Screening Methods Of Antioxidants Activity On Animal Models

Antioxidants are endogenous or exogenous substance which inactivate the free radical.

Example:

I) Natural Origin-

1) Enzymatic:-           Superoxide dismutase, Catalase, Glutathione peroxidase.

2) Non enzymatic:-   Carotenoids, Ascorbic acid, Selenium, Tocopherol.

II) Synthetic origin - BHA

Use of synthetic antioxidants are restricted (eg- BHT)because they are suspected to be carcinogenic. So natural antioxidants gain importance.

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Free Radicals

Free radicals are molecules or molecular species that contain one or more unpaired electron and capable of independent existence.

Example- Superoxide(O₂), hydroperoxy  radical (HOO¯), lipid peroxide radical (ROO¯), nitric oxide (NO¯).

Non radical derivatives do not contain unpaired electron. 

Example- H₂O₂

Free radicals are highly reactive , having short half life, cause destruction, generate new radicals by chain reaction. 

Origin Of Free Radical:

Origin Of Free Radical

Formation Of Free Radicals:

Formation Of Free Radicals

Generation Of Oxygen Free Radical:

Generation Of Oxygen Free Radical

Generation  of Nitrogen Containing Free Radical

NO has  very high affinity to superoxide free radical and react rapidly to form an unstable Peroxynitrite moiety (ONOO*) and this is decomposed to hydroxy and nitrite free radical which are responsible for cytotoxic action of NO.

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Cytotoxicity By Free Radicals:

Cytotoxicity By Free Radicals

Free radical mediated reactions play important role in many forms of cell injury :

1. Atherosclerosis
2. Cellular aging
3. Killing of microbial agent
4. Hypoxia

Cellular antioxidants status determines the susceptibility to oxidation damage.

Molecular oxygen is an essential component of all living organism but all aerobic species suffer from injury if exposed to conc. more than 21%.

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Relation Between Free Radical And Antioxidant: 

Relation Between Free Radical And Antioxidant:

Working Of Antioxidant:

Working of antioxidant 

 Need of antioxidant and their screening:

Free radicals are leading cause of various diseases so antioxidant therapy is required. Screening methods are necessary for identification of suitable antioxidants and measurement of their ability to work as antioxidant.

Screening Method of antioxidant

IN VITRO SCREENING METHODS Of ANTIOXIDANT ACTIVITY:DPPH Free Radical Scavenging Activity

Based on reduction of coloured DPPH free radical by antioxidant. Odd electron in DPPH free radical gives absorption maxima(9660) at 517 nm. After pairing of odd electron by hydrogen electron of antioxidants DPPH show yellow colour and absorption maxima(1640) at 517nm.

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Reaction involve:

Procedure of DPPH Free Radical Scavenging Test:

Procedure of DPPH Free Radical Scavenging Test 

Percentage of DPPH Scavenging Formula:

Conclusion:

With the help of this method we easily determine the percentage effectiveness of test substance and compare result with standard antioxidant preparation by using the colour change generated by test and standard antioxidant.

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Nitric Oxide Scavenging Activity

• Use Spectrophotometry method.
• Used Chemicals: Sodium nitroprusside (for free radical generation) Phosphate buffer (pH 7.4) Griess reagent (1 % sulphanilamide, 2% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride).

Procedure of Nitric Oxide Scavenging:

Procedure of Nitric Oxide Scavenging

Oxygen Radical Absorbance Capacity (ORAC)

This assay is based on generation of free radical using AAPH (2,2-azobis (2-amino propane) dihydrochloride) and measurement of decrease in fluorescence in the presence of antioxidant. Used for testing antioxidant power of food and other chemicals.

Procedure Of Oxygen radical absorbance capacity (ORAC) Test:

Procedure Of Oxygen radical absorbance capacity (ORAC) Test:

Superoxide Scavenging Activity 

Procedure Of Superoxide Scavenging Activity:

Procedure Of Superoxide Scavenging Activity

Evaluation Of Superoxide Scavenging Activity:

Generated superoxide radical reduces nitro blue tetrazolium to a blue colour compound. In presence of test and standard antioxidant,    generation of colour is inhibited.

Reducing Power Antioxidant Test

Principle of Reducing Power Antioxidant Test: 

Substance which has reduction potential, react with potassium ferric cyanide (Fe 3+) to form potassium ferrocyanide (Fe 2+), which then react with ferric chloride to form ferrous complex that has an absorption maxima at 700 nm.

Reaction involve: 

Procedure Of Reducing Power Antioxidant Test:

Procedure Of Reducing Power Antioxidant Test

IN VIVO SCREENING METHODS OF ANTIOXIDANT ACTIVITY Enzymatic Antioxidant system:

Superoxide Dismutase-

Catalase-

Glutathione Peroxidase-

Procedure OF Enzymatic Antioxidant Test:

Various enzyme assays are:

    1- Determination of Catalase activity

    2- Determination of SOD activity

    3- Reduce glutathione (GSH) determination

    4- Glutathione Reductase  activity determination

    5- Lipid peroxidation activity

Catalase Activity Determination

Procedure Of Catalase Test:

Procedure Of Catalase Test

Reduced Glutathione (GSH)

Glutathione is   low molecular weight thole present in almost all cells. It is a intra-cellular  reductant  and protect cells  against free radicals , peroxide & other toxins. Deficiency of GSH with in living  organism can lead to tissue disorder and injury like cataract.

Procedure of Reduced Glutathione: 

Procedure of Reduced Glutathione

LIPID PEROXIDATION

• Lipid peroxidation is oxidative degradation.
• Mainly affect polyunsaturated fatty acid.
• End product of lipid peroxidation is MDA. 
• That form basis for diagnostic tests such as TBARS assay  (thiobarbituric acid reactive substance assay). 
• TBA react  with MDA to yield fluorescent product.

Procedure Of Lipid Peroxidation

Procedure Of Lipid Peroxidation

Glutathione Reductase

Glutathione Reductase is an enzyme, responsible for generation of reduced glutathione from glutathione disulphide. A heat labile enzyme system capable of reducing GSSG was discovered in liver in abundant amount. So this enzyme assay was performed on liver. 

Preparation Of Liver Supernatant:

Preparation Of Liver Supernatant

Superoxide Dismutase Activity (SOD)

Superoxide Dismutase Activity (SOD) is a metalloprotein & Its first enzyme involve in the antioxidant defense by the lowering level of superoxide.

Procedure Of Superoxide Dismutase Test:

Flow Diagram Of Superoxide Dismutase Test