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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.
Antioxidants as biological response modifi ers of oxidative stress response in the cellular redox cycle. Superoxide radical (O • Ϫ ) 2 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. O • Ϫ is then disproportionated by superoxide dismutase 2 (SOD) to hydrogen peroxide (H O ), which can be further neutralized to water by catalase (cat), glutathione peroxidase (GPX) or can 2 2 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 detoxifi cation 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 modifi ers that maintain oxidative homeostasis.… 
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Natural and synthetic antioxidants: An updated overview
AGNIESZKA AUGUSTYNIAK
1 , GRZEGORZ BARTOSZ
2 , ANA C
ˇ IPAK
3 ,
GUNARS DUBURS
4 , LUBICA HOR Á KOV Á
5 , WOJCIECH Ł UCZAJ
1 ,
MAGDALENA MAJEKOVA
5 , ANDREANI D. ODYSSEOS
6,7 , LUCIA RACKOVA
5 ,
EL Z
. BIETA SKRZYDLEWSKA
1 , MILAN STEFEK
5 , MIRIAM Š TROSOV Á
5 ,
GUNARS TIRZITIS
3 , PETRAS RIMANTAS VENSKUTONIS
8 , JANA VISKUPICOVA
5 ,
PANAGIOTA S. VRAKA
6 & NEVEN Ž ARKOVI C
´
3
1 Department of Analytical Chemistry, Medical University of Bia ł ystok, 14, Poland,
2 Department of Molecular Biophysics,
University of Ł ó d z´ and Department of Biochemistry and Cell Biology, University of Rzesz ó w, Poland,
3 Rudjer Boskovic
Institute, Zagreb, Croatia,
4 Institute of Organic Synthesis, Latvian Academy of Sciences, Riga, Latvia,
5 Institute of
Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovakia,
6 Department of Chemistry,
University of Cyprus, Nicosia, Cyprus,
7 EPOS-Iasis, R&D, Nicosia, Cyprus, and
8 Department 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 antioxi-
dants. Representative studies presented in the review concern antioxidant properties of various kinds of tea, the search for
new antioxidants of herbal origin, modifi cation 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
to maintain oxidative homeostasis and assure the cell
survival, especially as ROS are produced under physi-
ologic conditions. Generally, antioxidative defense mech-
anisms are grouped as enzymatic and non-enzymatic
systems. Enzymatic mechanisms of ROS detoxifi ca-
tion are enzymatic cascades leading to complete
detoxifi cation of these reactive species. By their action,
they can be divided into two groups: one reacting dir-
ectly with ROS, with the other acting as redox regula-
tors [2]. For example, the importance of catalase could
thus be seen not only for detoxifi cation of hydrogen per-
oxide, but consequently in adaptation to endogenous
Introduction: Antioxidants modulators
of redox homeostasis
While oxygen is essential for aerobic organisms, it pro-
duces reactive oxygen species (ROS) and can cause
oxidative stress, defi ned as an imbalance in cell redox
reactions in favour of oxidative ones. Oxidative stress
can be the result of either ROS over-production or dec-
reased antioxidant defense [1]. Since oxygen is ubiqui-
tous 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
Correspondence: Grzegorz Bartosz, Department of Molecular Biophysics, University of Ł ó dz´, Banacha 12/16, 90-237 Ł ó d z´ , Poland.
Tel/Fax: 48 42 6354476. Email: gbartosz@biol.uni.lodz.pl
Free Radical Research, October 2010; 44(10): 1216–1262
ISSN 1071-5762 pri nt/ISSN 1029-2470 onli ne © 2010 Infor ma UK, Ltd.
DOI : 10. 3109/10715762. 2010.508 495
REVIEW ARTICLE
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Natural and synthetic antioxidants 1217
oxidative stress and lipid peroxidation [3], since the rem-
oval of H
2 O
2 can modulate signal transduction and trig-
ger proliferation of dormant tumour cells [4]. This gives
new insight into catalase as a fundamental biological
response modifi er of redox signalling and oxidative
homeostasis.
Non-enzymatic antioxidative systems are not as
specifi c as enzymatic ones, but, nevertheless, they are
in the fi rst line of antioxidative defense and are there-
fore of high importance in cellular response to oxida-
tive 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 inactiva-
tion of ROS, fi nally being regenerated by ascorbate.
Vitamin E has been shown to also be involved in sig-
nal transduction by modulating specifi c 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 A
2 (PLA
2 ), cyclooxygenase-2 (COX-
2) and the mitogen activated protein kinase (MAPK)
signal transduction pathway and also transcription fac-
tors like NF κ B [5]. Therefore, vitamin E is involved
in the control of various cellular functions, such as apop-
tosis, necrosis, survival, adhesion and differentiation.
Summarizing the knowledge of ROS and antioxida-
tive defence mechanisms one should notice a feed-
back 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 gen-
erated 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 modifi ers 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 detoxifi cation 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 modifi ers that
maintain oxidative homeostasis.
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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 anti-
oxidants which could be considered as biological
response modifi ers maintaining oxidative homeosta-
sis, both in health and in disease.
While it is certain that endogenous antioxidants,
both enzymatic and non-enzymatic, may be consid-
ered as biological response modifi ers to oxidative
stress and together with the products of lipid peroxi-
dation, 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 syn-
thetic antioxidants. The aim of the present review is
presentation of some results and perspectives of anti-
oxidant studies performed by participants of COST
B-35 Action between 2006 and 2010. They concerned
natural antioxidants of various origin, including semi-
synthetic 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. There-
fore, the scope of information in this area is extremely
large, diverse and rather diffi cult for systematic
reviewing and assessment. For instance, search in the
ISI WEB of Knowledge
SM database (http://apps.
isiknowledge.com/summary.do?qid 1&product
WOS&SID Q195a7cggB4a9IoG7GD&search_
mode GeneralSearch) by using keyword combina-
tion 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 var-
ious redox systems and consequently broad spectra of
possible applications: antioxidative phytochemicals
are considered as functional ingredients for pharma-
ceuticals, functional foods, dietary supplements, ani-
mal 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 spe-
cies. 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 anti-
oxidants 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 applica-
tions of well-established raw materials for the com-
mercialization of natural antioxidants; methods of
isolation, fractionation, separation and purifi cation of
antioxidatively active substances; elucidation of chem-
ical structures of natural compounds and characteriza-
tion 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
nal tasks in search and characterization of natural
antioxidants are either development of commercial
ingredients for the stabilization of oxidizable sub-
strates, particularly unsaturated fatty acids in foods, or
effective components for functional foods, dietary
supplements, pharmaceuticals and cosmetics demon-
strating benefi cial health effects. The achievements of
such tasks are associated with many analysis, assess-
ment 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, time-
consuming and in most cases provide only limited
information on their properties and health benefi ts.
Taking into account the diversity of the topics asso-
ciated with investigations of natural antioxidants as
well as a large amount of information which is regu-
larly reviewed, this chapter is not aiming at a compre-
hensive 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 antioxi-
dative 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 contribut-
ing compounds. On the other hand, the main bio-
chemical synthesis pathways are common for many
plant species; therefore, the main antioxidatively
active structures elucidated in various botanicals are
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Natural and synthetic antioxidants 1219
often similar. These structures include simple pheno-
lic compounds, phenolic acids, avonoids, coumarins,
sesquiterpene lactones, terpenoids and their deriva-
tives as well as other classes of phytochemicals. Con-
sidering many possibilities of substitutions and
intermolecular binding (esterifi cation, glycosidation),
as well as improvements in analytical methods, the
number of identifi ed natural antioxidants is rapidly
increasing. For instance, more than 4000 species of
avonoids, which are well-known natural antioxi-
dants, have been identifi ed, many of which are respon-
sible for the attractive colours of fl owers, fruit and
leaves [8].
