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Effects of interaction between α-tocopherol, oryzanol, and phytosterol on the antiradical activity against DPPH radical
LWT - Food Science and Technology 112 (2019) 108206
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Effects of interaction between α-tocopherol, oryzanol, and phytosterol on the antiradical activity against DPPH radical
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Lisha Zhang, Tao Zhang, Ming Chang, Mengyao Lu, Ruijie Liu∗, Qingzhe Jin, Xingguo Wang Synergetic Innovation Center of Food Safety and Nutrition, International Joint Research Laboratory for Lipid Nutrition and Safety, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
ARTICLE INFO
ABSTRACT
Keywords: α-Tocopherol γ-Oryzanol Phytosterol Antagonism Hydrogen bond Regeneration
Natural antioxidants are a complex system of molecules that interact to scavenge free radicals and often exist as minor compounds that are important for the nutritional benefits and functions of plant oils. In this study, the scavenging activity of α-tocopherol in the presence of γ-oryzanol and phytosterol in a radical medium was investigated using the 2,2-diphenyl-a-picrylhydrazyl assay. The compounds α-tocopherol, γ-oryzanol, and phytosterol were added to the reaction system separately and together at a range of concentrations. Interaction of the antioxidants was calculated by comparing the experimental and theoretical free radical scavenging activities. The results revealed an antagonistic interaction between α-tocopherol and γ-oryzanol, as well as between α-tocopherol and phytosterol at all concentrations investigated. Additionally, formation of hydrogen bonds and inhibition of α-tocopherol regeneration played important roles for the antagonistic interaction in the polar system. These findings provided insights that may be of interest for oil processing to ensure that the contents of the minor compounds are in a suitable range.
1. Introduction Minor constituents of dietary oils, such as phenolic compounds, have antioxidant properties and therefore play essential roles in the quality of oils. Hashempour-Baltork and colleagues showed that none of the pure plant oils examined in their study had excellent functional or nutritional characteristics or proper oxidative stability (HashempourBaltork, Torbati, Azadmard-Damirchi, & Savage, 2016). Phenolic compounds consist of at least one aromatic ring and one hydroxyl group (Fine et al., 2016), endowing them with the ability to scavenge free radicals through different mechanisms. α-Tocopherol is a crucial natural antioxidant that plays important roles in vegetable oils, including protection of polyunsaturated fatty acids against peroxidation and prevention of biological membrane damage (Evstigneeva, Volkov, & Chudinova, 1998). Furthermore, as the most abundant tocopherol among the four forms (i.e., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol) found in nature, α-tocopherol deficiency has been reported to cause neuropathy and encephalopathy (Wysota, Michael, Hiew, Dawson, & Rajabally, 2017). Although many studies have examined the functions of α-tocopherol, the mechanisms mediating these activities are still unclear (Lúcio et al., 2009). Whether the interactions between α-tocopherol and other antioxidants can be defined as equal to, greater than, or less than the simple ∗
sum of the single antioxidant components, namely whether they interact via addition, synergy, or antagonism, is a lingering question (Çelik, Rubio, & Gökmen, 2017). For example, antioxidant activity of αtocopherol was in synergy with other antioxidants, such as flavonoids (Altunkaya, Becker, Gökmen, & Skibsted, 2009), lecithin (Doert, Jaworska, Moersel, & Kroh, 2012), trans-resveratrol, quercetin (Mikstacka, Rimando, & Ignatowicz, 2010), and some natural active substances containing food matrices, including Centella Asiatica extracts (Yin et al., 2013) and strawberries (Reber, Eggett, & Parker, 2011), but showed additive interactions when combined with vitamin C (Im et al., 2014). However, the interactions between α-tocopherol and other lipidsoluble compounds, which represent many minor antioxidants in plant oils, have not yet been studied. γ-Oryzanol composed of ferulic acid esters of sterols and triterpene alcohols (Lemus, Angelis, Halabalaki, & Skaltsounis, 2014) is found in cereal grains, such as corn, wheat, and rice bran (Norton, 1995; Nyström, Mäkinen, Annamaija Lampi, & Piironen, 2005). γ-Oryzanol has various biological activities, including scavenging of free radicals (Massarolo, Denardi de Souza, Collazzo, Badiale Furlong, & Souza Soares, 2017), mediated primarily by ferulic acid moiety (Akiyama, Hori, Takahashi, & Yoshiki, 2005). Phytosterol is also a rich trace concomitant in plant oil, which reaches 12.655 mg/g in rice bran oil. (Pokkanta et al., 2019). Mixtures of bioactive molecules in plant oil can
Corresponding author. School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu Province, China. E-mail address: [email protected] (R. Liu).
https://doi.org/10.1016/j.lwt.2019.05.104 Received 31 October 2018; Received in revised form 21 May 2019; Accepted 22 May 2019 Available online 23 May 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Antioxidant capacity assay
result in interesting interactions (Perez-Ternero, Sotomayor, & Herrera, 2017). Indeed, tocopherols and γ-oryzanols show different activities depending on the lipid system evaluated, and a combination of various lipids could greatly enhance the antioxidant activity of rice bran oil (Hall & Gunstone, 2003, pp. 73–112). There are many methods for evaluating antioxidant properties and measuring interactions of antioxidants, including measurement of the 2,2-diphenyl-a-picrylhydrazyl (DPPH) radical, a prominent, stable compound that can be used to show a color reaction in the presence of antioxidants. DPPH assays are commonly used for evaluating interactions of antioxidants because they are feasible, sensitive, and simple, as well as more convenient than other assays (Deng, Cheng, & Yang, 2011; Li, Chen, Wang, & Lu, 2015). Several studies reported different mechanisms for free radical scavenging in different solvent systems (Zahalka et al., 1989), which may also affect the synergy between some antioxidants (Marteau, Favier, Nardello-Rataj, & Aubry, 2014). In this study, we aimed to evaluate the behaviors of α-tocopherol in a polar radical medium in the presence of γ-oryzanol and phytosterol. Considering the solubility problem, we used an absolute ethanol system to establish a model to pave the way for the study of oil systems. To this end, the DPPH scavenging capacities of α-tocopherol, γ-oryzanol, and phytosterol, alone or in combination (Table 1), were examined using a previously described method (Foti, 2015).
The DPPH method reported by Brandwilliams, Cuvelier, and Berset (1995) was performed, with slight modifications. Briefly, 2 mL DPPH (0.1 mM) solution was added to a 5-mL test tube, and the reaction was performed after addition of 100 μL of different concentrations of sample solutions (25 μM–1 mM). The solutions were mixed thoroughly and protected from light at room temperature for 30 min, and radical scavenging was estimated by determining the loss of absorbance at 517 nm using an ultraviolet–visible (UV–Vis) spectrophotometer. The experimental scavenging capacity (ESC) for the DPPH radical was calculated as follows: ESC = (1 – [Ai – A j] / A0) × 100 (1) Where A0 was the absorbance of ethanol (100 μL) and DPPH (2 mL), Ai was the absorbance of each sample (100 μL) and DPPH (2 mL), and Aj was the absorbance of samples (100 μL) and ethanol (2 mL), set as the blank. 2.4. Calculation of the synergistic effects (SEs) of antioxidant mixtures The theoretical scavenging capacity (TSC) reported by Luís, Duarte, Pereira, and Domingues (2017) was used in this study. TSC was defined as the sum of the separate scavenging capacities of each equimolar substance in the mixture and was calculated using the following equation:
2. Materials and methods 2.1. Chemicals
TSCmixture = (ESCA + ESCB) – (ESCA × ESCB / 100) (2)
Phytosterol (three major compounds, including stigmasterol [5%], β-sitosterol [75%], and campesterol [15%]), α-Tocopherol, and DPPH were obtained from Sigma-Aldrich (Bellefonte, PA, USA). A mixture of γ-oryzanol standard (three major compounds, including cycloartenyl ferulate [39%], 24-methylene cycloartanyl ferulate [50%] and campesterferulate [6%]) was purchased from Toronto Research Chemicals (Canada). All standards were stored at −20 °C in the dark. Analyticalgrade ethanol (100%, v/v) was purchased from Sinopharm Medicine Holding Co., Ltd. (Shanghai, China). The structures of the compounds used in this study are shown in Fig. 1.
where ESCA and ESCB are the ESC values of the individual compounds in the mixture. The antioxidant effects of the combinations of α-tocopherol and γoryzanol or α-tocopherol and phytosterol were determined by measuring the SEs, described as the ratio of the estimated scavenging capacity of ESCmixture and TSCmixture, as follows: SE = ESCmixture / TSCmixture 2.5. Kinetic measurements The reaction between α-tocopherol and DPPH radical and the behavior of α-tocopherol in the presence of γ-oryzanol and phytosterol were determined by monitoring the DPPH visible absorbance variation at the maximum wavelength as a function of time (Skroza, Mekinić, Svilović; Šimat, & Katalinić, 2015). The reactions were performed by Thermo Mulitskan Go. Kinetic analyses for samples were performed by adding an aliquot of 100 μL sample to 100 μL DPPH (1 mM) solution and monitoring the absorbance at 517 nm until a plateau was reached.
