Hindawi Publishing Co po a ion
BioMed Resea ch In e na ional
Volume 2013, A icle ID 408491, 11 pages
h p://dx.doi.o g/10.1155/2013/408491
Resea ch A icle
F ac iona ion and Pu i ica ion o Bioac i e Compounds
Ob ained om a B ewe y Was e S eam
Le icia Ba bosa-Pe ei a,1Aina a Poche ille,2Inmaculada Angulo,2
Pe ec o Pasei o-Losada,1and Jose M. C uz3
1Depa men o Analy ical Chemis y, Nu i ion and Food Science, Facul y o Pha macy, Uni e si y o San iago de Compos ela,
15782 San iago de Compos ela, Spain
2GAIKER Technological Cen e, 48170 Zamudio, Spain
3Depa men o Chemical Enginee ing, Indus ial Enginee ing School, Uni e si y o Vigo, 36310 Vigo, Spain
Co espondence should be add essed o Jose M. C uz; jmc uz@u igo.es
Recei ed 7 Feb ua y 2013; Accep ed 28 Ma ch 2013
Academic Edi o : Ana Moldes
Copy igh © 2013 Le icia Ba bosa-Pe ei a e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion
License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
The b ewe y indus y gene a es was e ha could be used o yield a na u al ex ac con aining bioac i e phenolic compounds. We
compa ed wo me hods o pu i ying he c ude ex ac —solid-phase ex ac ion (SPE) and supe c i ical luid ex ac ion (SFE)—
wi h he aim o imp o ing he quali y o he inal ex ac o po en ial use as sa e ood addi i e, unc ional ood ing edien , o
nu aceu ical. The p edominan ac ions yielded by SPE we e he mos ac i e, and he ac ion elu ed wi h 30% ( / ) o me hanol
displayed he highes an ioxidan ac i i y (0.20 g L−1), simila o ha o BHA. The mos ac i e ac ion yielded by SFE (EC50 o
0.23 g L−1) was ob ained unde he ollowing condi ions: empe a u e 40∘C, p essu e 140 ba , ex ac ion ime 30 minu es, e hanol
(6%) as a modi ie , and modi ie low 0.2 mL min−1. Finally, we ound ha SFE is he mos sui able p ocedu e o pu i ying he
c ude ex ac s and imp o es he o ganolep ic cha ac e is ics o he p oduc : he inal ex ac was odou less, did no con ain sol en
esidues, and was no s ongly colou ed. The e o e, na u al ex ac s ob ained om he esidual s eam and pu i ied by SFE can be
used as na u al an ioxidan s wi h po en ial applica ions in he ood, cosme ic, and pha maceu ical indus ies.
1. In oduc ion
Bioac i e phenolic compounds a e widely dis ibu ed in
na u e and a e he mos abundan an ioxidan s in he die ,
being common componen s o ui s, ege ables, and be e -
ages [1,2]. Nume ous s udies ha e associa ed he consump-
ion o oods ich in bioac i e compounds, such as phenolic
compounds, wi h he p e en ion o ca dio ascula diseases,
ce ain ypes o cance , and o he diseases ela ed o aging [3].
The bene icial e ec s de i ed om phenolic compounds ha e
been a ibu ed o hei an ioxidan ac i i y. These bioac i e
compounds may be a majo de e minan o he an ioxidan
po en ialso oods,and heymay he e o ebeana u alsou ce
o an ioxidan s [4]. An ioxidan s a e widely used in ood
p oduc s o p e en o delay he oxida ion o a s and oils [5].
The ecen wo ldwide end o a oid o a leas educe he
use o syn he ic addi i es, such as BHT and BHA, has c ea ed
he need o iden i y na u al (and possibly sa e ) al e na i e
sou ces o ood an ioxidan s [6,7]. In ecen yea s, he e has
been a g owing in e es in he use o na u al an ioxidan s in
he oodindus y,no only o applica ionasp ese a i esbu
also because o hei bene i s o human heal h [8,9].
Bee p oduc ion is an ex ensi ely s udied bio echnolog-
ical p ocess ha gene a es a ious by-p oduc s. The mos
commonbyp oduc sa egene a ed om hemain awma e-
ials used o make bee , ha is, ba ley mal , hop, and yeas .
These by-p oduc s can be used in bio echnological p ocesses,
such as e men a i e p ocesses o he p oduc ion o alue-
added compounds (e.g., xyli ol, e hanol) as subs a es o
cul u ing mic oo ganisms and as aw ma e ial o ex ac ion
o compounds such as an ioxidan s [10].
Bee con ains a la ge a ie y o phenolic compounds
which a e de i ed om he bio echnological e men a ion
o ba leymal (70%)andhop(30%)andwhicha e espon-
sible o he o e all an ioxidan ac i i y o he be e age
[11,12]. Nume ous s udies ha e shown ha polyphenols
2BioMed Resea ch In e na ional
a e ex emely impo an o he physical s abili y (a unda-
men al quali y pa ame e ) o bee [12]. Du ing s o age o
bee , colloidal haze o ms as a esul o he complexes ha
polyphenols o m wi h p o eins and polypep ides [13]. The
nega i e impac o mal and hop polyphenols on haze s abili y
can be minimized by using poly inylpolypy olidone (PVPP)
esin o s abilize bee and consequen ly ex end i s shel
li e. S abiliza ion wi h PVPP emo es a subs an ial po ion
o he haze ac i e and nonhaze ac i e polyphenols om
bee , and hese polyphenols can subsequen ly be eco e ed
om he PVPP by an alkaline ea men [14]. The e o e,
a na u al ex ac con aining bioac i e phenolic compounds
wi h high an ioxidan ac i i y can be ob ained om he
alkaline esidual s eam gene a ed a e cleaning he PVPP in
he b ewe y indus y, by ex ac ion in a sol en such as e hyl
ace a e [15].
