Chinese Journal of Natural Medicines 2013, 11(5): 0673−0675

Chinese Journal of Natural Medicines

A new diphenyl ether from the endophytic fungus Verticillium sp. isolated from Rehmannia glutinosa PENG Wei 1, 2, YOU Fei 1, LI Xiao-Li 2, JIA Min 1, ZHENG Cheng-Jian 1, HAN Ting 1*, QIN Lu-Ping 1, 3* 1

Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China； Department of Pharmacology, College of Pharmacy, Third Military Medical University, Chongqing 400038, China; 3 Shanghai Key Laboratory for Pharmaceutical Metabolite Research, Shanghai 200433, China Available online 20 Nov. 2013 2

[ABSTRACT] AIM: To investigate the chemical constituents of the endophytic fungus Verticillium sp. isolated from Rehmannia glutinosa. METHODS: The compounds were isolated and purified by repeated column chromatography, and their structures were determined on the basis of physicochemical properties and spectral analysis. Their cytotoxic and antifungal activities were evaluated. RESULTS: Ten compounds were obtained and their structures were identified as 2, 4-dihydroxy-2’, 6-diacetoxy-3’-methoxy-5’-methyl-diphenyl ether (1), paecilospirone (2), α-acetylorcinol (3), 2-methoxy-1,8-dimethyl-xanthen-9-one (4), 4-hydroxy-α-lapachone (5), enalin A (6), 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (7), 4-hydroxyethyl-phenol (8), 2,4-dihydroxy-3,5,6-trimethylmethylbenzoate (9), and 3-isopropenyl-(Z)-monomethyl maleate (10). CONCLUSIONS: Compound 1 is a new diphenyl ether, and showed cytotoxic activity against HL-60 cells (IC50 2.24 μg·mL−1), and antifungal activities against Candida albicans (MIC 8 μg·mL−1) and Aspergillus fumigatus (MIC 16 μg·mL−1). [KEY WORDS] Endophytic fungus; Rehmannia glutinosa; Diphenyl ether; Cytotoxic activity; Antifungal activities

[CLC Number] R284.1; R965

1

[Document code] A

Introduction

Endophytic fungi are known to possess various rare and novel natural products with significant biological activities [1-3] . In a previous study, a new antifungal metabolite of the endophytic fungus Verticillium sp., isolated from Rehmannia glutinosa (Gaertn) Libosch. ex Fisch. & C. A. Mey (Orobanchaceae), was reported [4]. As part of a systematic search for new bioactive compounds from endophytic fungi, this endophytic fungus was selected for chemical and biological investigation. In this study, a new diphenyl ether, 2,4- dihydroxy-2’, 6-diacetoxy-3’-methoxy-5’-methyl-diphenyl ether (1), was produced by the endophytic fungus Verticillium sp. isolated from Rehmannia glutinosa, and isolated together with nine known compounds (2-10) as shown in Fig. 1. [Received on] 09-Jul.-2012 [*Corresponding author] HAN Ting: Associate Prof., Tel/Fax: 86-21-81871306, E-mail: [email protected]; QIN Lu-Ping: Prof., Tel/Fax: 86-21- 81871300, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

[Article ID] 1672-3651(2013)06-0673-03 Compound 1 showed cytotoxic activity against HL-60 cell lines (IC50 2.24 μg·mL−1) and antifungal activities against Candida albicans (MIC 8 μg·mL−1) and Aspergillus fumigatus (MIC 16 μg·mL−1).

2

Results and Discussion

Compound 1 was obtained as a white powder with the molecular formula C18H18O8 from the positive HR-ESI-MS quasimolecular ion peak [M + H]+ at m/z 363.107 7 (calcd. 363.107 4), which was supported by the NMR data. The 1H and 13C NMR spectra of 1, together with DEPT analysis, indicated the presence of the general structural features of 2, 3’-dimethylosoate, which was previously isolated from a marine-derived fungal Aspergillus sp. [5]. The only difference was the substituent group at C-2, where compound 1 possesses a hydroxyl instead of a methyl group (in 2, 3’-dimethylosoate) which was deduced by the NMR and HRESIMS spectra. In the 1H NMR spectrum of 1, one methyl singlet at δ 2.10, and three methoxyl singlets at δ 3.74, 3.69, and 3.94 were observed, while the 13C NMR spectrum of 1 showed a total of 18 signals. Therefore, one methoxyl signal had disappeared in compound 1, compared with 2,

PENG Wei, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 673−675 Table 1

1

H NMR (600 MHz) and 13 C NMR (150 MHz) data of compound 1 in CDCl3

Position

δH (J in Hz)

1 2

δc (DEPT)

Position

136.4 (-C)

2'

153.7 (-C)

3'

6.70 (1H, d, 2.9)

105.1 (-CH)

4'

153.9 (-C)

5'

6.95 (1H, d, 2.9)

108.5 (-CH)

6'

6

125.1 (-C)

7'

7

165.3 (-C)

3 4 5

8

3.74 (s)

1’

Fig. 1

δH (J in Hz)

δc (DEPT) 100.5 (-C) 163.3 (-C)

6.95 (1H, br s)

110.0 (-CH)

5.82 (1H, br s)

105.8 (-CH)

8'

3.69 (s)

52.4 (-OCH3)

52.2 (-C)

9'

3.94 (s)

56.4 (-OCH3)

160.5 (-C)

10'

2.10 (s)

22.1 (-CH3)

146.0 (-C) 171.1 (-C)

Compounds isolated from the endophytic fungus Verticillium sp. HL-60 (IC50 0.83 μg·mL−1) and LOVO (IC50 2.31 μg·mL−1) cell lines; compound 3 showed significant antifungal activities against Trichophyton rubrum (MIC 0.25 μg·mL−1), Aspergillus fumigatus (MIC 0.25 μg·mL−1). The remaining compounds showed no significant cytotoxic (IC50 > 4 μg·mL−1) or antifungal activities (MIC > 32 μg·mL−1).

