Flavonoid glycosides from Viscum album - Vu Kim Thu

Vietnam Journal of Chemistry, International Edition, 54(4): 443-447, 2016 DOI: 10.15625/0866-7144.2016-00344 443 Flavonoid glycosides from Viscum album Vu Kim Thu 1 , Nguyen Thi Kim Thoa 1 , Phan Van Kiem 2* 1 Faculty of Basic Science, Hanoi University of Mining and Geology (HUMG) 2 Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST) Received 17 March 2016; Accepted for publication 12 August 2016 Abstract Using combined chromatographic methods, four flavonoid glycosides, (2S)-homoeriodictyol-7-O-β-D- apiofuranosyl-(1→2)-O-β-D-glucopyranoside (1), (2S)-5-hydroxy-7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl- (1→2)-O-β-D-glucopyranoside (2), homoflavoyadorinin-B (3), and 3′-methoxyapiin (4) were isolated from the methanol extract of the leaves and twigs of Viscum album. Their structures were elucidated by 1D- and 2D-NMR spectra and in comparison with those reported in the literature. Keywords. Viscum album, flavonoid glycoside. 1. INTRODUCTION Viscum album L. var. meridianum Dans. is a hemiparasitic shrub. It has been used as a remedy in traditional oriental medicine to treat swell spleen, wound, tumour and sore ears [1]. V. album exerts several biological effects such as antitumor, anticancer [2, 3], and anti-inflammatory activities [4]. It is well established that the extract of V. album inhibited tumour angiogenesis and metastasis of haematogenous and non-haematogenous tumour cells in mice [5]. Chemical investigation of V. album proved the presence of flavonoids [6], lignans, phenylpropanoids [7], and triterpenes [8]. This paper reported the isolation and structure elucidation of four flavonoid glycosides from the methanol extract of the leaves and twigs of V. album. 2. MATERIAL AND METHODS 2.1. Plant materials The leaves and twigs of V. album were collected in Cucphuong, Ninhbinh, Vietnam in October, 2012 and identified by Prof. Dr. Ninh Khac Ban, Institute of Marine Biochemistry, VAST. A voucher specimen (TG1012) was deposited at the Herbarium of the Institute of Marine Biochemistry. 2.2. General experimental procedures All NMR spectra were on a Bruker AM400 FT- NMR spectrometer (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR), and chemical shifts (δ) are reported in ppm using TMS as an internal standard. Column chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) and RP-18 resins. Thin layer chromatography was performed on DC Alufolien Kieselgel 60 F254 (Merck) or RP-18 F254s (Merck) plates. Compounds were visualized by spraying with aqueous 10 % H2SO4 and heating for 5 minutes. 