Introduction

Trichinellosis is a zoonotic parasitic disease caused by nematodes of the genus Trichinella [1]. Consuming raw or undercooked pork containing Trichinella infective larvae is the transmission route for various mammals [2]. T. spiralis is a parasite that completes its biological cycle in the same host; hence it is frequently used as a model to determine the efficacy of various anthelmintic drugs [3].

The primary anthelmintic drugs utilized in the clinical management of trichinellosis are benzimidazole derivatives. However, they have numerous drawbacks [4, 5], as none of these drugs is powerful enough to kill encapsulated and new-born larvae [6] due to their low bioavailability [7], and the development of drug resistance [8]. Likewise, most of them are contraindicated in pregnant women and children below two years of age [3]. Therefore, scientific research aims to discover a novel, secure, and effective anthelmintic agent against T. spiralis. In addition, drug repurposing has recently emerged as a tool for developing new indications for existing, failed, or abandoned drugs [9].

Nifedipine is a calcium channel blocker primarily used as an antihypertensive and antianginal medication. It inhibits the transmembrane influx of calcium into cardiac and smooth muscles affecting their contractility [10]. Nematode muscle cells are similar to smooth muscles, with an important distinguishing feature dependent on extracellular calcium for contraction [11]. On the other hand, there is a growing need to find new therapeutic natural agents as a simple replacement for synthetic chemical agents [12, 13]. The Chrysanthemum genus belongs to the Asteraceae family, with nearly 300 species. It has undergone extensive research on the biological functions and chemical composition of its species. For instance, C. morifolium, a popular dietary supplement in China, has been shown to exhibit antihepatotoxic and antigenotoxic effects [14, 15]. Other effects include anti-inflammatory, humoral, and cellular immunomodulatory [16]. Additionally, the flowers were demonstrated to have insecticidal and herbicide effects [17]. Moreover, C. coronarium has been tried as an antibacterial agent with promising results [18]. This study aims to assess the in vitro effects of nifedipine, a calcium channel blocker, and a methanol extract of flowers of C. coronarium as anthelminthic agents for different developmental stages of T. spiralis in comparison with the reference drug, albendazole.

Materials and methods

Parasite

Trichinella spiralis was kindly provided by the Parasitology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt, from laboratory-bred infected Swiss albino mice. Mice were orally infected with 200–300 T. spiralis larvae and maintained following the institutional and national guidelines in the animal house exposed to 12 h light/12 h dark cycles with a free access to standard pelleted diet) Delta group, Egypt) and water ad libitum [19].

Isolation of adult worms and muscle larvae

The collection of adult worms was typically performed at five days post-infection (dpi). Ten infected mice were sacrificed under light anesthesia by isoflurane inhalation (Forane®, UK). Then, the intestines of infected mice were removed and opened with scissors. The opened intestines were gently washed in phosphate-buffered saline (PBS) to remove the intestinal contents. Afterward, the small intestines were cut into 2 cm sections, slit longitudinally, and placed on a gauze in a beaker containing 250 ml of PBS for 3 h at 37 °C [20].

In addition, muscle larvae (ML) were obtained from infected mice on day 35 post-infection. Then, each mouse was dissected, and the muscles were digested in 200 ml of distilled water with 1% concentrated HCl and 1% pepsin. The mixture was continuously stirred with an electric stirrer for two hours at 37 °C. On a 200-mesh/inch screen, the larvae released from cysts were collected, washed twice in tap water, and then suspended in a conical flask in 150 ml of tap water. Finally, the supernatant fluid was removed, and a McMaster counting chamber was used to count the larvae in the sediment under a microscope [21].

Extraction and preparation of C. coronarium

Chrysanthemum coronarium fresh flowers were collected from El-Fayoum Governorate, Egypt, in January 2021. Botany specialists from the Department of Flora and Taxonomy, Faculty of Science, Alexandria, Egypt, established the identification and authentication of the collected plant. A voucher specimen number (C.c.f.2021) was deposited in the Medicinal Chemistry Department, TBRI. C. coronarium air-dried flowers (0.5 kg) were extracted four times via maceration using methanol (4L) at room temperature (22–26 ºC). The extract was concentrated via a rotary evaporator (BUCHI R-300, Switzerland) at 40 ºC to obtain 46 g of methanol extract. The extraction yield was equal to 9.2% calculated by the following equation: Yield (%) = Dry extract weight/ dry powder weight × 100 [22].

