Bioactive Alpha-Pyrone and Phenolic Glucosides from the Marine-Derived Metarhizium sp. P2100

Glycoside compounds have attracted great interest due to their remarkable and multifarious bioactivities. In this study, four hitherto unknown 4-methoxy-β-D-glucosyl derivatives were obtained and identified from the marine-derived fungus Metarhizium sp. P2100, including three alpha-pyrone glycosides (1–3) and one phenolic glycoside (4). Their planar structures were elucidated by comprehensive spectroscopic analysis, including 1D/2D NMR and HRESIMS. The absolute configurations of 1–3 were determined by a single-crystal X-ray crystallographic experiment, a comparison of the experimental, and a calculated electronic circular dichroism (ECD) spectra, respectively. Compounds 2 and 3 are a pair of rare epimeric pyranoside glycosides at C-7 with a core of aglycone as 2H-pyrone. Compounds 1–4 exhibited weak anti-inflammatory activities. In particular, compounds 1–3 displayed inhibitory activities against α-amylase, showing a potential for the development of a new α-amylase inhibitor for controlling diabetes.


Introduction
Glycosylation represents one of the most common and essential biochemical reactions in vivo. The resulting glycoconjugates possess diverse functions, including information storage and transfer, energy storage, maintenance of cell structural integrity, molecular recognition, signaling, virulence, and chemical defense [1]. Glycosylated modification of natural and synthetic small-molecular drugs could significantly alter the pharmacological properties of parent compounds [2]. Glycoside compounds fall into several main structure types by aglycone, including pyranone, flavone, alkaloid, macrolide, iridoid, etc. A significant number of these molecules have been clinically used as antibiotics, enzyme inhibitors, hormones, and drugs for the treatment of human diseases [1]. For example, streptomycin, gentamycin, and vancomycin are typical representatives of glycosidic antibiotics that have been used as antibacterial agents [3]. Amphotericin B [4] and nystatin A1 [5] have been used as potent antifungal drugs. Acarbose has been used to treat diabetes as an alpha-glucosidase inhibitor [6]. Cardiac glycosides have been used to treat various heart conditions [7].
In recent decades, with the development of marine biological resources, marine organisms-sourced secondary metabolites have played an important role in the discovery and development of novel drugs [8][9][10]. Among these bioactive natural products, secondary metabolites derived from marine fungi account for an increasing proportion, and more and more bioactive glycosides have been excavated [11,12]. These include 3-O-(6-O-α-L-arabinopyranosyl)-β-D-gluco-pyranosyl-1,4-dimethoxyxanthone, and xanthone O-glycoside, obtained from the mangrove endophytic fungus Phomopsis sp. and found to display cytotoxicity against HEp-2 and HepG2 cells with IC 50 values of 9 and 16 µM, respectively [13]. Aquastatin A was isolated from a marine-derived fungus Cosmospora sp., displaying potent and selective inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) with an IC 50 value of 0.19 µM in a competitive manner [14]. A series of virescenosides were obtained from Acremonium striatisporum, isolated from sea cucumber, among which virescenosides M-U displayed cytotoxic activity against the tumor cells, Ehrlich carcinoma, in vitro [15,16].
Metarhizium is a class of entomopathogenic filamentous fungi. It plays a vital role in the protection of the ecological environment and the development of green agriculture [17]. Its secondary metabolites perform many functions, such as mediating intra-and interspecies communication, and mitigating abiotic and biotic stresses in the process of insect infection [18]. There are abundant Metarhizium resources in nature, and novel Metarhizium species have been found continuously. It was reported that the genomic sequencing of existing Metarhizium revealed an abundance of secondary metabolite biosynthesis gene clusters with great potential in the mining of novel secondary metabolites [19]. Considering the diversity of the genome and secondary metabolome, it is expected that the structurally unique and bioactive compounds may be more prone to be identified in new species or strains than the known ones.
During our continuous research, aiming to explore bioactive natural products from the marine environment, a variety of new secondary metabolites with multiple biological activities have been obtained from marine microorganisms [20][21][22][23]. In the present study, the chemical investigation of the marine-derived fungal strain Metarhizium sp. P2100 was performed, which was isolated from the seawater of the Yellow Sea, China. Four new glucoside compounds were isolated and identified. Herein, we reported the isolation, structure elucidation, and biological activities of these compounds.

