Arch. Pharm. Res. DOI 10.1007/s12272-015-0588-5
RESEARCH ARTICLE
Versicolols A and B, two new prenylated isocoumarins from endophytic fungus Aspergillus versicolor and their cytotoxic activity Min Zhou • Jie Lou • Yin-Ke Li • Yue-De Wang Kun Zhou • Bing-Kun Ji • Wei Dong • Xue-Mei Gao • Gang Du • Qiu-Fen Hu
•
Received: 12 October 2014 / Accepted: 17 February 2015 Ó The Pharmaceutical Society of Korea 2015
Abstract Versicolols A and B (1 and 2), two rare prenylated isocoumarin derivatives, along with five known isocoumarins (3–7) were isolated from the fermentation products of an endophytic fungus Aspergillus versicolor. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D- and 2D-NMR techniques. Compounds 1 and 2 were evaluated for their cytotoxicity against five human tumor cell lines. The results showed that compounds 1 exhibited weak cytotoxicity against A549 and MCF7 cells with IC50 values of 9.4 and 8.8 lm, and compound 2 exhibited weak cytotoxicity against SHSY5Y and MCF7 cells with IC50 values of 8.2 and 6.8 lm, respectively. Keywords Prenylated isocoumarins Aspergillus versicolor Cytotoxicity
Electronic supplementary material The online version of this article (doi:10.1007/s12272-015-0588-5) contains supplementary material, which is available to authorized users. M. Zhou J. Lou Y.-D. Wang K. Zhou B.-K. Ji W. Dong X.-M. Gao G. Du (&) Q.-F. Hu (&) Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650031, People’s Republic of China e-mail: [email protected]
Introduction Aspergillus, a genus of filamentous fungi, is famous for its medical and commercial importance (Sanchez et al. 2012). Among them, Aspergillus versicolor, a well-studied species, has attracted particular attention as a prolific source of secondary metabolites with diverse structures and biological properties (Zhuang et al. 2011; Song et al. 2012; Zhou et al. 2014; Hawas et al. 2012; Ji et al. 2013; Greshock et al. 2008; Lee et al. 2010; Wang et al. 2009) (e.g., cottoquinazoline D, a new alkaloid with a 1-aminocyclopropane-1-carboxylic acid residue, brevianamide S, a antibacterial dimeric diketopiperazine, and aspergillines A–E, five antiviral highly oxygenated cyclopiazonic acid (CPA)-derived alkaloids bearing a rigid hexacyclic indole–tetrahydrofuran–tetramate scaffold) (Zhuang et al. 2011; Song et al. 2012; Zhou et al. 2014). Here, our further efforts on an endophytic strain (YNCA1266) of A. Versicolor (Zhou et al. 2014), isolated from the rhizome of Paris polyphylla var. yunnanensis, led to the isolation and identification of two rare prenylated isocoumarin derivatives, named versicolols A and B (1 and 2), together with five known compounds (3–7). Those new compounds were elucidated by means of spectroscopic methods, while the known compounds were identified by comparison with data in the literature. Compounds 1 and 2 were evaluated for cytotoxicity against five human tumor cell lines.
Materials and methods Phytochemical investigations
Q.-F. Hu e-mail: [email protected]
General experimental procedures
Y.-K. Li College of Resource and Environment, Yuxi Normal University, Yuxi 653100, People’s Republic of China
Optical rotations were measured in a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu
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UV-2401A spectrophotometer. IR spectra were obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter. ESI-MS were measured on a VG Auto Spec-3000 MS spectrometer. 1H, 13C and 2D NMR spectra were recorded on Bruker DRX-400 instrument with TMS as internal standard. Column chromatography was performed on silica gel (200–300 mesh), or on silica gel H (10–40 lm, Qingdao Marine Chemical Inc., China). Final purifications utilized an Agilent 1100 HPLC equipped with ZORBAX-C18 (21.2 mm 9 250 mm, 7.0 lm) column and DAD detector. Paclitaxel (purity [95 %) was obtained from Sigma-Aldrich Company.
(8.6 mg, tR = 18 min). Fraction B2-1 (81 mg) was subjected to preparative HPLC (CH3OH/H2O 70:30) to give 6 (8.5 mg). fraction D (6:4, 8.2 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (8:2, 7:3, 6:4, 1:1), yielded mixtures D1–D4. Fraction D3 (1.2 g) was subjected to RP-18 column chromatography (MeOH/ H2O 20:80–60:40 gradient) to provide four fractions, D31–D3-4. Fraction D3-1 (125 mg) was subjected to preparative HPLC (CH3OH/H2O 55:45) to give 7 (1.2 mg, tR = 8 min), 3 (18.5 mg, tR = 12 min), and 5 (2.3 mg, tR = 14 min). Fraction D3-3 (32 mg) was subjected to preparative HPLC (CH3OH/H2O 57:43) to give 4 (8.0 mg, tR = 11 min).
