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

Summary

This study evaluated the biological activities of Tithonia diversifolia extracts using radical-based methods and an in vitro egg hatching assay (EHA) to assess their effects on gastrointestinal nematodes in small ruminants. The methodology and key findings are described, highlighting the potential of these extracts for controlling parasites.

Abstract

Parasitic diseases caused by gastrointestinal nematodes (GIN) are considered important limiting factors for livestock production worldwide, as they significantly compromise animal welfare and increase production costs. For years, parasite control has relied exclusively on the use of synthetic drugs. Resistance of GIN to commercial anthelmintics has been increasingly reported throughout the world. Therefore, alternative approaches have emerged for controlling GIN in farm animals, including the exploration of plants with bioactive potential. Tithonia diversifolia can be harvested year-round, and all parts of the plant have been used in folk medicine for a wide range of diseases. This study evaluated the in vitro antioxidant properties of water and 70% aqueous acetone extracts of T. diversifolia aerial parts, including leaves, branches, and flowers, using radical-based methods, and assessed its anthelmintic potential through a parasite model, employing the egg hatching assay (EHA) with water extracts. Additionally, the total polyphenolic content of the extracts was determined. These findings highlight T. diversifolia as a promising natural source of antioxidant and anthelmintic compounds.

Introduction

Parasitic diseases caused by gastrointestinal nematodes (GIN) are considered important limiting factors for livestock production worldwide, as they significantly compromise animal welfare and increase production costs¹. The relationship between the parasite and livestock is influenced by several factors related to the host, namely the genetic characteristics of resistance and resilience of the animals, the nutritional status of the animals, the level of parasitism, and the pathogenicity of the parasite populations involved².

For years, parasite control has relied exclusively on synthetic antiparasitic drugs. However, prolonged and exclusive use has led to the emergence of resistance, reducing their effectiveness and urging the search for alternative strategies³. Resistance of nematodes to various anthelmintics has been increasingly described throughout the world, presenting a concerning scenario, especially in tropical regions⁴. In addition, consumers are increasingly aware of the need to reduce the residues of antiparasitic medications in animal meat and the environment⁵. Therefore, alternative approaches have emerged for the control of nematode parasites in farm animals⁶.

Tithonia diversifolia is a tropical shrub native to Central America, possessing unique characteristics that make it valuable for ruminant production systems. It can be harvested year-round, and all parts of the plant have been traditionally used in folk medicine by indigenous communities to treat a wide range of ailments. Topically, it has been applied to alleviate abdominal pain, wounds, dermatosis, and muscular disorders, while oral administration has been employed to manage infections, malaria, fever, hepatitis, and diabetes. These diverse therapeutic applications highlight its broad medicinal potential⁷.

Oxidative stress plays an important role in the development and progression of parasitic infections, both in the host organism and in the parasitic organism struggling to survive⁸. For this reason, detecting antioxidant activity is crucial for evaluating the biological activity of medicinal plant extracts with anthelmintic potential. Due to parasitic infection, the concentration of antioxidants in the host decreases, while the concentration of oxidation products of cellular components increases. In the case of parasites, maintaining oxidative balance is essential to prolong their interaction with the host and to defend against the oxidative stress generated in the host organism⁹.

Evidence of the development and rapid spread of resistance to anthelmintics raises concerns that commonly used treatments may become ineffective. Therefore, an in vitro test called the egg hatch assay (EHA) is used to assess susceptibility or resistance to benzimidazole (BZ) in gastrointestinal nematodes. This test is commonly used to evaluate the anthelmintic activity of plant extracts on eggs and to compare their activity as a function of dose. One of the main limitations of this assay is the variability in egg hatching between tests. The viability of the eggs is limited to a maximum of 3 h, requiring rapid recovery and prompt initiation. Despite this, the hatching percentage may vary. For this reason, it is established that at least 70% of the eggs must hatch in the control group for the result to be considered valid^10,11. It is a cheap, rapid, easy, and reproducible test^12,13,14.

The use of plant extracts to explore alternatives for GIN control is based on the presence of secondary metabolites and their bioactive potential. One of the challenges in this area of research is the quantity and stability of secondary metabolites, as these vary according to biotic and abiotic factors, as well as due to the chemical stress induced by the extraction process^15,16,17.

This study hypothesized that differences in biological activities (antioxidant and anthelmintic effects) could occur depending on the type of solvent used for extraction, which might influence the composition of polyphenols and, consequently, their bioactivity. The objective of this manuscript was to outline the steps taken in the evaluation of extracts using an in vitro model that could be easily replicated.

Protocol

The CEMIT animal ethics committee approves all experiments involving animal manipulation. The egg-donor animals belong to farms that collaborate with CEMIT by providing fecal samples for preventive diagnostic purposes. These animals are sheep or goats (no specific breed), of either sex, over one year of age, and must not have been dewormed within 15 days prior to sample collection. All participating farms are informed about the use of the samples for research purposes and provide their consent to be part of the study. For egg collection, nearby establishments are first notified, and upon receiving permission, field visits are organized to collect fresh fecal samples, ensuring processing within three hours. The reagents and equipment used in this study are listed in the Table of Materials.

