Characterization of Antioxidant Activity

Gangapatrula Hema Sudha*, Edarada Kavya Anisha,  Dongala Kanakamaha Lakshmi, Pasumarthi Phaneendra

 Vikas institute of pharmaceutical sciences, Rajahmundry

*Correspondence: sudhagangapatrula@mail.com; Tel.: (9381519289)

DOI: https://doi.org/10.71431/IJRPAS.2025.4403

INTRODUCTION

"Against oxidation" is what the term "antioxidant" signifies. An antioxidant is any material that, at low concentrations relative to an oxidizable substrate, considerably slows down or stops the oxidation of that substrate. To sustain human health and preserve food quality, antioxidants are essential.

Antioxidants are compounds that can protect your cells from free radicals, which are important for health. Your body produces free radicals when it breaks down food, smokes, or is exposed to radiation. These are either manufactured or natural compounds that can help avoid or postpone cell harm.^[1]

The effects of an oxidation reaction vary depending on the location of the event.^[1] "Pro-oxidants" are chemical substances and reactions that could produce potentially harmful oxygen species or free radicals. The substances and reactions that eliminate these species, scavenge them, restrict their creation, or oppose their actions are known as antioxidants. On the other hand, they target macromolecules such as protein, DNA, and lipid, causing damage to cells and tissue. There is a proper pro-oxidant: antioxidant equilibrium in a healthy cell. Nonetheless, when oxygen species production rises or antioxidant levels fall, this equilibrium may be tipped in favor of the pro-oxidant.^[2] Oxidation damages or kills the cell when it takes place in a biological cell system. When fats and oils are used as a component in food, their oxidative degradation causes rancid flavors and odour, which lowers the food's nutritional value, sensory appeal, and safety. This is brought on by the auto-oxidation of unsaturated fatty acids using a free radical chain mechanism, which produces primary hydroperoxides and secondary potentially hazardous chemicals.^[1]

Both oxygen-derived (ROS) and nitrogen-derived (RNS) free radicals are possible. Superoxide anion (O2), hydrogen peroxide (H2O2), peroxyl radicals (ROO), and reactive hydroxyl radicals (OH) are the most prevalent reactive oxygen species. Nitric oxide (NO), peroxy nitrite anion (ONOO), nitrogen dioxide (NO2), and dinitrogen trioxide (N2O3) are the free radicals that are produced from nitrogen. Endogenous sources of ROS include the mitochondrial electron transport chain and β-oxidation of fat, while external sources include electromagnetic radiation, cosmic radiation, UV light, ozone, cigarette smoke, and low wavelength electromagnetic radiations. "Oxidative stress" is the term for this condition, which, if severe or protracted, can cause major cell damage. Since ancient times, Indian alternative medical systems have effectively used herbal antioxidants as rejuvenators.^[2]

Antioxidants work by halting or postponing other molecules' oxidation. Antioxidants' involvement in biology was initially studied in relation to their ability to keep unsaturated fats from getting rancid. But the discovery of vitamins A, C, and E as well as the comprehension of how vitamin E prevents lipid peroxidation was the turning point that led to the realization of the importance of antioxidants for living things. Enzymatic and non-enzymatic antioxidants are the two primary types.

Various compounds with distinct modes of action, locations of action, and ultimate consequences are among them. Each one's unique function within the body is determined by this diversity. It should be highlighted that the network of antioxidant enzymes that interact, such as superoxide dismutase enzyme (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRd), shows the antioxidant defence effectiveness.^[3]

 Antioxidant potential in plants:

                                            Antioxidant classification ^[3]

Plants possess antioxidant potential because they naturally produce a variety of compounds to guard against oxidative damage from free radicals produced during their metabolic processes, especially in response to environmental stressors like UV radiation, extreme temperatures, and pathogens. By neutralizing harmful reactive species and preventing damage to vital plant tissues, these antioxidants help maintain cellular balance.

Within plant cells, reactive oxygen species (ROS) are primarily produced by mitochondria and chloroplasts. These substances also play a role in preserving a delicate equilibrium between energy-related processes and ROS generation regulation.^[4] These ROS are essential for oxidative signaling and, when combined with other elements such as plant hormones, trigger a prompt and efficient reaction that guarantees plant acclimation and, ultimately, adaption to a stressful environment. However, biological macromolecules including proteins, membrane lipids, and DNA can potentially be harmed by elevated ROS concentrations. Plants have a robust antioxidant defense mechanism that maintains oxidative signaling while regulating ROS levels to prevent oxidative damage. Dehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), and other enzymes of the AsA-GSH cycle, as well as those that provide reducing power, are crucial for recycling non-enzymatic antioxidants like AsA and GSH, even though they are directly involved in ROS detoxification. Both compounds are water-soluble and have a low molecular weight (LMW).^[10]