2.2. Preparation of single and mixture standard solutions α-Tocopherol, γ-oryzanol, and phytosterol solutions were dissolved in absolute ethanol to a final concentration of 1 mM and stored in a freezer at −8 °C. The prepared solutions were diluted to various concentrations (25, 50, 100, 200, 400, 500, and 800 μM) for use, and mixtures of compounds with different concentrations were prepared by mixing α-tocopherol with γ-oryzanol and phytosterol single solutions at a 1:1 ratio, yielding mixtures of compounds at equimolar concentrations.
2.6. Fourier transform infrared (FTIR) spectroscopy of hydrogen bonding In this study, a Nicolet Nexus FTIR spectrometer with a resolution of ± 0.5 cm−1 was used to evaluate the bonds formed. The FTIR spectrometer was controlled by OMNIC software. Samples were prepared by mixing with potassium bromide (KBr) powder in an agate mortar. An average of 32 scans was collected for each measurement in the region of 4000–400 cm−1.
Table 1 Two compound mixtures (content are expressed as μM). Mixtures
α-Tocopherol
γ-Oryzanol
Phytosterols
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12
50 100 200 400 800 1000 50 100 200 400 800 1000
50 100 200 400 800 1000 – – – – – –
– – – – – – 50 100 200 400 800 1000
(3)
2.7. Ultra-performance liquid chromatography (UPLC) quadrupole time-offlight (Q-TOF) mass spectrometry (MS) Detection was carried out using a Waters Acquity UPLC (Milford, MA, USA) coupled with an autosampler and binary solvent-transfer system. Chromatography was performed on a 10 cm × 2.1 mm 1.7-μm particle Waters Acquity C18 column. A mobile phase for elution was composed of 96% methanol (component A) and 4% formic acid 2
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Fig. 1. Chemical structures of the compounds.
aqueous solution (0.1%) (component B) with a constant gradient. And the flux rate and total operation time were 0.3 mL/min and 15 min, respectively. The samples for the UPLC analysis were the reacted products of α-tocopherol, γ-oryzanol with DPPH free radical. The injection volume was 5 μL. MS was then performed on a Waters Synapt Q-TOF system (Milford). The optimized conditions were as follows: cone gas, 50 L/h; desolvation gas, 700 L/h; temperature, 400 °C. The source temperature was 100 °C, and the capillary and cone potentials were 3000 and 50 V, respectively. The Q-TOF device was used in the wide pass quadrupole mode. Scan analyses were collected from m/z 50 to 1000 with a scan accumulation time of 0.2 s.
processed using SPSS software. One-way analysis of variance was performed to evaluate significant differences among means (p < 0.05). Duncan's post-hoc tests were performed using SPSS Statistics version 17. All graphs were drawn with Origin Pro2016. Additionally, synergistic and antagonistic effects were only accepted if there were significant differences between the mean values of ESCmixture and TSCmixture. 3. Results and discussion 3.1. Antioxidant activity of single compounds The DPPH radical is remarkably stable and intensely colored compared with other radicals, permitting its wide use in a variety of assays. Additionally, reduction of DPPH by antioxidants yields an H atom, resulting in the formation of DPPH-H and the disappearance of violet
2.8. Statistical analysis Results are presented as the percent inhibition values and were 3
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capacity and concentration of α-tocopherol and γ-oryzanol. Moreover, the DPPH scavenging capacity of α-tocopherol was higher than that of γ-oryzanol; at a concentration of 500 μM, α-tocopherol displayed 90.37% ± 0.04% scavenging capacity, whereas 1 mM γ-oryzanol showed 58.75% ± 0.15% scavenging capacity. In contrast, for phytosterol, only 3.07% ± 0.15% activity was observed at 1 mM. Accordingly, the antioxidant capacities of the compounds decreased in the following order: α-tocopherol > γ-oryzanol > phytosterol, indicating that α-tocopherol played a key role in the mixture. Consistent with our findings, fat-soluble α-tocopherol is thought to be the most effective radical scavenging compound among the minor substances in plant oils (Belo, Nolasco, Mateo, & Izquierdo, 2017). Additionally, γ-oryzanol is thought to have radical scavenging capacity based on its many bioactivities (Lavecchia, Rea, Antonacci, & Giardi, 2013). Indeed, the DPPH radical scavenging abilities of the three constituents of γ-oryzanol used in the study were similar to those of ferulic acid (Akiyama et al., 2005), demonstrating the importance of the hydroxyl group in γ-oryzanol. However, no antioxidant capacity was observed for phytosterol, consistent with previous results (Fu et al., 2014; Huang, Zhao, & Zhou, 2012). Fig. 2. The capacity of scavenging DPPH free radical of three single compounds.
