an ioxidan s
A icle
Op imiza ion o he Ex ac ion o Bioac i e
Compounds om Walnu (Juglans majo 209 x
Juglans egia) Lea es: An ioxidan Capaci y and
Phenolic P o ile
Adela Fe nández-Agulló, Aída Cas o-Iglesias, Ma ía Sonia F ei e and Julia González-Ál a ez *
Depa men o Chemical Enginee ing, School o Enginee ing, Uni e sidade de San iago de Compos ela,
15782 San iago de Compos ela, Spain; [email p o ec ed] (A.F.-A.); [email p o ec ed] (A.C.-I.);
ma iasonia. ei [email p o ec ed] (M.S.F.)
*Co espondence: [email p o ec ed]; Tel.: +34-881816761
Recei ed: 27 No embe 2019; Accep ed: 20 Decembe 2019; Published: 24 Decembe 2019
Abs ac :
This wo k s udies he ex ac ion o phenolic compounds om walnu lea es o he
hyb id Juglans majo 209 x Juglans egia based on ex ac an ioxidan capaci y. Once he solid/liquid
a io was selec ed (1/10 g/mL), by means o a Box-Benkhen expe imen al design, he in luence
o empe a u e (25–75
◦
C), ime (30–120 min), and aqueous e hanol concen a ion (10–90%) on
ex ac ion yield and e ic educing an ioxidan powe (FRAP), 2,2-diphenyl-1-pic ylhyd azyl (DPPH),
and 2,2’-azinobis-3-e hylbenzo hiazoline-6-sul onic acid (ABTS) an ioxidan ac i i ies we e analyzed.
In all cases, he quad a ic e ec o % E OH was he mos signi ican , ollowed by he linea e ec o
empe a u e and, o mos o he esponses, he e ec o ime was almos negligible. Response su ace
analysis allowed o selec he op imal ex ac ion condi ions: 75
◦
C, 120 min and 50% e hanol, which
led o he ollowing ex ac p ope ies: ex ac ion yield, 30.17%; FRAP, 1468 nmol asco bic acid
equi alen s (AAE)/mg ex ac d.b.; DPPH, 1.318 mmol T olox equi alen s (TRE)/g ex ac d.b.; DPPH
EC
50
, 0.11 mg/mL; ABTS, 1.256 mmol TRE/g ex ac (on d y basis) and ABTS EC
50
, 0.985 mg/mL.
Que ce in 3-
β
-D-glucoside, neochlo ogenic acid, and chlo ogenic acid, in his o de , we e he main
compounds iden i ied in his ex ac by ul a-pe o mance liquid ch oma og aphy coupled wi h
elec osp ay ioniza ion and ime-o - ligh mass spec ome y (UPLC/ESI-QTOF-MS), wi h a ious
po en ial applica ions ha suppo his alo iza ion al e na i e o walnu lea es.
Keywo ds:
walnu lea es; Juglans majo 209 x Juglans egia; mace a ion; phenolic compounds;
an ioxidan ac i i y; UPLC/ESI-QTOF-MS; esponse su ace me hodology
1. In oduc ion
Juglans majo 209 x Juglans egia is a walnu hyb id species in ended o p oduce good-quali y
wood. Al hough wood is he main p oduc , he use o o he ac ions ha can be conside ed as
was es, such as he lea es, would con ibu e o mo e p o i able p oduc ion and o a mo e sus ainable
plan a ion managemen .
Walnu lea es ha e been in ensi ely used in adi ional medicine, and a ious s udies ha e
demons a ed he an imic obial and an ioxidan p ope ies o he ex ac s om he lea es o se e al
walnu (Juglans egia) cul i a s [
1
–
4
]. Ca alho e al. [
3
] also demons a ed he an ihemoly ic and
human enal cance cell an ip oli e a i e ac i i ies o walnu lea me hanolic ex ac s. Quan i a i e and
quali a i e de e mina ions o he phenolic compounds p esen in walnu (Juglans egia) lea es ha e also
been ca ied ou [
1
,
4
–
6
] demons a ing qui e signi ican a ia ions in he ex ac composi ion. Ama al e
al. [
5
] iden i ied se en phenolic compounds in me hanol and acidi ied wa e ex ac s o walnu lea es,
An ioxidan s 2020,9, 18; doi:10.3390/an iox9010018 www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2020,9, 18 2 o 14
being que ce in 3-O-galac oside he majo compound while 4-p-couma oylquinic acid was he mino one.
