Intracellular reactive oxygen species scavenging activity and lipid peroxidation inhibition by secondary metabolites isolated from the endophytic fungus, Daldinia eschscholtzii

Fungal endophytes associated with medicinal plants are of research interests, since they provide a viable alternative source for molecules with medicinal value. In this study, we report for the rst time two fungal endophytic isolates, AI.EF 001 and AI.EF 002 belonging to the genus Daldinia from the leaves of medicinal plant, Abutilon indicum linn (AI). Both AI.EF were identied as Daldinia eschscholtzii (DE) species by ITS1-5.8-ITS2 sequence analysis and phylogenetic tree reconstruction by Neighbor-joining method. Crude extracts of DE (EFEA), generated by ethylacetate/water fractionation of the fungal methanol extracts when subjected to column chromatography separation yielded 5 compounds. NMR and other spectral data revealed the compounds to be (cid:0) -napthoavone, Syringaldehyde, 3,4,5-trimethoxy benzoic acid, 2-Furoic acid and Gossypetin 3′ O glycoside. All of these compounds are being reported for the rst time from DE. The isolated compounds showed promising free radical scavenging activities. The compounds also exhibited anti inammatory property by down regulating intracellular ROS as well as inhibiting LPS induced lipid peroxidation in AtT 20 mouse pituitary cells. Current nding demonstrates endophyte DE as a new source for the avanoid, Gossypetin-3′-O-glycoside along with other phytoconstituents with strong antioxidant and anti-inammatory activity.


