【作者】 杨光义；

【导师】 林励；

【作者基本信息】 广州中医药大学 ， 中药学， 2009， 博士

【摘要】 【目的】通过对武当山道地药材金果榄Tinospora sagittata（Oliv.）Gagnep.的资源调查、化学成分研究、药材质量研究、有效部位（成分）提取方法研究、主要药理作用研究、制剂工艺等的系统研究,确定其基源和药效成分,优选有效部位提取工艺,并对该资源进行新制剂开发利用研究。【方法】1.资源调查和药材鉴别采集武当山金果榄样品及其他产地如湖北神农架、江西、广西等地金果榄样品,进行对比研究,并对武当山金果榄原植物的地理生态环境、资源现状进行调查和品种鉴定整理。2.化学成分研究采用硅胶柱层析法对武当山产金果榄药材（青牛胆Tinospora sagittata（Oliv.）Gagnep.块根）的乙酸乙酯部位进行成分分离,采用¹³C-NMR、EI-MS法对洗脱物进行结构鉴定,确定该产地药材主要有效成分种类。3.金果榄药材质量研究采用《中国药典》2005年版一部附录规定方法分别对金果榄药材的水分、灰分、浸出物指标成分进行检查,建立金果榄药材薄层鉴别方法、薄层扫描法测定盐酸药根碱含量方法、TLC—S法测定盐酸药根碱含量方法、HPLC法测定盐酸巴马汀和盐酸药根碱含量方法,并运用所建立的新方法对不同产地10批金果榄药材质量进行比较,科学地评价武当山产金果榄药材质量。4.武当山金果榄Tinospora sagittata（Oliv.）Gagnep.有效部位提取方法研究采用药理实验示踪法考察药效部位与疗效指标的关系,寻求疗效指标达到最优时的金果榄药材活性部位,进行不同提取方法比较、正交试验设计优选回流提取工艺、均匀设计法优选半仿生提取工艺等研究。5.武当山金果榄Tinospora sagittata（Oliv.）Gagnep.药理作用研究通过观察武当山产金果榄对二甲苯所致小鼠耳肿胀的影响、对醋酸所致小鼠腹腔毛细血管通透性增高的影响、对组胺引起的大鼠皮肤毛细血管通透性增高的影响、对蛋清所致大鼠足爪肿胀的影响,对其抗炎作用进行评价。通过观察该金果榄对应激型胃溃疡大鼠的影响、对消炎痛所致大鼠胃溃疡的影响、对阿司匹林所致大鼠胃溃疡的影响、对大鼠幽门结扎型胃溃疡的影响、对大鼠乙酸型胃溃疡愈合的影响,对其抗胃溃疡作用进行评价。通过对四氧嘧啶复制糖尿病小鼠模型的降血糖作用研究,比较两种不同来源的金果榄药材（金果榄Tinospora capi llipes Gagnep和青牛胆Tinospora sagittata（Oliv.）Gagnep）降血糖作用的优劣。通过武当山金果榄含药血清指纹图谱研究、含药血清抑菌试验等含药血清药理学研究,初步寻找其在实验动物体内代谢规律。6.武当山金果榄Tinospora sagittara（Oliv.）Gagnep.药材的开发研究以武当山金果榄总生物碱为有效部位,对经方复方金果榄汤剂重新组方,优选剂型和制剂工艺,制备与临床使用相适应的复方金果榄喷雾剂,并进行了制剂质量控制方法研究。【结果】1.资源调查和药材鉴别对金果榄药材的两种原植物来源（金果榄T.capilllipes Gagnep.和青牛胆Tinospora sagittara（Oliv.）Gagnep.）的块根比较研究发现:青牛胆的外部形态和内部结构差异明显:金果榄外皮皱褶较多,内部质地较松软,色泽较浅,呈浅黄色;青牛胆外皮相对光滑,内部质地较致密,色泽较深,呈深黄色。形态结构差异提示二者化学成分和药理作用可能存在不同。采集于武当山地区的金果榄药材经专家鉴定均为防已科青牛胆属植物青牛胆Tinospora sagittara（Oliv.）Gagnep.块根。2.化学成分研究取武当山产金果榄药材（青牛胆Tinospora sagi ttata（Oliv.）Gagnep.块根）乙酸乙酯部位进行化学成分研究:将其乙醇提取物用水溶解,加入乙酸乙酯萃取,静置分层,在水和乙酸乙酯中间有一乳化层,收集乳化层,精制,得到一针状结晶;将乙酸乙酯部分浓缩得到浸膏。浸膏经硅胶柱层析洗脱（CHCl3:MeOH=95:5）,精制,得到4个化合物,分别为古伦宾（Columbin）、异古伦宾（Isocolumbin）、金果榄苷（TiRoside）、巴马汀（Palmatine）。3.金果榄药材质量研究按照《中国药典》2005版一部附录规定的方法,依法制定了包括鉴别TLC鉴别、检查（水分、灰分、酸不溶性灰分）、浸出物（水冷浸、热浸,不同浓度乙醇冷浸、热浸）、TLC-S含量测定、HPLC含量测定等金果榄药材较完善质量控制方法。结果金果榄药材水分不得超过5.83%;总狄分不得过6.33%,酸不溶性灰分不得过0.30%;水冷浸浸出物含量不得低于7.98%,醇冷浸浸出物含量不得低于2.83%,醇热浸浸出物含量不得低于13.41%,醇热浸浸出物含量不得低于11.20%。HPLC法测定盐酸巴马汀含量色谱条件为色谱柱为Diamonsiltm C18柱（4.6um×250mm×5um）;流动相为甲醇—0.35%四甲基乙二胺水溶液（用磷酸调pH=7.5）（1:1）;流速:1mL.min^-1;检测波长:345nm;柱温:25℃;通过不同产地金果榄药材质量比较发现武当山金果榄药材中盐酸巴马汀含量高于其他产地样品3倍以上。4.金果榄药材有效部位提取方法研究采用正交试验设计对武当山金果榄有效部位提取工艺进行了研究。以盐酸药根碱、盐酸巴马汀含量和浸膏率为评价指标,采用HPLC法测定含量,采用正交试验考察回流提取乙醇浓度、乙醇用量、回流时间、回流次数对提取效果的影响,得到最佳提取工艺为95%乙醇浓度,6倍量乙醇,回流2次,每次0.5h。5.