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Cyclooxygenase inhibitory and antioxidant compounds from the fruiting body of an edible mushroom, Agrocybe aegerita
Phytomedicine 10: 386–390, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Cyclooxygenase inhibitory and antioxidant compounds from the fruiting body of an edible mushroom, Agrocybe aegerita Y. Zhang1, G. L. Mills2, and M. G. Nair1 1 2
Bioactive Natural Products Laboratory, Department of Horticulture and National Food Safety and Toxicology Center Biosystems Engineering, Michigan State University, East Lansing, Michigan, USA
Summary In the search for bioactive natural products from edible mushrooms, we have investigated the fruiting body of Agrocybe aegerita. The methanol extract of this mushroom yielded a fatty acid fraction (FAF), along with palmitic acid (1), ergosterol (2), 5,8-epidioxy-ergosta-6,22-dien-3β-ol (3), mannitol (4) and trehalose (5). The composition of FAF was confirmed by GC-MS and by comparison to the retention values of authentic samples of palmitic, stearic, oleic and linoleic acids. The structures of 1–5 were established using spectroscopic methods. FAF and compounds 1–3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition values of liposome peroxidation by FAF, compounds 1 and 2 at 100 µg/ml were 75, 45, and 43%, respectively. The inhibition values of COX-I enzyme by FAF and 1–3 at 100 µg/ml were 80, 39, 19, and 57%, respectively. Similarly, COX-II enzyme activity was reduced by FAF and 1–3 at 100 µg/ml with values of 88, 45, 28, and 22%, respectively. Compounds 1, 3 and fatty acids were isolated here for the first time from the fruiting body of A. aegerita. Key words: Agrocybe aegerita, linoleic acid, oleic acid, stearic acid, palmitic acid, ergosterol, 5,8-epidioxy-ergosta-6,22-dien-3β-ol, mannitol and trehalose
Introduction It is well known that cancer has a close relationship with inflammation (DuBois et al. 1996) and cellular oxidation (Weitberg, 1989). Inflammatory cells are capable of inducing genotoxic effects such as DNA strand break and mutation, and of promoting neoplastic transformation in the nearby cells (Weitzman et al. 1985). Therefore, it is considered that anti-inflammatory and antioxidant agents may play an important role in cancer prevention. In the inflammatory process, two distinct isoforms of cyclooxygenase exist, namely COX-I and COX-II, which are both involved in the conversion of arachidonic acid to prostaglandins (Lipsky et al. 1998). It is documented that the inducible COX-II is associated with inflammatory conditions, 0944-7113/03/10/05-386 $ 15.00/0
whereas extensively expressed COX-I is responsible for the cytoprotective effects of prostaglandins (Seibert et al. 1994; Smith et al. 1998). Naturally-occurring selective COX-II inhibitors are significant since they can be consumed as supplements, reducing inflammation with fewer side-effects, and potential prevention of cancer. Therefore, some of our research efforts have been focused on the isolation of specific COX-II enzyme inhibitors from fungal products. A. aegerita is an edible mushroom, a basidiomycete, found in the order Agaricales. Indole derivatives with free radical scavenging activity (Stransky et al. 1992), cylindan with anticancer activity (Hyun et al. 1996), agrocybenine (Koshino et al. 1996), and several anti-
Cyclooxygenase inhibitory and antioxidant compounds of Agrocybe aegerita fungal, antibiotic compounds (Kim et al. 1997) have been isolated from the fruiting body of Agrocybe spp. This is the first report of the isolation of cyclooxygenase-inhibitory and antioxidant compounds 1–5 and the fatty acids from the fruiting body of A. aegerita.
Materials and Methods General Experimental 1
H- and 13C-NMR spectra were recorded on Varian INOVA 300 and 500 MHz spectrometers. Compounds 1–3 were dissolved in CHCl3 and are reported in δ (ppm) based on δ residual of CHCl3 at 7.24 for 1H NMR and 77.0 for 13C NMR. Compounds 4 and 5 were dissolved in DMSO-d6 and are reported in δ (ppm) based on δ residual of DMSO-d6 at 2.49 for 1H NMR and 39.5 for 13C NMR. Coupling constants, J, are in Hz. Silica gel (30–60 µm particle sizes) used for MPLC was purchased from Merck. TLC plates and Prep-TLC (GF Uniplate, with binder, 250 µm) were the products of Analtech, Inc., Newark, DE. Positive controls t-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) used in the anti-oxidant assay were purchased from Sigma Chemical Company. Vioxx® tablets and CelebrexTM capsules used in the cyclooxygenase inhibitory assay as positive controls were physician’s professional samples provided by Dr. Subash Gupta of Sparrow Pain Center, Sparrow Hospital, MI. All organic solvents were ACS reagent grade (Aldrich Chemical Co., Inc., Milwaukee, WI).
