Microsoft Word - Chem040209.doc SQU Journal For Science, 9 (2004) 19-23 © 2004 Sultan Qaboos University 19 Isolation of Aristolochic Acids from Aristolochia Bracteolata and Studies of their Antioxidant Activities Saleh Al-Busafi, Munir Al-Harthi and Bushra Al-Sabahi Department of Chemistry,College of Science, Sultan Qaboos University, P. O. Box 36, Al-Khod 123, Muscat, Sultanate of Oman, Email: Saleh1@squ.edu.om. ودراسـة انشـطتها المضـادة أريستولوكيا براكتيوالتا إستخالص أحماض االريستولوكيك من نبات لألكسدة صالح البوصافي ، منير الحارثي و بشرى الصباحي أريسـتولوكيا براكتيوالتـا لتركيب ألجزيئ ألحما ض أألريستولوكيك من نبـات ا إستخالص وكشف : خالصة إلـى Mo(VI) لمركبات الطبيعيه قد تم تقيمها بناء علـى قـدرتها إلختـزال األنشطه ألمضاده لألكسده لهذه ا .Mo(V) أ -د أكثر نشاطا من فيتامين ج بينما حمض أريسـتولوكيك -لدراسه أظهرت أن حمض أريستولوكيك ا .مماثل لنشاط فيتامين ج ABSTRACT: The isolation and structural elucidation of aristolochic acid-A and aristolochic acid-D from Omani Aristolochia bracteolata plant is reported. Antioxidant activities of these two natural products were evaluated for their capacity to reduce Mo(VI) to Mo(V). The study revealed that aristolochic acid-D is more active than vitamin C while aristolochic acid-A has activity similar to vitamin C. KEYWORDS: Aristolochia bracteolata; aristolochic acid-A; aristolochic acid-D; Antioxidant activities and Omani plants. 1. Introduction ristolochia bracteolata is a climbing perennial plant with cordate leaves and dark-purple tubular flowers with unpleasant smell (Ghazanfar, 1991). Traditionally, the plant has been used to treat wounds and for snakebites. The genus Aristolochia is a source of aristolochic acid, which has been evaluated in China for the treatment of wounds and infectious diseases; it was found to be useful for promoting wound healing in ulcers, burns, and scalds (Jiangsu, 1977). Recently, aristolochic acids, present in Aristolochia plants, have been shown to be nephrotoxic in rats (Matsuo, et al., 2003). A family of aristolochic acids have been isolated from Aristolochia argentina (Priestap, 1987), and from Aristolochia indica (Kupchan et al., 1968). Aristolactam alkaloids and amides were isolated from Aristolochia A SALEH AL-BUSAFI, MUNIR AL-HARTHI and BUSHRA AL-SABAHI 20 kankauensis (Tian-Shung Wu et al., 1994) and from Aristolochia auricularia (Houghton et al., 1990). Aristolochic acid-A was isolated from the root of Aristolochia bracteata from Sudan (Mohamed et al., 1999). Magnoflorine and aristolochic acid-A were isolated from A. bracteata from Saudi Arabia (El Tahir et al., 1991). Both compounds were found to induce contractions in the isolated pregnant rat uterus and stimulated the isolated guinea pig ileum. N-acetylnornuciferine and aristololactam were isolated from A. bracteata from India (Chakravarty, et al., 1988). Aristolochic acid-A was characterized as the principal tumer-inhibitory principle (Kupchan et al., 1968). In recent years, the antioxidative action of natural products received much attention, due to their possible role in disease prevention (Aruoma, 1998). In the course of continuing search for natural products with antioxidant properties from Omani plants we report herein the isolation and elucidation of two aristolochic acids (1, 2) from n-butanol extract of A. bracteolata leafs and stems and their antioxidant activities relative to vitamin C. The antioxidant activities were tested using the reduction of phosphomolybdenum complex system (Prieto, et al, 1999). Aristolochic acid-A (1) Aristolochic acid-D (2) O O OH O NO2 OMe H H H H H 12 4 5 6 7 8 9 10 3 O O OH O NO2 OMe H HO H H H 12 4 5 6 7 8 9 10 3 2. Results and Discussion 2.1 Isolation and Structural Elucidation of Aristolochic Acids 1 and 2 Isolation of the two compounds was carried out by a standard column chromatography with silica gel (0.13-0.25 mm, 60-120 mesh). The principal product was aristolochic acid-A (1), which was eluted with a mixture of