The composi ion o he ex ac will depend on he
sol en used and also on he quali y o he o iginal ma e ial,
i s composi ion, gene ic ac o s, en i onmen al condi ions,
s o age condi ions, and any p io ea men . In o de o
ob ain a high quali y ex ac wi h an ioxidan ac i i y ha
issui able o usein he ood,cosme ic,andpha maceu ical
indus ies, he ex ac mus be pu i ied o emo e all ine and
undesi able componen s, so as o imp o e he an ioxidan
ac i i y o he ex ac and minimize any odou , as e, and
colou [16].
A pu i ica ion p ocess ha emo es ac ions wi h limi ed
an ioxidan ac i i y enables a good le el o an ioxidan
ac i i y o be ob ained om ela i ely small amoun s o he
o iginal na u al ex ac . Mo eo e , i is also impo an o
ob ain pu e ex ac s o ensu e he iden i y and sa e y o
an ioxidan compounds o be used as ood addi i es [17].
In hep esen s udy,wee alua ed wome hodso
pu i ying he c ude ex ac —solid-phase ex ac ion (SPE)
and supe c i ical luid ex ac ion (SFE). SPE has been widely
used o clean-upandpu i ica iono ex ac saswellasp e-
concen a ion o juices, wines, and bee . Phenolic compounds
a e eadily ac iona ed by se e al o ma s o SPE in di e en
ma e ials o na u al o igin; elu ion wi h me hanol on e e se-
phase columns is he mos popula me hod o sepa a ing
hese compounds [18–20].
Ex ac ion and eco e y o aluable compounds a e he
mos common uses o SFE, which ope a e a low empe -
a u es,in heabsenceo oxygen,and ypicallyuseCO
2as
ex ac ion sol en (SC-CO2). These ea u es make SFE an
ideal echnique o ex ac ing bioac i e compounds [21]. The
mos ob ious ad an ages o SFE a e ha i is clean and
en i onmen ally iendly. Di ec SC-CO2ex ac ion is no
ecommended o by-p oduc s ob ained on a la ge scale and
ha con ain small amoun s o bioac i e compounds [17].
Howe e , SFE has been used o pu i y c ude ex ac s yielded
by o ganic sol en s, o imp o e hei pu i y and hei bio-
logical p ope ies wi hou he mal o chemical deg ada ion.
As CO2is a non- oxic, inexpensi e, nonin lammable, ola ile
sol en , i can be used in a a ie y o di e en condi ions [22,
23]. The ex ac ion e iciency o SC-CO2can be op imized
by changing he densi y o CO2( a ying p essu e and
empe a u e), he modi ie (e.g., o ganic sol en ), modi ie
pe cen age, o ime, among o he pa ame e s. Due o he
apola na u e o CO2, heuseo modi ie s(e.g.,e hanol)
can signi ican ly imp o e he eco e y o he phenolic com-
pounds due o he pola i y o hese compounds [17].
The aims o he p esen s udy we e (i) o e alua e he
e iciency o he SPE and SFE echniques o pu i y na u al
an ioxidan s ob ained om b ewe y was e and (ii) o de e -
mine he eco e y yield and he adical-sca enging ac i i y
o he ac ions ob ained. Chemical analysis o he ac ions
by e e sed-phase high-pe o mance liquid ch oma og aphy
(RP-HPLC) coupled o a diode a ay de ec o (DAD) was ca -
ied ou o iden i y and quan i y he polyphenols esponsible
o he an ioxidan ac i i y.
2. Ma e ials and Me hods
2.1. Reagen s, Sol en s, and S anda d Phenolics. E hyl ace a e
(GR o analysis), me hanol (≥99.9%), absolu e e hanol,
hyd ochlo ic acid (37%), glacial ace ic acid, and ace oni ile
(ACN, HPLC g ade) we e ob ained om Me ck (Da ms ad ,
Ge many). Ul apu e wa e was p epa ed using a Milli-Q
il e sys em (Millipo e, Bed o d, MA, USA). 2,2-diphenyl-
1-pic ylhyd azyl (DPPH, ≥85%) and gallic acid (≥98%)
we e supplied by Fluka Chemie AG (Buchs, Swi ze land).
2,6-Di- e -bu hyl-4-me hylphenol (BHT, 99.0%) and 2(3)-
e -bu yl-4-hyd oxyanisole (BHA, 98%) we e p o ided by
Sigma-Ald ich (S einhein, Ge many). Supe c i ical ca bon
dioxide, CO2SCF (pu i y : 99.998%), was supplied by Ai
Liquide (Spain).
Polyphenol s anda ds we e supplied as ollows: p o oca e-
chuic acid (≥97.0%), ca eic acid (≥98.0%), (−)-epica echin
(≥90%), ace osy ingone (97%), es e a ol (≥99%), (±)-
na ingenin (95%), epigalloca echin (≥90%), (+)-ca echin
hyd a e (98%), e ulic acid (99%), que ce in (≥98%),
kaemp e ol (≥97.0%), galloca echin (≥98%), p-couma ic
acid (≥98.0%), and apigenin (≥97%) by Sigma-Ald ich
(S einhein, Ge many); gallic acid (≥98.0%), sy ingic acid
(≥97%), isoque ce in, and salicylic acid (≥99.0%) by Fluka
Chemie AG (Buchs, Swi ze land); and homo anillic acid
(98%), 4-hyd oxybenzoic acid (99%), and ace o anillone
(98%) by Al a Aesa (Ka ls uhe, Ge many).
