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Effect of edible antioxidants on chemical stability of ß-carotene loaded nanostructured lipid carriers
LWT - Food Science and Technology 113 (2019) 108272
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Effect of edible antioxidants on chemical stability of ß-carotene loaded nanostructured lipid carriers
T
Abdolrasoul Hejria, Alireza Khosravia,∗, Kamaladin Gharanjigb, Mohammadreza Malekzade Davarania a b
Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, Hafez St., 15875-4413, Tehran, Iran Department of Organic Colorants, Institute for Color Science and Technology, Hossein Abad Sq., 1668814811, Tehran, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanostructured lipid carriers Response surface methodology β-carotene Antioxidant Degradation
Nanostructured lipid carriers (NLC) containing ß-carotene was produced using solvent diffusion method. Edible antioxidants of butylated hydroxytoluene (BHT), vitamin E and vitamin C were successfully incorporated in NLC to protect ß-carotene. Response surface methodology (RSM) was employed in order to evaluate the effect of different antioxidants on ß-carotene stability. A quadratic polynomial model was fitted to empirical data with high accuracy. The ß-carotene retention improved with raising the BHT concentration while addition of vitamin C resulted in ß-carotene instability. Increasing the vitamin E concentration first elevated the ß-carotene degradation rate and then decreased it. However, the total effect of vitamin E alone was less significant. The statistical analysis demonstrated that the interactions between BHT and two other antioxidants had important effect on ß-carotene stability. The optimum formulations predicted by the model contained BHT and vitamin E combinations. They had minimum ß-carotene degradations of 2.12–4.51% which were in a reasonable agreement with model predictions. Furthermore, particles size analysis indicated that addition of antioxidants to NLCs results in production of larger nanoparticles.
1. Introduction ß-carotene is one of the most common carotenoids in nature widely used as a natural colorant in food industry (Burri, 1997). Due to its polyene structure, ß-carotene is able to act as a notable free radical scavenging and singlet oxygen quenchers makes it an important natural antioxidant. Consequently, according to numerous studies, ß-carotene supplement may result in significant health benefits including reducing the risk of cardiovascular, pulmonary, ophthalmological diseases and certain types of cancer (Gaziano, Manson, Buring, & Hennekens, 1992; Krinsky & Johnson, 2005; Nishino, 1997; Stahl & Sies, 2005). However, this electron rich conjugated double bond structure which gave ß-carotene its special characteristics makes it unstable against oxidation, heat and light (Socaciu, 2007). Taking under consideration its water insolubility and sensitivity, ß-carotene's application in foods is facing with some limitations. Therefore, ß-carotene can be delivered via functional food carries which may improve its solubility, stability and bioavailability (Soukoulis & Bohn, 2018). Since their introduction in early 1990s, solid lipid nanoparticles (SLN) have got a great deal of attraction due to their unique properties
∗
which merge together advantages of traditional carriers such as bioavailability, biocompatibility, controlled release and membrane permeability. Classical carriers like emulsions, liposomes and polymeric nanoparticle have limitations including organic solvent usage, carrier bio toxicity, low drug payload and large scale production which SLNs could also reduce them (Müller et al., 1995; Müller, Radtke, & Wissing, 2002a,b). Because of their crystalline solid structure, polymorphic transitions lead to low drug payload, drug expulsion during storage and particle aggregation (Mehnert & Mäder, 2012; Weiss et al., 2008). These disadvantages resulted in introduction of the second generation of lipid nanoparticles “Nanostructured lipid carriers” (NLC) (Radtke & Müller, 2001). In NLCs, the lipid phase is made of both solid and liquid lipids leading to a less ordered crystalline or amorphous solid structure. Accordingly, NLCs have higher drug loading capacity and can avoid or minimize drug expulsion during storage (Müller et al., 2002a,b). Moreover, NLCs have potential to be used in transparent products; they are physically stable and can improve the bioavailability of lipophilic bioactive compounds (Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2013). These properties make NLCs a proper carrier for bioactive food components attracting the interest of food area researchers in recent
Corresponding author. E-mail address: [email protected] (A. Khosravi).
https://doi.org/10.1016/j.lwt.2019.108272 Received 29 January 2019; Received in revised form 7 May 2019; Accepted 16 June 2019 Available online 17 June 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Experimental design and statistical analysis
years (Hejri, Khosravi, Gharanjig, & Hejazi, 2013; Liu & Wu, 2010; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014a). Application of bioactive compounds in foods is being increased by growing the demand for functional foods (Goldberg, 2012). In this regard, one of the major difficulties is the chemical stability of bioactive component. For instance, degradation of the carotenoids in a food enriched with them will affect both the quality and bioactivity of the food (Boon, McClements, Weiss, & Decker, 2010; de Vos, Faas, Spasojevic, & Sikkema, 2010). NLCs are able to preserve these ingredients however this is not inclusive for all formulations and optimization is required (Hejri et al., 2013). Utilizing antioxidants is a major procedure to further prevent oxidation of lipids and bioactive ingredients in food dispersions. Since the lipid oxidation in dispersions depend on many factors comprising lipid and bioactive composition, oxygen concentration, type and concentration of antioxidants and pro-oxidants, lipid droplet characteristics, emulsion droplet interfacial properties and etc.; hence combination of antioxidative techniques and selection of antioxidants are crucial to obtain an efficient protection (Jee, Lim, Park, & Kim, 2006; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014b; Waraho, McClements, & Decker, 2011). In this study, nanostructured lipid carriers were used as a carrier for delivering ß-carotene. The purpose of this study is to minimize the degradation of ß-carotene as a model nutraceutical in NLCs using an antioxidant system. For this purpose both lipophilic antioxidants of vitamin E and BHT and hydrophilic antioxidant of vitamin C were utilized. The relations between the antioxidants concentration and ßcarotene degradation were evaluated applying response surface methodology (RSM) and the optimum conditions were obtained. At the end, the physical properties of the optimum ß-carotene loaded nanostructured lipid carriers were investigated. Formulating chemically stable bioactive loaded NLCs will contribute to expand the application of these carriers in foods and thus to be more benefited from the advantages of these systems in our diet in the future.
Response surface methodology (RSM) was employed to evaluate the relation between the response and independent variables where the ßcarotene degradation value is the response and BHT (A), vitamin E (B) and vitamin C (C) concentrations are independent variables. This method has been successfully used in previous literature on NLCs (Hejri et al., 2013; Liu & Wu, 2010). Utilizing a 3 factor Box–Behnken design, a set of 17 runs was designed including 5 replicates at central point to calculate the pure error. The independent variables values at coded levels of −1 and +1 were set to 0 and 2% w/w, respectively. Design-Expert® 7.0 software was employed for statistical analysis. In order to find the best fitted mathematical model the sequential model sum of squares, the multiple correlation coefficient (R2), the adjusted multiple correlation coefficient (adjusted R2), the predicted multiple correlation coefficient (predicted R2), predicted residual error sum of squares (PRESS) and lack-of-fit of the various polynomial models were evaluated. The F-test was performed to assess if there is a significant relation between the response and the model factors at the confidence 95% (α = 0.05). Utilizing student's t-tests the significant model factors and their interactions were specified at the confidence 95% (α = 0.05). In order to modify the mathematical model, insignificant model parameters were eliminated using backward stepwise regression. Contour plots for the fitted model were generated by Design-Expert® 7.0. Finally, predicted formulations with minimum ß-carotene degradation were obtained by numerical solution of the fitted model and compared with experimental data to evaluate the model accuracy.
2.4. ß-carotene stability analysis According to the experimental design, 17 samples were prepared and stored in dark at room temperature (25 ± 2 °C). The stability of ßcarotene was evaluated after 2 weeks. The absorbance of ß-carotene loaded NLCs were measured using UV–Vis spectroscopy (Jenway 6715, Staffordshire, UK) and employed to determine the ß-carotene stability (Triplett & Rathman, 2009). The NLC dispersion was dissolved in acetone (1:1 v/v) and absorbance was measured in λmax = 455 nm. Then, the ß-carotene concentration was calculated according to beerlambert law using calibration curve. The ß-carotene degradation was calculated using the relation:
2. Material and methods 2.1. Materials All the materials used in this study were food grade and used as received. Palmitic acid (PA), Polyoxyethylene sorbitan monooleate (Tween 80), ethanol and acetone were obtained from Merck (Darmstadt, Germany). Ascorbic acid (Vitamin C), DL -α-Tocopherol (Vitamin E) and Butylated hydroxytoluene (BHT) were purchased Sigma-Aldrich Chemie GmbH (Munich, Germany). Pure ß-carotene powder was provided by BASF (Ludwigshafen, Germany). Corn oil was from Behshahr industrial Co. (Tehran, Iran). Double distilled water was used in the experiments.
