THE SCAVENGING CAPACITY OF COMBINATIONS OFLYCOPENE, b-CAROTENE, VITAMIN E, AND VITAMIN C ONTHE FREE RADICAL 2,2-DIPHENYL-1-PICRYLHYDRAZYL

(DPPH)

J. CHEN1, J. SHI2,8, L. MACNAUGHTON3, Y. KAKUDA4, S.J. XUE2, Y. MA5,M. ZHANG6 and Y. JIANG7

1Department of Food Science and NutritionZhejiang University

Hangzhou, Zhejiang 310027

2Guelph Food Research Center, Agriculture and Agri-Food Canada

4Department of Food ScienceUniversity of Guelph

Guelph, Ontario N1G 5C9, Canada

3Department of Human Biology and Nutritional SciencesUniversity of Guelph

5School of Food Science and EngineeringHarbin Institute of Technology

Harbin 150090, China

6Biotechnological Research InstituteGuangdong Academy of Agricultural Sciences

Guangzhou 510650, China

7South China Botanical GardenChinese Academy of Sciences

Guangzhou 510650, China

Accepted for Publication August 27, 2007

ABSTRACT

Lycopene, a very powerful antioxidant present in fruits and vegetables, isthe most efficient quencher of singlet oxygen among the carotenoids. There areother natural antioxidants such as carotenoids, vitamin C, tocopherols andpolyphenolics in fruits and vegetables that are consumed together and thattherefore have potential for synergistic interactions. Synergistic effects were

8 Corresponding author. TEL: 1-519-7808035; FAX: 1-519-8292602; EMAIL: shij@agr.gc.ca

Journal of Food Biochemistry 33 (2009) 232–245.© 2009, Crown in the right of Canada

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determined by combining lycopene with other antioxidants (vitamin E, vitaminC, b-carotene) in various ratios. A synergistic effect was observed when allfour antioxidants were present in combination at high lycopene activity. Asynergistic effect was also observed when vitamin E and vitamin C were usedin combination at high vitamin E concentrations. It appears that certaincombination ratios and concentration levels of these antioxidants have effectsto enhance the antioxidant activity, compared to the individual compound,which suggests that similar combinations in the diet or in functional foodproducts might provide greater health beneficial effects.

PRACTICAL APPLICATIONS

The composition and concentration of individual bioactive components ina mixture would affect the total bioactivity and function in food system andhuman body. It is necessary to develop a model system for water- and lipid-soluble compounds to understand and assess the interactions and synergisticantioxidant effects of lycopene with b-carotene, vitamin C, vitamin E. Theexperimental results of synergistic effects as new approach could reveal theinteractions among these antioxidants. The results provide a useful guidancein the formulation and development of functional food products that havehigh antioxidant and functional potential. The results of present study areessentially useful to reveal the antioxidant mechanism, and as guidance forformulating functional foods. It would assist in understanding and controllingthe functionality of bioactive components during process. The experimentaldata is valuable to design the optimum concentrations and composition oflycopene mixed with other carotenoids for maximum antioxidative effects.

INTRODUCTION

Many fruits and vegetables contain mixtures of various antioxidants, andtheir interactions might play an important role in their total antioxidant poten-tial. It is well documented that the intake of fruits and vegetables can reducethe risk of chronic diseases such as cardiovascular disease and cancer (LeMarchand et al. 1989; Ziegler 1991; Block et al. 1992; Tinkler et al. 1994;Steinmetz and Potter 1996). With over 600 different carotenoids in nature andwith many of them having antioxidant properties, there are ample opportuni-ties for these antioxidants to interact with one another in a synergistic oradditive manner (Stahl and Sies 1996; Fuhrman et al. 2000).

