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Antioxidant activity of tomato lipophilic extracts and interactions between carotenoids and α-tocopherol in synthetic mixtures
LWT - Food Science and Technology 43 (2010) 67–72
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Antioxidant activity of tomato lipophilic extracts and interactions between carotenoids and a-tocopherol in synthetic mixtures Assunta Zanfini, Gianfranco Corbini, Caterina La Rosa, Elena Dreassi* Dipartimento Farmaco Chimico Tecnologico, Via Aldo Moro, 53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 November 2008 Received in revised form 15 May 2009 Accepted 16 June 2009
In the present study we assayed the antioxidant activity of lipophilic extracts obtained from different tomato varieties. The results showed that cherry tomatoes, characterized by a high carotenoid content, had the highest antioxidant activity. A quantitative analysis of lycopene, b-carotene, lutein and a-tocopherol was also performed and the correlation between the antioxidant content and the antioxidant activity was estimated. The highest correlation coefficient was found for lycopene (R2 ¼ 0.9236, P 0.001). The analysis of two-component mixtures containing a-tocopherol and carotenoids showed that significant synergism occurred for all the combinations which contained a-tocopherol and b-carotene mixed together. The highest synergistic effects were detected for a-tocopherol-lycopene mixtures, which were the most efficient combinations tested in the present study. The analysis of the carotenoid combinations indicated that synergism occurred for lycopene-b-carotene, lycopene-lutein and lutein-b-carotene mixtures. The analysis of four-component mixtures did not show statistically significant synergistic effects. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids a-Tocopherol Antioxidant activity Synergistic effect Synthetic mixtures
1. Introduction The great interest in studying antioxidant compounds is due to their ability to neutralize active oxygen species and free radicals that play an important role in the pathogenesis of such degenerative diseases. A large number of epidemiological and clinical studies have associated a lower incidence of some cancer types (Riboli & Norat, 2003; Steinmetz & Potter, 1996), cardiovascular diseases (Joshipura et al., 2001; Ness & Powles, 1997) etc with a high antioxidant dietary intake. The positive correlation between vegetable intake and cancer prevention has been attributed to the presence of antioxidant compounds (Machlin, 1995; Ziegler, 1991). Many studies have shown the beneficial effects produced by carotenoids and a-tocopherol on human health. Their biological roles have been described in many papers. The role of different carotenoids in the prevention of degenerative diseases has been attributed to their antioxidant properties (Conn, Schlach, & Truscott, 1991; Di Mascio, Kaiser, & Sies, 1989; Landrum & Bone, 2001; Rao & Agarwal, 1999; Sundquist, Briviba, & Sies, 1994). Other studies have described a-tocopherol (vitamin E) as one of the most important lipid-soluble radical scavenging antioxidant in membranes and in plasma (Burton, Joyce, & Ingold, 1983).
* Corresponding author. Tel.: þ39 0577 234321; fax: þ39 0577 234333. E-mail address: [email protected] (E. Dreassi). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.06.011
Tomatoes are important dietary sources of carotenoids, especially lycopene. The lipophilic fractions of these vegetables are a mixture of components including lycopene and other minor compounds such as b-carotene, lutein and a-tocopherol (Abushita, Daood, & Biacs, 2000;Abushita, Hebshi, Daood, & Biacs, 1997; Leonardi et al., 2000). Tomatoes are considered functional foods because of their high contents of physiologically active compounds. The antioxidant properties of single carotenoids and a-tocopherol were tested in many studies by using different approaches. However, limited information is available on the antioxidant properties of these compounds when they are combined in synthetic mixtures and contradictory data have been presented about synergistic effects. Carotenoid mixtures were shown to be more effective than the individual compounds in TBARS assay and the synergistic effects were particularly evident for mixtures containing lycopene or lutein (Stahl et al., 1998). Synergistic effects were also observed for lycopene-a-tocopherol combinations by using different experimental approaches such as LAME and AVMN model systems (Shi et al., 2007) or the inhibition of the LDL oxidation (Fuhrman, Volkova, Rosenblat, & Aviram, 2000). Other authors tested the scavenging activity of lycopene-a-tocopherol-bcarotene mixtures on the DPPH free radical and observed synergistic effects for lycopene-b-carotene and lycopene-a-tocopherol mixtures (Liu, Shi, Colina Ibarra, Kakuda, & Jun Xue, 2008). No synergism was observed for mixtures containing the same compounds when the percent inhibition of spontaneous oxidation
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in rat brain homogenate was used as model (Castro, Moraes Barros, Lanfer Marquez, Montizuki, & Higashi Sawada, 2005). Several studies on the antioxidant properties of the single compounds have been performed by using different experimental models. A recent review summarized the complex aspects of antioxidant reactions and analyzed the chemical principles of antioxidant capacity assays (Huang, Ou, & Prior, 2005). The mechanisms implicated in the interactions among antioxidant compounds have not been completely clarified. It seemed that the type of compounds, the concentration and the ratio at which they are mixed are important variables in defining the antioxidant capacity. A mechanism has been proposed to explain the interactions between carotenoids and a-tocopherol (Bo¨hm, Edge, Land, McGarvery, & Truscott, 1997). In this case the synergistic effects have been shown as a consequence of the transfer of electrons from the carotenoid to the a-tocopherolxyl radical to regenerate a-tocopherol. Similar considerations have been suggested to explain the interactions between vitamin C, carotenoids and a-tocopherol (Bo¨hm et al., 1997; Niki, Noguchi, Tsuchihashi, & Gotoh, 1995). We needed to investigate the interactions between carotenoids and a-tocopherol because only few and contradictory data are available. Moreover, the need to study and to clarify these effects is strongly associated with the emerging use of nutritional supplements which contain mixtures of two or more antioxidant compounds. The aims of the present work were (1) to evaluate the antioxidant activity of tomato lipophilic extracts and to establish a correlation coefficient between the antioxidant capacity and the content of single compounds such as lycopene, b-carotene, lutein and a-tocopherol; (2) to assay the antioxidant activity of a synthetic mixture which simulated the tomato lipophilic composition; (3) to test synthetic mixtures in which the compounds were mixed at different concentration levels, with the aim to verify if synergistic effects occurred. To measure the antioxidant activity, we used the ABTS assay because no papers are available on synergistic interactions between carotenoids and a-tocopherol by using this experimental approach. 2. Material and methods 2.1. Reagents and standards All solvents used were of HPLC grade from BHD (Poole, England).
