ABSTRACT Serious human health impacts have been observed worldwide due to several life-threatening diseases such as cancer, candidiasis, hepatic coma, and gastritis etc. Exploration of

nature for the treatment of such fatal diseases is an area of immense interest for the scientific community. Based on this idea, the genus _Aspergillus_ was selected to discover its hidden

therapeutic potential. The genus _Aspergillus_ is known to possess several biologically active compounds. The current research aimed to assess the biological and pharmacological potency of

the extracts of less-studied _Aspergillus ficuum_ (FCBP-DNA-1266) (_A. ficuum_) employing experimental and bioinformatics approaches. The disc diffusion method was used for the antifungal

albicans_ (_C. albicans_) with their zone of inhibitions (ZOI) values reported as 19 ± 1.06 mm and 25 ± 0.55 mm, respectively at a dose of 30 µg.mL−1. In vitro cytotoxicity assay against

HeLa, fibroblast 3T3, prostate PC3, and breast MCF-7 cancer cell lines was performed. The ethyl acetate extract of _A. ficuum_ was found to be significantly active against MCF-7 with its

IC50 value of 43.88 µg.mL−1. However, no substantial effects on the percent cell death of HeLa cancer cell lines were observed. In addition, the _A. ficuum_ extracts also inhibited the

urease enzyme compared to standard thiourea. The antispasmodic activity of _A. ficuum_ extract was assessed by an in vivo model and the results demonstrated promising activity at 150 

mg.kg−1. Molecular docking results also supported the antifungal, anticancer, and antiurease potency of _A. ficuum_ extract. Overall, the results display promising aspects of _A. ficuum_

extract as a future pharmacological source. SIMILAR CONTENT BEING VIEWED BY OTHERS SYNTHESIS, CHARACTERIZATION AND BIO-EVALUATION OF NOVEL SERIES OF PYRAZOLINE DERIVATIVES AS POTENTIAL

ANTIFUNGAL AGENTS Article Open access 28 April 2025 EXPLORING THE ANTIFUNGAL, ANTIBIOFILM AND ANTIENZYMATIC POTENTIAL OF ROTTLERIN IN AN IN VITRO AND IN VIVO APPROACH Article Open access 15

May 2024 NON-ANTIFUNGAL DRUGS INHIBIT GROWTH, MORPHOGENESIS AND BIOFILM FORMATION IN _CANDIDA ALBICANS_ Article 19 January 2021 INTRODUCTION New drugs are needed due to certain factors such

as globalization, the aging population, and microbial resistance to existing medicines. Only a small number of biologically active compounds are currently used in prescription drugs1.

Scientists are currently investigating emerging diseases to gain a better understanding of them and find their potential cures using various natural and chemical formulations. However, there

are still many areas that remain unexplored due to insufficient knowledge and techniques. To find new bioactive compounds that can be used for medicinal, agricultural, and industrial

purposes, researchers are focusing on isolating such compounds from new species of fungi2. In recent times, researchers have isolated several metabolites from fungi, including

anthraquinones, alkaloids, terpenes, steroids, flavonoids, and cyclic peptides. Some secondary metabolites produced by various fungi have been found to exhibit potent biological activities

such as antifungal, antitumor, anti-inflammatory antibacterial, antiparasitic, and antiviral properties. The chemical diversity of fungal secondary metabolites provides a significant

advantage for the development of new drugs3,4. Anofinic acid, a potent active metabolite produced by _Aspergillus tubingensis_, has shown strong antimicrobial activity against a variety of

hazardous bacteria in addition to its potent activity against various carcinoma cells, including Hep-G2, MCF-7, and HCT-1162. Sorbicillinoids possess the capability to be used as

pharmaceutical and agrochemical agents due to their antiviral, antimicrobial, and anticancer effects besides their use as food colorants, and pigments. Sorbicillin and sorbicatechol D have

been reported to exhibit antiproliferative effects against HT-29 tumor cells dose-dependently5. The metabolites gliotoxin, deoxytryptoquivaline, and patulin isolated from various

_Aspergillus_ species are known to induce apoptosis in cells6,7,8. A study performed on the secondary metabolites (terretonin N and butyrolactone I) of _A. terreus_ confirmed their promising

anticancer potential. Cellular apoptosis was induced at higher rates in cancer cells lacking a necrotic apoptotic pathway9. Similarly, recent studies on extracts of various fungi,

particularly _Aspergillus,_ have revealed their potent anticancer, antinociceptive, and anti-inflammatory activities10,11. In addition, the latest studies have also confirmed the strong

enzyme-inhibiting effects of numerous species of fungi9. _Aspergillus ficuum_ is a filamentous fungus that belongs to the _Niger_ clade in the _Aspergillus_ section _Nigri_. Its

morphological characteristics are the formation of spores and the display of colony characteristics. The taxonomy of the _Niger_ clade depends on the generation of various secondary

metabolites belonging to the five classes such as pyranone, alkaloid, cyclopentapeptide, polyketide, and sterol. These metabolites are known as naphthopyrones, malformins, bicoumarins