Antioxidants play distinctive roles in biological systems, which are deter-mined by their chemical reactions, placement in the plasma membrane and

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mobility within the membrane (Niki and Noguchi 2004). Tomatoes are a goodsource of lycopene and other antioxidants including vitamin E, b-carotene, andvitamin C in the Western diet (Beecher 1998). Carotenoids undergo a series ofenzymatic steps in plants that convert phytoene to lycopene, with each stepadding a double bond to the structure. Lycopene is an acyclic hydrocarboncarotene with eleven double bonds in its structure, and is considered to be themost efficient quencher of singlet oxygen among the carotenoids (Paiva andRussell 1999). b-Carotene is another hydrocarbon carotenoid and quencher ofsinglet oxygen at a low partial pressure of oxygen (Tsuchihashi et al. 1995).Unlike lycopene, b-carotene is a precursor of retinol and retinoic acid (Tsuchi-hashi et al. 1995). However, lycopene is much more effective at inhibiting thegrowth of cancer cells in culture (Levy et al. 1995; Narisawa et al. 1996) andis a more powerful inhibitor of lipid peroxidation in vitro (Stahl et al. 1998).Vitamin E is considered to be an efficient chain-breaking antioxidant thatproduces a relatively nonreactive chromanoxyl radical (Chan et al. 1991;Wrona et al. 2003). Vitamin C is a hydrophilic antioxidant, and is consideredto be a poor antioxidant within the lipophilic plasma membrane (Doba et al.1985). However, vitamin C plays a valuable role in the regeneration of vitaminE and thereby acts to reduce the rate of oxidative consumption of vitamin E(Doba et al. 1985; Chan et al. 1991; Wrona et al. 2003).

2, 2′-Diphenyl-1-picrylhydrazyl (DPPH) is a polar free radical that pro-duces a violet solution in ethanol. In the presence of an antioxidant the radicalis reduced and the color fades, which causes a drop in absorbance. The colorloss is depended on the number of electrons taken up by the DPPH (Brand-Willians et al. 1995; Mensor et al. 2001). Recent studies have shown that thescavenging of DPPH by various antioxidants depends on the number of avail-able hydroxyl groups on the antioxidant molecule (Brand-Willians et al.1995).

A good example is the tomato fruit, which has lycopene, vitamin E,b-carotene and vitamin C as the predominate naturally occurring antioxidants.The possibility that these naturally occurring antioxidants can interact withone another and enhance the total antioxidant potential has been a topic ofcurrent interest. However, there have been a limited number of studies thatinvestigated the combined effect of the antioxidants found in tomatoes. Arecent study found that short-term intake of a meal high in lycopene, lutein andb-carotene resulted in competitive incorporation into the chylomicron, whilelong-term intake resulted in a synergistic response amongst these antioxidants(Tyssandier et al. 2002). Stahl et al. (1998) studied the effects of carotenoidmixtures on inhibition of lipid peroxidation and found that lutein and lycopenewere responsible for the synergistic antioxidant effect, but only when used inspecific combinations. At the present time, there is very little information inthe literature on the competitive, additive and synergistic properties of lyco-

234 J. CHEN ET AL.

pene with other natural antioxidants. The aim of this study was to find optimalconcentrations and combination ratio of antioxidant mixture in a model foodsystem (vitamin E, lycopene, b-carotene, and vitamin C) that produce a syn-ergistic antioxidant effect.

MATERIAL AND METHODS

Materials

Reagent grade lycopene, vitamin E, vitamin C acid, b-carotene, and2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical were purchased fromSigma-Aldrich Co. (Oakville, ON, Canada). Ethanol (95%, v/v) waspurchased from Commercial Alcohols Inc. (Brampton, Ontario, Canada),hexane and other chemicals were HPLC grade from Fisher Scientific (Ottawa,Ontario, Canada).

Antioxidant Solutions

Stock solutions in hexane : tetrahydrofuran (3:1, v/v) were prepared at0.3 mM DPPH, 0.25 mM lycopene, 2.5 mM b-carotene, 0.05 mM vitamin Eand 0.05 mM vitamin C. All procedures were performed under dim light andall stock solutions were stirred at room temperature for 10 min before furtheruse.