3.2. Effects of the interactions between α-tocopherol, γ-oryzanol, and phytosterol on antioxidant properties
color at 517 nm generated by π-π* transitions (Jabbari & Jabbari, 2016). The process can be easily monitored via UV–vis spectroscopy. The scavenging activities of each three compounds (α-tocopherol: 25–1000 μM, γ-oryzanol: 25–1000 μM, and phytosterol: 25–1000 μM) were determined using DPPH radical scavenging assays (Fig. 2). A linear correlation was observed between free radical scavenging
In order to determine the nature of interactions among γ-oryzanol, phytosterol, and α-tocopherol, different concentrations of single antioxidants were chosen to contribute to the partial reduction of the DPPH radical (Table 1). The mixtures were prepared while considering the antioxidant effects of each component. For example, TSC of the mixture of each α-tocopherol and γ-oryzanol (25 μM of each when mixed at a
Fig. 3. Relative inhibition (%) of theoretical and experimental values as well as synergy effect. Asterisks denote a significant difference (theoretical VS experimental values). (*P < 0.05, **P < 0.01). (AB) interaction between α-tocopherol and γ-oryzanol, (CD) interaction between α-tocopherol and phytosterol, and “An” means “antagonism”. 4
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1:1 v/v ratio) for the DPPH radical was compared with the ESC calculated by the sum of 25 μM α-tocopherol and 25 μM γ-oryzanol. If the ESC was significantly higher than the TSC value, a synergistic relationship was considered to have occurred in the mixture. In contrast, when the ESC was significantly lower than the TSC, an antagonistic relationship was considered. An additive interaction was considered when there were no significant differences between the two results. Fig. 3 shows the results of the analysis of two component mixtures and the SE values. All SEs were less than 1 and were statistically significant (p < 0.05) for all combinations in which α-tocopherol was mixed with γ-oryzanol. Similar antagonistic interactions were observed for α-tocopherol and phytosterol. In the presence of γ-oryzanol and phytosterol, the DPPH radical scavenging abilities at a concentration of 800 μM were 86% and 77%, respectively, demonstrating only a slight decrease in antioxidant capacity compared with α-tocopherol alone (90% at a concentration of 500 μM). The highest SE value detected was 0.98 at 800 μM for the mixture of α-tocopherol with γ-oryzanol, whereas an SE value of 0.99 was calculated at 1 mM for the mixture of α-tocopherol with phytosterol. This result may be caused by the different abilities of γ-oryzanol and phytosterol to scavenge the DPPH radical. Many studies have described the effects of structure and concentration on the types of interactions between antioxidants (Hajimehdipoor, Shahrestani, & Shekarchi, 2014; Luís et al., 2017).We also found that the degree of antagonism was stronger at low concentrations (50–200 μM) than at high concentrations (400–1000 μM), which was consistent with a previous study demonstrating that as the concentration increased, the degree of antagonism decreased (Liu, Shi, Ibarra, Kakuda, & Xue, 2008; Zhi-Lu et al., 2015). The synergy effect (SE) value in this study is calculated by the ratio of ESC to TSC. Compounds concentrations may have an effect on this ratio. At low concentrations (50–200 μM), the behavior of scavenging DPPH· for α-tocopherol is greatly affected by γ-oryzanol, phytosterols and solvents, making the ESC smaller than the TSC, thus the obtained SE value is small. At high concentrations (400–1000 μM), the ability of α-tocopherol to scavenge free radical is less affected by γ-oryzanol, phytosterols and solvents, making the ESC closer to the TSC, thus resulting in a increased SE value that is close to 1. It can be seen in Fig. 3, the SE value is small at low concentrations (50–200 μM), which means the antagonism is strong; the SE value is large at high concentrations (400–1000 μM), meaning a weak antagonism. To further understand how γ-oryzanol and phytosterols affect the ability of α-tocopherol to scavenge free radicals, we have done the following experiments. As a highly useful instrument for investigating the performance of compounds, FTIR is very valuable for understanding the interactions between molecules (Geboes, Proft, & Herrebout, 2018; Sivagurunathan & Hajasharif, 2012). As shown in Fig. 4, 200 μM αtocopherol and 400 μM mixtures of α-tocopherol with γ-oryzanol or phytosterol were analyzed. Because absolute ethanol has its own hydroxyl bond, the solvent was first dried with nitrogen and then pelleted using a KBr-based standardized procedure (Lin, Kasper, & Kjaergaard, 2013). The highest frequency of α-tocopherol, observed at 3467 cm−1, could be attributed to OH stretching, and the peak at 2750–3000 cm−1 was caused by the stretching vibration of C–H3 and the C–H2 out-ofphase stretching vibration, consistent with a previous report (Singh, Sachdeva, Rai, & Saini, 2017). After mixing with γ-oryzanol and phytosterol, several peaks changed in the spectrum compared with that of α-tocopherol alone. The fundamental transition wavenumbers of the OH-stretching of the mixture of α-tocopherol and γ-oryzanol or α-tocopherol and phytosterol were 3452 and 3436 cm−1, respectively, indicating red-shifted bands compared with the typical OH of α-tocopherol moving at a wavenumber of 15 and 31, respectively. According to the FTIR result, the formation of hydrogen bonds have occurred. Considering the structures of the antioxidants and the polar system, we think hydrogen bonds may be formed among the antioxidants and solvent, which decreased the number of binding sites of the antioxidants with DPPH during the process of scavenging DPPH in the
Fig. 4. Transmittance of 200 μM of α-tocopherol, γ-oryzanol, phytosterol as well as 400 μM mixtures in the region of 4000–400 cm−1.
solvent system (D. Li, Li, & Liu, 2016). Moreover, at low concentrations (50–200 μM), when a hydrogen atom in an antioxidant molecule forms a hydrogen bond with a polar solvent or an antioxidant, two bulky hetero-atoms are generated in the hydrogen atom around, thus results in a large steric congestion. It is the steric congestion that prevents DPPH· from coming into contact with α-tocopherols that have formed hydrogen bonds with polar solvent or other antioxidants and reacts only with the free fraction of antioxidants, resulting in the ESC is smaller than the TSC(Foti, 2015). This resulted in a smaller ESC than TSC and thus a small SE, which means a strong antagonism. However, the ability of α-tocopherol to scavenge free radicals is less affected by γoryzanol, phytosterols and solvents at high concentrations (400–1000 μM). Although the hydrogen bonds are still generated in the system at high concentrations, the main antioxidant, α-tocopherol scavenges DPPH· more rapidly than at low concentrations (SánchezMoreno, Larrauri, & Saura-Calixto, 1998). Therefore, the addition of γ– oryzanol and phytosterol has less influence on the total free radical scavenging ability of the system, resulting in an SE value close to 1, which means a weaker antagonism compared with the SE value at low concentrations (50–200 μM). 3.3. Kinetics of antioxidant behaviors When antioxidant (A) comes into contact with DPPH, the following reactions may occur: A – H + DPPH· → A + DPPH-H (reaction 1) A· + DPPH· → closed shell products (reaction 2) 2 A· → closed shell products (reaction 3) To further explore the antioxidant properties of α-tocopherol in the presence of γ-oryzanol and phytosterol, the kinetic curves of DPPH scavenging by α-tocopherol or the mixtures were determined (Fig. 5). The reaction rate of α-tocopherol with the DPPH radical was first compared with that of the mixture. Notably, the O–H bond dissociation enthalpy (BDE) of α-tocopherol is equal to 77.2 kcal/mol (Valgimigli, Ingold, & Lusztyk, 1996), which is lower than that of many other compounds. The mathematical model that satisfactorily describes the 5
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tocopherol to scavenge DPPH. The results of this study were consistent with the results of previous reports in the literature when using excess DPPH radical (Marteau et al., 2014; Yang et al., 2008; Zahalka et al., 1989). Thus, 1 mol of α-tocopherol could reduce nearly 2 mol of DPPH radicals, although α-tocopherol has only one available hydroxyl group. The mechanism of the α-tocopherol reaction with DPPH has been described in previous studies (Yang et al., 2008; Yang, Liu, Nie, Gao-Fei, & Yan, 2009) and is summarized in Eq. (7).
Fig. 5. Plots of relative inhibition versus time during scavenging DPPH radical in (■) 200 μM α-tocopherol; (●) 400 μM α-tocopherol + oryzanol; (▲) 400 μM α-tocopherol + phytosterol.
One molecule of α-tocopherol first reacts with DPPH free radicals to form a molecule of tocopheroxyl radical, and then two molecules of tocopheroxyl radical generate a molecule of α-tocopherol and a part of tocopherol quinone by the disproportionation reaction (Yang et al., 2008). The product of tocopherol quinone identified by UPLC-Q-TOF/ MS and the probable pathway to scavenge the DPPH radical are shown in Fig. 6 and Fig. 7. Several excellent reviews have described the antioxidant mechanisms of α-tocopherol in polar solvents (Marteau et al., 2014; Zahalka et al., 1989), in which sequential proton-loss-electron-transfer is likely involved (Bakhouche, Dhaouadi, Jaidane, & Hammoutène, 2015; Zhang & Ji, 2006) (Fig. 6). This method for scavenging DPPH increases the reaction rate compared with pure hydrogen donation (Foti, 2015). Electrospray ionization for α-tocopheryl quinone (TQ) was enhanced by the addition of formic acid in the mobile phase to produce [M+H] +species (Fig. 6). Thus, ions at m/z 447.5 would be expected for the TQ. In fact, we observed that this mode of ionization also produced ions at m/z 429.5 for TQ, representing [M + H–H2O]+, and the full-scan product ion spectra of m/z 429 for TQ showed a predominant fragment ion at m/z 165, which was attributed to loss of the phytyl chain (Mottier, Gremaud, Guy, & Turesky, 2002). Clearly, the process of scavenging DPPH for α-tocopherol occurred via the α-tocopheroxyl radical as an intermediate. However, in the presence of γ-oryzanol and phytosterol, the stoichiometry of α-tocopherol scavenging free radicals was less than 1 (i.e., 0.60 and 0.52, respectively). Previous studies have explained this phenomenon (Yang et al., 2008); the antagonism between α-tocopherol and γ-oryzanol or phytosterol for scavenging the DPPH radical can be explained as follows: during the process of scavenging DPPH, the tocopheroxyl radical product combines with other free radicals, inhibiting the regeneration of α-tocopherol. Some studies have reported that nonradical products can be formed from the coupling of free radicals with the α-tocopheroxyl radical in the presence of other free radicals (Yamauchi, 1997), which may have been the main antagonistic mechanism observed in this study.