Plants naturally produce a variety of reactive oxygen species (ROS), including non-radical molecules like hydrogen peroxide (H2O2) and singlet oxygen (O2) and free radicals like superoxide anion (O2•−), hydroperoxyl radical (HO2•), alkoxy radical (RO•), and hydroxyl radical (•OH).^[5]

Catalase (CAT) and flavin oxidases, which produce hydrogen peroxide (H2O2), are basic enzyme components found in peroxisomes. Photosystem I and II (PS I and PS II) of the chloroplasts, the peroxisome's membrane and matrix, and complex I, ubiquinone, and complex III of the mitochondrial electron transport chain (ETC) are the sites in plant cells where ROS creation takes place. PS I and PS II of the chloroplasts, the mitochondrial ETC membrane, and the peroxisome all experience electron slippage under typical physiological conditions. A superoxide radical (O2-●) is subsequently created when these electrons interact with molecular oxygen.

Subsequently, the superoxide radical transforms into the hydroperoxyl radical (HO2●) and then H2O2. As with ROS, the cell's chloroplasts, mitochondria, and peroxisomes are among the compartments where reactive nitrogen species (RNS) like the nitric oxide radical (NO•) and peroxinitrite (ONOO-) are produced. Thiols are said to combine with reactive sulfur species (ROS) to create RSS, the third type of free radical. In live cells, these free radicals are continuously generated in the subcellular organelles. Since free radicals serve as signaling molecules, their production is typically genetically predetermined. On the other hand, an excess of free radicals can occasionally harm biomolecules including proteins, lipids, and DNA.^[4]

To prevent the harmful effects of free radicals, plants have effective complex enzymatic and non-enzymatic antioxidant defense systems. Among the non-enzymatic systems are high molecular weight secondary metabolites like tannins and low molecular weight antioxidants like ascorbic acid, glutathione, proline, carotenoids, phenolic acids, flavonoids, etc. Non-enzymatic antioxidant synthesis is a natural ability of plants. However, biotic and abiotic stressors cause plants to produce more reactive oxygen species (ROS), which in turn causes oxidative stress. Under in vitro studies, plants respond to increased oxidative stress by increasing the production and accumulation of several low molecular weight antioxidants (like vitamin C, vitamin E, phenolic acids, etc.) and high molecular antioxidant secondary metabolites, like tannins, which provide antioxidants to the majority of plants by acting as metal chelators, reducing agents, and free radical scavengers.^[4]

Plants with an antioxidant defense mechanism are essential in stressful situations because it prevents programmed cell death. Plants cannot effectively carry out their functions if they lack enough antioxidant enzymes to scavenge excessive ROS. This leads to lipid peroxidation, oxidative protein degradation, DNA and nucleic acid breakdown, and several enzyme inhibitions. The antioxidant defense system delays oxidation and regulates DNA and nucleic acid damage under stress, ensuring effective ROS detoxification, decreased lipid peroxidation in membranes, and prevention of protein damage. The plants' capacity to withstand stress is thus made possible by the general cellular protection this offers.^[5]

Antioxidant properties of banana leaves:

Organic antioxidants called polyphenols, which are found in banana leaves, offer antioxidant qualities and aid in the body's defense against free radicals. Polyphenols, which are antioxidants with possible health benefits, are found in banana leaves. Certain polyphenols may migrate onto the food when it is served hot on the leaf, offering some antioxidant advantages.^[7]

The methanol extract of banana leaves had a higher scavenging ability (44.50 µg/mL) than the acetone extract (27.81 µg/mL). The researchers hypothesized that the antioxidant activity of banana leaves was directly related to the presence and content of phenolic compounds. The antioxidant activity of banana leaves is influenced by the type of banana and the drying temperature. The aqueous extract of banana leaves had a trolox equivalent antioxidant capacity (TEAC) value that was 24–295 times lower than that of the acetone and methanol extracts, respectively.^[6]

The natural antibacterial qualities of banana leave aid in the destruction of dangerous microorganisms found in food. Thus, eating a banana leaf can lower your chance of contracting a foodborne illness. Vitamin A, vitamin C, and polyphenols are just a few of the vital nutrients found in banana leaves. Some of these nutrients are transmitted to the food when it is placed on a banana leaf, increasing the food's nutritious worth. Eating on the leaf of a banana helps improve digestion. Better digestion and nutrient absorption are facilitated by the polyphenols in banana leaves, which also increase the synthesis of digestive enzymes.^[7]

The banana is among the most important foods that are strong in antioxidants. When a chemical is present at low quantities and has the ability to delay, retard, or halt the oxidation or free radical-mediated oxidation of a substrate, resulting in the formation of stable radicals after scavenging, it is considered an antioxidant. The physiological defense of bananas against oxidative and free-radical stress is aided by the presence of bioactive substances with antioxidant potential.^[8]

Secondary metabolites as antioxidants:

