EVALUATION OF ANTIOXIDANT POTENTIAL OF TEST SAMPLE USING 1, 1, DIPHENYL-2-PICRYLHYDRAZYL (DPPH) RADICAL SCAVENGING ACTIVITIES

 

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 (0.0625, 0.125, 0.25, 0.5, and 1 mg/ml) of the test sample will be prepared in methanol (analytical grade).

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

· 1ml of each studied sample will be transferred into a clean test tube into which 0.5ml of 0.3mM DPPH in methanol will be added.

· The mixture will be shaken and left to stand in the dark place at room temperature for 15 minutes.

· Blank solutions comprising of the studied sample solutions (2.5ml) and 1ml of methanol will be used as the baseline.

· The negative control comprised 2.5ml of DPPH solution and 1ml of methanol, while L-ascorbic acid at the same concentrations as the studied extracts will be used as the positive control.

· After incubation in the dark, the absorbance values will be measured at 517 nm using a spectrophotometer.

· The experiments will be performed in triplicate and the DPPH radical scavenging activity will be estimated using the equation described by Brand-Williams et al.

 % Radical scavenging activity = A_c – A_s/A_c × 100

 

Where A_s is the absorbance of the sample and A_c is the absorbance of the control.

The half-maximal inhibitory concentration (IC50) of the test samples will be computed from a plot of percentage DPPH free radical inhibition versus the sample concentration.

3.0 ENDPOINT PARAMETER(S):

· % Radical scavenging activity

4.0 REFERENCE(S):

4.1 Beatrice Muthoni Guchu, Alex King’ori Machocho, Stephen Kiruthi Mwihia, and Mathew Piero Ngugi. In Vitro Antioxidant Activities of Methanolic Extracts of Caesalpinia volkensii Harms, Vernonia lasiopus O. Hoffm., and Acacia hockii De Wild. Evidence-Based Complementary and Alternative Medicine Volume 2020, Article ID 3586268, 10 pages https://doi.org/10.1155/2020/3586268.

4.2 Shahinuzzaman, M., Yaakob, Z., Anuar, F.H. et al. In vitro antioxidant activity of Ficus carica L. latex from 18 different cultivars. Sci Rep 10, 10852 (2020). https://doi.org/10.1038/s41598-020-67765-1.

4.3 Kenny O., Brunton N.P., Smyth T.J. (2015) In Vitro Protocols for Measuring the Antioxidant Capacity of Algal Extracts. In: Stengel D., Connan S. (eds) Natural roducts From Marine Algae. Methods in Molecular Biology, vol 1308. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2684-8_24. 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.

4.4 Shukla ST, Kulkarni VH, Habbu PV, Jagadeesh KS, Patil BS, Smita DM. Hepatoprotective and antioxidant activities of crude fractions of endophytic fungi of Ocimum sanctum Linn. in rats. Oriental Pharmacy and Experimental Medicine. 2012 Jun;12(2):81-91.

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