Antifungal constituents from the stems of dracaena cambodiana - Nguyen Hai Dang

Journal of Chemistry, Vol. 46 (4), P. 503 - 508, 2008 ANTIFUNGAL CONSTITUENTS FROM THE STEMS OF DRACAENA CAMBODIANA Received 6 September 2007, Revised 1 November 2007 Nguyen Hai Dang1, Phan Van Kiem1, Chau Van Minh1, and Tran Thu Huong2 Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology 1Faculty of Chemical Technology, Hanoi University of Technology, summary summary The stems of Dracaena cambodiana have been investigated for antifungal constituents, resulting in the isolation of a new steroidal saponin dracagenin A (1) together with two known components 25S-namogenin B (2) and spriroconazole A (3). Their structures were elucidated on the basis of extensive spectroscopic analysis including 1D, 2D NMR and ESI MS. The isolated compounds were evaluated for their antifungal activities. 1 and 3 exhibited inhibitory activity against Aspergilus niger at the MICs of 50 μg/ml. No inhibition effects on F. oxysporum, S. cerevisiae and C. albicans have been found for the tested compounds. Interestingly, toxicity of 3 towards several cancer cell lines has been demonstrated. The IC50 of 3 against Hep-2, Lu, and RD are 2.002, 4.727, and 4.029 μg/ml, respectively. Keywords: Dracaenaceae, Dracaena cambodiana, Steroidal saponin, Dracagenin A. I - INTRODUCTION Dracaena cambodiana (Dracaenaceae) is mostly distributed in the North of Vietnam. The plant has been found in ethnomedicine to treat menoschesis, ostealgia etc (Bich, 2006; Chi, 1997; Loi, 2001). Investigations on the chemical constituents of the genus revealed that the spirosterol and steroidal saponin are among the major components (Gonzalez, 2004; Mimaki, 1998). Interestingly, there has been no report on the phytochemical of D. cambodiana. On our continuing studies for antifungal agents, we have isolated a new steroidal saponin namely dracagenin A (1), one sprirosterol 25S- namogenin B (2) and one known steroidal saponin spriroconazole A (3). The antifungal activities of these compounds were also described. II - MATERIALS AND METHODS General experiment procedures The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer. Chemical shifts are referenced to δ using tetramethylsilan (TMS) as an internal standard. The Electron Spray Ionization (ESI) mass spectrum was obtained using an AGILENT 1100 LC-MSD Trap spectrometer. Column chromatography (CC) was performed on silicagel 230 - 400 mesh (0.040-0.063 mm, Merck) or YMC RP-18 resins (30-50 μm, FuJisilisa Chemical Ltd.). Thin layer chromatography (TLC) was performed on DC- Alufolien 60 F254 (Merck 1.05715) or RP18 F254s (Merck) plates. Plant material The roots of D. cambodiana were collected in Hoabinh province, Vietnam in January 2006 503 and identified by Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, VAST, Vietnam. An authentic sample was deposited at the Institute of Natural Products Chemistry, VAST, Vietnam. Extraction and isolation The dried roots of D. cambodiana (4 kg) were extracted with MeOH three times (7 days each time) and then concentrated under low pressure to obtain 150 g MeOH