3.1 General experimental procedures

Optical rotations were measured on a Perkin-Elmer 343 polarimeter in MeOH. UV spectra were recorded on a Shimadzu UV-1601PC spectrometer in MeOH. ECD spectra were measured on a Chirascan-Plus CD Spectrometer in MeOH. 1H and 13C NMR spectra were recorded in aceton-d6 on a Bruker Avance-300, Avance-500 and Avance III-700 spectrometers (Bruker BioSpin GmbH) operating at 300 and 75, 500 and 125 MHz and 700 and 176 MHz, respectively. HRESIMS spectra were obtained on a Bruker maXis Impact II mass spectrometer (Bruker Daltonics GmbH). Low-pressure liquid column chromatography was performed using C18-SiO2 Gel ODS-A (12 nm, S—75 um, YMC Co., Ishikawa, Japan) and a Buchi B-688 Chromatography Pump on a Buchi glass column using Si gel KSK (50/100 μm, Imid Ltd., Russia). Plates precoated with Si gel (5–17 μm, 4.5 × 6.0 cm, Imid Ltd., Russia) and Si gel 60 RP-18 F254S (20 × 20 cm, Merck KGaA, Germany) were used for thin-layer chromatography. Preparative HPLC was carried out on a Shimadzu LC-20 (Shimadzu, Kyoto, Japan) and Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) chromatographs using a Shimadzu RID-20A and Agilent 1100 refractometers and YMC ODS-AM (YMC Co, 5 μm, 250 × 10 mm), Ultrasphere Si (5 μm, 250 × 4.6 mm), and Hydro-RP (Phenomenex, 4 μm, 250 × 10 mm) columns.

3.2 Fungal strain

The fungal strain ZK-14 was isolated from the superficial mycobiota of the rhizome seagrass Zostera marina (the Sea of Japan) and stored in the Collection of Marine Microorganisms (PIBOC FEB RAS, Vladivostok, Russia).

The ZK-14 strain was identified as Penicillium velutinum based on four molecular genetic markers: ITS, beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) regions/gene sequences. BLAST analysis showed that the strain ZK-14 was 100% similar to Penicillium velutinum NRRL2069 in the GenBank. The sequences were deposited in the GenBank database under accession numbers OQ427361 for ITS and OQ466610 for partial β-tubulin.

3.3 DNA extraction and amplification

Genomic DNA was isolated from fungal mycelia (mycelium) grown on malt extract agar (MEA) at 25 °C for 7 days using the MagJET Plant Genomic DNA Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. PCR was conducted using GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA). For amplification of the ITS region were used the primer pair 1400-F (5’-CTGCCCTTTGTACACACCGCCCGTC-3’) [54] and D3B*-R (5’- ACTTCGGAGGGAACCAGCTAC-3’) [55]. The reaction profile was 95 °C for 300 s, 35 cycles of 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 90 s, and finally 72 °C for 300 s. For amplification of the partial BenA gene were used the standard primer pair Bt-2a and Bt-2b [56]. The reaction profile was 95 °C for 300 s, 35 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 60 s, and finally 72 °C for 300 s. For amplification of the partial CaM gene were used the degenerate primer pair cal_P/A_F (5’-TCYGAGTACAAGGAGGCSTT-3’) and cal_P/A_R (5’-CCRATGGAGGTCATRACGTG-3’). The reaction profile was 95 °C for 300 s, 35 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 90 s, and finally 72 °C for 300 s. For amplification of the partial RPB2 gene were used the degenerate primer pair rpb2_Pen_F (5’-GAGACYAAYCGBGARATYTA-3’) and rpb2_Pen_R (5’-GTCATSACAATCATRATDGT-3’). The reaction profile was 95 °C for 300 s, 35 cycles of 95 °C for 20 s, 50 °C for 30 s, and 72 °C for 90 s, and finally 72 °C for 300 s. The amplified ITS, BenA, CaM and RPB2 genes were purified with the ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was bidirectional performed with the same primers on an Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using the Big Dye Terminator reagent kit, version 3.1. Gene sequences were deposited in GenBank under the accession numbers OQ427361 for ITS, OQ466610 for the partial BenA, OR356207 for the partial CaM and OR356208 for the partial RPB2 (Additional file 1: Table S1).