Fig. 2

Key HMBC correlations of compound 1

3

Experimental

3.1

3’-dimethylosoate (detailed 1H and 13C NMR data are summarized in Table 1). Furthermore, these assumptions were confirmed by HMBC spectroscopic analysis (Fig. 2). The other nine known compounds were identified as paecilospirone (2) [6], α-acetylorcinol (3) [7], 2-methoxy-1,8- dimethyl-xanthen-9-one (4) [8], 4-hydroxy-α-lapachone (5) [9], enalin A (6) [10], 2, 3, 4-trimethyl-5, 7-dihydroxy-2, 3-dihydrobenzofuran (7) [11], 4-hydroxy-ethylphenol (8) [12], 2, 4-dihydroxy-3, 5, 6-trimethyl-methyl-benzoate (9) [13], and 3-isopropenyl-(Z)-monomethyl maleate (10) [14], by comparison of their spectral data with literature values. Among the ten isolates, compound 1 showed cytotoxic activity against HL-60 cell lines (IC50 2.24 μg·mL−1) and antifungal activities against Candida albicans (MIC 8 μg·mL−1) and Aspergillus fumigatus (MIC 16 μg·mL−1); compound 10 showed significant cytotoxic activity against

Extraction and isolation The roots of Rehmannia glutinosa were collected during March, 2007 from Wushe County, Henan Province, where R. glutinosa has a wide distribution, and identified by Professor QIN Lu-Ping. After digging out, the roots were taken to the laboratory within 24 h, and the surplus roots were planted in the Botanic Garden of the Second Military Medical University. The preparation of extracts from Verticillium sp. isolated from Rehmannia glutinosa was detailed in the previous study [4] . After extraction with 95% ethanol (AR), a crude extract (3.4 g) was obtained from the culture of Verticillium sp. (60 L). The extract was subjected to silica gel size exclusion chromatography (200-300 mesh), eluting with a gradient of chloroform (AR)-MeOH (AR) (15 : 1, 10 : 1, 5 : 1, 2 : 1, 1 : 1, and 1 : 5, V/V) to give fractions A-H. Fraction B was purified by gel filtration on Sephadex LH-20 (chloroform-MeOH, 1 : 2) to afford compounds 2 (17 mg), 6 (14 mg), and 10 (24

PENG Wei, et al. /Chinese Journal of Natural Medicines 2013, 11(6): 673−675

mg); and one sub-fraction was further separated by preparative TLC to afford compound 1 (15 mg) (Fig. 1). Fraction C was subjected to column chromatography over silica gel (200-300 mesh) eluting with a chloroform-MeOH gradient, and Sephadex LH-20 (MeOH) to afford 3 (20 mg), 4 (18 mg), and 5 (27 mg). Compounds 7 (32 mg), 8 (40 mg), and 9 (13 mg) were isolated from the fractions D-E in the same way, respectively (Fig 1). 3.2 Biological assays All compounds were evaluated for cytotoxic activities (MKN45, LOVO, HepG2, and HL-60 cell lines) and antifungal activities (Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, and Aspergillus fumigatus) in vitro [15].

[7]

[8]

[9]

[10]

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地黄内生真菌中一个新的二苯醚类代谢产物 彭

伟 1, 2，游

飞 1，李小丽 2，贾

敏 1，郑承剑 1，韩

1

第二军医大学药学院生药学教研室，上海 200433;

2

第三军医大学药学院药理学教研室，重庆 400038;

3

上海市药物代谢研究重点实验室，上海 200433

婷 1*，秦路平 1, 3*

【摘 要】 目的：研究地黄内生真菌的代谢产物。方法：采柱色谱方法进行分离纯化，并根据化合物的理化性质和波谱数 据鉴定其结构。结果：从地黄内生真菌代谢产物中分离并鉴定了 10 个化合物：2, 4-dihydroxy-2’, 6-diacetoxy-3’-methoxy-5’-methyldiphenyl ether (1), paecilospirone (2), α-axetylorcino (3), 2-methoxy-1, 8-dimethyl-xanthen-9-one (4), 4-hydroxy-α-lapachone (5), enalin A (6), 2, 3, 4-trimethyl-5, 7-dihydroxy-2, 3-dihydrobenzofuran (7), p-hydroxyethyl-phenol (8), 2, 4-dihydroxy-3, 5, 6- trimethyl-methylbenzoate (9), 3-isopropenyl-(z)-monomethyl maleate (10)。结论：化合物 1 为新的二苯醚类化合物，其显示了一定的细 胞毒和抗真菌活性。 【关键词】 内生真菌；地黄；二苯醚；细胞毒；抗真菌