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 plant mutualists living asymptomatically within plant tissues [1]. Endophytic fungi may promote the growth of their host plant by producing phytohormones or by increasing the plant's resistance to various stresses, and they can produce pesticides to protect plants from herbivores [2,3]. Plant endophytic fungi are an important and novel resource of natural bioactive compounds with their potential applications in agriculture, medicine and food industry. endophytic fungi for producing plant-derived bioactive compounds such as paclitaxel, podophyllotoxin, camptothecine, vinblastine, hypericin and diosgenin etc. The relations between the endophytic fungi and their host plants, some available strategies for efficiently promoting production of these bioactive compounds, as well as their potential applications. It is beneficial for us to better understand and take advantage of plant endophytic fungi [4]. Novel bioactive compounds are in high demand to combat challenges of microbial resistance. In recent years, secondary metabolites from endophytic bacteria have drawn attention from researchers due to their novel structures and significant biological activities [5]. Endophytes are known to produce metabolites such as alkaloids, terpenoids, steroids, quinones, isocoumarin derivatives, flavanoids, phenols, phenolic acids, and peptide. Recently endophytes are viewed as outstanding source of secondary metabolites bioactive natural products. Endophytes are able to produce secondary metabolites these may be an antibiotic, therapeutic agent, agrochemical and an enzyme. Endophytes are rich in bioactive compound which have an important in the fields of medicine, agriculture and industry. There are a large number of bioactive compounds that have been isolated from endophytic fungi and these bioactive natural products have demonstrated a broad range of biological activities. The production of metabolites is mainly depending on the environmental factor and some nature of host plant also because most of the endophytes have a mutual relationship with the plant to favour them. Endophytes are mostly unexplored but they have enormous number of sources as antioxidant, insecticidal, antitumor, antidiabetic, immunosuppressive and biocontrol compounds [6]. Fungal endophytes produce in valuable bioactive metabolic compounds beneficial to humans with antimicrobial, anticancer, antidiabetic, anti-inflammatory, antitumor properties [7]. Different extracellular enzymes secreted by endophytic fungi have applications in food, textile, leather, confectionery, agriculture, beverage, and human health [8].

MATERIALS AND METHODS

CLEANING OF GLASSWARES

Glassware’s were soaked in chromic acid solution (10% Potassium dichromate in 25% sulfuric acid) for few hours and washed thoroughly followed by with detergent solution.  Glass wares were again washed with tap water and dried on the hot air over.

STERILIZATION

Media and glassware’s were sterilized on autoclave at 121º C with 15lbs pressure for 20 minutes.  All the sugars were filter sterilized using a 0.45 micro meter pore sized Millipore filters.

COLLECTION OF PLANT MATERIAL

The plant was located in the Campus of Ayya Nadar Janaki Ammal College, Sivakasi. Healthy and mature plant of Lantena camera,Hhibiscus rosa-sinensis, Musa paradisiaca, Physalis minima, Ocimum tenuiflorum, Calotropis gigantean, Coriandrum sativum, Lilum and Saraca asoca leaves samples were collected from the Campus. To avoid contamination, the fresh plant leaf and stem were taken for the isolation process.

ISOLATION OF ENDOPHYTIC FUNGI

REMOVAL OF DEBRIS

The plant samples were initially washed with running tap water for 5 minutes for removal of debris and other particles and rinsed twice with double distilled water. 

SURFACE STERILIZATION 

The samples were cleaned by running water to get rid of any dust and debris that was stuck to them. The samples were then cut into 1 cm-long pieces and cleaned two or three times in sterile distilled water before being placed in a laminar airflow chamber. The samples were cleaned twice with distilled water after first being treated with a 5% sodium hypochlorite solution. 70% ethanol was used to treat the samples, and sterile distilled water was used to rinse them. Following that, the samples were cleaned with sterile distilled water and exposed to 1% mercuric chloride. The samples were once again rinsed twice with sterile distilled water after being rinsed with 79% ethanol for two minutes. To remove excess, the surface-sterilized samples were placed on sterile tissue paper. (ALKahtani et al., 2020 and Sheeba et al., 2019).  

INOCULATION AND INCUBATION

The surface sterilized leaves were trimmed using sterile blade and inoculated into plates containing sterile Potato Dextrose Agar medium. The plates were incubated at 28°C for 7 days and observed for growth of fungus from leaves [10].

