Caenorhabditis elegans is often used as a model organism for anthelmintic phenotypic screens. This nematode is favoured due to its ease of cultivation, high fecundity, and low maintainance cost. Moreover, recent worm motility phenotype screening campaigns employing the WMicrotracker instrument have demonstrated the combined utility of this model organism and high-throughput platform in natural products anthelminthic research.
Alpine cultivated plants, predominantly members of the Asteraceae family, have recently demonstrated anthelmintic activities in vitro and could be considered as alternative natural sources for deworming purposes. Liquid–liquid fractions were prepared from a methanolic extract of the aerial parts and were submitted in parallel to embryo development (ED), worm motility (WMT), and cytotoxicity assays for anthelmintic and toxicity evaluations. The objective of this study is twofold: (1) exploration whether the C. alpina extract and its subsequent fractions possess dual anthelmintic activity by targeting multiple helminth life cycle stages (i.e., worms, eggs); (2) annotation of the active principle(s) to provide the basis for future quality control. To achieve these goals, we also worked on the implementation of a fast and orthogonal screening method based on the cultivable helminth C. elegans.
Methods
Anthelmintic activity evaluation experiments
Anthelmintic worm motility assay (WMT) with C. elegans.
The wild-type C. elegans N2 Bristol strain was obtained from the Caenorhabditis Genetics Center. The anthelminthic screening assay was performed in a 96-well microplate (flat-bottom). Synchronised adult worms were collected in S-medium (V = 8–12 mL) with the suspension volume adjusted until the average number of worms was calculated (around 35–50 worms/per 100 μL, V ~ 10 mL). A liquid culture was prepared from the worm suspension by adding OP50-1/S-medium (cstock = 200 mg/mL, V = 184 μL) in (OD600= 0.6). Then, the final liquid culture suspension (V = ~ 10 mL) was distributed across the 96-well microplate (V = 100 μL per well). The worms/plate were left to shake gently on a wave shaker (t = 20 min, frequency = ~ 10 rpm, waving motion) and after, the microplate was submitted to the WMicrotracker (t = 30 min) to measure the basal worm motility. Each extract, fraction, or pure compound stock solution was prepared in DMSO and further diluted with S-medium. Subsequently, each diluted test solution or controls were added to the desired wells (V = 20 μL each) reaching the final concentrations of c = 150, 100, 50 μg/mL, or -where insoluble- of c = 75, 50, 25 μg/mL with DMSO (1%). Ivermectin (cfinal = 0.1 μM, in 1% DMSO) was used as a positive control and the vehicle negative control was DMSO (1%, v/v). After shaking (t = 2 min, frequency = ~ 10 rpm, waving motion), the 96-well microplate was placed into the WMicrotracker and incubated (up to t = 12 h, T = 24 °C). Worm motility (WMT) within each well was measured every thirty minutes and motility recorded as activity counts per defined time intervals by the WMicrotracker. The relative WMT anthelminthic activity of the test samples was estimated by normalising with average worm motility shown over four hours.
Anthelmintic egg hatch assay (EHA) with C. elegans
Worms were chunked from a stock plate containing a mixed population of N2 Bristol worms and transferred to a freshly prepared OP50-1 seeded NGM plate and left to incubate until the development of large numbers of gravid adults and eggs were visibly laid on the plate. Next, eggs were harvested and isolated by bleaching. The eggs were collected in S-medium and the volume was adjusted until an average of ~ 150 embryos/100 μL was counted. Thereafter, the egg/S-medium suspension (V = 100 μL) was distributed to the wells of a 96-well microplate (flat-bottom) and the positive control albendazole ( cfinal = 10 μM) or test sample was added (V = 20 μL). Next, the plate was gently shaken (t = 2 min, frequency = ~ 10 rpm, waving motion) and then placed into the WMicrotracker and incubated (t = 24 h, T = 24 °C). Egg hatching was monitored by the mean number of beam interruptions during the experiment.
Results
Worm Motility Assay with C.elegans
A 1-to-4-h time interval was chosen as a suitable time point range for screening C. alpina fractions for effect on the worm motility phenotype. Overall, this confirmed the validity of the motility device and suitability of ivermectin as a positive control as with previous insights. The preliminary worm motility results (SI, Sect. 2.1.1, Figure S2) revealed the parent MeOH extract (c = 150 μg/ml) to have a mild effect compared to the vehicle control between 1.5–3 h, with its highest motility reduction observed at two hours with 67.4% worm motility.
Next, soluble fractions (EtOAc, DCM, BuOH) of the MeOH extract were tested at c = 150 μg/ml. The apolar PE fraction was omitted due to solubility limitations under the test conditions (1% DMSO). From the results (SI, Sect. 2.1.1, Figure S2), the DCM fraction was considered inactive with 92–100% worm motility. The EtOAc fraction had a mild effect (36% reduction) on motility over 1.5–3 h. The BuOH fraction had the strongest effect on C. elegans adult worm motility with an average of 49% worm motility between 1.5–3 h (Fig. 3). Moreover, the BuOH fraction showed a slight time-dependant effect with a gradual decrease in motility observed from 1 h (WMT = 64.8%) to 2.5 h (WMT = 41.7%). As the most active sample in the worm motility assay, the BuOH fraction was subsequently tested at different concentrations (c = 50, 100, and 150 μg/ml) and showed worm motility inhibition in a concentration dependent manner (SI, Sect. 2.1.1, Figure S3).
Egg hatch assay with C. elegans
To further gain insight into the active phytochemical principle of the most interesting fractions (i.e., the non-cytotoxic and anthelminthic BuOH fraction as well as the anthelmintic EtOAc fraction) eight subfractions of the BuOH fraction (B1–B8) and thirteen EtOAc subfractions (E1–E13) were obtained via silica-gel flash column chromatography (FCC). Additional eight subfractions derived from E3 (E3.1–E3.8) were obtained with size exclusion chromatography (SEC).
To determine whether the observed embryo development inhibition effect of luteolin (E3.8) is not only limited to the ascarid A. galli eggs but ubiquitously active across other species of helminths, a complimentary assay measuring C. elegans egg hatching (% EH) with the WMicrotracker was employed. The anthelmintic albendazole (c = 10 μM) and DMSO (1%) were used as the positive and negative controls, respectively. The egg hatching assay results are presented below in Fig. 5. Albendazole (c = 10 μM) proved to be a suitable positive control and clearly demonstrated egg hatch inhibition over time (t = 10 h | 47.2% EH). When luteolin (c = 1 μM) was tested at the comparable concentration to the embryo development assay, little effect on C. elegans hatching was observed. However, the flavonoid did exert an anthelmintic effect when treated at a tenfold higher concentration (c = 10 μM), also demonstrating its egg hatch inhibition effect around 9–12 h (t = 10 and 12 h | 65.4 and 75.3% EH) compared to the DMSO control. Significance determined by unpaired t-test (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, n.s = not significant).
In this study, natural products derived from a C. alpina methanol extract demonstrated in vitro anthelmintic activity against two helminth species, A. galli and C. elegans. The compounds luteolin and dihydrolactucin were isolated and demonstrated as two of the major active principles responsible for the observed bioactivity.
Sci Rep. 2025 Feb 3;15(1):4108. doi: 10.1038/s41598-024-73958-9. PMID: 39900941; PMCID: PMC11791071.
Horgan MJ, Sigg I, Poulopoulou I, Rodriguez-Mejias FJ, Albertini E, Fusani P, Fischer F, Martinidou E, Schuster D, Martens S, Dürr PJ, Gauly M, Stuppner H, Weiss A, Temml V, Siewert B.