Fungal material
Versicolol A (1)
The culture of A. versicolor was isolated from the rhizome of Paris polyphylla var. yunnanensis, collected from Dali, Yunnan, People’s Republic of China, in 2012. The strain was identified by prof. Gang Du based on the analysis of the ITS sequence (GenBank Accession number KJ801852). It was cultivated at room temperature for 7 days on potato dextrose agar at 28 °C. Agar plugs were inoculated into 250 mL Erlenmeyer flasks each containing 100 mL potato dextrose broth and cultured at 28 °C on a rotary shaker at 180 rpm for 5 days. Unlike the previous research (Zhou et al. 2014), large scale fermentation was carried out in 100 Fernbach flasks (500 mL) each containing 300 mL of medium (glucose 5 %; peptone 0.15 %; yeast 0.5 %; KH2PO4 0.05 %; MgSO4 0.05 % in 1 L of deionized water; pH 6.5 before autoclaving). Each flask was inoculated with 5.0 mL of cultured broth and incubated at 27 °C for 14 days.
C16H20O3, obtained as a pale yellow gum; UV (MeOH) kmax (log e): 210 (3.87), 272 (3.58), 292 (3.18), 336 (3.47) nm; IR (KBr) mmax 3425, 3068, 2952, 2861, 1647, 1612, 1538, 1492, 1380, 1234, 1127, 1082, 860, 758 cm-1; 1H and 13C NMR (400 and 100 MHz, in CDCl3 see Table 1; ESIMS m/z (positive ion mode) 283 [M?Na]?; HRESIMS (positive ion mode) m/z 283.1318 [M?Na]? (calcd C16H20NaO3 for 283.1310). Versicolol B (2) C16H20O2, obtained as a pale yellow gum; UV (MeOH) kmax (log e): 210 (3.90), 268 (3.55), 290 (3.22), 335 (3.38) Table 1 1H and 13C NMR data of compounds 1 and 2 (in CDCl3 d in ppm, 400 and 100 MHz) No.
dC (m)
Extraction and isolation The whole culture broth of A. versicolor was extracted four times with ethyl acetate (4 9 10 L) at room temperature and filtered. The crude extract (120 g) was applied to silica gel column chromatography, eluting with a CHCl3– (CH3)2CO gradient system (9:1, 8:2, 7:3, 6:4, 5:5). Five fractions were obtained from the silica gel column and individually decolorized on MCI gel to yield fractions A– E. The further separation of fraction B (8:2, 12.5 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures B1– B5. Fraction B2 (1.2 g) was subjected to RP-18 column chromatography (MeOH/H2O 20:80–80:20 gradient) to provide five fractions, B2-1–B2-5. Fraction B2-4 (105 mg) was subjected to preparative HPLC (ZORBAX-C18, 7.0 lm, 21.2 mm 9 250 mm, flow rate 12 mL/min, UV detection at kmax = 210, 254, and 280 nm, eluted with CH3OH/H2O 75:25) to give 1 (7.2 mg, tR = 16 min) and 2
123
Compound 1 dH (m, J, Hz) 5.19 s
Compound 2 dC (m) 71.4 t
dH (m, J, Hz)
1
73.9 t
3
157.7 s
5.11 s
4 4a
99.9 d 130.0 s
5
132.7 s
133.2 s
6
158.2 s
157.5 s
7
147.8 s
114.9 d
6.52 (d) 8.2
8
111.3 d
125.0 d
7.03 (d) 8.2
8a
127.9 s
158.5 s 6.29 s
6.51 s
98.5 d 138.6 s
6.34 s
122.8 s
9
19.1 q
2.08 s
18.9 q
2.11 s
10
27.3 t
3.34 (d) 7.2
27.1 t
3.24 (d) 7.2
5.30 (t) 7.2
123.4 d
5.25 (t) 7.2
20
123.4 d
30
133.7 s
40
17.2 q
1.55 s
17.4 q
1.52 s
50
25.2 q
1.74 s
25.8 q
1.78 s
6-OMe
60.9 q
3.81 s
56.1 q
3.82 s
Ar–OH
133.8 s
9.18 s
Versicolols A and B, two new prenylated isocoumarins
nm; IR (KBr) mmax 3430, 30452, 2934, 2867, 1638, 1610, 1546, 1472, 1357, 1250, 1135, 1073, 882, 763 cm-1; 1H and 13C NMR (400 and 100 MHz, in CDCl3 see Table 1; ESIMS m/z (positive ion mode) 267 [M?Na]?; HRESIMS (positive ion mode) m/z 267.1365 [M?Na]? (calcd C16H20NaO2 for 267.1361).
continued for 4 h at 37 °C. The cells were lysed with 100 lL of 20 % SDS-50 % DMF after removal of 100 lL of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method (Reed et al. 1938).