Introduction
Endophytes are microorganisms that colonize inside the plant and form an important component in the plant-micro ecosystem. They reside in the tissue of plants in a symbiotic fashion without causing any negative impact on the host (Chatterjee et al. 2019). Endophytes mostly belong to the class Ascomycetes, Basidiomycetes and Deuteromycetes, forming a considerable proportion of the fungal species (Padhi et al. 2013). This makes endophytes a promising alternative source for mining of compounds with novel therapeutic molecules. Metabolites from endophytes have shown potential applications in agriculture, industries and in modern medicine as antibiotics, antimycotics, immunosuppressant and anticancer compounds (Jia M et al. 2016). Added advantage of utilizing endophytic fungi as a source for obtaining phytochemicals is, it helps to conserve the plant biodiversity. Since, endophytes mimic metabolites found in the host plant and can provide an alternative source for the same (Mollaei S et al. 2019).This also makes endophytes an attractive target for commercial utilization. Overall, endophytes serve as a better source for the increasing demand of biologically active drugs in an environmentally friendly process (Gupta D et al. 2014).
Abutilon indicum linn (AI) commonly known as thuthi is an important medicinal plant belonging to the family Malvaceae. In traditional system of medicine, the plant is used as a cure for human ailments like ulcers, piles, anxiety and as diuretic (Suryawanshi V.S and S.R Umate 2020). A number of bioactive secondary metabolites derived from this plant like beta sitosterol, caffeic acid, gallic acid, avonoids, saponins, alkaloids have been reported. Earlier reports indicate that, the plant possesses hepatoprotective, anti-in ammatory, anti-fungal, wound healing and anti-bacterial properties (Sharma et al. 2013). A few strains of endophytic fungi like Alternaria alternata, Aspergillus niger, Nigrospora sp, Cladosporium sp, Fusarium sp has been previously reported in this plant (Mahobiya D and Gupta A.K 2017;Suryanarayanan et al. 2007). Considering that extracts of AI is already in use as traditional medicine, it could be hypothesized that metabolites of endophytic fungi from AI could be of potential medicinal value.
In ammation is a major factor for the progression of various chronic disorders and these in ammatory disorders, in turn show correlation to the presence of free radicals in the biological system. Increase in free radicals leads to oxidative damage of the functional macromolecules, which is a precursor for increased expression of in ammatory cytokines. Hence, antioxidant compounds that are e cient in reducing the free radical levels are shown to have a signi cant effect on reducing in ammation, leading to the amelioration of in ammatory disorders (Hussain et al. 2016). One of the major pathways involved in the oxidative stress mediated in ammation is the lipid peroxidation. In this pathway, lipid macromolecules are oxidized to toxic lipid aldehydes by the free radicals. The chief instigators of lipid peroxidation are hydroxyl radical (OH − ), superoxide radical (O 2 − ), hydrogen peroxide (H 2 O 2 ), and nitric monoxide (NO − ). When present in excess, these oxidative species readily attack cellular lipid membrane resulting in their oxidative degradation. This leads to cellular and tissue damages, in ammation and cell death (Chang Y and Kim C.Y 2018). Identifying compounds which in general target ROS or speci cally prevents lipid peroxidation will be a valuable tool in our ght against in ammatory disorders. In this scenario, investigating the antioxidant ability of metabolites from endophytic fungi isolated from established medicinal plants could be of interest. Recent studies have shown that fungal metabolities, 3methoxy avone, Nobelitin, Scopoletin and Formononetin have been isolated from endophytic fungal strain CBL 12. The endophytic fungus was isolated from the medicinal plant, Conyza blinii H.Lev. (Tang et al. 2020). Apart from this, Quercetin, a avonoid has been reported from endophytic fungus, Psathyrella candolleana and Apigenin 8-C-β-D glucopyranoside from Colletotrichum sp (Toghueo 2020). Similarly, a avone, Chrysin (5,7 dihydroxy avone) from endophytic fungi A. alternate, C.capsici and C.taiwanense with anticancer property has been reported (Seetharaman et al. 2017). These studies clearly highlight the potential of endophytic fungi as a rich source of antioxidant metabolites like avonoids, with the added advantage of large scale microbial mass cultivation and production.
In the present study, attempts were made to isolate and identify new endophytic fungal species from the leaves and pods of AI. In the process, a noteworthy species of Daldinia was identi ed from leaf tissues whereas the pods did not show any presence of endophyte colonization. Literature search indicates that as on date there is no known report of DE from any parts of AI. Also, this study is evidenced by detailed morphological, molecular and phenotypic characterization. Further, attempts were made to characterize the bioactive fractions and identify the major metabolites and to investigate the inhibitory activity of the bioactive compounds on lipid peroxidation and ROS generation. Isolation of Endophytic fungi from leaves and pods of AI Endophytic fungi were cultured from the leaves (2 nos.) and pods (2 nos.) after appropriate surface sterilization of the plant parts. Surface sterilization protocol was carried out according to the method described by Pedra et al (2018) with minor modi cations. Fresh leaves and pods were washed thoroughly in running tap water, and then surface sterilized with 4% hypochlorite for 2-3 minutes and washed 3 times with autoclaved distilled water. The plant samples were further washed with 70% ethanol and allowed to air dry before further processing. The leaves were excised into small pieces (0.5 cm x 0.5 cm) using a sterile scalpel and placed on potato dextrose agar plates (PDA: 300g/L diced potatoes, 20g/L dextrose and 20 g/L agar under sterile conditions. Similarly, a single lea et from the pods were excised and placed on to the agar plates. 4 leaf and pod segments were placed on each plate. 50 µg/ml of chloramphenicol was added to the media to prevent bacterial growth. The samples were incubated for 5-7 days at 28°C. The fungal hyphae emerging from the leaves were sub cultured in PDA and the pure cultures were kept as PDA slants at 4°C for further studies. After nal identi cation of the endophytic fungi as DE, the results were further con rmed by plating the leaf samples in Oatmeal agar (OA: 60g/L oatmeal and 12.5 g/L Agar)

Collection of AI plant materials
Identi cation of the isolated Endophytic fungi

Morphological Analysis
The isolated endophytic fungi were identi ed based on the morphological characteristics. Morphological features such as color, texture and topography of the isolates were examined on PDA and OA. The cultures were incubated at 30°C with day to day examination of fungal growth. Morphological analysis was done by staining the isolates with lactophenol cotton blue as previously described by Kuan et al (2015). The stained fungi were observed and imaged using uorescent microscope -BF (Zeiss, Germany) at 100X magni cation. The ZEN 10 software was used to process the images.

