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Journal of Chemistry, Vol. 42 (4), P. 496 - 498, 2004 Apigenin 7-O- -glucoside from the leaves of Acanthus integrifolius T. Anders., Acanthaceae Received 18th-Sept.-2003 Phan Minh Giang, Phan Tong Son Faculty of Chemistry, College of Natural Science, Vietnam National University, Hanoi Summary The first investigation on chemical constituents of Acanthus integrifolius resulted in the isolation of apigenin-7-O- -glucoside from the leaves of this plant. Its chemical structure was determined by ESIMS, 1H NMR, 13C NMR, DEPT, HMBC and HMQC. Acanthus integrifolius T. Anders., Acanthaceae (local name: ¾c ã) is a shrub growing to the height of 1 - 2 m and possessing white flowers [1]. The leaves of A. integrifolius (Folium Acanthi) are used in the treatment of pain and rheumatism [2]. Our first phytochemical investigation on A. integrifolius was prompted by the need to identify the compounds responsible for anti-inflammatory therapeutic effects of this plant. Successive liquid-liquid fractionation of the aqueous MeOH extract of the leaves of A. integrifolius by solvents with increasing polarity gave n-hexane-, CH2Cl2-, ethyl acetateand n-BuOH-soluble fractions. The n-hexane soluble fraction contained mainly phytosterols as indicated in a TLC analysis and was not further investigated. Two-time column chromatography of the ethyl acetate soluble fraction on lipophilic Sephadex LH-20 gave apigenin-7-O- -glucoside (1) with 95% purity. We reported herein the structure elucidation of this flavone glucoside. The 1H-NMR spectrum of 1 in DMSO-d6 contained two distinctive groups of resonances. Those at 7.96 (2H) and 6.95 (2H) (both as d, J = 9 Hz), 6.87 (1H, s), 6.84 (1H) and 6.45 (1H) (both as d, J = 2.2 Hz), represented the flavone 496 3' HOCH2 HO HO 2' O OH 8 O 9 1' O 2 7 6 5 OH 10 OH 4' 5' 6' 3 4 O Figure 1: Chemical structure of apigenin 7-O- -glucoside (1) nucleus protons; the 13C NMR chemical shifts of this moiety were consistent with the identification of 1 as a derivative of apigenin [3]. The second group, appearing between 3.1 and 5.06, comprised the resonances of a glucosidic moiety. The anomeric proton resonance observed at 5.06 (1H, d, J = 7 Hz) indicated the -glucose. The proton signals at 12.96 (1H, s) (downfield-shifted due to the hydrogen bonding with 4-oxo group) and 10.41 (1H, s) were indicative for the hydroxyl groups located at C-5 and C-4’. The molecular formula C21H20O10 (from ESIMS quasimolecular ion peaks at m/z 433.4 ([M+H]+), 455.4 ([M+Na]+) and 431.4 ([M-H]+)) provided the identity of 1 as apigenin 7-O- -glucoside, the 1H and 13C NMR resonance signals of which are in good accordance with those reported for the apigenin 7-O-diglycosides [4]. This structure was firmly confirmed by the correlation between the sugar proton Glc-1 ( 5.06) and C-7 ( 163.0) in the HMBC spectrum (Fig. 1). The HPLC-UV spectrum of apigenin 7-O- -glucoside taken on line was shown in the Fig. 2. Peak A2 12.78 60 50 40 30 20 10 0 radical) and cumene hydroperoxide (peroxyl radical) [6]. The in vitro superoxide anion radical and peroxyl radical scavenging properties of apigenin 7-glucoside may contribute to its anti-inflammatory effect. There is accumulating evidence that natural antioxidants inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and their major regulator at the transcription level - the transcription factor NFB (nuclear factor kappa B) [7-10]. Taken together, the presence of apigenin 7-O- glucoside may, even in part, contribute to the traditional application of A. integrifolius as an anti-inflammatory herbal medication against pain and rheumatism. Experimental Section -10 100 300 400 500 600 700 800 Figure 2: HPLC-UV of apigenin 7-O- glucoside (1) [HPLC analytical condition: YMC HPLC column J’sphere ODS-H80, 150×4.6 mm I.D., S-4 µm, 8 nm; gradient 20% MeOH in water–100% MeOH (25 min), flow rate: 1 ml/min, injection volume: 10 µl] Recently, apignein 7-glucoside was demonstrated to inhibit the stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils. When the cells were preincubated with apigenin 7-glucoside, the superoxide generation induced by N-formyl-methionylleucyl-phenylalanine, by arachidonic acid and by phorbol 12-myristate 13-acetate was significantly suppressed in a concentration-dependent manner. In the presence of apigenin 7-glucoside, N-formyl-methionyl-leucyl-phenylalanine -induced tyrosyl phosphorylation of 45-kDa proteins of the cells was suppressed in parallel to the suppression of N-formyl-methionylleucyl-phenylalanine-induced superoxide generation [5]. Fuchs J. and Milbradt R. also reported that subsequent intradermal application of liposomal apigenin 7-glucoside inhibited in a dose dependent manner skin inflammation in rats induced by intradermal injection xanthine-oxidase (superoxide anion 1. General Experimental Procedures. Melting point was measured on an Electrothermal model 9100 and is uncorrected. NMR spectra were obtained on a Varian Unity NMR spectrometer (300 MHz for 1H NMR and 75 MHz for 13C NMR). ESIMS was measured on a Finigan Navigator mass spectrometer in DMSO as a solvent. Analytical highperformance liquid chromatography was performed on a Dionex HPLC system (Dionex Co., USA). Samples at a concentration of 10 mg/ml were injected onto analytical column (YMC ODS-H80 (150 × 4.6 mm I.D., S-4 µm) (YMC Co., Japan) using an AS1-100 Automated Sample Injector in a dose of 10 µl and detected with a PDA-100 Photodiode Array Detector. 2. Plant material The fresh leaves of A. integrifolius were collected in Can Gio, Ho Chi Minh city in April 2002 and identified by a botanical taxonomist Dr. Vo Van Chi. 3. Extraction and Isolation The fresh leaves of A. integrifolius were airdried in shadow and then further processed at 40oC in a temperature-controled heating oven. The dried leaves (812 g) were powdered and extracted with MeOH by percolation at room 497 temperature (three times, each for 2 days). The combined MeOH extract was evaporated under reduced pressure and the obtained residue was suspended in distilled water and partitioned with n-hexane, CH2Cl2, EtOAc and n-butanol, successively. Evaporation under reduced pressure to dryness yielded the following soluble fractions: n-hexane (12.7 g), CH2Cl2 (2.2 g), EtOAc (2.5 g) and n-butanol (20.5 g). The residual water extract was evaporated under reduced presssure to yield 49.5 g of a darkbrown water soluble fraction. Repeated chromatography of the ethyl acetate soluble fraction (2.5 g) on Sephadex LH-20 (2 times) eluted with MeOH gave apigenin-7-O- glucoside (1) (20 mg) with 95 % purity (on the basis of a HPLC analysis). Apigenin-7-O- -glucoside (1) Yellow powder, mp 204 - 205oC. HPLC Rt: 12.86 min (analytical condition: gradient 20 % - 100 % MeOH in H2O, run time 25 min, flow rate 1 ml/min). UV (MeOH) max nm (log ): 224.6 (4.1), 230.8 (4.1), 253.4 (3.9), 343 (4.1). ESIMS: positive-ion m/z 433.4 ([M+H]+), 455.4 ([M+Na]+); negative-ion m/z 431.4 ([MH]+). 1 H-NMR (300 MHz, DMSO-d6): 12.96 (1H, s, 5-OH), 10.41 (1H, s, 4’-OH), 7.96 (2H, d, J = 9 Hz, H-2’, H-6’), 6.94 (2H, d, J = 9 Hz, H-3’, H-5’), 6.87 (1H, s, H-3), 6.84 (1H, d, J = 2.2 Hz, H-8), 6.45 (1H, d, J = 2.2 Hz), 5.41 (1H, d, J = 3.6 Hz, OH), 5.14 (1H, d, J = 3 Hz, OH), 5.06 (1H, d, J = 7 Hz, Glc-1), 5.06 (1H, m, OH), 4.62 (1H, m, OH), 3.71 (1H, dd, J = 4.8 Hz, 9.6 Hz, Glc-6b), 3.1-3.52 (5H, m, Glc-2, Glc-3, Glc-4, Glc-5, Glc-6a). 13 C-NMR (75 MHz, DMSO-d6): 182.0 (s, C4), 164.3 (s, C-2), 163.0 (s, C-7), 161.4 (s, C4’), 161.1 (s, C-5), 157.0 (s, C-9), 128.6 (2d, C2’, C-6’), 121.0 (s, C-1’), 116.0 (2d, C-3’, C-5’), 105.4 (s, C-10), 103.1 (d, C-3), 99.9 (d, Glc-1), 99.5 (d, C-6), 94.9 (d, C-8), 77.2 (d, Glc-5), 76.4 (d, Glc-3), 73.1 (d, Glc-2), 69.0 (d, Glc-4), 498 60.6 (t, Glc-6). Acknowledgements: This research was supported by the International Foundation for Science, Stockholm, Sweden, through a grant to Phan Minh Giang and the National Basic Research Program in Natural Sciences. We thank Dr. Vo Van Chi for the collection and botanical identification of plant material. We thank Dr. Jung Joon Lee, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, for the help in recording the ESIMS and NMR spectra. References 1. Pham Hoang Ho. Cay co Vietnam, An Illustrated Flora of Vietnam, Tome III, p. 60, Publishing House Youth, Ho Chi Minh city (2000). 2. Vo Van Chi. Dictionary of Vietnamese Medicinal Plants, P. 35, Publishing House Medicine (1997). 3. T. Kaneko, M. Sakamoto, K. Ohtani, A. Ito, R. Kasai, K. Yamasaki and W. G. Padorina. Phytochemistry, 39, P. 115 - 120 (1995). 4. N. C. Veitch, R. J. Grayer, J. L. Irwin and K. Takeda. Phytochemistry, 48, P. 389 393 (1998). 5. J. Fuchs, R. Milbradt. Arzneimittelforschung, 43, P. 370 - 372 (1993). 6. 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