Free Radical Research

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Methods to Measure the Antioxidant Activity in

Plant Material. A Comparative Discussion

Marino B. Arnao, Antonio Cano & Manuel Acosta

To cite this article: Marino B. Arnao, Antonio Cano & Manuel Acosta (1999) Methods to
Measure the Antioxidant Activity in Plant Material. A Comparative Discussion, Free Radical
Research, 31:sup1, 89-96, DOI: 10.1080/10715769900301371

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 fn·e R11cl . R1•s ., Vol. 3 1, pp. 589- 96 (' 1999 OPA (Overseas Publis hers Associa tion) N.V.
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Methods to Measure the Antioxidant Activity

in Plant Material. A Comparative Discussion
MARINO B. ARNAO*, ANTON IO ANO and MANUEL A OSTA

Depn rtmc111 of Pln11l Biology (Pin11l Physiology), U11iversit y of M11rcin, 30100 M11rcia, Spn i11

(Received 20 November 1998; 111 revised form 2 j11ly 1999)

Free Radical Research 1999.31:89-96.

Method s to d e termine total antioxid a nt ac tivity (TAA) INTRODUCTION

are gene rally based o n the inhibition of certa in reactions
by the presence of a ntioxid a nts. The most wid ely used
method s a re those tha t involve the gene ratio n of radi a l Tn recent yea rs, a group o f vitamins has aroused
compound s, and it is the presence of a nti oxid a nts tha t special inter st for th eir ability to protec t aga inst:
d ete rmin es the d isa ppeam nce of these radicals. Stmtc- card iovascul ar diseases, a ncer, ata rac ts, neuro-
g ics for m easuring TAA a rc discussed , pa rti cula rl y the
diffe rent method ological a nd ins trum ental a pproa hes logica l dysfunction s, defici encies in immune
used . In additio n, our own method s a rc prese nted in re ponse, age-related probl ms, etc. These are
ord e r to facilita te a nd s peed u p the mani p ula tio n of th e so-ca ll ed a ntiox idant vitamins, w hi ch includ e
biologica l m a terial. The va lues of TAA by diffe rent
compou nds a re presented a nd compared . vitamin E (cHocopherol), vitamin A (retinoid s)
and vita min C (ascorbi c acid ). Also includ ed in
Keywords: Anti ox id ant activity, method s, ABTS, radica ls, the g roup is the compound /1-ca rot ne (kriown as
ascorbic acid, phenoli cs pro-vitamin A), the potential precursor of vitamin
A. Beneficia l effec ts have been illustrated by
Abbrevintio11s: AAPH, 2,2'-azobis(2-a midin o pro pa ne) dih y- means of three types of experimental ev idence:
d roc hlorid e; ABAP, 2,2'-azobis(2-a m id o pro pa ne) hydro-
chlorid e; ABT , 2,2'-azino-bis-(3-ethylbenzthi azolinc-6-
biochemica l studies (on DNA, proteins a nd
su lfo nic ac id ); BHA, Butyla ted hydroxyani sole; BHT, lipid s); epid emiologica l stt1di es a nd intervention
Butyla ted hyd roxy tolu e ne; DPPH , 1,1-diphc nyl-2-picrylhy- tri a ls. Mo re recently, some other natural com-
drazy l; HRP, Horseradish peroxid ase; O RA , Oxygen-
radical a bsorbin g ca pacity; TAA, To tal anti ox id a nt acti vity; pounds such as ca rotenoids, fl avonoid s, simple
TRAP, Total reacti ve a ntiox idant po tenti al; Tro lox, phenols and glu cobrasicins, have become objects
6-hydroxy-2,5,7,8- tetra- methylch ro man-2-ca rboxy lic acid
of interest du e to their beneficial properties for
health .11 ' 21 Practically all the above mentioned
an ti ox idant compound s are obtained through the
ingestion of plant products such as fruits and
vegetables, nuts, fl ours, vegetable oils, drinks a nd
infusions. Such ingesti on ca n be in the form of

•corres ponding author. Fa x: + 34-968-363963. E-ma il : ma [email protected] .es.