Degradation(%) =
C0 − C × 100 C0
(1)
where C0 and C are initial concentration and concentration after 2 weeks of ß-carotene, respectively. A NLC without ß-carotene was prepared and its absorption at λmax = 455 nm was measured. Since the absorption of the NLC without ß-carotene was negligible, therefore, the absorbance of the samples was related to ß-carotene. Furthermore, since there were no severe heating and illumination conditions during storage, the effect of stereoisomer transformation is ignored in this study (Chen, Chen, & Chien, 1994).
2.2. Preparation of ß-carotene loaded NLCs Nanostructured lipid carriers were produced by solvent diffusion method according to our previous work with some modifications (Hejri et al., 2013). In brief, an aqueous phase containing Tween 80 (3% w/ w), double distilled water and different amounts of vitamin C was preheated to 40 °C. Meanwhile, a lipid phase was prepared by mixing corn oil, palmitic acid and ß-carotene and various amounts of vitamin E and BHT. Then, 2 g/l lipid phase was dissolved in ethanol at 65–70 °C. Subsequently the resulting solution was added to the prepared aqueous phase under mechanical agitation at 1000 rpm and mixed for 10 min. The resulting dispersion was then cooled to room temperature to form NLCs. In all formulations, the ß-carotene concentration in lipid phase, corn oil to palmitic acid ratio and volume ratio of ethanol/water were fixed at 2% (w/w), 1:10 (w/w) and 1:10 (v/v), respectively.
2.5. Particle size analysis The particle size and size distribution of the NLCs were investigated by dynamic light scattering (DLS) using a Cordouan Vasco™ (Cordouan Technologies, France) instrument. The measurements were performed at 25 °C. In order to prevent multiple scattering effects, the samples were diluted to appropriate concentration with distillated water. The instrument reports the mean particle size (z-average) and polydispersity index (PDI) of the samples.
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the observed F-value if the null hypothesis is true. Hence, at the confidence level of 95% (α = 0.05) the p-value of less than 0.05 indicated strong evidence against the null hypothesis which denoted that the terms in the model have significant effect on response. The higher Fvalues result in lower p-values. As it is shown in Table 2, the model's Fvalue of 65.72 indicated the quadratic model is significant at the level of α = 0.001. The F-values for all model terms were calculated with similar procedure. According to Table 2, BHT (A) and vitamin C (C) concentrations had important effect on ß-carotene degradation (p < 0.001). Furthermore, quadric terms of vitamin E (B2) and vitamin C (C2) concentrations were significant model terms (p < 0.001). Also, the interactions between BHT and vitamin E (AB) and the interaction between BHT and vitamin C (AC) had considerable impact on ß-carotene stability (p < 0.05). Other model term have negligible effect on ß-carotene retention (p > 0.05). The quadratic model was modified by eliminating the insignificant model term applying backward stepwise regression procedure. It should be considered that R2 will increase by adding any terms to the model, even if it is due to chance. Thus, adjusted R2 was used to compare models with different number of terms. Adjusted R2 increases only when adding a term amends the model fit more than expected by chance. Accordingly, omitting the interactive term of BC from the model leaded to raising the adjusted R2 from 0.973 to 0.977. However, the term A2 was not deleted from the model because of the reduction in adjusted R2 value. Also, Vitamin E (B) parameter was not removed from the model in order to keep the hierarchical structure of the quadratic model. The modified second order polynomial model based on codded levels for the ß-carotene degradation is described as follow:
Table 1 The β-carotene degradation values of samples prepared according to Box–Behnken experimental design. Run
A: BHT concentration (% w/w)
B: Vitamin E concentration (% w/w)
C: Vitamin C concentration (% w/w)
β-carotene degradation (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0 1 1 2 1 0 1 1 1 1 0 2 1 2 1 0 2
1 1 1 1 2 2 1 0 0 1 0 0 1 2 2 1 1
0 1 1 2 0 1 1 2 0 1 1 1 1 1 2 2 0
71 90 93 87 9 91 87 76 17 87 86 79 92 58 69 84 2
3. Results and discussion 3.1. Establishing the model The NLC dispersions were prepared according to Box–Behnken design. 17 samples were kept at 25 ± 3 °C in dark for 2 weeks. The absorbances of the samples were measured at the day of preparation and after 2 weeks and the ß-carotene degradation values were calculated according to calibration curve. Data is shown in Table 1. The various statistical parameters of different fitted models were compared. The results indicated that the best fitted mathematical model to experimental data was quadratic polynomial model. The R2 = 0.988 for the quadratic model implies that the relationship between the independent variables and the response is well described by the model. The adjusted R2 and predicted R2 values were 0.973 and 0.843, respectively. The high value of the predicted R2 which is in good agreement with adjusted R2, means that the model is not overfitted and can be used to predict new observations for ß-carotene degradation. The analysis of variance (ANOVA) of the model terms performed by Design-Expert® 7.0 software is summarized in Table 2. F-test was performed to evaluate the significance of the model. F-value compares the model variance with the residual variance. If the variances are close to the same then it is less likely that any of the factors have a significant effect on the response. The null hypothesis of the F-test is that the all regression coefficients are zero. The p-value is the probability of finding
Beta carotene degradation(%) = 89.80 − 13.25A − 3.88B + 27.13C − 6.50AB + 18.00AC + 3.47A2 − 14.77B2 (2) − 32.28C 2 The model F-value was increased to 84.37 (p < 0.0001). The R2 and predicted R2 values of the modified model were 0.988 and 0.866, respectively. The lack of fit F-value of 4.55 (p > 0.05) indicated insignificant lack of fit of the modified model. In lack of fit test the null hypothesis is that the model fits well. Hence, p > 0.05 means that the null hypothesis is true. 3.2. Effect of antioxidants on particle size of NLC Since particle size of NLC have great effect on chemical stability of the loaded bioactive compound (Hejri et al., 2013), the particle size of the 5 NLC samples including 2% w/w BHT, vitamin E and vitamin C loaded NLCs together with an antioxidant free NLC and a central point run were measured by DLS (Table 3). As shown in Table 3, Incorporation of the antioxidants into NLC formulation increased the particle size of it (p < 0.05). The highest grow in particle size was observed for vitamin C (p < 0.0001) while the lowest was for BHT (p < 0.05). The effect of vitamin C could not be explained by the current data; nevertheless it would be because of an interaction between the Tween 80 and vitamin C and its impact on the surface chemistry of the particles. Also, The larger particle size of vitamin E loaded NLC compare to BHT loaded NLC could be due to the higher molecular size of vitamin E; however it was not statistically significant (p > 0.05).
Table 2 The Analysis of variance (ANOVA) of the model terms. Term
a
Model Intercept A B C AB AC BC A2 B2 C2
β -carotene degradation F -value
p-value
65.72
< 0.0001
57.646 4.930 241.588 6.936 53.193 0.010 2.087 37.726 180.018
0.0001 0.0618 < 0.0001 0.0337 0.0002 0.9222 0.1918 0.0005 < 0.0001
Coefficient
89.80 −13.25 −3.88 27.13 −6.50 18.00 0.25 3.47 −14.78 −32.28
3.3. Parameters effect on ß-carotene There are different ways that antioxidants intercept oxidation process. The effectiveness of antioxidants depend on various factors including oxidation reduction potential, rate constants, activation energy, solubility, volatility and heat susceptibility. The most efficient antioxidants interrupt the free radical chain reaction. The H-atom transfer and electron transfer are two pathways that an antioxidant can
a A: BHT concentration, B: Vitamin E concentration, C: Vitamin C concentration.
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Table 3 Effect of antioxidants on the particle size of β-carotene loaded nanostructured lipid carriers (NLC). Sample
BHT concentration (% w/w)
Vitamin E concentration (% w/w)
Vitamin C concentration (% w/w)
β-carotene concentration (% w/w)
Particle size (nm)a
Polydispersity Index (PDI)a
Antioxidant free BHT Vitamin E Vitamin C Central point
– 2.00 – – 1.00
– – 2.00 – 1.00
– – – 2.00 1.00
2.00 2.00 2.00 2.00 2.00
111 126 138 218 174
0.245 0.251 0.304 0.334 0.313
a
± ± ± ± ±
4 6 9 5 9
± ± ± ± ±
0.032 0.028 0.025 0.052 0.068
Data were driven from three replications and reported as mean ± standard deviation.
