Free radicals and non-reactive radicals of oxygen species.
Free radicals or reactive oxygen species (ROS) generated from various sources in the environment as well as from cellular processes in the body are of serious health challenges. Overwhelming levels of these free radicals disrupt the antioxidant defense system in the body thereby damaging cell membranes and cellular macromolecules such as proteins, lipids and nucleic acids leading to cell death or causing mutations leading to uncontrolled cell division. Once the cellular antioxidant system is disrupted and becomes deficient, oxidative stress emerges thereby promoting several diseases such as diabetes, arthrosclerosis, cancer, cardiovascular diseases, etc. Better management of oxidative stress requires antioxidants from external sources to supplement the body’s antioxidant defense system. Because of their natural origin and therapeutic benefits, plants have been considered as a major source of antioxidants. Certain non-enzymatic plant phytochemicals such as glutathione, polyphenols, bioflavonoids, carotenoids, hydroxycinnamates as well as some vitamins have shown to possess antioxidant properties in vitro and in vivo. These plant phytochemicals are now been used in the prevention and management of oxidative stress-related diseases.
- free radicals
- oxidative stress
- reactive oxygen species (ROS)
Man as a living creature has always indulged himself into several activities to ensure his survival and well-being. In so doing, he has induced the production or release of various reactive substances or free radicals which are either consumed or inhaled. Also, certain physiological processes in the body generate free radicals or proxidants. These free radicals or reactive species, because of their deficiency in electron and instability, attack electron rich centers such as lipid membranes, proteins and nucleic acids thereby damaging cells and tissues in the body. Eventual, the human body is adapted to remove these unstable molecules by a myriad of molecules including certain enzymes collectively known as antioxidants. This antioxidant defense system reduces the level of these free radicals in the body and maintains the homeostatic balance for proper functioning of the body. However, when these reactive species are overwhelming high in the body, it surpasses the capacity of the antioxidant defense system leading to a condition known as oxidative stress. This imbalance between antioxidant and proxidants is characteristic of certain disease conditions such as diabetes, atherosclerosis, cardiovascular diseases, cancer etc. One of the possible remedy for this condition is to supplement the endogenous antioxidant defense system with exogenous antioxidants. Plants have gained considerable interest in recent time in managing oxidative stress related diseases; firstly, because of their ethnopharmacological uses in managing diseases and secondly, due to their richness in phytochemicals which possess antioxidant properties. Hence, this chapter is aimed to give an overview of free radicals, their sources of origin and processes of generation in the environment and body. Also, it will highlight on the various mechanisms of free radical induced cellular damage and the associated diseases due to oxidative stress. The various mechanisms of the antioxidant defense system; both enzymatic and non-enzymatic antioxidants will be described as well as the contribution of plant phytochemicals as antioxidants. Emphasis will be laid on some plants and phytochemicals with antioxidant activities stating their mode of scavenging free radicals and prevention of oxidative stress-related diseases.
2. Free radicals
Free radicals are molecular species with unpaired electrons in their atomic orbital capable of independent existence. As such, these radicals are highly reactive and can either extract an electron from molecules or donate an electron to other molecules thus acting as a reductant or an oxidant. Though free radicals have high reactivity, most of them have a very short half-life of less than 10−6 s in biological systems . Some oxygen species known as reactive oxygen species (ROS) are non-reactive in their natural state but are capable of generating free radicals.
The idea of free radicals began in chemistry around the beginning of the twentieth century, where chemists initially described them as intermediate organic and inorganic compounds with several suggested definitions. A clear understand of these radicals was then proposed based on the work of Daniel Gilbert and Rebecca Gersham in 1954  in which these radicals were suggested to play important roles in biological environments but also responsible for certain deleterious processes in the cell. Thereafter by 1956, Herman Denham further suggested that these reactive species may play critical roles in physiological process particularly aging process . This hypothesis on the theory of free-radical on aging, inspired numerous research and studies which significantly contributed to the understanding of radicals and other related species such as ROS, reactive nitrogen species (RNS) and non-radical reactive species .
