Natural and synthetic antioxidants: An updated overview

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Natural and synthetic antioxidants: An updated overview Article in Free Radical Research · October 2010 DOI: 10.3109/10715762.2010.508495 · Source: PubMed

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Natural and synthetic antioxidants: An updated overview AGNIESZKA AUGUSTYNIAK1, GRZEGORZ BARTOSZ2, ANA CˇIPAK3, GUNARS DUBURS4, L’UBICA HORÁKOVÁ5, WOJCIECH ŁUCZAJ1, MAGDALENA MAJEKOVA5, ANDREANI D. ODYSSEOS6,7, LUCIA RACKOVA5, . ELZBIETA SKRZYDLEWSKA1, MILAN STEFEK5, MIRIAM ŠTROSOVÁ5, GUNARS TIRZITIS3, PETRAS RIMANTAS VENSKUTONIS8, JANA VISKUPICOVA5, PANAGIOTA S. VRAKA6 & NEVEN ŽARKOVIC´3 1Department

of Analytical Chemistry, Medical University of Białystok, 14, Poland, 2Department of Molecular Biophysics, University of Łódz´ and Department of Biochemistry and Cell Biology, University of Rzeszów, Poland, 3Rudjer Boskovic Institute, Zagreb, Croatia, 4Institute of Organic Synthesis, Latvian Academy of Sciences, Riga, Latvia, 5Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovakia, 6Department of Chemistry, University of Cyprus, Nicosia, Cyprus, 7EPOS-Iasis, R&D, Nicosia, Cyprus, and 8Department of Food Technology, Kaunas University of Technology, Lithuania ( Received date: 15 March 2010; In revised form date: 9 July 2010 )

Abstract The current understanding of the complex role of ROS in the organism and pathological sequelae of oxidative stress points to the necessity of comprehensive studies of antioxidant reactivities and interactions with cellular constituents. Studies of antioxidants performed within the COST B-35 action has concerned the search for new natural antioxidants, synthesis of new antioxidant compounds and evaluation and elucidation of mechanisms of action of both natural and synthetic antioxidants. Representative studies presented in the review concern antioxidant properties of various kinds of tea, the search for new antioxidants of herbal origin, modification of tocopherols and their use in combination with selenium and properties of two promising groups of synthetic antioxidants: derivatives of stobadine and derivatives of 1,4-dihydropyridine.

Keywords: Antioxidants, dihydropyridine derivatives, free radicals, lipid peroxidation, oxidative stress, polyphenols, reactive oxygen species, selenium, stobadine, tea, tocopherol

Introduction: Antioxidants—modulators of redox homeostasis While oxygen is essential for aerobic organisms, it produces reactive oxygen species (ROS) and can cause oxidative stress, defined as an imbalance in cell redox reactions in favour of oxidative ones. Oxidative stress can be the result of either ROS over-production or decreased antioxidant defense [1]. Since oxygen is ubiquitous and necessary for oxidative metabolism of any aerobic organism, oxidative stress response is a common process induced by various stressful conditions. The toxicity of oxygen requires effective defense mechanisms

to maintain oxidative homeostasis and assure the cell survival, especially as ROS are produced under physiologic conditions. Generally, antioxidative defense mechanisms are grouped as enzymatic and non-enzymatic systems. Enzymatic mechanisms of ROS detoxification are enzymatic cascades leading to complete detoxification of these reactive species. By their action, they can be divided into two groups: one reacting directly with ROS, with the other acting as redox regulators [2]. For example, the importance of catalase could thus be seen not only for detoxification of hydrogen peroxide, but consequently in adaptation to endogenous

Correspondence: Grzegorz Bartosz, Department of Molecular Biophysics, University of Łódz´, Banacha 12/16, 90-237 Łódz´, Poland. Tel/Fax: 48 42 6354476. Email: [email protected] ISSN 1071-5762 print/ISSN 1029-2470 online © 2010 Informa UK, Ltd. DOI: 10.3109/10715762.2010.508495


Natural and synthetic antioxidants 1217 oxidative stress and lipid peroxidation [3], since the removal of H2O2 can modulate signal transduction and trigger proliferation of dormant tumour cells [4]. This gives new insight into catalase as a fundamental biological response modifier of redox signalling and oxidative homeostasis. Non-enzymatic antioxidative systems are not as specific as enzymatic ones, but, nevertheless, they are in the first line of antioxidative defense and are therefore of high importance in cellular response to oxidative stress. Vitamin C quenches radicals and forms an ascorbyl radical, a stable radical which causes little oxidative damage. Vitamin E is a generic name given to a group of tocopherols and tocotrienols. Vitamin E protects lipid compartments of cells by terminating the lipid peroxidation chain reaction or by inactivation of ROS, finally being regenerated by ascorbate. Vitamin E has been shown to also be involved in signal transduction by modulating specific enzymes such as protein kinase C (PKC), protein phosphatase 2A

(PP2A), protein tyrosine phosphatase (PTP), protein tyrosine kinase (PTK), diacylglycerol kinase (DAGK), 5-, 12- and 15-lipoxygenases (5-, 12- and 15-LOX), phospholipase A2 (PLA2), cyclooxygenase-2 (COX2) and the mitogen activated protein kinase (MAPK) signal transduction pathway and also transcription factors like NFκB [5]. Therefore, vitamin E is involved in the control of various cellular functions, such as apoptosis, necrosis, survival, adhesion and differentiation. Summarizing the knowledge of ROS and antioxidative defence mechanisms one should notice a feedback in the maintenance of redox balance (oxidative homeostasis) in the cell, as presented in Figure 1, on the example of physical exercise. During exercise ROS and RNS (Reactive Nitrogen Species) are generated in muscle [5]. Numerous studies demonstrated that, although ROS/RNS were previously considered to be harmful, in the case of physical exercise they induce a hormetic response leading to adaptation to oxidative stress and increasing organism tolerance to

Figure 1. Antioxidants as biological response modifiers of oxidative stress response in the cellular redox cycle. Superoxide radical (O2•) is produced under physiological conditions by NAD(P)H oxidase (NAD(P)H-OX), cyclooxygenase (COX), lipoxygenase (LOX), xanthine oxidase (XO) and by mitochondrial ubisemiquinone-cytochrome b (Q-b) cycle. O2• is then disproportionated by superoxide dismutase (SOD) to hydrogen peroxide (H2O2), which can be further neutralized to water by catalase (cat), glutathione peroxidase (GPX) or can undergo Fenton reaction. Oxidized glutathione (GSSG) from GPX reaction is regenerated by glutathione reductase (GR), which cycles with glucose-6-phosphate dehydrogenase (G6PD). The most reactive and harmful hydroxyl radical (•OH) attacks PUFAs in cell membranes defended by lipid-soluble antioxidants such as tocopherol (recycled by water-soluble antioxidants like vitamin C). In the case of severe oxidative stress the lipids undergo self-catalysed lipid peroxidation (LPO) resulting in production of α,β-unsaturated aldehydes, especially 4-hydroxynonenal (HNE). HNE can induce further production of ROS on one hand, but on the other it can change gene expression through various signalling pathways such as Nrf2/ARE element, thereby stimulating its own detoxification and increasing overall antioxidative defense mechanisms necessary to maintain oxidative homeostasis. Therefore, reactive aldehydes together with ROS participate in a complex interplay of epigenomic signalling pathways and involve endogenous and exogenous antioxidants as biological response modifiers that maintain oxidative homeostasis.


1218 A. Augustyniak et al. stress [6]. These discoveries pointed out the necessity to maintain the natural oxidative homeostasis of the organism and the importance to help the organism to keep this homeostasis in illness. For this reason the efforts were raised to synthesize multi-functional antioxidants which could be considered as ‘biological response modifiers’ maintaining oxidative homeostasis, both in health and in disease. While it is certain that endogenous antioxidants, both enzymatic and non-enzymatic, may be considered as biological response modifiers to oxidative stress and together with the products of lipid peroxidation, in particular 4-hydroxynonenal (HNE), and ROS as factors maintaining oxidative homeostasis, it remains to be further evaluated if exogenous natural and synthetic antioxidants may act so too. World-wide studies of antioxidants concern both natural and synthetic antioxidants. The aim of the present review is presentation of some results and perspectives of antioxidant studies performed by participants of COST B-35 Action between 2006 and 2010. They concerned natural antioxidants of various origin, including semisynthetic oxidants obtained on the basis of natural compounds and fully synthetic antioxidants. Advances in the search and characterization of new natural antioxidants The search, characterization and application of natural antioxidants remain in the focus of numerous research teams all over the world. Therefore, the scope of information in this area is extremely large, diverse and rather difficult for systematic reviewing and assessment. For instance, search in the ISI WEB of KnowledgeSM database (http://apps. isiknowledge.com/summary.do?qid 1&product WOS&SID Q195a7cggB4a9IoG7GD&search_ modeGeneralSearch) by using keyword combination ‘natural antioxidant’ gave 6718 hits, including 657 review articles, while another database (PubMed http://www.ncbi.nlm.nih.gov/sites/entrez) gave 9584 hits including 1244 review articles (date of access: 7 June 2010). The interest in natural antioxidants is determined by the universality of their action in various redox systems and consequently broad spectra of possible applications: antioxidative phytochemicals are considered as functional ingredients for pharmaceuticals, functional foods, dietary supplements, animal feed, cosmetics and other products. For instance, the interest in natural antioxidants to be used for the stabilization of lipid containing foods has increased remarkably because of the emerging information about possible toxicity of synthetic antioxidants as well as consumer preferences towards natural food additives [7]. Phytochemicals are biosynthesized as secondary metabolites by the thousands of plant kingdom species. Therefore, the majority of publications on natu-

ral antioxidants have been reporting antioxidant properties, composition, bioactivities and applications of the preparations isolated from one or more plant species, which most frequently include berries, fruits, vegetables, medicinal, aromatic plants, spices and other botanicals. Generally, the studies of natural antioxidants are related to several important research tasks, such as exploratory assessment of unstudied and poorly investigated botanical sources; developments in agrotechnology of cultivating, processing and applications of well-established raw materials for the commercialization of natural antioxidants; methods of isolation, fractionation, separation and purification of antioxidatively active substances; elucidation of chemical structures of natural compounds and characterization of their properties in vitro, in vivo and in situ as well as the ways of applications in the production of healthy foods and other products. In most cases, the final tasks in search and characterization of natural antioxidants are either development of commercial ingredients for the stabilization of oxidizable substrates, particularly unsaturated fatty acids in foods, or effective components for functional foods, dietary supplements, pharmaceuticals and cosmetics demonstrating beneficial health effects. The achievements of such tasks are associated with many analysis, assessment and technological development steps, which are summarised in Figure 2, showing that comprehensive characterization of plant preparations requires a great number of assays, which might be expensive, timeconsuming and in most cases provide only limited information on their properties and health benefits. Taking into account the diversity of the topics associated with investigations of natural antioxidants as well as a large amount of information which is regularly reviewed, this chapter is not aiming at a comprehensive review of recent achievements in this area of research. Instead of that it is intended to discuss some important and challenging issues by selecting some typical examples from the most recent publications and our own research results. Botanicals as the main source of natural antioxidants Studies of antioxidants present in plants and foods are one of the most popular topics in the area of food and agriculture today. Hundreds of botanical species were studied until now and these studies discovered thousands of various compounds possessing antioxidative properties, which are used by the plants for different biological tasks, e.g. as a photoprotective defence systems. Consequently, every plant species, sub-species, variety and cultivar may be an object for the assessment of antioxidant potential and contributing compounds. On the other hand, the main biochemical synthesis pathways are common for many plant species; therefore, the main antioxidatively active structures elucidated in various botanicals are


Natural and synthetic antioxidants 1219

Figure 2. General scheme in the search, characterization and application of natural antioxidants from botanical sources.

often similar. These structures include simple phenolic compounds, phenolic acids, flavonoids, coumarins, sesquiterpene lactones, terpenoids and their derivatives as well as other classes of phytochemicals. Considering many possibilities of substitutions and intermolecular binding (esterification, glycosidation), as well as improvements in analytical methods, the number of identified natural antioxidants is rapidly increasing. For instance, more than 4000 species of flavonoids, which are well-known natural antioxidants, have been identified, many of which are responsible for the attractive colours of flowers, fruit and leaves [8]. It may be observed that in recent years many publications were focused on screening of unstudied or less studied genera and/or individual botanical

species. These studies resulted in identification of new natural compounds and selection of promising species in terms of their expected use for the isolation of bioactive constituents. For instance, the following classes of natural compounds were presented in the aerial parts of most recently reviewed Potentila species: flavonoid aglycones (22), flavonoid O-glycosides and O-glucuronides (36), hydrolysable tannins and related compounds (13), precursors of condensed tannins (4), triterpenoids (25), organic acids and phenol carboxylic acids (14), coumarins (4), carotenoids (2), sterols (6), megastigmanes (4) and others (3) [9]. In total, 120 structures were reported in the review of Rhaponticum carthamoides, including steroids, particularly ecdysteroids, and phenolics (flavonoids and phenolic acids) accompanied by polyacetylenes,

1220 A. Augustyniak et al. Table I. Investigation steps, methods and progress in characterization of botanicals: case study—sweet grass (Hierochloe odorata). Investigation step Assessment of available information


Extraction and preliminary assessment of antioxidant/ radical scavenging capacity by various chemical and/or biological assays in batch

Methods Literature survey, information on uses in folk medicine, foods and beverages

Result Very few records, used to flavour alcoholic beverages, no information on antioxidant properties, active compounds Sweet grass extract reported as a very strong antioxidant for the first time.

Extraction with acetone, measurement of total phenolics and antioxidant activity in rapeseed oil Extraction with methanol:water:acetic Acetone extract was stronger radical; scavenger acid or acetone and testing by DPPH however, the yield of polar extract was and ABTS radical scavenging assay remarkably higher Fractionation and isolation of Fractionation with hexane, tert-butyl TBME and B fractions were strongest radical active fractions/components methyl ether (TBME), butanol (B) scavengers; HPLC-UV-DPPH revealed one followed by original batch and water; testing by DPPH free very strong radical scavenging compound; assay until the separated radical scavenging assay. TBME and active fractions collected for further analysis compounds are pure enough B fractions were analysed by on-line to identify the active HPLC-UV-DPPH method and compound(s); further fractionated by TLC and chromatographical separation on silica gel column. of complex mixtures and Characterization of redox properties, Similar to quercetin; effectively neutralized the on-line assessment of the reactivity with peroxidase, effect of singlet oxygen to erythrocytes antioxidant/radical scavenging of singlet oxygen (retarding photo-oxidation); the products of scavenging peroxidase-catalysed oxidation of DHC react with the –SH groups and may act as oxidizing substrates for flavoenzymes 1H, 13C, 2D NMR, MS-EI, UV was Structure elucidation of the 5,8-dihydroxycoumarin (DHC) and 5-hydroxyactive components by used for structure elucidation 8-O--D-glucopyranosyl benzopyranone spectroscopic and chemical identified; the compounds were very strong methods radical scavengers Pharmacological, toxicological Cytotoxicity in bovine leukaemia DHC possessed the pro-oxidant character of testing, evaluation of other virus-transformed lamb kidney cytotoxicity, which correlated with the properties by using in vivo fibroblasts (line FLK) cytotoxicity of flavonoids and in vivo assays Assessment of genotoxicity using DHC was not genotoxic in CA SMART chromosome aberration (CA) and systems in vivo; however it induced sister chromatid exchange (SCE) significant increase of SCE and a slight tests in human lymphocytes in vitro increase of CA in human lymphocytes and Drosophila melanogaster somatic in vitro, and significant increase of mutation and recombination test micronucleus in rat bone marrow cells (SMART) in vivo in vivo Assessment of some pharmacological DHC rich fraction inhibited vascular smooth properties muscle contractility and was toxic for them in high concentrations; low doses only slightly reduced the contraction ability of small arteries and did not have any negative effect on relaxative function of endothelium and even muscles (possibly some anti-hypertensive effect) Technological developments, Isolation of fractions enriched with Optimization of extraction procedure, extracts in situ evaluation, application DHC and its glycoside by using containing up to 20% of active compounds; trials different combinations of solvent, possibility of industrial up scaling supercritical fluid and microwave assisted extractions. Addition of sweet grass carbon dioxide No significant effect on the formation of extract in Dutch style fermented peroxides. Determination of TBARS revealed sausages with the aim of retarding even some slight pro-oxidative effect, while in lipid oxidation hexanal assay only a negligible antioxidative effect was observed. The influence of sweet grass extract Significant increase of PhIP concentration was and its fraction rich in DHC on the observed in meat samples with plant origin formation of 2-amino-1-methyl-6ingredients as compared with the samples phenylimidazo[4,5-b]pyridine (PhIP) without additives in meat was studied The antioxidant activities of extract The antioxidant activity was about the same in were studied in emulsions of lard the two substrates. The stability against and rapeseed oil using soy lecithin as autoxidation was substantially increased by an emulsifier and addition of cupric sweet grass extract. The stability was acetate as an oxidation catalyst particularly high, if citric acid and/or ascorbyl palmitate were added to plant extract

