In vitro evaluation of the anthelmintic activity of citrus flavonoids against free-living and parasitic nematodes

Abstract

Helminth infections remain a significant global health and economic burden, and the growing emergence of resistance to frontline anthelmintic drugs such as albendazole and ivermectin underscores the urgent need for novel therapeutic strategies. Flavonoids, a diverse group of plant-derived polyphenolic compounds, have gained attention for their broad-spectrum biological activities, including potential antiparasitic properties. This study aimed to investigate the anthelmintic potential of orange-derived flavonoids using two complementary models: the free-living nematode Caenorhabditis elegans (wild-type, albendazole-resistant, and ivermectin-resistant strains) and the muscle-stage larvae of Trichinella spiralis as a representative parasitic nematode. Among the five flavonoids tested, quercetin exhibited the strongest anthelmintic activity across all C. elegans strains and against T. spiralis, while demonstrating minimal cytotoxicity in human cell lines, indicating a favorable safety profile. To investigate its potential mode of action, electron microscopy was employed to assess morphological changes, while a mass spectrometry-based metabolomics approach was used to examine molecular mechanisms in T. spiralis treated with quercetin. The results showed that quercetin did not induce detectable morphological alterations but significantly disrupted multiple key metabolic pathways, particularly those associated with energy production, lipid metabolism, and mitochondrial function, indicating a systemic metabolic disturbance. These findings offer new insights into the metabolic effects of orange-derived flavonoids and underscore quercetin as a promising lead candidate for anthelmintic development.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-34049-5.

Introduction

Parasitic nematodes pose significant global health, agricultural, and economic challenges, affecting both humans and animals. In humans, gastrointestinal nematodes such as Ascaris lumbricoides, hookworms (Necator americanus and Ancylostoma duodenale), and Trichuris trichiura infect billions worldwide, leading to anemia, malnutrition, growth retardation, and impaired cognitive development, particularly in children from low-resource settings¹. Other nematodes, including Trichinella spiralis, cause trichinellosis through the consumption of undercooked meat, leading to symptoms ranging from gastrointestinal distress to muscle inflammation and, in severe cases, cardiac or neurological complications². The global annual incidence of trichinellosis is estimated to be approximately 10,000 cases, with a mortality rate of about 0.2%. However, serological evidence suggests that up to 11 million individuals may be infected worldwide. Cases of trichinellosis have been documented in 55 countries, representing roughly 27.8% of all nations globally³. In 2014, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) listed T. spiralis among the top 10 most common foodborne parasites that eventually lead to serious health problems⁴. T. spiralis is transmitted through the consumption of raw or undercooked meat containing encysted larvae, primarily from pigs or wild animals. The parasite maintains both domestic and sylvatic cycles—pigs acquire infection by ingesting infected tissues, while wild carnivores and scavengers sustain transmission through predation and cannibalism. Humans are accidental hosts, acquiring infection via contaminated meat, thus representing a dead-end in the parasite’s life cycle.

In animals, parasitic nematodes significantly reduce livestock productivity by decreasing weight gain, milk production, and reproductive performance, resulting in considerable economic losses for farmers globally⁵. Moreover, several nematodes are zoonotic, such as T. spiralis and Toxocara spp., increasing the risk of transmission between animals and humans and highlighting the urgent need for integrated “One Health” approaches. Albendazole (ABZ) and ivermectin (IVM) are widely regarded as the gold standard for treating nematode infections due to their broad-spectrum activity and clinical efficacy⁶. ABZ, a benzimidazole derivative, functions by selectively binding to β-tubulin, thereby preventing its dimerization with α-tubulin and inhibiting microtubule formation within nematode cells, ultimately disrupting essential cellular processes⁷. In contrast, IVM, a macrocyclic lactone, targets the α-subunit of glutamate-gated chloride channel receptors in nematode nerve and muscle cells. This binding enhances chloride ion influx, causing hyperpolarization, leading to paralysis, impaired feeding, reduced fecundity, and eventual death of motile stages⁸. Despite their effectiveness, reliance on ABZ and IVM poses serious challenges. Widespread and repeated usage has led to the emergence of anthelmintic resistance across multiple nematode species, compromising drug efficacy and threatening sustainable control efforts⁹. This growing resistance, combined with persistent environmental contamination, limited access to clean water, and poor sanitation, underscores an urgent need for novel therapeutic strategies. To address these challenges, comprehensive approaches are required, including improved diagnostic tools, integrated control programs, and the development of new anthelmintic agents with distinct modes of action. Advancing drug discovery pipelines will be critical to mitigating the global health and economic burdens posed by parasitic nematodes.

