Phytochemical Profiling and Evaluation of Antibacterial, Anticancer, Anti‐inflammatory, and Wound‐Healing Activities of Endophytic Fungi Isolated From Atalantia racemosa

ABSTRACT

Endophytic fungi are recognized as prolific sources of structurally diverse secondary metabolites with significant pharmaceutical potential. In the present study, endophytic fungi were isolated from healthy tissues of Atalantia racemosa (Rutaceae), an ethnomedicinal plant traditionally used for respiratory disorders and blood purification. A total of three fungal isolates were obtained and identified as Aspergillus flavus, Pleosporales sp., and Endothia sp. based on morphological and molecular (ITS sequencing) analyses. Crude extracts of the isolates were subjected to phytochemical characterization using GC–MS, FTIR, and LC–MS techniques. GC–MS profiling revealed the presence of fatty acids, sterols, phenolics, and terpenoids, while FTIR analysis confirmed key functional groups such as hydroxyl, carbonyl, and aromatic moieties. Importantly, LC–MS analysis provided high‐resolution metabolite profiling, identifying diverse bioactive compounds including alkaloids, flavonoids, quinic acid derivatives, xanthones (mangostin derivatives), phenolic amides (N‐feruloyl‐octopamine), diterpenoids, chalcones, macrolides, and terpene lactones in both positive and negative ionization modes. Biological evaluation demonstrated notable antioxidant, antibacterial, anti‐inflammatory, anticancer, and wound healing activities, with Endothia sp. showing superior anticancer activity (IC₅₀: 19.37 µg/mL) and significant wound healing potential. The integration of LC–MS data highlights the metabolic diversity and pharmacological relevance of these fungal isolates. Overall, the study confirms that endophytic fungi associated with A. racemosa are promising sources of bioactive metabolites with potential applications in drug discovery.

1. Introduction

Endophytes are microorganisms that penetrate plant tissue beneath the epidermal cell layers, without causing any symptomatic infection to their host [1]. The term endophytes was coined by the German scientist Heinrich Anton de Bary in 1866. The word endophyte means “in the plant” (endon = within, phyton = plant). Endophytes are ubiquitous, colonizing all plants, and are isolated from most endophytic fungi. Fungi live inside their host available tissue, including the leaves, petioles, stems, twigs, bark, roots, fruit, flowers, and seeds. Fungal endophytes and their host plants range from being symbiotic to antagonistic or opportunistic pathogens [2, 3]. Plants limit endophyte growth but use various mechanisms to adapt to living environments [4, 5, 6]. Endophytic fungi are broadly classified into clavicipitaceous (Class I) and nonclavicipitaceous (Classes II and III) endophytes. Class I endophytes (family Clavicipitaceae) are predominantly associated with grasses, especially members of the Poaceae family, and are typically transmitted vertically through seeds. In contrast, nonclavicipitaceous endophytes (Classes II and III) comprise a diverse group of fungi that colonize a wide range of plant species, including both monocots and dicots. These endophytes are generally transmitted horizontally and exhibit varied ecological roles, including stress tolerance, growth promotion, and protection against pathogens [7]. Class II endophytes are diverse, ranging from pezizomycotina (Ascomycota), agaricomycotina, and pucciniomycotina (Basidiomycota) [8]. To maintain healthy symbiosis, endophytes produce many compounds that promote plant development and help them to adapt to environmental factors like temperature, humidity, geography location, and surrounding vegetation [9]. Endophytes protect host plant from pathogens and parasites, enhancing their resistance and tolerance to biotic and abiotic factors. Fungal endophytes have also been shown to be an important source of bioactive antimicrobial compounds such as alkaloids, peptides, steroids, and phenol, which have a wide range of applications in the medical field [10, 11]. Endophytic fungi produce secondary metabolites such as hypericin, which have been used to reduce depression and anxiety [12].

Atalantia racemosa, a plant belonging to the Rutaceae family, is known as Kattu naragam in Tamil. It is often found in Peninsular India and the Western Ghats, including the regions of South, Central, and Maharashtra, particularly the Sahyadri mountain range. The decoction of A. racemosa leaves is used to treat bronchitis, asthma, and cough, as well as to purify the blood [13]. The plant A. racemosa is commonly utilized by tribes in Tamil Nadu for the treatment of various diseases [14, 15]. A. racemosa (family Rutaceae) is an ethnomedicinally important plant known for its therapeutic applications and rich phytochemical composition, including alkaloids, flavonoids, and phenolic compounds. Medicinal plants are well recognized as reservoirs of endophytic microorganisms capable of producing diverse bioactive secondary metabolites, often similar to those of their host plants [16]. These endophytes play a significant role in enhancing plant fitness by improving stress tolerance and providing protection against pathogens through the production of biologically active compounds [17].

Despite its medicinal relevance, the endophytic microbiota of A. racemosa remains largely unexplored. This highlights the need to investigate its endophytes as potential sources of novel natural products. Endophytic fungi have been widely reported as promising candidates in drug discovery due to their metabolic diversity and ability to synthesize pharmacologically important compounds [18]. Studies on related members of the Rutaceae family further support this rationale. For instance, endophytes isolated from Citrus species have demonstrated notable antimicrobial and bioactive potential, suggesting that plants within this family harbor metabolically active endophytes of significant therapeutic interest [19]. Furthermore, previous studies on endophytes from Rutaceae and related species strengthen this rationale. For instance, endophytic fungi isolated from Aegle marmelos (Rutaceae) have shown significant antimicrobial and bioactive properties [20]. Similarly, endophytes from Citrus species have been reported to produce a wide range of secondary metabolites with antibacterial and antifungal activities [21]. Despite these advances, there are very limited or no detailed reports on endophytes associated with A. racemosa, underscoring the novelty of the present investigation and its importance in filling a critical gap in the existing literature.

Therefore, the selection of A. racemosa is justified based on its ethnomedicinal importance and the likelihood of discovering novel bioactive compounds from its associated endophytes. With this background the present study aimed to isolate and identify endophytic fungi from A. racemosa and screen for bioactive compounds using GC‐MS and FTIR analysis for biomedical applications.

2. Materials and Methods

2.1. Collection of Plant Sample

Healthy and mature plants were collected in March 2024 from Jamboti, Khanapur Taluk, Belgaum District, Karnataka. The plant was given for authentication. The material was processed in a laboratory within 24 h, and fresh material was used for isolation to reduce contamination.

2.2. Isolation of Endophytic Fungi

The plant material was thoroughly washed and cut into small pieces under aseptic conditions. The isolation of endophytic fungi was performed using a method described by Schulz et al. [16]. The samples were surface sterilized with sodium hypochlorite and ethanol, then rinsed three times with sterile distilled water. The samples were then plated on PDA media supplemented with streptomycin, sealed with parafilm, and incubated at 27°C. Fungi were subculture on PDA medium, and their colonies were characterized through microscopic observation and isolated fungi were molecularly identified using 18S rRNA internal transcribed spacer sequencing techniques. Genomic DNA was extracted from fungal isolates and amplified using universal primers ITS1 and ITS4. The PCR amplification process involved denaturation, annealing, extension, and extension at 95°C for 5 min. PCR amplicons were analyzed using agarose gel electrophoresis and purified using a commercial DNA purification kit. The purified ITS sequences were submitted to the NCBI GenBank database and aligned using Clustal W. Phylogenetic analysis was performed in MEGA 11 using the Neighbor‐Joining method with 1000 bootstrap replicates to assess branch reliability. The purified ITS sequences were then submitted to the National Center for Biotechnology Information (NCBI) GenBank database and aligned using Clustal W. The Neighbor‐Joining method was used to assess branch reliability.

2.3. Preparation of Fungal Crude Extract

The selected fungi were used for the extraction of secondary metabolites. Mycelia were inoculated with 100 mL of PDB and 5% molasses in a 250 mL Erlenmeyer flask, then the starter culture was transferred to a 100 mL Erlenmeyer flask for mass culture to the required biomass. After incubation, the culture broth was filtered with a muslin cloth. The fungal biomass was dried and crushed with a mortar and pestle and soaked in 75% ethanol and subjected to maceration for 24 h. The suspension was filtered using muslin cloth and evaporated at 60°C until a residue was obtained, which was then used as a crude extract.

2.4. FTIR Analysis

The Fourier transform infrared spectrophotometer (FTIR) is a highly useful tool to identify functional groups present in compounds. The extracts of endophytic fungi of various species were used for FTIR analysis. The extracts are mixed with a homogenized powder and placed in a sample container, and then the powdered extract is loaded into an FTIR spectrophotometer. To identify functional groups in metabolites with distinct peaks ranging from 400 to 4000 cm⁻¹.

2.5. Estimation of Total Flavonoid Content

The 10% aluminum chloride (AlCl₃) and 1 M sodium acetate were mixed with a known quantity of fungal extract (1 mg/mL) with respective solvents. Then, incubated in the dark for 45 min, and the absorbance was measured at 415 nm. The quantity was determined using the quercetin calibration curve. The findings were expressed as quercetin equivalent (QE) mg/100 mL of a sample [17]. The experiment was conducted in triplicate, and the results were presented as mean ± SD.

