Phytochemical Characterization, Molecular Docking Analysis, and In Vitro Antischistosomal Activity of Capsicum annuum Methanolic Extract

ABSTRACT

Human schistosomiasis is a neglected tropical disease affecting millions worldwide, highlighting the need for affordable alternative therapies. This study evaluated the in vitro antischistosomal activity of the Capsicum annuum methanolic extract against adult Schistosoma mansoni. Tegumental alterations were examined by scanning electron microscopy (SEM), while potential molecular targets were investigated using STITCH network analysis, molecular docking, and in silico pharmacokinetic prediction. Concentrations of C. annuum extract (20–320 µg/mL) and praziquantel (0.2 µg/mL) were evaluated for time‐dependent worm mortality at 1, 3, 12, 24, 48, and 72 h post‐treatment. Praziquantel induced 100% mortality within 24 h, whereas the dose (320 µg/mL) caused 50% mortality at 24 h and complete mortality at 48 h. Docking analysis suggested apoptosis‐related proteases and a Ser/Thr kinase as putative molecular targets that warrant further experimental validation. These findings demonstrate that C. annuum methanolic extract possesses promising antischistosomal activity against S. mansoni.

Crude methanolic extract of Capsicum annuum was phytochemically analyzed by HPLC to evaluate its antischistosomal activity against adult Schistosoma mansoni worms in vitro. The extract induced significant tegumental alterations, confirmed by scanning electron microscopy (SEM), indicating severe parasite damage. Molecular docking analysis demonstrated that the major identified phytochemicals exhibited favorable binding affinities toward key schistosome target proteins, supporting the observed biological activity and highlighting C. annuum as a promising natural source of antischistosomal compounds.

1. Introduction

Schistosomes, parasitic flatworms, are a major hazard to public health in many countries across the world. According to the World Health Organization (WHO), at least 253.7 million people required preventive treatment for schistosomiasis in 2024, of whom more than 100.5 million received treatment [1]. Schistosoma mansoni is a major trematode parasite that causes hepatointestinal schistosomiasis. Unlike other species that predominantly target the bladder or small intestine, S. mansoni adults often live in the inferior mesenteric veins, which drain the large intestine [2]. The host's prolonged inflammatory and granulomatous immunological reaction to the eggs trapped in tissues is what causes the disease's main clinical symptoms, not the adult worms themselves [3]. Clinical consequences of this pathology include hepatosplenomegaly (enlargement of the liver and spleen), bowel wall ulceration, and the development of esophageal varices due to the increased pressure in the portal circulation [2]. Lakes and rivers in East Africa provide perfect environments for snail intermediate hosts, making control efforts more difficult because snails are common in marshy and damp areas [4, 5]. Clerinx and Van Gompel [6] summarized the life cycle of Schistosoma as follows: adult worms migrate against the bloodstream to lay eggs in small veins by the female. Eggs are expelled with urine or feces. These eggs hatch in freshwater into miracidia, searching for their intermediate host snails. Cercariae, the final stage produced from asexual reproduction within snails, emerge from the snail hosts, penetrate human skin, and lead to disease development within 4–6 weeks. Chemotherapeutic interventions have been the cornerstone for managing this disease [7]. Since 1970, the preferred medication for treating Schistosoma species has been praziquantel (PZQ). This oral medication reduces the prevalence of schistosomiasis [8, 9]. Although PZQ is highly effective against adult worms, it has certain limitations, including reduced efficacy against juvenile stages (schistosomula) and the growing risk of drug resistance as a result of its widespread usage in mass drug administration programs [10]. Its mode of action is still poorly understood. It was suggested that the medication causes changes to the membranes, which result in the admission of Ca²⁺ into the muscle cells and paralysis when the muscle cells are squeezed, the bloodstream then carries away the paralyzed parasites [11]. The drug can only be used to treat the early stages of infection because it is mostly effective against the adult forms of the parasite [12]. Currently, the primary objectives of anti‐schistosomiasis drug research are the development of praziquantel analogues and the screening of compounds with therapeutic targets [13].

Many studies have examined the effectiveness of natural products against helminths [14, 15, 16, 17]. The discovery of natural products, particularly those derived from plants with schistosomicidal activity, represents a promising approach for developing new compounds to address this health problem [18]. Schistosomiasis‐associated liver fibrosis has been reported to be lessened by a variety of plant metabolites and extracts that target several pathways, such as lowering oxidative stress and inflammation, and worm egg burden, which are critical to disease pathology [13]. Capsicum annuum (chilli or pepper) has antiparasitic potential for its bioactive substances, including terpenoids and capsaicinoids, which disrupt parasite physiology [19]. The efficacy and phytochemical properties of Carcia papaya and C. annuum extracts against helminthiasis in chicken were assessed by Zirintunda et al. [19]. Scanning electron microscopy (SEM) is used as a vital diagnostic tool to monitor topographical alterations in the parasite's tegument in order to comprehend the physical impact of biological extracts [20, 21]. Eissa et al. [22] examined in vitro the biological activity of the drug Miltefosine against both S. mansoni and Schistosoma hematobium adult and larvae and emphasized their study by SEM. Abozeid et al. [14] and Rashed et al. [23] studied the in vitro antischistosomal activity of Allium sativum, Curcuma longa, and Punica granatum, respectively, on S. mansoni, and supported their study by SEM. Fadladdin [20] evaluated the effectiveness of three plant extracts, Artemisia annua, A. sativum, and Nigella sativa on S. mansoni in vitro.

