Bioactive Components and Health Potential of Endophytic Micro-Fungal Diversity in Medicinal Plants

The endophytic fungi that reside inside medicinal plants have the potential to produce various pharmaco-potential bioactive compounds. The endophytic fungi Graminicolous helminthosporium, Bipolaris australiensis and Cladosporium cladosporioides were isolated from different medicinal plants. The GC-MS analysis of intra- and extracellular products of endophytic fungi revealed the presence of various bioactive metabolites, such as Anthracene, Brallobarbital, Benzo [h] quinolone, Ethylacridine, 2-Ethylacridine, Cyclotrisiloxane, 5 methyl 2 phenylindolizine, and 1,4-Cyclohexadien-1-one, etc. The phytochemical composition analysis of endophytic fungus extracts also revealed the presence of flavonoids, phenols, saponins, carbohydrates, glycosides, and proteins. The intra- and extracellular endophytic extracts exhibited strong antibacterial and antioxidant activity, which was screened with the agar-well diffusion method and DPPH, H2O2, and nitric oxide scavenging activity, respectively. The bioactive compounds identified in the endophytic extracts from GC-MS profiling served as ligands for molecular-docking analysis to investigate the anticancer potential against non-small cell lung carcinoma receptor EGFR. Molecular docking results showed that compounds, such as Brallobarbital, and 5 methyl 2 phenylindolizine had the lowest E- min values, which suggests that these compounds could be used in anticancer drug development. Thus, the isolated endophytic fungal species can be used to produce various bioactive compounds that could be used in novel drug development from natural sources and reduce the environmental burden of synthetic chemical drugs.


Introduction
Endophytes are the microorganisms present in the innermost part of the plant ecosystem and play a vital role in their mechanisms. Most endophytes are isolated from the plant parts, such as the leaf, stem, root, flower, meristem, and seeds. Endophytes are The present work focused on the isolation of endophytic micro-fungi and the screening of their bioactive compounds by FTIR and GC-MS analysis. Furthermore, biochemical tests for protein, carbohydrates, flavonoids, tannins and alkaloids were employed to detect the presence of various phytoconstituents. Antibacterial and antioxidant activities were carried out to determine the bioactive potentials of the intra-and extracellular fungal extracts. In addition, molecular docking studies were employed to determine the anticancer properties of the identified compounds in the extracts.

Sample Collection
Plants with medicinal properties were collected, originating from Sivakasi (9.4533° N, 77.8024° E). Ten plant samples, namely, Leucas aspera, Tamarindus indica, Jatropha gossypifolia, Senna auriculata, Manikara zapota, Clitoriaternatea, Portulaca grandiflora, epipremnumaureum, Tridax procumbens and Acalypha indica with medicinal values were collected and placed in separate sterile zip lock bags followed by processing within 24 h of collection. To avoid contamination, the fresh plant leaf and stem were taken for the isolation process. The above medicinal plants have already been used for their antifungal, prostaglandin inhibitory, antioxidant, antimicrobial, antinociceptive and cytotoxic activities, and anticancer properties, within cases of wound curing, abdominal pain, diarrhea, dysentery, parasitic infection, fever, malaria and respiratory problems [9]. The workflow of the present work is represented in Figure 1.

Surface Sterilization
The dust and debris attached to the samples were removed by washing them in running water. Then the samples were sliced into tiny pieces of 1 cm, followed by washing two to three times with sterile distilled water before they were taken into a laminar airflow chamber. First, the samples were rinsed with sodium hypochlorite solution (5%) and then cleaned with distilled water twice. The samples were treated with 70% ethanol and

Surface Sterilization
The dust and debris attached to the samples were removed by washing them in running water. Then the samples were sliced into tiny pieces of 1 cm, followed by washing two to three times with sterile distilled water before they were taken into a laminar airflow chamber. First, the samples were rinsed with sodium hypochlorite solution (5%) and then cleaned with distilled water twice. The samples were treated with 70% ethanol and cleaned with sterile distilled water. After that, the samples were subjected to 1% mercuric chloride and washed with sterile distilled water. Once again, the samples were washed with 79% ethanol for two minutes and then washed with sterile distilled water twice. The surface-sterilized samples were placed on sterile tissue paper to remove excess moisture or water in the samples [10].

Sample Inoculation
The inoculation media was prepared with Potato Dextrose Agar (PDA) medium or Sabouraud Dextrose Agar (SDA) medium or Malt Extract Agar (MEA) medium. These media were prepared and sterilized in an autoclave for 20 min at 121 • C. While transferring the medium to a sterile Petri plate, antibiotics, such as tetracycline and amoxicillin, were included at a palm-bearable temperature to reduce Gram-positive and Gram-negative bacterial contamination. After solidification, surface-sterilized samples were inoculated with the help of clean forceps. The inoculated samples were incubated at 28 • C for 15 days [11].

Morphological Identification of Endophytic Fungi
The staining solutions, such as Lactophenol and Lactophenol cotton blue stain, were procured from Hi Media Laboratories Private Limited, India. The slides prepared were sealed with DPX mountant. Identification of the fungal species was conducted as prescribed in, "The Genera of Hyphomycetes from the soil" [12], in the Compendium of soil fungi [13].

Lactophenol Cotton Blue Mounting
With the use of a clean inoculation loop, a small amount of culture was taken, and semi-permanent slides were developed with lactophenol cotton blue. The slides were mildly heated in a spirit lamp to remove the air bubbles from inside the cover glass. The surplus dye was discharged using tissue paper, and the cover glass was sealed with white nail polish.

