Original Papers
Antiviral and Cytotoxic Isocoumarin Derivatives from an Endophytic Fungus Aspergillus oryzae
Authors
Min Zhou 1, 2, 3, Kun Zhou 1, 2, Pei He 3, Kun-Miao Wang 3, Rui-Zhi Zhu 3, Yue-De Wang 1, 2, Wei Dong 1, 2, Gan-Peng Li 1, 2, Hai-Ying Yang 1, Yan-Qing Ye 1, Gang Du 1, 2, Xue-Mei Li 3, Qiu-Fen Hu 1, 2
Affiliations
1
3
Key words " Aspergillus oryzae l " Trichocomaceae l " isocoumarin derivatives l " anti‑tobacco mosaic virus l activity " cytotoxicity l
received revised accepted
Sept. 14, 2015 Nov. 3, 2015 Nov. 9, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1558331 Published online Planta Med © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Qiu-Fen Hu Key Laboratory of Chemistry in Ethnic Medicinal Resources State Ethnic Affairs Commission & Ministry of Education Yunnan Minzu University 121 Road 134 Kunming 650031 P. R. China Phone: + 86 8 71 65 91 30 43 Fax: + 86 8 71 65 91 00 17 [email protected] Correspondence Prof. Xue-Mei Li Technology Center China Tobacco Yunnan Industry Company (Ltd.) Keyi Road 41 Kunming 650000 P. R. China Phone: + 86 8 71 68 31 96 56 Fax: + 86 8 71 68 31 90 18 [email protected]
Abstract !
Oryzaeins A–D (1-4), four new isocoumarin derivatives, along with five known ones (5-9) were isolated from solid cultures of an endophytic fungus Aspergillus oryzae. Their structures were elucidated by detailed spectroscopic analysis and by comparison with reported data of related derivatives. Among them, compounds 1 and 2 represent the first examples of isocoumarins possessing an unusual 2-oxopropyl group and a rare 3-hydroxy-
Introduction !
Aspergillus (Trichocomaceae), a genus of filamentous fungi, consists of approximately 180 species and has been found in various climates worldwide in both terrestrial and marine environments [1]. Species in Aspergillus have been a prolific source of bioactive secondary metabolites (e.g., alkaloids, polyketides, terpenes, peptides, and polyphenols) and mass-produced industrial enzymes (e.g., glucose oxidase and lysozyme) [1–3]. Among them, Aspergillus oryzae has been safely used in various East Asian cuisines for centuries to ferment soybeans, potatoes, rice, and other grains in the making of alcoholic beverages such as huangjiu, sake, koji, and makgeolli [4]. This species was also used to transform piceid in an extract of Polygonum cuspidatum. Sieb. & Zucc. (Polygonaceae) to resveratrol, a phenolic compound possessing a wide range of pharmacological properties [5]. A previous chemical investigation of this species from red alga Heterosiphonia japonica (Dasyaceae) has resulted in the discovery of a series of novel indoloditerpenes (e.g., asporyzins A–C) [6]. In this study, our efforts on an endophytic fungus A. oryzae, isolated from the rhizome of Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz. (Liliaceae), led to the isolation and identification of four new isocoumarin
propyl group. Compounds 1 and 2 displayed moderate anti-tobacco mosaic virus activities with inhibition rates of 28.4 % and 30.6 %, respectively, at the concentration of 20 µM. The new compounds showed moderate inhibitory activities against several human tumor cell lines with IC50 values in the range of 2.8–8.8 µM. Supporting information available online at http://www.thieme-connect.de/products
derivatives (1–4) and five known ones (5–9). Herein, we report the isolation and structure elucidation of the new compounds and their activities.
Results and Discussion !
The whole culture broth of A. oryzae was extracted with ethyl acetate. The extract was subjected repeatedly to column chromatography on silica gel, MCI, RP-18, preparative high-performance liquid chromatography (HPLC), and semipreparative HPLC to afford compounds 1–9, including four new isocoumarin derivatives, oryzaeins A–D (1–4), along with five known ones (5–9), tabaisocoumarin A (5) [7], caudacoumarin C (6) [8], versicolol B (7) [9], exserolide D (8) [10], and exserolide F (9) [10]. The structures of compounds 1–9 " Fig. 1, and the 1 H and 13 C NMR are shown in l (nuclear magnetic resonance) data of 1–4 are " Table 1. listed in l Compound 1 was obtained as a pale yellow gum. Its molecular formula C15H16O5 was established by positive high-resolution electrospray ionization mass spectrometry (HR‑ESI‑MS) and 13 C NMR data, requiring 8 degrees of unsaturation. The IR (infrared) absorptions revealed the presence of hydroxyl (3435 cm−1), carbonyl
Zhou M et al. Antiviral and Cytotoxic …
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2
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming, P. R. China School of Ethnic Medicine, Yunnan Minzu University, Kunming, P. R. China Technology Center, China Tobacco Yunnan Industry Company (Ltd.), Kunming, P. R. China
Original Papers
No.
