Effect of polyglycerol polyricinoleate on the inhibitory mechanism of sesamol during bulk oil oxidation

In this study, effects of sesamol on improving the oxidative stability of sunflower oil and its oil-in-water emulsion was investigated. To investigate the kinetic parameters related to the initiation and propagation stages of oxidation, a sigmoidal-model was used. Sesamol exhibited higher antioxidant activity in sunflower oil-in-water emulsion than that of sunflower oil. In both sunflower oil and sunflower oil-in-water emulsion, the inhibitory effect of sesamol against lipid oxidation continued even after the induction period. To improve the efficiency of sesamol in sunflower oil, polyglycerol polyricinoleate (PGPR) was incorporated into the functional environment of the sesamol. Sesamol exhibited a synergistic effect with PGPR during both initiation (synergistic effect of 68.87%) and propagation (synergistic effect of 36.84%) stages. Comparison of the size of reverse micelles in samples containing PGPR with those without PGPR revealed that PGPR can enhance the efficiency of sesamol by increasing the acceptance capacity of lipid hydroperoxides in reveres micelles structures. This can result in enhancing the effective collisions between sesamol and lipid hydroperoxides in the presence of PGPR. The water produced as a major byproduct of oxidation played a key role on the antioxidant activity of sesamol alone or in combination with PGPR during oxidation process.

www.nature.com/scientificreports/ organo-haloperoxyl radicals 9 . The efficiency of a selected antioxidant in the oil-based food products is not only determined by its chemical reactivity. Antioxidant activity of an antioxidant in oil-based food products is the result of more complex reactions and phenomena. Many factors such as chemical reactivity and interaction of an antioxidant with other food compounds, environmental conditions, and interfacial activity of the antioxidant compound can affect its efficiency in oil-based food products 2,10 . A highly reactive antioxidant needs to be located at water-oil interface of reverse micelles in bulk oil and at the surface of oil droplets in oil-in-water emulsion to encounter pro-oxidants and protect unsaturated fatty acids 11 . Incorporating surfactants into bulk oils at concentrations below their critical micelle concentration (CMC) can improve the interfacial activity of antioxidants. Considering the fact that surfactants can significantly reduce interfacial tension, the number and size of the reverse micelles are likely to increase significantly in the presence of surfactants. As a result, there can be an increase in the acceptance capacity of LOOHs in these structures. Accordingly, more interactions can occur between antioxidant molecules and LOOHs in the presence of surfactants. In addition, increasing the number of reverse micelles in the presence of surfactants can excite the movement of the nonpolar part of antioxidant molecules into the water-oil interface of reverse micelles 12,13 .
In this study, effects of sesamol on inhibiting the oxidation of sunflower oil and its oil-in-water emulsion was investigated. A sigmoidal kinetic model was used to evaluate the inhibitory effect of sesamol over the whole practical range of peroxidation, including the initiation and propagation stages. In addition, polyglycerol polyricinoleate (PGPR) which is considered as a surfactant with low HLB (hydrophilic lipophilic balance) value was incorporated into sunflower oil to improve the antioxidant activity of sesamol (based on the interfacial phenomena), so as to inhibit the oxidation of stripped sunflower oil. Furthermore, various kinetic parameters and rate constants arising from the initiation and propagation stages of lipid oxidation were evaluated to elucidate the details of physicochemical events that occurred during the oxidation process. This can be considered as the first attempt to use these evaluations in describing how PGPR can affect the interfacial activity of sesamol during the initiation and propagation stages of oxidation.