There are two core and secondary categories for plant metabolism. Sugars, fats, amino acids, and nucleic acids are examples of compounds that are created via primary metabolism and are commonly referred to as primary metabolites. Plant cell upkeep necessitates primary metabolites, but normal plant growth, development, and defense depend on secondary metabolites.^[4]  It has been observed that antioxidants guard against oxidative damage brought on by free radicals. Although defense mechanisms scavenge and stabilize free radicals, oxidative stress is a harmful process that can harm cell components, including proteins, DNA, and lipids, when the rate of free radical generation reaches a certain threshold. This process protects all organisms against the attack of free radicals.^[9]

The optimum system for classifying secondary metabolites in plants is up for debate. These are most categorized into three primary classes according to their biosynthetic origin: Compounds comprising 1. nitrogen and sulfur, 2. terpenoids, 3. polyphenols, and flavonoids. All these three groups contain compounds with antioxidant properties. A common component is present in the most abundant secondary metabolites of plants that exhibit antioxidant activity. This is an atom that has at least one free pair of electrons, such as nitrogen, sulphur, or oxygen, connected to an aromatic ring. The group with the fewest reported antioxidants is those that contain nitrogen and sulphur.^[10] The radical scavenging ability of alkaloids is said to be modest to non-existent under the conditions of an in vitro antioxidant measuring experiment. With over 40,000 distinct chemicals, terpenoids make up another sizable class of secondary metabolites. Various in vitro tests have revealed that monoterpenes, sesquiterpenes, and diterpenes exhibit significant antioxidant potential. In vitro and in vivo studies have demonstrated the strong antioxidant properties of tetraterpenes and carotenoids; nevertheless, at high oxygen pressure and concentration, several valued carotenoids, such beta-carotene, exhibited prooxidant effects. Given their encouraging antioxidant activity in both in vitro and in vivo studies, phenolic antioxidants seem to be the most significant of all secondary metabolites.^[4] Many medication formulations based on antioxidants are used to prevent and treat diseases whose processes involve the process of oxidative stress. Since synthetic antioxidants are prohibited because they cause cancer, there is a lot more interest in identifying naturally occurring antioxidants to replace synthetic ones in foods, cosmetics, and pharmaceutical materials.^[9]

Since there are some theories that the natural antioxidants found in many medicinal plants can neutralize free radicals within the body, there has been a lot of interest lately in natural foods and their own therapeutic potential to scavenge free radicals. As a result, there has been a lot of interest in adding natural antioxidants to food and biological-based systems of scavenging free radicals, which may offer a safe substitute for toxic and harmful synthetic antioxidants.^[9] In general, phenolic substances have one or more hydroxyl groups attached to aromatic rings. It has long been believed that the more free hydroxyls and side chain conjugation to aromatic rings there are in phenolics, the greater their antioxidant activity. The acetate and shikimate processes biosynthesize flavonoids and phenolic acids, the two main families of plant phenolics, from phenylalanine or tyrosine. As H2O2 scavengers, flavonoids and phenylopropanoids are likewise oxidized by peroxidase. Under experimental conditions, the ability of plant phenolics to donate electrons, reduce power, and chelate metal ions is always associated with their antioxidant potential.^[4]

Mechanism of antioxidant activity {free radical mechanism}:

There are several ways that phenolic chemicals can improve human health. Since they exhibit a high direct radical scavenging capacity against numerous nitrogen and oxygen reactive species, their antiradical qualities and capacity to modulate biological oxidative stress should result in the most obvious mechanism of action in preventing cellular oxidative damage. This should prevent damage to lipids, proteins, and DNA. Their interactions with singlet oxygen, lipid peroxyl radicals, and superoxide anions are advantageous from a thermodynamic perspective. As was previously stated, these substances may also indirectly exhibit their antioxidant properties by recycling endogenous antioxidants or by chelating metals, as in the case of quercetin and catechins. The reactivity of antioxidants with reactive oxygen species (RONs) has been extensively researched, as has the significance of RONs in biological systems. The superoxide anion is often obtained from non-enzymatic sources (potassium superoxide) and the enzymatic system (hypo)xanthine-xanthine oxidase for assessing antioxidant activity.^[11]

The mitochondrial electron transport chain, neutrophil myeloperoxidase and NADPH oxidase, and endothelial cell xanthine oxidase are the three main producers of reactive oxygen species (ROS). However, xanthine oxidase and the mitochondrial respiratory chain are the main producers of ROS. Additionally, the inflammatory response triggered by cytokines released from the injured cells results in a delayed and increased formation of ROS. The body has an efficient defense mechanism to deal with reactive species, which includes: high molecular weight antioxidants like albumin, ceruloplasmin, and ferritin; a variety of low molecular weight antioxidants like ascorbic acid, α-tocopherol, β-carotene, glutathione, and uric acid; and enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP), and glutathione reductase (GR). Maintaining equilibrium between the generation of ROS/RNS and antioxidant defense in health.^[12]