IDENTIFICATION OF ENDOPHYTIC FUNGI

The staining solution was lactophenol and lactophenol cotton blue stain (Hi Media laboratories private limited). The ready slides were sealed with DPX mountant. The Compendium of Soil Fungi [11] and "The Genera of Hyphomycetes from the Soil" [12] were used to identify the fungus species.

LACTOPHENOL COTTON BLUE MOUNTING

A loopful culture was picked up with the help of a sterile inoculation loop and semi-permanent slides were prepared using lactophenol cotton blue.

BIOACTIVE COMPOUND PRODUCTION

The fungi were cultured in appropriate media for the production of bioactive compounds. Primarily cultivation was done in small scale to perform bioassays for the detection of bioactive compounds. Both liquid state fermentation was performed for the production of endophytic fungi. Potato Dextrose broth was mainly used to culture the endophytic fungi. Liquid state fermentation was carried out for the production of bioactive compounds. Medium was prepared for the growth of endophytic fungi.

EXTRACTION OF PRODUCTION MEDIUM

The culture broths were filtered, and the culture media and mycelia were separated. The mycelia were soaked in methanol. Metabolite was extracted by solvent extraction procedure using ethyl acetate as organic solvent. Equal volume of the filtrate and ethyl acetate was taken in a separating funnel and shaken vigorously for 30 min. Ethyl acetate collected after extraction was evaporated and the resultant compound was dried at 37 C to yield the crude metabolite. After evaporation, a brown coloured crude extract was obtained. The crude extract was then dissolved in Dimethyl sulphoxide (DMSO) until analysis. The methanolic extract of mycelia was collected after 7 days of soaking [13].

GC-MS Analysis:

GCMS has been utilized to determine the intracellular and intercellular bioactive compounds found in the fungal extract. Particles and pollutants are absent from the liquid sample used for the GCMS test sample. To do this, the test samples were centrifuged at 3000 rpm for five minutes. From the clear supernatant, the test sample was extracted. Centrifuging the mixture for intracellular extract was done after dissolving the sample powder in methanol to get the clear supernatant [14].

FTIR ANALYSIS

FTIR analysis was performed on the endophytic fungal extracts in order to identify the functional group that the metabolites contained. The FTIR employed a range of 400 to 4000 cm-1 for both transmittance and absorption. The sample was combined with the KBR powder and sent through a hydraulic pellet press to create a pellet suitable for examination. Next, the fungal samples' peak range was utilized to forecast the functional groups [14].

ANTIBACTERIAL ACTIVITY

The test organisms were spread aseptically using a cotton swab on the surface of the already prepared Nutrient agar for the bacteria. The culture plates were allowed to dry for about five (5) minutes and four wells were made on the agar. The three wells were filled with 30, 50 and 70 µl concentration of the ethyl acetate and methanolic crude extract, The plates were kept on the work bench to allow the agents diffuse into the agar and incubated accordingly. DMSO and methanol were used as the negative control. The Nutrient agar plates were incubated at 37°C for twenty-four (24) hours. The antimicrobial activities were evaluated by measuring the diameter of the inhibition zones in millimeters and the readings were recorded [15].

MTT ASSAY

The methanol extracts of C. Cladosporioides (dried) on the cell response of the human cervical cancer cells (Hela cells) using the MTT assay [16]. The OD value was measured and tabulated (Table.7). The cell viability was measured by adding different concentration of methanol extract into the selected cancer cells. The untreated cells are used as a control (100?ll viability). The cells are treated by 1 µg/ml to 500 µg/ml of sample respectively. They gives various results of cell viability. In the first well contain 91.40% of viable cells because the cells treated with 1 µg/ml of sample. The last well contain 13.67?ll viability due to the presence of 500 g/ml sample. The cell viability is gradually reduced from the first to last well of the 96 well tissue culture plate. The results are mentioned in the table (Table.8) and (Fig. 13) The IC50 Value of tested sample is 183.5 µg/ml (Table.9). The microscopic views of treated cancer cells are given. (Fig.14)

CELL CULTURE

Hela cells (Human cervical cancer cells) cell line was purchased from NCCS, Pune and cultured in liquid medium (DMEM) supplemented 10?tal Bovine Serum (FBS), 100 ug/ml penicillin and 100 µg/ml streptomycin and maintained under an atmosphere of 5% CO₂ at 37^oC.