extract. The MeOH extract was suspended in water and partitioned with hexane, CHCl3 and BuOH to obtain n-hexane (60 g), CHCl3 (35 g), and BuOH (65 g) fractions. The CHCl3 fraction was then chromatographed on silicagel column eluting with CHCl3-MeOH gradient (from 10:1 to 1:1 v/v) to give fractions C1 (16 g), C2 (11 g), and C3 (7 g). C1 fraction was chromatographed on a silicagel column using CHCl3-EtOAc (3:1) as eluent to give subfractions C3A (6.5 g), C3B (3.4 g), C3C (6.0 g). The C3C subfraction (6.0 g) was chromatographed on a silica gel column using a CHCl3-MeOH (3:1 v/v) system as eluent yielded 2 (25 mg) as white powder. The BuOH fraction was chromatographed on silicagel column eluting with CHCl3-MeOH gradient (from 10:1 to 1:1 v/v) to obtain fractions B1 (25 g), B2 (20 g), and B3 (12 g). B2 fraction showed the most active antifungal activity then selected for further isolation of bioactive compounds. The B2 fraction (20 g) was chromatographed on an YMC RP-18 column using a MeOH-H2O (2:1 v/v) system as eluent yielded 1 (18.5 mg) and 3 (9 mg) as white amorphous powder. 25S-Namogenin B (2): White powder, positive ESI-MS m/z: 447 [M+H]+, 429 [M+H- H2O] +, 411 [M+H-2H2O] +; negative ESI-MS m/z: 445 [M-H]-; 1H-NMR (500 MHz, CDCl3) δ: 3.46 (dd, J = 4.0, 11.5 Hz, H-1), 1.57/2.06 (m, H-2), 3.53 (m, H-3), 1.89/2.24 (m, H-4), 5.57 (d, J = 5.5 Hz, H-6), 1.94/2.08 (m, H-7), 1.97 (m, H-8), 1.59 (m, H-9), 1.60/2.11 (m, H-11), 1.42/1.75 (m, H-12), 1.61/1.95 (m, H-15), 4.60 (m, H-16), 2.29 (m, H-17), 0.94 (s, H-18), 1.07 (s, H-19), 2.10 (m, H-20), 1.01 (d, J = 7.0 Hz, H-21), 1.94/2.02 (m, H-23), 1.40/2.11 (m, H- 24), 1.72 (m, H-25), 3.29 (d, J =2.5 Hz, H-26a), 3.93 (d, J = 11.0 Hz, H-26b), 0.90 (d, J = 7.0 Hz, H-27); 13C-NMR (125 MHz, CDCl3) δ: 77.7 (C-1), 42.0 (C-2), 67.9 (C-3), 42.6 (C-4), 137.6 (C-5), 125.5 (C-6), 26.2 (C-7), 35.7 (C-8), 43.7 (C-9), 43.2 (C-10), 22.6 (C-11), 32.0 (C-12), 44.0 (C-13), 87.6 (C-14), 39.4 (C-15), 81.9 (C- 16), 58.8 (C-17), 20.0 (C-18), 13.0 (C-19), 42.2 (C-20), 14.5 (C-21), 110.0 (C-22), 26.1 (C-23), 25.8 (C-24), 27.1 (C-25), 65.1 (C-26), 16.1 (C- 27). Spriroconazole A (3): White powder, positive ESI-MS m/z: 907 [M+Na]+; negative ESI-MS m/z: 883 [M-H]-; 1H-NMR (500 MHz, CDCl3) δ: 1.11/1.90 (m, H-1), 1.31/1.91 (m, H- 2), 3.66 (m, H-3), 2.29/2.47 (m, H-4), 5.40 (d, J = 5.0 Hz, H-6), 1.37/1.59 (m, H-7), 1.65 (m, H- 8), 0.98 (m, H-9), 1.54/1.63 (m, H-11), 1.65/2.03 (m, H-12), 1.73 (m, H-14), 1.25/1.71 (m, H-15), 4.01 (dd, J = 7.0, 8.0 Hz, H-16), 0.85 (s, H-18), 1.07 (s, H-19), 2.11 (m, H-20), 0.94 (d, J = 7.0 Hz, H-21), 1.70/2.03 (m, H-23), 1.45/1.62 (m, H-24), 1.62 (m, H-25), 3.36/3.47 (m, H-26), 0.81 (d, J = 6.5 Hz, H-27), 4.53 (d, J = 7.0 Hz, H-1Glc), 3.58 (t, J = 9.0 Hz, H-3 Glc), 4.99 (d, J = 1.5 Hz, H-1 RhaI), 1.26 (d, J = 6.5 Hz, H-6 RhaI), 4.92 (d, J = 1.5 Hz, H-1 RhaII), 1.28 (d, J = 6.5 Hz, H-6 RhaII); 13C- NMR (125 MHz, CDCl3) δ: 38.6 (C-1), 30.8 (C- 2), 79.1 (C-3), 39.3 (C-4), 141.9 (C-5), 122.9 (C-6), 32.8 (C-7), 33.3 (C-8), 51.5 (C-9), 38.0 (C-10), 21.7 (C-11), 33.2 (C-12), 45.8 (C-13), 53.1 (C-14), 32.1 (C-15), 90.6 (C-16), 91.3 (C- 17), 