Adult paralysis and mortality assay. In the male adult worms, quercetin was found to be most active at the concentration of 1 mM, at which 40 % of the worms were paralyzed after 1 h, and 80 % were paralyzed after 3 h of exposure. At 6 h, 40 % were dead, and after 12 h, we found 80 % mortality in the adult worms (Table 1). Furthermore, in adult males, quercetin at 1mM concentration caused 100 % mortality within 24h after the treatment. In the case of adult females, quercetin showed a slower effect than the males as 40 % paralysis of the adults took place after 3 h and only at 6 h, 80% of the worms were paralyzed. Mortality in the females was only visible at 12 h (60 %) however, 100 % mortality was found after 24 h of quercetin treatment (Table 1). The LD50 values for adult mortality calculated respectively for the adult male and female worms were 0.62 mM (95 % lower confidence limit 0.49, 95 % higher confidence limit 0.78) and 0.88 mM (95 % lower confidence limit 0.71, 95 % higher confidence limit 1.1) at 12 h of quercetin exposure. Physical damage under the bright field has also been demonstrated in control, Alb (0.2 mM) and Quercetin (1 mM) treated worms, respectively (Fig. S1A-C).
Table 1
Paralysis and Death time analysis in the adult H. contortus. Adult H. contortus worms were exposed to different doses of quercetin (0.125, 0.25, 0.5, 1, and 2 mM), 0.2 mM albendazole, and RPMI media (control condition) for 24 h. Paralysis and death time (viability of the worms) were calculated at different time points after the treatments. If no movement was observed, then worms were exposed to a warm saline solution (50 oC) for 1 min to stimulate movement. Worms were counted based on the number that showed movement. If the worm, which was previously immobile, started moving upon exposure to the warm saline, then it was considered paralyzed but otherwise considered dead in case of no movement in the warm saline solution.
Time of Exposure | Paralysis Time | Death Time |
Male Worms |
Con | Alb | 0.125 | 0.25 | 0.5 | 1 | 2 | Con | Alb | 0.125 | 0.25 | 0.5 | 1 | 2 |
0 hr | - | - | - | - | - | - | | - | - | - | - | - | - | - |
0.5 hr | - | - | - | - | - | - | 9 ± 0 | - | - | - | - | - | - | - |
1 hr | - | - | - | - | - | 6 ± 0 | 12 ± 0.5 | - | - | - | - | - | - | - |
3 hr | - | - | - | - | 9 ± 0 | 12 ± 0.5 | 3 ± 0.5 | - | - | - | - | - | - | 12 ± 0.5 |
6 hr | - | 3 ± 0.5 | - | - | 12 ± 0.5 | 9 ± 0 | 1.5 ± 1.0 | - | - | - | - | 3 ± 0.5 | 6 ± 0 | 13.5 ± 1 |
12 hr | - | 7.±0.5 | 3 ± 0 | 6 ± 0.5 | 9 ± 0 | 3 ± 0.5 | 0 | - | 1.5 ± 1 | - | - | 6 ± 0.5 | 12 ± 0.5 | 15 ± 0 |
24 hr | 1 ± 0 | 0 | 9 ± 0.5 | 12 ± 0.5 | 0 | 0 ± 0 | 0 | - | 15 ± 0 | - | 3 ± 0 | 15 ± 0 | 15 ± 0 | 15 ± 0 |
| Female Worms |
0 hr | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
0.5 hr | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
1 hr | - | - | - | - | - | 3 ± 0.5 | 6 ± 0 | - | - | - | - | - | - | - |
3 hr | - | - | - | - | 3 ± 0 | 6 ± 0.5 | 12 ± 0.5 | - | - | - | - | - | - | 3 ± 0 |
6 hr | - | - | - | - | 9 ± 0.5 | 12 ± 0 | 6 ± 1.5 | - | - | - | - | - | - | 9 ± 1.5 |
12 hr | - | - | - | 3 ± 0 | 15 ± 0 | 6 ± 0.5 | 0 | - | 3 ± 0 | - | - | - | 9 ± 0.5 | 15 ± 0 |
24 hr | 1 ± 0 | 9 ± 1 | 3 ± 0 | 3 ± 0.5 | 4 ± 0.7 | 0 | 0 | - | 6 ± 1 | - | 3 ± 0 | 11 ± 0.7 | 15 ± 0 | 15 ± 0 |