DNA extraction
Pure fungal mycelia were harvested carefully from the agar surface using a sterile forceps. These mycelia were transferred aseptically to sterile phosphate buffered saline (pH 7.4) and vortexed with glass beads for 5 minutes. 200 µl of lysate was subjected to DNA extraction using DNeasy Plant Mini kit, Qiagen, USA, according to the manufacturer's protocol. The extracted DNA samples were quanti ed and their quality con rmed using nanodrop for further sequencing analysis.
PCR ampli cation and DNA sequencing DNA isolated from the endophytic fungi was diluted in sterile water and stored at 4˚C.PCR was performed using the primersF:5′AATCAGTTATAGTTTATTTGATGGTGGT3′, R:3′TCTCAGGCTCCCTCTCCGGAACC5′. The reaction was performed in a 25 µl nal volume containing 0.1 µg of genomic DNA, 10 pM of each primer, 1X Taq.pol buffer, 1.5 mM Mgcl 2 , 0.2 mM dNTPs and 1U Taq DNA polymerase. Thermal parameters used for the PCR was, 94˚C(3 min) followed by 35 cycles with following parameter executed in each cycle, 94˚C (30s), 55˚C (40s) and 72˚C (35s) and a nal extension at 72˚C (7 min). PCR ampli ed products were examined by electrophoresis in 1.5% agarose gels in TAE buffer and puri ed using a PCR clean-up kit (QIAquick PCR Puri cation Kit, Qiagen, USA). The puri ed PCR products were sequenced using the Nextseq 500 DNA Analyzer. As an underlying basis to identify the fungi, the sequences were manually edited and compared with available data from Genbank databases (NCBI website; http://www.ncbi.nlm.nih.gov/) using the BLASTN program.
Phylogenetic analysis rDNA-ITS sequences from the fungal endophyte strains (AI.EF 001, AI.EF 002) were submitted to GenBank and accession numbers were obtained. Fungal rDNA-ITS sequences along with the closely identi ed sequences obtained from GenBank based on the BLAST search were used for phylogenetic analysis to identify the endophytes (Samarakoon S, 2019). A total of 67 closely related sequences were obtained, out of which 10 sequences were used for phylogenetic reconstruction. Original sequences chromatogram was edited using chromas version 2.6.6 software (Technelysiumpty ltd) and aligned using the Clustal W version 1.8 program. Phylogenetic tree was constructed using neighbor-joining (NJ) algorithm with Hypoxylon fragiforme as outgroup. NJ analysis was done using MEGA version 10.1.8 software with bootstrap values calculated for 1000 replicate runs.

Mass cultivation of Endophytic Fungi
Isolated strains of endophytic fungi were grown in Potato dextrose broth (PDA: 300g/L diced potatoes and 20g/L dextrose). 8mm discs of the fully grown fungal mycelium was punched with the cork borer and inoculated aseptically to the broth in 500 ml Erlenmeyer's asks (15 nos) each containing 200 ml of PDA broth. The cultures were grown for a period of 21 days (Yang et al. 2017 Cell lines and culture medium Mouse pituitary cell lines AtT20 were procured from National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in DMEM supplemented with 20% inactivated Fetal Bovine Serum (FBS), Penicillin (100 IU/ml), Streptomycin (100 mg/ml) and Amphotericin B (5 mg/ml) in a humidi ed atmosphere of 5% CO 2 at 37˚C until con uent.

Determination of cell viability of EFEA fractions
EFEA 001 and 002 were tested on AtT20 cell lines and cell viability was assessed using PI staining with the aid of ow cytometry (FACS) (BD FACS calibur) (Ramachandran et al, 2018). AtT20 cells (20,000cells/ml) were seeded and incubated for 18 hrs with EFEA (2.5mg/ml -0.15 mg/ml) or Doxorubicin (positive control, 0.1mg/ml).At the end of incubation, the cells were centrifuged, pelleted, re suspended in FACS buffer and stained with PI (10µl of 1mg/ml)for 10 min in the dark. Viability of cells was assessed using FACS -FL2 detector and analyzed using BD CELLQUESTPRO software.
Fractionation and puri cation of compounds from EFEA using Column chromatography Preliminary TLC investigation of EFEA 001 and 002 revealed a similar molecular pro le with few exceptions. Both EFEA were subjected to silica gel column chromatography for isolation of compounds. 4.8g of EFEA was loaded on to the packed column as admixture (4.8 g of silica). Silica gel G (60-120 mesh, 69 g) in hexane was used to pack the column. Increasing volume of EtOAc in Hexane was used as eluent. Fractions were monitored with TLC and those containing similar compound pro les were pooled, solvent evaporated under vacuum and weighed. Since EFEA 002 showed a similar compound pro le as EFEA 001 with some exceptions, EFEA 002 was also subjected to similar column chromatography separation and the compounds isolated. The isolated compounds were characterized using standard spectroscopic techniques.
Spectroscopic Characterization of isolated compounds EFEA 001 yielded 5 compounds (C1-C5) and EFEA 002 yielded 3 compounds (C1, C3 and C4) which were characterized using NMR, UV, IR and Mass spectrometry. UV characterization was done by dissolving compounds (C1-C5) in methanol (10 µg/ml) and analyzing them for maximum absorbance using UV-VIS spectrophotometer (Shimadzu). FT-IR analysis for the isolated compounds was carried out in KBr pellets using Bruker alpha-E&T spectrophotometer (Lab India). 1 H, 13 C, HMBC and HSQC spectra were recorded with the aid of NMR (500 MHz -BRUKER) using tetramethylsilane (TMS) as an internal standard. NMR for compounds C1-C3 was carried out in CDCl 3 while compounds C4 andC5 were dissolved in D 6 -DMSO solvent. MS analysis was done using SHIMADZU mass spectrophotometer to determine the molecular mass of the isolated compounds.