89
 90 M.B. ARNAO el nl .

fresh produce or processed produ cts in ca ns, MATERIALS AND METHODS

juices, frozen, cooked , sa u ces, etc. 131
A d eta iled ana lysis to verify the presence of a Reagents
certain a ntioxid a nt compound a nd its end ogen-
H o rseradi sh peroxidase (HRP) ty pe VI
ous qu a ntiti es in every foodstuff would require
(RZ = 2.76) was ob tained from Sigm a (Spa in ).
the use of very sophi sti ca ted and cos tl y identifica-
The enzym e concentra ti o n was determined by
tion a nd qua ntifi ca ti on techniqu es and as ri es of
measuring the abso rban ce at 403 nm
protocols a nd m e thod s to prepare the sa mpl es for
(c: = 100 mM - 1 cm - 1 ). 2,2'-Az ino-bis-(3-e thy l-
a na lys is. The m ost w id ely used separation, id en-
benz thi azo line-6-su lf<?..nic aci d) (A BTS), as th e
tifi ca tion and qu antifi ca ti o n techniqu es are hig h
crys talli zed di ammonium sa lt, hydrogen p e rox-
performa nee Iiqu id chrom a tography (HPL ) w ith
id e (30 % v j v) a nd L-ascorbic acid were obta ined
photometric, e lectrochemi ca l o r fluorescence
from Aldrich (Spain). ABTS conce ntra ti on was
d etection a nd m ass spectrome try with ca pill a ry
determined u sing f 34 onm = 36 mM - 1 cm - 1• H y-
gas chroma tography (MS-GC) or liquid chroma -
drogen peroxide concentra ti o n was d etermined
togra phy (MS-LC).
u sin g C:z4onm = 43.6 M 1 cm - 1 a nd L-a scorbi c acid
However, a know led ge of th e p o tenti a l a nti-
co nce ntra tion u sing c: 2 \lonm = 2.8 mM 1 em 1 .
ox id a nt compound s of a food does not neces a rily
Free Radical Research 1999.31:89-96.

Syn the ti c compo und were ob ta ined from

indi ca t its a ntioxid a nt ca p acity. Furth rm or , the
Aldrich o r Sigm a (Spain) . Buffer subs tances (ana-
oope ra ti ve eff ct which could ex ist betw en
ly ti ca l reagent g rade) were obta ined fro m Merck
different a nti oxid ants (t rmed syne rg is m) m ea n
(Spain).
th a t th tota l a ntioxid ant effect may be g r a ter
than the urn of th e indi v idu a l a ntioxid a nt acti v-
iti es and the isolation of one compound would not Plant Material
exa tly re fl ect the overa ll action. 14•51A dditi o na lly,
unid e ntifi ed j unm asu red compone nts in th e Fresh citru fruits a nd fruit juices were kindl y
sa mpl e could pot nti a ll y produ ce a simil ar ef- provid ed by ALVALLE S.A. (Murcia, Sp ain).
fect. For thi s reason, m ethod s to quantify th e so- Fresh fruits were squ eezed , cloth filte red a nd as-
ca ll ed total a ntioxid ant activity (TAA) ha ve been sayed imm ed iately for tota l a ntioxid a nt activity.
sought for man y y a rs.16•71 TAA i a p ara me ter
that quantifi s th ca pacity of a sa mpl e (in eith er
ABTS / HRP End-point Method
a na tura l or proces ed product) to scavenge free
radica l in aqu ous m edi a. Such m ethod s look Th e loss of a bsorba nce assay · was ca rri ed out
at th e antioxidant activity as a g loba l character- according to Cano et a/.,' 81 wi th som e m odifi ca-
is ti c of a produ ct, ind ep e nd ent of its p arti cul ar tion . Th e reaction mixture contained 2 mM ABTS,
compos ition. 15 pM hydrogen p eroxid e and 0.25 pM HRP in
We have d ev loped two m e thods to s tud y the 50 mM phospha te buffer (pH 7.5) in a total
antioxid a nt activity of fresh pl a nt ma teri a l a nd volume of 2 mL. Th e assay temperature was
foodstuffs and th e 'a ntioxid ant properti es of maintained a t 25 ± 0.1 °C using a H aa ke DlG
na tura l compound s. Our m thods a re pr sented tempe ra ture controller. The reaction was mon-
in an a ttempt to facilita te a nd sp ed up the itored at 730 nm usin g a Pe rkin-Elme r Lambd a-25
m a nipul a tion of biologica l material u sing simpl e UV-VIS s pectrophotometer until a s tabl e absor-
ins trum nta tion. Also, in thi s paper, stra tegies bance, du e to ABTS radi ca l formation was
for m easuring TAA are discu ssed, pa rticul arly the obtained. Differe nt a mounts of L-asco rbi c acid
differe nt me thodolog ica l and instrumenta l a p- or other an ti oxidan t compound were th en add ed
proach s u ed . and th e d ecrease in absorbance was determined .
 ANT IOX IDANT A T IVITY I PLANTS 91

