Free Radical Scavenging Assay

ABSTRACT: Strychnos potatorum Linn. is a medium sized deciduous tree belonging to the family Loganiaceae. The collected seeds of Strychnos potatorum were dried and powdered. Methanolic extract of these seeds was prepared by cold maceration. The preliminary phytochemical screening of the crude extract revealed the presence of flavonoids and phenolic compounds. The crude extract was partitioned (gradient) with different solvents like chloroform, ethyl acetate, acetone and water. After conducting preliminary in vitro DPPH free radical scavenging assay on these fractions, the one with maximum antioxidant activity (bioactive fraction) was selected and screened with different in vitro antioxidant methods. The different in vitro antioxidant methods include DPPH free radical scavenging assay, nitric oxide radical scavenging assay, superoxide radical scavenging assay, hydroxyl radical scavenging assay and inhibition of peroxide formation method. The percentage inhibition of free radicals in each method was compared with standard drug. Ascorbic acid and α- tocopherol were used as standard drugs. The seeds of Strychnos potatorum have shown significant antioxidant activity.

Flowers of higher plants have been used for centuries for several purposes such as medicine, food and garnishing food in many parts of the world. In the present study, we have determined the antioxidant and antimicrobial activity of methanol extract of flowers of Wendlandia thyrsoidea (Roemer & Schultes) Steudel (Rubiaceae), Olea dioica Roxb. (Oleaceae), Lagerstroemia speciosa L. (Lythraceae) and Bombax malabaricum DC. (Bombacaceae). Antioxidant efficacy of various concentrations of flower extracts was evaluated by DPPH free radical scavenging assay and Ferric reducing assay. Antimicrobial activity was determined against four bacteria and two fungi by agar well diffusion method. Total phenolic and flavonoid contents were determined by Folin - Ciocalteau reagent and Aluminium chloride colorimetric estimation methods respectively. The DPPH free radical scavenging effect of flower extracts was concentration dependent and was higher in case of extract of L. speciosa followed by W. thyrsoidea, B. malabaricum and O. dioica. In ferric reducing assay, it was shown that the absorbance of reaction mixture at 700nm increased on increasing the concentrations of flower extracts indicating reducing power of extracts. The reducing ability was also highest in L. speciosa extract. Extract of L. speciosa displayed marked inhibitory activity against bacteria and fungi than other flower extracts. Gram positive bacteria have shown more susceptibility than Gram negative bacteria. Among fungi, C. neoformans was more inhibited than C. albicans. Extracts of B. malabaricum and O. dioica were not effective against C. albicans. The phenolic and flavonoid contents were higher in L. speciosa and O. dioica respectively. A positive correlation has been observed between total phenolic content of flower extracts and antioxidant and antimicrobial activity. The flowers can be employed as a remedy for treatment of infectious diseases and oxidative damage. Further, isolation of active components from flower extracts and their biological activity determinations are under progress.

The present study was undertaken to determine antimicrobial and antioxidant activities of leaf and flower extracts of Caesalpinia pulcherrima, Delonix regia and Peltaphorum ferrugineum. Antimicrobial activity was tested against Staphylococcus aureus, Salmonella typhi, Candida albicans and Cryptococcus neoformans by Agar well diffusion assay. Antioxidant activity was determined by DPPH free radical scavenging assay, ABTS free radical scavenging assay, Ferric reducing assay and Total antioxidant capacity determination. Total phenolic content of extracts was estimated by Folin-Ciocalteau Reagent method. S. typhi and C. neoformans were susceptible to extracts to greater extent than S. aureus and C. albicans among bacteria and fungi respectively. Except C. pulcherrima extract, the leaf extracts were more effective in inhibiting bacteria than flower extracts. Leaf extracts have shown high antifungal activity than flower extracts. The extracts have shown dose dependent scavenging of DPPH and ABTS radicals. Scavenging of ABTS radicals was more efficient than that of DPPH radicals as revealed by low IC50 values. All leaf extracts except C. pulcherrima displayed stronger scavenging activities than flower extracts. Similar results were observed in ferric reducing assay and total antioxidant capacity determination. Total phenolic content was found to be higher in leaf extracts (except C. pulcherrima) than flower extracts. A correlation has been observed between phenolic content of leaf and flower extracts and the antioxidant activity. A marked antimicrobial and antioxidant activity of leaf and flower extracts was observed which may be attributed to the presence of phenolic compounds and other phytochemicals. The plants can be used to control infectious diseases and oxidative damage.

