Exogenous salicylic acid modulates the essential oil of Dizygostemon riparius and enhances anthelmintic efficacy against Haemonchus contortusDizygostemon riparius is a Brazilian aromatic subshrub of the Plantaginaceae family (Scatigna et al., 2020). This species has high economic potential due to the bioactive compounds present in its essential oil, particularly endo-fenchyl acetate, endo-fenchol, (E)-caryophyllene, and caryophyllene oxide (Brandão et al., 2020). These compounds exhibit larvicidal, fungicidal, and acaricidal activities, positioning D. riparius as a promising natural resource for pest and disease management (Brandão et al., 2020; Corrêa et al., 2023; Ferreira et al., 2025).
In general, plants produce volatile compounds in essential oils (EOs) to protect against pathogens and herbivores and to attract pollinators (Adenubi et al., 2018). However, the production and chemical composition of these oils can vary substantially in response to environmental factors and cultivation conditions (Anjali et al., 2023; Galvão et al., 2023). Such plasticity may directly affect EO stability and bioactivity, highlighting the need for cultivation strategies capable of enhancing metabolite consistency and functional performance. Elicitors such as exogenous salicylic acid (SA) have been shown to stimulate the accumulation of alkaloids, glycosides, anthraquinones, and phenolics (Jain et al., 2024). Thus, SA elicitation has emerged as a practical strategy to enhance the synthesis of bioactive metabolites on a commercial scale (Ali, 2021; Humbal and Pathak, 2023).
The anthelmintic properties of many EOs have been investigated against gastrointestinal nematodes (Camurça-Vasconcelos et al., 2007; Katiki et al., 2017; Macedo et al., 2015). Among these, Haemonchus contortus is a nematode that causes significant economic losses in small ruminant production (Adduci et al., 2022). Current control strategies rely mainly on synthetic anthelmintics, but their excessive use has led to drug-resistant H. contortus strains (Emery et al., 2016; Sallé et al., 2019). Despite the recognized role of SA in modulating secondary metabolism, its influence on the chemical profile of D. riparius EOs and the consequent effects on their anthelmintic potential remain unknown. Given this context, this study aimed to investigate the influence of exogenous SA on the chemical composition of D. riparius EOs and to evaluate their in vitro anthelmintic activity against H. contortus.
D. riparius plants were cultivated in a greenhouse at the Graduate Program in Agricultural Sciences, State University of Maranhão (UEMA), Brazil. Plants were propagated using cuttings taken from parent plants maintained in pots containing Carolina Soil® and black soil (1:1, v/v). All plant cultivation procedures and treatment applications with SA (100 µM) and distilled water were performed following Albuquerque et al. (2024).
Leaves were harvested 66 days after planting and dried in an oven at 45 °C for 72 hours. Dried D. riparius leaves (20 g) were then subjected to hydrodistillation using a Clevenger apparatus for 3 hours. After extraction, the EOs were centrifuged at 3000 rpm for 3 minutes and dried over anhydrous sodium sulfate. The EO yield was determined as described by Lima et al. (2022). Essential oil extracted from salicylic acid-treated plants was designated as DRSA, whereas oil from control plants was designated as DR0.
The chemical composition of the EOs was analyzed by gas chromatography coupled with mass spectrometry (GC-MS; Shimadzu QP 2010 Ultra system), using an oven temperature program ranging from 60 °C to 240 °C at a rate of 3 °C min−1 over 10 min. A 1 µL aliquot of essential oil diluted in hexane (3:500, v/v) was injected with an AOC-20i auto-injector. Chromatographic separation was performed on a 30 m × 0.25 mm Rtx-5MS silica capillary column coated with 5% diphenyl/95% dimethyl polysiloxane (0.25 µm film thickness). The injector (split 1:20), transfer line, and ion source temperatures were set at 250, 250, and 200 °C, respectively. Helium was used as the carrier gas at a constant flow rate of 1 mL min−1. Mass spectra were recorded under electron impact ionization at 70 eV, with automatic scanning from 35 to 400 Da at 0.30 scans per second. Compounds were identified by comparing retention times and linear retention indices (LRI) calculated from a homologous series of n-alkanes (C8-C40), and by matching mass spectra with the Adams (2007), NIST (2011), and FFNSC 2 libraries.
H. contortus eggs were obtained from donor sheep with a monospecific experimental infection of H. contortus isolated from a naturally infected goat and maintained in the Laboratorio de Controle de Parasitos (LCP-UFMA), São Luis, Brazil. H. contortus strain were maintained from artificially infected using a methodology described by Silva et al., 2021. All procedures for parasite maintenance and animal care followed the guidelines of the Ethics Committee of the Federal University of Maranhão and were approved under protocol number 23115.002630/2023-21. The eggs were collected following Coles et al. (1992). H. contortus eggs were recovered using a saturated NaCl solution, washed, and resuspended in distilled water. Approximately 100 eggs were transferred to each well of a 24-well plate, and 100 µL of each treatment (DRSA and DR0) was added. Treatments were prepared in 3% Tween 80 at 12 concentrations ranging from 10 to 0.005 mg mL−1. The 3% Tween 80 solution served as the negative control. Each treatment was tested in triplicate. Plates were incubated at 27 °C with relative humidity > 80%, and egg hatchability was assessed under an inverted microscope after 48 h. Data were analyzed by nonlinear regression using GraphPad Prism 8.0, and the concentration required to inhibit 50% of egg hatching (IC50) was calculated. DRSA and DR0 oils were considered significantly different (P ≤ 0.05) when the confidence intervals (CIs) of their IC50 values did not overlap (Roditakis et al., 2005).
