- Email: [email protected]
UV-induced changes in antioxidant capacities of selected carotenoids toward lecithin in aqueous solution
ARTICLE IN PRESS
Radiation Physics and Chemistry 77 (2008) 34–41 www.elsevier.com/locate/radphyschem
UV-induced changes in antioxidant capacities of selected carotenoids toward lecithin in aqueous solution Dragan Cvetkovic, Dejan Markovic Faculty of Technology, University of Nish, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia Received 17 January 2007; accepted 21 February 2007
Abstract Antioxidant action of four selected carotenoids (two carotenes, b-carotene and lycopene, and two xanthophylls, lutein and neoxanthin) on UV-induced lecithin lipid peroxidation in aqueous solution has been studied by thiobarbituric acid (TBA) test. TBA test is based on absorbance measurements of complex formed between malondialdehyde, secondary product of lipid peroxidation and thiobarbituric acid, at 532 nm. The antioxidant capacities of investigated carotenoids appeared to be strongly affected by UV-action. High energy input of the involved UV-photons plays major governing role, though a certain impact of the carotenoid structures cannot be neglected. The results suggest a minor remained contribution of selected carotenoids to prevention of lecithin peroxidation in the studied system as a result of UV-irradiation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Carotenoids; Lipids; UV-irradiation; Peroxidation
1. Introduction The depletion of the ozone shield, caused by a huge release of atmospheric pollutants (such as chlorofluorocarbons, chlorocarbons and organobromides), leads to increase of biologically damaging UV-light at ambient levels (mainly UV-B light, 280–320 nm). UV-light can generally influence many crucial biologically important processes, such as DNA replication (Ichihashi et al., 2003; Pfeifer et al., 2005), photosynthesis (Teramura and Ziska, 1996; Strid et al., 1990), etc. Also, UV-light can generally influence whole human immune system (Schwarz, 1996; Vermer et al., 1996), by initiating a lot of harmful free radicals mediated processes, lipid peroxidation (LP) among them. Lipid peroxidation is tightly connected with many pathological processes which finish in some form of cancer at the very end (Young, 1996), melanoma skin cancer among them (Ouhtit and Ananthaswamy, 2001). Lipid Corresponding author.
E-mail addresses: [email protected], [email protected] (D. Markovic). 0969-806X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2007.02.078
peroxidation is either free radicals chain reaction (type I), or alternatively, it occurs through a non radical pathway, by direct reaction with singlet oxygen in the presence of a photosensitizer (Girotti, 2001; Wheatley, 2000; Markovic and Patterson, 1993; Mimica-Dukic, 1997; Burton and Ingold, 1984; Haila, 1999). Reactive oxygen species (ROS), like hydroxy radicals (OHd) or peroxy radicals (ROOd), are typical lipid peroxidation initiators. They can be created through a variety of chemical reactions (Mimica-Dukic, 1997), some of them including typical lipid radicals producers (Aikens and Dix, 1993; Ross et al., 1994; Tian Li et al., 2000). Additionally, they can be induced through a variety of external stresses (Girotti, 2001) implying very commonly an external radiation (Heijman et al., 1985; Erben-Russ et al., 1987), which, in case of UV-irradiaton may include photosensitisers in very different media (Markovic and Patterson, 1989, 1993; Markovic et al., 1990; Markovic, 2001). Lipid peroxidation is mostly controlled by antioxidants action in vivo. Many biomolecules (and classes of biomolecules) serve as antioxidants, like enzymes, tocopherols
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
(like vitamin E), L-ascorbic acid (vitamin C), retinol (vitamin A), thiamin and riboflamin (vitamin B), flavonoids, etc. (Saija et al., 1995; Deng et al., 1997; McBride and Kraemer, 1999; Van der Sluis et al., 2000; Choi et al., 2002; Heim et al., 2002). In recent years, carotenoids have received wide research interest as potential antioxidants. There are a number of studies reporting that higher consumption of carotenoids leads to lower risk of cancer and cardiovascular disease, and relating antioxidant action of carotenoids with their conjugated chemical structures, having multiple potential sites approachable for attack by ROS species (Burton and Ingold, 1984; Palozza and Krinsky, 1992; Simic, 1992; Woodall et al., 1997a; Krinsky and Yeum, 2003; Peng Lim et al., 1992; Frank and Cogdell, 1996). UV-irradiation certainly affects carotenoids antioxidant function in vivo (Teramura and Ziska, 1996), though the involved mechanisms are not elucidated yet. Therefore, UV-irradiation effects on antioxidant capacities of four plants photosynthetic accessory pigments (b-carotene, lycopene, lutein and neoxanthin) in vitro, in the presence of lipoidal target (lecithin), have been studied in this paper. Lipid peroxidation has been induced by UV-light from three different ranges (UV-A, UV-B and UV-C). The antioxidant activities of investigated carotenoids (and its dependence on UV-irradiation) have been monitored by thiobarbituric–malondialdehyde (TBA–MDA) test. 2. Experimental Pigments were isolated from plant material (b-carotene, lutein and neoxanthin from spinach leaves and lycopene from tomato fruits) purchased at the local market. Lecithin Epikuron 100 P, a mixture of phospholipids, was gifted from ‘‘ICN Galenika’’, Beograd. It was manufactured by ‘‘Degusa Texturant Systems’’, Hamburg, Germany. The lipid content was: phosphatidylethanolamine 18.0%, phosphatic acid 8.3%, phosphatidylinositol 14.1% and phosphatidylcholin 21.7%. The acid value, peroxide number and iodine number were periodically checked out and found to be true. The lecithin mixture was kept in dark to prevent the autooxidation process to some extent. However, autooxidation could not be annihilated, and it has been taken into account during calculation of lipid peroxidation yield. 2.1. Pigments extraction from spinach (Spinacia oleracea) The photosynthetic pigments were extracted from spinach leaves by using modified method proposed by Hynninen (1991) and Svec (1991). Fresh spinach leaves free of midribs (0.030 kg) were dropped into boiling water, which was quickly replaced (after 1–2 min) with cooled water. Hot water inactivates enzymes preventing the pigment alteration and permitting coagulation of proteins and water-soluble substances. After drying between paper towels, leaves are separated and placed in mixture of methanol (60 cm3) and 40–75 1C petroleum ether (30 cm3); the stuff was occasionally agitated in next 30 min.
