Miguel 2010 FF (25) 291-312 Review Ac Esen

Review
Received: 19 October 2009; Accepted: 2 November 2009; Published online in Wiley Online Library: 30 November 2009
(wileyonlinelibrary.com) DOI 10.1002/ffj.1961
Antioxidant activity of medicinal and aromatic plants. A review.

M. G. Miguel*
ABSTRACT: Antioxidant properties have been attributed to the essential oils but a great diversity of results has been reported, mainly attributed to the chemical composition of the essential oils and the diversity of tests used. In fact, several in vitro assays for the determination of antioxidant ability have been used. They can be divided into two main groups: those that evaluate lipid peroxidation and that those that measure free radical scavenging ability. In the present study, a brief description of the mechanism involved in the majority of techniques used for the evaluation of antioxidant activity of essential oils is given. At the same time, the antioxidant activities of some essential oils, measured through several methodologies, are presented and compared. Copyright 2009 John Wiley & Sons, Ltd. Keywords: antioxidant; essential oils; lipid peroxidation; free radicals; phenols; aromatic plants
Antioxidants and Oxidation: General Concepts

Recently there has been growing interest in research into the role of plant-derived antioxidants in food and human health. However, other fields also need antioxidants, such as in polymerization control in the manufacture of rubber, plastics and paint and for the protection of clear plastics against ultraviolet light, or in the design of better automobile fuels and lubricating oils.[1] Therefore, the practical application of antioxidants has grown to be so wide that it is becoming difficult to find a proper definition for the term antioxidant itself. In the food industry, an antioxidant is defined as a substance that, in small amounts, is capable of preventing or delaying, in a significant way, the oxidation of easily oxidizable materials, such as fats. However, lipids are not the only macromolecules that may suffer oxidation. In biological systems, DNA and proteins are also susceptible to the oxidation processes. As a result, antioxidants can also be defined as molecules that, in low quantities when compared to those of an oxidizable substrate, are able to delay or prevent the substrate from becoming oxidized.[2] With such a broad definition, an antioxidant can be considered a substance that is capable of inhibiting a specific oxidizing enzyme, or a substance that reacts with oxidizing agents prior to them causing damages to other molecules, or a substance that sequesters metal ions, or even a substance capable of repairing systems such as iron-transporting proteins.[3] Not only it is difficult to define specifically the term antioxidant, but there is no accepted international definition for that term, as there is also no universal best antioxidant. For example, ascorbate protects well against plasma lipid peroxidation, caused by tobacco smoke, but not against plasma protein damage.[1] In mechanistic terms, an antioxidant can be defined as a hydrogen donor or an electron donor.[3] Thermodynamically, an antioxidants action depends on the bond energies and standard reduction potentials. These parameters allow deduction of whether or not a given radical can be quenched by a specific antioxidant.[2]
Reduction potential determines the feasibility that a certain compound has to chemically reduce another compound. A system with a negative standard reduction potential (E) should reduce (donate electrons to) a system with a less negative, zero, or positive E (Table 1).[1] Due to lower positive reduction potential, the ascorbate/ascorbyl radical system is capable of reducing the tocopheroxyl radical/-tocopherol system (Table 1). Therefore, the ascorbate reaction (ionic form, pH 7.4) is thermodynamically possible, enabling the ascorbate to regenerate vitamin E (tocopherol): H+ + ascorbate + -tocopheroxyl -tocopherol-H + ascorbate
The oxidizing action depends also upon the velocity constants. In that sense, the hydroxyl radical is an extremely strong oxidant, not just because of its strong reduction potential (+2310 mV), but also due to the relatively high velocity constants (Table 2).[4] The order of magnitude of the velocity constant characteristic of reactions limited only by the molecules movement is 1010 mol/l/s. This order of magnitude, verified for the hydroxyl radical with many substrates, reveals that there is a reaction as soon as a collision occurs between two entities, i.e. with a practically null activation energy. The hydroxyl radicals are extremely reactive, with a very small diffusion, and their reactivity is
* Correspondence to: M. G. Miguel, Faculdade de Cincias e Tecnologia, Edifcio 8, Universidade do Algarve, Centro de Desenvolvimento de Cincias e Tcnicas de Produo Vegetal, Campus de Gambelas, 8005-139 Faro, Portugal. E-mail: mgmiguel@ualg.pt Faculdade de Cincias e Tecnologia, Universidade do Algarve, Centro de Desenvolvimento de Cincias e Tcnicas de Produo Vegetal, Campus de Gambelas, 8005-139 Faro, Portugal
This article is part of the Special Issue of Flavour and Fragrance Journal entitled Aromatic Plants, Spices and Volatiles in Food and Beverages, edited by Ana Cristina Figueiredo and M. Graa Miguel
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Copyright 2009 John Wiley & Sons, Ltd.
M. G. Miguel antioxidants, whereas those acting by the last two mechanisms are considered primary antioxidants.[5]
Table 1. Standard reduction potentials of some biologically relevant species (pH 7) Speciesa Hydroxyl radical Aliphatic alkoxyl radical Alkyl peroxyl radical Glutathionyl radical Polyunsaturated fatty acid Vitamin E Vitamin C Iron complex Superoxide Oxidized glutathione Ionizing radiation
a

Half-cell OH , H+/H2O OR , H+/H2O OOR , H+/ROOH GS /GS PUFA , H+/PUFA-H OT , H+/TOH Asc , H+/Asc Fe3+EDTA/Fe2+EDTA O2/O2 RSSR/RSSR H2O/e aq

E/mV +2310 +1600 +1000 +920 +600 +480 +282 +120 330 1500 2870
In Vitro Determination of Antioxidant Activity

The antioxidant properties of different plant extracts, essential oils and pure compounds can be evaluated using various in vitro assays. Antioxidant assays in foods and biological systems can be divided in two groups: 1. Those that evaluate lipid peroxidation. 2. Those that measure free radical scavenging ability.[6] In assessing lipid peroxidation, several lipid substrates can be used. i.e. oils and fats, linoleic acid, fatty acid methyl esters and low-density lipoproteins (LDLs). The antioxidant activity in such systems can be detected by measuring the substrate and the oxidant consumption, and the intermediates or the final products formation. In food matrices, several measurements can be carried out: (a) (b) (c) (d) (e) (f ) Peroxide value. Thiobarbituric acid value (TBA). Iodine value. Free fatty acid content. Polymer content. Formation of conjugated dienes by absorption at 232 and 268 nm. (g) Colour. (h) Fatty acid composition. (i) Ratio of unsaturated : saturated fatty acids. Most of the tests based on lipid substrates need accelerated oxidation conditions: increased partial oxygen pressure and temperature; addition of transition metal catalysts; exposure to light; and variable shaking and free radical sources.[7] For measuring free radical scavenging ability, the methods are grouped in two groups, according to the chemical reactions involved: hydrogen atom transfer reaction-based methods and single electron transfer reaction-based methods.[3,8] Both types of reaction can occur simultaneously and the dominant reaction in the system can be determined according to the following features: the antioxidant structure and properties; the solubility and partition coefficients; and the solvent system. Bond dissociation energy and ionization potential are two factors that determine the mechanism and efficacy of antioxidants.[9,10] In addition, tests evaluating effectiveness against several reactive oxygen species and nitrogen reactive species (O2 , HO , ONOO, H2O2) are also needed and generally performed. In the hydrogen atom transfer reaction-based methods, the antioxidant is able to quench free radicals by hydrogen donation: X + AH XH + A . The relative reactivity of these methods is determined by the bond dissociation energy of the H-donating group of the antioxidant, which is characteristic for compounds with an interval of bond dissociation energy 10 kcal/mol and ionization potential <36 kcal/mol. These reactions are pH- and solvent-independent and are very fast (usually completed in seconds to minutes). The presence of reducing agents (i.e. metal ions) in such methods is not recommended because it can originate an apparently high reactivity.[10]

Species are displayed with the most oxidizing potential at the top and the strongest reducing potential at the bottom. The first species in the list can remove electrons from the latter ones.[127] Adapted from[127].
Table 2. Velocity constant values of hydroxyl radicals with several biological substrates Biological substrate Guanine Cytosine DNA Tryptophan (pH 6.9) Tyrosine (pH 7.8) Cysteine (pH 5.5) Albumin Haemoglobin Linoleate Ribose Glucose Ascorbate Adapted from[4]. Velocity constant k (HO + substrate) mol/l/s
9.2 109 4.9 109 4.0 108 1.4 1010 1.0 1010 4.0 1010 2.3 1010 3.6 1010 1.1 1010 1.6 109 7.4 108 1.1 1010
connected to the place of their formation or just some dozen nanometers away. Furthermore, hydroxyl radicals have a very short half-life (106 s).[4] These free-radical reactions are responsible for the extensive biological damage detected away from the site of their production.[5] In an oxidative sequence, antioxidants can act at different levels by: (a) decreasing localized oxygen concentrations; (b) preventing chain initiation by scavenging initiating radicals (HO , O2 ); (c) binding metal ions in such a manner that they will not generate species such as HO , ferryl or Fe2+/Fe3+/O2, and/or decompose lipid peroxides to peroxyl and alkoxyl radicals; (d) decomposing peroxides by converting them to non-radical products, such as alcohols; (e) chain-breaking, i.e. scavenging intermediate radicals, such as peroxyl and alkoxyl radicals, to prevent continued hydrogen abstraction. Chain-breaking antioxidants are often phenols or aromatic amines. Antioxidants acting by the first three mechanisms are designated preventive

