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Optimization of the antioxidant capacity of a mixture of carotenoidsand a-tocopherol in the development of a nutritional supplement

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

a Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sao Paulo, 05508-900 Sao Paulo, SP, Brazilb Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of Sa o Paulo, 05508-900 Sao 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 for-mulation of a nutritional supplement using a simplex-centroid design and response surface methodology (RSM). Mixtures con-taining different proportions of each compound had their antioxidant activity (AOA) evaluated by the percent inhibition ofspontaneous autoxidation. The regression model fitted to the experimental data demonstrated that the linear terms and only thequadratic terms containing the lycopene variable had a significant impact on the AOA response. A validation assay for the modelwas performed. The observed value obtained experimentally for AOA did not differ significantly from the value estimated by theadjusted model. The results showed that no synergism occurred between the three compounds when rat brain homogenate was usedas the oxidable substrate, and also suggested that RSM can be applied to estimate the behavior of mixed ingredients in nutritionalsupplements.

2005 Elsevier Ltd. All rights reserved.

Keywords: a-Tocopherol; b-Carotene; Lycopene; Antioxidant; RSM

1. Introduction

Accumulating evidences have suggested that oxida-tive stress is involved in a variety of physiological andpathological processes, such as cellular signal transduc-tion, cell proliferation and differentiation, apoptosis, aswell as arteriosclerosis, ischemia-reperfusion injury, can-

cer, stroke and many degenerative diseases (Cai et al.,2002; Halliwell & Gutteridge, 1998; Weinreb, Mandel,Amit, & Youdim, 2004). The beneficial influence ofmany foodstuffs and beverages including fruits, vegeta-bles, tea, red wine and nuts on human health has been

recognized. This beneficial effect is due to the antioxi-dant activity (AOA) property from the bioactive sub-stances naturally present in these products (Roginsky& Lissi, 2005; Stahl & Sies, 2005). These substancesknown as antioxidants can delay or inhibit oxidativedamage when present is small quantities compared toan oxidizable substrate. Therefore, antioxidants can

help in disease prevention by effectively quenching freeradicals or inhibiting damage caused by them (Chatter-jee, Poduval, Tilak, & Devasagayam, 2005).

One of the main risk factor for coronary artery dis-ease is the oxidation of the LDL (low density lipopro-teins cholesterol) in the sub-endothelial space of thearterial wall (Arrol, Mackness, & Durrington, 2000;Luostarinen, Laasonen, & Calder, 2001; Song &Miyazawa, 2001). Antioxidants can reduce LDL oxida-tion and thus reduce plaque growth (Lewis, 2004).

0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2005.02.010

* Corresponding author. Tel.: +55 11 30913652; fax: +55 1138154410.

E-mail address: [email protected](I.A. Castro).

www.elsevier.com/locate/foodres

Food Research International 38 (2005) 861866
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Attention has been paid to fat-soluble antioxidantssuch as a-tocopherol, b-carotene and lycopene, whichare present in LDL in quantities that can be modifiedby dietary intake or oral supplementation (Arrol et al.,2000). Some carotenoid pigments such as b-caroteneare 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). Carotenoidshave been reported to play a role as antioxidants in li-pid phases by trapping free radicals or quenching sin-glet oxygen (Subagio & Morita, 2001). Lycopene, acarotenoid naturally present in tomatoes and otherfruits, has been found to be the most effective quencherof singlet oxygen and peroxyl radicals in plasma, lipo-proteins and human lymphoid cells in vitro, with thehighest levels being found in LDL-cholesterol (Bram-ley, 2000; Fleshner & Kucuk, 2001; Stahl & Sies,

1996). In addition, a-tocopherol acts as a chain-break-ing antioxidant by donating its phenolic hydrogen tothe chain-propagating lipid peroxyl radical forming aless reactive a-tocopheroxyl radical, which can furtherreact with another lipid peroxyl radical to stop thepropagation of lipid peroxidation (Zhang & Omaye,2001). It is also known that interactions betweencarotenoids and a-tocopherol can occur in vitro bymechanisms that are still not completely understood.Various compounds show synergism in their antioxi-dant capacity, thus, permitting that mixtures can pro-mote more effective 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 wellestablished concept for planning and execution ofinformative experiments and it can be used in manyapplications. One main type of DOE applicationconcerns the preparation and modification of mix-tures. This involves the use of mixture designs forchanging mixture composition and exploring how suchchanges will affect some specific properties of the finalproduct. The use of this statistical technique, knownas response surface methodology (RSM), allows thevisualization of the modeling results in terms of coef-ficients by contour or 3D-response surface plots. Thisfacilitates the understanding of the model and alsohelps in decision making (Eriksson, Johansson, &Wikstrom, 1998).

