A glucosylceramide with antimicrobial activity from the edible mushroom Pleurotus citrinopileatus

Abstract

The golden oyster mushroom Pleurotus citrinopileatus is a popular edible mushroom with multifunctional biological activities, but there are a limited number of previous studies on its chemical composition. This is the first report of the isolation of glucosylceramide with antimicrobial activity from the fruiting body of this mushroom. This compound was identified as 1-O-β-d-glucopyranosyl-(2S,3R,4E,8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol. The IC₅₀ value of this compound for the growth of Escherichia coli and Staphylococcus aureus was 275.1 μM (200 μg/ml) and 323.2 μM (235 μg/ml), respectively.

Introduction

Mushrooms are a nutritionally functional food and a source of physiologically beneficial medicines. Pleurotus citrinopileatus is an edible mushroom (Synonymy: P. cornucopiae, P. cornucopiae var. citrinopileatus) belonging to the genus Pleurotus, Pleurotaceae family. The name of this mushroom in English is golden oyster mushroom, tamogitake in Japanese, yuhuangmo in Chinese, goldenseed in Korean, and weishenga limonaya in Russian. A half-dozen recent studies have focused on the cultivation of P. citrinopileatus for its numerous multifunctional biological activities, including antigenotoxicity [1], angiotensin-converting enzyme (ACE) inhibition activity [2], antihyperlipidemic activity [3], antiatopic dermatitis activity [4], antioxidant activities [5], anticancer activity [5], and HIV-1 reverse transcriptase inhibition activity [6]. There are a limited number of previous studies on the chemical composition, and there have been few reports identifying the lectin, peptide, and protein from water extracts of P. citrinopileatus [2, 7].

Our previous study showed that the fruiting body of P. citrinopileatus had melanin biosynthesis inhibition, antioxidant, antibacterial, and antihyaluronidase activities [8]. Thus, the investigation of these biologically active constituents in the fruiting bodies of P. citrinopileatus led us to isolate an antimicrobial compound, glucosylceramide.

Materials and methods

General experimental procedure

The nuclear magnetic resonance (NMR) spectra were obtained on a JNM-AL400 FT-NMR spectrometer (JEOL, 400 MHz) in CD₃OD. The structure was assigned by means of ¹H and ¹³C NMR, distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and ¹H–¹H correlation spectroscopy (COSY) experiments, in combination with liquid chromatography electrospray ionization-time of flight mass spectrometry (LC-ESI-IT-TOF–MS) (Shimadzu Corporation) and fast atom bombardment mass spectrometry (FAB-MS) (JEOL, JMS-700 mass spectrometer). Column chromatography was performed by silica-gel (Wakogel C-200 particle size 75–150 μm, Wako). Thin-layer chromatography (TLC) was carried out using Merck pre-coated silica-gel 60 F₂₅₄ plates (0.25 mm) and spots were detected with I₂ detection and under UV light. The compound was isolated by preparative high-performance liquid chromatography (HPLC) using a Waters™ 600 Controller, Waters™ 486 Tunable Absorbance Detector and Waters 600 Multisolvent Delivery System. Optical rotation was measured on a JASCO DIP-370 digital polarimeter. The absorbance was measured by Tecan Spectra microplate reader (Tecan Japan, Kanagawa, Japan) and UV/vis Spectrometer V-530 (JASCO Corporation). Fluorescence was recorded on FP-6500 Research Fluorescence Spectrometer (JASCO Corporation).

Mushroom materials

Fresh fruiting bodies of P. citrinopileatus were obtained from Tamogitake Pharmaceutics Co. Ltd (Nagano, Japan). The fruiting bodies were cleaned to remove any residual materials and then freeze-dried. The milled freeze-dried P. citrinopileatus (900.0 g) were extracted with methanol (2 × 9.0 l) at room temperature for 1 week and then filtered. The methanol extract was concentrated by a rotary evaporator. The yield of the methanol extract was 114.0 g (12.7%).

