ABSTRACT
The increased resistance of A. fumigatus to antifungal agents highlights the need for the development of novel therapeutics from natural sources. The present study aimed to assess the antifungal activity of four Swertia species (S. chirayita, S. purpurascens, S. paniculata and S. cordata) using both in vitro and in silico methods. Agar well diffusion and minimum inhibitory concentration (MIC) tests were used to measure in vitro antifungal activity. Gas chromatography‒mass spectrometry (GC‒MS) analysis was performed to identify phytochemicals, which were subsequently docked against glucosamine‐6‐phosphate N‐acetyltransferase (Gna1), an essential enzyme in fungal cell wall biosynthesis. The pharmacokinetics and toxicity of the top candidates were further assessed via absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses. The extract of S. paniculata presented the greatest antifungal activity, followed by that of S. chirayita, whose inhibition diameters were 16.50 ± 0.4 mm and 13.33 ± 0.47 mm, respectively, at 80 mg/mL. GC‒MS profiling revealed epilupeol, γ‐sitosterol, and obacunone, which exhibited stronger binding affinities toward Gna1 than amphotericin B, which was used as the positive docking control. ADMET analysis identified fifteen phytochemicals with favorable drug‐likeness and safety profiles. Overall, Swertia species exhibit significant antifungal potential and may provide promising lead compounds for antifungal drug development.
Keywords: ADMET, Aspergillus fumigatus, glucosamine‐6‐phosphate N‐acetyl transferase, molecular docking, Swertia
Ethyl acetate extracts of various species of Swertia are evaluated for antifungal activity against Aspergillus fumigatus through in vitro and in silico approaches. The findings demonstrate significant antifungal potential and favorable molecular interactions, suggesting that Swertia species may serve as promising sources of bioactive antifungal compounds for antifungal drug development.
1. Introduction
Over the past few decades, the global incidence of fungal infections has risen markedly, posing serious challenges to human health, agriculture, and the environment [1]. Aspergillus fumigatus, an opportunistic fungus, has become a leading cause of respiratory tract infections, accounting for approximately 80% of aspergillosis cases, especially in immunocompromised individuals [2, 3]. The pathogenic effectiveness of A. fumigatus is attributed to various biological traits, such as the presence of tiny conidia, which can penetrate deep into the pulmonary tract, where they may evade being cleared by the mucociliary system [4, 5, 6]. The melanin in the cell wall protects them against phagocytosis and reactive oxygen species [7]. Also, negatively charged sialic acid, which is expressed on the surface, helps the fungus adhere to basal lamina proteins in the lungs of the host [8]. Its hydrophobic cell wall and ability to tolerate a wide range of pH levels and temperatures also help it be transmitted via air and remain viable in harsh environments [9, 10]. Infection by A. fumigatus contributes to numerous diseases, such as allergic bronchopulmonary aspergillosis, chronic pulmonary aspergillosis, invasive pulmonary aspergillosis, invasive fungal rhinosinusitis, central nervous system (CNS) aspergillosis, and Aspergillus arthritis [11, 12, 13]. The annual number of A. fumigatus infections is up to 16 million lung infections per year, with several hundred thousand of those infections ending in fatal outcomes [14, 15, 16, 17]. Individuals with a previous history of chronic pulmonary inflammatory disorders or Mycobacterium tuberculosis are more likely to develop chronic Aspergillus infections. These infections may result in permanent damage to the lungs and worsening of respiratory function [18, 19]. The treatment of Aspergillus infections depends mostly on a small group of antifungal drugs, such as azoles (such as voriconazole, posaconazole, and isavuconazole), polyenes (like amphotericin B), echinocandins (like caspofungin), and allylamines. Triazoles, especially voriconazole, are the first‐line treatment for invasive A. fumigatus infections because of their wide‐spectrum efficacy and bioavailability [20, 21].
However, the effectiveness of these interventions has become less reliable due to the rise of azole‐resistant A. fumigatus (ARAF) strains [22]. The prolonged use of azoles in hospitals, agriculture, and industry contributes to the development and dispersal of resistant strains [23, 24]. These resistant spores have the ability to cause aspergillosis even in people who have never been treated with azole drugs. This has led to changes in treatment guidelines, which suggest the use of liposomal amphotericin B as an alternative. Several antifungal drugs exhibit side effects or are expensive. For instance, liver enzyme levels were found to be elevated in up to 15% of patients given voriconazole, which is an indicator of liver toxicity. Other drugs, such as voriconazole and posaconazole, have also been associated with multiple organ toxicity [22]. Although isavuconazole and oteseconazole (VT‐1161) are newer triazoles that have shown better safety profiles in clinical and preclinical settings, their high cost and limited availability hinder their widespread use. Given the high cost of current treatments, potential toxicity, and increasing resistance, safer, less expensive, and more effective treatment options are urgently needed. One of the strategies that can be explored is the natural product approach, specifically the use of plant‐derived compounds, which have been historically acknowledged as important sources of pharmacologically active molecules and could provide new avenues for the discovery of new antifungal drugs [25, 26].
A promising strategy for antifungal drug development involves targeting the fungal cell wall, a structure essential for fungal survival but absent in humans; thus, it is an ideal selective therapeutic target [27]. One of the key enzymes involved in fungal cell wall biosynthesis is glucosamine‐6‐phosphate N‐acetyltransferase (Gna1), which plays a crucial role in the hexosamine biosynthetic pathway by catalyzing the conversion of glucosamine‐6‐phosphate into N‐acetyl‐glucosamine‐6‐phosphate, a precursor essential for chitin synthesis. Studies have established that Gna1 is essential for fungal survival, as A. fumigatus deletion mutants exhibit severe growth defects that can be reversed only through external supplementation with N‐acetylglucosamine [28, 29]. Furthermore, these mutants exhibit markedly reduced virulence in murine models, further emphasizing the critical role of Gna1 in fungal pathogenesis. Structural studies have revealed a unique and druggable pocket at the Gna1 dimer interface that is conserved across fungal species but is absent in the human counterpart, making it an ideal target for selective inhibition and a promising candidate for molecular docking studies aimed at discovering novel antifungal agents with high specificity and minimum toxicity [30]. To explore the natural sources of potential Gna1 inhibitors, the present study focused on four medicinal plants from the genus Swertia (family: Gentianaceae), which have been extensively used in traditional Indian systems of medicine. More specifically, four species of Swertia (Swertia chirayita, S. purpurascens, S. paniculata, and S. cordata) were selected because of their well‐documented ethnomedicinal applications in the treatment of fevers, liver disorders, skin diseases, and microbial infections [31]. These species are known to possess a variety of pharmacological activities, such as hepatoprotective, anti‐inflammatory, antidiabetic, and antimicrobial properties. However, their antifungal potential, particularly against A. fumigatus, is still largely unexplored. Therefore, the current study was designed to systematically evaluate the antifungal efficacy of ethyl acetate extracts of S. chirayita, S. purpurascens, S. paniculata, and S. cordata against Aspergillus fumigatus. This study integrates in vitro assays with phytochemical analysis via GC‒MS and molecular docking studies to identify phytochemicals that target the fungal enzyme glucosamine‐6‐phosphate N‐acetyltransferase (Gna1), Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET)‐based pharmacokinetic and toxicity evaluation. This study seeks to identify essential bioactive metabolites that may serve as promising scaffolds for the development of novel plant‐based antifungal agents against A. fumigatus.
2. Experimental Section
2.1. Plant Material
During the flowering season (July–September, 2024), the selected Swertia species—Swertia chirayita, S. purpurascens, S. paniculata, and S. cordata were collected from various locations in the Garhwal region of the Indian Himalayas. Chakrata (30°48'50.5“, 77°45'48.8”), Chopta (30.48’3682″, 79.20’0907″) and Mandal (30°29′08.08″, 79°17′34.46″) were some of the places where samples were taken. Healthy plant materials from their natural habitat were collected and stored in sterile and well‐labelled polyethene bags. Voucher specimens were submitted to the Botanical Survey of India (BSI), Northern Regional Centre, Dehradun, Uttarakhand, for authentication (Voucher numbers: 1723 to 1726).
