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Drimane-type sesquiterpenoids from cultures of the fungus Xylaria polymorpha
Phytochemistry Letters 20 (2017) 13–16
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Drimane-type sesquiterpenoids from cultures of the fungus Xylaria polymorpha Ning Ning Yanga,b , Fan Dong Kongb , Qing Yun Mab , Sheng Zhuo Huangb , Du Qiang Luoc , Li Man Zhoub , Hao Fu Daib , Zhi Fang Yua,* , You Xing Zhaob,* a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China c College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China b
A R T I C L E I N F O
Article history: Received 19 October 2016 Received in revised form 27 February 2017 Accepted 3 March 2017 Available online xxx Keywords: Sesquiterpenoid Xylaria polymorpha X-ray diffraction analysis Anti-AChE inhibitory activity
A B S T R A C T
Three new drimane-type sesquiterpenoids named xylariaines A–C (1–3), together with one known analogue (4), were isolated from the ethyl acetate extract of cultures of the fungus Xylaria polymorpha (Pers.: Fr.) Grer. Their structures were elucidated unambiguously by NMR and single-crystal X-ray diffraction analysis. Compounds 1 and 2 exhibited weak anti-acetylcholinesterase activities at a concentration of 50 mg/mL with inhibition ratios of 12.4% and 18.0%, respectively. © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction
2. Result and discussion
Xylaria polymorpha (Pers.: Fr.) Grer. is a small black mushroom belonging to the genus Xylaria, which is widely distributed in the southeast of mainland China, mainly in Hainan, Guangdong, Fujian and Taiwan provinces. So far, a few researches on chemical constituents of this fungus were reported and led to the isolation of some different kinds of natural products including xylarinols A and B with moderate ABTS radical scavenging activity (Lee et al., 2009), three isopimarane diterpene glycosides with cytotoxicity against human cancer cell lines (Shiono et al., 2009), and xylarinic acids A and B with significant antifungal activity (Jang et al., 2007). In a search for new bioactive compounds from X. polymorpha, the deep chemical investigation on cultures of this fungus was thus undertaken, which led to the isolation of three new drimanetype sesquiterpenoids named xylariaines A–C (1–3) along with one known analogue (4). The AChE inhibitory activity of the isolates (1– 4) was tested. Herein, this paper describes the isolation, structure elucidation, and AChE inhibitory activities of these compounds.
Compound 1 was obtained as white amorphous powder, and its molecular formula was assigned to be C15H28O4 with two degrees of unsaturation according to its positive HRESIMS (m/z 295.1877 [M +Na]+, calcd. 295.1880 for C15H28O4Na) and NMR spectroscopic data (Table 1). The IR spectrum displayed absorption band (3433 cm1) for hydroxyls. The 1H NMR (Table 1) showed the presence of signals for three singlet methyls [d 0.89 (3H, s, H-14), 0.83 (3H, s, H-15), 0.90 (3H, s, H-13)], two pairs of oxymethylene protons, and one oxymethine proton. The 13C NMR spectrum displayed 15 carbon resonances for three methyls, six methlenes (two oxygenated), three methines (one oxygenated), and three quarternary carbons (one oxygenated). These data indicated that 1 might be characteristic as sesquiterpenoid. In addition, detailed analysis of NMR data suggested that compound 1 should be with a similar planar structure to that of agripilol A (Shan et al., 2011), a drimane-type sesquiterpenoid, bearing a hydroxyl group at C-1 (dC 79.8) rather than at C-3 as in agripilol A. This conjecture was evidenced by 1H–1H COSY correlations from the oxymehine proton H-1 [dH 3.38 dd (4.5, 11.5)] to H2-3 through H2-2 as well as HMBC correlations from H3-13 [dH (0.90, s)] to C-1, C-5 (dC 55.4), C-9 (dC 63.9), and C-10 (dC 44.6). Other correlations in the HMBC and 1 H–1H COSY spectra (Fig. 2) further supported the atom connectivity in compound 1. The relative configuration of the drimane sesquiterpenoid skeleton in compound 1 was elucidated
* Corresponding authors. E-mail addresses: [email protected] (Z.F. Yu), [email protected] (Y.X. Zhao).