It may be observed that in recent years many pub-
lications were focused on screening of unstudied or
less studied genera and/or individual botanical
species. These studies resulted in identifi cation of new
natural compounds and selection of promising spe-
cies 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 spe-
cies: fl avonoid aglycones (22), fl avonoid O -glycosides
and O -glucuronides (36), hydrolysable tannins and
related compounds (13), precursors of condensed
tannins (4), triterpenoids (25), organic acids and phe-
nol 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, par-
ticularly ecdysteroids, and phenolics (fl avonoids and
phenolic acids) accompanied by polyacetylenes,
Figure 2. General scheme in the search, characterization and application of natural antioxidants from botanical sources.
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1220 A. Augustyniak et al.
Table I. Investigation steps, methods and progress in characterization of botanicals: case study sweet grass ( Hierochloe odorata ).
Investigation step Methods Result References
Assessment of available
information
Literature survey, information on uses
in folk medicine, foods and beverages
Very few records, used to fl avour alcoholic
beverages, no information on antioxidant
properties, active compounds
[17]
Extraction and preliminary
assessment of antioxidant/
radical scavenging capacity
by various chemical and/or
biological assays in batch
Extraction with acetone, measurement
of total phenolics and antioxidant
activity in rapeseed oil
Extraction with methanol:water:acetic
acid or acetone and testing by DPPH
and ABTS radical scavenging assay
Sweet grass extract reported as a very strong
antioxidant for the fi rst time.
Acetone extract was stronger radical; scavenger
however, the yield of polar extract was
remarkably higher
[17]
[18]
Fractionation and isolation of
active fractions/components
followed by original batch
assay until the separated
compounds are pure enough
to identify the active
compound(s);
chromatographical separation
of complex mixtures and
on-line assessment of
antioxidant/radical
scavenging
Fractionation with hexane, tert-butyl
methyl ether (TBME), butanol (B)
and water; testing by DPPH free
radical scavenging assay. TBME and
B fractions were analysed by on-line
HPLC-UV-DPPH method and
further fractionated by TLC and
on silica gel column.
Characterization of redox properties,
the reactivity with peroxidase,
scavenging of singlet oxygen
TBME and B fractions were strongest radical
scavengers; HPLC-UV-DPPH revealed one
very strong radical scavenging compound;
active fractions collected for further analysis
Similar to quercetin; effectively neutralized the
effect of singlet oxygen to erythrocytes
(retarding photo-oxidation); the products of
peroxidase-catalysed oxidation of DHC react
with the SH groups and may act as
oxidizing substrates for fl avoenzymes
[18]
[19]
Structure elucidation of the
active components by
spectroscopic and chemical
methods
1 H,
13 C, 2D NMR, MS-EI, UV was
used for structure elucidation
5,8-dihydroxycoumarin (DHC) and 5-hydroxy-
8- O -
-D-glucopyranosyl benzopyranone
identifi ed; the compounds were very strong
radical scavengers
[18]
Pharmacological, toxicological
testing, evaluation of other
properties by using in vivo
and in vivo assays
Cytotoxicity in bovine leukaemia
virus-transformed lamb kidney
broblasts (line FLK)
Assessment of genotoxicity using
chromosome aberration (CA) and
sister chromatid exchange (SCE)
tests in human lymphocytes in vitro
and Drosophila melanogaster somatic
mutation and recombination test
(SMART) in vivo
Assessment of some pharmacological
properties
DHC possessed the pro-oxidant character of
cytotoxicity, which correlated with the
cytotoxicity of fl avonoids
DHC was not genotoxic in CA SMART
systems in vivo ; however it induced
signifi cant increase of SCE and a slight
increase of CA in human lymphocytes
in vitro, and signifi cant increase of
micronucleus in rat bone marrow cells
in vivo
DHC rich fraction inhibited vascular smooth
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)
[19]
[20]
[20]
Technological developments,
in situ evaluation, application
trials
Isolation of fractions enriched with
DHC and its glycoside by using
different combinations of solvent,
supercritical fl uid and microwave
assisted extractions.
Addition of sweet grass carbon dioxide
extract in Dutch style fermented
sausages with the aim of retarding
lipid oxidation
The infl uence of sweet grass extract
and its fraction rich in DHC on the
formation of 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP)
in meat was studied
The antioxidant activities of extract
were studied in emulsions of lard
and rapeseed oil using soy lecithin as
an emulsifi er and addition of cupric
acetate as an oxidation catalyst
Optimization of extraction procedure, extracts
containing up to 20% of active compounds;
possibility of industrial up scaling
No signifi cant effect on the formation of
peroxides. Determination of TBARS revealed
even some slight pro-oxidative effect, while in
hexanal assay only a negligible antioxidative
effect was observed.
Signifi cant increase of PhIP concentration was
observed in meat samples with plant origin
ingredients as compared with the samples
without additives
The antioxidant activity was about the same in
the two substrates. The stability against
autoxidation was substantially increased by
sweet grass extract. The stability was
particularly high, if citric acid and/or ascorbyl
palmitate were added to plant extract
[365]
[22]
[23]
[24]
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Natural and synthetic antioxidants 1221
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, com-
mon 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 fi rst marketed natural antioxi-
dants. 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 spe-
cies. 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 ber-
ries 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, avanols, avan-3-ols,
anthocyanins/anthocyanidins, condensed tannins/
proanthocyanidins, tocopherols and vitamin C. Patil
et al. [30] focused their review on historical perspec-
tives, opportunities and challenges of fl avonoids,
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 med-
icine, such as Ayurveda, which is supposed to be the
oldest medical system in the world and which pro-
vides potential leads to fi nd active and therapeuti-
cally 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 anti-
oxidants, discussed 24 plants reported to have anti-
oxidant properties. Some of the plants reviewed are
part of multi-herbal preparations while others are
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
identifi ed 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 , Rha-
ponticum 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 chromato-
graphic and spectroscopic techniques resulted in the
identifi cation of strong antioxidants, mainly phenolic
acid and fl avonoid derivatives [12 14]. Characteriza-
tion 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 prep-
arations 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 rape-
seed 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 capac-
ity is not always correlating with antioxidant activity
of the same preparations used in real foods [25], as
was demonstrated for H. odorata [22], G. macrorrhi-
zum and P. fruticosa [26] extracts added to Dutch style
fermented sausages.
Natural antioxidants from various botanical sources
have been regularly reviewed. Different approaches
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1222 A. Augustyniak et al.
used singly. Certain herbs like Amaranthus panicula-
tus , Aerva lanata , Coccinia indica and Coriandrum
sativum are used as vegetables, indicating that these
plants could be a source of dietary antioxidant sup-
plies. 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, fl a-
vonoids 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]. Isola-
tion and application of bioactive components from
inexpensive or residual sources, e.g. agricultural and
industrial wastes, may increase economical and tech-
nological feasibility of the commercialization of natu-
ral 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 biorefi nery 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 anti-
oxidants and they may be classifi ed into the three
major categories: (i) assays involving actual ROS-
oxidizable 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 elec-
tron 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 anti-
oxidant assays were critically assessed in several review
articles and it was attempted to propose standardized
methods for the determination of antioxidant capac-
ity and phenolics in foods and dietary supplements.
