Nematocidal Activity of a Variety of Plants Used in Mexico Against Strongyloides venezuelensis

Abstract

Strongyloidiasis represents a public health problem in tropical and subtropical regions. The medicinal plants demonstrate the potential of plants as a source of molecules with helminthic activity. In this research, we assessed the potential of five extracts medicinally used in Mexico against Strongyloides venezuelensis third-stage infective larvae (L3). Plant methanol (MeOH) extracts of Argemone mexicana (chicalote), Jatropha dioica (Sangre de Drago), Lippia graveolens (oregano), Thymus vulgaris (tomillo), and Kalanchoe daigremontiana (aranto) were prepared by the maceration technique. The toxicity of the extracts was evaluated in human red blood cells by the hemolysis test and in monkey kidney epithelial cells (Vero cells) using the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, we showed their antioxidant potential by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The methanolic extracts of A. mexicana, J. dioica, L. graveolens, and T. vulgaris exhibited significant activity against L3 cultures at 72 and 96 h post-incubation. None of the extracts showed hemolytic effects on red cells or Vero cells. L. graveolens demonstrated the strongest antioxidant activity, with an EC₅₀ of 19.80 µg/mL. Plant MeOH extracts used in this study showed a promising anthelmintic effect in vitro, making it a suitable candidate for future research in nematocidal therapies.

1. Introduction

Helminth-induced parasitosis remains a significant public health concern in developing countries, where these infections are closely associated with poverty and inadequate sanitation. Consequently, parasitic diseases are more prevalent in these regions [1]. Regarding strongyloidiasis, the World Health Organization (WHO) estimates that between 30 and 100 million people are infected globally [2]. Strongyloidiasis is a chronic infection caused by Strongyloides stercoralis that can lead to pulmonary symptoms, eosinophilia, and abdominal discomfort that have the feature of autoinfection. This disease is endemic in tropical and subtropical regions [3]. S. stercoralis is a soil-transmitted helminth (STH) that affects approximately 370 million people worldwide, predominantly in tropical and subtropical areas [4]. Disseminated and hyperinfection syndromes occur in immunocompromised individuals and involve widespread parasite massive dissemination, leading to high mortality rates [4,5]. Strongyloides venezuelensis is widely used as a laboratory model for the human parasite S. stercoralis [6]. Several studies involve the use of S. stercoralis as a model for in vitro and in vivo studies [7,8,9].

Moreover, S. stercoralis is a zoonotic nematode affecting canids and some primate species. Strongyloides papilosis has considerable importance in cattle, sheep, and goats around the world. Moreover, Strongyloides ransomi and Strongyloides westeri affect swine and horses, respectively. Finally, Strongyloides felis, Strongyloides tumefaciens, and Strongyloides planiceps affect cats and small wild carnivores [10].

Currently, no vaccines are available for strongyloidiasis; however, preventive measures such as proper sanitation, basic hygiene measures, and the use of appropriate footwear in endemic areas can help reduce the risk of infection [11]. The disease is primarily treated with ivermectin (IV) or albendazole (Alb) to reach total elimination in the patient to avoid autoinfection [12]. The recommended treatment involves oral ivermectin at 200 µg/kg/day for two consecutive days, with a second round of treatment after 14 days [13]. In immunocompromised patients, three treatment cycles are advised, each separated by a 14-day interval [14]. Since the introduction of IV, it has been considered the drug of choice first against veterinary nematode and arthropods and after against Onchocerca volvulus, Strongyloides stercolaris, and Sarcoptes scabiei [15]. However, IV-resistant nematode strains have been reported in grazing livestock, and there is concern about reduced susceptibility or potential resistance in human nematodes [16].

