FITOTE-03105; No of Pages 4 Fitoterapia xxx (2015) xxx–xxx
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Fitoterapia
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journal homepage: www.elsevier.com/locate/fitote
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Ruijian Zhong a, Qing Guo a,b, Guoping Zhou a, Huizheng Fu a,⁎, Kaihua Wan c,⁎
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Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell lines
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Jiangxi Provincial Institute for Drug and Food Control, Nanchang 330029, China Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China Drug Adverse Reaction Monitoring Center of Jiangxi, Nanchang 330046, China
a r t i c l e
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Article history: Received 27 November 2014 Accepted in revised form 7 January 2015 Available online xxxx
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Keywords: Rubus chingii Labdane-type Diterpene glycosides Cytotoxic activity
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1. Introduction
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Rubus chingii Hu, belonging to the family Rosaceae, is distributed widely in Jiangxi, Zhejiang, Fujian, and Guangxi Provinces of mainland China. Its fruits are used as a Chinese medicine for treating enuresis with renal asthenia, frequent urination, premature ejaculation, and impotence [1]. Previous phytochemical studies on R. chingii have led to the isolation of flavonoids [2], triterpenoids [3–6], organic acids [7,8], sterols [3,7], and alkaloids [9]. In our preliminary screening, the nBuOH part of the 70% EtOH extract of the fruits of R. chingii showed significant anticancer activity. As part of program to discover biologically significant anticancer properties from plant resources has led to the isolation of three new compounds, 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-7(8),13(14)diene-3β,15,18-triol (1), 15,18-di-O-β-D-glucopyranosyl-13(E)ent-labda-8(9),13(14)-diene-3β,15,18-triol (2), and 15-O-β-D-
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Three new labdane-type diterpene glycosides, 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda7(8),13(14)-diene-3β,15,18-triol (1), 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-8(9), 13(14)-diene-3β,15,18-triol (2), and 15-O-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranosyl-18O-β-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)-diene-3β,15,18-triol (3), were isolated from the fruits of Rubus chingii. Their structures were elucidated on the basis of spectroscopic data and chemical methods. The cytotoxic activities of compounds 1–3 were evaluated against five human tumor cell lines (HCT-8, BGC-823, A549, and A2780). Compounds 3 showed cytotoxic activity against A549 with an IC50 value of 2.32 μM. © 2015 Published by Elsevier B.V.
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⁎ Corresponding authors. E-mail addresses: [email protected] (H. Fu), [email protected] (K. Wan).
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apiofuranosyl-(1 → 2)-β-D-glucopyranosyl-18-O-β-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)-di-ene-3β,15,18-triol (3), were isolated from the fruits of R. chingii. Reported herein are the isolation, structure elucidation and biological activity of these compounds.
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2. Experimental
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2.1. General experimental procedures
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Optical rotations were measured on an Autopol IV-T/V (Rudolph Research Analytical, New Jersey, USA). UV spectra were recorded in MeOH on a Jasco V650 spectrophotometer (JASCO, Inc., Easton, Maryland, USA). The 1H (600 MHz), 13C (150 MHz), and 2D NMR spectra were recorded on a Bruker AVANCE III 600 instrument using TMS (Tetramethylsilane) as an internal reference (Bruker Company, Massachusetts, USA). HRTOFMS data were obtained on an Agilent 7890-7000A mass spectrometer (Agilent Technologies, Santa Clara, USA). Preparative HPLC (high performance liquid chromatography) was conducted with an Agilent Technologies 1200 series instrument with a MWD detector using a YMC-pack ODS (Octadecylsilyl)-A column (5 μm, 250 × 20 mm). Column chromatography was
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http://dx.doi.org/10.1016/j.fitote.2015.01.007 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
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The fruits of R. chingii were collected from Jinghua, Zhejiang, China, in June 2012 and identified by Prof. Cuisheng Fang at Jiangxi University of Traditional Chinese Medicine, China. A voucher specimen (no. 20120622) has been deposited in the Herbarium of Jiangxi Provincial Institute for Drug and Food Control.
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2.3. Extraction and isolation
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The powdered dried fruits of R. chingii (50.5 kg) were extracted three times with 70% EtOH under reflux (2 h each). The extracting solution was evaporated under reduced pressure to yield a dark brown residue (1.1 kg). The residue was suspended in water (20 L) and then successively partitioned with petroleum ether (2 × 20 L), EtOAc (3 × 20 L), and n-BuOH (3 × 20 L). After removing the solvent, the n-BuOH-soluble portion (175 g) was fractionated via silica gel column chromatography (CC), eluting with CHCl3–MeOH–H2O (17:7:0.5, v/v) to give twelve fractions (A1–A12). Fraction A10 (4.9 g) was separated by ODS CC (20–80%, MeOH−H2O) to give seven fractions (A10–1–A10–7). Fraction A10–3 (1.14 g) was subjected to macroporous resin CC and eluted with a EtOH–H2O gradient (0%, 20%, 40%, 60%, 80%, v/v). The fractions eluted with 40% EtOH (180 mg) were further separated by preparative HPLC (YMCODS-A 5 μM, 250 mm × 20 mm, detection at 210 nm) using 55% MeOH as mobile phase to yield 1 (4 mg), 2 (19 mg), and 3 (5 mg). 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-7(8),13(14)diene-3β,15,18-triol (1): white amorphous powder; [α]20D −124 (c 0.1, MeOH); UV (MeOH) λmax (logε): 206 (1.98) and 255 (1.70) nm; 1H NMR (600 MHz, C5D5N and 13C NMR (150 MHz, C5D5N see Table 1; positive ESIMS m/z: 669.3 [M + Na]+; negative ESIMS m/z: 645.3 [M−H]−; HRTOFMS m/z 645.3489 [M−H]− (calcd for C32H53O13, 645.3486). 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)diene-3β,15,18-triol (2): white amorphous powder; [α]20D −25 (c 0.14, MeOH); UV (MeOH) λmax (logε): 210 (2.05) and 255 (1.86) nm; 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N) see Table 1; positive ESIMS m/z: 669.3 [M + Na]+; negative ESIMS m/z: 645.3 [M−H]−; HRTOFMS m/z 645.3494 [M−H]− (calcd for C32H53O13, 645.3486). 15-O-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranosyl-18-Oβ-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)-di-ene-3β,15, 18-triol (3): white amorphous powder; [α]20D −52 (c 0.09, MeOH); UV (MeOH) λmax (logε): 207 (2.03) and 255 (1.64) nm; 1 H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N) see Table 1; positive ESIMS m/z: 801.4 [M + Na]+; negative ESIMS m/z: 777.4 [M−H]−; negative ESIMS m/z: 663.3 [M−H]−; HRTOFMS m/z 777.3955 [M−H]− (calcd for C37H61O17, 777.3909).