DPPH Free Radical-Scavenging Capacity Assay

The antioxidant activity was measured with a simple and rapid freeradical assay using DPPH. The absorbance of the free radical DPPH wasmonitored over time in the presence or absence of antioxidant compounds. Tomeasure scavenging capacity, 0.75 mL of DPPH solution was added to thestock solutions of antioxidants, which were diluted to a desired final concen-tration in a 3 mL cuvette. The mixtures were shaken and absorbance wasmeasured using a UV spectrophotometer (Shimadzu UV-Visible RecordingSpectrophotometer UV-260) at 518 nm and 540 nm. The wavelength ofmaximum absorbance is 518 nm for DPPH. Because of interference fromlycopene absorbance at this wavelength, all activity measurements wereshifted to 540 nm. Absorbance was also measured at 518 nm and used as ameasure of optimal concentration range. The concentration of the mixturesused at various combinations as model food system is shown in the Table 1.

The reaction mixtures were incubated in their cuvettes at room tempera-ture, taking care to prevent exposure to light by holding the cuvette in anopaque container. Absorbance was measured at time 0 and every 10 min for60 min time period.

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Calculation of Synergistic Effects of Antioxidant Mixtures

The scavenging capacity (%SC) of each mixture was calculated using thefollowing formula (Mensor et al. 2001):

%SC Abs Abs Abssample blank control= − −( ) ×[ ]{ }100 100

The antioxidant or antioxidant mixtures were used as the blank. DPPH solu-tion (0.75 mL) was used as the negative control. The expected values werecalculated from the scavenging capacity of the individual antioxidants at eachtime interval using the following equation, where A and B represent thescavenging capacity of the individual antioxidants (Fuhrman et al. 2000).

Expected value A 1 A B= + −( ) ×

A synergistic effect was considered to be present if the ratio of the measuredscavenging capacity is greater than 1.

Statistical Analysis

The experiments were done in triplicate. Student’s t-test and a one-wayanalysis of variance was used for comparison between two means. A differencewas considered statistically significant when P < 0.05.

RESULTS AND DISCUSSIONS

Antioxidant Activity

A synergistic response (P < 0.05) was seen when all four antioxidants(Group 4) were tested together (Fig. 1). In this trial, the %SC of lycopene

TABLE 1.THE CONCENTRATION OF SINGLE ANTIOXIDANTS IN EACH ANTIOXIDANT MIXTURE

Group Vitamin E Lycopene b-Carotene Vitamin C

1 (540 nm) 0.5 6.35 12.5 202 (540 nm) 0.5 6.35 12.5 5.03 (540 nm) 0.5 5.00 12.5 3.04 (540 nm) 1.0 6.35 12.5 5.05 (540 nm) 1.0 5.00 12.5 5.06 (540 nm) 1.0 6.35 25.0 5.07 (540 nm) 2.0 6.35 12.5 5.0

Final concentration (mM/mL) of each antioxidant.

236 J. CHEN ET AL.

alone was 43.2%. No synergism was observed in a second trial using the sameantioxidant combinations and concentrations (Group 4), but with a lycopenepreparation with a %SC of only 15.8% (Fig. 2).

Antioxidant concentrations used in Group 4 also showed a significantsynergistic response when vitamin E and vitamin C were combined (Fig. 3).This might provide insight about antioxidants that were most responsible forthe synergistic response when all four antioxidants were combined. However,this was difficult to determine because no other concentrations of vitamin Eand vitamin C showed this response. The individual scavenging capacities ofvitamin E and vitamin C were 26.1% SC � 2.05 and 38.64% SC � 0.54,respectively, averaged over the 60 min time period.