Sunflower oil stripping. Sunflower oil stripping was performed by an adsorption chromatography column. A glass column (36 cm length and 2.9 cm internal diameter) was packed with silica gel (20.04 g) and aluminum oxide 60 (140.04 g). Silica gel and aluminum oxide 60 were activated at 180 °C for 4 h. Sunflower oil (120 g) was passed through the column by a vacuum pump. The stripping procedure was performed twice to gain inconsiderable levels of indigenous antioxidative compounds and lipid hydroperoxides 14 . Preparation of sunflower oil samples. Sesamol was dissolved in acetone and added to the purified sunflower oil at concentration of 0.05% (w/w) oil. Then, acetone was evaporated under a stream of nitrogen. To provide samples containing PGPR, 0.05% (w/w oil) of PGPR was dissolved in ethyl acetate (1:10 w/v) for 1 h at 40 °C by a magnetic thermo-stirrer. Then, purified sunflower oil was slowly added to the cooled solution and the stirring process remained at ambient temperature for 10 min. Afterwards, ethyl acetate was eliminated by a rotary evaporator. In the next step, sesamol (0.05% w/w oil) was separately added to the purified sunflower oil containing PGPR. The CMC value of PGPR vary between 0.76 and 1.50% in the oil phase 15 . Since surfactants self-aggregate and form reverse micelles above their CMC value 16 , a concentration lower than the CMC of PGPR was used in this study.
Preparation of sunflower oil-in-water emulsion samples. Sunflower oil-in-water emulsion samples were prepared using the emulsion phase inversion method. Initially, purified sunflower oil and Tween 80 were mixed using a magnetic stirrer (750 rpm) for 30 min. Then, sesamol (0.5%, w/w oil) was dissolved in acetone and added to the purified sunflower oil. After that, the acetone was removed from purified sunflower oil samples using nitrogen stream. Finally, potassium phosphate buffer solution (0.04 mol L −1 , pH 7) was titrated into purified sunflower oil containing Tween 80 with a flow rate of 300 µL/min, while continuing to stir the system by magnetic stirrer (750 rpm). The Tween 80:oil ratio was 1:1 and the oil:water ratio was 1:10 17 . The particle size of the sunflower oil-in-water emulsion was 181.05 ± 0.49 nm.
Monitoring accumulation of LOOHs. Accumulating of LOOHs during storage at 55 °C was monitored by measuring peroxide value (PV) at certain time intervals. To determine PV, the oil samples (0.001-0.3 g) were mixed with 9.8 mL chloroform-methanol (7:3, v/v) using a vortex mixer for 2-4 s. Then, 50 μL of ammonium thiocyanate aqueous solution (30%, w/v) was added to the oil sample and shaked for 5 s. After that, 50 μL Iron (II) chloride solution ([0.25 g FeSO4.7H 2 O dissolved in 25 mL H 2 O] + [0.2 g barium chloride dehydrate dissolved in 25 mL H 2 O] + 1 mL HCl 10 N, and then the resultant solution was filtered to remove barium sulphate deposits) was added. After 5 min incubation at room temperature, absorption values of samples were determined at 500 nm 18 . For oil extraction from emulsions, 1.5 mL of chloroform:methanol (1:1, v/v) was blended with 0.3 mL emulsion and vortexed for 1 min. Then, the mixture was centrifuged for 5 min at 1300g. The lower lipid layer was collected and its solvent evaporated using nitrogen stream. where k c (h −1 ) is the pseudo-first order rate constant of LOOHs formation at the propagation stage, k d (kg meq −1 h −1 ) is the pseudo-second order rate constant of LOOHs decomposition at the propagation stage, C (kg meq −1 ) is an overall integration constant 19 . The Eq. (3) exhibits a sigmoidal characteristic as illustrated in Fig. 1.
The second derivative of the sigmoidal equation (d 2 [LOOH]/dt 2 ) at t = 0 provided the coordinates of a turning point (T max , h). In this point, the rate of LOOH accumulation approaches a maximum value (R max , meq kg -1 h −1 ) in the propagation stage (Fig. 1). T max was calculated according to Eq. (5).
The initiation oxidizability parameter (O i , h 2 meq −1 kg), which unifies k i and IP, could show well the resistance of the sunflower oil samples to the formation of LOOH during the initiation stage. The O i was calculated using Eq. (11).
