Evaluation of in vitro and in vivo antileishmanial potential of bergenin rich Bergenia ligulata (Wall.) Engl. root extract against visceral leishmaniasis in inbred BALB/c mice through immunomodulation

Abstract

Background: Medicinal plants with immunomodulatory properties can provide good alternative therapeutics for curing visceral leishmaniasis. Bergenia ligulata (Wall.) Engl. is an interesting plant with strong antioxidant, antimicrobial, immunomodulatory and hepatoprotective properties.

Aim: The present study was planned to determine the antileishmanial activity of plant extract by modulating the immune responses of inbred BALB/c mice.

Methodology: Bergenin, the principle active component of B. ligulata, was quantitated in crude extract by performing RP-HPLC. The therapeutic potential was assessed through in vitro antileishmanial activity and in mice model through parasite load, cytokine assays, IgG antibody levels, DTH responses, histopathology and biochemical enzyme assays.

Results: B. ligulata showed the presence of glycosides, saponins, carbohydrates, tannins, flavonoids and bergenin which contributed to the antileishmanial activity of extract with IC50 of 22.70 μg/mL. Furthermore, the higher dose significantly reduced the parasite load by 95.56 %. The reduction was further associated with significant enhancement of IL-12 and IFN-γ levels in comparison to IL-10 and IL-4 cytokines. The switching towards Th1 type of immune response was also confirmed by elevated antibody levels of IgG2a isotype as compared to IgG1 as well as increased DTH responses. The histology of liver and kidney further complimented the non toxic nature of plant extract in addition to its negligible toxicity on HeLa cells.

Conclusions: The current study revealed the significant antileishmanial and immunomodulatory properties of this plant extract against murine visceral leishmaniasis. Further, the bioactive components will be explored to assess their efficacy for the development of safe and cost effective drug.

Keywords: BLEE, Bergenia ligulata ethanolic extract; Biochemical; CMI, Cell mediated immune responses; DCT, Distal convoluted tubules; DMSO, Dimethyl sulphoxide; Extract; HPLC; Histopathological studies; Immunological; PCT, Proximal convoluted tubules; Parasite load; SRBC, Sheep red blood cells; SSG, Sodium stibogluconate; T.S, Transverse section; VL, Visceral leishmaniasis; p.i.d., Post infection days; p.t.d., Post treatment days.

Graphical abstract

Fig. 1

A, B. Chromatograms obtained from bergenin standard and crude extract of B. ligulata at 214 nm.

Fig. 2

A. Percentage growth inhibition of L. donovani promastigotes with B. ligulata ethanolic extract. B. Flow cytometry scatter plot of (a) only stained untreated promastigotes (b) promastigotes treated with 10 μg/mL of SSG (c) promastigotes treated with 10 μg/mL of BLEE (d) promastigotes treated with 20 μg/mL of BLEE.

Fig. 3

Parasite load in different groups of animals. P value: Infected vs infected + SSG 40 mg/kg b.wt./infected + BLEE 500 mg/kg b.wt./infected + BLEE 1000 mg/kg b.wt. p* < 0.05.

Fig. 4

A, B: Levels of IgG2a and IG1 antibodies in different groups of animals. P value: Infected vs infected + SSG/infected + BLEE 500 mg/kg b.wt./infected + BLEE 1000 mg/kg b.wt. *p < 0.05.

Fig. 5

A, B, C, D: Concentration of cytokines IFNγ, IL-12, IL-4, IL-10 in serum samples of different groups of animal. P value: Infected vs infected + SSG/infected + BLEE 500 mg/kg b.wt./infected + BLEE 1000 mg/kg b.wt. *p < 0.05.

Fig. 6

Delayed type hypersensitivity response in animals of different groups. P value: Infected vs infected + SSG/infected + BLEE 500 mg/kg b.wt./infected + BLEE 1000 mg/kg b.wt. *p < 0.05.

Fig. 7

T.S. of kidney of BALB/c mice stained with haematoxylin and eosin stain (A) Normal control (10X) (B) Infected Control (10X) (C) Infected mice treated with SSG (10X) (D) Mice treated with 1000 mg/kg b.wt. of BLEE (40X).

Fig. 8

T.S. of liver of BALB/c mice stained with haematoxylin and eosin stain (A) Normal control (10X) (B) Infected Control (40X) (C) Infected mice treated with SSG (10X) (D) Mice treated with 1000mg/kg b.wt. of BLEE (10X).