Saffron consists of bioactive compounds with health-promoting properties and is mainly used in medicine, flavoring and coloring. In this study, we aimed to investigate the effect of extraction methods on the antioxidant activity of saffron (Crocus sativus L.) extracts (SE) and to evaluate the antioxidant performance of SE in vegetable oils. Saffron stigmas were extracted in water, ethanol, methanol, and their combinations using maceration extraction (ME), ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), and the combination of UAE with MAE. The results showed that the sample extracted by methanol/water (50:50) using the combination of UAE with MAE methods had the highest amount of total phenolic content (31.56 mg/g GAE) and antioxidant activity (83.24% inhibition). The extract with the highest antioxidant activity was freeze-dried before incorporation into oil samples. Freeze-dried SE contained trans-crocin-4 and trans-crocin-3 (most abundant constituents), kaempferol, and picrocrocin. Moreover, the addition of SE at 1000 ppm resulted in a significant increase in the oxidative stability of canola (CAO), sunflower (SO), and corn oil (COO).
El azafrán contiene compuestos bioactivos con propiedades promotoras de la salud de uso destacado en medicina, saborizante y colorante. En este estudio, nuestro objetivo fue investigar el efecto de los métodos de extracción sobre la actividad antioxidante de los extractos (EA) de azafrán (Crocus sativus L.) y evaluar el rendimiento antioxidante de EA en aceites vegetales. Los estigmas de azafrán se extrajeron en agua, etanol, metanol y sus combinaciones, mediante extracción por maceración (EM), extracción asistida por ultrasonidos (EAU), extracción asistida por microondas (EAM) y la combinación de EAU con EAM. Los resultados mostraron que la muestra extraída con metanol/agua (50:50) usando la combinación de métodos EAU con EAM tuvo la mayor cantidad de fenoles totales (31.56 mg/g GAE) y actividad antioxidante (83.24 % de inhibición). El extracto que incluía la mayor actividad antioxidante se liofilizó antes de incorporarlo a las muestras de aceite. El SE liofilizado contenía
The detection of natural antioxidants has recently become an attractive area of research for both food and pharmaceutical applications. Natural antioxidants can be proposed as substitutes for synthetic antioxidants which have restricted applications due to the harmful health problems like cancerogenic effects which probably occur due to their long-term consumption. Antioxidants cover a broad range of compounds that can retard the degradation of lipids during oxidation and consequently prevent diseases caused by free radicals and enhance the quality and nutritional values of food products (
Saffron (
These health-promoting effects result from the valuable nutraceuticals present in saffron, including crocins, safranal, picrocrocin, crocetin, kaempferol, quercetin, α-carotene, β-carotene, and zeaxanthin. The three main bioactive compounds in saffron stigmas are crocins, picrocrocin, and safranal. Crocins (mainly crocin-4) are the water-soluble mono- and di-glycosyl esters of crocetin (a dicarboxylic acid named, C20H24O4). They are derived from zeaxanthin and have the ability to provide the outstanding golden-red color. Picrocrocin (C16H26O7), the second main component of saffron, is a monoterpene glycoside which is responsible for the bitter taste of saffron resulting from the thermal and enzymatic dissociation of zeaxanthin. Safranal (C10H14O), the volatile oil that contributes to saffron’s unique aroma is a product of the thermal or enzymatic degradation of picrocrocin (
The extraction methods influence extraction efficiency and the quality of the bioactive constituents obtained from saffron. Maceration extraction (ME), steam distillation and Soxhlet extraction have been traditionally used for the extraction of different bioactive compounds from saffron. Furthermore, several modern procedures like microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), ohmic-assisted extraction (OHAE), supercritical fluid extraction (SFE), subcritical water extraction (SWE) and pulsed electric field have been used to obtain various bioactive constituents from saffron (
Traditional extraction approaches suffer from several disadvantages such as prolonged extraction time, use of large volumes of solvents that are not safe for the environment, high energy consumption, low selectivity, and low extraction efficiency (
Conventional ME, includes several steps: firstly, samples are ground to increase the surface area exposed to solvent. Then, they are placed in closed vessels and the appropriate solvent is added. Finally, the solvent containing bioactive compounds is filtered. Time, temperature, and added solvents are defined as important factors for ME (
