FITOTE-03141; No of Pages 7 Fitoterapia xxx (2015) xxx–xxx
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Fitoterapia
F
journal homepage: www.elsevier.com/locate/fitote
Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum
2Q2
Haizhen Li 1, Zhun Hou 1, Chao Li, Yao Zhang, Tao Shen, Qingwen Hu, Dongmei Ren ⁎
3
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 Wenhuaxi Road, Jinan 250012, PR China
R O O
1Q1
4
a r t i c l e
7 8 9 10 11
Article history: Received 31 January 2015 Accepted in revised form 2 March 2015 Accepted 6 March 2015 Available online xxxx
21 22 23 20 24 25 26
Keywords: Viscum articulatum Flavanone glycosides Diastereoisomers Circular dichroism Cytoprotection
27
1. Introduction
28
Viscum articulatum Burm, belonging to the family Loranthaceae, is distributed widely in south and southwest of China. The leaves and stems of the plant have been commonly used for the treatment of hemorrhage, pleurisy, gout, heart disease, arthritis, and hypertension in traditional Chinese medicine [1]. Previous phytochemical investigations have revealed that triterpenoids, organic acids and flavonoids are the major secondary metabolites of this plant [2–5]. Although there are a variety of biological activities reports have been published on some species of the genus Viscum, only a few of these reports is about V. articulatum. The extracts of this plant have been demonstrated to possess significant diuretic activity in rats [6], promising wound healing potential in rats [7], and antihypertensive effect in the NO deficient type of hypertension [1]. In our preliminary studies, the total flavonoids of V. articulatum showed cytoprotective activity against H2O2-induced oxidative stress in EA.hy926 cells. In the course of our ongoing survey for biologically active flavonoids, this phytochemical study on the
35 36 37 38 39 40 41 42 43 44 45
D
E
T
12 13 14 15 16 17 18 19
C E R
R
O
33 34
Phytochemical examination of the leaves and stems of Viscum articulatum resulted in the isolation of three pairs of new flavanone glycosides, 2R/2S-viscarticulide A–C (1a/1b–3a/3b), together with eight known compounds (7–14). Their structures were established by extensive spectroscopic data analyses. The diastereoisomers were separated by HPLC on a chiral phase and the absolute configuration at C-2 was determined by circular dichroism (CD) spectra. The protective effects of compounds 1–3 against H2O2-induced cytotoxicity with EA.hy926 cells were tested. The results showed that compounds 1–3 improved the survival of EA.hy926 cells after H2O2 exposure at the tested concentrations. © 2015 Published by Elsevier B.V.
N C
31 32
a b s t r a c t
U
29 30
i n f o
P
6
⁎ Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382548. E-mail address: [email protected] (D. Ren). 1 These authors contributed equally to this work.
leaves and stems of V. articulatum yielded three pairs of new flavanone glycosides (1a/1b–3a/3b), together with eight known compounds (7–14) (Fig. 1). In this paper, we describe the isolation, structure elucidation, stereoanalysis and bioassay of the novel flavanone glycosides.
46
2. Experimental
51
2.1. General experimental procedures
52
Optical rotation value was measured on an Anton Paar MCP 200 polarimeter. IR spectra were recorded on a Thermo Nicolet NEXUS 670 FTIR spectrometer with KBr disks. NMR spectra were measured on a Brucker AV-600 spectrometer with TMS as internal standard. High-resolution ESI-MS mass spectra were carried out on an LTQ-Orbitrap XL instrument. Semipreparative HPLC was performed on an Agilent HP 1260 instrument, using an Agilent Eclipse XDB-C18 (250 × 9.4 mm I.D., 5 μM) column. The chiral columns (250 × 4.6 mm I.D.) were amylose tris-(3, 5-dimethylphenylcarbamate) immobilized on 5 μm silica gel (Chiralpak IA) and amylose tris-(3, 5-dimethylphenylcarbamate) (Chiralpak AD-H) coated on 5 μm silica gel. The chiral columns were obtained from Daicel (Tokyo, Japan). CD spectra were measured on an applied
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http://dx.doi.org/10.1016/j.fitote.2015.03.009 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
47 48 49 50
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H. Li et al. / Fitoterapia xxx (2015) xxx–xxx
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O
R
R
E
C
T
E
D
P
R O
O
F
2
Fig. 1. Structure of compounds isolated from V. articulatum.
74
2.2. Plant material
75
78 79
The whole plants of V. articulatum were collected from Xishuangbanna, Yunan Province and authenticated by one of the co-author, associate professor Tao Shen. A voucher specimen (No. 20101207-03) has been deposited in the School of Pharmaceutical Sciences, Shandong University.
80
2.3. Extraction and isolation
81
Air-dried and powdered whole plants of V. articulatum (10 kg) were extracted with EtOH–H2O (95:5, v/v, 20 L × 3,
76 77
82
N
U
70 71
C
72 73
photophysics Chirascan spectrometer, using 1 mm cell. Silica gel (200–300 mesh, Haiyang Co., Qingdao, China), YMC-GEL ODS-A (50 μM, YMC Co., Ltd., Japan), Sephadex LH-20 gel (Amersham Biosciences) and Toyopearl HW-40F (Tosoh, Japan) were used for column chromatography. Precoated silica GF254 plates (Haiyang Co., Qingdao, China) were used for TLC analysis.
68 69
each 3 days) at room temperature. After removal of solvent under reduced pressure, the crude extract (944 g) was suspended in H2O and partitioned successively with petroleum ether, EtOAc and n-BuOH to give three different polar parts. The EtOAc fraction was evaporated in vacuo to give a residue (90 g), which was subjected to silica gel column chromatography (CC) using a gradient system of CH2Cl2–MeOH (100:0–0:100, v/v) as an eluent to afford five major fractions (EA1–EA5). Fraction EA2 (3.7 g) was further separated into three major subfractions (EA2A–EA2C) by chromatographing over silica gel column with a step gradient system of petroleum ether–EtOAc (30:1, 20:1, 10:1, 5:1, 1:1, v/v). Subfraction EA2B (200 mg) was subjected to Sephadex LH-20 CC eluted with CH2Cl2–MeOH (1:1) to yield compounds 11 (15 mg). Subfraction EA2C (1.2 g) was submitted to Sephadex LH-20 CC (CH2Cl2–MeOH, 1:1) followed by semi-preparative HPLC eluted with MeOH–H2O (65:35, v/v) to produce compounds 8 (10 mg), 9 (30 mg) and 10 (15 mg). Fraction EA4 (6.3 g) was subjected to CC over silica gel and eluted with a CH2Cl2–MeOH gradient system (50:1 → 1:1), producing three subfractions (EA4A–EA4D). Subfraction EA4A
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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H. Li et al. / Fitoterapia xxx (2015) xxx–xxx
114 115 116 117 118 119 120 121 122 123 124 125 126 127 128
t1:1 t1:2
Table 1 1 H NMR (600 MHz) data for compounds 1–3 (in CD3OD, δ in ppm, J in Hz).
E
132 133
144 145
2.4. Cell culture and treatment
152
No.
