A new lactone from mangrove endophytic fungus Aspergillus sp. GXNU-A9

Abstract A new lactone, asperlactone A (1), and four known lactone derivatives 2–5 were isolated from the mangrove endophytic fungus Aspergillus sp. GXNU-A9. Their structures were elucidated based on high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) datum, extensive nuclear magnetic resonance (NMR) spectroscopic analysis, and comparison with literature data. The structure of 1 was further confirmed by single-crystal X-ray diffraction analysis, and the absolute configuration of 1 was established. Compounds 1–5 were evaluated for their anti-inflammatory activities against nitric oxide (NO) production, and compounds 1–5 showed moderate inhibitory activities with IC50 values ranging from 15.87 to 30.48 µM. Graphical abstract


Introduction
The family of lactone natural products is widely distributed in terrestrial and marinederived fungi and displays a diverse array of biological activities, including cytotoxic (Greve et al. 2008), antibacterial (Chaudhary et al. 2018), antiviral (Nothias-Esposito et al. 2019), antifungal (Wu et al. 2008), a-glucosidase inhibitory (Cai et al. 2018), insecticidal (Bai et al. 2019, anti-inflammatory (Tang Y et al. 2020), and immunosuppressive activities (Gao et al. 2021). Mangrove endophytic fungi are an important source of biologically active lactones (Carroll et al. 2020), which are produced mainly from genera Aspergillus (Liu et al. 2019), Penicillium Kaleem et al. 2021), Leptosphaerulina Chartarum (Zhang et al. 2018), Trichoderma (Tang X et al. 2020) and from Cladosporium species . For example, lactones such as cladocladosin A, penicoffeazine A and B were identified from various sourced fungi and were described to have potent antimicrobial activities Zhang et al. 2020).
In our continuing endeavor to search for novel and bioactive metabolites of mangrove endophytic fungi from Qinzhou city, China Zhou et al. 2020). The fungal strain Aspergillus sp. GXNU-A9 was collected from the leaves of a mangrove plant Acanthus ilicifolius L. The leaf extract exhibited nitric oxide (NO) production, and its HPLC profile revealed the presence of diverse chemical entities. This initial analysis prompted the rice medium fermentation of this strain and the liquid media of a new compound was isolated from this strain before . The fungal strain Aspergillus sp. GXNU-A9 was cultured on the rice medium containing 0.5% NaCl at 28 C for 28 days. Chemical investigation on the extracts resulted in the isolation of a new lactone, asperlactone A (1), together with four known metabolites 2-5 ( Figure 1). Compounds 1-5 were preliminarily screened for the ability to inhibit NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The results revealed that compounds 1-5 had moderate inhibitory activities with IC 50 values ranging from 15.87 to 30.48 mM. Herein, we report the details of the isolation, structure elucidation, and antiinflammatory evaluation of the isolates.
Compounds 1-5 were evaluated for their anti-inflammatory effects on the production of nitric oxide (NO) in the RAW 264.7 macrophage cell line exposed to the inflammatory stimulus by lipopolysaccharide (LPS) ( Table S2). Dexamethasone was used as a positive control (IC 50 ¼ 4.1 lM). Cell viability was measured by an MTT assay. At the effective concentration, all compounds showed no obvious cytotoxicity to the RAW 264.7 cells. The results showed that compounds 1-5 showed moderate inhibitory activities with IC 50 values ranging from 15.9 to 30.5 mM.

Fungal material and fermentation
The strain GXNU-A9 was collected from a leaf of mangrove Acanthus ilicifolius L., Oct., 2019, Qinzhou City, China. The fungal strain GXNU-A9 was identified as Aspergillus sp. based on the sequence of its internal transcribed spacer region (ITS) and morphology. ITS-rDNA of GXNU-A9 was submitted to GenBank and the accession number is: MT626059.

Anti-inflammatory assay
The anti-inflammatory effects of compounds 1-5 were examined on the production of nitric oxide (NO) in LPS-stimulated cells using a method according to our previously described method (Liu et al. 2020). The macrophage cell line was RAW 264.7, cells (5 Â 10 5 cells/mL) were grown in DMEM and cultured in 96-well plates at 37 C. Cells were pretreated with or without test compounds for 2 h, then incubated with LPS for 24 h. NO production was determined by measuring the nitrite concentration using the Griess reagent. Results are expressed as the means ± SD, n ¼ 3.

Conclusion
In this study, the chemical investigation of marine-derived fungus Aspergillus sp. GXNU-A9 resulted in the isolation of a new compound 1 and four known compounds 2-5. The evaluation of their anti-inflammatory activities was made by examining their effects on NO reduction in LPS-stimulated RAW 264.7 macrophage cells. Compounds 1-5 showed moderate inhibitory activities in these assays.

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