【作者】 周伟；

【导师】 周珮；

【作者基本信息】 复旦大学 ， 药物化学， 2008， 博士

【摘要】 人参（Panax ginseng）在我国的应用已有数千年历史,自古被列为上品,现作为名贵中药材也已闻名全世界。现代药理学研究表明,人参皂苷（ginsenoside）是人参的主要活性成分。人参皂苷compound K（CK）属二醇型稀有人参皂苷,其在天然的人参中并不存在,是其他二醇型人参皂苷在人肠道内的主要降解产物,是真正被吸收和发挥作用的实体,现已成为人参皂苷研究的新热点。但是,有关其制备和体内活性的研究还很少,更未见大规模制备的报道。本研究在上海-SK研究与发展基金（2003001-S）,上海市科委重点项目（No.05431927）和复旦大学研究生创新基金资助下,从菌种筛选开始,完整地建立了霉菌好气性发酵转化二醇型皂苷制备人参皂苷compound K的新方法。并成功把实验室的研究成果产业化,建立了中试发酵和纯化工艺,为大规模生产稀有人参皂苷compound K奠定了扎实的基础。研究中建立了同时检测多个转化中间体的HPLC方法,从理论上具体阐述了霉菌生物转化人参皂苷Rb1至CK的主要代谢途径。报道了CK的晶体结构,并从发酵液中分离得到了另外2个新化合物和2个已知化合物;在对CK的生物活性进行研究时,不仅探讨了CK抗肿瘤活性的作用机制;还系统地进行了CK抗急性炎症、佐剂性关节炎和Ⅱ型胶原诱导的类风湿性关节炎的药效研究,发现了CK在该方面的新的显著活性。以上研究结果均为CK一类新药开发奠定了基础。具体结果如下:1、本研究共从采集到的土样中分离得到306株霉菌,筛选到了新的高转化菌株Paecilomyces Bainier sp.229,并进行了紫外诱变。2、本研究采用单因素筛选和正交优化相结合的方法,对碳、氮源,生长因子及金属离子进行了配比优化,确定最佳培养基配方,CK克分子转化率从原始培养基的21%提高到了51%左右。并对其它发酵条件进行了考察,通过确定最佳发酵温度、pH、发酵时间、通气量、接种量、底物投料量及表面活性剂等,使转化率进一步提高到72.7%,大大高于文献报道值。3、本研究首次建立了发酵转化过程中,中间代谢产物同时检测的HPLC分析方法,并通过线性、灵敏度、准确性以及精密度进行了方法验证。利用该方法进行了代谢途径的研究,结果表明Paecilomyces Bainier sp.229发酵转化Rb1生成CK的主要代谢途径是Rb1→Rd→F2→CK,并首次报道了各个代谢物在转化过程中的变化曲线,为今后继续深入进行代谢调控研究奠定了基础。4、为适应工业化生产,本研究首次建立了三级发酵的中试工艺,筛选了一、二级种子培养基和发酵培养基。通过pH调节,溶氧-转速关联,以及底物诱导等一系列关键点控制,10L发酵罐转化率最终达到75.47%,连续三批50 L中试放大实验,转化率最高达到83.22%。同时本研究建立了适用于中试放大生产的CK纯化工艺,总收率达到65%以上,最终产品的纯度可以达到92%以上。以上研究建立了我国独特的工业化生产人参皂苷compound K的新工艺。5、本研究首次对CK进行了X-ray单晶衍射分析,研究了其晶体学结构。其A环和B环都是高度对称的椅式构象,C环由于受到C12羟基的拉伸和五元D环的张力,形成了扭曲的椅式构象,而D环采用13α,14β-半椅式构象。为进一步进行CK构效关系研究提供了重要数据。6、本研究运用常规硅胶柱分离和半制备高效液相分离相相结合的方法,从发酵液中分离到其他2个已知化合物,2个新化合物。经ESI-MS,¹H、¹³C-NMR,DEPT,HSQC及HMBC谱分析鉴定,两个已知化合物分别是PPT和Rh1。两个新化合物中,NewⅠ是C3位连有羰基的二醇型人参皂苷,NewⅡ则是在侧链C25上引入了过氧羟基,双键移到C23-C24位置的二醇型人参皂苷。7、本研究进行了CK体外肿瘤细胞增殖抑制试验,结果显示其对不同肿瘤细胞均有较为显著的增殖抑制作用,并呈明显的量效关系。体内抗肿瘤药效试验结果表明,CK单独用药抑制肿瘤生长的效果一般,但与化疗药物联用时具有明显的增效作用,抑制率可达66%。抗肿瘤机制研究表明,CK与CTX联用时,可以明显改善CTX对WBC、IL-2、TNF-γ和胸腺系数的抑制,CK既可提高化疗药物的药效,同时可提高机体免疫力,降低化疗药物引起的毒性。8、本研究考察了CK在抗关节炎方面的活性,发现其对大鼠足趾急性炎症的抑制率达到78.23%;对大鼠佐剂性关节炎预防作用的抑制率为31.26%,治疗作用的抑制率可达36.15%,均明显高于消炎痛和雷公藤。对Ⅱ型胶原诱导的大鼠类风湿性关节炎预防作用,抑制率最高可达48.29%;治疗作用,抑制率最高达36.15%,均显著优于Enbrel和雷公藤。本研究是首次发现了CK在治疗关节炎方面的显著活性,为CK的新药开发提供了新方向。更多还原

