key: cord-0771499-2xkdkr39 authors: Flieger, Jolanta; Flieger, Wojciech; Baj, Jacek; Maciejewski, Ryszard title: Antioxidants: Classification, Natural Sources, Activity/Capacity Measurements, and Usefulness for the Synthesis of Nanoparticles date: 2021-07-25 journal: Materials (Basel) DOI: 10.3390/ma14154135 sha: 60c110ace7a813df493697afd7450bd6077b3e11 doc_id: 771499 cord_uid: 2xkdkr39 Natural extracts are the source of many antioxidant substances. They have proven useful not only as supplements preventing diseases caused by oxidative stress and food additives preventing oxidation but also as system components for the production of metallic nanoparticles by the so-called green synthesis. This is important given the drastically increased demand for nanomaterials in biomedical fields. The source of ecological technology for producing nanoparticles can be plants or microorganisms (yeast, algae, cyanobacteria, fungi, and bacteria). This review presents recently published research on the green synthesis of nanoparticles. The conditions of biosynthesis and possible mechanisms of nanoparticle formation with the participation of bacteria are presented. The potential of natural extracts for biogenic synthesis depends on the content of reducing substances. The assessment of the antioxidant activity of extracts as multicomponent mixtures is still a challenge for analytical chemistry. There is still no universal test for measuring total antioxidant capacity (TAC). There are many in vitro chemical tests that quantify the antioxidant scavenging activity of free radicals and their ability to chelate metals and that reduce free radical damage. This paper presents the classification of antioxidants and non-enzymatic methods of testing antioxidant capacity in vitro, with particular emphasis on methods based on nanoparticles. Examples of recent studies on the antioxidant activity of natural extracts obtained from different species such as plants, fungi, bacteria, algae, lichens, actinomycetes were collected, giving evaluation methods, reference antioxidants, and details on the preparation of extracts. Recently, much research has been devoted to free radical chemistry. There are undeniable pieces of evidence that free radicals are responsible for the oxidative damage of biomolecules such as proteins, lipids, or nucleic acids in the structures of cell nuclei and molecular membranes. Maintaining the balance between free radicals and antioxidants is a prerequisite for staying healthy. Thus, the control of oxidative stress processes may turn out to be fundamental in both the prevention and treatment of many diseases, such as diabetes, atherosclerosis, coronary artery disease, cancer, inflammation, liver diseases, cardiovascular diseases, cataracts, nephrotoxicity, and neurodegenerative processes accompanying aging. In order to maintain redox homeostasis, excess free radicals are neutralized by enzymes and non-enzymatic antioxidants, which, with the exception of a few produced by the human body, e.g., glutathione, uric acid, and uricinol, must be supplied with the diet. Since synthetic antioxidants butylated hydroanisole (BHA), butylated hydrotoluene (BHT), n-propyl gallate (PG) pose a potential health risk due to contamination with chemical precursors, toxic solvents, and the formation of hazardous by-products, natural antioxidants are an attractive alternative. For this reason, there is an extensive search for effective, non-toxic, and natural antioxidants. According to PubMed, in the last 5 years, over three thousand review articles that prove the effectiveness of natural antioxidants in preventing Antioxidants act by delaying or preventing the oxidation of other chemicals. The first studies on the role of antioxidants in biology focused on their use in preventing unsaturated fats from going rancid [67] [68] [69] . However, the milestone that led to the understanding of the role of antioxidants for living organisms was the identification of vitamins A, C, and E [70] and the understanding of the mechanism of lipid peroxidation prevention by vitamin E [71] . The classification of antioxidants, along with the most representative examples, is shown in the diagram (Figure 1 ). Antioxidants are usually classified into enzymatic and non-enzymatic. Among them, there are various compounds with different modes and places of action and different final effects. This diversity determines the individual role of each of them in the body. It should be emphasized that the network of interacting antioxidant enzymes, such as superoxide dismutase enzymes (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRd), shows the highest antioxidant defense effectiveness [72] . Low-molecular-weight antioxidants, including vitamin C, E, coenzyme Q, carotenes, glutathione, and trace elements, are also responsible for inactivating reactive radicals. Some of them, including glutathione, ubiquinone, albumin and metallothioneins, and uric acid, are produced in the body [73] , but most are exogenous compounds derived from natural sources such as plants (flavonoids, phenolic acids, carotenoids, stilbenes, coumarins, lignans, organosulfur compounds, vitamins) or minerals (selenium, zinc, manganese) provided with the diet. When endogenous antioxidants involved in free radical defenses cannot protect the body against ROS, there is a need for exogenous antioxidants. Almost all living organisms, both prokaryotes and eukaryotes, are capable of producing bioactive compounds. Many of the naturally occurring antioxidants are now isolated, fully characterized, and available for various applications as prophylactic and therapeutic agents to inhibit the adverse effects generated by ROS [74, 75] . A good diet that includes fruit, tea, wine, vegetables, and grains is a rich source of antioxidants. Some drugs, apart from their therapeutic effect, also have antioxidant effects, e.g., captopril belonging to angiotensin-converting enzyme (ACE) inhibitors, Nacetylcysteine [76] , or dihydropyridine calcium antagonists [77] . However, the concentrations used in the therapy do not provide antioxidant activity in vivo. The source of antioxidants and other bioactive compounds are also microorganisms, including actinomycetes, bacteria [78] , cyanobacteria, fungi, and lichens [79] . Compared Low-molecular-weight antioxidants, including vitamin C, E, coenzyme Q, carotenes, glutathione, and trace elements, are also responsible for inactivating reactive radicals. Some of them, including glutathione, ubiquinone, albumin and metallothioneins, and uric acid, are produced in the body [73] , but most are exogenous compounds derived from natural sources such as plants (flavonoids, phenolic acids, carotenoids, stilbenes, coumarins, lignans, organosulfur compounds, vitamins) or minerals (selenium, zinc, manganese) provided with the diet. When endogenous antioxidants involved in free radical defenses cannot protect the body against ROS, there is a need for exogenous antioxidants. Almost all living organisms, both prokaryotes and eukaryotes, are capable of producing bioactive compounds. Many of the naturally occurring antioxidants are now isolated, fully characterized, and available for various applications as prophylactic and therapeutic agents to inhibit the adverse effects generated by ROS [74, 75] . A good diet that includes fruit, tea, wine, vegetables, and grains is a rich source of antioxidants. Some drugs, apart from their therapeutic effect, also have antioxidant effects, e.g., captopril belonging to angiotensin-converting enzyme (ACE) inhibitors, Nacetylcysteine [76] , or dihydropyridine calcium antagonists [77] . However, the concentrations used in the therapy do not provide antioxidant activity in vivo. The source of antioxidants and other bioactive compounds are also microorganisms, including actinomycetes, bacteria [78] , cyanobacteria, fungi, and lichens [79] . Compared to plants, these organisms can grow very quickly under strictly controlled conditions, which makes them a favorable source of natural bioactive molecules for industrial food, pharmaceuticals, nutraceuticals, and agricultural applications. Antioxidants can also be delivered to the body in the form of dietary supplements. The synthetic forms of antioxidants are bioequivalent to their natural forms, e.g., biovitamin C vs. chemically synthesized L-ascorbic acid, or synthetic and natural R, R, R-α-tocopherol. Antioxidants are also used as additives to prevent the oxidation of unstable ingredients in the food, cosmetic, and pharmaceutical industries. This mainly concerns synthetic antioxidants with a phenolic structure, such as butylated hydroanisole (BHA), butylated hydrotoluene (BHT), and tert-butylated hydroquinone (TBHQ), which are added to foodstuffs to prevent lipid rancidity [80] . Antioxidants differ in their ability to scavenge free radicals. It has been shown that antioxidant activity can be significantly correlated with the number of active groups such as OH or NH 2 and the position of these functional groups in the order ortho > para > meta, from the highest to the lowest active [81] . It should be remembered that antioxidants can act through various mechanisms, not only scavenging radicals, but also sequestering transition metal ions, decomposing hydrogen peroxide or hydroperoxides, quenching active pro-oxidants, and enhancing endogenous antioxidant defense but also by repairing the resulting cellular damage. Therefore, antioxidants are sometimes classified as primary or chain-breaking antioxidants and as secondary or preventive antioxidants [82] . Primary antioxidants actively inhibit oxidation reactions by scavenging ROS/RNS, while secondary antioxidants act indirectly through chelation of transition metal (iron) ions [83, 84] and other specific actions such as anti-inflammatory, induction of protective factors, inhibition of NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase), inhibition of xanthine oxidase, and regulation of redox-sensitive signal transduction pathways, including transcription factors and inhibition of poly (ADP-ribose) −1 (PARP-1) polymerase [81, 85, 86] . Another indirect way of antioxidant activity is the activation of transcription factors, including Nrf2, which in turn leads to the activation of endogenous antioxidant enzymes [87] . Currently, the role of exogenous antioxidants in preventing or delaying oxidative damage is becoming more and more controversial. The initial enthusiasm for their positive health effects was mainly based on in vitro experiments. In the initial studies, the in vivo bioavailability of the antioxidants, which is generally quite low, was neglected. In this context, the activity of scavenging free radicals by antioxidant metabolites seems to be more reliable [50, 88] . The high in vitro chemical reactivity of the antioxidant is therefore not evidence of its effectiveness in vivo. Moreover, as shown by individual studies [89, 90] , supplementation with antioxidants may be ineffective and even very dangerous. An example may be the disappointing research on the effectiveness of vitamin E in the risk of cardiovascular disease or hemorrhagic stroke [91] [92] [93] [94] [95] . Reports that the use of antioxidants not only prevent cancer but may also provoke it are also alarming [96] . As it turns out, it is especially dangerous to supplement with antioxidants in doses exceeding the daily intake. For example, supplementation with β-carotene over ten times the daily intake increased the incidence of lung cancer in smoking men by 18% [97] . Vitamin C supplementation is particularly controversial. Linus Pauling recommended health-promoting use of a high daily dose of 1000 mg [98] . Unfortunately, it turned out that even at low concentrations of ascorbic acid, a pro-oxidative effect can occur in the presence of transition metals, e.g., iron. An example of this effect is the effect of ascorbic acid on iron-induced lipid peroxidation [99] . In the review by Hrelia and Angeloni [100] , recent reports on new mechanisms of action of natural antioxidants are collected. Their study highlights the fact that natural antioxidants are heavily metabolized in vivo, a result of which is that their redox potential drops significantly at the physiological level. The authors observed a growing interest in the scientific community in the interactions of natural antioxidants with proteins that are involved in intracellular signaling cascades and modulation of the gut microflora. Currently, in research on natural antioxidants, research issues can be distinguished regarding (i) combination therapies using the synergistic effect of natural antioxidants, (ii) anti-aging effects of fermented preparations, (iii) enzyme research, (iv) genetic research, (v) studies on the effect of antioxidants on the intestinal microflora, and (vi) the effect of antioxidants on hormonal activity. Determination of antioxidant status attracts growing attention for clinical purposes [48, 101] . However, the determination of antioxidative potential, in this case, is difficult to establish due to the complex mechanisms of action for the individual anti-oxidants. Some of them act by scavenging free radicals, some by preventing the formation of ROS or inducing the signaling pathways or by repairing the oxidative damage. Cellular protection is ensured