The dried methanol extract of C. Cladosporioides sample was tested for in vitro cytotoxicity, using Hela cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cultured Hela cells were harvested by trypsinization, pooled in a 15 ml tube. Then, the Hela cells were plated at a density of 1×10⁵ cells/ml cells/well (200 µL) into 96-well tissue culture plate in DMEM medium containing 10 ?S and 1% antibiotic solution for 24-48 hour at 37°C. The wells were washed with sterile PBS and treated with various concentrations of the sample in a serum free DMEM medium. Each sample was replicated three times and the cells were incubated at 37°C in a humidified 5% CO2 incubator for 24 h. After the incubation period, MTT (20 µL of 5 mg/ml) was added into each well and the cells incubated for another 2-4 h until purple precipitates were clearly visible under an inverted microscope. Finally, the medium together with MTT (220 µL) were aspirated off the wells and washed with 1X PBS (200 µl). Furthermore, to dissolve formazan crystals, DMSO (100 µL) was added and the plate was shaken for 5 min. The absorbance for each well was measured at 570 nm using a micro plate reader (Thermo Fisher Scientific, USA) and the percentage cell viability and IC50 value was calculated using GraphPad Prism 6.0 software (USA).

MOLECULAR DOCKING

HEX 8.0 is an interactive molecular graphics program for calculating and displaying feasible docking modes of pairs of protein and DNA molecules. HEX can also calculate protein-ligand docking, assuming the ligand is rigid, and it can superpose pairs of molecules using only knowledge of their 3D shapes. It is the only docking and superposition program to use Spherical Polar Fourier (SPF) correlations to accelerate the calculations. The Fast Fourier transform (FFT) docking methods which use Cartesian grid representations of protein shape and other properties, and which then use translational FFTs to perform the docking correlations. Hex 6.3 Parameter set and distance dependent dielectric functions were used in the calculation of the Van Deer Waals and the electrostatic terms, respectively [17].

RESULT AND DISCUSSION

ISOLATION OF ENDOPHYTIC FUNGI

Plant endophytic fungi have been recognized as an important and novel resource of natural bioactive products, especially in anticancer application [18]. Nontoxic natural bioactive compounds from endophytic fungi in developing new drugs with multifunction mechanisms to meet human needs is essential. Fungal endophytes produce in valuable bioactive metabolic compounds beneficial to humans with antimicrobial, anticancer, antidiabetic, anti-inflammatory, antitumor properties [19, 20]. Secondary metabolites from endophytic fungi show important biological activities such as antioxidant, anticancer, immune modulatory, antivirus, antituberculosis, anti-parasite and insecticides [6].  Endophytes are presumably ubiquitous in plants, with populations dependent on host species and location [21]. As the endophytic fungi are a good source for bioactive compounds and a great demand arises for new drugs. The leaves were selected for this study because, according to Arnold et al.,[22] they are especially rich and abundant endophytic diversity.In present study totally six endophytic fungi Aspergillus flavus, Aspergillus japonicas, Aspergillus nedulans, Fusarium solani, caruvularia lunata and Cladosporium cladosporioides isolated from Lantena camera, hibiscus rosa-sinensis, Musa paradisiaca, Physalis minima, Ocimum tenuiflorum, Calotropis gigantean, Coriandrum sativum, Lilum and Saraca asoca. These six species belong to the class Hyphomycetes (Fig:1-2). The endophytic micro-fungi Cladosporium cladosporioides used for further screening of studies.

       
           
       
Fig:1- Front (left side) and back view (right side) of C. cladosporiodes in culture plate

       
           
       
Fig:2- The structure of C. Clodosporioides under light microscope

       
           
       
Fig:3 -Potato Dextrose Broth before inoculation (left side) and after incubation (right side) of C. Cladosporioides microfungal species

       
           
       
Fig:4-Separation of bioactive compounds by separating funnel

       
           
       
Fig: 5 Organic phase of methanol extract of C. Cladosporioides was separated by using separating funnel.

BIOACTIVE COMPOUNDS

Secondary metabolites from endophytic bacteria have drawn attention from researchers due to their novel structures and significant biological activities [5] . Endophytes are known to produce metabolites such as alkaloids, terpenoids, steroids, quinones, isocoumarin derivatives, flavanoids, phenols, phenolic acids, and peptide. Recently endophytes are viewed as outstanding source of secondary metabolites bioactive natural products. Endophytes are able to produce secondary metabolites these may be an antibiotic, therapeutic agent, agrochemical and an enzyme. Endophytes are rich in bioactive compound which have an important in the fields of medicine, agriculture and industry. There are a large number of bioactive compounds that have been isolated from endophytic fungi and these bioactive natural products have demonstrated a broad range of biological activities. The production of metabolites is mainly depending on the environmental factor and some nature of host plant also because most of the endophytes have a mutual relationship with the plant to favour them. Endophytes are mostly unexplored but they have enormous number of sources as antioxidant, insecticidal, antitumor, antidiabetic, immunosuppressive and biocontrol compounds [4].

