Two New Fatty Acid Derivatives, Omphalotols A and B and Anti-Helicobacter pylori Fatty Acid Derivatives from Poisonous Mushroom Omphalotus japonicus

As part of ongoing systematic research into the discovery of bioactive secondary metabolites with novel structures from Korean wild mushrooms, we investigated secondary metabolites from a poisonous mushroom, Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. belonging to the family Marasmiaceae, which causes nausea and vomiting after consumption. The methanolic extract of O. japonicus fruiting bodies was subjected to the fractionation by solvent partition, and the CH2Cl2 fraction was analyzed for the isolation of bioactive compounds, aided by an untargeted liquid chromatography mass spectrometry (LC–MS)-based analysis. Through chemical analysis, five fatty acid derivatives (1–5), including two new fatty acid derivatives, omphalotols A and B (1 and 2), were isolated from the CH2Cl2 fraction, and the chemical structures of the new compounds were determined using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high resolution electrospray ionization mass spectrometry (HR-ESIMS), as well as fragmentation patterns in MS/MS data and chemical reactions followed by the application of Snatzke’s method and competing enantioselective acylation (CEA). In the anti-Helicobacter pylori activity test, compound 1 showed moderate antibacterial activity against H. pylori strain 51 with 27.4% inhibition, comparable to that of quercetin as a positive control. Specifically, compound 3 exhibited the most significant antibacterial activity against H. pylori strain 51, with MIC50 and MIC90 values of 9 and 20 μM, respectively, which is stronger inhibitory activity than that of another positive control, metronidazole (MIC50 = 17 μM and MIC90 = 46 μM). These findings suggested the experimental evidence that the compound 3, an α,β-unsaturated ketone derivative, could be used as a moiety in the development of novel antibiotics against H. pylori.


Introduction
Mushrooms have been used to treat various diseases in traditional medicine [1], and a number of pharmacological and phytochemical studies on mushrooms have demonstrated that they are rich sources of various bioactive compounds that exhibit beneficial immunomodulatory, antioxidant, and angiostatic activities, as well as cytotoxicity against cancers [1][2][3][4][5]. Based on this evidence, mushrooms have emerged as potential valuable sources of bioactive natural products; however, most studies have focused on medicinal and edible mushrooms, and little is known regarding bioactive secondary metabolites produced from poisonous mushrooms.
As part of ongoing systematic research on Korean wild mushrooms for the discovery of bioactive secondary metabolites with novel structures [6], we investigated bioactive secondary metabolites from a poisonous mushroom; Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. O. japonicus is an orange-to-brown-colored gilled mushroom belonging to the family Marasmiaceae, which is found in Japan and Eastern Asia. It is a member of the genus Omphalotus, the members of which have bioluminescent fruit bodies that glow in dark [7]. This poisonous mushroom causes nausea and vomiting after consumption. Sesquiterpenoids have been identified as the major secondary metabolites in O. japonicus, the most well-known of which is illudin S, a representative toxic metabolite that exhibits potent cytotoxic and antiviral activities [8]. It has also displayed strong in vitro and in vivo antitumor activity against multi-drug-resistant tumors, and a novel anticancer drug, irofulven, was developed based on the structure and anticancer activity of illudin S [9][10][11][12][13]. Additionally, the potent cytotoxicity of illudin S has extended its application to other pharmacological effects, where its antiviral activity in an HSV-I/CV-1 assay and glutathione reductase inhibition have been confirmed [8,13,14]. Toxic illudane-type sesquiterpenes from O. japonicus, including dihydroilludin S and neoilludins A and B, have also been reported [15,16]. As other secondary metabolites, the luminescent substances, lampteroflavin [17], lampterol [18] have been identified from this mushroom, and polysaccharides [19] from O. japonicus have been reported to show antitumor activities.
In the present study, we conducted the fractionation of the methanolic extract of O. japonicus fruiting bodies, and chemical analysis of CH 2 Cl 2 fraction was carried out to isolate potential bioactive compounds aided by an untargeted liquid chromatographytandem mass spectrometry (LC-MS/MS)-based analysis. Five fatty acid derivatives (1-5), including two new fatty acid derivatives, omphalotols A and B (1 and 2), were isolated from the CH 2 Cl 2 fraction. Herein, we describe the isolation and structural determination of compounds 1-5, and evaluate their anti-H. pylori activity.

