Synergistic Effects of Phenanthroindolizidine Alkaloids from Tylophora indica Against Ascaridia galli: Phytochemical Characterization and In vitro Evaluation

The increasing resistance of helminths such as Ascaridia galli to conventional anthelmintics has necessitated the search for alternative treatments from natural sources. This study aimed to assess the anthelmintic properties of phenanthroindolizidine alkaloids derived from Tylophora indica leaves, a plant renowned for its medicinal value, including its traditional use in treating respiratory disorders, inflammation, and various infections. The leaves were subjected to sequential extraction with chloroform, ethanol, and water, which produced yields of 7.2%, 17.8%, and 11.2%, respectively. Phytochemical analysis revealed that the ethanol extract was rich in bioactive compounds, including significant amounts of alkaloids, flavonoids, phenols, and terpenoids. Quantitative analysis confirmed the ethanol extract's superiority, displaying the highest contents of phenolics (7.51 ± 0.62 mg/g), flavonoids (9.34 ± 1.63 mg/g), and alkaloids (17.65 ± 1.69 mg/g), underscoring its potential for various therapeutic applications. Further fractionation and High-Performance Liquid Chromatography (HPLC) purification isolated key phenanthroindolizidine alkaloids, including Tylophorinidine, Tylophorine, Septicine, Tylophorinol, and Antofine. Structural characterization via Nuclear Magnetic Resonance (NMR) and High-Resolution Mass Spectrometry (HRMS) validated these compounds. In vitro assays demonstrated significant dose-dependent anthelmintic activity against Ascaridia galli worms. Ethanol extracts exhibited the highest mortality rates, achieving 100% mortality within 24 hours at a concentration of 5 mg/mL. The mixture of all five alkaloids at 500 µg/mL showed a synergistic effect, leading to rapid and complete anthelmintic action. The egg embryonation assay further highlighted the efficacy of these alkaloids. The egg embryonation assay further demonstrated the potent efficacy of these alkaloids, with the mixture at 500 µg/mL inhibiting 92.67% of egg development, surpassing the positive control, i.e. piperazine citrate, which showed 87.25% inhibition. Among individual alkaloids, Tylophorinidine exhibited the highest inhibition of egg embryonation (80.76%), followed by Antofine (78.42%). These findings demonstrated the potent anthelmintic properties of phenanthroindolizidine alkaloids from Tylophora indica, particularly when used in combination (Tylophorinidine, Tylophorine, Septicine, Tylophorinol, and Antofine), compared to their individual effects. The study underscores the potential of these compounds as effective treatments for helminth infections and highlights the importance of further research to isolate specific mechanisms and optimize their therapeutic efficacy.