mainly by enzymes (glutathione peroxidase, SOD, catalase), whereas the nonenzymatic antioxidants act in the plasma. Additionally, the status of redox homeostasis differs significantly between the individuals; therefore, the reference values have not been established so far [102, 103] . Presently, there is also no direct method dedicated to accurate measurement of oxidative stress in vivo conditions. Therefore, oxidative stress is measured by the use of multiple in vitro assays [102] , which can identify free radicals directly like electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy, fluorescent probes, or indirect methods enabled to identify the stable products which are created as a consequence of the free radical attack, like chromatography, colorimetry, and immune, or enzymatic tests [104] . There is also some misunderstanding regarding specific terms that are used to describe antioxidants measurement assays. Bunaciu et al., in a critical review [105] , pointed out that the terms "antioxidant activity" and "antioxidant capacity" need some more clarification because they are often used interchangeably despite having different meanings. It should be emphasized that the term "antioxidant activity" refers to kinetic-based assays measuring the rate constant of a reaction between reactants or scavenging percentages per unit time. Thus, the term is characteristic of a specific antioxidant and oxidant, expressed as reaction rates value. In turn, the antioxidant capacity can be defined as the efficiency of antioxidants to inhibit the oxidative degradation of the various bio-compounds. The measurements are based on the reaction between studied antioxidants and free radicals (reactive species inactivation, quenching, or scavenging) or on the reaction of the sample with transition metals. Antioxidant capacity expresses the amount (in moles) of a given free radical that is scavenged by a sample. In the case of a heterogeneous mixture, the antioxidant capacity of each individual component is not possible to measure as all antioxidants react simultaneously to produce the total scavenging ability of the sample. In the case of the complex samples, the most reasonable way of their antioxidant capacity is using a variety of methods that can address the different mechanisms of action of individual components [106, 107] . The collaborative effect of all sample components (i.e., synergistic or antagonistic effects) is responsible for "total antioxidant capacity" (TAC) measured. Antioxidants' capacity can be estimated by considering the final effects of their presence, by the use of in vitro tests, or directly by more complex methods utilizing exogenic probes to detect oxidation. With such a variety of mechanisms involved in the action of antioxidants, determining the level of total antioxidant capacity (TAC) is one of the major challenges in antioxidant testing. Thus far, no universal method has been developed that would gain general and univocal acceptance. Therefore, when choosing a specific method, one should be aware of what kind of an antioxidant function is being measured [46, 108] . The measured activity of primary antioxidants reflects their ability to scavenge ROS/RNS throughout hydrogen atom (H • ) or electron (e − ) transfer or both species simultaneously (i.e., proton-coupled electron transfer). Secondary antioxidants, which are known as preventive ones, are evaluated by the chelating ability of selected transition metal ions e.g., Fe(II) or Cu(I). Preventive antioxidants act by inhibiting Fenton reactions as a source of hydroxyl radicals or a Lewis acid-base neutralization (metal ion-antioxidant). In turn, endogenous antioxidative enzymes, being "first-line defense antioxidants" such as SOD, CAT, and GPx, which are able to scavenge superoxide anion radicals and hydrogen peroxides, require enzymatic methods for evaluation of the antioxidants activity [108] . Nonenzymatic primary antioxidant assays can be non-competitive or competitive [109] . Competitive assays such as TRAP, ORAC, TOSC, crocin bleaching, peroxyl radical trapping antioxidant parameter, act due to the competition between a fluorogenic or chromogenic probe and antioxidants for the reactive species (ROS/RNS). In the presence of antioxidants, the probe undergoes weaker oxidation, which is reflected in the changes of its measurable properties (absorbance, fluorescence, luminescence) [110] (Figure 2 ). as a source of hydroxyl radicals or a Lewis acid-base neutralization (metal ionantioxidant). In turn, endogenous antioxidative enzymes, being "first-line defense antioxidants" such as SOD, CAT, and GPx, which are able to scavenge superoxide anion radicals and hydrogen peroxides, require enzymatic methods for evaluation of the antioxidants activity [108] . Nonenzymatic primary antioxidant assays can be non-competitive or competitive [109] . Competitive assays such as TRAP, ORAC, TOSC, crocin bleaching, peroxyl radical trapping antioxidant parameter, act due to the competition between a fluorogenic or chromogenic probe and antioxidants for the reactive species (ROS/RNS). In the presence of antioxidants, the probe undergoes weaker oxidation, which is reflected in the changes of its measurable properties (absorbance, fluorescence, luminescence) [110] (Figure 2 ). The non-competitive ( Figure 3 ) ones based on Folin−Ciocalteu reaction, ABTS/TEAC, CUPRAC, FRAP, DPPH, ABTS differ in the lack of the presence of any competing target molecule. TAC measurements are considered to be noncompetitive if they rely on electron transfer (ET) mechanism, whereas competitive measurements are usually based on a hydrogen atom transfer (HAT) [46] . The non-competitive ( Figure 3 ) ones based on Folin−Ciocalteu reaction, ABTS/TEAC, CUPRAC, FRAP, DPPH, ABTS differ in the lack of the presence of any competing target molecule. TAC measurements are considered to be noncompetitive if they rely on electron transfer (ET) mechanism, whereas competitive measurements are usually based on a hydrogen atom transfer (HAT) [46] . The measured activity of primary antioxidants reflects their ability to scavenge ROS/RNS throughout hydrogen atom (H • ) or electron (e − ) transfer or both species simultaneously (i.e., proton-coupled electron transfer). Secondary antioxidants, which are known as preventive ones, are evaluated by the chelating ability of selected transition metal ions e.g., Fe(II) or Cu(I). Preventive antioxidants act by inhibiting Fenton reactions as a source of hydroxyl radicals or a Lewis acid-base neutralization (metal ionantioxidant). In turn, endogenous antioxidative enzymes, being "first-line defense antioxidants" such as SOD, CAT, and GPx, which are able to scavenge superoxide anion radicals and hydrogen peroxides, require enzymatic methods for evaluation of the antioxidants activity [108] . Nonenzymatic primary antioxidant assays can be non-competitive or competitive [109] . Competitive assays such as TRAP, ORAC, TOSC, crocin bleaching, peroxyl radical trapping antioxidant parameter, act due to the competition between a fluorogenic or chromogenic probe and antioxidants for the reactive species (ROS/RNS). In the presence of antioxidants, the probe undergoes weaker oxidation, which is reflected in the changes of its measurable properties (absorbance, fluorescence, luminescence) [110] (Figure 2) . The non-competitive ( Figure 3 ) ones based on Folin−Ciocalteu reaction, ABTS/TEAC, CUPRAC, FRAP, DPPH, ABTS differ in the lack of the presence of any competing target molecule. TAC measurements are considered to be noncompetitive if they rely on electron transfer (ET) mechanism, whereas competitive measurements are usually based on a hydrogen atom transfer (HAT) [46] . In certain circumstances, ET/HAT mechanisms may not be easily identified like for 2,2azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, which are sometimes classified as mixed-mode assays (ET/HAT). Both free radicals react according to two mechanisms: HAT (1) and SET (single electron transfer) (2): Experimental investigations [111, 112] confirm that HAT and SET transformations may occur at the same time as a sequential proton-loss electron transfer (SPLET), which is also named as a proton-coupled electron transfer (PCET) [106] : It has been proven that the HAT mechanism dominates in aqueous solutions. In turn, the SET and SPLET may dominate in non-aqueous solutions due to the possibility of organic solvents forming hydrogen bonds with molecules of antioxidants [113] [114] [115] [116] . Among the SET methods, the most used are DPPH radical scavenging capacity assay, Trolox equivalent antioxidant capacity (TEAC or ABTS) assay, ferric reducing (FRAP) assay, reducing power assay (RP), and copper reduction (CUPRAC) assay. HAT assays include the total per-oxyl radical-trapping antioxidant parameter (TRAP) assay, the crocin bleaching assay, oxygen radical absorbance capacity (ORAC) assay, and total oxyradical scavenging capacity (TOSC) assay. Antioxidant activity can also be estimated using nanoparticle-based assays utilizing nanoparticles probes exhibiting localized surface Plasmon resonance (LSPR) absorption [117, 118] . It has been established that the LSPR absorption connected with the nanoparticles grove rises linearly depending on antioxidant concentration. Scampicchio et al. described such correlation for gold nanoparticles (AuNPs) generated under the influence of phenolic acid antioxidants being able to donate electrons. Özyürek et al. proved the same for silver nanoparticles (Ag-NPs), which were formed as a product of AgNO 3 reduction with polyphenolic antioxidants. Many studies are dedicated to the estimation of the antioxidant power of various individual chemicals, as well as food samples and natural extracts [119] . For this purpose, various tests were applied, including, among others, the oxygen radical absorbance capacity test, the Trolox equivalent antioxidant capacity, and the ability to reduce metal ions, such as copper or iron. Several reviews have been published that highlight the advantages and disadvantages of the available tests [120] [121] [122] [123] [124] . However, there is still no standard quantitative method for measuring antioxidant activity. Therefore, it is extremely difficult to compare the results obtained from different studies. The complexity and variety of research systems make it impossible to repeat and confirm experiments by independent laboratories. The most common methods related to the antioxidant assessment are summarized in Table 2 . Table 2 . Examples of the non-enzymatic assays used for in vitro determination of antioxidant capacity with distinguished chromogenic agents, observed changes, the principle, mode, and mechanism of the assay (Mech). A number of techniques are used to assess antioxidant capacities, such as UV-Vis spectroscopy, fluorescence spectroscopy, chemiluminescence, electron paramagnetic resonance (EPR), enzyme-catalyzed assays [164] [165] [166] [167] [168] , and cell culture assays. Moreover, there are some electrochemical techniques, including controlled potential techniques, electrochemical sensors, and biosensors, which are commonly applied [169] . However, the most widely used techniques for evaluating the ability of an antioxidant to scavenge e.g., ABTS •+ , DPPH • , O 2 •− , H 2 O 2 , a total antioxidant reducing capacity, e.g., TEAC, ORAC, and FRAP belong to spectrometric techniques. These methods have been commonly used to determine the antioxidant capacity of many plant extracts, foods, and dietary supplements [170] [171] [172] [173] [174] . These assays despite some drawbacks [129] are easy to use. To measure antioxidants' power, their ability to deactivate free radicals was used. One of the most frequently used stable free radicals is DPPH (1,1-diphenyl-2-picrylhydrazyl) discovered by Goldsmith and Renn in 1922 [175] . Due to the relocation of the unpaired electron, DPPH forms a stable radical cation and does not form dimers in alcohol solutions [176, 177] . The DPPH solution has a dark purple color with maximum absorbance at wavelength = 517 nm. By reaction with a substance that gives off a hydrogen atom, a reduced form of DPPH 2,2-diphenyl-1-picrylhydrazine is formed, and then the purple color of the solution changes to yellow with a concomitant decrease in absorbance ( Figure 4 ). the most widely used techniques for evaluating the ability of an antioxidant to scavenge e.g., ABTS •+ , DPPH • , O2 •− , H2O2, a total antioxidant reducing capacity, e.g., TEAC, ORAC, and FRAP belong to spectrometric techniques. These methods have been commonly used to determine the antioxidant capacity of many plant extracts, foods, and dietary supplements [170] [171] [172] [173] [174] . These assays despite some drawbacks [129] are easy to use. To measure antioxidants' power, their ability to deactivate free radicals was used. One of the most frequently used stable free radicals is DPPH (1,1-diphenyl-2picrylhydrazyl) discovered by Goldsmith and Renn in 1922 [175] . Due to the relocation of the unpaired electron, DPPH forms a stable radical cation and does not form dimers in alcohol solutions [176, 177] . The DPPH solution has a dark purple color with maximum absorbance at wavelength = 517 nm. By reaction with a substance that gives off a hydrogen atom, a reduced form of DPPH 2,2-diphenyl-1-picrylhydrazine