The authors have declared that no competing interests exist.
When lipids are exposed to heat, light and oxygen, it leads to oxidation. The addition of antioxidants is required to preserve colour, flavour and vitamin destruction. Present study was, therefore, planned to investigate pod coat of pigeon pea as possible sources of natural antioxidants and to assess their efficacy in stabilization of crude soybean oil during normal storage (28 days at 50°C). Study revealed that acetone pod coat extract of pigeon pea showed richness in total phenolics (17.72 mg/g), flavonoids (9.00 mg/g) and tannins (2.21 mg/g) while the extract of ethyl acetate was found enriched in tocopherols content (9.56 mg/g). The IC50 value of acetone extract was found to be lowest, exhibited potent antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric thiocyanate (FTC) methods. After adding synthetic and natural antioxidants in oil, Peroxide, p-Anisidine, Thiobarbituric acid value, Conjugated dienes, trienes and free fatty acids content were measured every 4 days. Acetone pod coat extract (2000ppm) of pigeon pea gave strong antioxidant efficacy in stabilization of crude soybean oil and hence could be recommended as natural antioxidants for food applications.The research explored the possibility of using pod coat of pigeon pea as imminent sources of green antioxidants and to evaluate their efficacy in stabilization of crude soybean oil.
The use of natural extracts for medicinal purpose has taken prominent dimensions in the past few decades, largely due to the discovery that these contain not only primary metabolites and minerals but also a great variety of secondary metabolites having antioxidant properties
Various plant extracts are being investigated throughout the world with rapidly rising awareness about the disease-preventive and health promoting properties of polyphenols and other antioxidants found in plants
Pigeon pea was grown in experimental field of CCS Haryana Agricultural University Hisar, Haryana (India) with a temperature range from 26ᵒC to 30ᵒC in rainy season (June to October). The threshed pod coats of pigeon pea were dried and ground into powder, using an electric grinder. The powdered sample was extracted by petroleum ether (60-80°C). Hundred grams of dried, defatted and powdered sample was then extracted separately by the Soxhlet method using acetone, ethyl acetate and chloroform for 8h. The extracts were evaporated under a vacuum at 40°C to the required dryness using a rotary evaporator. The dried extracts of acetone (AE), ethyl acetate (EAE) and chloroform (CE) were kept in a refrigerator until they were analyzed.
Total tocopherolswere determined by the method of Philip
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
p-Anisidine assay was carried out according to the procedure described in AOCS method
Thiobarbituric acid (meq/kg) value (TBA) was determined according to the method given by Marcuse and Johansson with slight modification
Conjugated diene and conjugated triene of oil samples were assessed according to method described by Frankel
Free fatty acids were determined as described by Rao method
The data obtained were analyzed using the analysis of variance (ANOVA) in Online Statistical Analysis (OPSTAT) available at
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
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 (
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
Sample | Incubation days | |||||||
0 | 4 | 8 | 12 | 16 | 20 | 24 | 28 | |
Control | 3.40±0.01 | 5.89±0.04 | 8.86±0.06 | 12.31±0.05 | 19.15±0.04 | 26.98±0.07 | 33.71±0.04 | 41.62±0.05 |
BHA(100ppm) | 3.40±0.01 | 4.51±0.01 | 6.61±0.03 | 7.76±0.03 | 9.51±0.03 | 10.87±0.04 | 12.13±0.02 | 13.38±0.03 |
BHA(200ppm) | 3.40±0.01 | 3.85±0.02 | 5.48±0.02 | 6.72±0.02 | 7.86±0.02 | 8.45±0.03 | 9.72±0.03 | 10.86±0.02 |
AE(1000ppm) | 3.40±0.01 | 4.82±0.02 | 7.92±0.04 | 10.68±0.02 | 14.67±0.03 | 19.61±0.04 | 25.67±0.06 | 31.12±0.07 |
AE(2000ppm) | 3.40±0.01 | 4.66±0.01 | 6.13±0.02 | 9.33±0.01 | 12.21±0.02 | 16.93±0.03 | 21.55±0.02 | 27.77±0.03 |
EAE(1000ppm) | 3.40±0.01 | 5.01±0.02 | 8.13±0.02 | 11.40±0.04 | 16.97±0.02 | 21.08±0.03 | 27.31±0.02 | 31.61±0.05 |
EAE(2000ppm) | 3.40±0.01 | 5.04±0.01 | 6.93±0.02 | 10.49±0.01 | 13.46±0.03 | 17.78±0.02 | 23.12±0.03 | 29.19±0.05 |
CE(1000ppm) | 3.40±0.01 | 5.32±0.01 | 8.46±0.02 | 11.86±0.02 | 17.31±0.03 | 23.68±0.02 | 28.08±0.04 | 33.12±0.14 |
CE(2000ppm) | 3.40±0.01 | 5.46±0.02 | 7.12±0.01 | 11.09±0.04 | 14.34±0.02 | 19.43±0.03 | 24.51±0.02 | 31.60±0.04 |
Notes. The values are expressed as mean ± SD;BHT- butylated hydroxyl toluene; AE-acetone pod coat extract of pigeon pea; EAE-ethyl acetate pod coat extract of pigeon pea ; CE-chloroform pod coat extract of pigeon pea .