No one antioxidant capacity assay will truly refl ect
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 antioxi-
dants. 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 equiva-
lent antioxidant capacity (TEAC) should be stan-
dardized for use in the routine quality control and
measurement of antioxidant capacity of dietary sup-
plements 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), malon-
dialdehyde/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 scaveng-
ing, 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, over-
all 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 per-
oxidation ( b -carotene bleaching, conjugated diene or
FTC); the other for monitoring the fi nal 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) bleach-
ing associated with linoleic acid hydroperoxidation
catalysed by the soybean lipoxygenase (LOX)-1 isoen-
zyme (LOX/RNO reaction). This method was used to
determine the antioxidant activity of pure hydrophilic
and lipophilic antioxidant compounds and of mix-
tures of antioxidants and was able to highlight syner-
gism (among extracts) three-times more than the
ORAC method, whereas TEAC did not measure syn-
ergism under used experimental conditions.
In terms of analysis performances, the methods of
antioxidant capacity assays may be classifi ed into a
batch and more effi cient 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 profi ling and identifi cation of individual anti-
oxidatively active components in mixtures to provide
a powerful method for natural product-based drug
discovery. This group of methods was recently com-
prehensively reviewed [37,44]. Some most recent
articles [45] reported the use of the on-line methods
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Natural and synthetic antioxidants 1223
for the characterization of some specifi c 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 electrochem-
ical 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 polyphe-
nols and vitamins C and E). The main electrochemi-
cal approaches used have been cyclic voltammetry
(CV) and fl ow injection analysis with amperometric
detection (FIA-ED). In addition, miniaturization is
going to break new frontiers in the evaluation of anti-
oxidant activity. Gazdik et al. [48] applied the multi-
functional HPLC-ED array method coupled with
a DPPH reference; according to the authors the
method appears to be the optimal analytical progress,
accurately refl ecting the nutritive-therapeutic proper-
ties of a fruit.
Although the approach of the standardized mea-
surement of antioxidant capacity has not been widely
implemented in the search and evaluation of natural
antioxidants, some new developments should be
mentioned. Magalh ã es et al. [49] developed an auto-
matic fl ow procedure based on multi-syringe fl ow
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 anti-
oxidant 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 assess-
ment of total antioxidant activity by monitoring kinet-
ics of hydrogen peroxide (H
2 O
2 ) scavenging after its
injection into a liquid sample under study. The
pseudo-fi rst order kinetic constants of H
2 O
2 scaveng-
ing in the presence of different food additives corre-
lated with total antioxidant activity of these samples
evaluated via standard procedure based on lipid per-
oxidation. 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 reac-
tivities 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 con-
tained in beverages.
Another important issue is associated with the rel-
evance 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 diffi cult to transfer antioxidant mechanisms estab-
lished in model systems and in foods to the in vivo
situation and that no simple relationship has been
recognized so far between antioxidant capacity deter-
mined for various foods and beverages and health
benefi ts 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 scientifi cally justifi ed. 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 antioxi-
dant 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 fl uorescent dichlorofl uorescein (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 activ-
ity assays because it accounts for some aspects of
uptake, metabolism and location of antioxidant com-
pounds within cells. The method was applied for
studies of structure activity relationships of fl avonoids
[55] and assessment of common fruits [56]. Results
obtained by using the CAA method were well cor-
relating 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 meth-
ods 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
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1224 A. Augustyniak et al.
the results obtained by such studies will highly depend
on the strategies of the evaluation of new natural prep-
arations. The main limitations in commercialization of
natural antioxidants from botanicals are associated
with the fi nal goals of their applications. In the case of
foods, technological and economical aspects are play-
ing the major role, while in the case of pharmaceuti-
cals and nutriceuticals the lack of sound scientifi c
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 effi cient antioxidants
Tea infusions, consumed by two-thirds of the world s
population, are obtained from the manufactured
leaves of plants Camellia sinensis and Camellia assa-
mica . Besides its unique taste and fl avour, which
have popularized tea all over the world, its therapeu-
tic 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), carbo-
hydrates (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 fl avanols, especially the catechins
(epigallocatechin gallate (EGCG), epigallocatechin
(EGC), epicatechin gallate (ECG) and epicatechin
(EC; Figure 4) [59,60]. Other groups of fl avanols
such as: chalkan-fl avans, biomolecular combinations
of a catechin attached to a chalcone derivative; bisfl a-
vanols 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
identifi ed in the tea leaves [59]. Other groups of poly-
phenols occurring in the tea leaves are fl avonols exist-
ing both in the free state and as glycosides [60] and
depsides which are condensation products of two dif-
ferent 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 respon-
sible for methylxanthine synthesis [59,61]. The popu-
larity 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 precur-
sors 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 com-
ponents. The composition of green tea beverage
(15 g leaves/L) is: catechins 3893.9 μ mol/L, theafl avins
2.59 μ mol/L, flavonols 212.9 μ mol/L, gallic acid
315.1 μ mol/L, carbohydrates 1136.8 μ mol/L, proteins
(per amino acid residues) 129.8 μ mol/L and minerals
763.9 μ mol/L [59].
Figure 4. Chemical structure of catechins (A) and theafl avins (B).
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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 theafl avins while the green tea bev-
erage (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
theafl avins (Figure 4), which are dimeric catechins
[68]. The characteristic element of their structure is
the seven-member benzotropolone ring [69]. In addi-
tion to the theafl avins other benzotropolone com-
pounds as well as bisfl avanols, theasinensin and
thearubigins were also identifi ed in black tea, but in
considerably smaller amounts [70,71]. Besides the
already mentioned components, black tea contains
avonols 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 μ mol/L, theafl avins 75.1 μ mol/L, thearubigins
210.86 μ mol/L, fl avonols 139.7 μ mol/L, gallic acid
527.3 μ mol/L, carbohydrates 289.6 μ mol/L, proteins
22.3 μ mol/L and minerals 199.8 μ mol/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, poly-
phenols, is connected with the bioavailability and
biotransformation of these compounds, especially
catechins and theafl avins, in the gastrointestinal tract
and in the liver. The bioavailability compounds and
distribution of tea components was investigated in the
biological fl uids (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 pre-
sumably ECG to EC is probably caused by microbial
catechin esterase. The cleavage of the remaining gal-
loyl groups takes place in the intestines [73]. Next
free hydroxyl groups of the catechins are conjugated
with glucuronic acid, sulphate, glycine or O -methy-
lated which occurs in the jejunal and ileal sections of
the small intestine. Catechins and their derivatives are
transported into the liver where unmodifi ed ones are
conjugated (methylated, sulphated and glucuroni-
dated). 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 sul-
phate groups facilitates the urinary and biliary excre-
tion of catechins and explains their rapid elimination.
The catechins conjugates are distributed to fl uids 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
tea
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 fi nally 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 rela-
tively well examined in the animal and human organ-
isms, little information concerning bioavailability and
biotransformation of black tea polyphenols is avail-
able. An increase in catechins as well as theafl avins
levels in the plasma and the liver after black tea inges-
tion 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 metabo-
lized [86].
Effects of antioxidant action of different kinds of tea . The
past few years have been rich in information concern-
ing the role of tea antioxidants concerning the health
benefi ts of tea consumption. Antioxidant properties
of the main tea polyphenols, catechins and theafl avins
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, signifi -
cantly prevent biologically important cellular compo-
nents such as lipid, protein and nucleic acids from
oxidative modifi cations [88,89].
Prevention of free radical generation by green tea
as well as catechins and TF
3 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 myeloperoxi-
dase [93]; tea has also been proved to inhibit the
activity of cyclooxygenase COX-2 and 5-, 12- and
15-lipoxygenases participating in enzymatic lipid per-
oxidation [94,95].
