A new sesquiterpenoid from the aconitum-derived fungus Aspergillus fumigatus M1

Abstract A new bergamotane sesquiterpenoid, fumigatanol (1), along with nine known compounds (2-10) were isolated from the Aconitum-derived fungus Aspergillus fumigatus M1. Their structures were established on the basis of extensive spectroscopic analyses, ECD experiment and NMR computational method. Antibacterial and cytotoxic activities of compound 1 were evaluated and no obvious antibacterial and cytotoxic activities were observed at concentrations of 256 μg/mL and 40.00 μM, respectively. Graphical Abstract


Introduction
Aconitum brevicalcaratum Diels is a traditional Chinese herbal medicine whose roots have been reputedly used for the treatment of cough, cold and various kinds of pain, and it is mainly distributed in the northwest of Yunnan Province, China (Wang et al. 2010). The main active compounds of A. brevicalcaratum are diterpenoid alkaloids (DAs) Meng et al. 2017;Guo et al. 2018). The fungal strain Aspergillus fumigatus has been widely studied (Frisvad et al. 2009;Li et al. 2019;Zhao et al. 2010;Zhang et al. 2021). Previous investigations found that metabolites of A. fumigatus have diverse biological activities, such as antitubercular (Song et al. 2021), cytotoxic (Zhao et al. 2009), antioxidant and a-glucosidase inhibitory activities . And its typical metabolites are diketopiperazines (Wang et al. 2008(Wang et al. , 2012Ishikawa et al. 2010), terpenoids (Wang et al. 2015), steroids (Liang et al. 2015), and alkaloids (Liang et al. 2015;Zou et al. 2021).
Previously, during the investigations on the secondary metabolites of endophytic fungi from Aconitum, we reported several novel bioactive secondary metabolites including sesterterpenoid from Penicillium roqueforti , isoprenoid epoxycyclohexenone from P. expansum (Wang et al. 2019), indole alkaloid from P. polonicum (Cai et al. 2022), cycloheptane from P. crustosum ) and cytochalasin from Arthrinium arundinis (Shu et al. 2022). As part of our continued search for new bioactive products produced by endophytic fungi from Aconitum, an investigation of the secondary metabolites of A. fumigatus separated from the roots of A. brevicalcaratum led to the isolation of a new bergamotane sesquiterpenoid, fumigatanol (1) (Figure 1). The structure of compound 1 was elucidated by HR-ESI-MS, 1 D/2D NMR data, ECD experiment and NMR computational method.

Results and discussion
Compound 1 was obtained as colorless oil. Its molecular formula was established as C 15  82) with C-1, C-5 and C-6 indicated the presence of 6-methylbicyclo[3.1.1] heptane skeleton. In addition, the main differences between compound 1 and known compound 2 were the presence of one additional oxygen-bearing carbon (d C 75.7 d) and the absence of two alkenyl carbons (d C 125.4 d, 140.5 d) in 1. These results, together with the molecular formula revealed 1 might share the same skeleton with compound 2. The HMBC correlations between H 3 -14 and the oxygenated carbons C-6 and C-8 established the connection between these two moieties. Therefore, the planar struture of compound 1 was established as 5,8,10,11-tetrahydroxybergamot.
In addition, the antibacterial and cytotoxic activities of compound 1 were evaluated. No obvious antibacterial and cytotoxic activities were observed at concentrations of 256 lg/mL and 40.00 lM, respectively.

General procedure
Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Nicolet Magna-IR 550 spectrometer (Thermo Nicolet Co., Madison, WI, USA) was used for scanning IR spectroscopy. NMR spectra including 1 D and 2 D spectra were recorded on Bruker Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. HR-ESI-MS analyses were recorded with Agilent G3250AA (Agilent, Santa Clara, CA, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, MA, USA). Silica gels (200-300 mesh or 300-400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Xciences AB) and Lichroprep RP-18 gel (40-63 lm, Merck, Darmstadt, Germany) were used for column chromatography. Fractions were monitored by TLC and visualized by dipping into vitriolated chromogenic agent or under UV light. All the solvents used in the experiment were redistilled before use.