http://dx.doi.org/10.1016/j.phytol.2017.03.003 1874-3900/© 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
14 Table 1 1 H (500 MHz) and Position
N.N. Yang et al. / Phytochemistry Letters 20 (2017) 13–16
13
C (125 MHz) NMR spectroscopic data for compounds 1–3 in CD3OD. 1
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
79.8, CH
3.38(dd, 4.5, 11.5)
80.4, CH
3.36 (dd, 7.5, 8.5)
40.8, CH2
2
28.2, CH2
37.1, CH2
41.1, CH2
1.76, m 1.74, m 3.27 (dd, 7.0, 8.5)
27.8, CH2
3
1.70, m 1.60, m 1.41, m 1.32, m
1.65, m 1.08, m 1.64, m 1.59, m 3.19 (dd, 4.5, 11.5)
4 5 6
33.9, C 55.4, CH 20.8, CH2
7
37.9, CH2
8 9 10 11
75.4, 63.9, 44.6, 58.8,
12
64.1, CH2
13 14 15
12.5, CH3 21.8, CH3 33.5, CH3
C CH C CH2
76.9, CH
0.96 (dd, 2.0, 12.0) 1.66, m 1.35, m 1.22, m 2.20 (ddd, 3.0, 3.0, 13.0) 1.53 (dd, 1.5, 8.5) 4.13 (dd, 1.5, 11.5) 3.70 (dd, 8.5, 11.5) 3.51 (d, 11.5) 3.23 (d, 11.5) 0.90 s 0.89 s 0.83 s
39.7, C 50.1, CH 19.0, CH2 32.8, CH2 74.0, C 57.5, CH 42.1, C 70.5, CH2
0.91 (dd, 2.0, 12.0) 1.68, m 1.29, m 2.39, m 1.58, m 2.06 (d, 5.5) 4.70 (br d, 9.5) 4.52 (dd, 5.5, 9.5
180.1, C 10.0, CH3 16.6, CH3 29.0, CH3
by ROESY experiment (Fig. 3) and determined to be the same as those of agripilol A. The a-orientation of H-1 was proposed from the ROESY correlation of H-1 [dH 3.38 dd (4.5, 11.5)] with H-9 [dH 1.53 dd (1.5, 8.5)]. Thus, compound 1 was assigned as shown in Fig. 1, and named xylariaine A. Compound 2 was purified as white crystal. Its molecular formula C15H24O5 was determined on the basis of the HRESIMS (m/ z 307.1513 [M+Na]+, calcd. 307.1516 for C15H24O5Na) and NMR spectroscopic data (Table 1). Its IR spectrum showed the presence of hydroxyls (3429 cm1) and lactone carbonyl (1762 cm1). The 1H NMR spectroscopic data of 2 (Table 1) revealed the presence of three singlet methyls [d 0.83 (3H, s, H-13), 0.77 (3H, s, H-14), 1.00 (3H, s, H-15)]. The 13C NMR and DEPT spectra (Table 1) of 2 displayed 15 carbon resonances, including three methyls, four methylenes (one oxygenated), four methines, and four quaternary carbons (one carbonyl and one oxygenated). These data suggested that 2 also had a sesquiterpenoid skeleton with a tricyclic system. Comparison of its NMR spectroscopic data with those of 3bhydroxypeniopholide (William and Latchezar, 1992) suggested that they were structurally close related, except for an additional hydroxyl located at C-1 (dC 80.4) in compound 2, as demonstrated by HMBC correlations from H1-9[dH 2.06 d (5.5)] and H3-13[dH (0.83, s)] to the same oxygenated carbon C-1. In the ROESY spectrum, correlations of Ha-11[dH 4.52 dd (5.5, 9.5)]/H3-13/H3-14 [dH (0.77, s)], H-1/H-9/H-3/H-5/H-1, and H-5/H3-15 indicated that
79.5, CH 39.7, C 53.4, CH 20.5, CH2 34.7, CH2 80.3, C 62.6, CH 36.7, C 70.0, CH2 77.5, CH2
0.83 s 0.77 s 1.00 s
14.9, CH3 16.3, CH3 28.8, CH3
0.98 (dd, 2.0, 11.5) 1.67, m 1.38, m 2.10, m 1.62, m 1.56 (d, 6.0) 3.80 (d, 8.5) 4.05 (dd, 6.0, 8.5) 3.71 (d, 9.0) 3.58 (d, 9.0) 0.92 s 0.80 s 1.02 s
the relative configuration of 2 was the same as 3b-hydroxypeniopholide, except for the a-oriented H-1 proton. To further verify the above deduction and determine the absolute configuration of 2, a single-crystal X-ray diffraction pattern was obtained using the anomalous scattering of Cu Ka radiation. Fig. 4 shows an ORTEP drawing and unambiguously demonstrates the absolute configuration of 1R, 3S, 5S, 8R, 9S, and 10S for 2. Thus, compound 2 was assigned as shown in Fig. 1, and named xylariaine B. Compound 3 was isolated as a white amorphous powder, and its molecular formula of C15H26O3 was deduced from its molecular ion peak of HRESIMS at m/z 277.1772 [M+Na]+ (calcd. 277.1774 for C15H26O3Na), equivalent to that of agripilol A by loss of one H2O unit. Besides, the 13C NMR data of 3 were nearly identical with those of the known compound agripilol A. The above data led to a deduction that 3 could be a dehydration derivative of agripilol A via losing a molecule of water between two hydroxyl groups located at C-11 and C-12 or C-8 in agripilol A to form a cyclic ether moiety. In the HMBC spectrum of 3, correlations from H-12 [dH 3.71 d (9.0), 3.58 d (9.0)] to C-11 (dC 70.0) confirmed the connection of C-12 (dC 77.5) with C-11 through an O atom. The assignment of compound 3 was also corroborated by the analysis of other correlations in HMBC and 1H–1H COSY spectra (Fig. 2). The relative configuration of compound 3 was also elucidated by ROESY experiment (Fig. 3), and assigned to be same as that of agripilol A by correlations of H-5 [dH 0.98 dd (2.0, 11.5)] with H-3[dH 3.19 dd (4.5, 11.5)], H-9[dH 1.56 d
Fig. 1. Structures of compounds 1–4.
