Background and Objective: The intense frying of oils causes an oxidizing thermal degradation with the formation of peroxides in high levels that are harmful to human health. Antioxidants have possibly been used to preserve fats and oil without degradation inhibiting oxidation deterioration. The purpose of this study is to evaluate the potent antioxidant stability of oil on repeated heating to frying temperature which helps us to choose the oil best for cooking. Methods: The anti-oxidative properties of heated and unheated sunflower and rice bran oil are examined by using different in-vitro antioxidant assays such as 2-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 1, 1'-diphenyl-2-picryl-hydrazyl free radical (DPPH) scavenging, peroxide ion radical because both methods are responsible for the same chemical property of abstraction of hydrogen or electron donation to the antioxidants in edible oil on heating the oil to frying temperature for five times.
Results: The radical scavenging activity IC 50 value varies with the concentration of heated and unheated sunflower and rice bran oil. The activity in the oils is due to the presence of antioxidants like Vitamin A, sitosterol, oryzanol, etc., that change with heating effect. The data were recorded and analysed by SPSS (version 12).One-way analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan's multiple range tests, p-Values <0.05 were regarded as significant and p-value<0.001 were very significant. Interpretation and Conclusion: Heated and unheated sunflower oil was positively correlated with antioxidant activity of ABTS and DPPH method (r = 0.333; p<0.01) and rice bran oil (r=0.013; p<0.01). The present study reveals that the antioxidant stability in rice bran oil is greater than sunflower oil even under repeated thermal fluctuations. Hence, it can be recommended that rice bran oil can be used for deep-frying without adverse effect.
Key words: ABTS, DPPH, antioxidants, Inhibition Concentration.
According to WHO's Global Health Statistics 2008, the most recent health statistics for WHO's 193 member states, "cases of cancer of the stomach, liver, colon, rectum and oesophagus will rise sharply by 2030 and will cause nearly 10% of all deaths as compared to 5% in 2004". Coronary artery disease (CAD) - a condition in which blood vessels get narrowed or blocked due to deposition of cholesterol, affecting blood supply to the heart which will be the number one killer in 2030, causing 14.2% of all deaths worldwide. Hence awareness of adverse effect of repeatedly heated edible oil is essential for the society.
Hydro- peroxide, which is the major oxidation product, decomposes to secondary products, such as esters, aldehydes, alcohols, ketones, lactones and hydrocarbons. The secondary products adversely affect flavour, aroma, taste, nutritional value and overall quality of foods. In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell (1).
Antioxidants are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with oxidants in the cell cytoplasm and the blood plasma, while lipid-soluble antioxidants protect cell membranes from lipid peroxidation (1). These compounds may be synthesized in the body or obtained from the diet (2). Rancidity of lipids in foods produces undesirable effects including loss of fat-soluble vitamins, generation of off-flavours, palatability problems and even production of toxins and cause food poisoning.
Sunflower oil contains high concentration of Palmitic acid (4 - 9%), Stearic acid (1 - 7%), Oleic acid (14 - 40%), Linoleic acid (48 - 74%), lecithin, tocopherols, carotenoids and waxes. Crude rice bran oil contains ~96% of saponifiable fractions and ~ 4% unsaponifiable fractions, which include phytosterols, sterolesters, triterpene alcohols, hydrocarbons, and tocopherols (3). Oryzanol content is about 2% in crude rice bran oil. The unsaponifiable fractions presence of plant sterols; ?-oryzanol and tocotrienols make the oil hypocholesterolaemic (3) and bioactive so that it provides positive nutritional and health benefits. ? -Oryzanol a major component of rice bran oil is used to decrease plasma cholesterol, platelet aggregation, cholesterol absorption from cholesterol-enriched diets and aortic fatty streaks (4, 5).
There is an increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body and to prevent the deterioration of fats and other constituents of foodstuffs. In biochemistry, 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) or ABTS is chemical compound used to observe the reaction kinetics of peroxidase. It can be used to quantify the amount of hydrogen peroxide in a sample. To find the stability of free radical, diphenylhydrazyl or DPPH method is used to estimate the activity of antioxidants. In the present study the parameter IC50 (Inhibition concentration to produce 50% reduction of ABTS and DPPH) is calculated for differentiation concentration of unheated and heated sunflower oil and rice bran oil which indicates the formation of peroxide on heating. The IC50 value shows that antioxidants in rice bran oil has more stability than sunflower oil on heating, reduce the compositional changes in the oil and retains its nutrition value.
