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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Bioactive metabolites from the mycelia of the basidiomycete Hericium erinaceum a
a
a
Qiang-Qiang Lu , Jun-Mian Tian , Jing Wei & Jin-Ming Gao
a
a
Department of Applied Chemistry, College of Science, Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, Northwest A&F University, Yangling 712100, Shaanxi, P.R. China Published online: 18 Mar 2014.
To cite this article: Qiang-Qiang Lu, Jun-Mian Tian, Jing Wei & Jin-Ming Gao (2014) Bioactive metabolites from the mycelia of the basidiomycete Hericium erinaceum, Natural Product Research: Formerly Natural Product Letters, 28:16, 1288-1292, DOI: 10.1080/14786419.2014.898145 To link to this article: http://dx.doi.org/10.1080/14786419.2014.898145
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Downloaded by [University of Tasmania] at 10:46 03 September 2014
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Natural Product Research, 2014 Vol. 28, No. 16, 1288–1292, http://dx.doi.org/10.1080/14786419.2014.898145
SHORT COMMUNICATION Bioactive metabolites from the mycelia of the basidiomycete Hericium erinaceum Qiang-Qiang Lu1, Jun-Mian Tian, Jing Wei and Jin-Ming Gao* Department of Applied Chemistry, College of Science, Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, Northwest A&F University, Yangling 712100, Shaanxi, P.R. China
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(Received 9 December 2013; final version received 16 February 2014) Seven known compounds, three diketopiperazine alkaloids, 12b-hydroxyverruculogen TR-2 (1), fumitremorgin C (2) and methylthiogliotoxin (5), two hetero-spirocyclic glactam alkaloids, pseurotin A (3) and FD-838 (4), and cerevisterol (6) and herierin IV (7), were isolated from the mycelia of the basidiomycete Hericium erinaceum and identified by spectroscopic analyses. The antioxidant and antifungal activities of compounds 1 – 6 were evaluated. The results indicated that compounds 1, 3 and 6 exhibited potential antioxidant activity against DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical with their IC50 data of ca. 12 mM, compared with positive control tertiary butylhydroquinone. In addition, compound 4 significantly inhibited the growth of two plant fungal pathogens Botrytis cinerea and Glomerella cingulata with an minimum inhibitory concentration of 6.25 mM for each, similar to that of the positive fungicide, carbendazim. Compounds 1 – 5 were isolated from the genus Hericium for the first time. Keywords: Hericium erinaceum; mushroom; diketopiperazine alkaloids; antioxidant activity; antifungal activity
1. Introduction Higher fungi have long been considered as an important source that produce a large and diverse variety of secondary metabolites (Gao 2006). Hericium erinaceum (Pers.) is known as an edible mushroom (family Hericiaceae) that has been used as medicine or food in China, Japan and Europe without harmful effects. Chemical studies of H. erinaceum have produced a variety of secondary metabolites, such as cyathane diterpenoids, irregular terpenoids, pyranones and sterols (Gao 2006; Ma et al. 2010; Ueda et al. 2008). Some of them have been reported to possess numerous bioactivities, including nerve growth factor (NGF)-like effects (Gao 2006), cytotoxicity against HeLa cells (Kawagishi et al. 1990) and protective activity against endoplasmic reticulum stress-dependent Neuro-2a cell death (Ueda et al. 2008). In the course of our continuing search for bioactive metabolites from this mushroom, chemical investigations of the mycelia of H. erinaceum were carried out, leading to the isolation and identification of seven compounds 1 –7. Compounds 1 – 6 were tested for their antioxidant and antifungal activities.