Flavonoids are naturally occurring plant-derived compounds that exhibit a broad spectrum of biological activities, including antioxidant, anti-inflammatory, antimicrobial, and antiparasitic effects^10–12. Citrus fruits, particularly oranges, are a rich source of flavonoids, predominantly concentrated in the peels, pulp, and seeds. Orange flavonoids are mainly classified into three principal types: flavanones, flavones, and flavonols¹³. Several studies have identified key flavonoids in oranges, such as apigenin, hesperetin, naringin, quercetin, and tangeretin^11,14. Apigenin, a compound found in traditional medicines, is recognized as a bioactive flavonoid with antioxidant, anti-inflammatory, antihypertensive, antimicrobial, and antiparasitic activities^15,16. Apigenin has antiprotozoal activity in vitro against Leishmania amazonensis and in vivo against visceral leishmaniasis by oral administration^17,18. Hesperetin is a bioactive flavonoid whose bioactivities include antioxidant, anti-inflammatory, antivirus, and antiparasitic effects^19–21. Naringin is a natural flavonoid that is abundant in vegetables and fruits. Studies revealed that naringin possesses multiple pharmacological activities, including antioxidant, anti-inflammatory, anti-apoptosis, and anti-diabetes^22,23. Naringin demonstrated a potent anti-plasmodial effect against Plasmodium falciparum through its anti-inflammatory properties²⁴. Quercetin is widely used in traditional medicine due to its therapeutic potential, such as antioxidant, anti-inflammatory, antiviral, anticancer, antimicrobial, antiprotozoal, and anthelmintic activities^25–28. Ethnopharmacological studies on tangeretin have revealed several properties, including antioxidant, anti-inflammatory, and anticarcinogenic^29,30. Throughout the prolonged history of consuming these flavonoid compounds as part of the daily diet, no harmful effects have been seen^30,31. The pharmacological potential of these polyphenolic compounds explained why they are among the most popular plant-derived flavonoids. Accumulating evidence highlights the potential of flavonoids as natural anthelmintic agents. Experimental in vitro and in vivo studies have demonstrated their efficacy against a range of helminths, including Haemonchus contortus, Schistosoma mansoni, Trypanosoma brucei brucei, and Toxocara canis^32–35. Notably, kaempferol treatment in Trichinella spiralis-infected mice significantly reduced parasite burden and ameliorated intestinal inflammation³⁶. Despite these promising findings, few studies have focused on the use of orange-derived flavonoids as potential anthelmintic agents, and the mechanisms by which they exert their effects against parasitic nematodes remain poorly understood. This knowledge gap highlights the need for further investigation, which this study aims to address.

Metabolomics integrates biological experimentation with high-throughput analytical technologies, particularly mass spectrometry, alongside bioinformatics for comprehensive metabolite profiling³⁷. By quantitatively and qualitatively characterizing metabolites—the intermediates and end products of cellular metabolism—metabolomics can distinguish between normal and pathological pathways, thereby aiding in disease diagnosis and prognosis prediction³⁸. Recent studies have successfully applied metabolomic profiling in parasitology to unravel host-parasite interactions and identify potential therapeutic targets in several helminths, including Onchocerca volvulus, Schistosoma japonicum, Toxocara canis, and T. spiralis^39–42. Leveraging metabolomics in the study of flavonoid activity against parasitic nematodes could therefore provide new insights into their mechanisms of action and metabolic vulnerabilities, facilitating the discovery of novel antiparasitic drug candidates.

In this study, we evaluated the anthelmintic potential of orange-derived flavonoids using Caenorhabditis elegans as a nematode model, including wild-type, albendazole (ABZ)-resistant, and ivermectin (IVM)-resistant strains. Additionally, their efficacy was assessed against T. spiralis larvae to establish activity in a parasitic nematode model. The cytotoxicity of orange flavonoids was also tested on human cell lines to evaluate safety. To investigate their systemic effects on T. spiralis, a mass spectrometry-based metabolomics approach was employed. The half-maximal effective concentration (EC₅₀) of the flavonoids was determined, and ultrastructural changes induced by flavonoid treatment were visualized using transmission electron microscopy (TEM). Furthermore, differential metabolite profiling and pathway analysis were conducted to elucidate the molecular mechanisms underlying the anthelmintic effects of orange flavonoids.

Materials and methods

Anthelmintic activity of orange flavonoids on C. elegans

Wild-type N2, albendazole-resistant CB347, and ivermectin-resistant JD608 strains of Caenorhabditis elegans, obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, USA), were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50⁴³. The NGM was prepared according to the standard protocol from the Caenorhabditis Genetics Center (CGC, University of Minnesota, USA) using reagents purchased from Sigma-Aldrich (St. Louis, US): 3 g NaCl (S9888), 2.5 g peptone (P5905), and 17 g agar (A5306) per liter of distilled water, supplemented with 1 mL of 1 M MgSO₄ (M7506), 1 mL of 1 M CaCl₂ (C4901), 25 mL of 1 M KPO₄ buffer, pH 6.0 (prepared from KH₂PO₄, P5655, and K₂HPO₄, P3786), and 1 mL of 5 mg/mL cholesterol solution (C8667) added after autoclaving. For synchronization, worms were collected in M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL 1 M MgSO₄, distilled water) and subjected to bleaching. Eggs were transferred to unseeded NGM plates and incubated at 20 °C until the L1 stage (~ 24 h).

For drug testing, apigenin (Cat. No. 10798), hesperetin (H4125), naringin (91842), quercetin (PHR1488), and tangeretin (91004) (Sigma-Aldrich, Switzerland) were dissolved in M9 buffer to achieve final concentrations of 10, 100, 1,000, 10,000, and 100,000 ng/mL, with 0.5% DMSO included in each well. This concentration range facilitates the identification of the therapeutic window and is consistent with established practices in pharmacological research⁴⁴.

Negative controls contained 0.5% DMSO, while albendazole and ivermectin were used as positive controls. Thirty L1 worms were dispensed into each well (triplicate) and incubated for 72 h at 20 °C. Worm motility was assessed under an inverted microscope by observing the movement of the worms after compound exposure. The degree of motility was scored visually on a qualitative scale, where a complete lack of movement was defined as death, and any visible movement was considered alive⁴⁵. EC₅₀ values were determined by nonlinear regression, and statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post-hoc test, with statistical significance set at P < 0.05 (GraphPad Prism 9). All experiments were performed in three independent replicates⁴⁶.