Determination of IC 50 values of isolated compounds by MTT assay
Isolated compounds (C1-C5) were tested on AtT20cell lines and cell viability was assessed using MTT assay as previously described by Shazia Anjum et al (2020)  Antioxidant assays DPPH, ABTS, Superoxide anion and hydroxyl radical scavenging activity assay was carried out to identify the antioxidant potential of all the compounds. All these assays were carried out as triplicates with the compounds (C1-C5) evaluated within the concentration range of (0.06-1.0 mg/ml), using ascorbic acid as the positive control. The following formula was used to calculate the antioxidant activity: Radical scavenging activity (%) = [(Acontrol − Asample)/Acontrol] × 100% DPPH radical scavenging activity assay 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was used to determine the free radical scavenging activity of the isolated compounds (C1-C5) using colorimetric method with slight modi cation (Sannasimuthu A et al, 2018). Brie y, an aliquot of 100 µL of individual compounds were mixed with 100 µL of DPPH solution (0.1 mM). The reaction mixture was incubated for 30 min at room temperature and its absorbance (A) measured at 517 nm using a Thermo Multiskan go 96 well Microplate reader. The radical scavenging e cacy of the isolated compounds was determined as mentioned above ABTS Radical Scavenging Assay ABTS radical scavenging activity was done based on the method described by Sannasimuthu et al (2019) with slight modi cations. Brie y, 7 mM ABTS solution was mixed with 2.45 mM potassium persulfate to produce ABTS radical cation (ABTS˙+). The reaction mixture was diluted to an absorbance of0.70 ± 0.02 at 734 nm using 0.2 M PBS (pH 7.4) at 30°C. 20µL of compounds were added to 180 µL of diluted ABTS˙+ solution and incubated at 30°C for 6 min. Decrease in absorbance at 734 nm was monitored using a spectrophotometer (UV1800, SHIMADZU, Kyoto, Japan). Concentration of the compounds giving the similar decrease of ABTS˙+ cation as that of positive control was calculated using the formula as mentioned above.
Superoxide anion radical scavenging activity assay The superoxide anion radical scavenging activity of the compounds was analyzed according to the method described by Chi et al (2014) with minor modi cations. Brie y, superoxide anion radical was generated in 50 all of nitrotetrazolium blue chloride (2.52 mM), 50 all of NADH (624 mM) along with 50 all of compounds. This was followed by the addition of 50 all of phenazine methosulfate solution (120µg/mL) to the reaction mixture. After incubation for 5 min at 25°C, the absorbance of the product formed was read at 560 nm (UV1800, SHIMADZU, Kyoto, Japan). Ability of the compounds to scavenging superoxide anion radical was calculated using the formula mentioned above.
Hydoxyl radical scavenging activity assay Hydroxyl radical scavenging activity was performed as described by Batool et al (2019)  Intracellular ROS scavenging activity of the isolated compounds Intracellular Reactive Oxygen Species (ROS) was measured using DCFH-DA uorescent dye in AtT 20 mouse pituitary cells using a standardized method with minor modi cations (Ramachandran S et al, 2018). The cells were cultured in a 6-well plate at a density of 7x10 4 cells/well/ml. Cells were treated with peroxide followed by the treatment of compounds C1-C5 at IC 50 concentration as mentioned above. H 2 O 2 alone treated cells were used as positive control and all the groups were incubated for 24h. At the end of incubation, the cells were exposed to 10 µM of DCFH-DA at for 30 min. Cells were then harvested and uorescence intensity measured using BD FACS calibur (Ex/Em -488 nm/530 nm). The analysis was done using BD Cell Quest Pro software.
In vitro nitric oxide inhibitory activity Intracellular nitric oxide inhibition activity was measured in AtT20 cells according to the method described by Adebayo et al (2019) with some modi cations. Brie y, 20,000 cells/well were seeded in 96 well plates followed by treatment with 50 µl of compounds C1-C5 (IC 50 ). After 2 hr of incubation at 37˚C, 50 µl of LPS (5 µg/ml) in DMEM was added to all wells containing the compounds. After 24 hrs of incubation, the cells were collected and pelleted and the supernatants from cells were collected for NO measurement using colorimetric Griess reaction method. 60 µl of supernatants were combined with equal volume of Griess reagent [1% sulphanilamide/0.1% N-(1napthyl) ethylene diamine (International Laboratory, USA), each in 2.5% H 3 PO 4 ] in a 96 well plate at room temperature for 10 mins, and the absorbance was read at 550 nm using a Thermo Multiskan96 well plate reader. Absorbance measurements were averaged and converted to µmol/L of NO per well using a standard curve of sodium nitrite.