The tota l tim e to ca rry out each assay was 30-50 s, ag ree men t w ith o ne of th e followi ng reacti on
includin g ABTS radi ca l ge ne ra ti o n by p erox id ase m od e ls:
foll owed by antiox id a nt additi o n a nd th e acquisi-
ti o n of the fin a l a bsorba n e va lu e. Th e sensiti vity (A) Radi ca ls a re u sed as a qua ntify in g facto r in
a nd accuracy o f the me th od we re cha rac teri zed th e pres nee o f a nti ox id ant compound s to
foll ow in g th e g ui delin es reco mm e nd ed by th e p rovoke a loss of s ig na l.
Am eri ca n C he mi ca l So iety' 91 de termining th e (B) Th e reagent is used as the d etectab le facto r.
LO D (limit o f d e tecti o n) a nd LOQ (limit of T he p resence of radi ca ls prod u e a lo s of
qu a ntifi ca ti o n). To ta l a nti ox id a nt ac ti v ity (TAA) sig n<ll . In this case, th e pre e nc of a nti ox i-
was calcul a ted as the co ncentra tio n, ex pressed as d ants p rovokes an increase in the s igna l.
asco rbi c acid equi va le nts (mo l/ L) (s tcmd a rd), thot Neverth e less, th me thods tha t have been m ost
produ ced the sa me a nti ox id a nt cffe t as th e w id e ly accepted fo r a ppli ation in food technol-
sa mpl e und e r stud y. ogy in volve the g nera ti o n of free radica ls, w hi h
a r eas il y d e tectabl e by flu orim e tric o r pho to-
me tri c techniqu es. Usin g these techniqu s, it is
RESULTS AND DISCUSSION po s ibl e to d efin e two stra tegi fo r stud ying the
TAA of a compound or bi olog i a l a mpl e (see
Free Radical Research 1999.31:89-96.

Th e d termina tio n of TAA is gene roll y based on Fig ure 1) .

th e inhibi tio n o f certa in reac tio ns by the presence
o f a nti ox id a nts. Th e mos t w id e ly used m eth od s Stra tegy 1 A di scolo ra ti on o r ig na l loss assay.
a rc th ose tha t in volve th e ge ne ra ti o n o f a radi ca l The reacti o n produ es a q ua ntity o f free ra dica ls
com pound a nd it is the p rese nce o f a nti ox id a nts fro m th e r age nt(s), w hi ch a re suffi cie ntl y sta bl e
tha t provo kes the di a ppea ronce o f thi s radi ca l, in th a t, a fter th e additi o n of a nti ox idant o r mixture

STRA1EGY1 STRA1EGY2

_/} :
Antioxidant addtion
Inhibition (%) Strategy 2A
Signal
(at t time)

a d

Tirre Jllltioxidant Concentration

Strateqy2B
- t.Signal

b e
Jllltioxidant Concentration JllltioxidantConcentration

FIGURE 1 Strategies for TAA determina tions of chemical compou nd s or b iologica l sa mples (sec tex t).
 92 M.B. ARNAO el nl.