Materials and Methods
This study was conducted in the Centre for Advanced Research in Indian System of Medicine, SASTRA University
Collection of oil:
Commonly available and popular branded sunflower and rice bran oil was collected from a local grocery shop located in Thanjavur district of Tamil Nadu, India to assess the possibility of usage of repeatedly heated oil by common people.
Frying of oil
Hundred milliliter of the sample oil was placed in a copper beaker and heated on an electric device, stirring manually with glass rod. A microcontroller based temperature controller was designed and was used to monitor the sample temperature. To mimic the oil oxidation process during frying, the sample was heated up to 250C for five times. Initially, the sample was heated to 250C for half an hour. Then, it was allowed to cool until room temperature is achieved. Similarly, the sample was subjected to heating up to 250C for 1 hour, 1 hour, 2 hour and 2 hour respectively ensuring that every time the sample is allowed to cool up to room temperature before heating it next time. In order to ensure that the sample has been heated to the temperature greater than its smoke point, it has been exposed to successive heating.
ABTS+. radical decolorisation assay
The ABTS*+scavenging test is used to determine the antioxidant activity (by estimating peroxide formation) of both hydrophilic and hydrophobic compounds. The reaction between ABTS and ammonium per sulphate directly generates the bluish green ABTS*+ chromophore, which can be reduced by antioxidants. The experiment was carried out according to an improved method as described by Re et al. (1999) (6) with slight modification. ABTS*+is generated by mixing 2.5 ml of 7 mM ABTS with 14.7 mM ammonium per sulphate and stored in the dark at room temperature for 16 hours. The solution is diluted with water to achieve an absorbance of 0.70.05 O.D at 734 nm. The radical-scavenging activity was assessed by mixing 2 ml of this ABTS*+solution with different concentrations of oil sample dissolved in chloroform (25, 50, 75, 100 l). 1.0 ml of chloroform along with 2.0 ml of ABTS*+was used as control. The reaction between ABTS*+and ammonium per sulfate directly generates the blue green ABTS*+chromophore, which can be reduced by an antioxidant, thereby resulting in a loss of absorbance at 734 nm. The antioxidant capacity is expressed as percentage inhibition, calculated using the following formula:
- Inhibition (%) = 100 x (A0 -A1))/A0 - (1)
Where A0 is the absorbance of the control, and A1 is the absorbance of the sample at 734 nm. The peroxide level was determined from the absorbance using UV- Spectrophotometer. Mixing 2 ml of this ABTS* assesses the radical-scavenging activity solution with different concentrations is measured. Low IC50 values indicate high radical - scavenging activity. All experiments were performed in triplicate. IC50 is the antioxidant concentration that inhibits the ABTS+? reaction by 50% under the experimental conditions. This is calculated using Graph pad software version 5.0.
DPPH*radical scavenging assay
DPPH* method is also used to study the scavenging activity of antioxidants in oils. This method is based on the reduction of a methanol solution of DPPH*in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H (7).This transformation results in a change in colour from purple to yellow, which was measured spectrophotometrically by the disappearance of the purple colour at 517 nm using UV- Spectrophotometer (Perkin Elmer). Chloroform solutions of oil (25, 50, 75,100 g/ml of chloroform) are added to 2 ml of a methanol solution of DPPH free radical or methanol alone (control). The reaction mixture is shaken by cyclomixer and then kept in the dark for 30 min under ambient conditions. The absorbance is measured at 517 nm, and the antioxidant capacity was expressed as percentage of inhibition, calculated using the following formula,
- Inhibition (%) = 100 x (A0 -A1))/A0 - (2)
Where, A0 is the absorbance of the control and A1 the absorbance of the sample at 517 nm. IC50 is the antioxidant concentration that inhibits the DPPH* reaction by 50% under the experimental conditions. Low IC50 values indicate high radical - scavenging activity. All analyses were run in triplicate and averaged for heated and unheated sunflower oil, and rice bran oil.
All data on total antioxidant activity are the average of triplicate. To examine the effect of type of compound and concentration on antioxidant activity, graph pad software version 5.0 was used (r2 =0. 0.9949, p<0.005, n>7). The data were recorded and analysed by SPSS (version 12).One-way analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Tukey multiple range tests, p-Values <0.05 were regarded as significant and p-value<0.001 were very significant.
