Phylogenetic Diversity and Antioxidant Activity of Selected Fungi From Ethno Medicinal Plants & Soil

Endophytic fungi are prolic generators of bioactive metabolites with natural antioxidant properties that have a range of potential uses. In the present work, the antioxidant activity and phylogenetic diversity of endophytic fungi obtained from semi-arid terrestrial plants and the fungi obtained from the mangrove soil samples is investigated. The fungal isolates were identied by employing morphological features and phylogenetic characterization by internal transcribed spacer (ITS) sequences. Six taxonomic orders of Ascomycota associated with nine genera were identied, these being Diaporthales, Eurotiales, Hypocreales, Onygenales, Pleosporales and Xylariales. The antioxidant activity methanolic extracts of the fungal isolates were determined using modied 2,2’-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) methods. Among the fungal isolates, Aspergillus sp. AREF023 and Fusarium sp. AREF014, obtained from different parts of Datura metel, displayed the highest level of radical inhibition (80.76% and 70.62% respectively). GC/-MS analysis of the active isolates conrmed the presence of known antioxidant phenolic compounds, including 2-tert-Butyl-5-hydroxymethyl-5-methyl-[1,3]dioxolan-4-one and Phenol, 2,5-bis(1,1-dimethylethyl), in the their crude extracts. Our ndings suggest that fungal isolates from D. metel can be a sustainable resource for natural antioxidants. two sub-clades, the two endophyte sequence Fusarium falciforme, Fusarium solani, Monascus kaoliang, Monascus purpureus, obtained from NCBI (NCBI (ID: KY318489, MF401578, KP132214, AB477250, AB477247, AB477252), with 98% bootstrapping. The second sub clade consisted of three endophyte grouped with nine sequences of Curvularia sp., Curvularia lunata, Alternaria spp., Alternaria alternata, Alternaria brassicae obtained from NCBI (ID: MN215713, NR_138223, HF934911, MG228423, MG228422, MN615420, MF356599, MG722823, MF356599). The last clade was the smallest with one endophyte sequence clustered with two sequences of Diaporthe detrusa and Dipothe sp. picked with NCBI (ID: KC343061, MH267914) with 71% bootstrapping, however, one sequence of Diaporthe eres, NCBI ID: MH003633, fall apart with 54% bootstrap value.


Introduction
Fungal endophytes are a group of diverse lamentous fungi belonging to ascomyta that grows within plant tissues without causing any immediate harmful effects (Hyde and Soytong 2008;Debbab et al. 2013). These endophytic fungi are proli c producers of secondary metabolites and can serve as a valuable reservoir of lead molecules in the hunt for drug candidates against infectious diseases, cancer and many other disorders (Rana et al. 2020;Mishra et al. 2021). Among various important leads obtained from endophytic fungi, cholesterol lowering drugs (statins), immunosuppressant drugs (cyclosporins), anti -cancer drug taxol and penicillin are well known for their pharmacological actions (El-Bondkly et al. 2020;Sharmila and Kalaiselvam 2014;El-Sayed et al. 2020). In addition, phomoterpene A, phomoisocoumarin D isolated from Diaporthe prunorum, an endophytic fungi isolated from Hypericum ascyron, has potential antimicrobial activity (Qu et al. 2020), while Curvularia sp. G6-32, an endophytic isolate of medicinal plant Sapindus saponaria, produces Asperpentyn, an epoxyquinone compound, which is a potential antioxidant (Polli et al. 2020).
Indian has a large biodiversity and diverse ecosystems of the landforms that are an excellent source of bioactive secondary metabolites. The present study screened endohpytic fungi isolated from medicinal plants and soil from two diverse regions for antioxidant activity. Firstly, the semi-arid region characterized by the intermediate climatic conditions between the desert and humid regions. Semi-arid land forms are comprised of succulent, non-succulent and xerophytic perennials . Secondly, mangroves sediments which are complex as compared to terrestrial ecosystems in terms of a characteristic marine biome. The vegetation is well equipped to survive saline stress (Guillén-Navarro et al. 2015). There is no up to date published study that describes the phylogenetic diversity of endophytic fungi and soil fungi obtained from these two sources. In the current study we have identify, establish the genetic relationship, and screened the antioxidant activity of endophytic fungi isolated from some semi-arid terrestrial plants and the fungi obtained from the soil of the mangrove region of a marine area. These endophytic and soil fungal isolates were found to be signi cantly active by inhibiting the DPPH, ABTS radicals and showed active spots on the DPPH bio -autography assay. These bioactive endophytic and soil fungi isolates were further subjected to molecular characterization and were their chemical constituents investigated using GC-MS.

