Triterpenes from the roots of codonopsis pilosula - Trinh Thi Thuy

Journal of Chemistry, Vol. 46 (4), P. 515 - 520, 2008 TRITERPENES FROM THE ROOTS OF CODONOPSIS PILOSULA Received 30 October 2007 Trinh Thi Thuy1, Tran Van Sung1, Katrin Frank2 and Ludger Wessjohann2 1Institute of Chemistry, Vietnam Academy of Science and Technology 2Leibniz-Institute of Plant Biochemistry,Weinberg 3, D-06120 Halle/Saale, Germany Summary From the roots of Codonopsis pilosula (Franch) Nannf (Campanulaceae) five triterpenoids: taraxerol, taraxeryl acetate, 14-α-taraxeran-3-one and D:B-friedoolean-5-ene-3-β-ol as well as α-spinasterone were isolated. Their structures have been identified by MS, 1H-, 13C-NMR spectroscopy and comparison with reported data. I - Introduction Codonopsis pilosula (Franch) Nannf is one of the most famous traditional medicine and sometimes as cheaper substitute like ginseng in Chinese and Vietnamese drugs. It has been used for a long time and still being used widely today as a remedy for appetite, psychoneurosis, fatigue, dyspepsia and possessing adaptogenic, anti-stress properties [1]. Our previous study on the roots of C. pilosula (Franch) Nannf has led to the isolation and structural identification of lobetyol, 5-hydroxymethyl-2-furandehyde as well as bis-(2-ethylhexyl)-phthalate [2]. In this paper we report the isolation and structural elucidation of four triterpenoids, taraxerol (1), taraxeryl acetate (2), 14-α-taraxeran-3-one (3- taraxeranone, 3), D:B-friedoolean-5-en-3-α-ol (4) and α-spinasterone (5). II - Experiment 1. General Optical rotation [α]D: Digital Polarimeter Jasco DIP 1000. EI-MS: ADM 402, 70 eV, Finnigan TSQ 700. NMR: VARIAN 300 spectrometer at 300 MHz (1H) and 75.5 MHz (13C, 13C-APT). Chemical shifts were referenced to internal TMS (δ = 0, 1H) and CDCl3 (δ = 77.0, 13C). CC: Silica gel 60, 0.06 - 0.20 mm (Merck) for the first column, silica gel 60, 40-63 μm (Merck) for the following columns. TLC: Silica gel 60 F-254 (Merck). 2. Plant material The roots of C. pilosula were bought in Hanoi market, Vietnam in May 2005. The species was identified by Dr. Ngo Van Trai, Institute of Materia Medica, Hanoi. A voucher specimen (Nr. 1) is deposited in the Institute of Chemistry, VAST, Hanoi. 3. Extraction and isolation The ground and dried roots of C. pilosula (2.4 kg) were extracted four time with MeOH (95%) at room temperature. MeOH was evaporated in vacuo, and the aq. solution (1.25 kg) was partitioned with n-hexane followed by EtOAc and n-BuOH (each four time), giving 45.0 g, 21.0 g and 29.3 g extracts, respectively. The n-hexane extract was separated on silica gel using n-hexane-CHCl3 (20:80→90:10) and then CHCl3-MeOH (98:2 → 90:10) to afford 24 fractions (F1-F24, 150 ml/Fr.). Compounds 1-5 were further purified by CC on silica gel or 515 crystallization. Table 1: 1H-NMR data of taraxeryl acetate (2) and α-spinasterone (5) (300 MHz, δppm, CDCl3, J in Hz) H 2 5 5 [8] 1 1.39 m 1.92 dd (14.6; 3.1) 1.46 m; 2.13 ddd (6.1; 14.6; 