Generation of the ABTS radical cation forms the basis of one of the spectrophotometric methods that have been applied to the measurement of the total antioxidant activity of the solutions of pure substances and aqueous mixtures. As seen in Tables 1 and 2, the IC50 values of sunflower oil is calculated using equation (1&2) for different concentration. The Inhibition concentration (IC50) of unheated is 25.42g (ABTS) and 28.81g (DPPH) shows strong antioxidant stability. On heating (IC50) value is 50.39g in ABTS and 40.45g using DPPH method. Hence it is observed that on heating the antioxidant in the oil becomes volatile (8).
The variation of antioxidant activity of rice bran oil on heating and its free radical scavenging activity is found with the concentration. The decolorisation ABTS+? and DPPH of heated and unheated rice bran oil appears to increase with the concentration as given in Tables 1 and 2. The Inhibition concentration (IC50) of unheated is 24.03 g in ABTS and 34.44 g using DPPH method. On heating (IC50) value is 24.53 g in ABTS and 39.83 g using DPPH method. The antioxidant activity in unheated and heated rice bran oil is almost the same and shows strong antioxidant stability due to the presence of natural antioxidants (9).
The food material at higher temperature looses certain part of their bioactivity and produces peroxides, triacyl glycerol and carbonyl compound which decreases the nutritional value of oil and also leads to diseases like colon cancer, arteriosclerosis, etc. Radical scavenging activities are very important due to the deleterious role of free radicals in foods and biological systems excessive formation of free radicals accelerates the oxidation of lipids in foods and decreases food quality. Measuring the ability to inhibit free radical can assess the scavenging activity of antioxidants.
The Inhibition concentration of unheated sunflower oil has 49.6% more scavenging activity than heated oil using ABTS method and 47.1% using DPPH method. Re et al have reported similar increase in decolorisation with concentration. Free radical scavenging activity of these samples also increases with an increasing concentration. The IC50 value of heated and unheated sunflower oil is given in Tables 1 and 2. It is found that IC50 of unheated oil is 49.6 % greater than that of heated sunflower oil using ABTS radical decolorisation assay and 41.7 % by DPPH radical decolorisation assay. At different concentration of the sample the scavenging activity of unheated oil is found to be more than heated oil. The scavenging activity of heated and unheated sunflower oil increases and the differences were statistically significant (p<0.01) using ABTS and DPPH methods.
Similarly, The Inhibition concentration of unheated rice bran oil has 2.03% more scavenging activity than heated oil using ABTS method and 13.5% using DPPH method. In rice bran oil Oryzanol is a group of ferulic acid esters of triterpene alcohols present in rice bran oil possess strong stabilization effects during frying application (9). It clearly reveals that the heated rice bran oil has been equally effective in antioxidant component properties as that of the unheated oil. At concentration25g/ml., the scavenging activity of heated and unheated oil using ABTS and DPPH methods increases (p<0.01) At the concentration of 100g/ml., the scavenging activity of heated and unheated rice bran oil increases and the differences were statistically significant (p<0.05) using ABTS and DPPH methods. This study reveal that antioxidant activity of rice bran oil even, on repeated heating is better than sunflower oil due to strong antioxidants present in the oil. Heated and unheated sunflower oil was positively correlated with antioxidant activity of ABTS and DPPH method (r = 0.333; p<0.01) and rice bran oil (r=0.013; p<0.01).The variencebetween the groups are tabulated in Table 3.
The ABTS and DPPH systems provide information on the reactivity of a test compound with a stable free radical. Bleaching of the reagents colour by the test sample represents the capacity for hydrogen or electron donation by the test compound. The IC 50 value of sunflower oil is (< 50 g) whereas it is (<39 g) for in rice bran oil. The present study has revealed the understanding that the percentage of conversion of saturated fatty acid into unsaturated fatty acid in the repeatedly heated Rice bran oil has been comparatively less and also the stability level of antioxidants (Oryzanol and Sitosterol) in the pre and post heated conditions leads to the appreciable effect of free radical scavenging activity even in the repeatedly used condition. This study has highlighted the fact that the rice bran oil can exhibit remarkable antioxidant activity and thermal stability on heating to frying temperature. Hence it would be a suitable oil for frying with less adverse effect.