Isolation and Characterization of New Anti-Inflammatory and Antioxidant Components from Deep Marine-Derived Fungus Myrothecium sp. Bzo-l062

In the present study, four new compounds including a pair of 2-benzoyl tetrahydrofuran enantiomers, namely, (−)-1S-myrothecol (1a) and (+)-1R-myrothecol (1b), a methoxy-myrothecol racemate (2), and an azaphilone derivative, myrothin (3), were isolated along with four known compounds (4–7) from cultures of the deep-sea fungus Myrothecium sp. BZO-L062. Enantiomeric compounds 1a and 1b were separated through normal-phase chiral high-performance liquid chromatography. The absolute configurations of 1a, 1b, and 3 were assigned by ECD spectra. Among them, the new compound 1a and its enantiomer 1b exhibited anti-inflammatory activity, inhibited nitric oxide formation in lipopolysaccharide-treated RAW264.7 cells, and exhibited antioxidant activity in the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and oxygen radical absorbance capacity assays.

Microorganisms of the deep-sea are an attractive source of candidate drugs. While screening inhibitors of lipopolysaccharide (LPS)-induced nitric oxide (NO) production, we recently isolated cyclopenol and cyclopenin from the extract of the fungal strain Aspergillus sp. SCSIOW2 collected from a depth of approximately 2000 m in the sea [14]. At non-toxic concentrations, these compounds inhibited LPS-induced NO production and IL-6 secretion in RAW264.7 cells. This inhibitory effect of cyclopenol and cyclopenin was attributed to the suppression of the upstream signal of NF-B activation. These compounds also suppressed the expression of IL-1β, IL-6, and iNOS in microglia cells (macrophages in the mouse brain) [14]. In Alzheimer's disease, amyloid β-peptide induces inflammation in the brain. Between the two compounds, cyclopenin showed ameliorative effects in an in vivo Alzheimer's model using flies [14].

Results and Discussion
The molecular formula of 1 was determined as C 12 Figure 2). The four aromatic carbon signals indicated the presence of one symmetrically substituted benzene ring. The HMBC experiment correlations confirmed the presence of a 3,5-dihydroxy-4-methyl benzoyl moiety (Table 1 and Figure 2). Finally, the key HMBC correlations from H 2 -2 to C-1 and from H-1 to C-2 allowed the linkage of the 3,5-dihydroxy-4-methyl benzoyl and tetrahydro-2-furanyl groups (Table 1 and Figure 2).
Accordingly, 1 was established as (3,5-dihydroxy-4-methylphenyl)-(tetrahydro-2-furanyl)methanone and denoted as a myrotheciol.  The absence of the Cotton effect in the CD spectrum and zero specific rotation indicated that 1 was a racemate. Generally, enantiomers are more advantageous than racemates for drug development. To detect the enantiomers of 1, chiral HPLC was performed using a Chiralpak IC column; the HPLC results showed two separate peaks ( Figure S9 Figure S10). The 1 H and 13 C NMR data of 2 ( Table 2, Figures S11-S16) closely resembled those of 1, except for three major differences: the presence of an additional methoxy group (δ H 3.09, δ C 50.2), the absence of a methine proton (δ H 5.09), and the chemical shift of C-1 (from δ C 79.1 to 109.5); these differences indicated the substitution of the methine proton at C-1 by a methoxy group. The position of the new methoxy group was confirmed by HMBC correlation from 1-OMe to C-1 (Table 2 and Figure 2). Thus, 2 was established as 1-methoxy-myrotheciol. The structure of 2 was validated through a detailed analysis of 2D NMR data (Table 2 and Figure 2).  Compound 2 was also considered as a racemic mixture based on the zero specific rotation and absence of the Cotton effect in its CD spectrum. The chiral HPLC performed using the same condition as that used for 1 revealed two peaks, attributable to 2a and 2b, at a ratio of approximately 1:1 ( Figure S17). However, due to the limited sample size, further isolation was not carried out.
LPS-induced NO production in RAW264.7 cells was used to evaluate the anti-inflammatory activity of different compounds [14]. NO is produced by NF-κB-dependent inducible NO synthase. All the isolated compounds were evaluated for cytotoxicity and for their effects on LPS-induced NO production. Among all the tested compounds, only two new compounds (1a and 1b) significantly inhibited LPS-induced NO production at non-toxic concentrations ( Figure 4).
In the present research, we isolated several compounds including new structures from a deep-sea fungus. We found cellular anti-inflammatory activity in 1a and 1b. Microorganisms often produce useful compounds for therapy. However, the role of these compounds on producing organisms is not clear. At the beginning of antibiotic research, antibiotics are considered to protect the producing organisms by killing their enemy microorganisms. But later, many enzyme inhibitors such as pepstatin and leupeptin were discovered from the secondary metabolites of Streptomyces, and they showed no antibiotic activity. Therefore, it is unlikely that these secondary metabolites are useful for the producers. From this point of view, new compounds, 1a and 1b, may be remnants of microorganisms in their evolution.

Fungal Material
The fungus Myrothecium sp. BZO-L062 used in this study was isolated from a deep-sea (2130 m depth) sediment sample collected from an area close to Yongxing Island, China. The strain was identified as Myrothecium sp. based on the morphological features and internal transcribed spacer sequence analysis. This strain was deposited at the Marine Natural Products Laboratory, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China.

Fermentation and Extraction
The fungus Myrothecium sp. BZO-L062 was activated on petri dishes containing potato dextrose agar supplemented with 3% sea salt at 28 • C for three days [24]. Agar plugs were inoculated in a 500 mL Erlenmeyer flask containing 150 mL of liquid potato dextrose culture medium [24] supplemented with 3% sea salt as seed cultures and were incubated at 28 • C on a rotary shaker at 180 rpm for three days. Large-scale fermentation (70 L) was conducted using the same medium as that for seed cultures at 28 • C and 180 rpm for seven days. After seven days, the fermentation broth was filtered through cheesecloth to separate the supernatant from the mycelia. The supernatant was then concentrated to 8 L and successively extracted three times with EtOAc (3 × 8 L), yielding a crude extract (40.0 g).

ECD Calculation
The conformational distribution search was conducted with the MMFF94 molecular mechanics force field in Spartan 12 software (Wavefunction Inc., Irvine, CA, USA). The lowest energy conformers within the 5-kcalmol −1 energy window were optimized using the Gaussian 09 program [25]. TDDFT calculations for all optimized conformers were performed at the B3LYP/6-31G (d, p) level. The ECD spectra were generated using the software SpecDis [26].

MTT and NO Production Assay
MTT and NO production inhibitory activities of the isolated compounds in RAW264.7 cells were determined as reported previously [14].

Antioxidant Activity
The ABTS and DPPH scavenging assays were carried out as reported earlier [23]. L-ascorbic acid and trolox were used as positive controls. The ORAC assay was conducted according to a previously reported protocol [27]. The results were expressed as µmol Trolox equivalents per µmol of sample solution.
Among these compounds, new compounds 1a and 1b showed anti-inflammatory and antioxidant activities at non-toxic concentrations. Derivatives of these compounds could be potent and safe and may be useful for the development of new anti-inflammatory agents.