measuring antioxidant activity in bioorganic...

Review ArticleMeasuring Antioxidant Activity in Bioorganic Samples bythe Differential Oxygen Uptake Apparatus Recent Advances

Riccardo Amorati Andrea Baschieri and Luca Valgimigli

Department of Chemistry ldquoG Ciamicianrdquo University of Bologna Via S Giacomo 11 40126 Bologna Italy

Correspondence should be addressed to Riccardo Amorati riccardoamoratiuniboit

Received 2 January 2017 Accepted 27 February 2017 Published 19 March 2017

Academic Editor Jacques Lalevee

Copyright copy 2017 Riccardo Amorati et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The measure of O2 consumption during the inhibited autoxidation of an easily oxidizable substrate is one of the most reliableand predictive methods to assess antioxidant activity especially for structure-activity relationship studies for food and industrialapplications The differential oxygen uptake apparatus described herein represents a powerful and cost-effective way to obtainantioxidant activity from inhibited autoxidation studies These experiments provide the rate constant and the stoichiometry of thereaction between antioxidants and peroxyl radicals (ROO∙) which are involved in the propagation of radical damage We showthe operation principles and the utility of this instrumentation in the bioorganic laboratory with regard to the recent advances inthis field ranging from the study of natural antioxidants in biomimetic system to the use of substrates generating hydroperoxylradicals and to the evaluation of novel nanoantioxidants

1 Introduction

The reaction of organic substrates with atmospheric oxygenunder mild conditions to form various oxygenated com-pounds such as hydroperoxides peracids epoxides andketones is called ldquoautoxidationrdquo [1] It can either be beneficialsuch as when it is used for the functionalization of organiccompounds [2] or it can be detrimental as in case of foodoils or polymers getting rancid or oxidized in the pre-sence of atmospheric O2 [3] Antioxidants are always presentin natural and man-made oxidizable materials as a strategyto preserve them from the damage of autoxidation [4] More-over the awareness of the importance of radical-mediatedreactions in biology and the theories relating oxidative stressto aging and degenerative diseases represent another majorfactor promoting the study of antioxidants [5] A correctand unbiased measure of the antioxidant activity is of funda-mental importance for the evaluation of natural antioxidantsand to guide for the design of new synthetic ones To adaptto the different testing needs an impressively large array ofrapid assays has been proposedThese includemethods based

on inhibited autoxidation studies which are performed bymonitoring O2 consumption or the formation of hydroper-oxides (the primary oxidation products) or of secondaryoxidation products like carbonyl compounds When resultsare analyzed with a proper kinetic treatment these methodsare the most reliable to assess antioxidant activities [6] Mosttesting methods however do not involve substrate autoxida-tion They rely on the competitive bleaching of a probe (egORAC luminol and120573-carotene bleaching assays) [7] or theyare indirect methods based on the reduction either of persis-tent radicals (eg DPPH and TEAC assays) or of inorganicoxidizing species (egFRAPandFollin-Ciocalteu assays) [8]All such methods that are not based on the kinetic studyof a substratersquos autoxidation should be used with cautiononly as preliminary indication after careful consideration ofthe chemistry behind them that is bearing in mind whatthey are actually measuring Failure to do so might result inunrealistic and scarcelymeaningful data [6ndash9] In this reviewwe describe how tomeasure the rate of inhibited autoxidationby using a differential oxygen uptake apparatus and how toobtain accurate quantitative data of antioxidant activity from

HindawiJournal of ChemistryVolume 2017 Article ID 6369358 12 pageshttpsdoiorg10115520176369358

2 Journal of Chemistry

RHROOH

Nonradicalproducts

AH

IH

Initiator

PropagationInitiation Termination

I∙ + RH R∙ROO∙

ROO∙

ROOH + A∙

kinh

kp

kt

O2

Scheme 1 Chain reaction of autoxidation for a substrate (RH) andmode of interference by chain-breaking antioxidants (AH)

the analysis of O2-consumption traces We show the utility ofthis instrumentation in the bioorganic laboratory with regardto the recent advances in this field ranging from the study ofnatural antioxidants in biomimetic systems to the evaluationof novel nanoantioxidants

2 How to Determine the Antioxidant Activityfrom Inhibited Autoxidation

Autoxidation follows a free-radical chain mechanismdepicted in its simplest form in Scheme 1

Autoxidation can in principle be initiated by any reac-tion that generates free radicalsThemost common initiatingsystems with practical relevance are the catalytic decom-position of hydroperoxides by transition metals ions (suchas Fe2+ and Cu+) and the interaction of UV-vis light withphotoinitiators (such as ketones and peroxides) Howeverbecause hydroperoxides (and peroxides) and ketones are alsoproducts of the autoxidation their accumulation may leadto autocatalytic behavior consisting in an increasing rate ofreaction with time [8] When a controlled rate of initiation isdesired thermal decomposition of azo-initiator is preferredThe use of azo-initiators such as the lipid-soluble AIBN(221015840-azobis-isobutyronitrile) or water-soluble AAPH (221015840-azobis(2-amidinopropane) dihydrochloride) is much morepractical than metal catalysis or photoinitiation becausethe homolytic decomposition of azo-initiators proceeds atconstant rate at a given temperature during the entire courseof autoxidation thereby providing a constant rate of initiation[10] Following the mechanism reported in Scheme 1 theinitiating radical (I∙) abstracts a H-atom from the substrategenerating a carbon-centered (alkyl) radical R∙ which reactswith O2 to form a peroxyl radical (ROO∙) Except in somespecific cases [11] this last reaction is very efficient [10] Forthis reason in the presence of atmospheric oxygen virtuallyall R∙ are converted to ROO∙ and peroxyl radical chemistrydominates autoxidation kinetics The propagation can eitherconsist of H-atom transfer (ROO∙+RHrarr ROOH+R∙) or ofaddition to a C=Cdouble bond (ROO∙ +X=Y rarr ROOX-Y∙where X and Y are substituted C atoms) Termination eventsconsist mainly in the radical-radical self-quenching of per-oxyl radicals to give nonradical products [1] If propagationis fast enough the overall process becomes a chain reactionand after the initiation event several propagation steps

Table 1 Rate constants for the autoxidation of selected organicsubstrates in PhCl at 303K

Substrate 119896119901 [Mminus1sminus1] 2119896119905 [Mminus1sminus1] 119896119901(2119896119905)12Methyl oleatea 088 10 times 106 88 times 10minus4Methyl linoleatea 62 88 times 106 0021Toluenea 024 30 times 108 14 times 10minus5Styrenea 41 42 times 107 63 times 10minus3Cumeneb 032 46 times 104 15 times 10minus3Tetrahydrofurancd 43 62 times 107 17 times 10minus414-Cyclohexadienee 1400 13 times 109 0038Heptanalf g 2800 54 times 107 038aSee [12] bsee [13] csee [14] dneat ethe propagating radical is hydroperoxyl(HOO∙) see [15] f the propagating radicals are an acylperoxyl see Ref [16]gat 273 K

can take place before the chain reaction is terminated Bysolving the kinetic equations that describe the individualreactions depicted in Scheme 1 and applying the steady stateapproximation oxygen consumption during the autoxidationof a generic substrate RH is described by (1) where 119896119901 and2119896119905 are respectively the propagation and termination rateconstant for the autoxidation of the oxidizable substrate and119877119894 is the initiation rate

minus119889 [O2]119889119905 =119896119901radic2119896119905 [RH] radic119877119894 + 119877119894 (1)

Since the autoxidation rate depends on the ratio 119896119901(2119896119905)12 this quantity is commonly referred to as the oxidiz-ability of the substrate Some examples of typical oxidizablesubstrates are given in Table 1 together with their respective119896119901 2119896119905 and 119896119901(2119896119905)12 values From the values reported inTable 1 it is evident that the oxidizability of organicmoleculesvaries considerably from very low values for saturatedhydrocarbons to 1000-fold higher values for polyunsaturatedhydrocarbons or for aldehydes

