Chinese Journal of Natural Medicines 2014, 12(1): 0043−0046
Chinese Journal of Natural Medicines
Triterpenoid saponins from Patrinia scabra FENG Feng1, 2, XU Xi-Yu1, LIU Fu-Lei1, LIU Wen-Yuan3, 4*, XIE Ning5 1
Department of Natural Products Chemistry, China Pharmaceutical University, Nanjing 210009, China;
2
Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, China;
3
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, China;
4
Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education,
Nanjing 210009, China; 5
Qingfeng Pharmaceutical Inc., Ganzhou 341000, China Available online 20 Jan. 2014
[ABSTRACT] AIM: To study the chemical constituents and bioactivity of the roots of Patrinia scabra Bunge. METHODS: The chemical constituents were isolated using various chromatographic methods, and the structures were elucidated on the basis of spectral analysis and chemical methods. In addition, cytotoxic activities toward HepG2 cells were tested by the MTT method. RESULTS: A new triterpenoid saponin, 3-O-(4'-isovaleryl)-O-β-D-xylose-12,30-dihydroxy-oleanane-28,13-lactone-22-Oβ-D-glucoside (1), along with two known triterpenoid saponins, acanthopanax saponin CP3 (2) and foetoside C (3), were isolated. CONCLUSION: The aglycone of compound 1 was a new skeleton derivative of oleanolic acid. Compound 2 showed strong cytotoxicity to HePG2 cells (IC50 1.49 µmol⋅L−1). [KEY WORDS] Patrinia scabra Bunge; Chemical constituents; Triterpenoid saponins; Cytotoxicity
[CLC Number] R284
[Document code] A
[Article ID] 2095-6975(2014)01-0043-04
Introduction Patrinia scabra Bunge (Valerianaceae) is indigenous to the northeastern part of China, and exhibited sedative, analgesia, antibacterial, and anti-tumor activities according to recent studies, and was used for the treatment of gynecological inflammation and cancer in some hospitals in China. In the course of examining the biologically active compounds, a new triterpenoid saponin, 3-O-(4'-isovaleryl)-β-D-xylose-12, 30- dihydroxy-oleanane-28, 13-lactone-22-O-β-D-glucoside (1, Fig. 1), along with two known triterpenoid saponins, acanthopanax saponin CP3 (2) and foetoside C (3), were isolated. This paper describes the isolation and structure elucidation of the new compound. Cytotoxic evaluations showed that compound 2 exhibited strong cytotoxicity to HePG2 cells (IC50 1.49 [Received on] 20-Oct.-2012 [*Corresponding author] FENG Feng: Prof., Tel: 86-25-83271447,
E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
µmol·L−1).
Results and Discussion Compound 1 was obtained as white, needle-like crystals from methanol, mp 246−248 °C, and was positive to Liebermann-Burchard and Molisch tests. The molecular formula of C46H74O16 was deduced on the basis of HR-ESI-MS ([M + HCOO]− m/z 927.496 2; Calcd. m/z 927.495 8). Its IR spectrum showed absorption bands at 3 452, 1 739, 1 720 and 1 050 cm−1, ascribable to hydroxyl, carboxyl, and lactone groups. The 13C NMR spectrum of compound 1 indicated 46 carbons, of which 30 carbons could be assigned to the aglycone. Compared with the NMR data of the aglycones of kochianosides III [1], patrirupin A [2], and