2.1. Types of free radicals or reactive oxygen species
ROS are classified into two major categories of compounds which includes the free radicals and the non-reactive radicals. The free radical includes nitric oxide radical (NO•), hydroxyl radical (OH•), superoxide ion radical (O•2), peroxyl (ROO•), alkoxyl radicals (RO•), and one form of singlet oxygen (1O2) as shown in Table 1 . These species are considered as free radicals since they contain at least one unpaired electron in the shells around the atomic nucleus which makes them unstable and therefore can easily donate or obtain another electron to attain stability. As such, they are highly reactive and capable of independent existence [6, 7]. On the other hand, the non-reactive radicals are a group of compounds which are not radicals but are extremely reactive or can easily be converted to reactive species. Examples of these substances include hypochlorous acid (HClO), hydrogen peroxide (H2O2), organic peroxides, aldehydes, ozone (O3), and O2 as shown in Table 1.
|Non-reactive oxygen radical|
2.2. Sources of free radicals
As reviewed from Sultan , free radicals can originate either from the environment, physiological processes or endogenous sources.
2.3. Generation and chemical reactions of free radicals
Free radicals are generated through various physiological processes in living organisms. Once generated, they can react with other biomolecules to attain stability.
At physiological pH, iron is usually oxidized to Fe3+ and chelates to biological molecules. Thus, for Fenton reaction to occur, iron must be converted to its reduced form Fe2+. Superoxide radicals can reduce Fe3+ to Fe2+ ions thereby enabling the Fenton reaction.
net reaction (Haber-Weiss reaction):
Protonated form of peroxynitrite (ONOOH) acts as a powerful oxidizing agent to sulfhydryl (SH) groups thereby causing oxidation of many molecules and proteins leading to cellular damage . It can also cause DNA damage such as breaks, protein oxidation and nitration of aromatic amino acid residues in proteins. Reactive oxygen species and their oxidative stress induced damaged is summarized in Figure 1.
3. ROS induced oxidative damage
Continual influx and generation of ROS from endogenous and exogenous sources lead to oxidative damage of cellular components and may impair many cellular functions . The most vulnerable biological targets to oxidative damage include proteins, enzymes, lipidic membranes and DNA .
Termination by another radical:
Termination by an antioxidant:
4. Oxidative stress and human diseases
When the concentration of ROS exceeds those of antioxidant neutralizing species, a condition known as oxidative stress occurs. As reviewed from Rahman et al. , oxidative stress has been implicated in several diseases including atherosclerosis, cancer, malaria, rheumatoid arthritis, chronic fatigue syndrome, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease . Evidence via monitoring biomarkers such as the presence of ROS and RNS as well as antioxidant defense has indicated oxidative damage may be implicated in the pathogenesis of these diseases . Elevated levels of free radicals such as 4-hydroxy-2,3-nonenal (HNE), acrolein, malondialdehyde (MDA) and F2-isoprostanes have been observed in Alzheimer’s disease [30, 31]. Oxidative stress also contributes to tissue injury following hyperoxia and irradiation. Evidence from studies have shown oxidative stress to play an important role in the pathogenesis and development of metabolic syndrome related disorders such as obesity, hypertension, diabetes, dyslipidemia etc. as well as in cardiovascular related diseases such as myocardial infarction, aortic valve stenosis, angina pectoris, atherosclerosis and heart failure [32, 33, 34, 35]. Cancer is another disease associated with ROS as ROS have been suggested to stimulate oncogenes such as Jun and Fos whose overexpression is directly associated with lung cancer . In lung cancers, p53 can be mutated by ROS thereby losing its function of apoptosis and functioning as an oncogene . Also, the development of gastric cancer has been thought to be due to increase production of ROS and RNS by
5. Defense mechanism against free radicals
In response to the prevailing level of free radicals both from exogenous and endogenous sources, the human body developed a defense mechanism for protection against cellular damages. These may involve direct and indirect mechanisms put in place by the body.
5.1. Indirect defense mechanisms
Firstly, the indirect mechanisms are those mechanisms that do not directly act on the free radicals to eliminate them or convert them to less reactive forms. Rather this indirect system can act in several ways. Certain regulatory mechanisms can control and regulate processes that lead to the endogenous production of ROS . This may be transcriptional control of the enzymes that are involved in the generation of endogenous ROS. Another indirect approach consists of certain molecules and enzymes that are transported to oxidative-damage sites for repair of macromolecules. This may include repair of damage DNA, protein or lipids. For examples damage oxidized adducts of DNA such as 8-hydroxy-2-deoxyguanosine, thiamine glycol, and apurinic can be removed from a nucleotide sequence and replaced by a normal nucleotide base . Also, certain molecules that can donate hydrogen atoms to damaged molecules are also considered as repair compounds. Molecules such as ascorbate or tocopherol can donate hydrogen atom to a fatty acid radical on cell membrane thereby repairing the membrane. Certain natural cellular or surface barriers such as the skin or cell membranes act as indirect defense system against ROS by preventing exogenous ROS from entering the body or preventing certain endogenous ROS from reaching the target macromolecules. Though these indirect defense mechanisms are helpful against ROS, they are usually non-specific and do not act directly on the ROS.
