|Year : 2013 | Volume
| Issue : 3 | Page : 214-223
In vitro antioxidant and H + , K + -ATPase inhibition activities of Acalypha wilkesiana foliage extract
Rajesh Kashi Prakash Gupta1, Pradeepa2, Manjunatha Hanumanthappa1
1 Department of PG Studies and Research in Biotechnology and Bioinformatics, Jnanasahyadri, Kuvempu University, Shankaraghatta, India
2 Department of Biotechnology, NMAM Institute of Technology, Nitte, Karkala, Udupi, Karnataka, India
|Date of Web Publication||23-Aug-2013|
Department of PG Studies and Research in Biotechnology and Bioinformatics, Jnanasahyadri, Kuvempu University, Shankaraghatta
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims: The aim of this study was to evaluate the antioxidant activty and anti-acid property of Acalypha wilkesiana foliage extract. Materials and Methods: Hot and cold aqueous extracts were prepared from healthy leaves of A. wilkesiana. Free radical scavenging activity and H + , K + -ATPase inhibition activities of aqueous foliage extracts was screened by in vitro models. Statistical Analysis Used: All experiments were performed in triplicate and results are expressed as mean ± SEM. Results: A. wilkesiana hot aqueous extract (AWHE) showed significant antioxidants and free radical scavenging activity. Further, AWHE has shown a potent H + , K + -ATPase inhibitory activity (IC 50 : 51.5 ± 0.28 μg/ml) when compare to standard proton pump inhibitor omeprazole (56.2 ± 0.64 μg/ml); however, latter activity is equal to A. wilkesiana cold aqueous extract (AWCE). Quantitative analysis of AWHE has revealed more content of phenols and flavonoids; this is found to be the reason for good antioxidant activity over AWCE. Molecular docking was carried out against H + , K + -ATPase enzyme crystal structure to validate the anti-acid activity of A. wilkesiana major phytochemicals. Conclusions: The present study indicates that the constituents of AWHE and AWCE have good antacid and free radical scavenging activity.
Keywords: Acalypha wilkesiana, Anti-acid, antioxidant, gallic acid, gastric ulcer, molecular docking, polyphenols,
|How to cite this article:|
Gupta RK, Pradeepa, Hanumanthappa M. In vitro antioxidant and H + , K + -ATPase inhibition activities of Acalypha wilkesiana foliage extract. J Pharm Bioall Sci 2013;5:214-23
|How to cite this URL:|
Gupta RK, Pradeepa, Hanumanthappa M. In vitro antioxidant and H + , K + -ATPase inhibition activities of Acalypha wilkesiana foliage extract. J Pharm Bioall Sci [serial online] 2013 [cited 2019 Oct 23];5:214-23. Available from: http://www.jpbsonline.org/text.asp?2013/5/3/214/116822
Gastric ulcer is a common global problem with an increasing incidence and the prevalence in modern society. It is a kind of heterogeneous disorder causing due to imbalance between defensive and aggressive factors such as stress,  exposure to bacterial infection,  and use of the non-steroidal anti-inflammatory drugs [Figure 1].  It is being one of the most rampant gastrointestinal disorders continues to occupy a key position in concern of both, clinical practitioners, and researchers.
A modest approach to prevent ulceration is through enhancement of antioxidants, gastric mucin synthesis, scavenging of reactive oxygen species (ROS), inhibition of H + , K + - ATPase and Helicobacter pylori growth in the stomach  although the anti-secretory drugs, such as proton potassium ATPase (PPA) pump inhibitors-omeprazole, pantoprazole etc. and histamine H2-receptor blockers-ranitidine, famotidine etc. are being used to control an acid secretion and acid related disorders.
Proton pump inhibitors (PPIs) are widely prescribed for acid-peptic disease. In general, safety of this class of drugs has been excellent. However, in the past several years, epidemiologic studies have indicated possible risks that are biologically plausible. , Hence, there is an urgent need of PPIs of plant or animal origin with the minimal side-effects since ulcer is the multi-step disease as the drugs available in the market fails to exert multi-step antiulcer effect with elevation of antioxidant levels, proton pump, and H. pylori growth inhibition. There is an increased interest in recent years to investigate antiulcer phytochemical drug with the multi-step activity.