2.2. Preparation of Extract
The collected samples were rinsed under running tap water, followed by sterile distilled water to remove any contaminants. The cleaned samples were then dried in the shade at room temperature for 15 to 20 days to preserve thermolabile compounds. After drying, the plant material was ground into a uniform powder and stored in airtight containers until further processing. For extraction, 25 grams of powdered material from each sample was subjected to Soxhlet extraction via ethyl acetate for a period of 6–10 h or until the solvent in the siphon tube appeared clear. The resulting extracts were filtered through a Whatman No. 1 filter and evaporated through a rotary evaporator. The crude dried extracts were then weighed, coded as SChEA, SPuEA, SPnEA and SCoEA for S. chirayita, S. purpurascens, S. paniculata, and S. cordata, respectively, and stored in sterile vials at 4°C for subsequent analyses.
2.3. Culture Maintenance
The fungal strain of Aspergillus fumigatus was procured from the Department of Microbiology at the Shri Guru Ram Rai Institute of Medical & Health Sciences, Dehradun, India. The fungus was sub‐cultured and kept on potato dextrose agar (PDA) slants at 4°C for maintenance. For antifungal susceptibility tests, conidia were taken from cultures that were 4 to 5 days old and cultivated on PDA plates at 28 ± 2°C. The surface of each colony was gently scraped using a sterile loop, and the conidia were suspended in sterilized normal saline. The spore concentration was adjusted to ∼1–5 × 104 spores/mL with the help of a hemocytometer to standardize the inoculation for further testing [32].
2.4. In Vitro Antifungal Activity
2.4.1. Agar Well Diffusion Method
The antifungal activity of the extracts was evaluated via the agar well diffusion method on PDA plates. A total of 20 mL of sterile potato dextrose agar (PDA) was poured into Petri dishes and allowed to solidify. The plates were surface inoculated with fungal spore suspensions (∼1–5 × 104 spores/mL) using a sterile cotton swab. Wells 6 mm in diameter were punched in the agar, and 50 µL of extract at four different concentrations (80, 40, 20, and 10 mg/mL) was added to the wells. Amphotericin B at 2 mg/mL was used as the positive control. The plates were incubated at 28 ± 2°C for 48–72 h, and zones of inhibition were measured in millimeters (mm) [33, 34].
2.4.2. Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of each ethyl acetate extract was determined by the broth microdilution method in a 96‐well microtiter plate in accordance with slightly modified Clinical and Laboratory Standards Institute (CLSI M38‐A2) guidelines for filamentous fungi. Initially, 100 µL of potato dextrose broth (PDB) was dispensed into each well of a 96‐well plate. Afterward, 100 µL of the plant extract at 80 mg/mL was added to the first well of each row (for a total volume of 200 µL). Twofold serial dilutions were then performed to obtain concentrations ranging from 40 to 0.039 mg/mL. After serial dilution, 100 µL of standardized fungal spore suspension (1–5 × 104 spores/mL) was added to each well, resulting in a final volume of 200 µL per well. For the positive control, Amphotericin B at 2 mg/mL was used under similar conditions; however, serial dilution was not performed for the standard. Wells with only PDB and fungal inoculum served as a negative control, while broth without fungal inoculum served as the sterility control. The plates were sealed and incubated at 28 ± 2°C for 48 h. The MIC was defined as the lowest concentration of the extract at which no visible fungal growth was observed [35]. The modification to the CLSI M38‐A2 guidelines included the use of PDB instead of RPMI 1640 media, while visual determination of fungal growth was used to determine the MIC instead of spectrophotometric measurement.
2.4.3. GC‐MS Analysis
To identify the phytochemical constituents of the extracts, gas chromatography–mass spectrometry (GC–MS) analysis was performed. The analysis was performed via an Agilent 5977C single quadrupole GC/MSD system chromatograph (Agilent Technologies, Wilmington, USA). For separation, an HP‐5MS UI column (30* 0.23 mm D* 0.25 µm) film was used. Helium served as the carrier gas, with a constant flow rate of 1.0 mL/min. For the first 8 min, the column temperature was maintained at 50°C and then gradually increased to 270°C at a steady rate of 5°C/min for 5 min. The injector and detector temperatures were 270°C and 220°C, respectively. One microliter of the sample was injected at a 50:1 split ratio. After 10 min of solvent delay, the detector was used in scan mode at m/z 50–500. The EI source was maintained at 230°C, and the MSD quadrupole was maintained at 150°C. By comparing the mass spectra and retention indices of the compounds with those found in the NIST mass spectral library, different compounds were identified [36].
2.4.4. Protein 3D Structure Preparation
The 3D structure of the protein from A. fumigatus, namely, glucosamine‐6‐phosphate N‐acetyl transferase, was retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/). Water molecules were removed while hydrogen molecules were added to the structure for molecular docking via PyMOL [37].
2.4.5. Ligand Preparation
A comprehensive catalog of natural compounds present in different species of Swertia was generated through GC‒MS profiling. The 3D structures of these compounds in SDF format were acquired from the PubChem compound database (https://pubchem.ncbi.nlm.nih.gov). PyMOL was subsequently used to convert the data into the Protein Data Bank (PDB file) format [37].
2.4.6. Molecular Docking
Molecular docking was used to evaluate the effectiveness of the interaction between the ligands (identified compounds) and glucosamine‐6‐phosphate N‐acetyl transferase (target protein). CB‐Dock2 was used to carry out the cavity search and blind docking procedures. (https://cadd.labsh are. cn/cb‐dock2/ php/ blind dock) [38].
2.4.7. In Silico Pharmacokinetic and Toxicity Assessment of Selected Phytocompounds
To predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the selected compounds, they were subjected to two online web tools (Swiss ADME and ProTox‐II). All the compounds under investigation were first converted into their canonical Simplified Molecular Input Line Entry System (SMILES) format and subsequently submitted to Swiss ADME and ProTox‐II [39].
2.4.8. Statistical Analysis
The tests were carried out in triplicate, and the means ± SDs were used to represent the data. The data were analyzed using one‐way ANOVA followed by Duncan's multiple range test (DMRT) to assess significant differences among treatment groups, with p < 0.05 considered to indicate statistical significance according to SPSS software version 16 [40].
3. Results
3.1. Antifungal Activity
The antifungal activity of ethyl acetate extracts from four Swertia species was evaluated against Aspergillus fumigatus via the agar well diffusion method at concentrations of 10, 20, 40, and 80 mg/mL. All the samples showed a concentration‐dependent increase in activity. Among them, SPnEA had the greatest and most consistent antifungal effect, with a zone of inhibition (ZOI) value of 16.50 ± 0.41 mm at 80 mg/mL, while SChEA also considerably inhibited the antifungal effect, with a ZOI value of 13.33 ± 0.47 mm at similar concentrations. Moreover, SCoEA and SPuEA demonstrated relatively lower inhibition, with SCoEA recording ZOIs of 12.80 ± 0.28 mm, and SPuEA produced the lowest inhibition values of 11.67 ± 0.47 mm at 80 mg/mL. These results clearly indicate that SPnEA was the most effective extract, whereas SPuEA was the least effective in terms of ZOI. The detailed results are presented in Table 1 and Figure 1.
TABLE 1.
