Bioflavonoid-Induced Apoptosis and DNA Damage in Amastigotes and Promastigotes of Leishmania donovani: Deciphering the Mode of Action

Abstract

Natural products from plants contain many interesting biomolecules. Among them, quercetin (Q), gallic acid (GA), and rutin (R) all have well-reported antileishmanial activity; however, their exact mechanisms of action are still not known. The current study is a step forward towards unveil the possible modes of action of these compounds against Leishmania donovani (the causative agent of visceral leishmaniasis). The selected compounds were checked for their mechanisms of action against L. donovani using different biological assays including apoptosis and necrosis evaluation, effects on genetic material (DNA), quantitative testing of nitric oxide production, ultrastructural modification via transmission electron microscopy, and real-time PCR analysis. The results confirmed that these compounds are active against L. donovani, with IC₅₀ values of 84.65 µg/mL, 86 µg/mL, and 98 µg/mL for Q, GA, and R, respectively. These compounds increased nitric oxide production and caused apoptosis and DNA damage, which led to changes in the treated cells' ultrastructural behavior and finally to the death of L. donovani. These compounds also suppressed essential enzymes like trypanothione reductase and trypanothione synthetase, which are critical for leishmanial survival. The selected compounds have high antileishmanial potentials, and thus in-vivo testing and further screening are highly recommended.

Keywords: DNA damage; Leishmania donovani; apoptosis; gallic acid; leishmaniasis; quercetin; rutin.

Figure 1

Percent growth inhibition of the Leishmania (Ds-Red L. donovani strain LV82) promastigotes caused by quercetin, gallic acid, and rutin at concentration equal to their IC₅₀ (µg/mL) values. Data represent the mean values of three replicates with ± standard error. Different labels on columns show significant difference at p < 0.05.

Figure 2

Graph showing amastigote count inside 1000 raw macrophages following treatment with quercetin, gallic acid, and rutin at 15.62 μg/mL–500 μg/mL after 72 h of incubation compared with vehicle control. Data represent the mean values of three replicates with ± standard error. Different labels on column data show significant difference at (p < 0.05).

Figure 3

Amastigotes inside macrophages shown with arrows: (a) untreated control, (b) treated with quercetin, (c) treated with gallic acid, and (d) treated with rutin at the highest concentration tested (500 μg/mL) after 72 h of incubation, stained with Giemsa stain. (e) SSG 250 μg/mL was used as positive control. Images were taken with a Nikon microscope.

Figure 4

BMDMs infected with transgenic L. donovani parasites expressing the red fluorescent protein Ds-Red for the evaluation of parasitic loads using flow cytometry. Treatment was conducted with (a) untreated control and cells treated with (b) quercetin, (c) gallic acid, and (d) rutin at their inhibitory concentrations (IC₅₀) (84.65 µg/mL, 86 µg/mL, and 98 µg/mL, respectively) for 72 h. (e) SSG 250 µg/mL was used as a positive control.

Figure 5

Percentage of amastigotes inside BMDMs infected with transgenic L. donovani parasites. Treatment was conducted with quercetin, gallic acid, and rutin at their 50% inhibitory concentrations (IC₅₀) (84.65 µg/mL, 86 µg/mL, and 98 µg/mL, respectively) for 72 h. SSG at 250 µg/mL was used as a positive control. Data represent the mean values of three replicates with ± standard error. Different labels on columns show significant difference at p < 0.05.

Figure 6

Images taken with fluorescent microscope: (a) untreated leishmania promastigotes and treated with (b) quercetin, (c) gallic acid, and (d) rutin. AB: apoptotic body, LA: late apoptosis, EA: early apoptosis, MB: membrane blebbing, N: necrosis, and NP: normal promastigotes.

Figure 7

Images of EtBr-stained DNA migration towards anode. Damaged DNA from L. donovani was differentiated via alkaline comet assay: (a) normal DNA (untreated control) and DNA from samples treated with (b) quercetin, (c) gallic acid, and (d) rutin at (500 μg/mL) after 72 h of incubation.

Figure 8

Total comet scores of leishmanial DNA damage following treatment with quercetin, gallic acid, and rutin, compared with control DNA (from untreated Leishmania). Data represent the mean values of three replicates with ± standard error. Different labels on columns show significant difference at p < 0.05.

Figure 9

Quantification of NO production by BMDMs infected with L. donovani and treated with Q, GA, and R at 250 µg/mL and 500 µg/mL for 72 h. Infected macrophages without treatment served as a control. Data represent the mean values of three replicates with ± standard error. Different labels on columns show significant difference at p < 0.05.

Figure 10

Fold change in expression level of trypanothione reductase enzyme in Leishmania donovani after treatment with two different concentrations (250 µg/mL and 500 µg/mL) of quercetin, gallic acid, and rutin. Data represent the mean values of three replicates with ± standard error. Different labels on columns show significant difference at p < 0.05.

Figure 11

Fold change in expression level of trypanothione synthetase enzyme in Leishmania donovani after treatment with two different concentrations (250 µg/mL and 500 µg/mL) of quercetin, gallic acid, and rutin. Data represent the mean values of three replicates with ± standard error. Different labels on columns show significant difference at p < 0.05.

Figure 12

Ultrastructural changes in L. donovani LV82 promastigotes treated with quercetin at 500 μg/mL for 72 h. (a) Normal cells, (b) nuclear condensation, (c) appearance of lipid reservoirs, (d) distortion of the flagellar pocket, and (e) disruption of the mitochondria–kinetoplast complex.

Figure 13

Ultrastructural changes in L. donovani LV82 promastigotes treated with gallic acid at 500 μg/mL for 72 h. (a) Distortion of the flagellar pocket, (b) acidocalcisomes, (c,d) large number of vacuoles, and (e) lipid reservoirs.

Figure 14

Ultrastructural changes in L. donovani LV82 promastigotes treated with rutin at 500 μg/mL for 72 h. (a) Distortion of the flagellar pocket, (b) acidocalcisomes, (c) large number of vacuoles, (d) lipid reservoirs, and (e) nuclear condensation.