Once the rate constants 119896119901 and 2119896119905 are known along with119877119894 values the effect of the addition of any modifier to theautoxidation can be studied Some compounds can accele-rate autoxidation for example by providing better chaintransfer than peroxyl radicals as observed in the case of N-hydroxyphthalimide (NHPI) class of compounds

It was shown that after the addition ofNHPI to the autox-idation of isopropylbenzene (cumene) the O2 consumptionis increased because PINO radicals react with RH fasterthan ROO∙ NHPI and related molecules can be used asmetal-free catalysts for the functionalization of hydrocarbons(Scheme 2) [17 18] Addition of easily oxidizable compoundsmay either decrease or increase the rate of O2 consumptionby cooxidizing with the substrate Some of these systemshave been investigated by us in the case of garlic sulfides andestragole [19 20]

However arguably the most important class of com-pounds that are used to modify the rate of autoxidation isantioxidants which are used byman and nature to slow downthe degradation of organicmaterials underO2 [1] Depending

Journal of Chemistry 3

R∙

ROO∙

RH

N N

O

OO

OPINO

ROOH

NHPI

OHO2O∙

Scheme 2 Acceleration of autoxidation of RH byNHPIPINO cata-lysis

on their mechanism of action two main classes of antioxi-dants preventive and chain-breaking can be recognized Pre-ventive antioxidants reduce the probability of initiation thatis they reduce the rate of initiation 119877119894 by eliminating theprimary source of free radicals For instance they can reduceor decompose hydroperoxides into nonradical products orthey can chelate transition metal ions removing them fromthe redox cycle (eg Fentonrsquos reaction) that would causethe decomposition of peroxides into radical species [8] Onthe other hand chain-breaking antioxidants (AH) react withperoxyl radicals to yield stabilized radicals (A∙) that are suf-ficiently unreactive toward the substrate RH to be unable tofurther propagate the oxidative chain Rather they are suffi-ciently long-lived to ldquowaitrdquo until they quench a secondperoxyl radical ((2)-(3)) [8] When this happens they are toldto have a stoichiometric factor of 2

AH + ROO∙119896inh997888997888997888rarr A∙ + ROOH (2)

A∙ + ROO∙ 997888rarr non-radical products (3)

In the case of monophenols (ArOH) such as 120572-toco-pherol and 26-di-tert-butyl-4-methylphenol (BHT) the sec-ond ROO∙ radical is trapped by addition on aromatic ringof ArO∙ whereas in the case of catechols (CatH2) by H-atom transfer from the CatH∙ radical to ROO∙ Also in thecase of hydroquinones (QH2) the second ROO∙ radical istrapped by H-atom transfer fromQH∙ but the expected stoi-chiometry of 2 is diminished by the competition between thereactions of the semiquinone (QH∙) with ROO∙ (to quench asecond chain) or with O2 to afford HOO∙ which propagatesthe autoxidation [21] The effectiveness of an antioxidantis therefore described by two independent parameters theinhibition rate constant 119896inh and the number of radicaltrapped 119899 (the stoichiometric factor) Analytical resolutionof the kinetic equations in the assumption that every ROO∙radical is trapped by AH and A∙ (see (2)-(3)) affords (4) for

the O2 consumption rate and (5) for the relationship betweenthe length of the inhibition period (120591) and 119877119894 [10]

minus119889 [O2]inh119889119905 =119896119901 [RH] 119877119894119899119896inh [AH] + 119877119894 (4)

119877119894 = 119899 [AH]120591 (5)

The typical oxygen consumption plots observed duringinhibited autoxidation are reported in Figure 1(a) Trace(A) represents the O2 consumption during an uninhibitedautoxidation while traces (B-D) are those typically observedin the presence of antioxidants with low (B) intermediate (C)or high (D) 119896inh values

Plots (C) and (D) of Figure 1(a) show a period inwhich the autoxidation of the substrate is strongly retardedby the antioxidant the so-called ldquoinduction (or inhibition)periodrdquo The length of the inhibition period graphicallycalculated as shown in Figure 1(a)(D) provides 120591 which inconjunction with 119877119894 and (5) affords the stoichiometric factorof the antioxidant The slope of the inhibited period providesminus119889[O2]inh119889119905 that can be used to obtain 119896inh by (4)

A usual problem encountered when using (4) is thatthe rate of O2 consumption minus119889[O2]inh119889119905 should be takenat the very beginning of the inhibited period where theconcentration of the antioxidant is actually equal to theinitial concentration [AH] used in the equation Howeverin practice this is often impossible due to the time requiredfor system equilibration so that the slope can be measuredonly after a certain time interval after the beginning of thereaction (see for instance the beginning of traces (C) and (D)in Figure 1(a)) While it is always possible to correct [AH] forthe antioxidant concentration effectively present at the pointin which the slope is measured a better solution consists inusing (6) that is the integrated form of (4) [22]

Δ [O2] = [O2]119905=0 minus [O2]119905= minus(119896119901 [RH]119896inh )[ln(1 minus

119905120591)]

(6)

In this equation the oxygen consumption after time 119905 isrelated to a logarithmic function of time in the assumptionthat 119899 = 2 When plotting Δ[O2] as a function of minus ln(1 minus119905120591) a straight line is obtained whose slope is 119896119901[RH]119896inhas shown in Figure 1(b)

When the antioxidant has a too low 119896inh to afford arecognizable induction period with a given substrate (see forinstance Figure 1(a) trace (B)) (4) and (6) are no longervalid because a certain fraction of ROO∙ radicals disappearsby self-termination Erroneous use of (4) and (6) in thesecases may lead to large overestimation of 119896inh Changing thesubstrate with another one having lower 119896119901 and 2119896119905 such ascumene usually overcomes this problem However when 119896inhis too low that also cumene is weakly inhibited 119896inh can beobtained by (7) where 119889[O2]0119889119905 and 119889[O2]inh119889119905 are the O2


Time (s)

minus15

minus10

minus05

00

(A)(B)

(C)

(D)minusΔ[O

2]

(mM

)

0 500 1000 1500 2000

휏

(a)

01

02

03

04

05

06

07

Δ[O

2]

(mM

)

minusln(1 minus t휏)

0 1 2 3 4 5

(b)

Figure 1 (a) Typical O2 consumption traces observed during the autoxidation of an organic substrate in the absence (A) or in the presence ofantioxidants with low (B) intermediate (C) and high (D) 119896inh values (b) Analysis of the O2 consumption by (6) for the antioxidants havingintermediate (∘) and high (e) 119896inh

consumption rates in the absence and in the presence ofantioxidants respectively

119889 [O2]0119889119905119889 [1198742]inh119889119905 minus119889 [1198742]inh119889119905119889 [1198742]0119889119905 =

119899119896inh [AH]radic2119896119905119877119894 (7)

On the other hand very effective antioxidant may gener-ate O2 consumption plots in which the slope of the inhibitedperiod is nearly zero in this case 119896inh values obtained eithervia (4) or (6) could be underestimated In this regard it isrecommended to perform autoxidation experiments by usingoxidizable substrates with different 119896119901 values to properlyanalyze antioxidants with various potencies Styrene with119896119901 of 41Mminus1sminus1 is used to measure 119896inh values in the range105ndash107Mminus1sminus1 while cumene (119896119901 = 032Mminus1sminus1) is bettersuited for 119896inh values in the range 103ndash105Mminus1sminus1 Kineticsfaster than 107Mminus1sminus1 can be determined by using methodsbased on the peroxyl radical clock developed by Pratt et al[23] while the development of oximetric methods for ldquofastrdquoantioxidants is in progress in our laboratory Autoxidationshould be performed in solvents that are not reactive towardperoxyl radicals such as chlorobenzene and acetonitrileSolvents should not contain traces of acids or bases that couldcatalyze the reaction of ROO∙ radicals [24ndash26]

