Anti-inflammatory Cerebrosides from Cultivated Cordyceps militaris

Feb 6, 2016 - The fungus Cordyceps belongs to the Ascomycota phylum, Sordariomycetes class, Hypocreales order, and Clavicipitaceae family.(1) More ...
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Anti-inflammatory Cerebrosides from Cultivated Cordyceps militaris Ching-Peng Chiu,†,∥ Shan-Chi Liu,‡,∥ Chih-Hsin Tang,‡ You Chan,§ Mohamed El-Shazly,# Chia-Lin Lee,⊗ Ying-Chi Du,† Tung-Ying Wu,⊥ Fang-Rong Chang,*,†,Δ,Π,Θ and Yang-Chang Wu*,Σ,◊,⊡,⬡ †

Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan Graduate Institute of Basic Medical Science, China Medical University, Taichung 404, Taiwan § Institute of Microbiology, Chung Shan Medical University, Taichung 402, Taiwan # Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street, Abassia, Cairo 11566, Egypt ⊗ Department of Cosmeceutics, China Medical University, Taichung 404, Taiwan ⊥ Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan Δ Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan Π Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Θ Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan Σ School of Pharmacy, College of Pharmacy, China Medical University, Taichung 40402, Taiwan ◊ Research Center for Chinese Herbal Medicine, China Medical University, Taichung 40402, Taiwan ⊡ Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 40402, Taiwan ⬡ Center of Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan ‡

S Supporting Information *

ABSTRACT: Cordyceps militaris (bei-chong-chaw, northern worm grass) is a precious and edible entomopathogenic fungus, which is widely used in traditional Chinese medicine (TCM) as a general booster for the nervous system, metabolism, and immunity. Saccharides, nucleosides, mannitol, and sterols were isolated from this fungus. The biological activity of C. militaris was attributed to the saccharide and nucleoside contents. In this study, the aqueous methanolic fraction of C. militaris fruiting bodies exhibited a significant anti-inflammatory activity. Bioactivity-guided fractionation of the active fraction led to the isolation of eight compounds, including one new and two known cerebrosides (ceramide derivatives), two nucleosides, and three sterols. Cordycerebroside A (1), the new cerebroside, along with soyacerebroside I (2) and glucocerebroside (3) inhibited the accumulation of pro-inflammatory iNOS protein and reduced the expression of COX-2 protein in LPS-stimulated RAW264.7 macrophages. This is the first study on the isolation of cerebrosides with anti-inflammatory activity from this TCM. KEYWORDS: Cordyceps militaris, cerebrosides, fungal fruiting bodies, LPS-stimulated RAW264.7 macrophages, anti-inflammatory

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INTRODUCTION The fungus Cordyceps belongs to the Ascomycota phylum, Sordariomycetes class, Hypocreales order, and Clavicipitaceae family.1 More than 400 species of Cordyceps have been identified.2 Cordyceps sp. are entomogenous Ascomycetes, which parasitize the larvae of Lepidopteran pupae. Some species of this fungus such as Cordyceps militaris are valued as traditional Chinese medicines or as biocontrol agents against pests.3 The wild fungi, with their plant-like fruiting bodies that originate from dead caterpillars after being filled with mycelia, have generally been called wormgrass due to their insect-shape appearance.4 C. militaris or the Himalayan Viagra has been used for centuries by the inhabitants of the Tibetan Plateau and central Asia to treat hyposexuality, hyperglycemia, hyperlipidemia, arrhythmias, and respiratory, renal, and liver diseases.4 In 1993, it received attention in the West after Chinese female athletes broke world records in long-distance races.5 Such achievement was attributed in part to a special © 2016 American Chemical Society

Cordyceps-containing diet that enhanced the runners’ endurance and physical performance. In 2006, the retail price for highquality C. sinensis reached over U.S. $30,000 per kilogram in the coastal cities of mainland China, as well as in the United States.6 Due to the high price and unreliability in supply, the usage of natural Cordyceps sp. has been limited to rich citizens in Asia and the West. Therefore, several research groups started investigating the possibility of Cordyceps sinensis cultivation to achieve large-scale production.7 Currently, health-care products derived from C. militaris are extremely popular in many parts of the world in various forms such as capsules, dietary supplements, and beverages.8 Most of these products are derived from submerged mycelia C. militaris cultures. Received: Revised: Accepted: Published: 1540

December 15, 2015 February 1, 2016 February 6, 2016 February 6, 2016 DOI: 10.1021/acs.jafc.5b05931 J. Agric. Food Chem. 2016, 64, 1540−1548