of antioxidants, th e ignal loss (loss of radical) TABL E I Classification of m ethods according to the d etcc-
ti o n and quantifica tion s trategy u sed (sec Fi g ure 1)
can be used as an ind icator of TAA (Figure l(a))
Quantification is by means of a calibration curve Me thod De tection Strategy Ref.
that re lates the loss of signal a nd the concentra- ABAP/ ABTS Absorbance 120 1
tion of antioxidant present (Figure 1(b)). DPPH Absorbance 11 8 1
ABTS/ HRP Absorba nce 18]
Stmtegy 2 An inhibition a say, in which the (end-point)
reaction course is estimated in the absence ABTS/ MnOz Absorbance 125 1
(control) or presence of different quantities of {3-phycocrythrin/ Fl uorc ccncc 2A 11 4,15 1
antioxidant (A 1, A 2 , . . . ) (Figure l(c)). The re- ORA
is-Parina ri c acid Fluorescence 2/\ 11 6 1
ag nts a nd the a ntioxid a nt sa mpl e are mi xed a t
ABTS/ m yoglobi n Absorban e 2A 11 7 1
the out e t of th r action. The presence of
TRAP Oxygen electrode 28 11 0 1
a ntioxidant affects the reaction in two ways:
TRAP modified hc miluminiscc n e 28 Ill]
(A) Inhibition of th e s ig nal per unit of tim e (f) Luminol / HRP Chemiluminiscence 28 11 2 1
{3-phycoery thrin Fluorescence 28 11 3 1
(Figure l(c)). Thus, we can make a ca libration 28
ABTS/ HRP (lag ) Absorba nce 122 1
curve which correla tes the % of inhibition
for very concentra tio n of a ntioxid a nt used, in w hich integra ted ,13-p hycoerythrin fluore ccnce
Free Radical Research 1999.31:89-96.

with re pec t to an assay ontrol without anti- versus time is mea ured. This method uses
oxida nt (Figure 1(d)). Stra tegy 2A, w he r by the inhibiti on of fluores-
(B) By regi te ring th our e of the reacti on, the en e (d<P / dt) by th e prcsenc of antioxidants is
appearance of a s low or lag phase ca n be quantifi d. Another method that uses this s trat-
observ d (Figure 1(c)). The duration of the gy is that of McKenna et a/. 11 61 where th e fluo-
lag phase (time before reaching th e tea dy rescence due to cis-parinaric acid is monitored,
s tat ) i proportional to th e co ne ntra tion of and the au tomated m ethod of Mill er et a/.11 71
a ntioxida nt in the sa mpl e (Figure l(e)) . Stra tegy 1 i pote ntially th mo t attractive
Thus, methods a n be Ia ified according to beca u of its simplicity but it is less widely used
the d tection a nd quantification trategy u ed in practice since it is difficult to obta in radica ls
(Tab! I) . In th original TRAP as ay' 101 wh re the that a r s tabl e und er different conditions and
th rmald c mpositionofABAP(peroxylradica l which respond adequately to the pre encc of
ou r e) is coup led to oxygen on umption, the antioxidant . Among these me thod , is that of
param te rm a ured i the lag phase (Strategy 28) Hogg et a/.1181 which uses the me tas tabl e radi ca l
of oxyg n uptake by pia ma material. Th sa me DPPH, which has an absorbance m ax imum at
param ter is measu red in it subseq uent adap- 517nm and yield s a colorl ess hyd razin e produ ct
tation , where a chemilumine ce nce-coupl ed in the presence of a ntiox id a nt . An important
r actionis d v loped .1111 White headeta/.1121 used limitation to the use of thi me th od is that sin ce
a n enha nced chemilumin e cen t reac tion involv- DPPH radicals are not water soluble, only organic
ing hors radi h peroxida a nd luminal to detect solvent med ia ca n b utili zed.
antioxidant , mea uring th lag-tim in light mis- Anoth r me thod employing Strategy 1 is tha t
sion du e to th pre encc of antioxidants. The developed by Lissi and co-workers,' 191 in which
origina l m ethods that used ,6-phyco rythrin fluo- th e radi ca l-g ne rating ca pacity of ABAP and the
rescence inhibition coupled to th e the rmal d ecom- excell e nt s pectral cha racte ri s tics of ABTS are
position of AAPH (peroxyl radi ca ls source) 1131 used. The ABTS radi ca l that is ge nera ted is
also e tima ted th lag phase. capa bl e of reac ting with a grea t va ri ety of
ne of the most widely u d me thod s is the a ntioxidant . One in conve ni e nce of the me thod
ORA m thad of ao e/ a/. 1141 Iater automated, 11 51 is th e hig h te mpe ra ture (45° ) necessary for the
 ANTIOX IDANT ACTI VITY IN PLANTS 93