is formed, and then the purple color of the solution changes to yellow with a concomitant decrease in absorbance ( Figure 4 ). The drop in absorbance is proportional to the amount of DPPH oxidized form that remains in solution. The color change from purple to yellow can be monitored spectrophotometrically and utilized for the assessment of the free radical scavenging potential of many antioxidants and natural products. For the first time, the colorimetric method was described by Blois [177] for the evaluation of the antioxidant properties of The drop in absorbance is proportional to the amount of DPPH oxidized form that remains in solution. The color change from purple to yellow can be monitored spectrophotometrically and utilized for the assessment of the free radical scavenging potential of many antioxidants and natural products. For the first time, the colorimetric method was described by Blois [177] for the evaluation of the antioxidant properties of the thiol-containing amino acid cysteine as the model antioxidant. Since that time, an easy and convenient colorimetric method has been extensively used to evaluate the antioxidant capacity of many products of natural origin [178] [179] [180] [181] [182] [183] . The reaction of DPPH with antioxidants was adapted for illustration and measuring the kinetics of radical quenching [184, 185] . Since the beginning of the 1960s, the method, as well as antioxidant activity calculations, have evolved into numerous modifications [186, 187] . DPPH free radical scavenging has been conducted by using at least two commonly practiced procedures (a) fixed reaction time, when the researcher imposes reaction times of 15, 30, or 60 min, and (b) steady-state saturation one, when the reaction time is related to the reaction kinetics. The reaction of DPPH radicals with antioxidants is a kinetically driven process. It has been proven that the time required to reach saturation state, i.e., the highest decrease in DPPH absorbance depends on concentration and the kind of antioxidant. To check out the kinetic behavior of the disappearance of DPPH radicals with individual antioxidants, kinetic scans should be performed at different concentration levels. Although at higher concentrations, the scavenging capacity is higher, sometimes the reaction cannot be completed quickly because of slow kinetics. For instance, the reaction of DPPH with ascorbic acid is fast and achieves completion within a minute [188] , whereas even 3 h is not enough to finish the reaction for curcumin at so small a concentration as from 5 to 15 µM. In turn, the reaction time for BHT was found to be around 6 h. Such antioxidants as lipoic acid, melatonin, and pentoxifylline demonstrate slow reaction with DPPH radical up to 2 mM. Such kinetic measurements have been performed for different chemicals used as reference antioxidants. Considering the time duration of reaction to achieve the steadystate, antioxidants can be divided into categories of fast (<30 min), medium (30 min to 1 h), and slow (>1 h) kinetics. In 2012, Mishra et al. [178] established the nature of individual chemicals such as alpha-tocopherol, ascorbic acid, sesamol, gallic acid, ferulic acid, and BHT-butylated hydroxytoluene, which are commonly used as references in the comparative evaluation of antioxidant properties. Among these reagents, there are examples of fast (ascorbic acid), medium (gallic acid), and slow reaction kinetics, which is observed for BHT. Despite the fact that the time to attain an equilibrium state depends on the nature of antioxidants, researchers have usually chosen a fixed reaction time mode where reaction time is pre-imposed to be 20-30 min instead of the real-time required to attain completion of the redox reaction [176] , ignoring their kinetic behavior and the fact that many antioxidants might react with different kinetics or might not react at all. Furthermore, some authors emphasize the reversibility of the free radical reduction by antioxidants, which results in underestimation of the antioxidant capacity of many antioxidants [106, 189] . Considering numerous methodologies of DPPH assay described in the literature, involving variation in (i) concentrations of reagents, (ii) sample' volume, (iii) the kind of reference molecules, (iv) antiradical parameters used, (v) units of applied parameters, and (vi) the kind of sample environment (methanol or semi-aqueous media), the antiradical potential of any sample assessed by DPPH assay, it is very difficult to compare results between laboratories. Mishra et al. [134] collected IC 50 values of reference standards such as butylated hydroxyl anisole (BHA), ascorbic acid, gallic acid, BHT, and Trolox that determined by different authors. It appeared that the reported IC 50 value of ascorbic acid was in the range from 11.85 to 629 µM. Unfortunately, such a large variation in IC 50 values was also observed for remaining antioxidants. Recently, Xie and Schaich [190] have reevaluated the DPPH assay considering the solvent kind and pH values. The DPPH free radical scavenging activity is commonly expressed in terms of the percentage of inhibition of the free radical by examined antioxidants. The EC 50 value relates to the antioxidant concentration required to achieve a 50% decrease in the DPPH absorbance. This parameter is typically employed not only to express the antioxidant capacity but also to compare the activity of different compounds with each other. To find the above parameter, antiradical curves are plotted, representing the relationship between the concentration of antioxidants on the x-axis and relative scavenging capacity (E%) on the y-axis. The radical scavenging capacity can be calculated using the following equation: However, to find the most credible EC 50 value, an assay should be done using several antioxidant concentrations located near the estimated ED 50 value. The above graph looks like a typical rectangular hyperbole, but it can be changed into a sigmoidal curve after the logarithmic transformation of the x-axis (log[mol/L]). The EC 50 value is usually located in a short linear range, and it may be calculated by the use of the right-angled triangle [191, 192] . This mathematical method must meet two assumptions: reaching the maximum response and recording at least two points located near the targeted point of the 50% maximal response. The following equation enables EC 50 value calculation: It should be noted that sigmoid curves based on the Hill equation are easier to interpret [193] . The logarithmic curve does not have to be symmetrical around its midpoint, thanks to the model using the Richards equation which provides a fitting thanks to the introduction of the S parameter, quantifying the asymmetry. Chen et al. [192] conducted a comparative study of several specialized computer programs based on various regression models towards the aim of EC 50 estimation. The EC 50 values obtained by the use of the statistical programs were similar to each other; however, GraphPad Prism@ five-parameter analysis showed the smallest variance in relation to the experimental estimated EC 50 . The authors claim that the observed differences in the results between the statistical processing programs GraphPad and SigmaPlot are due to the fact that the first one calculates actual EC 50 values, while the second gives the inflection point as the EC 50 . Antiradical power (ARP) is another parameter that can be used to define antioxidant activity. This parameter is defined as a reciprocal of EC 50 , which is why the higher value of EC 50 is related to smaller antiradical power: The antioxidant capacity can be expressed as reference chemical equivalent such as Trolox (µmol TE/g), ascorbic acid, gallic acid (GAE/g), etc. Unfortunately, comparison of results presented by different studies is difficult because of the variety of units used for the above recalculations. We can find mass/mass units such as milligrams per gram of dry material, µmol/g, or mass/volume ones. In the original DPPH assay, provided by batch experiments, several automation approaches based on flow injection analysis (FIA) [194, 195] and sequential injection analysis (SIA) [196] have been proposed in recent decades. An interesting approach inspired by HPLC-FIA [197] has been elaborated on by Koleva [198] . In this method, the HPLCseparated analytes react postcolumn with the DPPH solution, and the induced bleaching is detected as a negative peak by the second detector at 517 nm. Cerda et al. [199] described multi-syringe flow injection analysis (MSFIA) for determining the total antioxidant capacity of several food products. Flow injection analysis (FIA), similarly to sequential injection analysis (SIA), is beneficial for rapid testing of antioxidation/radical scavenging activity of large series of multicomponent samples [177] . Another advantage of automatic approaches in comparison to the standard spectrophotometric batch experiments lies in the visible improvement of measurement reproducibility. Another assay suitable for screening of either hydrophilic or lipophilic antioxidants is a high-throughput relative DPPH radical scavenging capacity (RDSC) assay elaborated by Cheng et al. [189] . The assay, which can be performed in aqueous and organic environments, utilizes a 96-well microplate reader with the spectrophotometric detector, ensuring acceptable accuracy, precision, and reproducibility. The sophisticated instruments are required not only for the rapid determination of the antioxidant activity of complex mixtures but also for providing separation and identification of the selected antioxidant compounds. The HPLC method appears to be the method of choice in this case. For this purpose, HPLC should be used in combination with an appropriate detector, which is usually connected online to chromatographic apparatus. However, simultaneous determining of antioxidant capacity requires additional coupling with another radical scavenging detection mode. Such systems have been described in the literature; unfortunately, they are not adopted commonly due to their complexity and the lack of commercial availability. As an example, in 2007, Wu et al. [200] developed HPLC-ESI-MS and NMR for estimation of antioxidant capacity of polyphenolic acids in the plant extract. In turn, Nuengchamnong et al. [201] proposed RP-HPLC coupled with an electrospray ionization MS/MS system for the identification of antioxidant compounds in an extract of a Thai medicinal plant. An interesting HPLC approach, suitable for searching natural antioxidants in plant extract of Flos Lonicerae Japonicae, was developed by Tang et al. in 2008 [202] . The method's idea assumes that the peak areas of compounds with antioxidant activity undergo reduction after reaction with DPPH. The authors performed additional identification of antioxidants by the HPLC-DAD-TOF/MS hyphenated technique. Traditional thin-layer chromatography with post chromatographic derivatization using DPPH solution for free radical scavenging activity evaluation, discovered by Glavind and Holmer in 1967 [203] , exists nowadays in the modern version owing to video scanning technology [204] . Electrochemical measurements possess some major advantages in comparison to spectrophotometric methods mainly due to the fact that they are fast, less tedious, cheaper, and safer for the environment. They include electrochemical techniques of antioxidant characterization as potentiometry, amperometry, biamperometry, cyclic voltammetry (CV), square-wave voltammetry (SWV), and differential pulse (DPV). These methods utilize the fact that antioxidants are involved in redox reactions acting as reducing agents. The electrochemical techniques are able to measure their redox potentials. The cyclic voltammetry method is applied to screen the reducing capacity of the samples. Cyclic voltammetry (CV) operates due to the combination of three electrodes, namely working electrode, reference, and auxiliary electrode. A polarogram representing the relationship between current intensity and an increasing potential applied to the working electrode is recorded. The obtained voltammograms show well-defined voltammetric peaks corresponding to the oxidation and reduction processes. Lower Epa values are associated with the higher reducing activity of the tested sample. Therefore, considering the first oxidation potential, the following classes of chemical compounds can be distinguished: if Ep is lower than 0.8 V, antioxidant power is high, and if Ep is between 0.8 and 1.3 V, antioxidant power is low [205] . The area under the curve of the voltammetric peak (AUC) corresponds to the concentration of antioxidants. Broad anodic peaks are usually observed due to the response of multiple reducing agents with different oxidation potentials present in the respective extracts. In such cases, Chevion et al. [206] . Martinez et al. [207] , and Zielińska and Zieliński [208] suggested that the area under the anodic current wave should be used for the evaluation of reducing the power of the samples. Lower AUC indicates a lower reducing capacity of the investigated extract. Usually, the reducing capacity is statistically significantly correlated with the active components of the extracts. Zielińska et al. [209] found the existence of a significant positive