Fe³⁺ Reducing Power as the Most Common Assay for Understanding the Biological Functions of Antioxidants

Abstract

Antioxidants counteract the harmful effects of free radicals on metabolism and prevent fatty food degradation during processing and storage. The Fe³⁺-reducing assay, based on reduction of ferric ions (Fe³⁺) to ferrous ions (Fe²⁺) in the presence of antioxidants acting as reducing agents, is widely recognized and used to evaluate the antioxidant capacity of various biological samples, including plant extracts, food, beverages, and pharmaceuticals. Reduction of Fe³⁺ to Fe²⁺ is also crucial in biogeochemical cycling, microbial metabolism, and industrial applications. This review comprehensively describes the Fe³⁺-reducing assay, its adaptation to different analytes, identification of the most potent antioxidants, and optimization of measurement techniques. It outlines the chemical and fundamental principles of Fe³⁺ reducing ability, along with an in-depth analysis of Fe³⁺-reducing activity, covering biochemical mechanisms, microbial contributions, analytical methods, and practical applications along with recent advances and future perspectives in Fe³⁺ reduction research. The assay is straightforward, testing compounds or plant extracts are mixed with an Fe³⁺ solution, and their absorbance is measured after a specific incubation period. Despite significant advancements in analytical instrumentation and techniques, this method remains largely unchanged.

1. Introduction

1.1. Reactive Oxygen Species (ROS)

Oxidation processes are crucial for metabolism and cell survival. In aerobic cellular respiration, organisms generate energy from organic molecules like glucose, also leading to the production of free radicals that can cause cellular damage via metabolic activity [1]. In aerobic organisms, the process of breaking down organic molecules like glucose to release energy involves a series of reactions occurring in the mitochondria. These reactions are important for synthesizing adenosine triphosphate (ATP), the primary energy currency of the cell. Although this process is necessary for cellular energy metabolism, it generates byproducts known as free radicals and reactive oxygen species (ROS). These highly reactive molecules cause significant damage to cellular structures and contribute to various degenerative diseases [2]. ROS are highly reactive molecules derived from molecular oxygen (

Table 1. Reactive oxygen (ROS) and nitrogen species (RNS).

Reactive Oxygen Species		Non-Free-Radical Species
Hydroxyl radical	HO·	Hydrogen peroxide	H2O2
Superoxide radical	O2·−	Singlet oxygen	1O2
Hydroperoxyl radical	HOO·	Ozone	O3
Lipid radical	L·	Lipid hydroperoxide	LOOH
Lipid peroxyl radical	LOO·	Hypochlorous acid	HOCl
Peroxyl radical	ROO·	Peroxynitrite	ONOO−
Lipid alkoxyl radical	LO·	Dinitrogen trioxide	N2O3
Nitrogen dioxide radical	NO2·	Nitrous acid	HNO2
Nitric oxide radical	NO·	Nitryl chloride	NO2Cl
Nitrosyl cation	NO+	Nitroxyl anion	NO−
Thiyl radical	RS·	Peroxynitrous acid	ONOOH
Protein radical	P·	Nitrous oxide	N2O

). They include various oxygen-containing molecules that are chemically reactive owing to the presence of unpaired electrons. ROS are by-products of normal cellular metabolism; however, their levels can increase under stress and other conditions [3]. Although ROS play essential roles in cellular signaling and defense, their excessive accumulation can cause oxidative damage to cells, tissues, and DNA, leading to disease and aging. ROS, including superoxide (O₂∙⁻) and hydroxyl radicals (∙OH), contribute to Fe³⁺ reduction. Superoxide, often produced by photochemical reactions involving dissolved organic matter, enhances Fe³⁺ reduction rates and alters iron solubility [4].

Reactive oxygen species (ROS) include free radicals such as superoxide anion (O₂^•−) and hydroxyl radical (·OH) and non-radical molecules like hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂) [5]. ROS play a dual role in biological systems, acting both as signaling molecules for physiological processes and as agents of oxidative damage, leading to various diseases. ROS are generated from both endogenous and exogenous sources. Endogenous sources of ROS include the mitochondrial electron transport chain (ETC), enzymatic reactions, and peroxisomes. Of these, the primary source of ROS in aerobic organisms is the mitochondrial ETC, where incomplete reduction of oxygen generates O₂^•− [6]. Furthermore, enzymes, such as NADPH oxidase and xanthine oxidase, also contribute to ROS production [7]. Oxidative reactions in peroxisomes generate H₂O₂ [8]. Among exogenous sources of ROS, the most important are ultraviolet (UV) radiation, ionizing radiation, pollutants, toxins, drugs, and chemicals. UV and ionizing radiation cause water radiolysis and production of OH radicals [9]. Furthermore, pollutants and toxins, including heavy metals, cigarette smoke, and environmental pollutants, enhance ROS production [10]. Drugs, chemicals, and some chemotherapeutics induce ROS production via cytotoxic mechanisms [11]. Notably, ROS play important roles in various biological processes including cell signaling, homeostasis, immune defense, cell proliferation, and cell differentiation. Moderate ROS levels regulate kinase pathways, transcription factors, and immune responses [12]. Furthermore, phagocytic cells produce ROS to destroy pathogens during infection [13]. ROS also modulate growth signals and differentiation in various cell types [14].

