Apigenin 7-O-B-glucoside from the leaves of Acanthus integrifolius T. Anders., Acanthaceae

Journal of Chemistry, Vol. 42 (4), P. 496 - 498, 2004<br /> <br /> <br /> Apigenin 7-O- -glucoside from the leaves of Acanthus<br /> integrifolius T. Anders., Acanthaceae<br /> Received 18th-Sept.-2003<br /> Phan Minh Giang, Phan Tong Son<br /> Faculty of Chemistry, College of Natural Science, Vietnam National University, Hanoi<br /> <br /> <br /> Summary<br /> The first investigation on chemical constituents of Acanthus integrifolius resulted in the<br /> isolation of apigenin-7-O- -glucoside from the leaves of this plant. Its chemical structure was<br /> determined by ESIMS, 1H NMR, 13C NMR, DEPT, HMBC and HMQC.<br /> <br /> Acanthus integrifolius T. Anders., 3'<br /> Acanthaceae (local name: ¾c ã) is a shrub OH<br /> HOCH2 2' 4'<br /> growing to the height of 1 - 2 m and possessing HO O 8 1'<br /> O O 5'<br /> white flowers [1]. The leaves of A. integrifolius HO 7<br /> 9<br /> 2 6'<br /> (Folium Acanthi) are used in the treatment of OH 3<br /> 6 10<br /> pain and rheumatism [2]. Our first phyto- 5 4<br /> chemical investigation on A. integrifolius was OH O<br /> prompted by the need to identify the<br /> compounds responsible for anti-inflammatory Figure 1: Chemical structure of apigenin<br /> therapeutic effects of this plant. 7-O- -glucoside (1)<br /> Successive liquid-liquid fractionation of the<br /> nucleus protons; the 13C NMR chemical shifts<br /> aqueous MeOH extract of the leaves of A.<br /> of this moiety were consistent with the<br /> integrifolius by solvents with increasing<br /> identification of 1 as a derivative of apigenin<br /> polarity gave n-hexane-, CH2Cl2-, ethyl acetate-<br /> and n-BuOH-soluble fractions. The n-hexane [3]. The second group, appearing between 3.1<br /> and 5.06, comprised the resonances of a<br /> soluble fraction contained mainly phytosterols<br /> glucosidic moiety. The anomeric proton<br /> as indicated in a TLC analysis and was not<br /> further investigated. Two-time column resonance observed at 5.06 (1H, d, J = 7 Hz)<br /> chromatography of the ethyl acetate soluble indicated the -glucose. The proton signals at<br /> fraction on lipophilic Sephadex LH-20 gave 12.96 (1H, s) (downfield-shifted due to the<br /> hydrogen bonding with 4-oxo group) and 10.41<br /> apigenin-7-O- -glucoside (1) with 95% purity.<br /> (1H, s) were indicative for the hydroxyl groups<br /> We reported herein the structure elucidation of<br /> located at C-5 and C-4’. The molecular formula<br /> this flavone glucoside. C21H20O10 (from ESIMS quasimolecular ion<br /> The 1H-NMR spectrum of 1 in DMSO-d6 peaks at m/z 433.4 ([M+H]+), 455.4 ([M+Na]+)<br /> contained two distinctive groups of resonances. and 431.4 ([M-H]+)) provided the identity of 1<br /> Those at 7.96 (2H) and 6.95 (2H) (both as d, J as apigenin 7-O- -glucoside, the 1H and 13C<br /> = 9 Hz), 6.87 (1H, s), 6.84 (1H) and 6.45 (1H) NMR resonance signals of which are in good<br /> (both as d, J = 2.2 Hz), represented the flavone accordance with those reported for the apigenin<br /> 496<br /> 7-O-diglycosides [4]. This structure was firmly radical) and cumene hydroperoxide (peroxyl<br /> confirmed by the correlation between the sugar radical) [6]. The in vitro superoxide anion<br /> proton Glc-1 ( 5.06) and C-7 ( 163.0) in the radical and peroxyl radical scavenging<br /> HMBC spectrum (Fig. 1). The HPLC-UV properties of apigenin 7-glucoside may<br /> spectrum of apigenin 