Cytotoxic constituents of the fungus kretzschmaria sandvicensis collected in Dien Bien, Vietnam

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Three fungal metabolites, (1) O-methylsilvaticol, (2) orthosporin and (3) diplosporin were isolated from a rare fungus, Kretzschmaria sandvicensis, collected in Dien Bien, in northwest Vietnam. Their structures were established using a combination of high resolution MS (FT-ICR-MS) and 1H-NMR, 13C-NMR, HSQC and HMBC spectroscopy. They were evaluated for anticancer properties against the KB cell line.

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Nội dung Text: Cytotoxic constituents of the fungus kretzschmaria sandvicensis collected in Dien Bien, Vietnam

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol. 58, No. 9, pp. 15-19 This paper is available online at http://stdb.hnue.edu.vn CYTOTOXIC CONSTITUENTS OF THE FUNGUS Kretzschmaria sandvicensis COLLECTED IN DIEN BIEN, VIETNAM Ha Thi Nhung1, Bui Thi Thu Hien1 , Luu Van Huyen2 and Dang Ngoc Quang1 1 Faculty of Chemistry, Hanoi National University of Education 2 Hanoi University for Natural Resources and Environment Abstract. Three fungal metabolites, (1) O-methylsilvaticol, (2) orthosporin and (3) diplosporin were isolated from a rare fungus, Kretzschmaria sandvicensis, collected in Dien Bien, in northwest Vietnam. Their structures were established using a combination of high resolution MS (FT-ICR-MS) and 1 H-NMR, 13 C-NMR, HSQC and HMBC spectroscopy. They were evaluated for anticancer properties against the KB cell line. It was found that compound 2 had moderate activity (IC50 = 78.57µg/mL) while compounds 1 and 3 had no cytotoxic activity (IC50 > 100µg/mL). This is a first report on the chemical constituents of Kretzschmaria sandvicensis. Keywords: Chemical constituents, Kretzschmaria sandvicensis, O-methylsilvaticol, orthosporin, diplosporin. 1. Introduction Fungi are among the largest groups of organisms of our globe. Out of an estimated 1.5 million species, it is thought that only 5% (75,000) are now known [3]. The Xylariaceae family is one of the most important fungal groups, especially with respect to the production of secondary metabolites. Of the Xylariaceae species, it has been seen that their biological diversity correlates with the chemical diversity of their characteristic secondary metabolites [10]. This biodiversity is very high in the tropics [6] but of the 2200 fungal species that have been found in Vietnam [9], relatively few are Xylariaceae. If one discounts some recent work [4, 5] it can be said that macrofingi, including the Xylariaceae of Vietnam, are still unknown. Kretzschmaria sandvicensis is a rare fungus of the Xylariaceae family that has never undergone chemical investigation. Its fruit bodies are black and they do not react in contact with a 10% KOH solution. A large amount of K. Received September 3, 2013. Accepted October 17, 2013. Contact Dang Ngoc Quang, e-mail address: quangdn@hnue.edu.vn 15
Ha Thi Nhung, Bui Thi Thu Hien, Luu Van Huyen and Dang Ngoc Quang sandvicensis fruitbodies were recently collected in our field trip in Dien Bien, Vietnam, that allowed us to study its chemical constituents. The isolation and structural elucidation of three fungal metabolites as well as their cytotoxicity is the subject of this paper. 2. Content 2.1. Experiments 2.1.1. General procedure TLC was carried out on precoated silica gel GF254 (Merck Co., Germany) and TLC spots were viewed at 254, 302 and 366 nm and visualized by spraying with a vanillin-10% H2 SO4 solution. Column chromatography was carried out on silica gel 60 (60 - 100 µM, Merck). Preparative HPLC was performed using a Jasco PU-2087 instrument with UV-2070 and RI-2031 detectors using a Waters 5 SL-II column (10.0 × 250 mm) with a flow rate of 1.0 mL/min. NMR (1 H, 13 C-NMR, DEPT, HSQC and HMBC) and spectra were recorded on a Brucker Avance 500 MHz. The chemical shift (δ) values are given in ppm with TMS as the internal standard, coupling constant J - by Hz. Mass spectra, including high resolution MS, were recorded on a HP 5989B mass spectrometer and a FT-ICR-MS (Varian 910-MS TQFTMS-7 Tesla). 2.1.2. Fungal material Fresh fruit bodies of Kretzschmaria sandvicensis (212 g) were collected in Muong Phang, Dien Bien, in June 2011 and identified by Dr. Duong Minh Lam, Faculty of Biology, Hanoi National University of Education. Voucher specimens (HTN-01) were deposited at the Faculty of Chemistry, Hanoi National University of Education. 