17.5 (C-18), 19.8 (C-19), 45.5 (C-20), 9.1 (C-21), 111.0 (C-22), 32.5 (C-23), 29.4 (C-24), 31.3 (C-25), 67.7 (C-26), 17.5 (C-27), Glc (1) 100.2, (2) 97.4, (3) 88.4, (4) 70.5, (5) 77.5, (6) 62.6, RhaI (1) 102.8, (2) 72.3, (3) 72.5, (4) 73.6, (5) 70.1, (6) 17.8, RhaII (1) 103.9, (2) 72.2, (3) 72.4, (4) 73.8, (5) 70.9, (6) 18.1. III - RESULTS AND DISCUSSION Bioassay guided fractionation led to the isolation of three bioactive components. Compound 1 was obtained as white amorphous powder, The ESI MS (positive mode) showed the pseudomolecular [M+Na]+ ion peak at m/z 1085 (corresponding to the molecular formula C51H82O23). The 13C-NMR spectrum of 1 showed 504 OOHO O O O OH HO OH HO OHOO O OH O OH OH OH CH3O OH HO HO H3C RhaIRhaII 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2526 27 GlcI GlcII 1 O O HO 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2526 27 OH OH 2 O OHO O OHOO O OH O OH OH OH CH3O OH HO HO H3C RhaIRhaII 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2526 27 GlcI 3 Figure 1: Structures of 1-3 51 signals, among which 27 were assigned to the aglycone, the remaining 24 signals were indicative of the presence of four hexoses due to two D-glucoses and two L-rhamnoses. The structure of the aglycone moiety was recognized to be penogenin (3β,17α-dihydroxyspirost-5en) by 1H- and 13C-NMR spectral analysis using connectivities observed in COSY, HSQC, and 505 HMBC. The 1H-NMR spectrum of 1 showed two three-proton singlet signals at δ 0.83 and 1.07 and two three-proton doublet signals at δ 0.91 (J = 7.5 Hz) and 1.00 (J = 6.5 Hz), which were characteristic of the spirostanol skeleton, as well as signals for four anomeric protons at δ 4.54 (d, J =7.5 Hz), 4.37 (d, J = 7.5 Hz), 5.00 (d, J = 1.5 Hz), 1.92 (d, J = 1.5 Hz). The salient features of this aglycone part were the 13C-NMR signals at δ 141.9 (C-5) and 122.6 (C-6), characteristic of Δ5-spirostene-type sapogenin. The proton coupling constant between H-25 and H-26ax (J = 11.5 Hz), and the 13C-NMR shifts of the F-ring part (C-25/δ 38.5) gave an evidence for the C-25R configuration. Table 1: NMR data of 1 No. δCa,b δHa,c (J, Hz) HMBC (H to C) 1 38.6 1.13 m; 1.90 m C-5 2 30.7 1.62 m; 1.91 m 3 79.1 3.65 m C-1 Glc 4 39.3 2.30 m; 2.47 m C-2, C-5, C-6, C-10 5 141.9 6 122.6 5.40 d (5.5) C-8, C-10 7 32.8 1.37 m; 1.68 m 8 33.3 1.65 m 9 51.5 0.97 m 10 38.0 11 21.7 1.49 m; 1.63 m 12 33.2 1.59 m; 2.03 m 13 45.7 14 53.9 1.74 m 15 32.0 1.31 m; 2.05 m C-15, C-17 16 90.9 3.99 t (6.5) 17 91.8 18 17.5 0.83 s C-12, C-13, C-14, C-17 19 19.8 1.07 s C-4, C-9 20 45.9 2.16 m C-14, C-17, C-21, C-23 21 9.0 0.91 d (7.5) C-17, C-20, C-22 22 113.8 23 41.0 1.69 m; 2.22 m C-24, C-25 24 82.0 3.62 m 25 38.5 1.67 m 26 65.8 3.38 m; 3.53 m C-24, C-27 27 13.5 1.00 d (6.5) C-24, C-26 GlcI (1 → C3) 1 100.2 4.54 d (7.5) C-3 2 79.4 3.47 m 3 88.4 3.60 m C-2 Glc, C-4, Glc 4 70.6 3.40 m 5 77.5 3.29 m 6 62.6 3.70 m; 3.88 m RhaI (1 → 2Glc) 1 102.8 5.00 d (1.5) C-2 Glc, C-3 Rha, C-5 Rha 2 72.3 3.64 m 506 No. δCa,b δH a,c (J, Hz) HMBC (H to C) 3 72.5 3.88 m 4 73.6 3.43 m 5 70.1 4.10 m 6 17.8 1.26 d (6.0) RhaII (1 → 3Glc) 1 103.9 4.92 d (1.5) C-3 Glc, C-3 