chloroform-methanol (95% : 5%). Elution with increasing polar mixtures of chloroform- methanol (88% : 12%) yielded aristolochic acid-D (2). Aristolochic acid-A (1) separated from methanol as dark-red solid with melting point of 260-265 ˚C. The mass spectrum ([M]+at m/z 341) establishes the molecular formula as C17H11NO7 with fragment ions at 327 [M-CH2] +, 281 [M-CH2-NO2] +, 253 [M-CH2-NO2-CO] +, 237 [M-CH2-NO2-CO2] +, 207 [M-CH2-NO2- CO2-OCH2] +. Ultraviolet (UV) absorptions at 218, 227, 255, and 312 nm suggest a highly conjugated system. The infrared (IR) spectrum showed the presence of hydroxyl group (strong broad signal at 2850 – 3579 cm-1), carbonyl group (sharp signal at 1714 cm-1), nitro group (signals at 1345 cm-1 symmetric stretch and at 1517,1594 cm-1 asymmetric stretch). The 1H nuclear magnetic resonance (1H NMR) spectrum of 1 showed signals for an aromatic methoxyl group at δ 4.04 (3H, s) and for methylenedioxy group at δ 6.47 (2H, s). Three mutually coupled aromatic protons appeared at δ 7.32 (1H, d, J=8.1Hz), 7.79 (1H, t, J=8.2 Hz), δ8.66 (1H, d, J=8.4 Hz); the signals were assigned to H-7, H-6 and H-5 respectively as revealed by 1H- 1H correlation spectroscopy (1H-1H COSY) and compared to literature values (Tian-Shung Wu et al., 1994). Two singlets at δ 7.75 and 8.47 (each 1H) were assigned to H-2 and H-9 respectively. The position of 8- OMe was deduced from nuclear Overhauser effect (NOESY spectrum) which showed H-7 (δ 7.32) to be within NOE distance from the 8-OMe. ISOLATION OF ARISTOLOCHIC ACIDS 21 Table 1. NMR spectral data of Aristolochic acids 1 and 2. Aristolochic acid-A (1) Aristolochic acid-D (2) Position 1H data (ppm) 13C data (ppm) 1H data (ppm) 13C data (ppm) 1 119.4 118.0 2 7.76, s 112.4 7.69, s 112.6 3 146.34 145.7 4 147.06 145.7 4’ 117.6 117.0 5 8.62, d, J 8.4 119.2 8.06, d, J 2.4 104.1 5’ 130.4 132.1 6 7.79,t, J 8.2 132.0 158.4 7 7.32, d, J 8.1 109.5 6.84, d, J 2.4 100.0 8 156.68 161.5 8’ 115.8 112.3 9 8.47, s 119.3 8.36, s 112.6 10 146.34 145.7 10’ 117.1 117.0 C=O 168.7 168.9 -OCH2O- 6.43, s 103.0 6.40, s 102.7 CH3O- 4.04, s 56.7 3.98, s 56.6 Aristolochic acid-D (2) was obtained as brown solid with melting point range of 280-288 ºC. The structure of 2 was inferred from its mass spectrum ([M-2]+ at m/z 355) which establishes the molecular formula as C17H11NO8 with fragment ions at 327 [M-OCH2] +, 281 [M-OCH2-NO2] +, 277 [M-2OH-NO2], 207 [M-CH2-NO2-CO2-OCH2O] +. UV absorptions at 225, 245, 289, and 364 nm show a close resemblance to that of 1. The IR spectrum showed the presence of hydroxyl group (strong broad signal at 2720 – 3691 cm-1), carbonyl group (at 1702 cm-1), nitro group (signals at 1517 cm-1). The aromatic region of the 1H NMR spectrum of 2 contained two doublets at δ 6.84 (1H, d, J=2.4 Hz) and 8.06 (1H, d, J=2.4 Hz) attributed to H-7 and H-5 respectively. The small J value suggests a meta coupling system. The low frequency signal of H-7 at δ 6.84 is due to the shielding effect of the ortho hydroxyl and methoxyl groups. Two singlets at δ 7.69 and 8.36 (each 1H) were assigned to the C-2 and C-9 protons. The 1H NMR spectrum showed the presence of methoxyl group at δ 3.98 (3H, s) and methylenedioxy group at δ 6.40 (2H, s). The position of 8-OMe was deduced from a NOESY spectrum which showed H-7 (δ 7.32) to be within NOE distance from the 8-OMe. The NMR spectral data of aristolochic acid-A (1) and aristolochic acid-D (2) are summarized in Table 1. 