Phytochemical analysis and Enzyme Inhibition Assay of Aerva javanica for Ulcer
© Khan et al.; licensee Chemistry Central Ltd. 2012
Received: 19 June 2012
Accepted: 17 July 2012
Published: 31 July 2012
Aerva javanica (Burm. f.) Juss. ex Schult. (Amaranthacea) is traditionally used for the treatment of wound healings, cough, diarrhoea, ulcer and hyperglycaemia. The current study was aimed to fractionate and isolate bioactive compounds and ultimately to evaluate their anti-ulcereogenic potential.
In order to achieve these aims, the fractionation, purifications and then biological potential determination of the isolated compounds was carried out. For purification purpose, initially extraction of the plant material was done with aqueous MeOH in the order of increasing polarity by using solvent-solvent extraction method. Phytochemical analysis revealed the presence of three compounds, 3-hydroxy-4 methoxybenzaldehyde (1), ursolic acid (2) and (E)-N-(4-hydroxy-3-methoxyphenethyl)-3-(4-hydroxy-3-ethoxyphenyl) acryl amide (3). Inhibition of urease activity of various fractions revealed that ethyl acetate fraction showed significant activity (P <0.05) as compared to other fractions. (E)-N-(4-hydroxy-3-methoxyphenethyl)-3-(4-hydroxy-3-ethoxyphenyl) acryl amide (3) showed marked anti ulcer activity (P <0.05).
These results suggested the mild potential of A. javanica against ulcer.
Medicinal plants play important role in the treatment of various disorders [1, 2]. The plants of genus Aerva (Amaranthaceae) are perennial herb under shrubs and are found in the North Temperate Zone, especially in the Mediterranean regions and Asia. About 20 species of genus Aerva are present in Pakistan and India; most of them are used in traditional medicinal system . A. javanica (Burm.f.) Juss. ex Schult., locally known as Khar Buta, is found over a broad range of sandy sediments. Various chemical constituents including steroids, triterpenes, lipids, flavonoids, tannins, saponins, alkaloids, sulphates, carbohydrates and glycosides have been isolated from this plant [4, 5]. The plant has been widely used for its therapeutic effects in relieving the swelling and pain due to kidney stones . The plant decoction is used for dysentery, gonorrhea and cutaneous infections . A. javanica showed antioxidant , antiviral , antiplasmodial  and antidiabetic activities .
Urease (urea amidohydrolase) is an enzyme that catalyzes the hydrolysis of urea to ammonia and carbamate, which is the final step of nitrogen metabolism in living organisms . Carbamate rapidly and spontaneously decomposes, yielding a second molecule of ammonia. These reactions may cause significant increase in pH and are responsible for negative effects of urease activity in human health and agriculture [13, 14]. Urease is responsible for urinary tract and gastrointestinal infections, possibly causing severe diseases such as peptic ulcers and stomach cancer as in the case of Helicobacter pylori. Ureases are also involved in the development of urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic coma and urinary catheter encrustation [15, 16]. Here, we investigated the antiulcer activity of different fractions as well as pure constituents of A. javanica.
Results and discussion
1 H and 13 C NMR spectral data for compound 1
d, J = 2.0
d, J = 8.7
dd, J = 8.7, 2.0
1 H and 13 CNMR spectral data for compound 2
dd, J = 10.0, 4.5
dd, J = 13.7, 3.5
t, J = 3.5
d, J = 11.3
d, J = 6.8
d, J = 6.6
Prominent 1 H and 13 CNMR spectral data for compound 3
d, J = 12.8
d, J = 12.8
d, J = 2.0
d, J = 8.4
dd, J = 8.4, 2.0
t, J = 7.2
t, J = 7.2
d, J = 2.0
d, J = 8.0
dd, J = 8.0, 2.0
The signals at δH 6.73 (1 H, d, J = 8.4 Hz), δH 6.93 (1 H, dd, J = s 8.4,2.0 Hz) and 7.36 (1 H, d, J = 2.0 Hz) were for H-8, H-9 and H-5 of ferulic acid moiety, respectively. The C-3 olefinic proton of the ferulic acid moiety exhibited a doublet at δH 6.61 (J = 12.8 Hz) which showed the trans-coupling with C-2 olefinic proton, having a doublet at δH 5.82 (J = 12.8 Hz). A further ABX was observed at δH 6.67 (1 H, dd, J = 188.8.131.52 Hz) for H-8´, δH 6.63 (1 H, d, J = 8.0 Hz) for H-7´ and 6.82 (1 H, d, J = 2.0 Hz) for H-4´ in the methoxytyramine moiety. Two coupled triplets of methylene protons at δH 2.77 and δH 3.49 (each 2 H, t, J = 7.2 Hz) were assigned to H-2´ and H-1´, in the methoxtyramine moiety, respectively. In 13C-NMR spectrum, the signal at 169.2 indicated an amide functionality. 13C-NMR spectrum of compound 3 showed resonances of two methyls; two methylenes, eight methines and seven quaternary carbons. The mass, UV, IR and 1 H-NMR data proposed compound 3 an amide with phenolic acid funtionality. The position of aromatic substituent was deduced by NOESY experiment. Detailed spectroscopic data established the structure of compound 3 as the (E)-N-(4-hydroxy-3-methoxyphenethyl)-3-(4-hydroxy-3-ethoxyphenyl) acryl amide. Similarly various bioactive compounds were isolated from A. javanica.
