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Food Research International 38 (2005) 861866 www.elsevier.

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Optimization of the antioxidant capacity of a mixture of carotenoids and a-tocopherol in the development of a nutritional supplement
I.A. Castro
a b

a,b,*

, S.B. Moraes Barros b, U.M. Lanfer Marquez a, M. Motizuki a, T.C. Higashi Sawada b

Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sa o Paulo, 05508-900 Sa o Paulo, SP, Brazil Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of Sa o Paulo, 05508-900 Sa o Paulo, SP, Brazil Received 1 June 2004; accepted 17 February 2005

Abstract The objective of the present study was to determine the interaction between a-tocopherol, b-carotene and lycopene in the formulation of a nutritional supplement using a simplex-centroid design and response surface methodology (RSM). Mixtures containing dierent proportions of each compound had their antioxidant activity (AOA) evaluated by the percent inhibition of spontaneous autoxidation. The regression model tted to the experimental data demonstrated that the linear terms and only the quadratic terms containing the lycopene variable had a signicant impact on the AOA response. A validation assay for the model was performed. The observed value obtained experimentally for AOA did not dier signicantly from the value estimated by the adjusted model. The results showed that no synergism occurred between the three compounds when rat brain homogenate was used as the oxidable substrate, and also suggested that RSM can be applied to estimate the behavior of mixed ingredients in nutritional supplements. 2005 Elsevier Ltd. All rights reserved.
Keywords: a-Tocopherol; b-Carotene; Lycopene; Antioxidant; RSM

1. Introduction Accumulating evidences have suggested that oxidative stress is involved in a variety of physiological and pathological processes, such as cellular signal transduction, cell proliferation and dierentiation, apoptosis, as well as arteriosclerosis, ischemia-reperfusion injury, cancer, stroke and many degenerative diseases (Cai et al., 2002; Halliwell & Gutteridge, 1998; Weinreb, Mandel, Amit, & Youdim, 2004). The benecial inuence of many foodstus and beverages including fruits, vegetables, tea, red wine and nuts on human health has been

Corresponding author. Tel.: +55 11 30913652; fax: +55 11 38154410. E-mail address: inar@usp.br (I.A. Castro). 0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.02.010

recognized. This benecial eect is due to the antioxidant activity (AOA) property from the bioactive substances naturally present in these products (Roginsky & Lissi, 2005; Stahl & Sies, 2005). These substances known as antioxidants can delay or inhibit oxidative damage when present is small quantities compared to an oxidizable substrate. Therefore, antioxidants can help in disease prevention by eectively quenching free radicals or inhibiting damage caused by them (Chatterjee, Poduval, Tilak, & Devasagayam, 2005). One of the main risk factor for coronary artery disease is the oxidation of the LDL (low density lipoproteins cholesterol) in the sub-endothelial space of the arterial wall (Arrol, Mackness, & Durrington, 2000; Luostarinen, Laasonen, & Calder, 2001; Song & Miyazawa, 2001). Antioxidants can reduce LDL oxidation and thus reduce plaque growth (Lewis, 2004).

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I.A. Castro et al. / Food Research International 38 (2005) 861866

Attention has been paid to fat-soluble antioxidants such as a-tocopherol, b-carotene and lycopene, which are present in LDL in quantities that can be modied by dietary intake or oral supplementation (Arrol et al., 2000). Some carotenoid pigments such as b-carotene are able to limit the oxidative damage induced by oxygen radical-generating systems (Polyakov, Kruppa, Leshina, Konovalova, & Kispert, 2001; Stahl & Sies, 2005; Tatsuzawa, Maruyama, Misawa, Fujimori, & Nakano, 2000; Zhang & Omaye, 2001). Carotenoids have been reported to play a role as antioxidants in lipid phases by trapping free radicals or quenching singlet oxygen (Subagio & Morita, 2001). Lycopene, a carotenoid naturally present in tomatoes and other fruits, has been found to be the most eective quencher of singlet oxygen and peroxyl radicals in plasma, lipoproteins and human lymphoid cells in vitro, with the highest levels being found in LDL-cholesterol (Bramley, 2000; Fleshner & Kucuk, 2001; Stahl & Sies, 1996). In addition, a-tocopherol acts as a chain-breaking antioxidant by donating its phenolic hydrogen to the chain-propagating lipid peroxyl radical forming a less reactive a-tocopheroxyl radical, which can further react with another lipid peroxyl radical to stop the propagation of lipid peroxidation (Zhang & Omaye, 2001). It is also known that interactions between carotenoids and a-tocopherol can occur in vitro by mechanisms that are still not completely understood. Various compounds show synergism in their antioxidant capacity, thus, permitting that mixtures can promote more eective antioxidant responses than when the compounds are applied individually to the substrate (Stahl et al., 1998; Yeum, Aldini, Chung, Krinsky, & Russell, 2003). Statistical experimental design (DOE) is a well established concept for planning and execution of informative experiments and it can be used in many applications. One main type of DOE application concerns the preparation and modication of mixtures. This involves the use of mixture designs for changing mixture composition and exploring how such changes will aect some specic properties of the nal product. The use of this statistical technique, known as response surface methodology (RSM), allows the visualization of the modeling results in terms of coefcients by contour or 3D-response surface plots. This facilitates the understanding of the model and also helps in decision making (Eriksson, Johansson, & Wikstrom, 1998). Therefore, the objective of the present study was to determine the interaction between mixtures containing a-tocopherol, b-carotene and lycopene using the response surface methodology statistical technique, and to identify the proportion of the three compounds that would maximize the antioxidant capacity in order to elaborate a nutritional supplement.

