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Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon
Fitoterapia 75 (2004) 451–456
Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon Y.M. Maa,b, Y. Lia, J.Y. Liua, Y.C. Songa, R.X. Tana,* a
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China b College of Forestry, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, PR China Received 24 September 2003; accepted 31 March 2004
Abstract A new benzophenone, named rhizoctonic acid (1), together with three known compounds monomethylsulochrin (2), ergosterol (3) and 3b,5a,6b-trihydroxyergosta-7,22-diene (4) were isolated through bioassay-guided fractionations from the culture of Rhizoctonia sp. (Cy064), an endophytic fungus in the leaf of Cynodon dactylon. The structure of the new acid 1 was elucidated to be 5-hydroxy-2-(2-hydroxy-6-methoxy-4-methylbenzoyl)-3-methoxybenzoic acid by a combination of spectral analyses. Furthermore, the structure of monomethylsulochrin 2 was confirmed by 13C-NMR analysis. All four metabolites were subjected to a more detailed in vitro assessment of their antibacterial action against five clinically isolated and one reference (ATCC 43504) Helicobacter pylori strains. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Rhizoctonia sp.; Cynodon dactylon; Endophyte; Rhizoctonic acid; Anti-Helicobacter pylori
1. Introduction Endophytes commonly present in almost all plants are frequently considered a rich source of bioactive metabolites w1x. As a follow-up to our previous communi*Corresponding author. Tel.: q86-25-3592945; fax: q86-25-3302728. E-mail address: [email protected] (R.X. Tan). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.03.007
452
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456
cations describing the bioactivity and chemical structures of endophyte metabolites w2–5x, we investigated the methanol extract of the solid-substrate culture of Rhizoctonia sp. (Cy064) harboring in the healthy elder leaf of Cynodon dactylon (Gramineae) used locally for treating hepatitis w6x. With fractionations directed by anti-Helicobacter pylori test, four anti-H. pylori compounds were characterized from the extract. We hereby wish to report the structure elucidation of the new metabolite 1, named rhizoctonic acid, and the anti-H. pylori action of all four isolates. 2. Experimental 2.1. Source of Rhizoctonia sp. Rhizoctonia sp. is an endophytic fungus isolated from surface-sterilized fresh leaves of an apparently healthy C. dactylon collected in November 2001 in Jiangsu Province, China, according to the method detailed elsewhere w2x. The fungus (strain Cy064) was identified by Dr. Y.C. Song via microscopic morphology w7x and a reference living culture was kept at Institute of Functional Biomolecules, PR China. The mycelium hyaline of the fungus was dark, cells of mycelium long, septa of branches set off from the main hyphae, asexual fruit bodies and conidia absent, sporodochium-like bodies and chlamydospore-like cells in chains produced in the species, sclerotia light colored. These morphological characteristics led to the identification of Cy064 as Rhizoctonia sp. 2.2. Cultivation The fresh mycelium grown on PDA medium at 28 8C for 5 days was inoculated into 1000 ml Erlenmeyer flasks containing 300 ml PD medium. After 2 days of incubation at 28 8C on rotary shaker at 150 rev.ymin, a 20 ml culture liquid was transferred as seed into each of 200–250 ml Erlenmeyer flasks containing 7.5 g grain, 7.5 g bran, 0.5 g yeast, 0.1 g sodium tartrate, 0.01 g FeSO4 7H2O, 0.1 g sodium glutamate, 0.1 ml pure corn oil and 30 ml water. The cultivation was kept for 40 days at 28 8C. 2.3. Extraction and isolation The 3 kg air-dried solid culture was extracted exhaustively with MeOH. The extract was concentrated in vacuo to give a residue (55.5 g), which was Si-gel CC eluting with petroleum ether-acetone (25:1, 8 l) and chloroform-MeOH gradient (0:1, 7.0 l; 80:1, 6.5 l; 40:1, 6.0 l; 20:1, 6.5 l; 10:1, 6.5 l; 1:1, 6.0 l; 0:1, 5.5 l) to give eight fractions (F-1, 21.8 g; F-2, 2.8 g; F-3, 2.6 g; F-4, 3.3 g; F-5, 3.5 g; F-6, 3.0 g; F-7, 9.8 g; F-8, 4.6 g). F-2, F-3 and F-6 were shown to be anti-H. pylori fractions. Repeated chromatography of the bioactive fractions (F-2, F-3 and F-6) on silica gel with gradient of MeOH in chloroform and Sephadex LH-20 with chloroform:MeOH (1:1) afforded 2 (60 mg) from F-3, 3 (15 mg) from F-2, 1 (9 mg) and 4 (8 mg) from F-6.