Molecular docking study revealed potential interactions between the target protein of S. mansoni and bioactive components from plant extracts. These interactions point to possible therapeutic benefits that might be investigated further through experimentation, directing the creation of novel therapies [24]. Few molecular docking experiments were conducted to determine the molecular targets of natural substances that provide helminth protection. Mahamba et al. [25] identified the potential inhibitors of G4IZI3 and suggested candidates for additional experimental verification. Employing computational tools. Paul et al. [26] used molecular dynamics simulation to determine the possible anthelmintic activity of syringaldehyde in pineapple. Additionally, Morales‐Bayuelo et al. [27] employed molecular docking to find possible Plasmodium drug targets. The present study aims to evaluate the in vitro antischistosomal activity of the methanolic extract of C. annuum against adult S. mansoni and to elucidate its potential molecular mechanisms of action through ultrastructural analysis, network pharmacology, molecular docking, and ADMET profiling.

2. Results and Discussions

2.1. Phytochemistry

For Capsaicinoids, HPLC analysis of the methanolic extract of C. annuum showed the presence of major capsaicinoids: nordihydrocapsaicin, capsaicin, and dihydrocapsaicin (Figure S1). The concentrations of the main constituents of capsaicinoids in the extract are shown in Table 1. In addition, a diverse profile of phenolic compounds; Gallic acid, chlorogenic acid, catechin, methyl gallate, coffeic acid, syringic acid, ellagic acid, coumaric acid, vanillin, ferulic acid, naringenin, rosmarinic acid, daidzein, quercetin, cinnamic acid, and kaempferol were represented by the 16 peaks in HPLC analysis (Figure S2). Capsaicin and dihydrocapsaicin are recognized as the principal bioactive constituents of chili peppers and have been widely associated with antioxidant, anti‐inflammatory, and antiparasitic activities. Moreover, the other identified phenolic compounds are known to possess potent antioxidant and immunomodulatory properties. The biological activities seen in this study may be enhanced by the coexistence of these bioactive metabolites, indicating that the therapeutic potential of C. annuum extract is likely attributable to the combined action of capsaicinoids and polyphenols rather than a single constituent.

TABLE 1.

HPLC‐identified capsaicinoids constituents in C. annuum methanolic extract.

Peak	Compound	RT	Concentration (µg/mL)
1	Nordihydrocapsaicin	5.461	6.87
2	Capsaicin	5.763	56.44
3	Dihydrocapsaicin	7.627	75.54

The concentrations of polyphenols in C. annuum extract, compared with standard values, are shown in Table S1.

2.2. Parasitological Results

2.2.1. In Vitro Anthelmintic Activity of Capsicum annuum Methanolic Extract

Table 2 showed the effect of different concentrations of C. annuum methanolic extract on the mortality of S. mansoni adult worms, dependent on time. The control group had no mortality at any time point. Treatment with praziquantel (0.2 µg/mL) caused 33.3% mortality at 3 h and reached 100% mortality by 24 h. The extract induced dose‐ and time‐dependent mortality, with the highest concentration (320 µg/mL) showing 50% and 100% mortality at 24 and 48 h, respectively, while lower concentrations (160, 80, and 40 µg/mL) exhibited less mortality. No mortality of worms was recorded at 20 µg/mL of the extract. Statistical analysis (two‐way ANOVA followed by Tukey post‐hoc test) revealed significant differences from the control group only for the higher extract concentrations, whereas lower concentrations (20 and 40 µg/mL) did not differ significantly from the control group. Zirintunda et al. [19] found that 0.08 gm/mL of C. annuum extract caused motility inhibition of half of Ascaridia galli adults in chickens after 5 h of treatment. Also, these results agree with those introduced by Fadladdin [20], who conducted the con. 500 mg of Artemisia annua was effective against adult worms of S. mansoni. We can suggest that this impact is due to the disturbance in the tegumental surface of the worm, signaling dysfunction and oxidative damage to the parasite [17]. Doenhoff et al. [28] postulated that the potent effect of PZQ is related to its mechanism that involves calcium influx–mediated spastic paralysis and tegumental vacuolization. On the other hand, Abozeid et al. [14] found that the plant extract P. granatum (50 mg/mL) is capable of killing S. mansoni worms in vitro after 5 days of exposure, while Rashed et al. [23] found that (50 and 100 mg/mL) of allicin and curcumin cause 100% and 91.3% mortality of S. mansoni after 24 h of exposure in vitro. These findings are therapeutically relevant because adult S. mansoni worms are responsible for continuous egg production, which is the principal cause of hepatointestinal schistosomiasis. Eggs retained within host tissues trigger granulomatous inflammation that progressively leads to periportal fibrosis, portal hypertension, and chronic liver damage. Therefore, effective elimination of adult worms can reduce egg deposition, interrupt disease progression, and limit parasite transmission, consistent with previous reports [29, 30, 31]. The observed concentration‐dependent activity suggests that increasing levels of bioactive constituents such as capsaicinoids, enhance antiparasitic efficacy.

