Evaluation and comparison of in vitro antioxidant activities of unsaponifiable fraction of 11 kinds of edible vegetable oils

Abstract The radical scavenging capabilities of the extracts from eleven edible vegetable oils were investigated by using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis‐3‐ ethylbenzothiazoline‐6‐sulfonic acid (ABTS), and ferric reducing ability of plasma (FRAP) assays. The results indicated that rapeseed oil and sesame oil showed higher radical scavenging abilities than other vegetable oils. When the radical scavenging capabilities of the extracts from virgin camellia oils and commercially available refined camellia oils were evaluated by FRAP assay, the results showed that the antioxidant capabilities of the former were higher than the latter. Therefore, it is recommended that moderate refining processes should be taken to minimize the loss of antioxidant components and people consume virgin oils or less processed edible vegetable oils for higher antioxidant activities.


| INTRODUC TI ON
Highly active free radicals (FRs) are generated from normal and essential metabolic processes or derived from external sources such as air pollutants and industrial chemicals. If excessive FRs exist, oxidant reactions may be induced and cause many diseases, such as cancers, autoimmune disorders and Alzheimer's and Parkinson's diseases (Bagchi et al., 2000;Devasagayam et al., 2004;Lobo, Patil, Phatak, & Chandra, 2010). Antioxidants can effectively scavenge FRs so as to decelerate human aging and prevent diseases. Lipid oxidation may also be induced by FRs, which may lead to bioactive nutrient loss and deteriorate the quality of edible vegetable oils (Bagchi et al., 2000;Castelo-Branco, Santana, Di-Sarli, Freitas, & Torres, 2016). Therefore, it is necessary to assess the radical scavenging capabilities of antioxidant active compounds in edible vegetable oils, which are essential in human daily life.
Edible vegetable oils contain a variety of nutritional components, such as phytosterols, tocopherols, and polyphenols (Yang et al., 2018). The radical scavenging capabilities of these bioactive ingredients have become hot research topics in recent years, and studies have showed that these nutritional components could provide antioxidant capabilities (Kris-Etherton et al., 2002). Gao (2009) investigated the antioxidant effects of different amounts of brassicasterol under high temperature after it was added to sunflower seed oils. It was found that brassicasterol in sunflower seed oil had antioxidant effects and an increased dose produced a more noticeable effect. β-carotenoid was found to inhibit the oxidation of rapeseed oil and could work with tocopherols to achieve higher antioxidative activities (Xu, Li, & Zhang, 1999). Xu, Hua, and Godber (2001) investigated the antioxidant activities of tocopherols, tocotrienols, and gamma-oryzanol in a cholesterol oxidation system and found that all these compounds exhibited notable antioxidant activities.
It is difficult for animal models to comprehensively reveal the total antioxidant capability due to their high cost and low usability in antioxidant capability measurements. In vitro antioxidant reactions to evaluate antioxidant activities of food are simple and rapid and therefore widely used. At least two different methods should be used to assess the antioxidant capability of a target since no standard in vitro antioxidant evaluation methods are available. All of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing ability of plasma (FRAP) assays were used in this work based on the concentrations of trace bioactive materials in edible vegetable oils to study the systematic antioxidant activities of these oils. Recently, the antioxidant capabilities of the edible oils including virgin coconut oil (Ghani et al., 2018), virgin olive oil (Xiang et al., 2017), sesame oil (Saleem, Chetty, & Kavimani, 2013), and sunflower oil (Aladedunye, Przybylski, Niehaus, Bednarz, & Matthäusa, 2014). It seems that edible oils possess more or less antioxidant capabilities. To discover the advantage of edible oils, it is necessary to detect the antioxidant capabilities of edible oils and compare them with each other. The results in this study reflect the nutritional values of 11 edible oils, which would help consumers comprehensively evaluate the quality and nutritional functions of edible oils.

