Bioactive polyketides and meroterpenoids from the mangrove-derived fungus Talaromyces flavus TGGP35

Six new polyketides, which includes three new lactones (talarotones A–C) (1–3), one new polyketide (talarotide A) (4), two new polyenes (talaroyenes A, B) (5, 6), together with one new meroterpenoid (talaropenoid A) (7) and 13 known compounds (8–20) were isolated from the mangrove-derived fungus Talaromyces flavus TGGP35. The structure and configuration of the compounds 1–7 were elucidated from the data obtained from HR-ESI-MS, IR, 1D/2D NMR spectroscopy, Mo2 (OAc)4-induced electronic circular dichroism (ECD), CD spectroscopy, and modified Mosher's method. Compounds 5 and 20 displayed antioxidant activity with IC50 values of 0.40 and 1.36 mM, respectively. Compounds 3, 6, 11, 16, and 17 displayed cytotoxic activity against human cancer cells Hela, A549, and had IC50 values ranging from 28.89 to 62.23 μM. Compounds 7, 10–12, and 14–18 exhibited moderate or potent anti-insect activity against newly hatched larvae of Helicoverpa armigera Hubner, with IC50 values in the range 50–200 μg/mL. Compound 18 showed antibacterial activity against Ralstonia solanacearum with the MIC value of 50 μg/mL.


GRAPHICAL ABSTRACT
that Talaromyces exhibits numerous beneficial bioactivities and exceptional biosynthetic capabilities, which makes it suitable for wide-ranging industrial applications.
As a part of our ongoing investigation to identify secondary metabolites from marine-derived fungus with a novel structure and potent bioactivity (Bai et al., 2019a(Bai et al., ,b, 2021;;Liao et al., 2019), we obtained six new isocoumarins from the fungus Talaromyces flavus TGGP35 (separated from the medicinal mangrove Acanthus ilicifolius) (Cai et al., 2022).With the aim of isolating more compounds with potent bioactivity from T. flavus TGGP35, the solid-state fermentation condition was changed (sea salt was replaced by sodium bromide).During our investigations, we also found that EtOAc extract, a modified fermentation extract from T. flavus TGGP35, showed potent anti-insect activity against newly hatched larvae of Helicoverpa armigera Hubner with IC 50 value of 200 µg/mL.We carried out bioassay-guided separation following the isolation of the EtOAc extract and isolated three new lactones (talarotones A-C) (1-3), one new polyketide (talarotide A) (4), two new polyenes (talaroyenes A and B) (5,6), one new meroterpenoid (talaropenoid A) (7), along with 13 known compounds (8-20) (Figure 1), and characterized them.In this paper, we describe the isolation, determination of the structure, and the bioactivities of these compounds.

