New Meroterpenoid Derivatives from the Pomegranate-Derived Endophytic Fungus Talaromyces purpureogenus

In this study, we report the isolation of two new meroterpenoids, miniolutelide D (1) and miniolutelide E (13-epi-miniolutelide C) (2), along with two meroterpenoidal analogues (3 and 4) and two phenolic compounds (5 and 6) from the endophytic fungus Talaromyces purpureogenus derived from Punica granatum fruits. Their structures were elucidated using extensive MS, 1D, and 2D NMR spectroscopic analyses as well as by comparing with data in the literature. The absolute configurations of 1 and 2 were determined using TDDFT-ECD calculations. Antimicrobial activity was evaluated. Compound 5 displayed significant activity against methicillin-resistant Staphylococcus aureus strain ATCC 700699 and moderate activity against S. aureus strain ATCC 29213.


Introduction
Nature has granted mankind plenty of drug leads since the beginning of history.Many of these are widely used now in clinical settings and in everyday use to ameliorate various diseases [1].Originally, plants were the major source for isolating bioactive compounds.However, endophytes have recently received much more attention from researchers due to the structural and biological diversity of their natural products.Endophytes are the micro-organisms that live within the healthy tissues of plants in a symbiotic relationship, causing no harm to their hosts [2].
Fungal endophytes represent outstanding sources of new secondary metabolites spanning different classes of secondary metabolites, including polyketides, steroids, alkaloids, peptides, terpenoids, and quinones, among others, displaying potential biological activities.
Although it has been estimated that approximately one million species of endophytic fungi exist, only 5% have been investigated.There are still many fungi yet to be explored that hold many undiscovered treasures [3][4][5].
As a subclass of terpenoids, meroterpenoids are composed of isoprene units with diverse chemical structures.This high diversity is accounted for by their mixed biosynthesis, which comprises both terpenoid and polyketide pathways [15].Various meroterpenoids have been reported to exhibit antimicrobial activity against different bacterial and fungal strains.For example, 11-hydroxychevalone E showed antibacterial activity against Escherichia coli and Salmonella enterica serovar Typhimurium.Chevalone C exhibited antibacterial activity against Bacillus cereus and Staphylococcus aureus [16].Antifungal activity was tested on formyl phloroglucinol meroterpenoids isolated from Eucalyptus robusta leaves.Among them, eucalrobusone J, eucalrobusone O, and macrocarpal C showed potent antifungal activity against Candida glabrata, whereas eucalrobusone O and macrocarpal C showed moderate antifungal activity against Candida albicans [17].Other examples worth mentioning are chrodrimanins A and B isolated from Talaromyces funiculosus, which exhibited both broad spectrum and some selectivity against bacterial strains, respectively.Chrodrimanin A showed broad spectrum antibacterial activity against S. aureus, Mycobacterium phlei, and E. coli, and was majorly selective against Micrococcus tetragenus.Chrodrimanin B was moderately selective against E. coli [18].Significant antibacterial activity against Helicobacter pylori and S. aureus was detected in aspergillactone obtained from the marine-derived fungus Aspergillus sp.CSYZ-1 [19].