Fig. 1. Contour plot showing (a) the effect BHT and vitamin E concentrations on β-carotene degradation and (b) the effect BHT and vitamin C concentrations on βcarotene degradation.
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radicals and ROS (Anguelova & Warthesen, 2000). The influence of the vitamin C on ß-carotene degradation is demonstrated in Fig. 1b (vitamin E concentration is fixed at central point). The same trend observed for vitamin E was obtained for vitamin C although its effect was more important compared to vitamin E. Vitamin C was dissolved in aqueous medium and presented in this phase as a water soluble antioxidant. In general, known as “antioxidant polar paradox”, nonpolar antioxidants are more effective than polar antioxidants in emulsions since they accumulate in lipid phase interface where the most oxidation occurs (Marquardt et al., 2013). Vitamin C can show pro-oxidant effect through an autoxidation mechanism. At high oxygen tensions, like this study, molecular oxygen and ascorbyl radical (VC˙−) are dominant. The interaction between them results in generation of superoxide radical which can then react with ascorbate molecule and oxidize it to another ascorbyl radical (Eqs. (5) and (6)) (Eghbaliferiz & Iranshahi, 2016; Zhang & Omaye, 2000):
deactivate a free radical (Brewer, 2011; Wright, Johnson, & DiLabio, 2001). BHT is one of the commonly used synthetic antioxidants in food industry. Fig. 1a shows the contour plot of the ß-carotene degradation as a function of BHT and vitamin E concentrations while other parameter is fixed at central point. BHT is a hindered-phenol antioxidant and can be refer as synthetic analogue of vitamin E. BHT act as a chain breaking antioxidant by H-atom transfer mechanism as follow (Burton & Ingold, 1984; Yehye et al., 2015):
ROO˙ + ArOH → ROOH + ArO ˙
3
ArO˙ + ROO˙ → nonradical products
4
As it is illustrated in Fig. 1a, the ß-carotene degradation was decreased by increasing the BHT concentration. BHT as a more reactive antioxidant first reacts with free radicals and the reactive oxygen species (ROS) and therefore protects the ß-carotene against deterioration (Anguelova & Warthesen, 2000; Jee et al., 2006). It should be noted that BHT possessed superior antioxidant activity than vitamin E to protect ß-carotene. This behavior has been observed in solid lipid nanoparticles containing all trans retinol (Jee et al., 2006) and could be due to the different chemical structure of BHT and vitamin E. Bond dissociation energy (BDE) is a physical parameter which plays a critical role in antioxidant activity of chemical compounds; The lower the BDE the higher the antioxidant efficiency. The BDE of phenolic O–H bond of vitamin E is about 2 kcal/mol less than BHT. However, substituents at ortho position result in steric hindrance to minimize undesirable reactions such as pro-oxidation (Yehye et al., 2015). Therefore, ortho tbutyl groups of BHT make its radicals unreactive and prevent any prooxidation effect unlike vitamin E. Elevating the vitamin E concentration initially lowered the ß-carotene stability and then improved it (Fig. 1a). However, it did not significantly protect ß-carotene even at high concentrations. It has been reported that α-tocopherol did not significantly increase the astaxanthin stability in NLCs after 10 and 15 days (Tamjidi et al., 2014b).Vitamin E can act as a pro-oxidant in food and biological systems. In the other word, increasing the vitamin E level under high oxidative stress conditions would lead in generation of high amounts of vitamin E radicals, which could not reduce to vitamin E in the absence of a co-antioxidant. These radicals could start lipid peroxidation process by themselves and therefore oxidize the ß-carotene present in the nanoparticles as a potent reactive bioactive compound (Rietjens et al., 2002). Furthermore, due to amphiphilic nature of vitamin E, its hydrophobic chain penetrates into nanoparticles while the hydrophilic head group tend toward the less hydrophobic environment. Therefore, vitamin E antioxidant activity take place at less hydrophobic microenvironment near the surface. Despite hydrophobic ß-carotene which would stays in the core of the particles (Aliaga, de Arbina, Pastenes, & Rezende, 2018; Marquardt et al., 2013). Thus, vitamin E would first react with radicals diffusing into nanoparticles which cause production of vitamin E radicals inside nanoparticles. Consequently, in absence of any reducing agent these radicals would oxidize ß-carotene. Another reason that may have been led to the observed results in low vitamin E concentrations was the competition between ß-carotene and vitamin E toward reaction with peroxyl radicals and ROS in which the ß-carotene was the substance reacted at first due to its dominant concentration (Anguelova & Warthesen, 2000). Adding the vitamin E to NLC formulation has grown the particle size of nanoparticles (Table 3). Larger particles provide lower surface area for diffusion of oxidative species (Hejri et al., 2013). By increasing the vitamin E concentration, this effect became predominant after a point and ß-carotene degradation rates started to fall down (Fig. 1a). Moreover, incorporation of vitamin E could enhance the ß-carotene loading efficiency and hence reducing the free ß-carotene amounts (Jee et al., 2006). In addition, as mentioned in past paragraph, at high vitamin E levels, this time it is vitamin E which initially reacts with peroxyl
VC·− + O2 → VC + O2⋅− O2⋅−
+
VCH−
→
HOO−
+
5
VC· −
6
This is an autoxidation process for vitamin C oxidizing it without showing a protective effect. In addition, since the superoxide radical produced in Eq. (5) is a very strong oxidizing agent, it could attack ßcarotene and degrade it. Another probable process would be the pro-oxidant effect of vitamin C in presence of trace metals (Kanner, Mendel, & Budowski, 1977; Podmore et al., 1998; Zhang & Omaye, 2000). Natural antioxidants generally act as pro-oxidant in presence of transition metals specially Iron and Copper. Ferric cations increase the oxidative stress by generating ROS via Fenton-type reaction (Eq. (7) and Eq. (8)). The ferrous cations react with hydrogen peroxide to produce hydroxyl radicals (Eq. (8)) (Eghbaliferiz & Iranshahi, 2016).
Fe3 + + O2⋅− → Fe 2 + + O2
7
Fe 2 + + H2 O2 → Fe 3 + + OH˙ + OH−
8
The generated ferrous species could also start lipid oxidation process directly (Eq. (9)) or interact with O2 to generate O2˙- (Eq. (10)). Vitamin C act as a catalyst in this process and keeps iron in its reduced state (Kanner et al., 1977; Rietjens et al., 2002; Zhang & Omaye, 2000). The O2˙- and OH˙ as strong oxidants would diffuse into NLC and decompose the incorporated ß-carotene.
Fe 2 + + ROOH → Fe3 + + RO˙ + OH− Fe 2 + + O2 → Fe3 + + O2⋅−
9 10
The particle size of NLC increased by rising the vitamin C concentration (Table 3) and accordingly ß-carotene degradation decreased at high vitamin C amounts. However, considering the strong oxidative species produced according to Eqs. (5)-(10), only a small reduction in ßcarotene degradation rate was observed (Fig. 1b). Yet, the degradation level is much more than the value yielded in the absence of vitamin C. One of the significant interactions was the interaction between BHT and vitamin E. Fig. 2a and b displays the effect of vitamin E on ßcarotene degradation at BHT concentrations of 0 and 2% w/w, respectively (vitamin C concentration fixed at 0). The higher the BHT concentration the better the vitamin E performance; so there was a perfect protection of ß-carotene at high BHT and vitamin E amounts (Fig. 2b). Both antioxidants are located inside the nanoparticles. Therefore, BHT can act as a reducing agent for vitamin E radicals and consume them to reproduce vitamin E (Burton & Ingold, 1984; Rietjens et al., 2002). Thus, the issue of the vitamin E radicals would be eliminated and vitamin E could still trap free radicals and ROS. Another major interaction was among BHT and vitamin C. The ßcarotene degradation as a function of BHT concentration at 0 and 2% w/w vitamin C concentrations is illustrated in Fig. 2c and d, respectively (vitamin E concentration fixed at 0). At high values of vitamin C, 5
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Fig. 2. Effect of vitamin E concentration on β-carotene degradation at (a) 0% w/w and (b) 2% w/w BHT concentrations at vitamin C concentration of 0 and effect of BHT concentration on β-carotene degradation at (c) 0% w/w and (d) 2% w/w vitamin C concentrations at vitamin E concentration of 0.