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Antioxidant activity of medicinal and aromatic plants Methods based on the hydrogen atom transfer reaction include: inhibition of induced LDL oxidation; oxygen radical absorbance capacity (ORAC); total radical trapping antioxidant parameter (TRAP); and crocin-bleaching assays.[8] Single electron transfer based-methods detect the ability of an antioxidant to transfer one electron to reduce any compound, including metals, carbonyl groups and radicals: X + AH X + AH+
+ AH+ A + H3O H2 O
3. Evaluation of the antioxidant ability to inhibit or stop lipid oxidation in adequate model systems.[2]
Hydrogen Atom Transfer Reaction-based Methods Inhibition of induced oxidation of low-density lipoprotein, crocin bleaching assay, oxygen radical absorbance capacity method (ORAC) and the total radical-trapping antioxidant parameter method (TRAP) are examples of methods based on the transfer of hydrogen. Only the last two methods will be taken into account in the present review. In both cases there is: (a) a thermal radical generator, usually 2,2 azobis(2-amidinopropane) dihydrochloride (AAPH), to give a steady flux of peroxyl radicals in airsaturated solution; (b) a substrate to monitor (UV or fluorescence) the reaction progress; and (c) the antioxidant that will compete with the substrate for the radicals and inhibit or delay the substrate oxidation.[8] In the ORAC method, a fluorescent protein (R-phycoerythrin), fluorescein or dichlorofluorescein are used as substrates and the reaction progress is followed by fluorescence. The peroxyl radicals will react with a fluorescent substrate to form a nonfluorescent compound. The ORAC assay monitors the reaction during a period of >30 min and the antioxidant activity is determined from the net integrated areas under the fluorescence decay curves. ORAC values are generally reported as Trolox equivalents [M Trolox equivalents (TE)/g].[8,10] In the TRAP method, the substrates include R-phycoerythrin or 2,2 azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and therefore the substrate oxidation is followed by fluorescence or spectrophotometry, respectively. TRAP values are usually expressed as a lag time or reaction time of the sample compared to corresponding times for Trolox.[10]
X + H3O + XH + H2O M(III) + AH AH+ + M(II) Relative reactivity of the single electron transfer method is based on deprotonation and in ionization potential of the reactive functional group.[911] Therefore, single electron transfer methods are pH-dependent. Generally, ionization potential decreases with increasing pH values, which reflects a higher electron-donating capacity with deprotonation. The antioxidant mechanism is predominantly of the electron transfer type when the ionization potential values are superior to 45 kcal/mol. The reactions based on the electron transfer are usually slow and calculations are based on product percentage decrease more than in kinetic terms.[10] The electron transfer reaction-based methods include: (a) (b) (c) (d) Total phenols assay by FolinCiocalteu reagent. Trolox equivalence antioxidant capacity (TEAC). Ferric ion reducing antioxidant power (FRAP). Total antioxidant potential assay, using a Cu(II) complex as an oxidant. (e) 2,2-Diphenyl-1-picrylhydrazyl (DPPH).[8] There are also other assays that measure the samples scavenging ability for oxidants, which interact and damage the major macromolecules either in biological systems or in foodstuffs, such as: (a) (b) (c) (d) (e) Superoxide anion. Hydrogen peroxide. Hydroxyl radical. Singlet oxygen. Peroxynitrite.[3,8]
Electron Transfer Reaction-based Methods Methods based on this principle include an oxidant (substrate) that abstracts an electron from the antioxidant, causing colour changes of the substrate. The degree of colour change is proportional to the antioxidant concentrations. The reaction end-point is reached when colour change stops. The slope of the curve of the change of absorbance plotted against the antioxidant concentration reflects the antioxidant activity. This group of the methods includes: (a) total phenols quantification; (b) the Trolox equivalent antioxidant capacity (TEAC) method; (c) the ferric ion reducing antioxidant power (FRAP) method; (d) the Cu(II) reduction ability method; and (e) the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method.[3] Total phenols quantification (FolinCiocalteu reagent). Quantification of total phenolic content is typically carried out by the FolinCiocalteu method, based on the number of phenolic groups or other potential oxidizable groups present in compounds in the sample.[2] The chemical nature of the Folin Ciocalteu reagent is not exactly known, but it is believed to contain heteropolyphosphotungstatesmolybdates. The electron-transfer reaction occurs between antioxidants and molybdenum, which is reduced in the complex, leading to blue species, which can be measured spectrophotometrically. This reaction only occurs in an alkaline environment (pH 10), therefore the addition of sodium carbonate is very important.[8]
As reported above, sometimes the results obtained by different methods are not comparable, due to several factors such as: the physical structure of the test system; the nature of substrate for oxidation; the presence of interacting components; the mode of initiating oxidation; and the analytical method of measuring oxidation.[2,12] There is a great need to standardize antioxidant assays in order to minimize the present disorder in the methodologies. For foodstuffs, some authors propose a scheme that includes three main steps in the antioxidant evaluation of samples: 1. Quantification and identification of the phenolic compounds. 2. Quantification of radical scavenging activity, using more than one method and considering the solvent effect in the antioxidant mechanism.
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M. G. Miguel Trolox equivalent antioxidant capacity (TEAC) or ABTS method. This method relies on the reduction of the blue-green cation radical of ABTS+ . The extent of decolorization, expressed as percentage inhibition of ABTS+ , is determined as a function of the concentration and the time and it is calibrating against Trolox as the reference standard.[3] The concentration of antioxidants giving the same percentage change of absorbance of ABTS+ as that of 1 mM Trolox is considered as TEAC.[2,8] There is a method similar to that of TEAC, where ABTS+ is replaced by the coloured and stable DMPD + (N,N-dimethyl-pphenylenediamine).[2]

Ferric ion reducing antioxidant power (FRAP) method. In this method, a ferric salt, Fe(III)(TPTZ)2Cl3 (TPTZ = 2,4,6-tripiridil-striazina), is used as an oxidant agent. The redox potential of this salt is approximately 0.70 V. TEAC and FRAP assays are quite similar, differing only in the pH of the assay: TEAC occurs in neutral pH, while the FRAP method needs an acid environment (pH 3.6). One FRAP unit is arbitrarily defined as the reduction of 1 M Fe(III) to Fe(II).[3] Cu(II) reduction capability method. This method is not used extensively, or it was not mentioned enough in the literature. It is based on reduction of Cu(II) to Cu(I) by the antioxidant present in the sample. The Cu(I) forms with bathocuproin (2,9-dimethyl4,7-diphenyl-1,10-phenanthroline), a complex that has a maximal absorbance at 490 nm; 1 M -tocopherol is capable of reducing 2 M Cu(II) to Cu(I).[8] DPPH (2,2-diphenyl-1-picrylhydrazyl) method. Colourchanging of DPPH from purple to yellow is the consequence of of the reducing ability of antioxidants toward DPPH stable radical. Initially, DPPH was thought to be reduced to the corresponding hydrazine when it reacted with the donating hydrogen substances. However, more recent studies have shown that what mainly occurs is a fast electron transfer from the sample to DPPH . The abstraction of hydrogen from the sample by DPPH is marginal, because it occurs very slowly and depends on the hydrogen-bond accepting solvent. Methanol and ethanol, solvents generally used for antioxidant ability assays, are strongly hydrogen bond-accepting, therefore the hydrogen-abstracting reaction occurs very slowly.[3] The presence of acids or bases in methanol may greatly influence the ionization equilibrium of phenols and cause either a reduction or an increase, respectively, of the measured rate constants.[8] In the case of reversible reactions, DPPH radical can give wrong low antioxidant ability values, as with eugenol or other phenolic compounds with a similar structure (ometoxyphenol).[8] Carotenoids interfere with DPPH at 515 nm, therefore data interpretation is complicated.[10]

Superoxide anion radical scavenging (O2 ). Xanthine oxidase is a dehydrogenase enzyme that transfers electrons to nicotinamide adenine dinucleotide (NAD+), reducing it to NADH, and oxidizes xanthine or hypoxanthine to uric acid. Nevertheless, under stress conditions, the dehydrogenase is converted to an oxidase enzyme and, under these conditions, the enzyme reduces oxygen instead of NAD+. On this way, there is a reduction of dioxygen to superoxide anion and hydrogen peroxide.[13] Superoxide anion can be generated by the hypoxanthine xanthine oxidase system or using a non-enzymatic reaction. In this case, superoxide anion is generated through the reaction of phenazine methosulphate in the presence of NADH and dioxygen. In both cases, superoxide anion reduces nitro-blue tetrazolium (NBT) into formazan at pH 7.4 and room temperature. The formazan generation is followed spectrophotometrically at 560 nm.[6] The scavenging activity towards superoxide anion of antioxidants is measured in terms of inhibition of generation of superoxide anion, and therefore with a reduction of formazan production. In addition, the scavenging ability of superoxide anion can be evaluated by reaction of the superoxide with -ketomethiolbutyric acid, which produces ethane, which can be measured by gas chromatography. Another method can use electron spin resonance spectrometry (ESR). In this case, the superoxide anion is trapped with 5,5-dimethyl-1-pirroline N-oxide (DMPO-OH), and the resulting DMPO-OH is detected by ESR, using manganese oxide as the internal standard.[3,6] After undergoing one-electron oxidation, luminol can react with superoxide, generating an unstable endoperoxide, whose decomposition into aminophthalate generates light emission at 430 nm. The presence of superoxide scavengers limits the correspondent light emission.[1]
Scavenging Reactive Oxygen Species and Reactive Nitrogen Species The main reactive oxygen and nitrogen species are: (a) (b) (c) (d) (e) (f ) (g) Superoxide anion. Peroxyl radical. Hydroxyl radical. Hydrogen peroxide. Singlet oxygen. Peroxynitrite. Nitric oxide.
Peroxyl radicals have already been discussed.
Hydroxyl radical scavenging (HO ). There are several ways to ascertain the ability to form hydroxyl radicals (i.e. the deoxyribose test). This method includes a mixture of ferric chloride (FeCl3) and ethylenediamine tetra-acetic acid (EDTA), which in the presence of ascorbic acid forms Fe2+EDTA, and oxidized form of ascorbic acid. After addition of hydrogen peroxide (H2O2), Fe3+ EDTA and HO are formed. This is the Fenton reaction, which generates the highly reactive hydroxyl radical (Fe2+ + H2O2 Fe3+ + HO + HO ). Hydroxyl radicals that are not scavenged by any component of the mixture attack the deoxyribose and degrade it into several fragments. Some of these fragments are capable of reacting with thiobarbituric acid after heating and in an acidic pH, originating a pink pigment that can be quantified by spectrophotometry.[6] Compounds capable of scavenging hydroxyl radicals inhibit chromagen formation. The deoxyribose method can be performed without ascorbic acid. It allows determination of whether the samples have pro-oxidant ability, because the compounds substitute the ascorbic acid in the Fentons reaction.[14] Some compounds inhibit chromagen formation not by reacting with the hydroxyl radicals but after forming stable complexes with the metallic ions that obstruct the hydroxyl radicals formation. To identify the compounds that chelate iron ions, it is recommended to avoid the use of EDTA. In the absence of metal chelation, iron ions are coupled to deoxyribose, causing sitespecific hydroxyl radical damage. In the presence of a substance with ability to form complexes with iron ions, the amount of hydroxyl radical is lower and characteristic pink coloration is decreased.[6]

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Antioxidant activity of medicinal and aromatic plants Furthermore, the hydroxyl radical can be detected using DMPO, which reacts with the hydroxyl radical, originating the most stable radical DMPO-OH , which exhibits a characteristic ESR response.[6] These adducts are stable and relatively easier to determine than hydroxyl radicals, which have a very short life. In the presence of compounds able to scavenge hydroxyl radicals, the DMPO-OH signal detected by ESR diminishes. Some authors[15] have developed the metal-chelating ability method known as hydroxyl radical averting capacity (HORAC). This method detects the hydroxylation of p-hydroxybenzoic acid, as the indicator of hydroxyl radical formation, by highperformance liquid chromatography coupled with mass spectrometry (HPLCMS). The scavenging ability is measured using fluorescein. The fluorescence decay of fluorescein is measured with and without an antioxidant (metal ion sequester). The HORAC value indicates the ability of the antioxidant to chelate metals, as well as the ability to form a stable complex between Co(II) and the antioxidant.[3]