35
Methanol removes water from the plant material and the petroleum ether picks up the pigments before undergoing secondary reactions. The deep green extract is decanted through a pad of cotton. The leaves are reextracted twice with same quantities of methanol and 40–75 1C petroleum ether (2:1). The extracts are diluted with 120 cm3 of saturated NaCl solution, keeping most of the pigments in the petroleum ether layer. The remained aqueous methanol layer is reextracted with 40 cm3 of mixture containing 40–75 1C petroleum ether and diethyl ether (1:1), ensuring solubility of pigments in the organic phase. The successive extracts are treated by the same procedure. The final extract is mixture of pigments and contains various chlorophyll forms as well as accessory pigments—carotenoids (carotenes and xanthophylls). 2.2. Isolation of carotenoids by column chromatography from spinach extract The carotenoid fractions were isolated using a modified procedure of Svec (1978) and Backman and Risch (1991)— column chromatography with silica gel (silica gel 60, Merck, 0.063–0.2 mm) as the adsorbent and benzene/ acetone mixture as the eluent. The benzene/acetone ratio was changed from initial 1:0 to final 1:1, to permit an easier eluation of the polar fractions. b-Carotene appears first (eluted by benzene only), followed by chlorophylls (benzene:acetone 7:1) and xanthophylls fractions—lutein and neoxanthin, (benzene:acetone 6:1–1:1). The fractions were dried and resolved in hexane. The fractions identification has been done by comparing their visible (VIS) spectra with standards spectra. 2.3. Pigments extraction from tomato fruits Eight grams of ground tomato fruit has been thoroughly mixed with 40 cm3 of ethanol. The slurry was stirred until the tomato paste material was no longer sticky (about 3 min). Ethanol was removed by vacuum filtration. The retained tomato residue was mixed with 60 cm3 of a mixture of acetone and petroleum ether (1:1). The extract was collected by vacuum filtration, and the filter residue was rewashed with the solvent mixture (20 cm3) in order to improve the yield. The filtrate was transferred to a small separatory funnel and mixed with 50 cm3 of saturated NaCl solution. The organic layer was rewashed twice, repeatedly, first with 50 cm3 of 10% potassium carbonate and then with 50 cm3 of water. At the end, approximately 1 g of anhydrous magnesium sulfate was added to dry the organic layer. After 10–15 min the solution was vacuum filtered to remove the drying agent. 2.4. Isolation of carotenoids by column chromatography from tomato extract The lycopene fraction was isolated by using column chromatography with alumina (aluminium oxide 90,
ARTICLE IN PRESS 36
D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
Merck, 0.063–0.2 mm) as the adsorbent and petroleum ether/acetone mixture as the eluent. The mixture ratio was changed from initial 10:0.1 to final 9:1, to permit an easier eluation of lycopene. b-Carotene appears first (the ratio of 10:0.1), followed by lycopene fraction (9:1). The fractions were dried and resolved in hexane. 2.5. HPLC analysis of carotenoids fractions High percentage of carotenoids in the separated fractions has been proved by HPLC analysis. The analysis was done on HP HPLC set-up under isocratic conditions; column: Zorbax Eclipse XDB-C18, mobile phase: acetonitrile/methanol/ethylacetate, 60:20:20; flow rate: 0.5 ml/min. The monitoring wavelenghts were 445 nm for b-carotene and lycopene, 430 and 447 nm for lutein and neoxanthin (maxima of the pigments absorptions, respectively). 2.6. UV-irradiation Continuous irradiations of samples were performed in cylindrical photochemical reactor ‘‘Rayonnet’’, with 14 symmetricaly placed lamps with emission maxima in three different ranges: 254 nm (UV-C), 300 nm (UV-B) and 350 nm (UV-A). The samples were irradiated in quartz cuvettes (1 1 4.5 cm) placed on rotating circular holder, in the middle of the cylinder, 10 cm from the walls. The total measured energy flux (hitting the samples) is 25 W/m2 for 254 nm, 21 W/m2 for 300 nm and 18 W/m2 for 350 nm. 2.7. VIS spectroscopy The samples spectra in VIS range, before and after irradiation with UV-light, were recorded on VARIAN Cary-100 Spectrophotometer. All spectra were recorded from 400 to 800 nm. 2.8. TBA test Lecithin peroxidation, as well as its inhibition in the presence of the carotenoids, were measured by TBA–MDA test (Choi et al., 2002; Fernandez et al., 1997). This method is based on the MDA (secondary product of lipid peroxidation) reaction with TBA to obtain a red colored complex with maximum absorption at 532 nm. The reaction mixture contained aqueous solution of lecithins (2.2 103 mol/ dm3) and methanolic solution of carotenoids (7 106 mol/
dm3), in 10:1 (v/v) ratio. Lecithin peroxidation was initiated by continuous UV-irradiation during increasing time periods. Two centimeter cube of aqueous trichloroacetic acid (5.5%), followed by 2 cm3 of TBA (4.2 102 mol/dm3 in 5 102 mol/dm3 NaOH) were added in the reaction mixture immediately after irradiation. The mixture was incubated for 10 min at 37 1C in the dark, and centrifuged for 10 min at 4000 rpm. VIS spectra of the TBA–MDA complex were recorded from 400 to 800 nm afterwards. The complex absorbances in the supernatant read at 532 nm were used to calculate the inhibition percentage of lecithin peroxidation by using the following equation: inhibition of lecithin peroxidation ð%Þ ¼ ðAC AS Þ
100 , AC AB
where AC is the absorbance of control (aqueous solution of pure lecithin) which is UV-irradiated and treated by TBA solution, AS the absorbance of sample (lecithin/carotenoids mixture) which is UV-irradiated and treated by TBA solution and AB is the absorbance of blank (aqueous solution of pure lecithin which is not UV-irradiated, but treated with TBA solution—monitoring MDA level in the lecithin before UVirradiation). 3. Results Structures of investigated carotenoids (two carotenes: bcarotene and lycopene, and two xanthophylls: lutein and neoxanthin) have been shown in Fig. 1. The decrease of carotenoids mediated inhibition of UVinduced lecithin peroxidation, and simultaneous increase of lipid peroxides production in control and sample (as a result of increasing UV-irradiation), have been shown in Fig. 2 (for b-carotene irradiated with UV-A), Fig. 3 (for lutein irradiated with UV-B), Fig. 4 (for lycopene irradiated with UV-A), and Fig. 5 (for neoxanthin irradiated with UV-A), in a form of log-plots. The analog plots obtained with UVB and UV-C light (for b-carotene, lycopene and neoxanthin), and UV-A and UV-C light (for lutein) have shown very similar shapes (not shown). The plots expressed linear fitting, with an average R values of about 0.97. The slopes calculated from kinetic log-plots represent the rate constants for decreased inhibition of UV-induced lecithin peroxidation (K1), increase of control absorbance at 532 nm (K2) and increase of sample absorbance at 532 nm (K3); the latter two are directly related to lipid peroxidation process. The rate constants are given in Table 1.
Fig. 1. Structures of investigated carotenoids.
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
37
B
C
Fig. 2. Results of TBA–MDA test: decrease of b-carotene-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-A intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance at 532 nm during increasing UV-A intervals (C).
Such presentation provides the slopes (rate constants) comparison, reflecting UV-induced changes in inhibition of lecithin peroxidation (by the involved carotenoids), and in peroxidation kinetics for pure lecithin solution and lecithin/pigments mixtures. It allows an insight to possible changes in protective function of the investigated carotenoids toward UV-induced lecithin peroxidation. 4. Discussion Carotenoids are usually C40 tetraterpenoids built from eight C5 isoprenoid units. The basic linear and symmetrical skeleton can be cyclized at one or both ends. Cyclization,
Fig. 3. Results of TBA–MDA test: decrease of lutein-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-B intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance of at 532 nm during increasing UV-B intervals (C).
hydrogenation, dehydrogenation, double-bond migration, chain shortening or extension, rearrangement, isomerization, introduction of oxygen functions or combinations of these processes, results in a countless structures of carotenoids (Scheer, 2003). A significant characteristic is a long conjugated double-bond system, providing an extended p-delocalization, leading to a substantial batochromic schift in VIS region. The shift is responsible for yellow, orange or red color of these compounds, including carotenes (made of carbon and hydrogen only) and xanthophylls (containing oxygen).
ARTICLE IN PRESS 38
D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
Fig. 4. Results of TBA–MDA test: decrease of lycopene-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-A intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance at 532 nm during increasing UV-A intervals (C).
Fig. 5. Results of TBA–MDA test: decrease of neoxanthin-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-A intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance at 532 nm during increasing UV-A intervals (C).