2. Results and discussion The EtOAc-soluble extract of the mycelia of H. erinaceum was subjected to column chromatography (CC) over silica gel, followed by purification on Sephadex LH-20,
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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reversed-phase silica gel CC and preparative thin-layer chromatography (PTLC) to yield seven secondary metabolites 1 –7 (Figure 1). These metabolites were identified by analysing their spectroscopic data (MS and NMR data) (see supplementary material) and by comparing with the literature data, which are 12b-hydroxyverruculogen TR-2 (1) (Li et al. 2012), fumitremorgin C (2) (Li et al. 2012), pseurotin A (3) (Li et al. 2011, 2012), FD-838 (4) (Hayashi et al. 2009; Yamada et al. 2010), methylthiogliotoxin (5) (Lee et al. 2001; Li et al. 2011), cerevisterol (6) (Gao et al. 2001) and herierin IV (7) (Qian et al. 1990). Compounds 1– 5 are ubiquitous diketopiperazines commonly found in nutrient-rich cultures of both terrestrial and marine fungi, most notably Aspergillus and Penicillium species (Li et al. 2012); compound 6 has been relatively frequently encountered in mushrooms (Gao 2006). To our knowledge, this is the first report of the isolation of 1 –5 from the genus Hericium. In the course of discovering the natural radical-scavenging and antifungal agents from edible mushroom, six isolated metabolites were tested for antioxidant activity against DPPH radical (2,2diphenyl-1-picrylhydrazyl), and the results are presented in Table S1. Of the tested compounds, 1, 3 and 6, were found to exhibit significant radical-scavenging activities with IC50 values of 12.56, 12.56 and 11.38 mM, respectively, as compared with the two positive controls, tertiary butylhydroquinone (TBHQ) and ascorbic acid, whilst the other compounds were all weak (IC50 . 50 mM). Furthermore, the six metabolites were also tested in vitro for the antifungal activity against the phytopathogens Fusarium graminearum, Glomerella cingulate, Alternaria solani and Botrytis cinerea, and the results are listed in Table S2. Of the tested compounds, compounds 1, 2, 4 and 6 were demonstrated to exert significant antifungal activities towards the four pathogens with minimum inhibitory concentrations (MICs) of 6.25 –50 mM; 4 exhibited almost the best
HO
H3CO
OH
O
13 12 N
N H
O N
N H3CO
N
N H
O
O
OH 2
1 OH
O
O O
NH
HO O
OH
O
O
O
O
O OH
CH3
NH
O
O CH3
4
3 Me S H OH O
N N S OH 5
O
O Me
Me
HO HO
O
OH OH 6
Figure 1. Structures of compounds 1 – 7 from H. erinaceum.
7
OH
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antifungal activity against the four pathogens tested, in comparison to the positive control, carbendazim. Especially, 4 significantly inhibited the growth of B. cinerea and G. cingulate, with MICs of 6.25 mM for each fungus, similar to that of the control. This may be because the furan ring was highly sensitive to the two plant pathogens. Diketopiperazines such as 1 and 2 belong to a large group of compounds with different biological activities. Fumitremorgin C (2), a mycotoxin isolated from Aspergillus fumigatus, was described as a potent and selective BCRP (breast cancer resistance protein) inhibitor (Rabindran et al. 2000), and had antifungal effects on B. cinerea, Alternaria alternata, Colletotrichum gloeosporioides and Gibberella saubinetti (Li et al. 2012). Both pseurotin A (3) and FD-838 (4) were characterised structurally by their unusual 1-oxa-7-azaspiro[4.4]non-2ene-4,6-dione core skeleton. Pseurotin A (3) was previously isolated from the culture filtrate of Pseudeurotium ovalis and later from several species of Aspergillus (Blotch & Tamm 1976; Martı´nez-Luis et al. 2012; Shaaban et al. 2013), and was reported to be a competitive inhibitor of chitin synthase, an inducer of nerve-cell proliferation (Martı´nez-Luis et al. 2012) and an effective nematicidal agent of Bursaphelenchus xylophilus without any plant growth inhibition (Hayashi et al. 2007). FD-838 (4), isolated from A. fumigatus Fresenius F-838, inhibited the growth of certain Gram-positive bacteria and fungi (Hayashi et al. 2009). The pyranone 7 exhibited cytotoxicity towards HeLa cells (Kawagishi et al. 1992). This study first isolated and characterised seven metabolites 1– 7 from the mushroom H. erinaceum and revealed that this mushroom contains antioxidant and antifungal substances. 