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A new triterpenoid saponin from the leaves and stems of Panax quinquefolium L.
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Chinese Chemical Letters 20 (2009) 1207–1210 www.elsevier.com/locate/cclet
A new triterpenoid saponin from the leaves and stems of Panax quinquefolium L. Guo Yu Li a, Yi Mei Zeng b, He Meng b, Xian Li b, Jin Hui Wang a,b,* a
b
School of Pharmacy, Shihezi University, Shihezi 832002, China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China Received 17 January 2009
Abstract One new triterpenoid saponin, quinquenoside L17 (1), was isolated from the leaves and stems of Panax quinquefolium L., and its structure was elucidated as 20-O-[(b-D-xylopyranosyl-(1-6)-O-b-D-glucopyranosyl)]-6-O-b-D-glucopyranosyl-dammar-24-ene3,6,12,20-tetraol, by the combination analysis of one-dimensional NMR and two-dimensional NMR, mass spectrometry, CD spectrum and chemical evidences. # 2009 Jin Hui Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Panax quinquefolium L.; Quinquenoside L17; Triterpenoid saponin
American ginseng (roots of Panax quinquefolium L.) is well known for its tonic value worldwide. The leaves and stems of P. quinquefolium L. also show similar medical effects in recent research [1]. Chemical investigations on saponin composition of the leaves and stems of P. quinquefolium [2–5] have been reported by us. This paper deals with the structural elucidation of the new constituent, quinquenoside L17 (1), by the chemical and spectroscopic methods. 1. Experimental The melting points were determined on Yamaco MP-S3 Micro-hot stage and were uncorrected. ESI-TOF-MS was performed on Waters LCT Premier KE399 mass spectrometer. A model MOS-450 Chiral Detector (Biologic Science instruments, Grenoble, French) was used for CD analysis. The IR spectra were recorded on Bruker IR S-55 spectrometer. The NMR spectra were recorded in pyridine-d5 on a Bruker AV-600 spectrometer (1H 600 MHz, 13C 150 MHz), using TMS as the internal standard. For HSQC-TOCSY, data matrix is 384 1024 points, applying a zero filling in the F1, F2 dimension and a 908 shifted qsine window function in both dimensions, consisting of 1024 1024 points, going along FT transform. Preparative HPLC was carried out on a Hitachi L-7420 UV–vis spectrophotometric detector at 210 nm and TEDA chrom YWG C18 reversed-phase column (250 mm 20 mm i.d. 10 mm). Silica gel for chromatography was produced by Qingdao Ocean Chemical Group Co. of China. * Corresponding author at: School of Pharmacy, Shihezi University, Shihezi 832002, China. E-mail address: [email protected] (J.H. Wang). 1001-8417/$ – see front matter # 2009 Jin Hui Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.05.017
