Polyketide Derivatives, Guhypoxylonols A–D from a Mangrove Endophytic Fungus Aspergillus sp. GXNU-Y45 That Inhibit Nitric Oxide Production

Four undescribed compounds, guhypoxylonols A (1), B (2), C (3), and D (4), were isolated from the mangrove endophytic fungus Aspergillus sp. GXNU-Y45, together with seven previously reported metabolites. The structures of 1–4 were elucidated based on analysis of HRESIMS and NMR spectroscopic data. The absolute configurations of the stereogenic carbons in 1–3 were established through a combination of spectroscopic data and electronic circular dichroism (ECD). Compounds 1–11 were evaluated for their anti-inflammatory activity. Compounds 1, 3, 4, and 6 showed an inhibitory activity against the production of nitric oxide (NO), with the IC50 values of 14.42 ± 0.11, 18.03 ± 0.14, 16.66 ± 0.21, and 21.05 ± 0.13 μM, respectively.


Introduction
Marine-derived endophytic fungi have drawn considerable attention for drug discovery, and have been shown to produce various constituents, including sesquiterpenes, alkaloids, and polyketides [1]. Fungi are prolific producers of a variety of biologically active secondary metabolites, including anti-inflammatory, antibiotics, and cytotoxic compounds [1,2]. Lately, the investigation of the constituents of a fungus Pleosporales sp., isolated from diverse marine environments has led to the discovery of broad-spectrum cytotoxic secondary metabolites, such as dipleosporalones A and B [3]. In recent years, metabolites discovered from marine-derived fungi have been shown to display a broad range of promising biological activities [1][2][3][4][5][6]. Our group has reported a series of polyketides and structurally related polyketide derivatives from the culture of mangrove endophytic fungi [7][8][9][10].
As part of our ongoing project to discover anti-inflammatory polyketide derivatives from mangrove endophytic fungi, modifications of the composition of the culture medium were employed to reinvestigate the secondary metabolites of Aspergillus sp. GXNU-Y45, isolated from a fresh branch of the mangrove plant Acanthus ilicifolius L. Chemical investigation of its culture extracts resulted in the isolation of four undescribed polyketides, guhypoxylonols A (1), B (2), C (3), and D (4), together with seven previously reported metabolites (5)(6)(7)(8)(9)(10)(11) (Figure 1). Preliminarily screening of 1-11 in Supplementary Materials for their ability to prevent NO production of lipopolysaccharide (LPS)-stimulated RAW264.7 cells showed that 1, 3, 4, and 6 have significant inhibitory potency. Herein we report the details of isolation, structure elucidation, and anti-inflammatory activity evaluation of 1, 3, 4, and 6.   9. Analysis of the 2D-NMR spectra ( Figure 2) revealed that the structure of 1 resembled that of the previously reported 6 [11] except for the chemical shift value of C-7 which appeared at δ C 76.4 CH, indicating that C-7 is oxygen-bearing.  The relative configuration of 1 was determined by the NOESY spectrum ( Figure 3) analysis. The NOESY correlations between H-1 (δ H 5.22) and OCH 3 -3 (δ H 3.29), OCH 3 -3 and H-6b (δ H 3.94), and H-6b and OH-7 (δ H 6.17) determined the relative configuration of 1 as 1S*3S*6bR*7S*. The experimental ECD spectrum of 1 was recorded ( Figure 4) and the calculated ECD spectrum of 1S3S6bR7S-1 fits well with the experimental ECD spectrum of 1, as shown in Figure 4. Since 1 has not been previously reported, it was named guhypoxylonol A.   Table 2) showed 12 carbon signals comprising six aromatic carbons of a benzene ring (δ C 157.5 C, 143.1 C, 128.5 CH, 124.8 C, 118.7 CH and 108.8 CH), two methoxyls (δ C 55.7 and 56.7), two methylene sp 3 (δ C 27.2 and 24.7), and two oxygenated methine sp 3 (δ C 69.8 and 67.8). The COSY spectrum (Table 2) of 2 displayed two isolated proton spin systems (H-1/H 2 -2/H 2 -3/H-4, and H-6/H-7/H-8). The HMBC spectrum showed correlations from the proton sinal at δ H 4.41 (1H, m, H-1) to δ C 24.7 (C-3), 118.7 (C-8), and 143.1 (C-8a), from δ H 4.35 (1H, t, J = 2.8 Hz, H-4) to δ C 157.5 (C-5), 27.2 (C-2), and 143.1 (C-8a). The 1 H and 13 C NMR spectra of 2 were very similar to those of nodulisporol [12]. The main difference between 2 and nodulisporol was the replacement of a hydroxyl group with a methoxy group at C-4. The relative configuration of 2 was determined from its NOESY spectrum, which showed correlations from H-1/H-3α (δ H 2.09), and H-4/H-3β (δ H 1.51) suggesting that H-1 and H-4 were on the opposite face. To establish the absolute configuration of C-1 and C-4, the ECD spectra of two simplified isomers (1S4S, and 1R4R) of 2 were calculated at the Cam-B3LYP/6-31+G(d,p) level of theory in methanol, and these calculated spectra were compared with the experimental spectrum of 2. The experimental ECD spectrum of 2 showed an excellent fit with the calculated ECD spectrum of 1S4S-2 (Figure 4), establishing the absolute configurations of C-1 and C-4 as 1S4S. Since 2 has never been reported, it was named guhypoxylonol B.

