Abstract: Background: β-carotene and naringenin are two plant derived molecules which posses antioxidant property. Naringenin is the predominant flavanone found in grape fruit. β-carotene is a natural fat-soluble pigment found principally in plants. Beta-carotene is converted by the body into vitamin A and acts a powerful antioxidant and helps support the immune system. Objective: Present studies were performed to compare the free radical scavenging property of β-carotene and naringenin with respect to vitamin C and N-acetyl cysteine which are the synthetic compounds and posses the potent antioxidant property. Materials and Methods: Various biochemical assay like DPPH, hydroxyl radical, nitric oxide and superoxide scavenging activity test were carried out by considering different concentration of the compounds. Results: It was found that β-carotene and naringenin showed a remarkable capacity to scavenge all the tested reactive species in a dose dependent manner and all the IC50 values (Mean±SEM) for three individual experiments being found at the μg mL-1 level (66±2.34-84.99±2.84 μg mL-1) which was comparable to synthetic compound vitamin C and N-acetyl cysteine. vitamin C and N-acetyl cysteine was a well established synthetic antioxidant used in different inflammatory disorder however, the prohibitive synthesis cost of these drugs coupled with the need for long-term therapy results in a high frequency of side-effects, making it imperative for better and safer drugs to be made available. So we are hopeful that naturally occurring antioxidant can also be a replacement for treatment of free radical mediated inflammatory diseases.
INTRODUCTION
Free radicals is defined as any atom or molecule possessing unpaired electrons and are highly reactive to destroy biomolecules like protein, lipid and fats by oxidation where the antioxidants are agents which scavenge the free radicals or inhibit the formation of prooxidant enzymes mediated free radicals or prevent the damage caused by free radicals (Naskar et al., 2011). Free radicals plays a pivotal role for the etiology of various diseases like cancer, diabetes, autoimmune disorders, etc (Beckman and Ames, 1998). Different therapeutic approaches using antioxidant from both natural and synthetic origin has been proposed for use in the treatment of various human diseases (Kundu et al., 2011). However, it has been suggested that these compounds have shown toxic effects like liver damage and mutagenesis. Different antioxidant plants secondary metabolites like flavonoids and some other phenolic compounds have been already reported therefore, it’s the time to find the natural safe antioxidant molecules that are gaining a great importance in the field of free radical biology (Naskar et al., 2011). One of such antioxidant molecules is naringenin which is a flavanone and considered to have a safe bioactive effect on human health showing antioxidant, anti-inflammatory and immunomodulatory activity. Additionally the predominant effect of this grapefruit flavanone to reduce oxidative damage to DNA has also been established (Edwards and Bernier, 1996). β-carotene, a known as carotenoids which are the principal pigments responsible for the red, orange, yellow and green colors of vegetables and fruits. Similarly beta-carotene is a natural fat-soluble plant pigment which is converted by the body into the powerful antioxidant vitamin A antioxidant and helps support to the immune system (Bjelakovic et al., 2007). N-acetylcysteine (NAC) an acetylated derivative of the amino acid L-cysteine has been commonly used as a chronic respiratory mucolytic agent. Subsequently, it is proved that NAC is an antidote for hepatotoxicity due to acetaminophen overdose.
During time NAC is becoming a powerful antioxidant and potential therapeutic agent in the treatment of oxidative inflammatory diseases (Borgstrom et al., 1986). Vitamin C, chemically known as L-ascorbic acid or L-ascorbate is an essential nutrient for living organisms having antioxidant property and thus protect the body against oxidative stress (Padayatty et al., 2003).
The aim of the present study was to compare the antioxidant potential as well as free radical scavenging activity of the plant derived compounds like β-carotene and naringenin with the synthetic compound like NAC and vitamin C towards the different reactive oxygen and nitrogen species.
MATERIALS AND METHODS
Chemicals: Vitamin C was obtained from Merck Mumbai, N- acetyl cysteine from SRL Mumbai and β-carotene and Naringenin was obtained from Sigma, USA. All the others reagents were used of high analytical grade.