2.3. Extraction and isolation The dried leaves and twigs of V. album (2.5 kg) were extracted with hot MeOH three times (3 × 5 L) under reflux for 12 h to yield 320 g extract after evaporation of the solvent. This extract was suspended in H2O and successively partitioned with CHCl3 and EtOAc to yield the CHCl3 (VA1, 45.0 g), EtOAc (VA2, 9.0 g), and H2O (VA3, 260.0 g) extracts after removal of the solvents in vacuo. The VA2 fraction was chromatographed on a silica gel column eluting with a gradient of CHCl3-MeOH (10:1→2:1, v/v) to give four fractions, VA2A- VA2D. The VA2C fraction was chromatographed on a Sephadex LH-20 column and eluting with MeOH to give compounds 3 (20.0 mg) and 4 (4.1 mg). The VA3 fraction (260 g) was chromatographed on a Diaion HP-20P column eluting with H2O containing increasing concentrations of MeOH (0, 25, 50, 75, and 100 %, v/v) to yield five sub-fractions, VA3A (120.0 g), VA3B (12.0 g), VA3C (14.0 g), VA3D (20.0 g), and VA3E (15.0 g). The VA3D fraction was chromatographed on a silica gel column eluting with gradient of CHCl3-MeOH (10:1→2:1, v/v) to VJC, 54(4) 2016 Phan Van Kiem, et al. 444 give five fractions, VA3D1-VA3D5. The VA3D2 fraction was chromatographed on a RP-18 Figure 1: The chemical structures of compounds 1-4 column eluting with MeOH-H2O (1:1, v/v) to yield 2 (9.0 mg). The VA3D3 fraction was chromatographed on a RP-18 column eluting with MeOH - H2O (1:1, v/v) to yield compound 1 (180.0 mg). (2S)-Homoeriodictyol-7-O-β-D-apiofuranosyl- (1→2)-O-β-D-glucopyranoside (1): yellowish powder; mp 143-146 ºC; 25][ D = -46.0 (c, 0.1, CH3OH); C27H32O15, ESI-MS m/z 597 [M+H] + ; 1 H- and 13 C-NMR data, see table 1. (2S)-5-Hydroxy-7,3′-dimethoxyflavanone-4′-O-β- D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside (2): yellowish powder; mp 219-221 o C; 25][ D = -85.0 (c, 0.1, MeOH); C28H34O15, ESI-MS m/z 611 [M+H] + ; 1 H- and 13 C-NMR data, see table 1. Homoflavoyadorinin-B (3): yellowish powder; mp 217-220 ºC; 25][ D = -13.0 (c 0.1, CH3OH); C28H32O15, ESI-MS m/z 609 [M+H] + ; 1 H- and 13 C- NMR data, see table 1. 3′-Methoxyapiin (4): yellowish powder; mp 200- 202 ºC; 25][ D = -38.0 (c 0.1, CH3OH); C27H30O15, ESI-MS m/z 595 [M+H] + ; 1 H- and 13 C-NMR data, see table 1. 3. RESULTS AND DISCUSSION Compound 1 (figure 1) was yielded as a yellowish powder. The 1 H-NMR of 1 showed the signals of one oxymethine proton at H 5.33 (1H, dd, J = 2.0, 12.5 Hz), three protons of ABX aromatic system at H 7.05 (1H, s), 6.81 (1H, d, J = 8.0 Hz), and 6.90 (1H, d, J = 8.0 Hz), suggested the presence of a flavanone moiety; two anomeric protons at δH 5.00 (1H, d, J = 7.6 Hz) and 5.42 (1H, d, J = 2.0 Hz), confirmed two sugar moieties, and one methoxy group at H 3.86 (3H, s). The 13 C-NMR and DEPT spectra displayed the signals of 27 carbons, including one methoxy, four methylene, thirteen methine, and nine quaternary carbons. Of which, 16 carbons were assigned to a flavanone with a methoxy group and 11 carbons to two sugar units. The HMBC correlations between methoxy protons (δH 3.86) and C-3 (δC 149.04) indicated the methoxy group was located at C-3. The HMBC correlations between glc H-1′′ (δH 5.00) and C-7 (δC 166.70), api H-1′′′ (δH 5.42) and glc C-2′′ (δC 78.53), and between glc H-2′′ (δH 3.61) and api C-1′′′ (δC 110.83) indicated the linkage of sugar moiety as O-β-D- apiofuranosyl (1→2)-O-β-D-glucopyranoside and this sugar linkage was connected to C-7 of flavanone (Figure 2). All NMR assignments of 1 were confirmed by detailed analyses of HSQC and HMBC spectra (table 1), which are in good agreement with those reported in the literature [6]. Thus compound 1 was identified as (2S)- homoeriodictyol-7-O-β-D-apiofuranosyl-(1→2)-O- β-D-glucopyranoside. The NMR spectra of 2 were similar to those of 1 with the presence of a flavanone skeleton, two sugar moieties but two methoxy groups. The HMBC correlations between methoxy group at δH 3.79 (3H, s) and C-7 (δC 167.41) confirmed the location of the methoxy group at C-7 (figure 1). The observed HMBC correlations between glc H-1′′ (δH 4.98) and C-4′ (δC 146.51) indicated the position of sugar moiety at C-4′ of the flavanone. The sugar moities were also confirmed by the good agreement of 13 C- NMR chemical shifts for sugar moieties previously reported in some flavonoid glycosides from V. album [6, 9]. Based on the evidence above and in comparison with those reported in the literature [10], compound 2 was determined to be (2S)-5-hydroxy- 7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl- (1→2)-O-β-D-glucopyranoside. Table 1: The 1 H- and 13 C-NMR data for compounds 1-4 Pos. 1 2 3 4 δC # δC a δH a (J in Hz) δC $ δC b δH b (J in Hz) δC ¥ δC b δH b (J in Hz) δC a δH a (J in Hz) Aglycon 2 78.8 80.86 5.33 (dd, 2.0, 12.5) 78.5 78.57 5.53 (d, 12.5) 163.4 163.46 - 166.71 - 3 42.2 44.29 2.70 (m) 3.14 (m) 42.0 42.04 2.78 (d, 12.5) 3.36 (m) 104.2 104.29 7.03 (s) 104.50 6.59 (s) 4 197.1 198.51 - 196.6 196.72 - 181.9 182.09 - 184.07 - 5 162.8 164.83 - 163.1 163.12 - 161.1 161.17 - 162.91 - 6 95.2 96.81 6.16 (s) 94.6 94.68 6.15 (s) 98.0 98.09 6.35 (d, 2.4) 101.06 6.37 (s) 7 164.9 166.70 - 167.4 167.41 - 165.2 165.24 - 164.68 - 8 96.4 97.86 6.13 (s) 93.7 93.81 6.09 (s) 92.7 92.83 6.81 (d, 2.4) 96.10 6.73 (s) 9 162.7 164.49 - 162.7 162.72 - 157.2 157.32 - 158.99 10 103.2 104.84 - 102.5 102.55 - 104.7 104.80 - 107.10 1′ 129.1 131.44 - 131.9 131.95 - 123.9 123.96 - 123.52 2′ 111.2 111.40 7.05 (s) 111.2 111.14 7.16 (s) 110.1 110.00 7.61 (d, 2.4) 110.91 7.42 (s) 3′ 147.5 149.04 - 148.8 148.77 - 149.6 149.09 - 149.55 - 4′ 147.0 148.14 - 146.5 146.51 - 149.0 149.65 - 152.33 - 5′ 115.1 116.13 6.81 (d, 8.0) 114.9 114.89 7.07 (d, 8.0) 114.8 114.79 7.21 (d, 8.4) 116.82 6.85 (d, 8.0) 6′ 119.7 120.70 6.90 (d, 8.0) 119.0 119.05 7.00 (d, 8.0) 119.7 119.81 7.66 (dd, 2.4, 8.4) 122.00 7.46 (d, 8.0) 7-OMe 55.7 55.67 3.79 (s) 55.9 59.99 3.86 (s) 3′-OMe 55.6 56.48 3.86 (s) 55.8 55.86 3.78 (s) 56.0 56.11 3.89 (s) 56.74 3.87 (s) 4′ or 7-O-Glc 1′′ 97.7 99.67 5.00 (d, 7.6) 98.4 98.34 4.98 (d, 7.5) 98.0 97.95 5.14 (d, 8.4) 100.27 5.06 (d, 7.5) 2′′ 76.8 78.53 3.61* 77.1 77.18 3.67 (m) 77.0 77.06 3.60* 78.77 3.67* 3′′ 75.7 78.30 3.60* 75.0 74.91 3.60* 74.8 74.75 3.45* 78.47 3.62* 4′′ 69.7 71.05 3.40* 69.9 69.97 3.41* 69.9 69.94 3.20 (m) 71.32 3.40 (t, 8.5) 5′′ 76.6 78.12 3.41* 76.8 76.83 3.46* 77.1 77.23 3.51 (m) 78.35 3.53* 6′′ 60.4 62.22 3.68 (m) 3.85 (m) 60.6 60.60 3.55 (m) 3.67 (m) 60.5 60.60 3.46* 3.71 (dd, 5.6, 10.0) 62.46 3.69 (dd, 5.0, 12.0) 3.92 (d, 12.0) 2′′-O-Apio 1′′′ 108.6 110.83 5.42 (d, 2.0) 108.2 108.28 5. 