Results and Discussion
Compounds (1 and 2) were isolated together with two known meroterpenoids, berkeleyone C (3) and berkeleydione (4) [20], and two known phenolic compounds, alternariol (5) [21] and 3-methylorsellinic acid (6) [22].Compounds 3-6 were identified by comparing their obtained spectral data with those reported in the literature.The molecular structures of isolated compounds are shown in Figure 1.Compound 1 was isolated as a white amorphous powder.HRESIMS revealed a molecular ion peak at m/z 489.1762 equivalent to C25H29O10 [M − H] − (Figure S1). 1 H and APT NMR data are summarized in Table 1. 1 H NMR, APT NMR and HSQC spectra are shown in Figures S2-S4. 1 H NMR spectrum of compound 1 (Figure S2) showed three methine Compound 1 was isolated as a white amorphous powder.HRESIMS revealed a molecular ion peak at m/z 489.1762 equivalent to C 25 H 29 O 10 [M − H] − (Figure S1). 1 H and APT NMR data are summarized in Table 1. 1 H NMR, APT NMR and HSQC spectra are shown in Figures S2-S4. 1 H NMR spectrum of compound 1 (Figure S2) showed three methine protons at δ H (ppm) 4.07 (1H, q, J = 6.3 Hz, H-9), 4.23 (1H, dd, J = 13, 3.75 Hz, H-13), and 5.97 (1H, s, H-23), where the former two methines are oxygenated, while the latter is di-oxygenated.Also, three methylene protons were found at δ H (ppm) 3. 34 By inspection of the COSY spectrum (Figure S5), it was noted that there were correlations between H-13 and H 2 -14, as well as between H-9 and H 3 -21.Additionally, the HMBC correlations (Figure S6) confirmed the planar structure of 1 where correlations of δ H 3. From the above-mentioned data of spectroscopic analyses, it was revealed that compound 1 showed a close similarity with the previously reported data of miniolutelide C [23], showing they have a similar planar structure with only two differences.Those differences were an additional hydroxy group on the quaternary carbon C-22, accounting for the downfield shift (δ c 80.68) compared to the methine group in miniolutelide C at (δ C 44.80) and the absence of the proton peak at (δ H 3.05), and also accounting for the downfield shift in C-11 from δ c 62.1 in miniolutelide C to δ c 72.38 in 1.Moreover, the disappearance of the methoxy group at C-20 suggests that compound 1 is a 20-demethoxy, 22-hydroxy derivative of miniolutelide C. Compound 1 was given the trivial name miniolutelide D. Some discrepancies in 13 C NMR data values were observed between them.C-13 found at δ c 92.91 in 1 was located at δ c 79.3 in miniolutelide C, in addition to C-19 found at δ c 27.91 in 1 was at δ c 14.9 in miniolutelide C, suggesting a possible difference in absolute configurations could be found between the two compounds.
After being measured in CDCl 3 (Figures S9 and S14), the 1 H NMR and NOESY spectra were measured in dimethylsulfoxide (DMSO) (Figures S15 and S16) to confirm the relative configuration of 2. NOE correlations were observed from H-22 to H 3 -24, H 3 -19, and H-23, from H-23 to H 3 -24, from H 3 -19 to H-13, and H 3 -26, from OH-10 to H-23, and H-9, indicating that they are on the same side.Since H-9 is on the prior plane and H-21 showed no correlations with any of that plane's protons, this suggests that H-21 is on the opposite face.By comparing the relative configuration of 2 with that of miniolutelide C [23], a difference in C-13 was observed, where its configuration is (S*) instead of (R*).Therefore, the relative configuration was determined as (7R*,9S*,10S*,11S*,12S*,13S*,22R*,23R*).Thus, 2 is a new metabolite for which the trivial name miniolutelide E (13-epi-miniolutelide C) was given.Key COSY, HMBC, and NOESY correlations are shown in Figure 5.
face.By comparing the relative configuration of 2 with that of miniolutelide C [23], a ference in C-13 was observed, where its configuration is (S*) instead of (R*).Therefore relative configuration was determined as (7R*,9S*,10S*,11S*,12S*,13S*,22R*,23R*).Th is a new metabolite for which the trivial name miniolutelide E (13-epi-miniolutelid was given.Key COSY, HMBC, and NOESY correlations are shown in Figure 5.The absolute configuration of 2 was also confirmed by TDDFT-ECD calculations puting for (7R,9S,10S,11S,12S,13S,22R,23R)-2.DFT re-optimization of the initial 9 M conformers resulted in a single major conformer with 99.3% Boltzmann population.spectra computed at various levels for this conformer gave acceptable to good agreem with the experimental ECD spectrum (Figures 6 and 7), allowing determination o absolute configuration as (7R,9S,10S,11S,12S,13S,22R,23R)-2.The emergence and prevalence of antimicrobial resistance are curr a worldwide public health threat.Most pathogens develop resistance to crobials, which risk treatment regimens' failure.Therefore, the urgent n microbial agents to combat this serious problem is steadily increasing [ activity of the total crude extract was evaluated against the pathogenic g terial strain S. aureus ATCC 29213, which causes food poisoning, toxic and scalded skin syndrome, among other diseases, as well as the Gram-n strain E. coli ATCC 25922 known to cause food-borne illnesses [28].The The emergence and prevalence of antimicrobial resistance are currently depicted as a worldwide public health threat.Most pathogens develop resistance to common antimicrobials, which risk treatment regimens' failure.Therefore, the urgent need for new antimicrobial agents to combat this serious problem is steadily increasing [26,27].Inhibitory activity of the total crude extract was evaluated against the pathogenic grampositive bacterial strain S. aureus ATCC 29213, which causes food poisoning, toxic shock syndrome, and scalded skin syndrome, among other diseases, as well as the Gram-negative bacterial strain E. coli ATCC 25922 known to cause food-borne illnesses [28].The extract was only significantly active against S. aureus ATCC 29213, with an MIC value of 6.3 µg/mL.Subsequently, all isolated compounds 1-6 were tested for antimicrobial activity against S. aureus ATCC 29213, as well as the methicillin-resistant S. aureus (MRSA) strain ATCC 700699.Only compound 5 exhibited considerable activity against S. aureus ATCC 700699 with an MIC of 25 µM, and modest activity against S. aureus ATCC 29213 with an MIC of 100 µM.None of the other compounds showed significant activity at a dose of 100 µM.
Tuberculosis, an infectious disease caused by M. tuberculosis, is the major cause of death by an infectious bacterium, accounting for more than a million deaths every year.Although huge efforts are being made by health communities, the morbidity and mortality rates are still high, especially in developing countries, along with the problem of emerging resistant strains, which put great importance in finding new anti-tubercular agents [29,30].Hence, the total extract as well as all isolated compounds were tested against M. tuberculosis.However, none of them showed any significant activity at 100 µM.