Biological evaluation Cytotoxicity assays
Results and discussion
The cytotoxicity of compounds was tested using a previously reported procedure (Hu et al. 2013). Colorimetric assays were performed to evaluate each compound’s activity. NB4 (human acute promyelocytic leukemia cells), A549 (Human lung adenocarcinoma epithelial cells), SHSY5Y (human neuroblastoma cells), PC3 (Human prostate cancer cell), and MCF7 (human breast adenocarcinoma cells) tumor cellcells were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT) supplemented with 10 % fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5 % CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Briefly, 100 lL of suspended adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition. In addition, suspended cells were seeded just before drug addition, with an initial density of 1 9 105 cells/mL in 100 lL of medium. Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h; paclitaxel (purity [95 %) was used as a positive control. After the incubation, MTT (100 lg) was added to each well, and the incubation was
The whole culture broth of A. versicolor was extracted with ethyl acetate. The extract was subjected repeatedly to column chromatography on Silic gel, MCI, RP-18 and preparative HPLC to afford compounds 1–7, including two prenylated isocoumarins, versicolols A and B (1 and 2), and five known isocoumarins, 6,8-dihydroxy-3-hydroxymethylisocoumarin (3) (McGraw and Hemingway 1977), 6,8-dihydroxy-3-methylisocoumarin (4) (Singh et al. 2013), 4,6-dihydroxy-3,9-dehydromellein (5) (Takenaka et al. 2011), fusariumin (6) (Yang et al. 2011), and penicimarin F (7) (Qi et al. 2013). The structures of the compounds 1–7 were shown in Fig. 1, and the 1H and 13C NMR data of 1 and 2 were listed in Table 1. Compound 1 was obtained as a pale yellow gum. Its molecular formula C16H20O3 was determined by positive HRESIMS at m/z 283.1318 [M?Na]? (calcd C16H20NaO3 for 283.1310), indicating 7° of unsaturation. The IR spectrum showed the absorption bands of hydroxyl and carbonyl groups at 3425 and 1647 cm-1, respectively. The 1H NMR spectrum (Table 1) showed signals assignable to a hydroxyl group at dH 9.18 (1H, s, Ar–OH), a prenyl group at dH 3.34 (2H, d, J = 7.2 Hz, H2-10 ), 5.30 (1H, t, J = 7.2 Hz, H-20 ), 1.55 (3H, s, H3-40 ), and 1.74 (3H, s, H350 ), two additional olefinic protons at dH 6.29 (1H, s, H-4) and 6.51 (1H, s, H-8), one aromatic methyl group at dH 2.08 (3H, s, H3-9), one methoxyl groups at dH 3.81 (3H, s,
Fig. 1 Chemical structures of isocoumarins 1–7 from A. versicolor
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M. Zhou et al. Table 2 Cytotoxic activity of compound 1 and 2
Compounds
Cell lines and IC50 (lm) NB4
SHSY5Y
PC3
MCF7
1
[10
9.4 ± 1.2
[10
[10
8.8 ± 1.0
2
[10
[10
8.2 ± 0.8
[10
6.8 ± 0.5
Taxol
0.03 ± 0.002
0.02 ± 0.002
0.2 ± 0.002
0.2 ± 0.002
0.1 ± 0.001
Fig. 2 1H–1H COSY and selected HMBC correlations of isocoumarins 1 and 2
6-OMe), and two methylene protons at dH 5.19 (2H, s, H21). In the 13C NMR and DEPT NMR spectra (Table 1), there were signals for four methyls (one oxygenated), two methylenes (one oxygenated), three methines, seven quaternary carbons. These data were closely related to chaetophenol B (Asai et al. 2013), an unusual prenylated isocoumarin, except for the substituents at C-6, C-7, and C-8 of the benzene ring. This structural assignment was further supported by the HMBC correlations from the methoxyl groups at dH 3.81 (3H, s, 6-OMe) to C-6 (dC 158.2), the hydroxyl group at dH 9.18 (1H, s, 7-OH) to C-7 (dC 147.8), and a olefinic proton at dH 6.51 (1H, s, H-8) to C-6 and C-7 (Fig. 2). Thus, the structure of 1 was established as shown in Fig. 1, and named as versicolol A. Compounds 2 was obtained as white amorphous powder with the molecular formula C16H20O2 determined by its HRESIMS at m/z 267.1365 [M?Na]? (calcd 267.1361). Analyses of the 1H and 13C NMR spectra (Table 1) revealed that the structure of 2 was very similar to that of 1, except for the absence of a hydroxyl group and the presence of a olefinic proton at dH 6.52 (1H, d, J = 8.2 Hz, H-7) in 2 (Table 2). The HMBC corrections from H-7 to C-5 (dC 133.2), C-6 (dC 157.5), and C-8a (dC 122.8), together with the 1H-1H COSY correlation of H-7 and H-8, provided further evidence for the structural assignment. The structure of 2 was therefore determined. Since many isocoumarins have been reported to possess potential cytotoxic properties (Singh et al. 2013; Thongbai et al. 2013), compounds 1 and 2 were screened for their cytotoxic activities against a panel of human cell lines, including NB4 promyelocytic leukemia, A549 lung epithelial carcinoma, SHSY5Y neuroblastoma, PC3 prostate