Antioxidant effectiveness in the initiation stage was calculated using Eq. (12).
where IP AH is the IP in the presence of antioxidant and IP C is the IP in the absence of antioxidant.
Oxidation rate ratio (ORR) in the initiation stage, which is an inverse measure of antioxidant strength, was calculated using Eq. (13).
where k i, AH is the value of k i in the presence of antioxidant and k i, C is the value of k i in the absence of antioxidant. Antioxidant activity (A) was calculated using Eq. (14).
Synergistic effect of sesamol with PGPR during the initiation stage (SE i ) was calculated according to Eq. (15).
where IP i,AH , IP i,P , IP i , C, and IP i, AH+P are initiation oxidizability parameter of the antioxidant per se, PGPR per se, control, and antioxidant + PGPR, respectively.
The end time of the propagation stage (ET pp , h) was calculated according to Eq. (16).
Antioxidant effectiveness during the propagation stage was calculated using Eq. (18).
where PP AH is the PP in the presence of antioxidant and PP C is the PP in the absence of antioxidant. The oxidation rate ratio of LOOHs formation during the propagation stage was calculated using Eq. (19).
where k c, AH is the value of k c in the presence of antioxidant and k c, C is the value of k c in the absence of antioxidant. The oxidation rate ratio of LOOHs decomposition during the propagation stage was calculated using Eq. (20). www.nature.com/scientificreports/ where k d, AH is the value of k d in the presence of antioxidant and k d, C is the value of k d in the absence of antioxidant. IA c that is the inhibitory activity against the LOOHs formation, and IA d that is the inhibitory activity against the LOOHs decomposition were calculated using Eqs. (21) and (22), respectively 21 .
Synergistic effect of sesamol with PGPR during the propagation stage (SE p ) was calculated according to Eq. (23): where T max,AH , T max,P , T max , C, and T max, AH+P are propagation oxidizability parameter of the antioxidant per se, PGPR per se, control, and antioxidant + PGPR, respectively.
Water content. Changes in water content of sunflower oil samples during lipid oxidation were determined by coulometric Karl Fischer titrator (KF Titrino, Metrohm, Herisau, Switzerland)) using the ASTM E1064 standard test method 22 . Particle size. Particle size of sunflower oil and sunflower oil-in-water emulsion samples were determined using dynamic light scattering instrument (SZ-100 nanopartica series, Horiba Ltd., Kyoto, Japan) at light scattering angle of 173°. Sunflower oil-in-water emulsion was diluted 100-times using potassium phosphate buffer (0.04 mol L −1 , pH 7) prior to analysis to avoid multiple scattering effects.
Viscosity. The viscosity of sunflower oil and its oil-in-water emulsion was measured using a capillary viscometer (Schott Gerate 51810; Germany). The dynamic viscosity was calculate at 25 °C using Eq. (24) 23 .

Statistical analysis.
All experiments were done in three independent tests. Significant differences among the mean values were measured using a one-way analysis of variance. Comparisons of the mean values were carried out using Duncan's multiple range test (P < 0.05). Statistical and regression analyses were performed using SPSS, CurveExpert, and Microsoft Office Excel software.