b-carotene and lycopene standards were produced by Sigma (St. Louis, USA); D-a-tocopherol and lutein standards were from ICN Biochemical Inc. (Ohio, USA). Ammonium persulfate was from Merk & Co. Inc. (Darmstadt, Germany) and 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) (ABTS) in the diammonium salt form was produced by Fluka Chemie (Buchs, Switzerland). Trolox (6 hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) was from Hoffman La Roche Aldrich Chem. Co. (Saint Louis, MO, USA). 2.2. Tomato sampling and sample preparation The samples analyzed in the present study were from three different tomato varieties: cluster type (cv. Ikram F1), cherry type (cv. Naomi F1) and salad type (cv. Eroe F1). Naomi is a cherry type tomato with small and red skin fruits (approximately 8–12 g in weight); Ikram is a tomato variety with fruits larger than a cherry tomato, normally commercialized at full ripeness (approximately 80–100 g in weight); Eroe is a salad variety with fruits of mediumlarge size (8–10 cm diameter range and 150–200 g in weight). Tomatoes were purchased in a local supermarket at commercial maturity. On the day of purchase, the samples were homogenized (IKA Labortechnik, model T25 basic) and lyophilized for 24 h
(Freeze Dryer Modulyo, Edwards equipped with a Motors BS 5000– 11 pump, Edwards, England). The lyophilized samples were then stored at 20 C until their analysis. 2.3. Quantitative analysis of tomato lipophilic extracts 2.3.1. Carotenoids extraction Carotenoids were extracted using a procedure previously published (Setiawan, Sulaeman, Giraud, & Driskell, 2001) with small variations. A sample of 275 mg of homogenized freeze-dried tomatoes was extracted using 10 ml THF in presence of 0.01% butylated hydroxytoluene (BHT) and then centrifuged at 3000 rpm for 10 min. This extraction was performed twice until the pellet became colourless. The organic fractions were collected and evaporated to dryness under nitrogen. The residue was dissolved in 3 ml of chloroform and appropriately diluted with the mobile phase mixture (methanol: acetonitrile: dichloromethane 50:48:2); 1 ml was filtered (0.45 mm Minisart SRP 4 filter, Sartorius, Germany) and analyzed by using HPLC. Three replications were carried out to examine each sample. 2.3.2. -a-tocopherol extraction a-tocopherol was extracted using base hydrolysis of 275 mg lyophilized tomatoes followed by the addition of 10 ml of C2H4Cl2 (Raffo, La Malfa, Fogliano, Maiani, & Quaglia, 2006). The organic fractions were collected, evaporated to dryness under nitrogen and the remaining residue was resuspended in methanol and diluted with the mobile phase mixture (methanol:acetonitrile:dichloromethane 50:48:2). The HPLC analysis was performed to quantify a-tocopherol contents in tomato extracts. For each sample, three replications were carried out. 2.3.3. HPLC analyses LC 410 Series Perkin-Elmer apparatus (Norwalk, Connecticut, USA) equipped with a UV/VIS LC295 Perkin-Elmer detector and with 1022 Plus integrator Perkin-Elmer was used. The column was a reversed-phase LiChrospher 100 RP 18 (5 mm, 125 4.6 mm) Merck. Elution was carried out using a mixture of methanol:acetonitrile:dichloromethane (50:48:2) at a flow rate of 1.0 ml/min and the run time was 35 min (Saleh & Tan, 1991). UV–VIS detector was set at 290 nm for the simultaneous detection of all investigated compounds. The quantitative analysis of lycopene, b-carotene, lutein and a-tocopherol was based on an external standard method. 2.4. Determination of antioxidant activity 2.4.1. Tomato lipophilic extracts The extraction was carried out using a method described in a previous work (Raffo et al., 2006). A sample of 275 mg of homogenized freeze-dried tomatoes was extracted with 10 ml of CH2Cl2 and then centrifuged at 3000 rpm for 10 min. The extraction was performed twice and the supernatant fractions were collected and evaporated to dryness under nitrogen. The residue was dissolved in 3 ml of CH2Cl2 and analyzed. The antioxidant activity was measured using ABTS radical cation (ABTS.þ) decolorization assay (Pellegrini, Re, Yang, & Rice-Evans, 1999). In brief, 1 ml of the ABTS.þ solution was added to different volumes of the lipophilic extract (20, 40 or 60 ml) and diluted at a final volume of 2 ml using ethanol. The solution was vortexed for 10 s and the decolourization produced by the presence of antioxidants was measured at 751 nm (UV/Visible Lambda 2 spectrophotometer, Perkin-Elmer, Norwalk, Connecticut, USA), 10 min after initial mixing. Trolox was used to prepare the standard curve and
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the activity was reported as equivalent millimolar Trolox (mM TEAC/100 g of fresh weight). 2.4.2. Single compounds and synthetic mixtures Single compounds (ten different concentrations analyzed in triplicate) and synthetic mixtures were analyzed using the ABTS assay as previously described for tomato extracts. A four component mixture which simulated tomato composition was prepared at the following concentrations: lycopene 3.155 mM, b-carotene 0.230 mM, lutein 0.007 mM, a-tocopherol 0.380 mM. The same components were also combined in two or four component synthetic mixtures as reported in Table 1.