Diversity Analysis
A fungal colony from the inoculated plant samples was used for the diversity analysis, which was stained and examined under a microscope. The white fungal strains were stained with lactophenol cotton blue dye, and dark color sporing, such as black or grey strains, were stained with paraffin oil. The fungal strain was taken with a sterile needle and placed on the microscopic slides loaded with a few drops of stain. Then the fungal strain in the slides was subjected to teasing to spread the conidia and spores of the fungi and then examined under a microscope [11].

Production of Fermentation Medium
The fungal strain was transferred to the production medium for the production of secondary metabolites. The fermentation medium was prepared with a combination of 50% potato extract and a 5% carbon source, such as dextrose, along with 20% of mineral sources, such as yeast extract, dipotassium hydrogen orthophosphate, magnesium sulphate with heptahydrate, sodium chloride, and ferrous sulphate. The production medium above this concentration was prepared in 200 mL of a 500 mL Erlenmeyer flask and autoclaved to maintain a sterile environment. After the production medium attained room temperature, antibiotics, such as tetracycline and amoxicillin, were added to it at a concentration of 200 mg/L. The fungal strain with spores was transferred to the prepared fermentation medium and incubated at 28 • C for 15 days [9].

Extraction of Metabolites
The extraction of secondary metabolites was the major process to screen out the bioactive compounds produced by the endophytic fungi present in the production medium. The liquid-liquid extraction method was utilized to extract the metabolites (intra/extracellular) from the production medium. For the extracellular metabolites, the liquid broth of the production medium was collected after 20 days of incubation. Initially, the production medium was filtered using a glass funnel and filter paper. The filtrate could not contain any mycelium or fungal culture. This filtrate was mixed with an equal amount of ethyl acetate and allowed to incubate for four hours at room temperature. Then the filtrate and ethyl acetate mixture were partitioned with a separating funnel. The organic layer was collected and used for the screening process of bioactive compounds [9]. For the intracellular metabolites, the mycelium from the production medium was harvested and disrupted with mortar and pestle, along with the solvent methanol. The ground mycelium extract was filtered with a grass cloth. The extract was lyophilized and then analyzed with the instruments, such as FTIR and GC-MS [14].

FTIR Analysis
The endophytic fungal extracts were analyzed with FTIR to predict the functional group present in the metabolites. The absorption and transmittance range used in the FTIR was 400 to 4000 cm −1 . The sample was mixed with powdered KBr and exhibited to a hydraulic pellet press to prepare the pellet for analysis. Then the peak range of the fungal samples was used to predict the functional groups [15].

GC-MS Analysis
Identification of bioactive compounds in the fungal extract of both extracellular and intercellular was conducted using GC-MS. The liquid sample without any contaminant or debris was used for the test sample of GC-MS. To achieve this, the test samples were centrifuged at 3000 rpm for 5 min. The clear supernatant was taken as the test sample. The sample powder was dissolved with methanol and centrifuged for the intracellular extract to obtain a clear supernatant [15].

Antibacterial Assay
The agar-well diffusion method was used to test the antibacterial activity of the test samples. The nutrient medium was prepared with an additional agar source with a concentration of 2%. The medium was sterilized with an autoclave. The Petri plates with nutrient agar were inoculated with bacterial cultures, such as Escherichia coli, Streptococcus pneumoniae, Shigella sps, and Streptomyces avermitilis. Then, with the sterile well puncher, a well with a diameter of 1 cm was created on the cultured agar plates. The test samples with different concentrations of 10 µL and 20 µL were added to the well in the Petri plate. The solvent used to dissolve or extract the fungal metabolites was used as the control for this agar-well diffusion method. The control solvents were ethyl acetate and methanol. The zone of inhibition was recorded after incubation to forecast the ability of the sample against bacterial colony growth [16].

Phytochemical Analysis
Both the extracellular and intracellular extracts were used for the phytochemical analysis. Ten phyto-compounds were analyzed in this work, such as flavonoids, alkaloids, saponins, tannins, carbohydrates, terpenoids, steroids, phenols, glycosides, and proteins [17].

Alkaline
A total of 2 mL of the fungal extract was added to 2 to 3 drops of 2% diluted NaOH solution. The yellow color change of the test indicated the presence of flavonoids [17].

Lead Acetate
A total of 2 mL of the extract was taken, and a few drops of lead acetate was added to it. The yellow color precipitate formation in the test indicated the presence of flavonoids in the extract [17].

Ferric Chloride
A total of 2 mL of the fungal extract was taken, and a few drops of ferric chloride were added to it. The green color change of the test indicated the presence of flavonoids [17].

Sulphuric Acid
A total of 2 mL of the fungal extract was taken, adding a few drops of concentrated H 2 SO 4 . The formation of an orange color in the test indicated the presence of flavonoids [17].

Saponins
A total of 1 mL of the fungal extract was taken and mixed with 5 mL of distilled water. The test tube was mixed efficiently for 15 to 20 s, followed by maintaining at room temperature for 10 min. The generation of a stable form indicated the presence of saponins in the fungal extract [17].
2.14. Tannins Test 2.14.1. Ferric Chloride A total of 2 mL of the fungal extract was taken along with 1 mL of 5% FeCl 2 . The color change indicated the presence of tannin in the sample. The dark green-black color formation indicated the presence of condensed tannins, and the dark blue-black color formation indicated the presence of hydrolyzed tannins in the sample [17].

