Two new antioxidant active polyketides from Penicillium citrinum , an endolichenic fungus isolated from Parmotrema species in Sri Lanka

Bioassay guided fractionation of the ethyl acetate extract of a laboratory culture of Penicillium citrinum, an endolichenic fungal strain isolated from a Parmotrema species in Sri Lanka, led to the isolation of two new fungal polyketides (1 and 2). The structures of these compounds were determined on the basis of their 1D NMR, 2D NMR and FABMS spectroscopic data. The two compounds (1 and 2) showed radical scavenging activity with IC50 values of 159.7 ± 22.3 μg/mL and 68.6 ± 4.3 μg/mL, respectively in DPPH antioxidant assay. The antioxidant activity of compound 2 was comparable to the standard antioxidant compound butylated hydroxytoluene (BHT).


InTroducTIon
Lichens are symbiotic associations of algae or cyanobacteria (photobiont) and filamentous fungi (mycobiont).Endolichenic fungi are microorganisms living in the thalli of lichens that are analogous to the plant endophytic species inhabiting the intercellular spaces of the hosts (Arnold, 2007;Arnold & Lutzoni, 2007).The diversity and prevalence of endolichenic fungi have not been studied extensively, and there are only a few recorded studies on the isolation and identification of endolichenic fungi (Miadikowska et al., 2004) and their metabolites (Paranagama et al., 2007).The endolichenic fungi in Sri Lanka remain an untapped source of bioactive natural products as their identity and the chemistry of their secondary metabolites have not been studied thoroughly.The first record of endolichenic fungi in Sri Lanka was by a group of researchers from the University of Kelaniya (Kannangara et al., 2009).Under the current research programme on the study of new bioactive compounds from endolichenic fungi (Kannangara et  A literature survey on bioactive secondary metabolites of endolichenic fungi from different geographic locations revealed that its isolation, identification and bioactivity have not been studied extensively.An ethyl acetate (EtOAc) extract of P. citrinum cultured on potato dextrose agar (PDA) exhibited a significant antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.We report here the bioassay-guided fractionation of ethyl acetate extract of P. citrinum leading to the isolation of two new polyketides (1 and 2).

Isolation of the fungal strain
The lichen host, Parmotrema sp., was collected from the Hakgala Botanical Garden situated at an elevation of about 1745 m above mean sea level within the Hakgala Strict Natural Reserve in the Nuwara Eliya District, Sri Lanka.The collected lichen samples were kept in sterilized polythene packs and transported to the laboratory at the Department of Chemistry, University of Kelaniya and processed within 24 h.Fungal isolations were carried out according to the surface sterilization method described by Kannagara et al. (2009).Healthy lichen thalli were cleaned in tap water and surface sterilized by consecutive immersion for 10 s in 95 % ethanol, 3 min in 0.5 % sodium hypochloride and 30 s in 75 % ethanol.The thalli were surface dried with sterile filter papers and aseptically cut into small segments.Lichen segments of size 1 × 1 cm were then placed (20 pieces from each of the 5 replicate samples of the lichen species) on 2 % malt extract agar (MEA) supplemented with 0.01 % streptomycin.The plates were sealed with parafilm and incubated up to 14 days at room temperature under ambient light.Fungi grown from each lichen particle were isolated into pure cultures.Slides containing pure cultures were prepared using the sticky tape method (Felgel, 1980) and identified using identification keys (Barron, 1968).The emergent fungi were sub-cultured on 2 % MEA, photographed and deposited as a living voucher in the Department of Chemistry, University of Kelaniya under the accession number US/PA/06.

Molecular identification of the isolated endolichenic fungus
Identification of isolated pure strain of the endolichenic fungus was carried out using a molecular biological protocol by genomic DNA extraction, amplification and sequencing.Promega Wizard ® Genomic DNA Purification Kit was used for the extraction of fungal DNA.The fungal strain was identified by analysis of the nuclear internal transcribed spacer (ITS) region of extracted DNA, and selectively amplified by polymerase chain reaction (PCR) using ITS 1 and ITS 4 primers (Arnold & Lutzoni, 2007).Before being subjected to sequencing, the excess nucleotides and remaining primers, enzymes etc., in the PCR product were removed according to a gel purification protocol (Arnold, 2007).First, the desired amplified DNA bands were separated from non specific bands using agarose gel electrophoresis.To isolate the DNA from the gel, Promega Wizard® SV Gel and PCR Clean-Up System was used (Betz & Strader, 2002).The amplified and purified DNA was subjected to DNA sequencing and the obtained sequences were submitted to the GenBank as a nucleotide query and compared with already existing DNA sequences using NCBI BLAST ® (http://www.ncbi.nlm.nih.gov/blast/).