 Antioxidant capabilities are evaluated using a variety of methods, including electron paramagnetic resonance (EPR), chemiluminescence, UV-Visible spectroscopy, fluorescence spectroscopy, enzyme-catalysed tests, and cell culture assays. Additionally, several electrochemical methods are frequently used, such as biosensors, electrochemical sensors, and controlled potential approaches. However, the most popular methods for assessing an antioxidant's scavenging activity, such as ABTS●+, DPPH●, O2●−, and H2O2, as well as its total antioxidant reduction capacity, such as TEAC, ORAC, and FRAP, are spectrometric methods. Numerous plant extracts, foods, and nutritional supplements have had their antioxidant capacity assessed using these techniques like hydrogen atom transfer (HAT), singlet electron transfer (SET), peroxynitrite scavenging activity.^[3]

Hydrogen atom transfer (HAT):

Adopting hydrogen atom transfer (HAT) in a photocatalytic technique, where substrate activation is accomplished by an excited catalyst, presents special prospects in organic synthesis by making it possible to easily activate R–H (R = C, Si, S) bonds in chosen reagents. There are two possible approaches: an indirect one that uses a photocatalytic cycle to create a thermal hydrogen abstractor, or a direct one that relies on the inherent reactivity of a small number of photocatalysts in the excited state.^[13]

The most frequent reaction involving the transfer of two elementary particles—a proton and an electron—is hydrogen atom transfer. From combustion and aerobic oxidations to enzymatic catalysis and the harmful effects of reactive oxygen species in vivo, HAT is an essential step in many different processes. More recently, metalloenzyme active sites have been demonstrated to oxidize substrates using HAT, which has prompted research into transition metal mediated HAT. We create a model based on Marcus Theory that provides new insight into HAT reactions and quantitative predictions of rate constants.^[14]

                                                     A-H + B -------> A + H-B    equation (1)

The abstracting group in classical organic HAT reactions is a p-block radical X•, like t-butoxyl. On the other hand, transition metal complexes that abstract H• (≡ H+ + e–) usually contain a basic ligand to receive the proton and an oxidizing metal core to accept the electron. Because the e– and H+ may separate, these reactions are sometimes referred to as proton-coupled electron transfer (PCET) reactions. Therefore, we believe that the same word (HAT) should be applied to almost all reactions where [H+ + e–] are moved from one group to another in a single kinetic step.^[14]

                                                         X + H-R -------> X-H + R    equation (2)

Singlet electron transfer (SET):

The most crucial energy component in assessing antioxidant action in the SET mechanism is the antioxidant's IP (ionization potential).^[15] Electron transfer from a nucleophile to a substrate that results in a radical intermediate is what defines single electron transfer (SET) reactions. A carbonyl is protonated before being attacked by a nucleophile. The carbonyl oxygen's pre-protonation greatly accelerates the nucleophile's rate of attack. The carbon now has a significantly stronger positive charge than the unprotonated carbonyl's simple resonance partial positive charge, which speeds up the reaction rate with the nucleophile.^[16]

Free radical species are extremely unstable intermediates with an unpaired electron that are formed and then react in single-electron pathways. A common process that results in the formation of free radicals is homolytic cleavage, which occurs when two electrons in a covalent bond break in opposing directions. Conversely, nearly every reaction we have examined thus far involves bond-breaking events known as heterolytic cleavage, in which both electrons travel in the same direction.^[17] SET reaction involves of four steps mainly, SET between the substrate (RX) and the nucleophile.

After the radical anion separates, the radical (R•) interacts with Nu–.

To create the radical anion (RNu–) product. continues the radical chain process and produces RNu as the result.^[16]

Techniques for evaluating plants' antioxidant potential:

The substance or extract's antioxidant potential could be assessed both in vitro and in vivo. The following principles are used to develop the in-vitro antioxidant estimations, depending on the mechanism:

1.Calculation using the capacity to scavenge free radicals.

2. Metal ion reduction estimation.

3. Calculation through suppression of plasma lipid peroxidation.

4. Estimation of oxidative stress resistance using cultivated cells.^[1]

One antioxidant test model should not be used to determine antioxidant activity. Additionally, several in vitro test techniques are used in practice to assess antioxidant activity in the relevant samples. Free radical traps are generally a fairly simple way to conduct in vitro antioxidant studies. When compared to other test models, the DPPH approach is also quick, easy, and affordable among free radical scavenging techniques.^[18]

DPPH scavenging assay:

The capacity to scavenge DPPH radicals produced in a model system is the measurement principle of the dpph scavenging test.^[1] The reason that 1, 1-diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH) is a persistent free radical is that the spare electron delocalizes over the entire molecule, preventing it from dimerizing like most other free radicals do. The deep violet color, which is defined by an absorption band in ethanol solution with a center at roughly 517 nm, is likewise caused by the delocalization of electrons. This violet hue is lost when a DPPH solution is combined with one of a substrate (AH) that can donate a hydrogen atom to create the reduced form.^[18] where A• is a free radical created in the first step,

