The use of animal-derived parasitic material was approved by the Institutional Animal Ethics Committee of Pachhunga University College, Aizawl, Mizoram, India (approval number PUC-IAEC-2022-Z02, dated 15/03/2022). The study involved the collection of adult Raillietina echinobothrida from the intestines of slaughtered chickens and did not involve experimental infection, live animal experimentation, or any invasive procedure on live animals.

All procedures for parasite collection, handling, maintenance, treatment, fixation, histological processing, and scanning electron microscopy were conducted in accordance with institutional ethical guidelines and standard laboratory biosafety practices. The use of parasite material was limited strictly to the objectives of evaluating the anthelmintic efficacy of A. scholaris bark extract. No human participants or clinical human samples were used in this study.

The study was conducted from November 2022 to May 2025. Plant specimens of A. scholaris were collected from Lungdai, Mizoram, India (latitude 23°52′ N, longitude 92°44′ E, altitude 1200 m above sea level). Antifungal, anthelmintic, histological, scanning electron microscopy, chemical profiling, and molecular docking analyses were performed at Pachhunga University College, Aizawl, Mizoram, India. Antimalarial and cytotoxicity assays were performed at the Central Drug Research Institute (CDRI), Lucknow, Uttar Pradesh, India.

This experimental laboratory-based study evaluated the anti-infective potential of A. scholaris bark extract against fungal, malarial, and helminth pathogens. The study included the extraction of plant material, antifungal assays, antimalarial assays against P. falciparum, cytotoxicity testing using Vero C1008 cells, anthelmintic evaluation against R. echinobothrida, histological and scanning electron microscopy observations, GC-MS-based chemical profiling, and molecular docking analysis.

Mature plant parts, including flowers of A. scholaris, were collected from a forest in Lungdai, Mizoram, India. Authentication of the specimens (voucher number PUC-A-23-01 dated 25/11/2022) was performed at the Eastern Regional Center of the Botanical Survey of India, Meghalaya, India (BSI/ERC/Tech/2023-24/102 dated 17/05/2023). The bark was cut into small pieces, washed in distilled water, and dried in shade under ambient conditions (25 ± 2°C).

The dried samples were extracted by maceration and Soxhlet extraction using chloroform and methanol. The solvents were evaporated and recycled under vacuum pressure using Buchi Rotavapor® R-100 (Büchi Labortechnik AG, Flawil, Switzerland). The extract yield was calculated using the formula: [W1 ÷ W2] × 100, where W1 is the weight of the final extract and W2 is the weight of the dried sample. The extract was refrigerated at 4°C until use.

The antifungal activity of A. scholaris extract was evaluated using the poison plate technique of Grover and Moore [27], with a modified potato-dextrose agar culture method [28]. Subcultures of Aspergillus fumigatus (ATCC 204305), Candida albicans (ATCC 26790), and Neocosmospora keratoplastica (ATCC 36031) were obtained from ATCC, Manassas, Virginia, USA. The fungal cultures were streaked evenly on sterilized agar media in Petri dishes. They were grown for seven days at 27 ± 2°C in a disinfected microbiological chamber (Igene IG-95I, Igene Labserve Pvt. Ltd., New Delhi, India).

Serial dilutions of the A. scholaris extract (10, 5, 2.5, and 1.25 mg/mL) were prepared in 20 mL potato-dextrose agar. The agar was kept molten at approximately 50°C. Fungi grown only in the growth medium were maintained as the control. Using a sterile cork borer, 6 mm disks of fully grown fungi were inoculated into the center of the culture plates containing extract-treated and control media. After solidification, the plates were hermetically sealed with parafilm and incubated for seven days at 27 ± 2°C. The growth zones formed radially around the inoculum were measured at two opposite circumferences every 24 h. The inhibitory activity was estimated using the standard formula.

