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伊贝母化学成分及其生物活性研究
李燕
学位类型博士
导师阿吉艾克拜尔·艾萨
2016-06-03
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词伊贝母 维吾尔药 化学成分 结构鉴定 生物活性评价
摘要

伊贝母(Fritillaria pallidiflora Schrenk)是多年生草本植物,是百合科贝母属伊犁贝母的干燥鳞茎,也是药材伊贝母的主流品种,另一种是新疆贝母(Fritillaria waluiewii Regel)。这两个贝母品种在《中国药典》2000年版本中收载;同时作为传统维药也被《维吾尔药志》收录,具有清热润肺、化痰止咳的功效,可用于肺热燥咳、干咳少痰、咳痰带血等症。古代维吾尔医用当地贝母治疗疾病,18世纪后期,在维吾尔医药中有临床使用历史。伊犁贝母(F. pallidiflora)、新疆贝母(F. walujewii)、托里贝母(F. tortifolia)、裕民贝母(F. yuminensis)和砂贝母(F. karelinii )等为新疆地区分布广泛的5个特有品种。伊犁贝母(F. pallidiflora)、砂贝母(F. karelinii )、裕民贝母(F. yuminensis)等也分布于中亚古丝绸之路这一带地区。关于贝母品种的药效物质基础研究,对川贝、浙贝、平贝、湖北贝母等的镇咳平喘的药效物质基础及作用机制研究已较为深入。到目前为止,已从贝母属植物中分离并鉴定了200多个化合物,包括生物碱、皂苷、萜类和甾体类等化合物。有关伊贝母中化学成分的提取分离和结构鉴定及其相关的药理学研究尚处于初始阶段。因此对伊贝母中具有生物活性的化学成分进行提取分离研究很有必要,对伊贝母镇咳、祛痰作用机理的探究不仅具有很大的学术价值,而且对伊贝母工业化生产以及开发应用具有现实意义。本论文包括伊贝母化学成分的提取分离、伊贝母鳞茎有效部位提取工艺和生物活性研究等三个方面内容。第一章对贝母属植物的分类及资源地理分布,其化学成分的研究和生物活性的研究进行了系统的阐述。第二章通过集成植物化学现代的提取分离方法和四大波谱学方技术,对伊贝母鳞茎进行系统的分离纯化和鉴定研究,从伊贝母中分离得到了17个化合物,其中生物碱类化合物14个,包括5个新化合物,4个首次本植物中得到,具体为如下:伊贝酮A(yibeinone A)、伊贝酮B(yibeinone B)、伊贝酮C(yibeinone C)、伊贝酮D(yibeinone D)、伊贝酮E(yibeinone E)、西贝母碱苷(imperialine 3β-D-glucoside)、西贝母碱(imperialine)、浙贝酮葡糖苷(zhebeinone 3β-D-glucoside)、西贝素氮氧化物(imperialine β-N-oxide)、dongbeinine、新贝甲素(sinpeinine A)、裕贝甲素(yubeinine)、鄂贝乙素(ebeinone)、梭砂贝母酮碱(delavinone);非生物碱化合物3个:3-3′-联苯二甲酸二丁酯、阿魏酰甘油酯、胡萝卜苷。其中伊贝酮A(yibeinone A)、伊贝酮B(yibeinone B)、伊贝酮C(yibeinone C)、伊贝酮D(yibeinone D)、伊贝酮E(yibeinone E)为新化合物;浙贝酮葡糖苷(zhebeinone 3β-D-glucoside)、dongbeinine、鄂贝乙素(ebeinone)、梭砂贝母酮碱(delavinone)为首次从伊贝母中分离得到。第三章对伊贝母鳞茎有效部位提取工艺和分离纯化工艺进行了优化。采用回流提取方法,通过单因素实验和正交试验确定了最佳提取工艺:75%乙醇溶液,20倍量液料比,70℃提取温度,1.5 h提取时间,提取3次。确定最佳纯化工艺:使用Amberlite FPC 22Na大孔强酸性阳离子树脂,上样液浓度为0.39 mg/mL,pH5,上样体积为4 BV,以流速为4 BV/h进行吸附,用去离子水洗脱吸附饱和的树脂,6 BV水洗。1 mol/L氨水的95%乙醇洗脱,洗脱8 BV,4 BV/h流速。提取物中含有2.16%生物碱,该方法简单可行,树脂可以重复使用,安全可靠,对开发伊贝母相关经济产品具有指导意义。第四章进行伊贝母单体和提取物的生物活性研究,涉及到四部分内容。第一部分:急毒实验。伊贝母提取物单次灌胃给药,确定小鼠的最大耐受量为2 g/kg;第二部分:对伊贝母药材中分离得到的单体化合物和有效部位进行了平喘初筛的研究。应用诱导剂诱发的大鼠气管平滑肌收缩模型筛选出具有舒张效应的活性化合物,得出结论:具有一定舒张作用的受试物分别为:FPSA11、FPSA07、FPSA03、FPSA02、FPSA06、提取物I、提取物Ⅱ;第三部分:在初筛基础上进行了平喘功效的研究,应用诱导剂诱发的大鼠气管平滑肌收缩离体模型研究活性化合物的拮抗参数(pA2)和半数有效量(EC50)。拮抗参数越大,说明受试物的拮抗作用越强,因此受试物的拮抗作用从强到弱依次为:FPSA02受试物>氨茶碱、提取物Ⅰ、提取物Ⅱ> FPSA07受试物> FPSA03受试物、硝苯地平;第四部分:对于伊贝母药材提取、纯化得到的有效部位,通过进行动物实验研究伊贝母有效部位对浓氨水引起小鼠咳嗽次数与咳嗽潜伏期的影响,以及对小鼠腹腔注射酚红后引起气管酚红排泌量的影响。得出结论:伊贝母提取物0.5 g/kg能够在延长咳嗽潜伏期、减少咳嗽次数、促进酚红分泌方面组间比较具有显著性差异(P < 0.01);此外,伊贝母提取物0.25 g/kg在延长咳嗽潜伏期方面组间比较具有统计学意义(P < 0.01)。伊贝母提取物具有一定镇咳化痰功效,具有进一步研究和开发的价值。