General Experimental Procedures
NMR spectra were recorded on a Bruker Avance III HD 400 MHz NMR spectrometer (Zurich, Switzerland).LC-MS Shimadzu 8045 spectrometer coupled with photodiode array (PDA) detector (LC-2030/2040) was used to record the mass spectra.HRESIMS spectra were acquired on FTHRMS-Orbitrap (Thermo-Finnigan, Weiler bei Bingen, Germany) mass spectrometer.Semi-preparative HPLC (Shimadzu, Kyoto, Japan) was utilized for purification, with Kromasil C-18 RP semi-preparative column (10 mm × 250 mm), with 5 mL/min flow rate, and UV detection at 254 nm with λ max absorption at 220-400 nm.Medium-pressure liquid chromatography was performed using the Puriflash 4125 system, Montlucon, France (Interchim software, Intersoft v5.0b09) coupled with a PDA detector.HiMedia silica gel GRM7484 (0.037-0.063 mm) was utilized for column chromatography.TLC analyses were carried out on precoated silica gel 60 F 254 plates (Merck, Darmstadt, Germany), followed by UV detection at 254 and 365 nm and spraying with vanillin sulphuric acid reagent.All solvents used were priorly distilled, and spectroscopic grade solvents were used for spectroscopic measurements.

Fungal Material and Identification
Pomegranate fruit collected in Cairo, Egypt, in the summer of 2019 was identified and authenticated as P. granatum, family Lythraceae.The fruit was cleaned and cut longitudinally.Inside the aseptic area, different fruit parts were cut by sterile blades, washed with distilled water, followed by treatment with 70% ethanol to exclude any contaminants and epiphytes that might arise.The sterile dissected parts were inoculated aseptically on malt extract agar medium (MEA).Plates were grown at room temperature (25 • C).After successive purification steps, the pure fungal strain was successfully isolated from the tissues of the P. granatum fruit.The same fungal strain was simultaneously isolated from the mesocarp, calyx, and exocarp, which confirms its indigeneity in P. granatum fruits.The fungus was identified as T. purpureogenus according to a molecular biological protocol using DNA amplification and sequencing of the ITS region [31].The obtained sequencing data were submitted to Genbank with accession number OM367903.A voucher plate for this fungal strain was deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, with the ID code AMP-R-2.

Fermentation, Fractionation, and Isolation of Bioactive Compounds
Solid rice medium was prepared in 22 1-L Erlenmeyer flasks, each containing 100 g of commercial rice and 110 mL of water.The solid rice medium was autoclaved at 121 • C for 20 min prior to fungal fermentation.The fungal strain was cultivated on the rice medium for 7 weeks at room temperature.Afterwards, fungal biomass was extracted using 700 mL ethyl acetate (EtOAc) for each flask.The flasks were left in static mode overnight.The following day, the flasks were put on electric shakers at 150 rpm for 8 h.Filtration and evaporation steps were followed to obtain a dried extract.The extraction step was run four successive times, yielding 75 g of crude extract.
While running the VLC on the total extract, an orange band F-3 was observed in a 60% n-hexane solvent system, which was collected separately from the original fraction F-3.F-3 (590 mg) was subjected to semi-preparative HPLC to yield 6 (7.4 mg).
All fractionation and isolation steps were continually tracked by TLCs of isolated fractions, sub-fractions, and compounds in different mobile phase systems.

Antimicrobial Susceptibility Tests
The broth micro-dilution method was applied to evaluate the antimicrobial activity against Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 700699, Escherichia coli ATCC 25922, and the minimum inhibitory concentration (MIC) values were measured according to the Clinical and Laboratory Standards Institute (CLSI) recommendations [32].The MIC against Mycobacterium tuberculosis H37Rv was determined in 96-well microtiter plates employing the resazurin reduction assay, as described previously [33].Moxifloxacin and ciprofloxacin were used as positive controls for S. aureus and E. coli, whereas rifampicin was used as a positive control for M. tuberculosis.DMSO was used as the solvent control.

Computational Section
Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl 3 [34].Geometry re-optimizations were carried out at the ωB97X/TZVP level [35] with the PCM solvent model for MeCN.TDDFT-ECD calculations were run with various functionals (B3LYP, BH&HLYP, CAMB3LYP, and PBE0) and the TZVP basis set as implemented in the Gaussian 09 package with the same solvent model as in the preceding DFT optimization step [36].ECD spectra were generated as sums of Gaussians with 2700 and 4200 cm −1 width at half-height, using dipole-velocity-computed rotational strength values [37].Boltzmann distributions were estimated from the ωB97X energies.The Molekel software package (v5.4) was used for the visualization of the results [38].

Conclusions
Chemical investigation of the endophytic fungus Talaromyces purpureogenus, derived from Punica granatum fruits, led to the isolation of two new meroterpenoids together with two other known meroterpenoids and two known phenolic compounds.The compounds were identified based on their spectral data and by comparison with previously reported data in the literature.The absolute configuration of newly isolated compounds was confirmed by TDDFT-ECD calculations.Additionally, antimicrobial activity was evaluated.Compound 5 displayed significant activity against the MRSA strain S. aureus ATCC 700699, as well as moderate activity against the drug-susceptible reference strain S. aureus ATCC 29213.Further biological investigations of the isolated compounds from T. purpureogenus are suggested for future research.

Table 1 .
1H and APT NMR data for compounds 1