123
A549
cancer, and MCF7 breast adenocarcinoma cells using the MTT method as previously described with paclitaxel as a positive control. The results showed that compounds 1 exhibited weak cytotoxicity against A49 and MCF7 cells with IC50 values of 9.4 and 8.8 lm, and compound 2 exhibited weak cytotoxicity against SHSY5Y and MCF7 cells with IC50 values of 8.2 and 6.8 lm, respectively. Versicolols A and B (1 and 2), two rare prenylated isocoumarin derivatives, together with five known isocoumarins (3–7) were isolated from the fermentation products of an endophytic fungus A. versicolor. Their structures were elucidated on the basis of extensive spectroscopic analysis, particularly 1D and 2D NMR techniques. Furthermore, the new isocoumarins were evaluated for their cytotoxicity against five human tumor cell lines. The results showed that compounds 1 exhibited weak cytotoxicity against A549 and MCF7 cells and compound 2 exhibited weak cytotoxicity against SHSY5Y and MCF7 cells. Although several isocoumarin derivatives have been reported from several members of the genus Aspergillus (Prompanya et al. 2014; Sun et al. 2014), this is the first report of isolation of their analogs from A. versicolor. Acknowledgments This Project was supported financially by the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan Minzu University Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan Minzu University) (2010XY08). Conflict of interest interest.
The authors declare that there is no conflict of
References Asai, T., T. Yamamoto, N. Shirata, T. Taniguchi, K. Monde, I. Fujii, K. Gomi, and Y. Oshima. 2013. Structurally diverse chaetophenol productions induced by chemically mediated epigenetic manipulation of fungal gene expression. Organic Letters 15: 3346–3349. Greshock, T.J., A.W. Grubbs, P. Jiao, D.T. Wicklow, J.B. Gloer, and R.M. Williams. 2008. Isolation, structure elucidation, and biomimetic total synthesis of versicolamide B, and the isolation of antipodal (-)-stephacidin A and (?)-notoamide B from Aspergillus versicolor NRRL 35600. Angewandte Chemie International Edition 47: 3573–3577.
Versicolols A and B, two new prenylated isocoumarins Hawas, U.W., A.A. El-Beih, and A.M. El-Halawany. 2012. Bioactive anthraquinones from endophytic fungus Aspergillus versicolor isolated from red sea algae. Archives of Pharmacal Research 35: 1749–1756. Hu, Q.F., B. Zhou, J.M. Huang, Z.Y. Jiang, X.Z. Huang, L.Y. Yang, X.M. Gao, G.Y. Yang, and C.T. Che. 2013. Cytotoxic deoxybenzoins and diphenylethylenes from Arundina graminifolia. Journal of Natural Products 76: 1854–1859. Ji, N.Y., X.H. Liu, F.P. Miao, and M.F. Qiao. 2013. Aspeverin, a new alkaloid from an algicolous strain of Aspergillus versicolor. Organic Letters 15: 2327–2329. Lee, Y.M., H. Li, J. Hong, H.Y. Cho, K.S. Bae, M.A. Kim, D.K. Kim, and J.H. Jung. 2010. Bioactive metabolites from the spongederived fungus Aspergillus versicolor. Archives of Pharmacal Research 33: 231–235. McGraw, G.W., and R.W. Hemingway. 1977. 6,8-Dihydroxy-3hydroxymethyl-isocoumarin, and other phenolic metabolites of Ceratocystis minor. Phytochemistry 16: 1315–1316. Prompanya, C., T. Dethoup, L.J. Bessa, M.M. Pinto, L. Gales, P.M. Costa, A.M.S. Silva, and A. Kijjoa. 2014. New isocoumarin derivatives and meroterpenoids from the marine sponge-associated fungus Aspergillus similanensis sp. nov. KUFA 0013. Marine Drugs 12: 5160–5173. Qi, J., C.L. Shao, Z.Y. Li, L.S. Gan, X.M. Fu, W.T. Bian, H.Y. Zhao, and C.Y. Wang. 2013. Isocoumarin derivatives and benzofurans from a sponge-derived Penicillium sp. fungus. Journal of Natural Products 76: 571–579. Reed, L.J., and H. Muench. 1938. A simple method of estimating fifty percent endpoints. American Journal of Epidemiology 27: 493–497. Sanchez, J.F., A.D. Somoza, N.P. Keller, and C.C.C. Wang. 2012. Advances in Aspergillus secondary metabolite research in the post-genomic era. Natural Product Reports 29: 351–371. Singh, B., R. Parshad, R.K. Khajuria, S.K. Guru, A.S. Pathania, R. Sharma, R. Chi, S. Aravinda, V.K. Gupta, I.A. Khan, S. Bhushan, S.B. Bharate, and R.A. Vishwakarma. 2013. Saccharonol B, a new cytotoxic methylated isocoumarin from Saccharomonospora azurea. Tetrahedron Letters 54: 6695–6699.