(kotanins), fumonisin B2 and B4, citric acid, diketopiperazine, asperazine, and other related compounds12. Extensive biological aspects have been reported for the _Niger_ clade13. In our

previous study, we investigated the metabolic profile of _A. ficuum_ extracts for the first time, which led to the discovery of various bioactive compounds, including hydroxyvittatine,

aurasperone D, choline sulfate, noruron, cetrimonium, heneicosane, kurilensoside, eicosane, and nonadecane. Furthermore, a pharmacological analysis of _A. ficuum_ extracts was also carried

out via in vitro and in vivo models14. In continuation of our previous study, extracts of the lesser-explored member of the Niger clade, _A. ficuum_ were further investigated for their

antifungal and anticancer activities via an in vitro model. Whereas their anti-inflammatory and antispasmodic effects were studied through an in vivo model. In addition, computational

analyses were performed to support the in vitro and in vivo results. These investigations of _A. ficuum_ extracts will offer new understandings into the area of the fungal diaspora.

MATERIALS AND METHODS CHEMICALS AND ANIMALS All standard chemicals and solvents were obtained from Sigma Aldrich. The (BALB/c) mice weighing 25–35 g were purchased from the National

Institute of Health (NIH), Islamabad, Pakistan. They were acclimated at 22 °C on a light/dark cycle (12 h/12 h) with adequate provision of water and food throughout the study. All methods

were performed following the relevant guidelines and regulations15. The in vivo studies were conducted under the number 7196/LM/UoA Ethical Committee FAHV&S, the University of

Agriculture Peshawar, Pakistan14. FUNGAL STRAIN The fungal strain (_A. ficuum_, FCBP-DNA-1266) was obtained from the fungal bank of the University of Punjab, Pakistan. CULTURE CULTIVATION

The fungal culture was cultured at 28 °C for 21 days in the static state. _A. ficuum_ spores (105 conidia/mL) were transferred to the multiple Erlenmeyer flask (500 mL) containing about 250 

mL of Potato Dextrose Broth (PDB). To inhibit bacterial growth in the medium, PDB was added with 25 mg.L−1 of streptomycin sulfate16. EXTRACTION AND FRACTIONATION After 21 days, the mycelia

formed in each flask were removed and processed for drying. The mycelia were washed several times with deionized water to remove any media ingredients. The constant dry weight of mycelia was

obtained after drying at 60 °C in an oven. The dried mycelia were crushed to powder using a mortar and pestle. The powdered mycelia weighing 60 g was extracted in triplicate using three

hundred milliliters of ethyl acetate (EtOAc) (3 × 300 mL). Subsequently, the obtained 4 g of ethyl acetate extract was fractionated by n-hexane (3 × 300 mL). Both portions were condensed via

a rotary evaporator (Buchi, Germany, Model R-300). The dried-up portions, ethyl acetate (2.3 g) and n-hexane (1.7 g) were kept at 4 °C in the refrigerator for further studies16,17.

ANTIFUNGAL ASSAY The microorganisms such as _Aspergillus niger_ (_A. niger_), _Fusarium oxysporum_ (_F. oxysporum_), _Trichoderma harzianum_ (_T. harzianum_)_, Candida albicans_ (_C.

albicans_)_,_ and _Aspergillus flavus_ (_A. flavus_) used for the antifungal assay were acquired from the Department of Agricultural Chemistry and Biochemistry, the University of

Agriculture, Peshawar, Pakistan. A sterilized malt extract agar (MEA) medium was prepared and poured into Petri dishes. The spores from each fungal inoculum were added to these plates. The

sterilized discs were soaked in _A. ficuum_ extracts at concentrations of 10, 20, and 30 µg.mL−1 and were then positioned on the plates. Subsequently, the plates were cultured at 28 °C and

then analyzed for antifungal assay after 72 h. Mancozeb was used as a drug control while DMSO was utilized as a negative control. The antifungal analysis was performed by measuring the zone

of inhibition in millimeters17. ANTISPASMODIC ACTIVITY Standard protocol was followed with minor modifications18,19. Mice weighing 20–30 g were used for the antispasmodic activity. Before

the experiments, five mouse groups were created and kept on fast. Ethyl acetate extract of _A. ficuum_ was introduced in triplicate at a dose of 10–20 mg.Kg−1. Two groups were orally

administered castor oil (0.5 mL) and normal saline water (negative control) at a dose of 10 mL.kg−1. The residual three groups were orally administered _A. ficuum_ extract at a dose of 10–20

 mg.kg−1. Each group of mice was orally administered with charcoal (1 mL) after 1 h of induction of castor oil, and extract. The passage of charcoal from the pylorus to the caecum was noted

by sacrificing mice through cervical dislocation after 50 min of charcoal induction. The percent inhibition caused by the _A. ficuum_ extract was determined as under: Percent inhibition = 

