Evaluation of antioxidant properties of lemon verbena (Lippia citriodora) essential oil and its capacity in sunflower oil stabilization during storage time

Abstract In this study, lemon verbena essential oil as a natural antioxidant was used to increase the stability of sunflower oil, and stabilization effects in terms of storage conditions were compared with synthetic antioxidant (BHT). For this purpose, the antioxidant activity of the essential oil was determined by DPPH assay and β‐carotene bleaching method. Then, lemon verbena essential oil (0, 400, 800, and 1,600 ppm) was added to sunflower oil without synthetic antioxidant and stored at 60°C for 60 days. Results from different parameters (peroxide value, free fatty acid, iodine value, total polar compound, carbonyl value, conjugated dienes, and oxidative stability index) were in agreement with each other, suggesting that lemon verbena essential oil (1,600 ppm) could act better than BHT in inhibition of lipid oxidation in sunflower oil and can be used as predominant alternative of synthetic antioxidants.

BHT used as standard antioxidant provided from TITRAN.

| Preparation of lemon verbena essential oil
Essential oils were extracted by hydrodistillation from the powdered lemon verbena by the Clevenger-type apparatus, and the obtained essential oils are stored in a dark container at 4°C until used.

| Analysis of lemon verbena essential oil
A GC-MS instrument (5973N; Agilent Technologies, Wilmington, DE, USA) equipped with a mass selective detector operating in the electron impact mode (70 eV) was used to study the compositions of the lemon verbena essential oil according to Hashemi, Khaneghah, Tavakolpour, Asnaashari, and Mehr (2015). For each compound on the chromatogram, the percentage of peak area relative to the total peak areas from all compounds was determined and reported as relative amount of that compound.

| Determination of radical scavenging activity
To evaluate the antioxidant activity of the lemon verbena essential oil, DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging method was used. DPPH free radicals scavenging were measured by the method described by Farahmandfar, Asnaashari, and Sayyad (2017). Initially, the samples were reacted with the stable DPPH radical in methanol solution. The solution kept for 20 min in the dark, and then its absorbance was measured at 517 nm using spectrophotometer (GBC, Cintra 20). Inhibition of free radical DPPH was calculated using the following equation: where A blank is the absorbance of the control reaction, and A sample is the absorbance of the test compound.

| β-Carotene bleaching method
Oxidation scavenging activity of lemon verbena essential oil was carried out using β-carotene bleaching method (Bektas, Serdar, Sokmen, & Sokmen, 2016). For this purpose, 5 mg of β-carotene was dissolved in 10 ml chloroform solvent (high-performance liquid chromatography grade). Then, 600 μl removed from the prepared solution and were mixed with 40 mg of linoleic acid and 400 mg of Tween 40. Chloroform need to completely evaporate.
For this purpose, the rotary vacuum evaporator was used. In the next step, 100 ml distilled water saturated with oxygen was added and gently stirred. 2.5 ml of the solution was transferred in the test tube. Then, 350 μl of each essential oil with a concentration of 2 g/L was added to the test tube. All of the above were performed for blanks. All samples were put into a water bath with temperature of 50°C for 120 min. Then, the samples' absorbance was read using a spectrophotometer at 470 nm at zero and 120 min. To determine the antioxidant capacity of essential oils, the following equation was used: where DR blank is the degradation rate of blank, and DR sample is the degradation rate of sample.

| Oxidative stability index (OSI) analysis
Three gram of sunflower oil was mixed separately with different concentrations of the lemon verbena essential oil (Zero,200,400,800,1,600,and 3,200 ppm) and BHT as control antioxidant and then exposed to Rancimat (Metrohm model 734, Herisan, Switzerland) at 120°C at an airflow of 15 L/hr. During heating process of sunflower oil containing different concentrations of lemon verbena essential oil and BHT at specific time interval, also OSI was determined at 120°C. Measuring vessels, electrodes, connecting tubes, and glassware were cleaned several times before the experiments.

| Peroxide value (PV) analysis
The PV (meqO 2 /kg oil) of sunflower oil samples containing different concentrations of lemon verbena essential oil and BHT was (1) (2) Antioxidant capacity (%) = (DR blank − DR sample ) DR blank × 100 measured spectrophotometrically at 500 nm by UV-VIS instrument (model 160A Shimadzu, Tokyo, Japan sulfate, filtered off to produce a clear solution]) were added, respectively, and after adding each of them, the sample was mixed on a vortex mixer for 2-4 s. Then, the absorbance of the sample was read, after 5 min incubation at room temperature (Asnaashari, Farhoosh, & Farahmandfar, 2016).

