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 µg/mL, shows strong cytotoxicity against the KB cell line. This paper describes the isolation and cytotoxicity of four compounds from this mushroom.
JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2014, Vol 59, No 9, pp 25-29 This paper is available online at http://stdb.hnue.edu.vn 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 Faculty of Chemistry, Hanoi National University of Education Department 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-3β-ol (4) through the use of various chromatographic and spectroscopic methods, including ESI-MS and 2D NMR In addition, compounds 1, and showed moderate cytotoxic activity toward KB cell lines with their IC50 values of 12.68, 10.72 and 87.95 µg/mL, respectively Keywords: Ganoderma neo-japonicum, cytotoxicity, lingzhi, steroid 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 µg/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 Content 2.1 Experiment * General procedure Column chromatography was carried out on silica gel 60 (60 - 100 µM, 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 SL-II column (10.0 × 250 mm), flow rate of 1.0 ml/min NMR (1 H-NMR, 13 C-NMR, DEPT, HSQC and HMBC) spectra were recorded on a Bruker Avance 500 MHz Instrument The chemical shift (δ) 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 sub-fractions Sub-fr (111 mg) was further purified by prep HPLC, n-hexane-EtOAc (8/1) to afford compound (3 mg), rt 44.96 Compound (3 mg) was obtained from sub-fr (53 mg) by prep HPLC, n-hexane-EtOAc (4/1), rt 12.69 Compound (5 mg) and (4 mg) were purified from sub-fr (227 mg) by prep HPLC, n-hexane-EtOAc (6/1) with rt 4.85 and 8.39 min, respectively Compound 1 H-NMR (CDCl3 ): δH 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); 13 C-NMR (CDCl3): δC 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 H-NMR (CDCl3 ): δH 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); 13 C-NMR (CDCl3 ): δC 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 H-NMR (CDCl3 ): δH 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) 13 C-NMR (CDCl3 ): δC 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 H-NMR (CDCl3 ): δH 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) 13 C-NMR (CDCl3 ): δC 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 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 C28 H40 O Its H-NMR spectrum showed the presence of five olefinic protons and six methyls Interpretation of the 13 C-NMR spectrum revealed that it contained 28 carbon atoms, including one carbonyl group (δC = 199.5 ppm) and eight olefinic carbons as described in the experimental section Interestingly, three olefinic protons are resonanced at low field with δH = 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 is found to be ergosta-4,6,8(14),22-tetraen-3-one [4] Compound 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 C30 H48 O2 due to its ESI-MS and 13 C-NMR spectra Its H-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 13 C-NMR spectrum shows the resonances of 30 carbon atoms including six olefinic carbons and seven methyls Then, the structure of compound 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 C7−8 and C9−11 by long range correlations between H-6 /C-7, C-8 and H-12/C-9, C-11 Another double bond deduced is C24−25 by HMBC correlations from H-23, H-26 and H-27 to C-24 and C-25 Finally, compound is elucidated as ganodermadiol [1] Figure Structures of compounds 1-4 Analysis of H-NMR and 13 C-NMR spectra of compound 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 C28 H44 O by a combined analysis of ESI-MS and 13 C-NMR spectra Comparing the spectral data of with that found in previous publications, compound is determined to be ergosterol [6] Compound has spectral data similar to that of compound except for the appearance of only two double bonds The first double bond is found at C7−8 by HMBC correlations between H-6, H-9 with C-7 and C-8 The second double bond is located at C22−23 by HMBC correlations from H-20, H-21 to C-22 and H-24, H-25, H-28 to C-23 Therefore, compound is established as ergosta-7,22-dien-3β-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, and showed good cytotoxic activity against KB cells with their IC50 values of 12.68, 10.72 and 87.95 µg/mL, respectively This is the first report on the chemical constituents of Vietnamese lingzhi Ganoderma neo-japonicum and its cytotoxicity Conclusion Four fungal metabolites, ergosta-4,6,8(14),22-tetraen-3-one (1), ganodermadiol (2), ergosterol (3) and ergosta-7,22-dien-3β-ol (4) were isolated from the fruit bodies of 28 Cytotoxic steroids found in Vietnamese lingzhi Ganoderma neo-japonicum Vietnamese lingzhi Ganoderma neo-japonicum Three of them (compounds 1, and 4) showed good cytotoxic activity against the KB cell line with their IC50 values of 12.68, 10.72 and 87.95 µg/mL, respectively Acknowledgements This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED), Grant No 104.01-2011.55 REFERENCES [1] M Arisawa, A Fujita, M Saga, H Fukumura, T Hayashi, M Shimizu, N Morita, 1986 Three new lanostanoids from Ganoderma lucidum, J Nat Prod., Vol 49, pp 621-625 [2] B T T Hien, L T P Hoa, L X Tham, D N Quang, 2013 Cattienoids A-C, three novel steroids from the mushroom Tomophagus cattienensis Fitoterapia, Vol 91, pp 125-127 [3] K Peter, A Ngo, T K Trinh, S Brigitte, M D Hans, H Albert, G Udo, 2001 Colossolactones, New triterpenoid metabolites from a Vietnamese mushroom Ganoderma colossum J Nat Prod., Vol 64, pp 236-239 [4] D N Quang, D D Bach, 2008 Ergosta-4,6,8(14),22-tetraen-3-one from Vietnamese Xylaria sp possessing inhibitory activity of nitric oxide production Natural Product Research, Vol 22, pp 901-906 [5] D N Quang, T T Nga, L X Tham, 2011 Chemical Composition of Vietnamese Black Lingzhi Amauroderma subresinosum Murr Research Journal of Phytochemistry, Vol 5, pp 216-221 [6] D N Quang, B T T Huong, L X Tham, A Porzel, 2011 Chemical compositions and the anticancer activity of red Lingzhi Ganoderma tropicum Journal of Chemistry, No 6, pp 693-696 (in Vietnamese) [7] D A Scudiero, R H Shoemaker, D P Kenneth, A Monks, S Tierney, T H Nofziger, M J Currens, D Seniff, M R Boyd, 1988 Evaluation of a soluable tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines Can Res., Vol 48, pp 4827-4833 [8] Y Takaishi, T Ohashi, T Tomimatsu, 1989 Ergosta-7,22-dien-3 β-ol glycoside from Tylopilus neofelleus Phytochemistry, Vol 28, pp 945-947 [9] L X.Tham, 2005 Ganodermataceae family Science and Technology Publishing House, Hanoi, Vietnam 29 ... of 28 Cytotoxic steroids found in Vietnamese lingzhi Ganoderma neo-japonicum Vietnamese lingzhi Ganoderma neo-japonicum Three of them (compounds 1, and 4) showed good cytotoxic activity against... (6H, H-28, H-30), 0.57 (3H, s, H-18); 13 C-NMR (CDCl3 ): δC 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... chemical constituents of Vietnamese lingzhi Ganoderma neo-japonicum and its cytotoxicity Conclusion Four fungal metabolites, ergosta-4,6,8(14),22-tetraen-3-one (1), ganodermadiol (2), ergosterol