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In Vitro Studies Regarding the Antioxidant Effect of Rhodoxanthin and Canthaxanthin

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In Vitro Studies Regarding the Antioxidant Effect of Rhodoxanthin and Canthaxanthin IN VITRO STUDIES REGARDING THE ANTIOXIDANT EFFECT OF RHODOXANTHIN AND CANTHAXANTHIN Associate professor Sanda ANDREI*, PhD Associate professor Adela PINTEA*, PhD Diana CRAINIC*, PhD Student Dumitrita RUGINA*, PhD Researcher Lecturer Adela BUNEA*, PhD Abstract Evaluation of antioxidant action of rhodoxanthin compared with that of canthaxanthin was performed using a human retinal cell line D407 (RPE), in which oxidative stress was induced by administration of H2O2. The incorporation of these carotenoids in the cells was determined by UV-Vis spectrophotometry, after extraction from cell pellet. RPE cultured cell can incorporate rhodoxanthin in a rate between 1.28 and 1.76% while for canthaxanthin, procents were lower, values being between 0.88 and 0.93%. The effects of carotenoids on the cells vialbility and cells antioxidant defense were also investigated. Cells viability was determined by MTT assay and antioxidant effect by intracellular ROS assay. In rhodoxanthin treated samples was observed a decrease in viability within first 24 hours, then, at 48 hours, cell viability increased, reaching high values, of 92.37%. In canthaxanthin, viability decreased at 24 hours, reached a value of 36.38% and then increased at 48 h reaching 97.81%. In the presence of carotenoids ROS concentration decrease, the antioxidant effect of rhodoxanthin is comparable to that observed in the case of canthaxanthin. Keywords: oxidative stress, rhodoxanthin, canthaxanthin, RPE cells Introduction Oxidative stress is a term commonly used to denote the imbalance between the concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the antioxidative defense mechanisms in all living cells [Lykkesfeldt at al., 2007]. The oxidative stress is the cause of occurrence and evolution of various pathological states, such as the degenerative diseases, cardiovascular diseases, immune-inflammatory lesions, nervous system diseases, cancer [Poulsen, 2005; Hwang and Kim, 2007]. The effect of ROS is balanced by the antioxidant action of non-enzymatic antioxidants, as well as by antioxidant enzymes.. Unlike the enzymatic antioxidant systems that contain a reduced number of antioxidant enzymes, the non enzymatic systems contain a large molecules variety. According to the solubility, non-enzymatic * University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, 3-5 Mănăstur Street, Cluj-Napoca, Romania, e-mail: [email protected] Cluj Veterinary Journal, 16(2)/2009, pp. 14-19 14 antioxidants can be classified in two big classes: liposoluble antioxidants (vitamin A, carotenoids, tocopherols etc.) and hydro soluble antioxidants (vitamin C, glutathione and other molecules). The carotenoid pigments are among the most widespread natural pigments. Although they are biosynthesized only by plants, they can be taken and stocked by animals, especially in the adipose tissue, ovaries, blood, hepatic tissue, milk, egg yolks, exoskeleton of marine animals etc. From the chemical structure point of view, the carotenoids are classified in hydrocarburic and oxygenized derivates or xanthophylls. Many of the researches regarding the antioxidant function, realized on biological systems, proved that both the hydrocarburic carotenoids and the xanthophylls, or even the apocarotenoids, function as antioxidants. The carotenoids from different animal tissues interact with oxygen reactive species such as: the singlet oxygen, peroxide radicals, hydroxyl radicals, alcoxyl radicals, and superoxide radical [Krinsky, 1994; Sarkar et al., 1995]. Many researches regarding the carotenoid pigments proved that these molecules function as antitumoral factors. In the beginning the studies focused on the antitumoral effect of -carotene, but now the xanthophylls are more studied. Numerous studies (epidemiological, in vitro or in vivo) proved the antioxidant / antitumoral capacities of asthaxantin and canthaxanthin [Ribaya et al., 2004; Agarwal, 2000, Andrei et al. 2003]. Rhodoxanthin represents a less studied carotenoid pigment. Chemically, it belongs to the xanthophylls group, along with the canthaxanthin and asthaxanthin, but it has a retro type polyenic system. Taxus baccata and Potamogeton natans are the only plants in Romania rich in carotenoid pigments with retro type polyenic system: rhodoxanthin, esholtxanhtin, and esholtxanthona [Andrei et al., 2005]. Although considered a toxic plant, Taxus baccata (Taxaceae family), contains in its shoots a substance named tacol, used with very good results in the treatment of ovarian cancer. The plant leaves can be used internally for asthmatic bronchitis and indigestion treatments, and externally in the rheumatism