Extraction and isolation
Penicillium citrinum was cultured on PDA for 2 weeks.The mycelium and the medium were cut into small pieces, extracted with EtOAc (6 × 500 mL) and the solvent was evaporated under reduced pressure, which afforded a dark brown semisolid (5.3 g) that was found to be active in antioxidant assays using DPPH method.A portion (3.6 g) of the EtOAc extract was next subjected to bioassay directed fractionation.It was first partitioned between hexane and 80 % aqueous MeOH and the bioactive aqueous MeOH fraction was diluted to 50 % aqueous MeOH by the addition of water and extracted with CHCl 3 .The evaporation of solvents under reduced pressure yielded hexane (489.9 mg), CHCl 3 (2.9 g) and 50 % aqueous MeOH (44.6 mg) fractions.A portion (2.8 g) of the bioactive CHCl 3 fraction was subjected to column chromatography on silica gel (86.0 g) by elution with CH 2 Cl 2 followed by increasing amounts of MeOH in CH 2 Cl 2 , which afforded 67 fractions.These fractions were combined on the basis of their TLC profiles to give seven fractions; F 1 (116.3mg), F 2 (160.0 mg), F 3 (76.7 mg), F 4 (476.8mg), F 5 (71.7 mg), F 6 (33.7 mg) and F 7 (175.8mg).Fraction F 1 was found to contain compounds active in the DPPH antioxidant assay.A portion of (110.0 mg) fraction F 1 was further fractionated by gel permeation chromatography over a column of Sephadex LH-20 (4.0 g) made up in hexane, and eluted with hexane containing increasing amounts of CH 2 Cl 2 , CH 2 Cl 2 only, followed by CH 2 Cl 2 containing increasing amounts of MeOH, and finally 100 % MeOH.Twenty two fractions (10 mL each) were collected and combined on the basis of their TLC profiles to obtain two fractions of which the fraction F 1A was found to be active in DPPH assay.
A portion (70 mg) of the resulting active fraction F 1A was next chromatographed on silica gel to give two active fractions F 1A-1 (12.3 mg) and F 1A-2 (20 mg).Final purification of each of these two fractions by normal phase preparative TLC (2 % MeOH in CH 2 Cl 2 ) led to the isolation of two pure compounds, 1 (8 mg) and 2 (12 mg).

Scavenging ability on 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH)
The assay was carried out in a flat bottom 96-well microtiter plate according to the method described by Chatatikun & Chiabchalard (2013) with slight modifications.160 μL of different concentrations of the sample or standard butylated hydroxytoluene (BHT) (3.13, 6.25, 12.5, 25, 50, 100 μg/mL) was added to 40 μL of 0.25 mM methanolic DPPH solution in the 96-well plate.All reagents were mixed and incubated for 15 min at room temperature under dark conditions.The absorbance of each well was measured at 517 nm with a Microplate Reader (Biotek, USA).The percentage inhibition was calculated based on the following equation x 100 The inhibition rate was calculated and plotted against test concentrations to determine the IC 50 .

Equipment and thin layer chromatography
1D and 2D NMR spectra of compounds 1 and 2 were recorded in CDCl 3 with a Bruker Avance III 400 spectrometer at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR using residual CHCl 3 as an internal reference.Low-resolution and high-resolution MS were recorded on Shimadzu LCMS-QP8000α and JEOL HX110A spectrometers, respectively.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 visualize the compounds on analytical TLC.