3. Experimental 3.1. General CC: silica gel (SiO2: 200– 300 mesh, Qingdao Marine Chemical Group Co., Qingdao, P.R. China); Sephadex LH-20 (Pharmacia Co., Stockholm, Sweden). Melting points were measured on an X-4 micromelting point apparatus. IR spectra: Bruker IFS-55 spectrophotometer (Bruker Corporation, Ettlingen, Germany). UV spectra: Shimadzu UV-2401A spectrophotometer (Shimadzu Corporation, Tokyo, Japan); 1H, 13C NMR and 2D NMR Spectra: Bruker AM-400 and Bruker AV 500 (Bruker Corporation, Fa¨llanden, Switzerland); d in ppm rel. to Me4Si as internal standard, J in Hz. 3.2. Fungal material and cultivation The fungal strain H. erinaceum H6 (accession no. DQ185914) was purchased from the Applied Research Institute of Microbiology, Academy of Agricultural Sciences, Xinjiang, China. It was identified based on 16S rDNA sequence analysis with 99% similarity by BeiJing Sunbiotech Co. and has been deposited at the College of Science, Northwest A&F University. The strain was inoculated into the medium (glucose 30 g, soluble starch 20 g, yeast extract 10 g, KH2PO4 1 g, MgSO4z7H2O 0.6 g, water 1000 mL, pH 5.5) in Erlenmeyer flasks and incubated for 14 days at 258C. 3.3. Extraction, isolation and purification The cultures (50 L) were filtrated and concentrated to give mycelia and then ultrasonically extracted three times with EtOAc, which was removed under reduced pressure to give a crude extract (22 g). The extract was fractionated by silica gel CC using CHCl3 – MeOH (10:0 to 2:1) gradient elution to provide fractions (Fr.1 –Fr.5). Fr.2 (CHCl3 – MeOH, 8:1) was chromatographed over silica gel (CHCl3 –MeOH, 10:1 –1:1) and then was subjected to repeated CC (Sephadex LH-20, reversed-phase column, silica gel) to yield compounds 1 (10 mg) and 2 (46 mg). Fr.3 (CHCl3 –MeOH, 6:1) was purified by CC on Sephadex LH-20 (MeOH), PTLC
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(petroleum ether –acetone, 1:1; CHCl3 – acetone, 2:1), and silica gel (CHCl3 – acetone, 10:1) to yield 3 (13 mg) and 4 (25 mg). Fr. 4 (CHCl3 – MeOH, 5:1) was subjected to CC on RP-18 silica gel (MeOH-H2O, 4:6) and Sephadex LH-20 (CHCl3 – MeOH, 1:1) to yield 5 (10 mg) and 6 (10 mg). Fr.5 (CHCl3 –MeOH, 2:1) was further purified by CC on Sephadex LH-20 (CHCl3 – MeOH, 1:1), PTLC (petroleum ether – EtOAc, 1:1; CHCl3 –acetone, 2:1) and Sephadex LH-20 (MeOH) to yield 7 (50 mg).
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3.4. Antifungal and antioxidant bioassays The test phytopathogenic fungi used in this study were F. graminearum, G. cingulate, A. solani and B. cinerea and determined by using the microbroth dilution assay (Li et al. 2012). All the fungi were isolated from infected plant parts at the Northwest A&F University. DPPH free radical-scavenging ability was measured to evaluate the antioxidant activity according to our reported method (Zheng et al. 2010). 4. Conclusion In conclusion, seven known compounds 1 –7 were isolated and characterised from H. erinaceum. The antioxidant and antifungal activities of compounds 1 –6 were tested. Compounds 1, 3 and 6 were found to exhibit potential antioxidant activity against DPPH radical, and 4 showed significant antifungal effects against two plant pathogens Botrytis cinerea and Glomerella cingulata. Compounds 1 –5 were first obtained from the genus Hericium. Supplementary material Supplementary material relating to this article is available online, alongside Tables S1 and S2. Acknowledgements This work is supported by the earmarked fund for National Key Technology R&D Program of the Ministry of Science and Technology of China (2012BAD36B04) and by the Program for New Century Excellent Talents in University (NCET-05-0852).
