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、~(13)C-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。
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、~(13)C-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.
引文
[1]张树臣卡编.《中国人参》[M].上海:上海科技敦育出版社,P93-105.
[2]王F筠默.人参约理研究的进展[J].人参研究,2001,13:2-13.
[3]Sanada S,Kondo N,Sgoji J,et al.Studies on the saponins of ginseng,I Structure of ginsenoside Ro.Rb,Rbl,Rd[J].Chem Pharm Bull,1974,22:421-428.
[4]Sanada S,Kondo N,Sgoji J,et al.Studies on the saponins of ginseng,1 Structure of ginsenoside Re.Rf,Rg[J].Chem Pharm Bull,1974,22,2407-2412.
[5]Nagai Y,Tanaka O,and Shibata S.Chemical studies on the oriental plant drugs,ⅩⅩⅣStructure of ginsenoside Rgl,A neutral saponin of ginseng root[J].Tetrahedron,1971,27:881-892.
[6]Yahara S,Tanaka O.and Komori T.Saponin of leaves of Panax ginseng C.A.Meyer[J].Chem Pharm Bull,1976,24:2204-2208.
[7]Yahara S.Matsuura K,Kasai R,et al.Saponin of buds and flowers of Panax ginseng C.A.Meyer,(1) Isolation of ginsenoside Rd,Re,Rgl[J].Chem Pharm Bull,1976,24:3212-3213.
[8]Yahara S,Tanaka O,and Nishioka I.Dammarane type saponins of leaves of Panax japonicus C.A.Meyer.(2) Saponins of the specimens collected in Tottori-ken,Kyto-shi,and Niigata-ken [J].Chem Pharm Bull,1978,26:3010-3016.
[9]Yahara S,Kaji K,and Tanaka O.Further study on dammarane-type saponins of roots,leaves,flowerbuds,and fruits of Panax ginseng C.A.Meyer[J].Chem Pharm Bull,1979,27:88-92.
[10]Kasai R,Besso H,Tanaka O,et al.Saponins of red ginseng[J].Chem Pharm Bull,1982,31:2120-2125.
[11]Koizumi H,Sanada S,Ida Y,et al.Studies on the saponins of ginseng.Ⅳ.On the structure and enzymatic hydrolysis ofginsenoside-Ral[J].Chem Pharm Bull,1982,30:2393-2396.
[12]Besso H,Kasai R,Wei JX,et al.Further studies on dammarane saponins of American ginseng roots of Panax quinquefolium L[J].Chem Pharm Bull,1982,30:4534-4538.
[13]王毅,蒋艳,王本祥等.人参皂苷Rg3及其肠内菌代谢产物Rhl对小鼠免疫细胞功能的影响[J].药学学报,2002,37:9 27-929.
[14]钟国赣,江岩,王雪清等.人参二醇与三醇组皂昔对正常与黄嘌呤一黄嘌呤氧化酶致损的培养心肌细胞动作电位的影响[J].中国药理学报,1999,12:256-260.
[15]Rudakewich M,Ba F,and Benisgen CG.Neurotrophic and nueroprotective actions of ginsenosides Rbl and Rgl[J].Planta Med,2001,67:509-515.
[16]Berek L,Szabo D,Pitri IB,et al.Effects of naturally occurring glucosides,and parishin derivatives on multidrug resistance of lymphoma cell and leukoeyte function[J].In ⅥⅤO,2001,15:151-156.
[17]赵越,苏适.人参皂苷Rh2抗肿瘤作用的研究[J].微生物杂志,2003,23:61-63.
[18]Yosioka I,Sugawara T,Imai K,et al.Soil bacterial hydrolysis leading to genuine aglycone.Ⅴ.On ginsenosides-Rbl,Rb2 and Rc of the ginseng rootsaponins[J].Chem Pharm Bull,1972,20:2418-2421.
