Cellular and humoral immunomodulation studies of Capparis spinosa, Pulicaria undulate and Echinacea purpurea in vitro and in vivo

Abstract

Herbal medicine is widely used in Saudi Arabia because of their effectiveness, little side effects and low cost, so investigations based on traditional plants has become more important. Although herbal medicine has long been used effectively in treating many immune diseases, the mechanism of action of most of the herbs used still not known precisely. Humoral and cellular immune response of methanolic extract of Capparis spinosa (C. spinosa) and Pulicaria undulate (P. undulata) was the aim of the present study. Rats, rabbits and chickens were used for assessment. The immunomodulation responses of methanolic extracts of the two plants were compared to either positive response of Echinacea purpurea (E. purpurea) or negative response of cyclophosphamide (CY). Animals were divided into seven groups. The first group was challenged with sheep erythrocytes (SRBC) at days 0 and 15 whereas animals of the second group were challenged with SRBC after 3 days of treatment with CY. The third group was challenged with SRBC after two weeks from treatment with standardized dried powder extract of E. purpurea. Animals in the fourth and fifth groups were pretreated with extract of P. undulata and C. spinosa for two weeks then challenged with SRBC. Animals in the last two groups were treated with CY and E. purpurea only. Blood samples were taken prior to challenge or on the fourth day of CY injection or after two weeks of E. purpurea, C. spinosa and P. undulate extracts (day 0) and on days 7, 21, and 42 post-immunization. The sera were obtained and then inactivated at 56°C for 30 min for removal the nonspecific agglutinins. Part of blood samples was used for counting the total leukocytic count (TLC) as well as neutrophil (N), lymphocyte (L) and N/L ratio within one hour after collection. The solid phase enzyme-linked immunosorbent assay (ELISA) using membrane of sheep red blood cells as antigen was performed to estimate immunoglobulin at days 0 (pre-challenge), 7, 14, 21, 28, 35 and 42 (post challenge). Viable splenocytes number and histopathology of spleen were also evaluated. Cellular immune response was done using in vivo and in vitro carbon clearance assay, phagocytosis index and lymphocyte transformation assay using glucose consumption test. Results obtained showed that: The microscopic criteria of the spleen of the control rats, rabbits and chickens groups were within the normal histologic limits. In comparison with such animals, all the remaining experimental groups showed microscopic immunological changes either good (immunostimulation) or adverse (immunosuppression). Generally, these immune reactions were completely similar for each species in relation to any of the tested materials meaning that any of the tested materials induced the same immunologic reaction in the spleen of rats, rabbits or chickens. TLC, N%, L%, spleen cellular viability %, in vivo and in vitro phagocytic index and glucose consumption of phytohemagglutinin (PHA) stimulated blood L of rats, rabbits and chickens challenged with SRBC and treated with CY were significantly decreased than challenged group. TLC and L% in rats, rabbits and chickens pretreated with E. purpurea were significantly increased than challenged with SRBC. Administration of Echinacea was significantly differ from the suppressive effects induced by CY. Rat and rabbit groups pretreated with E. purpurea showed significantly increased spleen cellular viability %, immunoglobulin, phagocytic index and glucose consumption of PHA stimulated blood L than that of the SRBC challenged animals. Generally, Echinacea is thought to create activity in the immune system by stimulating T-cell production, phagocytosis, and lymphocytic activity. The augmentation of the humoral immune response to SRBC by E. purpurea, increased the levels of circulating antibodies in rats, rabbits and chicken indicating the enhanced responsiveness of T and B lymphocyte subsets, involved in the antibody production. TLC and N% in rats or rabbits pretreated with P. undulate were significantly increased than challenged with SRBC. L% of rats pretreated with P. undulate was significantly lower than that of rats challenged with SRBC. At all sampling days, TLC in chickens pretreated with P. undulate was significantly increased than TLC in chickens challenged with SRBC. P. undulate pretreated groups of rats and rabbits showed significantly increased spleen cellular viability % than that of the SRBC challenged animals. No significant changes were observed on Igs, IgM and IgG in serum. It was found that the phagocytic index and glucose consumption of PHA stimulated blood L of P. undulate pretreated in rabbits and chickens was significantly higher than that of SRBC challenged group. TLC and N% of rats, rabbits and chickens pretreated with C. spinosa were significantly increased than challenged with SRBC. L% in rats pretreated with C. spinosa was significantly decreased than rats challenged with SRBC. Results revealed no significant change in N/L between rats pretreated with C. spinosa and challenged group. L% in rabbits and chickens pretreated with C. spinosa was significantly increased than SRBC challenged group. C. spinosa pretreated rats and rabbits showed significantly lower spleen cellular viability % than that of the SRBC challenged animals. No significant effect of C. spinosa pretreatment on Igs. It was found that in the day 21, 35 and 42 post challenge, IgM of C. spinose pretreated rats was significantly higher than that of SRBC challenged rats. In the day 14 post challenge, IgG in serum of C. spinose pretreated rabbits was significantly higher than that of SRBC challenged rabbits. Results showed that the phagocytic index and glucose consumption of PHA stimulated blood Lof C. spinosa pretreated rats and chickens were significantly higher than that of SRBC challenged group. However, the phagocytic index of C. spinose pretreated rabbits was significantly lower comparing with SRBC challenged group. The administration of Capparis significantly differ from the myelosuppressive effects induced by CY. The effect exhibited by C. spinose under our experimental conditions could be due to its polyphenolic active components, in particular flavonoids, which are known to possess immunomodulatory properties. TLC, N% and L% of E. purpurea treated rats, rabbits and chickens were significantly higher than that of CY treated animals. However, at day 14, E. purpurea treated chickens showed H/L significantly lower than CY treated chickens. SRBC-Igs, IgM and IgG of E. purpurea treated rats, rabbits and chickens were significantly increased than that of CY treated animals at the day 7, 14, 21, 28 and 42 post treatment. Phagocytic index, spleen cellular viability % and glucose consumption of PHA stimulated L of E. purpurea treated rats, rabbits and chickens was significantly increased than that of CY treated groups. On the other hand, in vitro phagocytic index of E. purpurea treated rabbits was significantly lower than that of CY treated rabbits. Finally, it could be concluded that administrations of extract of C. spinosa and to a lesser extent P. undulate significantly increased the TLC and also varied from the suppressive effects induced by CY. Pretreatment of animals with P. undulata and C. spinosa extracts showed a remarkable augmentation in the phagocytic function by exhibiting increase in the clearance rate of carbon particles. An increase in the viability of spleen cellularity % of C. spinosa was observed in the present study, which indicates that the extract may stimulate and increases the number of viable spleen cells and thus enhances the immune system. Also such findings were confirmed by histological studies. The present investigation suggests that extracts used may stimulate both cellular and humoral immune responses. Further studies to elucidate the exact immunostimulatory mechanisms are in progress. It is of interest to note here that extracts used are good candidates for alternative adjuvant chemotherapy and immunomodulatory in reducing the immunotoxicity of CY in animal models. However, clinical studies are needed to confirm the safety and efficacy of these plant components. Results expected may pave the road for further immune improvement for better healthy state. A more in-depth clinical approach is necessary to establish the clinical relevance of these changes. (We need to think differently).