Cytotoxicity and Nitric Oxide Production Inhibitory Activities of Compounds Isolated from the Plant Pathogenic Fungus Curvularia sp.

Chemical investigation of the mycelia of the pathogenic fungus Curvularia sp. which was isolated from a leaf of Dactyloctenium aegyptium (crowfoot grass), resulted in the isolation of a new compound, curvulariahawadride (5), along with five known compounds (1–4, and 6). Their structures were determined on the basis of spectroscopic data, including 1D and 2D NMR and HRESIMS. The absolute configuration of 5 was established from experimental and calculated electronic circular dichroism (ECD). Compounds 1, 3, and 5 showed nitric oxide (NO) production inhibitory activity with IC50 values of 53.7, 32.8, and 12.8 µM, respectively. Compounds 2 and 4 showed significant cytotoxicity against lung cancer A549, colorectal cancer SW480, and leukemic K562 cells with an IC50 ranging value of 11.73 to 17.59 µM.


Introduction
Endophytic fungi are well-known as promising sources of structurally diverse and novel biologically active compounds [1][2][3]. Endophytic fungi have become one of the most important sources for drug discovery from nature because of the short period to scale up and the diversity of production of secondary metabolites [4,5]. Curvularia species are phytopathogenic fungi that are commonly isolated from soil or infected grasses and food plants, such as maize (Zea mays), oil palm (Elaeis guineensis), or rice (Oryza sativa) [6][7][8].
A major cause of leaf blight and leaf spot is infection by Bipolaris and Curvularia fungi and is considered one commonly associated with oil palm and corn leaf spots in Thailand [7,9]. In addition, several species of Curvularia have been reported for opportunistic infections in humans [10][11][12]. Previously, C. lunata was believed to be the most frequently reported human pathogenic species, while C. hawaiiensis (previously Bipolaris hawaiiensis) was reported to infect the ear of a patient post-trauma [12,13]. Previously, cochlioquinones, polyketides, terpenoids, alkaloids, quinones, and peptides have been isolated from the genus of Curvularia [14]. However, differences in fungal collections and fermentation conditions have yielded different compounds. Various types of compounds isolated from Curvularia species have shown impressive biological activities, including phytotoxic [15], antimicrobial [16][17][18][19][20], and cytotoxic [17,21,22] activities. Herein, we describe the isolation and structure elucidation of a new compound (5) along with five known compounds (1-4, and 6) from the ethyl acetate extract of mycelia from laboratory cultures of Curvularia sp. which was isolated from a leaf of D. aegyptium. In addition, the in vitro nitric oxide (NO) production inhibitory and cytotoxicities of the isolated compounds against three cancer cell lines, including lung cancer A549, colorectal cancer SW480, and leukemic K562 cells, and mammalian cells (RAW 264.7) are also reported.

Fungal Material and Identification
A leaf spot symptom of crowfoot grass was collected from Hat Yai, Songkhla Province, in July 2012. Fresh specimens were incubated for 1-2 days in a moist chamber to induce sporulation. Fungi were isolated by a modified single spore suspension method [23]. Conidia were taken from the fungal sporulation from leaf spot samples and placed in sterilized water for spore suspension. The conidia were then transferred to water agar media and left overnight to germinate, and germinated conidia were individually transferred to PDA. The pure cultures of Curvularia sp. (MFLCC12-0192) were deposited in MFLUCC for future study. Genomic DNA was extracted from fungal mycelium grown on PDA media using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® , Hangzhou, China), following the instructions of the manufacturer. The DNA amplification was performed by polymerase chain reaction (PCR). Primers ITS1 and ITS4 (Glass and Donaldson, 1995) were used to amplify the 5.8S and ITS regions. The quality of PCR amplification was confirmed on 1% agarose gel electrophoresis stained with ethidium bromide. The amplified PCR fragments were sent to the commercial sequencing provider (Shanghai Sangon Biological Engineering Technology & Services Co., Shanghai, China).