2.2. Sampling. In bee p oduc ion, a cla i ica ion s ep is
essen ial o imp o e bee s abili y. As a esul o his p ocess,
a PVPP sludge is ob ained in he b ewing indus y. The PVPP
sludge loaded wi h polyphenolic compounds was washed
wi h a NaOH solu ion (2% w/w) a oom empe a u e. A e
he NaOH-PVPP was il e ed, a cleaned PVPP esin and
a PVPP washing solu ion (PVPP-WS) con aining phenolic
compounds we e ob ained (see Figu e 1). The esidual s eam
gene a ed a e he PVPP cleaning p ocess was kindly sup-
plied by Mahou-San Miguel, Spain.
2.3. Indus ial Plan Scale Ex ac ion o he An ioxidan s
om PVPP Sludge. The PVPP-WS (1000 L) was acidi ied
o pH 1.5 wi h HCl (37%), and polyphenolic compounds
we e ex ac ed wi h e hyl ace a e (2000 L) by s i ing o
30 minu es a oom empe a u e. The o ganic and aqueous
phases we e sepa a ed by decan a ion, and he o ganic phase
BioMed Resea ch In e na ional 3
B ewing
p ocess
Ba ley mal
Fe men a ion
Hops
Fil a ion (PVPP esin) PVPP sludge con aining phenolic compounds
Deso p ion
Cleaned PVPP esin
E hyl ace a e
Decan a ion Aqueous phase
O ganic phase
Pu i ica ion
SPE SFE
F ac ions
F .1 o F .11
Assay 1 Assay 2 Assay 3
F ac ions
A, B, C
F ac ions
D, E
F ac ions
F, G
Reused
Reused
Ex ac ion o
bioac i e
compounds
Pu i ica ion
o bioac i e
compounds
Yeas
(See Table 1)
(See Table 2)
Bee (e hanol, phenolic compounds p o eins)
Bee
S abilized PVPP-WS
PVPP-WS acidi ied
Shake 30 min a oom
empe a u e
E apo a ed o d yness a 40∘C
PVPP-WS ex ac
NaOH 2% (w/w)
HCl (37%) up o pH =1–1.5
Figu e 1: Schema ic ep esen a ion o he b ewing p ocess, ex ac ion, and pu i ica ion o he PVPP-WS ex ac con aining bioac i e
compounds.
was collec ed and e apo a ed o d yness a 40∘C. The esid-
ual wa e was emo ed om he ex ac by lyophilisa ion
be o e he eco e y yield was de e mined g a ime ically, and
he d y ex ac was used in ac iona ion expe imen s (see
Figu e 1).
2.4. F ac iona ion and Pu i ica ion o PVPP C ude Ex ac
2.4.1. Solid-Phase Ex ac ion (SPE). SPE was pe o med
wi h supe clean ca idges (LC-18 20 mL, om Supelco,
Ge many) and 5 g o e e sed-phase so ben (modi ied sil-
ica wi h oc adecyl g oups). The c ude ex ac (50 mg) was
dissol ed in 10 mL o wa e and loaded on he ca idge.
The na u al ex ac was elu ed wi h di e en pe cen ages o
me hanol ( / ): 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, and inally 100% o me hanol, so ha ele en
sepa a e ac ions we e ob ained a he end o he p ocess. All
ac ions we e e apo a ed o d yness, unde acuum a 40∘C,
in a o a y e apo a o , and inally edissol ed in me hanol
o u he analysis. The eco e y yield o each ac ion was
4BioMed Resea ch In e na ional
Table 1: SFE ope a ional condi ions es ed.
Assay 𝑇(∘C) Time
(min)
CO2 low
(g min−1)P essu e (ba ) Modi ie Modi ie
(%)
Modi ie low
(mL min−1)F ac ions ob ained
None None None A100,A
120,A
140,A
160,A
200,A
250,A
300
14030—
∗100, 120, 140,
160, 200, 250,
300 E hanol
—∗0.1 B100,B
120,B
140,B
160,B
200,B
250,B
300
—∗0.2 C100,C
120,C
140,C
160,C
200,C
250,C
300
24030 3
100, 120, 140,
160, 200, 250,
300
E hanol 3
6
0.1
0.2
D100,D
120,D
140,D
160,D
200,D
250,D
300
E100,E
120,E
140,E
160,E
200,E
250,E
300
34030 3 140 E hanol
Me hanol
0, 0.5, 1,
1.5, 2, 2.5, 3
0.0, 0.02,
0.03, 0.05,
0.06, 0.08, 0.1
F0,F
0.5,F
1,F
1.5,F
2,F
2.5,F
3
G0,G
0.5,G
1,G
1.5,G
2,G
2.5,G
3
∗In p essu e mode, nei he CO2 low no modi ie pe cen age we e con olled.
de e mined g a ime ically, and he an ioxidan ac i i y o
each ac ion was measu ed by he DPPH adical-sca enging
es . The phenolic compounds esponsible o he an ioxidan
ac i i y we e de e mined by HPLC-DAD.
2.4.2. Supe c i ical Fluid Ex ac ion. The c ude ex ac was
ac iona ed using a supe c i ical luid SCF R100 sys em (Tha
Technologies, Inc.) equipped wi h a 5 mL SFE cell (Tha
Technologies, Inc.).
Di e en ex ac ion condi ions we e es ed in h ee di -
e en assays. In each assay, 1 g o sample was submi ed o
he ac iona ion p ocedu e. The assay condi ions a e shown
in Table 1.
