Cytotoxic steroids found in Vietnamese lingzhi

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2014, Vol. 59, No. 9, pp. 25-29 This paper is available online at CYTOTOXIC STEROIDS FOUND IN VIETNAMESE LINGZHI Ganoderma neo-japonicum Bui Thi Thu Hien1, Nguyen Trang Thu1, Nguyen Quang Tuyen1, Le Xuan Tham2 and Dang Ngoc Quang1 1Faculty of Chemistry, Hanoi National University of Education 2Department of Science and Technology, Lam Dong Abstract. A chemical investigation of the ethylacetate extract of fruit bodies of Vietnamese lingzhi, Ganoderma neo-japonicum, collected in Cat Tien National Park, Lamdong Province, has resulted in the isolation and structural elucidation of four fungal metabolites, ergosta-4,6,8(14),22-tetraen-3-one (1), ganodermadiol (2), ergosterol (3) and ergosta-7,22-dien-3-ol (4) through the use of various chromatographic and spectroscopic methods, including ESI-MS and 2D NMR. In addition, compounds 1, 3 and 4 showed moderate cytotoxic activity toward KB cell lines with their IC50 values of 12.68, 10.72 and 87.95 g/mL, respectively. Keywords: Ganoderma neo-japonicum, cytotoxicity, lingzhi, steroid. 1. Introduction It is estimated that 60 species of the Ganodermataceae family can be found in Vietnam, however, only half of them have actually been found [9]. They have long been used in traditional Chinese, Japanese, Korean and Vietnamese medicine to treat a variety of conditions [3]. Our previous studies on the chemical constituents of Vietnamese lingzhi revealed that they contained various fungal metabolites, such as cattienoids A-C and schisanlactone A from Tomophagus cattienensis [2], sterols from Amauroderma subresinosum [5] and lanostane-type triterpenoids from Ganoderma tropicum [6]. Recently, we collected fruit bodies of Ganoderma neo-japonicum in Lamdong Province and found that the EtOAc crude extract obtained, having an IC50 value of 19.14 g/mL, shows strong cytotoxicity against the KB cell line. This paper describes the isolation and cytotoxicity of four compounds from this mushroom. Received August 24, 2014. Accepted September 29, 2014. Contact Dang Ngoc Quang, e-mail address: quangdn@hnue.edu.vn 25 Bui Thi Thu Hien, Nguyen Trang Thu, Nguyen Quang Tuyen, Le Xuan Tham and Dang Ngoc Quang 2. Content 2.1. Experiment * General procedure Column chromatography was carried out on silica gel 60 (60 - 100 M, Merck) and Sephadex LH-20 (Amersham Pharmacia Biotech). Preparative HPLC was performed on a Jasco PU-2087 instrument with UV-2070 and RI-2031 detectors using a Waters 5 SL-II column (10.0  250 mm), flow rate of 1.0 ml/min. NMR (1H-NMR, 13C-NMR, DEPT, HSQC and HMBC) spectra were recorded on a Bruker Avance 500 MHz Instrument. The chemical shift () values are given in ppm with TMS as an internal standard, coupling constant J - by Hz. * Fungal material Fruit bodies of Ganoderma neo-japonicum were collected in Cat Tien National Park, Lamdong Province, in January 2013 and identified by Dr. Le Xuan Tham. A voucher specimen (THU-01) has been deposited at the Faculty of Chemistry, Hanoi University of Education, Vietnam. * Extraction and Isolation Dried fruit bodies of Ganoderma neo-japonicum (62.0 g) were extracted with EtOAc using an ultrasonic bath. The EtOAc extract was concentrated using a rotary evaporator to give a residue (1.82 g) which