In vitro Lipid peroxidation assay
Lipid peroxidation assay is based on the production of malandialdehyde (MDA) in AtT20 cells as described by Hasanzadeh et al (2017) with some modi cations. For evaluation of MDA production rate, thiobarbituric acid (TBARS) assay was used. 70,000 cells/well were seeded in a 12 well plate followed by treatment with 50 µl of compounds C1-C5 (IC 50 ). After 2 hr of incubation at 37˚C, 50 µl of LPS (5 µg/ml) in DMEM was added. After 24 hrs of incubation, the cells were collected and centrifuged at 1800 rpm for 10 min. The pellet was resuspended in 500 µl of deionized water and lysed by sonication. 1 ml of TBA solution (15% trichloroacetic acid, 0.8% thiobarbituric acid, 0.25 N HCl) was added. The mixture was heated at 95˚C for 15 min to form MDA-TBA product. Optical density was measured at 532 nm spectrophotometrically.

Statistical analysis
Results obtained were analyzed and were then evaluated by analysis of variance (One-way and Two-way ANOVA). P-values less than 0.05 (p < 0.05) are considered to be signi cant.

Results And Discussion
Endophytic fungi ubiquitously inhabit most plant species and are recognized as a repository of novel compounds with immense applications. Added advantage of using the endophytic fungi is that they can be grown in a controlled environment in a sustainable manner to obtain secondary metabolites of medicinal value (Fouda A.H et al. 2015). Apart from this, endophytic fungi are also shown to mimic metabolites generated by the host plant. This makes exploration of endophytic fungi from traditional medicinal plants, a necessity for therapeutic advancements. In the present investigation, two fungal endophyte isolates (AI.EF 001, AI.EF 002) were isolated from the leaves of AI and were identi ed as Daldinia eschscholtzii (DE). Earlier reports indicate the presence of endophytic fungus Alternaria alternata in the root and petiole of AI (Mahobiya and Gupta 2017). However, as on date, only a few species of endophytic fungi are reported from the medicinal plant AI (TN, India). Hence, the current study based on endophytic fungi and its metabolites from leaves of a traditionally used medicinal plant AI will be of high value.

Morphological Study
Leaf samples of AI incubated on PDA and OA after surface sterilization showed rapid growth of fungi. Initially, growth of white cottony hyphae was observed at the inoculated site after 3 days in both PDA and OA (Fig. 1A & 1B). After 7 days, the fungal isolate was sub cultured in fresh PDA and OA plates. Subcultured fungal isolates turned into smoky grey in color after 2 weeks and the fully differentiated mycelium showed uffy texture. Fungal isolates showed black coloration on the reverse side with dense mycelial growth. On the other hand, the pod samples from the same AI plant, processed similarly as leaves, did not show growth of any isolate after 5 days (Fig S1). This indicates that the endophytic fungi might be localized to the plant leaves. Lack of fungal isolate from pods harvested from the same AI plant along with the leaves also served the secondary purpose of internal controls.
Light microscopic analysis of the fungi stained with Lactophenol and cotton blue clearly visualized the structural characteristics of the isolates. Fungal isolates from both the leaf samples showed similar morphological features. The hyphae were septate with thin and thick walls ( Fig. 2A), while the conidiogenous cells were hyaline and cylindrical (Fig. 2B). Conidiophores were irregularly branched with conidiogenous cells originating from each ends (Fig. 2C). The septate conidiophores form dichotomous and trichotomous branches with clusters of conidia at the terminus (Fig. 2D). Thick walled hyphae of the fugal isolates showed brownish black exudates on its surface (Fig. 2E). Conidia were hyaline and ellipsoid with attenuated base with approximately 4.3-4.7 µm in length and 2.0-2.25 µm in diameter (Fig. 2F). These speci c features indicate that the isolates belong to the family Hypoxylaceae and genus Daldinia. Both AI.EF 001 and AI.EF 002 fungal isolates shared similar morphological and structural characteristics indicating that they both belong to the same genus Daldinia.

