Jou nal o
APPLIED
BIOMEDICINE
J Appl Biomed. 10: 155–167, 2012
DOI 10.2478/ 10136-012-0006-y
ISSN 1214-0287
ORIGINAL ARTICLE
Au oma ed assay o he po ency o na u al an ioxidan s using
pipe ing obo and spec opho ome y
Mi osla Pohanka1, Jiří Socho 2, 3, B anisla Ru kay-Nedecký2, 3, Na alia Ce nei2, 3, Voj ěch Adam2, 3,
Ja omí Hubálek2, 3, Ma ie S ibo o á4, Tomáš Eckschlage 5, René Kizek2, 3
1Facul y o Mili a y Heal h Sciences, Uni e si y o De ence, H adec K álo é, Czech Republic
2Depa men o Chemis y and Biochemis y, Facul y o Ag onomy, Mendel Uni e si y in B no, B no,
Czech Republic
3Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czech Republic
4Depa men o Biochemis y, Cha les Uni e si y, Facul y Sci., P aha, Czech Republic
5Depa men o Paedia ic Haema ology and Oncology, 2nd Facul y o Medicine Cha les Uni e si y, P aha,
Czech Republic
Recei ed 12 h Oc obe 2011.
Re ised 13 h Decembe 2011.
Published online 15 h Decembe 2011.
Summa y
In he ood indus y, in he p ocess o c ea ing new ag icul u al plan p oduc s, and in he es ing o
an i-cance d ugs he e is o en a need o assay mul iple samples o low molecula weigh an ioxidan s, plan
samples and oods ich in an ioxidan s, wi h minimal addi ional cos s and low deg ees o unce ain y. Wi h
hese demands in mind, we decided o s udy he ully au oma ed assay o an ioxidan s using no only
au oma ed sample measu emen s bu also au oma ed p ocessing o samples and applica ion o eagen s. The
au oma ed pipe ing sys em epMo ion 5075 and he au oma ed spec opho ome e BS 400 we e chosen o
he assay pu poses. Fi e me hods we e in oduced o he au oma ion: 2-diphenyl-1-pic ylhyd azyl (DPPH)
es , e ic educing an ioxidan powe (FRAP) me hod, 2,2’-azino-bis(3-e hylbenzo hiazoline-6-sul onic acid
(ABTS) based es , N,N-dime hyl-1,4-diaminobenzene (DMPD) based es and he ee adicals me hod.
Samples con aining one o he ou an ioxidan s (s anda d u in, que ci in, e ulic and gallic acid) in a ange
1–1000 μg/ml we e used h oughou . All o he es ed me hods we e ound sui able o implemen a ion in an
au oma ed assay. Howe e , some o hem, such as he ABTS es ailed o assay all es ed an ioxidan s. The
coe icien s o de e mina ion we e also unequal. F om he analy ical poin o iew, FRAP me hods p o ided
he mos eliable esul s in he au oma ed assay; because o he capaci y o he me hod, app oxima ely
240 samples pe hou (one sample pe 15 seconds) can be assayed using he au oma ed p o ocol. We we e
encou aged by he da a ecei ed and we expec u he in e es in he p ac ical pe o mance o such
au oma ion. As a mean o es ing he obus ness o ou me hod, in he nex s ep o ou s udy, oxida i e s a us
was assessed in model cell lines de i ed om p os a e cance (PC-3, PNT1A and 22RV1) ha we e cul u ed
on ellip icine (0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15 μmol/l) supplemen ed aga . An ioxidan ac i i y was
assessed (DPPH, ABTS, FRAP, DMPD, FR) and
calcula ed on he phenolic an ioxidan le el ( u in,
que ci in, e ulic and gallic acid), and hus an
es ima ion was o mula ed o he oxida i e s ess as
a esul o he impac o an i-cance d ugs. I can be
demons a ed ha he new me hod has wide
applicabili y.