correlation between the total phenolic content (r = 0.867; p < 0.01) and total flavonoid content (r = 0.752, p < 0.01) with the reducing capacity of peels of the investigated apple cultivars. Determination of the antioxidant activity by biamperometric measurements is based on a high degree of reversibility redox couple potential, including Fe 3+ /Fe 2+ , I 2 /I − , 6 3− /Fe(CN) 6 4− , and Ce(IV)/Ce(III). The DPPH • /DPPH couple is also suitable for this purpose. The current intensity is proportional to the decreasing concentration of free radicals after reaction with the antioxidants. The obtained results of antioxidant activity are usually in very good agreement with those determined by the use of other conventional methods such as spectroscopic measurements. The biamperometric technique was applied by Milardovic et al. [210] for evaluation of the selected standard antioxidants (ascorbic acid, uric acid, gallic acid, N-acetyl-l-cysteine, glutathione, caffeic acid, ferulic acid, sinapic acid, catechin hydrate, quercetin) and food samples such as coffee, tea, wine, and juices. More recently, the new nanoparticle-based approach for evaluation of antioxidant activity has been reported. This approach utilizes the unique optical, electronic, and catalytic properties of metallic nanoparticles (1-100 nm) [211] [212] [213] [214] . For the first time, Scampicchio et al. [117] described a nanoparticle-based method for measuring antioxidant activity. The idea of the method was based on the catalytic growth of gold (Au) NPs mediated by phenolic acids as active reducing agents (vanillic acid, propyl gallate, protocatechuic acid, caffeic acid, ferulic acid). It appeared that the antioxidant (reducing) power of the phenolic acids was correlated with the optical properties of generated nanoparticles. The absorbance characteristic of the plasmon of the Au NPs (555 nm) was linearly dependent upon the concentration of the investigated phenolic acids. The authors confirmed the good agreement between the total phenolic content estimated by the Folin-Cicolteau spectrophotometric determination and the results of the Au NPs protocol. A few years later, Özyürek et al. [118] elaborated on a sensitive colorimetric method based on the reduction of Ag+ ions to silver nanoparticles (AgNPs) for the detection of polyphenols. The AgNPs revealed the absorption band at 423 nm, allowing the quantification of the polyphenols. The initial seeds were formed by the reduction of silver ions with trisodium citrate. The addition of antioxidants as secondary reductants caused the reduction of Ag+ ions on silver seeds and the deposition of more Ag atoms on the seeds, resulting in the final core−shell AgNP structures. The growth of AgNPs on monodisperse seed particles caused a linear, concentration-dependent absorbance increase. The method was named by the research group "Silver NanoParticle Antioxidant Capacity", abbreviated as the SNPAC method, which is recommended for measuring the total antioxidant capacity (TAC) of a wide range of plant samples ( Figure 5 ). Until now, most assays applied for antioxidant capacity determination have involved the use of NPS of gold, silver, Fe 3 O 4 , quantum dots, and titania nanoparticles. The estimation of antioxidant activity relies on the antioxidant-mediated growth of NPs, monitoring changes in NPs size, changes in surface oxidation states, the degree of agglomeration of nanostructures, and optical monitoring of the plasmon absorption bands. AuNPs are still the most commonly used for that purpose. AuNPs have a very characteristic absorbance peak at 517 nm. AuNPs are soluble and stable in different solvents such as water, dichloromethane, or methanol. NPs formation can be monitored visually owing to AuNPs' color, which depends on their shape and size, but also surface-adsorbed species, the refractive index of the dispersion medium, and interparticle interactions [215] . Different techniques have been engaged for detection and characterization of NPs such as the localized surface plasmon resonance (SPR), Surface-Enhanced Raman Scattering, spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), Resonance Light Scattering, Raman spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM) [216] . Selected methods suitable for measuring size, electric and mechanical properties, size distribution, hydrodynamic radius, elemental composition, and quantitative analysis of nanoparticles together with the methods' detection limits are illustrated in Figure 6 . measuring antioxidant activity. The idea of the method was based on the catalytic growth of gold (Au) NPs mediated by phenolic acids as active reducing agents (vanillic acid, propyl gallate, protocatechuic acid, caffeic acid, ferulic acid). It appeared that the antioxidant (reducing) power of the phenolic acids was correlated with the optical properties of generated nanoparticles. The absorbance characteristic of the plasmon of the Au NPs (555 nm) was linearly dependent upon the concentration of the investigated phenolic acids. The authors confirmed the good agreement between the total phenolic content estimated by the Folin-Cicolteau spectrophotometric determination and the results of the Au NPs protocol. A few years later, Özyürek et al. [118] elaborated on a sensitive colorimetric method based on the reduction of Ag+ ions to silver nanoparticles (AgNPs) for the detection of polyphenols. The AgNPs revealed the absorption band at 423 nm, allowing the quantification of the polyphenols. The initial seeds were formed by the reduction of silver ions with trisodium citrate. The addition of antioxidants as secondary reductants caused the reduction of Ag+ ions on silver seeds and the deposition of more Ag atoms on the seeds, resulting in the final core−shell AgNP structures. The growth of AgNPs on monodisperse seed particles caused a linear, concentration-dependent absorbance increase. The method was named by the research group "Silver NanoParticle Antioxidant Capacity", abbreviated as the SNPAC method, which is recommended for measuring the total antioxidant capacity (TAC) of a wide range of plant samples ( Figure 5 ). Until now, most assays applied for antioxidant capacity determination have involved the use of NPS of gold, silver, Fe3O4, quantum dots, and titania nanoparticles. The estimation of antioxidant activity relies on the antioxidant-mediated growth of NPs, monitoring changes in NPs size, changes in surface oxidation states, the degree of agglomeration of nanostructures, and optical monitoring of the plasmon absorption bands. AuNPs are still the most commonly used for that purpose. AuNPs have a very characteristic absorbance peak at 517 nm. AuNPs are soluble and stable in different solvents such as water, dichloromethane, or methanol. NPs formation can be monitored visually owing to AuNPs' color, which depends on their shape and size, but also surfaceadsorbed species, the refractive index of the dispersion medium, and interparticle interactions [215] . Different techniques have been engaged for detection and characterization of NPs such as the localized surface plasmon resonance (SPR), Surface-Enhanced Raman Scattering, spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), Resonance Light Scattering, Raman spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM) [216] . Selected methods suitable for measuring size, electric and mechanical properties, size distribution, hydrodynamic radius, elemental composition, and quantitative analysis of nanoparticles together with the methods' detection limits are illustrated in Figure 6 . Since nanoparticles-based assay is a new analytical tool, calibration is usually performed using control antioxidants [218] [219] [220] [221] , and additionally, the assay is compared with reference methods, e.g., ORAC and TEAC. Many authors achieved very good Figure 6 . Selected methods applied to detection of nanoparticles together with detection limits [217] . Since nanoparticles-based assay is a new analytical tool, calibration is usually performed using control antioxidants [218] [219] [220] [221] , and additionally, the assay is compared with reference methods, e.g., ORAC and TEAC. Many authors achieved very good agreement between the TAC values obtained by the nanoparticle-based approach and the Trolox Equivalent Antioxidant Capacity (TEAC), CUPRAC [222] , Folin-Ciocalteu, FRAP, and DPPH [218] as reference tests. Antioxidant capacity determination by nanoparticles-based method also involves other metallic or metal oxide NPs. Gatselou et al., in 2016 [223] , reported that phenolic compounds (i.e., gallates, catechins, dihydroxybenzoic acids, and cinnamates) generate changes in the localized surface plasmon resonance of rhodium NPs, causing characteristic spectral and color transitions in their suspensions. Under the influence of the reaction between phenolic compounds and rhodium, absorbance at 450 nm and 580 nm increased linearly together with increasing concentration of antioxidants in the range of 0-500 µM. Recently, antioxidant activity (AOA) assays using cerium oxide nanoparticles (CeO-NPs) as a novel colorimetric sensor were developed. Cerium oxide nanoparticles (CeO-NPs) may act as both an oxidant and an anti-oxidant, switching between trivalent and tetravalent oxidation states [224] . In 2018, Ozdemir Olgun [225] elaborated on a novel colorimetric sensor consisting of the poly(acrylic acid) sodium salt (PAANa)-coated CeO-NPs which oxidized a peroxidase substrate, namely tetramethyl benzidine (TMB) in acidic conditions to charge-transfer complex of a blue color. The analytical wavelength of the colored product was estimated at 651 nm. The antioxidant activity evaluation was based on the measurement of decreasing intensity of the nanoceria suspension absorbance caused by antioxidants. The authors demonstrated that the antioxidant capacities of hydrophilic and lipophilic antioxidants such as rutin, tetramethyl benzidine, quercetin, ascorbic acid, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), ferulic acid, BHT, caffeic acid, and catechin estimated by the above procedure were compatible with those of reference assays ABTS, CUPRAC, and CERAC [225] . Currently, portable nanoparticle-based tests for rapid detection of food antioxidants (NanoCerac) are being developed, e.g., for nanoparticles of immobilized cerium oxide [226] , or nanoparticles of metal oxides TiO 2 , Fe 2 O 3 , ZrO 2 , ZnO, and SiO 2 , which are immobilized on cellulose [227] . Several reviews regarding TAC determination by using NPs can be found in the literature [228] [229] [230] [231] [232] . Epidemiological research confirms that the conditions related to oxidative stress can be improved by the consumption of food products rich in numerous compounds with high antioxidant activity [233, 234] . Natural products containing at least 0.1% of antioxidants can be accepted as dietary supplements with antioxidant properties. As the total antioxidant capacity (TAC) covers the additive (synergistic/antagonistic) action of different antioxidants of complex samples, most researchers use this parameter to assess plant-based extracts rather than the separate determination of the concentrations of the individual constituents. It should be emphasized that antioxidant capacity reflects the thermodynamic conversion efficiency of reactive species by antioxidants in contrast to the antioxidant activity, which is related to the kinetics of this reaction, usually expressed scavenging percentages per unit time. Unfortunately, many phytochemical studies have reported conflicting results, which is why TAC assays still require consideration and standardization in the following issues: (i) procedures of sample preparation, (ii) expressing results, (iii) statistical validation (e.g., using certified reference compounds that take into account the different reaction kinetics), and (iv) establishing effects of solvent, concentration, pH, etc. The choice of extraction techniques has the greatest impact on the composition and concentration of the bio-composition of both active compounds and matrix components obtained from a wide range of plant materials (herbs, vegetables, berries, and fruits) [235] . It has been shown that different extraction methods lead to different extraction yields on the same plant material [236] . For example, Lisitsyn et al. [237] studied the plant extracts (rosemary, black pepper, thyme, and sage) obtained by the use of supercritical CO 2 extraction. Owing to this extraction method, they produced extracts with a significantly different composition in comparison to those obtained in traditional ways. It appeared that supercritical extracts were rich in a variety of substances with high antioxidant, and antimicrobial activities such as alkaloids, terpene, phytosterols, waxes, pigments, high molecular weight unsaturated and saturated fatty acids, and vitamins. Currently, classic extraction techniques, i.e., Soxhlet extraction, maceration, percolation, and distillation, which use large amounts of volatile organic solvents, or elevated temperature, are less frequently used due to the requirements of the so-called "green chemistry", poor efficiency, and possible thermolability of extracted analytes [238] . High extraction efficiency and effectiveness are possible thanks to the use of unconventional