Antioxidants: Classification, Natural Sources, Activity/Capacity Measurements, and Usefulness for the Synthesis of Nanoparticles

1Recently, 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 diseases caused by oxidative stress have been published. Therefore, antioxidants have become co-adjuvants utilized in conventional therapies with the aim of combating oxidative stress. Many natural antioxidants have been shown to have strong antiviral effects. The efficacy of flavonoids, i.e., (+)—catechin, luteolin, apigenin, quercetin, and quercetin 7-rhamnoside, has been proven in coronavirus infections (Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV). In the absence of effective therapies for the treatment of diseases caused by coronaviruses, antioxidants may prove to be an effective alternative to fight the SARS- and MERS-CoV pandemic. The site of action of antioxidants is the oxidative stress pathway, which plays a key role in coronavirus-induced pathogenesis. Diniz et al. reviewed different effects of natural antioxidants against coronavirus covering reduction nucleocapsid (N) protein expression, inhibition 3C-like protease (3CLpro) enzyme responsible for replication of SARS-CoV (quercetin and its derivatives), papain-like protease (PLpro) (isobavachalcone and psoralidin), and helicase protein by affected ATPase activity (myricetin and scutellarein). A recently published review demonstrated the usefulness of antioxidants in the treatment of neurological disorders caused by COVID-19. However, reports that do not confirm the effectiveness of antioxidants in vivo cannot be ignored. The activity of antioxidants is mainly limited by ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicology)processes related to poor absorption caused by restrictions in the penetration of cell membranes and degradation that occurs in the stomach and intestines. It has also been reported that low molecular weight antioxidants lose their ability to scavenge free radicals inside cells. This is especially true for the scavenging of the hydroxyl radical (OH•), superoxide (O2•−), and H2O2.

2The sources of natural antioxidants are mainly plants, i.e., edible vegetables, fruits, spices, and herbs, which are rich in vitamins, phenolic compounds, carotenoids, and microelements. However, it should be emphasized that the antioxidant activity is different for different varieties and morphological parts of natural resources. In addition, the activity of natural products is influenced by many other factors, such as climatic and soil conditions or harvest time. They hinder the standardization of natural products to a large extent. Due to the fact that natural antioxidants have the ability to inhibit the processes of oxidation and the growth of microorganisms, including many pathogenic ones, e.g., Salmonella spp. and Escherichia coli, they are more and more often used as preservatives in food products or as packaging ingredients for food. In recent years, a large body of evidence has been published that natural antioxidants increase the stability of edible oils, the stability of carotenoid dyes, and the aroma of fruit juices and that they work well as additives in meat products and even in bakery products, successfully replacing artificial preservatives and stabilizers. It should be emphasized that the choice of bioactive compounds for the food industry is significantly limited due to the obvious taste requirements and the need for approval by EFSA (European Food Safety Authority) or FDA (American Food and Drug Administration). Currently, we can observe an interesting trend in the strategy of using by-products of the processing industry. This is related not only to environmental protection or economic reasons but to the fact that they have a significant content of bioactive substances, exceeding that in the flesh of fruit, such as polyphenols in apple and olive pomace, lycopene in tomato pomace, phenolic compounds from the group of flavonoids (anthocyanins, catechins), and phenolic acids and stilbenes in grape skins or citrus fruits.

3In recent years, natural extracts have become attractive, also due to the rapid development of nanotechnology. As a source of substances with reducing potential, they have replaced the toxic reagents used in chemical synthesis and ushered in the era of the so-called biogenic synthesis and nanobiotechnology.

4Numerous spectroscopic, biochemical, and electrochemical assays are used to test antioxidant abilities, which are still modified so that they can effectively assess the potential of antioxidants, taking into account the variability of their mechanisms of action. They are usually based on a free radical scavenging reaction or the prevention of their formation by the addition of an antioxidant. Various techniques used for this purpose differ in terms of repeatability and costs associated with the necessity of using specialized equipment. Valuable reviews, published in recent years, describe problems related to (i) the mechanisms responsible for the antioxidant activity; (ii) the antioxidant activity of natural extracts prepared from various plant species, microorganisms, and food ingredients; and (iii) the preventive role of antioxidants in various diseases such as diabetes, human gut diseases, and cancer, as well as the use of natural extracts for the synthesis of nanoparticles.