R• + SH = RH + S•

and R• is the donor molecule and SH is the DPPH, culminating in the reduced form RH. The free radical will then go through additional reactions to produce RS-SR, a stable compound. Even while DPPH can stabilize as a diamagnetic molecule by accepting an electron or hydrogen radical, it can only be oxidized with difficulty and then irreversibly.^[19]

RS• + RS• = RS – SR

The following formula is used to determine the percentage of DPPH radical scavenging:

% inhibition of DPPH radical = ([Abr – Aar]/Abr) *100

Abr is the absorbance prior to the reaction, and Aar is the absorbance following the reaction.^[18]

Hydroxyl scavenging assay:

The capacity to scavenge hydroxyl radicals produced in a model system is the basis for measuring H2O2. The source of hydroxyl radicals is H2O2.^[1] The human body can absorb hydrogen peroxide through contact with the skin or eyes, as well as through inhaling vapor or mist. OH radical, which can start lipid peroxidation and damage DNA in the body, are produced when H2O2 breaks down quickly into oxygen and water.^[18] After mixing the test chemical with reaction buffer, it is incubated at 37°C for one hour. To the mixture, trichloroacetic acid and thiobarbituric acid are mixed and kept in boiling water bath for 10 min, followed by cooling to room temperature and measure the absorbance at 532 nm. FeCl3, ascorbic acid, ethylenediamine tetra acetate (EDTA), deoxyribose, and H2O2 in phosphate buffer (pH 7.4) were used to prepare the reaction mixture. After thoroughly mixing each of these solutions, they were each employed as an analytical reaction buffer.^[1]

This is how the hydrogen peroxide scavenging % is determined:

%scavenged(H2O2) = [(Ai-At)/Ai] ×100

where At represents the test absorbance and Ai represents the control absorbance.

Trolox equivalent antioxidant capacity (TEAC) /ABTS radial cation decolorization assay:

The ABTS/TEAC assays employ the highly colorful cation radicals of ABTS•+ as practical colorimetric probes that take either electrons or hydrogen atoms from antioxidant substances.^[20] TEAC is derived from trolox, a synthetic antioxidant (water-soluble vitamin E analogue) that is used as a benchmark. The millimolar concentration of a Trolox solution with an antioxidant capacity equal to a 1 mM solution of the material being studied is known as the TEAC. Therefore, in comparison to Trolox, TEAC indicates the relative capacity of hydrogen or donating-electron antioxidants to scavenge the ABTS radical cation.^[19] The antioxidant decolorizes and converts ABTS·+ to ABTS. The human body does not contain the stable radical ABTS·+. Solid manganese dioxide (80 mg) is added to a 5 mM aqueous stock solution of ABTS (20 mL) using a 75 mM Na/K buffer of pH 7 to create ABTS radical cations. A standard calibration curve is created for Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) at concentrations of 0, 50, 100, 150, 200, 250, 300, and 350 μM. Samples are suitably diluted based on the antioxidant activity in a pH 7 Na/K buffer. 200 μL of ABTS radical + radical cation solution is added to diluted samples on 96-well plates, and after five minutes, absorbance (at 750 nm) is measured in a microplate reader.^[18]

Total radial trapping antioxidant parameter (TRAP) method:

TRAP measured the overall peroxyl radical-trapping capacity of plasma by measuring the induction times in the oxidation of lipids. Peroxyl-radicals were produced by ABAP [2,2′-azo-bis- (2-amidipropropane hydrochloride)] thermolysis, which initiated the plasma peroxidation. The duration of oxygen uptake inhibition by plasma antioxidants was noted. The induction period, which is a quantitative measure of the inhibition time of O2 absorption, is expressed as the TRAP index (number of mols of ROO•/liter of fluid). This allows TRAP to be computed as follows:

TRAP = R ROO x τplasma,

 where RROO is the rate at which free radicals develop and τplasma is the oxygen consumption delay time.^[19] A TRAP measurement is performed on an organic substrate, which could be plasma or lipid. Here, AAPH is used to incubate the control (without test sample), experiment, and standard samples at 37°C. The amount of time needed for the oxidizable substrate to absorb oxygen is assessed once AAPH is added. The oxygen electrode is used to control the time. Trolox is used to express the activity. A different method for TRAP, however, uses the loss of fluorescence of the protein R phycoerythrin (R-PE) to track the rate of peroxidation brought on by AAPH. In the TRAP experiment, the lag-phase that Trolox and plasma generate in the same plasma sample are contrasted.^[1]

Ferric reducing antioxidant power (FRAP) assay:

 The basis of the FRAP assay, which was intially developed by Benzie and Strain to assess the antioxidant capacity of plasma and subsequently adapted for use with other matrices like tea and wine, is the reduction of Fe (III) to Fe (II) by antioxidants when tripyridyl triazine tridentate ligand is present and forms a coloured complex with Fe (II)^[20]