time dependence of scavenging DPPH is the exponential function: y = a (1 – e-bx)
(4)
Where x is the reaction time, y denotes the scavenging of DPPH radical capacity, a and b are coefficients of the fit functions listed in Table 2, showing a good fit (R2 and Adj. R2 > 0.99). The curves in Fig. 5 were plotted based on parameters a and b in Eq. (4), which were acquired by nonlinear regression analysis. The rate of DPPH radical scavenging (Rs) was determined to extrapolate the kinetics of scavenging of the DPPH radical. For all nonlinear kinetic reactions, the only useful rate is the maximum (Prieto, Curran, Gowen, & Vázquez, 2015). Hence, the rate of the corresponding process calculated at t = 2 s (the initial rate) as the first derivative of the function 4 described by Eq. (5) is listed. R = abe-bx
(5)
The rate of DPPH radical scavenging is presented in Table 2. Higher values indicated better responses when antioxidants were added to the system. The rate of DPPH radical scavenging was the highest for αtocopherol, reaching 4.25 s−1, followed by 3.95 and 2.12 s−1 for γoryzanol + α-tocopherol and phytosterol + α-tocopherol, respectively; thus, these compounds decreased the capacity of α-tocopherol to scavenge the DPPH radical. Moreover, we calculated the stoichiometry as previously described (Mendoza-Wilson et al., 2010), using the following equation: n = c0 (1 – At / A0) / c
(6)
Where c0 is the initial DPPH radical concentration, c is the initial antioxidant concentration (200 or 400 μM), At is the absorbance at the end of the kinetic run, and A0 is the initial absorbance. Stoichiometry is a parameter used to describe the moles of DPPH free radical reduced by 1 mol of antioxidant (Osman, 2011). Several studies have demonstrated that the stoichiometry is 2.1 for α-
Table 2 The determination coefficients of α-tocopherol and the mixture with γ-oryzanol and phytosterols in the reaction system determined by fitting data through Eq. (4) for kinetics of DPPH radical scavenging(r2 (DPPH radical)). The rate of free radical scavenging (Rs DPPH radical) was determined at t = 2s. sample
R2
Adj. R2
coefficients
α-tocophenol α-tocophenol+γ-oryzanol α-tocophenol + phytosterols
0.9984 0.9935 0.9984
0.9983 0.9928 0.9982
a 42.0600 22.6300 20.5353
Note:the value in parentheses indicates the standard error of stoichiometries. 6
b 0.1315 0.3544 0.1351
Rs DPPH radical(s−1) (t = 2s)
Stoichiometries
4.253 3.948 2.117
2.13(0.0062) 0.60(0.0364) 0.52(0.0135)
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Fig. 6. The mechanism stepwise sequential proton-loss-electron-transfer, proposed to elucidate α-tocopherol scavenging DPPH radical in ethanol.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.05.104. References Akiyama, Y., Hori, K., Takahashi, T., & Yoshiki, Y. (2005). Free radical scavenging activities of γ-oryzanol constituents. Food Science and Technology International Tokyo, 11(3), 295–297. Altunkaya, A., Becker, E. M., Gökmen, V., & Skibsted, L. H. (2009). Antioxidant activity of lettuce extract (Lactuca sativa) and synergism with added phenolic antioxidants. Food Chemistry, 115(1), 163–168. Bakhouche, K., Dhaouadi, Z., Jaidane, N., & Hammoutène, D. (2015). Comparative antioxidant potency and solvent polarity effects on HAT mechanisms of tocopherols. Computational and Theoretical Chemistry, 1060, 58–65. Belo, R. G., Nolasco, S., Mateo, C., & Izquierdo, N. (2017). Dynamics of oil and tocopherol accumulation in sunflower grains and its impact on final oil quality. European Journal of Agronomy, 89, 124–130. Brandwilliams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28(1), 25–30. Çelik, E. E., Rubio, J. M. A., & Gökmen, V. (2017). Behaviour of trolox with macromolecule-bound antioxidants in aqueous medium: Inhibition of auto-regeneration mechanism. Food Chemistry, 243, 428–434. Deng, J., Cheng, W., & Yang, G. (2011). A novel antioxidant activity index (AAU) for natural products using the DPPH assay. Food Chemistry, 125(4), 1430–1435. Doert, M., Jaworska, K., Moersel, J. T., & Kroh, L. W. (2012). Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol. European Food Research & Technology, 235(5), 915–928. Evstigneeva, R. P., Volkov, I. M., & Chudinova, V. V. (1998Doert, M., Jaworska, K., Moersel, J. T., & Kroh, L. W. (2012). Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol. European Food Research & Technology, 235(5), 915-928Doert, M., Jaworska, K., Moersel, J. T., & Kroh, L. W. (2012). Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol. European Food Research & Technology, 235(5), 915-928). Vitamin E as a universal antioxidant and stabilizer of biological membranes. Membrane and Cell Biology, 12(2), 151–172. Fine, F., Brochet, C., Gaud, M., Carre, P., Simon, N., Ramli, F., et al. (2016). Micronutrients in vegetable oils: The impact of crushing and refining processes on vitamins and antioxidants in sunflower, rapeseed, and soybean oils. European Journal of Lipid Science and Technology, 118(5), 680–697. Foti, M. C. (2015). Use and abuse of the DPPH radical. Journal of Agricultural and Food Chemistry, 63(40), 8765. Fu, Y., Zhang, Y., Hu, H., Chen, Y., Wang, R., Li, D., et al. (2014). Design and straightforward synthesis of novel galloyl phytosterol with excellent antioxidant activity. Food Chemistry, 163(20), 171–177. Geboes, Y., Proft, F. D., & Herrebout, W. A. (2018). Towards a better understanding of the parameters determining the competition between bromine halogen bonding and hydrogen bonding: An FTIR spectroscopic study of the complexes between bromodifluoromethane and trimethylamine. Journal of Molecular Structure, 1165, 349–355. Hajimehdipoor, H., Shahrestani, R., & Shekarchi, M. (2014). Investigating the synergistic antioxidant effects of some flavonoid and phenolic compounds. Research Journal of Pharmacognosy, 1(3), 35–40. Hall, C., Iii, & Gunstone, F. D. (2003). Other natural antioxidants - rice bran oil, sesame oil, rosemary extract. flavonoids: Elsevier Ltd. Hashempour-Baltork, F., Torbati, M., Azadmard-Damirchi, S., & Savage, G. P. (2016). Vegetable oil blending: A review of physicochemical, nutritional and health effects. Trends in Food Science & Technology, 57, 52–58. Huang, Y., Zhao, Y., & Zhou, Z. (2012). Scavenging effects on free radical by phytosterol.
Fig. 7. TOF MS spectra and proposed fragmentation of degradation products of α-tocopherol.