Plants and natural products with biological activity against various pathogens, including parasites [17] and nematodes [18], appear as a valuable source of bioactive metabolites with diverse therapeutic properties based on new treatments [19]. Plants contain a wide range of chemical constituents [20], many of which exhibit antifungal, antiparasitic, antiviral, and antibacterial activity [21,22]. In Mexico, medicinal plants have been used continuously from the pre-Hispanic era to the present. Among them, Argemone mexicana has demonstrated significant biological activity against Plasmodium falciparum and Plasmodium berghei [23,24]. Jatropha dioica is a plant widely distributed in the semi-desert regions of Mexico and the United States, traditionally used in folk medicine for pain and inflammation as well as to prevent hair loss and promote hair strengthening [25]. Additionally, this plant has been reported to exhibit nematocidal activity against Trichinella spiralis [18]. Lippia graveolens, Mexican oregano, is a rich source of flavonoids, sakuranetin, and cirsimaritin, useful in combating E. histolytica [26]. Thymus vulgaris (thyme), native to the Mediterranean basin, is widely utilized in Hispanic America as a culinary spice and is also appreciated for its anti-inflammatory and antimicrobial properties [27,28,29]. Moreover, thyme has demonstrated activity against Haemonchus contortus, T. spiralis, and Trypanosoma cruzi [30]. Kalanchoe daigremontiana, an introduced plant from Madagascar, has shown notable activity against Entamoeba histolytica and Trichomonas vaginalis [31].

In Mexico, the prevalence of intestinal parasitic infections (IPIs) remains high, especially in rural and marginalized communities, with national epidemiological surveys reporting frequent cases of Ascaris lumbricoides, Trichuris trichiura, and S. stercoralis among school-aged children [32,33]. These infections contribute significantly to the burden of disease in the country and reflect underlying socio-economic inequalities [32,34].

Therefore, this study aimed to evaluate the nematocidal activity of methanolic extracts from five medicinal plants traditionally used in Mexico against S. venezuelensis infective third-stage larvae (L3) as well as to assess their toxicity and antioxidant potential.

It is important to emphasize that the studies conducted are limited to the L3 larvae, which are the infective stage. This serves as a good model since it represents one of the stages that can be targeted in cases of autoinfection [35]. To determine the toxicity of the extracts, we evaluated their effects on two distinct cell lines: human erythrocytes and Vero cells [36].

2. Materials and Methods

2.1. Plant Material and Extraction

Between 2.5 and 3.5 kg of dry material was collected in Monterrey, Mexico (25°41′04″ N 100°19′05″ O at 540 m above sea level). An ethnobotanical and taxonomist expert, Prof. Marco Antonio Alvarado Vázquez from the Faculty of Biological Sciences, Universidad Autónoma de Nuevo León, carried out the collection and identification of the whole plant (

Table 1. Plants used to extract bioactive molecules from the leaves of Argemone mexicana, Jatropha dioica, Lippia graveolens, Thymus vulgaris, and Kalanchoe daigremontiana.

* Species	** Family	Popular Name	Hv
A. mexicana L. 1753	Papaveraceae	Chicalote or Mexican poppy	29,127
J. dioica Sessé 1887	Euphorbiaceae	Sangre de Drago	30,648
L. graveolens Kunth. 1818	Verbenaceae	Mexican oregano	25,554
T. vulgaris L. 1753	Lamiaceae	Tomillo or thyme	20,888
K. daigremontiana Raym.-Hamet & H. Perrier 1913	Crassulaceae	Aranto	29,130

Hv: herbarium voucher. * Plant species and ** botanical families were validated taxonomically using WFO and IPNI websites (accessed on 15 February 2025). The links reviewed were the following: A. mexicana https://www.worldfloraonline.org/taxon/wfo-0000547034 and https://www.ipni.org/n/18816-2; J. dioica https://www.worldfloraonline.org/taxon/wfo-0000219593 and https://www.ipni.org/n/317899-2; L. graveolens https://www.worldfloraonline.org/taxon/wfo-0000228865 and https://www.ipni.org/n/863641-1; T. vulgaris https://www.worldfloraonline.org/taxon/wfo-0000324951 and https://www.ipni.org/n/461765-1; K. daigremontiana https://www.worldfloraonline.org/taxon/wfo-0001299505 and https://www.ipni.org/n/274315-1.

). Specimens were identified and deposited in the herbarium of the Universidad Autónoma de Nuevo León. The botanical identifications were taxonomically validated using the WorldFlora Online (WFO) (http://www.worldfloraonline.org) (accessed on 15 February 2025) and the International Plant Names Index (IPNI) website (https://www.ipni.org) (accessed on 15 February 2025).