Comparison of the zones of inhibition and MIC values of different extracts of Swertia species.
| Concentration | Zone of Inhibition (in mm) ± Standard Deviation | MIC (mg/mL) | |||
|---|---|---|---|---|---|
| 10 mg/mL | 20 mg/mL | 40 mg/mL | 80 mg/mL | ||
| Sample | |||||
| SChEA |
3.07 ± 0.49b |
8.83±0.24b |
9.90 ± 0.14b |
13.33 ± 0.47b |
5.00 |
| SPuEA |
1.83 ± 0.24c |
6.17±0.62c |
8.17 ± 0.24c |
11.67 ± 0.47c |
10.00 |
| SPnEA | 5.33 ± 0.47a | 9.83±0.24a |
13.17 ± 0.24a |
16.50 ± 0.41a |
2.5 |
| SCoEA |
2.17 ± 0.24c |
5.50±0.41c |
8.33 ± 0.47c |
12.80 ± 0.28b |
10.00 |
| Amphotericin B * (2 mg/mL) | — | — | — | 18.23 ± 0.25 | NA |
Amphotericin B was used as the positive control at a fixed concentration of 2 mg/mL. The values are expressed as the mean ± standard deviation (SD) (n = 3). Means within the same column followed by different superscript letters are significantly different according to one‐way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) at p < 0.05.
FIGURE 1.
Antifungal activity of ethyl acetate extracts of four Swertia species against Aspergillus fumigatus determined by the agar well diffusion method. (A) SChEA, (B) SPuEA (C) SPnEA, (D) SCoEA, (E) positive and negative control.
3.2. Minimum Inhibitory Concentration
To support these findings, the minimum inhibitory concentration (MIC) was determined through broth microdilution. SPnEA had the lowest MIC of 2.5 mg/mL, highlighting its potency even at low concentrations. SChEA had an MIC of 5 mg/mL, whereas both SPuEA and SCoEA had the highest MIC values of 10 mg/mL, which is consistent with their lower ZOI values. The MIC results further confirmed the superior efficacy of SPnEA, which combines strong inhibition with the minimal required dosage. The complete ZOI and MIC data are summarized in Table 1.
3.3. GC‒MS Analysis
The ethyl acetate extracts of S. paniculata, S. purpurascens, S. cordata, and S. chirayata contain a diverse array of bioactive compounds, as revealed by GC‒MS analysis. S. paniculata presented the highest abundance of epilupeol (30.5%), followed by γ‐sitosterol and palmitic acid derivatives, indicating a sterol‐ and triterpenoid‐rich profile, whereas S. chirayita presented notable levels of corymbiferin (17.5%), α‐linolenic acid, and 9,12‐octadecadienoic acid, reflecting a composition rich in phenolic xanthones and polyunsaturated fatty acids. S. cordata features unique nitrogen‐containing heterocycles, such as tri‐methoxyindazole and dimethoxy benzyl pyrimidines, along with palmitic and oleic acid derivatives. In contrast, S. purpurascens presented a relatively less complex profile dominated by cyclic hydrocarbons and oxygenated terpenoids with lower percent (%) area values. Across all the species, fatty acids such as palmitic, stearic, and octadecadienoic acids were recurrent, supporting their role in antifungal activity. Various compounds, such as palmitic acid derivatives, 9,12‐octadecadienoic acid (Z, Z) derivatives, 1‐hydroxy‐3,7,8‐trimethoxyxanthen‐9‐one, and behenic acid derivatives, have been identified across multiple species. A detailed list and chromatograms of the GC‒MS analysis results are provided in Tables S1–S4 and Figure S1.
3.4. Molecular Docking of Selected Compounds
Following GC‒MS profiling, we identified multiple bioactive compounds from the extract. Based on their relative abundance (% occurrence), those present in minimal amounts were excluded to focus on the major constituents. The selected compounds were subsequently subjected to molecular docking analysis against the target protein of A. fumigatus. Using CB‐Dock2, five potential binding cavities were predicted in the target protein, each of which was associated with a corresponding binding score. Among these, we selected the cavity offering the most favorable binding energy for further analysis [41, 42].
Docking evaluations revealed that most of the compounds derived from Swertia species exhibited promising binding affinities toward the glucosamine‐6‐phosphate N‐acetyltransferase of A. fumigatus. From the overall results, we highlighted the top fifteen compounds based on their binding energies, as detailed in Table 2, while the docking of the positive control and the top two compounds is represented in Figure 2, and the docking of all 15 selected compounds is represented in Figure S2.
TABLE 2.
Binding energies of the best 15 hits against Gna1 using the CB‐Dock2 tool.
| S. no. | Sample name | % Area |
Score (kcal/mol) |
Cavity vol. |
|---|---|---|---|---|
| S. chirayita of ethyl acetate extract | ||||
| 1 | Androstan‐17‐one,3‐ethyl‐3‐hydroxy‐, (5.α.) | 3.62 | −8.4 | 859 |
| 2 | Stigmasterol, TMS derivative | 1.35 | −8.8 | 859 |
| 3 | Obacunone | 0.20 | −9.0 | 859 |
| 4 | Norethindrone Acetate | 0.13 | −8.8 | 324 |
| 5 | Androst‐5‐en‐4‐one | 0.11 | −8.8 | 859 |
| S. purpurascens of ethyl acetate extract | ||||
| 6 | 24‐Norursa‐3,12‐diene | 6.82 | −9.2 | 859 |
| 7 | γ ‐Sitosterol | 1.98 | −9.4 | 859 |
| 8 | Olean‐18‐ene | 0.97 | −9.1 | 324 |
| 9 | 5α‐cyano‐3‐methoxymethylenecholestane, (E) | 0.56 | −9.8 | 589 |
| S. paniculate of ethyl acetate extract | ||||
| 10 | Epilupeol | 30.49 | −9.8 | 859 |
| 11 | D‐Friedoolean‐14‐en‐3‐one | 2.29 | −9.5 | 859 |
| 12 | Campesterol | 1.88 | −9.7 | 859 |
| 13 | Lup‐20(29)‐en‐3‐one | 1.43 | −9.0 | 859 |
| S. cordata of ethyl acetate extract | ||||
| 14 | β‐Sitosterol | 1.04 | −8.7 | 859 |
| 15 | Cholesta‐5,7,9(11)‐trien‐3‐ol acetate | 0.35 | −8.9 | 324 |
| * | Amphotericin B (standard) | — | −7.7 | 324 |
FIGURE 2.
Molecular docking interactions of the top two compounds from ethyl acetate extracts of Swertia species with the target protein glucosamine‐6‐phosphate N‐acetyl transferase (Gna1). Representative 3D and 2D interaction profiles are shown for the docked compounds [A] 5α‐cyano‐3‐methoxymethylenecholestane, (E), [B] Epilupeol, and [standard] Amphotericin B.
Among the top docked compounds, epilupeol from S. paniculata presented the highest peak area (30.49%) and strong binding affinity, with a docking score of −9.8 kcal/mol. It interacts with key residues within chain A of the protein, including LEU69, ILE131, ALA132, VAL133, GLY143, PHE173, TYR174, GLN138, LYS141, and LEU144, indicating a strong interaction with the target protein. Other compounds, such as 24‐Norursa‐3,12‐diene from S. purpurascens and γ‐sitosterol, presented good docking scores of −9.2 and −9.4 kcal/mol, respectively, and interacted with conserved residues located within the large cavity (859 Å3), including GLN138, GLY139, LYS141, LEU144, PHE173, TYR174, ILE131, VAL133, and ALA132. These residues are crucial for substrate recognition and stabilization of ligand binding. Compounds such as stigmasterol, the TMS derivative, obacunone, and norethindrone acetate displayed docking scores ranging between ‐8.8 and ‐9.0 kcal/mol and bound to residues similar to those of epilupeol, indicating their structural compatibility with the protein's active site. Notably, Obacunone, despite a low % area (0.20%), exhibited a strong binding affinity (−9.0 kcal/mol).