Identification of the binding site of nifedipine to T. spiralis β-tubulin

Template-based model construction

The FASTA formatted amino acid sequence of T. spiralis β-tubulin was obtained from the NCBI GenBank database (GenBank: EFV50889) [23]. The I-TASSER service (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) was used to identify binding sites and templates for this sequence [24]. Due to the presence of amino acids associated with nematode resistance in the binding site discovered by I-TASSER, the D chain, which is present in the b-subunit of the heterotetrameric structure of Ovis aries (PDB ID: 3N2G D), was selected as a template [25]. T. spiralis β-tubulin residues 1–428 were used in the homology modeling method conducted with MODELLER 9v10 software [26].

Deep learning-based model construction

The Alphafold-2 deep-learning-based technique was used to create another model for T. spiralis β-tubulin [27]. Then, ten models were created, and the one with the lowest energy was selected for optimization. After that, a series of molecular dynamics simulations were carried out to refine the template-based and Alphafold-2-based models.

Molecular dynamic simulation

We essentially followed the iterative technique that had previously been reported. We simulated each generated model for 50 ns at 310 K, and ran five different trajectories. During the scoring process, we utilized RWplus [28].

Docking Study

The modeled structure of nifedipine was docked into the predetermined binding site in model-generated homology modeling using AutoDock Vina. The docking grid-box's coordinates were set to be: x = 115.43, y = 90.11, z = 7.90. The ligand to binding site shape matching root means square (RMSD) threshold was set to 2.0 Å. The interaction energies were determined using the Charmm force field (v.1.02) with 10.0 Å as a non-bonded cutoff distance and distance-dependent dielectric. Then, 5.0 Å was set as an energy grid extending from the binding site. The tested compound retinol was energy minimized inside the selected binding pocket. The editing and visualization of the generated binding poses were performed using PyMOL software [29].

Drug concentrations

Different concentrations of albendazole, nifedipine, and C. coronarium extract were investigated. Albendazole (Pharco Pharmaceuticals, Egypt) stock solution was prepared in 1% dimethyl sulfoxide (DMSO) at 400 µg /ml concentration. Then, serial dilutions were carried out to obtain 200, 100, 50, and 25 µg/ml concentrations [30]. Different concentrations of nifedipine (25, 12.5, 6.25, 3.125, 1.56, 0.78 µg/ml) were prepared from Epilat capsules (Epico, Egypt). Next, C. coronarium (400 µg/ml) was prepared by dissolving 400 µg of the extract into one ml of distilled water. After that, serial dilutions were conducted from this preparation to obtain 200, 100, 50, and 25 µg/ml concentrations.

Experimental design

All experiments were carried out in a sterile 24-well tissue culture plate (SoCal BioMed, USA). Fifty muscle larvae or 25 adult worms were incubated in 2 ml of Rapid Prototyping and Manufacturing Institute medium (RPMI)-1640 (Lonza, Belgium) containing 10% fetal calf serum, 200 U/ml penicillin, 200 μg/ml streptomycin (Omega Scientific, USA), and the desired drug concentration [31]. Negative control (blank) containing only the parasite (adult worms or ML) in pure culture media or DMSO control were included in all assays and subjected to the same conditions as the experimental cultures. Then, the plates were sealed and incubated at 37 °C in an atmosphere containing 5% CO2 for 1, 6, 24, 48, and 72 h. The observations were prolonged to 96 h for the ML. The experiments were carried out in duplicates, and data were compared with the DMSO control for albendazole and the corresponding blank control for nifedipine and C. coronarium. The efficacy of the studied drugs was evaluated by parasitological and scanning electron microscope examination [19].

Parasitological studies

The viability of Trichinella stages cultured in vitro in different concentrations was evaluated by assessing their shapes and mobility. They were counted either alive or dead. The average value of the two experiments was calculated [19].

Scanning electron microscope (SEM) examination

T. spiralis adult worms or ML were directly pipetted into a fresh fixation solution of 2.5% glutaraldehyde (w/v) in 0.1 M sodium cacodylate at pH 7.2 and left overnight at 37 °C. Trichinella stages were washed in 0.1 M sodium cacodylate buffer at pH 7.2 for 5 min, post-fixed in a 2% (w/v) osmium tetroxide in sodium cacodylate buffer for one hour. The specimens were dehydrated in an ethanol series and dried using liquid carbon dioxide. The dried parasite stages were sprinkled onto and mounted on stubs bearing double sided carbon adhesive tape. Then, samples were coated with gold using sputter coater (EIKO Engineering CO, Japan), and examined by SEM (Jeol Corp., Japan) at 5 to 20 kV in the scanning electron microscopy lab, at the Faculty of Science, Alexandria University, Egypt [32].