Gelatin
A total of 2 mL of the fungal extract was taken along with 1% gelatin solution, and 1 mL of 10% NaCl was added. The white color precipitate formation in the test indicated the presence of tannin in the sample [17].

Lead Acetate
A total of 2 mL of the fungal extract was taken, adding a few drops of lead acetate. The formation of a white color precipitate in the test indicated the presence of tannin in the sample [17].

Millon
A total of 2 mL of the fungal extract was taken along with 2 mL of the Millon reagent. The formation of a white color precipitate in the test indicated the presence of proteins in the sample [17].

Biuret
A total of 2 mL of the fungal extract was taken. A drop of 2% CuSO 4 and 1 mL of ethanol and a sodium hydroxide pellet was added to the extract, followed by incubation for 2 to 5 min without shaking. The formation of a pink color indicated the presence of protein in the sample [17].

Ninhydrin
A total of 2 mL of the fungal extract and 2 mL of the ninhydrin solution were taken in the test tube and placed in a water bath for 5 min at 50 • C. The formation of violet-blue or violet color in the test indicated the presence of protein in the solution [17].

Aminoacids
A total of 2 mL of the fungal extract was taken along with a few drops of lead acetate and diluted NaOH. The formation of a black color precipitate indicated the presence of protein in the sample [17]. A total of 2 mL of the fungal extract was taken along with 2 mL of Benedict reagent, then was placed in a water bath for 2 min at 50 • C. The formation of the orange and red colors indicated the presence of carbohydrates in the sample [17].

Iodine
A total of 2 mL of the fungal extract was taken along with 2 mL of the iodine solution. The formation of dark blue or purple color indicated the presence of carbohydrates in the sample [17].

Fehlings
Initially, the Fehling A and Fehling B solutions were diluted well and boiled for 1 min. After boiling, the clear blue-colored Fehling solution was obtained. A total of 2 mL of the fungal extract was taken along with 1 mL of the prepared Fehling solution. The test sample was placed in a water bath for 5 min at 50 • C. The formation of a dark brick-red color indicated the presence of carbohydrates in the sample [17].

Terpenoids
A total of 2 mL of the fungal extract was taken along with 2 mL of chloroform. Then 2 mL of con. H 2 SO 4 was added to the test tubes. The test sample was placed in a water bath for 2 min at 50 • C. The formation of a grey color suggested the presence of terpenoids in the sample [17].

Steroids
A total of 2 mL of the fungal extract was taken along with a few drops of chloroform and 1 mL of the con. H 2 SO 4 . The production of the brown ring specified the presence of steroids, and the emergence of the bluish-brown ring suggested the presence of phytosteroids [17].

Glycosides
A total of 2 mL of the fungal extract was taken along with a few drops of FeCl 2 as well as 1 mL of glacial acetic acid, and 1 mL of the con. H 2 SO 4 . The production of the brown ring indicated the presence of cardiac glycosides in the sample [17].

Alkaloids Mayer Test
A total of 2 mL of the fungal extract was taken with a few drops of Mayer reagent and 1 mL of the con. H 2 SO 4 was added. The formation of yellow color in the test indicated the presence of alkaloids in the sample [17].

Phenols Ferric Chloride Test
A total of 2 mL of the fungal extract was taken along with 3-4 drops of 10% ferric chloride solution and 1 mL of potassium ferro cyanide. The formation of a bluish-black color in the test indicated the presence of phenols in the sample [17].

Litmus Paper Test
The red color changes in the litmus paper confirmed the presence of phenols in the sample when the litmus paper was dipped in the sample to be tested [17].

Antioxidant Analysis
The fungal species contained a property to inhibit or reduce the cell detriment imposed by free radicals or other unstable molecules. The quantitative presence of antioxidant properties in the endophytic fungal extract was estimated by DPPH, H 2 O 2 , and nitric oxide scavenging analysis [18].

DPPH Radial Scavenging Activity
For DPPH scavenging activity, the test samples were taken at different concentrations from 10 µL to 50 µL. Then the test samples were made up to 100 µL with DPPH solution. After incubation, the violet color changed to yellow based on the concentration, indicating the physical presence of the antioxidant property. The test samples were examined under UV spectrophotometry with absorption of 517 nm. The standard was prepared with ascorbic acid. The standard was also taken at different concentrations, from 10 µL to 50 µL and made up with DPPH up to 100 µL, followed by incubation for up to 30 min at 37 • C. After incubation, the standard was examined under UV spectrophotometry with an absorption of 517 nm. Pure methanol was used as a blank, whereas the 3 mL DPPH solution was used as a control for DPPH scavenging activity [18]. The percentage of DPPH was calculated with the following equation.
where the Abs control and Abs sample were the absorption of control and absorption of the sample at 517 nm.

H 2 O 2 Radical Scavenging Activity
The test samples were taken at different concentrations of 10 µL to 50 µLand were made up to 100 µL with H 2 O 2 solution (4 mM H 2 O 2 was dissolved in phosphate buffer). The test sample was examined under a UV spectrophotometer with the absorption at 230 nm. Pure methanol was used as the control. The standard (ascorbic acid) was also prepared as a test sample and incubated for up to 10 min at 37 • C. After incubation, the standard was examined under UV spectrophotometry with absorption at 230 nm [18]. Pure methanol was used as the blank, whereas the 3 mL pure hydrogen peroxide solution was used as a control. The percentage of the H 2 O 2 scavenging activity was calculated with the following equation.
whereas the Abs control and Abs sample were the absorption of control and absorption of the sample at 230 nm.