The plant Acalypha wilkesiana commonly called as copper leaf, Joseph's coat, and fire-dragon belongs to the sole genus Acalyphinae of the family Euphorbiaceae. The plant found all over the geographic area mainly used as an oornamental plant because of its striking foliage color. It consists of different terpenoides,  gallic acid, geranin, corilogin,  saponins, tannins, and glycosides.  The plant also has shown antibacterial, antifungal,  analgesic, antimalarial,  and antidiabetic activity. 
The present study was aimed to evaluate the antioxidant properties of hot and cold aqueous extracts of A. wilkesiana leaves and to relate their antioxidant power to antiulcer effect through proton pump inhibition.
| Materials and Methods|| |
Chemicals and instruments
Petroleum ether, ethanol, butylated hydroxyl anisole (BHA), 1-amino-2-naphthol-6-sulphonic acid (ANSA), perchloric acid, bovine serum albumin (BSA) were purchased from Merck (Germany). Gallic acid, catechin hydrate, quercetin, thio barbituric acid (TBA), and 2.2-diphenyl-1-picrylhydrazyl (DPPH), nitro blue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS) were purchased from Sigma Aldrich (Poznan´,Poland). Trichloroacetic acid (TCA), Folin-Ciocalteu reagent was procured from Himedia (India). Water was purified on a Milli-Q system from Millipore (Millipore, Bedford, MA, USA). Absorbance was measured using PC based double beam spectrophotometer-2202 (Systronics, India). All the chemicals used were of analytical grade.
Plant material and preparation of plant extract
Healthy leaves of A. wilkesiana were collected from the orchard of bioscience complex, Kuvempu University, Shankaraghatta, Shivamogga, Karnataka, India. Plant was identified as A. wilkesiana by Dr. V. Krishna, Professor, Department of Biotechnology and Bioinformatics, Kuvempu University, Shankaraghatta. Healthy leaves were separated, shade dried, and pulverized mechanically (Sieve No. 10/44). 100 g of leaf powder was defatted using the petroleum ether in soxhlet apparatus. Defatted material was divided into two equal parts (50 g each). One part of material was used for hot aqueous extraction. Total 50 g of defatted leaf powder was taken in 500 ml millipore water and boiled for 60 min, cooled and filtered. The color solution was separated, dried, and referred as Acalypha wilkesiana hot aqueous extract (AWHE). Another part was used for cold aqueous extraction. Total 50 g of defatted leaf powder was mixed with 500 ml of millipore water mixed well and kept in the dark for an overnight. Then, filtered, dried, and referred as Acalypha wilkesiana cold aqueous extract (AWCE). These extracts were stored in desiccator to avoid oxidation and used for further studies.
Qualitative phytochemical screening
The crude extracts were qualitatively examined for the presence of various phytochemical constituents using standard tests as described by Khanna and Kannabiran,  and Harborne. 
Quantitative analysis of crude extracts
Determination of total phenolic content
Total phenolic content in crude extracts was measured by the Folin-Ciocalteu method using gallic acid as standard.  In brief, 2 ml of both AWHE and AWCE at different concentration (50-150 μg) in millipore water was mixed with 2.5 ml of Folin-Ciocalteu reagent (diluted 1:10 v/v) followed by the addition of 2 ml of Na 2 CO 3 (7.5% w/v). Tubes were mixed and allowed to stand for 90 min at room temperature and absorbance of the sample was measured against the blank at 750 nm. Total phenolic content of the extract was expressed in terms of μg equivalent to gallic acid. All estimations were performed in triplicate and the results were averaged.
Determination of total flavonoid
Total flavonoids content of the extracts was determined according to modified method  using catechin as standard. Briefly, 2 ml of extracts at different concentrations (50-150 μg) in millipore water was mixed with 0.3 ml of 5% NaNO 2 and 0.3 ml of 10% AlCl 3 followed by the addition of 2 ml of 1 M NaOH after the incubation of the reaction mixture at room temperature for 6 min. Later, the volume in each test tube was made up to 5 ml by adding 2.4 ml of millipore water. Absorbance was measured against the blank at 510 nm. Total flavonoid content of the extract was expressed in terms of μg equivalent to catechin. Test was performed in triplicate and the results were averaged.