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calculated as the ratio between the experimental antioxidant capacity and the theoretical antioxidant capacity. The synergistic action was shown if the ratio SE ¼ EAC/TAC was greater than 1. 2.6. Statistical analysis All analyses were run in triplicates and results were expressed with the standard deviation of the means. Statistical analyses were performed with Statgraphics plus 4.1. software package. 3. Results and discussion
2.5. Calculation of synergistic effects
3.1. Antioxidant activity of tomato extracts
For each mixture a theoretical antioxidant capacity (TAC) was calculated and compared with the experimental antioxidant capacity (EAC). The theoretical antioxidant capacity was calculated by using Fuhrman’s equation (Fuhrman et al., 2000):
It is well known that tomato is the main dietary source of lycopene and is one of the most frequently consumed vegetables in the Mediterranean area. In the present study we carried out a quantitative analysis of lycopene, b-carotene, lutein and a-tocopherol in tomato lipophilic extracts. The antioxidant activity of the same lipophilic extracts was also assayed. We analyzed three commercial varieties (cluster type, cherry type and salad type) that were sampled at the ripening stage at which they are normally used for culinary purposes: incomplete ripeness for salad type (greenorange skin color) and full ripeness for cluster type and cherry type (red skin color). Quantitative data on tomato composition are presented in Table 2. As previously reported in other works (Abushita et al., 2000, 1997; Leonardi et al., 2000), lycopene was the most abundant component ranging from 850 mg/100 g (recorded for salad tomatoes) to 11,270 mg/100 g of fresh weight (recorded for cherry tomatoes). The antioxidant activity of tomato lipophilic extracts was also assayed. Table 2 shows the TEAC values (mM Trolox/100 g) measured for each investigated variety. Cherry type tomatoes, characterized by a high carotenoid contents, showed the highest antioxidant activity. Similar results were obtained in previous studies which performed similar investigations (Raffo et al., 2006, 2002). A Pearson correlation was calculated to establish the relationships between the individual parameters and to ascertain their relative significance in determining antioxidant activity. This investigation was performed using the quantitative data obtained from the analysis of 40 tomato samples (tomatoes from the same varieties, sampled during a period of three months and analyzed in triplicate). Table 3 shows Pearson correlations between each pair of variables. The statistical significance of the estimated correlation was also reported. P-values below 0.05 (statistically significant correlations at the 95% confidence level) were found for the following pairs of variables: b-carotene and lycopene, b-carotene and lutein, b-carotene and a-tocopherol, lycopene and a-tocopherol. The correlation between the antioxidant content (independent variable) and the TEAC value (dependent variable) was also estimated (data from the analysis of 40 tomato samples as previously
EAC ¼
Absblank Abssample
. Absblank 100
TAC ¼ ðEAC1 þEAC2 Þ ðEAC1 EAC2 =100Þ where EAC1 and EAC2 represents the inhibition effect of antioxidants 1 and 2 respectively. The synergistic effects (SEs) were Table 1 Two and four-component mixtures (contents are expressed as mM). Mixtures
Lycopene
b-Carotene
Lutein
a-Tocopherol
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41
1.00 2.30 1.00 2.30 1.00 2.30 1.00 2.30 1.00 2.30 1.00 2.30 – – – – – – – – – – – – 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.30 2.30 2.30 2.30 2.30 2.30 2.30 2.30 1.65
2.50 5.00 5.00 2.50 – – – – – – – – – – – – 2.50 5.00 2.50 5.00 2.50 5.00 2.50 5.00 2.5 2.5 2.5 5.00 2.5 5.00 5.00 5.00 2.5 5.00 2.5 2.5 5.00 5.00 5.00 2.5 3.75
– – – – 1.90 3.70 3.70 1.90 – – – – 1.90 3.70 1.90 3.70 – – – – 1.90 3.70 3.70 1.90 1.90 3.70 1.90 1.90 3.70 1.90 3.70 3.70 1.90 1.90 3.70 1.90 3.70 3.70 1.90 3.70 2.80
– – – – – – – – 3.50 7.00 7.00 3.50 3.50 7.00 7.00 3.50 3.50 3.50 7.00 3.50 – – – – 3.50 3.50 7.00 3.50 7.00 7.00 3.50 7.00 3.50 3.50 3.50 7.00 7.00 3.50 7.00 7.00 5.25
Table 2 Carotenoid and a–tocopherol contents (mg/100 g of fresh tomato) and TEAC values obtained from the analysis of five samples analyzed in triplicate (Mean values Standard Deviation). Compound
Lutein Lycopene b-Carotene a-Tocopherol TEAC (mM Trolox/100 g)
Tomato varieties Salad
Cluster
Cherry
16 1.4 850 153 420 90 620 37 0.009 0.002
20 1.1 8470 217 620 22 820 39 0.021 0.002
21 1.3 11,270 682 710 12 740 41 0.028 0.004
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Table 3 Pearson correlations between each pair of variables (significant correlations are in bold). Variable
Lutein
Lycopene
b-Carotene
a-Tocopherol
Lutein Lycopene b-Carotene a-Tocopherol
– 0.2787 P-value ¼ 0.0739 0.4300 P-value ¼ 0.0045 0.2390 P-value ¼ 0.1274
– 0.8425 P-value ¼ 0.0000 0.5214 P-value ¼ 0.0004
– 0.5053 P-value ¼ 0.0006
–
described). Fig. 1 shows the correlation between TEAC (mM) and the antioxidant contents. The highest correlation coefficient (R2 ¼ 0.9236, P 0.001) was found for lycopene, indicating a relatively strong and significant relationship between the variables. A significant and moderately strong relationship was found between antioxidant capacity and b-carotene content (R2 ¼ 0.7303, P 0.001), and between antioxidant activity and a-tocopherol content (R2 ¼ 0.5732, P 0.001). A relatively weak but significant relationship was found between antioxidant activity and lutein content (R2 ¼ 0.3253, P 0.05). Similar correlation coefficients were observed in a previous study (Raffo et al., 2002), although no significant correlation was found for a-tocopherol. 3.2. Antioxidant activity of synthetic mixtures In the present study the ABTS.þ radical cation assay was performed to test the antioxidant activity. This assay is largely used in food analysis because easy to perform, not subject to pH variations and useful to analyze both hydrophilic and lipophilic compounds. In a first time, the antioxidant activities of single compounds were tested. We obtained the following mean TEAC (mM) values S.D.: lycopene 2.89 0.18, b-carotene 1.85 0.16, lutein 1.40 0.16, a-tocopherol 1.05 0.12. The results confirmed that lycopene scavenged the ABTS.þ radical more effectively than the other investigated compounds. As previously reported in other studies (Miller, Sampson, Candeias, Bramley, & Rice-Evans, 1996), the antioxidant ability follows the order: lycopene > b-carotene > lutein > a-tocopherol. We analyzed a mixture which simulated tomato lipophilic extracts. This one was a synthetic mixture in which the components were combined at the concentration levels at which they were
naturally mixed in tomato lipophilic fractions: a high lycopene content (more than the 83% of the total content) combined with low contents of the other compounds. The analysis of this mixture showed that TEAC values (mean value 10.12 0.52 mM) fundamentally resulted from lycopene, as consequence of its high content and of its relatively high antioxidant activity. The other compounds were present at low concentration ant their contribute to the antioxidant activity was less than the 10%. In order to investigate if synergistic interactions occurred when carotenoids and a-tocopherol were mixed, we analyzed two and four component mixtures in which the compounds were combined as reported in Table 1. Concentrations of the single compounds were chosen so to provide partial reduction of ABTS.þ. Consequently, the mixtures were prepared considering the antioxidant ability of each compound, so that TEAC values ranging from 2.9 (lycopene 1 mM) to 7.4 mM (a-tocopherol 7 mM). To verify if synergistic effects happened, the theoretical antioxidant capacity (TAC) was calculated and compared with the experimental antioxidant capacity (EAC). If the EAC was greater than the TAC value (EAC/TAC > 1), a synergistic interaction occurred. Table 4 presents the results obtained from the analysis of two-component mixtures. The results suggest interesting considerations about the interactions between a-tocopherol and carotenoids. The SEs were greater than 1 and statistically significant (P 0.05) for all the combinations in which a-tocopherol was mixed with b-carotene (M17–M20). Similar synergistic effects were detected in a concentration-depended manner for a-tocopherollycopene mixtures (M9–M12) and were not statistically significant for M9 and M10 mixtures. The highest SE values were detected for M11 and M12 that were found to be the most efficient mixtures
Fig. 1. Correlation between TEAC (mM) and the antioxidant contents (lycopene, b-carotene, lutein, a-tocopherol mg/100 g). The lines in scatter plot indicate regression lines, 95% confidence interval and 95% prediction interval.