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1226 A. Augustyniak et al.
those containing hydroxyl groups and decrease also
the fl uidity in the polar surface of the phospholipid
bilayer [105]. Green as well as black tea administra-
tion 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 infl uence on the
antioxidant capacity of biomembranes [110]. Tea
components prevent the oxidative consumption of
α -tocopherol, repairing tocopheryl radical and pro-
tecting 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 con-
tains large amounts of polyunsaturated fatty acids and
a high content of catalytically active metal ions, espe-
cially in the striatum and hippocampus [112].
The brain tissue is particularly vulnerable to mem-
brane lipid peroxidation that disturbs fundamental
functions of the brain. Lipid peroxidation may be
implicated in the irreversible loss of the neuronal tis-
sue 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 infl uence free radical generation and lipid
peroxidation [88].
It has been shown that teas may prevent cardiovas-
cular diseases, because drinking tea (oolong, green
and black tea) affects favourably lipid metabolism
[112]. Green and black tea as well as theafl avins and
catechins prevent LDL oxidation and at higher con-
centrations are more effective than tocopherol [113].
Theafl avins inhibit LDL oxidation [114,115]. Cate-
chins 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 his-
tydyl and lysyl residues of apolipoprotein B-100
[117,118]. Green and black tea protect also other
protein molecules against oxidative modifi cations.
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 administra-
tion [83,84].
Teas have recently obtained also signifi cant accep-
tance as cancer preventive substances. Epidemiologi-
cal and laboratory studies have revealed that green
Many studies have demonstrated that both cate-
chins and teafl avins, besides preventing free radical
generation, have strong free radical-scavenging abili-
ties both in vitro and in vivo [88,96]. The conversion
of catechins to theafl avins during tea manufacturing
does not affect their radical-scavenging potency [96].
Moreover, it was shown that the galloyl moiety of cat-
echins and theafl avins 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 infl uenced by the
low values of standard one-electron reduction poten-
tial of the polyphenols. The reduction potentials of
catechins and theafl avins are similar to those of vita-
min E, but higher than that of vitamin C, which is one
of the strongest free radical scavengers [98,99]. Con-
sidering the standard one-electron reduction potential
value, polyphenols may be expected to scavenge vari-
ous free radicals generated in the organism [100].
Several structures appear to be important in con-
ferring 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 delocaliza-
tion while the trihydroxyl group in the B ring stabi-
lizes the radical form of polyphenols. A gallate moiety
esterifi ed 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
TF
3 , TF
2 , TF
1 are all able to scavenge superoxide
radical (O
2
), but TF
3 acts the most effectively [90].
In addition theafl avins 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 (
1 O
2 )
and, of course, OH [90]. In addition, catechins have
been found also to effi ciently 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 gastrointesti-
nal tract [102]. Moreover they also infl uence the anti-
oxidant 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 com-
partment and diminish the oxidative attack on phos-
pholipids 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 fl uidity of this region
of the membrane [104]. Catechins can also interact
with phospholipid head groups, particularly with
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Natural and synthetic antioxidants 1227
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 adminis-
tered in drinking water inhibit carcinogenesis in vari-
ous organs in humans and rodents [119,120]. Teas
and their polyphenols inhibit the biochemical activa-
tion of genotoxic pro-carcinogens and carcinogens
metabolism. They inhibit cytochrome P450, in par-
ticular cytochrome P-450 1A1, 1A2 and 2B1 activi-
ties [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- κ B,
controlling of the activity of transforming growth fac-
tors TGF- α and TGF- β [73,128,129]. Tea polyphe-
nols have been found to attenuate the activation of
NF- κ B [128,130] and AP-1 activity [131], i. a. by
inhibiting kinases [114,131]. It has been shown that
theafl avins inhibit the c-jun protein phosphorylation
what in turn causes inhibition of the transcription fac-
tor AP-1 activity which plays a crucial role in cell
proliferation and transformation [132]. Studies on
human cancer cell lines showed the potency of inhibi-
tion of cell growth by catechins as well as theafl avins
[133,134].
Teas protect also cellular components of different
tissues against oxidative modifi cations appearing dur-
ing 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 fl uids
[102,135,136].
Teas are thus regarded as one of the most promis-
ing chemopreventive agents. However, several inves-
tigations indicate a signifi cant positive relationship
between tea consumption and cancer development. It
has been found that catechins and theafl avins, in par-
ticular when used at high non-physiological concen-
trations, may induce reactive oxygen species and
oxidative DNA damage [137,138]. These data suggest
that teas, like other natural antioxidants, can also unfor-
tunately 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).
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1228 A. Augustyniak et al.
effects may be important factors in the amelioration
of initiation and progression of atherosclerosis dem-
onstrated by epidemiological studies [142].
Drinking tea is also associated with decreased fre-
quency 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
specifi c types of cancer, including lung, breast, ovar-
ian and prostate cancer [150 154]. Intervention stud-
ies 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
confi rmed [87].
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 epidemio-
logical 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, cho-
lesterol and reduces LDL oxidation [148,149]. There
also appears to be an immediate effect of improving
the endothelial function and enhancing the blood fl ow
[149]. These combined biochemical and physiological
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).
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Natural and synthetic antioxidants 1229
Table II. Effect of tea consumption on the risk of cancer development.
Black tea Green tea
Disease Health effect References Health effect References
Cancer 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
Prostate cancer: No signifi cant 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
Bladder cancer: 1452 bladder cancer cases vs
406 kidney cancer cases vs 2434 controls.
No signifi cant 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, confl icting 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)
[366]
[367]
[368]
[369]
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
Breast cancer: Prospective study on
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
breast cancer patients and 594
controls; consumption: 85.7
mL/day 0–85.7 mL/day,
signifi cant trend of decreasing
risk of breast cancer with
increasing amount of green tea
intake
[150]
[154]
[370]
[151]
[152]
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 signifi cant
association between tea and colon or rectal
cancer, or positive association between
black tea and colon cancer
[139] Gastrointestinal cancer: Japan; 887
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
chronic atrophic gastritis, 133
gastric cancer and 433 controls,
up to 21 cups/week, signifi cant
inverse association between tea
drinking and gastric cancer
[371–373]
[374]
Cardiovascular
diseases (CVD)
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
[140,145,
375,376]
40 530 subjects, consumption of
green tea: up to 10 cups/day; the
benefi cial effects of green tea on
CVD risk profi le in more than
half of the controlled trials
[377–381]
Neurodegenerative
diseases
Chinese aged 45–74 years, consumption: up to
6 cups/day, black tea reduces Parkinson’s
disease risk
[141] Chinese aged 45–74 years,
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
cups/day or more; in prospective
cohort study of 30 000 Finnish
drinking 3 or more cups/day
[141]
[382,383]
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1230 A. Augustyniak et al.
oxygen [156,157]; and (iii) the Hydrophobic Domain ,
responsible for docking the agents in circulating lipo-
proteins and biological membranes [158]. Vitamin E
derivatives, such as the α -tocopheryl succinates (sa-
α -toc), in which the radical inhibitory activity has
been blocked by esterifi cation 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 signifi cantly
higher effi cacy than α -tocopherol alone [159 162].
Hydrolysis of sa- α -toc by activated esterases, how-
ever, after absorption into the cells and consequent
release of the negatively charged antioxidant α -
tocopheryl, endows signifi cant chemoprotective prop-
erties 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 anti-
apoptotic 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
amplifi cation of the pro-apoptotic signal (Figure 7).
The rate of hydrolysis of sa- α -toc to α -tocopheryl is
signifi cantly 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 prod-
ucts 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 signal-
ling pathways, such as those involving mitochondrial
Human epidemiological data suggest that tea
drinking may decrease the incidence of neurological
disorders, because ROS play a pivotal role in the age-
associated cognitive decline and neuronal loss in neu-
rodegenerative diseases including Alzheimer s and
Parkinson s diseases [141,143]. In particular, the main
polyphenol constituents of tea are now being consid-
ered as therapeutic agents in well controlled epidemio-
logical studies, aimed to alter the brain ageing processes
and to serve as possible neuroprotective agents in pro-
gressive neurodegenerative disorders [141,144].