techniques, i.e., Solid Phase Microextraction (SPME), Supercritical Fluid Extraction (SFE), Microwave-Assisted Extraction (MAE), Pulsed-Electric Field (PEF) Extraction, Ultrasound-Assisted Extraction (UAE), and Enzymatic Treatment or Pressurized Liquid Extraction (PLE). It should be remembered that antioxidant capacity changes not only in relation to the extraction techniques but varies with the growth period and drying methods and between plant parts. These factors' influence has been confirmed by a multivariate analysis performed by Buitrago et al. [239] using Chenopodium quinoa Willd. Plant-derived compounds possess well-known and established antioxidant activity. However, the microbes are also efficient producers of primary and secondary metabolites with specific antioxidant potential [240] . Thus far, microbial metabolites have been recognized as efficient remedies against fungal and bacterial infections (tetracyclines, amphotericin, penicillins, erythromycins, streptomycin, and vancomycin), cancer (daunorubicin, bleomycin, mitomycin, doxorubicin,), transplant rejection (rapamycin, cyclosporine), or high cholesterol (mevastatin, lovastatin) [241] . Almost all eubacteria possess the ability to produce a variety of extracellular metabolites with significant antioxidant activity, such as thiazostatins A, phenazoviridin, (Z)-1-((1hydroxypenta-2,4-dien-1-Yl)oxy)anthracene-9,10-dione, 5-(2,4-dimethyl benzyl) pyrrolidin-2-one, benthophoenin, benzastatins C, benthocyanins A, B, C, exopolysaccharides (EPS), and benzastatins A [78] . Exopolysaccharides (EPS) are produced by numerous strains of microorganisms belonging to the genera Lactobacillus, Leuconostoc, Lactococcus, and Streptococcus, which are abbreviated LAB (Lactic Acid Bacteria) [54] . EPS are characterized by the presence of reactive functional groups including aldehyde, hydroxyl, and ketone groups. They can efficiently react with free radicals. These compounds have a polymeric structure. They are made up of repeating subunits of connected carbohydrates αand β-glycosidic bonds. Homopolysaccharides are composed of one type of simple sugar, i.e., glucose or fructose, while heteropolysaccharides are more complex. The structure of individual heteropolysaccharides produced by particular species and strains of these bacteria can significantly vary. A common feature of most of them is occurrence in the composition of sugars e.g., rhamnose, arabinose, mannose, xylose, fructose, glucose, and galactose, in various ratios [242] . The EPS may exist in two forms: a cell-bound exopolysaccharide (c-EPS) that strongly binds to the bacterial surface and a released exopolysaccharide (r-EPS) that can be released into the medium [243] . Cyanobacteria and blue-green algae are sources of a significant amount of free radical scavengers such as carotenoids, phycocynanin which are water-soluble pigments possessing N-H reactive groups. Astaxanthin produced by microalga Haematococcus pluvialis possessing several times higher antioxidant activity in comparison to vitamin E [244, 245] . Algae are able to produce also other phenolic compounds with reactive OH moieties responsible for an antioxidant activity like carrageenan, bromophenol, fucophlorethols, galactan sulphate, phlorotannins, fucoxanthin, phycoerythrin, shinorine, catechin, por-phyran, epicatechin, gallate, laminaran, vitamin A, alginic acid, phloroglucinol, eckol, fucodiphlorethol G, 7-phloroeckol, dieckol, phlorofucofuroeckol A, 6,60-bieckol, 2,70-phloroglucinol-6,60bieckol, and triphlorethol-A, [246] [247] [248] . As a potential antioxidant, phycobilins rich with groups i.e., N-H, COOH, C-O, and O-H produced by cyanobacteria have been described. However, efficient extraction and purification are required for the recovery of phycobiliproteins on an industrial scale [249] . In 2019, the Special Issue of Antioxidants focused on recent investigations concerning marine algal antioxidants and specific antioxidant networks functioning in algae [248] [249] [250] [251] [252] [253] [254] [255] [256] [257] [258] [259] . Lichens produce various extracellular, secondary metabolites that can be used as potential sources of natural antioxidants [260] . At the beginning of the 21st century, the significant free radical scavenging activity of Cetraria islandica aqueous extracts [261] and Usnea ghattensis [262] methanolic extracts of Platismatia glauca, Parmelia saxatilis, Ramalina pollinaria, Umbilicaria nylanderiana, and Ramalina polymorpha [263] were described. Fernández-Morianoet et al. [264] prepared a systematic review concerning the key antioxidant compounds in lichens extracts. It appeared that flavonoids and phenols are mainly responsible for the antioxidant activity of the examined extracts [265] [266] [267] [268] . Some of them also exhibited beneficial antimicrobial and anticancer activities [269, 270] . Actinomycetes also produce chemically diverse and pharmaceutically useful compounds with antifungal, antibacterial, diabetogenic, antiviral, immunosuppressive, antiparasitic, antitumor, insecticidal, antioxidant, anti-inflammatory, enzyme inhibitory, and others [271] . Actinomycetes originating from different habitats usually manifest very different antioxidant activity [272] [273] [274] [275] [276] [277] . Published studies show that nitrogen-containing metabolites, such as the carbazole and phenazinylhetero cycles, constitute the main group of antioxidant compounds produced by Streptomyces spp. Stealthins contain OH, NH, and CO groups isolated from S. Aeriouvifer, S. Violaceus, and S. viridochromogenes showed even several dozen times stronger activity than vitamin E [278] . Other examples of antioxidant capacity assessment of the different extracts from plants, lichens, fungi, algae, and actinomycetes are collected in Table 3 . The dry plant material (20 g) was extracted using 100 mL of ethanol/water 80/20, (v/v) at 60 • C, for 60 min. The extract was filtered and dried under a vacuum at 50 • C. The dry residues were redissolved with 50% methanol-water and centrifuged at 5000 rpm for 10 min. [282] Ornithogalum billardieri A.auricula was extracted by hot water and ultrasonic-assisted extraction. The supernatants were precipitated with absolute ethanol (95%) and maintained at 4 • C overnight. The precipitate was centrifugated, dissolved in DI, and dialyzed. The non-dialyzed portion was lyophilized to give a crude polysaccharide extract. The separation of polysaccharides was performed using CTAB or CPC. [292] Cephalosporium sp. DPPH gallic acid The fermented material (1.75 kg) was extracted twice with EtOAc (9.0 L) for 3 days at RT, and the extract was evaporated under vacuum. [293] Cerrena unicolor ABTS, DPPH Trolox, ascorbic acid After 20 h of the incubation period, TCA was added to achieve 4% (w/v). The mixture was stirred for 30 min at RT. Cells were removed by centrifugation (10,000× g, 4 • C, 15 min). Crude EPS was precipitated by the addition of 2 volumes of cold ethanol. Crude EPS was collected by centrifugation. The pellet was dissolved in deionized water and dialyzed (MW cut-off 3500 Da) for 24 h against distilled water at 4 • C and then lyophilized. [303] Pseudomonas hibiscicola, Macrococcus caseolyticus, Enterobacter ludwigii, Bacillus anthracis The bacteria were cultured in 500 mL broth at 35 • C. Then, the culture was centrifuged at 8000× g for 5 min. The supernatant was extracted with ethyl acetate (ratio of 1:1). Then, the extract was concentrated to dryness in a rotatory evaporator at 37 • C. The solids were re-dissolved in 20% DMSO and filtered. [304] Alteromonas sp. Shewanella sp. Serratia sp., Citricoccus sp., Cellulophaga sp., Ruegeria sp. Staphylococcus sp. Isolated bacteria were cultured in 500 mL of Marine Broth for 3 days at 25 • C. Bacteria cells were centrifuged, and the pellet was lyophilized. Then, the lyophilizes were extracted with methanol and dichloromethane (1:1) for 12 h. The solvents were evaporated at 40 • C, and the extracts were re-dissolved in DMSO. [305] Methanol extraction: 20 mL methanol was added to 0.5 g dry biomass and sonicated (9 cycles, 50% power) for 20 min. Then, samples were centrifuged at 3500 rpm for 5 min. Pellets were re-extracted in 20 mL methanol 3 times and the supernatants were collected. The samples were filtered and evaporated at 40 • C. Hot water extraction: 1 g of dry sample was added to 100 mL DW and boiled for 30 min. After cooling, extracts were centrifuged at 3500 rpm for 10 min, and supernatants were freeze-dried. [314] Chlorella vulgaris Spirulina platensis ORAC, DPPH, FRAP Trolox The extracts were obtained by the use of ultrasound-assisted extraction by water/ethanol (50:50, v/v). Scenedesmus subspicatus DPPH catequin, gallic acid Different solvents such as ethanol, methanol, butanol, acetone, DMSO, and water were used for extraction. One gram of dried samples were mixed with 10 mL for each solvent. The extraction was carried out for 30 min by sonication (40 kHz) in an ultrasonic bath followed by a 2 h shake, and centrifugation for 10 min. [316] Tetraselmis suecica DPPH α-tocopherol A total of 100 mg of freeze-dried biomass was extracted with 1 mL ethanol/water (3:1, v/v) for 30 min. The mixture was centrifuged at 4500× g, for 10 min, at 20 • C. Then, the ethanolic phase was dried. [317] Lichens Cetraria islandica (L) Ach. The various types of metallic/metal oxides nanoparticles composed of silver, gold, platinum, palladium, cerium, copper, nickel, selenium, or iron have been described in the literature. Their unique physicochemical properties make them advanced materials for industry and biomedical applications [321] . There is also evidence that, besides natural extracts, some nanoparticles such as carbon nanotubes, metal, and metal oxides, and various types of polymer-loaded nanoparticles also possess antioxidant activity and can scavenge the reactive nitrogen and reactive oxygen species (RNS/ROS) [322, 323] . The iron nanoparticles (INPs), due to high catalytic activity, low toxicity, high magnetism, and microwave absorption ability [324] [325] [326] , have already found varied applications in pharmacy (drug delivery), clinical diagnostic (magnetic targeting, negative MRI contrast enhancement, pigments, stem cell sorting), therapy (gene therapy), and analytical chemistry (bio-separation), bioprocesses (environmental remediation, food preservation), industry (lithium-ion batteries) [247, 327] . Unfortunately, nanoparticles synthesized by chemical methods often require toxic reducing and stabilizing agents. These toxic substances adsorbed on the surfaces of the nanoparticles limit their applications in biomedical fields [143] . Thus to obtain nanomaterials, the natural synthesis methods involving the reduction of metallic cations by plant extracts, yeasts, fungus, and bacteria are used more and more often. The formation of NPs is achieved via two steps: in the first one, metal ions are reduced, and in the second one the agglomeration of colloidal suspension causing the formation of the oligomeric clusters [328] . So-called "green synthesis" or "biogenic synthesis" has gained more and more attention as an eco-friendly approach useful for synthesis of not only metal/metal oxide nanoparticles but also the production of other nanomaterials, such as hybrid materials, or a variety of bioinspired materials. Nanoparticles produced by green synthesis methods may be less stable compared to nanoparticles obtained as a product of chemical synthesis [329] [330] [331] . The stabilization of nanoparticles is mainly achieved by electrostatic repulsion. Unfortunately, this type of stabilization is only effective with low ionic strength extracts where the repulsion is facilitated by the highly dispersed double layer. In the case of high ionic strength, aggregation occurs under the influence of strong van der Waals interactions [332] . Another type of stabilization is the creation of an additional barrier on the surface of the NPs. Steric stabilization is provided by proteins, if they are components of the extracts, or by coating the surface with polymers such as PEG or PVP (polyethylene glycol, polyvinylpyrrolidone). Steric stoppers, thanks to their hydrophilic properties, provide an additional stabilizing element in the form of short-distance repulsive forces. The stages of NPs formation and stabilization are schematically illustrated in Figure 7 . Presently, one can observe an increasing interest in studies on the reactivity of nanoparticles compared to macroscopic objects and their cytotoxicity [333] [334] [335] [336] [337] accumulation in the body, which can generate reactive oxygen species (ROS) [338] [339] [340] [341] . A relatively new area of research is the use of nanoparticles with redox-active potential as radical scavengers. For example, cerium and yttrium oxides either act as antioxidants [342] or can prevent the increase of ROS [343, 344] by mimicking the activity of the oxidative enzymes, catalase, or superoxide dismutase [345] . It has been proven that silver nanoparticles (AgNPs) inhibit cell proliferation and modulate the activity of antioxidant enzymes [340, 346, 347] . Hirst et al. confirmed by an in vivo test on mice the effectiveness of cerium oxide nanoparticles (CONPs) in treating oxidative stress [348] . A comparative study conducted by Caputo et al. (2015) revealed that the