[19]Karikura M,Miyase T,Tanizawa H,et al.Studies on absorption,distribution,excretion and metabolism of ginseng saponinis.Ⅶ.Comparison of decomposition models of ginsenoside-Rbl and-Rb2 in the digestive tract of Rats[J].Chem Pharm Bull,1991,39:2357-2361.
[20] Hasegawa H, Sung JH, Matsumiya S, et al. Main ginseng saponins metabolites formed by intestinal bacteria [J]. Planta Med, 1996, 62: 453-457.
[21] Hasegawa H. Sung JH. and Benno Y. Role of human intestinal Prevotella Oris in hydrolyzing ginseng saponins [J]. Planta Med, 1997, 63: 436-440.
[22] Sung JH, Hasegawa H, Ha JY, et al. Metabolism of ginseng saponins by human intestinal bacteria (part II) [J]. Kor J Pharmacogn, 1997, 28: 35-41.
[23] Chen X, Zhou QL and Wang BX. The metabolism of ginsenoside Rb1 by intestinal bacteria [J]. Acta Pharm Sin. 1999, 34: 410-414.
[24] Bae EA, Choo MK, Park EK, et al. Metabolism of ginsenoside Rc by intestinal bacteria and its related antiallergic activity [J]. Biol Pharm Bull, 2002, 25: 743-747.
[25] Chi H. and Ji GE. Transformation of ginsenosides Rbl and Re from Panax ginseng by food microorganisms. Biotechnol Lett, 2005, 27: 765-771.
[26] Chi H, Kim DH, and Ji GE. Transformation of Ginsenosides Rb2 and Rc from Panax ginseng by Food Microorganisms. Biol Pharm Bull, 2005,28: 2102-2105.
[27] Jang IS, Kang HH and Kim DH. Preparing compound K and ginsenoside F1 from saponin of ginseng comprises dissolving purified saponin of ginseng in an aqueous solvent and adding naringinase and/or pectinase into the saponin solution: KR, 2003037005-A, [P]. 2003-5-12.
[28] Cho BG, Choi GJ and Kim YH. Preparing ginsenoside compound k using cellulase or lactase composition y-ao: KR, 2003043168-A, [P]. 2003-6-2.
[29] Park SY, Bae EA, Sung JH, et al. Purification and characterization of ginsenoside Rb1-Metabolizing β-glucosidase from Fusodobacterium K-60, a human intestinal anaerobic bacterium [J]. Biosci Biotechnol Biochem, 2001, 65: 1163-1169.
[30] Hasegawa H. Sung JH, Matsumiya S, et al. Main ginseng saponins metabolites formed by intestinal bacteria [J]. Planta Med, 1996, 62: 453-457.
[31] Akao T, Kida H, Hatori M and Kobashi K. Intestinal bacterial hydrolysis is required for the appearance of Compound K in rat plasma after oral administration of ginsenoside Rbl from Panax ginseg [J]. J Pharm Pharmacol, 1998, 50: 1155-1160.
[32] Sung JH, Huh JD, Hasegawa H, et al. Process for the preparation of metabolites of ginseng saponins: US, 5925537 [P]. 1999-7-20.
[33] Bae EA, Kim NY, Han MJ, et al. Transformation of ginsenosides to compound K (IH-901) by lactic acid bacteria of human intestine [J]. J Microbiol Biotechn, 2003,13: 9-14.
[34] Han Y, Zhao YQ and Jiang BH. Seletion of the microbe for preparing the anticancer constituent C-K by the transformation of microbe [J]. R & I on TCM, 2005, 7: 17-19
[35] Wakabayashi C, MuraKami K, Hasegawa H. An intestinal bacterial metabolite of ginseng protopanaxadiol saponins has the ability to induce apoptosis in tumor cells [J]. Biochem Biophys Res Commun, 1998,246: 725-730.
[36] Lee SJ, Ko WG, Kim JH, et al. Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c-mediated activation of capase-3 protease [J]. Biochem Pharmacol, 2000, 60: 667-685.
[37] Oh SH and Lee BH. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage [J]. Toxicol Appl Pharm, 2004, 194: 221-229.