contained it. Although, BHT itself could protect ß-carotene, combination of it with vitamin E had led to the best results. In this regard, Tamjidi et al. (2014b) investigated the effect of ethylenediaminetetraacetic acid (EDTA), ascorbic acid coenzyme Q10 and α-tocopherol on the chemical stability of astaxantin in NLCs. They concluded that combination of EDTA with high concentrations of α-tocopherol was more effective than other antioxidant systems and it could reduce the astaxantin degradation to about 20% after 15 days of storage in the dark at 35 °C. This was due to slower degradation of α-tocopherol in presence of EDTA. Furthermore, it has been reported that adding the BHT-BHA (Butylate hydroxyanisol) antioxidant combination to the alltrans retinol loaded SLNs increased the intact amount of all-trans retinol from almost zero to 89% after 72 h storage under shade at room temperature (Jee et al., 2006). The reasonable agreement between the predicted and actual data (p < 0.05) indicated that the fitted polynomial model is accurate and can be used to navigate in the design space. Moreover, the particle sizes and PDI indexes of the optimum formulations were evaluated
the BHT behavior changed and increasing the BHT concentration had negative effect on ß-carotene stability. The oxidative species generated in presence of vitamin C, which has discussed in this section, would have resulted in nearly complete oxidation of BHT and it could no longer protect ß-carotene. Consequently, the BHT radicals created in NLC could be a factor led to ß-carotene instability as well. 3.4. Determination of optimum formulations and model accuracy Three different antioxidants had three different influences on ßcarotene stability. In order to find the optimum formulations with the lowest ß-carotene degradation, numerical solution of the model together with overlay plots were utilized. Table 4 shows three formulations with low ß-carotene degradation as predicted by the model and the actual amounts measured for them. In comparison with ß-carotene degradation in antioxidant free NLC (51%), the results revealed a superb protection of ß-carotene in the optimized NLCs. Since the vitamin C has negative effect on ß-carotene stability, none of the formulas
Table 4 Comparison of predicted and actual values of β-carotene degradation for optimum formulations together with particle size and PDI index of them. Sample
1 2 3 a
BHT concentration (% w/ w)
1.98 2.00 1.30
Vitamin E concentration (% w/w)
1.19 0.24 1.94
Vitamin C concentration (% w/w)
0.0 0.0 0.0
Data were driven from three replications and reported as mean ± standard deviation. 6
β-carotene degradation (%) predicted
actual
1.00 2.00 3.00
2.37 2.12 4.51
Particle size (nm)a
Polydispersity Index (PDI)a
163 ± 8 132 ± 11 167 ± 4
0.291 ± 0.023 0.266 ± 0.038 0.282 ± 0.041
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employing DLS particle size analysis (Table 4). The NLCs had particle sizes of 132–167 nm and low PDI values (0.266–291) indicating the narrow distribution which could be a reason for physical stability of produced NLCs. Finally, since there is close relationship between the physical characteristics of NLC and chemical stability of loaded bioactive compound, more researches should be conducted to evaluate the physical properties of the obtained NLCs and to subsequently determine the exact mechanisms behind the observed results. Moreover, these NLCs should be employed in different food media in order to investigate the effect of the food matrices on function of the antioxidant system.
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Optimization of nanostructured lipid carriers for lutein delivery. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 353(2), 149–156. Marquardt, D., Williams, J. A., Kučerka, N., Atkinson, J., Wassall, S. R., Katsaras, J., et al. (2013). Tocopherol activity correlates with its location in a membrane: A new perspective on the antioxidant vitamin E. Journal of the American Chemical Society, 135(20), 7523–7533. Mehnert, W., & Mäder, K. (2012). Solid lipid nanoparticles: Production, characterization and applications. Advanced Drug Delivery Reviews, 64, 83–101. Müller, R., Mehnert, W., Lucks, J.-S., Schwarz, C., Zur Mühlen, A., Meyhers, H., ... Rühl, D. (1995). Solid lipid nanoparticles (SLN): An alternative colloidal carrier system for controlled drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 41(1), 62–69. Müller, R. H., Radtke, M., & Wissing, S. A. (2002a). Nanostructured lipid matrices for improved microencapsulation of drugs. International Journal of Pharmaceutics, 242(1–2), 121–128. Müller, R. H., Radtke, M., & Wissing, S. A. (2002b). Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Advanced Drug Delivery Reviews, 54, S131–S155. Nishino, H. (1997). Cancer prevention by natural carotenoids. Journal of Cellular Biochemistry, 67(S27), 86–91. Podmore, I. D., Griffiths, H. R., Herbert, K. E., Mistry, N., Mistry, P., & Lunec, J. (1998). Vitamin C exhibits pro-oxidant properties. Nature, 392(6676), 559. Radtke, M., & Müller, R. H. (2001). Nanostructured lipid drug carriers. New Drugs, 2, 48–52. Rietjens, I. M., Boersma, M. G., de Haan, L., Spenkelink, B., Awad, H. M., Cnubben, N. H., ... Koeman, J. H. (2002). The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids. Environmental Toxicology and Pharmacology, 11(3–4), 321–333. Socaciu, C. (2007). Food colorants: Chemical and functional properties. CRC Press (Chapter 2). Soukoulis, C., & Bohn, T. (2018). A comprehensive overview on the micro-and nanotechnological encapsulation advances for enhancing the chemical stability and bioavailability of carotenoids. Critical Reviews in Food Science and Nutrition, 58(1), 1–36. Stahl, W., & Sies, H. (2005). Bioactivity and protective effects of natural carotenoids. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1740(2), 101–107. Tamjidi, F., Shahedi, M., Varshosaz, J., & Nasirpour, A. (2013). Nanostructured lipid carriers (NLC): A potential delivery system for bioactive food molecules. Innovative Food Science & Emerging Technologies, 19, 29–43. Tamjidi, F., Shahedi, M., Varshosaz, J., & Nasirpour, A. (2014a). Design and characterization of astaxanthin-loaded nanostructured lipid carriers. Innovative Food Science & Emerging Technologies, 26, 366–374. Tamjidi, F., Shahedi, M., Varshosaz, J., & Nasirpour, A. (2014b). EDTA and α‐tocopherol improve the chemical stability of astaxanthin loaded into nanostructured lipid carriers. European Journal of Lipid Science and Technology, 116(8), 968–977. Triplett, M. D., & Rathman, J. F. (2009). Optimization of β-carotene loaded solid lipid nanoparticles preparation using a high shear homogenization technique. Journal of Nanoparticle Research, 11(3), 601–614. de Vos, P., Faas, M. M., Spasojevic, M., & Sikkema, J. (2010). Encapsulation for preservation of functionality and targeted delivery of bioactive food components. International Dairy Journal, 20(4), 292–302. Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in food dispersions. Trends in Food Science & Technology, 22(1), 3–13. Weiss, J., Decker, E. A., McClements, D. J., Kristbergsson, K., Helgason, T., & Awad, T. (2008). Solid lipid nanoparticles as delivery systems for bioactive food components. Food Biophysics, 3(2), 146–154. Wright, J. S., Johnson, E. R., & DiLabio, G. A. (2001). Predicting the activity of phenolic antioxidants: Theoretical method, analysis of substituent effects, and application to major families of antioxidants. Journal of the American Chemical Society, 123(6), 1173–1183. Yehye, W. A., Rahman, N. A., Ariffin, A., Hamid, S. B. A., Alhadi, A. A., Kadir, F. A., et al. (2015). Understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (BHT): A review. European Journal of Medicinal Chemistry, 101, 295–312. Zhang, P., & Omaye, S. T. (2000). β-Carotene and protein oxidation: Effects of ascorbic acid and α-tocopherol. Toxicology, 146(1), 37–47.