Larval mortality assay. Spindling shrinkage and tissue damage morphology of L-3 larvae were observed in the case of quercetin treatment, whereas no such change was found in the albendazole-treated samples. The survival of larvae was measured after 24 h of quercetin treatment using equation (i). The reduction in the percentage of larval survival with the increase in quercetin concentration was seen: 60.0 ± 6.6, 33.3 ± 3.3, 18.8 ± 3.8, 10.0 ± 3.3, 1.1 ± 1.9 and 0 % survival respectively for 0.125, 0.25, 0.50, 1, 2, and 5 mM of quercetin. In contrast, albendazole (Alb) showed 56.6 ± 3.3 % survival of the L-3 larvae at 0.2 mM (Fig. S2). We measured an LD50 value of 0.16 mM (95% lower confidence limit 0.09, 95% higher confidence limit 0.22) for the larval mortality for 24 h of quercetin treatment. A one way ANOVA test showed a significant interaction between the larval survival and concentration of quercetin (F(5, 12) = 111.31, p < 0.00001), which confirmed the dose-response effect for quercetin (Results of the posthoc test, comparing larval survival for the pairs of quercetin concentration, are given in the supplementary Table S1). Albendazole (Alb) treatment also showed significantly reduced survival of the L3 larvae compared to the control (T-test for independent samples: t = 19, df = 4, p = 0.00004). We also compared the survival of larvae treated with Alb with a comparable concentration of quercetin, i.e., 0.25 mM, and found significantly less survival in the quercetin treated than the Alb treated larvae (T-test for Independent Samples: t = -8.57, df = 4, p = 0.001).
Egg hatch assay. For the egg hatch assay, percentages of inhibition in egg hatching were measured after 48 h of quercetin treatment and quantified using equation (ii). The results, demonstrating the toxic effect of quercetin, showed reduced egg hatching with the increase in quercetin concentration: 69.5 ± 2.5, 35.3 ± 3.3, 29.5 ± 4.2, 19.5 ± 1.5, 15.6 ± 2.3, and 10 ± 1.8% of egg hatching (presence of larvae L1) respectively at the concentrations of 0.125, 0.25, 0.50, 1, 2, and 5 mM (Fig. S3). Contrastingly, albendazole (0.2 mM) showed 72.6 ± 3.01% egg hatching. LD50 measured for the inhibition of egg hatching was 0.19 mM (95% lower confidence limit 0.04, 95% higher confidence limit 0.37) for 48 h. A one-way ANOVA test showing a significant interaction between the egg hatching and concentration of quercetin (F(5, 12) = 177.78, p < 0.00001) has confirmed the dose-response effect for quercetin (80 % of the eggs fail to hatch at 1 mM). Results of the posthoc test, comparing the number of eggs hatched for the pairs of quercetin concentration, are given in the supplementary Table S2. Similar to the findings of the larval mortality assay, Alb treatment also showed significantly less egg hatching than the control (t = 7.68, df = 4, p = 0.001) whereas quercetin at 0.25 mM showed significantly less egg hatching compared to the Alb treatment (t = -14.39, df = 4, p = 0.0001).
Morphological damage inflicted by quercetin. Scanning electron microscopy was performed on the adult worms to understand the possible morphological changes caused by exposures to albendazole and quercetin. In the control group, treated with RPMI Media, smooth cuticles with well-developed body regions were seen in the adult female worm (Fig. 1A). In the anterior portion, intact sharp end and blood-sucking mouth region with no disruption (Fig. 1B) were identified. In the posterior end of the worm, a sharp end with an intact tail region was identified (Fig. 1C). In the case of Alb-treated (0.2 mM for 3 h) worm, folds, partial shrinkage, and disorganization of the cuticle were observed throughout the body (Fig. 1D) as well as at the anterior (Fig.. 1E) and posterior (Fig. 1F) ends. However, in the quercetin-treated worm (1 mM for 3 h), complete disorganization and disruption of body regions along with the loss of the cuticle were observed (Fig. 1G). Also, shrinkage of the body ends, a complete loss of the blood-sucking anterior mouth region, and rupturing of the cuticle at the tail region were observed (Fig. 1H-I).