formation); or (b) transfer of its excitation energy to other molecules and a return to the ground state. This last phenomenon is designated quenching of singlet oxygen. In laboratory experiments, the following molecules are frequently used for quenching/ scavenging of singlet oxygen: histidine; 1,4-diazabicyclooctane (DABCO); diphenylisobenzofuran; and azide.[1,8] Singlet oxygen emits characteristic phosphorescence at 1270 nm. The decay rates of light intensity have been used to measure singlet oxygen quenching ability by a given compound.[8] Peroxynitrite scavenging. Peroxynitrite (ONOO) is considered a strong oxidant but, on the other hand, its protonated form, peroxynitrous acid (ONNOH), is known as very powerful oxidant. At a physiological pH, peroxynitrous acid rearranges itself in order to form nitrate, a much less oxidizing form. Both peroxynitrite and peroxynitrous acid may cause the nitration or hydroxylation of aromatic compounds, such as tyrosine, forming nitro-tyrosine. Under physiological conditions, peroxynitrite also forms an adduct with the carbon dioxide dissolved in body fluids. This adduct seems to be responsible for oxidative damage to protein.[8] There are two main ways to measure peroxynitrite scavenging ability: (a) by inhibition of tyrosine nitration; or (b) by inhibition of dihydro-rhodamine 123 (123DHR) oxidation, to fluorescent rhodamine.[8] Some authors[23] measured the antioxidant ability of catechin and other polyphenolic compounds by determining the changes in tyrosine concentration at 275 nm and the changes of 3nitrotyrosine (the main product of tyrosine nitration) at 430 nm in the presence of several antioxidants. Simultaneously, nitrotyrosine was identified and quantified by HPLC. The oxidation of dihydro-rhodamine to fluorescent rhodamine is measured spectrophotometrically using 500 and 536 nm, respectively, as the excitation and emission wavelengths.[24] Antioxidants inhibit the oxidation of dihydro-rhodamine. The reaction is dose-dependent and does not require a metal-ion catalyst. Nitric oxide scavenging. The nitric oxide-induced oxidation of 4,5-diaminofluorescein to triazofluorescein can be followed spectrophotometrically using excitation and emission wavelengths of 495 and 521 nm, respectively.[25]
Hydrogen peroxide scavenging. One of the most common methods for evaluating the scavenging ability of hydrogen peroxide uses horseradish peroxidase and hydrogen peroxide to oxidize scopoletin into a non-fluorescent product. The presence of a molecule with scavenging hydrogen peroxide prevents scopoletin oxidation.[6] The decrease of scopoletin fluorescence is measured at 460 nm.[1] However, some authors consider that the detailed mechanism of this inhibition is ambiguous, for the following reasons: (a) the antioxidant can directly react with peroxide hydrogen; (b) the antioxidant can react with intermediates that are formed from enzyme and peroxide hydrogen; and (c) the antioxidant can inhibit horseradish peroxidase from binding hydrogen peroxidase.[8,16] There are also some non-enzymatic methods to evaluate hydrogen peroxide scavenging ability, which are based on the chemiluminescence of luminol or lucigenin in the presence of hypochlorite. This reaction is based on the oxidation of luminol to diazaquinone by sodium hypochlorite. Diazaquinone is then converted by hydrogen peroxide to an excited aminophthalate. This reaction has a very short luminescence signal (2 s) at a maximal wavelength of 431 nm.[3] The described assay measures the concentration of hydrogen peroxide. In the case of lucigenin, an excited N-methylacridone is formed in the presence of hydrogen peroxide.[17] There are other assays that evaluate the hydrogen peroxide scavenging ability. Some authors[18] have used a method that measured the direct reaction of hydrogen peroxide with titanium(IV). Precipitated TiH2O2 complex is then dissolved in sulphuric acid and measured at about 410 nm. Singlet oxygen scavenging. Singlet oxygen is usually formed in the presence of light and photosensitizers. It is thought that singlet oxygen is responsible for UV radiation skin damage, cataract formation in the lenses of the eyes, macular degenerationand photosensitivity derived from absorption or ingestion of phytochemicals, pharmaceuticals and pesticides that act as photosensitizers.[1922] The in vivo singlet oxygen formation, without the presence of light, seems to be result of the spontaneous dismutation of the superoxide anion. Chemically, the singlet oxygen can be generated from the non-photochemical decomposition of hydrogen peroxide by metals or hypochlorite.[3,8] Singlet oxygen can interact with molecules in two ways: (a) direct reaction with the molecules (i.e. endoperoxides
Evaluation of the Ability to Inhibit or Prevent Lipid Oxidation Lipid oxidation is a complex reaction that can be generated via three different pathways: 1. Non-enzymatic free radical-mediated chain reaction. 2. Non-enzymatic, non-radical photo-oxidation. 3. Enzymatic reaction. An example of the second pathway is the stoichiometric oxidation of oleic acid by singlet oxygen to produce two allylic hydroperoxides via the addition of oxygen at either end of the double bond. The third pathway involves the action of lipoxygenases on diverse substrates, whereas the first pathway leads to initiation of rapidly progressing, destructive chain reactions, generating hydroperoxides and volatile compounds, generally through a three-phase process: initiation, propagation and termination.[7,26]
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M. G. Miguel The initiation phase involves homolytic breakdown of hydrogen in the -position relative to the fatty acid chain double bond, leading to the formation of an allyl radical. These species with an unpaired electron are highly unstable, short-lived intermediates that stabilize themselves by abstracting hydrogen from another chemical species or rapidly react with oxygen to form a peroxyl radical (propagation phase). The initiation phase is induced by external agents such as heat, light or ionizing radiation or by chemical agents (i.e. metal ions or metalloproteins and free radicals).[7,26] In the propagation phase, peroxyl radicals are formed and they can further oxidize the lipid, producing hydroperoxides. Many hydroperoxide isomers can be formed during this phase, which is generally accompanied by the stabilization of the radical via double-bond rearrangement (electron delocalization), originating conjugated dienes and trienes. These intermediates decompose, originating compounds such as alcohols, aldehydes, alkyl formates, ketones and hydrocarbons, and even alkoxyl radicals.[7] The formation of these compounds, which are known as secondary products of lipid oxidation, is characteristic for the termination phase. In an auto-oxidation process (non-enzymatic free radicalmediated chain reaction), it is possible to distinguish three main stages, corresponding to the three main phases of lipid oxidation: (a) depletion of the oxidation substrates (oxygen, fatty acids); (b) appearance of primary oxidation products (hydroperoxides and peroxides); and (c) appearance of secondary oxidation products, obtained by recombination and scission from peroxides (epoxydes, volatile compounds).[27] In contrast to tests based on free-radical scavenging, tests for the evaluation of inhibition of lipid oxidation need pure or mixed lipid substrates. The evaluation of antioxidant activity in such systems can be performed in at least three ways: (a) by measuring the concentration changes of reducing agent; (b) by measuring the depletion of oxygen; (c) by measuring the formation of oxidation products.[2,27] Sometimes the correlation between the methods (oxygen absorption, peroxide index, formation of secondary oxidation products) is extremely low, due to the fact that they cannot reflect the same stage of the oxidation process. Therefore, antioxidant activity should be determined by more than one method and should measure different oxidation products (initial and decomposition products of lipid oxidation).[28] This means that the evaluation of the antioxidant activity requires the use of a multiple-method approach, with different mechanisms of inhibition of lipid oxidation.[12] As lipid substrates, various types of oils and fats can be used, as well as linoleic acid, fatty acid methyl esters, liposomes and LDLs.[7] The evaluation of oxidative stability of a studied lipid system can be made under normal storage conditions (stability tests in real time) or using accelerated oxidation conditions (increased temperature and partial pressure of oxygen, addition of transition metal catalysts, exposure to light, variable shaking to enhance reactant contact).[7,29] Heating is the most common and effective means to accelerate the oxidation process. In the presence of antioxidants, the activation energy of lipid oxidation increases because antioxidants decrease the rate of oxidation reactions by increasing the complete energy of activation. For some antioxidants, the global protection index, measured at high temperatures, will generally be lower than those measured at lower temperatures.[28] Consequently, the efficacy of the antioxidants can depend on the temperature (e.g. -tocopherol is more effective as an antioxidant when exposed to temperatures < 60 C, in comparison with its - and -tocopherol isomers). In addition, ascorbic acid cannot inhibit the oxidation of linoleic acid at high temperatures, but at room temperature it can act as an anti-radical agent in methanolic solution.[27] Oxidative stability tests at room temperature are not very practical, in spite of the fact that they reflect real conditions of food storage. They are extremely slow and have low reproducibility, due to several factors that cannot be easily controlled during long storage conditions.[28] On the other hand, accelerated oxidative stability tests are often used for food analysis and their results are expressed as the induction period. This term is defined as the time required to reach an end-point of oxidation, accompanied by a sharp change of the oxidation rate.[28] The results are quantified by measuring the induction period of the control with and without antioxidant. The higher induction period of the lard with antioxidant added, compared to the control (pure lard), means a better antioxidant activity of that compound. Measurements can also be at a fixed time point, i.e. after a predetermined time interval.[7] Oxygen depletion. This method is based on the fact that the oxidation of fats and oils is accompanied by an uptake of atmospheric oxygen and an increase of the sample mass. The measurements can be performed by manometer (Warburg manometer) or by gravimetric (detection of the mass of fatty acids upon oxygen fixation) or polarographic (using a Clark electrode) means.[26,30] Generally, the weight-gain methods, based on the increase in weight due to oxygen absorption, are not very sensitive. The end-point requires a level of oxidation that is beyond the point where flavour deterioration is detectable in polyunsaturated fatty oils.[28] Substrate loss. The detection of oxidative stability by substrate loss includes methods that determine the residual non-oxidized unsaturated fatty acids after extraction and derivatization by GC.[27] There are other methods which use a single fully characterized substrate, a simple medium or labelled systems (fluorophorelabelled biological particles or living cells) that can be followed by a spectral measurement system. Most of these substrates have a conjugated polyenic structure, possessing, therefore, absorption and/or fluorescence properties in the UV-Vis spectrum.[26] -Carotene bleaching method. The -carotene bleaching method (coupled oxidation of -carotene and linoleic acid) estimates the relative ability of antioxidant compounds in plant extracts to scavenge the radical of linoleic acid peroxide (LOO*) that oxidizes -carotene in the emulsion phase. Linoleic acid oxidation is not specifically catalysed with heat (50 C). Sometimes it is difficult to interpret the results obtained because -carotene itself is an oxygen-sensitive antioxidant.[26] This method can be combined with thin-layer chromatography. In this case, after chromatographic separation, a mixture of -carotene and linoleic acid is pulverized on the plate and exposed to daylight or UV radiation for some hoursuntil the yellow colour disappears. Antioxidant compounds are identified by those bands that have remained yellow in colour.[26] Fluorescence decay of cis-parinaric acid. cis-Parinaric acid is a polyunsaturated fatty acid with four conjugated double bonds, which are responsible for its high fluorescence properties as well
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Antioxidant activity of medicinal and aromatic plants as high sensitivity to free radical attack. Oxidation of this conjugated acid is responsible for its irreversible loss of fluorescence. cis-Parinaric acid can be metabolically integrated in membranes of diverse cell types, such as erythrocytes, sarcoplasmic reticulum, lens membranes and macrophages.[26] Fluorescence decay of BODIPY probes. C11-BODIPY is a fluorescent fatty acid analogue and therefore an oxidizable substrate, mainly in the presence of peroxyl radicals. This molecule presents a phenyl moiety combined to a conjugated diene, displaying a bright red fluorescence. Oxidation of C11-BODIPY by peroxyl radicals leads to a gradual extinction of the fluorescence.[26] Formation of primary oxidation products. Hydroperoxides are considered to be primary products of lipid oxidation. Several techniques have been developed in order to evaluate their formation, and these techniques are well adapted for foods or in vitro biological samples. Iodometric hydroperoxide measurement. The hydroperoxides or peroxides react with the iodide ion to generate iodine, which is titrated with a standard sodium thiosulphate solution in the presence of starch solution. The peroxide value is expressed in milliequivalent units of oxygen per kilogram of the fat. Peroxide value can be determined colorimetrically, based on the oxidation of ferrous to ferric ion, with the latter determined as ferric thiocyanate.[30] Iodometric hydroperoxide measurements have some limitations, such as poor sensitivity and selectivity, since it is possible the addition of some iodine across unsaturated bonds, oxidation of iodine by dissolved molecular oxygen and variations in reactivity of different peroxides.[7] Ultraviolet measurement of conjugated dienes. The abstraction of hydrogen from a CH2 group of a polyunsaturated lipid is usually stabilized by a molecular rearrangement to form a conjugated diene or triene. The UV absorption method measures the hydroperoxides and the conjugated dienes and trienes, which absorb in the range 232278 nm. The usual substrates for the determination of these compounds are polyunsaturated fatty acids, which. after heating or in the presence of copper or iron metal ions, AAPH or DPPH, generate conjugated dienes. Quantificantion of dienes can be obtained by calculating the increase in absorbance per mass of sample at a fixed time.[7] Formation of secondary oxidation products. Several assays that monitor the formation of secondary oxidation products are primarily based on spectrophotometric and chromatographic methods. Thiobarbituric acid reactive substances (TBARS). This method measures the malondialdehyde (MDA) formed after lipid hydroperoxide decomposition, which forms a pink chromophore with thiobarbituric acid (TBA). This coloured complex, which absorbs at 532 nm, results in the condensation of 2 M TBA and 1 M malondialdehyde in an acidic environment.[7,26] This method is not very specific, because 4-hydroxyalkenals, 2,4-alkadienals and 2-alkenals, protein and sugar degradation products, amino acids, nucleic acids and anthocyanins are also able to react with TBA, forming a chromophore.[7,27,31] Selectivity of the TBARS method can be improved using HPLC to characterize the individual species.[26] Malondialdehyde only forms from fatty acid chain containing at least three double bonds. The results may be expressed as percentage inhibition of oxidation.[7] Aldehyde measurement by the anisidine assay. This method is based on the reaction of the aldehyde carbonyl group (especially 2-alkenals) with the amine group of p-anisidine, originating the formation of a Schiff base that absorbs at 350 nm. The colorimetric response with p-anisidine varies according to the extent of aldehyde unsaturation, i.e. the response is more intense with di-unsaturated aldehydes than with mono-unsaturated aldehydes, considering that they are at identical concentrations.[26] Chromatographic measurement of volatile compounds. Decomposition of the primary products of lipid oxidation forms epoxides, ketones, hydrocarbons and saturated and unsaturated aldehydes (hexanal). Some of these compounds are volatiles and they are considered to be the major contributors of off-flavours associated with the rancidity of foods. Some of these volatile compounds are highly specific to the oxidative degradation of a particular polyunsaturated fatty acid.[7,26] Hexanal and pentanal are markers of fatty acids of the n-6 family, whereas propanal is the main marker of oxidation of fatty acids of the n-3 family. Hexanal is the most frequently measured secondary oxidation product.[26,3234] The volatile constituents are generally measured by GC, and headspace GC (static headspace, dynamic purge-and-trap headspace or headspacesolid phase microextraction) or GC coupled to mass spectrometry (GCMS).[26] Formic acid measurement. The Rancimat method is an automated test that measures the conductivity of low molecular weight fatty acids (formic acid) produced during the auto-oxidation of lipids at 100 C or above. This method presents some drawbacks because it includes the use of high temperatures to detect organic acids: levels of rancidity exceed those in normal conditions, thermolability or volatility of some products.[28]
Antioxidants and Food

