The Polyketides with Antimicrobial Activities from a Mangrove Endophytic Fungus Trichoderma lentiforme ML-P8-2

Five new polyketides, including two chromones (1–2), two phenyl derivatives (4–5), and a tandyukusin derivative (6), along with five known polyketides (3 and 7–10) were isolated from mangrove endophytic fungus Trichoderma lentiforme ML-P8-2. The planar structures of compounds were elucidated via detailed 1D, 2D NMR, and HR-ESI-MS analysis. ECD spectra, optical rotation values calculation, and alkali hydrolysis were applied in the determination of the absolute configuration of the new compounds. In bioassays, 6 and 9 exhibited promising antifungal activities against Penicillium italicum, with an MIC value of 6.25 μM for both compounds. Moreover, 3 displayed moderate AChE inhibitory activity with an IC50 value of 20.6 ± 0.3 μM.


Introduction
Mangrove endophytic fungi are an important source to provide biologically active lead compounds due to their unique living environment.Increasing numbers of secondary metabolites from mangrove-associated fungi have been newly reported in recent decades [1,2].Trichoderma species have been widely discovered in marine environments, including soil, decaying wood, and living plants in mangrove forests [3,4].Over 450 metabolites have been structurally documented from the species of Trichoderma genus [4,5], wherein polyketides are considered a significant group of these metabolites.Lai et al. reported the isolation of two new chromone polyketides with a broad spectrum of antimicrobial activities from Trichoderma sp.JWM29-10-1 [6].Yamada et al. documented six decalin polyketides named tandyukusins from Trichoderma harzianum OUPS-111D-4, some of which exhibited significant cytotoxicities against human cancer cell lines [7][8][9].Polyketides have attracted extensive attention due to their diverse chemical structures and wide range of biological activities [3,4,10].
Herein, we report the detailed structural identification for the new compounds (1-2 and 4-6) and bioactivities results.
Compound 6 was isolated as a pale-yellow oil.Its molecular formula C 25 H 38 O 7 with seven degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na] + 473.2509 (calcd.for C 25 H 38 O 7 Na + , 473.2510).Analyses of the 1 H and 13 C NMR data aided with HSQC revealed the presence of three carbonyls, two quaternary carbons, ten methines, five methylenes, and five methyls (NMR data in Table 1).A comparison with the NMR data of tandyukusin D (8) [8] indicated that 6 and 8 shared the same core structure, eujavanicol A [16], and the main difference occurred on the side chain.With the key HMBC from H-2' to C-4' and C-6'; H 2 -4' to C-3', C-5', and C-6'; H 3 -6' to C-2', C-3', and C-4'; and NOESY correlation H-2'/H 3 -6', the side chain was established as a (2'Z)-3'-methyl-2'-pentenedioic acid moiety.The 13 C resonance of C-6' at δ C 26.2 (larger than 20 ppm) additionally supported the Z configuration of the double bond on the side chain [6,17].The HMBC from H-8 to C-1' attached the side chain at C-8 of the eujavanicol A fragment.
Further, the relative configuration of the eujavanicol A fragment was identified using the key NOESY correlations of H 3 -19 with H-6, H-10, and H-13 and of H-13 with H 3 -17, indicating these protons were on one face (Figure 4).The NOESY correlations of H-5 with H-9 and H 3 -18 suggested these protons were on the other face, and the decalin ring was trans.And 1 H coupling constants J 7α,8 = J 7 β,8 = J 9,8 = 2.5 Hz showed that 9-OH was oriented cis to the esterified side chain at C-8 [7][8][9]15,16].As for the configuration of C-14 at the sec-butyl group, the observed NOESY correlations in 6 were equal to those of 7-10 [6,8,15], which revealed that the rotation of the sec-butyl group in its pseudo-axial arrangement was restricted.Therefore, the relative configuration of C-14 was identified as R*.As a result, the relative configuration of 6 was determined as 4S*, 5S*, 6R*, 8R*, 9S*, 10R*, 13S*, and 14R*, in line with that in 7-10.To determine the absolute configuration of 6, treatments of 6 and tandyukusin D (8) with NaOH aqueous in MeOH were carried out, and the reactions resulted in the acquisition of eujavanicol A (Figure 4), which was identified according to its 1 H, 13 C NMR and HR-ESI-MS data (Figures S40-S42).Comparing the optical rotation values of products (eujavanicol A) from 6 and 8 with previous article [16] (respectively, for [α] 25 D +41.8, [α] 25 D +39.7, and [α] 25 D +49.9), it could be confirmed that the absolute configuration of 6 was 4S, 5S, 6R, 8R, 9S, 10R, 13S, and 14R.Finally, the structure of 6 was determined and named Tandyukisin J. which revealed that the rotation of the sec-butyl group in its pseudo-axial arrangement was restricted.Therefore, the relative configuration of C-14 was identified as R*.As a result, the relative configuration of 6 was determined as 4S*, 5S*, 6R*, 8R*, 9S*, 10R*, 13S*, and 14R*, in line with that in 7-10.To determine the absolute configuration of 6, treatments of 6 and tandyukusin D (8) with NaOH aqueous in MeOH were carried out, and the reactions resulted in the acquisition of eujavanicol A (Figure 4), which was identified according to its 1 H, 13 C NMR and HR-ESI-MS data (Figures S40-S42).Comparing the optical rotation values of products (eujavanicol A) from 6 and 8 with previous article [16] (respectively, for [α] 25 D +41.8, [α] 25 D +39.7, and [α] 25 D +49.9), it could be confirmed that the absolute configuration of 6 was 4S, 5S, 6R, 8R, 9S, 10R, 13S, and 14R.Finally, the structure of 6 was determined and named Tandyukisin J.