Nitric Oxide Scavenging Activity
A nitric oxide scavenging assay was performed as reported previously by Palanichamy et al. [2]. The test sample and standard (ascorbic acid) was taken atdifferent concentrations of 10 µL to 50 µl and made up with methanol solution up to 100 µL. Then 1.5 mL of 10 mM sodium nitroprusside was added to all the tubes, then the reaction was incubated at 25 • C for 30 min. After incubation, the samples were equally mixed with the Griess reagent and subsequently examined under UV spectrophotometry with an absorption of 546 nm. Pure methanol was used as the blank, whereas the 3 mL of sodium nitroprusside and Griess reagent mixture was used as a control for nitric oxide scavenging activity [18]. The percentage of nitric oxide scavenging activity was calculated with the below equation.
Nitric oxide (scavenging effect %) = Abs control − Abs sample Abs control × 100 where the Abs control and Abs sample were the absorption of control and absorption of the sample at 546 nm.

Molecular Docking
EGFR kinase (Epidermal Growth Factor Receptor kinase) is the receptor for nonsmall cell lung cancer (NSCLC), which has a high risk of death. The entire description of the receptor was collected from the site of the Therapeutic Target Database. The 3D structure of EGFR for humans was retrieved from the PDB database as PDB ID-1M14 (EGFR_HUMAN). The PDB form of the receptor was obtained by Online Smile Translator and transferred to the docking tool Hex 8.0. Then the ligand for docking was chosen by the peak range of GC-MS. The molecular structure of the ligand was obtained from the PubChem database, and the PDB form of the 3D structure of the ligand was retrieved from Online Smile Translator and transferred to the docking tool Hex 8.0. The docking was done with Hex 8.0 with receptors and ligands. The Hex message with E-minimum values was observed to predict the interaction of the ligand with receptor EGFR [19].

FT-IR Spectrophotometer Analysis
Infrared spectra (IR) were also used to identify the bioactive compounds. Micro-fungal extracts were tested using a SHIMADZU-FT-IR instrument to analyze the functional groups in the bioactive constituents.

Statistical Analysis
All the trials were carried out in triplicate with mean standard deviation (SD). Each experiment was repeated twice with the level of significance at p ≤ 0.05. Means were separated using Duncan's multiple range test.

