Biological Activity Study Of Ginsenosides And Fusion Toxins

Part I.Chemical study of ginsenosides and their attenuation effectsThe follow-up chemical study was carried out for the better development and use of the traditional Chinese medicinal material resources of Panax ginseng and Panax quinquefolium in northeastern China.Additionally,several dammarane dihydro-ginsenosides were obtained to enrich the ginsenoside derivatives library.Finally,in vitro and in vivo studies were performed to study the toxic-attenuation effects of ginsenosides and their derivatives.The innovative results are as follows:1.Panax ginseng C.A.Meyer cv.Silvatica(PGS),which is also known as"Lin-Xia-Shan-Shen",or "Zi-Hai"in China,is grown in forests and mountains by broadcasting the seeds of ginseng.In this study,four new dammarane-type triterpenoids,ginsengenin-S 1(1),ginsengenin-S2(2),ginsenoside-S3(3),ginsenoside-S4(4),along with one known compound were isolated from pearl knots of PGS.Ginsengenin-S2 significantly alleviated oxidative damage on A549 cells when they were exposed to cigarette smoke(CS)extract.In addition,ginsengenin-S2 could inhibit the CS-induced inflammatory reaction in A549 cells.The protective effects of ginsengenin-S2 against CS-induced oxidative stress and the inflammatory response in A549 cells may involve the Nrf2 and HDAC2 pathways.2.A new ocotillol-type ginsenoside,namely 12-one-pseudoginsenoside F11(12-one-PF11),was isolated from stems and leaves of Panax quinquefolium.Its structure was elucidated as 6-O-[?-L-rhamnopyranosyl-(1-2)-?-D-glucopyranosyl]-dammar-12-one-20S,24R-epoxy-3?,6?,25-triol(21).12-one-PFii could significantly suppress hydrogen-peroxide-induced oxidative stress in human lung carcinoma A549 cells.Compared with the model group,12-one-PFii improved cell viability of A549 cells in a dose-dependent manner.Morever,it significantly decreased the generation of malondialdehyde and increased the production of superoxide dismutase and glutathione and protein expression levels of Nrf2 and HO-1 in A549 cells.3.To enrich the chemical information of dammarane dihydro-ginseosides,eighteen dihydro-derivatives of dammarane ginsenoside were semi synthesized by hydrogenation reaction,including 10 novel dihydro-ginsenosides:2H-Rb2(23),2H-Rb3(24),2H-Rc(25),(20S)-2H-Rh2(28),2H-F2(29),2H-gypenoside XVII(30),2H-gypenoside LXXV(31),2H-Rg1(35),2H-Rg2(36),and 2H-Rh1(37).In addition,ginsenoside Rh2,its dihydro-derivative dihydro-ginsenoside Rh2,and its oxidized products(24R)-pseudo-ginsenoside HQ and(24S)-pseudo-ginsenoside HQ were chemically prepared using the stems and leaves of Panax ginseng as raw material,which provided material basis for the in vivo toxic-attenuation effects study.4.Ginsenoside Rh2(Rh2),an important ingredient from Panax ginseng,has received much attention due to a range of pharmacological actions.The aim of the study was to investigate the therapeutic potential Rh2 on cisplatin(CDDP)-induced nephrotoxicity and to elucidate involved mechanisms.An in vivo mice model of CDDP-induced nephrotoxicity was established by a single intraperitoneal injection of CDDP to assess the effects of Rh2 on renal biochemical parameter,oxidative stress,inflammation tubular cell apoptosis and serum metabolic profiles.Rh2 protected against CDDP-induced renal dysfunction and ameliorated CDDP-induced oxidative stress,histopathological damage,inflammation and tubular cell apoptosis in kidney.Rh2 treatment had significantly increased expression of Bcl-2 and decreased expression of p53,Bax,cytochrome c,caspase-8,caspase-9,and caspase-3 in kidney tissues.Metabolomic analysis identified 29 altered serum metabolites in Rh2 treatment mice.These results suggest that Rh2 protects against CDDP-induced nephrotoxicity via action on caspase-mediated pathway.5.(24R)-pseudo-ginsenoside HQ(R-PHQ)and(24S)-pseudo-ginsenoside HQ(S-PHQ)are the main metabolites of(20S)-ginsenoside Rh2(Rh2)in vivo.In this study,we found that Rh2,R-PHQ,and S-PHQ upregulated the innate and adaptive immune response in cyclophosphamide(CTX)induced-immunocompromised mice as evidenced by the number of leukocytes,cellular immunity,and phagocytosis of macrophages.Spleen T-lymphocyte subpopulations and the serum cytokines level were also balanced in these immunosuppressed mice.Furthermore,co-administration with R-PHQ or S-PHQ did not compromise the antitumor activity of CTX in the hepatoma H22-bearing mice.Treatment with R-PHQ and S-PHQ clearly induced the apoptosis of tumor cells,significantly increased the expression of Bax,and remarkably inhibited the expression of Bcl-2 and vascular endothelial growth factor(VEGF)in H22 tumor tissues.The anti-tumor activity of R-PHQ and S-PHQ could be related to the promotion of tumor apoptosis and inhibition of angiogenesis and may involve the caspase and VEGF signaling pathways.This study provides a theoretical basis for further study on R-PHQ and S-PHQPart II.Fusion toxins targets head and neck squamous cell carcinomaEpidermal growth factor receptor(EGFR)is often overexpressed in head and neck squamous cell carcinoma(HNSCC),and targeting EGFR represents the first FDA approved drug for treatment of HNSCC.However,the clinical effectiveness is moderate and the overall survival rate of HNSCC remains low.It is urgently needed to develop novel treatment options especially targeted therapies for HNSCC patients In this study,we have developed a diphtheria toxin-based recombinant bivalent human epidermal growth factor fusion toxin(bEGF IT).Both bEGF IT and mEGF IT showed a very high selectivity in binding and depletion of EGFR+HNSCC cell lines,without binding and depletion of EGFR-cancer cell lines in vitro.The bEGF IT has an improved in vitro binding affinity than the monovalent EGF fusion toxin(mEGF IT).bEGF IT,but not mEGF IT exhibited equivalent potency as the FDA approved EGFR-targeted drug,Erlotinib in inhibition of flank-xenograft HNSCC tumor growth in vivo.Similar finding was also observed in the HNSCC tongue-orthotopic mouse model,with significant prolonged survival in either bEGF IT or the Erlotinib treated groups.Interestingly,mice with the experimental metastasis significantly survived longer in both bEGF IT treatment groups than the rest groups.Our results demonstrated that the bEGF IT is superior in inhibition of primary tumor growth to the mEGF IT,and in inhibition of metastasis to Erlotinib.Thus,the novel bEGF IT has a great potential as a better EGFR-targeted drug candidate for treatment of HNSCCs.