2.2 Antioxidant Activities of Aristolochic Acids 1 and 2 The phosphomolybdenum method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compound and the formation of a green phosphate/Mo(V) complex with a maximal absorption at 695 nm. The assay was successfully used to quantify vitamin E in seeds (Prieto et al., 1999) and measuring antioxidant activity of the sage polyphenols (Yinrong et al, 2001). The method is simple and independent of other antioxidant measurements commonly employed. Our value for vitamin C (A695 = 1.625) at a concentration of 5 mM, was well comparable with the reported molar absorption coefficient of vitamin C [ε = (3.4 ± 0.1) x 103 M-1 cm-1] (Prieto et al., 1999). The results in Table 2 show that the antioxidant activity of aristolochic acid-A (1) is almost equal to that of vitamin C. On the other hand, aristolochic acid-D (2) has better antioxidant activity than vitamin C. SALEH AL-BUSAFI, MUNIR AL-HARTHI and BUSHRA AL-SABAHI 22 This high antioxidant activity of aristolochic acid-D (2) is presumably due to the presence of the hydroxyl group at C-6. Table 2. Antioxidant activities of Aristolochic acids 1 and 2 relative to vitamin C. Compound Am a Aristolochic acid-A (1) 1.03 Aristolochic acid-D (2) 1.20 Vitamin C 1 a Am , activity relative to vitamin C on a molar basis. 3. Experimental 3.1 General The UV spectra were obtained with a Varian model Cary 50 conc spectrophotometer. The IR spectra were obtained as KBr discs with a Nicolet model Magna 560 spectrometer; absorption bands are recorded in wave number (cm-1). The 1H NMR and 13C NMR spectra were recorded at 400 MHz using Bruker spectrometer in DMSO-d6. Chemical shifts are expressed as δ values (ppm) downfield from TMS. Thin-layer chromatography (TLC) was carried out on Merck F254 silica gel plates (0.2 mm thickness) with mobile phase CHCl3 – MeOH, (80 : 20) and spots were detected under UV 254 nm. Melting points were recorded using Gallen Kamp apparatus. Electron impact mass spectroscopy (EIMS) was performed on a Shimadzu GCMS QP5050 spectrometer. 3.2 Plant Material Aristolochia bracteolata was collected from Mazara, wilayat of Quraiyat, Sultanate of Oman in 2001 and was authenticated by specialists in the Department of Biology, Sultan Qaboos University. 3.3 Extraction and Isolation 450 g of shade dried powdered leafs and stems of A. bracteolata was repeatedly extracted with methanol at room temperature. The combined extract was concentrated under reduced pressure to get 45 g of greenish viscous material which was first partitioned between hexane/H2O, then EtOAc/H2O and finally n-Butanol/H2O to give a hexane extract, an ethyl acetate extract and n-Butanol extract. The concentrated n- Butanol extract (23 g) was chromatographed over a silica gel column and eluted with chloroform then with chloroform-methanol mixture. Aristolochic acid-A (1) (40 mg) was isolated from chloroform-methanol mixture (95% : 5%) fractions. Aristolochic acid-D (2) (20 mg) was isolated from chloroform-methanol mixture (88% : 12%). 3.4 Evaluation of Antioxidant Activity The antioxidant activity of aristolochic acids was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al. (1999). An aliquot of 0.1 ml sample solution (5 mM in MeOH) was combined in a 4-ml vial with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials were capped and incubated in a water bath at 95 ºC for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank. The antioxidant activity was expressed relative to that of ascorbic acid. 4. Acknowledgements This research was supported by Sultan Qaboos University grant number IG/SCI/CHEM/03/01. The authors are grateful to Salem Al-Saidi, Department of Chemistry, Sultan Qaboos University for EI-Ms ISOLATION OF ARISTOLOCHIC ACIDS 23 spectra, and to Wafa Al-Shuaily, Department of Chemistry, Sultan Qaboos University for the NMR spectra. The authers wish to thank Dr. Hisham Kader for helpful discussions. 5. References ARUOMA, O.I. 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. J. Am.Oil Chemists’ Soc., 75: 199-212. CHAKRAVARRY, M.; CHAUDHURI, C.; ACHARI, B. and PAKRASHI, S.C. 1988. Studies on Indian medicinal plants. Part 90. N-acetylnornuciferine and other constituents of Aristolochia bracteata. Planta Med., 54(5): 467-468. El TAHIR, K.E. 1991. Pharmacological actions of magnoflorine and aristolochic acid-1 isolated from the seeds of Aristolochia bracteata. Int. J. Pharmacogn, 29(2): 101-110. 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