4. Conclusion In conclusion, we examined the interactions among α-tocopherol, γoryzanol, and phytosterol using DPPH assays. Our findings showed that interactions among the three bioactive compounds may have negative effects. The γ-oryzanol and phytosterol present in α-tocopherol could decrease the capacity of α-tocopherol to scavenge the DPPH radical. This antagonism may result from inhibition of the auto regeneration of α-tocopherol. The results provided insights that may be of interest in oil processing in order to ensure that the contents of these minor compounds are in a suitable range. Of course, the contents of γ-oryzanol and phytosterol in rice bran oil are 10 times that of α-tocopherol; thus, further studies are needed to explore the interactions of bioactive compounds in nonpolar systems and oil systems. Additionally, the SEs and interactions among various antioxidants should be studied to determine the net antioxidant capacity of plant oils, highlighting the importance of choosing the right combination and concentration of compounds to promote synergism and avoid antagonistic reactions. Conflicts of interest There was no conflict of interest exits in the submission of this manuscript, and all authors approve the publication of the manuscript. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant No. 31872895) & national first-class discipline program of Food Science and Technology (JUFSTR20180202). 7
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Effects of interaction between α-tocopherol, oryzanol, and phytosterol on the antiradical activity against DPPH radical
T
Lisha Zhang, Tao Zhang, Ming Chang, Mengyao Lu, Ruijie Liu∗, Qingzhe Jin, Xingguo Wang Synergetic Innovation Center of Food Safety and Nutrition, International Joint Research Laboratory for Lipid Nutrition and Safety, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
ARTICLE INFO
ABSTRACT
Keywords: α-Tocopherol γ-Oryzanol Phytosterol Antagonism Hydrogen bond Regeneration
Natural antioxidants are a complex system of molecules that interact to scavenge free radicals and often exist as minor compounds that are important for the nutritional benefits and functions of plant oils. In this study, the scavenging activity of α-tocopherol in the presence of γ-oryzanol and phytosterol in a radical medium was investigated using the 2,2-diphenyl-a-picrylhydrazyl assay. The compounds α-tocopherol, γ-oryzanol, and phytosterol were added to the reaction system separately and together at a range of concentrations. Interaction of the antioxidants was calculated by comparing the experimental and theoretical free radical scavenging activities. The results revealed an antagonistic interaction between α-tocopherol and γ-oryzanol, as well as between α-tocopherol and phytosterol at all concentrations investigated. Additionally, formation of hydrogen bonds and inhibition of α-tocopherol regeneration played important roles for the antagonistic interaction in the polar system. These findings provided insights that may be of interest for oil processing to ensure that the contents of the minor compounds are in a suitable range.
1. Introduction Minor constituents of dietary oils, such as phenolic compounds, have antioxidant properties and therefore play essential roles in the quality of oils. Hashempour-Baltork and colleagues showed that none of the pure plant oils examined in their study had excellent functional or nutritional characteristics or proper oxidative stability (HashempourBaltork, Torbati, Azadmard-Damirchi, & Savage, 2016). Phenolic compounds consist of at least one aromatic ring and one hydroxyl group (Fine et al., 2016), endowing them with the ability to scavenge free radicals through different mechanisms. α-Tocopherol is a crucial natural antioxidant that plays important roles in vegetable oils, including protection of polyunsaturated fatty acids against peroxidation and prevention of biological membrane damage (Evstigneeva, Volkov, & Chudinova, 1998). Furthermore, as the most abundant tocopherol among the four forms (i.e., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol) found in nature, α-tocopherol deficiency has been reported to cause neuropathy and encephalopathy (Wysota, Michael, Hiew, Dawson, & Rajabally, 2017). Although many studies have examined the functions of α-tocopherol, the mechanisms mediating these activities are still unclear (Lúcio et al., 2009). Whether the interactions between α-tocopherol and other antioxidants can be defined as equal to, greater than, or less than the simple ∗
sum of the single antioxidant components, namely whether they interact via addition, synergy, or antagonism, is a lingering question (Çelik, Rubio, & Gökmen, 2017). For example, antioxidant activity of αtocopherol was in synergy with other antioxidants, such as flavonoids (Altunkaya, Becker, Gökmen, & Skibsted, 2009), lecithin (Doert, Jaworska, Moersel, & Kroh, 2012), trans-resveratrol, quercetin (Mikstacka, Rimando, & Ignatowicz, 2010), and some natural active substances containing food matrices, including Centella Asiatica extracts (Yin et al., 2013) and strawberries (Reber, Eggett, & Parker, 2011), but showed additive interactions when combined with vitamin C (Im et al., 2014). However, the interactions between α-tocopherol and other lipidsoluble compounds, which represent many minor antioxidants in plant oils, have not yet been studied. γ-Oryzanol composed of ferulic acid esters of sterols and triterpene alcohols (Lemus, Angelis, Halabalaki, & Skaltsounis, 2014) is found in cereal grains, such as corn, wheat, and rice bran (Norton, 1995; Nyström, Mäkinen, Annamaija Lampi, & Piironen, 2005). γ-Oryzanol has various biological activities, including scavenging of free radicals (Massarolo, Denardi de Souza, Collazzo, Badiale Furlong, & Souza Soares, 2017), mediated primarily by ferulic acid moiety (Akiyama, Hori, Takahashi, & Yoshiki, 2005). Phytosterol is also a rich trace concomitant in plant oil, which reaches 12.655 mg/g in rice bran oil. (Pokkanta et al., 2019). Mixtures of bioactive molecules in plant oil can
Corresponding author. School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu Province, China. E-mail address: [email protected] (R. Liu).
https://doi.org/10.1016/j.lwt.2019.05.104 Received 31 October 2018; Received in revised form 21 May 2019; Accepted 22 May 2019 Available online 23 May 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Antioxidant capacity assay
result in interesting interactions (Perez-Ternero, Sotomayor, & Herrera, 2017). Indeed, tocopherols and γ-oryzanols show different activities depending on the lipid system evaluated, and a combination of various lipids could greatly enhance the antioxidant activity of rice bran oil (Hall & Gunstone, 2003, pp. 73–112). There are many methods for evaluating antioxidant properties and measuring interactions of antioxidants, including measurement of the 2,2-diphenyl-a-picrylhydrazyl (DPPH) radical, a prominent, stable compound that can be used to show a color reaction in the presence of antioxidants. DPPH assays are commonly used for evaluating interactions of antioxidants because they are feasible, sensitive, and simple, as well as more convenient than other assays (Deng, Cheng, & Yang, 2011; Li, Chen, Wang, & Lu, 2015). Several studies reported different mechanisms for free radical scavenging in different solvent systems (Zahalka et al., 1989), which may also affect the synergy between some antioxidants (Marteau, Favier, Nardello-Rataj, & Aubry, 2014). In this study, we aimed to evaluate the behaviors of α-tocopherol in a polar radical medium in the presence of γ-oryzanol and phytosterol. Considering the solubility problem, we used an absolute ethanol system to establish a model to pave the way for the study of oil systems. To this end, the DPPH scavenging capacities of α-tocopherol, γ-oryzanol, and phytosterol, alone or in combination (Table 1), were examined using a previously described method (Foti, 2015).
The DPPH method reported by Brandwilliams, Cuvelier, and Berset (1995) was performed, with slight modifications. Briefly, 2 mL DPPH (0.1 mM) solution was added to a 5-mL test tube, and the reaction was performed after addition of 100 μL of different concentrations of sample solutions (25 μM–1 mM). The solutions were mixed thoroughly and protected from light at room temperature for 30 min, and radical scavenging was estimated by determining the loss of absorbance at 517 nm using an ultraviolet–visible (UV–Vis) spectrophotometer. The experimental scavenging capacity (ESC) for the DPPH radical was calculated as follows: ESC = (1 – [Ai – A j] / A0) × 100 (1) Where A0 was the absorbance of ethanol (100 μL) and DPPH (2 mL), Ai was the absorbance of each sample (100 μL) and DPPH (2 mL), and Aj was the absorbance of samples (100 μL) and ethanol (2 mL), set as the blank. 2.4. Calculation of the synergistic effects (SEs) of antioxidant mixtures The theoretical scavenging capacity (TSC) reported by Luís, Duarte, Pereira, and Domingues (2017) was used in this study. TSC was defined as the sum of the separate scavenging capacities of each equimolar substance in the mixture and was calculated using the following equation:
2. Materials and methods 2.1. Chemicals
TSCmixture = (ESCA + ESCB) – (ESCA × ESCB / 100) (2)
Phytosterol (three major compounds, including stigmasterol [5%], β-sitosterol [75%], and campesterol [15%]), α-Tocopherol, and DPPH were obtained from Sigma-Aldrich (Bellefonte, PA, USA). A mixture of γ-oryzanol standard (three major compounds, including cycloartenyl ferulate [39%], 24-methylene cycloartanyl ferulate [50%] and campesterferulate [6%]) was purchased from Toronto Research Chemicals (Canada). All standards were stored at −20 °C in the dark. Analyticalgrade ethanol (100%, v/v) was purchased from Sinopharm Medicine Holding Co., Ltd. (Shanghai, China). The structures of the compounds used in this study are shown in Fig. 1.
where ESCA and ESCB are the ESC values of the individual compounds in the mixture. The antioxidant effects of the combinations of α-tocopherol and γoryzanol or α-tocopherol and phytosterol were determined by measuring the SEs, described as the ratio of the estimated scavenging capacity of ESCmixture and TSCmixture, as follows: SE = ESCmixture / TSCmixture 2.5. Kinetic measurements The reaction between α-tocopherol and DPPH radical and the behavior of α-tocopherol in the presence of γ-oryzanol and phytosterol were determined by monitoring the DPPH visible absorbance variation at the maximum wavelength as a function of time (Skroza, Mekinić, Svilović; Šimat, & Katalinić, 2015). The reactions were performed by Thermo Mulitskan Go. Kinetic analyses for samples were performed by adding an aliquot of 100 μL sample to 100 μL DPPH (1 mM) solution and monitoring the absorbance at 517 nm until a plateau was reached.