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2.4. Examination of the sterile medium for presence of the compounds 1–4 The sterile medium was extracted following exactly the procedure as with that of the solid-substrate culture. LC-MS comparisons demonstrated that the medium did not contain any of the fungal metabolites 1–4. 2.5. Anti-Helicobacter pylori bioassay Bacterial strains: a reference strain (ATCC 43504) and five randomly selected clinical strains from antral biopsies from children and adults were used in this study. Growth inhibition assay of H. pylori was performed according to agar dilution method w8x. Each of the four isolated fungal metabolites was dissolved at 5 mgyml in DMSO and diluted with sterile distilled water to obtain a series of stock solutions at given concentrations. Every stock solution (1 ml) was added into a Petri dish containing 7 ml of unsolidified columbia agar base supplemented with 7% horse serum. Final concentrations of each compound in the medium were set to be 40.0, 35.0, 30.0, 25.0, 20.0, 15.0, 10.0, 5.0 and 2.5 mgyml with the pre-used DMSO concentration in the medium kept -1%. The H. pylori strains taken from the seed cultures was shortly diluted with sterile medium to approximately 1=108 CFUyml followed by immediate inoculation into the agar plates which were cultured microaerobically for 3 days at 37 8C in anaerobic jar (containing: 85% N2, 10% CO2 and 5% O2). The MICs were determined by judging visually the H. pylori growth. Ampicillin was co-assessed as a positive control at concentrations of 8.0, 4.0, 2.0, 1.0, 0.5, 0.25 and 0.125 mgyml. All experiments were conducted in triplicate. 2.6. Rhizoctonic acid (1) 5-Hydroxy-2-(2-hydroxy-6-methoxy-4-methylbenzoyl)-3-methoxybenzoic acid (1) yellowish crystals (CHCl3 yMeOH, 1:1), mp 230 8C (dec.); IR bands (KBr): 3322, 3014, 2965, 2941, 1719, 1680, 1612,1574, 1505, 1465, 1411, 1220, 1108, 1060, 912, 817 and 722 cmy1; HRESI-MS (negative mode) myz: 331.0835 (calcd. for C17H16O7-H 331.0818); 1H and 13C-NMR data: see Table 1. 2.7. Monomethylsulochrin (2) Yellowish crystals (CHCl3 yacetone, 1:1), mp 197–198 8C; 1H-NMR (500 MHz, CDCl3): d 6.99 (1H, d, J 2.0 Hz, H-6), 6.58 (1H, d, J 2.0 Hz, H-4), 6.45 (1H, br s, H-39), 6.06 (1H, br s H-59), 3.67 (3H, s, COOCH3 ), 3.66 (3H, s, H-8), 3.36 (3H, s, H-99), 2.28 (3H, s, H-79); 13C-NMR (125 MHz, CDCl3): d 199.8 (C-89), 166.3 (C-7), 164.2 (C-29), 160.9 (C-69), 157.0 (C-5), 156.5 (C-3), 148.1(C-49), 128.3 (C-1), 127.6 (C-2), 110.9 (C-19), 110.4 (C-39), 107.9 (C-6), 103.2 (C-59), 102.9 (C-4), 56.1 (C-8), 55.6 (C-99), 52.2 (COOCH3), 22.5 (C-79).