TABLE 2.

Values represent the number of dead worms out of the total (n) per group.

Concentration (µg/mL)	1 h	3 h	12 h	24 h	48 h	72 h
Control	0/12 (0%)	0/12 (0%)	0/12 (0%)	0/12 (0%)	0/12 (0%)	0/12 (0%)
PZQ 0.2	0/15 (0%)	5/15 (33.3%)	9/15 (60%)	15/15 (100%)	15/15 (100%)	15/15 (100%)
Extract 320	0/12 (0%)	0/12 (0%)	4/12 (33.3%)	6/12 (50%)	12/12 (100%)	12/12 (100%)
Extract 160	0/11 (0%)	0/11 (0%)	0/11 (0%)	2/11 (18.2%)	6/11 (54.6%)	6/11 (54.6%)
Extract 80	0/14 (0%)	0/14 (0%)	0/14 (0%)	0/14 (0%)	2/14 (14.3%)	2/14 (14.3%)
Extract 40	0/13 (0%)	0/13 (0%)	0/13 (0%)	0/13 (0%)	1/13 (7.7%)	1/13 (7.7%)
Extract 20	0/12 (0%)	0/12 (0%)	0/12 (0%)	0/12 (0%)	0/12 (0%)	0/12 (0%)

Note: Percent mortality is shown in parentheses. Significant difference compared with Control (p < 0.05, Tukey test).

2.2.2. Scanning Electron Microscopy of Schistosoma mansoni

Figure 1A,D,G shows the scanning electron micrographs of control S. mansoni worms. The adult male worm possesses ventrally a gynaecophoric canal with a normal appearance (Figure 1A). In addition, the oral sucker rim is provided with sharp and intact spines (Figure 1D), and the body is covered with tubercles, which are provided with apical spines (Figure 1G).

FIGURE 1.

Scanning electron micrographs illustrating the surface topography of S. mansoni. (A, D, and G) Adult worms from the control group. (A) Adult male worm showing normal morphology of the body. (D) Spines at oral sucker rim. (G) Tubercles provided with apical spines (blue arrow). (B, E, and H) Worms exposed to PZQ. (B) Adult worm exhibiting extensive body coiling. (E) Oral sucker spines. (H) Tubercles with complete loss of apical spines (blue arrow). (C, F, and I) Worms exposed to the methanolic extract of C. annuum. (C) Adult contracted worm. (F) Spines at the oral sucker rim are showing shortening and a reduction in number. (I) Flattened tubercles with reduction in apical spines (blue arrow). Scale bar (A, B, and C) = 500 µm, (D, E, and F) = 2 µm, (G, H, and I) = 10 µm.

On the other hand, mature worms that were exposed to PZQ exhibited pronounced morphological damage. It showed extensive coiling to the adult worm (Figure 1B). Oral sucker spines were scattered in different orientations (Figure 1E). Complete loss of regular tubercles and spines was observed after treatment of worms with PZQ (Figure 1H). Following a 48‐h in vitro incubation period, tegumental alterations in adult S. mansoni exposed to the methanolic extract of C. annuum were observed. The extract induced significant morphological alterations in the tegument of adult S. mansoni worms compared with the control group. These changes were observed in the contraction of worms, erosion, and distortion of tubercles (Figure 1C). Furthermore, oral sucker spines became distinctly shorter, and some spines were observed to decrease in number after exposure to the extract (Figure 1F). In addition, some tubercles appeared flattened with a reduction in the number of spines and a wider intertubercular area (Figure 1I).

Since the parasite's tegument serves as its primary interface with the host environment, examining this structure after drug exposure helps us understand the therapeutic mechanism. In addition to the intake of nutrients like amino acids, carbohydrates, lipids, and other nutrients of similar size, it is essential for the defense system to avoid the immunological response and for the excretion of catabolic products like lactic acid [32, 33, 34]. Results showed extensive coiling of the adult worm and significant tegumental damage, including tubercle and spine deformation and erosion. These results suggest that C. annuum methanolic extract has direct antischistosomal action. Such changes, as described by Abozeid et al. [14] after exposure of S. mansoni in vitro to 50 mg/mL of Punica granatum and by Rashed et al. [23], after exposure of S. mansoni to allicin (8.66 µl/mL) and curcumin (87.25 µl/mL) in vitro. Damage to the tegument's structure can seriously reduce parasite survival as it is essential for immune evasion, food absorption, and host–parasite contact [35]. These morphological changes in the worm's body surface are the main cause of parasite death. Therefore, one of the main structural mechanisms driving higher mortality is probably surface erosion and the abnormalities observed.

2.3. In Silico Analysis

2.3.1. STITCH Network Pharmacology Analysis

The STITCH chemical–protein interaction network generated from the HPLC‐identified constituents of C. annuum comprised 29 nodes (19 phytochemicals and 10 S. mansoni proteins/transcripts) connected by 102 edges (Figure 2). Compound–compound interactions predominated (66/102; 64.7%), whereas unique compound–protein and protein–protein interactions accounted for 20/102 (19.6%) and 16/102 (15.7%) of the total edges, respectively. All 19 HPLC‐identified phytochemicals were represented within the network; however, dihydrocapsaicin and coumaric acid were mapped to the closely related homodihydrocapsaicin and p‐coumaric acid, respectively. Although the three capsaicinoids were retained as individual network nodes, they did not generate high‐confidence compound–protein interactions under the applied confidence threshold.