N.N. Yang et al. / Phytochemistry Letters 20 (2017) 13–16
15
Fig. 2. Key COSY and HMBC correlations of 1–3.
Fig. 3. Key NOE correlations of 1–3.
(6.0)], and H-15[dH (1.02, s)] and correlations of H3-13[dH (0.92, s)] with H3-14[dH (0.80, s)], Hb-11[dH 3.80 d (8.5)] and Hb-12[dH 3.58 d (9.0)]. Thus, compound 3 was elucidated as shown in Fig. 1, and named xylariaine C. The known compound (4) was determined to be sulphureuine B (He et al., 2015) by comparison of its spectroscopic data with that of literature. Besides, the absolute configuration of 4 was originally established as 3S, 5R, 8R, 9R, and 10S by Cu-Ka X-ray diffraction in our study (Fig. 4). The acetylcholinesterase inhibitory activity of compounds 1–4 were tested by previous method as described in the literature. The result showed that compounds 1 and 2 exhibited weak anti-AChE activities with inhibition rates of 12.4% and 18.0%, respectively, at a concentration of 50 mg/mL (Tacrine as positive control, inhibitory rate: 56.7%). 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Rudolph Autopol III polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Shimadzu UV-2550 spectrometer (Beckman, Brea, CA, USA) was used for scanning UV spectroscopy. IR spectra were obtained on a Tensor 27 spectrometer, as KBr pellets (Thermo, Pittsburgh, PA, USA). HR-ESI-MS were performed on an API QSTAR Pulsar mass spectrometer (Bruker, Bremen, Germany). NMR spectra were
recorded on a AV-500 spectrometer (Bruker, Bremen, Germany) with TMS (Tetramethylsilane) as an internal standard. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 (40–70 mm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography (CC). Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm 25 cm, column. Fractions were monitored by TLC (thin-layer chromatography) and spots were visualized by heating after spraying with 5% H2SO4 in ethanol. 3.2. Fungal material The fungus Xylaria polymorpha was collected in the valley of Yinggeling, Hainan province of China, in June 2015, and identified by Associate Professor Sheng-zhuo Huang, the Institute of Tropical Bioscience and Biotechnology. The mycelium was isolated from the X. polymorpha and its strain was maintained on potato dextrose agar (PDA) slant at 4 C. A voucher specimen (YGL-017) was deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China. 3.3. Fermentation, extraction and isolation The fungus was cultured on rice medium in conical flasks (1 L) at 25 C for a week. Two pieces of mycelial agar plugs (0.5 0.5 cm2) were inoculated into 1 L Erlenmeyer flasks containing damp white
Fig. 4. ORTEP drawings of compounds 2 and 4.
16
N.N. Yang et al. / Phytochemistry Letters 20 (2017) 13–16
rice (50 g rice plus 100 mL H2O). The fermentation was carried out on a shaker at 25 C and 150 rpm for 7 days, and allowed to proceed under static conditions at 25 C for 28 days. The culture broth (100 L) was filtered to give the filtrate and mycelia. The filtrate was evaporated in vacuo to a small volume and then suspended in H2O and partitioned successively with EtOAc and n-BuOH. The EtOAc solution was evaporated under reduced pressure to give a crude extract (30.0 g), which was separated into fractions 1–5 on silica gel CC using a gradient eluent of petroleum ether-EtOAc (15:1–0:1, v/v, each 3 L). Fr. 2 (5.0 g) was subjected to repeated RP-18 CC (eluted with MeOH/H2O from 3:7 to 10:0, v/v, each 500 mL) and silica gel CC (eluted with petroleum ether–EtOAc from 3:1 to 1:1, v/v, each 600 mL) to afford compounds 1 (5 mg) and 4 (10 mg); Fr. 3 (1 g) was purified by ODS column (50% MeOH/H2O, v/v, 800 mL) and HPLC over a pNAP column (75% MeOH/H2O, v/v, 500 mL) to give compound 2 (30 mg). Fr. 5 (3.0 g) was submitted to silica gel CC with petroleum ether–EtOAc (1:1, v/v, 1200 mL) as eluent and further purified by Sephadex LH-20 CC with CHCl3/MeOH (1:1, v/v, 600 mL) as eluent, yielding compound 3 (2.0 mg). Xylariaine A (1): white amorphous powder; ½a32 D 10.0 (c 0.10, MeOH); IR (KBr) nmax 3433, 2946, 2930, 1454, 1377, 1039; For 1H and 13C NMR spectroscopic data, see Table 1; ESIMS positive m/z [M +Na]+ 295; HRESIMS m/z [M+Na]+ 295.1877 (calcd for C15H28O4Na, 295.1880). Xylariaine B (2): white crystal; ½a32 D 20.0 (c 0.02, MeOH); IR (KBr) nmax 3429, 2955, 2940, 1762, 1381, 1200, 1003; For 1H and 13C NMR spectroscopic data, see Table 1; ESIMS positive m/z [M+Na]+ 307; HRESIMS m/z [M+Na]+ 307.1513 (calcd for C15H24O5Na, 307.1516). Xylariaine C (3): white amorphous powder; ½a32 D 17.4 (c 0.10, MeOH); IR (KBr) nmax 3357, 2936, 1370, 1045; For 1H and 13C NMR spectroscopic data, see Table 1; ESIMS positive m/z [M+Na]+ 277; HRESIMS m/z [M+Na]+ 277.1772 (calcd for C15H26O3Na, 277.1774). 