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G.Y. Li et al. / Chinese Chemical Letters 20 (2009) 1207–1210
The leaves and stems of P. quinquefolium L. were collected from Canada by Dalian Tianma Pharmacy Co. LTD, and identified by Prof. Tian-De Qin. A voucher specimen (No. 20061009) has been deposited in the school of Chinese medicine, Shenyang Pharmaceutical University in China. Dried leaves and stems of P. quinquefolium L. (2.0 kg) were extracted with hot water (20 L 3) and the water soluble fraction was extracted with CHCl3 and n-BuOH. The n-BuOH extract was subjected to column chromatography on reversed-phase highly porous polymer column D101, and was eluted with H2O (40 L) and 95% EtOH (40 L), and then afforded the EtOH fraction (312 g). A part of the EtOH fraction (100 g) was chromatographed Table 1 1 H 13C NMR, HMBC and HSQC-TOCSY data for compound 1. Position
dC
dH (J in Hz)
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 28 29 30 20-glc-10 20 30 40 50 60 60 -xyl(p)-100 200 300 400 500 6-glc-1000 2000 3000 4000 5000 6000
39.5 28.0 78.7 40.4 61.4 80.2 45.1 41.1 50.1 39.7 30.7 70.2 49.2 51.5 30.9 26.6 51.4 17.5 17.6 83.5 22.2 36.2 23.2 126.1 131.0 18.0 25.8 31.8 16.4 17.2 98.1 74.9 79.4 71.6 76.9 70.2 105.9 74.9 78.0 71.1 67.0 106.0 75.5 79.7 71.9 78.2 63.1
1.72, 0.96 1.91, 1.83 3.49 (dd, 11.0, 4.5) 1.40 (d, 1H, 10.4) 4.40 2.48, 1.92
HMBC
HSQC-TOCSY
C-4, 28, 29
C-1,2,3 C-1,2,3 C-1,2,3
C-4, 6, 7, 8, 9, 10, 28, 29 C-1000
1.51 1.90, 1.50 4.20 1.50 1.90, 1.50 1.25, 0.95 1.50 1.15 (s, 3H) 1.02 (s, 3H) 1.59 (s, 3H) 1.80, 1.60 2.55, 2.35 5.30 (t, 1H, 6.6) 1.63 (s, 3H) 1.60 (s, 3H) 2.06 (s, 3H) 1.60 (s, 3H) 0.80 (s, 3H) 5.10 (7.5) 3.80 4.30 4.20 4.30 4.05, 4.72 (br.d, 11.7) 4.97 (7.20) 4.00 4.10 4.20 4.30, 3.70 (dd, 10.8, 9.6) 5.02 (7.5) 3.90 4.30 4.10 4.20 4.35, 4.51 (dd, 11.7, 2.5)
C-5, 6, 7 C-5, 6, 7 C-5, 6, 7 C-9
C-9, 12, 13 C-11, 12, 14, 17, 20
C-11, 12, 13 C-11, 12, 13 C-11, 12, 13
C-12, 14, 20, 21 C-7, 8, 9, 14 C-1, 5, 9, 10
C-15, 16, 17 C-15, 16, 17 C-15, 16, 17 C-18 C-19
C-17, 20, 22
C-22, 25, 26, 27
C-21 C-22, 23, 24 C-22, 23, 24 C-22, 23, 24
C-24, 25, 27 C-24, 25, 26 C-3, 4, 5, 29 C-3, 4, 5, 28 C-8, 13, 14, 15 C-20
C-26 C-27 C-28 C-29 C-30 20-glc-10 , 20 , 30 , 40 , 50 , 60
C-100 C-60
20-glc-10 , 20 , 30 , 40 , 50 , 60 10 -xyl(p)-100 , 200 ,300 , 400 , 500
C-6
10 -xyl(p)-100 , 200 , 300 , 400 , 500 6-glc-1000 , 2000 , 3000 , 4000 , 5000 , 6000
6-glc-1000 , 2000 , 3000 , 4000 , 5000 , 6000
G.Y. Li et al. / Chinese Chemical Letters 20 (2009) 1207–1210
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Fig. 1. The CD spectrum of compound 1.
