Sus ainable Chemis y and Pha macy 24 (2021) 100544
A ailable online 4 No embe 2021
2352-5541/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Ex ac ion o la onoid compounds om ba k using sus ainable
deep eu ec ic sol en s
Ley e Sille o
a
, Raquel P ado
b
, Tom Wel on
b
, Jalel Labidi
a
,
*
a
Uni e si y o Basque Coun y UPV/EHU, Depa men o Chemical and En i onmen al Enginee ing, Bio e ine y P ocesses Resea ch G oup, Plaza
Eu opa 1, 20018, Donos ia-San Sebas ian, Spain
b
Impe ial College London, Chemis y Depa men , 80 Wood Lane, W12 OZ, London, UK
ARTICLE INFO
Keywo ds:
Fla onoids
Deep eu ec ic sol en s
Ba k
Ex ac ion
ABSTRACT
The use o g een sol en s in ex ac ion p ocesses, especially o applica ions o lignocellulosic
biomass, has been ex ensi ely s udied o e he las yea s. Among he ange o di e en g een
sol en s, deep eu ec ic sol en s (DES) show p omising esul s o ex ac ion p ocesses. The e o e,
he aim o his wo k was he use o DES as addi i es in aqueous mix u es o he selec i e
ex ac ion o la onoid compounds om he ba k o La ix decidua. Fo his pu pose, ba k has been
ea ed using di e en sol en a ios consis ing o a DES/H
2
O mix u e (0, 25, 50 and 75 w %).
Two DES we e s udied, choline chlo ide:u ea and choline chlo ide:1,4-bu anediol. In o de o
s udy he success o he ex ac ions, he ex ac s and he emaining solid ac ion we e cha ac-
e ised. F om he esul s, i was concluded ha he choline chlo ide:1,4-bu anediol (75 w %) ga e
he bes esul s, ob aining he iches ex ac s in la onoids (383 mg CE/g d ied ba k ex ac ), as
well as hose wi h he highes an ioxidan capaci y. These good esul s con i m he capaci y o
his DES o ob ain ac i e biomolecules o u he applica ion.
1. In oduc ion
Bio e ine ies, de ined by he In e na ional Ene gy Agency (IEA) as “ he sus ainable ans o ma ion o biomass in o a spec um o
ma ke able bio-based p oduc s (chemicals, ma e ials) and bioene gy (bio uels, powe , hea )" (Rombau e al., 2014), a e seen as a key
pilla o he de elopmen o a bioeconomy-based socie y. In his way, he wide ange o bio uels and biop oduc s ha can be ob ained
om a wide a ie y o biomass sou ces, especially lignocellulosic biomass, opens up he possibili y o de eloping new, mo e sus-
ainable p ocesses and p oduc s o d i e he ans o ma ion om he cu en si ua ion, which is mainly based on pe oleum and i s
de i a i es, o one based on he ci cula bioeconomy. The e o e, all ac ions o lignocellulosic biomass mus be s udied o be alo ised,
om he s uc u al compounds (cellulose, lignin and hemicelluloses) o he mino compounds such as ex ac i es and ino ganic
compounds, among o he s.
T ee ba ks a e an impo an sou ce o phenolic compounds (Sk ypnik e al., 2019). Ne e heless, he ex ac ion and sepa a ion o
he di e en compounds in lignocellulosic biomass is no an easy ask, mainly because o i s s uc u al complexi y (Mi anda e al.,
2012). As a esul , he selec ion o a success ul ex ac ion p ocess becomes e y impo an .
Al hough i is ue ha ee ba k is ich in ex ac i es, especially in compa ison wi h wood, his alue no mally does no exceed 30%
in weigh o he ba k (Sille o e al., 2019). Mo eo e , his ac ion is composed o a la ge a ie y o di e en compounds (Dou e al.,
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (J. Labidi).
Con en s lis s a ailable a ScienceDi ec
Sus ainable Chemis y and Pha macy
jou nal homepage: www.else ie .com/loca e/scp
h ps://doi.o g/10.1016/j.scp.2021.100544
Recei ed 25 May 2021; Recei ed in e ised o m 11 Sep embe 2021; Accep ed 4 Oc obe 2021
Sus ainable Chemis y and Pha macy 24 (2021) 100544
2
2016), which means ha he p opo ion o each compound is small. Fo ins ance, low pe cen ages o phenolic compounds we e
quan i ied by Sille o e al. (2019), who epo ed pe cen ages anging om 2 o 9% o phenolic compounds in he ex ac s. The e o e, in
o de o make he ex ac ion p ocess e icien , i is necessa y o choose no only he app op ia e ex ac ion echnique, bu also he mos
selec i e sol en . The use o a selec i e sol en can imp o e he e iciency o he ex ac ion, educing he subsequen pu i ica ion
s ages. A his poin , he p inciples o g een chemis y mus also be conside ed wi h he aim o ca ying ou mo e en i onmen ally
iendly p ocesses ha elimina e he use o haza dous subs ances. Fo hese pu poses, he mos commonly used sol en s should be
eplaced by mo e en i onmen ally iendly al e na i es such as H
2
O, e hanol o no el mode n sol en s ha a e being s udied, such as
ionic liquids (ILs) o deep eu ec ic sol en s (DES).
The inc easing in e es in he use o na u al compounds in eplacemen o ossil uel de i a i es has led o a conside able inc ease in
he in e es o he applica ion o DES o he ex ac ion o bioac i e compounds om lignocellulosic biomass (Cunha and Fe nandes,
2018; Pai a e al., 2014). DES a e compounds o med by combining a hyd ogen bond accep o (HBA) and a hyd ogen bond dono
(HBD) o yield a mix u e wi h a mel ing poin below hose o ei he o he pu e componen s. They a e o med by mixing wo o mo e
non- oxic compounds ha a e also cheap, enewable and biodeg adable, which o m an eu ec ic mix u e (Cunha and Fe nandes, 2018;
Zhang e al., 2012), which con e s upon i unique p ope ies. Some o hei mos ele an p ope ies a e good chemical s abili y,
negligible apou p essu e, and uneable solubili y, among o he s (C je ko Bubalo e al., 2018). Due o hei p ope ies, DES a e
conside ed as g een and designe sol en s (Mbous e al., 2017), which has led o hem becoming one o he mos popula sol en s,
along wi h ILs. The e a e many di e en combina ions o he syn hesis o ILs and DES, which esul s in a e y la ge amily o
compounds. This allows he adap a ion o p ope ies such as iscosi y, pola i y, mel ing poin and solubili y (Veka iya, 2017) in o de
o acili a e and op imise he ex ac ion o he a ge compounds (Passos e al., 2014).