When vitamin E (1.0 mM) was mixed with lycopene (6.35 mM), thecombination showed a significant (P < 0.05) synergistic effect (Fig. 4). Otherstudies have also reported similar results. Fuhrman et al. (1997) found that thecombination of lycopene, b-carotene, and vitamin E had only an additiveeffect on the oxidative protection of low-density lipoprotein (LDL). However,a similar study by Fuhrman et al. (2000) found that the combination of lyco-pene plus vitamin E, garlic or glabridin produced a synergistic antioxidanteffect on LDL oxidation. Tsuchihashi et al. (1995) studied the effect of vitaminE and b-carotene in combination in many different free radical mediums.These investigators discovered that the effect was dependent on the mediumand the site of radical generation in the liposomal membrane. They found thatb-carotene had a low antioxidant activity in comparison with vitamin E, which

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FIG. 1. PERCENT SCAVENGING CAPACITY (%SC) OF GROUP 6 AT 60 MIN WHENLYCOPENE ACTIVITY TOTALED 43.25 � 2% � SEM

*Represents statistical significance of measured compared to expected at P < 0.05. Ly, lycopene;VE, vitamin E; VC, vitamin C; b-Car, b-carotene (�: expected %SC; : measured %SC).

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was also shown in this study. They also discovered that b-carotene was sparedover vitamin E, and might be involved in the regeneration of vitamin E.Palozza and Krinsky (1991) found similar results to as mentioned earlier.Because of the nature of the assay used in this study, the physical interaction

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FIG. 2. PERCENT SCAVENGING CAPACITY (%SC) OF GROUP 6 AT 60 MIN WHENLYCOPENE ACTIVITY TOTALED 15.82% � 0.5 � SEM (�: EXPECTED %SC; :

MEASURED %SC)

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FIG. 3. PERCENT SCAVENGING CAPACITY (EXPECTED AND MEASURED VALUES) OFTHE COMBINATION OF VITAMIN E (1.0 MM) + VITAMIN C (5.0 MM) � SEM OVER 60 MIN

The increase in %SC of the measured value over the expected value was statistical significant atP < 0.05 (�: expected %SC; �: measured %SC).

238 J. CHEN ET AL.

of the antioxidants within liposomal membranes did not show significant,however, they might play a role in producing an additive or synergistic anti-oxidant effect.

The four antioxidants with Group 7 concentration levels also producedstatistically significant synergistic responses after 10 min (Fig. 5). In this trial,the vitamin-E level was doubled to 2.0 mM while holding the other threeantioxidants at the same level as those in Group 4. In phenomena similar toGroup 4, the combination of all four antioxidants in Group 6 produced astatistically significant synergistic response (Fig. 6).

The other combinations of antioxidants in Table 1 showed only additiveeffects. With Group 2 experiment, concentrations of lyopene, vitamin E,b-carotene, and vitamin C are 6.35, 0.5, 12.5 and 5.0 mM, respectively. Themeasured %SC values for the antioxidant combinations were the same as thesum of the individual %SC values for the single antioxidants (Fig. 7).

In this free radical assay, it was previously recognized that the scavengingof DPPH depended in past on the number of available hydroxyl groups on theantioxidant (Mensor et al. 2001). Vitamin E contains one hydroxyl group in itsstructure and vitamin C contains three hydroxyl groups; whereas, b-caroteneand lycopene do not contain any hydroxyl groups. DPPH is a polar freeradical, therefore polar hydrophilic antioxidants have a higher scavengingcapacity. This might also explain why all four antioxidants must be incombination in order to see a synergistic effect, and why vitamin E at highconcentration with vitamin C, showed a synergistic response. A higher number

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FIG. 4. PERCENT SCAVENGING CAPACITY (EXPECTED AND MEASURED VALUES) OFTHE COMBINATION OF VITAMIN E (1.0 MM) + LYCOPENE (6.35 MM)

*Represents statistical significance of measured value compared to expected value at P < 0.05 (�:expected %SC; : measured %SC).