5.2. Direct defense mechanism
This category of defense system which constitutes the antioxidant system is the most important because they directly act on free radicals either by decomposing, scavenging or converting free radicals to less reactive forms. This defense mechanism constitute two groups; the enzymatic and non-enzymatic antioxidants.
5.2.1. Enzymatic antioxidants
The enzymatic antioxidants include superoxide dismutase (SOD), catalase, glutathione reductase (GRx) and glutathione peroxidase (GPx).
The hydrogen peroxide can then be removed by catalase or glutathione peroxidase.
The fact that GPx also acts on lipid hydroperoxides suggest it may be involved in repairing cellular damages due lipid peroxidation . The activity of GPx is dependent on the constant availability of reduced glutathione which is regenerated from oxidized glutathione (GSSG).
The NADPH required by this enzyme to replenish the supply of reduced glutathione is provided by Glucose-6-phosphate dehydrogenase (G-6-PD) in the pentose phosphate pathway. Competing pathway that utilizes NADPH such as the aldose reductase pathway may lead to a deficiency of reduced glutathione thereby limiting the action of glutathione peroxidase.
5.2.2. Non-enzymatic antioxidants
The non-enzymatic antioxidants are usually low-molecular-weight antioxidant (LMWA) compounds capable of preventing oxidative damage either by directly interacting with ROS or indirectly by chelating metals . Transition metals are directly chelated by some of this LMWA thereby preventing them from participating in metal-mediated Haber-Weiss reaction . Other direct acting LMWA molecules scavenge free radicals by donating electrons to free radicals to make them stable thereby preventing attacks of biological targets. These LMWA molecules also called scavengers may be advantageous over enzymatic antioxidants as they can penetrate cellular membranes and be localized in close proximity to the biological target due to their small size. More so, these non-enzymatic antioxidants can interact together to scavenge free radicals and their scavenging activity may be synergic. Most scavengers originate from endogenous sources, such as biosynthetic processes and waste-product generation by the cell. However, the number of LMWA synthesized by the living cell or generated as waste products such as histidine dipeptides, glutathione, uric acid, lipoic acid and bilirubin is limited . More so, the concentration of scavenger must be sufficiently high to compete with the biological target on the deleterious species . As such, exogenous sources of non-enzymatic antioxidants especially from plant diet and phytochemicals are needed to supplement the endogenous non-enzymatic antioxidants. The oxidative stress defense mechanism in humans is summarized in Figure 2.
6. Plants as source of antioxidants
Plants have long been consumed as food which is rich in vitamins and other nutrients that are useful for the body. Also, various plants were used in folk medicine for various therapeutic purposes. Though these uses, the notion of plant as a source of antioxidant became more evident in recent time as oxidative stress was considered a major attribute to most diseases in humans and the antioxidant defense system in human was usually not sufficient to overcome the free radical level in the body. As such, plants have gained considerable interest as a source of antioxidants and so much research has been done to identify plants substances with antioxidant activities.
Like other humans, plants do have enzymatic and non-enzymatic antioxidant defense systems to protect them against free radicals. The enzymatic system includes catalase, SOD, glutathione peroxidase(GPx), and glutathione reductase (GRx) , while non-enzymatic systems consist of low molecular weight antioxidants (LMWA) such as ascorbic acid, proline, glutathione, carotenoids, flavonoids, phenolic acids, etc. and the high molecular weight antioxidants (HMWA) which are mostly secondary metabolites such as tannins . The possible reason for the presence of these antioxidants in plants is that plants lack an immune system unlike animals thus, utilize the antioxidant defense system to protect them against microbial pathogens and animal herbivores. Also, these phytochemicals serve as a defense system against environmental stress.
6.1. Non-enzymatic plant antioxidants and their mode of action
Though plants have enzymatic antioxidants, it is usually difficult to isolate these enzymes for therapeutic uses in humans. Also, they are usually denatured during food processing, preparation and not sufficiently present in diets such as fruits and vegetables. On the contrary, non-enzymatic antioxidants are readily present in plants leaves, fruits and food in sufficient amounts and can easily be extracted from plants. For these reasons, this section will focus on the non-enzymatic plant antioxidants.