3 Differential Oxygen Uptake Apparatus

Inhibited autoxidation kinetics can be determined by mon-itoring the disappearance of reactants usually O2 or theformation of oxidation products (such as hydroperoxides orconjugated dienes) [27] alternatively it can be monitored bytracking the cooxidation of an easily detectable probe [28]Oxygen consumption experiments need to be performedin a closed system and the reaction can be monitored byone of several methods such as by a polarographic probe[29] fluorescence quenching [30] or a pressure gauge [31]

The best-established method for bioorganic studies in whichaccurate kinetic measurements are required is based on theuse of a differential pressure transducer [22] The apparatusshown in Figure 2 consists of two identical reaction vessels(reference and sample) connected through high performanceliquid chromatography (HPLC) tubing to the opposite sidesof the membrane of a variable reluctance differential pressuretransducer [32]

The equilibration or the isolation of the two channelsis made possible by a three-way valve and all equipmentis placed in a thermostatic bath A mixture containing theoxidizable substrate (typically from 15 to 75 volvol) andthe initiator in a suitable high-boiling solvent (typicallychlorobenzene or acetonitrile) are loaded in both reactionvesselsThe reference flask also contains a large excess (1mM)of a very good antioxidant (often the 120572-tocopherol ana-logue 22578-pentamethyl-6-chromanol) to completely stopautoxidation (except the initiation step) Solutions are contin-uously stirred to ensure equilibration with air inside the sys-tem at the temperature of the experiment (30ndash50∘C) and aftera fewminutes the 3-way valve is closed and the O2 consump-tion during the uninhibited autoxidation is recorded Whena constant consumption is reached the 3-way valve is openedand a concentrated solution of antioxidant is rapidly injectedinto the sample vessel bymeans of amicrosyringe through theopening of a glass tap then the 3-way valve is closed againFrom this point on the recording of the effect of the antioxi-dant on the autoxidation starts A typical plot of an autoxida-tion measured in this way is reported in Figure 3 where thetime-point of antioxidant injection is marked by an arrow

The response of the sensor (in mVV) is transformedin oxygen consumption (M) by using a conversion factorwhich is determined in separate calibration experiments Cal-ibration consists in performing autoxidation of an oxidizablesubstrate with known oxidizability (ie styrene) inhibited bythe reference antioxidant 22578-pentamethyl-6-chromanol(PMHC) whose stoichiometry of radical trapping is 2


Syringe

AD conversion

HPLC tubing

Magnetic stirrers

Reference flask Sample flask

Glass tap

Thermostated bath

Pressure transducer

3-way valve

30∘C

Time (s)O

xyge

n

Low-volume tee

Figure 2 Differential oxygen uptake apparatus for autoxidation kinetics Reprinted with permission from reference [32]

Time (s)

Diff

pre

ssur

e (m

VV

)

minus12

minus10

minus8

minus6

minus4

minus2

0

AH

0 500 1000 1500 2000 2500 3000 3500

Figure 3 Differential pressure profile measured during the autoxi-dation of styrene inhibited by PMHC at 30∘C in chlorobenzeneThearrow indicates the injection of the antioxidant Reaction conditionswere styrene 43M AIBN 005M PMHC 50120583M

The induction period length (120591) provides 119877119894 (5) while theexpected 119889[O2]119889119905 for the uninhibited autoxidation can becalculated from the known oxidizability of the substrate byusing (1) The conversion factor (119886) is therefore defined as119889[O2]119889119905 = 119886 times 119889 (mVV)119889119905

It should be noted that this instrumental set-up ensuresthat the pressure effects given by N2 evolution which isdue to azo-initiator decomposition are compensated by thepresence of the same amount of azo-initiator in the referenceflask For the same reason also O2 consumption by initiatingI∙ radicals is compensated and additive term (+119877119894) in (1)

and (4) has to be skipped when analyzing O2 slopes Theinstrument described above has been used in the last yearsin our laboratory to develop among others new classes ofsulfur-containing heterocyclic antioxidants [33 34] novelpyridine and pyrimidine radical-trapping derivatives [13]and to study the reactions with peroxyl radicals of naturalpolyphenols [20]

4 Autoxidation Kinetics in Water Solution

A considerable number of chain-breaking antioxidants iswater-soluble but little of their chemistry with peroxylradicals is known in water because of the limited availabilityof practical investigation methods While oximetry mayrepresent a promising way to address this lack of know-ledge as a matter of facts this method is limited by the poorwater-solubility of typical oxidizable substrates Previousefforts consisted in incorporating the substrate into micellesor liposomes [29 31 35] Unfortunately in heterogeneoussystems the observed rate constants reflect the rate of reac-tants exchange between the particles In these systems thereactivity of different biomolecules with peroxyl radicalsmostly depends on their lipophilicity [36] We have recentlyreported that tetrahydrofuran (THF) presents most of theideal features for an oxidizable substrate to be used in aqueoussolutions as it is miscible with buffered water it is readilyavailable in high purity (distillation can be used to removethe stabilizer) and it undergoes reasonably rapid autoxidation[37] Autoxidation of THF proceeds by hydrogen abstractionfrom 120572 position as exemplified in Scheme 3 and the reactioncan be conveniently initiated by the water-soluble azo-initiator AAPH

The oxidizability of THF is independent from pH and119896119901 and 2119896119905 in water were measured by us as 48 and


O H O H

O

O H

O+ +

O HH

O HO H

O OH

+O H

O

O

HOCOOH

O H

O OH

OCOOH

O∙

kinh

kp

∙

∙

O∙

O∙

+O∙

+ O2

Scheme 3 Propagation steps of the autoxidation of tetrahydrofuran in water and inhibition by Trolox

Table 2 Values of 119896inh (Mminus1sminus1) in buffered water and in organic solvents of different polarity

Antioxidant Water pH 21 Water pH 74 PhCl MeCNTrolox (14 plusmn 03)times 105a (41 plusmn 07)times 105a 11times 106b (16 plusmn 02)times 105cPMHC (19 plusmn 03)times 105a (20 plusmn 03)times 105a 32times 106d 68times 105eDBHAf (33 plusmn 04)times 104a (32 plusmn 04)times 104a 11times 105a 25times 104eCatechol (3 plusmn 1)times 103a (7 plusmn 2)times 103a 55times 105g 25times 104daSee [37] bsee [38] csee [39] dsee [13] esee [24] f26-di-tert-butyl-4-methoxyphenol gsee [40]

66 times 107Mminus1sminus1 at 30∘C [37] This method allowed us tomeasure for the first time 119896inh values for antioxidants ofbiological relevance at various pH as reported in Table 2while the investigation of the deuterium kinetic isotope effect(DKIE) provided insight into the mechanism of proton-coupled electron transfer from phenols to peroxyl radicals

5 Kinetics Studies with Hydroperoxyl Radicals

Hydroperoxyl radicals (HOO∙) aremediators ofmany impor-tant (bio)chemical processes but their reactivity with pheno-lic antioxidants has often been neglected mainly because thisradical is hard to be monitored by common spectroscopicmethods [41] We have recently shown that autoxidation of14-cyclohexadiene in conjunction with oximetry performedby a differential pressure transducer represents an easy way tostudy the reaction of this radical It is known that under air14-cyclohexadiene undergoes autoxidation to benzene thepropagating radical being HOO∙ instead of an alkylperoxylradical (see Scheme 4) and that antioxidants can slow downthe process [15]

However the measure of 119896inh was precluded by thecomplex kinetics that were observed due to the unusualshape of the O2 consumption plots and to the strong solventdependence of 119896119901 and 2119896119905 (see Table 3)

