Antitumor And Immunological Adjuvant Activity Of Saponins From Roots Of Panax Notoginseng

Panax notoginseng (Burk.) F. H. Chen (Araliaceae), a well-known traditional Chinese medicinal plant, is commonly used for treatment of cardiovascular diseases, inflammation, different body pains, trauma, and internal and external bleeding due to injury. It has also been used as a tonic and haemostatic agent. Phytochemical and pharmacological studies on this plant have proved the dammarane-type saponins to be the main bioactive principles. Saponins from Panax notoginseng were found to possess extensive pharmacological activities on immune, hematology, cardiovascular, cerebrovascular and neural systems,as well as on metabolism, inflammation and cancer.In this thesis, ginsenoside Rb₁ (Rb₁), ginsenoside Rd (Rd), notoginsenoside K (K), notoginsenoside R₄ (R₄), ginsenoside Re (Re), ginsenoside Rg₁ (Rg₁), ginsenoside Rh| (Rh₁) and notoginsenoside R₁ (R₁) were screened for the cytotoxic activity on human cervical cancer HeLa cells and human colorectal carcinoma COLO 205 cells with MTT assay. Of the eight saponins tested, Rd was found to possess the strongest activity in inhibiting tumor cell proliferation. The effects of Rd on cytotoxicity, induction of apoptosis, and putative pathways of its actions using HeLa cells were investigated. Meanwhile, eleven saponins from Panax notoginseng were evaluated for their haemolytic activities and adjuvant potentials on the cellular and humoral immune responses of ICR mice to ovalbumin (OVA). Effects of the substitution pattern of these saponins on their biological activities were investigated and structure-activity relationship established. The effect of Rd on mice splenocyte proliferation in vitro and its adjuvant mechanisms on immune responses of ICR mice was studied by MTT assay, Semi-quantitative RT-PCR and flow cytometric analysis.1. Cytotoxicity and apoptosis induction of HeLa cells by ginsenoside Rd from roots of Panax notoginseng in HeLa cellsEight saponins Rbi, Rd, K, R4, Re, Rgi, Rhi, and R\ all showed certain inhibitory effect on HeLa and COLO 205 cells at higher concentrations. Of the eight saponins tested, Rd was found to possess the strongest inhibitory against tumor cell proliferation. Rd significantly inhibited the growth of HeLa cells in a concentration- and time-dependent manner. The IC50 value of Rd against HeLa cell was 150.5 ± 0.80 ug/ml following 48 h incubation. Rd also significantly reduced total protein content of HeLa cells in a dose-dependent manner after 48h incubation.Rd-treated cells stained with Giemsa showed clear apoptotic characteristics, such as volume reduction, condensation of nuclei, and nuclear fragmentation, membrane blebbing. Under fluorescence microscope, the DNA in the nucleus of control cells showed homogeneously Kelly fluorescence, while Rd-treated cells showed typical apoptosis features characterized by volume reduction , chromatin condensation, nuclear fragmentation with dense Kelly fluorescence stain, and appearance of apoptotic bodies. Electron microscopic examination revealed that numerous Rd-treated cells had reduced volume and shrunk cytoplasm, well-defined plasma membrane, condensed chromatin, located along nuclear envelope or formed irregularly shaped crescents at nuclear edges, as compared with untreated cells. DNA isolated from HeLa cells cultured with 180 and 210 ug/ml of Rd for 48 h showed a characteristic "ladder" pattern of apoptosis.After treatment of HeLa cells with different concentrations (120, 180, and 210 u,g/ml) of Rd for 48 h, the obvious changes in cell cycle distribution of the Rd-treated cells were characterized by decrease of G0/G1 phase and increase of S phase cells in a dose-dependent manner. However, there were no marked changes of G2/M phase cells. After treatment of HeLa cells with Rd 120, 180, and 210 ug/ml for 48h, a hypodiploid peak (apoptotic peak) of DNA characteristic of apoptosis was observed, and the apoptosis rate was 7.36%, 15.89%, and 35.82 % respectively. In the control, there was only a minor cell population (0.94%) underwent apoptosis .Expression of bcl-2 and bax proteins were evaluated in Rd-treated HeLa cells using streptavidin-biotin complex (SABC) immunohistochemical method. Compared to control cells, bcl-2 protein expression was markedly decreased and bax protein expression highly increased, suggesting Rd down-regulated bcl-2 protein level and up-regulated bax protein level.The Av|/m in HeLa cells treated with Rd 180 and 210 (ig/ml for 48 h was decreased significantly, suggesting that Rd decreased the A\|/m without altering plasma membrane permeability in HeLa cells. According to MTT assay, DEVD-CHO at 2 u.M could significantly increase the viability of HeLa cell treated with Rd (^240 u.g/ml) for 48h.In summary, the above data provided the first experimental evidence that Rd showed potent inhibitory activity on HeLa cells proliferation and could induce HeLa cells apoptosis through down-regulating bcl-2 expression, up-regulating bax expression, lowering A\j/m, and activating caspase-3 pathway. Thus, Rd could have the potential as a novel chemotherapeutic or chemopreventive agent for human cervical cancer.2. Relationship between haemolytic or adjuvant activity and structure of saponins from roots of