Despite numerous studies in recent years, the
understanding of the biological activities and health
benefi ts of tea polyphenols is still very limited. Fur-
ther in-depth studies are needed to investigate the
safety and effi cacy 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 mecha-
nisms have important public health implications.
Refi ning 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 nor-
mal cells. The past decade has experienced the evolution
of a promising group of selective agents, comprised of
(a) organic selenium compounds, (b) redox-silent vita-
min E analogues and (c) their combination. This chap-
ter highlights the propensities of these agents to
modulate critical signals and affect selectively a multi-
tude of molecular targets in pre-malignant and malig-
nant lesions. It further provides an insight into how
modifi cations of their structural characteristics in novel
selenoanalogues of vitamin E esters may alter dramati-
cally their biological activities due to the evolution of
signal-generating active intermediates and metabolites.
Structural interventions in the functional domain
of vitamin E
The term vitamin E refers to one or more structurally-
related 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, epito-
mized by the hydroxyl group in α -tocopherol; (ii) the
Signalling Domain , comprised of the aromatic rings
(phenol-, chromanol-) and activated by the monoes-
terifi cation of dicarboxylic acids with the phenol
Figure 7. Proposed model of early biochemical events and signal
generation and transduction following the introduction of sa-
α-tocopherol into the cells and its hydrolysis by active esterases.
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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 can-
cer cells [175 178], induce differentiation [167], halt
metastasis [179] or sensitize them to other non-target
selective anti-cancer agents [177,180]. The require-
ment 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 ( α -
TOA) or dicarboxylic diesters. On the contrary, α -
tocopheryl-lysine, which bears a cationic Functional
Moiety at physiological pH, exhibits a very potent pro-
apoptotic activity [181]. Enhancement of lipophilicity
reduces apoptogenic activity of tocopheryl dicarboxy-
lates [163], most likely due to perturbation of the
amphiphilic balance that maintains the conforma-
tional characteristics required for signalling modula-
tion. 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 organose-
lenium 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 contribut-
ing to the hydrogen selenide pool with excess of sele-
noprotein 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 stud-
ies, 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 effi -
cacy. 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 identifi cation of synergistic effect of the meth-
ylselenol 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 anti-
cancer potential of selenium and Vitamin E analogues.
These mainly involve (a) the increase of the bioavail-
ability of selenium with concurrent decrease of its
toxicity, (b) modulation of antioxidant activity of vita-
min E analogues without compromising their apop-
totic and immunoregulatory properties, (c) delineation
of the relationship between structures, lipophilicity,
acidity and biological activity and (d) enhancement
of apoptotic vs necrotic properties of selenocom-
pounds by directing the molecules towards the meth-
ylselenol 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-2-
phenylselanyl-succinate (pssa- α -toc), γ -tocopheryl-
2-phenylselanyl-succinate (pssa- γ -toc) and
γ -tocotrienyl-2-phenylselanyl-succinate (pssa- γ -tot)
have been the fi
rst selenium-containing derivatives of
sa- α -toc, of γ -tocopherol and γ -tocotrienol, respec-
tively (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 assess-
ment of the DPPH scavenging activity of these com-
pounds 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 pssa-
esters protected against H
2 O
2 -induced damage to
DNA in a standard Comet Assay (Figure 9, Ia).
Quantifi cation of oxidative DNA damage confi rmed
that pssa-esters are more potent antioxidants than the
ptsa- and sa-esters and all the esterifi ed forms are
more potent than free tocopherol (Figure 9, Ib).
This effect is attributed to the high activity of
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1232 A. Augustyniak et al.
When applied, as an approach to detect apoptotic
nuclei in colon cancer cells exposed to the same com-
pounds, SYBR Green staining revealed signifi cant
pro-apoptotic induction of apoptotic nuclei by these
esters. This effect was not shared by non-esterifi ed
α -tocopherol (Figure 9, IIA). The specifi city of the
pro-apoptotic effect was further confi rmed by the
staining of normal nuclei in samples co-treated with
a pan-caspase inhibitor (Figure 9, IIB).
The impact of esterifi cation in succinate
monoesters and the co-existence of phenylselenyl-
or phenylthioyl-moieties on the induction of pro-
apoptotic responses was further correlated with the
effect of structural modifi cations identifi ed in the
chromanyl moiety, driving the generation and
transduction of molecular signals. Apoptotic cell
death is disclosed by DAPI-stained nuclei and is
defi ned by condensed chromatin, apoptotic bodies
and rings (Figure 10A). Apoptotic cell death was
quantifi ed 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 com-
pared to their α -counterparts and higher pro-apop-
totic 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 exten sively documented. We identifi ed recently
765 proteins differentially modulated by γ -tocot-
rienol in prostate cancer cells [169]; Odysseos et
al., unpublished). Notably, the effect of phenylth-
ioyl 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 cas-
pase-3 enzymatic activity by the sa-, phenylselenyl-
and 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 amplifi cation of the signals greatly
depends on the number of esterifi ed moieties and
the structural characteristics of the chromanol ring.
The augmented apoptogenicity of the tocotrienyl
compounds is greatly attributed to the better mem-
brane docking effi cacy of the unsaturated phytyl
chain leading to potentially higher intracellular
concentrations.
The correlations between structural modifi cations,
in vitro free radical scavenging activity and peroxida-
tion inhibition effi cacy in normal living cells and pro-
apoptotic activity in cancer cells are depicted in
Figure 11. Ongoing studies with fl uorescent/biolumi-
nescent ester compounds and in vivo molecular imag-
ing intend to reveal the active products of hydrolysis
in tumour cell populations and lead to the identifi ca-
Figure 8. (A) Structure of α-tocopherol, γ-tocopherol and
γ-tocotrienol. Green indicates the chromanol ring with the free OH-
functional domain. Sa, pssa and ptsa are the structural units
replacing OH- during esterifi cation 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 fi rst 10 min of the reaction. Concentrations were
calculated from absorption at 305 nm. bCalculated from the slope
of the plot of pseudo-fi rst-order rate kobsd. Concentrations were
calculated from the absorbance at 515 nm. Modifi ed from [182].
non-specifi c esterases in normal colon cells which
hydrolyse sa- α -toc and which apparently have similar
effect on the selenyl- and thioyl-esters.
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Natural and synthetic antioxidants 1233
tion of new molecular targets that would serve as effi -
cacy biomarkers in future trials.
Synthetic molecules with antioxidant action
In vitro studies and experiments in animal models
sug gest a plethora of synthetic antioxidant com-
pounds 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, hydro-
gen peroxide [194], GPX mimetics (ebselen, BXT-
51072), coenzyme Q analogues (e. g. ibedenone)
[195] or aminosterols (lazaroids) [196]. In this
review, two groups of promising synthetic antioxi-
dants 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 esterifi ed forms of α-tocopherol. Arrows indicate different degree of oxidative
damage (comet index). White: 0; pink: 1; blue: 2; yellow: 3; red: 4. (b) Quantifi cation of oxidative DNA damage with calculation of the
comet index shows statistically signifi cant 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 esterifi ed
compounds. DNA damage was not observed with caspase inhibition.
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1234 A. Augustyniak et al.
Figure 10. Structural modifi cations 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 fl uorescence microscopy. The apoptotic effi cacy 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 specifi c fl uorigenic 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.