Antimicrobial Assays
The isolated compounds 1-10 along with the alkali hydrolysis treatment product eujavanicol A were evaluated for antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas aeruginosa and for antifungal activities against Candida albicans and an agricultural plant pathogenic fungus Penicillium italicum.The results showed that the de-Mar.Drugs 2023, 21, 566 6 of 11 calin derivatives (6-10 and eujavanicol A) exhibited promising inhibitory activities against the fungi, with a minimal inhibition concentration (MIC) value in the range of 6.25 to 50 µM (Table 2), wherein 6 and 9 showed significant antifungal activities against P. italicum, with an MIC value of 6.25 µM for both compounds.And the chromone derivative 3 displayed moderate inhibitory activity against C. albicans, with an MIC value of 25 µM.

AChE Inhibitory Activity Assays
The isolated compounds 1-10, along with the alkali hydrolysis treatment product eujavanicol A, were also evaluated for AChE inhibitory activities.The results showed that the chromone derivative 3 moderately inhibited AChE with IC 50 values for 20.6 ± 0.3 µM, but the other tested compounds mostly exhibited weak inhibitory activities toward AChE (Table 3).65.5 ± 1.5 (nM) 1 Positive control.

Cytotoxic Assays
The isolated compounds 1-10, along with the alkali hydrolysis treatment product eujavanicol A, were also tested for cytotoxicities against six human cancer cell lines, which were MDA-MB-435, MDA-MB-231, HCT116, A549, SNB19, and PC3.But, only compound 1 exhibited a weak cytotoxicity against A549 with an IC 50 value of 47.2 ± 5.5 µM, and the other compounds were inactive to the tested cell lines (IC 50 > 50 µM).

General Experimental Procedures
The 1D and 2D NMR spectra were obtained on a Bruker Advance 400 MHz spectrometer (Billerica, MA, USA) at room temperature.HR-ESI-MS spectra were acquired from a Thermo Fisher LTQ-Orbitrap-LCMS spectrometer (Palo Alto, Santa Clara, CA, USA).Optical rotation values were measured on an MCP500 modular polarimeter (Anton Paar, North Ryde, Austria) at 25 • C. UV-Vis and ECD curves were achieved on an Applied Photophysics Chirascan spectropolarimeter (Surrey, UK).Semi-preparative HPLC was utilized on an Ultimate 3000 separation module combined with a DAD detector produced by Thermo Fisher, and a ChiralPak AY-H column (5 µm, 4.6 × 250 mm) was applied for separation at 22 • C. Organic solvent was evaporated using a vacuum pump with a Heidolph rotavapor.