Results and discussion
Evaluating kinetic parameters related to the initiation stage of lipid oxidation. The predicted sigmoidal kinetic model fitted well on the curve of LOOHs production (R 2 ≥ 0.985) and distinguished the different stages of the oxidation process (Fig. 2). During lipid oxidation process, a variety of free radicals with different redox potentials (Eh) such as alkoxyl (LO · : 1600 mV), hydroxyl ( · OH: 2320 mV), peroxyl (LOO · : 1000 mV), and alkyl (L · : 600 mV) can be produced in the lipid systems 24,25 . At the beginning of the oxidation process, the only pathway of LOOHs production is the conversion of L · to LOO · (due to its low Eh) and its attack on the hydrogen attached to allylic or bis-allylic carbon 26 . The initiation stage can be basically characterized by IP, k i , and [LOOH] IP . In both sunflower oil and its oil-in-water emulsion, sesamol significantly increased the IP, E i , and A values and reduced the ORR and O i values, compared to the control sample. Also, in both sunflower oil and sunflower oil-in-water emulsion, the [LOOH] IP value of sample containing sesamol was higher than the control sample (Table 1). This indicates that sesamol has participated in chain termination reaction (AH + LOO · → LOOH + A · ) and increased hydroperoxide concentration. Sesamol exhibited higher efficiency in increasing the A value of sunflower oil-in-water emulsion than the sunflower oil. The location of antioxidant molecules or, indeed, their interfacial distribution in lipid system is an important parameter that can affect their efficiency on inhibiting the lipid oxidation. In sunflower oil, hydrophilic antioxidants that are able to locate at the interface of the reverse micelles can inhibit the lipid oxidation more efficiently than hydrophobic antioxidants, while in sunflower oil-in-water emulsion, hydrophobic antioxidants that are able to locate at the surface of the oil droplets are more effective than hydrophilic antioxidants 11 . Sesamol is a moderately lipophilic compound that has low solubility in water (~ 38.8 mg mL −1 ). The partition coefficient (log P) value of sesamol is 1.29. The log P value is positive if a substance is more soluble in fat-like solvents, and is negative if it is more soluble in water 27 . Accordingly, sesamol has higher affinity for locating at the surface of oil droplets of sunflower oil-in-water emulsion than locating at the interface of reverse micelles of sunflower oil that result in its higher antioxidant activity in sunflower oil-in-water emulsion. Another important factor that determines the efficiency of antioxidants in lipid systems is their ability to be transferred to the active site of oxidation. A highly reactive antioxidant needs to be mobile and to diffuse easily to the site of action. Viscosity is an important factor that can govern the transfer rate and mobility of antioxidants in lipid systems. A decrease in viscosity can improve the mass transfer of antioxidants in lipid systems 16 . In this study, the dynamic viscosity of sunflower oil and sunflower oil-in-water emulsion were 47.57 ± 0.70 and 2.11 ± 0.50 mPa s, respectively. The lower viscosity of sunflower oil-in-water emulsion www.nature.com/scientificreports/ is expected to result in a higher transfer rate of sesamol toward the actual sites of oxidation than that of sunflower oil. In addition, in this study Tween 80 was used in sunflower oil-in-water emulsion at concentration higher than its CMC value. When surfactants are used at concentration higher than their CMC value, they form micelles in the continuous phase of oil-in-water emulsions 28 . Surfactant micelles can act as carriers of lipophilic compounds, and thereby can cause an increase in the transfer rate of sesamol toward the actual site of oxidation 29 . As a consequence, the probability of effective collisions between sesamol molecules and peroxyl radicals or ROOHs in actual sites of oxidation in sunflower oil-in-water emulsion is higher than that of sunflower oil. PGPR was incorporated into the sunflower oil to improve the interfacial activity of sesamol in sunflower oil. Incorporating PGPR into the functional environment of sesamol resulted in a remarkable change in the mechanism of hydrogen donating of sesamol, which ultimately increased the E i and A values and decreased the ORR and O i values (Fig. 3a). PGPR did not show any significant antioxidant activity in the stripped sunflower oil, but synergistically improved the inhibitory effect of sesamol (SE i of 41.50%). This implies the ability of PGPR in transferring sesamol to the water-oil interfaces created by reverse micelles, which results in improving the accessibility of sesamol to the reactive free radicals.
According to Fig. 3b, the O i value was decreased by increasing the amount of produced water in the initiation stage of lipid oxidation. A linear relationship was found between the O i value and the amount of water being produced during the initiation stage of the lipid oxidation (R 2 = 0.964).