3.4 Phylogenetic analysis

The ITS region, the partial BenA, CaM and RPB2 gene sequences the fungal strain ZK-14 and members of genus Penicillium section Exilicaulis, series Lapidosa were aligned by MEGA X software version 11.0.9 [57] using the Clustal W algorithm. The ex-type homologs were searched in the GenBank database (http://ncbi.nlm.nih.gov) using the BLASTN algorithm (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 20 February 2024). The phylogenetic analysis was conducted using MEGA X software [57]. The ITS region and partial BenA, CaM, and RPB2 gene sequences were concatenated into one alignment. A phylogenetic tree was constructed according to the Maximum Likelihood (ML) algorithm based on the Kimura 2-parameter model [58]. The tree topology was evaluated by 1000 bootstrap replicates. Talaromyces marneffei CBS 388.87T was used in the phylogenetic analysis as an outgroup (Additional file 1: Table S1).

3.5 Molecular identification of the fungal strain

The strain ZK-14 was identified using molecular markers such as ITS and partial BenA, CaM, and RPB2 regions. Approximately 1600 bp fragment of the ITS region, about 500 bp fragments of the partial BenA and CaM regions, and a 770 bp fragment of the partial RPB2 gene were successfully amplified. BLAST search showed that the ITS and partial BenA regions sequences were 100% identical with the sequences of the ex-type strain Penicillium velutinum NRRL 2069, whereas partial CaM and RPB2 genes were more than 99% identical. The phylogenetic ML tree of the concatenated ITS-BenA-CaM-RPB2 gene sequences clearly showed that the strain ZK-14 clusters with the ex-type strain Penicillium velutinum NRRL 2069 (Additional file 1: Fig. S1).

3.6 Cultivation of fungus Penicillium velutinum

The fungal strains were cultured for 21 days at 22 °C in Erlenmeyer flasks (500 mL) for each condition on the rice medium (RM) and various amounts of natural sea salt or metal ions (the fungus was cultured in two flasks for each culture condition). RM containing rice (20.0 g, white round-grain polished rice Oryza sativa grade extra "Japonka", Primorsky region, Russia), yeast extract powder (0.02 g, Himedia RM027, HiMedia Laboratories LLC., India), KH2PO4 (0.01 g, Lenreactiv Ltd., Russia), KNaC4H4O6*4H2O (0.01 g, Reakhim Ltd., Russia), natural seawater (40 mL, Vodolaznaya bay, Troitsa bay, the Sea of Japan, September 2022), metal salt (100 μM) [48]. The natural salinity of used water was 37.8 g/L. ZnCl2 (Lenreactiv Ltd., Russia), Ni(NO3)2·6H2O (Lenreactiv Ltd., Russia), Fe(NO3)3·9H2O (Lenreactiv Ltd., Russia), MgCl2·6H2O (Lenreactiv Ltd., Russia). The fermentation conditions are listed in Table 4. Control of fungus was cultivated on the RM.

Table 4 Cultivation conditions for Penicillium velutinum ZK-143.7 Extraction of the Penicillium velutinum and extract preparation

At the end of the incubation period, each fungal culture, together with the medium, was extracted with EtOAc (2 × 100 mL). The obtained extracts were filtered, concentrated to dryness using a rotary evaporator, and then weighed (Table 4). For the obtained crude extracts, extract preparation was carried out for further LC/UV analysis. The dry crude extract residues were dissolved in methanol (30 mL) and purified by flash column chromatography in 100% methanol (total volume of 250 mL for each extract). Low-pressure liquid column chromatography was performed using an empty glass column (2 × 7 cm) and the Gel ODS-A (Table 4).