药理作用研究二甲苯所致小鼠耳肿胀影响实验结果显示,与模型对照组比较,武当山金果榄中、高剂量组可明显减轻二甲苯所致的小鼠耳廓肿胀度,阳性对照组也可减轻耳廓肿胀度（p＜0.01）,低剂量组与模型组比较无统计学差异（p＞0.05）。醋酸所致小鼠腹腔毛细血管通透性增高影响实验结果显示,与模型对照组比较,武当山金果榄低、中、高剂量组可明显降低醋酸所致小鼠腹腔毛细血管通透性（p＜0.05-0.01）,阳性对照组也可降低小鼠腹腔的毛细血管通透性（p＜0.01）。组胺引起的大鼠皮肤毛细血管通透性增高的影响实验结果显示,与模型对照组比较,武当山金果榄中、高剂量组可明显降低组胺所致的小鼠腹腔毛细血管通透性（p＜0.05）,阳性对照组也可降低小鼠腹腔的毛细血管通透性（p＜0.01）,低剂量组与模型组比较无统计学差异（p＞0.05）。蛋清所致大鼠足爪肿胀影响实验结果显示,与模型对照组比较,武当山金果榄高剂量组可明显降低蛋清所致的大鼠2h、3h、4h、5h的足肿胀率（p＜0.05-0.01）,金果榄中剂量组可明显降低蛋清所致的大鼠3h、4h、5h的足肿胀率（p＜0.05-0.01）,阳性对照组也可明显降低蛋清所致的大鼠2h、3h、4h的足肿胀率（p＜0.05-0.01）,低剂量组与模型组比较无统计学差异（p＞0.05）。应激型胃溃疡大鼠的影响实验结果表明,与模型对照组比较,武当山金果榄低、中、高剂量组均可明显降低应激型胃溃疡大鼠溃疡指数（p＜0.05-0.01）,阳性对照组也可明显降低大鼠溃疡指数（p＜0.01）;与模型对照组比较,金果榄中、高剂量组可明显减少应激型胃溃疡大鼠囊状扩张腺体数量和炎症细胞浸润数量（p＜0.05-0.01）,阳性对照组也可明显减少大鼠囊状扩张腺体数量和炎症细胞浸润数量（p＜0.01）,低剂量组与模型组比较无统计学差异（p＞0.05）;与正常对照组比较,模型对照组大鼠血中PGE2、NO含量明显下降（p＜0.01）:与模型对照组比较,金果榄中、高剂量组可明显增加应激型胃溃疡大鼠血中PGE2和NO的含量（p＜0.05-0.01）,低剂量组可增加血中PGE2含量,阳性对照组也可明显增加大鼠血中PGE2和NO的含量（p＜0.05-0.01）,低剂量组血中NO含量与模型组比较无统计学差异（p＞0.05）。消炎痛所致大鼠胃溃疡影响实验结果表明,与模型对照组比较,武当山金果榄低、中、高剂量组可明显降低消炎痛所致胃溃疡大鼠溃疡指数（p＜0.05-0.01）,阳性对照组也可明显降低胃溃疡大鼠溃疡指数（p＜0.01）。阿司匹林所致大鼠胃溃疡影响实验结果表明,与模型对照组比较,武当山金果榄中、高剂量组可明显降低阿司匹林所致胃溃疡大鼠溃疡指数（p＜0.05-0.01）,阳性对照组也可明显降低胃溃疡大鼠溃疡指数（p＜0.01）,低剂量组与模型组比较无统计学差异（p＞0.05）。大鼠幽门结扎型胃溃疡的影响实验结果表明,与模型对照组比较,武当山金果榄低、中、高剂量组可明显降低幽门结扎型胃溃疡大鼠溃疡指数（p＜0.05-0.01）,阳性对照组也可明显降低胃溃疡大鼠溃疡指数（p＜0.01）。大鼠乙酸型胃溃疡愈合的影响实验结果结果表明,与正常对照组比较,模型对照组大鼠血中PGE2、EGF含量明显下降（p＜0.01）;与模型对照组比较,武当山金果榄低、中、高剂量组可明显增加乙酸型胃溃疡大鼠血中PGE2和EGF的含量（p＜0.05-0.01）,阳性对照组也可明显增加大鼠血中PGE2和EGF的含量（p＜0.05-0.01）。四氧嘧啶复制糖尿病小鼠模型的降血糖作用研究结果显示,与模型对照组比较,青牛胆Tinospora sagittata（Oliv.）Gagnep.低、中、高剂量组、金果榄Tinosporacapillipes Gagnep.低、中、高剂量组、二甲双胍组血糖下降百分率均显著升高（P＜0.01）;青牛胆高剂量组血糖下降百分率显著高于金果榄高剂量组（P＜0.05）;青牛胆高剂量组与二甲双胍组相比无统计学差异（P＞0.05）。6.武当山金果榄Tinospora sagittata（Oliv.）Gagnep.制剂开发研究以金果榄总生物碱为有效部位制备了复方金果榄喷雾剂。依次经浸渍、超声、回流法进行提取分离总生物碱,提取较完全,得率较高,工业化生产可操作性强。以总生物碱为有效成分为对象重新组方,实现了对复方金果榄汤剂的剂型创新和工艺改进。制剂中金果榄、山豆根TLC法鉴别操作简便,可行性强。HPLC法所测喷雾剂中盐酸巴马汀含量为0.06334 mg·mL^-1,苦参碱含量为0.1937mg·mL^-1,为临床前研究提供了准确可靠的定量依据。【结论】1.资源调查和药材鉴别武当山金果榄Tinospora sagittata（Oliv.）Gagnep.资源急骤减少,野生资源濒危,亟须进行可持续开发利用。武当山地区的金果榄药材原植物为防已科青牛胆属植物青牛胆Tinospora sagittata（Oliv.）Gagnep.块根。两个原植物来源金果榄T.capillipesGagnep.和青牛胆Tinospora sagittata（Oliv.）Gagnep.形态结构差异明显,提示二者化学成分和药理作用可能存在不同。1.化学成分研究首次从武当山产会果榄药材（青牛胆Tinospora sagi ttata（Oliv.）Gagnep.块根）的乙酸乙酯部位分离出5个化合物,已鉴定4个化合物,分别为古伦宾（Columbin）、异古伦宾（Isocolumbin）、金果榄苷（Tinoside）、巴马汀（Palmatine）。2.金果榄药材质量研究依法增补了TLC鉴别、检查（水分、灰分、酸不溶性灰分）、浸出物（水冷浸、热浸,不同浓度乙醇冷浸、热浸）、TLC—S法测定盐酸药根碱含量、HPLC含量测定等金果榄药材质量控制手段。