Materials And Methods
Sampling sites and collection of samples -

Terrestrial plants:
Healthy terrestrial plant parts such as stem, leaves and roots were collected from Aravalli Bio-diversity Park (28°28'59.9"N 77°06'34.7"E) Gurugram, Haryana, India. The identi cation and validation of plant species were done as previously described (Gamble, J.S. 1925), and samples were preserved in our laboratory. Healthy plant parts were collected by sampling from different parts of the plant. Plant parts such as leaf, stem and roots were randomly cut with an ethanol-disinfected sickle and placed independently in sterile polythene bags. Endophytic fungi were isolated within 48 hours of collecting the samples.

Soil samples:
The mangrove soil samples were collected randomly in July 2018 from the marine area of Alibaugh (18°39' 23.9544''N, 72°52'47.5248''E) Maharashtra, India. 09 soil samples each with 1 km distance from the sea bed were randomly collected and the soil from the surface to a depth of 5-10 cm was collected in sterile zip a lock bag which is placed in an ice box and taken to the laboratory. The samples were then stored in the refrigerator (4 °C) for fungal isolation.
Isolation of fungal samples -1. Endophytic fungi: Isolation of the fungal isolates was done using a previous method of Qadri et al with some modi cations (Qadri et al. 2013). Brie y, mature and excided small Bacopa monnieri, Cymbopogon citratus, Datura metel and Ocimum tenui orum plant parts (leaves, stems, roots) were used for isolating the fungal endophytes. The samples were washed for 1 min with 70% ethanol (v/v) and disinfected for 2 min using 2% sodium hypochlorite solution (v/v) followed by 20s of brief rinsing with 70% (v/v) ethanol. Later they were rinsed with sterile distilled water. These plant parts were placed on potato dextrose agar (PDA) plate amended with 50µg/ml chloramphenicol. PDA plates containing three pieces of plant material were incubated for 7-10 days at 28 °C. Plating was done in triplicates.

Fungi from soil:
Endophytic fungi were isolated on water agar (WA) by employing soil plate method, incubation was done at 28°C for 3-5 days, and checked for fungal colonies appearing in culture, then a single colony was transferred on PDA plate and further incubated for growing pure culture. The cultures in petri dishes were incubated at 28°C for 7 -10 days for observation and further identi cation was done on the basis of morphological characteristics. All the isolates were deposited in the TERI Deakin Nano-biotechnology culture collection.
Growth kinetic analysis of potential bioactive fungal isolate Ergosterol estimation and analysis methods were adapted from Axelsson et al. (Axelsson et al. 1995). 50 mg of dried fungal mycelium (collected regularly at 24 hr interval) was mixed with 1 ml ethanol. Sterol was extracted and pooled twice (30 s agitation, 1 hr incubation and centrifugation at 14,000 rpm for 5 mins), The pooled and ethanol partitioned sterol was ltered using 0.22 mm nitrocellulose lter and 10 µL injection volume was injected into a high performance liquid chromatography (Shimadzu, Japan). A ve point calibration curve was plotted with the standard in concentration range (10-50 ppm) in ethanol employing an absorption detection at 282 nm (Total run time was 20 min).
Variation of ergosterol content was compared by referring to the standard graph (R 2 -0.97).