anemoside B [3], it was shown that compound 1 was a triterpene saponin possessing a 13, 28-lactone skeleton. This was based on the 1H NMR spectral signals assigned to six tertiary methyl groups at δ 0.81, 0.96, 1.08, 1.21, 1.27 and 1.64 (each 3H, s), together with six corresponding sp3 carbon signals, and signals for an oxygenated quaternary carbon at δ 91.88 and a carboxyl carbon at δ 179.8 in the 13C NMR spectrum. The presence of a hydroxyl group at C-12 was proved by the NMR resonance at
– 43 –
FENG Feng, et al. / Chin J Nat Med, 2014, 12(1): 43−46
δC 75.17 and δH 4.14 (1H, br. s), as well as the correlation signal between δH 4.14 (1H, br s) and C-13 (δ 91.88) in the HMBC experiment. The saccharide moiety at C-3 and the hydroxy group at C-30 could be explained by the 13C NMR resonances at δ 88.64 (C-3) and δ 66.39 (C-30), respectively. The α-configuration for the 12-OH and the β-configuration for the 3-O-sugar moiety were determined by the correlations of
Fig. 2 Key correlations of HMBC and ROE of compound 1
H-12 (δ 4.14) with H-18 (δ 2.60), and H-3 (δ 3.25) with CH3-23 (δ 1.27) and H-5 (δ 0.81) observed in the ROESY spectrum (Fig. 2). Furthermore, a sugar moiety at C-22 was deduced from the NMR resonance at δC 77.73 and δH 4.48 (1H, dd, J = 5.0, 12.0 Hz), the long range coupling between the latter and the quaternary carbon C-17 at δ 49.60, and the anomeric carbon C-1 of glucose at δ 105.7 in the HMBC spectrum. The α-configuration at C-22 was proved by the correlation cross-peak between H-22 (δ 4.48) and H-18 (δ 2.60). The assignments of the NMR signals associated with the aglycone moiety (Table 1) were derived from 1H-1H COSY, HSQC, HMBC, and ROESY experiments. These data revealed a new aglycone for compound 1, i.e. 3β, 12α, 22α, 30-tetrahydroxy -oleanane-13β, 28-lactone. The 1H NMR spectrum of compound 1 displayed two doublets ascribable to anomeric protons (δ 4.79 and 5.24) which correlated in the HSQC experiment with the carbon signals at δ 107.3 and 105.8, respectively. By careful analysis of the 1H-1H COSY and NOESY spectra, further confirmed by Fig. 1
Structures of compounds 1, 2 and 3 1
Table 1 Key H NMR and 13C NMR data of compound 1a, b No. No. δH (mult, J in Hz) δC δH (mult, J in Hz) 1 1.62 m, 1.02 m 38.88 24 0.96 s 2 2.02 m, 1.83 m 26.67 25 0.81 s 3 3.25 dd (4.5,12.0) 88.64 26 1.21 s 4 39.63 27 1.64 s 5 0.81 m 55.70 28 6 1.47 m, 1.34 m 17.96 29 1.08 s 7 1.62 m, 1.18 m 34.30 30 3.72 d (20.5), 3.74 d (20.5) 8 42.79 Xyl-1 4.79 d (7.5) 9 1.91 m 44.91 Xyl-2 3.99 dd (7.5, 8.5) 10 36.47 Xyl-3 4.24 m 11 1.91 m, 1.65 m 29.22 Xyl-4 5.37 dt (5.0, 9.0, 9.0) 12 4.14 br s 75.17 Xyl-5 4.32 dd (5.0, 11.0), 3.63 m 13 91.88 Glc-1 5.24 d (7.5) 14 42.79 Glc-2 4.09 t (8.5) 15 1.90 m, 1.19 m 27.98 Glc-3 4.22 m 16 2.20 m 16.02 Glc-4 4.20 m 17 49.60 Glc-5 3.92 m 18 2.60 dd (2.5,14.0) 51.05 Glc-6 4.29 dd (5.5,11.5), 4.46 dd (2.5,11.5) 19 2.80 br d (14.0), 2.18 m 34.07 1’’’ 20 38.37 2’’’ 21 2.47 dd (13.5, 5.0), 1.70 m 38.82 3’’’ 2.08 m 22 4.48 dd (5.0,12.0) 77.73 4’’’ and 5’’’ 0.87 d (7.5) 23 1.27 s 27.98 Notes: a. Assignments were based on 1D and 2D NMR experiments (COSY, HSQC, and HMBC).