对《中国药典》2005版一部金果榄药材质量控制方法进行了必要的补充、完善和提高。武当山产金果榄药材中盐酸巴马汀含量较高,为所测其他产地样品3倍以上。4.金果榄药材提取方法研究与其他提取方法比较,回流提取法所得盐酸巴马汀,盐酸药根碱含量均为最高。金果榄的最佳提取工艺为95%乙醇浓度,6倍量乙醇,回流2次,每次0.5h。本实验通过正交试验法对金果榄的提取工艺条件进行了优化,并按最佳工艺进行了验证试验,结果证明该工艺简单、稳定性好、提取率高,为武当山金果榄进一步开发利用提供了科学依据。5.药理作用研究武当山金果榄药材水煎液对急性炎症、免疫性炎症均有明显的抑制作用。该药材能明显抑制小鼠腹腔毛细血管通透性,能使蛋清致大鼠足爪肿胀度降低,药物组与空白组相比,有显著性差异。武当山金果榄药材水煎液通过提高再生粘膜腺体成熟度,减少炎症细胞浸润,提高血浆PGE2水平,降低溃疡指数,从而达到促进溃疡愈合,对应激性胃溃疡有明显的改善作用。武当山金果榄药材（青牛胆Tinospora sagittara（Oliv.）Gagnep.块根）与金果榄（金果榄T.capillipes Gagnep.的块根）均具有显著的降血糖作用,且青牛胆降血糖作用优于金果榄。6.武当山金果榄Tinospora sagi ttata（Oliv.）Gagnep.新制剂的开发研究以武当山产金果榄药材总生物碱为有效部位,对经方复方金果榄汤剂重新组方进行开发利用研究,结合工业化生产,最终确立提取方法为以复方金果榄汤剂原处方配比,依次经浸渍、超声、回流法进行提取分离总生物碱,制备出与临床使用相适应的复方金果榄喷雾剂,并建立了科学、简便、准确的制剂质量控制方法。更多还原

【Abstract】 Objective:The project is a system research on Tinospora capillipes Gagnep.crude drugs,which include studying on quality standard,extraction process,chemical components, pharmacological actions et al..According to the system research on Tinospora capillipes Gagnep,the project will clarify the relations between components and pharmacological actions,establish the optimum extraction process for Tinospora capillipes Gagnep,institute preparation and quality control of Jinshan yansukang spray.Method:1.Study on the chemical constituentsSeparate extract of Ethyl acetate of Tinospora sagittata（Oliv.） Gagnep by chrotamography method.identify the extract of Ethyl acetate by ¹³C-NMR.EI-MS.2.Study on quality standard detecte ingredients of Tinospora capillipes Gagnep with the methods of Pharmacopoeia 2005 edition adopted in Appendix,such as water,ash ect. Establish TCL identificaqtion for inospora capillipes Gagnep,Establish the method for the content determination of jatrorrhizine in inospora capillipes Gagnep by TLC scanning and the content of Palmatine hydrochloride and jatrorrhizine hydrochloride by HPLC.detect 10 Tinospora capillipes Gagnep by developed method.3.Study on the extraction process Research the relation between Pharmacodynamics site and Pharmacological effect With Pharmacological Tracer,search high efficiency Pharmacodynamics site,Compare different extraction methods of Tinospora capillipes Gagnep,study on the heat reflux extraction method for Tinospora capillipes Gagnep by Orthogonal test,and semi-bionic extraction method by homogeneous design method.4.Study on the pharmacology functionStudy on anti-inflammatory effect of Tinospora capillipes Gagnep