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New phenolic glycosides isolated from Penthorum chinense Pursh
Phytochemistry Letters 11 (2015) 163–167
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New phenolic glycosides isolated from Penthorum chinense Pursh Doudou Huang a,1, Yun Jiang b,1, Wansheng Chen c, Fengyan Yao a, Dan Xue a, Lianna Sun a,* a
Department of Identification of Traditional Chinese Medicine, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Science, University of Macau, Macau, PR China c Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 September 2014 Received in revised form 5 December 2014 Accepted 9 December 2014 Available online 20 December 2014
A phytochemical investigation from 80% ethanol extract of Penthorum chinense Pursh (Saxifragaceae) resulted in the isolation of three new phenolic glycosides (1–3), together with three known phenolic glycosides (4–6). The structures of the new compounds were deduced from their comprehensive spectroscopic analysis including IR, HR-EI-MS, 1H NMR, 13C NMR, DEPT, COSY, HMBC and HMQC. And the structures of known compounds 4–6 were identified by comparison of their spectral data with those reported in the literature. ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Penthorum chinense Pursh Saxifragaceae Phenolic glycosides
1. Introduction Penthorum chinense Pursh (Saxifragaceae) is widely distributed in eastern Asia and traditionally used as a diuretic, promoting blood circulation, soothing the liver, protecting the spleen, removing jaundice (Zhang et al., 2007a,b). The aerial parts of P. chinense are available from September to October and are used as an ingredient in foods and Chinese tea (Zhang et al., 2013). Several studies concerning the bioactivities of metabolic products of P. chinense showed that they have antioxidant, antitumor, and antifibrosis activities (Mahesh and Menon, 2004; Sigurdsson et al., 2005; Wang et al., 2008). Ethyl acetate and n-butanol fractions obtained from the ethanol extract of P. chinense exert inhibition on HSC-T6 cells proliferation (Huang et al., 2014). Meanwhile, the ethyl acetate part of 70% ethanol extract of P. chinense possesses remarkable hepatoprotective activity in HL-7702 cells (Zhang et al., 2013). Gansu, a Chinese prepared medicine, was derived from the extracts of P. chinense and has been used in clinics as a remedy for chronic hepatitis B and acute virus hepatitis by its efficacy against the hepatitis B, C, and D viruses (Lu et al., 2012). As part of our continuing chemical studies of P. chinense (Huang et al., 2014), herein we describe the chromatographic separation of ethanol extract of P. chinense which resulted in the isolation of two
* Corresponding author. Tel.: +86 21 81871308; fax: +86 21 81871308. E-mail address: [email protected] (L. Sun). 1 These authors contributed equally to this work.
phenolic glycosides and a flavanone glycosides together with three known phenolic glycosides. 2. Results and discussion The 80% ethanol extract of P. chinense was suspended in water (1.5 L) and partitioned successively with petroleum ether, ethyl acetate, n-BuOH, respectively. The ethyl acetate extract was separated by column chromatography (Silica gel, Sephadex LH20 gel and RP-C18 gel) and high-performance liquid chromatography (HPLC) to give six compounds, including three new compounds (1–3). Compound 1, isolated as a faint yellow powder, was determined to have a molecular formula of C23H26O11 by its HRESIMS (m/z 501.1360, [M+Na]+ calc. 501.1367) and NMR data analysis. The IR absorptions suggested the presence of hydroxyl (3423 cm1), carbonyl (1698.9 cm1) and phenyl groups (1612.2, 1511.9 cm1). The 1H NMR spectrum (Table 1) of 1 exhibited signals characteristic for a methoxy at dH 3.75 (s, 3H) and an (E)-propenyl at dH 6.27 (1H, d, J = 15.6 Hz), dH 6.14 (1H, m) and dH 1.81 (3H, d, J = 6.6 Hz). In addition, one ABX system at dH 6.96 (1H, d, J = 1.2 Hz), dH 6.98 (1H, d, J = 8.4 Hz), dH 6.77(1H, dd, J = 1.2, 8.4 Hz) suggested that compound 1 exhibited a 1,2,4-trisubstituted benzene ring. The 13C NMR (Table 1) signals at dC 100.0, 73.1, 76.7, 69.8, 73.8 also suggested the presence of the sugar moiety. The glucopyranose moiety was determined to have a b-configuration, supported by the coupling constant of the glucose anomeric proton H-100 (J = 7.2 Hz). In the 13C NMR spectrum, the six sp2-hybridized carbons include two equivalent quaternary carbons (dC 108.7, C-20 /60 ), and two oxygenated quaternary carbons
http://dx.doi.org/10.1016/j.phytol.2014.12.012 1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
D. Huang et al. / Phytochemistry Letters 11 (2015) 163–167
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Table 1 1 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 1 in DMSOd6. Position
dH
dC
HMBC (H–C)
1 2 3 4 5 6 7 8 9 10 20 , 60 30 , 50 40 100 200 300 400 500 600
– 6.96(1H,d, 1.2) – – 6.98(1H, d, 8.4) 6.77(1H, dd, 1.2, 8.4) 6.27(1H, d, 15.6) 6.14(1H, m) 1.81(3H, d, 6.6) – 6.98(2H, s,) – – 4.90(1H, d, 7.2) 3.28(1H, m) 3.33(1H, m) 3.27(1H, m) 3.67(1H, m) 4.21(1H, dd, 12.0, 1.8) 4.42(1H, dd, 12.0, 7.2) – 3.75(3H, s)
148.7 109.7 131.6 118.3 115.2 145.4 130.4 123.8 18.2 119.2 108.7 145.5 138.6 100.0 73.1 76.7 69.8 73.8 63.5
– C-1, C-3, C-4, C-6, C-7 – – C-1, C-2, C-3, C-4, C-6 C-1,C-2, C-4, C-5, C-7 C-1, C-2, C-6, C-8, C-9 C-1, C-7, C-9 C-7, C-8 – C-10 , C-30 , C-40 , C-50 , C-70
70 (C5 5O) 3-OCH3
165.7 55.6
C-4, C-200 , C-500 C-100 , C-300 C-200 , C-400 C-300 , C-500 C-100 , C-400 , C-600 C-500 , C-700
C-3
(dC 145.5, C-30 /50 ) as well as two further quaternary carbons at dC 119.2 (C-10 ) and 138.6 (C-40 ), and their chemical shifts were indicative of a galloyl group when the carbonyl group (dC 165.7, C-70 ) was taken into consideration. This hypothesis was supported by the HMBC spectrum, in which the correlations from the aromatic proton(s) H-20 /60 (dH 6.98) to C-10 , C-30 /C-50 , C-40 and C-70 were observed. The galloyl group was linked to C-600 via an ester bond, while the glucose was connected to C-4, as evidence by the HMBC (Fig. 2) correlations from H-600 (dH 4.21) to C-70 (dC 165.7), and from H100 (dH 4.90) to C-4 (dC 118.3). All proton and carbon signals were assigned based on HMQC, HMBC, and 1H–1H COSY spectra. Thus, based on the above evidences, compound 1 was established as (E)phenylpropene-3-methoxyphenyl-[600 -O-galloy]-4-O-b-D-glucopyranoside (Fig. 1).