– 44 –
δC 16.62 16.54 18.95 18.64 179.8 28.29 66.39 107.31 75.66 74.92 72.83 63.21 105.75 76.23 78.19 71.62 78.45 62.86 172.7 43.38 25.84 22.26
FENG Feng, et al. / Chin J Nat Med, 2014, 12(1): 43−46 b. δ in pyridine-d5, 500 MHz.
the HSQC and HMBC spectra, all of the proton signals due to the sugars were identified, and the sugar moieties of compound 1 were determined to be the pyranose form D-xylose (Xyl) and D-glucose (Glc). The β-anomeric configuration for the xylose and glucose units were determined from their 3JH-1/H-2 coupling constants as 7.5 Hz. Beside the above-mentioned aglycone and saccharide moieties, an isovaleryl fragment was deduced from five 13C NMR resonances at δ 172.7 (C), 43.38 (CH2), 25.84 (CH), and 22.26 (2 × CH3), as well as two methyl signals at δH 0.87 (6H, d, J = 7.5 Hz). Detailed inspection of the HMBC and ROESY spectra led to the determination of the sequence and binding sites of the saccharide chain (Fig. 2). In the HMBC spectrum, a cross-peak between C-3 of the aglycone and H-1 of Xyl indicated that the Xyl unit was connected to C-3 of the aglycone. The linkage of glucose at C-22 was indicated by the cross-peak between H-1 of Glc and C-22. Similarly, the isovaleryl group was located at C-4 of Xyl because of the correlation between the carboxyl carbon (δ 172.7) of the isovaleryl group and H-4 of Xyl (δ 5.37, 1H, dt, J = 5.0, 9.0, 9.0 Hz). Therefore, compound 1 was established as 3-O-(4'-isovaleryl)-O-β-D-xylose-12, 30-dihydroxy-oleanane-28, 13-lactone- 22-O-β-D-glucoside. Compounds 2 and 3 were identified as acanthopanax saponin CP3 [4] and foetoside C [5-6] by analysis of their ESI-MS, NMR spectra, and comparison with literature data.
Experimental General experimental procedures Melting points were measured on a XT-4 Microscope melting point apparatus. IR spectra were measured on a Nicolet Impact 410 Infrared Spectrophotometer (KBr). 1D and 2D NMR spectra were recorded on a Bruker AV500 NMR spectrometer (TMS as internal standard). ESI-MS analyses were performed on Agilent LC-MSD-Trap-1100E system (ESI model) and HR-ESI-MS were conducted on an Agilent 1100LC/TOF/MSD system (ESI model). Sephadex LH-20
(Pharmacia); MCI chromatography material (CHP 20P, Mitsubishi Chemical Co, Japan); Column chromatography and TLC silica gel (Qingdao Chemical Factory, China); D101 macroporous resin (Haiguang Chemical Co., Tianjin, China) were used in the isolation work. All the solvents used in the isolation and purification studies were analytical grade and were purchased from Nanjing Chemical Reagents Company, Nanjing, China. Plant material The roots of P. scabra were collected in September 2008 in Bozhou, An Hui Province, China, and were identified by Dr. FENG Feng (Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, China). A voucher specimen (No. 20080901-01) is deposited in the Department of Natural Products Chemistry, China Pharmaceutical University. Extraction and isolation The air-dried roots (15 kg) of P. scabra were crushed and refluxed five times with aqueous EtOH (30 L) of different concentrations (75%, 75%, 75%, 95%, 95% V/V) for 2 h each time, then concentrated in vacuo. The residue was suspended in H2O, and then successively partitioned with petroleum ether, EtOAc and n-BuOH to afford 465, 453 and 661 g of the extracts, respectively. The n-BuOH soluble extract (600 g) was fractionated by D-101 macroporous resin column (60 cm × 8 cm) with an elution gradient of EtOH-H2O. Fraction 50% (99.6 g) was further separated by silica gel chromatography with elution of increasing polarities of CHCl3-MeOH. After repeated column chromatography (Sephadex LH-20, MCI, and silica gel) and recrystallization, compound 1 (6 mg), compounds 2 (1.5 g), and compound 3 (30 mg) were obtained. Cytotoxicity assay The cytotoxicity tests of compounds 2 and 3 toward the tumor cell line HePG2 were measured by the MTT method in vitro [7], using gambogic acid as positive control. Compound 2 exhibited strong cytotoxicity (IC50 1.49 µmol⋅L−1), and the activity of the compound 3 was much weaker (Table 2).