3.8 LC/UV

Analysis was performed using an Agilent 1260 Infinity II chromatograph with UV detector Agilent 1260 VWD (Agilent Technologies, USA) using column YMC ODS-AM C18 (YMC Co, 5 μm, 250 × 10 mm). The mobile phases were H2O (eluent A) and MeCN (eluent B). The gradient program was as follows: from 0 to 100% eluent B from 0 to 60 min. Chromatographic separation was performed at a 1.5 mL/min flow rate at 40 °C. The extract concentration was 30 µg/µL. The injection volume was 80 μL. The detection was performed at 220 and 290 nm. A qualitative and quantitative analysis of LC/UV chromatograms was carried out using Agilent OpenLab software (version 2.4). Obtained LC/UV data were exported into MS Excel software and calculated total area peaks and their number. Peaks with a relative area of less than 1% were considered noise and were eliminated.

3.9 LC/MS

Analysis was performed using Bruker Elute UHPLC chromatography (Bruker Daltonics, Bremen, Germany) connected to a Bruker Impact II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). InfinityLab Poroshell 120 SB-C18 column (2.1 × 150 mm, 2.7 μm, Agilent Technologies, Santa Clara, CA, USA) was used for chromatographic separation. The mobile phases were 0.1% formic acid in H2O (eluent A) and 0.1% formic acid in MeCN (eluent B). The gradient program was same as in previews work [59].

The mass spectrometry detection has been performed using an ESI ionization source in positive ion mode. Optimized ionization parameters for ESI were as follows: a capillary voltage of 4.5 kV, nebulization with nitrogen at 2.5 bar, and dry gas flow of 6 L/min at a temperature of 200 °C. The mass spectra were recorded within the m/z mass range of 50–2000 (scan time 1 s). Collision-induced dissociation (CID) product ion mass spectra were recorded in auto-MS/MS mode with a collision energy ranging from 15 eV at 100 m/z to 120 eV at 1500 m/z (an exact collision energy setting depended on the molecular masses of precursor ions). The precursor ions were isolated with an isolation width of 4 Th.

The mass spectrometer was calibrated using the ESI-L Low Concentration Tuning Mix (Agilent Technologies, Santa Clara, CA, USA). The instrument was operated using the otofControl (ver. 4.1, Bruker Daltonics, Bremen, Germany) and data were analyzed using the Data Analysis Software (ver. 4.4, Bruker Daltonics, Bremen, Germany).

LC/MS data were converted from Bruker “.d” to “mzXML” format using MSConvert 3.0 (part of ProteoWizard 3.0 package, Palo Alto, California, USA) [60] and further processing was performed with MZmine (version 2.53) [61]. The MZmine processing settings are given in Additional file 1: Fig. S24. Resulted in data was exported to GNPS by the Export/Submit module with the Merge MS/MS function for the identification of detected features.

PCA analysis, hierarchical dendrogram, and visualization of the resulting graphs were performed using the “google colab” web resource based on Python 3.8 using Pandas, Seaborn, and Matplotlib libraries. Below is a link to the notepad with the code used in the analysis (https://drive.google.com/drive/folders/120W5BaIs0p4gLrhpEnx18EY6afy85NU2).

3.10 Separation of the extract fungus Penicillium velutinum ZK-14 cultivated with Fe(NO3)3·9H2O

At the end of the incubation period, the mycelia and medium were extracted with EtOAc (5 L). The obtained extract was concentrated to dryness. The residue was dissolved in H2O − EtOH (4:1) (300 mL) and was extracted with n-hexane (0.2 L × 3) and EtOAc (0.2 L × 3). After evaporation of the EtOAc layer, the residual material (7 g.) was purified via low-pressure liquid column chromatography on a Buchi B-688 Chromatography Pump using a Buchi glass column (49 × 230 mm) passed over a Si gel column, which was eluted, followed by a step gradient from 100% n-hexane to n-hexane – EtOAc (50:50) (total volume 25 L). Fractions (250 mL) were collected, dried on a rotary evaporator, weighed, and combined based on the TLC results.

The n-hexane – EtOAc (75:25) eluate (493 mg) was purified via low-pressure liquid column chromatography using an empty glass column (2 × 7 cm) and the Gel ODS-A with MeOH – H2O (90:10) to yield subfraction ZK-14 + Fe-25 (425 mg).