References [17]

[17]

[18]

[18]

[19]

[18]

[19]

[20]

[20]

[365]

[22]

[23]

[24]



Figure 3. Effect of 0.1% plant acetone extract additives on the formation of peroxides in rapeseed oil at 80°C. PV, peroxide value; BHT concentration of 0.02% (left). The main antioxidants identified in sweet grass (right): 5,8-dihydroxybenzopyranone and 5-hydroxy-8-O-β-D-glucopyranosyl-benzopyranone.

sesquiterpene lactones, triterpenoid glycosides and terpenes [10]. The extracts of Potentilla fruticosa, Rhaponticum carthamoides and another, less studied plant Geranium macrorrhizum were shown to possess strong radical scavenging properties [11]; further studies of these species using batch type and on-line chromatographic and spectroscopic techniques resulted in the identification of strong antioxidants, mainly phenolic acid and flavonoid derivatives [12–14]. Characterization of new plant preparations was continued by applying preliminary toxicological assessment [15], which is also of great importance in terms of their possible applications as it is outlined in the guidance for the safety assessment of botanicals and botanical preparations for use in food and food supplements [16]. As a rule, the way from preliminary screening of plant species until commercialization of their preparations is a very long and laborious process (Figure 2). This process is chronologically illustrated by a case study example, showing progress in characterization of antioxidants in sweet grass (Hierochloe odorata), the plant which 10 years ago was completely unknown from the point of view of its antioxidative properties [17–24] (Table I). Already preliminary screening of the antioxidant power of various herb extracts in rapeseed oil revealed distinctive properties of sweet grass, which was more effective compared with such a well known species as sage; further analysis of this herb resulted in discovery of new natural compounds which were proved to be very strong antioxidants (Figure 3). However, good radical scavenging capacity is not always correlating with antioxidant activity of the same preparations used in real foods [25], as was demonstrated for H. odorata [22], G. macrorrhizum and P. fruticosa [26] extracts added to Dutch style fermented sausages. Natural antioxidants from various botanical sources have been regularly reviewed. Different approaches

were used in the published review articles; they were focused on a single species, genus, origin, popularity, applications, bioactivities, selected phytochemical groups of antioxidants, etc. For instance, Yanishlieva et al. [27], in their review of natural antioxidants from herbs and spices, presented the results on stabilization of lipids and lipid containing foods with different herbs and spices (ground materials or extracts) and reported the structure of the main antioxidatively active compounds isolated from popular spices and aromatic herbs such as rosemary, sage, oregano, thyme, ginger, summer savoury, black pepper, red pepper, clove, marjoram, basil, peppermint, spearmint, common balm, fennel, parsley, cinnamon, cumin, nutmeg, garlic, coriander, etc. Among the herbs of the Labiatae family, rosemary has been more extensively studied and its extracts are the first marketed natural antioxidants. Sage and oregano, which belong to the same family, have also gained the interest of many research groups as potential antioxidants. The review of Carbone et al. [28] is focused on the advances in functional research of antioxidants and organoleptic traits in berry crops belonging to the families Rosaceae, Ericaceae and Grossulariaceae. The fruits of the plants belonging to these families were reported as very important sources of dietary antioxidants in many articles, demonstrating that the concentration of active constituents in berries and other anatomical parts of plants depends not only on the species but also on various other factors, which may cause high variations even within the same species. Therefore, plant cultivar, climatic conditions, harvesting time, various agrotechnological practices as well as processing methods should be optimized to increase the yield of bioactive compounds in berries and other sources of antioxidants. Amarowicz and Pegg [29] reviewed research published mostly in the last 10 years on biologically-active compounds found in leguminous seeds, including phenolic acids as well as their derivatives, flavanols, flavan-3-ols, anthocyanins/anthocyanidins, condensed tannins/ proanthocyanidins, tocopherols and vitamin C. Patil et al. [30] focused their review on historical perspectives, opportunities and challenges of flavonoids, carotenoids, curcumin, ascorbic acid and citrus limonoids. Search and characterization of natural antioxidants in medicinal and aromatic herbs and spices very often are related to their uses in folk medicine, such as Ayurveda, which is supposed to be the oldest medical system in the world and which provides potential leads to find active and therapeutically useful compounds from plants. Thus, Ali et al. [31], considering the growing interest in assessing the antioxidant capacity of herbal medicines in their review of Indian medicinal herbs as sources of antioxidants, discussed 24 plants reported to have antioxidant properties. Some of the plants reviewed are part of multi-herbal preparations while others are


1222 A. Augustyniak et al. used singly. Certain herbs like Amaranthus paniculatus, Aerva lanata, Coccinia indica and Coriandrum sativum are used as vegetables, indicating that these plants could be a source of dietary antioxidant supplies. Suhaj [32] reviewed some information about the most common and most-used spice antioxidants and methods of their preparation and described their antioxidant/anti-radical properties, while Kiokias et al. [33] reviewed in vitro activity of vitamins, flavonoids and natural phenolic antioxidants against the oxidative deterioration of oil-based systems. Search of natural antioxidants in residual sources is another important trend in the area of research and development of plant origin preparations [34]. Isolation and application of bioactive components from inexpensive or residual sources, e.g. agricultural and industrial wastes, may increase economical and technological feasibility of the commercialization of natural antioxidants. For instance, it was shown that the residues collected after isolation of very expensive essential oil from black currant buds, which is used mainly in perfumery, contain strong antioxidants [35] and possibly could be used for their isolation. It seems that so-called agro and biorefinery approaches will be important trends in search and development of natural antioxidants and other bioactive constituents in the nearest future. Problems and prospects in the assessment of natural antioxidants A great number of antioxidant activity assays have been applied for the characterization of natural antioxidants and they may be classified into the three major categories: (i) assays involving actual ROSoxidizable substrate interactions; (ii) assays involving a relatively stable single oxidizing reagent; and (iii) assays relating antioxidant activity to electrochemical behaviour [36–38]. With respect to the mechanism, antioxidants can deactivate radicals by two major mechanisms, hydrogen atom transfer and single electron transfer [39]. A great number of different assay methods have been developed and applied until now and therefore it is rather complicated to compare the results reported in various publications on natural antioxidants from different sources [40]. So far as the kinetic approach provides the basis of the majority of these methods, only a few of them have been analysed from the viewpoint of chemical kinetics. The review of Roginsky and Lissi [41] was intended to close down this gap, at least partly. The methods of antioxidant assays were critically assessed in several review articles and it was attempted to propose standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. No one antioxidant capacity assay will truly reflect the ‘total antioxidant capacity’ of a particular sample [39]. Different protocols will have to be used for

evaluation of the protection of food by antioxidants and for evaluation of the health effects of antioxidants. However, taking into account many factors, Prior et al. [39] suggested that three methods, namely Oxygen Radical Absorbing Capacity (ORAC), Folin Ciocalteu phenolics assay (F-C) and Trolox equivalent antioxidant capacity (TEAC) should be standardized for use in the routine quality control and measurement of antioxidant capacity of dietary supplements and other botanicals. A recent review [42] re-evaluated various types of assays for antioxidative capacity, by grouping them into two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidation, including the thiobarbituric acid assay (TBA), malondialdehyde/high-performance liquid (MDA/HPLC) or gas (MDA/GC) chromatography assays, b-carotene bleaching assay and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the DPPH• and ABTS• reduction, FRAP, ferrous oxidation-xylenol orange (FOX), ferric thiocyanate (FTC) and aldehyde/carboxylic acid (ACA) assays. The authors of this review emphasized that most widely used spectrophotometric assays may have problems with substances exhibiting UV wavelengths similar to that of the test chemical, overall causing interference of the chemical being tested. They recommend using at least two different types of assays; one for monitoring the early stage of lipid peroxidation (b-carotene bleaching, conjugated diene or FTC); the other for monitoring the final stage of lipid peroxidation (TBA, MDA/HPLC or MDA/GC). Regardless, a big choice of various antioxidant assays new methods are being developed.Thus, most recently Pastore et al. [43] proposed a new tool to evaluate a comprehensive antioxidant activity in food extracts by using 4-nitroso-N,N-dimethylaniline (RNO) bleaching associated with linoleic acid hydroperoxidation catalysed by the soybean lipoxygenase (LOX)-1 isoenzyme (LOX/RNO reaction). This method was used to determine the antioxidant activity of pure hydrophilic and lipophilic antioxidant compounds and of mixtures of antioxidants and was able to highlight synergism (among extracts) three-times more than the ORAC method, whereas TEAC did not measure synergism under used experimental conditions. In terms of analysis performances, the methods of antioxidant capacity assays may be classified into a batch and more efficient on-line methods. The on-line methods coupling HPLC and less frequently GC to on-line, post-column (bio)chemical assays and parallel chemical analysis have proved to be very useful for rapid profiling and identification of individual antioxidatively active components in mixtures to provide a powerful method for natural product-based drug discovery. This group of methods was recently comprehensively reviewed [37,44]. Some most recent articles [45] reported the use of the on-line methods


Natural and synthetic antioxidants 1223 for the characterization of some specific antioxidant sources; however, to the best of our knowledge there were no essential developments of such assays in the last 2 years. The use of multi-well microplates is another approach for increasing the speed and the effectiveness of antioxidant capacity measurements. High-throughput methods to assess lipophilic and hydrophilic antioxidant capacity (ORAC, FRAP and iron chelating activity) of food extracts in vitro by using 96-well microplates were recently reported [46]. Blasco et al. [47] reviewed the role of electrochemical approaches in the sensing of antioxidants and their antioxidant capacity with special attention to the analytical possibilities of electrochemistry in the direct evaluation of antioxidant capacity exhibited by food and biological samples due to the termed dietary, natural or biological antioxidants (mainly polyphenols and vitamins C and E). The main electrochemical approaches used have been cyclic voltammetry (CV) and flow injection analysis with amperometric detection (FIA-ED). In addition, miniaturization is going to break new frontiers in the evaluation of antioxidant activity. Gazdik et al. [48] applied the multifunctional HPLC-ED array method coupled with a DPPH• reference; according to the authors the method appears to be the optimal analytical progress, accurately reflecting the nutritive-therapeutic properties of a fruit. Although the approach of the standardized measurement of antioxidant capacity has not been widely implemented in the search and evaluation of natural antioxidants, some new developments should be mentioned. Magalhães et al. [49] developed an automatic flow procedure based on multi-syringe flow injection analysis for the assessment of F-C reagent reducing capacity in several types of food products using gallic acid as the standard. The application of the proposed method to compounds with known antioxidant activity (both phenolic and non-phenolic) and to samples (wines, beers, teas, soft drinks and fruit juices) provided results similar to those obtained by the conventional batch method. Karyakina et al. [50] recently proposed a novel approach for assessment of total antioxidant activity by monitoring kinetics of hydrogen peroxide (H2O2) scavenging after its injection into a liquid sample under study. The pseudo-first order kinetic constants of H2O2 scavenging in the presence of different food additives correlated with total antioxidant activity of these samples evaluated via standard procedure based on lipid peroxidation. Omata et al. [51] proposed a simple method in which the total radical scavenging capacity is assessed from the bleaching of pyranine and pyrogallol red induced by free radicals generated from an azo initiator. The total content of antioxidants contained in red wine, green tea and capsis drink and their reactivities toward peroxyl radicals were measured from

the lag phase and rate of bleaching using pyranine and pyrogallol red as a probe, respectively. It was found that this method to follow spectrophotometrically the bleaching of two probes is convenient and applicable for assessment of total radical scavenging capacity of both content and activity of the antioxidants contained in beverages. Another important issue is associated with the relevance of bio(chemical) assays in vitro with expected effects of dietary antioxidants in vivo. Becker et al. [52] evaluated various types of assays for antioxidative capacity, focusing on the antioxidant mechanism of natural dietary antioxidants, particularly phenolic compounds, in lipid oxidation and concluded that it is difficult to transfer antioxidant mechanisms established in model systems and in foods to the in vivo situation and that no simple relationship has been recognized so far between antioxidant capacity determined for various foods and beverages and health benefits for humans. It seems that the main problem of such methods is related to their irrelevance to the processes taking place in the real biological systems. Consequently, screening of antioxidant capacity using simple assays in order to predict positive health effects of food is not scientifically justified. The attempts to improve in vivo predictability of stable free radical decolouration assay for antioxidant activity by using a medium which includes an aqueous buffer at physiological pH was applied by Bartasiute et al. [53]. Wolfe and Liu [54] developed a new cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts and dietary supplements. The method measures the ability of compounds to prevent the formation of fluorescent dichlorofluorescein (DCF) by 2,2′azobis(2-amidinopropane) dihydrochloride (AAPH)generated peroxyl radicals in human hepatocarcinoma HepG2 cells and it is considered as more biologically relevant than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism and location of antioxidant compounds within cells. The method was applied for studies of structure–activity relationships of flavonoids [55] and assessment of common fruits [56]. Results obtained by using the CAA method were well correlating with F-C values, however less correlating with ORAC values [56]. Separation of plant phytochemicals is another important issue, both in analysis and development of natural antioxidants. Various chromatographic methods remain the main separation techniques of natural antioxidants [57]. Electromigration based techniques are another methods of choice for the separation of natural antioxidants [58]. It may be concluded that various botanicals as a sources of natural antioxidants will remain in the focus of research in the nearest future; however, the value of

1224 A. Augustyniak et al. the results obtained by such studies will highly depend on the strategies of the evaluation of new natural preparations. The main limitations in commercialization of natural antioxidants from botanicals are associated with the final goals of their applications. In the case of foods, technological and economical aspects are playing the major role, while in the case of pharmaceuticals and nutriceuticals the lack of sound scientific evidence on their health effects in the human body is the main drawback for the wider application of new natural antioxidants.


Black and green tea as efficient antioxidants Tea infusions, consumed by two-thirds of the world’s population, are obtained from the manufactured leaves of plants Camellia sinensis and Camellia assamica. Besides its unique taste and flavour, which have popularized tea all over the world, its therapeutic potential has been proved over the last few years. Tea is manufactured in three basic forms: green, black and oolong tea and approximately from the 2.5 million metric tons of manufactured dried tea, 76– 78% is black, 20–22% green and less than 2% oolong tea [59].

Composition of tea leaves and tea beverages On average, fresh tea leaves contain in wt% of extract solids: polyphenolic compounds (13–32% wt), carbohydrates (25% wt), proteins (15% wt), lignin (6.5% wt), ash (5% wt), amino acids (4% wt), lipids (2% wt), organic acids, chlorophyll as well as carotenoids and volatile substances [59]. From the biological point of view, polyphenols make up the largest and most important group of tea leaves components which mainly contain flavanols, especially the catechins

(epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC; Figure 4) [59,60]. Other groups of flavanols such as: chalkan-flavans, biomolecular combinations of a catechin attached to a chalcone derivative; bisflavanols dimeric gallocatechins linked by C-C bonds at the B rings; dimeric proantocyanidins, condensation products of the catechins linked by C-C bonds between an A ring and a pyrane ring have also been identified in the tea leaves [59]. Other groups of polyphenols occurring in the tea leaves are flavonols existing both in the free state and as glycosides [60] and depsides which are condensation products of two different hydroxy acids, e.g. theogallin (derivatives of gallic and quinic acids, respectively) [59]. Tea leaves contain also free gallic and quinic acids as well as various amino acids including an unusual amino acid known as theanine. The protein fraction of green tea leaves includes mainly enzymes such as polyphenol oxidase, glucosidases, lipoxidases and enzymes responsible for methylxanthine synthesis [59,61]. The popularity of tea is partly due to the presence of moderate amounts of caffeine (2.5–4%). Tea leaves contain also low levels of carotenoids which are important precursors of the tea aroma. The volatile fraction of fresh green tea contains more than 60 volatile components. Tea is relatively rich in Al, Mn, K, Ca, Mg and F. Green tea is prepared by dehydration of Camellia sinensis or assamica leaves that precludes the oxidation of the contained polyphenols. For this reason the composition of dried green tea leaves is similar to that of the fresh tea leaves with regard to the major components. The composition of green tea beverage (15 g leaves/L) is: catechins 3893.9 μmol/L, theaflavins 2.59 μ mol/L, flavonols 212.9 μ mol/L, gallic acid 315.1 μmol/L, carbohydrates 1136.8 μmol/L, proteins (per amino acid residues) 129.8 μmol/L and minerals 763.9 μmol/L [59].