Among he di e en bioac i e compounds belonging o he ex ac i e ac ion o biomass, la onoids a e gaining in impo ance.
They a e becoming popula as pa o ba k ex ac i es because hey a e bioac i e compounds, which means ha hey a e capable o
modula ing di e en biological ac i i ies (O ega and Campos, 2019). As a esul o his p ope y, la onoids ha e a g ea numbe o
bene i s, such as an i-alle gic, an ioxidan , asop o ec i e, and an i-in lamma o y p ope ies, among o he s (Kesa ka e al., 2009).
An ioxidan capaci y is one o he mos impo an o hese, as i p e en s he oxida ion p ocess, allowing he p o ec ion o o he
compounds such as lipids, DNA and p o eins in biological sys ems (C eji´
c e al., 2017). Thanks o hei p ope ies, hei applica ion in
di e en ields is inc easing, om ood o pe sonal ca e indus ies, and e en in he c ea ion o new bio-based ma e ials (Feng e al.,
2013; Maimoona e al., 2011; M´
a mol e al., 2019).
The e o e, knowing he excellen po en ial o ee ba k as a na u al sou ce o la onoids, he combina ion o his and ex ac ion wi h
DES becomes a ac i e wi h he aim o mo e sus ainable ex ac ion p ocesses. Hence, in e es in la onoids ex ac ion om na u al
sou ces coupled wi h he eno mous po en ial ha DES ha e o he selec i e ex ac ion o hese compounds, has esul ed in he ecen
g ow h o his a ea. I is impo an no o igno e he ac ha he DES p ope ies allow ope a ing a lowe empe a u es wi h he
consequen ad an ages ha i can gene a e, om he p o ec ion o ola ile compounds o he educ ion o ene gy consump ion.
The main objec i e o his wo k was o s udy he use o di e en DES as selec i e sol en s o he ex ac ion o la onoid compounds
om La ix decidua ba k. Fo his pu pose, di e en ex ac ions we e ca ied ou , and he achie ed esul s we e compa ed in o de o
choose he bes selec i e sol en om he poin o iew no only o he ex ac ion yield, bu also o he composi ion o he ex ac s. The
ex ac s we e cha ac e ised by measu ing bo h, o al la onoid con en and di e en an ioxidan capaci ies (DPPH, ABTS and FRAP).
2. Ma e ial and me hods
2.1. Raw ma e ial
La ix decidua ( om now on ‘pine’) ee ba k was supplied by he E ekondo Egu -Ze a company (Basque Coun y, Spain). A e
collec ion, he aw ma e ial was p epa ed o ob ain a homogeneous ba ch as g anules wi h a size o less han 0.5 mm. Fo his, he s eps
ollowed we e d ying a oom empe a u e, cleaning and g inding using a Re sch Cu ing Mill SM 100. The chemical composi ion o he
used eeds ock was epo ed in ou p e ious wo k (Sille o e al., 2020). B ie ly, pine ba k was 2.0 w % sube in, 20.0 w % ex ac i es,
25.7 w % cellulose, 7.6 w % hemicelluloses, 36.8 w % o al lignin, and 3.5 w % ash.
2.2. Syn hesis o deep eu ec ic sol en s
Fo he selec i e ex ac ion o la onoids, wo DES we e selec ed, choline chlo ide:u ea (1:2) (DES 1) and choline chlo ide:1,4-
bu anediol (1:2) (DES 2). In bo h cases, he same syn hesis me hod was used. B ie ly, he eagen s we e d ied unde acuum o e -
nigh , a oom empe a u e. Then choline chlo ide (ChCl) was mixed wi h he chosen HBD (u ea o 1,4-bu anediol) in a a io o 1:2
(ChCl:HBD). Then, he mix u e was hea ed a 80 ◦C unde cons an s i ing o 2 h, whe eupon a clea and homogeneous liquid was
ob ained.
2.3. Deep eu ec ic sol en s cha ac e isa ion
The success ul syn hesis o bo h DES was con i med by s udying hei s uc u al cha ac e is ics using Nuclea Magne ic Resonance
(NMR),
1
H NMR and
13
C NMR, and A enua ed To al Re lec ance-Fou ie T ans o m In a ed Spec oscopy (ATR-FTIR).
ATR-FTIR was collec ed using a Pe king Elme Spec um Two spec ome e wi h a Uni e sal A enua ed To al Re lec ance
accesso y. The de ined esolu ion was 4 cm
−1
wi h 12 scans wo king in he ange 700-4000 cm
−1
.
The NMR spec a we e eco ded a 30 ◦C on a B uke Ul ashield 400 MHz equipped wi h a z g adien BBI p obe. Typically, 40 mg
o sample we e dissol ed in DMSO-d
6
. 2D-NMR (HSQC) spec a we e eco ded wi h a elaxa ion delay o 1.43 o e 32 scans. The
spec al wid hs we e 5000 and 25,000 Hz o he
1
H and
13
C dimensions, espec i ely. Fo NMR acquisi ion, Top Spin so wa e was
L. Sille o e al.
Sus ainable Chemis y and Pha macy 24 (2021) 100544
3
employed, and o u he da a p ocessing, Mes e No a ( .9.1) was used.
