Direct Incorporation of Ginger and Oregano Antioxidants in Canola Oil

Antioxidants are used to increase the shelf life of food and oils. The most common extraction method of antioxidants from natural sources is the use of organic solvents, like methanol and ethanol. Evaporation of all the organic solvent is necessary to prevent health diseases. In this study, we present a new “organic solvent-free” method to extract antioxidants, which uses canola oil as extraction solvent to incorporate natural antioxidants from oregano and ginger without the use of organic solvent. The oil solvent extraction capacity was assessed by assays of oxygen radical absorbance capacity method for hydrophilic (721.9 ± 10.5 and 89.0 ± 2.5 μmol TE g to canola oil with oregano and ginger, respectively) and lipophilic (118.8 ± 7.4 and 48.2 ± 9.7 μmol TE g to canola oil with oregano and ginger, respectively) compounds, oxidation test (Oxitest) (1,434 and 1,018 minutes to canola oil with oregano and ginger, respectively) and thiobarbituric acid reactive substances (0.01 ± 0.02 and 0.11 ± 0.06 mg MA g to canola oil with oregano and ginger, respectively). Results provide evidence of the efficiency of canola oil as antioxidants extractor. Nine antioxidant compounds from canola oil incorporated with antioxidants compounds were identified using high performance liquid chromatography tandem mass spectrometry.


Introduction
The use of antioxidants is the simplest method to reduce fat oxidation in food and oils. 1 In most cases, synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) are used as additives.However, there is evidence that synthetic antioxidants have harmful effects on human health. 2,3ecause of this, research for suitable natural sources of antioxidants for use in the food and oil industries have been performed. 4atural antioxidants from plants have many advantages when compared to synthetic antioxidants. 1Overall, natural antioxidants have functional and sensory properties, are considered safe, and have greater acceptability by the consumers. 1On the other hand, different organic solvents such as methanol, ethanol and acetone are used to extract antioxidants from fruits, vegetables and other types of foods.These solvents have hazardous and toxic properties and can cause adverse health effects. 5is study aimed to develop a new "organic solventfree" method to extract antioxidants from oregano and ginger using canola oil.The antioxidant capacity from the canola oil with spices was evaluated by oxygen radical absorbance capacity method (ORAC), using the fluorescein (FL) decay curve (ORAC FL ) of hydrophilic compounds (H-ORAC FL ) and lipophilic compounds (L-ORAC FL ).The lipid oxidation of the samples was measured by two different methods: a new method known as Oxitest, and by thiobarbituric acid reactive substances (TBARS) assay.Antioxidants compounds incorporated in the canola oil were identified by high performance liquid chromatography (HPLC) tandem mass spectrometry using a fingerprint analysis (MS) and daughters scan mode (MS/MS).Furthermore, we evaluated the canola oil extraction efficiency compared to methanolic extraction of spices.rhizomes and dried oregano leaves were purchased at a local market in Maringá (Brazil).Ginger rhizome was dried in a drying oven at 50 °C for 24 h.Both spices were grounded and sieved in a 35 mesh.Ginger or oregano was added individually to canola oil at three different concentrations: 2.5, 5.0, and 10.0 g in 100.0 g of canola oil.Samples were agitated in magnetic stirrer for 1 h and thermostatic bath at 30 °C to incorporate antioxidants in the canola oil.The samples were stored at 8 °C in dark until analyses were performed.

ORAC FL assay
L-ORAC FL and H-ORAC FL were determined separately as described by Wu et al. 6 with modifications.ORAC FL analysis was conducted in a microplate reader (Victor X4, PerkinElmer, USA) with fluorescence filters with excitation and emission wavelengths of 485 and 535 nm, respectively.In each recipient, inside the microplate, 20 µL of sample extract and 200 µL of FL (12.0 mmol L -1 in potassium phosphate buffer 75.0 mmol L -1 , pH 7.0) were added.The microplate was heated to 37 °C for 5 min, and 75 µL of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in potassium phosphate buffer 75.0 mmol L -1 , pH 7.0 (63.5 mmol L -1 to lipophilic assay and 32.0 mmol L -1 to hydrophilic assay), was added.Fluorescence was immediately measured after addition of radical and the following measurements were obtained at intervals of 1 min, for 30 min.A blank with only phosphate buffer was used and the quantification was performed using linear regression (y = ax + b) and coefficient of determination (r 2 ) between Trolox (T) concentration (5-50 µmol T L -1 ) and the net area under the FL decay curve. 7The results are expressed in µmol Trolox equivalent per gram of sample (µmol TE g -1 ).The total antioxidant capacity (TAC) was calculated by summing the L-ORAC FL and H-ORAC FL .