In S. cordata, β‐sitosterol and Cholesta‐5,7,9(11)‐trien‐3‐ol acetate also showed promising docking interactions, with scores of −8.7 and −8.9 kcal/mol, respectively. These compounds engaged with residues in both the large cavity (859 Å3) and the alternate pockets (324 Å3), further confirming the presence of multiple potential binding sites within the target protein. Compounds such as 5α‐cyano‐3‐methoxymethylenecholestane (E) and D‐Friedoolean‐14‐en‐3‐one also revealed favorable docking scores of ‐9.8 and −9.5 kcal/mol, respectively, with interaction patterns similar to those of epilupeol and γ‐sitosterol. Overall, most of the top‐performing compounds targeted the same conserved binding residues (VAL68 to LYS176), especially around the 859 Å3 cavity, indicating a common binding mode and suggesting that these residues play a pivotal role in ligand recognition. Compounds such as epilupeol, 24‐norursa‐3,12‐diene, γ‐sitosterol, and obacunone have emerged as promising candidates on the basis of their binding energy, cavity occupation, and interaction profile. Compared with the positive control, all the top‐hit compounds presented greater negative binding affinities, indicating stronger and more stable interactions with the target protein.
3.5. Pharmacokinetic Profiling of Selected Compounds
The drug‐likeness profiles and physicochemical properties of the selected compounds were evaluated to assess their suitability as potential antifungal agents. The molecular weights (MWs) of the compounds ranged from 272.43 to 484.87 g/mol, which is an acceptable range for oral drugs, as suggested by Lipinski's rule of five.
Compounds such as Obacunone and Norethindrone Acetate had multiple hydrogen bond acceptors (HBAs), indicating the potential for polar interactions, whereas several compounds (e.g., 24‐Norursa‐3,12‐diene, Olean‐18‐ene, 5α‐cyano‐3‐methoxymethylenecholestane, (E)‐ had zero donors and acceptors, suggesting limited hydrogen bonding capacity. The topological polar surface area (TPSA) values also varied, with Obacunone having the highest TPSA (95.34 Å2), potentially contributing to good solubility and cell permeability, whereas several compounds, such as 24‐Norursa‐3,12‐diene and olean‐18‐ene, had TPSA values close to zero, suggesting high lipophilicity and poor aqueous solubility.
In terms of lipophilicity, most compounds presented logP values (iLOGP, XLOGP3, WLOGP, and MLOGP) within the acceptable drug‐like range. However, compounds such as stigmasterol, the TMS derivative 24‐Norursa‐3,12‐diene, olean‐18‐ene and 5α‐cyano‐3‐methoxymethylenecholestane (E)‐ exhibited very high logP values, particularly XLOGP3 and WLOGP (exceeding 8 in some cases), indicating extreme lipophilicity, which might affect solubility and bioavailability.
All the compounds presented zero pan‐assay interference compounds (PAINS) alerts, suggesting a low likelihood of producing false positives in bioassays and thus having better pharmacological specificity. Further evaluation of absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties provided critical insights into their pharmacokinetic behavior. Compounds such as Androstan‐17‐one, 3‐ethyl‐3‐hydroxy‐, (5.α.), Obacunone, Norethindrone Acetate and Androst‐5‐en‐4‐one demonstrated high gastrointestinal (GI) absorption, which is favorable for orally administered drugs. Permeability of the blood–brain barrier (BBB) was observed for compounds like Androstan‐17‐one,3‐ethyl‐3‐hydroxy‐, (5.α.), Norethindrone acetate and Androst‐5‐en‐4‐one, which suggests their potential to act on central nervous system (CNS) targets or possibly lead to CNS‐related side effects. The P‐glycoprotein (P‐gp) substrate prediction was positive only for obacunone, suggesting its susceptibility to efflux and a potential reduction in intracellular concentration. CYP enzyme inhibition profiling revealed minimal interactions overall, with a few exceptions. For example, androst‐5‐en‐4‐one inhibited CYP2C19 and CYP2C9, whereas norethindrone acetate inhibited CYP2C9. Notably, none of the compounds inhibited CYP3A4, which is favorable since inhibition of this enzyme is often associated with clinically relevant drug–drug interactions. The skin permeability coefficient (log Kp) ranged from ‐1.06 for olean‐18‐ene to ‐6.83 for obacunone, with more negative values indicating poorer skin permeability. Most of the compounds complied well with the Lipinski, Veber, Egan, Ghose, and Muegge rules. However, Stigmasterol, a TMS derivative with four Ghose parameters and one Lipinski rule, showed two Muegge violations, indicating its deviation from the ideal drug‐like space. A detailed list of the different pharmacological properties of the selected compounds determined via Swiss ADMET analysis is provided in Table 3.
TABLE 3.
Different parameters of the pharmacological properties of the selected compounds determined by using Swiss ADMET analysis.
| Compound | GI absorption | BBB permeant | Pgp substrate | CYP1A2 inhibitor | CYP2C19 inhibitor | CYP2C9 inhibitor | CYP2D6 inhibitor | CYP3A4 inhibitor | log Kp (cm/s) | Lipinski #violations | Ghose #violations | Veber #violations | Egan #violations | Muegge #violations |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Androstan‐17‐one,3‐ethyl‐3‐hydroxy‐, (5.α.) | High | Yes | No | No | No | No | No | No | −5.12 | 1 | 0 | 0 | 0 | 0 |
| Stigmasterol, TMS derivative | Low | No | No | No | No | No | No | No | −1.79 | 1 | 4 | 0 | 1 | 2 |
| Obacunone | High | No | Yes | No | No | No | No | No | −6.83 | 0 | 0 | 0 | 0 | 0 |
| Norethindrone Acetate | High | Yes | No | No | No | Yes | No | No | −5.79 | 0 | 0 | 0 | 0 | 0 |
| Androst‐5‐en‐4‐one | High | Yes | No | No | Yes | Yes | No | No | −4.06 | 1 | 0 | 0 | 0 | 2 |
| 24‐Norursa‐3,12‐diene | Low | No | No | No | No | No | No | No | −2.25 | 1 | 2 | 0 | 1 | 2 |
| γ‐Sitosterol | Low | No | No | No | No | No | No | No | −2.2 | 1 | 3 | 0 | 1 | 2 |
| Olean‐18‐ene | Low | No | No | No | No | No | No | No | −1.06 | 1 | 3 | 0 | 1 | 2 |
| 5 α‐cyano‐3‐methoxymethylenecholestane, (E) | Low | No | No | No | No | No | No | No | −2.47 | 1 | 3 | 0 | 1 | 1 |
| Epilupeol | Low | No | No | No | No | No | No | No | −1.9 | 1 | 3 | 0 | 1 | 2 |
| D‐Friedoolean‐14‐en‐3‐one | Low | No | No | No | No | No | No | No | −2.51 | 1 | 3 | 0 | 1 | 2 |
| Campesterol | Low | No | No | No | No | No | No | No | −2.5 | 1 | 2 | 0 | 1 | 2 |
| Lup‐20(29)‐en‐3‐one | Low | No | No | No | No | No | No | No | −2.1 | 1 | 3 | 0 | 1 | 2 |
| β ‐Sitosterol | Low | No | No | No | No | No | No | No | −2.2 | 1 | 3 | 0 | 1 | 2 |
| Cholesta‐5,7,9(11)‐trien‐3‐ol acetate | Low | No | No | No | No | Yes | No | No | −3.24 | 1 | 3 | 0 | 1 | 1 |
Moreover, the compounds Androstan‐17‐one,3‐ethyl‐3‐hydroxy‐, (5.α.), Obacunone, norethindrone acetate, and Androst‐5‐en‐4‐one showed favorable drug‐likeness profiles and good pharmacokinetic behavior, particularly in terms of high GI absorption, low or no CYP inhibition, acceptable hydrogen bonding and polarity, and limited rule violations. These compounds merit further consideration for in vitro and in vivo evaluation. Other compounds with low GI absorption and multiple rule violations (such as the compounds stigmasterol, TMS derivatives, olean‐18‐ene and 5α‐cyano‐3‐methoxymethylenecholestane, (E)), may require structural optimization or formulation strategies to enhance their drug‐like properties. The physicochemical properties of the selected compounds were visualized via a radar plot (Figure S3). The plot highlights key parameters, such as molecular size (SIZE), lipophilicity (LIPO), solubility (INSOLU), polarity (POLAR), molecular flexibility (FLEX) and unsaturation (INSATU). This graphical representation provides an overview of the compound's drug‐likeness and structural balance.
