Activity of Armillarisin B in Vitro Against Plant Pathogenic Fungi

Activity of Armillarisin B in vitro against Plant Pathogenic Fungi Jin-Wen Shena, Bing-Ji Mab,*, Wen Lib, Hai-You Yua, Ting-Ting Wua, and Yuan Ruanb a College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, P. R. China b Department of Traditional Chinese Medicine, Agronomy College of Henan Agricultural University, Zhengzhou 450002, P. R. China. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 64 c, 790 – 792 (2009); received August 6, 2009 The methanolic extract of the fruiting bodies of the mushroom Armillariella tabescens was found to show antifungal activity against Gibberella zeae. The active compound was isolated from the fruiting bodies of A. tabescens by bioassay-guided fractionation of the extract and identiﬁ ed as armillarisin B. Armillarisin B eventually corresponds to 2-hydroxy-2- phenylpropanediamide and its structure was conﬁ rmed on the basis of spectroscopic studies including 2D NMR experiments. Key words: Armillariella tabescens, Armillarisin B, Antifungal Activity

Introduction by bioassay-guided fractionation of the extract and identifi ed as armillarisin B. In this report, we Research during the last decade has convinc- describe the isolation, structural elucidation, and ingly shown that natural products isolated from antifungal activity of armillarisin B. mushrooms play an important role, not only in pharmacology, but also in agriculture, as a rich Results and Discussion source of bioactive components that can be used in crop protection (Wink, 1993; Luo et al., 2005). The MeOH extract of A. tabescens showed Strobilurin A and oudemansin A are fungicidal fungitoxic activity against Gibberella zeae, Col- natural products found in the basidiomycete fungi letotrichum ophiopogonis and Gloesporum fructi- Strobilurus tenacellus (Pers. Ex Fr.) Singer (Anke genum. Gibberella zeae was the most sensitive to et al., 1977) and Oudemansiella mucida Hoehn this fungal extract, and the active principle was (Musilek et al., 1969), respectively. The mush- armillarisin B. Armillarisin B showed a strong an- rooms, one of the main biological resources, are tifungal effect on mycelial growth of Gibberella still an unexplored source for new agricultural zeae. However, it just exhibited weak antifungal chemicals. activity against the other two plant pathogenic Armillariella tabescens is a mushroom belong- fungi. ing to the family Tricholomataceae which is also Armillarisin B was fi rst isolated in China by called “Luminous Fungus” in China. A. tabescens “Luminous Fungus” Cooperative Research Group has been used for the treatment of cholecystitis in of Kiangsu (1977) and proposed to be 2-hydroxy- traditional Chinese medicine, and armillarisin A 2-phenylpropanediamide; however, its accurate (3-acetyl-5-hydroxymethyl-7-hydroxycoumarin) chemical structure has not yet been elucidated is the active component for curing cholecystitis owing to the defi ciency of spectroscopic data. In (Wang et al., 2007). In continuation of our stud- our experiment, armillarisin B was obtained as ies on basidiomycete-derived bioactive secondary white needles. Its HRESI-mass spectrum gave metabolites employed as antifungal agents. The an ion [M+Na]+ peak at m/z 217.0605 (calcd.

methanolic extract of the fruiting bodies of the for C9H10N2O3Na 217.0589) and corresponded mushroom A. tabescens was found to show anti- to a molecular formula of C9H10N2O3, requiring fungal activity against Gibberella zeae, Colletotri- six degrees of unsaturation. The IR spectrum of –1 chum ophiopogonis and Gloesporum fructigenum. armillarisin B showed peaks of -NH2 (3436 cm ), –1 –1 The active compound against Gibberella zeae was -OH (3352 cm ), -CONH2 (1680 cm ), and mono- isolated from the fruiting bodies of A. tabescens substituted benzene (1580, 1452, 763, 692 cm–1).

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1 13 Table I. H and C NMR (in C5D5N) data of armillarisin CONH2 B.

Position δC δH C OH 1, 3 174.2 2 80.2 1’ 142.1 2’, 4’ 128.6 8.22 (1H, dd, 1.4, 7.2) CONH2 3’, 5’ 126.8 7.35 (m)

-NH2 9.03 (br.s) Fig. 1. Key HMBC correlations of armillarisin B.

The 1H NMR (Table I) spectrum of armillarisin People’s Republic of China, in July 2008. The B showed signals of ﬁ ve aromatic H atoms at fungal identiﬁ cation was made by Prof. Jin-Wen

δH 8.22 (2H, dd, J = 1.4, 7.2 Hz, H-2’, 6’), δH 7.35 Shen. A dried specimen was deposited in the

(2H, m, H-3’, 5’), and δH 7.25 (1H, m, H-4’), and Herbarium of Henan Agricultural University, 13 an -NH2 group at δH 9.03 (br.s). The C NMR Zhengzhou, P. R. China. (Table I) spectrum of armillarisin B showed six

signals in all including three doublets at δC 174.2 Antifungal activity of A. tabescens and