2.4. Ex ac ion me hod
The con en ional ex ac ions we e ca ied ou in an o bi al shake wi h empe a u e con ol (Heidolph Unimax 1010 +Heidolph
Incuba o 1000) using a di e en mix u e o DES/H
2
O (0, 25, 50 and 75 w %) as sol en . The ope a ion condi ions we e 58 ◦C o 94
min, which we e based on ou p e ious wo k abou ex ac ions om pine ba k (Sille o e al., 2018). These condi ions we e selec ed o
a oid he deg ada ion/e apo a ion o he DES. Fo his pu pose, he wo k conduc ed by Delgado-Mellado e al. (2018) on he he mal
s abili y o di e en DES was conside ed. They obse ed ha he weigh loss o he s udied DES, in he wo s case, was lowe han 3%
a e 2 h a 70 ◦C. Thus, i con i ms ha he chosen pa ame e s a e sui able. Fu he mo e, i was also no ed ha he selec ed wo king
condi ions a e wi hin he ange o hose ha ha e been used by o he au ho s o he ex ac ion o la onoids wi h DES (Dai e al., 2013;
ˇ
Skulco ´
a e al., 2018; Wan e al., 2019).
A e he ex ac ions, he solid and liquid phases we e sepa a ed by acuum il a ion. A e wa ds, he solids we e washed wi h
abundan dis illed wa e , and ai -d ied. Finally, he ex ac ion yields we e calcula ed g a ime ically wi h Equa ion (1).
Ex ac ion yield (%) = 100 −[(Wd ied solid wi hou ex ac s (g)
wd y ba k(g))x100](1)
The esul s we e exp essed as he mean ±s anda d de ia ion o he h ee pe o med measu emen s. Analysis o a iance (ANOVA)
in IBM SPSS S a is ic 24 so wa e was used o conduc he s a is ical analysis o he ex ac ion yield. Fo he signi icance s udy, Tukey’s
ange es was used, conside ing s a is ically signi ican alues hose wi h p- alues <0.05.
The pH o all he used DES/H
2
O mix u es we e measu ed wi h a pH-2005 SELECTA. In addi ion, he ex ac ions we e compa ed o
he pola i ies o he DES using he sol a och omic pola i y scale desc ibed by Kamle and Ta . This me hod is buil on h ee solu e-
sol en in e ac ions: hyd ogen bond dona ing abili y,
α
(Ta and Kamle , 1976); pola isabili y,
π
* (Kamle e al., 1977); and hyd ogen
bond accep ing abili y, β (Kamle and Ta , 1976).
2.5. Chemical cha ac e isa ion o he solid phase a e he ex ac ion
The emaining clean and d ied solid a e he ex ac ions we e submi ed o a quan i a i e acid hyd olysis (QAH) (NREL/TP-510-
42618) in o de o quan i y hei glucan, lignin, and hemicelluloses con en acco ding o he me hodology epo ed by Sille o e al.
(2019).
2.6. Ba k ex ac s cha ac e isa ion
The cha ac e isa ion o he ex ac s was ca ied ou di ec ly on he liquid phase ob ained a e sepa a ion by il a ion om he solid
phase. This means ha he cha ac e ised ex ac s we e a mix u e o wa e , DES and ex ac ed compounds. The o al la onoids con en
(TFC) was de e mined by he spec opho ome y echnique epo ed by Blasa e al. (2006), ollowing he p ocedu e desc ibed by
Sille o e al. (2019). Ca echin was used as a s anda d, and he esul s we e exp essed as ca echin equi alen s (CE)/g o d ied ba k
ex ac .
The po en ial o he ba k ex ac s was s udied by measu ing hei an ioxidan capaci y using DPPH, ABTS and FRAP assays. DPPH is
a adical sca enging assay (B and-Williams e al., 1995), FRAP measu ed he e ic educing powe (Benzie and S ain, 1996), and
ABTS de e mines he 2,2′-azinobis(3-e hylbenzo hiazoline-6-sul onic acid) equi alen an ioxidan capaci y (Re e al., 1999). All o
hese spec opho ome y measu emen s we e pe o med wi h a Jasco V-630 UV-VIS spec opho ome e acco ding o he p ocedu e
explained by Sille o e al. (2018). T olox was used as a s anda d in he h ee me hods, and he esul s we e epo ed as mg o T olox
equi alen (TE)/g o d ied ba k ex ac .
Finally, in o de o ha e a be e comp ehension o he s uc u e o he ex ac s, he d ied ex ac s we e cha ac e ised by ATR-FTIR
ollowing he me hodology desc ibed in sec ion 2.3.
3. Resul s and discussion
3.1. DES cha ac e isa ion
The DES we e cha ac e ised by
1
H and
13
C NMR spec oscopy. The images o he spec a a e p o ided in he supplemen a y
ma e ials, while he assignmen is summa ised below.
Peak assignmen o DES 1was pe o med acco ding o he s uc u al da a o o he au ho s (D’Agos ino e al., 2011; Delso e al.,
2019). The peaks o he
1
H NMR spec um o DES 1 a e assigned as ollow; δ
H
(270 MHz, DMSO-d
6
)/ppm: 3.25 (s, 9H, NCH3), 3.39 (s,
2H, NCH2CH2) 3.42 ( , 2H, CH2 CH2O), 3.83 (m, 2H, CH2O), 5.51 (s, 8H, CNH2). The peaks o he
13
C NMR spec um o DES 1a e
assigned as ollows; δ
C
(68 MHz, DMSO-d
6
)/ppm: 54,0 ( , 3C, NCH3), 55,3 (s, CH2CH2O), 67,7 (s, N CH2CH2), 160.1 (s, 2C,
NH2CONH2).