Ultrasonication extraction involves special kinds of sound waves with high-frequency (20 kHz and 100 MHz), passing through a medium which causes the formation, developing, and collapsing of bubbles based on the cavitation phenomenon. The collapse of bubbles close to the plant cell wall leads to disruption of the cell. Then, the solvent washes out the cell contents including bioactive components. This method has some advantages such as the possibility of extraction at ambient temperature, enhancing the mass transfer, being simple and rapid, high extraction rate, and high purity of the extract (
In the Maceration extraction process, the transfer of mass and heat occurs in opposite directions; while in MAE, it happens in the same direction from inside plant material to the solvent medium. Consequently, during microwave extraction, solute transfer is accelerated as a result of the one-pot heat-mass transfer, and the extraction rate of bioactive compounds increases. On the other hand, conventional extraction methods lead to the collection of high amounts of undesirable components in the extracted solution which cause the quality and purity of the extract to decrease (
Vegetable oils are sensitive to oxidation due to containing high levels of polyunsaturated fatty acids, and therefore the use of antioxidants is necessary to retard their oxidation (
Several studies have been conducted to evaluate the antioxidant properties of food products supplemented with saffron extract (SE). For example, the incorporation of saffron into wheat flour pasta and fresh ovine cheese enhanced their antioxidant activity and their sensory properties (
Dried saffron stigmas were purchased from Jamshidi Marandi producer (Khorasan-e-Razavi, Iran). Refined, bleached and deodorized (RBD), canola oil (CAO), sunflower oil (SO) and corn oil (COO), without added antioxidant, were supplied from Cargill Co., Istanbul, Turkey. Crocin-4 with 98% purity was purchased from Biopurify Phytochemicals Ltd (Sichuan, China). Gallic acid, Folin-Ciocalteu’s phenol reagent, and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS)) reagent were purchased from Merck (Darmstadt, Germany). All solvents were obtained from Merck and were of HPLC-grade.
Five grams of dried saffron stigmas were completely ground using a porcelain mortar and screened with a sieve with mesh size of 0.5 mm. Then, the dried powder from saffron stigmas (0.2 g) was extracted using 15 mL of solvents (water, ethanol, methanol, the mixture of ethanol: water, 50:50 v/v and methanol: water, 50:50 v/v) with different extraction methods including ME, UAE, MAE and combinations of UAE and MAE. Then, the extracts were filtered using a stainless-steel Buchner funnel (Sartorius AG 3400 Gottingen, Germany). Finally, the extracts were completed to 25 mL with extraction solvent, and kept at -20 °C until further analysis.
ME was carried out using a shaker incubator (IKA® KS 4000 I control, Germany). Ground saffron stigmas (0.2 g) were extracted with different previously described solvents for 24 h, at a speed of 100 rpm at room temperature (25 ±1.0 ºC).
UAE was performed using an ultrasonic probe with automatic control of time, cycles, and power (Bandelin Sonoplus HD 3100, 20 kHz frequency with an MS 73 probe). The same amount of saffron was extracted with the same extraction solvents for 3 min using 4 cycles at a power between 50-60% of the maximum power (
For MAE, the prepared samples were located in a conventional microwave oven (Arçelik MD 565 S, Turkey). Ground saffron stigmas (0.2 g) were extracted with extraction solvents (as mentioned above) using a microwave power level of 30% for 2 min (
Ground saffron stigmas (0.2 g) were weighed and the extraction solvents were added. The extraction was performed using combined methods of UAE and MAE as described in the previous section. Firstly, UAE was performed, followed by MAE.
The TPC of all extracted solutions was determined calorimetrically at 725 nm using the Folin-Ciocalteau reagent according to the method described by
The antioxidant property of the extract was analyzed according to the ABTS method (
Saffron samples were analyzed according to the ISO 3632 trade standard (
Where D is the specific absorbance; m is the mass of the saffron sample in grams; H is the moisture and volatile content of the sample, expressed as a mass fraction. For our sample, the H value was about 5%.
After the characterization of the extracts obtained by different solvents and extraction methods, the extract which had the highest levels of bioactive components was selected for the second part of the study. This extract was freeze-dried to be inserted into the oils for the evaluation of their oxidative stability.