Rha’, C-5 Rha’ 2 72.2 3.86 m 3 72.4 3.66 m 4 73.8 3.43 m 5 70.9 3.95 m 6 18.1 1.28 d (6.0) GlcII (1 → C24) 1 106.0 4.37 d (7.5) C-24 2 75.5 3.17 m 3 78.1 3.39 m 4 71.7 3.33 m C-5 Glc’ 5 77.7 3.31 m 6 62.8 3.70 m; 3.88 m aMeasured in CD3OD, b125 MHz, c500 MHz, Chemical shift (δ) in ppm. The connection of the sugars part to C-3 of the aglycone was confirmed by the correlation of an anomeric proton (δH 4.54) and C-3 position (δC 79.1) in the HMBC spectrum. This carbohydrate consisted of one disubstituted glucose and two terminal rhamnose moieties. The linkage positions of the sugars were determined by HMBC spectrum. Accordingly, the anomeric proton signal observed in 1H-NMR spectrum at δH 5.00 correlated with C-2 (δC 79.4) of glucose, the anomeric proton signal observed in 1H-NMR spectrum at δH 4.92 correlated with C-3 (δ 88.3) of glucose. Furthermore, the presence of two upfield signals of methyl protons at δ 1.26 (d, J = 1.5 Hz), and 1.28 (d, J = 1.5 Hz) in its 1H-NMR spectrum confirmed that the two deoxyhexopyranose units were rhamnose moieties. The rhamnopyranosyl residues were shown to be terminal units as suggested by the absence of any glysosydation shift for their carbon resonances. The 2,3 distribution of the glucopyranosyl moiety was shown by downfield chemical shifts at δ 79.4 (C-2 GlcI) and 88.3 (C- 3 GlcI) in the 13C-NMR spectrum. The NMR data were in good agreement with literature data (Tepono, 2001; Yokosuka, 2002). The remaining sugar moiety was also determined by extensive spectroscopic studies. The strong downfield shift signal at C-24 (δ 82.0) in 13C-NMR spectrum was indicative of the presence of one sugar moiety at this position. The correlation observed in HMBC spectrum between the anomeric proton (δH 4.37 (d, J = 7.5 Hz)) and C-24 (δC 82.0) confirmed that one glucose unit connected to C-24 of the F-ring. By above evidence and comparison with published data (Mimaki, 2001), 1 was identified as 24-O-β-D-glucopyranosyl-3-O-α-L-rhamno- pyranosyl-(1→2)-[α-L-rhamnopysanosyl- (1→3)]-β-D-glucopyranosylpennogenin which were named as dracagenin A. This is the first report of this compound from the nature. Compound 2 was obtained as white powder. Inspecting the NMR data of 2 revealed that 2 was a spirostane-type sterol. The NMR data of 2 were similar to those of the aglycone of 1 except for the appearance of two hydroxyl group at C-1 (δH 3.46 (dd, J = 4.0, 11.5 Hz)/ δC 77.7) and C- 14 (δC 87.6) and the upfield shifts of the signals of F-ring from C-23 to C-27. This evidence suggested to the 25S configuration of 2. The 13C-NMR spectrum of 2 showed the upfield 507 shift of the oxygenated signal (δ 58.8) assigned to C-17 in 1. Therefore, C-17 was considered as a methine group. The spectroscopic data of 2 were compared with those of 25S-namogenin B and found to match (Tran, 2001). By carried out the same methods on structure elucidation using spectroscopic evidence, compound 3 were determine to be spriroconazole A, which were isolated from D. manii, D. arborea, and Dioscorea bulbifera L.var sativa (Tepono, 2001). 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