3.4. X-ray crystal data for 2 and 4 Colorless crystals of 2 and 4 were obtained in MeOH–H2O. Crystal data of 2 and 4 were recorded using a Bruker D8 QUEST diffractometer (Bruker) with graphite monochromatic Cu-Ka radiation (l = 0.71073 Å). 3.4.1. Crystal data for xylariaine B (2) Orthorhombic, C15H24O5H2O; space group P21 with a = 7.1140 (3) Å, b = 11.6648(6) Å, c = 17.2573(8) Å, V = 1432.07(12) Å3, Z = 4, Dcalcd = 1.4023 Mg m3, m = 0.891 mm1, and F(000) = 658.3437; unique cell angle (b) = 90; T = 273(2) K. The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of the full-matrix leastsquares refinement was based on 2608 unique reflections (2u < 50 ) and 198 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0413, wR2 = 0.1082, and R = 0.0426 for I > 2s (I) data. The absolute structure parameter is 0.2 (2). Crystallographic information file of compound 2 with Cambridge Crystallographic Data Centre (CCDC) reference number 1497293 has been deposited at the CCDC, and can be obtained free of charge from the CCDC via https:// www.ccdc.cam.ac.uk/deposit/. 3.4.2. Crystal data for sulphureuine B (4) Triclinic, C15H28O4; space group P1 with a = 6.0589(12) Å, b = 9.963(2) Å, c = 12.954(3) Å, V = 717.1(2) Å3, Z = 1, Dcalcd = 1.302 Mg
m3, m = 1.105 mm1, and F(000) = 292; unique cell angle (b) = 82.555(4); T = 273.15 K. The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of the full-matrix least-squares refinement was based on 5041 unique reflections (2u < 50 ) and 357 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0385, wR2 = 0.1065, and R = 0.0386 for I > 2s (I) data. The absolute structure parameter is 0.11 (3). Crystallographic information file of compound 4 with Cambridge Crystallographic Data Centre (CCDC) reference number 1497292 has been deposited at the CCDC, and can be obtained free of charge from the CCDC via https://www.ccdc.cam.ac.uk/deposit/. 3.5. Bioassay for AChE inhibitory activity AChE inhibitory activity of these compounds was assayed by the spectrophotometric method developed by Ellman (Ellman et al., 1961). Acetylthiocholine iodide (Sigma, St. Louis, MO, USA) was used as substrate in the assay. Compounds were dissolved in DMSO. The reaction mixture, consisting of 110 mL phosphate buffer (pH 8.0), 10 mL of tested compounds solution (2000 mmol L1), and 40 mL AChE solution (0.04 U/100 mL), was mixed and incubated for 20 min (30 C). The reaction was initiated by the addition of 20 mL 5,5-dithiobis-2-nitrobenzoic acid (6.25 mmol L1) and 20 mL acetylthiocholine. The hydrolysis of acetylthiocholine was monitored at 405 nm after 30 min. Tacrine (Sigma–Aldrich, St. Louis, MO, USA, 99%) was used as positive control. All the reactions were done in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E S)/E 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compounds). Acknowledgment This work was supported by Special Fund for Agro-Scientific Research in the Public Interest (201303117), Fundamental Scientific Research Funds for CATAS (ITBB2017) and Natural Science Foundation of Hainan Province (20152035). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.03.003. References Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–90. He, J.B., Tao, J., Miao, X.S., Bu, W., Zhang, S., Dong, Z.J., Li, Z.H., Feng, T., Liu, J.K., 2015. Seven new drimane-type sesquiterpenoids from cultures of fungus Laetiporus sulphureus. Fitoterapia 102, 1–6. Jang, Y.W., Lee, I.K., Kim, Y.S., Lee, S.K., Lee, H.J., Yu, S.H., Yun, B.S., 2007. ChemInform Abstract: xylarinic acids A and B, new antifungal polypropionates from the fruiting body of Xylaria polymorpha. J. Antibiot. 60, 696–699. Lee, I.K., Jang, Y.W., Kim, Y.S., Yu, S.H., Lee, K.J., Park, S.M., Oh, B.T., Chae, J.C., Yun, B.S., 2009. Xylarinols A and B, two new 2-benzoxepin derivatives from the fruiting bodies of Xylaria polymorpha. J. Antibiot. 