A secondary metabolite with in vitro radical scavenging activity from endolichenic fungus Daldinia eschscholzii found in lichen, Parmotrema sp. in Sri Lanka

: Endolichenic fungi, an unexplored group of microorganisms, are a promising source of bioactive compounds. Secondary metabolites were isolated from the chloroform fraction of crude ethyl acetate extract of endolichenic fungus Daldinia eschscholzii inhabiting the lichen, Parmotrema sp. in Sri Lanka. Two pure compounds, 1 and 2 were isolated and the structures were identiﬁ ed using 1 H-, 13 C-, 2D- nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) data. Compound 1 did not show any radical scavenging activity in DPPH assay. Compound 2, identiﬁ ed as 8-methoxynaphthalen-1-ol showed strong radical scavenging ability in the DPPH assay with a half maximal inhibitory concentration (IC 50 ) of 10.2 ± 5.8 µg/ mL. The activity of compound 2 was higher than that of the standard, butylated hydroxy toluene (BHT).


INTRODUCTION
Natural products with biological activities are promising leads for novel therapeutic agents (Koehn & Carter, 2005). Isolation and characterisation of bioactive natural compounds as pharmocophores have been the focus of ongoing research. Fungi are well known producers of naturally occurring bioactive compounds (Samanthi et al., 2014). Endolichenic fungi (ELF), analogous to plant endophytes (Wang et al., 2010) are one of the ecological groups of fungi that occur asymptomatically within the lichen thalli (Kannangara et al., 2009). Although they have shown a great potential to be a rich source of structurally diverse bioactive compounds, there are only a few studies reported (Kulasekera et al., 2013;Pary et al., 2013;Samanthi et al., 2013;2015a;2015b) on isolation and identifi cation of secondary metabolites from ELF (Paranagama et al., 2007). ELF in Sri Lanka still remains almost untapped as a source of bioactive compounds (Samanthi et al., 2015a).
The objective of this study was to isolate secondary metabolites with radical scavenging activity from the ELF Daldinia eschscholzii inhabiting the lichen Parmotrema sp. in the Hakgala Botanical Garden, Sri Lanka and screen them for biological activity. Two major compounds were isolated. The isolation procedures and the results of bioactivity screening are reported here.
METHODOLOGY 1 H-, 13 C-and 2D-NMR spectra for the isolated compounds were recorded in CDCl 3 with Bruker Ascend TM 400 Spectrometer at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. LCMS data were recorded on June 2020 Journal of the National Science Foundation of Sri Lanka 48 (2) Waters e 2695 separations module using 2998 PDA detector and Zspray TM SQ mass detector 2. Thin layer chromatography (TLC) (analytical and preparative) was performed on 0.2 mm pre-coated silica (60 Å pore size, Fluka, Germany) plates with fl uorescent indicator 254 nm.

Isolation of the endolichenic fungal species
The lichen Parmotrema sp., was collected from Hakgala Botanical Garden located at an altitude of about 1745 m. Samples were transported in sterile polythene bags to the laboratory at the Department of Chemistry, University of Kelaniya and were processed within 24 h.
Surface sterilisation method (Kannangara et al., 2009) was used to isolate fungal species from the lichen thalli. Healthy lichen thalli cleaned up with tap water were surface-sterilised by sequential immersion in 95 % ethanol, 0.5 % sodium hypochloride and 75 % ethanol for 10 s, 3 min and 30 s, respectively. The surface of the thalli was dried with sterile fi lter papers. Sterilised lichen thalli were aseptically cut into small pieces of 1×1 cm and plated on 2 % malt extract agar containing 0.01 % streptomycin (fi ve replicate thalli samples of the selected lichen species, 20 small segments from each). Plates were incubated at room temperature (28 ± 2 0 C) for 14 d. Pure cultures of diff erent types of fungi growing from each lichen segment were isolated by sub-culturing from actively growing edge.
The growing fungi sub-cultured on 2 % MEA were photographed and vegetative mycelia of each fungus in sterile water were deposited at room temperature in the Department of Chemistry, University of Kelaniya.

