The Role Of Free Radicals And Polyphenols In Oxidative Stress And Cardiovascular Diseases

The Free Radical Theory and Reactive Oxygen Species

The detection of free radicals (chemical entities possessing highly reactive unpaired electrons) in biological systems gave rise to the free radical theory, which was supported by the discovery of superoxide dismutase (SOD) and by pioneering concepts such as oxidative stress and antioxidants (COMMONER, TOWNSEND and PAKE, 954). All these findings have placed aerobic metabolism and reactive oxygen species (ROS) as the most accepted cause of aging and numerous inflammatory diseases. ROS comprise both free radical and non-free radical oxygen intermediates (peroxides), like superoxide radicals (O2•-), hydrogen peroxide (H2O2), hydroxyl radicals (OH•), and singlet oxygen (O2) (Pizzino et al., 207). These molecules are generated by plasma membrane proteins, such as the growing family of NADPH oxidases, by lipid metabolism within the peroxisomes(Hu and Ren, 206), and by the activity of various cytosolic enzymes such as cyclooxygenases (Martínez-Revelles et al., 203). Although all these sources contribute to the overall oxidative burden, the vast majority of cellular ROS (approximately 90%) come from mitochondria due to oxidative phosphorylation

Oxidative stress is described as an imbalance between oxidants and antioxidants that favors the oxidants, resulting in redox processes being disrupted and/or molecular damage. explains the redox idea, which is involved in essential processes in living cells and is referred to as “redox signaling” and “redox control” (Sies, 205). Increased amounts of ROS, on the other hand, may cause more widespread and irreversible cell damage by oxidation of DNA, RNA, carbohydrates, proteins, and lipids, ultimately leading to cell death via apoptosis or necrosis (Benhar, 2020). Oxidative stress can be photooxidative, nitrosative, or reductive at the molecular level. Finally, many scales ranging from normal oxidative stress to excessive and hazardous oxidative load might be evaluated.

ROS are created in excess in pathological circumstances and when exposed to harsh environmental stressors (Srivastava and Kumar, 205). Ion channel opening, lipid peroxidation, protein changes, and DNA oxidation are generally involved in these pathogenic outcomes. Inflammation is caused by pro-inflammatory chemicals produced by ROS in stressful settings, which plays a vital role in aging and the development of a range of illnesses. Vascular diseases (heart disease, hypertension, metabolic syndrome, atherosclerosis), autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease), neurodegenerative diseases (Alzheimer’s, Parkinson’s, age-related macular degeneration), and respiratory diseases (asthma, chronic obstructive pulmonary disease, acute lung injury, cystic fibrosis) are among them (Paola Rosanna and Salvatore, 202).

The biggest cause of mortality globally is cardiovascular disease, which includes coronary artery disease, hypertensive heart disease, and stroke (Kim, Yun and Kwon, 206). Overproduction of oxidative stress-related factors like ROS has been linked to myocardial infarction, atherosclerosis, and diabetes in recent research (Ray, Huang and Tsuji, 202).

ROS participate in cell signaling as mediators and regulators of vascular function. ROS include free radicals such as superoxide anion (O2–), lipid radicals (ROO–), hydroxyl radical (HO–), nitric oxide (NO) and not free radicals such as hydrogen peroxide (H2O2), hypochlorous acid (HClO) and peroxynitrite (ONOO–), that have oxidizing effects and contribute to oxidative stress (Ray, Huang and Tsuji, 202).

All vascular layers, including endothelium, smooth muscle, and adventitia, generate reactive oxygen species (ROS) (Ray, Huang and Tsuji, 202). The regulation of vascular tone, modulation of inflammation, and stimulation or inhibition of vascular growth, platelet aggregation, and coagulation are all important functions of endothelium (Ray, Huang and Tsuji, 202).  Endothelial dysfunction and other cardiovascular disease disorders are caused by a balance between ROS (oxidants) and antioxidants that is induced by pathophysiological situations. Because of their direct oxidizing effects on macromolecules including proteins, lipids, and DNA, ROS have been linked to cell damage, necrosis, and apoptosis.