1a
1b
t1:4 t1:5
2 3
t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18
6 8 2′ 3′ 5′ 6′ 3′-OCH3 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6
5.08 (dd, 3.0, 13.2) 2.61 (dd, 3.0, 16.8) 3.02 (dd, 13.2, 16.8) 6.06 (d, 2.4) 6.07 (d, 2.4) 7.01 (d, 1.2)
5.23 (dd, 3.0, 13.2) 2.62 (dd, 3.0, 16.8) 2.97 (dd, 13.2, 16.8) 6.05 (d, 1.8) 6.06 (d, 1.8) 7.01 (brs)
t1:19 t1:20 t1:21
Api-1 Api-2 Api-4
t1:22
Api-5
t1:23 t1:24 t1:25
3″, 7″ 4″, 6″ 5″
N C
O
t1:3
U
6.81 (d, 7.8) 6.85 (dd, 1.2, 7.8) 3.87 (s) 5.04 (d, 7.2) 3.67 (m) 3.43 (t, 9.0) 3.38 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.99 (brs) 3.88 (d, 10.2) 4.23 (d, 10.2) 4.27 (d, 11.4) 4.32 (d, 11.4) 7.86 (m) 7.35 (t, 7.8) 7.52 (m)
6.81 (d, 8.4) 6.84 (brd, 8.4) 3.87 (s) 5.05 (d, 7.8) 3.67 (m) 3.43 (m) 3.38 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.48 (brs) 4.00 (brs) 3.89 (d, 9.6) 4.23 (d, 9.6) 4.27 (d, 11.4) 4.33 (d, 11.4) 7.87 (m) 7.35 (t, 7.8) 7.52 (m)
138 139 140 141 142 143
146 147 148 149 150 151
EA.hy926 (human endothelial-like immortalized) cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a humidified incubator containing 5% CO2 [9].
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158 159
2.5. Measurement of cell viability
161
154 155 156 157
160
Cell viability was monitored by 3-(4, 5-dimethylthiazol-2- 162 yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In 163 brief, 1 × 104 cells per well were seeded in a 96-well plate 164
R
130 131
R
129
C
134 135
2.3.1. Viscarticulide A (1) Amorphous yellow powder; IR (KBr) νmax.cm−1: 3428, 2925, 1722, 1643, 1578, 1519, 1452, 1273, 1073, and 714; and HRESIMS: m/z 701.2086 [M + H]+, and (calcd. for 701.2082). 1 2S-viscarticulide A (1a), [α]20 D -30.8 (c 0.5, CH3OH); H NMR 13 data see Table 1 and C NMR data see Table 2. 1 2R-viscarticulide A (1b), [α]20 D -22.3 (c 0.53, CH3OH); H 13 NMR data see Table 1 and C NMR data see Table 2.
2.3.3. Viscarticulide C (3) Amorphous yellow powder; IR (KBr) νmax.cm−1: 3420, 2925, 1717, 1641, 1578, 1523, 1451, 1275, 1072, 714; and HRESIMS: m/z 687.1921 [M + H]+, and (calcd. for 687.1925). 1 2S-viscarticulide C (3a), [α]20 D -28.3 (c 0.23, CH3OH); H NMR data see Table 1 and 13C NMR data see Table 2. 1 2R-viscarticulide C (3b), [α]20 D -40.2 (c 0.28, CH3OH); H 13 NMR data see Table 1 and C NMR data see Table 2.
F
112 113
R O O
110 111
P
108 109
136 137
D
107
2.3.2. Viscarticulide B (2) Amorphous yellow powder; IR (KBr) νmax.cm−1: 3424, 2924, 1719, 1641, 1579, 1519, 1451, 1273, 1080, and 714; HRESIMS: m/z 671.1976 [M + H]+, and (calcd. for 671.1976). 1 2S-viscarticulide B (2a), [α]20 D -23.2 (c 0.25, CH3OH); H 13 NMR data see Table 1 and C NMR data see Table 2. 1 2R-viscarticulide B (2b), [α]20 D -23.9 (c 0.27, CH3OH); H NMR data see Table 1 and 13C NMR data see Table 2.
E
105 106
(2.1 g) was chromatographed on C18 column eluted with MeOH–H2O (20:80–90:10, v/v) to yield compound 4 (70 mg). Subfraction EA4B (900 mg) was chromatographed on C18 column eluted with MeOH–H2O (20:80–90:10, v/v) and then separated by semipreparative RP-18 HPLC column (CH3CN/ H2O 35:65, v/v) to yield compounds 1 (6 mg), 2 (5 mg) and 3 (6 mg). Separation of the two single stereoisomers of 1 and 3 was achieved by HPLC on a Chiralpak IA column [8]. The solvent system used for the isolation of 1a and 1b was EtOH-i-PrOH (55:45, v/v) and the flow rate was 0.5 ml/min. The solvent system used for the isolation of 3a and 3b was n-hexane-EtOH (30:70, v/v) and the flow rate was 0.7 ml/min. Separation of 2a and 2b was achieved by HPLC on a Chiralpak AD-H column eluted with 100% EtOH at a flow rate of 0.5 ml/min. After concentration, the n-BuOH fraction (70 g) was separated by Toyopearl HW-40F CC, eluted with a gradient of MeOH–H2O (20:80 → 100:0) to give five major fractions (BU1–BU5). Fraction BU3 (15 g) was chromatographed on C18 column eluted with MeOH–H2O (30:70–90:10) to yield three subfractions (BU3A–BU3C). Subfraction BU3A (2.4 g) was purified by Sephadex LH-20 (CH2Cl2–MeOH, 1:1) CC repeatedly to yield compound 4 (100 mg). Subfraction BU3B (5.0 g) was chromatographed on C18 column eluted with MeOH–H2O (20:80–90:10, v/v) to yield compounds 5 (30 mg), 6 (12 mg) and 7 (11 mg).