[Distance covered by charcoal/Total length of intestine] × 100. MTT ASSAY The anticancer studies were carried out at Hussain Ejaz Institute, Department of Chemistry, University of Karachi. A

standard colorimetric MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) analysis was performed to determine the cytotoxic potential of crude extract of _A. ficuum_ by

using 96-well flat-bottomed microplates20. In this study, HeLa cells (Cervical Cancer), 3T3 (mouse fibroblast), PC3 cells (Prostrate Cancer), and MCF-7 (Breast Cancer) cells were examined.

These different cancer cells were cultivated in Minimum Essential Medium Eagle (MEME), added with fetal bovine serum (5%) (FBS), penicillin (100 IU.mL−1), and streptomycin (100 µg.mL−1) in

flasks (75 cm2). The flasks were cultured at 37 °C in a CO2 (5%) incubator. The substantially growing cells were counted with a hemocytometer followed by dilution with a specific medium. A

volume of 100 µL of each cell culture (HeLa, 3T3, MCF-7, and PC3) having 1 × 104 cells/well was seeded into 96-well plates at a concentration of 100 µL/well. The cell cultures were incubated

overnight and the medium was separated from them. A newly harvested medium (200 µL) was administered to the well of each cell culture along with different concentration doses of extract of

_A. ficuum_ (15–60 µg.mL−1). After incubation for 48 h, each well was added MTT solution (200 µL) having a concentration of 5 mg.mL−1. The cell cultures were further nurtured for 4 h. Later,

DMSO (100 µL) was introduced to each well. The synthesis of formazan by the decrease of MTT was determined using a microplate reader (Spectra Max Plus, Molecular Devices, CA, USA) through

absorbance maximum at a wavelength of 570 nm. Doxorubicin and DMSO were used as a positive and negative control, respectively. The 50% growth inhibition (IC50) was measured for cytotoxicity

by using the straight-line equation y = mx + c. By using Soft-Max Pro software (Molecular Device, USA), the following formula was applied for the measurement of percent inhibition: %

inhibition = 100 − (mean of O.D of _A ficuum_ extract − mean of O.D of negative control)/(mean of O.D of positive control – mean of O.D of negative control) *100). UREASE INHIBITION ASSAY

The urease inhibitory effect of the crude extracts of _A. ficuum_ was evaluated by following the standard protocol with little modifications21. A solution of 20 µL of Jack bean urease (2.5

units/mL) and 50 µL of urea (100 mM) was prepared. The solution was dissolved in phosphate buffer, pH 8.2 [K2HPO4·3H2O (0.01 M), EDTA (1.0 m), and LiCl2 (0.01 M)] and was mixed with 30 µL of

250 µg.mL−1 concentrations of EtOAc and n-hexane extracts. The mixture was subjected to incubation at 37 °C in a 96-well plate for 10 min. The reaction mixture was incubated at 30 °C in a

96-well plate for 15 min. The indophenol method was used to determine the amount of ammonia production to measure the suppression of urease activity. The phenol reagent [phenol (1%), sodium

nitroprusside (0.005%), 40 µL] and the alkaline reagent [60 µL, NaOH (0.5%), NaOCl (0.1%)] were added to each well. Thiourea (100 µg.mL−1) was taken as a standard and data were obtained in

triplicate by measuring the absorption maximum at 630 nm. Percent inhibition was determined via the equation: Percent inhibition = 100 − (OD _A. ficuum extracts_/OD control) × 100. IN SILICO

ANALYSIS Mycocompounds (Fig. S1) tentatively reported for _A. ficuum_ extracts14 were chosen for docking assay against heat shock protein 90 (hsp90) (PDB ID: 6CJI) of _C. albicans_, jack

bean urease (PDB ID: 3LA4) and human HER2 protein (PDB ID: 3PP0) at a resolution of 1.64, 2.05 and 2.25 Å, respectively17,22,23. High-resolution X-ray crystal structures of the chosen

proteins were obtained from the Protein Data Bank (http://www.rcsb.org/pdb). By using a preparation program integrated into the Molecular Operating Environment (MOE) software, protein

molecules were optimized for docking study by eliminating water molecules, introducing missing hydrogen atoms, allocating the correct hybridization state to each atom in each residue, and

fixing charges. Active sites of the designated proteins were located using the active site finder tool embedded in MOE software. Using the docking tool of the MOE software, mycocompounds

were docked to the active binding sites of the chosen pathogenic proteins. For each molecule, 30 conformations were produced using specific torsion angles for all the rotatable bonds. The