Screening of endolichenic fungi for antioxidant activity
A small-scale analysis was carried out for the crude ethyl acetate extracts of 6 endolichenic fungi sp. selected based on the absence of previous studies for isolation and identifi cation of antioxidant active compounds. Each fungus was grown on 5 PDA plates (150 × 25 mm) and secondary metabolites were extracted after 1 week of incubation at room temperature (28 ± 2 0 C). The crude ethyl acetate extracts of the selected endolichenic fungi were evaluated for their radical scavenging activity using DPPH assay to identify promising candidates for further studies.

Extraction and isolation of bioactive compounds from selected fungus
Daldinia eschcholzii, which showed a higher in vitro radical scavenging activity in DPPH assay during smallscale analysis was cultivated on 48 PDA plates (150 × 25 mm) and incubated at room temperature (28 ± 2 0 C). After one week of incubation, mycelia were cut into small pieces along with the PDA medium and secondary metabolites were extracted into ethyl acetate. The extract was dried with anhydrous Na 2 SO 4 and the solvent was evaporated under reduced pressure. The dark brown semisolid crude extract (1.5 g) was subjected to antioxidant assay using DPPH method. The extract was found to be an active radical scavenger. A portion (1.4 g) of the bioactive crude extract was dissolved in 100mL of aqueous 80 % methanol and extracted with hexane (2 × 100 mL). The methanol fraction was diluted with distilled water to a 60 % aqueous methanolic solution and extracted with chloroform (2 × 100 mL). The hexane, chloroform and aqueous methanol fractions were separately dried with anhydrous Na 2 SO 4 and solvents were evaporated under reduced pressure to yield 222 mg, 660 mg and 37 mg of the fractions, respectively. The insoluble sticky material from the crude extract that could be particles from fungal cultures was removed during partitioning. All three fractions were subjected to DPPH assay. The chloroform fraction which had the highest antioxidant activity was then subjected to bioassay guided fractionation using silica gel column chromatography.
Silica gel column (70 -230 mesh, 19.2 g, 2.5 cm × 53 cm) was packed with dichloro methane (CH 2 Cl 2 ). A slurry of sample (510 mg) was packed on top of the column and was eluted with 100 % CH 2 Cl 2 followed by CH 2 Cl 2 containing increasing amounts of methanol and fi nally with 100 % methanol. Fractions were analysed by TLC (developed in 4% methanol in CH 2 Cl 2 and observed for UV absorption at 254 nm). The solvent was evaporated and eight fractions, F1 to F8 were obtained. Fraction F2 (25 mg) which had radical scavenging activity was then subjected to gel-fi ltration column chromatography on Sephadex LH-20 made up in hexane (1.0 g, 1 cm × 30 cm). The sample was eluted with 100% hexane, hexane containing increasing amounts of CH 2 Cl 2 , 100 % CH 2 Cl 2 , followed by CH 2 Cl 2 containing increasing amounts of methanol and fi nally with 100 % methanol. Column fractions were combined after running the TLC (developed in 50% hexane in CH 2 Cl 2 and observing under UV absorption at 254) to obtain three combined Journal of the National Science Foundation of Sri Lanka 48 (2) June 2020 fractions F2-1, F2-2 and F2-3. Fraction F2-1 (16 mg), which showed the presence of a compound with radical scavenging ability upon qualitative analysis by spraying DPPH on TLC plate, was further purifi ed using normal phase preparative thin layer chromatography (PTLC) (40 % CH 2 Cl 2 in hexane) to isolate pure compound 1 (6 mg) and the fraction F2-1-2 (5 mg) with slight impurity. The fraction F2-1-2 was subjected to PTLC (30 % CH 2 Cl 2 in hexane) to remove the impurity and isolate pure compound 2 (4 mg).
Structure elucidation of the isolated pure compounds was done with the aid of 1D-, 2D-NMR and mass spectroscopic data.
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability Assay was carried out in a 96-well plate according to the method described by Chatatikun and Chiabchalard (2013) with slight modifi cations. Butylated hydroxy toluene (BHT) and methanol were used as the positive and negative controls, respectively. A volume of 160 µL of each dilution of test sample was mixed with 0.25 mM methanolic DPPH solution (40 µL). Absorbance measurements were taken at 517 nm with the micro plate reader (Thermo Scientifi c Multiskan GO, Finland) after 20 min of incubation at room temperature in the dark. The percentage inhibition was calculated and plotted against the concentration to determine the IC 50 .