antioxidant potential of Nacetyl-cysteine and Trolox (soluble analogues of vitamin E) was significantly lower in comparison to CONPs [349] . The authors highlighted the CONPs regenerative redox cycle influencing the stability of the antioxidants molecules. The first experiments on AgNPs biosynthesis using bacteria were carried out in the culture of Pseudomonas stutzeri AG 259, Morganella sp. Bacillus subtilis [350] . Using microscopic and spectral techniques SEM, TEM, EDX, and EDS, it was possible to identify Presently, one can observe an increasing interest in studies on the reactivity of nanoparticles compared to macroscopic objects and their cytotoxicity [333] [334] [335] [336] [337] accumulation in the body, which can generate reactive oxygen species (ROS) [338] [339] [340] [341] . A relatively new area of research is the use of nanoparticles with redox-active potential as radical scavengers. For example, cerium and yttrium oxides either act as antioxidants [342] or can prevent the increase of ROS [343, 344] by mimicking the activity of the oxidative enzymes, catalase, or superoxide dismutase [345] . It has been proven that silver nanoparticles (AgNPs) inhibit cell proliferation and modulate the activity of antioxidant enzymes [340, 346, 347] . Hirst et al. confirmed by an in vivo test on mice the effectiveness of cerium oxide nanoparticles (CONPs) in treating oxidative stress [348] . A comparative study conducted by Caputo et al. (2015) revealed that the antioxidant potential of N-acetyl-cysteine and Trolox (soluble analogues of vitamin E) was significantly lower in comparison to CONPs [349] . The authors highlighted the CONPs regenerative redox cycle influencing the stability of the antioxidants molecules. The first experiments on AgNPs biosynthesis using bacteria were carried out in the culture of Pseudomonas stutzeri AG 259, Morganella sp. Bacillus subtilis [350] . Using microscopic and spectral techniques SEM, TEM, EDX, and EDS, it was possible to identify various shapes of nanoparticles, i.e., triangular, hexagonal, and spherical with sizes ranging from a few to several hundred nm. The synthesis process was initiated within the first hour of cultivation. The obtained NPs were coated with protein, which allowed them to maintain stability and avoid their aggregation. It has been shown that the enzyme nitrate reductase is responsible for the reduction of silver ions. Thus far, the participation of other groups of enzymes, whose role is electron donation and their further transfer, has been described and proven, i.e., nitrate and iron reductases, dependent on the nicotinamide adenine dinucleotide (NAD+)/NADH and the nicotinamide adenine dinucleotide phosphate NADP+/NADPH redox couples hydrogenase, and oxidase. Silver ions, due to their interaction with cytochromes and inhibition of electron transport, lead to disturbances in the functioning of the respiratory chain. The mechanism of silver nanoparticle synthesis in lactic acid bacteria was investigated in detail [351] . It was noted that the alkaline environment clearly favors the formation of nanoparticles as it catalyzes the enolization of monosaccharides. The resulting aldehyde is oxidized to carboxylic acid, while the metal ions are reduced to nanoparticles. The effect of nanoparticles on bacteria is complex and not fully understood due to the existence of numerous mechanisms of action (Figure 8 ). Bacteria and other microorganisms such as viruses, fungi, flagella, yeasts, and actinomycetes possess the ability to produce metallic NPs intracellularly as well as extracellularly. The studies showed the action of AgNPs on Escherichia coli and Staphylococcus ureus [352] . The appearance of pits in the bacterial envelope has been observed, which lead to a change in the electrostatic potential, an increase in the permeability of membranes, and damage to the DNA of the cell. Later, research was extended to other species of bacteria, which allowed for the emergence of two more mechanisms of action in the form of overproduction of free radicals (ROS) and the formation of complexes with various intracellular compounds, i.e., nucleic acids [353, 354] . It is particularly interesting that even a short incubation with nanoparticles leads to the accumulation of chaperones and the S6 protein [355] and inhibition of the bacterial communication system (quorum sensing, QS), which is associated with a change in gene expression controlled by transcription regulators. It is known that disturbances in the functioning of these genes cause a change in the behavior of cells in the environment, e.g., the ability to create biofilms. This applies even to pathogenic bacterial strains such as Pseudomonas aeruginosa and Staphylococcus aureus. Biofilms create populations of microorganisms (bacteria, fungi, protozoa) that live at the interface [356] . They are surrounded by a protective substance composed of polysaccharides, proteins and nucleic acids, called a matrix. Biofilm formation is a multi-step process but is always initiated by adhesion. Within the biofilm, there may be synergistic or antagonistic interactions between the species inhabiting it, which may lead to the matrix disintegration [357] . The most dangerous are biofilms composed of pathogenic bacteria E. faecalis, S. aureus, Staphylococcus epidermidis, E. coli, Klebsiella pneumoniae, and P. aeruginosa [358] , which are mostly responsible for hospital infections that are difficult to cure and are characterized by increased resistance to therapies [359] . The antibiotic resistance of biophimes is the result of, among other things, the presence of a matrix that is a barrier to drug penetration and the production of enzymes responsible for the hydrolysis of ß-lactam antibiotics [360, 361] . Research on the potential of nanoparticles to combat biofilms meets the expectations of modern medicine. However, the number of publications on this topic has so far been rather small. It was shown that P. aeruginosa and S. epidermidis biofilms were inhibited in over 95% of cases by silver nanoparticles with a spherical shape and an average diameter of 50 nm [362] . The inhibition of biofilms formed by Multidrug-Resistant Klebsiella pneumoniae [363] , Methicillin-Resistant Staphylococcus aureus [364] , and Mycobacterium tuberculosis [365] has been proven. Unfortunately, the aggregation of nanoparticles inhibits their effective activity. Consequently, various stabiliz-ers such as starch, citrate, and amino silica are used, and numerous composites composed of nanoparticles and other compounds have been identified. There are also reports on the inhibition of biofilm formation by AgNPs on medical devices, i.e., urological catheters, the surface of which was covered with nanosilver, were characterized by resistance to E. coli, S. aureus, and Candida albicans, even under continuous fluid flow conditions. [366] . Metallic/metal oxide nanoparticles, i.e., silver, gold, magnesium, titanium, zinc, aluminum, tantalum, and zirconium have been tested in orthopedics [367] [368] [369] [370] . Nanoparticles embedded in implants and orthopedic scaffolds provide mechanical strength and antimicrobial protection. However, it should be remembered that many nanoparticles exhibit cytotoxicity and genotoxicity, especially in the case of their small size and higher concentrations [371] . Interesting examples of NPs that were produced by microorganisms are iron oxide nanoparticles which were produced by aquatic magnetotactic bacteria (MTB). These bacteria are able to biomineralize, magnetic magnetite, or greigite nanocrystallites called magnetosomes. When isolated from the MTB, magnetosomes exceed synthetic magnetic nanoparticles exhibiting promising anti-tumor efficacy against glioblastoma tumors in vivo tests [372, 373] . It should be emphasized, however, that the anticancer activity is based on various mechanisms of action (heat, the release of chemotherapeutic drugs under a pH variation, nanoparticle excitation by radiation, and apoptotic tumor cell death). Magnetic nanoparticles are useful for targeted cancer therapies because they can be manipulated by external magnetic fields. Moreover, they are attracted toward hypoxic areas, such as the tumor regions, while retaining the therapeutic and imaging capacities of the isolated magnetosomes [374] . In nature, we can find other examples of a variety of nanomaterials synthesized by biological processes like example diatoms, which synthesize siliceous materials or S-layer bacteria forming NPs of gypsum and calcium carbonate layers. Plant extracts contain diverse compounds, which can be utilized as potent reducing agents, stabilizing agents, and precursor molecules for NPs formation [375, 376] . In order to prepare the extracts, both the biomass of the whole plant and selected parts such as leaves, fruit, seeds, and above-ground parts can be used. The plant material can be fresh or powder-dried. Various techniques are used to prepare the extracts, but most consist of Interesting examples of NPs that were produced by microorganisms are iron oxide nanoparticles which were produced by aquatic magnetotactic bacteria (MTB). These bacteria are able to biomineralize, magnetic magnetite, or greigite nanocrystallites called magnetosomes. When isolated from the MTB, magnetosomes exceed synthetic magnetic nanoparticles exhibiting promising anti-tumor efficacy against glioblastoma tumors in vivo tests [372, 373] . It should be emphasized, however, that the anticancer activity is based on various mechanisms of action (heat, the release of chemotherapeutic drugs under a pH variation, nanoparticle excitation by radiation, and apoptotic tumor cell death). Magnetic nanoparticles are useful for targeted cancer therapies because they can be manipulated by external magnetic fields. Moreover, they are attracted toward hypoxic areas, such as the tumor regions, while retaining the therapeutic and imaging capacities of the isolated magnetosomes [374] . In nature, we can find other examples of a variety of nanomaterials synthesized by biological processes like example diatoms, which synthesize siliceous materials or S-layer bacteria forming NPs of gypsum and calcium carbonate layers. Plant extracts contain diverse compounds, which can be utilized as potent reducing agents, stabilizing agents, and precursor molecules for NPs formation [375, 376] . In order to prepare the extracts, both the biomass of the whole plant and selected parts such as leaves, fruit, seeds, and above-ground parts can be used. The plant material can be fresh or powder-dried. Various techniques are used to prepare the extracts, but most consist of classical maceration with various solvents including water or water-alcohol mixtures. Nanoparticle synthesis is mediated by extract components with reducing potential including alkaloids, terpenoids, polyphenols, phenols, flavonoids, and proteins, which have additionally been identified as nanoparticle stabilizers. As compared with ordinary metal salts or initial materials alone, biologically synthesized nanoparticles have been found to be better scavengers of free radicals [377] . The antioxidant activity of NPs frequently depends on their size [378, 379] as well as shape [380, 381] . So far, many examples of the phytogenic synthesis of NPs have been described, including, among others, copper oxide and copper nanoparticles by the use of the leaf extract of Cissus arnotiana with antioxidant ability [382, 383] . Apart from zinc oxide (ZnONPs), selenium (SeNPs), and nickel oxide nanoparticles (NiONPs), one of the biggest groups of plant-mediated NPs is iron nanoparticles (INPs). This group is divided into (a) iron oxide nanoparticles (IONs), (b) iron oxide hydroxide (FeOOH) nanoparticles, and (c) zero-valent iron (ZVI) nanoparticles [384] [385] [386] [387] . Iron oxide (magnetite Fe 3 O 4 , magemite Fe 2 O 3 ) NPs of certain sizes have superparamagnetic properties; therefore, they are useful as contrast agents and drug carriers. The main problems encountered in the biogenic synthesis of nanoparticles concern achieving their appropriate shape, size, and monodispersity in the solution phase. Undoubtedly, the size and shape of NPs depend on the synthesis conditions and the chemical composition of the extract. Usually, optimization of synthesis conditions concerns such factors as the extract concentration, pH, temperature, and reaction or incubation time [388] [389] [390] [391] . The reports on a plant-mediated approach to synthesize NPs by the use of different extracts are collected in Table 4 . Increasing attention is paid to nanoparticles functionalized by various antioxidants obtained from various natural sources, such as algae, bacteria, fungi, lichens, and plants. It should be emphasized that most authors reported that the functionalized NPs exhibit a few times greater antiradical activity. The effective transport across the cell membrane through pinocytosis and the possibility of targeted localization give rise possibility of NPs utilization also as carriers for antioxidants. In those cases, inert metalcore and antioxidants attached to the nanoparticle surface can exert also independent activity [63] . In 2020, a review on antioxidant functionalized NPs was published [247] . Most papers present the synthesis of gold and silver nanoparticles that are easily