Note 1. Present address: Phytochemistry Laboratory, Xi’an Botanical Garden, Institute of Botany of Shaanxi Province, Xi’an 710061, P.R. China.
References Blotch P, Tamm C. 1976. Pseurotin, a new metabolite of Pseudeurotium ovalis Stolk having an unusual heterospirocyclic system. Helv Chim Acta. 59:133–137. Gao JM. 2006. New biologically active metabolites from Chinese higher fungi. Curr Org Chem. 10:849–871. Gao JM, Dong ZL, Liu JK. 2001. A new ceramide from basidiomycetes Russula cyanoxantha. Lipids. 36:175–180. Hayashi A, Fujioka S, Nukina M, Kawano T, Shimada A, Kimura Y. 2007. Fumoquinones A and B, nematocidal quinones produced by Aspergillus fumigatus. Biosci Biotechnol Biochem. 71:1607–1702. Hayashi Y, Sankar K, Ishikawa H, Nozawa Y, Mizoue K, Kakeya H. 2009. Total synthesis and determination of the absolute configuration of FD-838, a naturally occurring azaspirobicyclic product. Bioorg Med Chem Lett. 19:3863–3865. Kawagishi H, Ando M, Mizuno T. 1990. Hericenone A and B as cytotoxic principles from the mushroom of Hericium erinaceum. Tetrahedron Lett. 31:373–376. Kawagishi H, Shirai R, Sakamoto H. 1992. Erinapyrones A and B from the cultured mycelia of Hericium erinaceum. Chem Lett. 21:2475–2476. Lee HJ, Lee JH, Hwang BY, Kim HS, Lee JJ. 2001. Anti-angiogenic activities of gliotoxin and its methylthio-derivative, fungal metabolites. Arch Pharm Res. 24:397–401.
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Li HQ, Li XJ, Wang YL, Zhang Q, Zhang AL, Gao JM, Zhang XC. 2011. Antifungal metabolites from Chaetomium globosum, an endophytic fungus in Ginkgo biloba. Biochem Systemat Ecol. 39:876–879. Li XJ, Zhang Q, Zhang AL, Gao JM. 2012. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with melia azedarach, and their antifungal, antifeedant, and toxic activities. J Agric Food Chem. 60:3424–3431. Ma BJ, Shen JW, Yu HY, Yuan R, Wu TT, Zhao X. 2010. Hericenones and erinacines: stimulators of nerve growth factor (NGF) biosynthesis in Hericium erinaceus. Mycology. 1:92–98. Martı´nez-Luis S, Cherigo L, Arnold E, Spadafora C, Gerwick WH, Cubilla-Rios L. 2012. Antiparasitic and anticancer constituents of the endophytic fungus Aspergillus sp. strain F1544. Nat Prod Commun. 7:165–168. Qian FG, Xu GY, Du S J. 1990. Isolation and identification of two new pyrone compounds from the culture of Hericium erinaceum. Yaoxue Xuebao. 25:522– 525. Rabindran S, Ross D, Doyle L, Yang W, Greenberger L. 2000. Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein. Cancer Res. 60:47–50. Shaaban M, Magdy El-Metwally M, Nasr H. 2013. A new diketopiperazine alkaloid from Aspergillus oryzae. Nat Prod Res, doi:10.1080/14786419.2013.841687. Ueda K, Tsujimori M, Kodani S, Chiba A, Kubo M, Masuno K, Sekiya A, Nagai K, Kawagishi H. 2008. An endoplasmic reticulum (ER) stress-suppressive compound and its analogues from the mushroom Hericium erinaceum. Bioorg Med Chem. 16:9467–9470. Yamada T, Kitada H, Kajimoto T, Numata A, Tanaka R. 2010. The relationship between the CD cotton effect and the absolute configuration of FD-838 and its seven stereoisomers. J Org Chem. 75:4146–4153. Zheng CD, Li G, Li HQ, Xu XJ, Gao JM, Zhang AL. 2010. DPPH-scavenging activities and structure– activity relationships of phenolic compounds. Nat Prod Commun. 5:1759–1765.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Bioactive metabolites from the mycelia of the basidiomycete Hericium erinaceum a
a
a
Qiang-Qiang Lu , Jun-Mian Tian , Jing Wei & Jin-Ming Gao
a
a
Department of Applied Chemistry, College of Science, Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, Northwest A&F University, Yangling 712100, Shaanxi, P.R. China Published online: 18 Mar 2014.