Ojinnaka et al.,[23] reported that many bioactive compounds in petroleum ether extract and chloroform crude extract of seed (fruit) of Buchholzzia coriacea recorded in  50 different bioactive compounds such as (E)-9- eicosene, 2-methyl-trans-decalin, 1,2,3-trimethyl- benzene, 1,2,4-trimethyl-benzene, dodecane, azulene, palmotoleic acid, oleic acid, (E) 9- octadecenoic acid and stigmasterol. In chloroform crude extract has 40 different bioactive compounds such as 2-pyrrolidinone, tetrahdropyran, trimyristin, palmotoleic acid, n-hexadecenoic acid, oleic acid, (E)-9-octadecenoic acid, octac-9- enoic acid, n- propyl 9-octadecenoate, n-propyl 11-octadecenoate and. Similarly in present study also similar compounds from ethyl acetate extract and methanol extract of C. Cladosporioides, the compounds such as oleic acid, (E) 9-octadecenoic acid, n-propyl 11- octadecenoate, octac-9-enoic acid respectivelyoleic acid, n-Propyl 11-octadecenoate, 9- octadecenoic acid [E], 9-octadecenoic acid [Z]-2,3-dihydroxypropyl ester, 3,4- Difluorobenzyl alcohol, tert-butyldimethylsilyl ether, Oleic acid, 3- hydroxypropyl ester, 6- octadeceloic acid, [Z], 13- octadecenal [Z], Fumaric acid, 4-cyanophenyl dodecyl ester, 9-octadecenoic acid [Z], octadecyl ester, 2- Butenamide, N, 2,3-trimethyl, Hexadecenoic acid, 2-hydroxy-methyl ester, Oleyl alcohol, heptafluorobutyrate, Fumaric acid, and pent-4-en-2-yl tridecyl ester. The methanol  extract of C.cladosporioides mycelia mat contain tributyrin, Propanoic acid, 2- methyl-1,2,3-propanetriyl ester, Erucic acid, 9-octadecenoic acid, [E]-, 9-octadecenoic acid, [E]-,2,3- dihydroxypropylelaidate, n-propyl 11-octadecenoate, Octadec-9-enoic acid and 6- octadecenoic acid, [Z] (Fig:4,5,6,7 and Table:1,2).

FTIR Analysis:

Sundar et al., [24] 2021 reported utilizing FTIR to estimate the metabolites of Cladosporium endophytic fungus in both (intracellular) methanol mat and (extracellular) ethyl acetate or dichloro-methane extract based on functional groups. Elango et al., [14] 2020 discovered that functional groups such N-N stretching, C-H stretching, O-H stretching, CH2 bending, and C-H bending were recorded in the various absorption peaks of the ethyl acetate crude extract of Aspergillus sojae after it was subjected to FTIR analysis. Similarly present analysis of the ethyl acetate crude extracts of C. Cladosporioides showed the major peaks with intensity of 516.89cm (Strong, C-Br stretching, halo compound), 674.07cm -1 (Strong, C=C bending, alkene), 707.83cm -1 (Strong, C=C bending, alkene), 1140.82cm -1 ( Strong, C-O stretching, aliphatic ether), 1216.03cm -1 (Strong, C-O stretching, vinyl ether), 1315.36cm -1 (Strong, S=O stretching sulfone), 1373.22cm -1 ( Medium, O-H bending, phenol), 1406.01cm -1 (Strong, S=O stretching, sulfonyl chloride). 1437.83cm -1 (Medium, O-H bending, carboxylic acid), (Fig.6 and Table.3).

The FTIR spectral analysis of the Methanol crude extracts of C. Cladosporioides showed the major peaks with intensity of 585.36cm -1 (Strong, C-Br stretching, halo compound), 621.04 cm -1 (Strong, C-Br stretching, halo compound), 1112.85cm -1 (Strong, C-O stretching, aliphatic ether), 1191.93cm -1 (Strong, C-O stretching, tertiary alcohol), 1383.83cm -1 ( Strong, S=O stretching, sulfonyl chloride), 1586.34cm -1 (Medium, N-H bending, amine), 1744.49cm -1 ( Strong, C=O stretching, esters, 6-membered lactone), 2921.96cm -1 (Medium, C-H stretching, alkane), 3461.02cm -1 (Strong, Broad, O-H stretching, alcohol, intermolecular bonded) (Fig.7 and Table-4).

