Vol. 33, No. 1, 2011 114 J.Chem.Soc.Pak., Vol. 33, No. 1,J.Chem.Soc.Pak., 2011 Urease Inhibitors and Antioxidants from Vernonia cinerascens 1,2 IRSHAD AHMAD*, 2BASHIR AHMAD CHAUDHARY, 2KHALID HUSSAIN JANBAZ, 2 MUHAMMAD UZAIR AND 1MUHAMMAD ASHRAF 1 Faculty of Pharmacy & Alternative Medicine, The Islamia University of Bahawalpur, Pakistan. 2 Faculty of Pharmacy, Bahauddin Zakariya University Multan, Pakistan. (Received on 1st February 2010, accepted in revised form 29th June 2010) Summary: Eight compounds have been isolated for the first time from Vernonia cinerascens namely, 2-hydroxy-3-methoxy-5-(2-propenyl)-phenol (1), vanillic acid (2), isoferulic acid (3), caffeic acid (4), methyl gallate (5), uridine (6), 3'-methylquercetin (7) and quercetin (8), respectively. Their structures have been elucidated based on EI-MS, HREI-MS, FAB-MS, HRFABMS, 1H-, 13C-NMR spectroscopic data and in comparison with the reported data. All these compounds exhibited promising antioxidant and significant urease inhibitory activities. Introduction The genus Vernonia belongs to family “Asteraceae” which consists of about 1300 genera and 25,000 species distributed all over the world and in almost all habitats [1]. The genus is enriched with pharmacological properties. V. amygdalina showed antimicrobial [2], immunomodulating [3], lipids lowering [4], antidiabetic [5], antioxidant and hepatoprotective [6] activities. V. cinerea displayed antimalarial [7], immunomodulating, anticancer [8], anti-arthritis [9], analgesic, anti-pyretic and antiinflammatory [10] activities. Various species of this genus are used for the treatment of schistosomiasis, amoebic dysentery, gastrointestinal problems, malaria, venereal diseases, wounds, hepatitis, diabetes, colic, fever, stomachache, toothache, cough, nasal and bronchial pain along with anti-ulcer [11] and anti-leishmanial [12] activities. Literature survey revealed that only a sesquiterpene is reported from V. cinerascens [13]. The diverse medicinal applications of genus Vernonia has prompted us to carry out the phytochemical investigations on this species. Herein we report the isolation and structure elucidation of 2hydroxy-3-methoxy-5-(2-propenyl)-phenol (1), vanillic acid (2), isoferulic acid (3), caffeic acid (4), methyl gallate (5), uridine (6), 3'-methylquercetin (7) and quercetin (8), respectively, on the basis of their respective spectral data (Fig. 1). All these compounds are reported for the first time from this plant and exhibited promising antioxidant and significant urease inhibitory activities. Results and Discussion The methanolic extract of the aerial parts of V. cinerascens were subjected to a series of column and flash chromatographic techniques as described in the experimental to obtain eight compounds (1-8) reported for the first time from this source. All these * To whom all correspondence should be addressed. compounds showed promising antioxidant and significant urease inhibitory activities with IC50 values ranging between 51.4-600.9 M for antioxidant and 37.4-56.6 M for urease inhibition (Table-1). Table-1: Antioxidant and urease inhibitory activities of compounds 1-8. All compounds were dissolved in methanol