After then this subfraction was purified via preparative HPLC on a Shimadzu LC-20 (Shimadzu, Kyoto, Japan) chromatograph using a Shimadzu RID-20A (Shimadzu, Kyoto, Japan) refractometer on a YMC ODS-AM (YMC Co, 5 μm, 250 × 10 mm) column eluting with CH3CN – H2O (80:20) at 1.5 ml/min to yield subfractions ZK-14 + Fe-25–0 (292 mg) and ZK-14 + Fe-25–1 (224 mg).

Subfraction ZK-14 + Fe-25–0 (292 mg) was purified by low-pressure liquid column chromatography using an empty glass column (2 × 7 cm) and Gel ODS-A with EtOH – H2O (50:50; 100:0) to yield subfractions ZK-14 + Fe-25–0-50 (163.6 mg) and ZK-14 + Fe-25–0-100 (125.2 mg). After then ZK-14 + Fe-25–0-100 (125.2 mg) was purified via preparative HPLC on an Agilent 1100 chromatograph using an Agilent 1100 refractometer on a Hydro-RP column eluting with CH3CN − H2O (70:30) and Ultrasphera Si column eluting with toluene-isopropanol (9:1) at 1 ml/min to yield 4 (0.9 mg). Subfraction ZK-14 + Fe-25–0-50 (163.6 mg) was purified via preparative HPLC on a Shimadzu LC-20 chromatograph using a Shimadzu RID-20A refractometer on a YMC ODS-AM column eluting with CH3CN – H2O (80:20) at 1.5 ml/min to subfraction ZK-14 + Fe-25–0-50–2 (52 mg). Subfraction ZK-14 + Fe-25–0-50–2 was purified on an Agilent 1100 chromatograph using an Agilent 1100 refractometer on a Hydro-RP column eluting with CH3CN − H2O (70:30) at 2.5 ml/min to yield subfraction ZK-14 + Fe-25–0-50–2-3 (1.5 mg) which was purified using HyperClone column eluting with CH3CN − H2O (50:50) at 0.8 ml/min to yield 3 (0.5 mg).

Subfraction ZK-14 + Fe-25–1 (224 mg) was purified via preparative HPLC on a Shimadzu LC-20 chromatograph using a Shimadzu RID-20A refractometer on a YMC ODS-AM column eluting with CH3CN – H2O (80:20) at 2.0 ml/min to subfraction ZK-14 + Fe-25–1 (130 mg) and this subfraction was purified on an Agilent 1100 chromatograph using an Agilent 1100 refractometer on a Hydro-RP column eluting with CH3CN − H2O (70:30) 2.5 ml/min to yield compound 2 (5.1 mg) and compound 1 (21.7 mg).

3.11 Spectral data of isolated compounds

Helvamide B (1): colorless crystal needles (MeOH); mp 144 − 145 °C; \([\alpha]_}^\) + 68.7 (c 0.115 MeOH); UV (MeOH) λmax (log ε) 210 (4.80), 199 (4.83), 205 (4.79) nm; CD (c 0.000217 M, MeOH) λmax (∆ε) 204 (+ 3.50), 216 (+ 12.05), 252 (−1.37), 283 (+ 0.31) nm; 1H and 13C NMR data, see Table 3, Additional file 1: Figs. S5–S9; HRESIMS [M + Na]+m/z 571.2193 (calcd for C34H32N2O5Na 571.2203, ∆ + 1.8 ppm), [M − H]−m/z 547.2215 (calcd. for C34H31N2O5 547.2238, ∆ + 4.2 ppm) (Additional file 1: Fig. S2).

Helvamide C (2): colorless amorphous; \([\alpha]_}^\) + 67.6 (c 0.327 MeOH); UV (MeOH) λmax (log ε) 198 (4.78), 204 (4.75), 209 (4.75) nm; CD (c 0.000175 M, MeOH) λmax (∆ε) 201 (+ 23.83), 227 (-2.92), 246 (-2.07) nm; 1H and 13C NMR data, see Table 3, Additional file 1: Figs. S15–S17; HRESIMS [M + Na]+m/z 537.2358 (calcd for C31H34N2O5Na 537.2360, ∆ + 0.4 ppm), [M − H]−m/z 513.2405 (calcd. for C31H34N2O5 513.2395, ∆ − 1.9 ppm) (Additional file 1: Fig. S10).