4. Conclusion The ß-carotene loaded into NLC was successfully stabilized using combination of BHT and vitamin E as antioxidant system. The statistical analysis revealed that BHT and vitamin C had significant effect on ßcarotene degradation while influence of vitamin E was evaluated less important. Addition of vitamin C into the formulations of the NLC affected ß-carotene stability negatively while BHT improved it. Also, there were two considerable interactions, one between the BHT and vitamin E and another between BHT and vitamin C. High concentrations of vitamin C reversed the positive performance of BHT. The quadratic polynomial model derived using response surface methodology (RSM) was properly fitted to experimental data. The polynomial model had high accuracy and employed to determine the optimum conditions with minimum ß-carotene degradation. The optimized NLCs had very low ß-carotene degradation rates compared with antioxidant free NLC. Moreover, the particle size of the NLCs was increased by incorporation of the antioxidants (p < 0.05). Nowadays, with growing demand for healthy and functional foods, protecting bioactive ingredients become more predominant in order to preserve the appearance and functionality of the product. In this study, it was shown that ßcarotene can be retained in valued NLC systems using combination of antioxidants. This system can also be used to protect other bioactive compounds incorporated into NLC. Acknowledgements We would like to thank the Center of Excellence for Color Science and Technology, Institute for Color Science and Technology, TehranIran for providing us a good environment and facilities to complete this project. References Aliaga, C., de Arbina, A. L., Pastenes, C., & Rezende, M. C. (2018). Antioxidant-spotting in micelles and emulsions. Food Chemistry, 245, 240–245. Anguelova, T., & Warthesen, J. (2000). Degradation of lycopene, α‐carotene, and β‐carotene during lipid peroxidation. Journal of Food Science, 65(1), 71–75. Boon, C. S., McClements, D. J., Weiss, J., & Decker, E. A. (2010). Factors influencing the chemical stability of carotenoids in foods. Critical Reviews in Food Science and Nutrition, 50(6), 515–532. Brewer, M. (2011). Natural antioxidants: Sources, compounds, mechanisms of action, and potential applications. Comprehensive Reviews in Food Science and Food Safety, 10(4), 221–247. Burri, B. J. (1997). Beta-carotene and human health: A review of current research. Nutrition Research, 17(3), 547–580. Burton, G. W., & Ingold, K. (1984). Beta-carotene: An unusual type of lipid antioxidant. Science, 224(4649), 569–573. Chen, B., Chen, T., & Chien, J. (1994). Kinetic model for studying the isomerization of. alpha.-and. beta.-carotene during heating and illumination. Journal of Agricultural and Food Chemistry, 42(11), 2391–2397. Eghbaliferiz, S., & Iranshahi, M. (2016). Prooxidant activity of polyphenols, flavonoids, anthocyanins and carotenoids: Updated review of mechanisms and catalyzing metals. Phytotherapy Research, 30(9), 1379–1391. Gaziano, J. M., Manson, J. E., Buring, J. E., & Hennekens, C. H. (1992). Dietary antioxidants and cardiovascular disease. Annals of the New York Academy of Sciences,
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effect of edible antioxidants on chemical stability of ß-carotene loaded nanostructured lipid carriers
T
Abdolrasoul Hejria, Alireza Khosravia,∗, Kamaladin Gharanjigb, Mohammadreza Malekzade Davarania a b
Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, Hafez St., 15875-4413, Tehran, Iran Department of Organic Colorants, Institute for Color Science and Technology, Hossein Abad Sq., 1668814811, Tehran, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanostructured lipid carriers Response surface methodology β-carotene Antioxidant Degradation
Nanostructured lipid carriers (NLC) containing ß-carotene was produced using solvent diffusion method. Edible antioxidants of butylated hydroxytoluene (BHT), vitamin E and vitamin C were successfully incorporated in NLC to protect ß-carotene. Response surface methodology (RSM) was employed in order to evaluate the effect of different antioxidants on ß-carotene stability. A quadratic polynomial model was fitted to empirical data with high accuracy. The ß-carotene retention improved with raising the BHT concentration while addition of vitamin C resulted in ß-carotene instability. Increasing the vitamin E concentration first elevated the ß-carotene degradation rate and then decreased it. However, the total effect of vitamin E alone was less significant. The statistical analysis demonstrated that the interactions between BHT and two other antioxidants had important effect on ß-carotene stability. The optimum formulations predicted by the model contained BHT and vitamin E combinations. They had minimum ß-carotene degradations of 2.12–4.51% which were in a reasonable agreement with model predictions. Furthermore, particles size analysis indicated that addition of antioxidants to NLCs results in production of larger nanoparticles.
1. Introduction ß-carotene is one of the most common carotenoids in nature widely used as a natural colorant in food industry (Burri, 1997). Due to its polyene structure, ß-carotene is able to act as a notable free radical scavenging and singlet oxygen quenchers makes it an important natural antioxidant. Consequently, according to numerous studies, ß-carotene supplement may result in significant health benefits including reducing the risk of cardiovascular, pulmonary, ophthalmological diseases and certain types of cancer (Gaziano, Manson, Buring, & Hennekens, 1992; Krinsky & Johnson, 2005; Nishino, 1997; Stahl & Sies, 2005). However, this electron rich conjugated double bond structure which gave ß-carotene its special characteristics makes it unstable against oxidation, heat and light (Socaciu, 2007). Taking under consideration its water insolubility and sensitivity, ß-carotene's application in foods is facing with some limitations. Therefore, ß-carotene can be delivered via functional food carries which may improve its solubility, stability and bioavailability (Soukoulis & Bohn, 2018). Since their introduction in early 1990s, solid lipid nanoparticles (SLN) have got a great deal of attraction due to their unique properties
∗
which merge together advantages of traditional carriers such as bioavailability, biocompatibility, controlled release and membrane permeability. Classical carriers like emulsions, liposomes and polymeric nanoparticle have limitations including organic solvent usage, carrier bio toxicity, low drug payload and large scale production which SLNs could also reduce them (Müller et al., 1995; Müller, Radtke, & Wissing, 2002a,b). Because of their crystalline solid structure, polymorphic transitions lead to low drug payload, drug expulsion during storage and particle aggregation (Mehnert & Mäder, 2012; Weiss et al., 2008). These disadvantages resulted in introduction of the second generation of lipid nanoparticles “Nanostructured lipid carriers” (NLC) (Radtke & Müller, 2001). In NLCs, the lipid phase is made of both solid and liquid lipids leading to a less ordered crystalline or amorphous solid structure. Accordingly, NLCs have higher drug loading capacity and can avoid or minimize drug expulsion during storage (Müller et al., 2002a,b). Moreover, NLCs have potential to be used in transparent products; they are physically stable and can improve the bioavailability of lipophilic bioactive compounds (Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2013). These properties make NLCs a proper carrier for bioactive food components attracting the interest of food area researchers in recent
Corresponding author. E-mail address: [email protected] (A. Khosravi).
https://doi.org/10.1016/j.lwt.2019.108272 Received 29 January 2019; Received in revised form 7 May 2019; Accepted 16 June 2019 Available online 17 June 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Experimental design and statistical analysis
years (Hejri, Khosravi, Gharanjig, & Hejazi, 2013; Liu & Wu, 2010; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014a). Application of bioactive compounds in foods is being increased by growing the demand for functional foods (Goldberg, 2012). In this regard, one of the major difficulties is the chemical stability of bioactive component. For instance, degradation of the carotenoids in a food enriched with them will affect both the quality and bioactivity of the food (Boon, McClements, Weiss, & Decker, 2010; de Vos, Faas, Spasojevic, & Sikkema, 2010). NLCs are able to preserve these ingredients however this is not inclusive for all formulations and optimization is required (Hejri et al., 2013). Utilizing antioxidants is a major procedure to further prevent oxidation of lipids and bioactive ingredients in food dispersions. Since the lipid oxidation in dispersions depend on many factors comprising lipid and bioactive composition, oxygen concentration, type and concentration of antioxidants and pro-oxidants, lipid droplet characteristics, emulsion droplet interfacial properties and etc.; hence combination of antioxidative techniques and selection of antioxidants are crucial to obtain an efficient protection (Jee, Lim, Park, & Kim, 2006; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014b; Waraho, McClements, & Decker, 2011). In this study, nanostructured lipid carriers were used as a carrier for delivering ß-carotene. The purpose of this study is to minimize the degradation of ß-carotene as a model nutraceutical in NLCs using an antioxidant system. For this purpose both lipophilic antioxidants of vitamin E and BHT and hydrophilic antioxidant of vitamin C were utilized. The relations between the antioxidants concentration and ßcarotene degradation were evaluated applying response surface methodology (RSM) and the optimum conditions were obtained. At the end, the physical properties of the optimum ß-carotene loaded nanostructured lipid carriers were investigated. Formulating chemically stable bioactive loaded NLCs will contribute to expand the application of these carriers in foods and thus to be more benefited from the advantages of these systems in our diet in the future.