Fig. 2. Key HMBC (H ! C) and 1H–1H COSY correlations of compounds 1–3.
Compound 2 was obtained as white amorphous solid and was suggested to have the molecular formula C19H22O11 based on HRESIMS [m/z 449.1043 [M+Na]+ (calc. for C19H22O11Na, 449.1045)]. The IR spectrum also showed the presence of hydroxyl group (3623 cm1), carbonyl group (1737 cm1), a conjugated carbonyl group (1628 cm1) and benzene ring (1602, 1523 cm1). The 1H NMR spectrum of 2 (Table 2) showed the presence of one 1,2,3,4,5-penta-substituted aromatic ring and one methyl group at dH 7.06 (1H, s) and dH 2.65 (3H, s). The 2(5H)-furanone unit in the molecule was deduced from the 1H and 13C NMR spectrum (dH 6.28, 1H, t, J = 1.8 Hz, H-30 and dH 4.75, 2H, t, J = 1.8 Hz, H-40 ; dC-20 133.5, dC-30 148.4, dC-40 72.2, dC-50 177.3) (Table 2), confirmed by 1 H–1H COSY [between H-30 (dH 6.28) and H-40 (dH 4.75)] and HMBC [between H-30 (dH 6.28) and C-20 (dC 133.5), C-40 (dC 72.2), C-50 (dC 177.3); between H-40 (dH 4.75) and C-20 (dC 133.5), C-30 (dC 148.4), C-50 (dC 177.3)]. The HMBC correlations beteween H-10 (dH 3.51, s) to C-20 (dC 133.5) and C-5 (dC 106.3) suggested the 2(5H)-furanone was linked to C-5 through a methylene (C-10 ). The 13C NMR (Table 2) and DEPT spectrum showed that compound 2 exhibited
Fig. 1. Compounds 1–6 isolated from P. chinense.
D. Huang et al. / Phytochemistry Letters 11 (2015) 163–167 Table 2 1 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 2 in DMSOd6.
165
Table 3 1 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 3 in DMSOd6.
Position
dH
dC
HMBC (H–C)
Position
dH (J Hz)
dC
HMBC (H–C)
1 2 3 4 5 6 7 8 10 20 30 40 50 100 200 300 400 500 600
– – 7.06(1H, s) – – – – 2.65(3H, s) 3.51(2H, s) – 6.28(1H, t, 1.8) 4.75(2H, d, 1.8) – 4.90(1H, d, 7.8) 3.44(1H,m) 3.45(1H, m) 3.35(1H, m) 3.38(1H, m) 3.93(1H, dd, 12.0, 1.8) 3.71(1H, dd, 12.0, 6.0)
107.0 162.7 94.6 162.8 106.3 164.3 205.6 33.1 19.2 133.5 148.4 72.2 177.3 101.7 74.6 78.1 71.2 78.5 62.6
– – C-1, C-2, C-4, C-5 – – – – C-1, C-7 C-20 , C-30 , C-50 , C-4, C-5, C-6 – C-10 , C-20 , C-40 , C-50 C-20 , C-30 , C-50 – C-4, C-200 , C-500 C-100 , C-300 C-200 , C-400 C-300 , C-500 C-100 , C-300 C-400 , C-500
1 2, 6 3, 5 4
– 7.28(2H, 7.25(2H, 7.27(1H, 2.91(1H, 3.35(1H, – – – 6.07(1H, – 6.07(1H,
141.0 128.2 128.2 125.7 45.1 30.0 204.7 105.4 163.9 95.0 163.1 95.0 163.9 99.1 71.2 77.4 67.3 76.7 60.0
– C-1, C-2, C-3, C-1, C-1,
one methylene at dC 19.2 (C-10 ) and a sugar moiety at dC 101.7 (C-100 ), 74.6 (C-200 ), 78.1 (C-300 ), 71.2 (C-400 ), 78.5 (C-500 ) and 62.6 (C-600 ). The glucopyranose moiety was determined to have a b-configuration at C-100 with the large coupling constant of H-100 (J = 7.8 Hz). Correlations from H-100 to C-4 in the HMBC spectrum (Fig. 2), indicated that the b-glycopyranoside group was located at C-4. In the HMBC spectrum, correlations from H-8 (dH 2.65, 3H, s) to C-7 (dC 205.6) and C-1 (dC 107.0) suggested the acetyl group (C7–C8) was located at C-1. On the basis of the molecular formula and chemical shifts, the rest two aromatic carbons were hydroxyl group substituted (dC-2 162.7; dC-2 164.3). Thus, compound 2 was established as 2,6-dihydroxyacetophenone-5-(20 -methylene2(5H)-furanone)-4-O-b-D-glucopyranoside. Compound 3, a salmon pink powder, had the molecular formula of C28H28O13 deduced from the positive HRESIMS for the peak at m/ z 595.1409 [M+Na]+ (calc. for C28H28O13Na 595.1422). The IR spectrum also showed the presence of hydroxyl group (3398 cm1), carbonyl group (1697 cm1), phenyl ketone (1630 cm1) and benzene ring (1597 cm1). In the 1H NMR spectrum, the symmetrical benzene ring at dH 7.00 (H-2000 and H6000 ) suggested the occurrence of a galloyl group (Wang et al., 2010). The occurrence of a dihydrochalcone skeleton (Calanasan and Macleod, 1998; Tanimoto et al., 2009) in the molecule could be easily deduced from the 1H NMR spectrum (Table 3), in which compound 3 showed the signals for a mono-substituted aromatic protons at dH [7.28 (H-2 and H-6), 7.25 (H-3 and H-5), 7.27 (H-4)] on A-ring, together with one singlet in the aromatic region dH 6.07 (s, H-30 , 50 ) (She et al., 2011). The 13C NMR (Table 3) and DEPT spectrum showed that compound 3 exhibited two methylene at dC 45.1 (C-a) and dC 30.0 (C-b), a sugar moiety at dC 99.1 (C-100 ), 71.2(C-200 ), 77.4(C-300 ), 67.3(C-400 ), 76.7(C-500 ) and 60.0 (C-600 ). Acid hydrolysis of 3 released D-glucopyranose. The glucopyranose moiety was determined to have a b-configuration at C-100 with the large coupling constant of H-100 (J = 7.8 Hz). In the HMBC (Fig. 2) spectrum, the galloyl group was shown to be at C-300 due to a down field shift of the dH 5.07 (H-300 ) and the HMBC correlation between the dH 3.49 (H-300 ) and the dC 165.3 (C-7000 ). In addition, the HMBC spectrum showed that dH 5.07 (H-100 ) was correlated to dC 163.1 (C-40 ), indicating that the b-glucopyranosyl group was attached to the C-40 position. Thus, compound 3 was established as 20 ,60 -dihydroxydihydrochalcone-40 -O-[300 -O-galloyl]-b-D-glucopyranoside.