Table 2 Cytotoxicity to HePG2 of compounds 2 and 3 in vitro Sample
The mortality rate at different concentrations /(µmol·L−1)
IC50/(µmol·L−1)
10−4
10−5
10−6
10−7
Compound 2
99.3%
98.2%
44.5%
17.4%
1.49
Compound 3
53.9%
12.0%
11.4%
2.3%
98.4
Gambogic acid
98.8%
71.9%
11.4%
2.1%
3.53
In the 3D structure of most oleanolic acid derivatives, the 28-COOH is always highly sterically hindered [8] and could not be the active group. Therefore, the reason that the activity of compound 3 was much weaker than 2 was possibly that the suitable relative position of the active groups were changed by adding another chain of β-D-glu-(1-6)β-D-glu in C-28. It is worthwhile to pursue further research
of the anticancer effects and mechanism of action of compound 2.
References [1]
– 45 –
Yoshikawa M, Dai Y, Shimada H, et al. Studies on kochiae fructus II. On the saponin constituents from the fruit of Chinese Kochia scoparia (Chenopodiaceae): Chemical structures of
FENG Feng, et al. / Chin J Nat Med, 2014, 12(1): 43−46
[2] [3]
[4]
[5]
kochianosides I, II, III, and IV[J]. Chem Pharm Bull, 1997, 45(6): 1052-1055. Yang XP, Yuan CS, Jia ZJ. Chemical constituents from the roots of Patrinia rupestris [J]. J Chin Chem Soc, 2007, 54(2): 459-463. Liao X, Li BG, Wang MK, et al. The chemical constituents from Anemone rivularis [J]. Chem J Chin Univ, 2001, 22(8): 1338-1341. Liu XQ, Kim IS, Yook CS, et al. Oleanene glycosides from the leavesl of Acanthopanax sieboldianus forma albeofolium Yook [J]. Nat Prod Res Dev, 2008, 20(1): 66-69. Ganenko TV, Isaev MI, Gorovits TT, et al. Triterpene glycosides and their genins from Thalictrum foetidum. I. The struc-
[6]
[7]
[8]
ture of foetoside C [J]. Khim Prir Soedin, 1984, 20(4): 433-438. Shao Y, Zhou BN, Lin LZ, et al. Two new oleanolic acid saponins from Aster yunnanensis[J]. Chin Chem Lett, 1994, 61(5): 843-846. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays [J]. J Immunol Methods, 1983, 65(1-2): 55-63. Ma CM, Nakamura N, Hattori M. Chemical modification of oleanane type triterpenes and their inhibitory activity against HIV-I protease dimerisation [J]. Chem Pharm Bull, 2000, 48(11): 1681-1688.
Cite this article as: FENG Feng, XU Xi-Yu, LIU Fu-Lei, LIU Wen-Yuan, XIE Ning. Triterpenoid saponins from Patrinia scabra [J]. Chinese Journal of Natural Medicines, 2014, 12(1): 43-46
– 46 –
Chinese Journal of Natural Medicines
Triterpenoid saponins from Patrinia scabra FENG Feng1, 2, XU Xi-Yu1, LIU Fu-Lei1, LIU Wen-Yuan3, 4*, XIE Ning5 1
Department of Natural Products Chemistry, China Pharmaceutical University, Nanjing 210009, China;
2
Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, China;
3
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, China;
4
Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education,
Nanjing 210009, China; 5
Qingfeng Pharmaceutical Inc., Ganzhou 341000, China Available online 20 Jan. 2014
[ABSTRACT] AIM: To study the chemical constituents and bioactivity of the roots of Patrinia scabra Bunge. METHODS: The chemical constituents were isolated using various chromatographic methods, and the structures were elucidated on the basis of spectral analysis and chemical methods. In addition, cytotoxic activities toward HepG2 cells were tested by the MTT method. RESULTS: A new triterpenoid saponin, 3-O-(4'-isovaleryl)-O-β-D-xylose-12,30-dihydroxy-oleanane-28,13-lactone-22-Oβ-D-glucoside (1), along with two known triterpenoid saponins, acanthopanax saponin CP3 (2) and foetoside C (3), were isolated. CONCLUSION: The aglycone of compound 1 was a new skeleton derivative of oleanolic acid. Compound 2 showed strong cytotoxicity to HePG2 cells (IC50 1.49 µmol⋅L−1). [KEY WORDS] Patrinia scabra Bunge; Chemical constituents; Triterpenoid saponins; Cytotoxicity