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Table 5 EAC and SE values for four component mixtures.
Table 4 EAC and SE values for two component mixtures. Mixtures
EACa
SEb
P-valuec
Mixtures
EACa
SEb
P-valuec
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24
6.02 0.14 13.24 0.39 9.74 0.17 10.54 0.22 3.98 0.10 12.72 0.36 7.36 0.14 9.06 0.20 6.48 0.12 13.68 0.48 11.63 0.26 11.14 0.36 5.97 0.12 12.43 0.68 10.02 0.51 7.68 0.19 7.73 0.19 15.14 0.39 11.77 0.27 11.14 0.26 5.87 0.13 11.62 0.26 8.95 0.11 9.57 0.20
0.94 0.99 0.99 1.05 0.72 1.11 0.93 0.99 1.01 1.01 1.15 1.12 0.97 1.02 1.01 0.90 1.09 1.08 1.09 1.06 0.95 0.97 1.04 0.99
0.05 ns ns 0.05 0.001 0.05 0.01 ns ns ns 0.01 0.05 ns ns ns ns 0.05 0.05 0.05 0.05 0.05 ns 0.05 ns
M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41
9.93 0.51 12.29 0.62 15.01 1.10 14.62 0.51 17.72 1.12 18.23 0.82 16.24 0.85 20.71 1.29 15.22 0.69 17.86 0.99 18.36 0.44 19.79 0.62 21.74 1.47 20.08 0.88 21.76 1.02 21.90 0.81 19.30 0.86
0.81 0.85 0.96 0.95 0.99 0.97 0.93 0.99 0.98 0.96 1.04 1.04 0.91 0.97 0.99 1.04 1.03
0.01 0.05 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
a
EAC values are reported as mean (n ¼ 3) S.D. b For SE > 1 synergistic effect found. c The significance of the observed differences is reported as P-values; ns: no statistically significant.
a
EAC values are reported as mean (n ¼ 3) S.D. For SE > 1 synergistic effect found. c The significance of the observed differences is reported as P-values; ns: no statistically significant. b
when these components are combined in a two-component mixture (same concentration levels for each compound) is removed by the addition of other antioxidants. 4. Conclusions
tested in the present study. The observed synergistic interactions between a-tocopherol and lutein were not statistically significant. Other authors observed the synergistic effects for lycopene-atocopherol mixtures by using different experimental approaches (Fuhrman et al., 2000; Liu et al., 2008; Shi et al., 2007). Synergistic effects for a-tocopherol-b-carotene mixtures were not previously observed (Liu et al., 2008; Shi et al., 2007). Experimental data by using the same assay are not available, so that it is not possible to do a direct comparison. The analysis of carotenoid mixtures indicated that synergistic effects occurred for lycopene-b-carotene, lycopene-lutein and lutein-b-carotene mixtures. The EAC/TAC ratios were greater than 1 for M4, M6, M23 (Table 4) and the SEs were found to be statistically significant at the 95% confidence level (P 0.05). The highest SE value was detected for M6 (lycopene-lutein), the most efficient carotenoid mixture in scavenging the ABTS.þ radical cation. As the results suggested, the concentration of the single compounds had an effect on SE: significant synergistic effects were detected for the mixtures which contained lycopene (M4 and M6) and lutein (M6 and M23) at high concentrations and for that in which b-carotene (M4 and M23) was present at the minimum concentration level. Contradictory data on carotenoids interactions have been reported in previous studies. Lycopene-b-carotene mixtures showed synergistic effects in a work in which the free radical DPPH method was used (Liu et al., 2008), while synergism was not observed using LAME and AMVN model systems (Shi et al., 2007). Synergism was also found measuring the lipid peroxidation in multilamellar liposomes (Stahl et al., 1998): significant synergistic effects were detected for lycopene-b-carotene, lycopene-lutein (the most efficient mixture) and lutein-b-carotene mixtures. Table 5 shows the results obtained from the analysis of fourcomponent mixtures. M35, M36, M40 and M41 showed synergistic effects which were not statistically significant. M36 and M40 were combinations of various two-component mixtures for which significant SEs were detected. Consequently, it seemed that the antioxidant activity was reduced when the four components were mixed together In other words, the synergistic effect detected
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Raffo, A., Leonardi, C., Fogliano, V., Ambrosino, P., Salucci, M., Gennaro, L., et al. (2002). Nutritional value of cherry tomatoes (Lycopersicon esculentum Cv. Naomi F1) harvested at different ripening stages. Journal of Agricultural and Food Chemistry, 50(22), 6550–6556. Rao, A. V., & Agarwal, S. (1999). Role of lycopene as antioxidant carotenoid in the prevention of chronic diseases: a review. Nutritional Research, 19(2), 305–323. Riboli, E., & Norat, T. (2003). Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. American Journal for Clinical Nutrition, 78(3), 559S–569S. Saleh, M. H., & Tan, B. (1991). Separation and identification of cis/trans carotenoid isomers. Journal of Agricultural and Food Chemistry, 39(8), 1438–1443. Setiawan, B., Sulaeman, A., Giraud, D. W., & Driskell, J. A. (2001). Carotenoid content of selected Indonesian fruits. Journal of Food Composition and Analysis, 14(2), 169–176. Shi, J., Qu, Q., Kakuda, Y., Jun Xue, S., Jiang, Y., Koide, S., et al. (2007). Investigation of the antioxidant and synergistic activity of lycopene and other natural antioxidants using LAME and AMVN model systems. Journal of Food Composition and Analysis, 20, 603–608. Stahl, W., Junghans, A., De Boer, B., Driomina, E. S., Briviba, K., & Sies, H. (1998). Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein. FEBS Letters, 427, 305–308. Steinmetz, K. A., & Potter, J. D. (1996). Vegetables, fruit and cancer prevention. A review. Journal of American Dietetic Association, 96(10), 1027–1039. Sundquist, A. R., Briviba, K., & Sies, H. (1994). Singlet oxygen quenching by carotene. Methods in Enzymology, 234, 384–388. Ziegler, R. G. (1991). Vegetables, fruits, and carotenoids and the risk of cancer. American Journal of Clinical Nutrition, 53, 251S–259S.