To cite this article: Qiang-Qiang Lu, Jun-Mian Tian, Jing Wei & Jin-Ming Gao (2014) Bioactive metabolites from the mycelia of the basidiomycete Hericium erinaceum, Natural Product Research: Formerly Natural Product Letters, 28:16, 1288-1292, DOI: 10.1080/14786419.2014.898145 To link to this article: http://dx.doi.org/10.1080/14786419.2014.898145
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2014 Vol. 28, No. 16, 1288–1292, http://dx.doi.org/10.1080/14786419.2014.898145
SHORT COMMUNICATION Bioactive metabolites from the mycelia of the basidiomycete Hericium erinaceum Qiang-Qiang Lu1, Jun-Mian Tian, Jing Wei and Jin-Ming Gao* Department of Applied Chemistry, College of Science, Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, Northwest A&F University, Yangling 712100, Shaanxi, P.R. China
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(Received 9 December 2013; final version received 16 February 2014) Seven known compounds, three diketopiperazine alkaloids, 12b-hydroxyverruculogen TR-2 (1), fumitremorgin C (2) and methylthiogliotoxin (5), two hetero-spirocyclic glactam alkaloids, pseurotin A (3) and FD-838 (4), and cerevisterol (6) and herierin IV (7), were isolated from the mycelia of the basidiomycete Hericium erinaceum and identified by spectroscopic analyses. The antioxidant and antifungal activities of compounds 1 – 6 were evaluated. The results indicated that compounds 1, 3 and 6 exhibited potential antioxidant activity against DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical with their IC50 data of ca. 12 mM, compared with positive control tertiary butylhydroquinone. In addition, compound 4 significantly inhibited the growth of two plant fungal pathogens Botrytis cinerea and Glomerella cingulata with an minimum inhibitory concentration of 6.25 mM for each, similar to that of the positive fungicide, carbendazim. Compounds 1 – 5 were isolated from the genus Hericium for the first time. Keywords: Hericium erinaceum; mushroom; diketopiperazine alkaloids; antioxidant activity; antifungal activity
1. Introduction Higher fungi have long been considered as an important source that produce a large and diverse variety of secondary metabolites (Gao 2006). Hericium erinaceum (Pers.) is known as an edible mushroom (family Hericiaceae) that has been used as medicine or food in China, Japan and Europe without harmful effects. Chemical studies of H. erinaceum have produced a variety of secondary metabolites, such as cyathane diterpenoids, irregular terpenoids, pyranones and sterols (Gao 2006; Ma et al. 2010; Ueda et al. 2008). Some of them have been reported to possess numerous bioactivities, including nerve growth factor (NGF)-like effects (Gao 2006), cytotoxicity against HeLa cells (Kawagishi et al. 1990) and protective activity against endoplasmic reticulum stress-dependent Neuro-2a cell death (Ueda et al. 2008). In the course of our continuing search for bioactive metabolites from this mushroom, chemical investigations of the mycelia of H. erinaceum were carried out, leading to the isolation and identification of seven compounds 1 –7. Compounds 1 – 6 were tested for their antioxidant and antifungal activities.