Peak assignmen o DES 2 was acco ding o he s uc u al da a o ano he au ho (Delso e al., 2019). The peaks o he
1
H NMR
spec um o DES 1 a e assigned as ollows; δ
H
(270 MHz, DMSO-d
6
)/ppm: 1.42 ( , 4H, CH
2
CH
2
CH
2
) 3.14 (s, 9H, NCH3), 3.35 ( , 8H,
CH2CH2O) 3.42 ( , 2H, NCH2 CH2), 3.83 (m, 2H, CH2CH2O), 4.47 (s, 4H, CH2OH), 5.59 ( , 1H, CH2OH). The peaks o he
13
C NMR
spec um o DES 2 a e assigned as ollow; δ
C
(68 MHz, DMSO-d
6
)/ppm: 29.5 ( , CH
2
CH
2
CH
2
) 53,5 ( , 3C, NCH3), 55,3 (s, CH2CH2O),
61.2 (s, 2C, OCH2CH2) 67,7 (s, NCH2CH2).
ATR-FTIR analysis o he DESs was also pe o med. Fig. 1 shows he spec a o he wo DES, whe e s uc u al di e ences can be
L. Sille o e al.
Sus ainable Chemis y and Pha macy 24 (2021) 100544
4
seen. The band assignmen is based on he assignmen s gi en by o he au ho s (Ma and Row, 2017; Meng e al., 2018; Piasek and
U banski, 1962), and a summa y o his assignmen is gi en in Table 1.
As shown in Fig. 1 and Table 1, he e a e many simila i ies be ween he s uc u es o he wo syn hesised DES. In ac , many o he
bands iden i ied co espond o hose o ChCl, which, al hough when he DES is syn hesised, loses some o i s cha ac e is ic bands, i has
been demons a ed ha he e a e o he s ha emain ega dless o he HBD (Delgado-Mellado e al., 2018). The bands ypically
p ese ed a e hose a 2900-2850 and 1484-1420 cm
−1
which co espond o ib a ions o he alkyl g oup, wi h he mos p onounced
band a 1485 cm
−1
co esponding o he bending o he CH
2
g oup; 1200-800 cm
−1
a ibu ed o N–H s e ching and C–N symme ic
s e ching; and he band de ec ed a 3200 cm
−1
assigned o he hyd oxyl g oups. When he DES is o med, hese bands con inue, bu
o he bands appea o he exis ing ones a e a ec ed in in ensi y when he DES is gene a ed, which means ha he spec a o he
di e en DES a e no equal.
The main di e ences be ween he spec a o he wo DES a e in he anges 3500-2500 cm
−1
and 1750-1250 cm
−1
. The DES 1 has,
apa om he peak a 3300 cm
−1
associa ed wi h –OH ib a ion o he pu e choline chlo ide, ano he a 3189 cm
−1
which is iden i ied
as –NH s e ching, coming om u ea. Fu he mo e, i also has wo high-in ensi y peaks in he ange o 1600–1700 cm
−1
ha co e-
spond o he s e ching o he –CN o u ea. DES 2, howe e , has he bands associa ed wi h –CH s e ching wi h highe in ensi y (2900-
2700 cm
−1
), due o he p esence o 1,4-bu anediol. The es o he bands a e cha ac e is ic o choline chlo ide, so hey a e simila ,
al hough hey a y in in ensi y.
These spec a oge he con i m ha he syn heses we e comple ed success ully, and ha he ob ained compounds we e he desi ed
ones.
3.2. Fla onoid ex ac ion wi h DES
In his wo k, he ex ac ions we e ca ied ou using he di e en DES as addi i es in combina ion wi h wa e . In gene al, wa e is
conside ed he mos sus ainable op ion o he ex ac ion o di e en compounds om lignocellulosic biomass, al hough i is no
always sui able when use alone. This is he case o la onoids, whose p ope ies lead hem o exhibi limi ed solubili y (Ali e al.,
2019). The e o e, in ecen yea s many s udies ha e been ca ied ou o imp o e he ex ac ion o la onoid compounds by using
wa e /o ganic sol en mix u es (Li e al., 2018; Sille o e al., 2018; Yu e al., 2017; Zhao e al., 2017). In his wo k, howe e , hese
o ganic sol en s ha e been eplaced by g eene sol en s such as DES. We selec ed h ee composi ions, 25 w %, 50 w % and 75 w %
DES in wa e in o de o be able o compa e o pine ba k ex ac ions wi h ionic liquid/wa e mix u es ha had p e iously used hese
composi ions (Sille o e al., 2020). We did no conduc he ex ac ions wi h d y DES, since hese a e solid a oom- empe a u e.
E en hough wa e has limi ed chemical a ini y o la onoid compounds, i s use wi h DES dec eases he iscosi y o he sol en ,
acili a ing he ex ac ion (Dai e al., 2013). Howe e , iscosi y is no he only pa ame e ha a ies, since pola i y is also a ec ed,
which is di ec ly ela ed o he ex ac ion capaci y o hese compounds. This pa ame e is desc ibed as he sum o he o al possible
in e ac ions ha occu be ween he solu e and he sol en . Thus, he pola i ies o bo h DES, and o hei mix u es, would no be equal,
as can be seen in Table 2 whe e he sol a och omic pa ame e s calcula ed o bo h DES a e summa ised.
The pa ame e
α
is p incipally in luenced by he HBD, u ea and 1,4-bu anediol, wi h some addi ional con ibu ion om he –OH
unc ionali y o he choline ca ion. The alues o
π
* a e a ec ed by bo h HBD and HBA, and DES 2 had he highes alue. β pa ame e is
mo e dependen on he HBA, which in bo h o hese cases is he chlo ide ion. Howe e , i is known ha al hough he hyd ogen bond
accep o p ope y o he DES a ises p incipally om he anion, i is mode a ed by he HBD. DES 1 has he lowes alue, because u ea is
Fig. 1. ATR-FTIR spec a o he wo DES, choline chlo ide:u ea (DES 1) and choline chlo ide:1,4-bu anediol (DES 2).
L. Sille o e al.
Sus ainable Chemis y and Pha macy 24 (2021) 100544
5
he mo e basic compound o he wo HBD. In o de o de e mine he pola i y o he mix u es used in his wo k, an es ima ion was made,
aking he wa e alues (Table 2) as a e e ence, which will be a ec ed o a g ea e o lesse deg ee depending on he DES pe cen age
used in each mix u e. This discussion will be held in combina ion wi h he discussion o he ex ac ion yields.