Extraction was performed by subjecting 100 g of milled dry plant leaves to 500 mL of acetone-free absolute methanol (MeOH, Merck KGaA, Darmstadt, Germany, DE) in a 1 L ball-bottom flask for 48 h at 200 rpm on an orbital shaker at room temperature (24.0 ± 2.0 °C). Extracts were filtered and rotaevaporated (Yamato Scientific Co., Ltd., Harumi, Chuo-ku, Tokyo, Japan, JPN) followed by vacuum application for 96 h using a desiccator loaded with analytical silica gel absorbent beads with a moisture indicator (Merck) and stored at 4 °C in amber bottles until use. The extraction yield percentage (%) was determined as follows (1):

$$Yield \%={\displaystyle \frac{Final weight}{Initial weight}}\times 100$$

(1)

2.2. Qualitative Phytochemical Tests

The following phytochemical tests were performed to identify the functional groups of each extract based on the methodology reported by Rodríguez-Garza et al. (2023) [37]: KMnO₄ (unsaturations), anthrone (carbohydrates), Lieberman–Buchard (sterols), Shinoda (flavonoids), NaOH (coumarins), Baljet (sesquiterpene lactones), saponins, sulfuric acid (quinones), ferric chloride (tannins), and Dragendorff (alkaloids).

2.3. Activity Against S. venezuelensis (L3)

The S. venezuelensis life cycle and the L3 larvae harvesting were described in previous research [18]. The S. venezuelensis life cycle was maintained through successive passages in 4-week-old male Wistar rats (150–200 g). Rats were subcutaneously injected with 12,000 infective third-stage larvae (L3) of S. venezuelensis in 500 μL of phosphate-buffered saline (PBS, pH~7.4). Fecal samples were collected from days 5 to 18 post-infection and cultured in 250 mL polyethylene containers with vermiculite and distilled water at 28 °C in a humid atmosphere (SANYO Electric Co. Ltd., Gunma, Japan) for 4 to 7 days. L3 larvae were harvested using the Baermann method and washed three times with distilled water, and their viability was assessed by light microscopy before experiments [18].

For the assays, 100–150 L3 per well were placed in a 96-well culture plate (Corning Incorporated, Corning, NY, USA) and incubated at 28 °C for 1 h for adaptation before treatment with methanolic extracts. The extracts and the positive control ivermectin (IV, Sigma-Aldrich^®, Merck KGaA, Darmstadt, Germany, DE) were dissolved in 5% dimethyl sulfoxide (DMSO, Merck) and appropriately diluted. L3 cultures were treated with extract concentrations ranging from 1 to 500 µg/mL. All assays were conducted in a final volume of 200 µL per well at 28 °C in a humid atmosphere. Nematode mortality was defined as the inability to move within three minutes of observation under 40× magnification for at least 1 min, using visible light to stimulate movement at 0, 24, 48, 72, and 96 h post-treatment. The positive control (C+) consisted of 10 μM of IV, while the negative control (C−) included distilled water and a vehicle control of 0.4% DMSO. LC₅₀ determination was performed at test concentrations of 1, 10, 100, 250, and 500 μg/mL at 0, 24, 48, 72, and 96 h.

2.4. Antioxidant Activity

The antioxidant potential of the plant extracts was tested by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [38]. For this, we incubated 100 μL of 125 μM DPPH to 100 μL of each sample at different concentrations (31.25 to 500 µg/mL) for 30 min at 37 °C in darkness. Absorbance (Abs) at 517 nm was then measured with a spectrophotometer, Genesys 20 (Thermo-Fisher Scientific, Waltham, MA, USA), using 50 µM ascorbic acid as a positive control (C+) and MeOH as a blank. IC₅₀ values were calculated as the concentration of the sample required to scavenge 50% of DPPH. The reduction % was calculated using Formula (2):

$$DPPH scavenging \%={\displaystyle \frac{{Abs}_{517nm} Control-{Abs}_{517nm} Sample}{{Abs}_{517nm} Control}}\times 100$$