During the oxidation process, the water contents of sunflower oil samples were increased significantly due to the occurrence of bimolecular reactions of LOOHs (LOOH + LH → LO · + L · + H 2 O). In samples containing sesamol, the amounts of water increased to the higher extent than that of the control sample (Table 2). It has been shown that sesamol participates in the side reactions of chain initiation 14 . Accordingly, the higher amounts of water in samples containing sesamol can be related to the participation of a part of this molecule in side reactions of the chain initiation (AH + LOOH → LO · + A · + H 2 O), which produces water. The produced water plays a key role in the interfacial activity of sesamol during the oxidation process 13 .
In all sunflower oil samples, the size of reverse micelles increased by increasing the amounts of water until the end of the initiation stage (Table 2). After the point of IP, a significant reduction was observed in the reverse micelles size, which can be attributed to the production of a large amount of water through bimolecular reaction (2LOOH → LOO · + LO · + H 2 O). The produced water can migrate to the core of the reverse micelles of sunflower oil and cause their collapse due to the volume increase 28 . During the propagation stage of lipid oxidation, the reverse micelles were regenerated and their size was increased again. In samples containing PGPR, size of the reverse micelles increased to the higher extent than those samples without PGPR. This may be due to the greater reduction of interfacial tension in the presence of PGPR. The effect of this behavior change is well observable in increasing the reverse micelles size at the IP point ( Table 2). As previously mentioned, the addition of PGPR into the sunflower oil containing sesamol improved the kinetic parameters related to the initiation stage of the lipid oxidation. This can be attributed to the increase in the size of reverse micelles in the presence of PGPR, which  Table 2, increasing the size and number of reverse micelles was parallel with the amount of water produced in the system. The water molecules produced during the oxidation process can reduce the interfacial tension by attaching to the hydrophilic part of PGPR 13 . Accordingly, the water molecules can create preliminary cores and accelerate the formation of reverse micelles in the presence of PGPR. By increasing the number of the reverse micelles in the presence of PGPR, sesamol will have higher chance to locate at the oil-water interface of reverse micelles 30,31 . Evaluating kinetic parameters related to the propagation stage of lipid oxidation. The propagation stage was characterized by various kinetic parameters and rate constants ( Table 1). The T max value can use instead of IP during the propagation stage. In both sunflower oil and sunflower oil-in-water emulsion, the T max value of samples treated with sesamol was significantly higher than that of control sample (P < 0.05). In addition, the k c and k d values of sunflower oil and sunflower oil-in-water emulsion samples containing sesamol were lower than that of control sample (P < 0.05). The R n value, which is a measure of propagation oxidizability, can be taken into account as a more comprehensive kinetic parameter encompassing the values of every single kinetic parameter and the rate constant mentioned above 20 . The lower value of R n indicates a higher resistance of the system against lipid oxidation. Sesamol decreased the R n value of sunflower oil and sunflower oil-in-water emulsion by 17.10% and 18.99%, respectively, compared to the control sample. These results indicate that all sesamol molecules were not consumed in the initiation stage of the lipid oxidation and a part of them was remained active in the propagation stage of the lipid oxidation.  www.nature.com/scientificreports/ The [LOOH] max value of sunflower oil and sunflower oil-in-water emulsion is affected by the k c and k d values at the same time. A high correlation (R 2 > 0.99) was found between [LOOH] max value and the ratio between R max and k d (Fig. 4a). In both sunflower oil and sunflower oil-in-water emulsion samples, no significant difference was found between the parameter related to the inhibitory activity of sesamol against formation of LOOHs (IA c ) with the parameter related to the inhibitory activity of sesamol against decomposition of LOOHs (IA d ). Therefore, trend of variations in the formation and decomposition of LOOHs were completely interdependent during the propagation stage 32 .