MOE-implanted London dock scoring function was used to determine the binding energy for each protein–ligand complex system24. STATISTICAL ANALYSIS Statistical analysis of all data obtained

was performed using GraphPad Prism version 8.0. Results were evaluated using a mean n = 3 with standard deviation. Antifungal analyses were evaluated through one-way ANOVA while means were

separated by applying LSD at _P_ ≤ 0.05. Similarly, the results of the in vivo pharmacological investigation were evaluated through one-way ANOVA, followed by post hoc analysis (Dunnett’s

test). ETHICS APPROVAL All experimental protocols were approved by the ethical committee FAHV&S, the University of Agriculture Peshawar, Pakistan under the number 7196/LM/UoA. All

methods are reported following ARRIVE guidelines (https://arriveguidelines.org). RESULTS AND DISCUSSIONS ANTIFUNGAL ACTIVITY The antifungal analysis of ethyl acetate and n-hexane extracts of

_A. ficuum_ was determined versus five fungal species (Table 1). _C. albicans_ is a mutual fungus that causes death by colonizing the human skin and gastrointestinal tract. It is the

causative agent of multidrug resistance in individuals with compromised immune systems due to multiple diseases. Several studies have been conducted on its control using natural

products25,26. In this study, the antifungal investigations reveal that ethyl acetate extract was significantly active (_P_ ≤ 0.05) against _C. albicans_ with a ZOI of 25 ± 0.55, at a dose

of 30 µg.mL−1. It was also recorded that ethyl acetate extract was substantially active (_P_ ≤ 0.05) against all fungal species at a dose concentration of 30 µg.mL−1. Likewise, potent

antifungal activity (_P_ ≤ 0.01–0.05) was also exhibited by n-hexane extract against other fungal species (Fig. 1). The n-hexane extract was also profoundly active (_P_ ≤ 0.05) against the

tested fungal strains at high concentrations. The fungal species are known to be great synthesizers of antifungal compounds. Several classes of compounds produced by the genus _Aspergillus_,

such as statins and polyketides, are considered effective antifungal compounds27. A study conducted on the extract isolated from the endophytic fungus _Colletotrichum gloeosporioides_

displayed potent antifungal activity against _C. albicans_28. In addition, a similar study was conducted on various fungal strains of different genera such as _Acremonium_, _Fusarium_,

_Aspergillus,_ and _Penicillium_ which showed potent antifungal activities29. In another study, the n-hexane extract was found to be effective against _A. flavus_ with a ZOI of 21.63 mm and

less significant against _F. oxysporum_ with a ZOI of 15.31 mm. The genus _Aspergillus_ is considered a good producer of antimicrobial agents without any significant toxicity30,31.

ANTISPASMODIC ACTIVITY Inflammatory bowel disorders and gastritis are very common in humans and are induced by histamine and acetylcholine. The gastrointestinal tract becomes irritated and

inflamed by smooth muscle contractions, causing uneasiness and discomfort. Such conditions are usually treated with antispasmodics which also show numerous side effects18. A charcoal

motility assay was performed to assess the inhibition of ethyl acetate extract of _A. ficuum_ (Table 2). The three different dosages of extracts from _A. ficuum_ were able to show an

antispasmodic effect. Smooth muscle contractions were reduced in a dose-dependent manner by ethyl acetate extract compared to mice treated with castor oil. It was observed that as

concentrations of _A. ficuum_ extract increased, there was a gradual increase in spasmodic activity, which is not a good indication. However, _A. ficuum_ extracts possessed antispasmodic

activity compared to the percent mobility of charcoal in castor oil, providing evidence that _A. ficuum_ extracts can relieve spasms (Fig. 2). According to the literature, an antispasmodic

effect against three different spasmogens was shown in a study of the n-hexane extract from endophytic fungi32. The literature review showed that bio-transformed diterpenes from _A. niger_

displayed significant antispasmodic activity33. Although this study was conducted using a few concentrations of the extract, further investigation of the mycochemicals of this fungus will

lead to the development of the most standardized and effective drug. ANTICANCER ACTIVITY The anticancer activity of ethyl acetate extracts of _A. ficuum_ was measured versus HeLa, 3T3, PC3,