2.6. GCMS Analysis

The ethanolic extract of endophytic fungi was dissolved in methanol and subjected to GC‐MS to determine its phytochemical constituents. The GC‐MS‐QP 2010 Plus instrument was used for sample analysis, with an ionization voltage of 70 eV, injector temperature of 250°C, and split injector mode of 36.5 cm/s and pressure of 57.5 kPa. A sample of 1 µL was injected into a mobile phase of helium at a flow rate of 1 mL/min. The oven temperature was adjusted to 100°C, then 280°C for 9 min, resulting in a total run time of 34 min. The compounds were identified by comparing unknown and known spectrums and specifying their names, molecular weights, molecular formulas, retention times, peak areas, and compound nature. The identification of chemical constituents was carried out by comparing the obtained mass spectra and retention indices with reference spectra from the NIST and Wiley Mass Spectral Libraries.

2.7. LCMS Analysis

LC–MS analysis of fungal crude extracts was carried out using an Acquity UPLC system coupled with an Acquity SQD mass spectrometer (Waters, USA). Metabolite separation was achieved on a BEH C18 reversed‐phase column (50 × 2.1 mm, 1.7 µm) using a gradient elution system consisting of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). A 10 µL sample was injected, and the flow rate was maintained at 0.50 mL min^{− 1}. The gradient program started with 95% A and 5% B, which was gradually shifted to 5% A and 95% B by 3 min, held until 4 min for elution of nonpolar compounds, and then returned to initial conditions at 5 min, followed by equilibration until 6 min. Detection was performed at wavelengths of 210, 254, 280, and 320 nm to capture a broad range of secondary metabolites. Mass spectrometric analysis was conducted using electrospray ionization (ESI) in both positive and negative modes, with a capillary voltage of 2.8 kV and cone voltage of 30 V. The ion source temperature was set at 140°C, and desolvation was carried out at 400°C with a gas flow of 600 L h^{− 1}. Data acquisition and processing were performed using MassLynx software (version 4.1). Metabolite identification was based on retention time, m/z values, isotopic distribution, and comparison with spectral libraries and literature reports. LC–MS is a highly sensitive and selective technique widely used for rapid characterization of fungal secondary metabolites such as alkaloids, flavonoids, terpenoids, and phenolics.

2.8. Antioxidant Activity

2.8.1. DPPH Radical Scavenging Activity

The DPPH radical's scavenging ability was used to assess the antioxidant activity. The DPPH test is conducted using the method described by Rice–Evans et al. [18]. The endophytic fungal extract was tested at 100 µg and combined in various concentrations with 100 µL of freshly prepared DPPH solution (40 mM). The samples were thoroughly mixed and stored for 30 min at room temperature and in the dark to get the desired color. The mixtures were analyzed for their DPPH radical‐scavenging activity using a UV–vis spectrophotometer, observing their absorbance at 517 nm. The antioxidant activity was calculated using the formula:

$$\mathrm{Antioxidant}\mathrm{activity}\left(\%\right)=\left(\frac{\left(Ac-Ae\right)}{Ac}\right)\times 100$$

Ac is the absorbance of the control reaction

Ae is of the test extract

2.9. Antibacterial Activity

The antimicrobial activity of the fungal extracts was screened using the agar well diffusion method [19] using Escherichia coli and Staphylococcus aureus as test organisms. The inoculum was spread evenly over the sterile agar plates. The extract was added to the sterile agar plates at different concentrations (30, 60, 90, and 120 µL/well). In addition to the control drug Ciprofloxacin (30 µg), and incubated for 24 h at 37°C. The inhibition zones were measured in millimeters.

2.10. Cytotoxicity and Anticancer Activity

2.10.1. Cell Line

A375 skin cancer cell line, and L929 fibroblast cell lines where grown in DMEM (Dulbecco's Modified Eagle Medium) media with 10% FBS provided by the National Centre for Cell Science in Pune, India. The cells were maintained at 37°C and 5% CO₂.

2.10.2. MTT Cell Viability Assay

The cytotoxicity of the compound was assessed through an MTT assay. A375 and L929 cells were seeded in 96 microtiter plates at 1.4 × 10⁴ cells/well and incubated at 37°C for 24 h. 200 µL of different test concentrations (50, 100, 150, 200, and 250 µg/mL from stock) of test drugs were added to the well and incubated at 37°C and 5% CO₂ atmosphere for 24 h. After 24 h of treatment, 0.5 mg/mL MTT reagent was added to the medium and incubated for 3 h. After removing the MTT solution, 100 µL of DMSO was added to each well to dissolve the formazan crystals [20]. After complete solubilization, the absorbance at 570 and 630 nm was measured. IC₅₀ was determined to be 50% inhibition of viability, and the sample's impact on A375 and L929 cell proliferation was expressed as a percentage of cell viability.

$$\mathrm{Inhibition}\mathrm{Percentage}=\frac{\mathrm{OD}\mathrm{of}\mathrm{test}\mathrm{sample}}{\mathrm{OD}\mathrm{of}\mathrm{test}\mathrm{control}}\times 100$$

2.11. Anti‐Inflammatory

The protein denaturation method by the standard methodology to evaluate the anti‐inflammatory activity of the ethanolic extract of the Aspergillus flavus, Pleosporales sp., and Endothia sp., [21]. The reaction mixture was with phosphate‐buffered saline (1 mL) and 50 µL of bovine serum albumin (BSA). Aspirin is used as a standard drug. The sample and aspirin were incubated for 15 min at room temperature. Denaturation was induced by maintaining the mixture at 70°C in a hot water bath for 15 min. Absorbance was measured at 660 nm using a Labman UV‐vis spectrophotometer, and the percentage inhibition of protein denaturation was calculated.

$$\%\mathrm{inhibition}=[(Ac-At)/Ac]$$

where, Ac = absorbance of control; At = absorbance of test samples.

2.12. Wound Healing

The study used a wound‐healing technique to examine cell migration and proliferation in samples. The cells were seeded in 12‐well plates and grown for 24 h at 37°C in a humidified environment, resulting in a confluent cell monolayer. After adhesion, the media was removed, and the monolayer was scraped with a micropipette, and reference points were marked. Cellular debris was removed from the wells by washing with PBS. Ascorbic acid was used as a positive control, and untreated cells were used as a negative control and incubated at 37°C with a 5% CO₂ atmosphere. Cell migration, wound closure, and morphological changes in the cells were observed by capturing images using an inverted microscope equipped with a digital camera at different time intervals (12 and 24 h). The width of the scratch and wound closure was examined quantitatively at 4X resolution with Mag Vision Software. The migration rate and % wound closure were calculated using the formula, respectively [22, 23].

$$\mathrm{Rm}=\frac{W_i-W_f}{T}$$

$$\%\mathrm{of}\mathrm{wound}\mathrm{closure}=\frac{{\mathrm{A}}_0-A_t}{A_t}\times 100$$

where A ₀ = area of the wound at time zero, A _t = area after time t, W _i = early wound width, W _f = closing wound width measured in µm, and T = time taken for migration in hours.

2.13. Statistical Analysis

All the results (triplicates) were represented as mean ± standard deviation. One‐way ANOVA was carried out to check the variation between the samples using IBM SPSS Statistics 20, Ink, India.

3. Results

3.1. Isolation and Molecular Identification of Endophytic Fungi

The three endophytes were isolated from leaves and identified based on their characteristics, reproductive structure, and molecular basis. The isolates were slow‐growing, exhibiting dark, round‐shaped colonies with mycelia on PDA medium. The 7‐day‐old fungal culture was observed, and the mycelia were stained with lactophenol cotton blue, showing the presence of conidiophores and spores. The phylogenetic tree was constructed based on the ITS sequence. The fungal isolates were identified as A. flavus, Pleosporales sp., and Endothia sp. The accession numbers of the isolates were PV569472, PV569473, and PV569474 (Figures 1 and 2).

FIGURE 1.

Isolated endophytic fungal colony and its microscopic structure of (A) Aspergillus flavus, (B) Pleosporales sp., and (C) Endothia sp.

FIGURE 2.

Phylogenetic tree of endophytic fungi isolated from Aspergillus flavus, Pleosporales sp., and Endothia sp. based on the internal transcribed spacer.

3.2. Crude Extraction

The phytochemical study of the ethanolic fungal extract showed a significant amount of plant secondary metabolites. The total yield of the ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp., was determined by 31.26%, 40.41%, and 11.66% (w/w), respectively.

3.3. FTIR Analysis

The FTIR spectrum was used to identify the functional groups of active components present in the fungal extract based on their IR radiation peaks. The result of the FTIR analysis revealed the presence of alcohol, Alkene, Amine, Alkane, Vinyl ether, Sulfonyl chloride, and secondary amine (Tables 1, 2, 3).

TABLE 1.

FTIR interpretation of the ethanolic extract of Aspergillus flavus.