2.1.3. Extraction and isolation An extraction of fresh fruit bodies of Kretzschmaria sandvicensis (212 g) was carried out using MeOH. The methanolic extract was concentrated using a rotary evaporator which yielded a residue (7.7 g) which was further subjected to silica gel column, using a hexane-EtOAc gradient to obtain seven fractions. Sub-fraction 3 (94 mg) was purified using a Sephadex LH-20 column with CHCl3 - MeOH (1:1) and this was followed by prep. HPLC, hexane-EtOAc (4/1) to yield compound 1 (5 mg). Sub-fraction 6 (391 mg) was chromatographed on a silica gel column using a hexane-EtOAc gradient and finally using prep. HPLC eluted with hexane-EtOAc (1/3) to yield compound 2 (10 mg) and compound 3 (7 mg). Compound 1: 1 H − NMR(CDCl3 ) : δH 7.09 (1H, s, H-7), 5.39 (2H, s, H-3), 3.89 (6H, s, 4-OCH3 and 4-OCH3 ), 2.20 (3H, s, 5-CH3 ); 13 C-NMR (CDCl3 ): δC 171.2 (C-1), 160.1 (C-6), 152.9 (C-4), 128.3 (C-4a), 125.5 (C-5), 124.9 (C-7a), 100.8 (C-7), 68.3 (C-3), 59.3 (4-OMe), 56.1 (6-OMe), 9.6 (5-Me). FT-ICR-MS: m/z 231.0626 [M + Na]+ (Calcd for C11 H12 O4 Na : 231.0633). 16
Cytotoxic constituents of the fungus Kretzschmaria sandvicensis... Compound 2: 1 H-NMR (CDCl3 ): δH 6.37 (1H, d, J = 2.0 Hz, H-7), 6.26 (1H, d, J = 2.0 Hz, H-5), 6.25 (1H, s, H-4), 2.59 (2H, m, H-9), 4.22 (1H, m, H-10), 1.28 (3H, d, J = 6.0 Hz, H-11); 13 C NMR (CDCl3 ): δC 166.5 (C-1), 165.3 (C-6), 163.1 (C-8), 154.4 (C-3), 139.4 (C-4a), 106.0 (C-4), 102.8 (C-5), 101.9 (C-7), 98.9 C(-8a), 65.1 (C-10), 42.8 (C-9), 22.9 (C-11). FT-ICR-MS: m/z 259.0576 [M + Na]+ (Calcd. for C12 H12 O5 Na: 259.0581). Compound 3: 1 H-NMR (CDCl3 ): δH 7.78 (1H, s, H-2), 4.60 (1H, d, J = 7.5 Hz, H-5), 4.47 (2H, d, J = 3.5 Hz, H-13), 2.60 (2H, m, H-8), 2.05 (1H, m, H-7), 1.83 (1H, m, H-11), 1.65 (1H, m, H-6), 1.47 (1H, m, H-7), 1.28 (1H, m, H-11), 0.98 (3H, t, J = 7.5 Hz, H-12). 13 C-NMR (CDCl3 ): δC 180.7 (C-4), 166.2 (C-9), 152.0 (C-2), 126.7 (C-3), 123.7 (C-10), 68.7 (C-5), 541.3 (C-6), 58.2 (C-13), 26.8 (C-8), 24.2 (C-11), 23.1 (C-7), 11.2 (C-12). FT-ICR-MS: m/z 247.0938 [M + Na]+ (Calcd. for C12 H16 O4 Na : 247.0946). 2.1.4. Cytotoxic assay Cytotoxic assays of compounds 1-3 were tested against KB cell lines from an American Type Culture Collection according to the method described by Scudiero [7]. Cell lines were cultured in an RPMI 1640 medium supplemented with a 10% foetal bovine serum (FBS) in standard condition, sterile with 5% CO2 at 37 0 C, 98% humidity and harvested at log phase for assays. In this assay, 200 µL volumes of cells at a concentration of 3 × 104 cells mL−1 were inoculated into a 96-well plate in a RPMI 1640 medium. Compounds 1-3 were applied at final concentrations 128, 32, 8, 2 and 0.5 µL−1 and the cultures were incubated for 3 days at 37 0 C with 5% CO2 . Then, 50 µL of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, prepared at 1mg mL−1 in FBS, was added to the microculture. After 4 h of incubation, 250 µL of the supernatant was removed from each well and 100 µL of DMSO was added, mixing thoroughly. Absorbance was measured at 540 nm in a Genios TECAN spectrophotometer. The IC50 value was calculated based on percent growth inhibition (ODcontrol −ODsample )/ODcontrol . 2.2. Results and discussion The molecular formula of compound 1 was established as C11 H12 O4 by FT-ICR-MS (m/z 231.06264; [M + Na]+ ) and NMR data, which corresponded to 6 degrees of unsaturation. Its 1 H-NMR spectrum showed the presence of one aromatic proton with δH = 7.09 ppm, one vinylic methyl group at 2.20 ppm, and one methoxyl group (δH = 3.89 ppm). Analysis of the 13 C-NMR revealed that it contained 11 carbon atoms, including one carbonyl group (δC = 171.22 ppm), the six carbon signals corresponding to an aromatic ring. The structure of compound 1 was then determined by 2D NMR spectra. There were cross signals from −CH2 − to C-4, C-4a and C-7a in the HMBC spectrum indicating that this –CH2 – group was attached to the aromatic ring at C-4a. Furthermore, the methylene −CH2 − group also coupled to the carbonyl group suggesting the presence of a five membered lactone ring. Detailed analysis of the HMBC spectrum revealed that H-7 correlated to C=O, C-4a, C-5, C-6 and C-7a, thus, it was located at C-7 and the carbonyl group connected with C-4a of the aromatic ring. The methyl group has HMBC correlations with C-4a, C-4, C-5, C-6 and C-7, thus it was at C-5. Finally, two methoxyl 17