FIGURE 2.

STITCH‐derived interaction network linking HPLC‐identified C. annuum constituents to S. mansoni proteins. The network contains 29 nodes (19 phytochemicals; 10 parasite proteins/transcripts) and 102 edges (66 compound–compound, 20 compound–protein, 16 protein–protein). Compound–protein hubs include Smp_028500 (Caspase‑3‑like), Smp_172010 (Caspase‑7‑like), and Smp_080730 (Ser/Thr kinase).

Within the compound–protein subnetwork, three parasite proteins emerged as the principal multi‐ligand hubs based on degree and combined interaction score. The caspase‐3‐like protease Smp_028500 was associated with five phytochemicals that reached the high‐confidence threshold, including quercetin (0.913), gallic acid (0.716), kaempferol (0.716), ellagic acid (0.716), and daidzein (0.700). Similarly, the caspase‐7‐like protease Smp_172010 interacted with five phytochemicals, although only quercetin (0.875) and gallic acid (0.709) met the high‐confidence threshold, whereas kaempferol (0.632), daidzein (0.588), and ellagic acid (0.588) remained as contextual associations. The Ser/Thr kinase Smp_080730 exhibited high‐confidence interactions with kaempferol (0.873) and quercetin (0.741), while daidzein (0.606) was retained as a contextual association. Additional high‐confidence single‐target interactions were predominantly associated with quercetin, including ATP synthase subunits (Smp_038100, Smp_002880.1__mRNA, and Smp_033670.5__mRNA), the PI3K catalytic subunit (Smp_164960), a Sir2‐like sirtuin (Smp_138640), and metabolic enzymes (Smp_123710 and Smp_127730). These findings suggest that proteins involved in apoptosis and intracellular signaling may constitute important interaction hubs for the phytochemicals identified in C. annuum [36]. The predominance of high‐confidence interactions involving polyphenolic constituents, together with the absence of comparable interactions for capsaicinoids under the selected STITCH confidence threshold, suggests that polyphenols may play a major role in the predicted molecular interaction profile of the extract. The identification of caspase‐3‐like (Smp_028500), caspase‐7‐like (Smp_172010), and Ser/Thr kinase (Smp_080730) as the major multi‐ligand hubs further highlights apoptosis‐ and signaling‐related pathways as potential targets underlying the antischistosomal activity of the extract. Evidence decomposition of the exported STITCH interactions further demonstrated that the predicted caspase associations were supported mainly by curated database annotations and automated text mining, whereas several quercetin interactions with mitochondrial energy‐related proteins and PI3K were additionally supported by experimental evidence (interaction scores 0.752–0.773). Collectively, these findings support a polyphenol‐centered interaction network in which multiple phytochemicals may cooperatively target proteins involved in apoptosis and signal transduction rather than relying on a single dominant constituent.

2.3.2. Molecular Docking, Binding‐Site Interpretation, and Domain Mapping

Molecular docking is a valuable computational approach in computer‐aided drug design and structural molecular biology, enabling the prediction of three‐dimensional interactions between bioactive compounds and their molecular targets [24, 37]. The integration of deep learning‐based structural prediction has further improved the reliability of docking studies, even for proteins with limited experimental structural information [38]. In schistosome research, molecular docking has been widely applied to predict the binding affinities of phytochemicals toward essential parasite proteins, including thioredoxin glutathione reductase (SmTGR) and acetylcholinesterase (AChE), thereby providing mechanistic support for their potential antiparasitic activity [39].

Docking of the HPLC‐identified phytochemicals against the three STITCH‐prioritized targets revealed distinct target‐specific affinity profiles (Figure 3). For the caspase‐3‐like protease Smp_028500, rosmarinic acid exhibited the strongest predicted binding affinity (−7.94 kcal/mol), followed closely by quercetin (−7.90 kcal/mol), chlorogenic acid (−7.88 kcal/mol), catechin (−7.58 kcal/mol), kaempferol (−7.51 kcal/mol), and naringenin (−7.49 kcal/mol), whereas ellagic acid showed a slightly lower affinity (−7.17 kcal/mol). In contrast, quercetin displayed the highest affinity toward the caspase‐7‐like protease Smp_172010 (−8.65 kcal/mol), followed by ellagic acid (−8.56 kcal/mol), catechin (−8.42 kcal/mol), kaempferol (−8.34 kcal/mol), rosmarinic acid (−8.19 kcal/mol), and chlorogenic acid (−7.89 kcal/mol). For the Ser/Thr kinase Smp_080730, quercetin again demonstrated the strongest predicted affinity (−7.35 kcal/mol), followed by ellagic acid (−7.21 kcal/mol), catechin (−7.10 kcal/mol), rosmarinic acid (−7.06 kcal/mol), kaempferol (−7.02 kcal/mol), and naringenin (−6.84 kcal/mol).

FIGURE 3.