Oxidative Stress and Cellular Damage

Increased oxidative stress on cellular structures and alterations in molecular pathways underlying the pathophysiology of cardiovascular illnesses are caused by abnormal free radical generation (Kim, Yun and Kwon, 206).

Polyphenols are non-nutrient natural products of plants, also known as plant secondary metabolites, which may be found in the fruits, vegetables, and seeds we eat every day. Polyphenols are a vast class of secondary metabolism-derived chemicals found throughout the plant world. The phenylpropanoid pathway produces the majority of them from l-phenylalanine. Polyphenols are defined by the presence of at least two phenolic groups linked together in more or less complicated structures, usually of high molecular weight, although simple phenolics, which might be polyphenol precursors, are also included in this category.

The word “polyphenol” is used to identify substances entirely produced from the shikimate/phenylpropanoid and/or polyketide pathways, comprising more than one phenolic unit and void of nitrogen-based functionalities,” according to the most generally used definition (Ray, Huang and Tsuji, 202). Polyphenols are made up of phenolic acids, coumarins, flavonoids, stilbenes, and lignans, among others. Tannins and lignins, among other polymerized forms, are also included. The scent, color, and antioxidant capabilities of the fruits, vegetables, seeds, and nuts we eat are all due to them. Polyphenols are becoming increasingly essential, especially because of their health benefits. Their importance as natural antioxidants in the prevention and treatment of cancer, inflammatory, cardiovascular, and neurological illnesses is growing (Ray, Huang and Tsuji, 202).

Synthetic antioxidants have been employed instead of natural antioxidants because they have better stability and performance, are less expensive, and are more widely available . Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tert-butyl hydroquinone are the most often used synthetic antioxidants in the food business (TBHQ). Also often used in fruits and vegetables are 2-naphthol (2NL), 4-phenylphenol (OPP), and 2,4-dichlorophenoxyacetic acid (2,4-DA). Despite the widespread usage of synthetic antioxidants, concerns about their safety have arisen over time.

Several studies have found a link between long-term consumption of synthetic antioxidants and a variety of health concerns, including skin allergies, gastrointestinal disorders, and, in some circumstances, an increased risk of cancer. Synthetic antioxidants in high amounts can damage DNA and cause premature aging. In animal studies, BHA and BHT have already been linked to liver toxicity and cancer. Furthermore, nothing is known about the presence and fate of these chemicals in the environment. Natural antioxidants are increasingly being used to replace synthetic antioxidants (Lourenço, Moldo-Martins, and Alves, 209).

The need for alternative plant antioxidants has necessitated the need to identify samples with high amount of polyphenols and antioxidant activity. The study embarked on the evaluation of the antioxidant action and quantity in extracts from juices and rinds of lemon and orange samples.

All assays were performed in 3ml cuvettes in phosphate buffer (pH 8.8) containing 75µM NADH and 50µM NBT with 10µl of test agents. Reaction was started by the addition of 10µM PMS and the absorbance recorded with time till an endpoint was reached at 560nm. The experiment was performed with four replicates with an appropriate control agent.

Pathological Conditions and the Role of ROS in Inflammation

To find the quantity of polyphenols in the samples, a Gallic acid calibration curve is prepared by running sequentially diluted Gallic acid from 0-500mg/L at 765 nm. SAamples were analysed by adding 100uL of Folin-Ciocalteau reagent and diluting the resulting solution in various ratios and running at 765 nm.

To assess the super oxide scavenging, the percentage inhibition of NBT reduction was performed by running the assay in the presence of control and in the presence of Lemon 1 juice and skin. The percentage inhibition was presented as a bar graph plot depicting a higher superoxide scavenging from lemon skin compared to lemon juice (Figure 1).

Figure 1. Superoxide scavenging of Lemon 1 juice and skin.

Assays were prepared in 3ml cuvettes in phosphate buffer (pH 8.8) containing 75µM NADH and 50µM NBT with 10µl of test agents. Reaction was started by the addition of 10µM PMS and the extent of NBT reduction was measured after 4 minutes at 560nm. Results expressed as mean (+/-SEM) % inhibition of NBT reduction as compared to control.