The T max value of sunflower oil sample containing combination of sesamol and PGPR was significantly higher than that of sunflower oil sample containing sesamol alone (Table 2). Accordingly, PGPR efficiently enhanced the antioxidant activity of sesamol in sunflower oil during the propagation stage (SE p of 36.84%) as in the initiation stage (SE i of 68.87%). This can be related to the physical role of PGPR on inhibiting the oxidation reactions. Surface-active LOOH molecules produced during the propagation stage of the lipid oxidation have a high affinity to migrate toward the reverse micelles 27 . During the propagation stage, the size of reverse micelles in samples containing sesamol + PGPR was significantly higher than those samples containing sesamol alone (Table 2). Accordingly, the acceptance capacity of LOOHs in the reveres micelles was increased in the presence of PGPR, which resulted in higher accessibility of sesamol to LOOHs. This physical role of PGPR can postpone the occurrence of turning point in the propagation stage.
The water produced in sunflower oil during the lipid oxidation process exhibited various linear relationships with some kinetic parameters related to the initiation or propagation stage. A high correlation (R 2 = 0.944) was found between water content at IP point and the ratio between LOOH IP and k 1 (Fig. 4b). This indicates  Table 2. Reverse micelles size and water content of sunflower oil samples. In each row and for each factor means with different uppercase letters are significantly different (P < 0.05). In each column, means with different lowercase letter are significantly different (P < 0.05). BIO at the beginning of the oxidation, IP at the induction period, AIP after the induction period, PP at the propagation period. *Mean ± SD (n = 3). www.nature.com/scientificreports/ that enhancing the amount of water during the initiation stage reduced the lipid oxidation rate at this stage. In addition, a high correlation between the kinetic parameters related to the propagation stage (k f × ([LOOH] Tmax / Tmax) and water content at IP points indicates that the water produced during the initiation stage can alter the lipid oxidation rate at the propagation stage (Fig. 4c). According to Fig. 4d, a high correlation exists between the end time of the propagation stage and the ratio of occurrence time of turning point and LOOHs concentration at the turning point (T max /[LOOH] Tmax ). This reveals that postponing the occurrence time of maximum rate of LOOHs formation can enhance the duration of the propagation period.

Conclusion
In this study, antioxidant activity of sesamol in sunflower oil and its oil-in-water emulsion was investigated over the whole practical range of peroxidation, including the initiation and propagation stages. In both sunflower oil and sunflower oil-in-water emulsion, the inhibitory effect of sesamol was not limited to the initiation stage of lipid oxidation and a part of this molecule was also active in the propagation stage. Sesamol exhibited better antioxidant performance in sunflower oil-in-water emulsion than that of sunflower oil. This was attributed to the higher interfacial activity of sesamol in sunflower oil-in-water emulsion than that of sunflower oil. In addition, the lower viscosity of sunflower oil-in-water emulsion than that of sunflower oil indicates the higher transfer rate of sesamol toward the actual sites of oxidation in sunflower oil-in-water emulsion than that of sunflower oil. PGPR was incorporated into sunflower oil to enhance the interfacial performance of sesamol in sunflower oil. Incorporating PGPR into the functional environment of sesamol lead to a significant change in the mechanism of hydrogen donating of sesamol which ultimately improved the kinetic parameters related to the initiation stage and propagation stage. Comparison of the size of reverse micelles in samples containing PGPR with those without PGPR showed that PGPR can improve the antioxidant performance of sesamol by increasing the acceptance capacity of LOOHs in reveres micelles structures. Therefore, the effective collision between sesamol and LOOHs www.nature.com/scientificreports/ is expected to increase in the presence of PGPR. The water produced as a major byproduct of lipid oxidation played a key role on the efficiency of sesamol alone or in combination with PGPR during the initiation stage of lipid oxidation. In general, sesamol exhibits better interfacial activity in sunflower oil-in-water emulsion than that of sunflower oil. In addition, PGPR can improve the interfacial activity of sesamol in sunflower oil. The results of this study can help manufacturers of food industry to reduce lipid oxidation by using the most adapted antioxidative strategies for their specific products.

Data availability
The data used to support the findings of this study are included within the article.