A new butenolide derivative from the deep-sea fungus Aspergillus terreus SCSIO FZQ028

Abstract A new butenolide derivative (±)-asperteretal F (1) and related congener (2) recently reported containing an unusual 2-benzyl-3-phenyl substituted lactone core, together with five known compounds (3–7) were isolated and characterized from the fungus Aspergillus terreus. SCSIO FZQ028 derived from a deep-sea sediment of South China Sea. Their chemical structures were established on the basis of 1D- and 2D-NMR spectroscopic data, and HR-ESI-MS analysis. Additionally, all the compounds were evaluated for the antioxidative activities against DPPH, cytotoxic activities against two tumor cell lines (SF-268 and HepG-2), and antimicrobial activities. Compounds 2-4, and 7 showed significant activities against DPPH with IC50 ranging from 5.89 to 10.07 μg/mL. Compounds 2 and 4 showed moderate antimicrobial activities against all four tested bacteria.


Introduction
The fungus Aspergillus terreus is ubiquitous in the marine benthos and terrestrial plants, which has been reported to be one of the most incredible chemical factories of secondary metabolites with intriguing pharmaceutical activities (Sun et al. 2013). Specifically, the famous statins drug lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase for lowering cholesterol to prevent cardiovascular disease, was mainly produced by A. terreus (Alberts et al. 1980;Manzoni and Rollini 2002). In addition, a series of compounds such as alkaloids (Cai et al. 2014;Lhamo et al. 2015), terpenoids (Liu et al. 2016;Chaiyosang et al. 2016;Liu et al. 2017;Shaaban et al. 2018;Wu et al. 2018), polyketides (Yoo et al. 2005), and butenolides (Gao et al. 2013;Elkhayat et al. 2016) were also isolated from these fungus. Butenolides, among all the above compounds, have attracted widespread attention for their prominent bioactivities, such as antimicrobial (Ibrahim et al. 2015), antioxidant (Dewi et al. 2015), cytotoxic (Gu and Qiao 2012), and a-glucosidase inhibitory activities (Wang et al. 2016). During exploring bioactive fungal strains from deep sea (Wang et al. 2012;Huang et al. 2012;Zhong et al. 2018aZhong et al. , 2018b, A. terreus SCSIO FZQ028 was isolated and identified from the sediment collected in the South China Sea. With the aim of searching for structurally novel and biologically potent compounds, a comprehensive chemical investigation was performed on A. terreus SCSIO FZQ028, which led to the discovery of a new butenolide derivative (1), along with six known ones (2-7) ( Figure 1). Herein, the isolation, structure elucidation, and bioactivities of these compounds are described.

Result and discussion
(±)-Asperteretal F (1) was obtained as a yellowish oil and assigned the molecular formula C 22 H 22 O 6 from HR-ESI-MS data (m/z [M þ Na] þ 405.1318, calcd for 405.1308), indicating 12 degrees of unsaturation. Its IR spectrum showed absorption bands at max 3336, 1737, 1608 cm À1 , indicating the presence of hydroxyl group, and a,b-unsaturated lactone rings (Parvatkar et al. 2009). The 1 H NMR spectrum showed an A 2 B 2 system at d H 7.47 and 6.82 (d, 2H, J ¼ 8.8 Hz), revealing the presence of a typical para-disubstituted benzene moiety (Rao et al. 2000). Three aromatic signals (d H 6.93, d, 1H, J ¼ 8.3 Hz; d H 6.90, s, 1H; d H 6.64, d, 1H, J ¼ 8.3 Hz) were indicative of the presence of a 1,3,4-trisubstituted benzene ring in the molecule. A methylene group was inferred from the signals at d H 3.74 (2H, s), and 2.92, 2.64 (each 1H, d, J ¼ 16.7 Hz), and two methyl groups were inferred from the signals at d H 1.28 (3H, s) and d H 1.21 (3H, s). The 13 C NMR and DEPT spectra (Table S1) suggested the presence of 22 carbon signals, which were attributable to two methyls, two methylenes, nine methines (two oxygenated methines, seven olefinic carbons), and nine quaternary carbons (one carbonyl carbon, one oxygenated quaternary carbon, two oxygenated olefinic carbons, five olefinic carbons). These data showed close similarity to those of asperteretal E (2) (Sun et al. 2018) except for the replacement of the dihydropyran moiety (d H 2.66, d C 21.9; d H 1.72, d C 32.1; d C 73.8) of 2 with a three-membered epoxy ring (d H 2.64 and 2. 92, d C 32.1; d H 3.71,d C 70.5; d C 77.9), implying compound 1 was a homologue of 2 which had a rare 2-benzyl-3-phenyl substituted pattern. In addition, the key HMBC correlations ( Figure S1) from H 2 -5 (d H 3.74) to C-1 (d C 175.16), C-2 (d C 125.69) and C-3 (d C 158.45), from H-2' (d H 7.47) to C-3 (d C 158.45) and C-4' (d C 160.96) substantiated that a benzyl and a phenyl group were attached at C-3 and C-2 of the c-butenolide, respectively. Therefore, the planar structure of 1 was identified as shown and named as asperteretal F. Compound 1 was the first report of 2,3-disubstituted butenolide derivative with an unexpected three-membered epoxy ring in side chain.
Several butenolides had been reported as racemate manners, such as (±) asperteretones A-D (Liu et al. 2018a), and (±) versicolactone B (Liu et al. 2018b).The low optical rotation value and inapparent Cotton effect in ECD spectrum of 1 implied that it could be also existed as a racemate. Unfortunately, by using various chiral columns and mobile phase systems, the enantiomeric separation of 1 was still not successful. Maybe the interconversion of the two enantiomers in the solvent was too fast to be separated (Sun et al. 2018).
All the compounds were evaluated for their antioxidative activities by DPPH method (Table S2), cytotoxic activities against two tumor cell lines (SF-268 and HepG-2), and inhibitory activities against four microbial strains (Table S3). Compounds 2-4, and 7 exhibited significant activities against DPPH with IC 50 values ranging from 5.89 to 10.07 lg/mL, which were comparable to the positive control ascorbic acid (IC 50 : 5. 13 lg/mL). Compounds 2 and 4 showed antimicrobial activities against all four tested bacteria. No compounds showed potent cytotoxic activities.