[CLC Number] R284
[Document code] A
[Article ID] 2095-6975(2014)01-0043-04
Introduction Patrinia scabra Bunge (Valerianaceae) is indigenous to the northeastern part of China, and exhibited sedative, analgesia, antibacterial, and anti-tumor activities according to recent studies, and was used for the treatment of gynecological inflammation and cancer in some hospitals in China. In the course of examining the biologically active compounds, a new triterpenoid saponin, 3-O-(4'-isovaleryl)-β-D-xylose-12, 30- dihydroxy-oleanane-28, 13-lactone-22-O-β-D-glucoside (1, Fig. 1), along with two known triterpenoid saponins, acanthopanax saponin CP3 (2) and foetoside C (3), were isolated. This paper describes the isolation and structure elucidation of the new compound. Cytotoxic evaluations showed that compound 2 exhibited strong cytotoxicity to HePG2 cells (IC50 1.49 [Received on] 20-Oct.-2012 [*Corresponding author] FENG Feng: Prof., Tel: 86-25-83271447,
E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
µmol·L−1).
Results and Discussion Compound 1 was obtained as white, needle-like crystals from methanol, mp 246−248 °C, and was positive to Liebermann-Burchard and Molisch tests. The molecular formula of C46H74O16 was deduced on the basis of HR-ESI-MS ([M + HCOO]− m/z 927.496 2; Calcd. m/z 927.495 8). Its IR spectrum showed absorption bands at 3 452, 1 739, 1 720 and 1 050 cm−1, ascribable to hydroxyl, carboxyl, and lactone groups. The 13C NMR spectrum of compound 1 indicated 46 carbons, of which 30 carbons could be assigned to the aglycone. Compared with the NMR data of the aglycones of kochianosides III [1], patrirupin A [2], and anemoside B [3], it was shown that compound 1 was a triterpene saponin possessing a 13, 28-lactone skeleton. This was based on the 1H NMR spectral signals assigned to six tertiary methyl groups at δ 0.81, 0.96, 1.08, 1.21, 1.27 and 1.64 (each 3H, s), together with six corresponding sp3 carbon signals, and signals for an oxygenated quaternary carbon at δ 91.88 and a carboxyl carbon at δ 179.8 in the 13C NMR spectrum. The presence of a hydroxyl group at C-12 was proved by the NMR resonance at
– 43 –
FENG Feng, et al. / Chin J Nat Med, 2014, 12(1): 43−46
δC 75.17 and δH 4.14 (1H, br. s), as well as the correlation signal between δH 4.14 (1H, br s) and C-13 (δ 91.88) in the HMBC experiment. The saccharide moiety at C-3 and the hydroxy group at C-30 could be explained by the 13C NMR resonances at δ 88.64 (C-3) and δ 66.39 (C-30), respectively. The α-configuration for the 12-OH and the β-configuration for the 3-O-sugar moiety were determined by the correlations of
Fig. 2 Key correlations of HMBC and ROE of compound 1