Comparative binding affinity and pose confidence analysis from molecular docking simulations. For each compound, the mean binding affinity (kcal/mol) was calculated from the top 10 affinity‐ranked docking poses; error bars represent the standard deviation (SD).

Overall, quercetin exhibited consistent multi‐target binding across all three proteins, whereas ellagic acid showed comparatively stronger affinity toward the caspase‐7‐like protease. Rosmarinic acid demonstrated the highest affinity for the caspase‐3‐like target, while several additional phenolic compounds displayed binding energies within a comparable range, suggesting that the predicted biological activity of C. annuum extract may result from the complementary action of multiple phytochemicals rather than a single dominant constituent. These observations are consistent with the findings of Mahamba et al. [25].

Domain annotation by InterProScan further supported the biological relevance of the selected targets. Smp_028500 contained the characteristic caspase p20 (55–179; IPR001309) and p10 (196–293; IPR002138) domains, together with conserved histidine and catalytic cysteine motifs. Likewise, Smp_172010 possessed a catalytic caspase domain (70–318; IPR015917) comprising both p20 and p10 subdomains and the conserved catalytic cysteine signature. The Ser/Thr kinase Smp_080730 contained the characteristic protein kinase catalytic domain (12–300; IPR000719), ATP‐binding region (18–41; IPR017441), and Ser/Thr kinase active‐site signature (132–144; IPR008271), supporting its annotation as a functional protein kinase.

Representative docking complexes further illustrated the predicted ligand–protein interactions (Figure 4A–C). In the Smp_028500–ellagic acid complex (Figure 4A), ellagic acid established hydrophobic contacts with Tyr150 and Ala153, hydrogen bonds with Arg159, and salt bridges involving Arg156 and Arg159, indicating stable interactions within the predicted binding pocket. Similarly, in the Smp_172010–ellagic acid complex (Figure 4C), ellagic acid formed multiple hydrophobic interactions together with several hydrogen bonds involving Asp172, Arg179, Arg199, and Tyr239, supporting its high predicted affinity for this target. In contrast, the representative quercetin binding pose in the Smp_080730 complex (Figure 4B) interacted with residues located beyond the InterPro‐annotated kinase catalytic domain (12–300), indicating that the predicted binding mode requires further experimental validation before confirming the functional binding pocket.

FIGURE 4.

Predicted binding modes and interaction fingerprints of representative lead complexes. (A) Ellagic acid bound to Smp_028500 (Caspase‑3‑like) showing hydrophobic contacts, hydrogen bonds, and salt bridges. (B) Quercetin bound to Smp_080730 (Ser/Thr kinase) showing hydrophobic and hydrogen‐bond contacts. (C) Ellagic acid bound to Smp_172010 (Caspase‑7‑like) highlighting a hydrogen‐bond network engaging ASP172/ARG179/ARG199/TYR239.

The favorable docking profiles observed for quercetin, ellagic acid, rosmarinic acid, and related polyphenols support the experimental findings of concentration‐dependent worm mortality and severe tegumental damage observed in vitro, suggesting that interference with apoptosis‐ and signaling‐related proteins may contribute to the schistosomicidal activity of the extract. Protein kinases regulate essential biological processes in schistosomes, including metabolism, cytoskeletal organization, and stress adaptation [40]. Therefore, simultaneous disruption of kinase‐mediated signaling and apoptosis‐related pathways could impair parasite cellular homeostasis and accelerate worm death, providing a plausible molecular explanation for the observed biological activity.

2.3.3. Pharmacokinetic and ADMET Considerations

The pharmacokinetic and ADMET profiles of the three representative phytochemicals, ellagic acid, quercetin, and rosmarinic acid, are summarized in Table 3. All three compounds exhibited moderate lipophilicity, with logP values of 0.95, 1.45, and 2.00, respectively, together with relatively high polarity, as reflected by TPSA values of 141.34, 131.36, and 144.52 Ų. Their predicted human intestinal absorption probabilities were 50.0% for ellagic acid, 13.4% for quercetin, and 31.7% for rosmarinic acid. Drug‐likeness (QED) values were estimated at 0.434 for quercetin, 0.298 for rosmarinic acid, and 0.183 for ellagic acid. In agreement with their relatively high polarity, all three compounds displayed low predicted Caco‐2 permeability and limited oral bioavailability (F 20%–30%).

TABLE 3.

Comprehensive in silico ADMET profiling of top polyphenolic ligands identified from STITCH network analysis.

