Phytochemical Profiling, Antioxidant, Cytotoxicity, and Anthelmintic Activity of the Bark Extracts of Terminalia superba Against the Murine Intestinal Nematode Heligmosomoides polygyrus: An Integrated In Vitro and In Silico Approach.

Abstract

Background: Intestinal helminthiases are major global health concerns affecting over a billion people, causing significant morbidity, mortality, and economic losses in both humans and livestock. The growing emergence of drug-resistant parasites has intensified the search for new, safe anthelmintic alternatives. This study evaluated the anthelmintic potential of bark extracts of Terminalia superba against intestinal nematode infections.

Methods: Aqueous and ethanolic extracts were tested in vitro for ovicidal and larvicidal effects against Heligmosomoides polygyrus eggs and larval stages at concentrations ranging from 0.3125 to 5 mg/mL, in triplicate, using a high-throughput worm microtracker. Albendazole served as positive control, and distilled water served as negative control. IC₅₀ values were derived from concentration-response curves via nonlinear regression in GraphPad Prism. Antioxidant activity was assessed through DPPH, reducing power, nitric oxide, and hydrogen peroxide scavenging assays, while cytotoxicity was evaluated by hemolysis assay on human red blood cells with 1.0% Tween 20 as positive control. Phytochemical screening and UHPLC-MS/MS fingerprinting were performed to characterize bioactive compounds. Molecular docking against succinate dehydrogenase (SDH) and β-tubulin, alongside ADME predictions, were conducted using Schrödinger Maestro, with toxicity assessed via ProTox-III.

Results: Both extracts exhibited concentration-dependent ovicidal and larvicidal activity. The aqueous extract was more effective against eggs and L1 larvae (IC₅₀: 1.2 and 0.3 mg/mL), while the ethanolic extract outperformed the aqueous extract against L2 and L3 larvae (IC₅₀: 0.09 and 0.8 mg/mL). The ethanolic extract demonstrated superior DPPH radical scavenging activity IC₅₀: 0.75 μg/mL (95% CI: 0.14-1.62), compared to the aqueous extract, 6.26 μg/mL (4.07-9.50), and vitamin C, 1.91 μg/mL (1.38-2.55). Both extracts showed minimal cytotoxicity, with hemolysis of 7.6% and 2% at 1000 μg/mL, respectively. Phytochemical analysis revealed a rich profile of tannins, flavonoids, and phenolic compounds, with UHPLC-MS/MS confidently identifying 82 of over 300 detected features. Top docking candidates against β-tubulin included kaempferol (-8.033), brevifolin-carboxylic acid (-7.985), luteolin (-7.954), scutellarein (-7.373), and (-)-epicatechin (-6.980), all surpassing albendazole (-3.728). Against SDH, leading compounds were termilignan (-6.767), cyanidin (-6.659), hesperetin (-6.314), combretastatin A-1 (-6.274), and luteolin (-6.166), outperforming albendazole (-5.541). ADME profiling of these leading compounds indicated favorable oral absorption and bioavailability. ProTox-III classified all top compounds at low toxicity levels (class ≥ 4).

Conclusion: This study offers preliminary scientific support for the traditional use of T. superba bark extracts in managing intestinal helminthiases, warranting further in vivo validation.