Antioxidants substances assay

Recently, Maruta et al. [112] have found that methanol extracts of roots of burdock show a significant antioxidant activity in an in vitro lipid peroxidation assay, and have isolated five caffeoylquinic acid derivatives (CQAs) from the roots of burdock (Arctium lappa L ), an edible plant in Japan. Antioxidant activities of DCQAs and related compounds have been investigated by measuring the hydroperoxidation of methyl linolate via radical chain reaction. This study indicates that in this particular system caffeic acid and CQAs are more effective than a-tocopherol. These results approximately agree with our findings [38], Additionally, CQAs as the principle antioxidative substance in burdock root have been characterized. 

The free-radical-scavenging activity of compounds was evaluated in the TEAC and CL assays. The first measures the relative abiUty of antioxidant substances to scavenge the radical cation 2,2 -azinobis(3-ethylbenzothiozoline-6-sulfonate) (ABTS" ) as compared to a standard amount of the synthetic antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The CL assay measures the inhibition of iodophenol-enhanced chemiluminescence by a horseradish peroxidase/perborate/luminol system. Trolox was used as the reference antioxidant. The results showed that xanthones exhibited free-radical-scavenging activity at potency levels comparable to those of reference antioxidant compounds quercetin and rutin, while the benzophenone and phloroglucinol type compoimds had more moderate activities [87]. 

The photochemiluminiscence (PCL) assay was initially used by Popov and others (1987). Popov and Lewin (1994 1996) have extensively studied this technique to determine water-soluble and lipid-soluble antioxidants. The PCL assay measures the antioxidant capacity, toward the 02 radical, in lipidic and water phase. This method allows the quantification of both the antioxidant capacity of hydrophilic and/or lipophilic substances, either as pure compounds or complex matrices from different origin synthetic, vegetable, animal, human, etc. The PCL method is based on an approximately 1,000-fold acceleration of the oxidative reactions in vitro by the presence of an appropriate photosensitizer. The PCL is a very quick and sensitive method. Chua and others (2008) used this assay to determine the antioxidant potential of Cin-namomum osmophloeum, whereas Kaneh and Wang and others (2006) determined the antioxidant capacity of marigold flowers. The antioxidant activity of tree nut oil extracts was also assessed by this method (Miraliakbari and Shahidi 2008). 

Analysis of antioxidant properties relative to the DPPH" radical involves observation of colour disappearance in the radical solution in the presence of the solution under analysis which contains antioxidants. A solution of extract under analysis is introduced to the environment containing the DPPH radical at a specific concentration. A methanol solution of the DPPH radical is purple, while a reaction with antioxidants turns its colour into yellow. Colorimetric comparison of the absorbance of the radical solution and a solution containing an analysed sample enables one to make calculations and to express activity as the percent of inhibition (IP) or the number of moles of a radical that can be neutralised by a specific amount of the analysed substance (mmol/g). In another approach, a range of assays are conducted with different concentrations of the analysed substance to determine its amount which inactivates half of the radical in the test solution (ECso). The duration of such a test depends on the reaction rate and observations are carried out until the absorbance of the test solution does not change [4]. If the solution contains substances whose absorbance disturbs the measurement, the concentration of DPPH radical is measured directly with the use of electron paramagnetic resonance (EPR) spectroscopy. 

Analysis of antioxidant activity by performing a FRAP assay was proposed by Benzie and Strain [23]. It involves colorimetric determination of the reaction mixture in which the oxidants contained in the sample reduce Fe ions to Fe. At low pH, Fe(in)-TPTZ (ferric-tripyridltria-zine) complex is reduced to the ferrous (Fe ) form and intense blue colour at 593 nm can be observed. The FRAP reagent is prepared by mixing 2.5 ml of TPTZ (2,4,6-tris (l-pyridyl)-5-triazine) solution (10 mM in 40mM HCl), 25 ml acetate buffer, pH 3.6, and 2.5 ml FeCl3 H20 (20 mM). The colour of Fe(II)(TPTZ)2 which appears in the solution is measured colorimetri-cally after incubation at 37°C. The measurement results are compared to those of a blank sample, which contains deionised water instead of the analysed sample. The duration of the assay differs from one study to another 4 min [23, 24], 10 min [25] to 15 min [26]. The analysis results are converted and expressed with reference to a standard substance, which can be ascorbic acid [26], FeS04 [23, 25], Trolox [27,18]. 