1 3 4 4a 5 6 7 8 8a 9 10 11 1′ 2′ 3′ 4′ 5′ -OMe Ar-OH a
1
H NMR and 13 C NMR spectroscopic data for compounds 1–4 (δ in ppm and J in Hz). Compound 1a
Compound 2a
Compound 3b
δC (m)
δC (m)
δC (m)
δH (m, J, Hz)
δC (m)
δH (m, J, Hz)
73.4 t 156.2 s 98.3 d 136.9 s 133.5 s 155.0 s 115.4 d 125.5 d 127.9 s 62.8 t
5.15 s
73.7 t 156.0 s 98.5 d 136.5 s 132.3 s 157.0 s 114.0 d 125.2 d 127.6 s 62.4 t
5.19 s
25.6 t 126.0 d 133.0 s 67.7 t 13.1 q
3.36 (d) 7.2 5.29 (t) 7.2
25.5 t 126.1 d 133.1 s 67.5 t 13.4 q 56.0 q
3.37 (d) 7.1 5.33 (t) 7.1
161.0 s 154.7 s 100.1 d 126.5 s 136.2 s 117.2 d 157.0 s 113.2 d 131.0 s 48.2 t 203.0 s 29.9 q 32.4 t 36.3 t 62.7 t
δH (m, J, Hz)
6.40 s
6.74 (d) 2.2 7.47 (d) 2.2 3.68 s 2.14 s 2.68 (t) 7.8 1.84 m 3.57 (t) 6.6
161.2 s 154.3 s 101.0 d 136.3 s 130.2 s 133.7 d 113.8 d 157.9 s 114.8 s 48.1 t 202.8 s 29.5 q 32.6 t 36.2 t 62.5 t
56.0 q
δH (m, J, Hz)
6.37 s
6.79 (d) 8.2 7.39 (d) 2.2
3.67 s 2.15 s 2.66 (t) 7.8 1.83 m 3.54 (t) 6.6
Compound 4b
6.20 s
6.52 (d) 8.2 6.74 (d) 8.2 4.28 s
3.93 s 1.48 s
3.82 s
10.07 s
6.22 s
6.59 (d) 8.2 6.82 (d) 8.2 4.29 s
3.95 s 1.44 s 3.82 s
9.83 s
500 and 125 MHz, in CDCl3; b 400 and 100 MHz, in CDCl3
(1728 cm−1), and aromatic or olefinic (1660 cm−1) functionalities. The 1H NMR spectrum showed characteristic signals of a hydroxyl group at δH 10.07 (1 H, s, Ar-OH), a 1,2,3,5-tetrasubstituted benzene moiety, one trisubstituted olefinic proton at δH 6.40 (1 H, s, H-4), four methylene groups, and a methyl signal at δH 2.14. The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) data exhibited fifteen carbon signals, consisting of one methyl, four methylenes (of which one was oxygenated), one olefinic and two aromatic methines, and seven quaternary carbons (including two oxygenated ones and two carbonyls). The initial analysis of these 1D NMR data indicated that compound 1 had an isocoumarin skeleton [7–10] with a hydroxyl group, a 2-oxopropyl group, and a 3-hydroxypropyl group, which were further established by the heteronuclear multiple-bond correlation (HMBC) and 1 H-1 H correlation spectroscopy (COSY) spectra analysis. Firstly, the isocoumarin skeleton was assigned by the key HMBC correlations from H-4 to C-1 (4JCH), C-3, C-4a, C-5, and C-8a, from H-6 to C-4a, C-5, C-7, and C-8, and from H-8 to C-4a, C-6, C-7, and C-8a. The HMBC correlations from H3-11 to C-9 and C-10 confirmed the presence of the 2-oxopropyl group. Similarly, the 3-hydroxypropyl group was also determined by the key HMBC correlations from H2-3′ to C-1′, along with a linear spin system of H2-1′/H2-2′/H2-3′ presented in the 1 H-1 H COSY spectrum. Furthermore, the locations of the three substituents on the isocoumarin nucleus were confirmed at C-3, C-5, and C-7 based on the HMBC correlations from H2-9 to C-1 (4JCH)/C-3/C‑4, from H2-1′ to C-4a/C-6, and from Ar-OH to C-6/C-7/C-8, respectively. Therefore, the structure of compound 1 was established as 7-hydroxy-3-(2-oxopropy)-5-(3-hydroxypropyl)isocoumarin with proposed name oryzaein A. Compound 1 is the first naturally occurring isocoumarin possessing an uncommon 2-oxopropyl moiety and a rare 3-hydroxypropyl moiety. Oryzaein B (2) was assigned the molecular formula C16H18O5 by HRESIMS. The 1D NMR spectroscopic data of 2 were similar to those of oryzaein A (1), except for the presence of the signals of ortho-coupled aromatic protons (H-6 and H-7) in 2 in place of the meta-coupled protons (H-6 and H-8) in 1. This structural as-
Zhou M et al. Antiviral and Cytotoxic …
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signment was further supported by the HMBC correlations from the methoxyl protons (δH 3.82) to C-8 (δC 157.9), as well as a linear spin system of H-6/H-7 observed in the 1 H-1 H COSY spectrum. The structure of 2 was therefore assigned as 8-methoxy-3(2-oxopropy)-5-(3-hydroxypropyl)isocoumarin. Compound 3, a pale yellow gum, had a molecular formula of " Table C15H18O4. The 1 H and 13 C NMR spectroscopic data of 3 (l 1) resembled those of co-occurring versicolol B (7) [9]. The main difference was the replacement of two methyl groups and a methoxyl group in 7 with two oxymethylene groups and a hydroxyl group in 3. The HMBC correlations from H2-9 to C-3 and C-4, from H2-4′ to C-2′, C-3′, and C-5′, and from Ar-OH to C-6, C7, and C-5 permitted the assignment of the three hydroxyl groups at C-9, C-4′, and C-6, respectively. The E geometry of the double bond between C-3′ and C-4′ was easily established from the rotating-frame nuclear Overhauser effect spectroscopy (ROESY) correlation of H2-4′ with H-2′. Consequently, compound 3 was " Fig. 1. characterized as shown in l The spectroscopic data of compound 4 indicated it to be an analogue of 3 with the molecular formula C16H20O4. Comparison of their NMR spectra revealed that a hydroxyl group in 3 was replaced with a methoxyl group (δH 3.82 and δC 56.0) in 4. In the HMBC spectrum, the correlations between the methoxy protons (δH 3.82) and C-6 (δC 157.0) also demonstrated the presence of a methoxyl group at C-6. Accordingly, compound 4 was assigned as " Fig. 1, and named oryzaein D. shown in l Since many isocoumarin derivatives have been reported to exhibit potential antivirus [8] and antitumor properties [9], the new compounds 1–4 (purities > 95 %) were tested for their antitobacco mosaic virus (anti-TMV) and cytotoxic activities. The anti-TMV activities were tested using the half-leaf method [11]. Ningnanmycin, a commercial product for plant disease in China, was used as a positive control. Their antiviral inhibition rates at the concentration of 20 µM are summarized in Table S1, Supporting Information. The results showed that compounds 1 and 2 exhibited moderate anti-TMV with inhibition rates of 28.4 % and 30.6 %, respectively. The new compounds 1–4 were also tested