We have recently demonstrated that the reason of the firstanomaly is the two-faced behavior of the HOO∙ radical thatcan both abstract a H-atom from the antioxidant and reducethe corresponding phenoxyl radical back to the starting phe-nol as indicated in Scheme 4Thanks to Gepasi software (seeSection 6) we could disentangle this complex kinetic behav-ior and obtain 119896inh values for the analogue of 120572-tocopherolPMHC (119896inh in Scheme 4) and the ratio between two possiblereactions between PMHC radical and HOO∙ (119896Reg119896Ter in

Table 3 Propagation (119896119901Mminus1sminus1) and termination (2119896119905 times 10minus6Mminus1sminus1) rate constants for the autoxidation of 14-cyclohexadiene indifferent solvents and corresponding rate constants for the reactionof PMHC with hydroperoxyl radicals (119896inhMminus1sminus1 and 119896Reg119896Ter) at30∘C

Solvent 119896119901 2119896119905 119896inhb (HOO∙) 119896Reg119896TerbCCl4 3800a 5600a (16 plusmn 04) times 107 99 plusmn 35PhCl 1400a 630a (16 plusmn 01) times 106 83 plusmn 05MeCN 350a 43a (68 plusmn 07) times 104 22 plusmn 10Dioxane 385b 63b (11 plusmn 01) times 105 52 plusmn 09THF 230b 13b (25 plusmn 05) times 104 05 plusmn 01Me2SO 160c 0017caSee [15] bsee [42] csee [43]

Scheme 4) which lead either to regeneration or to termi-nation (see Table 3) These experiments showed for the firsttime that theH-atomabstracting ability ofHOO∙ is decreasedby H-bond formation (S---HOO∙) with polar solvents (S)as the strength of this noncovalent interaction is weaker inthe transition state than in the reactants H-bond formationis also the reason why 119896119901 and 2119896119905 of 14-cyclohexadiene aresmaller in polar solvents than in nonpolar ones [42]

6 Complex Kinetics Analysis byUsing Gepasi Software

Extraction of kinetic information from oximetry studiesduring inhibited autoxidation is straightforward when theantioxidant and the oxidizable substrate are both ldquowellbehavedrdquo and obey (2)-(3) described above When this is nolonger true amore sophisticated analysis based on numericalfitting of kinetic data should be used by means of dedicated


HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +

Scheme 4 Autoxidation of 14-cyclohexadiene inhibited by theanalogue of 120572-tocopherol PMHC

software such as Gepasi which is freely available on the webat the developer site httpwwwgepasiorg This softwaresimulates the kinetics of (bio)chemical reactions and withthe help of numerical algorithms enables us to fit modelsto data and optimize any function of the model Modelbuilding is straightforward as it only requires the list ofthe reactions that take place in the system [44] In thissection we report an example of the application of Gepasi tothe interpretation of the complex autoxidation experimentscollected during the study the antioxidant activity of anallicin (1) analogue isolated fromPetiveria alliacea (S)-benzylphenylmethanethiosulfinate (2) (Scheme 5)

As shown in Figure 4 the rate of oxygen consumptionand the length of the inhibition period were not related to theantioxidant concentration as predicted instead by (4) and (5)[45]

However in a series of studies aimed at clarifying theantioxidant activity of garlic-derived thiosulfinates similar toallicin (1) it was discovered that the active molecule ableto trap peroxyl radical was not (1) itself but the sulfenicacid that derives from its decomposition by Cope elimination

(Scheme 5) [46 47] Therefore we hypothesized that theodd kinetic traces depended on a preliminary equilibrium toyield the ldquotruerdquo antioxidant species By using the reporteddata for the decomposition of (2) the autoxidation curveswere analyzed by the Gepasi software which provided theunknown 119896inh constant for the sulfenic acid (3) as (28plusmn 12)times107Mminus1sminus1 (at 30∘C) that is about 10 times larger than that of120572-tocopherol [38 45] Interestingly Gepasi software allowedus to calculate the concentrations of (2) and (3) during thecourse of the reaction as reported in Figure 4(b) At the endof the inhibition period (which lasted about 8000 seconds)more than 50 of (2) was still present in solution whilethe concentration of the ldquoactiverdquo sulfenic acid (3) droppedto zero This showed that differently to ldquowell-behavedrdquoantioxidants the inhibition period did not end when allantioxidant was depleted but when the production rate of theactive specie (sulfenic acid) by the thiosulfinate is insufficientto keep the pace and compete effectively with propagation

7 Analysis of Nanoantioxidants

Nanoantioxidants are a class of emerging materials that arebased on nanoparticles having intrinsic radical scavengingactivity or that are functionalized with antioxidants The effi-cacy of nanoantioxidants can easily be assessed by oximetryusing a differential pressure transducer as this method is notinfluenced by the color of the solution or by the presenceof suspended particles [48] As an example we will describethe study of CoNPsTOH magnetic nanoparticles based ona graphite-coated cobalt core having the diameter of 50 nm(CoNPs) [49] that were functionalized by click chemistrywith a phenolic antioxidant derived from Trolox (TOH) [50]Their antioxidant activity was investigated and compared tothat of nanoparticles lacking the pendant antioxidant andto TOH alone as depicted in Figure 5(a) Results showedthat such nanoantioxidants were very active in inhibitingthe autoxidation of styrene with 119896inh values higher thanTOH However the length of the induction period wasshorter than expected presumably because the proximityof the phenolic moieties favors the self-recombination oftwo phenoxyl radicals instead of the quenching of a secondperoxyl radicalThe photographs in Figure 5(b) show that themagnetic nanoantioxidants stick on the stir bar while afterthe stirring is turned on they are well dispersed in solution

Two additional examples of nanoantioxidants wererecently studied by oximetry methods They both consistedin halloysite nanotubes (NHT) [51] that had been graftedor grafted and loaded with phenols In the first study thenatural antioxidant curcumin was grafted on the surfaceof halloysite nanotubes by thiol-disulfide chemistry (HNT-cur) with the aim of obtaining a prodrug with a controlledcurcumin release on dependence of both intracellular glu-tathione (GSH) and pH conditions HNT-cur showed goodantioxidant activity toward cumene autoxidation with 119896inhcomparable to that of curcumin alone (17 times 104Mminus1sminus1 inPhCl) Similar to CoNPsTOH the stoichiometric coefficientwas shorter than that of curcumin [52] The second exampleis represented by a synergic nanoantioxidant obtained by dif-ferent functionalization of the external surface and the inner


ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+

Scheme 5 Formation of the active radical-trapping sulfenic acid (3) from decomposition of benzyl phenylmethanethiosulfinate (2) ananalogue of allicin (1)

minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)

Figure 4 Analysis of autoxidation traces by the Gepasi software (a) Experimental (solid lines) and simulated (dotted lines) O2 consumptionduring the autoxidation of cumene in the presence of variable amounts of thiosulfinate (2) (b) Variations of the concentration of thiosulfinate(2) and sulfenic acid (3) obtained by simulation based on kinetic data determined in (a) at the concentration of 60120583m Reprinted withpermission from [45]

Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)

Figure 5 (a) Oxygen consumption during the autoxidation of styrene (43M) in PhCN initiated by AIBN (25mm) at 30∘Cwithout inhibitor38 120583M of TOH or with 015mgmLminus1 of various CoNPs without the phenolic moiety and CoNPsTOH (corresponding to a concentration ofTOH groups of 144 120583M) 119877119894 = 36 times 10minus9Msminus1 (b) Photographs of the reaction vessels of the pressure transducer apparatus containing theCoNPs in the absence (left) and in the presence (right) of magnetic stirring Reprinted with permission from [50]


O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox

HNT-Trolox HNT-Troloxque

NH

Figure 6 Schematic representation of quercetin loading into HNT-Trolox Reproduced from [39] with permission from the Royal Society ofChemistry

minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)