1. Preparation of plant material

1. Select a suitable location for collecting the plant species.
2. Collect at least two specimens of Tithonia diversifolia for taxonomic identification and herbarium preparation.
3. Collect a minimum of 1000 g of T. diversifolia aerial parts.
4. Dry the collected biomass at room temperature (approximately 26  °C; see Figure 1).
5. Grind the dried biomass using an electric grinder.
6. Store the ground material in a refrigerator at 4 °C.

2. Preparation of extracts

1. Prepare the water extract:
   1. Mix 10 g of dried biomass with 100 mL of distilled water at 100 °C.
   2. Allow the mixture to macerate for 24 h under continuous shaking at 120 rpm.
   3. Filter the extract through filter paper (Whatman no. 5 or equivalent).
   4. Freeze-dry the filtered extract.
2. Prepare the acetone extract:
   1. Mix 10 g of dried plant material with 70% aqueous acetone in a 1:3 ratio.
   2. Allow the mixture to macerate for 24 h under continuous shaking at 120 rpm.
   3. Filter the extract through filter paper (Whatman no. 5 or equivalent).
   4. Freeze-dry the filtered extract.
      NOTE: Store the lyophilized extracts at -20 °C for better preservation (see Figure 2). Ensure at least 5 g of lyophilized extracts are obtained.

3. Preparation of stock solution

1. For the antioxidant assay:
   1. Solubilize the dried extracts in the corresponding solvents (distilled water or 70% aqueous acetone) to a final concentration of 10 mg/mL.
2. For the egg-hatching assay:
   1. Dilute 12 mg of lyophilized extracts in 5 mL of distilled water to obtain a stock solution of 2.4 mg/mL.
   2. Prepare five concentrations: 1.2 mg/mL, 0.6 mg/mL, 0.3 mg/mL, and 0.15 mg/mL (see Figure 3).
      NOTE: Adjust the volume of the stock solution based on the number of wells and plates used. For example, if 48 wells are used instead of 12, reduce the volume accordingly; prepare 6 mg in 2.5 mL instead of 12 mg in 5 mL.

4. Phytochemical composition

1. Total phenolic content of extracts (TPC)
   1. Mix 5 µL of the samples with 100 µL ofFolin-Ciocalteu (F-C) reagent diluted 10× in distilled water in 96-well transparent plastic plates.
   2. Incubate at room temperature (RT) for 10 min.
   3. Add 100 µL of sodium carbonate solution (75 g/L in water).
   4. Incubate at RT for 90 min.
   5. Measure the absorbance at 650 nm.
      NOTE: Express TPC as gallic acid equivalents (GAE). Build a calibration curve using at least five concentrations of gallic acid.
2. Total flavonoid contents (TFC)
   1. Mix 50 µL of the extracts with 50 µL of 2% aluminum chloride in methanol in 96-well transparent plastic plates.
   2. Incubate for 10 min at RT.
   3. Measure the absorbance at 405 nm.
      NOTE: Express TFC as quercetin equivalents (QE). Build a calibration curve using at least five concentrations of quercetin.
3. Total tannin content (TTC)
   1. Mix 10 µL of the extracts with 200 µL of 1% DMACA in methanol and 100 µL of 37% HCl in 96-well transparent plastic plates.
   2. Incubate for 15 min at RT.
   3. Measure the absorbance at 640 nm.
      NOTE: Express TTC in terms of mg/mL of catechin.

5. Antioxidant assay - ABTS

1. Add 0.003514 g of potassium persulfate and 0.0203 g of ABTS to 5 mL of distilled water.
2. Wrap the solution in aluminum foil and incubate at 4 °C in a refrigerator for 12-16 h.
3. Measure the absorbance at 734 nm and adjust it to approximately 0.7 by adding ethanol. As a starting point, mix 5 µL of ABTS solution with 195 µL of ethanol.
4. In 96-well transparent plastic plates, mix 10 µL of the extracts with 190 µL of ABTS solution.
5. For the positive control (C⁺), mix 10 µL of Trolox (determine the concentration using a standard curve based on the sample's absorbance) with 190 µL of ABTS solution.
6. For the negative control (C^-), mix 10 µL of the solvent used to dissolve the extracts (water or aqueous acetone) with 190 µL of ABTS solution.
7. Incubate the plates for 6 min at room temperature (RT).
8. Measure the absorbance at 734 nm.
   NOTE: Figure 4 shows an example of an ABTS assay.