reaction to take place and the problem of radica l more sens itive (LOQ: 0.65 nmol for L-ascorbic
s tability. N everth eless, these authors applied the acid); (iv) it di scriminates between antioxid ants
method to s tud y th e antioxidant capacity o f and inhibitors o f th e enzymatic system; and (v) it
va ri ous pure antioxidants and of sa mpl es of is easi ly reproduced manu a lly. 122 1 However, the
human serum and herbal infusions. 1201 presence o f end ogenous peroxidase acti vity in
One of the most interes ting m ethods is tha t the plant sa mpl es could possibly interfere with
presented by Ri ce-Eva ns' group.1171 The method the TAA determinations.
was firs t used to ascertain TAA in blood and other More recentiy we have developed a modifi ca-
hum an fluid s. This method is based on th e ti on of the method u sing the sa m e enzym ati c
capacity of antioxidants to elimina te free radi ca ls system but in a n assay which measures the loss of
of ABTS. Free rad ica ls a re generated by mea ns of a absorba nce (Strategy 1).1" 1 In this ase, the ABTS
reaction that uses the m et-myoglobi n/ H 20 z/ radica l is pre-generated enzymati ca ll y and th
ABTS system, and the ex tent to w hi ch this anti oxidant or sa mpl e to be analyzed is added to
reac ti on is inhibited by a nti ox id a nts is evaluated the reaction m ed ium . This results in the dis-
(Strategy 2A) . Alth oug h this method has been appea ra n e of th e rad ica l, es timated by a d ecrease
patented and marketed (Ra nd ox La b., UK) as a kit in absorba nce at 414nm (to avoid exogenou
to measure antioxida nt sta tu s, it prese nts some absorption interferences, a wavelength between
Free Radical Research 1999.31:89-96.

compli cations in its appli cation s uch as the need 400 and 750 nm ca n be selected). Th m o t
for a chro matog rap hi c purification of the met- importa nt limiting fac tor in thi s type o f s trat gy
myog lobin and some ad diti onal spectrophoto- is the fact th at ABTS radical s mu s t be s tab!
metric calcul a ti ons. Subs tra te concentra ti ons are duri ng th e our e of ana lys is. With thi s in mind,
s toichi ometri c: met-myoglobin fo ll ows firs t-ord er assay o nditi ons were optimi z d to ensure > 99%
kin eti cs, w hi h m ea ns th at the conce ntrati on of s tability of th e ABTS radi ca l a nd it was verified
ubstrates in the assay mu st be known exactly. A that the concentrati on ratio betwee n rad ica l
hi gh degree of accuracy a nd precision in the (ABTS• ' ) and s ubstrate (ABTS) i a det rminin g
protocol is required in order to obtain th e des ired factor for s tab ility of th e ABTS rad ica l. Henc , a
level of reprod ucibi lity. The enzy mati c assay does ca libration urve for differe nt anti oxida nt com-
not permit studi es of anti oxida nt activity over a pound s, corr lating the loss of absorbance with
wide pH ra nge. the anti ox id a nt concentra ti on ca n be obtaitiled . For
We have previous ly developed a me thod to L-asco rbic acid, th e o ptimi zed method presents
measure anti ox id a nt activity based on the inhibi- excell ent reproducibi lity, a LOD of 0.15 nm ol and
tion of th e ox id ati on of the ABTS chro mophore by a LOQ of 0.38 nmo l were obtain ed . The pa tented
ascorbic acid. 122 1 This is a s pec trophotometri c m ethod presents numerous adva ntages: it avo ids
method tha t d etermines th e lng p hase (Strategy un wa nted reacti ons of th e a nti ox id a nt w ith the
28) ca used by different anti ox id a nts in th e reagents (hydrogen peroxid e, oxygen, enzyme,
production of ABTS rad ica ls. Thus, the anti ox- etc.); th e ABTS rad ica l generati o n ca n be con-
id ant capaci ties of natural compound s such as troll ed by th e a mount of hyd rogen peroxide
ascorbic acid , glutathio ne, cysteine o r synth etic added; hig h temperatures are not required to
a ntioxida nts such as BHT, BHA or Trolox® ca n be generate rad ica ls; it permits s tudies of anti ox id ant
estima ted, as ca n th ose of plant ex tracts or activity over a w id e range of pH va lu es; and it is
sa mples from o ther sources. Th e m ethod pos- easy and rapid to perform sin ce it does not require
sesses a number of adva ntages with res pect to that con tinu ous spec trophotometri c m onitoring but
of Miller et a/. :1171(i) it uses comm ercia ll y ava il ab le ra ther estimates th e fina l absorbance va lu e. This
HRP; (ii) peroxidase oxid izes ABTS more effi- meth od also presents th e adva ntage of avoi din g
cientl y than met-myoglob in; (iii) our meth od is interference du e to peroxidase acti vity in sa mpl es,
 94 M.B. ARNAO et nl.