Song, F.H., X.R. Liu, H. Guo, B. Ren, C.X. Chen, A.M. Piggott, K. Yu, H. Gao, Q. Wang, M. Liu, X.T. Liu, H.Q. Dai, L.X. Zhang, and R.J. Capon. 2012. Brevianamides with antitubercular potential from a marine-derived isolate of Aspergillus versicolor. Organic Letters 14: 4770–4773. Sun, K.L., Y. Li, L. Guo, Y. Wang, P.P. Liu, and W.M. Zhu. 2014. Indole diterpenoids and isocoumarin from the fungus, Aspergillus flavus, isolated from the Prawn, Penaeus vannamei. Marine Drugs 12: 3970–3981. Takenaka, Y., N. Morimoto, N. Hamada, and T. Tanahashi. 2011. Phenolic compounds from the cultured mycobionts of Graphis proserpens. Phytochemistry 72: 1431–1435. Thongbai, B., F. Surup, K. Mohr, E. Kuhnert, K.D. Hyde, and M. Stadler. 2013. Gymnopalynes A and B, chloropropynyl-isocoumarin antibiotics from cultures of the basidiomycete Gymnopus sp. fungus. Journal of Natural Products 76: 2141–2144. Wang, W.L., P.P. Liu, Y.P. Zhang, J. Li, H.W. Tao, Q.Q. Gu, and W.M. Zhu. 2009. 2-Hydroxydiplopterol, a new cytotoxic pentacyclic triterpenoid from the halotolerant fungus Aspergillus variecolor B-17. Archives of Pharmacal Research 32: 1211–1214. Yang, S.X., J.M. Gao, Q. Zhang, and H. Laatsch. 2011. Toxic polyketides produced by Fusarium sp., an endophytic fungus isolated from Melia azedarach. Bioorganic & Medicinal Chemistry Letters 21: 1887–1889. Zhou, M., M.M. Miao, G. Du, X.N. Li, S.Z. Shang, W. Zhao, Z.H. Liu, G.Y. Yang, C.T. Che, Q.F. Hu, and X.M. Gao. 2014. Aspergillines A–E, highly oxygenated hexacyclic indole–tetrahydrofuran–tetramic acid derivatives from Aspergillus versicolor. Organic Letters 16: 5016–5019. Zhuang, Y.B., X.C. Teng, Y. Wang, P.P. Liu, G.Q. Li, and W.M. Zhu. 2011. New quinazolinone alkaloids within rare amino acid residue from coral-associated fungus, Aspergillus versicolor LCJ-5-4. Organic Letters 13: 1130–1133.
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RESEARCH ARTICLE
Versicolols A and B, two new prenylated isocoumarins from endophytic fungus Aspergillus versicolor and their cytotoxic activity Min Zhou • Jie Lou • Yin-Ke Li • Yue-De Wang Kun Zhou • Bing-Kun Ji • Wei Dong • Xue-Mei Gao • Gang Du • Qiu-Fen Hu
•
Received: 12 October 2014 / Accepted: 17 February 2015 Ó The Pharmaceutical Society of Korea 2015
Abstract Versicolols A and B (1 and 2), two rare prenylated isocoumarin derivatives, along with five known isocoumarins (3–7) were isolated from the fermentation products of an endophytic fungus Aspergillus versicolor. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D- and 2D-NMR techniques. Compounds 1 and 2 were evaluated for their cytotoxicity against five human tumor cell lines. The results showed that compounds 1 exhibited weak cytotoxicity against A549 and MCF7 cells with IC50 values of 9.4 and 8.8 lm, and compound 2 exhibited weak cytotoxicity against SHSY5Y and MCF7 cells with IC50 values of 8.2 and 6.8 lm, respectively. Keywords Prenylated isocoumarins Aspergillus versicolor Cytotoxicity
Electronic supplementary material The online version of this article (doi:10.1007/s12272-015-0588-5) contains supplementary material, which is available to authorized users. M. Zhou J. Lou Y.-D. Wang K. Zhou B.-K. Ji W. Dong X.-M. Gao G. Du (&) Q.-F. Hu (&) Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650031, People’s Republic of China e-mail: [email protected]
Introduction Aspergillus, a genus of filamentous fungi, is famous for its medical and commercial importance (Sanchez et al. 2012). Among them, Aspergillus versicolor, a well-studied species, has attracted particular attention as a prolific source of secondary metabolites with diverse structures and biological properties (Zhuang et al. 2011; Song et al. 2012; Zhou et al. 2014; Hawas et al. 2012; Ji et al. 2013; Greshock et al. 2008; Lee et al. 2010; Wang et al. 2009) (e.g., cottoquinazoline D, a new alkaloid with a 1-aminocyclopropane-1-carboxylic acid residue, brevianamide S, a antibacterial dimeric diketopiperazine, and aspergillines A–E, five antiviral highly oxygenated cyclopiazonic acid (CPA)-derived alkaloids bearing a rigid hexacyclic indole–tetrahydrofuran–tetramate scaffold) (Zhuang et al. 2011; Song et al. 2012; Zhou et al. 2014). Here, our further efforts on an endophytic strain (YNCA1266) of A. Versicolor (Zhou et al. 2014), isolated from the rhizome of Paris polyphylla var. yunnanensis, led to the isolation and identification of two rare prenylated isocoumarin derivatives, named versicolols A and B (1 and 2), together with five known compounds (3–7). Those new compounds were elucidated by means of spectroscopic methods, while the known compounds were identified by comparison with data in the literature. Compounds 1 and 2 were evaluated for cytotoxicity against five human tumor cell lines.