42 (br s) 108.3 108.36 5.44 (s) 110.91 5.35 (s) 2′′′ 76.0 78.03 3.95 (d, 2.0) 76.0 76.04 3.79 (s) 76.0 76.04 3.78 (s) 78.15 3.95 (s) 3′′′ 79.1 80.70 - 79.9 79.29 - 79.3 79.44 - 80.69 - 4′′′ 73.8 75.35 3.77 (d, 9.6) 3.96 (d, 9.6) 73.8 73.90 3.59 (d, 9.5) 4.06 (d, 9.5) 73.0 74.02 3.62 (d, 9.6) 4.07 (d, 9.6) 75.50 3.81 (d, 10.0) 4.03 (d, 10.0) 5′′′ 64.1 65.88 3.51 (s) 64.4 64.45 3.30 (s) 64.4 64.53 3.29 (s) 65.89 3.53 (s) arecorded in CD3OD, bDMSO-d6, *overlapped signals, #δC of (2S)-homoeriodictyol-7-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside [6], $δC of (2S)-5-hydroxy-7,3′-dimethoxyflavanone-4′- O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside [10], ¥δC of homoflavoyadorinin-B [6]. V JC , 5 4 (4 ) 2 0 1 6 F la vo n o id g lyco sid es fro m V iscu m a lb u m . 4 4 5 VJC, 54(4) 2016 Phan Van Kiem, et al. 446 1 H-NMR spectrum of 3 showed characteristic flavone proton signals with three protons at δH 7.03 (1H, s, H-3), 6.35 (1H, d, J = 2.4 Hz, H-6), 6.81 (1H, d, J = 2.4 Hz, H-8), and three protons of ABX aromatic system at δH 7.61 (1H, d, J = 2.4 Hz, H-2′), 7.21 (1H, d, J = 8.4 Hz, H-5′), and 7.66 (1H, dd, J = 2.4, 8.4 Hz, H-6′), two anomeric protons of sugar units at 5.14 (1H, d, J = 8.4 Hz) and 5.44 (1H, s), and two methoxy groups at H 3.86 and 3.89 (each 3H). The 13 C-NMR data showed the presence of a D-glucopyranose moiety and a D-apiofuranose moiety with the chemical shifts of anomeric carbons at δC 97.95 (C-1′′) and 108.36 (C-1′′′). The coupling constant of H-1′′ and H-2′′ (J = 8.4 Hz) indicated the  configuration for the glucopyranose moiety. The HMBC correlations from methoxy groups at H 3.86 and H 3.89 to C-7 (165.24), C-3′ (149.09), respectively, proved the locations of two methoxy groups at C-7 and C-3′. Moreover, the HMBC correlations between glc H-1′′ (δH 5.14) and C-4′ (δC 149.65); api H-1′′′ (δH 5.44) and glc C-2′′ (δC 77.06); between glc H-2′′ (δH 3.60) and api C-1′′′ (δC 108.36) indicated the sugar moiety of 3 to be [O-β-D- apiofuranosyl (1→2)-O-β-D-glucopyranoside] and its location at C-4′. All NMR assignments of 3 were confirmed by detailed analyses of HSQC and HMBC spectra, which are in good agreement with those reported in the literature [6]. Thus compound 3 was identified as homoflavoyadorinin-B. Compound 4 was yielded as yellowish powder and its molecular formula was determined as C27H30O15 by the ESI-MS at m/z 595 [M+H] + and 13 C-NMR data. Analysis the NMR spectra of 4 indicated that the structure of 4 was very similar to those of 3 except for the position of sugar linkage and methoxy group. 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Corresponding author: Phan Van Kiem Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam E-mail: phankiem@yahoo.com.