3-O-ca eoylquinic acids and que ce in O-pen oside we e he main phenolic compounds among he
25 phenolic compounds iden i ied in me hanol and decoc ion ex ac s by San os e al. [
4
]. Pe ei a e al. [
1
]
iden i ied 10 compounds in aqueous ex ac s: 3- and 5-ca eoylquinic acids, 3- and 4-p-couma oylquinic
acids, p-couma ic acid, que ce in 3-O-galac oside, que ce in 3-O-pen oside de i a i e, que ce in
3-O-a abinoside, que ce in 3-O-xyloside and que ce in 3-O- hamnoside. In addi ion o phenolic acids
and la onoids, Nou e al. [
6
] also epo ed he p esence o juglone (5-hyd oxy-1,4-nap hoquinone) in
walnu lea es, ellagic acid as he domina ing phenolic acid and my ice in, ca echin hyd a e and u in
as he main la onoids.
Di e en sol en s ha e been used o he ex ac ion o phenolic compounds om walnu lea es:
Aqueous e hanol [
2
,
7
], me hanol [
4
,
5
], me hanol wi h 1% bu yla ed hyd oxy oluene (BHT) [
6
],
wa e [
1
,
4
], acidi ied wa e [
5
], and chlo o o m [
5
]. In his wo k, looking o a g een ex ac ion p ocess,
ex ac ion was pe o med by mace a ion wi h aqueous e hanol, a bio-sol en p oduced om biomass,
comple ely biodeg adable [
8
], and gene ally ecognized as sa e (GRAS) sol en . In addi ion o he ype
o sol en , o he ac o s ha can a ec he e iciency o he ex ac ion p ocess ha we e examined in
his wo k a e he ex ac ion empe a u e, he ex ac ion ime, and he solid o sol en a io [9].
The aim o his wo k was o p o ide a means o he disposal and alo iza ion o walnu lea es
based on ex ac an ioxidan ac i i y, gi ing he walnu plan a ion an addi ional alue o ha p o ided
by wood. As a as we know, his is he i s wo k on he ex ac ion and cha ac e iza ion o phenolic
compounds o walnu lea es o he hyb id Juglans majo 209 x Juglans egia. An expe imen al
design combined wi h esponse su ace me hodology was used o op imize he ex ac ion o
phenolic compounds om walnu lea es based on ex ac an ioxidan ac i i y. The phy ochemical
cha ac e iza ion o he ex ac s was ca ied ou and he esul s compa ed wi h hose p e iously ob ained
o lea es om he Juglans egia species.
2. Ma e ials and Me hods
2.1. Reagen s and S anda ds
Ace ic acid, sodium ace a e, FeCl
3·
6H
2
O, HCl, l-asco bic acid, me hanol, e hanol, and po assium
pe sul a e we e pu chased om Pan eac (Ba celona, Spain). 2,4,6-T ipy idyl-s- iazine (TPTZ), T olox
(6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid) and DPPH (2,2-diphenyl-1-pic ylhyd azyl) we e
pu chased om Fluka (S einheim, Ge many). ABTS (2,2’-azinobis-3-e hylbenzo hiazoline-6-sul onic acid)
and HPLC s anda ds: (–)-epigalloca echin, (–)-galloca echin, ca echin hyd a e, chlo ogenic acid, ellagic
acid, epica echin, e ulic acid, gallic acid, iso ha mne in, kaemp e ol, neochlo ogenic acid, p-couma ic
acid, p ocyanidin B2, que ce in, que ce in 3-
β
-D glucoside, and axi olin, we e ob ained om Sigma
(S einheim, Ge many).
2.2. Ma e ials
Walnu (Juglans majo 209 x Juglans egia) lea es we e collec ed in July 2018 in a plan a ion loca ed
in A Co uña, Spain, d ied a oom empe a u e, milled, sie ed, and he ac ion o pa icle size be ween
0.1 and 1 mm was selec ed and s o ed p o ec ed om ligh ill analysis.
2.3. Ex ac ion
Walnu lea es we e ex ac ed wi h aqueous e hanol in an o bi al shake (UNITRONIC-OR, Selec a
(Ba celona, Spain) wi h empe a u e con ol a a shaking speed o 90 pm. The solid/liquid a io,
empe a u e, ime, and E OH concen a ion we e ixed o each expe imen acco ding o he es ablished
expe imen al planning (Tables 1and 2). The ex ac was sepa a ed by acuum il a ion, concen a ed
in a Büchi R-210 o a apo and inally d ied unde acuum o ob ain a d y powde . Ex ac ion yield
was de e mined as he weigh loss pe cen age o he ini ial walnu lea es.
An ioxidan s 2020,9, 18 3 o 14
The choice o he independen a iables o be analyzed and hei espec i e a ia ion in e als was
based on p e ious in es iga ions on he ex ac ion o phenolic compounds om a ious lignocellulosic
ma e ials using aqueous alcohols [
10
–
12
]. In he i s s age, he in luence o he solid/liquid a io (1/5,
1/7.5 and 1/10 g/mL) was analyzed o ixed alues o he o he a iables: Tempe a u e, 50
◦
C, ime, 60 min,
and e hanol concen a ion, 50% (Table 1). Ex ac ion yield and ex ac e ic educing an ioxidan powe
(FRAP) an ioxidan ac i i y we e de e mined. All he assays we e eplica ed, and he esul s exp essed as
mean alue and s anda d de ia ion. The exis ence o signi ican di e ences among he esul s depending
on he solid/liquid a io used was analyzed by applying one-way ANOVA oge he wi h he Tukey’s es a
a con idence le el o 95% using he IBM SPSS S a is ics 24 so wa e (New Yo k, NY, USA).