Response surface methodology (RSM) was employed to evaluate the relation between the response and independent variables where the ßcarotene degradation value is the response and BHT (A), vitamin E (B) and vitamin C (C) concentrations are independent variables. This method has been successfully used in previous literature on NLCs (Hejri et al., 2013; Liu & Wu, 2010). Utilizing a 3 factor Box–Behnken design, a set of 17 runs was designed including 5 replicates at central point to calculate the pure error. The independent variables values at coded levels of −1 and +1 were set to 0 and 2% w/w, respectively. Design-Expert® 7.0 software was employed for statistical analysis. In order to find the best fitted mathematical model the sequential model sum of squares, the multiple correlation coefficient (R2), the adjusted multiple correlation coefficient (adjusted R2), the predicted multiple correlation coefficient (predicted R2), predicted residual error sum of squares (PRESS) and lack-of-fit of the various polynomial models were evaluated. The F-test was performed to assess if there is a significant relation between the response and the model factors at the confidence 95% (α = 0.05). Utilizing student's t-tests the significant model factors and their interactions were specified at the confidence 95% (α = 0.05). In order to modify the mathematical model, insignificant model parameters were eliminated using backward stepwise regression. Contour plots for the fitted model were generated by Design-Expert® 7.0. Finally, predicted formulations with minimum ß-carotene degradation were obtained by numerical solution of the fitted model and compared with experimental data to evaluate the model accuracy.
2.4. ß-carotene stability analysis According to the experimental design, 17 samples were prepared and stored in dark at room temperature (25 ± 2 °C). The stability of ßcarotene was evaluated after 2 weeks. The absorbance of ß-carotene loaded NLCs were measured using UV–Vis spectroscopy (Jenway 6715, Staffordshire, UK) and employed to determine the ß-carotene stability (Triplett & Rathman, 2009). The NLC dispersion was dissolved in acetone (1:1 v/v) and absorbance was measured in λmax = 455 nm. Then, the ß-carotene concentration was calculated according to beerlambert law using calibration curve. The ß-carotene degradation was calculated using the relation:
2. Material and methods 2.1. Materials All the materials used in this study were food grade and used as received. Palmitic acid (PA), Polyoxyethylene sorbitan monooleate (Tween 80), ethanol and acetone were obtained from Merck (Darmstadt, Germany). Ascorbic acid (Vitamin C), DL -α-Tocopherol (Vitamin E) and Butylated hydroxytoluene (BHT) were purchased Sigma-Aldrich Chemie GmbH (Munich, Germany). Pure ß-carotene powder was provided by BASF (Ludwigshafen, Germany). Corn oil was from Behshahr industrial Co. (Tehran, Iran). Double distilled water was used in the experiments.
Degradation(%) =
C0 − C × 100 C0
(1)
where C0 and C are initial concentration and concentration after 2 weeks of ß-carotene, respectively. A NLC without ß-carotene was prepared and its absorption at λmax = 455 nm was measured. Since the absorption of the NLC without ß-carotene was negligible, therefore, the absorbance of the samples was related to ß-carotene. Furthermore, since there were no severe heating and illumination conditions during storage, the effect of stereoisomer transformation is ignored in this study (Chen, Chen, & Chien, 1994).
2.2. Preparation of ß-carotene loaded NLCs Nanostructured lipid carriers were produced by solvent diffusion method according to our previous work with some modifications (Hejri et al., 2013). In brief, an aqueous phase containing Tween 80 (3% w/ w), double distilled water and different amounts of vitamin C was preheated to 40 °C. Meanwhile, a lipid phase was prepared by mixing corn oil, palmitic acid and ß-carotene and various amounts of vitamin E and BHT. Then, 2 g/l lipid phase was dissolved in ethanol at 65–70 °C. Subsequently the resulting solution was added to the prepared aqueous phase under mechanical agitation at 1000 rpm and mixed for 10 min. The resulting dispersion was then cooled to room temperature to form NLCs. In all formulations, the ß-carotene concentration in lipid phase, corn oil to palmitic acid ratio and volume ratio of ethanol/water were fixed at 2% (w/w), 1:10 (w/w) and 1:10 (v/v), respectively.
2.5. Particle size analysis The particle size and size distribution of the NLCs were investigated by dynamic light scattering (DLS) using a Cordouan Vasco™ (Cordouan Technologies, France) instrument. The measurements were performed at 25 °C. In order to prevent multiple scattering effects, the samples were diluted to appropriate concentration with distillated water. The instrument reports the mean particle size (z-average) and polydispersity index (PDI) of the samples.
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the observed F-value if the null hypothesis is true. Hence, at the confidence level of 95% (α = 0.05) the p-value of less than 0.05 indicated strong evidence against the null hypothesis which denoted that the terms in the model have significant effect on response. The higher Fvalues result in lower p-values. As it is shown in Table 2, the model's Fvalue of 65.72 indicated the quadratic model is significant at the level of α = 0.001. The F-values for all model terms were calculated with similar procedure. According to Table 2, BHT (A) and vitamin C (C) concentrations had important effect on ß-carotene degradation (p < 0.001). Furthermore, quadric terms of vitamin E (B2) and vitamin C (C2) concentrations were significant model terms (p < 0.001). Also, the interactions between BHT and vitamin E (AB) and the interaction between BHT and vitamin C (AC) had considerable impact on ß-carotene stability (p < 0.05). Other model term have negligible effect on ß-carotene retention (p > 0.05). The quadratic model was modified by eliminating the insignificant model term applying backward stepwise regression procedure. It should be considered that R2 will increase by adding any terms to the model, even if it is due to chance. Thus, adjusted R2 was used to compare models with different number of terms. Adjusted R2 increases only when adding a term amends the model fit more than expected by chance. Accordingly, omitting the interactive term of BC from the model leaded to raising the adjusted R2 from 0.973 to 0.977. However, the term A2 was not deleted from the model because of the reduction in adjusted R2 value. Also, Vitamin E (B) parameter was not removed from the model in order to keep the hierarchical structure of the quadratic model. The modified second order polynomial model based on codded levels for the ß-carotene degradation is described as follow:
Table 1 The β-carotene degradation values of samples prepared according to Box–Behnken experimental design. Run
A: BHT concentration (% w/w)
B: Vitamin E concentration (% w/w)
C: Vitamin C concentration (% w/w)
β-carotene degradation (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0 1 1 2 1 0 1 1 1 1 0 2 1 2 1 0 2
1 1 1 1 2 2 1 0 0 1 0 0 1 2 2 1 1
0 1 1 2 0 1 1 2 0 1 1 1 1 1 2 2 0
71 90 93 87 9 91 87 76 17 87 86 79 92 58 69 84 2
3. Results and discussion 3.1. Establishing the model The NLC dispersions were prepared according to Box–Behnken design. 17 samples were kept at 25 ± 3 °C in dark for 2 weeks. The absorbances of the samples were measured at the day of preparation and after 2 weeks and the ß-carotene degradation values were calculated according to calibration curve. Data is shown in Table 1. The various statistical parameters of different fitted models were compared. The results indicated that the best fitted mathematical model to experimental data was quadratic polynomial model. The R2 = 0.988 for the quadratic model implies that the relationship between the independent variables and the response is well described by the model. The adjusted R2 and predicted R2 values were 0.973 and 0.843, respectively. The high value of the predicted R2 which is in good agreement with adjusted R2, means that the model is not overfitted and can be used to predict new observations for ß-carotene degradation. The analysis of variance (ANOVA) of the model terms performed by Design-Expert® 7.0 software is summarized in Table 2. F-test was performed to evaluate the significance of the model. F-value compares the model variance with the residual variance. If the variances are close to the same then it is less likely that any of the factors have a significant effect on the response. The null hypothesis of the F-test is that the all regression coefficients are zero. The p-value is the probability of finding
Beta carotene degradation(%) = 89.80 − 13.25A − 3.88B + 27.13C − 6.50AB + 18.00AC + 3.47A2 − 14.77B2 (2) − 32.28C 2 The model F-value was increased to 84.37 (p < 0.0001). The R2 and predicted R2 values of the modified model were 0.988 and 0.866, respectively. The lack of fit F-value of 4.55 (p > 0.05) indicated insignificant lack of fit of the modified model. In lack of fit test the null hypothesis is that the model fits well. Hence, p > 0.05 means that the null hypothesis is true. 3.2. Effect of antioxidants on particle size of NLC Since particle size of NLC have great effect on chemical stability of the loaded bioactive compound (Hejri et al., 2013), the particle size of the 5 NLC samples including 2% w/w BHT, vitamin E and vitamin C loaded NLCs together with an antioxidant free NLC and a central point run were measured by DLS (Table 3). As shown in Table 3, Incorporation of the antioxidants into NLC formulation increased the particle size of it (p < 0.05). The highest grow in particle size was observed for vitamin C (p < 0.0001) while the lowest was for BHT (p < 0.05). The effect of vitamin C could not be explained by the current data; nevertheless it would be because of an interaction between the Tween 80 and vitamin C and its impact on the surface chemistry of the particles. Also, The larger particle size of vitamin E loaded NLC compare to BHT loaded NLC could be due to the higher molecular size of vitamin E; however it was not statistically significant (p > 0.05).