Thanks to their structural features, carotenoids have a lot of functions in nature. One of their major functions is to protect photosynthetic apparatus in excess of light (Teramura and Ziska, 1996; Strid et al., 1990; Middleton and Teramura, 1993). The other very important function of carotenoids, of much more global character than the one related to photosynthesis (but including it!) is antioxidant function (that is one of the reasons for a wide use of carotenoids in food industry, besides the coloring effect (Paust, 1991; Baker and Gunther, 2004). For such a purpose, carotenoids can act in a preventive manner: they may inhibit formation of ROS species by reacting directly with oxygen, or, if radicals are already created, they may
scavenge them acting as a chain-breaking antioxidants (Burton and Ingold, 1984; Haila, 1999; Palozza and Krinsky, 1992). All biological antioxidants, concerning the lipid peroxidation process, are grouped into two categories: preventive antioxidants, which reduce the initiation of peroxidation by suppressing the generation of chain-initiating radicals, and chain-breaking antioxidants which disrupt the chain propagation by trapping the chain-initiating and/or chainpropagating peroxyl radicals (Haila, 1999; Peng Lim et al., 1992). Carotenoids are generally classified as preventive antioxidants because of physical and chemical quenching of toxic singlet oxygen, but also may act as chain-breaking
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41 Table 1 Kinetics of lipid peroxides inhibition (K1), and production, in a solution containing lecithin only (K2) or mixture of carotenoids (7 107 mol/dm3) and lecithin (2 103 mol/dm3) (K3), during UV-irradiation with emission maxima in three different ranges: 254 nm (UV-C), 300 nm (UV-B) and 350 nm (UV-A)—results of TBA–MDA test UV-irradiation K1 (min1) wavelength (nm)
K2 (min1)
K3 (min1)
b-Carotene 254 300 350
0.18093 0.17945 0.04531
0.19304 0.05345 0.01043
0.11346 0.03940 0.00938
Lycopene 254 300 350
0.19878 0.15004 0.04054
0.18012 0.04189 0.00956
0.17318 0.05805 0.01789
Lutein 254 300 350
0.34654 0.11893 0.05199
0.12506 0.04938 0.01145
0.16368 0.05718 0.01670
Neoxanthin 254 300 350
0.08151 0.05448 0.00877
0.11407 0.03817 0.00958
0.10475 0.04262 0.01065
y1 ¼ K1x+n; y1—log (% of inhibition of lecithin peroxidation); x— irradiation time y2 ¼ K2x+n; y2—log (control absorbance at 532 nm); x—irradiation time y3 ¼ K3x+n; y3—log (sample absorbance at 532 nm); x—irradiation time
antioxidants (Haila, 1999; Palozza and Krinsky, 1992; Krinsky and Yeum, 2003; Frank and Cogdell, 1996). 4.1. TBA–MDA test One of the most prominent and currently used assay for lipid peroxidation detection is thiobarbituric acid assay (TBA–MDA test). This method is based on the MDA reaction with TBA to obtain a red colored complex. MDA is a three-carbon dialdehyde, with carbonyl groups at the C-1 and C-3 positions generated through degradation of hydroperoxides formed from polyunsaturated fatty acids with three double bonds, or more (Fernandez et al., 1997). The complex strongly absorbs at 532–535 nm. The complex absorbance read at 532 nm was used to estimate changes of carotenoids mediated inhibition of lecithin peroxidation during continuous UV-irradiation. The initiation of lipid peroxidation in this paper was performed by UV-light only, which is not to often used approach; UV-light is usually used in combination with photosensitizers (Girotti, 2001; Rontani, 2001; Wrona et al., 2003), transition-metal salts (ferrous salts—Fenton reaction) (Tian Li et al., 2000; Deng et al., 1997; Halliwell and Chirico, 1993), etc. Table 1 gives a comparative review of the slopes calculated from kinetic log-plots (as those in Figs. 2–5) representing the rates of inhibition decrease of UV-induced lecithin peroxidation (K1), increase of control absorbance at
39
532 nm (K2) and increase of sample absorbance at 532 nm (K3), during continuous irradiation in the three investigated UV-ranges. The second column shows decrease of lipid peroxidation inhibition by carotenoids during prolonged UV-irradiation: there is noticeable drop in K1 values following changes from UV-C to UV-B, and from UV-B to UV-A photons. This fact confirms that decrease of lecithin peroxidation inhibition by carotenoids, during prolonged UV-irradiation, depends on UV-photons energy input (ranging from 18 to 25 W/m2). The K1,UVC/K1,UVB ratio is 1.0 for b-carotene, 1.3 for lycopene, 2.9 for lutein and 1.5 for neoxanthin. At the same time the K1,UVB/ K1,UVA ratio is 4 for b-carotene, 3.7 for lycopene, 2.3 for lutein and 6.2 for neoxanthin. For each particular carotenoid the ratio K1,UVB/K1,UVA is bigger than the corresponding ratio K1,UVC/K1,UVB, but lutein makes a little exception. The ratio of the two ratios—[(K1,UVB/ K1,UVA)]/[(K1,UVC/K1,UVB]) is quite different for the involved carotenoids: the [(K1,UVB/K1,UVA)]/[(K1,UVC/ K1,UVB)] ratio is 4 for b-carotene, 2.8 for lycopene, 0.8 for lutein and 4.1 for neoxanthin. Neoxanthin, with ratios K1,UVB/K1,UVA ¼ 6.2, and [(K1,UVB/K1,UVA)]/ [(K1,UVC/K1,UVB)] ¼ 4.1, is the most sensitive to the change of UV-photons energy input. Since neoxanthin contains four oxygens (including the one closing epoxy moiety—Fig. 1), it looks like the differences between the chemical structures among the investigated carotenoids (Fig. 1) play a non-negligible role. At the same time, neoxanthin has the lowest K1 values (Table 1), compared to all investigated carotenoids and all three UV-irradiation ranges, which indicates that neoxanthin antioxidant function is the most resistant to UV-irradiation inside the same UV-range. UV-induced peroxidation of carotenoids free lecithin aqueous solution has been monitored by TBA–MDA test, too. Rate constants for this reaction (K2), for all three UVranges, have been presented in third column of Table 1. The UV-photons energy input plays again a crucial role in the peroxides production: irradiation by UV-C photons led to the fastest lecithin peroxidation, followed by UV-B and UV-A irradiation, respectively. The UV-induced peroxidation of lecithin in the presence of carotenoids (e.g. in lecithin/carotenoids mixtures) has been also monitored by TBA–MDA test. The belonging rate constants (K3), for all three UV-ranges and all investigated carotenoids, are presented in fourth column of Table 1. The UV-photons energy input plays again major role in the lecithin peroxide production. UV-C photons induce the fastest lecithin peroxidation, followed by UV-B and UV-A photons, respectively, and the same answer has been noticed in the presence of all investigated carotenoids. The K3,UVC/ K3,UVB ratio is 2.9 for b-carotene, 3.0 for lycopene, 2.8 for lutein and 2.4 for neoxanthine, while rate constants ratio K3,UVB/K3,UVA is 4.2 for b-carotene, 3.2 for lycopene, 3.4 for lutein and 4 for neoxanthine. The ratio of the two ratios—[(K3,UVB/K3,UVA)]/[(K3,UVC/K3,UVB]) is 1.4 for b-carotene, 1.1 for lycopene, 1.2 for lutein and 1.7 for
ARTICLE IN PRESS 40
D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
neoxanthine. The cited ratios for the rate constant K3 are very similar to the K2 constant’s ratios (peroxidation of pure lecithin—Table 1)—K2,UVC/K2,UVB (3.6, 4.3, 2.5 and 3); K2,UVB/K2,UVA (5.1, 4.4, 4.3 and 4); [(K2,UVB/ K2,UVA)]/[(K2,UVC/K2,UVB] (1.4, 1.0, 1.8 and 1.3, respectively). Estimation of antioxidant activities of the four carotenoids in the presence of lecithin comes from comparison of K2 and K3 rate constants (Table 1). Even a brief look shows that these values (obtained for the same UV-range and for the same carotenoid) are generally very close. That means that carotenoids antioxidative control of the lecithin lipid peroxidation process is of marginal importance in a (highly unordered) and UV-irradiated homogeneous solution where all present radicals are free to move in any direction; this certainly leads to smaller probabilities for at least some radical interactions and the consequent scavenging impact. Only b-carotene has shown some antioxidant activity under these conditions (K2/K3 ratio is 1.7, 1.3 and 1.1 for UV-C, UV-B and UV-A photons, respectively—Table 1). The antioxidant activity of b-carotene is proportional to the UV-photons energy input, i.e. decrease of UV-photons energy input leads to the lower antioxidant activity. However, it is reasonable to expect that in a very constraint, space-limited systems like micelles or monolayers, where movement of free radicals is highly steric dependent due to predominant ‘‘cage effect’’ (Markovic and Patterson, 1993; Markovic et al., 1990; Markovic, 2001, 2004) scavenging capacities of the employed carotenoids toward lipid radicals should be more expressed (Woodall et al., 1997b; Liebler et al., 1997; Matsushita et al., 2000). To conclude, (i) decrease of the carotenoids mediated inhibition of UV-induced lecithin peroxidation, i.e. decrease of antioxidant activity of investigated carotenoids, highly depends on UV-photons energy input and their chemical structures; (ii) the investigated carotenoids (bcarotene, lycopene, lutein and neoxanthin) have a marginal remained antioxidant activity to prevent UV-induced lecithin peroxidation in highly unordered homogeneous solution and (iii) antioxidant activity of neoxanthin appears to be the most resistant toward UV-irradiation, but, at the same time, its antioxidant activity is the most affected by change of UV-photons energy input (from UVC via UV-B to UV-A). Acknowledgment Dragan Cvetkovic is a recipient of a fellowship granted by Ministry of Science and Environmental Protection, Republic of Serbia. References Aikens, J., Dix, T., 1993. Hydrodioxyl (perhydroxyl), peroxyl, and hydroxyl radical-initiated lipid peroxidation of large unilamellar vesicles (liposomes): comparative and mechanistic studies. Arch. Biochem. Biophys. 305 (2), 516–525.