Assay 1—P essu e Mode (P essu e Range). The expe imen
was un a 40∘C, and CO2was au oma ically ed in o he
sys em by he CO2pump o main ain a cons an p essu e. Fo
ac iona ed sepa a ion o he di e en compounds p esen
in he c ude ex ac , p essu e o be ween 100 and 300 ba
(100,120,140,160,200,250,and300ba )wasapplied.The
ex ac ion ime was 30 minu es a each p essu e es ed.
Expe imen s we e ca ied ou wi h and wi hou modi ie .
E hanol was used as a modi ie , and wo di e en low a es
(0.1 mL min−1 and 0.2 mL min−1) we e es ed.
Assay 2—Flow Mode (P essu e Range). The ial was un
a 40∘C, and CO2was ed in o he sys em a a low a e
o 3 g min−1. The p essu e ange and ex ac ion ime a
each p essu e es ed we e he same as desc ibed ea lie . The
modi ie (e hanol) was applied unde di e en condi ions:
3% and 6% o modi ie a low a e o 0.1 mL min−1 and
0.2 mL min−1, espec i ely.
Assay 3—Flow Mode (Pe cen age o Modi ie Range). The
expe imen was un a 40∘C, and CO2was ed in o he
sys em a a low a e o 3 g min−1. In his es , p essu e was
main ained cons an a 140 ba . Two modi ie s, e hanol and
me hanol,we e es edwi hina angeo 0and3%(0%,0.5%,
1.0%, 1.5%, 2.0%, 2.5%, 3.0%).
Single ex ac s ob ained unde he h ee di e en es
condi ions we e e apo a ed o d yness unde ni ogen s eam.
The eco e y yield o each ac ion was de e mined g a ime -
ically. F ac ions we e cha ac e ized by HPLC-DAD, and he
an ioxidan ac i i y o each was de e mined by he ee adical
me hod DPPH.
2.5. Sepa a ion and Quan i ica ion o Bioac i e Phenolic Com-
pounds (RP-HPLC-DAD). Ch oma og aphic analysis was
pe o med on an HPLC sys em model 1200 HP (Hewle -
Packa d, Waldb onn, Ge many), equipped wi h a diode
a ay de ec o (DAD) and con olled by HP Chems a ion
ch oma og aphic so wa e.
Ch oma og aphic sepa a ion o polyphenols was ca -
iedou ona e e sephaseK omasilC18column(250×
3.2 mm in e nal diame e , 5 𝜇m pa icle size) (Phenomenex,
Ba celona,Spain).Thesol en scons i u ing hemobilephase
we e milli-Q wa e 0.1% ace ic acid (sol en A) and 100%
ACN (sol en B). The g adien p og am was as ollows: 0–
5 min , 90% A and 10% B; 5–35 min, linea g adien un il
eaching 50% B a 35 min; 35–43 min, 50% B isoc a ic; 43–
45 linea g adien om 50% o 10% B; and inally, he
column was washed and econdi ioned. The mobile phase
low a ewas0.5mLmin
−1 du ing he en i e analy ical un,
he column empe a u e was se a 38∘C, and he sample
injec ion olume was 20 𝜇L.Ascanin he angeo 190 o
700 nm was con inuously pe o med, by DAD.
Indi idual phenolic compounds we e iden i ied by com-
pa ing hei e en ion ime and hei UV spec um wi h
hoseob ainedbyinjec ings anda dsin hesameHPLC
condi ions. Phenolic acids we e moni o ed and quan i ied a
225 nm, la an-3-ols, la anones, la ones, and ace ophenone
de i a es a 280 nm, hyd oxycinnamic acids and es e a ol
a 325 nm, and la onols a 372 nm.
2.6. An ioxidan Ac i i y, DPPH Assay. The an ioxidan ac i -
i y o phenolics in he c ude ex ac and i s ac ions,
ob ained du ing he pu i ica ion p ocesses, we e de e mined
by he DPPH (2,2-diphenyl-1-pic ylhyd azyl) adical sca -
enging me hod desc ibed by on Gadow e al. (1997) wi h
sligh modi ica ions [24].
S anda d solu ions o he di e en an ioxidan ac ions
and o wo syn he ic compounds wi h an ioxidan p ope ies,
BioMed Resea ch In e na ional 5
BHA and BHT, which a e commonly used in he ood indus-
y, we e p epa ed in me hanol. An aliquo o an ioxidan
(50 𝜇L) was added o 2 mL o DPPH adical me hanolic
solu ion (3.6 ×10−5 M),shaken igo ouslyona o exshake
(MS2 Mini Vo ex Shake IKA), and le o s and in he da k a
oom empe a u e. The abso bance was measu ed a 515 nm,
a e 16mina oom empe a u e,inadual-beamspec opho-
ome e (U ikon XL, Bio-Tek Ins umen s, Milan, I aly). All
de e mina ions we e pe o med in iplica e. The dec ease
in abso bance was con e ed o inhibi ion pe cen age o he
DPPH (IP), acco ding o he ollowing equa ion:
IP =𝐴0−𝐴16
𝐴0×100, (1)
whe e 𝐴0is he abso bance o he con ol a ini ial ime; 𝐴16
is he abso bance o he sample a e 16 minu es.
The concen a ion o an ioxidan compound o ac ion
equi ed o achie e 50% inhibi ion o he adical DPPH
(equi alen concen a ion = EC50) was de e mined om he
linea eg ession cu e ob ained by plo ing he di e en
concen a ions o an ioxidan compound o ac ion used
(wi hin he ange 0.1 o 3.5 g L−1) agains he inhibi ion
pe cen age o he DPPH (IP).
3. Resul s and Discussion
In his s udy, a new by-p oduc (PVPP-WS) was conside ed
as a na u al sou ce o an ioxidan - ich bioac i e compounds
wi h se e al po en ial applica ions.