Cytotoxicity testing

HepG2 and Caco-2 cell lines were employed to evaluate the cytotoxic effects of orange-derived flavonoids. Cells were seeded in 96-well plates (20,000 cells/well) and maintained in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Science, USA) at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h. The culture medium was subsequently replaced with the test compounds—apigenin, hesperetin, naringin, quercetin, tangeretin, albendazole (ABZ), and ivermectin (IVM)—at final concentrations of 10, 100, 1,000, 10,000, and 100,000 ng/mL, prepared in MEM with 0.5% DMSO per well. Negative controls contained 0.5% DMSO, while albendazole and ivermectin were used as positive controls. After a further 24 h of incubation, 10 µL of MTT solution (5 mg/mL; AppliChem GmbH, Germany) was added to each well, followed by 4 h incubation under the same culture conditions. The medium was subsequently replaced with a solubilization buffer (4 mM HCl and 0.1% Nonidet P-40 in isopropanol) to dissolve the formazan crystals. Absorbance was recorded at 590 nm with a reference at 620 nm. Cell viability was expressed relative to untreated controls, and the 50% cytotoxic concentration (CC₅₀) was determined using nonlinear regression. Statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post-hoc test, with statistical significance set at P < 0.05 in GraphPad Prism version 9. All assays were conducted in three independent replicates⁴⁶.

Anthelmintic activity of apigenin and quercetin on T. spiralis muscle stage

The mice used in this study were purchased from Mahidol University National Laboratory Animal Center, Thailand. The T. spiralis strain used in this study was a clinical isolate that has been maintained long-term in the Animal Care Unit, Faculty of Tropical Medicine, Mahidol University. Eight-week-old female ICR mice were orally inoculated with 100 T. spiralis larvae. After 60 days of infection, the animals were euthanized by CO₂ exposure, and muscle-stage larvae (L1) were recovered from skeletal tissue by enzymatic digestion with 0.7% pepsin and 0.7% HCl. The isolated larvae were maintained in RPMI medium supplemented with 10% fetal calf serum, 200 U/mL penicillin, and 200 µg/mL streptomycin.

For EC₅₀ determination, apigenin and quercetin were prepared in RPMI medium (supplemented as described above) at final concentrations of 10, 100, 1,000, 10,000, and 100,000 ng/mL, with 0.5% DMSO included in each well. Negative controls contained 0.5% DMSO, while albendazole (ABZ) and ivermectin (IVM) were included as positive controls. Thirty larvae were transferred into each well in triplicate and incubated for 72 h at 37 °C. Larval motility was scored under an inverted microscope, with live larvae exhibiting free movement, whereas dead larvae showed a C-shaped or linear body and showed no movement. EC₅₀ values were calculated using nonlinear regression. Statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post-hoc test, with statistical significance set at P < 0.05 in GraphPad Prism version 9. All experiments were performed in three independent replicates (adapted from⁴⁶.

Preparation of specimen for transmission electron microscopy

T. spiralis-treated were initially treated with quercetin at the EC₅₀ value, and sequentially fixed with 2.5% glutaraldehyde and 1% osmium tetroxide (1 h each). The fixed samples underwent dehydration through a graded ethanol series, infiltrated with increasing concentrations of LR white resin (EMS^®, USA), embedded in pure LR white resin, and then polymerized at 60 °C for 48 h. After polymerization, thin sections were cut into 100 nm sections using an ultramicrotome for subsequent immunogold labeling.

Immunogold labeling technique

To investigate the effects of quercetin treatment and explore the correlation between metabolomic and morphological alterations in T. spiralis, immunoelectron microscopy was performed as described in our previous studies⁴⁷ using specific antibodies. The primary antibodies targeted markers of mitochondrial function, including translocase of outer mitochondrial membrane 20 (TOM20), a mitochondrial outer membrane receptor that plays a crucial role in mitochondrial biogenesis⁴⁸, and succinate dehydrogenase subunit D (SDHD), a subunit of mitochondrial complex II that associates with muscular dysfunction⁴⁹, along with Bcl-2 associated X (BAX), a pro-apoptotic protein initiating apoptosis⁵⁰. Briefly, the thin sections were blocked for non-specific binding with 50 mM glycine in phosphate-buffered saline (PBS) and incubated in 5% bovine serum albumin (BSA; EMS^®, USA) in PBS. Primary antibodies targeting mitochondrial function, including anti-succinate dehydrogenase (SDHD; MyBioSource, USA, MBS7000340), anti-TOM20 (MyBioSource, USA, MBS2400038), and anti-BAX (MyBioSource, USA, MBS9600035), were diluted 1:50 and added to the sections for 1 h. After incubation, GAR IgG conjugated with 25 nm gold particles (EMS^®, USA) was applied for 1 h. To enhance the visibility of the gold particles, a silver enhancement was used for 30 min using the Aurion R-Gent SE-EM kit (EMS^®, USA). The sections were then counterstained with uranyl acetate and lead citrate. Ultrastructural features and the distribution of immunogold labeling in T. spiralis were examined under TEM (HT7700 model, Hitachi, Japan).

Metabolomics analysis of quercetin-treated T. spiralis muscle larvae

T. spiralis larvae treated with 0.5% DMSO served as controls, while larvae exposed to the EC₅₀ concentration of quercetin were processed for metabolite extraction as described by⁴¹. Samples were homogenized in 500 µL of cold methanol, snap-frozen in liquid nitrogen, thawed, and centrifuged at 800 ×g for 1 min at 4 °C. The supernatant was collected, and the extraction was repeated. Supernatants from both extractions were combined. The remaining pellet was resuspended in 250 µL of deionized water, frozen in liquid nitrogen, thawed, and centrifuged at 15,000 ×g for 1 min at 4 °C. This supernatant was pooled with the previous extracts. The combined extracts were centrifuged again at 15,000 ×g for 1 min at 4 °C to remove residual debris, and the clarified supernatant was collected and dried using a speed vacuum concentrator. Three biological replicates and two technical replicates were performed for metabolomics analysis.

Identification of metabolome using mass spectrometry

Metabolomic analysis was conducted following an established protocol⁵¹. Briefly, chromatographic separation was achieved using a high-performance liquid chromatography system (Agilent 1260 Quaternary Pump, Agilent 1260 Autosampler, and Agilent 1290 Thermostatted Column Compartment SL; Agilent Technologies, CA, USA) coupled to a quadrupole time-of-flight mass spectrometer (TripleTOF 5600+, SCIEX, USA) equipped with a DuoSpray electrospray ionization (ESI) source. Mobile phase A consisted of 0.1% formic acid in water, while mobile phase B contained 0.1% formic acid in acetonitrile.