In his wo k, se en di e en solu ions, we e p epa ed and es ed o he ex ac ion o pine ba k, and Table 3 shows he de e mined
ex ac ion yields. These esul s ange om 9% o almos 22% o he d y weigh o he ba k. The lowes alue measu ed was eco ded
o wa e as sol en , while he highes yield was ob ained o DES 1/H
2
O (75 w %). All he ex ac ions ca ied ou a e signi ican ly
di e en om he one wi h wa e as shown in Table 3.
The da a clea ly show ha he concen a ion o DES in he DES/H
2
O mix u e has a signi ican e ec , wi h lowe ex ac ion yields
measu ed o he DES/H
2
O mix u es wi h he lowes DES concen a ion. Acco ding o he da a, he pH o he mix u es had no di ec
in luence on he ex ac ion yield, while he pe cen age o wa e has a di ec in luence. Howe e , i can also be seen ha he di e ence
in he ex ac ion yields o he 50 w % and 75 w % DES/H
2
O mix u es a e much less han he di e ence be ween hese and he 25 w %
DES/H
2
O mix u es. Wan e al. (2019) ha e sugges ed ha a p opo ion o wa e highe han 70% in he mix u e dec eases he
ex ac ion yield due o he des uc ion o he DES s uc u e, which may be esponsible o he esul s desc ibed he e.
Looking a he measu ed ex ac ion yield o he di e en DES mix u es, i can be seen ha he e a e only small di e ences be ween
he DES 1/H
2
O and DES 2/H
2
O mix u es a he same concen a ion. Hence, no clea end can be obse ed in hese small di e ences.
DES 1 has alues o bo h β and
π
* ha a e e y close o hose o wa e , bu has a highe alue o
α
(1.42) han does wa e (1.23). In his
case, he highe yield can be en a i ely a ibu ed o he highe
α
alue. Howe e , in he case o DES 2 all pa ame e s a e di e en o
hose o wa e , β and
π
* inc eased while, con a y o DES 1,
α
dec eases. Thus, he e is no clea end on how he di e en pola i y
pa ame e s can a ec he ex ac ion yield o he pine ba k in he s udied condi ions. The inc eased ex ac ion o la onoid compounds
is likely he esul o many in e ac ions be ween hese and he DES.
A signi ican inc ease in he ex ac ion yield is obse ed when compa ing he yields ob ained in his wo k wi h hose epo ed wi h
con en ional sol en s (E OH/H
2
O) o he same aw ma e ial. Sille o e al. (2018) ca ied ou he op imisa ion o h ee di e en
ex ac ion me hods, con en ional, assis ed by ul asonic and assis ed by mic owa e (8.2%, 6.1% and 8.3% ex ac ion yield, espec-
i ely). These esul s a e g ea ly imp o ed in his wo k, as well as hose ob ained o he same eeds ock using simul aneous
mic owa e-ul asound assis ed ex ac ion (15.7% ex ac ion yield) (Sille o e al., 2020).
Looking a o he wo ks ha in es iga ed he use o DES o ob ain la onoids, he i s o be men ioned is by ˇ
Skulco ´
a e al. (2018),
who ca ied ou he ex ac ion o Sp uce ba k using nine di e en DES. The ex ac ion yields epo ed in his s udy anged om 11.4%
(ChCl:glyce ol, 1:1) o 27.7% (ChCl: a a ic acid (1:1), so i can be said ha all ou esul s a e wi hin simila ange. In he case o he
esul s epo ed by Haz e al. (2018) o he same aw ma e ial using h ee di e en DES, he epo ed ex ac ion yield was lowe . The
esul s a ied be ween 11.40% (ChCl:glyce ol, 1:1) and 14.68% (ChCl:malic acid, 1:1).
Table 1
FTIR spec a band gene al assignmen .
Wa enumbe (cm
−1
) DES 1 DES 2 Band assignmen Re e ence
3317 X x symme ic NH
2
s e ching Ma and Row (2017)
3200 X -OH s e ching Delgado-Mellado e al. (2018)
2970–2850 X x -CH s e ching om an alkyl g oup Delgado-Mellado e al. (2018)
1662–1606 X -NH de o ma ion ib a ion Du e al. (2016)
1485–1420 X x -CH s e ching om an alkyl g oup Delgado-Mellado e al. (2018)
1200–800 X x N–H s e ching and C–N symme ic s e ching Delgado-Mellado e al. (2018)
Table 2
Kamle -Ta coe icien s o he pu e sol en s used in his wo k, measu ed using he dyes N,N-die hyl-4-ni oaniline, 4-ni oaniline,and Reicha d .
Abb e ia ion Sol en
α
β
π
* Re e ence
H
2
O H
2
O 1.23 0.47 1.14 Jessop e al. (2012)
DES 1 ChCl:U ea 1.42 0.50 1.14 Flo indo e al. (2018)
DES 2 ChCl:1,4-bu adienol 0.65 0.79 1.74 Ha is (2008)
Table 3
Ex ac ion yield de e mined o he di e en DES/H
2
O mix u es, and esul s o he cha ac e isa ion o he ex ac .
Sol en [DES] (w .%) pH Yield* (w .%) TFC (mg CE/g DBE) DPPH (mg TE/g DBE) ABTS (mg TE/g DBE) FRAP (mg TE/g DBE)
H
2
O 0 5.80 9.3 ±0.2
a
34 ±2 22.3 ±0.3 106 ±2 31 ±4
DES 1/H
2
O 25 9.49 16.2 ±0.7
b,c
159 ±7 215 ±20 391 ±17 98 ±2
50 9.77 19.4 ±0.1
d
305 ±9 297 ±17 593 ±16 155 ±8
75 10.16 21.9 ±0.8
e
275 ±1 215 ±4 637 ±45 120 ±2
DES 2/H
2
O 25 4.63 16.5 ±0.7
c
376 ±3 460 ±17 850 ±54 309 ±7
50 5.70 20.0 ±0.5
d,e
376 ±12 453 ±8 736 ±22 314 ±6
75 5.29 20.4 ±0.1
d,e
383 ±7 452 ±19 799 ±19 289 ±11
DBE: d ied ba k ex ac .