was further subjected to a Sephadex LH-20 column, using MeOH-CHCl3 (1/1) to give three fractions (Fr. A-C). Fr-B (0.83 g) was purified by silica gel column, using n-hexane-EtOAc gradient to yield 9 sub-fractions. Sub-fr. 1 (111 mg) was further purified by prep. HPLC, n-hexane-EtOAc (8/1) to afford compound 1 (3 mg), rt. 44.96 min. Compound 2 (3 mg) was obtained from sub-fr. 3 (53 mg) by prep. HPLC, n-hexane-EtOAc (4/1), rt. 12.69 min. Compound 3 (5 mg) and 4 (4 mg) were purified from sub-fr. 2 (227 mg) by prep. HPLC, n-hexane-EtOAc (6/1) with rt. 4.85 min and 8.39 min, respectively. Compound 1. 1H-NMR (CDCl3): H 6.61 (1H, d, J = 9.5 Hz, H-7), 6.03 (1H, d, J = 9.5 Hz, H-6), 5.74 (1H, s, H-4), 5.27 (1H, dt, J = 8.0, 15.0 Hz, H-23), 5.21 (1H, dt, J = 8.0, 15.0 Hz, H-22), 1.07 (3H, d, J = 5.5 Hz, H-21), 0.99 (3H, s, H-19), 0.96 (3H, s, H-18), 0.94 (3H, d, J = 7.0 Hz, H-28), 0.87 (3H, d, J = 7.0 Hz, H-27), 0.84 (3H, d, J = 7.0 Hz, H-26); 13C-NMR (CDCl3): C 199.5 (C-3), 164.5 (C-5), 156.0 (C-14), 135.0 (C-22), 133.9 (C-7), 132.6 (C-23), 124.5 (C-6), 124.4 (C-8), 123.0 (C-4), 55.8 (C-17), 44.4 (C-9), 44.0 (C-13), 42.9 (C-24), 39.3 (C-20), 36.8 (C-10), 35.7 (C-12), 34.2 (C-1), 34.1 (C-2), 33.1 (C-25), 27.7 (C-16), 25.4 (C-15), 21.2 (C-21), 20.0 (C-26), 19.7 (C-27), 19.0 (C-11, C-18), 17.6 (C-28), 16.7 (C-19). ESI-MS: m/z 393.21 [M + H]+. Compound 2. 1H-NMR (CDCl3): H 5.47 (1H, d, J = 5.5 Hz, H-11), 5.40 (1H, t, J = 7.0 Hz, H-24), 5.32 (1H, d, J = 6.0 Hz, H-7), 4.00 (2H, s, H-26), 3.25 (1H, dd, J = 3.5, 11.5 Hz, H-3), 1.67 (3H, s, H-27), 0.99 (3H, s, H-19), 0.98 (3H, H-29), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.88 (6H, H-28, H-30), 0.57 (3H, s, H-18); 13C-NMR (CDCl3): C 146.0 26 Cytotoxic steroids found in Vietnamese lingzhi Ganoderma neo-japonicum (C-9), 142.7 (C-8), 134.4 (C-25), 127.0 (C-24), 120.3 (C-7), 116.3 (C-11), 78.9 (C-3), 69.1 (C-26), 50.9 (C-17), 50.4 (C-5), 49.2 (C-14), 43.8 (C-13), 38.7 (C-4), 37.9 (C-12), 37.4 (C-10), 36.1 (C-20), 36.0 (C-22), 35.8 (C-1), 31.5 (C-16), 28.2 (C-2), 27.9 (C-15, C-19), 25.6 (C-28), 24.6 (C-23), 23.0 (C-6), 22.8 (C-19), 18.4 (C-21), 15.8 (C-30), 15.7 (C-18), 13.7 (C-27). ESI-MS: m/z 463.21 [M+Na]+. Compound 3. 1H-NMR (CDCl3): H 5.57 (1H, d, J = 5.0 Hz, H-6), 5.38 (1H, d, J = 5.0 Hz, H-7), 5.23 (1H, m, H-23),5.20 (1H, m, H-22), 3.64 (1H, m, H-3), 1.03 (3H, d, J = 6.5 Hz, H-21), 0.95 (3H, s, H-18), 0.92 (3H, d, J = 6.5 Hz, H-28), 0.85 (3H, d, J = 7.0 Hz, H-27), 0.82 (3H, d, J = 7.0 Hz, H-26), 0.63 (3H, s, H-19). 13C-NMR (CDCl3): C 141.4 (C-5), 139.9 (C-8), 135.5 (C-23), 132.0 (C-22), 119.6 (C-6), 116.3 (C-7), 70.5 (C-3), 55.8 (C-17), 54.6 (C-14), 46.5 (C-9), 42.9 (C-13, C-24), 40.5 (C-16), 40.4 (C-4, C-20), 39.1 (C-1), 38.4 (C-10), 33.1 (C-25), 32.0 (C-2), 23.0 (C-15), 21.1 (C-11, C-27), 19.9 (C-21), 19.8 (C-12), 19.6 (C-26), 17.6 (C-28), 16.3 (C-19), 12.0 (C-18). ESI-MS: m/z 396.20 [M]+. Compound 4. 1H-NMR (CDCl3): H 5.19 (1H, dd, J = 2.5, 8.0 Hz, H-23), 5.15 (1H, m, H-22), 3.59 (1H, m, H-3), 1.02 (3H, d, J = 6.5 Hz, H-21), 0.92 (3H, d, J = 6.5 Hz, H-28), 0.84 (3H, d, J = 7.0 Hz, H-26), 0.82 (3H, d, J = 7.0 Hz, H-27), 0.80 (3H, s, H-19), 0.55 (3H, s, H-18). 