functionalized with different small molecules of antioxidants, for instance, gold nanoparticles functionalized with tocopherol [440, 441] , gold nanoparticles coated with chitosan [442] , silver NPs with glutathione [443] , or more complex ones like graphite layered 30 nm cobalt nanomagnets with attached tocopherol derivatives [444] . Konopko et al. [440] and Nie et al. [441] prepared and characterized gold nanoparticles (AuNPs) coated with α-tocopherol-like residues. Both research groups proved that the assembly of chromanol groups on gold nanoparticles could efficiently enhance the activity of the vitamin E-derived antioxidant. In 2019, Mohd Taib et al. [445] synthesized Au-NPs utilizing water extract of Hibiscus sabdariffa leaves. Owing to the UV-VIS, FTIR, and HPLC analysis, chlorogenic acid was identified as the major antioxidant compound involved in the reduction of Au 3+ ions. Moreover, the thiol groups can interact directly with the gold core to form gold-sulfur bonds (Au-S) responsible for the mucoadhesion properties of the synthetized AuNPs [446] . In 2017, Choi et al. [447] described nanoparticles modified by caffeic acid, which was immobilized on the surfaces of micro-dielectric barrier discharge (DBD) plasma-treated ZnO nanoparticles. Obtained nanoparticles showed strong antioxidant (ABTS), antibacterial activity against Gram-positive bacteria (Staphylococcus aureus), including resistant bacteria such as methicillin-resistant S. aureus, and against Gram-negative bacteria (Escherichia coli). Nanostructural materials such as nanotubes have been described as novel synergistic nano-antioxidants [448] , for example, ascorbic acid loaded into the inner lumen of natural halloysite nanotubes [449] or halloysite externally deco-rated with tocopherol-like moieties and containing quercetin inside the nanotube [450] . Many studies have described functionalized silver and gold nanoparticles derived from fungal or bacterial extracts obtained from species Ganoderma lucidium [408] [409] [410] , Aspergillus versicolor, Cladosporium cladosporioides, Pestalotiopsis microspore [401, 403, 451] , and bacteria Lactobacillus kimchicus [419] . Another promising trend of nanobiotechnology represents the development of nanodrug delivery systems composed of biocompatible and biodegradable polymeric nanomaterials (polylactide-PLA, poly-lactic-co-glycolic acid-PLGA) that are able to encapsulate the therapeutic agent and progressively release it at the target site. Chlorogenic acid entrapped in hybrid materials composed of SiO 2 and polyethylene glycol has been identified as a system able to control the overproduction of RNS/ROS [452] . Another example is curcumin encapsulated in a nanocarrier and covered with chitosan. Authors observed a protective effect of chitosan on the antioxidant activity of curcumin [453, 454] . While inorganic nanoparticles, especially those with semiconductor properties, have found applications in in vitro diagnostics and imaging, nano-drugs ensure effective biodistribution thanks to the ability to overcome biological barriers. Thus far, many medicinal preparations in the form of nanoparticles have been developed, belonging to different classes of NPs (polymeric, inorganic, and lipid-based), such as polymer-drug conjugate, protein-drug conjugate, polymer-protein conjugate, antibody-drug conjugate, dendimeric drug, polymeric micelle, polymersome, liposome, PEGylated liposome, organic/inorganic colloid, quantum dot, Si-NPs, Au-NPs, and INPs. Extensive reviews on this subject have already been published [455] [456] [457] [458] ; unfortunately, it is beyond the scope of the current study. Interesting nano-formulas are also doubly hydrophilic self-assembling block copolymers (DHBC), which in recent years have aroused more and more interest not only for the production of nanoparticles, but also as controlled drug distribution systems. A valuable review on DHBC was published in 2020 by Jundi et al. [459] . Considering the key role of antioxidants to treat oxidative diseases, the development of reliable antioxidant activity assays of different products with high antioxidant content, as potential drugs or supplements, is needed. Several analytical techniques can be applied for this purpose such as spectroscopic, chromatographic, and electrochemical ones. At the beginning of the 21st century, antioxidant assays based on NPs were developed. The use of NPs as optical or electrochemical probes appears to be a very promising approach; however, this technique has still been scarcely followed. Over 5 years of research on the NPs-based method has resulted in a negligible number of publications, which illustrates the fact that in the PubMed database, the phrases "antioxidant capacity", "nanoparticles", and "plant extracts" are associated with no more than 70 scientific papers. One should emphasize that performing the comparative analysis of antioxidant potentials on the basis of results published by different research groups is very difficult. The antioxidant potential of natural products, and even single chemicals, depends on many factors such as conditions of samples collections, as well as the extracts preparation method and the way of expressing results. On the other hand, the plant extracts rich in antioxidants that act as both reducing and stabilizing agents appear to be useful for creating metallic nanoparticles. Green synthesis surpasses classical methods, providing such benefits as low-cost, environmentally friendly strategies not requiring high pressure, energy, temperature, or external toxic chemical agents. Furthermore, green synthesis ensures the formation of nanoparticles free of toxic contaminants, which makes them suitable in therapeutic applications such as antimicrobial agents in bandages, applications in targeted drug delivery, or clinical diagnostics as contrast agents (MRI-Magnetic Resonance Imaging). The popularity of green nanoparticle synthesis toward bio and medical applications is reflected in the number of around 5000 publications that have appeared in the PubMed database in the last five years. A promising trend that has been developing dynamically in recent years is the synthesis of antioxidant functionalized nanoparticles. Such modification improves the bioavailability of antioxidants providing the benefits of biocompatibility, high stability, and targeted delivery. Author Contributions: J.F. and W.F. wrote-original draft preparation, J.B. and W.F. collected and reviewed the data; W.F. edited the paper and prepared visualization of the paper., J.F. and R.M. wrote-review and edited, R.M. was responsible for supervision, R.M. was responsible for project administration, and J.F., J.B., and R.M. dealt with funding acquisition. All authors have read and agreed to the published version of the manuscript. 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Asian Pac Production of Bioactive Compounds by Actinomycetes and Their Antioxidant Properties Characterization of antioxidant activity of Streptomyces species VITTK3 isolated from Puducherry coast Cytotoxicity and antioxidant activity of 5-(2,4-dimethylbenzyl)pyrrolidin-2-one extracted from marine Streptomyces VITSVK5 spp Isolation of isoflavonoids possessing antioxidant activity from the fermentation broth of Streptomyces spp Flavonoid Analysis and Antioxidant Activities of the Bryonia alba L Quantification of the Antioxidant Activity of Plant Extracts: Analysis of Sensitivity and Hierarchization Based on the Method Used Evaluation of antioxidant activity and polyphenol content of leaves from some fruit species Phenolic Profile, Antioxidant Capacity, and Antimicrobial Activity of Leaf Extracts from Six Vitis vinifera, L. Varieties Antioxidant Activity and Biocompatibility of Fructo-Polysaccharides Extracted from a Wild Species of Ornithogalum from Lebanon Phenolic Composition of Hydrophilic Extract of Manna from Sicilian Fraxinus angustifolia Vahl and its Reducing, Antioxidant and Anti-Inflammatory Activity in Vitro Chemical Properties, Fatty-Acid Composition, and Antioxidant Activity of Goji Berry (Lycium barbarum L. and Lycium chinense Mill.) Fruits. Antioxidants HPLC-DAD Method on Tracking the Antioxidant Activity of Pure Antioxidants and Goutweed (Aegopodium podagraria L.) Hydroalcoholic Extracts. Molecules Antioxidant and hepatoprotective potentials of novel endophytic fungus Achaetomium sp., from Euphorbia hirta. Asian Pac Assay of Antioxidant Potential of Two Filamentous Fungi Isolated from the Indonesian Fermented Dried Cassava In vitro antioxidant potential of some soil fungi: Screening of functional compounds and their purification from Penicillium citrinum In vitro antioxidant activity and total phenolic content of endophytic fungi isolated from Eugenia jambolana Lam. Asian Pac Antioxidant coumarin and pyrone derivatives from the insect-associated fungus Aspergillus versicolor In vitro antioxidant activities of sulfated derivatives of polysaccharides extracted from Auricularia auricular A novel antioxidant isobenzofuranone derivative from fungus Cephalosporium sp.AL031. Molecules New bioactive fungal molecules with high antioxidant and antimicrobial capacity isolated from Cerrena unicolor idiophasic cultures Evaluation of antioxidant and antibacterial activities of the stems of Flammulina velutipes and Hypsizygus tessellates (white and brown var.) extracted with different solvents Evaluation of Selected Culinary-Medicinal Mushrooms for Antioxidant and ACE Inhibitory Activities Antioxidant Potential of Penicillium expansum and Purification of its Functional Compound Biodelignification of wheat straw and its effect on in vitro digestibility and antioxidant properties Antioxidant and electrochemical properties of cultivated Pleurotus spp. and their sporeless/low sporing mutants Antioxidative, antifungal, cytotoxic and antineurodegenerative activity of selected Trametes species from Serbia Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium Production, characterization and in vitro antioxidant activities of exopolysaccharide from Weissella cibaria GA44 Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88 Diversity, antimicrobial and antioxidant activities of culturable bacterial endophyte communities in Aloe vera Antioxidant and antimicrobial potential of the Bifurcaria bifurcata epiphytic bacteria Phenazoviridin, a novel free radical scavenger from Streptomyces sp. Taxonomy, fermentation, isolation, structure elucidation and biological properties Modulatory Effect of the Intracellular Content of Lactobacillus casei CRL 431 Against the Aflatoxin B 1 -Induced Oxidative Stress in Rats Physicochemical characteristics and in vitro and in vivo antioxidant activity of a cell-bound exopolysaccharide produced by Lactobacillus fermentum S1 Immunomodulatory and antioxidant potential of Lactobacillus exopolysaccharides Evaluation of the Antioxidant Capacities of Antarctic Macroalgae and Their Use for Nanoparticles Production Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity. Mar. Drugs Oxygen radical absorbance capacity (ORAC) of cyclodextrin-solubilized flavonoids, resveratrol and astaxanthin as measured with the ORAC-EPR method Antioxidant activity of three microalgae Dunaliella salina, Tetraselmis chuii and Isochrysis galbana clone Tahiti Evaluation of several microalgal extracts as bioactive metabolites as potential pharmaceutical compounds Antioxidant Potential of Extracts Obtained from Macro-(Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) and Micro-Algae (Chlorella vulgaris and Spirulina platensis) Assisted by Evaluation of antioxidant and antibacterial capacity of green microalgae Scenedesmus subspicatus The green microalga Tetraselmis suecica reduces oxidative stress and induces repairing mechanisms in human cells In vitro and ex vivo free radical scavenging activities of carazostatin, carbazomycin b and their derivatives Isolation and structural elucidation of an antioxidative agent, naphterpin Antioxidant activity of newly discovered lineage of marine actinobacteria. Asian Pac Gold nanoparticles in nanomedicine: Preparations, imaging, diagnostics, therapies and toxicity The promising future of nano-antioxidant therapy against environmental pollutants induced-toxicities Plant-mediated green synthesis of iron nanoparticles Synthesis, properties, and applications of iron nanoparticles Synthesis of Fe nanoparticles@ graphene composites for environmental applications Plant-mediated synthesis and applications of iron nanoparticles Flower-Based Green Synthesis of Metallic Nanoparticles: Applications beyond Fragrance Current Challenges toward In Vitro Cellular Validation of Inorganic Nanoparticles What are the emerging concepts and challenges in NANO Nanoarchitectonics, hand-operating nanotechnology and mechanobiology Silver-Assisted Synthesis of Gold Nanorods: The Relation between Silver Additive and Iodide Impurities Specific ion effects: Why DLVO theory fails for biology and colloid systems Toxic potential of materials at the nanolevel Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles Anisotropic Platinum Nanoparticle-Induced Cytotoxicity, Apoptosis, Inflammatory Response, and Transcriptomic and Molecular Pathways in Human Acute Monocytic Leukemia Cells Resveratrol Inhibits Oxidative Stress and Prevents Mitochondrial Damage Induced by Zinc