over a silica gel column (400 g) to yield fraction A (CHCl3–CH3OH, 100:15) and fraction B (CHCl3–CH3OH, 100:30). Fraction B was rechromatographed over an ODS column eluting with 70% MeOH to give fraction B-1 and 80% MeOH to give fraction B-2. Fraction B-2 was then subjected to preparative RP-HPLC CH3OH–H2O (70:30) to yield 1 (15 mg, tR = 46 min). Compound 1. White amorphous solid, IR (KBr), cm1: 3420 (nOH), 2925 (nCH), 1652 (nC C), 1384, 1078, 1040, 624. ESI-TOF-MS: m/z [M + Na]+ (calcd. for 955.5242), [M + H]+ (calcd. for 933.5423); 1H NMR (600 MHz, pry-d5) and 13C NMR (150 MHz, pyr-d5) spectra data, see Table 1. The CD spectrum (c = 5 105, MeOH): 4.10 (196 nm), +4.28 (204 nm), showed by Fig. 1. A solution of compound 1 (3.0 mg) in 2 mol/L trifluoroacetic acid (TFA, 1 mL) was heated at 110 8C for 2.0 h, and then fried with N2 gas. After cooling, the reaction mixture was neutralized with Amberlite IRA-400 resin (OH-form) and the resin was filtered. After removal of the solvent under pressure from the filtrate, the residue was passed through a Sep-Pak C18 cartridge with H2O and MeOH. The H2O eluate was concentrated and the residue was treated with L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine (1.0 mL) at 60 8C for 2.0 h. After drying the by N2 gas, the residue was treated with N-(trimethylsily)imidazol (0.2 mL) at 60 8C for 1.0 h. The reaction was ended by adding water (1.0 mL), and extracted with cyclohexane (1.0 mL, three times). The cyclohexane layer was collected and concentrated to 1.0 mL for GC analysis to identify the derivatives of D-glucose (2), D-xylose (3) from compound 1. GC chromatographic analysis was performed with a OV-17 column and an H2 flame ionization detector at a column temperature of 220 8C, vaporizer temperature of 280 8C, and detector temperature of 280 8C. The Rt of 2 and 3, are 12.9 min and 9.7 min, respectively. 2. Results and discussion Compound 1 was obtained as white amorphous solid, responded positively to Liebermann-Burchard and Molish reagent. HR-ESI-MS: m/z [M + Na]+ (calcd. for 955.5242), [M + H]+ (calcd. for 933.5423), indicated the molecular formula of 1 to be C47H80O18. The sugars were identified as D-glucose and D-xylose by acid hydrolysis and GC compared with authentic samples. The sugars were determined as three b-linked sugars (D-glucose and D-xylose) by the coupling constants of the anomeric protons [d 5.10 (d, 1H, J = 7.5 Hz, 20-glc-10 ), d 5.02 (d, 1H, J = 7.5 Hz, 6-glc1000 ) and d 4.97 (d, 1H, J = 7.2 Hz, 60 -xyl(p)-100 )] in the 1H NMR spectrum. The 1H and 13C NMR data (Table 1) of 1 suggested the presence of a 20(S)-protopanaxatriol moiety as ginsenoside Rg1 [1]. In the HMBC spectrum of 1 (Fig. 2 and Table 1), the anomeric proton of 20-glc [d 5.10 (d, 1H, J = 7.5 Hz, 20-glc10 )] showed 1H–13C long-range correlations to 83.5 (C-20), and the anomeric proton of 6-glc [d 5.02 (d, 1H, J = 7.5 Hz,
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G.Y. Li et al. / Chinese Chemical Letters 20 (2009) 1207–1210
Fig. 2. Important correlations in HMBC and HSQC-TOCSY spectra of compound 1.
6-glc-1000 )] showed 1H–13C long-range correlations to 80.2 (C-6), indicated that two glucopyranosyl moieties were linked to C-20 and C-6 position of 20(S)-protopanaxatriol moiety, respectively. Acknowledgments This work was financially supported by 973 Program (No.2006CB708517) and Program for New Century Excellent Talents in University of Peoples Republic of China (No.NCET-04-0289). We are grateful to Professor Tian-De Qin for identification of the plant material. Thanks also for Dalian Tianma Pharmacy Co. LTD. References [1] [2] [3] [4] [5]
J.H. Wang, Y. Sha, W. Li, Y. Tezuka, S. Kadota, X. Li, J. Asian Nat. Prod. Res. 3 (2001) 293. Z. Li, Y.Y. Guo, C.F. Wu, X. Li, J.H. Wang, J. Pharm. Pharmacol. 51 (1999) 435. J.H. Wang, X. Li, Chinese J. Med. Chem. 7 (1997) 130. J.H. Wang, W. Li, Y. Sha, Y. Tezuka, S. Kadota, X. Li, J. Asian Nat. Prod. Res. 3 (2000) 123. O. Tanaka, S. Yahara, Phytochemistry 17 (1978) 353.