Property	Category	Ellagic acid	Quercetin	Rosmarinic acid	Acceptable range/threshold
Physicochemical properties
Molecular weight (g/mol)	Size	302.01	302.04	360.08	160–480 (optimal)
logP	Lipophilicity	0.95	1.45	2.00	0–3 (oral drugs)
logD (pH 7.4)	Ionization	0.75	1.42	1.95	1–3 (optimal)
logS (solubility)	Aqueous solubility	−3.37	−3.72	−3.03	>−4 (good)
TPSA (Ų)	Polarity	141.34	131.36	144.52	≤140 (oral bioavail.)
H‐bond donors	Polarity	4	5	5	≤5 (Lipinski)
H‐bond acceptors	Polarity	8	7	8	≤10 (Lipinski)
Rotatable bonds	Flexibility	0	1	7	≤10 (oral bioavail.)
Drug‐likeness
QED score	Overall drug‐likeness	0.216	0.434	0.298	0.5–1.0 (desirable)
Synthetic accessibility	Synthesis ease	2.98	2.54	2.90	1–3 (easy)
Fsp3	Scaffold saturation	0.000	0.000	0.111	>0.25 (CNS drugs)
Absorption
Caco‐2 (log cm/s)	Intestinal permeability	−5.17	−6.18	−6.51	>−5.15 (high perm.)
HIA (probability)	Human intestinal absorption	0.500	0.134	0.025	>0.30 (well absorbed)
F20% (probability)	Bioavailability ≥20%	0.953	0.504	0.976	>0.50 (moderate‐high)
F30% (probability)	Bioavailability ≥30%	0.967	0.991	0.985	>0.70 (high)
P‐gp substrate	Efflux transporter	0.847	0.944	0.615	<0.50 (non‐substrate)
P‐gp inhibitor	DDI potential	0.001	0.009	0.000	<0.30 (low DDI risk)
Distribution
BBB penetration	CNS distribution	0.001	0.000	0.000	>0.30 (CNS active)
PPB (%)	Plasma protein binding	68.5	98.7	77.3	<90% (adequate free fraction)
VDss (log L/kg)	Tissue distribution	−0.35	−0.88	−0.35	−0.15 to 0.45 (typical)
Fu (%)	Fraction unbound	24.2	1.1	12.5	>1% (adequate for efficacy)
Metabolism
CYP1A2 inhibition	DDI—CYP1A2 substrates	0.904	0.998	0.534	<0.50 (low DDI)
CYP2C9 inhibition	DDI—CYP2C9 substrates	0.028	0.432	0.000	<0.50 (low DDI)
CYP2D6 inhibition	DDI—CYP2D6 substrates	0.000	0.937	0.000	<0.50 (low DDI)
CYP3A4 inhibition	DDI—CYP3A4 substrates	0.004	0.937	0.000	<0.50 (low DDI)
CYP1A2 substrate	Primary metabolism	0.000	0.000	0.000	—
CYP3A4 substrate	Primary metabolism	0.000	0.000	0.000	—
Excretion
Plasma clearance (mL/min/kg)	Elimination rate	14.7	8.3	13.2	5–15 (moderate)
t½ (hours)	Elimination half‐life	1.65	1.59	1.91	>4 h (QD dosing)
Toxicity
hERG inhibition	Cardiotoxicity	0.028	0.053	0.056	<0.50 (low risk)
DILI	Hepatotoxicity (general)	0.984	0.783	0.961	<0.50 (low risk)
H‐HT	Hepatotoxicity (specific)	0.616	0.337	0.699	<0.50 (low risk)
Ames test	Mutagenicity	0.670	0.586	0.549	<0.30 (non‐mutagenic)
Carcinogenicity	Long‐term toxicity	0.015	0.015	0.128	<0.50 (non‐carcinogenic)

Additional ADMET predictions indicated plasma protein binding values of 78.7% for quercetin, 92.1% for rosmarinic acid, and 57.0% for ellagic acid. None of the compounds was predicted to penetrate the blood–brain barrier, while the predicted DILI probabilities were 0.703, 0.809, and 0.670 for quercetin, ellagic acid, and rosmarinic acid, respectively. The predicted hERG blockage risk scores ranged from 0.182 to 0.491, suggesting no marked cardiotoxicity concerns among the evaluated compounds.

Collectively, the network pharmacology, molecular docking, and ADMET analyses support a polyphenol‐centered, multi‐target interaction profile involving caspase‐ and kinase‐associated proteins in S. mansoni, with multiple phytochemical constituents contributing to target engagement rather than a single dominant compound [41]. When considered together with the phenotypic and ultrastructural findings, these computational data support a multi‐target mechanism of action in which the identified phytochemicals may cooperatively affect parasite survival through mechanisms that remain to be experimentally validated, including possible effects on oxidative stress, tegumental integrity, apoptosis‐related proteases and kinase signaling pathways.

These data suggest the potential of C. annuum methanolic extract as a viable natural antischistosomal option. Consequently, there is an urgent global need to discover novel, safe, and effective antischistosomal agents, with natural products serving as a primary reservoir for drug discovery.

3. Conclusions

Capsicum annuum methanolic extract exhibited promising in vitro antischistosomal activity against adult S. mansoni in a concentration‐ and time‐dependent manner. The observed tegumental damage, together with the phytochemical profile and molecular docking results, suggests that the identified bioactive compounds may contribute to the extract's antiparasitic effects. In silico analyses further suggested that major phytoconstituents may target apoptosis‐related proteases and signaling proteins, providing computational hypotheses that may explain the observed experimental findings and require further validation. These findings support the potential of C. annuum methanolic extract as a natural source of antischistosomal agents and a potential complementary strategy for schistosomiasis control; however, further in vivo and safety studies are required to confirm its therapeutic applicability.