tone (4:1, 400 ml), hexane-acetone (2:1, 400 ml), CHCl3-MeOH (9:1, 400 ml), CHCl3-MeOH (8 :2, 400 ml), CHCl3-MeOH (1:1, 400 ml), and methanol (400 ml), respectively. Fractions were collected in aliquots of 25 mL in each test tube. The fractions with a similar TLC profile were combined and yielded fractions A (37.9 mg), B (51.2 mg), C (129.8 mg), D (46.8 mg), E (167.5), F (188.9), G (338.0 mg) and H (1204.3 mg). Fractions B, C and D showed COX enzyme inhibitory and antioxidant activities. Therefore, these fractions were further purified. A white solid was precipitated from fraction C (compound 1, 7.0 mg). Its NMR spectrum was identical with that of palmitic acid (Pouchert, 1983). White needle-like crystals were precipitated from fraction B (compound 2, 30.5 mg). Detailed NMR studies revealed that compound 2 (Fig. 1) was identical to ergosterol (Zhou et al. 2000). The mother liquor (121.9 mg) from compound 1 was further purified by preparative TLC (20 × 20, 250 µm) using hexane-ether (4 :1) as the mobile phase. An oily residue (FAF, 59.6 mg) was obtained and gave one spot on TLC plate using hexane-acetone as the developing solvent. The components of the fatty acid fraction were determined by GC-MS analyses. Compound 3 (7 mg) was obtained from the purification of fraction D by preparative TLC (20 × 20, 250 µm)
Plant Materials
The fruiting bodies of A. aegerita were grown in the Bioactive Natural Product and Phytoceutical green house at Michigan State University. After harvest, the fruiting bodies of A. aegerita were air-dried and stored in –20 °C freezers in sealed bags until extraction. Extraction and Isolation
The fruiting bodies of A. aegerita (928 g) were airdried to obtain 94.6 g of dried materials. Air-dried fruiting bodies of A. aegerita (94.6 g) were ground to a fine powder and extracted sequentially with hexane (500 ml × 3, 24 h), ethyl acetate (500 ml × 3, 24 h) and methanol (500 ml × 3, 24 h) in an extraction column. The extracts were evaporated in vacuo and yielded 0.99, 0.36, and 15.03 g of residues, respectively. The methanol extract (5.03 g) was suspended in 200 ml of methanol and filtered, and yielded methanol-soluble (2.74 g) and -insoluble fractions (2.28 g). The methanol-soluble portion (2.74 g) was subjected to silica gel medium-pressure liquid chromatography (MPLC) and eluted with hexane (400 ml), hexane-ace-
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Fig. 1. Structures of Compounds 1, 2 and 3.
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using hexane-acetone (4 :1) as the mobile phase. NMR studies revealed that compound 3 (Fig. 1) was identical to 5,8-epidioxy-ergosta-6-22-dien-3β-ol (Yue et al. 2001). A white solid was precipitated from fraction E and recrystallized from methanol to yield compound 4 (32 mg). The NMR spectrum of 4 was identical to that reported for mannitol (Fujita et al. 1984). Similarly, the fraction G, a white precipitate, upon crystallization from methanol yielded white needle-like crystals (compound 5, 38.6 mg). The NMR spectrum of compound 5 was identical to that reported for trehalose (Batta et al. 1999).
(Kelm et al. 1998). The diazomethane generated was stored in ether and used to methylate the fatty acids. The fatty acids fraction (FAF, 2 mg) was dissolved in ether and reacted with diazomethane in ether until the solution become pale yellow. The resulting yellow solution was kept at room temperature for 1 h and evaporated; the residue was dissolved in ether and analyzed by GC-MS. GC-MS analysis
Diazomethane was prepared by reacting N-nitroso-Nmethylurea with concentrated KOH solution in ether
Analysis of the FAF methyl ester was carried out on a JEOL AX-505H double-focusing mass spectrometer coupled to a Hewlett-Packard 5890J gas chromatograph via a heated interface. GC separation of the FAF methyl ester was accomplished on a DBWAX fusedsilica capillary column (30 m length × 6.35-mm ID
Fig. 2a. In vitro COX-I and COX-II inhibitory activities of Vioxx, Celebrex, Aspirin, Naproxen and Ibuprofen. Vioxx and Celebrex were tested at 1.67 µg/ml. Aspirin, Naproxen and Ibuprofen were tested at 180, 2.52 and 2.06 µg/ml, respectively. Vertical bars represent standard deviation of each data point (n = 2). Fig. 2b. In vitro COX-I and COX-II inhibitory activities of FAF and Compounds 1–3 at 100 µg/ml. Vertical bars represent standard deviation of each data point (n = 2).
