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BETANIN INHIBITS MYELOPEROXIDASE/NITRITE-MEDIATED PEROXIDATION OF HUMAN LOW-DENSITY LIPOPROTEIN M. Allegra, L. Tesoriere, D. Butera, A.M. Pintaudi, M.A. Livrea Dipartimento Farmacochimico Tossicologico e Biologico - Facoltà di Farmacia - Università di Palermo - Via C. Forlanini 1 - 90134 Palermo INTRODUCTION: Betanin, the betalain red pigment occurring in the Cariophillalae order plants, including cactus pear, has recently been reported to posses reducing properties and to behave as lipoperoxyl radical-scavenger in vitro (1). In addition, this phytochemical is bioavailable, accumulates in human LDL after ingestion of cactus pear fruits, and is able to protect LDL against copper-induced oxidation in vitro (2,3). Myeloperoxidase (MPO) has been implicated in the in vivo LDL modification and atherogenesis (4). The enzyme, in the presence of nitrite, generates two powerful oxidizing agents, the tyrosyl radical and the nitrosyl one, both of which promote LDL lipid oxidation (4). Taking all this into account we have decided to investigate weather betanin could counteract MPO/nitrite-induced oxidation of LDL. MATERIALS AND METHODS: Human MPO was purchased from Calbiochem and Glucose oxidase from Sigma. All other chemical and solvents were purchased from Sigma Aldrich or Merck. Preparation of LDL. LDL was prepared from blood serum of healthy volunteers according to Kleinveld et al. (5) with minor modification and stored in the presence of 4mM EDTA at -80°C. Lipid peroxidation of LDL. Reactions were carried out according to Kostyuk et al. (6). RESULTS: Our results, indicate that betanin is able to inhibit the MPO/nitrite-induced LDL lipid peroxidation, in a dose-dependent manner in the range 1 to 10 μM (Fig.1). We have compared the effectiveness of betanin with that of two well-known physiological antioxidants: α-tocopherol and ascorbic acid. As reported (4), α-tocopherol, the most powerful radical-scavenger, is only able to partially protect LDL lipids from oxidation by the MPO/nitrite system and has scarce or no effect on the powerful hydrophilic oxidants generated by nitrite. On the contrary, vitamin C, which is able to scavenge the peroxidase-generated nitrating species, was very efficient in counteracting the MPO/nitrite-sustained lipid peroxidation. Betanin, was even more effective than vitamin C, at inhibiting the oxidative damage to LDL. The IC50 calculated for betanin (1.4 μM) was more than 10-fold lower than that for ascorbic acid (15.6 μM). Nitrite, an oxidation product of nitric oxide metabolism, and MPO are considered mediators of the LDL oxidation process in vivo. Our study shows that betanin, a phytochemical occurring in the cactus pear fruit, is able to protect LDL, in an experimental set-up of physiological relevance. In addition, the molecule acts at micromolar concentrations, and appears much more effective than ascorbic acid. Our data collectively indicate a favourable modulation of the oxidation process of LDL and may contribute to the supposed beneficial effect of cactus fruit (2). 1. Lievrea M.A. et al. (2003) in Herbal Medicines, Marcel Dekker, Inc. 537-556. 2. Tesoriere L. et al. The American Journal of Clinical Nutrition (in press). 3. Tesoriere L. et al. (2003). Free Radical Research 37, 689-696. 4. Carr A.C. et al. (2000). Arterioscler Thromb Vasc Biol. 1716-1723. 5. Kleinveld, H.A. et al. (1992). Clin. Chem. 38, 2066-2072. 6. Kostyuk V.A. et al. (2003) FEBS Letters 537:146-50. 7. Kleinveld H.A. et al. (1992). Clinical Chemistry, 38. 2006-2072. 8. Kostyuk V.A. et al. (2003). FEBS Letters, 146-153.

It is known amateur female athletes show an altered redox status [1] and the consumption of Opuntia Ficus Indica (OFI) decreases oxidative stress (OS) in healthy humans [2]. Therefore, the aim of this study was to investigate whether the supplementation with Sicilian OFI juice affected plasma redox balance following a maximal effort test in young physically active women . This study was randomized, double blind, placebo controlled and crossover design. Eight women (23.25±2.95 years old, weight of 54.13±9.05 kg, height of 157.75±0.66 cm and BMI of 21.69±0.66 kg/m2) were randomly divided into 2 groups and each group was supplied with either 50 ml OFI, diluted to 170 ml with water, or 170 ml Placebo containing the same concentration of fruit juice ingredients except for Vitamin C and indicaxanthin. They consumed OFI or Placebo every day for 3 days before of effort test on cycle ergometer and continued to take it for 2 consecutive days after testing. Blood samples were taken before and after the effort test without supplementation (baseline), pre- and post-test, 24 h and 48 h post-test with OFI or Placebo supplementation. H2O2 levels and total antioxidant capacity (PAT) were measured with photometer and resonance Raman spectroscopy [1,2]. The differences within and between groups were calculated with ANOVA analysis and considered significant with P, Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