7-O- -glucoside taken on contribute to its anti-inflammatory effect. There<br /> line was shown in the Fig. 2. is accumulating evidence that natural<br /> antioxidants inhibit the expression of inducible<br /> Peak A2 12.78<br /> 60 nitric oxide synthase (iNOS), cyclooxygenase-2<br /> (COX-2) and their major regulator at the<br /> 50<br /> transcription level - the transcription factor NF-<br /> 40 B (nuclear factor kappa B) [7-10]. Taken<br /> together, the presence of apigenin 7-O- -<br /> 30 glucoside may, even in part, contribute to the<br /> 20<br /> traditional application of A. integrifolius as an<br /> anti-inflammatory herbal medication against<br /> 10 pain and rheumatism.<br /> 0<br /> Experimental Section<br /> -10<br /> 100 300 400 500 600 700 800 1. General Experimental Procedures.<br /> Figure 2: HPLC-UV of apigenin 7-O- - Melting point was measured on an<br /> glucoside (1) Electrothermal model 9100 and is uncorrected.<br /> [HPLC analytical condition: YMC HPLC column NMR spectra were obtained on a Varian Unity<br /> J’sphere ODS-H80, 150×4.6 mm I.D., S-4 µm, 8 nm; NMR spectrometer (300 MHz for 1H NMR and<br /> gradient 20% MeOH in water–100% MeOH (25 75 MHz for 13C NMR). ESIMS was measured<br /> min), flow rate: 1 ml/min, injection volume: 10 µl] on a Finigan Navigator mass spectrometer in<br /> Recently, apignein 7-glucoside was DMSO as a solvent. Analytical high-<br /> demonstrated to inhibit the stimulus-induced performance liquid chromatography was<br /> superoxide generation and phosphorylation of performed on a Dionex HPLC system (Dionex<br /> tyrosine residues of protein in human Co., USA). Samples at a concentration of 10<br /> neutrophils. When the cells were preincubated mg/ml were injected onto analytical column<br /> with apigenin 7-glucoside, the superoxide (YMC ODS-H80 (150 × 4.6 mm I.D., S-4 µm)<br /> generation induced by N-formyl-methionyl- (YMC Co., Japan) using an AS1-100 Automated<br /> leucyl-phenylalanine, by arachidonic acid and Sample Injector in a dose of 10 µl and detected<br /> by phorbol 12-myristate 13-acetate was signifi- with a PDA-100 Photodiode Array Detector.<br /> cantly suppressed in a concentration-dependent 2. Plant material<br /> manner. In the presence of apigenin 7-gluco-<br /> side, N-formyl-methionyl-leucyl-phenylalanine The fresh leaves of A. integrifolius were<br /> -induced tyrosyl phosphorylation of 45-kDa collected in Can Gio, Ho Chi Minh city in April<br /> proteins of the cells was suppressed in parallel 2002 and identified by a botanical taxonomist<br /> to the suppression of N-formyl-methionyl- Dr. Vo Van Chi.<br /> leucyl-phenylalanine-induced superoxide 3. Extraction and Isolation<br /> generation [5]. Fuchs J. and Milbradt R. also<br /> reported that subsequent intradermal appli- The fresh leaves of A. integrifolius were air-<br /> cation of liposomal apigenin 7-glucoside dried in shadow and then further processed at<br /> inhibited in a dose dependent manner skin 40oC in a temperature-controled heating oven.<br /> inflammation in rats induced by intradermal The dried leaves (812 g) were powdered and<br /> injection xanthine-oxidase (superoxide anion extracted with MeOH by percolation at room<br /> <br /> 497<br /> temperature (three times, each for 2 days). The 60.6 (t, Glc-6).