Frequency	Functional group	Bond strength	Nature of the bond
3287.85	Alcohol	O─H stretching	Covalent bond
2925.14	Alkane	C─H stretching	Nonpolar
1621.66	Conjugated alkene	C═C stretching	Nonpolar
1456.53	Alkane	C─H bending	Covalent bond
1083.13	Primary alcohol	C─O stretching	Polar covalent bond

TABLE 2.

FTIR interpretation of the ethanolic extract of Pleosporales sp.

Frequency	Functional group	Bond strength	Nature of the bond
3353.3	Secondary amine	N─H stretching	Nonpolar
2924.35	Amine salt	N─H stretching	Nonpolar
1629.15	Alkene	C═C stretching	Nonpolar
1405.28	Sulfonyl chloride	S═O stretching	Covalent bond

TABLE 3.

FTIR interpretation of the ethanolic extract of Endothia sp.

Frequency	Functional group	Bond strength	Nature of the bond
2925.97	Alkane	C─H stretching	Covalent bond
1625.36	Conjugated alkene	C═C stretching	Nonpolar
1406.71	Sulfonyl Chloride	S═O stretching	Covalent bond
1085.41	Primary alcohol	C─O stretching	Polar covalent bond
1026.23	Amine	C─N stretching	Basic
931.70	Alkene	C═C bending	Nonpolar

3.4. Total Flavonoid Content

The total flavonoid content was estimated using the AlCl₃ method, with Quercetin as the standard drug. The total flavonoid content in the crude extract of A. flavus (23.32%), Pleosporales sp. (35.32%), and Endothia sp. (31.72%).

3.5. GC‐MS Analysis

The ethanolic fungal extracts of GC‐MS analysis revealed the phytoconstituent that may contribute to its therapeutic effects. The phytochemical compounds were identified based on their peak area, molecular formula, molecular weight, and retention time. The major compounds of various ethanolic extracts of A. flavus, Pleosporales sp., and Endothia sp. Arbiraterone and Ergosterol was found to be major compounds in the A. flavus. Hexadeconoic acid, ethyl ester and (E)‐9‐Octadecenoic acid, ethyl ester was found to be a major compounds in the Pleosporales, Sorbitol and Carbamic acid, N‐[10,11‐dihydro‐5‐(2‐methylamino‐1‐oxoethyl)‐3‐5H‐dibenzo[b,f]azepinyl]‐, ethyl ester was found to be a major compound in Endothia sp., overall, the GC‐MS results of the ethanolic extract showed the presence of 30, 30, and 30 phytocompounds, respectively and the results are shown in (Tables 4, 5, 6).

TABLE 4.

GC‐MS analysis of phytochemical compounds in the ethanolic extract of Aspergillus flavus.

Compound name	Molecular formula	Molecular weight	Retention time	Peak area in %	Nature of the compound	Uses
Hydrazine, (2‐methyl‐1‐propenyl)‐	C4H10N2	86	2.710	1.63	Alkene	Anticancer, antimicrobial, and antifungal
Cyclooctasiloxane, hexadecamethyl‐	C16H48O8Si8	592	14.042	4.12	Alkaloids	Antibacterial, wound healing
.alpha.‐D‐Galactopyranoside, methyl	C7H14O6	194	15.747	3.48	Glycosides	Anticancer, anti‐inflammatory
Cyclononasiloxane, octadecamethyl‐	C18H54O9Si9	666	17.238	3.28	Fatty acid	Antibacterial
Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15‐hexadecamethyl	C16H50O7Si8	578	19.685	1.36	Fatty acid ester	Supporting metabolic and heart health
n‐Hexadecanoic acid	C16H32O2	256	20.331	1.67	Palmitric acid (saturated fatty acid)	Antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant
Hexadecanoic acid, ethyl ester	C18H36O2	284	20.919	2.19	Palmitic acid ester	Antioxidant, hemolytic, hypocholesterolemic, flavor, nematicide, antiandrogenic.
Cyclononasiloxane, octadecamethyl‐	C18H54O9Si9	666	22.194	1.53	Fatty acids	Antibacterial
9‐octadecenoic acid, 2,2,2‐trifluoroethyl ester	C20H35F3O2	364	23.307	1.97	Fatty acid ester	Antimicrobial
9,12‐Octadecadienoic acid (Z,Z)‐	C18H32O2	280	23.718	3.22	Linoleic acid	Anticoronary, antialopecic, antiarteriosclerotic, antieczemic, cancer preventive,
(E)‐9‐Octadecenoic acid ethyl ester	C20H38O2	310	23.837	4.19	Ester	Antimicrobial, antiparasitic, cosmetic
Octadecanoic acid, ethyl ester	C20H40O2	312	24.295	1.35	Ester	Inflammatory, anticancer, insectifuge
Cyclononasiloxane, octadecamethyl‐	C18H54O9Si9	666	32.942	1.06	Essential oil	Cosmetics and personal care products due to its desirable properties
Squalene	C30H50	410	33.886	1.09	Triterpene	Neutralize different xenobiotics, anti‐ inflammatory, antiatherosclerotic
Cyclodecasiloxane, eicosamethyl‐	C20H60O10Si10	740	34.740	1.18	Cyclic methyl siloxane	Cosmetic formulations
5‐Cholesten‐3beta‐yl isobutyl carbonate	C32H54O3	486	34.921	1.57	Sterol	Antimicrobial, drug delivery
Ergosterol	C28H44O	396	38.943	8.05	Sterol	Anticancer
4‐Hydroxy‐2‐methylbenzoic acid, TMS	C14H24O3Si2	296	39.575	1.73	Phenol	Antioxidant, anti‐inflammatory and antimicrobial
Murolan‐3,9(11)‐diene‐10‐peroxy	C15H24O2	236	40.436	1.03	Terpene	Antioxidant, antimicrobial
Anthraergostatetraenol	C28H42O	394	40.729	2.59	Steroid	Anticancer, antioxidant, anti‐inflammatory and antimicrobial
Heptasiloxane, hexadecamethyl‐	C16H48O6Si7	532	41.246	1.02	Organosilicon	Antibacterial
4‐[1‐(1‐Hydroxyethyl)‐1H‐indol‐4‐yl]‐2‐methylbut‐2‐en‐1‐	C15H19NO2	245	42.218	4.38	Alkaloid	Neuroactive, anti‐inflammatory and anticancer
Abiraterone	C24H31NO	349	42.461	33.08	Sterol	Anticancer
3‐(4,7‐Dimethylocta‐3,7‐dienyl)‐1H‐indole	C18H23N	253	42.784	3.79		Antioxidant, anti‐inflammattory, anticancer

TABLE 5.

GC‐MS analysis of compounds in the ethanolic extract of Pleosporales sp.

