The wide scientific recognition that a greater consumption of foods rich in antioxidants results in clear benefits for the health of the population has led consumers to become increasingly interested in knowing what is the antioxidant richness of the products that the market offers them?
Although until recently the mere mention of the term "antioxidant", whether through a promotional campaign or inscribed in the packaging of a product, was sufficiently attractive for consumers to be inclined towards this product, recently, and increasingly , consumers seek to distinguish between those products whose marketing "simply claims to have antioxidants" ... and those in which "the content of these compounds is duly supported and quantitatively described in their labeling".
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Within the framework of the aforementioned, this section addresses, among others, questions such as the following: What Antioxidants should be measured in a Food? What are the main tests and analytical methods available for its measurement? What is important to measure and label the content of polyphenols and the ORAC value in Foods? What is the difference between measuring and certifying the content and the antioxidant activity of a food?
To face the question : What Antioxidants should be measured in a Food? , it is necessary to respond previously to the following:
What are the main antioxidants present in food? The antioxidant richness of food is generally given by the addition and interaction of numerous molecules. Although the chemical structure of such molecules can be significantly different, among the main antioxidants present in foods it is possible to distinguish:
- i) Antioxidant Vitamins , which include Ascorbic Acid (or Vitamin C); Vitamin E, a term that includes not only alpha-tocopherol, but also, isoforms, alpha, beta, gamma and delta, tocopherols and tocotrienols; and the Pro-Vitamin A compounds (represented by beta-carotene, alpha-carotene and beta-cryptoxanthin).
- ii) Carotenoids , including lutein, lycopene, zeaxanthin and astaxanthin. Also referred to as carotenoids are those compounds which are Pro-Vitamin A (mentioned above). From a chemical point of view, the carotenoids include the carotenes (alpha-carotene, beta-carotene and lycopene), which do not include oxygen atoms in their structure, and the xanthophylls (beta-cryptoxanthin, lutein, astaxanthin and zeaxanthin), which they do present it, mostly in the form of hydroxyls.
- iii) Polyphenols . The polyphenols account for the antioxidant richness of the greater part of the foods usually consumed by the population. All polyphenols exhibit in their structure, at least, one or more hydroxyl groups attached to an aromatic ring. Among the polyphenols it is possible to distinguish two major types of compounds: flavonoids, for which several thousands have been described in the plant kingdom) and whose structure comprises two aromatic rings joined a heterocycle of three carbon atoms and one of oxygen (C6- C3-C6), and the so-called non-flavonoids (some hundreds) that comprise, mostly, mono-phenolic alcohols, phenolic acids and stilbenes.
Detailed information regarding the antioxidant composition of the main foods ingested by the population, is the section " Antioxidants in food: Main sources and their contents ".
Now, knowing what are the types of antioxidants that predominate in a given food, it is possible to return to the question of what antioxidants should be measured in a food? Although the answer to this question is subject to the nature of the food to be analyzed, in general terms it will be possible to measure in food:
- (I) the specific content of those antioxidants that said food concentrates more, or of those whose presence is more relevant for its distinction as a source or contribution of said compound; for example, the content of ascorbic acid, that of alpha-tocopherol, that of lycopene, or that of some flavonoid in particular;
- (II) the total content of a certain type of antioxidant, for example, the total content of polyphenols or the total content of flavonoids present in a food;
- (III) the antioxidant activity of the food. Unlike the single measurement of the content of a given antioxidant, measuring the "antioxidant activity" of a food allows to quantify the "capacity that all antioxidant compounds present in it" (vitamins + carotenoids + polyphenols + others that do not respond) to the previous categories) to act simultaneously as a mixture of antioxidant compounds.
But then, what should be measured?
If you intend to make a characterization of a food from the point of view of its antioxidant richness, ideally you should quantify the individual content of each of those antioxidants that a priori is known to contain such food, and you should measure the antioxidant activity that -as a result of the sum and interaction of its antioxidant components- said food presents.