<br /> combined MeOH extract was evaporated under<br /> reduced pressure and the obtained residue was Acknowledgements: This research was<br /> suspended in distilled water and partitioned supported by the International Foundation for<br /> with n-hexane, CH2Cl2, EtOAc and n-butanol, Science, Stockholm, Sweden, through a grant to<br /> successively. Evaporation under reduced Phan Minh Giang and the National Basic<br /> pressure to dryness yielded the following Research Program in Natural Sciences. We<br /> soluble fractions: n-hexane (12.7 g), CH2Cl2 thank Dr. Vo Van Chi for the collection and<br /> (2.2 g), EtOAc (2.5 g) and n-butanol (20.5 g). botanical identification of plant material. We<br /> The residual water extract was evaporated under thank Dr. Jung Joon Lee, Korea Research<br /> reduced presssure to yield 49.5 g of a dark- Institute of Bioscience and Biotechnology,<br /> brown water soluble fraction. Repeated Daejeon, Korea, for the help in recording the<br /> chromatography of the ethyl acetate soluble ESIMS and NMR spectra.<br /> fraction (2.5 g) on Sephadex LH-20 (2 times)<br /> eluted with MeOH gave apigenin-7-O- - References<br /> glucoside (1) (20 mg) with 95 % purity (on the<br /> basis of a HPLC analysis). 1. Pham Hoang Ho. Cay co Vietnam, An<br /> Apigenin-7-O- -glucoside (1) Illustrated Flora of Vietnam, Tome III, p.<br /> 60, Publishing House Youth, Ho Chi Minh<br /> Yellow powder, mp 204 - 205oC. city (2000).<br /> HPLC Rt: 12.86 min (analytical condition: 2. Vo Van Chi. Dictionary of Vietnamese<br /> gradient 20 % - 100 % MeOH in H2O, run time Medicinal Plants, P. 35, Publishing House<br /> 25 min, flow rate 1 ml/min). Medicine (1997).<br /> UV (MeOH) max nm (log ): 224.6 (4.1), 3. T. Kaneko, M. Sakamoto, K. Ohtani, A. Ito,<br /> 230.8 (4.1), 253.4 (3.9), 343 (4.1). R. Kasai, K. Yamasaki and W. G. Padorina.<br /> Phytochemistry, 39, P. 115 - 120 (1995).<br /> ESIMS: positive-ion m/z 433.4 ([M+H]+),<br /> 455.4 ([M+Na]+); negative-ion m/z 431.4 ([M- 4. N. C. Veitch, R. J. Grayer, J. L. Irwin and<br /> H]+). K. Takeda. Phytochemistry, 48, P. 389 -<br /> 1<br /> 393 (1998).<br /> H-NMR (300 MHz, DMSO-d6): 12.96 (1H,<br /> 5. J. Fuchs, R. Milbradt. Arzneimittel-<br /> s, 5-OH), 10.41 (1H, s, 4’-OH), 7.96 (2H, d, J =<br /> forschung, 43, P. 370 - 372 (1993).<br /> 9 Hz, H-2’, H-6’), 6.94 (2H, d, J = 9 Hz, H-3’,<br /> H-5’), 6.87 (1H, s, H-3), 6.84 (1H, d, J = 2.2 6. J. Lu, X. Feng, Q. Sun, H. Lu, M. Manabe,<br /> Hz, H-8), 6.45 (1H, d, J = 2.2 Hz), 5.41 (1H, d, K. Sugahara, D. Ma, Y. Sagara, H. Kodama.<br /> J = 3.6 Hz, OH), 5.14 (1H, d, J = 3 Hz, OH), Clin Chim Acta., 316, P. 95 - 99 (2002).<br /> 5.06 (1H, d, J = 7 Hz, Glc-1), 5.06 (1H, m, 7. Y. -J. Surh. Food and Chemical Toxicology,<br /> OH), 4.62 (1H, m, OH), 3.71 (1H, dd, J = 4.8 40, P. 1091 - 1097 (2002).<br /> Hz, 9.6 Hz, Glc-6b), 3.1-3.52 (5H, m, Glc-2, 8. H. K. Kim, B. S. Cheon, Y. H. Kim, S. Y.<br /> Glc-3, Glc-4, Glc-5, Glc-6a). Kim and H. P. Kim. Biochem Pharmacol,<br /> 13<br /> C-NMR (75 MHz, DMSO-d6): 182.0 (s, C- 58, P. 759 - 765 (1999).<br /> 4), 164.3 (s, C-2), 163.0 (s, C-7), 161.4 (s, C- 9. Y. -C. Liang, Y. -T. Huang, S. -H. Tsai, S. -<br /> 4’), 161.1 (s, C-5), 157.0 (s, C-9), 128.6 (2d, C- Y. Lin-Shiau, C. -F. Chen and J. -K. Lin.<br /> 2’, C-6’), 121.0 (s, C-1’), 116.0 (2d, C-3’, C-5’), Carcinogenesis, 20, P. 1945 - 1952 (1999).<br /> 105.4 (s, C-10), 103.1 (d, C-3), 99.9 (d, Glc-1), 10. A. -H. Lo, Y. -C. Liang, S. -Y. Lin-Shiau,<br /> 99.5 (d, C-6), 94.9 (d, C-8), 77.2 (d, Glc-5), C. -T. Ho and J. -K. Lin. Carcinogenesis,<br /> 76.4 (d, Glc-3), 73.1 (d, Glc-2), 69.0 (d, Glc-4), 23, P. 983 - 991 (2002).<br /> <br /> <br /> <br /> 498<br />