Urease inhibitors from Hypericum oblongifolium WALL

Journal of Enzyme Inhibition and Medicinal Chemistry, 2010; 25(2): 296–299 research article Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by Yonsei University on 04/11/11 For personal use only. Urease inhibitors from Hypericum oblongifolium WALL. Mohammad Arfan1, Mumtaz Ali1, Habib Ahmad2, Itrat Anis3, Ajmal Khan4, M. Iqbal Choudhary4, and Muhammad Raza Shah4 Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan, 2Department of Botany, Hazara University, Dudial, Mansehra, Pakistan, 3Department of Chemistry, University of Karachi, Karachi, Pakistan, and 4H.E.J. Research Institute of Chemistry; International Center for Chemical and Biological Sciences; University of Karachi; Karachi; Pakistan 1 Abstract The bioassay-guided fractionation of H. oblongifolium has led to the isolation of potent urease inhibitors 1–3. The structures were elucidated by NMR and mass spectroscopic techniques. Compound 2 showed a potent enzyme inhibition activity (IC50 20.96 ± 0.93), which is comparatively higher than that for the standard thiourea (IC50 21.01 ± 0.51 µM). Compounds 1 and 3 also showed a significant activity, with IC50 37.95 ± 1.93 and 138.43 ± 1.23 µM, respectively. The sub crude fractions (F1, F2, F3, and F4) were tested in vitro for their urease inhibition activity. Fractions F2 and F4 showed significant activity with IC50 140.37 ± 1.93 and 167.43 ± 3.03 µM, respectively. Keywords: Hypericum oblongifolium; guttiferae; urease inhibitors Introduction Hypericum oblongifolium WALL. is an erect evergreen shrub, 6–12 m high, that occurs on Khasia Hill at an altitude of 5000–6000 m, in China, and in the Himalayan hills1. It has been used in traditional Chinese medicine for the treatment of hepatitis, bacterial infections, and nasal hemorrhage, and as a remedy for dog-bites and bee-stings. The plants of the Hypericum genus have shown antidepressant, anxiolytic, antiviral, wound healing, and antimicrobial activities2. Due to their widespread use in folk medicine, the chemical constituents and sub crude fractions of Hypericum oblongifoliun were evaluated for their urease inhibitory activities. Urease (urea amidohydrolase, EC 3.5.15) catalyzes the hydrolysis of urea to ammonia and carbon dioxide3 and has been shown to be an important factor in the pathogenesis of many clinical conditions4. Urease inhibitors have attracted major attention as potent anti-ulcer drugs5. Due to the diverse functions of this enzyme, its inhibition by potent and specific compounds could lead to the treatment of infections caused by urease-producing bacteria6. A number of synthetic compounds including imidazoles, hydroxamic acids, and phosphazenes are effective urease inhibitors, but limited studies have been conducted on natural products5,7. Herein we report the urease inhibitory activity of various fractions and compounds isolated for the first time from H. oblongifolium. Experimental General Ultraviolet (UV) spectra were obtained on a Hitachi-U-3200 UV-visible spectrometer with CHCl3 as solvent. Infrared (IR) spectra were recorded on a Vector 22 (Bruker) Fouriertransform infrared (FT-IR) spectrometer using CH2Cl2 as solvent. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance AV-500 spectrometer. Chemical shifts () are expressed in ppm relative to tetramethylsilane (TMS) as internal standard and coupling constants are given in Hz. 1H NMR spectra were referenced against the CHCl3 + CH3OH signal at H 7.27, 4.9 and 13C NMR spectra against the corresponding signal at C 77.04. Mass spectra (electron ionization (EI-) and high resolution (HR)-EI-MS) were measured in electron impact mode on Finnigan MAT312 and MAT-95 XP spectrometers, and ions are denoted as m/z (%). Thin layer chromatography (TLC) was performed on pre-coated silica gel F-254 plates (E. Merck); the detection was done at 254 nm, by spraying with ceric sulfate reagent. Column silica gel (E. Merck, 70–230 mesh) and flash silica gel (E. Merck, 230–400 mesh) were used for column chroma- Address for Correspondence: Dr Muhammad Raza Shah PhD, H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. E-mail: [email protected] (Received 12 February 2009; revised 04 June 2009; accepted 11 June 2009) ISSN 1475-6366 print/ISSN 1475-6374 online © 2010 Informa UK Ltd DOI: 10.3109/14756360903179385 http://www.informahealthcare.com/enz Urease inhibitors from Hypericum oblongifolium WALL.   297 Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by Yonsei University on 04/11/11 For personal use only. tography. Melting points were determined on a Buchi-535 apparatus and are uncorrected. Plant material Hypericum oblongifolium was authenticated by Professor Dr Habib Ahmad, Dean of the Faculty of Science, Hazara University. The plant material was collected during the flowering period in June 2007 from Buner District, Northwest Frontier Province (NWFP). A voucher specimen (HUH-002) was retained for verification purposes in the Department of Botany, Hazara University, NWFP, Pakistan. Extraction and isolation The air-dried, powdered twig material (12 kg) was exhaustively extracted with hexane, ethyl acetate, and methanol (3 × 25 L, each for 3 days) at room temperature. Extracts were concentrated under vacuum to obtain a residue, as fractions F1 (hexane) and F2 (ethyl acetate). The concentrated methanolic fraction was suspended in water and extracted with n-butanol to afford fractions F3 (butanol) and F4 (water). The ethyl acetate fraction (F2, 260 g) was subjected to ­column chromatography over silica gel and eluted with n-hexane– ethyl acetate and ethyl acetate–MeOH, in increasing order of polarity, to afford 200 fractions that were combined according to similarity on TLC profiles, and 30 major fractions were obtained. Fraction 14 was further subjected to column chromatography over flash silica gel (chloroform–hexane, 30:70) and led to the isolation of compound 2 ( 22 mg). Fractions 15 and 16 were combined and loaded over a flash silica gel O O OH OH OH 1 O HO CH3 O HO (CH2)22 2 O OH HO O 3 Figure 1. Structure of compounds 1–3. OH chromatography column (chloroform–hexane, 40:60) to afford 1 ( 6 mg). Similarly, fractions 18 and 19 were combined and subjected to flash silica gel column chromatography (methanol–chloroform, 1:99) to afford 3 ( 11 mg). 3,4,5-Trihydroxy xanthone (1) Yellow amorphous powder solid; m.p. 280–283°C; IR, max (chloroform) cm−1 3599, 3512, 3462 (O-H), 2928, 2843, 1644 (C=O), 1580, 1443, 1328, 1257, 1137, 1047; max (CHCl3) nm (log ε): 240 (4.38), 308 (3.81), 346 (3.71); EI-MS (70 eV) m/z 244.0 (calc. for [C13H8O5]+); 1H NMR (400 MHz, CD3OD + CDCl3): H 7.7 (1H, dd, J = 7.9 Hz, J = 1.4 Hz, H-8), 7.39 (1H, dd, J = 7.9 Hz and 1.45 Hz, H-6), 7.35 (1H, d, J = 8.9 Hz, H-2), 7.28 (1H, t, J = 7.9 Hz, H-7), 6.96 (1H, d, J = 8.9, H-1); 13C NMR (100 MHz, CD3OD + CDCl3): C 183.0 (C-9), 161.0 (C-4), 149.0 (C-3), 148.0 (C-4), 147.0 (C-5a), 141.0 (C-4a), 124.0 (C-2, 7), 122.0 (C-8a), 121.0 (C-6), 116.0 (C-8), 110.0 (C-1a), and 107.0 (C-1). The physical and spectral data showed complete agreement with those reported in the literature8. Tetracosyl 3-(3,4-dihydroxyphenyl) acrylate (2) White solid; m.p. 202–205°C; UV, max (MeOH): 235 (log ε 4.1), 325 nm (4.0); IR