Oxidation test
Oxidation tests were performed as described by Verardo et al. 8 using Oxitest ® (VelpScientifica, Usmate, Milan, Italy) and 5 g of each sample (oils incorporated with antioxidants).The Oxitest ® assay determines induction periods (IPs) to detect rancidity of lipids, by accelerating the oxidation process using high temperatures (until 110 °C) and high pressure ultra-pure oxygen.The IPs of the samples were obtained by two-tangent method.When a compound which delays oxidation is added to the system, latency to a measurable decrease in oxygen pressure will increase. 4emperature was set to 90 °C and oxygen pressure to 600 kPa (99.9999% purity).The oxidation of canola oil alone (control) was also evaluated.

TBARS analysis
The assays of TBARS was conducted according to the method described by Adolfo Lutz Institute Standards. 9amples of canola oil (incorporated with antioxidants) were stored for 100 days at 4 °C and in dark prior to the analysis.Malonaldehyde (MA) solutions was prepared with 5 different concentrations (3-200 mg MA L -1 ) and a standard curve was prepared.The concentrations of MA in the samples were plotted and were expressed in mg malonaldehyde per gram of sample (mg MA g -1 ).
Preparation of samples for HPLC analysis 0.25 g of ginger rhizomes and oregano leaves were submitted to extraction procedure in 6.3 mL (equivalent to 5 g) of methanol, under magnetic agitation for 1 h and thermostatic bath at 30 °C.5.0 g of canola oil (incorporated with antioxidants) was mixed to 2 mL of methanol (3 times) to obtain the methanolic extract.A control was prepared using canola oil (without spices) extracted with methanol.The samples were shaken and centrifuged for 5 min.The solvent of all extracts was evaporated under nitrogen flow.
The MS determination was performed in positive electrospray mode.The conditions were as follows: capillary temperature of 150 °C; dessolvation temperature of 400 °C, capillary voltage of 2.0 kV, cone voltage of 25 V. Argon at 0.32 Pa was used for collision-induced dissociation at collision energy of 10-35 eV.

Statistics
Statistical analysis was carried out using the Tukey's test (Statistica) with the statistical significance level fixed at p < 0.05.The data are expressed as means ± standard deviation for samples in triplicate.