reSuLTS
The fungus, US/PA/06 was identified based on the morphological and molecular characteristics.Its colony is fast-growing, velvety and blue-green with a white mycelium edge.Conidiophores arise from the mycelium singly, bearing brush-like sporulating structures, which are characteristic to the genus Penicillium.The ITS sequence matched with P. citrinum sequences from GenBank with a sequence identity of 99 %.This endolichenic fungus was thus identified as Penicillium citrinum.
The antioxidant capacity is widely used as a parameter for medicinal bioactive components in natural extracts.In this study, the total antioxidant activity of the EtOAc extract of P. citrinum was determined by DPPH assay and compared with BHT.In the antioxidant assay, the positive control, BHT showed the best results (~68 % at 100 µg/mL).All the test concentrations and the results are presented in Figure 1.The activity increased in a concentration dependant manner compared to BHT.At the concentrations of 100 μg/mL, effect of the crude extract of P. citrinum was found to be 35 % for DPPH (Figure 1) when compared with the same concentration of BHT.
The results of the antioxidant assay of EtOAc extract of the solid culture of P. citrinum and hexane, CHCl 3 and 50 % MeOH fractions obtained after partitioning with the EtOAc extract are shown in Figure 1.The results revealed that the CHCl 3 fraction has the highest activity.Bioassayguided sequential fractionation of the antioxidant active CHCl 3 fraction of P. citrinum with normal-phase and gel  The IR spectrum of compound 1 (Figure 2) showed absorption bands at 1770 and 1681 cm -1 , which suggested the presence of lactone and conjugated carbonyl groups, respectively.The UV λ max at 240 nm indicated the presence of carbonyl chromophore.The 1 H NMR spectrum of compound 1, when analyzed with the help of DQF-COSY indicated the presence of two spin systems in addition to four 3H singlets due to three OCH 3 groups (δ H 3.64, 3.67 and 3.93) and a CH 3 (δ H 2.42) on a quaternary carbonyl carbon.One of the spin systems consisting of two 1H doublets was shown to be due to the two protons δ H 7.08 (d, J = 3 Hz) and δ H 5.75 (d, J = 3 Hz).The two protons, δ H 6.35 (d) and 6.53 (d) in the 1 H NMR of compound 1 were due to the two protons attached to the aromatic ring system and indicate the second spin system of compound 1 (Table 1).In the HMBC correlations of compound 1 (Figure 2), the proton at δ H With the help of HSQC data the three methoxyl protons, δ H 3.67, 3. unsaturation.The UV λ max at 238 nm indicated the presence of a carbonyl chromophore.The IR spectrum had absorption bands at 3489, 1706 and 1641 cm −1 , suggesting the presence of a OH group, carboxylic acid carbonyl and carbonyl functionalities.The 1 H and 13 C NMR spectra together with HSQC data of compound 2 (Table 2) showed the presence of an acetyl group attached to the aromatic ring similar to compound 1.The 1 H and 13 C NMR data of compound 2 (Table 2) were similar to those of compound 1 and the major difference in NMR data was found to be the absence of an ester carbonyl carbon group at δ C 174.4, which was assigned to C-2 in compound 1; instead compound 2 showed the presence of a carboxylic acid group (C-10, δ C 163.9), which exhibited an HMBC correlation to H-3 (δ H 6.38).
Further, the ketone carbonyl group (C-6, δ C 185.7) in compound 1 was absent; instead the 13 C NMR spectrum of compound 2 showed oxygenated C-2' (δ C 152.2).The 1 H NMR spectrum of compound 2 indicated the occurrence of two meta-coupled aromatic protons (δ C 7.51 and 7.14, J = 3 Hz), four 3H singlets of which three were due to OCH 3 groups (δ C 3.58, 3.62 and 3.23) and the fourth due to a CH 3 group on a quaternary carbon (δ H 2.42), and two 1H singlets at δ H 6.38 and 5.99 accounting for 16 out of 18 protons in compound 2. The remaining two protons were suspected to be that of OH groups.The 13 C

Methylation
Oxidation to a quinone acid group attached to C-2.The structural features of compounds 1 and 2 implies that both compounds have the same polyketide origin, and the biosynthetic pathway proposed in Figure 6 suggests that compound 2 is the precursor for compound 1.The proposed biosynthetic pathway given in Figure 5 indicates that compound 2 originated from a heptaketide via condensation, reduction, oxidation, carboxylation and other reactions to form the final polyketide product compound 2. Compound 2 is the precursor for compound 1 as compound 2 could undergo oxidation to a quinone to which the carboxylate group can add in a Michael-type reaction as proposed in Figure 6.
been reported from the methanol extract of P. citrinum isolated from the soil in Sri Lanka.It was reported that citriquinone A exhibited antibacterial activity against Bacillus sp. and cell migration inhibitory activity against human cancer cell line HEp 2 (Ranji et al., 2013).In the present study, two new polyketides (1 and 2) have been isolated with strong radical scavenging activity when compared to the control, BHT.
A plausible biosynthetic pathway for the two new polyketides (1 and 2) has been proposed in Figures 5  and 6.Compound 2 contains a similar carbon skeleton as compound 1 except for the absence of a carboxylic al., 2009; Kulasekera et al., 2013; Pary et al., 2013; Samanthi et al., 2013 a & b), investigations have been conducted on the endolichenic fungus, Pennicillium citrinum isolated from the lichen, Parmotrema sp.collected from the Hakgala Botanical Garden, Sri Lanka in December 2010.