2. Results and discussion The EtOAc-soluble extract of the mycelia of H. erinaceum was subjected to column chromatography (CC) over silica gel, followed by purification on Sephadex LH-20,
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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reversed-phase silica gel CC and preparative thin-layer chromatography (PTLC) to yield seven secondary metabolites 1 –7 (Figure 1). These metabolites were identified by analysing their spectroscopic data (MS and NMR data) (see supplementary material) and by comparing with the literature data, which are 12b-hydroxyverruculogen TR-2 (1) (Li et al. 2012), fumitremorgin C (2) (Li et al. 2012), pseurotin A (3) (Li et al. 2011, 2012), FD-838 (4) (Hayashi et al. 2009; Yamada et al. 2010), methylthiogliotoxin (5) (Lee et al. 2001; Li et al. 2011), cerevisterol (6) (Gao et al. 2001) and herierin IV (7) (Qian et al. 1990). Compounds 1– 5 are ubiquitous diketopiperazines commonly found in nutrient-rich cultures of both terrestrial and marine fungi, most notably Aspergillus and Penicillium species (Li et al. 2012); compound 6 has been relatively frequently encountered in mushrooms (Gao 2006). To our knowledge, this is the first report of the isolation of 1 –5 from the genus Hericium. In the course of discovering the natural radical-scavenging and antifungal agents from edible mushroom, six isolated metabolites were tested for antioxidant activity against DPPH radical (2,2diphenyl-1-picrylhydrazyl), and the results are presented in Table S1. Of the tested compounds, 1, 3 and 6, were found to exhibit significant radical-scavenging activities with IC50 values of 12.56, 12.56 and 11.38 mM, respectively, as compared with the two positive controls, tertiary butylhydroquinone (TBHQ) and ascorbic acid, whilst the other compounds were all weak (IC50 . 50 mM). Furthermore, the six metabolites were also tested in vitro for the antifungal activity against the phytopathogens Fusarium graminearum, Glomerella cingulate, Alternaria solani and Botrytis cinerea, and the results are listed in Table S2. Of the tested compounds, compounds 1, 2, 4 and 6 were demonstrated to exert significant antifungal activities towards the four pathogens with minimum inhibitory concentrations (MICs) of 6.25 –50 mM; 4 exhibited almost the best
HO
H3CO
OH
O
13 12 N
N H
O N
N H3CO
N
N H
O
O
OH 2
1 OH
O
O O
NH
HO O
OH
O
O
O
O
O OH
CH3
NH
O
O CH3
4
3 Me S H OH O
N N S OH 5
O
O Me
Me
HO HO
O
OH OH 6
Figure 1. Structures of compounds 1 – 7 from H. erinaceum.
7
OH
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antifungal activity against the four pathogens tested, in comparison to the positive control, carbendazim. Especially, 4 significantly inhibited the growth of B. cinerea and G. cingulate, with MICs of 6.25 mM for each fungus, similar to that of the control. This may be because the furan ring was highly sensitive to the two plant pathogens. Diketopiperazines such as 1 and 2 belong to a large group of compounds with different biological activities. Fumitremorgin C (2), a mycotoxin isolated from Aspergillus fumigatus, was described as a potent and selective BCRP (breast cancer resistance protein) inhibitor (Rabindran et al. 2000), and had antifungal effects on B. cinerea, Alternaria alternata, Colletotrichum gloeosporioides and Gibberella saubinetti (Li et al. 2012). Both pseurotin A (3) and FD-838 (4) were characterised structurally by their unusual 1-oxa-7-azaspiro[4.4]non-2ene-4,6-dione core skeleton. Pseurotin A (3) was previously isolated from the culture filtrate of Pseudeurotium ovalis and later from several species of Aspergillus (Blotch & Tamm 1976; Martı´nez-Luis et al. 2012; Shaaban et al. 2013), and was reported to be a competitive inhibitor of chitin synthase, an inducer of nerve-cell proliferation (Martı´nez-Luis et al. 2012) and an effective nematicidal agent of Bursaphelenchus xylophilus without any plant growth inhibition (Hayashi et al. 2007). FD-838 (4), isolated from A. fumigatus Fresenius F-838, inhibited the growth of certain Gram-positive bacteria and fungi (Hayashi et al. 2009). The pyranone 7 exhibited cytotoxicity towards HeLa cells (Kawagishi et al. 1992). This study first isolated and characterised seven metabolites 1– 7 from the mushroom H. erinaceum and revealed that this mushroom contains antioxidant and antifungal substances. 