(s), δC 128.6 (d) and δC 126.8 (d). The construction armillarisin B in vitro of the molecular framework was deduced from 400 g air-dried fruiting bodies of A. tabescens 1H,1H COSY, HMQC, and HMBC experiments. were immersed in MeOH and left at room tem- The HMBC correlations C (2) (δ 80.2)/H-C (2’, C perature for 48 h. Then the MeOH extract was 6’) (δ 8.22), C (2) (δ 80.2)/H -N (δ 9.03), and C H C 2 H decanted and evaporated. The MeOH extract (1’) (δ 142.1)/H-C (3’, 5’) (δ 7.35) were consist- C H dissolved in DMSO was tested for antifungal ac- ent with the structure shown in Fig. 1. Therefore, tivity in vitro by the poison food technique. Po- armillarisin B eventually corresponds to 2-hy- tato dextrose agar (PDA) medium was used as droxy-2-phenylpropanediamide. the medium for all test fungi. The test pathogenic fungi were Gibberella zeae, Colletotrichum ophi- Experimental opogonis, Gloesporum fructigenum, Fusarium General graminearum Schw., Alternaria alternata f. sp. UV spectra were recorded on a Shimadzu Mali, Colletotrichum gloeosporioides (Penz.) Sacc. UV-2401PC spectrophotometer. IR spectra were and Sclerotinia sclerotiorum. measured on a Bio-Rad FTS-135 spectrometer The MeOH extract at a concentration of 20 mg/ using KBr pellets. NMR spectra were recorded on ml (DMSO content 1%) was inoculated at the Bruker AM-400 and DRX-500 spectrometers in centre of agar discs containing the test fungi (4 mm diameter). Three replicate plates for each C5D5N, with TMS as the internal standard. Mass spectra were recorded on VG Auto-spec-3000 and fungus were included. After incubation for 2 ~ 6 d, API QSTAR Pulsar spectrometers (Manchester, until the fungal growth in the control dishes was UK). almost complete, the mycelial growth of fungi Silica gel (200 – 300 mesh, Qingdao Marine (mm) in both treated (T) and control (C) Petri Chemical Inc., China) and reversed-phase silica dishes was measured diametrically in three differ- gel (Fuji Silica Chemical Ltd., Kasugai, Japan) ent direction. The percentage of growth inhibition were used for column chromatography. Fractions (I) was calculated using the following formula were monitored by TLC, and spots were visual- (Sztejnberg et al., 1983): ized by heating the silica gel plates sprayed with I (%) = [(C – T)/C] · 100. 10% H2SO4 in ethanol. The MeOH extract was found to show fungitoxic activity against Gibberella zeae, Colletotri- Mushroom material chum ophiopogonis and Gloesporum fructigenum. The fresh fruiting bodies of A. tabescens were The percentages of growth inhibition were 81.9%, collected at Funiu Mountain of Henan Province, 65.3% and 66.5%, respectively, at 20 mg/ml.

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In order to isolate the active principle against Armillarisin B showed a signiﬁ cant antifungal Gibberella zeae, the MeOH extract (20 g) was effect on mycelial growth of Gibberella zeae, and fractionated by column chromatography (silica the percentage of growth inhibition was 78.7% at gel) and eluted with petroleum ether, petroleum 100 μg/ml, in vitro.

ether/acetone (9:1, 8:2, v/v), CHCl3/MeOH (9:1, 8:2, v/v) and MeOH to give six fractions. The Physicochemical properties fraction (1.8 g) eluted with CHCl3/MeOH (9:1, White needles, m.p. 162 – 164 ºC. – UV (EtOH): v/v) was conﬁ rmed by the mycelial growth inhi- λ = 250.5, 256.5, 263.0 nm. – IR (KBr): ν = 3436, bition test in vitro to be the active fraction, with 3352, 1680, 1580, 1452, 1127, 763, 692 cm–1. – 1H a growth inhibition 76.3% at 1 mg/ml. Then the and 13C NMR: see Table I. – HRESI-MS: m/z = active fraction was submitted for further puriﬁ ca- + 217.0605 [M+Na] (calcd. for C9H10N2O3Na m/z = tion to reversed-phase column chromatography 217.0589). (RP-8, MeOH/H2O 85:15) to give a pure compound (90 mg), namely armillarisin B.

Anke T., Oberwinkler F., Steglich W., and Schramm G. omycete Oudemansiella mucida. Folia Microbiol. 14, (1977), The strobilurins – new antifungal antibiotics 377 – 387. from the basidiomycete Strobilurus tenacellus. J. An- Sztejnberg A., Azaizia H., and Chet I. (1983), The pos- tibiot. 30, 806 – 810. sible role of phenolic compounds in resistance of Chemical Section, “Luminous Fungus” Cooperative Re- horticulture crops to Dematophora necatrix Hartig. search Group of Kiangsu (1974), Studies on Armil- Phytopathology 107, 318 – 326. lariella tabescens II. The isolation and determination Wang Y., Li P., Tang Y., Fawcett J. P., and Gu J. (2007), of the structures of armillarisins. Acta Microbiol. Sin. Quantitation of armillarisin A in human plasma by liq- 14, 9 – 16. uid chromatography-electrospray tandem mass spec- Luo D. Q., Wang F., Bian X. Y., and Liu J. K. (2005), Ru- trometry. J. Pharm. Biomed. Anal. 43, 1860 – 1863. fuslactone, a new antifungal sesquiterpene from the Wink M. (1993), Production and application of phy- fruiting bodies of the basidiomycete Lactarius rufus. tochemicals from an agricultural perspective. In: J. Antibiot. 58, 456 – 459. Phytochemistry and Agriculture (van Beek T. A. Musilek V., Cerna J., Sasek V., Semerdziev M., and Von- and Breteler H., eds.). Clarendon Press, Oxford, pp. dracek M. (1969), Antifungal antibiotic of the basidi- 171 – 212.

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