H-12 (δ 4.14) with H-18 (δ 2.60), and H-3 (δ 3.25) with CH3-23 (δ 1.27) and H-5 (δ 0.81) observed in the ROESY spectrum (Fig. 2). Furthermore, a sugar moiety at C-22 was deduced from the NMR resonance at δC 77.73 and δH 4.48 (1H, dd, J = 5.0, 12.0 Hz), the long range coupling between the latter and the quaternary carbon C-17 at δ 49.60, and the anomeric carbon C-1 of glucose at δ 105.7 in the HMBC spectrum. The α-configuration at C-22 was proved by the correlation cross-peak between H-22 (δ 4.48) and H-18 (δ 2.60). The assignments of the NMR signals associated with the aglycone moiety (Table 1) were derived from 1H-1H COSY, HSQC, HMBC, and ROESY experiments. These data revealed a new aglycone for compound 1, i.e. 3β, 12α, 22α, 30-tetrahydroxy -oleanane-13β, 28-lactone. The 1H NMR spectrum of compound 1 displayed two doublets ascribable to anomeric protons (δ 4.79 and 5.24) which correlated in the HSQC experiment with the carbon signals at δ 107.3 and 105.8, respectively. By careful analysis of the 1H-1H COSY and NOESY spectra, further confirmed by Fig. 1
Structures of compounds 1, 2 and 3 1
Table 1 Key H NMR and 13C NMR data of compound 1a, b No. No. δH (mult, J in Hz) δC δH (mult, J in Hz) 1 1.62 m, 1.02 m 38.88 24 0.96 s 2 2.02 m, 1.83 m 26.67 25 0.81 s 3 3.25 dd (4.5,12.0) 88.64 26 1.21 s 4 39.63 27 1.64 s 5 0.81 m 55.70 28 6 1.47 m, 1.34 m 17.96 29 1.08 s 7 1.62 m, 1.18 m 34.30 30 3.72 d (20.5), 3.74 d (20.5) 8 42.79 Xyl-1 4.79 d (7.5) 9 1.91 m 44.91 Xyl-2 3.99 dd (7.5, 8.5) 10 36.47 Xyl-3 4.24 m 11 1.91 m, 1.65 m 29.22 Xyl-4 5.37 dt (5.0, 9.0, 9.0) 12 4.14 br s 75.17 Xyl-5 4.32 dd (5.0, 11.0), 3.63 m 13 91.88 Glc-1 5.24 d (7.5) 14 42.79 Glc-2 4.09 t (8.5) 15 1.90 m, 1.19 m 27.98 Glc-3 4.22 m 16 2.20 m 16.02 Glc-4 4.20 m 17 49.60 Glc-5 3.92 m 18 2.60 dd (2.5,14.0) 51.05 Glc-6 4.29 dd (5.5,11.5), 4.46 dd (2.5,11.5) 19 2.80 br d (14.0), 2.18 m 34.07 1’’’ 20 38.37 2’’’ 21 2.47 dd (13.5, 5.0), 1.70 m 38.82 3’’’ 2.08 m 22 4.48 dd (5.0,12.0) 77.73 4’’’ and 5’’’ 0.87 d (7.5) 23 1.27 s 27.98 Notes: a. Assignments were based on 1D and 2D NMR experiments (COSY, HSQC, and HMBC).
– 44 –
δC 16.62 16.54 18.95 18.64 179.8 28.29 66.39 107.31 75.66 74.92 72.83 63.21 105.75 76.23 78.19 71.62 78.45 62.86 172.7 43.38 25.84 22.26
FENG Feng, et al. / Chin J Nat Med, 2014, 12(1): 43−46 b. δ in pyridine-d5, 500 MHz.
the HSQC and HMBC spectra, all of the proton signals due to the sugars were identified, and the sugar moieties of compound 1 were determined to be the pyranose form D-xylose (Xyl) and D-glucose (Glc). The β-anomeric configuration for the xylose and glucose units were determined from their 3JH-1/H-2 coupling constants as 7.5 Hz. Beside the above-mentioned aglycone and saccharide moieties, an isovaleryl fragment was deduced from five 13C NMR resonances at δ 172.7 (C), 43.38 (CH2), 25.84 (CH), and 22.26 (2 × CH3), as well as two methyl signals at δH 0.87 (6H, d, J = 7.5 Hz). Detailed inspection of the HMBC and ROESY spectra led to the determination of the sequence and binding sites of the saccharide chain (Fig. 2). In the HMBC spectrum, a cross-peak between C-3 of the aglycone and H-1 of Xyl indicated that the Xyl unit was connected to C-3 of the aglycone. The linkage of glucose at C-22 was indicated by the cross-peak between H-1 of Glc and C-22. Similarly, the isovaleryl group was located at C-4 of Xyl because of the correlation between the carboxyl carbon (δ 172.7) of the isovaleryl group and H-4 of Xyl (δ 5.37, 1H, dt, J = 5.0, 9.0, 9.0 Hz). Therefore, compound 1 was established as 3-O-(4'-isovaleryl)-O-β-D-xylose-12, 30-dihydroxy-oleanane-28, 13-lactone- 22-O-β-D-glucoside. Compounds 2 and 3 were identified as acanthopanax saponin CP3 [4] and foetoside C [5-6] by analysis of their ESI-MS, NMR spectra, and comparison with literature data.