Fig. 3a. Antioxidant activities of TBHQ, BHT and BHA in a liposomal model system. TBHQ, BHT and BHA were evaluated at 1.8, 2.2, 1.66 µg/ml, respectively. Vertical bars represent the standard deviation of each data point (n = 2). Fig. 3b. Antioxidant activities of FAF and compounds 1–3 in a liposomal model system. Samples were tested at 100 µg/ml. Vertical bars represent the standard deviation of each data point (n = 2).
Preparation of diazomethane and methylation of the fatty acids fraction
Cyclooxygenase inhibitory and antioxidant compounds of Agrocybe aegerita with a 0.25-µm film coating) purchased from J & W. Direct (splitless) injection was used and the carrier gas was Helium at 1 ml/min. The GC temperature program consisted of initial temperature 50 °C; 10 °C/min to reach a final temperature of 250 °C. MS conditions were interface temperature 210 °C, ion source temperature ca. 200 °C, electron energy 100 µA, and the scan rate was 1 scan/s over the m/z range 45–750. Cyclooxygenase inhibitory assay
The COX-I enzyme inhibitory assay was conducted with an enzyme preparation from ram seminal vesicles. COX-II activity was determined using a preparation from insect cell lysate. COX assays were measured at 37 °C and at pH 7.0 according to the published procedure (Wang et al. 1999). Antioxidant assay
This assay was conducted by analysis of model liposome oxidation using fluorescence spectroscopy according to the procedure reported previously (Wang et al. 1999). Peroxidation was initiated by addition of FeCl2 for positive controls of BHA, BHT, TBHQ (1.80, 2.20, 1.66 µg/ml, respectively) and test samples. Fluorescence was measured at 384 nm and monitored at 0, 1, 3 and every 3 min thereafter up to 21 min using a Turner Model 450 Digital Fluorometer (Barnstead Thermolyne, Dubuque, IA). The decrease of relative fluorescence intensity with time indicated the rate of peroxidation and these data are reported for 21 min after the initiation of peroxidation. Relative fluorescence (Ft/F0) was calculated by dividing the fluorescence value at a given point (Ft) by that at t = 0 min (F0) (Arora et al. 1997).
Results Compounds isolated from the fruiting body of the edible mushroom A. aegerita were characterized using 1 H- and 13C-NMR experiments. The composition of fatty acids was confirmed by GC-MS and by the comparison of retention times of the methyl esters of authentic fatty acids. The methyl ester of FAF showed peaks at m/z 298, 296, 294 and 270, corresponding to retention times of 19.00, 20.95, 21.17 and 21.57 min and represented the methyl esters of palmitic, stearic, oleic and linoleic acids, respectively. Cyclooxygenase inhibitory activities are shown in Figs. 2a and 2b. FAF and compounds 1–3 inhibited the activities of both COX-I and COX-II enzymes. The inhibition levels of COX-I enzyme by FAF and compounds 1–3 at 100 µg/ml were 80, 39, 19, 57%, respectively (Fig. 2b). The positive controls Vioxx® (1.67 µg/ml), CelebrexTM (1.67 µg/ml), Aspirin (180 µg/ml), Naprox-
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en (2.52 µg/ml) and Ibuprofen (2.06 µg/ml) showed 23, 46, 92, 43 and 30% inhibition, respectively (Fig. 2a). FAF and compounds 1–3 inhibited COX-II enzyme at 100 µg/ml at 88, 45, 28, 22%, respectively (Fig. 2b). The positive controls Vioxx® (1.67 µg/ml), Celebrex TM (1.67 µg/ml), Aspirin (180 µg/ml), Naprox en (2.52 µg/ml) and Ibuprofen (2.06 µg/ml) showed 67, 88, 24, 61 and 43% inhibition, respectively (Fig. 2a). Compounds 4 and 5 were not active in COX-I and COX-II enzyme-inhibitory assays. An iron-catalyzed liposome model and fluorescence spectroscopy was employed to evaluate the inhibition of lipid peroxidation by FAF and compounds 1–5 (Arora et al. 1997). Antioxidant properties of FAF and compounds 1 and 2 at 100 µg/ml were 75, 45, and 43%, respectively (Fig. 3b). The positive controls TBHQ (1.66 µg/ml, BHA (1.80 µg/ml) and BHT (2.20 µg/ml) showed peroxidation inhibition at 90, 88, and 92%, respectively (Fig. 3a). Compounds 3–5 did not show antioxidant activity in our assay system.