a b C–C5 5O 10 0 2 30 40 50 60 100 200 300 400 500 600 1000 2000 , 6000 3000 , 5000 4000 7000
m) m) br s) t, 7.8) t, 7.8)
s) s)
5.07(1H, d, 7.8) 3.45(1H, dd, 9.0,7.8) 3.49(1H,m) 3.48(1H,m) 3.50(1H, m) 3.69(1H, dd, 11.4,1.2) 3.54(1H, dd, 11.4,3.6) – 7.00(2H, s) – – –
119.9 108.7 145.4 138.1 165.3
C-3, C-5, C-a C-4, C-6 C-2, C-5, C-6 C-2, C-6, C-b, C–C5 5O C–C5 5O, C-10 , C-a
C-10 , C-20 , C-40 , C-50 C-10 , C-30 , C-40 , C-50 C-40 , C-200 C-100 , C-300 C-200 , C-400 , C-7000 C-300 , C-600 C-100 , C-400 , C-600 C-400 , C-500
C-1000 , C-3000 ,C-5000 ,C-7000 – –
The structures of the known compounds were identified based on MS, NMR data and comparison with literature: 1-O-sinapoyl-bD-glucopyranoside (4) (Gao et al., 2014), 2,6-dihydroxyacetophenone-4-O-b-D-glucopyranoside (5) (Jeanette, 1973), ferulic acid glucopyranoside (6) (Stranda˚s et al., 2008). 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a PE 341 polarimeter at 22 8C. Infrared (IR) spectra were recorded on a Nicolet FTIR 6700 spectrometer with a KBr pellet. The NMR spectra, including 1 H, 13C, DEPT and 2D-NMR, were recorded on a Bruker AM-400 and AC-600 spectrometer with chemical shifts reported as d values, using TMS as internal standard. HRESIMS data were obtained on an Agilent Technologies 6538 UHD Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, MA, USA). Thin layer chromatography was performed on TLC plates (Silica gel HSGF254. Jiangyou Company of Yantai; RP-18 F254. Merck) and spots were visualized by heating after dipping into 10% H2SO4. Silica gel (100–200 and 200–300 mesh, Jiangyou Company of Yantai), Sephadex LH-20 (Pharmacia), RP-C18 (43–60 mm, Merck), and MCI gel (75–150 mm, Mitsubishi Chemical Corporation) were used for column chromatography. 3.2. Plant materials The dry aerial parts of P. chinense were provided by Gulin Gansu Pharmaceutical Co., Ltd. (Sichuan, China). The plant was identified by Prof. Wansheng Chen, and a voucher specimen was deposited in the Department of Pharmacognosy of the Second Military Medical University in Shanghai, China. 3.3. Extraction and isolation 10 kg of dry aerial parts of P. chinense were extracted three times with 80% EtOH (3 80 L, 2 h each) at 80 8C, and concentrated
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in vacuum to give a residue (1.167 g). The residue was suspended in water (1.5 L) and then successively partitioned with petroleum ether (3 1.0 L), EtOAc (3 2.0 L), and n-BuOH (2 1.5 L) to give petroleum ether-soluble (99 g), EtOAc-soluble (245 g), and nBuOH-soluble (239 g) extracts. The ethyl acetate fraction (100 g) was subjected to silica gel (200–300 mesh) column chromatography, eluted with CH2Cl2/CH3OH (50:1, 30:1, 20:1, 10:1, 5:1, 0:1 v/ v) to afford four fractions Fr. 1–Fr. 4, which were combined based on TLC profiles. Fr. 3 (23 g) was refined over MCI gel column chromatography (75–150 mm, 200 g, 8 cm 100 cm), eluted with H2O, followed by increasing concentrations of MeOH (10%, 20%, 50%, 100%) to give four subfractions (Fr. 3.1–Fr. 3.4). Fr. 3.2 (3.7 g) was further subjected to a Sephadex LH-20 column eluted with MeOH/H2O (1:9, 3:7, 5:5, 1:0) and divided into four main subfractions Fr. 3.2a–Fr. 3.2d. Fr. 3.2b (210 mg) was then purified to HPLC chromatography (MeOH/H2O from 40:60 to 100% MeOH in 30 min, flow rate 2 ml/min) to yield compound 1 (6.8 mg). Fr. 3.2c (1210 mg) was subjected to Sephadex LH-20 (80 g, 1.5 cm 150 cm) eluting with MeOH/H2O (4:6, 200 ml) to obtain compound 2 (21 mg). Fr.4 (32 g) was separated by silica gel (320 g, 200–300 mesh, 8 cm 100 cm) eluting with CH2Cl2/CH3OH (10:1, 5:1, 0:1 v/v) to give five subfractions, Fr. 4.1–Fr. 4.5. Fr 4.2 (4.8 g) applied to ODS column with MeOH/H2O (2.5 cm 150 cm) (0:1, 1:9, 5:5, 8:2, 1:0, each 350 ml) to give five subfractions Fr. 4.2a–Fr. 4.2e. Fr. 4.2a (721 mg) was subjected to Sephadex LH-20 eluting with MeOH/H2O (3:7, 5:5, each 200 ml) to obtain compound 5 (12.8 mg) and compound 4 (8.2 mg). Fr. 4.2c (1210 mg) was subjected to Sephadex LH-20 eluting with MeOH/H2O (8:2, each 200 ml) to afford five fractions, and compound 3 (113 mg) was obtained from the second fraction according to the TLC profile. Fr. 4.2e (117 mg) was purified by HPLC chromatography (MeOH/H2O from 40:60 to 90:10 in 30 min, flow rate 1 ml/min) to yield compound 6 (12.8 mg). 