两种菌的最低抑制浓度均为50μg/mL,化合物6对两种菌的最低抑制浓度均为100μg/mL,但是化合物6的两个乙酰化产物在200μg的剂量下没有表现出抑菌活性。这是首次报道天然新木脂素类化合物的抗细菌作用。
其他摘要The qualitative LC-ESI-MS detection methods of diterpene alkaloids (DAs) of atisine-and hetisine-type and quantitative measurement of DAs in tissue cultures of Spiraea japonica were established. The two different skeleton types of DAs from S. japonica var. were distinguished by the difference of ESI-MS2 collision energy of their MS fragmentations. Under the optimized instrumental conditions, MS fragmentations of 9 diterpene alkaloids from S. japonica var. were analyzed, which indicated that the collision energy of the secondary dissociation of atisine-type DAs was obviously lower than that of hetisine-type ones. So the difference of collision energy of ESI-MS2 could be used as evidence to simply and rapidly identify the structural types of DAs isolated from S. japonica. DAs were isolated by Xtarre Cjs column eluted with methanol-water (95: 5, v/v, 0.1% aqueous trienthylamine), and detected by ESI ion trap mass spectrometer. A quantitative analysis method of DAs of S. Japonica was established, which showed that the stem suspension cultures of Spiraea japonica could produce atisine-type DAs under normal condition, and that the content of atisine-type DAs was increased 8-flod by the inducement of chitosan at 150 mg/L compared with the controls. The double labeled 2- C, N-serine was fed into the stem suspension cultures which incorporated spiramines A/B and C/D to produce 2-13C, I5N-spiramines A/B and 2-13C5 15N-spiramines C/D indicated by ESI-LC-ESI-MS and tandem MS analysis. The content of 2-13C, 15N-spiramine A/B was 1/20 of spiramine A/B, indicating that serine was the nitrogen source of atisine-type diterpene alkaloids and the biosynthesis pathway of atisine-type DAs was proved to be from diterpene to DAs for the first time. Eleven compounds were isolated from the seed crusts of Trewia nudiflora. The structures were determined to be 9'-methoxy-7'-en-3',8:4',7-diepoxyneolignan-3,4,9- triol (1), 91-methoxy-7I-en-3l)7:4's8-diepoxyneolignan-3,4,9-triol (2), 9'-ethyoxyl- 71-en-3'8:41,7-diepoxyneolignan-3,4,9-triol (3), 9'-butyloxyl-7'-en-3',8:4',7-diepoxy- neolignan-3,4,9-triol (4), 3,6,7-trihydroxy-l-benzopyran-4-one (5), 9'-aldehyde-7'-en- 3'57:4',8-diepoxyneolignan-3,4,9-triol (6), 9'-aldehyde-7'-en-3',8:4',7-diepoxyneo- lignan-3,4,9-triol (7), cc,2',4,4'-tetrahydroxydidrochalaone (8), isoliquiritigenin (9), 3,4-dihydroxy-7-en-phenylpropyl aldehyde (10) and p-sitosterol (11). Compounds 1~5 were determined to be new. Two acetylated products of compound 6, 9'-aldehyde-3I,7,4',8-diepoxy- neolignan-3,4,9-triacetyl (6a) and 9'-aldehyde-3',7:4',8-diepoxyneolignan-3J4- diacetyl-9-hydroxy (6b) were prepared. The antibacterial activities of twelve compounds (1、2、3、4、5、6、6a、6b、7、8、9 and 10) were assayed. Compounds 4 and 6 exhibited antibacterial activity against Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Mycobacterium tuberculosis at the minimum inhibitory concentration (MIC) of 50μg/mL and 100 μg/mL, respectively. The two acetylated products of 6 did not exhibit antibacterial activity at 200μg/disk, and compound 9 also showed weak activity against the two tested strains by disk diffusion method. The antibacterial activity of neolignans was reported for the first time in our work.
页数70
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/722
专题昆明植物所硕博研究生毕业学位论文
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赵沛基. 绣线菊二萜生物碱生物合成的研究及滑桃树种壳的化学成分分析[D]. 中国科学院昆明植物研究所. 中国科学院昆明植物研究所,2004.
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