Publisher Summary This chapter discusses the analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Analyses of the Folin-Ciocalteu (FC) type are convenient, simple, and require only common equipment and have produced a large body of comparable data. Under proper conditions, the assay is inclusive of monophenols and gives predictable reactions with the types of phenols found in nature. Because different phenols react to different degrees, expression of the results as a single number—such as milligrams per liter gallic acid equivalence—is necessarily arbitrary. Because the reaction is independent, quantitative, and predictable, analysis of a mixture of phenols can be recalculated in terms of any other standard. The assay measures all compounds readily oxidizable under the reaction conditions and its very inclusiveness allows certain substances to also react that are either not phenols or seldom thought of as phenols (e.g., proteins). Judicious use of the assay—with consideration of potential interferences in particular samples and prior study if necessary—can lead to very informative results. Aggregate analysis of this type is an important supplement to and often more informative than reems of data difficult to summarize from various techniques, such as high-performance liquid chromatography (HPLC) that separate a large number of individual compounds .The predictable reaction of components in a mixture makes it possible to determine a single reactant by other means and to calculate its contribution to the total FC phenol content. Relative insensitivity of the FC analysis to many adsorbents and precipitants makes differential assay—before and after several different treatments—informative.

Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent

Methods available for the measurement of antioxidant capacity are reviewed, presenting the general chemistry underlying the assays, the types of molecules detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equivalent antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biology. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method. Other assays may need to be considered in the future as more is learned about some of the other radical sources and their importance to human biology.

https://faculty.missouri.edu/~glaserr/3700s14/Antioxidant-Capacity_jf0502698.pdf

Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements

The antioxidant activities and total phenolics of 28 plant products, including sunflower seeds, flaxseeds, wheat germ, buckwheat, and several fruits, vegetables, and medicinal plants were determined. The total phenolic content, determined according to the Folin−Ciocalteu method, varied from 169 to 10548 mg/100 g of dry product. Antioxidant activity of methanolic extract evaluated according to the β-carotene bleaching method expressed as AOX (Δ log A470/min), AA (percent inhibition relative to control), ORR (oxidation rate ratio), and AAC (antioxidant activity coefficient) ranged from 0.05, 53.7, 0.009, and 51.7 to 0.26, 99.1, 0.46, and 969.3, respectively. The correlation coefficient between total phenolics and antioxidative activities was statistically significant. Keywords: Antioxidant activity; phenolics; medicinal plants; oilseeds; buckwheat; vegetables; fruits; wheat products

Antioxidant Activity and Total Phenolics in Selected Fruits, Vegetables, and Grain Products

The antioxidative activity of a total of 92 phenolic extracts from edible and nonedible plant materials (berries, fruits, vegetables, herbs, cereals, tree materials, plant sprouts, and seeds) was examined by autoxidation of methyl linoleate. The content of total phenolics in the extracts was determined spectrometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents (GAE). Among edible plant materials, remarkable high antioxidant activity and high total phenolic content (GAE > 20 mg/g) were found in berries, especially aronia and crowberry. Apple extracts (two varieties) showed also strong antioxidant activity even though the total phenolic contents were low (GAE < 12.1 mg/g). Among nonedible plant materials, high activities were found in tree materials, especially in willow bark, spruce needles, pine bark and cork, and birch phloem, and in some medicinal plants including heather, bog-rosemary, willow herb, and meadowsweet. In addition, potato peel and beetroot peel extracts showed strong antioxidant effects. To utilize these significant sources of natural antioxidants, further characterization of the phenolic composition is needed.

Antioxidant activity of plant extracts containing phenolic compounds

The main dietary sources of polyphenols are reviewed, and the daily intake is calculated for a given diet containing some common fruits, vegetables and beverages. Phenolic acids account for about one third of the total intake and flavonoids account for the remaining two thirds. The most abundant flavonoids in the diet are flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products. The main polyphenol dietary sources are fruit and beverages (fruit juice, wine, tea, coffee, chocolate and beer) and, to a lesser extent vegetables, dry legumes and cereals. The total intake is approximately 1 g/d. Large uncertainties remain due to the lack of comprehensive data on the content of some of the main polyphenol classes in food. Bioavailability studies in humans are discussed. The maximum concentration in plasma rarely exceeds 1 microM after the consumption of 10-100 mg of a single phenolic compound. However, the total plasma phenol concentration is probably higher due to the presence of metabolites formed in the body's tissues or by the colonic microflora. These metabolites are still largely unknown and not accounted for. Both chemical and biochemical factors that affect the absorption and metabolism of polyphenols are reviewed, with particular emphasis on flavonoid glycosides. A better understanding of these factors is essential to explain the large variations in bioavailability observed among polyphenols and among individuals.