Dried metabolite extracts were dissolved in 200 µL of a 1:1 (v/v) mixture of mobile phases A and B, and 100 µL was transferred to LC vials and maintained at 6 °C in the autosampler. A 5 µL aliquot was injected onto a reversed-phase C18 column (ACQUITY UPLC BEH, 2.1 × 100 mm, 1.7 μm; Waters, USA) operated at 40 °C with a flow rate of 0.3 mL/min. The gradient program was as follows: 5% B for 0–2.0 min, 5–60% B from 2.0 to 2.5 min, 60–80% B from 2.5 to 4.0 min, 80–100% B from 4.0 to 12.0 min, held at 100% B until 17.0 min, decreased to 5% B at 17.1 min, and re-equilibrated at 5% B until 20.0 min.

Mass spectrometric detection was carried out in both positive and negative ESI modes using Analyst Software v1.7 (SCIEX). Data were acquired in information-dependent acquisition (IDA) mode, consisting of a TOF-MS survey scan (m/z 100–1000) followed by ten dependent product ion scans (m/z 50–1000) in high-sensitivity mode with dynamic background subtraction. To ensure reproducibility and system stability, pooled quality control (QC) samples were injected at the beginning, intermittently throughout, and at the end of the analytical sequence.

Data processing and statistical analysis

Metabolomic mass spectra files obtained from LC–MS/MS (.wiff and .wiff.scan) in both + ESI and -ESI modes were separately processed using MS-DIAL software⁵². Alignment parameters were set to a retention time tolerance of 0.1 min and a mass accuracy of 0.015 Da. The compound detection, alignment, and identification were performed using two databases: ESI(+)-MS/MS from authentic standards and ESI(-)-MS/MS from authentic standards. The peak tables containing accurate m/z values, retention times, and peak intensities were exported and subsequently analyzed using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/) under the Statistical Analysis (one factor) module. Data were filtered using the interquartile range (IQR), normalized by quantile normalization, transformed by cube root transformation, and scaled by data range scaling. For visualization, Principal Component Analysis (PCA), Partial Least Squares–Discriminant Analysis (PLS-DA), volcano plot, and pathway analysis were generated. PCA and PLS-DA were displayed with 95% confidence intervals, while volcano plots were constructed using log₂ fold-change and –log p-values. Differential metabolites were identified based on a cut-off of fold change > 1.5 and p < 0.01. Pathway analysis was conducted using MetaboAnalyst, and pathways with p < 0.01 were considered statistically significant. Annotation of putative metabolites was performed using MS-DIAL software.

Results

In vitro nematocidal activities of orange flavonoids against C. elegans

In vitro assays were conducted to evaluate the anti-nematode activity of orange flavonoids against C. elegans, including the wild-type N2 strain, the albendazole-resistant CB347 strain, and the ivermectin-resistant JD608 strain. For the C. elegans N2 strain, EC_50​ values for apigenin, hesperetin, naringin, quercetin, and tangeretin were 218.50 ± 11.16, 282.97 ± 23.87, > 1,000, 80.45 ± 20.34, and > 1,000 µM, respectively. The positive controls (ABZ and IVM) exhibited EC₅₀ values of 672.17 ± 72.97 and 17.67 ± 1.85 µM, respectively (Table 1). Among the tested compounds, the nematocidal activity against the N2 strain was highest for ivermectin (IVM), followed by quercetin, apigenin, hesperetin, albendazole (ABZ), and the lowest activity was observed for naringin and tangeretin.

Table 1.

In vitro anthelmintic activity on C. elegans (N2, CB347, JD608 strain) of orange flavonoids.

Flavonoid	EC50 (µM)
	N2	CB347	JD608
Apigenin	218.50 ± 11.16	55.82 ± 4.89	51.33 ± 1.46
Hesperetin	282.97 ± 23.87	> 1,000	51.71 ± 2.92
Naringin	> 1,000	> 1,000	> 1,000
Quercetin	80.45 ± 20.34	76.25 ± 7.40	45.28 ± 4.92
Tangeretin	> 1,000	> 1,000	43.11 ± 11.25
ABZ	672.17 ± 72.97	> 1,000	788.35 ± 22.64
IVM	17.67 ± 1.85	15.40 ± 6.07	642.03 ± 56.66

EC₅₀ is the concentration of flavonoid associated with 50% of the maximum effect against the strain of C. elegans evaluated. Results are reported as mean ± S.E.

Against the C. elegans CB347 strain, EC₅₀​ values were 55.82 ± 4.89 µM for apigenin and 76.25 ± 7.40 µM for quercetin, while hesperetin, naringin, and tangeretin all showed EC₅₀​ values greater than 1,000 µM. The positive controls (ABZ and IVM) exhibited EC₅₀ values of > 1,000 and 15.40 ± 6.07 µM, respectively (Table 1). The nematocidal activity against the CB347 strain was in the order: IVM > apigenin > quercetin > hesperetin, naringin, tangeretin, and ABZ.

In the assessment against the C. elegans JD608 strain, EC_50​ values for apigenin, hesperetin, naringin, quercetin, and tangeretin were 51.33 ± 1.46, 51.71 ± 2.92, > 1,000, 45.28 ± 4.92, and 43.11 ± 11.25 µM, respectively. The positive controls (ABZ and IVM) exhibited the EC₅₀ values of 788.35 ± 22.64 and 642.03 ± 56.66 µM, respectively (Table 1). Among the tested compounds, the nematocidal activity against the JD608 strain was highest for tangeretin, followed by quercetin, apigenin, hesperetin, ivermectin (IVM), albendazole (ABZ), and the lowest activity was observed for naringin.

