Antioxidant Activity of Pod Coat Extracts of Pigeon Pea (Cajanus Cajan L.) and Their Efficacy in Stabilization of Soybean Oil

Total tocopherolswere determined by the method of Philip¹¹. Tannin content was determined by Pearson method using tannic acid as standard ¹². Total phenolics were estimated spectrophotometrically by Folin-Ciocalteu method keeping gallic acid as a standard ¹³. The antioxidant activity of different extracts was determined by percent inhibition of DPPH method ¹⁴ and by Ferric thiocyanate (FTC) method ¹⁵ while aluminum chloride colorimetric method ¹⁶ was used for the determination of flavonoid content.

Oil was extracted by Soxhlet method using petroleum ether (60-80°C) as a solvent for 8 h. Oil extraction is a three step process which includes: preparation, extraction, and desolventization.

Each of the dried pod extracts was added into soybean oil at 1000 and 2000 ppm concentrations. Experiments were also carried out with synthetic antioxidant, Butylated hydroxy anisole (BHA), and control set without added antioxidants. All the samples were homogenized thoroughly and were incubated, in triplicate, at 50°C in thermostat. Required quantity of the samples was removed periodically and studied for their oxidative quality indices.

Peroxide value (meq/kg) of oil samples were estimated by AOAC method ¹⁷.

p-Anisidine assay was carried out according to the procedure described in AOCS method ¹⁸. The absorbance was measured at 350 nm.

Thiobarbituric acid (meq/kg) value (TBA) was determined according to the method given by Marcuse and Johansson with slight modification ¹⁹. Absorbance was measured at 530 nm. At the same time, a reagent blank (without TBA reagent) was also done.

Conjugated diene and conjugated triene of oil samples were assessed according to method described by Frankel ²⁰. The absorbance of the solution was measured at 234 nm and 268 nm for conjugated dienes and conjugated trienes, respectively.

Free fatty acids were determined as described by Rao method ²¹.

Among three solvents, acetone gave highest yield (4.88g/100g) followed by ethyl acetate (4.35g/100g) and chloroform extract (3.68g/100g). The disparity in yield of different extracts is a result of differences in polarities of compounds present in pod coat ²². Acetone extract showed highest amount of total phenolics ((17.72 mg Gallic Acid Equivalent per gram (GAE)/g)) followed by ethyl acetate extract (15.51 mg GAE/g) and chloroform (11.46 mg GAE/g) extract. Flavonoid content was also found in decreasing order in the extracts of acetone ((9.00 mg Catechin Equivalent per gram (CAE)/g)), ethyl acetate (7.19 mg CAE/g) and chloroform (6.01 mg CAE/g). Similarly, pod coats of Pigeon pea yielded highest amount of tannins, 2.21 mg Tannic Acid Equivalent per gram (TAE/g) followed by2.04 mg TAE/g and 1.80 mg TAE/g, when extracted with acetone, ethyl acetate and chloroform, respectively. Tocopherols content, on the other hand, was maximum in the extract retrieved using ethyl acetate (9.56 mg tocopherol/g) followed by acetone (7.01 mg tocopherol/g) and chloroform (4.31 mg tocopherol/g).

Antioxidant activities were evaluated by 2,2’-diphenyl-1-picrylhydrazyl radical (DPPH) method. Maximum DPPH free radical scavenging activity exhibited by BHA (91.14%) at 0.9 mg/ml concentration and IC₅₀ value was 0.28 mg/ml (Figure 1). Maximum antioxidant activity was exhibited by the extracts of acetone (78.21%), ethyl acetate (66.08%) and chloroform (60.18%) at 1.0 mg/ml concentration. Acetone extract per ml depicted lowest IC₅₀ value equals to 0.52 mg followed by 0.60 mg and 0.66 mg, in case of ethyl acetate and chloroform, respectively. Acetone extract exhibited highest free radical scavenging activity. The results of correlation analysis in acetone extract showed that total phenols show positive and significant correlation with flavonoids (r = 0.968*) and tocopherols (r = 0.985*). Flavonoids were significantly correlated with tocopherols (r = 0.996**) and IC₅₀ value by DPPH method (r = 0.985*). Tocopherols show positive and significant correlation with IC₅₀ value of DPPH methods (r = 0.967*).

Figure 1.IC50 values of various pod coat extracts of pigeon pea by DPPH and FTC method

The FTC method was used to evaluate the level of lipid peroxidation by measuring the absorbance of hydroperoxide of linoleic acid. In this method, the concentration of peroxide decreases as the antioxidant activity increases. The highest per cent inhibition was shown by extracts of acetone (68.22) followed by ethyl acetate (62.09) and chloroform (57.01). Higher free radical scavenging activity and strong antioxidant activity in FTC method of acetone extract is clearly due to higher amount of phenolics, flavonoids and tocopherol content as compared to other extracts.

Concentration of peroxides and hydroperoxides formed during the initial stages of lipid oxidation determines the peroxide value of any sample. Hydroperoxides are produced as primary oxidation product during lipid oxidation ²³. Further breakdown of oil into nonvolatile and volatile secondary products may deteriorate its quality ²⁴. Therefore the level of peroxides gave a clear indication of prevention in lipid oxidation by chemical or natural antioxidants. A continuous increase in peroxide value was observed in all the oil samples with increasing incubation period (Table 1).