and breast cancer cells (MCF-7) (Table 3). In the case of MCF-7, the IC50 value was noted to be 43.88 µg.mL−1, significantly potent compared to other cancer cell lines, HeLa, 3T3 & PC3

with their IC50 noted to be 250.1, 75.54, and 70.07 µg.mL−1, respectively. From the analysis, it was visible that percent cell viability decreases with the increase in the concentration of

the extract. The linear pattern of cell viability against the dose concentrations of the extracts was evident (Fig. 3). At a high dose of 60 µg.mL−1, no significant difference was observed

for the positive control, doxorubicin. A similar pattern was observed for all doses of _A. ficuum_ extract against the 3T3 cancer cell line. Moreover, no significant effect of _A. ficuum_

extract was found against the HeLa cell line. It was also evident that high doses of _A. ficuum_ extract caused significant cell death (Fig. 4). Previously, a study showed that more than 100

anticancer drugs associated to 19 diverse chemical groups were obtained from fungi34. Artika et al_._ studied the anticancer activity of endophytic fungi. Their data showed that only one

isolated chemical at a concentration of 400 µg.mL−1 exhibited potency comparable to the standard drug35. Felczykowska and coworkers found that fungi possess antiproliferative materials. They

studied that the fungus, _Protoparmeliopsis muralis_ isolated from lichen exhibited potent activity versus MCF-7 cancer cells36. Tincho et al_._ studied the cytotoxic potential of numerous

fungi isolated from _Terminalia cattapa_. They discovered that an extract of _F. oxysporum_ exhibited a potent 50 percent inhibitory potential versus cancer cell lines37. Thomas et

al.examined twenty-one fungal extracts versus MCF-7 cancer cells. Their results revealed that sample F-21 possessed strong anticancer activity with its IC50 value of 44.75 µg.mL−138.

Nevertheless, several species of _Aspergillus_ are highlighted as potent producers of anticancer metabolites39. The biosynthesis of secondary metabolites in fungi is highly reliant on the

substrate and other ecological conditions40. Since this study was performed on a PDB medium, mycelial growth on other substrates and optimized conditions could contribute to the potent

anticancer activities of fungal extracts. UREASE INHIBITION ASSAY The conversion of urea into harmful products by the urease enzyme has serious effects on plants, animals, and humans. Its

action causes urinary stones, gastritis, gastric cancer, hepatic coma, and other serious diseases in living organisms. The ureolytic activity of urease was suppressed by the natural products

obtained from various sources41. The Urease inhibition assay of different extracts of _A. ficuum_ was assessed in this study. A significant percent urease inhibitory potential was exhibited

by ethyl acetate extract of _A. ficuum_. The ethyl acetate and n-hexane extracts of _A. ficuum_ suppressed urease by 71.58 and 53.22%, respectively. The result agreed well with standard

thiourea (Fig. 5). The difference between the ethyl acetate extract and the standard thiourea was not significant, which is a good sign as the commercially available ureases are toxic and

less stable. The study regarding the suppression of the urease enzyme by fungi is very limited. Rauf et al.determined that the ethyl acetate fraction of _Screlotium rolfsii_ and _A. flavus_

was significantly inactive to urease enzymes with less than 50% inhibition42. A literature review suggested that fungal metabolites can inhibit the urease enzyme at different dose levels43.

A research group isolated two compounds from _Paecilomyces formosus_ which exhibited significant urease inhibitory activity with their potencies of 75.8 and 190.5 µg.mL−144. Another group of

researchers found the urease inhibitory activity of a metabolite isolated from fungi, Bipolaris sorokiniana LK12. In a dose-dependent study, the metabolites showed an IC50 value of 81.62 

µg.mL−1 against the urease enzyme45. DOCKING ANALYSIS Ethyl acetate extract of _A. ficuum_ was found significantly active against all tested fungi in general and _C. albicans_ in particular.

Therefore, the mycocompounds were cautiously identified from ethyl acetate extract14 [Fig. S1] were docked versus the Hsp90 protein of _C. albicans_ to support the antifungal activity of