| Chemicals and reagents
HPLC-grade methanol and acetonitrile, analytical-grade acetone, ether, petroleum ether, n-hexane, and the FRAP kit were supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

| Test sample preparations
Flaxseed oil, olive oil, grape seed oil, corn oil, soybean oil, sunflower seed oil, walnut oil, perilla oil, rapeseed oil, sesame oil, and camellia oil were numbered in the laboratory and stored in the dark. Camellia oils numbered 1-12 were self-prepared, and No. 13-39 were purchased from the market.
A total of 0.2 g (accurate to 0.005 g) vegetable oil was weighed and placed into a glass tube with a plug. A volume of 6 ml 2 mol/L KOH ethanol solution was added for dissolution and evenly mixed by a liquid mixer for 1 min. Saponification was performed in an ultrasonic bath with the ultrasonic conditions as follows: temperature: 75°C; time: 40 min; power: 300 W.
After saponification, the sample was cooled at room temperature and then 7 ml distilled water and n-hexane (1:1, v/v) were added.
The unsaponifiable matter was extracted three times with 3.5 ml hexane. The combined hexane fractions were washed 2-4 times with 10% ethanol/water (v/v) until the washing solution was neutral. The hexane phase was evaporated under a stream of nitrogen, and the residue was dissolved in 1 ml methanol and filtered through a 0.22 μm membrane filter. Subsequently, the obtained liquid was stored at −20°C in a refrigerator for later use. The measurements were performed three times to obtain average values.

| Measurement of radical scavenging capabilities by DPPH assay
The experiment was carried out according to the report of Brand-Williams (Brand-Williams, Cuvelier, & Berset, 1995) with slight modification. Before the experiment, the reagents saved in the refrigerator at −20°C were thawed under room temperature. A volume of 5 ml GENMED dye liquor B (Reagent C2) was added to a bottle filled with GENMED dye liquor A (Reagent C1), and then the mixture was vortexed evenly and labeled as GENMED dye working solution.
It should be noted that the GENMED dye working solution must be saved in the dark before use.
The standard curve solution was prepared as follows: Five centrifugal tubes (1.5 ml) were labeled 1-5. GENMED buffer (Reagent B) and GENMED standard solution (Reagent D) listed in Table 1 were added into each tube, shaken thoroughly, and then stored in the dark.

| Statistical analyses
All antioxidant capability tests were performed in triplicate during the entire course, and all statistical results were analyzed by the SAS software.

| Standard curves for different antioxidant assays
The antioxidant capabilities were measured by DPPH, ABTS, and FRAP assays to construct standard curves. The measurements were conducted in triplicate to obtain average values, and the data were sent for processing using Origin 7.5. Then,

| Evaluation of the antioxidant capabilities of eleven edible vegetable oils
FR chain reactions involve a series of complicated oxidative reactions such as hydrolysis, oxidation, and polymerization, which eventually lead to rancidity of edible vegetable oils and deteriorated nutritional quality of oils (Johnson & Decker, 2015). In this paper, FR chain reactions based on hydroxyl oxygen radicals, superoxide radicals, and hydrogen peroxide were selected to evaluate the antioxidant functions of vegetable oils. The DPPH, ABTS, and FRAP methods were employed to evaluate the radical scavenging capabilities of the extracts from edible vegetable oils.

| Comparison of radical scavenging capabilities of virgin camellia oils with commercially available refined camellia oils
Ferric reducing ability of plasma was used to compare the radical scavenging capabilities of the extracts from virgin camellia oils with those of commercially available refined camellia oils. added before being sold. Earlier literatures indicated that certain amounts of phytosterols and tocopherols remained in the solid residues after a series of edible vegetable oil processing procedures including pressing, heating, alkali refining, and decoloring (Ferrari, Schulte, Esteves, Brühl, & Mukherjee, 1996;Ghazani & Marangoni, 2013). Some weak acid antioxidant materials such as polyphenols may react with alkali substances during the alkali refining procedure, which decreases the antioxidant material concentrations (Deng, Deng, Hu, Li, & Fan, 2015). Therefore, the antioxidant capabilities of the measured virgin camellia oils were higher than the refined oils due to the oversimplified processing procedures and retention of many antioxidant materials to produce better antioxidant effects for the virgin camellia oils. It indicated that virgin vegetable oils had higher nutritional values than refined vegetable oils.

| CON CLUS IONS
In this study, ABTS, FRAP, and DPPH assays were employed to evaluate the radical scavenging capabilities of unsaponifiable fraction of edible vegetable oil. It was discovered that rapeseed oil, and sesame oil had higher total antioxidant capabilities than other oils.
Moreover, the FRAP method was used to compare the radical scavenging capabilities of the extracts from virgin camellia oil with those of commercially available refined camellia oils. The results showed that the antioxidant capabilities of virgin camellia oils were higher than refined camellia oils. Therefore, it is recommended that virgin edible vegetable oils be used rather than refined oils with their radical scavenging capabilities considered.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.