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
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2.2. Plant material
122 123 124 125 126 127 128 129 Q9 130 131 132 133 134 135 136 137
2.5. Cytotoxicity assay
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Compounds 1−3 were tested for cytotoxicity against HCT-8 (human colon cancer cell line), Bel-7402 (human hepatoma cancer cell line), BGC-823 (human gastric cancer cell line), A549 (human lung cancer cell line), and A2780 (human ovarian cancer cell line) by MTT assay [11].
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Based on the reported procedure [10], each (2 mg) of compounds 1−3 was dissolved in 2 M HCl (dioxane–H2O, 1:1 v/v) and refluxed for 10 h. After removal of the HCl by evaporation and extraction with EtOAc, the H2O extract was again evaporated and dried in vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine (1 ml) to which 2 mg Lcysteine methyl ester hydrochloride was added. The mixture was kept at 60 °C for 2 h, evaporated under an N2 stream, and dried in vacuo, then trimethylsilylated with Ntrimethylsilylimidazole (0.2 ml) for 2 h. The mixture was partitioned between n-hexane and H2O (2 ml each), and the n-hexane extract was analyzed by GC. In the acid hydrolysate of 1− 3, D-glucose and D-apiose were verified by comparison with retention times of their derivatives and those of corresponding control samples prepared in the same way.
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2.4. Determination of absolute configurations of the sugar moieties 120 in 1−3 121
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performed with silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Develosil ODS (50 μm, Nomura Chemical Co. Ltd., Osaka, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). TLC (thin layer chromatography) was carried out with glass precoated with silica gel GF254. Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
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3. Results and discussion
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The 70% EtOH extract of the fruits of R. chingii was fractionated with petroleum ether, EtOAc and n-BuOH. The nBuOH-soluble portion was separated by a combination of silica gel, ODS column chromatography and preparative HPLC which afforded three new compounds (1−3) (Fig. 1). Their structures were elucidated by extensive NMR techniques mainly including 1D NMR (1H, 13C NMR), 2D NMR (COSY, NOESY, HSQC and HMBC) (Fig. 2) and ESIMS. Compound 1 was obtained as a white amorphous powder. The molecular formula, C32H54O13, was determined by HRTOFMS at m/z 645.3489 [M−H]−, in agreement with the NMR spectroscopic data. The 1H NMR spectrum of 1 in C5D5N showed four methyl singlets at δH 0.81 (3H, s), 1.04 (3H, s), 1.65 (3H, s), and 1.69 (3H, s), four oxymethylene protons at δH 3.62 and 4.31 (1H each, d, J = 10.2 Hz), δH 4.45 and 4.73 (1H each, d, J = 6.6 Hz), two olefinic protons at δH 5.38 (1H, br s) and 5.63 (1H, t, J = 6.6 Hz), and two anomeric protons at δH 4.89 (1H, d, J = 7.8 Hz) and 4.98 (1H, d, J = 7.8 Hz) correlated in the HSQC spectrum with two anomeric carbons at δC 106.0 and 104.4 in the 13C NMR spectrum, respectively (Table 1). The 13C NMR spectrum of 1 displayed 32 carbon signals, of which 12 were assigned to the sugar moieties and the remaining 20 to the aglycone, which was very similar to those of 7,13E-labdadien3β,15-diol [12]. However, the signal of the methyl group at C18 of 7,13E-labdadien-3β,15-diol was replaced by a signal of hydroxymethylene group (δC 75.2) in 1. From the foregoing evidences, it was concluded that 1 was a glycoside of labdanetype diterpene. Acid hydrolysis of 1 with 2 M HCl afforded monosaccharides, which were identified as β-D-glucose by GC analysis of their trimethylsilyl L-cysteine derivatives [13] and by the coupling constant of the anomeric proton. In the HMBC
145 146
Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
147 148 Q10 149 Q11 150 151 152 Q12 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175
R. Zhong et al. / Fitoterapia xxx (2015) xxx–xxx Table 1 1 H and 13C NMR data of compounds 1−3 at 600/150 MHz in pyridine-d5, respectively (δ in ppm, J in Hz).