Excessive ROS production or insufficient antioxidant defense leads to oxidative stress, which can cause lipid peroxidation, protein oxidation, and DNA damage. ROS react with membrane lipids, leading to loss of membrane integrity [15,16]. Furthermore, oxidative modification of proteins can impair their function [17]. ROS-induced DNA damage contributes to mutations and carcinogenesis [18]. Overall, ROS are important in both physiological and pathological processes. Although they contribute to essential signaling pathways, excessive ROS can cause oxidative stress and damage biomolecules, leading to various diseases, such as cancer, neurodegeneration, and cardiovascular disorders. Thus, maintaining a balance between ROS production and antioxidant defense is the key to cellular health [19,20].

A free radical has an unpaired electron, which exhibits a quantum-mechanical property known as spin. Free radicals are generally highly reactive because of their open-shell structure [21]. However, numerous free radicals remain stable under laboratory conditions, even in the presence of air and at room temperature [22]. Free radicals are often linked with oxidative stress [23], a relatively modern concept that has gained significant attention in medical research [24]. Oxidative stress arises when excessive ROS are produced within cellular mitochondria. Free radicals are known to be involved in various degenerative diseases, including cancer, acute inflammation, hypertension, diabetes, preeclampsia, acute kidney failure, atherosclerosis, Alzheimer’s disease, Parkinson’s disease, mutagenesis, aging, and cardiovascular disorders [25]. Multiple environmental factors, such as UV radiation and pollutants, contribute to oxidative stress and continuously impact human health [26]. Cellular oxygen metabolism leads to the formation of potentially harmful ROS. Under normal physiological conditions, oxidant production is counterbalanced by their elimination. However, oxidative stress occurs when the equilibrium between antioxidants and pro-oxidants is disrupted [27].

Recently, extensive scientific research has classified reactive species and free radicals into three primary categories: reactive nitrogen species (RNS), ROS, and reactive sulfur species (RSS). These groups comprise nitrogen-, oxygen-, and sulfur-containing molecules, respectively [28,29]. Among these, hydroxyl (HO·), superoxide anion (O₂·⁻), nitric oxide (NO·), alkoxyl (RO·), and peroxyl (ROO·) radicals are classified as free radicals. Furthermore, nitrogen monoxide (NO), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), ozone (O₃), nitrous acid (HNO₂), nitrous oxide (N₂O), lipid hydroperoxide (LOOH), and hypochlorous acid (HOCl) are classified as non-radical reactive oxygen species [30].

Reactive species are naturally present in living organisms and are of great importance to the immune system. Phagocytic cells, including monocytes, macrophages, and neutrophils, produce large amounts of O₂·⁻ or NO· to eliminate foreign pathogens as part of their cellular defense mechanisms [31]. Antioxidants mitigate oxidative reactions and reduce the harmful effects of reactive species, thus playing a crucial role in maintaining health [32]. Excessive accumulation of free radicals in the human body can cause severe damage to various tissues [33,34]. Lipid peroxidation in the plasma membrane is one of the most critical consequences that promotes the formation of RNS and ROS. Additionally, transition metals such as iron and copper catalyze the Fenton and Haber–Weiss reactions, leading to the generation of highly reactive OH· [35,36,37]. In the presence of metal ions and oxygen, H₂O₂ undergoes the Fenton reaction to produce OH radicals. Similarly, the Haber–Weiss reaction, first proposed by Fritz Haber, generates OH· from O₂·⁻ and H₂O₂ with an iron catalyst [38,39]. Subsequent studies have confirmed that both reactions serve as major sources of free radicals and are primarily responsible for cellular damage [40,41].

$${\mathrm{F}\mathrm{e}}^{2+}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{F}\mathrm{e}}^{3+}+{\mathrm{O}\mathrm{H}}^{-}+{\mathrm{O}\mathrm{H}}^{•}\left(\mathrm{F}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)$$

$${\mathrm{O}}_2^{•-}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}+{\mathrm{O}\mathrm{H}}^{•}(\mathrm{H}\mathrm{a}\mathrm{b}\mathrm{e}\mathrm{r}–\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{s}\mathrm{s}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n})$$

1.2. Oxidative Stress

Oxidative stress is characterized by an imbalance between ROS production and the ability of biological systems to detoxify reactive intermediates or repair the resulting damage. This phenomenon plays a dual role in cellular biology, acting both as a signaling mechanism for physiological functions and as a pathological factor contributing to various diseases, including cancer, aging, and neurodegenerative and cardiovascular diseases [42,43]. Oxidative stress is induced when ROS levels overwhelm the cellular antioxidant defenses. This imbalance causes cellular damage and disrupts normal cellular function. Oxidative stress is implicated in numerous diseases, including cancer, neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s diseases), cardiovascular diseases, and diabetes [44].

The term “oxidative stress” was first introduced by Helmut Sies in 1985, who defined it as a disturbance in the pro-oxidant–antioxidant balance that leads to cellular and tissue damage [45]. Under normal physiological conditions, cells maintain a delicate balance between ROS production and antioxidant defense. ROS, which include O₂·⁻, H₂O₂, ·OH, and ROO· radicals, are primarily generated as by-products of mitochondrial respiration and some enzymatic reactions [46,47]. Low ROS levels act as crucial signaling molecules in processes such as cell proliferation, differentiation, and immune responses, excessive ROS accumulation leads to oxidative stress, resulting in damage to lipids, proteins, and DNA [48,49].