Determination of antioxidant activity
Total antioxidant capacity
Total antioxidant capacity of AWHE and AWCE was performed by phosphomolybdenum method as described by Prieto et al.,  Both extracts at different concentrations (50-150 μg) in millipore water were taken in separate test tubes. To this, 3 ml of reagent mixture containing 4 mM ammonium molybdate, 0.6 M sulfuric acid and 28 mM of sodium phosphate was added. Test tubes were kept for incubation at 95°C for 90 min. and allowed to cool. Absorbance of the content in each test tube was measured at 695 nm against blank. Antioxidant capacity of each extract is expressed as equivalents of ascorbic acid. Ascorbic acid equivalents are calculated using the standard graph of ascorbic acid. Test was performed in triplicate and the results were averaged.
Total reductive capability
Total reductive capacity of AWHE and AWCE was determined according to the method of Oyaizu,  Both extracts at different concentrations (50-150 μg) in millipore water mixed with phosphate buffer (2.5 ml, 0.2 mol/L, pH 6.5) and potassium ferricyanide (2.5 ml, 1%). Then, the mixture was incubated at 50°C for 20 min. At the end of the incubation, TCA (2.5 ml, 10%) was added to the mixture, which was centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was collected and mixed with 2.5 ml of millipore water and ferric chloride (0.5 ml, 0.1%). The absorbance was measured at 700 nm against a blank. Increased absorbance of the reaction mixture indicates increased reducing power. Test was performed in triplicate and the results were averaged. Reducing the capacity of each extract is expressed as equivalents of quercetin.
DPPH radical scavenging activity
AWHE and AWCE were screened for free radical scavenging activity by DPPH method of Braca et al.,  Free radical scavenging activity of the extracts was carried based on the scavenging activity of stable DPPH. Extracts in millipore water at different concentration (5-15 μg) were added to each test tube and volume was made up to 2 ml using the millipore water. To this 3 ml of 0.004% DPPH in 95% ethanol was added and the mixtures were incubated at room temperature under dark condition for 30 min. The scavenging activity on the DPPH radical was determined by measuring the absorbance at 517 nm. Radical scavenging activity was calculated using the formula: % of radical scavenging activity = ([(A control−A test )/A control ]) × 100, where A control is the absorbance of the control sample and A test is the absorbance of the test sample. The DPPH radical scavenging activity of BHA was also assayed for comparison. Test was performed in triplicate and the results were averaged.
Superoxide radical scavenging assay
Super oxide anion radical scavenging activity of AWHE and AWCE was measured according to the method of Nishikimi et al., 1972  with slight modification. All the solutions used in this experiment were prepared in phosphate buffer (pH 7.4). 1 ml of NBT (156 μM), 1 ml of NADH (468 μM) and 3 ml of extracts at different concentration (50-150 μg) were added. The reaction was started by adding 100 μl of PMS (60 μM) and the mixture was incubated at 25°C for 5 min. fallowed by the measurement of absorbance at 560 nm. Decreased absorbance of the reaction mixture indicated increased super oxide anion radical scavenging activity. The percentage inhibition was calculated from the formula, % of radical scavenging activity = ([(A control−A test )/A control ]) × 100, where A control is the absorbance of the control sample and A test is the absorbance of the test sample. The Super oxide anion radical scavenging of ascorbic acid was also assayed for comparison. Test was performed in triplicate and the results were averaged.
Lipid peroxidation inhibition assay
TBA reacts with malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected spectrophotometrically at 532 nm.  10% of egg homogenate was prepared using 0.15 M KCl. 0.5 ml of egg homogenate and 2 ml of AWHE and AWCE in millipore water at different concentrations (50-150 μg) were taken in test tubes. 100 μl of ferric chloride (0.2 mM) was added to each test tube and incubated at room temperature for 30 min. to induce lipid peroxidation. The reaction was stopped by adding 2 ml of ice-cold HCl (0.25 N) containing 15% TCA, 0.38% TBA, and 0.5% BHA. The content was mixed thoroughly and heated on boiling water bath for 60 min. Reaction mixture was cooled and centrifuged at 3000 rpm for 10 min. Absorbance of the supernatant was measured at 532 nm. Percentage of inhibition was calculated from the formula, % of inhibition = ([(A control−A test )/A control ]) × 100, where A control is the absorbance of the control sample and A test is the absorbance of the test sample. Test was performed in triplicate and the results were averaged.