Molecular Study
Identi cation of AI.EF001 and AI.EF002 at species level based on these morphological characteristics seemed di cult and could lead to ambiguity, hence PCR based ITS sequence analysis was carried out to further con rm the species of fungal isolates Ng et al. 2016). DNA sequences from the isolates were subjected to molecular phylogenetic identi cation. ITS1-5.8-ITS2 sequences obtained from the fungal DNA were compared with the closely related sequences from GENBANK to reconstruct the phylogenetic tree. Ncbi-BLAST search of AI.EF001 and AI.EF002 DNA sequences identi ed them as DE.
BLAST results also showed that the DNA sequence obtained from the isolates had several close matches.
Phylogenetic reconstruction for the isolates (Table 1) was done by neighbourhood joining (NJ) method (kimura 2 parameter model for 1000 bootstrap replications) with reference isolates of DE and other closely related species of DE from BLAST which formed a cluster within DE group (Fig. 3). Hypoxylon fragiforme was used as outgroup. DNA sequences of both the fungal isolates shared a maximum identity of 100% with available data in NCBI and were grouped under the genera Daldinia. The isolates AI.EF 001 and AI.EF 002 showed high bootstrap values (100%) with the DE strain FR852577 (Fig. 3), which indicates the level of con dence within the clade. Hence, the phylogenetic analysis con rmed the isolates as DE. ITS sequences of AI.EF 001 and AI.EF 002 were submitted to GenBank with accession numbers MT712203 and MT712204 respectively  (Table 2).These family of phytochemical constituents identi ed in both the fungi are well known for their different types of bioactivity. It is of interest that both endophytic fungi showed a varying phytochemical pro le in spite of sharing a common host.  Stadler et al. 2001;Tarman et al. 2012;Zhang et al. 2011). These phytochemicals have shown strong immunomodulatory effects (Helaly et al. 2018). In this study, the focus is on mid polar, EtOAc extract (partitioned using H 2 O/EtOAc). The EtOAc extract was subjected to silica gel column chromatography method using various combinations of Hexane and Ethylacetate, for separation of compounds identi ed using TLC. EFEA was subjected to column chromatography puri cation and compounds isolated by these means characterized using spectroscopic techniques.
EFEA 001 and EFEA 002 were subjected to further puri cation by silica gel-column chromatography. Column chromatographic separation of the EFEA 001 fraction yielded 7 major fractions, of which 5 fractions were identi ed as pure compounds (C1-C5). Similarly, the chromatographic separation of EFEA 002 fraction yielded 3 major compounds which were similar to compounds C1, C3 and C4 of EFEA 00.
However, compounds C2 and C5 were absent or maybe present in undetectable amounts in EFEA 002.
However, detailed investigation is needed before con rming the biosynthetic link between AI and DE. Also, the compounds syringaldehyde and Gossypetin 3′O glycoside are absent in the fraction EFEA 002. This could be the basis for difference in bioactivity of EFEA 001 compared to EFEA 002. Overall, the identi cation of DE from the plant AI serves as a valuable source for the production of several of these compounds, especially the avone derivatives like gossypetin.
Quanti cation of isolated compounds The 5 phytoconstituents isolated for the rst time from the endophytic fungi DE were quanti ed using HPTLC. HPTLC validation and quanti cation of phytoconstituents has become an effective and valuable tool (Noman et al. 2020). Ease of use, as well as the exibility of using multiple solvent systems, makes it an ideal tool for quantifying several constituents simultaneously in the EFEA. Linearity for each compound was obtained by plotting different concentrations (7.5-1000 µg/ml) against their respective peak area in the chromatogram. Individual compounds showed unique R f value as followsnaptho avone (0.93), syringaldehyde (0.70), 3,4,5 trimethoxy benzoic acid (0.68), 2-Furoic acid (0.39) and Gossypetin 3′ O glycoside (0.90) (Fig S7 & S8 ). R f values of the compounds were used to match with EFEA and peak areas used to quantify the amount of compound present in the extract (Table S1).
Regression equations and correlation co-e cients for the individual compounds obtained from the linearity plot were used for accurately calculating the amount of compounds present in EFEA (Fig. 6). The amount of compounds C1-C5 in crude fraction EFEA 001 were estimated to be -naptho avone -3.29 mg, Syringaldehyde -2.24 mg,3,4,5 trimethoxy benzoic acid -4.8 mg,2-Furoic acid -6.4 mg and Gossypetin 3′ O glycoside -4.6 mg of dried weight of 30 mg of crude fraction respectively whereas the amount of compounds C1,C3 and C4 in crude fraction EFEA 002 were estimated as 1.2 mg, 1.6 mg and 2.1 mg respectively. These results indicate that although the compounds present in EFEA 002 were similar to that of EFEA 001, the yield of compounds -naptho avone, and 2-Furoic acid was very less in EFEA 002 when compared to EFEA 001.