[38] Steve Helms, ND. Cancer Prevention and Therapeutics: Panax Ginseng. Alter Med Rev, 2004, 9: 259-274.
[39]Hasegawa H and Uchiyama M.Antimetastatic efficacy of orally administered ginsenoside Rbl in dependence on intestinal bacterial hydrolyzing potential and significance of treatment with an active bacterial metabolite[J].Planta Med,1998,64:696-700
[40]Kang JK,Lee YJ.No KO,et al.Ginseng intestinal metabolite-1(GIM-I) reduces doxorubicin toxicity in the mouse testis[J].Reprod Toxicol,2002,16:291-298.
[41]Shin YW,Bae EA,Kim SS,et al.Effect of ginsenoside Rbl and compound K in chronic oxazolone-induced mouse dermatitis[J].Int Imnunopharmacol,2005,5:1183-1191.
[42]Shin YW and Kim DH.Antipruritic effect ofginsenoside Rbl and compound K in scratching behavior mouse models[J].J Pharmacol Sci,2005,99:83-88.
[43]Lee JY,Shin JW.Chun S,et al.Antitumor promotional effects of a novel intestinal bacterial metabolite(IH-901) derived from the protopanaxadiol-type ginsenosides in mouse skin[J].Carcinogenesis,2005,26:359-367.
[44]Choo MK,Park EK,Hart M J,et al.Antiallergic activity of ginseng and its ginsenosides[J].Planta Med,2003,69:518-522.
[45]Lee HU,Bae EA,Hart M J,et al.Hepatoprotective effect of ginsenoside Rbl and compound K on tert-butyl hydroperoxide-induced liver injury[J].Liver Int,2005,25:1069-1073.
[46]Jang S,Ryu JH,Kim DH,et al.Changes of[3H]MK-801,[~3H]muscimol and[~3H]flunitrazepam binding in rat brain by the prolonged ventricular infusion of transformed ginsenosides[J].Neur Res,2004,29:2257-2266.
[47]马吉胜,周秋丽,费晓方等.真菌对人参皂苷Rbl及人参二醇系皂苷的代谢作用[J].药学学报,2001,36:603-605.
[48]Zhou W,Luo ZS and Zhou P.Determination of ginsenoside compound K by reserved-phase high performance liquid chromatography[J].Chin JChromatography,2005,23:270-272.
[49]Wan JB,Li P,Li SP,et al.Simultaneous determination of 11 saponins in Panax notoginseng using HPLC-ELSD and pressurized liquid extraction[J].JSep Sci.2006,29:2190-2196.
[50]Xie HT,Wang G J,Sun JG,Tucker l,et al.High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies[J].J Chromatogr B,2005,818:167-173.
[51]周佩,李继杨.那西肽突变生物合成的研究[J].中国抗生素杂志,1995,20:159.
[52]万睿,刘晓会,李继杨等.那西肽产生菌S.actuosus选育及发酵条件的优化[J].复旦报(医学版),2004,31:579-581.
[53]中国科学院微生物研究所主编.《常见与常用真菌》[M].北京:科学出版社,P199-20
[54]魏锦超主编.《真菌鉴定手册》[M].上海:上海科学技术出版社,P495-501.
[55]张曙光和张发群.β-葡萄糖苷酶产生菌产酶条件研究.纤维素科学与技术[J].1995,37-41
[56]Bakhtiari MR,Faezi MG.,Fallahpour M,et al.Medium optimization by orthogonal array designs for urease production by Aspergillus niger PTCC5011[J].Process Biochem,2006,41:547-551.
[57]Xu ZN,and Yang ST.Production of mycophenolic acid by Penicillium brevicompactum immobilized in a rotating fibrous-bed bioreactor.Enzyme Microb Tech,2007,40,623-628.
[58]孙力军,沈爱光,熊晓辉等.β-葡萄糖苷酶产酶发酵条件的优化研究[J].食品工业科技,1998.5:22-24.