| Acid value (AV) analysis
The AV was determined according to the AOCS (1993) Official Method Cd 3d-63 . In this method,

| Total polar compounds (TPC) determination
The TPC was determined according to Saoudi et al. (2016) method. At first, silica gel 60, dried (12 hr) at 160 °C, was added five parts of water to 95 parts of it and was shaken vigorously for about 1 min and stay overnight. Then, the silica gel 60 (1 g) was compressed and filled between two cotton wool balls into a pipette tip. Oil sample (500 mg) and toluene (4 ml) were mixed, and finally, the solution (1 ml) was pipetted on top of the pipette tip and used toluene as the eluent. After the solvent was eliminated, weighing TPC% was calculated by the equation of 100 (w−w 1 )/w, in which w and w 1 are the sample weight and the weight of nonpolar compounds in milligrams, respectively.

| Carbonyl value (CV) determination
For CV analysis of sunflower oil samples including different concentrations of lemon verbena essential oil and BHT, one kilogram of 2-propanol with 0.5 g of sodium borohydride was refluxed for 1 hr to remove any carbonyl components of solvent. Then, 2,4-dinitrophenylhydrazine (DNPH) (50 g) was dissolved in 100 ml of the solvent including 3.5 ml of 37% HCL. 0.04-1 gr of the oil sample was made by adding the solvent including triphenylphosphine (0.4 mg/ml) to reach 10 ml. Moreover, the solutions of 50-500 μmol/L of 2,4-decadienal in 2-propanol were prepared. Then, standard carbonyl compound solution or oil solution (1 ml) and DNPH solution (1 ml) were mixed in a tube. After that, the tube was heated (20 min, 40°C) and cooled in water bath after adding 2% KOH (8 ml). Finally, the absorbance of upper layer after centrifuging (2,000×g for 5 min) was read at 420 nm.

| Conjugated dienes analysis
The absorbance of sunflower oil samples solutions in hexane (1:600 v/v) was read at 234 nm against a hexane as blank for conjugated dienes.

| Statistical analysis
All determinations were carried out in duplicate, and data were subjected to analysis of variance (ANOVA). ANOVAs were performed according to SAS software. Significant differences between means were determined by Duncan's multiple range tests; p values less than .05 were considered statistically significant.

| Composition of lemon verbena essential oil
The yield of essential oil extracted from lemon verbena with hydrodistillation method is 20.14 ± 0.27% (v/w). The percentage of

| Determination of radical scavenging activity (DPPH assay)
The absorption range of DPPH free radical is maximum within 515-528 nm . Immediately after receiving proton from any hydrogen donor, especially from phenolics, it instantly loses its chromophore and becomes yellow . As the concentration of antioxidative compounds increases, their DPPH radical scavenging activity also increases and can be defined as antioxidant activity. DPPH radical scavenging test is applied as a rapid method to determine antioxidant activity of chemical substances. Bleaching is known to occur via abstraction of hydrogen by this stable free radical with a maximum absorption band around 515-528 nm and can easily be monitored spectrophotometrically. The antioxidant activity of plant essential oil such as lemon verbena essential oil related to their ability to donate hydrogen atoms or electrons and free electrons (Sharopov, Wink, & Setzer, 2015). DPPH radical scavenging of lemon verbena essential oil was measured in triplicate and is shown in Figure 1. As can be seen, as concentration of the essential oil raised, the DPPH radical inhibition also enhanced. Moreover, the sunflower oil samples including 3,600 ppm of lemon verbena essential oil also act better than BHT in radical scavenging ability. This can be a result of the capability of components within lemon verbena essential oil for scavenging free radicals via electron or hydrogen-donating mechanisms.

| β-Carotene/linoleic acid bleaching
To indicate the potential antioxidant activity of plant essential oil, synthetic free radical scavenging (DPPH) model will be valuable tool. However, this system does not use a food or biologically relevant oxidizable substrate, so no direct information on an essential oil's protective action can be determined (Kreps, Vrbiková, & Schmidt, 2014). Therefore, assessing the essential oil in β-carotene/linoleic acid was considered important: water emulsion assay despite its reported limitations. This assay applies that the oxidation of linoleic acid would generate peroxyl free radicals according to the abstraction of hydrogen atom from methylene groups of linoleic acid. The highly unsaturated β-carotene will be oxidized by the free radicals. The oxidation of β-carotene will be minimized by the presence of antioxidants in the lemon verbena essential oil. The antioxidants from the essential oil will decompose hydroperoxides formed in this system. So it can be inferred that the degradation rate of β-carotene is dependent on the antioxidant activity of the essential oils. As can be seen in Figure