Growth inhibition (%) = [(Growth of control − Growth of treated) ÷ Growth of control] × 100

Two isolates of P. falciparum, chloroquine-sensitive 3D7 and multidrug-resistant K1, were obtained from the Biodefense and Emerging Infections Research Resources Repository of the National Institute of Allergy and Infectious Diseases, U.S. National Institutes of Health, and maintained at CDRI, Lucknow, India. They were cultivated in complete RPMI 1640 medium containing 8% parasitaemia at the erythrocytic trophozoite stage and 2% erythrocyte volume [29]. The culture medium was supplemented with HEPES, 0.5% AlbuMAX™ II lipid-rich bovine serum albumin (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.2% D-glucose, 0.2% sodium bicarbonate, 45 mg/L hypoxanthine, 0.25 mg/L fungizone, 50 mg/L gentamycin, and 15 μM hypoxanthine. The cultures were maintained at 37 ± 1°C in a humidified 5% CO₂ incubator (Forma™ Series II, Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Antimalarial susceptibility was tested using malaria SYBR Green I-based fluorescence (MSF) [30]. Parasitized erythrocytes at 0.8% parasitaemia and 1% hematocrit in RPMI were exposed to A. scholaris extract prepared in serial dilution from 50 μg/mL stock solution. They were cultured in 96-well plates at 37 ± 1°C in a humidified 5% CO₂ incubator.

Uninfected erythrocytes were maintained as the negative control, and infected cells without drug treatment were maintained as the positive control. One group of parasitized erythrocytes was treated with chloroquine, a reference drug, at 0.05–0.3 ng/μL. After 72 h of culture, 100 μL of lytic buffer containing 1× SYBR Green was added to all samples, and the samples were incubated again for 2 h. Fluorescence intensity was detected at 485 ± 20 nm excitation and 530 ± 20 nm emission using a fluorescence reader (BioTek Synergy™, Agilent Technologies, Inc., Santa Clara, California, USA). IC₅₀ was calculated using programmed statistical analysis [31].

Cytotoxicity of A. scholaris extract was assessed using the MTT reduction assay [31]. Vero C1008 cells were acquired from ATCC (CRL-1586™) and maintained at CDRI. They were seeded in a 96-well plate at 10⁴ cells per well and maintained under the same conditions as the malarial parasites. The cells were treated with different dilutions of the extract. Positive control cells were exposed to podophyllotoxin.

After 72 h of incubation, the samples were mixed with 25 µL of MTT at a stock concentration of 5 mg/mL. After 2 h, absorbance was recorded at 570 nm, from which CC₅₀ was calculated. SI was calculated as the ratio of CC₅₀ to IC₅₀.

Anthelmintic activity was tested on the intestinal cestode R. echinobothrida Mégnin, 1880, collected from chicken intestines [32]. The parasites were maintained in 0.9% phosphate buffered saline (PBS) supplemented with 1% dimethyl sulfoxide (DMS) at 37 ± 1°C. Parasites maintained only in PBS + DMS served as the negative control.

Experimental groups were treated with different concentrations of A. scholaris extract: 5, 10, and 20 mg/mL. Positive control groups consisted of parasites treated with albendazole at similar concentrations. After each experiment, the parasites were washed in PBS and fixed in Bouin solution. Dehydration was performed using 30–100% ethanol, then cleared in xylene. Paraffinized blocks were cut at a thickness of 4–5 µm using an auto-microtome (MRM-ST, Medimeas Instruments, Ambala, Haryana, India). The sections were dehydrated again, stained with eosin and hematoxylin, fixed on glass slides, and observed under a Nikon Eclipse image analyzer (Nikon Corporation, Tokyo, Japan).

For scanning electron microscopy, the parasites were fixed in 10% neutral-buffered formaldehyde at 4°C for 4 h. They were dehydrated in increasing grades of acetone. The specimens were immersed in tetramethylsilane for 10 min and evaporated to dryness at 25°C in an air-drying chamber. After coating with gold using an MC1000 ion sputter coater (Hitachi, Ltd., Tokyo, Japan), the electron micrographs were taken with a TM4000Plus II scanning electron microscope (Hitachi, Ltd.).

Identification of bioactive compounds in A. scholaris extract was performed using GC-MS with a TRACE™ 1300 ISQ™ LT system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). One microliter of extract dissolved in methanol was injected with helium as the carrier gas. A non-polar TR-5MS column (30 m × 0.25 mm × 0.25 μm) was used. The injection port temperature was set at 280°C, the transfer line and ion source temperatures at 220°C, and the oven temperature was increased from 70°C to 250°C. Thermo Scientific™ Xcalibur™ software (Thermo Fisher Scientific) was used to generate the data. The data were compared with the National Institute of Standards and Technology (NIST) database.

The compounds identified in A. scholaris extract were docked against vital receptors in pathogenic fungi, P. falciparum, and helminth parasites. The three-dimensional structures of the compounds were retrieved from PubChem, NCBI. The chemical conformations were optimized and the cumulative energies minimized using ChemBio3D Ultra 12.0 (CambridgeSoft Corporation, Cambridge, Massachusetts, USA) with the MMFF94 force field.