Figure 4. Chemical structure of catechins (A) and theaflavins (B).


Natural and synthetic antioxidants 1225 Black tea obtained through fermentation of tea leaves is oxidized and contains mainly multimeric polyphenols. In the black tea production process, ∼ 75% of catechins contained in the tea leaves undergo enzymatic transformation consisting in oxidation and partial polymerization [62]. We found that the black tea beverage (3 g/L) contains 19.57 mg/L catechins and 468.48 mg/L theaflavins while the green tea beverage (3 g/L) contains 852 mg/L catechins [63–67]. Polyphenol oxidase together with other enzymes in tea leaves takes part in the catechin oxidation process to form seven-member ring compounds known as theaflavins (Figure 4), which are dimeric catechins [68]. The characteristic element of their structure is the seven-member benzotropolone ring [69]. In addition to the theaflavins other benzotropolone compounds as well as bisflavanols, theasinensin and thearubigins were also identified in black tea, but in considerably smaller amounts [70,71]. Besides the already mentioned components, black tea contains flavonols and phenolic acids [72]. Tea contains also caffeine, amino acids including theanine and many aromatic compounds [73]. The black tea beverage (15 g leaves/L) composition is as follows: catechins 260.0 μmol/L, theaflavins 75.1 μmol/L, thearubigins 210.86 μmol/L, flavonols 139.7 μmol/L, gallic acid 527.3 μmol/L, carbohydrates 289.6 μmol/L, proteins 22.3 μmol/L and minerals 199.8 μmol/L [59]. The third form of tea is oolong tea, which is a partially oxidized product. Metabolism and bioavailability of tea polyphenols. The biological action of the main tea components, polyphenols, is connected with the bioavailability and biotransformation of these compounds, especially catechins and theaflavins, in the gastrointestinal tract and in the liver. The bioavailability compounds and distribution of tea components was investigated in the biological fluids (mainly blood) and in the tissues (including liver and brain) after treatment of rats with green as well as black tea [74–78]. It is known that catechin metabolism begins in the mouth, where conversion of EGCG to EGC and presumably ECG to EC is probably caused by microbial catechin esterase. The cleavage of the remaining galloyl groups takes place in the intestines [73]. Next free hydroxyl groups of the catechins are conjugated with glucuronic acid, sulphate, glycine or O-methylated which occurs in the jejunal and ileal sections of the small intestine. Catechins and their derivatives are transported into the liver where unmodified ones are conjugated (methylated, sulphated and glucuronidated). The catechins are readily biotransformed in the liver, although it is known that the small intestine also plays an important role. The formation of anionic derivatives by conjugation with glucuronides and sulphate groups facilitates the urinary and biliary excretion of catechins and explains their rapid elimination.

The catechins conjugates are distributed to fluids and different organs with blood and excreted into the duodenum. It has been shown that catechins occur mainly as conjugates in the blood [73]. After green t e a consumption, substantial amounts of EGC and EC were found in the oesophagus, large intestine, kidney, bladder, lung and prostate, while lower amounts were observed in different tissues [79,80]. Catechins that are not absorbed in the small intestine, as well as conjugated catechins excreted into the bile, reach the large intestine where they may be metabolized by colonic bacteria and finally absorbed [81,82]. Many studies on the humans and rats indicated that EGCG is mainly excreted through the bile while EGC and EC are excreted through the urine and bile [80], which is consistent with the observation that EGC and EC, but not EGCG are recovered from human urine samples [79]. It was shown that 47–58% of the total tea catechins is excreted to urine and excretion of the unchanged catechins in the urine is only 0.1–2% [81]. Although metabolism and bioavailability of the major antioxidants of green tea, catechins, are relatively well examined in the animal and human organisms, little information concerning bioavailability and biotransformation of black tea polyphenols is available. An increase in catechins as well as theaflavins levels in the plasma and the liver after black tea ingestion was shown [75–78,83–85]. However, these results suggest that catechins of black tea are not very well absorbed in animal organisms or are rapidly metabolized [86]. Effects of antioxidant action of different kinds of tea. The past few years have been rich in information concerning the role of tea antioxidants concerning the health benefits of tea consumption. Antioxidant properties of the main tea polyphenols, catechins and theaflavins are manifested particularly in their ability to diminish free radical generation, chelate transition metal ions, scavenge free radicals [72,87] and protect antioxidant systems [88]. In consequence, tea polyphenols, due to their multidirectional antioxidant action, significantly prevent biologically important cellular components such as lipid, protein and nucleic acids from oxidative modifications [88,89]. Prevention of free radical generation by green tea as well as catechins and TF3 appears to be due to effective inhibition of xanthine oxidase activity [90,91]. Moreover black and green tea as well as their components alone inhibit the activity of inducible nitric oxide synthase (iNOS) [79,92]. Catechins have also the ability to inhibit the activity of myeloperoxidase [93]; tea has also been proved to inhibit the activity of cyclooxygenase COX-2 and 5-, 12- and 15-lipoxygenases participating in enzymatic lipid peroxidation [94,95].


1226 A. Augustyniak et al. Many studies have demonstrated that both catechins and teaflavins, besides preventing free radical generation, have strong free radical-scavenging abilities both in vitro and in vivo [88,96]. The conversion of catechins to theaflavins during tea manufacturing does not affect their radical-scavenging potency [96]. Moreover, it was shown that the galloyl moiety of catechins and theaflavins is essential for their scavenging ability because it increases the total number of hydroxyl groups and improves the ability to donate a proton due to the resonance delocalization [97]. The ability to scavenge free radicals is partially influenced by the low values of standard one-electron reduction potential of the polyphenols. The reduction potentials of catechins and theaflavins are similar to those of vitamin E, but higher than that of vitamin C, which is one of the strongest free radical scavengers [98,99]. Considering the standard one-electron reduction potential value, polyphenols may be expected to scavenge various free radicals generated in the organism [100]. Several structures appear to be important in conferring the radical scavenging activity of catechins. The catechol in the A ring is of primary importance for the scavenging of free radicals.The orto-dihydroxyl group in the B ring participates in electron delocalization while the trihydroxyl group in the B ring stabilizes the radical form of polyphenols. A gallate moiety esterified at the 3rd position in the C ring adds next three hydroxyl groups to the polyphenol molecule. Generally, the number of hydroxyl groups determines the antioxidant capacity [101]. Therefore, EGCG and TF3, TF2, TF1 are all able to scavenge superoxide radical (O2•), but TF3 acts the most effectively [90]. In addition theaflavins have been proved to react with the superoxide radical over 10-times faster than EGCG [97]. Investigations in vitro have shown that a solution of black tea is able to scavenge also other reactive oxygen species such as singlet oxygen (1O2) and, of course, •OH [90]. In addition, catechins have been found also to efficiently scavenge •NO in vitro. Green tea is an ∼ 5-times more potent •NO scavenger than black tea [79]. Independently of the direct antioxidant action teas act indirectly by chelating metal ions and also by diminishing metal absorption from the gastrointestinal tract [102]. Moreover they also influence the antioxidant system of different tissues [75,85,103]. Antioxidant action of teas results in inhibition of lipid peroxidation. Water-soluble antioxidants cause a decrease in the free radical level in the aqueous compartment and diminish the oxidative attack on phospholipids from the aqueous phase. In addition, tea components can reduce the mobility of free radicals in the lipid bilayer since they can incorporate into the hydrophobic core of the membrane where they cause a dramatic decrease in the lipid fluidity of this region of the membrane [104]. Catechins can also interact with phospholipid head groups, particularly with

those containing hydroxyl groups and decrease also the fluidity in the polar surface of the phospholipid bilayer [105]. Green as well as black tea administration leads to changes in the content of individual phospholipids and diminution of the surface of charge density of hepatocytes and erythrocytes [106,107]. Moreover, black tea protects the membrane protein composition and of liver cells against changes caused by oxidative stress [108,109]. The protective action of tea is also manifested in its influence on the antioxidant capacity of biomembranes [110]. Tea components prevent the oxidative consumption of α-tocopherol, repairing tocopheryl radical and protecting the hydrophilic antioxidant, ascorbate [83,84], which also repairs this radical. Protection of membrane phospholipids (Figure 5) and proteins (Figure 6) by green as well as black tea was observed in the rat liver, erythrocytes and brain [66,77,111] and is particularly important for the brain that contains large amounts of polyunsaturated fatty acids and a high content of catalytically active metal ions, especially in the striatum and hippocampus [112]. The brain tissue is particularly vulnerable to membrane lipid peroxidation that disturbs fundamental functions of the brain. Lipid peroxidation may be implicated in the irreversible loss of the neuronal tissue following the brain or spinal cord injury [111]. The green tea polyphenols have been demonstrated to inhibit iron-induced oxidation of synaptosomes. Moreover, the chelating effect of green tea results in a lowering of the free form of iron and in consequence is likely to influence free radical generation and lipid peroxidation [88]. It has been shown that teas may prevent cardiovascular diseases, because drinking tea (oolong, green and black tea) affects favourably lipid metabolism [112]. Green and black tea as well as theaflavins and catechins prevent LDL oxidation and at higher concentrations are more effective than tocopherol [113]. Theaflavins inhibit LDL oxidation [114,115]. Catechins may suppress or inhibit the proliferation of smooth muscle cells of the bovine aorta which leads to luminal narrowing and sclerosis of the arteries [116]. Apart from the protection of the lipid fraction of LDL, these compounds prevent oxidation of histydyl and lysyl residues of apolipoprotein B-100 [117,118]. Green and black tea protect also other protein molecules against oxidative modifications. Suppression of the increase in the level of protein carbonyl groups and bistyrosine residues as well as of the decrease in the level of sulphydryl groups and tryptophan residues, induced by UVB irradiation, alcohol intoxication, cigarette smoking or ageing, was observed after green as well as black tea administration [83,84]. Teas have recently obtained also significant acceptance as cancer preventive substances. Epidemiological and laboratory studies have revealed that green



Figure 5. The level of thiobarbituric acid-reactive substances in the liver, brain and blood serum of 2-month old rats drinking black/green tea, chronically intoxicated with ethanol and rats chronically intoxicated with ethanol and drinking black/green tea [102]. Green tea experiment: Control group was fed control Lieber de Carli liquid diet for 5 weeks; green tea group was fed control Lieber de Carli liquid diet containing green tea (7 g/L) for 5 weeks; ethanol group was fed control Lieber de Carli liquid diet for 1 week, followed by feeding of Lieber de Carli liquid diet containing ethanol for the next 4 weeks; ethanol green tea group was fed control Lieber de Carli liquid diet containing green tea (7 g/L) for 1 week, followed by feeding of Lieber de Carli liquid diet containing ethanol as well as green tea (7 g/L) for the next 4 weeks. Data points represent mean SD, n 6 (ap 0.05 in comparison with values for control group; bp 0.05 in comparison with values for green tea group; cp 0.05 in comparison with values for ethanol group; xp 0.05 in comparison with values for 2-months group; yp 0.05 in comparison with values for 12-months group). Black tea experiment: Rats were fed a granular standard diet and water or black tea; control group was treated intragastrically with 1.8 mL of physiological saline each day for 4 weeks; black tea group has been given black tea solution (3 g/L) adlibitum for 1 week and then treated intragastrically with 1.8 mL of physiological saline and received black tea solution (3 g/L) each day for 4 weeks; alcohol group was treated intragastrically with 1.8 mL of ethanol

and black tea as well as their polyphenols administered in drinking water inhibit carcinogenesis in various organs in humans and rodents [119,120]. Teas and their polyphenols inhibit the biochemical activation of genotoxic pro-carcinogens and carcinogens metabolism. They inhibit cytochrome P450, in particular cytochrome P-450 1A1, 1A2 and 2B1 activities [73,121] and induce phase II detoxifying enzymes [122]. Metabolism of carcinogens and of anti-cancer drugs leads to formation of free radicals that may attack DNA [123,124]. Green, black as well as oolong tea have been recently reported to prevent oxidative DNA damage, measured by the level of 8-OHdG, induced by different xenobiotics [93,125–127]. Tea polyphenols affect the action of tumour promotors and transcription factors such as AP-1 or NF-κB, controlling of the activity of transforming growth factors TGF-α and TGF-β [73,128,129]. Tea polyphenols have been found to attenuate the activation of NF-κB [128,130] and AP-1 activity [131], i. a. by inhibiting kinases [114,131]. It has been shown that theaflavins inhibit the c-jun protein phosphorylation what in turn causes inhibition of the transcription factor AP-1 activity which plays a crucial role in cell proliferation and transformation [132]. Studies on human cancer cell lines showed the potency of inhibition of cell growth by catechins as well as theaflavins [133,134]. Teas protect also cellular components of different tissues against oxidative modifications appearing during ageing [82]. The protective action of green as well as black tea in physiological as well as pathological conditions is also related to the enhancement of the antioxidant capacity of the cells and body fluids [102,135,136]. Teas are thus regarded as one of the most promising chemopreventive agents. However, several investigations indicate a significant positive relationship between tea consumption and cancer development. It has been found that catechins and theaflavins, in particular when used at high non-physiological concentrations, may induce reactive oxygen species and oxidative DNA damage [137,138]. These data suggest that teas, like other natural antioxidants, can also unfortunately reveal pro-oxidative properties. This property of teas may have pathological consequences.

at doses from 2.0–6.0 g/kg b.w. every day for 4 weeks; alcohol black tea group has been given black tea solution (3 g/L) ad libitum for 1 week and then treated intragastrically with 1.8 mL of ethanol at doses from 2.0–6.0 g/kg b.w. and received black tea solution each day for 4 weeks. Data points represent mean SD, n 6 (ap 0.05 in comparison with values for control group; bp 0.05 in comparison with values for green tea group; cp 0.05 in comparison with values for ethanol group; xp 0.05 in comparison with values for 2-months group; yp 0.05 in comparison with values for 12-months group).


1228 A. Augustyniak et al.

Figure 6. The level of carbonyl group in the brain, of 2-months, 12-months and 24-months old rats drinking black/green tea [76]. Green tea experiment: Control group was fed a control Lieber de Carli liquid diet for 5 weeks; green tea group was fed control Lieber de Carli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber de Carli liquid diet for 1 week, followed by feeding of Lieber de Carli liquid diet containing ethanol for the next 4 weeks; ethanol green tea group was fed control Lieber de Carli liquid diet containing green tea (7 g/L) for 1 week, followed by feeding of Lieber de Carli liquid diet containing ethanol as well as green tea (7 g/L) for the next 4 weeks. Data points represent mean SD, n 6 (ap 0.05 in comparison with values for control group; bp 0.05 in comparison with values for green tea group; cp 0.05 in comparison with values for ethanol group; xp 0.05 in comparison with values for 2-months group; yp 0.05 in comparison with values for 12-months group). Black tea experiment: Rats were fed a granular standard diet and water or black tea; control group was treated intragastrically with 1.8 mL of physiological saline each day for 4 weeks; black tea group has been given black tea solution (3 g/l) ad libitum for 1 week and then treated intragastrically with 1.8 mL of physiological saline and received black tea solution (3 g/L) each day for 4 weeks; alcohol group was treated intragastrically with 1.8 mL of ethanol at doses from 2.0–6.0 g/kg b.w. every day for 4 weeks; alcohol black tea group has been given black tea solution (3 g/L) ad libitum for 1 week and then treated intragastrically with 1.8 mL of ethanol at doses from 2.0–6.0 g/kg b.w. and received black tea solution each day for 4 weeks. Data points represent mean SD, n 6 (ap 0.05 in comparison with values for control group; bp 0.05 in comparison with values for green tea group; cp 0.05 in comparison with values for ethanol group; xp 0.05 in comparison with values for 2-months group; yp 0.05 in comparison with values for 12-month group).