Chinese Chemical Letters 20 (2009) 1207–1210 www.elsevier.com/locate/cclet
A new triterpenoid saponin from the leaves and stems of Panax quinquefolium L. Guo Yu Li a, Yi Mei Zeng b, He Meng b, Xian Li b, Jin Hui Wang a,b,* a
b
School of Pharmacy, Shihezi University, Shihezi 832002, China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China Received 17 January 2009
Abstract One new triterpenoid saponin, quinquenoside L17 (1), was isolated from the leaves and stems of Panax quinquefolium L., and its structure was elucidated as 20-O-[(b-D-xylopyranosyl-(1-6)-O-b-D-glucopyranosyl)]-6-O-b-D-glucopyranosyl-dammar-24-ene3,6,12,20-tetraol, by the combination analysis of one-dimensional NMR and two-dimensional NMR, mass spectrometry, CD spectrum and chemical evidences. # 2009 Jin Hui Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Panax quinquefolium L.; Quinquenoside L17; Triterpenoid saponin
American ginseng (roots of Panax quinquefolium L.) is well known for its tonic value worldwide. The leaves and stems of P. quinquefolium L. also show similar medical effects in recent research [1]. Chemical investigations on saponin composition of the leaves and stems of P. quinquefolium [2–5] have been reported by us. This paper deals with the structural elucidation of the new constituent, quinquenoside L17 (1), by the chemical and spectroscopic methods. 1. Experimental The melting points were determined on Yamaco MP-S3 Micro-hot stage and were uncorrected. ESI-TOF-MS was performed on Waters LCT Premier KE399 mass spectrometer. A model MOS-450 Chiral Detector (Biologic Science instruments, Grenoble, French) was used for CD analysis. The IR spectra were recorded on Bruker IR S-55 spectrometer. The NMR spectra were recorded in pyridine-d5 on a Bruker AV-600 spectrometer (1H 600 MHz, 13C 150 MHz), using TMS as the internal standard. For HSQC-TOCSY, data matrix is 384 1024 points, applying a zero filling in the F1, F2 dimension and a 908 shifted qsine window function in both dimensions, consisting of 1024 1024 points, going along FT transform. Preparative HPLC was carried out on a Hitachi L-7420 UV–vis spectrophotometric detector at 210 nm and TEDA chrom YWG C18 reversed-phase column (250 mm 20 mm i.d. 10 mm). Silica gel for chromatography was produced by Qingdao Ocean Chemical Group Co. of China. * Corresponding author at: School of Pharmacy, Shihezi University, Shihezi 832002, China. E-mail address: [email protected] (J.H. Wang). 1001-8417/$ – see front matter # 2009 Jin Hui Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.05.017
