Studies On The Preparation And Activity Of Ginsenoside Compound K

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(Newâ… ) was a protopanaxadiol ginsenoside with a carbonyl group connected to C3.Another new compound(Newâ…¡) 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 byâ…¡collagen,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.