28.0 72.9 43.6 42.9 24.4
1.27 (1H, m) 1.53 (1H, m) 1.92 (1H, m) 1.94 (1H, m)
140.8 122.0 66.6
12.9 14.9
4.97 (1H, d, 7.8) 4.29 (1H, m) 4.01 (1H, m) 4.10 (1H, m) 4.24 (1H, m) 4.34 (1H, m) 4.62 (1H, m)
104.4 75.8 79.1 72.7 79.2 63.6
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R
R
106.0 75.4 79.0 72.3 79.1 63.4
127.0 140.3 39.6 27.6 40.9
5.66 (1H, t, 6.6) 4.48 (1H, dd, 11.4, 6.6) 4.75 (1H, dd, 11.4, 6.6) 1.68 (3H, s) 1.53 (3H, s) 3.63 (1H, d, 10.2) 4.50 (1H, d, 10.2) 1.06 (3H, s) 1.00 (3H, s)
17.1 22.7 75.2
4.89 (1H, d, 7.8) 4.26 (1H, m) 4.05 (1H, m) 4.09 (1H, m) 4.27 (1H, m) 4.44 (1H, m) 4.62 (1H, m)
34.2
2.03 (1H, m) 2.04 (1H, m) 1.99 (1H, m) 2.15 (1H, m)
43.2
5.63 (1H, t, 6.6) 4.45 (1H, dd, 12.0, 6.6) 4.73 (1H, dd, 12.0, 6.6) 1.65 (3H, s) 1.69 (3H, s) 3.62 (1H, m) 4.31 (1H, d, 10.2) 1.04 (3H, s) 0.81 (3H, s)
72.5 43.7 44.5 19.6
δC
1.22 (1H, m) 1.72 (1H, m) 1.89 (1H, m) 1.94 (1H, m) 4.26 (1H, m) 1.86 (1H, m) 1.72 (1H, m) 1.80 (1H, m) 1.89 (1H, m) 2.25 (1H, m)
R O O
135.4 54.9 37.1 26.4
1.48 (1H, m)
28.6
1.98 (1H, m) 1.79 (1H, m) 1.97 (1H, m) 1.92 (1H, m) 2.36 (1H, m)
123.2
δH
35.7
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2.18 (1H, m) 2.10 (1H, m) 1.95 (1H, m) 5.38 (1H, br s)
δC
1.28 (1H, m) 1.77 (1H, m) 1.96 (1H, m) 2.00 (1H, m) 4.05 (1H, m)
141.0 121.4 66.6 17.2 20.1 75.1 13.7 21.3
1.92 (1H, m) 2.08 (1H, m) 1.96 (1H, m) 1.97 (1H, m)
5.60 (1H, t, 6.6) 4.40 (1H, dd, 11.4, 6.6) 4.71 (1H, dd, 11.4, 6.6) 1.63 (3H, s) 1.44 (3H, s) 3.58 (1H, d, 10.2) 4.37 (1H, d, 10.2) 0.90 (3H, s) 0.97 (3H, s)
35.0 27.5 71.6 43.0 43.7 18.8 33.5 126.3 139.7 38.9 26.9 40.2 140.3 120.8 65.9 16.5 19.4 74.0 12.9 20.6
4.89 (d, 7.8) 4.23 (1H, m) 4.02 (1H, m) 4.05 (1H, m) 4.21 (1H, m) 4.38 (1H, dd, 11.4, 5.4) 4.61 (1H, d, 11.4)
106.1 75.5 79.0 72.3 79.2 63.4
4.76 (1H, d, 7.8) 4.15 (1H, m) 4.12 (1H, m) 3.97 (1H, m) 4.00 (1H, m) 4.18 (1H, m) 4.30 (1H, d, 10.2)
105.1 74.6 76.5 72.1 78.3 62.6
4.98 (1H, d, 7.8) 4.29 (1H, m) 4.04 (1H, m) 4.11 (1H, m) 4.25 (1H, m) 4.44 (1H, dd, 11.4, 3.6) 4.61 (1H, d, 11.4)
104.3 75.8 79.1 72.5 79.2 63.5
4.93 (1H, d, 7.8) 3.96 (1H, m) 4.74 (1H, m) 4.15 (1H, m) 3.96 (1H, m) 4.40 (1H, m) 4.53 (1H, d, 11.4)
103.6 75.1 85.9 71.8 78.5 62.7
5.67 (1H, d, 2.4) 4.73 (1H, m) 4.86 (1H, m) 4.74 (1H, m) 4.09 (1H, m)
110.0 78.3 83.0 74.6 65.9
U
Api 1‴ 2‴ 3‴ 4‴ 5‴
δH
37.7
N C
Glc 1″ 2″ 3″ 4″ 5″ 6″
1.05 (1H, m) 1.76 (1H, m) 1.89 (1H, m) 1.91 (1H, m) 4.23 (1H, m)
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δC
δH
3
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1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16 17 18a 18b 19 20 Glc 1′ 2′ 3′ 4′ 5′ 6′
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t1:4 t1:5 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 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49 t1:50 t1:51 t1:52 t1:53
1
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Proton
C
t1:3
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t1:1 t1:2 Q1
3
Fig. 1. Chemical structures of compounds 1–3.
Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
R. Zhong et al. / Fitoterapia xxx (2015) xxx–xxx
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Acknowledgments
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This study was supported financially by the National Natural Science Foundation of China (NSFC, grant no. 81460589) and Jiangxi province science and technology support program, China (no. 2013BBG0039). We thank Prof. A.H. Liu at Center of Analysis and Testing Nanchang University for NMR measurements.