4. Experimental Section

4.1. Collection of Plant Material and Preparation of the Extract

Fresh fruits of C. annuum were collected from a cultivated farm in Abu Sultan, Ismailia Governorate, Egypt. The plant was authenticated by a qualified taxonomist from the Department of Botany, Faculty of Science, Helwan University, Egypt. Before being shade‐dried at room temperature, the gathered fruits were carefully cleaned with tap water, cut open, and sliced into little pieces. An electric grinder was then used to grind the dried material into a fine powder. For 24 h, 50 g of the powdered fruits were macerated in 450 mL of 70% methanol while being constantly shaken at 4°C. Whatman No. 1 filter paper was used to filter the resultant combination. The crude methanolic extract was obtained by concentrating the filtrate under reduced pressure using a rotary evaporator (IKA, HBIO basic, RV10B S99, Deutschland, Germany). This extract was then stored at 4°C for additional analysis.

4.2. Analysis of the Extract Using High‐Performance Liquid Chromatography (HPLC)

Capsaicinoids were analyzed using an Agilent C18 column (4.6 mm × 250 mm i.d., 5 µm). The flow rate was 1.5 mL/min, and the mobile phases were (A) 1% acetic acid and (B) acetonitrile (50:50, v/v). Each sample solution had an injection volume of 20 µL. Detection was performed at 280 nm, and the column temperature was maintained at 40°C. For the polyphenolic fraction, a separate HPLC method using authentic reference standards was applied, and the complete analytical parameters, including mobile phase, elution program, detection wavelength, retention time, calibration range, regression equation, correlation coefficient, replicate injections, LOD, LOQ, and compound concentration, are reported in Tables 1 and Table S1. Peak identification was based on comparison of retention times with reference standards, and quantification was performed using calibration curves prepared from standard solutions. All standards and samples were analyzed in replicate, and concentrations are reported as mean ± SD.

4.3. Parasitological Study

4.3.1. Isolation of Adult Worms of Schistosoma mansoni

Adult S. mansoni worms used in the in vitro antischistosomal assay were recovered from experimentally infected Swiss albino mice (Mus musculus), weighing 20–25 g, obtained from the Schistosome Biological Supply Center (SBSC) at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. The authors did not infect the mice; instead, they were experimentally infected at TBRI before being transferred. To remove adult worms from the mesenteric veins, the hepatic portal system is perfused retrogradely. This is accomplished by euthanizing the mouse and opening its abdomen to reveal its viscera. The thoracic inferior vena cava is cut to create an exit site for the perfusate, and a cannula is placed into the hepatic portal vein. After that, a physiological saline solution is pushed through the portal system at a regulated pressure, frequently with an anticoagulant like heparin added [42]. The mature worms are collected, then repeatedly cleaned with sterile phosphate‐buffered saline (PBS) and kept in RPMI‐1640 medium supplemented with 20% fetal calf serum, penicillin (300 U/mL), streptomycin (300 µg/mL), and gentamicin (160 µg/mL). The in vitro antischistosomal tests were conducted solely on mature S. mansoni worms.

4.3.2. In Vitro Effect of Capsicum annuum Methanolic Extract Against Schistosoma mansoni Adult Worms Viability

The extracts were evaluated in vitro against adult S. mansoni worms following a protocol similar to that described by Yousif et al. [43]. Freshly recovered worms were washed in phosphate buffer using a 20 µm sieve to remove blood, then rinsed with culture medium under sterile laminar flow conditions. A stock solution of the extract was prepared in 10% DMSO (1 mg/mL), and serial concentrations of 320, 160, 80, 40, and 20 µg/mL were obtained. The final DMSO concentrations corresponding to each extract concentration were (0.2%, 0.4%, 0.8%, 1.6%, and 3.2%). Worms were incubated with these concentrations in 24‐well sterile tissue culture plates. The culture medium used was RPMI 1640 supplemented with 20% fetal calf serum, streptomycin (300 µg/mL), penicillin (300 U/mL), and gentamycin (160 µg/mL). Each treatment was performed in duplicate using approximately three pairs of viable adult S. mansoni worms per well. The total number of worms per treatment varied slightly (11–15 worms) according to the number of viable adult worms recovered by portal perfusion and available for allocation at the time of the experiment. Negative controls contained the same concentration of DMSO without extract, whereas the positive control included praziquantel (0.2 µg/mL), which is known to cause complete worm mortality within 24 h.

Worm viability was assessed under a stereomicroscope for 24 h by observing spontaneous movement. Worms showing no spontaneous movement were gently stimulated mechanically using a fine needle and monitored for an additional observation period. Worms that failed to respond to mechanical stimulation and exhibited no detectable movement throughout the observation period were considered dead. Mortality rates were calculated relative to the total number of worms and compared with both negative and positive controls.

4.3.3. Scanning Electron Microscopy

The adult worms were fixed in 3% buffered glutaraldehyde for 3 h, then specimens were post‐fixed in 2% osmium tetroxide (OsO4) in phosphate buffer solution at 4°C for 2 h. Afterward, specimens were dehydrated at 4°C in a graded series of ethanol, then dried using a CO2 critical point drier, coated with gold, and finally examined and photographed using a scanning electron microscope (IT 200, Jeol).