_A. ficuum_ extract. Hsp90 plays a vital role in pathogenesis by performing protein biogenesis as well as interacting with various cellular proteins46 and is referred to as a molecular

chaperone; its inhibition will halt fungal infection. Docking results reveal that all selected ligands (L1-L9) can inhibit the Hsp90 protein by forming a stable protein–ligand complex. Among

all docked ligands, L3 was found to be more efficient since it developed two physical interactions with the catalytic residues of Hsp90 resulting in the highest binding energy of − 7.9738 

kcal.mol−1 (Table 4). One conventional H-bond was generated between residue Lys47 and the polar oxygen atom of L3; the second Pi-cation interaction was formed between the benzene ring of L3

and residue Lys47 (Fig. 6). L1 formed two physical interactions with Hsp90 causing a binding affinity of − 5.7161 kcal.mol−1 (Table 4). Catalytic residues Gly86 and Lys47 were involved in

developing two conventional H-bonds with two different hydroxyl groups of L1 (Fig. S2a,b). Ligand L2 was associated with amino acid Asn95 of Hsp90 through one H-bond; it resulted in the

binding energy of − 5.8924 kcal.mol−1 (Table 4) (Fig. S2c,d). L4 formed a stable protein–ligand complex by developing two conventional H-bond interactions; two different oxygen atoms of the

sulfonate group of L4 were found associated with the catalytic residue Lys47 (Fig. S2e,f). The binding energy was noted to be − 4.8836 kcal.mol−1 (Table 4). L5 bound to the active sites of

Hsp90 generated the second-highest binding affinity of − 7.8284 kcal.mol−1 (Table 4) among all screened ligands. Residue Lys47 developed an H-bond interaction with the carbonyl oxygen atom

of L5; the second H-bond originated between residue Lys47 and the NH group of L5 (Fig. S2g,h). L6 was linked with Hsp90 through two H-bond interactions originating binding affinity of − 

6.7134 kcal.mol−1 (Table 4). Residue Asp43 formed two H-bonds with two different hydroxyl groups of L6 (Fig. S2i,j). Ligand L7 formed a stable protein–ligand complex through three H-bond

interactions. The Hydroxyl moiety of L7 participated in developing H-bond interaction with residue Glu36. Two different carbonyl groups of L7 formed two H-bond interactions with residue

Lys47 and Gly126, respectively (Fig. S2k,l). The binding energy value was calculated to be − 7.5397 kcal.mol−1 (Table 4). L8 formed two physical interactions with Hsp90 originating with the

third highest binding affinity of -7.7921 kcal.mol−1 (Table 4). One H-bond was developed between residue Asp91 and the hydroxyl moiety of L8; a second H-bond was generated between the

carbonyl oxygen atom of L8 and residue Lys47 (Fig. S2m, n). In the case of L9, two physical forces were responsible for the formation of a protein–ligand complex; the binding energy value

was noted to be − 7.0975 kcal.mol−1 (Table 4). Catalytic residue Asp91 generated two conventional H-bond interactions with L9 (Fig. S2o,p). Docking data display three major findings:

firstly, all ligands (L1-L9) can bind to catalytic residues located in the active pocket of Hsp90; secondly, L3 has the highest binding affinity with Hsp90 among all screened ligands;

thirdly, residue Lys47 is actively involved in binding to six ligands. Keeping in view the chemistry of Lys47, future drug candidates can be designed. These findings support the antifungal

activity of _A. ficuum_ extracts_._ Similarly, ligands (L1-L9) were docked against the HER2 protein to support the anticancer activity of _A. ficuum_ extracts_._ HER2 is a membrane tyrosine

kinase enzyme; it plays a major part in tumor growth and the spreading of breast cancer on activation47. Docking results indicate that ligand L6 showed the highest affinity toward the HER2

protein among all screened ligands. It is associated with HER2 protein through four H-Bond interactions resulting in the highest binding energy of − 7.8907 kcal.mol−1 (Table 5). Residue

Ser728 developed two H-Bond interactions with the same hydroxyl group of L6; each amino acid Asn850 and Asp808 formed a single H-Bond interaction with two different hydroxyl groups of L6

(Fig. 7). Ligand L1 generated three physical interactions with HER2 protein stemming binding affinity of − 5.2498 kcal.mol−1 (Table 5). Catalytic residue Asp808 participated in developing

two H-Bond interactions with the same hydrogen atom of the hydroxyl moiety of L1. Residue Ser728 formed Pi-H interaction with the benzene ring of L1 (Fig. S3a,b). L2 developed two

non-covalent interactions with HER2 protein; these interactions originated a binding affinity of − 5.1722 kcal.mol−1 (Table 5). Each of the residues (Cys805 and Ser728) formed a single

H-bond with the polar hydrogen atom of the NH moiety and the carbonyl oxygen atom of L2, respectively (Fig. S3c,d). Ligand L3 formed a stable protein–ligand complex by developing two

physical interactions with binding affinity reported to be − 6.8174 kcal.mol−1 (Table 5). Each of the amino acids (Leu726 and Cys805) generated a single H-Bond with the polar hydrogen of the

hydroxyl moiety and carbonyl oxygen atom of L3, respectively (Fig. S3e,f). L4 generated a single interaction with the HER2 protein resulting in a binding affinity of − 6.1479 kcal.mol−1