(2)

2.5. Red Cell Cytotoxicity

The activity of plant MeOH extracts was evaluated by the hemolysis test. The red cell cytotoxicity test was used as a model of cell toxicity, as previously reported by our research group [39]. The degree of hemolysis was determined by measuring Abs at 540 nm, whereas the IC₅₀ values were calculated as the concentration of sample required to hemolyze 50% of human red blood cells. Sterile distilled water was used as a positive control for hemolysis because it causes erythrocyte lysis and total hemolysis in erythrocytes. The % of hemolysis was calculated with Formula (3):

$$Hemolysis \%={\displaystyle \frac{{Abs}_{540nm} Treatment}{{Abs}_{540nm} Positive control}}\times 100$$

(3)

2.6. Cytotoxic on Vero Cells

The African green monkey kidney epithelial cell line (Vero; ATCC CCL-81) was cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2 g/L sodium bicarbonate (NaHCO₃), and a 1% penicillin–streptomycin solution (Gibco), referred to as complete DMEM medium. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ [40]. Vero cells were seeded at a density of 1 × 10⁴ cells per well in flat-bottomed 96-well microplates (Corning Incorporated) containing complete DMEM medium. After 24 h of incubation at 37 °C with 5% CO₂, cells were treated with methanolic extracts at concentrations ranging from 10 to 1000 µg/mL. Culture medium with or without 1% DMSO served as negative controls; the antineoplastic vincristine sulfate (Hospira, Warwickshire, UK) at 100 µg/mL was used as a positive control. Following 48 h of incubation, cell viability was assessed using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Affymetrix, Cleveland, OH, USA) reduction assay. MTT was added to each well at a final concentration of 0.5 mg/mL and incubated at 37 °C for an additional 3 h. The plates were then decanted, and formazan crystals were dissolved in 100 µL of DMSO. Optical density (OD) was measured at 570 nm [41] using a MULTISKAN GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Growth inhibition (%) was calculated using Formula (4):

$$Cell viability \%={\displaystyle \frac{ {Abs}_{570nm}Treatment}{ {Abs}_{570nm} Negative control}}\times 100$$

(4)

2.7. Statistical Analysis

All assays were performed in triplicate and repeated at least three times. Results were expressed as means and standard error (SD) of the mean. Significant differences between groups were determined by a one-way ANOVA test. The post hoc Dunnett’s test was performed to determine statistical differences between treated and untreated controls. The post hoc Tukey’s honest significance test (HSD) was performed to determine any statistical differences between treatments. The Probit test was used to determine the mean cell lethal concentration (LC₅₀), the immobilization of 50% of the L3 concentration (IC₅₀), and the half-maximal effective concentration (EC₅₀) of the radical scavenging activity. The analyses were performed with SPSS software Ver. 24 (IBM—SPSS, Armonk, NY, USA). All statistical analyses were considered significant at the p < 0.05 level.

3. Results and Discussion

3.1. Phytochemical Analysis

The extraction yield determined using Formula 1 of the total dry mass of the MeOH extracts of the leaves from A. mexicana was 11.30%, for J. dioica it was 15.99%, for L. graveolens was 43.33%, for T. vulgaris was 11.36%, and for K. daigremontiana was 10.04% (

Table 2. Phytochemical results and extraction yield of methanolic extracts of dry leaves of Argemone mexicana, Jatropha dioica, Lippia graveolens, Thymus vulgaris, and Kalanchoe daigremontiana.

Plant	Yield %	Chemical Group
Species		Uns	Car	Str	Flv	Cou	Sql	Sap	Qns	Tan	Alk
A. mexicana	11.30	+	−	+	+	+	+	−	−	−	+
J. dioica	15.99	+	+	+	−	+	+	−	+	−	−
L. graveolens	43.33	+	+	+	+	+	+	+	+	+	−
T. vulgaris	11.36	+	+	+	+	−	−	+	−	+	−
K. daigremontiana	10.04	+	+	+	+	−	−	+	−	+	−

Uns: unsaturations, Car: carbohydrates, Str: sterols, Flv: flavonoids, Cou: coumarins, Sql: sesquiterpene lactones, Sap: saponins, Qns: quinones, Tan: tannins, Alk: alkaloid, +: present, −: absent.