3.6. Toxicity Study
The results of the toxicity study indicated that the majority of the tested compounds are nontoxic, with no predicted hepatotoxicity, mutagenicity or cytotoxicity. However, Norethindrone acetate, Androst‐5‐en‐4‐one, and Cholesta‐5,7,9(11)‐trien‐3‐ol acetate showed a potential risk of carcinogenicity, whereas the remaining compounds were predicted to be inactive across all toxicity parameters. This suggests a generally safe profile for most compounds, with only a few requiring careful consideration due to their carcinogenic predictions [43, 44]. The detailed list is given in Table 4.
TABLE 4.
Toxicity prediction and the probability of selected compounds by using Protox‐II.
| Toxicity report (probability/prediction) | |||||
|---|---|---|---|---|---|
| Compound | Hepatotoxicity | Carcinogenicity | Mutagenicity | Cytotoxicity | LD50 (mg/kg) |
| Androstan‐17‐one,3‐ethyl‐3‐hydroxy‐, (5.α.) | Inactive | Inactive | Inactive | Inactive |
3000 |
| Stigmasterol, TMS derivative | Inactive | Inactive | Inactive | Inactive | 2000 |
| Obacunone | Inactive | Inactive | Inactive | Inactive | 555 |
| Norethindrone Acetate | Inactive | Active | Inactive | Inactive | 4000 |
| Androst‐5‐en‐4‐one | Inactive | Active | Inactive | Inactive | 2450 |
| 24‐Norursa‐3,12‐diene | Inactive | Inactive | Inactive | Inactive | 5000 |
| γ‐Sitosterol | Inactive | Inactive | Inactive | Inactive | 890 |
| Olean‐18‐ene | Inactive | Inactive | Inactive | Inactive | 5000 |
| 5 α‐cyano‐3‐methoxymethylenecholestane, (E) | Inactive | Inactive | Inactive | Inactive | 5000 |
| Epilupeol | Inactive | Inactive | Inactive | Inactive | 2000 |
| D‐Friedoolean‐14‐en‐3‐one | Inactive | Inactive | Inactive | Inactive | 5000 |
| Campesterol | Inactive | Inactive | Inactive | Inactive | 890 |
| Lup‐20(29)‐en‐3‐one | Inactive | Inactive | Inactive | Inactive | 5000 |
| β ‐Sitosterol | Inactive | Inactive | Inactive | Inactive | 890 |
| Cholesta‐5,7,9(11)‐trien‐3‐ol acetate | Inactive | Active | Inactive | Inactive | 1185 |
| *Amphotericin B | Inactive | Inactive | Active | Inactive | 100 |
4. Discussion
Owing to the extensive use of antifungal drugs, Azole‐resistant A. fumigatus (ARAF) strains have emerged as serious threats to global health. Plant‐based compounds act through multiple mechanisms, such as disruption of the cell wall, changes in membrane permeability and inhibition of biofilm formation, making them suitable candidates for the development of antifungal drugs [39, 45].
The results of the antifungal tests confirmed that all the extracts inhibited fungal growth in a dose‐dependent manner, with S. paniculata showing the highest activity (MIC of 2.5 mg/mL). Its potency was strongly related to its phytochemical composition, as GC‒MS confirmed the high contents of epilupeol (30.5%), γ‐sitosterol (10.8%), and palmitic acid (9%). Notably, epilupeol has both abundant and effective docking activity (−9.8 kcal/mol) and was previously reported to have antimicrobial and anti‐inflammatory activities [46, 47]. In combination with γ‐sitosterol (−9.4 kcal/mol), these compounds are most likely responsible for the increased antifungal activity of S. paniculata. Research has also shown that β‐sitosterol, a structurally similar γ‐sitosterol, inhibits the germination of spores and reduces germ‐tube elongation by 65% at 50 µg/mL and an effective dosage (ED50) of approximately 31 µg/mL in the fungi Aspergillus niger and Botryodiplodia theobromae [48]. These observations further support the possible role of phytosterols such as γ‐sitosterol in inhibiting fungal development and morphogenesis, thus supporting their possible role in antifungal action, as observed in this research.
In the case of S. chirayita and S. purpurascens, activity is seemingly the result of synergy between fatty acids and xanthones. Their fatty acid extracts were characterized by corymbiferin (14–17%) and fatty acids such as palmitic (12–13%) and α‐linolenic acids (6–13%). Other fatty acids, such as 9,12‐octadecadienoic acid, stearic acid, and behenic acid, have been reported to impair fungal membranes and diminish virulence features, such as biofilm formation [49, 50], and xanthones, such as corymbiferin, have antimicrobial and antioxidant properties [31]. Notably, Obacunone, despite being found at trace levels (0.2%) in the extracts of Swertia, demonstrated high docking affinity (−9.0 kcal/mol) toward the Gna1 enzyme of A. fumigatus. This observation highlights the promising role of minor compounds in promoting antifungal activity. Recent studies on Obacunone have established it as a broad‐spectrum antimicrobial agent. It interferes with important virulence pathways and microbial signaling, inhibits enzyme function, and disrupts membrane integrity [51, 52, 53]. In addition, obacunone has been shown to have antagonistic effects on the growth of the pathogenic fungus Candida albicans [52]. In combination with abundant compounds, such as epilupeol or γ‐sitosterol, obacunone can augment general antifungal activity via synergistic interactions. The contribution of obacunone in this research, therefore, advances the general idea that even low‐abundance metabolites are capable of disproportionate biological action, especially when used in the integration of the intricate chemical landscape of plant extracts.
S. cordata was also quite different, with a predominance of palmitic acid (15.5%) and indazole derivatives (14.7%) and minor levels of sterols such as β‐sitosterol (1%). Its reduced antifungal activity was consistent with the relative lack of high‐affinity docking molecules. However, cholesta‐5,7,9(11)‐trien‐3‐ol acetate and β‐sitosterol, among other compounds, exhibited intense docking interactions, indicating that even though they are less common, they still play important roles in antifungal activity. The possible carcinogenicity of cholesta‐5,7,9(11)‐trien‐3‐ol acetate, however, highlights the necessity of safety screening during natural product drug discovery.
Docking studies highlighted the importance of glucosamine‐6‐phosphate N‐acetyltransferase (Gna1) as a viable target in A. fumigatus. Several compounds, such as epilupeol, γ‐sitosterol, and obacunone, interact with conserved residues of the large 859 Å3 pocket of Gna1 with affinities that exceed those of amphotericin B (−7.7 kcal/mol). This implies a mechanism other than ergosterol binding, the conventional target of polyenes and azoles, suggesting a potentially unique antifungal mechanism [29]. In addition, several other compounds were identified across all the extracts as having established antifungal properties. Fatty acids such as palmitic acid, 9,12‐octadecadienoic acid, stearic acid, and behenic acid disrupt fungal membranes and inhibit virulence pathways [46, 47]. Terpenoids and phytosterols such as epilupeol, γ‐sitosterol, D‐fryoolean‐14‐en‐3‐one, and stigmasterol have shown antifungal activity in previous studies, often by disrupting membrane integrity or interfering with fungal metabolism [39]. Although the antifungal potential of corymbiferin has not been documented, its structural similarity to other bioactive xanthones suggests that it could be explored as an antifungal agent in the future.