Discussion The bioassay-guided fractionation of the methanol extract of A. aegerita led to the isolation of a fatty acid fraction and GC-MS analysis revealed that the composition of this fatty acid fraction contained palmitic, stearic, oleic and linoleic acids. The height of the GC peaks revealed that linoleic, oleic and, palmitic acids were the main components in equal proportions. Stearic acid was present only in trace quantity. Our results indicated that the fatty acids were the most effective components of A. aegerita, demonstrating both COX-I and COX-II enzyme inhibition. Although compound 1, palmitic acid, exhibited lower COX-enzyme-inhibitory activity than the fatty acid fraction (FAF), the COX-II/COX-I inhibitory ratio was similar to that of FAF (Fig. 2b). Fatty acids are ubiquitous in mushrooms and may be responsible for some of the phytoceutical properties attributed to many of the edible mushrooms. The structure-activity relationship among fatty acids related to COX-I and COX-II enzyme inhibitory activities has been reported (Ringborn et al. 2001). It was suggested that a fatty acid with 20 carbons or more showed higher inhibition of COX enzyme than those with less than 16 carbons. Ergosterol, compound 2, showed higher inhibitory activity towards the COX-II enzyme than towards COX-I, but compound 3, the peroxidation product of ergosterol, showed higher COX-I than COX-II enzyme inhibitory activity (Fig. 2b). The antioxidant results indicated that FAF containing unsaturated fatty acids demonstrated 75% peroxidation inhibition, comparable to those of the commer-
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cial antioxidants, BHA, BHT and TBHQ. However, palmitic acid and ergosterol showed only a mild antioxidant activity. The antioxidant and cyclooxygenase enzyme activities of the compounds isolated from A. aegerita suggest that the consumption of A. aegerita as daily food or an ingredient in food preparation may contribute a potential health benefit as an anti-oxidant, reducing inflammation and promoting cancer prevention. Acknowledgment
This is a contribution from Michigan State University Agricultural Experiment Station. Funding for this project was partially provided by the Center for Plant Products and Technologies (CPPT), MSU, MI. The NMR data were obtained on instrumentation purchased in part with funds from NIH Grant 1-S10-RR04750, NSF Grant CHE-88-00770, and NSF Grant CHE-92-13241. GC-MS was performed at the Michigan State University Mass Spectrometry Facility. The COX-II enzyme was supplied by Dr. Dave Dewitt, Department of Biochemistry, Michigan State University, MI.
References Arora A, Nair MG, Strasburg GM (1997) A novel fluorescence assay to evaluate antioxidants efficiency: application to flavonoids and isoflavonoids. Ed Aruma I, Cuppett SC, AOCS Press, Illinois, pp 205–222 Batta G, Koever KE, Gervay J, Hornyak M, Roberts GM (1997) Temperature Dependence of Molecular Conformation, Dynamics, and Chemical Shift Anisotropy of a,a-Trehalose in D2O by NMR Relaxation. J Am Chem Soc 119: 1336–1345 DuBois RN, Giardiello FM, Smalley WE (1996) Nonsteroidal anti-inflammatory drugs, eicosanoids, and colorectal cancer prevention. Gastroenterology Clinics of North America 25: 773–791 Fujita M, Yamada M, Nakajima S, Kawai K, Nagai M (1984) O-Methylation effect on the carbon-13 nuclear magnetic resonance signals of ortho-disubstituted phenols and its application to structure determination of new phthalides from Aspergillus silvaticus. Chem Pharm Bull 32: 2622–2627 Hyun YW, Kim CK, Park SH, Yoon JM, Shim MJ, Kang CY, Choi EC, Kim BK (1996) Antitumor components of Agrocybe cylindracea. Arch Pharm Res 19: 207–212 Kelm MA, Nair MG (1998) Mosquitocidal compounds and a triglyceride, 1,3-dilinoleneoyl-2-palmitin, from Ocimum sanctum. J Agric Food Chem 46: 3092–3094 Kim WG, Lee IK, Kim JP, Ryoo IJ, Koshino H, Yoo ID (1997) New indole derivatives with free radical scaveng-
ing activity from Agrocybe cylindracea. J Nat Prod 60: 721–723 Koshino H, Lee IK, Kim JP, Kim WG, Uzawa J, Yoo ID (1996) Agrocybenine, novel class alkaloid from the Korean mushroom Agrocybe cylindracea. Tetrahedron Lett 37: 4549–4550 Lipsky LP, Abramson SB, Crofford L, Dubois RN, Simon LS, Van de Putte LB (1998) The classification of cyclooxygenases inhibitors. J Rheumatol 25: 2298–2303 Pouchert CJ (1983) The Aldrich Library of NMR Spectra, Volume I, Edition II, Aldrich Chemical Company, Inc. pp 422 Ringborn T, Huss U, Stenholm L, Flock S, Skattebol L, Perera P, Bohlin L (2001) COX-2 Inhibitory effects of naturally occurring and modified fatty acids. J Nat Prod 64: 745–749 Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L, Isakson P (1994) Pharmacological and biochemical demonstration of the role of cyclooxygenase-2 in inflammation and pain. Proc Natl Acad Sci USA 91: 12013–12017 Smith CJ, Zhang Y, Koboldt CM, Muhammad J, Zweifel