so tha t the determination of TAA in plant and inhibited by phenolic compo unds in the sa mple
other ex tracts, is more accurate and rigorous. in the method of Ri ce-Eva ns (since fl avonoids
Table li summari zes the principal characteri stics and phenoli c acids, ca n serve as substrate of
of th e most w id ely used m thod s in their applica- peroxid ases). In the reacti on ca talyzed by m et-
tion to a nti ox id ant activity determina tions. Th e m yoglobin, this hem oprotein acts as a perox id ase;
m ost important differences are rela ted to reagent thus, ABTS radi ca ls may be prod uced m ore
1
manipul atio ns, da ta processing, autom ati on pro- slowly beca use of phenoli c competition and ,
cess and possible interferences. th erefore TAA va lues would be overes tima ted .
Th e most ex tensive stud y on anti oxid ant acti v- Simila r competiti ve behavior by fl avonoids has
ity of plant-derived compound s has been ca rri ed been d escribed by other auth ors. 121 ' 24 1 Neverthe-
out by Ri ce-Eva ns and co-wo rkers. Flavonoid s less, w hen Miller and Ri ce-Eva ns 125 1 compared
s uch as qu ercetin, mirice tin, cya nidin, ca techin their method w ith a m ethod involving chemi ca l
and rutin, all have high antioxid ant ca pacities. generation of ABTS rad ica ls by m anga nese di ox-
Others su h as naringin, hes perid in, ,a pigenin, id e no differences in the anti ox id ant acti vi ty
kaemph erol, genis t in and perla rgonidin have va lues were found . In our end -point meth od , 181
b n shown to have lower TAAs. 123 1 Our data th e inhibitory actions of anti oxid ants would not
Free Radical Research 1999.31:89-96.

indica te tha t phenoli c compounds such as ca te- ap pea r beca use anti oxid ant is add ed w hen the
chol, syrin gic acid and ca ffeic acid present TAA ABTS radi cal has been s tabi lized in th reac tion
va lu es similar to ascorbic acid, w hile feru lic acid , m edium and , since th ere i no hydrogen pe roxid e,
p-coum aric acid and n-prop ylga ll a te have va lu es no p roxid a e acti vity ca n occur.
twic th at o f L-ascorbic acid. om pounds su ch a To summari ze, the m ethods for meas urin g TAA
ga ll ic acid , r svera trol and qu ercetin pr se nt the th at currentl y ex is t, present important meth odo-
hi g h s t va lu s (Tabl ITJ). The a nti ox id ant acti v- logica l and instrum ental differences. Broa dl y
itie hown by ascorbi c aci d, gluta thi on , Trolox® imila r methods give di stin ct va lue of TAA for
and to oph rol ar the sa me as those obta ined by th sa me compound or biological sa mple. Th ese
Ri c -Eva ns. 1171 In our method, simple ph noli c differen es could be du e to s ta ti sti cal errors,
camp un d r suit in lower TAA va lu es ( ~ 10- internal di ffe rences in sa mpl s o r to un controll ed
15%) b ca us th perox id ase acti vity may be va ri ables of the method. We present a method

hara teris tics of the most widely used methods for dete rmi ni ng a nti ox ida nt activity