Materials and methods Phytochemical investigations
Q.-F. Hu e-mail: [email protected]
General experimental procedures
Y.-K. Li College of Resource and Environment, Yuxi Normal University, Yuxi 653100, People’s Republic of China
Optical rotations were measured in a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu
123
M. Zhou et al.
UV-2401A spectrophotometer. IR spectra were obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter. ESI-MS were measured on a VG Auto Spec-3000 MS spectrometer. 1H, 13C and 2D NMR spectra were recorded on Bruker DRX-400 instrument with TMS as internal standard. Column chromatography was performed on silica gel (200–300 mesh), or on silica gel H (10–40 lm, Qingdao Marine Chemical Inc., China). Final purifications utilized an Agilent 1100 HPLC equipped with ZORBAX-C18 (21.2 mm 9 250 mm, 7.0 lm) column and DAD detector. Paclitaxel (purity [95 %) was obtained from Sigma-Aldrich Company.
(8.6 mg, tR = 18 min). Fraction B2-1 (81 mg) was subjected to preparative HPLC (CH3OH/H2O 70:30) to give 6 (8.5 mg). fraction D (6:4, 8.2 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (8:2, 7:3, 6:4, 1:1), yielded mixtures D1–D4. Fraction D3 (1.2 g) was subjected to RP-18 column chromatography (MeOH/ H2O 20:80–60:40 gradient) to provide four fractions, D31–D3-4. Fraction D3-1 (125 mg) was subjected to preparative HPLC (CH3OH/H2O 55:45) to give 7 (1.2 mg, tR = 8 min), 3 (18.5 mg, tR = 12 min), and 5 (2.3 mg, tR = 14 min). Fraction D3-3 (32 mg) was subjected to preparative HPLC (CH3OH/H2O 57:43) to give 4 (8.0 mg, tR = 11 min).
Fungal material
Versicolol A (1)
The culture of A. versicolor was isolated from the rhizome of Paris polyphylla var. yunnanensis, collected from Dali, Yunnan, People’s Republic of China, in 2012. The strain was identified by prof. Gang Du based on the analysis of the ITS sequence (GenBank Accession number KJ801852). It was cultivated at room temperature for 7 days on potato dextrose agar at 28 °C. Agar plugs were inoculated into 250 mL Erlenmeyer flasks each containing 100 mL potato dextrose broth and cultured at 28 °C on a rotary shaker at 180 rpm for 5 days. Unlike the previous research (Zhou et al. 2014), large scale fermentation was carried out in 100 Fernbach flasks (500 mL) each containing 300 mL of medium (glucose 5 %; peptone 0.15 %; yeast 0.5 %; KH2PO4 0.05 %; MgSO4 0.05 % in 1 L of deionized water; pH 6.5 before autoclaving). Each flask was inoculated with 5.0 mL of cultured broth and incubated at 27 °C for 14 days.