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Appendix A. Supplementary data
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bone. This makes the structures of compounds 1−3 more stable. The aglycone of compounds 1−3 was isolated from Rubus plants for the first time. Compounds 1−3 may be the characteristic metabolites of R. chingii. It may provide evidence for chemical classification of R. chingii. Compounds 1−3 were evaluated for cytotoxic activities against five human cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) with paclitaxel as a positive control. Compounds 1 and 2 were inactive (IC50 N 10 μM) to HCT-8, Bel-7402, BGC823, A549, and A2780 cell lines. Compound 3 showed cytotoxic activity only against A549, with an IC50 value of 2.32 μM.
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spectrum of 1, the two oxymethylene proton signals at δH 3.62 and 4.31, which correlated with the carbon resonance at δC 75.2 in the HSQC spectrum, showed HMBC correlations with C-3, C-4, 179 C-5 and C-19, justifying its assignment to C-18. HMBC correla180 tions between the anomeric proton at δH 4.89 (1H, d, J = 7.8 Hz, 181 H-1′) and the carbon signal at δC 75.2 (C-18) and between H-1″ 182 at δH 4.97 (1H, d, J = 7.8 Hz, H-1′) and C-15 at δC 66.6 indicated 183 that two β-D-glucopyranosyl units were attached to C-15 and C184 18 positions of the aglycone, respectively. The β-orientation of 3185 OH in 1 was deduced by analysis of the NOESY spectrum which 186 showed NOE correlations between H-3 and H2-19. Thus, the 187 structure of 1 was elucidated as 15,18-di-O-β-D-glucopyranosyl188 13(E)-ent-labda-7(8),13(14)-diene-3β,15,18-triol. 189 Compound 2 gave the same molecular formula as 1, namely 190 C32H54O13, as deduced by HRTOFMS (m/z 645.3494 [M−H]− 191 calcd for 645.3486) and supported by the NMR spectroscopic 192 data. Comparison of the NMR data of 2 (Table 1) and 1 showed 193 that the two compounds were almost identical except that the 194 7(8)-olefin in 1 is isomerized as 8(9)-olefin in 2. A series 195 of HMBC correlations from H3-17 to C-7, C-8, and C-9, from 196 H3-20 to C-1, C-5, C-9, and C-10 further confirmed the 197 aglycone with a 8(9)-double bone. Thus, compound 2 was 198 determined as 15,18-di-O-β-D-glucopyranosyl-13(E)-ent199 labda-8(9),13(14)-diene-3β,15,18-triol. 200 Compound 3 had the molecular formula C37H62O17, as 201 indicated from the HRTOFMS (m/z 777.3955 [M−H]− calcd for 202 777.3909). The NMR spectroscopic data of compound 3 203 resembled those of 2, except for an additional set of β-D204 apiofuranosyl resonances with an anomeric proton signal at δH 205 5.67 (1H, d, J = 2.4 Hz) and a downfield shift of C-2 (from δC 79.1 206 to δC 85.9) of the D-glucopyranosyl unit due to a glycosidation 207 shift (Table 1). The linkage position of the sugar units with 208 the aglycone was established from the following HMBC 209 correlations: H-1′ (δH 4.76) of Glc′ with C-18 (δC 74.0) of 210 aglycone, H-1″ (δH 4.93) of Glc″ with C-15 (δC 65.9) of aglycone, 211 and H-1‴ (δH 5.67) of Api with C-3 (δC 85.9) of Glc″. Thus, 212 Q13 compound 3 was identified as 15-O-β-D-apiofuranosyl-(1 → 2)213 β-D-glucopyranosyl-18-O-β-D-glucopyranosyl-13(E)-ent-labda214 8(9),13(14)-di-ene-3β,15,18-triol. 215 Q14 Until now, only kaurane- and labdane-type terpenoids were 216 get from R. chingii, and all of labdane-type terpenoids obtained 217 were 8(17) double bone, which could undergo hydrogenation 218 and dehydrogenation to form 7(8) double bone and 8(9) double 176
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Fig. 2. Key HMBC (H → C) correlations of compounds 1–3.
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233 234 Q15 235
Supplementary data to this article can be found online at 237 http://dx.doi.org/10.1016/j.fitote.2015.01.007. 238 References
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[1] Jiangsu New Medical College. Dictionary of Chinese traditional drugs. Shanghai: Shanghai Scientific and Technical Publishers; 2006 3812. [2] Guo QL, Yang JS. China J Chin Mater Med 2005;30:198. [3] Hattori M, Kuo KP, Shu YZ, Tezuka Y, Kikuchi T, Namba T. Phytochemistry 1988;27:3975. [4] Ohtani K, Yang CR, Miyajima C, Zhou J, Tanaka O. Chem Pharm Bull 1991; 39:2443. [5] Ohtani K. Pharmacobio-Dynamics 1990;13:45. [6] Tanaka T, Kohda H, Tanaka O, Chen FH, Chou WH, Leu JL. Agric Biol Chem 1981;45:2165. [7] Xu ZW, Zhao JJ. Chin Tradit Herbal Drugs 1981;12:19. [8] Xie YH, Miao JR, Liu WQ. J Chin Med Mater 2005;28:99. [9] Chen D, Li J, Zhou B, Zheng PW. J Chin Med Mater 2012;35:1873. [10] Kinjo J, Araki K, Fukui K, Higuchi H, Ikeda T, Nohara T, et al. Chem Pharm Bull 1992;40:3269. [11] Li CL, Zhang DM, Luo YM, Yu SS, Li Y, Lu Y. Phytochemistry 2008;69:2867. [12] Pascual Teresa JD, Urones JG, Basabe P, Llanos A. An Quire, 73; 1977 1029. [13] Zhang DM, Miyase I, Kuroyanagi M, Umehara K, Ueno A. Chem Pharm Bull 1996;44:810.