3.4. Chemical characterization Compound 1: Faint yellow powder; IR (KBr): ymax 3423.0, 2921.6, 1698.9, 1612.2, 1511.9, 1072.2, 767.5 cm1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z 501.1360 [M+Na]+ (calc. for C22H24O9Na, 501.1367). Compound 2: White amorphous solid; IR (KBr): ymax 3623.6, 3399.9, 2923.6, 2854.1, 1737.6, 1627.6, 1602.6, 1523.5, 1455.9, 1428.9, 871.7 cm1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 2; HRESIMS m/z 449.1043 [M+Na]+ (calc. for C22H24O9Na, 449.1045). Compound 3: Salmon pink powder; IR (KBr): ymax 3398.0, 2927.4, 1697.1, 1629.6, 1596.8, 1523.5, 1436.7, 1348.0, 1205.3, 1174.4, 1078.0, 1039.4, 823.5, 752.1, 698.1 cm1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 3; HRESIMS m/z 595.1409 [M+Na]+ (calc. for C28H28O13Na, 595.1422). Compound 4: White powder; ESI-MS: m/z 387.1 [M+H]+, 408.2 [M+Na]+; 1H NMR(600 MHz, DMSO-d6, d ppm): 7.05 (2H, s, H-2/6), 9.00 (1H, s, 4-OH), 7.64 (1H, d, J = 15.6 Hz, H-7), 6.55 (1H, d, J = 15.6 Hz, H-8), 3.81 (6H, s, 3/5-OCH3), 5.47 (1H, d, J = 7.8 Hz, H10 ), 3.27–3.37 (4H, m, H-20 –50 ), 3.75 (1H, d, J = 2.0, 12.0 Hz, H-60 ), 3.59 (1H, d, J = 12.0, 5.4 Hz, H-60 ); 13C NMR (150 MHz, DMSO-d6, d ppm): 124.2 (C-1), 106.3 (C-2/6), 148.0 (C-3/5), 138.5 (C-4),146.5 (C-7), 114.3 (C-8), 165.3 (C-9), 56.0 (3/5-OCH3), 94.2 (C-10 ), 72.5 (C20 ), 76.5 (C-30 ), 69.5 (C-40 ), 77. 8(C-50 ), 60.6 (C-60 ). Compound 5: White powder; ESI-MS: m/z 331.5[M+H]+; 1H NMR (400 MHz, DMSO-d6, d ppm): 6.07 (2H, s, H-3/5), 2.59 (3H, s, H-8), 4.86 (1H, d, J = 7.2 Hz, H-10 ), 3.17–3.45 (4H, m, H-20 –50 ), 3.49 (1H, m, H-60 ), 3.67 (1H, d, J = 17.4 Hz, H-60 ); 13C NMR (100 MHz, DMSO-d6, d ppm): 105.5 (C-1), 163.9 (C-2/6), 94.9(C-3/5), 163.5
(C-4), 203.4 (C-7), 32.6 (C-8), 99.4 (C-10 ), 73.0 (C-20 ), 76.4 (C-30 ), 69.4 (C-40 ), 77.1 (C-50 ), 60.4 (C-60 ). Compound 6: ESI-MS: m/z 357.2 [M+H]+; 1H NMR (400 MHz, DMSO-d6, d ppm): 6.48 (1H, d, J = 15.6 Hz, H-2), 7.63 (1H, d, J = 15.6 Hz, H-3), 3.81 (3H, s, H-OCH3), 7.31 (1H, d, J = 1.6 Hz, H-20 ), 6.81 (1H, d, J = 8.4 Hz, H-50 ), 7.14 (1H, dd, J = 8.4, 1.6 Hz, H-60 ), 5.45 (1H, d, J = 8.4 Hz, H-100 ), 3.07–3.51 (4H, m, H-200 –500 ), 3.45 (1H, m, H600 ), 3.67 (1H, m, H-600 ); 13C NMR (100 MHz, DMSO-d6, d ppm): 174.6 (C-1), 113.9 (C-2), 146.4 (C-3), 55.7 (C-OCH3), 125.4 (C-10 ), 111.3 (C-20 ), 149.6 (C-30 ), 148.0 (C-40 ), 115.6 (C-50 ), 123.4 (C-60 ), 94.2 (C-100 ), 72.5 (C-200 ), 76.5 (C-300 ), 69.5 (C-400 ), 77.8 (C-500 ), 60.6 (C-600 ). 4. Acid hydrolysis A solution of compound 1 (5 mg), compound 2 (5 mg) and compound 3 (10 mg) in 2N aqueous CF3COOH (10 ml) was refluxed at 80 8C for 2 h, respectively. The mixture was then diluted in water (10 ml) and extracted with EtOAc (3 ml 3 ml). The combined water layers and evaporated to dryness to afford the glycoside. The residue was purified over ODS column to afford D-glucopyranose [1.2 mg, [a´]22D +38.2 (c 0.15, H2O); 0.8 mg, [a´]22D +40.3 (c 0.20, H2O), 2.1 mg, [a´]22D +35.3 (c 0.20, H2O)], respectively. The glucopyranose was identified on the basis of their specific rotation. Declaration of interest The authors report no declarations of interest. Acknowledgements This research project was supported by the Science Foundation of Shanghai (No. 13401900106 the National Science Fund for Distinguished Young Scholars (No.81325024), and the Second Military Medical University Stem Cell and Medical Research Center’s Innovation Research Program (SCMRC1201). The NMR spectra were conducted by the Second Military University School of Pharmacy. The authors also thank the Shanghai Institute of Pharmaceutical Industry of Academia Sinica for their aid in obtaining polarimetry and IR spectra.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
New phenolic glycosides isolated from Penthorum chinense Pursh Doudou Huang a,1, Yun Jiang b,1, Wansheng Chen c, Fengyan Yao a, Dan Xue a, Lianna Sun a,* a
Department of Identification of Traditional Chinese Medicine, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Science, University of Macau, Macau, PR China c Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 September 2014 Received in revised form 5 December 2014 Accepted 9 December 2014 Available online 20 December 2014
A phytochemical investigation from 80% ethanol extract of Penthorum chinense Pursh (Saxifragaceae) resulted in the isolation of three new phenolic glycosides (1–3), together with three known phenolic glycosides (4–6). The structures of the new compounds were deduced from their comprehensive spectroscopic analysis including IR, HR-EI-MS, 1H NMR, 13C NMR, DEPT, COSY, HMBC and HMQC. And the structures of known compounds 4–6 were identified by comparison of their spectral data with those reported in the literature. ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Penthorum chinense Pursh Saxifragaceae Phenolic glycosides