Therefore, the objective of the present study was todetermine the interaction between mixtures containinga-tocopherol, b-carotene and lycopene using the re-sponse surface methodology statistical technique, andto identify the proportion of the three compounds thatwould maximize the antioxidant capacity in order toelaborate a nutritional supplement.

2. Materials and methods

2.1. Materials

a-Tocopheryl acetate (Vitamin E-acetate SD50), b-carotene (Lucarotin 10CWD) and lycopene (Lycovit

10CWD) were obtained from BASF Health & Nutri-tion, Ludwigshafen, Germany.

2.2. Experimental design

The characteristic feature of a mixture is that the sumof all its components add up to 100%

Pqi1xi 1. This

means that these components (xi) mixture factors, can-not be manipulated completely independently of one an-other and that their proportions must lie somewherebetween 0 and 1. A classical simplex-centroid experi-mental design was proposed for the present study (Table

1), considering a mixture of three variables, wherex1= a-tocopherol, x2= b-carotene and x3= lycopene,representing pure, binary and tertiary mixtures of thevariables, with dextrin being used as control in the nutri-tional supplement formulation (Cornell, 2002). Eachmixture was based on the recommended daily allow-ances for a-tocopherol and b-carotene, while the con-centration adopted for lycopene was based in theliterature (Southon, 2000). No absolutely pure mixtureswere applied to this design in order to guarantee a min-imum of 25% and maximum of 100% RDA (Recom-mended Dietary Allowances) concentrations in the

nutritional supplement. Thus, the values coded in thedesign should be interpreted as the percentage of contri-bution of each compound to complete the formulation.Antioxidant activity (AOA) of each point from theexperimental simplex-centroid design was evaluatedin duplicate.

2.3. Determination of antioxidant activity

Adult male Wistar rats (297.88 25.64 g) fed on astandard diet were perfused with 60.0 ml of a cold0.9% saline solution, under light ether anesthesia. Brainwas immediately removed, weighted and homogenizedin 1:4 proportion with phosphate-buffered saline solu-tion (8.18 g of NaCl, 2.75 g of NaH2PO4, 2.83 g ofNa2HPO4, to 1.0 l, pH 7.4). Tubes were centrifuged at1.000gfor 15 min at 4 C and the supernatant was usedas substrate for lipoperoxidation. This preparation wascarried out daily with fresh brain tissue. Fifty microlitersof the phosphate-buffered saline solution containing fivedifferent concentrations ofa-tocopherol,b-carotene andlycopene were added to 1 ml of the diluted homogenateand 2.95 ml of phosphate-buffered saline, and incubatedfor 1 h at 37 C in an orbital water shaker. 1.0 ml of

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TBA/TCA/BHT solution (3.75 g of thiobarbituric acid,

93.75 mg of trichloroacetic acid and 5.00 mg of butyl-ated hydroxyl toluene dissolved slowly into 25 ml ofHCl 0.25 N) was added and the tubes were incubatedat 100 C/15 min. After cooling, the tubes were centri-fuged at 1.000g for 15 min at 25 C and the oxidationproducts represented by thiobarbituric acid reactive sub-stances (TBARS) were determined by spectrophotome-try at 532 nm according to Stocks, Gutteridge, Sharp,and Dormandy (1974). The AOA of the brain homoge-nate was expressed as percent inhibition of spontaneousautoxidation or IC50 (as measured in the controlhomogenate), determined by the curve obtained by

non-linear regression. Each point from the simplex-centroid design was evaluated in duplicate for eachone of the five concentrations. Three rats were usedfor each assay (brain weight @ 1.6 g/rat). In the end ofthis study, the same procedure was applied to the pro-portion selected for the quadratic model validation. Intotal, 48 animals were used in this study (3 rats/8 sam-ples/2 repetition).

