Evaluating the antioxidant capacity of natural products: A review on chemical and cellular-based assays

Abstract

Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different chemical methods have been developed including the popular ORAC that evaluates the potential of a sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening, they are low cost, high-throughput and yield an index value (expressed as equivalents of Trolox) that allows comparing and ordering different products. More recently, nanoparticles-based assays have been developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal models and human studies are more appropriate but also more expensive and time-consuming, making the cell culture assays very attractive as intermediate testing methods. Cellular antioxidant activity (CAA) assays, activation of redox transcription factors, inhibition of oxidases or activation of antioxidant enzymes are reviewed and compared with the classical in vitro chemical-based assays for evaluation of antioxidant capacity of natural products.

Introduction

During the last decades it has been proposed that oxidative stress, defined as the unbalance between reactive oxygen and nitrogen species (ROS/RNS) production and the antioxidant defense, plays a pivotal role in different pathophysiological conditions [1], [2]. Oxidative stress, originated from an increase in ROS/RNS production or from a decrease in the antioxidant network, is characterized by the inability of endogenous antioxidants to counteract the oxidative damage on biological targets [3]. In this context, it has been suggested that an intake of a rich antioxidant diet is inversely associated with the risk to develop some pathologies like cardiovascular diseases [4], [5], [6]. Thus, attention has been paid on the antioxidant capacity of natural products, with particular interest on those that are frequently (or potentially) consumed by people. Different in vitro chemical-based assays have been developed to determine the antioxidant capacity of natural products, including the popular ORAC, DPPH scavenging method, ferric reducing capacity, etc. and more recently the use of nanoprobes to evaluate the metal-reducing capacity of antioxidants [7], [8], [9], [10], [11], [12], [13], [14]. These assays are based on different strategies and endow different information about the ROS/RNS-sample interaction. However, to study the potential antioxidant health-protecting effects of natural products, considering the complexity involved in their in vivo mechanisms of action, a single in vitro chemical method is not enough to evaluate and compare their antioxidant properties. In addition, these chemical assays do not consider relevant parameters involved in biological environments such as lipophilicity and bioavailability; therefore, the obtained antioxidant capacity indexes not necessary reflect the antioxidant effects that would be associated with a particular sample in vivo. The ability of natural products to induce an antioxidant response at the cellular level also has to be evaluated. A good antioxidant is not just a good radical scavenger and reducing compound but a molecule that can exert its antioxidant activity by activating transcription factors that induce the expression of antioxidant enzymes, thus, ameliorating oxidative stress [15]. To evaluate a new product as antioxidant, as a compound able to change the redox cellular state, we must use a cellular antioxidant assay since participation of different components of the cell are critical to finally develop an antioxidant response.

In the present work we review the chemical-based methodologies employed for screening antioxidant capacity of natural products discussing the classical and non-classical mechanisms of action of antioxidant-containing natural products. In particular, we highlight the advantages and drawbacks of each method; introduce the importance of pursuing the evaluation of antioxidant capacity at the cellular level and compare reported results between chemical- and cellular-based assays.

The classical definition of antioxidant as “any substance that delays, prevents or removes oxidative damage to a target molecule” [2] is usually understood as the ability of these compounds to neutralize free radicals, acting for example, as chain-breaking derivatives in agreement with Reaction (1).

$$\text{AH}+\text{FR}\to \text{A}+\text{FRH}$$

where AH and FR represent an antioxidant and a free radical, respectively.

Thus, Reaction (1) is considered the base of the classical mechanism of action of antioxidants and explains their ability to inhibit (or delay) many damaging processes induced by FR on lipids, proteins, or DNA. However, Reaction (1) does not represent all the factors affecting the antioxidant activity of a compound or an antioxidant-containing mixture. Among these factors, the most relevant are: the reactivity of antioxidants toward FR, the number of FR molecules neutralized by each antioxidant molecule (stoichiometric factor, n), the liposolubility of the antioxidant, and the presence of secondary reactions.

Taking into account Reaction (1) as a bimolecular process, the rate of the reaction, r, expressed as:

$$r=k_1\left[\text{AH}\right]{\left[\text{FR}\right]}_{\text{ss}}$$

depends on the kinetic rate constant k₁, the antioxidant concentration [AH], and the steady state concentration of FR ([FR]_ss). Accordingly, from a kinetic point of view, a compound with a high k₁ value would be a good antioxidant. Nonetheless, for antioxidants expected to act in biological environments (for example human blood plasma) the latter is relevant only if their reactions with FR are faster or comparable with the rate of endogenous antioxidants (for example human serum albumin or uric acid) reacting with FR. In that case, the stoichiometry of Reaction (1), defined as the number of FR molecules that each antioxidant is able to neutralize (n), could also be a relevant factor in a food or beverage preservative.

The capacity of a particular antioxidant to reach the place where FR are being generated is also a critical aspect. In this context, antioxidants with high in vitro antioxidant capacity are not necessarily efficient neutralizers of FR in compartmentalized systems. This is a pivotal point when trying to inhibit lipid peroxidation processes in cell membranes, where antioxidants should have an appropriate liposolubility to be incorporated into the membrane and to react with FR through chain-breaking reactions [16]. For instance, α-tocopherol is liposoluble and is the most important biological antioxidant in membranes, but its solubility in water is very low. One particular case is ascorbic acid, which in spite of its well-known hydrophilic character is able to work additively with membrane-immersed α-tocopherol to prevent lipid peroxidation, by reducing the surface-exposed α-tocopheroxyl radical back to α-tocopherol [17].