| Peroxide value analysis
Peroxides are the main initial products of oil oxidation and can be determined using the peroxide value (PV) (Asnaashari, Hashemi, Mohammad, Mehr, & Asadi Yousefabad, 2015). A higher peroxide value implies a lower oxidative stability (Farahmandfar, Asnaashari, & Sayyad, 2015). The changes in peroxide values are shown in

| Free fatty acids analysis
One important measure of rancidity of foods might be recognized as formation of free fatty acids (Asnaashari, Asnaashari, Ehtiati, & Farahmandfar, 2015).

| Iodine value analysis
A huge amount of polyunsaturated fatty acids (PUFAs) can be found in sunflower oil, more than 70%. These PUFAs are prone to lipid oxidation. During storage, the double bonds of these PUFAs are attacked by free radicals, which results in the formation of conjugated bonds (Avramović et al., 2015). Hence, measuring the amount of unsaturated fatty acids present in sunflower oil can be used as TA B L E 2 Oxidative stability index (OSI) and iodine value of the sunflower oil as affected by the different concentrations of lemon verbena essential oil (0, 400, 800, and 1,600 ppm) during storage time Means ± SD (standard deviation) within a column with the same lowercase letters are not significantly different at p < .05. Means ± SD within a row with the same uppercase letters are not significantly different at p < .05. a reference to determine the freshness of the oil. Adding iodine monochloride to the oil samples can be used for determining the freshness of sunflower oil. The unsaturated fatty acids react with iodine monochloride and release free iodine. The free iodine can then react with sodium thiosulfate (Martínez, Sánchez, Encinar, & González, 2014). The changes in iodine values are shown in Table 2

| Total polar compounds analysis
In here, all of the degradation products without the nonpolar fraction (unaltered triglycerides) will collectively be referred as the total polar compounds (TPC). The changes in total polar compounds are shown in Table 3. These data demonstrated a significant increase in TPCs for all the treatments during storage time. It was concluded from the results that the most stable formulation in terms of the TPCs would be obtained by addition of the lemon verbena essential oil (1,600 ppm). On day 60, higher values of TPCs of the essential oil of lemon verbena are attributed to control with 11.48%, followed by BHT (11.04%), 400 ppm of the lemon verbena essential oil (8.90%).
TA B L E 3 Total polar content (TPC) and conjugated diene of the sunflower oil as affected by the different concentrations of lemon verbena essential oil (0, 400, 800, and 1,600 ppm) during storage time  While lipid oxidation is taking place, primary products transform into secondary products such as carbonyls. Secondary products of oxidation can be estimated by measuring carbonyl value (CV) (Kiralan et al., 2016). The changes in carbonyl values are shown in Figure 3.
The oils with 800 and 1,600 ppm of lemon verbena essential oil showed significantly (p < .05) lower CV values (11.96 ± 0.53 and 11.21 ± 0.53 μmol/g, respectively) than other samples. It was concluded from the results that 800 and 1,600 ppm of lemon verbena essential oil were more efficient to inhibit oil oxidation than BHT (16.25 ± 0.25 μmol/g). It can be seen that increase in CV in the presence lemon verbena essential oil is slightly lower than sunflower oil samples without any essential oil.

| Conjugated diene analysis
Absorbance at 234 nm provides a measure of the content of conjugated dienes (CD), which represent the degree of production of the primary oxidation products (Dridi et al., 2017). The changes in conjugated dienes are shown in Table 3

| CON CLUS ION
The presence of bioactive compounds that possess antioxidant properties causes lemon verbena essential oil to inhibit the formation of oxidation products in sunflower oil. Oxidative Stability Index (OSI) test revealed the superiority of essential oil of lemon verbena than the BHT, in preventing of oil oxidation. More reduction in peroxide value, free fatty acid, total polar compounds, carbonyl value, and conjugated dienes in sunflower oil by adding lemon verbena essential oil than BHT was observed by test results. In this study, it is clearly indicated that 1,600 ppm of this essential oil in stabilization of sunflower oil can act as the same effective as BHT. So, lemon verbena may be considered a cheap potential source of bioactive compounds for application as antioxidants in lipid foods.

ACK N OWLED G M ENTS
This study does not involve any human or animal testing. We are grateful to Sari Agricultural Sciences & Natural Resources University (SANRU) and Iran's National Elites Foundation for support of this research.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interests.