Backman, H., Risch, N., 1991. Chlorophylls. CRC Press, Boca Raton, FL, p. 103. Baker, R., Gunther, C., 2004. The role of carotenoids in consumer choice and the likely benefits from their inclusion into products for human consuption. Trends Food Sci. 15, 484–488. Burton, G., Ingold, K., 1984. b-Carotene: an unusual type of lipid antioxidant. Science 224, 569–573. Choi, C., Kim, S., Hwang, S., Choi, B., Ahn, H., Lee, M., Park, S., Kim, S., 2002. Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-guided comparison. Plant Sci. 163, 1161–1168. Deng, W., Fang, X., Wu, J., 1997. Flavonoids function as antioxidants: by scavenging rective oxygen species or by chelating iron? Radiat. Phys. Chem. 50 (3), 271–276. Erben-Russ, M., Michel, C., Bors, W., Winter, R., Saran, M., 1987. Absolute rate constants of radical reactions in aqueous solution. J. Phys. Chem. 91, 2362–2365. Fernandez, J., Perez-Alvarez, J., Fernindez-Lopez, J., 1997. Thiobarbituric acid test for monitoring lipid oxidation in meat. Food Chem. 59 (3), 345–353. Frank, H., Cogdell, R., 1996. Carotenoids in photosynthesis. Photochem. Photobiol. 63 (3), 257–264. Girotti, A., 2001. Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms. J. Photochem. Photobiol. B 63, 103–113. Haila, K., 1999. Effects of carotenoids and carotenoid-tocopherol interaction on lipid oxidation in vitro. Academic Dissertation, University of Helsinki, Department of Applied Chemistry and Microbiology. Halliwell, B., Chirico, S., 1993. Lipid peroxidation: its mechanism, measurement, and significance. Am. J. Clin. Nutr. 57 (5), 715–725. Heijman, M., Nauta, H., Levine, Y., 1985. A pulse radiolysis study of the dienyl radical in oxigen-free linoleate solution: time and concentration dependence. Radiat. Phys. Chem. 26, 73–82. Heim, K., Tagliaferro, A., Bobilya, D., 2002. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J. Nutr. Biochem. 13, 572–584. Hynninen, P., 1991. Modifications. In: Scheer, H. (Ed.), Chlorophylls. CRC Press, Boca Raton, FL, pp. 145–209. Ichihashi, M., Ueda, M., Budiyannto, A., Bito, T., Oka, M., Fukunaga, M., Tsuru, K., Horikawa, T., 2003. UV-induced skin damage. Toxicology 189, 21–39. Krinsky, N., Yeum, K., 2003. Carotenoid–radical interaction. Biochem. Biophys. Res. Commun. 305, 754–760. Liebler, D., Straton, S., Kaysen, K., 1997. Antioxidant actions of b-carotene in liposomal and microsomal membranes: role of carotenoid-membrane incorporation and a-tocopherol. Arch. Biochem. Biophys. 338, 244–250. Markovic, D., 2001. Benzophenone-sensitized peroxidation in compressed lipid monolayers at airwater interface. Collect. Czech. Chem. Commun. 66, 1603–1614. Markovic, D., 2004. Photochemistry of aromatic cetones in sodium dodecyl sulphate micelles in the presence of unsaturated fatty acids. J. Serb. Chem. Soc. 69 (2), 107–115. Markovic, D., Patterson, L., 1989. Radical processes in lipids. Selectivity of hydrogen abstraction from lipids by benzophenone triplet. Photochem. Photobiol. 49, 531–535. Markovic, D., Patterson, L., 1993. Benzophenone-sensitized lipid peroxidation in linoleate micelles. Photochem. Photobiol. 58, 329–334. Markovic, D., Durand, T., Patterson, L., 1990. Hydrogen abstraction from lipids by triplet states of derivatized benzophenone photosensitizers. Photochem. Photobiol. 51, 389–394. Matsushita, Y., Suzuki, R., Nara, E., Yokoyama, A., Miyashita, K., 2000. Antioxidant activity of polar carotenoids including astaxanthinb-glucoside from marine bacterium on PC liposomes. Fisheries Sci. 66, 980–985. McBride, J., Kraemer, W., 1999. Free radicals, exercise, and antioxidants. J. Strength Cond. Res. 13 (2), 175–183.
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41 Middleton, E., Teramura, A., 1993. The role of flavonol glycoside and carotenoids in protecting soybean from ultraviolet-B damage. Plant Physiol. 103, 741–752. Mimica-Dukic, N., 1997. In vivo i in iitro ispitivanja antioksidantnih svojstava biljnih ekstrakata. Arh. Farma. 5 (7), 475–493. Ouhtit, A., Ananthaswamy, H., 2001. A model for UV-induction of skin cancer. J. Biomed. Biotechnol. 1 (1), 5–6. Palozza, P., Krinsky, N., 1992. Antioxidant effects of carotenoids in vivo and in vitro: an overview. Methods Enzymol. 213, 403–420. Paust, J., 1991. Recent progress in commercial retinoids and carotenoids. Pure Appl. Chem. 63 (1), 45–58. Peng Lim, B., Nagao, A., Terao, J., Tanaka, K., Suzuki, T., Takama, K., 1992. Antioxidant activity of xanthophylls on peroxyl radicalmediated phospholipid peroxidation. Biochim. Biophys. Acta 1126, 178–184. Pfeifer, G., You, Y., Besaratinia, A., 2005. Mutations induced by ultraviolet light. Mutat. Res. 571, 19–31. Rontani, J., 2001. Visible light-dependent degradation of lipidic phytoplantonic components during senescence: a review. Photochemistry 58, 187–202. Ross, L., Barclay, C., Vinqvist, M., 1994. Membrane peroxidation: inhibiting effects of water-soluble antioxidants on phospholipids of different charge types. Free Radic. Biol. Med. 16 (6), 779–788. Saija, A., Scalese, M., Lanza, M., Marzzullo, D., Bonina, F., Castelli, F., 1995. Flavonoids as antioxidanta agents: importance of their interaction with biomembranes. Free Radic. Biol. Med. 19 (4), 481–486. Scheer, H., 2003. The pigments. In: Green, B., Parson, W. (Eds.), Light Harvesting Antenas in Photosynthesis. Kluwer Academic Publishers, Dordrecht, pp. 29–81. Schwarz, T., 1996. Effects of UVA light on the immune system. A settled issue. Eur. J. Dermatol. 6 (3), 227–228. Simic, M., 1992. Carotenoid free radicals. Methods Enzymol. 213, 444–454.
41
Strid, A., Chow, W., Anderson, J., 1990. Effects of supplementary ultraviolet-B radiation on photosynthesis in Pisum sativum. Biochim. Biophys. Acta 1020, 260–268. Svec, W., 1978. The Porphyrins. Academic Press, New York, p. 342. Svec, W., 1991. Chlorophylls. CRC Press, Boca Raton, FL, p. 89. Teramura, A., Ziska, L., 1996. Ultraviolet-B ratiation and photosynthesis. In: Photosynthesis and the Environment. Kluwer Academic Publishers, Dordrecht, pp. 435–450. Tian Li, Q., Huang Yeo, M., Kheng Tan, B., 2000. Lipid peroxidation in small and large phospholipid unilamellar vesicles induced by watersoluble free radical sources. Biochem. Biophys. Res. Commun. 273, 72–76. Van der Sluis, A., Dekker, M., Verker, R., Jongen, W., 2000. An improved, rapid in vitro method to measure antioxidant activity. Application on selected flavonoids and apple juice. J. Agric. Food Chem. 48, 4116–4122. Vermer, B., Wintzen, M., Claas, F., Schothorst, A., Hurks, H., 1996. UVinduced immunosuppression; the critical role of wavelength. Eur. J. Dermatol. 6 (3), 231–233. Wheatley, R., 2000. Some recent trends in the analytical chemistry of lipid peroxidation. Trends Anal. Chem. 19 (10), 617–628. Woodall, A., Lee, S., Weesie, R., Jackson, M., Britton, G., 1997a. Oxidation of carotenoids by free radicals: relationship between structure and reactivity. Biochim. Biophys. Acta 1336, 33–42. Woodall, A., Britton, G., Jackson, M., 1997b. Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals: relationship between carotenoid structure and protective ability. Biochim. Biophys. Acta 1336, 575–586. Wrona, M., Korytowski, W., Rozanowska, M., Sarna, T., Truscott, G., 2003. Cooperation of antioxidants in protection against photosensitized oxidation. Free Radic. Biol. Med. 35 (10), 1319–1329. Young, A., 1996. Does UVA exposure cause human malignant melanoma? Eur. J. Dermatol. 6 (3), 225–226.