Fungal Material
The fungus Trichoderma lentiforme ML-P8-2 was isolated from a fresh leaf of the mangrove plant Bruguiera gymnorrhiza, which was collected in July 2022 from Dong Zhai Gang National Nature Reserve in Hainan Province, China.The fungus strain was identified according to sequencing of the internal transcribed spacer, and the results of a BLAST search on National Center for Biotechnology Information (NCBI) revealed it was most similar (99%) to the sequence of Trichoderma lentiforme (compared to MK714910.1).The sequence data have been uploaded and deposited at GenBank with accession No. OR617437.And the fungus specimen was kept in our laboratory at −20 • C.

Fermentation, Extraction, and Isolation
The fungus ML-P8-2 was proliferated in potato dextrose broth (PDB) in 4 × 500 mL Erlenmeyer flasks at 28 • C for 4 days in a shaker and then cultured in 150 × 1 L Erlenmeyer flasks, each containing 60 mL of 0.3% saline and 60 g of rice.After fermentation for 28 days, the culture media were soaked with MeOH and extracted with EA three times after concentration.Then, the extracts were condensed under 45 • C with a vacuum pump to finally obtain a crude extract (61 g).The crude extract was separated using a silica gel column, eluting with a gradient of petroleum ether (PE)/EA from 1:0 to 0:1 to afford 7 fractions (Frs.1-7).

ECD and Optical Rotation Computation Methods
Initial conformational analysis was carried out using the Merck molecular force field method with the Spartan 14' software (Wavefunction Inc., Irvine, CA, USA).The conformation with a Boltzmann population larger than 1% was selected for optimization and calculation in MeOH at B3LYP/6-31+G(d,p) level with the density functional theory (DFT) executed via Gaussian 09 [18].The ECD spectra and optical rotation values were extracted and generated via the SpecDis 1.6 software (University of Würzburg, Würzburg, Germany).The Gibbs free energy, Boltzmann population and Cartesian coordinates for low-energy conformers of 1 and 5 for calculation in Tables S1-S6.

Antimicrobial Assays
The compounds to be tested were dissolved individually in dimethyl sulfoxide (DMSO), and the antimicrobial activity assays were performed in 96-well plates via a serial dilution test in the range of 0.1-100 µM, according to the methods previously reported [11,19].All measurements were conducted in triplicate.Ampicillin and ketoconazole were applied as positive controls for antibacterial and antifungal assays, respectively, and DMSO was utilized as a blank control.

AChE Inhibition Assays
Compounds 1-10 and eujavanicol A were evaluated for AChE inhibitory activity, following the same method previously described [12].Donepezil hydrochloride was taken as a positive control.All measurements were conducted in triplicate from two independent experiments.The reported IC 50 was the average value of two independent experiments.

Conclusions
In summary, five new polyketides, including two chromones (1-2), two phenyl derivatives (4-5), and a tandyukusin derivative (6), along with five known polyketides (3 and 7-10), were isolated from mangrove endophytic fungus Trichoderma lentiforme ML-P8-2, and it is the first time to report secondary metabolites from this specific species.The planar structures of the isolated compounds were elucidated via detailed 1D, 2D NMR, and HR-ESI-MS analysis.ECD spectra, optical rotation values calculation, and alkali hydrolysis were applied in the determination of the absolute configuration of the new compounds.In bioassays, antimicrobial, AChE inhibitory, and cytotoxic activities tests for compounds 1-10, together with the alkali-hydrolysis treatment product eujavanicol A, were carried out.Compounds 6 and 9 exhibited promising antifungal activities against Penicillium italicum, with MIC, both for 6.25 µM.Moreover, 3 displayed moderate AChE inhibitory activity with an IC 50 value of 20.6 ± 0.3 µM.

Figure 3 . 5 .
Figure 3. (i) Experimental and calculated ECD spectra of 1; (ii) experimental and calculated ECD spectra of 5. Compound 4 was acquired as a yellow oil.Its molecular formula C13H14O5 with seven degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na] +

Figure 3 . 5 .Figure 3 . 5 .
Figure 3. (i) Experimental and calculated ECD spectra of 1; (ii) experimental and calculated ECD spectra of 5. Compound 4 was acquired as a yellow oil.Its molecular formula C13H14O5 with seven degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na] +

Table 2 .
MIC for antibacterial and antifungal activities of compounds 1-10 and eujavanicol A.

Table 3 .
IC 50 for AChE inhibitory activities of compounds 1-10 and eujavanicol A.
H (J in Hz) δ C , Typeδ H (J in Hz)