14.6) 1.47 m; 2.12 ddd (6.1; 14.6; 14.6) 2 1.66 m; 2.02 m 2.28 br d (14.5) 2.28 br d (15) 3 4.45 dd (10.3; 6.2) - - 4 - 2.24 m 2.23 m 5 1.33 m 1.80 m 1.81 m 6 1.43 m, 1.46 m 1.83 m 1.82 m 7 1.29 m,1.62 m 5.18 m 5.18 m 9 1.54 m 1.77 m 1.76 m 11 1.42-1.46 m 1.56, 1.75 m 1.55, 1.75 m 12 0.93 - 1.33 m 1.27, 2.04 1.27, 2.04 m 14 - 1.83 m 1.83 m 15 5.53 dd (7.0; 3.2) 1.39 m, 1.50 m 1.40 m, 1.52 m 16 1.65 m, 2.01 m 1.29 m, 1.77 m 1.29 m, 1.67 m 17 - 1.30 m 1.30 m 18 1.03 m 0.58 s 0.58 s 19 1.43 m, 2.05 m 1.02 s 1.02 s 20 - 2.04 m 2.05 m 21 1.32 - 1.42 m 1.03 d (6.8) 1.03 d (6.7) 22 1.32 - 1.42 m 5.13 dd (8.4; 15.0) 5.16 dd (8.5; 15.2) 23 0.95 s 5.02 dd (8.5; 15.0) 5.02 dd (8.8; 15.3) 24 0.82 s 1.55 m 1.56 m 25 0.91 s 1.56 m 1.57 m 26 1.09 s 0.82 d (6.2) 0.82 d (6.1) 27 0.88 s 0.84 d (6.4) 0.84 d (6.7) 28 0.86 s 1.19 m; 1.41 m 1.18 m; 1.41 m 29 0.95 s 0.81 t (6.4) 0.81 t (7.3) 30 0.91 s - - COCH3 2.04 s - - 516 Table 2: 13C-NMR data of compounds 1-5 (CDCl3, 75.5 MHz, δppm) C 1* 2 3 4 5 1 38.56 37.73 38.31 18.30 38.79 2 26.60 28.86 28.22 27.88 38.15 3 78.65 80.98 212.96 76.32 211.68 4 38.83 39.01 42.16 40.86 44.26 5 55.39 55.63 53.08 141.45 42.88 6 18.68 18.76 18.30 121.96 30.10 7 35.63 35.15 35.36 23.71 116.88 8 38.55 37.92 39.28 47.45 139.33 9 48.56 48.76 42.79 34.88 48.85 10 37.81 37.72 37.45 49.70 34.44 11 17.39 17.59 18.30 34.65 21.76 12 36.51 35.83 35.65 30.41 39.35 13 37.54 37.41 36.03 39.33 43.37 14 157.76 157.78 58.19 37.87 55.02 15 116.56 116.84 30.03 32.12 23.02 16 37.40 36.70 32.79 36.07 28.55 17 37.62 37.58 39.70 30.15 55.85 18 49.68 49.20 59.44 43.08 11.98 19 41.17 41.23 41.55 35.12 12.34 20 28.64 28.86 28.22 28.32 40.86 21 34.95 33.72 32.44 33.16 21.76 22 33.55 33.13 30.54 38.99 137.92 23 27.75 28.04 18.74 29.02 129.37 24 15.33 15.59 6.93 25.53 51.25 25 15.33 16.67 14.73 16.30 31.91 26 29.63 29.99 18.74 19.70 19.06 27 25.76 25.99 32.13 18.52 21.45 28 29.73 29.89 31.83 32.10 25.45 29 33.13 33.41 35.07 34.59 12.34 30 21.15 21.42 20.33 32.47 - C=O - 170.82 - - - COCH3 - 21.36 - - - * CDCl3:CD3OD (95:5) a) Taraxerol (1) Fractions 12-13 (Fr.12-14, 1.35g) were separated on silica gel, eluted with hexane- EtOAc (90:10) and then crystallization to give 360 mg (0.0150 %). [α]24D -22O (c 1.0, CHCl3). EI-MS 70 eV, m/z (rel. int.): 426 [M]+ (16), 411 517 [M-15]+ (14), 302 (52), 287 (40), 218 (37), 205 (38), 204 (100), 189 (30), 135 (19), 121 (24), 107 (22), 95 (23); ). 1H-NMR (CDCl3, 300 MHz, δ ppm): 0.97 (Me-23), 0.82 (Me-24), 0.95 (Me- 25), 1.09 (Me-26), 0.91 (Me-27), 0.80 (Me-28), 0.93 (Me-29), 0.91 (Me-30). 13C-NMR (125 MHz, CDCl3+CD3OD): data see table 2. b) Taraxeryl acetate (2) Compound 2 was isolated from Fr. 4+5 by CC (silica gel, n-hexane-EtOAC 98:2). White needles from EtOAc, yield 60 mg (0.0032 %). ESI-MS (m/z): 491 [M+Na]+ (C32H52O2). EI-MS, 70 eV, m/z (rel. int.): 468 [M]+ (41), 453 (24), 408 (20), 344 (60), 329 (26), 269 (24), 218 (40), 204 (100), 189 (30), 135 (18), 121 (17), 109 (21) 107 (10), 95 (24). 