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456
454 Table 1 1 H- and
13
C-NMR data of rhizoctonic acid (1) (DMSO-d6)
Carbon
dC
1 2 3 4 5 6 7 8 19 29 39 49 59 69 79 89 99 5-OH 29-OH
130.2 126.7 157.2 103. 6 158.7 108.4 167.7 56.8 111.0 164.2 110.9 148.5 104.3 161.7 22.7 200.8 56.7
*
dH*
6.64 (1H, d, Js2.0 Hz) 6.89 (1H, d, Js2.0 Hz)
HMBC H-4, H-6 H-4, H-8 HO-5, H-6 HO-5, H-6 H-4, HO-5 H-6
3.62 (3H, s) 6.36 (1H, br s) 6.25 (1H, br s) 2.25 (3H, s)
HO-29, H-39, H-59 HO-29, H-39 HO-29, H-59, H-79 H-79 H-39, H-79 H-59, H-99 H-39, H-59
3.33 (3H, s) 9.98 (1H, s) 13.08 (1H, s)
Assigned by DEPT and HMQC experiments.
3. Results and discussion In addition to known metabolites 2–4, a new compound 1 was afforded by repeated chromatography on Si-gel and Sephadex LH-20 of the methanol extract of the solid-substrate culture of Rhizoctonia sp. Cy064. LC-MS analyses of both culture and the substrate used showed that these four compounds were metabolites of the Rhizoctonia sp. Cy064 endophytic fungus. By comparing the melting points, MS, and 1H-NMR data with the published data, compounds 2–4 were readily identified as monomethylsulochrin w9x, ergosterol w10x and 3b,5a,6b-trihydroxyergosta-7,22-diene w11x, respectively. Furthermore, the structure of monomethylsulochrin 2 was confirmed by its 13C-NMR data, along with its 1H-NMR data. In the negative-mode high resolution electrospray ionization mass spectrum of 1, a quasi-molecular ion was displayed at myz 331.0835 wM-Hxcorresponding to the molecular formula C17H16O7 (calcd. for C17H15O7: 331.0818). This molecular formula was well consistent with the 17 carbon resonances in its 13 C-NMR spectrum (Table 1). The IR spectrum of 1 showed typical absorption bands at 3322, 1719 and 1686 cmy1 indicative of hydroxyl and carbonyl groups, respectively. The 1H-NMR spectrum of 1 gave an aromatic methyl singlet at d 2.25, two methoxyl singlets at d 3.33 and d 3.62, four benzene hydrogen signals at d 6.25, 6.36, 6.64 and 6.89 and two hydroxyl protons at d 9.98 and d 13.08, being hydrogen-bonded to an ortho-carbonyl group presumably through a six-membered ring w12x. These 1H-NMR spectral data were similar to those of monomethylsulochrin 2. However, a methoxy singlet at d 3.67 in the 1 H-NMR spectrum of 2 was missing
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456
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in that of 1, suggesting that 1 could be a demethyl derivative of monomethylsulochrin 2. This hypothesis was further confirmed by the absence of an ester methoxy signal around d 52 in the 13C-NMR spectrum of 1. This observation demonstrated that compound 1 was an acid which gave a positive result of bromocresol green test. In the HMBC spectrum of 1, a correlation of H-6 with C-7 demonstrated the position of the carboxylic group. Furthermore, HMBC correlations between H-8 and C-3 as well as between H-99 and C-69 required the formulated connections of the two methoxy groups. In addition, the presence of a methyl at C-49 was confirmed by the HMBC correlations of C-79 to both H-39 and H-59. On the basis of spectral data 1 was determined as 5-hydroxy-2-(2-hydroxy-6methoxy-4-methylbenzoyl)-3-methoxybenzoic acid. Both 1 and 2 are benzophenone derivatives a type of metabolites previously reported from the fungi belonging to the genera Aspergillus w9,13,14x, Penicillium w15x and Oospora. w16x. The present characterization of 1 and 2 from Rhizoctonia sp. suggested another fungal source of benzophenone analogs. The in vitro growth inhibition assay aganist H. pylori was carried out according to the agar dilution method w8x. The results indicated that the minimum inhibitory concentrations (MICs) of compounds 1–4 against all of the five clinical and a reference (ATCC 43504) strains were 25.0,10.0, 30.0, and 25.0 mgyml, respectively. The MIC of ampicillin used as the positive control against these strains was 2.0 mgyml.