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of hydroxyl groups in the system resulted in higher scavenging capacity.Soares et al. (2003) used several antioxidants and tested the inhibition of theseantioxidants using different free radical assays, including DPPH. They foundthat vitamin C had a high scavenging capacity and vitamin E had a very low

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FIG. 5. PERCENT SCAVENGING CAPACITY (EXPECTED AND MEASURED VALUES) OFGROUP 10 ANTIOXIDANT MIXTURE

*Represents significant difference between measured value and expected value at p < 0.05(�: expected %SC; �: measured %SC).

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FIG. 6. PERCENT SCAVENGING CAPACITY (EXPECTED AND MEASURED VALUES) OFGROUP 8 WITH ALL FOUR ANTIOXIDANTS IN COMBINATION

*Represents statistical significance of measured value compared to expected value at P < 0.05(�: expected %SC; : measured %SC).

240 J. CHEN ET AL.

scavenging capacity, as a result of the aqueous nature of the system. Thiscoincides with the results found in our study. However, the antioxidant activity,as well as the concentration of lycopene, had an effect on the synergisticresponse. At low antioxidant activity, an additive effect was observed;whereas, at high antioxidant activity, a synergistic response was observed. Inaddition, high and low lycopene concentrations showed no synergistic effectwhen in combination with the other antioxidants when their concentrationswere held constant. Pastori et al. (1998) found that vitamin E and lycopene hada synergistic effect in inhibiting prostate carcinoma cells. However, this effectwas dose dependant. The investigators stated that this synergistic inhibition ofcancer cells may occur because the antioxidants affected different signaltransduction pathways. Similar to Pastori et al. (1998), our study showed asynergistic response when vitamin E and lycopene were combined at specificconcentrations and mixing ratios. This was significant when one of the repli-cations was removed from the statistical analysis.

Synergistic Effects

The synergistic effect was dependent on the combination ratio, varietiesand content of antioxidants. It was discovered that when all four antioxidantswere used in the assay mixture, there was a synergistic response, but only withcertain concentration of the antioxidants and mixing ratios. A significantsynergistic effect was observed using concentrations of 1.0–2.0 mM vitaminE, 6.35 mM lycopene, 12.5 mM b-carotene, and 5.0 mM vitamin C. The

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FIG. 7. PERCENT SCAVENGING CAPACITY OF MEASURED VALUES OF GROUP 4 OFSINGLE ANTIOXIDANTS AND SOME OF THE COMBINATIONS � SEM (�: VE; : LY; �:B-CAR; ¥: VC; �: VE + LY; �: VE + LY + B-CAR; D: LY + B-CAR; |: VE + LY + B-CAR + VC)

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scavenging capacity of the antioxidants was dose dependant. In general, higherantioxidant concentration resulted in a higher percent of scavenging capacity.However, this did not always correlate to a higher synergistic response(Table 2). These results demonstrate that the synergistic response can beaffected by changes in the concentration of a single antioxidant within themixture. It also appears that a certain level is required to produce an optimumsynergistic response. This phenomena was true for all antioxidants in themixture. Vitamin E and vitamin C also showed a synergistic effect when usedin combination with a higher vitamin E concentration. All four antioxidantshad a synergistic effect at a vitamin E level of 2.0 mM after 10 min. Otherantioxidant combinations only had an additive effect.

CONCLUSIONS AND DISCUSSIONS

There are many factors that might contribute to synergistic effects andinteraction of mixed antioxidants in a biological system. The concentrationand combination ratio of mixed antioxidants are important factors. Concen-tration is a factor because of the antioxidant/pro-oxidant interaction thatoccurs with various concentration ranges. The synergistic effect of mixedantioxidants as a result of their interaction is dose dependent. There mightalso be competitive inhibition of antioxidants at high concentration levels,which will lower synergistic effects. These factors might also work incombination. To produce synergistic effects with mixed antioxidants, it isimportant to find an optimal concentration level. The results from this pilotstudy are not comprehensive and represent preliminary findings that provide