(Table 5). Residue Thr862 formed a single H-Bond interaction with the polar hydrogen of the CH2 group of L4 (Fig. S3g,h). Ligand L5 was associated with HER2 protein through two non-covalent

interactions; these forces generated the second-highest binding energy of − 7.7000 kcal.mol−1 (Table 5). Catalytic residue Ser728 formed a single H-Bond interaction with the oxygen atom of

the methoxy group of L5; residue Gly727 developed a single Pi-H interaction with the pyrrole moiety of L5 (Fig. S3i,j). Ligand L7 formed a stable protein–ligand complex by generating two

physical interactions; it resulted in a binding energy of − 6.5073 kcal.mol−1 (Table 5). Residue Ser728 originated two H-Bond interactions with the polar hydrogen atom of the hydroxyl group

and carbonyl oxygen atom of L7; Lys724 developed a single H-Bond with the hydroxyl moiety of L7 (Fig. S3k,l). L8 displayed single H-Bond interaction with HER2 protein producing the third

highest binding affinity of − 6.8651 kcal.mol−1 (Table 5). The Hydroxyl moiety of L8 was involved in the creation of the protein–ligand complex (Fig. S3m,n). Ligand L9 generated two physical

interactions with HER2 protein; these non-covalent forces resulted in a binding energy value of − 6.7778 kcal.mol−1 (Table 5). Residue Asp808 developed H-Bond and Pi-Cation interactions

with two different methyl groups of L9, respectively (Fig. S3o,p)48. In the case of HER2 protein, three new aspects emerged: firstly, all ligands (L1-L9) form a stable protein–ligand

complex; secondly, ligand L6 has a greater ability to bind to the active pocket of HER2 protein among all tested ligands; thirdly, the catalytic role of residue Ser728 is found prominently

by binding to five ligands. Structural features of L6 and residue Ser728 should be considered for drugs designed against breast cancer. Herein, docking results support the anticancer

activity of _A. ficuum_ extracts. Similarly, ligands (L1-L9) were also screened against the urease enzyme through the molecular docking technique; the results indicated that all ligands

could inhibit the urease enzyme by forming a stable protein–ligand complex. Based on binding affinity, L8 was found to have the highest binding affinity of − 7.7921 kcal.mol−1 (Table 6)

among all selected ligands. It developed four physical interactions with urease protein. Both residues (His593 and His585) generated a single Pi–Pi interaction with two different benzene

rings of L8; in addition, two residues namely Met637 and Cme592 also formed a single H-Bond interaction with the hydroxyl moiety and carbonyl oxygen atom of L8, respectively (Fig. 8). L1

originated two non-covalent interactions with the urease enzyme resulting in the binding energy of − 5.0588 kcal.mol−1 (Table 6). Residue Arg835 developed a single H-Bond interaction with

the hydroxyl moiety of L1; Arg575 formed a single H-Bond interaction with the methyl moiety of the heterocyclic ring of L1 (Fig. S4a,b). Ligand L2 generated a single non-covalent interaction

with the urease enzyme; it resulted in a binding energy value of − 5.4100 kcal.mol−1 (Table 6). Catalytic residue Val831 participated in the development of a single H-Bond interaction with

the carbonyl oxygen of L2 (Fig. S4c,d). L3 formed a stable protein–ligand complex by developing two physical interactions resulting in the binding affinity of − 6.9148 kcal.mol−1 (Table 6).

Residue Arg835 generated Pi-H and Pi-Cation interactions with the benzene ring and oxygen heterocyclic ring of L3, respectively (Fig. S4e,f). L4 generated four physical forces with the

urease enzyme; these forces resulted in a binding affinity of − 4.5903 kcal.mol−1 (Table 6). Residue Glu584 developed two H-Bond interactions with two different methyl groups of L4. Gln649

formed a single H-Bond interaction with the oxygen atom of L4 (Fig. S4g,h). Ligand L5 was involved in complex formation with urease through two physical interactions; these interactions

originated the second-highest binding affinity of − 7.6472 kcal.mol−1 (Table 6). Both residues (Glu642 and Val831) developed single H-Bond interactions with OH and NH groups of L5,

respectively (Fig. S4i,j). Ligand L6 developed a single non-covalent interaction with the active spot of the urease enzyme yielding a binding affinity of − 7.1083 kcal.mol−1 (Table 6).