Once S/L a io was ixed a 1/10, a Box-Behnken expe imen al design was applied o analyze he
in luence o empe a u e (x
1
; 25, 50, and 75
◦
C), ime (x
2
; 30, 75, and 120 min) and aqueous e hanol
concen a ion (x
3
; 10%, 50%, and 90%) on ex ac ion yield (Y
1
, g ex ac /100 g lea es on d y basis (d.b.))
and ex ac an ioxidan ac i i y de e mined acco ding o he FRAP (Y
2
, nmol AAE/mg ex ac d.b.), DPPH
(Y
3
, mmol TRE/g ex ac d.b.), and ABTS (Y
4
, mmol TRE/g ex ac d.b.) assays. The phenolic p o ile o he
ex ac selec ed as he op imum was analyzed by ul a-pe o mance liquid ch oma og aphy coupled wi h
elec osp ay ioniza ion and ime-o - ligh mass spec ome y (UPLC/ESI-QTOF-MS).
Table 1.
In luence o he solid/liquid a io on ex ac ion yield and e ic educing an ioxidan powe
(FRAP) an ioxidan ac i i y o walnu lea ex ac s (50 ◦C, 60 min, and 50% aqueous e hanol).
Exp. S/L Ra io (g/mL) Ex ac ion Yield (%) FRAP (nmol AAE/mg Ex ac )
A 1/5 25.82 ±0.25 a1246 ±48 a
B 1/7.5 27.04 ±0.42 b1350 ±53 b
C 1/10 27.86 ±0.04 b1512 ±61 c
Values a e p esen ed as mean
±
s anda d de ia ion.
a–c
In each column, alues wi h di e en le e s a e signi ican ly
di e en (p<0.05).
Table 2.
Box-Benkhen expe imen al design wi h he expe imen al and p edic ed alues o he esponses.
Exp x*
1x*
2x*
3Y1 exp Y1 p ed Y2 exp Y2 p ed Y3 exp Y3 p ed Y4 exp Y4 p ed
1−1−1 0 21.03 ±0.89 22.35 1181 ±39 1262 1.114 ±0.026 1.050 0.951 ±0.026 0.964
2−1 0 −1 20.35 ±0.18 19.86 842 ±46 908 0.663 ±0.044 0.666 0.671 ±0.027 0.774
3−1 0 1 19.15 ±2.13 18.71 930 ±47 908 0.873 ±0.013 0.852 0.807 ±0.017 0.774
4−1 1 0 23.89 ±0.28 24.16 1250 ±58 1262 1.008 ±0.159 1.050 1.053 ±0.049 0.964
5 0 −1−1 23.13 ±0.27 22.85 1160 ±37 1041 0.913 ±0.024 0.932 0.893 ±0.041 0.875
6 0 −1 1 20.03 ±1.16 19.96 1138 ±17 1041 0.919 ±0.039 0.932 0.866 ±0.050 0.875
7 0 1 −1 24.52 ±0.11 24.66 943 ±65 1041 0.873 ±0.037 0.932 0.869 ±0.030 0.875
8 0 1 1 21.93 ±1.76 21.77 876 ±18 1041 1.003 ±0.036 0.932 0.875 ±0.028 0.875
9 1 −1 0 29.08 ±1.43 28.40 1364 ±42 1529 1.493 ±0.106 1.396 1.166 ±0.047 1.250
10 1 0 −1 27.01 ±0.30 27.64 1199 ±50 1174 1.193 ±0.021 1.198 1.089 ±0.035 1.060
11 1 0 1 22.35 ±0.88 23.02 1238 ±32 1174 1.031 ±0.023 1.012 1.095 ±0.032 1.060
12 1 1 0 30.17 ±0.09 30.21 1468 ±20 1529 1.318 ±0.049 1.396 1.256 ±0.017 1.250
13 0 0 0 25.85 ±0.05 26.28 1389 ±78 1395 1.239 ±0.043 1.223 1.299 ±0.049 1.315
14 0 0 0 27.19 ±0.51 26.28 1560 ±49 1395 1.190 ±0.031 1.223 1.330 ±0.032 1.315
Independen Va iables Le els
−1 0 +1
x1, Tempe a u e (◦C) 25 50 75
x2, Time (min) 30 75 120
x3, % E OH 10 50 90
Expe imen al alues a e p esen ed as mean
±
s anda d de ia ion. Y
1
, ex ac ion yield (g ex ac /100 g lea es d.b.);
an ioxidan ac i i y: Y
2
, FRAP (nmol AAE/mg ex ac d.b.); Y
3
, DPPH (mmol TRE/g ex ac d.b.); Y
4
, ABTS (mmol
TRE/g ex ac d.b.); x∗
1, codi ied empe a u e; x∗
2, codi ied ime; x∗
3, codi ied e hanol concen a ion.