Fungal Material
The fungal strain Metarhizium sp. P2100 was isolated from the seawater collected from the Qingdao Huiquan Bay, Yellow Sea, in August 2019. The fungus was identified as Metarhizium sp. fungus based on its morphological features and the sequence analysis of the internally transcribed spacer (ITS) region (GenBank accession number: OP028052) of the rRNA gene, as well as its derived phylogenetic analysis [24]. This fungal strain was deposited in the Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

ECD Calculation of Metabolites
Monte Carlo conformational searches were carried out via the Spartan's software (Wavefunction&Q-Chem, Irvine&,Pleasanton USA) using the Merck Molecular Force Field (MMFF). The conformers with a Boltzmann population of over 5% were chosen for ECD calculations, and initially optimized at B3LYP/6-311+G (d) level. The theoretical calculation of ECD was conducted using the Time Dependent Density Functional Theory (TDDFT) at the B3LYP/6-311+ +G (2d, p) level for all conformers. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany), according to Boltzmann distributions.

Bioassay
The antibacterial activity was evaluated following the standards recommended by Yang [25]. Three marine-derived pathogenic bacterial strains, Vibriovulnificus MCCC E1758, V. rotiferianus MCCC E385 and V. campbellii MCCC E333, were used, and ampicillin sodium was tested as a positive control.
The antifungal bioassay was conducted following the standards recommended by Yang [25]. Three pathogenic fungal strains, Candida albicans ATCC 24433, C. tropicalis ATCC 20962, and C. parapsilosis ATCC 22019 were tested, and with amphotericin B as a positive control.
The 1,1-diphenyl-2-picryl-hydazyl (DPPH) scavenging assay was performed using the method described by Aquino [26]. The reaction mixture consisted of freshly prepared 100 µmol/L DPPH in ethanol and different concentrations of the tested compound. The reaction mixtures were incubated for 20 min at room temperature in the dark, and the absorbance was recorded at 517 nm.
The Fe 3+ reduction assay was performed using the method described by Aktumsek [27]. The reaction mixture consisted of freshly prepared 100 µmol/L 2,4,6-tripyridin-2-yl-1,3,5-triazine (TPTZ) in ultrapure water and different concentrations of tested compounds. The reaction mixture was incubated for 20 min at room temperature in the dark, and the absorbance was recorded at 593 nm.
The bioassay for NO production inhibitory activity was conducted as described by Xia [28]. The mouse macrophage was seeded in 96-well plates. In each well, lipopolysaccharide (LPS) (1 µg/mL) was added after treating it with or without the tested compound for 24 h. The NO production in the supernatant was detected by the Griess reaction. The absorbance at 540 nm was measured with a microplate reader. The NO concentration and the inhibitory rate were calculated through a calibration curve. Dexamethasone was used as the positive control. Experiments were operated in triplicate, and the data were described as the mean ± SD of three independent experiments.
The α-amylase inhibition activity was measured by Milella [29] with minor modifications. Tested compounds and 1-Deoxynojirimycin (1-DNJ) were dissolved in methanol at different concentrations. A substrate solution was prepared with 0.25% starch dissolved in 20 mM phosphate buffer solution (PBS). The enzyme (10 units/mL of α-amylase in PBS, 25 µL) and sample solutions (25 µL) were mixed in Eppendorf vials, and the mixtures were incubated at 37 • C for 10 min. Then, 25 µL of substrate solution was added to each tube, and the reaction mixture was incubated at 37 • C for 10 min. The reaction was terminated by adding 150 µL of dinitrosalicylic acid color reagent. Subsequently, the test tubes were heated for 5 min at 100 • C. After cooling at room temperature, the absorbance was measured at 540 nm. The absorbance of the blank (enzyme solution was added during the boiling) and negative controls (methanol) were recorded. Analyses were performed in triplicate, and the final sample absorbance was obtained by subtracting its corresponding blank reading. The inhibitory activity (%) was calculated as follows: The half-maximal inhibitory concentration (IC 50 ) values were calculated according to the inhibition curve and the data were shown in the layout of mean ± SEM by GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Results and Discussions