[59]顾卫民,郭海风,沈爱光.β-葡萄糖苷酶产生凶、发酵条件的优化、粗分离及其特性研究[J].江办食品与发酵,2001,4:12-16.
[60]Gokhale DV.Hyperproduction of β-glucosidase by an Asperillanssp[J].Biotechenol Lett,1984,8:719-922.
[61]Li W,Koike K,Asada Y,et al.Biotransformation of Korean Panax ginseng by Pectinex[J].Biol Pharm Bull,2006,29:2472-2478.
[62]Li GH,Shen YM,Liu Y,et al.Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation of saponin by Bacillus subtilis[J].Annal Mivrobiol,2006,56:151-153.
[63]高峰,鱼洪闪,金凤燮.柱层析法在人参皂甙组分分离中的应用[J].大连工业学院学报,2000,19:274-276
[64]郑长龙,鱼红闪,金凤燮.人参稀有皂苷CK的分离与纯化[J].大连累工业学院学报,2003.22:167-169.
[65]彭树林,肖蓉,肖倬殷.大叶珠子参化学成分研究(1)[J].中草药,1987,18:10-11.
[66]Yu PZ,Hu CQ,Meehan E J,et al.X-Ray Crystal Structure and Antioxidant Activity of Salidroside,a Phenylethanoid Glycoside[J].Chem Biodivers,2007,4:058-513.
[67]Ko SR,Choi KJ,Suzuki K,et al.Enzymatic Preparation of Ginsenosides Rg2,Rhl,and F1[J].Chem Pharm Bull,2003,51:404-408.
[68]Ko SR,Suzuko Y,Choi K J,et al.Enzymatic preparation of genuine prosapogenin,20(S)-ginsenoside Rhl,from ginsenoside Re and Rgl[J].Biosci Biotechnol Bioehem,2000,64:2739-2743.
[69]Yoshikawa M,Sugimoto S,Nakamura S,et al.Medicinal flowers.XI.Structures of new dammarane-type triterpene diglycosides with hydroperoxide group from flower buds of Panax ginseng[J].Chem Pharm Bull,2007,55:571-576.
[70]Nakamura S,Sugimoto S,Matsuda H,et al.Medicinal flowers.XVII.New dammarane-type triterpene Glycosides from flower buds of american ginseng,Panax quinquefolium L[J].Chem Pharm Bull,2007,55:1342-1348.
[71]Wang ZH,Zheng QS,Liu K,et al.Ginsenoside Rh-2 enhances antitumour activity and decreases genotoxic effect of cyclophosphamide[J].Basic Clin Pharmacol Tixicol,2006,98:411-415.
[72]Kim JH,Cho SY,Lee,JH,et al.Neuroprotective effects of ginsenoside Rg(3) against homocysteine-induced excitotoxicity in rat hippocampus[J].Brain Res,2007,1136:190-199.
[73]Liu Y,Ma H,Zhang JW,et al.Influence of ginsenoside Rh-1 and F-1 on human cytochrome P450 enzymes[J].Planta Med,2006,72:126-131.
[74]Yao Y,Han WW,Zhou YH,et al.The inhibition effect of 20(S)-Protopanaxadiol(PPD) and Ginsenoside Rh2 for CYP2C9 and CYP3A4[J].Polymer,2007,48:2644-2648.
[75]徐叔云,卞如濂,陈修主编.《药理实验方法学》[M].北京:人民卫生出版社,P1757-1827.
[76]喻林华,曾献,王元清.三七伤约片的抗炎消肿约理作用研究[J].中国实用医药,2007,2:16-18.
[77]张佳红,王晋平,王慧娟.三七总皂苷调整类风湿关节炎免疫相关内环境失衡状态的临床研究[J].中国中西医结合杂志,2007,27:589-592.
[78]刘刚,刘育辰,鲍建才等.三七约理作用的研究进展[J].人参研究,2005,3:12-17.