Figure 7 Inhibition of the autoxidation of cumene in acetonitrile (a) or chlorobenzene (b) by 01mgmLminus1 of pristine HNT (A) HNTQue(B) HNT-Trolox (C) and HNT-TroloxQue (D) Reproduced from [39] with permission from the Royal Society of Chemistry

lumen of halloysite nanotubes Trolox a mimic of natural120572-tocopherol was selectively grafted on the HNT externalsurface while quercetin a natural polyphenolic antioxidantwas loaded into the inner lumen to afford HNT-TroloxQuea bifunctional nanoantioxidant (see Figure 6) [39]

Autoxidation studies in cumene reported in Figure 7showed that NHT functionalized by Trolox (HNT-Trolox)had a good antioxidant activity (lines (C)) while quercetinloaded into HNT (HNTQue) had a poor performance(traces (B)) due to its slow kinetic of release

Interestingly the presence of both grafted Trolox andloaded quercetin in HNT-TroloxQue improved the over-all antioxidant performance more than expected from anadditive contribution of the two antioxidants as it can beinferred by comparing traces (B) (C) and (D) in Figure 7Accurate kinetic analysis by the oximetry method allowed usto conclude that the synergy between Trolox and quercetin

was due to the regeneration of Trolox from its radical byquercetin occurring at the nanotube external surface [39]

8 Challenges and Perspectives

The main limitations of the technique described herein arelow productivity (one sample at time) long reaction times(gt2 h for a run) and relatively large volumes of reagents(05ndash4mL) In our experience in research routine thesedrawbacks are largely overcome by many advantages whichare among others the strength of the instrumentation the lowrunning costs and the wide range of chemical compatibility(from fully organic to aqueous solvents) which make differ-ential uptake devices more versatile than O2 optical sensors

Future improvements should be directed toward themin-iaturization of the reaction vessels and toward the possibilityof running several samples at one time Such improvements


have been achieved in case of O2-optical sensing by usingmicrofluidic devices [53] Besides the quest of novel bio-logically relevant oxidizable substrates is an active researchfield that should be more deeply investigated In this contextthe possibilities of easily performing experiments in waterenvironment (see Section 4) andworkingwithHOO∙ radicals(see Section 5) are just two examples of the capabilities of thedifferential uptake apparatus

9 Conclusion

The differential oxygen uptake apparatus described hereinrepresents a powerful and cost-effective way to obtain antiox-idant activity from inhibited autoxidation studies Amongthe many assays available in the literature the measurementof O2 consumption during the inhibited autoxidation of astandard substrate is one of the most reliable and predictivemethods especially for structure-activity relationship studies[54ndash61] These experiments provide the rate constants of thereaction between antioxidants with peroxyl radicals (ROO∙)which are involved in the propagation of radical damage [62]The versatility of this method allows one to measure theactivity of awide variety of antioxidants (natural polyphenolsnanoantioxidants bioactive sulfides etc) in organic andmixed water-organic solvents We hope that this review willinspire researchers working in the field of antioxidants toadopt this powerful technique for their studies

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by a grant to Luca ValgimigliAndrea Baschieri and Riccardo Amorati from the ItalianMIUR (PRIN 2010-2011 2010PFLRJR PROxi Project) LucaValgimigli and Riccardo Amorati acknowledge funding fromthe University of Bologna (FARB Project FFBO123154)

References

[1] K U Ingold ldquoPeroxy radicalsrdquo Accounts of Chemical Researchvol 2 no 1 pp 1ndash9 1969

[2] U Neuenschwander and K F Jensen ldquoOlefin autoxidation inflowrdquo Industrial and Engineering Chemistry Research vol 53 no2 pp 601ndash608 2014

[3] H Yin L Xu and N A Porter ldquoFree radical lipid peroxidationmechanisms and analysisrdquoChemical Reviews vol 111 no 10 pp5944ndash5972 2011

[4] K U Ingold andD A Pratt ldquoAdvances in radical-trapping anti-oxidant chemistry in the 21st century a kinetics and mecha-nisms perspectiverdquo Chemical Reviews vol 114 no 18 pp 9022ndash9046 2014

[5] J M C Gutteridge and B Halliwell ldquoAntioxidants moleculesmedicines and mythsrdquo Biochemical and Biophysical ResearchCommunications vol 393 no 4 pp 561ndash564 2010

[6] V Roginsky and E A Lissi ldquoReview of methods to determinechain-breaking antioxidant activity in foodrdquo Food Chemistryvol 92 no 2 pp 235ndash254 2005

[7] M Takashima M Horie M Shichiri Y Hagihara Y Yoshidaand E Niki ldquoAssessment of antioxidant capacity for scavengingfree radicals in vitro a rational basis and practical applicationrdquoFree Radical Biology and Medicine vol 52 no 7 pp 1242ndash12522012

[8] R Amorati and L Valgimigli ldquoAdvantages and limitationsof common testing methods for antioxidantsrdquo Free RadicalResearch vol 49 no 5 pp 633ndash649 2015

[9] T A Dix and J Aikens ldquoMechanisms and biological relevanceof lipid peroxidation initiationrdquo Chemical Research in Toxicol-ogy vol 6 no 1 pp 2ndash18 1993

[10] M Lucarini and G F Pedulli ldquoFree radical intermediates inthe inhibition of the autoxidation reactionrdquo Chemical SocietyReviews vol 39 no 6 pp 2106ndash2119 2010

[11] D A Pratt andN A Porter ldquoRole of hyperconjugation in deter-mining carbon-oxygen bond dissociation enthalpies in alkyl-peroxyl radicalsrdquo Organic Letters vol 5 no 4 pp 387ndash3902003

[12] J A Howard and K U Ingold ldquoAbsolute rate constants for hyd-rocarbon autoxidation VI Alkyl aromatic and olefinic hyd-rocarbonsrdquo Canadian Journal of Chemistry vol 45 no 8 pp793ndash802 1967

[13] L Valgimigli D Bartolomei R Amorati et al ldquo3-Pyridinolsand 5-pyrimidinols tailor-made for use in synergistic radical-trapping co-antioxidant systemsrdquo Beilstein Journal of OrganicChemistry vol 9 no 1 pp 2781ndash2792 2013

[14] J A Howard and K U Ingold ldquoAbsolute rate constants for hyd-rocarbon autoxidation XVII The oxidation of some cyclicethersrdquoCanadian Journal of Chemistry vol 47 no 20 pp 3809ndash3815 1969

[15] J A Howard and K U Ingold ldquoAbsolute rate constants for hyd-rocarbon autoxidation V The hydroperoxy radical in chainpropagation and terminationrdquo Canadian Journal of Chemistryvol 45 no 8 pp 785ndash792 1967

[16] G E Zaikov J A Howard and K U Ingold ldquoAbsolute rate con-stants for hydrocarbon autoxidation XIII Aldehydes photo-oxidation co-oxidation and inhibitionrdquo Canadian Journal ofChemistry vol 47 no 16 pp 3017ndash3029 1969

[17] L Melone S Prosperini G Ercole N Pastori and C Punta ldquoIsit possible to implement N-hydroxyphthalimide homogeneouscatalysis for industrial applications A case study of cumeneaerobic oxidationrdquo Journal of Chemical Technology and Biotech-nology vol 89 no 9 pp 1370ndash1378 2014

[18] F Recupero and C Punta ldquoFree radical functionalizationof organic compounds catalyzed by N-hydroxyphthalimiderdquoChemical Reviews vol 107 no 9 pp 3800ndash3842 2007

[19] R Amorati and G F Pedulli ldquoDo garlic-derived allyl sulfidesscavenge peroxyl radicalsrdquoOrganic amp Biomolecular Chemistryvol 6 no 6 pp 1103ndash1107 2008

[20] R Amorati J Zotova A Baschieri and L Valgimigli ldquoAntioxi-dant activity ofmagnolol and honokiol kinetic andmechanisticinvestigations of their reaction with peroxyl radicalsrdquo TheJournal of Organic Chemistry vol 80 no 21 pp 10651ndash106592015