BS, Shaffer A, Talley JJ, Masferrer JL, Seibert K, Isakson PC (1998) Pharmacological analysis of cyclooxygenase-1 in inflammation. Proc Natl Acad Sci USA 95: 13313–13318 Stransky K, Semerdzieva M, Otmar M, Prochazka Z, Budesinsky M, Ubik K, Kohoutova J, Streinz L (1992) Antifungal antibiotic from the mushroom Agrocybe aegerita (Brig.) Sing Collect Czech Chem Commun 57: 590–603 Wang H, Nair MG, Strasburg GM, Chang YC, Booren AM, Gray IJ, Dewitt DL (1999) Antioxidant and anti-inflammatory activities of anthocyanins and their aglycone, cyanidin, from tart cherries. J Nat Prod 62: 294–296 Weitberg AB (1989) Effect of combination of antioxidants on phagocyte-induced sister chromatid exchanges. Mutat Res 224: 1–4 Weitzman SA, Weitberg AB, Clark EP, Stossel TP (1985) Phagocytes as carcinogenous: malignant transformation produced by human neutrophils. Science 227: 1231–1233 Yue JM, Chen SN, Li ZW, Sun HD (2001) Sterols from the fungus Lactarium volemus. Phytochemistry 56: 801–806 Zhou WX, Nes WD (2000) Stereochemistry of hydrogen introduction at C-25 in ergosterol synthesized by the mevalonateindependent pathway. Tetrahedron Lett 41: 2791–2795
Address Muraleedharan G. Nair, Bioactive Natural Products Laboratory, Department of Horticulture and National Food Safety and Toxicology Center. Michigan State University, East Lansing, Michigan, 48824, USA Tel.: ++1-517-353-2915; Fax: ++1-517-432-2310; e-mail: [email protected]
Phytomedicine
Cyclooxygenase inhibitory and antioxidant compounds from the fruiting body of an edible mushroom, Agrocybe aegerita Y. Zhang1, G. L. Mills2, and M. G. Nair1 1 2
Bioactive Natural Products Laboratory, Department of Horticulture and National Food Safety and Toxicology Center Biosystems Engineering, Michigan State University, East Lansing, Michigan, USA
Summary In the search for bioactive natural products from edible mushrooms, we have investigated the fruiting body of Agrocybe aegerita. The methanol extract of this mushroom yielded a fatty acid fraction (FAF), along with palmitic acid (1), ergosterol (2), 5,8-epidioxy-ergosta-6,22-dien-3β-ol (3), mannitol (4) and trehalose (5). The composition of FAF was confirmed by GC-MS and by comparison to the retention values of authentic samples of palmitic, stearic, oleic and linoleic acids. The structures of 1–5 were established using spectroscopic methods. FAF and compounds 1–3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition values of liposome peroxidation by FAF, compounds 1 and 2 at 100 µg/ml were 75, 45, and 43%, respectively. The inhibition values of COX-I enzyme by FAF and 1–3 at 100 µg/ml were 80, 39, 19, and 57%, respectively. Similarly, COX-II enzyme activity was reduced by FAF and 1–3 at 100 µg/ml with values of 88, 45, 28, and 22%, respectively. Compounds 1, 3 and fatty acids were isolated here for the first time from the fruiting body of A. aegerita. Key words: Agrocybe aegerita, linoleic acid, oleic acid, stearic acid, palmitic acid, ergosterol, 5,8-epidioxy-ergosta-6,22-dien-3β-ol, mannitol and trehalose
Introduction It is well known that cancer has a close relationship with inflammation (DuBois et al. 1996) and cellular oxidation (Weitberg, 1989). Inflammatory cells are capable of inducing genotoxic effects such as DNA strand break and mutation, and of promoting neoplastic transformation in the nearby cells (Weitzman et al. 1985). Therefore, it is considered that anti-inflammatory and antioxidant agents may play an important role in cancer prevention. In the inflammatory process, two distinct isoforms of cyclooxygenase exist, namely COX-I and COX-II, which are both involved in the conversion of arachidonic acid to prostaglandins (Lipsky et al. 1998). It is documented that the inducible COX-II is associated with inflammatory conditions, 0944-7113/03/10/05-386 $ 15.00/0
whereas extensively expressed COX-I is responsible for the cytoprotective effects of prostaglandins (Seibert et al. 1994; Smith et al. 1998). Naturally-occurring selective COX-II inhibitors are significant since they can be consumed as supplements, reducing inflammation with fewer side-effects, and potential prevention of cancer. Therefore, some of our research efforts have been focused on the isolation of specific COX-II enzyme inhibitors from fungal products. A. aegerita is an edible mushroom, a basidiomycete, found in the order Agaricales. Indole derivatives with free radical scavenging activity (Stransky et al. 1992), cylindan with anticancer activity (Hyun et al. 1996), agrocybenine (Koshino et al. 1996), and several anti-
Cyclooxygenase inhibitory and antioxidant compounds of Agrocybe aegerita fungal, antibiotic compounds (Kim et al. 1997) have been isolated from the fruiting body of Agrocybe spp. This is the first report of the isolation of cyclooxygenase-inhibitory and antioxidant compounds 1–5 and the fatty acids from the fruiting body of A. aegerita.
