Aspergivones A and B, two new flavones isolated from a gorgonian-derived Aspergillus candidus fungus

Abstract Two new flavones, aspergivones A (1) and B (2), were isolated from the fungus Aspergillus candidus cultured from the gorgonian coral Anthogorgia ochracea collected from the South China Sea. The structures of 1 and 2 were elucidated by NMR and MS methods and comparison with relatively known compounds. Only 2 showed slight inhibitory activity against alpha-glucosidase with an IC50 value of 244 μg/mL. Compounds 1 and 2 were also evaluated for their cytotoxic and antibacterial activities.


Introduction
Flavonoids are a group of natural compounds with variable phenolic structures and are found in plants (Hichri et al. 2011;Kumar & Pandey 2013;Gholami et al. 2014). Till date, around 8000 naturally occurring flavonoids have been identified and characterised which are abundantly deposited in vegetables, stems, fruits, seeds and other organs (Ververidis et al. 2007). However, flavonoids from marine fungi are relatively uncommon. Flavonoids become one of the important agents to treat cancer, oxidant stress, pathogenic bacteria, inflammations and cardiovascular dysfunctions (Veitch & Grayer 2011;Koirala et al. 2016), which have attracted much interests of many chemists and pharmacists (Simkhada et al. 2009;Bansal et al. 2012;Stahlhut et al. 2015;Hou et al. 2016).
Marine micro-organisms have proven to be rich sources of structurally novel and biologically active secondary metabolites that have become interesting and significant resources for drug discovery (Blunt et al. 2016). In the course of our ongoing research for bioactive secondary metabolites from marine invertebrates, especially from corals collected from the South China Sea (Shao et al. 2010;Yang et al. 2012;Chen, Fu, et al. 2014;Liu et al. 2014;Sun et al. 2014;Jia et al. 2015;Hou et al. 2015). Two new flavones, aspergivones A (1) and B (2), were isolated from fungus Aspergillus candidus. Herein, we report the isolation, structure elucidation and biological activity of these two compounds.

Results and discussion
The fungus A. candidus was cultured for two months on sea water-added rice solid medium. The culture was extracted by etOAc to obtain an organic extract, which was separated and purified by a combination of silica gel column chromatography, Sephadex LH-20 and semi-preparative HPLC to yield 1 and 2 ( Figure 1).
Aspergivone A (1) was obtained as an yellowish powder. Its molecular formula was established as C 19 H 17 ClO 7 (11° of unsaturation) on the basis of the HReSIMS ion at m/z 393.0736 [M + H] + . Furthermore, the ratio of [M + Na] + isotopic peaks (415:417/3:1) clearly indicated the presence of one chlorine atom. The 1 H NMR (500 MHz, CdCl 3 ) spectrum (Table S1) revealed three double doublets (δ H 7.56, δ H 7.04 and δ H 7.69) and a singlet (δ H 6.44) in the aromatic part, and four methoxy protons (δ H 4.00, δ H 4.00, δ H 3.91 and δ H 3.86). These spectroscopic features indicate 1,2,3-tri-substituted benzenic ring. The 13 C NMR (125 MHz, CdCl 3 ) spectrum data indicated that there are 19 carbon resonances including four methoxyl group, four sp 2 methine groups and 11 quaternary carbon atoms in 1. These spectroscopic features suggest that 1 belongs to the family of flavones. Comprehensive interpretation of the 1 H and 13 C NMR data indicating that compound 1 was similar to chlorflavonin ( Figure 2) which was isolated from the fungus Acanthostigmella sp. (Watanabe et al. 2001) and Aspergillus sp. AF119 (Liu et al. 2012). The most obvious differences were the presence of one more methoxyl group (δ H 4.00) and the absence of one hydrogen bond hydroxyl group (δ H 12.42) in 1 compared with those in chlorflavonin. The key HMBC ( Figure S1) correlations from H-6 (δ H 6.44) to C-4a (δ c 109.1) and C-8 (δ c 130.6); from H-5′ (δ H 7.04) to C-3′ (δ c 124.1), C-1′ (δ c 119.9), C-2′ (δ c 151.0) indicated that the location of the methoxyl group was attached to C-5 instead of C-2′. On the basis of other key HMBC correlations ( Figure S1), the structure of compound 1 was determined as shown in Figure 1.  (Table S2) revealed four double doublets (δ H 7.69, δ H 7.35, δ H 6.99 and δ H 6.99) and a singlet (δ H 6.34) in the aromatic part and four methoxy protons (δ H 3.90, δ H 3.90, δ H 3.78, δ H 3.78). These spectroscopic features indicate there is one 1,2-disubstituted benzenic ring. The chemical shift at δ H 8.00 indicated that a hydroxyl group was presented in compound 2. The 13 C NMR (125 MHz, CdCl 3 ) spectrum data showed that there are 19 carbon resonances including 4 methoxyl group, 5 sp 2 methine groups and 10 quaternary carbon atoms in 2. Careful analysis on the 1 H NMR, 13 C NMR and MS data suggested that compound 2 was similar to 1. The only difference is that the chlorine atom in 1 was substituted by one aromatic proton (δ H 7.35) in 2. This was further confirmed that no isotopic peaks were observed in 2. detailed inspection of the 1 H and 13 C NMR data, and the structure of compound 2 was determined as shown in Figure 1. There are no references about compound 2, although its structure was listed in SciFinder Scholar.

Conclusion
The paper reported two new flavones, aspergivones A (1) and B (2), which were isolated from the fungus A. candidus. Compound 2 showed slight inhibitory activity against alpha-glucosidase with an IC 50 value of 244 μg/mL.

Supplementary material
experimental details related to this article along with Figures S1-S19 and Tables S1 and S2 are available online.

Disclosure statement
No potential conflict of interest was reported by the authors.