Effects of various fractions on Urease inhibition
Name of the fraction
0.2 mg/ml Hexane
15.3 ± 1.2a
0.2 mg/ml Dichloromethane
33.4 ± 3.2b
0.2 mg/ml Ethyl acetate
54.6 ± 4.1c
0.2 mg/ml Water
5.1 ± 0.9a
0.2 mg/ml Thiourea
98.2 ± 5.1d
Effects of isolated compounds 1–3 on Urease inhibition
Name of the compounds
0.2 mg/ml Compound 1
15.3 ± 0.8a
0.2 mg/ml Compound 2
33.4 ± 2.7b
0.2 mg/ml Compound 3
64.6 ± 4.2c
0.2 mg/ml Thiourea
98.2 ± 4.3d
Electron Impact Mass Spectra (EI-MS) were measured on Finnigan MAT-311 mass Spectrometer. High-Resolution Electron Impact Mass spectra (HREI-MS) were obtained on Jeol HX mass spectrometer. IR spectra were obtained in chloroform by using JASCO IRA-1 and JASCO A-302 spectrophotometers. KBr discs as supporting suface and CHCl3 was used as dissolving solvent. Ultraviolet (UV) spectra of UV active compounds were recorded in methanol by using a Shimadzu UV-240 and U-3200 Hitachi spectrophotometer. The 1H and 13CNMR, COSY 45°, HMQC and HMBC spectra were recorded on Bruker AV-400 and AV-500 spectrometer.
The A. javanica (whole plant) was collected from Bannu Township, Bannu, Khyber Pakhtunkhwa in March 2010. The plant was identified by Professor Abdur Rehman, Department of Botany, Government Post Graduate College Bannu, Pakistan and a specimen (W-12) was submitted at Herbarium of Biotechnology and Botany University of Science and Technology Bannu KPK, Pakistan.
Extraction and isolation
The air-dried plant (20 kg) was chopped and was initially extracted with methanol/water (4:1) at room temperature. The hydromethanolic extract (700 g) was initially partitioned into n-hexane (84 g). The defatted MeOH extract was dried and then suspended in H2O (2 L), was successively partitioned with dichloromethane (110 g), and ethyl acetate (27 g). The EtOAc soluble fraction was subjected to CC by using normal silica gel. The eluent used were n-hexane, EtOAc, DCM and MeOH in gradient manner to obtain compounds 1–3.
White Powder; m.p 116–117°C; IR (KBr, CHCl3) v max (cm-1): 3600–2500, 2685; 1H and 13C NMR spectral data, see Table 1; EI-MS m/z (rel. int.): [M]+, 152.0 (100), 151 (94), 137 (5), 109 (24), 95 (3), 81 (27), 43(20); HR-EI-MS: m/z [M]+ 152.0 (calcd for C8H8O3, 152.05).
White crystals; m.p 283–285°C; [α]D20 + 62.5° (c = 0.2, CHCl3); IR (KBr, CHCl3) v max (cm-1): 3510, 3050, 1697, 1635, 820; 1H and 13C NMR spectral data, see Table 2; EI-MS m/z (rel. int.): [M]+, 456 (M+, 10), 411 (22), 248 (34), 203 (100), 189 (16); HR-EI-MS: m/z [M]+ 456.3599 (calcd for C30H48O3, 456.3603).
Yellowish white amorphous powder; m.p 111-113°C, IR (KBr, CHCl3) v max (cm-1): 3440, 3350, 1680, 1650; 1H and 13C NMR spectral data, see Table 3; EI-MS m/z (rel. int.): [M]+, 343 (20), 193 (30), 177 (94), 151 (25), 150 (100), 145 (30), 55 (6); HR-EI-MS: m/z [M]+ 343.1411 (calcd for C19H21NO5, 343.1420).
Urease enzyme inhibition assay
Urease (Jack bean) solution (25 μl) was mixed with the 5 mg (500 μg) extracts and the mixture was incubated at 30°C. Aliquot were taken after 15 min and then was transferred to assay mixtures having urea (100 mM) in buffer (40 μl) and again incubated for 30 min in 96 well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described . Briefly, 50 μ1 each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μ1 of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCI) were added to each well. The increasing absorbance was measured after 50 min at wavelength of 630 nm using microtitre plate reader (Spectramax plus 384 Molecular Device, USA). All reactions were performed in triplicates in final volume of 200 μl. All the assays were performed at pH 8.2 (0.01 M K2HPO4. 3H2O, 1 mM EDTA and 0.01 M LiC12). Thiourea was used as standard and percentage inhibitions were calculated from formula, 100 - (OD test/OD control) x 100. In this study, the reductive ability was measured by investigating the Fe+3 → Fe transformation in the presence of various extracts of plants and standard antioxidant (BHA) by using the Oyaizu method .
The parametric data were expressed as the mean ± SEM for the 03 replicates in each group. To determine the differences between groups one-way analysis of variance (ANOVA) was carried out by using the SPSS software (version 13.0) using the least significant difference (LSD) test at P<0.01. Means not sharing the same letter are significantly different (LSD) at P < 0.01 probability level in each column.
The current phytochemical study provided preliminary data for the first time that the A. javanica possesses significant anti-ulcer activity. This might be contributed towards the presence of some bioactive constituents contributed towards the various biological activities including the in the treatment of gastric ulcer. The compound 3 shows mild activity which might be due to the presence of amide group. Further study on the plant and specifically on the compound 3 could provide many chemically interesting and biologically active drugs, including, some with potential anti-ulcer properties.
Provision of research facility at the premier research center, the H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi is highly acknowledged.
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