Table 2 The Analysis of variance (ANOVA) of the model terms. Term
a
Model Intercept A B C AB AC BC A2 B2 C2
β -carotene degradation F -value
p-value
65.72
< 0.0001
57.646 4.930 241.588 6.936 53.193 0.010 2.087 37.726 180.018
0.0001 0.0618 < 0.0001 0.0337 0.0002 0.9222 0.1918 0.0005 < 0.0001
Coefficient
89.80 −13.25 −3.88 27.13 −6.50 18.00 0.25 3.47 −14.78 −32.28
3.3. Parameters effect on ß-carotene There are different ways that antioxidants intercept oxidation process. The effectiveness of antioxidants depend on various factors including oxidation reduction potential, rate constants, activation energy, solubility, volatility and heat susceptibility. The most efficient antioxidants interrupt the free radical chain reaction. The H-atom transfer and electron transfer are two pathways that an antioxidant can
a A: BHT concentration, B: Vitamin E concentration, C: Vitamin C concentration.
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Table 3 Effect of antioxidants on the particle size of β-carotene loaded nanostructured lipid carriers (NLC). Sample
BHT concentration (% w/w)
Vitamin E concentration (% w/w)
Vitamin C concentration (% w/w)
β-carotene concentration (% w/w)
Particle size (nm)a
Polydispersity Index (PDI)a
Antioxidant free BHT Vitamin E Vitamin C Central point
– 2.00 – – 1.00
– – 2.00 – 1.00
– – – 2.00 1.00
2.00 2.00 2.00 2.00 2.00
111 126 138 218 174
0.245 0.251 0.304 0.334 0.313
a
± ± ± ± ±
4 6 9 5 9
± ± ± ± ±
0.032 0.028 0.025 0.052 0.068
Data were driven from three replications and reported as mean ± standard deviation.
Fig. 1. Contour plot showing (a) the effect BHT and vitamin E concentrations on β-carotene degradation and (b) the effect BHT and vitamin C concentrations on βcarotene degradation.
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radicals and ROS (Anguelova & Warthesen, 2000). The influence of the vitamin C on ß-carotene degradation is demonstrated in Fig. 1b (vitamin E concentration is fixed at central point). The same trend observed for vitamin E was obtained for vitamin C although its effect was more important compared to vitamin E. Vitamin C was dissolved in aqueous medium and presented in this phase as a water soluble antioxidant. In general, known as “antioxidant polar paradox”, nonpolar antioxidants are more effective than polar antioxidants in emulsions since they accumulate in lipid phase interface where the most oxidation occurs (Marquardt et al., 2013). Vitamin C can show pro-oxidant effect through an autoxidation mechanism. At high oxygen tensions, like this study, molecular oxygen and ascorbyl radical (VC˙−) are dominant. The interaction between them results in generation of superoxide radical which can then react with ascorbate molecule and oxidize it to another ascorbyl radical (Eqs. (5) and (6)) (Eghbaliferiz & Iranshahi, 2016; Zhang & Omaye, 2000):
deactivate a free radical (Brewer, 2011; Wright, Johnson, & DiLabio, 2001). BHT is one of the commonly used synthetic antioxidants in food industry. Fig. 1a shows the contour plot of the ß-carotene degradation as a function of BHT and vitamin E concentrations while other parameter is fixed at central point. BHT is a hindered-phenol antioxidant and can be refer as synthetic analogue of vitamin E. BHT act as a chain breaking antioxidant by H-atom transfer mechanism as follow (Burton & Ingold, 1984; Yehye et al., 2015):
ROO˙ + ArOH → ROOH + ArO ˙
3
ArO˙ + ROO˙ → nonradical products
4
As it is illustrated in Fig. 1a, the ß-carotene degradation was decreased by increasing the BHT concentration. BHT as a more reactive antioxidant first reacts with free radicals and the reactive oxygen species (ROS) and therefore protects the ß-carotene against deterioration (Anguelova & Warthesen, 2000; Jee et al., 2006). It should be noted that BHT possessed superior antioxidant activity than vitamin E to protect ß-carotene. This behavior has been observed in solid lipid nanoparticles containing all trans retinol (Jee et al., 2006) and could be due to the different chemical structure of BHT and vitamin E. Bond dissociation energy (BDE) is a physical parameter which plays a critical role in antioxidant activity of chemical compounds; The lower the BDE the higher the antioxidant efficiency. The BDE of phenolic O–H bond of vitamin E is about 2 kcal/mol less than BHT. However, substituents at ortho position result in steric hindrance to minimize undesirable reactions such as pro-oxidation (Yehye et al., 2015). Therefore, ortho tbutyl groups of BHT make its radicals unreactive and prevent any prooxidation effect unlike vitamin E. Elevating the vitamin E concentration initially lowered the ß-carotene stability and then improved it (Fig. 1a). However, it did not significantly protect ß-carotene even at high concentrations. It has been reported that α-tocopherol did not significantly increase the astaxanthin stability in NLCs after 10 and 15 days (Tamjidi et al., 2014b).Vitamin E can act as a pro-oxidant in food and biological systems. In the other word, increasing the vitamin E level under high oxidative stress conditions would lead in generation of high amounts of vitamin E radicals, which could not reduce to vitamin E in the absence of a co-antioxidant. These radicals could start lipid peroxidation process by themselves and therefore oxidize the ß-carotene present in the nanoparticles as a potent reactive bioactive compound (Rietjens et al., 2002). Furthermore, due to amphiphilic nature of vitamin E, its hydrophobic chain penetrates into nanoparticles while the hydrophilic head group tend toward the less hydrophobic environment. Therefore, vitamin E antioxidant activity take place at less hydrophobic microenvironment near the surface. Despite hydrophobic ß-carotene which would stays in the core of the particles (Aliaga, de Arbina, Pastenes, & Rezende, 2018; Marquardt et al., 2013). Thus, vitamin E would first react with radicals diffusing into nanoparticles which cause production of vitamin E radicals inside nanoparticles. Consequently, in absence of any reducing agent these radicals would oxidize ß-carotene. Another reason that may have been led to the observed results in low vitamin E concentrations was the competition between ß-carotene and vitamin E toward reaction with peroxyl radicals and ROS in which the ß-carotene was the substance reacted at first due to its dominant concentration (Anguelova & Warthesen, 2000). Adding the vitamin E to NLC formulation has grown the particle size of nanoparticles (Table 3). Larger particles provide lower surface area for diffusion of oxidative species (Hejri et al., 2013). By increasing the vitamin E concentration, this effect became predominant after a point and ß-carotene degradation rates started to fall down (Fig. 1a). Moreover, incorporation of vitamin E could enhance the ß-carotene loading efficiency and hence reducing the free ß-carotene amounts (Jee et al., 2006). In addition, as mentioned in past paragraph, at high vitamin E levels, this time it is vitamin E which initially reacts with peroxyl
VC·− + O2 → VC + O2⋅− O2⋅−
+
VCH−
→
HOO−
+
5
VC· −
6
This is an autoxidation process for vitamin C oxidizing it without showing a protective effect. In addition, since the superoxide radical produced in Eq. (5) is a very strong oxidizing agent, it could attack ßcarotene and degrade it. Another probable process would be the pro-oxidant effect of vitamin C in presence of trace metals (Kanner, Mendel, & Budowski, 1977; Podmore et al., 1998; Zhang & Omaye, 2000). Natural antioxidants generally act as pro-oxidant in presence of transition metals specially Iron and Copper. Ferric cations increase the oxidative stress by generating ROS via Fenton-type reaction (Eq. (7) and Eq. (8)). The ferrous cations react with hydrogen peroxide to produce hydroxyl radicals (Eq. (8)) (Eghbaliferiz & Iranshahi, 2016).