To affirm results from the first lemon sample used, the percentage inhibition of NBT reduction was performed by running the assay in the presence of control and in the presence of Lemon 2 juice and skin. The percentage inhibition was presented as a bar graph plot depicting a higher superoxide scavenging from lemon skin compared to lemon juice (Figure 2). 

Figure 2. Superoxide scavenging of Lemon 2 juice and skin.

Assays were prepared in 3ml cuvettes in phosphate buffer (pH 8.8) containing 75µM NADH and 50µM NBT with 10µl of test agents. Reaction was started by the addition of 10µM PMS and the extent of NBT reduction was measured after 4 minutes at 560nm. Results expressed as mean (+/-SEM) % inhibition of NBT reduction as compared to control.

To quantify the super oxide scavenging, the percentage inhibition of NBT reduction was performed by running the assay in the presence of control and in the presence of Orange 1 juice and skin. The percentage inhibition was presented as a bar graph plot depicting a higher superoxide scavenging from orange skin compared to orange juice (Figure 3).

Figure 3. Superoxide scavenging of Orange 1 juice and skin.

Assays were prepared in 3ml cuvettes in phosphate buffer (pH 8.8) containing 75µM NADH and 50µM NBT with 10µl of test agents. Reaction was started by the addition of 10µM PMS and the extent of NBT reduction was measured after 4 minutes at 560nm. Results expressed as mean (+/-SEM) % inhibition of NBT reduction as compared to control.

To affirm results from the first orange sample used, the percentage inhibition of NBT reduction was performed by running the assay in the presence of control and in the presence of Orange 2 juice and skin. The percentage inhibition was presented as a bar graph plot depicting a higher superoxide scavenging from orange skin compared to orange juice (Figure 4). 

Figure 4. Superoxide scavenging of Orange 2 juice and skin.

Cardiovascular Diseases and Abnormal Free Radical Generation

Assays were prepared in 3ml cuvettes in phosphate buffer (pH 8.8) containing 75µM NADH and 50µM NBT with 10µl of test agents. Reaction was started by the addition of 10µM PMS and the extent of NBT reduction was measured after 4 minutes at 560nm. Results expressed as mean (+/-SEM) % inhibition of NBT reduction as compared to control. 

To quantify the total polyphenols in the samples, a calibration curve was prepared by using diluted standards of Gallic acid. Figure 5 shows a plot of a calibration curve with a good correlation (R²-0.9948). The equation of the line was used to calculate the concentration of the samples from their absorbance values.

Figure 5. Calibration curve of Gallic acid concentration expressed in mg/L Gallic acid equivalent.

Assay prepared by diluting 500mg/L standard and the addition of 1.58 ml of water to a standard or test agent. On the addition of 100uL of Folin-Ciocalteau reagent and the incubation at room temperature for 30 minutes. The absorbance was recorded at 765nm against 0mg/L Gallic acid blank.

To quantify the total polyphenols in the samples, the equation of line from the ca;libration curve was used by substituting for the values of y in the equation (Table 1).

Table 1: Total polyphenol concentration of test samples. Determined using the Folin-Ciolateu assay, expressed as mg/L gallic acid equivalents (mg/L /GAE)

To determine how the super oxide scavenging compares in all the samples, a one-way ANOVA test was conducted with a Tukey Pairwise Comparison at (p<0.05). It was noted that juices from lemon and orange samples had no significant differences. They contained close to the same activity. This was also found to be true for samples from Lemon and Orange skins except for one sample. (Figure 6) 

Figure 6. Tukey Pairwise Comparisons. Grouping Information Using the Tukey Method and 95% Confidence of the samples after a one-way ANOVA (N=4).

As postulated by (Hano and Tungmunnithum, 2020), citrus fruits possess a substantial amount of antioxidants. It was also noted that the activity and presence of antioxidants was more concentrated in the rinds(skin) compared to the juices from the fruits: lemon rinds 3280.875mg/L and lemon juice 1094.625mg/L, Orange skin 50% activity while that of orange juice is 9% activity.

In results from an experiment conducted by (Forrester et al., 2018), extracts from rinds were found to be more potent in their activity compared to the juice extracts. The concentration of polyphenols was also recorded to be significantly higher (p<0.05) in lemon and orange rinds when compared to their respective juices.

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