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Table 1
Original Papers
for their in vitro cytotoxicities against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method, as described previously in the literature [11]. Previously, the known compound 7 has been reported to possess mild cytotoxicity [9]. In this study, the results revealed that compounds 1–4 showed moderate to weak inhibitory activities against some tested human tumor cell lines with IC50 values in the range of 2.8–8.8 µM (see Table S2, Supporting Information).
Materials and Methods !
General UV (ultraviolet) spectra were obtained using a Shimadzu UV2401 A spectrophotometer. IR spectra were obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ESI‑MS were measured on a VG Auto Spec-3000 MS spectrometer. 1 H, 13 C, and 2D NMR spectra were recorded on Bruker DRX-400 or 500 instruments with TMS (tetramethylsilane) as the internal standard. Column chromatography was performed on silica gel (200–300 mesh) or on silica gel H (10–40 µm, Qingdao Marine Chemical, Inc.). Final purifications utilized an Agilent 1100 HPLC equipped with a ZORBAX‑C18 (21.2 mm × 250 mm, 7.0 µm) column and DAD detector (diode array detector). Ningnanmycin (purity > 98%) was provided by Chengdu Institute of Biology, Chinese Academy of Sciences. Paclitaxel (purity > 95 %) was obtained from Sigma-Aldrich Company.
Fungal material The culture of A. oryzae was isolated from the rhizome of P. polyphylla var. yunnanensis, collected from Dali, Yunnan, Peopleʼs Republic of China, in 2012. A voucher specimen (Ynun-12-10-1) has been deposited in the Key Laboratory of Chemistry in Ethnic Medicinal Resources, Yunnan Minzu University. The strain was identified by one of the authors (Prof. Gang Du) based on the analysis of the ITS sequence (Genbank Accession number KM999948, see Supporting Information). 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
Chemical structures of compounds 1–9.
containing 100 mL potato dextrose broth and cultured at 28 °C on a rotary shaker at 180 rpm for 5 days. Large-scale fermentation was carried out in 150 Fernbach flasks (500 mL) each containing 100 g of rice and 120 mL of distilled H2O. Each flask was inoculated with 5.0 mL of cultured broth and incubated at 25 °C for 30 days.
Extraction and isolation The fermentation products were extracted three times with ethyl acetate (3 × 10 L) at room temperature and filtered. The crude extract (250 g) was applied to silica gel (80–100 mesh, 12 × 130 cm, 2.2 kg) column chromatography, eluting with a CHCl3-acetone gradient system (10 : 1, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5) to produce fractions A–F. Fraction B (9 : 1, 45 g) was decolorized on MCI gel after which it was eluted with 90 : 10 MeOH/H2O and was further purified by silica gel column chromatography (200–300 mesh, 2.5 × 120 cm, 0.3 kg) eluted with petroleum ether-EtOAc (10 : 1, 9 : 1, 8 : 2, 7 : 3, 1 : 1) to yield five fractions, B1–B5. Fraction B3 (CHCl3/acetone, 8 : 2; 5.25 g) was subjected to RP-18 column chromatography (8 × 50 cm, MeOH/H2O 20 : 80 to 80 : 20 gradient) to provide four fractions, B31–B34. Fraction B33 (350 mg) was subjected to preparative HPLC (ZORBAX‑C18, 7.0 µm, 21.2 mm × 250 mm, flow rate 12 mL/min, UV detection at λmax = 202, 210, 254, and 280 nm, eluted with CH3OH/H2O 68 : 32 to 75:25) to give 1 (8.5 mg, purity > 95 %), 2 (6.5 mg, purity > 95 %), 4 (4.2 mg, purity > 95%), 5 (1.8 mg, purity > 90 %), 6 (3.3 mg, purity > 90%), and 7 (14.8 mg, purity > 95 %). Fraction B4 (CHCl3/acetone, 7 : 3; 1.98 g) was subjected to RP-18 column chromatography (8 × 50 cm, MeOH/H2O 20 : 80 to 70 : 40 gradient) to provide six fractions, B41–B46. Fraction B43 (250 mg) was subjected to preparative HPLC (ZORBAX‑C18, 7.0 µm, 21.2 mm × 250 mm, flow rate 12 mL/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3OH/H2O 60 : 40) to give 3 (11.2 mg, purity > 95%), 8 (2.8 mg, purity > 90%), and 9 (1.1 mg, purity > 90 %). Oryzaein A (1), C15H16O5, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.85), 268 (3.52), 290 (3.37), 328 (3.42) nm; IR (KBr) νmax 3435, 3076, 2942, 2854, 1728, 1660, 1618, 1573, 1462, 1357, 1149, 1056 cm− 1; 1 H and 13 C NMR (500 " Table 1; ESIMS (positive ion mode) and 125 MHz, in CDCl3) see l
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Fig. 1
Original Papers
Anti-tobacco mosaic virus assays TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, P. R. China. The virus was multiplied in Nicotiana tabacum L. (Solanaceae) cv. K326 and purified as described [3]. For the half-leaf method [11], the virus was inhibited by mixing with the solution of compound. After 30 min, the mixture was inoculated on the left side of the leaves of Nicotiana glutinosa L., whereas the right side of the leaves was inoculated with the mixture of DMSO (dimethyl sulfoxide) solution and the virus as a control. The local lesion numbers were recorded 3–4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula: inhibition rate (%) = [(C–T)/C] × 100 % where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment. Ningnanmycin, a commercial virucide for plant disease in China, was used as a positive control.