Article

Journal of Agricultural and Food Chemistry

a potato dextrose agar (PDA) plate at 18 °C for 7 days. After the plate was full of mycelia, one piece of mycelia was cut and placed into the growth medium. The mycelia were fermented in 1000 mL of growth medium containing potato juice (200 g), glucose (10 g), peptone (10 g), KH2PO4 (3 g), MgSO4 (1.5 g), vitamin B1 (100 mg), yeast extract (5 g), and water (1000 mL) at 18 °C for 14 days (150 rpm). The inoculum preparation of C. militaris to solid-state fermentation was carried out in the bottle by inoculating the fungus in 15 mL of raw rice with 10 mL of the medium containing glucose (10 g), peptone (10 g), vitamin B1 (100 mg), yeast extract (5 g), water (1000 mL), and agar (15−20 g) and incubated in the dark at 18 °C for 7 days after inoculation. Then, the bottle was moved under continuous fluorescent lamp illumination. The C. militaris fruiting bodies were dried in an oven at 40 °C for 24 h and stored at 4 °C for further use.15 Isolation and Purification of C. militaris Secondary Metabolites. The fresh fungal material was lyophilized for 72 h, and C. militaris powder was accurately weighed (4 kg). The powder was sonicated with 20 L of EtOH (Branson 5510, Branson Ultrasonic, Ontario, Canada) for 120 min. The ethanolic extract was then decanted, filtered under vacuum, and concentrated using a rotary evaporator to yield the crude extract (663.9 g). This crude extract was partitioned with H2O/EtOAc (1:1, v/v) to afford the EtOAc fraction (109.6 g). The EtOAc fraction was partitioned with n-hexane/90% methanol(aq) (1:1, v/v) to afford the 90% methanol(aq) fraction (31.0 g). The 90% methanol(aq) fraction was purified on silica gel column chromatography eluted by a mixture of dichloromethane (DCM) and MeOH in increasing polarity (pure dichloromethane, 40:1, 30:1, 20:1, 15:1, 10:1, 5:1) to yield 13 fractions. Fraction 10 (280.8 mg) was chromatographed with Sephadex LH20 (2.5 × 55 cm) using acetone/MeOH (1:1) as the eluent, yielding three subfractions (10-1−10-3). Subfraction 10-2 was purified on reversed-phase HPLC (RI detector, Supelco C-18, 250 mm × 10 mm i.d., 5 μm) eluted with isocratic mobile phase consisting of MeOH/ H2O 80:20, at a flow rate of 2 mL/min, to afford 3β-O-5,8epidioxyeroxiergosta-6,22-diene (6) (13.9 mg, tR 13.4 min), cerevisterol (7) (18.9 mg, tR 15.4 min), and ergosterol (8) (8.9 mg, tR 18.4 min). Fraction 12 (890.32 mg) was the cerebroside-rich fraction. On TLC developed by the solvent system DCM/MeOH 5:1, it showed a clear purple spot after spraying 10% H2SO4 aqeuoue and heating on the hot plate. However, it showed complicated signals on the 1H NMR spectrum. It was chromatographed on Sephadex LH-20 (2.5 × 55 cm) using acetone/MeOH (1:1) as the eluent, yielding six subfractions (12-1−12-6). Subfraction 12-2 (393.2 mg) was chromatographed on reversed-phase CC using 95% MeOH as the eluent to afford a major fraction. The major fraction (173.4 mg) was purified with a reversedphase HPLC (RI detector; Supelco C-18, 250 mm × 10 mm i.d., 5 μm; MeOH/H2O 9:1; flow rate = 2 mL/min) to afford one new cerebroside compound, cordycerebroside A (1) (10.8 mg; tR 21.2 min), soyacerebroside (2) (54.8 mg; tR 22.7 min), and glucocerebroside (3) (201.8 mg; tR 24.1 min). The cordycerebroside A (1) is a new cerebroside compared to other isolated cerebrosides of C. militaris. Fraction 13 (2.5 g) was separated on Sephadex LH-20 (2.5 × 55 cm) using acetone/MeOH (1:1) as the eluent to provide adenosine (4) (340.4 mg) and cordycepin (5) (895.4 mg). Anti-inflammatory Material. Mouse monoclonal antibody specific to β-actin was purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit polyclonal antibodies specific to COX-2 and iNOS from Santa Cruz Biotechnology (Santa Cruz, CA, USA); DMEM, fetal bovine serum (FBS), and all other cell culture reagents from Gibco-BRL Life Technologies (Grand Island, NY, USA); LPS and all other chemicals from Sigma-Aldrich (St. Louis, MO, USA). Cell Culture. Murine RAW264.7 cells (a mouse macrophage cell line obtained from American Type Culture Collection) were grown in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 units/mL) at 37 °C under a humidified atmosphere of 5% CO2. Western Blot Analysis. Lysates were prepared as described previously,16 and proteins were resolved by sodium dodecyl sulfate− polyacrylamide gel electrophoresis and transferred to Immobilon