FIGURE
The structures of compounds -.
was generated as an auxiliary chromophore after the addition of Mo 2 (OAc) 4 to a DMSO solution of 1.The observation of Cotton effect at ∼300 nm in the induced spectrum originates from the chirality of the vic-diol, as exhibited by the O-C-C-O torsion angle in the favored conformation, which led to the assignment of the absolute configuration.A negative Cotton effect observed at 316 nm ( ε = −0.42) in the induced CD spectrum of 1 confirmed the 9R, 10R configurations (Figure 4).To determine the absolute configuration of 1, the theoretical ECD spectra of two possible stereoisomers of 4R, 9R, 10R and 4S, 9R, 10R were created by the TDDFT calculations, and the calculated ECD curve of the isomer 4R, 9R, 10R was found to have a good agreement with the experimental one (Figure 5).Therefore, the absolute configuration of 1 was determined as 4R, 9R, 10R, and compound was named talarotone A.
The HR-ESI-MS data showed that compound 2 also has same molecular formula C 11 H 16 O 4 as 1. Analysis of 1D NMR data (Tables 1, 2) and HR-ESI-MS data suggested that 2 has a similar structure as 1 with the same carbon skeleton.A further investigation of 2 by DEPT135 • , 1 H-1 H COZY, HMQC, and HMBC spectra established its planar structure, which is similar to 1.The NOESY correlations of H-6/H-8 and H-8/H-11 (Figure 3), combined with the coupling constants of 3 J H−6,H−7 = 11.2Hz, pointed to the 6Z, 8Z configurations of the two double bonds.The coupling constants of H-8/H-9 (5.2 Hz) suggested that 9,10-diols are oriented at a threo configuration (Wang et al., 2016;Zou et al., 2020).Likewise, the absolute configurations of C-9 and C-10 in 2 were also determined using an in situ dimolybdenum CD method (Dewapriya et al., 2018;Bai et al., 2019a).The positive Cotton effect at 311 nm ( ε = +1.58)confirmed the 9S, 10S configurations for C-9 and C-10 (Figure 4).The absolute configuration at C-4 was determined to be R through TDDFT ECD calculation (Figure 5).Thus, the absolute configuration of 2 was 4R, 9S, 10S, and the compound was named talarotone B.    The C-5 and C-6 atoms in 3 were found to be oriented in a trans configuration, which was determined based on a large coupling constant of 3 J H−5,H−6 = 16.4Hz.The NOESY correlation of H-6 and H-8 indicated a Z configuration of the double bond (Figure 3).
Mosher's method was used to determine the absolute configuration of C-2 (Bai et al., 2019a).Unfortunately, because of the excessive humidity in the environment, this reaction was unsuccessful and we did not have enough amount of the compound to perform this

FIGURE
Experimental CD spectra of -and and Experimental ECD spectra of the Mo + complex of -and with the inherent CD spectral subtracted.
reaction again.The absolute configuration on C-9 was determined as S by ECD calculation (Figure 5).Therefore, 3 was identified as talarotone C.
Compound 7 was isolated as a white powder.Its molecular formula was deduced as C 26 H 32 O 7 (11 degrees of unsaturation) from the HR-ESI-MS spectral data.The 1 H and 13 C NMR data (Table 3) revealed that 7 had a austin meroterpenoid skeleton and also a similar structure to brasilianoid G (Zhang et al., 2019).The obvious differences though were the presence of one methoxyl group at [δ H 3.57 (s), δ C 56.3 (CH 3 )] and one methyl group at [δ H 1.62 (s), δ C 15.8 (CH 3 )], and the absence of an aldehyde group at [δ H 9.44 (s), δ C 199.9 (C)] in 7. The key HMBC correlations from H-11 to C-9/11-OMe, H-13 to C-5/C-9/C-10 pointed out that the aldehyde group for C-11 in brasilianoid G was replaced by the methoxyl group in 7, and the double bond at [δ H 5.24 (brs) and 5.74 (brs), δ C 128.8 (CH 2 )] for C-13 in brasilianoid G was replaced by a methyl group in 7. The 1 H-1 H COZY and HMBC spectra established the complete structure of 7 (Figure 2).The ROESY correlations of H-1 with H-9′/11-OMe, H-9′ with H-15, and H-12 with H-14, and H-14 with H-7′, confirmed the relative configuration of 7 (Figure 3).The absolute configuration of 7 was determined as 5R, 8S, 11S, 1′S, 5′R, 6′R by ECD quantum chemical calculations (Figure 5).Thus, the structure of 7 was named talaropenoid A.
Compounds 5 and 20 were found exhibit a strong antioxidant activity with IC 50 values of 0.40 and 1.36 mM, respectively, while the IC 50 value of the positive control trolox is 0.29 mM.
Compounds 3, 6, 11, and 16 and 17 showed cytotoxic effects on Hela and A549 human cancer cells, with their IC 50 values ranging from 28.89 to 62.23 µM (Table 4).All compounds exhibited no activity against the gastric cancer cell line RKO even at a concentration of 100 µM.
Compounds 7, 10-12, and 14-18 displayed moderate or strong anti-insect activity against newly hatched larvae of H. armigera Hubner, with their IC 50 values ranging from 50 to 200 µg/mL (Table 5), while the IC 50 value of the positive control azadirachtin is 50 µg/mL.Other compounds showed no growth inhibition activity against newly hatched larvae of H. armigera Hubner even at a concentration of 200 µg/mL.
Structure-activity relationships of antiinsect activity against the newly hatched larvae of H. armigera Hubner with regard to interaction with meroterpenoids has been discussed.The substitution of hydroxyl and acetyl groups and the ether ring moiety on the skeleton of meroterpenoids influences their antiinsect activity.Compound 10, which has a hydroxyl group on C-7, exhibits a better anti-insect activity superior to that of 11, suggesting the hydroxyl group at C-7 contributes to an increased growth inhibition potency.The growth inhibition activity data of 12 and 13 indicates that the central ether ring moiety was a nonessential functional group for anti-insect activity in the structure of meroterpenoids.Compound 15, which has a hydroxyl group on C-13, showed a higher anti-insect activity than compounds 11 and 16, suggesting that 13-OH group enhances anti-insect activity, and the acetylation of 13-OH decreases the anti-insect activity.
The antibacterial activity of all compounds was assessed against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Ralstonia solanacearum.Compound 18 displayed a weak antibacterial activity against R. solanacearum even at an MIC value of 50 µg/mL, while the MIC value of the positive control streptomycin is 12.5 µg/mL.Other compounds showed no antibacterial activity against the bacterial species tested even at a concentration of 100 µg/mL.
These results suggest that anstin meroterpenoids are capable of contributing to the development of novel biopesticides such as microbial insecticides and antibiotics.