Free-radical intermediates are involved in several metabolic disturbances that are responsible for cell and disease injuries, and are therefore implicated in many human diseases (cancer, cardiovascular diseases, inflammatory processes, cataracts, and even the normal ageing process).[5] Food has a primordial role in the prevention of free radicals production. The nutritional prevention of oxidizing stress and its consequences implies the optimization of the antioxidant concentration in food. The benefits of a diet rich in fruits and vegetables are recognized and attributed to the relatively high amounts of antioxidants. In this sense, a diet rich in antioxidant micronutrients (vitamins C, E, carotenoids, selenium and zinc) and other complementary micronutrients (vitamins B2, B3, B9), as well as phytochemicals (polyphenols, allyl sulphide), decrease the incidence of cancer and cardiovascular and degenerative diseases.[3537] In spite of the importance of these antioxidants in the diet, some of them are also used in the food industry as preservatives for preventing or delaying the oxidation process (i.e. vitamin E and ascorbic acid). These natural compounds could replace the synthetic ones, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), suspected to be dangerous for animal health.[38]
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M. G. Miguel
HOOC OH
COOH
COOH
COOH
OR OH Benzoic acid
OR OH
OR
HO
R=H, Protocatechic acid R=H, gallic acid R=CH3, vanilic acid R=CH3, syringic acid COOH
homogentisic acid
CH3
CH2
CH2OH
H3CO OCH3 (E)-Anethole O Myristicine
O OH
R H3CO OH
OCH3
R=H, p-Cumaric acid R=OH, Caffeic acid R=OCH3, ferulic acid
Sinapylic alcohol
Figure 1.
Some examples of phenolic acids
Plant-derived Antioxidants Allyl sulphides. Sulphur compounds are compounds characteristic of garlic and they have strong antioxidant properties. The antioxiant activity of allyl sulphides is associated with their ability to reduce reactive oxygen species and their activation of antioxidative enyzmes (superoxide dismutase, catalase, glutathione peroxidase).[39] Due to these properties, allyl sulphide compounds are important in the prevention of DNA damage as well as the prevention of cancer and cardiovascular disease.[37] Plant phenols. Plants are known as very rich sources of phenolic compounds (containing an OH group attached to a benzene ring). In nature, they occur mostly as glycosides. Phenols represent a huge group of natural compounds, among which phenolic acids and flavonoids are the most important. The structures of phenolic acids are diverse (Figure 1). Hydroxybenzoic acids are based on a C6C1 skeleton, while cinnamic acids are a series of trans-phenyl-3-propenoic acids with C6C3 structures differing in their ring substitution. Caffeic acid and its esters and ferulic acid are the most frequently encountered phenolic acids in plant foods.[40] Flavonoids can be dividied into several classes, according to the degree of oxidation in the oxygen heterocycle and the degree of saturation in the C-ring.[40] All flavonoids derived from a tricyclic structure (flavane), whose modifications originate flavan-3ols (e.g. epicatechin), flavanones (e.g. naringenin), flavonols (e.g. quercetin), flavones (e.g. apigenin) and anthocyanidins (e.g. cyanidin) (Figure 2).[41] Plant phenols exhibit in vitro antioxidant activity, inhibiting lipid peroxidation by acting as chain-breaking peroxyl-radical
scavengers. Phenols with two adjacent hydroxyl groups can bind transition metal ions, such as iron and copper. In addition, phenols directly scavenge reactive oxygen species (hydroxyl radicals, peroxynitrite and hypochlorous acid). Sometimes phenols can act as pro-oxidants by reducing transition metal ions.[1] Some phenolic compounds exhibit higher antioxidant activity than tocopherols. It was reported that after the intake of food rich in polyphenols, there is an increase of the antioxidant pool in plasma.[37] It is considered that the antioxidant activity of phenolic compounds is due to their high redox potentials, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. The antioxidant activity of the phenolics is essentially determined by their structures, in particular the electron delocalization over an aromatic nucleus. When these compounds react with free radicals, the delocalization of the gained electron over the phenolic antioxidant occurs, and the stabilization by the resonance effect of the aromatic nucleus, which prevents the continuation of the free radical chain reaction.[42] The presence of hydroxyl groups in positions 3-, 4- and 5 in the B ring of flavonoids increases the antioxidant activity when compared with compounds with one hydroxyl group. Also, the presence of a hydroxyl group in position 3, and a 2,3-double bond conjugated with the 4-oxo group seems to have importance in the antioxidant properties.[43,44] Generally aglycones are more effective as antioxidants than the corresponding glycosides. Beyond the mere presence of molecules of sugars and the total amount of units attached to the flavonoid, their positions and structures are also important. For example, the aglycones luteolin and quercetin are more effective
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Antioxidant activity of medicinal and aromatic plants

R
3' 2' 4' 5' 6'
R OH HO O OH HO O H OH OH O OH O Flavanones R=H, Naringenin R=OH, Eriodictyol R OH OH HO O H H OH Flavan-3-ols or catechins R=H, Afzalechin R=OH, Catechin R OH HO O
+
R OH
HO
7 6
O
1 2 3
1'
OH Flavones
Flavonols R=H, Kaempferol R=OH, Quercetin R
R=H, Apigenin R=OH, Luteolin
R OH HO O H OH OH H H OH
HO
O H H OH O Dihydroflavonols R=H, Dihydrokaempferol R=OH, Dihydroquercetin OH
OH
Flavan-3,4-diols R=H, Leucopelargonidin R=OH, Leucocyanidin
OH OH Anthocyanidins R=H, Pelargonidin R=OH, Cyanidin
Figure 2. Some examples of flavonoids
than the corresponding 3-, 4- and 7-O-glycosides in delaying hydroperoxide formation in the lipid double layer of membranes. However, these glycosides have different abilities to prevent hydroperoxide accumulation.[44] Other studies have shown that flavonoids may also exhibit pro-oxidant activity, especially those compounds that possess multiple hydroxyl groups, viz. in ring B. Some studies connect this with the presence of a double bond between the carbon atoms in positions 2 and 3, as well as the presence of a carbonyl group in position 4 of the flavonoid, which promote the formation of oxygenated reactive species induced by the presence of divalent copper and oxygen.[44,45] Phenolic acids with strong antioxidant activities include cinnamic, ferulic, caffeic, sinapic, salicylic and vanillic acids, among others.[30] Aromatic plants. The addition of spices to food is largely used in most cultures to improve the flavour and to preserve the food.[30,46] Many herbs and spices (anise, fennel, basil, mint,
tarragon, marjoram, rosemary, thyme, parsley, juniper, laurel and black pepper) make food more pleasant and, at the same time, serve as a rich source of phenolic compounds with strong antioxidant activity.[47,48] The flavour of herbs and spices derives from essential oil components, which also show good antioxidant activity. Beyond the beneficial effect on human health, those plants may also serve as natural food preservatives.[49] The utilization of antioxidants in the food industry is important because they prevent oxidation, one of the major causes of chemical spoilage, preventing rancidity and/or deterioration of the nutritional quality, colour, flavour and texture.[7] Essential oils are aromatic oily liquids obtained from raw plant materials (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots) by distillation (steam, steam/water and water), expression (cold pressing, used for citrus peel oils), fermentation or enfleurage.[50] Essential oils can contain more than 100 different compounds. Major components can constitute up to 85% of the essential oils, while the remaining components can be present only in trace amounts.[51]
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M. G. Miguel There are many data about the antioxidant activity of essentials oils extracted from plants belonging to various species, harvested in different places and at different development stages.[5289] The chemical composition of these oils depends on several factors (plant age, plant part, development stage, growing place, harvesting period, chemotype) and the correlation between the biological activity (i.e. the antioxidant activity) of essential oils and their chemical composition is often very complicated. Furthermore, due to the many different methods for in vitro evaluation of the antioxidant activity based on completely different mechanisms, data interperation is not simple. Consequently, results on the antioxidant activity of essential oils from the same plant, which are reported in numerous studies, may greatly vary.[52,9093] It is necessary to point out that there is no perfect system available that evaluates the true antioxidant activity of a single compound or a complex mixture. Differences in used analytical methods and measurement conditions can be responsible for such variations in same samples.[3] strongest antioxidant activitiy to the presence of relative high amounts of phenolic compounds, such as eugenol (30%) in L. nobilis oil and thymol (30%) and carvacrol (15%) in Thymus vulgaris. Interestingly, the oils of F. vulgare and E. globulus, which also exhibited good antioxidant activity, did not contain phenolic compounds. Contrary to other oils, essential oils from Tagetes minuta, Satureja parvifolia and Lippia polystachya Gris exhibited pro-oxidant activity. The main components were: (E)-ocimenone (35%), (Z)-ocimenone (32%) and -ocimene (17%) in T. minuta oil; piperitone oxide (31%), menthol (16%) and piperitenone oxide (14%) in S. parvifolia oil; and -thujone (69%) constituted the main component in L. polystachya oil (Figures 3, 4). Maestri et al.[95] noted the relationship between essential oil concentration and antioxidant activity. They showed that as the concentration of essential oils in peanut oil increased, the antioxidant activity decreased. The essential oils of Origanum majorana L., Acantholippia seriphioides Mold. and Tagetes filifolia Lag. presented the best activities, mainly at lower concentrations (0.02%). This activity was attributed to carvacrol and thymol, present in O. majorana and A. seriphioides, and anethole as the major component in T. tiifolia. The essential oils of Origanum vulgare subsp. hirtum, O. onites L., Coridothymus capitatus (L.) Reichenb. fil. and Satureja thymbra L. showed antioxidant activity when tested by two methods (the -carotene bleaching test and determination of the oxidative stability of lard).[96] Carvacrol and thymol, the main constituents of the essential oils, were equally effective as antioxidants, independent of the assayed method. Some authors[97] evaluated the antioxidant activity of extracts obtained by different isolation procedures, essential oils and dried deodorized aqueous extracts of Majorana hortensis Moench, Nepeta cataria L., Origanum vulgare L., Lavandula angustifolia Mill., Thymus vulgaris L., Hyssopus officinalis L., Lophantus anisatus Benth. and Salvia officinalis L. by the -carotene bleaching test. According to their results, the essential oils of the studied plants did not delay the oxidation of the -carotenelinoleate system, except for thyme essential oil, which exhibited some antioxidant activity. In contrast, the essential oils of Origanum ehrenbergii Boiss. and O. syriacum L. inhibited the oxidation of linoleic acid evaluated by the -carotene-linoleic acid bleaching test. The authors[98] attributed these activities to the high content of phenolic components (carvacrol, carvacrol methyl ether and thymol methyl ether). The essential oils obtained from Satureja cuneifolia Ten. in different maturations stages (preflowering, flowering and postflowering), also mainly constituted by the phenols thymol and carvacrol as well as p-cymene, were able to inhibit linoleic acid peroxidation at almost the same level as BHT. [87] However, the presence of phenolic compounds in the essential oils to prevent lipid oxidation was not necessary in three Salvia species from Turkish flora (S. aucheri var. aucheri, S. aramiensis and S. pilifera).[82] The authors reported that the activities of these oil samples were even comparable to that of curcumine, an efficient antioxidative substance for food systems containing lipids. Some authors[92] evaluated the ability of O. vulgare L. spp. hirtum, Th. vulgaris L., Th. serpyllum L., Satureja montana L. and S. cuneifolia Ten. essential oils and their fractions to inhibit lard oxidation, using the Rancimat method. They found that the studied essential oils and their fractions (hydrocarbons CH fraction, oxygen-containing compounds fraction and phenolic fraction) showed a poor inhibitory effect against the lard oxidation process compared to the references BHA, ascorbic acid and
Essential oils as natural antioxidants