Ha Thi Nhung, Bui Thi Thu Hien, Luu Van Huyen and Dang Ngoc Quang groups were at C-4 and C-6 because of HMBC correlations between the methoxyl groups and C-4 and C-6. From the above discussion, compound 1 was found as shown in Figure 1. Compound 1 was already isolated from the fungus Aspergillus silvaticus and named 4,6-dimethoxy-5-methylphthalide or O-methylsilvaticol [1]. Figure 1. The structures of compounds 1 - 3 The molecular formula of compound 2 was found to be C12 H12 O5 by FT-ICR-MS with 7 degrees of unsaturation. Analysis of its 1 H-NMR spectrum revealed that it contained two proton signals at δH = 6.37 (J = 2 Hz) and 6.26 (J = 2 Hz) indicating that they are at meta-position. In addition, one multiplet peak was at δH = 2.59, corresponding to the methylene group −CH2 . The 13 C-NMR spectrum showed 12 carbon signals, including one carbonyl group at δC = 166.5 ppm and eight olefinic carbons. The presence of a 2-hydroxyl-propyl partial structure was determined by the HMBC correlation between i) H-11 and C-9, C-10 and ii) H-9 and C10, C-11, C-3, C-4. Furthermore, compound 2 has spectral data similar to that of orthosporin. Consequently, compound 2 was characterized as being orthosporin [2]. Compound 3 has a molecular formula of C12 H16 O4 with five degrees of unsaturation. The 1 H-NMR spectrum of compound 3 has proton signals for a methyl (δH = 0.98 ppm), one olefinic proton (δH = 7.78 ppm), and two protons connected to carbon bearing oxygen at δH = 4.60 and 4.47 ppm. Its 13 C-NMR spectrum showed 12 carbon signals, including a carbonyl at δC = 180.7 ppm. Our interpretation of the HMBC spectrum is that there is a possible presence of an ethyl group at C-6 due to HMBC correlations of H-12 and C-11, C-6; H-11 and C-5, C-6, C-7, C-12. In addition, −CH2 OH was determined at C-3 since H-13 are coupled to C-2, C-3, C-4; and C-13 has a cross peak with H-2. Based on the above, compound 3 was determined to be diplosporin [8]. The anticancer properties of compounds 1-3 against the KB cell line were examined. The results show that compound 2 had moderate activity (IC50 = 78.57µg/mL), and compounds 1 and 3 lacked cytotoxic activity (IC50 > 100µg/mL). 18
Cytotoxic constituents of the fungus Kretzschmaria sandvicensis... 3. Conclusion A phytochemical examination of the methanolic extract of Kretzschmaria sandvicensis collected in Dien Bien, Vietnam, has for the first time led to the isolation and structural elucidation of three compounds: O-methylsilvaticol (1), orthosporin (2) and diplosporin (3). Of the three, compound 2 showed moderate cytotoxicity against the KB cell line. Acknowledgements. This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2011.55. REFERENCES [1] M. Fujita, M. Yamada, S. Nakajima, K.I. Kawai, M. Nagai, 1984. O-Methylation effect on the carbon-13 nuclear magnetic resonance signal of ortho-disubstituted phenols and its application to structure determination of new phthalides from Aspergillus silvaticus. Chem. Phar. Bull., Vol. 32, pp. 2622-2627. [2] Y. F. Hallock, J. Clardy, D. S. Kenfield, G. Strobel, 1988. De-O-methyldiaporthin, a new phytotoxin from Drechslera siccans. Phytochemistry, Vol. 27, pp. 3123-3125. [3] D. L. Hawksworth, 1991. The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycological Research, Vol. 95, pp. 641-655. [4] D. N. Quang, D. D. Bach, T. Hashimoto, Y. Asakawa, 2006. Chemical constituents of the Vietnamese inedible mushroom Xylaria intracolorata. Natural Product Research, Vol. 20, pp. 317-321. [5] D. N. Quang, D.D. Bach, Y. Asakawa, 2007. Sterols from a Vietnamese wood-rotting Phellinus sp. Z. Naturforschu., Vol. 62b, pp. 289-292. [6] J.D. Rogers, 2000. Thoughts and musings about tropical Xylariaceae. Mycol Res., Vol. 104, pp. 1412-1420. [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. Cancer Res., Vol. 48, pp. 4827-4233. [8] P.S. Steyn, 1979. The biosynthesis of polyketide-derived mycotoxins. Pure Appi. Chem., Vol. 52, pp.189-204. [9] H. Tran, 2005. Pharma IndoChina IV; Proceeding; Ho Chi Minh, Vietnam, November, 2005, p. 59. [10] A. J. S. Whalley, R. L. Edwards, 1995. Secondary metabolites and systematic arrangement within the Xylariaceae. Can. J. Bot., Vol. 73, pp. 802-810. 19