New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum

Eight trichothecenes, including four new compounds 1–4 and four known entities 5–8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1–9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 μM, respectively. Compounds 1–2, 4–6, and 7–8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.


Introduction
Fungi are a potential source of drug leads that researchers are still seeking [1,2]. The discovery of new compounds with unique structural diversity and low molecular weight presents opportunities for discovering bioactive natural products from fungi [2]. Although less explored, marine fungi are an important and rich source for the discovery of new compounds. A number of new compounds from marine fungi have been discovered from various sources, including extreme sea environment. These compounds showed various activities including anticancer, antimicroalgal, antibacterial, and antiviral effects [3][4][5]. The vast symbiotic relationships and diversity of many marine organisms has caused marine fungi to distribute in almost all marine habitats, including from marine ray-finned fish, sponges, mangroves, and algae [3,[5][6][7]. Among them, marine algae-derived fungi offer opportunities and attract attention because they produce secondary metabolites with unique chemical diversity and various pharmacological properties [4,6,[8][9][10][11].
Compound 4 was obtained as a colorless gum with elemental formula of C 15 H 24 O 4 determined by 13 (Table 2), and HMBC ( Figure 2) spectrum data confirmed that compound 4 was almost similar to compound 7, indicating that 4 possessed an identical skeleton to that of 7 except for acetoxy group signal in 7 replaced by a hydroxy in C-2; these results, together with the appearance of the oxymethine carbons (C-4, δ C 72.3) in 4, were much more upfield than that in 7. The COSY spectrum data supported these results, in that the correlations of H-9 (δ H 1.75) to H ab -16 (δ H 3.74 and 3.47), H ab -10 (δ H 1.92 and 1.59), and H ab -8 (δ H 1.70 and 1.64); Hab-8 to H ab -7 (δ H 1.98 and 1.17); H ab -10 to H-11 (3.39). The NOESY correlation peaks of compound 4 were similar to those of compound 7, which is also a tricothecene-based compound [12].

Discussion
Trichothecenes comprise a group of sesquiterpenes that have been reported both from fungal cultures such as those of Myrothecium spp., Trichothecium spp., and Fusarium spp., as well as from some higher plants such as Bacchairis coridifolia, B. artemisioides, Ficus fistulosa, and Rhaphidophora decursiva [7,15,16,18,23,24]. Among these, some of the trichothecene-producing fungal species were marine-derived, such as Myrothecium sp.

Discussion
Trichothecenes comprise a group of sesquiterpenes that have been reported both from fungal cultures such as those of Myrothecium spp., Trichothecium spp., and Fusarium spp., as well as from some higher plants such as Bacchairis coridifolia, B. artemisioides, Ficus fistulosa, and Rhaphidophora decursiva [7,15,16,18,23,24]. Among these, some of the trichotheceneproducing fungal species were marine-derived, such as Myrothecium sp. and Trichoderma sp. [7,12]. Structurally, trichothecenes are a family of sesquiterpenoids composed of a tricyclic 12,13-epoxytrichothec-9-ene (trichothecene) ring. On the basis of substitutions on the tricyclic moiety, trichothecenes are subcategorized into four types (A, B, C, and D), and over 200 compounds have been isolated [25]. Type A is the simplest structure, being nonsubstituted, hydroxylated, or esterified. All compounds that we have isolated in this report can be categorized as type A. The structure-cytotoxic activity relationship of trichothecenes has been extensively researched previously [2,25]. In particular, trichothecene with the C-12,13-epoxy ring, the double bond between C-9 and C-10 in A ring, and OH-4 have been identified as key structural features contributing to their toxicity [26][27][28]. In this study, we observed that in compounds 1, 3, and 6, the C-12,13-epoxy ring is hydrolyzed to be opened as well as the double bond between C-9 and C-10 in A ring that led to no cytotoxic activity against three cancer cell lines. On the contrary, compounds 4 and 7 with the C-12,13-epoxy ring, without a double bond between C-9 and C-10 in A ring, showed reduced cytotoxicity, but the presence of -OAc instead of -OH at C-4 could increase cytotoxic activity [28]. On the other hand, the new compounds 2 and 4 and the know compounds 5, 7, and 8 potently inhibited LPS-induced NO production in BV-2 cells. Further, the presence of a double bond between C-9 and C-10, and OH-4 could also increase activity in the inhibition of MMP-9 gelatinolysis, especially in compound 8. These findings provide evidence that compounds 5, 7, and 8 may serve as potential drugs for neuroinflammation-related diseases and for anticancer treatment.

