Synergistic Effects of Extracts of Aloe secundiflora Eng L. and Callistemon citrinus William C. in Leishmania major Infected BALB/c Mice

Abstract

Leishmania major causes painful skin sores in humans medically known as cutaneous leishmaniasis. The current drugs against the disease required prolonged use, is toxic and expensive. The prolonged use has led to emergence of drug resistant by the parasite. Studies have reported antileishmanial activity of several plants with low toxicity and thus such plants can be used as alternative medicine. For instance, Aloe secundiflora and Callistemon citrinus individual aqueous extracts of have been shown to have antileishmanial activities. Combination therapy using different plant extracts can potentially reduce drug resistance. Efficacy of A. secundiflora and C. citrinus combination therapy (1:1 ratio) against BALB/c mice L. major infected and treated intraperitoneally and orally was evaluated. Positive and negative controls were Pentostam administered intraperitoneally, and phosphate buffered saline intraperitoneally and orally treated respectively. Dry ground test materials were soaked in H2O at 800 C for 1 hour, filtered, and freeze dried. Data was analyzed using student t-test and ANOVA with significance level of p value < 0.05. A. secundiflora had of MIC 2mg/ml while C. citrinus had 5mg/ml with IC50 levels 467.09μg/ml and 457.88μg/ml respectively. The MIC for combination therapy was at 1: 1 ratio with 58.45μg/ml of IC50. The Infection rate (IR) and multiplication index percentage (MI) of the combination therapy was at 19% and 52.81% respectively. Combination therapy cell viability was at 58.45 μg/ml compared to 467.09μg/ml and 457.88μg/ml for A. secundiflora and C. citrinus respectively. No statistically significant difference (t = 2.481, p = 0.089) observed even though combination therapy had high cytotoxicity. The IR of combination therapy (1:1 ratio) at 125μg/ml concentration was 19% compared to 46% for C. citrinus and 23% for A. secundiflora. ANOVA comparison of the IR% showed no statistically significant difference (F (1, 3) = 2.242 and p = 0.446). The MI% for A. secundiflora, C. citrinus and combination therapy (1:1) were 49%, 189% and 127% respectively. There was statistically significant difference (p = 0.05) among the MI% using ANOVA analysis. The Nitric Oxide could not be determined since the OB produced was less than 0.1 and therefore antileishmanial activity in these extracts is not related to NO production. Lesions sizes decreased after treatment with combination therapy and there was statistically significant difference (F (4,16) = 9.127 and p = 0.001) in decrease of the lesion sizes among the treatments. Post hoc analysis using Games-Howell for multiple comparison of lesion sizes showed statistically significant difference (p = 0.042 and p = 0.044) between oral combination treated and both PBS oral and ip treated groups respectively. The average total parasite load in BALB/c mice treated with the combination therapy reduced and there was significant statistical (F (4, 12) = 113, p = 0.0001) when comparing with controls. The spleen parasite load reduced significantly (t = 2.653, p = 0.057) after treatment with combination therapy. In conclusion, combination therapy was effective against L. major parasites, significantly reduced both lesion size and showed possibility of synergistic effects both in vitro and in vivo. This study recommends further research on natural products of test plants to support use of their extracts in management of VL in poverty stricken leishmaniases endemic areas of Kenya.