Saroclazin A (3): white powder; \([\alpha]_}^\) − 4.5 (c 0.044 MeOH); 1H and 13C NMR data, see Additional file 1: Table S2, Additional file 1: Figs. S19, S20; HRESIMS [M + Na]+m/z 417.1278 (calcd for C19H26N2O3S2Na 417.1278, ∆ −0.2 ppm), [M − H]−m/z 393.1313 (calcd for C19H25N2O3S2 393.1312, ∆ − 0.3 ppm) (Additional file 1: Fig. S18).

(4S,5R,7S)-4,11-Dihydroxy-guaia-1(2),9(10)-dien (4): colorless oil; \([\alpha]_}^\) + 4.4 (c 0.023 MeOH); 1H and 13C NMR data, see Additional file 1: Table S3, Figs. S22–S24; HRESIMS [M + Na]+m/z 259.1669 (calcd. for C15H24O2Na 259.1669, ∆ 0.0 ppm), [M − H]−m/z 235.1701 (calcd for C15H23O2 235.1704, ∆ + 1.3 ppm) (Additional file 1: Fig. S21).

3.12 X-ray crystallographyc data of 1

X-ray analysis which was carried out for a single light-yellow crystal obtained by recrystallization from MeOH–H2O (70:30). Experimental intensity data for 1 were collected at T = 100(2)K on a BRUKER Kappa APEX2 diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). Intensity data were corrected for absorption using the multi-scan method. The structure was solved using direct methods and refined by least-squares calculation in anisotropic approximation for non-hydrogen atoms. Hydrogen atoms were placed in geometrically idealized positions and refined in the riding-model approximation. Data collection, reduction, and refinement of the lattice parameters were performed using the Apex2 software package (Bruker. APEX 2 V.7, Bruker AXS Inc., Madison, Wisconsin, U.S.A. (2010)). All calculations were performed with SHELXL/PC program [62, 63]. Main crystallographic data and details of refinement of the crystal structure of 1 are shown in Additional file 1: Tables S4-S8, Figs. S25.

The crystallographic data (accession numbers CCDC 2326660) can be obtained free of charge from the Cambridge Crystallographic Data Center via http://www.ccdc.cam.ac. uk/data_request/cif (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, UK; fax: + 44 1223 336 033 or email: deposit@ccdc.cam.uk).

C34H32N2O5, M = 548, Crystal size: 0.48 × 0.13 × 0.11 mm3, monoclinic, space group C2, a = 46.095(4) Å, b = 6.1121(6) Å, c = 20.2447(19) Å, β = 99.780(5)°, V = 5620.8(9) Å3, Z = 8, Dcalc. = 1.297 g/cm3, μ = 0.087 mm-1. F000 = 2320, θmax = 27.242°, 44,378 reflections collected, 12,335 unique (R(int) = 0.0374). Final GooF = 1.029; for I > 2 σ (I) R1 = 0.0424, wR2 = 0.0957; for all data R1 = 0.0597, wR2 = 0.1050, |Δρ|max = 0.214 e/Å3.

3.13 Quantum-chemical modeling

All quantum-chemical calculations were performed using the B3LYP exchange–correlation functional, the polarization continuum model (PCM) and 6-311G(d) basis set implemented in the Gaussian 16 package of programs [20]. The statistical weights (gim) of the individual conformations were calculated according to equation:

where ΔGim = Gi – Gm are the relative Gibbs free energies and index “m” denotes the most stable conformation.

The ECD spectra were calculated using time-dependent density functional theory (TDDFT), B3LYP functional, PCM model, and 6-311G(d) basis set for conformations, where the relative Gibbs free energies satisfied the relation ΔGim ≤ 4 kcal/mol. To describe well the short-wave region of ECD spectra 95 electronic transitions were calculated for each conformation of 1. The Gauss-type functions were used to simulate the individual bands in the theoretical spectra. The bandwidths ζ = 0.16 eV and the UV shifts Δλ =  + 1 nm were used for best correspondence between experimental and calculated spectra for 1.