Extraction of O. japonicus and Isolation of Compounds
Dried O. japonicus fruiting bodies were extracted with 80% methanol, and the crude methanolic extract was extracted by rotary evaporation. The resultant MeOH extract was sequentially applied to solvent partitioning using n-hexane, dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), and n-butanol (BuOH) as four organic solvents with increasing polarity. As a result, four main solvent fractions were obtained: n-hexane, CH 2 Cl 2 , EtOAc, and BuOH-soluble fractions. Based on the data from LC/MS and thin-layer chromatography (TLC) analysis for the four solvent fractions where major peaks characteristic of fatty acid derivatives were observed in CH 2 Cl 2 -soluble fraction, the CH 2 Cl 2 fraction was subjected to chemical analysis since the fatty acid derivatives from O. japonicus have rarely been investigated in terms of their chemical constituents. The chemical analysis using sequential column chromatography, as well as preparative and semi-preparative HPLC, resulted in the isolation of five fatty acid derivatives (1-5) (Figure 1).

Antibacterial Activity Evaluation of Isolated Compounds against H. pylori
H. pylori is a Gram-negative and microaerophilic bacterium, which causes major public health problems worldwide, affecting approximately 50% of the global population [29]. Eradication of H. pylori leads to resolution of both gastritis and gastric ulcers, and even gastric cancer [30]. Although a combination prescription of antibiotics with a proton pump inhibitor is effective, the efficacy has decreased mainly due to the increasing resistance of H. pylori strains against antibiotics such as clarithromycin and metronidazole [31][32][33][34]. Therefore, there has been a pressing need to look for new compounds, which can overcome this resistance and provide an effective therapy against H. pylori infection. Natural products with less adverse effects can be alternative approaches for the intervention of gastric disorders caused by this bacterium. The isolated compounds 1-5 were tested for their antibacterial activity against H. pylori strain 51 at the final concentration of 100 µM ( Table 2). Among the isolates, compound 1 showed moderate antibacterial activity against H. pylori strain 51 with 27.4% inhibition, comparable to that of quercetin as a positive control. Specifically, compound 3 exhibited the most significant antibacterial activity against the strain with 97.5% inhibition ( Table 2). Its inhibitory activity, with the minimal inhibitory concentrations (MIC) 50 and MIC 90 values of 9 and 20 µM, respectively, was more potent than those of a positive control and metronidazole (MIC 50 = 17 µM and MIC 90 = 46 µM). In addition, the minimum bactericidal concentration (MBC) values of compound 3 and metronidazole were 12.5 and 12.5 µM, respectively. The other compounds failed to show anti-H. pylori activity. Based on these findings, it is suggested that the α,β-unsaturated carbonyl moiety of compound 3 can play a role in the inhibition of H. pylori growth, and the hydroxyl group of compound 2 may decrease the activity. H. pylori produces a urease which catalyzes the hydrolysis of urea to produce ammonia for neutralizing the acidic condition of the stomach. It has been known that simple α,β-unsaturated ketones inhibited urease activity by binding to the cysteinyl residue in the active sites of the enzyme [35]. Further study is required to elucidate the exact mechanism of compound 3 to inhibit the growth of H. pylori. Specificity to H. pylori and toxicity to other cells of this compound are also required in the following study.

General Experimental Procedure
The information on general experimental procedure is provided in Supplementary Materials.

Mushroom Material
Fresh fruiting bodies of O. japonicus were collected from Pocheon, Gyeonggi-do, Korea in September 2019. This material was identified by DNA analysis, depending on the modified method [36]. The fungal-specific PCR primers ITS1 and ITS4 were used to amplify the internal transcribed spacer (ITS) region according to a modified method [37]. This sequence homology corresponded to that of O. japonicus (syn. Omphalotus guepiniiformis), with the highest matching score in the NCBI BLAST network server. A voucher specimen (SKKU-HK-2019-09) of the mushroom was deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Korea.

Extraction of O. japonicus and Isolation of Compounds
The dried fruiting bodies of O. japonicus (0.6 kg) were extracted with 80% aqueous MeOH three times (each 3 L × 24 h) at room temperature. The resultant extracts were filtered, and the filtrate was evaporated under reduced pressure using a rotary evaporator to obtain a crude MeOH extract (43.6 g). The extract was suspended in distilled water (700 mL) and MeOH (30 mL) and successively solvent-partitioned three times with n-hexane, dichloromethane, ethyl acetate, and n-butanol, yielding soluble layers of nhexane (6.3 g), CH 2 Cl 2 (6.7 g), EtOAc (2.4 g), and n-butanol (15.6 g). The CH 2 Cl 2 fraction (6.7 g) was subjected to silica gel column chromatography (CC) (a gradient solvent system; CH 2 Cl 2 /MeOH, from 70:1 to 1:1) to yield five fractions (Fr. C1-C5). Fr. C3 (1.2 g) was subjected to reverse phase (RP) C 18   Parallel reactions for the CEA reaction were performed as reported by Lee et al. [21], using Sand R-HBTM. Compound 1 (0.5 mg, 1.61 µmol) was transferred to two transparent, capped 5 mL vials at room temperature, and dimethylformamide (DMF) (90 µL) was added as the organic solvent for the CEA reaction. Both Sand R-HBTM (10 µL, 0.38 µmol) were added, and N,N-diisopropylethylamine (1.0 µL, 5.3 µmol) was successively transferred. Propionic anhydride (0.6 µL, 5.3 µmol) was added to start the CEA reaction. After 10 min, 2 µL aliquots from each reaction were acquired for LC/MS analysis and quenched with 98 µL of MeOH to make a total volume of 100 µL.