A Fe3+ complex reduction's capacity serves as the foundation for the FRAP assay chemical reaction. Antioxidant characteristics cause a colorless complex to transform into a blue one called Prussian blue, or [Fe2+(TPTZ)2 ]2+ in an acidic media. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a soluble analogue of vitamin E, is employed as a positive control in this colorimetric test, which measures color changes at 593 nm using a spectrophotometer. This standard is a ferrous ion solution. The outcome is given in FRAP units because the FRAP unit is associated with the reduction of one M ferric ion to one ferrous ion.^[19] Measured in mM of Fe2+ equivalents per kg (solid food) or per L (beverage) of sample, FRAP values are produced by comparing the absorption change in the test mixture with those obtained from increasing concentrations of Fe3+.^[18]

A few drawbacks of the FRAP assay include the redox potential of the Fe2+/Fe3+ pair, which can cause a false Fe3+ reduction when testing compounds with a lower redox potential, the rapid reaction time, which assumes that redox reactions are quick but aren't always, and the hydrophilic reaction environment, which makes it difficult to test hydrophobic compounds effectively. The FRAP assay is a commonly used technique to evaluate the overall antioxidant capacity of foods, drinks, plant extracts, essential oils, and biological fluids. It can also be used as a prognostic biomarker for several conditions, including chronic kidney disease, hypertension, and acute myocardial infarction followed by cardiogenic shock.^[19]

Superoxide dismutase (SOD) radical scavenging activity:

Despite being a mild oxidant, superoxide anion eventually generates singlet oxygen and strong, hazardous hydroxyl radicals, both of which exacerbate oxidative stress.^[18] Superoxide dismutase or metal ion-dependent superoxide dismutase, are vital elements in the antioxidant system's defense. In mammalian physiology, SODs come in three different varieties with highly controlled localization patterns. CuZnSOD (SOD1), which is found in the cytosol, mitochondrial intermembrane space, and nucleus, and EcSOD (SOD3), which is the main antioxidant enzyme which is secreted into the extracellular space, are the two members that comprise copper and zinc. The mitochondrial matrix contains manganese-containing SOD (SOD2), which differs little from other SODs.^[19] The SOD enzyme was mounted onto carbon fiber microelectrodes coated with cysteine-functionalized Au nanoparticles (via self-assembled monolayers), allowing direct electron transfer with O2•– and catalyzing their dismutation to O2 and H2O2.^[20]

Oxygen radical absorbance capacity (ORAC):

The "Antioxidant Power" of foods and other chemicals can be tested using ORAC, a novel and interesting new test tube technique.^[18] The capacity to scavenge oxygen radicals (peroxy radicals) is the fundamental idea behind ORAC. Using 2,2'-azobis(2-amidino-propane) dihydrochloride, oxygen radicals are produced.^[1] The target molecule for this test can be either fluorescein or β-phycoerythrin (β-PE). Therefore, it appears that in the ORAC test, fluorescein is taking the place of β-PE as the target molecule. Trolox, a water-soluble analog of vitamin E, is used as a reference in the test to calculate the Trolox Equivalent (TE). After that, the Trolox Equivalent is used to determine the ORAC value, which is then converted to ORAC units or value. The "Antioxidant Power" increases with the ORAC number.^[18] β-phycoerythrin (β-PE), which has an excitation wavelength of 540 nm and an emission wavelength of 565 nm, was employed as a fluorescent probe in the first technique. Because of its high fluorescence production and ability to absorb visible light, β-PE is extremely sensitive to ROS. Furthermore, different fluorescent probes were proposed to avoid the interaction between β-PE and samples containing phenols and polyphenols, as well as to address the inconsistent emission of β-PE fluorescent light (it is photo-sensitive, meaning it loses its fluorescence even in the absence of free radicals). The new probes, which were most commonly fluorescein and 6-carboxyfluorescein, required to have high molar absorption coefficients, significant fluorescence yields, and photochemical stabilities. Based on the in situ generation of peroxyl free radicals using the azo-compound 2,2,-azobis(2-methylpropionamidine) dihydrochloride (AAPH), the ORAC assay^[21]

Role and significance of Reactive Oxygen Species & Antioxidant defense system in plants:

 In the age of climate change, plants face a number of challenges in the field. Plant development, growth, and sustainable crop production are significantly impacted by abiotic stressors such as xenobiotics, severe temperatures, metal/metalloid toxicity, salinity, and water stress (drought and waterlogging). One of the most significant effects of abiotic stress is the disruption of the balance between ROS production and antioxidant defense mechanisms, which leads to an excessive build-up of ROS and oxidative stress in plants.^[22] However, during stress, plant cells produce too many ROS. Because ROS are extremely reactive, they disrupt plant metabolism and seriously harm vital cellular constituents like proteins, lipids, carbohydrates, DNA, and others. Consequently, oxidative stress in plants results from this disturbance of the equilibrium between typical ROS formation and antioxidant activity.^[5]