Compound name	Molecular formula	Molecular weight	Retention time	Peak area in %	Nature of the compound	Uses
Argon	Ar	40	2.877	1.18		Anti‐inflammatory, wound healing,
Allene	C3H4	40	3.092	1.32	Alkene	Anticancer, anti‐inflammatory
2‐(Aminomethyl)‐4‐methylpentanoic acid, N‐methyl‐, methyl	C9H19NO2	173	15.269	1.77	Amino acids	Drug delivery, enzyme inhibitor
Acetamide, N‐methyl‐N‐heptyl‐	C10H21NO	171	15.411	1.24	Amide	Anti‐inflammatory, antimicrobial, and antifungal
.alpha.‐D‐Galactopyranoside, methyl	C7H14O6	194	15.746	2.38	Glycoside	Antimicrobial, antidiabetic
Diimidotricarbonic diamide	C3H6N4O3	146	15.896	1.19	Amide	Antimicrobial, antidiabetic, anti‐inflammatory, anticancer
3‐(Prop‐2‐en‐1‐yloxy)propan‐1‐ol, TMS	C9H20O2Si	188	16.225	1.43	Alcohol	Tissue engineering, anticancer, antimicrobial
d‐Glycero‐d‐galacto‐heptose	C7H14O7	210	18.041	2.22		Antimicrobial, anti ‐inflammatory
n‐Hexadecanoic acid	C16H32O2	256	20.355	7.77	Fatty acid	Antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant, antiandrogenic, hemolytic
Hexadecanoic acid, ethyl ester	C18H36O2	284	20.925	18.76	Palmitic acid ester	Antioxidant, hemolytic, hypocholesterolemic, flavor, nematicide, antiandrogenic.
9,12‐Octadecadienoic acid (Z,Z)‐	C18H32O2	280	23.214	3.64	Fatty Acid	Anti‐inflammatory, hypocholesterolemic, cancer preventive, insectifuge,
Oleic Acid	C18H34O2	282	23.327	4.67	Fatty acid	allergenic, anti‐inflammatory, anticancer, insectifuge
trans, trans‐9, 12‐Octadecadienoic acid, propyl ester	C21H38O2	322	23.724	8.06	Ester	Anti‐inflammatory, anticancer, antioxidant.
(E)‐9‐Octadecenoic acid ethyl ester	C20H38O2	310	23.841	12.00	Ester	Anti‐inflammatory, antimicrobial
Octadecanoic acid, ethyl ester	C20H40O2	312	24.300	3.71	Ester	Inflammatory, anticancer, insectifuge
9,12‐Octadecadienoic acid (Z,Z)‐, 2,3‐dihydroxypropyl ester	C21H38O4	354	32.037	1.50	Ester	Anti‐inflammatory, antioxidant.
Oleoyl chloride	C18H33ClO	300	32.113	1.94		Anticancer, wound healing,
5,7,9(11)‐Androstatriene, 3‐hydroxy‐17‐oxo‐	C19H24O2	284	35.801	0.98	steroid.	Anticancer, anti‐inflammatory
Mannonic acid (Et‐TFA)	C18H11F15O12	704	36.175	1.03	Carboxylic acid	Anti‐inflammatory, anticancer
Dehydroergosterol 3,5‐dinitrobenzoate	C35H44N2O6	588	38.433	1.30	Sterol	Antifungal, Anti‐inflammatory, membrane interaction study
4 15‐nor‐Prezizaan‐7‐one			38.515	1.73	Steroid	Anti‐inflammatory, anticancer
2‐(4‐(1‐Hydroxyethyl)‐2,5‐dimethoxyphenyl)ethylamine, N	C17H25NO5	323	38.667	1.52	Amine	Neuropharmacology, Antioxidant
Ergosterol	C28H44O	396	38.940	7.21	Sterol	Antifungal, antioxidant, anti‐inflammatory, anticancer
6‐Dimethyl(chloromethyl)silyloxytetradecane	C17H37ClOSi	320	39.233	1.96	Silicon	Wound healing, anticancer, antibacterial
3‐Hydroxypiperidin‐2‐one	C5H9NO2	115	39.390	1.34	Lactam	Antimicrobial, enzyme inhibitors
2‐Methyl‐N‐(4‐methylphenyl)benzene‐1‐carboximidic acid,	C18H23NOSi	297	39.475	1.07	Carboximidic acid	Antimicrobial, anticancer
2,4,6‐Cycloheptatrien‐1‐one, 3,5‐bis‐trimethylsilyl‐	C13H22OSi2	250	39.570	2.34	Ketone	Antimicrobial, anticancer, anti‐inflammatory, antioxidant
1‐Pentene, 1,3‐diphenyl‐1‐(trimethylsilyloxy)‐	C20H26OSi	310	39.733	1.84	Alkene	Drug delivery, anticancer, anti‐inflammatory
4‐Chloro‐8‐methoxy‐N,N‐dimethyl‐5‐nitroquinolin‐2‐amine	C12H12ClN3O3	281	40.498	1.01		Antimalaria, antibacterial, anticancer, anti‐inflammatory
Ergosta‐4,6,8(14),22‐tetraen‐3‐one	C28H40O	392	41.315	1.89	Steroid	Antifungal, anticancer, antioxidant, cosmetic

TABLE 6.

GC‐MS analysis of compounds in the ethanolic extract of Endothia sp.

Compound name	Molecular formula	Molecular weight	Retention time	Peak area in %	Nature of the compound	Uses
Propyne	C3H4	40	3.051	1.63		Anticancer, antiviral
Glycerin	C3H8O3	92	6.176	2.50	Alcohol	Flavor
Methyl[(1‐methyl‐2,3‐dihydropyrrol‐3‐yl)methyl]amine	C7H14N2	126	10.117	2.37	Alkaloid	Antimicrobial, anticancer, neurodegenerative, anti‐inflammatory
Methyl[(1‐methyl‐2,3‐dihydropyrrol‐3‐yl)methyl]amine	C14H22O	206	14.490	2.40	Phenol	Antioxidant, Anti‐inflammatory, anticancer
2R,3S‐9‐[1,3,4‐Trihydroxy‐2‐butoxymethyl]guanine	C10H15N5O5	285	14.685	4.28		Antiviral, anticancer
Pentane‐1,2,3,4,5‐pentaol	C5H12O5	152	14.756	4.09	Alcohols	Antidiabetic, wound healing
.alpha.‐D‐Galactopyranoside, methyl	C7H14O6	194	15.679	1.15	Glycosides	Anticancer, anti‐inflammatory
.alpha.‐L‐Galactopyranoside, methyl 6‐deoxy‐2‐O‐(trimethylsilyl)	C11H23BO5Si	274	15.890	1.59	Glycoside	Antimicrobial, Anticancer
3‐Deoxy‐d‐mannitol	C6H14O5	166	16.399	1.41	Alcohol	Drug delivery
Octadecanoic acid	C18H36O2	284	17.390	1.37	Stearic acid	Antifungal, antitumor, antibacterial, cosmetic
Heptadecanoic acid, heptadecyl ester	C34H68O2	508	17.870	2.05	Fatty acid ester	Wound healing, anti‐inflammatory, antioxidant
4‐Diisopropylsilyloxytridecane	C19H42OSi	314	18.070	3.33		Anticancer
Ethyl 2‐hexenoate, trans‐	C8H14O2	142	18.150	1.74	Ester	Antimicrobial, flavour
1,1‐Cyclopropanedicarboxamide	C5H8N2O2	128	18.330	5.00	Amide	Anticancer, antimicrobial, enzyme inhibition
Sorbitol	C6H14O6	182	18.683	19.81	Alcohol	Antidiabetic, wound healing
n‐Hexadecanoic acid	C16H32O2	256	20.344	2.41	Fatty acid	Antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant, antiandrogenic, hemolytic
Oleyl alcohol, trifluoroacetate	C20H35F3O2	364	23.316	2.53	Ester	Anticancer, anti‐inflammatory
9,12‐Octadecadienoic acid (Z,Z)‐	C18H32O2	280	23.727	3.19	Linoleic acid	Anticoronary, antialopecic, antiarteriosclerotic,
Octadecanoic acid, ethyl ester	C20H40O2	312	24.298	1.76	Ester	Anti‐inflammatory, anticancer, insectifuge
4‐Piperidinone, 1,2,5‐trimethyl‐, o‐(4‐nitrophenyl)oxime	C14H19N3O3	277	25.053	2.26	Oxime	Anticancer, antimicrobial, enzyme inihibition
5‐Bromo‐N,N‐dimethyl‐2H‐1,2,4‐triazole‐3‐carboxamide	C5H7BrN4O	218	26.124	2.20	Amide	Antimicrobial, anticancer,
Pentasiloxane, dodecamethyl‐	C12H36O4Si5	384	36.380	1.95	Silicon	Wound healing, cosmetics,
3‐(2‐Benzyl‐benzoimidazol‐1‐yl)‐propane‐1,2‐diol	C17H18N2O2	282	38.455	1.56		Antimicrobial, anticancer, anti‐inflammmatory
Ergosterol	C28H44O	396	38.951	3.57	Sterol	Antifungal, anticancer.
Methyl 2‐oxo‐5,6,7,8‐tetrahydro‐1H‐quinoline‐3‐carboxylate	C11H13NO3	207	39.155	1.68		Antimicrobial, anticancer, anti‐inflammmatory
1‐(3,4‐Dihydroxyphenyl)‐2‐(methylamino)ethan‐1‐one, 2TM	C15H27NO3Si2	325	39.705	5.87	ketone	Antioxidant, anticancer, neurological
3‐Methyl‐7‐(4‐methyl‐piperazin‐1‐yl)‐3H‐thiazolo[4,5‐d]pyrimidine‐2‐thione	C11H15N5S2	281	39.930	2.19		Antimicrobial, anticancer, antioxidant
Carbamic acid, N‐[10,11‐dihydro‐5‐(2‐methylamino‐1‐oxoeth	C20H23N3O3	353	40.205	6.75	Amide	Antimicrobial, anticancer, anti‐inflammatory
5 Trimethylsilyl‐di(timethylsiloxy)‐silane	C9H27O2Si4	279	41.353	2.73	Silane	Wound healing, antimicrobial, cosmetic

3.6. LCMS Analysis

The LC–MS analysis of ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp. revealed the presence of a wide range of secondary metabolites with varying chemical characteristics. Chromatographic detection at multiple wavelengths (210, 254, 280, and 320 nm) showed multiple peaks corresponding to compounds with different polarities and structural classes. The total ion chromatograms obtained in both positive and negative ionization modes confirmed the presence of both polar and non‐polar metabolites distributed across different retention times. Early eluting peaks represented polar compounds, whereas later retention times corresponded to relatively hydrophobic metabolites.

In the ethanolic extract of A. flavus, several compounds were detected including 4‐methyl‐5‐thiazoleethanol, exemestane derivatives, nomegestrol acetate derivatives, mangostin trimethyl ether, N‐feruloyl‐octopamine, flavonol derivatives, and quinic acid derivatives. Additional metabolites such as pomiferin, voacristine, honokiol, and 4‐methylabyssinone V were observed in negative ion mode. These findings indicate that extract contains a mixture of phenolics, flavonoids, alkaloids, and steroid‐like compounds. The results are shown in Table 7.