Materials and Methods General Experimental 1
H- and 13C-NMR spectra were recorded on Varian INOVA 300 and 500 MHz spectrometers. Compounds 1–3 were dissolved in CHCl3 and are reported in δ (ppm) based on δ residual of CHCl3 at 7.24 for 1H NMR and 77.0 for 13C NMR. Compounds 4 and 5 were dissolved in DMSO-d6 and are reported in δ (ppm) based on δ residual of DMSO-d6 at 2.49 for 1H NMR and 39.5 for 13C NMR. Coupling constants, J, are in Hz. Silica gel (30–60 µm particle sizes) used for MPLC was purchased from Merck. TLC plates and Prep-TLC (GF Uniplate, with binder, 250 µm) were the products of Analtech, Inc., Newark, DE. Positive controls t-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) used in the anti-oxidant assay were purchased from Sigma Chemical Company. Vioxx® tablets and CelebrexTM capsules used in the cyclooxygenase inhibitory assay as positive controls were physician’s professional samples provided by Dr. Subash Gupta of Sparrow Pain Center, Sparrow Hospital, MI. All organic solvents were ACS reagent grade (Aldrich Chemical Co., Inc., Milwaukee, WI).
tone (4:1, 400 ml), hexane-acetone (2:1, 400 ml), CHCl3-MeOH (9:1, 400 ml), CHCl3-MeOH (8 :2, 400 ml), CHCl3-MeOH (1:1, 400 ml), and methanol (400 ml), respectively. Fractions were collected in aliquots of 25 mL in each test tube. The fractions with a similar TLC profile were combined and yielded fractions A (37.9 mg), B (51.2 mg), C (129.8 mg), D (46.8 mg), E (167.5), F (188.9), G (338.0 mg) and H (1204.3 mg). Fractions B, C and D showed COX enzyme inhibitory and antioxidant activities. Therefore, these fractions were further purified. A white solid was precipitated from fraction C (compound 1, 7.0 mg). Its NMR spectrum was identical with that of palmitic acid (Pouchert, 1983). White needle-like crystals were precipitated from fraction B (compound 2, 30.5 mg). Detailed NMR studies revealed that compound 2 (Fig. 1) was identical to ergosterol (Zhou et al. 2000). The mother liquor (121.9 mg) from compound 1 was further purified by preparative TLC (20 × 20, 250 µm) using hexane-ether (4 :1) as the mobile phase. An oily residue (FAF, 59.6 mg) was obtained and gave one spot on TLC plate using hexane-acetone as the developing solvent. The components of the fatty acid fraction were determined by GC-MS analyses. Compound 3 (7 mg) was obtained from the purification of fraction D by preparative TLC (20 × 20, 250 µm)
Plant Materials
The fruiting bodies of A. aegerita were grown in the Bioactive Natural Product and Phytoceutical green house at Michigan State University. After harvest, the fruiting bodies of A. aegerita were air-dried and stored in –20 °C freezers in sealed bags until extraction. Extraction and Isolation
The fruiting bodies of A. aegerita (928 g) were airdried to obtain 94.6 g of dried materials. Air-dried fruiting bodies of A. aegerita (94.6 g) were ground to a fine powder and extracted sequentially with hexane (500 ml × 3, 24 h), ethyl acetate (500 ml × 3, 24 h) and methanol (500 ml × 3, 24 h) in an extraction column. The extracts were evaporated in vacuo and yielded 0.99, 0.36, and 15.03 g of residues, respectively. The methanol extract (5.03 g) was suspended in 200 ml of methanol and filtered, and yielded methanol-soluble (2.74 g) and -insoluble fractions (2.28 g). The methanol-soluble portion (2.74 g) was subjected to silica gel medium-pressure liquid chromatography (MPLC) and eluted with hexane (400 ml), hexane-ace-
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Fig. 1. Structures of Compounds 1, 2 and 3.