Morphological and molecular identifi cation of the endolichenic fungus
Identifi cation keys (Barron, 1968) were used in morphological identifi cation of pure fungus culture prepared on slides using sticky tape method (Felgel, 1980). Genomic DNA extraction and amplifi cation followed by sequencing was used in molecular identifi cation of the pure strain of endolichenic fungus used for the study (Samanthi et al., 2015a). Fungal DNA extraction was carried out using Promega Wizard ® Genomic DNA Purifi cation Kit. Ribosomal internal transcribed spacer (ITS) region analysis of extracted DNA, and selective amplifi cation by polymerase chain reaction (PCR) with the use of ITS 1 and 4 primers (Arnold & Lutzoni, 2007) were used to identify the fungus. The excess nucleotides, remaining primers and enzymes etc., in the PCR product (500-600 bp) were removed using a gel purifi cation protocol before sequencing. First, the amplifi ed DNA bands that are of interest were separated from undesired bands using agarose gel electrophoresis. Promega Wizard ® SV Gel and PCR Clean-Up System were used (Promega, 2002) to isolate DNA from the gel. Nucleotide sequence of the amplifi ed, purifi ed DNA was submitted to GenBank as a nucleotide query. The nucleotide sequence was compared with existing nucleotide sequences using NCBI BLAST ® (https://www.ncbi.nlm.nih.gov/nuccore/ MN862623) to identify the fungal strain.

RESULTS AND DISCUSSION
Out of a total of 11 diff erent fungi species isolated and identifi ed from the lichen Parmotrema sp. based on the morphological and molecular studies, the fungus BM/ F7, identifi ed as D. eschscholzii revealed a higher in vitro radical scavenging activity in small-scale analysis and was selected for further analysis. The lichen identifi cation was done as described in Kannangara et al. (2009) and this paper presents results of further study. The fungus has a white colour hyphae that grows from  The in vitro radical scavenging activity of the crude extract of D. eschscholzii was determined using DPPH assay and the activity was compared with that of the standard antioxidant, BHT. The standard showed higher activity (~ 67 % inhibition at 50 µg/ mL) than the crude extract (~ 36 % inhibition at 50 µg/ mL). Figure 3 shows the DPPH radical scavenging activity for hexane, chloroform and methanol fractions separated from ethyl acetate extract of D. eschscholzii and pure compound 2.
According to results, the chloroform fraction showed the highest activity (IC 50 = 63.8 ± 4.8 µg/ mL) out of all three fractions. However, activity of chloroform fraction was higher than that of ethyl acetate extract (IC 50 = 77.9 ± 5.1 µg/ mL) and less than that of BHT (IC 50 = 38.2 ± 4.0 µg/ mL). The hexane fraction showed the lowest activity.
Bioassay guided fractionation of the chloroform fraction using column chromatography and PTLC yielded two pure compounds; 1 and 2 and only compound 2 showed radical scavenging activity.
Molecular formula for compound 2 was determined as C 11 H 10 O 2 with six degrees of unsaturation on the basis of pseudomolecular ion at m/z 175.05 (calculated for C 11 H 11 O 2 ). NMR data are given in Table 2. The compound ( Figure 2) is a slightly yellow coloured solid.
The 13 C NMR along with DEPT 90, DEPT 135 and HMQC data indicated the presence of ten aromatic/ olefi nic carbons of which six were protonated (δ c 103.8, 110.4, 118.9, 121.9, 125.6 and 127.7), two were oxygenated (δ c 156.1, δ c 154.5) and two were quaternary carbons (δ c 115.1 and 136.7). In addition 13 C NMR data also revealed the presence of one methoxy carbon (δ c 56.1).
The pure compound 2 showed strong radical scavenging activity with an IC 50 value of 10.2 ± 5.8 µg/ mL, compared to BHT (Figure 3).

CONCLUSION
In this study, two major compounds were isolated from the ethyl acetate extract of D. eschscholzii using bioassay guided fractionation. The structures of compounds 1 and 2 were identifi ed using spectroscopic data, of which only one compound (8-methoxynaphthalen-1-ol) showed high radical scavenging activity in DPPH assay. The activity was even higher than that of the standard BHT.