In this work we approach the relationship between redox state and iron overload by noninvasive instrumental techniques. Intracardiac, liver iron and liver fibrosis have been monitored in transfusion-dependent thalassemia patients by magnetic resonance imaging and hepatic transient elastography examinations. These measurements have been matched with a non-invasive, and yet unexplored in clinical practice, evaluation of body’s oxidative stress through measurement of antioxidant carotenoids in skin, by a spectroscopic method based on Raman technology (RRS). The global body’s antioxidant status results from a balance between the level of antioxidants in cells and body fluids, including blood, and pro-oxidant species endogenously produced or coming from external sources. On this basis, the level of skin carotenoids can be considered a biomarker of the entire antioxidant status. In our work the use of RRS method provided information on the redox state of thalassemia patients, which was correlated with the iron status of the patients. Due to the highly adverse effects of accumulated iron, the novel, simple, non-invasive RRS to monitor dermal carotenoids with high compliance of the patients may be a useful tool for the management of thalassemia patients.

Background: Cactus pear (Opuntia ficus-indica) fruit contains vitamin C and characteristic betalain pigments, the radical-scavenging properties and antioxidant activities of which have been shown in vitro. Objective: We investigated the effects of short-term supplementation with cactus pear fruit compared with vitamin C alone on total-body oxidative status in healthy humans. Design: In a randomized, crossover, double-treatment study, 18 healthy volunteers received either 250 g fresh fruit pulp or 75 mg vitamin C twice daily for 2 wk, with a 6-wk washout period between the treatments. Before (baseline) and after each treatment, 8-epiprostaglandin F2α (8-epi-PGF2α) and malondialdehyde in plasma, the ratio of reduced to oxidized glutathione (GSH:GSSG) in erythrocytes, and lipid hydroperoxides in LDL were measured as biomarkers of oxidative stress; plasma Trolox-equivalent antioxidant activity (TEAC) and vitamins A, E, and C were evaluated as indexes of antioxidant status. Results: Both treatments caused comparable increases compared with baseline in plasma concentrations of vitamin E and vitamin C (P < 0.05); vitamin A and TEAC did not change significantly. After supplementation with cactus pear fruit, 8-epi-PGF2α and malondialdehyde decreased by ≈30% and 75%, respectively; GSH:GSSG shifted toward a higher value (P < 0.05); and LDL hydroperoxides were reduced by almost one-half. Supplementation with vitamin C did not significantly affect any marker of oxidative stress. Conclusions: Consumption of cactus pear fruit positively affects the body's redox balance, decreases oxidative damage to lipids, and improves antioxidant status in healthy humans. Supplementation with vitamin C at a comparable dosage enhances overall antioxidant defense but does not significantly affect body oxidative stress. Components of cactus pear fruit other than antioxidant vitamins may play a role in the observed effects. © 2004 American Society for Clinical Nutrition.

Sicilian cultivars of prickly pear (Opuntia ficus indica) produce yellow, red, and white fruits, due to the combination of two betalain pigments, the purple-red betanin and the yellow-orange indicaxanthin. The betalain distribution in the three cultivars and the antioxidant activities of methanolic extracts from edible pulp were investigated. In addition, the reducing capacity of purified betanin and indicaxanthin was measured. According to a spectrophotometric analysis, the yellow cultivar exhibited the highest amount of betalains, followed by the red and white ones. Indicaxanthin accounted for about 99% of betalains in the white fruit, while the ratio of betanin to indicaxanthin varied from 1:8 (w:w) in the yellow fruit to 2:1 (w:w) in the red one. Polyphenol pigments were negligible components only in the red fruit. When measured as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalents per gram of pulp, the methanolic fruit extracts showed a marked antioxidant activity. Vitamin C did not account for more than 40% of the measured activity. In addition, the extracts dose-dependently inhibited the organic hydroperoxide-stimulated red cell membrane lipid oxidation, as well as the metal-dependent and -independent low-density lipoprotein oxidation. The extract from the white fruit showed the highest protection in all models of lipid oxidation. Purified betanin and indicaxanthin were more effective than Trolox at scavenging the [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt cation radical. Cyclic voltammetric measurements show two anodic waves for betanin and indicaxanthin, and differential pulse voltammetry shows three anodic waves for betanin, with calculated peak potentials of 404, 616, and 998 mV, and two anodic waves for indicaxanthin, with peak potentials of 611 and 895 mV. Betanin underwent complex formation through chelation with Cu(2+), whereas indicaxanthin was not modified. These findings suggest that the above betalains contribute to the antioxidant activity of prickly pear fruits.