TABLE 2.SYNERGISTIC EFFECT OF ANTIOXIDANTS WITH

INCREASING LEVELS OF VITAMIN E. THECONCENTRATIONS OF LYCOPENE (6.35 mM),

b-CAROTENE (12.5 mM), AND VITAMIN C (5.0 mM) WEREHELD CONSTANT

Vitamin Econcentration

Synergistic effect(Measured%SC/Expected %SC)

Standarddeviation

0.5 mM 1.06 �0.041.0 mM 1.19* �0.062.0 mM 1.06** �0.02

Values are the mean (n = 3) � S.D.Asterisks denote a significant difference compared with the respec-tive synergistic effect value (*P < 0.05, **P < 0.01).

242 J. CHEN ET AL.

evidence supporting the assumption that consuming a mixture of antioxidantswill produce a synergistic antioxidant effect. The synergistic effects of anti-oxidants play an important role in the field of functional foods and nutraceu-ticals. This theory can also play a significant role in the formulation anddevelopment of functional food products, with greater antioxidant capacity.The results of this study might also provide clues to investigators studyingbiological system. The incorporation of a combination of antioxidants mighthave greater health benefits than that of single antioxidants. Further studiesare required to systematically determine the optimum concentrations andcombinations for these antioxidants and to better understand the mechanismof the synergistic effect.

ACKNOWLEDGMENTS

The authors wish to thank the Guelph Food Research Center, Agricultureand Agri-Food Canada (AAFC Journal Series No. S295).

REFERENCES

BEECHER, G. 1998. Nutrient content of tomatoes and tomato products.PSEB. 218, 98–100.

BLOCK, G., PATTERSON, B. and SUBAR, A. 1992. Fruit, vegetables, andcancer prevention: A review of the epidemiological evidence. Nutr.Cancer 18, 1–29.

BRAND-WILLIANS, W., CUVELIER, M. and BERSET, C. 1995. Use of afree radical method to evaluate antioxidant activity. Lebensm. Wiss.Technol. 28, 25–30.

CHAN, A., TRAN, K., RAYNOR, T., GANZ, P. and CHOW, C. 1991. Regen-eration of Vitamin E in human platelets. J. Biol. Chem. 266, 17290–17295.

DOBA, T., BURTON, G. and INGOLD, K. 1985. Antioxidant andco-antioxidant activity of vitamin C. The effect of vitamin C, either aloneor in the presence of vitamin E or a water-soluble vitamin E analogue,upon the peroxidantion of aqueous multilamellar phospholipid lipo-somes. Biochim. Biophys. Acta. 835, 298–303.

FUHRMAN, B., ELIS, A. and AVIRAM, M. 1997. Hypocholesterolemiceffect of lycopene and b-carotene is related to suppression of cholesterolsynthesis and augmentation of LDL receptor activity in macrophages.Biochem. Biophys. Res. Commun. 223, 658–662.

243SCAVENGING CAPACITY

FUHRMAN, B., VOLKOVA, N., ROSENBLAT, M. and AVIRAM, M. 2000.Lycopene synergistically inhibits LDL oxidation in combination withvitamin E, glabridin, rosmarinic acid, carnosic acid, or garlic. Antioxid.Redox. Signal. 2, 491–506.

LE MARCHAND, L., YOSHIZAWA, C.N., KOLONEL, L.N., HANKIN, J.H.and GOODMAN, M.T. 1989. Vegetable consumption and lung cancerrisk: A population-based case-control study in Hawaii. J. Natl. CancerInst. 81(15), 1158–1164.

LEVY, J., BOSIN, E., FELDMAN, B., GIAT, Y., MIINSTER, A.,DANILENKO, M. and SHARONI, Y. 1995. Lycopene is a more potentinhibitor of human cancer cell proliferation than either a-carotene orb-carotene. Nutr. Cancer 24, 257–266.