Generation of Singlet Oxygen from Chlorophyll Biosynthesis Intermediates:

When plants are exposed to light, intermediates of chlorophyll production, such as protoporphyrin IX or protochlorophyllide, generate oxygen and induce oxidative damage. Reactive oxygen species formation from stages of chlorophyll biosynthesis was another theory put forth. The majority of 1O2 production occurs in the thylakoids. As a result of their partial hydrophobicity, the intermediates of chloroplast biogenesis are weakly affixed to the thylakoid membranes. Since these tetrapyrroles do not form pigment protein complexes, they are not linked to the reaction center even though they are associated with the thylakoid membranes. Even while the lipid bilayer contains some carotenoids, the majority are found in pigment-protein complexes, which are too far away from intermediates of chlorophyll production to quench their triplet states. The production of Chl biosynthetic intermediates is strictly controlled to prevent plants from producing too many of them. Under high light and other stress circumstances, however, the intermediates of chlorophyll production that are typically found in plants can produce O2 that leads to oxidative damage.^[23] One of the ways that severe desiccation brought on by water stress, salt stress, etc., might cause apoptosis is by ROS. When excited Chl molecules are present in desiccated photosynthetic tissues but carbon fixation is restricted by water scarcity, certain challenges occur. The excitation energy can be transferred from photo-excited chlorophyll pigments to ground state oxygen (O2), creating singlet oxygen (O2), while electron transport continues under these circumstances. Through non-photochemical quenching (NPQ), photosynthetic organisms can release surplus energy and prevent the production of O2. This most likely involves the xanthophyll cycle, wherein solar radiation is lost as heat and violaxanthin is gradually de-epoxidized to antheraxanthin and then to zeaxanthin. Energy dissipation is another function of other carotenoids, in addition to their role as accessory pigments in photosynthesis.^[23]

Oxidative stress in plants and downstream implications:

ROS are produced by redox reactions, which are highly common in living things and involve the transfer of electrons between a donor and an acceptor. Redox homeostasis develops in plant cells as a result of the balance between ROS formation and antioxidant enzyme activity. An effective defense system in plants maintains the right ratio between ROS generation and elimination. Therefore, appropriate ROS or redox signaling in cells requires a basal level of ROS that is maintained above cytostatic or below cytotoxic concentration. The equilibrium between ROS generation and scavenging keeps this level stable. The balance between low ROS levels that serve as signals to trigger signaling cascades that modify regular plant functions and high ROS levels that result in oxidative cellular damage has been identified as redox signaling. With the help of the cellular redox-sensitive components, a stable equilibrium between ROS formation and ROS scavenging systems is therefore well synchronized throughout time and space, helping to mold and fine-tune downstream signaling processes in a context-specific and cell-specific manner. On the other hand, under a variety of abiotic stress situations, any disruption in the balance between ROS formation and ROS scavenging by antioxidants causes ROS to over accumulate and cause oxidative stress.^[22]

Abiotic stress results in the generation of excess ROS because it disrupts several metabolic processes and causes physiological diseases. When NADPH is used up, antioxidant defense

pathways like the AsA-GSH pathway cannot prevent ROS damage. However, during the close of the 20th and the start of the 21st centuries, the roles of ROS (particularly H2O2) in plant responses to stressors gained attention. Electrons from the photosynthetic machinery may be diverted by reactive oxygen species produced in the chloroplast under stress, preventing antenna overload and eventual damage. In a similar manner, reactive oxygen species also shield mitochondria. ROS production may be aided by cell wall peroxidase in the direction of signaling, where H2O2 uses the Ca2+ and MAPK pathway as a downstream signaling cascade. The dual role of ROS under stress conditions is further supported by the fact that plant hormones, particularly ethylene (ET) and abscisic acid (ABA), improve stress tolerance and interact with ROS to influence stress responses.^[22] ROS have the ability to control metabolic fluxes under abiotic stress conditions in addition to signal transduction and hormone interaction. These processes work together to control plant acclimation processes, where redox reactions govern the transcription and translation of stress acclimation proteins and enzymes, ultimately preventing damage to plant cells. Additionally, under a variety of abiotic stressors, H2O2 regulates the NO and Ca2+ signaling pathways, which govern plant growth and development in addition to other cellular and physiological reactions. Given that endogenous H2O2 contributes to enhancing resistance to abiotic stress, exogenous H2O2 application is becoming more and more popular and has largely demonstrated its effectiveness.^[22] Numerous strategies have been employed by researchers to boost antioxidant defences and lessen the detrimental effects of oxidative damage. strategies spanning from exogenous protectant introduction in plants to genetic modification. Antioxidant enzyme characterization and profiling are two of the targeted strategies.^[5]

Therapeutic relevance and plant antioxidant behavior:

