The optical rotations, CD, and FT-IR spectra were measured with a Perkin-Elmer 341 polarimeter (PerkinElmer, Waltham, MA, USA), JASCO J-810 spectrometer (Jasco Corporation, Japan), Bruker Vertex 70 FT-IR spectrophotometer (Bruker, Karlsruhe, Germany), respectively. The UV spectrum was recorded using a Waters e2695 spectrophotometer (Waters, Massachusetts, USA) equipped with a DAD and a 1-cm-path length cell. Samples in methanol solution were scanned from 190 to 400 nm in 1-nm steps. The structure characterization of the obtained compound was based on 1D NMR (¹H, ¹³C) and 2D NMR (COSY, HSQC, HMBC, and NOESY) data, recorded on the Bruker AM-400, AM-500, and AM-700 NMR spectrometers (Bruker, Karlsruhe, Germany) with TMS as internal standard, respectively. The detailed parameters for the NMR data of all isolates are provided (see Supporting Information, Supplementary Figures S3–S9; Supplementary Figures S11–S30). Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HRESIMS data were acquired on a Thermo Fisher LTQ XL LC/MS (Thermo Fisher, Palo Alto, CA, USA). Semi-preparative HPLC was performed on an Agilent 1220 apparatus equipped with a UV detector with a semi-preparative column (RP-C₁₈, 5 μm, 250 × 10 mm, Welch Materials, Inc.). Column chromatography was performed using silica gel (200–300 mesh and 80–120 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and Sephadex^TM LH-20 gel (40–70 μm; Merck, Darmstadt, Germany). Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co., Ltd., Qingdao), and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH. All solvents were of analytical grade (Guangzhou Chemical Regents Company, Ltd., Guangzhou, China).

The fungal strain Pseudofusicoccum sp. J003 was isolated from the fruit of Sonneratia apetala Buch.-Ham., which was collected at a wetland of Nansha district, Guangzhou, China, in September 2020. The sequence data for this strain have been submitted to the GenBank under accession no. MZ854244. The fungal strain was deposited on 20% aqueous glycerol stock in a −80°C freezer at the School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China. The strain was cultured on potato dextrose agar for 5 days at 28°C. Agar plates, including the strain, were cut into small pieces, and then these pieces were inoculated in a tissue culture bottle (150 × 350 ml) on a solid rice medium (40 g of rice and 35 ml of distilled water) and cultured at room temperature for 30 days.

Cultural media were extracted with methanol three times. Methanol was removed by reduced pressure evaporation at 45°C, and the remaining aqueous phase was extracted 4 times with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to yield a brown extract (60.0 g). The crude extract was introduced to a silica gel chromatography column (CC) and eluted with petroleum ether/ethyl acetate (35:1→0:1) to obtain seven fractions (Fr. 1–Fr. 7). Fr. 2 (7.3 g) was separated into 7 subfractions (Fr. 2.1–Fr. 2.7) using silica gel CC and eluted with n-hexane/2-propanol. Fr. 2.2 (102.3 mg) was purified by semi-preparative HPLC (100% MeOH, 3.0 ml/min) to yield 4 (21.6 mg, t _R 24.5 min). Fr. 2.6 (800.5 mg) was purified by semi-preparative HPLC (45% MeOH/H₂O, v/v, 3.0 ml/min) to yield 5 (1.3 mg, t _R 8.5 min). Fr. 2.4 (113.0 mg) and Fr. 2.7 (220.7 mg) were separated by repeated CC over silica gel to yield 10 (3.4 mg) and 9 (6.7 mg). Fr. 4 (500.5 mg) was separated with repeated silica gel CC to yield six fractions (Fr. 4.1–Fr. 4.6) and then subjected subfraction Fr. 4.3 (268.3 mg) to a Sephadex LH-20 CC (CHCl₂–MeOH, 1:1) to afford three parts (Fr. 4.4a–Fr. 4.4c). Fr. 4.4b (43.2 mg) was purified by semi-preparative HPLC (100% MeOH/H₂O, v/v, 3.0 ml/min) to yield 8 (14.0 mg, t _R 14.5 min). Fr. 4.4c (55.8 mg) was purified by semi-preparative HPLC (70% MeOH/H₂O, v/v, 3.0 ml/min) to yield 1 (2.0 mg, t _R 23.1 min). Fr. 5 (18.9 g) was separated with repeated Sephadex LH-20 CC (MeOH) to yield 6 (7.1 mg) and 7 (90.0 mg). Fr. 7 (7.7 g) was separated with repeated silica gel CC and eluted with CH₂Cl₂/MeOH and n-hexane/2-propanol to yield 2 (22.0 mg), 3 (20.0 mg), and 11 (10.0 mg).

