Immunological Adjuvant Activity Of Saponins From Roots Of Platycodon Grandiflorum A.DC

Platycodi Radix is the root of Platycodon grandiflorum A.DC.(Campanulaceae).With the aim of selecting saponin with relatively low haemolytic saponins and high adjuvant activity which may be used as adjuvant in future large-scaled studies of vaccination, the total saponin from roots of P. grandiflorum was isolated and purified to afford four triterpenoid saponins. The haemolytic activities of the isolated saponins and their adjuvant potentials on the cellular and humoral immune responses of 1CR mice against ovalbumin (OVA), recombinant hepatitis B surface antigen (HBsAg) and Newcastle disease virus (NDV)-based avian influenza vaccine were evaluated.1. Isolation and identification of immunological adjuvant active saponins from P. grandiflorumThe roots of P. grandiflorum were extracted with 70%EtOH. and then concentrated in a small volume. EtOH extract was suspended in water and defatted with ether followed by partitioning with n-BuOH. The combined BuOH layers were concentrated to dryness. The dried extract was subjected to D101 resin column chromatography, washed with H₃O. and eluted with EtOH to afford a total saponin (PGS). PGS was divided into four fractions PGSA. PGSB, PGSC and PGSD by silica gel column chromatography. PGSC and PGSD were further isolated and purified with silica gel. Rp-Cl8. and Sephadex LH-20 gel column chromatography to affoiti saponin compound 1-â…£.Based of extensive spectroscopic methods including HRESI-MS,¹H-NMR.¹³C-NMR. DEPT.'H-'H COSY. HMQC and HMBC. Four saponins were identified to be platycodin D (PD). platycodin D2 (PD2). platycodin D3 (PD3) and platycoside E (PE). All shared a piatycodigenin skeieton and the same sugar side chains attached to C-28 of the aglycone. only differ from one another by the number of glycosyl units in sugar moieties attached to C-3.2. Haemolytic activities and adjuvant effect of of platycodigenin-type saponins on the immuneresponses to ovalbumin in mice and their structure-activity relationshipsThe HD₅₀ values for PD. PD2. PD3 and PE were 11.79±0.55.18.57±1.37.224.3±10.36 and >250μg/ml using 0.5%rabbit red blood cell suspensions, respectively. However, the HD₅₀ value of Quil A was 5.76±0.23μg/ml determined on the same condition. It implicated the higher haemolytic activity for Quil A than platycodigenin-type saponins. Among three saponins. the haemolytic activity with the following order:PD> PD2> PD3> PE. The differences were significant on one another (P＜O.001). Four platycodigenin-type saponins could significantly enhance Con A, LPS and OVA-induced splenocyte proliferation in the OVA-immunized mice (P＜O.001). The order in increasingOVA-stimuiated splenocyte proliferation was PD> PD2>PD3> PE (P＜O.05. P＜O.01.or P＜O.001). The sera OVA-specific 1gG 1gGl. 1gG2a and 1gG2b antibody levels in the OVA-immunized mice were significantly enhanced by PD, PD2 and PD3. However, PE only significantly promoted the production of the sera OVA-specific lgG2a and lgG2b antibody in the OVA-immunized mice, Adjuvant potentials of PD and PD2 on antibody responses were higher than those of PD3 and PE (P＜O.05, P＜O.01, or P＜O.001).The structure-activity relationship studies suggested that the number and the linkage of of sugar residues in the glycidic chains attached to C-3 of aglycone could affect the haemolytic and adjuvant activities of plarycodigenin-type saponins.4. Adjuvant activity of PD and PD2 on recombinant hepatitis B surface antigen (rHBsAg) vaccinePD and PD2 were evaluated for their adjuvant potentials on the cellular and humoral immune responses of mice against rHBsAg vaccine. Con A-. LPS-, and rHBsAg-stimulated splenocytes proliferation in the mice immunized with rHBsAg/PD or PD2 showed a greater proliferative response than that observed for the mice immunized with rHBsAg alone, while there is no significant difference between mice immunized with rHBsAg/Alum and rHBsAg alone, indicating the significant improvement of cell mediated immunity (CMI) induced by specific or non-specific antigen stimulation (P＜O.05, P＜O.01 or P＜O.001). PD and PD2 could significantly not only enhance rHBsAg-specific 1gG and 1gGl antibody levels, but increase lgG2a and lgG2b antibody titers in rHBsAg-immunized mice as compared with rHBsAgcontrol group (P＜O.001). Alum increased the rHBsAg-specific lgG and lgGl antibody responses when given with rHBsAg. There were, however, no significant differences in lgG2a and lgG2b antibody levels between the rHBsAg group and Alum/rHBsAg group. This clearly demonstrated that Alum, PD and PD2 modulated differentially the type of immune response to rHBsAg in mice. Alum induces almost exclusively a Th2 response, while PD and PD2 elicits both Th1 and Th2 responses as associated with an enhancement of lg2a, lgG2b and IgGl levels.The effects of PD on natural killer (NK) cell and rHBsAg-specific cytotoxie T lymphocyte (CTL) activity from splenocytes in rHBsAg-immunized mice were measured. PD could significantly NK and CTL activity, suggesting that the usage of PD in rHBsAg vaccine can help to improve specific and non-specific ability to kill target cell.To further explain the mechanism of adjuvant activities of PD. we then applied real-time PCR assay to evaluate Thl cytokine expression of splenocytes from the mice immunized with rHBsAg. The result showed that the mRNA levels of Thl cytokines IL-2 and IFN-γin the mice immunized with rHBsAg were significantly elevated by PD(P＜O.001). However, no significant difference was observed between mice immunized with rHBsAg/Alum and rHBsAg alone. 5. Adjuvant activity of PD on Newcastle disease viruses (NDV)-based avian influenza vaccinePD significantly increased Con A-, LPS-, and avian influenza antigen-stimulated splenocytes proliferation in the mice immunized with NDV-based avian influenza vaccine compared with the mice immunized with vaccine alone.The avian influenza antigen-specific IgG 1gGl. 1gG2a and 1gG2b antibody titers in mice immunized with NDV-based avian influenza vaccine were also significantly enhanced by PD compared with vaccine alone control group (P＜O.01). The improvement effect of PD on Thl specific antibody titers show no significant difference with that of QuilA. but significantly stronger than that of Alum.RT-PCR assay showed that the addition of PD in the vaccine can improve mRNA expression level of both IFN-y and IL-10. which are indicated cytokine of Thl and Th2 immune response respectively. And the result of mRNA level of transcription factor further proved that PD can lead to a mixed Th1/Th2 response in mice immunized with NDV-base HIV vaccine.