Antioxidant and antiradical activity of polyphenols

Antioxidants are substances which, when present in small amounts relative to the substrate susceptible to oxidation (lipids, proteins, carbohydrates, DNA), inhibit or completely prevent their oxidation. Polyphenolic compounds are, together with alpha-tocopherol and beta-carotene, a group of non-enzymatic antioxidants. First of all, the antioxidant activity of polyphenolic compounds is believed to be the result of their ability to be hydrogen donors thus yielding less reactive phenoxyl radicals. The relatively high stability of the phenoxyl radicals can be attributed to the delocalization of electrons with the existence of multiple resonance forms.

Furthermore, it is believed that some of antioxidant potential of many species of plants can be attributed to polyphenolic compounds. Plant polyphenols are not always considered to be real antioxidants, but many in vitro studies have demonstrated the potential of antioxidant phenolic compounds in the aqueous phase, namely they are capable of "scavenging" radicals as well as boosting resistance to oxidation of low density lipoprotein (LDL) which indicates pathogenesis in the case of coronary disease.
Polyphenol compounds demonstrate their antioxidant properties in biological systems in several forms, namely:

  • as H-atom donors, through direct bonding ( "scavenging") of free oxygen and nitrogen radicals;
  • by chelating prooxidant metal ions (Fe2 +, Cu2 +, Zn2 + and Mg2 +);
  • by activatingantioxidant enzymes;
  • by inhibiting prooxidant enzymes (lipoxygenase, NAD (P) H oxidase, xanthine oxidase, oxidase, cytochrome P-450).

As molecular weight increases, antioxidant activity of the polyphenols is reduced. Flavonoids have of late attracted special attention of the scientific community due to their extraordinary antioxidant and antiradical activity. Studies have shown that flavonoids are good "catchers" of free radicals so they have an important role in pharmaceutical and food industries. The protective effects of these compounds were demonstrated both in vitro and ex vivo. Flavonoids inhibit the oxidation of lipids, which are in biological systems associated with the emergence of chronic diseases and aging cells. Flavonoids inhibit certain enzyme systems, they react with the peroxyl radical as vitamin E and thereby terminate auto oxidation of unsaturated fatty acids.

Antioxidant activity of polyphenols, meaning of flavonoids depends on their structure. By inhibiting certain enzymes Flavonoids have been reported to inhibit the enzymes responsible for the production of superoxide anions such as xanthine oxidase, protein kinase C. Some flavonoids are also capable of inhibiting cyclooxygenase, lipoxygenase, microsomal monooxygenase, glutathione S-transferase, mitochondrial succinoxidase, and NADH oxidase, all involved in radical oxygen species (ROS) generation.

Flavonoids exhibit antioxidant activity both in hydrophilic and in lipophilic systems. Due to lower redox potential (0.23 - 0,75V) flavonoids may reduce free radicals (R *), with a higher redox potential (2.13 - 1.0V), such as superoxide anion radical, peroxyl, alkoxy, hydroxyl radical, by yielding hydrogen atoms. Flavonoids also have a wide range of other biochemical functions and the synergistic effects in antioxidant activity of flavonoids are also well known. Flavonoids under conditions of oxidative stress can act as prooxidants, but instead of "capturing", can form free radicals. The cells of aerobic organisms are constantly exposed to the activity of prooxidative species resulting in the destruction of their DNA, proteins and lipids. Free radicals are atoms, ions, or molecules that have one or more unpaired electrons in their structure, which are the cause of their highnon-selective reactivity. Free radicals are formed by the process of:

  • Thermolysis;
  • electromagnetic radiation;
  • redox reactions;
  • enzymatic reactions;
  • chemical processes.

Free radicals are ranked as one of the most reactive chemical species, as well as non radical forms, which are oxidizing agents and are easily converted into radicals. Most free radicals easily succumb to monomolecular and bimolecular reactions of decomposition. They be positively (radical cations) and negatively charged (radical anions). The unpaired electron can be in the atoms of different elements, so free radicals can be divided into free radicals of oxygen, chlorine, nitrogen, etc. Reactive species are divided into:

  • reactive free radical;
  • non-radical (oxidizing agent, which can easily turn into free radicals).

Reactive free radicals can occur in a number of reactions, which can be reduced to four basic types: thermolysis, photolysis, oxidation-reduction processes and high-energy radiation. The formation of toxic oxygen species and other free radicals is balanced against the antioxidant defense system of the body, and can be induced by a variety of endogenous (pro-oxidative enzyme systems, the process of cell respiration, phagocytosis, etc.) and exogenous (radiation, contaminated air, etc.) factors. The state in which there is an imbalance between prooxidant and antioxidants with a greater prevalence of prooxidants is called oxidative stress. Oxidative stress causes oxidative damage to biomolecules and leads to primary development of many diseases, such as atherosclerosis, cancer, cardiovascular disease, asthma, arthritis, gastritis, dermatitis, diabetes, liver diseases, kidney diseases, inflammatory processes, Alzheimer's disease, Parkinson's disease and so on. Oxidative stress can lead to the decomposition of primary biomolecules: proteins, lipids, nucleic acids and carbohydrates, which can be the cause of a number of disturbances in the metabolism and lead to dysfunction and cell death.

