Two new bioactive polyketides from Curvularia trifolii, an endolichenic fungus isolated from Usnea sp., in Sri Lanka

Two new polyketides (compounds 1 and 2) were isolated from the EtOAc extract of an endolichenic fungus, Curvularia trifolii obtained from Usnea sp. in Sri Lanka. The structures of these compounds were elucidated on the basis of spectroscopic methods (UV, IR, MS, 1D NMR and 2D NMR). The bioactivity of the compounds 1 and 2 were studied using DPPH antioxidant assays and they showed radical scavenging activity with IC 50 values of 4.0 ± 2.6 and 1.3 ± 0.2 mg/mL, respectively. The radical scavenging activity of compound 2 was higher than that of compound 1 and it was comparable to BHT. Moreover, the new compounds 1 and 2 were evaluated for their anti-inflammatory activity, and only compound 2 exhibited a significant activity comparable to the standard antiinflammatory drug, aspirin. Compound 1 was evaluated for the inhibition of cell proliferation in a panel of five cancer cell lines NCI-H460, MCF-7, SF-268, PC-3M and MIA Pa Ca-2, and exhibited > 90 % inhibitory activity at 5 μg/mL with all of the above cell lines.


INTRODUCTION
Endolichenic fungi are considered as a new source of bioactive fungal metabolites (Paranagama et al., 2007). Only a few investigations have been reported on the isolation and characterisation of secondary metabolites of endolichenic fungi, but they have shown great potential to be a new source for structurally diverse and biologically active natural products (Paranagama et al., 2007;Ding et al., 2009;Zhang et al., 2009;Wang et al., 2010). The endolichenic fungi available in Sri Lanka are still an untapped source of bioactive natural products since their identity and the chemistry of their secondary metabolites have not been explored thoroughly. The first report on isolation and identification of endolichenic fungi from the lichens in Sri Lanka was published by a research team from the University of Kelaniya (Kannangara et al., 2009). New bioactive compounds from endolichenic fungi from Sri Lanka have been isolated and identified (Kannangara et al., 2009;Kulasekera et al., 2013;Pary et al., 2013;Samanthi et al., 2013a;2013b;2014;2015). In the ongoing study on isolation of bioactive secondary metabolites from endolichenic fungi, the fungal strain US/US/06 was found inhabiting the lichen, Usnea sp. collected from the Hakgala Botanical Garden, Central province, Sri Lanka in December 2010. This study was aimed at determining the bioactive compounds present in the ethyl acetate (EtOAc) extract of US/US/06 cultured on potato dextrose agar (PDA) and their potential for the production of bioactive secondary metabolites. This led to the isolation of two new polyketides (compounds 1 and 2). The details of isolation, structure elucidation, and bioactivity screening of compounds 1 and 2 are described here.

Isolation of the fungal strain
Usnea sp. was collected from the Hakgala Botanical Garden in Sri Lanka. Sterilised polythene bags were used to store the lichen samples for transport to the laboratory of the Department of Chemistry, University of Kelaniya. Surface sterilisation method was used to isolate the fungi from the lichen (Kannagara et al., September 2015 Journal of the National Science Foundation of Sri Lanka 43 (3) 2009). Samples of Usnea sp. were cleaned in tap water and surface sterilised by consecutive immersion for 10 s in 95 % ethanol, 3 min in 0.5 % sodium hypochloride and 30 s in 75 % ethanol. Sterile filter papers were used to dry the cleaned lichens, which were cut aseptically into small segments (1 mm × 1 mm). Twenty pieces of the lichen were placed on 2 % malt extract agar (MEA) supplemented with 0.01 % streptomycin. The plates were sealed with parafilm and incubated up to 14 d at room temperature under ambient light. Pure cultures were prepared using fungi growing from each lichen particle. The identification of fungi was initially carried out using the sticky tape method (Felgel, 1980) and identification keys (Barron, 1988). The pure culture of the fungus was photographed and deposited as a living voucher at the Department of Chemistry, University of Kelaniya, Sri Lanka under the accession number US/US/06.

Molecular identification of isolated endolichenic fungus
Molecular identification of the isolated pure strain of the endolichenic fungus was performed using a molecular biological protocol by genomic DNA extraction, amplification and sequencing as described by Samanthi et al. (2015). The extraction of fungal DNA was carried out using Promega Wizard ® Genomic DNA Purification Kit, and the fungal strain was identified by analysis of the nuclear internal transcribed spacer (ITS) region of the extracted DNA and selective amplification by polymerase chain reaction (PCR) using ITS 1 and ITS 4 primers (Arnold & Lutzoni, 2007). Before being subjected to sequencing, excess nucleotides and the remaining primers and enzymes in the PCR product were removed according to a gel purification protocol. Agarose gel electrophoresis was carried out to separate the desired amplified DNA bands from non specific bands.

