Bioactive Metabolites from Mangrove Endophytic Fungus Aspergillus sp. 16-5B

Chemical investigation of the endophytic fungus Aspergillus sp. 16-5B cultured on Czapek’s medium led to the isolation of four new metabolites, aspergifuranone (1), isocoumarin derivatives (±) 2 and (±) 3, and (R)-3-demethylpurpurester A (4), together with the known purpurester B (5) and pestaphthalides A (6). Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The absolute configuration of Compound 1 was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra, and that of Compound 4 was revealed by comparing its optical rotation data and CD with those of the literature. The structure of Compound 6 was further confirmed by single-crystal X-ray diffraction experiment using CuKα radiation. All isolated compounds were evaluated for their α-glucosidase inhibitory activities, and Compound 1 showed significant inhibitory activity with IC50 value of 9.05 ± 0.60 μM. Kinetic analysis showed that Compound 1 was a noncompetitive inhibitor of α-glucosidase. Compounds 2 and 6 exhibited moderate inhibitory activities.


Introduction
Diabetes is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period and can cause many serious complications, including diabetic ketoacidosis, non-ketotic hyperosmolar coma, cardiovascular disease, stroke, kidney failure, etc. [1][2][3]. The world Health Organization reported that there are 387 million people living with diabetes worldwide in 2014, and the number would increase to 592 million in 2035 [4]. Being a huge and growing global public health problem, diabetes dictates the urgent need to discover and develop new chemotherapeutic approaches to medical treatment.
Endophytic fungi have been demonstrated to be a rich and reliable source of biologically-active and/or chemically-novel compounds [5][6][7]. In the past decade, our research group has focused on the exploration of new bioactive metabolites from mangrove endophytic fungi collected from the South China Sea [8][9][10][11], including some potential α-glucosidase inhibitors [12][13][14][15]. Recently, a chemical investigation of the mangrove-derived fungus Aspergillus sp. 16-5B, from the leaves of Sonneratia apetala, had led to the isolation and characterization of four new compounds (1)(2)(3)(4), as well as two previously reported compounds (5,6) ( Figure 1). All compounds were evaluated for their α-glucosidase inhibitory activities. Details of the isolation, structural elucidation, as well as evaluation of the biological activity of these compounds are reported herein.
Compound 3 was obtained as a white amorphous powder. Its molecular formula was determined to be C12H12O6 on the basis of its HREIMS molecular ion cluster at m/z 252.0630 [M] + (calcd. for C12H12O6, 252.0628). Analysis of the 1 H and 13 C NMR data for 3 revealed the presence of nearly the same identical structural features as those found in 2, except that the carbon signal of C-4 (δC 69.8) in 2 was absent and replaced by a carboxyl group signal (δC 190.1), which indicated that Compound 3 was the oxidative product of Compound 2 ( Figure 4). However, the absence of any CD spectrum and zero optical rotation indicated that 3 was also a racemic mixture. Unfortunately, separation of 3's enantiomers also failed.    The structures of the known compounds were identified as purpuresters B (5) [18] (Table 3) and pestaphthalides A (6) [19] by comparison of their spectroscopic and optical rotation data with those reported in the literature. The structure of Compound 6 was further confirmed by single-crystal X-ray diffraction experiment using CuKα radiation [20]  All compounds were tested for their in vitro inhibitory activities against α-glucosidase along with the clinical α-glucosidase inhibitor acarbose (positive control) [21]. As a result (Table 4), Compound 1 was the most active and showed better inhibitory potential (IC50 = 9.05 ± 0.60 μM) than acarbose (IC50 = 553.7 ± 6.8 μM). In order to examine the inhibition type of Compound 1, further kinetic studies were carried out by the Lineweaver-Burk plot method. The results are shown in Figure 6, indicating that 1 was a noncompetitive inhibitor of α-glucosidase. Compounds 2 and 6 exhibited more efficacy than that of the positive control, with IC50 values of 90.4 ± 2.9 and 69.6 ± 3.5 μM, respectively. More details are given in the Supplementary Information.

General
Melting points were measured on an X-4 micro-melting-point apparatus (Cany Precision Instruments Co., Ltd., Shanghai, China) and are uncorrected. Optical rotations were recorded with an MCP 300 (Anton Paar, Shanghai, China) polarimeter at 25 °C. UV data were measured on a UV-240 spectrophotometer (Shimadzu, Beijing, China). IR spectra were recorded on a Nicolet Nexus 670 spectrophotometer (Thermo Fisher Scientific, Inc., Hudson, NH, USA) in KBr discs. CD data were measured on a Chirascan™ CD spectrometer (Applied Photophysics, London, UK). 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer (Bruker BioSpin Corporation, Bellerica, MA, USA) in CDCl3, acetone-d6 or methanol-d4. Chemical shifts were reported in δ (ppm), using Tetramethylsilane (TMS) as the internal standard, and coupling constants (J) were reported in Hertz (Hz). EIMS spectra were measured on a Thermo DSQ EIMS spectrometer and HREIMS on a Thermo MAT95XP high-resolution mass spectrometer. Single-crystal data were measured on an Agilent Gemini Ultra diffractometer (CuKα radiation). Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Huang Hai Chemical Group Co., Qingdao, China; G60, F-254) and Sephadex LH-20 (GE Healthcare, Buckinghamshire, UK) stationery phases.

Fungal Material
The mangrove endophytic fungal strain 16-5B used in this study was isolated from the leaves of Sonneratia apetala, which was collected from Dongzhaigang Mangrove National Nature Reserve in Hainan Island, China, in April, 2009. The strain was identified by Yayue Liu as Aspergillus sp., according to morphologic traits and molecular identification. Its 444 base pair internal transcribed spacer (ITS) sequence had 99% sequence identity to that of Aspergillus sp. (No. JF312217). The sequence data had been submitted to GenBank with Accession Number KP059102. A voucher specimen (Registration Number 16-5B) has been deposited at the School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, China.

Quantum Mechanical Calculation
In theoretical calculations, the preliminary conformational distribution search was performed by Spartan'10 software (Wavefunction, Inc., Irvine, CA, USA) using the MMFF94S force field. The corresponding minimum geometries were further fully optimized with the Gaussian 03 (Gaussian, Wallingford, CT, USA) program package at the B3LYP/6-31G (d) computational level as frequency calculations. The obtained stable conformers were submitted to CD calculation by the TDDFT cam-b3lyp/6-311+g (2d, p) method. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and OriginPro 8.5 (OriginLab, Ltd., Northampton, MA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.19 electron volt (ev). All calculations were performed with the High-Performance Grid Computing Platform of Sun Yat-Sen University.

Conclusions
Chemical investigation of Aspergillus sp. 16-5B, a marine endophytic fungus isolated from the leaves of Sonneratia apetala, led to the discovery of six compounds (1-6), including new compound aspergifuranone (1), two new pairs of enantiomers of isocoumarin derivatives (±) 2 and (±) 3, and (R)-3-demethylpurpurester A (4). The structures of the isolates were established by 1D and 2D NMR spectroscopic data, as well as ECD calculation and optical rotation data. Meanwhile, the previously reported Compound 6 was obtained as a suitable crystal and confirmed by single-crystal X-ray diffraction experiment using CuKα radiation for the first time. All isolated compounds were evaluated for their α-glucosidase inhibitory effects. Compounds 1, 2 and 6 exhibited more potent inhibitory effects against α-glucosidase activity than the clinical α-glucosidase inhibitor acarbose. Meanwhile, further mechanistic analysis showed that Compound 1 exhibited noncompetitive inhibition characteristics.