However, the purpose of measuring individually the content of each of the antioxidant components of a food is excessively expensive and analytically complex, since in those foods that are richer in antioxidants, these compounds comprise, regularly, a large number and a huge diversity of structures.
What alternative exists to measure the individual content of each of the antioxidant components of a food? As an alternative to the individual measurement, the total content of a food can be quantified in terms of a certain type of antioxidant. For example, when the antioxidant richness of a food resides mainly in a high presence of polyphenols, the measurement is limited to the characterization of the antioxidant content in the form of total polyphenols (PFT). If required, together with the measurement of PFT it is possible to measure, in a more precise way, the total flavonoid content, and even more specific, the content of certain flavonoid subtypes, such as total anthocyanidins, total flavonols or flavanols. -3-oles total. Further details regarding the chemistry and presence of these compounds in foods are described in the section " Antioxidants in food: Main sources and their contents ".
How is the total polyphenol content determined? The content of PFT is determined through an assay using the Folin-Ciocalteu (FC) reagent. All previously published methods that use this reagent measure the ability of polyphenols to reduce (donate an electron) the Mo (VI) to Mo (V) present in the complex molybdichotostats that characterizes the FC reagent. As a result of such reduction, the reagent, of yellow color, acquires an intense blue color, which is quantified spectrophotometrically at 765 nm. Although all the available methods that use the FC reagent ensure the total oxidation of all those compounds capable of reducing it, these differ in terms of the concentrations of said reagent used, the type of base and concentration used to alkalize the medium (carbonate versus sodium hydroxide), at the incubation times necessary to quantify the reagent reduction (3-120 min), and the incubation temperatures of the samples during their analysis (20-50 ºC).
How is the total polyphenol content of a sample expressed? The total polyphenol content resulting from the application of any method based on the use of the Folin-Ciocalteu reagent is regularly expressed as mg of gallic acid equivalents (EAG) / 100 g of food. Gallic acid is a simple phenolic compound widely used in this test as a comparison standard. However, polyphenols such as catechin or tannic, chlorogenic, caffeic, vanillic and ferulic acids are occasionally used as a standard for comparison and expression of results.
To find a complete list of PFT content values in fruits produced and / or consumed in Chile, visit our section " Antioxidant Database ".
Are the methods that use the specific Folin-Ciocalteu reagent for the determination of polyphenols? As mentioned above, in the Folin-Ciocalteu test, the capacity of polyphenols to act as reducing agents of Mo (VI) in the molybdichottate complex is measured. Although the reagent reduction test is simple, sensitive and precise, it should be noted that said reagent can be reduced not only by all polyphenols, but also by reducing agents such as ascorbic acid, sodium metabisulfite, iron (II) salts , EDTA, certain amino acids, fructose and glucose, among others. The latter is extremely important because when this method is applied to samples containing one or more of these interferences, without taking the corrective measures, a result that represents an overestimation of the actual value of total polyphenols will be mistakenly obtained.
For example, it has been seen that the application of the test in the presence of fructose (5 g / L), a sugar abundantly present in fruits, results in a recovery of gallic acid content (10 mg / L) that is wrongly increased at around 58%.
As a result of the interfering effect that would have compounds such as fructose, glucose or ascorbic acid, normally present in fruits, vegetables, and in numerous processed foods, it is of great importance that, in the application of any method that employs the FC reagent, present the incorporation of adequate controls, ideally, using modifications to the original method that allow to discriminate analytically between the contribution made to the reduction of the said reagent polyphenols to be measured and those "interferentes" non-polyphenolic components present in a sample.
What limitations of interpretation supposes the single measurement of PFT in the characterization of the antioxidant richness of a food? Although the measurement of PFT is widely used and recognized as a preliminary way to characterize the antioxidant richness of a food, it must be borne in mind that, as such, the PFT measurement does not distinguish the measure or the proportion in which the various polyphenol subtypes present in a food are individually contributing to the total polyphenolic content. Said essay does not evaluate the value that the interaction between the different polyphenols present in a food would have.