Radiation Physics and Chemistry 77 (2008) 34–41 www.elsevier.com/locate/radphyschem
UV-induced changes in antioxidant capacities of selected carotenoids toward lecithin in aqueous solution Dragan Cvetkovic, Dejan Markovic Faculty of Technology, University of Nish, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia Received 17 January 2007; accepted 21 February 2007
Abstract Antioxidant action of four selected carotenoids (two carotenes, b-carotene and lycopene, and two xanthophylls, lutein and neoxanthin) on UV-induced lecithin lipid peroxidation in aqueous solution has been studied by thiobarbituric acid (TBA) test. TBA test is based on absorbance measurements of complex formed between malondialdehyde, secondary product of lipid peroxidation and thiobarbituric acid, at 532 nm. The antioxidant capacities of investigated carotenoids appeared to be strongly affected by UV-action. High energy input of the involved UV-photons plays major governing role, though a certain impact of the carotenoid structures cannot be neglected. The results suggest a minor remained contribution of selected carotenoids to prevention of lecithin peroxidation in the studied system as a result of UV-irradiation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Carotenoids; Lipids; UV-irradiation; Peroxidation
1. Introduction The depletion of the ozone shield, caused by a huge release of atmospheric pollutants (such as chlorofluorocarbons, chlorocarbons and organobromides), leads to increase of biologically damaging UV-light at ambient levels (mainly UV-B light, 280–320 nm). UV-light can generally influence many crucial biologically important processes, such as DNA replication (Ichihashi et al., 2003; Pfeifer et al., 2005), photosynthesis (Teramura and Ziska, 1996; Strid et al., 1990), etc. Also, UV-light can generally influence whole human immune system (Schwarz, 1996; Vermer et al., 1996), by initiating a lot of harmful free radicals mediated processes, lipid peroxidation (LP) among them. Lipid peroxidation is tightly connected with many pathological processes which finish in some form of cancer at the very end (Young, 1996), melanoma skin cancer among them (Ouhtit and Ananthaswamy, 2001). Lipid Corresponding author.
E-mail addresses: [email protected], [email protected] (D. Markovic). 0969-806X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2007.02.078
peroxidation is either free radicals chain reaction (type I), or alternatively, it occurs through a non radical pathway, by direct reaction with singlet oxygen in the presence of a photosensitizer (Girotti, 2001; Wheatley, 2000; Markovic and Patterson, 1993; Mimica-Dukic, 1997; Burton and Ingold, 1984; Haila, 1999). Reactive oxygen species (ROS), like hydroxy radicals (OHd) or peroxy radicals (ROOd), are typical lipid peroxidation initiators. They can be created through a variety of chemical reactions (Mimica-Dukic, 1997), some of them including typical lipid radicals producers (Aikens and Dix, 1993; Ross et al., 1994; Tian Li et al., 2000). Additionally, they can be induced through a variety of external stresses (Girotti, 2001) implying very commonly an external radiation (Heijman et al., 1985; Erben-Russ et al., 1987), which, in case of UV-irradiaton may include photosensitisers in very different media (Markovic and Patterson, 1989, 1993; Markovic et al., 1990; Markovic, 2001). Lipid peroxidation is mostly controlled by antioxidants action in vivo. Many biomolecules (and classes of biomolecules) serve as antioxidants, like enzymes, tocopherols
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
(like vitamin E), L-ascorbic acid (vitamin C), retinol (vitamin A), thiamin and riboflamin (vitamin B), flavonoids, etc. (Saija et al., 1995; Deng et al., 1997; McBride and Kraemer, 1999; Van der Sluis et al., 2000; Choi et al., 2002; Heim et al., 2002). In recent years, carotenoids have received wide research interest as potential antioxidants. There are a number of studies reporting that higher consumption of carotenoids leads to lower risk of cancer and cardiovascular disease, and relating antioxidant action of carotenoids with their conjugated chemical structures, having multiple potential sites approachable for attack by ROS species (Burton and Ingold, 1984; Palozza and Krinsky, 1992; Simic, 1992; Woodall et al., 1997a; Krinsky and Yeum, 2003; Peng Lim et al., 1992; Frank and Cogdell, 1996). UV-irradiation certainly affects carotenoids antioxidant function in vivo (Teramura and Ziska, 1996), though the involved mechanisms are not elucidated yet. Therefore, UV-irradiation effects on antioxidant capacities of four plants photosynthetic accessory pigments (b-carotene, lycopene, lutein and neoxanthin) in vitro, in the presence of lipoidal target (lecithin), have been studied in this paper. Lipid peroxidation has been induced by UV-light from three different ranges (UV-A, UV-B and UV-C). The antioxidant activities of investigated carotenoids (and its dependence on UV-irradiation) have been monitored by thiobarbituric–malondialdehyde (TBA–MDA) test. 2. Experimental Pigments were isolated from plant material (b-carotene, lutein and neoxanthin from spinach leaves and lycopene from tomato fruits) purchased at the local market. Lecithin Epikuron 100 P, a mixture of phospholipids, was gifted from ‘‘ICN Galenika’’, Beograd. It was manufactured by ‘‘Degusa Texturant Systems’’, Hamburg, Germany. The lipid content was: phosphatidylethanolamine 18.0%, phosphatic acid 8.3%, phosphatidylinositol 14.1% and phosphatidylcholin 21.7%. The acid value, peroxide number and iodine number were periodically checked out and found to be true. The lecithin mixture was kept in dark to prevent the autooxidation process to some extent. However, autooxidation could not be annihilated, and it has been taken into account during calculation of lipid peroxidation yield. 2.1. Pigments extraction from spinach (Spinacia oleracea) The photosynthetic pigments were extracted from spinach leaves by using modified method proposed by Hynninen (1991) and Svec (1991). Fresh spinach leaves free of midribs (0.030 kg) were dropped into boiling water, which was quickly replaced (after 1–2 min) with cooled water. Hot water inactivates enzymes preventing the pigment alteration and permitting coagulation of proteins and water-soluble substances. After drying between paper towels, leaves are separated and placed in mixture of methanol (60 cm3) and 40–75 1C petroleum ether (30 cm3); the stuff was occasionally agitated in next 30 min.