The reported chemical constituents of C. sinensis are saccharides, polysaccharides, nucleotides, steroids, heterocyclic compounds, fatty acids, cyclopeptides, and amino acids.9 Studies showed that the fruiting bodies of C. militaris have many active principles, which possess a wide range of pharmacological actions such as aphrodisiac, antioxidant, antitumor, antimicrobial, anti-inflammatory, immunomodulatory, hepatoprotective, and nephroprotective activities.10−12 Although many activities had been reported for this fungus, the research focused only on certain groups of secondary metabolites including adenosine, cordycepin, and steroids. The optimum utilization of C. sinensis can be achieved only with the full investigation of its secondary metabolite content along with the determination of their biological activity. In this study, the cultivated C. militaris fruiting bodies ethanolic crude extract was partitioned with H2O/EtOAc to afford EtOAc fraction. The EtOAc fraction was partitioned with n-hexane/90% methanol(aq) to afford the 90% methanol(aq) fraction. The 90% methanol(aq) exhibited significant antiinflammatory activity. Bioactivity-guided fractionation of the 90% methanol(aq) fraction led to the isolation of eight secondary metabolites, including one new and two known cerebrosides (ceramide derivatives), two nucleosides, and three sterols from C. militaris. The anti-inflammatory activity of the three cerebrosides was evaluated through studying the inhibition of pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins by lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Ceramide derivatives have never been isolated from Cordyceps sp. The isolation of ceramides with anti-inflammatory activity lends further evidence to the ethnopharmacological use of this treasured fungus in treating tissue injuries, bronchitis, asthma, and emphysema.13,14

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were taken on a JASCO-P-1020 polarimeter (JASCO Inc., Tokyo, Japan) (cell length = 10 mm). IR spectra were recorded on a Shimadzu model IR Prestige-21 Fourier transform infrared spectrophotometer (Shimadzu Inc., Tokyo, Japan). 1D (1H, 13C, DEPT) and 2D (COSY, HSQC, HMBC, NOESY) NMR spectra were measured on a Bruker ULTRASHIELD 500 PLUS instrument (Bruker, Rheinstetten, Germany). Chemical shift (δ) values are presented in parts per million (ppm) with pyridine-d5 as the internal standard, and coupling constants (J) are reported in hertz. Low-resolution ESIMS was measured on a Bruker Daltonics Esquire HCT ultrahigh-capacity trap mass spectrometer (Bruker). HRESIMS was obtained on an Orbitrap mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific, Waltham, MA, USA). The GC-MS analyses were performed with a DSQ II GC-MS (Thermo Fisher Scientific, Waltham, MA, USA), and a DB-5MS (30 m, 0.25 mm i.d., 0.25 μm film thickness) column was used for GC-MS. TLC was performed on Kieselgel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) or RP-18 F254S (0.25 mm, Merck), and spots were viewed under UV light at 254 and 356 nm and then stained by spraying with 10% H2SO4 and heated on a hot plate. For column chromatography (CC) silica gel (SILICYCLE 70−230 and 230−400 mesh), RP-18 (LiChroprep 40−63 μm, Merck), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used. A Shimadzu LC-20AT pump, a Shimadzu RID-10A refractive index detector, and a Supelco Ascentis 5 μm (250 × 10 mm i.d.) column were used for HPLC. C. militaris Fruiting Body Culture Condition. C. militaris fruiting bodies were cultured in the Laboratory of Microbiology and Immunology, Chung-Shan Medical University, between June 2009 and June 2010. This strain of C. militaris was provided by Prof. You Chan (Chung-Shan Medical University, Taichung, Taiwan). One piece of C. militaris fruiting bodies was maintained and inoculated at the center of 1541

