Characterization of cytochalasins and steroids from the fruiting bodies of daldinia concentrica in Viet Nam

Vietnam Journal of Science and Technology 56 (4A) (2018) 83-88 CHARACTERIZATION OF CYTOCHALASINS AND STEROIDS FROM THE FRUITING BODIES OF Daldinia concentrica IN VIET NAM Hoang Van Trung 1 , Nguyen Thi Bich Ngoc 2 , Nguyen Ngoc Tuan 3 , Nguyen Tan Thanh 1 , Tran Dinh Thang 1, * 1 School of Chemistry, Biology and Enviroment, Vinh University, 182 Le Duan str., Vinh City, Nghe An province 2 School of Natural Sciences Education, Vinh University, 182 Le Duan str., Vinh City, Nghe An province 3 Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao str., Ward 4, Go Vap dist., Ho Chi Minh City * Email: thangtd@vinhuni.edu.vn Received: 8 August 2018; Accepted for publication: 10 October 2018 ABSTRACT [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18,19-trihydroxy-16,18-dimethyl-10-phenyl- (7S*,13E,16S*,18S*,19R*) (1), [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18-dihydroxy- 16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*) (2), ergosterol (3) and ergosterol peroxide (4) were isolated from the methanolic extract of the fruit body of Daldinia concentrica. The structures of these compounds were elucidated using a combination of UV, IR, 1D and 2D NMR techniques ( 1 H-, 13 C-NMR, COSY, HSQC and HMBC) and MS analyses. Keywords: Daldinia concentrica, Xylariaceae, cytochalasin, ergosterol, ergosterol peroxide. 1. INTRODUCTION Daldinia is a genus of fungi and an inedible wood-rotting fungus in the family Xylariaceae. There are more than 80 species, but only 25 species have medicinal value. However, the ascomycete genus Daldinia (Xylariaceae) have been shown to be a good medicinal fungi of unique bioactive secondary metabolites [1]. During more recent studies on the chemical constituents of genus Daldinia more than 20 novel compounds have been discovered, including cytochalasins, aromatic steroids, terpenoids, polyketides, lactones, azaphilone derivatives, benzophone, benzoquinones, binaphthyl derivatives etc. [2-5]. The compounds showed cytotoxicity against cancer cells, such as KB (a human epidermal carcinoma), SK-LU-1 (human lung carcinoma), MCF7 (human breast carcinoma) and HepG2 (hepatocellular carcinoma) [4, 5]. Hoang Van Trung et al. 84 Recently, as part of our ongoing study on extraction and isolation the fruiting bodies of D. concentria, we collected it from Nghe An, Vietnam, and we reported herein the isolation procedure and structural determination of [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18,19- trihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18S*,19R*), [11]-cytochalasa-6(12),13- diene-1,21-ione-7,18-dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*), ergosterol and ergosterol peroxide. The structures of these compounds were elucidated using a combination of UV, IR, 1D and 2D NMR techniques (1H-, 13C-NMR, COSY, HSQC and HMBC) and MS analyses. 2. MATERIAL AND METHODS 2.1. Fungal material The fungus Daldinia concentrica (Bolton) Ces. & De Not. were collected at the Pumat National Park of Nghe An Province, Viet Nam and identified by Prof. Dr. Ngo Anh, Department of Biology, Hue University. A voucher specimen was deposited at the herbarium of the School of Chemistry, Biology and Environment, Vinh University. 