2.4. Statistical analysis and regressions optimization

The results obtained by assessing the AOA in the se-ven assays were analyzed statistically on the basis of thedependent variable, considering that the mixture factorsare correlated (Cornell, 2002). The models quality wasdetermined by analysis of variance (ANOVA) of regres-sion which is useful for checking the adequacy of aregression model in terms of lack of fit test, throughthe evaluation of the model residuals using a normalprobability plot and finally by the Coefficient of Deter-mination (R2). Before analysis, data were screenedgraphically for normality and for homogeneity of vari-ance by Levene test. Calculations and graphs were ob-tained with the STATISTICA software, v.6 (StatSoftInc., Tulsa, OK).

3. Results and discussion

3.1. Antioxidant capacity of the mixtures

Table 1 shows the results regarding the antioxidantcapacity (AOA) of the mixtures proposed in the experi-mental design using brain homogenate as substrate.

3.2. Regression model

A quadratic polynomial regression model was as-sumed for prediction purpose of the response (y). Themodel proposed was

where b0, bi, bii and bijare intercept, linear, quadraticand interaction regression coefficient of the model,respectively, xi and xjare coded independent variablesand e the error associated to the y determination. Theregression model fitted to the experimental data (Eq.(1)) demonstrated that the linear terms and only the qua-dratic terms containing the lycopene variable had a sig-nificant impact on the response evaluated ^y1 AOA

^y1 53.89x12.38

63.88x22.38

21.71x32.59

28.16x1x311.93

39.90x2x311.93

.

1

Fig. 1shows that the most expressive results were ob-tained with the maximum presence of lycopene in the mix-ture and that none of the interactions between the threecompounds showed synergism, in contrast to the originalhypothesis of the present study.

Miller, Sampson, Candeias, Bramley, and Rice-Evans(1996) assessing the relative AOA of a range of

Table 1Antioxidant capacity (AOA) of the mixtures

Mix Experimental designa a-Tocopherolb (mg/d) b-Caroteneb (mg/d) Lycopeneb (mg/d) mg Mixture/ml (IC50)c

01 (100, 0, 0) 3.37 0.00 0.00 52.61 4.7902 (0, 100, 0) 0.00 3.60 0.00 62.59 0.2103 (0, 0, 100) 0.00 0.00 11.25 21.82 1.5004 (50, 50, 0) 1.69 1.80 0.00 61.23 8.68

05 (50, 0, 50) 1.69 0.00 5.62 44.40 0.8406 (0, 50, 50) 0.00 1.80 5.62 32.38 0.9307 (33, 33, 33) 1.12 1.20 3.75 46.18 0.29

OTMd (57, 0, 43) 1.92 0.00 4.84 49.55 1.57

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

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carotenes, concluded that the sequence of relative radi-cal scavenging abilities was lycopene >b-carotene >lutein and zeaxanthin, and that the AOA of lycopenewas almost three times higher than that ofa-tocopherol.Stahl et al. (1998)evaluated the effects of single carote-

noids and a-tocopherol on lipid peroxidation inducedby 2,2 0-azo-bis(2,4-dimethylvaleronitrile), and observedthat lycopene was the most effective carotenoid, reduc-ing TBARS formation in multilamellar liposomes toabout 25% of the control, with the other carotenoidssuch as b-carotene and a-tocopherol being less active.These authors also evaluated carotenoid mixtures andtheir results demonstrated that only lutein and lycopenewere responsible for synergistic antioxidant effects.Using triacylglycerol as substrate in an in vitro system,Subagio and Morita (2001) observed that carotenoidsmay also promote lipid oxidation because of the insta-bility against heat and light, and that this inductioncould be blocked by the presence of tocopherols.According to Zhang and Omaye (2001), mixture ofb-carotene anda-tocopherol had no significant interactioneffect on AOA measured in rat liver microsome suspen-sions. The authors also suggested that the antioxidant orpro-oxidant activities of these compounds are related toseveral factors, including antioxidant concentrations,governed by complex interactions. Southon (2000) inthe Core Study found no significant effect on LDL oxid-izability and other biomarkers of oxidative and antioxi-dant status when carotenoids were provided incombination with a-tocopherol.