Preparation of methanolic extract:
Methanolic extracts of all the fungal isolates were prepared using a Diaion HP20 column (Merck). Three fungal plugs (8-9 mm in diameter) from 10 day old culture (in PDB medium) were inoculated in 1000 ml Erlenmeyer asks containing 250 ml of the medium. The asks were incubated at 28º C for 8 -10 days at 150 rpm. For preparation of the extract, methanol was added slowly in the grown culture (1:1) (to avoid over heating), for metabolite extraction and kept at shaking for 6 hrs. The media was then ltered thorough sterile muslin cloth to separate mycelia. The ltrate obtained was then concentrated to 1/5 volume using rotary evaporator and diluted to 100 mL with distilled water. The diluted solution was then passed through Dianion HP20 column and was eluted using 70% methanol. The obtained elute was concentrated in a peer shape asks, diluted with distilled water and lyophilized to obtain a powdered extract.
Thin layer chromatography (TLC) bio -autography: Each fungal extract was spotted and eluted with CHCl 3 : MeOH (85: 15 v/v) on silica 60 F 254 TLC plate (Merck), followed by spraying with 0.2 % DPPH solution in methanol and incubated for 30 minutes in dark. Active compounds appeared as yellow spots against a purple background.
Total antioxidant capacity assay: DPPH assay -DPPH radical scavenging assay was conducted according to the previous method of Blois (Blois 1958) with modi cations. DPPH (Sigma) was dissolved in 100 mL of MeOH. The investigated samples were prepared by dissolving 1mL of 0.2 mM 1,1-diphenyl -2-picrylhydrazyl (DPPH) in methanol and 0.5mL of test sample at different concentrations (5 µg/mL to 100 µg/mL) at room temperature. The absorbance at 517 nm was measured after 30 min versus the blank (0.5 mL of methanol instead of test sample in 1mL DPPH solution). Positive controls ascorbic acid, tert-Butylhydroquinone (TBHQ) and Trolox were also subjected to the same procedure for comparison. The analysis was carried out in triplicate, and the percentage (%) of inhibition was calculated by the following formula: % inhibition = absorbance of control -absorbance of sample x 100 / absorbance of control The DPPH radical scavenging activity of each sample was expressed as Trolox equivalent antioxidant capacity (TEAC) (Shimamura et al. 2007). TEAC was calculated as follows: TEAC = IC 50 of Trolox (µg ml -1 )/ IC 50 of sample (µg ml -1 ) ABTS assay - The ABTS radical scavenging assay of the fungal crude extracts performed in accordance with method reported by Re et al ).
Brie y, 2.45 mM potassium persulfate and 7 mM ABTS aqueous solution was mixed to generate ABTS + radical. The initial absorbance mixture solution was adjusted to 0.70 ± 0.02 at 745 nm. Different sample concentrations (5 µg/mL to 100 µg/mL) and standard Trolox (1 µM to 50 µM) with ABTS + working solution was adjusted to a nal volume of 1 mL and reacted at room temperature (25º C) for 6 min. The decrease in absorbance at 745nm was recorded and calculated as half maximal inhibitory concentration (IC 50 ) for test samples and trolox was calculated by plotting the scavenging capacity against the concentration. Trolox standard solution ( nal concentration 0 -50 µM) was used. Results were expressed in terms of TEAC (Shimamura et al. 2007). TEAC was calculated as follows: TEAC = IC 50 of Trolox (µg ml -1 )/ IC 50 of sample (µg ml -1 ) Molecular identi cation of the fungal isolates:

Genomic DNA Extraction
The pure culture obtained after isolation of the fungal isolates were grown in 250 ml Erlenmeyer asks containing 50 ml of the medium, incubated at 28º C for 8 -10 days at 150 rpm . The fungal mycelia obtained were freeze-dried and the genomic DNA was extracted by the CTAB (Cetyl trimethylammonium bromide) method (Ausubel et al. 1999).
PCR ampli cation was performed according to the methods of Reajeendran et al (Rajeendran et al., 2017). The amplicons obtained were gel puri ed by amicon ultra columns (Millipore, USA) and 20-40 ng were used for sequencing at Euro ns Laboratory Pvt, Ltd (Bengaluru, India).
Finch TV software (http://www.geospiza.com/Products/ nchtv.shtml) was used for assembling sequences and homology was determined using BLASTn against the NCBI Gen Bank database (Altschul et al. 1997). A phylogenetic tree was constructed using the sequences displaying maximum homology. CLUSTAL W was employed for multiple sequence alignment and MEGA X was used for phylogenetic and molecular evolutionary analyses . Following the Tamura -Nei model and Maximum Likelihood algorithm phylogenetic reconstruction was done with bootstrap values calculated from 1000 replicate runs (Tamura and Nei 1993).