*Supe sc ip le e s depic signi ican di e ences (Tuke y es , p <0.05)).
L. Sille o e al.

Sus ainable Chemis y and Pha macy 24 (2021) 100544
6
Once he o e all ex ac ion yield had been s udied, he nex s ep was o con i m ha he ex ac ed compounds belonged only o he
ex ac i e ac ion o he lignocellulosic biomass. Fo his pu pose, chemical cha ac e isa ion o he solids emaining a e ex ac ion
was pe o med by QAH. In his way, he a ia ion o he hemicelluloses, cellulose (exp essed as glucan) and acid-insoluble lignin
con en o he solids was s udied (Table 4).
The i s hing o poin ou is ha no glucan solubilisa ion was obse ed in any o he pe o med ex ac ions. The e olu ion o he
solubili y in each DES/H
2
O mix u e e ealed ha he e was p ac ically no a ia ion be ween hem ( a ia ion o ±0.8 w %), no in
compa ison wi h he ex ac ion ca ied ou only wi h wa e . This con i ms ha in he s udied condi ions he e is no glucan solubili-
sa ion. Simila ly, he e is also no solubilisa ion o hemicelluloses, wi h a maximum a ia ion o 0.8 w %.
Acid insoluble lignin (AIL) appea s o be he main componen o he solid ac ion, and his is he ac ion in which mos a ia ion
has been measu ed. I is obse ed ha he highes alue was de e mined o he solid ea ed wi h wa e , 50.2 w %, while he alues o
he es o he solids we e in he ange o 40–45 w %. In he compa ison, i is obse ed ha o he ex ac ions ca ied ou wi h he DES/
H
2
O mix u es, he solubilisa ion o he AIL inc eases wi h he inc ease o he DES concen a ion. Howe e , hese di e ences in he
esul s may be due o he solubilisa ion o sube in o ex ac s since he measu emen o AIL can be a ec ed by hese compounds (Slui e
e al., 2012). The e o e, i canno be s a ed wi h ce ain y ha solubilised lignin is p esen in he ex ac ions pe o med unde hese
condi ions.
3.3. Fla onoid quan i ica ion
I has been con i med ha he ex ac ion yield obse ed o he DES/H
2
O mix u es is conside ably highe han ha epo ed o
wa e /o ganic sol en mix u es o pine ba k, so he nex s ep was o e i y he selec i i y o he ex ac ion. Fo his pu pose, he o al
la onoid con en (TFC) o he liquid phase was measu ed. Table 3 illus a es ha wa e did no ex ac la onoids a all, which
con i ms he poo a ini y o hese compounds o wa e (Meng e al., 2018).
The TFC a ies om 159 o 383 mg CE/g d ied ba k ex ac . In gene al, he amoun o la onoids ob ained wi h he DES 2 mix u es
a e mo e han 25% highe han hose ob ained wi h DES 1. The highes TFC was de e mined o DES 2 (75%), al hough he di e ence
wi h he o he wo DES 2 mix u es was sligh . On he o he hand, he e we e la ge di e ences in he TFC alues o he DES 1/H
2
O
mix u es a di e en concen a ions. I is obse ed ha he wo s alues we e ob ained wi h he lowes DES concen a ions. This could
indica e ha highe DES concen a ion a ou s la onoid ex ac ion up o a poin a which no u he imp o emen is achie ed.
Compa ing he TFC alues ob ained in his wo k wi h he alues epo ed by o he au ho s, in gene al i can be said ha good
esul s ha e been achie ed. The alues de e mined wi h DES 2/H
2
O a e in he ange o he ones epo ed o ace one/H
2
O ex ac s
om Pinus du angensis (379 mg CE/g ex ac ) (Rosales-Cas o e al., 2017), and he ones epo ed o hyd oalcoholic ex ac s om
Qe cus side oxyla (386 mg CE/g ex ac ) (So o-Ga cía and Rosales-Cas o, 2016). Howe e , he alues measu ed wi h DES 1/H
2
O a e
no ably lowe . Compa ing he esul s ob ained he e wi h hose ob ained o he same aw ma e ial wi h E OH/H
2
O, by bo h con-
en ional and di e en in ensi ica ion me hods (ul asound, mic owa e and simul aneous mic owa e-ul asound assis ed ex ac ion),
e en he alues de e mined o DES 2 a e lowe , al hough no g ea ly so. Sille o e al. (2018) epo ed alues be ween 412 and 430 mg
CE/g d ied ba k ex ac o con en ional, ul asound assis ed and mic owa e assis ed ex ac ions. While, Sille o e al. (2020) epo ed
alues o 433 mg CE/g d ied ba k ex ac o he E OH/H
2
O ex ac s ob ained by simul aneous mic owa e-ul asound assis ed
ex ac ion. The di e ence o he TFC may be due, on he one hand, o he di e en iscosi ies o he applied sol en s, and on he o he
hand, o he sol en ’s pola i y. I is also impo an o conside he possible s e ic hind ance o DES, especially o DES 2.
S udying di e en wo ks pe o med o he ex ac ion o la onoid compounds om di e en lignocellulosic ma e ials wi h DES,
bo h Wang e al. (2019) and Cui e al. (2018) conclude ha ChCl:1,4-bu anediol was he bes . Al hough in he case o Ciu e al. (2018)
he bes a io was 1:3, while in he case o Wang e al. (2019) he bes a io was 1:2. This is in acco dance wi h he esul s ob ained he e,
whe e among all he ex ac ions done wi h DES, he bes TFC was ob ained o DES 2 (75%) ex ac . The use o 25% wa e o acili a e
he ex ac ion o la onoid compounds is in acco dance wi h ha epo ed by Wang e al. (2019). Ma and Row (2017) s udied he
ex ac ion o h ee di e en la onoids om He ba A emisiae Scopa iae using di e en ILs and DES as sol en s, including he wo DES
s udied he e. The bes measu ed alues o DES we e ob ained wi h ChCl: o mic acid (10234.43
μ
g/g u in, 1032.23
μ
g/g que ce in,
and 211.87
μ
g/g scopa on), al hough bo h DES 1 and DES 2 ex ac ed high con en s o hese la onoids, wi h DES 2 showing sligh ly
be e esul s.