Key wo ds: DPPH; FRAP; ABTS; DMPD; ee
adicals; an ioxidan
René Kizek, Depa men o Chemis y and
Biochemis y, Facul y o Ag onomy,
Mendel Uni e si y in B no, Zemědělská 1,
613 00 B no, Czech Republic
[email p o ec ed]
+420 545 133 350
+420 545 212 044
© Jou nal o Applied Biomedicine
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
156
Abb e ia ions
ABTS, 2,2’-azino-bis(3-e hylbenzo hiazoline-6- sul-
onic acid;
DMPD, N,N-dime hyl-1,4-diaminobenzene;
DMSO, dime hyl sul oxide;
DPPH, 2,2-diphenyl-1-pic ylhyd azyl;
FeCl3, e ic chlo ide;
FR, ee adical;
FRAP, e ic educing an ioxidan powe ;
FTB, oe al bo ine se um;
TPTZ, 2,4,6- ipy idyl-s- iazine;
UV-VIS, ul a iole - isible spec um.
INTRODUCTION
In a body, homeos asis, he main enance o sui able
inne condi ions, is a p ima y ask o many
biochemical pa hways. Equilib ium be ween he
p oduc ion o eac i e oxygen (ni ogen) species and
he abili y o be p o ec ed om hem is a
physiological unc ion o an ioxidan s. When
an ioxidan s become deple ed, oxida i e s ess can
de elop. Some pa hological p ocesses a e con-
sequences o he unco e ed p oduc ion o eac i e
oxygen and ni ogen species. Alzheime ’s disease,
Pa kinson disease and o he age ela ed diso de s a e
examples o he pa hological p ocesses, in which
oxida i e s ess is suspec ed o playing a c ucial ole
(Pohanka 2011). Alimen al adminis a ion o
an ioxidan s is conside ed as a way o p e en ing he
pa hological consequences o oxida i e s ess ela ed
dys unc ions. Plan ex ac s and plan en iched
sou ces especially a e conside ed sui able and easily
a ailable p oduc s o he p e en ion o hese
pa hologies, as epo ed by e.g. Ka ia asan e al.
(2008).
Un o una ely, esea ch in o low molecula weigh
an ioxidan s is no well s anda dized and di e ing
p o ocols a e used o he assay o an ioxidan s.
Resul s can be in luenced because o me hodological
e o s as well as aul s caused by human ac o s. The
impac o me hodological di e ences has been well
epo ed. by e.g Mülle e al. (2011), and he
implemen a ion o s anda d p o ocols wi h an
au oma ed assay p ocedu e is no only sui able o
simpli ica ion and cos sa ing bu i is also necessa y
o he lessening o assay unce ain y and
imp o emen o da a alidi y. This expe imen is
aimed a he pe o mance o a ully au oma ed
p ocedu e sui able o as and eliable assay o low
molecula weigh an ioxidan s in biological samples.
The p ocedu e uses ully au oma ed manipula ion
wi h samples and eagen s in o de o ecei e a alue
wi h minimal cos and unce ain y.
MATERIAL AND METHODS
De ice and chemicals
The de ice used was composed o wo basic pa s: an
au oma ed pipe ing sys em epMo ion 5075
(Eppendo , Ge many) and an au oma ed
spec opho ome e BS 400 (Mind ay, China). The
pipe ing p o ides a obo ic a m wi h adap e s (TS 50,
TS 300 and TS 1000) and G ippe (TG-T). The emp y
mic o ubes a e placed in he posi ion B3 (scheme o
depic ed as Fig. 1) in adap e Ep0.5/1.5/2 ml. A
Module Rese oi is loca ed in he posi ion B1, whe e
s ock solu ions a e a ailable. The de ice is con olled
by he epMo ion con ol panel. Tips sized 300 and
1000 μl (Eppendo – Ge many) a e loca ed in he A4
(eP ips 50), A3 (eP ips 300) and A2 (eP ips 1000)
posi ions.