bands (KBr): 3500, 1700, 1670, 1600, 1510, 1460, 1280, 1160, 715 cm−1; EI-MS (70 eV) m/z 516.0 (calc. for [C33H56O4]+); 1H NMR (400 MHz, CDCl3): H 7.6 (1H, d, J = 15.0 Hz, H-7), 7.2 (1H, s, H-6), 7.1 (1H, d, J = 10.0 Hz, H-3), 6.98 (1H, d, J = 10.0 Hz, H-2), 6.26 (1H, d, J = 15.0 Hz, H-8), 4.1 (2H, m, H-10), 1.0–1.3 (44H, m, H-11-24), 0.97 (3H, brs, Me-25); 13C NMR (100 MHz, CDCl3): C 168.5 (C-9), 148.5 (C-5), 147.4 (C-7), 146.27 (C-4), 123.49 (C-1), 122.61 (C-6), 120.41 (C-2), 119.43 (C-8), 118.54 (C-3), 63.4 (C-10), 24.44–30.4 (C-11-32), 23.92 (C-25). The physical and spectral data showed complete agreement with those reported in the literature9. 1,3,7-Trihydroxy xanthone (3) Yellow solid; m.p. 318–320°C; IR, max (chloroform) cm−1 3519, 3502, 3442 (O-H), 2928, 2843, 1654 (C=O), 1580, 1443; max (CHCl3) nm (log ε): 244 (4.38), 318 (3.81), 356 (3.71); EI-MS (70 eV) m/z 244.0 (calc. for [C13H8O5]+; 1H NMR (400 MHz, CD3OD + CDCl3): H 7.45 (1H, d, J = 8.9, H-8), 7.35 (1H, d, J = 8.9 Hz, H-5), 7.25 (1H, dd, J = 8.9 Hz and 2.8 Hz, H-6), 6.39 (1H, d, J = 1.9 Hz, H-4), 6.19 (1H, d, J = 8.9 Hz, H-2); 13C NMR (100 MHz, CD3OD + CDCl3): C 176.7 (C-9), 167.3 (C-3), 164.6 (C-1), 159.3 (C-4a), 155.8 (C-7), 151.0 (C-5a), 125.0 (C-6), 122.0 (C-8a), 119.8 (C-5), 109.0 (C-8), 98.8 (C-2), and 96.9 (C-4) The physical and spectral data showed complete agreement with those reported in the literature10. Urease inhibition assay Reaction mixtures comprising 25 L of enzyme (jack bean urease) solution and 55 L of buffer containing 100 mM urea were incubated with 5 L of test compound (0.5 mM) at 30°C for 15 min in 96-well plates. Urease activity was determined by measuring the ammonia production using the indophenol method, as described by Weatherburn11. Briefly, 45 L of each phenol reagent (1% (w/v) phenol and 0.005% (w/v) sodium nitroprussside) and 70 L of alkali reagent (0.5% (w/v) NaOH and 0.1% active chloride, NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). All reactions were performed in triplicate in a final volume of 200 L. The results (change in absorbance per min) were processed using SoftMax Pro software (Molecular Devices). The entire assay was performed at pH 6.8. Percentage inhibitions were measured from the formula 100 – (ODtestwell/ ODcontrol) × 100. Thiourea was used as the standard inhibitor of urease12. For kinetic studies, the concentration of compounds 1 and 2 that inhibited the hydrolysis of substrates (jack bean urease) by 50% (IC50) was determined by monitoring the inhibition effect of various concentrations of both compounds in the assay. The IC50 (inhibitor concentration that inhibits 50% activity of enzyme) values were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, MA, USA). Graphs were plotted using the GraFit program13. Values of correlation coefficients, slopes, intercepts, and their standard errors were obtained by linear regression analysis using the same program. Each point in the constructed graphs represents the mean of three experiments. The Ki values were calculated from the slopes of each line in the Lineweaver–Burk plot versus different concentrations of both compounds. and 3 also showed significant activity, with IC50 37.95 ± 1.93 and 138.43 ± 1.23 µM, respectively. The activities of 1, 2, and 3 can be attributed to their co-ordinating capabilities with the metallocenter (i.e. nickel) of the enzyme14. The greater activity of compound 2 can be conceived to be due to the presence of two aromatic hydroxyl groups and , unsaturated carboxylic in the backbone of the molecule, which can strongly bind to the active sites of the enzyme15. Compounds 