Health effects of tea consumption: Epidemiological data. Tea as a beverage is considered to be one of the most promising dietary agents for the prevention and treatment of many diseases. Several epidemiological studies have shown beneficial effects of tea in cancer, cardiovascular and neurological diseases [139–144]. Prospective cohort studies generally indicate that habitual green as well as black tea consumption has been associated with lower incidence of heart disease/ cardiac death and a reduction in the risk factor [145– 147]. Inclusion of tea in a diet decreases LDL, cholesterol and reduces LDL oxidation [148,149]. There also appears to be an immediate effect of improving the endothelial function and enhancing the blood flow [149]. These combined biochemical and physiological

effects may be important factors in the amelioration of initiation and progression of atherosclerosis demonstrated by epidemiological studies [142]. Drinking tea is also associated with decreased frequency of cancer development (Table II). However, large cohort studies show that green tea consumption provides no protection against gastric and pancreatic cancers. Green tea reveals some protective effect in specific types of cancer, including lung, breast, ovarian and prostate cancer [150–154]. Intervention studies show that green tea consumption may prevent relapse after surgical removal of colorectal adenomas and enhance survival rates in epithelial ovarian cancer [153,155]. However, the role of black tea in cancer of the gastrointestinal tract, liver and prostate was confirmed [87].

Natural and synthetic antioxidants 1229 Table II. Effect of tea consumption on the risk of cancer development. Black tea Disease


Cancer

Cardiovascular diseases (CVD)

Neurodegenerative diseases

Health effect

Green tea References

Health effect

References

Lung cancer: 427 lung cancer cases matched with 428 hospitalized controls, black tea associated with lower risk of lung cancer intake of 2 cups/day

[366]

[150]

Prostate cancer: No significant difference in risk between subjects who drank 500 mL/ day vs non-tea consumers, decrease in risk associated with tea intake of 4500 mL/day

[367]

Bladder cancer: 1452 bladder cancer cases vs 406 kidney cancer cases vs 2434 controls. No significant association between tea and risk of kidney cancer. Tea consumption of 45 cups/day (490th percentile) linked to reduced severity of bladder cancer. No evidence of a dose–response Breast cancer: for black tea, conflicting results were observed in case–control vs cohort studies, minor inverse association between black tea consumption and risk of breast cancer (1–5 cups/day)

[368]

Lung cancer: case-control study of 649 lung cancer women and 675 controls women, consumption of green tea was associated with a reduced risk of lung cancer, the risks decreased with increasing consumption Prostate cancer: Case-control study of 130 prostate adenocarcinoma patients and 274 controls. The prostate cancer risk declined with those consuming more than 1 L tea/day Bladder cancer: 14 873 men and 23 667 women Hiroshima atomic bomb survivors, green tea consumption is not related to risk of bladder cancer

Gastrointestinal cancer: Moscow; 663 cases vs 323 controls, black tea associated with lower risk of rectal cancer. Dose–response with higher concentrations of tea related to stronger associations. No significant association between tea and colon or rectal cancer, or positive association between black tea and colon cancer

[139]

Strong evidence from meta-analysis and cohort studies concerning a reduction in myocardial infarction. Supported by evidence from epidemiology, case control studies and one RCT, black tea consumption: 1 cup/day Chinese aged 45–74 years, consumption: up to 6 cups/day, black tea reduces Parkinson’s disease risk

[369]

[140,145, 375,376]

[141]

[154]

[370]

Breast cancer: Prospective study on [151] 1160 Japan females, consumption up to 6 cups/day, A decrease of the risk of cancer recurrence is observed with a consumption of 3 cups/day of tea. Asian-american women, 501 [152] breast cancer patients and 594 controls; consumption: 85.7 mL/day 0–85.7 mL/day, significant trend of decreasing risk of breast cancer with increasing amount of green tea intake Gastrointestinal cancer: Japan; 887 [371–373] gastric cancer and 28619 control age: 20–79 y and other cases; consumption of more than 6 cups/day vs never drinking decreased the risk of gastric cancer. China; study 166 [374] chronic atrophic gastritis, 133 gastric cancer and 433 controls, up to 21 cups/week, significant inverse association between tea drinking and gastric cancer 40 530 subjects, consumption of [377–381] green tea: up to 10 cups/day; the beneficial effects of green tea on CVD risk profile in more than half of the controlled trials Chinese aged 45–74 years, [141] consumption: up to 6 cups/day, no effect of green tea on PD riskDecreased risk of PD was observed: in a case-control study in the US—people consumed 2 [382,383] cups/day or more; in prospective cohort study of 30 000 Finnish drinking 3 or more cups/day


1230 A. Augustyniak et al. Human epidemiological data suggest that tea drinking may decrease the incidence of neurological disorders, because ROS play a pivotal role in the ageassociated cognitive decline and neuronal loss in neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases [141,143]. In particular, the main polyphenol constituents of tea are now being considered as therapeutic agents in well controlled epidemiological studies, aimed to alter the brain ageing processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders [141,144]. Despite numerous studies in recent years, the understanding of the biological activities and health benefits of tea polyphenols is still very limited. Further in-depth studies are needed to investigate the safety and efficacy of tea in humans and to determine their different mechanisms in health protection. Because tea is widely consumed, classifying it as a potential factor that may reduce the risk of different diseases and understanding its underlying mechanisms have important public health implications.

Refining the nature: Modulation of signalling and antioxidant activity of chromanols and selenium with chemical interventions Preventive and therapeutic interventions with naturally occurring and synthetic analogues of vitamin E and selenium have received great attention due to their wide therapeutic windows and minimally toxic effects on normal cells. The past decade has experienced the evolution of a promising group of selective agents, comprised of (a) organic selenium compounds, (b) redox-silent vitamin E analogues and (c) their combination. This chapter highlights the propensities of these agents to modulate critical signals and affect selectively a multitude of molecular targets in pre-malignant and malignant lesions. It further provides an insight into how modifications of their structural characteristics in novel selenoanalogues of vitamin E esters may alter dramatically their biological activities due to the evolution of signal-generating active intermediates and metabolites.

oxygen [156,157]; and (iii) the Hydrophobic Domain, responsible for docking the agents in circulating lipoproteins and biological membranes [158]. Vitamin E derivatives, such as the α-tocopheryl succinates (saα-toc), in which the radical inhibitory activity has been blocked by esterification of the phenolic oxygen with a dicarboxylic acid, have anti-cancer effects in a variety of malignant cell lines by suppressing DNA synthesis and inducing apoptosis with significantly higher efficacy than α-tocopherol alone [159–162]. Hydrolysis of sa-α-toc by activated esterases, however, after absorption into the cells and consequent release of the negatively charged antioxidant αtocopheryl, endows significant chemoprotective properties against several toxic factors, including toxic chemicals, chemotherapeutic drugs, peroxides and UV radiation [163]. In cancer cells the α-tocopheryl moiety is involved in protein phosphatase 2A (PP2A) activation, leading to the inactivation of protein kinase C (PKC) and the dephosphorylation of the antiapoptotic mitochondrial protein bcl-2, while the charged succinyl moiety causes destabilization of both lysosomal and mitochondrial membranes leading to additional enhancement of cytochrome c release and amplification of the pro-apoptotic signal (Figure 7). The rate of hydrolysis of sa-α-toc to α-tocopheryl is significantly reduced in cancer compared to normal cells, thus allowing for ROS accumulation and further enhancement of the mitochondrial pathway [164]. Over the past decade considerable evidence has been provided supporting that active hydrolysis products of sa-α-toc trigger apoptosis directly through cell components associated with death receptors [165– 169] and execution phase mediators [157,169, 170] or amplify and complement parallel apoptotic signalling pathways, such as those involving mitochondrial

Structural interventions in the functional domain of vitamin E The term vitamin E refers to one or more structurallyrelated phenolic compounds called tocopherols (Toc) and tocotrienols (Tot). Vitamin E derivatives have three main distinct domains, described as: (i) the Functional Domain, responsible for the antioxidant activity and, therefore, Vitamin E properties, epitomized by the hydroxyl group in α-tocopherol; (ii) the Signalling Domain, comprised of the aromatic rings (phenol-, chromanol-) and activated by the monoesterification of dicarboxylic acids with the phenol

Figure 7. Proposed model of early biochemical events and signal generation and transduction following the introduction of saα-tocopherol into the cells and its hydrolysis by active esterases.


Natural and synthetic antioxidants 1231 destabilization [157,171–174] and modulation of transcription factor mediators of apoptosis [165,168,175]. Inhibition of cell cycle progression is further implicated as a means by which sa-α-toc may block survival [176,177], inhibit proliferation of cancer cells [175–178], induce differentiation [167], halt metastasis [179] or sensitize them to other non-target selective anti-cancer agents [177,180]. The requirement for a charged group within the Functional Domain and the key role of the amphiphilicity in the biological functions of these compounds are further supported by the lack of apoptogenic properties in esters deprived of acid functionality, such as acetate analogues (αTOA) or dicarboxylic diesters. On the contrary, αtocopheryl-lysine, which bears a cationic Functional Moiety at physiological pH, exhibits a very potent proapoptotic activity [181]. Enhancement of lipophilicity reduces apoptogenic activity of tocopheryl dicarboxylates [163], most likely due to perturbation of the amphiphilic balance that maintains the conformational characteristics required for signalling modulation. The role of a free hydroxyl group on tocopheryl monoesters of carboxylic acids has been explored in recently synthesized methylated analogues of a novel series of selenoanalogues of succinate esters [182]. Anti-cancer properties of selenium compounds are greatly related to their structural characteristics Biological effects of selenium greatly depend on the wide variety of its chemical forms and not the element per se. There has been growing interest in the synthesis of organoselenium compounds for enzymology and bioorganic chemistry because these compounds are much less toxic compared to the inorganic selenium species. Several of these organoselenium compounds, including selenomethionine and aromatic selenium molecules, have been found to inhibit tumourogenesis in a variety of animal models [183–185] and to arrest the growth and induce death of human tumour cells in vitro [186]. Molecular mechanisms of cancer cell death are associated with structural characteristics of organoselenium molecules [187]. Transformation of selenium to a monomethylated metabolite is a crucial step in achieving cancer prevention through induction of apoptosis and decreased proliferation of pre-malignant cells [188]. In contrast, inorganic selenium contributing to the hydrogen selenide pool with excess of selenoprotein synthesis can lead to DNA single-strand breaks and necrosis-like, caspase-independent cell death [189]. Based on a large body of data from these studies, it is articulated that cancer chemoprevention by Se is independent of the antioxidant activity of plasma or tissue selenoproteins. The methylselenol metabolite pool has many desirable attributes of chemoprevention by targeting transformed cells and matastasis-related vascular endothelial cells, while excess in the hydrogen selenide

pool can lead to DNA single strand breaks, mediated by ROS and caspase-independent apoptosis. Blocking the conversion of hydrogen selenide to methylselenol decreased the anti-cancer activity, whereas inhibiting further methylation of methylselenol increased the efficacy. The compelling need for safe selenocompounds for long-term chemopreventive applications has led to the synthesis of minimally toxic selenoureas [190] and has further indicated the need for new chemical forms of selenium in chemopreventive and therapeutic agents. Selenium analogues of vitamin E: Combining the synergism in a single molecule The identification of synergistic effect of the methylselenol precursor methylseleninic acid with sa-α-toc in apoptosis induction through activation of initiator caspases [191] has made it even more compelling to address a multitude of challenges underlying the anticancer potential of selenium and Vitamin E analogues. These mainly involve (a) the increase of the bioavailability of selenium with concurrent decrease of its toxicity, (b) modulation of antioxidant activity of vitamin E analogues without compromising their apoptotic and immunoregulatory properties, (c) delineation of the relationship between structures, lipophilicity, acidity and biological activity and (d) enhancement of apoptotic vs necrotic properties of selenocompounds by directing the molecules towards the methylselenol pool vs the hydrogen selenide pool. A new insight into the synergism between selenium and Vitamin E was provided via a strategy involving the introduction of organoselenium and succinate moieties into the functional domains of vitamin E derivatives [182]. α-Tocopheryl-2phenylselanyl-succinate (pssa-α-toc), γ-tocopheryl2-phenylselanyl-succinate (pssa-γ-toc) and γ-tocotrienyl-2-phenylselanyl-succinate (pssa-γ-tot) have been the first selenium-containing derivatives of sa-α-toc, of γ-tocopherol and γ-tocotrienol, respectively (Figure 8A). To further decipher the impact of the presence of selenium in these esters, a similar series of thioyl-compounds where selenium was replaced by sulphur was synthesized. In vitro assessment of the DPPH scavenging activity of these compounds disclosed a 10-fold decrease of this activity for both succinate- and pssa- or ptsa-esters compared to free chromanols (Figure 8B). However, incubation of primarily cultured normal colon epithelial cells with free α-tocopherol and its sa-, ptsa- and pssaesters protected against H2O2-induced damage to DNA in a standard Comet Assay (Figure 9, Ia). Quantification of oxidative DNA damage confirmed that pssa-esters are more potent antioxidants than the ptsa- and sa-esters and all the esterified forms are more potent than free tocopherol (Figure 9, Ib). This effect is attributed to the high activity of



Figure 8. (A) Structure of α-tocopherol, γ-tocopherol and γ-tocotrienol. Green indicates the chromanol ring with the free OHfunctional domain. Sa, pssa and ptsa are the structural units replacing OH- during esterification and depriving chromanols from antioxidant activity. (B) In vitro free radical scavenging activity. The table shows the reaction rates (V0) of the reduction of H2O2 (2 mM) by PhSH (1 mM) and the second-order rate constants (k2) of the reaction of DPPH.(20.0 μM) with the antioxidants (60.0–300 μM) in methanol at 25°C. Standard deviation is given in parentheses. aCalculated from the slopes of the plots of concentration against time during the first 10 min of the reaction. Concentrations were calculated from absorption at 305 nm. bCalculated from the slope of the plot of pseudo-first-order rate kobsd. Concentrations were calculated from the absorbance at 515 nm. Modified from [182].

non-specific esterases in normal colon cells which hydrolyse sa-α-toc and which apparently have similar effect on the selenyl- and thioyl-esters.

When applied, as an approach to detect apoptotic nuclei in colon cancer cells exposed to the same compounds, SYBR Green staining revealed significant pro-apoptotic induction of apoptotic nuclei by these esters. This effect was not shared by non-esterified α-tocopherol (Figure 9, IIA). The specificity of the pro-apoptotic effect was further confirmed by the staining of normal nuclei in samples co-treated with a pan-caspase inhibitor (Figure 9, IIB). The impact of esterification in succinate monoesters and the co-existence of phenylselenylor phenylthioyl-moieties on the induction of proapoptotic responses was further correlated with the effect of structural modifications identified in the chromanyl moiety, driving the generation and transduction of molecular signals. Apoptotic cell death is disclosed by DAPI-stained nuclei and is defined by condensed chromatin, apoptotic bodies and rings (Figure 10A). Apoptotic cell death was quantified by (i) the percentage of apoptotic cells in a Trypan Blue exclusion assay and (b) caspase-3 enzymatic activity. Both assays disclosed higher pro-apoptotic activity of γ-tocopheryl esters compared to their α-counterparts and higher pro-apoptotic acticity of tocotrienyl-compounds compared to their tocopheryl conterparts (Figure 10B). The potential of the γ-chromanyl-moiety to activate pro-apoptotic and anti-proliferative pathways has been extensively documented. We identified recently 765 proteins differentially modulated by γ-tocotrienol in prostate cancer cells [169]; Odysseos et al., unpublished). Notably, the effect of phenylthioyl is comparable to that of phenylselenyl diesters, strongly supporting that structural conformations within these organic selenocompounds are those responsible for the induction of apoptotic responses since substitution of selenium with sulphur does not compromise this effect. Modulation of caspase-3 enzymatic activity by the sa-, phenylselenyland phenylthioyl-esters has followed the same trend as the number of apoptotic cells. It is thus evident that these compounds share common pathways where the amplification of the signals greatly depends on the number of esterified moieties and the structural characteristics of the chromanol ring. The augmented apoptogenicity of the tocotrienyl compounds is greatly attributed to the better membrane docking efficacy of the unsaturated phytyl chain leading to potentially higher intracellular concentrations. The correlations between structural modifications, in vitro free radical scavenging activity and peroxidation inhibition efficacy in normal living cells and proapoptotic activity in cancer cells are depicted in Figure 11. Ongoing studies with fluorescent/bioluminescent ester compounds and in vivo molecular imaging intend to reveal the active products of hydrolysis in tumour cell populations and lead to the identifica-

Natural and synthetic antioxidants 1233 tion of new molecular targets that would serve as efficacy biomarkers in future trials. Synthetic molecules with antioxidant action


In vitro studies and experiments in animal models suggest a plethora of synthetic antioxidant compounds which can be potentially useful in therapy. Various classes of synthetic antioxidants include nitroxides, spin traps [192], Mn-porphyrin superox-

ide dismutase mimics (like M40403 and M40419, AEOL-10113 and AEOL-10150) [193], salens (e.g. EUK134), able not only to dismutate superoxide but also decompose products of this reaction, hydrogen peroxide [194], GPX mimetics (ebselen, BXT51072), coenzyme Q analogues (e. g. ibedenone) [195] or aminosterols (lazaroids) [196]. In this review, two groups of promising synthetic antioxidants will be discussed: derivatives of stobadine and derivatives of dihydropyridine.