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Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
Contents lists available at ScienceDirect
Fitoterapia
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journal homepage: www.elsevier.com/locate/fitote
3Q3
Ruijian Zhong a, Qing Guo a,b, Guoping Zhou a, Huizheng Fu a,⁎, Kaihua Wan c,⁎
4 5 6
a b c
R O O
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Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell lines
1Q2
Jiangxi Provincial Institute for Drug and Food Control, Nanchang 330029, China Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China Drug Adverse Reaction Monitoring Center of Jiangxi, Nanchang 330046, China
a r t i c l e
10 14 11 12 13
Article history: Received 27 November 2014 Accepted in revised form 7 January 2015 Available online xxxx
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Keywords: Rubus chingii Labdane-type Diterpene glycosides Cytotoxic activity
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1. Introduction
29 30
Rubus chingii Hu, belonging to the family Rosaceae, is distributed widely in Jiangxi, Zhejiang, Fujian, and Guangxi Provinces of mainland China. Its fruits are used as a Chinese medicine for treating enuresis with renal asthenia, frequent urination, premature ejaculation, and impotence [1]. Previous phytochemical studies on R. chingii have led to the isolation of flavonoids [2], triterpenoids [3–6], organic acids [7,8], sterols [3,7], and alkaloids [9]. In our preliminary screening, the nBuOH part of the 70% EtOH extract of the fruits of R. chingii showed significant anticancer activity. As part of program to discover biologically significant anticancer properties from plant resources has led to the isolation of three new compounds, 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-7(8),13(14)diene-3β,15,18-triol (1), 15,18-di-O-β-D-glucopyranosyl-13(E)ent-labda-8(9),13(14)-diene-3β,15,18-triol (2), and 15-O-β-D-
35 36 37 38 39 40 Q7 41 42 43
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Three new labdane-type diterpene glycosides, 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda7(8),13(14)-diene-3β,15,18-triol (1), 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-8(9), 13(14)-diene-3β,15,18-triol (2), and 15-O-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranosyl-18O-β-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)-diene-3β,15,18-triol (3), were isolated from the fruits of Rubus chingii. Their structures were elucidated on the basis of spectroscopic data and chemical methods. The cytotoxic activities of compounds 1–3 were evaluated against five human tumor cell lines (HCT-8, BGC-823, A549, and A2780). Compounds 3 showed cytotoxic activity against A549 with an IC50 value of 2.32 μM. © 2015 Published by Elsevier B.V.
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a b s t r a c t
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i n f o
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9
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7
⁎ Corresponding authors. E-mail addresses: [email protected] (H. Fu), [email protected] (K. Wan).
15 16 Q4 17 18 19 20 21 22
apiofuranosyl-(1 → 2)-β-D-glucopyranosyl-18-O-β-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)-di-ene-3β,15,18-triol (3), were isolated from the fruits of R. chingii. Reported herein are the isolation, structure elucidation and biological activity of these compounds.
44 Q8 45
2. Experimental
49
2.1. General experimental procedures
50
Optical rotations were measured on an Autopol IV-T/V (Rudolph Research Analytical, New Jersey, USA). UV spectra were recorded in MeOH on a Jasco V650 spectrophotometer (JASCO, Inc., Easton, Maryland, USA). The 1H (600 MHz), 13C (150 MHz), and 2D NMR spectra were recorded on a Bruker AVANCE III 600 instrument using TMS (Tetramethylsilane) as an internal reference (Bruker Company, Massachusetts, USA). HRTOFMS data were obtained on an Agilent 7890-7000A mass spectrometer (Agilent Technologies, Santa Clara, USA). Preparative HPLC (high performance liquid chromatography) was conducted with an Agilent Technologies 1200 series instrument with a MWD detector using a YMC-pack ODS (Octadecylsilyl)-A column (5 μm, 250 × 20 mm). Column chromatography was
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http://dx.doi.org/10.1016/j.fitote.2015.01.007 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
46 47 48
52 53 54 55 56 57 58 59 60 61 62 63
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The fruits of R. chingii were collected from Jinghua, Zhejiang, China, in June 2012 and identified by Prof. Cuisheng Fang at Jiangxi University of Traditional Chinese Medicine, China. A voucher specimen (no. 20120622) has been deposited in the Herbarium of Jiangxi Provincial Institute for Drug and Food Control.