1H- and 13C-NMR data see Table 1 and 2. c) 3-Taraxeranone (14-α-taraxeran-3-one, 3) Compound 3 was isolated from Fr.10 by CC (silica gel, n-hexane-EtOAC-CHCl3, 90:10:1). White needles from EtOAc, yield 56mg (0.0023 %). [α]24D – 45 (c 2, CHCl3, lit. [5] + 31O). ESI- MS, m/z: 449 [M+Na]+ (C30H50O). EI-MS, 70 eV, m/z (rel. int.): 426 [M]+ (87), 411 (29), 341 (14), 302 (32), 273 (63), 205 (54), 191 (17), 179 (46), 163 (51), 123 (100), 109 (77), 95 (82), 69 (47); 1H-NMR (CDCl3, 300 MHz, δ ppm): 1.05 (Me-23), 1.00 (9H, Me-24, Me-25, Me-29), 1.18 (Me-26), 0.87 (Me-27), 0.88 (Me-28), 0.95 (Me-30). 13C-NMR data see table 2. d) D:B-friedoolean-5-ene-3-β-ol (4) Compound 4 was isolated from Fr.10 and purified by CC [silica gel, n-hexane-EtOAc- CHCl3 (90:10:1)]. Powder from EtOAc, yield 28 mg (0.0012 %). EI-MS, 70 eV, m/z (rel. int.): 426 [M]+ (18), 409 (30), 274 (C20H34, 100), 259 (92), 205 (C14H21O, 47), 137 (C10H17, 30), 134 (45), 109 (46), 95 (42), 81 (23); 1H-, 13C-NMR data see Table 1 and 2. e) α-Spinasterone [(22E)-5α-stigmasta-7,22- diene-3-one (5)] Compound 5 was isolated from Fr.10 and purified by CC [silica gel, n-hexane-EtOAC- CHCl3 (90:10:5)]. White needles from EtOAc, yield 45 mg (0.0019 %). EI-MS 70 eV, m/z (rel. int.): 410 [M]+ (56), 397 (32), 395 [M-Me]+, 367 [M-C3H7] + (52), 298 [M-C8H16] + (32), 271, 269 (loose of the side chain, 75), 257 (13), 244 (38), 229 (77), 95 (40), 83 (22), 55 (24). 13C-NMR data see table 2. III - Results and Discussion The residue of an ethanol extract of C. pilosula was partitioned with n-hexane, EtOAc, n-BuOH, successively. The n-hexane extract, after evaporation of solvent was chromatographed on column over silica gel and then crystallization to afford compounds 1-5. Compounds 1-5 showed no fluorescence under UV light with λmax 254 and 366 nm. The EI-MS spectrum of 1 gave a mol peak at m/z 426 [M]+, corresponding to the molecular formula C30H50O. The APT and 13C-NMR spectra showed the presence of 30 carbons (CH3x8, CH2x10, CHx5, Cqx7), suggested that 1 has a triterpene skeleton. This was further confirmed by the signals of 8 tertiary methyl signals in the 1H-NMR spectrum. One double bond was confirmed by olephinic methine signal at δH 5.53 (dd, J = 7.0; 3.2 Hz) and δC 157.76 (C-14), 116.56 (C-15). The structure of 1 was identified as taraxerol (14-taraxeren-3β-ol) by comparison of its 1H- and 13C-NMR spectra (Table 1) with reported data [3, 4]. Taraxerol showed antiulcer activity and is a cancer chemopreventive agent. It was isolated for the first time from Taraxacum officinale and then frequently found in other plants (Rhododendron spec., Euphorbia spec.) [3, 4]. Compound 2 was obtained as white needles from n-hexane extract using column chromatography on silica gel. The EI-MS spectrum gave a mol peak at m/z 468 [M]+ (41), corresponding to the molecular formula C32H52O2. The 1H- and 13C-NMR spectral data were similar to those of 1, except the presence of an acetyl group (δH 2.04 and δC 170.28, 21.36), therefore 2 was identified as taraxeryl