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Oxidative stability of canola oil by Biarum bovei bioactive components during storage at ambient temperature

Abstract In this study, antioxidative activities of aqueous extract of Biarum bovei (BBE) in stabilizing of canola oil during storage at 20°C was evaluated. For this purpose, the total phenolic (TP), flavonoids (TFC), and tocopherol content (TTC) of the extract were determined and β‐carotene bleaching system was used to assess the antioxidant efficacy of BBE. The amount of TP, TFC, and TTC in BBE indicated high antioxidant activity. So, different concentrations (0, 200, 800, and 1400 ppm) of BBE and butylatedhydroxyanisole (BHA; 100 ppm), were added to canola oil for 60 days at 20°C. Peroxide value (PV), carbonyl value (CV), Total polar compounds (TPC), acid value (AV), iodine values (IV), and conjugated dienes (CD) were employed to evaluate the BBE effect on canola oil stabilizing. Results indicated that 1,400 ppm of BBE exhibited stronger antioxidant activity in canola oil than BHA.


FARAHMANDFAR AND RAMEZANIZADEH
There is no report describing efficiency of B. bovei extract for the stabilization of food materials, especially canola oil. The purpose of this study was to determine the effect of B. bovei extract on the stabilization of canola oil and comparing its antioxidant activity with commercially available antioxidant (BHA) during storage at 20 ± 1°C.

| Materials
Refined-bleached-deodorized canola oil (without any synthetic antioxidants) was obtained from Ghoncheh Co. (Sari, Iran). The plant B. bovei was collected from the south of Iran (Izeh, Kozestan, Iran) in April 2016 and authenticated by a botanist (School of Pharmacy, Sari University of Medical Sciences, Sari, Iran). They were washed, airdried (in the shade) and powdered fine using an electric device then stored at 4 ± 1°C until use. All other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Frankfurt, Germany). BHA was also purchased from Sigma-Aldrich (St. Louis, MO, USA).

| Preparations of B. bovei extract (BBE)
The powdered B. bovei was added into water (1:50 wt/vol) and the resulting mixture was shaken in a dark place at room temperature for 24 hr. Then, the extract was filtered using Whatman No. 42 filter paper and residue was again extracted. The solvent was completely evaporated at 40°C. The obtained extract was weighted to calculate the yield and was stored in a dark container in refrigerator (4 ± 1°C) until used ).

| Determination of total phenolic, flavonoids, and tocopherol content
Total phenolic content (TP) of extract was measured according to the Folin-Ciocalteu method and result was expressed as mg/g Gallic acid equivalents (GAE) (Farahmandfar et al., 2015). The total flavonoid content (TFC) of extract was determined according to the method described by Sayyari and Farahmandfar (2017) and the results were expressed as catechin equivalents (mg/100 g of dry weight). The total tocopherol content (TTC) was measured spectrophotometrically in keeping with method described by Asnaashari et al. (2014) and the results are expressed as microgram of α-tocopherol equivalents in milliliter extract (μg α-tocopherol/ml extract).

| Antioxidant activity (β-carotene bleaching system)
Lipid peroxidation inhibition activity of different concentrations of BBE was determined using the β-carotene bleaching method (Farahmandfar et al., 2015). For this purpose, 0.5 mg β-carotene was dissolved in 1 ml chloroform (high-performance liquid chromatography grade). Then, 25 μl linoleic acid and 200 mg Tween 40 were added. Chloroform was completely evaporated using rotary vacuum evaporator. At that moment, 100 ml of distilled water saturated with oxygen was added and the contents were shaken vigorously, 2.5 ml of the above solution was transferred to the test tube and 350 μl of each extract (with a concentration of 2 g/L dissolved in their own solvent) was added. All samples were put into a water bath for 120 min at 50°C. The absorbance values of samples were read spectrophotometrically at 470 nm at zero time and after 120 min. Antioxidant capacity of the extracts were expressed by applying following equation: where A control is the absorbance of the control (without extract) and A extract is the absorbance of extract.

| Preparation of canola oil
The solubility of the water extract of a plant is low in oil. For that reason, we added 0.3% polyglycerol polyricinoleate (PGPR) content to the oily phase. To ensure complete solution, the oily phase with the respective amount of PGPR was stirred for 30 min with a magnetic stirrer at 300 rpm. While stirring, the aqueous phase was slowly added to prepare uniform solution. Therefore, canola oil containing different concentrations of BBE (0, 200, 800, and 1400 ppm) and 100 ppm of BHA as control antioxidant were placed in dark brown colored bottles and stored at 20 ± 1°C for 60 days. Samples (20 g) were removed periodically at specific interval (0, 15, 30, 45, and 60 days) for analysis.