Endogenous ROS sources include mitochondrial oxidative phosphorylation, peroxisomal metabolism, and enzymatic reactions involving NADPH oxidases, xanthine oxidase, and cytochrome P450 enzymes [50,51]. External factors such as UV radiation, pollution, smoking, heavy metals, and certain drugs also contribute to ROS production and oxidative stress [52,53]. The persistent oxidative burden can overwhelm cellular antioxidant defense mechanisms, leading to cellular dysfunction and the initiation of pathological conditions. Numerous studies have linked oxidative stress with the pathogenesis of various chronic diseases. Oxidative damage contributes to neuronal dysfunction and cell death in neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease [54,55]. In cardiovascular diseases, ROS mediates endothelial dysfunction, inflammation, and atherosclerosis [56]. Furthermore, oxidative stress plays a significant role in cancer progression by inducing DNA mutations and promoting tumor growth [57,58]. Thus, understanding oxidative stress mechanisms provides opportunities for therapeutic interventions aimed at enhancing antioxidant defenses and reducing disease risks. Overall, oxidative stress is a fundamental biological process with physiological and pathological implications. Although ROS perform essential cellular functions, their excessive accumulation leads to oxidative damage and contributes to numerous chronic diseases [59,60]. Future research should thus focus on the development of targeted antioxidant therapies and lifestyle modifications to mitigate oxidative stress and improve health outcomes.

1.3. The Importance of Iron in Biological Systems

Iron is an essential element in biological and geochemical processes, participating in numerous redox reactions that influence both microbial metabolism and environmental transformations [61,62]. In nature, iron predominantly exists in two oxidation states: ferric iron (Fe³⁺) and ferrous iron (Fe²⁺). The reduction of Fe³⁺ to Fe²⁺ is a crucial step in the biogeochemical cycling of iron, with significant implications in microbial respiration, metal detoxification, and industrial applications [63].

Fe³⁺ reduction is a key process in anaerobic environments, where it serves as a terminal electron acceptor for various iron-reducing bacteria, such as Geobacter and Shewanella species [64]. These microorganisms utilize Fe³⁺ ions in respiration, playing a vital role in carbon and nutrient cycling [65]. The electron transfer processes facilitating Fe³⁺ reduction is mediated by cytochromes, electron shuttles, and conductive pili, allowing efficient energy generation even in oxygen-limited conditions [66,67]. Beyond microbial systems, Fe³⁺ reduction is influenced by abiotic factors, including natural organic matter, pH variations, and the presence of reducing agents such as flavins and quinones [68]. Photochemical reactions, particularly in sunlit aquatic systems, also contribute to Fe³⁺ reduction, affecting the bioavailability of iron for phytoplankton growth [69]. These redox transformations are essential for maintaining iron homeostasis in both terrestrial and marine environments. From an industrial perspective, Fe³⁺ reduction has applications in bioremediation, wastewater treatment, and corrosion control [70,71]. The ability of Fe³⁺-reducing bacteria to immobilize heavy metals and degrade organic pollutants makes them valuable tools for environmental sustainability [72]. Additionally, Fe³⁺-reducing ability plays an important role in medical applications, particularly in iron supplementation therapies and drug formulations [73,74]. Thus, understanding Fe³⁺ reduction mechanisms will facilitate studies on antioxidant and an understanding of the relevant mechanisms. Despite its importance, Fe³⁺ reduction is a complex and multifaceted process influenced by various biological and physicochemical factors. Recent advances in molecular biology, spectroscopy, and electrochemical techniques have provided deeper insights into the mechanisms governing Fe³⁺-reducing activity [66,75]. However, many aspects of Fe³⁺ reduction, particularly its interactions with emerging contaminants and nanomaterials, remain poorly understood, warranting further investigation.

Thousands of review articles that evaluate antioxidants can be found in a general literature search. This review is the first to focus only on reduction power. This review aims to provide a comprehensive overview of Fe³⁺-reducing ability, detailing its mechanisms, influencing factors, and applications in diverse fields. By synthesizing the current knowledge and identifying future research directions, this study seeks to enhance our understanding of iron redox chemistry and its broader implications.

2. Antioxidants

Antioxidants can be categorized in various ways depending on their environment and functional roles [1,76]. An antioxidant is defined as a compound that can significantly slow down or completely inhibit the oxidation of substrates, even when present in low concentrations [77]. These molecules neutralize free radicals by donating electrons, thereby reducing oxidative damage to biological systems [78,79,80]. In addition, they prevent free radical formation and interrupt oxidative processes at any of the three key stages: initiation, propagation, and termination [81,82].

Several factors influence antioxidant efficacy. The key parameters include the physical state of the system, temperature, molecular structure, characteristics of the oxidation-sensitive substrate, concentration, synergistic interactions, and the presence of prooxidant molecules [83]. The chemical composition of an antioxidant determines its reactivity and efficiency in neutralizing free radicals and ROS, its reducing potential, singlet oxygen quenching, and hydrogen peroxide decomposition [84]. Moreover, its effectiveness depends on its concentration and localization in the system, such as its distribution at the phase interfaces. The kinetics of the reaction also play a crucial role in the long- and short-term protective functions of antioxidants. These include the thermodynamic feasibility of reactions with oxidants, reaction rates, and overall antioxidant capacity [85,86].