DOI: 10.1021/acs.jafc.5b05931 J. Agric. Food Chem. 2016, 64, 1540−1548

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of the compounds isolated from C. militaris. polyvinyl difluoride membranes (Immobilon P, Millipore). Blots blocked with 4% BSA for 1 h at room temperature were probed with rabbit anti-human antibodies against COX-2 and iNOS (1:1000) for 1 h at room temperature. After a third wash, blots were incubated for 1 h at room temperature with donkey anti-rabbit peroxidase-conjugated secondary antibody (1:3000), then visualized by enhanced chemiluminescence with Kodak X-OMATLS film (Eastman Kodak, Rochester, NY, USA). Measurement of Nitric Oxide(NO)/Nitrite. NO production was indirectly assessed by measuring the nitrite levels in the culture medium and serum as determined according to a previous study.17 The cells were pre-incubated with all pure compounds (1−8) (0, 1, 5, and 10 μM) for 1 h and then cotreated with 100 ng/mL LPS at 37 °C for 24 h. Subsequently, 100 μL of each collected culture medium was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance of the mixture was measured at 540 nm with a Micro-Reader (Molecular Devices, Sunnyvale, CA, USA). Homogenized tissue samples were diluted four times with distilled water and deproteinized by adding 1/20 volume of zinc sulfate (300 mg/mL) to a final concentration of 15 mg/mL. After centrifugation at 10000g for 5 min at room temperature, 100 μL of the supernatant was added into a microtiter plate, followed by 100 μL of Griess reagent. After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader. Using sodium nitrite to generate a standard curve, the concentration of nitrite was measured by absorbance at 540 nm. Transfection and Reporter Gene Assay. RAW264.7 cells were cotransfected with 0.8 mg of luciferase plasmid and 0.4 mg of βgalactosidase expression vector.16 RAW264.7 cells were grown to 80% confluence in 12-well plates and were transfected on the following day by Lipofectamine 2000 (LF2000; Invitrogen). DNA and LF2000 were premixed for 20 min and then applied to the cells. After 24 h of transfection, the cells were incubated with the indicated agents. After a further 24 h of incubation, the media were removed, and cells were washed once with cold PBS. To prepare lysates, 100 mL of reporter lysis buffer (Promega, Madison, WI, USA) was added to each well, and the cells were scraped from dishes. The supernatant was collected after centrifugation at 13000 rpm for 2 min. Aliquots of cell lysates (20 mL) containing equal amounts of protein (20−30 mg) were placed into wells of an opaque black 96-well microplate. An equal volume of luciferase substrate was added to all samples, and luminescence was measured in a microplate luminometer. The value of luciferase activity was normalized to transfection efficiency monitored by the cotransfected β-galactosidase expression vector. Chromatin Immunoprecipitation Assay. Chromatin immunoprecipitation analysis was performed as described previously.18 DNA

immunoprecipitated with an anti-p65 was purified and extracted with phenol−chloroform. The purified DNA pellet was subjected to PCR. PCR products were then resolved by 1.5% agarose gel electrophoresis and visualized with UV light. The primers 59-CAAGACATGCCAAAGTGCTG-39 and 59-TTGAGACTCATGGGAAAATCC-39 were utilized to amplify across the human COX-2 promoter region containing NF-κB binding sit. The primers 59-GAACTGACCTGACTTACATA-39 and 59-TTGAGACTCATGGGAAAATCC-39 were utilized to amplify across the human iNOS promoter region containing NF-κB binding site. Statistics. The results were expressed as mean ± standard deviation (SD). Comparison in each experiment was performed using an unpaired Student’s t test, and a p value of 10 μg/mL after calculation. Furthermore, soyacerebroside I (2) showed significant inhibitory effect on NO production from LPSstimulated cells (Figure 4). On the basis of the assay results, cerebrosides (1−3), especially soyacerebroside I (2), the most potent derivative, were selected for further analyses aiming to understand their anti-inflammatory mechanism of action. Effect of Cerebrosides on iNOS and COX-2 Protein Expression. Next, we investigated the effect of the isolated cerebrosides on iNOS and COX-2 protein expression.20 LPS stimulation of RAW264.7 macrophages induces the accumulation of pro-inflammatory iNOS and COX-2 proteins resulting in increased NO production.35 The results showed that soyacerebroside I (2) exhibited the most potent inhibitory effect on iNOS and COX-2 protein expression (Figure 5). Compound 2 Inhibited NF-κB Activation Phosphorylation in Macrophages. The regulation of the inflammatory cytokines and inflammatory responses are transcriptionally governed by NF-κB transcription factors. The transcription of p65, a subunit of NF-κB, can adjust the activation of NF-κB.36 This transcription factor participates in the regulation of inflammatory-related genes through binding to DNA to induce the transcription of the inflammatory mediators.37 We found that LPS stimulation increased p65 phosphorylation, which was inhibited by soyacerebroside I (2) in a dose-dependent manner (Figure 6). Taken together, these findings demonstrated that soyacerebroside I (2) suppressed the expression of iNOS and COX-2 in part via an NF-κB-dependent mechanism. NF-κB plays a pivotal role in the regulation of iNOS expression and COX-2.37 NF-κB activation was further evaluated by analyzing the NF-κB luciferase activity. Soyacerebroside I (2) inhibited NF-κB activation, which was stimulated by LPS, in a dose-dependent manner (Figure 7A). We next investigated whether p65 bindd to the NF-κB element on the iNOS and COX-2 promoter after LPS stimulation. The in vitro recruitment of p65 to the iNOS and COX-2 promoter was assessed by the chromatin immunoprecipitation assay. The in vitro binding of p65 to the NF-κB element of the iNOS and COX-2 promoter occurred after LPS stimulation. The binding of p65 to the NF-κB element by LPS was attenuated by soyacerebroside I (2) in a dose-dependent manner (Figure 7B). Previous studies on the anti-inflammatory effect of C. militaris focused on components other than cerebrosides. It was found that ergosterol inhibited NO production in LPS-

Figure 5. Effect of 1−3 on the LPS-induced pro-inflammatory iNOS and on COX-2 protein expression of RAW264.7 macrophages as demonstrated by immunoblot analysis. The relative intensity of the control alone stimulated group was taken as 100%. Significantly different from LPS-stimulated (control) group (∗) p < 0.01 compared to LPS.