Figure 9. (I) SYBR Green staining of normal colon epithelial cells DNA. Primarily cultured normal human colon epithelial cells were incubated with isomolar concentrations of the indicated compounds over 48 h and subsequently exposed to 100 μmol/L of H2O2 for 20 min at 4°C. (a) Fluorescent microscopy following single cell alkaline electrophoresis and nuclear staining with SYBR Green disclosed inhibition of the oxidative DNA damage by both free and esterified forms of α-tocopherol. Arrows indicate different degree of oxidative damage (comet index). White: 0; pink: 1; blue: 2; yellow: 3; red: 4. (b) Quantification of oxidative DNA damage with calculation of the comet index shows statistically significant protective effect for all compounds. Pssa-esters are more potent free radical scavengers in normal cells that ptsa-esters and ptsa-esters more potent than sa-esters. (II) SYBR Green staining reveals apoptotic cell death in colon cancer cells. The grade-IV metastatic colon cancer cell line LoVo was incubated with isomolar concentrations of the indicated compounds either alone (A) or with 50 μmol/L pan-caspase inhibitor Z-VADfmk (B). Fluorescent microscopy revealed fragmented nuclei in cells treated with esterified compounds. DNA damage was not observed with caspase inhibition.



Figure 10. Structural modifications modulate apoptotic properties of vitamin E analogues and selenium. (A) Morphologic representation of apoptosis by nuclear staining with DAPI. LoVo cells were seeded on plastic chamber slides and treated as indicated with each compound at IC50 concentrations. Apoptotic cell death is assessed based on nuclear morphology. Apoptotic rings, apoptotic bodies and condensed chromatin (arrows) were visualized by fluorescence microscopy. The apoptotic efficacy of each compound is expressed as (B) the percentage of apoptotic cells determined by Trypan Blue exclusion assay and (C) DEVD-caspase proteolytic activity. Apoptotic cells were distinguished by their rough membranes, different shapes and nuclear condensation. Cell lysates were subjected to assessment of caspase-3 activity, using a caspase-3 specific fluorigenic substrate. Caspase activity is expressed in arbitrary units of proteolytic cleavage elicited by equimolar concentrations of the compounds. Pssa-esters were compared to pssa (∗), sa-esters (#) and ptsa-esters (♦). Single symbol indicates values 0.01 p 0.05. Double symbol indicates 0.05 p 0.001. Triple symbol indicates p 0.005.

Stobadine as an indole-type antioxidant standard: Physicochemical properties, mechanism of action and efficiency in comparison with Trolox Trolox (Figure 12A), a water-soluble analogue of αtocopherol, represents a popular reference antioxidant. This carboxylic acid chromane has been broadly used as a standard when screening the antioxidant efficacy of prospective antioxidants in studies involving chemical, sub-cellular, cellular and tissue experimental models of oxidative damage [39,40,197]. However, a fairly large and specific group of prospective substances with beneficial biological effects is represented by the indole-type antioxidants [198]. This puts a demand on the reassessment of the suitability of Trolox as a phenol-type reference for antioxidant studies focusing on nitrogen heterocyclic compounds. The pyridoindole stobadine (Figure 12A) has been postulated as a chain-breaking antioxidant exerting its ability to scavenge effectively a variety of reactive oxygen species [199–201]. There are more than 200 PubMed references on stobadine

Figure 11. Structure–activity relations in free tocols, sa-, pssa- and ptsa-esters: (A) apoptotic activity in malignant cells increases with esterification; binary Se esters are more potent than binary S esters and S esters are more potent than succinate monoesters; (B) in vivo antioxidant activity in normal epithelial cells increases with esterification: binary Se esters are more potent antioxidant in normal cells than binary S esters and S esters are more potent than succinate monoesters; (C) in vitro antioxidant activity decreases with esterification: binary Se esters are less potent antioxidant in vitro than binary S esters and S esters are less potent than succinate monoesters.



Figure 12. (A) Structures of stobadine and Trolox; (B) Mechanisms of free radical scavenging by stobadine and vitamin E; biologicallyrelevant coupled reactions that might recycle stobadine [201] and Trolox [214].

and other indole-type antioxidants. Several comprehensive reviews cover stobadine action in various simple chemical systems, biological models at subcellular, cellular or organ level and extensive studies in vivo in a number of free-radical disease models [202–205]. Trolox and stobadine: Physico-chemical properties. Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, is an organic acid, while stobadine, (-)-cis-2,8dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3b] indole, is an organic base. In spite of the fact that Trolox is more lipophilic than stobadine, based on log P-values 2.83 and 1.95, respectively, at physiological pH 7, stobadine preferentially distributes into a lipid compartment, while Trolox preferentially resides in the aqueous phase. The acid-basic behaviour accounts for this apparent discrepancy. With the pKa value of the carboxyl group 3.89 [206], Trolox undergoes virtually complete dissociation at physiological pH (99.92% in the COO form). On the other hand, stobadine with the pKa value of the tertiary nitrogen

of 8.5 [207] has ∼ 92% of the basic nitrogen in protonated form at pH 7. As a result of the acid-base equilibria, the corresponding distribution ratios at pH 7 of Trolox and stobadine, D 0.33 [208] and 3.72 [201], respectively, clearly favour partitioning of stobadine but not of Trolox, into the lipid phase. This may explain the low apparent antioxidant efficiency of Trolox in experimental models involving membraneous systems [209–213]. Trolox and stobadine: Redox properties. Early pulse radiolysis studies indicated differences with regard both to the site of antioxidant activity (residing in the indolic nitrogen and phenolic moiety of stobadine and Trolox, respectively) and to deprotonation mechanism following the oxidation of the parent molecules (Figure 12B). One-electron oxidation of stobadine leads to the radical cation which deprotonates from the indolic nitrogen to give a resonance stabilized nitrogen-centred radical [200].With regard to the pKa value of ∼ 5 of Trolox-derived phenoxyl radical cation [214] and its expected extremely rapid

1236 A. Augustyniak et al. Table III. Second-order rate constants of stobadine and Trolox interaction with reactive oxygen species and DPPH stable free radical. Rate constant (M1 s1) Reactive species HO• CH3COO• Cl3COO• DPPH• C6H6O• O2•− 1O 2

Stobadine 7 15.9 5 6.6 4.9 5.1 7.5

109 109 106 108 102 108 102

[200] [199] [200] [200] [210] [200] [201]

1.3 108 [200]

Trolox 8.5 1010 [219] 2.5 106 [384] 3.7 108 [214] 1.6 103 [210] 4.1 108 [214] 1.7 104 [217] 0.1 [218] pH 6 3.5 108 [385]


DPPH•, 1,1’-diphenyl-2-picrylhydrazyl.

deprotonation, no spectral evidence for generation of Trolox radical cation was obtained. However, depending on the reaction conditions, electron transfer followed by proton shift or even sequential proton loss and electron transfer (SPLET) has been suggested as a radical scavenging mechanism of phenolic antioxidants involving Trolox and α-tocopherol [215,216]. As shown in Table III, stobadine and Trolox are characterized by comparable rate constants of their interactions with the majority of individual reactive oxygen species tested. The major differences concern the second order rate constants of their reactions with superoxide and hydroxyl radicals. The data by Nishikimi and Machlin [217] point to considerably higher ksuperoxide value for Trolox than that reported for stobadine [201] (Table III). However, the study by Bielski [218] showed a notably low second order rate constant of Trolox for its reaction with superoxide (ksuperoxide 0.1 M1 s1), while Davies et al. [214] reported an apparent absence of reaction of Trolox with superoxide. Regarding the hydroxyl radical scavenging, Davies et al. [214] reported the value k•OH. for Trolox comparable with that of stobadine (Table III). Nevertheless, according to the study of Aruoma et al. [219], the second order rate constant of Trolox for scavenging HO• radicals is almost one order of magnitude higher than that of stobadine. This finding is in a close agreement with our data obtained in a study where the efficacy of stobadine and Trolox in the inhibition of hydroxyl-radical-induced cross-linking of bovine serum albumin (BSA) were compared [220]. The redox potential of stobadine (E 0.58 V) [200] is more positive than that of vitamin E (E 0.48 V) and thus, at pH 7, stobadine radical formed as a consequence of its free radical scavenging activity may subtract proton from the Trolox molecule resulting in regeneration of the parent stobadine molecule. Indeed, Steenken et al. [200] demonstrated the ability of Trolox to recycle stobadine from its one-electron oxidation product, to give a corresponding Trolox phenoxyl

radical. When stobadine and Trolox were present simultaneously in oxidatively stressed liposomes, Trolox spared stobadine in the system in a dose-dependent manner [211]; a direct interaction of Trolox with stobadinyl radical appears to be a plausible explanation. Thus, under physiological conditions, the antioxidant potency of stobadine may be increased by its interaction with vitamin E. The antioxidant action of stobadine was indeed profoundly diminished in tocopherol-deficient rat liver microsomes [221]. Analogically, in biological systems, vitamin E (E 0.48 V) can be regenerated from its phenoxyl radical by interaction with ascorbate [214] with a more negative redox potential (E 0.30 V) [222], as shown in Figure 12B. In the same way, stobadinyl radical was shown to be quenched by ascorbate, as demonstrated by the increased magnitude of the ascorbyl radical ESR signal generated in the presence of stobadine in the system of lipoxygenase arachidonate [201]. Thus, the antioxidant potency of both Trolox and stobadine within biological systems may be modulated by their interaction with other lipid- or watersoluble antioxidants. Trolox and stobadine: Antioxidant efficacies in the assay systems. In a homogeneous system, antioxidant activity stems from an intrinsic chemical reactivity towards radicals. In membranes, however, the relative reactivities may be different since they are determined also by additional factors such as location of the antioxidant and radicals, ruled predominantly by their distribution ratios between water and lipid compartments. As already mentioned, a notably lower distribution ratio of Trolox than that of stobadine may account for their different efficacies in systems involving lipid interface (membranes) in comparison to homogenous units (true solutions). In the ethanolic solution, Trolox scavenged the DPPH• radical more efficiently than stobadine, based on the initial velocity measurements [211] and comparison of rate constants [210] (Table III). In the models of oxidative damage comprising soluble proteins in buffer solutions, the water-soluble antioxidants stobadine and Trolox have free access both to free radical initiator and to protein-derived radicals. Stobadine inhibited the process of albumin oxidative cross-linking induced by the Fenton reaction system of Fe2/EDTA/H2O2/ascorbate less effectively than did Trolox [220]. The experimental IC50 values correlated well with the reciprocal values of the corresponding second order rate constants for scavenging •OH radicals. Trolox, in comparison with stobadine, was also found to be a more efficient inhibitor of AAPH-induced precipitation of the soluble eye lens proteins [223]. Conversely, protein oxidation yielding free carbonyls was more efficiently inhibited by stobadine. Both stobadine and Trolox showed comparable efficacies

Natural and synthetic antioxidants 1237 Table IV. Summary of antioxidant and protective efficacies of stobadine and Trolox in experimental models of oxidative damage. Assay system

Parameter measured

Trolox

AAPH induced LPO in DOPC liposomes [211]

IC50 (μmol/L)

25.3 14.6

93.5 8.5

BSA cross-linking induced Fe2/EDTA/H2O2/ ascorbate [220]

IC50 (mmol/L)

0.651 0.078

0.131 0.019

AAPH-induced oxidative Inhibition of protein modification of soluble precipitation eye lens proteins [223] Inhibition of protein oxidation Oxidative modification Glucose attachment into of BSA in an the molecule of BSA experimental glycation model [224]

IC50 (μmol/L)

121 15

79 8

44 8

131 20

Glycation-induced fluorescence changes of BSA Free Radic Res Downloaded from informahealthcare.com by 117.174.36.229 on 05/20/14 For personal use only.

Stobadine

Formation of DNPHreactive carbonyl groups in BSA

Cu2mediated oxidation of LDL

Inhibition of Fe2/ascorbate induced TBARS oxidative damage of rat brain homogenate [227] AAPH-induced haemolysis of rat erythrocytes [212]

Amadori product (with respect to 8.2 0.4 nmol/mg BSA for control without inhibitor) Relative fluorescence (with respect to 11.2 0.7 for control without inhibitor) Carbonyl groups (with respect to 5.6 0.4 nmol/mg BSA for control without inhibitor) Δtlag (min) (The increase in lag time given by one stobadine molecule per single LDL particle) IC50 (μmol/L)

tlag (min) (88.6 2.2 for control erytrocytes)

8.1 0.5 (0.25 mmol/L)

7.4 0.7 (0.25 mmol/L)

7.9 0.7 (0.25 mmol/L)

6.5 0.4 (0.25 mmol/L)

3.4 0.5 (0.25 mmol/L)

3.3 0.2 (0.25 mmol/L)

1.5

0.38

35

98

300 (100 μmol/L)

143.5 (100 μmol/L)

LDL, low density lipoprotein; LPO, lipid peroxidation; DOPC, dioleoyl phosphatidylcholine; BSA, bovine serum albumin; AAPH, 2,2′azobis (2-amidinopropane)hydrochloride; DNPH, dinitrophenylhydrazine; TBARS, thiobarbituric acid reactive substances.

in an experimental glycation model in preventing glycation-related fluorescence changes of BSA as well as in lowering the yield of 2,4-dinitrophenylhydrazine-reactive carbonyls as markers of glycooxidation (Table IV) [224]. On the other hand, Trolox was found to be much less effective in inhibiting AAPH-induced peroxidation of DOPC liposomes with respect to stobadine [209,210, 225] (Table IV). Stobadine, in comparison with Trolox, more effectively prolonged the lag phase of Cu2-induced LDL oxidation measured by diene formation [226]. The same pattern of efficacy in prevention of the lipid oxidation boost was shown in the system of tissue homogenate. Stobadine showed a more potent inhibitory effect than Trolox on lipid peroxidation in rat brain homogenates exposed to Fe2/ ascorbate as documented by TBARS levels (Table IV) [227]. Interestingly, in the case of alloxan-induced lipid peroxidation of heat denaturated rat liver microsomes, the inhibitory efficacy of stobadine and Trolox was comparable [228]. This finding may indicate that the critical competition of the scavengers with the alloxan-derived initiating reactive oxygen

species takes place outside the membrane in the bulk solution. In the cellular system of intact erythrocytes exposed to peroxyl radicals generated by thermal degradation of the azo initiator AAPH in vitro, stobadine protected more powerfully erythrocytes from haemolysis, as judged from the lag phase prolongation [212]. In another cellular model, stobadine increased the viability of hydrogen-peroxide treated PC12 cells more effectively than did Trolox, while both compounds reduced the content of malondialdehyde with a comparable efficiency [213]. In summary, these data underscore the structural and physicochemical differences between Trolox and stobadine as respective representatives of phenolicand indole-type antioxidants. The structural variance explains their different mechanisms of antioxidant action and variable efficacies in the range of assay systems studied, suggesting that stobadine may represent a promising indole-type reference antioxidant. In studies of indole compounds, stobadine antioxidant standard may thus be used as a more acceptable alternative to the structurally diverse Trolox.