ADMET predictions also corroborated the drug‐likeness of a number of compounds. Obacunone exhibited excellent gastrointestinal absorption, low toxicity, and a lack of PAINS alerts, which is indicative of good pharmacological characteristics, whereas epilupeol and γ‐sitosterol showed relatively low GI absorption but favorable docking affinities and toxicity profiles. The relatively benign profiles of these compounds contrast with the limitations of amphotericin B, which, despite its broad spectrum, is constrained by nephrotoxicity and infusion‐related adverse reactions [54]. Moreover, the low likelihood of CYP450 inhibition among the tested phytochemicals is advantageous compared with azoles, which often cause drug–drug interactions. Together, these features support the therapeutic potential of Swertia‐derived metabolites. From a socio‐economic perspective, these findings underscore the potential of Swertia‐derived metabolites as cost‐effective antifungal alternatives. Current antifungal therapies are often expensive and associated with adverse effects that increase the economic burden on healthcare systems [55], particularly in low‐ and middle‐income countries [56]. The use of medicinal plants that are regionally available and traditionally utilized offers a sustainable route for drug development [57, 58]. These phytochemicals could improve treatment accessibility, support local cultivation and reduce drug costs. This approach, in the long term, may contribute to affordable antifungal strategies while simultaneously promoting conservation [59, 60].
Despite these promising findings, certain limitations should be noted. The current research was limited to in vitro assays and computational predictions, which do not fully replicate the conditions of real infection. in vivo validation is thus critical for determining efficacy, pharmacokinetics, and safety under physiological conditions. Additionally, the antifungal activity of individual compounds was not assessed and should be examined in future studies. Furthermore, only ethyl acetate extracts were examined. Subsequent studies should compare various solvents for extraction, explore synergistic interactions, and use in vivo models to elucidate the true therapeutic potential of these compounds. Also, compounds like epilupeol, γ‐sitosterol, and obacunone could be promising candidates for future studies because they are either highly abundant or have good docking and ADMET predictions. To increase solubility, formulation strategies and screening against other pathogens would further increase the importance of such compounds. In addition, to ensure sustainable use, conservation methods for Swertia species should be integrated into future work.
Overall, this investigation highlights the antifungal potential of Swertia species. Evidence from phytochemical profiling, molecular docking, and ADMET prediction revealed that both major and minor metabolites are responsible for antifungal activity. Although additional confirmation is needed, these results constitute an important step toward the integration of ethnomedicinal knowledge and contemporary antifungal drug discovery.
5. Conclusion
This study revealed that species of Swertia are potential sources of antifungal agents against A. fumigatus. In particular, S. paniculata had the highest zone of inhibition (16.50 ± 0.41 mm) at 80 mg/mL and the lowest MIC (2.5 mg/mL), followed by S. chirayita. Phytochemical and in silico analyses revealed that phytochemicals such as epilupeol, γ‐sitosterol, obacunone, and norethindrone acetate exhibited significant binding affinities with the Gna1 enzyme, frequently exceeding those of amphotericin B. Compounds such as epilupeol and γ‐sitosterol were present in abundance and were effective against the target protein. While obacunone was present in less quantity, it was associated with significant interactions in terms of docking. These findings suggest that both major and minor metabolites might play a significant role in antifungal activity.
ADMET and toxicity studies have also indicated the drug‐likeness and safety of several key compounds, especially epilupeol and obacunone, which have strong binding and favorable pharmacokinetics. These outcomes suggest that metabolites from Swertia could potentially be promising candidates for antifungal drug development. Beyond the scientific relevance of the study, the results carry socio‐economic significance by underscoring the potential for affordable, plant‐derived antifungal agents. Specially benefiting the low and middle‐income regions where the cost and toxicity of current antifungal drugs remain major concerns. Future studies must focus on the evaluation of the antifungal activity of individual phytoconstituents with in vivo validation, the enhancement of their pharmacological attributes, and the sustainable sourcing or cultivation of Swertia species to facilitate drug development initiatives.
Author Contributions
Divyansh Panthari and Vinita Gouri: validation, methodology, investigation, formal analysis, data curation.Kanchan Bhardwaj, Subhash Chandra and Sarkar M. A. Kawsar: writing – review and editing, writing – original draft, conceptualization, supervision.
Funding
The authors have nothing to report.
Ethics Statement
The authors have nothing to report.
Consent
The authors have nothing to report
Conflicts of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Generative AI Statement
AI‐assisted tools were used for language editing and generation of the graphical abstract during manuscript preparation. The authors take full responsibility for the originality and content of the manuscript.
Supporting information
The supplementary material, Tables S1–S4 and Figures S1, S2 and S3, is provided along with the manuscript submission.
Supporting File 1: cbdv71468‐sup‐0001‐SuppMat.docx
Acknowledgments
The authors sincerely thank the Department of Botany and Chemistry at Shri Guru Ram Rai University in Dehradun, Uttarakhand, for its laboratory and logistical support.
Contributor Information
Kanchan Bhardwaj, Email: kanchankannu1992@gmail.com.
Subhash Chandra, Email: subhashkothiyal@gmail.com.
Sarkar M. A. Kawsar, Email: akawsarabe@yahoo.com.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
References
- 1. Colley T., Sehra G., Chowdhary A., et al., “In Vitro and In Vivo Efficacy of a Novel and Long‐Acting Fungicidal Azole, PC1244, on Aspergillus fumigatus Infection,” Antimicrobial Agents and Chemotherapy 62, no. 5 (2018): 1941–2017, 10.1128/AAC.01941-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Pimienta D. A., Cruz Mosquera F. E., Palacios Velasco I., Giraldo Rodas M., Oñate‐Garzón J., and Liscano Y., “Specific Focus on Antifungal Peptides Against Azole Resistant Aspergillus fumigatus: Current Status, Challenges, and Future Perspectives,” Journal of Fungi 9, no. 1 (2022): 42, 10.3390/jof9010042. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Lamoth F., “ Aspergillus fumigatus‐related Species in Clinical Practice,” Frontiers in Microbiology 7 (2016): 683, 10.3389/fmicb.2016.00683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Shi C., Shan Q., Xia J., et al., “Incidence, Risk Factors and Mortality of Invasive Pulmonary Aspergillosis in Patients With Influenza: A Systematic Review and Meta‐Analysis,” Mycoses 65 (2022): 152–163, 10.1111/myc.13410. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Calderón‐Parra J., Mills‐Sanchez P., Moreno‐Torres V., et al., “COVID‐19‐Associated Pulmonary Aspergillosis (CAPA): Risk Factors and Development of a Predictive Score for Critically Ill COVID‐19 Patients,” Mycoses 65 (2022): 541–550, 10.1111/myc.13434. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Latgé J.‐P. and Chamilos G., “ Aspergillus fumigatus and Aspergillosis in 2019,” Clinical Microbiology Reviews 33 (2019): e00140–e00218, 10.1128/CMR.00140-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Chamilos G., Akoumianaki T., Kyrmizi I., Brakhage A., Beauvais A., and Latge J.‐P., “Melanin Targets LC3‐Associated Phagocytosis (LAP): A Novel Pathogenetic Mechanism in Fungal Disease,” Autophagy 12, no. 5 (2016): 888–889, 10.1080/15548627.2016.1157242. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Warwas M. L., Watson J. N., Bennet A. J., and Moore M. M., “Structure and Role of Sialic Acids on the Surface of Aspergillus fumigatus Conidiospores,” Glycobiology 17, no. 4 (2007): 401–410, 10.1093/glycob/cwl085. [DOI] [PubMed] [Google Scholar]
