Alcoholic Fractions F5 and F6 from Withania somnifera Leaves Show a Potent Antileishmanial and Immunomodulatory Activities to Control Experimental Visceral Leishmaniasis

Abstract

Visceral leishmaniasis (VL) causes fatal life-threatening disease, if left untreated. The current drugs have various limitations; hence, natural products from medicinal plants are being focused in search of new drugs to treat leishmaniasis. The aim of the present study was to evaluate the antileishmanial and immunomodulatory activities of F5 and F6 alcoholic fractions from Withania somnifera leaves and purified withaferin-A in Leishmania donovani-infected peritoneal macrophages and BALB/c mice. We observed that F5 (15 µg/mL), F6 (10 µg/mL), and withaferin-A (1.5 µM) reduce amastigote count in peritoneal macrophages and induce reactive oxygen species and significant decrease in IL-10 mRNA expression compared to control upon treatment. Subsequently, in vivo study mice were treated with F5 (25 and 50 mg/kg b.wt.), F6 (25 and 50 mg/kg b.wt.) orally, and withaferin-A (2 mg/kg b.wt.) intraperitoneally for 10 consecutive days and a drastic reduction in parasite burden in both spleen and liver were observed. The treatment resulted in the reduction in IL-10, IL-4, and TGF-β mRNA expression and a significant increase in IFN-γ/IL-10 expression ratio in the treated group compared to control. The humoral response of these alcoholic fractions and withaferin-A shows increased IgG2a levels when compared with IgG1 in treated mice. Taken together, our result concludes that withanolides in alcoholic fractions demonstrate a potent antileishmanial and immunomodulatory activities in experimental VL.

Keywords: IL-10; Withania somnifera; peritoneal mouse macrophages; visceral leishmaniasis; withaferin-A.

Figure 1

(A) Cytotoxic assay and parasite burden. MTT assay with fraction F5, F6, and withaferin-A. The results are represented as percentage of viability compared to control. (B) Antileishmanial activity of F5, F6, and withaferin-A on intracellular amastigotes using ex vivo peritoneal mouse macrophages (PMM) was counted using Giemsa staining. The graph shows the number of amastigote/100 macrophages treated with F5 (15 µg/mL), F6 (10 µg/mL), and withaferin-A (0.5 µM, 1.0 µM, and 1.5 µM). The values are from three independent experiments, and significance values were calculated using unpaired Student’s t-test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 2

Reactive oxygen species (ROS) production from peritoneal mouse macrophages (PMM) was estimated using a fluorescence dye H₂DCFDA. The graph shows the mean fluorescence intensity (MFI) values of uninfected, infected, F5 (15 µg/mL), F6 (10 µg/mL), withaferin-A (1.5 µM), miltefosine (3.2 µM), and LPS (100 ng/mL) treated PMM.

Figure 3

Immunomodulatory effect of the withanolides in infected control and treated peritoneal mouse macrophages (PMM) ex vivo. (A) RT-qPCR analysis of IFN-γ and IL-10 cytokines. (B) IFN-γ/IL-10 ratio representing the parasite clearance from PMM. (C) IFN-γ and TNF-α protein levels in PMM culture supernatants were assessed using CBA analysis. Unpaired Student’s t-test was performed (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 4

Antileishmanial effect of withanolides in BALB/c model of visceral leishmaniasis. (A) Leishman-Donovan units (LDU) in spleen and liver of infected control and treated mice groups after 45-day postinfection/posttreatment. (B) The bar graph represents the percentage of parasite reduction compared to infected control. The two independent experiments were done with five mice per group, and the significance between infected control and treated groups was calculated using one-way ANOVA with Dunnett’s posttest (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 5

Immunomodulatory effects of F5, F6, and withaferin-A in BALB/C mice spleen tissue using RT-qPCR analysis. (A) mRNA expression analysis of Th1, Th2 cytokines, and iNOS gene expression in F5- and F6-treated mice after 45-day postinfection/posttreatment. (B) mRNA expression analysis of Th1, Th2 cytokines, and iNOS gene expression in withaferin-A-treated mice. (C) The graph depicts the IFN-γ/IL-10 ratio in F5-, F6-, and withaferin-A-treated mice. The significance between infected control and treated groups was calculated using one-way ANOVA with Dunnett’s posttest (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 6

The IgG1 and IgG2a antibody titer levels in different treatment groups of mice. The results are expressed as mean ± SD of five mice. The significance was calculated between infected control and treated mice groups using one-way ANOVA with Dunnett’s posttest (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 7

Effect of F5, F6, and withaferin-A on histological changes in liver and hematoxylin and eosin-stained liver sections of different treatment mice groups.