Conside ing all he abo e, i can be concluded ha he ex ac ion yield is no associa ed wi h he la onoid ex ac ion. Acco ding o
Table 3, he bes ex ac ion yield was ob ained wi h DES 1 (75%), howe e , his is no consis en wi h he highes TFC. This may be a
consequence o a lowe selec i i y o hese sol en s o wo easons. On he one hand, he use o wa e in he mix u es may ha e
esul ed in he ex ac ion o o he non- la onoid compounds, since i alone ex ac s subs ances om pine ba k as i is shown in Table 3.
Table 4
Chemical composi ion o he pine ba k a e he ex ac ions (all esul s a e exp essed as w .%).
Sol en [DES] Acid-insoluble lignin Glucan Hemiceluloses
H
2
O 0 50.2 ±3.2 27.7 ±0.6 11.1 ±0.1
DES 1/H
2
O 25 45.4 ±1.4 27.6 ±0.3 11.7 ±0.1
50 41.3 ±1.3 27.7 ±0.1 11.2 ±0.5
75 40.38 ±4.7 28.0 ±0.2 11.0 ±0.7
DES 2/H
2
O 25 45.1 ±1.9 27.8 ±0.4 11.1 ±0.3
50 43.5 ±0.3 27.7 ±0.3 11.0 ±0.9
75 41.8 ±0.6 27.2 ±0.1 10.9 ±0.1
L. Sille o e al.
Sus ainable Chemis y and Pha macy 24 (2021) 100544
7
On he o he hand, i is impo an o men ion ha DES 1 has also been s udied o he deligni ica ion o di e en lignocellulosic
ma e ials (Espinoza-Acos a e al., 2014; Kalho and Ghandi, 2019; P ado e al., 2018). This indica es ha he use o DES 1 mix u e o
la onoid ex ac ion may also solubilise pa o he lignin. The e o e, epo ing a high ex ac ion yield whe eas TFC would no be
consis en . This is con i med by he dec ease o he AIL con en measu ed in he solids a e ex ac ion (Table 4).
3.4. Cha ac e isa ion o ba k ex ac s: an ioxidan capaci y
A u he poin o conside o he possible applica ion o he ex ac s is hei an ioxidan capaci y. Th ee an ioxidan capaci y
measu emen s ha e been pe o med in his wo k, p o iding a mo e accu a e idea o he capaci y o hese ex ac s. The alues measu ed
o he s udied an ioxidan capaci ies a e di e en o he same ex ac , which is no mal because hey measu e di e en aspec s.
Table 3 shows he alues de e mined o he 3 measu ed pa ame e s, and he i s hing o poin ou is he low an ioxidan capaci y
o he aqueous pine ba k ex ac s. This is in line wi h he low TFC, which con i ms he bene i o he use o DES o he ex ac ion o
la onoid compounds. The ange o alues measu ed o he DPPH o he di e en es ed ex ac s we e be ween 215 and 460 mg TE/g
d ied ba k ex ac . The highes de e mined alue was o DES 2 (25%) ex ac , bu he e was no signi ican di e ence wi h he o he
wo DES 2 ex ac s. The ex ac s ob ained wi h DES 1 all exhibi ed lowe DPPH alues, in acco dance wi h hei lowe TFC. In he case
o ABTS, as wi h DPPH, he ex ac s ob ained wi h DES 2 had he highes esul s, al hough in gene al all ABTS alues we e highe han
DPPH alues. The bes alue de e mined was he one measu ed o DES 2 (25%) ex ac s, 850 mg TE/g d ied ba k ex ac . Finally,
ega ding he FRAP assay, DES 1 ex ac gi e he lowes an ioxidan capaci ies, wi h he wo s , as o he o he an ioxidan capaci ies,
we e ob ained wi h 25 w % DES 1/H
2
O. The alues in his case a e especially low, since hey did no e en each 100 mg TE/g d ied
ba k ex ac . This is consis en wi h he ac ha DES 1/H
2
O mix u es a e he ones wi h he lowes la onoid ex ac ion, which sugges s
ha his sol en is no e y selec i e o he ex ac ion o la onoids.
Compa ing he alues p esen ed in his s udy wi h hose p e iously ob ained o pine ba k using E OH/H
2
O as sol en (Sille o e al.,
2018), i can be concluded ha all he measu ed alues he e a e in he ange (677–906 mg TE/g d ied ba k ex ac ), excep o he 25
w % DES 1/H
2
O ex ac . Rega ding he FRAP assay, howe e , only he ex ac s ob ained wi h DES 2 app oaches he esul s ob ained
wi h E OH/H
2
O (330–390 mg TE/g d ied ba k ex ac ).
Addi ionally, a cau ious compa ison has also been conduc ed wi h he esul s eco ded o o he aw ma e ials. Bibi Sadee e al.
(2019) ob ained me hanolic ex ac s om 3 di e en ee ba ks, whe e he lowes ABTS and DPPH alues we e measu ed o Zan-
hoxylum gille ii (178 and 82 mg TE/g ex ac , espec i ely). These alues a e a exceeded in his wo k. In he case o DPPH, alues
ha e been epo ed o Maca anga hu i olia and S e culia agacan ha close o 495 mg TE/g d ied ba k ex ac , which is in he ange o
hose calcula ed o he ex ac s ob ained wi h DES 2/H
2
O mix u es (452–460 mg TE/g d ied ba k ex ac ). Analysing he alues
measu ed by Bibi Sadee e al. o FRAP, he alue epo ed o he me hanolic ex ac o Zan hoxylum gille ii (163 mg TE/g ex ac ) was
lowe han he alues epo ed o DES 2/H
2
O ex ac s.