This extract was obtained using methanol/water (50:50) and combinations of UAE and MAE. Then, the obtained extract was concentrated by a rotary evaporator, filtered and kept at -20 °C for 24 h. Finally, the extract was freeze-dried using a LyoAlfa 6-50 freeze-dryer (Telstar, Terrassa, Spain) for 16 h.
The Shimadzo HPLC system (Kyoto, Japan) with two pumps (LC-20AD) was applied for the detection of SE bioactive components. This system was equipped with a photodiode array detector (UV-Vis PDA, SPD-M20A). The freeze-dried SE in the concentration of 100 µg/mL was re-dissolved in 50:50 methanol/water, then filtered through a 0.2 µm (Millipore) filter and injected into the system at a volume of 20 µL at 30 °C. The column was Alltima (C18 zorbax, 250 mm × 5 m; 4.6 mm id). The mobile phase consisted of solvent A (Water containing 0.1% formic acid) and solvent B (Acetonitrile containing 0.1% formic acid). A gradient program was performed to analyse the SE components: 20% B for 5 min, then increased linearly until 80% B in 30 min, then adjusted to 98 % B and kept for 5 min and then decreased to 20 % B and kept for 15 min at a constant flow rate of 0.8 mL·min-1. The detection wavelengths were set at 440 nm and 250 nm, i.e., the maximum absorbance for crocins and picrocrocin, respectively.
Standard solutions of crocin-4 were prepared in 50% methanol/water (v/v) at concentrations of 1-20 µg/mL. (Y=156331X, R2 = 0.999). Picrocrocin was identified by LC-MS, while its quantification was performed using the regression equation from the literature, Y = 1952830X - 3808.1 (
The identification of each compound was performed with the LC system including solvent delivery pump and PDA detector. It was coupled to a LTQ Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, USA). The HPLC conditions were the same as the conditions described above for HPLC-UV. The peaks were detected at wavelengths of 279.5-280.5 nm. Mass spectra were obtained in positive (ESI+) and negative ion modes (ESI−) in scan ranges of 120-2000 m/z (
Peroxide value (PV) and acid value (AV) for the oil samples were determined according to the AOCS methods (Cd-8-53 and AOCS Cd-3a-63) (
The fatty acid composition of the oils was determined by the conversion of oil to fatty acid methyl esters (FAMEs) according to the modified method of
Before measuring the OSI of the oil samples, the saffron extract was blended homogenously with the oil samples. At first the freeze-dried saffron extract was dissolved in an appropriate volume of 1,2-propanediol (20% w/w). Then, the solution was placed in the test tubes containing oil samples (10 mL) and subjected to the sonication (4 cycles, power of 50-60% maximum power) for 2 min using an ultrasonic probe.
The SE at concentrations of 1000 and 1500 ppm (w/w) were examined for the stabilization of oil samples. Since no significant differences were observed in the OSI values of the oil samples enriched with both concentrations of SE, 1000 ppm level was selected to be inserted into the oil samples. The OSI of control oils and oils treated with SE and BHT (200 ppm) was measured using the Metrohm Rancimat model 743 (Herisau, Switzerland) according to (
All experiments were conducted in triplicate. The values of the means were statistically analyzed by IBM SPSS statistics software package (version 17.0). The results were analyzed by one-way ANOVA and followed by the TUKEY test. The cluster analysis was used to classify objects into relative groups according to Minitab® 16 Statistical Software, 2010. Data were presented as mean ± standard deviation (SD).