5This review gathers together issues related to antioxidants (classification, natural sources, measurement of antioxidant activity) as well as their application in nanotechnology. Within the review, two main issues can be distinguished:

6The wide range of industrial and biomedical applications of antioxidants requires effective and rapid in vitro tests to evaluate total antioxidant activity. Various methods were collected in the review, i.e., chromatographic, spectrometric, and electrochemical. Particular attention was paid to the method based on metallic nanoparticles, which are used as optical probes (SNPAC). The method is useful for measuring the antioxidant activity of both simple chemical compounds and mixtures of natural origin. The SNAPC tests are effective in assessing electron transfer but are not used very often. The review includes information on the extracts from plants, lichens, fungi, algae, and actinomycetes (reference antioxidants, extraction process, antioxidant activity tests, and activity parameters).

7Natural extracts as a source of both reducing and stabilizing substances are used for the green synthesis of nanoparticles. The review includes examples of the synthesis of metallic/metal oxides of nanoparticles using extracts from various plant species and microorganisms (yeast, algae, cyanobacteria, fungi, and bacteria). The information collected allows us to trace the links between the type of antioxidant, its origin, activity, and suitability for the efficient synthesis of nanoparticles. Extensive data were collected on the methods of extract preparation, antioxidant activity tests, detection methods, NPs synthesis conditions, and the morphology of the obtained nanoparticles.

8This review highlights recent trends in antioxidant research, measurement of antioxidant activity, biogenic nanoparticle synthesis, and nano-drug delivery systems.

9Free radicals can be defined as highly reactive species that contain an unpaired electron in the valence shell. They can donate this electron but also accept it from other molecules, acting as an oxidant or reducing agent. In the human body, reactive forms (RS) come from metabolic processes involved in the respiratory chain, phagocytosis, prostaglandin synthesis, and the cytochrome P-450 system.

10The most reactive species found in biological systems include the hydroxyl radical (OH●), which is formed by attaching three electrons to an oxygen molecule, e.g., as a result of the Fenton reaction, and the superoxide radical (O2●−), which is formed mainly in mitochondria, as a byproduct of electron transport in the respiratory chain. Other reactive forms of oxygen (ROS), nitrogen (RNS), and chlorine occurring as free radicals and nonradicals that as oxidizing agents can be easily converted into radicals are listed in Table 1.

11ROS/RNS generated in oxygen metabolism are necessary in the regulation of gene expression, cell proliferation, apoptosis, the processes of protein phosphorylation or calcium concentration in cells, activation of proteins controlling cell division, and elimination of microorganisms. Free radicals are also generated under the influence of external sources, such as exposure to X-rays, ozone, smoking, air pollution, and industrial chemicals. There is a balance in the cell between RS production and its neutralization by defense systems. Under physiological conditions, this balance is slightly shifted in favor of pro-oxidative conditions, providing continuous, mild oxidative stress.

12Each disturbance of this particular balance may lead to the development of oxidative stress, i.e., a state in which the oxidizing potential increases to a level that threatens the stability of cellular structures. Under oxidative stress, biologically important macromolecules such as DNA, proteins, carbohydrates, and lipids are damaged. The excess of free radicals changes their structure and thus the physiological functioning of the cell by disrupting redox signaling and the accumulation of cytotoxic compounds, such as malonyl dialdehyde or 4-hydroxynonenal.

13There is evidence that free radicals can accumulate throughout the body with age, initiating the aging process, as well as various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, muscular dystrophy, and atherosclerosis. An imbalance between ROS and the antioxidant defense system has also been recognized in the induction of diabetes and age-related eye disease. Currently, it is believed that oxidative stress has a significant negative impact also on inflammatory diseases, cancer, ischemic diseases, immunodeficiency syndrome, hypertension, alcoholism, smoking-related diseases, and many others. Oxidative stress was first described and defined by Sies in 1991.