In he wo k conduc ed by Tanase e al. (2019), Fagus syl a ica ba k ex ac s ob ained wi h di e en sol en s by mic owa e-assis ed
ex ac ion we e cha ac e ised. The ABTS an ioxidan capaci y epo ed o Fagus syl a ica ba k ex ac s ob ained wi h 80% e hanol in
wa e was 472 mg TE/g d ied ex ac . These esul s we e exceeded in all expe imen s in his wo k excep o he DES 1 ex ac (25%).
All he esul s epo ed o DPPH and ABTS we e be e han hose de e mined in his wo k.
A compa ison o he an ioxidan capaci ies o Ch ysophyllum pe pulch um ex ac s ob ained using di e en sol en s by Baloglu e al.
(2019) shows ha he highes DPPH alue ob ained was o MeOH ex ac s (73.23 mg TE/g ex ac ). This alue is signi ican ly lowe
han hose ob ained he e o pine ba k ex ac s (Table 3). Some hing simila occu s wi h he aqueous ex ac s o ABTS an ioxidan
capaci y (491 mg TE/g ex ac ), whe e only he DES 1 (25%) ex ac s p esen ed a lowe alue.
Conside ing he a o emen ioned, i can be concluded ha he use o DES p omo es he ex ac ion o an ioxidan compounds,
especially he use o DES 2.
Fig. 2. ATR-FTIR spec a o di e en ba k ex ac s. a) Ex ac s ob ained wi h di e en DES 1 concen a ions. b) Ex ac s ob ained wi h di e en DES 2 concen a ions.
L. Sille o e al.
Sus ainable Chemis y and Pha macy 24 (2021) 100544
8
3.5. S uc u al cha ac e isa ion o ex ac s
The p esence o la onoid compounds in he di e en ex ac s was con i med by ATR-FTIR analysis. The ollowing igu es show he
ex ac s wi h di e en concen a ions o sol en s compa ed o he spec um o he pu e DES used as a sol en . In he Fig. 2 i can be
seen how he in ensi ies o he di e en bands change depending on he concen a ion o sol en used, as well as he appea ance o new
bands.
In he wo s udied cases, a signi ican inc ease in in ensi y is obse ed in he band a ibu ed o –OH s e ch ib a ion in phenolic
and alipha ic s uc u es (be ween 3400 and 3300 cm
−1
). Fig. 2b o DES 2 ex ac s shows ha he band a wa enumbe 1630 cm
−1
unde goes a conside able inc ease in in ensi y, which is assigned o he alence ib a ions C
–
–
O, ypical o la onoid compounds
(T i unschi e al., 2015). Thus, he ex ac ion o hese compounds is con i med. In he case o DES 1, as shown in Fig. 2a, ins ead o he
ypical C
–
–
O band o he la onoids, ano he band appea s a a wa enumbe o 1705 cm
−1
. This band is ela ed o he p esence o
lignin, since i co esponds o he s e ching ib a ion o non-conjuga ed ca bonyl g oups om he a oma ic lignin skele on (Boe iu
e al., 2004). This con i ms he capaci y o his sol en o solubilise lignin, as i was men ioned in abo e.
4. Conclusions
Two DES ha e been success ully syn hesised in his wo k o be used as g een sol en s o he ex ac ion o bioac i e compounds.
They ha e been used as addi i es in aqueous mix u es o enhance he selec i e ex ac ion o la onoid compounds om pine ba k.
In his wo k, i is con i med ha he use o aqueous mix u es o DES can be an al e na i e sol en o la onoids ex ac ion om
pine ba k, since all he cases s udied showed an imp o emen in ex ac ion yield in compa ison wi h aqueous ex ac ion. Mo eo e , he
ex ac ion yields ha e been imp o ed compa ed o he esul s ob ained wi h con en ional sol en (E OH/H
2
O). This u he dem-
ons a es he po en ial o DES o his use. Howe e , o TFC, only DES 2 mix u es achie ed esul s simila o hose epo ed wi h
con en ional sol en s. Fu he mo e, his wo k e i ies ha DES 1 canno be conside ed a selec i e sol en o la onoids, since
al hough he highes ex ac ion yield is ob ained wi h DES 1 (75%), he TFC con en is low, as well as he an ioxidan capaci y o he
ex ac s. This can be explained by he solubilisa ion o lignin when DES 1 is used, which is e i ied in he ATR-FTIR esul s.
DES 2/H
2
O (75% w %) was selec ed as he bes sol en due no only o i s good abili y o ex ac la onoids, bu also o he high
an ioxidan p ope ies o he ex ac . The good la onoid ex ac ion capaci y was con i med by he ATR-FTIR analysis o he ex ac s,
which showed a la ge inc ease in he band ypically assigned o he la onoids a 1630 cm
−1
. Consequen ly, i can be con i med ha
biologically ac i e ex ac s ha e been ob ained. This is e y sui able o hei use in di e en applica ions, om ood indus y o
cosme ics, o biologically ac i e bio-based ma e ials. Ne e heless, u he cha ac e isa ion o he ob ained compounds and hei
pu i ica ion p io o hei applica ion should be ca ied ou be o e hei use.
CRediT au ho ship con ibu ion s a emen
Ley e Sille o: Me hodology, In es iga ion, Visualiza ion, W i ing – o iginal d a , P epa a ion. Raquel P ado: Concep ualiza ion,
Da a cu a ion, Supe ision, W i ing – e iew & edi ing. Tom Wel on: Resou ces, Valida ion, W i ing – e iew & edi ing. Jalel Labidi:
Resou ces, Supe ision, W i ing – e iew & edi ing, Funding acquisi ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o
in luence he wo k epo ed in his pape .
Acknowledgemen s
Au ho s would like o hank he Depa men o Economic De elopmen and In as uc u es o he Basque Go e nmen o he
inancial suppo (schola ship o young esea che s aining). Ley e Sille o would also like o hank Impe ial College London o hei
suppo du ing he s ay.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.o g/10.1016/j.scp.2021.100544.
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