T
103 104
3
2a
2b
3a
3b
5.08 (dd, 3.0, 12.6) 2.61 (dd, 3.0, 16.8) 3.09 (dd, 12.6, 16.8) 6.05 (brs) 6.06 (brs) 7.24 (d, 8.4) 6.81 (d, 8.4) 6.81 (d, 8.4) 7.24 (d, 8.4)
5.23 (dd, 3.0, 12.6) 2.60 (dd, 3.0, 16.8) 3.03 (dd, 12.6, 16.8) 6.05 (brs) 6.06 (brs) 7.23 (d, 8.4) 6.82 (d, 8.4) 6.82 (d, 8.4) 7.23 (d, 8.4)
5.01 (dd, 3.0, 13.2) 2.63 (dd, 3.0, 16.8) 2.99 (dd, 13.2, 16.8) 6.04 (brs) 6.05 (brs) 6.86 (brs)
5.17 (dd, 3.0, 13.2) 2.62 (dd, 3.0, 16.8) 2.92 (dd, 13.2, 16.8) 6.04 (brs) 6.05 (brs) 6.86 (brs)
6.76 (d, 8.4) 6.74 (dd, 1.8, 8.4)
6.76 (d, 8.4) 6.73 (dd, 1.8, 8.4)
5.06 (d, 7.8) 3.66 (m) 3.42 (m) 3.37 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.99 (brs) 3.88 (d, 9.6) 4.23 (d, 9.6) 4.28 (d, 11.4) 4.32 (d, 11.4) 7.87 (m) 7.37 (m) 7.53 (m)
5.05 (d, 7.8) 3.67 (m) 3.42 (m) 3.37 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.98 (brs) 3.89 (d, 9.6) 4.25 (d, 9.6) 4.29 (d, 11.4) 4.31 (d, 11.4) 7.87 (m) 7.37 (m) 7.53 (m)
5.06 (d, 7.8) 3.66 (m) 3.42 (m) 3.37 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.46 (brs) 3.98 (brs) 3.89 (d, 10.2) 4.23 (d, 10.2) 4.29 (d, 11.4) 4.32 (d, 11.4) 7.88 (m) 7.37 (m) 7.53 (m)
5.05 (d, 7.8) 3.66 (m) 3.43 (m) 3.36 (m) 3.62 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.99 (brs) 3.89 (d, 10.2) 4.23 (d, 10.2) 4.30 (d, 11.4) 4.31 (d, 11.4) 7.88 (m) 7.36 (m) 7.53 (m)
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
4
3a
3b
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2:35
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3′-OCH3 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6 Api-1 Api-2 Api-3 Api-4 Api-5 1″ 2″ 3″, 7″ 4″, 6″ 5″
79.3 42.8 196.8 163.5 96.2 165.2 95.2 163.0 103.4 130.0 109.8 147.7 146.7 114.7 119.1 55.1 98.0 77.2 76.8 69.8 76.3 60.9 108.9 77.3 77.7 73.8 66.6 166.1 129.5 129.1 128.1 132.8
79.3 42.7 196.8 163.5 96.1 165.1 95.1 163.0 103.4 130.0 109.8 147.7 146.8 114.7 119.2 55.1 98.0 77.2 76.8 69.7 76.3 60.8 108.9 77.3 77.7 73.8 66.5 166.1 129.5 129.1 128.1 132.7
79.0 42.3 196.9 163.5 96.1 165.0 95.1 163.1 103.4 129.5 127.6 114.9 157.7 114.9 127.6
78.9 42.3 196.9 163.5 96.1 165.1 95.1 163.0 103.4 129.5 127.7 114.9 157.7 114.9 127.7
79.2 42.6 196.8 163.5 96.1 165.0 95.0 163.1 103.4 130.0 113.4 145.6 145.0 114.8 117.9
79.1 42.8 196.7 163.5 96.0 165.1 95.1 163.0 103.4 130.1 113.3 145.5 145.0 114.8 117.8
97.9 77.2 76.7 69.7 76.3 60.8 108.8 77.3 77.7 73.8 66.7 166.1 129.3 129.1 128.1 132.8
98.0 77.2 76.7 69.7 76.3 60.8 108.9 77.3 77.7 73.8 66.6 166.1 129.3 129.0 128.1 132.8
97.9 77.2 76.8 69.7 76.4 60.8 108.9 77.2 77.7 73.8 66.7 166.1 129.5 129.1 128.1 132.8
98.0 77.2 76.8 69.7 76.4 60.8 108.9 77.3 77.7 73.7 66.6 166.0 129.4 129.1 128.1 132.8
E
165
O
2b
R O
2a
P
1b
D
1a
E
No.
C
t2:3
176
3. Results and discussion
177 178
Compound 1 was isolated as a yellow amorphous powder. The molecular formula was determined as C34H36O16 by the positive mode HRESIMS ([M + H]+ found at m/z 701.2086, calcd. 701.2082). The IR spectrum showed bands due to hydroxyl (3428 cm−1), carbonyl (1710 cm−1) and benzyl (1578 and 1579 cm−1) functions. The UV spectrum showed maxima at 290 nm. Although 1 showed a single peak detected by HPLC using a reversed-phase C18 column, some of the 1H and 13 C NMR signals of 1 were splitting into two sets of peaks with almost equal intensities, revealed that 1 might be a mixture of two compounds in a ratio of ca. 1:1 [10]. 1H and 13C NMR spectra of 1 suggested that the compound was a flavanone glycoside, because of the presence of the chiral center in the aglycone (C-2) and the optically sugar residue, it may exist as a mixture of a pair of diastereoisomers [11]. This assumption was
179 180 181 182 183 184 185 186 187 188 189 190 191
R
O
173 174
C
171 172
N
170
U
168 169
R
175
and incubated overnight. Cells were pretreated with several concentrations of compound for 2 h before exposure to H2O2 for 24 h. The cells were incubated for an additional 4 h after addition of 20 μl 2.0 mg/mL MTT solution, the plate was then centrifuged and the medium was removed. 100 μl DMSO was added into each well, and crystals were dissolved by shaking the plate at room temperature. Absorbance was measured at 570 nm by a microplate reader (Biorad, Model 680, USA). Triplicate wells were used for each sample and the experiments were repeated at least three times to get means and standard deviations [9].
166 167
confirmed by HPLC investigation using a chiral-phase column, which showed two peaks with nearly equal areas (Fig. 2). In order to unequivocally clarify the structures of the two diastereoisomers of compound 1, 1a (corresponding to the faster peak) and 1b (corresponding to the slower peak) were prepared by using HPLC on a chiral-phase column. 1a was obtained as a yellow solid. The 1H NMR and 13C NMR spectra of 1a comprised resonances corresponding to aromatic and glycosidic protons and carbons, and one methoxy group. The 1H NMR spectrum showed a typical three protons ABX spin system at δH 3.02 (1H, dd, J = 16.8, 13.2 Hz, H-3a), 2.61 (1H, dd, J = 16.8, 3.0 Hz, H-3b) and 5.08 (1H, dd, J = 13.2, 3.0 Hz, H-2), suggesting a flavanone skeleton. A 5, 7-disubstituted ring A was represented by two meta-coupled protons at δH 6.06 (1H, d, J = 2.4 Hz) and 6.07 (1H, d, J = 2.4 Hz), assigned to H-6 and H-8, respectively. A 3′, 4′-disubstituted ring B was revealed by resonances at δH 7.01 (1H, d, J = 1.2 Hz, H-2′), 6.81 (1H, d, J = 7.8 Hz, H-5′) and 6.85 (1H, dd, J = 1.2, 7.8 Hz, H-6′). The location of the methoxy group was established at C-3′ from the correlations between H-2′, H-5′ and 3′-OMe to C-3′ (δH 147.7) (Fig. 3). Complete assignment of the remaining resonances of the aglycone in the 13C NMR spectrum of 1a was discerned by analysis of the HSQC and HMBC data. These evidences suggested the flavanone moiety was homoeriodictyol [12,13]. In addition, proton signals at δH 7.86 (2H, m), 7.35 (2H, t, J = 7.8 Hz) and 7.52 (1H, m) for a monosubstituted aromatic ring as well as a carboxyl carbon signal at δC 166.1 were obtained, which gave evidence for 1a as possessing a benzoyl moiety. Furthermore, two anomeric proton resonances corresponding to O-linked sugars were displayed in 1H NMR spectrum as one doublet at δH 5.04 (1H, d, J = 7.2 Hz) and one broad singlet at δH 5.47 (1H, brs). Based on the analysis of 1D and 2D NMR data of 1a, one of the two sugar units was elucidated as glucose. The other sugar residue showed NMR signals corresponding to two methine and two methylene groups, in addition to a quaternary carbon, which were in agreement with the apiosyl moiety [13,14]. Since only the D-configuration is known to exist in naturally occurring glucose and apiose, the sugars in 1a were tentatively assigned the D-configuration. The C-1 of the glucose should be in a β-configuration, due to the large coupling constant value (J = 7.2 Hz) of the anomeric proton. The β-anomeric configuration for the apiose was indicated from the anomeric carbon signal at δC 108.5 and a small coupling constant value of the anomeric proton. The HMBC correlation between Api-H-1 and Glu-C-2 (δC 77.2) suggested the interglycosidic linkage. A correlation between Glu-H-1 and C-7 (δC 165.2) in the HMBC spectrum defined the site of O-glycosylation. The HMBC correlations observed between Api-H2-5 (δH 4.27, 4.32) and C-1″(δC 166.1) indicated the attachment of the benzoyl moiety to Api-C-5 (Fig. 3). In the CD spectrum of 1a, a positive Cotton effect (CE) at 335 nm and a negative CE at 290 nm established an S-configuration at C-2 (Fig. 2) [10,15]. Therefore, the structure of 1a was determined as 2S-homoeriodictyol-7-O-(5O-benzoyl)-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, named to be 2S-viscarticulide A. 1b was obtained as amorphous yellow solid. The 1H NMR and 13C NMR data of 1b were almost superimposable to those of 1a, except for some minor differences (Tables 1 and 2). The HMQC and HMBC spectra of 1b revealed the same correlations as 1a. The CD curve of 1b showed a negative CE at 335 nm and a
F
Table 2 13 C NMR (150 MHz) data for compounds 1–3 (in CD3OD, δ in ppm).