Materials and methods . General experimental procedures
The melting points of the isolated compounds were determined on a WRX-4 micromelting point apparatus (Shanghai YiCe Apparatus and Equipment Co., Ltd., Shanghai, China).CD spectra of the compounds were recorded on a Mos-500 spectrometer.IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer.PERSEE TU-1990 spectrophotometer was used for recording the UV spectra.Optical rotations were measured using a JASCO P-1020 digital polarimeter (JASCO, Tokyo, Japan).1D and 2D NMR spectra were recorded from a Bruker AV spectrometer (400 MH Z for 1 H and 100 MH Z for 13 C) and a JNM-ECZS spectrometer (600 HM Z for 1 H and 150 MH Z for 13 C).HR-ESI-MS spectra were obtained from a Q-TOF Ultima Global GAA076 LC mass spectrometer.ESI-MS spectra were recorded on a MAT-95-MS mass spectrometer.Agilent 1100 prep-HPLC system with an Agilent C18 analytical (9.4 × 250 mm, 5 µm) HPLC column was utilized for performing highperformance liquid chromatography (HPLC).Silica gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were employed in column chromatography (CC) and Sephadex LH-20 gel column (Amersham Blosclences manage) were used for recording CC.Biological activities were tested in ultra-clean workbench (Suzhou Sujing Company) and these results were tested with a full wavelength multifunctional microplate reader (BioTek, USA).Methanol, ethyl acetate, petroleum ether, chloroform, dimethyl sulfoxide, and other conventional chemical reagents used in the experimental investigations (Guangzhou Xilong Chemical Reagent Factory) (Cai et al., 2022). .

Fungal materials
The fungus TGGP35 was isolated from the stem of the mangrove plant Acanthus ilicifolius and the sequence data have been deposited in GenBank (accession number MT071116).The fungal strain was identified as Talaromyces flavus (Eurotiales: Trichocomaceae) (Cai et al., 2022).