2.2. Preparation of single and mixture standard solutions α-Tocopherol, γ-oryzanol, and phytosterol solutions were dissolved in absolute ethanol to a final concentration of 1 mM and stored in a freezer at −8 °C. The prepared solutions were diluted to various concentrations (25, 50, 100, 200, 400, 500, and 800 μM) for use, and mixtures of compounds with different concentrations were prepared by mixing α-tocopherol with γ-oryzanol and phytosterol single solutions at a 1:1 ratio, yielding mixtures of compounds at equimolar concentrations.
2.6. Fourier transform infrared (FTIR) spectroscopy of hydrogen bonding In this study, a Nicolet Nexus FTIR spectrometer with a resolution of ± 0.5 cm−1 was used to evaluate the bonds formed. The FTIR spectrometer was controlled by OMNIC software. Samples were prepared by mixing with potassium bromide (KBr) powder in an agate mortar. An average of 32 scans was collected for each measurement in the region of 4000–400 cm−1.
Table 1 Two compound mixtures (content are expressed as μM). Mixtures
α-Tocopherol
γ-Oryzanol
Phytosterols
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12
50 100 200 400 800 1000 50 100 200 400 800 1000
50 100 200 400 800 1000 – – – – – –
– – – – – – 50 100 200 400 800 1000
(3)
2.7. Ultra-performance liquid chromatography (UPLC) quadrupole time-offlight (Q-TOF) mass spectrometry (MS) Detection was carried out using a Waters Acquity UPLC (Milford, MA, USA) coupled with an autosampler and binary solvent-transfer system. Chromatography was performed on a 10 cm × 2.1 mm 1.7-μm particle Waters Acquity C18 column. A mobile phase for elution was composed of 96% methanol (component A) and 4% formic acid 2
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Fig. 1. Chemical structures of the compounds.
aqueous solution (0.1%) (component B) with a constant gradient. And the flux rate and total operation time were 0.3 mL/min and 15 min, respectively. The samples for the UPLC analysis were the reacted products of α-tocopherol, γ-oryzanol with DPPH free radical. The injection volume was 5 μL. MS was then performed on a Waters Synapt Q-TOF system (Milford). The optimized conditions were as follows: cone gas, 50 L/h; desolvation gas, 700 L/h; temperature, 400 °C. The source temperature was 100 °C, and the capillary and cone potentials were 3000 and 50 V, respectively. The Q-TOF device was used in the wide pass quadrupole mode. Scan analyses were collected from m/z 50 to 1000 with a scan accumulation time of 0.2 s.
processed using SPSS software. One-way analysis of variance was performed to evaluate significant differences among means (p < 0.05). Duncan's post-hoc tests were performed using SPSS Statistics version 17. All graphs were drawn with Origin Pro2016. Additionally, synergistic and antagonistic effects were only accepted if there were significant differences between the mean values of ESCmixture and TSCmixture. 3. Results and discussion 3.1. Antioxidant activity of single compounds The DPPH radical is remarkably stable and intensely colored compared with other radicals, permitting its wide use in a variety of assays. Additionally, reduction of DPPH by antioxidants yields an H atom, resulting in the formation of DPPH-H and the disappearance of violet
2.8. Statistical analysis Results are presented as the percent inhibition values and were 3
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capacity and concentration of α-tocopherol and γ-oryzanol. Moreover, the DPPH scavenging capacity of α-tocopherol was higher than that of γ-oryzanol; at a concentration of 500 μM, α-tocopherol displayed 90.37% ± 0.04% scavenging capacity, whereas 1 mM γ-oryzanol showed 58.75% ± 0.15% scavenging capacity. In contrast, for phytosterol, only 3.07% ± 0.15% activity was observed at 1 mM. Accordingly, the antioxidant capacities of the compounds decreased in the following order: α-tocopherol > γ-oryzanol > phytosterol, indicating that α-tocopherol played a key role in the mixture. Consistent with our findings, fat-soluble α-tocopherol is thought to be the most effective radical scavenging compound among the minor substances in plant oils (Belo, Nolasco, Mateo, & Izquierdo, 2017). Additionally, γ-oryzanol is thought to have radical scavenging capacity based on its many bioactivities (Lavecchia, Rea, Antonacci, & Giardi, 2013). Indeed, the DPPH radical scavenging abilities of the three constituents of γ-oryzanol used in the study were similar to those of ferulic acid (Akiyama et al., 2005), demonstrating the importance of the hydroxyl group in γ-oryzanol. However, no antioxidant capacity was observed for phytosterol, consistent with previous results (Fu et al., 2014; Huang, Zhao, & Zhou, 2012). Fig. 2. The capacity of scavenging DPPH free radical of three single compounds.