Determination H + , K + -ATPase inhibition
Preparation of H + , K + -ATPase enzyme
To prepare H + , K + - ATPase enzyme sample, fresh sheep stomach was obtained from a local slaughterhouse at Lakkavalli, Karnataka, India. The stomach was cut opened, the mucosa at gastric fundus was cut-off, and the inner layer was scraped out for parietal cells.  Thus obtained cells were homogenized in 16 mM Tris buffer (pH 7.4) containing 10% Triton X-100 and centrifuged at 6000 g for 10 min. The supernatant (enzyme extract) was used to determine the H + , K + - ATPase inhibition. Protein content of the cell extract was determined according to Bradford's method  using the BSA as standard.
Assessment of H + , K + -ATPase inhibition
The reaction mixture containing 0.1 ml of enzyme extract (300 μg) and plant extract at different concentrations was pre-incubated for 60 min at 37°C. The reaction was initiated by adding substrate 2 mM ATP (200 μL), in addition to this 2 mM MgCl 2 (200 μL) and 10 mM KCl (200 μL) was added. After 30 min of incubation at 37°C, the reaction was stopped by the addition of assay mixture containing 4.5% ammonium molybdate and 60% perchloric acid followed by centrifugation at 2000 g for 10 min and inorganic phosphate released was measured spectrophotometrically at 660 nm by following Fiske-Subbarow method.  Briefly, to the 1 ml of supernatant 4 ml of millipore water, 1 ml of 2.5% ammonium molybdate, 0.4 ml of ANSA was added and allowed to stand for 10 min at room temperature. Absorbance of released inorganic phosphate was measured at 660 nm. Enzyme activity was calculated as micromoles of Pi released per hour at various doses (0-100 μg) of AWCE and AWHE. Results were compared with the known antiulcer PPA inhibitor drug omeprazole and expressed as Mean ± SEM.
Percentage of enzyme inhibition was calculated by using the formula;
Percentage of inhibition= [Activity (control) - Activity (test) /Activity (control) ] × 100.
Molecular docking studies
As reports of earlier investigator suggests, A. wilkesiana consists of a major pharmacologically active antioxidant molecule signatures such as gallic acid, corilagin, geranin, quercetin, kaempferol, artemetin, luteolin, and vitexicarpin. , Molecular docking was performed for these reported phytochemicals of A. wilkesiana against PPA to substantiate the in vitro PPA inhibition by extract and their eventual comparison with standard omeprazole, being used in the treatment of gastric acid-secretory related disorder. A Lamarckian genetic algorithm method implemented in the program AutoDock 4.2 was employed to determine the orientation of phytochemicals with the active site of PPA. The ligand molecules [Table 1] were designed and the structure was analyzed by using ChemDraw Ultra 6.0. 3D coordinates prepared using PRODRG (http://davapc1.bioch.dundee.ac.uk/prodrg/) server.  The protein structure file PDB ID: 2XZB [Figure 2] was taken from PDB (www.rcsb.org/pdb) and edited by removing the heteroatoms with simultaneous adding of C terminal oxygen.  For docking calculations, Gasteigere-Marsili partial charges  were assigned to the ligands, non-polar hydrogen atoms were merged and all torsions were allowed to rotate during docking. Active pockets were identified and ligplot of PDB Sum provided in the external links of PDB for the proteins was downloaded from PDB. Computed atlas of surface topography of proteins server was used to cross-check the active pockets on the target protein molecules. The Lamarckian genetic algorithm and the pseudo-Solis and Wets methods were applied for energy minimization using default parameters.
Results are expressed as Mean ± SEM of three parallel measurements. Statistical analysis was carried out by using the SPSS 17.0.
| Results|| |
Many studies have shown the potential benefits of antioxidants from plant sources. The present study showing the antioxidant activity and PPA inhibition property of foliage extracts of A. wilkesiana.
Qualitative analysis of crude extracts
The AWHE and AWCE was extracted from the foliage part of A. wilkesiana and the preliminary phytochemical screening indicates the presence of flavonoids, glycosides, triterpenoids, saponins, and tannins [Table 2].