Extraction techniques can be used alone or in combination with other methods for the separation and purification of different bioactive ingredients from various parts of saffron. The quality of the extracted bioactive ingredients is important for further applications in the formulation of nutraceutical and functional food products (
As can be seen from
Treatments | Sample code | Total phenolic content (TPC) (mg GAE/g of DW*) | Antioxidant activity using ABTS+ (% inhibition) | ||||
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Maceration for 24 h | |||||||
Water | 1 | 13.08 | ± | 0.82j | 63.28 | ± | 3.31f |
Ethanol | 2 | 18.83 | ± | 0.16h | 57.37 | ± | 1.51i |
Methanol | 3 | 29.54 | ± | 0.38bc | 75.50 | ± | 3.31bc |
Ethanol/Water (50:50) | 4 | 24.15 | ± | 0.22fg | 68.32 | ± | 0.40ef |
Methanol/Water (50:50) | 5 | 29.46 | ± | 0.71bc | 78.76 | ± | 1.91ab |
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Water | 6 | 28.03 | ± | 0.88cd | 72.16 | ± | 0.20cde |
Ethanol | 7 | 4.43 | ± | 0.44l | 47.03 | ± | 1.71hi |
Methanol | 8 | 27.25 | ± | 0.44de | 71.66 | ± | 0.10cde |
Ethanol/Water (50:50) | 9 | 29.27 | ± | 0.33bc | 72.16 | ± | 0.20cde |
Methanol/Water (50:50) | 10 | 30.86 | ± | 0.49ab | 79.83 | ± | 0.60ab |
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Water | 11 | 11.22 | ± | 0.82j | 75.64 | ± | 0.30bc |
Ethanol | 12 | 16.65 | ± | 0.05i | 50.71 | ± | 1.41g |
Methanol | 13 | 27.29 | ± | 0.49de | 68.75 | ± | 1.81def |
Ethanol/Water (50:50) | 14 | 25.62 | ± | 0.22ef | 74.50 | ± | 1.10bcd |
Methanol/water (50:50) | 15 | 30.05 | ± | 0.44ab | 79.62 | ± | 0.30ab |
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Water | 16 | 22.98 | ± | 0.33g | 80.54 | ± | 1.00ab |
Ethanol | 17 | 8.66 | ± | 0.05k | 56.46 | ± | 1.51gh |
Methanol | 18 | 25.93 | ± | 0.11ef | 74.57 | ± | 1.41bcd |
Ethanol/Water (50:50) | 19 | 24.18 | ± | 0.16fg | 69.96 | ± | 1.51cde |
Methanol/water (50:50) | 20 | 31.56 | ± | 0.38a | 83.24 | ± | 0.60a |
Values are means ± standard deviation (n = 3); Different superscript letters in the same column represent significant difference (p < 0.05, one-way ANOVA with Tukey test) *DW, dry weight of saffron stigmas.
The TPC of the ethanol/water (50:50, v/v) extract was higher than that obtained with absolute ethanol. According to the previous studies, the blending of organic solvents with water increases the polarity of the extraction medium and may allow easier extraction of the components which are soluble in water or in organic solvents (
In the case of UAE with pure solvents, the TPC of the extracts was in the range of 4.43 to 28.03 mg GAE /g in the following decreasing order: water > methanol > ethanol. Using solvent and water mixtures (50:50, v/v) for extraction led to a significant increase in TPC. The most prominent increase was observed for ethanol; the TPC of 4.43 mg GAE /g obtained with pure ethanol increased to 29.27 mg GAE/g when 50:50 (v/v) ethanol/water mixture was used. The solvent properties can affect the extraction of bioactive components from plant cells. Since ethanol has higher viscosity compared to other solvents, the mass transfer rate of these compounds reduces and moreover, the very short extraction time of the UAE method (3 min) can also result in less extraction of phenolic compounds with ethanol. Extraction with a mixture of water and ethanol enhances mass transfer and therefore accelerates the extraction of TPC due to its lower viscosity (
For MAE, a similar tendency as for ME was observed. The TPC increased significantly (p < 0.05) when the combination of UAE and MAE was used, which ranged from 8.66 to 31.56 mg GAE /g for ethanol and methanol/water, respectively. Moreover, the combination of UAE and MAE was more efficient especially in the case of using methanol/water as the solvent.
