S Prahalad Umachigi, K Jayaveera, K Ashok, G Kumar
S Prahalad Umachigi, K Jayaveera, K Ashok, G Kumar. Antioxidant Potential of galls of Quercus infectoria . The Internet Journal of Pharmacology. 2007 Volume 5 Number 2.
Nowadays, the fact of harmful effect of reactive oxygen species on human health is well-known. The capability of natural defense systems of living organisms against excess production of these species decreases when influenced with negative environmental factors or aging. As a result, different cellular and extracellular components, and especially nucleic acids, are damaged, causing or enhancing a number of degenerative diseases. Therefore, antioxidants that scavenge free radicals are of great value in preventing such “oxidative” pathologies. That is why natural products with antioxidant properties become more and more popular all over the world. Natural phenolic phytochemicals in fruits and vegetables have been receiving increased interest from consumers and researchers for their beneficial health effects on coronary heart diseases and cancers mainly due to their antioxidant activity. 1 As plants produce a lot of antioxidants to control the oxidative stress caused by sunbeams and oxygen, they can represent a source of new compounds with antioxidant activity. Among natural antioxidants, phenolic antioxidants are in the forefront since all the phenolic classes (simple phenolics, phenolic acids, anthocyanins, hydroxycinnamic acid derivatives, and flavonoids) have the structural requirements of free radical scavengers and antioxidants 2
Report suggest the presence of tannoid principles 7 which are known antioxidants, we studied the antioxidant potential of the extract and also the percentage of gallic acid by high performance thin layer chromatography (HPTLC), which may be responsible for the antioxidant activity. HPLC and GC are efficient but time consuming methods; HPTLC on the other hand is relatively simple and a non expensive assay method, which does not require any experience, equipment or complex derivatization process.
Thus present study aims to assess the antioxidant potential of methanolic extract of
Material and Method
Gallic acid, 1,1-diphenyl 2-picryl hydrazyl (DPPH), 1,1,3,3-tetraethoxypropane, 2-nitrobenzoic acid (DTNB) and pottasium superoxide were obtained from Sigma Chemical Co. (St. Louis, U.S.A.), ferrous sulphate (FeSO4), trichloroacetic acid (TCA), thiobarbituric acid (TBA), acetic acid, ethylenediaminetetraacetic acid (EDTA),
sodium nitroprusside, sulfanilamide, phosphoric acid, naphthyl ethylene diamine, ammonium molybdate, sodium phosphate,sodium hypochlorite, hydrogen peroxide and dimethyl sulfoxide (DMSO) were obtained from Sd. fine chemicals (Mumbai, India). All other reagents used were of analytical grade.
Plant material and Extraction
The air-dried galls of
Hydrogen- Donating Activity
Hydrogen donating activity was quantified in presence of stable DPPH radical on the basis of Blois method 8 . Briefly, to a methanolic solution of DPPH (100?M, 2.95 ml), 0.05ml of test compounds dissolved in methanol was added at different concentrations (2—10 mg/ml). Reaction mixture was shaken and absorbance was measured at 517 nm at regular intervals of 30 s for 5 min. Ascorbic acid was used as standard. The degree of discoloration indicates the scavenging efficacy of the extract
Total Antioxidant Activity
Total antioxidant capacity was measured according to spectrophotometric method 9 .
0.1 Ml of the extract (10 mg/ml) dissolved in water was combined in an eppendorf tube with 1 ml of reagent solution (0.6 M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The tubes were capped and incubated in a thermal block at 95°C for 90 min. After cooling to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against a blank. Ascorbic acid was used as the standard and the total antioxidant capacity is expressed as equivalents of ascorbic acid.
Nitric Oxide Scavenging
Nitric oxide scavenging activity was measured spectrophotometrically 10 . Sodium nitroprusside (5mM) in phosphate buffered saline was mixed with different concentrations of extract (2—10 mg/ml) dissolved in methanol and incubated at 25°C for 30 min, then 1.5 ml of the incubation solution were removed and diluted with 1.5 ml of Griess reagent (1% Sulfanilamide, 2% phosphoric acid, and 0.1% naphthyl ethylene diamine dihydrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with naphthylethylene diamine was measured at 546 nm along with a control.
Hydrogen Peroxide Decomposition
Hydrogen peroxide decomposition was determined according to standard method. 11 The assay mixture contained 4 ml of H2O2 solution (80mM) and 5 ml of phosphate buffer (pH 7.4). One milliliter of the extract (10 mg/ml) in water was rapidly mixed with the reaction mixture by a gentle swirling motion at room temperature. One milliliter portion of the reaction mixture was then blown into 2 ml of dichromate/acetic acid reagent at 60 s intervals. The decomposition of the hydrogen peroxide was determined based on the standard plot for H2O2 and the monomolecular velocity constant K for the decomposition of H2O2 was determined by the use of the following Formula
K=1/t log10 S0/S
Where, S0 is the initial concentration and S is the final concentration of H2O2
Super Oxide Scavenging Activity
Super oxide scavenging was carried out by using alkaline DMSO method 12 Solid potassium superoxide was allowed to stand in contact with dry DMSO for at least 24 h and the solution was filtered immediately before use. Filtrate (200ml) was added to 2.8ml of an aqueous solution containing nitroblue tetrazolium (56 m M), EDTA (10m M) and potassium phophate buffer (10mM, pH 7.4). Sample extract (1 ml) at various concentrations (30—1500m g/ml) in water was added and the absorbance was recorded at 560 nm against a control in which pure DMSO has been added instead of alkaline DMSO.