Extraction and isolation
A culture of C. trifolii was grown in PDA for ten days. The secondary metabolites were extracted with EtOAc (6 × 500 mL) and the solvent was evaporated under reduced pressure. This afforded a dark brown solid (1.3 g), which was found to be active in antioxidant assay using the 2,2-diphenyl-1-picrylhydrdrazyl (DPPH) method. taken any anti-inflammatory drugs for 2 months prior to the experiment. The blood was transferred to heparinised centrifuge tubes and centrifuged at 3,000 rpm followed by washing three times with an equal volume of normal saline and reconstituted as 10 % v/v suspension with normal saline. The reaction mixture (5.5 mL) consisted of 5 mL of the test solution and 0.5 mL of 10 % RBCs suspension. Saline was also added to the control test tube instead of the sample. Aspirin was taken as the standard drug. All the centrifuge tubes containing the reaction mixtures were incubated in a water bath at 56 o C for 30 min and cooled under running tap water. The reaction mixtures were centrifuged at 3000 rpm for 10 min and the absorbances of the supernatants were taken at 560 nm. The test was performed in triplicate. Percentage membrane stabilisation activity was calculated (Sakat et al., 2010) and plotted against test concentrations to determine the IC 50 .

Cytotoxicity assay
The in vitro assay of cytotoxicity to human nonsmall cell lung cancer (NCI-H460), CNS glioma (SF-268), breast cancer (MCF-7), human metastatic breast adenocarcinoma (MDAMB-231), prostate adenocarcinoma (PC-3), metastatic prostate adenocarcinoma (PC-3M) and pancreatic cancer (MIAPaCa-2), and normal human primary fibroblast cells (WI-38) was carried out using the resazurin-based colourimetric (alamarBlue) assay (Wang et al., 2011). The cancer cells were all cultured under standard culture conditions and the test compound or vehicle control (DMSO) was added to appropriate wells, and the cells were incubated for 72 h. Then 20 μL/well of alamarBlue solution was added into the assay plates for a final assay volume of 200 μL/well, yielding a final concentration of 10 % alamarBlue. After they were shaken for 10 s, the plates were returned to the incubator and kept for 4 h. The plates were then exposed to an excitation wavelength of 560 nm, and the fluorescence emitted at 590 nm was read. The percentage viability was expressed as fluorescence counts in the presence of the test compound as a percentage of that in the vehicle control. Doxorubicin and DMSO were used as positive and negative controls, respectively. 1 H-and 13 C-NMR spectra were recorded on Bruker Avance III 400 instrument using CDCl 3 as a solvent. Shimadzu LCMS-QP8000α was used for MS analysis. Analytical and preparative thin layer chromatography (TLC) were performed on precoated 0.25 mm thick plates of silica gel 60 F254 sprayed with a solution of anisaldehyde in EtOH, followed by heating to visualise the compounds on analytical TLC. The melting point of compound 1 was determined using a melting point apparatus (MEL-TEMP ® , USA).

RESULTS AND DISCUSSION
The fungus, US/US/06 was identified based on the morphological and molecular characteristics. The ITS sequence matched with C. trifolii sequences from Gene-Bank with a sequence identity of 99 %. This endolichenic fungus was identified as Curvularia trifolii. The 1 H NMR spectrum of compound 1 indicated the occurrence of one 3H singlet due to CH 3 groups (δ H 1.2) and three 1H double doublets at δ H 7.33, 5.80 and 5.22 due to three olefinic protons attached to C2, C3, C11, and the olefinic C attached to C12 showed a multiplet as it was coupled with the two protons attached to C13 at δ C 130.2. DEPT spectrum of compound 1 indicated the occurrence of five CH 2 (δ C 34, 31.8, 26.7, 41.0 and 42.9) groups accounting for 17 protons in compound 1 ( Table 1) (Table 3). However compound 1 did not show any significant activity against the antioxidant and anti-inflammatory assays (Figure 3). Compound 2 obtained as a fluorescent active semi -solid when analysed for C 23 H 30 O 6 by a combination of FABMS and NMR data indicated 9 degrees of unsaturation with an isocoumarin ring. The UV λ max at 238 nm indicated the presence of an α,β-unsaturated lactone carbonyl chromophore (Schubert & Sweeney, 1955). Its IR spectrum had absorption bands at 3438, 1706 and 1641 cm −1 , suggesting the presence of a OH group, carboxylic acid carbonyl, and α,β-unsaturated lactone functionalities.
The 1 H NMR spectrum of compound 2 ( Table 2) indicated the occurrence of four 3H singlets, of which three were due to CH 3 groups attached to carbons 4, 8 and 21 (δ H 1.1, 1.4 and 2.1) and the fourth due to a OCH 3 group (δ H 3.8), and one 1H singlet at δ H 6.5 due to an aromatic proton. DEPT spectrum of compound 2 indicated the occurrence of seven CH 2 groups accounting for 27 out of 30 protons. The remaining three protons were suspected to be that of the two CH and COOH groups.    (2) its 1 H− 1 H COSY spectrum ( Figure 4). The connectivity of the above two spin systems, tertiary methyls, and nonprotonated carbons was established by the analysis of the HMBC correlations ( Figure 2) to constitute a structure with a twelve membered ring connected to a dihydroisocoumarin ring skeleton.