[1]Yosioka I,Sugawara T,lmai K and Kitagawa I.Soil bacterial hydrolysis leading to genuine aglycone.V.On ginsenosides-Rbl,Rb2 and Rc of the ginseng rootsaponins[J].Chem Pharm Bull,1972,20(11):2418-2421.
[2]Kohda H and Tanaka O.Enzymatic hydrolysis of ginseng saponins and their related glycosides [J]. Yakugaku Zasshi, 1975, 95(2): 246-249.
[3] Koizumi H. Sanada S, Ida Y and Shoji J. Studies on the saponins of ginseng .IV. On the structure and enzymatic hydrolysis of ginsenoside-Ral [J]. Chem Pharm Bull, 1982, 30(7): 2393-2398.
[4] Karikura M. Miyase T, Tanizawa H, et al. Studies on absorption, distribution, excretion and metabolism of ginseng saponinis. V. The decomposition production products of ginsenosides Rb2 in the large intestine of rats [J]. Chem Pharm Bull, 1990, 38(10): 2859-2861.
[5] Karikura M, Miyase T, Tanizawa H, et al. Studies on absorption, distribution, excretion and metabolism of ginseng saponinis. VII. Comparison of decomposition models of ginsenoside-Rb1 and -Rb2 in the digestive tract of Rats [J]. Chem Pharm Bull, 1991, 39(9): 2357-2361.
[6] Hasegawa H, Sung JH, Matsumiya S, et al. Main ginseng saponins metabolites formed by intestinal bacteria [J]. Planta Med, 1996, 62: 453-457.
[7] Hasegawa H, Sung JH and Benno Y. Role of human intestinal Prevotella Oris in hydrolyzing ginseng saponins [J]. Planta Med, 1997, 63: 436-440.
[8] Sung JH, Hasegawa H, Ha JY, et al. Metabolism of ginseng saponins by human intestinal bacteria (part II) [J]. Kor J Pharmacogn, 1997, 28(1): 35-41
[9] Akao T, Kanaoka M and Kobashi K. Appearance of compound K, a major metabolite of ginsenoside Rb1by intestinal bacteria, in rat plasma after oral administration- measurement of compound K by enzyme immunoassay [J]. Biol Pharm Bull, 1998, 21(3):245-249.
[10] Akao T, Kida H, Hatori M and Kobashi K. Intestinal bacterial hydrolysis is required for the appearance of Compound K in rat plasma after oral administration of ginsenoside Rb1 from Panax ginseg [J]. J Pharm Pharmacol, 1998, 50: 1155-1160.
[11] Chen X, Zhou QL and Wang BX. The metabolism of ginsenoside Rb1 by intestinal bacteria [J]. Acta Pharm Sin (药学学报) , 1999,34(6):410-414.
[12] Bae EA, Choo MK, Park EK, et al. Metabolism of ginsenoside Rc by intestinal bacteria and its related antiallergic activity [J]. Biol Pharm Bull, 2002, 25(6): 743-747.
[13] Jang IS, Kang HH and Kim DH. Preparing compound K and ginsenoside F1 from saponin of ginseng comprises dissolving purified saponin of ginseng in an aqueous solvent and adding naringinase and/or pectinase into the saponin solution: KR, 2003037005-A, [P]. 2003-5-12.
[14] Cho BG, Choi GJ and Kim YH. Preparing ginsenoside compound k using cellulase or lactase composition y-ao: KR, 2003043168-A, [P]. 2003-6-2.
[15] Jiang BH, Han Y and Zhao YQ. Optimization of enzymatic tran slation for preparati on ginsenoside compound K in total saponins of panaxnotoginseng [J]. Chin Tradit Herb Drugs (中草药), 2004, 35 (9): 986-988.
[16] Chi H and Ji GE. Transformation of ginsenosides Rbl and Re from Panax ginseng by food microorganisms [J]. Biotechnol Lett, 2005, 27: 765-771.
[17] Chi H, Kim DH and Ji GE. Transformation of ginsenosides Rb2 and Rc from Panax ginseng by food microorganisms [J]. Biol Pharm Bull, 2005, 28(11):2102-2105.
