Isolation And Evaluation Of Immunological Adjuvant Active Saponins From Polygala Tenuifolium Willd.

The roots of Polygala tenuifolium Willd., "Yuanzhi", is a well-known traditional Chinese medicine used as an expectorant, tonic, sedative and dementia preventing agent. Extensive phytochemical and pharmacological studies on this plant proved the saponins to be the main bioactive principles. With the aim of selecting non-haemolytic saponins which could be possibly used as adjuvant in future large-scaled studies of vaccination, in previous work, the saponins from the root of Achyranthes bidentata, Astragalus membranaceus, Bupleurum chinense, Glycyrrhiza uralensis, Panax ginseng, Panax notoginseng, Polygala tenuifolium and the herb of Gynostemma pentaphyllum, which share a triterpenoid aglycone moiety, were screened for their haemolytic activity on rabbit erythrocytes, and their adjuvant potentials on the cellular and humoral immune response of ICR mice against OVA. We found that the best adjuvant saponins were obtained from P. tenuifolium. In this thesis, we wish to report here the isolation and structural elucidation of isolated saponins, as well as the immunological adjuvanticity of these isolated saponins on the humoral immune responses of ICR mice against OVA.1. Extraction and isolationThe roots of P. tenuifolium were extracted with 70% EtOH, and then concentrated in a small volume. After extracting with ether, the EtOH extract was partitioned with n-BuOH saturated with water to give total saponin. The total saponin was divided into three fractions Fr-A, Fr-B and Fr-C by silica gel column chromatography. The Fr-B were further isolated and purified with silica gel, Rp-C18 gel column chromatography and semi-HPLC to afford compounds 1-18.2. Determination of structuresThe structures of compounds 1-18 were determined on the basis of chemical evidence and extensive spectroscopic methods including ESI-MS, HRESI-MS, ¹H-NMR, ¹³C-NMR, ¹H-¹HCOSY, DEPT, HMQC and HMBC techniques. Tenuifolisaponin A (4), B (5), tenuifoliose R (12) and tenuifoliside F (14) were new compounds, and polygalasaponin XXVIII (3), XXXII (6) and glomeratose B (16) were first isolated from P. tenuifolium.3. Haeinolytic activitiesThe haemolytic activity of compounds 1-5 were significantly lower than that of Quil A. However, there were not significant differences between compound 6 and Quil A. Compound 3 showed a slight haemolytic effect, with its HD50 value being was 399.70+1. 93 ug/ml.4. Splenocyte proliferation in OVA-immunized mice4.1 Effect of fraction B and its subfractions on splenocyte proliferation in OVA-immunized mice Con A-, LPS- and OVA-stimulated splenocyte proliferation in the mice immunized withOVA/Frs B, Bl, B2 and B3 showed a greater proliferative response than that observed for the mice immunized with OVA alone. The enhancing effects of subfraction B3 on splenocyte proliferation in OVA-immunized mice were significantly stronger than those of other subfractions.4.2 Effect of compounds 1 - 6 on splenocyte proliferation in OVA-immunized miceCompounds 1-6 significantly enhanced the Con A-, LPS- and OVA-stimulated splenocyte proliferation in OVA-immunized mice (P<0.05, PO.01 or PO.001). Compound 4 and 5 stimulated splenocyte proliferation more significantly than Quil A and other componds5. OVA-specific serum antibody response in OVA-immunized mice5.7 Effect ofPolygala tenuifolium saponins (PTS) and its Fractions on OVA-specific antibody in OVA-immunized miceThe serum OVA-specific IgG, IgGl and IgG2b level in mice immunized with OVA was significantly enhanced by PTS, Fr-B and Fr-C (P<0.01 or PO.001). Moreover, PTS, Fr-B andFr-C stimulated total-IgG and IgGl antibody responses more significantly than Quil A (P<0.05, P<0.0\ or /><0.001). Among extracts, the order in terms of stimulating total-IgG, IgGl and IgG2b antibody responses was Fr-B > PTS > Fr-C.5.2 Effect of fraction B and its subfractions on OVA-specific antibody in OVA-immunized mice Frs. B, Bl, B2 and B3 significantly improved the serum OVA-specific IgG, IgGl and IgG2blevels in OVA-immunized mice (/><0.01 or PO.001). Moreover, they stimulated total-IgG, IgGl and lgG2b antibody responses more significantly than Quil. Among subfractions, the order in terms of stimulating total-IgG, IgGl and IgG2b antibody responses was B3 > B2> Bl.5.3 Effect of compounds 1- 6 on OVA-specific antibody in OVA-immunized miceSignificant enhancements in serum OVA-specific IgG, IgGl and IgG2b levels were observed in groups of compounds 1- 6 (P<0.05, P<0.01 or PO.001). Moreover, compounds 5 and 4 stimulated total-IgG, IgGl and IgG2b antibody responses more significantly than Quil A.6. Effect of compounds 4 and 5 on Thl/Th2 type cytokines in the OVA-immunized mice.Compound 4 and 5 significantly enhanced the production of the Thl type cytokines IL-2, tumor necrosis factor-/? (TNF-P) and interferon-y (IFN-y), and the Th2 type cytokines IL-4 and IL-5 in OVA-immunized mice.In addition, the effect of the substitution pattern of these isolated saponins on their haemolytic and adjuvant activities was investigated and structure-activity relationships were established.