Ceramides, containing a double-chain lipid with LCB and LCA, are highly hydrophobic structures.29 The ceramide family is a very heterogeneous group with different LCB and LCA structures.30 Ceramides are the lipid moieties of sphingolipids, such as sphingomyelin and glycolipids, which are the amphiphilic components of cell membranes. Ceramides are minor components of all cell membranes, but they are abundant in skin tissues.31 In mammalian cells, ceramides are produced from sphingomyelin and glycosphingolipids by plasma membrane sphingomyelinase and glycosphingolipid hydrolases, respectively, and may act as apoptotic compounds under certain conditions.32 Recent studies reported ceramides’ molecular mechanism of action in regulating chronic 1545

DOI: 10.1021/acs.jafc.5b05931 J. Agric. Food Chem. 2016, 64, 1540−1548

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Journal of Agricultural and Food Chemistry

Figure 6. Effect of soyacerebroside I (2) on LPS-induced NF-κB transcriptional activity via the suppression of nuclear translocation of the p65 subunit in RAW264.7 cells.

and 1.68 ± 0.32 μM, respectively.39 A recent paper attributed the anti-inflammatory activity of the fungal aqueous extract to the presence of β-(1→3)- D -glucan. It suppressed the inflammatory phase of formalin-induced nociceptive response as well as the migration of total leukocytes but not the neutrophils stimulated by LPS.40 Most of the literature related to C. militaris focused on the anti-inflammatory property of cordycepin and its active mechanism. Cordycepin was found to inhibit the phosphorylation of Akt, p38, TNF-α, and IκBα in LPS-activated macrophages.41 Moreover, the expressions of iNOS and COX-2 in RAW264.7 cells were suppressed by cordycepin (5) through inhibiting the production of NO and pro-inflammatory cytokines such as IL1β and IL6.42 In the current study, one new and two known cerebrosides, two nucleosides, and three sterols were obtained from the selected 90% methanol(aq) fraction of C. militaris fruiting bodies by a bioactivity-guided fractionation method. Notably, it is the first time that the cerebrosides were found in C. militaris. Among the cerebrosides, the new isolate, cordycerebroside A (1), and glucocerebroside (3) showed moderate inhibitory ability, whereas soyacerebroside I (2) exhibited the most potent anti-inflammatory activity. According to the COX-2 and iNOS results of 1−3, the increase of unsaturated degree at the LCA and the substituent group at the LCB led to the level reduction of protein expression. Furthermore, compound 2 ameliorated iNOS and COX-2 expression by blocking NO production and the NF-κB (p65) signaling pathway. Except for adenosine, cordycepin and sterols demonstrated weak inhibitory activity on NO production. Cordycepin (5) was also reported to suppress NF-κB through Akt and p38 inhibition in RAW264.7 macrophage cells.43 A similar cordycepin derivative, N6-(2hydroxyethyl)adenosine, was reported for its anti-inflammatory activity by suppressing TLR4-mediated NF-κB signaling pathways.44 On the other hand, ergosterol peroxide (6) was found to inhibit the production of MyD88 and VCAM-1 expression and pro-inflammatory mediators, IL-1β, IL-6, and TNF-α cytokines in LPS-stimulated THP-1 cells.45 All of these compounds contributed to the anti-inflammatory activity of the 90% methanol(aq) fraction. Our results revealed the key role of cerebrosides in the anti-inflammatory activity of C. militaris and open a new avenue to understand the full therapeutic potential of this fungus.

Figure 7. COX-2 and iNOS pathways are involved in LPS-induced NF-κB activation. (A) The inflammatory promotors were transfected with NF-κB-luciferase expression vector and then pretreated with LPS for 24 h, and luciferase activity was then assayed. (B) The inflammatory promoters were pretreated with LPS for 120 min, and the chromatin immunoprecipitation assay was then performed. Chromatin was immunoprecipitated with p65. One percent of the precipitated chromatin was assayed to verify equal loading (input).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05931. Experimental procedures and compound characterization data (PDF)

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triggered BV2 microglia cells. In another study, 1-(5hydroxymethyl-2-furyl)-β-carboline inhibited superoxide anion generation and elastase release with IC50 values of 0.45 ± 0.15 1546