Acorenone C 1) Colorless oil; [ α ] D 29 –34.6 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 233 (1.2) nm; CD (0.10 mM, MeOH) λ _max (Δε) 213 (−1.72), 244 (+9.25) nm; IR v _max 3,429, 2,951, 2,922, 2,872, 1,663, 1,456, 1,381, 1,369, 1,248, 1,034 cm⁻¹; ¹H NMR and ¹³C NMR data (see Table 1 ); HRESIMS [M + Na]⁺ m/z 259.1679 (calcd. for C₁₅H₂₄O₂Na, 259.1669).

Acetylcholinesterase (AChE) inhibitory activity of the compounds isolated was assayed by the spectrophotometric method with slight modification (Ellman et al., 1961). S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′-dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman’s reagent), and acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical. The compounds were dissolved in DMSO. The reaction mixture (totally 200 μL) containing phosphate buffer (pH 8.0), a test compound (50 μM), and acetyl cholinesterase (0.02 U/mL) was incubated for 20 min (37°C). Then the reaction was initiated by the addition of 40 μL of a solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s for 1 h. Tacrine was used as a positive control with a final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E–S)/E × 100 (E is the activity of the enzyme without the test compound and S is the activity of the enzyme with the test compound).

The RAW 264.7 cells (2 × 10⁵ cells/well) were incubated in 96-well culture plates with or without 1 μg/ml LPS (Sigma Chemical Co., USA) for 24 h in the presence or absence of the test compounds. Aliquots of supernatants (50 µL) were then reacted with 100 µL Griess reagent (Sigma Chemical Co., USA). The absorbance was measured at 570 nm by using the Synergy TMHT Microplate Reader (BioTek Instruments Inc., USA). In the study, L-NMMA (Sigma Chemical Co., USA) was used as a positive control. In the remaining medium, an MTT assay was carried out to determine whether the suppressive effect was related to cell viability. The inhibitory rate of NO production = (NO level of blank control – NO level of test samples)/NO level of blank control. The percentage of NO production was evaluated by measuring the amount of nitrate concentration in the supernatants with Griess reagent, as described previously (Wu et al., 2017).

Five human cancer cell lines, including the A549 lung cancer cell line, the HL-60 human myeloid leukemia cell line, the MCF-7 breast cancer cell line, the SMMC-7721 human hepatocarcinoma cell line, and the SW-480 human pancreatic carcinoma were used. Cells were cultured in RPMI-1640 or DMEM medium, supplemented with 10% fetal bovine serum and 5% CO₂ at 37°C. The cytotoxicity assay was performed using an MTTS 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium) method in 96-well microplates, as reported previously (Liu et al., 2012), with slight modification. In brief, 100 μL of adherent cells were seeded into each well of the 96-well culture plates and allowed to adhere for 12 h before adding the test compounds, while suspended cells were seeded into wells at a density of 1 × 10⁵ cells/mL just prior to the addition of the test compounds. Each tumor cell line was exposed to the test compound at concentrations of 40 μM in triplicates for 48 h. Wells with DMSO were used as negative controls, and Taxol and DDP were used as positive controls. After treatment of the compounds, cell viability was detected by a microplate reader at λ = 492 nm.