Essential oils contain volatile aroma compounds from aromatic plants. They are complex mixtures of compounds belonging to diverse chemical families (terpenes, alcohols, aldehydes, phenolic compounds, esters, ethers, ketones). As already mentioned, several factors are responsible for variations in the level of the antioxidant activity of essential oils. One of the factors concerns the methodology for evaluating the antioxidant activity. There are two main approaches to determining the in vitro antioxidant activity of essential oils: 1. The inhibition of lipid oxidation in different systems (oils, solutions of lipids in organic solvents, oil-in-water emulsions, micelles, liposomes, etc.). 2. The ability for scavenging free radical species. The chemical complexity of essential oils additionally complicates the interpretation of their in vitro antioxidant activity. Inhibition of Lipid Oxidation There are many reports on the ability of essential oils to inhibit lipid oxidation (Table 3). The data diversity in relation to various factors (oil samples, antioxidant concentration, methodology) is discussed here. Some authors[46] report on the high antioxidant activity of several essential oils compared with commerical antioxidants (BHA) and they found that essential oils from Rosmarinus officinalis L., Salvia fruticosa Mill. and Foeniculum dulce Mill. exhibited the highest inhibitory activity of lipid oxidation. The peroxide values ranged from 101 mEq/kg for R. officinalis L. to 450 mEq/kg for Foeniculum dulce Mill. These authors did not determine the chemical composition of the oils but they explained these results by the probable presence of phenolic compounds (carvacrol, thymol and eugenol), whose presence in the studied essential oils had already been detected by other authors Zygadlo et al.[94] studied the inhibitory activity of lipid oxidation by several essential oils in other lipid substrates (e.g. soybean oil) at a lower temperature (60 C). They found that essential oils from Thymus vulgaris L. and Laurus nobilis L. showed the highest activity, while the activity of Foeniculum vulgare var. dulce and Eucalyptus globulus was little lower. The authors attributed the
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Table 3. Examples of essential oils assayed for inhibition of lipid oxidation capacity by diverse tests Essential oil Artemisia dracunculus L. Foeniculum dulce Mill. Ocimum basilicum L. Origanum majorana L. Origanum vulgare L. Petroselinum sativum Hoffm. Pimpinella anisum L. Rosmarinus officinalis L. Salvia fruticosa Mill. Satureja hortensis L. Sinapis arvensis L. Eucalyptus globulus Labill. Foeniculum vulgare var. dulce Laurus nobilis L. Lippia polystachya Gris. Satureja parvifolia (Phil.) Epl. Tagetes minuta L. Thymus vulgaris L. Acantholippia seriphioides Mold. Eucalyptus cinerea F. von Mueller Myrcianthes cisplatensis Camb. Origanum majorana L. Rosmarinus officinalis L. Tagetes filifolia Lag. Coridothymus capitatus (L.) Reichenb. fil. Origanum onites L. Origanum vulgare ssp. hirtum Satureja thymbra L. Hyssopus officinalis L. Lavandula angustifolia Mill. Lophantus anisatus Benth Majorana hortensis Moench Nepeta cataria L. Origanum vulgare L. Salvia officinalis L. Thymus vulgaris L. Origanum ehrenbergii Boiss. O. syriacum L. Satureja cuneifolia Ten. Salvia aucheri var. aucheri S. aramiensis S. pilifera Origanum vulgare L. spp. hirtum Satureja cuneifolia Ten. S. montana L. Thymus serpyllum L. Th. vulgaris L. Origanum vulgare L. ssp. hirtum BHA Standard Assay Peroxide value Lipid substrate/T (C) Sunflower oil/70 C References 46
BHT
Peroxide value
Soybean oil/60 C
94
BHT
Peroxide value
Peanut oil/60 C
95
BHT Thymol Carvacrol Thymol + carvacrol BHT
Peroxide value -Carotene bleaching test -Carotene bleaching
Lard/35 C
96
Linoleic acid/100 C
97
Propyl gallate BHT Ascorbic acid BHT Curcumine Ascorbic acid BHT BHA Ascorbic acid -Tocopherol Thymol Thymoquinone -Tocopherol
-Carotenebleaching -Carotenebleaching -Carotenebleaching Rancimat
Linoleic acid/45 C Linoleic acid/50 C Linoleic acid/room temperature Lard/100 C
98 87 82
92
Peroxide value
Lard/60 C
100
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Table 3. Continued Essential oil Thymbra capitata Thymus mastichina, 1,8-cineole chemotype Th. mastichina, linalool chemotype Th. albicans Th. carnosus Cinnamomum zeylanicum Nees Coriandrum sativum L. Foeniculum vulgare Muller Helichrysum italicum G. (Roth) Don Laurus nobilis L. Marjorana hortensis Moench. Mentha piperita L. Myristica fragrans Houtt. Ocimum basilicum L. Piper nigrum L. Salvia officinalis L. Syzygium aromaticum L. Boswellia thurifera Roxb. ex Colebr. Canaga odorata Hook. F. et Thw Cinnamomum zeylanicum Nees Citrus limon L. Coriandrum sativum Cymbopogon citratus Stapf. Laurus nobilis Marjorana hortensis Moench. Ocimum basilicum L. Origanum vulgare Rosmarinus officinalis Rosmarinus officinalis L. Salvia officinalis Monarda citriodora var. citriodora Myristica fragrans Origanum vulgare ssp. hirtum Pelargonium sp. Thymus vulgaris Origanum vulgare L. ssp. hirtum BHT -Tocopherol Ascorbic acid BHT -Tocopherol BHT BHT -Tocopherol Standard Carvacrol BHT Assay Peroxide value Peroxide value Peroxide value Acid value Acid value Rancimat TBARS Lipid substrate/T (C) Olive oil/60 C Sunflower oil/60 C Peanut oil/60 C References 56,57,65
Lard/110 C
101
TBARS in the absence and presence of a radical inducer (AAPH)
Egg yolk/95 C Rat liver homogenates/95 C
102,103
TBARS
Egg yolk/95 C Chick liver homogenates/95 C Chicken muscle/95 C Egg yolk/95 C Linoleic acid/50 C Egg yolk/95 C Linoleic acid/50 C Liposomes
104
TBARS -Carotene bleaching test TBARS -Carotene bleaching test TBA
105
Satureja montana L.
106
Ocimum basilicum L. Origanum vulgare L. Thymus vulgaris L.
107
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Table 3. Continued Essential oil Artemisia dracunculus L. Cinnamomum zeylanicum blum. Coryodotymus capitatus L. Cuminum cyminum L. Eugenia caryophyllus Spreng. Foeniculum vulgare Miller Illicum verum Hook. Ocimum basilicum L. O. basilicum L. var. album O. gratissimum L. Origanum heracleoticum L. Petroselinum sativum Hoffm. Pimenta racemosa miller Pimpinella anisum L. Satureja hortensis L. S. montana L. Styrax benzoe dryander Thymus serpyllum L. Th. vulgaris L. Th. vulgaris L. var. thymol Th. zygis L. Vanilla planifolia Andrews Thymus serpyllum L. Th. vulgaris L. Thymus spathulifolius (Hausskn. and Velen.) Thymus eigii M. Zohary et P.H. Davis Thymus sipyleus subsp. sipyleus var. sipyleus Th. sipyleus subsp. sipyleus var. rosulans Thymbra capitata L. (Cav.) Origanum vulgare L. BHT BHT BHA -Tocopherol BHT -Tocopherol Ascorbic acid BHT Standard Assay Hexanal (headspaceGC) Lipid substrate/T (C) Human LDL References 108
TBARS -Carotenebleaching test -Carotene bleaching test -Carotenebleaching test -Carotenebleaching test TBARS (in the absence and presence of AAPH) Micellar model system TBARS (in the absence and presence of AAPH)
Egg yolk/95 C Linoleic acid/50 C Linoleic acid/50 C Linoleic acid/50 C Linoleic acid/50 C Egg yolk/95 C Linoleic acid /50 C
93
113 114 70 64
Origanum floribundum O. glandolosum Thymus guyonii Th. munbyanus Th. numidicus Th. pallescens Thymbra capitata Thymus caespititius Brot. Th. camphoratus Hoffmanns. & Link Th. mastichina (L.) L. subsp. mastichina
BHT BHA -Tocopherol
Egg yolk/95 C
115
BHT BHA BHT BHA -Tocopherol
TBARS TBARS (in the absence and presence of AAPH)
Egg yolk/95 C Egg yolk/95 C
71 59
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Table 3. Continued Essential oil Thymus albicans Hoffmanns. & Link, 1,8-cineole chemotype Th. albicans, 1,8-cineole/linalool chemotype Th. camphoratus Hoffmanns. & Link, 1,8-cineole chemotype Th. camphoratus, linalool/borneol chemotype Th. camphoratus, trans-sabinene hydrate chemotype Th. carnosus Boiss., borneol chemotype Th. carnosus, cis-sabinene hydrate/borneol chemotype Th. carnosus, cis-sabinene/borneol chemotype Th. mastichina, 1,8-cineole chemotype Th. mastichina, linalool chemotype Standard BHT BHA -Tocopherol Assay TBARS (in the absence and presence AAPH) Micellar model system Lipid substrate/T (C) Egg yolk/95 C Linoleic acid /50 C References 72
CH3
CH3
CH3
CH2
CH3
CH2
CH3 CH2 H3C CH3 H3C CH3
+
H3C CH3 H3C CH3 H3C CH3 H3C CH3
p-Cymene
a -Phellandrene
CH2
b -Ocimene (Z/E)
CH3 CH3
a-Pinene
b -Pinene
H3C
CH3 H3C
CH3
H3C
CH3
Sabinene
a-Terpinene
b -Terpinene
Figure 3. Selected hydrocarbon monoterpenes
-tocopherol. The antioxidant activity of the tested oils was very similar to those obtained for pure thymol and carvacrol and decreased in the following order, which is probably related to the amount of thymol and carvacrol in the samples: O. vulgare L. spp. hirtum > Th. vulgaris L. > Th. serpyllum L. > S. montana L. > S. cuneifolia Ten. The authors showed that the antioxidant activity of the tested oils and their fractions was dose-dependent. These results are different from those presented elsewhere;[95] in that study, using other oil samples and methods, an inverse relation was noticed between the antioxidant activity and its concentration. Other authors also reported the poor ability of thymol and carvacrol to inhibit lard oxidation, using the Rancimat method, in contrast to their derivatives previously synthesized in the laboratory.[99] Some authors[100] studied for the first time the antioxidant activity of glycosidically bound volatile compounds of oregano (O. vulgare L. ssp. hirtum). The authors found that the aglycones and the essential oil showed a similar capacity to inhibit hydroperoxide formation in lard, even after 80 days. This ability was
superior to that verified for reference compounds (-tocopherol, thymol and thymoquinone), therefore suggesting an effect of synergy among minor components present in the aglycones and essential oil. The antioxidant activity of the essential oils isolated from thyme species from Portugal was evaluated by measuring their ability to inhibit lipid peroxidation in the presence of olive, peanut and sunflower oils.[56,57,65] Thymbra capitata oil, with carvacrol as the main component, demonstrated a good capacity for preventing lipid oxidation when olive oil and peanut oil were used as lipidic substrates.[56,57] The authors indicated the role of the substrate on the evaluation of the antioxidant ability, since the antioxidant activity of T. capitata oil and carvacrol was higher in olive oil than in sunflower oil. According to the authors,[56] such a result could be related to the relatively high amount of linoleic acid in sunflower oil in comparison with that in olive oil. Other T. capitata oils showed weak capacity to prevent lipid peroxidation in peanut oil as well as in sunflower oil after 78 days of storage at 60 C in the dark.[65] This is in contradiction to
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CH2 CH3 CH3 O H3C Borneol CH3 O CH2 O OH CH3
CH3 OH H3C CH3
CH3
OH CH3 H3C CH3 H3C CH3 Thymol H3C CH2OH CH2 OH
+
CH3 H3C Ocimenone (Z/E) CH2OH CH3 CHO Carvacrol CH3
H3C
CH3
H3C
CH3
H3C
CH3
H3C
CH3
H3C
CH3
1,8-Cineole CH3
Cuminol CH3
Geranial CH3
Geraniol CH3
Linalyl acetate CH3
CHO OH H3C CH3 H3C CH3 H3C O CH3 H3C Neral CH3
CHO
CH2OH
CH3
H3C Nerol CH3 O
CH3
Citronellal CH2OH
Menthol CH3
Menthone CH3
OH
OH H3C CH2 H3C CH3 H3C CH3 H3C
OH CH3 H3C
CH3
Perillyl alcohol trans-Sabinene hydrate
Terpinen-4-ol
a-Terpineol
CH3
Thujone
CH3 CH3 H3C OH
CH3 CH3 O H3C
CH3
H3C
O CH3 H3C
O CH3
cis-Verbenol
Verbenone
Piperitenone
Piperitone
Figure 4. Selected oxygenated monoterpenes
previously reports. The authors[65] attributed this to the different nature of the lipid substrate used and/or the essential oil composition, in spite of its carvacrol richness. This also suggests that, besides carvacrol, other oil components may contribute to exhibiting synergistic or antagonistic effects in the oil. In addition, the authors showed that peanut or sunflower oil, with addition of T. mastichina oil, had lower acid values than the control (without this essential oil) after 78 days of storage. The authors therefore stressed the importance of using several methods for evaluating lipid oxidation, as different methods provide different antioxidant results.
The antioxidant ability of essential oils is generally associated with phenolic compounds such as carvacrol and thymol, as reported for T. capitata oil. Nevertheless, it has also been shown[57] that Th. albicans, Th. mastichina and Th. carnosus oils, mainly constituted by 1,8-cineole, linalool, borneol and terpinen-4-ol, showed a capacity to prevent lipid peroxidation of sunflower oil and that their antioxidant activity was even higher than that of BHT. Assessing the antioxidant activity of essential oils of 12 spice plants (Table 3), it was shown [101] that the Rancimat method was inappropriate for the investigation of volatile compounds
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M. G. Miguel because of the relatively drastic conditions used for oxidation (110 C and airflow of 20 l/h). However, other methods that also used the lipid substrate allowed the conclusion that essential oils from some spice plants could be considered as a good source of natural antioxidants. The TBARS method, using egg yolk as the lipid substrate, showed that the essential oils of clove (Syzygium aromaticum L.), basil (Ocimum basilicum L.) and laurel (Laurus nobilis L.) could be potential sources of natural antioxidants, due to their relative high antioxidant activities. The antioxidant activity of the essential oils of basil and laurel was unexpected, due to relatively low amount of phenolic compounds in these oils. They were predominantly constituted of estragole and linalol (basil) and 1,8-cineole (laurel). Therefore the authors suggested that such antioxidant activities could be explained by the presence of eugenol (12%) and methyl eugenol (4%) in basil oil and of methyl eugenol (14%) in laurel oil. Some authors[102,103] also reported the antioxidant activitiy of O. basilicum and L. nobilis oils by the TBARS method in different lipidic substrates (egg yolk and rat liver homogenates) (Table 3). At higher concentration (1000 ppm) the ability to prevent lipid peroxidation by essential oils was similar to that of BHT. When egg yolk was replaced by rat liver homogenate, the antioxidant activity decreased, especially for basil and laurel. The presence of AAPH decreased the activity of all oil samples, provoking the pro-oxidant activity of some samples, especially in the presence of rat liver homogenates. In this lipidic substrate, -tocopherol showed higher antioxidant activity than BHT. The different antioxidant behaviour of the oil samples as well as references could be related to different qualitative and quantitative lipid compositions of the substrate. The authors confirmed, once again, that the antioxidant activity of the oils could be attributed to the high concentration of the phenolic isomers thymol (32%) and carvacrol (17%) in oregano oil, the presence of borneol (21%), -terpinene (14%) and sabinene (9%) in marjoram oil (9%), and the presence of estragole and 1,8-cineole in laurel oil, which are not, contrary to thymol and carvacrol, phenolic compounds. Furthermore, Dorman et al. (1995)[104] reported that the amount of lipid substrate had an effect on the inhibitory rate of lipid oxidation. In that sense, the egg yolk TBARS assay showed that monarda (Monarda citriodora var. citriodora), nutmeg (Myristica fragrans) and thyme (Th. vulgaris) essential oils are the most effective, while the chick liver assay revealed the nutmeg oil to be the most active. When the assay used chicken muscle, the best antioxidant activities were found for monarda, nutmeg, oregano (O. vulgare ssp. hirtum) and thyme without significant differences among them, revealing once again how the chemical composition of the lipidic substrate plays an important role in antioxidant assays. Some authors[105] concluded that oregano essential oil, mainly constituted by thymol and carvacrol, the oxygenated and nonoxygenated fractions and the pure references thymol and carvacrol exhibited almost the same antioxidant power when evaluated by TBARS method. However, the -carotene bleaching method showed different results for the same samples. The oxygenated fraction exhibited the highest antioxidant activity in comparison with the phenolic fraction or the pure constituents thymol and carvacrol, suggesting that the antioxidant activity of oregano essential oil is due to more polar constituents as well as to a possible synergistic effect among minor oxygen-containing compounds. Both methods proved that the antioxidant activity was dose-dependent. The authors indicated that the antioxidant activity depends on the chosen method, the concentration and the nature and physicochemical properties of the studied antioxidants. In contrast to the results reported above[105], other authors[106] found that in both methods (TBARS and -carotenelinoleic acid), the total savory (Satureja montana