Isolation and molecular identification of strain KNUFCPF01
Among the different fungal isolates, the endophytic fungal strain KNUFCPF01 (Fig. 1A) was selected and identified by sequencing of the ITS region (ITS1, 5.8S, and ITS2 of gDNA. The gene sequencing of strain KNUFCPF01 was identified as P. brasiliense (MT269522). A study reported that endophytic fungus Paraconiothyrium sp. was isolated from the root of Capsicum annum . Those finding was supported the existence of Paraconiothyrium sp. in Capsicum annum. Further, the phylogenetic relationship among the P. brasiliense strains and other genera was studied and shown in Fig. 1B. The neighbor-joining method of MT269522, a phylogenetic tree constructed using gene sequences of partial ITS1 and ITS2, complete 5.8S rRNA in MEGA X. The phylogenetic tree showed that strain MT269522 was clustered the similarity with Paraconiothyrium strains whereas R. oryzae was used as an outgroup.
The TPC and TFC of PB-EAE showed 2.59 ± 0.06 mg/g of gallic acid equivalent and 31.53 ± 0.90 mg/g of quercetin equivalent (Table 1). The results indicated that the TPC of PB-EAE was lower than TFC. Similarly, EA extract of endophytic fungal strain Fritillaria unibracteata var. exhibits the different concentrations of phenolic, flavonoid saponin that responsible for the biological properties . The phenolics, flavonoids, and terpenoids are widely reported as antioxidant, antibacterial, and anticancer compounds [34–36]. Further, the volatile compound profile of PB-EAE was determined by GC-MS analysis (Fig. 2). Table 2 shows the retention time, peak area, compound name, molecular formula, and molecular weight of the volatile compound from EAE of P. brasiliense. The results predicted that existence of 12 major bioactive volatile compounds such as 2,3-butylene glycol (CH3CH(OH)CH(OH)CH3), isopentyl acetate (CH3COOCH2CH2CH(CH3)2), o-cymene (CH3C6H4CH(CH3)2), dipentene (C10H16), γ-terpinene (C10H16), 3,5-nonadien-7-yn-2-ol (C9H12O), 2-phenylethanol (C6H5CH2CH2OH), 4-carvomenthenol (C10H18O), 2-phenethyl acetate (CH3COOCH2CH2C6H5), heneicosane (CH3(CH2)19CH3), 2,4-di-tert-butylphenol ([(CH3)3C]2C6H3OH), and 2,5-piperazinedione (C4H6N2O2). The 2,3-butylene glycol was reported a substantial presence in the seeds of Dimocarpus longan Lour. which is used for wine production and facilitates antioxidant properties . 2, 3-butylene glycol helpful for plant-microbe interactions and act as an elicitor . Also, the derivative of 1, 3-butylene glycol exhibited potent antibacterial and antifungal activities . Similarly, the compound isopentyl acetate from endophytic Bacillus subtilis inhibits the growth and sporulation of Curvularia lunata . Cymene isomers are reported to possess insecticidal and repellent properties . Also, the recent studies confirmed that o-cymene has an inhibitory effect against the H1N1 influenza virus  and coronavirus 2 . The study assumes that the maximum existence same molecular weight derivative of dipentene, and γ-terpinene in black pepper was the reason for antioxidant activity . The 4-carvomenthenol is a fragrance molecule  and it has the property to recover from allergic rhinitis and asthma syndrome . 2,5-Piperazinedione derivatives from Penicillium griseofulvum exhibited immunosuppressive activity in anti-CD3/anti-CS28 stimulated murine splenocytes . Also, diketopoperazines derivatives were isolated from endophytic fungus Bionectria sp. Y1085 showed significant antibacterial activity. Further, ascotoxin reported from EA extract of Capsicum annum root endophytic fungus Paraconiothyrium sp. inhibit the seed germination of wild grass (Echinochloa crus-galli) . Also, the study reported that 1,8-dihydroxyanthraquinone from endophytic Paraconiothyrium sp. from Zingiber officinale Rosc. and it was shown in broad spectrum of antimicrobial activity against human pathogenic bacteria and phytopathogenic fungi .