In vitro cytotoxicity of orange flavonoids against Caco-2 cells

This assay has been widely used to measure in vitro cytotoxicity effects of compounds and standard drugs in cell lines. The results showed a CC₅₀ of apigenin, hesperetin, naringin, quercetin, and tangeretin of 53.31 ± 4.52, > 1,000, > 1,000, > 1,000, and 251.30 ± 34.96 µM on the HepG2 cell line, respectively. The positive controls of ABZ and IVM resulted in a CC₅₀ of > 1000 and 14.92 ± 1.39 µM, respectively, on the same cell line (Table 2). In addition, the results showed a CC₅₀ of apigenin, hesperetin, naringin, quercetin, and tangeretin of > 1,000, 56.30 ± 6.18, 249.03 ± 65.30, > 1,000, and 531.27 ± 70.31 µM, respectively, on the Caco-2 cell line while the positive control (ABZ and IVM) resulted in a CC₅₀ of > 1000 and 19.98 ± 5.28 µM, respectively (Table 2). Based on these findings, apigenin and quercetin exhibited the most potent anti-nematode activity (Table 1), while simultaneously showing the lowest cytotoxicity among all compounds. Therefore, apigenin and quercetin were chosen for subsequent T. spiralis testing.

Table 2.

Cytotoxicity of orange flavonoids against HepG2 and Caco-2 cells.

Flavonoid	CC50 (µM)
	HepG2	Caco-2
Apigenin	53.31 ± 4.52	> 1,000
Hesperetin	> 1,000	56.30 ± 6.18
Naringin	> 1,000	249.03 ± 65.30
Quercetin	> 1,000	> 1,000
Tangeretin	251.30 ± 34.96	531.27 ± 70.31
ABZ	> 1000	> 1000
IVM	14.92 ± 1.39	19.98 ± 5.28

CC₅₀ is the flavonoid concentration associated with 50% of maximum cytotoxic effect against the HepG2 and Caco-2 cell lines. Results are reported as mean ± S.E.

In vitro nematocidal activities of apigenin and quercetin against T. spiralis

In vitro testing assessed the anti-nematode activity of apigenin and quercetin against T. spiralis. The results showed an EC₅₀ of apigenin at > 1,000 µM and quercetin at 146.54 ± 28.24 µM on the T. spiralis. In addition, the positive control (ABZ and IVM) showed an EC₅₀ of 128.67 ± 34.09 and 106.69 ± 7.89 µM, respectively, on the T. spiralis (Table 3). The nematocidal activity of our chosen compounds and positive controls against T. spiralis was in the order IVM > ABZ > quercetin and apigenin.

Table 3.

In vitro anthelmintic activity on T. spiralis of apigenin and quercetin.

Flavonoid	EC50 (µM)
Apigenin	> 1,000
Quercetin	146.54 ± 28.24
ABZ	128.67 ± 34.09
IVM	106.69 ± 7.89

The EC₅₀ represents the concentration of each flavonoid that produces 50% of the maximum effect against T. spiralis. Results are expressed as the mean ± standard error (S.E.). The selectivity index (SI) is defined as the ratio of CC₅₀ to EC₅₀, while “n/a” indicates data not available.

Effects of quercetin treatment on mitochondrial function, apoptosis, and morphological changes

To assess the effects of quercetin treatment on mitochondrial function, apoptosis, and morphological changes in T. spiralis, immunogold TEM was conducted. In summary, no apparent ultrastructural changes were observed in the treatment group across the cuticular layer, muscular area, or coelomic space compared to the non-treatment. Immunoreactivity of TOM20 and SDHD was more prominent than that of BAX, with signals detected throughout all three regions. Quantitative immunolocalization was examined across various regions of the parasite, including the cuticle, muscle, and internal structures. The results showed that the expression levels of TOM20, SDHD, and BAX were comparable between the quercetin-treated and control groups. Interestingly, although muscle tissue is typically prone to structural alterations, the muscle ultrastructure appeared well-preserved in both groups. The qualitative examination of each marker between the treated and control groups was illustrated in Figs. 1 and 2, 3 and 4.

Fig. 1.

Electron microscopic immunogold labeling of TOM20 in Trichinella spiralis from control and quercetin-treated groups. Transmission electron microscopy (TEM) images show the control group (A–C) and quercetin treatment group (D–F). Yellow arrows indicate immunolocalization of TOM20 in the T. spiralis samples.

Fig. 2.

Immunogold localization of SDHD in T. spiralis from control and quercetin-treated groups. The images show the control group (A–C) and quercetin treatment group (D–F). Yellow arrows indicate the SDHD immunolabelling in the T. spiralis samples.

Fig. 3.

Immunogold labelling of BAX in T. spiralis from control and quercetin-treated groups. The TEM images show T. spiralis from the control group (A–C) and the quercetin-treated group (D–F). BAX protein localization is indicated by yellow arrows.

Fig. 4.

Multivariate analysis of metabolite profiles from T. spiralis treated with DMSO (red circles) or quercetin (green circles), using partial least squares-discriminant analysis (PLS-DA).

Metabolite profiles of T. spiralis treated with quercetin

A volcano plot was created to display the statistical significance and the fold change between DMSO control and quercetin treatment (Fig. 5). After exposure to quercetin, 272 differential features showed a p-value < 0.01 and > 1.5-fold change. Around 204 features were up-regulated, and 68 were down-regulated. The most up-regulated metabolites, following treatment with quercetin were N-Acetyl-D-Galactosamine, lignoceric acid, and linolenic acid, as measured by the fold change (Table 4). The most down-regulated metabolites after treatment with quercetin were inosine, deoxyguanosine, and tetradecanoylcarnitine (Table 5).

Fig. 5.