The ex ac ion p ocedu e used o ob ain he c ude ex ac
has al eady been es ed a labo a o y scale and pilo plan
scale in p e ious s udies. Wi h he o e all aim o enabling
he b ewing indus y o implemen his ex ac ion p ocess in
indus ial plan s, he p esen s udy in es iga ed he scaling-
up o he ex ac ion p ocess and he pu i ica ion o he
bioac i e phenolic compound ex ac ed. The ex ac ion yield
was app oxima ely 0.1%. In he b ewe y indus y, a ound one
li e o his was e s eam (PVPP-WS) can be gene a ed om
e e y 138 L o bee p oduced. App oxima ely 403 million
hec oli es o bee we e p oduced in Eu ope in 2010, which
means ha up o 400 ons o his c ude ex ac could be
ob ained in Eu ope e e y yea [25].
The c ude ex ac mus be p ocessed (by pu i ica ion
and ac iona ion) as i s b own colou would hinde i s use
as a ood addi i e. In addi ion, mo e in o ma ion abou
he composi ion o he c ude ex ac in bioac i e phenolic
compounds could be ob ained om di e en ac ions o
de e mine he co ela ion be ween he an ioxidan ac i i y
and he phenolic compounds o g oup o phenolic com-
pounds p esen in he ac ions.
3.1. Solid-Phase Ex ac ion Resul s. Solid-phase ex ac ion is
gene ally used o sample clean-up, ac iona ion, pu i ica-
ion and/o p econcen a ion o na u al ex ac s. In his s udy,
ele en di e en ly colou ed ac ions con aining phenolic
compounds we e ob ained (see Figu e 1).
3.1.1. Ex ac ion Yield and An ioxidan Ac i i y. The eco e y
yield and he adical sca enging ac i i y we e de e mined
o each ac ion ob ained a each sol en a io applied o
heca idge.The esul sa eshowninTable 2,alongwi h
he colou o each ac ion. The an ioxidan ac i i y o he
syn he ic an ioxidan s commonly used in he ood indus y
was also e alua ed o compa e he po en ial o he na u al
compounds as ood addi i es. The c ude ex ac was also
es ed o e alua e whe he he ac iona ion p ocess yielded
ac ions ha we e mo e o less ac i e han he c ude ex ac .
The mos ac i e ac ions and he bes yields ob ained
in he SPE p ocess co esponded o he i s se en ac ions
elu ed wi h a sol en mix u e om 0%–60% o me hanol.
This showed ha he na u al ex ac and he polyphenolic
compounds a e wa e soluble bu he addi ion o me hanol
yielded he mos ac i e ac ions. The e o e, he ac ion
ob ained wi h 30% ( / ) o me hanol exhibi ed he highes
an ioxidan ac i i y. All he ac ions ha display a no able
le el o an ioxidan ac i i y ( ac ions 1–7) we e colou ed,
pa icula ly ac ions F . 4, F . 5, and F . 6, which also
displayed he highes deg ee o an ioxidan ac i i y agains
he ee adical DPPH. This is consis en wi h he esul s
desc ibed by Wo endem e al. in a s udy e alua ing he ela-
ionship be ween an ioxidan ac i i y and colou o c ys al
mal ex ac s [26].F ac ions8,9,10,and11we ecolou less,
and he an ioxidan ac i i y was lowe han ha o he o he
ex ac s.
The EC50 alues o F . 3, F . 4, F . 5, and F . 6 we e
simila o ha o he syn he ic an ioxidan BHA used in
ood indus y. Excep o he las 4 ac ions yielded, all he
ac ions ob ained om he c ude ex ac showed a highe
DPPH adical sca enging capaci y han he an ioxidan BHT,
also commonly used in ood indus y. In his s udy, he
an ioxidan capaci y o he c ude ex ac (EC50 =0.32gL
−1)
was also calcula ed: (a) o compa e he capaci y o his ex ac
and he ac ions ob ained; and (b) o e alua e whe he he
an ioxidan ac i i y inc eased as a esul o he pu i ica ion
p ocess. Pu i ica ion o he c ude ex ac yielded ac ions
wi h highe an ioxidan ac i i y han he c ude ex ac .
3.1.2. HPLC-DAD-UV Analysis o he An ioxidan F ac ions.
The esul s o he iden i ica ion and quan i ica ion o majo
polyphenolic compounds p esen in each ac ion ob ained
in he ac iona ion p ocess (SPE) by HPLC-DAD-UV a e
shown in Table 3.
The di e en a ios o sol en s yielded di e en ac ions.
These ac ions displayed di e en le els o an ioxidan ac i -
i y because he polyphenolic con en a ies conside ably wi h
solubili y.
The i s ac ions, which exhibi ed he highes le el
o an ioxidan ac i i y (see Table 2), con ained he highes
amoun s o polyphenolic compounds (Table 3). F ac ions 1
and 2 con ain la ge amoun s o galloca echin, which la gely
accoun s o he high adical sca enging ac i i y exhibi ed
by hese wo ac ions. Compounds wi h la onoid s uc u e
such as ca echin gene ally display a highe le el o an ioxidan
ac i i y han non la onoid compounds [27].
F ac ion 4 displayed he highes le el o an ioxidan
ac i i y, mainly due o he high con en o e ulic acid, a
phenolic compound. Howe e , F ac ion 3, which displayed
a simila le el o an ioxidan ac i i y o F ac ion 4, also
6BioMed Resea ch In e na ional
Table 2: Reco e y yield, adical sca enging ac i i y (DPPH), and colou a ion o each ac ion (F .) ob ained by SPE wi h di e en % o
me hanol.