62, 163–165. Shan, W.G., Chen, X.X., Ying, Y.M., Zhan, Z.J., 2011. Sesquiterpenoids from Fusarium, sp. an endophytic fungus in Agriminia pilosa. HeIv. Chim. Acta 94, 1254–1259. Shiono, Y., Motoki, S., Kosek, iT., Murayama, T., Tojima, M., Kimura, K.I., 2009. Isopimarane diterpene glycosides, apoptosis inducers, obtained from fruiting bodies of the ascomycete Xylaria polymorpha. Phytochemistry 70, 935–939. William, A.A., Latchezar, S.T., 1992. Drimane sesquiterpene lactones from Peniophora polygonia. J. Nat. Prod. 55, 1454–1461.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Drimane-type sesquiterpenoids from cultures of the fungus Xylaria polymorpha Ning Ning Yanga,b , Fan Dong Kongb , Qing Yun Mab , Sheng Zhuo Huangb , Du Qiang Luoc , Li Man Zhoub , Hao Fu Daib , Zhi Fang Yua,* , You Xing Zhaob,* a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China c College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China b
A R T I C L E I N F O
Article history: Received 19 October 2016 Received in revised form 27 February 2017 Accepted 3 March 2017 Available online xxx Keywords: Sesquiterpenoid Xylaria polymorpha X-ray diffraction analysis Anti-AChE inhibitory activity
A B S T R A C T
Three new drimane-type sesquiterpenoids named xylariaines A–C (1–3), together with one known analogue (4), were isolated from the ethyl acetate extract of cultures of the fungus Xylaria polymorpha (Pers.: Fr.) Grer. Their structures were elucidated unambiguously by NMR and single-crystal X-ray diffraction analysis. Compounds 1 and 2 exhibited weak anti-acetylcholinesterase activities at a concentration of 50 mg/mL with inhibition ratios of 12.4% and 18.0%, respectively. © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction
2. Result and discussion
Xylaria polymorpha (Pers.: Fr.) Grer. is a small black mushroom belonging to the genus Xylaria, which is widely distributed in the southeast of mainland China, mainly in Hainan, Guangdong, Fujian and Taiwan provinces. So far, a few researches on chemical constituents of this fungus were reported and led to the isolation of some different kinds of natural products including xylarinols A and B with moderate ABTS radical scavenging activity (Lee et al., 2009), three isopimarane diterpene glycosides with cytotoxicity against human cancer cell lines (Shiono et al., 2009), and xylarinic acids A and B with significant antifungal activity (Jang et al., 2007). In a search for new bioactive compounds from X. polymorpha, the deep chemical investigation on cultures of this fungus was thus undertaken, which led to the isolation of three new drimanetype sesquiterpenoids named xylariaines A–C (1–3) along with one known analogue (4). The AChE inhibitory activity of the isolates (1– 4) was tested. Herein, this paper describes the isolation, structure elucidation, and AChE inhibitory activities of these compounds.
Compound 1 was obtained as white amorphous powder, and its molecular formula was assigned to be C15H28O4 with two degrees of unsaturation according to its positive HRESIMS (m/z 295.1877 [M +Na]+, calcd. 295.1880 for C15H28O4Na) and NMR spectroscopic data (Table 1). The IR spectrum displayed absorption band (3433 cm1) for hydroxyls. The 1H NMR (Table 1) showed the presence of signals for three singlet methyls [d 0.89 (3H, s, H-14), 0.83 (3H, s, H-15), 0.90 (3H, s, H-13)], two pairs of oxymethylene protons, and one oxymethine proton. The 13C NMR spectrum displayed 15 carbon resonances for three methyls, six methlenes (two oxygenated), three methines (one oxygenated), and three quarternary carbons (one oxygenated). These data indicated that 1 might be characteristic as sesquiterpenoid. In addition, detailed analysis of NMR data suggested that compound 1 should be with a similar planar structure to that of agripilol A (Shan et al., 2011), a drimane-type sesquiterpenoid, bearing a hydroxyl group at C-1 (dC 79.8) rather than at C-3 as in agripilol A. This conjecture was evidenced by 1H–1H COSY correlations from the oxymehine proton H-1 [dH 3.38 dd (4.5, 11.5)] to H2-3 through H2-2 as well as HMBC correlations from H3-13 [dH (0.90, s)] to C-1, C-5 (dC 55.4), C-9 (dC 63.9), and C-10 (dC 44.6). Other correlations in the HMBC and 1 H–1H COSY spectra (Fig. 2) further supported the atom connectivity in compound 1. The relative configuration of the drimane sesquiterpenoid skeleton in compound 1 was elucidated
* Corresponding authors. E-mail addresses: [email protected] (Z.F. Yu), [email protected] (Y.X. Zhao).