C16H20O3, obtained as a pale yellow gum; UV (MeOH) kmax (log e): 210 (3.87), 272 (3.58), 292 (3.18), 336 (3.47) nm; IR (KBr) mmax 3425, 3068, 2952, 2861, 1647, 1612, 1538, 1492, 1380, 1234, 1127, 1082, 860, 758 cm-1; 1H and 13C NMR (400 and 100 MHz, in CDCl3 see Table 1; ESIMS m/z (positive ion mode) 283 [M?Na]?; HRESIMS (positive ion mode) m/z 283.1318 [M?Na]? (calcd C16H20NaO3 for 283.1310). Versicolol B (2) C16H20O2, obtained as a pale yellow gum; UV (MeOH) kmax (log e): 210 (3.90), 268 (3.55), 290 (3.22), 335 (3.38) Table 1 1H and 13C NMR data of compounds 1 and 2 (in CDCl3 d in ppm, 400 and 100 MHz) No.
dC (m)
Extraction and isolation The whole culture broth of A. versicolor was extracted four times with ethyl acetate (4 9 10 L) at room temperature and filtered. The crude extract (120 g) was applied to silica gel column chromatography, eluting with a CHCl3– (CH3)2CO gradient system (9:1, 8:2, 7:3, 6:4, 5:5). Five fractions were obtained from the silica gel column and individually decolorized on MCI gel to yield fractions A– E. The further separation of fraction B (8:2, 12.5 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures B1– B5. Fraction B2 (1.2 g) was subjected to RP-18 column chromatography (MeOH/H2O 20:80–80:20 gradient) to provide five fractions, B2-1–B2-5. Fraction B2-4 (105 mg) was subjected to preparative HPLC (ZORBAX-C18, 7.0 lm, 21.2 mm 9 250 mm, flow rate 12 mL/min, UV detection at kmax = 210, 254, and 280 nm, eluted with CH3OH/H2O 75:25) to give 1 (7.2 mg, tR = 16 min) and 2
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Compound 1 dH (m, J, Hz) 5.19 s
Compound 2 dC (m) 71.4 t
dH (m, J, Hz)
1
73.9 t
3
157.7 s
5.11 s
4 4a
99.9 d 130.0 s
5
132.7 s
133.2 s
6
158.2 s
157.5 s
7
147.8 s
114.9 d
6.52 (d) 8.2
8
111.3 d
125.0 d
7.03 (d) 8.2
8a
127.9 s
158.5 s 6.29 s
6.51 s
98.5 d 138.6 s
6.34 s
122.8 s
9
19.1 q
2.08 s
18.9 q
2.11 s
10
27.3 t
3.34 (d) 7.2
27.1 t
3.24 (d) 7.2
5.30 (t) 7.2
123.4 d
5.25 (t) 7.2
20
123.4 d
30
133.7 s
40
17.2 q
1.55 s
17.4 q
1.52 s
50
25.2 q
1.74 s
25.8 q
1.78 s
6-OMe
60.9 q
3.81 s
56.1 q
3.82 s
Ar–OH
133.8 s
9.18 s
Versicolols A and B, two new prenylated isocoumarins
nm; IR (KBr) mmax 3430, 30452, 2934, 2867, 1638, 1610, 1546, 1472, 1357, 1250, 1135, 1073, 882, 763 cm-1; 1H and 13C NMR (400 and 100 MHz, in CDCl3 see Table 1; ESIMS m/z (positive ion mode) 267 [M?Na]?; HRESIMS (positive ion mode) m/z 267.1365 [M?Na]? (calcd C16H20NaO2 for 267.1361).
continued for 4 h at 37 °C. The cells were lysed with 100 lL of 20 % SDS-50 % DMF after removal of 100 lL of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method (Reed et al. 1938).
Biological evaluation Cytotoxicity assays
Results and discussion
The cytotoxicity of compounds was tested using a previously reported procedure (Hu et al. 2013). Colorimetric assays were performed to evaluate each compound’s activity. NB4 (human acute promyelocytic leukemia cells), A549 (Human lung adenocarcinoma epithelial cells), SHSY5Y (human neuroblastoma cells), PC3 (Human prostate cancer cell), and MCF7 (human breast adenocarcinoma cells) tumor cellcells were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT) supplemented with 10 % fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5 % CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Briefly, 100 lL of suspended adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition. In addition, suspended cells were seeded just before drug addition, with an initial density of 1 9 105 cells/mL in 100 lL of medium. Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h; paclitaxel (purity [95 %) was used as a positive control. After the incubation, MTT (100 lg) was added to each well, and the incubation was
The whole culture broth of A. versicolor was extracted with ethyl acetate. The extract was subjected repeatedly to column chromatography on Silic gel, MCI, RP-18 and preparative HPLC to afford compounds 1–7, including two prenylated isocoumarins, versicolols A and B (1 and 2), and five known isocoumarins, 6,8-dihydroxy-3-hydroxymethylisocoumarin (3) (McGraw and Hemingway 1977), 6,8-dihydroxy-3-methylisocoumarin (4) (Singh et al. 2013), 4,6-dihydroxy-3,9-dehydromellein (5) (Takenaka et al. 2011), fusariumin (6) (Yang et al. 2011), and penicimarin F (7) (Qi et al. 2013). The structures of the compounds 1–7 were shown in Fig. 1, and the 1H and 13C NMR data of 1 and 2 were listed in Table 1. Compound 1 was obtained as a pale yellow gum. Its molecular formula C16H20O3 was determined by positive HRESIMS at m/z 283.1318 [M?Na]? (calcd C16H20NaO3 for 283.1310), indicating 7° of unsaturation. The IR spectrum showed the absorption bands of hydroxyl and carbonyl groups at 3425 and 1647 cm-1, respectively. The 1H NMR spectrum (Table 1) showed signals assignable to a hydroxyl group at dH 9.18 (1H, s, Ar–OH), a prenyl group at dH 3.34 (2H, d, J = 7.2 Hz, H2-10 ), 5.30 (1H, t, J = 7.2 Hz, H-20 ), 1.55 (3H, s, H3-40 ), and 1.74 (3H, s, H350 ), two additional olefinic protons at dH 6.29 (1H, s, H-4) and 6.51 (1H, s, H-8), one aromatic methyl group at dH 2.08 (3H, s, H3-9), one methoxyl groups at dH 3.81 (3H, s,