Method IRef. l

T RAP RA DPP H ABTS/ Myoglobi n ABTS/ Mn 2 ABTS/ HR P lag AlHS/ HRP e nd -point
[1 01 114,15] 11 81 [1 71 125 1 122 1 [81

Rad ica l wa ter so lu bi lity Yes Yes No Yes Yes Yes Yes
Radica l s tabi lity No No Yes Ye ? Yes Yes
lnterfcrenc s d ue lo
Enzy me com petitio n ? No Yes No Yes No
Enzy me conta mina tion No Yes No Yes No
li m e of a na lysis Low H igh Low Low Low Low Low
Reag nt Ma ni pulations" No No No Yes Yes No No
l~eprod u c i b il i ty ( .V.) Low H ighb Hi gh Hig hb < 4% < 5%
Se nsiti vity (LOD)c 0.52 0. 15
Precision (LOQ)c 0.65 0.38
Versa tili ty with res pec t to
pH No No No No ? Yes Yes
No No
.
Solvent No No

hroma tograp hy, centri fuga tio n, etc. b Au toma ted m ethods. c LOD a nd LOQ (a s nmo l of L-ascorbic acid) .
 ANT IOX IDANT ACT IVITY IN !'LA TS 95

TABLE Ill TAA of different compound s determined by 13 1 D. Mackcrras (1995) Antioxidants and h ea lth . Fruits
the ABTS/ HRP end -point m ctlwd 1HI and vegetab les o r s upplements? Food Attstmlia 475,
3- 23.
Compound TAA" Co mpound TAA 14 1 B. Pocggclcr, R.) . Re ite r, R. Hard e la nd , E. Seweryn ek,
D. Melchi orri a nd L.R. Ba rl ow-Wa lden (1995) Mela tonin, a
L-as orbic acid 1.00 Gil lli cacid 3.02 mediator o f ele tron tra nsfe r a nd r pair reacti on, acts
Trolox"' 1.03 p-coumaric acid 1.8 1 synergis ti ca ll y w ith the hain-breaking a n tioxid ants as-
Tocopherol 0.99 Caffei c acid 1.14 co rba te, tro lox and glutathione. Neuroclldocrillologicnl
Glutat hi one (GS H) 0.97 f.e ruli c acid 1.94 Letters 17,87-92.
L-cys te inc 0.65 Syrin g ic acid 1.1 5
lSI B. Poegge lcr, M .A. Pappolla, R. Hard eland ,
/1.. Rassoulpour, P.S. ll od gkins, P. .uid ctti and
Pheno l 0.50 Vilni lli ilCid 0.76
R. Schwa r z (1999) lnd o lc-3-p ropio na lc: a pote nt hy-
G uaiilco l 1.47 Sinapini itcid 2.'10 droxy l radi ca l scavenge r in rat brain . Brai11 Research 815,
Cillccho l 1.10 n- l'ropylga ll atc 1.90 382-388.
Res ve ra trol 2.34 Quercetin 4.30 161 B. Halli well and j.M. . u ttcrid ge (1990) Role o f free
radi ca ls and ca taly ti c m til l io ns in humil n disease.
"Values arc expressed as th e TAA ~om pt,und / TAI\ ,,..,u,rhh .ll ld
Mc tlwds i11 EII Zylllology 186, ·1-85.
ratio . Errors arc less thil n 4'Yt,. 171 ). Stilnl cy a nd A. Mogg (1 995) MAFF antioxid a nt rcsca r h
programme. Tlte Bioclw111ist Feb / Mar, 22- 24..
181 A. il no, J. Hcrm1nd cz-R uiz, F. GilrcJil - .1novils, M. Acos ta
and M.B. Arnao (1998) An end -point me l hod for estima-
w hi ch is easy a nd rapid to app ly, in volves littl e ti on of the tota l a ntiox id a nt acti vi ty in plil nt ma teri a l.
manipul a tion of th e biolog icnl m ntcria l nnd P!tytoc!tclltical Atta lysis 9, 196- 202.
191 1\ S o mmittcc on Environme nta l Improvement il nd
Free Radical Research 1999.31:89-96.