). All extracts tested were positive for the unsaturation and sterol tests. For carbohydrate and flavonoid tests, the extracts of A. mexicana and J. dioica exhibited negative reactions, respectively. The extracts from A. mexicana, J. dioica, and L. graveolens were positive for coumarins and sesquiterpene lactones. Additionally, the extracts of L. graveolens, T. vulgaris, and K. daigremontiana showed positive results for saponins and tannins.

3.2. Anti-Strongyloides Venezuelensis Activity

We observed the concentration–response effects of the different extracts evaluated against S. venezuelensis (Figure 1). It can be observed that the extracts of A. mexicana (Figure 1a) and L. graveolens (Figure 1c) demonstrated significant activity at 250 and 500 μg/mL. J. dioica (Figure 1b) showed significant activity at 72 h post-treatment at 250 and 500 μg/mL, while T. vulgaris (Figure 1d) showed activity up to 96 h at the same concentrations. K. daigremontiana (Figure 1e) did not demonstrate any significant effect against the L3 larvae at any of the evaluated concentrations.

The nematocidal activity of the extracts at different post-infection times is shown in

Table 3. Nematocidal activities expressed in LC₅₀ of methanolic extracts from dry leaves of Argemone mexicana, Jatropha dioica, Lippia graveolens, Thymus vulgaris, and Kalanchoe daigremontiana.

Plant Extract	LC50 in µg/mL
	24 h	48 h	72 h	96 h
A. mexicana	>250	<250	<250	<200
J. dioica	>500	>500	<200	<200
L. graveolens	>500	>250	<100	<100
T. vulgaris	>500	>500	<250	<200
K. daigremontiana	>1000	>1000	>500	>500

Positive control (C+) consisted of 10 μM IV.

. At 24 and 48 h, none of the treatments exhibited activity against L3 larvae. However, at 72 h, J. dioica and L. graveolens demonstrated activity with LC₅₀ values below 200 µg/mL and 100 µg/mL, respectively. Finally, at 96 h, L. graveolens showed remarkable nematicidal activity with an LC₅₀ < 100 µg/mL, while A. mexicana, J. dioica, and T. vulgaris exhibited LC₅₀ values < 200 µg/mL. In contrast, K. daigremontiana was the extract with the lowest activity across all time points, with an LC₅₀ > 500 µg/mL.

3.3. Hemolytic, Cytotoxic, and Antioxidant Test

The toxicity of the different methanolic extracts was evaluated on human erythrocytes and normal Vero cell cultures (

Table 4. Hemolytic, cytotoxic, and antioxidant activities of methanolic extracts of dry leaves of Argemone mexicana, Jatropha dioica, Lippia graveolens, Thymus vulgaris, and Kalanchoe daigremontiana.

Plant Extract	IC50 (LL − UL) in µg/mL	IC50 (LL − UL) in µg/mL	EC50 (LL − UL) in µg/mL
	Red-Cells	Vero Cells	DPPH
A. mexicana	>1000 e	202.99 (187.74–218.26) b	565.98 (526.77–605.19) d
J. dioica	565.80 (518.74–612.86) c	>1000 d	116.80 (93.28–140.32) b
L. graveolens	432.60 (412.52–452.68) b	ND	19.80 (15.77–23.83) a
T. vulgaris	229.77 (217.60–241.95) a	404.52 (369.56–438.82) c	418.72 (377.33–460.06) c
K. daigremontiana	671.81 (658.18–685.49) d	111.39 (102.12–120.66) a	699.05 (660.59–737.51) e

Different letters within the same column are significantly different by Tukey’s post hoc test. LL: lower limit, UL: upper limit, ND: not determined since it caused a mitogenic effect. The positive controls evaluated in the hemolysis test, Vero cell cytotoxicity, and antioxidant capacity assay are indicated in the Materials and Methods section: distilled water, vincristine sulfate (100 µg/mL), and ascorbic acid (50 µM), respectively.