The scaled theoretical and experimental ECD spectra were obtained according to equation:

$$\Delta _\left(\lambda \right)=\Delta \varepsilon \left(\lambda \right)/\left|\Delta \varepsilon \left(_\right)\right|$$

(2)

where the denominator |Δε (λpeak)| is the modulo of the peak value for the positive characteristic band at λ ≈ 216 nm in the corresponding ECD spectrum.

3.14 Bioassays3.14.1 Cell culture

The human prostate cancer cells PC-3 CRL-1435 and the human embryonic kidney cells HEK-293 CRL-1573™ were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). PC-3 and HEK-293 cells were cultured in DMEM medium containing 10% fetal bovine serum (Biolot, St. Petersburg, Russia) and 1% penicillin/streptomycin (Biolot, St. Petersburg, Russia) at 37 °C in a humidified atmosphere with 5% (v/v) CO2. The cells were incubated in cultural flasks until sub-confluent (~ 80%).

3.14.2 Cell viability assay

The PC-3 cells (5 × 103 cells/well) and HEK293 cells (7 × 103 cells/well) were seeded in a 96-well plate and incubated overnight. Then, the extracts at a concentration of 100 μg/mL or compounds 1–4 at concentrations of 100 µM were added and the cells were further incubated for an additional 24 h. After that, cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method based on the manufacturer’s instructions (M5655-500MG, Sigma-Aldrich, St. Louis, MO, USA). The absorbance of the converted formazan was measured using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA) at λ = 570 nm. The results were presented as percentages of control data. All experiments were carried out twice in triplicate.

3.14.3 Antimicrobial assay

Antimicrobial activity was determined against yeast-like fungi Candida albicans KMM 455 from the Collection of Marine Microorganisms PIBOC FEBRAS in liquid nutrient media. Test culture of C. albicans was cultured in a Petri dish at 37 °C for 24 h on solid medium Mueller Hinton broth with agar—16.0 g/L.

The assays were performed in 96-well microplates in appropriate Mueller Hinton broth. Each well contained 90 µL of yeast-like fungi C. albicans suspension (109 CFU/mL). Then, was added 10 µL of the extracts diluted at 10% DMSO at final concentrations of 100 μg/mL or compounds 1–4. Culture plates were incubated overnight at 37 °C, and the OD620 was measured using a Multiskan Spectrum spectrophotometer (Thermo Labsystems Inc., Beverly, MA, USA). All experiments were carried out twice in triplicate. The antimicrobial activity of the extracts was evaluated in comparison with the negative control by the change in optical density and expressed as % inhibition of bacterial growth. Nitrofungin was used as a positive control in a concentration of 1 mg/mL; 1% DMSO solution in dH2O was used as a negative control.

3.14.4 Radical scavenging assay

DPPH radical scavenging activity of compounds or fungal extracts or compounds 1–4 were tested as described [64] with minor modifications. The compounds or fungal extracts were dissolved in DMSO, and the solutions the compounds, fungal extracts, or quercetin (Sigma-Aldrich, Steinheim, Germany) as a positive control (120 µL) were dispensed into wells of a 96-well microplate. In all of them, 30 µL of the DPPH (Sigma-Aldrich, Steinheim, Germany) solution in MeOH (0.75 mM) was added to each well. The concentrations of compounds and quercetin in mixture were 0.1–100.0 µM. The concentrations of fungal extracts in mixture were 0.1–100.0 µg. The plates were incubated in the dark at room temperature for 30 min, and then the absorbance was measured at 517 nm with a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA). The negative control contained no test compound. The results are presented as percentages of the negative control (DMSO) data.

3.14.5 Statistical data evaluation

All data were obtained twice in three independent replicates and calculated values were expressed as a mean ± standard error mean (SEM). Student’s t-test was performed using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) to determine statistical significance. The differences were considered statistically significant at p < 0.05.