The au oma ed spec opho ome e is composed o
a cu e e space empe ed o 37±1 °C, eagen space
wi h a ca ousel o eagen s ( empe ed o 4±1 °C),
sample space wi h a ca ousel o p epa a ion o
samples and an op ical de ec o . The ans e o
samples and eagen s is p o ided by a obo ic a m
equipped wi h a dosing needle (e o o dosage up o
5% o olume). Cu e e con en s a e mixed by an
au oma ic mixe , including a s i e , immedia ely a e
he addi ion o eagen s o samples. Con amina ion is
educed due o he insing sys em, which includes
insing o he dosing needle as well as he s i e by
MilliQ wa e .
The chemicals used in his s udy include
deionized wa e , u in, que ci in, e ulic and gallic
acid, 2.2-diphenyl-1-pic ylhyd azyl (DPPH), dime hyl
sul oxide (DMSO), 2.2’-azino-bis(3-e hylbenzo-
hiazoline-6-sul onic acid (ABTS), po assium
pe oxodisulpha e, 2,4,6- ipy idyl-s- iazine (TPTZ),
hyd ochlo ic acid, e ic chlo ide hexahyd a e,
sodium ace a e ihyd a e, N,N-dime hyl-
1,4-diaminobenzene (DMPD), ace ic acid and we e
pu chased om Sigma Ald ich (S . Louis, MO, USA).
The eac ion bu e , chlo ophyllin concen a e and i s
ca alys we e pu chased om Sedium R&D (Czech
Republic).
S anda d
As s anda ds, u in, que ci in, e ulic and gallic acid
in a calib a ion ange: 1; 2; 3; 4; 5; 6; 7; 8; 9; 10;
12.5; 15; 17.5; 20; 25; 30; 40; 50; 60; 70; 80; 90; 100;
125; 150; 175; 200; 250; 300; 350; 400; 450; 500;
750; 1000 μg/ml we e used. The s anda d calib a ion
solu ions we e achie ed by au oma ed dilu ion o
s ock solu ions 1000, 100 and 10 μg/ml using
deionized wa e .
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
157
Fig. 1. epMo ion 5075 au oma ed pipe ing sys em om on al pa .
De e mina ion o an ioxidan ac i i y by he DPPH
es
The DPPH es was used in compliance wi h a pape
by Pa ejo e al. (2000). The me hod is based on he
abili y o he DPPH eagen o eac wi h hyd ogen
dono s.
Reagen p epa a ion: 0.95 mmol/ml solu ion o
adical DPPH was p epa ed by dissol ing DPPH in
50 ml o DMSO ollowed by mixing wi h 100 ml o
deionized wa e . The solu ion was kep in a da k lask
and could be used o 7 days when s o ed a 4 °C.
Measu emen p ocedu e using he au oma ed
de ice: A 150 μl olume o eagen is incuba ed wi h
15 μl o sample. Abso bance was measu ed a 505 nm
o 12 minu es, and he ou pu a io was es ablished
by calcula ing he di e ence be ween abso bance a
he las (12 h) minu e and he second minu e o he
assay p ocedu e.
De e mina ion o an ioxidan ac i i y by he ABTS
es
The ABTS me hod is based on neu aliza ion o a
adical-ca ion a ising om he one-elec on oxida ion
o he ch omopho e 2,2’-azino-bis(3-e hylbenzo-
hiazoline-6-sul onic) acid. The eac ion can be
simply moni o ed spec opho ome ically (Re e al.
1999).
Reagen p epa a ion: 7 mmol/l ABTS and
4.95 mmol/l po assium we e mixed and dissol ed in
deionized wa e . The solu ion was hen dilu ed wi h
deionized wa e in a a io o 1:9 / . The solu ion was
incuba ed o 12 hou s in he da k and he eagen was
kep sui able in he da k a 4 °C o one week.
Measu emen p ocedu e o an au oma ed
analyse : A 150 μl olume o eagen was pou ed
wi h 3 µl o sample. Abso bance was measu ed a
660 nm. An ioxidan ac i i y was calcula ed as he
di e ence be ween abso bance a he las (12 h)
minu e and a he second minu e o he assay
p ocedu e.
De e mina ion o an ioxidan ac i i y by he FRAP
me hod
The e ic educing an ioxidan powe (FRAP)
me hod is based on he educ ion o e ic o e ous
sal by an ioxidan s and he ollowing eac ion o
e ous sal wi h 2,4,6- ipy idyl-s- iazine (TPTZ)
eagen p o iding con as blue (Ou e al. 2002).