The molecular target proteins were acquired from RCSB-PDB, including Candida albicans sterol 14α-demethylase (CYP51), C. albicans farnesyltransferase, C. albicans geranylgeranyltransferase type 1 (GGTase-I), Plasmodium falciparum erythrocyte membrane protein 1 (VAR2CSA), and S-adenosyl-L-homocysteine hydrolase (SAHH), Caenorhabditis elegans β-tubulin, and C. elegans glutamate-gated chloride channel (GluCl). Ligand binding was simulated using AutoDock Vina v1.2.4 (Molecular Graphics Lab, The Scripps Research Institute, La Jolla, California, USA) [33]. MGLTools 1.5.6 (Molecular Graphics Lab) was used to add Kollman charges and polar hydrogens to the proteins. The final configurations were saved in PDBQT files. BIOVIA Discovery Studio Visualizer 2016 (Dassault Systèmes, Vélizy-Villacoublay, France) was used to analyze the final structures.

Comparison of mean differences between treatments and controls was performed using Student’s t-test. Group comparisons were conducted using analysis of variance followed by Tukey’s honest significant difference test. The level of significance was set at p < 0.05. Statistical analysis and graphs were generated using GraphPad Prism version 10.4.1 (Dotmatics, Boston, Massachusetts, USA).

The extract yields of A. scholaris bark were 5.15% by maceration, 6.36% with chloroform, and 18.35% with methanol. The best-yielding methanol extract was found to contain the greatest variety of compounds and was used for all experiments.

The antifungal activity of A. scholaris bark extract was assessed based on the growth patterns of A. fumigatus, C. albicans, and N. keratoplastica, as tabulated in Table 1. There was a steady proliferation of the three fungi in the culture media for 7 days, and the proliferation rate was determined from the growth zones formed around the sample wells. The growth rate of the control sample, maintained without the plant extract, was highest, as indicated by the increasing number of growth zones each day across all species. The relative growth rate was in the order A. fumigatus > N. keratoplastica > C. albicans. In the plant extract-treated groups, there was no significant inhibition of growth (p ≥ 0.05) on the first day in all species compared to the growth rate of control, and a significant difference appeared after the first day. The percentage of inhibition was calculated for each species from the growth zones of the respective control. Statistical comparison between treatment groups, as shown in Figure 1, indicates that N. keratoplastica was evidently the most susceptible species against the plant extract, followed by C. albicans. The inhibitory effect on A. fumigatus was minimal after the second day.

A. scholaris bark extract was almost equally effective against both the drug-sensitive and drug-resistant isolates of P. falciparum as shown in Table 2. The IC₅₀ values were 43.40 µg/mL against 3D7 and 45.60 µg/mL against K1, both within the antimalarial efficacy category of good or moderate activity. It was remarkable that the plant extract was effective against the multidrug-resistant isolate, while the reference drug, chloroquine, had no effect. The antimalarial resistance index (Ri), calculated as the ratio of the relative effectiveness of drug-sensitive to drug-resistant isolates, was 1.05. This indicates that the plant extract has an extremely low level of potential resistance in the parasites. In comparison, chloroquine shows an extreme level of resistance in K1, with an Ri of 39.85, indicating that it has no pharmaceutical value against this isolate.

The cytotoxicity of A. scholaris bark extract against Vero C1008 cells is also shown in Table 2. CC₅₀ values above 100 µg/mL indicate that the plant extract is non-toxic and safe for normal cells. In contrast, the reference toxin, podophyllotoxin, was extremely toxic with a CC₅₀ of 1.45 µg/mL. However, the selectivity indices of the plant extract were low: 6.12 for 3D7 and 5.81 for K1.

The anthelmintic efficacy of albendazole and A. scholaris bark extract is summarized in Table 3. Both treatments resulted in concentration-dependent responses in the cestode. The parasites were significantly more sensitive to the drug, albendazole, as indicated in Figure 2. A. scholaris bark extract, albeit with less potency, showed significant efficacy even at the lowest concentration tested. However, albendazole and the plant extract were equally effective, i.e., no significant differences (p > 0.05) across all concentrations, with p values of 0.22 for 5 mg/mL, 0.14 for 10 mg/mL, and 0.07 for 20 mg/mL.