[21] V Roginsky T Barsukova D Loshadkin and E Pliss ldquoSub-stituted p-hydroquinones as inhibitors of lipid peroxidationrdquoChemistry and Physics of Lipids vol 125 no 1 pp 49ndash58 2003

[22] G W Burton and K U Ingold ldquoAutoxidation of biologicalmolecules 1 The antioxidant activity of vitamin E and relatedchain-breaking phenolic antioxidants in vitrordquo Journal of theAmerican Chemical Society vol 103 no 21 pp 6472ndash6477 1981


[23] D A Pratt K A Tallman and N A Porter ldquoFree radical oxi-dation of polyunsaturated lipids new mechanistic insights andthe development of peroxyl radical clocksrdquo Accounts of Chem-ical Research vol 44 no 6 pp 458ndash467 2011

[24] L Valgimigli R Amorati S Petrucci et al ldquoUnexpected acidcatalysis in reactions of peroxyl radicals with phenolsrdquo Ange-wandte ChemiemdashInternational Edition vol 48 no 44 pp8348ndash8351 2009

[25] R Amorati L Valgimigli G F Pedulli S A Grabovskiy NN Kabalrsquonova and C Chatgilialoglu ldquoBase-promoted reactionof 5-hydroxyuracil derivatives with peroxyl radicalsrdquo OrganicLetters vol 12 no 18 pp 4130ndash4133 2010

[26] E A Haidasz D Meng R Amorati et al ldquoAcid is key to theradical-trapping antioxidant activity of nitroxidesrdquo Journal ofthe American Chemical Society vol 138 no 16 pp 5290ndash52982016

[27] J J Hanthorn E Haidasz P Gebhardt and D A Pratt ldquoAversatile fluorescence approach to kinetic studies of hydrocar-bon autoxidations and their inhibition by radical-trapping anti-oxidantsrdquo Chemical Communications vol 48 no 81 pp 10141ndash10143 2012

[28] E A Haidasz A T M Van Kessel and D A Pratt ldquoA con-tinuous visible light spectrophotometric approach to accuratelydetermine the reactivity of radical-trapping antioxidantsrdquo Jour-nal of Organic Chemistry vol 81 no 3 pp 737ndash744 2016

[29] K Jodko-Piorecka and G Litwinienko ldquoAntioxidant activity ofdopamine and L-DOPA in lipid micelles and their cooperationwith an analogue of 120572-tocopherolrdquo Free Radical Biology andMedicine vol 83 pp 1ndash11 2015

[30] X H Kai V YWaisundara M Timmins et al ldquoHigh-through-put quantitation of peroxyl radical scavenging capacity in bulkoilsrdquo Journal of Agricultural and Food Chemistry vol 54 no 15pp 5299ndash5305 2006

[31] E AMengele I G Plashchina andO T Kasaikina ldquoKinetics oflecithin oxidation in liposomal aqueous solutionsrdquoColloid Jour-nal vol 73 no 6 pp 815ndash821 2011

[32] L Valgimigli and D A Pratt ldquoAntioxidants in chemistry andbiologyrdquo in Encyclopedia of Radicals in Chemistry Biology andMaterials C Chatgilialoglu andA Studer Eds vol 3 pp 1623ndash1677 John Wiley amp Sons Chichester UK 2012

[33] SMenichetti R Amorati VMeoni L Tofani G Caminati andC Viglianisi ldquoRole of noncovalent sulfursdot sdot sdotoxygen interactionsin phenoxyl radical stabilization synthesis of super tocopherol-like antioxidantsrdquoOrganic Letters vol 18 no 21 pp 5464ndash54672016

[34] C Viglianisi R Amorati L Di Pietro and S Menichetti ldquoAstraightforward route to potent phenolic chain-breaking anti-oxidants by acidminuspromoted transposition of 1 4-benzo[b]oxa-thiines to dihydrobenzo[b]thiophenesrdquo Chemistry vol 21 no46 pp 16639ndash16645 2015

[35] M Musialik M Kita and G Litwinienko ldquoInitiation of lipidautoxidation by ABAP at pH 4ndash10 in SDS micellesrdquo Organic ampBiomolecular Chemistry vol 6 no 4 pp 677ndash681 2008

[36] L Castle and M J Perkins ldquoInhibition kinetics of chain-break-ing phenolic antioxidants in SDS micelles Evidence that inter-micellar diffusion rates may be rate-limiting for hydrophobicinhibitors such as alpha-tocopherolrdquo Journal of the AmericanChemical Society vol 108 no 20 pp 6381ndash6382 1986

[37] R Amorati A Baschieri G Morroni R Gambino and LValgimigli ldquoPeroxyl radical reactions in water solution a gymfor proton-coupled electron-transfer theoriesrdquo ChemistrymdashAEuropean Journal vol 22 no 23 pp 7924ndash7934 2016

[38] G W Burton T Doba E J Gabe et al ldquoAutoxidation of bio-logical molecules 4 Maximizing the antioxidant activity ofphenolsrdquo Journal of the American Chemical Society vol 107 no24 pp 7053ndash7065 1985

[39] MMassaro S Riela S Guernelli et al ldquoA synergic nanoantiox-idant based on covalently modified halloysite-trolox nanotubeswith intra-lumen loaded quercetinrdquo Journal of Materials Chem-istry B vol 4 no 13 pp 2229ndash2241 2016

[40] A Tarozzi M Bartolini L Piazzi et al ldquoFrom the dual functionlead AP2238 to AP2469 a multi-target-directed ligand for thetreatment of Alzheimerrsquos diseaserdquo Pharmacology Research ampPerspectives vol 2 no 2 Article ID e00023 2014

[41] Z Kozmer E Arany T Alapi E Takacs L Wojnarovits andA Dombi ldquoDetermination of the rate constant of hydroperoxylradical reaction with phenolrdquo Radiation Physics and Chemistryvol 102 pp 135ndash138 2014

[42] J Cedrowski G Litwinienko A Baschieri and R AmoratildquoHydroperoxyl radicals (HOO∙) vitamin E regeneration andH-bond effects on the hydrogen atom transferrdquo ChemistrymdashAEuropean Journal vol 22 no 46 pp 16441ndash16445 2016

[43] D T Sawyer M S McDowell and K S Yamaguchi ldquoReactivityof perhydroxyl (HOObul) with 14-cyclohexadiene (modelfor allylic groups in biomembranes)rdquo Chemical Research inToxicology vol 1 no 2 pp 97ndash100 1988

[44] P Mendes ldquoBiochemistry by numbers simulation of biochemi-cal pathways with Gepasi 3rdquo Trends in Biochemical Sciences vol22 no 9 pp 361ndash363 1997

[45] RAmorati P T Lynett LValgimigli andDA Pratt ldquoThe reac-tion of sulfenic acids with peroxyl radicals insights into theradical-trapping antioxidant activity of plant-derived thio-sulfinatesrdquo ChemistrymdashA European Journal vol 18 no 20 pp6370ndash6379 2012

[46] V Vaidya K U Ingold and D A Pratt ldquoGarlic source of theultimate antioxidantsmdashsulfenic acidsrdquo Angewandte ChemiemdashInternational Edition vol 48 no 1 pp 157ndash160 2009

[47] P T Lynett K Butts V Vaidya G E Garrett and D APratt ldquoThe mechanism of radical-trapping antioxidant activ-ity of plant-derived thiosulfinatesrdquo Organic and BiomolecularChemistry vol 9 no 9 pp 3320ndash3330 2011

[48] N L Pacioni V FilippenkoN Presseau and J C Scaiano ldquoOxi-dation of copper nanoparticles in water mechanistic insightsrevealed by oxygen uptake and spectroscopic methodsrdquo DaltonTransactions vol 42 no 16 pp 5832ndash5838 2013

[49] RNGrass E KAthanassiou andW J Stark ldquoCovalently func-tionalized cobalt nanoparticles as a platform for magnetic sepa-rations in organic synthesisrdquo Angewandte ChemiemdashInterna-tional Edition vol 46 no 26 pp 4909ndash4912 2007

