Fig 1.
Anti-promastigote efficacy of C. cassia fractions.
(a) Growth kinetics assay. Growth inhibitory potential of C. cassia fractions (500 μg ml-1) was assessed against L. donovani promastigotes (2×106 ml-1). The parasite density was evaluated after every 24h as described in methods. Control = parasite control without any treatment; solvent control = 0.25% DMSO; CBD = C. cassia bark DCM fraction; CBE = C. cassia bark ethanolic fraction; CBA = C. cassia bark aqueous fraction. ***P<0.001 with respect to parasite control. (b) Morphological analysis of CBD treated promastigotes. Untreated or treated L. donovani parasites were stained with Erythrosin B and visualized under a phase contrast microscope at 100X. (c) Growth reversibility assay. L. donovani promastigotes (2× 106 ml-1) were incubated with or without test fractions for 7 days and growth reversal after drug withdrawal was ascertained post 96 h. ***P<0.001with respect to parasite control. (d) IC50 determination. L. donovani promastigotes (2× 106 ml-1) were incubated with CBD or pentamidine (500 μg ml-1) or medium alone at 22°C for 96 h after which the cell viability was determined as described in methods.
Fig 2.
Induction of PS externalization in L. donovani promastigotes.
CBD or pentamidine (34 μg ml-1) treated promastigotes (2×106 ml-1) were co-stained with Annexin-V-FLUOS and PI. The cells were acquired on a flow cytometer and dot plots generated. The lower left quadrant represents the percentage of live cells (Annexin-V and PI dual negative), whereas apoptotic cells (Annexin-V positive) can be seen in lower right quadrant. The upper left quadrant represents apoptotic (or PI positive) cells whereas upper right quadrant represents late apoptotic cells (Annexin-V and PI dual positive). The percentage of stained cells is mentioned in respective quadrants.
Fig 3.
Apoptotic events in L. donovani promastigotes.
(a) Study of mitochondrial membrane potential. L. donovani promastigotes (2×106 ml-1) were subjected to treatment with C. cassia bioactive fraction and pentamidine (34 μg ml-1) for 72 h at 22°C. The untreated and treated parasites were stained with JC-1 as described in methods. ***P<0.001 with respect to parasite control. (b) Determination of ROS generation. Parasites (2× 106 ml-1) were incubated with or without CBD and pentamidine (34 μg ml-1, 72 h, 22°C) and stained with H2DCFDA for detection of ROS production. Shift in mean fluorescence intensity (MFI) in P2 gated region depicted the extent of ROS production. (c) Bar graph representing changes in ROS production. The recorded MFI in untreated or treated samples are represented graphically to divulge the changes in ROS production. ***P<0.001 with respect to parasite control.
Fig 4.
DNA fragmentation and cell cycle analysis.
(a) Detection of DNA fragmentation by TUNEL assay. Parasites (2×106 ml-1) were cultured with CBD and pentamidine (34 μg ml-1) for 72 h at 22°C. The TUNEL staining was carried out as described in methods. ***P<0.001 with respect to parasite control. (b) Cell cycle analysis. L. donovani promastigotes (2×106 ml-1) were treated with test fraction and pentamidine (34 μg ml-1). Post 72 h, the cells were processed for PI staining and flow cytometry analysis as described in methods. (c) Bar graph depicting changes in cell population in different stages of interphase in treated as well as untreated samples. ***P<0.001 with respect to parasite control.
Fig 5.
Effect of CBD treatment on L. donovani infected/ normal peritoneal macrophages.
(a) Anti-amastigote potential of CBD. Peritoneal macrophages (5×106 ml-1) were infected with Leishmania parasites (10:1). Promastigotes were allowed to transform into amastigotes under optimum ex vivo conditions (37°C, 5% CO2) and were treated with CBD (0–200 μg ml-1). Post 48 h, the amastigote number and percent infectivity was calculated as described in methods. ***P<0.001 with respect to parasite control. (b) Detection of NO generation. L. donovani infected macrophages were treated with CBD and culture supernatants were collected after 48 h. NO production was quantified in terms of nitrite by means of Griess reagent as depicted in methods. ns = P>0.05 with respect to infection control group. (c) Microscopic imaging of Giemsa stained Leishmania infected peritoneal macrophages. The infected macrophages were subjected to treatment with CBD or pentamidine for 48 h after which the cells washed, fixed and giemsa stained as described in methods. The infection control group bore high amastigote burden whereas CBD or pentamidine treatment profoundly reduced the amastigote burden (as indicated by arrows).