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Antioxidant activity of tomato lipophilic extracts and interactions between carotenoids and a-tocopherol in synthetic mixtures Assunta Zanfini, Gianfranco Corbini, Caterina La Rosa, Elena Dreassi* Dipartimento Farmaco Chimico Tecnologico, Via Aldo Moro, 53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 November 2008 Received in revised form 15 May 2009 Accepted 16 June 2009
In the present study we assayed the antioxidant activity of lipophilic extracts obtained from different tomato varieties. The results showed that cherry tomatoes, characterized by a high carotenoid content, had the highest antioxidant activity. A quantitative analysis of lycopene, b-carotene, lutein and a-tocopherol was also performed and the correlation between the antioxidant content and the antioxidant activity was estimated. The highest correlation coefficient was found for lycopene (R2 ¼ 0.9236, P 0.001). The analysis of two-component mixtures containing a-tocopherol and carotenoids showed that significant synergism occurred for all the combinations which contained a-tocopherol and b-carotene mixed together. The highest synergistic effects were detected for a-tocopherol-lycopene mixtures, which were the most efficient combinations tested in the present study. The analysis of the carotenoid combinations indicated that synergism occurred for lycopene-b-carotene, lycopene-lutein and lutein-b-carotene mixtures. The analysis of four-component mixtures did not show statistically significant synergistic effects. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids a-Tocopherol Antioxidant activity Synergistic effect Synthetic mixtures
1. Introduction The great interest in studying antioxidant compounds is due to their ability to neutralize active oxygen species and free radicals that play an important role in the pathogenesis of such degenerative diseases. A large number of epidemiological and clinical studies have associated a lower incidence of some cancer types (Riboli & Norat, 2003; Steinmetz & Potter, 1996), cardiovascular diseases (Joshipura et al., 2001; Ness & Powles, 1997) etc with a high antioxidant dietary intake. The positive correlation between vegetable intake and cancer prevention has been attributed to the presence of antioxidant compounds (Machlin, 1995; Ziegler, 1991). Many studies have shown the beneficial effects produced by carotenoids and a-tocopherol on human health. Their biological roles have been described in many papers. The role of different carotenoids in the prevention of degenerative diseases has been attributed to their antioxidant properties (Conn, Schlach, & Truscott, 1991; Di Mascio, Kaiser, & Sies, 1989; Landrum & Bone, 2001; Rao & Agarwal, 1999; Sundquist, Briviba, & Sies, 1994). Other studies have described a-tocopherol (vitamin E) as one of the most important lipid-soluble radical scavenging antioxidant in membranes and in plasma (Burton, Joyce, & Ingold, 1983).
* Corresponding author. Tel.: þ39 0577 234321; fax: þ39 0577 234333. E-mail address: [email protected] (E. Dreassi). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.06.011
Tomatoes are important dietary sources of carotenoids, especially lycopene. The lipophilic fractions of these vegetables are a mixture of components including lycopene and other minor compounds such as b-carotene, lutein and a-tocopherol (Abushita, Daood, & Biacs, 2000;Abushita, Hebshi, Daood, & Biacs, 1997; Leonardi et al., 2000). Tomatoes are considered functional foods because of their high contents of physiologically active compounds. The antioxidant properties of single carotenoids and a-tocopherol were tested in many studies by using different approaches. However, limited information is available on the antioxidant properties of these compounds when they are combined in synthetic mixtures and contradictory data have been presented about synergistic effects. Carotenoid mixtures were shown to be more effective than the individual compounds in TBARS assay and the synergistic effects were particularly evident for mixtures containing lycopene or lutein (Stahl et al., 1998). Synergistic effects were also observed for lycopene-a-tocopherol combinations by using different experimental approaches such as LAME and AVMN model systems (Shi et al., 2007) or the inhibition of the LDL oxidation (Fuhrman, Volkova, Rosenblat, & Aviram, 2000). Other authors tested the scavenging activity of lycopene-a-tocopherol-bcarotene mixtures on the DPPH free radical and observed synergistic effects for lycopene-b-carotene and lycopene-a-tocopherol mixtures (Liu, Shi, Colina Ibarra, Kakuda, & Jun Xue, 2008). No synergism was observed for mixtures containing the same compounds when the percent inhibition of spontaneous oxidation
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in rat brain homogenate was used as model (Castro, Moraes Barros, Lanfer Marquez, Montizuki, & Higashi Sawada, 2005). Several studies on the antioxidant properties of the single compounds have been performed by using different experimental models. A recent review summarized the complex aspects of antioxidant reactions and analyzed the chemical principles of antioxidant capacity assays (Huang, Ou, & Prior, 2005). The mechanisms implicated in the interactions among antioxidant compounds have not been completely clarified. It seemed that the type of compounds, the concentration and the ratio at which they are mixed are important variables in defining the antioxidant capacity. A mechanism has been proposed to explain the interactions between carotenoids and a-tocopherol (Bo¨hm, Edge, Land, McGarvery, & Truscott, 1997). In this case the synergistic effects have been shown as a consequence of the transfer of electrons from the carotenoid to the a-tocopherolxyl radical to regenerate a-tocopherol. Similar considerations have been suggested to explain the interactions between vitamin C, carotenoids and a-tocopherol (Bo¨hm et al., 1997; Niki, Noguchi, Tsuchihashi, & Gotoh, 1995). We needed to investigate the interactions between carotenoids and a-tocopherol because only few and contradictory data are available. Moreover, the need to study and to clarify these effects is strongly associated with the emerging use of nutritional supplements which contain mixtures of two or more antioxidant compounds. The aims of the present work were (1) to evaluate the antioxidant activity of tomato lipophilic extracts and to establish a correlation coefficient between the antioxidant capacity and the content of single compounds such as lycopene, b-carotene, lutein and a-tocopherol; (2) to assay the antioxidant activity of a synthetic mixture which simulated the tomato lipophilic composition; (3) to test synthetic mixtures in which the compounds were mixed at different concentration levels, with the aim to verify if synergistic effects occurred. To measure the antioxidant activity, we used the ABTS assay because no papers are available on synergistic interactions between carotenoids and a-tocopherol by using this experimental approach. 2. Material and methods 2.1. Reagents and standards All solvents used were of HPLC grade from BHD (Poole, England).