Figure 11. Structure–activity relations in free tocols, sa-, pssa- and
ptsa-esters: (A) apoptotic activity in malignant cells increases with
esterifi cation; 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
esterifi cation: 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 esterifi cation: binary Se esters are less potent antioxidant in
vitro than binary S esters and S esters are less potent than succinate
monoesters.
Stobadine as an indole-type antioxidant standard:
Physicochemical properties, mechanism of action and
effi ciency in comparison with Trolox
Trolox (Figure 12A), a water-soluble analogue of α -
tocopherol, represents a popular reference antioxi-
dant. This carboxylic acid chromane has been broadly
used as a standard when screening the antioxidant
effi cacy of prospective antioxidants in studies involv-
ing chemical, sub-cellular, cellular and tissue experi-
mental models of oxidative damage [39,40,197].
However, a fairly large and specifi c group of prospec-
tive substances with benefi cial biological effects is
represented by the indole-type antioxidants [198].
This puts a demand on the reassessment of the suit-
ability 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
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Natural and synthetic antioxidants 1235
of 8.5 [207] has 92% of the basic nitrogen in pro-
tonated 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 sto-
badine but not of Trolox, into the lipid phase. This
may explain the low apparent antioxidant effi ciency
of Trolox in experimental models involving membra-
neous systems [209 213].
Trolox and stobadine: Redox properties . Early pulse radi-
olysis 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 deproto-
nates from the indolic nitrogen to give a resonance
stabilized nitrogen-centred radical [200]. With regard
to the pK
a value of 5 of Trolox-derived phenoxyl
radical cation [214] and its expected extremely rapid
and other indole-type antioxidants. Several compre-
hensive reviews cover stobadine action in various
simple chemical systems, biological models at sub-
cellular, 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-2,8-
dimethyl-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 pK
a value of
the carboxyl group 3.89 [206], Trolox undergoes vir-
tually complete dissociation at physiological pH
(99.92% in the COO
form). On the other hand,
stobadine with the pK
a value of the tertiary nitrogen
Figure 12. (A) Structures of stobadine and Trolox; (B) Mechanisms of free radical scavenging by stobadine and vitamin E; biologically-
relevant coupled reactions that might recycle stobadine [201] and Trolox [214].
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1236 A. Augustyniak et al.
radical. When stobadine and Trolox were present simul-
taneously in oxidatively stressed liposomes, Trolox
spared stobadine in the system in a dose-dependent
manner [211]; a direct interaction of Trolox with sto-
badinyl 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-defi cient
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 neg-
ative 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 demon-
strated by the increased magnitude of the ascorbyl
radical ESR signal generated in the presence of sto-
badine in the system of lipoxygenase arachidonate
[201].
Thus, the antioxidant potency of both Trolox and
stobadine within biological systems may be modu-
lated by their interaction with other lipid- or water-
soluble antioxidants.
Trolox and stobadine: Antioxidant effi cacies in the assay sys-
tems . In a homogeneous system, antioxidant activity
stems from an intrinsic chemical reactivity towards rad-
icals. In membranes, however, the relative reactivities
may be different since they are determined also by addi-
tional factors such as location of the antioxidant and
radicals, ruled predominantly by their distribution ratios
between water and lipid compartments. As already men-
tioned, a notably lower distribution ratio of Trolox than
that of stobadine may account for their different effi ca-
cies in systems involving lipid interface (membranes) in
comparison to homogenous units (true solutions).
In the ethanolic solution, Trolox scavenged the
DPPH
radical more effi ciently than stobadine,
based on the initial velocity measurements [211]
and comparison of rate constants [210] (Table III).
In the models of oxidative damage comprising sol-
uble 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 reac-
tion system of Fe
2
/EDTA/H
2 O
2 /ascorbate less
effectively than did Trolox [220]. The experimental
IC
50 values correlated well with the reciprocal val-
ues of the corresponding second order rate con-
stants for scavenging
OH radicals. Trolox, in
comparison with stobadine, was also found to be a
more effi cient inhibitor of AAPH-induced precipita-
tion of the soluble eye lens proteins [223]. Con-
versely, protein oxidation yielding free carbonyls
was more effi ciently inhibited by stobadine. Both
stobadine and Trolox showed comparable effi cacies
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 phe-
nolic 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 k
superoxide 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 super-
oxide ( k
superoxide 0.1 M
1 s
1 ), 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 com-
parable with that of stobadine (Table III). Neverthe-
less, according to the study of Aruoma et al. [219],
the second order rate constant of Trolox for scaveng-
ing HO
radicals is almost one order of magnitude
higher than that of stobadine. This fi nding is in a close
agreement with our data obtained in a study where
the effi cacy 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
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 Stobadine Trolox
HO7 109 [200]
15.9 109 [199]
8.5 1010 [219]
CH3COO
Cl3COO
5 106 [200]
6.6 108 [200]
2.5 106 [384]
3.7 108 [214]
DPPH4.9 102 [210] 1.6 103 [210]
C6H6O5.1 108 [200] 4.1 108 [214]
O2
•− 7.5 102 [201] 1.7 104 [217]
0.1 [218]
1O21.3 108 [200] pH 6 3.5 108 [385]
DPPH, 1,1’-diphenyl-2-picrylhydrazyl.
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Natural and synthetic antioxidants 1237
Table IV. Summary of antioxidant and protective effi cacies of stobadine and Trolox in experimental models of oxidative damage.
Assay system Parameter measured Stobadine 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
modifi cation of soluble
eye lens proteins [223]
Inhibition of protein
precipitation
IC50 (μmol/L) 121 15 79 8
Inhibition of protein
oxidation
44 8 131 20
Oxidative modifi cation
of BSA in an
experimental glycation
model [224]
Glucose attachment into
the molecule of BSA
Amadori product (with
respect to 8.2 0.4
nmol/mg BSA for
control without
inhibitor)
8.1 0.5
(0.25 mmol/L)
7.4 0.7
(0.25 mmol/L)
Glycation-induced
uorescence changes
of BSA
Relative fl uorescence (with
respect to 11.2 0.7
for control without
inhibitor)
7.9 0.7
(0.25 mmol/L)
6.5 0.4
(0.25 mmol/L)
Formation of DNPH-
reactive carbonyl
groups in BSA
Carbonyl groups (with
respect to 5.6 0.4
nmol/mg BSA for
control without
inhibitor)
3.4 0.5
(0.25 mmol/L)
3.3 0.2
(0.25 mmol/L)
Cu2mediated oxidation of LDL Δtlag (min) (The increase
in lag time given by
one stobadine molecule
per single LDL
particle)
1.5 0.38
Fe2/ascorbate induced
oxidative damage of rat
brain homogenate [227]
Inhibition of
TBARS
IC50 (μmol/L) 35 98
AAPH-induced haemolysis of rat erythrocytes [212] tlag (min) (88.6 2.2 for
control erytrocytes)
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.
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 via-
bility of hydrogen-peroxide treated PC12 cells more
effectively than did Trolox, while both compounds
reduced the content of malondialdehyde with a com-
parable effi ciency [213].
In summary, these data underscore the structural
and physicochemical differences between Trolox and
stobadine as respective representatives of phenolic-
and indole-type antioxidants. The structural variance
explains their different mechanisms of antioxidant
action and variable effi cacies in the range of assay
systems studied, suggesting that stobadine may rep-
resent a promising indole-type reference antioxidant.
In studies of indole compounds, stobadine antioxi-
dant standard may thus be used as a more acceptable
alternative to the structurally diverse Trolox.
in an experimental glycation model in preventing
glycation-related fl uorescence changes of BSA as
well as in lowering the yield of 2,4-dinitrophenylhy-
drazine-reactive carbonyls as markers of glyco-
oxidation (Table IV) [224].