. Fermentation, extraction, and isolation
The fungal strain TGGP35 was grown on solid rice cultures in 1 L Erlenmeyer flasks (100 flasks; 50 mL of rice and 1.0 gram of sodium bromide per Erlenmeyer flask, autoclave sterilization) at 28 • C for 32 days.The fermentation was extracted three times with ethyl acetate (EtOAc), followed by vacuum concentration, thus generating EtOAc extracts weighing 90.7 g.

. Biological assays . . Antioxidant activity
The antioxidant activity assay was performed following a previously reported method (Zeng et al., 2022).The assay was performed in a 96-well microplate by adding 10 µL of the sample solution to 200 µL of ABTS working solution.Concentration gradients of 2.0, 1.0, 0.5, and 0.25 mg/mL were prepared for all the test groups, including a positive control.The blank control consisted of PBS buffer, DMSO served as the negative control, and Trolox was used as the positive control, with the IC 50 value of 0.29 mM.The measurement of antioxidant effect was done using a full wavelength multifunctional microplate reader at a specific wavelength of 734 nm.The inhibition rate of each sample was calculated using the formula: inhibition rate = [(A blank -A compound )/A blank ] * 100%.Finally, the IC 50 value was calculated using the SPSS software.

. . Cytotoxic activity
All compounds were assessed for their cytotoxic activity against three human cell lines (A549, Hela, RKO) using the MTT method (Mosmann, 1983).The sample concentrations were prepared in five gradient levels: 100, 80, 60, 40, and 20 µM.The experiment was repeated more than three times, and the experimental data were measured using a full-wavelength microplate reader with a test wavelength of 492 nm.DMSO was used as the negative control, and doxorubicin hydrochloride served as the positive control.The inhibition rate of each sample was calculated using the formula: inhibition rate = [(OD compound -OD DMSO )/OD DMSO ] * 100%.The IC 50 value was determined using GraphPad Prism software.

. . Anti-insect activity
The growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner was tested using methods from literature (Bai et al., 2019a).The tested compounds and the positive control azadirachtin were dissolved in DMSO at a concentration of 1 mg/mL.The activity was evaluated by adding serial dilutions of the tested compounds and azadirachtin (concentrations of 200, 100, 50, 25, and 12.5 µL/well) as an artificial diet for the newly hatched larvae.Each treatment was replicated three times, and the bioassay diet was placed in six-well plates.The larvae were then incubated at a controlled temperature of 25 ± 1 • C at a relative humidity of 80%.DMSO was used as the negative control, azadirachtin was used as the positive control, and the artificial diet was used as the blank control.The mortality rate of the larvae was recorded on the 2nd, 4th, 6th, and 8th day after treatment.

. . Antibacterial activity
The antibacterial activity of 1-20 was assessed against five pathogenic bacteria, including Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Ralstonia solanacearum by the microplate assay method (Pierce et al., 2008).The broth medium containing pathogenic bacteria was used as the blank group, DMSO as the negative control, and ciprofloxacin and streptomycin were used as positive control.

Conclusions
In summary, 20 secondary metabolites, including six new polyketides (1-6) and one new meroterpenoid (7), and 13 known compounds were isolated from mangrove-derived fungus T. flavus TGGP35.Compounds 5 and 20 demonstrated moderate antioxidant capability, with IC 50 values of 0.40 and 1.36 mM, respectively.Compounds 3, 6, 11, and 16 and 17 exhibited weak cytotoxic activity on Hela and A549 human cancer cells, with IC 50 values ranging from 28.89 to 62.23 µM.Compounds 7, 10-12, and 14-18 displayed moderate or significant anti-insect activity, with the IC 50 values ranging from 50 to 200 µg/mL.Compound 18 showed antimicrobial activity against R. solanacearum, with an MIC value of 50 µg/mL.The biosynthetic pathway and structureactivity relationship with regard to the anti-insect activity of meroterpenoids were explained in detail.
FIGUREH-H COZY and key HMBC correlations for compounds -.

FIGURE
FIGUREExperimental ECD spectral of -and .