b-carotene and lycopene standards were produced by Sigma (St. Louis, USA); D-a-tocopherol and lutein standards were from ICN Biochemical Inc. (Ohio, USA). Ammonium persulfate was from Merk & Co. Inc. (Darmstadt, Germany) and 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) (ABTS) in the diammonium salt form was produced by Fluka Chemie (Buchs, Switzerland). Trolox (6 hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) was from Hoffman La Roche Aldrich Chem. Co. (Saint Louis, MO, USA). 2.2. Tomato sampling and sample preparation The samples analyzed in the present study were from three different tomato varieties: cluster type (cv. Ikram F1), cherry type (cv. Naomi F1) and salad type (cv. Eroe F1). Naomi is a cherry type tomato with small and red skin fruits (approximately 8–12 g in weight); Ikram is a tomato variety with fruits larger than a cherry tomato, normally commercialized at full ripeness (approximately 80–100 g in weight); Eroe is a salad variety with fruits of mediumlarge size (8–10 cm diameter range and 150–200 g in weight). Tomatoes were purchased in a local supermarket at commercial maturity. On the day of purchase, the samples were homogenized (IKA Labortechnik, model T25 basic) and lyophilized for 24 h
(Freeze Dryer Modulyo, Edwards equipped with a Motors BS 5000– 11 pump, Edwards, England). The lyophilized samples were then stored at 20 C until their analysis. 2.3. Quantitative analysis of tomato lipophilic extracts 2.3.1. Carotenoids extraction Carotenoids were extracted using a procedure previously published (Setiawan, Sulaeman, Giraud, & Driskell, 2001) with small variations. A sample of 275 mg of homogenized freeze-dried tomatoes was extracted using 10 ml THF in presence of 0.01% butylated hydroxytoluene (BHT) and then centrifuged at 3000 rpm for 10 min. This extraction was performed twice until the pellet became colourless. The organic fractions were collected and evaporated to dryness under nitrogen. The residue was dissolved in 3 ml of chloroform and appropriately diluted with the mobile phase mixture (methanol: acetonitrile: dichloromethane 50:48:2); 1 ml was filtered (0.45 mm Minisart SRP 4 filter, Sartorius, Germany) and analyzed by using HPLC. Three replications were carried out to examine each sample. 2.3.2. -a-tocopherol extraction a-tocopherol was extracted using base hydrolysis of 275 mg lyophilized tomatoes followed by the addition of 10 ml of C2H4Cl2 (Raffo, La Malfa, Fogliano, Maiani, & Quaglia, 2006). The organic fractions were collected, evaporated to dryness under nitrogen and the remaining residue was resuspended in methanol and diluted with the mobile phase mixture (methanol:acetonitrile:dichloromethane 50:48:2). The HPLC analysis was performed to quantify a-tocopherol contents in tomato extracts. For each sample, three replications were carried out. 2.3.3. HPLC analyses LC 410 Series Perkin-Elmer apparatus (Norwalk, Connecticut, USA) equipped with a UV/VIS LC295 Perkin-Elmer detector and with 1022 Plus integrator Perkin-Elmer was used. The column was a reversed-phase LiChrospher 100 RP 18 (5 mm, 125 4.6 mm) Merck. Elution was carried out using a mixture of methanol:acetonitrile:dichloromethane (50:48:2) at a flow rate of 1.0 ml/min and the run time was 35 min (Saleh & Tan, 1991). UV–VIS detector was set at 290 nm for the simultaneous detection of all investigated compounds. The quantitative analysis of lycopene, b-carotene, lutein and a-tocopherol was based on an external standard method. 2.4. Determination of antioxidant activity 2.4.1. Tomato lipophilic extracts The extraction was carried out using a method described in a previous work (Raffo et al., 2006). A sample of 275 mg of homogenized freeze-dried tomatoes was extracted with 10 ml of CH2Cl2 and then centrifuged at 3000 rpm for 10 min. The extraction was performed twice and the supernatant fractions were collected and evaporated to dryness under nitrogen. The residue was dissolved in 3 ml of CH2Cl2 and analyzed. The antioxidant activity was measured using ABTS radical cation (ABTS.þ) decolorization assay (Pellegrini, Re, Yang, & Rice-Evans, 1999). In brief, 1 ml of the ABTS.þ solution was added to different volumes of the lipophilic extract (20, 40 or 60 ml) and diluted at a final volume of 2 ml using ethanol. The solution was vortexed for 10 s and the decolourization produced by the presence of antioxidants was measured at 751 nm (UV/Visible Lambda 2 spectrophotometer, Perkin-Elmer, Norwalk, Connecticut, USA), 10 min after initial mixing. Trolox was used to prepare the standard curve and
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the activity was reported as equivalent millimolar Trolox (mM TEAC/100 g of fresh weight). 2.4.2. Single compounds and synthetic mixtures Single compounds (ten different concentrations analyzed in triplicate) and synthetic mixtures were analyzed using the ABTS assay as previously described for tomato extracts. A four component mixture which simulated tomato composition was prepared at the following concentrations: lycopene 3.155 mM, b-carotene 0.230 mM, lutein 0.007 mM, a-tocopherol 0.380 mM. The same components were also combined in two or four component synthetic mixtures as reported in Table 1.