Fungal material and fermentation
The fungus Aspergillus fumigatus was isolated from the healthy roots of Aconitum brevicalcaratum Diels, collected from Kunming, Yunnan Province, China in November 2018. A BLAST search result indicated that the sequence was most similar 99% with Aspergillus fumigatus (compared to MG659651.1). It was preserved in the Key Laboratory of Medicinal Chemistry for Natural Resources at Yunnan University. The strain was activated in potato dextrose agar (PDA; 1 L of water, 200 g of potato, 20 g of dextrose, and 15 g of agar, natural pH) slant culture medium in a constant-temperature incubator at 28 C for 3 days. And then the fungus was cultivated at 28 C for 7 days while shaking at 160 rpm in 500 mL flask containing 200 mL potato dextrose broth per flask (PDB; 1 L of water, 200 g of potato, 20 g of dextrose, natural pH) which had been sterilised at 121 C for 30 min before.

Antibacterial activity assay
Antibacterial activity assays were performed in sterilized 96-well microplates using a microdilution method described previously (Dong et al. 2015). The 18-h-old bacterial cultures from Staphylococcus aureus (ATCC 25917), Bacillus subtilis (ATCC 6633), and Escherichia coli (ATCC 25922) was added to LB broth medium (1 L of water, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl) to reach 1 Â 10 5 colony forming units/ mL. Compound 1 and the positive control were dissolved in dimethylsulfoxide (DMSO), and their final concentrations ranged from 0.125 to 512 lg/mL as deter-mined by using a 2-fold serial dilution method. The wells containing strains and diluted samples were incubated at 37 C (24 h). Kanamycin has prominent antibacterial activity against B. subtilis, E. coli and S. aureus was introduced in the experiments as a positive control. All experiments were repeated two times. CX21BIM-set 5 microscope was used to monitor the growth of test strains. The MIC was defined as the lowest antibiotic concentration that produced complete growth inhibition of the microorganisms.

Cytotoxic activity assay
Colorimetric assays were performed to evaluate compound activity. The following human tumor cell lines were used: the SMMC-7721 human hepatoma cell line, the MCF-7 human breast cancer cell line, the A549 lung cancer cell line, the SW480 colon cancer cell line, and the HL-60 human Leukemia cell line. Cells were seeded in 96-well plates at a density of 1.0 Â 10 4 cells/well in 100 lL of complete culture medium. After cell attachment 3 overnight, the medium was removed, and each well was treated with 100 lL of medium containing 0.1% DMSO or appropriate concentrations of the test compound and the positive control taxol and incubated with cells at 37 C for 48 h in a 5% CO 2 -containing incubator. Proliferation was assessed by adding 20 lL of MTS (Promega) to each well in the dark, after 90 min of incubation at 37 C. The optical density was recorded on a microplate reader at 490 nm. Three duplicate wells were used for each concentration, and all the tests were repeated three times. The IC 50 value of each compound was calculated by Reed and Muench's method.

NMR computational methods
The theoretical calculation of 1 was performed using the Gaussian 09 Program (Frisch et al. 2013). NMR computational methods, an effectively empirical rule, were established to analyze the most likely relative configuration of compound 1 . First of all, the conformers which showed more than 1% Boltzmann populations of each diastereomer (energy cutoff of 5.0 kcal/mol) of compound 1 were optimized at the PCM/mPW1PW91/6-311 þ G(d,p) level of theory. Then, the conformers were subjected to 13 C NMR calculations using the gauge including the atomic orbital (GIAO) method at the PCM/mPW1PW91/6-311 þ G(d,p) level of theory for DP4þ calculations. The calculations in the solution were carried out using the polarizable continuum model, PCM, for methanol (the solvent used experimentally).
The magnetic shielding constants (r) were computed using the gauge including atomic orbitals (GIAO) method, the method of choice among the different approaches to solve the gauge origin problem, with the mPW1PW91 functional (one of the most reliable DFT functionals for NMR calculations). Single-point NMR calculations were carried out in the gas phase (with the 6-31 G Ã basis set) and in solution (with the 6-31 G ÃÃ basis set) using the polarizable continuum model (PCM) with methanol as a solvent for artificial neural networks pattern recognition analysis . The NMR shielding constants were subjected to Boltzmann averaging over all conformers. The ANN training was done using the Neural Network Toolbox incorporated in MATLAB 7.0.22.

Conclusion
A new bergamotane sesquiterpenoid, fumigatanol (1), along with nine known compounds (2-10) possessing different structures were isolated from the fungus A. fumigatus M1 which was separated from the roots of A. brevicalcaratum. The structure of 1 was established on the basis of extensive spectroscopic analyses. Although the absolute configurations of C-8 and C-10 could not be confirmed by ECD experiment, strict NMR calculations including DP4þ and ANN methods have assigned their most possible absolute configurations. Compound 1 showed no antibacterial and cytotoxic activities.

Supplementary material
Supplementary material relating to this article is available online.