Endophytic Microfungal Isolation
Endophytic fungi naturally occur in plant tissue and play an essential role in plant development by the secretion of novel bioactive compounds, phytopathogenic activity, and phytohormones. Khalil et al. [20] reported that endophytes, such as penicillium crustosum, Penicillium chrysogenum, Penicillium commune, Penicillium corylophilum, Alternaria infectoria, Penicillium caseifulvum, Alternaria alternata, Alternaria tenuissima, Aspergillus flavus, and Aspergillus niger were isolated from the plant, Ephedra pachyclada. Similarly, Saroj et al. [21] demonstrated the isolation of endophytic microfungi as Bipolarisaustraliensis from Senna angustifolia plants in India.
Jannath et al. [22] reported that the medicinal plant Baliospermum montanum (Willd.) Muell was distributed throughout India, and twenty-nine endophytic fungal species were isolated from the tissues of this medicinal plant. The phytochemical analysis recorded that 70% of endophytic isolates showed alkaloids and flavonoids, and 13% were positive for phenols, saponins, and terpenoids. GC-MS profile detected twenty-five bioactive compounds from ethyl acetate extracts. Among endophytic fungi, Trichoderma reesei isolated from the flowers exhibited nine bioactive compounds, namely 2-Cyclopentenone, 2-(4chloroanilino)-4-piperidino, Oxime-methoxy-Phenyl, Methanamine N-hydroxy-N-methyl, Strychane, Cyclotetrasiloxane, Octamethyl, and 1-Acetyl-20a-hydroxy-16-methylene [22]. The endophytic bioactive compounds are explored to develop a novel drug with commercial value. Similarly, in the present study, three species of endophytic fungi, such as Graminicolous helminthosporium, Bipolaris australiensis and Cladosporium cladosporioides were spotted and isolated from the leaves and stems of medicinal plant samples, such as Leucas aspera, Tamarindus indica, Jatropha gossypifolia, Senna auriculata, Manikara zapota, Clitoriaternatea, Portulaca grandiflora, Epipremnumaureum, Tridax procumbens, and Acalypha indica. The endophytic fungi emerged from the plant sample after 18 days of incubation. Different staining methods using paraffin oil and lactophenol cotton blue dyes displayed diversified endophytic fungi under microscopical observation. The mature Graminicolous helminthosporium was a greenish-black color colony with dark color spores in the SDA medium. The microscopic examination of G. helminthosporium showed a brown color elongated spore with ten segments along with conidia ( Figure 2a). The mature colony of Bipolaris australiensis was a blackish brown colony with brown color spores. The microscopic examination of Bipolaris australiensis showed brown color oval-shaped spores with five segments along with curved conidia (Figure 2b). The mature colony of Cladosporium cladosporioides was a grayish black colored small colony with brown color spores. Cladosporium cladosporioides grew slower and formed a small colony in MEA medium. The microscopic examination of Cladosporium cladosporioides showed that it was grey in color, with tiny round spores with segmented conidia (Figure 2c). thyl, Strychane, Cyclotetrasiloxane, Octamethyl, and 1-Acetyl-20a-hydroxy-16-methylene [22]. The endophytic bioactive compounds are explored to develop a novel drug with commercial value. Similarly, in the present study, three species of endophytic fungi, such as Graminicolous helminthosporium, Bipolaris australiensis and Cladosporium cladosporioides were spotted and isolated from the leaves and stems of medicinal plant samples, such as Leucas aspera, Tamarindus indica, Jatropha gossypifolia, Senna auriculata, Manikara zapota, Clitoriaternatea, Portulaca grandiflora, Epipremnumaureum, Tridax procumbens, and Acalypha indica. The endophytic fungi emerged from the plant sample after 18 days of incubation. Different staining methods using paraffin oil and lactophenol cotton blue dyes displayed diversified endophytic fungi under microscopical observation. The mature Graminicolous helminthosporium was a greenish-black color colony with dark color spores in the SDA medium. The microscopic examination of G. helminthosporium showed a brown color elongated spore with ten segments along with conidia ( Figure 2a). The mature colony of Bipolaris australiensis was a blackish brown colony with brown color spores. The microscopic examination of Bipolaris australiensis showed brown color oval-shaped spores with five segments along with curved conidia (Figure 2b). The mature colony of Cladosporium cladosporioides was a grayish black colored small colony with brown color spores. Cladosporium cladosporioides grew slower and formed a small colony in MEA medium. The microscopic examination of Cladosporium cladosporioides showed that it was grey in color, with tiny round spores with segmented conidia (Figure 2c). More than 15 bioactive compounds were recorded in the extracellular and intracellular products of the isolated endophytic micro-fungi, namely Graminicolous helminthosporium, Bipolaris australiensis, and Cladosporium cladosporioides. The endophytic extract comprised the bioactive compounds, such as Benzo [h] quinoline, 2-Ethylacridine, Cyclotrisiloxane, Anthracene, 5-methyl-2-phenylindolizine, Indole-2-one, Tris (tert- More than 15 bioactive compounds were recorded in the extracellular and intracellular products of the isolated endophytic micro-fungi, namely Graminicolous helminthosporium, Bipolaris australiensis, and Cladosporium cladosporioides. The endophytic extract comprised the bioactive compounds, such as Benzo [h] quinoline, 2-Ethylacridine, Cyclotrisiloxane, Anthracene, 5-methyl-2-phenylindolizine, Indole-2-one, Tris (tert-butyldimethylsilyloxy)arsane, 3-quinolinecarboxylic acid, Benzene, n-Hexadeconic acid, Oleic acid, Oxacyclotetradecan-2one, Cis-Vaccenic acid, Oxacyclotetradecan-2-one, Cis-Vaccenic acid, and Tetrasiloxane.
Aspergillus brasiliensis, which was isolated from a root, and Fusarium oxysporum, which was isolated from seed, each produced nine and seven bioactive chemicals from the endophyte. Overall, it was found that the various endophytic fungi associated with B. montanum contributed significantly to the production of bioactive chemicals, which might be useful for the potential development of novel drugs with marketable benefits [22]. Similarly obtained endophytic bioactive compounds possess various medicinal and industrial values, such as antitumor, antioxidant, wound healing, antibacterial, antimalarial, antiviral, anticonvulsant, anti-HIV, anti-inflammatory, reduction of coronary heart disease (CHD), industrial manufacturing dyes, lubricant, drug development, fuel, anti-polymerization agent, insecticidal, wood preservative, preparation of cool-tar, fossil fuel, and cosmetics.

FTIR Analysis
Sundar et al. [23] reported the prediction of metabolites of Cladosporium endophytic fungi of both (extracellular) ethyl acetate or dichloro-methane extract and (intracellular) methanol mat based on functional groups using FTIR. Elango et al. [15] found that the ethyl acetate crude extract of Aspergillus sojae was analyzed with FTIR and recorded the functional groups, such as N-N stretching, C-H stretching, O-H stretching, CH 2 bending, and C-H bending in the different peaks of absorption. In accordance with this, in our study, functional groups in the fungal extracts, such as methanol mat (intracellular) and ethyl acetate (extracellular) of Graminicolous helminthosporium, Bipolaris australiensis, and Cladosporium cladosporioides were analyzed by FTIR and recorded many functional groups in the extracellular extract of Cladosporium cladosporioides which included C-O stretching and C-H stretching as a major functional group. In Bipolaris australiensis CO-O-CO stretching, C-O stretching, N-O stretching, and C=O stretching wereidentified as the major functional groups. In Graminicolous helminthosporium, the predicted major functional groups were CO-O-CO stretching, C-N stretching, S=O stretching, N-O stretching, and C=O stretching. The analysis of intracellular methanol mat extract from Graminicolous helminthosporium revealed major functional groups, such as O-H bending, C=C stretching, and C-H stretching. While in Bipolaris australiensis the noticed major functional groups were C-Br stretching, C-Br stretching, C=C bending, S=O stretching, and N=O stretching were the major functional groups identified in FTIR. In Cladosporium cladosporioides the observed functional groups were CO-O-CO stretching, S=O stretching, O-H bending, C=C stretching, O-H stretching, and N-H stretching. Mehdi et al. [24] reported that the FT-IR spectrum confirmed the presence of the alcohol group, alkene group, amine group, carbonates, ethers, carboxylic acid, and disulfides in leaf extracts of Tamarindus indica. Similarly, in our study, we noticed the alkene, aldehyde, alcohol, anhydride, and amine groups. Different peaks of absorption were obtained in this study, and the functional groups were tabulated (Table 1).