T
t2:1 t2:2
H. Li et al. / Fitoterapia xxx (2015) xxx–xxx
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252
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T
Fig. 2. HPLC separation of viscarticulide A–C (1–3) using chiral columns, and assignment of the absolute configurations of C-2 by CD spectra.
Fig. 3. Key HMBC (H → C) and 1H-1H COSY (bold) correlations of 1–3.
positive CE at 290 nm, completely opposite to those of 1a (Fig. 2), which confirmed an R-configuration at C-2 [10]. Thus, 1b was identified as 2R-homoeriodictyol-7-O-(5-O-benzoyl)β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, named to be 2R-viscarticulide A. Compound 2 was obtained as a yellow amorphous powder with a molecular formula of C33H34O15 by the positive mode HRESIMS ([M + H]+ found at m/z 671.1976, calcd. 671.1976), which was 30 amu less than that of compound 1. IR, UV, 1H NMR and 13C NMR spectroscopic data of 2 were very similar to those of compound 1. The split of some of the 1H and 13C NMR signals into doublets also suggested that compound 2 was a diastereoisomeric mixture. The two diastereoisomers, 2a and 2b corresponding to the two peaks in a chiral HPLC analysis (Fig. 2), were prepared, and their 1H and 13C NMR spectra were recorded separately (Tables 1 and 2). Comparison of the 1H and 13 C NMR data of compound 2 with that of compound 1 suggested that the two compounds have a similar structure except for the signals assigned to the flavanone moiety. The presence of a naringenin unit was revealed by a typical ABX spin system (δH 2.61, dd, J = 3.0, 16.8 Hz; 2.97, dd, J = 13.2, 16.8 Hz; and 5.23, dd, J = 3.0, 13.2 Hz), a set of AA′BB′ protons (δH 6.81 and 7.24, each d, J = 8.4 Hz), as well as two proton signals for H-6 (δH 6.05, brs) and H-8 (δH 6.06, brs) [16]. This partial structure was further confirmed by the 13C NMR data. The presence of glucose, an apiose and a benzoyl moiety was deduced from the similar NMR data as those of compound 1. The HMBC correlations defining the site of acylation, glycosylation and interglycosidic linkages were similar to those detected for 1 (Fig. 3). Thus, compound 2 was concluded to be naringenin-7-O-(5-O-benzoyl)-β-D-apiofuranosyl-(1 → 2)β-D-glucopyranoside, named to be viscarticulide B. 2a was
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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Conflict of interest
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The authors declare no conflict of interest.
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Financial support from the National Natural Science 330 Foundation of China (Nos. 81173528 and 31470419) are 331 Q3 gratefully acknowledged. 332
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determined as 2S-viscarticulide B according to a positive CE at 335 nm and a negative CE at 290 nm, while 2b was established an R-configuration at C-2 based on the opposite CEs to 2a (Fig. 2). Compound 3 was obtained as a yellow amorphous powder with a molecular formula of C33H34O16 by the positive mode HRESIMS ([M + H]+ found at m/z 687.1921, calcd. 687.1925), which suggested an additional hydroxyl group when compared with 2. IR, UV, 1H NMR and 13C NMR data (Tables 1 and 2) of 3 were similar with those of 2, except for the presence of an eriodictyol unit [17] instead of a naringenin unit as the aglycone in 3. Analysis of 2D NMR data and comparison of the 1H NMR and 13C NMR values of the oligosaccharide part with those of 1 and 2, revealed that 3 contained the same disaccharide chain β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside linked to C-7 of eriodictyol. The HMBC correlations of Api-H2-5 (δH 4.29, 4.32) with the carbonyl carbon (δC 166.1) of benzoyl moiety confirmed the linkage of the aromatic ester at Api-C-5 (Fig. 3). The structure of 3 was therefore elucidated as eriodictyol-7-O(5-O-benzoyl)-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, named to be viscarticulide C. Similar to 1 and 2, compound 3 was also a diastereoisomeric mixture. The two diastereoisomers, 3a and 3b, were obtained by using a chiral HPLC column, and their 1H and 13C NMR spectra were measured separately (Tables 1 and 2). Analysis of the CD spectra determined the configuration of C-2 to be R for 3a, while 3b was assigned to be 2S-configured (Fig. 2). In addition, eight known compounds were also isolated: homoeriodictyol-7-O-β-D-glucoside (4) [18], eriodictyol-7-Oβ-D-glucoside (5) [19], naringenin-7-O-β-D-glucoside (6) [20], pinocembrin-7-O-β-D-glucoside (7) [21,22], eriodictyol (8) [23], homoeriodictyol (9) [12], naringenin (10) [16], and p-hydroxyphenylacetic acid (11) [24]. Their structures were identified by comparison of the NMR and MS spectroscopic data with those previously reported in the literatures. To investigate whether compounds 1–3 protect cells from H2O2-induced cell death, EA.hy926 cells were treated with 300 μM H2O2 in the presence or absence of compounds 1–3 (1, 2, 5, 10 μM), and the cell viability was assessed by performing MTT assay. The cytotoxic effects of H2O2 on EA.hy926 cells were blocked in different degree by pretreatment with different concentrations of compounds 1–3 (Fig. 4).
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Fig. 4. Protective effects of 1–3 on H2O2-induced cytotoxicity in EA.hy926 cells, the cell viability were determined by MTT assay.
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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[22] Massaro CF, Katouli M, Grkovic T, Vu H, Quinn RJ, Heard TA, et al. Antistaphylococcal activity of C-methyl flavanones from propolis of Australian stingless bees (Tetragonula carbonaria) and fruit resins of Corymbia torelliana (Myrtaceae). Fitoterapia 2014;95:247–57. [23] Miyake Y, Yamamoto K, Osawa T. Metabolism of antioxidant in lemon fruit (Citrus limon Burm. f.) by human intestinal bacteria. J Agric Food Chem 1997;45:3738–42. [24] Abe H, Uchiyama M, Sato R. Isolation of phenylacetic acid and its p-hydroxy derivative as auxin-like substances from Undaria pinnatifida. 1974;38(4):897–8.