1. Introduction Penthorum chinense Pursh (Saxifragaceae) is widely distributed in eastern Asia and traditionally used as a diuretic, promoting blood circulation, soothing the liver, protecting the spleen, removing jaundice (Zhang et al., 2007a,b). The aerial parts of P. chinense are available from September to October and are used as an ingredient in foods and Chinese tea (Zhang et al., 2013). Several studies concerning the bioactivities of metabolic products of P. chinense showed that they have antioxidant, antitumor, and antifibrosis activities (Mahesh and Menon, 2004; Sigurdsson et al., 2005; Wang et al., 2008). Ethyl acetate and n-butanol fractions obtained from the ethanol extract of P. chinense exert inhibition on HSC-T6 cells proliferation (Huang et al., 2014). Meanwhile, the ethyl acetate part of 70% ethanol extract of P. chinense possesses remarkable hepatoprotective activity in HL-7702 cells (Zhang et al., 2013). Gansu, a Chinese prepared medicine, was derived from the extracts of P. chinense and has been used in clinics as a remedy for chronic hepatitis B and acute virus hepatitis by its efficacy against the hepatitis B, C, and D viruses (Lu et al., 2012). As part of our continuing chemical studies of P. chinense (Huang et al., 2014), herein we describe the chromatographic separation of ethanol extract of P. chinense which resulted in the isolation of two
* Corresponding author. Tel.: +86 21 81871308; fax: +86 21 81871308. E-mail address: [email protected] (L. Sun). 1 These authors contributed equally to this work.
phenolic glycosides and a flavanone glycosides together with three known phenolic glycosides. 2. Results and discussion The 80% ethanol extract of P. chinense was suspended in water (1.5 L) and partitioned successively with petroleum ether, ethyl acetate, n-BuOH, respectively. The ethyl acetate extract was separated by column chromatography (Silica gel, Sephadex LH20 gel and RP-C18 gel) and high-performance liquid chromatography (HPLC) to give six compounds, including three new compounds (1–3). Compound 1, isolated as a faint yellow powder, was determined to have a molecular formula of C23H26O11 by its HRESIMS (m/z 501.1360, [M+Na]+ calc. 501.1367) and NMR data analysis. The IR absorptions suggested the presence of hydroxyl (3423 cm1), carbonyl (1698.9 cm1) and phenyl groups (1612.2, 1511.9 cm1). The 1H NMR spectrum (Table 1) of 1 exhibited signals characteristic for a methoxy at dH 3.75 (s, 3H) and an (E)-propenyl at dH 6.27 (1H, d, J = 15.6 Hz), dH 6.14 (1H, m) and dH 1.81 (3H, d, J = 6.6 Hz). In addition, one ABX system at dH 6.96 (1H, d, J = 1.2 Hz), dH 6.98 (1H, d, J = 8.4 Hz), dH 6.77(1H, dd, J = 1.2, 8.4 Hz) suggested that compound 1 exhibited a 1,2,4-trisubstituted benzene ring. The 13C NMR (Table 1) signals at dC 100.0, 73.1, 76.7, 69.8, 73.8 also suggested the presence of the sugar moiety. The glucopyranose moiety was determined to have a b-configuration, supported by the coupling constant of the glucose anomeric proton H-100 (J = 7.2 Hz). In the 13C NMR spectrum, the six sp2-hybridized carbons include two equivalent quaternary carbons (dC 108.7, C-20 /60 ), and two oxygenated quaternary carbons
http://dx.doi.org/10.1016/j.phytol.2014.12.012 1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
D. Huang et al. / Phytochemistry Letters 11 (2015) 163–167
164
Table 1 1 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 1 in DMSOd6. Position
dH
dC
HMBC (H–C)
1 2 3 4 5 6 7 8 9 10 20 , 60 30 , 50 40 100 200 300 400 500 600
– 6.96(1H,d, 1.2) – – 6.98(1H, d, 8.4) 6.77(1H, dd, 1.2, 8.4) 6.27(1H, d, 15.6) 6.14(1H, m) 1.81(3H, d, 6.6) – 6.98(2H, s,) – – 4.90(1H, d, 7.2) 3.28(1H, m) 3.33(1H, m) 3.27(1H, m) 3.67(1H, m) 4.21(1H, dd, 12.0, 1.8) 4.42(1H, dd, 12.0, 7.2) – 3.75(3H, s)
148.7 109.7 131.6 118.3 115.2 145.4 130.4 123.8 18.2 119.2 108.7 145.5 138.6 100.0 73.1 76.7 69.8 73.8 63.5
– C-1, C-3, C-4, C-6, C-7 – – C-1, C-2, C-3, C-4, C-6 C-1,C-2, C-4, C-5, C-7 C-1, C-2, C-6, C-8, C-9 C-1, C-7, C-9 C-7, C-8 – C-10 , C-30 , C-40 , C-50 , C-70
70 (C5 5O) 3-OCH3
165.7 55.6
C-4, C-200 , C-500 C-100 , C-300 C-200 , C-400 C-300 , C-500 C-100 , C-400 , C-600 C-500 , C-700
C-3
(dC 145.5, C-30 /50 ) as well as two further quaternary carbons at dC 119.2 (C-10 ) and 138.6 (C-40 ), and their chemical shifts were indicative of a galloyl group when the carbonyl group (dC 165.7, C-70 ) was taken into consideration. This hypothesis was supported by the HMBC spectrum, in which the correlations from the aromatic proton(s) H-20 /60 (dH 6.98) to C-10 , C-30 /C-50 , C-40 and C-70 were observed. The galloyl group was linked to C-600 via an ester bond, while the glucose was connected to C-4, as evidence by the HMBC (Fig. 2) correlations from H-600 (dH 4.21) to C-70 (dC 165.7), and from H100 (dH 4.90) to C-4 (dC 118.3). All proton and carbon signals were assigned based on HMQC, HMBC, and 1H–1H COSY spectra. Thus, based on the above evidences, compound 1 was established as (E)phenylpropene-3-methoxyphenyl-[600 -O-galloy]-4-O-b-D-glucopyranoside (Fig. 1).