Antioxidant properties of carboxymethylated pyridoindoles: Theoretical study of structure–activity relationships The depletion of NADPH cell stores by aldose reductase (ALR2, EC 1.1.1.21), the first enzyme of the polyol pathway, may inhibit the activity of other NADPH-requiring enzymes, including those of the glutathione redox cycle.The decreased levels of reduced glutathione increase the susceptibility of cells to damage by reactive oxygen species. Indeed, various studies have documented elevated blood and tissue levels of markers of oxidative stress in diabetic patients and demonstrated the ability of antioxidant supplementation to attenuate complications in diabetic animals. Recently novel carboxymethylated pyridoindoles (Figure 13), analogues of stobadine, have been designed, synthesized and characterized as bifunctional compounds with joint antioxidant/aldose reductase inhibitory activities, with the potential of preventing diabetic complications [229]. Here we report the results of our theoretical study on structure–activity relationships performed for their antioxidant action. The optimal geometries of the structures were obtained by the program package Spartan’08 [230]. The systematic MMFF94 conformational search was performed for all molecules given in and subsequently the low conformers were reoptimized by the AM1 and DFT methods using the B3LYP functional and the 6-31G∗ basis set. The piperidine ring was taken in the chair conformation with nitrogen in equatorial position, while nitrogen in the tetrahydropyridine ring, in the conformation derived from the former one, was in axial position. DFT results were used for EHOMO and BDErel calculation. Values of BDErel were obtained by differences of the energies of individual structures E and their relevant indolyl radical ER, i.e. BDE E ER, and BDErel were related to the lowest value (stobadine). Activa-

tion barriers EA were calculated by AM1 method as EA Ereact ETS, where Ereact was the energy of reactants (actually, it was the energy of the hydrogen bonded complex of HO• with corresponding H-N species) and ETS was the energy of the transition state. The results calculated are summarized in Table V together with the values of anti-radical activities obtained earlier in DPPH• test [209]. As expected, the values of calculated parameters for unsaturated pyridoindoles differed markedly from those for saturated compounds, in agreement with their measured activities of −ΔA5min. Using the values of EHOMO and BDErel we calculated the linear regression equations −ΔA5min 0.0031·(EHOMO) 1.7284, n 7; R2 0.92 −ΔA5min 0.0052·(BDErel) 0.2094, n 7; R2 0.94 As seen, the higher computational level led to better estimation of the structure–activity relationships, compared with our former results based only on semiempirical level [225]. According to the Arrhenius equation and using the values of activation energies we obtained the exponential regression equation: −ΔA5min 203.9·exp(0.1 EA), n 7; R2 0.96. The use of activation energies provided better results in comparison with calculations employing other parameters. The main reason is probably approaching the physical meaning of the rate constant approximated with the values of −ΔA5min. To conclude, the structure–activity relationships obtained may thus be used for a rational design of more efficient antioxidant compounds in the series of carboxymethylated pyridoindoles potentially effective in prevention of diabetic complications. Peculiarities of 1,4-dihydropyridines as hydrophobic antioxidants

Figure 13. General chemical structure of carboxymethylated hexahydro- (A) and tetrahydro-pyridoindoles (B) related to stobadine.

Modulation of lipid peroxidation reactions in biological objects include several possibilities of action: (i) influence on chemical reactions contributing to peroxidation by the use of exogenous antioxidants, (ii) influence on enzymatic reactions inducing peroxidation (by the use of enzyme inhibitors or activators), (iii) influence on the compartmentalization of peroxidation and integrity of cellular membranes and (iv) influence on the biosynthesis of endogenous antioxidant proteins and low-molecular mass antioxidants and pro-oxidants.

Natural and synthetic antioxidants 1239 Table V. Values of activation barriers EA for the reaction of the pyridoindoles with a hydroxyl radical, highest occupied molecular orbital energies EHOMO, relative bond-dissociation energies BDErel and experimental scavenging activities (−ΔA5 min). EAa (kJ/mol) stobadine 1a 2a dehydrostobadine 1b 2b 3b

67.1 69.7 68.2 82.9 84.7 83.8 88.3

EHOMOb (kJ/mol)

BDErelb (kJ/mol)

474.4 508.9 509.2 531.7 546.2 543.8 547.7

0.00 6.54 5.80 32.62 35.51 34.62 35.71

−ΔA5minc 0.239 0.132 0.187 0.033 0.031 0.030 0.031

aactivation

barriers EA calculated by AM1 method. highest occupied molecular orbital energies EHOMO and relative bond dissociation energies BDErel calculated by DFT (B3LYP/ 6-31G∗) method. canti-radical activity in DPPH test [209]; measured as absorbance decrease; recorded at λ max 518 nm; during the first 5-min interval.


bthe

There are numerous studies of the influence of drugs, potential drugs and other synthetic or natural compounds on peroxidation processes in cells, tissues, organisms or simple in vitro models. Quite often molecular mechanisms are not studied in the initial phase and the ‘black box’ approach is employed to find compounds which are efficient in modulating lipid peroxidation. It is important that methods used for such studies are appropriate, to avoid artifacts, which can easily appear in studies of multicomponent systems.

1,4-Dihydronicotinamide is the active part NAD(P)H, a coenzyme involved in a plethora of enzymatic redox reactions of hydrogen and electron transfer. 1,4-Dihydropyridines (1,4-DHP) are analogues of 1,4-dihydronicotinamide and model compounds for NAD(P)H. 1,4-DHP derivatives are synthetically available due to the convenient Hantzsch type cyclic condensations. Derivatives of 1,4DHP have been objects of numerous studies, the more that some of these compounds have been stud-

Figure 14. Structures of some 1,4-dihydropyridine derivatives.


1240 A. Augustyniak et al. ied and registered as anti-hypertensive and antianginal drugs. There is a lot of data on their neurotropic, anti-inflammatory, anti-diabetic, antimutagenic, growth stimulating, anti-ageing and antioxidant activities [231]. The antioxidant activity (AOA) of 1,4-dihydropyridine (1,4-DHP) derivatives was first demonstrated for 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine (Hantzsch ester, Diludin; Figure 14). This compound was found to stabilize β-carotene in solutions, in grass meal and other carotene-containing materials [232]. 1,4-DHP, especially Diludin, have high AOA stabilizing effectively plant oils and plant oil containing products [233,234]. The ability of 1,4DHP to inhibit free radical reactions was also documented [235]. In 3,5-dicarbonyl-1,4-dihydropyridine derivatives strong bis-β-dicarbonylvinyl-amino conjugation exists and consequently they cannot be considered as amino antioxidants, but rather as of C-H antioxidants. The abstraction (donation) of electron and/or H takes place from all 3,5-dicarbonyl-1,4dihydropyridine systems and results in the formation of corresponding pyridine derivatives (Figure 15). Investigation of AOA of the compounds relative to Diludin showed that the introduction of a substituent, in position 4 of the dihydropyridine ring, except for the COO group, strongly diminishes the AOA [236]. Comparative studies of the behaviour of these compounds in model and biological membranes of various degrees of complexity (emulsions of unsaturated fatty acids esters, phospholipids liposomes and erythrocyte membranes) revealed the influence of length of alkyl groups of DHP esters, as well as of the nature of heterogenous systems on AOA of 1,4-DHP derivatives [237–239]. 4-Alkyl- and especially 4-aryl-3,5dialkoxycarbonyl-1,4-DHPs have lower AOA, but of 3,5-dicarbamoyl-1,4-DHPs not substituted in position 4 are more prone to oxidation so 4-aryl derivatives have higher AOA. It was found that 1,4-DHP derivatives in model systems are less active antioxidants than BHT. However, Diludin was found to be a good synergist of the natural antioxidant α-tocopherol and synthetic antioxidant BHT. Unexpectedly, no synergism was found with respect to the similar synthetic antioxidant BHA (Table VI) [240,241]. Besides, 1,4-DHP revealed also a singlet oxygen quenching activity, close to that of α-tocopherol [242]. Some 1,4-DHP derivatives possess good Fe3-reducing ability [243]

Table VI. Additivity of the antioxidant action of Diludin with BHT and BHA.

Figure 15. Reaction of 1,4-dihydropyridines leading to the formation of pyridine derivatives.

Figure 16. A 1,4-dihydroisonicotinic acid derivative (Compound I) showing high Fe3-reducing activity.

Substrate and antioxidants Methyl oleate (60°C) Diludin BHT BHT Diludin BHA BHA Diludin Sunflower oil (20°C) Diludin BHT BHT Diludin Cooking fat (20°C) Diludin BHT BHT Diludin

AOA (τ/τ0) — 1.0 38.0 51.0 25.0 25.0 — 1.2 1.4 1.8 — 1.2 1.2 1.9

(Table VI). The Fe3-reducing ability of a 1,4-dihydroisonicotinic acid derivative shown in Figure 16 (Compound I) is comparable to those of BHT and Trolox, but Nifedipin (4-o-nitrophenyl derivative of 1,4-DHP) does not reduce Fe3. Compound I protects also liver cells from copper-induced lipid peroxidation and increases hepatocyte viability [244]. Studies of 1,4-DHP in metal-ion catalysed peroxidation of liposomes showed that AOA of Diludin is associated with its lipophilicity and consequently ability to incorporate into liposomes [245]. It was found that Diludin easily incorporates into the outer monolayer of erythrocyte membranes [246]. Diludin and relative 1,4-DHPs were found to be relatively non-toxic. LD50 value of Diludin administered per os exceeds 10 000 mg/kg (mice) and repeated daily administration of this compound to rats at doses of 20 mg/kg during 6 months causes no toxic symptoms in the animals [232]. Compound I (Figure 16) and its derivatives were also found to be non-toxic. Recent studies of the reactivity of 1,4-DHP with alkylperoxyl radicals and ABTS radical cation [247] revealed similar dependence on the structure of these compounds as those found previously in other systems. Diludine was again found to be the most active compound, the photodegradation product of nifedipine (nitrosophenyl derivative of pyridine) showed a high reactivity and kinetic rate constants for the reaction between 1,4-DHP compounds and alkylperoxyl radicals exhibited a fairly good linear correlation


Natural and synthetic antioxidants 1241 with the reduction potential of DHP derivatives. The physico-chemical mechanism of AOA of 1,4-DHP has been studied and discussed [248], but details of this mechanism still await elucidation. Many 4-aryl (heteryl) derivatives of 3,5-dialkoxycarbonyl-1,4-DHPs have L-type Ca2 channel antagonist (blocker) properties. Several such compounds have been proposed as drugs for the treatment of hypertension and other cardiovascular diseases; they also revealed anti-atherosclerotic properties in animal experiments. Their influence on lipid peroxidation processes has been studied and AOA have been determined for several of such compounds. Numerous studies are devoted to the AOA of 1,4DHP derivatives bearing calcium antagonist properties. PubMed database shows that this problem has been discussed in at least 19 reviews. Our studies of 1,4-DHPs substituted in position 4 with phenyl and/or substituted phenyl residues (except for o-nitrophenyl) do not show their considerable reactivity with radicals or ability of iron reduction (Table VII). Antioxidant activity of nifedipine (3,5-dimethoxycarbonyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine; Figure 14) was demonstrated already in 1982 [249] and the mechanism of its action, involving formation of 4-(2-nitrosophenyl)-pyridine derivative as a result of intramolecular redox reaction, has been proposed [250]. Nitroso aromatic compounds in the presence of unsaturated lipids can form nitroxyl radicals, exhibiting antioxidant activity. Later on, AOA of nifedipine and its oxidized nitroso analogue was studied [251,252]. It has been revealed that Ca2 antagonists (especially derivatives of 1,4-DHP) and also some 1,4DHPs of lower activity of Ca2 antagonists, ameliorate low density lipoprotein (LDL) oxidation induced by copper ions or human monocytes [253]. The order of potency is: vitamin E felodipine 2-Cl analogue of nifedipine nifedipine amlodipine, nitrendipine, verapamil diltiazem. In the cell-induced oxidation system nifedipine and felodipine induced significant reductions in the TBARS content of LDL compared with amlodipine, verapamil and the 4-nitro isomer of nifedipine. In this oxidation system nifedipine was a more effective antioxidant than felodipine. So, 2-substitution of the phenyl ring is quite important and also the presence of the 1,4-dihydropyridine ring has an essential role. It should be remembered, however, that the order of potency of the drugs depends on the oxidation system and the assay used to measure the antioxidant effect. Dihydropyridine derivatives amlodipine and nisoldipine attenuate extra- and intracellular superoxide formation stimulated by high glucose concentration. Interestingly, L-type calcium channel agonist BayK 8644 revealed the same type of activity. As a result:

dihydropyridines limit high glucose-induced superoxide formation and improve NO• bioavailability in human endothelial cells [254]. 1,4-DHP derivatives are metabolized by the cytochrome P-450 system, localized mainly in the hepatic endoplasmic reticulum. Cytochrome P-450 metabolism of many lipophilic drugs generates ROS and induces oxidative stress. A group of 3- and 4-nitrophenyl-1,4DHP derivatives inhibit the microsomal lipid peroxidation and microsomal thiols oxidation induced by Fe3/ ascorbate system, a generator of oxygen free radicals. It has been concluded that when drugs which are activated by biotransformation are administered together with antioxidant drugs, such as dihydropyridines, oxidative stress in situ may be prevented [255]. Mitochondria are potential targets to pharmacological and toxicological actions of membrane-active agents, including some 1,4-DHP derivatives. Out of a group of compounds, 3-acetyl(carbamoyl)6-methylsulfanyl-1,4-DHP-5-carbonitriles and 4-pchlorophenyl derivative OSI-1146 displayed antioxidant and anti-radical activities in vitro. All studied compounds protected mitochondria against lipid peroxidation induced by ADP/Fe2, OSI-1146 being the most potent [256]. A lot of compounds with AOA have anti-inflammatory properties [257], among them the cardiovascular drugs amlodipine [258], benidipine [259], felodipine [260] and cerebrocrast [261,262]. It has been suggested that felodipine may exert vascular protective effects by suppressing free radical generation in human smooth muscle cells during activation of inflammatory mechanisms and diabetic conditions [260]. Amlodipine inhibited IL-1α release [258], cerebrocrast showed an anti-inflammatory effect by reducing inflammation in the rat paw oedema model and inhibiting secretion of neurotoxic cytokines interleukins IL-1β and IL-6 in human monocyte (THP-1) cell line [261]. Nifedipine was demonstrated to upregulate the biosynthesis of Mn-superoxide dismutase [263]. ROS formation in bovine aorta endothelial cell induced by oxidized LDL was significantly reduced only with lacidipine and lercanidipine. Amlodipine, nimodipine and nifedipine had no effect on ROS formation. Strong AOA of lacidipine may be related to the lipophilic cinnamic acid side chain, which confers higher stability to the lipid moiety of cell membranes [264]. Several DHP derivatives inhibited the 1-methyl-4phenylpyridinium iodide (MPP) induced ROS production in cerebellar granule cells with a distinct potency order: foridone (2,6-dimethyl-3,5-dimethoxycarbonyl4-(o-difluoromethoxyphenyl)-1,4-dihydropyridine) 2, 6-dimethyl-3, 5-diethoxycarbonyl-4-phenyl-1,4-dihydropyridine diludine [265]. These DHP derivatives reversed the MPP-induced decrease in the mitochondrial membrane potential in the same order.



Figure 17. Some other 1,4-dihydropyridine derivatives.

