Evaluation of Antioxidant and Anthelmintic Properties of Tithonia diversifolia Extracts Against Gastrointestinal Nematode Eggs Using In Vitro Assays

My research focuses on parasitology, currently studying natural components from plants to control internal parasites in small ruminants. We want to integrate traditional knowledge and alternative method to control small ruminants world population. Nowadays, we are increasingly using technologies like predictive modeling, automatic counters, and molecular tools for detecting specific alerts.

Moreover, several institutes around the world are developing vaccine prototypes against Haemonchus contortions. This is a major step forward. Others are using organic motors to reduce reliance on laboratory animals, which is ethically and scientifically valuable.

Overall, there is a strong and coordinated global effort to advance diagnostic tools and research, aiming to develop innovative and sustainable strategies for parasite control worldwide, and prevent drug resistance. To begin, mix 30 grams of each fecal sample with 50 milliliters of saturated salt solution with a density of 1.2 grams per milliliter to prepare for coprology assays. Homogenize three grams of fecal sample in 50 milliliters of saturated sodium chloride solution, and filter the mixture through a cheesecloth.

Examine the slide under a microscope at 10x magnification, and count strongyle-type eggs. Calculate eggs per gram by using the given formula. Filter the feces twice using sterile gauze.

Then, filter the resulting suspension and transfer it to a sieve. Wash the material retained on the sieve with distilled water, and collect the washed suspension into 15 milliliter tubes. Place the samples in a tabletop centrifuge with a swinging bucket rotor, and spin at approximately 4, 800 G for five minutes at room temperature.

Resuspend the eggs and PBS, and adjust the egg concentration to 100 eggs per milliliter for assay setup. Prepare the positive control by dissolving Fenbendazole in PBS to obtain a 1, 000 micrograms per milliliter stock solution. After adding the one to one mixture of extract and egg suspension, add approximately 100 eggs per milliliter to each well of a 48 well plate.

The main challenge of this technique lies in the variability of the egg hatching response. Even when following all the protocol steps, the eggs hatching may occur between 48 to 72 hours, or in some cases, not at all. The inhibitory effect of Tithonia diversifolia extract on egg hatching, showed a dose-dependent pattern reflected in the EC50 value of 521.6 micrograms per milliliter.

This result indicates that moderate concentrations of the crude extract are needed to achieve 50%inhibition, suggesting the presence of bioactive compounds with anthelmintic potential. At low doses of 150 to 300 micrograms per milliliter, inhibition was minimal, and variability remained low, consistent with insufficient exposure to interfere with embryogenesis or larval emergence. Variability peaked at the intermediate dose of 1, 200 micrograms per milliliter, consistent with the nonlinear transition zone of a dose response curve, where small biological or experimental differences can cause large outcome shifts.

This is expected with crude plant extracts containing multiple active compounds. Overall, the results confirm the anti-hatching activity of Tithonia diversifolia with increasing efficacy at higher doses. Refining or fractionating the extract may enhance potency, and further phytochemical and in vivo studies are recommended to assess its usefulness in parasite control programs.

Our findings show in vitro activity, confirming the anti-parasitic potential. Those representing a meaningful discovery and the potential clinical application to enhance animal health. Additionally, the low cost and ethical approach of this assay validate the use of traditional knowledge regarding future studies.

Our study result delivering evidence-based insights into anthelmintic strategies, validated eco-friendly components to support control strategies to reduce drug dependence.