at given concentrations and experiments were carried out in triplicate. Antiurease activity (% DPPH scavenging activity (IC50 µg/ mL) inhibition) at given conc. 1 85.05 ± 4.6 48.4 ±1.2 (236µg/mL) 2 210.8 ± 4.7 37.4 ± 1.5 (236µg/mL) 3 298.2 ± 8.2 51.2 ± 1.1 (59µg/mL) 4 600.9 ± 6.7 49.5 ± 1.2 (59µg/mL) 5 108.4 ± 1.9 38.7 ± 1.3 (236µg/mL) 6 439.2 ± 8.7 44.6 ± 0.9 (118µg/mL) 7 186.5 ± 7.2 56.6 ± 2.2 (236µg/mL) 51.45 ± 1.1 47.5 ± 2.3 (236µg/mL) Quercetina ca. 98% (55 µg/mL) Thioureab  a Standard inhibitor for DPPH scavenging activity b Standard inhibitor for urease inhibition Compounds Experimental General Column chromatography was carried out using silica gel of 70-230 and 230-400 mesh. Aluminium sheets precoated with silica gel 60 F254 (2020 cm, 0.2 mm thick; E-Merck) were used for TLC to check the purity of the compounds and were visualized under UV light (254 and 366 nm) followed by ceric sulfate as spraying reagent. Optical rotations were measured on a Jasco DIP-360 digital polarimeter. The UV spectra were recorded on a Hitachi UV-3200 spectrometer (max in nm). IR spectra were recorded on Shimadzu IR-460 spectrophotometer ( in cm−1). EI-MS, HREI-MS, J.Chem.Soc.Pak., Vol. 33, No. 1, 2011 115 IRSHAD AHMAD et al., FAB-MS and HRFAB-MS spectra were recorded on Jeol JMS-HX 110 spectrometer with data system. The 1H-NMR spectra were recorded on Bruker AMX-400 and 500 MHz instruments using TMS as an internal reference. The chemical shift values are reported in ppm () units and the scalar coupling constants (J) are in Hz. The 13C-NMR spectra were recorded on Bruker AMX-100 and 125 MHz instruments. For DPPH scavenging and urease inhibition assays, all chemicals used were purchased from Sigma (St. Louis, MO). OH CH2 8 7 1 9 COOH OH 6 2 1 2 6 5 7 3 OCH3 4 5 3 OH 2 O 7COOCH3 6 9 7 5 RO OCH3 4 1 1 8 OH 1 2 4 2 6 3 HO OH 3 5 4 OH OH 3 R = CH3 4R=H 5 O 5' 4 NH 5 6 2 1 N O 4' 5' 2' 3' HO 8 8a 7 O 1' 1 2 4a 5 OH 3 4 OH 4' 3' 2' OR OH O OH 6 Fig. 1: O 6 1' HO HO 6' 3 7 R = CH3 8R=H Structure of Compounds isolated from Vernonia cinerascens. Plant Material The aerial parts of V. cinerascens Sch. Bip. (2 kg) were collected from Peruwal (District Khaniwal) in May 2006 and identified by Prof. Dr. Altaf Ahmad Dasti, Plant Taxonomist, Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan, Pakistan, where a voucher specimen (VC09/IPAB/06) is deposited. Isolation The shade dried, ground aerial parts of V. cinerascens (2 kg) were extracted with MeOH (3×6L). The combined MeOH extract was concentrated, and the residue (20 g) was subjected to column chromatography over silica gel eluting with n-hexane, n-hexane-ethylacetate, ethylacetate, ethylacetate-methanol along with water in increasing order of polarity. The fraction obtained by n-hexaneethylacetate (5.5:4.5), gave two spots on TLC was subjected to Sephadex LH-20 eluted with methanol and finally purified by preparative TLC using solvent system n-hexane-ethylacetate (3.5:6.5) gave 2hydroxy-3-methoxy-5-(2-propenyl)-phenol (1) (26 mg) and vanillic acid (2) (3 mg), respectively. The fraction obtained by n-hexane-ethylacetate (4.5:5.5), gave two spots on TLC which was subjected to Sephadex LH-20 eluted with methanol and final