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G.Y. Li et al. / Chinese Chemical Letters 20 (2009) 1207–1210
The leaves and stems of P. quinquefolium L. were collected from Canada by Dalian Tianma Pharmacy Co. LTD, and identified by Prof. Tian-De Qin. A voucher specimen (No. 20061009) has been deposited in the school of Chinese medicine, Shenyang Pharmaceutical University in China. Dried leaves and stems of P. quinquefolium L. (2.0 kg) were extracted with hot water (20 L 3) and the water soluble fraction was extracted with CHCl3 and n-BuOH. The n-BuOH extract was subjected to column chromatography on reversed-phase highly porous polymer column D101, and was eluted with H2O (40 L) and 95% EtOH (40 L), and then afforded the EtOH fraction (312 g). A part of the EtOH fraction (100 g) was chromatographed Table 1 1 H 13C NMR, HMBC and HSQC-TOCSY data for compound 1. Position
dC
dH (J in Hz)
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 28 29 30 20-glc-10 20 30 40 50 60 60 -xyl(p)-100 200 300 400 500 6-glc-1000 2000 3000 4000 5000 6000
39.5 28.0 78.7 40.4 61.4 80.2 45.1 41.1 50.1 39.7 30.7 70.2 49.2 51.5 30.9 26.6 51.4 17.5 17.6 83.5 22.2 36.2 23.2 126.1 131.0 18.0 25.8 31.8 16.4 17.2 98.1 74.9 79.4 71.6 76.9 70.2 105.9 74.9 78.0 71.1 67.0 106.0 75.5 79.7 71.9 78.2 63.1
1.72, 0.96 1.91, 1.83 3.49 (dd, 11.0, 4.5) 1.40 (d, 1H, 10.4) 4.40 2.48, 1.92
HMBC
HSQC-TOCSY
C-4, 28, 29
C-1,2,3 C-1,2,3 C-1,2,3
C-4, 6, 7, 8, 9, 10, 28, 29 C-1000
1.51 1.90, 1.50 4.20 1.50 1.90, 1.50 1.25, 0.95 1.50 1.15 (s, 3H) 1.02 (s, 3H) 1.59 (s, 3H) 1.80, 1.60 2.55, 2.35 5.30 (t, 1H, 6.6) 1.63 (s, 3H) 1.60 (s, 3H) 2.06 (s, 3H) 1.60 (s, 3H) 0.80 (s, 3H) 5.10 (7.5) 3.80 4.30 4.20 4.30 4.05, 4.72 (br.d, 11.7) 4.97 (7.20) 4.00 4.10 4.20 4.30, 3.70 (dd, 10.8, 9.6) 5.02 (7.5) 3.90 4.30 4.10 4.20 4.35, 4.51 (dd, 11.7, 2.5)
C-5, 6, 7 C-5, 6, 7 C-5, 6, 7 C-9
C-9, 12, 13 C-11, 12, 14, 17, 20
C-11, 12, 13 C-11, 12, 13 C-11, 12, 13
C-12, 14, 20, 21 C-7, 8, 9, 14 C-1, 5, 9, 10
C-15, 16, 17 C-15, 16, 17 C-15, 16, 17 C-18 C-19
C-17, 20, 22
C-22, 25, 26, 27
C-21 C-22, 23, 24 C-22, 23, 24 C-22, 23, 24
C-24, 25, 27 C-24, 25, 26 C-3, 4, 5, 29 C-3, 4, 5, 28 C-8, 13, 14, 15 C-20
C-26 C-27 C-28 C-29 C-30 20-glc-10 , 20 , 30 , 40 , 50 , 60
C-100 C-60
20-glc-10 , 20 , 30 , 40 , 50 , 60 10 -xyl(p)-100 , 200 ,300 , 400 , 500
C-6
10 -xyl(p)-100 , 200 , 300 , 400 , 500 6-glc-1000 , 2000 , 3000 , 4000 , 5000 , 6000
6-glc-1000 , 2000 , 3000 , 4000 , 5000 , 6000
G.Y. Li et al. / Chinese Chemical Letters 20 (2009) 1207–1210
1209
Fig. 1. The CD spectrum of compound 1.