[50] C Viglianisi V Di Pilla S Menichetti et al ldquoLinking an 120572-tocopherol derivative to cobalt (0) nanomagnets magneticallyresponsive antioxidants with superior radical trapping activityand reduced cytotoxicityrdquo ChemistrymdashA European Journal vol20 no 23 pp 6857ndash6860 2014

[51] M Du B Guo and D Jia ldquoNewly emerging applications ofhalloysite nanotubes a reviewrdquo Polymer International vol 59no 5 pp 574ndash582 2010

[52] M Massaro R Amorati G Cavallaro et al ldquoDirect chemicalgrafted curcumin on halloysite nanotubes as dual-responsiveprodrug for pharmacological applicationsrdquo Colloids and Sur-faces B Biointerfaces vol 140 pp 505ndash513 2016

[53] B Kuswandi Nuriman J Huskens and W Verboom ldquoOpticalsensing systems for microfluidic devices a reviewrdquo AnalyticaChimica Acta vol 601 no 2 pp 141ndash155 2007


[54] L R C Barclay M R Vinqvist K Mukai S Itoh and H Mori-moto ldquoChain-breaking phenolic antioxidants steric and elec-tronic effects in polyalkylchromanols tocopherol analogs hyd-roquinones and superior antioxidants of the polyalkylben-zochromanol and naphthofuran classrdquo Journal of OrganicChemistry vol 58 no 26 pp 7416ndash7420 1993

[55] S C Mojumdar D A Becker G A DiLabio J J Ley L R CBarclay and K U Ingold ldquoKinetic Studies on Stilbazulenyl-bis-nitrone (STAZN) a nonphenolic chain-breaking antioxidant insolution micelles and lipid membranesrdquo Journal of OrganicChemistry vol 69 no 9 pp 2929ndash2936 2004

[56] L R C Barclay and M R Vinqvist ldquoMembrane peroxidationinhibiting effects ofwater soluble antioxidants on phospholipidsof different charge typesrdquo Free Radical Biology amp Medicine vol16 no 6 pp 779ndash788 1994

[57] L R C Barclay C E Edwards and M R Vinqvist ldquoMediaeffects on antioxidant activities of phenols and catecholsrdquo Jour-nal of the American Chemical Society vol 121 no 26 pp 6226ndash6231 1999

[58] F Antunes L R C Barclay K U Ingold et al ldquoOn the anti-oxidant activity of melatoninrdquo Free Radical Biology and Med-icine vol 26 no 1-2 pp 117ndash128 1999

[59] G L Hatfield and L R C Barclay ldquoBilirubin as an antioxidantkinetic studies of the reaction of bilirubin with peroxyl radicalsin solution micelles and lipid bilayersrdquo Organic Letters vol 6no 10 pp 1539ndash1542 2004

[60] M Frenette P D MacLean L R C Barclay and J C ScaianoldquoRadically different antioxidants thermally generated carbon-centered radicals as chain-breaking antioxidantsrdquo Journal of theAmerican Chemical Society vol 128 no 51 pp 16432ndash164332006

[61] C Viglianisi M G Bartolozzi G F Pedulli R Amorati andS Menichetti ldquoOptimization of the antioxidant activity ofhydroxy-substituted 4-thiaflavanes a proof-of-concept studyrdquoChemistrymdashA European Journal vol 17 no 44 pp 12396ndash12404 2011

[62] P Mulder H-G Korth and K U Ingold ldquoWhy quantum-thermochemical calculationsmust be usedwith caution to indi-cate lsquoa promising lead antioxidantrsquordquoHelvetica Chimica Acta vol88 no 2 pp 370ndash374 2005

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


RHROOH

Nonradicalproducts

AH

IH

Initiator

PropagationInitiation Termination

I∙ + RH R∙ROO∙

ROO∙

ROOH + A∙

kinh

kp

kt

O2

Scheme 1 Chain reaction of autoxidation for a substrate (RH) andmode of interference by chain-breaking antioxidants (AH)

the analysis of O2-consumption traces We show the utility ofthis instrumentation in the bioorganic laboratory with regardto the recent advances in this field ranging from the study ofnatural antioxidants in biomimetic systems to the evaluationof novel nanoantioxidants

2 How to Determine the Antioxidant Activityfrom Inhibited Autoxidation

Autoxidation follows a free-radical chain mechanismdepicted in its simplest form in Scheme 1

Autoxidation can in principle be initiated by any reac-tion that generates free radicalsThemost common initiatingsystems with practical relevance are the catalytic decom-position of hydroperoxides by transition metals ions (suchas Fe2+ and Cu+) and the interaction of UV-vis light withphotoinitiators (such as ketones and peroxides) Howeverbecause hydroperoxides (and peroxides) and ketones are alsoproducts of the autoxidation their accumulation may leadto autocatalytic behavior consisting in an increasing rate ofreaction with time [8] When a controlled rate of initiation isdesired thermal decomposition of azo-initiator is preferredThe use of azo-initiators such as the lipid-soluble AIBN(221015840-azobis-isobutyronitrile) or water-soluble AAPH (221015840-azobis(2-amidinopropane) dihydrochloride) is much morepractical than metal catalysis or photoinitiation becausethe homolytic decomposition of azo-initiators proceeds atconstant rate at a given temperature during the entire courseof autoxidation thereby providing a constant rate of initiation[10] Following the mechanism reported in Scheme 1 theinitiating radical (I∙) abstracts a H-atom from the substrategenerating a carbon-centered (alkyl) radical R∙ which reactswith O2 to form a peroxyl radical (ROO∙) Except in somespecific cases [11] this last reaction is very efficient [10] Forthis reason in the presence of atmospheric oxygen virtuallyall R∙ are converted to ROO∙ and peroxyl radical chemistrydominates autoxidation kinetics The propagation can eitherconsist of H-atom transfer (ROO∙+RHrarr ROOH+R∙) or ofaddition to a C=Cdouble bond (ROO∙ +X=Y rarr ROOX-Y∙where X and Y are substituted C atoms) Termination eventsconsist mainly in the radical-radical self-quenching of per-oxyl radicals to give nonradical products [1] If propagationis fast enough the overall process becomes a chain reactionand after the initiation event several propagation steps

Table 1 Rate constants for the autoxidation of selected organicsubstrates in PhCl at 303K

Substrate 119896119901 [Mminus1sminus1] 2119896119905 [Mminus1sminus1] 119896119901(2119896119905)12Methyl oleatea 088 10 times 106 88 times 10minus4Methyl linoleatea 62 88 times 106 0021Toluenea 024 30 times 108 14 times 10minus5Styrenea 41 42 times 107 63 times 10minus3Cumeneb 032 46 times 104 15 times 10minus3Tetrahydrofurancd 43 62 times 107 17 times 10minus414-Cyclohexadienee 1400 13 times 109 0038Heptanalf g 2800 54 times 107 038aSee [12] bsee [13] csee [14] dneat ethe propagating radical is hydroperoxyl(HOO∙) see [15] f the propagating radicals are an acylperoxyl see Ref [16]gat 273 K

can take place before the chain reaction is terminated Bysolving the kinetic equations that describe the individualreactions depicted in Scheme 1 and applying the steady stateapproximation oxygen consumption during the autoxidationof a generic substrate RH is described by (1) where 119896119901 and2119896119905 are respectively the propagation and termination rateconstant for the autoxidation of the oxidizable substrate and119877119894 is the initiation rate

minus119889 [O2]119889119905 =119896119901radic2119896119905 [RH] radic119877119894 + 119877119894 (1)