14The reasons for the occurrence of oxidative stress may be (i) an increase in the rate of ROS production, (ii) deficiencies of low-molecular-weight antioxidants, and (iii) inactivation of enzymes with antioxidant activity. Increased and/or prolonged state of oxidative stress may cause serious damage to the cell and even lead to its death. Therefore, the current discussions focus on the role of free radicals in the pathogenesis of many diseases and the usefulness of antioxidants in their potential therapy.

15Antioxidants are produced by the protective system of various organisms in order to respond to the destructive effects of free radicals. Antioxidants are able to reduce the damage caused by ROS/RNS and even chlorine. The action of the protective system may limit the negative effects of free radicals by preventing the formation of reactive radicals or by interrupting free radical reactions.

16Antioxidants 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. 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 and the understanding of the mechanism of lipid peroxidation prevention by vitamin E. 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.

17Low-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, 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.

18Many 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.

19A 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, N-acetylcysteine, or dihydropyridine calcium antagonists. However, the concentrations used in the therapy do not provide antioxidant activity in vivo.

20The source of antioxidants and other bioactive compounds are also microorganisms, including actinomycetes, bacteria, cyanobacteria, fungi, and lichens. 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.

21Antioxidants 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.

22Antioxidants 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 NH2 and the position of these functional groups in the order ortho > para > meta, from the highest to the lowest active. 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. Primary antioxidants actively inhibit oxidation reactions by scavenging ROS/RNS, while secondary antioxidants act indirectly through chelation of transition metal (iron) ions 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. 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.

23Currently, 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. The high in vitro chemical reactivity of the antioxidant is therefore not evidence of its effectiveness in vivo. Moreover, as shown by individual studies, 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. Reports that the use of antioxidants not only prevent cancer but may also provoke it are also alarming. 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%. Vitamin C supplementation is particularly controversial. Linus Pauling recommended health-promoting use of a high daily dose of 1000 mg. 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.

24In the review by Hrelia and Angeloni, 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.

25The 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.

26Currently, 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.

27Determination of antioxidant status attracts growing attention for clinical purposes. 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 non-enzymatic 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. 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, 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.

28There is also some misunderstanding regarding specific terms that are used to describe antioxidants measurement assays. Bunaciu et al., in a critical review, 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.

29In 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. The collaborative effect of all sample components (i.e., synergistic or antagonistic effects) is responsible for “total antioxidant capacity” (TAC) measured.

30Antioxidants’ 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.

31The 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.

32Nonenzymatic primary antioxidant assays can be non-competitive or competitive. 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) (Figure 2).

33The 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).

34In certain circumstances, ET/HAT mechanisms may not be easily identified like for 2,2′-azinobis-(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):

35Experimental investigations 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):

36It 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. 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.

37Antioxidant activity can also be estimated using nanoparticle-based assays utilizing nanoparticles probes exhibiting localized surface Plasmon resonance (LSPR) absorption. 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 AgNO3 reduction with polyphenolic antioxidants.

38Many studies are dedicated to the estimation of the antioxidant power of various individual chemicals, as well as food samples and natural extracts. 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. 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.

39A number of techniques are used to assess antioxidant capacities, such as UV-Vis spectroscopy, fluorescence spectroscopy, chemiluminescence, electron paramagnetic resonance (EPR), enzyme-catalyzed assays, and cell culture assays. Moreover, there are some electrochemical techniques, including controlled potential techniques, electrochemical sensors, and biosensors, which are commonly applied. However, 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. These assays despite some drawbacks are easy to use.

40To 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. Due to the relocation of the unpaired electron, DPPH forms a stable radical cation and does not form dimers in alcohol solutions. 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).

41The 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 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. The reaction of DPPH with antioxidants was adapted for illustration and measuring the kinetics of radical quenching. Since the beginning of the 1960s, the method, as well as antioxidant activity calculations, have evolved into numerous modifications.

42DPPH 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, 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 steady-state, 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. 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, 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.

43Considering 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. collected IC50 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 IC50 value of ascorbic acid was in the range from 11.85 to 629 µM. Unfortunately, such a large variation in IC50 values was also observed for remaining antioxidants. Recently, Xie and Schaich have reevaluated the DPPH assay considering the solvent kind and pH values.