TABLE 7.

LCMS analysis of ethanol extract of Aspergillus flavus.

RT (min)	Compound	m/z (approx.)	Mode	Nature (Class)	Reported biological activity
0.312	4‐Methyl‐5‐thiazoleethanol	∼144 [M + H]+	Positive	Thiazole derivative	Antimicrobial, antioxidant
0.642	Exemestane	∼297 [M + H]+	Positive	Steroidal compound	Anticancer
0.888	Nomegestrol acetate	∼413 [M + H]+	Positive	Steroid hormone	Hormonal activity
0.972	Quassinoid derivative	∼400+ [M + H]+	Positive	Quassinoids	Antimalarial, anticancer
1.042	7‐beta‐Hydroxylathyrol	∼345 [M + Na]+	Positive	Diterpenoid	Anti‐inflammatory
1.077	Mangostin trimethyl ether	∼425 [M + H]+	Positive	Xanthone	Antioxidant, anticancer
1.615	Isoquinoline derivative	∼350 [M + H]+	Positive	Alkaloid	Neuroactive
2.518	N‐feruloyl‐octopamine	∼332 [M + Na]+	Positive	Phenolic amide	Antioxidant
2.640	Aporphine derivative	∼342 [M + H]+	Positive	Alkaloid	Antimicrobial
2.761	Flavonol derivative	∼600+ [M + H]+	Positive	Flavonoid	Anti‐inflammatory
2.970	Valine derivative	∼500+ [M + H]+	Positive	Amino acid derivative	Metabolic role
3.178	Urapidil	∼387 [M + H]+	Positive	Phenylpiperazine	Antihypertensive
3.838	Hydroxybenzaldehyde derivative	∼300+ [M + Na]+	Positive	Phenolic aldehyde	Antioxidant
4.585	1,3,5‐Tricaffeoylquinic acid	∼515 [M + H]+	Positive	Quinic acid	Hepatoprotective
5.557	10‐Hydroxyusambarine	∼350+ [M + H]+	Positive	Harmala alkaloid	Neuroactive
0.286	4‐O‐Methylphloracetophenone	∼180 [M‐H]‐	Negative	Phenolic ketone	Antioxidant
1.971	Lagochilin	∼350 [M − H]−	Negative	Diterpenoid	Anti‐inflammatory
2.527	Alpha‐guaiaconic acid	∼300 [M − H]−	Negative	Phenolic compound	Antioxidant
2.961	Valine derivative	∼500+ [M − H]−	Negative	Amino acid derivative	Metabolic role
3.048	Calopocarpin	∼350 [M − H]−	Negative	Pterocarpan	Antimicrobial
3.291	Dofetilide	∼440 [M − H]−	Negative	Sulfanilide	Cardiac drug
3.360	Voacristine	∼368 [M − H]−	Negative	Indole alkaloid	Anticancer
3.794	Pomiferin	∼453 [M − H]−	Negative	Isoflavone	Anti‐inflammatory
4.420	4‐Methylabyssinone V	∼435 [M − H]−	Negative	Flavanone	Antioxidant

Ethanolic extract of Pleosporales sp. exhibited a comparatively moderate number of metabolites. The major compounds identified included mangostin trimethyl ether, alibendol, N‐feruloyl‐octopamine, hydroxybenzaldehyde derivatives, and quinic acid derivatives. In addition, isosakuranetin, linoleic acid, elaidic acid, and honokiol were detected in negative ion mode. These results suggest that extract mainly consists of phenolic compounds, flavonoids, and fatty acids (Table 8).

TABLE 8.

LCMS analysis of ethanol extract of Pleosporales sp.

RT (min)	Compound	m/z (approx.)	Mode	Nature (Class)	Reported biological activity
1.077	Mangostin trimethyl ether	∼425 [M + H]+	Positive	Xanthone	Anticancer
1.632	Alibendol	∼239 [M + H]+	Positive	Salicylamide	Anti‐inflammatory
2.518	N‐feruloyl‐octopamine	∼332 [M + Na]+	Positive	Phenolic amide	Antioxidant
3.855	Hydroxybenzaldehyde derivative	∼300+ [M + Na]+	Positive	Phenolic aldehyde	Antimicrobial
4.567	1,3,5‐Tricaffeoylquinic acid	∼515 [M + H]+	Positive	Quinic acid	Hepatoprotective
0.321	Obscurolide A1	∼250 [M − H]−	Negative	Aminobenzoic acid	Antimicrobial
0.842	Methoxychromone derivative	∼260 [M − H]−	Negative	Chromone	Antioxidant
1.624	Methoxybenzoic acid derivative	∼300 [M − H]−	Negative	Phenolic acid	Antioxidant
2.336	Pyranocoumarin	∼350 [M − H]−	Negative	Coumarin	Anticancer
2.648	Coumarin derivative	∼320 [M − H]−	Negative	Coumarin	Anti‐inflammatory
3.030	Isosakuranetin	∼285 [M − H]−	Negative	Flavonoid	Antioxidant
3.169	Linoleic acid	∼279 [M − H]−	Negative	Fatty acid	Anti‐inflammatory
3.360	Elaidic acid	∼281 [M − H]−	Negative	Fatty acid	Lipid metabolism
3.829	Coumarin glycoside	∼500+ [M − H]−	Negative	Glycoside	Antioxidant
4.263	Honokiol	∼265 [M − H]−	Negative	Biphenyl	Anticancer

Ethanolic extract of Endothia showed a higher number of metabolites distributed across the chromatographic gradient. Identified compounds included 4‐methyl‐5‐thiazoleethanol, mangostin trimethyl ether, N‐feruloyl‐octopamine, (‐)‐eburnamonine, saliniketal A, Mexicanolide derivatives, quinic acid derivatives, aphidicolin, trimethoxychalcone, linoleic acid, macrolide derivatives, and terpene lactones. These findings indicate that extract contains a diverse group of metabolites including alkaloids, terpenoids, chalcones, macrolides, and fatty acids (Table 9).

TABLE 9.

LCMS analysis of ethanol extract of Endothia sp.

RT (min)	Compound	m/z (approx.)	Mode	Nature (Class)	Reported biological activity
0.312	4‐Methyl‐5‐thiazoleethanol	∼144 [M + H]+	Positive	Thiazole	Antimicrobial
1.077	Mangostin trimethyl ether	∼425 [M + H]+	Positive	Xanthone	Antioxidant
1.754	Terpene glycoside	∼600+ [M + H]+	Positive	Glycoside	Antioxidant
2.518	N‐feruloyl‐octopamine	∼332 [M + Na]+	Positive	Phenolic amide	Neuroprotective
2.622	(‐)‐Eburnamonine	∼352 [M + Na]+	Positive	Alkaloid	Neuroprotective
2.778	Triazole derivative	∼350 [M + H]+	Positive	Heterocycle	Antifungal
3.022	Terpene glycoside	∼600+ [M + Na]+	Positive	Glycoside	Antioxidant
3.456	Aporphine derivative	∼342 [M + H]+	Positive	Alkaloid	Neuroactive
3.508	Saliniketal A	∼400 [M + Na]+	Positive	Sesquiterpenoid	Cytotoxic
3.612	Mexicanolide derivative	∼500 [M + Na]+	Positive	Terpenoid	Anticancer
3.873	Hydroxybenzaldehyde derivative	∼300+ [M + Na]+	Positive	Phenolic	Antioxidant
4.567	1,3,5‐Tricaffeoylquinic acid	∼515 [M + H]+	Positive	Quinic acid	Anti‐inflammatory
0.321	Obscurolide A1	∼250 [M − H]−	Negative	Aminobenzoic acid	Antimicrobial
1.641	Aphidicolin	∼337 [M − H]−	Negative	Diterpenoid	Antiviral
1.693	Trimethoxychalcone	∼330 [M − H]−	Negative	Chalcone	Anticancer
2.110	Linoleic acid	∼279 [M − H]−	Negative	Fatty acid	Anti‐inflammatory
2.370	Indole‐3‐acetyl‐L‐phenylalanine	∼350 [M − H]−	Negative	Amino acid derivative	Growth regulator
2.544	Dihydropyranone derivative	∼300 [M − H]−	Negative	Lactone	Antimicrobial
2.631	Aspartame	∼293 [M − H]−	Negative	Peptide	Metabolic relevance
3.169	Macrolide derivative	∼400 + [M − H]−	Negative	Macrolide	Antibacterial
3.360	Sesquiterpenoid derivative	∼300 [M − H]−	Negative	Terpenoid	Anti‐inflammatory
4.350	Terpene lactone	∼300 [M − H]−	Negative	Terpenoid	Anti‐inflammatory
4.454	4‐Methylabyssinone V	∼435 [M − H]−	Negative	Flavanone	Antioxidant

3.7. Antioxidant Activity

3.7.1. Radical Scavenging Assay Using 2,2‐diphenyl‐2‐picrylhydrazyl (DPPH)

In case of the DPPH assay, the ethanol extract of A. flavus, Pleosporales sp., and Endothia sp. using the DPPH free radical scavenging assay. The three fungal extracts exhibited good scavenging activity, but the inhibition percentage was less compared to the standard ascorbic acid. The percentage inhibition of the A. flavus was (58.01 ± 0.386); Pleosporales sp. (50.66 ± 0.18); Endothia sp. (43.23 ± 0.23 and the standard ascorbic acid showed around 81.59 ± 0.5 (Table 10 and Figure 3).