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
p-Anisidine value represents the extent of secondary oxidation products
Sample | Incubation days | |||||||
0 | 4 | 8 | 12 | 16 | 20 | 24 | 28 | |
Control | 2.49±0.04 | 11.83±0.06 | 16.44±0.08 | 22.42±0.06 | 26.67±0.09 | 33.71±0.05 | 40.18±0.09 | 48.41±0.13 |
BHA(100ppm) | 2.49±0.04 | 6.44±0.02 | 8.81±0.05 | 10.02±0.06 | 13.32±0.08 | 17.18±0.10 | 21.21±0.07 | 24.94±0.10 |
BHA(200ppm) | 2.49±0.04 | 3.45±0.04 | 5.93±0.05 | 8.23±0.03 | 11.27±0.06 | 13.11±0.07 | 17.33±0.06 | 21.12±0.09 |
AE(1000ppm) | 2.49±0.01 | 6.70±0.03 | 10.21±0.05 | 14.34±0.06 | 19.81±0.08 | 25.42±0.11 | 28.82±0.14 | 33.61±0.16 |
AE(2000ppm) | 2.49±0.04 | 5.79±0.04 | 9.01±0.06 | 12.92±0.05 | 15.62±0.08 | 18.75±0.06 | 22.38±0.10 | 26.08±0.10 |
EAE(1000ppm) | 2.49±0.04 | 7.14±0.04 | 11.89±0.06 | 16.37±0.07 | 22.48±0.09 | 26.84±0.12 | 30.30±0.13 | 32.24±0.17 |
EAE(2000ppm) | 2.49±0.04 | 6.37±0.05 | 11.41±0.06 | 14.37±0.07 | 17.38±0.08 | 20.39±0.09 | 24.43±0.11 | 27.61±0.14 |
CE(1000ppm) | 2.49±0.04 | 8.58±0.05 | 12.94±0.06 | 18.68±0.09 | 24.67±0.16 | 28.49±0.10 | 32.08±0.14 | 35.19±0.19 |
CE(2000ppm) | 2.49±0.04 | 7.29±0.05 | 13.07±0.06 | 15.79±0.08 | 19.43±0.10 | 22.09±0.11 | 26.51±0.14 | 29.98±0.16 |
Notes. The values are expressed as mean ± SD;BHT- butylated hydroxyl toluene; AE-acetone pod coat extract of pigeon pea ; EAE-ethyl acetate pod coat extract of pigeon pea ; CE-chloroform pod coat extract of pigeon pea
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
Sample | Incubation days | |||||||
0 | 4 | 8 | 12 | 16 | 20 | 24 | 28 | |
Control | 4.47±0.02 | 12.16±0.03 | 22.78±0.05 | 37.82±0.07 | 49.77±0.09 | 48.52±0.08 | 44.23±0.12 | 41.67±0.17 |
BHA(100ppm) | 4.47±0.02 | 8.57±0.04 | 12.79±0.06 | 20.64±0.06 | 27.44±0.08 | 26.16±0.10 | 25.62±0.12 | 25.18±0.13 |
BHA(200ppm) | 4.47±0.02 | 7.31±0.03 | 10.48±0.05 | 17.26±0.07 | 24.61±0.08 | 23.77±0.11 | 23.05±0.12 | 22.75±0.13 |
AE(1000ppm) | 4.47±0.02 | 9.95±0.10 | 15.58±0.05 | 21.67±0.08 | 28.25±0.09 | 33.38±0.12 | 36.43±0.14 | 34.42±0.16 |
AE(2000ppm) | 4.47±0.02 | 8.68±0.04 | 12.59±0.06 | 19.32±0.09 | 25.28±0.08 | 29.22±0.13 | 33.41±0.11 | 32.38±0.14 |
EAE(1000ppm) | 4.47±0.02 | 10.22±0.08 | 16.28±0.11 | 22.49±0.14 | 29.12±0.07 | 33.89±0.10 | 36.94±0.05 | 34.99±0.16 |
EAE(2000ppm) | 4.47±0.02 | 9.07±0.05 | 13.05±0.04 | 20.16±0.07 | 25.87±0.06 | 29.96±0.08 | 33.98±0.10 | 32.93±0.14 |
CE(1000ppm) | 4.47±0.02 | 10.66±0.05 | 17.65±0.06 | 23.75±0.08 | 30.25±0.10 | 35.19±0.11 | 38.13±0.12 | 36.46±0.14 |
CE(2000ppm) | 4.47±0.02 | 9.76±0.05 | 14.31±0.07 | 20.86±0.05 | 27.12±0.06 | 31.89±0.08 | 35.26±0.10 | 35.05±0.12 |
Notes. The values are expressed as mean ± SD; BHT- butylated hydroxyl toluene; AE-acetone pod coat extract of pigeon pea ; EAE-ethyl acetate pod coat extract of pigeon pea ; CE-chloroform pod coat extract of pigeon pea .