35
Methanol removes water from the plant material and the petroleum ether picks up the pigments before undergoing secondary reactions. The deep green extract is decanted through a pad of cotton. The leaves are reextracted twice with same quantities of methanol and 40–75 1C petroleum ether (2:1). The extracts are diluted with 120 cm3 of saturated NaCl solution, keeping most of the pigments in the petroleum ether layer. The remained aqueous methanol layer is reextracted with 40 cm3 of mixture containing 40–75 1C petroleum ether and diethyl ether (1:1), ensuring solubility of pigments in the organic phase. The successive extracts are treated by the same procedure. The final extract is mixture of pigments and contains various chlorophyll forms as well as accessory pigments—carotenoids (carotenes and xanthophylls). 2.2. Isolation of carotenoids by column chromatography from spinach extract The carotenoid fractions were isolated using a modified procedure of Svec (1978) and Backman and Risch (1991)— column chromatography with silica gel (silica gel 60, Merck, 0.063–0.2 mm) as the adsorbent and benzene/ acetone mixture as the eluent. The benzene/acetone ratio was changed from initial 1:0 to final 1:1, to permit an easier eluation of the polar fractions. b-Carotene appears first (eluted by benzene only), followed by chlorophylls (benzene:acetone 7:1) and xanthophylls fractions—lutein and neoxanthin, (benzene:acetone 6:1–1:1). The fractions were dried and resolved in hexane. The fractions identification has been done by comparing their visible (VIS) spectra with standards spectra. 2.3. Pigments extraction from tomato fruits Eight grams of ground tomato fruit has been thoroughly mixed with 40 cm3 of ethanol. The slurry was stirred until the tomato paste material was no longer sticky (about 3 min). Ethanol was removed by vacuum filtration. The retained tomato residue was mixed with 60 cm3 of a mixture of acetone and petroleum ether (1:1). The extract was collected by vacuum filtration, and the filter residue was rewashed with the solvent mixture (20 cm3) in order to improve the yield. The filtrate was transferred to a small separatory funnel and mixed with 50 cm3 of saturated NaCl solution. The organic layer was rewashed twice, repeatedly, first with 50 cm3 of 10% potassium carbonate and then with 50 cm3 of water. At the end, approximately 1 g of anhydrous magnesium sulfate was added to dry the organic layer. After 10–15 min the solution was vacuum filtered to remove the drying agent. 2.4. Isolation of carotenoids by column chromatography from tomato extract The lycopene fraction was isolated by using column chromatography with alumina (aluminium oxide 90,
ARTICLE IN PRESS 36
D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
Merck, 0.063–0.2 mm) as the adsorbent and petroleum ether/acetone mixture as the eluent. The mixture ratio was changed from initial 10:0.1 to final 9:1, to permit an easier eluation of lycopene. b-Carotene appears first (the ratio of 10:0.1), followed by lycopene fraction (9:1). The fractions were dried and resolved in hexane. 2.5. HPLC analysis of carotenoids fractions High percentage of carotenoids in the separated fractions has been proved by HPLC analysis. The analysis was done on HP HPLC set-up under isocratic conditions; column: Zorbax Eclipse XDB-C18, mobile phase: acetonitrile/methanol/ethylacetate, 60:20:20; flow rate: 0.5 ml/min. The monitoring wavelenghts were 445 nm for b-carotene and lycopene, 430 and 447 nm for lutein and neoxanthin (maxima of the pigments absorptions, respectively). 2.6. UV-irradiation Continuous irradiations of samples were performed in cylindrical photochemical reactor ‘‘Rayonnet’’, with 14 symmetricaly placed lamps with emission maxima in three different ranges: 254 nm (UV-C), 300 nm (UV-B) and 350 nm (UV-A). The samples were irradiated in quartz cuvettes (1 1 4.5 cm) placed on rotating circular holder, in the middle of the cylinder, 10 cm from the walls. The total measured energy flux (hitting the samples) is 25 W/m2 for 254 nm, 21 W/m2 for 300 nm and 18 W/m2 for 350 nm. 2.7. VIS spectroscopy The samples spectra in VIS range, before and after irradiation with UV-light, were recorded on VARIAN Cary-100 Spectrophotometer. All spectra were recorded from 400 to 800 nm. 2.8. TBA test Lecithin peroxidation, as well as its inhibition in the presence of the carotenoids, were measured by TBA–MDA test (Choi et al., 2002; Fernandez et al., 1997). This method is based on the MDA (secondary product of lipid peroxidation) reaction with TBA to obtain a red colored complex with maximum absorption at 532 nm. The reaction mixture contained aqueous solution of lecithins (2.2 103 mol/ dm3) and methanolic solution of carotenoids (7 106 mol/
dm3), in 10:1 (v/v) ratio. Lecithin peroxidation was initiated by continuous UV-irradiation during increasing time periods. Two centimeter cube of aqueous trichloroacetic acid (5.5%), followed by 2 cm3 of TBA (4.2 102 mol/dm3 in 5 102 mol/dm3 NaOH) were added in the reaction mixture immediately after irradiation. The mixture was incubated for 10 min at 37 1C in the dark, and centrifuged for 10 min at 4000 rpm. VIS spectra of the TBA–MDA complex were recorded from 400 to 800 nm afterwards. The complex absorbances in the supernatant read at 532 nm were used to calculate the inhibition percentage of lecithin peroxidation by using the following equation: inhibition of lecithin peroxidation ð%Þ ¼ ðAC AS Þ
100 , AC AB
where AC is the absorbance of control (aqueous solution of pure lecithin) which is UV-irradiated and treated by TBA solution, AS the absorbance of sample (lecithin/carotenoids mixture) which is UV-irradiated and treated by TBA solution and AB is the absorbance of blank (aqueous solution of pure lecithin which is not UV-irradiated, but treated with TBA solution—monitoring MDA level in the lecithin before UVirradiation). 3. Results Structures of investigated carotenoids (two carotenes: bcarotene and lycopene, and two xanthophylls: lutein and neoxanthin) have been shown in Fig. 1. The decrease of carotenoids mediated inhibition of UVinduced lecithin peroxidation, and simultaneous increase of lipid peroxides production in control and sample (as a result of increasing UV-irradiation), have been shown in Fig. 2 (for b-carotene irradiated with UV-A), Fig. 3 (for lutein irradiated with UV-B), Fig. 4 (for lycopene irradiated with UV-A), and Fig. 5 (for neoxanthin irradiated with UV-A), in a form of log-plots. The analog plots obtained with UVB and UV-C light (for b-carotene, lycopene and neoxanthin), and UV-A and UV-C light (for lutein) have shown very similar shapes (not shown). The plots expressed linear fitting, with an average R values of about 0.97. The slopes calculated from kinetic log-plots represent the rate constants for decreased inhibition of UV-induced lecithin peroxidation (K1), increase of control absorbance at 532 nm (K2) and increase of sample absorbance at 532 nm (K3); the latter two are directly related to lipid peroxidation process. The rate constants are given in Table 1.
Fig. 1. Structures of investigated carotenoids.
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
37
B
C
Fig. 2. Results of TBA–MDA test: decrease of b-carotene-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-A intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance at 532 nm during increasing UV-A intervals (C).
Such presentation provides the slopes (rate constants) comparison, reflecting UV-induced changes in inhibition of lecithin peroxidation (by the involved carotenoids), and in peroxidation kinetics for pure lecithin solution and lecithin/pigments mixtures. It allows an insight to possible changes in protective function of the investigated carotenoids toward UV-induced lecithin peroxidation. 4. Discussion Carotenoids are usually C40 tetraterpenoids built from eight C5 isoprenoid units. The basic linear and symmetrical skeleton can be cyclized at one or both ends. Cyclization,
Fig. 3. Results of TBA–MDA test: decrease of lutein-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-B intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance of at 532 nm during increasing UV-B intervals (C).
hydrogenation, dehydrogenation, double-bond migration, chain shortening or extension, rearrangement, isomerization, introduction of oxygen functions or combinations of these processes, results in a countless structures of carotenoids (Scheer, 2003). A significant characteristic is a long conjugated double-bond system, providing an extended p-delocalization, leading to a substantial batochromic schift in VIS region. The shift is responsible for yellow, orange or red color of these compounds, including carotenes (made of carbon and hydrogen only) and xanthophylls (containing oxygen).
ARTICLE IN PRESS 38
D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
Fig. 4. Results of TBA–MDA test: decrease of lycopene-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-A intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance at 532 nm during increasing UV-A intervals (C).
Fig. 5. Results of TBA–MDA test: decrease of neoxanthin-mediated inhibition of UV-induced lecithin peroxidation during increasing UV-A intervals (A); increase of control absorbance at 532 nm (B); and increase of sample absorbance at 532 nm during increasing UV-A intervals (C).