44The DPPH free radical scavenging activity is commonly expressed in terms of the percentage of inhibition of the free radical by examined antioxidants. The EC50 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:

45However, to find the most credible EC50 value, an assay should be done using several antioxidant concentrations located near the estimated ED50 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 EC50 value is usually located in a short linear range, and it may be calculated by the use of the right-angled triangle. 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 EC50 value calculation:

46It should be noted that sigmoid curves based on the Hill equation are easier to interpret. 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. conducted a comparative study of several specialized computer programs based on various regression models towards the aim of EC50 estimation. The EC50 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 EC50. 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 EC50 values, while the second gives the inflection point as the EC50.

47Antiradical power (ARP) is another parameter that can be used to define antioxidant activity. This parameter is defined as a reciprocal of EC50, which is why the higher value of EC50 is related to smaller antiradical power:

48The 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.

49In the original DPPH assay, provided by batch experiments, several automation approaches based on flow injection analysis (FIA) and sequential injection analysis (SIA) have been proposed in recent decades. An interesting approach inspired by HPLC-FIA has been elaborated on by Koleva. In this method, the HPLC-separated 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. 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. 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.. 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.

50The 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. developed HPLC-ESI-MS and NMR for estimation of antioxidant capacity of polyphenolic acids in the plant extract. In turn, Nuengchamnong et al. 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. 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.

51Traditional thin-layer chromatography with post chromatographic derivatization using DPPH solution for free radical scavenging activity evaluation, discovered by Glavind and Holmer in 1967, exists nowadays in the modern version owing to video scanning technology.

52Electrochemical 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.

53The 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. 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.. Martinez et al., and Zielińska and Zieliński 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. 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.

54Determination of the antioxidant activity by biamperometric measurements is based on a high degree of reversibility redox couple potential, including Fe3+/Fe2+, I2/I−, Br2/Br−, VO3−/VO2− Fe(CN)63−/Fe(CN)64−, 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. 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.

55More 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).

56For the first time, Scampicchio et al. 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.

57A few years later, Özyürek et al. 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).

58Until 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 surface-adsorbed species, the refractive index of the dispersion medium, and interparticle interactions. 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). 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.

59Since nanoparticles-based assay is a new analytical tool, calibration is usually performed using control antioxidants, 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, Folin-Ciocalteu, FRAP, and DPPH as reference tests.

60Antioxidant capacity determination by nanoparticles-based method also involves other metallic or metal oxide NPs. Gatselou et al., in 2016, 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.

61Recently, 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. In 2018, Ozdemir Olgun 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. Currently, portable nanoparticle-based tests for rapid detection of food antioxidants (NanoCerac) are being developed, e.g., for nanoparticles of immobilized cerium oxide, or nanoparticles of metal oxides TiO2, Fe2O3, ZrO2, ZnO, and SiO2, which are immobilized on cellulose. Several reviews regarding TAC determination by using NPs can be found in the literature.

62Epidemiological 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. Natural products containing at least 0.1% of antioxidants can be accepted as dietary supplements with antioxidant properties.

63As 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.

64The 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). It has been shown that different extraction methods lead to different extraction yields on the same plant material. For example, Lisitsyn et al. studied the plant extracts (rosemary, black pepper, thyme, and sage) obtained by the use of supercritical CO2 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.

65Currently, 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. 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. using Chenopodium quinoa Willd.

66Plant-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. 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).

67Almost all eubacteria possess the ability to produce a variety of extracellular metabolites with significant antioxidant activity, such as thiazostatins A, phenazoviridin, (Z)-1-((1-hydroxypenta-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. 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). 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 α- and 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. 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.

68Cyanobacteria 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. 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-6,60-bieckol, and triphlorethol-A,. 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. In 2019, the Special Issue of Antioxidants focused on recent investigations concerning marine algal antioxidants and specific antioxidant networks functioning in algae.

69Lichens produce various extracellular, secondary metabolites that can be used as potential sources of natural antioxidants. At the beginning of the 21st century, the significant free radical scavenging activity of Cetraria islandica aqueous extracts and Usnea ghattensis methanolic extracts of Platismatia glauca, Parmelia saxatilis, Ramalina pollinaria, Umbilicaria nylanderiana, and Ramalina polymorpha were described. Fernández-Morianoet et al. 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. Some of them also exhibited beneficial antimicrobial and anticancer activities.