Reagen p epa a ion: Solu ion 1: 10 mmol/l
solu ion o TPTZ, in 40 mmol/l o hyd ochlo ic acid.
Solu ion 2: 20 mmol/l solu ion o e ic chlo ide
hexahyd a e in deionized wa e . Solu ion 3: 20
mmol/l ace a e bu e , pH 3.6 (weigh o sodium
ace a e ihyd a e was 0.272 g in 100 ml deionized
wa e , adjus ed by HCl). These h ee solu ions
(TPTZ, FeCl3, ace a e bu e ) a e pou ed in a 1:1:10
a io. The eagen could be used o se en days i
s o ed a 4 °C in he da k.
Measu emen p ocedu e o an au oma ed
analyse : A 150 μl olume o eagen is injec ed in o
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
158
a plas ic cu e e wi h he subsequen addi ion o a
3 μl sample (gallic acid solu ion). Abso bance is
measu ed a 605 nm o 12 minu es. The di e ence
be ween abso bance a he las (12 h) minu e and
second minu e o he assay p ocedu e was used o
calcula ing he an ioxidan ac i i y.
De e mina ion o an ioxidan ac i i y by he DMPD
me hod
The compound N,N-dime hyl-1,4-diaminobenzene
(DMPD) was con e ed o a ela i ely sui able and
colou ed adical o m in he cou se o he e ic sal
ac ion. A e addi ion o he an ioxidan solu ion, he
colou ed solu ion o DMPD eagen was decolo ized
(Gulcin 2008, Gulcin e al. 2010).
Reagen p epa a ion: Solu ion 1: ace a e bu e
(0.2 mol/l, pH 5.25); 1a) 2.17 g o sodium ace a e
ihyd a e was dissol ed in 80 ml o deionized wa e ;
1b) 300 μl o concen a ed ace ic acid (>99.5 %, / )
was dilu ed o a olume o 20 ml wi h deionized
wa e . Sho ly a e mixing, pH = 5.5 was
spon aneously eached. Solu ion 2: 0.74 mmol/l o
e ic chlo ide: 1 mg o e ic chlo ide hexahyd a e
was dissol ed wi h deionized wa e o a olume o
5 ml. Solu ion 3: 36.7 mmol/l DMPD: 25 mg o
DMPD was dissol ed in 5 ml o deionized wa e . The
eagen s we e used immedia ely and p epa ed esh
o each day. Solu ions 1,2 and 3 we e mixed in a
20:1:1 ( / / ) a io.
Measu emen p ocedu e o an au oma ed
analyse : A 160 μl olume o eagen was injec ed
in o a plas ic cu e e wi h he subsequen addi ion o
4 μl sample. Abso bance was measu ed a 505 nm.
The di e ence be ween abso bance a he las (12 h)
minu e and second minu e o he assay p ocedu e was
used o calcula ing he an ioxidan ac i i y.
De e mina ion o an ioxidan ac i i y by he FR
me hod
This me hod was based on abili y o chlo ophyllin
( he sodium-coppe sal o chlo ophyll) o accep and
dona e elec ons. This e ec was condi ioned by an
alkaline en i onmen and he addi ion o a ca alys
and i was ollowed by a s ong change in he
abso bance maximum (Vo uba e al. 1999).
Reagen p epa a ion: 5 ml o eac ion bu e (100
mmol/l o hyd ochlo ic acid) was dilu ed wi h 45 ml
deionized wa e . A e ha , 100 µl o chlo ophyllin
was added. Once i became sol ed, 0.25 ml o ca alys
was added. Reac ion bu e was sui able o one
mon h when s o ed a 2–8 °C in he da k.
Measu emen p ocedu e o an au oma ed
analyse : A 150 μl olume o eagen is injec ed in o
a plas ic cu e e wi h he subsequen addi ion o a
6 μl sample. Abso bance was measu ed a 450 nm o
he second minu e o assay and he las (12 h) minu e.
