| Ginsenosides are the mainly effective components in the plants of Araliaceae,Panax genus, Panax ginseng C. A. Meyer, Panax quinquefolium L. and Panax notoginseng(Burk.) F.H.Chen. It has been reported that ginsenosides have many kinds of pharmacological effects, such as central nerve regulation, immune regulation,cardiovascular protection, antitumor, antiaging, et al. Ginsenosides are classified into five types, i.e. protopanaxatriol type, protopanoxatriol type, ocotillol type, oleanane type and other types.With the research in-depth, it was found that the degraded secondary ginsenosides and its aglycone possessed better pharmacological effects, especially ginsenosides Rg3, Rh2 and PPD showed stronger antitumor activity. Ginsenoside Rg3 as anti-cancer drug has been on listed sale, and ginsenosides Rh2 and PPD have entered the stage of clinical research.In recent years, the metabolic researches of the secondary ginsenosides without glycosyl group at C-20(ginsenoside Rg3, Rh2, Rg2 and Rh1) and their aglycones(PPD and PPT) in vitro or vivo hint that their corresponding ocotillol type ginsenosides or aglycones maybe the active ingredients in vivo.In order to carry out the above research on metabolism process and metabolic products of secondary ginsenosides and their aglycones, reveal biological activities of ocotillol type ginsenosides, and explore the truly effective ingredients of ginsenosides in vivo, we oxidated twelve kinds of above-mentioned ginsenosides and aglycone(20(S)-/20(R)-ginsenoside Rg3, 20(S)-/20(R)-ginsenoside Rh2, 20(S)-/20(R)-ginsenoside Rg2, 20(S)-/20(R)-ginsenoside Rh1, 20(S)-/20(R)-PPD, 20(S)-/20(R)-PPT)which were provided by the preliminary preparations, synthesised the corresponding ocotillol type ginsenosides, and then evaluated the antitumor and antioxidant activities of these oxidative products.Firstly in this paper, we oxidated the twelve secondary ginsenosides and theirVI aglycones with hydrogen peroxide as oxidant. The oxidative products were isolated and purified by using the methods of column chromatography and preparative liquid chromatography. Forty compounds were isolated in total, and their chemical structure were identified by the means of MS, 1D and 2D-NMR spectroscopies. Among them,twenty-four compounds were confirmed as ocotillol type ginsenosids, and sixteen oxidative products of non-ocotillol type ginsenosids with the cyclized structure in the side chain. Fifteen new compounds were identified as20(S)-dammar-20,25-epoxy-3β,12β,24α-triol, 20(R)-dammar-20,25-epoxy-3β,12β,24α-triol,(20R,24R)-3-O-formyl-dammar-20,24-epoxy-3β,25-diol,(20R,24S)-3-Oformyl-dammar-20,24-epoxy-3β,25-diol, 20(R)-3-O-β-D-glucopyranosyl-dammar-20,25-epoxy-3β,12β,24β-triol, 20(R)-3-O-β-D-glucopyranosyl-dammar-20,25-epoxy-3β,12β,24α-triol, 20(S)-3-O-[β-D-glucopyranosyl-(1â†'2)-β-D-glucopyranosyl]-dammar-20,25-epoxy-3β,12β,24α-triol, 20(R)-3-O-[β-D-glucopyranosyl-(1â†'2)-β-D-glucopyr-anosyl]-dammar-12-one-20,24-lactone-3β-ol], 20(S)-dammar-20,24-lactone-3β,6α,12β-triol,(20S,24R)-dammar-22-one-20,24-epoxy-3β,6α,12β,25-tetraol,(20R,24R)-dam-mar-20,24-epoxy-3β,6α,12β,25-tetraol, 20(R)-dammar-20,24-lactone-3β,6α,12β- triol,(20R,24S)-dammar-20,24-epoxy-3β,6α,12β,25-tetraol,(20R,24R)-6-O-β-D-glucopyra-nosyl-dammar-20,24-epoxy-3β,6α,12β,25-tetraol,(20R,24S)-6-O-β-D-glucopyranosy-l-dammar-20,24-epoxy-3β,6α,12β,25-tetraol, respectively. Moreover, the last four products are new ocotillol type ginsenosides.Under the reaction condition, the configuration of each starting material at C-20 did not be changed. According to the SN1 reaction mechanism, they got a pair of ocotillol type isomers at C-24.By the analysis of 13 C NMR data in pyridine, the chemical shifts at C-20 and C-24 of all the four ocotillol type ginsenosides are as follows: 20 S,24R(δC86, δC85);20S,24S(δC87, δC88); 20 R,24R(δC86, δC86); 20 R,24S(δC86, δC87). The above carbon spectrum data can provide the spectroscopic evidence for the analysis of stereochemical structure of ocotillol type ginsenosides.This paper for the first time evaluated the antitumor activity of a series of oxidative products in vitro by MTT, and found all of them could inhibit cellsVII proliferation to a certain extent. Eleven compounds of them obtained from protopanaxatriol type ginsenosides and aglycones showed better inhibitory effects against He La and Hep G2 cells(IC50<40 μg/m L). The compound PDQ had the strongest inhibition on the proliferation of He La cells(IC50=2.18 μg/m L). The results showed the oxidative products from protopanaxadiol type ginsenosides have better effects than those from protopanaxatriol type ginsenosides. At the same time the number of glycosyl and the configuration at C-20 and C-24 also have certain influence on antitumor activity.To explore the antitumor mechanism of the compound PDQ, this paper investigated the cell morphology, Caspase activity and western blot analysis. The results suggested PDQ can induce He La cells apoptosis, and activated Caspase-8-dependent the extrinsic apoptotic pathways mediated by cell surface death receptor and Caspase-9-dependent intrinsic apoptosis pathway mediated by mitochondria.As we known, excess free radicals can cause cancer by various pathways including causing DNA damage to lead gene replicated by error, attacking unsaturated fatty acid on the cell membrane to destroy its structure and function and further affect signal transduction, and damaging DNA repair enzymes to gene mutations. In this paper, we took antioxidant model in vitro including three kinds of free radicals clearance and two kinds of inhibiting DNA damage experiments to evaluate antioxidant capacity of twelve oxidative products with better antitumor activities. The results showed that the tested compounds had weak abilities in terms of clearing free radicals in vitro, but possessed the abilities of inhibiting hydroxyl radical in DNA damage experiments. Hydroxyl radical is a kind of high reactive free radicals in vivo,thus these oxidative products are worthy of further research and application to the prevention and treatment for tumor as antioxidant reagent.Given the compound PDQ and its isomer great activities on antitumor and antioxidation, we studied its synthetic conditions by univariate analysis method combined with HPLC-ELSD detection, and established the optimum reaction conditions. When starting material initial concentration is 4.6 mg/m L, m-CPBA isused as oxidant, molar proportion of oxidant and starting material is 9:1, reaction temperature is set at 25℃, reaction time is 1.0 h, the yield of PDQ and its isomer is highest.On the basis of the preparations of twelve kinds of 20(S/R)-secondary ginsenosides without glycosyl at C-20 and aglycones, this article firstly comprehensively synthesized their corresponding ocotillol type ginsenosides,identified their chemical structure, discussed a portion of antitumor mechanism, and analyzed the relationships between the activities and structures. The results of this paper provide new scientific basis for the research of pharmacokinetics and efficacy material base of ginsenosides, reveal the metabolite and active ingredients of ginsenosides, and develop new ginsenosides and their derivatives with better medicine applications. |
PDF Full Text Request