purification by PTLC using n-hexane-EtOAc (3:7) to provide isoferulic acid (3) (18 mg) and caffeic acid (4) (16 mg), respectively. The fraction obtained by nhexane-ethylacetate (3.5:7.5) gave two spots on TLC again subjected to Sephadex LH-20 eluted with methanol and final purification by PTLC using ethylacetate-methanol (9.8:0.8) provided methyl gallate (5) (4 mg) and uridine (6) (11 mg), respectively. The fraction obtained by ethylacetatemethanol (9.8:0.2) gave two spots on TLC subjected to Sephadex LH-20 eluted with methanol and finally purified by PTLC using ethylacetate-methanol-water (9.4:0.5:0.1) provided 3'-methylquercetin (7) (17 mg) and quercetin (8) (13 mg), respectively. 2-Hydroxy-3-methoxy-5-(2-propenyl)-phenol (1) Yellow oil (26 mg); UV (MeOH) max (log ε) nm: 330 (3.7), 280 (3.9), 236 (4.1); IR (KBr) max cm-1: 3362, 2968, 1604, 1284, 1054; 1H-NMR (CD3OD, 500 MHz) : 6.80 (1H, br s, H-6), 6.77 (1H, br s, H-4), 5.92 (1H, m, H-8), 5.02 (2H, m, H9), 3.81 (3H, s, OCH3), 3.28 (2H, d, J = 7.0 Hz, H-7); 13 C-NMR (CD3OD, 125 MHz) : 150.1 (C-3), 141.4 (C-1), 138.9 (C-8), 138.2 (C-2), 132.1 (C-5), 116.5 (C-6), 115.9 (C-9), 110.5 (C-4), 40.7 (C-7); HREIMS, m/z: 180.0779 (calcd. for C10H12O3, 180.0786). The physical and spectral data completely matched with the literature values for 2-hydroxy-3-methoxy-5(2-propenyl)-phenol [14]. Vanillic acid (2) Crystallized from methanol (3 mg), m.p. 209-211°C; UV (MeOH) max (log ε) nm: 283 (3.7), 251 (3.8), 219 (4.0); 1R (KBr) max cm-1: 3445, 2925, 1741, 1599-1515, 1281, 1032; 1H-NMR (CD3OD, 500 MHz) : 7.56 (1H, d, J = 1.5 Hz, H-2), 7.46 (1H, dd, J = 8.0, 1.5 Hz, H-6), 6.73 (1H, d, J = 8.0 Hz, H- IRSHAD AHMAD et al., 6), 3.87 (3H, s, OCH3); 13C-NMR (CD3OD, 125 MHz) : 175.0 (C-7), 149.2 (C-4), 148.0 (C-3), 130.0 (C-1), 124.2 (C-6), 115.1 (C-5), 114.0 (C-2), 56.2 (OCH3); HREI-MS, m/z: 168.0415 (calcd. for C8H8O4, 168.0422). The physical and spectral data completely overlapped over literature value for vanillic acid [15]. Isoferulic Acid (3) Crystallized in methanol (18 mg), m.p. 229231°C; UV (MeOH) max (log ε) nm: 335 (3.9), 300 (4.01), 220 (4.1); IR (KBr) max cm-1: 3627, 34272500, 1695, 1596, 1264; 1H-NMR (CD3OD, 400 MHz) : 7.60 (1H, d, J = 16.0 Hz, H-7), 7.04 (1H, d, J = 1.5 Hz, H-2), 6.98 (1H, dd, J = 8.4, 1.5 Hz, H-6), 6.71 (1H, d, J = 8.4 Hz, H-5), 6.25 (1H, d, J = 16.0 Hz, H-8), 3.95 (3H, s, OCH3); 13C-NMR (CD3OD, 100 MHz) : 168.3 (C-9), 149.0 (C-4), 147.5 (C-3), 147.1 (C-7), 127.7 (C-1), 123.1 (C-6), 116.4 (C-5), 115.1 (C-2), 115.1 (C-8), 56.0 (OCH3); HREI-MS m/z: 194.0570 (calcd. for C10H10O4, 194.0579). The physical and spectral data matched completely with the literature [16]. Caffeic Acid (4) Crystallized in methanol (16 mg), m.p. 223225°C; UV (MeOH) max (log ε) nm: 334 (4.0), 245 (4.15), 220 (3.8); IR (KBr) max cm-1: 3666, 34222395, 1694, 1598, 1264; 1H-NMR (CD3OD, 400 MHz) : 7.62 (1H, d, J = 15.5 Hz, H-7), 7.08 (1H, d, J = 2.0 Hz, H-2), 6.96 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.78 (1H, d, J = 8.0 Hz, H-5), 6.42 (1H, d, J = 15.5 Hz, H-8); 13C-NMR (CD3OD, 100 MHz) : 168.9 (C9), 149.0 (C-4), 146.7 (C-3), 147.6 (C-7), 128.0 (C1), 123.3 (C-6), 116.7 (C-5), 115.5 (C-8), 114.5 (C2); HREI-MS