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
The increase in conjugated dienes and trienes signifies the increased oxidative degradation and decreased stability of the oil
Conjugated dienes | ||||||||
Sample | Incubation days | |||||||
0 | 4 | 8 | 12 | 16 | 20 | 24 | 28 | |
Control | 0.36±0.02 | 3.42±0.04 | 8.43±0.07 | 13.62±0.10 | 18.42±0.13 | 24.02±0.14 | 30.49±0.13 | 36.44±0.16 |
BHA(100ppm) | 0.36±0.02 | 1.58±0.03 | 4.56±0.08 | 8.29±0.06 | 12.34±0.08 | 16.26±0.13 | 20.63±0.10 | 24.57±0.16 |
BHA (200ppm) | 0.36±0.02 | 1.07±0.02 | 3.97±0.04 | 6.93±0.06 | 10.28±0.09 | 13.75±0.11 | 17.38±0.13 | 21.27±0.16 |
AE(1000ppm) | 0.36±0.02 | 2.09±0.02 | 5.69±0.05 | 9.88±0.06 | 13.86±0.09 | 18.36±0.12 | 23.83±0.15 | 29.51±0.18 |
AE(2000ppm) | 0.36±0.02 | 1.73±0.05 | 5.19±0.06 | 9.22±0.08 | 13.29±0.08 | 17.85±0.10 | 23.13±0.12 | 28.97±0.13 |
EAE (1000ppm) | 0.36±0.02 | 2.36±0.03 | 6.08±0.06 | 10.43±0.05 | 15.04±0.10 | 19.79±0.12 | 25.11±0.10 | 30.79±0.15 |
EAE(2000ppm) | 0.36±0.02 | 1.94±0.05 | 5.55±0.10 | 9.59±0.07 | 13.89±0.13 | 18.33±0.10 | 23.78±0.11 | 29.44±0.09 |
CE(1000ppm) | 0.36±0.02 | 2.49±0.04 | 6.38±0.05 | 10.78±0.06 | 15.83±0.08 | 20.51±0.13 | 25.81±0.11 | 31.42±0.16 |
CE(2000ppm) | 0.36±0.02 | 2.17±0.04 | 5.95±0.05 | 10.19±0.07 | 15.15±0.06 | 20.03±0.08 | 25.18±0.08 | 30.98±0.10 |
Conjugated trienes | ||||||||
Control | 0.16±0.02 | 1.79±0.02 | 3.48±0.04 | 5.89±0.04 | 8.89±0.02 | 11.58±0.05 | 13.99±0.06 | 17.36±0.06 |
BHA(100ppm) | 0.16±0.02 | 1.09±0.03 | 2.19±0.02 | 3.44±0.04 | 4.96±0.05 | 7.28±0.06 | 9.77±0.08 | 12.44±0.05 |
BHA (200ppm) | 0.16±0.02 | 0.89±0.02 | 2.05±0.03 | 3.25±0.02 | 4.81±0.03 | 6.49±0.06 | 8.42±0.04 | 10.56±0.04 |
AE(1000ppm) | 0.16±0.02 | 1.22±0.02 | 2.65±0.02 | 4.45±0.03 | 6.72±0.04 | 9.56±0.06 | 11.61±0.05 | 15.03±0.05 |
AE(2000ppm) | 0.16±0.02 | 1.17±0.02 | 2.71±0.04 | 4.31±0.06 | 6.53±0.05 | 9.28±0.08 | 11.42±0.05 | 14.39±0.08 |
EAE(1000ppm) | 0.16±0.02 | 1.31±0.03 | 2.79±0.05 | 4.61±0.05 | 7.05±0.06 | 9.78±0.08 | 11.88±0.06 | 15.32±0.09 |
EAE(2000ppm) | 0.16±0.02 | 1.22±0.02 | 2.78±0.05 | 4.42±0.06 | 6.82±0.08 | 9.59±0.09 | 11.72±0.08 | 14.77±0.09 |
CE(1000ppm) | 0.16±0.02 | 1.39±0.02 | 2.91±0.06 | 4.82±0.05 | 7.62±0.05 | 10.23±0.06 | 12.39±0.07 | 15.96±0.09 |
CE(2000ppm) | 0.16±0.02 | 1.27±0.02 | 2.84±0.04 | 4.54±0.05 | 7.27±0.06 | 10.24±0.05 | 12.19±0.06 | 15.57±0.09 |
Notes. The values are expressed as mean ± SD;BHT- butylated hydroxyl toluene; AE-acetone pod coat extract of pigeon pea ; EAE-ethyl acetate pod coat extract of pigeon pea ; CE-chloroform pod coat extract of pigeon pea.
The free fatty acid content is considered as a sign of oil hydrolysis. It usually increases with oxidative degradation of lipids
Addition of pod coat extracts of pigeon pea in crude soybean oil gave strong antioxidative efficiency and hence could be used as alternative natural antioxidants for food applications. It was quite clearly depicted that all the extracts considerably reduced the rate of oxidative deterioration of crude soybean oil but acetone extract (2000ppm) was most effective. As future prospects, an understanding of mechanisms involved and the factors affecting the antioxidant activity of these compounds would be of significant importance in exploitation of such natural antioxidants to control lipids oxidation in food.