 The human body, like plants, is continuously exposed to free radicals and/or oxidants produced by physiological functions including mitochondrial respiration. While plants produce more free radicals in response to biotic and abiotic stressors, humans produce more free radicals in response to pathophysiological situations such inflammation, foreign substance metabolism, and radiation.^[4] Many human diseases, including cancer, stroke, neurological diseases, and many more, are thought to be caused by or made worse by reactive oxygen species (ROS). Antioxidants are thought to prevent or treat diseases linked to oxidative stress by neutralizing the negative effects of ROS.^[24] Many neurological, neurodegenerative, and psychiatric conditions share oxidative stress. Thus, neuroprotective and oxidative stress-reducing chemicals could be promising new treatments. The following were investigated: phenolic, flavonoid, and anthocyanin content; free radical scavenging by ORAC and DPPH; and Cu2+ and Fe2+ chelating capabilities.^[25] A small number of antioxidants, such as edaravone (used in Japan to treat ischemic stroke), acetaminophen toxicity, diabetic neuropathy, alfa-lipoic acid, and certain flavonoids (polyphenolic compounds found in dietary plants), such as oxerutins (used to treat chronic venous insufficiency), baicalein, and catechins (used to treat osteoarthritis), as well as micronized purified flavonoid fraction (diosmin and hesperidin) and oxerutins (used to treat chronic venous insufficiency), have accepted clinical use.^[24] The potential of plant-derived chemicals, including phenolic, flavonoid, and anthocyanic derivatives, to improve several facets of health by having antioxidant and anti-inflammatory properties is being studied more and more. Phenols are a broad class of chemicals found in many plants, including fruits and vegetables, that are physically defined by at least one aromatic ring to which one or more hydroxyl groups are connected. Through the transfer of hydrogen atoms, the transfer of a single electron, successive proton loss electron transfer, and the chelation of pro-oxidant metal ions, phenolic compounds react with free radicals to provide antioxidant effects.^[26] In addition to low-molecular-weight scavenging antioxidants like glutathione, ascorbic acid, tocopherols, proline, carotenoids, and phenolic compounds, plants also contain strong enzymatic antioxidants like catalase, superoxide dismutase, glutathione peroxidase, NADPH dehydrogenase, and peroxiredoxin, as well as high-molecular-weight compounds like tannins. Carotenoids and phenolic compounds are found in many fruits, vegetables, and other plants. Their antioxidant properties provide them anti-inflammatory, anti-carcinogenic, anti-atherogenic, antithrombotic, cardioprotective, and vasodilatory properties, which have some health benefits.^[25] The pathophysiology of diseases associated to free radicals can be improved by supplementing with external antioxidants or increasing the body's endogenous antioxidant defenses, notwithstanding the debates around the benefits and drawbacks of plant antioxidants. The most effective exogenous antioxidants have been found to be plant-based antioxidants such flavonoids and ascorbic acid. Indeed, by scavenging free radicals, these chemicals not only limit the generation of ROS but also support the body's natural antioxidant defenses. However, there are still concerns that need to be thoroughly examined regarding the potential benefits of endogenous antioxidants as therapeutic agents.^[4] The potential of plant-derived chemicals, including phenolic, flavonoid, and anthocyanic derivatives, to improve several facets of health by having antioxidant and anti-inflammatory properties is being studied more and more. Phenols are a broad class of chemicals found in many plants, including fruits and vegetables, that are physically defined by at least one aromatic ring to which one or more hydroxyl groups are connected. Through the transfer of hydrogen atoms, the transfer of a single electron, successive proton loss electron transfer, and the chelation of pro-oxidant metal ions, phenolic compounds react with free radicals to provide antioxidant effects.^[26] Plants have been used by humans from the beginning of time, and today between 70 and 95 percent of people in poor nations use them. High-molecular-weight compounds like tannins and low-molecular-weight scavenging antioxidants like glutathione, ascorbic acid, tocopherols, proline, carotenoids, and phenolic compounds are found in plants, along with potent enzymatic antioxidants like catalase, superoxide dismutase, glutathione peroxidase, NADPH dehydrogenase, and peroxiredoxin. Carotenoids and phenolic compounds are found in many fruits, vegetables, and other plants. Their antioxidant properties provide them anti-inflammatory, anti-carcinogenic, anti-atherogenic, antithrombotic, cardioprotective, and vasodilatory properties, which have some health benefits.^[25]

Conclusion:

Currently, little is known about the abundance of bioactive chemicals and antioxidants found in plant secondary metabolites. The vast chemical variety of this class of antioxidant phytochemicals offers a wealth of knowledge about the plant species' nutraceutical or medicinal potential as well as their evolutionary adaption to challenging environmental conditions.^[10]  It is crucial to use a range of techniques for evaluating antioxidant capacity due to the different mechanisms underlying lipid and antioxidant interactions, complex heterogeneous biological and food-related systems, and inconsistent analytical results regarding antioxidant potency. Further study is necessary to better understand and set regulatory standards because antioxidants have also been demonstrated to have toxicity and pro-oxidant effect.

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