TABLE 10.

DPPH assay of different ethanolic fungal extracts.

Concentration (µg/mL)	Ascorbic acid	Aspergillus flavus	Pleosporales sp.	Endothia sp.
50	34.13 ± 0.19	6.64 ± 0.22	10.05 ± 0.21	4.56 ± 0.23
100	49.86 ± 0.91	13.87 ± 0.31	15.77 ± 0.29	18.45 ± 0.14
150	63.46 ± 0.41	29.93 ± 0.29	26.06 ± 0.30	29.53 ± 0.14
200	76.84 ± 0.44	42.53 ± 0.23	37.97 ± 0.19	35.24 ± 0.26
250	81.59 ± 0.52	58.01 ± 0.38	50.66 ± 0.18	43.23 ± 0.23
IC50 (µg/mL)	104	225	255	276

The results are expressed as mean ± standard error.

FIGURE 3.

DPPH scavenging assay for the std Ascorbic acid and funal extract.

3.8. Antibacterial Activity

Ethanol extract of the A. flavus, Pleosporales sp., and Endothia sp., had the greatest zone of inhibition against E. coli and S. aureus of all the fungal extracts tested for antibacterial activity. With a maximum inhibitory zone of 19 mm in A. flavus extract was found to be more effective against S. aureus than the Pleosporales sp. (14 mm), and Endothia sp. (12 mm). A maximum inhibitory zone of 16 mm in A. flavus was found to be more sensitive to E. coli than the Pleosporales sp. (12 mm), and Endothia sp. (5 mm) (Table 11 and Figure 4).

TABLE 11.

Zone of inhibition (in mm) for different ethanolic fungal extracts.

Types of fungi	Inhibition zone (mm)
	Staphylococcus aureus				Escherichia coli
	30 µg	60 µg	90 µg	120 µg	30 µg	60 µg	90 µg	120 µg
Aspergillus flavus	14	16	14	19	10	11	13	16
Pleosporales sp.	3	4	12	14	9	12	13	12
Endothia sp.	10	15	20	12	1	1	2	5
Control	30				28

FIGURE 4.

Antibacterial activity of the fungal extract against S. aureus and E. coli.

3.9. Cytotoxicity and Anticancer Activity

The ethanol extract of A. flavus, Pleosporales sp., and Endothia sp., was used to treat the A375 (skin melanoma) cell line and L929 (noncancerous cell line) along with standard drug cisplatin as a positive and untreated cells as a negative control. The percentage of cell viability in the non‐cancerous (L929) cells decreased as the concentration of the extract increased. All the tested extracts showed cytotoxic nature at higher concentrations. At higher concentration, the ethanol extracts of A. flavus, Pleosporales sp., and Endothia sp. showed cell viability as 32.70 ± 2.82, 28.63 ± 0.62, and 31.66 ± 1.14, respectively. The results are shown in Table 12 and Figures 5 and 6.

TABLE 12.

Growth inhibitory activity of different ethanolic fungal extracts against the L929 cell line.

Concentration (µg/mL)	Percentage of cell viability
	Aspergillus flavus	Pleosporales sp.	Endothia sp.
50	91.43 ± 1.77	86.20 ± 1.35	90.38 ± 0.94
100	78.16 ± 1.25	70.42 ± 0.83	78.78 ± 1.46
150	72.62 ± 0.52	58.20 ± 1.56	64.36 ± 1.88
200	59.14 ± 1.88	45.24 ± 1.77	53.39± 1.56
250	32.064 ± 2.82	28.63 ± 0.62	31.66 ± 1.14
Cisplatin	10.44 ± 0.62

The results are represented as mean ± standard error.

FIGURE 5.

Percentage inhibition of cell viability of different ethanolic fungal extracts in the non‐cancerous (L929) cell line.

FIGURE 6.

Morphological changes in the L929 cell line after treatment with the different ethanolic fungal extracts.

After the toxicity study, the extract was subjected to anticancer activity against the skin cancer (A375) cell line using cisplatin as a standard drug for comparison. In the tested cell line the fungal extract showed a significant activity by suppressing the cells growth through a decrease in the cell viability. At the initial concentration of A. flavus was observed to be 88.40 ± 1.01; Pleosporales sp. was observed to be 81.75 ± 1.35, and Endothia sp. was observed to be 91.10 ± 0.78. The standard drug cisplatin at a given concentration of 15 µg/mL significantly affects the cancer cell line with a lower percentage of cell viability of 8.67 ± 0.03. The results are shown in Table 13 and Figures 7 and 8.

TABLE 13.

Growth inhibitory activity of different ethanolic fungal extracts against A375 cell line.

Concentration (µg/mL)	Percentage of cell viability
	Aspergillus flavus	Pleosporales sp.	Endothia sp.
50	88.40	81.75	91.10
100	77.36	64.52	73.64
150	65.76	54.84	61.14
200	55.63	31.64	50.45
250	36.93	20.38	31.30
Cisplatin	8.67

The results are represented as mean ± standard error.

FIGURE 7.

Percentage of inhibition of cell viability of the different ethanolic fungal extracts in skin cancer (A375) cell line.

FIGURE 8.

Morphological changes in the A375 cell line after treatment with different ethanolic fungal extracts.

The cellular morphology differed when untreated A375 cells were compared to treated cells. The untreated cells’ morphology was clear without any intercellular spaces. In case of the treated test samples, the number of cells gradually decreased as the concentration increased, and intercellular gaps were observed between the cells.

The IC₅₀ value was determined using the standardized calibration curve for the fungal extract. For the A375 cell line, the IC₅₀ value of the A. flavus was 209.46 µg/mL, whereas in the Pleosporales sp. results were 152 µg/mL, and the IC₅₀ value of the Endothia sp. was 19.37 µg/mL.

3.10. Anti‐Inflammatory

The study evaluated the anti‐inflammatory activity of the ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp. were subjected to an anti‐inflammatory activity through protein denaturation assay. The in vitro anti‐inflammatory activity of the extracts was comparable to that of Aspirin, a reference drug. The results revealed that the standard drug, aspirin, exhibited significant anti‐inflammatory activity with a percentage of inhibition of 85.81% compared to other fungal extracts. Whereas the A. flavus showed 49.26%, Pleosporales sp. showed 67.25% and Endothia sp. showed 58.61%. The results are shown in Table 14 and Figure 9.

TABLE 14.

The anti‐inflammatory activity of ethanolic fungal extracts by protein denaturation assay.

Concentration (µg/mL)	Percentage of protein inhibition
	Standard aspirin	Aspergillus flavus	Pleosporales sp.	Endothia sp.
50	34.72 ± 0.88	10.99 ± 0.84	34.32 ± 1.29	15.03 ± 1.41
100	54.25 ± 1.27	18.18 ± 0.61	42.64 ± 0.98	27.21 ± 1.14
150	65.28 ± 0.69	32.83 ± 1.33	48.57 ± 1.36	37.78 ± 1.11
200	74.07 ± 0.64	40.57 ± 0.97	58.29 ± 1.10	46.39 ± 1.02
250	85.81 ± 0.70	49.26 ± 1.46	67.25 ± 0.72	58.61 ± 1.11

The results are represented as mean ± standard error.

FIGURE 9.

Anti‐inflammatory activity of ethanolic fungal extracts.

3.11. Wound Healing Activity

Wound healing activity for the ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp., was studied in two ways by studying cell migration and the percentage of wound closure. In comparison, the standard drug ascorbic acid showed higher cell migration activity, followed by the fungal extract and the untreated group. For the standard ascorbic acid at 12, 24 h, the cell migration was found to be 27.70 and 19.44 µm, respectively. Similarly, the fungal extract of A. flavus, Pleosporales sp., and Endothia sp., at 12 h was found to be 7.8, 9.27, and 14.94 µm, whereas in the 24 h the cell migration was found to be 14.25, 14.11, and 12.59 µm, respectively. In case of wound closure at 12, 24 h, the standard ascorbic acid showed a value of 68.09% and 95.17%. In contrast, the ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp. at 12 h showed 19.89%, 23.96%, and 44.54% and whereas in the 24 h the wound closure shown to be 72.74%, 72.95% and 75.10%, respectively, and for the untreated group, it was found to be 7.50% at 12 h and 17.17% for 24 h (Tables 15 and 16 and Figure 10).

TABLE 15.

Cell Migration of different fungal extracts at different durations.