by significant inhibitory effect on the xylene induced ear edema,by the effect on the acetic acid induced permeability Increased of Peritoneal Capillary,by the effect on the Histamine induced permeability Increased of Skin Capillary,by the effect on the Egg white induced foot edema.Study on Anti- Gastric ulcer of Tinospora capillipes Gagnep by the effect of disease caused by the fail in emotion in Tinospora capillipes Gagnep,by the effect of xiaoyantong induced peptic antiulcer in rats,by the effect of Aspirin induced peptic antiulcer in rats,by the effect of pylori Ligation induced peptic antiulcer in rats,by the effect of Ulcer healing induced by acetic acid.Study on the effect of blood glucose depressing induced by Alloxan in rats,compare the effect of blood glucose depressing between Tinospora capillipes Gagnep and Tinospora sagittata（Oliv.） Gagnep.Search the rule of vivo metabolism of Tinospora eapillipes Gagnep in animals by blood fingerprint and Antibacterial test.5.The preparation technology of SprayTo optimize formulation and preparation technology at the basis of Total alkaloids of Tinospora capillipes Gagnep,and made a study on the Quality control Standard for the optimized formulation.Results:1 Herbalism study on Radix Tinosporae has been accomplished.According to the result of the herbal works,the real origine of the sampleshas has been identified as Tinospora sagittata（Oliv.） Gagnep.2.A elementary syudy on Chemical constituent has been accomplished.The study isolated pure compounds by using repeated column chromatography,while the structure of new compounds were determined by detailed spectral analysis.Four compounds were isolated from the decoction of the roots of Tinospora sagittata（Oiiv.） Gagnep,which are Columbin,Palmatine,Isocolumbin and Tinoside.3 A system quality standard has been established.The primary contents include ingredients water,ash,acid insoluble ash,extract,identify and assay method,and other related quality standards.4 Compared Palmatine in Tinospora sagittata（Oliv.）Gagnep from different Habitats,the sample from Wudang mountain in Hubei province was the best.It can be cultivated as the best seed quality resourse for Good Agricultural Practice.5 Study on the optimum extraction process of Tinospora sagittata（Oliv.） Gagnep.crude drugs by Orthogonal Design.In order to optimize the conditions of the extraction technique for Tinospora