2.4. Box-Behnken Expe imen al Design
The Box-Behnken expe imen al design applied consis ed o 12 eplica ed expe imen s and
2 eplica es in he cen al poin (Table 2). The expe imen s we e andomized o a oid unp edic able
e ec s on he esponses. Expe imen al esul s we e analyzed using he IBM SPSS S a is ics 24 so wa e
and i ed o polynomials o he o m:
An ioxidan s 2020,9, 18 4 o 14
Y=a0+
3
X
i=0
aix∗
i+
2
X
i=1
3
X
j=2
j>i
aijx∗
ix∗
j+
3
X
i=1
aii x∗2
i(1)
whe e Yis he dependen a iable o esponse, a
0
is a scaling cons an , a
i
ep esen s he linea coe icien s,
a
ij
he in e ac ion coe icien s, a
ii
he quad a ic coe icien s, and
x∗
i
he independen a iables coded a h ee
le els:
−
1 (lowe limi ), 0 (cen al poin ), and +1 (uppe limi ) (Table 2). Analysis o a iance (ANOVA)
was applied o de e mine he alidi y o he quad a ic model as well as he s a is ical signi icance o he
eg ession coe icien s a a 95% con idence le el. Mo eo e , o con i m he model’s accu acy, p edic ed
alues o each dependen a iable we e calcula ed and compa ed wi h he expe imen al ones (Table 2).
The equa ions ob ained o each dependen a iable we e isualized as esponse su ace plo s.
2.5. Analy ical Techniques
In o de o ha e a mo e comple e cha ac e iza ion o he an ioxidan ac i i y o he ex ac s,
a ious me hods we e used, namely, he DPPH and ABTS assays based on he capaci y o sca enge
ee adicals and he FRAP assay ha measu es he capaci y o educe a me al ion.
The DPPH adical sca enging abili y o he ex ac s was de e mined ollowing he me hod p oposed
by Ba ei a e al. [
13
] modi ied as desc ibed in V
á
zquez e al. [
11
]. The esul s we e exp essed as mmol
T olox equi alen (TRE) pe g ex ac d.b. and as he EC
50
alue, o ex ac concen a ion necessa y o
achie e a 50% DPPH adical inhibi ion. ABTS sca enging ac i i y was de e mined acco ding o he me hod
o Re e al. [
14
], and he esul s exp essed as mmol T olox equi alen (TRE) pe g ex ac d.b. and as he
EC
50
alue. The FRAP assay was done acco ding o Szöllösi and Szöllösi-Va ga [
15
]. The esul s we e
exp essed as nmol asco bic acid equi alen (AAE) pe mg ex ac d.b.
Phenolic compounds in he ex ac selec ed as he op imum we e de e mined by
UPLC/ESI-QTOF-MS using a B uke Elu e UHPLC (Bille ica, MA, USA) and a B uke TimsTOF
(Bille ica, MA, USA). Sepa a ions we e pe o med using a B uke In ensi y Solo C18 2
µ
m (2.1 mm
×
100 mm) column (Bille ica, MA, USA) and a bina y g adien o 0.1% aqueous o mic acid o mobile
phase A and 0.1% o mic acid in me hanol o mobile phase B a a low a e o 0.25 mL/min. The LC
g adien was 5% B om 0 o 0.4 min, om 5% o 35% B om 0.4 o 0.5 min, om 35% o 100% B om
0.5 o 7 min, 100% B om 7 o 12 min, om 100% o 5% B om 12 o 12.1 min and 5% B om 12.1 o
15 min. Table 3shows he eg ession equa ion o each s anda d compound analyzed. As obse ed,
all he compounds showed good linea i y in a ela i ely wide concen a ion ange.
Table 3.
Calib a ion cu es o he s anda d compounds analyzed by ul a-pe o mance liquid
ch oma og aphy coupled wi h elec osp ay ioniza ion and ime-o - ligh mass spec ome y
(UPLC/ESI-QTOF-MS).
