New C13 lipids from the marine-derived fungus Trichoderma harzianum.

Chemical examination of the fermentation broth of a sponge-associated fungus Trichoderma harzinum HMS-15-3 led to the isolation of four pairs of new C13 lipid enantiomers namely harzianumols A–H (1a–4b). Their structures were elucidated on the basis of extensive spectroscopic (IR, MS, 1D, and 2D NMR) data analysis, including the modified Mosher's method for the assignment of their absolute configurations. The new compounds were evaluated for antihyperlipidemic effects in HepG2 cells.


Introduction
Fungal secondary metabolites are a rich source for mining new bioactive molecules with potential pharmaceutical applications, while some fungus-derived metabolites have been developed as antibiotics, antiviral, antitumor, and cholesterol-lowering drugs [1]. Polyketides encompass a large group of structurally diverse fungal natural products, while polyketide-based compounds are reported to be potential leads used for molecular probes, new drug candidates, in addition to the ecological functions such as toxins and virulence factors of pathogens [2,3]. During the past decades, a number of polyketide-derived drugs or lead compounds have been found from fungi, such as the cholesterol-lowering lovastatin [4], the immunosuppressive mycophenolic acid [5], the antifungal griseofulvin [6], and the histone deacetylase (HDAC) inhibitor depudecin [7,8].
In the course of our search for new bioactive secondary metabolites from marine-derived microorganisms, a fungus Trichoderma harzianum HNS-15-3 isolated from the sponge Petrospongia nigra HNS-15 was undertaken for cultivation. Screening the functional gene clusters using bioinformatics tools including antiSMASH [1,9] revealed the fungus T. harzianum possessing many synthetic genes related to PKS (polyketide synthase) biosynthetic pathways. The 1 H NMR spectrum of the EtOAc extract of the cultured material featured the resonances for unsaturated lipids.

Results and discussion
Chromatographic separation of the EtOAc extract of the solid fermentation material of T. harzianum HNS-15-3 resulted in the isolation of four HPLC (C 18 ) pure components (1 -4) ( Figure 1). Each component displayed a pair of inseparable shoulder peaks in chiral column HPLC [10] and the duplicated NMR data, suggesting the presence of enantiomeric mixture.
The enantiomeric mixture of harzianumols A (1a) and B (1b) has the molecular formula of C 13   1a (6R,7S ) 1b (6S,7R )  ), bearing three degrees of unsaturation. The COSY correlations established two spin systems, of which the moiety from C-1 to C-7 was identical to that of 1. The second spin system afforded a linear chain from C-9 to C-13, in which a double bond was resided at C-11 (d C 130.8) and C-12 (d C 125.1) according to the terminal methyl protons H 3 -13 (d H 1.60, d, J ¼ 6.5 Hz) correlated to C-11 and C-12 in the HMBC spectrum. In addition, a carbonyl carbon was located at C-8 based on the HMBC interactions from C-8 ( (Table 2) revealed R configuration for C-6 and C-7 of 4a, and S configuration for C-6 and C-7 of 4b.
These lipids were tested for antihyperlipidemic effects in HepG2 cells, whereas none of them showed significant lipidlowering activity. The C 13 -polyketide metabolites are rarely found from nature. Oshima and coworkers first reported a number of C 13 polyketides from a fungus Chaetomium mollipilium through the epigenetic regulation approach to inhibit NAD þ -dependent HDAC activity by the inhibitor nicotinamide [12]. This finding implied that C 13 polyketides and relative polyketides are a group of unexplored metabolites embedded in fungi. Uncovering structurally unprecedented metabolites by genomics guidance and by stimulation of silent biosynthetic gene clusters may provide additional approaches to discover chemical diversity.

General experimental procedures
IR spectra were determined on a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Fisher Scientific Inc., New York, NY, USA). 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker Advance 400 NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C, respectively) (Bruker Corporation, Zurich, Switzerland). Chemical shifts are expressed in d (ppm) referenced to the solvent peaks at d H 2.50 and d C 39.8 for DMSO-d 6 , and d H 7.28 and d C 77.0 for CDCl 3 , respectively, and coupling constants are in Hz. ESI-MS and HR-ESI-MS spectra were obtained from a Thermo Scientific LTQ Orbitrap XL instrument (Thermo Fisher Scientific Inc., New York, NY, USA). Silica gel (160 -200 and 200-300 mesh, Qingdao Marine Chemistry Co., Ltd, Qingdao, China) and ODS (50 mm, YMC Co., Ltd, Kyoto, Japan) were used for column chromatography. Precoated silica gel plates (Kieselgel 60 F 254 , 0.25 mm, Merck, Darmstadt, Germany) were used for TLC analysis. HPLC chromatography was performed on an Alltech instrument (426-HPLC pump) equipped with an Alltech uvis-200 detector (Alltech Inc., Chicago, IL, USA) at 210 nm and semipreparative reversed-phased columns (YMC-packed C 18 , 5mm, 250 mm £ 10 mm).

Fungal material
Fungus T. harzinum HNS-15-3 was isolated from the sponge P. nigra HNS-15 collected from South China Sea. The species was identified by Prof. Tian Li from the Qingdao Chemical and Technology University using morphological method and analysis of the ITS region of the rDNA, whose sequence data have been deposited at GenBank with the accession number KJ716469. The voucher specimen is deposited in State Key Laboratory of Natural and Biomimetic Drugs of China at 2808C. The producing strain was prepared on potato dextrose agar (PDA) slants and stored at 48C.

Fermentation and extraction
The fungal strain was cultured on slants of PDA at 258C for 7 days. Fermentation was carried out in fifty 500 ml Erlenmeyer flasks each containing 100 g of rice for 30 days at 258C. The fermentation broth was extracted three times with EtOAc, and then was concentrated under reduced pressure to give an extract (23.2 g).

Cell-based lipid accumulation assay
HepG2 cells were maintained in DMEM (Dulbecco's modification of Eagle's medium) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 mg/ml). The cells with 70 -80% confluence were incubated in DMEM plus oleic acid (100 mM) for 12 h, then were treated with the compounds (each, 10 mM), while the positive control lovastatin in DMEM/oleic acid (100 mM) was used as a blank for an additional 6 h. Subsequently, the cells were subjected to Oil Red O staining or total cholesterol (TC) and triglyceride (TG) determination as described previously [13]. Each experiment (n ¼ 8 for Oil Red O staining or n ¼ 3 for TC and TG determination) was repeated in triplicate.

Disclosure statement
No potential conflict of interest was reported by the authors.