Experimental section
3.1. General experimental procedures NMR spectra were recorded on an AVANCE III HD-700 spectrometer (Bruker, Billerica, MA, USA) with TMS as internal standard. HR-ESI-MS spectra data were performed on a MaXis quadrupole-time-of-flight mass spectrometer. Optical rotations were measured on an MCP 500 automatic polarimeter (Anton Paar, Graz, Austria) with MeCN as solvent. IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu, Tokyo, Japan). UV spectra were recorded on a UV-2600 spectrometer (Shimadzu). HPLC purification was performed on an Agilent 1260 HPLC equipped with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 Â 250 mm, 5 mm, 3 mL/min). Silica gel (200-300 and 300-400 mesh) and Sephadex LH-20 for column chromatography (CC) were purchased from Qingdao Marine Chemical Group Co. (Qingdao, China) and GE Healthcare (Uppsala, Sweden), respectively. All solvents used in CC and HPLC were of analytical grade (Tianjin Damao Chemical Plant, Tianjin, China) and chromatographic grade (Oceanpak, Goteborg, Sweden), respectively.

Materials
The strain A. terreus SCSIO FZQ028 was isolated from a deep-sea sediment sample (117 03"43' E, 20 04"38' N) at the depth of 1718 m collected from an open voyage to the South China Sea in April 2016. The strain was identified as A. terreus SCSIO FZQ028 by sequence analysis of rDNA ITS (internal transcribed spacer) region, which shared a similarity of 100% with A. terreus (accession no. KX792117). The strain was deposited in RNAM center, South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Fermentation and extraction
The strain A. terreus SCSIO FZQ028 was sub-cultured in 250 mL Erlenmeyer flasks each containing 50 mL of the seed medium (2.0% glucose, 1.0% peptone, 0.5% yeast extract, 3.0% sea salt, with the pH adjusted to 7.5). After growth at 28 C, 220 rpm for 2 days, 50 mL of seed cultures was inoculated into 1000 mL Â 180 Erlenmeyer flasks containing the liquid medium (20 g/L of maltose, 10 g/L of glucose, 20 g/L of mannitol, 1 g/L of corn syrup, 10 g/L of sodium glutamate, 3 g/L of yeast extract, 0.5 g/L of KH 2 PO 4 , 0.3 g/L of MgSO 4 Á7H 2 O, 2 g/L of CaCO 3 , 30 g/L of sea salt, and the pH was adjusted to 6.5 before sterilization) and incubated at 28 C under static stations for 1 month. The whole fermented broth (55 L) was filtered through cheesecloth to separate into filtrate and mycelia. The filtrate was concentrated under vacuum to about a quarter of the original volume and then extracted three times with EtOAc, while the mycelia were extracted three times with 80% Acetone/H 2 O. The acetone solution was evaporated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted three times with EtOAc to get another EtOAc solution. Two EtOAc solutions were combined and evaporated to afford 78 g crude extract.

Bioassays
3.5.1. Antioxidative assay 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay was used to measure the free radical scavenging activities of the compounds. Specific experimental operation used the methods of Yen and Chen (1995) with little modifications. 100 lL of 0.2 mM DPPH prepared in EtOH was mixed with 100 lL each of test sample ranging from 10 to 1000 lg/mL in 96-cell plates. The reaction mixture was incubated at room temperature in the dark for 30 min. The absorbance of the resulting solution was then measured spectrophotometrically at 517 nm. Ascorbic acid was used as positive control and EtOH was used as blank. A decrease in absorbance of the reaction mixture indicates higher free radical scavenging activity. The scavenging ability of the compounds was calculated using the standard equation (Liyana-Pathirana & Shahidi 2005). IC 50 values were calculated with the SPSS software using a non-linear curve-fitting method.

Cytotoxic assay
Cytotoxicity against SF-268 and HepG2 cell lines was assayed by the SRB (Sulforhodamine B) method (Zhong et al. 2018a). Taxol was used as a positive control.

Conclusion
We investigated the chemical constituents of the fungal strain A. terreus SCSIO FZQ028 isolated from a deep-sea sediment sample of the South China Sea, which led to the isolation and identification of a new butanolide derivative (±)-asperteretal F (1), together with six known compounds (2-7). All the compounds were evaluated for the antioxidant activities by DPPH methods, cytotoxic activities against two tumor cell lines (SF-268 and HepG-2), and antimicrobial activities. Compounds 2-4, and 7 showed significant activities against DPPH (IC 50 : 5.89-10.07 lg/mL). Compounds 2 and 4 showed moderate antimicrobial activities against all four tested bacteria. No compounds showed potent cytotoxic activities.

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