Figure 2 :
Figure 2: Structure and selected HMBC correlations for compound 1

Figure 1 :
Figure 1: DPPH radical scavenging activity of the crude extract of P. citrinum and the resulting hexane, chloroform and methanol fractions compared with the positive control, BHT after 15 minutes of reaction.Each value is expressed as mean ± SD (n = 3)

Figure 4 :
Figure 4: DPPH radical scavenging activity of the chloroform extract of P. citrinum and the pure compounds (1 and 2) isolated from the chloroform extract compared with the positive control, BHT after 15 minutes of reaction.Each value is expressed as mean ± SD (n = 3) NMR spectrum assigned with the help of the HSQC data contained signals due to a carbonyl carbon (δ C 199.0), 12 aromatic/olefinic carbons of which four were oxygenated (δ C 166.3, 156.4,152.2 and 160.9) and four protonated (δ C 103.0, 110.8, 109.5 and 114.2), three methoxyl carbons (δ C 56.3, 52.4 and 55.5), one methyl carbon (δ C 22.5), and a carbonyl carbon C of the carboxylic acid (δ C 163.9).The HMBC spectrum indicated the presence of two partial structures (Figure2) that contained two aromatic rings with meta-coupled protons.The proton at δ H 6.38 of compound 2 showed HMBC correlations with methyl C of the acetyl group (δ C 22.5), C-5 (δ C 103.0), C-2 (δ C 110.0), and carboxylic acid carbonyl carbon (δ C 163.9) suggesting that this proton is attached to the aromatic ring at C-3 and the remaining proton (δ H 5.99) in the aromatic ring should be attached to C-5.The proton at H-5 δ H 5.99 of compound 2 showed HMBC correlations with C-2 (δ C 110.0), C-3 (δ C 110.8), C-6 (δ C 160.9), C-8 (δ C 199.0) and C-9 (δ C 22.5) confirming the presence of the proton (δ H 5.99) attached to C-5 (δ C 103.0).These HMBC correlations together with 1 H and 13 C NMR data established the partial structure B (C 10 H 9 O 4 ) for compound 2 (Figure 4).The cross-peaks between δ H 7.51 with C-1'(δ C 131.9), C-3'(δ C 166.3), C-2' (δ C 152.2), C-2 (δ C 110.0); δ H 7.14 with C-5' (δ C 156.4), C-2' (δ C 152.2), C-1' (δ C 131.9), C-6' (δ C 114.2); δ H 3.58 with C-3' (δ C 166.3); δ H 3.62 with C-5' (δ C 156.4) in the HMBC spectrum and the 1 H and 13 C NMR data for the remainder of the molecule established the presence of the partial structure A (C 8 H 9 O 3 ) in compound 2 (Figure 3).The proton (δ H 7.51) showed a correlation with C-2 (δ C 110.0) indicating that partial structures A and B are connectedto each other establishing the structure of compound 2, which contains an additional carboxylic acid group in the aromatic ring A and a hydroxyl group in the aromatic ring B and disappearance of ketone carbonyl group and ester carbonyl group in compound 1 suggesting that the partial structures A and B of compound 2 are linked to each other as shown in Figure3.The structure of new compound 2 was thus established as 4-acetyl-2'hydroxy-3',5',6-trimethoxy biphenyl-2-carboxylic acid.Antioxidant assay results of compound 2 showed strong radical scavenging activity in the DPPH assay with an IC 50 value of 68.6 ± 4.3 µg/mL (Figure4).dIScuSSIonand concLuSIonPenicillium citrinum is a mycotoxin producing fungus known to produce citrinin, a well-known mycotoxin both nephrotoxic and carcinogenic.Bioactivity of citrinin has been studied extensively and it also possesses antibiotic, bacteriostatic, antifungal and antiprotozoal properties (Abou-Zeid, 2012).There have been several reports on the isolation of bioactive metabolites, and the investigation of the ethyl acetate extracts from the broth and mycelia of P. citrinum has led to the isolation of eighteen metabolites(Chen et al., 2011), antioxidant JBIR-124(Kawahara et al., 2012), cytotoxic penicitrinols C and E, topo IIαinhibitory tricitrinol B and the cytotoxic dicitrinone B(Khamthong et al., 2012).The isolation of five polyketides including two benzopyranones, one isochroman and two anthraquinone-citrinin derivatives was reported from the sea fan-derived fungus P. citrinum(Khamthong et al., 2012).Two benzoquinones and citriquinone A and B have