Initially rate was very slow became fast after 12th day of incubation following which it continued to increase further, and reached to a maximum value after 28 days of incubation. The peroxide value (meq O₂/kg) of control sample reached a maximum of 41.62±0.05 after 28 days of incubation. A significant (p < 0.05) difference in peroxide value was perceived between the control and soybean oil containing chemical (BHA) and natural antioxidants which slowed down the formation of peroxides edifying good antioxidant efficacy of different pod coat extracts in stabilizing oil.

The antioxidants under study can be arranged in following descending order depending upon their competence in preserving the standards of the studied oil: BHA (200ppm), BHA (100ppm), acetone extract (2000ppm), ethyl acetate extract (2000ppm), acetone extract (1000ppm), chloroform extract (2000ppm), ethyl acetate extract (1000ppm) and chloroform extract (1000ppm). Among all pod coat extracts, the acetone extract of pigeon pea revealed low peroxide values and better antioxidant activity because of the existence of extortionate phenolics quantity. However, among all the samples, BHA (200ppm) was found to be the most effective in reducing the peroxide value of oil. Similar kind of results showing the antioxidant potential of agro wastes extracts and their application in stabilization of corn oil ²⁵. They also observed a significant (p < 0.05) stabilization in peroxide value of corn oil in the presence of BHT (200ppm) and methanolic extracts of different plant materials (600ppm).

p-Anisidine value represents the extent of secondary oxidation products i.e. aldehyde in fats and oils²⁶. The p-Anisidine value of control sample increased from 2.49±0.04 to 48.41±0.13 after 28 days of incubation (Table 2). Differences observed between the p-Anisidine values of control and experimental samples were statistically significant. However inhibitory effect of pod coat extracts of pigeon pea was lower than BHA. p-Anisidine values of various crude soybean oil samples on 28^th day of incubation were arranged in descending order as follows: control, CE (1000ppm), AE (1000ppm), EAE (1000ppm), CE (2000ppm), EAE (2000ppm), AE (2000ppm, BHA (100ppm), BHA (200ppm). Among all extracts, the acetone extract (2000ppm) had higher antioxidant activity than ethyl acetate and chloroform

During oxidation process, peroxides decompose to lower molecular weight compounds such as malonaldehyde. TBA value measures the rate of oxidative rancidity in terms of formation of a non-volatile compound, malonaldehyde ²⁷. TBA value of all the samples initially increased and reached to a maximum value followed by a slight decrease towards the end (Table 3). It could be due to oxidation of secondary oxidation products and formation of carboxylic acids ²⁸. TBA value (meq of malonaldehyde/g) of control increased from 4.47±0.02 (at zero time) to 49.77±0.09 (16^th day) and then decreased to 41.67 ±0.17 (28^th day). All the varying concentrations of pod coat extracts were effective in lowering the TBA value of different samples. The capability of these extracts to reduce TBA value of different samples slightly increased as the concentration of the extract increased. Maximum TBA value, was observed on 16^th day of incubation, in case of control and soybean oil sample supplemented with BHA (100 and 200ppm), while in case of soybean oil supplemented with all the other extracts the maxima was observed on 24^th day of incubation.

After the formation of hydroperoxides from polyunsaturated fatty acids present in oil, non-conjugated double bonds undergo rearrangement and forms conjugated dienes which absorbs at 234 nm. Conjugated trienes are formed as a result of oxidation and rearrangement of polyunsaturated fatty acids having three or more double bonds which can be estimated as absorption at 268 nm ^{29, 30}. The change in absorbance at 234 and 268 nm, quantified with absorbance coefficient (K₀) is used as a measure of oxidative degradation of oil.

The increase in conjugated dienes and trienes signifies the increased oxidative degradation and decreased stability of the oil ³¹. There was a consistent increase in conjugated dienes and conjugated trienes of all the samples over the storage period (Table 4). Per cent conjugated dienes and trienes of control sample increased from 0.36±0.02 and 0.16±0.02 to 36.44±0.16 and 17.36±0.06, respectively during incubation period of 28 days. Highest percentage contents of CD and CT were observed for control after 28 days of incubation ,indicating greater intensity of oxidation, followed by chloroform extract (1000ppm), chloroform extract (2000ppm), ethyl acetate extract (1000ppm), acetone extract (1000ppm), ethyl acetate extract (2000ppm), acetone extract (2000ppm), BHA (100ppm) and BHA (200ppm). The superior antioxidant activity has been indicated by reduced CD and CT of all the stabilized oil samples as compared to control.

The free fatty acid content is considered as a sign of oil hydrolysis. It usually increases with oxidative degradation of lipids ²⁶. Free fatty acids measured in terms of percentage oleic acid content of all samples had shown a consistent increase along the storage period (Figure 2). In control sample, the free fatty acids content increased from 0.567±0.002 to 4.22±0.023 % after 28 days of incubation which are significantly higher than other sample established with various extracts and BHA. The free fatty acid content of soybean oils containing different extracts of pod coat of pigeon pea were lower than the control having no stabilizer. In contrast to other extracts, acetone (2000ppm) pod coat extract manifested superior antioxidant activity in terms of free fatty acid but lower than BHA.

Figure 2.Free fatty acid content of crude soybean oil samples stabilized with BHA and pod coat extracts of pigeon pea retrieved using acetone (A), ethyl acetate and chloroform (B)