Lipid Peroxidation Inhibition. Liver Homogenate
Male Sprague-Drawley rats (160—180 g) were purchased from the animal house of the Gold Mohur Lipton India Ltd, India. Ethical clearance for the animal study was obtained from the institutional animal ethics committee These were kept in the departmental animal house at 26 ± 2°C and relative humidity 44—55% light and dark cycles of 10 and 14 h respectively for one week before the experiment. Animals were provided with rodent diet (Amruth, India) and water ad libitum. Randomly selected male rats were fasted overnight and were sacrificed by cervical dislocation, dissected and abdominal cavity was perfused with 0.9% saline. Whole liver was taken out and visible clots were removed and weighed amount of liver was processed to get 10% homogenate in cold phosphate buffered saline, pH 7.4 using glass teflon homogeniser and filtered to get a clear homogenate.
Assay Of Lipid Peroxidation
The degree of lipid peroxidation was assayed by estimating the thiobarbituric acid-reactive substances (TBARS) by using the standard method 13 In brief, different concentration of extracts (200—1000m g/ml) in water was added to the liver homogenate. Lipid peroxidation was initiated by adding 100m l of 15mM FeSO4 solution to 3 ml of liver homogenate (final concentration was 0.5mM). After 30 min, 100m l of this reaction mixture was taken in a tube containing 1.5 ml of 10% TCA. After 10 min tubes were centrifuged and supernatant was separated and mixed with 1.5 ml of 0.67% TBA in 50% acetic acid. The mixture was heated in a hot water bath at 85 °C for 30 min to complete the reaction. The intensity of pink coloured complex formed was measured at 535 nm. The values of TBARS were calculated from a standard curve (absorption against concentration of Tetraethoxy propane) and expressed as nmol/mg of protein. The percentage inhibition of lipid peroxidation was calculated by comparing the results of the test with those of controls not treated with the extracts.
For the determination of the integral antioxidative capacity (AC) of the water soluble substances in
(PCL) was used. Apparatus used was Photochem® with Standard kit ACW (Analitik jena AG), where the luminol plays a double role of photosensitizer as well as the radical detecting agent. Lyophilized extract was measured at 10 ?g g/ml concentration. A standard plot was plotted and the results were calculated in ascorbic acid equivalents (?mol/g).
Quantitative Densitometric Assay for Estimation of Gallic Acid
1 mg of pure gallic acid standard was dissolved in 10 ml of methanol. 1 g of powdered drug material was macerated with 10ml of 80% methanol. This mixture was refluxed at 60 °C for 3h and decanted through filter paper. Same process was repeated for 2 more times and all the filtrates were pooled and made up to 250 ml. From this 10µl was applied on TLC plates. 10µl of the standard and the sample respectively were then applicated using the CAMAG Linomat V applicator onto the precoated silica gel F 254 plates (Merck) of 0.2 mm thickness plates. The plate was then eluted in solvent system. Toluene: Ethyl acetate: Formic acid (5:4:1). After elution, the plate was dried and scanned densitometrically using CAMAG TLC scanner 3 at 272 nm. The percentage of gallic acid in the extract was calculated by calibration using peak height ratio.
Result And Discussion
Free radical oxidative stress has been implicated in the pathogenesis of a wide variety of clinical disorders, resulting usually from deficient natural antioxidant defenses. In most diseases, increased oxidant formation is a consequence of the disease activity. Potential antioxidant therapy therefore should include either natural free radical scavenging antioxidant principles or agents, which are capable of augmenting the activity of the antioxidant enzymes. ROS are capable of damaging biological macromolecules such as DNA, carbohydrates or proteins. ROS is a collective term, which includes not only the oxygen radicals (O2 ·_ , and OH · ) but also some non-radical derivatives of oxygen these include H2O2, HOCl and ozone (O3). If human disease is believed to be due to the imbalance between oxidative stress and antioxidative defense, it is possible to limit oxidative tissue damage and hence prevent disease progression by antioxidant defense supplements 14
DPPH is stable nitrogen centered free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agents, then losing colour stoichometrically with the number of electrons consumed, which is measured spectrophotometrically at 517 nm. 15 As shown in
The total antioxidant capacity of the extract was calculated based on the formation of the phosphomolybdenum complex which was measured spectrophotometrically at 695 nm. The total antioxidant capacity of the extract was found to be 152.91 nmol/g ascorbic acid. Thus establishing the extract as an antioxidant.
Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H2O2 can probably react with Fe 2+ and possibly Cu 2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects 17 . It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate.Two types of enzymes exist to remove hydrogen peroxide within cells. They are the catalases and the peroxidases, which leads to ground state oxygen without any singlet oxygen. The extract may have decomposition of H2O2 activity due to any of these enzymes 17 .
Initiation of the lipid peroxidation by ferrous sulphate takes place either through ferryl–perferryl complex or through OH · radical by Fenton's reaction.
The present study aimed to evaluate the possible antioxidant activity of the