). None of the extracts showed toxicity toward human erythrocytes. However, in Vero cells, J. dioica exhibited an IC₅₀ greater than 1000 µg/mL, while K. daigremontiana, A. mexicana, and T. vulgaris presented IC₅₀ values of 111.39, 202.99, and 404.52 µg/mL, respectively. Table 4 also displays the antioxidant activity of the extracts, assessed using the DPPH radical scavenging assay. The extract with the highest antioxidant capacity was L. graveolens (EC₅₀ = 19.80 µg/mL), while those with the lowest antioxidant activities were A. mexicana (EC₅₀ = 565.98 µg/mL) and K. daigremontiana (EC₅₀ = 699.05 µg/mL).

4. Discussion

Strongiloides stercoralis is a medically significant parasitic nematode with a high annual incidence worldwide, particularly in tropical and subtropical regions [42,43]. Also, this nematode can develop chronic cryptic low-level infections in a process called autoinfection. Frequently, immunocompromised patients develop disseminated or hyperinfection syndromes with high mortality rates. This situation, linked to the limited availability of effective nematocidal drugs [44], underscores the need for novel treatment alternatives.

Plants synthesize a diversity of secondary metabolites with a wide range of biological activities, including significant antiparasitic properties [45]. The anthelmintic potential of plant extracts is primarily attributed to the presence of bioactive compounds such as alkaloids, flavonoids, coumarins, and terpenoids, among others [45,46]. These metabolites target helminths through various mechanisms that vary across different stages of the parasite’s life cycle. They can damage the cuticle, disrupt motility, or interfere with feeding, ultimately impairing the growth and reproductive capacity of the helminths [47]. For example, the alkaloid berberine has demonstrated the ability to intercalate into microtubules [48], disrupting chloride channels and subsequently affecting helminth motility [49]. However, caution is warranted, as improper use of berberine may result in toxicity [50].

The present study screened five methanolic extracts from Mexican plants against S. venezuelensis L3. We observed that L. graveolens, commonly known as Mexican oregano, showed the best results against L3 larvae, presenting a mean immobilization rate of 100 µg/mL at 72 and 96 h. These results combine low toxicity in contact with erythrocytes, Vero cells, and high antioxidant activity. This aligns with previous studies demonstrating the antiparasitic effects against T. cruzi cultures [51], H. contortus [52,53], and E. histolytica. Specifically, significant amoebicidal activity has been reported for its extracts, subfractions, the monoterpene carvacrol [54] and the isolated flavonoids pinocembrin, sakuranetin, cirsimaritin, and naringenin [55].

Previous studies conducted by Boyko, O. and Brygadyrenko, V. in 2023 [46] indicate the potential of several natural organic groups in vitro against S. papillosus and H. contortus, highlighting the potential of plant compounds to decrease their lethality [46]. In another study conducted by the same authors in 2023, they indicated the nematocidal potential of some essential oils, specifically eugenol, isoeugenol, thymol, and carvacrol, against S. papillosus and H. contortus. Therefore, the plants evaluated could represent a source of nematocidal compounds [56].

The antioxidant capacity of the extracts was evaluated using the DPPH radical scavenging assay, a widely employed method for determining the antiradical effects of various biological components, including plant extracts. In this study, L. graveolens demonstrated the strongest DPPH radical scavenging activity, as shown in Table 4. This effect may be attributed to its secondary metabolites (Table 2), such as flavonoids, polyphenols, monoterpenes, sesquiterpenes, and tannins [55,57,58], which are commonly found in extracts of the Lippia genus. Our findings align with those reported by Soto-Domínguez et al. in 2012 [59], where the aqueous extract of L. graveolens exhibited antioxidant activity without causing toxicity in an in vitro model using Artemia salina and an in vivo murine model. Histological and histochemical studies of liver and kidney tissues in these models revealed no adverse effects [59]. Additionally, the primary secondary metabolites of Mexican oregano, as previously mentioned, have been reported to possess antioxidant, anti-inflammatory, antiglycemic, and nutritional potential [26]. These findings underscore the beneficial effects of L. graveolens, as highlighted in the present study.