http://dx.doi.org/10.1016/j.phytol.2017.03.003 1874-3900/© 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
14 Table 1 1 H (500 MHz) and Position
N.N. Yang et al. / Phytochemistry Letters 20 (2017) 13–16
13
C (125 MHz) NMR spectroscopic data for compounds 1–3 in CD3OD. 1
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
79.8, CH
3.38(dd, 4.5, 11.5)
80.4, CH
3.36 (dd, 7.5, 8.5)
40.8, CH2
2
28.2, CH2
37.1, CH2
41.1, CH2
1.76, m 1.74, m 3.27 (dd, 7.0, 8.5)
27.8, CH2
3
1.70, m 1.60, m 1.41, m 1.32, m
1.65, m 1.08, m 1.64, m 1.59, m 3.19 (dd, 4.5, 11.5)
4 5 6
33.9, C 55.4, CH 20.8, CH2
7
37.9, CH2
8 9 10 11
75.4, 63.9, 44.6, 58.8,
12
64.1, CH2
13 14 15
12.5, CH3 21.8, CH3 33.5, CH3
C CH C CH2
76.9, CH
0.96 (dd, 2.0, 12.0) 1.66, m 1.35, m 1.22, m 2.20 (ddd, 3.0, 3.0, 13.0) 1.53 (dd, 1.5, 8.5) 4.13 (dd, 1.5, 11.5) 3.70 (dd, 8.5, 11.5) 3.51 (d, 11.5) 3.23 (d, 11.5) 0.90 s 0.89 s 0.83 s
39.7, C 50.1, CH 19.0, CH2 32.8, CH2 74.0, C 57.5, CH 42.1, C 70.5, CH2
0.91 (dd, 2.0, 12.0) 1.68, m 1.29, m 2.39, m 1.58, m 2.06 (d, 5.5) 4.70 (br d, 9.5) 4.52 (dd, 5.5, 9.5
180.1, C 10.0, CH3 16.6, CH3 29.0, CH3
by ROESY experiment (Fig. 3) and determined to be the same as those of agripilol A. The a-orientation of H-1 was proposed from the ROESY correlation of H-1 [dH 3.38 dd (4.5, 11.5)] with H-9 [dH 1.53 dd (1.5, 8.5)]. Thus, compound 1 was assigned as shown in Fig. 1, and named xylariaine A. Compound 2 was purified as white crystal. Its molecular formula C15H24O5 was determined on the basis of the HRESIMS (m/ z 307.1513 [M+Na]+, calcd. 307.1516 for C15H24O5Na) and NMR spectroscopic data (Table 1). Its IR spectrum showed the presence of hydroxyls (3429 cm1) and lactone carbonyl (1762 cm1). The 1H NMR spectroscopic data of 2 (Table 1) revealed the presence of three singlet methyls [d 0.83 (3H, s, H-13), 0.77 (3H, s, H-14), 1.00 (3H, s, H-15)]. The 13C NMR and DEPT spectra (Table 1) of 2 displayed 15 carbon resonances, including three methyls, four methylenes (one oxygenated), four methines, and four quaternary carbons (one carbonyl and one oxygenated). These data suggested that 2 also had a sesquiterpenoid skeleton with a tricyclic system. Comparison of its NMR spectroscopic data with those of 3bhydroxypeniopholide (William and Latchezar, 1992) suggested that they were structurally close related, except for an additional hydroxyl located at C-1 (dC 80.4) in compound 2, as demonstrated by HMBC correlations from H1-9[dH 2.06 d (5.5)] and H3-13[dH (0.83, s)] to the same oxygenated carbon C-1. In the ROESY spectrum, correlations of Ha-11[dH 4.52 dd (5.5, 9.5)]/H3-13/H3-14 [dH (0.77, s)], H-1/H-9/H-3/H-5/H-1, and H-5/H3-15 indicated that
79.5, CH 39.7, C 53.4, CH 20.5, CH2 34.7, CH2 80.3, C 62.6, CH 36.7, C 70.0, CH2 77.5, CH2
0.83 s 0.77 s 1.00 s
14.9, CH3 16.3, CH3 28.8, CH3
0.98 (dd, 2.0, 11.5) 1.67, m 1.38, m 2.10, m 1.62, m 1.56 (d, 6.0) 3.80 (d, 8.5) 4.05 (dd, 6.0, 8.5) 3.71 (d, 9.0) 3.58 (d, 9.0) 0.92 s 0.80 s 1.02 s
the relative configuration of 2 was the same as 3b-hydroxypeniopholide, except for the a-oriented H-1 proton. To further verify the above deduction and determine the absolute configuration of 2, a single-crystal X-ray diffraction pattern was obtained using the anomalous scattering of Cu Ka radiation. Fig. 4 shows an ORTEP drawing and unambiguously demonstrates the absolute configuration of 1R, 3S, 5S, 8R, 9S, and 10S for 2. Thus, compound 2 was assigned as shown in Fig. 1, and named xylariaine B. Compound 3 was isolated as a white amorphous powder, and its molecular formula of C15H26O3 was deduced from its molecular ion peak of HRESIMS at m/z 277.1772 [M+Na]+ (calcd. 277.1774 for C15H26O3Na), equivalent to that of agripilol A by loss of one H2O unit. Besides, the 13C NMR data of 3 were nearly identical with those of the known compound agripilol A. The above data led to a deduction that 3 could be a dehydration derivative of agripilol A via losing a molecule of water between two hydroxyl groups located at C-11 and C-12 or C-8 in agripilol A to form a cyclic ether moiety. In the HMBC spectrum of 3, correlations from H-12 [dH 3.71 d (9.0), 3.58 d (9.0)] to C-11 (dC 70.0) confirmed the connection of C-12 (dC 77.5) with C-11 through an O atom. The assignment of compound 3 was also corroborated by the analysis of other correlations in HMBC and 1H–1H COSY spectra (Fig. 2). The relative configuration of compound 3 was also elucidated by ROESY experiment (Fig. 3), and assigned to be same as that of agripilol A by correlations of H-5 [dH 0.98 dd (2.0, 11.5)] with H-3[dH 3.19 dd (4.5, 11.5)], H-9[dH 1.56 d
Fig. 1. Structures of compounds 1–4.
