Study On Analysis, Preparation Artwork And Anti-tumor Activity Of Heat-processed Ginsenosides

Ginsenosides, the major active ingredients from the roots of Panax ginseng, can almost reflect all biological functions of the whole ginseng. According to the chemical structures, ginsenosides were generally classified into two groups, namely protopanaxadiol-type saponins(ginsenosides Rb1, Rb2, Rc and Rd) and protopanaxotril-type saponins(ginsenosides Rg1, Re, Rg2 and Rh1). Previous Studies have confirmed that secondary ginsenosides via treatment exerted stronger biological activity. These secondary ginsenosides are comsisted of Rg3 and Rh2 which generated from processing red ginseng, and compound K(ginsenoside M1) isolated form intestinal bacteria metabolisms of protopanaxodiol-type ginsenosides.Steaming, an important processing methods of Traditional Chinese Medicine(TCM), plays a vital roles on effect-enhancing and toxicity-reducing, changing of drug properties, and convenient preservation and so on. Red ginseng process is a typical representative steaming processing of TCM. Increasing evidences showed secondary ginsenosides including Rg3, Rh2 and other small molecules generated during steaming processing of fresh ginseng to red ones, has more powerful bioactivities including anti-tumor, anti-inflammation and so on. In order to get more secondary ginsenosides from the processed ginseng, black ginseng was prepared by repeated steaming treatment. Generally, black ginseng needs "steaming for nine times and solarization for nine times". These secondary ginsenosides, namely “thermal cracking ginsenosides”, were not only generated via hydrolyzing sugar group, but also dehydration and isomerization at C-20 position. In the past several years, these thermal cracking ginsenosides including Rg5 and Rk1 received more attentions on bioactivities and chemical isolation.Based on the theory that heated-processed ginseng can result in “thermal cracking ginsenosides”, in the present thesis, the extracts contained abundant ginsenosides were used to investigate secondary ginsenosides. The findings indicated that there are much more thermal cracking ginsenosides in the extracts from the stem and leaves of Panax ginseng, Panax notoginseng, and Panax quinquefolium. These “thermal cracking ginsenosides” was consisted of ginsenoside Rg5 and Rk1. Moreover, LC/ESI-MS was employed to analyze “thermal cracking ginsenosides” after steaming the extract from the stems and leaves of Panax quinquefolium. After the heat-process treatment, the main secondary saponins including Rg6, F4, Rk3 and Rh4 were significantly increased and ginsenoside Rg3, Rk1 and Rg5 were also increased significantly.In this thesis, ginsenosides Rg1 was used to further investigate its generation mechanism of “thermal cracking ginsenosides”. The results from LC/ESI-MS analysis clearly exhibited that ginsenosides 20(S)-Rh1, 20(R)-Rh1, Rk3 and Rh4 were generated from heated-process production of ginsenoside Rg1. In addition, response surface methodology(RSM) was employed to optimized preparation conditions of ginsenoside Rk3 and Rh4 from ginsenoside Rg1 using heated-process treatment. The optimum preparation conditions were as follows: 0.02% of formic acid concentration, 116.4 ℃ of reaction temperature and 2.22 h of reaction time.The secondary ginsenoside exerted stronger and better anti-tumor activity. In the present thesis, the in vivo anti-tumor activity of heated-processed products of Rg1(HPPRg1) on H22 tumor bearing mice was investigated and the molecular mechanism was also studied. The results showed that HPPRg1 can significantly inhibit tumor growth in H22 bearing mice and prolong survival time of experimental animals after 2-weeks’ treatment. In addition, the levels of TNF-α, IFN-γ and IL-2 in serum of H22-bearing mice was significantly increased after HPPRg1 treatment. The antitumor effect was achieved by increasing the immune response. The results from H&E and Hoechst 33258 staining of tumor tissues shown that tumor cells in HPPRg1 group with a loose organization and cell shrinkage, suggesting that HPPRg1 is able to induce H22 tumor cells to be apoptosis after 2-weeks treatment.To further reveal molecular mechanisms of tumor-induced apoptosis of HPPRg1, the cytotoxicity and apoptosis mechanisms of ginsenosides Rh4 on HepG2 cells were investigated for the first time. The results showed that ginsenoside Rh4 can inhibit the proliferation of HepG2 cells in dose- and time-dependent manner. In addition, treatment with ginsenosides Rh4 for 24 h can induce cell apoptosis with apoptosis rates are 11.47% of 10 μM, 15.29% of 20 μM and 21.33% of 40 μM, respectively. The results of Hoechst 33258 staining shown that HepG2 cells with ginsenosides Rh4 treatment exerted nuclear condensation, fragmentation and formation of apoptotic bodies and other apoptotic state. Also, TUNEL staining further confirmed these results. Finally, western blot technique was used to analyze apoptotic proteins. The results showed that ginsenosides Rh4 can activate caspase signaling pathway to enhance the protein expression of Bax and inhibits the protein expression of Bcl-2, suggesting that the anti-tumor activity of ginsenoside Rh4 was partly via inducing apoptosis.