Statistical analysis

Data were presented as the mean ± standard deviation (S.D.). The results were analyzed using IBM SPSS software package version 20.0 (IBM Corp, USA) [33]. The student's t-test analyzed the significance of the differences between the experimental and the control groups. Moreover, the one way ANOVA and post hoc Tuky HSD with Bonferroni correction tests were applied for multigroup comparisons using the Rstatix package in Rstudio (R version 4.2.3). P-value < 0.05 was significant, and P-value < 0.001 was highly significant. All graphs were generated by the ggplot2 package. The half-lethal concentration (LC50) at 48 h after incubation with different drugs was calculated based on the Quest Graph online program https://www.aatbio.com/tools/ic50-calculator.

Results

Homology modelling and binding site prediction

Due to the lack of a crystallographic structure for T. spiralis β-tubulin, we generated a homology model by mining the known repertoire of helminth resistance mutations for a suitable template [34]. The structure of T. spiralis β-tubulin was generated by the O. aries β-tubulin (PDB ID: 3N2G; D subunit) as a starting point. There were 406 completely identical places and 18 partially identical positions aligned with the template and target sequences. With the I-TASSER platform, MODELLER software, and the PDB ID: 3N2G; D subunit as a starting point, we could create a 3D model of the T. spiralis β-tubulin (Fig. 1).

Fig. 1
figure 1

Structural alignment of two generated T. spiralis β-tubulin models showing the ligand-binding site. The cyan-colored structure is the Alphafold2-generated model, and the brick-red-colored structure is the template-generated model (RMSD = 1.16 Å)

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For more confirmation, the β-tubulin sequence was also subjected to Alphafold-2 to build another 3D model using a different method (i.e., deep neural networking-based de novo model construction) [2]. The resulting best-scoring and the first template-based models were subjected to 50 ns-long M.D simulations to relax the generated structures. In addition, the most populated structures for each model from each trajectory were extracted and compared. Upon alignment of the generated structures to each other, they were almost identical (RMSD = 1.16 Å). The Ramachandran plot analysis showed a reliability of 96.7% and placed all the amino acids corresponding to the co-crystallized ligand's binding site inside the permissible zones (Fig. 2). This model is high quality enough to be used in molecular docking. Figure 1 reveals that Phe167, Glu198, and Phe200, three of the most significant amino acids in helminth resistance, form the binding pocket [1]. Near the monomer–monomer interface of the heterodimer (in the N-terminal domain of the B monomer), the proposed binding site in β-tubulin models consists of several highly conserved hydrophobic amino acids (Leu240, Leu250, Leu253, and Phe266), and a few hydrophilic residues (Thr237, and Asn256).

Fig. 2
figure 2

The Ramachandran plot highlights the amino acids' preferred and forbidden zones

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Molecular docking

To investigate their binding modes, the O. aries β-tubulin co-crystallized inhibitor and nifedipine structures were docked into the generated T. spiralis β-tubulin model (i.e., the template-based one). The structure of colchicine, a well-known β-tubulin inhibitor, was also docked as a reference inhibitor. The three ligands got comparable docking scores (-7.67, -7.54, and -7.41 kcal/mol, respectively).

Figure 3 shows that hydrophobic interactions are the key to the three ligands. The binding mode of the nifedipine structure was more convergent to that of the colchicine than that of the O. aries β-tubulin co-crystallized inhibitor, where they established hydrophobic interactions with LEU-248, LEU-255, MET-259 (Figs. 3A and C). Accordingly, it can be concluded that nifedipine might act as a T. spiralis β-tubulin polymerization inhibitor.

Fig. 3
figure 3

Binding modes of nifedipine inside the T. spiralis β-tubulin's binding site alongside the two reference inhibitors (i.e., O. aries β-tubulin co-crystallized inhibitor and colchicine) (A-C, respectively)

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The effect of the studied drugs

The studied drugs showed a remarkable parasiticidal effect on T. spiralis. The effects on muscle larvae are shown in Table 1. At 25 µg/ml, albendazole showed a statistically significant effect after 96 h. It also killed all larvae after 48 h in concentrations starting at 100 µg/ml. The statistically significant larvicidal effect was evident at a concentration of 100 µg/ml and above from the first hour of exposure. Moreover, the LC50 of albendazole was calculated to be 81.25 µg/ml. As regards nifedipine, a statistically significant larvicidal effect was obtained at a concentration of 0.78 µg/ml after 4 h. In addition, it killed all larvae after 72 h in concentrations starting from 3.125 µg/ml. The LC50 of nifedipine was calculated to be 1.24 µg/ml. C. coronarium killed 100% of larvae at a high concentration (400 µg/ml) after 96 h and it needed at least 48 h to exert its effect on incubated ML. A statistically significant difference was recorded initially at 200 µg/ml after 48 h (P ≤ 0.05). Then, the LC50 was calculated to be 229.48 µg/ml.