13C-NMR (CDCl3): C 139.6 (C-8), 135.7 (C-22), 131.9 (C-23), 117.5 (C-7), 71.1 (C-3), 55.1 (C-14), 56.0 (C-17), 49.5 (C-9), 43.3 (C-13), 42.8 (C-24), 40.4 (C-5), 40.3 (C-20), 39.5 (C-12), 38.0 (C-4), 37.2 (C-1), 34.3 (C-10), 33.1 (C-25), 31.4 (C-2), 29.7 (C-6), 28.1 (C-16), 22.9 (C-15), 21.6 (C-11), 21.1 (C-21), 20.0 (C-27), 19.7 (C-26), 17.6 (C-28), 13.0 (C-19), 12.1 (C-18). ESI-MS: m/z 398.15 [M]+. 2.2. Results and discussion Compound 1 was isolated in the form of white crystals. Its ESI-MS has a quasi-molecular ion peak at m/z 393.21 [M + H]+, indicating the molecular formula of C28H40O. Its 1H-NMR spectrum showed the presence of five olefinic protons and six methyls. Interpretation of the 13C-NMR spectrum revealed that it contained 28 carbon atoms, including one carbonyl group (C = 199.5 ppm) and eight olefinic carbons as described in the experimental section. Interestingly, three olefinic protons are resonanced at low field with H = 6.61 (d, J = 9.5 Hz), 6.03 (d, J = 9.5 Hz) and 5.74 (s), suggesting that they are linked with carbons in a conjugated system. In comparison with previous publications, compound 1 is found to be ergosta-4,6,8(14),22-tetraen-3-one [4]. Compound 2 was obtained in the form of white crystals. The ESI-MS shows a quasi-molecular ion peak at m/z 463.21 [M + Na]+. Therefore, the compound has a molecular formula of C30H48O2 due to its ESI-MS and 13C-NMR spectra. Its 1H-NMR spectrum indicates the presence of three olefinic protons at 5.47 (d, J = 5.5 Hz), 5.40 (t, J = 7.0 Hz) and 5.32 (d, J = 6.0 Hz, H-7) and one methylene connected with oxygen (4.00 ppm). The 13C-NMR spectrum shows the resonances of 30 carbon atoms including six olefinic carbons and seven methyls. Then, the structure of compound 2 was determined 27 Bui Thi Thu Hien, Nguyen Trang Thu, Nguyen Quang Tuyen, Le Xuan Tham and Dang Ngoc Quang by HSQC and HMBC spectra and two doubles are located at C78 and C911 by long range correlations between H-6 /C-7, C-8 and H-12/C-9, C-11. Another double bond deduced is C2425 by HMBC correlations from H-23, H-26 and H-27 to C-24 and C-25. Finally, compound 2 is elucidated as ganodermadiol [1]. Figure 1. Structures of compounds 1-4 Analysis of 1H-NMR and 13C-NMR spectra of compound 3 indicates that it has 28 carbons, including three double bonds and one oxygen bearing carbon at C-3 together with other signals revealing a steroid skeleton. In addition, its molecular formula was deduced to be C28H44O by a combined analysis of ESI-MS and 13C-NMR spectra. Comparing the spectral data of 3 with that found in previous publications, compound 3 is determined to be ergosterol [6]. Compound 4 has spectral data similar to that of compound 3 except for the appearance of only two double bonds. The first double bond is found at C78 by HMBC correlations between H-6, H-9 with C-7 and C-8. The second double bond is located at C2223 by HMBC correlations from H-20, H-21 to C-22 and H-24, H-25, H-28 to C-23. Therefore, compound 4 is established as ergosta-7,22-dien-3-ol [8]. The anticancer property of compounds 1-4 against the KB cell line was investigated using the method described in [7]. The result showed that compounds 1, 3 and 4 showed good cytotoxic activity against KB cells with their IC50 values of 12.68, 10.72 and 87.95 g/mL, respectively. This is the first report on the chemical constituents of Vietnamese lingzhi Ganoderma neo-japonicum and its cytotoxicity. 3. 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