Cytotoxicity assays The cytotoxicity of the compounds was tested using the MTT method, as reported previously [11], with paclitaxel as the positive control. Each tumor cell line was exposed to each test compound at various concentrations for 48 h. After the incubation, MTT was added to each well as described [11]. The optical density of the lysate was measured in a 96-well microtiter plate reader.
Zhou M et al. Antiviral and Cytotoxic …
Planta Med
The IC50 value of each compound was calculated by Reed and Muenchʼs method [12].
Supporting information The spectroscopic data of the new compounds 1–4 are available as Supporting Information.
Acknowledgements !
This research was supported by the National Natural Science Foundation of China (No. 31 560 099), 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 start-up funds of Yunnan Minzu University.
Conflict of Interest !
The authors declare no conflict of interest.
References 1 Sanchez JF, Somoza AD, Keller NP, Wang CC. Advances in Aspergillus secondary metabolite research in the post-genomic era. Nat Prod Rep 2012; 29: 351–371 2 Zhou YM, Debbab A, Mándi A, Wray V, Schulz B, Müller WEG, Kassack M, Lin WH, Kurtán T, Proksch P, Aly AH. Alkaloids from the sponge-associated fungus Aspergillus sp. Eur J Org Chem 2013; 32: 894–906 3 Zhou M, Miao MM, Du G, Li XN, Shang SZ, Zhao W, Liu ZH, Yang GY, Che CT, Hu QF, Gao XM. Aspeverin, Aspergillines A–E, highly oxygenated hexacyclic indole-tetrahydrofuran-tetramic acid derivatives from Aspergillus versicolor. Org Lett 2014; 16: 5016–5019 4 Rokas A. The effect of domestication on the fungal proteome. Trends Genet 2009; 25: 60–63 5 Wang H, Liu L, Guo YX, Dong YS, Zhang DJ, Xiu ZL. Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae. Appl Microbiol Biotechnol 2007; 75: 763–768 6 Qiao MF, Ji NY, Liu XH, Li K, Zhu QM, Xue QZ. Indoloditerpenes from an algicolous isolate of Aspergillus oryzae. Bioorg Med Chem Lett 2010; 20: 5677–5680 7 Shang SZ, Xu WX, Li L, Tang JG, Zhao W, Lei P, Miao MM, Sun HD, Pu JX, Chen YK, Yang GY. Antiviral isocoumarins from the roots and stems of Nicotiana tabacum. Phytochem Lett 2015; 11: 53–56 8 Ye YQ, Xia CF, Yang JX, Yang YC, Gao XM, Du G, Yang HY, Li YK, Hu QF. Isocoumarins from the back of Lindera caudate. Heterocycles 2014; 89: 2365–2374 9 Zhou M, Lou J, Li YK, Wang YD, Zhou K, Ji BK, Dong W, Gao XM, Du G, Hu QF. Versicolols A and B, two new prenylated isocoumarins from endophytic fungus Aspergillus versicolor and their cytotoxic activity. Arch Pharm Res, advance online publication 7 March 2015; DOI: 10.1007/ s12272-015–0588-5 10 Li RX, Chen SX, Niu SB, Guo LD, Yin J, Che YS. Exserolides A–F, new isocoumarin derivatives from the plant endophytic fungus Exserohilum sp. Fitoterapia 2014; 96: 88–94 11 Zhou M, Du G, Yang HY, Xia CF, Yang JX, Li XN, Ye YQ, Gao XM, Du G, Hu QF. Antiviral butyrolactones from the endophytic fungus Aspergillus versicolor. Planta Med 2015; 81: 235–240 12 Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Epidemiol 1938; 27: 493–497
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m/z 299 [M + Na]+; HRESIMS (positive ion mode) m/z 299.0890 [M + Na]+ (calcd. 299.0895 for C15H16O5Na). Oryzaein B (2), C16H18O5, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.78), 270 (3.57), 288 (3.32), 325 (3.36) nm; IR (KBr) νmax 3430, 3085, 2957, 2848, 1724, 1654, 1615, 1568, 1469, 1350, 1182, 1128, 1071 cm− 1; 1 H and 13 C NMR " Table 1; ESIMS (positive ion (500 and 125 MHz, in CDCl3) see l + mode) m/z 313 [M + Na] ; HRESIMS (positive ion mode) m/z 313.1058 [M + Na]+ (calcd. 313.1052 for C16H18O5Na). Oryzaein C (3), C15H18O4, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.65), 270 (3.36), 292 (3.14), 315 (3.27) nm; IR (KBr) νmax 3422, 3068, 2963, 2856, 1612, 1573, 1463, 1354, 1165, 1054 cm− 1; 1 H and 13 C NMR (500 and " Table 1; ESIMS (positive ion mode) m/z 125 MHz, in CDCl3) see l 285 [M + Na]+; HRESIMS (positive ion mode) m/z 285.1109 [M + Na]+ (calcd. 285.1103 for C15H18O4Na). Oryzaein D (4), C16H20O4, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.72), 272 (3.41), 294 (3.18), 318 (3.30) nm; IR (KBr) νmax 3415, 3063, 2960, 2859, 1615, 1562, 1457, 1342, 1160, 1068 cm− 1; 1 H and 13 C NMR (500 and " Table 1; ESIMS (positive ion mode) m/z 125 MHz, in CDCl3) see l 299 [M + Na]+; HRESIMS (positive ion mode) m/z 299.1252 [M + Na]+ (calcd. 299.1259 for C16H20O4Na).