Oxide Nanoparticles in Zebrafish (Danio rerio) The Toxic Effects and Mechanisms of Nano-Cu on the Spleen of Rats Nanotoxicity: An Interplay of Oxidative Stress Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Mechanisms of nanoparticle-induced oxidative stress and toxicity Redox-active nanomaterials for nanomedicine applications Cerium and yttrium oxide nanoparticles are neuroprotective Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles Lynch, I. Mechanisms of Silver Nanoparticle Release, Transformation and Toxicity: A Critical Review of Current Knowledge and Recommendations for Future Studies and Applications Cytotoxicity and genotoxicity of silver nanoparticles in human cells Bio-distribution and in vivo antioxidant effects of cerium oxide nanoparticles in mice Cerium oxide nanoparticles, combining antioxidant and UV shielding properties, prevent UV-induced cell damage and mutagenesis Microbe-Mediated Biosynthesis of Nanoparticles: Applications and Future Prospects. Biomolecules 2021 Lactobacillus species mediated synthesis of silver nanoparticles and their antibacterial activity against opportunistic pathogens in vitro Synthesis and study of silver nanoparticles for antibacterial activity against Escherichia coli and Staphylococcus aureus Silver nanoparticles as a new generation of antimicrobials Antimicrobial activity of metals: Mechanisms, molecular targets and applications Silver nanoparticles: Partial oxidation and antibacterial activities A review of the scientific evidence for biofilms in wounds Multi-species biofilms: How to avoid unfriendly neighbors Biofilms: Survival mechanisms of clinically relevant microorganisms Mechanisms of biofilm resistance to antimicrobial agents Mechanisms of Antibiotic Resistance in Important Gram-Positive and Gram-Negative Pathogens and Novel Antibiotic Solutions Biofilms facilitate cheating and social exploitation of β-lactam resistance in Escherichia coli Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis Effect of Silver Nanoparticles on Biofilm Formation and EPS Production of Multidrug-Resistant Klebsiella pneumoniae Novel Biogenic Silver Nanoparticle-Induced Reactive Oxygen Species Inhibit the Biofilm Formation and Virulence Activities of Methicillin-Resistant Staphylococcus aureus The Role of Metallic Nanoparticles in Inhibition of Mycobacterium Tuberculosis and Enhances Phagosome Maturation into the Infected Macrophage Surface engineering of biomaterials in orthopedic and dental implants: Strategies to improve osteointegration, bacteriostatic and bactericidal activities Bacterial adhesion on orthopedic implants Review of titanium surface modification techniques and coatings for antibacterial applications Functions and applications of metallic and metallic oxide nanoparticles in orthopedic implants and scaffolds Nabiev, I. Dependence of Nanoparticle Toxicity on Their Physical and Chemical Properties Natural Metallic Nanoparticles for Application in Nano-Oncology Bio-synthesized iron oxide nanoparticles for cancer treatment Magnetotactic bacteria for cancer therapy Green Synthesis of Highly Dispersed Ni/SiO2 Catalysts Using Natural Biomass of Sesbania Powder Green and Chemical Syntheses of CdO NPs: A Comparative Study for Yield Attributes, Biological Characteristics, and Toxicity Concerns Silver nanoparticle synthesis using Clerodendrum phlomidis leaf extract and preliminary investigation of its antioxidant and anticancer activities Generation of reactive oxygen species induced by gold nanoparticles under X-ray and UV Irradiations Cytotoxicity and ROS production of manufactured silver nanoparticles of different sizes in hepatoma and leukemia cells Silver nanoparticles: Electron transfer, reactive oxygen species, oxidative stress, beneficial and toxicological effects. Mini review Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells Antibacterial and antioxidant potential of biosynthesized copper nanoparticles mediated through Cissus arnotiana plant extract Amaranthus spinosus leaf extract mediated FeO nanoparticles: Physicochemical traits, photocatalytic and antioxidant activity Synthesis, structural and spectroscopic characterizations of maghemite-Fe 2 O 3 prepared by one-step coprecipitation route Ionic strength and composition a_ect the mobility of surface-modified Fe0 nanoparticles in water-saturated sand columns Polyphenol/FeIII complex coated membranes having multifunctional properties prepared by a one-step fast assembly Development of receptor targeted magnetic iron oxide nanoparticles for effcient drug delivery and tumor imaging Synthesis of Metallic Nanoparticles by Plant Extracts Biological synthesis of metal nanoparticles Biofabrication of discrete spherical gold nanoparticles using the metal-reducing bacterium Shewanella oneidensis Shape control in gold nanoparticle synthesis Antioxidant, antibacterial and cytotoxic potential of silver nanoparticles synthesized using terpenes rich extract of Lantana camara L. leaves Silver Nanoparticles Mediated by Costus afer Leaf Extract: Synthesis, Antibacterial, Antioxidant and Electrochemical Properties. Molecules Bio-fabrication of silver nanoparticles using the leaf extract of an ancient herbal medicine, dandelion (Taraxacum o_cinale), evaluation of their antioxidant, anticancer potential, and antimicrobial activity against phytopathogens Biogenic synthesis of silver nanoparticles using S1 genotype of Morus alba leaf extract: Characterization, antimicrobial and antioxidant potential assessment Green synthesis of silver nanoparticlesusing Thymus kotschyanus extract and evaluation of their antioxidant, antibacterial and cytotoxic effects Antioxidant and antibacterial activity of silver nanoparticles synthesized by Cestrum nocturnum A study of the bactericidal, anti-biofouling, cytotoxic and antioxidant properties of actinobacterially synthesised silver nanoparticles Biosynthesis, characterization, antibacterial and antioxidant activity of silver nanoparticles produced by lichens Antimicrobial, antioxidant, and anticancer activities of biosynthesized silver nanoparticles using marine algae Ecklonia cava Biogenesis of silver nanoparticles using endophytic fungus Pestalotiopsis microspora and evaluation of their antioxidant and anticancer activities Biogenic synthesis of gold nanoparticles by marine endophytic fungus-Cladosporium cladosporioides isolated from seaweed and evaluation of their antioxidant and antimicrobial properties Endophytic fungal isolate mediated biosynthesis of silver nanoparticles and their free radical scavenging activity and anti microbial studies Trichoderma based synthesis of anti-pathogenic silver nanoparticles and their characterization, antioxidant and cytotoxicity properties Mycosynthesis of silver nanoparticles using extract of endophytic fungi, Penicillium species of Glycosmis mauritiana, and its antioxidant, antimicrobial, anti-inflammatory and tyrokinase inhibitory activity Mycosynthesis: Antibacterial, antioxidant and antiproliferative activities of silver nanoparticles synthesized from Inonotus obliquus (Chaga mushroom) extract Endophyte fungi, Cladosporium species-mediated synthesis of silver nanoparticles possessing in vitro antioxidant, anti-diabetic and anti-Alzheimer activity Photocatalytic, antioxidant, antibacterial and anti-inflammatory activity of silver nanoparticles synthesised using forest and edible mushroom Synthesis and characterization of Reishi mushroom-mediated green synthesis of silver nanoparticles for the biochemical applications Biosynthesis of silver nanoparticles using Ganoderma Lucidum and assessment of antioxidant and antibacterial activity Biosynthesis of silver nanoparticles by Streptomyces griseorubens isolated from soil and their antioxidant activity In Vitro and In Vivo Evaluation of Silver Nanoparticles Phytosynthesized Using Raphanus sativus L. Waste Extracts Biogenic synthesis of antioxidant, shape selective gold nanomaterials mediated by high altitude lichens Green synthesis of gold nanoparticles by the marine microalga Tetraselmis suecica Antibacterial and antioxidant activity of protein capped silver and gold nanoparticles synthesized with Escherichia coli The extra cellular synthesis of gold and silver nanoparticles and their free radical scavenging and antibacterial properties Screening of different algae for green synthesis of gold nanoparticles Eco-friendly synthesis of gold nanoparticles (AuNPs) using Inonotus obliquus and their antibacterial, antioxidant and cytotoxic activities Intracellularsynthesis of goldnanoparticles with antioxidantactivity by probiotic Lactobacillus kimchicus DCY51T isolated from Koreankimchi Enterococcus species for the one-pot biofabrication of gold nanoparticles: Characterization and nanobiotechnological applications A novel extracellular synthesis of monodisperse gold nanoparticles using marine alga, Sargassum wightii Greville Synthesis of gold nanoparticles by blue-green algae Spirulina platensis Green synthesis of gold nanoparticles from Vitex negundo leaf extract: Characterization and in vitro evaluation of antioxidant-antibacterial activity Biosynthesis of ZnO nanoparticles by a new Pichia kudriavzevii yeast strain and evaluation of their antimicrobial and antioxidant activities Phyto-mediated synthesis of zinc oxide nanoparticles of Berberis aristata: Characterization, antioxidant activity and antibacterial activity with special reference to urinary tract pathogens Biomedical potential of actinobacterially synthesized selenium nanoparticles with special reference to anti-biofilm, anti-oxidant, wound healing, cytotoxic and anti-viral activities Biosynthesis of selenium nanoparticles by Pantoea agglomerans and their antioxidant activity The Antimicrobial, Antioxidant, and Anticancer Activity of Greenly Synthesized Selenium and Zinc Composite Nanoparticles Using Ephedra aphylla Extract Antidiabetic and antioxidant properties of copper nanoparticles synthesized by medicinal plant Dioscorea bulbifera Green synthesized copper oxide nanoparticles ameliorate defence and antioxidant enzymes in Lens culinaris Antioxidant and catalytic activity of biosynthesized CuO nanoparticles using extract of Galeopsidis herba Green and Traditional Synthesis of Copper Oxide Nanoparticles-Comparative Study Biogenic synthesis of iron nanoparticles using Amaranthus dubius leaves extract as reducing agents Structural characterization, antioxidant and cytotoxic e_ects of iron nanoparticles synthesized using Phytogenic generation of NiO nanoparticles using Stevia leaf extract and evaluation of their in-vitro antioxidant and antimicrobial properties Synthesis of Nickel Oxide Nanoparticles from Berberis balochistanica Stem for Investigating Bioactivities. Molecules Green synthesis of Manganese nanoparticles using the aqueous extract of Ctenolepis garcini Green Synthesis of MnO Nanoparticles Using Abutilon indicum Leaf Extract for Biological, Photocatalytic, and Adsorption Activities An Eco-Friendly Approach to the Control of Pathogenic Microbes and Anopheles stephensi Malarial Vector Using Magnesium Oxide Nanoparticles (Mg-NPs) Fabricated by Penicillium chrysogenum Antioxidant Activity of Metal Nanoparticles Coated with Tocopherol-Like Residues-The Importance of Studies in Homo-and Heterogeneous Systems Enhanced radical scavenging activity by antioxidantfunctionalized gold nanoparticles: A novel inspiration for development of new artificial antioxidants. Free Radic Antioxidant-potentiality of gold-chitosan nanocomposites Glutathione promoted expeditious green synthesis of silver nanoparticles in water using microwaves Linking an α-tocopherol derivative to cobalt(0) nanomagnets: Magnetically responsive antioxidants with superior radical trapping activity and reduced cytotoxicity Electrooxidation of nitrite based on green synthesis of gold nanoparticles using Hibiscus sabdariffa leaves Insights into gold nanoparticles as a mucoadhesive system Antioxidant Potential and Antibacterial Efficiency of Caffeic Acid-Functionalized ZnO Nanoparticles Antioxidant activity of nanomaterials Enhanced Antioxidant Activity under Biomimetic Settings of Ascorbic Acid Included in Halloysite Nanotubes A synergic nanoantioxidant based on covalently modified halloysite-trolox nanotubes with intra-lumen loaded quercetin Characterization, antioxidant and antimicrobial activity of silver nanoparticles synthesized using marine endophytic fungus-Cladosporium cladosporioides Chlorogenic Acid Entrapped in Hybrid Materials with High PEG Content: A Strategy to Obtain Antioxidant Functionalized Biomaterials? Materials Bioaccessibility and antioxidant activity of curcumin after encapsulated by nano and Pickering emulsion based on chitosan-tripolyphosphate nanoparticles Nanoparticles with dual responses to oxidative stress and reduced ph for drug release and anti-inflammatory applications Core-Crosslinked Polymeric Micelles: Principles, Preparation, Biomedical Applications and Clinical Translation Block copolymer micelles as long-circulating drug vehicles Recent advances in polymeric micelles for anti-cancer drug delivery Engineering precision nanoparticles for drug delivery Double hydrophilic block copolymers self-assemblies in biomedical applications Funding: This research received no external funding. Informed Consent Statement: Not applicable. The authors declare no conflict of interest.