First Chemical Investigation of Korean Wild Mushroom, Amanita hemibapha subsp. javanica and the Identification of Anti-Helicobacter pylori Compounds

Amanita hemibapha subsp. javanica (Amanitaceae) is an edible Korean wild mushroom. A. hemibapha subsp. javanica is often confused with A. subjunquillea, known as the East Asian death cap, which is potentially fatal when ingested. This study aimed to conduct the first chemical investigation of A. hemibapha subsp. javanica, which resulted in the isolation of seven fatty acid derivatives (1–7) and three steroids (8–10) from the MeOH extract of its fruiting bodies, and their structures were determined by comparing their NMR spectroscopic data with those previously reported, along with the data from LC/MS. Compound 1 was reported previously without the identification of its absolute configuration; its structure, including the absolute configuration was confirmed for the first time, in this study, by using 1H NMR and its fragmentation patterns in MS/MS data, and LC/MS analysis. A recently developed method using competing enantioselective acylation (CEA) coupled with LC/MS analysis was applied for determining the absolute configuration of compound 1, which revealed the 11S-configuration. In the anti-Helicobacter pylori activity test, compound 3 showed antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition) as a positive control. Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 μm with a MIC50 value of 72 μm. These findings suggested that the active compound 4 is a natural antibiotic that may be used in the development of novel antibiotics against H. pylori. In addition, the first chemical investigation of A. hemibapha subsp. javanica revealed that this mushroom can serve as a promising natural source for the bioactive natural products.


Introduction
Mushrooms in the genus Amanita are the most well-known psychoactive and poisonous basidiomycete fungi [1]. The Amanita genus consists of cyclopeptide-containing mushrooms, which are responsible for over 90% of all fatal mushroom poisoning instances [2]. The major species that contain cyclopeptide toxins such as α-amanitin, β-amanitin, phalloidin, and phallacidin include A. phalloides, A. virosa, A. verna, A. ocreata, A. bisporigera, A. suballiacea, A. tenuifolia, and A. hygroscopica [2,3]. Among the over 600 species of this genus that are poisonous to humans, A. hemibapha subsp. javanica is an edible mushroom. The fruiting bodies of A. hemibapha subsp. javanica are white before maturation but turn yellowish red with their caps turning from a convex to a flat shape as they mature. A. hemibapha subsp. javanica usually grows throughout summer to fall, and it is often confused with A. subjunquillea Imai, also known as the East Asian death cap, which is potentially fatal when ingested [2]. Although a number of studies have reported the biological activities of A. hemibapha, there are limited studies on A. hemibapha subsp. Javanica. A recent study demonstrated that A. hemibapha subsp. javanica is known to scavenge hydroxyl radicals [4]. A. hemibapha subsp. javanica also secretes mucilage polysaccharides which activate RAW264.7 cells to release nitric oxide and cytokine mitogenactivated protein kinase pathways, thereby enhancing immunity [4,5]. Nevertheless, to the best of our knowledge, A. hemibapha subsp. javanica has never been evaluated in terms of its chemical constituents.
In our continuous efforts to chemically study these uninvestigated Korean wild mushrooms as well as to discover novel bioactive compounds from natural sources [6][7][8], we conducted the first chemical analysis of A. hemibapha subsp. javanica, which resulted in the isolation of seven fatty acid derivatives (1-7) and three steroids (8)(9)(10) from the methanol (MeOH) extract of the fruiting bodies. These compounds were isolated using successive column chromatography and preparative and semi-preparative HPLC purification. The structures of the isolated compounds were determined using magnetic resonance (NMR) spectroscopy, physical data interpretation, and liquid chromatography-mass spectrometry (LC/MS) analyses. In the current study, the isolation of compounds 1-10, their structural determination, and evaluation of their anti-Helicobacter pylori activity was also reported.

Isolation of Compounds
The dried fruiting bodies of A. hemibapha subsp. javanica were extracted with 80% MeOH/H 2 O, which provided the resultant MeOH extract after rotary evaporation. The MeOH extract was solvent-partitioned with hexane, dichloromethane, ethyl acetate, and n-butanol to obtain four major fractions according to the order of polarity ( Figure S4). The LC/MS analysis of the four fractions and thin-layer chromatography (TLC) analysis revealed that the hexane and ethyl acetate-soluble fractions were promising for chemical analysis as we observed major peaks characteristic of fatty acid derivatives and sterols, which are major constituents in mushrooms, in the hexane and ethyl acetate-soluble fractions. The chemical composition of A. hemibapha subsp. javanica was identified by intensive chemical analysis of the hexane and ethyl acetate-soluble fractions using successive column chromatography and preparative and semi-preparative HPLC purification ( Figure S4). During the isolation procedure, the subfractions and isolated compounds were monitored by LC/MS, and seven fatty acid derivatives (1-7) and three steroids (8-10) were isolated ( Figure 1).