2. Materials and methods 2.1. Materials a-Tocopheryl acetate (Vitamin E-acetate SD50), bcarotene (Lucarotin 10CWD) and lycopene (Lycovit 10CWD) were obtained from BASF Health & Nutrition, Ludwigshafen, Germany.

2.2. Experimental design The characteristic feature of a mixture Pqis that the sum of all its components add up to 100% i1 xi 1. This means that these components (xi) mixture factors, cannot be manipulated completely independently of one another and that their proportions must lie somewhere between 0 and 1. A classical simplex-centroid experimental design was proposed for the present study (Table 1), considering a mixture of three variables, where x1 = a-tocopherol, x2 = b-carotene and x3 = lycopene, representing pure, binary and tertiary mixtures of the variables, with dextrin being used as control in the nutritional supplement formulation (Cornell, 2002). Each mixture was based on the recommended daily allowances for a-tocopherol and b-carotene, while the concentration adopted for lycopene was based in the literature (Southon, 2000). No absolutely pure mixtures were applied to this design in order to guarantee a minimum of 25% and maximum of 100% RDA (Recommended Dietary Allowances) concentrations in the nutritional supplement. Thus, the values coded in the design should be interpreted as the percentage of contribution of each compound to complete the formulation. Antioxidant activity (AOA) of each point from the experimental simplex-centroid design was evaluated in duplicate.

2.3. Determination of antioxidant activity Adult male Wistar rats (297.88 25.64 g) fed on a standard diet were perfused with 60.0 ml of a cold 0.9% saline solution, under light ether anesthesia. Brain was immediately removed, weighted and homogenized in 1:4 proportion with phosphate-buered saline solution (8.18 g of NaCl, 2.75 g of NaH2PO4, 2.83 g of Na2HPO4, to 1.0 l, pH 7.4). Tubes were centrifuged at 1.000g for 15 min at 4 C and the supernatant was used as substrate for lipoperoxidation. This preparation was carried out daily with fresh brain tissue. Fifty microliters of the phosphate-buered saline solution containing ve dierent concentrations of a-tocopherol, b-carotene and lycopene were added to 1 ml of the diluted homogenate and 2.95 ml of phosphate-buered saline, and incubated for 1 h at 37 C in an orbital water shaker. 1.0 ml of

I.A. Castro et al. / Food Research International 38 (2005) 861866 Table 1 Antioxidant capacity (AOA) of the mixtures Mix 01 02 03 04 05 06 07 OTMd
a b c d

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Experimental designa (100, 0, 0) (0, 100, 0) (0, 0, 100) (50, 50, 0) (50, 0, 50) (0, 50, 50) (33, 33, 33) (57, 0, 43)

a-Tocopherolb (mg/d) 3.37 0.00 0.00 1.69 1.69 0.00 1.12 1.92

b-Caroteneb (mg/d) 0.00 3.60 0.00 1.80 0.00 1.80 1.20 0.00

Lycopeneb (mg/d) 0.00 0.00 11.25 0.00 5.62 5.62 3.75 4.84

mg Mixture/ml (IC50)c 52.61 4.79 62.59 0.21 21.82 1.50 61.23 8.68 44.40 0.84 32.38 0.93 46.18 0.29 49.55 1.57

Proportion (%) of a-tocopherol, b-carotene and lycopene added in the mixture. The values correspond to mg of a-tocopherol, b-carotene and lycopene in the variable part of the supplement formulation. Concentration (mg mixture/ml phosphate buer) sucient to inhibit 50% of the spontaneous autoxidation (IC50). Optimized proportion obtained by the RSM in the end of this study.