Compound Linea Range (mg/L) Calib a ion Cu e R2
(–)-Galloca echin 1–200 y=8068x+17,932 0.9941
Ca echin hyd a e 1–200 y=15,461x+52,943 0.9958
Chlo ogenic acid 1–1000 y=8181x+22,563 0.9936
Ellagic acid 1–1000 y=9647x+38,987 0.9959
Epica echin 1–1000 y=9780x+17,102 0.9976
Fe ulic acid 1–200 y=3455x+22,085 0.9936
Gallic acid 1–1000 y=3814x+4250 0.9977
Iso ha mne in 1–200 y=61,453x+78,552 0.9915
Kaemp e ol 1–200 y=61,712x+75,935 0.9923
Neochlo ogenic acid 1–200 y=10,675x+15,125 0.9989
p-Couma ic acid 1–200 y=4442x+4535 0.9972
P ocyanidin B2 1–200 y=4014x+7252 0.9925
Que ce in 1–1000 y=45,006x+111,541 0.9922
Que ce in 3-β-d-glucoside 1–1000 y=13,239x+42,498 0.9836
Taxi olin 1–200 y=21,398x+28,956 0.9903
An ioxidan s 2020,9, 18 5 o 14
3. Resul s and Discussion
3.1. E ec o he Solid/Liquid Ra io on Ex ac ion Yield and Ex ac An ioxidan Ac i i y
The in luence o he sol en o walnu lea es a io on he ex ac ion yield and ex ac FRAP
an ioxidan ac i i y is shown in Table 1. The ex ac ion yield was posi i ely in luenced by dec easing S/L
om 1/5 o 1/7.5. Howe e , a u he dec ease o 1/10 didn’ imp o e he ex ac ion yield signi ican ly.
On he o he hand, he ex ac FRAP an ioxidan ac i i y inc eased wi h dec easing he S/L a io in
he whole ange analyzed. These esul s we e in ag eemen wi h p e ious esea ch on he ex ac ion
o phenolic compounds om a ious plan ma e ials [
7
,
16
,
17
] and can be explained by analyzing
he concen a ion g adien o phenolics be ween walnu lea es and he sol en . The lowe he S/L
a io, he g ea e he concen a ion g adien and consequen ly he ex ac ion a e. The e o e, a S/L
a io o 1/10 was selec ed o subsequen expe imen s on analyzing he impac o empe a u e, ime,
and e hanol concen a ion on he ex ac ion o phenolic compounds om walnu lea es. A g ea e
dec ease in he S/L a io was no conside ed since i would mean no only a highe sol en consump ion
bu also a highe ene gy cos in he ex ac ion and sol en eco e y s ages.
3.2. Rela ionship be ween Ex ac An ioxidan P ope ies
An ioxidan s may espond in a di e en manne o di e en adical o oxidan sou ces [
18
].
Fo his eason, h ee me hods based on di e en eac ion mechanisms we e used o de e mine
he an ioxidan ac i i y o walnu lea ex ac s and he ela ionship be ween hem was analyzed.
A eg ession analysis was pe o med be ween he alues o he FRAP, DPPH, and ABTS an ioxidan
ac i i ies o he expe imen s in Table 2. As shown in Figu e 1, qui e good posi i e linea ela ionships
we e ound be ween he h ee me hods used o measu e he an ioxidan ac i i y, which sugges s ha
only one me hod could be used in p ac ice o p o ide eliable in o ma ion on he an ioxidan p ope ies
o walnu lea ex ac s.
An ioxidan s 2020, 9, x FOR PEER REVIEW 5 o 14
Kaem
p
e ol 1–200
y
= 61
,
712x + 75
,
935 0.9923
Neochlo o
g
enic acid 1–200
y
= 10
,
675x + 15
,
125 0.9989
p
-Couma ic acid 1–200
y
= 4442x + 4535 0.9972
P oc
y
anidin B2 1–200
y
= 4014x + 7252 0.9925
Que ce in 1–1000
y
= 45
,
006x + 111
,
541 0.9922
Que ce in 3-
β
-D-
g
lucoside 1–1000
y
= 13
,
239x + 42
,
498 0.9836
Taxi olin 1–200
y
= 21
,
398x + 28
,
956 0.9903
3. Resul s and Discussion
3.1. E ec o he Solid/Liquid Ra io on Ex ac ion Yield and Ex ac An ioxidan Ac i i y
The in luence o he sol en o walnu lea es a io on he ex ac ion yield and ex ac FRAP
an ioxidan ac i i y is shown in Table 1. The ex ac ion yield was posi i ely in luenced by dec easing
S/L om 1/5 o 1/7.5. Howe e , a u he dec ease o 1/10 didn’ imp o e he ex ac ion yield
signi ican ly. On he o he hand, he ex ac FRAP an ioxidan ac i i y inc eased wi h dec easing he
S/L a io in he whole ange analyzed. These esul s we e in ag eemen wi h p e ious esea ch on he
ex ac ion o phenolic compounds om a ious plan ma e ials [7,16,17] and can be explained by
analyzing he concen a ion g adien o phenolics be ween walnu lea es and he sol en . The lowe
he S/L a io, he g ea e he concen a ion g adien and consequen ly he ex ac ion a e. The e o e,
a S/L a io o 1/10 was selec ed o subsequen expe imen s on analyzing he impac o empe a u e,
ime, and e hanol concen a ion on he ex ac ion o phenolic compounds om walnu lea es. A
g ea e dec ease in he S/L a io was no conside ed since i would mean no only a highe sol en
consump ion bu also a highe ene gy cos in he ex ac ion and sol en eco e y s ages.