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using hexane-acetone (4 :1) as the mobile phase. NMR studies revealed that compound 3 (Fig. 1) was identical to 5,8-epidioxy-ergosta-6-22-dien-3β-ol (Yue et al. 2001). A white solid was precipitated from fraction E and recrystallized from methanol to yield compound 4 (32 mg). The NMR spectrum of 4 was identical to that reported for mannitol (Fujita et al. 1984). Similarly, the fraction G, a white precipitate, upon crystallization from methanol yielded white needle-like crystals (compound 5, 38.6 mg). The NMR spectrum of compound 5 was identical to that reported for trehalose (Batta et al. 1999).
(Kelm et al. 1998). The diazomethane generated was stored in ether and used to methylate the fatty acids. The fatty acids fraction (FAF, 2 mg) was dissolved in ether and reacted with diazomethane in ether until the solution become pale yellow. The resulting yellow solution was kept at room temperature for 1 h and evaporated; the residue was dissolved in ether and analyzed by GC-MS. GC-MS analysis
Diazomethane was prepared by reacting N-nitroso-Nmethylurea with concentrated KOH solution in ether
Analysis of the FAF methyl ester was carried out on a JEOL AX-505H double-focusing mass spectrometer coupled to a Hewlett-Packard 5890J gas chromatograph via a heated interface. GC separation of the FAF methyl ester was accomplished on a DBWAX fusedsilica capillary column (30 m length × 6.35-mm ID
Fig. 2a. In vitro COX-I and COX-II inhibitory activities of Vioxx, Celebrex, Aspirin, Naproxen and Ibuprofen. Vioxx and Celebrex were tested at 1.67 µg/ml. Aspirin, Naproxen and Ibuprofen were tested at 180, 2.52 and 2.06 µg/ml, respectively. Vertical bars represent standard deviation of each data point (n = 2). Fig. 2b. In vitro COX-I and COX-II inhibitory activities of FAF and Compounds 1–3 at 100 µg/ml. Vertical bars represent standard deviation of each data point (n = 2).
Fig. 3a. Antioxidant activities of TBHQ, BHT and BHA in a liposomal model system. TBHQ, BHT and BHA were evaluated at 1.8, 2.2, 1.66 µg/ml, respectively. Vertical bars represent the standard deviation of each data point (n = 2). Fig. 3b. Antioxidant activities of FAF and compounds 1–3 in a liposomal model system. Samples were tested at 100 µg/ml. Vertical bars represent the standard deviation of each data point (n = 2).
Preparation of diazomethane and methylation of the fatty acids fraction
Cyclooxygenase inhibitory and antioxidant compounds of Agrocybe aegerita with a 0.25-µm film coating) purchased from J & W. Direct (splitless) injection was used and the carrier gas was Helium at 1 ml/min. The GC temperature program consisted of initial temperature 50 °C; 10 °C/min to reach a final temperature of 250 °C. MS conditions were interface temperature 210 °C, ion source temperature ca. 200 °C, electron energy 100 µA, and the scan rate was 1 scan/s over the m/z range 45–750. Cyclooxygenase inhibitory assay
The COX-I enzyme inhibitory assay was conducted with an enzyme preparation from ram seminal vesicles. COX-II activity was determined using a preparation from insect cell lysate. COX assays were measured at 37 °C and at pH 7.0 according to the published procedure (Wang et al. 1999). Antioxidant assay
This assay was conducted by analysis of model liposome oxidation using fluorescence spectroscopy according to the procedure reported previously (Wang et al. 1999). Peroxidation was initiated by addition of FeCl2 for positive controls of BHA, BHT, TBHQ (1.80, 2.20, 1.66 µg/ml, respectively) and test samples. Fluorescence was measured at 384 nm and monitored at 0, 1, 3 and every 3 min thereafter up to 21 min using a Turner Model 450 Digital Fluorometer (Barnstead Thermolyne, Dubuque, IA). The decrease of relative fluorescence intensity with time indicated the rate of peroxidation and these data are reported for 21 min after the initiation of peroxidation. Relative fluorescence (Ft/F0) was calculated by dividing the fluorescence value at a given point (Ft) by that at t = 0 min (F0) (Arora et al. 1997).