| Analytical procedures
Peroxide value (PV) was determined spectrophotometric as said by the method of Farhoosh, Johnny, Asnaashari, Molaahmadibahraseman, and Sharif (2016) and the results are expressed as meq O 2 /Kg oil.
The carbonyl value (CV) of the canola oil was assessed according to the method described by Farahmandfar et al. (2015) using 2-propanol and 2, 4-decadienal as solvent and standard, respectively, and the results are expressed as μmol/gr. Acid value (AV) was determined in keeping with the method of Sayyari and Farahmandfar (2017) and the results are expressed as mg/g. Iodine value (IV) was determined according to the method described by Eshghi et al. (2014) and the results are expressed as g Iodine/100 g oil.
Total polar compounds (TPC) content of canola oil was measured in line with the economical micro method described by Farahmandfar et al. (2015) and the results are expressed as %.
Conjugated diene value (CDV) was specified based on the method of Sharayei and Farhoosh (2016). Canola oil samples were diluted to 1:600 with hexane and measured spectrophotometrically at 234 nm.
An extinction coefficient of 29,000 mol/L was used to quantify the concentration of conjugated dienes formed in canola oil samples during oxidation. (1)

| Statistical analysis
Analysis of variance (ANOVA) was used and means comparison was performed by Duncans' new multiple range test. SPSS statistic program (SPSS 16.0 for Windows, SPSS Inc., Chicago, IL, USA) was used for data analysis. All analyses were performed in triplicate and data reported as means ± standard deviation (SD).  (Farahmandfar et al., 2015). Moreover, these components are responsible for great free radical scavenging and antioxidant activities of fruits and vegetables (Maleki, Ariaii, & Fallah, 2015

| Antioxidant activity by β-carotene bleaching system
In this analyze, the oxidation of linoleic acid produces peroxyl free radicals by reason of the abstraction of hydrogen atom from diallylic methylene groups of linoleic acid. These free radicals will oxidize unsaturated β-carotene. In the presence of an antioxidant such as bioactive components of plant extract and essential oil, β-carotene could scavenge the free radicals formed in the system and retains its original yellow color (Farahmandfar et al., 2015). The inhibition percentage of antioxidant activity (β-carotene bleaching system) of water extract of B. bovei is presented in

| Oxidative stability of canola oil samples as affected by addition of BBE
PV is an index for peroxides and hydroperoxides measurement produced in the initial stages of lipid oxidation. Besides, it is one of the most widely-used methods for oils oxidative rancidity evaluation . Figure 1a shows  as aldehydes and ketones. Peroxides and hydroperoxides are transformed into secondary products, which are more stable than peroxides (Asnaashari, Farhoosh, & Farahmandfar, 2016). Figure  CDV is a good parameter for the oxidative deterioration measurement of oils, hence indicates the effectiveness of antioxidants in food products. As seen in Figure 5, the CDV raised with increasing storage period owing to formation hydroperoxides that possess conjugated diene structures. Increase in CDV in the presence of antioxidant is slightly lower than control samples. So that, at the end of storage period, the CDV of the control samples reached 9.23, whereas CDV of canola oil samples including 200, 800, and 1,400 ppm BEE and samples including BHA was 8. 48, 8.04, 6.97, and 8.13, respectively. This demonstrated the antioxidant potential of BHA and BBE in stabilizing of canola oil. On the other hand, BHA with the higher CDV showed incompatible antioxidant properties in preventing lipid oxidation compared to BBE at 800 and 1,400 ppm.

| CONCLUSION
In this study, it is found that B. bovei water extract had high TP, TFC, and TTC. Therefore, the antioxidant effect of this extract was evaluated to stabilize canola oil during storage at 20°C. The results could be summarized that all the concentrations of B. bovei extract can stabilize canola oil effectively. The antioxidant activity of B. bovei extract at concentration of 1,400 ppm generally greater than synthetic antioxidant (BHA) at its legal limit. Therefore, B. bovei water extract may be considered a source of natural antioxidant; hence, it may act as an alternative to synthetic ones for the stabilization of food formulation, possibly contributing to improve edible oils quality.