Histological section of the cestode treated with the plant extract indicated several tissue damages as shown in Figure 3. Numerous corrugations on the body surface (the tegument) indicate severe shrinkage that has formed creases and folds. Internally, the most affected anatomical parts are the parenchymatous tissue that hold the muscle and subtegumental layers. The tissues are marked with irregular dots and stains indicating the disintegration of the proteinaceous filaments. Several eggs were empty, indicating loss of cytoplasmic materials, including the nuclei. There are also some parts of the circular muscle that have disappeared, indicating the disintegration of the muscle fibers.

Signature anthelmintic effects of the plant extract were evident under scanning electron microscopy. The anterior bulbous end, the scolex, is full of wrinkles due to shrinkage of the body surface tegument (Figure 4A). Even the two eye-like suckers are completely wrinkled. A magnification of one sucker shows a circular rim having rows of spines (whitish, pointed, and curved structures) at some portion, but some of which are detached at the bottom and totally vanished on the left side (Figure 4B). However, all the spines on one sucker are completely lost (Figure 4C). The tegument in the neck region is totally eroded (Figure 4D), and the smooth surface is entirely replaced by tissue lumps and creases (Figure 4E). Tegumental shrinkage extends throughout the mature body segments, proglottids. Massive folds are visible on all the proglottids (Figure 4F). A closer view of the tegumental surface of a mature proglottid reveals contorted folds due to constriction and the complete loss of surface filaments and microtriches (Figure 4G).

The gas chromatogram of the extract for identification of bioactive compounds is shown in Figure 5. The peak intensity and mass were used to compare compounds in the NIST library (at similarity score > 90%), which showed that the plant extract is rich in triterpenoids (Table 4). The three most abundant compounds, α-amyrin, kolavenol, and lup-20(29)-en-3-yl acetate, were all triterpenoids. Kolavenol at retention time (RT) 26.54 and α-amyrin at RT 27.39 were identified as the two main bioactive compounds. Kolavenol was additionally detected at RT 26.39, and α-amyrin at RT 31.16. Although at a lower abundance value, lup-20(29)-en-3-yl alcohol was detected at seven RT.

The identified compounds of A. scholaris bark extract, α-amyrin and kolavenol, were docked against the vital proteins of pathogenic fungi, malaria, and helminth parasites to gain insight into the probable molecular mechanism and binding efficiency. The protein grid boxes were prepared according to the coordinates specified in Table 5. Exhaustiveness of analysis was set at 8 to dock the ligands to the target proteins of fungi (Table 6), malaria (Table 7), and helminth parasites (Table 8). The molecular structures depicting the ligand–receptor interactions and the exact binding sites are shown in Figures 6–8.

α-Amyrin was found to have exceptional binding efficiency to the major proteins. It interacts most efficiently with C. albicans target proteins. The highest binding efficiency was observed against CYP51 with a binding energy as low as −10.4 kcal/mol and amino acid interactions at seven residues. Its binding energy against farnesyltransferase and GGTase-I was −9.1 kcal/mol each. However, given equal binding energy, GGTase-I showed a higher capacity for molecular interaction, with binding sites at nine amino acid residues, compared with farnesyltransferase, which showed only four amino acid interaction sites. Its binding efficiency was almost equally high against P. falciparum VAR2CSA (binding energy −9.4 kcal/mol) and SAHH (binding energy −10 kcal/mol), but with fewer amino acid interactions. It also indicated high-affinity binding to C. elegans GluCl, with a binding energy of −9.4 kcal/mol, but comparatively lower binding to β-tubulin, with a binding energy of −9.4 kcal/mol.

Kolavenol was less efficient than α-amyrin, showing moderate binding across all proteins except GGTase-I, against which it was highly efficient (binding energy −9.1 kcal/mol).

We found that A. scholaris bark contains bioactive compounds that exhibit cross-ranging activities against pathogenic fungi, malarial, and helminth parasites. While many anti-infective medications have only specific activity against selected pathogens, the most important antimalarials are demonstrably broad-spectrum. Quinine and its derivatives, quinine, quinidine, and quinacrine, significantly reduced the worm burden and egg-laying of the blood helminth Schistosoma mansoni, the most prevalent helminth parasite of humans [34]. Several quinoline derivatives were shown to inhibit the motility and development of Haemonchus contortus, an intestinal nematode of ruminants [35]. There are reports that quinoline-containing plants, such as Acmella and Zanthoxylum species, effectively kill various helminth parasites [36]. Chemically modified quinoline compounds exhibit effective antimicrobial activity against important pathogens, including the bacterium that causes tuberculosis [37], as well as cancer cells [37].