Table 1 Survival numbers of T. spiralis muscle larvae incubated with different concentrations of studied drugs (n = 50)
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Table 2 shows the effects of the tested drugs on adult worms. At low doses of 25 and 50 µg/ml, albendazole showed a non-statistically significant effect even after 72 h. The statistically significant effect was evident starting from 4 h of exposure at 100 µg/ml (P ≤ 0.05) or higher concentrations (P ≤ 0.01). Albendazole killed all worms after 24 h in concentrations starting from 200 µg/ml. The LC50 of albendazole was calculated to be 89.77 µg/ml. Regarding nifedipine, a statistically significant effect was obtained at a concentration of 1.56 µg/ml after 4 h (P ≤ 0.05). Nifedipine killed all worms after 4 h in concentration starting from 6.25 µg/ml. The LC50 of nifedipine was calculated to be 1.87 µg/ml. C. coronarium caused the death of all adult worms at high concentrations (200 µg/ml and 400 µg/ml) after 72 h. A statistically significant difference was recorded initially after incubation at 100 µg/ml for 72 h (P ≤ 0.05). The LC50 of C. coronarium was calculated to be 124.66 µg/ml.

Table 2 Survival numbers of T. spiralis adult worms incubated with different concentrations of studied drugs (n = 25)
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The effects of the studied drugs on muscle larvae and adult worms were dose (Fig. 4) and time (Fig. 5) dependant. However, incubating muscle larvae with different concentrations of C. coronarium does not affect their survival in the first 24 h. There was no significant difference between blank and DMSO controls at all test incubation periods. A comparison of the newly tried drugs, nifedipine and C. coronarium with the reference drug, albendazole at the closest concentrations to the calculated LC50 is shown in (Table 3). Statistically significant differences were recorded between the studied drugs and albendazole on muscle larvae and adult worms.

Fig. 4
figure 4

Proportions of T. spiralis ML and adult worms survival at the different concentrations of the studied drugs. Data normalization relative to the corresponding control was performed

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Fig. 5
figure 5

Proportions of T. spiralis ML and adult worms survival at different incubation times with studied drugs. Data normalization relative to the corresponding control was performed

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Table 3 The effect of nifedipine and C. coronarium on T. spiralis developmental stages compared to the reference drug, albendazole, 48 h after incubation
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Scanning electron microscope findings

SEM examination of the tegument of T. spiralis ML from the control groups showed the typical coiling behaviour and the characteristic pattern of normal tegument with longitudinal ridges and transverse creases. Conversely, tegument destruction in the treated groups was apparent since multiple degenerative changes, such as the appearance of blebs, multiple vesicles, and loss of normal annulation (Fig. 6). This destruction was especially evident in the subgroup treated with albendazole.

Fig. 6
figure 6

SEM findings of the cultured T. spiralis muscle larvae after 48 h incubation. A, B: blank control group showing normal cuticle with transverse creases and longitudinal ridges, C, D: albendazole (100 µg/ml) treated group, E,F: nifedipine (1.56 µg/ml) treated group, G, H: C. coronarium extract (200 µg/ml) treated group

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Examination of adult worms by the SEM revealed that the tegument of T. spiralis adult worms from the control group showed normal cuticles with hypodermal glands opening and tapering ends. In contrast, the incubation of adult worms with either albendazole or nifedipine caused marked destruction of the adult worms. The tegument showed areas with marked swellings, multiple large blebs, fissures, and vesicles. In addition, sloughing of some areas of the tegument was observed. C. coronarium treated groups showed body collapse as well as the appearance of areas of decreased opacity (Fig. 7).