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[18] Wang XL, Li MR, Li LQ. Studies on the chemical compounds of Viscum articulatum Burm (I). West China J Pharm Sci 1990;5(2):63–8. [19] Hu QW, Zhang DD, Wang LM, Lou HX, Ren DM. Eriodictyol-7-O-glucoside, a novel Nrf2 activator, confers protection against cisplatin-induced toxicity. Food Chem Toxicol 2012;50:1927–32. [20] Tuntiwachwuttikul P, Pootaeng-on Y, Phansa P, Limpachayaporn P, Charoenchai P, Taylor WC. Constituents of the leaves of Holarrhena pubescens. Fitoterapia 2007;78:271–3. [21] Hanawa F, Yamada T, Nakashima T. Phytoalexins from Pinus strobus bark infected with pinewood nematode, Bursaphelenchus xylophilus. Phytochemistry 2001;57:223–8.
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Contents lists available at ScienceDirect
Fitoterapia
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journal homepage: www.elsevier.com/locate/fitote
Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum
2Q2
Haizhen Li 1, Zhun Hou 1, Chao Li, Yao Zhang, Tao Shen, Qingwen Hu, Dongmei Ren ⁎
3
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 Wenhuaxi Road, Jinan 250012, PR China
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a r t i c l e
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Article history: Received 31 January 2015 Accepted in revised form 2 March 2015 Accepted 6 March 2015 Available online xxxx
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Keywords: Viscum articulatum Flavanone glycosides Diastereoisomers Circular dichroism Cytoprotection
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1. Introduction
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Viscum articulatum Burm, belonging to the family Loranthaceae, is distributed widely in south and southwest of China. The leaves and stems of the plant have been commonly used for the treatment of hemorrhage, pleurisy, gout, heart disease, arthritis, and hypertension in traditional Chinese medicine [1]. Previous phytochemical investigations have revealed that triterpenoids, organic acids and flavonoids are the major secondary metabolites of this plant [2–5]. Although there are a variety of biological activities reports have been published on some species of the genus Viscum, only a few of these reports is about V. articulatum. The extracts of this plant have been demonstrated to possess significant diuretic activity in rats [6], promising wound healing potential in rats [7], and antihypertensive effect in the NO deficient type of hypertension [1]. In our preliminary studies, the total flavonoids of V. articulatum showed cytoprotective activity against H2O2-induced oxidative stress in EA.hy926 cells. In the course of our ongoing survey for biologically active flavonoids, this phytochemical study on the
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Phytochemical examination of the leaves and stems of Viscum articulatum resulted in the isolation of three pairs of new flavanone glycosides, 2R/2S-viscarticulide A–C (1a/1b–3a/3b), together with eight known compounds (7–14). Their structures were established by extensive spectroscopic data analyses. The diastereoisomers were separated by HPLC on a chiral phase and the absolute configuration at C-2 was determined by circular dichroism (CD) spectra. The protective effects of compounds 1–3 against H2O2-induced cytotoxicity with EA.hy926 cells were tested. The results showed that compounds 1–3 improved the survival of EA.hy926 cells after H2O2 exposure at the tested concentrations. © 2015 Published by Elsevier B.V.
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⁎ Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382548. E-mail address: [email protected] (D. Ren). 1 These authors contributed equally to this work.
leaves and stems of V. articulatum yielded three pairs of new flavanone glycosides (1a/1b–3a/3b), together with eight known compounds (7–14) (Fig. 1). In this paper, we describe the isolation, structure elucidation, stereoanalysis and bioassay of the novel flavanone glycosides.
46
2. Experimental
51
2.1. General experimental procedures
52
Optical rotation value was measured on an Anton Paar MCP 200 polarimeter. IR spectra were recorded on a Thermo Nicolet NEXUS 670 FTIR spectrometer with KBr disks. NMR spectra were measured on a Brucker AV-600 spectrometer with TMS as internal standard. High-resolution ESI-MS mass spectra were carried out on an LTQ-Orbitrap XL instrument. Semipreparative HPLC was performed on an Agilent HP 1260 instrument, using an Agilent Eclipse XDB-C18 (250 × 9.4 mm I.D., 5 μM) column. The chiral columns (250 × 4.6 mm I.D.) were amylose tris-(3, 5-dimethylphenylcarbamate) immobilized on 5 μm silica gel (Chiralpak IA) and amylose tris-(3, 5-dimethylphenylcarbamate) (Chiralpak AD-H) coated on 5 μm silica gel. The chiral columns were obtained from Daicel (Tokyo, Japan). CD spectra were measured on an applied
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http://dx.doi.org/10.1016/j.fitote.2015.03.009 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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Fig. 1. Structure of compounds isolated from V. articulatum.
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2.2. Plant material
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The whole plants of V. articulatum were collected from Xishuangbanna, Yunan Province and authenticated by one of the co-author, associate professor Tao Shen. A voucher specimen (No. 20101207-03) has been deposited in the School of Pharmaceutical Sciences, Shandong University.
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2.3. Extraction and isolation
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Air-dried and powdered whole plants of V. articulatum (10 kg) were extracted with EtOH–H2O (95:5, v/v, 20 L × 3,
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82
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photophysics Chirascan spectrometer, using 1 mm cell. Silica gel (200–300 mesh, Haiyang Co., Qingdao, China), YMC-GEL ODS-A (50 μM, YMC Co., Ltd., Japan), Sephadex LH-20 gel (Amersham Biosciences) and Toyopearl HW-40F (Tosoh, Japan) were used for column chromatography. Precoated silica GF254 plates (Haiyang Co., Qingdao, China) were used for TLC analysis.
68 69
each 3 days) at room temperature. After removal of solvent under reduced pressure, the crude extract (944 g) was suspended in H2O and partitioned successively with petroleum ether, EtOAc and n-BuOH to give three different polar parts. The EtOAc fraction was evaporated in vacuo to give a residue (90 g), which was subjected to silica gel column chromatography (CC) using a gradient system of CH2Cl2–MeOH (100:0–0:100, v/v) as an eluent to afford five major fractions (EA1–EA5). Fraction EA2 (3.7 g) was further separated into three major subfractions (EA2A–EA2C) by chromatographing over silica gel column with a step gradient system of petroleum ether–EtOAc (30:1, 20:1, 10:1, 5:1, 1:1, v/v). Subfraction EA2B (200 mg) was subjected to Sephadex LH-20 CC eluted with CH2Cl2–MeOH (1:1) to yield compounds 11 (15 mg). Subfraction EA2C (1.2 g) was submitted to Sephadex LH-20 CC (CH2Cl2–MeOH, 1:1) followed by semi-preparative HPLC eluted with MeOH–H2O (65:35, v/v) to produce compounds 8 (10 mg), 9 (30 mg) and 10 (15 mg). Fraction EA4 (6.3 g) was subjected to CC over silica gel and eluted with a CH2Cl2–MeOH gradient system (50:1 → 1:1), producing three subfractions (EA4A–EA4D). Subfraction EA4A
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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t1:1 t1:2
Table 1 1 H NMR (600 MHz) data for compounds 1–3 (in CD3OD, δ in ppm, J in Hz).
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2.4. Cell culture and treatment
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No.