The results of the Pearson correlation analysis shown in
The saffron samples, extracted with different techniques and solvents, were analyzed by spectrophotometric analysis in order to evaluate the absorbance values due to the presence of their secondary metabolites, crocins, picrocrocin, and safranal according to the ISO 3632 trade standard (
Treatment | Sample Code | Crocin content |
Safranal content |
Picrocrocin content |
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Water | 1 | 331.2 | ± | 3.0i | 127.4 | ± | 9.9def | 273.1 | ± | 12.2fg |
Ethanol | 2 | 513.4 | ± | 41.8h | 89.8 | ± | 2.3fg | 227.4 | ± | 11.4g |
Methanol | 3 | 1084.9 | ± | 9.1bcd | 148.4 | ± | 4.6de | 366.1 | ± | 9.9de |
Ethanol/Water (50:50) | 4 | 994.1 | ± | 12.9def | 152.2 | ± | 3.8de | 354.8 | ± | 4.6de |
Methanol/water (50:50) | 5 | 1019.4 | ± | 21.3cdef | 224.2 | ± | 6.8a | 467.7 | ± | 10.6bc |
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Water | 6 | 924.7 | ± | 53.2f | 199.5 | ± | 6.8abc | 415.6 | ± | 23.6cd |
Ethanol | 7 | 331.2 | ± | 9.1i | 39.8 | ± | 3.0h | 346.8 | ± | 29.7e |
Methanol | 8 | 1191.4 | ± | 39.5ab | 147.3 | ± | 15.2de | 381.2 | ± | 23.6de |
Ethanol/Water (50:50) | 9 | 1178.0 | ± | 34.2ab | 162.9 | ± | 11.4bcde | 459.1 | ± | 15.2c |
Methanol/water (50:50) | 10 | 995.7 | ± | 10.6def | 168.8 | ± | 15.2bcd | 372.0 | ± | 18.2de |
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Water | 11 | 1031.2 | ± | 45.6cdef | 204.3 | ± | 19.8ab | 463.4 | ± | 33.5bc |
Ethanol | 12 | 674.7 | ± | 9.9g | 72.0 | ± | 3.0gh | 249.5 | ± | 3.0g |
Methanol | 13 | 1055.4 | ± | 2.3cde | 125.8 | ± | 6.1ef | 326.9 | ± | 3.0ef |
Ethanol/Water (50:50) | 14 | 1054.3 | ± | 2.3cde | 150.5 | ± | 7.6de | 525.8 | ± | 15.2ab |
Methanol/water (50:50) | 15 | 1050.5 | ± | 1.5cde | 159.1 | ± | 7.6cde | 369.4 | ± | 8.4de |
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Water | 16 | 1124.2 | ± | 14.4abc | 194.6 | ± | 15.2abc | 470.4 | ± | 16.0bc |
Ethanol | 17 | 636.6 | ± | 4.6g | 59.7 | ± | 9.9gh | 361.8 | ± | 34.2e |
Methanol | 18 | 1137.6 | ± | 85.2abc | 140.3 | ± | 17.5de | 334.4 | ± | 9.1ef |
Ethanol/Water (50:50) | 19 | 959.1 | ± | 13.7ef | 141.9 | ± | 12.2de | 526.3 | ± | 3.8ab |
Methanol/water (50:50) | 20 | 1226.3 | ± | 9.9a | 226.3 | ± | 5.3a | 536.0 | ± | 3.8a |
Values are means ± standard deviation (n = 3); Different superscript letters in the same column represent significant difference (p < 0.05, one-way ANOVA with Tukey test).
The absorbance values for the crocin component of the extracts were higher than picrocrocin and safranal. They ranged from 331.2 for water extract (ME) to 1226.3 for methanol/water extract using UAE in combination with MAE. The absorbance values for the picrocrocin component at 257 nm ranged from 227.4 for ethanolic extract by ME to 536.0 for methanol/water extract using combinations of UAE and MAE (
Ethanol-water solvent mixtures were found to be the best media for the extraction of crocin and polyphenols from saffron with traditional extraction methods (
From the obtained data, it could be concluded that the MAE method, as well as UAE affected crocins, picrocrocin, and safranal contents. In addition, the extraction solvent was the most important factor in the efficiency of the extraction process.