By regulating the balance of oxidants, antioxidant compounds contribute to metabolic homeostasis. Additionally, antioxidants slow down lipid peroxidation in stored and processed foods, prevent spoilage, preserve pharmaceuticals, and extend the shelf life of products. Both synthetic and natural antioxidants are commonly used in food and pharmaceutical industries. The pharmaceutical industry has primarily focused on the use of synthetic antioxidants to decrease intracellular ROS and RNS levels. Synthetic antioxidants are widely utilized because of their high purity, low cost, and strong activity even at low concentrations. However, some studies have reported potential adverse effects associated with their use [87].

Consequently, natural antioxidants are increasingly favored over synthetic antioxidants, leading to the development of a growing number of methods for assessing antioxidant effectiveness. Among these, Fe³⁺ reduction ability is one of the most widely employed techniques. These assay results are generally compared to commonly used synthetic antioxidants which include butylated hydroxytoluene (BHT), propyl gallate (PG), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), which are frequently added to food and pharmaceutical products to prevent oxidative degradation [88]. Despite their known adverse effects, manufacturers continue to use synthetic antioxidants, leaving consumers with limited options. The chemical structures of these synthetic antioxidants are shown in Figure 1. Notably, recent studies have raised concerns regarding the safety of these synthetic additives because they have been found to inhibit various enzymes, leading to potential health risks [85,86]. Owing to these toxicological concerns, researchers are actively searching for alternative natural antioxidant compounds that offer similar protective benefits with fewer side effects. In this regard, there has been a significant shift toward replacing synthetic antioxidants with natural alternatives that are characterized by lower toxicity, higher biodegradability, and safer mechanisms of action [1,87].

Prolonged use of synthetic antioxidants has been linked with several health problems, including carcinogenesis, skin allergies, fatty liver, and gastrointestinal discomfort. Consequently, informed consumers are increasingly wary of these negative effects and tend to opt for natural antioxidants. Fruits, vegetables, herbs, and spices are the most readily available and abundant sources of antioxidants. Plants such as tea, linden, cinnamon, cloves, fennel, anise, and rosemary are widely used because of their high concentrations of tannin, catechin, theine, phenolic, and flavonoid compounds, all of which exhibit strong antioxidant properties [88]. Regular consumption of phenolic-rich herbal products with antioxidant effects lowers disease risk and helps prevent the onset of degenerative conditions. However, the antioxidant potency and overall quality of natural antioxidants and their extracts are influenced not only by the plant source but also by the methods used for their extraction and isolation [89,90].

3. Antioxidant Evaluation Methods

Recent studies have extensively examined the oxidation of free radicals and the general mechanisms by which antioxidants counteract them. This interest stems from the fact that despite being neutral, free radicals exert a significant impact on biological systems. Certain lipid-derived compounds, such as aldehydes, which naturally occur during food processing, may pose health risks. These harmful compounds are easily produced when food is subjected to heat treatment [85]. Various antioxidant assays have been developed to directly measure hydrogen atom transfer or electron donation from antioxidants to free radicals and ROS [91]. Over time, significant advances have been made in techniques for measuring antioxidant capacity. Earlier methods primarily assessed antioxidant efficacy based on the formation of specific oxidation products, such as in lipid peroxidation. More recent approaches employ highly automated and sensitive detection technologies, enabling the precise evaluation of antioxidant activity through multiple mechanisms, including ROS scavenging, reducing power, and metal chelation. Initially introduced as a purely chemical concept, the idea of antioxidant capacity has since been adapted to various disciplines, including medicine, biology, food science, and epidemiology [92,93].

Understanding the antioxidant profiles of food and pharmaceutical products is important for maintaining their commercial and nutritional value during processing and storage. Therefore, a rapid and straightforward method to determine antioxidant capacity is essential. Numerous techniques for antioxidant evaluation have been developed and implemented [94,95]. Among the most commonly used methods are the inhibition of linoleic acid emulsion autoxidation, β-carotene bleaching test, total radical-trapping antioxidant parameter (TRAP), and oxygen radical absorbance capacity (ORAC) assay [1]. Other widely applied approaches include ferric (Fe³⁺) and cupric (Cu²⁺) ion reduction assays [96], as well as scavenging experiments targeting the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH∙), N,N-dimethyl-p-phenylenediamine radicals (DMPD·⁺), and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals (ABTS∙⁺) and O₂∙⁻, along with metal chelation tests. Most of these methods rely on similar principles and spectrophotometric detection to evaluate antioxidant potential. However, assessing antioxidant capacity using a single method is insufficient. At least three different in vitro assays should be performed concurrently to ensure an accurate evaluation, because no single test can fully reflect antioxidant activity. Considering the complexity of these methods, a direct comparison between them is often challenging. Therefore, selecting and applying the most appropriate techniques for research purposes is essential [1,26]. A key objective of this review is to explore the chemistry, mechanisms, and applications of Fe³⁺-reducing ability, following an overview of the antioxidant evaluation methods commonly used in research. Additionally, the antioxidant capacities of indirect methods may differ when used in foods because of the complexity of matrices [97]. In recent years, despite growing interest in various antioxidant assays, many studies have predominantly focused on Fe³⁺ reducing ability to meet the needs of researchers. Furthermore, among antioxidant methods, reducing power has great importance as it is the most used and low-cost antioxidant evaluation method.