1a
1b
t1:4 t1:5
2 3
t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18
6 8 2′ 3′ 5′ 6′ 3′-OCH3 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6
5.08 (dd, 3.0, 13.2) 2.61 (dd, 3.0, 16.8) 3.02 (dd, 13.2, 16.8) 6.06 (d, 2.4) 6.07 (d, 2.4) 7.01 (d, 1.2)
5.23 (dd, 3.0, 13.2) 2.62 (dd, 3.0, 16.8) 2.97 (dd, 13.2, 16.8) 6.05 (d, 1.8) 6.06 (d, 1.8) 7.01 (brs)
t1:19 t1:20 t1:21
Api-1 Api-2 Api-4
t1:22
Api-5
t1:23 t1:24 t1:25
3″, 7″ 4″, 6″ 5″
N C
O
t1:3
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6.81 (d, 7.8) 6.85 (dd, 1.2, 7.8) 3.87 (s) 5.04 (d, 7.2) 3.67 (m) 3.43 (t, 9.0) 3.38 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.99 (brs) 3.88 (d, 10.2) 4.23 (d, 10.2) 4.27 (d, 11.4) 4.32 (d, 11.4) 7.86 (m) 7.35 (t, 7.8) 7.52 (m)
6.81 (d, 8.4) 6.84 (brd, 8.4) 3.87 (s) 5.05 (d, 7.8) 3.67 (m) 3.43 (m) 3.38 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.48 (brs) 4.00 (brs) 3.89 (d, 9.6) 4.23 (d, 9.6) 4.27 (d, 11.4) 4.33 (d, 11.4) 7.87 (m) 7.35 (t, 7.8) 7.52 (m)
138 139 140 141 142 143
146 147 148 149 150 151
EA.hy926 (human endothelial-like immortalized) cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a humidified incubator containing 5% CO2 [9].
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2.5. Measurement of cell viability
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Cell viability was monitored by 3-(4, 5-dimethylthiazol-2- 162 yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In 163 brief, 1 × 104 cells per well were seeded in a 96-well plate 164
R
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R
129
C
134 135
2.3.1. Viscarticulide A (1) Amorphous yellow powder; IR (KBr) νmax.cm−1: 3428, 2925, 1722, 1643, 1578, 1519, 1452, 1273, 1073, and 714; and HRESIMS: m/z 701.2086 [M + H]+, and (calcd. for 701.2082). 1 2S-viscarticulide A (1a), [α]20 D -30.8 (c 0.5, CH3OH); H NMR 13 data see Table 1 and C NMR data see Table 2. 1 2R-viscarticulide A (1b), [α]20 D -22.3 (c 0.53, CH3OH); H 13 NMR data see Table 1 and C NMR data see Table 2.
2.3.3. Viscarticulide C (3) Amorphous yellow powder; IR (KBr) νmax.cm−1: 3420, 2925, 1717, 1641, 1578, 1523, 1451, 1275, 1072, 714; and HRESIMS: m/z 687.1921 [M + H]+, and (calcd. for 687.1925). 1 2S-viscarticulide C (3a), [α]20 D -28.3 (c 0.23, CH3OH); H NMR data see Table 1 and 13C NMR data see Table 2. 1 2R-viscarticulide C (3b), [α]20 D -40.2 (c 0.28, CH3OH); H 13 NMR data see Table 1 and C NMR data see Table 2.
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136 137
D
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2.3.2. Viscarticulide B (2) Amorphous yellow powder; IR (KBr) νmax.cm−1: 3424, 2924, 1719, 1641, 1579, 1519, 1451, 1273, 1080, and 714; HRESIMS: m/z 671.1976 [M + H]+, and (calcd. for 671.1976). 1 2S-viscarticulide B (2a), [α]20 D -23.2 (c 0.25, CH3OH); H 13 NMR data see Table 1 and C NMR data see Table 2. 1 2R-viscarticulide B (2b), [α]20 D -23.9 (c 0.27, CH3OH); H NMR data see Table 1 and 13C NMR data see Table 2.
E
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(2.1 g) was chromatographed on C18 column eluted with MeOH–H2O (20:80–90:10, v/v) to yield compound 4 (70 mg). Subfraction EA4B (900 mg) was chromatographed on C18 column eluted with MeOH–H2O (20:80–90:10, v/v) and then separated by semipreparative RP-18 HPLC column (CH3CN/ H2O 35:65, v/v) to yield compounds 1 (6 mg), 2 (5 mg) and 3 (6 mg). Separation of the two single stereoisomers of 1 and 3 was achieved by HPLC on a Chiralpak IA column [8]. The solvent system used for the isolation of 1a and 1b was EtOH-i-PrOH (55:45, v/v) and the flow rate was 0.5 ml/min. The solvent system used for the isolation of 3a and 3b was n-hexane-EtOH (30:70, v/v) and the flow rate was 0.7 ml/min. Separation of 2a and 2b was achieved by HPLC on a Chiralpak AD-H column eluted with 100% EtOH at a flow rate of 0.5 ml/min. After concentration, the n-BuOH fraction (70 g) was separated by Toyopearl HW-40F CC, eluted with a gradient of MeOH–H2O (20:80 → 100:0) to give five major fractions (BU1–BU5). Fraction BU3 (15 g) was chromatographed on C18 column eluted with MeOH–H2O (30:70–90:10) to yield three subfractions (BU3A–BU3C). Subfraction BU3A (2.4 g) was purified by Sephadex LH-20 (CH2Cl2–MeOH, 1:1) CC repeatedly to yield compound 4 (100 mg). Subfraction BU3B (5.0 g) was chromatographed on C18 column eluted with MeOH–H2O (20:80–90:10, v/v) to yield compounds 5 (30 mg), 6 (12 mg) and 7 (11 mg).