However, the measurement of PFT, when it is properly performed (this is with interferences correction), constitutes a good, simple and practical approach to the purpose of initially characterizing a food in terms of its antioxidant content, especially when it comes to those in which polyphenols mostly account for their antioxidant composition. However, when polyphenols only partially account for the antioxidant richness of a food, the mere measurement of PFTs as a way to show such richness could suppose a sub-estimate proportional to the contribution made by compounds of a non- polyphenolic to the total antioxidant richness of the analyzed food.
To avoid such underestimation, and given that many foods rich in antioxidants possess not only phenolic compounds, but also non-phenolic antioxidants (various antioxidant vitamins and carotenoids), it is very important that the characterization of the antioxidant richness of the food Understand, in addition, the measurement of your "antioxidant activity".
But, really , what is measured when the antioxidant activity of a food is determined? The first thing to note is that the measurement of the antioxidant activity of a food supposes the quantification of "virtually" all the antioxidant molecules present in it.
Most of the assays used to determine the antioxidant activity of a food are based on the measurement of: (1) the ability of antioxidant compounds to react with a given free radical, or (2) the potential for such compounds would have to reduce a complex formed between Fe (III) ions and the TPTZ (2,4,6-tripyridyl-s-triazine) reagent.
Among those trials that are based on measuring the ability of antioxidants to react with a free radical, include the following:
- - ORAC Test (Oxygen Radical Absorbance Capacity, or Oxygen Radical Absorbance Capacity)
- - TEAC Trial (Trolox Equivalent Antioxidant Capacity, or Antioxidant Capacity as Trolox Equivalents)
- - DPPH assay (2,2-Diphenyl-1-picrilhydrazil).
There is a consensus that to characterize the antioxidant activity of a food, the ORAC test stands out among all the available assays due to its high sensitivity, precision and reproducibility.
What is the ORAC trial?
Unlike the simple measurement of the content of antioxidants present in a food, the ORAC assay measures the overall activity or capacity of all antioxidants present in a sample to "turn off or neutralize" (scavenging) peroxyl radicals.
The latter are reactive species comparable and therefore relevant to those ROS biologically generated in the body. In the ORAC assay, the peroxyl radicals, generated from the azo-compound AAPH or ABAP ([2,2'-azobis (2-amidinopropane)), react with fluorescein as a substrate, as a result of such a reaction, the fluorescence of this The last compound decreases over time, configuring an area under the curve (fluorescence versus time) When this reaction takes place in the presence of antioxidant compounds, the area under the curve increases linearly and proportional to the concentration of antioxidants.
To act as such, antioxidants must donate either an electron (SET), or a hydrogen atom to them (HAT) free radicals that they are intended to stabilize. The ORAC assay measures the ability of all antioxidants present in a food (or sample of it) to donate hydrogen atoms to the peroxyl radicals. Therefore, the ORAC method quantifies the ability of a food to act as an antioxidant through the HAT mechanism.
The ORAC assay includes the measurement of the contribution made to antioxidant activity by both polyphenols and those compounds of a non-polyphenolic nature present in a given food, and therefore allows to compare the antioxidant activity, ORAC value, of foods that do not necessarily have polyphenols as its main components with those who do. For example, it is possible to compare the ORAC value of a tomato (rich in lycopene but poor in polyphenols) with that of an apple (which is rich in polyphenols but does not contain lycopene).
The ORAC test not only reflects the total content of the antioxidant compounds, but also the additive, synergistic or potentiation interaction resulting from the simultaneous presence of these, resulting in a value that reflects the overall capacity or antioxidant activity of a food .
How is the ORAC value of a sample expressed? The ORAC value is expressed as micromoles of Trolox® equivalents / 100 g of sample. Trolox® is an analogue of vitamin E which, due to its easy solubility in water, is used as a comparison standard.