In a study evaluating the methanolic extract of J. dioica against Trichinella spiralis and S. venezuelensis, the extract exhibited an IC₅₀ of 134.80 µg/mL against T. spiralis and an IC₅₀ of 382.40 µg/mL at 72 h against S. venezuelensis [18]. The J. dioica MeOH extract demonstrated greater activity in the present investigation, with an LC₅₀ < 200 µg/mL at 72 and 96 h post-treatment (Table 3). Aranda-López, in 2021, reported the effect against Taenia crassiceps of the naphthoquinone 4a, one component of J. dioica [60]; this demonstrated the antiparasitic potential of J. dioica. Jatropha dioica, commonly known as “Sangre de Drago” in Mexico, has also demonstrated cytotoxic, antimicrobial, and antifungal activities [61,62].

Previous research demonstrated the antiparasitic effects of the plants evaluated in the present study. A. mexicana has shown activity against E. histolytica [63] and T. vaginalis [64] as well as nematocidal effects against Meloidogyne incognita and M. juvanica [65] and activity against Schistosoma mansoni [66]. These biological effects have been attributed to isoquinoline alkaloids, such as berberine [66,67].

The MeOH extract of T. vulgaris demonstrated significant activity only after 96 h of culture at a 200 µg/mL concentration also with low toxicity to erythrocytes and Vero cells and high antioxidant activity. This activity is lower than that demonstrated against H. contortus and T. spiralis [30], probably due to its principal components such as carvacrol and thymol [26,68] that likely have less nematocidal activity.

In addition, K. daigremontiana has demonstrated activity against E. histolytica and T. vaginalis [31], with the flavonoid quercetin being the main compound with antiparasitic activity [69]. In this study, no significant effect was observed when evaluating it against S. venezuelensis L3 cultures.

Regarding the cytotoxic effect of the extracts against Vero cells, these have been previously evaluated in several investigations where it has been determined that some of these extracts show activity against various cells of tumor lineage without affecting healthy cells such as Vero and erythrocytes [70]. These cellular models are widely utilized by various authors to determine the toxicological or safety profiles of natural products, including plant extracts [41,71]. None of the extracts evaluated exhibited toxic effects on these cell types; this is based on several investigations where concentrations higher than 100 µg/mL were considered as non-toxic [72,73]. In the present investigation, all the determinations presented ranges higher than these. In addition, several plant-based studies on the use of plants against parasites have used Vero cells as a model to determine toxicity as well as to determine the selectivity indexes of such treatments [18,72].

This study provides a preliminary evaluation of the nematocidal, antioxidant, and cytotoxic activities of selected plant extracts; however, it has several limitations. First, the nematocidal activity was only assessed in vitro using S. venezuelensis third-stage infective larvae (L3), and further studies are needed to confirm these effects in vivo.

We adapted to the characteristics of our experimental model of S. venezuelensis and to our objective, which was to characterize plant extracts in terms of efficacy against the infective phase and toxicity to mammalian cells and to be able to propose new studies. We did not perform studies on L1 or L2 because the intention was to look for active principles on the host, and L1 and L2 are found in the soil. Also, we can cultivate the L3 phase for a week, conserving motility up to 85% for a week. In our experiments, we conserved the untreated controls for a week, but our results were recorded at 24, 48, 72 and 96 h following the general use to report information in three days after the treatment.

Second, while we evaluated cytotoxicity using hemolysis and the MTT assay in Vero cells, additional toxicological tests in different cell lines or animal models are required to fully assess the safety profile of these extracts. Third, it is necessary to identify the phytochemical composition of the extracts. Future studies incorporating a detailed chemical characterization and in vivo validation will be essential to further explore the potential of these plant extracts for nematocidal therapy.

5. Conclusions

This study demonstrated the high nematocidal activity of crude methanolic extracts from L. graveolens compared to the other evaluated extracts against S. venezuelensis in a time- and concentration-dependent manner, exhibiting low cytotoxic activity in red cells and high antioxidant activity.

Our findings highlight the potential of Lippia graveolens as a nematocidal agent, warranting further studies on its bioactive compounds and in vivo efficacy to support future therapeutic applications.