4. Chemical Reduction Pathways

The chemical reduction of Fe³⁺ occurs through various mechanisms, primarily influenced by environmental conditions, electron donors, and catalytic agents. The conversion of Fe³⁺ to Fe²⁺ is facilitated by reducing agents such as organic acids, thiols, flavins, and phenolic compounds [68]. These molecules donate electrons to Fe³⁺, leading to its reduction and subsequent participation in biogeochemical cycles. One of the most well-studied pathways for Fe³⁺ reduction involves ascorbic acid, which acts as a potent reductant by directly transferring electrons to Fe³⁺ [98]. Additionally, flavins and hydroquinones, which possess biological activity are known to facilitate Fe³⁺ reduction, particularly in aqueous systems where their redox potential plays a critical role. The influence of pH on Fe³⁺ reduction is another key aspect. At lower pH levels, Fe³⁺ remains in a soluble form, allowing more efficient electron transfer [99]. However, in alkaline conditions, Fe³⁺ ions tend to precipitate as insoluble oxides, limiting their bioavailability and reduction potential [100]. Furthermore, iron-complexing ligands such as EDTA and citrate significantly enhance Fe³⁺ reduction by stabilizing Fe³⁺ in solution and preventing precipitation [101]. This effect is particularly relevant in environmental and industrial processes where Fe³⁺ solubility dictates reaction efficiency. In summary, Fe³⁺ reduction involves a complex interplay of electron donors, pH effects, and ligand interactions. Advances in spectroscopic and electrochemical techniques have provided deeper insight into these processes, with significant implications for environmental and industrial applications.

5. Biological Sources of Reducing Power

Reducing power influences several key biological processes, including energy production, biosynthesis, oxidative stress management, and immune functions. NADH donates electrons to the mitochondrial electron transport chain and drives ATP synthesis via oxidative phosphorylation [102]. NADPH provides reducing equivalents for anabolic pathways such as fatty acid and cholesterol biosynthesis [103]. NADPH is also crucial for the regeneration of antioxidant systems, including glutathione and thioredoxin, which protect cells from oxidative damage [104]. NADPH oxidase plays a role in generating ROS used by immune cells to kill pathogens [105]. Therefore, the reducing power of biological systems is indispensable for maintaining cellular function and homeostasis. NADH, NADPH, and glutathione are central to metabolism, biosynthesis, and oxidative stress defense. Understanding the mechanisms and roles of reducing power can provide insights into metabolic disorders and potential therapeutic targets for oxidative stress-related diseases [106]. The primary sources of reducing power in biological systems are glutathione, ascorbic acid, NADH, and NADPH, which act as electron carriers in numerous metabolic processes.

5.1. NADH

Reduced nicotinamide adenine dinucleotide (NADH) is a coenzyme that plays a significant role in cellular metabolism and energy production. It represents the reduced form of oxidized NAD⁺ and exerts vital functions primarily in redox reactions, transferring electrons from one molecule to another. This process is essential for ATP synthesis, the main cellular energy carrier [107]. NADH is generated during glycolysis, the tricarboxylic acid cycle, and β-oxidation of fatty acids. It transfers electrons to the mitochondrial electron transport chain and facilitates ATP production via oxidative phosphorylation [108]. In addition to energy metabolism, NADH is involved in several cellular functions, including DNA repair, gene expression regulation, and immune system modulation. It has been studied for its potential anti-aging effects as it contributes to the activity of sirtuins, a class of enzymes associated with longevity [109]. In the clinical and nutritional contexts, NADH supplementation is occasionally used to improve cognitive function, reduce fatigue, and enhance athletic performance. However, further studies are required to confirm these benefits [110].

5.2. NADPH

NADPH is primarily generated through the pentose phosphate pathway and the activity of malic enzyme. It is the reduced form of nicotinamide adenine dinucleotide phosphate (NADP⁺) and serves as an electron donor in several biosynthetic processes. NADPH plays a key role in supporting biosynthetic reactions like the synthesis of fatty acids and nucleotides, and it helps maintain redox balance by replenishing glutathione [111]. One of the primary functions of NADPH is its involvement in anabolic reactions such as fatty acid and cholesterol synthesis. These processes require a high-energy reducing agent, which NADPH provides by donating electrons. NADPH is also essential for the regeneration of glutathione, a key antioxidant that protects cells from oxidative stress by neutralizing ROS [103]. The PPP is the major cellular source of NADPH. In this pathway, glucose-6-phosphate is converted to ribulose-5-phosphate, generating NADPH as a byproduct. Other sources include malic enzyme activity and isocitrate dehydrogenase reactions in mitochondria [112]. NADPH is crucial for the function of NADPH oxidases, which mediate immune defense by generating ROS to combat pathogens [113]. Additionally, NADPH supports the activity of cytochrome P450 enzymes in drug metabolism and detoxification [114].