S no.	Test samples	Duration	Cell migration in µm
1	Untreated	12 h	3.21
		24 h	3.66
2	Standard	12 h	27.70
		24 h	19.44
3	Aspergillus flavus	12 h	7.8
		24 h	14.25
4	Pleosporales sp.	12 h	9.27
		24 h	14.11
5	Endothia sp.	12 h	14.94
		24 h	12.59

TABLE 16.

Percentage of wound closure of the different fungal extract at different duration.

S no.	Test samples	Duration	Percentage of wound closure
1	Untreated	12 h	7.50
		24 h	17.17
2	Standard	12 h	68.09
		24 h	95.17
3	Aspergillus flavus	12 h	19.89
		24 h	72.74
4	Pleosporales sp.	12 h	23.96
		24 h	72.95
5	Endothia sp.	12 h	44.54
		24 h	75.10

FIGURE 10.

The morphological wound healing of the ethanolic fungal extract using the scratch assay.

4. Discussion

Endophytes, comprising a diverse group of microorganisms residing asymptomatically within plant tissues, significantly contribute to the host plant's biochemical repertoire, thereby influencing its medicinal properties [24, 25]. Indeed, approximately 80% of populations in Africa, Asia, and Latin America still rely on traditional medicine, underscoring the enduring significance of these botanical resources [26]. This reliance highlights the urgent need for comprehensive research into these plants, particularly concerning the bioactive compounds produced by their associated endophytes, which offer a promising avenue for drug discovery and development [27, 28]. These microorganisms are prolific producers of secondary metabolites, many of which exhibit potent biological activities relevant to pharmaceutical applications [29]. This symbiotic relationship allows plants to produce a diverse array of biologically active chemicals [30]. Historically, various herbal medicines have been integral to traditional medical systems globally, with their unique secondary metabolites providing potential candidates for novel drug development [31].

Endophytic fungi, often known as plant hidden fungi, live naturally in plant tissues and do not cause any harm to the host. Endophytic microorganisms are those that live within the tissues of plants. Endophytic fungi are microorganisms often found in plants [32]. Endophytic fungi isolated from the leaves, stems, flowers, and seeds [33]. According to Selim et al. [34] and Zuber [35] the identification of fungi was also done by using the standard protocol of Barnett and Hunter [36]. Identification is based on the morphological characteristics of a fungus growing on PDA media. Microscopic characterization was done by observing the shape and size of conidia and hyphae. According to Anita and Sridar [37] they obtained 36 isolates of endophytic fungi from the Canavalia carthatica. The internal transcribed spacer region of nuclear ribosomal DNA is widely recognized as the primary genetic barcode for the precise identification and taxonomic classification of fungal species, including endophytic fungi [38, 39]. This region, commonly referred to as ITS, is comprised of ITS1, 5.8S rRNA, and ITS2 subunits, with ITS1 and ITS2 serving as particularly valuable targets for sequencing due to their variability and phylogenetic signal [40, 41]. This characteristic variability, coupled with conserved flanking regions, facilitates the design of universal primers suitable for broad‐range fungal amplification, making ITS sequencing an indispensable tool for characterizing fungal endophyte communities [42]. In the present study, three isolates of Endophytic fungi from the Atlantia racemosa. The phylogenetic tree was constructed based on the ITS sequence. The fungal isolates were identified as A. flavus, Pleosporales sp., and Endothia sp.

This investigation aims to identify and characterize the diverse array of secondary metabolites produced by endophytic fungi, which are recognized for their significant pharmaceutical potential [43]. Endophytic fungi, known for their enhanced metabolic capabilities compared to soil fungi, are prolific producers of a wide spectrum of bioactive compounds, including steroids, alkaloids, phenols, and terpenoids [44]. These compounds frequently exhibit a range of biological activities such as anticancer, antimicrobial, antioxidant, and anti‐inflammatory properties, making them valuable candidates for drug discovery and development [31, 45]. Specifically, techniques such as gas chromatography‐mass spectrometry are employed to profile the volatile organic metabolites present in these extracts, enabling a comprehensive understanding of their chemical composition [46]. This detailed analysis allows for the elucidation of both known and novel compounds, providing insights into the biosynthetic pathways and potential therapeutic applications of these fungal endophytes [46]. According to Khattab et al. [47] GC‐MS analysis of the A. flavus showed eight major compounds namely, tributylacetylcitrate (22.955), hexadecane (7.87%), pentadecane (7.81%), 1,2‐benzene dicarboxylic acid, diisoctyl ester (6.43%), 2‐methyl benzyl amine, N‐heptylN‐octyl (5.23%) benzene, (1‐methyl undecyl) (4.81%), heptadecane (4.56%) and 1,2‐benzene dicarboxylic acid, buty (4.05%). According to Samy et al. (2024) secondary metabolites produced by the endophytic fungus Pleosporales sp. isolated from the Artemisia annua were identified by using GC‐MS analysis revealed the presence of 5 compounds 13‐octadecenoic acid (19.36%), methyl palmitate (18.72%), ethyl palmitate (12.60%), and methyl linoleate (11.92%) were the major compounds and they six compounds in the methanol extracts of which methyl oleate (26.90%), methyl palmitate (25.48%), and methyl linoleate (23.39%) were the major compounds. In the present study the ethanol extract of A. flavus, Pleosporales sp., and Endothia sp. Arbiraterone and Ergosterol were found to be major compounds in A. flavus. Hexadeconoic acid, ethyl ester, and (E)‐9‐Octadecenoic acid, ethyl ester were found to be major compounds in the Pleosporales sp., Sorbitol and Carbamic acid, N‐[10,11‐dihydro‐5‐(2‐methylamino‐1‐oxoethyl)‐3‐5H‐dibenzo[b,f]azepinyl]‐, ethyl ester was found to be a major compound in Endothia sp.

Fourier transform infrared spectroscopy offers a distinctive fingerprint of biomarkers, enabling the discrimination of individual components and mixtures [48, 49]. High‐performance liquid chromatography further complements these techniques by enabling the separation and quantification of specific polyphenolic compounds, such as rutin and catechin equivalents, alongside other phenolic and flavonoid constituents within the extracts [50, 51]. This multipronged analytical strategy is critical for unravelling the chemical diversity of fungal metabolites, which encompass a wide array of bioactive compounds with significant biotechnological and pharmaceutical potential [52]. This is particularly relevant for the characterization of fungal biomass ethanol extracts, where the interplay of various compounds contributes to their overall bioactivity and warrants detailed structural elucidation and purity assessment. Furthermore, the application of chemometric tools to these spectroscopic and chromatographic data facilitates the descriptive analysis of process trends and the identification of key molecular features indicative of specific compound classes [53, 54]. Ultraviolet‐visible spectroscopy, for instance, serves as a common analytical tool to detect chromophores and conjugated systems in compounds, providing spectral information crucial for identifying complex mixtures typical in food samples and fungal extracts [55]. Fourier Transform Infrared spectroscopy, conversely, provides characteristic absorption bands indicative of functional groups such as hydroxyls and double bonds present in polygenic compounds, which is critical for structural identification and can be used to assess lipid extraction efficiency from fungal biomass [56, 57]. Meanwhile, high‐performance liquid chromatography is invaluable for isolating and quantifying individual components, even in intricate samples, offering high sensitivity and resolution for complex matrices like fungal extracts [58].

The LC–MS profiling of the ethanol extract of A. flavus, Pleosporales sp., and Endothia sp. demonstrates the ability of the fungal isolate to produce structurally diverse secondary metabolites belonging to multiple biosynthetic classes. The detection of both polar and nonpolar compounds across retention times suggests the presence of complex metabolic pathways operating within the fungal system.

The identification of thiazole‐containing metabolites such as 4‐methyl‐5‐thiazoleethanol indicates active microbial biosynthesis involving sulfur‐containing intermediates. Such compounds are commonly associated with microbial metabolism and are known to exhibit biological activities, including antimicrobial and antioxidant properties [59]. Their presence in multiple extracts suggests a conserved metabolic feature of the fungal strain.

Mangostin trimethyl ether, a prenylated xanthone detected in all extracts, indicates the involvement of polyketide biosynthetic pathways. Xanthones are well documented for their diverse pharmacological activities including antioxidant, antimicrobial, and anticancer effects [60]. The repeated detection of this compound across extracts suggests that it may represent a core metabolite produced by the fungal isolate under the given culture conditions.

The occurrence of N‐feruloyl‐octopamine highlights the role of phenolic amide biosynthesis in the fungal system. Phenolic amides are known to be associated with defense‐related mechanisms and antioxidant activity and are often linked to phenylpropanoid metabolism [61]. This indicates possible metabolic interactions between fungal pathways and plant‐derived precursors, particularly in endophytic fungi.

The detection of quinic acid derivatives such as 1,3,5‐tricaffeoylquinic acid suggests that the fungal strain may mimic plant‐like metabolic pathways. Such compounds are widely reported for their antioxidant, anti‐inflammatory, and hepatoprotective activities [62]. This phenomenon is commonly observed in endophytic fungi, which can produce host‐like bioactive metabolites [63].