Fe3 + + O2⋅− → Fe 2 + + O2
7
Fe 2 + + H2 O2 → Fe 3 + + OH˙ + OH−
8
The generated ferrous species could also start lipid oxidation process directly (Eq. (9)) or interact with O2 to generate O2˙- (Eq. (10)). Vitamin C act as a catalyst in this process and keeps iron in its reduced state (Kanner et al., 1977; Rietjens et al., 2002; Zhang & Omaye, 2000). The O2˙- and OH˙ as strong oxidants would diffuse into NLC and decompose the incorporated ß-carotene.
Fe 2 + + ROOH → Fe3 + + RO˙ + OH− Fe 2 + + O2 → Fe3 + + O2⋅−
9 10
The particle size of NLC increased by rising the vitamin C concentration (Table 3) and accordingly ß-carotene degradation decreased at high vitamin C amounts. However, considering the strong oxidative species produced according to Eqs. (5)-(10), only a small reduction in ßcarotene degradation rate was observed (Fig. 1b). Yet, the degradation level is much more than the value yielded in the absence of vitamin C. One of the significant interactions was the interaction between BHT and vitamin E. Fig. 2a and b displays the effect of vitamin E on ßcarotene degradation at BHT concentrations of 0 and 2% w/w, respectively (vitamin C concentration fixed at 0). The higher the BHT concentration the better the vitamin E performance; so there was a perfect protection of ß-carotene at high BHT and vitamin E amounts (Fig. 2b). Both antioxidants are located inside the nanoparticles. Therefore, BHT can act as a reducing agent for vitamin E radicals and consume them to reproduce vitamin E (Burton & Ingold, 1984; Rietjens et al., 2002). Thus, the issue of the vitamin E radicals would be eliminated and vitamin E could still trap free radicals and ROS. Another major interaction was among BHT and vitamin C. The ßcarotene degradation as a function of BHT concentration at 0 and 2% w/w vitamin C concentrations is illustrated in Fig. 2c and d, respectively (vitamin E concentration fixed at 0). At high values of vitamin C, 5
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Fig. 2. Effect of vitamin E concentration on β-carotene degradation at (a) 0% w/w and (b) 2% w/w BHT concentrations at vitamin C concentration of 0 and effect of BHT concentration on β-carotene degradation at (c) 0% w/w and (d) 2% w/w vitamin C concentrations at vitamin E concentration of 0.
contained it. Although, BHT itself could protect ß-carotene, combination of it with vitamin E had led to the best results. In this regard, Tamjidi et al. (2014b) investigated the effect of ethylenediaminetetraacetic acid (EDTA), ascorbic acid coenzyme Q10 and α-tocopherol on the chemical stability of astaxantin in NLCs. They concluded that combination of EDTA with high concentrations of α-tocopherol was more effective than other antioxidant systems and it could reduce the astaxantin degradation to about 20% after 15 days of storage in the dark at 35 °C. This was due to slower degradation of α-tocopherol in presence of EDTA. Furthermore, it has been reported that adding the BHT-BHA (Butylate hydroxyanisol) antioxidant combination to the alltrans retinol loaded SLNs increased the intact amount of all-trans retinol from almost zero to 89% after 72 h storage under shade at room temperature (Jee et al., 2006). The reasonable agreement between the predicted and actual data (p < 0.05) indicated that the fitted polynomial model is accurate and can be used to navigate in the design space. Moreover, the particle sizes and PDI indexes of the optimum formulations were evaluated
the BHT behavior changed and increasing the BHT concentration had negative effect on ß-carotene stability. The oxidative species generated in presence of vitamin C, which has discussed in this section, would have resulted in nearly complete oxidation of BHT and it could no longer protect ß-carotene. Consequently, the BHT radicals created in NLC could be a factor led to ß-carotene instability as well. 3.4. Determination of optimum formulations and model accuracy Three different antioxidants had three different influences on ßcarotene stability. In order to find the optimum formulations with the lowest ß-carotene degradation, numerical solution of the model together with overlay plots were utilized. Table 4 shows three formulations with low ß-carotene degradation as predicted by the model and the actual amounts measured for them. In comparison with ß-carotene degradation in antioxidant free NLC (51%), the results revealed a superb protection of ß-carotene in the optimized NLCs. Since the vitamin C has negative effect on ß-carotene stability, none of the formulas
Table 4 Comparison of predicted and actual values of β-carotene degradation for optimum formulations together with particle size and PDI index of them. Sample
1 2 3 a
BHT concentration (% w/ w)
1.98 2.00 1.30
Vitamin E concentration (% w/w)
1.19 0.24 1.94
Vitamin C concentration (% w/w)
0.0 0.0 0.0
Data were driven from three replications and reported as mean ± standard deviation. 6
β-carotene degradation (%) predicted
actual
1.00 2.00 3.00
2.37 2.12 4.51
Particle size (nm)a
Polydispersity Index (PDI)a
163 ± 8 132 ± 11 167 ± 4
0.291 ± 0.023 0.266 ± 0.038 0.282 ± 0.041
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employing DLS particle size analysis (Table 4). The NLCs had particle sizes of 132–167 nm and low PDI values (0.266–291) indicating the narrow distribution which could be a reason for physical stability of produced NLCs. Finally, since there is close relationship between the physical characteristics of NLC and chemical stability of loaded bioactive compound, more researches should be conducted to evaluate the physical properties of the obtained NLCs and to subsequently determine the exact mechanisms behind the observed results. Moreover, these NLCs should be employed in different food media in order to investigate the effect of the food matrices on function of the antioxidant system.
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4. Conclusion The ß-carotene loaded into NLC was successfully stabilized using combination of BHT and vitamin E as antioxidant system. The statistical analysis revealed that BHT and vitamin C had significant effect on ßcarotene degradation while influence of vitamin E was evaluated less important. Addition of vitamin C into the formulations of the NLC affected ß-carotene stability negatively while BHT improved it. Also, there were two considerable interactions, one between the BHT and vitamin E and another between BHT and vitamin C. High concentrations of vitamin C reversed the positive performance of BHT. The quadratic polynomial model derived using response surface methodology (RSM) was properly fitted to experimental data. The polynomial model had high accuracy and employed to determine the optimum conditions with minimum ß-carotene degradation. The optimized NLCs had very low ß-carotene degradation rates compared with antioxidant free NLC. Moreover, the particle size of the NLCs was increased by incorporation of the antioxidants (p < 0.05). Nowadays, with growing demand for healthy and functional foods, protecting bioactive ingredients become more predominant in order to preserve the appearance and functionality of the product. In this study, it was shown that ßcarotene can be retained in valued NLC systems using combination of antioxidants. This system can also be used to protect other bioactive compounds incorporated into NLC. Acknowledgements We would like to thank the Center of Excellence for Color Science and Technology, Institute for Color Science and Technology, TehranIran for providing us a good environment and facilities to complete this project. References Aliaga, C., de Arbina, A. L., Pastenes, C., & Rezende, M. C. (2018). Antioxidant-spotting in micelles and emulsions. Food Chemistry, 245, 240–245. Anguelova, T., & Warthesen, J. (2000). Degradation of lycopene, α‐carotene, and β‐carotene during lipid peroxidation. Journal of Food Science, 65(1), 71–75. Boon, C. S., McClements, D. J., Weiss, J., & Decker, E. A. (2010). Factors influencing the chemical stability of carotenoids in foods. Critical Reviews in Food Science and Nutrition, 50(6), 515–532. Brewer, M. (2011). Natural antioxidants: Sources, compounds, mechanisms of action, and potential applications. Comprehensive Reviews in Food Science and Food Safety, 10(4), 221–247. Burri, B. J. (1997). Beta-carotene and human health: A review of current research. Nutrition Research, 17(3), 547–580. Burton, G. W., & Ingold, K. (1984). Beta-carotene: An unusual type of lipid antioxidant. Science, 224(4649), 569–573. Chen, B., Chen, T., & Chien, J. (1994). Kinetic model for studying the isomerization of. alpha.-and. beta.-carotene during heating and illumination. Journal of Agricultural and Food Chemistry, 42(11), 2391–2397. Eghbaliferiz, S., & Iranshahi, M. (2016). Prooxidant activity of polyphenols, flavonoids, anthocyanins and carotenoids: Updated review of mechanisms and catalyzing metals. Phytotherapy Research, 30(9), 1379–1391. Gaziano, J. M., Manson, J. E., Buring, J. E., & Hennekens, C. H. (1992). Dietary antioxidants and cardiovascular disease. Annals of the New York Academy of Sciences,
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