ANTIOXIDANT CAPACITY ANALYSIS

ByShune Lei Thu

Cahyuning IsnainiMSc Food Technology Program School of Agro Industry

Mae Fah Luang University

Antioxidants are compounds capable to either delay or inhibit the oxidation processes which occur under the influence of atmospheric oxygen or reactive oxygen species.

ANTIOXIDANT

ANALYSIS OF ANTIOXIDANT

ORAC (oxygen radical absorption capacity) assay

The ORAC assay provides a controllable source of peroxyl radicals that model reactions of antioxidants with lipids in both food and physiological systems, and it can be adapted to detect both hydrophilic and hydrophobic antioxidants by altering the radical source and solvent

Method measures the antioxidant scavenging activity against the peroxyl radicalInduced by 2,2’- azobis-(2-amidino-propane) dihydrochloride (AAPH), at 37oC

Fluorescein --------> fluorescent probe ---------> reaction with the peroxyl radical ------> loss of fluorescence (indicator)

Determine the quantity of antioxidant decomposition amount

Use to study the AOC of many compounds and food samples. Industry has accepted the method to the point that some nutraceutical manufacturers are beginning to include ORAC values on product labels. 4

ROS (relative oxygen species) Free Radical (if over necessary intermediate) cancer, againg [degenerative diseasecardiovascular, Alzheimer]

HORAC (hydroxyl radical antioxidant capacity) assay

The initial fluorescence was measured, after which the readings were taken every minute after shaking. Gallic acid solutions were used for building the standard curve.

Measurement of the antioxidant capacity of biological fluids, bio cells, and extracts under the conditions of Fenton-like reactions

Use a Co(II) complex to evaluate the protecting ability against the formation of hydroxyl radical

Fluorescein is incubated with the sample to be analyzed

added

Fenton mixture (generating hydroxyl radicals)

HORAC antioxidant capacity cell lysate, plasma, serum, tissue homogenates, and food extracts (fast and reliable)

CUPRAC (cupric reducing antioxidant power) assay

• CUPRAC method for polyphenol and flavonoid number and plasma serum antioxidant capacity

• Standard antioxidants or extracts are mixed with CuSO4 and neocuproine. • After 30min, the absorbance was measured at 450nm. • In the assay, Cu(II) is reduced to Cu(I) through the action of electrondonating

antioxidants. • Results are expressed in milligrams of Trolox per liter of extract.

DPPH method (standard 2,2-Diphenyl-1-picrylhydrazyl)

Formation of DPPH upon absorption of hydrogen from an antioxidant. Therefore, the antioxidant concentration effect can be easily evaluated by following the decrease of UV absorption at 517 nm. Trolox is used as standard antioxidant

Measure the antioxidant activity of tissue extracts Act as free radical scavengers or hydrogen donors

DPPH• reacts with a hydrogen donor of antioxidant/reducing compound

Reduced (molecular) form (DPPH) is generatedcolor changing from purple (strong absorption, 517nm) to yellow

Stable free radical

Antioxidant capacity determination in fruit juices and fruit (guava) extracts

The PFRAP (Potassium ferricyanide reducing power) method

• An absorbance increase can be correlated to the reducing ability of antioxidants/antioxidant extracts. The compounds with antioxidant capacity react with potassium ferricyanide, to form potassium ferrocyanide. The latter reacts with ferric trichloride, yielding ferric ferrocyanide, a blue coloured complex, with a maximum absorbance at 700 nm.

Potassium ferricyanide reduction by antioxidants and subsequent reaction of

potassium ferrocyanide with Fe3+

Fluorometry• The fluorescence spectroscopy has been applied for the determination of phenolic

compounds in oils. A method based on fluorescence is proposed to quantify the butylhydroxyanisole (BHA) and tert-butylhydroquinone (TBHQ) antioxidant concentration in biodiesel produced from sunfower and soybean oils.

• It was verified that the fluorescence intensity around 330nm increases linearly as function of antioxidant concentration with correlation coefficient of about 1, regardless of the oil source and antioxidants

Fluorescence and excitation spectra of the solutions were recorded at room

temperature using a spectrofluorimeter. The emission spectra were obtained under

excitation at about 310 nm and fluorescence in the 320-800nm range was

evaluated.

Biodiesel samples without BHA and TBHQ showed fluorescence band at

about 420nm, which can be attributed to tocopherols, inherent to the vegetable

oils used in the biodiesel production. The addition of BHA and/or TBHQ is

responsible for the occurence of a fluorescence band around 330nm.

FRAP (Ferric Reducing Antioxidant Power) assay

The reaction detects compounds with redox potentials of <0.7 V (the redox potential of Fe3+-TPTZ), so FRAP is a reasonable screen for the ability to maintain redox status in cells or tissues.

The standard curve was linear between 25 and 800 mM Trolox. Results are expressed in mM TE/g fresh mass. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve.

The FRAP assay was originally developed by Benzie and Strain to measure reducing power in plasma, but the assay subsequently has also been adapted and used for the assay of antioxidants in botanicals.

The reaction measures reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a colored product taken at 593 nm.

Reducing power appears to be related to the degree of hydroxylation and extent of conjugation in polyphenols.However, FRAP cannot detect compounds that act by radical quenching (H transfer), particularly thiols and proteins. This causes a serious underestimation in serum.

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ABTS assay (2, 2’-Azinobis (3-etilbenzatiazolin)-6-sulfonat acid)

A simpler and more frequently applied approach, is the decolorization of preformed ABTS. An obvious drawback of ABTS-based assays is the promiscuity of reactions of ABTS• which is a nonphysiological free radical

The peroxidase substrate 2,2'-azino-bis(3-ethylbenzthiazoline- 6-sulphonic acid) (ABTS), forming a relatively stable radical (ABTS) upon one-electron oxidation, has become a popular substrate for estimation of total antioxidant capacity. Kinetic assays, including the commercialized TAS assay (Randox), are based on the inhibition of the formation of ABTS by one-electron oxidants. .

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TRAP (Total Peroxyl Radical Trapping Antioxidant Parameter) assay

According to Ciz (2010), It was impossible to detect the antioxidant capacity using TRAP method in samples where the quantity of polyphenols was equal to or less than 35 mg per 100 g of fresh weight.

The luminol-enhanced chemiluminescence (CL) was used to follow up the peroxyl radical reaction. The CL signal is driven by the production of luminol derived radicals from thermal decomposition of AAPH. The TRAP value is determined from the duration of the time period (Tsample) during which the sample quenched the CL signal due to the present antioxidants. A known quantity (8.0 nM) of trolox, a water-soluble analogue of tocopherol was used as a reference inhibitor (Ttrolox) instead of the sample.

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Methods of Total Antioxidant Capacity Assessment

Reference

• Pisoschi A.M. 2011, Methods for Total Antioxidant Activity Determination: A Review, Pisoschi and Negulescu, Biochem & Anal Biochem, 1:1.• Reşat Apak, 2007, Mechanism of antioxidant capacity assays and the

CUPRAC (cupric ion reducing antioxidant capacity) assay, Microchimica Acta, April 2008, Volume 160, Issue 4, pp 413-419 (http://link.springer.com/article/10.1007%2Fs00604-007-0777-0)• www.cellbiolabs.com/sites/default/files/STA-346-horac-assay-kit.pdf• www.bmglabtech.com/en/technology/assay-technologies/orac/

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