Quantitative analysis of crude extracts
Total phenolic contents of AWHE and AWCE are expressed as μg equivalent to gallic acid. Among the extracts, AWHE has a significant amount of phenolic content (0.888 g/g of extract) when compare to AWCE (0.666 g/g of extract).
Flavonoids are important secondary plant metabolites, which increase with plant stress. , The total flavonoid content of AWHE and AWCE was found to be 0.427 and 0.424 g/g of dry extract respectively.
Determination of antioxidant activity
Total antioxidant capacity
Total antioxidant capacity of both extracts was performed by phosphomolybdenum method as described by earlier.  Antioxidant capacities are expressed as equivalents of ascorbic acid. Both extracts had shown significant antioxidant activity as equivalents to ascorbic acid. The results of total antioxidant activity were presented in [Figure 3].
|Figure 3: Antioxidant activity of AWHE and AWCE (a) Total antioxidant capacity, (b) total reductive capability, (c) DPPH radical scavenging activity, (d) Superoxide radical scavenging activity and (e) Lipid peroxidation inhibition activity|
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Total reductive capacity
The reductive ability of extracts was determined according to the method of Oyaizu, 1986.  Quercetin was used as a standard to compare the activity of extracts. Among the extracts, AWCE showed excellent activity, whereas AWHE showed moderate activity [Figure 3]b.
DPPH radical scavenging activity
Both extracts were screened for free radical scavenging activity using the DPPH method and it is based on the measurement of the reducing ability of antioxidants on DPPH free radical. , [Figure 3]c represents the percentage of DPPH scavenging activity of AWHE, AWCE and BHT. AWHE and AWCE showed significant radical scavenging activity (79.9 ± 0.89 and 78.8 ± 0.52%) at 15 μg when compare to similar concentration of BHT (64.52 ± 0.56%). The variation exhibited in DPPH scavenging capacity was attributed to the synergistic contribution of mixture of more phenolics, flavonoids, and other molecules in AWHE and AWCE.
Superoxide radical scavenging assay
AWHE and AWCE were screened for superoxide radical scavenging activity using ascorbic acid as a standard. Superoxide radical is known to be very harmful as a precursor of more reactive oxidative species that have potential to react with biological macromolecules and thereby inducing tissue damage.  Superoxide radical scavenging activity of extracts is presented in [Figure 3]d. Both the extracts showed significant dose dependent activity. At 150 μg, AWHE and AWCE showed 73.2 ± 1.02 and 68.6 ± 0.78% of inhibition respectively when compare to BHT (71.0 ± 0.25%).
Lipid peroxidation inhibition assay
The results of the AWHE and AWCE to prevent lipid peroxidation were shown in [Figure 3]e. At 150 μg, AWHE and AWCE showed 72.9% and 70.2% of inhibition respectively. Lipid peroxidation, a well-established mechanism of cellular injury in plants and animals is used as an indicator of oxidative stress in cells and tissues. Polyunsaturated fatty acid peroxides generate MDA and the measurement of MDA has been used as an indicator of lipid peroxidation by means of derivatizing with TBA at high temperature and acidic condition. 
H + , K + -ATPase inhibition study
[Figure 4] suggests the PPA inhibition activity of AWCE and AWHE as well as omeprazole as the reference standard. Both extracts showed dose depended activity. At a concentration of 100 μg/ml AWHE, AWCE, and omeprazole showed 88.7 ± 1.54, 79.99 ± 1.43 and 86.7 ± 0.56% of inhibition respectively. The concentration required to inhibit 50% of H + , K + - ATPase activity is designated as IC 50 , thereby AWHE and AWCE showed IC 50 values of 51.5 ± 0.28 and 56.3 ± 0.89 μg/ml respectively, in comparison with omeprazole IC 50 value of 56.2 ± 0.64 μg/ml
|Figure 4: H+, K+ -ATPase inhibition study by AWHE and AWCE in comparison to Omeprazole|
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Validation of H + , K + -ATPase inhibition through molecular docking studies
Automated docking was used to assess the orientation of inhibitors bound with the active pockets of PPA. In general, binding of inhibitor to enzyme leads to change in the confirmation of enzyme and consequently arrest its activity. [Figure 5] clearly indicates, the interaction of phytoconstituents of A. wilkesiana with the active pocket of PPA. Interestingly, all the phytoconstituents showed minimum binding energy with PPA through non-covalent interaction [Table 3]. The in silico observation confirm the in vitro PPA inhibitory activity of AWHE and AWCE. The activity of extracts is due to stable and active synergistic role of major phytoconstituents present in A. wilkesiana.