5.3. Glutathione (GSH)

GSH is a tripeptide involved in the neutralization of ROS and detoxification of xenobiotics. Studies have shown that glutathione reductase and NADPH maintain cellular redox balance [115]. GSH is a vital tripeptide composed of glutamate, cysteine, and glycine. It serves as a major antioxidant, detoxifying ROS and maintaining cellular redox homeostasis. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). The GSH/GSSG ratio is a key indicator of oxidative stress in cells [116]. One of the most important functions of glutathione is detoxification. It conjugates with toxic compounds via glutathione S-transferase (GST) enzymes, facilitating their excretion [117,118]. Additionally, GSH plays a critical role in immune function by modulating cytokine production and lymphocyte activity. GSH is synthesized in a two-step enzymatic process. First, glutamate–cysteine ligase catalyzes the formation of γ-glutamylcysteine; glutathione synthetase then adds glycine to complete this process [1]. The primary sites of GSH synthesis are the liver and kidneys, where it supports detoxification. Glutathione is also vital for mitochondrial function, protection against oxidative damage, and maintenance of electron transport chain efficiency. Deficiencies in GSH are associated with various diseases, including neurodegenerative disorders, cancer, and cardiovascular diseases [119].

5.4. Ascorbic Acid (Vitamin C)

Ascorbic acid is a water-soluble vitamin with effective antioxidant ability for various physiological functions in humans. It plays critical roles in collagen synthesis, immune function, and ROS neutralization [120]. One of the primary roles of ascorbic acid is acting as a cofactor for prolyl and lysyl hydroxylases, which are necessary for collagen hydroxylation. This function is crucial for wound healing, connective tissue formation, and overall skin health. Ascorbic acid deficiency leads to scurvy, a disease characterized by weakened connective tissue, bleeding gums, and impaired wound healing [121].

As an antioxidant, ascorbic acid scavenges free radicals and ROS, and regenerates other antioxidants such as vitamin E, thereby maintaining cellular redox balance. It also enhances the absorption of non-heme iron from plant-based foods, reducing the risk of iron-deficiency anemia [1]. Ascorbic acid is involved in immune function by stimulating the production and activity of white blood cells and enhancing the skin defense system. Regular ascorbic acid intake has been shown to reduce the duration and severity of colds. Humans cannot synthesize ascorbic acid and must obtain it from dietary sources such as citrus fruits, berries, and leafy greens. Excessive intake may lead to gastrointestinal discomfort but is generally non-toxic [122].

6. Biochemical Mechanisms of Fe³⁺ Reduction

6.1. Non-Enzymatic Reduction

Non-enzymatic reduction of Fe³⁺ occurs through electron transfer from various organic and inorganic reducing agents, including natural organic matter, sulfides, and hydroquinones. These processes play crucial roles in geochemical iron cycling, particularly in environments where microbial activity is limited or absent. Abiotic reduction of Fe³⁺ has significant implications for iron mobility, mineral transformation, and redox-driven biogeochemical reactions [123].

6.1.1. Organic Reducing Agents

Organic compounds such as hydroquinones, ascorbic acid, and catechol are known to facilitate Fe³⁺ reduction. Hydroquinones, for instance, act as electron donors in both natural and synthetic systems, significantly enhancing Fe³⁺ reduction rates. These compounds are often found in humic substances, which contribute to iron redox cycling in sediments and soils [124]. Ascorbic acid, also known as vitamin C, is another potent Fe³⁺-reducing agent. It donates electrons to Fe³⁺, forming Fe²⁺ and dehydroascorbic acid. This reaction is particularly relevant in biological systems, where ascorbic acid enhances iron bioavailability by converting Fe³⁺ from dietary sources into its more soluble Fe²⁺ form [125]. Furthermore, organic substrates such as acetate, lactate, and hydrogen fuel microbial respiration and drive Fe³⁺ reduction [126]. High concentrations of sulfate, nitrate, or oxygen can suppress Fe³⁺ reduction owing to the preferential use of alternative electron acceptors [127].

6.1.2. Inorganic Reducing Agents

Several inorganic compounds also contribute to Fe³⁺ reduction. Sulfides (e.g., hydrogen sulfide, H₂S) react with Fe³⁺ to form Fe²⁺, often leading to the precipitation of iron sulfide minerals such as pyrite (FeS₂). This reaction plays a key role in anoxic environments, particularly in marine and freshwater sediments where sulfate-reducing bacteria produce-H₂S as a metabolic byproduct [128]. Furthermore, green rust-mixed valence iron minerals can mediate Fe³⁺ reduction through electron transfer processes. These minerals are formed under reducing conditions and act as both electron donors and acceptors, thereby influencing iron and contaminant mobility in subsurface environments [129].

6.1.3. Photochemical Reduction

Light-driven Fe³⁺ reduction is another significant abiotic process, particularly in surface waters. Fe³⁺ complexes with organic ligands, such as citrate and oxalate, undergo photochemical reduction when exposed to sunlight. This mechanism is especially significant in iron-limited aquatic ecosystems, where photoreduction of Fe³⁺ to Fe²⁺ enhances bioavailable iron concentrations [4]. Overall, non-enzymatic Fe³⁺ reduction is a vital pathway in iron biogeochemistry and interacts with biological and geochemical processes to influence iron availability, mineral stability, and environmental redox conditions. Photochemical reduction of Fe³⁺ is an essential natural process that contributes to the bioavailability of iron, particularly in aquatic systems. Sunlight-driven reduction of Fe³⁺ influences iron cycling, impacting both primary productivity and metal mobility in environmental systems [130]. This process occurs via direct photolysis, ligand-mediated reduction, and interactions with ROS [131]. Furthermore, in the absence of organic ligands, Fe³⁺ can be reduced through direct absorption of solar radiation, particularly in the UV spectrum. This reaction results in the formation of Fe²⁺ and oxygen radicals, significantly affecting iron speciation in surface waters [132].