1 and 2 inhibited the urease enzymes (Figure 2) in a concentration-dependent manner, with Ki value of 31 ± 0.010 and 18 ± 0.014 mM against the jack bean ureases, respectively. Lineweaver–Burk plots and their replots indicated that 2 is a mixed type of inhibitor of jack bean urease, as a change in both Vmax and affinity (Km value) of urease toward the substrate (urea) was observed. On the other hand, compound 1 showed a competitive type of inhibition (Figure 2), causing an increase in Km without affecting the Vmax value. Mechanistic studies of both compounds are expected to provide useful information about the design of new inhibitors of jack bean urease. 2 1.8 Results and discussion The bioassay-guided fractionation of H. oblongifolium led to the isolation of potent urease inhibitors. Various fractions (F1, F2, F3, and F4) were obtained from the air-dried, powdered twigs of H. oblongifolium (see “Experimental” section). These fractions (F1, F2, F3, and F4) were tested in vitro for their urease inhibition activity. Among the fractions, F2 and F4 showed significant activity with IC50 140.37 ± 1.93 and 167.43 ± 3.03 µM, respectively. Therefore, F2 was subjected to column chromatography over silica gel, eluting with n-hexane–ethyl acetate and ethyl acetate– MeOH in increasing order of polarity, to afford compounds 1–3. All these compounds were evaluated for urease inhibitory activity. The IC50 values with percent inhibition of urease by various fractions and compounds are summarized in Table 1. Compound 2 showed potent activity (IC50 20.96 ± 0.93), which is comparatively higher than that for standard thiourea (IC50 21.01 ± 0.51 µM). Compounds 1 Table 1. The IC50 values by percent inhibition of urease to the fractions and compounds. Compound/extract % Inhibition at 1000 g/ml IC50 (µM) ± SEM F1 26.9 — F2 68.3 140.37 ± 1.93 F3 23.7 — F4 67.5 167.43 ± 3.03 1 99.3 37.95 ± 1.93 2 96.96 20.96 ± 0.93 3 71.4 138.43 ± 1.23 Thiourea 98.82 21.01 ± 0.51 1/V 1.6 1.4 1.2 1 −1 0 −0.5 1 0.5 1/S (1) 3 2.8 2.6 2.4 2.2 1/V Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by Yonsei University on 04/11/11 For personal use only. 298   Mohammad Arfan et al. 2 1.8 1.6 1.4 1.2 1 −1 0 1 1/S (2) Figure 2. Inhibition of jack bean urease by compounds 1 and 2. Lineweaver–Burk plots of the reciprocal of initial velocities vs. reciprocal of four fixed substrate concentrations in absence (○) and presence of 100 mM (▲), 80 mM (△), 60 mM (■), 40 mM (□), 20 mM (●). Urease inhibitors from Hypericum oblongifolium WALL.   299 Table 2. In vitro inhibition of urease by compounds 1 and 2. Kia ± SEMb Type of (mM) Compound Enzyme inhibition IC50 (µM) ± SEM 1 Jack bean Mixed type 37.50 ± 0.94 31 ± 0.010 urease 2 Jack bean Competitive 20.96 ± 0.47 18 ± 0.014 urease a Ki is the mean of three values calculated using Lineweaver–Burk secondary plots. b Standard mean error of three assays. 3. 4. 5. 6. Journal of Enzyme Inhibition and Medicinal Chemistry Downloaded from informahealthcare.com by Yonsei University on 04/11/11 For personal use only. 7. Acknowledgements 8. Declaration of interest 9. Higher Education Commission of Pakistan is acknowledged for financial support under the 5000 indigenous PhD fellowship scheme. References 1. 2. Chopra RN, Chopra IC, Verma BS. Supplement to Glossary of Indian Medicinal Plants Council of Scientific and Industrial Research, Publications and Information Directorate, New Delhi, 1998. Hu LH, Sim KY. Sampsoniones A–M, a Unique Family of Caged Polyprenylated Benzoylphloroglucinol Derivatives, from Hypericum sampsonii. 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