DOI: 10.1021/acs.jafc.5b05931 J. Agric. Food Chem. 2016, 64, 1540−1548

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Journal of Agricultural and Food Chemistry

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(8) Zhou, X.; Gong, Z.; Su, Y.; Lin, J.; Tang, K. Cordyceps fungi: natural products, pharmacological functions and developmental products. J. Pharm. Pharmacol. 2009, 61, 279−291. (9) Zhao, J.; Xie, J.; Wang, L. Y.; Li, S. P. Advanced development in chemical analysis of Cordyceps. J. Pharm. Biomed. Anal. 2014, 87, 271− 289. (10) Patel, S.; Goyal, A. Recent developments in mushrooms as anticancer therapeutics: a review. 3 Biotech 2012, 2, 1−15. (11) Das, S. K.; Masuda, M.; Sakurai, A.; Sakakibara, M. Medicinal uses of the mushroom Cordyceps militaris: current state and prospects. Fitoterapia 2010, 81, 961−968. (12) Drewes, S. E.; George, J.; Khan, F. Recent findings on natural products with erectile-dysfunction activity. Phytochemistry 2003, 62, 1019−1025. (13) Jo, W. S.; Choi, Y. J.; Kim, H. J.; Lee, J. Y.; Nam, B. H.; Lee, J. D.; Lee, S. W.; Seo, S. Y.; Jeong, M. H. The anti-inflammatory effects of water extract from Cordyceps militaris in murine macrophage. Mycobiology 2010, 38, 46−51. (14) Holliday, J. C.; Cleaver, M. P. Medicinal value of the caterpillar fungi species of the genus Cordyceps (Fr.) Link (Ascomycetes). A review. Int. J. Med. Mushrooms 2008, 10, 219−234. (15) Hsu, C. H.; Sun, H. L.; Sheu, J. N.; Ku, M. S.; Hu, C. M.; Chan, Y.; Lue, K. H. Effects of the immunomodulatory agent Cordyceps militaris on airway inflammation in a mouse asthma model. Pediatr. Neonatol. 2008, 49, 171−178. (16) Liu, S. C.; Hsu, C. J.; Chen, H. T.; Tsou, H. K.; Chuang, S. M.; Tang, C. H. CTGF increases IL-6 expression in human synovial fibroblasts through integrin-dependent signaling pathway. PLoS One 2012, 7, e51097. (17) Schmid, D.; Gruber, M.; Piskaty, C.; Woehs, F.; Renner, A.; Nagy, Z.; Kaltenboeck, A.; Wasserscheid, T.; Bazylko, A.; Kiss, A. K.; Moeslinger, T. Inhibition of NF-kappaB-dependent cytokine and inducible nitric oxide synthesis by the macrocyclic ellagitannin oenothein B in TLR-stimulated RAW 264.7 macrophages. J. Nat. Prod. 2012, 75, 870−875. (18) Chiu, Y. C.; Lin, C. Y.; Chen, C. P.; Huang, K. C.; Tong, K. M.; Tzeng, C. Y.; Lee, T. S.; Hsu, H. C.; Tang, C. H. Peptidoglycan enhances IL-6 production in human synovial fibroblasts via TLR2 receptor, focal adhesion kinase, Akt, and AP-1- dependent pathway. J. Immunol. 2009, 183, 2785−2792. (19) Tang, J.; Meng, X.; Liu, H.; Zhao, J.; Zhou, L.; Qiu, M.; Zhang, X.; Yu, Z.; Yang, F. Antimicrobial activity of sphingolipids isolated from the stems of cucumber (Cucumis sativus L.). Molecules 2010, 15, 9288−9297. (20) Cheng, S. Y.; Wen, Z. H.; Chiou, S. F.; Tsai, C. W.; Wang, S. K.; Hsu, C. H.; Dai, C. F.; Chiang, M. Y.; Wang, W. H.; Duh, C. Y. Ceramide and cerebrosides from the octocoral Sarcophyton ehrenbergi. J. Nat. Prod. 2009, 72, 465−468. (21) Furuya, T.; Hirotani, M.; Matsuzawa, M. N6-(2-hydroxyethyl)adenosine, a biologically active compound from cultured mycelia of Cordyceps and Isaria species. Phytochemistry 1983, 22, 2509−2512. (22) Kawahara, N.; Sekita, S.; Satake, M.; Udagawa, S.-I. A nucleoside derivative from Emericella nidulans. Phytochemistry 1992, 31, 1409− 1410. (23) Takaishi, Y.; Uda, M.; Ohashi, T.; Nakano, K.; Murakami, K.; Tomimatsu, T. Glycosides of ergosterol derivatives from Hericum erinacens. Phytochemistry 1991, 30, 4117−4120. (24) Matsuda, H.; Akaki, J.; Nakamura, S.; Okazaki, Y.; Kojima, H.; Tamesada, M.; Yoshikawa, M. Apoptosis-inducing effects of sterols from the dried powder of cultured mycelium of Cordyceps sinensis. Chem. Pharm. Bull. 2009, 57, 411−414. (25) Mansoor, T. A.; Shinde, P. B.; Luo, X.; Hong, J.; Lee, C. O.; Sim, C. J.; Son, B. W.; Jung, J. H. Renierosides, cerebrosides from a marine sponge Haliclona (Reniera) sp. J. Nat. Prod. 2007, 70, 1481− 1486. (26) Costantino, V.; Fattorusso, E.; Mangoni, A. Glycolipids from sponges, III. Glycosyl ceramides from the marine sponge Agelas conifera. Liebigs Ann. 1995, 1995, 2133−2136.