TBA/TCA/BHT solution (3.75 g of thiobarbituric acid, 93.75 mg of trichloroacetic acid and 5.00 mg of butylated hydroxyl toluene dissolved slowly into 25 ml of HCl 0.25 N) was added and the tubes were incubated at 100 C/15 min. After cooling, the tubes were centrifuged at 1.000g for 15 min at 25 C and the oxidation products represented by thiobarbituric acid reactive substances (TBARS) were determined by spectrophotometry at 532 nm according to Stocks, Gutteridge, Sharp, and Dormandy (1974). The AOA of the brain homogenate was expressed as percent inhibition of spontaneous autoxidation or IC50 (as measured in the control homogenate), determined by the curve obtained by non-linear regression. Each point from the simplexcentroid design was evaluated in duplicate for each one of the ve concentrations. Three rats were used for each assay (brain weight @ 1.6 g/rat). In the end of this study, the same procedure was applied to the proportion selected for the quadratic model validation. In total, 48 animals were used in this study (3 rats/8 samples/2 repetition). 2.4. Statistical analysis and regressions optimization The results obtained by assessing the AOA in the seven assays were analyzed statistically on the basis of the dependent variable, considering that the mixture factors are correlated (Cornell, 2002). The models quality was determined by analysis of variance (ANOVA) of regression which is useful for checking the adequacy of a regression model in terms of lack of t test, through the evaluation of the model residuals using a normal probability plot and nally by the Coecient of Determination (R2). Before analysis, data were screened graphically for normality and for homogeneity of variance by Levene test. Calculations and graphs were obtained with the STATISTICA software, v.6 (StatSoft Inc., Tulsa, OK).

3. Results and discussion 3.1. Antioxidant capacity of the mixtures Table 1 shows the results regarding the antioxidant capacity (AOA) of the mixtures proposed in the experimental design using brain homogenate as substrate. 3.2. Regression model A quadratic polynomial regression model was assumed for prediction purpose of the response (y). The model proposed was

where b0, bi, bii and bij are intercept, linear, quadratic and interaction regression coecient of the model, respectively, xi and xj are coded independent variables and e the error associated to the y determination. The regression model tted to the experimental data (Eq. (1)) demonstrated that the linear terms and only the quadratic terms containing the lycopene variable had a signicant impact on the response evaluated ^ y 1 AOA ^ y 1 53.89x1 63.88x2 21.71x3 28.16x1 x3 39.90x2 x3 . 2.38 2.38 2.59 11.93 11.93 1 Fig. 1 shows that the most expressive results were obtained with the maximum presence of lycopene in the mixture and that none of the interactions between the three compounds showed synergism, in contrast to the original hypothesis of the present study. Miller, Sampson, Candeias, Bramley, and Rice-Evans (1996) assessing the relative AOA of a range of

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I.A. Castro et al. / Food Research International 38 (2005) 861866


LYC(x3) 0.00 1.00

0.25

0.75

0.50

0.50

0.75

0.25

1.00 0.00

0.25

0.50

0.75

0.00 1.00

TOC(x1)

CAR(x2)

60 50 40 30

Fig. 1. Response surfaces for antioxidant activity in a brain homogenate [mg mixture/ml (IC50)].

carotenes, concluded that the sequence of relative radical scavenging abilities was lycopene > b-carotene > lutein and zeaxanthin, and that the AOA of lycopene was almost three times higher than that of a-tocopherol. Stahl et al. (1998) evaluated the eects of single carotenoids and a-tocopherol on lipid peroxidation induced by 2,2 0 -azo-bis(2,4-dimethylvaleronitrile), and observed that lycopene was the most eective carotenoid, reducing TBARS formation in multilamellar liposomes to about 25% of the control, with the other carotenoids such as b-carotene and a-tocopherol being less active. These authors also evaluated carotenoid mixtures and their results demonstrated that only lutein and lycopene were responsible for synergistic antioxidant eects. Using triacylglycerol as substrate in an in vitro system, Subagio and Morita (2001) observed that carotenoids may also promote lipid oxidation because of the instability against heat and light, and that this induction could be blocked by the presence of tocopherols. According to Zhang and Omaye (2001), mixture of bcarotene and a-tocopherol had no signicant interaction eect on AOA measured in rat liver microsome suspensions. The authors also suggested that the antioxidant or pro-oxidant activities of these compounds are related to several factors, including antioxidant concentrations, governed by complex interactions. Southon (2000) in the Core Study found no signicant eect on LDL oxidizability and other biomarkers of oxidative and antioxidant status when carotenoids were provided in combination with a-tocopherol.