Antiviral and Cytotoxic Isocoumarin Derivatives from an Endophytic Fungus Aspergillus oryzae
Authors
Min Zhou 1, 2, 3, Kun Zhou 1, 2, Pei He 3, Kun-Miao Wang 3, Rui-Zhi Zhu 3, Yue-De Wang 1, 2, Wei Dong 1, 2, Gan-Peng Li 1, 2, Hai-Ying Yang 1, Yan-Qing Ye 1, Gang Du 1, 2, Xue-Mei Li 3, Qiu-Fen Hu 1, 2
Affiliations
1
3
Key words " Aspergillus oryzae l " Trichocomaceae l " isocoumarin derivatives l " anti‑tobacco mosaic virus l activity " cytotoxicity l
received revised accepted
Sept. 14, 2015 Nov. 3, 2015 Nov. 9, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1558331 Published online Planta Med © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Qiu-Fen Hu Key Laboratory of Chemistry in Ethnic Medicinal Resources State Ethnic Affairs Commission & Ministry of Education Yunnan Minzu University 121 Road 134 Kunming 650031 P. R. China Phone: + 86 8 71 65 91 30 43 Fax: + 86 8 71 65 91 00 17 [email protected] Correspondence Prof. Xue-Mei Li Technology Center China Tobacco Yunnan Industry Company (Ltd.) Keyi Road 41 Kunming 650000 P. R. China Phone: + 86 8 71 68 31 96 56 Fax: + 86 8 71 68 31 90 18 [email protected]
Abstract !
Oryzaeins A–D (1-4), four new isocoumarin derivatives, along with five known ones (5-9) were isolated from solid cultures of an endophytic fungus Aspergillus oryzae. Their structures were elucidated by detailed spectroscopic analysis and by comparison with reported data of related derivatives. Among them, compounds 1 and 2 represent the first examples of isocoumarins possessing an unusual 2-oxopropyl group and a rare 3-hydroxy-
Introduction !
Aspergillus (Trichocomaceae), a genus of filamentous fungi, consists of approximately 180 species and has been found in various climates worldwide in both terrestrial and marine environments [1]. Species in Aspergillus have been a prolific source of bioactive secondary metabolites (e.g., alkaloids, polyketides, terpenes, peptides, and polyphenols) and mass-produced industrial enzymes (e.g., glucose oxidase and lysozyme) [1–3]. Among them, Aspergillus oryzae has been safely used in various East Asian cuisines for centuries to ferment soybeans, potatoes, rice, and other grains in the making of alcoholic beverages such as huangjiu, sake, koji, and makgeolli [4]. This species was also used to transform piceid in an extract of Polygonum cuspidatum. Sieb. & Zucc. (Polygonaceae) to resveratrol, a phenolic compound possessing a wide range of pharmacological properties [5]. A previous chemical investigation of this species from red alga Heterosiphonia japonica (Dasyaceae) has resulted in the discovery of a series of novel indoloditerpenes (e.g., asporyzins A–C) [6]. In this study, our efforts on an endophytic fungus A. oryzae, isolated from the rhizome of Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz. (Liliaceae), led to the isolation and identification of four new isocoumarin
propyl group. Compounds 1 and 2 displayed moderate anti-tobacco mosaic virus activities with inhibition rates of 28.4 % and 30.6 %, respectively, at the concentration of 20 µM. The new compounds showed moderate inhibitory activities against several human tumor cell lines with IC50 values in the range of 2.8–8.8 µM. Supporting information available online at http://www.thieme-connect.de/products
derivatives (1–4) and five known ones (5–9). Herein, we report the isolation and structure elucidation of the new compounds and their activities.
Results and Discussion !