Glavind and Holmer proposed a method of determination of antioxidants by TLC using the DPPH radical. They sprinkled a plate with separated substances with methanol solutionofthe radical and observed discoloration where substances able to quench radicals were present [57]. The TLC-DPPH assay allows a researcher to access the analysed substances and to assess the biological activity of individual compounds. Another advantage of the method is the possibili-... 

Initially, the use of HPLC in analysis of antioxidant properties with the DPPH" radical was restricted to chromatographic analysis of the radical content in solution. An assay was performed in which a solution of the radical was treated with the extract under analysis. The reaction ran in a reaction tube and the remainder of the radical after the reaction was analysed chroma-tographically. A comparison of the radical content in the blank sample and in the extract sample showed the amormt of radical that was quenched by antioxidants in the analysed sample [62, 63].However, themethoddidnotprovidemoreinformation than the colorimetricmethod. Much better results are obtained in a post-column on-line reaction in which substances separated on a chromatographic column react with a radical in a reaction coil. 

Figure 8.2 Pomegranate juice polyphenols and antioxidant potency in comparison to other fruit juices. Total polyphenol concentration in the different juices was determined using quercetin as a standard. LDL (100 pg of protein/milliliter) was preincubated with increasing volume concentration (0-25 pL) of the juices. Then, 5 pmol/L of CuS04 was added, and the LDL was further incubated for 2 hours at 37°C. The extent of LDL oxidation was measured by the thiobar-bituric acid reactive substance (TBAR) assay, and the ICh, values (the concentration needed to get 50% inhibition) are given. Results are given as mean S.D. of three different experiments.

One type of inhibition assay is based on the formation of a colored or fluorescent product from a chromogenic or fluorogenic substrate and inhibition of formation of color or fluorescence by antioxidants. A popular chromogenic substance used... 

Numerous methods are required to characterize drug substances and drug products (Chapter 10). Specifications may include description identification assay (of composite sample) tests for organic synthetic process impurities, inorganic impurities, degradation products, residual solvents, and container extractables tests of various physicochemical properties, chiral purity, water content, content uniformity, and antioxidant and antimicrobial preservative content microbial tests dissolution/disintegration tests hardness/friability tests and tests for particle size and polymorphic form. Some of these tests may be precluded, or additional tests may be added as dictated by the chemistry of the pharmaceutical or the dosage form. 

A preservative is a substance that extends the shelf-life of drug products by preventing oxidation or inhibiting microbial growth.14 Preservatives must be monitored in the products since they are considered to be active components. Generic HPLC assays are typically developed for preservatives such as buty-lated hydroxytoluene (BHT), an antioxidant for solid dosage forms, and antimicrobials such as parabens, sodium benzoate, or sorbic acid in liquid formulations. For these additive components, typical assay specifications are 85-115% of label claim. 

Antioxidant molecules themselves are not free radicals and are not EPR-active. However, these compounds do react rapidly with reactive oxygen species, ROS, and other free radicals to render them harmless. The EPR-based DPPH assay described below can be used to provide quantitative information concerning the relative effectiveness of various dietary substances and their antioxidant capacities. 

Antioxidant activity is also a measure for substance ability to prevent free radical concentration increment, oxidative stress, and risk for development-related diseases. And for the purpose of measuring antioxidant activity, many assays are applied such as 2,2-diphenyl-l-picrylhydrazyl (DPPH) assay, 2,2 -azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid (ABTS) assay, p-carotene bleaching test, ferric and cupric reducing power, and linoleic acid assay (Akrout et ah, 2011 Chabir et ah, 2011 Jia et al, 2010 Serrano et al, 2011). 

---