2.2. General Melting points were determined by Yanagimoto MP-S3 apparatus without corrections, optical rotations- by JASCO DIP-370 polarimeter, UV spectra measured on an Agilent UV-VIS spectrophotometer, and IR spectra taken with a Bruker 270-30 spectrophotometer. 1D- and 2D- NMR spectra were measured on Bruker AV-III 500 NMR spectrometer, with tetramethylsilane (TMS) as the internal standard and chemical shifts were reported in δ values (ppm). The electrospray ionization (ESI) and high resolution electrospray ionization mass spectra (HR-ESI- MS) were performed with an Agilent 1200 LC-MSD Trap spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, E. Merck). TLC: visualization by spraying with 10 % (v/v) H2SO4 followed by heating at 110 °C for 10 min. 2.3. Extraction and isolation The dried fruiting bodies of D. concentrica (2.0 kg) was extracted with methanol 70 % at ambient temperature, and concentrated under reduced pressure to give a dark brown syrup (225 g). The crude extract was suspended in water and partitioned with ethyl acetate and butanol to afford ethyl acetate (95 g), butanol (47 g) and water soluble (43 g) fractions, respectively. The ethyl acetate soluble extracts were applied to silica gel column chromatography with a mixture of chloroform and methanol step gradient system (100:0, 40:1: 30:1; 20:1; 10:1: 4:1; 2:1, v/v) to afford minor fractions. These fractions were monitored by TLC to combine into seven major fractions (Frs. DC1-DC7). Fraction DC1 (1.2 g) was subjected to the preparative thin layer chromatography (Prep TLC) (typical plate dimensions: 20 cm × 20 cm, 2.5 mm SiO2 thickness) eluting with a mixture of chloroform and methanol (15:1, v/v) to afford compound 1 (23 mg). Fraction DC3 (2.6 g) was subjected to the silica gel column chromatography (300 g, 60 × 3 cm) eluting with a hexane and ethyl acetate solvent mixture (15:1, v/v) to yield compound 4 (138 mg). Fraction DC4 (2.5 g) was subjected to the preparative thin layer chromatography (Prep TLC) (typical plate dimensions: 20 cm × 20 cm, 2.5 mm SiO2 thickness) eluting with a mixture of chloroform and methanol (9:1, v/v) to produce compound 2 (15 mg) and 3 (34 mg). Characterization of cytochalasins and steroids from the fruiting bodies of Daldinia concentrica 85 Compound 1: colorless needles (CHCl3), m.p. 216-217 °C; HR-ESI-MS m/z 467.2669 [M] + (C28H37O5N, cal. m/z 467.2672); 1 H-NMR (500 MHz, CDCl3) ( ppm): 7.28 (2H, m, H-3’,5’), 7.25 (1H, m, H-4’), 7.13 (2H, m, H-2’,6’), 6.67 (1H, m, H-13), 6.35 (1H, br s, H-2), 5.50 (1H, m, H-14), 5.43 (1H, s, H-12b), 5.10 (1H, s, H-12a), 5.01 (1H, s, H-20a), 4.76 (1H, d, J = 3.0 Hz, H- 7), 4.31 (1H, d, J = 5.0 Hz, H-19), 3.59 (1H, m, H-3), 3.25 (1H, d, J = 4.5 Hz, H-4), 3.15 (1H, m, H-8), 3.10 (1H, m, H-5), 2.71 (1H, m, H-10a), 2.65 (1H,s, H-20b), 2.62 (1H, m, H-10b), 2.25 (1H, s, H-17a), 1.95 (1H,m, H-15a), 1.73 (1H, m, H-15b), 1.63 (3H, s, H-23), 1.52 (1H, dd, J = 4.0, 5.5 Hz, H-17b), 1.39 (1H, s, H-16), 0.91 (3H, m, H-11), 0.92 (3H, s, H-22); 13 C-NMR (125MHz, CDCl3) ( ppm): 212.3 (C-21), 173.1 (C-1), 148.5 (C-6), 136.5 (C-1’), 136.0 (C-14), 129.6 (C-2’,6’), 128.9 (C-13), 128.8 (C-3’,5’), 127.1 (C-4'), 114.4 (C-12), 75.4 (C-18), 71.5 (C- 7), 71.4 (C-19), 63.7 (C-9), 52.5 (C-3), 51.5 (C-8), 45.2 (C-4), 45.1 (C-17), 43.7 (C-10), 43.0 (C- 15), 42.8 (C-20), 31.9 (C-5), 29.9 (C-16), 25.7 (C-22), 23.1 (C-23), 13.1 (C-11). Compound 2: colorless needles (CHCl3), m.p. 120-121 °C; HR-ESI-MS m/z 451.2717 [M] + (C28H37O4N, cal.m/z 451.2712); 1 H-NMR (500MHz, CDCl3) ( ppm): 7.30 (2H, m, H-3’,5’), 7.24 (1H, m, H-4’), 7.08 (2H, m, H-2’,6’), 6.07 (1H, dd, J = 8.5, 15.5 Hz, H-13), 5.64 (1H, br s, H-2), 5.36 (1H, ddd, J = 4.5, 11.0, 11.0 Hz, H-14), 5.25 (1H, s, H-12a), 5.07 (1H, s, H-12b), 4.12 (1H, br d, J = 10.0 Hz, H-7), 3.70 (1H, ddd, J = 18.5, 9.5 Hz, H-20a), 3.31 (1H, m, H-3), 2.97 (1H, dd, J = 6.5, 2.0 Hz, H-4), 2.84 (1H, brqd, H-5), 2.64 (1H, dd, J = 13.5, 5.0, H-10a), 2.47 (1H, dd, J = 10.8, 6.5 Hz, H-10b), 2.46 (1H, t, J = 10.0 Hz, H-8), 2.03 (1H, dd, J = 4.5, 12.5 Hz, H-15a), 1.93 (1H, br d, J = 13.5 Hz, H-17a), 1.82 (1H, dt, J = 18.5, 9.5 Hz, H-20b), 1.80 (1H, m, H-15b), 1.65 (2H, m, H-19), 1.19 (3H, s, H-23), 1.13 (1H, m, H-16), 1.09 (1H, brddd, J = 13.5, 5.0, 1.5 Hz, H-17b), 1.03 (3H, d, J = 6.5 Hz, H-22), 0.97 (3H, d, J = 6.5 Hz, H-11); 13 C- NMR (125MHz, CDCl3) ( ppm): 211.0 (C-21), 174.0 (C-1), 148.8 (C-6), 136.6 (C-1’), 135.8 (C-14), 129.5 (C-2’,6’), 128.8 (C-3’,5’), 128.4 (C-13), 127.0 (C-4'), 114.3 (C-12), 73.2 (C-18), 71.7 (C-7), 63.8 (C-9), 52.6 (C-3), 51.4 (C-8), 46.3 (C-4), 45.1 (C-17), 43.9 (C-10), 43.0 (C-15), 34.3 (C-20), 31.8 (C-5), 31.1 (C-19), 30.5 (C-16), 28.2 (C-23), 25.5 (C-22), 12.9 s(C-11). Compound 3: white powder, m.p. 166-167 o C; EI-MS m/z 396 [M] + ; 1 H-NMR (500MHz, CDCl3) ( ppm): 5.49 (1H, m, H-7), 5.35 (1H, m, H-6), 5.28 (1H, dd, J = 15.5, 7.5 Hz, H-22), 5.25 (1H, dd, J = 15.5, 7.0 Hz, H-23), 3.48 (1H, m, H-3), 1.05 (3H, d, J = 7.0 Hz, H-28), 0.96 (3H, s, H-19), 0.93 (3H, d, J = 7.0 Hz, H-27), 0.85 (3H, d, J = 6.5 Hz, H-26), 0.82 (3H, d, J = 6.5 Hz, H-21), 0.61 (3H, s, H-18); 13 C-NMR (125MHz, CDCl3) ( ppm): 11.5 (C-18), 15.8 (C- 19), 17.0 (C-28), 19.1 (C-21), 19.4 (C-26), 20.4 (C-27), 20.6 (C-11), 22.2 (C-15), 27.4 (C-16), 31.5 (C-2), 32.2 (C-25), 37.7 (C-10), 38.2 (C-12), 39.0 (C-1), 40.0 (C-20), 40.4 (C-4), 41.8 (C- 24), 42.2 (C-13), 45.5 (C-9), 53.6 (C-14), 55.0 (C-17), 68.3 (C-3), 115.8 (C-7), 118.2 (C-6), 131.2 (C-22), 135.0 (C-23), 139.8 (C-8), 140.3 (C-5). Compound 4: white powder, m.p.: 177-178 o C; EI-MS m/z 428 [M] + ; 1 H-NMR (500MHz, CDCl3) ( ppm): 6.44 (1H, d, J = 8.5 Hz, H-7), 6.23 (1H, d, J = 8.5 Hz, H-6), 5.28 (1H, m, H- 22), 5.18 (1H, m, H-23), 3.58 (1H, m, H-3), 1.05 (3H, d, J = 6.5 Hz, H-21), 0.96 (3H, d, J = 7.0 Hz, H-28), 0.95 (3H, s, H-19), 0.89 (3H, d, J = 6.5 Hz, H-27), 0.87 (3H, s, H-18), 0.87 (3H, d, J = 6.5 Hz, H-26); 13 C-NMR (125MHz, CDCl3) ( ppm): 12.5 (C-18), 17.2 (C-28), 17.9 (C-19), 19.4 (C-26), 19.7 (C-27), 20.2 (C-21), 21.7 (C-11), 22.8 (C-15), 28.2 (C-16), 29.9 (C-2), 32.4 (C-25), 34.5 (C-1), 36.5 (C-10), 36.9 (C-4), 38.7 (C-12), 40.1 (C-20), 42.0 (C-24), 44.0 (C-13), 50.9 (C-9), 51.2 (C-14), 55.4 (C-17), 64.6 (C-3), 78.4 (C-8), 81.4 (C-5), 130.1 (C-7), 131.5 (C- 23), 135.2 (C-6), 135.6 (C-22). Hoang Van Trung et al. 86 3. RESULTS AND DISCUSSION The dried fruiting bodies of Daldinia concentrica was powdered and refluxed with methanol, and the resulted extracts were partitioned with ethyl acetate