L.) essential oil as well as its different fractions or pure constituents containing a hydroxyl group exhibited a relatively strong antioxidant effect. These authors also reported that the total essential oil, phenolic fraction and oxygenated fraction, as well as the pure isomers thymol and carvacrol, showed similar antioxidant effects, which allows the conclusion that there was no synergism among the minor oxygen-containing compounds. The authors therefore concluded that the antioxidant effect of the savory oil was due probably only to the presence of an elevated percentage of thymol (45%) in the total oil. Bozin et al. (2006)[107] characterized the volatile composition of essential oils of some Lamiaceae spices (O. basilicum L., O. vulgare L. and Th. vulgaris L.), which showed very strong protective activity in liposomes. However, some differences in the biological activity of the studied oils have been detected, relating to the induction system (Fe2+/ascorbate and Fe2+/H2O2) of the peroxidation of membrane lipids. Oregano oil exhibited higher activity in the Fe2+/ascorbate system than thyme oil and the reference BHT. In the Fe2+/H2O2 system, all the tested essential oils and BHT showed similar antioxidant activities. Teissedre and Waterhouse (2000)[108] studied another lipid substrate (human LDL) to evaluate the antioxidant activity of 23 different essential oils. The method used in this study was based on the inhibition of copper-induced oxidation of freshly prepared human LDL from plasma. The effects of the essential oils on the oxidative susceptibility of LDL were investigated by measuring the hexanal formed. The authors concluded that the inhibition of oxidation of the LDL depended on two factors, the phenolic concentration in the studied samples and their structures. This study also allowed classification of the essential oils into four categories: (a) oils with very low inhibitory activity on the inhibition of LDL ( < 2%), which contained methyl chavicol (estragole), anethole, p-cymene or vanillin; (b) oils with low inhibitory activity (610%), which contained carvacrol, thymol, p-cymene or vanillin; (c) oils with intermediate inhibitory activity (1050%) containing moderate amounts of thymol, carvacrol, cuminol or eugenol; and (d) oils with high inhibitory activity (50100%) containing eugenol as the major component (Figures 4, 5). There are many studies on the antioxidant activity of Thymus essential oils, mainly as inhibitors of lipid peroxidation. Some authors[93] showed that thyme (Thymus vulgaris L.) essential oil, mainly constituted of thymol (80%), showed a better antioxidant activity than wild thyme (Thymus serpyllum L.), which contained carvacrol (49%) and thymol (30%) as the major compounds. The antioxidant activities of the oil fractions demonstrated that the oxygenated fraction of wild thyme was slightly better than that of complete thyme oil. Non-polar fraction exhibited high antioxidant activity that could be attributed to the presence of relative amounts of -terpinene, p-cymene and -terpinene. The antioxidant activity of these monoterpene hydrocarbons had already been demonstrated by other authors.[109] Kulisic et al. (2004)[105] also attributed the antioxidant activity of this non-polar fraction to the possible retention of this fraction in the oil droplets and/ or its accumulation at the oilwater interface of the egg yolk water emulsion (the so-called polar paradox), already shown by other authors.[110112]
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CH3 CH2 CH2 CH2 CHO
OCH3 OCH3 Anethole OCH3 Estragole or Methylchavicol OH Eugenol OCH3
OCH3 OH Vanillin
OCH3
Methyleugenol
Figure 5. Selected phenylpropanoid derivatives
Some studies[113] reported the inhibitory activity of essential oil from endemic species from Inner Anatolia (Turkey), viz. Thymus spathulifolius, by the -carotene bleaching method. The studied oil contained thymol (37%) and carvacrol (30%) as the predominant compounds. Such activity was similar to that of BHT and even superior to the non-polar extract. This study showed that the essential oil of Th. spathulifolius is a better inhibitor of the formation of conjugated dienes than the free radical scavenger. Polar extracts and fractions of methanol extracts of deodorized material as well as deodorized hot water exracts of Thymus eigii M. Zohary et P. H. Davis, from Turkey, exhibited a higher ability to prevent the formation of conjugated dienes than the plant essential oil, in spite of the relatively high amounts of carvacrol and thymol in the oil.[114] Some authors[70] investigated the antioxidant activity of Th. sipyleus subsp. sipyleus var. sipyleus and Th. sipyleus subsp. sipyleus var. rosulans oils from Turkey and demonstrated that the oxidation of linoleic acid was suppressed by var. rosulans, while other oils did not exhibit any activity. The inhibitory rate of the var. rosulans oil was similar to that of the synthetic antioxidantBHT. Such ability of var. rosulans to inhibit the formation of conjugated dienes was expected, due to the relatively high amount of thymol (21%) and carvacrol (58%), which were not present in var. sipyleus. T. capitata and O. vulgare essential oils were detected as equally good antioxidants by the TBARS method. However, under oxidative process conditions, i.e. in the presence of the radical inducer AAPH, the antioxidant capacity of T. capitata essential oil was significantly better than that of oregano oil.[64] This could relate to results previously reported,[109] according to which carvacrol showed a higher antioxidant activity than thymol (the main component of T. capitata and O. vulgare) as shown by the TBARS method, in the presence of the radical inducer AAPH. Those authors[64] suggested that the essential oils of T. capitata and O. vulgare could act preferentially by reducing the formation of hydroperoxydienes, therefore preventing the primary oxidation. The antioxidant activity of Thymus and Origanum spp. oils from Algeria were evaluated and compared by Hazzit et al.[115] They concluded that the significant differences detected for some essential oils could not be explained only by the presence of the isomers thymol and carvacrol, because in spite of the low amount of carvacrol and thymol, the essential oils of Th. guyonii and Th. numidicus oils also exhibited good ability to prevent lipid peroxidation, mainly in the absence of the radical inducer AAPH. Some authors[71] demonstrated that Thymbra capitata oils from Tunisia, collected at different developmental stages, with concentrations of carvacrol ranging from 63% (in the post-flowering
phase) to 83% (in the vegetative phase) possessed different abilities for preventing lipid peroxidation. The oils isolated during the vegetative phase were less effective in the prevention of the lipid peroxidation process, despite the highest amount of carvacrol in that phase. The authors attributed that behaviour to the diverse relative amounts of the minor compounds present in the essential oils. The antioxidant activity of selected Thymus essential oils from Portugal, evaluated by the TBARS method, revealed some capacity for preventing lipid oxidation even in the absence of phenolic compounds.[59] The tested oils showed the following antioxidant activity: Th. caespititius (76%) > Th. camphoratus (52%) > Th. mastichina (39%). -Terpineol dominated in Th. caespititius essential oil, while linalool, linalyl acetate and 1,8-cineole dominated Th. camphoratus oil. 1,8-Cineole was the main component of Th. mastichina oil. Several chemotypes of Th. albicans, Th. camphoratus, Th. carnosus and Th. mastichina, can be found in Portugal. Miguel et al.[72] assessed the antioxidant activities of these chemotypes collected in different regions of Portugal and compared them with those of BHT, BHA and -tocopherol. Two methods were assayed, the TBARS and micellar model system methods. With the TBARS method (in the absence of the radical inducer AAPH), the antioxidant activity of Th. carnosus essential oils did not significantly differ, independent of the relative amount of the main components trans-sabinene hydrate and borneol. In the case of Th. camphoratus essential oils, a high content of 1,8-cineole (33%) seemed to be responsible for the lower antioxidant activity (35%). Similar results were achieved for Th. mastichina essential oils: the lower the percentage of 1,8-cineole, the higher the antioxidant activity. Nevertheless, this behaviour was not so evident for Th. albicans oils. Generally the presence of AAPH decreased the antioxidant activity, with the exception of control and Th. camphoratus essential oils. The use of the micellar model system also proved that the chemical composition of the oils was responsible for the differences observed in the antioxidant activity. Those authors[72] indicated that the antioxidant activity of essential oils depends on its origin and composition as well as on the methodology, which had already been shown by other authors.[105,109,111] Ruberto and Baratta[109] studied the antioxidant activity of 98 pure essential oil components by two model systems, the thiobarbituric acid reactive species (TBARS) method, using egg yolk as lipidic substrate, and measuring the formation of hydroperoxydienes from linoleic acid in a micellar system. In both systems, the AAPH was used as a radical inducer. The components were
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M. G. Miguel representative of the main classes of compounds typical of the essential oils: monoterpene hydrocarbons, oxygenated monoterpenoids, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, one diterpene, benzene derivatives and non-isoprenoid components. Terpinolene, -terpinene, -terpinene and, to some extent, sabinene, were the sole monoterpene hydrocarbons with remarkable antioxidant activity, having a comparable activity to that of -tocopherol at higher concentrations. Concerning the oxygenated monoterpenes, the most active components were thymol and carvacrol, whose antioxidant activity was confirmed by two methods and which was similar to that of -tocopherol. Other components from this class of compounds also showed antioxidant activity, mainly allylic alcohols (perillyl alcohol, nerol, cis-verbenol, geraniol), whose activity was especially expressed by the TBARS method. On the other hand, linalool, an allylic alcohol, showed a pro-oxidant effect by both the methods used (TBARS and the micellar model system). Generally, sesquiterpenes showed low antioxidant activity in this study. Among the benzene derivatives, phenols (eugenol and guaiacol) showed higher efficacy in preventing the formation of primary oxidation products (micellar model system) than the formation of secondary oxidation products (TBARS method). These results clearly demonstrate the importance of the method chosen as well as the chemical structure of the components on the antioxidant effectiveness of the essential oils. Ability for Scavenging Free Radicals Species Essential oils isolated from many aromatic plants have also exhibited good free radical scavenging capacity (Table 4). However, some authors[113] showed that the essential oil components of Th. spathulifolius are more effective as inhibitors of conjugated dienes and linoleic acid oxidation than free radical scavengers. Other authors[70,114] found an accordance of data resulting from both the -carotenelinoleic acid and the DPPH test systems, independent of the Thymus oils assayed. Chemat et al.[51] asserted that good inhibitors of lipid peroxidation are able to rapidly scavenge lipid peroxyl radicals and therefore have to possess the ability to scavenge stable DPPH radicals. However, the authors reported that some discrepancies appeared with geraniol, thymol and carvacrol. These phenolic compounds had stronger DPPH-scavenging activity than their ability to inhibit lipid peroxidation. In contrast, - and -terpinene, which possess 1,3- and 1,4-diene moities, were much weaker H-donors to DPPH than lipid peroxidation inhibitors. Their efficiency in the prevention of the lipid peroxidation process was equal to those of the phenolic compounds carvacrol and thymol. Besides the DPPH radical scavenging test, there are other assays for the evaluation of free radical-scavenging activity. Sacchetti et al.[67] made a comparative antioxidant and antiradical evaluation of 11 different essential oils. They used the DPPH test and the luminolphotochemiluminescence assay to evaluate free radical-scavenging activity and found that, by the DPPH method, Cananga odorata, Rosmarinus officinalis, Curcuma longa, and Cymbopogon citratus oils showed major effectiveness, with radical inhibition in the range 6064%, lower than that of the reference Thymus vulgaris oil (76%). By the luminol-photochemiluminescence assay, good results were obtained with R. officinalis oil (66 mM Trolox/l) and C. odorata oil (32 mM Trolox/l), distant from the value detected for the reference oil Th. vulgaris (342 mM Trolox/l). The chemical composition of the tested oils was quite different. C. odorata oil was mainly constituted by benzyl benzoate (34%) and linalool (25%), while in Cymbopogon citratus oil, neral (32%) and geranial (41%) predominated. -Phellandrene (20%), -turmerone (20%) shared with 1,8-cineole (10%) and -turmerone (7%), among other components, the oil of Curcuma longa oil. R. officnalis presented as its major components verbenone (22%) and borneol (10%). Using TEAC and DPPH assays for determining in vitro radical scavenging activity of essential oils from Columbian plants as well as fractions from Origanum vulgare L. oil, some authors[90] found that Cananaga odorata oil was the most effective as an anti-radical scavenger, using the TEAC method, whereas O. vulgare showed high activity by the DPPH method. In the same study, the authors demonstrated that Foeniculum vulgare Mill. and Coriander sativum L. had weak ability to scavenge the ABTS radical cation, which is in contradiction to results presented by other authors[52,102] (Table 3), who reported high antioxidant activity of these species using the TBARS assay. Concerning O. vulgare oil, the authors concluded that one of its fractions was particularly effective as an antioxidant, even superior when compared to the references thymol, carvacrol and a mixture of thymol and carvacrol. In contrast with the results of Puertas-Meja et al.,[90] who showed good radical scavenging activity of Cananga odorata essential oil, other authors[116] reported weak radical scavenging activity. The same authors also showed the weak radical scavenging activity of Th. vulgaris and R. officinalis essential oils, which is also in contradiction with previously published results. In the same study, the authors merely found good antioxidant activity against ABTS (using the TEAC method) by essential oils isolated from Cinnamomum verun, Commiphora molmol and Th. vulgaris. Bozin et al. (2006)[107] concluded that Origanum vulgare L., Th. vulgaris L. and Ocimum basilicum oils were able to reduce the stable DPPH radical even better than BHT. The method, coupled to TLC, demonstrated that the compounds most responsible for this activity were the oxygenated monoterpenes carvacrol, thymol and methyl chavicol (estragole) as well as the mixture of mono- and sesquiterpene hydrocarbons. However, Ocimum basilicum harvested in different seasons of the year in Pakistan exhibited good antioxidant activities, as measured by DPPH free radical-scavenging ability, even though linalool constituted the major component of the oils.[84] Concerning the scavenging capacity of essential oils for hydroxyl radicals, evaluated through the degradation of 2-deoxyribose, this was higher than that of BHT, especially in the case of thyme essential oil.[107] Mimica-Dukic et al. (2004)[117] showed that the essential oil of Melissa officinalis L. had very strong free radical scavenging capacity, mainly due to the presence of neral/geranial, citronellal, isomenthone, menthone and (E)-caryophyllene. The oil capacity for reducing the stable radical DPPH was similar to that of BHT. High inhibition of degradation of 2-deoxyribose was also observed for all of the tested essential oil concentrations. Leaf oils of Eucalyptus tereticornis exhibited HO and O2 scavenging activities parallel to the commercial antioxidant activity of BHT/ascorbic acid. However, their major components, such as (+)--citronellol and -citronellal, among others, had significantly less scavenging activity.[80] Karioti et al.[118] evaluated the antioxidant ability of the essential oils of Xylopia aethiopica leaves, stem bark, root bark and fresh and dried fruits from Ghana. Quantitative differences were detected in the main compounds (germacreneD, - and -pinenes, trans-m-mentha-1(7),8-diene) that constituted the oils of the different parts of the plant (Figures 3, 6). Such differences could probably explain the different capacity to

