【作者】 赵春芳；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2005， 博士

【摘要】 紫杉烷是以抗癌药物紫杉醇为首的、具有五甲基十五碳烯骨架的一类二萜类化合物的总称,其中有些紫杉烷可作为半合成紫杉醇的原料,有些具有多方面的药理活性。紫杉烷化学合成困难,大规模培养植物细胞和种植红豆杉,是获得紫杉醇和其它重要紫杉烷的可行途径,但目前面临着产量不稳定的问题。深刻认识紫杉醇的生物合成机制,通过遗传改造对植物或细胞实施代谢工程是解决这一问题的根本出路。本论文主要从紫杉烷类物质的积累规律和代谢轮廓分析的侧面切入到紫杉醇的生物合成的研究主题。通过构建中国红豆杉植物和细胞提取物中紫杉烷的代谢特征图谱, 利用液质联用方法鉴定谱图中的组分,结合不同的生态条件调查和诱导手段,试图从植物代谢组学的观点和角度研究紫杉烷的生物合成、代谢和积累的规律,在此基础上,探讨可持续性的利用红豆杉资源提高紫杉醇及有药用价值的紫杉烷产量的新技术、新途径。首先研究红豆杉植物或细胞样中微量紫杉烷的精确检测方法。发现用甲醇浸提,固相萃取( SPE)方法制样,以乙腈-甲醇-水三元流动相等度洗脱和C18 柱反相色谱体系、检测波长为227nm,对紫杉醇、10-去乙酰巴卡亭III 、巴卡亭III 、巴卡亭VI 和1-乙酰-5,7,10-去乙酰巴卡亭I 等5 种紫杉烷同时检测,最低检测限为0.5～4.5 mg/L,紫杉醇在2.0～180 mg/L (r= 0.9993)、10-DAB 在2.0～166.0 mg/L (r=0.9975)、B III 在6 ～170 mg/L (r=0.9937)、B-VI 在7.0～200.0 mg/L(r=0.9921)、DAB-I 在1.0～130 mg/L (r=0.9945)范围内具有良好的线性关系; 紫杉醇回收率为98%±1.5%,日内RSD 为1.8%,日间RSD 为2.5%。在此基础上,经固定提取和粗分离方法,获得了植物和细胞样中紫杉醇极性相关的一组紫杉烷的HPLC 色谱图,并通过了稳定性、精密性等方法学验证; 接着,进一步探讨了利用特征图谱对不同品种、不同植物组织部位的紫杉烷化学组进行动态分析的方法。利用液质联用确定了中国红豆杉细胞培养物所产生的主要紫杉烷。通过遗传相关性和广泛的文献查阅,可以确定分子结构的有Taxuyunnanine C 及其C14位丙酰和异戊酰取代衍生物,和yunnanxane、taxol、B III、DAB-I、B-VI 等8种紫杉烷,另外5个化合物获得了分子量信息,并根据质谱裂解规律归属了它们分子结构类型、取代基种类和数量,提出了可能的分子结构式。更多还原

【Abstract】 Taxanes are a group of specific diterpenoid compounds with a pentamethyl [9.3.1.0] 3,8 tricycopentadecane taxane skeleton including taxol and taxotere, a type of the most effective anticancer drugs. There are over 350 members in this family, classified according to their structural differences. Each class of the taxanes has different polar and pharmaceutical properties and special biological activities. It is very difficult to synthesize taxanes by chemical means. The increasing clinical demands for active taxanes and the exhausted supply of natural Taxus, initial source of taxanes, have spurred to produce these drugs or useful precursors by means of large-scale Taxus cell culture and Taxus planting, which however were hampered by low and unstable taxol yields. Presently, the manipulation and regulation of the metabolic pathway at molecular level for the Taxus cell culture system are believed to be a promising approach to increase the yields of taxol and its related taxanes. Any attempt to manipulate genes of the metabolic pathway requires a full understanding of taxol biosynthesis, including key enzymes, reactions, occurrent sites, and mechanisms of exudation, transport and storage after syhthesis. The dissertation presented here emphasized the study of taxol biosynthesis on metabolic profiling analysis of Taxus chinensis , and the new approaches to improve the yields of taxol and related taxanes in plant cuttings as well as in extracts by incubating clipplings and selective oxidation-hydrolysis were also studied. Firstly, the methods to determine minim taxanes which present in methanol extracts of Taxus chinensis plant or cell cultures were established. Solid-phase extraction (SPE) method was used for sampling after plant materials were ground with the help of liquid nitrogen and then extracted by methanol. And the chromatographic conditions of a C18 column (Hypersil ODS2 , 250 mm ×4.60 mm, 5 μm), the mobile phase being composed of acetonitrile-methanol-water, which eluted isocratically at flow rate 1 ml/min) and detection at 227 nm were employed. The calibration curve showed a good linearity in the mass concentration range of 2.0～180 mg/L for taxol (r= 0.9993), 2.0～166.0 mg/L for 10 –DAB-III(r=0.9975), 6~170 mg/L for B III (r=0.9937), 7.0～200.0 mg/L for B-VI(r=0.9921), 1.0～130 mg/L for DAB-I(r=0.9945), respectively. The detection limits of this method were 0.5～4.5 mg/L. The recovery for taxol was 98% with relative standard deviation (RSD) 1.5%.The inter days RSD of this method was 1.8% and between days 2.5%. The characteristic chromatograms including taxane compounds as more as possible were obtained following the above sampling methods and the chromatographic conditions. The metabolic profiling of the taxanes presented in Taxus chinensis cell culture extracts were characterized. About 13 taxane compounds were identified or characterized by means of liquid chromatography mass. Eight of them were identified as taxuyunanine C , yunnanxane , 2α,5α,10β-triacetoxy-14β-propionyloxytaxa-4(20),11-diene, 2α,5α,10β-triacetoxy-14β-(2-methyl)butyryloxytaxa-4(20) 11-diene , taxol , and Baccatin III, DAB-I, B-VI, respectively. Others were given the molecular weight, taxane skeletons, types and numbers of substituents. The dynamic profiling analysis of the taxanes by methyl jasmonate (MJ) elicitation and fungal elicitor revealed that the cell lines had the capability of production of a large assortment of other compounds possessing positionally different acylation patterns, especially formation of taxanes acetylation at C14-side, which may lead to the low production of taxol. The metabolism and accumulation of taxanes in plants was also investigated. It was found that (1) the needle of yew was a important organ or part to biosynthesize taxanes; (2) the synthesis of taxanes seemed to be non-tissue specificity; (3) taxanes accumulation tended to be polar trend, the non-polar taxanes preferred to accumulation in the sites of plant where have thick cell walls such as bast, in contrast, and polar taxanes could be detected frequently in leaves; (4) the distribution of taxanes in a part of plant related to the changes of seasons; (5) high content taxol often were found in the areas where Taxus good-growth. The variations of taxane contents in plant after harvest were also studied. It was observation that the accumulation of taxol in needles of the clippings was found increase in the initial stages of the stored period and then decreased gradually, the variation was related to the manner of preservation, timing and plant tissue, indicating taxol biosynthesis in response to mechanical wounding of harvest. Based the results, a novel method for in vitro incubating Taxus clippings to improve taxol yields was proposed. An application of selective oxidation-hydrolysis reactions was conducted to convertxyloside or glucoside taxanes into corresponding free taxanes for enhancement of taxol and it related taxane yields. With the new method, recovery of 168% could be achieved for taxol, 142% for 10-DAT, indicating the significance and potential of the method to make full use of plant sources.更多还原