       
           
       
Fig:6. GCMS analysis of ethyl acetate broth extract of C. Cladosporioides

Subbulakshmi et al., [25] reported that methanol extracts of the endophytic fungi responsible for antimicrobial activity. Endophytic fungal species are now considered as exciting novel sources for obtaining new bioactive compounds and have been reported from several hosts [26,27]. In present study ethyl acetate and methanol crude extract of the bioactive compounds were screened from the entophytic fungi C. Cladosporioides and antibacterial activity also recorded (Fig:10,11,12 and Table 5,6).

Zone of inhibition on Staphylococcus aureus, front view (right side) back view (left side)

       
           
       
Fig:11. Zone of inhibition on Pseudomonas auroginosa front view (right side), back view (left side)

Antibacterial activity of methanol extraction of C. Cladosporioides:

Zone of inhibition on E. coli, front view (left side), back view (right side)

        
           
       
Zone of inhibition on Klebsiella pneumoniae, front view (left side), back view (right side)

       
           
       
Zone of inhibition on Pseudomonas auroginosa, front view (left side), Back view (right side)

       
           
       
Zone of inhibition on Staphylococcus aureus, front view (left side), Back view (right side)

       
           
       
       
           
       
Fig:12. Zone of inhibition on E. coli, front view (right side), back view (left side)

 
Table-5. Antibacterial activity of ethyl acetate broth extracts of C. Cladosporioides (dried and stored in DMSO)

       
           
       
Table-6. Antibacterial activity of methanol mycelia mat extract of C. Cladosporioides

       
           
       
In present study of  ethyl acetate broth extract of C. Cladosporioides recorded  16 compounds and it’s have many applications such as oleic acid used as emulsifying agent, anticancer effect, reduce blood pressure, reduction in the risk of coronary heart disease, antioxidant, antipolymerization agent and used in asthma inhaler, 9-octadecenoic acid (E) used as preparation of lotion and pharmaceutical solvent, 9-octadecenoic acid (Z)-2,3-dihydroxypropyl ester uses as antifoam in juice processing, lipophilic emulsifier for water in oil application, cosmetics, encapsulating agent, sun care products, moisturizer, emulsifier and flavouring agent followed by 6-octadecenoic acid (Z) used as anti-irritant, hydrating agent, anti-aging agent, anti-oxidant and used in cosmetics. In present study of methanol mycelia mat extract of C. Cladosporioides recorded 8 compounds and it have some application in industries and medical field.  The compound tributyrin used as antineoplastic activity and used in the treatment of prostate cancer and inflammatory bowel disease.  Erucic acid used as a binder for oil paints.

MTT Assay:

The methanol extracts of C. Cladosporioides (dried) on the cell response of the human cervical cancer cells (Hela cells) using the MTT assay. The OD value was measured and tabulated (Table.7). The cell viability was measured by adding different concentration of methanol extract into the selected cancer cells. The untreated cells are used as a control (100?ll viability). The cells are treated by 1 µg/ml to 500 µg/ml of sample respectively. They gives various results of cell viability. In the first well contain 91.40% of viable cells because the cells treated with 1 µg/ml of sample. The last well contain 13.67?ll viability due to the presence of 500 g/ml sample. The cell viability is gradually reduced from the first to last well of the 96 well tissue culture plate. The results are mentioned in the table (Table.8) and (Fig. 13) The IC50 Value of tested sample is 183.5 µg/ml (Table.9). The microscopic views of treated cancer cells are given. (Fig.14)

IC50 Value

Table: 9 The IC50 Value of tested sample log(inhibitor) vs. normalized response -- Variable slope)

Fig:15. Microscopic view of control cells and treated cells

MOLECULAR DOCKING

In present study GCMS analysis on ethyl acetate broth extract and methanol mycelium mat extract of Cladosporium cladosporioides broth given 6 compounds respectively they are 9octadecenoic acid, Fumaric acid, Oleic acid, 9octadecenoic acid, 6octadecenoic acid and tributyrin.  These compounds might have anticancer activity and was tested in Hex Molecular docking tools [17]. Among these six compounds belonging to Oleic acid and 6octadecenoic acid has given the best energy minimization and maximization values as oleic acid (Emin -249.16, EMax372.94) and 6octadecenoic acid (Emin -327.20 EMax578.37) and these compounds has been selected for the further studies in the future.