Thanks to their structural features, carotenoids have a lot of functions in nature. One of their major functions is to protect photosynthetic apparatus in excess of light (Teramura and Ziska, 1996; Strid et al., 1990; Middleton and Teramura, 1993). The other very important function of carotenoids, of much more global character than the one related to photosynthesis (but including it!) is antioxidant function (that is one of the reasons for a wide use of carotenoids in food industry, besides the coloring effect (Paust, 1991; Baker and Gunther, 2004). For such a purpose, carotenoids can act in a preventive manner: they may inhibit formation of ROS species by reacting directly with oxygen, or, if radicals are already created, they may
scavenge them acting as a chain-breaking antioxidants (Burton and Ingold, 1984; Haila, 1999; Palozza and Krinsky, 1992). All biological antioxidants, concerning the lipid peroxidation process, are grouped into two categories: preventive antioxidants, which reduce the initiation of peroxidation by suppressing the generation of chain-initiating radicals, and chain-breaking antioxidants which disrupt the chain propagation by trapping the chain-initiating and/or chainpropagating peroxyl radicals (Haila, 1999; Peng Lim et al., 1992). Carotenoids are generally classified as preventive antioxidants because of physical and chemical quenching of toxic singlet oxygen, but also may act as chain-breaking
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41 Table 1 Kinetics of lipid peroxides inhibition (K1), and production, in a solution containing lecithin only (K2) or mixture of carotenoids (7 107 mol/dm3) and lecithin (2 103 mol/dm3) (K3), during UV-irradiation with emission maxima in three different ranges: 254 nm (UV-C), 300 nm (UV-B) and 350 nm (UV-A)—results of TBA–MDA test UV-irradiation K1 (min1) wavelength (nm)
K2 (min1)
K3 (min1)
b-Carotene 254 300 350
0.18093 0.17945 0.04531
0.19304 0.05345 0.01043
0.11346 0.03940 0.00938
Lycopene 254 300 350
0.19878 0.15004 0.04054
0.18012 0.04189 0.00956
0.17318 0.05805 0.01789
Lutein 254 300 350
0.34654 0.11893 0.05199
0.12506 0.04938 0.01145
0.16368 0.05718 0.01670
Neoxanthin 254 300 350
0.08151 0.05448 0.00877
0.11407 0.03817 0.00958
0.10475 0.04262 0.01065
y1 ¼ K1x+n; y1—log (% of inhibition of lecithin peroxidation); x— irradiation time y2 ¼ K2x+n; y2—log (control absorbance at 532 nm); x—irradiation time y3 ¼ K3x+n; y3—log (sample absorbance at 532 nm); x—irradiation time
antioxidants (Haila, 1999; Palozza and Krinsky, 1992; Krinsky and Yeum, 2003; Frank and Cogdell, 1996). 4.1. TBA–MDA test One of the most prominent and currently used assay for lipid peroxidation detection is thiobarbituric acid assay (TBA–MDA test). This method is based on the MDA reaction with TBA to obtain a red colored complex. MDA is a three-carbon dialdehyde, with carbonyl groups at the C-1 and C-3 positions generated through degradation of hydroperoxides formed from polyunsaturated fatty acids with three double bonds, or more (Fernandez et al., 1997). The complex strongly absorbs at 532–535 nm. The complex absorbance read at 532 nm was used to estimate changes of carotenoids mediated inhibition of lecithin peroxidation during continuous UV-irradiation. The initiation of lipid peroxidation in this paper was performed by UV-light only, which is not to often used approach; UV-light is usually used in combination with photosensitizers (Girotti, 2001; Rontani, 2001; Wrona et al., 2003), transition-metal salts (ferrous salts—Fenton reaction) (Tian Li et al., 2000; Deng et al., 1997; Halliwell and Chirico, 1993), etc. Table 1 gives a comparative review of the slopes calculated from kinetic log-plots (as those in Figs. 2–5) representing the rates of inhibition decrease of UV-induced lecithin peroxidation (K1), increase of control absorbance at
39
532 nm (K2) and increase of sample absorbance at 532 nm (K3), during continuous irradiation in the three investigated UV-ranges. The second column shows decrease of lipid peroxidation inhibition by carotenoids during prolonged UV-irradiation: there is noticeable drop in K1 values following changes from UV-C to UV-B, and from UV-B to UV-A photons. This fact confirms that decrease of lecithin peroxidation inhibition by carotenoids, during prolonged UV-irradiation, depends on UV-photons energy input (ranging from 18 to 25 W/m2). The K1,UVC/K1,UVB ratio is 1.0 for b-carotene, 1.3 for lycopene, 2.9 for lutein and 1.5 for neoxanthin. At the same time the K1,UVB/ K1,UVA ratio is 4 for b-carotene, 3.7 for lycopene, 2.3 for lutein and 6.2 for neoxanthin. For each particular carotenoid the ratio K1,UVB/K1,UVA is bigger than the corresponding ratio K1,UVC/K1,UVB, but lutein makes a little exception. The ratio of the two ratios—[(K1,UVB/ K1,UVA)]/[(K1,UVC/K1,UVB]) is quite different for the involved carotenoids: the [(K1,UVB/K1,UVA)]/[(K1,UVC/ K1,UVB)] ratio is 4 for b-carotene, 2.8 for lycopene, 0.8 for lutein and 4.1 for neoxanthin. Neoxanthin, with ratios K1,UVB/K1,UVA ¼ 6.2, and [(K1,UVB/K1,UVA)]/ [(K1,UVC/K1,UVB)] ¼ 4.1, is the most sensitive to the change of UV-photons energy input. Since neoxanthin contains four oxygens (including the one closing epoxy moiety—Fig. 1), it looks like the differences between the chemical structures among the investigated carotenoids (Fig. 1) play a non-negligible role. At the same time, neoxanthin has the lowest K1 values (Table 1), compared to all investigated carotenoids and all three UV-irradiation ranges, which indicates that neoxanthin antioxidant function is the most resistant to UV-irradiation inside the same UV-range. UV-induced peroxidation of carotenoids free lecithin aqueous solution has been monitored by TBA–MDA test, too. Rate constants for this reaction (K2), for all three UVranges, have been presented in third column of Table 1. The UV-photons energy input plays again a crucial role in the peroxides production: irradiation by UV-C photons led to the fastest lecithin peroxidation, followed by UV-B and UV-A irradiation, respectively. The UV-induced peroxidation of lecithin in the presence of carotenoids (e.g. in lecithin/carotenoids mixtures) has been also monitored by TBA–MDA test. The belonging rate constants (K3), for all three UV-ranges and all investigated carotenoids, are presented in fourth column of Table 1. The UV-photons energy input plays again major role in the lecithin peroxide production. UV-C photons induce the fastest lecithin peroxidation, followed by UV-B and UV-A photons, respectively, and the same answer has been noticed in the presence of all investigated carotenoids. The K3,UVC/ K3,UVB ratio is 2.9 for b-carotene, 3.0 for lycopene, 2.8 for lutein and 2.4 for neoxanthine, while rate constants ratio K3,UVB/K3,UVA is 4.2 for b-carotene, 3.2 for lycopene, 3.4 for lutein and 4 for neoxanthine. The ratio of the two ratios—[(K3,UVB/K3,UVA)]/[(K3,UVC/K3,UVB]) is 1.4 for b-carotene, 1.1 for lycopene, 1.2 for lutein and 1.7 for
ARTICLE IN PRESS 40
D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41
neoxanthine. The cited ratios for the rate constant K3 are very similar to the K2 constant’s ratios (peroxidation of pure lecithin—Table 1)—K2,UVC/K2,UVB (3.6, 4.3, 2.5 and 3); K2,UVB/K2,UVA (5.1, 4.4, 4.3 and 4); [(K2,UVB/ K2,UVA)]/[(K2,UVC/K2,UVB] (1.4, 1.0, 1.8 and 1.3, respectively). Estimation of antioxidant activities of the four carotenoids in the presence of lecithin comes from comparison of K2 and K3 rate constants (Table 1). Even a brief look shows that these values (obtained for the same UV-range and for the same carotenoid) are generally very close. That means that carotenoids antioxidative control of the lecithin lipid peroxidation process is of marginal importance in a (highly unordered) and UV-irradiated homogeneous solution where all present radicals are free to move in any direction; this certainly leads to smaller probabilities for at least some radical interactions and the consequent scavenging impact. Only b-carotene has shown some antioxidant activity under these conditions (K2/K3 ratio is 1.7, 1.3 and 1.1 for UV-C, UV-B and UV-A photons, respectively—Table 1). The antioxidant activity of b-carotene is proportional to the UV-photons energy input, i.e. decrease of UV-photons energy input leads to the lower antioxidant activity. However, it is reasonable to expect that in a very constraint, space-limited systems like micelles or monolayers, where movement of free radicals is highly steric dependent due to predominant ‘‘cage effect’’ (Markovic and Patterson, 1993; Markovic et al., 1990; Markovic, 2001, 2004) scavenging capacities of the employed carotenoids toward lipid radicals should be more expressed (Woodall et al., 1997b; Liebler et al., 1997; Matsushita et al., 2000). To conclude, (i) decrease of the carotenoids mediated inhibition of UV-induced lecithin peroxidation, i.e. decrease of antioxidant activity of investigated carotenoids, highly depends on UV-photons energy input and their chemical structures; (ii) the investigated carotenoids (bcarotene, lycopene, lutein and neoxanthin) have a marginal remained antioxidant activity to prevent UV-induced lecithin peroxidation in highly unordered homogeneous solution and (iii) antioxidant activity of neoxanthin appears to be the most resistant toward UV-irradiation, but, at the same time, its antioxidant activity is the most affected by change of UV-photons energy input (from UVC via UV-B to UV-A). Acknowledgment Dragan Cvetkovic is a recipient of a fellowship granted by Ministry of Science and Environmental Protection, Republic of Serbia. References Aikens, J., Dix, T., 1993. Hydrodioxyl (perhydroxyl), peroxyl, and hydroxyl radical-initiated lipid peroxidation of large unilamellar vesicles (liposomes): comparative and mechanistic studies. Arch. Biochem. Biophys. 305 (2), 516–525.