Prevention of damage caused by ROS is also an important antioxidant effect. Sodium 3,5-bisethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4carboxylate (AV-153; Figure 17) was found to stimulate rejoining of DNA strand breaks induced by hydrogen peroxide [266]. The lymphoblastoid Raji cells treated with AV-153 at concentration 1 nM ÷ 10 μM, showed a transient increase in poly(ADP-ribose) level and the rate and efficiency of DNA strands break rejoining. AV-153 was shown to have anti-mutagenic properties [267], reduce DNA damage, decrease 8-oxo-7,8dihydro-2’-deoxyguanosine content and lower mutation frequency [268,269]. A model system to study the biological action of antioxidants: Protection of sarcoplasmic reticulum membrane, sarcoplasmic Ca2-ATPase and plasma membrane Ca2-ATPase by natural and synthetic antioxidants Sarco-/endoplasmic reticulum Ca2-ATPase (SERCA) plays a key role in the relaxation of smooth, cardiac and skeletal muscle through the transport of cytosolic Ca2 into the sarco-/endoplasmic reticulum [270]. SERCA 1 from fast-twitch skeletal muscle is a single-chain transmembrane protein with easily measurable function, present in

high concentration in SR vesicles [271]. SERCA 1 is highly homologous with the other isoforms SERCA 2 and SERCA 3, present in cardiac and smooth muscles [272]. Some physiological and pathological processes, such as cell proliferation and apoptosis, are associated with abnormal activity or expression of SERCA [273]. Modulation of SERCA activity may be a contributing factor in the development of some cardiovascular, neurodegenerative or skeletal muscle diseases. An important feature of SERCA is its high sensitivity towards modification by ROS. Some phenolic substances are able to modulate specifically SERCA activity, most of them are inhibitors; however, certain substances are known to stimulate SERCA of both skeletal and cardiac muscle [274]. Phenolic antioxidants studied. Studies by the Bratislava group concerned the effect of phenolic antioxidants, synthetic antioxidants (Trolox, pyridoindole stobadine and its derivative SMe1), plant standardized extracts of Pinus pinaster bark (Pycnogenol®; Pyc) and leaves of Ginkgo biloba (Egb761) on the activity SERCA of rabbit skeletal muscle to assess their potency to modulate the activity of this enzyme in the presence or absence of oxidants in vitro [275]. Several of these antioxidants were tested also in vivo in experimentally-



Figure 18. Some pyridoindole antioxidants synthesized at the Institute of Experimental Pharmacology and Toxicology, Bratislava. Stobadine ()-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1Hpyrido[4,3b]indole and SMe1 methylated racemic derivate of stobadine, 8-methoxy-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3b] indolinium dichloride.

induced adjuvant arthritis, an animal model of rheumatoid arthritis, where redox imbalance is involved. Stobadine is a unique compound among carbolines since, unlike α- and β-carbolines, it does not reveal any obvious toxic effects and possesses a key antioxidative activity [276]. With the aim to improve antioxidant properties and to change lipophility and basicity of stobadine, 70 of its new derivatives were synthetized [277,278]. Stobadine and its methylated derivative SMe1 are depicted in Figure 18. Compared with stobadine, SMe1 possesses similar basicity, significantly lower lipophilicity and ∼ 2-times higher free radical scavenging activity [279]. Effect of phenolic antioxidants on SERCA activity. When studied in the absence of oxidants, Pyc (5 and 40 μg/mL) significantly decreased the activity of Ca2-ATPase in SR vesicles, as well as the activity of purified Ca2-ATPase, with respect to both enzyme

substrates, Ca2 and ATP (Figure 19). Trolox, stobadine, SMe1 (50 and 250 μmol/L), and Egb 761 (20 and 40 μg/mL) exerted no significant effect. The ability of Pyc to inhibit SR Ca2-ATPase may be linked to its ability to induce apoptosis. Inhibition of Ca2ATPase activity may lead to an increase of cytosolic Ca2, which is tightly controlled due to its importance in the regulation of many cellular processes [275]. Other authors found that incubation with Pyc in vitro actually induced apoptosis in human mammary cancer cells, whereas normal mammary cells were not affected [280]. Similarly, inhibition of Ca2-ATPase by the antioxidant curcumin was reported [281]. This compound has anti-cancerogenic properties [282] and is also able to affect a number of cellular processes including activation of apoptosis [283] or inhibition of platelet aggregation [284], which are known to be regulated by Ca2. Reactive oxygen species (ROS) in inflammation and SERCA. In many inflammatory diseases, phagocytes release HOCl, superoxide and H2O2. Release of the two latter agents into a fluid with free iron promotes the production of hydroxyl radical in the Fenton reaction and extensive damage of membranes, thus causing accumulation of intracellular calcium (Cai). Chronically-increased Cai is a final common pathway in cell injury and death [285,286]. Plasma membrane and sarcoplasmic/endoplasmic Ca2pump are mechanisms for removal of Cai. Therefore, we focused our studies on oxidative damage of SERCA by HOCl and the Fenton system and possible protective or modulating effects of the abovementioned antioxidants.

Figure 19. Effects of Pyc on purified Ca2-ATPase activity as a function of the concentrations of free Ca2 and ATP. Ca2-ATPase activity was measured as a function of free Ca2 in the absence (●) and presence of 5 μg/mL (◆) and 40 μg/mL (▲) Pyc. Each data point is the mean SEM of at least three independent experiments. pCafree and pATP were calculated from the concentrations of free Ca2 or ATP.


1244 A. Augustyniak et al. Oxidation of sarcoplasmic reticulum (SR) by the Fenton system. SR of rabbit skeletal muscle (70–90% of its protein being SERCA) was oxidized by Fe2/H2O2/ ascorbic acid (AA), a system which generates HO radicals according to the Fenton reaction under conditions similar to those occurring during inflammation. A 50% decrease of SERCA activity was observed, accompanied by a significant decrease of SH-groups and an increase of protein carbonyl groups and lipid peroxidation in SR. Two new bands, of ∼ 50 and 75 kDa, whose density was time-dependent, appeared in SERCA protein electrophoregrams after incubation with the Fenton system, probably due to structural changes as supported also by trypsin digestion. Immunoblotting of DNPH-derivatized protein bound carbonyls detected a time-dependent increase. Phenolic antioxidants and the Fenton system. Trolox is a water-soluble analogue of vitamin E which is the most important hydrophobic antioxidant in living organisms, while stobadine is a pyridoindole compound with known antioxidant effects in vitro as well as in vivo [202,204,276,287,288]. Several studies have shown that vitamin E can prevent most of the ironmediated damage, both in in vitro systems and in ironloaded animals [289]. According to our knowledge, Trolox and stobadine are not able to chelate Feions

Figure 20. Protection of SERCA against oxidation in the Fenton system by antioxidants. The figure shows the influence of the antioxidants used: Stobadine (Sto), Trolox (Tro), Pyc and EGb761 on SERCA activity, content of reduced SH groups, protein carbonyls and TBARS after oxidation in the Fenton reaction. The SR was incubated with the antioxidants 2 min before exposure to the Fenton system (50 μmol/L Fe2, 1.5 mM H2O2, 6 mM AA). Trolox and stobadine were used at a concentration of 50 μmol/L, Pyc and EGb761 at 40 mg/mL. All data are expressed as mean SEM of three independent measurements with at least three parallels. ∗p 0.05, ∗∗p 0.01, statistical significance of differences between the unprotected and the antioxidant protected sample, both oxidized by the Fenton system.

or scavenge hydrogen peroxide which are components of the Fenton system. In our experiments, Trolox and stobadine at a concentration of 50 μM were able to protect the SR against a decrease in SH-group content as well as TBARS increase after oxidative stress induced by Fe2/H2O2/AA. However, in spite of these effects, neither Trolox nor stobadine were able to prevent the decrease of Ca2-ATPase activity induced by the Fenton system (Figure 20) which may support the idea that structural changes are involved in the decrease of the Ca2 pump activity. The standardized plant flavonoid extracts of EGb761 and Pyc (40 μg/mL), prevented SH-group oxidation and protein carbonyl group formation. Additionally, Pyc prevented TBARS formation in SR oxidized by the Fenton system. The smaller antioxidant effect of EGb761 compared with Pyc may be associated with the fact that -OH groups of the compounds in the extract, effective in free radicals scavenging, are bound to glucose [290]. Mixtures of many compounds included in Pyc and EGb761 may have multiple and synergistic effects, which may be the reason for the effective decrease of protein carbonyl formation in contrast to the single compounds Trolox and stobadine, which were not effective.These extracts are also able to chelate Fe ions and scavenge hydrogen peroxide and hydroxyl radicals [280,291]. In spite of these antioxidant and scavenging effects, EGb761 and Pyc exerted no protective effects on Ca2-ATPase activity but even decreased the enzyme activity. This effect may be caused by the interaction of flavonoids with free radicals derived from the Fenton system, including secondary radicals. In addition, Pyc is able to inhibit Ca2-ATPase activity also in the absence of oxidants as was found for other enzymes [280,292]. Flavonoid constituents of EGb761 are able to bind to proteins. Their binding to the nucleotide-binding site of Ca2-ATPase [293], as well as interaction with ion channels was reported [294]. Oxidation of sarcoplasmic reticulum (SR) and pure SERCA by HOCl. HOCl inhibited both Ca2-ATPase activity in the SR membrane and pure enzyme in a concentration-dependent manner with IC50 values of 100 μmol/L and 150 μmol/L, respectively. Concentration of 13.5 or 6.6 μmol/L of HOCl, respectively, reduced the content of sulphydryl (SH) groups of SR by 50%; yet, HOCl did not influence the enzyme activity [295]. These data indicate that oxidation of SH groups is not critical for impairment of the Ca2ATPase activity. In comparison with SH group oxidation and enzyme activity inhibition, a significantly longer time was necessary for the generation of protein carbonyls in HOCl-treated SR. Studies of changes of kinetic parameters of Ca2ATPase revealed a significant decrease of Vmax for both Ca2 and ATP under the influence of 150



μmol/L HOCl and no changes in the enzyme affinity for both substrates [296]. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) interacting with SR decreased, indicating binding of HOCl to the nucleotide binding site of SERCA. This probe binds covalently to Lys515, which is close to the ATP binding site and competitively inhibits ATP binding [297]. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Immunoblotting of DNPH derivatized protein bound carbonyls detected a time-dependent increase after incubation of SERCA with the Fenton system. Phenolic antioxidants and HOCl injury. Trolox, EGb 761 and SMe1 exerted a protective effect on the Ca2-ATPase activity. Trolox and EGb761 protected also –SH groups of SR against oxidative modification by HOCl. Stobadine and Pyc inhibited markedly protein carbonyl formation. Stobadine, the only antioxidant able to scavenge HOCl among those tested, was without any effect on Ca2-ATPase activity and –SH group content, while Pyc significantly decreased the enzyme activity. Other authors also did not find any significant effect of stobadine on Ca2-ATPase activity in the liver of control and diabetic animals [298]. In the heart of normal control rats, stobadine treatment led to an increase in the Ca2-ATPase activity, though in diabetic rats it did not produce any protection against the decrease of the this activity [298]. A significant concentration-dependent protective effect of Trolox on SERCA oxidized by HOCl [295] up to the maximal Trolox concentration of 250 μmol/L was observed. Under the same conditions of HOCl oxidation, no protective effects of Trolox with respect to protein carbonyls formation and SH groups oxidation in the SR were observed [295] and no scavenging effect of HOCl by Trolox was found [299]. Trolox changed the kinetic parameters of SERCA with respect to Ca2 as well as to ATP. Using the fluorescent label FITC, specific for nucleotide binding sites, we found that Trolox was

able to induce conformational changes in SERCA (Figure 21). These results suggest that the protective effects of Trolox are due to its ability to alter structural properties of Ca2-ATPase. A protective effect of α-tocopherol on the Ca2ATPase activity was reported in several animal models, like thermal ischaemia of rat kidney [300], hypercholesterolaemic rabbits [301], catecholamine treatment of the heart [302], streptozotocin-induced diabetes (rat kidney) [303] or acute inflammation of rat pancreas, liver and kidney [304]. It may be supposed that also these effects are at least partly due to induction of conformational changes of SERCA. In our study, Egb 761 (40 µg/mL) significantly prevented the decrease of SR Ca2-ATPase activity, protected –SH groups and inhibited protein carbonyl formation induced by HOCl. The preventive effect of EGb 761 on the Ca2-ATPase activity decrease might be at least partially associated with the –SH group protection. On the other hand, the possibility that components of this extract bind to the enzymatic protein cannot be excluded. We observed a strong inhibiting effect of Pyc protein carbonyl formation induced by HOCl in SR. Pyc inhibited Ca2-ATPase activity in the absence and even more in the presence of HOCl. As mentioned, this may be induced by the ability of Pycnogenol to bind to proteins, thus altering their structure and activity [292]. To summarize, modulation of Ca2 pump function by antioxidants depends on the mode of oxidative injury. Both HOCl and radicals generated by the Fenton reaction participate in the processes of inflammation and are able to induce in proteins structural changes resulting in a decrease of Ca2 pump function. The protective effect of Trolox, EGb761 and SMe1 on SERCA activity may be based on the interaction of specific (HOCl related) secondary oxidative products with antioxidants. On the other hand, the same antioxidants, in spite of the fact that they protected –SH groups and prevented TBARS or carbonyl generation, were not effective in preventing SERCA activity decrease induced by the Fenton reaction.

Figure 21. FITC fluorescence of SERCA treated with HOCl in the absence and in the presence of Trolox. SR (25 µg prot./ml) was exposed HOCl, at various concentrations, for 3 min at 25°C and pH 7.2 (A). (B) SR was pre-treated by 250 μmol/L Trolox at 37°C, for 2 min. Values are means SD of 10 measurements. ∗∗p 0.01, statistically significant differences between control and HOCl-treated samples. Figure is used with permission [296].


1246 A. Augustyniak et al. SERCA and phenolic antioxidants in vivo. In contrast to human rheumatoid arthritis, experimental adjuvant arthritis is partially reversible. Although the active inflammatory responses gradually subside, the swelling and apparent anatomical deformities may last for a longer period [305]. The mechanisms of reversibility may be at least partially based on the modification of SERCA Ca2-ATPase activity. Selective reversible oxidation at critical sites of SERCA, involving free radicals, controls its function. In SR of rats with experimental adjuvant arthritis induced by injection of Mycobacterium butyricum to the tail of the animals, we found no alterations of protein carbonyls, SH groups and lipid composition, analysed by GC-MS. The only significant alteration was the modulation of Ca2-ATPase activity in the SR over the time course of adjuvant arthritis, which may indicate possible structural changes of SERCA [306]. Studies with fluorescence probes specific for cytosolic nucleotide (ATP) binding sites (FITC) and for SR membrane Ca2-binding sites of SERCA (NCD-4) suggest the occurrence of conformational changes of SERCA. These observations were supported also by the Trp fluorescence intensity ratio (I358 nm/I336 nm) of the plasma membrane, which may be a marker of conformational changes of SERCA [307]. Stobadine and SMe1 modified SERCA activity in adjuvant arthritis (Figure 22) [308]. Stobadine, of redox potential of 0.58 V, is able to protect directly amino acids (redox potential of ∼ 1.00 V) against oxidation [309,310]. Nevertheless, stobadine was found to have no protective effect against the decrease of Ca2-ATPase activity induced by HOCl [275,295]. A higher antioxidant activity of SMe1 was supposed by quantitative analysis between structure and biological effects of pyridoindole derivatives (QSAR) [311] and according to measurement with DPPH [279]. Compared to stobadine, SMe1 was also more effective in modulating SERCA activity in SR from adjuvant arthritis rats. Both pyridoindole derivatives slightly influenced the conformation of SERCA in the cytosolic nucleotide

Figure 22. SERCA activity in SR of skeletal muscles in animals with adjuvant arthritis, treated with pyridoindole derivatives. Ca2+ATPase activity was measured in four animals. AA, adjuvant arthritis; AA-STB, treated with stobadine; AA-SMe1, treated with a stobadine derivative. Each point is the mean SEM of four independent experiments measured in three parallels. Figure is used with permission [308].

binding site. Significant conformational alterations were found in the transmembrane part of SERCA where the binding site for Ca2 is situated. Vitamin E protected SERCA activity against alterations induced by adjuvant arthritis probably by conformational changes in the nucleotide binding site and, especially, in the Ca2 binding site (Figure 22). No significant changes in any binding sites were induced in adjuvant arthritis animals treated with Pyc. Therefore, modulation of SERCA activity by phenolic antioxidants in rats with adjuvant arthritis may be associated with conformational changes of SERCA mainly in its transmembrane part. Modulating effect of flavonoids and their lipophilized derivatives on biological membranes Various studies have shown that the structure and function of cellular membranes can be seriously affected by the action of reactive oxygen and/or nitrogen species. These species cause peroxidation of membrane lipids, oxidation of proteins, including membrane-bound enzymes, and they are able to alter the integrity of cellular components [312–314]. They cause oxidation of thiol groups, carbonylation and nitration of membrane proteins, leading to loss of protein function [314]. Flavonoids, a group of natural phenolic compounds, are known for a wide range of pharmacological properties which are beneficial for human health. Many of these effects may be explained by their interaction with enzymes, receptors, transporters and signal transduction systems [315]. In many cases, they interact with cell membranes prior to binding to receptors [316]. The bioactivity of flavonoids seems to be connected with their ability to interact with membranes [317,318] and with the protection of membrane lipids and proteins from free radical-induced injury [319]. Membrane interactions of flavonoids are connected with the modification of physico-chemical and thermodynamic properties of membranes [317,318, 320–322]. The incorporation of flavonoids into the lipid bilayer is affected by electrostatic interactions, formation of hydrogen bonds with polar headgroups of phospholipids, hydrophobic interactions with fatty acyl chains and the molecular geometry of phospholipids [323]. Flavonoids can modify membrane permeability and membrane-dependent processes [320], change fatty acid composition or phospholipid content in the membranes or interact with membrane proteins [318].The above-mentioned changes induced by flavonoids in cells may be crucial for their pharmacological activity employed in the treatment of certain diseases, including cancer [324]. Synthesis of rutin fatty acid esters. Selective flavonoid acylation was carried out with the purpose of modification of their biological and physicochemical prop-



Figure 23. Preparation of lipophilic rutin esters via lipase-catalysed acylation of rutin with fatty acids.

erties [325–330]. It has been proposed that selectively acylated flavonoids with different acyl donors may not only improve physicochemical properties of these molecules [326], but also introduce various beneficial properties to the maternal compound such as penetration through the cell membrane [325,331]. They improve existing or confer novel bioactivities, including antioxidant [328,329], antimicrobial [329], antiproliferative [330] and cytostatic [325] properties (reviewed in [332]). Novel rutin derivatives were synthesized to evaluate their possible membrane interactions [333]. The amphiphilic property would allow flavonoids to act, not only on membrane surfaces by electrostatic interaction with phospholipid polar heads, but also on deeper regions by hydrophobic interaction with phospholipid acyl chains [317]. Rutin esters were prepared via lipase-catalysed esterification of rutin with fatty acids of C4–C22 in 2-methylbutan-2-ol at 60°C (Figure 23). The highest yields of rutin esters

were reached when short and medium fatty acids of C4–C12 were introduced [334]. These results are partially in accordance with the findings of Ardhaoui et al. [335] who observed increasing conversion of rutin when esterified with fatty acids from C6 to C12. Kontogianni et al. [336] found no correlation between the fatty acid chain length and rutin conversion. Besides fatty acid chain length, the regioselectivity and performance of the enzymatic synthesis of flavonoid esters is influenced by structure, concentration and ratio of the acyl donor and acyl acceptor, by the type and concentration of the enzyme, reaction media, water content in the media, reaction temperature and type of reaction (reviewed in [337]). Antioxidant properties of modified flavonoids. Many biological properties of a drug may be related to their capacity to penetrate into the cell membrane and so to affect membrane-dependent processes, such as arachidonic acid metabolism, exocytotic histamine

Figure 24. Antioxidant activity of rutin and rutin fatty acid esters determined by DPPH• and β-carotene linoleate method. Final concentrations of the compounds tested was 2 and 1.8 mmol/L in the DPPH• and β-carotene linoleate systems, respectively. Results are mean SD.