GC/MS analysis
The volatiles and major chemical components of the fungal extracts were analysed using Agilent 6890 GC-MS coupled to Agilent 5873 (EI mode, 70 eV). The GC conditions were: 1 min split less time, helium carrier at 1.0 ml/min, oven temperature from 70 o C to 135 o C at 2 o C/min, for 10 min; then to 220 o C at 4 o C/min, 10 min; and nally to 270 o C at 3.5 o C /min, for 20 min. For analysis HP-5MS capillary column (0.32 mm x 30 m x 0.25 µm), GC injector with temperature set at 280 o C and MS transfer line temperature at 290 o C was used. The detected compounds were compared with the mass spectra from the NIST library for their identi cation (Proestos et al. 2006).

Isolation of endophytic fungi:
In total 43 fungal isolates were obtained from different parts of the plant and soil. The sources were given lab codes and a portion was used for isolation. Ethno-medicinal values of the sources (wherever applicable) of 17 antioxidant related endophytic fungi (AREF) isolates (10 cultivable endophytic fungi and 07 soil fungi) obtained after initial screening of the pure isolates are illustrated in Table S1. Subsequently, these sub-cultured pure isolates were further used for preparation of extracts for bioactivity guided screening. Ergosterol content was estimated using standardised control. The inoculated medium displayed signi cant growth and continual increase in ergosterol content from the 7 th to 10 th day. However, growth saturation was observed after the 10 th day. The growth kinetics of the fungal isolate and standard graph are shown in Figure S1.
Screening of isolated endophytic fungi for antioxidant activity: Methanol extracts of the cultivable fungal isolates were screened for antioxidant potential using two assays. Extract antioxidant activity was tested at a concentration of 1000 µg/ml. TLC bio-autography analysis -Among the various solvent elution tested, chloroform/methanol/acetic acid (8.5:1.5 by volume) gave the best separation of compounds from the 70% methanol extracts with retention factor values (R f ) ranging from 0.15 to 0.87. The existence of strong adsorbing sites, as depicted on TLC plate ( Figure S2), con rm the suitability of this solvent system for separation and mobility of the fungal extracts. The baseline of all the methanolic fractions indicated a range of deep to light yellow colour, indicating the presence of antioxidant constituents in varying amounts. The spots of R f 0.87 -0.71 displayed bright yellow spot on the TLC plate con rming the presence of antioxidant compounds in these extracts. Radical scavenging activity of methanol extracts from all fungal isolates against DPPH, ABTS + radical are shown in Table 1. The IC 50 values were obtained in the range of 33 µg/ml -616 µg/ml for DPPH assay and 14 µg/ml -167µg/ml for ABTS assay. The lower the IC 50 and the higher the TEAC value, the greater is the antioxidant activity.
Amongst all the 17 fungal crude extracts, isolate AERF023 identi ed as Aspergillus sp. obtained from the internal tissues of roots of D. metel, exhibited promising DPPH scavenging activity (IC 50 33.49 µg/ml and TEAC of 0.3) and also against ABTS (IC 50 14.33 µg/ml). The highest DPPH scavenging activity was from AERF023, which exhibited 80.76% of DPPH radical inhibition. The result of ABTS + scavenging activity was also maximum from AERF023, which exhibited 75.18 % of ABTS + radical inhibition. The second most potent isolate was AREF014 which showed moderate antioxidant activity (IC 50 35.39 µg/ml against DPPH and TEAC of 0.28; and IC 50 33.50 µg/ml against ABTS + ).

Phylogenetic diversity of cultivable fungi
Out of the 17 fungal isolates, the 11 isolates (Figure 1) exhibiting the highest antioxidant activities were subjected to sequencing for their molecular characterization. A further categorization of these isolates were carried out into 9 genera, namely Alternaria, Curvularia, Fusarium, Monascus, Diaporthe, Talaromyces, Aspergillus, Auxarthron and Xylaria which belongs to six orders Pleosporales, Hypocreales, Diaporthales, Eurotiales, Onygenales and Xylariales, found in phylum Ascomycota. Among isolated endophytic fungi Alternaria and Aspergillus were the predominant genus, with relative frequency of 18.18%, while the remaining genera Auxarthron, Fusarium, Monascus, Talaromyces, Xylaria, Curvularia, Phomopsis were found with relative frequency of 9.99%.