The currently used antimalarials, artemisinin and its related compounds, have been extensively shown to have a wide range of anthelmintic activity, including efficacy against a host of helminth parasites such as nematodes, trematodes, and cestodes [39]. They are not only shown in clinical trials to be useful against all forms of schistosomiasis, but they are also effective against carcinogenic helminths, Clonorchis sinensis and Opisthorchis viverrini [40], as well as several pathogenic fungi and bacteria [41, 42]. On the other hand, standard anthelmintics such as albendazole and mebendazole are known to have anticancer and antiprotozoal activities [43]. Recent studies have shown that albendazole and its derivatives are effective against various life-cycle stages of P. falciparum [44].

Our findings indicate that the antimicrobial, antimalarial, and anthelmintic activities of A. scholaris bark extract can be understood from the pharmacological properties of these antimalarial and anthelmintic drugs. The plant extract showed good activity against the cestode and fungi tested. It is notable for its efficacy against pathogenic fungi designated as “fungal priority pathogens” [45], which are largely underexplored in medicinal plants. In particular, the most susceptible species, N. keratoplastica, causes a range of diseases, from skin infections to organ failure, in different animals [46]. It was consistently as efficacious as albendazole (p > 0.05) at all concentrations tested against the cestode. Anthelmintic resistance is most prevalent in veterinary animals [47]; hence, the plant offers a promising solution to the major economic burden in animal farming. However, its antimalarial activity was of moderate efficacy (IC₅₀ between 10–50 µg/mL). This could be due to geographical variations and the mode of extraction of the plant species, as variable antimalarial efficacy has been reported from different parts of the world. Ethanolic extract of the specimen from West Timor, Indonesia, showed an IC₅₀ of 15.6 µg/mL against 3D7, with lupenyl acetate as its major compound [48], whereas the specimen from Sumatra showed weak activity (IC₅0 > 50 µg/mL) against 3D7 [49]. The methanolic extract of the Thai specimen exhibited negligible activity, with an IC₅₀ of 181.4 µg/mL against K1 [50]. The major bioactive compounds in different Alstonia species are indole alkaloids, with echitamine among the best-studied. These alkaloids exhibited only moderate antimalarial efficacy, with IC₅₀ values ranging from 11 to 45 µg/mL against various P. falciparum strains [51, 52]. Thus, the broad-ranging anti-infective activity of A. scholaris variety is a novel finding and distinct from studies from other parts of India and Asia. Furthermore, a low resistance index of 1.05 is a novel finding for A. scholaris extracts against multidrug-resistant P. falciparum, underscoring the potential to overcome antimalarial resistance.

We identified kolavenol and α-amyrin as the major compounds of A. scholaris. Kolavenol is a diterpenoid known to be an active principle isolated from plants such as Eupatorium adenophora, Entada abyssinica, and Kaempferia species. It is highly potent as an antiprotozoal compound against Trypanosoma brucei rhodesiense, the deadliest form of trypanosomiasis [53]. It has been shown to have anticancer effects on different carcinomas in mice [54], and to be highly cytotoxic against cancer cell lines while also exhibiting antibacterial activity [55]. α-Amyrin is a pentacyclic triterpenoid identified in several plants. It is widely recognized for its strong antihyperglycemic effect [56, 57] and anti-inflammatory action [58]. Other effects, including analgesic, antidepressant, gastroprotective, immunomodulatory, and hypolipidemic activities, are also documented [59, 60]. The predominance of α-amyrin and kolavenol, rather than indole alkaloids in other chemotypes, may explain the moderate but consistent multi-target activity and non-toxicity of the Mizoram variety, suggesting adaptation to the Indo-Burma hotspot environment.

Although it is not conclusive that the compounds identified are directly responsible for the multifaceted pharmacological activities of A. scholaris, the computational data support the general effects. Particularly, the high binding affinity of α-amyrin to key enzymes across kingdoms, such as −10.4 kcal/mol on fungal CYP51, −10.0 kcal/mol on malarial SAHH, and −9.4 kcal/mol on helminth GluCl, provides the first computational rationale for triterpenoid-driven broad-spectrum effects in the Mizoram variety of A. scholaris. Further molecular modeling, fractionation with isolation and individual-compound testing, including in vivo assays, will be essential for understanding precise binding interactions and other pharmacological properties. Our findings thus provide evidence that the Mizoram variety of A. scholaris is a source of lead molecules for the development of efficacious pharmaceutical drugs against a broad range of pathogens.