3. Experimental 3.1. General CC: silica gel (SiO2: 200– 300 mesh, Qingdao Marine Chemical Group Co., Qingdao, P.R. China); Sephadex LH-20 (Pharmacia Co., Stockholm, Sweden). Melting points were measured on an X-4 micromelting point apparatus. IR spectra: Bruker IFS-55 spectrophotometer (Bruker Corporation, Ettlingen, Germany). UV spectra: Shimadzu UV-2401A spectrophotometer (Shimadzu Corporation, Tokyo, Japan); 1H, 13C NMR and 2D NMR Spectra: Bruker AM-400 and Bruker AV 500 (Bruker Corporation, Fa¨llanden, Switzerland); d in ppm rel. to Me4Si as internal standard, J in Hz. 3.2. Fungal material and cultivation The fungal strain H. erinaceum H6 (accession no. DQ185914) was purchased from the Applied Research Institute of Microbiology, Academy of Agricultural Sciences, Xinjiang, China. It was identified based on 16S rDNA sequence analysis with 99% similarity by BeiJing Sunbiotech Co. and has been deposited at the College of Science, Northwest A&F University. The strain was inoculated into the medium (glucose 30 g, soluble starch 20 g, yeast extract 10 g, KH2PO4 1 g, MgSO4z7H2O 0.6 g, water 1000 mL, pH 5.5) in Erlenmeyer flasks and incubated for 14 days at 258C. 3.3. Extraction, isolation and purification The cultures (50 L) were filtrated and concentrated to give mycelia and then ultrasonically extracted three times with EtOAc, which was removed under reduced pressure to give a crude extract (22 g). The extract was fractionated by silica gel CC using CHCl3 – MeOH (10:0 to 2:1) gradient elution to provide fractions (Fr.1 –Fr.5). Fr.2 (CHCl3 – MeOH, 8:1) was chromatographed over silica gel (CHCl3 –MeOH, 10:1 –1:1) and then was subjected to repeated CC (Sephadex LH-20, reversed-phase column, silica gel) to yield compounds 1 (10 mg) and 2 (46 mg). Fr.3 (CHCl3 –MeOH, 6:1) was purified by CC on Sephadex LH-20 (MeOH), PTLC
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(petroleum ether –acetone, 1:1; CHCl3 – acetone, 2:1), and silica gel (CHCl3 – acetone, 10:1) to yield 3 (13 mg) and 4 (25 mg). Fr. 4 (CHCl3 – MeOH, 5:1) was subjected to CC on RP-18 silica gel (MeOH-H2O, 4:6) and Sephadex LH-20 (CHCl3 – MeOH, 1:1) to yield 5 (10 mg) and 6 (10 mg). Fr.5 (CHCl3 –MeOH, 2:1) was further purified by CC on Sephadex LH-20 (CHCl3 – MeOH, 1:1), PTLC (petroleum ether – EtOAc, 1:1; CHCl3 –acetone, 2:1) and Sephadex LH-20 (MeOH) to yield 7 (50 mg).
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3.4. Antifungal and antioxidant bioassays The test phytopathogenic fungi used in this study were F. graminearum, G. cingulate, A. solani and B. cinerea and determined by using the microbroth dilution assay (Li et al. 2012). All the fungi were isolated from infected plant parts at the Northwest A&F University. DPPH free radical-scavenging ability was measured to evaluate the antioxidant activity according to our reported method (Zheng et al. 2010). 4. Conclusion In conclusion, seven known compounds 1 –7 were isolated and characterised from H. erinaceum. The antioxidant and antifungal activities of compounds 1 –6 were tested. Compounds 1, 3 and 6 were found to exhibit potential antioxidant activity against DPPH radical, and 4 showed significant antifungal effects against two plant pathogens Botrytis cinerea and Glomerella cingulata. Compounds 1 –5 were first obtained from the genus Hericium. Supplementary material Supplementary material relating to this article is available online, alongside Tables S1 and S2. Acknowledgements This work is supported by the earmarked fund for National Key Technology R&D Program of the Ministry of Science and Technology of China (2012BAD36B04) and by the Program for New Century Excellent Talents in University (NCET-05-0852).
Note 1. Present address: Phytochemistry Laboratory, Xi’an Botanical Garden, Institute of Botany of Shaanxi Province, Xi’an 710061, P.R. China.
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