The whole culture broth of A. oryzae was extracted with ethyl acetate. The extract was subjected repeatedly to column chromatography on silica gel, MCI, RP-18, preparative high-performance liquid chromatography (HPLC), and semipreparative HPLC to afford compounds 1–9, including four new isocoumarin derivatives, oryzaeins A–D (1–4), along with five known ones (5–9), tabaisocoumarin A (5) [7], caudacoumarin C (6) [8], versicolol B (7) [9], exserolide D (8) [10], and exserolide F (9) [10]. The structures of compounds 1–9 " Fig. 1, and the 1 H and 13 C NMR are shown in l (nuclear magnetic resonance) data of 1–4 are " Table 1. listed in l Compound 1 was obtained as a pale yellow gum. Its molecular formula C15H16O5 was established by positive high-resolution electrospray ionization mass spectrometry (HR‑ESI‑MS) and 13 C NMR data, requiring 8 degrees of unsaturation. The IR (infrared) absorptions revealed the presence of hydroxyl (3435 cm−1), carbonyl
Zhou M et al. Antiviral and Cytotoxic …
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2
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming, P. R. China School of Ethnic Medicine, Yunnan Minzu University, Kunming, P. R. China Technology Center, China Tobacco Yunnan Industry Company (Ltd.), Kunming, P. R. China
Original Papers
No.
1 3 4 4a 5 6 7 8 8a 9 10 11 1′ 2′ 3′ 4′ 5′ -OMe Ar-OH a
1
H NMR and 13 C NMR spectroscopic data for compounds 1–4 (δ in ppm and J in Hz). Compound 1a
Compound 2a
Compound 3b
δC (m)
δC (m)
δC (m)
δH (m, J, Hz)
δC (m)
δH (m, J, Hz)
73.4 t 156.2 s 98.3 d 136.9 s 133.5 s 155.0 s 115.4 d 125.5 d 127.9 s 62.8 t
5.15 s
73.7 t 156.0 s 98.5 d 136.5 s 132.3 s 157.0 s 114.0 d 125.2 d 127.6 s 62.4 t
5.19 s
25.6 t 126.0 d 133.0 s 67.7 t 13.1 q
3.36 (d) 7.2 5.29 (t) 7.2
25.5 t 126.1 d 133.1 s 67.5 t 13.4 q 56.0 q
3.37 (d) 7.1 5.33 (t) 7.1
161.0 s 154.7 s 100.1 d 126.5 s 136.2 s 117.2 d 157.0 s 113.2 d 131.0 s 48.2 t 203.0 s 29.9 q 32.4 t 36.3 t 62.7 t
δH (m, J, Hz)
6.40 s
6.74 (d) 2.2 7.47 (d) 2.2 3.68 s 2.14 s 2.68 (t) 7.8 1.84 m 3.57 (t) 6.6
161.2 s 154.3 s 101.0 d 136.3 s 130.2 s 133.7 d 113.8 d 157.9 s 114.8 s 48.1 t 202.8 s 29.5 q 32.6 t 36.2 t 62.5 t
56.0 q
δH (m, J, Hz)
6.37 s
6.79 (d) 8.2 7.39 (d) 2.2
3.67 s 2.15 s 2.66 (t) 7.8 1.83 m 3.54 (t) 6.6
Compound 4b
6.20 s
6.52 (d) 8.2 6.74 (d) 8.2 4.28 s
3.93 s 1.48 s
3.82 s
10.07 s
6.22 s
6.59 (d) 8.2 6.82 (d) 8.2 4.29 s
3.95 s 1.44 s 3.82 s
9.83 s
500 and 125 MHz, in CDCl3; b 400 and 100 MHz, in CDCl3
(1728 cm−1), and aromatic or olefinic (1660 cm−1) functionalities. The 1H NMR spectrum showed characteristic signals of a hydroxyl group at δH 10.07 (1 H, s, Ar-OH), a 1,2,3,5-tetrasubstituted benzene moiety, one trisubstituted olefinic proton at δH 6.40 (1 H, s, H-4), four methylene groups, and a methyl signal at δH 2.14. The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) data exhibited fifteen carbon signals, consisting of one methyl, four methylenes (of which one was oxygenated), one olefinic and two aromatic methines, and seven quaternary carbons (including two oxygenated ones and two carbonyls). The initial analysis of these 1D NMR data indicated that compound 1 had an isocoumarin skeleton [7–10] with a hydroxyl group, a 2-oxopropyl group, and a 3-hydroxypropyl group, which were further established by the heteronuclear multiple-bond correlation (HMBC) and 1 H-1 H correlation spectroscopy (COSY) spectra analysis. Firstly, the isocoumarin skeleton was assigned by the key HMBC correlations from H-4 to C-1 (4JCH), C-3, C-4a, C-5, and C-8a, from H-6 to C-4a, C-5, C-7, and C-8, and from H-8 to C-4a, C-6, C-7, and C-8a. The HMBC correlations from H3-11 to C-9 and C-10 confirmed the presence of the 2-oxopropyl group. Similarly, the 3-hydroxypropyl group was also determined by the key HMBC correlations from H2-3′ to C-1′, along with a linear spin system of H2-1′/H2-2′/H2-3′ presented in the 1 H-1 H COSY spectrum. Furthermore, the locations of the three substituents on the isocoumarin nucleus were confirmed at C-3, C-5, and C-7 based on the HMBC correlations from H2-9 to C-1 (4JCH)/C-3/C‑4, from H2-1′ to C-4a/C-6, and from Ar-OH to C-6/C-7/C-8, respectively. Therefore, the structure of compound 1 was established as 7-hydroxy-3-(2-oxopropy)-5-(3-hydroxypropyl)isocoumarin with proposed name oryzaein A. Compound 1 is the first naturally occurring isocoumarin possessing an uncommon 2-oxopropyl moiety and a rare 3-hydroxypropyl moiety. Oryzaein B (2) was assigned the molecular formula C16H18O5 by HRESIMS. The 1D NMR spectroscopic data of 2 were similar to those of oryzaein A (1), except for the presence of the signals of ortho-coupled aromatic protons (H-6 and H-7) in 2 in place of the meta-coupled protons (H-6 and H-8) in 1. This structural as-