【Abstract】 Ginseng radix,the root of Panax ginseng C.A.Meyer,has been used as a traditional medicine in Asian countries for thousands of years,and now it is used worldwidely for preventive and therapeutic purpose.The main molecular components responsible for the actions of ginseng are ginsenosides,which are also known as ginseng saponins.Protopanaxadiol ginsenosides,such as Rbl,Rb2,Rb3,and Rc, have previously been shown to be metabolized by human intestinal bacteria to their final derivative,ginsenoside compound K（CK）.This metabolite is rapidly absorbed into the bloodstream and acts as an active compound and is currently a hot subject of ginsenoside research.The present dissertation deals with the biotransformation of protopanaxadiol ginsenosides by Paecilomyces Bainier sp.229 to prepare ginsenoside compound K and scale-up fermentation,with the financial support from the Shanghai SK R&D Foundation（No.2003001-S）,the Shanghai Scientific Fund Committee（No.05431927）, and Innovation Fund of Fudan University.The Mol conversion quotient increased to 75%.A simple and rapid gradient RP-HPLC method for simultaneous separation and determination of related ginsenosides during the process of biotransformation was first developed in order to study the metabolic pathway.The crystal structure of CK was first discussed,and two new compounds and two known compounds were also isolated.The anti-tumor mechanism of CK in combination therapy with chemotherapeutics was studied.Moreover,we first reported the anti-inflammatory activity of CK in acute inflammation,adjuvant-induced arthritis,and RhA.The main results are listed as following:1、306 strains of fungus were initially isolated from the soil samples collected from several ginseng plantation localities.Paecilomyces Bainier sp.229 was selected as the fermentation strain and mutated by UV irradiation.2、To find the optimal medium,individual constituent present within cultural medium was investigated using the one-factor-at-a-time method and the orthogonal matrix method,and the Mol conversion quotient of ginseng saponins increased from 21.2%to 51%.Furthermore,different cultural requirements,namely temperature,pH, fermentation time,agitation rate,inoculum size,substrate concentration,andsurfactant were investigated in the optimized medium.The Mol conversion quotient was elevalted to 72.7%finally.3、A simple and rapid gradient RP-HPLC method for simultaneous separation and determination of related ginsenosides during the process of biotransformation was developed in order to study the metabolic pathway.The method was validated in terms of linearity,sensitivity,precision,and accuracy.The result showed that the metabolic pathway of the biotransformation of ginsenoside Rb1 by Paecilomyces Bainier sp.229 was Rb1→Rd→F2→CK.4、Three-grade scale up fermentation of 10 L was set up.The seed culture and the fermentation culture was screened again.The Mol conversion quotient increased to 75.47%by the control of the key points,such as pH adjust,dissolved oxygen and agitation rate correlation,and substrate induction.Three batches fermentation of 50 L scale were carried out continuously,and the highest Mol conversion quotient rose to 83.22%.The purification technology of CK for scale-up production was devised.The total yield was about 65%and the purity was more than 92%.5、The crystal structure of CK was first discussed.Ring A and Ring B had an ideal chair conformation.The overall shape of ring C was close to the chair conformation. However,some distortion from the ideal form could be expressed as due to the strain at the junction with the five-membered D ring and some degree of flattening by 12β-hydroxyl.Ring D exhibited a 13a,14β-half chair conformation.6、Another two new compounds and two known compounds（PPT and Rh1） were isolated from the fermentation broth and the structures were identified by ESI-MS, ¹H、¹³C-NMR,DEPT,HMQC,and HMBC methods.A new compound（NewⅠ） was a protopanaxadiol ginsenoside with a carbonyl group connected to C3.Another new compound（NewⅡ） was also a protopanaxadiol ginsenoside,with a hydroperoxyl group connected to C25 and the double bond located at C23-C24.7、In vitro,CK showed conspicuous cytostasis to several cancer cell lines in a dose-dependent way.In vivo,the inhibition of Lewis lung carcinoma growth of CK was not significant on monotherapy.But the antitumor efficacy of combination with chemotherapeutics was much greater,with the maximum inhibition of 66%.The profiles of WBC,IL-2,INF-γ,and thymus coefficient were distinctly improved in CK combination therapy.These results indicate that CK combination therapy is of less toxicity and better immunoloregulation activity.8、The anti-inflammatory activity of CK was first studied,the inhibition of acute inflammation was up to 78.23%.The preventional and therapeutical effects on adjuvant-induced arthritis of rat were both much better than indomethacin and Tripterygium wilfordii,and the inhibition was about 36%.In the rat rheumatoid arthritis（RhA） model induced byⅡcollagen,CK shouwed conspicuous preventional and therapeutical effects.The inhibition rate of prevention and therapeutics were 48.29%and 36.15%,respectively.The activity is much better than the new anti-RhA Mab drug Enbrel,which indicates that CK is potential anti-RhA drug.更多还原