Flavonoids such as pomiferin and isosakuranetin detected in the extracts are known for their antioxidant and anti‐inflammatory activities, while alkaloids such as voacristine and (‐)‐eburnamonine are associated with significant pharmacological properties, particularly in neurological applications [64]. The presence of these compounds supports the potential therapeutic relevance of the fungal extracts.

Additionally, the detection of fatty acids such as linoleic acid and elaidic acid indicates the contribution of lipid‐derived metabolites to the overall bioactivity profile. Fatty acids are known to exhibit antimicrobial and anti‐inflammatory properties and may act synergistically with other secondary metabolites [65].

The presence of diterpenoids such as aphidicolin and sesquiterpenoids such as saliniketal A further highlights the metabolic versatility of the fungal strain. These compounds are widely reported for their bioactive properties, including cytotoxic and antimicrobial activities, suggesting potential applications in pharmaceutical research.

According to Prabavathy and Nachiyar [66], UV–vis spectroscopic analysis of ethyl acetate extract of Aspergillus sp. isolated from Ficus carica showed a peak at 260 nm. The FTIR spectrum of the Aspergillus sp. peak in the origin 2929.46 may be due to the stretching vibration of C─H group and the peak in the region 1715.36 may be due to the stretching vibration of the C─O. The peaks at 1042.64 and 813.83 indicate the presence of C─O stretch and the phenyl ring, respectively. In the present study, UV–vis spectroscopic analysis of A. flavus, Pleosporales sp., and Endothia sp., profiles showed the sharp peak at 290, 212, and 291. FTIR analysis of the ethanolic extract of A. flavus, 3287.85 due to the stretching vibration of O─H stretching, Pleosporales sp., 3553.3 due to the stretching vibration N─H stretching and Endothia sp., 2925.97 due to the stretching vibration C─H stretching. FTIR analysis revealed the presence of alcohol, Alkene, Amine, Alkane, Vinyl ether, Sulfonyl chloride, and secondary amine.

This study investigates the multifaceted therapeutic potential of an ethanol extract derived from fungal biomass, encompassing its antioxidant, anti‐inflammatory, and cytotoxic effects against L929 and A375 skin cancer cell lines, alongside an assessment of its wound healing capabilities via scratch assay. Mushrooms have long been recognized for their medicinal properties, serving as a rich source of bioactive compounds applicable to pharmaceuticals, cosmeceuticals, and nutricosmetics, with traditional uses often extending to skin conditions [67]. Specifically, numerous macrofungi have been explored for their antiaging, antioxidant, photoprotective, and anti‐inflammatory properties, attributed to their diverse biochemical constituents such as polysaccharides, phenolic compounds, and triterpenes [68]. This growing interest stems from their ability to inhibit key enzymes implicated in skin aging and hyperpigmentation, such as elastase, tyrosinase, and collagenase [69]. The cosmetic industry increasingly seeks natural compounds with significant bioactive properties for cosmeceutical formulations, highlighting the potential of mushroom extracts in this area [70]. The varied chemical structures of compounds such as terpenes and terpenoids found in fungi contribute to a wide array of biological activities, including anticancer, anti‐inflammatory, and antimicrobial effects, underscoring their therapeutic applicability in dermatological contexts [71].

The crude fungal extracts showed varying levels of antioxidant efficacy for DPPH radical scavenging. The antioxidant activity of the fungal extracts was assessed by discoloration to yellow following formation of the nonradical (2,2‐diphenyl‐1‐hydrazine) molecule [72]. According to Mazumdar [73] reported that the ethyl acetate extract of Diaporthe sp. exhibited a high scavenging activity of 12.91 ± 10.32. The ethanolic fungal extract of A. flavus showed the percentage inhibition of (58.01 ± 0.386); Pleosporales sp. (50.66 ± 0.18); Endothia sp. (43.23 ± 0.23 and the standard ascorbic acid showed around 81.59 ± 0.5.

According to Al‐Fakih and Almaqtri [74] the antibacterial activity of the ethyl acetate extract of A. flavus showed a zone of inhibition against S. aureus (5.6 mm) and no zone of inhibition in E. coli. In the present study, the ethanol extract of A. flavus showed the largest zone of inhibition against S. aureus (19 mm) than the Pleosporales sp. (14 mm), and Endothia sp. (12 mm). With a maximum inhibitory zone of 16 mm in A. flavus was found to be more sensitive to E. coli than the Pleosporales sp. (12 mm), and Endothia (5 mm).

According to Lowenthal et al. [75] the MTT test was used to examine the ethanol extract of Trametes versicolor fruting body and mycelium towards malignant melanoma cells. The IC₅₀ value of the fruiting body and mycelium toward malignant melanoma cells. The IC₅₀ value of the fruiting body extract towards both A375 (IC₅₀ = 114.5 and 663.3 µg/mL, respectively) and SK‐MEL‐5 (IC₅₀ = 88.6 and 358.4 µg/mL, respectively) cell lines. In the present study, the cytotoxic activity of the ethanol extract of the A. flavus, Pleosporales sp., and Endothia sp., showed IC₅₀ values of non‐cancerous L929 cell line was 211.61 µg/mL, 177.56 µg/mL, and 198 µg/mL. The IC₅₀ value of the A. flavus, Pleosporales sp., and Endothia sp. extracts against A375 cell line was 209.46 µg/mL, 152.00 µg/mL, and 190.37 µg/mL.

According to Moharram et al. [76] reported that the Emericella nidulans, Pleospora tarda, and Penicillium funiculosum extracts showed higher activities, with percentage inhibition of protein denaturation reaching 83%, 82.5% and 81.4%. In our study, the anti‐inflammatory activity of the ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp. showed the percentage inhibition of 49.26 ± 1.46, 67.25 ± 0.72, and 58.61 ± 1.11. whereas the standard aspirin exhibited significant anti‐inflammatory activity with a percentage inhibition of 85.81%.

According to Ameen et al. [77] the wound healing activity of the crude extract had a cell migration rate of 42.6% for 48 h, whereas the control nitrofurazone had 30% of cell migration. Beetroot extract at a minimum concentration 25 µg with a maximum wound closure of 78% after 24 h [78]. According to Al et al. [79] Aspergillus terreus extract was tested for wound healing activities using in vitro cell migration with HSF cell line. Wound healing activity of the A. terreus extract had a cell migration rate of 0.84 ± 0.0006 for 24 h and for 48 h 0.82 ± 0.0006. In the present study, the ethanolic extract of A. flavus, Pleosporales sp., and Endothia sp. was studied in two ways by studying cell migration and the percentage of wound closure. Compared to the ethanol extract, the standard ascorbic acid showed good results. The cell migration of the standard ascorbic acid at 12, 24 h was found to be 27.70 and 19.44 µm, respectively. Similarly, the fungal extract of A. flavus, Pleosporales sp., and Endothia sp., at 12 h was found to be 7.8, 9.27, and 14.94 µm, whereas in the 24 h the cell migration was found to be 14.25, 14.11, and 12.59 µm, respectively. In case of wound closure at 12, 24 h, the standard ascorbic acid showed a value of 68.09% and 95.17%. In contrast, the ethanolic extract of A flavus, Pleosporales sp., and Endothia sp. at 12 h showed 19.89%, 23.96%, and 44.54% and whereas in the 24 h the wound closure shown to be 72.74%, 72.95%, and 75.10%, respectively, and for the untreated group, it was found to be 7.50% at 12 h and 17.17% for 24 h.

5. Conclusion

The present study successfully demonstrated that endophytic fungi isolated from Atalantia racemosa represents a rich and underexplored source of pharmacologically important secondary metabolites. Comprehensive phytochemical profiling using GC–MS, FTIR, and LC–MS confirmed the presence of diverse bioactive compounds, including fatty acids, sterols, phenolics, flavonoids, alkaloids, quinic acid derivatives, and terpenoids. Notably, LC–MS analysis revealed a broader spectrum of metabolites such as mangostin derivatives, N‐feruloyl‐octopamine, eburnamonine, aphidicolin, chalcones, and terpene lactones, highlighting the metabolic versatility of the fungal isolates. Among the isolates, Endothia sp. exhibited the highest chemical diversity and significant biological activities, particularly in anticancer and wound healing assays.

The biological evaluations further supported the chemical findings, demonstrating appreciable antioxidant, antibacterial, anti‐inflammatory, cytotoxic, and wound healing properties of the fungal extracts. The presence of multiple classes of bioactive compounds suggests possible synergistic effects contributing to these activities. Overall, the integration of LC–MS data strengthens the evidence that these endophytic fungi possess significant therapeutic potential. Future studies should focus on the isolation, purification, structural elucidation, and mechanistic evaluation of individual compounds, along with in vivo validation, to advance their application in pharmaceutical development.

Author Contribution

S.M. conducted the experiment and drafted the data. A.S. has helped in making methodology standardize and execution of the work. J.H. helped in the correction and V.A.B. has completed the drafting with corrections and made upload.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

The present research did not receive any specific grant from funding agencies, either public or commercial.

Data Availability Statement

Data will be made available on request.