Fig. 2. Key HMBC (H ! C) and 1H–1H COSY correlations of compounds 1–3.
Compound 2 was obtained as white amorphous solid and was suggested to have the molecular formula C19H22O11 based on HRESIMS [m/z 449.1043 [M+Na]+ (calc. for C19H22O11Na, 449.1045)]. The IR spectrum also showed the presence of hydroxyl group (3623 cm1), carbonyl group (1737 cm1), a conjugated carbonyl group (1628 cm1) and benzene ring (1602, 1523 cm1). The 1H NMR spectrum of 2 (Table 2) showed the presence of one 1,2,3,4,5-penta-substituted aromatic ring and one methyl group at dH 7.06 (1H, s) and dH 2.65 (3H, s). The 2(5H)-furanone unit in the molecule was deduced from the 1H and 13C NMR spectrum (dH 6.28, 1H, t, J = 1.8 Hz, H-30 and dH 4.75, 2H, t, J = 1.8 Hz, H-40 ; dC-20 133.5, dC-30 148.4, dC-40 72.2, dC-50 177.3) (Table 2), confirmed by 1 H–1H COSY [between H-30 (dH 6.28) and H-40 (dH 4.75)] and HMBC [between H-30 (dH 6.28) and C-20 (dC 133.5), C-40 (dC 72.2), C-50 (dC 177.3); between H-40 (dH 4.75) and C-20 (dC 133.5), C-30 (dC 148.4), C-50 (dC 177.3)]. The HMBC correlations beteween H-10 (dH 3.51, s) to C-20 (dC 133.5) and C-5 (dC 106.3) suggested the 2(5H)-furanone was linked to C-5 through a methylene (C-10 ). The 13C NMR (Table 2) and DEPT spectrum showed that compound 2 exhibited
Fig. 1. Compounds 1–6 isolated from P. chinense.
D. Huang et al. / Phytochemistry Letters 11 (2015) 163–167 Table 2 1 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 2 in DMSOd6.
165
Table 3 1 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 3 in DMSOd6.
Position
dH
dC
HMBC (H–C)
Position
dH (J Hz)
dC
HMBC (H–C)
1 2 3 4 5 6 7 8 10 20 30 40 50 100 200 300 400 500 600
– – 7.06(1H, s) – – – – 2.65(3H, s) 3.51(2H, s) – 6.28(1H, t, 1.8) 4.75(2H, d, 1.8) – 4.90(1H, d, 7.8) 3.44(1H,m) 3.45(1H, m) 3.35(1H, m) 3.38(1H, m) 3.93(1H, dd, 12.0, 1.8) 3.71(1H, dd, 12.0, 6.0)
107.0 162.7 94.6 162.8 106.3 164.3 205.6 33.1 19.2 133.5 148.4 72.2 177.3 101.7 74.6 78.1 71.2 78.5 62.6
– – C-1, C-2, C-4, C-5 – – – – C-1, C-7 C-20 , C-30 , C-50 , C-4, C-5, C-6 – C-10 , C-20 , C-40 , C-50 C-20 , C-30 , C-50 – C-4, C-200 , C-500 C-100 , C-300 C-200 , C-400 C-300 , C-500 C-100 , C-300 C-400 , C-500
1 2, 6 3, 5 4
– 7.28(2H, 7.25(2H, 7.27(1H, 2.91(1H, 3.35(1H, – – – 6.07(1H, – 6.07(1H,
141.0 128.2 128.2 125.7 45.1 30.0 204.7 105.4 163.9 95.0 163.1 95.0 163.9 99.1 71.2 77.4 67.3 76.7 60.0
– C-1, C-2, C-3, C-1, C-1,
one methylene at dC 19.2 (C-10 ) and a sugar moiety at dC 101.7 (C-100 ), 74.6 (C-200 ), 78.1 (C-300 ), 71.2 (C-400 ), 78.5 (C-500 ) and 62.6 (C-600 ). The glucopyranose moiety was determined to have a b-configuration at C-100 with the large coupling constant of H-100 (J = 7.8 Hz). Correlations from H-100 to C-4 in the HMBC spectrum (Fig. 2), indicated that the b-glycopyranoside group was located at C-4. In the HMBC spectrum, correlations from H-8 (dH 2.65, 3H, s) to C-7 (dC 205.6) and C-1 (dC 107.0) suggested the acetyl group (C7–C8) was located at C-1. On the basis of the molecular formula and chemical shifts, the rest two aromatic carbons were hydroxyl group substituted (dC-2 162.7; dC-2 164.3). Thus, compound 2 was established as 2,6-dihydroxyacetophenone-5-(20 -methylene2(5H)-furanone)-4-O-b-D-glucopyranoside. Compound 3, a salmon pink powder, had the molecular formula of C28H28O13 deduced from the positive HRESIMS for the peak at m/ z 595.1409 [M+Na]+ (calc. for C28H28O13Na 595.1422). The IR spectrum also showed the presence of hydroxyl group (3398 cm1), carbonyl group (1697 cm1), phenyl ketone (1630 cm1) and benzene ring (1597 cm1). In the 1H NMR spectrum, the symmetrical benzene ring at dH 7.00 (H-2000 and H6000 ) suggested the occurrence of a galloyl group (Wang et al., 2010). The occurrence of a dihydrochalcone skeleton (Calanasan and Macleod, 1998; Tanimoto et al., 2009) in the molecule could be easily deduced from the 1H NMR spectrum (Table 3), in which compound 3 showed the signals for a mono-substituted aromatic protons at dH [7.28 (H-2 and H-6), 7.25 (H-3 and H-5), 7.27 (H-4)] on A-ring, together with one singlet in the aromatic region dH 6.07 (s, H-30 , 50 ) (She et al., 2011). The 13C NMR (Table 3) and DEPT spectrum showed that compound 3 exhibited two methylene at dC 45.1 (C-a) and dC 30.0 (C-b), a sugar moiety at dC 99.1 (C-100 ), 71.2(C-200 ), 77.4(C-300 ), 67.3(C-400 ), 76.7(C-500 ) and 60.0 (C-600 ). Acid hydrolysis of 3 released D-glucopyranose. The glucopyranose moiety was determined to have a b-configuration at C-100 with the large coupling constant of H-100 (J = 7.8 Hz). In the HMBC (Fig. 2) spectrum, the galloyl group was shown to be at C-300 due to a down field shift of the dH 5.07 (H-300 ) and the HMBC correlation between the dH 3.49 (H-300 ) and the dC 165.3 (C-7000 ). In addition, the HMBC spectrum showed that dH 5.07 (H-100 ) was correlated to dC 163.1 (C-40 ), indicating that the b-glucopyranosyl group was attached to the C-40 position. Thus, compound 3 was established as 20 ,60 -dihydroxydihydrochalcone-40 -O-[300 -O-galloyl]-b-D-glucopyranoside.