4.4. In Silico Study

4.4.1. Network‐Guided Target Selection and AlphaFold Structural Preparation

Chemical‐protein association analysis implemented in STITCH v5 was used to prioritize putative protein targets in S. mansoni using SMILES or PubChem CIDs for bioactive metabolites identified by HPLC as query inputs [44]. The organism was restricted to S. mansoni (Taxonomy ID: 6183) when available. The node numbers, edge numbers, edge categories, and confidence scores reported were obtained from the STITCH web. Interactions with scores ≥0.500 were retained as medium‐confidence or higher network associations for exploratory target prioritization, whereas only interactions ≥0.700 were described as high‐confidence interactions. Proteins with multi‐metabolite connections and strong topological influence were ranked using (i) maximum combined confidence score, (ii) number of interacting metabolites, and (iii) network centrality metrics, including degree and betweenness.

Three proteins were chosen for structure‐based docking based on convergence of STITCH confidence and biological relevance: Smp_028500 (caspase‐3‐like; UniProt accession C4Q500; AlphaFold model AF‐C4Q500‐F1), Smp_172010 (caspase‐7‐like; UniProt accession C7T4Z2; AlphaFold model AF‐C7T4Z2‐F1), and Smp_080730 (serine/threonine kinase; UniProt accession G4VLX9; AlphaFold model AF‐G4VLX9‐F1). Predicted 3D structures were obtained from the AlphaFold Protein Structure Database using the corresponding UniProt accessions [38, 45]. Per‐residue confidence values (pLDDT) were retrieved from the AlphaFold DB, where pLDDT is reported on a 0–100 scale and higher values indicate higher confidence. Particular attention was given to pLDDT values surrounding the selected docking pockets rather than only the global model confidence. Low‐confidence terminal or disordered regions spatially distant from catalytic pockets were excluded. Protein domain architecture and catalytic regions were confirmed using InterProScan 5 to ensure that docking boxes were constrained to biologically relevant domains and that active‐site signatures were present within the modeled core.

4.4.2. Ligand Identification and SMILES‐Based Library Preparation

The ligand set was strictly restricted to the phytochemicals confirmed to be present in the C. annuum methanolic extract. HPLC analysis identified the retained constituents (capsaicinoids and polyphenols). Canonical SMILES for these compounds were retrieved from PubChem, using compound names and/or PubChem CIDs to ensure unambiguous structure mapping [46]. Ligands were converted to 3D conformations and energy‐minimized with the MMFF94 force field; stereochemistry was preserved according to PubChem annotations. Structure files were converted and standardized for docking using Open Babel v3.1.1 [47].

4.4.3. Molecular Docking With GNINA

Docking was performed using GNINA (v1.1), which extends the AutoDock Vina search procedure with convolutional neural network (CNN) pose evaluation and rescoring [48, 49]. For each ligand‐target pair, 20 poses were generated using exhaustiveness 20 and a fixed random seed for reproducibility. Docking boxes were centered on catalytic or functional pockets using InterProScan‐mapped motifs and structural inspection. For caspases, boxes covered the catalytic groove containing active‐site signatures; for the kinase model, the interpreted binding region was checked against the annotated kinase domain and ATP‐binding or active‐site signatures. Vina affinity values, CNN pose scores, and CNN affinity scores are provided in the Supporting Information.

4.4.4. Protein–Ligand Interaction Profiling (PLIP)

Protein–ligand interaction fingerprints for prioritized docked complexes were generated using PLIP [50]. PLIP was used to annotate hydrogen bonds, hydrophobic contacts, π‐stacking, π‐cation interactions, and salt bridges using its geometry‐based rules. Interaction tables were parsed to extract bond types and quantitative geometry (e.g., hydrogen bond distances/angles; hydrophobic contact distances, aromatic centroid distances and offsets), which were reported in the Results section to support mechanistic interpretation.

4.4.5. ADMET Prediction

Pharmacokinetic and toxicity properties were predicted using ADMETlab 2.0 [51]. SMILES were submitted to obtain physicochemical descriptors (MW, logP, logS, TPSA), absorption and permeability predictors (HIA probability, Caco‐2), distribution predictors (BBB class/probability, plasma protein binding), transporter liabilities (P‐gp substrate/inhibitor), CYP interaction probabilities, and safety endpoints (hERG inhibition probability, Ames, DILI, carcinogenicity). Outputs were recorded as probabilities (0–1) or continuous values as provided by the platform.

Author Contributions

Amina A. Ghedan: writing – original draft, writing – review and editing, software, methodology, data curation, resources. Mohamed A. Dkhil: conceptualization, validation, visualization, software, supervision, data curation, writing – review and editing, investigation, methodology, resources. Irene S. Gamil: conceptualization, investigation, formal analysis, supervision, software, methodology, resources. Felwa A. Thagfan: project administration, funding acquisition, resources, software, writing – review and editing. Mona F. Khalil: investigation, writing – review and editing, supervision, methodology, software, data curation, resources. Rania G. Taha: writing – original draft, writing – review and editing, software, formal analysis, supervision, methodology.

Ethics Statement

The authors have followed the rules of the ethical committee of the Institutional Animal Care and Use Committee (HU‐IACUC), Faculty of Science, Helwan University, Approval Number: HU‐IACUC/Z/YE0911‐53.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71610‐sup‐0001‐SuppMat.zip

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.