3.2. Rela ionship be ween Ex ac An ioxidan P ope ies
An ioxidan s may espond in a di e en manne o di e en adical o oxidan sou ces [18]. Fo
his eason, h ee me hods based on di e en eac ion mechanisms we e used o de e mine he
an ioxidan ac i i y o walnu lea ex ac s and he ela ionship be ween hem was analyzed. A
eg ession analysis was pe o med be ween he alues o he FRAP, DPPH, and ABTS an ioxidan
ac i i ies o he expe imen s in Table 2. As shown in Figu e 1, qui e good posi i e linea ela ionships
we e ound be ween he h ee me hods used o measu e he an ioxidan ac i i y, which sugges s ha
only one me hod could be used in p ac ice o p o ide eliable in o ma ion on he an ioxidan
p ope ies o walnu lea ex ac s.
(a) (b)
Figu e 1. Linea ela ionships be ween FRAP and DPPH (2,2-diphenyl-1-pic ylhyd azyl) and ABTS
(2,2-azinobis-3-e hylbenzo hiazoline-6-sul onic acid) an ioxidan ac i i ies (a) and be ween DPPH
and ABTS an ioxidan ac i i ies (b).
Figu e 1.
Linea ela ionships be ween FRAP and DPPH (2,2-diphenyl-1-pic ylhyd azyl) and ABTS
(2,2-azinobis-3-e hylbenzo hiazoline-6-sul onic acid) an ioxidan ac i i ies (
a
) and be ween DPPH and
ABTS an ioxidan ac i i ies (b).
3.3. Op imiza ion o he Ex ac ion Condi ions by a Box-Behnken Design
In o de o de e mine he bes ex ac ion condi ions o he ex ac ion o phenolic compounds
om walnu lea es, a Box-Behnken expe imen al design was applied. The independen a iables
selec ed we e empe a u e, ime, and e hanol concen a ion. The ex ac ion condi ions oge he wi h
he expe imen al esul s ob ained o he dependen a iables: Ex ac ion yield (Y
1
), FRAP (Y
2
), DPPH
(Y
3
), and ABTS (Y
4
) an ioxidan ac i i ies a e shown in Table 2, ha also shows he good ag eemen
be ween expe imen al and p edic ed alues. The i ing model (Equa ion (1)) was ound app op ia e
o ep esen all he esponses analyzed (p<0.05). Table 4shows he signi ican eg ession coe icien s
oge he wi h he s a is ical pa ame e s used o e alua e he i ing esul s. Fo all he esponses,
An ioxidan s 2020,9, 18 6 o 14
he quad a ic e ec o %E OH (in nega i e mode) was he mos signi ican , ollowed by he linea e ec
o empe a u e (in posi i e mode), and he e ec s on which ex ac ion ime was in ol ed we e he
leas signi ican o no signi ican a all. Equa ions (2)–(5) show he dependence o he esponses Y
1
o
Y4on he signi ican independen a iables, double in e ac ions, and quad a ic e ec s:
Y1(%)=26.28 +3.024 x∗
1+0.905 x∗
2−1.444 x∗
3−0.865 x∗
1x∗
3−3.973 x∗2
3(2)
Y2(nmol AAE/mg ex ac d.b.)=1395.25 +13.25 x∗
1−354.50 x∗2
3(3)
Y3(mmol TRE/g ex ac d.b.)=1.223 +0.173 x∗
1−0.093 x∗
1x∗
3−0.291 x∗2
3(4)
Y4(mmol TRE/g ex ac d.b.)=1.315 +0.143 x∗
1−0.083 x∗2
1−0.125 x∗2
2−0.315 x∗2
3(5)
Figu e 2a–c show he esponse su aces o he ex ac ion yield (Y
1
) in he unc ion o empe a u e
and e hanol concen a ion o ex ac ion imes o 30 (a), 75 (b), and 120 min (c). Ex ac ion yield a ied
be ween 17.80% and 30.55% depending on he ex ac ion condi ions. Rega ding he in luence o he
linea e ec s, empe a u e and ime we e signi ican in he posi i e mode, whe eas %E OH in he
nega i e mode. Only he quad a ic e ec o %E OH and he double in e ac ion empe a u e x%E OH
had signi ican in luences on he esponse, bo h in he nega i e mode. The highes ex ac ion yield in
he anges essayed o 30.55% was a ained a he highes empe a u e and ex ac ion ime essayed,
75 ◦C and 120 min, and a an in e media e e hanol concen a ion, 38%.