From the ethyl acetate extracts of the fermented rice solid and rice-wheat solid media of Metarhizium sp. P2100, four new glucoside compounds (1-4) (Figure 1) were isolated by column chromatography and HPLC. Compound 1 is a pyranone glucoside whose C4 position is substituted by a 4-O-methyl-β-D-glucose. Compounds 2 and 3 are a pair of epimeric pyranoside glycosides with a 4-O-methyl-β-D-glucose attached to their C7 positions. Compound 4 is a phenolic glucoside.
Compound 3 had the same molecular formula as 2 (C 18 H 28 O 9 ), which was deduced from its HRESIMS with 389.1808 [M+H] + (calcd for 389.1767). Comparing the 1D-and 2D-NMR spectra of 3 with those of 2 (see Table 1) suggested that they might be a pair of epimers. The main differences were the 1 H and 13 C NMR signals of positions at C-7, C-5, and C-1 . Notably, the Cotton effect at 280 nm in the ECD spectrum of 3 was opposite to that of 2 ( Figure 4). Compound 3 was presumed to adopt the opposite configuration to compound 2 at C7. Thus, the planar structure of 3 was completely defined (For spectrums of compound 3 see Figures S16-S23). The absolute configurations of 2 and 3 were determined by ECD calculation. Due to the present of an alkyl side chain in compounds 2 and 3, their structures possess high conformational flexibility and low convergence, so it is difficult to search and calculate for all their conformations. In fact, the alkyl side chain has a negligible impact on the ECD spectrum, since there is no chirality in the alkyl side chain. Thus, the simplified truncated model compounds 2a and 3a ( Figure 5) were adopted by removing the alkyl side chains of 2 and 3, which were used for conformational analysis and ECD calculations. Truncated model compounds of the original structures are often used in TDDFT-ECD calculations to simplify the conformational analysis without changing the computed Boltzmann-weighted ECD spectrum [31,32]. The calculations for the 2a and 3a electronic circular dichroism (ECD) spectra were performed by using the time-dependent density functional theory (TDDFT) at the B3LYP/6-311+G (d, p) level in methanol. The results showed that the calculated ECD spectra of 7R and 7S for 2a and 3a had positive and negative Cotton effects in the range of 250−330 nm, respectively, which were in accordance with the experimental spectra of 2 and 3 ( Figure 4). Thus, the absolute configurations of compounds 2 and 3 were defined as 7R and 7S, respectively. Thus, compounds 2 and 3 were determined as a pair of epimeric pyranoside glycosides, and named as 7(R)-(4 -methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (2) and 7(S)-(4 -methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (3), respectively. Lecaniside D (4) was obtained as a yellow oil. Its molecular formula (C 16 (Table 2) were very similar to the reported data of lecaniside C, isolated from Lecanicillium attenuatum [33]. Compared to lecaniside C, the 13   These isolated glucoside compounds were evaluated for their antibacterial, antifungal, DPPH scavenging activities, Fe 3+ reduction, cell proliferation inhibition, α-amylase inhibition, and anti-inflammatory activities. No compounds showed antibacterial, antifungal, DPPH scavenging activity and Fe 3+ reduction ability. Compounds 1-4 exhibited weak anti-inflammatory activities, and compounds 2-3 showed weak cell proliferation inhibition activities (Table 3). Compounds 1-3 exhibited inhibitory activities against α-amylase with the IC 50 values of 128, 64 and 64 µM, respectively, slightly weaker than acarbose with the IC 50 values of 26.3 ± 1.2 µM [29]. Several anologs of 1-4 had been reported previously. 4 -O-methyl-β-mannopyranoside, isolated from Xylaria feejeensis, showed α-glucosidase inhibitory activity [34]. Lecaniside C, an analog of 4, has PTP1B inhibitory activity with the IC50 > 50 µM [29].