Fig. 1 Chemical structures of isocoumarins 1–7 from A. versicolor
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M. Zhou et al. Table 2 Cytotoxic activity of compound 1 and 2
Compounds
Cell lines and IC50 (lm) NB4
SHSY5Y
PC3
MCF7
1
[10
9.4 ± 1.2
[10
[10
8.8 ± 1.0
2
[10
[10
8.2 ± 0.8
[10
6.8 ± 0.5
Taxol
0.03 ± 0.002
0.02 ± 0.002
0.2 ± 0.002
0.2 ± 0.002
0.1 ± 0.001
Fig. 2 1H–1H COSY and selected HMBC correlations of isocoumarins 1 and 2
6-OMe), and two methylene protons at dH 5.19 (2H, s, H21). In the 13C NMR and DEPT NMR spectra (Table 1), there were signals for four methyls (one oxygenated), two methylenes (one oxygenated), three methines, seven quaternary carbons. These data were closely related to chaetophenol B (Asai et al. 2013), an unusual prenylated isocoumarin, except for the substituents at C-6, C-7, and C-8 of the benzene ring. This structural assignment was further supported by the HMBC correlations from the methoxyl groups at dH 3.81 (3H, s, 6-OMe) to C-6 (dC 158.2), the hydroxyl group at dH 9.18 (1H, s, 7-OH) to C-7 (dC 147.8), and a olefinic proton at dH 6.51 (1H, s, H-8) to C-6 and C-7 (Fig. 2). Thus, the structure of 1 was established as shown in Fig. 1, and named as versicolol A. Compounds 2 was obtained as white amorphous powder with the molecular formula C16H20O2 determined by its HRESIMS at m/z 267.1365 [M?Na]? (calcd 267.1361). Analyses of the 1H and 13C NMR spectra (Table 1) revealed that the structure of 2 was very similar to that of 1, except for the absence of a hydroxyl group and the presence of a olefinic proton at dH 6.52 (1H, d, J = 8.2 Hz, H-7) in 2 (Table 2). The HMBC corrections from H-7 to C-5 (dC 133.2), C-6 (dC 157.5), and C-8a (dC 122.8), together with the 1H-1H COSY correlation of H-7 and H-8, provided further evidence for the structural assignment. The structure of 2 was therefore determined. Since many isocoumarins have been reported to possess potential cytotoxic properties (Singh et al. 2013; Thongbai et al. 2013), compounds 1 and 2 were screened for their cytotoxic activities against a panel of human cell lines, including NB4 promyelocytic leukemia, A549 lung epithelial carcinoma, SHSY5Y neuroblastoma, PC3 prostate
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A549
cancer, and MCF7 breast adenocarcinoma cells using the MTT method as previously described with paclitaxel as a positive control. The results showed that compounds 1 exhibited weak cytotoxicity against A49 and MCF7 cells with IC50 values of 9.4 and 8.8 lm, and compound 2 exhibited weak cytotoxicity against SHSY5Y and MCF7 cells with IC50 values of 8.2 and 6.8 lm, respectively. Versicolols A and B (1 and 2), two rare prenylated isocoumarin derivatives, together with five known isocoumarins (3–7) were isolated from the fermentation products of an endophytic fungus A. versicolor. Their structures were elucidated on the basis of extensive spectroscopic analysis, particularly 1D and 2D NMR techniques. Furthermore, the new isocoumarins were evaluated for their cytotoxicity against five human tumor cell lines. The results showed that compounds 1 exhibited weak cytotoxicity against A549 and MCF7 cells and compound 2 exhibited weak cytotoxicity against SHSY5Y and MCF7 cells. Although several isocoumarin derivatives have been reported from several members of the genus Aspergillus (Prompanya et al. 2014; Sun et al. 2014), this is the first report of isolation of their analogs from A. versicolor. Acknowledgments This Project was supported financially by the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan Minzu University Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan Minzu University) (2010XY08). Conflict of interest interest.
The authors declare that there is no conflict of
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