1248 A. Augustyniak et al. release, phosphodiesterase activity and free radicalinitiated lipid peroxidation [338]. Flavonoids were reported to provide protection of the lipid bilayer from oxidants [319]. The capacity of flavonoids to influence free radical-induced lipid peroxidation in membranes is related not only to their structural characteristics but also to their ability to interact with and/or to penetrate the lipid bilayer [317,320], which may be associated with their hydrophobicity. Arora et al. [103] have suggested that the ability of flavonoids to localize in cell membranes and the resulting restriction of their fluidity could sterically hinder diffusion of free radicals and thereby decrease the rates of free radical reactions. In order to investigate free radical scavenging activity and inhibition of lipid peroxidation related to antioxidant action of rutin derivatives in the cell membrane, esters of rutin with C4–C18 fatty acids were tested both by the the DPPH• and β-carotene linoleate methods. It was assumed that amphiphilic rutin derivatives could more readily transfer electron into hydrophobic media and thus facilitate inhibition of lipid peroxidation reactions. The results obtained by both methods are summarized in Figure 24. The antioxidant activity of lipophilic rutin esters (R4–R18:3) varied from 60–79% and from 75–96% in the DPPH• and β-carotene linoleate systems, respectively. Rutin fatty acid esters proved to be both strong DPPH• scavengers and effective lipid peroxidation inhibitors. Lipophilization of the rutin skeleton had no effect on the DPPH• scavenging activity. On the other hand, the inhibition of lipid peroxidation in the β-carotene linoleate system was higher for rutin esters with longer fatty acid chain length than for rutin itself or for short chain fatty acid esters. Rutin palmitate, rutin stearate and rutin linolenate were found to be the most potent oxidation inhibitors of lipid peroxidation, with efficiency comparable to that of BHT [333]. Oil–water or lipid–water partition coefficients are basic parameters used to describe the ability of drugs

to interact with biological membranes. The relative hydrophobicity of flavonoids depends on the number and position of substituents, mainly hydroxyl groups [339]. It seems that the ability of flavonoids to interact with membranes is structure-dependent and is associated with their capacity to partition into the lipid–water phase. The ability of flavonoids to interact with membranes at the water–lipid interface may contribute to their antioxidant capacity [319]. Quercetin, a rather lipophilic molecule, interacts with the polar headgroups of phospholipids and penetrates the lipid bilayer, thus reaching its maximal antioxidant capacity [322]. Movileanu et al. [322] also reported that the scavenging activity of quercetin may depend on its interaction with the lipid bilayer of cell membranes. Effect of flavonoids and their derivatives on sarco/ endoplasmic reticulum Ca2-ATPase. The modulating effect of rutin and lipophilic rutin derivatives on the sarcoplasmic reticulum Ca2-ATPase (SERCA) was studied. This Ca2 pump has been found to be inhibited by a wide spectrum of hydrophobic molecules including flavonoids [340]. Flavonoids affect the membrane ion transport, Ca2 influx and Ca2 metabolism [341]. Strong inhibitory properties of rutin derivatives on the Ca2ATPase specific activity were observed, with IC50 values of 20–60 μmol/L (Figure 25). On the contrary, the rutin itself slightly stimulated Ca2-ATPase activity [342]. The mechanism of enzyme inhibition seems to involve the interaction of flavonoids with both the ATP-binding site and the hydrophobic region of ATP-binding proteins [343]. Increased hydrophobicity of fatty acid-modified rutin derivatives could facilitate their incorporation into the phospholipid bilayer and the resulting change in membrane properties but can cause disturbances of the fluidity of the lipid matrix, which may be the reason for inhibition of Ca2-ATPase

Figure 25. Inhibitory effect of rutin arachidonate on the plasma membrane Ca2-ATPase activity (left) and sarcoplasmic reticulum Ca2ATPase activity (right). Erythrocyte membranes (1.5 mg protein/mL) were incubated with rutin arachidonate (10–250 μmol/L) at 37°C in borate buffer (pH 7.4) for at least 30 min. Sarcoplasmic reticulum vesicles (0.1 mg protein/mL) were incubated with rutin arachidonate (10–250 μmol/L) at 37°C in phosphate buffer (pH of 7.4) for 2 min. Values are means SD of two independent measurements with at least two parallels. ∗∗∗p 0.001 with respect to the control. Right plot: SD smaller than the symbols used.


Natural and synthetic antioxidants 1249 activity. Moreover, flavonoid esters bound in the lipid bilayer may also interact with SERCA, affecting its conformation by creating pressure on the transmembrane region of the protein and changing membrane thickness. Hydrophilic rutin (log p 1.06) with low affinity to the SR membrane was found to possess a slightly stimulating effect on enzyme activity, unlike hydrophobic esters (log p 4.38–7.41) with strong inhibitory properties [342]. Structure– activity analysis revealed that the inhibitory potency of flavonoids depend on the polyhydroxylation pattern. Especially effective inhibitory agents were those with a hydroxyl group in position 3 on ring C, followed by those with a hydroxyl group in position 6 on ring A [340]. The differences in modulation properties of rutin and its derivatives on conformational changes of SERCA were studied using fluorescent probes. Of the compounds tested, only rutin caused a small decrease of FITC fluorescence, indicating possible interaction with the nucleotide domain. On the contrary, significant changes induced by lipophilic rutin derivatives were observed in the transmembrane region of SERCA. Rutin esters esterified with long chain fatty acids were found to decrease considerably both tryptophan (Trp) and N-cyclohexyl-N ’ -[4(dimethylamino)-α-naphthyl] carbodiimide (NCD4) fluorescence, by 70–80% on average. However, no relationship was found between the structure, lipophilicity or degree of fatty acid unsaturation and the corresponding decrease in fluorescence. Since the NCD-4 probe binds in the vicinity of the Ca2-binding sites in the transmembrane region of SERCA [344], the fluorescence drop observed may be connected with conformational alterations of the protein. These alterations are probably directly associated with the loss of the catalytic function of the protein [342]. Since SERCA is very vulnerable to redox regulation and oxidative stress, it is one of the main membrane targets for free radical-induced injury [345]. Inactivation of the SR Ca2-ATPase as a result of free radical exposure has been documented [342,346– 348]. Free radicals are involved in SERCA denaturation, aggregation or conformational changes of the protein [346–348]. ROS/RNS non-specifically react with a wide range of amino acids, including Met, Cys, Tyr, Trp, His, Pro and Lys [312]. The modulating effect of rutin and lipophilic rutin derivatives on SERCA activity and modifications of the protein by hypochloric acid (HOCl) and peroxynitrite (ONOO) was studied. Rutin showed strong ability to protect SERCA against HOCl- and ONOO-induced oxidation at low concentrations (EC50 5–20 μmol/L). It also prevented Ca2-ATPase from carbonyl formation, thiol group oxidation and tyrosine nitration. Upon oxidation, lipophilic rutin derivatives showed protective effects on enzyme activity only at low concentrations (5–50 μmol/L) and inhibitory properties

at higher concentrations (100–250 μmol/L). All the derivatives tested significantly protected SERCA against protein carbonyl formation and reduced tyrosine nitration (except rutin arachidonate and rutin erucate which may be connected with the number of carbon atoms and unsaturation degree of the attached fatty acids). Rutin arachidonate (R20:4) and rutin erucate (R22:1) belong to the derivatives with the longest fatty acid chains and the strongest inhibitory action on SERCA [349]. Effect of rutin and rutin fatty acid esters on red blood cells. Flavonoids were also reported to influence physicochemical properties of model membranes, such as fluidity and electrical properties [322]. The presence of a drug in the ordered bilayer structure can modify the lipid packing, cause variations in the transition temperature of the pure lipid and/or changes in enthalpy of chain melting [350,351]. To study the effect of lipophilic rutin esters on some physical and biochemical functions of red blood cells (RBCs), erythrocyte membranes were used. The structure and functions of erythrocytes are susceptible to alterations due to interactions with exogenous compounds [352]. The effect of rutin and lipophilized rutin derivatives on the erythrocyte membrane Ca2-ATPase (PMCA) activity was investigated. This transport system pumps Ca2 from the cytosol to the extracellular environment. PMCA, together with SERCA and the sodium calcium exchanger, is responsible for the maintenance of low cytosolic Ca2. Like in experiments on SERCA, rutin derivatives displayed a strong concentration-dependent inhibitory action on PMCA activity (Figure 25), while rutin had no effect. In the presence of a free radical

Figure 26. Effect of rutin and rutin arachidonate on TMA-DPH fluorescence anisotropy of erythrocyte membranes. Erythrocyte membranes (1.5 mg protein/mL) were treated with rutin and rutin arachidonate (50–250 μmol/L) at 37°C in borate buffer (pH of 7.4) for at least 30 min. Values are means SD of two independent measurements with at least two parallels. ∗∗∗p 0.001 with respect to the control.

1250 A. Augustyniak et al. Table VII. Rate of the reduction of Fe3 with some 1,4-dihydropyridyl compounds.

R


H CH3 COONa Nifedipin BHT Trolox

Reduction rate 104 [mol L1 s1] 2.0 0.5 28.0 0.0 11.0 39.0

generating system, the inhibiton of PMCA activity was enhanced (not shown). To evaluate changes in membrane fluidity, fluorescence anisotropy of trimethylammonium-diphenylhexatriene (TMA-DPH) was measured [353]. Lipophilic rutin esters affected membrane fluidity in a dose-dependent manner, while rutin caused significant changes only at the highest concentrations tested (Figure 26). Tsuchiya [354] showed that flavonoids affect the fluidity of deeper regions of lipid bilayers using fluorescence polarization. He found that alterations of membrane fluidity by flavonoids were due to the

modification of lipid composition (the content of unsaturated fatty acid residues and cholesterol) and has suggested that structure-dependent membrane interaction of flavonoids, which modifies membrane fluidity, may be mechanistically associated with flavonoid bioactivity in the membrane lipid phase. The obtained results indicate that selective flavonoid lipophilization may provide a useful tool for SERCA activity modulation. The inhibition of Ca2ATPase activity is responsible for elevation of cytosolic Ca2 concentrations, which is associated with induction of apoptosis. The protective effect of rutin and its derivatives on SERCA and their antioxidant activity could be useful in anti-inflammatory defense and may be potential enhancers of drug penetration into the cells.

Perspectives Antioxidants are an indispensable component of the ROS-dependent signalling network and a means of defence against excessive production of ROS. Although recent studies cast some doubt on the benefits of longterm antioxidant supplementation [355–357], which can be partly attributed to the tendency of the organism

Table VIII. Examples of therapeutic applications of antioxidants. Antioxidant N-acetylcysteine

Coenzyme Q10

Ibedenone (Coenzyme Q analogue) Carnitine Glutathione α-Tocopherol

Melatonin Resveratrol (Derivatized) Superoxide dismutase Edaravone (3-methyl-1phenyl-2-pyrazolin-5-one) Nitroxides

Probucol AGI-1067 (a probucol derivative) Ebselen

Field of application

References

Protection against ototoxicity after the administration of aminoglycoside antibiotics like gentamycin, against radiocontrast-induced nephropathy, preservation of endothelial function in patients with end-stage renal failure Treatment of chronic liver diseases, chronic obstructive pulmonary disease, pulmonary fibrosis, over-doses of acetaminophen, improvement of bronchial mucous fluidity Protection against adriamycin-induced cardiotoxicity Improvement of effects of coronary artery bypass graft surgery Therapy of hypertrophic cardiomyopathy Improvement of redox balance in Down’s syndrome Prophylaxis of migraine Attenuation of effects of Friedreich ataxia

[386–389]

Improvement of sperm quality after infection Treatment of chronic liver diseases and cataract Amelioration of cardiac complications in diabetes Treatment of infertility, increase in pregnancy rate Some positive effects in Alzheimer disease Attenuation of oxidative stress in newborns Amelioration of cardiac complications in diabetes Arthritis (application to synovial fluid), oxidative stress in premature babies

[398] [391] [399] [400] [401] [402] [399] [391,403]

Treatment of cerebral infarctions

[391]

Potential protection against ionizing radiation, cancer prevention and treatment, control of hypertension and weight, protection from damage resulting from ischemia/reperfusion injury Prevention of coronary artery disease in patients with familial hypercholesterolemia Reduction of post-angioplasty re-stenosis Reduction of restenosis

[404]

Protection against brain function deterioration in patients with cerebral infarctions or subarachnoid haemorrhages

[192,390–392] [393] [394] [395] [396] [397] [195]

[405] [406] [407] [408]


Natural and synthetic antioxidants 1251 to maintain redox homeostasis, harmful sequelae of antioxidant deficiency are obvious. There are numerous examples of successful use of antioxidants to ameliorate pathologic sequelae of oxidative stress (Table VIII). New ways of antioxidant delivery, i.e. including nanoparticles, have been proposed [358,359] and new antioxidants, targeted specifically to mitochondria, the main cellular source of ROS, have been synthesized and are tested [360,361]. Another approach can consist of stimulation of expression of genes coding for antioxidant proteins or antioxidant gene therapy [362]. Careful studies of their effects and side-effects are needed. On the other hand, the broad preference for compounds of natural origin cannot be ignored and is a stimulus for the search of new efficient antioxidants in the nature. Taking into account that oxidative stress depends both on the ROS production and their removal by antioxidants, inhibitors of ROS-producing enzymes are also tested and may find applications for amelioration of effects of oxidative stress [363,364]; such compounds may also be treated as antioxidants in a broader (biomedical) sense. Considering all these aspects, one may expect that the field of antioxidant research is likely to remain quite active in the years to come. Declaration of interest: Support by the COST Action B35 and the Croatian Ministry of Science, Education and Sports, Agency of the Ministry of Education of the Slovak Republic for the Structural Funds of EU, Project: ‘Evaluation of natural substances and their selection for prevention and treatment of lifestyle diseases’ ITMS 26240220040, Slovak National grants AV 4/0013/07, APVV-51-017905, VEGA 2/0001/08, VEGA2/7074/27, VEGA 2/0083/09 and APVV 51-017905, and Polish grant 83/N-Cost/2007/0 grants is gratefully acknowledged.

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