DPPH radical scavenging activity: DPPH radical scavenging activity was measured using the method of Cotelle et al. (1996) with some modifications. Three milliliter of reaction mixture containing 0.2 mL of DPPH (100 μM in methanol) and 2.8 mL of test solution, at various concentrations of the compounds was incubated at 37°C for 30 min and absorbance of the resulting solution was measured at 517 nm spectrophotometer. The percentage inhibition of DPPH radical was calculated by comparing the results of the test with those of the control (not incubated with compounds) using the following formula:
where, C is Absorbance of control and T is Absorbance of test.
Nitric oxide: Sodium nitroprusside is commonly used as a laboratory reagent to generate Nitric Oxide (NO). Being a unstable molecules, NO immediately interacts with dissolved oxygen to produce nitrite ions in physiological solution and presence of nitrite can be measured by the Griess reaction (Bala et al., 2009). One milliliter of 10 mM sodium nitroprusside was mixed with 1 mL of test solution of different concentrations in phosphate buffer (pH 7.4) and the mixture was incubated at 25°C for 150 min. From the incubated mixture, 1 mL was taken out and 1 mL of Griess’ reagent (1% sulphanilamide, 2% o-phosphoric acid and 0.1% naphthyl ethylene diamine dihydrochloride) was added to it. Absorbance of the chromophore formed by the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthyl ethylene diamine dihydrochloride was read at 546 nm and percentage inhibition was calculated by comparing the results of the test with those of the control using the formula mentioned in the DPPH radical scavenging assay.
Hydroxyl radical scavenging activity: The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell (1992). Stock solutions of EDTA (1 mM), FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM) and deoxyribose (10 mM), were prepared in distilled deionized water. The assay was performed by adding 0.1 mL EDTA, 0.01 mL of FeCl3, 0.1 mL H2O2, 0.36 mL of deoxyribose, 1.0 mL of the extract (of different concentration) dissolved in distilled water, 0.33 mL of phosphate buffer (50 mM, pH 7.4) and 0.1 mL of ascorbic acid in sequence. The mixture was then incubated at 37°C for 1 h. A 1.0 mL portion of the incubated mixture was mixed with 1.0 mL of 10% TCA and 1.0 mL of 0.5% TBA (in 0.025 M NaOH containing 0.025% BHA) to develop the pink chromogen measured at 532 nm. The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxyribose degradation and is calculated as mention above in the DPPH radical scavenging assay (Bala et al., 2009).
Superoxide radical scavenging activity: Superoxide anion scavenging activity was measured according to the reported method with some modifications (Robak and Gryglewski, 1998). All the solutions were prepared in 100 mM phosphate buffer (pH 7.4). One milliliter of nitroblue tetrazolium (NBT, 156 μM), 1 mL of reduced nicotinamide adenine dinucleotide (NADH, 468 μM) and 3 mL of test solution were mixed. The reaction was initiated by adding 100 μL of phenazine methosulphate (PMS, 60 μM). The reaction mixture was incubated at 25°C for 5 min, followed by measurement of absorbance at 560 nm. The percentage inhibition was calculated by using same formula mentioned in the DPPH radical scavenging assay (Bala et al., 2009).
Data analysis: All the data were shown as average±SEM IC50 value was determined to be the effective concentration at which free radicals were scavenged by 50%. The IC50 value was obtained by interpolation from linear regression using Graph Pad Prism software, version 4.03 (GraphPad Software Inc, San Diego, CA).
RESULTS
The compounds showed a remarkable capacity to scavenge all the tested reactive species in a dose dependent manner and all the IC50 values (Mean±SEM) for three individual experiments being found at the μg mL-1 level.
| Fig. 1: | 1,1-diphenyl-2-picrylhydrazil (DPPH) scavenging activity
of β-carotene, naringenin, NAC and vitamin C. Each point represents
the values obtained from three experiments, performed in triplicate (Mean±SEM) |
|
| Fig. 2: | Nitric oxide radical scavenging activity of β-carotene,
naringenin, NAC and vitamin C. Each point represents the values obtained
from three experiments, performed in triplicate (Mean±SEM) |
|
The more toxic RNS present in our body is NO which is significantly scavenged by the β-carotene and naringenin comparable with synthetic antioxidant vitamin C and NAC (Fig. 1). To check the H+ donating capability of the compounds we used DPPH assay where a concentration dependent proton donating capability was observed (Fig. 2). The plant derived compounds β-carotene and naringenin showed good scavenging activity against all the Reactive Oxygen Species (ROS) like hydroxyl and superoxide radicals tested biochemically.