Acknowledgments The work was co-financed by grants for RXT from National Natural Science Foundation of China (No.30171104) and from the Ministry of Science and Technology—National Marine 863 projects (Nos. 2001AA624010 and 2001AA620411). References w1x Tan RT, Zou WX. Nat Prod Rep 2001;48:448. w2x Lu H, Zou WX, Meng JC, Hu J, Tan RX. Plant Sci 2000;151:67.
456 w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x w15x w16x
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456 Zou WX, Meng JC, Lu H, Chen GX, Shi GX, Zhang TY, Tan RX. J Nat Prod 2000;63:1529. Liu JY, Liu CH, Zou WX, Tian X, Tan RX. Helv Chim Acta 2002;85:2664. Liu JY, Liu CH, Zou WX, Tan RX. Helv Chim Acta 2003;86:657. Xie ZW, Fan CS, Zhu ZY. Atlas of National Traditional Chinese Medicinal Herbs, Book II. 2nd ed. Beijing, PR China: People’s Hygiene Press, 1996. Barnett HL, Hunter BB. Illustrated Genera of Imperfect Fungi. 4th ed. Minnesota, USA: APS press, 1998. Bae EA, Han MJ, Kim NJ, Kim DH. Biol Pharm Bull 1999;21:990. Turner WB. J Chem Soc 1965;6658. Cushley RJ, Filipenko JD. Org Magn Res 1976;8:308. Takaishi Y, Und M, Ohashi T, Nakano K, Murakami K, Tomimatsu T. Phytochemistry 1991;30:4117. Shen CC, Syu WJ, Li SY, Lin CH, Lee GH, Sun CM. J Nat Prod 2002;65:1857. Inamori Y, Kato Y, Kubo M, Kamiki T, Takemoto T. Chem Pharm Bull 1983;31:4543. Kiriyama N, Nitta K, Sakaguchi Y, Taguchi Y, Yamamoto Y. Chem Pharm Bull 1977;25:2593. Mahmoodian A, Stickings CE. Biochem J 1964;92:369. Curtis RF, Harries PC, Levi JD, Phillips DM. J Chem Soc (C) 1966;168.
Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon Y.M. Maa,b, Y. Lia, J.Y. Liua, Y.C. Songa, R.X. Tana,* a
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China b College of Forestry, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, PR China Received 24 September 2003; accepted 31 March 2004
Abstract A new benzophenone, named rhizoctonic acid (1), together with three known compounds monomethylsulochrin (2), ergosterol (3) and 3b,5a,6b-trihydroxyergosta-7,22-diene (4) were isolated through bioassay-guided fractionations from the culture of Rhizoctonia sp. (Cy064), an endophytic fungus in the leaf of Cynodon dactylon. The structure of the new acid 1 was elucidated to be 5-hydroxy-2-(2-hydroxy-6-methoxy-4-methylbenzoyl)-3-methoxybenzoic acid by a combination of spectral analyses. Furthermore, the structure of monomethylsulochrin 2 was confirmed by 13C-NMR analysis. All four metabolites were subjected to a more detailed in vitro assessment of their antibacterial action against five clinically isolated and one reference (ATCC 43504) Helicobacter pylori strains. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Rhizoctonia sp.; Cynodon dactylon; Endophyte; Rhizoctonic acid; Anti-Helicobacter pylori
1. Introduction Endophytes commonly present in almost all plants are frequently considered a rich source of bioactive metabolites w1x. As a follow-up to our previous communi*Corresponding author. Tel.: q86-25-3592945; fax: q86-25-3302728. E-mail address: [email protected] (R.X. Tan). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.03.007
452
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456