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calculated as the ratio between the experimental antioxidant capacity and the theoretical antioxidant capacity. The synergistic action was shown if the ratio SE ¼ EAC/TAC was greater than 1. 2.6. Statistical analysis All analyses were run in triplicates and results were expressed with the standard deviation of the means. Statistical analyses were performed with Statgraphics plus 4.1. software package. 3. Results and discussion
2.5. Calculation of synergistic effects
3.1. Antioxidant activity of tomato extracts
For each mixture a theoretical antioxidant capacity (TAC) was calculated and compared with the experimental antioxidant capacity (EAC). The theoretical antioxidant capacity was calculated by using Fuhrman’s equation (Fuhrman et al., 2000):
It is well known that tomato is the main dietary source of lycopene and is one of the most frequently consumed vegetables in the Mediterranean area. In the present study we carried out a quantitative analysis of lycopene, b-carotene, lutein and a-tocopherol in tomato lipophilic extracts. The antioxidant activity of the same lipophilic extracts was also assayed. We analyzed three commercial varieties (cluster type, cherry type and salad type) that were sampled at the ripening stage at which they are normally used for culinary purposes: incomplete ripeness for salad type (greenorange skin color) and full ripeness for cluster type and cherry type (red skin color). Quantitative data on tomato composition are presented in Table 2. As previously reported in other works (Abushita et al., 2000, 1997; Leonardi et al., 2000), lycopene was the most abundant component ranging from 850 mg/100 g (recorded for salad tomatoes) to 11,270 mg/100 g of fresh weight (recorded for cherry tomatoes). The antioxidant activity of tomato lipophilic extracts was also assayed. Table 2 shows the TEAC values (mM Trolox/100 g) measured for each investigated variety. Cherry type tomatoes, characterized by a high carotenoid contents, showed the highest antioxidant activity. Similar results were obtained in previous studies which performed similar investigations (Raffo et al., 2006, 2002). A Pearson correlation was calculated to establish the relationships between the individual parameters and to ascertain their relative significance in determining antioxidant activity. This investigation was performed using the quantitative data obtained from the analysis of 40 tomato samples (tomatoes from the same varieties, sampled during a period of three months and analyzed in triplicate). Table 3 shows Pearson correlations between each pair of variables. The statistical significance of the estimated correlation was also reported. P-values below 0.05 (statistically significant correlations at the 95% confidence level) were found for the following pairs of variables: b-carotene and lycopene, b-carotene and lutein, b-carotene and a-tocopherol, lycopene and a-tocopherol. The correlation between the antioxidant content (independent variable) and the TEAC value (dependent variable) was also estimated (data from the analysis of 40 tomato samples as previously
EAC ¼
Absblank Abssample
. Absblank 100
TAC ¼ ðEAC1 þEAC2 Þ ðEAC1 EAC2 =100Þ where EAC1 and EAC2 represents the inhibition effect of antioxidants 1 and 2 respectively. The synergistic effects (SEs) were Table 1 Two and four-component mixtures (contents are expressed as mM). Mixtures
Lycopene
b-Carotene
Lutein
a-Tocopherol
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41
1.00 2.30 1.00 2.30 1.00 2.30 1.00 2.30 1.00 2.30 1.00 2.30 – – – – – – – – – – – – 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.30 2.30 2.30 2.30 2.30 2.30 2.30 2.30 1.65
2.50 5.00 5.00 2.50 – – – – – – – – – – – – 2.50 5.00 2.50 5.00 2.50 5.00 2.50 5.00 2.5 2.5 2.5 5.00 2.5 5.00 5.00 5.00 2.5 5.00 2.5 2.5 5.00 5.00 5.00 2.5 3.75
– – – – 1.90 3.70 3.70 1.90 – – – – 1.90 3.70 1.90 3.70 – – – – 1.90 3.70 3.70 1.90 1.90 3.70 1.90 1.90 3.70 1.90 3.70 3.70 1.90 1.90 3.70 1.90 3.70 3.70 1.90 3.70 2.80
– – – – – – – – 3.50 7.00 7.00 3.50 3.50 7.00 7.00 3.50 3.50 3.50 7.00 3.50 – – – – 3.50 3.50 7.00 3.50 7.00 7.00 3.50 7.00 3.50 3.50 3.50 7.00 7.00 3.50 7.00 7.00 5.25
Table 2 Carotenoid and a–tocopherol contents (mg/100 g of fresh tomato) and TEAC values obtained from the analysis of five samples analyzed in triplicate (Mean values Standard Deviation). Compound
Lutein Lycopene b-Carotene a-Tocopherol TEAC (mM Trolox/100 g)
Tomato varieties Salad
Cluster
Cherry
16 1.4 850 153 420 90 620 37 0.009 0.002
20 1.1 8470 217 620 22 820 39 0.021 0.002
21 1.3 11,270 682 710 12 740 41 0.028 0.004
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Table 3 Pearson correlations between each pair of variables (significant correlations are in bold). Variable
Lutein
Lycopene
b-Carotene
a-Tocopherol
Lutein Lycopene b-Carotene a-Tocopherol
– 0.2787 P-value ¼ 0.0739 0.4300 P-value ¼ 0.0045 0.2390 P-value ¼ 0.1274
– 0.8425 P-value ¼ 0.0000 0.5214 P-value ¼ 0.0004
– 0.5053 P-value ¼ 0.0006
–
described). Fig. 1 shows the correlation between TEAC (mM) and the antioxidant contents. The highest correlation coefficient (R2 ¼ 0.9236, P 0.001) was found for lycopene, indicating a relatively strong and significant relationship between the variables. A significant and moderately strong relationship was found between antioxidant capacity and b-carotene content (R2 ¼ 0.7303, P 0.001), and between antioxidant activity and a-tocopherol content (R2 ¼ 0.5732, P 0.001). A relatively weak but significant relationship was found between antioxidant activity and lutein content (R2 ¼ 0.3253, P 0.05). Similar correlation coefficients were observed in a previous study (Raffo et al., 2002), although no significant correlation was found for a-tocopherol. 3.2. Antioxidant activity of synthetic mixtures In the present study the ABTS.þ radical cation assay was performed to test the antioxidant activity. This assay is largely used in food analysis because easy to perform, not subject to pH variations and useful to analyze both hydrophilic and lipophilic compounds. In a first time, the antioxidant activities of single compounds were tested. We obtained the following mean TEAC (mM) values S.D.: lycopene 2.89 0.18, b-carotene 1.85 0.16, lutein 1.40 0.16, a-tocopherol 1.05 0.12. The results confirmed that lycopene scavenged the ABTS.þ radical more effectively than the other investigated compounds. As previously reported in other studies (Miller, Sampson, Candeias, Bramley, & Rice-Evans, 1996), the antioxidant ability follows the order: lycopene > b-carotene > lutein > a-tocopherol. We analyzed a mixture which simulated tomato lipophilic extracts. This one was a synthetic mixture in which the components were combined at the concentration levels at which they were
