Furobenzotropolones A, B and 3-Hydroxyepicoccone B with Antioxidative Activity from Mangrove Endophytic Fungus Epicoccum nigrum MLY-3

Three new metabolites, furobenzotropolones A, B (1–2) with unusual benzene and dihydrofuran moieties and 3-hydroxyepicoccone B (3), together with seven known compounds (4–10) were obtained from the endophytic fungus Epicoccum nigrum MLY-3 isolated from the fresh leaf of mangrove plant Bruguiear gymnorrhiza collected from Zhuhai. Their structures were assigned by the analysis of UV, IR, NMR, and mass spectroscopic data. Compound 1 was further confirmed by single-crystal X-ray diffraction experiment using Cu Kα radiation. In antioxidant activities in vitro, compounds 2, 3, 5, and 8 showed promising DPPH· scavenging activity with IC50 values ranging from 14.7 to 29.3 µM. Compounds 2, 3, 5, 7, and 8 exhibited promising potent activity in scavenging ABTS· with IC50 values in the range of 18–29.2 µM, which was stronger than that of the positive control ascorbic acid (IC50 = 33.6 ± 0.8 µM).


Structure Elucidation
Compound 1 was obtained as yellow amorphous powder. Its molecular formula was assigned as C 16 (Table 1). In addition, according to the DEPT 135 and HSQC (Heteronuclear Single Quantum Correlation) data, the 13 C NMR data showed the presence of 16 carbon signals, including one carbonyl (δ C 184.3), ten sp 2 -hybridized quaternary carbons, two oxygenated sp 3 -hybridized carbons (δ C 75.6 and δ C 78.0), and two methyls (δ C 14.7 and 15.1). The HMBC (Heteronuclear Multiple Bond Correlation) correlations from 4-OH to C-3, C-4 and C-4a, from 3-OMe to C-3, and from H-13 to C-1, C-2, and C-9a constructed a 3-methoxy-1-methylbenzene ring fragment ( Figure 2). Additionally, the HMBC correlations from H-10 to C-5, C-6, C-7, and C-8, from the H-11 to C-7, C-8, and C-9, from H-12 to C-8, C-9, from H-13 to C-9a, C-12, and from 4-OH to C-4a, C-5 assembled a tropolone ring, which connected with the benzene ring at C-4a and C-9a. Furthermore, HMBC correlations of H-11 to C-12, and H-12 to C-11 and unsaturation information indicated that 1 had a dihydrofuran ring, which was assigned to connect with the benzotropolone ring at C-8 and C-9. Thus, with the assistance of single-crystal X-ray (Figure 3), the structure of 1 was deduced and named furobenzotropolone A.   Table 1, which suggested that structure of 2 was similar with that of 1, except that the methoxy group was substituted with the hydroxyl group at C-3. Combined with HMQC and HMBC (Figure 2), the structure of compound 2 was clearly confirmed, which was named furobenzotropolone B.
Compound 3 was isolated as white amorphous powder. Its molecular formula was determined as C 9 H 8 O 6 (six degrees of unsaturation) in terms of HREIMS analysis at m/z 211.02518 [M−H] − (calcd. for C 9 H 7 O 6 , 211.02481). Analysis of the 1 H and 13 C NMR spectroscopic data of 3 (Table 2) revealed most similarities to those in the literature, rather than the methoxyl signal substituted at C-3 by a hydroxyl group [30]. Combined with HMQC and HMBC (Figure 2), compound 3 was determined as 3-hydroxyepicoccone B. The specific optical rotation value [α] 20 D + 1.2) of 3 indicates it to be a scalemic mixture.

Antioxidant Activities In Vitro
Compounds 1-10 were tested for their antioxidant activities in vitro. As seen in Table 3 Compounds 1, 4, and 6 also showed weak ABTS· scavenging activity with IC 50 values of 46.4, 43.1, and 93.5 µM, respectively. Through the analysis of the structure-activity relationship, we found that antioxidant activity increased with the increase of phenolic hydroxyl groups. If the phenolic hydroxyl group is replaced with a methoxy group, the antioxidant activity will significantly decrease.

General Experimental Procedures
Optical rotations were tested on an MCP300 (Anton Paar, Shanghai, China). UV data were recorded using a Shimadzu UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan). The NMR spectra were recorded on a Bruker Avance spectrometer (Bruker, Beijing, China) (compounds 2: 500 MHz for 1 H and 125 MHz for 13

Fungal Material
The fungal strain MLY-3 used in this study was isolated from fresh leaf of Bruguiear gymnorrhiza, which was collected from the Dongzhaigang Mangrove National Nature Reserve in Zhuhai, China, in April 2018. The strain was identified as Epicoccum nigrum (compared to no. MW081246.1) upon the analysis of ITS sequence data of the rDNA gene. The ITS sequence data obtained from the fungal strain have been submitted to GenBank with accession no. MZ407636. A voucher strain was deposited in our laboratory.

Fermentation, Extraction, and Isolation
The fungus Epicoccum nigrum MLY-3 was fermented on solid cultured medium (sixty 1000 mL Erlenmeyer flasks, each containing 50 g of rice and 50 mL 3‰ of saline water) for 30 days at 25 • C. The cultures were extracted three times with MeOH to yield 10.5 g of residue. Then, the crude extract was eluted by using gradient elution with petroleum ether/EtOAc from 9:1 to 0:10 (v/v) on silica gel CC to get six fractions (Fr.1-Fr.  Table 1.  Table 2.

X-Ray Crystallographic Data
Yellow crystal of compound 1 was obtained from MeOH-CH 2 Cl 2 at room temperature by slow evaporation and measured on an Agilent Xcalibur Nova single crystal diffractometer with Cu Kα radiation.
The The DPPH· radical scavenging capacities of compounds 1-10 were determined utilizing the reported method [31]. The DPPH· radical scavenging test was performed in 96-well microplates. Samples (100 µL) with a final concentration range of 6.25-100 µM were added to 100 µL of 0.16 mM DPPH· in MeOH. An ascorbic acid positive control was prepared at the same concentrations as the test samples (Table 3). Absorbance was measured at λ = 517 nm after 30 min of incubation in the dark. The DPPH· radical scavenging activity was calculated using the formula: DPPH· radical scavenging activity (%) = [(Abs control − Abs sample )/Abs control ] × 100.
3.5.2. ABTS· (2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) Radical Cation Scavenging Activity The ABTS· scavenging activity of compounds 1-10 was also resolved according to the ABTS· method (Beyotime Institute of Biotechnology, China) with a slight modification. In brief, ABTS· radical cation solution was prepared by mixing ABTS· solution with oxidant solution in equal quantities and allowing them to react in the dark at room temperature for 16 h before use. Then, the solution was diluted by mixing 1 mL working solution with 20 mL of 80% ethanol. A fresh ABTS· solution was prepared for each assay. Samples (100 µL) with a final concentration range of 12.5-100 µM were mixed with 100 µL of fresh ABTS· solution, and the mixture was left at room temperature for 6 min. Then, the absorbance was measured at 734 nm. Ascorbic acid was used as a reference compound. The ABTS· radical scavenging activity was calculated as follows: ABTS· radical scavenging activity (%) = [(Abs control − Abs sample )/Abs control ] × 100.