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2.3. Extraction and isolation
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The powdered dried fruits of R. chingii (50.5 kg) were extracted three times with 70% EtOH under reflux (2 h each). The extracting solution was evaporated under reduced pressure to yield a dark brown residue (1.1 kg). The residue was suspended in water (20 L) and then successively partitioned with petroleum ether (2 × 20 L), EtOAc (3 × 20 L), and n-BuOH (3 × 20 L). After removing the solvent, the n-BuOH-soluble portion (175 g) was fractionated via silica gel column chromatography (CC), eluting with CHCl3–MeOH–H2O (17:7:0.5, v/v) to give twelve fractions (A1–A12). Fraction A10 (4.9 g) was separated by ODS CC (20–80%, MeOH−H2O) to give seven fractions (A10–1–A10–7). Fraction A10–3 (1.14 g) was subjected to macroporous resin CC and eluted with a EtOH–H2O gradient (0%, 20%, 40%, 60%, 80%, v/v). The fractions eluted with 40% EtOH (180 mg) were further separated by preparative HPLC (YMCODS-A 5 μM, 250 mm × 20 mm, detection at 210 nm) using 55% MeOH as mobile phase to yield 1 (4 mg), 2 (19 mg), and 3 (5 mg). 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-7(8),13(14)diene-3β,15,18-triol (1): white amorphous powder; [α]20D −124 (c 0.1, MeOH); UV (MeOH) λmax (logε): 206 (1.98) and 255 (1.70) nm; 1H NMR (600 MHz, C5D5N and 13C NMR (150 MHz, C5D5N see Table 1; positive ESIMS m/z: 669.3 [M + Na]+; negative ESIMS m/z: 645.3 [M−H]−; HRTOFMS m/z 645.3489 [M−H]− (calcd for C32H53O13, 645.3486). 15,18-di-O-β-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)diene-3β,15,18-triol (2): white amorphous powder; [α]20D −25 (c 0.14, MeOH); UV (MeOH) λmax (logε): 210 (2.05) and 255 (1.86) nm; 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N) see Table 1; positive ESIMS m/z: 669.3 [M + Na]+; negative ESIMS m/z: 645.3 [M−H]−; HRTOFMS m/z 645.3494 [M−H]− (calcd for C32H53O13, 645.3486). 15-O-β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranosyl-18-Oβ-D-glucopyranosyl-13(E)-ent-labda-8(9),13(14)-di-ene-3β,15, 18-triol (3): white amorphous powder; [α]20D −52 (c 0.09, MeOH); UV (MeOH) λmax (logε): 207 (2.03) and 255 (1.64) nm; 1 H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N) see Table 1; positive ESIMS m/z: 801.4 [M + Na]+; negative ESIMS m/z: 777.4 [M−H]−; negative ESIMS m/z: 663.3 [M−H]−; HRTOFMS m/z 777.3955 [M−H]− (calcd for C37H61O17, 777.3909).
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
C
89 90
E
87 88
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86
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73 74
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2.2. Plant material
122 123 124 125 126 127 128 129 Q9 130 131 132 133 134 135 136 137
2.5. Cytotoxicity assay
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Compounds 1−3 were tested for cytotoxicity against HCT-8 (human colon cancer cell line), Bel-7402 (human hepatoma cancer cell line), BGC-823 (human gastric cancer cell line), A549 (human lung cancer cell line), and A2780 (human ovarian cancer cell line) by MTT assay [11].
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Based on the reported procedure [10], each (2 mg) of compounds 1−3 was dissolved in 2 M HCl (dioxane–H2O, 1:1 v/v) and refluxed for 10 h. After removal of the HCl by evaporation and extraction with EtOAc, the H2O extract was again evaporated and dried in vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine (1 ml) to which 2 mg Lcysteine methyl ester hydrochloride was added. The mixture was kept at 60 °C for 2 h, evaporated under an N2 stream, and dried in vacuo, then trimethylsilylated with Ntrimethylsilylimidazole (0.2 ml) for 2 h. The mixture was partitioned between n-hexane and H2O (2 ml each), and the n-hexane extract was analyzed by GC. In the acid hydrolysate of 1− 3, D-glucose and D-apiose were verified by comparison with retention times of their derivatives and those of corresponding control samples prepared in the same way.
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2.4. Determination of absolute configurations of the sugar moieties 120 in 1−3 121
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performed with silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Develosil ODS (50 μm, Nomura Chemical Co. Ltd., Osaka, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). TLC (thin layer chromatography) was carried out with glass precoated with silica gel GF254. Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
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R. Zhong et al. / Fitoterapia xxx (2015) xxx–xxx
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3. Results and discussion
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The 70% EtOH extract of the fruits of R. chingii was fractionated with petroleum ether, EtOAc and n-BuOH. The nBuOH-soluble portion was separated by a combination of silica gel, ODS column chromatography and preparative HPLC which afforded three new compounds (1−3) (Fig. 1). Their structures were elucidated by extensive NMR techniques mainly including 1D NMR (1H, 13C NMR), 2D NMR (COSY, NOESY, HSQC and HMBC) (Fig. 2) and ESIMS. Compound 1 was obtained as a white amorphous powder. The molecular formula, C32H54O13, was determined by HRTOFMS at m/z 645.3489 [M−H]−, in agreement with the NMR spectroscopic data. The 1H NMR spectrum of 1 in C5D5N showed four methyl singlets at δH 0.81 (3H, s), 1.04 (3H, s), 1.65 (3H, s), and 1.69 (3H, s), four oxymethylene protons at δH 3.62 and 4.31 (1H each, d, J = 10.2 Hz), δH 4.45 and 4.73 (1H each, d, J = 6.6 Hz), two olefinic protons at δH 5.38 (1H, br s) and 5.63 (1H, t, J = 6.6 Hz), and two anomeric protons at δH 4.89 (1H, d, J = 7.8 Hz) and 4.98 (1H, d, J = 7.8 Hz) correlated in the HSQC spectrum with two anomeric carbons at δC 106.0 and 104.4 in the 13C NMR spectrum, respectively (Table 1). The 13C NMR spectrum of 1 displayed 32 carbon signals, of which 12 were assigned to the sugar moieties and the remaining 20 to the aglycone, which was very similar to those of 7,13E-labdadien3β,15-diol [12]. However, the signal of the methyl group at C18 of 7,13E-labdadien-3β,15-diol was replaced by a signal of hydroxymethylene group (δC 75.2) in 1. From the foregoing evidences, it was concluded that 1 was a glycoside of labdanetype diterpene. Acid hydrolysis of 1 with 2 M HCl afforded monosaccharides, which were identified as β-D-glucose by GC analysis of their trimethylsilyl L-cysteine derivatives [13] and by the coupling constant of the anomeric proton. In the HMBC
145 146
Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
147 148 Q10 149 Q11 150 151 152 Q12 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175
R. Zhong et al. / Fitoterapia xxx (2015) xxx–xxx Table 1 1 H and 13C NMR data of compounds 1−3 at 600/150 MHz in pyridine-d5, respectively (δ in ppm, J in Hz).
