Anthelmintic Potential of Thymoquinone and Curcumin on Fasciola gigantica

Abstract

Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 μM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence potential. Thus, both thymoquinone and curcumin appeared to be promising anthelmintic compounds for further investigations.

Fig 1

Mean worm motility of Fasciola gigantica treated with Curcumin (A) and Thymoquinone (B). Both compounds showing a time and concentration dependent decrease in worm motility and the highest concentration (60 μM) of both C and T results in significant inhibition (p < 0.05) in the motility of the worms as compared to the untreated controls.

Fig 2

Scanning electron micrographs of adult Fasciola gigantica (Control) worms incubated in 0.2% DMSO for 3h at 37±1°C showing smooth oral sucker (OS), normal tegumental infoldings (a); intact and sharply pointed spines (a, inset); rounded and smooth ventral sucker (VS) surrounded by spines (b, inset); The posterior end of ventral surface with uniformly distributed tegumental spines (c); The dorsal surface showing tegumental infoldings interspersed with prominent spines (d, inset).

Fig 3

Scanning electron micrographs showing the effect of 40 μM (a, b, c) and 60 μM (d, e, f) curcumin on the tegumental surface of F. gigantica. The oral sucker (a) and the spines around it appeared normal, however, tegumental disruptions (b), swollen or bulging spines (b, inset) around disrupted acetabulum (VS), lesions around the excretory pore (c, arrows) and the erosions showing damaged posterior region (e) and dislodgement of huge number of spines from their sockets leaving pits (f, arrow).

Fig 4

Scanning electron micrographs showing the effect of 40 μM (a, b, c) and 60 μM (d, e, f) thymoquinone on the tegumental surface of F. gigantica. Tegument appeared to be sunken around the oral sucker (a and d). Severely swollen spines and corrugated tegument were evident. Acetabulum appeared normal with wrinkled regions around them having deep furrows (b and e). The posterior regions showing severe dislodgement of spines (c, arrow) revealing the syncitium (f).

Fig 5

The glutathione-S-transferase (GST) levels in Fasciola gigantica worms treated with Curcumin (A) and Thymoquinone (B). The enzyme activity was initially stimulated but then declined significantly with the increasing concentration of curcumin, while the inhibitory effect of thymoquinone was statistically insignificant. NC: RPMI control, D: DMSO control; Curcumin at 20 μM (C20), 40 μM (C40) and 60 μM (C60) concentrations and thymoquinone at 20 μM (T20), 40 μM (T40) and 60 μM (T60) concentrations. All the experiments were carried out in triplicate ± S.E.M. **: p<0.01.

Fig 6

The reduced glutathione (GSH) levels in Fasciola gigantica worms treated with curcumin (A) and thymoquinone (B). The treatment of liver flukes with both curcumin and thymoquinone caused a significant decrease in the level of GSH that may be disrupting the redox-balance within the parasite. NC: RPMI control, D: DMSO control, Curcumin at 20 μM (C20), 40 μM (C40) and 60 μM (C60); and thymoquinone at 20 μM (T20), 40 μM (T40) and 60 μM (T60) concentrations. All the experiments were carried out in triplicate ±S.E.M. *: p<0.05, ***: p<0.001

Fig 7

Superoxide dismutase (SOD) activity in the Fasciola gigantica adult worms treated with Curcumin (A) and Thymoquinone (B). Significant inhibition of SOD, particularly by curcumin will greatly impair the ability of worms to scavenge free radicals that will ultimately help in the elimination of parasites. NC: RPMI control, D: DMSO control, Curcumin at 20 μM (C20), 40 μM (C40) and 60 μM (C60) and Thymoquinone at 20 μM (T20), 40 μM (T40) and 60 μM (T60) concentrations. All the experiments were carried out in triplicate ±S.E.M., *: p<0.05

Fig 8

The level of protein carbonylation (PC) in Fasciola gigantica worms treated with different concentrations (20, 40 and 60 μM) of Curcumin (A) and Thymoquinone (B). Both the compounds produced significant increase in PC level in a concentration dependent manner. NC: RPMI control, D: DMSO control, Curcumin at 20 μM (C20), 40 μM (C40) and 60 μM (C60) concentration; Thymoquinone at 20 μM (T20), 40 μM (T40) and 60 μM (T60) concentration. Each experiment was repeated thrice + S.E.M. *: p<0.05, **: p<0.01

Fig 9. The effect of curcumin and thymoquinone on the expression of CatL gene of Fasciola gigantica.

Thymoquinone at 40 μM significantly inhibited (lane 3) CatL gene expression in F. gigantica, while curcumin at this concentration appeared to be comparatively ineffective, reflecting a differential effect. M: DNA ladder (100bp), Lane 1: RPMI control, 2: DMSO control, 3: Thymoquinone treated sample, 4: Curcumin treated sample, 5: GAPDH was used as internal control.

Fig 10

Computational model showing the binding interactions of Curcumin (a) (Lca, Rca) and Thymoquinone (b)(Ltb, Rtb) with CathepsinL of Fasciola gigantica. The hydrogen-bonding or electrostatic interaction acts as an "anchor" in the binding pocket (Lca, Ltb) and facilitate hydrophobic interaction of the chemical rings with the side chain of protein. Higher number of amino acids involved in the interaction placed curcumin (Rca) a little deeper in the binding site with CathepsinL compared to Thymoquinone (Rtb).