Since the autoxidation rate depends on the ratio 119896119901(2119896119905)12 this quantity is commonly referred to as the oxidiz-ability of the substrate Some examples of typical oxidizablesubstrates are given in Table 1 together with their respective119896119901 2119896119905 and 119896119901(2119896119905)12 values From the values reported inTable 1 it is evident that the oxidizability of organicmoleculesvaries considerably from very low values for saturatedhydrocarbons to 1000-fold higher values for polyunsaturatedhydrocarbons or for aldehydes

Once the rate constants 119896119901 and 2119896119905 are known along with119877119894 values the effect of the addition of any modifier to theautoxidation can be studied Some compounds can accele-rate autoxidation for example by providing better chaintransfer than peroxyl radicals as observed in the case of N-hydroxyphthalimide (NHPI) class of compounds

It was shown that after the addition ofNHPI to the autox-idation of isopropylbenzene (cumene) the O2 consumptionis increased because PINO radicals react with RH fasterthan ROO∙ NHPI and related molecules can be used asmetal-free catalysts for the functionalization of hydrocarbons(Scheme 2) [17 18] Addition of easily oxidizable compoundsmay either decrease or increase the rate of O2 consumptionby cooxidizing with the substrate Some of these systemshave been investigated by us in the case of garlic sulfides andestragole [19 20]

However arguably the most important class of com-pounds that are used to modify the rate of autoxidation isantioxidants which are used byman and nature to slow downthe degradation of organicmaterials underO2 [1] Depending


R∙

ROO∙

RH

N N

O

OO

OPINO

ROOH

NHPI

OHO2O∙








119877119894 = 119899 [AH]120591 (5)





119905120591)]

(6)




Time (s)

minus15

minus10

minus05

00

(A)(B)

(C)

(D)minusΔ[O

2]

(mM

)

0 500 1000 1500 2000

휏

(a)

01

02

03

04

05

06

07

Δ[O

2]

(mM

)


0 1 2 3 4 5

(b)



119889 [O2]0119889119905119889 [1198742]inh119889119905 minus119889 [1198742]inh119889119905119889 [1198742]0119889119905 =

119899119896inh [AH]radic2119896119905119877119894 (7)








Syringe

AD conversion

HPLC tubing

Magnetic stirrers


Glass tap

Thermostated bath

Pressure transducer

3-way valve

30∘C

Time (s)O

xyge

n

Low-volume tee


Time (s)

Diff

pre

ssur

e (m

VV

)

minus12

minus10

minus8

minus6

minus4

minus2

0

AH

0 500 1000 1500 2000 2500 3000 3500









O H O H

O

O H

O+ +

O HH

O HO H

O OH

+O H

O

O

HOCOOH

O H

O OH

OCOOH

O∙

kinh

kp

∙

∙

O∙

O∙

+O∙

+ O2















HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +










ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


R∙

ROO∙

RH

N N

O

OO

OPINO

ROOH

NHPI

OHO2O∙








119877119894 = 119899 [AH]120591 (5)





119905120591)]

(6)




Time (s)

minus15

minus10

minus05

00

(A)(B)

(C)

(D)minusΔ[O

2]

(mM

)

0 500 1000 1500 2000

휏

(a)

01

02

03

04

05

06

07

Δ[O

2]

(mM

)


0 1 2 3 4 5

(b)



119889 [O2]0119889119905119889 [1198742]inh119889119905 minus119889 [1198742]inh119889119905119889 [1198742]0119889119905 =

119899119896inh [AH]radic2119896119905119877119894 (7)








Syringe

AD conversion

HPLC tubing

Magnetic stirrers


Glass tap

Thermostated bath

Pressure transducer

3-way valve

30∘C

Time (s)O

xyge

n

Low-volume tee


Time (s)

Diff

pre

ssur

e (m

VV

)

minus12

minus10

minus8

minus6

minus4

minus2

0

AH

0 500 1000 1500 2000 2500 3000 3500









O H O H

O

O H

O+ +

O HH

O HO H

O OH

+O H

O

O

HOCOOH

O H

O OH

OCOOH

O∙

kinh

kp

∙

∙

O∙

O∙

+O∙

+ O2















HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +










ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Time (s)

minus15

minus10

minus05

00

(A)(B)

(C)

(D)minusΔ[O

2]

(mM

)

0 500 1000 1500 2000

휏

(a)

01

02

03

04

05

06

07

Δ[O

2]

(mM

)


0 1 2 3 4 5

(b)



119889 [O2]0119889119905119889 [1198742]inh119889119905 minus119889 [1198742]inh119889119905119889 [1198742]0119889119905 =

119899119896inh [AH]radic2119896119905119877119894 (7)








Syringe

AD conversion

HPLC tubing

Magnetic stirrers


Glass tap

Thermostated bath

Pressure transducer

3-way valve

30∘C

Time (s)O

xyge

n

Low-volume tee


Time (s)

Diff

pre

ssur

e (m

VV

)

minus12

minus10

minus8

minus6

minus4

minus2

0

AH

0 500 1000 1500 2000 2500 3000 3500









O H O H

O

O H

O+ +

O HH

O HO H

O OH

+O H

O

O

HOCOOH

O H

O OH

OCOOH

O∙

kinh

kp

∙

∙

O∙

O∙

+O∙

+ O2















HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +










ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Syringe

AD conversion

HPLC tubing

Magnetic stirrers


Glass tap

Thermostated bath

Pressure transducer

3-way valve

30∘C

Time (s)O

xyge

n

Low-volume tee


Time (s)

Diff

pre

ssur

e (m

VV

)

minus12

minus10

minus8

minus6

minus4

minus2

0

AH

0 500 1000 1500 2000 2500 3000 3500









O H O H

O

O H

O+ +

O HH

O HO H

O OH

+O H

O

O

HOCOOH

O H

O OH

OCOOH

O∙

kinh

kp

∙

∙

O∙

O∙

+O∙

+ O2















HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +










ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


O H O H

O

O H

O+ +

O HH

O HO H

O OH

+O H

O

O

HOCOOH

O H

O OH

OCOOH

O∙

kinh

kp

∙

∙

O∙

O∙

+O∙

+ O2















HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +










ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


HOOH +

AIBN

+ +

CN

CNCOOHCN

O

O

O

OOH

O

HO

O

HO

(PMHC)

O

+ O2

+ O2

kinh

kTer

kReg

∙

O∙

O2

∙

∙

H3C

H3CH3C

CH3

CH3CH3

COO∙

COO∙

OO∙

HOO∙+

HOO∙ +

H2O2 + O2

∙O

2HOO∙

HOO∙+

H2O2+

HOO∙ +










ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


ROO∙

O∙

kinh

O

O

OS

SS

SS

SSAllicin (1)

(2) (3)

H

ROOH

+


minus35

minus30

minus25

minus20

minus15

minus10

minus05

00

05

Oxy

gen

cons

umpt

ionminusΔ[O

2](m

M)

Time (s)

0휇M

40휇M 60휇M

0 4000 8000 12000 16000

(a)

0

10

20

30

40

50

60

70

00

02

04

06

08

10

Time (s)

[PhC

H2S

OH

](휇

M)

[2](휇

M)

0 4000 8000 12000 16000

(b)


Time (s)0 2000 4000 6000 8000

minus20

minus15

minus10

minus05

00

No inhibitor

TOH

minusΔ[O

2]

(mM

)

Co

Co

Co

Co

5

9

NN N

NNN

N3

OO

O

HO

(a) (b)



O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


O

O

OHOH

OHOH

HOO

OOO Si

OMe

OH

QueHNT-Trolox


NH


minus4

minus3

minus2

minus1

0

1

(A)

(B)

(C) (D)

Time (103 s)0 5 10 15 20 25 30 35

minusΔ[O

2]

(mM

)

(a)

minus05

minus04

minus03

minus02

minus01

00

01minusΔ[O

2]

(mM

)

Time (103 s)0 5 10 15 20 25 30

(A)(B) (C)

(D)

(b)











9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



9 Conclusion




Acknowledgments


References










































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

measuring antioxidant activity in bioorganic...

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