General Experimental Procedures
Optical rotations data were measured on a JASCO P-2000 polarimeter (Tokyo, Japan). 1D and 2D NMR spectrum data were recorded on Agilent DD2 600 MHz spectrometer (Agilent Technologies, Santa Clara, CA, USA). High-resolution ionization mass spectra were acquired on a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). Infrared (IR) spectra data were recorded on a JASCO FT/IR 4100 spectrometer (Tokyo, Japan). Open column chromatography was using Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), and thin-layer chromatography was performed using silica gel 60 F 254 plates (0.2 mm) (Merck, Darmstadt, Germany). An HPLC pump L-7100 (Hitachi, Naka, Japan) equipped with a refractive index detector (Bischoff, Leonberg, Germany) was used for compound purification. All the organic solvents were purchased from Merck (Darmstadt, Germany).

Strain Isolation and Fermentation
T. brevicompactum NTU439 was isolated from M. rosea marine alga, which was collected from Yilan coast (24 • 57 13.9" N 121 • 54 49.3" E), Taiwan, in June 2016, and was identified on the basis of sequencing of the internal transcribed spacer (ITS) regions of the rDNA. The sequence of fungus NTU439 matched as T. brevicompactum by A BLAST search sequence (GenBank accession no. OK217197). The mycelium of T. brevicompactum NTU439 was inoculated into 250 mL flasks, each containing 50 g of brown rice (Santacruz, Taipei, Taiwan) and 15 mL deionized water with 1% KH 2 PO 4 , 1% sodium tartrate, and 2% yeast extract (Becton, Dickinson and Company, Sparks, MD, USA). The fermentation process was conducted under aeration for 30 days at 27-30 • C.

Cell Culture
The colorectal cancer cell line HCT-116, prostate cancer cell line PC-3, and hepatocellular carcinoma cell line SK-Hep-1 were purchased from the American Type Cell Culture Collection (Manassas, VA, USA). Cell culture was performed following the procedure of our previous reports [6]. In summary, the cells were maintained in DMEM medium containing fetal bovine serum (FBS), penicillin, and streptomycin in humidified air containing 5% CO 2 at 37 • C.

Biologic Assay for Cytotoxic Activity
The SRB assay was used to determine the cytotoxic activity according to previously described procedures [6]. The HCT-116, PC-3, and SK-Hep-1 cancer cells were seeded onto 96-well plates in a density of 5 × 10 3 cells per well. Overnight, cells were treated with the tested compounds for 48 h.

Biologic Assay for Relative Gelatinolysis by MMP-9
The relative gelatinolysis of by in human THP-1 monocytic cells MMP-9 was performed following the procedure of our previous reports [29]. Briefly, the THP-1 cells were subcultured and developed for 24 h in 24-well plates using serum-free medium. After the cell's adhesion and growth, they were treated with 10 µM of compounds or vehicle (DMSO) followed by S1 protein (0.5 µg/mL) stimulation for 24 h before analysis of the MMP-9 gelatinolysis. The medium was collected and mixed with a non-reducing buffer that contains Tris-HCl, glycerol, SDS, and bromophenol blue (500 mM, 25%, 10%, and 0.32%, respectively), pH 6.8, and electrophoresed on gels containing 1 mg/mL of gelatin. The gels were washed with 2% Triton X-100 after electrophoresis and then incubated with reacting buffer containing Tris-base, NaCl, CaCl 2 , and Brij 35, pH 7.5, for 17 h at 37 • C. After incubation, the gels were fixed with 7% acetic acid and 40% methanol (v/v) for 30 min and then stained with Colloidal Brilliant Blue G in 25% methanol for 40 min. Clear zones (bands) against the blue background indicated the presence of gelatinolysis by MMP-9.
Gelatinolytic zones were imaged and analyzed. The viability of THP-1 monocytic cells was measured using MTT assay after incubation with compounds or vehicle (DMSO) for 24 h.

Biologic Assay for Anti-Neuroinflammatory Activity
Culturing procedure and media composition for culturing of the mouse microglial BV-2 cell line was performed as described in our previous report [30]. The cells were pretreated with a concentration of compounds or vehicle (DMSO) for 15 min and then stimulated with LPS for 24 h. Cellular viability of BV-2 cells treated for the 24 h with compounds was measured by a colorimetric assay of MTT reduction [31]. The levels of nitrite were measured at 550 nm using a microplate reader (MRX) for evaluation of nitric oxide production as we have previously described [32]. Sodium nitrite was used as a standard, and curcumin was used as the positive control.

Conclusions
In this report, eight trichothecenes, including four new trichothecenes 1-4 and four known compounds 5-8, along with one known cyclonerane, 9, were isolated from the marine algae M. rosea-derived fungus T. brecicompactum NTU439. Functional characterization of all the isolates was evaluated by cytotoxic activity against three cancer cell lines (HCT-116, PC-3, and SK-Hep-1), inhibition of LPS-induced NO production, and inhibition of gelatinolysis by MMP-9. Of the compounds identified, trichoderminol (5), trichodermarin E (7), and trichodermol (8) exhibited promising cytotoxicity against three cancer cell lines and inhibition of both MMP-9 gelatinolysis and LPS-induced NO production.