Figu es 3–5show, espec i ely, he esponse su aces o FRAP (Y
2
), DPPH (Y
3
), and ABTS
(Y
4
) an ioxidan ac i i ies in unc ion o empe a u e and e hanol concen a ion. All show a simila
end wi h espec o he in luence o empe a u e and %E OH, showing he p esence o a maximum
loca ed in he icini y o he design space o FRAP and DPPH and in he case o ABTS wi hin i .
Addi ionally, he esponses o FRAP and DPPH we e independen o ime. FRAP anged be ween
908 and 1529 nmol AAE/mg ex ac d.b. (Figu e 3) and depended only on he nega i e quad a ic e ec
o % E OH and he linea e ec o empe a u e in posi i e mode, eaching he highes alue in he
ange assayed, a 75
◦
C and 50% e hanol. Wi h espec o DPPH, i depended addi ionally on he
in e ac ion empe a u e x%E OH in he nega i e mode, and a alue o 1.40 mmol TRE/g ex ac d.b.
was eached a 75
◦
C and 44% e hanol (Figu e 4). ABTS depended on he quad a ic e ec s o %E OH,
ime and empe a u e, in his o de o impo ance, all in he nega i e mode, and on he posi i e linea
e ec o empe a u e. Fo ABTS, a maximum loca ed in he egion s udied, 1.38 mmol TRE/g ex ac
d.b., was eached a 71.3 ◦C, 75 min, and 50% e hanol (Figu e 5).
In summa y, sol en concen a ion, empe a u e, and ex ac ion ime, in his o de , a e impo an
ac o s in luencing he ex ac ion o phenolic compounds om walnu lea es. Bo h ex ac ion yield
and ex ac an ioxidan ac i i y depended signi ican ly on he % e hanol used and aqueous solu ions
in he ange o 38% o 50% e hanol we e ound o be supe io o he mo e concen a ed ones in
any o he sol en s, beha io ha migh be explained by he “likes dissol es like” p inciple [
19
].
High e hanol concen a ions dissol e mo e lipophilic compounds, whe eas highe p opo ions o
hyd ophilic compounds a e ex ac ed a low e hanol concen a ions [
20
]. O he wise, ex ac ion
yield inc eases when dec easing %E OH up o 38%, whe eas ex ac an ioxidan capaci y diminishes,
which can be explained due o he solubiliza ion o o he ypes o compounds such as p o eins and
polysaccha ides ha impai selec i i y [
21
]. Acco ding o hese esul s, he e hanol concen a ion
selec ed as he op imum was 50%. Conce ning ex ac ion empe a u e, a empe a u e ise o up o 75
◦
C
inc eased he ex ac ion yield as he ex ac ion p ocess was a o ed by empe a u e as he solubili y
o phenolic compounds and he mass ans e a e we e enhanced [
22
]. Mo eo e , FRAP, DPPH,
and ABTS an ioxidan ac i i y o he ex ac s also inc eased wi h inc easing ex ac ion empe a u e o
75
◦
C. Howe e , g ea e ex ac ion empe a u es we e no ecommended as oxida ion, epime iza ion,
and deg ada ion o phenolic compounds was p omo ed [
17
] wi h he consequen dec ease o he ex ac
an ioxidan ac i i y. The e o e, 75
◦
C was selec ed as he op imum empe a u e. Finally, 120 min
was he selec ed ime o a o he ex ac ion yield, as i s in luence on an ioxidan p ope ies was
An ioxidan s 2020,9, 18 7 o 14
almos negligible. Longe ex ac ion imes we e no p oposed since he e ec s we e he same as hose
p e iously indica ed o high empe a u es [17].
In b ie , he op imal condi ions o he ex ac ion o phenolic compounds om Juglans majo
209 x Juglans egia lea es selec ed by analyzing oge he he esponse su aces o all he dependen
a iables (Figu es 2–5) we e hose co esponding o Expe imen 12 (Table 2): 75
◦
C, 120 min, and 50%
e hanol. Unde hese condi ions, he p edic ed esponses we e: Y
1, p ed
=30.21%, Y
2, p ed
=1529 nmol
AAE/mg ex ac d.b., Y
3, p ed
=1.396 mmol TRE/g ex ac d.b. and Y
4, p ed
=1.25 mmol TRE/g ex ac
d.b. These alues we e equal o e y close o hose co esponding o he model op imum o each
esponse ha we e Y
1,p ed
=30.55% a 75
◦
C, 120 min, and 38% E OH, Y
2,p ed
=1529 nmol AAE/mg
ex ac d.b. a 75
◦
C and 50% E OH, Y
3,p ed
=1.40 mmol TRE/g ex ac d.b. a 75
◦
C and 44% E OH and
Y
4,p ed
=1.38 mmol TRE/g ex ac d.b. a 71.3
◦
C, 75 min and 50% E OH. Mo eo e , hese ex ac ion
condi ions we e close o hose ound by Viei a e al. [
7
] as he global op imum condi ions o he
ex ac ion o some o he main phenolic compounds ound in his wo k, namely, 3-O-ca eoylquinic
acid and que ce in 3-O-glucoside, om walnu (Juglans egia) lea es by mace a ion wi h aqueous
e hanol (61.3
◦
C, 112.5 min, and 50.4% e hanol). 50% e hanol also led o highe an ioxidan ac i i ies o
walnu (Juglans egia) g een husk ex ac s han pu e wa e and e hanol, al hough he highes ex ac ion
yield was ob ained wi h wa e [23].