3.2. Effects of the interactions between α-tocopherol, γ-oryzanol, and phytosterol on antioxidant properties
color at 517 nm generated by π-π* transitions (Jabbari & Jabbari, 2016). The process can be easily monitored via UV–vis spectroscopy. The scavenging activities of each three compounds (α-tocopherol: 25–1000 μM, γ-oryzanol: 25–1000 μM, and phytosterol: 25–1000 μM) were determined using DPPH radical scavenging assays (Fig. 2). A linear correlation was observed between free radical scavenging
In order to determine the nature of interactions among γ-oryzanol, phytosterol, and α-tocopherol, different concentrations of single antioxidants were chosen to contribute to the partial reduction of the DPPH radical (Table 1). The mixtures were prepared while considering the antioxidant effects of each component. For example, TSC of the mixture of each α-tocopherol and γ-oryzanol (25 μM of each when mixed at a
Fig. 3. Relative inhibition (%) of theoretical and experimental values as well as synergy effect. Asterisks denote a significant difference (theoretical VS experimental values). (*P < 0.05, **P < 0.01). (AB) interaction between α-tocopherol and γ-oryzanol, (CD) interaction between α-tocopherol and phytosterol, and “An” means “antagonism”. 4
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1:1 v/v ratio) for the DPPH radical was compared with the ESC calculated by the sum of 25 μM α-tocopherol and 25 μM γ-oryzanol. If the ESC was significantly higher than the TSC value, a synergistic relationship was considered to have occurred in the mixture. In contrast, when the ESC was significantly lower than the TSC, an antagonistic relationship was considered. An additive interaction was considered when there were no significant differences between the two results. Fig. 3 shows the results of the analysis of two component mixtures and the SE values. All SEs were less than 1 and were statistically significant (p < 0.05) for all combinations in which α-tocopherol was mixed with γ-oryzanol. Similar antagonistic interactions were observed for α-tocopherol and phytosterol. In the presence of γ-oryzanol and phytosterol, the DPPH radical scavenging abilities at a concentration of 800 μM were 86% and 77%, respectively, demonstrating only a slight decrease in antioxidant capacity compared with α-tocopherol alone (90% at a concentration of 500 μM). The highest SE value detected was 0.98 at 800 μM for the mixture of α-tocopherol with γ-oryzanol, whereas an SE value of 0.99 was calculated at 1 mM for the mixture of α-tocopherol with phytosterol. This result may be caused by the different abilities of γ-oryzanol and phytosterol to scavenge the DPPH radical. Many studies have described the effects of structure and concentration on the types of interactions between antioxidants (Hajimehdipoor, Shahrestani, & Shekarchi, 2014; Luís et al., 2017).We also found that the degree of antagonism was stronger at low concentrations (50–200 μM) than at high concentrations (400–1000 μM), which was consistent with a previous study demonstrating that as the concentration increased, the degree of antagonism decreased (Liu, Shi, Ibarra, Kakuda, & Xue, 2008; Zhi-Lu et al., 2015). The synergy effect (SE) value in this study is calculated by the ratio of ESC to TSC. Compounds concentrations may have an effect on this ratio. At low concentrations (50–200 μM), the behavior of scavenging DPPH· for α-tocopherol is greatly affected by γ-oryzanol, phytosterols and solvents, making the ESC smaller than the TSC, thus the obtained SE value is small. At high concentrations (400–1000 μM), the ability of α-tocopherol to scavenge free radical is less affected by γ-oryzanol, phytosterols and solvents, making the ESC closer to the TSC, thus resulting in a increased SE value that is close to 1. It can be seen in Fig. 3, the SE value is small at low concentrations (50–200 μM), which means the antagonism is strong; the SE value is large at high concentrations (400–1000 μM), meaning a weak antagonism. To further understand how γ-oryzanol and phytosterols affect the ability of α-tocopherol to scavenge free radicals, we have done the following experiments. As a highly useful instrument for investigating the performance of compounds, FTIR is very valuable for understanding the interactions between molecules (Geboes, Proft, & Herrebout, 2018; Sivagurunathan & Hajasharif, 2012). As shown in Fig. 4, 200 μM αtocopherol and 400 μM mixtures of α-tocopherol with γ-oryzanol or phytosterol were analyzed. Because absolute ethanol has its own hydroxyl bond, the solvent was first dried with nitrogen and then pelleted using a KBr-based standardized procedure (Lin, Kasper, & Kjaergaard, 2013). The highest frequency of α-tocopherol, observed at 3467 cm−1, could be attributed to OH stretching, and the peak at 2750–3000 cm−1 was caused by the stretching vibration of C–H3 and the C–H2 out-ofphase stretching vibration, consistent with a previous report (Singh, Sachdeva, Rai, & Saini, 2017). After mixing with γ-oryzanol and phytosterol, several peaks changed in the spectrum compared with that of α-tocopherol alone. The fundamental transition wavenumbers of the OH-stretching of the mixture of α-tocopherol and γ-oryzanol or α-tocopherol and phytosterol were 3452 and 3436 cm−1, respectively, indicating red-shifted bands compared with the typical OH of α-tocopherol moving at a wavenumber of 15 and 31, respectively. According to the FTIR result, the formation of hydrogen bonds have occurred. Considering the structures of the antioxidants and the polar system, we think hydrogen bonds may be formed among the antioxidants and solvent, which decreased the number of binding sites of the antioxidants with DPPH during the process of scavenging DPPH in the
Fig. 4. Transmittance of 200 μM of α-tocopherol, γ-oryzanol, phytosterol as well as 400 μM mixtures in the region of 4000–400 cm−1.
solvent system (D. Li, Li, & Liu, 2016). Moreover, at low concentrations (50–200 μM), when a hydrogen atom in an antioxidant molecule forms a hydrogen bond with a polar solvent or an antioxidant, two bulky hetero-atoms are generated in the hydrogen atom around, thus results in a large steric congestion. It is the steric congestion that prevents DPPH· from coming into contact with α-tocopherols that have formed hydrogen bonds with polar solvent or other antioxidants and reacts only with the free fraction of antioxidants, resulting in the ESC is smaller than the TSC(Foti, 2015). This resulted in a smaller ESC than TSC and thus a small SE, which means a strong antagonism. However, the ability of α-tocopherol to scavenge free radicals is less affected by γoryzanol, phytosterols and solvents at high concentrations (400–1000 μM). Although the hydrogen bonds are still generated in the system at high concentrations, the main antioxidant, α-tocopherol scavenges DPPH· more rapidly than at low concentrations (SánchezMoreno, Larrauri, & Saura-Calixto, 1998). Therefore, the addition of γ– oryzanol and phytosterol has less influence on the total free radical scavenging ability of the system, resulting in an SE value close to 1, which means a weaker antagonism compared with the SE value at low concentrations (50–200 μM). 3.3. Kinetics of antioxidant behaviors When antioxidant (A) comes into contact with DPPH, the following reactions may occur: A – H + DPPH· → A + DPPH-H (reaction 1) A· + DPPH· → closed shell products (reaction 2) 2 A· → closed shell products (reaction 3) To further explore the antioxidant properties of α-tocopherol in the presence of γ-oryzanol and phytosterol, the kinetic curves of DPPH scavenging by α-tocopherol or the mixtures were determined (Fig. 5). The reaction rate of α-tocopherol with the DPPH radical was first compared with that of the mixture. Notably, the O–H bond dissociation enthalpy (BDE) of α-tocopherol is equal to 77.2 kcal/mol (Valgimigli, Ingold, & Lusztyk, 1996), which is lower than that of many other compounds. The mathematical model that satisfactorily describes the 5
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tocopherol to scavenge DPPH. The results of this study were consistent with the results of previous reports in the literature when using excess DPPH radical (Marteau et al., 2014; Yang et al., 2008; Zahalka et al., 1989). Thus, 1 mol of α-tocopherol could reduce nearly 2 mol of DPPH radicals, although α-tocopherol has only one available hydroxyl group. The mechanism of the α-tocopherol reaction with DPPH has been described in previous studies (Yang et al., 2008; Yang, Liu, Nie, Gao-Fei, & Yan, 2009) and is summarized in Eq. (7).
Fig. 5. Plots of relative inhibition versus time during scavenging DPPH radical in (■) 200 μM α-tocopherol; (●) 400 μM α-tocopherol + oryzanol; (▲) 400 μM α-tocopherol + phytosterol.
One molecule of α-tocopherol first reacts with DPPH free radicals to form a molecule of tocopheroxyl radical, and then two molecules of tocopheroxyl radical generate a molecule of α-tocopherol and a part of tocopherol quinone by the disproportionation reaction (Yang et al., 2008). The product of tocopherol quinone identified by UPLC-Q-TOF/ MS and the probable pathway to scavenge the DPPH radical are shown in Fig. 6 and Fig. 7. Several excellent reviews have described the antioxidant mechanisms of α-tocopherol in polar solvents (Marteau et al., 2014; Zahalka et al., 1989), in which sequential proton-loss-electron-transfer is likely involved (Bakhouche, Dhaouadi, Jaidane, & Hammoutène, 2015; Zhang & Ji, 2006) (Fig. 6). This method for scavenging DPPH increases the reaction rate compared with pure hydrogen donation (Foti, 2015). Electrospray ionization for α-tocopheryl quinone (TQ) was enhanced by the addition of formic acid in the mobile phase to produce [M+H] +species (Fig. 6). Thus, ions at m/z 447.5 would be expected for the TQ. In fact, we observed that this mode of ionization also produced ions at m/z 429.5 for TQ, representing [M + H–H2O]+, and the full-scan product ion spectra of m/z 429 for TQ showed a predominant fragment ion at m/z 165, which was attributed to loss of the phytyl chain (Mottier, Gremaud, Guy, & Turesky, 2002). Clearly, the process of scavenging DPPH for α-tocopherol occurred via the α-tocopheroxyl radical as an intermediate. However, in the presence of γ-oryzanol and phytosterol, the stoichiometry of α-tocopherol scavenging free radicals was less than 1 (i.e., 0.60 and 0.52, respectively). Previous studies have explained this phenomenon (Yang et al., 2008); the antagonism between α-tocopherol and γ-oryzanol or phytosterol for scavenging the DPPH radical can be explained as follows: during the process of scavenging DPPH, the tocopheroxyl radical product combines with other free radicals, inhibiting the regeneration of α-tocopherol. Some studies have reported that nonradical products can be formed from the coupling of free radicals with the α-tocopheroxyl radical in the presence of other free radicals (Yamauchi, 1997), which may have been the main antagonistic mechanism observed in this study.