GC-MS Analysis for Bioactive Compound Screening
Recently, Tran et al. [25] reported three bioactive compounds, such as maydisone, 7-hydroxy-2,5-dimethylchromone, and 2,5-dimethylbenzoic acid in microfungal species of Bipolaris by using GC-MS analysis. Similarly, the bioactive compounds in three endophytic micro-fungal species, such as Graminicolous helminthosporium, Bipolaris australiensis, and Cladosporium cladosporioides were analyzed by GC-MS analysis. Bioactive compounds, such as 1,4-bis (trimethylsilyl) benzene, Arsenous acid, 1,2-bis (trimethylsilyl) benzene, Tris (tert-butyldimethylsilyloxy) arsane, Cyclotrisiloxane, and 2-Ethylacridine were commonly present in three fungal species of the ethyl acetate and methanol extract of endophytic micro fungal species. The endophytes create a variety of anti-cancerous compounds, such as taxol which has great potential to be used as an anticancer metabolite [26]. Because of this, there has been plenty of opportunity to use these metabolites against breast, uterine, and ovarian cancers [27]. These discoveries have shed light on endophytic fungi and their metabolites, which have the potential to be effective treatments for a variety of diseases. An endophytic fungus Phoma medicaginis associated with the medicinal plants Medicago sativa and Medicago lupulina produced the antibiotic brefeldine A, which also initiated apoptosis in cancer cells [28]. About 1500 fungal metabolites have shown antitumor and antibiotic activity [29], and some have been approved as drugs. Endophytes synthesize bioactive metabolites that may be involved in the host-endophyte relationship [26] and may function as primary sources of novel natural products for exploitation in medicine, agriculture, and industry [30]. Kanjana et al. [31] reported the ethyl acetate extracts of Chaetomium globosum, Cladosporium tenuissimum and Penicillium janthinellumn were shown to contain Hexadecanoic acid, Octadecanoic acid, Octadecanoic acid, 9-Octadecenoic acid, and 9,12-Octadecadienoic acid. Similarly, in our study, Benzo [h] quinoline was observed in the ethyl acetate extract of Bipolaris australiensis, Cladosporium cladosporioides, and the methanol extract of Cladosporium cladosporioides. The metabolites, such as n-Hexadeconic acid, and Cis-Vaccenic acid were noted in the ethyl acetate extract of Bipolaris australiensis.
Hateet [32] reported that an endophytic fungus (Acremonium coenophialum) was isolated from the medicinal plant Myrtus communis, and the microfungal extracts were analyzed by GC-MS to determine the chemical composition of fungal extract and their results showed the presence of nineteen compounds. In line with this, GC-MS analysis of the ethyl acetate and methanol mat extract of Graminicolous helminthosporium, Bipolaris australiensis and Cladosporium cladosporioides revealed several bioactive compounds, which was presented in Table 2 and the Supplementary Files.

Antibacterial Potential of the Microfungal Extract
Amelia et al. [33] described the antimicrobial efficacy of the ethyl acetate extract of Penicillium against the bacterial species of Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Pseudomonas aeruginosa and the results indicated that the highest level of the zone of inhibition (13 mm) was observed against Staphylococcus aureus. Ding et al. [34] also reported that the antimicrobial activity was performed with the microfungal extracts of Cladosporium sp. Against eight different microorganisms, such as Phoma sp., Staphylococcus aureus, Sarcina lutea, Escherichia coli, Bacillus pyocyaneus, Streptomyces viridochrmogenes, Candida albicans, and Mucor miehei and obtained the highest level of the zone of inhibition (25 mm) against Streptomyces sp. and followed by (24 mm) against Escherichia coli through the agar diffusion method. Similarly, we performed antimicrobial activity analysis with the ethyl acetate extract as well as the methanol mat extract of Graminicolous helminthosporium, Bipolaris australiensis, and Cladosporium cladosporioides endophytic fungi against pathogenic bacterial species, such as Escherichia coli, Shigella sp, Streptococcus pneumoniae, and Streptomyces avermitilis. The zone of inhibition pattern was different between the bacterial species as well as in the extracts used for the antimicrobial assay. For instance, the highest range of zone of inhibition (3.5 cm) was observed in the methanol mat extract of Cladosporium cladosporioides against Escherichia coli and a moderate range of zone of inhibition (1.3 cm) was observed in the ethyl acetate extract of Cladosporium cladosporioides against E. coli. The ethyl acetate and methanol mat extracts of Bipolaris australiensis exhibited the maximum zone of inhibition (1.9 cm) against S. pneumoniae. Moderate inhibition (2 cm) was noticed against E.coli when treated with the ethyl acetate extract and methanol mat extracts of G. helminthosporium (Figures 3a-c and 4a-c). Similarly, Hateet [32] reported that the ethyl acetate extract of the endophytic fungus Acremonium coenophialum exhibited antimicrobial activity against seven Gram-positive bacteria: S. aureus, Staphylococcus epidermidis, Streptococcus sp., Pneumoniae sp., Streptococcus pyogenes, Enterococcus faecalis, Bacillus cereus, and Bacillus subtilis, which ranged between (10.0-17.0 mm) whereas the range of microbial inhibition against seven Gram-negative bacteria, namely E.coli, S. typhi, P. aeruginosa, Klebsiella pneumoniae, Morganella morganii, Serratia sp., and Proteus vulgaris was between (10.0-18.0 mm). In line with this, in this study, the ethyl acetate extract and the methanol mat extract of G. helminthosporium, C. cladosporioides, and B. australiensis revealed the zone of inhibition against E. coli, Shigella sp, Streptocmyces avermitilis, and Streptococcus pneumoniae (0.3-3.5 cm) and the results are represented in Figures 3a-c and 4a-c. Bipolaris australiensis, and Cladosporium cladosporioides endophytic fungi against pathogenic bacterial species, such as Escherichia coli, Shigella sp, Streptococcus pneumoniae, and Streptomyces avermitilis. The zone of inhibition pattern was different between the bacterial species as well as in the extracts used for the antimicrobial assay. For instance, the highest range of zone of inhibition (3.5 cm) was observed in the methanol mat extract of Cladosporium cladosporioides against Escherichia coli and a moderate range of zone of inhibition (1.3 cm) was observed in the ethyl acetate extract of Cladosporium cladosporioides against E. coli. The ethyl acetate and methanol mat extracts of Bipolaris australiensis exhibited the maximum zone of inhibition (1.9 cm) against S. pneumoniae. Moderate inhibition (2 cm) was noticed against E.coli when treated with the ethyl acetate extract and methanol mat extracts of G. helminthosporium (Figure 3a-c and Figure 4a-c). Similarly, Hateet [32] reported that the ethyl acetate extract of the endophytic fungus Acremonium coenophialum exhibited antimicrobial activity against seven Gram-positive bacteria: S. aureus, Staphylococcus epidermidis, Streptococcus sp, Pneumoniae sp, Streptococcus pyogenes, Enterococcus faecalis, Bacillus cereus, and Bacillus subtilis, which ranged between (