treatment, as infusion. The fruits are red, gelatinous, 10 mm in diameter, and contained only one seed. This fruits are rich in carotenoids, especially with retro structure, but also in lycopine and zeaxanthin. The separation of retro-carotenoid can be realized using thin layer chromatography (TLC) and HPLC chromatography [Andrei et a.., 2005; Ren and Zhang, 2008]. The antioxidant function of canthaxanthin was certified by differet studies but the biological functions of rhodoxanthin haven`t been studied before. The objectives of this study consist in a comparative study regarding the antioxidant activity of oxygenized carotenoids, with both normal (canthaxanthin) and retro type structure (rhodoxanthin), and also in making evident the possible differences that could appear in this activity according to the structure. 15 Materials and Methods Cells culture (RPE line) The retinal pigment epithelium (RPE) is a monolayer of cells located between choroids and the photoreceptors. RPE has an important role in the functioning of retina by providing oxygen and nutrients and by removing the debris and metabolites of photoreceptors. The cells were grown in DMEM medium rich in glucose (4.5 mg / l), with glutamine, sodium bicarbonate and sodium pyruvate. A mixture of antibiotics and antifungal was added to the medium. It was used a medium with 10% fetal calf serum, then the culture was kept in an incubator with carbon dioxide (5%) at 37˚C. Cultivation was performed in sterile culture vessels. Media was changed every three days and passage was made in 5 days. At confluence, cells were trypsinized with 0.05% trypsin solution in PBS (phosphate buffered saline), incorporated into the media, centrifuged for 5 minutes at 300 g and 4˚C. Pellets were re-suspended in medium and cells were counted in the presence of Trypan Blue (0.4%). This dye allows the assessment of cell viability by counting the living and the dead cells, and then calculating the concentration of cells [Pintea et al., 2008]. Carotenoids incorporation of in RPE cells Carotenoids were purified, dissolved in a very small amount of tetrahydrofuran (so that the total concentration of tetrahydrofuran in culture medium not to exceed 0.5%) and then spectrophotometrically dosed. The required amount of pigment solution and the fetal serum culture medium were added. Culture of RPE cells located in subconfluence was treated with medium containing 5 μM carotenoids. Cells were incubated with medium containing carotenoids for 24 h and then were trypsinized after the protocol described above. Pellets of cells obtained after trypsinization were washed 3 times with phosphate buffered saline by vortex motion, centrifugation and removal of PBS. After the final wash the cell pellet was treated for 5 minutes, with cold Triton X100 2% solution. The extraction of carotenoids achieved by treatment with ethanol and hexane was repeated. Hexane phases were combined and the concentration of carotenoids was determined in cells by spectrophotometric method. Determination of cell viability - MTT method MTT method detects the activity of mitochondrial succinate dehydrogenase (SDH). SDH activity may indicate changes in cell viability when the cell undergoes a chemical or physical stress. The color yellow MTT (3 - (4,5-dimetiltiazol-2-yl) -2,5-difeniltetrazoliu bromide) is reduced to purple formazane in mitochondria of living cells. Solubility solution usually dimetilsulfoxidul (DMSO), is added to dissolve the insoluble purple formazane. Absorbance of the solution obtained is measured spectrophotometrically [Pintea et al., 2008]. For hydrogen peroxide-treated cells, the medium containing fetal serum is removed and H2O2 solution under the following recipe is added. A stock solution by diluting 30 ml of 30% H2O2 to 100 ml with PBS is prepared, obtaining a solution with a concentration of 10 mg ml H2O2/100ml. From this solution 1,7 ml H2O2 is removed, then 8,3 ml medium is added, the final concentration being 500 μM in the medium. Determination of intracellular reactive oxygen species (ROS) The method measures the levels of ROS accumulated intracellularly. Fluorescent compound: 2´,7´- diclorofluorescin diacetate (DCF) running through cell membranes rapidly and enzymatically hydrolyzed by intracellular esterases a compound nefluorescent diclorofluorescein (DCFH), which is then rapidly oxidized by reactive oxygen species in diclorofluorescein (DCF ). Fluorescence intensity 16 is proportional to the amount of reactive oxygen species formed intracellularly [Yamamoto et al., 2003; Pintea et al., 2008]. Results and Discussion As a first step we determined the proprotion in which the cells can incorporate the carotinoids present in culture medium. The results are shown in Figure 1. Rhodoxanthin presented an incorporation rate between 1.28 and 1.76% while for canthaxanthin, procents were lower,
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