and butanol to afford ethyl acetate and butanol fractions successively. The ethyl acetate layer was subjected into purification by a combination of conventional chromatographic techniques to result in four compounds including [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18,19-trihydroxy-16,18- dimethyl-10-phenyl-(7S*,13E,16S*,18S*,19R*) (1); [11]-cytochalasa-6(12),13-diene-1,21- dione-7,18-dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*) (2); ergosterol (3) and ergosterol peroxide (4). Compound 1 was isolated as optically active colorless needles, m.p. 216-217 °C. The HR ESI MS analytical data of 1 revealed the molecular formula as C28H37O5N (m/z 467.2669, [M] + ). The 1 H-NMR spectrum of 1 displayed the characteristic resonances for proton amide NH (δH 6.35, H-2), four proton methylene protons (δH 2.25, 1.85, 1.75), benzyl protons H-2’,6’ (δH 7.13), H-3’,5’ (δH 7.25) and H-4’ (δH 7.28), two secondary alcohol protons H-7 (δH 4.76) and H- 19 (δH 4.31). Moreover, the 1 H-NMR confirmed the presence of two exomethylene protons (δH 5.10, 5.43), two olefinic proton signals (δH 6.67, 5.50), three methyl groups (δH 0.92, 0.91, 1.63). The 13 C-NMR displayed the signals of 28 carbons, including two carbonyl carbons C-21 (δC 213.2), C-1 (δC 175.2); six aromatic carbons (δC 126.9, 128.8, 130.2, 138.0, four olefinic carbons (δC 112.7, 131.9, 133.8, 151.8), two oxygenated carbons (δC 72.4, 70.4), two quaternary carbons (δC 65.0, 75.8), five methine carbons, four methylene carbons (δC 44.2, 43.4, 46.3, 45.0) and three methyl carbons (δC 25.5, 24.1, 13.1). Comparison of the above spectral data with those of [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18,19-trihydroxy-16,18-dimethyl-10-phenyl- (7S*,13E,16S*,18S*,19R*) [6], the compound 1 was identified as [11]-cytochalasa-6(12),13- diene-1,21-dione-7,18,19-trihydroxy-16,18-dimethyl-10-phenyl-(7S*, 13E, 16S*, 18S*, 19R*). This compound has been isolated from fungi Daldinia [6]. Compound 2 was isolated as optically active colorless needles, m.p. 120-121 °C. The HR ESI MS analytical data of 2 revealed the molecular formula as C28H37O4N (m/z 451.2717 [M] + ). Its NMR data is similar to 1 except for the presence of a methylene group in replace of an oxygenated methine. The 1 H-NMR spectrum of 2 displayed the characteristic resonances for proton amide NH (δH 5.64), benzyl protons H-2’,6’ (δH 7.08), H-3’,5’ (δH 7.30) and H-4’ (δH 7.24), a secondary alcohol proton H-7 (δH 4.12), two exomethylene protons (δH 5.25 (1H, s), 5.07 (1H, s)), two olefinic proton signals (δH 6.07 (1H, dd), 5.36 (1H, ddd)), three methyl groups (δH 0.92, 0.91, 1.63). According to the MS, 1 H-, 13 C-NMR, DEPT spectral analysis and comparison of the spectral data between 2 and [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18- dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*) [6] the structure of 2 was identified as [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18-dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E, 16S*,18R*). This compound has been isolated from fungi Daldinia [6]. Compound 3 was obtained as optically active white powder. The molecular formula of 3 was established through the EI-MS analysis. The EI-MS showed a pseudo molecular ion peak at m/z 