Efficacy of Crude Extracts from Allium Sativum, Callistemon Citrinus and Moringa Stenopetala of Kenya against Leishmania Major

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Kinuthia, Geoffrey Kariuki
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Cutaneous leishmaniasis (CL) is endemic in more than 80 countries worldwide and it causes skin ulcers and disfigurement. Leishmania major causes CL in Kenya and its drugs are expensive, toxic and require prolonged use. Multidrug combination therapy prevents drug resistance and reduces toxicity. Herbal extracts can be safe and cheaper. This study investigated in vitro and in vivo efficacy of single and blends of crude aqueous and methanolic extracts from Moringa stenopetala, Callistemon citrinus, and Allium sativum against L. major. Controls were contemporary Leishmania drugs pentostam and liposomal amphotericin B, and phosphate buffered saline. Dry ground test materials were soaked in H2O at 70oC for 1½ hours, filtered, and freeze dried. Similarly, ground test materials were soaked in 500 ml of analytical grade methanol for 72 hours at room temperature, filtered and concentrated using rotary evaporator. T-test and ANOVA were used for data analysis. P-value of < 0.05 was considered significant. The minimum inhibitory concentrations (MICs) of aqueous extracts of M. stenopetala (A), C. citrinus (B), and A. sativum (C) ranged from 3 to 5mg/ml while IC50 from 297.79 to 575.75μg/ml against L. major promastigotes as compared to MICs of 12.50 and 6.25μg/ml and IC50 of 0.26 and 0.82μg/ml for pentostam and liposomal amphotericin B respectively. MICs of methanolic extracts of M. stenopetala (H), C. citrinus (G), and A. sativum (F) ranged from 1 to 5mg/ml with IC50 of 572.69 to 1752.92μg/ml against L. major promastigotes. The extracts’s cytotoxicity against vero cells ranged from 467.11 to 2105.93μg/ml as compared to 60.95μg/ml and 108.58μg/ml for pentostam and liposomal amphotericin B respectively. Methanolic extracts of C. citrinus and aqueous A. sativum extracts stimulated production of 20μM nitric oxide in BALB/c mice peritoneal macrophages signifying their immuno-modulatory role. Blends of M. stenopetala & C. citrinus (AB), M. stenopetala & A. sativum (AC) and C. citrinus & A. sativum (BC) at concentrations based on MICs of individual extracts, were active at ratios 1:1, 1:9 and 1:1 with promastigotes’ viabilities of 33.82%, 17.41% and 60.74 % respectively. The ratios and promastigotes viabilities for methanolic blends were, FG (1:1; 31.32%), FH (1:9; 34.59%) and GH (9:1; 7.44%). The IC50 for any blends of two extracts ranged from 174μg/ml to 1314μg/ml against L. major promastigotes. There was strong synergistic (1:9) and additive (1:1 and 2:8) interactions for the blend AC. Blend BC interacted additively at ratio 1:1. Blend AC at 125μg/ml had infection rate (IR) of 71% and multiplication index (MI) of 48.20% for L. major amastigotes in vitro, and compared well to pentostam at 12.50μg/ml with IR of 67% and MI of 47.51%. Methanolic blends of three different extracts were more efficacious with MIs of 33.48 to 38.24%. Oral aqueous and methanolic A. sativum extracts (A and F) reduced the foot pad lesion sizes significantly (P < 0.05) in infected BALB/c mice. Oral blend BC reduced the footpad lesion size significantly (P < 0.05) like Leishmania control drugs. Oral blends BC and AC reduced spleen amastigotes in mice by 48.33% and 60.94% corresponding to total LDUs of 6.35±0.66 and 4.80±0.95 respectively. Oral/ip blend HGF (2:2:1) had amastigotes inhibition rate of 63.95% compared to 66.40% and 60.62% for pentostam and liposomal amphotericin B respectively. In conclusion, aqueous and methanolic crude extracts of C. citrinus, A. sativum and M. stenopetala were less toxic but active against L. major in vitro and in vivo and their blends that had additive or synergistic interaction lowered L. major survival. This study recommends that Kenyatta University in collaboration with relevant stakeholders to consider developing natural products based on these results for the management of CL in poverty stricken leishmaniases endemic areas of Kenya.
Department of Pathology, 171p. 2013, RM 666 .H33K5