Cedrus deodara: In vitro antileishmanial efficacy & immumomodulatory activity

Abstract

Background & objectives: The existing antileishmanial drugs for complete cure of visceral leishmaniasis (kala-azar) are limited. The available drugs are either toxic or less effective leading to disease relapse or conversion to post-kala-azar dermal leishmaniasis. Several herbal extracts have been shown to have antileishmanial activity, but a herbal drug may not always be safe. In the present study, the extract of Cedrus deodara leaves has been standardized and tested for immunomodulatory antileishmanial activities.

Methods: The extracts of C. deodara leaves with different solvents such as benzene, chloroform, ethyl acetate and methanol were made by soxhlation process. Solvents were removed under reduced pressure and temperature using rotary evaporator. The antileishmanial bioassay test was performed with in vitro maintained parasites. Immunomodulatory activity of different extracts was tested by flow cytometry. Standardization of the effective fraction was performed with Linalool as a marker compound through reverse-phase high-performance liquid chromatography.

Results: The extract with the use of benzene solvent showed strong antileishmanial activities within a dose 25-200 μg/ml culture with non-significant haemolytic activities and significant immunomodulant activities against the host cells. Linalool was found to be 1.29 per cent in the effective extract of C. deodara.

Interpretation & conclusions: The antileishmanial activity of C. deodara, as assessed by bioassay testing on.

Leishmania donovani: parasites and immunomodulatory effect of benzene extract of leaves on host cells indicated that it might be a potential new safe therapeutic target to cure the visceral leishmaniasis.

Keywords: Arginase - Cedrus deodara - linalool - reverse-phase high-performance liquid chromatography - visceral leishmaniasis.

Fig. 1

Benzene fraction of Cedrus deodara extract (CDB) showing antileishmanial activities. (A) Leishmania donovani promastigotes of late log phase challenged with different concentration of extract showed IC₅₀ and IC₉₀ concentrations of 25 and 200 μg/ml, respectively. (B) IC₅₀ and IC₉₀ concentrations of Leishmania donovani intracellular amastigotes were found at 25 and100 μg/ml, respectively. CDB, Cedrus deodara benzene; CDC, Cedrus deodara chloroform; CDE, Cedrus deodara ethanol; CDM, Cedrus deodara methanol.

Fig. 2

Intracellular parasite burden in the absence or presence of benzene extract of IC₅₀ concentration. (A) Macrophages (×100) without the infection treated as control, (B) macrophages (×100) infected with 2×10³ ± 85/100 cells, (C) infected macrophages (×100) treated with benzene extract to inhibit parasitic load 2×10² ± 25/100 cells.

Fig. 3

MTT assay to confirm antileishmanial activity of benzene extract of Cedrus deodara. Parasites treated with IC₅₀ concentration of extract. Only the 50 per cent parasites showed insoluble purple (P<0.001), indicating decreased dehydrogenase activities. CDB, Cedrus deodara benzene; CDC, Cedrus deodara chloroform; CDE, Cedrus deodara ethanol; CDM, Cedrus deodara methanol.

Fig. 4

Nitric oxide (NO) production in human cells after treating with Cedrus deodara extracts. In comparison to unstimulated cells (UNS) the nitric oxide production was 1.3-fold more with effective (benzene) extract (P<0.01); however, it was less than lipopolysaccharide (LPS) stimulated cells (P<0.001).

Fig. 5

Percentage of T helper cells producing (A) interferon-gamma (IFN-γ) and (B) interleukin-10 (IL-10) after PBMCs were stimulated with effective concentration of Cedrus deodara extracts and compared with unstimulated control and LPS stimulated positive control. The IFN-γ producing CD4⁺ cells were 2-fold higher (P<0.05) than unstimulated and 1.6-fold higher (P<0.05) than LPS. IL-10 production within the CD4⁺ cells was less (not significant) than unstimulated cells and significantly less (P<0.05) than lipopolysaccharide. CDB, Cedrus deodara benzene; LPS, lipopolysachride; UNS, unstimulated.

Fig. 6

MTT assay showing no effect on dehydrogenase activity in different extracts treated host cells compared to control. The absorbance values of test samples after exposure of effective extract of Cedrus deodara to the peripheral blood mononuclear cells for 24 h were almost similar to the negative control which indicated the effective concentration of extract did not inhibit mitochondrial dehydrogenase (P<0.01).

Fig. 7

Hemolytic activities of Cedrus deodara against human RBC. The final concentration of red blood cells number was calculated 5-10 per cent less compared to the healthy control cells (P<0.05).

Fig. 8

Reverse phase-HPLC chromatograms. The percentage amount of standard constituent (linalool) present in benzene fractions of plant extracts was found to be 1.29 per cent. Retention time of linalool had been generated by the author which was found to be 5.32 min. (A) Linalool, (B) Cedrus deodara.