A cluster analysis was carried out to classify the results of the three extraction methods and five extraction model systems into relative groups. The Dendrogram classified various crocins, picrocrocin, and safranal contents in this study into four main groups as shown in
Compound | Quantity (mg/g of Extract) |
---|---|
Picrocrocin | 3.28±0.00 |
Campherol diglucoside | nda |
Trans-crocine-4 (2 gen) | 168.17±0.28 |
Trans-crocin-3(gen, glu) | 60.55±1.99 |
Cis-crocin-4 | 10.47±0.06 |
Cis -crocin-3 | 29.49±0.47 |
Cis-crocin-2’ (2 glu) | nd |
a nd: not determined, value = means ± standard deviation (n = 3)
The total crocin content in our freeze-dried saffron extract was 268.7 mg of crocin-4 eq/g of extract which was similar to the findings of
Vegetable oils are rich in unsaturated fatty acids, particularly monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). The fatty acid composition of COO, CAO and SO, expressed as saturated and unsaturated (mono and polyunsaturated) fatty acids are summarized in
Parameter | Oil samples | ||
---|---|---|---|
Corn oil | Canola oil | Sunflower oil | |
Acid value (mg/g) | 0.12 ±0.01 | 0.23 ±0.02 | 0.17 ±0.01 |
Peroxide value (meqO2/kg) | 0.82 ±0.03 | 0.85 ±0.03 | 0.78 ±0.07 |
|
Area % | ||
Myristic acid (C14:0) | 0.04 | 0.05 | 0.09 |
Palmitic acid (C16:0) | 11.46 | 5.1 | 7.9 |
Palmitoleic acid (C16:1) | 0.53 | 0.21 | 0.14 |
Margaric acid (C17:0) | nd* | nd | 0.04 |
Heptadecanoic acid (C17:1) | nd | nd | 0.03 |
Stearic acid (C18:0) | 2.14 | 1.7 | 2.7 |
Oleic acid (C18:1) | 33.4 | 60.2 | 35.33 |
Linoleic acid (C18:2) | 50.5 | 23.7 | 52.3 |
Linolenic acid (C18:3) | 0.93 | 6.6 | 0.16 |
Arachidic acid (C20:0) | 0.44 | 0.52 | 0.26 |
Arachidonic acid (C20:1) | 0.41 | 1.2 | 0.27 |
Behenic acid (C22:0) | 0.15 | 0.6 | 0.6 |
Erucic acid (C22:1) | nd | 0.13 | nd |
Lignoceric acid (C24:0) | nd | nd | 0.21 |
∑ SFA | 14.23 | 7.97 | 11.55 |
∑ UFA | 85.77 | 91.91 | 88.2 |
∑ MUFA | 34.34 | 61.60 | 35.74 |
∑ PUFA | 51.43 | 30.3 | 52.46 |
*nd= not detected; value = mean ± SD (n=3)
The induction time for the control CAO was 17.38 h, and it increased significantly (p < 0.05) to 20.82 h in the case of samples treated with BHT (200 ppm), and samples treated with SE at 1000 ppm (19.41 h) (
The values provided in the figures are the mean values of triplicate analyses with standard deviation.
There were no significant differences (p < 0.05) between the induction times of SO treated with BHT and SE (12.50 and 12.12 h, respectively). The difference was clear with the control (9.61 h). On the other hand, the results for COO showed a protective effect against oxidation for the samples containing BHT (21.43 h) and SE at 1000 ppm (17.55 h) compared to the control (15.28 h). The protection factor (PF) was calculated (
Some studies investigated the antioxidant activity of SE against synthetic antioxidants by the DPPH and ABTS methods (
For corn oil without antioxidant, the OSI value was reported as 9.8 h by
According to the results, among the different extraction methods used in this study, UAE combined with the MAE method revealed the highest TPC as well as antioxidant activity. The type of extraction solvent was found to be important to enhance the efficiency of extraction of bioactive compounds from saffron stigmas. The LC-MS analysis showed trans-crocin-4 and trans-crocin-3 as the main constituents of freeze-dried SE and this extract contained high a level of total crocin content (268 mg/g). Therefore, it exhibited significant potential in the inhibition of CAO, SO and COO oxidation in comparison with BHT as a synthetic antioxidant. Based on the oxidative stability analysis, SE (1000 ppm) had the same effect (p < 0.05) as BHT (200 ppm), in preventing the oxidation of sunflower oil used in the study. In the future, further studies could be conducted with different concentrations of SE on the stability of vegetable oils during accelerated storage and frying.
This work was supported by a grant from Coordination Unit of Scientific Research Projects, ITU, Turkey (BAP) (Project No: 41582). We thank the Cargill Co., Turkey for supplying the vegetable oils used in this study.