396 (corresponding to a molecular formula of C28H44O), indicating seven indices of hydrogen deficiency. The 1 H-NMR exhibited the signals of one oxygenated methine proton [ H 3.48 (H- 3)], six methyl groups [ H 1.05 (H-28), 0.96 (H-19), 0.93 (H-27), 0.85 (H-26), 0.82 (H-21), and 0.61 (H-18)], and four olefinic protons [ H 5.28 (1H, dd, J = 15.5, 7.5 Hz, H-22), 5.25 (1H, dd, J = 15.5, 7.0Hz, H-23), 5.35 (1H, m, H-6), and 5.49 (1H, m, H-7)]. Moreover, the 13 C-NMR spectrum displayed the signals of 28 carbons, including six olefinic carbons [ C 115.8 (C-7); 118.2 (C-6); 131.2 (C-22); 135.0 (C-23); 139.8 (C-8) and 140.3 (C-5)] and one oxygenated Characterization of cytochalasins and steroids from the fruiting bodies of Daldinia concentrica 87 carbon [ c 68.3 (C-3) ppm]. According to the UV, IR, EI-MS, 1 H-, 13 C-NMR, DEPT spectral analysis and comparison of the spectral data between 3 and ergosterol, the structure of 3 was identified as ergosterol [7, 8, 9]. Compound 4 was obtained as optically active white powder. The 1 H-NMR displayed the presence of one oxygenated methine proton [ H 3.58 (H-3)], six methyl groups [ H 1.05 (H-21), 0.96 (H-28), 0.95 (H-19), 0.89 (H-27), 0.87 (H-18), 0.87 (H-26)], and four olefinic protons [ H 6.23 (H-6), 6.44 (H-7), 5.28 (H-22) and 5.18 (H-23)]. The 13 C-NMR and DEPT spectrum of 4 displayed signals for six methyl carbons, seven methylenes, eleven methines, and four quaternary carbons. In addition, the 13 C-NMR spectrum of compound 4 showed signals consistent with the presence of three oxygenated carbons [δC 81.4 (C-5); 78.4 (C-8); 64.6 (C-3)] and four olefinic carbons [ C 135.6 (C-22) and 131.5 (C-23); 135.2 (C-6) and 130.1 (C-7)], which were similar to those observed for compound 3, except for the absence of one C=C double bond. Notably, these spectroscopic data were consistent with those reported in the literature for a known compound ergosterol peroxide (5α,8 -epidioxyergosta-6,22-dien-3 -ol) [8]. OH N H O O HO OH 12 3 4 5 611 8 9 10 12 13 14 15 16 17 18 19 2021 4' 1' 22 23 7 1 2 HO CH3 CH3 CH3 CH3 H3C CH3 H 1 2 3 4 5 6 7 8 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 9 10 HO CH3 CH3 CH3 CH3 H3C CH3 H 1 2 3 4 5 6 7 8 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 O O 9 10 3 4 4. CONCLUSION In the present study, as part of our ongoing study on extraction and isolation the fruiting bodies of Daldinia concentria, we have succeeded in purification by a combination of conventional chromatographic techniques to result in five known compounds were identified as [11]-cytochalasa-6(12), 13-diene-1,21-dione-7,18,19-trihydroxy-16,18-dimethyl-10-phenyl- (7S*,13E,16S*,18S*, 19R*) (1), [11]-cytochalasa-6(12),diene-1,21-dione-7,18-dihydroxy- 16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*) (2), ergosterol (3) and ergosterol peroxide (4), respectively, based on the comparison of their spectroscopic and spectrometric profiles (NMR, UV, IR, and MS). Acknowledgements. The authors gratefully acknowledge grants from the Ministry of Education and Training (MOET), Vietnam (No. B2018-TDV10) for the financial support of the present research. REFERENCES 1. Lumbsch T. H. and Huhndorf S. M. - Outline of Ascomycota – 2007, Myconet. 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