T
103 104
3
2a
2b
3a
3b
5.08 (dd, 3.0, 12.6) 2.61 (dd, 3.0, 16.8) 3.09 (dd, 12.6, 16.8) 6.05 (brs) 6.06 (brs) 7.24 (d, 8.4) 6.81 (d, 8.4) 6.81 (d, 8.4) 7.24 (d, 8.4)
5.23 (dd, 3.0, 12.6) 2.60 (dd, 3.0, 16.8) 3.03 (dd, 12.6, 16.8) 6.05 (brs) 6.06 (brs) 7.23 (d, 8.4) 6.82 (d, 8.4) 6.82 (d, 8.4) 7.23 (d, 8.4)
5.01 (dd, 3.0, 13.2) 2.63 (dd, 3.0, 16.8) 2.99 (dd, 13.2, 16.8) 6.04 (brs) 6.05 (brs) 6.86 (brs)
5.17 (dd, 3.0, 13.2) 2.62 (dd, 3.0, 16.8) 2.92 (dd, 13.2, 16.8) 6.04 (brs) 6.05 (brs) 6.86 (brs)
6.76 (d, 8.4) 6.74 (dd, 1.8, 8.4)
6.76 (d, 8.4) 6.73 (dd, 1.8, 8.4)
5.06 (d, 7.8) 3.66 (m) 3.42 (m) 3.37 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.99 (brs) 3.88 (d, 9.6) 4.23 (d, 9.6) 4.28 (d, 11.4) 4.32 (d, 11.4) 7.87 (m) 7.37 (m) 7.53 (m)
5.05 (d, 7.8) 3.67 (m) 3.42 (m) 3.37 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.98 (brs) 3.89 (d, 9.6) 4.25 (d, 9.6) 4.29 (d, 11.4) 4.31 (d, 11.4) 7.87 (m) 7.37 (m) 7.53 (m)
5.06 (d, 7.8) 3.66 (m) 3.42 (m) 3.37 (m) 3.61 (m) 3.67 (m) 3.84 (m) 5.46 (brs) 3.98 (brs) 3.89 (d, 10.2) 4.23 (d, 10.2) 4.29 (d, 11.4) 4.32 (d, 11.4) 7.88 (m) 7.37 (m) 7.53 (m)
5.05 (d, 7.8) 3.66 (m) 3.43 (m) 3.36 (m) 3.62 (m) 3.67 (m) 3.84 (m) 5.47 (brs) 3.99 (brs) 3.89 (d, 10.2) 4.23 (d, 10.2) 4.30 (d, 11.4) 4.31 (d, 11.4) 7.88 (m) 7.36 (m) 7.53 (m)
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
4
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3b
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2:35
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3′-OCH3 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6 Api-1 Api-2 Api-3 Api-4 Api-5 1″ 2″ 3″, 7″ 4″, 6″ 5″
79.3 42.8 196.8 163.5 96.2 165.2 95.2 163.0 103.4 130.0 109.8 147.7 146.7 114.7 119.1 55.1 98.0 77.2 76.8 69.8 76.3 60.9 108.9 77.3 77.7 73.8 66.6 166.1 129.5 129.1 128.1 132.8
79.3 42.7 196.8 163.5 96.1 165.1 95.1 163.0 103.4 130.0 109.8 147.7 146.8 114.7 119.2 55.1 98.0 77.2 76.8 69.7 76.3 60.8 108.9 77.3 77.7 73.8 66.5 166.1 129.5 129.1 128.1 132.7
79.0 42.3 196.9 163.5 96.1 165.0 95.1 163.1 103.4 129.5 127.6 114.9 157.7 114.9 127.6
78.9 42.3 196.9 163.5 96.1 165.1 95.1 163.0 103.4 129.5 127.7 114.9 157.7 114.9 127.7
79.2 42.6 196.8 163.5 96.1 165.0 95.0 163.1 103.4 130.0 113.4 145.6 145.0 114.8 117.9
79.1 42.8 196.7 163.5 96.0 165.1 95.1 163.0 103.4 130.1 113.3 145.5 145.0 114.8 117.8
97.9 77.2 76.7 69.7 76.3 60.8 108.8 77.3 77.7 73.8 66.7 166.1 129.3 129.1 128.1 132.8
98.0 77.2 76.7 69.7 76.3 60.8 108.9 77.3 77.7 73.8 66.6 166.1 129.3 129.0 128.1 132.8
97.9 77.2 76.8 69.7 76.4 60.8 108.9 77.2 77.7 73.8 66.7 166.1 129.5 129.1 128.1 132.8
98.0 77.2 76.8 69.7 76.4 60.8 108.9 77.3 77.7 73.7 66.6 166.0 129.4 129.1 128.1 132.8
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3. Results and discussion
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Compound 1 was isolated as a yellow amorphous powder. The molecular formula was determined as C34H36O16 by the positive mode HRESIMS ([M + H]+ found at m/z 701.2086, calcd. 701.2082). The IR spectrum showed bands due to hydroxyl (3428 cm−1), carbonyl (1710 cm−1) and benzyl (1578 and 1579 cm−1) functions. The UV spectrum showed maxima at 290 nm. Although 1 showed a single peak detected by HPLC using a reversed-phase C18 column, some of the 1H and 13 C NMR signals of 1 were splitting into two sets of peaks with almost equal intensities, revealed that 1 might be a mixture of two compounds in a ratio of ca. 1:1 [10]. 1H and 13C NMR spectra of 1 suggested that the compound was a flavanone glycoside, because of the presence of the chiral center in the aglycone (C-2) and the optically sugar residue, it may exist as a mixture of a pair of diastereoisomers [11]. This assumption was
179 180 181 182 183 184 185 186 187 188 189 190 191
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C
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and incubated overnight. Cells were pretreated with several concentrations of compound for 2 h before exposure to H2O2 for 24 h. The cells were incubated for an additional 4 h after addition of 20 μl 2.0 mg/mL MTT solution, the plate was then centrifuged and the medium was removed. 100 μl DMSO was added into each well, and crystals were dissolved by shaking the plate at room temperature. Absorbance was measured at 570 nm by a microplate reader (Biorad, Model 680, USA). Triplicate wells were used for each sample and the experiments were repeated at least three times to get means and standard deviations [9].
166 167
confirmed by HPLC investigation using a chiral-phase column, which showed two peaks with nearly equal areas (Fig. 2). In order to unequivocally clarify the structures of the two diastereoisomers of compound 1, 1a (corresponding to the faster peak) and 1b (corresponding to the slower peak) were prepared by using HPLC on a chiral-phase column. 1a was obtained as a yellow solid. The 1H NMR and 13C NMR spectra of 1a comprised resonances corresponding to aromatic and glycosidic protons and carbons, and one methoxy group. The 1H NMR spectrum showed a typical three protons ABX spin system at δH 3.02 (1H, dd, J = 16.8, 13.2 Hz, H-3a), 2.61 (1H, dd, J = 16.8, 3.0 Hz, H-3b) and 5.08 (1H, dd, J = 13.2, 3.0 Hz, H-2), suggesting a flavanone skeleton. A 5, 7-disubstituted ring A was represented by two meta-coupled protons at δH 6.06 (1H, d, J = 2.4 Hz) and 6.07 (1H, d, J = 2.4 Hz), assigned to H-6 and H-8, respectively. A 3′, 4′-disubstituted ring B was revealed by resonances at δH 7.01 (1H, d, J = 1.2 Hz, H-2′), 6.81 (1H, d, J = 7.8 Hz, H-5′) and 6.85 (1H, dd, J = 1.2, 7.8 Hz, H-6′). The location of the methoxy group was established at C-3′ from the correlations between H-2′, H-5′ and 3′-OMe to C-3′ (δH 147.7) (Fig. 3). Complete assignment of the remaining resonances of the aglycone in the 13C NMR spectrum of 1a was discerned by analysis of the HSQC and HMBC data. These evidences suggested the flavanone moiety was homoeriodictyol [12,13]. In addition, proton signals at δH 7.86 (2H, m), 7.35 (2H, t, J = 7.8 Hz) and 7.52 (1H, m) for a monosubstituted aromatic ring as well as a carboxyl carbon signal at δC 166.1 were obtained, which gave evidence for 1a as possessing a benzoyl moiety. Furthermore, two anomeric proton resonances corresponding to O-linked sugars were displayed in 1H NMR spectrum as one doublet at δH 5.04 (1H, d, J = 7.2 Hz) and one broad singlet at δH 5.47 (1H, brs). Based on the analysis of 1D and 2D NMR data of 1a, one of the two sugar units was elucidated as glucose. The other sugar residue showed NMR signals corresponding to two methine and two methylene groups, in addition to a quaternary carbon, which were in agreement with the apiosyl moiety [13,14]. Since only the D-configuration is known to exist in naturally occurring glucose and apiose, the sugars in 1a were tentatively assigned the D-configuration. The C-1 of the glucose should be in a β-configuration, due to the large coupling constant value (J = 7.2 Hz) of the anomeric proton. The β-anomeric configuration for the apiose was indicated from the anomeric carbon signal at δC 108.5 and a small coupling constant value of the anomeric proton. The HMBC correlation between Api-H-1 and Glu-C-2 (δC 77.2) suggested the interglycosidic linkage. A correlation between Glu-H-1 and C-7 (δC 165.2) in the HMBC spectrum defined the site of O-glycosylation. The HMBC correlations observed between Api-H2-5 (δH 4.27, 4.32) and C-1″(δC 166.1) indicated the attachment of the benzoyl moiety to Api-C-5 (Fig. 3). In the CD spectrum of 1a, a positive Cotton effect (CE) at 335 nm and a negative CE at 290 nm established an S-configuration at C-2 (Fig. 2) [10,15]. Therefore, the structure of 1a was determined as 2S-homoeriodictyol-7-O-(5O-benzoyl)-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, named to be 2S-viscarticulide A. 1b was obtained as amorphous yellow solid. The 1H NMR and 13C NMR data of 1b were almost superimposable to those of 1a, except for some minor differences (Tables 1 and 2). The HMQC and HMBC spectra of 1b revealed the same correlations as 1a. The CD curve of 1b showed a negative CE at 335 nm and a
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Table 2 13 C NMR (150 MHz) data for compounds 1–3 (in CD3OD, δ in ppm).