Volcano plot analysis, illustrating up-regulated and down-regulated metabolites after treatment with quercetin. The two vertical lines indicate a ± 1.5-fold change compared to DMSO control. The horizontal line represents a statistically significant difference at a p-value < 0.01. Blue and red circles refer to down- and up-regulated metabolites, respectively.

Table 4.

Top-ten up-regulated metabolites of T. spiralis after treatment with quercetin.

No.	Potential metabolites	m/z	Mass Error (Da)	Log2 (FC)	p-Value
1	N-Acetyl-D-Galactosamine	222.09782	0.00061	4.5233	0.0070919
2	Lignoceric acid	369.38321	0.01050	4.0956	0.0001014
3	Linolenic acid	277.21588	0.00144	2.7081	0.0004623
4	LacCer(d18:1/16:0)	862.64905	0.02405	2.6728	0.0006945
5	Menaquinone 4	445.31357	0.00345	2.2163	0.0060384
6	Linoleic acid	279.23306	0.00012	2.1922	0.0000702
7	7alpha-Hydroxy-4-cholesten-3-one	423.33130	0.00794	2.1364	0.0037187
8	PC(P-18:1(9Z)/P-16:0)	760.57977	0.00531	2.1307	0.0000043
9	Coenzyme q6	597.45886	0.00989	1.8385	0.0021506
10	Vitamin K1	473.34482	0.00482	1.8127	0.0017177

Table 5.

Top-ten down-regulated metabolites of T. spiralis after treatment with quercetin.

No.	Potential metabolites	m/z	Mass Error (Da)	Log2 (FC)	p-Value
1	Inosine	291.07080	0.00079	-2.6373	0.0008150
2	Deoxyguanosine	290.08514	0.00083	-2.4539	0.0024828
3	Tetradecanoylcarnitine	372.30997	0.00085	-2.0664	0.0074963
4	LysoPE(0:0/16:0)	452.27704	0.00122	-1.8636	0.0077011
5	1-Heptadecanoyl-sn-glycero-3-phosphocholine	510.35422	0.00119	-1.6835	0.0008150
6	LysoPE(0:0/18:2(9Z,12Z))	476.27423	0.00403	-1.4991	0.0018140
7	LysoPC(0:0/18:0)	546.35083	0.00220	-1.4886	0.0085352
8	Arachidonic acid	303.23309	0.00015	-1.1472	0.0003581
9	Phosphatidylethanolamine	528.30853	0.00104	-1.0876	0.0001535
10	3-Phosphoglyceric Acid	186.99202	0.00819	-1.0514	0.0059161

Ranking the top three metabolites based on their impact on metabolic pathways revealed significant enrichment in the following processes: RORA-mediated activation of gene expression, BMAL1:CLOCK/NPAS2-driven circadian gene expression, and SREBF (SREBP)-regulated gene expression (Fig. 6).

Fig. 6.

Pathway analysis of the differential T. spiralis metabolite between DMSO and quercetin treatment.

Discussion

Current antinematode drugs, including benzimidazoles, macrocyclic lactones, imidazothiazoles, and tetrahydropyrimidines, are limited in number and have been in use for decades, leading to increasing drug resistance in livestock parasites and emerging reduced efficacy in human nematodes. Many agents show stage-specific activity, with poor effectiveness against larval or encysted stages, and variable cure rates across species, often resulting in treatment failures in mass drug administration programs. Adverse effects, contraindications in pregnancy, and pharmacokinetic limitations such as low solubility, poor absorption, and inadequate tissue penetration further hinder their utility. Moreover, the absence of combination therapies and a weak drug development pipeline, driven by low commercial incentives, restrict innovation and exacerbate the reliance on aging drugs with diminishing effectiveness. In the urgent search for alternative therapeutics against parasitic nematodes, significant attention has turned to plant-derived metabolites, particularly flavonoids, which have demonstrated promising anthelmintic potential¹⁰. In this study, we evaluated the anthelmintic activity of orange flavonoids using a C. elegans model to assess their effects on both drug-sensitive and drug-resistant strains. C. elegans is widely recognized as a preferred model for anthelmintic research due to its ease of cultivation under laboratory conditions, well-characterized genetics, and extensive molecular toolkit⁵³. In this study, we employed three C. elegans strains including N2 (wild type), CB347 (albendazole-resistant), and JD608 (ivermectin-resistant) to evaluate the anthelmintic activity of orange flavonoids. As expected, the positive controls, IVM and ABZ, exhibited EC₅₀ values consistent with their respective resistance profiles. Notably, our ABZ findings align with previous reports indicating that C. elegans displays limited sensitivity to ABZ⁵⁴. Remarkably, all three strains showed lower EC₅₀ values when treated with apigenin and quercetin, with both flavonoids demonstrating greater potency than ABZ (Table 1). Apigenin and quercetin, a member of the flavonoid family, are present in a wide variety of plant-derived foods such as oranges, lemons, berries, onions, apples, or black tea^17,55. Apigenin has a wide range of reported biological effects including antioxidant, cancer chemopreventive, antihypertensive, anti-inflammatory, antimicrobial and antiprotozoal activities^15,56. Dietary quercetin consumption has been associated to different health benefits, including anticancer, anti-inflammatory, antioxidant, and neuroprotective activities^55,57,58. According to previous reports, the crude extract of Artemisia campestris, which contains apigenin and quercetin, exhibited anthelminthic activity against both the eggs and adult stages of Haemonchus contortus⁵⁹. In both in vitro and in vivo studies, apigenin significantly enhanced the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae, including mcr-1-positive strains⁶⁰. Quercetin has been reported to be effective against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum by targeting the lactate dehydrogenase active site⁶¹. In addition, quercetin induced mortality and ultrastructure changes in T. canis third-stage larvae compared with the control and ABZ-treated groups³³.