Determination of cell viability and IC 50 concentrations of the isolated compounds
Earlier cytotoxic assays showed least cytotoxic activity for EFEA. It would be of interest to see if any or all of the 5 compounds isolated from EFEA share this activity. Compounds C1-C5 at a concentration range of 0.03 mg -1 mg/ml was used to assess the cytotoxicity in AtT 20 cells using MTT assay. All the 5 compounds showed signi cant viability ranging from 42% − 85%, 61% − 92%, 42% -89%, 49% − 81% and 64% -95% respectively in AtT 20 cells (Fig. 7). Some of the ndings were in line with existing literature reports, which indicate that syringaldehyde exerts neuroprotective effect through anti-oxidant and anti-apoptotic activities (Bozkurt et al. 2014). Similarly, Gossypetin 3′O glycoside has also been reported to exhibit strong anti oxidant activity (Haugrin et al. 2016). These ndings suggest that compounds isolated from the AI leaf endophytic fungus DE could be valuable source for bioactive compounds.
Also, from these studies the IC 50 values of all the compounds were determined as C1-0.1, C2-0.15, C3-0.15, C4 -0.125 and C5-0.1 mg/ml. These IC 50 concentrations were used for further in vitro assays.

Antioxidant activities of the isolated compounds
To evaluate the antioxidant ability of the isolated compounds to scavenge the free radicals DPPH, ABTS, Superoxide anions and hydroxyl ions radical scavenging assays were carried out. Ascorbic acid was used as positive control for all the assays.
DPPH is a stable, nitrogen centered free radical which upon reduction changes from violet to yellow colour. The change in the colour depends upon the scavenging abilities of antioxidant extracts or pure compounds as it reduces the DPPH radical by donating hydrogen (Batool et al. 2019). All the compounds isolated showed strong radical scavenging activities. Compound C1 showed scavenging activities at a range of 41% -74%, compound C2 31% -68%, compound C3-50% -81%, compound C4 42% -76 % and compound C5 51% − 86% (Fig. 8a). The compounds showed signi cant (p < 0.01) concentration dependent antioxidant activities, the higher concentration exhibited highest radical scavenging activity.
This was on par with the positive control ascorbic acid which possessed 61% − 94% antioxidant activity.
Superoxide anion is one of the crucial free radicals which are formed by the addition of an electron to molecular oxygen. Superoxide radical easily reacts with nucleic acids, amino acids, lipids in the cell membrane to exert a robust toxic effect (Luo et al. 2019). Hence, it is very essential to study the e cacy of a compound to scavenge superoxide radical. Our results showed concentration dependent scavenging activity of superoxide radicals at all tested concentrations. The compound C1 showed 28% -69% scavenging activity, C2 showed 40% -75%, C3 showed 31% -64%, C4 showed 39% -79%, C5 possessed 51% − 83% (p < 0.01) scavenging activities (Fig. 8c). This was similar to that of the scavenging activities of ascorbic acid (61% -91%).
Hydroxyl radical is a potent reactive species which cause severe pathogenesis to cell membrane phospholipids and react with polyunsaturated fatty acids. It is very short lived toxic free radicals which cause severe damage to cellular integrity (Batool et al. 2019). Therefore compounds that exhibit antioxidant against the hydroxyl radicals are needed for balancing the redox state in the cells and to deactivate the radical. In this study, the compounds C1-C5 showed strong scavenging potential against hydroxyl radicals. Compound C1 showed 28% − 69% scavenging activity at concentrations 0.06 -1 mg/ml. Compound C2 showed 40% − 75%, C3 31% -64%, C4 39% − 79% and C5 showed 51% − 83% (p < 0.01) hydroxyl ions scavenging activity (Fig. 8d). Ascorbic acid showed 64% − 91% activity.