acetate. Its 1H- and 13C-NMR spectral data were identical to reported data [4]. Compound 3 was obtained as white needles. The molecular formula of 3 was established as 518 C30H50O by combination of 13C-NMR and mol peak at m/z 426 [M]+(87). The 13C-NMR spectrum showed the presence of a keton group (δC 212.96) and an absence of olephinic signals, suggesting that 3 is taraxeranone. In combination of its MS and NMR spectra, the structure of 3 was determined as 14α-taraxeran- 3-one (3-taraxeranone) [5]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 H H 25 26 27 28 29 30 RO 1: R = H Taraxerol 2: R = Ac Taraxeryl acetate H H H H 3:14-α-Taraxeran-3-one H O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29 30 HO H HH 4: D:B-fiedoolean-5-ene-3-β-ol 28 5: α-Spinasterone H O 3 5 17 18 22 7 23 24 25 26 27 28 21 19 20 29 2 4 6 89 10 11 12 13 14 15 16 1 The EI-MS spectrum of 4 gave a mol peak at m/z 426 [M]+(18), indicating a same molecular formula (C30H50O) as 1. The APT and 13C-NMR spectra showed the presence of 30 carbons (CH3x8, CH2x10, CHx5, Cqx7). In comparison of the 1H- and 13C-NMR spectral data (table 2) with reported data [6], the structure of 4 was identified as D: B-friedoolean-5-ene-3-β-ol, which was isolated for the first time from Securinega tinctorena and its isomer (D:B- friedoolean-5-ene-3-α-ol) was found in Euphorbia royleana (Euphorbiaceae) [6, 7]. D:B-friedoolean-5-ene-3-β-ol is a relatively rare triterpene alcohol, which can be an intermediate in the biosynthesis of friedeline. The molecular formula of 5 (C29H46O) was determined by combination of molecular ion peak at m/z 410 [M]+ in EI-MS as well as its 13C- NMR spectra. The 1H-NMR spectrum displayed two methyl doublets at δH 0.82, 0.84 (each 3H, d, J = 6.4 Hz), corresponding to one isopropyl group. The mass spectrum showed a fragment at m/z 269 (loss of the side chain) and fragments were common to related steroids, suggested that 5 is a stigmasta-diene skeleton. This was further confirmed by the presence of three olephinic methine carbons at δC 116.88 (C-7), 137.92 (C- 22), 129.37 (C-23) and quaternary carbon at δC 139.33 (C-8) in the 13C-NMR spectrum. The 1H- NMR spectrum showed the presence of two olephinic protons at δH 5.13, 5.02 with trans configuration (J = 15.0 Hz). Combination of the MS, 1H- and 13C-NMR spectra (tables 1&2), the structure of 5 was identified as (22E)-5α- stigmasta-7,22-diene-3-one (α-spinasterone), which was isolated from the heartwood of Albizzia julibrissin and the bark of Acacia concinna [8]. 519 The presence of taraxerol as main component and its derivatives is a chemical support for the taxonomy of Codonopsis species used in Vietnamese traditional medicine. Acknowledgements: We thank BMBF, Germany, for financial support in form of a project. We are indebted Dr. Ngo Van Trai, Institute of Materia Medica, Hanoi for the identification of the plant material. References 1. Heidrun Noerr, H. Wagner. 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