Detection of bioactive metabolites of the methanol extracts by GC-MS analysis:
Considering the highest antioxidant activity obtained from the methanol extracts of Aspergillus sp. AREF023 and Fusarium sp. AREF014, a tentative identi cation of the metabolites were carried out using GC-MS. Twenty metabolites were found in the extract of AREF023 and seven were identi ed in the extract of AREF014. The identi ed myco-constituents belonged to diverse classes of alkenes, di-ethers, esters, fatty acids, phenols, polysaccharides, organic acids and hydrocarbons. Chemical diversity was observed in the constituents of both the extracts. A total of 20 compounds constituting 96.3% of the relative area in the methanol extract and 95% similarity with the standard mass spectra were revealed from the GC/-MS analysis of the AREF023 extract (Figure 3a) (Table 4a). The methanol extract of AREF023 indicated the presence of phenolics, including 2-tert-Butyl-5-hydroxymethyl-5-methyl-[1,3]dioxolan-4-one (8.97%); a common antioxidant molecule which is used in a range of applications. Similarly, analysis of AREF014 showed the presence of 7 compounds, having resemblance almost 97% with the standard masses in the library and representing 100% of the relative area in the extract (Figure 3b) (Table 4b). The extract clearly showed the presence of the phenolic compound, Phenol, 2, 5-bis (1, 1-dimethylethyl) (36.45 %), which is widely used as a dietary antioxidant compound.

Discussion
Endophytic fungi are a novel source of natural bio actives which can sustainably help to overcome the negative impact of diminishing plant diversity on the discovery of bioactive natural products. Moreover, plants with the history of use as traditional medicine can become excellent scaffolds for growing and isolating endophytic fungi that can produce similar bioactive metabolites to those of the host plant (Strobel 2002). With this rationale, the ethno-medicinal plants, D. metel, B. monnirei, C. citratus, O. tenui orum and soil sediments from marine ecosystem were selected for the isolation of endophytic fungi. The methanolic extracts of these isolated fungi were subjected to two antioxidant assays.
Varying activities between these isolates is due to varying metabolite content, as indicated by Scavenging actvity being directly proportional to the extract's concentration. This is the rst report of the antioxidant activities of endophytic fungi identi ed as Aspergillus sp.AREF023 obtained from D. metel.
Selected for their higher antioxidant capacity, 11 fungal isolates (08 endophytic and 03 soil fungi) representing the phylum Ascomycota, were identi ed. It is the largest phylum of fungi with over 64,000 species reported (Crozier et al. 2006;He et al. 2012;Koukol et al. 2012). Alternaria, Aspergillus and Fusarium sp. are the common endophytes reported from medicinal plants (El-Said et al. 2020;Kamel et al. 2020). The phylogenetic tree developed in this study represents the relations amongst the isolated fungal samples (Figure 2) (Table 3). Characterisation data from this study have been submitted in the NCBI GenBank, accession codes MT013399 to MT013409 (Table 2).

Conclusion
Discovering and developing new antioxidant compounds of natural origin that can be used safely in pharmaceutical as well as food industry is an important area of research. The unique phenomenon of cross talk existing between the colonizing endophytic fungi and the plant host results in the production of related and often the same bioactives as originally produced by the host plant. This symbiosis is harnessed as a useful reservoir of lead compounds for multiple applications. The most signi cant advantage is the relative ease of mass cultivation of endophytic fungi that can potentially replicate valuable metabolites of their hosts, without diminishing the plant biodiversity in natural ecosystems.
The results in this study have established that the endophytic fungal isolates, Aspergillus sp. AREF023 and Fusarium sp. AREF014, contain metabolites rich in phenolics and avonoids which are the major contributors to antioxidant activities in extracts from these strains. The extract with the highest phenolic content displayed the highest antioxidant capacity. The endophytic fungi Aspergillus terreus and Fusarium solani are excellent sources of naturally derived antioxidants. However, the toxicity of these fungal extracts having antioxidant activity needs further investigation, particularly in regard to their applicability as neutraceutics and pharmaceutics. In addition, the mechanisms of antioxidant activity of these extracts required investigation in order to gain more insight into their scavenging behaviour in food and biological systems.