Histopathology caused by quercetin treatment. Massive changes in the morphology, caused by quercetin treatment, were observed under the light microscope (Fig. 2C), whereas Alb (Fig. 2B) showed little to no change in the morphology in comparison to the control (Fig. 2A). Detailed magnified image analysis revealed intact anterior and posterior ends with unblemished (ai) isthmus, (aii) brut, (aiii) pseudocoele, (aiv) globular leukocytes (surrounded by small-sized cells), (av) muscle cells, (avi) intestinal epithelial region, (avii) ovum and growth zone of the ovary, and (aviii) intact skin tissue in control adult worm (Fig. 2A). In the 0.2 mM Alb-treated worms (Fig. 2B), moderate disruptions were visible at the (bi) isthmus and (biii) pseudocoele along with the (bvi) partially-punctured muscle cells, (bv) globular leukocytes with fewer surrounding cells, and a (bviii) ruptured ovary. On the other hand, quercetin treatment (Fig. 2C) has caused complete disruption of the anterior end, a near-total disruption of the ovary, and loss of eggs, splitting of the pseudocoele, punctured muscle cells, and damaged globular leukocytes with limited counts of surrounding cells. Figure 2, ci-cviii shows the histopathology of different body parts in the adult female H. contortus treated with quercetin.
Generation of reactive oxygen species in the nervous system due to quercetin treatment. We investigated the mechanism underlying the toxicity elicited by quercetin and checked whether the compound is producing oxidative stress in the adult H. contortus. Reactive oxygen species (ROS) was found to be generated in the adult worm when treated with quercetin (1 mM) for 3 h (Fig. 3C, 3F, 3I, and 3L), whereas ROS was not detected in control (Fig. 3A, 3D, 3G, and 3J) and Alb (Fig. 3B, 3E, 3H, and 3K) groups. Figure 3C, 3F, 3I, and 3L are respectively showing the generations of ROS in the adult worms, induced by quercetin in the anterior part of the body, ventral cord and tail ganglia (at the posterior end), nerve ring (at the anterior end), and commissural connections (middle of body region). No ROS was detected over the levels of autofluorescence at the same locations in the other two experimental groups. We also measured the increase in ROS generation in the nerve ring of the adult worm, caused by the treatment with 1 mM quercetin for 3 h, by repeated-measures ANOVA at different time points (after 1, 3, 5, and 7 min of DCFDA-treatment) and found a significant increase in pixel intensity (indicating increased staining with the ROS-detecting dye, DCFDA) with time (Fig. S4). Results of the repeated measures ANOVA showed a significant pixel-intensity × time effect (F(60, 1600) = 1.67, p = 0.001) and a significant time effect (F(3, 80) = 875.69, p < 0.0001). Tukey’s HSD posthoc test revealed significant difference in pixel intensities between the following pairs of time points: 1 min vs. 5 min (p = 0.0001), 1 min vs. 7 min (p = 0.0001), 3 min vs. 5 min (p = 0.0001), 3 min vs. 7 min (p = 0.0001) 5 min vs. 7 min (p = 0.0001).