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signment was further supported by the HMBC correlations from the methoxyl protons (δH 3.82) to C-8 (δC 157.9), as well as a linear spin system of H-6/H-7 observed in the 1 H-1 H COSY spectrum. The structure of 2 was therefore assigned as 8-methoxy-3(2-oxopropy)-5-(3-hydroxypropyl)isocoumarin. Compound 3, a pale yellow gum, had a molecular formula of " Table C15H18O4. The 1 H and 13 C NMR spectroscopic data of 3 (l 1) resembled those of co-occurring versicolol B (7) [9]. The main difference was the replacement of two methyl groups and a methoxyl group in 7 with two oxymethylene groups and a hydroxyl group in 3. The HMBC correlations from H2-9 to C-3 and C-4, from H2-4′ to C-2′, C-3′, and C-5′, and from Ar-OH to C-6, C7, and C-5 permitted the assignment of the three hydroxyl groups at C-9, C-4′, and C-6, respectively. The E geometry of the double bond between C-3′ and C-4′ was easily established from the rotating-frame nuclear Overhauser effect spectroscopy (ROESY) correlation of H2-4′ with H-2′. Consequently, compound 3 was " Fig. 1. characterized as shown in l The spectroscopic data of compound 4 indicated it to be an analogue of 3 with the molecular formula C16H20O4. Comparison of their NMR spectra revealed that a hydroxyl group in 3 was replaced with a methoxyl group (δH 3.82 and δC 56.0) in 4. In the HMBC spectrum, the correlations between the methoxy protons (δH 3.82) and C-6 (δC 157.0) also demonstrated the presence of a methoxyl group at C-6. Accordingly, compound 4 was assigned as " Fig. 1, and named oryzaein D. shown in l Since many isocoumarin derivatives have been reported to exhibit potential antivirus [8] and antitumor properties [9], the new compounds 1–4 (purities > 95 %) were tested for their antitobacco mosaic virus (anti-TMV) and cytotoxic activities. The anti-TMV activities were tested using the half-leaf method [11]. Ningnanmycin, a commercial product for plant disease in China, was used as a positive control. Their antiviral inhibition rates at the concentration of 20 µM are summarized in Table S1, Supporting Information. The results showed that compounds 1 and 2 exhibited moderate anti-TMV with inhibition rates of 28.4 % and 30.6 %, respectively. The new compounds 1–4 were also tested
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Table 1
Original Papers
for their in vitro cytotoxicities against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method, as described previously in the literature [11]. Previously, the known compound 7 has been reported to possess mild cytotoxicity [9]. In this study, the results revealed that compounds 1–4 showed moderate to weak inhibitory activities against some tested human tumor cell lines with IC50 values in the range of 2.8–8.8 µM (see Table S2, Supporting Information).
Materials and Methods !
General UV (ultraviolet) spectra were obtained using a Shimadzu UV2401 A spectrophotometer. IR spectra were obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ESI‑MS were measured on a VG Auto Spec-3000 MS spectrometer. 1 H, 13 C, and 2D NMR spectra were recorded on Bruker DRX-400 or 500 instruments with TMS (tetramethylsilane) as the internal standard. Column chromatography was performed on silica gel (200–300 mesh) or on silica gel H (10–40 µm, Qingdao Marine Chemical, Inc.). Final purifications utilized an Agilent 1100 HPLC equipped with a ZORBAX‑C18 (21.2 mm × 250 mm, 7.0 µm) column and DAD detector (diode array detector). Ningnanmycin (purity > 98%) was provided by Chengdu Institute of Biology, Chinese Academy of Sciences. Paclitaxel (purity > 95 %) was obtained from Sigma-Aldrich Company.
Fungal material The culture of A. oryzae was isolated from the rhizome of P. polyphylla var. yunnanensis, collected from Dali, Yunnan, Peopleʼs Republic of China, in 2012. A voucher specimen (Ynun-12-10-1) has been deposited in the Key Laboratory of Chemistry in Ethnic Medicinal Resources, Yunnan Minzu University. The strain was identified by one of the authors (Prof. Gang Du) based on the analysis of the ITS sequence (Genbank Accession number KM999948, see Supporting Information). 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
Chemical structures of compounds 1–9.
containing 100 mL potato dextrose broth and cultured at 28 °C on a rotary shaker at 180 rpm for 5 days. Large-scale fermentation was carried out in 150 Fernbach flasks (500 mL) each containing 100 g of rice and 120 mL of distilled H2O. Each flask was inoculated with 5.0 mL of cultured broth and incubated at 25 °C for 30 days.