Anti-Inflammatory Activity
Compounds 1-11 were evaluated for their anti-inflammatory effects on the production of the NO in the RAW 264.7 macrophage cell line exposed to the inflammatory stimulus by lipopolysaccharide (LPS) ( Table 5) . Compounds 1, 3, 4, and 6 showed inhibitory activity against the production of NO, with the IC 50 values 14.42 ± 0.11, 18.03 ± 0.14, 16.66 ± 0.21, and 21.05 ± 0.13 µM, respectively. Dexamethasone was used as a positive control with IC 50 value of 16.12 ± 1.41 µM, while 2, 5, and 7-11 did not show any inhibitory activity under their safe concentrations. Table 5. Inhibitory activities of 1-11 on NO production in LPS-induced RAW 264.7 cells a .

Fungal Material
The strain GXNU-Y45 was isolated from a leaf of a mangrove tree Acanthus ilicifolius, October 2019, in Beihai City, China. The fungal strain GXNU-Y45 was identified as Aspergillus sp. based on the sequence of its internal transcribed spacer region (ITS) and morphology. ITS-rDNA of GXNU-Y45 was submitted to GenBank and the accession number is MT626059.

Fermentation, Extraction, and Isolation
The fungus was cultured in 60 × 1000 mL Erlenmeyer flasks each containing 50 g cooked rice and 60 mL of water (30 g sea salt, per liter pure water) or 300 mL medium (liquid media, 20.0 g dextrose, 20.0 g potatoes, 30 g sea salt, per liter pure water). The fungus was cultured in the medium and incubated at room temperature for 35 days.

Extraction and Isolation
The fermented material was extracted three times with EtOAc to obtain 16.8 g crude extract (liquid medium) and 20.2 g (solid medium). The crude extract was subjected to a silica gel VLC column, eluting with a stepwise gradient of petroleum ether-EtOAc

Anti-Inflammatory Assay
The anti-inflammatory effects of compounds 1-11 were examined on the production of the NO in LPS-stimulated cells using a method described in the literature [16].

Conclusions
The chemical investigation of a marine-derived fungus Aspergillus sp. GXNU-Y45 resulted in the isolation of four undescribed compounds (1)(2)(3)(4), and seven previously reported metabolites (5)(6)(7)(8)(9)(10)(11). Based on modifications of the culture medium strategy, the fungus Aspergillus sp. GXNU-Y45 was cultured in different media to stimulate a production of its metabolites. It was found that the fungus Aspergillus sp. GXNU-Y45 produced different metabolites in two culture media. The liquid medium can stimulate the fungus to produce a series of metabolites, 1, 5, 6, 7, 8, 9, 10, and 2 (a new precursor of 1). On the contrary the solid medium yeiled 3 and 4. Different compositions of the culture media represented a powerful tool to induce new metabolites from microorganisms. Preliminarily screening of 1-11 for their ability to prevent NO production of LPS-induced RAW264.7 cells showed that 1, 3, 4, and 6 exhibited significant inhibitory effects against NO release with IC 50 values of 14.42 ± 0.11, 18.03 ± 0.14, 16.66 ± 0.21, and 21.05 ± 0.13 µM, respectively. The inhibition of NO production by 1 and 6 was stronger than 5 and 7, which showed the same skeleton but differ only the presence of -OCH 3 at C-3. Compounds 2 and 8-11, which are precursors of 1, 5, 6, and 7, did not exhibit inhibitory effects against NO release. Compounds 3 and 4 exhibited remarkable inhibitory effects against NO release suggesting that the fully substituted benzene ring was essential for inhibition of the production of NO release. In summary, this study revealed that 1, 3, 4, and 6 could be considered as potential metabolites for further anti-inflammatory studies.