Fig. 7
figure 7

SEM findings of the cultured T. spiralis adult worms after 48 h incubation. A, B: blank control group showing normal adult worm cuticle with hypodermal glands openings and tapering end, C, D: albendazole (100 µg/ml) treated group, E,F: nifedipine (1.56 µg/ml) treated group, G, H: C. coronarium extract (200 µg/ml) treated group

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Discussion

T. spiralis is a worldwide nematode infecting various mammalian hosts, including humans, with possible serious complications [35]. The limitations of benzimidazole derivatives to treat the parasite's encapsulated larval stages, in addition to the negative drug reactions, including mortality, encephalitis, convulsions, and severe drug eruptions, have motivated medical researchers to discover novel, safe, and efficient anthelminthic agents [2, 36, 37]. Repurposing the currently available drugs and the use of medicinal plants have emerged as two powerful strategies in the search for new treatment options.

In the present study, albendazole was used as the reference drug for the treatment of T. spiralis. The effect of albendazole was the strongest compared to other drugs. LC50 was nearly similar between muscle larvae and adults (104.21 µg/ml vs. 96.03 µg/ml). However, higher LC50 value against adult worms was reported by Kaiser et al. (> 200 µg/ml) against the adult worms. Also, Priotti et al. studied the effect of albendazole on adult worms and reported that worms' viability was 72% 48 h after incubation with albendazole.

Nifedipine is a potent calcium channel antagonist used to treat hypertension by blocking calcium input into endothelial cells, causing intense relaxation [38]. Nifedipine gained attention due to the discovery of its anti-parasitic properties, mainly against Plasmodium spp. and also against other parasites such as Schistosoma, Leishmania, and Microsporidia [39,40,41,42]. The results of the present in silico studies indicate that nifedipine might act as a T. spiralis β-tubulin polymerization inhibitor. Moreover, it has a lethal activity against muscle larvae and apostdult stages of T. spiralis in vitro. This remarkable effect was found to occur in a dose and time-dependent manner. The adult worms seemed more susceptible than muscle larvae since no viable adult worms were found after 4 h incubation with nifedipine at a concentration of 6.25 μg/ml. Similarly, Silva-Moraes et al. evaluated the effect of nifedipine on schistosomula, and adult worms cultures to provide new therapeutic strategies for Schistosoma mansoni treatment. Results displayed a significant antischistosomal effect even on the initial life cycle stages of the parasite [39]. Also, a previous study has evaluated the effect of the same drug on the filarial nematode; Acanthocheilonema viteae, and reported that it reduces calcium influx across the muscle membrane [43].

Nowadays, there is a broad consensus that diverse plant-derived products have inhibitory effects on many infectious agents. The chemical heterogeneity of the C. coronarium's composition was thought to be responsible for its numerous biological activities [44,45,46]. Though it has been shown to exhibit antibacterial, antifungal, and antiviral properties [18, 47, 48], not much is known about its antiparasitic activity. In the present study, C. coronarium showed a potentially lethal activity against T. spiralis muscle larvae and adult worms in vitro. The adults were more susceptible than the muscle larvae, as a lower LC50 after 48 h incubation was computed (124.66 µg/ml vs. 229.48 µg/ml). These results are in agreement with Bar-Eyal et al. [49], who found that adding C. coronarium to the soil as a green manure to suppress the root-knot nematodes Meloidogyne incognita and M. javanica was fatal. Other studies assessed the antibacterial properties of C. coronarium essential oil [48, 50]. These investigations showed that the essential oil has antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria, which goes in hand with previous literature reports that Gram-positive bacteria are more responsive to essential oil therapies than Gram-negative bacteria [51, 52].

Changes in the tegument of helminths are considered a good indicator for the possible anthelmintic activity of a drug [53]. Blebbing occurs as the parasite attempts to replace the damaged surface membrane in response to drug action. Albendazole disrupts the parasite's metabolic pathways, resulting in diminished ATP production, interfering with cell motility and maintaining cell shape [54, 55] In the present study, the ultra-structural effects of the studied drugs on T. spiralis were evident. Similar results were reported by Fahmy et al. [56], who applied clove oil (Syzygium aromaticum) against adults and muscle larvae of T. spiralis.

As far as we know, this is the first report presenting the results of the anthelmintic activity of nifedipine and C. coronarium against T. spiralis. Both drugs had remarkable lethal effects on adult forms and muscle larvae. However, one limitation of this study is that in vivo experiments are more useful than in vitro experiments, as many in vitro active drugs are inactive in living organisms. Therefore, further in vitro and in vivo studies should be performed to confirm the described findings and fully assess the antiparasitic efficacy of these drugs. HPLC–MS metabolite profiling of C. coronarium extract followed by bio-guided fractionation and isolation to purify its major components is strongly recommended in future perspectives.