cations describing the bioactivity and chemical structures of endophyte metabolites w2–5x, we investigated the methanol extract of the solid-substrate culture of Rhizoctonia sp. (Cy064) harboring in the healthy elder leaf of Cynodon dactylon (Gramineae) used locally for treating hepatitis w6x. With fractionations directed by anti-Helicobacter pylori test, four anti-H. pylori compounds were characterized from the extract. We hereby wish to report the structure elucidation of the new metabolite 1, named rhizoctonic acid, and the anti-H. pylori action of all four isolates. 2. Experimental 2.1. Source of Rhizoctonia sp. Rhizoctonia sp. is an endophytic fungus isolated from surface-sterilized fresh leaves of an apparently healthy C. dactylon collected in November 2001 in Jiangsu Province, China, according to the method detailed elsewhere w2x. The fungus (strain Cy064) was identified by Dr. Y.C. Song via microscopic morphology w7x and a reference living culture was kept at Institute of Functional Biomolecules, PR China. The mycelium hyaline of the fungus was dark, cells of mycelium long, septa of branches set off from the main hyphae, asexual fruit bodies and conidia absent, sporodochium-like bodies and chlamydospore-like cells in chains produced in the species, sclerotia light colored. These morphological characteristics led to the identification of Cy064 as Rhizoctonia sp. 2.2. Cultivation The fresh mycelium grown on PDA medium at 28 8C for 5 days was inoculated into 1000 ml Erlenmeyer flasks containing 300 ml PD medium. After 2 days of incubation at 28 8C on rotary shaker at 150 rev.ymin, a 20 ml culture liquid was transferred as seed into each of 200–250 ml Erlenmeyer flasks containing 7.5 g grain, 7.5 g bran, 0.5 g yeast, 0.1 g sodium tartrate, 0.01 g FeSO4 7H2O, 0.1 g sodium glutamate, 0.1 ml pure corn oil and 30 ml water. The cultivation was kept for 40 days at 28 8C. 2.3. Extraction and isolation The 3 kg air-dried solid culture was extracted exhaustively with MeOH. The extract was concentrated in vacuo to give a residue (55.5 g), which was Si-gel CC eluting with petroleum ether-acetone (25:1, 8 l) and chloroform-MeOH gradient (0:1, 7.0 l; 80:1, 6.5 l; 40:1, 6.0 l; 20:1, 6.5 l; 10:1, 6.5 l; 1:1, 6.0 l; 0:1, 5.5 l) to give eight fractions (F-1, 21.8 g; F-2, 2.8 g; F-3, 2.6 g; F-4, 3.3 g; F-5, 3.5 g; F-6, 3.0 g; F-7, 9.8 g; F-8, 4.6 g). F-2, F-3 and F-6 were shown to be anti-H. pylori fractions. Repeated chromatography of the bioactive fractions (F-2, F-3 and F-6) on silica gel with gradient of MeOH in chloroform and Sephadex LH-20 with chloroform:MeOH (1:1) afforded 2 (60 mg) from F-3, 3 (15 mg) from F-2, 1 (9 mg) and 4 (8 mg) from F-6.
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2.4. Examination of the sterile medium for presence of the compounds 1–4 The sterile medium was extracted following exactly the procedure as with that of the solid-substrate culture. LC-MS comparisons demonstrated that the medium did not contain any of the fungal metabolites 1–4. 2.5. Anti-Helicobacter pylori bioassay Bacterial strains: a reference strain (ATCC 43504) and five randomly selected clinical strains from antral biopsies from children and adults were used in this study. Growth inhibition assay of H. pylori was performed according to agar dilution method w8x. Each of the four isolated fungal metabolites was dissolved at 5 mgyml in DMSO and diluted with sterile distilled water to obtain a series of stock solutions at given concentrations. Every stock solution (1 ml) was added into a Petri dish containing 7 ml of unsolidified columbia agar base supplemented with 7% horse serum. Final concentrations of each compound in the medium were set to be 40.0, 35.0, 30.0, 25.0, 20.0, 15.0, 10.0, 5.0 and 2.5 