Experimental General experimental procedures Melting points were measured on a XT-4 Microscope melting point apparatus. IR spectra were measured on a Nicolet Impact 410 Infrared Spectrophotometer (KBr). 1D and 2D NMR spectra were recorded on a Bruker AV500 NMR spectrometer (TMS as internal standard). ESI-MS analyses were performed on Agilent LC-MSD-Trap-1100E system (ESI model) and HR-ESI-MS were conducted on an Agilent 1100LC/TOF/MSD system (ESI model). Sephadex LH-20
(Pharmacia); MCI chromatography material (CHP 20P, Mitsubishi Chemical Co, Japan); Column chromatography and TLC silica gel (Qingdao Chemical Factory, China); D101 macroporous resin (Haiguang Chemical Co., Tianjin, China) were used in the isolation work. All the solvents used in the isolation and purification studies were analytical grade and were purchased from Nanjing Chemical Reagents Company, Nanjing, China. Plant material The roots of P. scabra were collected in September 2008 in Bozhou, An Hui Province, China, and were identified by Dr. FENG Feng (Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, China). A voucher specimen (No. 20080901-01) is deposited in the Department of Natural Products Chemistry, China Pharmaceutical University. Extraction and isolation The air-dried roots (15 kg) of P. scabra were crushed and refluxed five times with aqueous EtOH (30 L) of different concentrations (75%, 75%, 75%, 95%, 95% V/V) for 2 h each time, then concentrated in vacuo. The residue was suspended in H2O, and then successively partitioned with petroleum ether, EtOAc and n-BuOH to afford 465, 453 and 661 g of the extracts, respectively. The n-BuOH soluble extract (600 g) was fractionated by D-101 macroporous resin column (60 cm × 8 cm) with an elution gradient of EtOH-H2O. Fraction 50% (99.6 g) was further separated by silica gel chromatography with elution of increasing polarities of CHCl3-MeOH. After repeated column chromatography (Sephadex LH-20, MCI, and silica gel) and recrystallization, compound 1 (6 mg), compounds 2 (1.5 g), and compound 3 (30 mg) were obtained. Cytotoxicity assay The cytotoxicity tests of compounds 2 and 3 toward the tumor cell line HePG2 were measured by the MTT method in vitro [7], using gambogic acid as positive control. Compound 2 exhibited strong cytotoxicity (IC50 1.49 µmol⋅L−1), and the activity of the compound 3 was much weaker (Table 2).
Table 2 Cytotoxicity to HePG2 of compounds 2 and 3 in vitro Sample
The mortality rate at different concentrations /(µmol·L−1)
IC50/(µmol·L−1)
10−4
10−5
10−6
10−7
Compound 2
99.3%
98.2%
44.5%
17.4%
1.49
Compound 3
53.9%
12.0%
11.4%
2.3%
98.4
Gambogic acid
98.8%
71.9%
11.4%
2.1%
3.53
In the 3D structure of most oleanolic acid derivatives, the 28-COOH is always highly sterically hindered [8] and could not be the active group. Therefore, the reason that the activity of compound 3 was much weaker than 2 was possibly that the suitable relative position of the active groups were changed by adding another chain of β-D-glu-(1-6)β-D-glu in C-28. It is worthwhile to pursue further research
of the anticancer effects and mechanism of action of compound 2.
References [1]
– 45 –
Yoshikawa M, Dai Y, Shimada H, et al. Studies on kochiae fructus II. On the saponin constituents from the fruit of Chinese Kochia scoparia (Chenopodiaceae): Chemical structures of
FENG Feng, et al. / Chin J Nat Med, 2014, 12(1): 43−46
[2] [3]
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Cite this article as: FENG Feng, XU Xi-Yu, LIU Fu-Lei, LIU Wen-Yuan, XIE Ning. Triterpenoid saponins from Patrinia scabra [J]. Chinese Journal of Natural Medicines, 2014, 12(1): 43-46
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