Adaptive immune response-modifying and antimicrobial properties of Andrographis paniculata and andrographolide

Andrographis paniculata (AP) is a traditional herbal medicine which is widely used for the treatment of many diseases in Asia. Recently, various biological activities of AP extract or andrographolide (AND), such as immunostimulatory activity, anti-inflammatory effect, cytokine induction or deduction, a potential cancer therapeutic agent and T cell activation suppression, have been reported. However the potential of AP extract or AND to stimulate the specific or adaptive immune response using microbial vaccines has not been determined. In this project, AND has been purified from AP and its identity confirmed by the melting point, colour test, TLC, UV absorption spectrum, ESI-MS and H-NMR. An aqueous and two ethanolic extracts of Andrographis paniculata and AND, an active principle of Andrographis paniculata, were investigated for their antimicrobial activity against nine bacterial species in vitro using the disc diffusion method. It was discovered that neither the aqueous extract nor AND were bacteriostatic or bactericidal against S. typhimurium, E. coli, S. sonnei, S. aureus, P. aeruginosa, S. pneumoniae, S. pyogenes, L. pneumophila or B. pertussis but the two ethanolic extracts of AP were bacteriostatic against L. pneumophila and B. pertussis. It was also observed that the ethanol extract of AP and AND stimulated both antibody and cell-mediated immunity (CMI) responses to a killed S. typhimurium vaccine. Mice were vaccinated with either one dose or two doses of killed S. typhimurium vaccine. They were fed two different quantities of an ethanol extract of AP or AND for 14 days in mice immunised with one dose of the vaccine, and for 28 days in mice immunised with two doses of vaccine, respectively. Both the extract and AND significantly increased the IgG antibody titres against S. typhimurium, with the increase in antibody titres being statistically significant in the two dose vaccine group. Although not statistically significant, there was also a substantial increase in the IgG antibody titres in the one dose vaccine group. Splenocyte cultures from mice from both the immunisation groups treated with the extract or AND stimulated with the S. typhimurium lysate showed a significant increase in the production of IFN-γ in both 14 and 28 day AP extract or AND treatment groups. The increase indicates the induction of a cell-mediated immune response. To confirm the immunomodulatory potential of AP extract and determine the immunomodulatory potential of AND, experiments were conducted using mouse salmonellosis as a model system. Mice were vaccinated with two doses of killed S. typhimurium vaccine by intraperitoneal (i/p) route and orally dosed with AP extract at 25 mg/kg bodyweight or AND at 4 mg/kg bodyweight for total 28 days, followed by oral challenge with virulent S. typhimurium. Both AP extract and AND substantially increased the survival rate by 50% after mice were challenged with 10 fold of a sublethal dose (1.5x10 6 cfu) of virulent S. typhimurium. They also promoted clearance of S. typhimurium from challenged mice by days 8 or 12 post-challenge with 1.5x10 cfu virulent S. typhimurium respectively. Sera IgG, IgA antibody titres against S. typhimurium and IFN-γ or IL-2 were detected after the mice were challenged by the oral route with a sublethal dose (1.5x10 cfu) of virulent S. typhimurium for 12 days. It was thus concluded that both Andrographis paniculata and AND not only elicited both humoral and cell-mediated immune responses in the mouse model, but also increased the protective efficacy against salmonellosis on mice vaccinated with inactivated S. typhimurium. Therefore the ability of Andrographis paniculata and AND to promote acquired immunity, particularly in inducing CMI may be important in protection against intracellular pathogen infection.