[18] Park SY, Bae EA, Sung JH, et al. Purification and characterization of Ginsenoside Rb1-Metabolizing β-Glucosidase From Fusodobacterium K-60, a Human Intestinal Anaerobic Bacterium [J]. Biosci Biotechnol Biochem, 2001, 65(5): 1163-1169.
[19] Shin HY, Lee JH and Lee JY. Purification and characterization of ginsenoside Ra-hydrolyzing β-D-xylosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium [J]. Biol Pharm Bull, 2003, 26(8): 1170-1173.
[20] Shin HY, Park SY, Sung JH, et al. Purification and characterization of a-L-Arabinopyranosidase and β-L-Arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc [J]. Appl Environ Microbiol, 2003, 69(12): 7116-7123.
[21] Sung JH, Huh JD, Hasegawa H, et al. Process for the preparation of metabolites of ginseng saponins: US, 5925537 [P]. 1999-7-20.
[22] Bae EA, Kim NY, Han MJ, et al. Transformation of ginsenosides to compound K (IH-901) by lactic acid bacteria of human intestine [J]. J Microbiol Biotechn, 2003,13(1): 9-14.
[23] Han Y, Zhao YQ and Jiang BH. Seletion of the microbe for preparing the anticancer constituent C-K by the transformation of microbe [J]. R&I on TCM( 中药研究与信息) , 2005,7(2): 17-19.
[24] Zhou W, Luo ZS and Zhou P. Determination of ginsenoside compound K by reserved-phase high performance liquid chromatography [J]. Chin J Chromatography, 2005, 23(3): 270-272.
[25] Zhou W, Feng MQ, Li JY, et al. Studies on the preparation,crystal structure and bioactivity of ginsenoside Compound-K [J]. J Asian Nat Prod Res, 2006, 8(6): 519-527.
[26] Wakabayashi C, MuraKami K, Hasegawa H. An intestinal bacterial metabolite of ginseng protopanaxadiol saponins has the ability to induce apoptosis in tumor cells [J]. Biochem Biophys Res Commun, 1998, 246: 725-730.
[27] Lee SJ, Ko WG, Kim JH, et al. Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome omediated activation of capase-3 protease [J]. Biochem Pharmacol, 2000, 60: 667-685.
[28] Kang KA, Lim HY, Kim SU, et al. Induction of apoptosis by gingseng saponin metabolite in U937 human monocytic leukemia cell [J]. J Food Biochem, 2005, 29: 27-40.
[29] Oh SH and Lee BH. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage [J]. Toxicol Appl Pharm, 2004,194: 221-229.
[30] Choi HY, Jong HS, Park JH, et al. A novel ginseng saponin metabolite induces apoptosis and downregulates fibroblast growth factor receptor 3 in myeloma cells [J]. Int J Oncol, 2003,23: 1087-1093.
[31] Hasegawa H, Sung JH and Matsumiya S. Reversal of daunomycin and vinblastine resistance in multidrug-resistant P338 leukemia in vitro through enhanced cytotoxicity by triterpenoids [J]. Planta Med, 1995, 61: 409-413.
[32] Lee SJ, Sung JH, Lee SJ, et al. Antitumor activity of a novel ginseng saponin metabolite in human pulmonary adenocarcinoma cells resistant to cisplatin [J]. Cancer Lett, 1999, 144: 39-43.
[33] Lee BH, Lee SL, Hui JH, et al. In vitro antigenotoxic activity of novel ginseng saponin metabolites formed by intestinal bacteria [J]. Planta Med, 1998, 64: 500-503.
[34] Hasegawa H and Uchiyama M. Antimetastatic efficacy of orally administered ginsenoside Rb1 in dependence on intestinal bacterial hydrolyzing potential and significance of treatment with an active bacterial metabolite [J]. Planta Med, 1998, 64: 696-700.