N.N. Yang et al. / Phytochemistry Letters 20 (2017) 13–16
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Fig. 2. Key COSY and HMBC correlations of 1–3.
Fig. 3. Key NOE correlations of 1–3.
(6.0)], and H-15[dH (1.02, s)] and correlations of H3-13[dH (0.92, s)] with H3-14[dH (0.80, s)], Hb-11[dH 3.80 d (8.5)] and Hb-12[dH 3.58 d (9.0)]. Thus, compound 3 was elucidated as shown in Fig. 1, and named xylariaine C. The known compound (4) was determined to be sulphureuine B (He et al., 2015) by comparison of its spectroscopic data with that of literature. Besides, the absolute configuration of 4 was originally established as 3S, 5R, 8R, 9R, and 10S by Cu-Ka X-ray diffraction in our study (Fig. 4). The acetylcholinesterase inhibitory activity of compounds 1–4 were tested by previous method as described in the literature. The result showed that compounds 1 and 2 exhibited weak anti-AChE activities with inhibition rates of 12.4% and 18.0%, respectively, at a concentration of 50 mg/mL (Tacrine as positive control, inhibitory rate: 56.7%). 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Rudolph Autopol III polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Shimadzu UV-2550 spectrometer (Beckman, Brea, CA, USA) was used for scanning UV spectroscopy. IR spectra were obtained on a Tensor 27 spectrometer, as KBr pellets (Thermo, Pittsburgh, PA, USA). HR-ESI-MS were performed on an API QSTAR Pulsar mass spectrometer (Bruker, Bremen, Germany). NMR spectra were
recorded on a AV-500 spectrometer (Bruker, Bremen, Germany) with TMS (Tetramethylsilane) as an internal standard. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 (40–70 mm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography (CC). Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm 25 cm, column. Fractions were monitored by TLC (thin-layer chromatography) and spots were visualized by heating after spraying with 5% H2SO4 in ethanol. 3.2. Fungal material The fungus Xylaria polymorpha was collected in the valley of Yinggeling, Hainan province of China, in June 2015, and identified by Associate Professor Sheng-zhuo Huang, the Institute of Tropical Bioscience and Biotechnology. The mycelium was isolated from the X. polymorpha and its strain was maintained on potato dextrose agar (PDA) slant at 4 C. A voucher specimen (YGL-017) was deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China. 3.3. Fermentation, extraction and isolation The fungus was cultured on rice medium in conical flasks (1 L) at 25 C for a week. Two pieces of mycelial agar plugs (0.5 0.5 cm2) were inoculated into 1 L Erlenmeyer flasks containing damp white
Fig. 4. ORTEP drawings of compounds 2 and 4.
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N.N. Yang et al. / Phytochemistry Letters 20 (2017) 13–16
rice (50 g rice plus 100 mL H2O). The fermentation was carried out on a shaker at 25 C and 150 rpm for 7 days, and allowed to proceed under static conditions at 25 C for 28 days. The culture broth (100 L) was filtered to give the filtrate and mycelia. The filtrate was evaporated in vacuo to a small volume and then suspended in H2O and partitioned successively with EtOAc and n-BuOH. The EtOAc solution was evaporated under reduced pressure to give a crude extract (30.0 g), which was separated into fractions 1–5 on silica gel CC using a gradient eluent of petroleum ether-EtOAc (15:1–0:1, v/v, each 3 L). Fr. 2 (5.0 g) was subjected to repeated RP-18 CC (eluted with MeOH/H2O from 3:7 to 10:0, v/v, each 500 mL) and silica gel CC (eluted with petroleum ether–EtOAc from 3:1 to 1:1, v/v, each 600 mL) to afford compounds 1 (5 mg) and 4 (10 mg); Fr. 3 (1 g) was purified by ODS column (50% MeOH/H2O, v/v, 800 mL) and HPLC over a pNAP column (75% MeOH/H2O, v/v, 500 mL) to give compound 2 (30 mg). Fr. 5 (3.0 g) was submitted to silica gel CC with petroleum ether–EtOAc (1:1, v/v, 1200 mL) as eluent and further purified by Sephadex LH-20 CC with CHCl3/MeOH (1:1, v/v, 600 mL) as eluent, yielding compound 3 (2.0 mg). Xylariaine A (1): white amorphous powder; ½a32 D 10.0 (c 0.10, MeOH); IR (KBr) nmax 3433, 2946, 2930, 1454, 1377, 1039; For 1H and 13C NMR spectroscopic data, see Table 1; ESIMS positive m/z [M +Na]+ 295; HRESIMS m/z [M+Na]+ 295.1877 (calcd for C15H28O4Na, 295.1880). Xylariaine B (2): white crystal; ½a32 D 20.0 (c 0.02, MeOH); IR (KBr) nmax 3429, 2955, 2940, 1762, 1381, 1200, 1003; For 1H and 13C NMR spectroscopic data, see Table 1; ESIMS positive m/z [M+Na]+ 307; HRESIMS m/z [M+Na]+ 307.1513 (calcd for C15H24O5Na, 307.1516). Xylariaine C (3): white amorphous powder; ½a32 D 17.4 (c 0.10, MeOH); IR (KBr) nmax 3357, 2936, 1370, 1045; For 1H and 13C NMR spectroscopic data, see Table 1; ESIMS positive m/z [M+Na]+ 277; HRESIMS m/z [M+Na]+ 277.1772 (calcd for C15H26O3Na, 277.1774). 