Results Compounds isolated from the fruiting body of the edible mushroom A. aegerita were characterized using 1 H- and 13C-NMR experiments. The composition of fatty acids was confirmed by GC-MS and by the comparison of retention times of the methyl esters of authentic fatty acids. The methyl ester of FAF showed peaks at m/z 298, 296, 294 and 270, corresponding to retention times of 19.00, 20.95, 21.17 and 21.57 min and represented the methyl esters of palmitic, stearic, oleic and linoleic acids, respectively. Cyclooxygenase inhibitory activities are shown in Figs. 2a and 2b. FAF and compounds 1–3 inhibited the activities of both COX-I and COX-II enzymes. The inhibition levels of COX-I enzyme by FAF and compounds 1–3 at 100 µg/ml were 80, 39, 19, 57%, respectively (Fig. 2b). The positive controls Vioxx® (1.67 µg/ml), CelebrexTM (1.67 µg/ml), Aspirin (180 µg/ml), Naprox-
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en (2.52 µg/ml) and Ibuprofen (2.06 µg/ml) showed 23, 46, 92, 43 and 30% inhibition, respectively (Fig. 2a). FAF and compounds 1–3 inhibited COX-II enzyme at 100 µg/ml at 88, 45, 28, 22%, respectively (Fig. 2b). The positive controls Vioxx® (1.67 µg/ml), Celebrex TM (1.67 µg/ml), Aspirin (180 µg/ml), Naprox en (2.52 µg/ml) and Ibuprofen (2.06 µg/ml) showed 67, 88, 24, 61 and 43% inhibition, respectively (Fig. 2a). Compounds 4 and 5 were not active in COX-I and COX-II enzyme-inhibitory assays. An iron-catalyzed liposome model and fluorescence spectroscopy was employed to evaluate the inhibition of lipid peroxidation by FAF and compounds 1–5 (Arora et al. 1997). Antioxidant properties of FAF and compounds 1 and 2 at 100 µg/ml were 75, 45, and 43%, respectively (Fig. 3b). The positive controls TBHQ (1.66 µg/ml, BHA (1.80 µg/ml) and BHT (2.20 µg/ml) showed peroxidation inhibition at 90, 88, and 92%, respectively (Fig. 3a). Compounds 3–5 did not show antioxidant activity in our assay system.
Discussion The bioassay-guided fractionation of the methanol extract of A. aegerita led to the isolation of a fatty acid fraction and GC-MS analysis revealed that the composition of this fatty acid fraction contained palmitic, stearic, oleic and linoleic acids. The height of the GC peaks revealed that linoleic, oleic and, palmitic acids were the main components in equal proportions. Stearic acid was present only in trace quantity. Our results indicated that the fatty acids were the most effective components of A. aegerita, demonstrating both COX-I and COX-II enzyme inhibition. Although compound 1, palmitic acid, exhibited lower COX-enzyme-inhibitory activity than the fatty acid fraction (FAF), the COX-II/COX-I inhibitory ratio was similar to that of FAF (Fig. 2b). Fatty acids are ubiquitous in mushrooms and may be responsible for some of the phytoceutical properties attributed to many of the edible mushrooms. The structure-activity relationship among fatty acids related to COX-I and COX-II enzyme inhibitory activities has been reported (Ringborn et al. 2001). It was suggested that a fatty acid with 20 carbons or more showed higher inhibition of COX enzyme than those with less than 16 carbons. Ergosterol, compound 2, showed higher inhibitory activity towards the COX-II enzyme than towards COX-I, but compound 3, the peroxidation product of ergosterol, showed higher COX-I than COX-II enzyme inhibitory activity (Fig. 2b). The antioxidant results indicated that FAF containing unsaturated fatty acids demonstrated 75% peroxidation inhibition, comparable to those of the commer-
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cial antioxidants, BHA, BHT and TBHQ. However, palmitic acid and ergosterol showed only a mild antioxidant activity. The antioxidant and cyclooxygenase enzyme activities of the compounds isolated from A. aegerita suggest that the consumption of A. aegerita as daily food or an ingredient in food preparation may contribute a potential health benefit as an anti-oxidant, reducing inflammation and promoting cancer prevention. Acknowledgment
This is a contribution from Michigan State University Agricultural Experiment Station. Funding for this project was partially provided by the Center for Plant Products and Technologies (CPPT), MSU, MI. The NMR data were obtained on instrumentation purchased in part with funds from NIH Grant 1-S10-RR04750, NSF Grant CHE-88-00770, and NSF Grant CHE-92-13241. GC-MS was performed at the Michigan State University Mass Spectrometry Facility. The COX-II enzyme was supplied by Dr. Dave Dewitt, Department of Biochemistry, Michigan State University, MI.
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Address Muraleedharan G. Nair, Bioactive Natural Products Laboratory, Department of Horticulture and National Food Safety and Toxicology Center. Michigan State University, East Lansing, Michigan, 48824, USA Tel.: ++1-517-353-2915; Fax: ++1-517-432-2310; e-mail: [email protected]