The antioxidant activity of NSP was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. NSP was mixed with 95% methanol to prepare the stock solution in the required concentration (10mg/100ml or 100μg/ml). From this stock solution, 1ml, 2ml, 4ml, 6ml, 8ml and 10ml was taken in six test tubes and by serial dilution with the same solvent, the final volume of each test tube was up to 10 ml whose concentration was then 10 μg/ml, 20 μg/ml, 40μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml respectively. Ascorbic acid was used as a standard which was prepared in the same concentration as that of the sample extract by using methanol as solvent. Final reaction mixture containing 1 ml of 0.3 mm DPPH methanol solution was added to 2.5 ml of sample solution of different concentrations and allowed to react at room temperature. Absorbance in the presence of test sample at different concentration (10 µg, 20 µg, 40 µg, 60 µg, 80 µg and 100µg/ml) was noted after 15 mins incubation period at 37 0 C. Absorbance was read out at 517 nm using double-beam U.V Spectrophotometer by using methanol as blank. The effective concentration of test sample required to scavenge DPPH radical by 50% (IC 50 value) was

Antioxidant activity of X. sagittifolium was tested using DPPH (2,2-Diphenyl-1- picrylhydrazyl) free radical scavenging assay method. Different concentrations (1-512 μg /mL) of the extract were added to 3 mL of a 0.004% w/v solution of DPPH. The absorbance was then determined at 517 nm. Then % inhibitions were plotted against log concentration and IC50 was calculated from the graph. The experiment was performed in triplicate and average absorption was noted for each concentration. Tested bacterial strains and reagents

The antioxidant activity of HAESO fruits was investigated using total antioxidant activity capacity, DPPH free radical scavenging assay, and NO scavenging assay. The free radical DPPH possesses a characteristic absorption at 517 nm. The radical scavengers cause reduction of DPPH by providing proton, which is indicated by color change (from purple to yellow) and a decrease in its absorption. The HAESO proved to be almost equivalent in offering protection against DPPH free radicals as compared to the standard ascorbic acid. NO, and reactive nitrogen species (RNS) have been reported to be involved in the oxidative cellular damage 29 . The major reactive species of NO are nitrous anhydride (N 2 O 3 ) and peroxynitrite

Results: In the DPPH free radical scavenging assay and superoxide radical scavenging assay, the ethyl acetate soluble fraction of ethanolic extract revealed the highest free radical scavenging activity with IC 50 value of 2.65 and 155.62 μg/ ml, respectively as compared to standard ascorbic acid (IC 50 value of 5.8 and 99.66 μg/ml). Ethyl acetate fraction also possessed highest reducing power activity with an EC50 value of 15.27 μg/ml compared to ascorbic acid (EC 50 0.91 μg/ml). On the other hand, the carbon tetrachloride fraction exhibited most significant NO scavenging activity with IC 50 value of 277.8 μg/ml that was even higher than that of standard ascorbic acid (IC 50 value 356.04 μg/ml). In addition, the total phenolic contents of these extract and fractions were evaluated using Folin-Ciocalteu reagent and varied from 7.93 to 50.21 mg/g dry weight expressed as gallic acid equivalents (GAE).

Aim: The aim of the present study was to find the antioxidant and anti-inflammatory potential of Phaseolus vulgaris L. seeds ethanol extract using in- vitro models. Method: The antioxidants effects of Phaseolus vulgaris L. seeds ethanol extract was determined using free radical scavenging assay, reducing power assay, hydrogen peroxide radical scavenging and metal chelating activity. Inhibition of protein denaturation, anti-proteinase action and membrane stabilization methods which includes heat induced haemolysis, hypotonicity-induced haemolysis and anti-lipoxygenase activity were used to resolve the power of anti-inflammatory agents. IC 50 values are calculated by linear regression method. Different concentrations (100 - 500 µg/ml)

extracts found alkaloids, sterols, carbohydrate and glycosides, tannins and flavonoids. The fraction inhibition of lipid peroxide at the first stage of oxidation illustrated antioxidant activity of O. lamiifolium and O. basilicum as 90% and 88% compared to those of gallic acid (97%) and BHT (84%) respectively. Also, the aqueous leaves extract of O. lamiifolium and O. basilicum exhibited significant DPPH free radical scavenging activity, nitric acid free radical scavenging activity assay, superoxide anion scavenging activity, ABTS scavenging activity and hydrogen peroxide free radical scavenging assay. Our findings provide confirmation that the aqueous leaves extract of O. lamiifolium and O. basilicum are potential source of natural antioxidants, and this warranted its uses in traditional medicine systems.