Alterations in the activities of catalase, superoxide dismutase, and glutathione peroxidase enzymes after quercetin treatment. Next, we have tested whether high ROS generation due to quercetin-mediated oxidative stress within the worm's body has altered the activity levels of the enzymes involved in reducing oxidative stress, such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). An increase in the catalase activity along with the increase in the concentration of quercetin was observed. The activity levels of CAT calculated respectively were 5.2 ± 0.5, 5.9 ± 0.1, 7.2 ± 0.52, 16.1 ± 0.18, and 16.6 ± 0.2 U/mg proteins at the concentrations of quercetin, 0.125, 0.25, 0.5, 1.0 and 2.0 mM after 3 h of treatment (Table 2). A one-way ANOVA test found an increase in the activity level of CAT depending on the concentration of quercetin; significant interaction between the CAT-activity and quercetin-concentration (F(5, 12) = 88765, p < 0.0001). Results of Tukey’s HSD posthoc test, comparing between the pairs of CAT-activity values, are given in Table S3. A significant increase in CAT-activity was also found in the Alb-treated worms compared to the control (t = -25.84, df = 4, p = 0.00001), whereas no difference was found between the worms treated with 0.2 mM Alb and 0.25 mM quercetin (t = -2.14, df = 4, p = 0.09). A similar dose-response effect was also found for superoxide dismutase (SOD) as we calculated the activity levels respectively 2.13 ± 0.56, 3.09 ± 0.47, 4.02 ± 0.56, 6.68 ± 0.21, and 7.61 ± 0.34 U/mg protein for the same set of concentrations of quercetin after 3 h of treatment (Table 2). One way ANOVA found a significant SOD-activity × quercetin-concentration effect (F(5, 12) = 1398.3, p < 0.0001). Results of Tukey’s HSD posthoc test, comparing between the pairs of SOD-activity values, are given in Table S4. Significant increase in SOD-activity was found in the Alb-treatment compared to the control (t = -26.08, df = 4, p = 0.00001), however no difference in activity levels was found between the groups, 0.2 mM Alb and 0.25 mM quercetin (t = 0.95, df = 4, p = 0.39). We also found the increasing activity of glutathione peroxidase, with increasing concentration of quercetin. The activity levels of GPx were found to be 6.32 ± 0.78, 9.39 ± 0.51, 15.05 ± 0.31, 17.28 ± 0.56, and 18.47 ± 0.2 U/mg protein respectively concentrations of quercetin, 0.125, 0.25, 0.5, 1.0 and 2.0 mM after 3 h of treatment (Table 2). A one-way ANOVA has confirmed the dose-response for quercetin; significant GPx-activity × quercetin-concentration effect (F(5, 12) = 25181, p < 0.0001) (Results of the Tukey’s HSD posthoc test, comparing between the pairs of GPx-activity values, are given in Table S5). Increased activity was also found for Alb-treatment compared to the control (t = -23.25, df = 4, p = 0.00001) and unlike the previous two enzymes, for GPx, we found significantly higher activity in the Alb-treated worms than in the worms treated with 0.25 mM quercetin (t = -3.6, df = 4, p = 0.02).
Table 2
Increase in the activity levels of enzymes involved in coping with oxidative stress, induced by quercetin treatment. The oxidative stress caused by quercetin treatment in the adult worm has increased the activities of the following enzymes, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), as measured by biochemical and spectrophotometric assays. All three enzymes showed an increase in activities with an increase in quercetin concentration (0.125, 0.25, 0.5, 1.0, and 2.0 mM). Higher activities of these enzymes are also found in the Alb-treated worms compared to the controls. The enzymatic activity was measured as the enzyme unit per mg of protein (U/mg protein).
Experimental Groups | U/mg protein |
CAT | SOD | GPx |
Control | 8.85 ± 0.46 | 1.65 ± 0.11 | 19.9 ± 1.75 |
0.2 mM Alb | 134.48 ± 8.4 | 24.89 ± 1.53 | 319.58 ± 22.25 |
0.125 mM | 107.86 ± 0.64 | 18.36 ± 0.08 | 183.61 ± 0.97 |
0.25 mM | 123.97 ± 1.11 | 26.40 ± 2.26 | 272.76 ± 2.56 |
0.50 mM | 149.6 ± 0.42 | 34.63 ± 0.08 | 436.47 ± 1.68 |
1 mM | 332.26 ± 0.66 | 57.49 ± 0.3 | 503.47 ± 0.48 |
2 mM | 342.08 ± 0.72 | 63.30 ± 0.19 | 518.33 ± 1.75 |
5 mM | 363.93 ± 0.42 | 65.47 ± 0.23 | 536.27 ± 1.28 |