Extraction and isolation

A portion of the methanol extract (100.0 g) was applied to a silica gel column [Wakogel C-200 (4.0 kg), 19 cm i.d. × 50 cm] and eluted with n-hexane/chloroform (7:3, 5:5, 3:7, 0:10), ethyl acetate, acetone, ethanol, and methanol (each 8.0 l), followed by methanol/water (50:1, 6.0 l; 11:1, 2.0 l; 8:1, 3.0 l), affording eight fractions (Fr 1 to Fr 8). Fr 4 (1.0 mg/ml) and Fr 5 (1.4 mg/ml) showed antimicrobial activity with growth inhibition of 98.5 and 100.0% against Escherichia coli IFO3301 for 8 h, respectively. Based on TLC analysis, using hexane/chloroform/methanol (1:9:1), Fr 4 (R _f = 0.34, 0.42, 0.60, 0.72, 0.84) and Fr 5 (R _f = 0.34, 0.47, 0.60, 0.72, 0.84) were combined to yield Fr 4′ (6.9 g) (R _f = 0.34, 0.42, 0.47, 0.60, 0.72, 0.84). A portion of the Fr 4′ (4.5 g) was then applied to a silica gel column [Wakogel C-200 (750 g), 5.5 cm i.d. × 120 cm] and eluted with n-hexane/chloroform (9:1, 0:10), chloroform/ethyl acetate (5:5), chloroform/ethyl acetate/ethanol (4:8:2), ethyl acetate/ethanol (5:5), n-butanol/ethanol (5:5), dichloromethane/n-butanol/ethanol (2:2:10), ethanol, ethanol/methanol (5:5), and methanol (each 1.4 l) to give eight fractions (Fr 4′-1 to Fr 4′-8). The Fr 4′-6 (303.5 mg, R _f = 0.06, 0.16, 0.30, 0.82; chloroform/ethyl acetate = 5/5) was purified by preparative HPLC and eluted with an Inertsil Prep-ODS column (20 mm i.d. × 250 mm) at a flow rate of 8.0 ml/min of methanol and a retention time (t _R) was 25.5 min to give compound 1, which was identified by NMR and MS analysis as a glucosylceramide (38.4 mg) as follows.

1-O-β-d-glucopyranosyl-(2S,3R,4E,8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol. (1): White amorphous powder. $[\alpha ]_{\text{D}}^{23}$ + 5.0° (c = 0.3, CH₃OH). FAB-MS (positive ion mode; matrix: nitrobenzyl alcohol) m/z: 710 [M+H–H₂O]⁺, 548 [M+H–H₂O–Glu]⁺; LCMS-IT-TOF m/z: 750.5488 [M+Na]⁺, 726.5535[M–H]⁻ (calcd for C₄₁H₇₆NO₉: 726.5520).

¹H-NMR (in CD₃OD): δ 3.25 (m, 1H, H-1a), 3.78 (m, 1H, H-1b), 3.63 (m, 1H, H-2), 3.88 (m, 1H, H-3), 5.39 (dd, J = 15.3, 7.3 Hz, 1H, H-4), 5.64 (m, 1H, H-5), 1.88 (m, 2H, H-6), 1.97 (m, 2H, H-7), 4.95 (m, 1H, H-8), 1.97 (m, 2H, H-10), 1.19–1.31 (m, 14H, H-11/17), 0.80 (t, J = 6.9 Hz, 3H, H-18), 1.50 (s, 3H, H-19), 7.25 (1H, d, J = 8.7 Hz, NH), 4.04 (m, 1H, H-2′), 1.62 (m, 1H, H-3′a), 1.45 (m, 1H, H-3′b), 1.19/1.31 (m, 24H, H-4′/15′), 0.80 (t, J = 6.9 Hz, 3H, H-16′), 4.17 (d, J = 7.7, 1H, H-1″), 3.10 (dd, J = 9.1, 7.8 Hz, 1H, H-2″), 3.17/3.19 (m, 3H, H-3″/5″), 3.60 (m, 2H, H-6″).

¹³C NMR (in CD₃OD): δ 69.73 (C-1), 54.67 (C-2), 72.94 (C-3), 134.60 (C-4), 131.10 (C-5), 33.05 (C-6), 33.76 (C-7), 124.82 (C-8), 136.79 (C-9), 40.75 (C-10), 23.70–30.79 (C-11/C-17), 14.41 (C-18, CH₃), 16.15 (C-19), 177.18 (C-1′), 73.15 (C-2′), 35.88 (C-3′), 23.70–30.79 (C-4′/15′), 14.41 (C-16′), 104.72 (C-1″), 75.01 (C-2″), 77.99 (C-3″), 71.60 (C-4″), 77.90 (C-5″), 62.73 (C-6″).

