BIOACTIVITY OF Ricinus communis AND Azadirachta indica EXTRACTS ON Leishmania major INFECTION IN BALB/c MICE

Abstract

Leishmaniasis is endemic in over 88 countries in the world and 350 million people are considered at risk. The standard drugs for treatment of leishmaniases are toxic, expensive and resistance against them is increasing. They require protracted administration, and currently, there is no vaccine. This study examined both in vivo and in vitro response of Leishmania major infection to combined therapy of Ricinus communis and Azadirachta indica crude extracts in BALB/c mice. BALB/c mice were inoculated intradermally on the left hind footpad (LHFD) with 106 L. major infective promastigotes. The mice were treated intraperitoneally with different drug combinations of daily doses for 28 days. Pentostam and amphotericin B were used as the reference drugs under the same experimental conditions. Negative controls were treated with Phosphate Buffered Saline (PBS) and Roswell Park Memorial Institute 1640 Medium (RPMI) in vivo and in vitro respectively. All experiments were performed in triplicate, the mean standard deviation of at least three experiments were determined, statistical analysis of the differences between mean values obtained for the experimental groups was done by the students t-test. P. values of 0.05 or less (p≤0.05) were considered to be significant. BALB/c mice, treated with combination therapy resulted in significantly (P < 0.0003) larger reduction of lesions than those treated with monotherapies. The spleen and spleno-somatic index was found to be significantly low with combination therapy than monotherapies. However, standard drugs had better efficacy for reduction of parasite load. Antiparasitic effect of A. indica and R. communis on amastigote with a 50% inhibitory concentration (IC50) was of 11.5μgml–1 and 16.5μgml–1 respectively while combination therapy gave 9.0μgml–1 compared to the standard drugs, pentostam and amphotericin B which had an IC50 of 6.5μgml–1 and 4.5μgml–1, respectively. Optimal efficacy of A. indica and R communis was 72% and 59.5%, respectively, combination therapy gave 88%, while pentostam and amphotericin B had 98% and 92%, against amastigotes respectively. Against promastigotes A. indica and R. communis gave an IC50 of 10.1μgml–1, 25.5μgml–1respectively, while combination therapy was 12.2 μg ml–1 against 4.1 μg ml–1 and 5.0 μg ml–1 for pentostam and amphotericin B, respectively. The optimal efficacy of the compounds against promastigotes was 78.0%, 61.5% and 91.2% (A. indica, R. communis and A. indica + R. communis respectively) against 96.5% and 98% for pentostam and amphotericin B respectively. The concentrations at optimal efficacy were significantly different (p<0.002) among the test compounds. Toxicity level for combined therapy was lower than standard drugs. The Leishman Donovan Units (LDU) of combination therapy was not significantly different (P<0.001) from that of pentostam though amphotericin B was better. These results showed that combination therapy of A. indica and R. communis had better antileishmanial activity than the monotherapies. It was also better than the standard drugs since it had lower toxicity to body cells. Further tests on the two plant extracts need to be done using non-human primates that are susceptible to leishmaniasis such as vervet monkey, Cercopithecus aethiops.