MENSOR, L.L., MENEZES, F.S., LEITAO, G.G., REIS, A.S., DOSSANTOS, T.C., COUBE, C.S. and LEITAO, S.G. 2001. Screening ofbrazilian plant extracts for antioxidant activity by the use of DPPH. FreeRadic. Method. Phytother. Res. 15, 127–130.

NARISAWA, T., FUKAURA, Y., HASEBE, M., ITO, M., AIZAWA, R.,MURAKOSHI, M., SHINGO, U., KHACHIK, F. and NISHINO, H.1996. Inhibitory effects of natural carotenoids, a-carotene, b-carotene,lycopene and lutein, on colonic aberrant crypt foci formation in rats.Cancer Lett. 107, 137–142.

NIKI, E. and NOGUCHI, N. 2004. Dynamics of antioxidant action of vitaminE. Acc. Chem. Res. 37, 45–51.

PAIVA, S. and RUSSELL, R. 1999. b-carotene and other carotenoids asantioxidants. J. Am. Coll. Nutr. 18, 426–433.

PALOZZA, P. and KRINSKY, N.I. 1991. The Inhibition of radical-initiatedperoxidation of microsomal lipids by both a-tocopherol and b-carotene.Free Radic. Biol. Med. 11, 407–414.

PASTORI, M., PFANDER, H., BOSCOBOINIK, D. and AZZI, A. 1998.Lycopene in association with a-tocopherol inhibits at physiological con-centrations proliferation of prostate cancer carcinoma cells. Biochem.Biophys. Res. Commun. 250, 582–585.

SOARES, D., ANDREAZZA, A. and SALVADOR, M. 2003. Sequesteringability of butylated hydroxytoluene, propyl gallate, resveratrol, and vita-mins C and E against ABTS, DPPH, and hydroxyl free radicals in chemi-cal and biological systems. J. Agric. Food Chem. 51, 1077–1080.

STAHL, W. and SIES, H. 1996. Lycopene: A biologically important caro-tenoid for humans? Arch. Biochem. Biophys. 336, 1–9.

STAHL, W., JUNGHANS, A., DE BOER, B., DRIONINA, E., BRIVIBA, K.and SIES, H. 1998. Carotenoid mixtures protect multilamellar liposomesagainst oxidative damage: Synergistic effects of lycopene and lutein.FEBS Lett. 427, 305–308.

244 J. CHEN ET AL.

STEINMETZ, K.A. and POTTER, J.D. 1996. Vegetables, fruits, and cancerprevention: A review. J. Am. Diet. Assoc. 96, 1027–1039.

TINKLER, J.H., BOHM, F., SCHALCH, W. and TRUSCOTT, T.G. 1994.Dietary carotenoids protect human cells from damage. J. Photochem.Photobiol. 26, 283–285.

TSUCHIHASHI, H., KIGOSHI, M., IWATSUKI, M. and NIKI, E. 1995.Action of b-carotene as an antioxidant against lipid peroxidation. Arch.Biochem. Biophys. 323, 137–147.

TYSSANDIER, V., CARDINAULT, N., CARIS-VEYRAT, C., AMIOT, M.J.,GROLIER, P., BOUTELOUP, C., AZAIS-BRAESCO, V. and BOREL,P. 2002. Vegetable-borne lutein, lycopene, and b-carotene compete forincorporation into chylomicrons, with no adverse effect on the medium-term (3-wk) plasma status of carotenoids in humans. Am. J. Clin. Nutr.75, 526–534.

WRONA, M., KORYTOWSKI, W., ROZANOWSKA, M., SARNA, T. andTRUSCOTT, T.G. 2003. Cooperation of antioxidants in protectionagainst photosensitized oxidation. Free Radic. Biol. Med. 35, 1319–1329.

ZIEGLER, R. 1991. Vegetables, fruits, and carotenoids and the risk of cancer.Am. J. Clin. Nutr. 53, 251S–259S.

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