sagittata（Oliv.） Gagnep.crude drugs,HPLC Method was used to analyze the content of atrorrhizine Hydrochloride and Palmation Hydrochioride,and the orthogonai experimental design was introduced to optimize the conditions of the extraction technology.The experiment of this part included ethanol,amount of ethanol,reflux timeand times of extraction,involved in four factors,which carried out by orthogonal design.The following Results have been obtained:The conditions of the ethanol extraction technology of extracting the constituents from Radix Tinosporae were 95%ethanol,6-fold solvent,0.5 hour of reflux and 2 times of extraction.The results show that the process is reasonable and reliable.6 Pharmacological actions study on Tinospora sagittata（Oliv.） Gagnep.The pharmacological actions of Tinospora sagittata（Oliv.） Gagnep.were preliminarily investigated in the dissertation.The experiment showed that aqueous extract of Tinospora sagittata（Oliv.） Gagnep.has significant anti-inflammatory effects against acute inflammation and immunity inflammation.It remarkedly decreased the parameters of ulcer,and possessing promotion of ulcer agglutination.It has a obviously amelioration effect against gastric stress ulcer. The results show that the effects of reducing blood sugar of Radix Tinosporae of is better than ofTinospora sagittata（Oliv.） Gagnep.7 preparation and quality control of Jinshan yansukang spray.To prepare Jinshan yansukang spray and establish its quality control methods,the preparation based on the original drug extract,dissolved,filterde,adjusted the pH value,packaged in spray containers.TLC identification and HPLC methods were used.Conclusion:After system studied on Tinospora capillipes Gagnep,The project preliminary identified the Choiceness Varieties of Tinospora sagittata（Oliv.） Gagnep,developed Quality control Standard methods of Tinospora sagittata（Oliv.） Gagnep,improved safety and effectiveness of the crude drug for Tinospora sagittata（Oliv.） Gagnep,examined Pharmacological activity of Tinospora capillipes Gagnep in Antiulcer,anti-inflammatory and blood glucose depressing,established the best process of heat reflux extraction for Tinospora sagittata（Oliv.） Gagnep,developed the process of Jinshan yansukang spray and the quality control standard for it,above all the research lay a solid foundation for resource development of Tinospora capillipes Gagnep.更多还原