time dependence of scavenging DPPH is the exponential function: y = a (1 – e-bx)
(4)
Where x is the reaction time, y denotes the scavenging of DPPH radical capacity, a and b are coefficients of the fit functions listed in Table 2, showing a good fit (R2 and Adj. R2 > 0.99). The curves in Fig. 5 were plotted based on parameters a and b in Eq. (4), which were acquired by nonlinear regression analysis. The rate of DPPH radical scavenging (Rs) was determined to extrapolate the kinetics of scavenging of the DPPH radical. For all nonlinear kinetic reactions, the only useful rate is the maximum (Prieto, Curran, Gowen, & Vázquez, 2015). Hence, the rate of the corresponding process calculated at t = 2 s (the initial rate) as the first derivative of the function 4 described by Eq. (5) is listed. R = abe-bx
(5)
The rate of DPPH radical scavenging is presented in Table 2. Higher values indicated better responses when antioxidants were added to the system. The rate of DPPH radical scavenging was the highest for αtocopherol, reaching 4.25 s−1, followed by 3.95 and 2.12 s−1 for γoryzanol + α-tocopherol and phytosterol + α-tocopherol, respectively; thus, these compounds decreased the capacity of α-tocopherol to scavenge the DPPH radical. Moreover, we calculated the stoichiometry as previously described (Mendoza-Wilson et al., 2010), using the following equation: n = c0 (1 – At / A0) / c
(6)
Where c0 is the initial DPPH radical concentration, c is the initial antioxidant concentration (200 or 400 μM), At is the absorbance at the end of the kinetic run, and A0 is the initial absorbance. Stoichiometry is a parameter used to describe the moles of DPPH free radical reduced by 1 mol of antioxidant (Osman, 2011). Several studies have demonstrated that the stoichiometry is 2.1 for α-
Table 2 The determination coefficients of α-tocopherol and the mixture with γ-oryzanol and phytosterols in the reaction system determined by fitting data through Eq. (4) for kinetics of DPPH radical scavenging(r2 (DPPH radical)). The rate of free radical scavenging (Rs DPPH radical) was determined at t = 2s. sample
R2
Adj. R2
coefficients
α-tocophenol α-tocophenol+γ-oryzanol α-tocophenol + phytosterols
0.9984 0.9935 0.9984
0.9983 0.9928 0.9982
a 42.0600 22.6300 20.5353
Note:the value in parentheses indicates the standard error of stoichiometries. 6
b 0.1315 0.3544 0.1351
Rs DPPH radical(s−1) (t = 2s)
Stoichiometries
4.253 3.948 2.117
2.13(0.0062) 0.60(0.0364) 0.52(0.0135)
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Fig. 6. The mechanism stepwise sequential proton-loss-electron-transfer, proposed to elucidate α-tocopherol scavenging DPPH radical in ethanol.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.05.104. References Akiyama, Y., Hori, K., Takahashi, T., & Yoshiki, Y. (2005). Free radical scavenging activities of γ-oryzanol constituents. Food Science and Technology International Tokyo, 11(3), 295–297. Altunkaya, A., Becker, E. M., Gökmen, V., & Skibsted, L. H. (2009). Antioxidant activity of lettuce extract (Lactuca sativa) and synergism with added phenolic antioxidants. Food Chemistry, 115(1), 163–168. Bakhouche, K., Dhaouadi, Z., Jaidane, N., & Hammoutène, D. (2015). Comparative antioxidant potency and solvent polarity effects on HAT mechanisms of tocopherols. Computational and Theoretical Chemistry, 1060, 58–65. Belo, R. G., Nolasco, S., Mateo, C., & Izquierdo, N. (2017). Dynamics of oil and tocopherol accumulation in sunflower grains and its impact on final oil quality. European Journal of Agronomy, 89, 124–130. Brandwilliams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28(1), 25–30. Çelik, E. E., Rubio, J. M. A., & Gökmen, V. (2017). Behaviour of trolox with macromolecule-bound antioxidants in aqueous medium: Inhibition of auto-regeneration mechanism. Food Chemistry, 243, 428–434. Deng, J., Cheng, W., & Yang, G. (2011). A novel antioxidant activity index (AAU) for natural products using the DPPH assay. Food Chemistry, 125(4), 1430–1435. Doert, M., Jaworska, K., Moersel, J. T., & Kroh, L. W. (2012). Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol. European Food Research & Technology, 235(5), 915–928. Evstigneeva, R. P., Volkov, I. M., & Chudinova, V. V. (1998Doert, M., Jaworska, K., Moersel, J. T., & Kroh, L. W. (2012). Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol. European Food Research & Technology, 235(5), 915-928Doert, M., Jaworska, K., Moersel, J. T., & Kroh, L. W. (2012). Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol. European Food Research & Technology, 235(5), 915-928). Vitamin E as a universal antioxidant and stabilizer of biological membranes. Membrane and Cell Biology, 12(2), 151–172. Fine, F., Brochet, C., Gaud, M., Carre, P., Simon, N., Ramli, F., et al. (2016). Micronutrients in vegetable oils: The impact of crushing and refining processes on vitamins and antioxidants in sunflower, rapeseed, and soybean oils. European Journal of Lipid Science and Technology, 118(5), 680–697. Foti, M. C. (2015). Use and abuse of the DPPH radical. Journal of Agricultural and Food Chemistry, 63(40), 8765. Fu, Y., Zhang, Y., Hu, H., Chen, Y., Wang, R., Li, D., et al. (2014). Design and straightforward synthesis of novel galloyl phytosterol with excellent antioxidant activity. Food Chemistry, 163(20), 171–177. Geboes, Y., Proft, F. D., & Herrebout, W. A. (2018). Towards a better understanding of the parameters determining the competition between bromine halogen bonding and hydrogen bonding: An FTIR spectroscopic study of the complexes between bromodifluoromethane and trimethylamine. Journal of Molecular Structure, 1165, 349–355. Hajimehdipoor, H., Shahrestani, R., & Shekarchi, M. (2014). Investigating the synergistic antioxidant effects of some flavonoid and phenolic compounds. Research Journal of Pharmacognosy, 1(3), 35–40. Hall, C., Iii, & Gunstone, F. D. (2003). Other natural antioxidants - rice bran oil, sesame oil, rosemary extract. flavonoids: Elsevier Ltd. Hashempour-Baltork, F., Torbati, M., Azadmard-Damirchi, S., & Savage, G. P. (2016). Vegetable oil blending: A review of physicochemical, nutritional and health effects. Trends in Food Science & Technology, 57, 52–58. Huang, Y., Zhao, Y., & Zhou, Z. (2012). Scavenging effects on free radical by phytosterol.
Fig. 7. TOF MS spectra and proposed fragmentation of degradation products of α-tocopherol.
4. Conclusion In conclusion, we examined the interactions among α-tocopherol, γoryzanol, and phytosterol using DPPH assays. Our findings showed that interactions among the three bioactive compounds may have negative effects. The γ-oryzanol and phytosterol present in α-tocopherol could decrease the capacity of α-tocopherol to scavenge the DPPH radical. This antagonism may result from inhibition of the auto regeneration of α-tocopherol. The results provided insights that may be of interest in oil processing in order to ensure that the contents of these minor compounds are in a suitable range. Of course, the contents of γ-oryzanol and phytosterol in rice bran oil are 10 times that of α-tocopherol; thus, further studies are needed to explore the interactions of bioactive compounds in nonpolar systems and oil systems. Additionally, the SEs and interactions among various antioxidants should be studied to determine the net antioxidant capacity of plant oils, highlighting the importance of choosing the right combination and concentration of compounds to promote synergism and avoid antagonistic reactions. Conflicts of interest There was no conflict of interest exits in the submission of this manuscript, and all authors approve the publication of the manuscript. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant No. 31872895) & national first-class discipline program of Food Science and Technology (JUFSTR20180202). 7
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