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Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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Fig. 2. HPLC separation of viscarticulide A–C (1–3) using chiral columns, and assignment of the absolute configurations of C-2 by CD spectra.
Fig. 3. Key HMBC (H → C) and 1H-1H COSY (bold) correlations of 1–3.
positive CE at 290 nm, completely opposite to those of 1a (Fig. 2), which confirmed an R-configuration at C-2 [10]. Thus, 1b was identified as 2R-homoeriodictyol-7-O-(5-O-benzoyl)β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, named to be 2R-viscarticulide A. Compound 2 was obtained as a yellow amorphous powder with a molecular formula of C33H34O15 by the positive mode HRESIMS ([M + H]+ found at m/z 671.1976, calcd. 671.1976), which was 30 amu less than that of compound 1. IR, UV, 1H NMR and 13C NMR spectroscopic data of 2 were very similar to those of compound 1. The split of some of the 1H and 13C NMR signals into doublets also suggested that compound 2 was a diastereoisomeric mixture. The two diastereoisomers, 2a and 2b corresponding to the two peaks in a chiral HPLC analysis (Fig. 2), were prepared, and their 1H and 13C NMR spectra were recorded separately (Tables 1 and 2). Comparison of the 1H and 13 C NMR data of compound 2 with that of compound 1 suggested that the two compounds have a similar structure except for the signals assigned to the flavanone moiety. The presence of a naringenin unit was revealed by a typical ABX spin system (δH 2.61, dd, J = 3.0, 16.8 Hz; 2.97, dd, J = 13.2, 16.8 Hz; and 5.23, dd, J = 3.0, 13.2 Hz), a set of AA′BB′ protons (δH 6.81 and 7.24, each d, J = 8.4 Hz), as well as two proton signals for H-6 (δH 6.05, brs) and H-8 (δH 6.06, brs) [16]. This partial structure was further confirmed by the 13C NMR data. The presence of glucose, an apiose and a benzoyl moiety was deduced from the similar NMR data as those of compound 1. The HMBC correlations defining the site of acylation, glycosylation and interglycosidic linkages were similar to those detected for 1 (Fig. 3). Thus, compound 2 was concluded to be naringenin-7-O-(5-O-benzoyl)-β-D-apiofuranosyl-(1 → 2)β-D-glucopyranoside, named to be viscarticulide B. 2a was
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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Conflict of interest
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The authors declare no conflict of interest.
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Financial support from the National Natural Science 330 Foundation of China (Nos. 81173528 and 31470419) are 331 Q3 gratefully acknowledged. 332
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Acknowledgments
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determined as 2S-viscarticulide B according to a positive CE at 335 nm and a negative CE at 290 nm, while 2b was established an R-configuration at C-2 based on the opposite CEs to 2a (Fig. 2). Compound 3 was obtained as a yellow amorphous powder with a molecular formula of C33H34O16 by the positive mode HRESIMS ([M + H]+ found at m/z 687.1921, calcd. 687.1925), which suggested an additional hydroxyl group when compared with 2. IR, UV, 1H NMR and 13C NMR data (Tables 1 and 2) of 3 were similar with those of 2, except for the presence of an eriodictyol unit [17] instead of a naringenin unit as the aglycone in 3. Analysis of 2D NMR data and comparison of the 1H NMR and 13C NMR values of the oligosaccharide part with those of 1 and 2, revealed that 3 contained the same disaccharide chain β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside linked to C-7 of eriodictyol. The HMBC correlations of Api-H2-5 (δH 4.29, 4.32) with the carbonyl carbon (δC 166.1) of benzoyl moiety confirmed the linkage of the aromatic ester at Api-C-5 (Fig. 3). The structure of 3 was therefore elucidated as eriodictyol-7-O(5-O-benzoyl)-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, named to be viscarticulide C. Similar to 1 and 2, compound 3 was also a diastereoisomeric mixture. The two diastereoisomers, 3a and 3b, were obtained by using a chiral HPLC column, and their 1H and 13C NMR spectra were measured separately (Tables 1 and 2). Analysis of the CD spectra determined the configuration of C-2 to be R for 3a, while 3b was assigned to be 2S-configured (Fig. 2). In addition, eight known compounds were also isolated: homoeriodictyol-7-O-β-D-glucoside (4) [18], eriodictyol-7-Oβ-D-glucoside (5) [19], naringenin-7-O-β-D-glucoside (6) [20], pinocembrin-7-O-β-D-glucoside (7) [21,22], eriodictyol (8) [23], homoeriodictyol (9) [12], naringenin (10) [16], and p-hydroxyphenylacetic acid (11) [24]. Their structures were identified by comparison of the NMR and MS spectroscopic data with those previously reported in the literatures. To investigate whether compounds 1–3 protect cells from H2O2-induced cell death, EA.hy926 cells were treated with 300 μM H2O2 in the presence or absence of compounds 1–3 (1, 2, 5, 10 μM), and the cell viability was assessed by performing MTT assay. The cytotoxic effects of H2O2 on EA.hy926 cells were blocked in different degree by pretreatment with different concentrations of compounds 1–3 (Fig. 4).
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Fig. 4. Protective effects of 1–3 on H2O2-induced cytotoxicity in EA.hy926 cells, the cell viability were determined by MTT assay.
Please cite this article as: Li H, et al, Three pairs of diastereoisomeric flavanone glycosides from Viscum articulatum, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.009
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[18] Wang XL, Li MR, Li LQ. Studies on the chemical compounds of Viscum articulatum Burm (I). West China J Pharm Sci 1990;5(2):63–8. [19] Hu QW, Zhang DD, Wang LM, Lou HX, Ren DM. Eriodictyol-7-O-glucoside, a novel Nrf2 activator, confers protection against cisplatin-induced toxicity. Food Chem Toxicol 2012;50:1927–32. [20] Tuntiwachwuttikul P, Pootaeng-on Y, Phansa P, Limpachayaporn P, Charoenchai P, Taylor WC. Constituents of the leaves of Holarrhena pubescens. Fitoterapia 2007;78:271–3. [21] Hanawa F, Yamada T, Nakashima T. Phytoalexins from Pinus strobus bark infected with pinewood nematode, Bursaphelenchus xylophilus. Phytochemistry 2001;57:223–8.
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