其他摘要

Fritillaria pallidiflora Schrenk belonging to the Fritillaria genus of Liliaceae family. The dried bulbs of this plant are used as one of important source of Fritillaria pallidiflora for antiasthmatic, anti-tussive and expectorant agents in traditional Chinese medicine (TCM), the other is Fritillaria waluiewii Regel. Both of them are recorded in Chinese Pharmacopoeia (2000), and in Uighur Medicine as traditional medicine. Ancient uygur mediciner used local Fritillaria to treat diseases. In the late 18 century, the clinical application has a history in uygur medicine.F. pallidiflora, F. walujewii, F. tortifolia, F. yuminensis and F. karelinii were special knids of plant in Xinjiang. F. pallidiflora, F. karelinii and F. yuminensis were distributed on the historic silk route in Central Asia.On the material foundation of antiasthmatic, anti-tussive afficacy, Fritillaria cirrhosae, Fritillaria thunbergii, Fritillaria ussuriensis, Fritillaria hupehensis have been studied deeply. To date, above 200 compounds have been isolated and identified from Fritillaria plants, including alkaloids, saponins, terpenoids and steroid compounds,et al. At present, the research of chemical constituents and pharmacocology from F. pallidiflora is still at an initial stage. So far it is necessary to study extraction and isolation of constituents with bioactivity from F. pallidiflora. Antitussive and expectorant effect mechanism to explore not only of great academic value, but also a realistic significance to industrial production and application of F. pallidiflora.This paper contains three aspects: isolation and separation of chemical constituents, extraction process of active ingrent and bioactivities from F. pallidiflora.In the first chapter, we introduced the species of the Fritillaria genus and their distribution, further chemical composition and biological activities.In the second chapter, 17 compounds were isolated and identified by the modern spectroscopy methods and techniques. Five new alkaloids, yibeinone A, yibeinone B, yibeinone C, yibeinone D, yibeinone E, together with nine known alkaloids, imperialine 3β-D-glucoside, imperialine, zhebeinone 3β-D-glucoside, imperialine β-N-oxide, dongbeinine, sinpeinine A, yubeinine, ebeinone, delavinone, three non alkaloid, were isolated from F. pallidiflora. Compounds zhebeinone 3β-D-glucoside, dongbeinine, ebeinone and delavinone were isolated in the first time from this gene.The research of extraction, isolation and purification of F. pallidiflora was reviewed in the third chapter. From the reflux extraction, single factor test and orthogonal test, the optimum extraction conditions were extracted 3 times by 20 BV of 75% ethanol at 70℃ degree. The optimum purification process conditions were subjected to the Amberlite FPC 22Na cationic resin. The sample concentration was 0.39 mg/mL, adjusted pH to 5. The solution treatment capacity was 4 BV and the flow rate was 4 BV/h. After washing with 6 BV water, 95% ethanol with 1 mol/L ammonium hydroxide was used as eluent, and 8 BV at the flow rate 4 BV/h. After purification, total alkaloids content was 2.16%. This method was simple, safe and reliable and the resin was used repeatly. It provided guiding significance for economic products development of F. pallidiflora.In the forth chapter, we studies on the bioactivities of alkaloids and extract from F. pallidiflora. An acute toxicity test was carried out in the first part. Results in the experiment of acute toxicity, the maximum tolerance dosage by gavage in mice were 2 g/kg. In the second part, we investigated the relaxant effect on compounds and extract from F. pallidiflora against the contraction of the rat′s isolated tracheal preparation which was pre-contracted in the tissues with inducer. We found compounds FPSA11, FPSA07, FPSA03, FPSA02, FPSA06 and extract with relaxant effect. Further we gained pA2 and EC50 of compounds FPSA07, FPSA03, FPSA02 and extractⅠ, Ⅱ in the third part. From the pA2 values, we obtained the relative relaxant effects were FPSA02 > aminnophylline, extract Ⅰ, extract Ⅱ> FPSA07 > FPSA03 > nifedipine. In the fourth part, we studied the effect on cough frequency, cough latency in the inducer of ammonia and the effect on mice tracheal phenol red output to the extract from F. pallidiflora. We found the extract could prolong the latent period of cough, decrease the frequency of cough and increase phenol red output (P < 0.01) at 0.5 g/kg dose. Moreover it can prolong the latent period of cough at 0.25 g/kg dose (P < 0.01). Therefore, further efforts to search for anti-tussive ingredients from F. pallidiflora are worthy.

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4583
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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李燕. 伊贝母化学成分及其生物活性研究[D]. 北京. 中国科学院大学,2016.
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