28.0 72.9 43.6 42.9 24.4
1.27 (1H, m) 1.53 (1H, m) 1.92 (1H, m) 1.94 (1H, m)
140.8 122.0 66.6
12.9 14.9
4.97 (1H, d, 7.8) 4.29 (1H, m) 4.01 (1H, m) 4.10 (1H, m) 4.24 (1H, m) 4.34 (1H, m) 4.62 (1H, m)
104.4 75.8 79.1 72.7 79.2 63.6
O
R
R
106.0 75.4 79.0 72.3 79.1 63.4
127.0 140.3 39.6 27.6 40.9
5.66 (1H, t, 6.6) 4.48 (1H, dd, 11.4, 6.6) 4.75 (1H, dd, 11.4, 6.6) 1.68 (3H, s) 1.53 (3H, s) 3.63 (1H, d, 10.2) 4.50 (1H, d, 10.2) 1.06 (3H, s) 1.00 (3H, s)
17.1 22.7 75.2
4.89 (1H, d, 7.8) 4.26 (1H, m) 4.05 (1H, m) 4.09 (1H, m) 4.27 (1H, m) 4.44 (1H, m) 4.62 (1H, m)
34.2
2.03 (1H, m) 2.04 (1H, m) 1.99 (1H, m) 2.15 (1H, m)
43.2
5.63 (1H, t, 6.6) 4.45 (1H, dd, 12.0, 6.6) 4.73 (1H, dd, 12.0, 6.6) 1.65 (3H, s) 1.69 (3H, s) 3.62 (1H, m) 4.31 (1H, d, 10.2) 1.04 (3H, s) 0.81 (3H, s)
72.5 43.7 44.5 19.6
δC
1.22 (1H, m) 1.72 (1H, m) 1.89 (1H, m) 1.94 (1H, m) 4.26 (1H, m) 1.86 (1H, m) 1.72 (1H, m) 1.80 (1H, m) 1.89 (1H, m) 2.25 (1H, m)
R O O
135.4 54.9 37.1 26.4
1.48 (1H, m)
28.6
1.98 (1H, m) 1.79 (1H, m) 1.97 (1H, m) 1.92 (1H, m) 2.36 (1H, m)
123.2
δH
35.7
P
2.18 (1H, m) 2.10 (1H, m) 1.95 (1H, m) 5.38 (1H, br s)
δC
1.28 (1H, m) 1.77 (1H, m) 1.96 (1H, m) 2.00 (1H, m) 4.05 (1H, m)
141.0 121.4 66.6 17.2 20.1 75.1 13.7 21.3
1.92 (1H, m) 2.08 (1H, m) 1.96 (1H, m) 1.97 (1H, m)
5.60 (1H, t, 6.6) 4.40 (1H, dd, 11.4, 6.6) 4.71 (1H, dd, 11.4, 6.6) 1.63 (3H, s) 1.44 (3H, s) 3.58 (1H, d, 10.2) 4.37 (1H, d, 10.2) 0.90 (3H, s) 0.97 (3H, s)
35.0 27.5 71.6 43.0 43.7 18.8 33.5 126.3 139.7 38.9 26.9 40.2 140.3 120.8 65.9 16.5 19.4 74.0 12.9 20.6
4.89 (d, 7.8) 4.23 (1H, m) 4.02 (1H, m) 4.05 (1H, m) 4.21 (1H, m) 4.38 (1H, dd, 11.4, 5.4) 4.61 (1H, d, 11.4)
106.1 75.5 79.0 72.3 79.2 63.4
4.76 (1H, d, 7.8) 4.15 (1H, m) 4.12 (1H, m) 3.97 (1H, m) 4.00 (1H, m) 4.18 (1H, m) 4.30 (1H, d, 10.2)
105.1 74.6 76.5 72.1 78.3 62.6
4.98 (1H, d, 7.8) 4.29 (1H, m) 4.04 (1H, m) 4.11 (1H, m) 4.25 (1H, m) 4.44 (1H, dd, 11.4, 3.6) 4.61 (1H, d, 11.4)
104.3 75.8 79.1 72.5 79.2 63.5
4.93 (1H, d, 7.8) 3.96 (1H, m) 4.74 (1H, m) 4.15 (1H, m) 3.96 (1H, m) 4.40 (1H, m) 4.53 (1H, d, 11.4)
103.6 75.1 85.9 71.8 78.5 62.7
5.67 (1H, d, 2.4) 4.73 (1H, m) 4.86 (1H, m) 4.74 (1H, m) 4.09 (1H, m)
110.0 78.3 83.0 74.6 65.9
U
Api 1‴ 2‴ 3‴ 4‴ 5‴
δH
37.7
N C
Glc 1″ 2″ 3″ 4″ 5″ 6″
1.05 (1H, m) 1.76 (1H, m) 1.89 (1H, m) 1.91 (1H, m) 4.23 (1H, m)
F
δC
δH
3
D
1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16 17 18a 18b 19 20 Glc 1′ 2′ 3′ 4′ 5′ 6′
2
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t1:4 t1:5 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 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49 t1:50 t1:51 t1:52 t1:53
1
T
Proton
C
t1:3
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t1:1 t1:2 Q1
3
Fig. 1. Chemical structures of compounds 1–3.