Antibacterial activity

The E. coli IFO 3301 or Staphylococcus aureus IFO 13276 bacterial strain was used. The antibacterial assay was performed as previously described by Meng et al. [8]. Eighty microliters of NB medium and 10 μl of 20% DMSO were added with or without (control) the test samples at various concentrations into 96-well plastic cell culture plates. Pre-cultured bacteria in the NB medium (10 μl) at a density of 1.0 × 10⁵ CFU/ml were then added into each well, and incubated for 8 h. The absorbance was measured at 630 nm.

Inhibitory effect on melanogenesis using cultured B16 melanoma cells

Determination of melanin content

This assay was performed as previously described by Arung et al. [9]. The melanin content of cells after treatment was determined as follows. After removing the medium and washing the cells, the cell pellet was dissolved in 1.0 ml of 1 M NaOH. The crude cell extracts were assayed using a micro plate reader (Bio-Tek, USA) at 405 nm to determine melanin content. The results from the samples were analyzed as a percent of the control culture. Arbutin was used as a positive control.

Cell viability

Cell viability was determined by use of the microculture tetrazolium technique (MTT assay). A culture was initiated, and after incubation, 50 μl of MTT [(3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide)] in phosphate buffered saline (5 mg/ml) was added to each well. The plates were incubated for 4 h. After removing the medium, formazan crystals were dissolved in 1.0 ml of 0.04 M HCl and the absorbance was measured at 570 nm relative to 630 nm.

Antioxidant assay

Oxygen radical absorbance capacity (ORAC) assay

The ORAC assay was performed as described previously by Shimamura et al. [10]. Data are expressed as milligram of Trolox equivalents (TE) per milligram of sample (mg TE/mg).

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay

This assay was performed as described previously by Meng et al. [8]. The free radical scavenging activity of the sample was quantified by the decolorization of DPPH at 517 nm.

SOD-like activity

SOD-like activity was evaluated using the SOD assay kit-WST (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the method described in previous studies [11]. The absorbance of each sample was measured at 450 nm in a Tecan Spectra microplate reader (Tecan Japan Co., Ltd., Kanagawa, Japan).

Antihyaluronidase activity

Antihyaluronidase activity was measured using the previously described method [8]. The absorbance was measured at 585 nm. Cromolyn interrupts the physiological response to nasal antigens and, when used prophylactically, can prevent the onset of symptoms as well as treat nasal allergy symptoms once they occur. In addition to its effects on mast cells, cromolyn inhibits macrophages, eosinophils, monocytes, and platelets believed to play a role in the inflammatory response [12]. Thus, cromolyn (Sigma Aldrich, USA) was used as a positive control.

Results and discussion

Our previous study has reported that the extract of the fruiting body of P. citrinopileatus have melanin biosynthesis inhibition, antioxidant, antibacterial, and antihyaluronidase activities [8]. In addition, the general category of cosmetic skin care products includes sunscreens to protect the skin from UV damage, antioxidant products to repair or hide skin imperfections (e.g., wrinkles, dark circles), and antibacterial products to treat pathogenic bacteria. For example, S. aureus is widespread, potentially pathogenic types of bacteria that cause some skin conditions such as acne, comedo, papules, pustules, and cellulitis. These led us to investigate the biologically active components in the fruiting bodies of P. citrinopileatus, with a special focus here on the antimicrobial activity. The methanol extract of P. citrinopileatus was successfully chromatographed on a silica gel column (Wakogel C-200, 19 cm i.d. × 50 cm and 5.5 cm i.d. × 120 cm), preparative HPLC [Inertsil Prep-ODS column (20 mm i.d. × 250 mm)] to lead to the isolation of compound 1 with antimicrobial activity. The structure was established based on the NMR and mass spectral data, as well as by comparison with the data reported in the literature [13, 14].