AUTHOR INFORMATION

Corresponding Authors

*(Y.-W.C.) Phone: +886-4-22057153. Fax: +886-4-22060248. E-mail: [email protected]. *(F.-R.C.) Phone: +886-7-3121101, ext. 2162. Fax: +886-73114773. E-mail: [email protected]. Author Contributions ∥

C.-P.C. and S.-C.L. contributed equally to this work.

Author Contributions

C.-P.C. and S.-C.L. performed most of the experimental work. C.-H.T., Y.-C.W, M.E.-S. and F.-R.C. conceived the project and designed the experiments. Y.C. introduced the technology of fungus culture to our group and provided the needed materials. C.-L.L., Y.-C.D., and T.-Y.W. designed and implemented the separation and purification protocols. M.E.-S., Y.-C.W., and F.R.C. drafted and revised the manuscript. All authors read and approved the final manuscript. Funding

This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 102-2628-B-037003-MY3 and 103-2320-B-037-005-MY2 awarded to F.-R.C.; MOST 103-2911-I-002-303, 104-2911-I-002-302, 103-2325-B039-008, 103-2325-B-039-007-CC1, and 102-2320-B-037-012MY2, awarded to Y.-C.W.), the National Health Research Institutes (NHRI-EX103-10241BI), and in part by a grant from the Chinese Medicine Research Center, China Medical University (Ministry of Education, Aim for the Top University Plan). This study is also supported partially by Kaohsiung Medical University (Aim for the Top Universities Grant KMUTP104E39), the Ministry of Health and Welfare of Taiwan (MOHW105-TDU-B-212-134-005), and the Health and Eelfare surcharge on tobacco products. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine receptor 5

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REFERENCES

(1) Yue, K.; Ye, M.; Zhou, Z.; Sun, W.; Lin, X. The genus Cordyceps: a chemical and pharmacological review. J. Pharm. Pharmacol. 2013, 65, 474−493. (2) Pruett, S. T.; Bushnev, A.; Hagedorn, K.; Adiga, M.; Haynes, C. A.; Sullards, M. C.; Liotta, D. C.; Merrill, A. H., Jr. Biodiversity of sphingoid bases (“sphingosines”) and related amino alcohols. J. Lipid Res. 2008, 49, 1621−1639. (3) Zheng, P.; Xia, Y.; Zhang, S.; Wang, C. Genetics of Cordyceps and related fungi. Appl. Microbiol. Biotechnol. 2013, 97, 2797−2804. (4) Lo, H.-C.; Hsieh, C.; Lin, F.-Y.; Hsu, T.-H. A systematic review of the mysterious caterpillar fungus Ophiocordyceps sinensis in DongChongXiaCao (Do̅ ng Chóng Xià Cǎo) and related bioactive ingredients. J. Tradit. Complement. Med. 2013, 3, 16−32. (5) Zhu, J. S.; Halpern, G. M.; Jones, K. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis: part I. J. Altern. Complement. Med. (New York, N.Y.) 1998, 4, 289−303. (6) Winkler, D. Yartsa Gunbu (Cordyceps sinensis) and the fungal commodification of Tibet’s rural economy. Econ. Bot. 2008, 62, 291− 305. (7) Li, S. P.; Yang, F. Q.; Tsim, K. W. K. Quality control of Cordyceps sinensis, a valued traditional Chinese medicine. J. Pharm. Biomed. Anal. 2006, 41, 1571−1584. 1547