m/z: 180.0416 (calcd. for C9H8O4, 180.0422). The physical and spectral data corresponded to the reported values for caffeic acid [16]. Methyl Gallate (5) Colorless amorphous powder (4 mg), m.p. 201-203°C; UV (MeOH) max (log ε) nm: 330 (3.7), 260 (3.9), 236 (4.1); IR (KBr) max cm-1: 3597, 2963, 1742, 1697, 1237, 1016; 1H-NMR (CD3OD, 400 MHz) : 7.01 (2H, s, H-2,6), 3.80 (3H, s, OCH3); 13 C-NMR (CD3OD, 100 MHz) : 169.0 (C-7), 146.5 (C-3,5), 139.9 (C-4), 121.3 (C-1), 110.0 (C-2,6), 52.2 (OCH3); HREI-MS m/z: 184.0364 (calcd. for C8H8O5, 184.0371). The physical and spectral data J.Chem.Soc.Pak., Vol. 33, No. 1, 2011 116 showed complete resemblance with those reported values for methyl gallate [17]. Uridine (6) Colorless amorphous powder (11 mg); UV (MeOH) max (log ε) nm: 264 (3.8), 214 (3.9); IR (KBr) max cm-1: 3451, 2926, 1670, 1595, 1096; 1HNMR (CD3OD, 500 MHz) : 8.00 (1H, d, J = 8.0 Hz, H-6), 5.88 (1H, d, J = 5.0 Hz, H-1'), 5.68 (1H, d, J = 8.0 Hz, H-5), 4.16 (1H, t, J = 5.0 Hz, H-2'), 4.13 (1H, t, J = 5.0 Hz, H-3'), 4.00 (1H, m, H-4'), 3.83 (1H, dd, J = 12.0, 2.5 Hz, H-5'), 3.72 (1H, dd, J = 12.0, 3.0 Hz, H-5'); 13C-NMR (CD3OD, 125 MHz) : 166.0 (C-4), 152.0 (C-2), 142.7 (C-6), 102.6 (C-5), 90.7 (C1'), 86.3 (C-4'), 75.7 (C-2'), 71.3 (C-3'), 62.5 (C-5'); HRFAB-MS m/z: 245.0770 [M+H]+ (calcd. for C9H13N2O6, 245.0773). The physical and spectral data were in agreement with same compound [18]. 3'-Methylquercetin (7) Yellow powder (17 mg); UV (MeOH) max (log ε) nm: 366 (4.02), 254 (4.1), 209 (3.7); IR (KBr) max cm-1: 3270, 2928, 1730, 1657-1509, 1168, 1033; 1 H-NMR (CD3OD, 400 MHz) : 8.00 (1H, d, J = 2.0 Hz, H-2'), 7.56 (1H, dd, J = 8.5 Hz, 2.0, H-6'), 6.89 (1H, d, J = 8.5 Hz, H-5'), 6.40 (1H, d, J = 1.2 Hz, H8), 6.17 (1H, d, J = 1.2 Hz, H-6), 3.94 (3H, s, OCH3); 13 C-NMR (CD3OD, 100 MHz) : 179.1 (C-4), 166.1 (C-7), 163.0 (C-8a), 159.3 (C-5), 151.1 (C-4'), 148.6 (C-3'), 148.6 (C-2), 133.6 (C-3), 123.7 (C-6'), 122.8 (C-1'), 116.0 (C-5'), 114.2 (C-2'), 105.7 (C-4a), 100.0 (C-6), 94.8 (C-8), 56.9 (OCH3); HREI-MS m/z: 316.0575 (calcd. for C16H12O7, 316.0583). The physical and spectral data complete agreement to the reported values 3'-methylquercetin [19]. Quercetin (8) Yellow powder (13 mg); UV (MeOH) max (log ε) nm: 360 (4.1), 257 (4.01), 211 (3.9); IR (KBr) max cm-1: 3423, 2925, 1658-1598, 1271, 1058; 1HNMR (CD3OD, 400 MHz) : 7.65 (1H, d, J = 2.0 Hz, H-2'), 7.63 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 6.84 (1H, d, J = 8.5 Hz, H-5'), 6.26 (1H, d, J = 1.5 Hz, H-8), 6.09 (1H, d, J = 1.5 Hz, H-6); 13C-NMR (CD3OD, 400 MHz) : 180.0 (C-4), 166.1 (C-7), 164.0 (C-8a), 158.0 (C-5), 150.0 (C-4'), 146.1 (C-3'), 146.0 (C-2), 133.0 (C-3), 123.2 (C-6'), 123.1 (C-1'), 116.9 (C-2'), 116.2 (C-5'), 104.0 (C-4a), 101.5 (C-6), 95.9 (C-8); HREI-MS m/z: 302.0419 (calcd. for C15H10O7, 302.0426). The physical and spectral data showed complete resemblance with those reported in literature [20]. J.Chem.Soc.Pak., Vol. 33, No. 1, 2011 117 IRSHAD AHMAD et al., Determination of DPPH Radical Scavenging Activity The free radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) using the method described by Lee et al [21]. 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