Compound 1 was isolated as an optically active white amorphous solid ($[\alpha ]_{\text{D}}^{23}$ + 5.0°; c = 0.3, CH₃OH). Positive FABMS (positive ion mode; matrix: nitrobenzyl alcohol) exhibited a molecular ion peak at m/z 710 [M+H–H₂O]⁺ and the characteristic ion peak at m/z 548 [M+H–H₂O–Glc]⁺ with the loss of a hexose residue from the quasi-molecular ion. The molecular formula of C₄₁H₇₇NO₉ for 1 was determined by the negative ion high-resolution LCMS-IT-TOF spectrum at m/z 726.5535 [M-H]⁻ (C₄₁H₇₆NO₉, calcd. 726.5520). In its ¹³C NMR spectrum, derived through DEPT, a d-glucopyranose moiety was indicated by the signals at δ 104.72 (C-1″, CH), 75.01 (C-2″, CH), 77.99 (C-3″, CH), 71.60 (C-4″, CH), 77.90 (C-5″, CH), and 62.73 (C-6″, CH₂). The sugar part of the molecular was indicated by the cross-peak between H-1″ (δ 4.17, 1H, d, J = 7.7 Hz) and C-1 (δ 69.73) exhibited in the HMBC spectrum. The ¹³C NMR spectrum displayed the signal of an anomeric carbon atom at δ 104.72 (C-1″) and anomeric proton at δ 4.17 (d, J = 7.7, 1H, H-1″), indicative of a β-configuration of C-1″. [15].

Its ¹H NMR spectrum presented an amide NH doublet at δ 7.25 (1H, d, J = 8.7 Hz). The ¹³C NMR and DEPT spectrum of compound 1 showed the signals at δ 177.18 (C-1′) of a carbonyl carbon and δ 54.67 (C-2) of a methine carbon; both carbons were connected to amide nitrogen. Correlation peaks between NH and H-2 (δ 3.63, m, 1H) in ¹H–¹H COSY, and crosspeaks between NH and C-1′ and H-2 and C-1′ in HMBC were observed. The existence of a methyl side chain and two long aliphatic chains in the molecule was shown by the presence of signals of three methyl groups [δ _C 14.41 (C-18), 14.41 (C-16′), 16.15 (CH₃-19); δ _H 0.80 (6H, t, CH₃-18, 16′), 1.50 (3H, s, CH₃-19)] and a complex region with overlapping signals characteristic of methylenes of the long alkyl chain in the NMR spectra that were essentially identical with those of ceramide, strongly suggesting the glycosphingolipid nature of this molecule [16, 17].

Detailed analysis of the ¹H–¹H COSY spectrum was conducted, and the correlation peaks between H-4 and H-3, H-4 and H-5, H-5 and H-6, H-6 and H-7, and H-7 and H-8 were observed. The above correlation analysis unambiguously assigned the positions of the two double bonds at C-4 and C-8, respectively. This was further supported by the HMBC spectrum of compound 1, which displayed the correlation between H-6 and C-4, H-3 and C-5, H-7 and C-9, and H-8 and C-10. On the other hand, the presence of a C-19 allylic methyl group (δ _C 16.15) in the branched long-chain base was also confirmed by the HMBC spectrum, in which the correlation between H-8 and C-19 was observed. The geometry of the C-4/C-5 alkene bond was determined to be E by the large vicinal coupling constant (J = 15.3 Hz) displayed between H-4 and H-5, as also evidenced by the ¹³C NMR chemical shift of the methylene carbon C-6 (δ _C 33.05) next to the olefinic carbon [18], and the signals of olefinic protons (H-4 and H-5) appeared in the range of δ _H 5.39–5.64 as a multiplet [19]. Furthermore, the ¹³C NMR chemical shift of the C-19 methyl group (δ _C 16.15) in turn supported the assignment of the Δ8-trans isomer, as demonstrated by comparison of the chemical shifts of the C-3 methyl groups in the E (δ _C 15.4) and Z (δ _C 22.7) isomers of 3-methyl-3-hexene [20]. It is thus clear that compound 1 possesses a branched sphingoid moiety with (4E,8E) geometry, 2-amino-1,3-dihydroxyl-9-methyl-4,8-octadecanediene. Moreover, the optical rotations of compound 1 ($[\alpha ]_{\text{D}}^{23}$ + 5.0°, c = 0.3, CH₃OH) is also in accordance with that of glucosyl-(2S,3R)-sphingadienine ($[\alpha ]_{\text{D}}^{23}$ + 5.4°) [21]. On the basis of the combined evidence presented above, the structure of compound 1 was established as 1-O-β-d-glucopyranosyl-(2S,3R,4E,8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol (Fig. 1) and was confirmed by the comparison with the published NMR data [13–16].

