Implication
(i) Assessment of antioxidant potency
Focus
(i) Scavenging of free radicals
(ii) Prevention of lipid peroxidation
Models
(i) Screening of potency of the test compound in vitro
(ii) In vivo screening ® demonstration of the effect on an animal model (rat, mouse) ® placebo controlled studies with human volunteers
(iii) The use of oxidized LDL as bioindicators
Why antioxidant testing?
Purposeful and directed production of reactive oxygen species (ROS) is a key element of important physiological functions (signal transduction, apoptosis). Excessive production of ROS (e.g. due to chronic inflammation, ischemia, drugs, pollutants, irradiation, cigarette smoke etc.) may cause oxidative damage to cell constituents. Such changes may lead to mutagenesis, membrane disruption, receptor malfunction, altered enzyme activities and mitochondrial damage. ROS are known to be implicated in ageing processes, and in diseases the incidence of which typically increases with age (cancer, cardiovascular disease, neurodegenerative disorders). The potential beneficial effects of antioxidant compounds have led to intensive search for novel antioxidant molecules to be used as components of dietary supplement/functional food.
Assessment of antioxidant effect in vitro
Tests:
(i) Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH)
(ii) Scavenging of superoxide anion
(iii) Scavenging of peroxyl radical
(iv) Metal-chelating properties
(v) Electrochemical behaviour
(vi) Prevention of lipid peroxidation
Aim: To find out if the test compound/extract/phytopreparation can inactivate free radicals and/or prevent lipid peroxidation.
Procedure: DPPH radical, superoxide anion and peroxyl radical are produced by standard methods. Detection of free radicals is based on sensitive chemiluminescense techniques. Studies will be done with several concentrations of the test compound and repeated on separate days. The results will be presented as comparison of the potency of the test compound with that of a simultaneously tested known antioxidant. As the model of lipid peroxidation, peroxidation is induced chemically in biological lipids (e.g. lipids from rat liver).Assesment of antioxidant effect in vivo
Tests:
(i) Total antioxidant capacity of plasma and liver tissues
(ii) Level of reduced glutathione in erythrocyte and liver cells
(iii) Level of lipid peroxidation products
(iv) Activities of antioxidant enzyme in erythrocytes and liver (SOD, glutathione peroxidase)
Aim: To find out if the test substance/extract/phytopreparation has antioxidant effects in vivo.
Procedure: Effect of the test compound/extract/phytopreparation will be investigated after induction of experimental oxidative stress (by vitamin E deficiency and/or chemical prooxidant) to mice/rats. The test compound will be administered with feed or drinking water. Markers of oxidative stress in various tissues will be determined at the end of the test period. Determination of test compounds in tissues, cells and extracellular fluid. Determination of total antioxidant capacity of plasma and tissues.