Results and Discussion
ORAC is one of the most popular methods used to evaluate the antioxidant capacities of oils. 7The total antioxidant capacity values for concentrations of 5.0 and 10.0 g of oregano incorporated in canola oil (Table 1) are similar and significantly higher than values for total antioxidant capacity of 2.5 g of oregano incorporated in 100 g of canola oil.In addition, values for total antioxidant capacity concentrations of 5.0 and 10.0 g are similar (p > 0.05).This is probably due to saturation of canola oil when 5.0 g of spices was used.Similar assumption may be extrapolated for the canola oil containing ginger (Table 2).Therefore, the optimum concentration used in subsequent experiments was 5.0 g spices in 100.0 g of canola oil.The antioxidant capacity of the canola oil containing oregano was higher than the antioxidant capacity of canola oil with ginger and the control, and both spices had higher antioxidant capacity than canola oil control.Furthermore, in the present study we were able to extract antioxidants from ginger and oregano using canola oil as solvent extractor without the use of any organic solvent extraction.
Oxitest is a new method used to evaluate the oxidative process of lipids.In this experiment, canola oils with ginger or oregano had greater protection against lipid oxidation compared to control (Figure 1).The IPs was greater for the canola oil incorporated with both spices compared to canola oil (control).The IP of canola oil with oregano was increased by approximately 811 minutes compared to IP of canola oil (623 minutes), and the IP of canola oil with ginger was increased by approximately 395 minutes compared to canola oil.Both spices provided protection to canola oil against lipid oxidation, however oregano was more efficient than ginger.These results provide evidence that the canola oil was a good solvent for the extraction of antioxidants and the extracted antioxidants delayed lipid oxidation, which is an intrinsic characteristic of 78.9 a ± 9.8 59.5 a ± 5.3 138.4 a ± 11.1 Average ± standard deviation (n = 3).Means followed by different letters in the same column showed significant difference by Tukey's test (p < 0.05).TAC: total antioxidant capacity.antioxidants.The main antioxidants action mechanism is based on the donation of an active hydrogen atom from antioxidant (AH) to a free radicals (ROO • and R • ).This donation stops the oxidation and after that antioxidants become radicals resonance stabilized (A • ), which are less active than those radicals they have neutralized (equations 1 and 2). 15,16 Thus, canola oil is a potential extractor solvent of antioxidants from oregano and ginger and these compounds inhibit the degradation of lipids.
One of the most common methods to measure lipid oxidation in foods is the quantification of malonaldehyde by reaction with 2-thiobarbituric acid, in the so-called TBA-reactive substances assay.All samples were stored in similar conditions for 100 days under refrigeration and protected from light.The action of antioxidants from oregano in canola oil was very impressive.Samples from canola oil with oregano had discrete oxidation, and canola oil alone (control) had ca.20-fold increase in oxidation (Table 3).The canola oil with ginger also had protection against oxidation, but less efficient than canola oil with oregano (ca.2-fold increase).Results of TBARS assay corroborate results obtained by ORAC FL and Oxitest ® methods.
Antioxidant compounds from oregano and ginger were identified by HPLC-MS analysis (Figure 2), and the efficiency of extraction using canola oil compared to methanol was assessed.The chromatograms of methanolic extracts of canola oil with oregano or ginger (Figures 2a and 2b, respectively) have a different profile (fingerprint) compared to the canola oil control (Figure 2c).This difference in chromatograms is probably from the incorporation of antioxidants from spices in canola oil in the solvent free process that was used.
The chromatographic profile of canola oil incorporated with antioxidants from either oregano (Figure 2a) or ginger (Figure 2b) was similar to the chromatograms of methanolic extracts of oregano (Figure 3a) or ginger (Figure 3b).HPLC-MS and HPLC-MS/MS analysis demonstrated that the incorporation of antioxidants directly in the canola oil provides similar results to the extraction carried out with methanol.Methanol is one of the most used solvents for antioxidant extraction, because it is cheap and reusable.However, it has hazardous and toxic properties, which can cause adverse health effects (e.g., intoxication, visual insufficiency and dermatitis). 17The methanol toxicity is due to the rate in which it is eliminated from the body, and also due to the toxicity of the products of its metabolic oxidation, formaldehyde and formic acid. 18,19Due to its toxicity, methanol extraction is not favored in the food industry.When employed, the solvent must be removed completely from the extract prior to its application in food products. 17Therefore, it can be suggested canola oil to be used as solvent extractor, as it was as good as methanol to extract antioxidants from oregano and ginger.][19][20] The Figures 4 and 5 show the fragmentation spectrum of apigenin (an oregano antioxidant), and 6-shogaol (a ginger antioxidant), respectively.
Antioxidants from oregano and ginger incorporated in canola oil and identified by study of their fragmentation spectra, published data, and searches in MassBank and mzCloud databases, are described in Table 4. Oregano and ginger antioxidants belong to different classes.The constituents responsible for the antioxidant activity of ginger are compounds belonging to the gingerol family and its derivatives. 10Oregano has a variety of classes of antioxidants, such as phenolic acids, flavones, flavonols and flavonoids. 21The antioxidant capacity and antioxidant compounds are mainly affected by season and planting region factors. 22

Conclusions
In this study, we successfully extracted antioxidants from oregano and ginger using canola oil as solvent extractor (i.e., solvent free), obtaining an oil with natural antioxidants, contributing to environmental sustainability and human health.The extraction efficiency is corroborated by assays of oxygen radical absorbance capacity method, oxidation test by Oxitest and thiobarbituric acid reactive substances.The results obtained using HPLC-MS and HPLC-MS/MS confirmed the efficiency of canola oil as solvent extractor to antioxidants compounds, as well as  methanol.In addition, a total of 9 antioxidant compounds were identified from oregano and ginger.Therefore, oregano and ginger are great sources of antioxidant compounds that protect the canola oil against lipid oxidation.

Figure 2 .
Figure 2. HPLC-ESI-MS analyses of methanolic extracts from: (a) canola oil with addition of oregano; (b) canola oil with addition of ginger; (c) control canola oil.

Table 1 .
Antioxidant capacity of canola oil incorporated with oregano antioxidants, analysed by the ORAC FL method

Table 2 .
Antioxidant capacity of canola oil incorporated with ginger antioxidants analysed, by the ORAC FL method

Table 3 .
Thiobarbituric acid reactive substances (TBARS) assay of canola oil control and canola oil with 5 g of spices in 100 g of canola oil Means followed by different letters in the same column showed significant difference by Tukey's test (p < 0.05).

Table 4 .
List of compounds identified in methanolic canola oil with oregano and ginger extracts a Retention time; b protonated ion m/z; c daughters of parent ion fragmentation.