Phytochemical Analysis of the Endophytic Microfungal Extract
Mehdi et al. [24] reported that phytochemical screening of the Tamarindus indica extracts revealed the presence of secondary metabolite classes that are responsible for causing the obtained antibacterial activity. Sundar et al. [23] reported investigations of the methanol mat extract and ethyl acetate extract of endophytic microfungal species of Cladosporium and noticed various chemical constituents, such as phenolic compounds, saponins, flavonoids, tannins, and terpenoids. In association with this, we observed various bioactive compounds, such as flavonoid, saponin, tannin, carbohydrate, phenol, terpenoid, steroids, glycoside, alkaloids, and protein in fungal extracts of both the ethyl acetate extract and methanol mat extract. The ethyl acetate and methanol extracts of C. cladosporioides contain a high concentration of flavonoid, phenol, and protein, whereas a higher level of tannin, carbohydrate, terpenoids, steroids, glycoside, and alkaloids were found to be observed in the methanol mat extracts. Higher levels of flavonoid, phenol, terpenoid, and steroids were observed in the ethyl acetate extracts of Bipolaris australiensis, while the methanol mat extracts contained the maximal level of tannin, phenol, glycoside, carbohydrate, and protein. Ethyl acetate and methanol mat extracts of G. helminthosporium were shown to contain increased levels of flavonoid, phenol, terpenoid, steroids, glycoside, alkaloids, and protein, whereas the methanolic extract had only a higher level of tannin and carbohydrates. Flavonoids were observed in all the extracts of endophytic