Sample % o me hanol Reco e y yield (% w/w)aDPPH (EC50)bColou
F . 1 0 10.4 0.44 Whea
F . 2 10 18.1 0.30 Bu n o ange
F . 3 20 16.2 0.27 B own
F . 4 30 19.4 0.20 Ma oon
F . 5 40 18.3 0.26 Da k b own
F . 6 50 8.19 0.23 Da k b own
F . 7 60 6.71 0.89 Och e
F . 8 70 1.59 7.02 Colou less
F . 9 80 0.324 6.64 Colou less
F . 10 90 0.615 8.25 Colou less
F . 11 100 0.194 14.3 Colou less
C ude ex ac — — 0.32 Da k b own
BHA — — 0.24 —
BHT — — 2.67 —
aValues exp essed as %o d y c ude ex ac .
bValues exp essed as g L−1 o ex ac ( ac ion).
Table 3: Phenolic compounds p esen in he di e en ac ions, ob ained wi h a LC-18 column, iden i ied and quan i ied by HPLC-DAD.
F ac ion C ude ex ac
An ioxidan compound 1 2 3 4 5 6 7 8 9 10 11 —
mg g−1o ac ion mg g−1
Gallic acid 193 — — — — — — — — — — 20.1
Galloca echin 178 641 — — — — — — — — — 132
P o oca echuic acid — 72.9 9.51 — — — — — — — — 15.5
Epigalloca echin 86.2 96.4 30.6
Ca echin — 114 75.5 — — — — — — — — 29.9
4-Hyd oxybenzoic acid — 6.51 7.49 — — — — — — — — 2.54
Ca eic acid — 13.7 66.0 — — — — — — — — 14.1
Epica echin — — 132 — — — — — — — — 21.4
p-couma ic acid — — 73.9 1.83 — — — — — — — 11.4
Isoque ce in — — 166 — — — — — — — — 28.3
Fe ulic acid — — 29.6 138 — — — — — — — 33.7
Ace osy ingone — — 35.2 50.9 — — — — — — — 14.6
Res e a ol — — — — — 51.6 14.5 — — — — 5.35
Que ce in — — — — 31.7 117 — — — — — 14.5
Apigenin — — — — — 58.8 59.7 — — — — 8.10
Kaemp e ol — — — — — 25.3 23.4 120 — — — 6.06
Na ingenin — — — — — 38.7 171 11.8 — — — 14.6
To al (mg g−1) 371 934 692 191 31.7 292 269 132 — — — 403
con ains la ge amoun s o an ioxidan compounds such
as epigalloca echin, ca eic acid, p-couma ic acid, and iso-
que ce in [11]. Hyd oxycinnamic and hyd oxybenzoic acids
a e known an ioxidan s ha ac as ee adical accep o s
and chain b eake s [28]. Ca eic, e ulic, and p-couma ic
acids ha e been widely s udied and epo ed o be majo
con ibu o s o he an ioxidan ac i i y o bee [27,29]. In
he p esen s udy, he na u al ex ac was ob ained a e he
ea men o he bee , and, as expec ed, his c ude ex ac
was loaded wi h hese bioac i e compounds. F ac ions 2 and
3, which con ain hese compounds, we e he mos ac i e
ee adical sca enge s. These indings may be a ibu ed o
a combined, syne gis ic, and/o addi i e ac ion. This ype o
ac ionhasp e iouslybeenobse ed o esul inaninc eased
an ioxidan po en ial [30].
The eco e y yields and he phenolic con en s o ac ions
6 and 7 we e lowe han hose o he o he ac ions. Howe e ,
bo h o hese ac ions displayed some an ioxidan ac i i y,
mainly due o he la onols. F ac ions 6 and 7 con ain he
la onols que ce in and kaemp e ol a simila concen a ions.
BioMed Resea ch In e na ional 7
Table 4: (a) SFE yield de e mined g a ime ically (w/w) and colou o ac ions A, B, and C. (b) SFE yield de e mined g a ime ically (w/w)
and colou o ac ions D and E. (c) SFE yield de e mined g a ime ically (w/w) and colou o ac ions F and G.
(a)
𝑃(ba ) AB C
Yield (%) Colou Yield (%) Colou Yield (%) Colou
100 0.02 Colou less 0.24 Whea 0.02 Colou less
120 0.02 Colou less 7.71 Da k b own 23.04 Da k b own
140 0.02 Colou less 4.27 Bu n o ange 6.39 B own
160 0.00 Colou less 2.77 Bu n o ange 0.06 Whea
200 0.02 Colou less 12.7 Ma oon 0.01 Colou less
250 0.02 Colou less 8.09 Da k b own 0.07 Och e
300 0.02 Colou less 3.29 Ma oon 0.03 Colou less
To al 0.12% 39.1% 29.6%
(b)
𝑃(ba ) DE
Yield (%) Colou Yield (%) Colou
100 0.89 B own 3.27 Bu n o ange
120 0.73 B own 19.83 Da k b own
140 0.76 B own o ange 5.67 Bu n o ange
160 1.42 Bu n o ange 3.65 Bu n o ange
200 1.13 Bu n o ange 3.84 Bu n o ange
250 0.79 Bu n o ange 2.56 Bu n o ange
300 0.98 Bu n o ange 2.64 Bu n o ange
To al 6.7% 41.5%
(c)
% Modi ie FG
Yield (%) Colou Yield (%) Colou
00.04 O ange 0.13 Whea
0.5 0.07 Co al 0.08 Whea
1.0 0.33 Whea 0.31 Whea
1.5 0.58 Whea 0.28 Whea
20.44 Whea 0.58 Whea
2.5 0.61 Whea 0.41 Whea
31.08 Whea 0.59 Whea
To al 3.15% 2.38%
F ac ions 8, 9, 10, and 11 we e ex ac ed using high con en s o
me hanol, and no phenolic compounds we e de ec ed in he
ac ions.