Extraction and isolation The fermentation products were extracted three times with ethyl acetate (3 × 10 L) at room temperature and filtered. The crude extract (250 g) was applied to silica gel (80–100 mesh, 12 × 130 cm, 2.2 kg) column chromatography, eluting with a CHCl3-acetone gradient system (10 : 1, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5) to produce fractions A–F. Fraction B (9 : 1, 45 g) was decolorized on MCI gel after which it was eluted with 90 : 10 MeOH/H2O and was further purified by silica gel column chromatography (200–300 mesh, 2.5 × 120 cm, 0.3 kg) eluted with petroleum ether-EtOAc (10 : 1, 9 : 1, 8 : 2, 7 : 3, 1 : 1) to yield five fractions, B1–B5. Fraction B3 (CHCl3/acetone, 8 : 2; 5.25 g) was subjected to RP-18 column chromatography (8 × 50 cm, MeOH/H2O 20 : 80 to 80 : 20 gradient) to provide four fractions, B31–B34. Fraction B33 (350 mg) was subjected to preparative HPLC (ZORBAX‑C18, 7.0 µm, 21.2 mm × 250 mm, flow rate 12 mL/min, UV detection at λmax = 202, 210, 254, and 280 nm, eluted with CH3OH/H2O 68 : 32 to 75:25) to give 1 (8.5 mg, purity > 95 %), 2 (6.5 mg, purity > 95 %), 4 (4.2 mg, purity > 95%), 5 (1.8 mg, purity > 90 %), 6 (3.3 mg, purity > 90%), and 7 (14.8 mg, purity > 95 %). Fraction B4 (CHCl3/acetone, 7 : 3; 1.98 g) was subjected to RP-18 column chromatography (8 × 50 cm, MeOH/H2O 20 : 80 to 70 : 40 gradient) to provide six fractions, B41–B46. Fraction B43 (250 mg) was subjected to preparative HPLC (ZORBAX‑C18, 7.0 µm, 21.2 mm × 250 mm, flow rate 12 mL/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3OH/H2O 60 : 40) to give 3 (11.2 mg, purity > 95%), 8 (2.8 mg, purity > 90%), and 9 (1.1 mg, purity > 90 %). Oryzaein A (1), C15H16O5, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.85), 268 (3.52), 290 (3.37), 328 (3.42) nm; IR (KBr) νmax 3435, 3076, 2942, 2854, 1728, 1660, 1618, 1573, 1462, 1357, 1149, 1056 cm− 1; 1 H and 13 C NMR (500 " Table 1; ESIMS (positive ion mode) and 125 MHz, in CDCl3) see l
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Fig. 1
Original Papers
Anti-tobacco mosaic virus assays TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, P. R. China. The virus was multiplied in Nicotiana tabacum L. (Solanaceae) cv. K326 and purified as described [3]. For the half-leaf method [11], the virus was inhibited by mixing with the solution of compound. After 30 min, the mixture was inoculated on the left side of the leaves of Nicotiana glutinosa L., whereas the right side of the leaves was inoculated with the mixture of DMSO (dimethyl sulfoxide) solution and the virus as a control. The local lesion numbers were recorded 3–4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula: inhibition rate (%) = [(C–T)/C] × 100 % where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment. Ningnanmycin, a commercial virucide for plant disease in China, was used as a positive control.
Cytotoxicity assays The cytotoxicity of the compounds was tested using the MTT method, as reported previously [11], with paclitaxel as the positive control. Each tumor cell line was exposed to each test compound at various concentrations for 48 h. After the incubation, MTT was added to each well as described [11]. The optical density of the lysate was measured in a 96-well microtiter plate reader.
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The IC50 value of each compound was calculated by Reed and Muenchʼs method [12].
Supporting information The spectroscopic data of the new compounds 1–4 are available as Supporting Information.
Acknowledgements !
This research was supported by the National Natural Science Foundation of China (No. 31 560 099), 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 start-up funds of Yunnan Minzu University.
Conflict of Interest !
The authors declare no conflict of interest.
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m/z 299 [M + Na]+; HRESIMS (positive ion mode) m/z 299.0890 [M + Na]+ (calcd. 299.0895 for C15H16O5Na). Oryzaein B (2), C16H18O5, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.78), 270 (3.57), 288 (3.32), 325 (3.36) nm; IR (KBr) νmax 3430, 3085, 2957, 2848, 1724, 1654, 1615, 1568, 1469, 1350, 1182, 1128, 1071 cm− 1; 1 H and 13 C NMR " Table 1; ESIMS (positive ion (500 and 125 MHz, in CDCl3) see l + mode) m/z 313 [M + Na] ; HRESIMS (positive ion mode) m/z 313.1058 [M + Na]+ (calcd. 313.1052 for C16H18O5Na). Oryzaein C (3), C15H18O4, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.65), 270 (3.36), 292 (3.14), 315 (3.27) nm; IR (KBr) νmax 3422, 3068, 2963, 2856, 1612, 1573, 1463, 1354, 1165, 1054 cm− 1; 1 H and 13 C NMR (500 and " Table 1; ESIMS (positive ion mode) m/z 125 MHz, in CDCl3) see l 285 [M + Na]+; HRESIMS (positive ion mode) m/z 285.1109 [M + Na]+ (calcd. 285.1103 for C15H18O4Na). Oryzaein D (4), C16H20O4, obtained as a pale yellow gum; UV (MeOH) λmax (log ε): 210 (3.72), 272 (3.41), 294 (3.18), 318 (3.30) nm; IR (KBr) νmax 3415, 3063, 2960, 2859, 1615, 1562, 1457, 1342, 1160, 1068 cm− 1; 1 H and 13 C NMR (500 and " Table 1; ESIMS (positive ion mode) m/z 125 MHz, in CDCl3) see l 299 [M + Na]+; HRESIMS (positive ion mode) m/z 299.1252 [M + Na]+ (calcd. 299.1259 for C16H20O4Na).