mgyml with the pre-used DMSO concentration in the medium kept -1%. The H. pylori strains taken from the seed cultures was shortly diluted with sterile medium to approximately 1=108 CFUyml followed by immediate inoculation into the agar plates which were cultured microaerobically for 3 days at 37 8C in anaerobic jar (containing: 85% N2, 10% CO2 and 5% O2). The MICs were determined by judging visually the H. pylori growth. Ampicillin was co-assessed as a positive control at concentrations of 8.0, 4.0, 2.0, 1.0, 0.5, 0.25 and 0.125 mgyml. All experiments were conducted in triplicate. 2.6. Rhizoctonic acid (1) 5-Hydroxy-2-(2-hydroxy-6-methoxy-4-methylbenzoyl)-3-methoxybenzoic acid (1) yellowish crystals (CHCl3 yMeOH, 1:1), mp 230 8C (dec.); IR bands (KBr): 3322, 3014, 2965, 2941, 1719, 1680, 1612,1574, 1505, 1465, 1411, 1220, 1108, 1060, 912, 817 and 722 cmy1; HRESI-MS (negative mode) myz: 331.0835 (calcd. for C17H16O7-H 331.0818); 1H and 13C-NMR data: see Table 1. 2.7. Monomethylsulochrin (2) Yellowish crystals (CHCl3 yacetone, 1:1), mp 197–198 8C; 1H-NMR (500 MHz, CDCl3): d 6.99 (1H, d, J 2.0 Hz, H-6), 6.58 (1H, d, J 2.0 Hz, H-4), 6.45 (1H, br s, H-39), 6.06 (1H, br s H-59), 3.67 (3H, s, COOCH3 ), 3.66 (3H, s, H-8), 3.36 (3H, s, H-99), 2.28 (3H, s, H-79); 13C-NMR (125 MHz, CDCl3): d 199.8 (C-89), 166.3 (C-7), 164.2 (C-29), 160.9 (C-69), 157.0 (C-5), 156.5 (C-3), 148.1(C-49), 128.3 (C-1), 127.6 (C-2), 110.9 (C-19), 110.4 (C-39), 107.9 (C-6), 103.2 (C-59), 102.9 (C-4), 56.1 (C-8), 55.6 (C-99), 52.2 (COOCH3), 22.5 (C-79).
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456
454 Table 1 1 H- and
13
C-NMR data of rhizoctonic acid (1) (DMSO-d6)
Carbon
dC
1 2 3 4 5 6 7 8 19 29 39 49 59 69 79 89 99 5-OH 29-OH
130.2 126.7 157.2 103. 6 158.7 108.4 167.7 56.8 111.0 164.2 110.9 148.5 104.3 161.7 22.7 200.8 56.7
*
dH*
6.64 (1H, d, Js2.0 Hz) 6.89 (1H, d, Js2.0 Hz)
HMBC H-4, H-6 H-4, H-8 HO-5, H-6 HO-5, H-6 H-4, HO-5 H-6
3.62 (3H, s) 6.36 (1H, br s) 6.25 (1H, br s) 2.25 (3H, s)
HO-29, H-39, H-59 HO-29, H-39 HO-29, H-59, H-79 H-79 H-39, H-79 H-59, H-99 H-39, H-59
3.33 (3H, s) 9.98 (1H, s) 13.08 (1H, s)
Assigned by DEPT and HMQC experiments.
3. Results and discussion In addition to known metabolites 2–4, a new compound 1 was afforded by repeated chromatography on Si-gel and Sephadex LH-20 of the methanol extract of the solid-substrate culture of Rhizoctonia sp. Cy064. LC-MS analyses of both culture and the substrate used showed that these four compounds were metabolites of the Rhizoctonia sp. Cy064 endophytic fungus. By comparing the melting points, MS, and 1H-NMR data with the published data, compounds 2–4 were readily identified as monomethylsulochrin w9x, ergosterol w10x and 3b,5a,6b-trihydroxyergosta-7,22-diene w11x, respectively. Furthermore, the structure of monomethylsulochrin 2 was confirmed by its 13C-NMR data, along with its 1H-NMR data. In the negative-mode high resolution electrospray ionization mass spectrum of 1, a quasi-molecular ion was displayed at myz 331.0835 wM-Hxcorresponding to the molecular formula C17H16O7 (calcd. for C17H15O7: 331.0818). This molecular formula was well consistent with the 17 carbon resonances in its 13 C-NMR spectrum (Table 1). The IR spectrum of 1 showed typical absorption bands at 3322, 1719 and 1686 cmy1 indicative of hydroxyl and carbonyl groups, respectively. The 1H-NMR spectrum of 1 gave an aromatic methyl singlet at d 2.25, two methoxyl singlets at d 3.33 and d 3.62, four benzene hydrogen signals at d 6.25, 6.36, 6.64 and 6.89 and two hydroxyl protons at d 9.98 and d 13.08, being hydrogen-bonded to an ortho-carbonyl group presumably through a six-membered ring w12x. These 1H-NMR spectral data were similar to those of monomethylsulochrin 2. However, a methoxy singlet at d 3.67 in the 1 H-NMR spectrum of 2 was missing