No gold standard assay exists for measuring antioxidant activity. Therefore, it has been suggested that these data should be obtained by dierent methods such as by measurement of endogenous antioxidant levels, determination of the products of oxidized macromolecules, and direct detection of free radicals (Fang, Yang, & Wu, 2002). Although limited by the RDA values and the assessment of AOA by a single method, our results conrm that interactions between pigments and a-tocopherol will basically depend on the concentration and solubility of these compounds, as well as on the choice of the oxidable substrate. 3.3. Optimization of the general function Analysis of variance for Eq. (1) (Table 2) indicated that the proposed model presented a good t to the experimental data as observed both by the non-signicance of the lack of t and by Coecient of Determination 2 R 2 adjust 0.93. The R value can be interpreted as the proportion of variability around the mena for the dependent variable, that can be accounted for by the respective model. This R2 value should be as close to unity as possible. By applying the general function proposed by Derringer and Suich (1980), we performed optimization of the regression (y1), which resulted in the proportion x1 = 0.56947, x2 = 0 and x3 = 0.43053, with an estimated value of 46.94 mg mixture/ml (IC50) for AOA within the condence interval predicted by the adjusted model

I.A. Castro et al. / Food Research International 38 (2005) 861866 Table 2 Analysis of variance for the reduced quadratic model Source Model Total error Lack of t Pure error Total adjusted SS 2626.773 122.488 20.334 102.154 2749.261 df 4 9 2 7 13 MS 656.6933 13.6098 10.1670 14.5934 211.4817 F 48.25154 0.69669 p 0.000004 0.529743

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(41.5752.32 mg mixture/ml) with a = 0.05. This proportion corresponded to a mixture containing 3.04 mg a-tocopherol, 1.20 mg b-carotene and 8.59 mg lycopene for use in the supplement formulation, in which antioxidant eects were seemed to be additive. One single daily dose would contribute with 20% and 22% of the RDA (2000) for a-tocopherol and b-carotene, respectively, and with 8.55 mg for lycopene. The construction of the model for the AOA response (Eq. (1)) was considered only as an indicator of the behavior of the variables. The adjusted model was validated by repeating the entire experimental procedure for the optimal proportion and a value of 49.55 1.57 mg mixture/ml (IC50) was obtained. This experimental value showed no signicant dierence (p < 0.558960) compared to the model estimated value using a v2 test. In the area of food research, statistical techniques such as RSM have been extensively applied mainly to the treatment of data obtained by physicochemical and sensory analyses (Castro, Tirapegui, & Silva, 1998; Castro, Tirapegui, Silva, & Cutrim, 2004). With respect to nutritional parameters, the application of this technique is still unexploited, possibly because the identication of good models highly depends on the eciency in isolating independent variables and reducing the interference of random variables. In practice, this procedure is much more dicult to apply to assays involving in vivo or ex vivo models than to in vitro assays, often leading to the non-validation of already adjusted polynomial equations and, consequently, losing their predictive capacity. The current study certainly presented an example of an experimental design using RSM in biological systems, encouraging the elaboration of future studies conducted on the basis of the combined models adjustment and optimization. It is essential for the investigation of compounds that act protecting the oxidable substrates in which the use of a single assessment method is considered to be insucient. Although specically referring to the higher oxidative stress experienced by astronauts during space missions, Fang et al. (2002) emphasized the importance of studying mixtures as an alternative in future investigations. The experimental model used in the present study illustrates the application of RSM for the development of functional supplements, thus,

broadening the possibility of a more ecient use of carotenoid pigment mixtures in human nutrition. In summary, in the present study no synergism was observed in the antioxidant capacity of mixtures containing lycopene, b-carotene and a-tocopherol evaluated by the percent inhibition of spontaneous oxidation (IC50) of rat brains compared to control. The behavior of the three compounds with respect to the AOA response could be graphically represented by a quadratic polynomial equation. In addition, the estimated AOA value for an optimized proportion of 3.04 mg a-tocopherol, 1.20 mg b-carotene and 8.59 mg lycopene used in the supplement formulation, did not signicantly dier from the experimentally observed value.

Acknowledgment This research was supported by Fundac a o de Am` Pesquisa do Estado de Sa paro a o Paulo (FAPESP- Process number 03/00570-0), Brazil.

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