70Actinomycetes also produce chemically diverse and pharmaceutically useful compounds with antifungal, antibacterial, diabetogenic, antiviral, immunosuppressive, antiparasitic, antitumor, insecticidal, antioxidant, anti-inflammatory, enzyme inhibitory, and others. Actinomycetes originating from different habitats usually manifest very different antioxidant activity. 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.

71Other examples of antioxidant capacity assessment of the different extracts from plants, lichens, fungi, algae, and actinomycetes are collected in Table 3.

72The 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. 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). The iron nanoparticles (INPs), due to high catalytic activity, low toxicity, high magnetism, and microwave absorption ability, 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).

73Unfortunately, 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. 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. 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. 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. 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.

74Presently, one can observe an increasing interest in studies on the reactivity of nanoparticles compared to macroscopic objects and their cytotoxicity accumulation in the body, which can generate reactive oxygen species (ROS). 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 or can prevent the increase of ROS by mimicking the activity of the oxidative enzymes, catalase, or superoxide dismutase. It has been proven that silver nanoparticles (AgNPs) inhibit cell proliferation and modulate the activity of antioxidant enzymes. Hirst et al. confirmed by an in vivo test on mice the effectiveness of cerium oxide nanoparticles (CONPs) in treating oxidative stress. 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. The authors highlighted the CONPs regenerative redox cycle influencing the stability of the antioxidants molecules.

75The first experiments on AgNPs biosynthesis using bacteria were carried out in the culture of Pseudomonas stutzeri AG 259, Morganella sp. Bacillus subtilis. 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. 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.

76The 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. 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. It is particularly interesting that even a short incubation with nanoparticles leads to the accumulation of chaperones and the S6 protein 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. 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. The most dangerous are biofilms composed of pathogenic bacteria E. faecalis, S. aureus, Staphylococcus epidermidis, E. coli, Klebsiella pneumoniae, and P. aeruginosa, which are mostly responsible for hospital infections that are difficult to cure and are characterized by increased resistance to therapies. 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. 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. The inhibition of biofilms formed by Multidrug-Resistant Klebsiella pneumoniae, Methicillin-Resistant Staphylococcus aureus, and Mycobacterium tuberculosis has been proven. Unfortunately, the aggregation of nanoparticles inhibits their effective activity. Consequently, various stabilizers 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.. Metallic/metal oxide nanoparticles, i.e., silver, gold, magnesium, titanium, zinc, aluminum, tantalum, and zirconium have been tested in orthopedics. 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.

77Interesting 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. 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. 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.

78Plant extracts contain diverse compounds, which can be utilized as potent reducing agents, stabilizing agents, and precursor molecules for NPs formation. 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. The antioxidant activity of NPs frequently depends on their size as well as shape.

79So 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. 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. Iron oxide (magnetite Fe3O4, magemite Fe2O3) NPs of certain sizes have superparamagnetic properties; therefore, they are useful as contrast agents and drug carriers.

80The 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. The reports on a plant-mediated approach to synthesize NPs by the use of different extracts are collected in Table 4.

81Increasing 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. In 2020, a review on antioxidant functionalized NPs was published. 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, gold nanoparticles coated with chitosan, silver NPs with glutathione, or more complex ones like graphite layered 30 nm cobalt nanomagnets with attached tocopherol derivatives. Konopko et al. and Nie et al. 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. 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 Au3+ 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. In 2017, Choi et al. 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, for example, ascorbic acid loaded into the inner lumen of natural halloysite nanotubes or halloysite externally deco-rated with tocopherol-like moieties and containing quercetin inside the nanotube. Many studies have described functionalized silver and gold nanoparticles derived from fungal or bacterial extracts obtained from species Ganoderma lucidium, Aspergillus versicolor, Cladosporium cladosporioides, Pestalotiopsis microspore, and bacteria Lactobacillus kimchicus.

82Another promising trend of nanobiotechnology represents the development of nano-drug 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 SiO2 and polyethylene glycol has been identified as a system able to control the overproduction of RNS/ROS. 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. 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; unfortunately, it is beyond the scope of the current study.

83Interesting 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..

84Considering 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.

85On 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.

86A 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.