EVALUATION OF NITRATE RADICAL SCAVENGING POTENTIAL OF TEST SAMPLE

                                                                       STUDY PROTOCOL 

1.0  INTRODUCTION:

Oxidative stress is the major driving factor responsible for the initiation and progression of cancer, diabetes mellitus, cardiovascular diseases, neurodegenerative diseases, and inflammatory diseases among other syndromes]. The condition is brought by excessive generation of free oxygen and nitrogen species or their inefficient quenching in the cell. Free oxygen and nitrogen species are unstable molecules that are present in the environment (exogenous) and are also generated in the body (endogenous) during the normal aerobic metabolic processes in the body. The body possesses a complex antioxidant defense system, comprising enzymatic and non-enzymatic pathways, which in the normal physiologic state, maintain a steady equilibrium between prooxidants and antioxidants, thereby ensuring well-being.

2.0 METHODOLOGY:

· Five different concentrations (From 100−1000 µg/mL) of the test sample will be prepared in methanol (analytical grade).

· The same concentrations will also be prepared for Gallic acid, which will use as a standard antioxidant.

· Nitric oxide will be generated from sodium nitroprusside (SNP) and measured by the Griess reaction.

· Griess reagent will be prepared by mixing equal amounts of 1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthyl ethylene diamine dihydrochloride in 2.5% phosphoric acid immediately before use.

· The reaction mixture (5.0ml) containing SNP (5mM) in phosphate-buffered saline (pH 7.3), with or without the test sample at different concentrations, will be incubated at 25˚C for 150min in front of a visible polychromatic light source (25 Watt tungsten lamp).

· The NO radical thus generated, interacted with oxygen to produce the nitrate ion (NO2– ) and will be assayed at 30 min intervals by mixing 1.0 ml of incubation mixture with an equal amount of Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% Nnaphthylethylenediamine dihydrochloride).

· The absorbance of the chromophore (purple azo dye) formed during the diazotization of nitrite ions with sulphanilamide and subsequent coupling with naphthyl ethylenediamine dihydrochloride will be measured at 546nm using a spectrophotometer.

· The nitrite generated in the presence or absence of the test sample Will be estimated using a standard curve based on sodium nitrite solutions of known concentrations.

· The percentage nitrite radical scavenging activity of the test sample and gallic acid will be calculated using the following formula:

% Radical scavenging activity = Ac – As/Ac × 100

Where As is the absorbance of the sample and Ac is the absorbance of the control.


3.0 ENDPOINT PARAMETER(S):

· % Radical scavenging activity

4.0 REFERENCE(S):

4.1 Fadzai Boora, Elaine Chirisa, and Stanley Mukanganyama. Evaluation of Nitrite Radical Scavenging Properties of Selected Zimbabwean Plant Extracts and Their Phytoconstituents. Journal of Food Processing Volume 2014, Article ID 918018, 7 pages http://dx.doi.org/10.1155/2014/918018.

4.2 Hardik Joshi, Manoj Pagare, Leena Patil, Vilasrao Kadam. In–Vitro Antioxidant Activity of Ethanolic Extract of Leaves of Buchanania lanzan Spreng. Research J. Pharm. and Tech. 4(6): June 2011; Page 920-924.

4.3 Owen Kenny, Nigel P. Brunton, and Thomas J. Smyth. In Vitro Protocols for Measuring the Antioxidant Capacity of Algal Extracts. Methods in Molecular Biology, vol. 1308, DOI 10.1007/978-1-4939-2684-8_24.

4.4 Habu JB, Ibeh BO. In vitro, antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia leave ethanolic leaf extract. Biol Res. 2015;48(1):16. Published 2015 Mar 14. doi:10.1186/s40659-015-0007-x

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