- 9. Kwon‐Chung K. J. and Sugui J. A., “Aspergillus fumigatus—What Makes the Species a Ubiquitous Human Fungal Pathogen?,” PLoS Pathogens 9, no. 12 (2013): e1003743, 10.1371/journal.ppat.1003743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Fernando L. D., Pérez‐Llano Y., Dickwella Widanage M. C., et al., “Structural Adaptation of Fungal Cell Wall in Hypersaline Environment,” Nature Communications 14, no. 1 (2023): 7082, 10.1038/s41467-023-42693-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Rudramurthy S. M., Paul R. A., Chakrabarti A., Mouton J. W., and Meis J. F., “Invasive Aspergillosis by Aspergillus Flavus: Epidemiology, Diagnosis, Antifungal Resistance, and Management,” Journal of Fungi 5, no. 3 (2019): 55, 10.3390/jof5030055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Gago S., Denning D. W., and Bowyer P., “Pathophysiological Aspects of Aspergillus Colonization in Disease,” Medical Mycology 57, no. S2 (2019): S219–S227, 10.1093/mmy/myy076. [DOI] [PubMed] [Google Scholar]
- 13. Bocci M. G., Cascarano L., Capecchi G., et al., “Pulmonary Aspergillosis in Immunocompromised Critically Ill Patients: Prevalence, Risk Factors, Clinical Features and Diagnosis—A Narrative Review,” Journal of Fungi 11 (2025): 617, 10.3390/jof11090617. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Lowes D., Al‐Shair K., Newton P. J., et al., “Predictors of Mortality in Chronic Pulmonary Aspergillosis,” European Respiratory Journal 49, no. 2 (2017): 1601062, 10.1183/13993003.01062-2016. [DOI] [PubMed] [Google Scholar]
- 15. Denning D. W., Cadranel J., Beigelman‐Aubry C., et al., “Chronic Pulmonary Aspergillosis: Rationale and Clinical Guidelines for Diagnosis and Management,” European Respiratory Journal 47 (2016): 45–68, 10.1183/13993003.00583-2015. [DOI] [PubMed] [Google Scholar]
- 16. Denning D. W. and Chakrabarti A., “Pulmonary and Sinus Fungal Diseases in Non‐Immunocompromised Patients,” The Lancet Infectious Diseases 17 (2017): e357–e366, 10.1016/S1473-3099(17)30309-2. [DOI] [PubMed] [Google Scholar]
- 17. Breuer O., Schultz A., Garratt L. W., et al., “Aspergillus Infections and Progression of Structural Lung Disease in Children With Cystic Fibrosis,” American Journal of Respiratory and Critical Care Medicine 201, no. 6 (2020): 688–696, 10.1164/rccm.201908-1585OC. [DOI] [PubMed] [Google Scholar]
- 18. Kaur K., Sharma S. K., and Aggarwal A., “ Aspergillus Species and Lung Disease in Children,” Mycopathologia 188, no. 2 (2023): 225–238, 10.1007/s11046-023-00794-4. [DOI] [Google Scholar]
- 19. Agarwal A., Vishwanath S., Aggarwal S., et al., “Itraconazole vs Prednisolone in Patients With Allergic Bronchopulmonary Aspergillosis: A Randomized Trial,” Clinical Respiratory Journal 12, no. 1 (2018): 247–254, 10.1111/crj.12512.27401776 [DOI] [Google Scholar]
- 20. Agarwal R., Chakrabarti A., Shah A., et al., “Allergic Bronchopulmonary Aspergillosis: Review of Literature and Proposal of New Diagnostic and Classification Criteria,” Clinical & Experimental Allergy 43, no. 8 (2013): 850–873, 10.1111/cea.12141. [DOI] [PubMed] [Google Scholar]
- 21. Fairs A., Agbetile J., Hargadon B., et al., “IgE Sensitization to Aspergillus fumigatus Is Associated With Reduced Lung Function in Asthma,” American Journal of Respiratory and Critical Care Medicine 182, no. 10 (2010): 1362–1368, 10.1164/rccm.201003-0422OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Chakrabarti A. and Kaur S., “Allergic Aspergillosis of the Respiratory Tract,” European Respiratory Review 25, no. 139 (2016): 119–132, 10.1183/16000617.00009515. [DOI] [Google Scholar]
- 23. Agarwal A. and Chakrabarti A., “Allergic Bronchopulmonary Aspergillosis in Asthma: Epidemiological, Clinical and Therapeutic Issues,” Future Microbiology 8, no. 2 (2013): 1463–1474, 10.2217/fmb.13.112. [DOI] [PubMed] [Google Scholar]
- 24. Agarwal R., Aggarwal A., Dhooria A., et al., “Role of Oral Corticosteroids in Management of Allergic Bronchopulmonary Aspergillosis: Systematic Review and Meta‐Analysis,” Mycopathologia 181, no. 5–6 (2016): 343–356, 10.1007/s11046-016-0045-y. [DOI] [Google Scholar]
- 25. Marfatia M., Nakhawa A., and Patel P., “Allergic Fungal Sinusitis: A Review,” Indian Journal of Otolaryngology and Head & Neck Surgery 76, no. S1 (2024): S85–S92, 10.1007/s12070-023-03505-9. [DOI] [Google Scholar]
- 26. Pant A., Sharma R., and Dhiman N., “Allergic Fungal Rhinosinusitis: Clinical Presentation, Diagnosis and Management,” Egyptian Journal of Otolaryngology 38 (2022): 20, 10.1186/s43163-022-00222-0. [DOI] [Google Scholar]
- 27. Agarwal A., Chakrabarti A., and Shah M., “Allergic Bronchopulmonary Aspergillosis: Review of 20 Years' Experience,” Indian Journal of Chest Diseases and Allied Sciences 39, no. 1 (1997): 25–32. [Google Scholar]
- 28. Shah A. and Panjabi R., “Allergic Bronchopulmonary Aspergillosis: A Review of a Disease With a Worldwide Distribution,” Journal of Asthma 39, no. 4 (2002): 273–289, 10.1081/JAS-120002200. [DOI] [PubMed] [Google Scholar]
- 29. Greenberger S., “Allergic Bronchopulmonary Aspergillosis,” Journal of Allergy and Clinical Immunology 110, no. 5 (2002): 685–692, 10.1067/mai.2002.129733. [DOI] [PubMed] [Google Scholar]
- 30. Agarwal A., Khan D., Aggarwal A., et al., “Clinical Significance of Aspergillus Sensitization in Bronchial Asthma,” Mycoses 57, no. 11 (2014): 645–655, 10.1111/myc.12202. [DOI] [PubMed] [Google Scholar]
- 31. Agarwal A., Behera R., Aggarwal A., et al., “Clinical Significance of Hyperattenuating Mucus in Allergic Bronchopulmonary Aspergillosis: An Analysis of 155 Patients,” Chest 146, no. 5 (2014): 1106–1113, 10.1378/chest.13-2720. [DOI] [PubMed] [Google Scholar]
- 32. Pant H., Negi S., Saklani K., Chandra S., de Lima L. R., and Coutinho H. D. M., “Chemical Composition and Pharmacological Activities of Ficus Racemosa Fruit Methanolic Extract,” Pharmacological Research—Natural Products 6 (2025): 100163, 10.1016/j.prenap.2025.100163. [DOI] [Google Scholar]
- 33. Qureshi K. A., Parvez A., Ismatullah H., Almahasheer H., and Al Rugaie O., “Exploring the Antimicrobial and Antibiofilm Potency of Four Essential Oils Against Selected Human Pathogens Using in Vitro and in Silico Approaches,” PLoS ONE 20, no. 4 (2025): e0315663, 10.1371/journal.pone.0315663. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Saklani S., Chandra S., Mishra A. P., et al., “Nutritional Evaluation, Antimicrobial Activity and Phytochemical Screening of Wild Edible Fruit of Myrica nagi Pulp,” International Journal of Pharmacy and Pharmaceutical Sciences 4, no. 3 (2012): 407–411, 10.7897/2277-4343.04363. [DOI] [Google Scholar]