No gold standard assay exists for measuring antioxi-dant activity. Therefore, it has been suggested that thesedata should be obtained by different methods such as bymeasurement of endogenous antioxidant levels, determi-nation of the products of oxidized macromolecules, and

direct detection of free radicals (Fang, Yang, & Wu,2002). Although limited by the RDA values and theassessment of AOA by a single method, our results con-firm that interactions between pigments and a-tocoph-erol will basically depend on the concentration andsolubility of these compounds, as well as on the choiceof the oxidable substrate.

3.3. Optimization of the general function

Analysis of variance for Eq. (1) (Table 2) indicated thatthe proposed model presented a good fit to the experimen-tal data as observed both by the non-significance of thelack of fit and by Coefficient of DeterminationR2adjust 0.93. The R

2 value can be interpreted as theproportion of variability around the mena for the depen-dent variable, that can be accounted for by the respectivemodel. This R2 value should be as close to unity aspossible.

By applying the general function proposed byDerrin-ger and Suich (1980), we performed optimization of theregression (y1), which resulted in the proportion x1=0.56947,x2= 0 andx3= 0.43053, with an estimated va-lue of 46.94 mg mixture/ml (IC50) for AOA within theconfidence interval predicted by the adjusted model

60

50

40

30

0.00

0.25

0.50

0.75

1.000.00

0.25

0.50

0.75

1.00

TOC(x1)

0.00 0.25 0.50 0.75 1.00

CAR(x2)

LYC(x3)

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



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(41.5752.32 mg mixture/ml) with a= 0.05. This pro-portion corresponded to a mixture containing 3.04 mga-tocopherol, 1.20 mg b-carotene and 8.59 mg lycopenefor use in the supplement formulation, in which antiox-idant effects were seemed to be additive. One single dailydose would contribute with 20% and 22% of theRDA(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 consid-ered only as an indicator of the behavior of the vari-ables. The adjusted model was validated by repeatingthe entire experimental procedure for the optimalproportion and a value of 49.55 1.57 mg mixture/ml(IC50) was obtained. This experimental value showedno significant difference (p< 0.558960) compared tothe model estimated value using a v2 test.

In the area of food research, statistical techniquessuch as RSM have been extensively applied mainly tothe treatment of data obtained by physicochemical andsensory analyses (Castro, Tirapegui, & Silva, 1998; Cas-

tro, Tirapegui, Silva, & Cutrim, 2004). With respect tonutritional parameters, the application of this techniqueis still unexploited, possibly because the identification ofgood models highly depends on the efficiency in isolatingindependent variables and reducing the interference ofrandom variables. In practice, this procedure is muchmore difficult to apply to assays involving in vivo orex vivo models than to in vitro assays, often leading tothe non-validation of already adjusted polynomialequations and, consequently, losing their predictivecapacity.

The current study certainly presented an example ofan experimental design using RSM in biological sys-tems, encouraging the elaboration of future studies con-ducted on the basis of the combined models adjustmentand optimization. It is essential for the investigation ofcompounds that act protecting the oxidable substratesin which the use of a single assessment method is consid-ered to be insufficient. Although specifically referring tothe higher oxidative stress experienced by astronautsduring space missions, Fang et al. (2002) emphasizedthe importance of studying mixtures as an alternativein future investigations. The experimental model usedin the present study illustrates the application of RSMfor the development of functional supplements, thus,

broadening the possibility of a more efficient use ofcarotenoid pigment mixtures in human nutrition.

In summary, in the present study no synergism wasobserved in the antioxidant capacity of mixtures con-taining lycopene,b-carotene anda-tocopherol evaluatedby the percent inhibition of spontaneous oxidation

(IC50) of rat brains compared to control. The behaviorof the three compounds with respect to the AOA re-sponse could be graphically represented by a quadraticpolynomial equation. In addition, the estimated AOAvalue for an optimized proportion of 3.04 mg a-tocoph-erol, 1.20 mg b-carotene and 8.59 mg lycopene used inthe supplement formulation, did not significantly differfrom the experimentally observed value.

Acknowledgment

This research was supported by Fundacao de Am-paro aPesquisa do Estado de Sao Paulo (FAPESP- Pro-cess number 03/00570-0), Brazil.

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Table 2Analysis of variance for the reduced quadratic model

Source SS df MS F p

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Total adjusted 2749.261 13 211.4817

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