Panax notoginsengThe haemolytic activity of Rd, Rbh R4, and K were 474.7±12.82, 394.3±9.89, 364.8±6.45, 317.8±12.80 ug/ml respectively using 0.5% rabbit red blood cell suspensions. Among four protopanaxadiol-type saponins (PDS), the haemolytic activity had the following order: K > R4 > Rbi > Rd. The differences were significant on one another (PO.Ol). The haemolytic activity of seven protopanaxatriol-type saponins (PTS) were 362.1±6.74, 407.3±12.32, 461.2±22.27, 420.3±22.92, 433.1±17.54, 469.6±16.85 and 397.3±15.44 ug/ml for Rhi, RI14, Rgi, Ri, R2, Re and U respectively. There weresignificant differences between Rhi and other six compounds {P <0.01). The HD5o values of Rh4 and U was significantly higher than those of R2, Rgi and Re (P <0.05 or P <0.01). However, there were no significant differences among RI14, U and Ri (P >0.O5). The haemolytic activity of R2 was higher than that of Re (P <0.05). However, the HD50 value of QuilA was 19.9±0.57 |ig/ml determined on the same condition, indicating the higher haemolytic activity for QuilA than these above compounds.Three representative purified saponins (Rru, Rgi and, Ri for monosaccharide, disaccharide, and trisaccharide glycosides, respectively) significantly enhanced OVA-specific IgG, IgGl, and IgG2b antibody levels in the OVA-immunized mice at the dose of 25 ^g. The doses of 10 and 50 |^g also induced these responses similarly to that of 25 |J.g, but with less significant differences compared with OVA control group.Con A-stimulated splenocyte proliferation in the mice immunized with 11 saponins was significantly higher than that in the OVA group and OVA/Alum group (/><0.01). Among four PDS, the order in terms of stimulation index was Rd > Rbi > K > R4. There were significant differences among these saponins (PO.01). Among seven PTS, the stimulation index of Rgi was higher than that of other compounds except for RI14. There were no significant differences between RI14 and Rht. However, there were significant differences among Rhi, Ri, R2, Re and U.Splenocytes isolated from mice immunized with OVA and Rd, Rbi, K and seven PTS showed a greater proliferative response upon LPS or OVA stimulation than that observed for the mice immunized with OVA alone and OVA/Alum (P<0.0\). Among three PDS, the order in terms of stimulation index was Rd > Rbi > K CP<0.05 or PO.01). The order of seven PTS in terms of stimulating LPS- or OVA-induced proliferative responsewas similar to that stimulated by Con A.The serum IgG and IgGl level in OVA-immunized mice was significantly enhanced by eleven saponins. Among four PDS, the stimulation order for IgG and IgGl was Rd > K > Rbi > R4 and Rd > Rbi > R4> K respectively. Among these PTS, the order was Re > Rgi > RJ14 > Ri> Rhi> R2 > U. However, there were no significant differences among three neighboring compounds. Significant enhancement in total sera IgG2a and IgG2b levels was observed in saponins-immunized mice compared with control group (PO.01). Among four PDS, the stimulation order for total-IgG2a and IgG2b was Rd > Rbi > K > R4, and among seven PTS, that was Rgi, RI14, Ri, Re, Rhi, R2 and U. However, there were no significant differences among three neighboring compounds among these PTS.The structure-activity relationship studies suggested that the number, length and position of sugar side chains as well as the type of glucosyl group in the structure of these saponins could not only affect their haemolytic activities and adjuvant potentials, but have significant effects on the nature of the immune responses. The information about this structure-function relationship might be useful for developing semisynthetic tetracyclic triterpenoid saponin derivatives with immunological adjuvant activity, and as a reference to distribution of the functional groups composing the saponin molecules.3. Mechanisms of adjuvant activity of ginsenoside Rd from roots of Panax notoginsengCon A- and LPS-stimulated mice splenocyte proliferation was significantly enhanced by ginsenoside Rdat concentrations from 0.01 ug/ml to 100 ng/ml (P<0.05 or PO.01) after 48h incubation in vitro. In RT-PCR, enhancement of IL-2, INF-y, IL-4 and IL-10 mRNA expression was shown in mouse spleen cells by treatment of Rd(0. l¹0 ng/ml) (PO.01)for 12h.Sera and whole blood prepared from OVA-immunized mice were analyzed using flow cytometer with CBA software for the amounts of IFN-y, TNF-a, IL-2, IL-4, IL-5, CD4+ and CD8+. The contents of cytokines IFN-y, TNF-a, IL-2, IL-4, and IL-5 in sera of mice immunized with OVA/Rd were significantly higher than those in OVA control mice (P<0.05 or P<0.01), and the content CD4+ in whole blood of mice immunized with OVA/Rd and OVA/Alum were higher than those in OVA control mice ((P>0.05). These studies provide the basis for further reseach the on mechanism of ginsenoside Rd on murine immunity.In summary, we studied the antitumor activity as well as haemolytic and adjuvant effects of saponins from Panax notoginseng. The structure-function relationship of saponins from Panax notoginseng and the mechanisms of Rd for inducing HeLa cells apoptosis and its adjuvant activity were further explored. The above results provided the references for developing novel efficient antitumor and immune adjuvant medicines, with low side-effects, from Panax notoginseng.