The biological activity of the extract of Calamus travancoricus Bedd.ex. Becc. & Hk.f. has so far remained unknown. The present work on the in vitro antioxidant activity of the ethanolic fruit extract of this plant is the first investigation. The study deals with phytochemical screening and antioxidant activity of the ethanolic fruit extract of C. travancoricus. Phytochemical analysis of the extract revealed the presence of terpenoids, steroids, tannins and Flavanoids. The total phenolic content of the extract was found to be 85.3mg per gram of the extract. The free radical scavenging activity of the extract was confirmed in a DPPH assay, which showed the stronger radical scavenging effect with IC 50 value of 25 μg /ml. The extract exhibited

Free radicals are implicated for many diseases including diabetes mellitus, arthritis, cancer, ageing etc. In the treatment of these diseases, antioxidant therapy has gained utmost importance. Ethanolic extract of Alternanthera sessilis (EEAS) was studied for its in vitro antioxidant activity using different models viz. Free radical scavenging activity assay (DPPH method), Reducing power assay, Nitric oxide scavenging activity, Superoxide radical scavenging activity and Hydroxyl radical scavenging activity. Total phenolic content was determined by using Pyrocatechol as a standard. The results were analyzed statistically by the regression method. Its antioxidant activity was estimated by IC 50 value and the values are 74.05 ± 1.44 μg/ml (Superoxide radical scavenging), 31.50 ± 1.04

1) DPPH assay: Melanin particle were found to possess antioxidant property in biological systems. It can scavenge free radicals and has the ability to sequester redox active metal Ions [14]. Free radical scavenging activity was evaluated by performing in- vitro DPPH assay. The colored DPPH solution faded reducibly to half during the course of incubation of 54 hours (3 rd day), 21 hours (1 st day), and 16 hours (1 st day) for 14.9, 29.8 and 44.7μg/mL of melanin concentrations respectively (Table II). This may be due to the reduction of the DPPH molecules and electron transfer from melanin suspension. Graph of dose dependant percent scavenging activity at residual period (Fig. 3) also indicates a linear pattern of DPPH reduction for various melanin doses used. The residual period was obtained after 84 hours (Table I) as there was a remarkable decrease in % DPPH contents (Fig. 2).

tubes containing either 500 µl of standard solutions of gallic acid (50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 µg/ml) or crude extracts (diluted 400-fold with distilled deionised water) was prepared, 500 µl of 10% folin-ciocalteu’s phenol reagent (in double distilled water) was added into each test tube and mixed. After incubation for 20 min at room temperature, the absorbance was determined at 750 nm against the parallel prepared blank (500 µl of DDW+500 µl of 10% FC reagent+350 µl of 1 M Na 2 CO 3 solution). In the present investigation different solvent extracts of Morinda tinctoria leaves were examined for their total phenolic content and free radical scavenging activity. The total phenolic content is presented in Table 1. The highest phenolic content was obtained in methanol followed by chloroform, ethyl acetate and hexane. The antioxidant assay was performed in the whole extract as a bio active individual component can change its properties in the presence of other compounds due to addition and synergistic effects of phytochemicals present in the extract. In the present

Free radicals are implicated for many diseases including Diabetes mellitus, arthritis, cancer, ageing. etc. In treatment of these diseases, antioxidant therapy has gained utmost importance. Phyllanthus reticulatus Poir (Euphrobiaceae) popularly known as ‘potato- bush’ is an important medicinal plant and useful in burning sensation, strangury, gastropathy, ophthalmodynia, burns, diarrhoea, skin eruption, diabetes and obesity. Keeping in view of the cited activity, it is contemplated to screen the plant for in vitro antioxidant activity using different models viz. DPPH radical scavenging, ABTS radical scavenging, iron chelating activity and lipid peroxidation assay, nitric oxide scavenging assay, alkaline DMSO assay, total antioxidant capacity and non-enzymatic haemoglobin glycosylation assay . The results were analyzed statistically by regression method. Its antioxidant activity was estimated by IC 50 value and the values are 20.36 µgm/ml (DPPH