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456
455
in that of 1, suggesting that 1 could be a demethyl derivative of monomethylsulochrin 2. This hypothesis was further confirmed by the absence of an ester methoxy signal around d 52 in the 13C-NMR spectrum of 1. This observation demonstrated that compound 1 was an acid which gave a positive result of bromocresol green test. In the HMBC spectrum of 1, a correlation of H-6 with C-7 demonstrated the position of the carboxylic group. Furthermore, HMBC correlations between H-8 and C-3 as well as between H-99 and C-69 required the formulated connections of the two methoxy groups. In addition, the presence of a methyl at C-49 was confirmed by the HMBC correlations of C-79 to both H-39 and H-59. On the basis of spectral data 1 was determined as 5-hydroxy-2-(2-hydroxy-6methoxy-4-methylbenzoyl)-3-methoxybenzoic acid. Both 1 and 2 are benzophenone derivatives a type of metabolites previously reported from the fungi belonging to the genera Aspergillus w9,13,14x, Penicillium w15x and Oospora. w16x. The present characterization of 1 and 2 from Rhizoctonia sp. suggested another fungal source of benzophenone analogs. The in vitro growth inhibition assay aganist H. pylori was carried out according to the agar dilution method w8x. The results indicated that the minimum inhibitory concentrations (MICs) of compounds 1–4 against all of the five clinical and a reference (ATCC 43504) strains were 25.0,10.0, 30.0, and 25.0 mgyml, respectively. The MIC of ampicillin used as the positive control against these strains was 2.0 mgyml.
Acknowledgments The work was co-financed by grants for RXT from National Natural Science Foundation of China (No.30171104) and from the Ministry of Science and Technology—National Marine 863 projects (Nos. 2001AA624010 and 2001AA620411). References w1x Tan RT, Zou WX. Nat Prod Rep 2001;48:448. w2x Lu H, Zou WX, Meng JC, Hu J, Tan RX. Plant Sci 2000;151:67.
456 w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x w15x w16x
Y.M. Ma et al. / Fitoterapia 75 (2004) 451–456 Zou WX, Meng JC, Lu H, Chen GX, Shi GX, Zhang TY, Tan RX. J Nat Prod 2000;63:1529. Liu JY, Liu CH, Zou WX, Tian X, Tan RX. Helv Chim Acta 2002;85:2664. Liu JY, Liu CH, Zou WX, Tan RX. Helv Chim Acta 2003;86:657. Xie ZW, Fan CS, Zhu ZY. Atlas of National Traditional Chinese Medicinal Herbs, Book II. 2nd ed. Beijing, PR China: People’s Hygiene Press, 1996. Barnett HL, Hunter BB. Illustrated Genera of Imperfect Fungi. 4th ed. Minnesota, USA: APS press, 1998. Bae EA, Han MJ, Kim NJ, Kim DH. Biol Pharm Bull 1999;21:990. Turner WB. J Chem Soc 1965;6658. Cushley RJ, Filipenko JD. Org Magn Res 1976;8:308. Takaishi Y, Und M, Ohashi T, Nakano K, Murakami K, Tomimatsu T. Phytochemistry 1991;30:4117. Shen CC, Syu WJ, Li SY, Lin CH, Lee GH, Sun CM. J Nat Prod 2002;65:1857. Inamori Y, Kato Y, Kubo M, Kamiki T, Takemoto T. Chem Pharm Bull 1983;31:4543. Kiriyama N, Nitta K, Sakaguchi Y, Taguchi Y, Yamamoto Y. Chem Pharm Bull 1977;25:2593. Mahmoodian A, Stickings CE. Biochem J 1964;92:369. Curtis RF, Harries PC, Levi JD, Phillips DM. J Chem Soc (C) 1966;168.