Phytochemical Analysis of the Endophytic Microfungal Extract
Mehdi et al. [24] reported that phytochemical screening of the Tamarindus indica extracts revealed the presence of secondary metabolite classes that are responsible for causing the obtained antibacterial activity. Sundar et al. [23] reported investigations of the methanol mat extract and ethyl acetate extract of endophytic microfungal species of Cladosporium and noticed various chemical constituents, such as phenolic compounds, saponins, flavonoids, tannins, and terpenoids. In association with this, we observed various bioactive compounds, such as flavonoid, saponin, tannin, carbohydrate, phenol, terpenoid, steroids, glycoside, alkaloids, and protein in fungal extracts of both the ethyl acetate extract and methanol mat extract. The ethyl acetate and methanol extracts of C. cladosporioides contain a high concentration of flavonoid, phenol, and protein, whereas a higher level of tannin, carbohydrate, terpenoids, steroids, glycoside, and alkaloids were found to be observed in the methanol mat extracts. Higher levels of flavonoid, phenol, terpenoid, and steroids were observed in the ethyl acetate extracts of Bipolaris australiensis, while the methanol mat extracts contained the maximal level of tannin, phenol, glycoside, carbohydrate, and protein. Ethyl acetate and methanol mat extracts of G. helminthosporium were shown to contain increased levels of flavonoid, phenol, terpenoid, steroids, glycoside, alkaloids, and protein, whereas the methanolic extract had only a higher level of tannin and carbohydrates. Flavonoids were observed in all the extracts of endophytic fungi. On the whole, the phytochemical analysis of the extracellular and intracellular extracts of G. helminthosporium, C. cladosporioides, and B. australiensis revealed the presence of a variety of secondary metabo-lites, which include Flavanoids, Saponin, Tannin, Carbohydrate, Phenolic compounds and terpenoid (Table 3). The effects of the fungal extract's radical-scavenging abilities were investigated using DPPH, a stable free radical. Antioxidants reduce absorption by providing this radical with a proton. The reaction could be seen as a purple color turning yellow. The ethyl acetate and methanolic extracts of the mycelial mats of endophytes (Daldinia eschscholtzii and Trichoderma asperellum) isolated from Ananus comosus L. wereexamined for antioxidant potential in vitro using DPPH [35]. The lowest EC50 value of the free-radical scavenging activity in the ethyl acetate extract of fungal endophyte Trichoderma asperellum indicates a better ability of the mycelial mat extract to act as a DPPH radical scavenger. The Unknown sp. (ML5) demonstrated the least antioxidant activity, with only 33.95%, whereas the EA extract of Alternaria sp. (ML4) showed a high scavenging activity of 85.20%. As a benchmark, ascorbic acid demonstrated 96.91% antioxidant activity, the percentage of ascorbic acid and endophytic fungus that may scavenge DPPH radicals [36]. Similarly, an improved antioxidant activity was observed in the extracellular and intracellular extracts of Graminicolous helminthosporium, Cladosporium cladosporioides, and bipolaris australiensis, which was determined by DPPH. The highest scavenging activity was recorded in the ethyl acetate extract of Cladosporium cladosporioides (70.23%) and methanol extract of Bipolaris australiensis (95.97%) ( Tables 4 and 5). The ethyl acetate and methanol mat extracts of B. australiensis possess enhanced DPPH scavenging activity compared to other fungal extracts, such as Cladosporium cladosporioides and Graminicolous helminthosporium.  In the analysis of H 2 O 2 scavenging activity, the extracellular extracts of Graminicolous helminthosporium, Bipolaris australiensis, and Cladosporium cladosporioides had the maximum scavenging activity when compared to the methanol mat extract of G. helminthosporium, B. australiensis, and C. cladosporioides (Tables 6 and 7). In particular, the extracellular extracts of B. australiensis and C. cladosporioides recorded maximum scavenging activity (96%) when compared to standard ascorbic acid. However, moderate nitric oxide scavenging activity (86%) was observed in both the ethyl acetate and methanol extracts of Graminicolous helminthosporium, B. australiensis, and C. cladosporioides (Tables 8 and 9). In particular, the extracellular extracts of B. australiensis and C. cladosporioides have a better antioxidant scavenging percentage.  Molecular docking was performed with the Hex 8.0 software, highly interactive software to calculate the interactions between ligands and receptors. The 3D structure of the lung cancer EGFR receptor was retrieved from the PDB database. Several studies reported that the various cancers, including NSCLC, were correlated with the EGFR mutations [8]. The 3D biostructure of the bioactive compounds (ligands), such as Benzo (h) quinolone, Ethylacridine, Cyclotrisiloxane, 5 Methyl 2 Phenylindolizine, and Brallobarbital were obtained from the analytical results of the GC-MS analysis of endophytic fungi and retrieved from the PubChem database ( Figure 5). Through docking the E-minimization and E-maximization values of the receptor and ligands were calculated. Among the various bioactive compounds, the lowest E (E min) values were recorded for Brallobarbital (−227.89), and 5 methyl 2 phenylindolizine (−224.8), which suggest that these compounds might serve as potential candidates to target the EGFR receptor which is a vital signaling molecule for various types of cancers (Table 10 and Figure 6). Similarly, Meenambiga and Rajagopal [37] reported that the molecular docking with ligands of bioactive metabolites, such as Taxifolin, Saltillin and 6-methoscyflavone obtained from the endophytic fungi Aspergillus nidulans showed maximum binding energy against the receptor of the N-myristoyl transferase enzyme in a pathogenic fungus Candida albicans.

Conclusions
Three different endophytic fungi, Cladosporium cladosporioides, Graminicolous helminthosporium, and Bipolaris australiensis were isolated from various medicinal plants. The ethyl acetate (extracellular) and methanol mat (intracellular) extracts of endophytic fungi (G. helminthosporium, B. australiensis, and C. cladosporioide) were subjected to various antioxidant assays, such as DPPH, nitric oxide and H 2 O 2 scavenging as well as antibacterial assays to determine the potential of bioactive compounds present in the extracts of endophytic fungi. Furthermore, the bioactive compounds were predicted using GC-MS, which served as ligands for in silico molecular docking of the lung cancer receptor EGFR. The extracts of endophytic fungi showed potential antioxidant activity and better antibacterial activity against several bacterial pathogens due to the presence of various bioactive compounds in the extracts identified through GC-MS. Phytochemcial composition analysis of endophytic extracts revealed the presence of various compounds, such as flavonoids, phenols, glycosides, carbohydrates, saponins, and proteins, etc. The anticancer potential of bioactive metabolites, which served as ligands was analyzed through molecular docking, which suggested that some compounds, such asBrallobarbital and 5 methyl 2 phenylindolizine had the lowest E min values that indicated these compounds may be used in anticancer drug development. However, further in vitro and molecular studies are warranted to harvest the benefits of the endophytic extracts in the drug development.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics11111533/s1, Figure S1: GC-MS spectral analysis of the extracellular ethyl acetate extract of Bipolaris australiensis. The corresponding peaks for the bioactive compounds showed in arrow marks; Figure S2: GC-MS spectral analysis of the intracellular methanolic extract of Bipolaris australiensis. The corresponding peaks for the bioactive compounds showed in arrow marks; Figure S3: GC-MS analysis of the extracellular ethyl acetate extract of Cladosporium cladosporioides. The corresponding peaks for the bioactive compounds showed in arrow marks; Figure S4: GC-MS analysis of the methanolic intracellular mat extract of Cladosporium cladosporioides. The corresponding peaks for the bioactive compounds showed in arrow marks; Figure