HepG2 is a widely used hepatic cell line which is utilized in various research areas, including liver oncogenesis and the evaluation of substance-induced cytotoxicity⁶². The human Caco-2 cell line is widely used as an in vitro model of the intestinal epithelial barrier that is mainly used for assessing the bioavailability of drugs or test compounds⁶³. This study found that the orange flavonoid exhibited CC₅₀ values comparable to those of the anthelmintic agents ivermectin (IVM) and albendazole (ABZ) in both HepG2 and Caco-2 cells (Table 2). In clinical trials, ABZ treatment induced hepatitis in infant and adult humans by elevating liver enzymes^64,65. Case studies report IVM-induced liver injury as hepatitis, hepatocellular injury, cholestasis, increased levels of alanine aminotransferase and aspartate aminotransferase, and abnormal liver function tests^66,67. Furthermore, there have been reported that IVM-treated rats show thinner circular and longitudinal muscular layers in the small intestine, which can result in slower intestinal transit times⁶⁸. Interestingly, quercetin showed the highest CC₅₀ value among the orange flavonoids in both HepG2 and Caco-2 cells (Table 2). This suggests that quercetin is least likely to induce hepatotoxicity or intestinal cytotoxicity compared to apigenin, ABZ, and IVM at the same concentration. Previous studies have demonstrated that quercetin exhibits hepatoprotective activity through several mechanisms such as antioxidant, anti-inflammatory, anti-apoptotic, and lipid metabolism regulation^69,70. Dietary quercetin enhances the intestinal barrier function through the assembly of tight junction proteins and the expression of claudin-4, which results from inhibition of a protein kinase C isoform⁷¹. Furthermore, dietary quercetin could alleviate inflammatory damage, maintain intestinal barrier function, and modulate gut microbiota in lipopolysaccharide-induced laying hens⁷². Similar to our study, mice treated with the high dose of apigenin showed liver damage through oxidative stress^73,74.

In the T. spiralis assay, quercetin demonstrated a lower EC₅₀ than apigenin and exhibited potency within the same range as ABZ. (Table 2). According to the flavonoid structures (Supplementary Fig. 1), glycosylated flavonoids such as naringin are bulkier and exhibit lower bioavailability due to reduced membrane permeability, while methoxylated derivatives like tangeretin are highly lipophilic and form fewer hydrogen bonds with biological targets. In contrast, quercetin’s intermediate polarity facilitates efficient membrane diffusion while retaining sufficient hydrogen-bonding capacity for strong interactions with enzyme active sites and other biomolecular targets. This balanced physicochemical profile likely enhances its absorption, distribution, and binding affinity within nematode systems, thereby contributing to its pronounced anthelmintic efficacy. The molecular effect of quercetin on the T. spiralis has not been previously investigated, although the anthelmintic effect of quercetin has been demonstrated in other helminth infections. Quercetin showed cestocidal activity against Hymenolepis diminuta, with an EC₅₀ of 12.03 mg/mL after 2 h exposure. Electron microscopy revealed extensive damage to the suckers and neck region, as well as deformed, shrunken, wrinkled proglottids in treated worms²⁷. Quercetin showed anthelmintic activity against all of the development stages of Haemonchus contortus though generation of oxidative stress¹⁰. Moreover, quercetin against Toxocara canis, with exposure at LC₉₀ (0.55 mM) causing marked body shrinkage, swelling of the cuticle and caudal papillae, and erosion of cuticular annulations under electron microscope⁷⁵. However, the EC₅₀ of quercetin did not cause ultrastructure changes across the cuticular layer, muscular area, or coelomic space in T. spiralis. In addition, the expression levels of TOM20, SDHD, and BAX were not significantly different between the quercetin-treated and control groups (Figs. 1, 2 and 3). Consistent with previous study, no ultrastructure changes were observed despite the ability to eliminate the parasites⁷⁶.

Metabolomic analysis revealed that the most up-regulated metabolite following quercetin treatment was N-acetyl-D-galactosamine (GalNAc), the initiating sugar in most O-linked glycans present in helminth glycoproteins⁷⁷. O-linked glycans are particularly abundant in the muscle-stage larvae (L1) of T. spiralis⁷⁸. Surface carbohydrates in helminths play crucial roles in adhesion to host cells, immune evasion, and host–parasite communication^79,80. Drug-induced damage to the parasite’s surface coat can lead to cleavage of GalNAc-rich glycoproteins and glycolipids, releasing GalNAc into the surrounding environment or cytosol. Therefore, Thus, the observed upregulation of GalNAc likely reflects a quercetin-induced disruption of surface glycan structures, which compromises essential cellular interactions and contributes to nematode death. Lignoceric acid, linolenic acid, and linoleic acid were also up-regulated in T. spiralis following quercetin treatment. Helminth fatty acids are vital components of membrane lipid composition⁸¹. The observed up-regulation likely represents a parasite stress response, as antiparasitic drugs can compromise membrane integrity either directly or indirectly, triggering increased synthesis of structural fatty acids such as the very-long-chain saturated lignoceric acid and polyunsaturated fatty acids like linoleic and linolenic acids to restore or strengthen damaged membranes. However, prolonged metabolic imbalance in lipid remodeling can disrupt membrane homeostasis, ultimately leading to loss of cellular integrity and parasite death. The most down-regulated metabolites following quercetin exposure were inosine and deoxyguanosine. Inosine and other small molecules have been identified in helminth parasites⁸². Inosine, a purine nucleoside, is generated either through adenosine degradation or via 5′-nucleotidase activity on inosine monophosphate (IMP)⁸³. IMP is a central precursor for guanosine monophosphate, which is subsequently converted to deoxyguanosine monophosphate, an essential DNA building block⁸⁴. Starvation for even a single DNA precursor can be sufficient to induce cell death⁸⁵. Therefore, the observed depletion of both DNA precursors may contribute to the parasite death, induced by quercetin treatment.