|Figure 5: Molecular docking studies of major phytoconstituent (a) artemetin, (b) corilagin, (c) gallic acid, (d) geranin, (e) kaempferol, (f) lutein, (g) luteolin, (h) quercetin, (i) vitexicarpin and (j) omeprazole against H+, K+-ATPase|
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|Table 3: Molecular docking studies of phytoconstituents of A. wilkesiana |
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| Discussion|| |
Metabolic pathway of the human body is continuously exposures to several degradative stresses, such as ROS and free radicals. These reactive species extensively cause the oxidative damage to the biomolecules nucleic acids, proteins and lipids  and contribute to the pathogenesis of oxidative stress related diseases such as cancer, ageing, heart failure, ulcer etc.  Antioxidants are considered as possible protective agents against oxidative damage of the human body. Consequently, there is a growing interest in the substances exhibiting antioxidant properties that are supplied to human and animal organisms. In that concern, natural antioxidants have become one of the major areas of scientific research. ,
The effective antioxidants from natural sources are the only alternative to synthetic molecules in scavenging the free radicals during the prevention of accelerating pathogenicity. The present study demonstrates the in vitro free radical scavenging activity and PPA inhibition activity of aqueous extracts of A. wilkesiana. The study was further validated by in silico approach.
In this study, A. wilkesiana hot and cold leaf extracts were prepared and preliminary phytochemical investigation indicates the presence of various metabolites having good antioxidant activity such as phenolics, flavonoids, tannins, terpenes etc. In general, phenol and flavonoid content of plant plays a significant role in scavenging free radicals by acting as an electron donor and serves as antioxidants with considerable health benefits. Interestingly, both AWHE and AWCE consist of the enormous amount of phenol and flavonoid content and showed significant free radical scavenging activity at a minimum concentration and the result is in agreement with the presence of various antioxidant molecules in A. wilkesiana has revealed by Adesina et al.  and Eugene et al.  of which, AWHE showed significant antioxidant, reducing and free radical scavenging activity compare to AWCE since in hot condition most of the secondary metabolites dissolve readily into the water.
Along with free radical scavenging activity and antioxidant activity, H + , K + - ATPase inhibition by AWHE and AWCE was also assessed. The human stomach is found with the numerous gastric pits from which acid get secret. One of the cells, which lining the gastric pits is parietal cell, which is responsible for the acidification of stomach. PPA, the proton pump present in parietal cell is responsible for acid, which locates in the gastric membrane vesicle and actively transports protons into the lumen of stomach with the hydrolysis of the cytoplasmic ATP.  Hyper secretion of this enzyme leads to acidity and ulcer. Therefore, this regulatory enzyme has found to be a pharmacological target to treat ulcer. Presently, ulcer is treated by providing allopathic PPIs, antioxidants, H 2 -receptor antagonist etc., but these are associated with side- effects. Hence, there is an urgent need of PPI of plant origin. Consequently, our present study is on in vitro PPA inhibition activity of foliage extract of A. wilkesiana. The study was evaluated by using sheep parietal cells where omeprazole was used as positive standard. AWHE showed a high degree of PPA inhibition. To further validate and for better understanding of PPA inhibition activity by A. wilkesiana, an in silico molecular docking approach was done with major reported phytoconstituents present in A. wilkesiana against the crystal structure of PPA. All the molecules showed good interaction with minimum binding energy in the enzyme active pocket through non-covalent interaction.
Analysis of in-vitro and in silico results indicates that AWHE and AWCE has good free radical scavenging activity by virtue of total antioxidant property, pertaining to this it also showed significant PPA inhibition activity indicating its presumed anti-gastric ulcer property.
Furthermore, detailed studies on the isolation and characterization of the plant extract as well as in vivo assays will be necessary in discovering new biological antioxidants and anti-gastric ulcer drug.
| Acknowledgments|| |
Authors are grateful to DBT, New Delhi for financial assistance and also thankful to Registrar, Kuvempu University, Shankaraghatta, Karnataka, India for providing necessary facilities to carry out the study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]
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