Egg phosphotidylcholine (20mg) in chloroform (2ml) was dried under vacuum in a rotary evaporator to give a thin homogenous film and further dispersed in normal saline (5ml) with a vortex mixture. The mixture was sonicated to get a homogeneous suspension of liposome. Lipid peroxidation was initiated by adding 0.05 mM ascorbic acid to a mixture containing liposome (0.1ml). 150 mM potassium chloride, 0.2 mM ferric chloride, drug solution (2-100 µg/ml) were added separately in a total volume of 1ml. The reaction mixture was incubated for 40 min at 37ºC. After incubation, the reaction was terminated by adding 1ml of ice cold 0.25M sodium hydroxide containing 20% w/v TCA, 0.4% w/v TBA and 0.05% w/v BHT. After keeping in boiling water bath for 20 min, the samples were cooled. The pink chromogen was extracted with constant volume of n-butanol and absorbance of the upper organic layer was measured at 532nm. The experiment was performed in triplicate. Superoxide scavenging assay [19]

DPPH radical scavenging assay uses DPPH to check the ability of the antioxidative compounds (algal extract) acting as radical scavengers of proton. Substances possessing antioxidative activity will bring about the change of color from purple chromogen radical to the pale yellow hydrazine (Arguelles et al., 2017; Goh et al., 2010). This method has been used considerably as an initial method for screening of novel antioxidants because of its simplicity, reproducibility, and stability (Chen et al., 2008; Jiménez et al., 2010). Sargassum siliquosum exerted a potent radical scavenging activity against DPPH free radical showing inhibition percentage that is dose dependent (Table 1 and Fig. 1). The activity of the seaweed extract to scavenge DPPH increases when the tested algal extract concentration is also increased. Furthermore, S. siliquosum extract exhibited greater antioxidant activity than the positive control (ascorbic acid) with an IC 50 of 0.19 mg GAE ml −1 and 0.23 mg GAE ml −1 , respectively.

The free radical scavenging activities of the samples were measured using the stable DPPH radical assay method with some modifications. [8] Sample extracts of different concentrations were added to 1ml of 0.1mM methanolic DPPH solution. The mixture were shaken vigorously and allowed to stand for 20 min in the dark at room temperature and the absorbance was monitored at 517 nm. DPPH solution without samples served as the control. Ascorbic acid at a concentration range of 1-5 μg/ml was considered as standard. DPPH radical scavenging activity percentage was calculated for the samples and the standard using following formula;

NO acts as a free radical and inhibits several physiological processes such as smooth muscle relaxation and neuronal signaling inhibition of platelet aggregation and regulation of cell mediated toxicity (Banerjee et al., 2011; Singh et al., 2012). NO scavenging assay is based on the scavenging ability of the extracts as well as ascorbic acid, which is used as standard. The scavenging of NO was found to increase in dose dependent manner. Overproduction and accumulation of NO are associated with cytotoxic effects observed in various disorders like cancer, alzheimer’s disease, arthritis and AIDS (Sainani et al., 1997). Excess generation of NO can mediate toxic effects such as DNA fragmentation, cell damage and neuronal cell death (Dawson et al., 1992).

Oxidative stress (OS) caused by generation of reactive oxygen species (ROS) in course of biological activity results in various detrimental effect to the viability of the cell. To counter this effect antioxidants defense mechanisms exist. An imbalance between these necessitates extraneous antioxidant supplementation. Ionidum suffruticosum has been in use in traditional medicine mainly as aphrodisiac among various divergent uses. The plant was subjected to cold extraction method with ethanol, chloroform and aqueous solvents. Ascorbic acid was used as control. The efficacy of this plant as an antioxidant has been analyzed by DPPH free radical scavenging, ABTS free radical scavenging, ferric reducing power assay, superoxide anion radical scavenging, hydrogen peroxide scavenging and hydroxyl radical scavenging activity. Interpretation of IC 50 values showed maximum efficacy in DPPH assay

Methods: Shade dried and powdered flower material was extracted by maceration process using methanol. Antibacterial activity of flower extract was determined by agar well diffusion assay against gram positive and gram negative bacteria. Antifungal activity was tested against two molds namely Rhizopus sp. and Curvularia sp. by poisoned food technique. Antioxidant activity was evaluated by DPPH free radical scavenging and ABTS free radical scavenging assays and ferric reducing assay. Insecticidal activity was assessed in terms of larvicidal activity against I, II and III instar larvae of Aedes species and Anopheles species. Elemental analysis was carried out to estimate the content of major and minor elements.