a b C–C5 5O 10 0 2 30 40 50 60 100 200 300 400 500 600 1000 2000 , 6000 3000 , 5000 4000 7000
m) m) br s) t, 7.8) t, 7.8)
s) s)
5.07(1H, d, 7.8) 3.45(1H, dd, 9.0,7.8) 3.49(1H,m) 3.48(1H,m) 3.50(1H, m) 3.69(1H, dd, 11.4,1.2) 3.54(1H, dd, 11.4,3.6) – 7.00(2H, s) – – –
119.9 108.7 145.4 138.1 165.3
C-3, C-5, C-a C-4, C-6 C-2, C-5, C-6 C-2, C-6, C-b, C–C5 5O C–C5 5O, C-10 , C-a
C-10 , C-20 , C-40 , C-50 C-10 , C-30 , C-40 , C-50 C-40 , C-200 C-100 , C-300 C-200 , C-400 , C-7000 C-300 , C-600 C-100 , C-400 , C-600 C-400 , C-500
C-1000 , C-3000 ,C-5000 ,C-7000 – –
The structures of the known compounds were identified based on MS, NMR data and comparison with literature: 1-O-sinapoyl-bD-glucopyranoside (4) (Gao et al., 2014), 2,6-dihydroxyacetophenone-4-O-b-D-glucopyranoside (5) (Jeanette, 1973), ferulic acid glucopyranoside (6) (Stranda˚s et al., 2008). 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a PE 341 polarimeter at 22 8C. Infrared (IR) spectra were recorded on a Nicolet FTIR 6700 spectrometer with a KBr pellet. The NMR spectra, including 1 H, 13C, DEPT and 2D-NMR, were recorded on a Bruker AM-400 and AC-600 spectrometer with chemical shifts reported as d values, using TMS as internal standard. HRESIMS data were obtained on an Agilent Technologies 6538 UHD Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, MA, USA). Thin layer chromatography was performed on TLC plates (Silica gel HSGF254. Jiangyou Company of Yantai; RP-18 F254. Merck) and spots were visualized by heating after dipping into 10% H2SO4. Silica gel (100–200 and 200–300 mesh, Jiangyou Company of Yantai), Sephadex LH-20 (Pharmacia), RP-C18 (43–60 mm, Merck), and MCI gel (75–150 mm, Mitsubishi Chemical Corporation) were used for column chromatography. 3.2. Plant materials The dry aerial parts of P. chinense were provided by Gulin Gansu Pharmaceutical Co., Ltd. (Sichuan, China). The plant was identified by Prof. Wansheng Chen, and a voucher specimen was deposited in the Department of Pharmacognosy of the Second Military Medical University in Shanghai, China. 3.3. Extraction and isolation 10 kg of dry aerial parts of P. chinense were extracted three times with 80% EtOH (3 80 L, 2 h each) at 80 8C, and concentrated
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in vacuum to give a residue (1.167 g). The residue was suspended in water (1.5 L) and then successively partitioned with petroleum ether (3 1.0 L), EtOAc (3 2.0 L), and n-BuOH (2 1.5 L) to give petroleum ether-soluble (99 g), EtOAc-soluble (245 g), and nBuOH-soluble (239 g) extracts. The ethyl acetate fraction (100 g) was subjected to silica gel (200–300 mesh) column chromatography, eluted with CH2Cl2/CH3OH (50:1, 30:1, 20:1, 10:1, 5:1, 0:1 v/ v) to afford four fractions Fr. 1–Fr. 4, which were combined based on TLC profiles. Fr. 3 (23 g) was refined over MCI gel column chromatography (75–150 mm, 200 g, 8 cm 100 cm), eluted with H2O, followed by increasing concentrations of MeOH (10%, 20%, 50%, 100%) to give four subfractions (Fr. 3.1–Fr. 3.4). Fr. 3.2 (3.7 g) was further subjected to a Sephadex LH-20 column eluted with MeOH/H2O (1:9, 3:7, 5:5, 1:0) and divided into four main subfractions Fr. 3.2a–Fr. 3.2d. Fr. 3.2b (210 mg) was then purified to HPLC chromatography (MeOH/H2O from 40:60 to 100% MeOH in 30 min, flow rate 2 ml/min) to yield compound 1 (6.8 mg). Fr. 3.2c (1210 mg) was subjected to Sephadex LH-20 (80 g, 1.5 cm 150 cm) eluting with MeOH/H2O (4:6, 200 ml) to obtain compound 2 (21 mg). Fr.4 (32 g) was separated by silica gel (320 g, 200–300 mesh, 8 cm 100 cm) eluting with CH2Cl2/CH3OH (10:1, 5:1, 0:1 v/v) to give five subfractions, Fr. 4.1–Fr. 4.5. Fr 4.2 (4.8 g) applied to ODS column with MeOH/H2O (2.5 cm 150 cm) (0:1, 1:9, 5:5, 8:2, 1:0, each 350 ml) to give five subfractions Fr. 4.2a–Fr. 4.2e. Fr. 4.2a (721 mg) was subjected to Sephadex LH-20 eluting with MeOH/H2O (3:7, 5:5, each 200 ml) to obtain compound 5 (12.8 mg) and compound 4 (8.2 mg). Fr. 4.2c (1210 mg) was subjected to Sephadex LH-20 eluting with MeOH/H2O (8:2, each 200 ml) to afford five fractions, and compound 3 (113 mg) was obtained from the second fraction according to the TLC profile. Fr. 4.2e (117 mg) was purified by HPLC chromatography (MeOH/H2O from 40:60 to 90:10 in 30 min, flow rate 1 ml/min) to yield compound 6 (12.8 mg). 