Residue Arg835 displayed a single H-Bond interaction with the hydroxyl moiety of L6 (Fig. S4k,l). L7 showed three physical interactions with catalytic residues of urease enzyme generating

the third highest binding energy of − 7.5593 kcal.mol−1 (Table 6). Two residues, namely, Ser645 and Thr829 formed single H-bond interaction with two different hydroxyl groups of L7;

catalytic residue Arg835 displayed Pi-H interaction with the benzene ring of L7 (Fig. S4m,n). Ligand L9 was noticed attached to the active site of urease through two physical interactions

that resulted in a binding affinity value of − 6.7125 kcal.mol−1 (Table 6). Catalytic residue Glu642 showed two H-Bond interactions with two different methyl groups of L9 (Fig. S4o,p). Some

interesting findings were obtained from docking data: all ligands form a stable protein–ligand complex with urease enzyme; L8 shows the highest binding affinity among all screened ligands

and catalytic residue Arg835 is noticed to be more active by forming a protein–ligand complex with four ligands. CONCLUSION In this research, urease inhibition, antifungal, anticancer, and

antispasmodic activities of _A. ficuum_ extracts were explored for the first time by applying in vivo and in vitro models followed by in silico models. Like other fungi, both n-hexane and

ethyl acetate extracts of _A. ficuum_ were noticed active against five tested fungal strains. However, both extracts displayed significant activity against the _C. albicans._ Potent

antispasmodic, as well as urease inhibitory activities, were recorded for _A. ficuum_ extracts_._ Similarly, the promising anticancer effect of _A. ficuum_ against PC3, 3T3, and especially

MCF-7 cancer cell lines was documented. Furthermore, the antiurease, antifungal, and anticancer activities of _A. ficuum_ extracts were supported by molecular docking results. Among all

docked ligands, L3, L6, and L8 displayed the highest binding affinity for Hsp90, HER2, and urease proteins, respectively. Similarly residues namely, Lys47, Ser728, and Arg835 in the active

pockets of Hsp90, HER2, and urease proteins, respectively emerged as catalytically active residues generating H-Bond interactions with a greater number of ligands. Keeping in view the

structural features of ligands (L3, L6, and L8) and residues (Lys47, Ser728, and Arg835), potential drug candidates could be designed in the future for the inhibition of pathogenic proteins

such as Hsp90, HER2, and urease. In addition, the cultivation of _A. ficuum_ extracts in a controlled environment with the progress of new approaches will lead to the discovery of new

bioactive chemical scaffolds in the drug discovery process. DATA AVAILABILITY All data generated or analyzed during this study are included in this published article and its supplementary

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_J. Mol. Struct._ 1258, 132652 (2022). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS The authors are grateful to the Department of Chemistry, Islamia

College, Peshawar, KP, Pakistan for the provision of all facilities needed to execute the project. The authors are also thankful to Universidade Federal de Pelotas—UFPel, Pelota, RS, Brazil

for providing support to perform this project. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Chemistry, Islamia College University, Peshawar, Khyber Pakhtunkhwa, Pakistan Zafar

Ali Shah, Khalid Khan, Nasir Ahmad & Akhtar Muhammad * Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pakhtunkhwa, Pakistan Tanzeel Shah * Center of

Chemical, Pharmaceutical and Food Sciences, Federal University of Pelotas, Pelotas RS, Brazil Haroon ur Rashid * Institute of Chemistry, Sao Paulo State University, Araraquara, Sao Paulo,

Brazil Haroon ur Rashid Authors * Zafar Ali Shah View author publications You can also search for this author inPubMed Google Scholar * Khalid Khan View author publications You can also

search for this author inPubMed Google Scholar * Tanzeel Shah View author publications You can also search for this author inPubMed Google Scholar * Nasir Ahmad View author publications You

can also search for this author inPubMed Google Scholar * Akhtar Muhammad View author publications You can also search for this author inPubMed Google Scholar * Haroon ur Rashid View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Z.A.S.: conceptualization, formal analysis, investigation, and methodology; K.K.: conceptualization,

supervision, project administration, data curation, and resources; T.S.: formal analysis, validation, and writing—review, and editing; N.A.: formal analysis and software; A.M.: formal

analysis, methodology, and writing—review and editing; H.R.: data curation, software, and writing—review, and editing. All authors agree to publish the data. CORRESPONDING AUTHORS

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Z.A., Khan, K., Shah, T. _et al._ Biological investigations of _Aspergillus ficuum_ via in vivo_, _in vitro and in silico analyses. _Sci Rep_ 13, 17260 (2023).

https://doi.org/10.1038/s41598-023-43819-y Download citation * Received: 03 May 2023 * Accepted: 28 September 2023 * Published: 11 October 2023 * DOI:

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