DOI: 10.1021/acs.jafc.5b05931 J. Agric. Food Chem. 2016, 64, 1540−1548

Article

Journal of Agricultural and Food Chemistry (27) Chen, J. H.; Cui, G. Y.; Liu, J. Y.; Tan, R. X. Pinelloside, an antimicrobial cerebroside from Pinellia ternata. Phytochemistry 2003, 64, 903−906. (28) Jin, W.; Rinehart, K. L.; Jares-Erijman, E. A. Ophidiacerebrosides: cytotoxic glycosphingolipids containing a novel sphingosine from a sea star. J. Org. Chem. 1994, 59, 144−147. (29) Karlsson, K. A. On the chemistry and occurrence of sphingolipid long-chain bases. Chem. Phys. Lipids 1970, 5, 6−43. (30) Lynch, D. V.; Dunn, T. M. An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function. New Phytol. 2004, 161, 677−702. (31) Aureli, M.; Loberto, N.; Chigorno, V.; Prinetti, A.; Sonnino, S. Remodeling of sphingolipids by plasma membrane associated enzymes. Neurochem. Res. 2011, 36, 1636−1644. (32) Alessenko, A. V. The role of sphingomyelin cycle metabolites in transduction of signals of cell proliferation, differentiation and death. Membr. Cell Biol. 2000, 13, 303−320. (33) Maceyka, M.; Spiegel, S. Sphingolipid metabolites in inflammatory disease. Nature 2014, 510, 58−67. (34) Choi, W.-S.; Shin, P.-G.; Lee, J.-H.; Kim, G.-D. The regulatory effect of veratric acid on NO production in LPS-stimulated RAW264.7 macrophage cells. Cell. Immunol. 2012, 280, 164−170. (35) Liu, Z.; Fan, Y.; Wang, Y.; Han, C.; Pan, Y.; Huang, H.; Ye, Y.; Luo, L.; Yin, Z. Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice. FEBS Lett. 2008, 582, 1643−1650. (36) Yeom, M.; Kim, J. H.; Min, J. H.; Hwang, M. K.; Jung, H. S.; Sohn, Y. Xanthii Fructus inhibits inflammatory responses in LPSstimulated RAW 264.7 macrophages through suppressing NF-kappaB and JNK/p38 MAPK. J. Ethnopharmacol. 2015, 176, 394−401. (37) Chu, P. Y.; Chien, S. P.; Hsu, D. Z.; Liu, M. Y. Protective effect of sesamol on the pulmonary inflammatory response and lung injury in endotoxemic rats. Food Chem. Toxicol. 2010, 48, 1821−1826. (38) Nallathamby, N.; Guan-Serm, L.; Vidyadaran, S.; Abd Malek, S. N.; Raman, J.; Sabaratnam, V. Ergosterol of Cordyceps militaris attenuates LPS induced inflammation in BV2 microglia cells. Nat. Prod. Commun. 2015, 10, 885−886. (39) Yang, M. L.; Kuo, P. C.; Hwang, T. L.; Wu, T. S. Antiinflammatory principles from Cordyceps sinensis. J. Nat. Prod. 2011, 74, 1996−2000. (40) Smiderle, F. R.; Baggio, C. H.; Borato, D. G.; Santana-Filho, A. P.; Sassaki, G. L.; Iacomini, M.; Van Griensven, L. J. Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1→3)-β-D-glucan. PLoS One 2014, 9, e110266. (41) Kim, H. G.; Shrestha, B.; Lim, S. Y.; Yoon, D. H.; Chang, W. C.; Shin, D. J.; Han, S. K.; Park, S. M.; Park, J. H.; Park, H. I.; Sung, J. M.; Jang, Y.; Chung, N.; Hwang, K. C.; Kim, T. W. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NFkappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur. J. Pharmacol. 2006, 545, 192−199. (42) Yang, X.; Li, Y.; He, Y.; Li, T.; Wang, W.; Zhang, J.; Wei, J.; Deng, Y.; Lin, R. Cordycepin alleviates airway hyperreactivity in a murine model of asthma by attenuating the inflammatory process. Int. Immunopharmacol. 2015, 26, 401−408. (43) Kim, H. G.; Shrestha, B.; Lim, S. Y.; Yoon, D. H.; Chang, W. C.; Shin, D.-J.; Han, S. K.; Park, S. M.; Park, J. H.; Park, H. I.; Sung, J.-M.; Jang, Y.; Chung, N.; Hwang, K.-C.; Kim, T. W. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-κB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur. J. Pharmacol. 2006, 545, 192−199. (44) Lu, M. Y.; Chen, C. C.; Lee, L. Y.; Lin, T. W.; Kuo, C. F. N(6)(2-Hydroxyethyl)adenosine in the medicinal mushroom Cordyceps cicadae attenuates lipopolysaccharide-stimulated pro-inflammatory responses by suppressing TLR4-mediated NF-kappaB signaling pathways. J. Nat. Prod. 2015, 78, 2452−2460. (45) Wu, S. J.; Lu, T. M.; Lai, M. N.; Ng, L. T. Immunomodulatory activities of medicinal mushroom Grifola f rondosa extract and its bioactive constituent. Am. J. Chin. Med. 2013, 41, 131−144.

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