Since the ORAC method allows us to compare foods of a very diverse nature in terms of their antioxidant richness, the ORAC assay currently represents the most used way to evaluate the antioxidant activity of foods. As such, the ORAC value is the most recognized index at the moment of defining the potential contribution that the consumption of a food could imply to the antioxidant capacity of our organism.
Although the confidence that has around the values of antioxidant activity generated through the use of the ORAC method is derived, to a large extent, from the high sensitivity, precision and reproducibility of the method, it is clear that to ensure such characteristics the test it must be executed by a laboratory that is equipped not only with an adequate instrumentation that allows its automation, but also, that ensures compliance with the standardized analytical protocol of the method.
To find a complete list of ORAC antioxidant activity values of fruits produced and / or consumed in Chile, visit our section " Antioxidant Database ".
How does the ORAC method differ from other methods of determining antioxidant activity? While other methods, such as TEAC and DPPH, also evaluate the ability of antioxidants in a sample to "quench or neutralize" a free radical, both TEAC and DPPH use free radicals as molecules that differ completely from any free radical or Reactive species generated by our organism. While in the first case the radical cation ABTS • + is used, in the second the radical DPPH • is used. Although the high stability of both radicals makes their use simpler, the same condition places the TEAC and DPPH methods as analytical approaches very distant from the high reactivity that typically characterizes ROS normally generated in biological systems. Therefore, the relevance of these methods is frequently questioned. Both methods are usually useful to perform a "ranking of antioxidant compounds / preparations" within a batch, experiment or study, but, beyond their technical simplicity, the relatively low sensitivity and reproducibility of both methods limits the desirable possibility of comparing the TEAC or DPPH values.
Can an equality of ORAC values between two different foods suppose equal nutritional value? It is essential to clarify that a possible equality in the ORAC value between one food and another (whether they are of the same nature or not), does not necessarily mean equality in the nutritional value of both. In fact, the latter will be given by the presence (content and type) of numerous nutrients, among others, proteins, fat, carbohydrates, vitamins, and micro / macro minerals.
Similarly, an equality in antioxidant activity would not allow an equivalence in the potential benefit that by ingestion of the antioxidants present in these foods would have for human health the indistinct consumption of these. As described in the section " Antioxidants and health: Scientific evidence ", while all polyphenols share their capacity to act as antioxidants, the existence of even small differences in their structures often often result in significant differences in both bioavailability (absorption and subsequent availability of these in the blood) as in the biological action profile of said compounds (since they act not only as free radical scavengers).
Why is it important to measure and label the polyphenol content and ORAC value in Foods?
In our country, warned of the excellent disposition that consumers also have to opt for products in which the presence of antioxidants stands out, several companies actively promote the consumption of products that, although they do not certify their content and antioxidant activity, label them as "Rich in antioxidants." Examples of the latter are certain brands of tea, coffee and beers that claim to be "naturally rich in antioxidants", as well as certain brands of mineral waters and other beverages that, after the addition of antioxidants to their formulation, are also generally promoted. as "drinks with antioxidants." According to the opinion of experts in the area of antioxidants, in order to continue advancing seriously in the promotion of the consumption of foods rich in antioxidants, it is essential to distinguish between those products whose marketing "simply claims to have antioxidants ..." and those in which " the content of these is sustained and quantitatively described in its labeling ". Increasingly, certain actors of the national industry have already become aware of the latter, endorsing through independent analysis the content and antioxidant activity in the label of some of their products. Examples of the latter are certain chocolates made by important companies in the food industry, such as Costa-Carozzi and Nestlé, which, because they are made with a high content of cocoa, are currently duly and validly marketed as "sources of natural antioxidants". Similarly, companies such as Corpora Tresmontes have also validated the PFT content and the ORAC antioxidant activity of their Livean-Antiox® powder juice products, which incorporate green tea extracts as a source of natural antioxidants. In order to have complete certainty about the real and continuous presence of a value of total polyphenols and ORAC in a given product, it is essential that it has the backing of its regular analysis and / or certification.