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Table 4. Examples of essential oils assayed for free radical scavenging by diverse tests Essential oil Thymus spathulifolius (Hausskn. and Velen.) Thymus eigii M. Zohary et P.H. Davis Thymus sipyleus subsp. sipyleus var. sipyleus Th. sipyleus subsp. sipyleus var. rosulans Cananga odorata Cupressus sempervirens Curcuma longa Cymbopogon citrates Eucalyptus globulus Pinus radiata Piper crassinervium Psidium guayana Rosmarinus officinalis Thymus vulgaris Thymus citriodorus Zingiber officinale Cananga odorata Hook. Fil. et Thomson Coriander sativum L. Foeniculum vulgare Mill. Lepechinia schiedeana (Schlecht) Vatke Lippia alba Mill. Origanum vulgare L. Pimpinela anisum L. Rosmarinus officinalis L. Anthemis nobilis Boswellia carterii Cananga odorata Cedrus atlantica Cinnamomum camphora C. verun Citrus aurantium C. limonum C. medica Commiphora molmol Cupressus sempervirens Cymbopogon nardus C. nartinii Hyssopus officinalis Lavandula vera Melaleuca alternifolia Melissa officinalis Origanum vulgaris Pelargonium roseum Pimpinella anisum Rosmarinus officinalis Thymus vulgaris Vervena officinalis Zingiber officinale Ocimum. basilicum L. Origanum vulgare L. Thymus vulgaris L. Melissa officinalis L. BHT BHT Trolox BHT Standard DPPH DPPH DPPH DPPH Photochemiluminescence Assay References 113 114 70 67
Thymol Carvacrol Thymol + carvacrol (1:1)
DPPH TEAC
90
TEAC
116
DPPH Hydroxyl radical scavenging (2-deoxyribose) DPPH Hydroxyl radical scavenging (2-deoxyribose)
84,107
BHT
117
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Table 4. Continued Essential oil Eucalyptus tereticornis Standard -Pinene 1,8-Cineole -Citronellal ()-Isopulegol (+)--Citronellol BHT Ascorbic acid Acetylsalicylic acid Caffeic acid Assay DPPH Hydroxyl radical scavenging (2-deoxyribose) Superoxide radical scavenging [phenazinemethosulphate (PMS)NADH] method DPPH Superoxide radical scavenging (xanthinexanthine oxidase system) DPPH FRAP References 80
Xylopia aethiopica (Dun) A. Rich.
118
Thymus vulgaris L., non-phenol chemotype Th. vulgaris L., non-phenol chemotype after catalytic oxidation Th. vulgaris L., phenol chemotype Th. vulgaris L., phenol chemotype after catalytic oxidation Carumi carvi Coriandrum sativum Curcuma zedoaria Foeniculum vulgare Laurus nobilis Lavandula spica latifoglia Mentha spicata Origanum majorana Pimpinella anisum Rosmarinus officnalis Sinapis alba Salvia officinalis Thymus vulgaris Zingiber officinalis Origanum vulgare L. Satureja montana L.
Thymol Carvacrol Thymoquinone Dithymoquinone Thymohydroquinone Trolox Vitamin C 1,8-Cineole Ascorbic acid
79,119
Inhibition of tyrosine nitration
120
Thymol Carvacrol Cymene -Terpinene Trolox Ascorbic acid Eugenol (E)-Cinnamaldehyde Linalool Ascorbic acid
121
Cinnamomum zeylanicum (Blume)
122
CH3
CH3
H H3C CH3
CH2 H2C H3C CH3
b -Caryophyllene
Germacrene D
Figure 6. Examples of hydrocarbon sesquiterpenes
scavenge free radicals. The essential oil of fresh fruits possessed the highest capacity for reducing DPPH, whereas the oil isolated from leaves showed the highest ability to scavenge superoxide anion radical. Essential oils of Th. vulgaris presented antioxidant activity as measured by the DPPH and FRAP tests, mainly in those rich in thymol.[79] Studying the antioxidant properties of the essential oils from two chemotypes of Th. vulgaris L. oils, some authors[119] demonstrated, by two methods (DPPH and FRAP), that the phenolic chemotype possessed stronger antioxidant properties than the non-phenolic phenotype. In the same study, the authors
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Antioxidant activity of medicinal and aromatic plants carried out catalytic oxidation of the essential oils. After this reaction, the essential oils of both chemotypes showed stronger antioxidant effects than before oxidation, owing to the conversion of thymol and carvacrol to thymoquinone and thymohydroquinone. Chericoni et al.[120] reported for the first time the activity of essential oils as inhibitors of peroxynitrite-mediated processes. The authors screened 15 essential oils and concluded that Laurus nobilis oil, mainly constituted by 1,8-cineole (50%), provided the highest in vitro protection against tyrosine nitration. Nevertheless, this compound was revealed to be inactive, the authors proposing that minor compounds and/or complex interactions between the components of the essential oil could play an important role. Other authors[121] proved that Origanum vulgare and Satureja montana oils were able to prevent in vitro peroxynitrite-induced formation of 3-nitrotyrosine better than reference antioxidants, such as ascorbic acid and Trolox. The authors considered that carvacrol and thymol were responsible for such activity. Cinnamomum zeylanicum oil isolated from bark, and its major component eugenol, were tested in two in vitro models of peroxynitrite-induced nitration and lipid peroxidation and proved to have powerful activities.[122]
5. S. S. Deshpande, U. S. Deshpande, D. K. Salunkhe. In Food Antioxidants, D. L. Madhavi, S. S. Deshpande, D. K. Salunkhe (eds). Marcel Dekker: New York, Basel, Hong Kong, 1995, 361. 6. C. Snchez-Moreno. Food Sci. Tech. Int. 2002, 8, 121. 7. M. Antolovich, P. D. Prenzler, E. Patsalides, S. McDonald, K. Robards. Analyst 2002, 127, 183. 8. D. Huang, B. Ou, R. L. Prior. J. Agric. Food Chem. 2005, 53, 1841. 9. J. S. Wright, E. R. Johnson, G. A. Dilabio. J. Am. Chem. Soc. 2001, 123, 1173. 10. R. L. Prior, X. Wu, K. Schaich. J. Agric. Food Chem. 2005, 53, 4290. 11. K. Lemaska, B. Tyrakowska, R. Zieliski, A. E. M. Soffers, M. C. M. Rietjens. Free Rad. Biol. Med. 2001, 31, 869. 12. E. N. Frankel, A. S. Meyer. J. Sci. Food Agric. 2000, 80, 1925. 13. H. Nohl, A. V. Kozlov, L. Gille, K. Staniek. In Oxidants and Antioxidant Defense Systems. The Handbook of Environmental Chemistry, G. Tilman (ed.). Springer-Verlag: Berlin, Heidelberg, New York, 2005, 1. 14. A. E. Hagerman, K. M. Riedl, G. A. Jones, K. N. Sovik, N. T. Ritchard, P. W. Hartzfeld, T. L. Richel. J. Agric. Food Chem. 1998, 46, 1887. 15. B. Ou, M. Hampsch-Woodhill, J. Flanagan, E. K. Deemer, R. L. Prior, D. Huang. J. Agric. Food Chem. 2002, 50, 2772. 16. M. Martnez-Tom, F. Garcia-Carmona, M. A. Murcia. J. Sci. Food Agric. 2001, 81, 1019. 17. D. Costa, A. Gomes, S. Reis, J. L. F. C. Lima, E. Fernandes. Life Sci. 2005, 76, 2841. 18. S. Y. Wang, H. Jiao. J. Agric. Food Chem. 2000, 48, 5677. 19. R. Ebermann, G. Alth, M. Kreitner, A. Kubin. J. Photochem. Photobiol. B 1996, 36, 95. 20. M. Rozanowska, J. Wessels, M. Boulton, J. M. Burke, M. A. Rodgers, T. G. Truscott, T. Sarna. Free Rad. Biol. Med. 1998, 24, 1107. 21. S. Zigman. J. Ocul. Pharmacol. Th. 2000, 16, 161. 22. H. Sies, W. Stahl. Photochem. Photobiol. Sci. 2004, 3, 749. 23. A. S. Pannala, R. Razaq, B. Halliwell, S. Singh, C. A. Rice-Evans. Free Rad. Biol. Med. 1998, 24, 594. 24. N. W. Kooy, J. A. Royall, H. Ischiropoulos, J. S. Beckman. Free Rad. Biol. Med. 1994, 16, 149. 25. E. Fernandes, S. A. Toste, J. L. F. C. Lima, S. Reis. Free Rad. Biol. Med. 2003, 35, 1008. 26. M. Laguerre, J. Lecomte, P. Villeneuve. Prog. Lipid Res. 2007, 46, 244. 27. C. Berset, M.-E. Cuvelier. Sci. Aliment. 1996, 16, 219. 28. E. N. Frankel. Trends Food Sci. Technol. 1993, 4, 220. 29. F. A. M. Silva, M. F. M. Borges, M. A. Ferreira. Quim. Nova 1999, 22, 94. 30. D. Rajalakshmi, S. Narasimhan. In Food Antioxidants, D. L. Madhavi, S. S. Deshpande, D. K. Salunkhe (eds). Marcel Dekker: New York, Basel, Hong Kong, 1995, 65. 31. D. M. Hodges, J. M. DeLong, C. F. Foney, R. K. Prange. Planta 1999, 207, 604. 32. E. N. Frankel, S.-W. Huang, J. Kanner, J. B. German. J. Agric. Food Chem. 1994, 42, 1054. 33. E. N. Frankel, S.-W. Huang, R. Aeschbach, E. Prior. J. Agric. Food Chem. 1996, 44, 131. 34. S.-W. Huang, E. N. Frankel, J. B. German. J. Agric. Food Chem. 1994, 42, 2108. 35. F. B. Hu. Am. J. Clin. Nutr. 2003, 78, 544. 36. E. Riboli, T. Norat. Am. J. Clin. Nutr. 2003, 78, 559. 37. A. M. Roussel, J. Nve, I. Hininger. In Radicaux Libres et Stress Oxydant. Aspects Biologiques et Pathologiques, J. Delattre, J.-L. Beaudeux, D. Bonnefont-Rousselot (eds). Editions TEC & DOC, Lavoisier: Paris, 2005, 261. 38. D. Madhavi, D. K. Salunkhe. In Food Antioxidants, D. L. Madhavi, S. S. Deshpande, D. K. Salunkhe (eds). Marcel Dekker: New York, Basel, Hong Kong, 1995, 267. 39. C. Borek. J. Nutr. 2001, 131, 1010S. 40. F. A. Tomas-Barberan, M. N. Clifford. J. Sci. Food Agric. 2000, 80, 1024. 41. T. Grune, P. Schrder, H. K. Biesalski. In Oxidants and Antioxidant Defense Systems. The Handbook of Environmental Chemistry, G. Tilman (ed.). Springer-Verlag: Berlin, Heidelberg, New York, 2005, 77. 42. R. Tsao, Z. Deng. J. Chromatogr. B 2004, 812, 85. 43. C. A. Rice-Evans, N. Y. Miller, P. G. Bolwell, P. M. Bramley, J. B. Pridham. Free Rad. Res. 1995, 22, 375. 44. K. E. Heim, A. R. Tagliaferro, D. J. Bobilya. J. Nutr. Biochem. 2002, 13, 572. 45. G. Cao, E. Sofic, R. L. Prior. Free Rad. Biol. Med. 1997, 22, 749. 46. M. zcan, A. Akgl. Acta Aliment. 1995, 24, 81. 47. H. L. Madsen, G. Bertelsen. Trends Food Sci. Tech. 1995, 6, 271.
Conclusions
Lipid oxidation has a importance for the food industry, due to the formation of undesirable off-flavours and potentially toxic compounds. A way to prevent food oxidation is the use of antioxidants. The synthetic antioxidants currently used in the food industry (BHA and BHT) have been found to possess several pernicious health effects. In that sense, there is an increasing interest in natural antioxidants, especially plant-derived antioxidant compounds. Numerous studies have shown that essential oils represent a huge source of compounds exhibiting strong antioxidant activity, and that they can be used as natural antioxidants in the food, pharmaceutical and cosmetic industries. However, there are some drawbacks, due to the chemical complexity of the essential oils. Some authors propose that the essential oils have to be characterized by the molar concentrations of their main components, used as a reference antioxidant, and tested by their H-donating properties and ability to inhibit lipid autoxidation.[51] More recently, the ability to inhibit lipid oxidation has been studied in real foods (e.g. in refrigerated stored pats or in salted cultured sea bream fillets stored under refrigeration).[123,124] Studies on the effect of dietary essential oils on lipid oxidation in raw and cooked meat during refrigerated storage offered very important and interesting results.[125,126] Therefore, the link between the preservative role and the pleasant odour and favourable taste of food, which can be achieved by the used of essential oils, offers a good alternative to synthetic antioxidants.
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(wileyonlinelibrary.com) DOI 10.1002/ffj.1961