GSH generally acts as a cofactor for glutathione peroxidase, thus serving as an indirect antioxidant by donating the necessary electrons for the decomposition of H2O2. GSH can directly scavenge ROS such as ROO•, OH• and RO• radicals as well as •O2 and HCLO•. Upon reacting with ROS, GSH becomes a glutathione radical, which can be reconverted to its reduced form . Glutathione also has other cellular functions such metabolism of ascorbic acid . Also, glutathione prevents the oxidation of SH protein groups and acts as a chelating agent for copper preventing its participation in the Haber-Weiss reaction .
Also, α-tocopherol can scavenge other ROS, such as •O2 to become tocopherolquinone and subsequently tocopherylquinone. However, it is not an efficient scavenger of OH• and alkoxyl (•OR) radicals in vivo . The resultant tocopheroxyl radical in these reactions can be recycled to its active form but this radical is relatively stable in normal circumstances and insufficiently reactive to initiate lipid peroxidation itself, which makes it a good antioxidant .
In general, flavonoids are oxidized by radicals, resulting in a more stable, less-reactive radical. In this reaction, flavonoids stabilize the ROS by reacting with them to become a flavonoids radical. This is achieved due to high reactive hydroxyl group of the flavonoids as shown below.
where R• is a free radical and O• is an oxygen free radical.
As reviewed from Nijveldt et al. , certain flavonoids can directly scavenge superoxides as well as peroxynitrite. Other flavonoids may act as antioxidants by inhibiting the activity of free radical generating enzymes such as xanthine oxidase and nitric-oxide synthase. Quercetin, rutin and silibin have shown to inhibit xanthine oxidase activity while silibin has been reported to inhibit nitric` oxide dose dependently. By scavenging radicals, flavonoids can inhibit LDL oxidation in vitro. This action protects the LDL particles and, theoretically, flavonoids may have preventive action against atherosclerosis.
6.2. Plants with antioxidant properties
Several plants are known to possess antioxidant properties due to the presence of certain phytochemicals that have been shown to exhibit antioxidant activities in
|Plant||In vitro antioxidant||In vivo antioxidant activity||Protective against Damage in vivo||Ref|
|Oxidative stress diseases||Plant||Phytochemical||Mechanism of action||References|
|Flavonoids||Vasodilating effects and protected vasodilator reactivity|||
|Stilbenoids||Antioxidant and anti-inflammation activities|||
|Polyphenols||Crocin, carotenoid||protected oxidative stress-induced apoptosis of platelets|||
|Anti-obesity||Anthocyanins, proanthocyanidins||Limits adipogenesis and inflammatory pathways in vitro|||
|Grape products||Polyphenol||Antioxidant action, blocking proinflammatory cytokines|||
|Diabetes||Phenolics||Antioxidant activity and anti-diabetic effect|||
|Polyphenolics||Strong antioxidant action and reduction of glycemia in rats|||
|------------------||Curcumin||Anti-inflammatory and anti-oxidant activities|||
|Polyphenol||Butein||Inhibit formation of nitric oxide in vitro and protecting pancreatic β-cells against cytokine-induced toxicity|||
|Cancer||Polyphenols||Ellagitannins and epicatechin||Anticarcinogenic properties|||
|Green tea, grape seeds||Polyphenols, proanthocyanidins||Protect the skin from the adverse effects of UV radiation preventing risk of skin cancers|||
|Aging||Methanol extract||High antioxidant activities and potential ability as an anti-aging agent|||
|Crude extract||Extended lifespan of healthy rats by reducing the damage of liver and kidney and improving age-associated inflammation and oxidative stress through inhibiting NF-β signaling|||
|Alzheimer’s disease||Crude extract||Potential neuroprotective activity for preventing oxidative-related disorders in vitro|||
|Ethyl acetate extract||Antioxidant activity as well as potential acetylcholinesterase inhibitory property|||
|Curcumin||Reduced levels of oxidative stress and attenuated increased acetylcholinesterase in mice|||
Obvious deleterious effects of free radicals as regards man’s heath cannot be over emphasized. Oxidative stress due to overwhelming levels of free radicals has promoted the progression of diseases such as diabetes, cancer, cardiovascular diseases, atherosclerosis etc. and even aging. Plants phytochemicals and some vitamins have shown to possess antioxidant properties capable of scavenging free radicals, preventing cellular damages and related diseases via several mechanisms. As such, plants phytochemicals are now being considered as the most sustainable alternative source of antioxidants to supplement the endogenous oxidative stress defense system in humans. Continuous efforts are needed to characterize plants phytochemicals for their antioxidant potentials and mode of action for various therapeutic uses against oxidative stress-related diseases while regular consumption of fruits and vegetables are encouraged for the prevention of these diseases.