Three methods widely employed in the evaluation of antioxidant activity, namely 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, static headspace gas chromatography (HS-GC) and β-carotene bleaching test (BCBT), have been compared with regard to their application in the screening of plant extracts. The strengths and limitations of each method have been illustrated by testing a number of extracts, of differing polarity, from plants of the genus Sideritis, and two known antioxidants (butylated hydroxytoluene and rosmarinic acid). The sample polarity was important for the exhibited activity in the BCBT and HS-GC methods but not for the DPPH method. The complex composition of the extracts and partition phenomena affected their activity in each assay. The value of the BCBT method appears to be limited to less polar samples. Although slow, the HS-GC method is preferable for assessing the antioxidant inhibitory properties on the formation of unwanted secondary volatile products. Being rapid, simple and independent of sample polarity, the DPPH method is very convenient for the quick screening of many samples for radical scavenging activity. Copyright © 2001 John Wiley & Sons, Ltd.

Screening of Plant Extracts for Antioxidant Activity: a Comparative Study on Three Testing Methods

Total equivalent antioxidant capacity (TEAC) and phenolic content of 26 common spice extracts from 12 botanical families were investigated. Qualitative and quantitative analyses of major phenolics in the spice extracts were systematically conducted by reversed-phase high-performance liquid chromatography (RP-HPLC). Many spices contained high levels of phenolics and demonstrated high antioxidant capacity. Wide variation in TEAC values (0.55−168.7 mmol/100 g) and total phenolic content (0.04−14.38 g of gallic acid equivalent/100 g) was observed. A highly positive linear relationship (R2 = 0.95) obtained between TEAC values and total phenolic content showed that phenolic compounds in the tested spices contributed significantly to their antioxidant capacity. Major types of phenolic constituents identified in the spice extracts were phenolic acids, phenolic diterpenes, flavonoids, and volatile oils (e.g., aromatic compounds). Rosmarinic acid was the dominant phenolic compound in the six spices of the family La...

Antioxidant Capacity of 26 Spice Extracts and Characterization of Their Phenolic Constituents

α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method offers the first approach for evaluating the antioxidant potential of a compound, an extract or other biological sources. This is the simplest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded after a defined period. However, with the advancement and sophistication in instrumental techniques, the method has undergone various modifications to suit the requirements, even though the basic approach remains same in all of them. This article presents a critical review on various developments to the DPPH method.

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Genesis and development of DPPH method of antioxidant assay

Antioxidant activity has been assessed in many ways. The limitation of many newer methods is the frequent lack of an actual substrate in the procedure. The combination of all approaches with the many test methods available explains the large variety of ways in which results of antioxidant testing are reported. The measurement of antioxidant activities, especially of antioxidants that are mixtures, multifunctional or are acting in complex multiphase systems, cannot be evaluated satisfactorily by a simple antioxidant test without due regard to the many variables influencing the results. Several test procedures may be required to evaluate such antioxidant activities. A general method of reporting antioxidant activity independent of the test procedure is proposed.

http://ucce.ucdavis.edu/files/datastore/608-46.pdf

Methods for testing antioxidant activity

Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods

A rapid on-line method for screening of complex mixtures for radical scavenging components was developed using a methanolic solution of 2,2‘-diphenyl-1-picrylhydrazyl (DPPH) stable free radical. The HPLC-separated analytes react postcolumn with the DPPH solution, and the induced bleaching is detected as a negative peak by an absorbance detector at 517 nm. An optimized instrumental setup is presented. The method is suitable for both isocratic and gradient HPLC runs with mobile-phase compositions ranging from 10 to 90% organic solvent in water or buffer (pH 3−6). The method is simple, has a broad applicability, and uses common instruments, inexpensive and stable reagents, and a time-saving and nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The limits of detection were 0.33−94 μg/mL, depending on the compound tested.

An on-line HPLC method for detection of radical scavenging compounds in complex mixtures

Antioxidant activity assays on-line with liquid chromatography

The radical cation 2,2‘-azinobis-(3-ethylbenzothiazoline-6-sulfonate), (ABTS•+) was utilized in an on-line HPLC method for the detection of radical scavengers in complex matrixes. The HPLC-separated analytes react postcolumn with the preformed ABTS•+, and the induced bleaching is detected as a negative peak by an absorbance detector at 734 nm. An optimized instrumental and experimental setup is presented. The method is suitable for both isocratic and gradient HPLC runs using mobile phases containing 100% organic solvent or its solution in water, weak acids, or buffers (pH 3−7.4). The method is sensitive, selective, relatively simple, applicable to compounds of different chemical natures; uses common instruments and inexpensive reagents; and has a time-saving, nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The minimum detectable concentration varied from 0.02 to 0.13 μg/mL, depending on the c...

Application of ABTS Radical Cation for Selective On-Line Detection of Radical Scavengers in HPLC Eluates

Alkaloid-containing plants are an intrinsic part of the regular Western diet. The present paper summarizes the occurrence of alkaloids in the food chain, their mode of action and possible adverse effects including a safety assessment. Pyrrolizidine alkaloids are a reason for concern because of their bioactivation to reactive alkylating intermediates. Several quinolizidine alkaloids, β-carboline alkaloids, ergot alkaloids and steroid alkaloids are active without bioactivation and mostly act as neurotoxins. Regulatory agencies are aware of the risks and have taken or are considering appropriate regulatory actions for most alkaloids. These vary from setting limits for the presence of a compound in feed, foods and beverages, trying to define safe upper limits, advising on a strategy aiming at restrictions in use, informing the public to be cautious or taking specific plant varieties from the market. For some alkaloids known to be present in the modern food chain, e.g., piperine, nicotine, theobromine, theophylline and tropane alkaloids risks coming from the human food chain are considered to be low if not negligible. Remarkably, for many alkaloids that are known constituents of the modern food chain and of possible concern, tolerable daily intake values have so far not been defined.

I. Koleva

Papers

Screening of Plant Extracts for Antioxidant Activity: a Comparative Study on Three Testing Methods

An on-line HPLC method for detection of radical scavenging compounds in complex mixtures

Antioxidant activity assays on-line with liquid chromatography

Application of ABTS Radical Cation for Selective On-Line Detection of Radical Scavengers in HPLC Eluates

Alkaloids in the human food chain - Natural occurrence and possible adverse effects