The di e ence be ween he wo abso bencies was
conside ed as an ou pu ing alue.
Cell line expe imen s
In ou s udy, we used he ollowing p os a ic cell lines
PC-3, PNT1A, and 22RV1. The PC-3 was de i ed
om he 4 h le el o p os a ic adenoca cinoma. The
PNT1A is a cell line de i ed om human p os a ic
epi helial cells. The las , 22RV1, is a human epi helial
cell line de i ed om an epi helial g a o he cance
issue. Cul u ing ook place in lasks wi h su ace
100 cm2 and las ed 72 hou s. Cell g ow h media
HAM’s F12 wi h 7% FVS o PC-3, RPMI-1640 wi h
10% FBS o 22RV1 and PNT1A we e used.
Ellip icine was dosed in he cell g ow h media up o
le el (0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 10 and 15 μmol/l).
The cul u ed cell lines we e washed wice by PBS
(6 ml o PBS and cen i uga ion on 2,700 RPM a
4 °C o 10 minu es). Supe na an was collec ed and
used o assay o an ioxidan s in as desc ibed abo e.
RESULTS AND DISCUSSION
Calib a ion o he me hods o an ioxidan ac i i y
de e mina ion
The i e me hods desc ibed in he expe imen al
chap e we e used o he assay pu poses. A summa y
o he sui abili y o he me hods o assay o low
molecula weigh an ioxidan s used h oughou
expe imen s is depic ed in Tables 1–4. The ABTS
es , he DPPH es and FRAP me hod we e sui able
o assay o all he es ed an ioxidan s. When
eg ession o calib a ion cu es ca ied ou , good
coe icien s o de e mina ion we e ound o he h ee
me hods. The ee adicals me hod was ound
unsui able o assay o e ulic acid. The las me hod,
DMPD, was ound sui able o he assay o e ulic
acid and gallic acid only, bu he que ci in assay by
DMPD es was limi ed in compa ison o he o he
me hods. The low ele ancy o DMPD is qui e
su p ising as i has been used in p e ious expe imen s
wi hou any di icul ies o assay o low molecula
weigh an ioxidan s in ap ico samples (Socho e al.
2011). Mo eo e , no c ucial disad an ages we e
ecognized when he me hod was es ed on a olox
model (Socho e al. 2010). The eason o he
ine ec i eness o he DMPD es is no clea , bu he
low a ini y o que ci in and u in owa d he DMPD
eagen and he equilib ium shi ed o eac an s can be
in e ed.
We selec ed he ollowing s anda ds – u in,
que ci in, e ulic and gallic acid – o es he
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
159
Table 1. Summa y o he pa ame e s o indi idual me hods ela ed o he s anda d gallic acid.
Me hod Wa eleng h
[nm]
Measu ing ange
[μg/ml]
Calib a ion equa ion Con idence
coe icien [R2]
S anda d
de ia ion [%]
DPPH 505 1–17.5 y = -0.1533x - 1.6252 0.9945 1.96
ABTS 660 1–25 y = -0.071x - 0.9841 0.9977 2.26
FRAP 605 1–350 y = 0.0978x + 0.9774 0.9989 1.19
DMPD 505 1–30 y = -0.0509x - 0.2913 0.9982 2.68
FR 450 1–750 y = 0.0029x - 0.085 0.9991 1.11
Table 2. Summa y o he pa ame e s o indi idual me hods ela ed o he s anda d e ulic acid.
Me hod Wa eleng h
[nm]
Measu ing ange
[μg/ml]
Calib a ion equa ion Con idence
coe icien [R2]
S anda d
de ia ion [%]
DPPH 505 1–100 y = -0.0548x - 1.1467 0.9927 1.53
ABTS 660 1–150 y = -0.038x - 0.7683 0.9915 2.28
FRAP 605 1–175 y = 0.0284x + 0.2702 0.9973 1.42
DMPD 505 17.5–800 y = -0.458ln (x) + 0.1955 0.9852 2.31
FR 450 – – – –
Table 3. Summa y o he pa ame e s o indi idual me hods ela ed o he s anda d que ci in.