These assays show that the compounds isolated from D.eschscholtzii possess strong antioxidant activity. Antioxidants are crucial substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress. Our study showed that all the isolated compounds possess the ability to trap and scavenge the free radicals and thus it can inhibit the oxidative stress that lead to several degenerative diseases.
Intracellular free radical scavenging activity in AtT 20 mouse pituitary cell lines To understand the in uence of isolated compounds on intracellular oxidative stress, DCFDA stained AtT 20 cells were used for FACS analysis. The non uorescent DCFH-DA dye that easily penetrates into the cells gets hydrolyzed by intracellular esterase to become DCFH, and this compound is trapped inside the cells further gets oxidized by peroxide (Sannasimuthu A et al. 2019). FACS analysis revealed that the compounds C1-C5 at their IC 50 concentrations signi cantly reduced the extent of free radical generation.
AtT 20 cells when treated with compounds C1-C5 along with peroxide (50 µm) showed a signi cant (p < 0.001) reduction in the uorescence intensity when compared to cells treated with peroxide alone (94%).
Among the 5 compounds, C5 exhibited most promising intracellular radical scavenging activity (approx 80%). These results con rmed that the isolated compounds exhibited the potential to reduce the intracellular oxidative stress in AtT 20 cells. Earlier work in our lab has shown that inhibition of ROS generation in cells activated by peroxide plays a crucial role in down regulating the cellular in ammatory process (V. Cheeran and G.Munuswamy-Ramanujam 2020). Hence the ability of the compounds to scavenge the intracellular ROS serves as an indicator for identifying potential anti-in ammatory activity of the isolated compounds.
In vitro nitric oxide inhibitory activity of isolated compounds in AtT 20 cells Nitric oxide (NO) is an intracellular mediator produced by inducible Nitric oxide synthase (iNOS) in various mammalian cells. NO is known to be responsible for the vasodilation and hypotension observed in septic shock and in ammation. However, overproduction of NO could result in tissue damage and activation of proin ammatory mediators associated with acute and chronic in ammation (Dzoyem JP et al. 2016). In the present study, the NO scavenging capacity of the compounds was determined by decrease in the absorbance at 550 nm, as a result of reduction of NO production. The inhibitory activity of NO was demonstrated in all the compounds C1-C5 at their IC 50 concentrations in AtT 20 mouse pituitary cells.
The cells when treated with compounds C1-C5 along with LPS showed a signi cant (p < 0.01) decrease in the generation of NO when compared to the LPS induced cells (92% NO generation). The compounds C1-C5 showed 32%, 39%, 36%, 41% and 22% NO generation respectively (Fig. 10). Once again the best inhibitory activity against NO was seen in compound C5. These results were in line with our previous results supporting the antioxidant and anti-in ammatory activity of the isolated compounds. Hence, the anti-in ammatory activity of these compounds isolated from DE targeting NO inhibition might serve as potential candidates for the treatment of in ammatory diseases caused due to the overproduction of Nitric oxide.
In vitro lipid peroxidation activity of isolated compounds in AtT 20 mouse pituitary cells

Con icts Of Interest
The authors declare no con ict of interest.

Availability of data and material
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Sequence data is deposited in the public repository NCBI-GENBANK and accession numbers are included in the manuscript Phylogenetic consensus tree based on the Neighborhood joining (NJ) analysis inferred from the nearest neighbors of endophytic fungi, DE isolated from AI with maximum Bootstrap values indicated above each branch. Hypoxylon fragiforme was used as outgroup. Both isolates AI.EF 001 and AI.EF 002 formed a cluster within the clade