Dietary Intake and Bioavailability of Polyphenols

Several details of the assay of total phenolic substances have been investigated and an improved procedure developed. The improvements include the use of Folin-Ciocalteu reagent rather than the Folin-Denis reagent, gallic acid as a reference standard, and a more reproducible time-temperature color development period. The values obtained are less subject to variation and interference from several nonphenols, yet are directly comparable to the "tannin" values obtained by the previously standard method.

Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents

The reaction of oxygen with wine in its normal acidic condition was in proportion to the total phenol content if the wine samples differed considerably in phenol content, but the reaction was fairly slow and did not reach an end-point in a convenient length of time even at an elevated temperature. Maximum oxygen consumption by wines made very alkaline was rapid, and reproducible values were obtained with a Warburg technique. These values were of the order of 200-600 cc O2/l for red table wines and 50-100 cc O2/l for white table wines. The oxygen consumption per unit phenol of wines or different purified phenol fractions under alkaline conditions was not constant, indicating differences related to the relative content of specific phenolic substances. All phenolic fractions tested did, however, absorb considerable oxygen, and white wines took up relatively more oxygen per unit phenol than did red wine.

Studies of both enzymatic and non-enzymatic browning indicated that the catechin fraction was most important in browning. The catechin fraction's oxidation products appear to be capable of accounting for the color of most white wines, whether very light or a dark amber. An analytical technique using the vanillin versus Folin-Ciocalteu assays has also been proposed as a criterion of a wine's tendency to brown.

Contributions of Grape Phenols to Oxygen Absorption and Browning of Wines

Grape seed phenolics were extracted and separated into three distinct groups of compounds. The separated fractions were evaluated in turn for their sensory effects in water and in wine with respect to their thresholds for astringency, bitterness, and effect on taste interaction with acidity. If was found that the three fractions have considerably different flavor effects, both qualitatively and quantitatively. The catechin fraction was bitter but not astringent at levels likely to occur in wine. The leucoanthocyanin fraction and the condensed tannin fraction were both bitter and astringent. All three affected the judges9 reaction to acidity by moderating the apparent sour taste when added at levels near threshold. All three fractions appear capable of important effects on these quality factors at levels which may occur in white wines and certainly do occur in rose and red wines. The condensed tannin fraction contributed the most intense flavor effects on a weight basis, and the evident flavor effects of the phenolic substances in wine were found to be 10 to 100 times as intense as previously noted. The levels present in a red wine are about 10 to 100 times the level barely detectable in a white wine. A comparison was made of the adsorptive properties of casein, gelatin, isinglass, nylon, and insoluble polyvinylpyrrolidone with respect to these phenolic fractions. Large differences were found in relative affinity for the different phenolic fractions. The PVP had a greater capacity in phenol per unit weight than did nylon, and the proteins were greater yet. The synthetic resins had a greater affinity for the smaller molecules than for the condensed tannins, and the proteins were the reverse, under the conditions tested. The resins had considerable affinity for all the fractions in column tests, but, as fining agents the proteins showed no affinity for the catechin fraction under our conditions.

Flavor Effects and Adsorptive Properties of Purified Fractions of Grape-Seed Phenols

A simple standardized technique for two-dimensional paper chromatographic "mapping" and estimation of the relative amounts of the phenolic substances in grape extracts was presented. This technique was applied to the study of weekly harvests of 12 grape varieties. The berry pulp tended to reflect the juice and seeds in phenolic composition. The juice phenolics, as a rule, were predominantly blue-fluorescing substances resembling chlorogenic acid and its analogs. Calzin and one of its parents, Refosco, were unique among 34 varieties tested in having a considerable content of astringent tannin in the juice. Skin extracts were quite variable in qualitative phenolic composition by variety, especially considering, but in addition to, the presence or absence of anthocyanins. The phenolic composition of the seeds, however, followed a similar pattern in red or white grapes and in varieties with parentage from two species of Vitis . Among a total of about 25 differentiated substances readily oxidized by FeCl 3 -K 3 Fe-(CN) 6 and considered as phenols, the majority were present in the seeds of all varieties at similar stages, and usually in roughly similar proportions. Varietal differences did occur with respect to the presence or absence of a few of the substances and the relative amounts of those present per unit of total phenol. The nature of several of the substances was outlined, and, in particular, evidence was presented that one was catechin, one was epicatechin, and a third was epicatechin gallate. Although these flavanols have been reported in grapes (4, 6) previously, epicatechin gallate has not always been found in seeds (6). A considerable metabolism of phenolic substances in seeds during ripening was found even up to the last stages, as shown by the appearance or increase of some and the decrease of others in relation to a unit of total phenol.

Joseph A. Rossi

Papers

Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents

Contributions of Grape Phenols to Oxygen Absorption and Browning of Wines

Flavor Effects and Adsorptive Properties of Purified Fractions of Grape-Seed Phenols

Paper Chromatography of Phenolic Compounds from Grapes, Particularly Seeds, and Some Variety-Ripeness Relationships