On the other hand, Trolox was found to be much
less effective in inhibiting AAPH-induced peroxida-
tion of DOPC liposomes with respect to stobadine
[209,210, 225] (Table IV). Stobadine, in comparison
with Trolox, more effectively prolonged the lag phase
of Cu
2
-induced LDL oxidation measured by diene
formation [226]. The same pattern of effi cacy in pre-
vention of the lipid oxidation boost was shown in the
system of tissue homogenate. Stobadine showed a
more potent inhibitory effect than Trolox on lipid per-
oxidation in rat brain homogenates exposed to Fe
2
/
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 effi cacy of stobadine and
Trolox was comparable [228]. This fi nding may indi-
cate that the critical competition of the scavengers
with the alloxan-derived initiating reactive oxygen
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1238 A. Augustyniak et al.
tion barriers E
A were calculated by AM1 method as
E
A E
react E
TS , where E
react was the energy of
reactants (actually, it was the energy of the hydrogen
bonded complex of HO
with corresponding H-N
species) and E
TS 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 pyri-
doindoles differed markedly from those for saturated
compounds, in agreement with their measured activ-
ities of Δ A5min.
Using the values of E
HOMO and BDErel we calcu-
lated the linear regression equations
− Δ A5min 0.0031 · (E
HOMO )
1.7284, n 7; R
2 0.92
−Δ A5min 0.0052 · (BDErel)
0.2094, n 7; R
2 0.94
As seen, the higher computational level led to bet-
ter estimation of the structure activity relationships,
compared with our former results based only on semi-
empirical level [225].
According to the Arrhenius equation and using the
values of activation energies we obtained the expo-
nential regression equation:
−Δ A5min 203.9 · exp( 0.1 EA),
n 7; R
2 0.96.
The use of activation energies provided better results
in comparison with calculations employing other para-
meters. The main reason is probably approaching the
physical meaning of the rate constant approximated with
the values of − Δ A5min.
To conclude, the structure activity relationships
obtained may thus be used for a rational design of
more effi cient antioxidant compounds in the series of
carboxymethylated pyridoindoles potentially effective
in prevention of diabetic complications.
Peculiarities of 1,4-dihydropyridines as hydrophobic
antioxidants
Modulation of lipid peroxidation reactions in bio-
logical objects include several possibilities of action:
(i) infl uence on chemical reactions contributing to
peroxidation by the use of exogenous antioxidants,
(ii) infl uence on enzymatic reactions inducing per-
oxidation (by the use of enzyme inhibitors or activa-
tors), (iii) infl uence on the compartmentalization of
peroxidation and integrity of cellular membranes and
(iv) infl uence on the biosynthesis of endogenous anti-
oxidant proteins and low-molecular mass antioxidants
and pro-oxidants.
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 fi rst 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 dam-
age 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 supplementa-
tion to attenuate complications in diabetic animals.
Recently novel carboxymethylated pyridoindoles
(Figure 13), analogues of stobadine, have been designed,
synthesized and characterized as bifunctional com-
pounds with joint antioxidant/aldose reductase inhibi-
tory 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 subse-
quently the low conformers were reoptimized by the
AM1 and DFT methods using the B3LYP func-
tional and the 6-31G
basis set. The piperidine ring
was taken in the chair conformation with nitrogen
in equatorial position, while nitrogen in the tetrahy-
dropyridine ring, in the conformation derived from
the former one, was in axial position. DFT results
were used for E
HOMO and BDE
rel calculation. Values
of BDE
rel were obtained by differences of the ener-
gies of individual structures E and their relevant
indolyl radical E
R , i.e. BDE E E
R , and BDE
rel
were related to the lowest value (stobadine). Activa-
Figure 13. General chemical structure of carboxymethylated
hexahydro- (A) and tetrahydro-pyridoindoles (B) related to
stobadine.
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Natural and synthetic antioxidants 1239
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 ana-
logues of 1,4-dihydronicotinamide and model com-
pounds for NAD(P)H. 1,4-DHP derivatives are
synthetically available due to the convenient Hantz-
sch type cyclic condensations. Derivatives of 1,4-
DHP have been objects of numerous studies, the
more that some of these compounds have been stud-
There are numerous studies of the infl uence of
drugs, potential drugs and other synthetic or natural
compounds on peroxidation processes in cells, tissues,
organisms or simple in vitro models. Quite often molec-
ular mechanisms are not studied in the initial phase
and the black box approach is employed to fi nd com-
pounds which are effi cient in modulating lipid peroxi-
dation. It is important that methods used for such
studies are appropriate, to avoid artifacts, which can
easily appear in studies of multicomponent systems.
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) EHOMOb (kJ/mol) BDErelb (kJ/mol) −ΔA5minc
stobadine 67.1 474.4 0.00 0.239
1a 69.7 508.9 6.54 0.132
2a 68.2 509.2 5.80 0.187
dehydrostobadine 82.9 531.7 32.62 0.033
1b 84.7 546.2 35.51 0.031
2b 83.8 543.8 34.62 0.030
3b 88.3 547.7 35.71 0.031
aactivation barriers EA calculated by AM1 method.
bthe 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 λmax 518 nm; during the fi rst 5-min interval.
Figure 14. Structures of some 1,4-dihydropyridine derivatives.
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1240 A. Augustyniak et al.
(Table VI). The Fe
3
-reducing ability of a 1,4-dihy-
droisonicotinic 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 Fe
3
. Compound I pro-
tects also liver cells from copper-induced lipid per-
oxidation and increases hepatocyte viability [244].
Studies of 1,4-DHP in metal-ion catalysed peroxi-
dation 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]. Dilu-
din and relative 1,4-DHPs were found to be rela-
tively non-toxic. LD
50 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 sys-
tems. Diludine was again found to be the most active
compound, the photodegradation product of nife-
dipine (nitrosophenyl derivative of pyridine) showed
a high reactivity and kinetic rate constants for the
reaction between 1,4-DHP compounds and alkylper-
oxyl radicals exhibited a fairly good linear correlation
ied and registered as anti-hypertensive and anti-
anginal drugs. There is a lot of data on their
neurotropic, anti-infl ammatory, anti-diabetic, anti-
mutagenic, growth stimulating, anti-ageing and anti-
oxidant activities [231].
The antioxidant activity (AOA) of 1,4-dihydropyri-
dine (1,4-DHP) derivatives was fi rst demonstrated
for 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydro-
pyridine (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 1,4-
DHP to inhibit free radical reactions was also docu-
mented [235]. In 3,5-dicarbonyl-1,4-dihydropyridine
derivatives strong bis- β -dicarbonylvinyl-amino conju-
gation exists and consequently they cannot be con-
sidered 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-1,4-
dihydropyridine 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 com-
pounds in model and biological membranes of vari-
ous degrees of complexity (emulsions of unsaturated
fatty acids esters, phospholipids liposomes and eryth-
rocyte membranes) revealed the infl uence of length
of alkyl groups of DHP esters, as well as of the nature
of heterogenous systems on AOA of 1,4-DHP deriva-
tives [237 239]. 4-Alkyl- and especially 4-aryl-3,5-
dialkoxycarbonyl-1,4-DHPs have lower AOA, but of
3,5-dicarbamoyl-1,4-DHPs not substituted in posi-
tion 4 are more prone to oxidation so 4-aryl deriva-
tives have higher AOA.
It was found that 1,4-DHP derivatives in model
systems are less active antioxidants than BHT. How-
ever, 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 antioxi-
dant 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 Fe
3
-reducing ability [243]
Figure 15. Reaction of 1,4-dihydropyridines leading to the
formation of pyridine derivatives.
Table VI. Additivity of the antioxidant action of Diludin with BHT
and BHA.