6.2. Enzymatic Reduction

The enzymes involved in Fe³⁺ reduction include cytochromes c, hydrogenases, and dehydrogenases. These proteins, including MtrA in Shewanella, are crucial in transferring electrons from the inner mitochondrial membrane to extracellular Fe³⁺ acceptors [133]. Enzymes also generate reducing equivalents to fuel electron transfer pathways [134].

6.3. Microbial Mediators

Microbial Fe³⁺ reduction is a biologically mediated process that plays a crucial role in iron cycling and anaerobic respiration. Certain microorganisms, particularly dissimilatory iron-reducing bacteria, have evolved mechanisms to use Fe³⁺ as a terminal electron acceptor. This process is fundamental in sedimentary environments, wastewater treatment, and biogeochemical cycling [135]. Several environmental parameters affect microbial Fe³⁺ reduction, including pH, redox conditions, electron donor availability, and presence of competing electron acceptors. Optimal Fe³⁺ reduction occurs in neutral to slightly acidic conditions, as extreme pH values can inhibit bacterial activity [136]. Microbial reduction of Fe³⁺ has importance in biotechnological and environmental applications, such as bioremediation, microbial fuel cells, and climate impact.

7. Procedure for Determining Reducing Power in Antioxidants

The reductive potential of antioxidants was first evaluated by Oyaizu [137]. For this aim, various concentrations of antioxidants were prepared in 1 mL of distilled water, followed by mixing with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide ([K₃Fe(CN)₆]) (1%). The resulting mixture was incubated at 50 °C for 20 min. Subsequently, 2.5 mL of 10% trichloroacetic acid (TCA) was added and the solution was centrifuged at 1000× g for 10 min. After centrifugation, the upper layer (2.5 mL) was collected and mixed with distilled water and 0.5 mL of 0.1% FeCl₃. The absorbance of the final solution was measured at 700 nm using a spectrophotometer. A higher absorbance value indicated a greater reductive potential of the tested materials [138]. The most suitable solution for evaluating reducing power is distilled and deionized water.

8. Reducing Power of Various Antioxidants

The reducing antioxidant power assay is based on the principle that an increase in absorbance corresponds to an increase in antioxidant activity. Reduction refers to the gain of electrons, whereas oxidation involves their loss [139]. A reducing agent (or reductant) donates electrons, facilitating the reduction of another substance, whereas an oxidizing agent (or oxidant) accepts electrons, leading to the oxidation of another reactant. As oxidation and reduction are interdependent, they occur simultaneously within a system. Chemical reactions involving both processes are classified as redox reactions. Redox reactions play a fundamental role in biological oxidation, a series of chemical reactions in which oxygen from air oxidizes molecules derived from food metabolism, ultimately generating energy for living organisms. While reductants and oxidants are chemical terms, antioxidants and pro-oxidants are used in the context of biological systems. Furthermore, an antioxidant capable of effectively reducing pro-oxidants may not necessarily exhibit the same efficiency in reducing Fe³⁺. In the Fe³⁺-reducing assay, the presence of reductants mediates the conversion of Fe³⁺ to Fe²⁺. The reducing potential of a bioactive compound is assessed by monitoring the direct reduction of [Fe(CN)₆]³⁻ to [Fe(CN)₆]⁴⁻. When free Fe³⁺ is introduced to the reduced product, it forms an intense blue-colored complex known as Perl’s Prussian blue, Fe₄[Fe(CN)₆]₃, which exhibits strong absorbance at 700 nm. The Fe³⁺-reducing assay thus involves an electron transfer mechanism in which a ferric salt functions as the oxidant [1].

$$\begin{gathered} \mathrm{R}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{e}\mathrm{d}{\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}+\mathrm{F}\mathrm{e}}^{3+}\to {\mathrm{O}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}+\mathrm{F}\mathrm{e}}^{2+} \\ {\mathrm{F}\mathrm{e}}^{2+}+{\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{3-}\to {\mathrm{F}\mathrm{e}\left[{\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6\right]}^{-} \\ \mathrm{o}\mathrm{r} \\ \mathrm{R}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}+{\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{3-}\to \mathrm{O}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}+{\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{4-} \\ {\mathrm{F}\mathrm{e}}^{3+}+{\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{4-}\to {\mathrm{F}\mathrm{e}\left[{\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6\right]}^{-} \end{gathered}$$

An antioxidant is a reductant, but a reductant is not necessarily an antioxidant. As reported in many studies, the activity of natural antioxidants in diseases is closely related to their ability to reduce DNA damage, mutagenesis, and carcinogenesis, and to inhibit pathogenic bacterial growth [1]. The Fe³⁺-reducing ability assay is a spectrophotometric method widely used for assessing the antioxidant capacity of beverages, pure compounds, food products, and herbal extracts. Because of its simplicity, sensitivity, speed, and reproducibility, this technique is one of the most convenient and commonly applied methods for evaluating the radical scavenging potential of various compounds and plant extracts. The absorbance values of herbal extracts and pure substances from recent studies on Fe³⁺-reducing ability are presented in

Table 2. The absorbance values of reducing ability of some pure antioxidant molecules.