naturally mixed in tomato lipophilic fractions: a high lycopene content (more than the 83% of the total content) combined with low contents of the other compounds. The analysis of this mixture showed that TEAC values (mean value 10.12 0.52 mM) fundamentally resulted from lycopene, as consequence of its high content and of its relatively high antioxidant activity. The other compounds were present at low concentration ant their contribute to the antioxidant activity was less than the 10%. In order to investigate if synergistic interactions occurred when carotenoids and a-tocopherol were mixed, we analyzed two and four component mixtures in which the compounds were combined as reported in Table 1. Concentrations of the single compounds were chosen so to provide partial reduction of ABTS.þ. Consequently, the mixtures were prepared considering the antioxidant ability of each compound, so that TEAC values ranging from 2.9 (lycopene 1 mM) to 7.4 mM (a-tocopherol 7 mM). To verify if synergistic effects happened, the theoretical antioxidant capacity (TAC) was calculated and compared with the experimental antioxidant capacity (EAC). If the EAC was greater than the TAC value (EAC/TAC > 1), a synergistic interaction occurred. Table 4 presents the results obtained from the analysis of two-component mixtures. The results suggest interesting considerations about the interactions between a-tocopherol and carotenoids. The SEs were greater than 1 and statistically significant (P 0.05) for all the combinations in which a-tocopherol was mixed with b-carotene (M17–M20). Similar synergistic effects were detected in a concentration-depended manner for a-tocopherollycopene mixtures (M9–M12) and were not statistically significant for M9 and M10 mixtures. The highest SE values were detected for M11 and M12 that were found to be the most efficient mixtures
Fig. 1. Correlation between TEAC (mM) and the antioxidant contents (lycopene, b-carotene, lutein, a-tocopherol mg/100 g). The lines in scatter plot indicate regression lines, 95% confidence interval and 95% prediction interval.
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Table 5 EAC and SE values for four component mixtures.
Table 4 EAC and SE values for two component mixtures. Mixtures
EACa
SEb
P-valuec
Mixtures
EACa
SEb
P-valuec
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24
6.02 0.14 13.24 0.39 9.74 0.17 10.54 0.22 3.98 0.10 12.72 0.36 7.36 0.14 9.06 0.20 6.48 0.12 13.68 0.48 11.63 0.26 11.14 0.36 5.97 0.12 12.43 0.68 10.02 0.51 7.68 0.19 7.73 0.19 15.14 0.39 11.77 0.27 11.14 0.26 5.87 0.13 11.62 0.26 8.95 0.11 9.57 0.20
0.94 0.99 0.99 1.05 0.72 1.11 0.93 0.99 1.01 1.01 1.15 1.12 0.97 1.02 1.01 0.90 1.09 1.08 1.09 1.06 0.95 0.97 1.04 0.99
0.05 ns ns 0.05 0.001 0.05 0.01 ns ns ns 0.01 0.05 ns ns ns ns 0.05 0.05 0.05 0.05 0.05 ns 0.05 ns
M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41
9.93 0.51 12.29 0.62 15.01 1.10 14.62 0.51 17.72 1.12 18.23 0.82 16.24 0.85 20.71 1.29 15.22 0.69 17.86 0.99 18.36 0.44 19.79 0.62 21.74 1.47 20.08 0.88 21.76 1.02 21.90 0.81 19.30 0.86
0.81 0.85 0.96 0.95 0.99 0.97 0.93 0.99 0.98 0.96 1.04 1.04 0.91 0.97 0.99 1.04 1.03
0.01 0.05 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
a
EAC values are reported as mean (n ¼ 3) S.D. b For SE > 1 synergistic effect found. c The significance of the observed differences is reported as P-values; ns: no statistically significant.
a
EAC values are reported as mean (n ¼ 3) S.D. For SE > 1 synergistic effect found. c The significance of the observed differences is reported as P-values; ns: no statistically significant. b
when these components are combined in a two-component mixture (same concentration levels for each compound) is removed by the addition of other antioxidants. 4. Conclusions
tested in the present study. The observed synergistic interactions between a-tocopherol and lutein were not statistically significant. Other authors observed the synergistic effects for lycopene-atocopherol mixtures by using different experimental approaches (Fuhrman et al., 2000; Liu et al., 2008; Shi et al., 2007). Synergistic effects for a-tocopherol-b-carotene mixtures were not previously observed (Liu et al., 2008; Shi et al., 2007). Experimental data by using the same assay are not available, so that it is not possible to do a direct comparison. The analysis of carotenoid mixtures indicated that synergistic effects occurred for lycopene-b-carotene, lycopene-lutein and lutein-b-carotene mixtures. The EAC/TAC ratios were greater than 1 for M4, M6, M23 (Table 4) and the SEs were found to be statistically significant at the 95% confidence level (P 0.05). The highest SE value was detected for M6 (lycopene-lutein), the most efficient carotenoid mixture in scavenging the ABTS.þ radical cation. As the results suggested, the concentration of the single compounds had an effect on SE: significant synergistic effects were detected for the mixtures which contained lycopene (M4 and M6) and lutein (M6 and M23) at high concentrations and for that in which b-carotene (M4 and M23) was present at the minimum concentration level. Contradictory data on carotenoids interactions have been reported in previous studies. Lycopene-b-carotene mixtures showed synergistic effects in a work in which the free radical DPPH method was used (Liu et al., 2008), while synergism was not observed using LAME and AMVN model systems (Shi et al., 2007). Synergism was also found measuring the lipid peroxidation in multilamellar liposomes (Stahl et al., 1998): significant synergistic effects were detected for lycopene-b-carotene, lycopene-lutein (the most efficient mixture) and lutein-b-carotene mixtures. Table 5 shows the results obtained from the analysis of fourcomponent mixtures. M35, M36, M40 and M41 showed synergistic effects which were not statistically significant. M36 and M40 were combinations of various two-component mixtures for which significant SEs were detected. Consequently, it seemed that the antioxidant activity was reduced when the four components were mixed together In other words, the synergistic effect detected
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