According to pathway analysis, quercetin’s effect on the RORA–circadian system pathway may contribute to parasite death by disrupting key regulatory processes that maintain parasite homeostasis. RORA, through its control of circadian rhythm genes such as BMAL1, coordinates metabolism, stress responses, and immune modulation^86,87. In parasites, circadian regulation influences essential functions including nutrient utilization, replication timing, and adaptation to host immune cycles⁸⁸. Therefore, quercetin-induced alterations in the RORA–circadian signaling pathway could impair metabolic synchronization and weaken the parasite’s ability to adapt to host immunity, ultimately leading to cellular dysfunction and death.

Quercetin’s impact on the PPARA pathway may contribute to parasite death by disrupting the tight link between energy metabolism and stress adaptation. PPARA is a transcription factor that regulates genes involved in glucose and lipid metabolism as well as inflammation-modulating pathways⁸⁹. In parasites, efficient lipid utilization and metabolic flexibility are essential for growth, reproduction, and adaptation to host environments. By altering PPARA activity, quercetin may impair fatty acid oxidation, disrupt energy balance, and reduce the availability of key lipid-derived structural components for membranes. Additionally, modulation of inflammation-related pathways could shift the host–parasite interaction toward conditions that favor immune clearance. In Leishmania infections, for example, activation of PPARγ by curcumin suppresses IFNγ and nitric oxide production, thereby increasing parasite burden⁹⁰. Together, these metabolic and immune disruptions can compromise parasite survival and lead to death.

Quercetin was found to affect sterol regulatory element-binding proteins (SREBPs). The SREBPs are key transcription factors that control the expression of genes involved in lipid metabolism⁹¹. Lipids are important components of the cell and organelle membrane of T. spiralis. They act as signaling molecules in the intracellular information transmission of parasites⁹². The transport and utilization of fatty acids is critical for their survival and proliferation within the host. A previous study indicated that long-chain fatty acid transport protein 1 (FATP1) plays an essential role in lipid metabolism, moulting, and the development of T. spiralis⁹³. Another study found that C. elegans treated with alpha-linoleic acids showed increased lifespan via activation of the NHR-49/PPARα and SKN-1/Nrf2 transcription factor⁹⁴. Helminths tend to concentrate linoleic acid series in their tissues relative to host serum, suggesting selective synthesis or accumulation to regulate membrane composition during growth⁹⁵. Lipid metabolism enzymes have been proposed as “chokepoint” for helminth survival and potential targets for anthelmintic drug discovery⁹⁶.

Quercetin was found to influence the mitochondrial biogenesis pathway, the process of generating new mitochondria, which is essential for repairing tissue damage and restoring cellular energy by replacing impaired mitochondria⁹⁷. Given that mitochondrial injury can disrupt energy production, activation of this pathway may represent a stress response to quercetin treatment. Notably, depletion of TbTob55, a protein involved in mitochondrial biogenesis, has been shown to cause death of Trypanosoma brucei at the procyclic stage⁹⁸, suggesting that quercetin’s impact on this pathway could contribute to parasite lethality.

Although this study successfully demonstrated the anthelmintic effects of quercetin against T. spiralis through the disruption of multiple key metabolic pathways, several limitations should be acknowledged. First, quantitative evaluation of immunogold labeling based on gold particle density measurements was not feasible due to inherent technical variability in sample preparation, section thickness, and antibody accessibility within T. spiralis larvae. To ensure interpretative accuracy, the immunogold TEM analysis was therefore performed qualitatively, emphasizing the presence, spatial distribution, and relative density of gold particles rather than absolute quantification. Second, the absence of in vivo validation limits the assessment of the compound’s efficacy and safety within a physiological context. Third, pharmacokinetic constraints, including variability in absorption and metabolism, may influence quercetin’s bioavailability and therapeutic potential. Notably, quercetin exhibits poor oral bioavailability (< 5%), while the observed EC₅₀ values (45–146 µM) may exceed achievable plasma concentrations. Fourth, the mechanistic investigations were conducted at a single concentration, which may not fully capture the dose–response relationship under in vivo conditions. Finally, combination therapy was not explored in this study, although it could provide valuable insights into potential synergistic effects. These limitations should be carefully considered and addressed in future research to strengthen the translational relevance of the findings.

In conclusion, this study demonstrates that quercetin have profound anthelmintic effects against T. spiralis through multiple pathways, including inflammation, lipid metabolism, and mitochondrial biogenesis. Interestingly, quercetin exhibited no cytotoxicity effects on the human cell. Taken together, quercetin can be a lead molecule for the development of a new drug for the treatment of T. spiralis and its associated complications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

K.S. wrote the original draft, conducted review and editing, and performed investigation, validation, formal analysis, and data curation. N.N. contributed to review and editing and carried out investigation. P.A. provided resources, contributed methodology, and performed investigation. N.U. conducted investigation, formal analysis, and visualization. K.F. performed investigation, visualization, and formal analysis. T.K. carried out investigation and visualization. S.A. provided resources and contributed methodology. T.T. contributed methodology and performed investigation. P.T. contributed methodology and investigation. J.T. contributed methodology and investigation. O.R. contributed to review and editing, supervised the work, provided resources, oversaw project administration, contributed methodology, secured funding, and conceptualized the study. All authors reviewed the manuscript.

Funding

This research project is supported by Mahidol University (MU’s Strategic Research Fund: 2023) to O.R. This research project was also supported by postdoctoral fellowships awarded by Mahidol University to N.N. and O.R.

Data availability

All the mass spectrometry data have been deposited in the Science Data Bank repository with accession number 10.57760/sciencedb.27176 (https://www.scidb.cn/en/anonymous/VnJ1WUZi).

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Animal experiments were approved by the Faculty of Tropical Medicine Animal Care and Use Committee (FTM-ACUC 032/2020), Mahidol University. All animal experiments were conducted in accordance with ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations.