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Secondary Metabolites with α-Glucosidase Inhibitory Activity from the Mangrove Fungus Mycosphaerella sp. SYSU-DZG01

Four new metabolites, asperchalasine I (1), dibefurin B (2) and two epicoccine derivatives (3 and 4), together with seven known compounds (5–11) were isolated from a mangrove fungus Mycosphaerella sp. SYSU-DZG01. The structures of compounds 1–4 were established from extensive spectroscopic data and HRESIMS analysis. The absolute configuration of 1 was deduced by comparison of ECD data with that of a known structure. The stereostructures of 2–4 were further confirmed by single-crystal X-ray diffraction. Compounds 1, 8 and 9 exhibited significant α-glucosidase inhibitory activity with IC50 values of 17.1, 26.7 and 15.7 μM, respectively. Compounds 1, 4, 6 and 8 showed antioxidant activity by scavenging DPPH· with EC50 values ranging from 16.3 to 85.8 μM.


Introduction
Diabetes mellitus (DM), a chronic metabolic disorder disease, is caused by the lack of insulin secretion (type Idiabetes mellitus) or insufficient insulin sensitivity (type IIdiabetes mellitus) [1,2], and the typical characteristic of the latter is post-prandial hyperglycemia. α-Glucosidase is a kind of membrane-bounded enzyme which is mainly found in intestinal epithelium cells and leads to the increase of blood glucose levels by hydrolyzing the glycosidic bonds of a polysaccharide [3][4][5]. As a result of that, α-Glucosidase inhibitors (AGIs), such as acarbose, miglitol and voglibose have become a widespread medical treatment in type IIdiabetes mellitus according to their glycemic control ability [6,7]. Nevertheless, existing AGIs often cause many side effects including abdominal pain, flatulence, diarrhea and other gastrointestinal disorders [8,9]. Hence, many natural medicine chemists were attracted to develop α-glucosidase inhibitors with lower toxicity and side effects for potential use. Some new α-glucosidase inhibitors have been researched like flavipesolides A-C [10], asperteretal E [11] and so on [12,13], and the discovery of better α-glucosidase inhibitors is still an urgent need.

Structure Elucidation
Compound 4 was deduced to have a molecular formula of C10H10O6 from its HRESIMS spectrum with a deprotonated molecular ion at m/z 225.0407. The 1 H NMR (Table 3) in MeOH-d4 showed three singlets at δH 9.71, 3.90 and 2.08, according to the 13 C NMR and HSQC data, which were attributed to an aldehyde group (δC 194.7), a methoxy group (δC 52.9) and a methyl (δC 12.4), respectively. In addition, resonances of a carbonyl and an aromatic ring were observed in the 13 C NMR data. In the HMBC spectrum, the correlations of H-9 to C-2, C-3 and C-4 supported the connection of Me-9 to C-3, the correlations of H-7 to C-1 and C-6 indicated the linkage of aldehyde group and C-1. Meanwhile, the carbonyl (δC 169.9, C-8) had the HMBC correlation from H-10 (δH 3.90), further indicated the presence of methyl ester. With the assistance of single-crystal X-ray (Figure 4), the structure of compound 4 was clearly confirmed. Compound 3 was purified as a colorless crystal whose molecular formula was deduced as C 10 (Table 3) displayed three aromatic proton resonances (δ H 7.13, 6.82 and 6.69), an oxygenated methylene (δ Ha 5.12, δ Hb 5.02), two oxygenated methines (δ H 5.06 and 3.97), and a methyl (δ H 1.20). The 13 C spectrum revealed 10 signals, indicating an aromatic ring, a methylene, two methines and a methyl. In the 1 H-1 H COSY spectrum, the ortho-trisubstitution on the aromatic ring was confirmed by the cross-peaks of H-4/H-5/H-6. Moreover, the 1 H-1 H COSY spectrum showed correlations from H-10 to H-9 and H-11, and the chemical shift of C-10 (δ C 70.6) showed the hydroxyl was attached to C-10. Subsequently, the HMBC correlations between H-6 and C-1/C-2 determined the linkage of 1-OH to C-1, and the correlations between H-7 and C-1/C-2/C-3, H-9 and C-2/C-3/C-10 established the presence of a phthalan ring. The same relative configuration of C-9 and C-10 was clearly deduced under the guidance of single-crystal X-ray ( Figure 4). Hence, the absolute configuration of 3 was determined as 9R, 10R.
Compound 4 was deduced to have a molecular formula of C 10 H 10 O 6 from its HRESIMS spectrum with a deprotonated molecular ion at m/z 225.0407. The 1 H NMR (Table 3) in MeOH-d 4 showed three singlets at δ H 9.71, 3.90 and 2.08, according to the 13 C NMR and HSQC data, which were attributed to an aldehyde group (δ C 194.7), a methoxy group (δ C 52.9) and a methyl (δ C 12.4), respectively. In addition, resonances of a carbonyl and an aromatic ring were observed in the 13 C NMR data. In the HMBC spectrum, the correlations of H-9 to C-2, C-3 and C-4 supported the connection of Me-9 to C-3, the correlations of H-7 to C-1 and C-6 indicated the linkage of aldehyde group and C-1. Meanwhile, the carbonyl (δ C 169.9, C-8) had the HMBC correlation from H-10 (δ H 3.90), further indicated the presence of methyl ester. With the assistance of single-crystal X-ray (Figure 4), the structure of compound 4 was clearly confirmed.

Biological Evaluation
Compounds 1-11 were tested for their inhibitory effects against α-glucosidase, and antioxidant activity. As seen in Table 4

General Experimental Procedures
UV data were measured on a UV-Vis-NIR spectrophotometer (Perkin Elmer, Waltham, UK). IR spectrum data were recorded using a Bruker Vector spectrophotometer 22. Melting points were tested on a Fisher-Johns hot-stage apparatus which were uncorrected. Optical rotations were recorded using an MCP300 (Anton Paar, Shanghai, China). HRESIMS data were conducted on an Ion Mobility-Q-TOF High-Resolution LC-MS (Synapt G2-Si, Waters). The ECD experiment data were measured with J-810 spectropolarimeter (JASCO, Tokyo, Japan). The NMR spectra were recorded on Bruker Avance spectrometer (Bruker, Beijing, China) (Compounds 1 and 3: 500 MHz for 1 H and 125 MHz for 13 C, respectively; compounds 2 and 4: 400 MHz for 1 H and 100 MHz for 13 C). Column chromatography (CC) was carried out on silica gel (200-300 mesh, Marine Chemical Factory, Qingdao, China) and sephadex LH-20 (Amersham Pharmacia, Piscataway, NJ, USA).

Fungal Materials
The fungus used in this research was isolated from the fruit of the marine mangrove plant Bruguiera collected in 2014 in Hainan Dongzhai Harbor Mangrove Reserve by using the standard protocol. The strain was identified as Mycosphaerella sp. (compared to no. KX067865.1) upon the analysis of ITS sequence data of the rDNA gene. The ITS sequence data obtained from the fungal strain has been submitted to GenBank with accession no. MN194208. A voucher strain was deposited in our laboratory.

X-Ray Crystallographic Data
Colorless crystals of compounds 2-4 were obtained from MeOH-CH 2 Cl 2 at room temperature by slow volatilization, and examined on an Agilent Xcalibur Nova single crystal diffractometer with Cu Kα radiation. The

Inhibitory Activity of α-Glucosidase
The α-glucosidase inhibitory activity was assayed according to the reported method [29]. The inhibitory activity of α-glucosidase was tested in the 96-well plated with 100 mm PBS (KH 2 PO 4 -K 2 HPO 4 , pH 7.0) buffer solution each. Compounds 1-11, acarbose and 1-deoxynojirimycin (positive control) were dissolved in DMSO, the substrate (p-nitrophenyl glycoside, 5 mM) were dissolved in PBS buffer solution and enzyme solutions (2.0 units/mL) were prepared. The assay was conducted in a 100 µL reaction system containing 20 µL enzyme stock solution, 69 µL PBS buffers and 1 µL of DMSO or testing materials. After 10 min incubation at 37 • C, 10 µL of the substrate was added and incubated for 20 min at 37 • C. The Absorbance which measured by a BIO-RAD (iMark) microplate reader at 405 nm was used to calculate the inhibitory activity according to the equation: η (%) is the percentage of inhibition; B is the assay medium with DMSO; S is the assay medium with compound. The results of IC 50 values were calculated by the nonlinear regression analysis. Acarbose and 1-deoxynojirimycin were used as positive controls.

Antioxidant Activity
The DPPH· scavenging was assayed according to the reported method [30]. The DPPH radical scavenging test was performed in 96-well microplates. Testing materials (compounds 1-11) were added to 150 µL (0.16 mmol/L) DPPH solution in MeOH at a range of 50 µL solutions of different concentrations (2, 25, 50 and 100 µM). After 30 min, absorbance at 517 nm was measured and the percentage of activity was calculated. Ascorbic acid was used as a positive control.

Conclusions
In summary, four new metabolites, including one new asperchalasine I (1), dibefurin B (2), two epicoccine derivatives (3,4) and seven known compounds were isolated from the fungus Mycosphaerella sp. SYSU-DZG01. The structures of 1-11 were established by spectroscopic data and the absolute configuration of compounds 1-3 was determined in this research. Compound 1 possesses a unique T-shaped skeleton. All of the compounds were tested for their biological activities.  Figure  S29: The LC-HRESIMS analysis profile of crude extract.
Author Contributions: P.Q. contributed to isolation, structure elucidation and wrote the paper; Z.L. contributed to the analysis of the NMR data and structure elucidation. Y.C. contributed to the analysis of the NMR data and biological tests. R.C. contributed to the spectral analysis. Z.S. and G.C. guided the whole experiment and revised the manuscript.