Backman, H., Risch, N., 1991. Chlorophylls. CRC Press, Boca Raton, FL, p. 103. Baker, R., Gunther, C., 2004. The role of carotenoids in consumer choice and the likely benefits from their inclusion into products for human consuption. Trends Food Sci. 15, 484–488. Burton, G., Ingold, K., 1984. b-Carotene: an unusual type of lipid antioxidant. Science 224, 569–573. Choi, C., Kim, S., Hwang, S., Choi, B., Ahn, H., Lee, M., Park, S., Kim, S., 2002. Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-guided comparison. Plant Sci. 163, 1161–1168. Deng, W., Fang, X., Wu, J., 1997. Flavonoids function as antioxidants: by scavenging rective oxygen species or by chelating iron? Radiat. Phys. Chem. 50 (3), 271–276. Erben-Russ, M., Michel, C., Bors, W., Winter, R., Saran, M., 1987. Absolute rate constants of radical reactions in aqueous solution. J. Phys. Chem. 91, 2362–2365. Fernandez, J., Perez-Alvarez, J., Fernindez-Lopez, J., 1997. Thiobarbituric acid test for monitoring lipid oxidation in meat. Food Chem. 59 (3), 345–353. Frank, H., Cogdell, R., 1996. Carotenoids in photosynthesis. Photochem. Photobiol. 63 (3), 257–264. Girotti, A., 2001. Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms. J. Photochem. Photobiol. B 63, 103–113. Haila, K., 1999. Effects of carotenoids and carotenoid-tocopherol interaction on lipid oxidation in vitro. Academic Dissertation, University of Helsinki, Department of Applied Chemistry and Microbiology. Halliwell, B., Chirico, S., 1993. Lipid peroxidation: its mechanism, measurement, and significance. Am. J. Clin. Nutr. 57 (5), 715–725. Heijman, M., Nauta, H., Levine, Y., 1985. A pulse radiolysis study of the dienyl radical in oxigen-free linoleate solution: time and concentration dependence. Radiat. Phys. Chem. 26, 73–82. Heim, K., Tagliaferro, A., Bobilya, D., 2002. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J. Nutr. Biochem. 13, 572–584. Hynninen, P., 1991. Modifications. In: Scheer, H. (Ed.), Chlorophylls. CRC Press, Boca Raton, FL, pp. 145–209. Ichihashi, M., Ueda, M., Budiyannto, A., Bito, T., Oka, M., Fukunaga, M., Tsuru, K., Horikawa, T., 2003. UV-induced skin damage. Toxicology 189, 21–39. Krinsky, N., Yeum, K., 2003. Carotenoid–radical interaction. Biochem. Biophys. Res. Commun. 305, 754–760. Liebler, D., Straton, S., Kaysen, K., 1997. Antioxidant actions of b-carotene in liposomal and microsomal membranes: role of carotenoid-membrane incorporation and a-tocopherol. Arch. Biochem. Biophys. 338, 244–250. Markovic, D., 2001. Benzophenone-sensitized peroxidation in compressed lipid monolayers at airwater interface. Collect. Czech. Chem. Commun. 66, 1603–1614. Markovic, D., 2004. Photochemistry of aromatic cetones in sodium dodecyl sulphate micelles in the presence of unsaturated fatty acids. J. Serb. Chem. Soc. 69 (2), 107–115. Markovic, D., Patterson, L., 1989. Radical processes in lipids. Selectivity of hydrogen abstraction from lipids by benzophenone triplet. Photochem. Photobiol. 49, 531–535. Markovic, D., Patterson, L., 1993. Benzophenone-sensitized lipid peroxidation in linoleate micelles. Photochem. Photobiol. 58, 329–334. Markovic, D., Durand, T., Patterson, L., 1990. Hydrogen abstraction from lipids by triplet states of derivatized benzophenone photosensitizers. Photochem. Photobiol. 51, 389–394. Matsushita, Y., Suzuki, R., Nara, E., Yokoyama, A., Miyashita, K., 2000. Antioxidant activity of polar carotenoids including astaxanthinb-glucoside from marine bacterium on PC liposomes. Fisheries Sci. 66, 980–985. McBride, J., Kraemer, W., 1999. Free radicals, exercise, and antioxidants. J. Strength Cond. Res. 13 (2), 175–183.
ARTICLE IN PRESS D. Cvetkovic, D. Markovic / Radiation Physics and Chemistry 77 (2008) 34–41 Middleton, E., Teramura, A., 1993. The role of flavonol glycoside and carotenoids in protecting soybean from ultraviolet-B damage. Plant Physiol. 103, 741–752. Mimica-Dukic, N., 1997. In vivo i in iitro ispitivanja antioksidantnih svojstava biljnih ekstrakata. Arh. Farma. 5 (7), 475–493. Ouhtit, A., Ananthaswamy, H., 2001. A model for UV-induction of skin cancer. J. Biomed. Biotechnol. 1 (1), 5–6. Palozza, P., Krinsky, N., 1992. Antioxidant effects of carotenoids in vivo and in vitro: an overview. Methods Enzymol. 213, 403–420. Paust, J., 1991. Recent progress in commercial retinoids and carotenoids. Pure Appl. Chem. 63 (1), 45–58. Peng Lim, B., Nagao, A., Terao, J., Tanaka, K., Suzuki, T., Takama, K., 1992. Antioxidant activity of xanthophylls on peroxyl radicalmediated phospholipid peroxidation. Biochim. Biophys. Acta 1126, 178–184. Pfeifer, G., You, Y., Besaratinia, A., 2005. Mutations induced by ultraviolet light. Mutat. Res. 571, 19–31. Rontani, J., 2001. Visible light-dependent degradation of lipidic phytoplantonic components during senescence: a review. Photochemistry 58, 187–202. Ross, L., Barclay, C., Vinqvist, M., 1994. Membrane peroxidation: inhibiting effects of water-soluble antioxidants on phospholipids of different charge types. Free Radic. Biol. Med. 16 (6), 779–788. Saija, A., Scalese, M., Lanza, M., Marzzullo, D., Bonina, F., Castelli, F., 1995. Flavonoids as antioxidanta agents: importance of their interaction with biomembranes. Free Radic. Biol. Med. 19 (4), 481–486. Scheer, H., 2003. The pigments. In: Green, B., Parson, W. (Eds.), Light Harvesting Antenas in Photosynthesis. Kluwer Academic Publishers, Dordrecht, pp. 29–81. Schwarz, T., 1996. Effects of UVA light on the immune system. A settled issue. Eur. J. Dermatol. 6 (3), 227–228. Simic, M., 1992. Carotenoid free radicals. Methods Enzymol. 213, 444–454.
41
Strid, A., Chow, W., Anderson, J., 1990. Effects of supplementary ultraviolet-B radiation on photosynthesis in Pisum sativum. Biochim. Biophys. Acta 1020, 260–268. Svec, W., 1978. The Porphyrins. Academic Press, New York, p. 342. Svec, W., 1991. Chlorophylls. CRC Press, Boca Raton, FL, p. 89. Teramura, A., Ziska, L., 1996. Ultraviolet-B ratiation and photosynthesis. In: Photosynthesis and the Environment. Kluwer Academic Publishers, Dordrecht, pp. 435–450. Tian Li, Q., Huang Yeo, M., Kheng Tan, B., 2000. Lipid peroxidation in small and large phospholipid unilamellar vesicles induced by watersoluble free radical sources. Biochem. Biophys. Res. Commun. 273, 72–76. Van der Sluis, A., Dekker, M., Verker, R., Jongen, W., 2000. An improved, rapid in vitro method to measure antioxidant activity. Application on selected flavonoids and apple juice. J. Agric. Food Chem. 48, 4116–4122. Vermer, B., Wintzen, M., Claas, F., Schothorst, A., Hurks, H., 1996. UVinduced immunosuppression; the critical role of wavelength. Eur. J. Dermatol. 6 (3), 231–233. Wheatley, R., 2000. Some recent trends in the analytical chemistry of lipid peroxidation. Trends Anal. Chem. 19 (10), 617–628. Woodall, A., Lee, S., Weesie, R., Jackson, M., Britton, G., 1997a. Oxidation of carotenoids by free radicals: relationship between structure and reactivity. Biochim. Biophys. Acta 1336, 33–42. Woodall, A., Britton, G., Jackson, M., 1997b. Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals: relationship between carotenoid structure and protective ability. Biochim. Biophys. Acta 1336, 575–586. Wrona, M., Korytowski, W., Rozanowska, M., Sarna, T., Truscott, G., 2003. Cooperation of antioxidants in protection against photosensitized oxidation. Free Radic. Biol. Med. 35 (10), 1319–1329. Young, A., 1996. Does UVA exposure cause human malignant melanoma? Eur. J. Dermatol. 6 (3), 225–226.