Compounds 1–11 were evaluated for their antimicrobial activities against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa. The antimicrobial assay was conducted by the previously described method (Zhang et al., 2019). The sample to be tested was added into a 96-well culture plate, and the maximum concentration of the used compounds was 250 μg/ml. Bacteria liquid was added to each well until the final concentration is 5 × 10⁵ CFU/ml. It was then incubated at 37°C for 24 h, and the OD value at 595 nm was measured by the microplate reader, and the medium blank control was used in the experiment.

Compound 1 was obtained as colorless oil. Its molecular formula was determined to be C₁₅H₂₄O₂ based on the deprotonated ion peak [M + Na]⁺ at m/z 259.1679 [M + Na]⁺ (calcd for 259.1669) in the (+)-HRESIMS, indicating 4 degrees of unsaturation. The IR spectrum showed characteristic absorption bands of hydroxyl (3,429 cm⁻¹) and the carbonyl groups (1,662 cm⁻¹). The ¹³C NMR and DEPT spectra of 1 (Table 1) showed 15 carbon signals, including three methyls, five methylenes (including oxygenated methylene at δc 68.5), three methine groups, two olefinic carbon signals (δc 147.1 and 136.1), an aliphatic quaternary carbon, and a carbonyl carbon (δ _C 203.2). The ¹H NMR spectrum of 1 (Table 1) displayed the presence of an olefinic proton resonated at δ _H 6.82 (t like, J = 3.9 Hz), two methyl group doublets at 0.91 (d, J = 6.7 Hz) and 0.84 (d, J = 6.8 Hz), and two oxygenated methine protons at δ _H 3.36 and 3.38. Apart from the two degrees of unsaturation occupied by the carbonyl group and a double bond, the remaining degrees of unsaturation suggested that compound 1 should be a dicyclic sesquiterpenoid (Amandine et al., 2017).

The ¹H-¹H COSY spectrum of 1 indicated the presence of spin systems of (HO)CH₂(12)-CH(11)-CH₃(13) and CH(1)-CH₂(2)-CH₂(3)-CH(4)-CH₃(14) (Figure 2). In the HMBC spectrum, the HMBC interactions from H-13 (δ _H 0.91) and H-12 (δ _H 3.36 and 3.38) to C-11 (δ _C 36.3) and C-1 (δ _C 52.2) revealed the direct C–C linkage from C-11 to C-1 (Figure 2). Subsequently, the HMBC correlations of H-2 and H-11 with C-5 (δ _C 50.0) and of H-14 with C-3, C-4, and C-5 indicated the presence of a methyl cyclopentane substructure with a 1-propanol substituted at C-1. The spin system of = CH(9)–CH₂(10) observed from the ¹H–¹H COSY spectrum of 1, together with the key HMBC correlations from H₃-15 (δ _H 1.75) to C-7 (δ _C 203.2)/C-8 (δ _C 136.1)/C-9 (δ _C 147.1), from H-6 (δ _H 2.24 and 2.63) to C-5/C-8/C-10, collaborated with the methyl cyclohexane substructure decorated by an α,β-unsaturated ketone functionality. Based on the aforementioned pieces of evidence, the crucial HMBC correlations from H-6 and H-10 to C-1, C-4, and C-5 and from H-1 and H-4 to C-5, C-6, and C-10 constructed the gross structure of 1, featuring a spiro[4,5]decane scaffold. The planar structure of 1 was thus deduced as shown (Figure 2), resembling the (3S)-1,4-epi-3-hydroxyacorenone (Calva et al., 2017).