Nitric Oxide (NO) Production Inhibitory Assay
This assay was performed as previously described [24]. RAW 264.7 cells were seeded at 4 × 10 4 cells/well in 96-well plates and incubated at 37 • C and 5% CO 2 overnight. Cells were incubated with 1 µg/mL LPS for 1 h and treated with various concentrations of tea extract, including 3.125, 6.25, 12.5, 25, 50, and 100 µg/mL, for 24 h. After 24 h, 100 µL of Griess reagent was added to the samples for 10 min. Nitric oxide was measured at 570 nm with a Biochrom EZ Read 400 ELISA microplate reader (Biochrom Ltd., Cambridge, UK). Additionally, the data are presented as the IC 50 , which was calculated with GraphPad Prism 6.0 software. Indomethacin was used as a positive control with an IC 50 value of 73.4 µM.

Cytotoxicity Assay in Mammalian Cells (RAW 264.7 Cells)
This assay was performed as previously described [25]. Cell viability was measured by the MTT assay. RAW 264.7 cells were seeded at 4 × 10 4 cells/well in 96-well plates and incubated at 37 • C and 5% CO 2 overnight. Cells were treated with different concentrations of tea extracts, including 3.125, 6.25, 12.5, 25, 50, and 100 µg/mL, for 24 h. After 24 h, cells were washed with PBS and incubated with 0.5 mM MTT reagent for 4 h. The detection of formazan at 570 nm was performed with a Biochrom EZ Read 400 ELISA microplate reader (Biochrom Ltd., Cambridge, UK). The data were calculated as IC 50 values with GraphPad Prism 6.0 software.

Cytotoxicity Assay against Lung Cancer A549, Colorectal Cancer SW480, and Leukemic K562 Cells
These assays were performed as previously described [25]. Lung cancer A549 and colorectal cancer SW480 cells were maintained with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Leukemic K562 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were cultured in 96-well plates at 37 • C in 5% CO 2 , followed by treatment with the sample for 24 h. After the incubation period, 0.5 mg/mL MTT was added to the cells and left for 4 h. The formazan was dissolved in DMSO and measured at 570 nm using Biochrom EZ Read 400 ELISA microplate reader (Biochrom Ltd., Cambridge, UK).

Computational Methods
The electronic circular dichroism (ECD) calculations of compound 5 were carried out by using TD-DFT at the CAM-B3LYP functional with 6-311++G(d,p) basis. All structures were optimized by the DFT method at the B3LYP/6-31G (d,p) level of theory. Geometry optimization and TD-DFT computations were both performed with Continuum Model (PCM) solvation model with methanol. The rotary strengths of 100 excited states were calculated. All calculations were performed using Gaussian 09 program package [26]. Gaussian band shape with a bandwidth of 0.25 eV was used to simulate ECD spectra. The ECD curves were generated by the software SpecDis 1.64 (University of Wurzburg, Wurzburg, Germany).

Isolated Compounds from Curvularia sp.
The mycelia of Curvularia sp. were cultured on potato dextrose agar, and the mycelia were harvested, extracted, and subjected to repeated column chromatography to afford six compounds ( Figure 1).

Nitric Oxide Production Inhibitory Activity of Isolated Compounds 1-6
Compounds 1-6 were screened for their NO production inhibitory activity. At the concentration of 100 µg/mL, compounds 1, 3, 5, and 6 showed NO production inhibitory with the percentage inhibition ranging from 41.03 to 74.01% and the IC 50 values ranging from 12.9-53.7 µM (Table 4). Cochlioquinone A (2) and anhydrocochlioquinone A (4) were not tested due to these compounds leading to cell death. Curvulariahawadride (5) showed the best NO production inhibition activity with an IC 50 value of 12.9 µM, which was better than the positive control, indomethacin (IC 50 value of 73.4 µM). Cochlioquinone N (1) and stemphone (3) also showed NO production inhibition activity better than the positive control with IC 50 values of 53.7 and 32.8, respectively. The other compounds (2, 4, and 6) were inactive at the concentration of 100 µg/mL. Notably, this is the first publication on the NO production inhibitory activity of these compounds.

Conclusions
The chemical investigation of plant pathogenic fungus Curvularia sp. led to the isolation and identification of a new nonadride derivative, curvulariahawadride (5), together with five known compounds. Cochlioquinones were found as major compounds from this study. All compounds were evaluated for their cytotoxicities against the three cancer cell lines, lung cancer A549, colorectal cancer SW480, and leukemic K562 cells, and NO production inhibitory activity. Only two compounds (2 and 4) showed cytotoxicities against the three cancer cell lines. In the case of NO production inhibitory activity, compound 5 showed the best NO production inhibitory activity, which better than the positive control.