Antioxidant activity of medicinal and aromatic plants. A review.

Antioxidants and Oxidation: General Concepts

Flavour Fragr. J. 2010, 25, 291312

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In Vitro Determination of Antioxidant Activity

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Flavour Fragr. J. 2010, 25, 291312

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Peroxyl radicals have already been discussed.

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Flavour Fragr. J. 2010, 25, 291312

Flavour Fragr. J. 2010, 25, 291312

Copyright 2010 John Wiley & Sons, Ltd.

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Copyright 2010 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2010, 25, 291312

Antioxidants and Food

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H3CO OCH3 (E)-Anethole O Myristicine

R=H, p-Cumaric acid R=OH, Caffeic acid R=OCH3, ferulic acid

Some examples of phenolic acids

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Antioxidant activity of medicinal and aromatic plants

Flavonols R=H, Kaempferol R=OH, Quercetin R

R=H, Apigenin R=OH, Luteolin

O H H OH O Dihydroflavonols R=H, Dihydrokaempferol R=OH, Dihydroquercetin OH

Flavan-3,4-diols R=H, Leucopelargonidin R=OH, Leucocyanidin

OH OH Anthocyanidins R=H, Pelargonidin R=OH, Cyanidin

Figure 2. Some examples of flavonoids

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Essential oils as natural antioxidants

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Antioxidant activity of medicinal and aromatic plants

BHT Thymol Carvacrol Thymol + carvacrol BHT

Peroxide value -Carotene bleaching test -Carotene bleaching

-Carotenebleaching -Carotenebleaching -Carotenebleaching Rancimat

Linoleic acid/45 C Linoleic acid/50 C Linoleic acid/room temperature Lard/100 C

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TBARS in the absence and presence of a radical inducer (AAPH)

Egg yolk/95 C Rat liver homogenates/95 C

TBARS -Carotene bleaching test TBARS -Carotene bleaching test TBA

Ocimum basilicum L. Origanum vulgare L. Thymus vulgaris L.

Antioxidant activity of medicinal and aromatic plants

BHT BHA -Tocopherol

BHT BHA BHT BHA -Tocopherol

TBARS TBARS (in the absence and presence of AAPH)