over a silica gel column (400 g) to yield fraction A (CHCl3–CH3OH, 100:15) and fraction B (CHCl3–CH3OH, 100:30). Fraction B was rechromatographed over an ODS column eluting with 70% MeOH to give fraction B-1 and 80% MeOH to give fraction B-2. Fraction B-2 was then subjected to preparative RP-HPLC CH3OH–H2O (70:30) to yield 1 (15 mg, tR = 46 min). Compound 1. White amorphous solid, IR (KBr), cm1: 3420 (nOH), 2925 (nCH), 1652 (nC C), 1384, 1078, 1040, 624. ESI-TOF-MS: m/z [M + Na]+ (calcd. for 955.5242), [M + H]+ (calcd. for 933.5423); 1H NMR (600 MHz, pry-d5) and 13C NMR (150 MHz, pyr-d5) spectra data, see Table 1. The CD spectrum (c = 5 105, MeOH): 4.10 (196 nm), +4.28 (204 nm), showed by Fig. 1. A solution of compound 1 (3.0 mg) in 2 mol/L trifluoroacetic acid (TFA, 1 mL) was heated at 110 8C for 2.0 h, and then fried with N2 gas. After cooling, the reaction mixture was neutralized with Amberlite IRA-400 resin (OH-form) and the resin was filtered. After removal of the solvent under pressure from the filtrate, the residue was passed through a Sep-Pak C18 cartridge with H2O and MeOH. The H2O eluate was concentrated and the residue was treated with L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine (1.0 mL) at 60 8C for 2.0 h. After drying the by N2 gas, the residue was treated with N-(trimethylsily)imidazol (0.2 mL) at 60 8C for 1.0 h. The reaction was ended by adding water (1.0 mL), and extracted with cyclohexane (1.0 mL, three times). The cyclohexane layer was collected and concentrated to 1.0 mL for GC analysis to identify the derivatives of D-glucose (2), D-xylose (3) from compound 1. GC chromatographic analysis was performed with a OV-17 column and an H2 flame ionization detector at a column temperature of 220 8C, vaporizer temperature of 280 8C, and detector temperature of 280 8C. The Rt of 2 and 3, are 12.9 min and 9.7 min, respectively. 2. Results and discussion Compound 1 was obtained as white amorphous solid, responded positively to Liebermann-Burchard and Molish reagent. HR-ESI-MS: m/z [M + Na]+ (calcd. for 955.5242), [M + H]+ (calcd. for 933.5423), indicated the molecular formula of 1 to be C47H80O18. The sugars were identified as D-glucose and D-xylose by acid hydrolysis and GC compared with authentic samples. The sugars were determined as three b-linked sugars (D-glucose and D-xylose) by the coupling constants of the anomeric protons [d 5.10 (d, 1H, J = 7.5 Hz, 20-glc-10 ), d 5.02 (d, 1H, J = 7.5 Hz, 6-glc1000 ) and d 4.97 (d, 1H, J = 7.2 Hz, 60 -xyl(p)-100 )] in the 1H NMR spectrum. The 1H and 13C NMR data (Table 1) of 1 suggested the presence of a 20(S)-protopanaxatriol moiety as ginsenoside Rg1 [1]. In the HMBC spectrum of 1 (Fig. 2 and Table 1), the anomeric proton of 20-glc [d 5.10 (d, 1H, J = 7.5 Hz, 20-glc10 )] showed 1H–13C long-range correlations to 83.5 (C-20), and the anomeric proton of 6-glc [d 5.02 (d, 1H, J = 7.5 Hz,
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G.Y. Li et al. / Chinese Chemical Letters 20 (2009) 1207–1210
Fig. 2. Important correlations in HMBC and HSQC-TOCSY spectra of compound 1.
6-glc-1000 )] showed 1H–13C long-range correlations to 80.2 (C-6), indicated that two glucopyranosyl moieties were linked to C-20 and C-6 position of 20(S)-protopanaxatriol moiety, respectively. Acknowledgments This work was financially supported by 973 Program (No.2006CB708517) and Program for New Century Excellent Talents in University of Peoples Republic of China (No.NCET-04-0289). We are grateful to Professor Tian-De Qin for identification of the plant material. Thanks also for Dalian Tianma Pharmacy Co. LTD. References [1] [2] [3] [4] [5]
J.H. Wang, Y. Sha, W. Li, Y. Tezuka, S. Kadota, X. Li, J. Asian Nat. Prod. Res. 3 (2001) 293. Z. Li, Y.Y. Guo, C.F. Wu, X. Li, J.H. Wang, J. Pharm. Pharmacol. 51 (1999) 435. J.H. Wang, X. Li, Chinese J. Med. Chem. 7 (1997) 130. J.H. Wang, W. Li, Y. Sha, Y. Tezuka, S. Kadota, X. Li, J. Asian Nat. Prod. Res. 3 (2000) 123. O. Tanaka, S. Yahara, Phytochemistry 17 (1978) 353.