3.2. Supe c i ical Fluid Ex ac ion. SC-CO2has been used
success ully o pu i y c ude ex ac s by concen a ing he
bioac i e compounds (e.g., an ioxidan s) in he ex ac and
also by emo ing con aminan s. In he p esen s udy, se e al
ac ions we e ob ained om he SFE ac iona ion assay
unde di e en es condi ions (see Figu e 1).
3.2.1. SFE F ac ions (Ex ac ion Yield, Colou , and An ioxi-
dan Ac i i y o Each F ac ion). The ex ac ion yield o he
di e en an ioxidan ac ions ob ained unde he di e -
en ope a ional condi ions (see Table 1)and hecolou o
each ac ion a e shown in Table 4. The ex ac ion yield
anged om 0.12% ( ac iona ion es A), o he ex ac ion
wi hou cosol en (see Table 4(a)), o 41.5% ( ac iona ion
es E) o he ex ac ions in which e hanol was used as
amodi ie (seeTable 4(b)). The esul s showed ha he
use o a modi ie is manda o y essen ial o success ul
ac iona ion o he c ude ex ac . This is consis en wi h da a
epo ed by a ious au ho s who ob ained high ex ac ion
yields om di e se na u al ma ices by using modi ie s
[17].
Resul s showed ha he inc ease in he pe cen age o
modi ie inc eases he amoun o ex ac (see Table 4(b)).
The olume o he ex ac collec ed a each p essu e is mo e
cons an in he low mode (see Table 4(b), es s D and E) han
in p essu e mode (see Table 4(a), es s B and C). Mo eo e ,
8BioMed Resea ch In e na ional
Table 5: An ioxidan ac i i y o SFE ex ac s de e mined by DPPH. Resul s a e exp essed as EC50 (g L−1).
F ac ion
𝑃(ba ) P essu e mode 𝑃(ba ) Flow mode Flow mode
A B C D E % modi ica ion F G
Cell — — 0.23 Cell 0.20 0.22 Cell 0.26 0.20
100 n.d. 2.65 37.73 100 0.33 0.74 021.99 0.64
120 n.d. 0.36 0.41 120 0.51 0.36 0.5 1.57 0.68
140 n.d. 0.27 0.25 140 0.62 0.23 1.0 0.59 3.52
160 n.d. 0.29 0.28 160 0.46 0.21 1.5 0.97 1.42
200 n.d. 0.34 1.00 200 0.74 0.22 20.88 0.80
250 n.d. 0.21 0.20 250 0.34 0.23 2.5 0.64 0.67
300 n.d. 0.10 3.12 300 0.42 0.34 30.54 0.42
Table 6: Phenolic p o ile o each ac ion ob ained unde SFE condi ions o es E: empe a u e 40∘C, ex ac ion ime 30 minu es, modi ie
e hanol (6%), and modi ie low 0.2 mL min−1. Phenolic compounds a e exp essed as mg g−1o ac ion.
Peak no. Phenolic compound SFE ac ions (mg g−1)
ECell E100 E120 E140 E160 E200 E250 E300
1 Gallic acid 1.04 0.24 1.05 1.68 2.61 3.02 3.44 3.07
2 Galloca echin 171 10.7 14.8 33.9 41.0 33.3 29.8 20.9
3 P o oca echuic acid — 1.99 4.12 9.19 12.8 14.9 16.7 16.0
4 Epigalloca echin 1.43 10.7 10.2 — — — — —
5 Ca echin 6.10 3.40 3.36 6.88 11.8 11.7 15.7 16.7
6 4-Hyd oxybeinzoic acid — 3.58 1.96 4.78 4.22 2.72 2.36 1.55
7 Ca eic acid — 7.29 7.38 17.1 19.65 14.8 13.3 9.52
8 Epica echin — — — — 0.23 0.14 0.17 0.40
9 p-couma ic acid — 7.97 5.89 13.5 13.9 9.66 8.45 6.26
10 Isoque ce in 2.51 — — — — 3.71 3.75 1.43
11 Fe ulic acid — 35.7 27.6 69.0 70.1 47.0 38.9 22.0
12 Ace osy ingone — 2.92 0.83 3.62 2.07 1.18 3.10 2.55
13 Res e a ol — — 0.54 1.68 0.37 0.46 0.60 0.80
14 Que ce in 0.57 0.65 0.98 1.91 2.61 2.83 3.13 2.96
15 Apigenin — — 0.04 0.66 0.78 0.72 0.79 0.93
16 Kaemp e ol 1.48 0.94 1.65 3.17 4.42 3.18 6.24 6.64
17 Na ingenin 0.39 0.40 0.78 1.69 2.45 2.75 3.26 3.52
To al (mg g−1) 11.0 88.9 82.5 172.7 192.8 155.9 152.3 117.0
when highe amoun s o he modi ie a e used, less p essu e
is equi ed, as obse ed in es s C ( low a e o 0.2 mL min−1
wice ha o B) and E (6% o modi ie wice ha o D), in
which almos all compounds we e yielded a a p essu e o
120 ba . These esul s indica ed ha he op imal condi ions o
he ex ac ion p ocess o he c ude ex ac a e hose used in
es E, which yielded mos phenolic compounds and ac ions
wi h he highes an ioxidan ac i i ies (see Table 5)simila o
BHA and highe han ha o BHT (see Table 2). Mo eo e ,
unde hese condi ions, he ac ions we e pale , which is an
ad an ageas hes ongb owncolou o hec udeex ac
may hinde i s use as a ood addi i e. Howe e , he ac ions
ob ained in high amoun s we e always da ke , as, o example,
E120 (see Table 4(b)).