Antioxidant molecules are functioning as the substrate to quenching the incomplete oxygen reduction of reactive oxygen species (ROS) formation in mitochondria thereby protects the cells from oxidative damage [50, 51]. Also, the increased level of ROS oncogenic signaling molecules initiates uncontrolled cell proliferation . To reduce chronic and degenerative diseases, antioxidant molecules have been used widely from different sources (bacteria, algae, fungi, and plants) [52, 53]. The present study showed that EAE of P. brasiliense contains a wide range of antioxidant molecules which are identified from biochemicals content and volatile compounds profile analysis. Hence, EAE of P. brasiliense was tested for inhibition of ROS formation by DPPH and ABTS radical scavenging assay and ferric reducing power assay (Table 1). According to the experimental result, the EAE of P. brasiliense showed the IC50 concentration for DPPH radical scavenging at 383.51 ± 6.97 µg/mL compared to ascorbic acid at 69.11 ± 0.99 µg/mL. Similarly, a previous study reported that EA extract of Paraconiothyrium sp. P83F4/1 showed potential DPPH activity . Also, the EA extract of P. brasiliense showed the IC50 concentration for ABTS radical scavenging at 29.57 ± 1.56 µg/mL compared to ascorbic acid 41.84 ± 1.37 µg/mL. The same way EA extract of endophytic fungus Diaporther sp. showed ABTS radical scavenging activity . In addition, EA extract of P. brasiliense showed the IC50 concentration for ferric reducing power at 358 ± 10.33 µg/mL compared to ascorbic acid 317.6 ± 4.11 µg/mL. Similarly, endophytic fungal (Fritillaria unibracteata var.) extract reported that required a higher concentration for reduction of ferric ion to ferrous . Based on the different antioxidant assays, we concluded that EA extract of P. brasiliense could be the potential source for the isolation of antioxidant molecules.
Cell viability assay is used to determine how the metabolites or drug molecules affect cell proliferation and its biochemical changes. In order to test the cell viability effect, EAE of P. brasiliense, HEK-293 cells, and PC3 cells were used, and cell viability was determined by the WST assay method . The EAE of P. brasiliense treated PC3 cells were showed the IC50 concentration at 187.3 ± 9.29 µg/mL while HEK293 cells did not show any IC50 concentration up to a tested concentration of 1000 µg/mL (Fig. 3). Similarly, EA extract of endophytic Phomopsis sp. did not affect the normal cell (HeLa cells) proliferation while showed the anticancer activity against breast cancer cell line (MCF-7) . Hence, the results revealed that EAE of P. brasiliense showed toxicity against cancer cells but not in a normal cell. Further, EAE of P. brasiliense treated PC3 cell viability and apoptotic changes evaluated using different fluorescent staining. The results of nuclear staining (PI and DAPI) demonstrated that more dead cells population in EAE of P. brasiliense treatment compared to untreated control PC3 cells (Fig. 4). The mitochondrial membrane potential of cells was determined using a rhodamine 123 fluorescent stain. The results indicated that EAE of P. brasiliense treated cells were lost the mitochondrial membrane potential compared to control. Also, the higher ROS elevation was observed in EAE of P. brasiliense treated cells but not observed in control cells evaluated by DCFH-DA staining. The overall results revealed that EAE of P. brasiliense had the excellent cytotoxic behavior on PC3 cells but less toxic to normal cells. Similarly, the study reported that brasilamide E (1) from P. brasiliense showed the toxicity to cancer cells (HO8910, MCF-7, and HeLa) while non-toxic to normal cell (NIH3T3). Also, ketal compound sporulosol exhibited considerable cytotoxicity to HeLa, T24, A549, HCT116, and SH-SY5Y cells . But isolated eight furanone compounds (paraconfuranones A-H) from P. brasiliense MZ-1 did not show any toxicity to A549, HepG2, and CaSki cells .