Other aspect to consider in the antioxidant behavior of a particular sample is the ability of secondary free radicals (A) generated in Reaction (1) to damage biological targets (BTH in Reaction (2)).

$$\text{A}+\text{BTH}\to \text{AH}+\text{BT}$$

It has been observed that secondary phenoxyl radicals can, in some conditions, trigger oxidative modifications on proteins, DNA, as well as on cell membrane lipids [18]. Furthermore, depending on the antioxidant compound and/or the place where A is generated, it can react with O₂ to form a secondary peroxyl radical or superoxide anion (Reaction (3) and Reaction (4), respectively). In the latter case, dismutation of superoxide (usually catalyzed by superoxide dismutase, SOD) yields hydrogen peroxide (H₂O₂), which in the presence of metals generates hydroxyl radical (Fenton mechanism), a strongly oxidant free radical.

$$\text{A}+{\text{O}}_2\to \text{AOO}$$

$$\text{A}+{\text{O}}_2\to {\text{A}}_{\text{ox}}+{\text{O}}_2^{-}$$

In 1985 Helmut Sies introduced the concept of oxidative stress as “a disturbance in the prooxidant-antioxidant balance in favor of the former” [3]. Experimental evidence supported the idea that oxidative stress contributes to the development of several pathologies including cardiovascular disease, neurodegenerative diseases, cancer and also aging. ROS such as superoxide, singlet oxygen, H₂O₂ and hydroxyl radical were mainly considered responsible for these damaging oxidative reactions. More recently, new radical species were identified, now centered on nitrogen, thus termed reactive nitrogen species RNS, derived from the biologically produced radical, nitric oxide (NO). The concept of ROS/RNS as just biologically damaging species has begun to change. Enzyme systems produce reactive species not only for chemical defense or detoxification, but also for cell signaling and biosynthetic reactions. The presence of both, toxic and beneficial effects of ROS/RNS precludes a simple definition of oxidative stress. At the same time, the intervention with antioxidant supplements did not show the results expected [19], [20], [21], in essence because most pathologies are multifactorial and oxidative stress is just one of many contributing factors. A new concept of oxidative stress was emerging, not limited to free radical damage of the macromolecular machinery but to perturbation of cellular redox status. Based on new accumulating data on redox signaling pathways, antioxidant intervention trials and oxidative stress markers, Dean Jones in 2006 re-defined oxidative stress as “a disruption in redox signaling and control” [15]. It is clear today that the mechanism of action of antioxidants is more complex than just intercepting reactive free radicals [15], [22]. Free radicals can be deleterious to life depending on the type of radical produced and the level and site of production, but at the same time, low concentrations of these reactive species are essential to perform normal physiological functions like gene expression, cellular growth and defense against infection. The redox cellular network is finely regulated and its perturbation provokes oxidative stress. The idea behind antioxidant supplementation is to restore redox cellular status. Some studies have indicated that antioxidants could also have deleterious effects on human health depending on dosage and bioavailability [19], [20]. It is therefore necessary to explore the mechanism of action of a potential antioxidant at the cellular level in order to extrapolate its therapeutic potential (Fig. 1).

Evidence has accumulated showing that endogenous oxidants like H₂O₂ can act as second messengers and trigger a cascade of intracellular responses resulting in the expression of antioxidant and detoxifying enzymes in order to control the cellular redox status [23]. Much research is still needed in this area but some redox signaling pathways have already been identified. Two transcription factors associated with redox control are: Nrf-2 (nuclear factor E2-related protein 2) and NF-κB (nuclear factor kappa B) with defined redox control compartimentalized in cytosol and nucleus.

Nrf-2 is a redox-sensitive transcription factor that is activated by an oxidative signal in the cytoplasm that causes its translocation to the nucleus where it binds to DNA ARE-regions (antioxidant response elements) inducing the expression of cytoprotective enzymes like glutathione S-transferase GST, superoxide dismutase SOD, heme oxigenase-1 HO-1, NADPH-quinone oxidase NQO (ARE-regulated genes) [24]. In the cytoplasm, Nrf-2 is associated with Keap1 (Kelch-like ECH associated protein) that facilitates its ubiquitination and degradation, thus, keeping down the levels of Nrf-2. Activation of Nrf-2 by ARE-inducers provokes dissociation of Nrf-2/Keap1 complex, less ubiquitination and the corresponding accumulation of Nrf-2 that reach the nucleus leading to increase transcription of genes under control of ARE. Not only Keap1 has critical cysteine residues that are modified under oxidative stress, but also Nrf-2 needs a reduced cysteine to bind to DNA.

Compounds that increase the expression of Nrf-2 and/or facilitate the dissociation of Nrf-2/Keap1 and translocate Nrf-2 to the nucleus, provoke an antioxidant response since ARE-genes are induced, i.e. the expression of antioxidant enzymes increased.

The NF-κB family consists of a group of inducible transcription factors which regulate immune and inflammatory responses and protect cells from undergoing apoptosis in response to cellular stress (including oxidative stress). NF-κB is kept inactive in the cytoplasm by association with inhibitors IκB proteins. In response to inflammatory stimulus, such as oxidants, IκB proteins are rapidly degraded by the proteasome liberating NF-κB protein to the nucleus where it binds to specific DNA sequences, activating the expression of specific pro-inflammatory and anti-apoptotic genes [25].

Compounds that decrease the expression of NF-κB and/or inhibit its activation prevent its translocation to the nucleus and the induction of pro-inflammatory/pro-oxidant genes.