Immunomodulatory activity and safety profile of prunus Africana in an animal model: a common medicinal plant used in management of respiratory virus infections in local communities in Mpigi District, Uganda

Abstract

Prunus africana is a common medicinal plant used in modulating the immune system in the treatment of respiratory infections by the local communities and herbalists in Mpigi district. However, limited scientific information exists on the immunomodulatory properties of the plant extracts in animal models. To achieve this, immunomodulatory activities and safety profiles of P. africana extracts were undertaken in Wistar albino rats and Swiss albino mice respectively. Experimental laboratory-based study was conducted to determine the effects of aqueous extracts (AQ) and total crude extracts (TC) of P. africana on the cell-mediated and humoral-mediated immune responses and their safety in animal models. Low dose (200 mg/Kg bwt) and high dose (1000 mg/Kg bwt) were used as test substances of both extracts and 0.2 mL of distilled water as a control. Healthy Wistar albino rats (8 weeks, 100-150 g) and Swiss mice of either sex were used in the experiments. The study was carried out in accordance to ARRIVE Guidelines 2.0 for reporting animal research. Complete Blood Cell Count (CBC) and neutrophil adhesion response of Witstar albino rats were determined for 14 days following daily dosing of 200 mg/kg and 1000 mg/kg of respective extracts. Whole blood sampling was done on day 0, 7 and 14. Complete Blood Cell Count was analyzed using an automated coulter machine with standard methods and procedures. Neutrophil adhesion was determined by incubating whole blood with 80 mg nylon fibers /mL for 15 minutes at 37oC and then analyzed like for CBC. Phagocytic index was determined using carbon clearance in Wistar albino rats. Hemagglutination antibody titer assay was determined using sheep red blood cells as antigens. The oral acute toxicity study was carried out using the “up and down” method of testing in eleven Swiss mice. Single doses ranging from 50mg/kg-5000 mg/kg were administered orally in three stages. The general behavioral effects and mortality rates were monitored for a period of 24 hours. For sub-acute test study, the TC (most active) was administered orally to adult Wistar albino rats at a dose rate of 1000mg/kg on a daily basis for 28 days. Selected biochemical and hematological parameters were measured, and histological examinations of the kidney, liver and heart were conducted to assess any signs of organ damage at the end of the treatment period. Statistical analysis was done using STATA version 13. One-way ANOVA followed by Bonferroni post hoc test were used to statistically compare the treatment groups. Results were considered significant at p < 0.05. No significant changes among the mean WBC counts, mean lymphocytes and mean LIC (Large Immature Cell) counts in all doses of plant extracts was observed throughout test period (p =0.05). However, eosinophil and neutrophils significantly increased (p=001) on D7 and D14, respectively, for AQ1000 compared to the control. Similarly, AQ1000 demonstrated a rise in basophils values. Total crude extracts significantly reduced neutrophil levels on D14 as well as eosinophil on D7 compared to AQ1000 extract. The TC200 extracts produced a low mean % hematocrit count throughout the study period, whereas the AQ1000 had a marginal reduction of the same. Significantly low mean MCV and MCH values were demonstrated by AQ1000 extracts on D7-D12, and D14, respectively. The TC1000 produced a reduction of the same values but increasing RDW-CV on D7 and D14. The AQ200 exhibited an increase in platelets on D7-D14, and TC1000 on D14. A statistically significant increase in mean % neutrophil adhesion was observed with TC1000 extracts on D7 compared to the control and much higher than TC200. Although, the phagocytic index significantly increased for both total crude extracts, the value was quite below 1. The test plant samples exhibited comparable antibody titers to levamisole and the negative control. The results of the acute toxicity study demonstrated that the LD50 value following oral administration of TC1000 of P. africana was greater than 5000 mg/kg. In the sub -acute toxicity study, TC1000 compared to the control, a statistically significant decrease in WBC counts and monocytes was observed on D28. Similarly, a reduction on neutrophils, lymphocytes, monocytes and basophils was observed. Reductions were also demonstrated in MCH, haematocrit and MCV D28. No changes in biochemical parameters were observed. Microscopic examination of vital organs such as the liver, heart and kidney revealed no significant injuries except that the kidney had mild multifocal mononuclear inflammatory cells in the interstitium. In conclusion, the findings of the present study demonstrated that the aqueous extracts P. africana stimulated the innate immune response by increasing neutrophils, monocytes, eosinophil and basophil. The extracts may as well cause blood clots due to increasing levels of platelet counts. The total crude extracts caused a reduction in the inflammatory cells and a less than 1 phagocytic index was observed, hence acting as an anti-inflammatory and immunosuppressive medicinal plant. The study findings also showed that P. africana was safe at an LD50 of 5000mg/kg. Unfortunately, long- term use of this medicinal plant may induce toxicities as supported by the hematological parameters. The extracts demonstrated an immunosuppressive (anti-inflammatory) effect and a strong support on innate immune response and offers prospects in the treatment of inflammatory disorders and infectious conditions. Therefore, there is a need to isolate and elucidate the bioactive compounds and to find out their mode of action. The communities should be advised on the use P. africana cautiously to avoid the adverse effects exhibited by the plant.