3.4. Chemical characterization Compound 1: Faint yellow powder; IR (KBr): ymax 3423.0, 2921.6, 1698.9, 1612.2, 1511.9, 1072.2, 767.5 cm1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z 501.1360 [M+Na]+ (calc. for C22H24O9Na, 501.1367). Compound 2: White amorphous solid; IR (KBr): ymax 3623.6, 3399.9, 2923.6, 2854.1, 1737.6, 1627.6, 1602.6, 1523.5, 1455.9, 1428.9, 871.7 cm1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 2; HRESIMS m/z 449.1043 [M+Na]+ (calc. for C22H24O9Na, 449.1045). Compound 3: Salmon pink powder; IR (KBr): ymax 3398.0, 2927.4, 1697.1, 1629.6, 1596.8, 1523.5, 1436.7, 1348.0, 1205.3, 1174.4, 1078.0, 1039.4, 823.5, 752.1, 698.1 cm1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 3; HRESIMS m/z 595.1409 [M+Na]+ (calc. for C28H28O13Na, 595.1422). Compound 4: White powder; ESI-MS: m/z 387.1 [M+H]+, 408.2 [M+Na]+; 1H NMR(600 MHz, DMSO-d6, d ppm): 7.05 (2H, s, H-2/6), 9.00 (1H, s, 4-OH), 7.64 (1H, d, J = 15.6 Hz, H-7), 6.55 (1H, d, J = 15.6 Hz, H-8), 3.81 (6H, s, 3/5-OCH3), 5.47 (1H, d, J = 7.8 Hz, H10 ), 3.27–3.37 (4H, m, H-20 –50 ), 3.75 (1H, d, J = 2.0, 12.0 Hz, H-60 ), 3.59 (1H, d, J = 12.0, 5.4 Hz, H-60 ); 13C NMR (150 MHz, DMSO-d6, d ppm): 124.2 (C-1), 106.3 (C-2/6), 148.0 (C-3/5), 138.5 (C-4),146.5 (C-7), 114.3 (C-8), 165.3 (C-9), 56.0 (3/5-OCH3), 94.2 (C-10 ), 72.5 (C20 ), 76.5 (C-30 ), 69.5 (C-40 ), 77. 8(C-50 ), 60.6 (C-60 ). Compound 5: White powder; ESI-MS: m/z 331.5[M+H]+; 1H NMR (400 MHz, DMSO-d6, d ppm): 6.07 (2H, s, H-3/5), 2.59 (3H, s, H-8), 4.86 (1H, d, J = 7.2 Hz, H-10 ), 3.17–3.45 (4H, m, H-20 –50 ), 3.49 (1H, m, H-60 ), 3.67 (1H, d, J = 17.4 Hz, H-60 ); 13C NMR (100 MHz, DMSO-d6, d ppm): 105.5 (C-1), 163.9 (C-2/6), 94.9(C-3/5), 163.5
(C-4), 203.4 (C-7), 32.6 (C-8), 99.4 (C-10 ), 73.0 (C-20 ), 76.4 (C-30 ), 69.4 (C-40 ), 77.1 (C-50 ), 60.4 (C-60 ). Compound 6: ESI-MS: m/z 357.2 [M+H]+; 1H NMR (400 MHz, DMSO-d6, d ppm): 6.48 (1H, d, J = 15.6 Hz, H-2), 7.63 (1H, d, J = 15.6 Hz, H-3), 3.81 (3H, s, H-OCH3), 7.31 (1H, d, J = 1.6 Hz, H-20 ), 6.81 (1H, d, J = 8.4 Hz, H-50 ), 7.14 (1H, dd, J = 8.4, 1.6 Hz, H-60 ), 5.45 (1H, d, J = 8.4 Hz, H-100 ), 3.07–3.51 (4H, m, H-200 –500 ), 3.45 (1H, m, H600 ), 3.67 (1H, m, H-600 ); 13C NMR (100 MHz, DMSO-d6, d ppm): 174.6 (C-1), 113.9 (C-2), 146.4 (C-3), 55.7 (C-OCH3), 125.4 (C-10 ), 111.3 (C-20 ), 149.6 (C-30 ), 148.0 (C-40 ), 115.6 (C-50 ), 123.4 (C-60 ), 94.2 (C-100 ), 72.5 (C-200 ), 76.5 (C-300 ), 69.5 (C-400 ), 77.8 (C-500 ), 60.6 (C-600 ). 4. Acid hydrolysis A solution of compound 1 (5 mg), compound 2 (5 mg) and compound 3 (10 mg) in 2N aqueous CF3COOH (10 ml) was refluxed at 80 8C for 2 h, respectively. The mixture was then diluted in water (10 ml) and extracted with EtOAc (3 ml 3 ml). The combined water layers and evaporated to dryness to afford the glycoside. The residue was purified over ODS column to afford D-glucopyranose [1.2 mg, [a´]22D +38.2 (c 0.15, H2O); 0.8 mg, [a´]22D +40.3 (c 0.20, H2O), 2.1 mg, [a´]22D +35.3 (c 0.20, H2O)], respectively. The glucopyranose was identified on the basis of their specific rotation. Declaration of interest The authors report no declarations of interest. Acknowledgements This research project was supported by the Science Foundation of Shanghai (No. 13401900106 the National Science Fund for Distinguished Young Scholars (No.81325024), and the Second Military Medical University Stem Cell and Medical Research Center’s Innovation Research Program (SCMRC1201). The NMR spectra were conducted by the Second Military University School of Pharmacy. The authors also thank the Shanghai Institute of Pharmaceutical Industry of Academia Sinica for their aid in obtaining polarimetry and IR spectra.
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