Substrate and antioxidants AOA (τ/τ0)
Methyl oleate (60°C)
Diludin 1.0
BHT 38.0
BHT Diludin 51.0
BHA 25.0
BHA Diludin 25.0
Sunfl ower oil (20°C)
Diludin 1.2
BHT 1.4
BHT Diludin 1.8
Cooking fat (20°C)
Diludin 1.2
BHT 1.2
BHT Diludin 1.9
Figure 16. A 1,4-dihydroisonicotinic acid derivative (Compound I)
showing high Fe3-reducing activity.
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Natural and synthetic antioxidants 1241
dihydropyridines limit high glucose-induced superox-
ide formation and improve NO
bioavailability in
human endothelial cells [254].
1,4-DHP derivatives are metabolized by the cyto-
chrome 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-nitro phenyl-1,4-
DHP derivatives inhibit the microsomal lipid peroxida-
tion and microsomal thiols oxidation induced by Fe
3
/
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 pharmaco-
logical 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-p-
chlorophenyl derivative OSI-1146 displayed antioxidant
and anti-radical activities in vitro . All studied compounds
protected mitochondria against lipid peroxidation
induced by ADP/Fe
2
, OSI-1146 being the most
potent [256].
A lot of compounds with AOA have anti-infl amma-
tory properties [257], among them the cardiovas-
cular 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 gen-
eration in human smooth muscle cells during acti-
vation of infl ammatory mechanisms and diabetic
conditions [260]. Amlodipine inhibited IL-1 α
release [258], cerebrocrast showed an anti-infl am-
matory effect by reducing infl ammation in the rat
paw oedema model and inhibiting secretion of neu-
rotoxic cytokines interleukins IL-1 β and IL-6 in
human monocyte (THP-1) cell line [261]. Nife-
dipine was demonstrated to upregulate the biosyn-
thesis of Mn-superoxide dismutase [263]. ROS
formation in bovine aorta endothelial cell induced
by oxidized LDL was signifi cantly 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-4-
phenylpyridinium iodide (MPP
) induced ROS pro-
duction in cerebellar granule cells with a distinct pot ency
order: foridone (2,6-dimethyl-3,5-dimetho xy carbonyl-
4-( o -difl uoromethoxyphenyl)-1,4-dihyd ropyridine) 2,
6-dimethyl-3, 5-diethoxy carbonyl-4-phenyl-1,4-dihy-
dropyridine diludine [265]. These DHP derivatives
reversed the MPP
-induced decrease in the mitochon-
drial membrane potential in the same order.
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-dialkoxy-
carbonyl-1,4-DHPs have L-type Ca
2
channel
antagonist (blocker) properties. Several such com-
pounds have been proposed as drugs for the treat-
ment of hypertension and other cardiovascular
diseases; they also revealed anti-atherosclerotic prop-
erties in animal experiments. Their infl uence 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 1,4-
DHP derivatives bearing calcium antagonist prop-
erties. PubMed database shows that this problem
has been discussed in at least 19 reviews. Our stud-
ies of 1,4-DHPs substituted in position 4 with phe-
nyl and/or substituted phenyl residues (except for
o-nitrophenyl) do not show their considerable reac-
tivity with radicals or ability of iron reduction
(Table VII).
Antioxidant activity of nifedipine (3,5-dimethoxy-
carbonyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihy-
dropyridine; 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 rad-
icals, exhibiting antioxidant activity. Later on, AOA of
nifedipine and its oxidized nitroso analogue was stud-
ied [251,252].
It has been revealed that Ca
2
antagonists (espe-
cially derivatives of 1,4-DHP) and also some 1,4-
DHPs of lower activity of Ca
2
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 amlo-
dipine, nitrendipine, verapamil diltiazem. In the
cell-induced oxidation system nifedipine and felo-
dipine induced signifi cant reductions in the TBARS
content of LDL compared with amlodipine, vera-
pamil and the 4-nitro isomer of nifedipine. In this
oxidation system nifedipine was a more effective anti-
oxidant 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 sys-
tem and the assay used to measure the antioxidant
effect.
Dihydropyridine derivatives amlodipine and nisol-
dipine 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:
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1242 A. Augustyniak et al.
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 activ-
ity or expression of SERCA [273]. Modulation of
SERCA activity may be a contributing factor in the
development of some cardiovascular, neurodegen-
erative or skeletal muscle diseases. An important
feature of SERCA is its high sensitivity towards
modifi cation by ROS. Some phenolic substances
are able to modulate specifi cally SERCA activity,
most of them are inhibitors; however, certain sub-
stances 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 mod-
ulate 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-
Prevention of damage caused by ROS is also an
important antioxidant effect. Sodium 3,5-bisethoxy-
carbonyl-2,6-dimethyl-1,4-dihydropyridine-4-
carboxylate (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 μ M, showed
a transient increase in poly(ADP-ribose) level and the
rate and effi ciency of DNA strands break rejoining.
AV-153 was shown to have anti-mutagenic properties
[267], reduce DNA damage, decrease 8-oxo-7,8-
dihydro-2 -deoxyguanosine content and lower muta-
tion frequency [268,269].
A model system to study the biological action
of antioxidants: Protection of sarcoplasmic reticulum
membrane, sarcoplasmic Ca
2
-ATPase and
plasma membrane Ca
2
-ATPase by natural
and synthetic antioxidants
Sarco-/endoplasmic reticulum Ca
2
-ATPase
(SERCA) plays a key role in the relaxation of
smooth, cardiac and skeletal muscle through the
transport of cytosolic Ca
2
into the sarco-/endo-
plasmic reticulum [270]. SERCA 1 from fast-twitch
skeletal muscle is a single-chain transmembrane
protein with easily measurable function, present in
Figure 17. Some other 1,4-dihydropyridine derivatives.
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Natural and synthetic antioxidants 1243
substrates, Ca
2
and ATP (Figure 19). Trolox, stoba-
dine, SMe1 (50 and 250 μ mol/L), and Egb 761 (20
and 40 μ g/mL) exerted no signifi cant effect. The abil-
ity of Pyc to inhibit SR Ca
2
-ATPase may be linked
to its ability to induce apoptosis. Inhibition of Ca
2
-
ATPase activity may lead to an increase of cytosolic
Ca
2
, 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 can-
cer cells, whereas normal mammary cells were not
affected [280]. Similarly, inhibition of Ca
2
-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 pro-
cesses including activation of apoptosis [283] or inhi-
bition of platelet aggregation [284], which are known
to be regulated by Ca
2
.
Reactive oxygen species (ROS) in infl ammation and
SERCA . In many infl ammatory diseases, phagocytes
release HOCl, superoxide and H
2 O
2 . Release of the
two latter agents into a fl uid with free iron promotes
the production of hydroxyl radical in the Fenton
reaction and extensive damage of membranes, thus
causing accumulation of intracellular calcium
(Ca
i ). Chronically-increased Ca
i is a fi nal common
pathway in cell injury and death [285,286]. Plasma
membrane and sarcoplasmic/endoplasmic Ca
2
-
pump are mechanisms for removal of Ca
i . Therefore,
we focused our studies on oxidative damage of
SERCA by HOCl and the Fenton system and
possible protective or modulating effects of the above-
mentioned antioxidants.
induced adjuvant arthritis, an animal model of rheu-
matoid 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 antioxi-
dative activity [276]. With the aim to improve anti-
oxidant 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, sig-
nifi cantly 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 μ g/mL) signifi cantly decreased the activity of
Ca
2
-ATPase in SR vesicles, as well as the activity of
purifi ed Ca
2
-ATPase, with respect to both enzyme
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-1H-
pyrido[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.