6. Parasitological procedures

1. Collect fecal samples directly from the rectum of naturally infected goats.
   NOTE: Collect fecal samples directly from the rectum of naturally infected goats and sheep to ensure freshness and avoid environmental contamination. Gently restrain animals using trained personnel to minimize stress. Using clean disposable gloves, insert a lubricated, gloved finger into the rectum and retrieve approximately 5-10 g of fecal material. Place samples into clean, labeled plastic containers and store at 4-8 °C. Process all samples within 3 h of collection to preserve the viability of helminth eggs. Follow institutional animal welfare guidelines and obtain informed consent from farm owners.
2. Identify the animal species associated with each sample.
3. Mix 30 g of each fecal sample with 50 mL of saturated salt solution (density = 1.2) to prepare for coprology assays.
4. Use a McMaster slide to count the eggs.
   1. Homogenize 3 g of fecal sample in 50 mL of saturated NaCl solution. Filter the mixture through cheesecloth, fill both chambers of a slide, and allow it to stand for 5 min.
   2. Examine the slide under a microscope at 10× magnification and count strongyle-type eggs. Calculate eggs per gram (EPG) using the formula: EPG = (egg count) × 50. This method standardizes the quantification of gastrointestinal nematodes in small ruminants.
5. Express the egg count as eggs per gram (EPG).
6. Select fecal samples with an EPG of at least 800 for fecal culture preparation.
7. Obtain L3 larvae from fecal cultures and identify their genera or species.
   NOTE: Gastrointestinal nematode eggs are deposited in grass during the life cycle (see Figure 5). Livestock small ruminants commonly exhibit polyparasitism. Figure 6 depicts the steps for performing coprological análisis.

7. In vitro tests - Egg Hatch Assay (EHA)

1. Collect fresh fecal samples.
2. Filter the feces twice using sterile gauze.
3. Filter the resulting suspension and transfer it to a sieve.
4. Wash the material on the sieve with distilled water.
5. Collect the washed suspension into 15 mL tubes.
6. Centrifuge the samples at ~4800 × g for 5 min at room temperature using a tabletop centrifuge equipped with a swinging bucket rotor. Centrifuge twice with distilled water and once with saturated saline solution to concentrate the eggs at the bottom (see Figure 7 for egg morphology).
7. Resuspend the eggs in PBS.
8. Adjust the egg concentration to 100 eggs/mL for assay setup.
9. Prepare the positive control by dissolving thiabendazole (TBZ) in PBS at 0.025 mg/mL.
10. Prepare the negative control using the egg suspension in PBS.
11. Use 48-well multiwell plates for the assay.
12. Prepare a final volume of 250 µL in each well using a 50/50 (v/v) mixture of extract and egg suspension.
13. Add approximately 100 eggs/mL to each well.
14. Test each concentration in quadruplicate (four replicates).
15. Count the eggs under an inverted microscope at 40× magnification.
16. Use only plates with a hatch rate of ≥70%.
17. Count larvae (L1), unhatched eggs (UnE), eggs containing larvae (LFE), and morulated eggs (ME) for each concentration.
    NOTE: A representative summary of the egg hatch assay protocol is shown in Figure 8.

8. Statistical análisis

1. Perform descriptive statistical analysis using Excel.
2. Use one-way analysis of variance (ANOVA) and t-tests to evaluate differences between concentrations at a significance level of 0.05.
3. Calculate the 50% Effective concentration (EC50) using Probit analysis based on log₁₀-transformed concentration data (see Supplementary File 1).

Results

Total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC)
Results of TPC, TTC, TFC, and ABTS on Tithonia diversifolia are shown in Table 1. Values are expressed as mg of gallic acid equivalents per gram of dry matter (mg GAE/g DM) for TPC, mg of quercetin equivalents per gram of dry matter (mg QE/g DM) for TFC, and mg of catechin equivalents per gram of dry matter (mg CE/g DM) for TTC. Antioxidant capacity is expressed as the half-maximal in...

Discussion

This article outlines several essential steps for advancing research on medicinal plants. In vitro tests, such as the egg hatch assay (EHA), are particularly attractive because they can be conducted under controlled laboratory conditions. The EHA is designed to evaluate the activity of plant extracts at various concentrations on nematode eggs, comparing the outcomes against both positive and negative controls. The results obtained offer valuable insights into the potential of plant-derived compounds for developi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL tubes			On site
48-hole plate			On site
96 well plates	VWR	734-0126
ABTS	Enzymatic	A1088.0005	On site
Academic optical microscope and inverted microscope			On site
Acetone	Enzymatic	A/0600/17
Aluminum chloride	Sigma Aldrich	6911
DMACA reagent	Sigma Aldrich	49825
Ethanol 96%	Enzymatic	VR11202
Feces from animals			Collected in farms near to university
Folin Ciocalteau	Sigma Aldrich	F9252
Gallic acid	Sigma Aldrich	91215
HCl	Sigma Aldrich	258148
McMaster camera			On site
Methanol	Enzymatic	M/4000/17
PBS			On site
Plant extracts			On site
Potassium persulfate	Sigma Aldrich	216224
Quercetin	Sigma Aldrich	Q4951
Salt			On site
Sodium carbonate	Sigma Aldrich	1063920500
Thiabendazole			On site
Trolox	Sigma Aldrich	648471	On site
Water			On site
Whatmann no. 5	VWR	WHAT97039654