[35] Hasegawa H, Lee KS and Nagaoka T. Pharmacokinetics of ginsenoside deglycosylated by intestinal bacteria and its transformation to biologically active fatty acid ester [J]. Biol Pharm Bull 2000, 23(3): 298-304.
[36] Kim S, Kang BY, Cho SY, et al. Compound K induces expression of hyaluronan synthase 2 gene in transformed human keratinocytes and increases hyaluronan in hairless mouse skin [J]. Biochem Biophys Res Commun, 2004, 316: 348-355.
[37] Choo MK, Park EK, Han MJ, et al. Antiallergic activity of ginseng and its ginsenosides [J]. Planta Med, 2003, 69(6): 518-522.
[38] Park EK, Shin YW, Lee HU, et al. Inhibitory effect of ginsenoside Rb1 and compound K on NO and prostaglandin E2 biosyntheses of RAW264.7 cells induced by lipopolysaccharide [J]. Biol Pharm Bull, 2005, 28(4): 652-656.
[39] Shin YW, Bae EA, Kim SS, et al. Effect of ginsenoside Rbl and compound K in chronic oxazolone-induced mouse dermatitis [J]. Int Immunopharmacol, 2005, 5: 1183-1191
[40] Shin YW and Kim DH. Antipruritic effect of ginsenoside Rbl and compound K in scratching behavior mouse models [J]. J Pharmacol Sci, 2005, 99: 83-88.
[41] Lee JY, Shin JW, Chun S, et al. Antitumor promotional effects of a novel intestinal bacterial metabolite (IH-901) derived from the protopanaxadiol-type ginsenosides in mouse skin [J]. Carcinogenesis, 2005, 26(2): 359-367.
[42] Lee HU, Bae EA, Han MJ, et al. Hepatoprotective effect of ginsenoside Rbl and compound K on tert-butyl hydroperoxide-induced liver injury [J]. Liver Int, 2005, 25: 1069-1073.
[43] Jang S, Ryu JH, Kim DH, et al. Changes of [~3H]MK-801, [~3H]muscimol and [~3H]flunitrazepam binding in rat brain by the prolonged ventricular infusion of transformed ginsenosides [J]. Neur Res, 2004, 29(12): 2257-2266.
[44] Tohda C, Matsumoto N, Zou K, et al. Abeta(25-35)-induced memory impairment, axonal atrophy, and synaptic loss are ameliorated by Ml, a metabolite of protopanaxadiol-type saponins [J]. Neuropsychopharmacology, 2004, 29(5): 860-868.
[45] Kang JK, Lee YJ, No KO, et al. Ginseng intestinal metabolite-I (GIM-I) reduces doxorubicin toxicity in the mouse testis [J]. Reprod Toxicol, 2002,16: 291-298.
[46] Kim DH, Jung JS, Moon YS, et al. Inhibition of intracerebroventricular injection stress-induced plasma corticosterone levels by Intracerebroventricularly administered compound K, a ginseng saponin metabolite, in mice [J]. Biol Pharm Bull, 2003, 26(7): 1035-1038.
[47] Kim JH, Lee BH, Jeong SM, et al. The inhibitory effects of Korean red ginseng saponins on 5-HT3_A receptor channel activity are coupled to anti-nausea and anti-vomiting action [J]. J Ginseng Res, 2005, 29(1): 37-43.
[48] Paek IB, Moon Y, Kim J, et al. Pharmacokinetics of a ginseng saponin metabolite compound K in rats [J]. Biopharm Drug Dispos, 2006, 27: 39-45.
[49] Lee PS, Song TW, Sung JH, et al. Pharmacokinetic characteristics of IH-901, a novel intestinal metabolite of ginseng saponin, in rat [J]. Planta Med, 2006, 72: 204-210.
[50] Hasegawa H, Lee KS, Nagaoka T, et al. Pharmacokinetics of ginsenoside deglycosylated by intestinal bacteria and its transformation to biologically active fatty acid esters [J]. Biol Pharm Bull, 2000, 23(3):298-304.