The relative configurations of 1 were elucidated by the observation of its NOESY spectrum. The NOESY correlations of H-10a with H₃-13 and H₃-14, and Me-13/H-2a revealed that H-10a, H₃-13, and H₃-14 were co-facial and provisionally assigned to be α-oriented. Accordingly, the NOESY cross-peaks of H-11/H-10b, H-1/H-6b, H-6a/H-4, and H-1/H-12a indicated the β-orientation of H-1, H-4, and H-11. The relative stereochemistries at C-1, C-4, C-5, and C-11 of 1 were thus determined (Figure 3). Therefore, as for the absolute configuration of 1, two possible enantiomers (Figures 4A,B) were presented (Figure 4).

To further determine the absolute stereochemistry of 1, the electronic circular dichroism (ECD) (for detailed procedures, see SI) calculation of the two possible enantiomers (Figures 5A,B; Figures 4, 5) was performed using Gaussian 09 and figured using GaussView 5.0 (Dennington et al., 2009; Frisch et al., 2009). Conformation search via molecular mechanics calculations was conducted in Discovery Studio 3.5 Client, with an MMFF force field with a 20 kcal/mol upper energy limit (Smith and Goodman, 2010). The optimized conformation geometries and thermodynamic parameters of the selected conformations were provided. The predominant conformers were subsequently optimized at the B3LYP/6-31G(d,p) level. The theoretical calculation of ECD was performed using a time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d,p) level in MeOH with the PCM model. The calculated spectrum of 1b (1S,4S,5R,11S) agreed with the experimental data, showing a negative Cotton effect (CE) at 213 nm and a strong positive CE at 244 nm (Figure 5). Consequently, the structure of 1 was determined to be (1S,4S,5R)-1-((S)-1-hydroxypropan-2-yl)-4,8-dimethylspiro [4.5]dec-8-en-7-one, and a trivial acorenone C was given.

Ten known compounds, uracil (2) (Xing et al., 2020), cyclo-(L-Pro-L-Tyr) (3) (Jayatilake et al., 1996), bis-(2-ethylhexyl) terephthalate (4) (Firdovsi et al., 2007), 4-hydroxybenzaldehyde (5) (Shataer et al., 2020), 2-phenylethanol (6) (Guerrini et al., 2011), 4-hydroxyphenethyl-alcohol (7) (Wei et al., 2013), estigmast-4-en-6β-ol-3-ona (8) (Correia et al., 2003), ergosterol (9) (Zhang et al., 2002), ergosterol peroxide (10) (Hybelbauerová et al., 2008), and cerevisterol (11) (Kang et al., 2017) were also isolated from Pseudofusicoccum sp. J003. The structures of these compounds (2–11) were elucidated by comparing the spectral data to those reported in the references.

According to the literature, acorenone analogs usually have AChE inhibitory activity (Calva et al., 2017). The AChE inhibition effect of new compound 1 was tested. It exhibited mild inhibitory activity against AChE with an inhibition rate of 23.34% ± 3.53 at the concentration of 50 μM (Table 2). To further test in vitro anti-inflammatory activity, compounds 1–4, 6–9, and 11 were evaluated for their inhibitory activities against LPS-induced nitric oxide (NO) production in RAW 264.7 mouse macrophages, of which compound 9 showed obvious inhibitory activity, with an inhibition rate of 72.89% ± 0.71 at the concentration of 25 μM (Table 3). Since steroid derivatives were reported to have cytotoxic properties against tumor cells (Bok et al., 1999), compounds 8, 9, and 11 were selected to test their cytotoxic activities against five human cancer cell lines, including HL-60, A549, MCF-7, SMMC-7721, and SW480, of which compound 9 inhibited the proliferation of tumor cells HL-60, with an inhibition rate of 98.68% ± 0.97 and SW480 with an inhibition rate of 60.40% ± 4.51 at a concentration of 40 μM, respectively (Table 4). The antimicrobial activity of compounds 1–11 was also evaluated against the bacteria S. aureus, B. subtilis, P. aeruginosa, and E. coli. However, all of them were found to be devoid of significant activity (MIC >250 μg/ml).