3.4. X-ray crystal data for 2 and 4 Colorless crystals of 2 and 4 were obtained in MeOH–H2O. Crystal data of 2 and 4 were recorded using a Bruker D8 QUEST diffractometer (Bruker) with graphite monochromatic Cu-Ka radiation (l = 0.71073 Å). 3.4.1. Crystal data for xylariaine B (2) Orthorhombic, C15H24O5H2O; space group P21 with a = 7.1140 (3) Å, b = 11.6648(6) Å, c = 17.2573(8) Å, V = 1432.07(12) Å3, Z = 4, Dcalcd = 1.4023 Mg m3, m = 0.891 mm1, and F(000) = 658.3437; unique cell angle (b) = 90; T = 273(2) K. The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of the full-matrix leastsquares refinement was based on 2608 unique reflections (2u < 50 ) and 198 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0413, wR2 = 0.1082, and R = 0.0426 for I > 2s (I) data. The absolute structure parameter is 0.2 (2). Crystallographic information file of compound 2 with Cambridge Crystallographic Data Centre (CCDC) reference number 1497293 has been deposited at the CCDC, and can be obtained free of charge from the CCDC via https:// www.ccdc.cam.ac.uk/deposit/. 3.4.2. Crystal data for sulphureuine B (4) Triclinic, C15H28O4; space group P1 with a = 6.0589(12) Å, b = 9.963(2) Å, c = 12.954(3) Å, V = 717.1(2) Å3, Z = 1, Dcalcd = 1.302 Mg
m3, m = 1.105 mm1, and F(000) = 292; unique cell angle (b) = 82.555(4); T = 273.15 K. The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of the full-matrix least-squares refinement was based on 5041 unique reflections (2u < 50 ) and 357 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0385, wR2 = 0.1065, and R = 0.0386 for I > 2s (I) data. The absolute structure parameter is 0.11 (3). Crystallographic information file of compound 4 with Cambridge Crystallographic Data Centre (CCDC) reference number 1497292 has been deposited at the CCDC, and can be obtained free of charge from the CCDC via https://www.ccdc.cam.ac.uk/deposit/. 3.5. Bioassay for AChE inhibitory activity AChE inhibitory activity of these compounds was assayed by the spectrophotometric method developed by Ellman (Ellman et al., 1961). Acetylthiocholine iodide (Sigma, St. Louis, MO, USA) was used as substrate in the assay. Compounds were dissolved in DMSO. The reaction mixture, consisting of 110 mL phosphate buffer (pH 8.0), 10 mL of tested compounds solution (2000 mmol L1), and 40 mL AChE solution (0.04 U/100 mL), was mixed and incubated for 20 min (30 C). The reaction was initiated by the addition of 20 mL 5,5-dithiobis-2-nitrobenzoic acid (6.25 mmol L1) and 20 mL acetylthiocholine. The hydrolysis of acetylthiocholine was monitored at 405 nm after 30 min. Tacrine (Sigma–Aldrich, St. Louis, MO, USA, 99%) was used as positive control. All the reactions were done in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E S)/E 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compounds). Acknowledgment This work was supported by Special Fund for Agro-Scientific Research in the Public Interest (201303117), Fundamental Scientific Research Funds for CATAS (ITBB2017) and Natural Science Foundation of Hainan Province (20152035). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.03.003. References Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–90. He, J.B., Tao, J., Miao, X.S., Bu, W., Zhang, S., Dong, Z.J., Li, Z.H., Feng, T., Liu, J.K., 2015. Seven new drimane-type sesquiterpenoids from cultures of fungus Laetiporus sulphureus. Fitoterapia 102, 1–6. Jang, Y.W., Lee, I.K., Kim, Y.S., Lee, S.K., Lee, H.J., Yu, S.H., Yun, B.S., 2007. ChemInform Abstract: xylarinic acids A and B, new antifungal polypropionates from the fruiting body of Xylaria polymorpha. J. Antibiot. 60, 696–699. Lee, I.K., Jang, Y.W., Kim, Y.S., Yu, S.H., Lee, K.J., Park, S.M., Oh, B.T., Chae, J.C., Yun, B.S., 2009. Xylarinols A and B, two new 2-benzoxepin derivatives from the fruiting bodies of Xylaria polymorpha. J. Antibiot. 62, 163–165. Shan, W.G., Chen, X.X., Ying, Y.M., Zhan, Z.J., 2011. Sesquiterpenoids from Fusarium, sp. an endophytic fungus in Agriminia pilosa. HeIv. Chim. Acta 94, 1254–1259. Shiono, Y., Motoki, S., Kosek, iT., Murayama, T., Tojima, M., Kimura, K.I., 2009. Isopimarane diterpene glycosides, apoptosis inducers, obtained from fruiting bodies of the ascomycete Xylaria polymorpha. Phytochemistry 70, 935–939. William, A.A., Latchezar, S.T., 1992. Drimane sesquiterpene lactones from Peniophora polygonia. J. Nat. Prod. 55, 1454–1461.