| Fig. 3: | Hydroxyl radical scavenging activities of β-carotene,
naringenin, NAC and vitamin C. Each point represents the values obtained
from three experiments, performed in triplicate (Mean±SEM) |
|
| Fig. 4: | Superoxide radical scavenging assay of β-carotene,
naringenin, NAC and vitamin C. Each point represents the values obtained
from three experiments, performed in triplicate (Mean±SEM) |
|
The assay showed the inhibitory ability of compounds on hydroxyl radical in a concentration dependent manner (Fig. 3).
| Table 1: | IC50 values of the compounds in different
biochemical in vitro scavenging experiments |
|
| Values are Mean±SEM | ||
The another toxic ROS, superoxide was generated by the chemical reaction between phenazine methosulphate and nicotinamide adenine dinucleotide and corresponding scavenging activity was tested spectrophotometrically using nitroblue tetrazolium salt (Fig. 4) where a concentration dependent superoxide scavenging property was observed in case of β-carotene and naringenin which is comparable to synthetic antioxidant vitamin C and NAC. All the results were finally expressed as concentration vs., %inhibition and 50% inhibitory concentrations were calculated accordingly (Table 1).
DISCUSSION
Oxidative stress refers to a situation where in the production of oxidants exceeds the capacity to neutralize them, leading to damage to cell membranes, lipids, nucleic acids, proteins and constituents of the extracellular matrix such as proteoglycans and collagens. Different therapeutic approaches can be used to decrease the oxidative stress and include scavenging of free radicals, inhibition of free radical producing enzymes, enhancing the antioxidant system or by targeting the signaling routes and expression of molecules involved in the inflammatory cascade (Karmakar et al., 2011). Although, such approach can easily be available due to synthetic drugs which protect against oxidative damage but still safety is an important aspect now a day. Therefore, an alternative solution to solve this problem is the abundant area of research that mainly focused the readily available non toxic natural antioxidants from food supplement.
DPPH can easily accept an electron or hydrogen radical to become a stable molecule. Then the free electrons become paired off and the color of DPPH is lost accordingly with the number of electrons taken up (KunduSen et al., 2010). This principle has been widely used to test the ability of the compound or extracts to act as free radical scavengers. Reduction of the DPPH radicals can be observed by the decrease in absorbance at 517 nm. Nitric oxide is recognized to be an inter and intra cellular mediator of several cell functions. It acts as a signal molecule in immune, nervous and vascular systems. Being a unstable molecule, nitric oxide immediately converted into nitrite and react with superoxide radical to form the highly reactive and toxic peroxynitrite anion (ONOO-) (Kundu et al., 2011). Two targeted plant derived molecules beta carotene and naringenin inhibits nitrite formation by directly competing with oxygen in the physiological solution and pH and the capacity is further comparable with synthetic antioxidant used in this comparative study. Thus, the present study proved that the compounds have good nitric oxide scavenging activity (Bala et al., 2011).
During oxygen utilization by the cell, toxic superoxide radical is formed by the action of NADPH oxidase enzyme thus start the numerous biological and physicochemical reactions. Flavonoids are effective antioxidant mainly because they scavenge superoxide anions which is the initiator of toxic free radical mediated biochemical reaction in the body. The results suggest that beta carotene and naringenin concentration dependently increase the% inhibition (Ischiropoulos et al., 1995). Superoxide radicals initially dismutase to hydrogen peroxide which further generate toxic hydroxyl radical by Fenton reaction thus causing tremendous lipid peroxidation and enormous biological damage (Kundu et al., 2011). Ferric-EDTA was incubated with H2O2 and ascorbic acid at pH 7.4 which is produced the hydroxyl radicals detected by their ability to degrade 2-deoxy-2-ribose into Malondialdehyde (MDA) fragments that formed a pink chromogen upon heating with TBA at low pH (Yazdanparast and Ardestani, 2007). When the natural compounds were added to the reaction mixture, it removed hydroxyl radicals from the sugar and prevented their degradation.
ACKNOWLEDGMENTS
Financial assistance was provided by All India Council of Technical Education (AICTE) Government of India and Department of Pharmaceutical Technology, Jadavpur University Kolkata India is highly acknowledged.