Antioxidants	Absorbance (nm)	Concentration (µg/mL)	References
L-Dopa	2.443	20	[142]
L-Tyrosine	0.388	20	[142]
Resveratrol	2.156	30	[143]
Coumestrol	0.739	30	[144]
Magnofluorine	0.966	30	[145]
Taxifoline	2.847	30	[146]
Cynarine	3.613	30	[147]
Pelargonin	2.064	30	[148]
Silychristin	1.181	30	[148]
Callistephin	2.328	30	[148]
Oenin	2.351	30	[148]
Malvin	2.189	30	[148]
Baicalin	1.249	30	[149]
Isofraxidin	0.547	30	[150]
Spiraeoside	1.012	30	[151]
Acteoside	1.387	30	[152]
Isoacteoside	1.682	30	[152]
Echinacoside	0.820	30	[152]
Arenarioside	0.544	30	[152]
Hamamelitannin	1.030	30	[153]
Caffeic acid	2.769	20	[154]
Tannic acid	2.737	45	[155]

and

Table 3. The absorbance values of similar concentrations of water and ethanol extracts of some antioxidant-containing plants.

Antioxidant Plants	Absorbance (nm)		Concentration (µg/mL)	References
	Water Extract	Ethanol Extract
Sage (Salvia pilifera)	1.636	1.762	30	[156]
Thyme (Thymus vulgaris)	2.020	1.889	30	[157]
Cherry stem (Cerasus avium)	0.926	1.517	30	[158]
Galanga (Alpinia officinarum)	1.332	1.976	30	[159]
Ginger (Zingiber offcinale)	0.332	1.122	30	[160]
Cinnamon (Cinnamomum verum)	0.719	0.886	30	[161]
Pennyroyal (Mentha pulegium)	1.433	1.362	30	[162]
Avocado (Folium perseae)	0.401	0.606	30	[163]
Kınkor (Ferulago stellata)	0.456	0.643	30	[164]
Pomegranate (Punica granatum)	1.278	1.219	30	[165]

. Notably, some natural antioxidants, such as essential oils, have an efficient antioxidant capacity under high-temperature conditions, even in extremely demanding thermal conditions such as deep-frying processes [140,141].

9. Advantages of the Fe³⁺ Reducing Ability Assay

The advantages of the Fe³⁺-reducing ability assay can be determined in terms of simplicity, speed, reproducibility, and no requirement for free radicals. The assay procedure is straightforward, requiring minimal sample preparation and providing results within minutes. Owing to its well-defined reaction conditions, this method offers high reproducibility and reliability. Unlike other antioxidant assays, such as DPPH or ABTS, the Fe³⁺-reducing ability assay does not involve free radicals, making it a more stable and less variable method [1].

10. Limitations of the Fe³⁺ Reducing Ability Assay

The Fe³⁺ reducing ability assay has some limitations in terms of selectivity for reducing agents, pH dependency, and the need for water-soluble antioxidants. The Fe³⁺ reducing ability assay is primarily used to analyze compounds that act via single-electron transfer mechanisms. It does not account for hydrogen-atom transfer-based antioxidants, such as thiols and some polyphenols. Furthermore, this assay requires an acidic environment, which may not reflect the physiological conditions under which the antioxidant activity occurs. In addition, the Fe³⁺ reducing ability assay primarily detects hydrophilic antioxidants, making it less effective for lipophilic compounds [1].

According to our review, reducing ability tends to yield stronger responses, particularly with phenolic compounds. For polar and phenolic substances, incorporating water into the reaction medium, such as aqueous methanol, enhance the accuracy of the results. Conversely, for low-polarity compounds, ethyl acetate is more suitable for interactions with radicals. These findings indicate that reducing ability is influenced more by the steric accessibility of the radical site than by the inherent chemical properties of the tested antioxidant compounds.

The reaction rate of reducing ability with antioxidants is determined by the varying contributions of single-electron or hydrogen atom transfer mechanisms. Additionally, environmental and experimental conditions can significantly affect the reaction mechanisms and response outcomes. The interaction of antioxidants with reducing agents is known to be lower than the total activity of the individual antioxidant compounds, suggesting that radical scavenging by extracts from mixtures may be suppressed in reducing ability assays.

11. Conclusions

Antioxidant compounds play a crucial role in minimizing the oxidative damage caused by ROS. The reducing ability of these compounds is thus important for determining their antioxidant profiles. Among the various antioxidant evaluation methods, the Fe³⁺-reducing ability assay is one of the most commonly utilized techniques in both food and pharmaceutical applications. Fe³⁺-reducing power can have significant health and industrial implications, affecting disease risk, food quality, and product stability. By understanding the mechanisms underlying this reduction and adopting strategies for its optimization, we can enhance health outcomes and improve the longevity of antioxidant-rich products. Further research is required to develop advanced techniques for preserving and restoring antioxidant power in various applications.