Me hod Wa eleng h
[nm]
Measu ing ange
[μg/ml]
Calib a ion equa ion Con idence
coe icien [R2]
S anda d
de ia ion [%]
DPPH 505 7–70 y = -0.0484x - 1.3425 0.9992 1.63
ABTS 660 7–70 y = -0.0309x - 1.1151 0.9959 2.03
FRAP 605 1–1000 y = 0.0342x + 0.4564 0.9996 1.02
DMPD 505 – – – –
FR 450 1–1000 y = 0.0039x + 0.127 0.9996 1.27
au oma ion o me hods o de e mina ion o
an ioxidan ac i i y. All es ed molecules can be used
as a s anda d o calib a ion o an ioxidan assays and
hei selec ion mainly depends on he ype o ma e ial
analysed and he labo a o y equipmen used.
Howe e , au oma ed spec ome ic assays es ed in
his s udy mus be e ec i e wi h all hese subs ances
o show he e sa ili y o he sys em. Fe ulic acid was
he i s compound es ed (Fig. 2). The dependencies
o abso bance on he concen a ion o e ulic acid
measu ed by he DPPH es , ABTS es , FRAP
me hod, DMPD me hod, and he ee adicals me hod
a e shown in Figs 2A, B, C, D and E. The s uc u e o
gallic acid is shown in Fig. 2F. The same esul s we e
ob ained o e ulic acid (Fig. 3), que ci in (Fig. 4)
and u in (Fig. 5).

Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
160
Table 4. Summa y o he pa ame e s o indi idual me hods ela ed o he s anda d u in.
Me hod Wa eleng h
[nm]
Measu ing ange
[μg/ml]
Calib a ion equa ion Con idence
coe icien [R2]
S anda d
de ia ion [%]
DPPH 505 1–25 y = -0.0354x - 0.3071 0.9966 1.15
ABTS 660 60–1000 y = -0.0053x - 0.5518 0.9982 2.08
FRAP 605 1–175 y = 0.0212x + 0.3304 0.9983 1.28
DMPD 505 – – – –
FR 450 1–1000 y = 0.0039x - 0.1429 0.9994 1.01
Fig. 2. Assay o gallic acid using he DPPH es (A), he ABTS es (B), he FRAP me hod (C), he DMPD me hod (D), and he
FR me hod (E). The las pa o igu e is he s uc u e o gallic acid (F).
Calib a ion plo s o he es ed me hods a e shown
in Figs 2–5. We ound good coe icien s o
de e mina ions in a ange 0.9927–0.9992 o he
DPPH es , 0.9915–0.9982 o he ABTS es ,
0.9973–0.9996 o he FRAP me hod, 0.9852 and
0.9982 o he DMPD me hod and he wo success ul
calib a ions, and 0.9991 – 0.9996 o he ee adicals
me hod and he h ee success ul calib a ions. I
clea ly ollows om he esul s ob ained ha he bes
co ela ions we e ound o he FRAP and ee
adicals me hods hus a ou able adap a ion o
au oma ion can be in e ed.
The calib a ions shown had di e ing linea
anges. The FRAP and ee adicals me hods
p o iding linea calib a ion as high as 1,000 μg/ml o
some an ioxidan s, had he longes linea anges, bu
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
161
Fig. 3. Assay o e ulic acid using he DPPH es (A), he ABTS es (B), he FRAP me hod (C), he DMPD me hod (D), and
he FR me hod (E). The las pa o igu e is he s uc u e o e ulic acid (F).
Fig. 4. Assay o que ci in using he DPPH es (A), he ABTS es (B), he FRAP me hod (C), he DMPD me hod (D), and he
FR me hod (E). The las pa o igu e is s uc u e o que ci in (F).