Table 4. Coe icien s o he models (Equa ion (1)) and s a is ical pa ame e s.
Y1Y2Y3Y4
Coe . SE pCoe SE pCoe . SE pCoe . SE p
a026.281 0.274 0.000 1395.250
40.672
0.000 1.223 0.019 0.000 1.315 0.028 0.000
a13.024 0.274 0.000 13.25
40.672
0.006 0.173 0.019 0.000 0.143 0.019 0.000
a20.905 0.274 0.008 - - NS - - NS - - NS
a3−1.444 0.274 0.000 - - NS - - NS - - NS
a12 - - NS - - NS - - NS - - NS
a13 −0.865 0.387 0.049 - - NS −
0.093
0.027 0.005 - - NS
a23 - - NS - - NS - - NS - - NS
a11 - - NS - - NS - - NS −
0.083
0.028 0.012
a22 - - NS - - NS - - NS −
0.125
0.028 0.001
a33 −3.973 0.387 0.000 −354.500
57.519
0.000 −
0.291
0.027 0.000 −
0.315
0.028 0.000
R20.964 0.789 0.947 0.951
R
2
co .
0.947 0.757 0.934 0.933
SE 0.774 115.038 0.053 0.055
p0.000 0.000 0.000 0.000
NS: Non-signi ican o a 95% con idence le el; SE: S anda d e o ; p: P obabili y; R
2
: Reg ession coe icien ; R
2c
:
Co ec ed eg ession coe icien ; a
0
: Scaling cons an ; a
i
: Linea coe icien s; a
ij
: In e ac ion coe icien s; a
ii
: Quad a ic
coe icien s; Y
1
, ex ac ion yield (g ex ac /100 g lea es d.b.); an ioxidan ac i i y: Y
2
, FRAP (nmol AAE/mg ex ac
d.b.); Y3, DPPH (mmol TRE/g ex ac d.b.); Y4, ABTS (mmol TRE/g ex ac d.b.).
An ioxidan s 2020,9, 18 8 o 14
An ioxidan s 2020, 9, x FOR PEER REVIEW 8 o 14
Figu e 2.
Response su aces o ex ac ion yield (Y
1
, %) in unc ion o empe a u e and e hanol
concen a ion o ex ac ion imes o (a) 30, (b) 75, and (c) 120 min.
An ioxidan s 2020,9, 18 9 o 14
An ioxidan s 2020, 9, x FOR PEER REVIEW 9 o 14
Figu e 2. Response su aces o ex ac ion yield (Y1, %) in unc ion o empe a u e and e hanol
concen a ion o ex ac ion imes o (a) 30, (b) 75, and (c) 120 min.
Figu e 3. Response su aces o FRAP an ioxidan ac i i y (Y2, nmol AAE/mg ex ac d.b.) in he
unc ion o empe a u e and e hanol concen a ion.
Figu e 4. Response su aces o DPPH an ioxidan ac i i y (Y3, mmol TRE/g ex ac d.b.) in he
unc ion o empe a u e and e hanol concen a ion.
Figu e 3.
Response su aces o FRAP an ioxidan ac i i y (Y
2
, nmol AAE/mg ex ac d.b.) in he
unc ion o empe a u e and e hanol concen a ion.
An ioxidan s 2020, 9, x FOR PEER REVIEW 9 o 14
Figu e 2. Response su aces o ex ac ion yield (Y1, %) in unc ion o empe a u e and e hanol
concen a ion o ex ac ion imes o (a) 30, (b) 75, and (c) 120 min.
Figu e 3. Response su aces o FRAP an ioxidan ac i i y (Y2, nmol AAE/mg ex ac d.b.) in he
unc ion o empe a u e and e hanol concen a ion.
Figu e 4. Response su aces o DPPH an ioxidan ac i i y (Y3, mmol TRE/g ex ac d.b.) in he
unc ion o empe a u e and e hanol concen a ion.
Figu e 4.
Response su aces o DPPH an ioxidan ac i i y (Y
3
, mmol TRE/g ex ac d.b.) in he unc ion
o empe a u e and e hanol concen a ion.