The digestive enzymes play a major role in converting complex sugar molecules into simple sugar molecules through enzymatic digestion . Even if in the case of diabetic patients, blood sugar might be increased than normal people due to the enzymatic action. Hence, the inhibition of enzyme function subsequently decreased the blood sugar level. In the present study, P. brasiliense EA extract did not show considerable digestive enzyme inhibitory activity against α-amylase and α-glucosidase (Data not shown).
Antibacterial agents are used to killing the bacteria (bactericidal) or slow down the bacterial growth (bacteriostatic). Most of the antibacterial agents were initially isolated from natural sources and then they have been synthesized their own or derivative form for the applications [59, 60]. However, prolonged use of drugs creates antibiotic-resistant bacterial strains which urge the discovery of novel antibiotics. Hence, EAE of P. brasiliense was evaluated for its antibacterial efficacy with bacterial pathogens such as B. cereus, S. aureus, L. monocytogenes, E. coli, and S. enterica (Fig. 5). The results showed that EAE of P. brasiliense had substantial inhibitory activity against both Gram-positive and Gram-negative bacteria. The inhibitory activity depending on the concentration that means when increasing/decreasing the EA extract concentration, the bacterial inhibition activity was increased/decreased, respectively. Among the different concentrations of EAE of P. brasiliense, 50 µL of maximum tested concentration (1000 µg/mL) showed the zone of inhibition at 17 ± 1.2 mm for B. cereus, 22 ± 1.4 mm for S. aureus, 24 ± 1.4 mm for L. monocytogens, 8 ± 0.4 mm for E. coli, and 21 ± 1.6 mm for S. enterica (Table 3). Also, the positive control 20 µL TCH (1000 µg/mL) exhibited the zone of inhibition 21 ± 1.2 mm for B. cereus, 22 ± 1.2 mm for S. aureus, 23 ± 1.4 mm for L. monocytogens, 12 ± 1.2 mm for E. coli, and 21 ± 1.4 mm for S. enterica. Interestingly, the EAE of P. brasiliense showed significant antibacterial activity (p < 0.05), which is the same as the antibiotic TCH. The minimum inhibitory concentration of EAE of P. brasiliense showed B. cereus at 31.25 µg/mL, for S. aureus at 15.62 µg/mL, L. monocytogens at 15.62 µg/mL, E. coli at 62.5 µg/mL, and S. enterica at 15.62 µg/mL. Similarly, endophytic Paraconiothyrium sp. isolated from Zingiber officinale Rosc. showed considerable antibacterial activity against clinical pathogens . But the antibacterial properties of Paraconiothyrium sp. were studied inadequately the research gap could be filled by future studies.
Molecular docking analysis
Molecular docking analysis was performed to identify the interaction of metabolites with the molecular target of anti-bacterial activity. The inhibition of dihydropteroate synthase (DHPS) is one of the key mechanisms to kill the bacterial or inhibit bacterial growth through pterin-sulfonamide conjugation . According to those strategies, the identified volatile compounds from EA extract of P. brasiliense interact with DHPS. The results indicated that out of 12 volatile compounds, 10 compounds completely satisfied Lipinski’s rules (Table 4). Further, molecular docking analysis of major volatile compounds was tested with DHPS (5JQ9) was shown in Table 5 and Fig. 6. The docking results showed that 5 volatile compounds such as o-cymene, dipentene, γ-terpinene, heneicosane, and 2,4-di-tert-butylphenol had the higher molecular interaction according to the binding energy. But those volatile compounds had no hydrogen bonding, which indicated that antibacterial mechanism might be done other than inhibition of DHPS mechanisms. Interestingly, one of the volatile compounds 2,5-piperazinedione showed less binding energy (-5.20 kcal/mol) with greater hydrogen bonding (PRO15; THR53; VAL17; GLY51) (Fig. 5) comparing to other volatiles tested in the present study.