The essential oil yield of D. riparius was 1.64% in SA-treated plants (DRSA) and 1.72% in control plants (DR0). A total of 41 compounds were identified by GC-MS and are listed in Table 1. The major constituents were endo-fenchyl acetate (39.8% in DRSA and 38.4% in DR0), endo-fenchol (36.1% in DRSA and 35.8% in DR0), (E)-caryophyllene (7.9% in DRSA and 6.6% in DR0), caryophyllene oxide (3.16% in DRSA and 2.85% in DR0), and terpinen-4-ol (1.07% in DRSA and 1.02% in DR0). Together, these compounds accounted for 88.03% of the total oil composition in DRSA and 84.67% in DR0.
Yield and chemical composition (%) of essential oils from D. riparius treated with salicylic acid (DRSA) and untreated (DR0), identified by GC–MS.
Previous studies have characterized the essential oil composition of D. riparius leaves. In a seasonal and circadian study, Galvão et al. (2023) reported that oils collected at different times of the year contained endo-fenchyl acetate (30.5-41.3%), endo-fenchol (31.3-37.4%), (E)-caryophyllene (2.8-7.6%), α-fenchene (3.3-6.5%), and p-cymene (0.9-4.5%) as the major constituents. Similarly, Brandão et al. (2020) analyzed oils from D. riparius collected in São Benedito do Rio Preto, Maranhão, and found high levels of endo-fenchyl acetate (42.8-48.0%) and endo-fenchol (33.3-35.0%), followed by (E)-caryophyllene (4.2-6.8%) and caryophyllene oxide (2.3-3.3%). In the present study, the major chemical classes and predominant constituents were comparable to those reported by Galvão et al. (2023) and Brandão et al. (2020).
Despite cultivation under greenhouse conditions, the essential oil profile remained comparable to that observed in plants grown under natural conditions. Notably, DRSA samples exhibited subtle changes in chemical composition relative to control plants (DR0), including the appearance of several minor compounds (Table 1). Some of these compounds were previously detected in D. riparius grown under natural conditions (Galvão et al., 2023). Their exclusive detection in SA-treated plants suggests that SA may have triggered physiological pathways associated with the reactivation of their biosynthesis. SA is a well-established elicitor that enhances plant defense responses and induces the production of secondary metabolites (Jeyasri et al., 2023).
The egg hatchability results demonstrated that both DRSA and DR0 oils exhibited anthelmintic activity against H. contortus eggs, with hatchability decreasing in a dose-dependent manner (Figure 1). DRSA oil showed greater efficacy, achieving over 80% inhibition at a concentration of 5 mg/mL. The inhibitory concentration required to prevent 50% of egg hatching (IC50) was 0.58 mg/mL for DRSA and 1.99 mg/mL for DR0 (Table 2). The negative control showed a 100% hatching rate for the eggs. Dizygostemon riparius is a recently described aromatic species, and reports on its biological activities remain limited. However, its essential oil has demonstrated larvicidal activity against Aedes albopictus larvae (Brandão et al., 2020; Brandão et al., 2025). Here, DRSA essential oil exhibited greater anthelmintic activity against H. contortus eggs than DR0. At lower concentrations (0.62 mg/mL), DRSA oil was 3.4-fold more effective, reaching 50% inhibition, whereas DR0 oil achieved only 19%.
Percentage inhibition of Haemonchus contortus egg hatching in response to increasing concentrations of essential oils from Dizygostemon riparius without (DR0) and with salicylic acid treatment (DRSA).
Inhibitory concentration (IC50) of essential oils from Dizygostemon riparus without (DR0) and with salicylic acid treatment (DRSA) against Haemonchus contortus egg hatching.
The enhanced activity of DRSA oil may be directly related to its chemical composition, including increased levels of neral and geranial (citral), as well as the reappearance of hexenol, α-pinene, and cis-piperitone epoxide. Citral and α-pinene have documented antiparasitic activity against H. contortus and the nematode model Caenorhabditis elegans (Silva et al., 2021; Lima et al., 2022). In addition, the observed anthelmintic activity may result from the combined action of multiple constituents through synergistic interactions. Synergistic effects among plant-derived compounds have previously been demonstrated against H. contortus (Katiki et al., 2017).
These findings suggest that the use of SA as an elicitor to regulate secondary metabolite production in greenhouse-grown plants may represent a viable strategy to compensate for reduced environmental stress stimuli while maintaining or enhancing biological activity. In Mentha piperita, treatment with SA (0.5 and 1 mM) altered both the yield and composition of EOs, increasing the levels of menthol and menthofuran (Afkar, 2023). Similar effects have been reported in other species, including Thymus kotschyanus and Thymus vulgaris (Mohammadi et al., 2019), Thymus daenensis Celak. (Pirbalouti et al., 2013), and Eryngium foetidum (Santos et al., 2024).
Essential oils of D. riparius, with and without SA application, shared the same major constituents, mainly endo-fenchyl acetate, endo-fenchol, and (E)-caryophyllene. However, SA influenced the relative abundance of minor compounds. Both EOs showed significant anthelmintic activity against H. contortus, with the SA-treated oil (DRSA) exhibiting the highest nematicidal activity. The results support the hypothesis that exogenous SA modulates essential oil composition while preserving the core chemical profile characteristic of plants grown in their natural habitat. This modulation may enhance biological activity and further support the potential of D. riparius essential oil as a plant-based anthelmintic.
Data Availability Statement
The research data is available upon prior request via email to the corresponding author.
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Edited by
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Editor:
Takako Matsumura Tundisi