LC/MS Analysis
An aliquot (5 µL) of the sample (100 µL) acquired from each parallel reaction was directly injected into the LC/MS (Phenomenex Luna C 18 , 4.6 × 100 mm, 3.5 µm, flow rate: 0.3 mL/min; Torrance, CA, USA), and full scans in positive-and negative-ion modes (scan range m/z 100−1000) were applied to identify the desired acylated derivative. The mobile phase consisted of 0.1% (v/v) formic acid in distilled water (A) or acetonitrile (B) with a gradient solvent system as follows: 10−100% B for 10 min, 100% B (isocratic) for 5 min, and then 10% B (isocratic) for 5 min for the post-run washing procedure of the column. The reaction rate catalyzed by both Sand R-HBTM was determined by measuring the peak areas of the acylated derivatives.

Absolute Configuration of the 1,2-diol Functionalities in Compound 2
According to a published procedure [22,23], 2 (0.5 mg) and Mo 2 (OAc) 4 (0.75 mg) were mixed in 1.0 mL of dry DMSO with a ligand-to-metal molar ratio of approximately 1.0:1.2, and the solution was directly subjected to ECD measurements. The first circular dichroism (CD) spectrum was recorded immediately after mixing, and its time evolution was monitored until it was stationary (approximately 30 min after mixing). The inherent CD was subtracted. The observed signs of the diagnostic band at approximately 310 nm in the induced CD spectra were correlated with the absolute configuration of the 1,2-diol moiety.

Anti-Helicobacter pylori Activity
A clinical strain of H. pylori 51 isolated from a Korean patient with a duodenal ulcer (HPKTCC B0006) was provided by the H. pylori Korean Type Culture Collection, School of Medicine, Gyeongsang National University, Korea. The strain was grown and maintained on Brucella agar medium (BD Co., Sparks, MD, USA) supplemented with 10% horse serum (Gibco, New York, NY, USA). The culture conditions were 37 • C, 100% humidity, and 10% CO 2 for 2-3 days.
MICs were determined by the broth dilution method previously reported [38,39]. Twenty microliters of bacterial colony suspension equivalent to 2-3 × 10 8 cfu/mL and twenty microliters of two-fold diluted samples and controls were added to each well of a 6-well plate containing Brucella broth medium supplemented with 10% horse serum. The final volume was brought to 2 mL. After 24 h of incubation, bacterial growth was evaluated by measuring the optical density at 600 nm on a spectrophotometer (Optizen POP, Mecasys, Daejeon, Korea). MIC 50 and MIC 90 values were defined as the lowest concentrations of samples at which bacterial growth was inhibited by 50% and 90%, respectively, and were calculated using GraphPad Version 5.01 (GraphPad Software, Inc., San Diego, CA, USA).
MBC was determined by re-culturing broth dilution that inhibits the growth of H. pylori on the agar plate. Twenty microliter of broth dilution was streaked onto Brucella agar plate and incubated for 48 h. The MBC value was defined as the lowest concentration that showed no colonies of bacteria on agar plates.

Statistical Analysis
One-way analysis of variance was performed using Excel 2019 (Microsoft, Redmond, WA, USA). Values with p < 0.05 were considered statistically significant.

Conclusions
In this study, we isolated and identified five fatty acid derivatives (1)(2)(3)(4)(5), including two new fatty acid derivatives, omphalotols A and B (1 and 2), from the methanolic extracts of O. japonicus fruiting bodies. The structures of the new compounds were established using NMR spectroscopy and LC-MS analysis, as well as fragmentation patterns in MS/MS data and chemical reactions followed by the application of Snatzke's method and competing enantioselective acylation (CEA). In the anti-H. pylori activity test, we demonstrated that compound 1 showed moderate antibacterial activity against H. pylori strain 51 comparable to that of quercetin, a positive control. Specifically, compound 3 displayed the most significant anti-H. pylori activity with 97.5% inhibition, and its inhibitory activity with MIC 50 and MIC 90 values of 9 and 20 µM, respectively, was more potent than those of metronidazole (MIC 50 = 17 µM and MIC 90 = 46 µM). Based on these findings, we conclude that compound 3, an α,β-unsaturated ketone derivative, could be used as a moiety in the development of novel antibiotics against H. pylori; however, further studies on its mechanism, antibacterial activity against another species, and toxicity to normal and cancerous cell lines are needed.