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
162
Fig. 5. Assay o u in using DPPH es (A), ABTS es (B), FRAP me hod (C), DMPD me hod (D), FR me hod (E). The las pa
o igu e is s uc u e o u in (F).
in con as , he DPPH es was limi ed by
app oxima ely 100 µg/ml o he e ulic acid assay
and e en 20 μg/ml o gallic acid. The good esul s
o he FRAP me hod a e no su p ising conside ing
i s applicabili y o low molecula weigh an ioxidan s
assays in issue samples (Pohanka e al. 2011),
assessmen o an ioxidan s in ui (Bouayed e al.
2010), and in i o assessmen o an ioxidan s such as
u ic acid assay (Duplancic e al. 2011). Conside ing
he au oma ion p ocess, he leng hs o linea anges
a e no dependen on au oma ion bu on he eac ion
p inciple and molecula mechanism o me hods
p e-limi ing hem o applicabili y in he au oma ion
o assays.
The majo ad an age o he au oma ed assay is he
sho ime needed pe sample. Though he o al ime
om inpu ing he sample o ecei ing he measu ed
alue is nea ly a qua e o an hou , he o al ime pe
sample is less han one minu e. When he au oma ed
de ice is used o an ex ensi e se ies o samples, he
o al ime pe sample is app oxima ely 15 seconds. I
means ha 240 samples can be assayed pe hou by
one selec ed me hod wi hou any addi ional e o o
special need o skilled manipula ion o he samples.
The pe o mance o an au oma ed pipe ing de ice
wi h au oma ed spec opho ome e is sui able o a
as and eliable assay o low molecula weigh
an ioxidan s. The au oma ed pipe ing sys em
epMo ion 5075 and he au oma ed spec opho ome e
BS 400 we e bo h sui able o he assay pu poses and
all o he selec ed expe imen al p o ocols we e
adap able o analy ical pu poses. The ailu e in he
assay o u in by he DMPD me hod, and in he assay
o e ulic acid by he ee adicals me hod was no
caused by he inapplicabili y o he au oma ed assay
bu by sho comings in he applicabili y o p o ocols
o some compounds. This was no disclosed in he
da abases sea ched. The easy a ailabili y o he
DMPD me hod has been epo ed in he wo k o
Damien Do man e al. (2011). On he o he hand,
samples a e commonly assayed by one o wo
me hods, so disc epancies in na u al sample assays
can s ay hidden. When he pe o mance o he
au oma ed p o ocol is conside ed using all o he
desc ibed me hods, he FRAP me hod mee s he bes
c i e ia as i was success ully pe o med o all
an ioxidan s, i has a long linea ange and good
coe icien s o de e mina ions. On he o he hand, no
Pohanka e al.: Au oma ed assay o he po ency o na u al an ioxidan s
163
Fig. 6. Assayed an ioxidan s in cell lines using he DPPH es (A), he ABTS es (B), he FRAP me hod (C), he DMPD me hod
(D), and he FR me hod (E). Calcula ed on gallic acid.
se ious eason a ose o disc imina e agains he o he
me hods and he las ou p o ocols a e applicable o
he cons uc ion o a ully au oma ed p o ocol also.
The signi icance o low molecula weigh
an ioxidan s in biological samples has been desc ibed
by se e al in es iga o s (Ka ia asan e al. 2008,
Salehi e al. 2012). The me hods p oposed he e can be
used o he as and eliable p ocessing o na u al
samples o judge hei an ioxidan capaci y. When he
au oma ed pipe ing sys em and he au oma ed
spec opho ome e a e used simul aneously, na u al
samples can be assessed wi h good e ec i eness and
low unce ain y.
Applica ion o he op imized me hods on eal
sample analyses
The es o sui abili y o he au oma ed assay o
e alua ion o an ioxidan s in cell lines was pe o med
in p os a ic cance cells exposed o ellip icine. The
d ug damages he DNA o cance cells and s ops
ansc ip ion and eplica ion. The cells a e no able o
di ide, and as a esul , he cells a e di ec ed o
apop osis (S ibo o á e al. 2011, Kizek e al. 2012).
Howe e , he d ug does no ac speci ically on cance
cells and no mal cells can be a ec ed, oo. The aim o
ou expe imen was o es ima e he scale o oxida i e
s ess – exp essed as he an ioxidan ac i i y – in