Free radicals and other reactive oxygen species, including peroxide radical-anion, hydroxyl radical, hydro-peroxide radical, hydrogen peroxide and lipidperoxide radicals, with endogenous and surrounding ozone being referred to as another form of free radical lately, are markedly involved in numerous disease processes, such as asthma , tumors, cardiovascular disease, cataracts, diabetes, gastrointestinal inflammatory diseases, liver diseases, macular degeneration, periodontal disease etc. Oxidants and radicals cause cellular aging, mutagenesis, carcinogenesis, and coronary heart disease through the destabilization of membranes, DNA damage and oxidation of low density lipoprotein (LDL). Reactive oxygen species are formed as a result of biochemical processes in the body, but also as a result of increased exposure to xenobiotics. Free radicals are extremely reactive thus causing damage to lipid membranes, forming carbon radicals, which react with oxygen to form peroxide radical, then further reacting with fatty acid creating new carbon radicals. As a result of these chain reactions lipid peroxidation products are formed, which means that one radical alone can damage many molecules in biological systems.

In order to prevent the chain reaction of free radicals in the body various antioxidant defense mechanisms are at work including enzymes, proteins, antioxidants, and flavonoids as scavengers of free radicals. The importance of antioxidants in diet and their value in the prevention of cardiovascular disease have of late sparked special interest among the scientific community. Therefore, for a flavonoid to be an antioxidant of good quality, two conditions must be met:

  • that particular flavonoid present in low concentrations in relation to the material subject to oxidation must slow down or prevent the oxidation reaction;
  • the radical resulting from a flavonoid has to be stable so as not to initiate a chain reaction.

The main structural characteristics of flavonoids crucial for scavenging free radicals are:

  • o-dihydroxy (catechol) structure of the B-ring, which provides stability and enables radical to be stable as well as delocalization of an electron;
  • 2,3-double bond in conjugation to 4-keto group to delocalize electrons from the B-ring;
  • hydroxyl groups at the 3- and 5- position ensure the creation of hydrogen bond with keto-group.

The radicals can be stabilized by:

  • delocalization of electrons;
  • formation of intramolecular hydrogen bonds;
  • subsequent reaction with other lipid radicals.

The positive effects of the increased antioxidant activity of flavonoids may also be the result of their interactions with other physiological antioxidants-vitamin C or vitamin E. Ascorbic acid protects polyphenols from oxidative degradation. Antioxidant activity (AO) is today measured in TEAC value (Trolox equivalent antioxidant activity), which is defined as mmol / dm3 solution concentration in a water-soluble analogue of Vitamin E, Trolox (6-hydroxy-2,5,7,8-tetrametylchroman -2-carboxylic acid), of equivalent antioxidant activity as the 1 mmol dm-3 solution of the tested flavonoids. Antiradical activity (AR) of flavonoids is determined by their ability to react with specific radicals, for example by measuring the discoloration of stable 2,2-diphenyl-1-picrylhydrazyl(DPPH).

The ability of monomeric phenols to act as antioxidants depends on the degree of conjugation, number and arrangement of substituents (functional group) and molecular weight. The antioxidant activity of phenolic acids is important for the stability of food, and the defense mechanisms of biological systems. Monohydric benzoic acids are very weak antioxidants, with the exception of m-hydroxybenzoic acid. Activity is significantly increased in dihydroxil substituted acids, whose antioxidant response depends on the position of the hydroxyl groups in the ring. Gallic acid is the best antioxidant of all hydroxybenzoicacids. The antioxidant activity of the phenolic acids depends on its substituents and their position on the aromatic ring structure and side chain. The presence of CH = CH-COOH group at the hydroxy derivative of cinnamic acid is the reason for a much higher antioxidant activity than the activity of COOH group in hydroxy benzoic acid derivative. A greater number of hydroxyl and methoxyl groups on the phenyl ring increases antioxidant activity, for example in coffee acids. The antiradical activity of individual phenolic acids was tested showing that the presence of the methoxyl groups in the ortho position on phenyl ring significantly affects the increase in antiradical activities towards the superoxide anion and the DPPH radicals, but not towards hydroxyl radicals.

Research has shown that tannins or polymeric polyphenols have higher antioxidant activity compared with a simple monomeric phenols with little, or no pro-oxidative activity, while many small phenolic molecules are pro-oxidants.

Total antioxidant activity of various portions of fruit and vegetables has been measured with potatoes ranking unexpectedly high. Based on the total antioxidant capacity in the list of 20 types of fruit and vegetables, potatoes ranked at 18 place (measured by the portion of boiled potatoes 299 g). Interestingly, the red beans are first on the list (portion of 92 g), followed by blueberries (portion 144 g), cranberry (portion of 95 g), artichoke (portion of 84 g).

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