requires simpl e ins trume ntntion . Our end -point Sub ommittec on ·nvironm cnta l Ana ly ti a l hcmistry
method is currently used to characteri ze differ nt (1 980) uid clin cs fo r d a ta 11cqui siti on ilnd da ta qu il lity
cva luiltion in en vironm ental hcmi s try. A11alytical Cltcllt-
plant ma teria ls (fresh fruits, vege tab les, juices,
ist n; 52, 2242- 2249.
wi nes, etc.) and to ascertain the effec t of industriol 11 0 1 D.D. M. Way nc r, G.W. Burton, K.U. Ingold and S. Locke
process in g a nd diffe rent s tora ge conditions (room ('1985) Q ua ntita tive meas ure me nt o f th e total pcroxy l
radi cil l-trappin g a nti ox id a nt apa bility of huma n bl ood
temperature, refrigeration , freezing ) o n antioxi- pia ma by contro ll ed lipid pcroxidation. FEBS Lrff ers 187,
dant a nd orga nolcp ti properti es. The method 33- 37.
1'111 T. Mctsa-Ketc la a nd A.L. Kirkkola (1 992) Tota l pc roxyl
may be of grea t usc in indus tri es w hi ch produ ce rild ica l-tra ppin g apab ility o f hum a n LDL. Free Radical
fruit juices a nd d e rivatives, w ines, beers, soft Research Ol//111/ tllicatiotts 165, 215.
1121 T.r. Whitchead ,G .II. . Thorpe a nd S. R.j . Maxwe ll (1 992)
drinks, infus ions, e tc. En hanced hcmilumin cs cnt assay for a nti oxida nt ca pil-
city in biologica l fluid s. Attalyticn lti11tim t\ctn 266,
265-277.
Acknowlerfgmen ts 113 1 R.j. D la nge a nd A.N . Glazer (1989) Ph ycoerythrin
flu orescence-based assily for pe roxy l radicil ls: a scree n
This work wa s suppo rted by n g ra nt (Project fo r bi o logica ll y releva nt protec ti ve age nts.' A11nlytical
ALI98-0524) from I YT (Spain) and in part from Biocltctll i try 177, 300- 306.
11 4] G. iiO, H.M. Alessio a nd R... Cutl er (1 993) xygcn-
the E.U. Trainin g and Mobility Researc h Pro- rad i ill absorbance ca pac ity assay for a nti ox idants. Free
gramme "Peroxidases in Agri cu lture, th e Environ - /~ndicals in Biology a11d Mcdici11e 14,303- 311.
11 5 1 G. ao, C.P. Verd on, A. I-I .B. Wu , H. Wang 11 nd R.L. Pri or
ment a nd Indu stry" (FM RX- T98-0200) . A. a na (1995) Automated assay of oxygen rad ica l ilbsorbance
has a g rant from lns tituto de Fomento ( Fundaci6n Cil pacity with th e oba Fara II. Clitticnl Cltc111istry 41 ,
1738-1744.
SENECA) of Co munidad Aut6noma de Murcia 11 6 1 R. M Kenna, F.). Kezd y a nd D.E. Epps (1991) Kineti c
(Spain) . Th a nks are g iven to Dr. A.N .P Hin er for ana lys is of the free radi ca l-indu ced lipid peroxi d a tion in
huma n ery th rocyte membra nes: eva lua tion of poten ti al
helpful s ugges tion s a nd correction s. iln tioxidants usin g cis-parinaric acid to monito r pcroxida-
tion . Attalytical Biochemistry 196,443-450.
11 71 N.j . Mi ll e r, .Rice- vans, M.j .Davies, V.Gopinatha nand
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