Please cite this article as: Zhong R, et al, Three new labdane-type diterpene glycosides from fruits of Rubus chingii and their cytotoxic activities against five humor cell li..., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.007
R. Zhong et al. / Fitoterapia xxx (2015) xxx–xxx
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Acknowledgments
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This study was supported financially by the National Natural Science Foundation of China (NSFC, grant no. 81460589) and Jiangxi province science and technology support program, China (no. 2013BBG0039). We thank Prof. A.H. Liu at Center of Analysis and Testing Nanchang University for NMR measurements.
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Appendix A. Supplementary data
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bone. This makes the structures of compounds 1−3 more stable. The aglycone of compounds 1−3 was isolated from Rubus plants for the first time. Compounds 1−3 may be the characteristic metabolites of R. chingii. It may provide evidence for chemical classification of R. chingii. Compounds 1−3 were evaluated for cytotoxic activities against five human cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) with paclitaxel as a positive control. Compounds 1 and 2 were inactive (IC50 N 10 μM) to HCT-8, Bel-7402, BGC823, A549, and A2780 cell lines. Compound 3 showed cytotoxic activity only against A549, with an IC50 value of 2.32 μM.
U
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177 178
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spectrum of 1, the two oxymethylene proton signals at δH 3.62 and 4.31, which correlated with the carbon resonance at δC 75.2 in the HSQC spectrum, showed HMBC correlations with C-3, C-4, 179 C-5 and C-19, justifying its assignment to C-18. HMBC correla180 tions between the anomeric proton at δH 4.89 (1H, d, J = 7.8 Hz, 181 H-1′) and the carbon signal at δC 75.2 (C-18) and between H-1″ 182 at δH 4.97 (1H, d, J = 7.8 Hz, H-1′) and C-15 at δC 66.6 indicated 183 that two β-D-glucopyranosyl units were attached to C-15 and C184 18 positions of the aglycone, respectively. The β-orientation of 3185 OH in 1 was deduced by analysis of the NOESY spectrum which 186 showed NOE correlations between H-3 and H2-19. Thus, the 187 structure of 1 was elucidated as 15,18-di-O-β-D-glucopyranosyl188 13(E)-ent-labda-7(8),13(14)-diene-3β,15,18-triol. 189 Compound 2 gave the same molecular formula as 1, namely 190 C32H54O13, as deduced by HRTOFMS (m/z 645.3494 [M−H]− 191 calcd for 645.3486) and supported by the NMR spectroscopic 192 data. Comparison of the NMR data of 2 (Table 1) and 1 showed 193 that the two compounds were almost identical except that the 194 7(8)-olefin in 1 is isomerized as 8(9)-olefin in 2. A series 195 of HMBC correlations from H3-17 to C-7, C-8, and C-9, from 196 H3-20 to C-1, C-5, C-9, and C-10 further confirmed the 197 aglycone with a 8(9)-double bone. Thus, compound 2 was 198 determined as 15,18-di-O-β-D-glucopyranosyl-13(E)-ent199 labda-8(9),13(14)-diene-3β,15,18-triol. 200 Compound 3 had the molecular formula C37H62O17, as 201 indicated from the HRTOFMS (m/z 777.3955 [M−H]− calcd for 202 777.3909). The NMR spectroscopic data of compound 3 203 resembled those of 2, except for an additional set of β-D204 apiofuranosyl resonances with an anomeric proton signal at δH 205 5.67 (1H, d, J = 2.4 Hz) and a downfield shift of C-2 (from δC 79.1 206 to δC 85.9) of the D-glucopyranosyl unit due to a glycosidation 207 shift (Table 1). The linkage position of the sugar units with 208 the aglycone was established from the following HMBC 209 correlations: H-1′ (δH 4.76) of Glc′ with C-18 (δC 74.0) of 210 aglycone, H-1″ (δH 4.93) of Glc″ with C-15 (δC 65.9) of aglycone, 211 and H-1‴ (δH 5.67) of Api with C-3 (δC 85.9) of Glc″. Thus, 212 Q13 compound 3 was identified as 15-O-β-D-apiofuranosyl-(1 → 2)213 β-D-glucopyranosyl-18-O-β-D-glucopyranosyl-13(E)-ent-labda214 8(9),13(14)-di-ene-3β,15,18-triol. 215 Q14 Until now, only kaurane- and labdane-type terpenoids were 216 get from R. chingii, and all of labdane-type terpenoids obtained 217 were 8(17) double bone, which could undergo hydrogenation 218 and dehydrogenation to form 7(8) double bone and 8(9) double 176
O
Fig. 2. Key HMBC (H → C) correlations of compounds 1–3.
220 221 222 223 224 225 226 227 228 229
233 234 Q15 235
Supplementary data to this article can be found online at 237 http://dx.doi.org/10.1016/j.fitote.2015.01.007. 238 References
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