Determination of the Structure of Compounds
Compound 1 was isolated as an amorphous gum. The molecular formula was established as C 18 Figure S1). The IR spectrum showed an absorption band of the hydroxyl group (3331 cm −1 ). The 1 H NMR spectrum of compound 1 ( Figure S2) showed signals of an olefinic pair at δ H 5.59 (1H, dt, J = 15.5, 7.0 Hz) and 5.40 (1H, dd, J = 15.5, 7.0 Hz), oxygenated methine at δ H 3.94 (1H, q, J = 7.0 Hz), terminal methyl group at δ H 0.89 (3H, t, J = 7.0 Hz), and deshielded methylenes at δ H 2.24 (2H, t, J = 7.5 Hz) and 2.03 (2H, m), and the overlapping signals are attributed to the remaining methylenes from 1.24 to 1.59 ppm. The coupling pattern of one of the olefinic protons [δ H 5.40 (dd, J = 15.5, 7.0 Hz)] indicated that the oxygenated methine could be located next to the olefinic proton, and the olefinic protons were determined to have a trans-form. The overall 1 H NMR data showed that the Compound 1 was a fatty acid derivative [9]. The double bond was located at C-9/C-10, as shown by the MS/MS data  Figure 2). Thus, the structure of compound 1 was determined to be (E)-11-hydroxy-9-octadecenoic acid, which has been reported previously without determining its absolute configuration [10].

Determination of the Structure of Compounds
Compound 1 was isolated as an amorphous gum. The molecular formula was established as C18H34O3 from the molecular ion peak [M − H] − at m/z 297.2427 (calculated for C18H33O3, 297.2430) in the negative-ion mode of HR-ESIMS ( Figure S1). The IR spectrum showed an absorption band of the hydroxyl group (3331 cm −1 ). The 1 H NMR spectrum of compound 1 ( Figure S2) showed signals of an olefinic pair at δH 5.59 (1H, dt, J = 15.5, 7.0 Hz) and 5.40 (1H, dd, J = 15.5, 7.0 Hz), oxygenated methine at δH 3.94 (1H, q, J = 7.0 Hz), terminal methyl group at δH 0.89 (3H, t, J = 7.0 Hz), and deshielded methylenes at δH 2.24 (2H, t, J = 7.5 Hz) and 2.03 (2H, m), and the overlapping signals are attributed to the remaining methylenes from 1.24 to 1.59 ppm. The coupling pattern of one of the olefinic protons [δH 5.40 (dd, J = 15.5, 7.0 Hz)] indicated that the oxygenated methine could be located next to the olefinic proton, and the olefinic protons were determined to have a trans-form. The overall 1 H NMR data showed that the Compound 1 was a fatty acid derivative [9]. The double bond was located at C-9/C-  Figure 2). Thus, the structure of compound 1 was determined to be (E)-11-hydroxy-9-octadecenoic acid, which has been reported previously without determining its absolute configuration [10].     Figure 2). Thus, the structure of compound 1 was determined to be (E)-11-hydroxy-9-octadecenoic acid, which has been reported previously without determining its absolute configuration [10].  To assign the absolute configuration of C-11 in compound 1, a recently developed chemical-derivative method, competing enantioselective acylation (CEA) coupled with LC/MS analysis [11], was applied. The method uses homobenzotetramisole (HBTM) catalysts, in which the reaction rates of the parallel reactions are compared using LC/MS. For each parallel acylation reaction, two sets of compound 1 (each 0.2 mg) and Sand R-HBTM catalysts (each 10 µL) were reacted. Samples of each reaction were quantitatively analyzed using LC/MS to measure the reaction rate catalyzed by Sand R-HBTM. The acylated derivative (1A, [M − H] − peak at m/z 353), esterified by propionic anhydride at the hydroxyl group of C-11, was expected because of the CEA reaction ( Figure 3). The anticipated derivatives could be directly detected through the extracted ion chromatogram (EIC) of the LC/MS, where the peak areas of the acylated derivatives were compared ( Figure S3) in samples of both parallel reactions to determine the reaction rate. The results revealed that the esterification reaction with R-HBTM was faster than that with S-HBTM (Figure 3), suggesting that compound 1 has an 11S-configuration, according to the Mnemonic to predict the configuration of secondary alcohols in the CEA reaction ( Figure 3) [11]. Collectively, the complete structure of compound 1 was determined to be (9E,11S)-hydroxyoctadecenoic acid ( Figure 1) and it was termed as amanitahemic acid A.