Fig. 1

Structure of compound 1

Our previous study has reported that some soluble fractions prepared from the methanol extract of P. citrinopileatus, for example, n-hexane-soluble (50 μg/ml), diethylether-soluble (75 μg/ml), and ethyl acetate-soluble (100 μg/ml) fractions inhibited the melanin production in B16 melanoma cells at 63.1, 64.0, and 58.8%, with cell viability at 61.3, 65.8, and 70.7%, respectively. The ORAC value of n-hexane-soluble and diethyl ether-soluble fractions were 0.25 and 0.2 mg TE/mg. On the other hand, the n-butanol-soluble, aqueous-soluble fractions, and methanol extract exhibited moderate antioxidant potential; their inhibitory concentration (IC₅₀) that caused 50% scavenging of the DPPH radical scavenging were 7.9, 5.2, and 5.4 mg/ml, respectively. The aqueous-soluble fraction and methanol extract exhibited 1.9 and 0.3 of SOD-like activity (U/mg), respectively. The n-hexane-soluble (0.5 mg/ml) and ethyl acetate-soluble (0.3 mg/ml) fractions showed high antibacterial activity with growth inhibition of 79.6 and 75.6% against S. aureus, and 87.0 and 60.0% against E. coli, respectively. The n-butanol-soluble (2.0 mg/ml), aqueous-soluble (1.1 mg/ml) fractions, and methanol extract (4.1 mg/ml) showed antihyaluronidase activity of 9.7, 10.8, and 25.4%, respectively [8].

Although the extract of P. citrinopileatus has the multifunctional biological activities mentioned above, we first focused on its antimicrobial activity. Antimicrobial-activity-guided fractionation led to isolation of glucosylceramide (1). This compound showed growth inhibitory activity against S. aureus and E. coli with IC₅₀ at 323.2 μM (235 μg/ml) and 275.1 μM (200 μg/ml) for 8 h, respectively (Table 1). It should be noted that the IC₅₀ of sorbic acid known as an antimicrobial compound against S. aureus and E. coil was 1560.0 μM (175 μg/ml) and 1516.1 μM (170 μg/ml), respectively.

Table 1 Biological activity of compound 1 from methanol extract, the fruiting bodies of P. citrinopileatus

Also, we evaluated the effect of compound 1 in several bioassays. The DPPH radical scavenging activity was 0.7% at concentrations of 724.8 μM, 9.4 U/mg of SOD-like activity (852.8 μM) and antihyaluronidase activity of 4.7% (433.3 μM), respectively. Our results suggested that compound 1 has no distinguishing activities other than antimicrobial activity (Table 1). It is not known what other active ingredients besides those responsible for antimicrobial activity are present in P. citrinopileatus, and that this needs to be investigated in the future.

Glucosylceramide is a sphingolipid. Sphingolipids are members of membrane lipids and are widely distributed in the animal, plant, and fungi kingdoms [22, 23]. Sphingolipids, e.g., ceramides, cerebrosides, sphingomyelin, and gangliosides, are important building blocks of the plasma membrane of eukaryotic cells. Their function is to anchor lipid-bound carbohydrates to cell surfaces and to create an epidermal water permeability barrier, as well as to participate in antigen–antibody reactions and transmission of biological information. [24] Some have shown antiulcerogenic, antihepatotoxic, antitumor, and immunostimulatory activities [25–27].

A large number of studies have revealed that sphingolipids exert a significant effect on the structure of biological membranes and lipoproteins [28]. The biological activities of ceramide vary with the chemical structure: the carbon-chain structure of the sphingoid base and/or composition of amide-linked fatty acids. It has been reported that glycosphingolipids exert their biological activities by interacting with the receptor proteins on the cell membrane, with consequences to the modulation of the signal transduction [29].

In conclusion, glucosylceramide (1) isolated from P. citrinopileatus showed antimicrobial activity against E. coli and S. aureus. This is the first report of the isolation of glucosylceramide from P. citrinopileatus, and the first to show its antimicrobial activity. An efficacious dose of this compound has potential health benefits, including controlling infection, and the compound can also be used as a food additive to prevent decay.