- 35. Khan H., Kaleem M., Khan A., et al., “GC‐MS/MS Based Identification of Bioactive Principles of Chloroform Fraction of Swertia Chirayatia (Chirata),” Oriental Journal of Chemistry 32 (2016): 875–883, 10.13005/ojc/320213. [DOI] [Google Scholar]
- 36. Bisht V. K., Bhandari A. K., Bisht R. S., et al., “HPLC Analysis and Antibacterial Activity of Methanolic Extract of Centella asiatica (L.) Urban,” Pharmacological Research—Natural Products 5 (2024): 100106, 10.1016/j.prenap.2024.100106. [DOI] [Google Scholar]
- 37. Gouri V., Roy G., Kanojia A., et al., “Identification of Novel Inhibitors From Rubus ellipticus as Anti‐Leishmanial Agents Targeting DDX3‐DEAD Box RNA Helicase of Leishmania donovani ,” BioTechniques 15, no. 1 (2025): 18, 10.1007/s13205-024-04183-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Gouri V., Patne K., Singh S., Kanojia A., Muthuswami R., and Samant M., “Molecular Docking of Berberis asiatica‐Derived Compounds Targeting DDX3 DEAD Box RNA Helicase of Leishmania donovani,” In Silico Pharmacology 13, no. 2 (2025): 92, 10.1007/s40203-025-00377-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Tan L. F., Yap V. I. L., Rajagopal M., et al., “Plant as an Alternative Source of Antifungals Against Aspergillus Infections: A Review,” Plants 11, no. 22 (2022): 3009, 10.3390/plants11223009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40. Chandra S., Saklani S., and Mathur A., “Study on Nutritional and Phytochemical Profile of Seven Edible Food Supplements of Uttarakhand (Garhwal Himalaya),” Vegetos 34, no. 3 (2021): 678–683, 10.5958/2229-4473.2021.00083. [DOI] [Google Scholar]
- 41. Karim T., Almatarneh M. H., Rahman S., et al., “ In Silico Prediction of Antibacterial Activity of Quinolone Derivatives,” ChemistrySelect 9 (2024): e202402780, 10.1002/SLCT.202402780. [DOI] [Google Scholar]
- 42. Saha S. and Kawsar S. M. A., “Natural Flavonoids as Primary Amoebic Meningoencephalitis Inhibitor: Virtual Screening, Molecular Docking, MD Simulation, MMPBSA, Density Functional Theory, Principal Component, and Gibbs Free Energy Landscape Analyses,” Chemistry & Biodiversity 22 (2025): e202402521, 10.1002/CBDV.202402521. [DOI] [Google Scholar]
- 43. Chandra S. and Saklani S., “Evaluation of the Anti‐Inflammatory Activity of the Methanolic Extract of the Fruits of Ficus palmata ,” British Journal of Pharmaceutical Research 12, no. 4 (2016): 1–5, 10.9734/BJPR/2016/25727. [DOI] [Google Scholar]
- 44. Kawsar S. M. A., Huq E., and Nahar N., “Cytotoxicity Assessment of the Aerial Parts of Macrotyloma Uniflorum Linn,” International Journal of Pharmacology 4, no. 4 (2008): 297–300, 10.3923/ijp.2008.297.300. [DOI] [Google Scholar]
- 45. Long N. and Li F., “Antifungal Mechanism of Natural Products Derived From Plants: A Review,” Natural Product Communications 19, no. 8 (2024): 1–12, 10.1177/1934578X241271747. [DOI] [Google Scholar]
- 46. Szreder J., Woźniak K., Erfurt K., Grymel M., and Pastuch‐Gawołek G., “Synthesis and Preliminary Cytotoxicity Evaluation of 3‐Lup‐20(29)‐Ene‐3β,28‐Diol Glycoconjugates Containing a Succinic Linker and a 1,2,3‐Triazole Ring,” Cancers 17, no. 11 (2025): 1737, 10.1016/j.bioorg.2018.11.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47. Bakrim S., Benkhaira N., Bourais I., et al., “Health Benefits and Pharmacological Properties of Stigmasterol,” Antioxidants 11, no. 10 (2022): 1912, 10.3390/antiox11101912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48. Aderiye B. I., Ogundana S. K., Adesanya S. A., and Roberts M. F., “The Effect of β‐Sitosterol on Spore Germination and Germ‐Tube Elongation of Aspergillus niger and Botryodiplodia Theobromae ,” International Journal of Food Microbiology 8, no. 1 (1989): 73–78, 10.1016/0168-1605(89)90082-2. [DOI] [PubMed] [Google Scholar]
- 49. Prasath K. G., Tharani H., Kumar M. S., and Pandian S. K., “Palmitic Acid Inhibits the Virulence Factors of Candida tropicalis: Biofilms, Cell Surface Hydrophobicity, Ergosterol Biosynthesis, and Enzymatic Activity,” Frontiers in Microbiology 11 (2020): 864, 10.3389/fmicb.2020.00864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50. Kapoor A., Mishra D. N., and Narasimhan B., “Antibacterial and Antifungal Evaluation of Synthesized 9,12‐Octadecadienoic Acid Derivatives,” Pharmaceutical Letters 6 (2014): 5. [Google Scholar]
- 51. Vikram A., Jayaprakasha G. K., Jesudhasan P. R., Pillai S. D., and Patil B. S., “Obacunone Represses Salmonella Pathogenicity Islands 1 and 2 in an envZ ‐Dependent Fashion,” Applied and Environmental Microbiology 78, no. 19 (2012): 7012–7022, 10.1128/AEM.01326-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52. Patra A. K., Dietary Phytochemicals and Microbes (Springer Science & Business Media, 2012), 1–400, 10.1007/978-94-007-3926-0. [DOI] [Google Scholar]
- 53. Zhou Y., Gu J., Li J., et al., “Obacunone, a Promising Phytochemical Triterpenoid,” Molecules 29, no. 8 (2024): 1791, 10.3390/molecules29081791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54. Ellis D., “Amphotericin B: Spectrum and Resistance,” Journal of Antimicrobial Chemotherapy 49 (2002): 7–10, 10.1093/jac/49.suppl_1.7. [DOI] [PubMed] [Google Scholar]
- 55. Benedict K., Gold J. A. W., Chiller T., and Lyman M., “Economic Burden of Fungal Diseases in the United States,” Medical Mycology 63, no. 6 (2025): myaf049, 10.1093/mmy/myaf049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56. Chakrabarti A., Patel A. K., Soman R., and Todi S., “Overcoming Clinical Challenges in the Management of Invasive Fungal Infections in Low‐ and Middle‐Income Countries (LMIC),” Expert Review of Anti‐Infective Therapy 21, no. 10 (2023): 1057–1070, 10.1080/14787210.2023.2257895. [DOI] [PubMed] [Google Scholar]
- 57. Singh A. K., Rana H. K., Sharma R., and Pandey A. K., “Chlorogenic Acid, a Dietary Phenolic Acid Ameliorates Hepatorenal Injury in Streptozotocin‐Induced Diabetic Rats Through Regulation of Oxidative Stress and Inflammation,” Food Bioscience 66 (2025): 106244, 10.1016/j.fbio.2025.106244. [DOI] [Google Scholar]
- 58. Sharma R., Banerjee S., and Sharma R., “Role of Mandukparni (Centella asiatica Linn Urban) in Neurological Disorders: Evidence From Ethnopharmacology and Clinical Studies to Network Enrichment Analysis,” Neurochemistry International no. 180 (2024): 105865, 10.1016/j.neuint.2024.105865. [DOI] [PubMed] [Google Scholar]
- 59. Ghosh S., Das R., Mohanty J. P., et al., “Current Perspectives on Fungal Infections: Pathogenesis, Diagnosis and Herbal Treatment,” Quantum Journal of Medical and Health Sciences 3, no. 4 (2024): 1–15. [Google Scholar]
- 60. Alkhamessi R. N., Abbas M. I., Bahedh S. B., et al., “Harnessing Medicinal Plants and Their By‐Products to Combat Fungal Pathogens in the Face of Climate Change,” Journal of Medicinal Plants By‐Products 15 (2026): 102–110, 10.22034/jmpb.2025.369007.1934. [DOI] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
The supplementary material, Tables S1–S4 and Figures S1, S2 and S3, is provided along with the manuscript submission.
Supporting File 1: cbdv71468‐sup‐0001‐SuppMat.docx
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.