Fig 6.
Cytotoxicity of CBD against peritoneal macrophages.
BALB/c mice peritoneal macrophages (2×106 ml-1) were isolated and seeded in 96 well plates for determination of cell viability post CBD treatment (0–500 μg ml-1, 48h) by MTT assay. The plate was read at 570 nm and absorbance was recorded to determine percent viability as described in methods.
Table 1.
Major plant secondary metabolites present in CBD.
Fig 7.
Therapeutic efficacy of CBD in L. donovani infected BALB/c mice.
L. donovani infected BALB/c mice were either untreated (INF, infection control group) or treated with VC (vehicle control), AmB (Amphotericin B, 5 mg/kg bw), CBD50 (C. cassia bark DCM fraction at 50 mg/kg bw) and CBD100 (C. cassia bark DCM fraction at 100 mg/kg bw). The parasite burden in host liver and spleen was calculated and expressed as Leishman Donovan Units. *P<0.05 and ***P<0.001 with respect to infection control.
Fig 8.
Evaluation of pro-inflammatory potential of CBD in L. donovani infected BALB/c mice.
(a) DTH response post CBD treatment. Magnitude of DTH was evaluated post 24 h of FT antigen inoculation in uninfected (normal group), untreated and variously treated groups. ***P<0.001 with respect to infection control. (b) Assessment of lymphoproliferation. Lymphoproliferative responses generated in unstimulated or SLA stimulated splenocytes from different treatment groups was analyzed by MTT assay as described in methods. ***P<0.001 in comparison to infection control. (c) Analysis of Th1/Th2 cytokine production. Post CBD (100 mg/kg bw) treatment levels of Th1 and Th2 cytokines were evaluated in culture supernatants of splenocytes post 48h of SLA stimulation. *P<0.05, **P<0.001 and ***P<0.001 and ns = non-significant in comparison to infection control.
Fig 9.
Analysis of NO production and IgG subclass response.
(a) Estimation of NO production in BALB/c mice splenocytes. NO production was quantified in terms of nitrite in culture supernatants of SLA stimulated splenocytes as described in methods. ***P<0.001 with respect to infection control. (b) Determination of serum levels of IgG isotypes. IgG2a and IgG1 levels were determined in mice sera from different experimental groups by ELISA as explained in methodology. IgG1 was particularly enhanced in VC and INF group in comparison to normal mice (##P<0.01). *P<0.05 and**P<0.001 in comparison to infection control.
Fig 10.
Antileishmanial efficacy of C. cassia bioactive fraction in L. donovani infected hamsters.
4–6 week old male hamsters were infected with L. donovani promastigotes and post six weeks of infection the hamsters were treated orally with CBD for ten days. One week post-treatment animals were sacrificed and parasite burden was estimated in giemsa stained impression smears of host liver and spleen. *P<0.05 and ***P<0.001 with respect to infection control.
Fig 11.
Evaluation of correlates of CMI in L. donovani infected hamsters.
(a) DTH response in CBD treated hamsters. Hamsters were inoculated with FT and PBS in right left hind footpads respectively. Post 24 h, differences in the footpad swelling (right-left) were calculated and mean±SEM were plotted to analyze the generation of DTH responses. ***P<0.001 in comparison to infection control. (b) NO production in L. donovani infected hamsters. NO generation in different experimental groups was studied by means of Griess reaction as described in methods. ***P<0.001 with respect to infection control. (c) In vitro recall responses in SLA stimulated splenocytes. Hamster splenocytes were labeled with CFSE and cultured in the absence or presence of SLA for 48 h. CFSE fluorescence was analyzed on BD LSR II flow cytometer and BD FACS DIVA software was used to generate histograms where percentage CFSE positive cells (as indicated) was deduced from histogram statistics.
Table 2.
In vivo toxicity of CBD in BALB/c mice.
Table 3.
In vivo toxicity of CBD in L. donovani infected mice.
Table 4.
Evaluation of hepatic and renal toxicity of CBD treatment in hamsters.
Table 5.
Assessment of hepatic and renal toxicity of CBD in L. donovani infected hamsters.