The da kes ac ions, which con ained mo e phenolic
compounds pe weigh o ex ac ac ion (see Tables 4(a),
4(b) and 4(c)), displayed a high adical sca enging capaci y
(see Table 5). Colou less ac ions, which did no appea o
con ain phenolic compounds, did no display any sca enging
ac i i y o he adical DPPH. This is u he indica ion ha
ac ions colou depends on he con en o ac i e compounds,
which a e also esponsible o he an ioxidan ac i i y o he
ac ions.
In he p esen s udy, me hanol was also e alua ed as a
modi ie in es G (see Table 4(b)). The esul s showed ha
e hanol is mo e sui able o he ex ac ion p ocess because
he phenolic compounds o in e es we e ob ained wi h a
lowe pe cen age o e hanol (1.5%), while 2% me hanol was
equi ed o ob ain compounds such as e ulic acid and
galloca echin (da a no shown), he main compounds p esen
in he c ude ex ac . Selec ion o a sui able modi ie and/o
adjus men o he modi ie pe cen age is essen ial o suc-
cess ul ac ional sepa a ion o he an ioxidan compounds
p esen in he c ude ex ac .
BioMed Resea ch In e na ional 9
(min)
(mAU)
2500
2000
1500
1000
500
0
1
23
4
56
7
8
9
10
11
12 13 14 15
16
17
5 10152025303540
(a)
(mAU)
(min)
510 15 20 25 30 35 40
300
250
200
150
100
50
0
1
34
56
10 13
1617
(b)
(min)
5 10152025303540
2000
1500
1000
500
0
(mAU)
1
234
56
7
8
9
10
11
12 13 14 15 1617
(c)
Figu e 2: Ch oma og ams acqui ed a 280 nm by HPLC-DAD-UV. (a) Ch oma og am o he c ude ex ac wi hou ac iona ion p ocess. (b)
Ch oma og am o he esidue o he c ude ex ac ha emains in he ex ac ion cell a e he ac iona ion p ocess (ECell). (c) Ch oma og am
o he ac ion ob ained unde op imal condi ions (E140).
3.2.2. HPLC-DAD-UV Analyses o he An ioxidan F ac ions.
The ac ions ob ained by SFE we e analysed by HPLC-DAD-
UV o he cha ac e iza ion o bioac i e compounds in he
c ude ex ac .
Figu e 2 shows, as a ep esen a i e example, he ch o-
ma og am o he c ude ex ac be o e ac iona ion by SFE
(Figu e 2(a)) and he ch oma og ams ob ained a e he ac-
iona ion p ocedu e unde op imal condi ions, he ex ac-
ion cell (Figu e 2(b)) and he ac ion E140 (Figu e 2(c)).
Ch oma og am C (Figu e 2(c)) shows ha SFE ac iona ion
imp o es he pu i y o he phenolic compounds (clea ly
obse ed om hebaselineo hech oma og am) ela i e
o he c ude ex ac (Figu e 2(a)). I is also e iden ha he
impu i ies in he c ude ex ac emain in he ex ac ion cell,
as e i ied by he peak ob ained in ch oma og am B. How-
e e , phenolic compounds and p obably o he compounds
no iden i ied in he p esen s udy emain ac i e in he
ex ac ion cell (ECell), which con e s an ioxidan ac i i y o
he esidue in he ex ac ion cell (see Table 5).
The phenolic compounds p esen in he ac ions
ob ained unde op imal condi ions in his s udy ( ac iona-
ion E) a e shown in Table 6.Thenumbe o phenolic
compounds shown in he able co esponds o he numbe s
in he ch oma og ams in Figu es (2(a),2(b),and2(c)). The
phenolic compounds we e iden i ied and quan i ied in his
s udy o all he ac ions ob ained unde he di e en assay
condi ions (da a no shown). The quali a i e composi ion
o phenolic compounds in he ac ions yielded by SFE
is he same in almos e e y ac ion ob ained unde he
di e en es condi ions assayed (see Table 6). The main
bioac i e phenolic compounds iden i ied in he ac ions
we e as ollows: e ulic acid, p-couma ic acid, ca eic acid,
p o oca echuic acid, ca echin, gallic acid, galloca echina,
and epigalloca echin. These esul s a e consis en wi h hose
ob ained wi h he SPE p ocedu e (see Sec ion 3.1.2.).
The phenolic compounds we e no success ully sepa a ed
by he SFE ac iona ion p ocedu e as in he SPE ac iona ion
(LC-18 column). Howe e , he pu i y o he ac ions yielded
by he SFE was g ea e han ha o he ac ions yielded by
SPE. Mo eo e , la ge amoun s o pu e ac ions con aining
hemainbioac i ephenoliccompoundswe eob ainedby
SFE, which makes his echnique he mos p omising o
pu i ica ion o he c ude ex ac .
Rega ding he an ioxidan ac i i y, he ac ions wi h he
highes con en s o polyphenolic compounds a e hose wi h
he highes an ioxidan ac i i y. Howe e , he an ioxidan
ac i i ies o he di e en ac ions did no di e signi ican ly
because he composi ion o bioac i e compounds was also
e y simila .
4. Conclusions
Indus ial ex ac ion o a na u al ex ac con aining bioac i e
compounds was success ul and could be implemen ed in
he b ewing indus y o eco e a esidue wi h added alue.
The d ied e hyl ace a e c ude ex ac was pu i ied by wo
al e na i e p ocedu es o imp o e i s quali y (an ioxidan
ac i i y and o ganolep ic p ope ies) o po en ial use as a
ood addi i e.