Evaluation of Antibacterial Activity of the Isolated Compounds against H. pylori
Helicobacter pylori is a major public health issue worldwide, affecting approximately 50% of the global population [20]. Eradication of H. pylori helps treat both gastritis and gastric ulcers, and even gastric cancer because the presence of H. pylori was associated with the gastric pathologies [21]. However, clinical failures due to antibiotic resistance are of increasing concern [22,23]. Thus, there is an urgent need to develop novel antibiotics against H. pylori. Prior to the test for the isolated compounds, the anti-H. pylori activity of the MeOH extract and solvent-partitioned fractions was evaluated using a clinical strain of H. pylori 51. Although the MeOH extract showed no inhibitory activity against H. pylori strain 51, hexane fractions showed weak inhibitory activity (Table S3). Next, the isolated compounds 1-10 were evaluated for antibacterial activity against H. pylori strain 51 (Table 1). Among the isolates, compound 3 exhibited antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition) as a positive control. Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 µm (Table 1), and it showed a MIC 50 value of 72 µm. The other compounds failed to show anti-H. pylori activity. Based on these findings, it was found that the presence of an α,β-unsaturated carbonyl moiety in fatty acid derivatives can be important for the anti-H. pylori activity and the hydroxyl group may decrease the inhibitory activity. In fact, it has been known that H. pylori produces a potent urease, which catalyzes the hydrolysis of urea to produce ammonia for neutralizing the acidic condition of stomach, and simple α,β-unsaturated ketones inhibit urease activity by binding to the cysteinyl residue in the active sites of the enzyme [24]. Further study is required to elucidate the exact mechanism of compounds 3 and 4 to inhibit the growth of H. pylori.

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

Fungal Material
The fruiting bodies of A. hemibapha subsp. javanica were collected from Yunggeolleung, Hwaseong, GyeongGi-do, Korea, in August 2015. The samples were identified by one of the authors (R. Ryoo). The DNA identification of this material was confirmed by the modified method of Lee and Taylor [25]. The nuclear ribosomal internal transcribed spacer (ITS) region of the DNA sequence was amplified by the fungal-specific PCR primers ITS1 and ITS4 [26]. This sequence was matched with A. hemibapha subsp. javanica with the highest score searched in NCBI BLAST database. A voucher specimen (SKKU 2015-08-AH) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

LC/MS Analysis
An aliquot (5 µL) of the sample (100 µL) acquired from each parallel reaction was directly injected into the LC/MS and analyzed using an analytical Kinetex C18 100 Å Pharmaceuticals 2022, 15, 152 7 of 9 column (Phenomenex) (C 18 , 4.6 × 100 mm, 3.5 µm, flow rate: 0.3 mL/min), 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.

H. pylori Culture
A clinical strain of H. pylori 51 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.

Anti-H. pylori Activity
Minimal inhibitory concentrations (MICs) were determined by broth dilution method previously reported [27]. 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 (BD Co., Sparks, MD, USA) 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 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). All of the values were obtained from three independent experiments.

Conclusions
In this study, the first chemical investigation of the fruiting bodies of A. hemibapha subsp. javanica, an edible mushroom among Amanita species, led to the isolation and identification of seven fatty acid derivatives (1-7) and three steroids (8)(9)(10). The structure of compound 1 (amanitahemic acid A), including its absolute configuration, was elucidated using 1 H NMR, MS/MS fragmentation data, and the application of CEA reaction coupled with LC/MS. In the anti-H. pylori activity test, compound 3 showed antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition). Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 µm with the MIC 50 value of 72 µm. Based on these findings, we conclude that compound 4 could be used to develop novel antibiotics against H. pylori.