Ci a ion: Pe o I anko i´c, A.;
´
Co o i´c, M.; Mili oje i´c, A.;
Blagoje i´c, S.; Radulo i´c, A.; Pjano i´c,
R.; Bezb adica, D. Assessmen o
Enzyma ically De i ed Blackcu an
Ex ac as Cosme ic
Ing edien —An ioxidan P ope ies
De e mina ion and In Vi o Di usion
S udy. Pha maceu ics 2024,16, 1209.
h ps://doi.o g/10.3390/
pha maceu ics16091209
Academic Edi o : An onio Vassallo
Recei ed: 8 Augus 2024
Re ised: 30 Augus 2024
Accep ed: 30 Augus 2024
Published: 14 Sep embe 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
pha maceu ics
A icle
Assessmen o Enzyma ically De i ed Blackcu an Ex ac as
Cosme ic Ing edien —An ioxidan P ope ies De e mina ion and
In Vi o Di usion S udy
Anja Pe o I anko i´c 1, Ma ija ´
Co o i´c 2,*, Ana Mili oje i´c 2, S e an Blagoje i´c 3, Aleksand a Radulo i´c 3,
Rada Pjano i´c 2and Dejan Bezb adica 2
1Inno a ion Cen e o Facul y o Technology and Me allu gy, 11000 Belg ade, Se bia; [email p o ec ed]
2Facul y o Technology and Me allu gy, Uni e si y o Belg ade, 11000 Belg ade, Se bia;
[email p o ec ed] (A.M.); [email p o ec ed] (R.P.); [email p o ec ed] (D.B.)
3Ins i u e o Gene al and Physical Chemis y, Uni e si y o Belg ade, 11000 Belg ade, Se bia;
[email p o ec ed] (S.B.); [email p o ec ed] (A.R.)
*Co espondence: [email p o ec ed]
Abs ac : Blackcu an is an an hocyanin- ich be y wi h p o en an ioxidan and pho op o ec i e
ac i i y and eme ging p ebio ic po en ial, widely applied in cosme ic p oduc s. He eby, highly
e icien enzyme-assis ed ex ac ion o blackcu an polyphenols was pe o med, gi ing ex ac
wi h e y high an ioxidan ac i i y. Ob ained ex ac was cha ac e ized in e ms o an hocyanin
composi ion, inco po a ed in o h ee di e en cosme ic o mula ions and subjec ed o F anz cell
di usion s udy. Expe imen al alues ob ained using cellulose ace a e memb ane o all ou dominan
an hocyanins (delphinidin 3-glucoside, delphinidin 3- u inoside, cyanidin 3-glucoside and cyanidin
3- u inoside) we e success ully i ed wi h he Ko smeye –Peppas di usion model. Calcula ed
e ec i e di usion coe icien s we e highe o hyd ogel compa ed o oil-in-wa e c eam gel and
oil-in-wa e emulsion, whe eas he highes alue was de e mined o cyanidin 3- u inoside. On he
o he hand, a e a 72 h long expe imen wi h ansde mal skin di usion model (S a -M
®
memb ane),
no an hocyanins we e de ec ed in he ecep o luid, and only 0.5% o he ini ial quan i y om he
dono compa men was ex ac ed om he memb ane i sel a e expe imen wi h hyd ogel. P esen
s udy e ealed ha hyd ogel is a sui able ca ie sys em o he opical deli e y o blackcu an
an hocyanins, while de mal and ansde mal deli e y o hese molecules is e y limi ed, which
implies i s applicabili y o ea men s a ge ing skin su ace (i.e., p ebio ic, pho op o ec i e).
Keywo ds: blackcu an ex ac ; an ioxidan ac i i y; an hocyanins; cosme ic o mula ion; di usion;
opical deli e y
1. In oduc ion
The skin is he la ges human o gan, sui able o adminis a ion and bo h opical
and sys emic deli e y o di e en d ugs [
1
]. Ha ing a ba ie unc ion o pa hogenic
ac o s (e.g., mic oo ganisms o alle gens) and p e en ing excessi e wa e loss, human
skin, pa icula ly i s uppe laye , s a um co neum, ep esen s limi a ion o he pene a ion
o desi ed subs ances in o deepe skin laye s o in o he body, which a e o en desi ed
a ge s o exhibi ing hei ull he apeu ic po en ial [
2
]. While d ugs ha a e adminis a ed
in o de o be sys emically abso bed need o be ansde mally deli e ed, hose a ge ing
di e en skin condi ions should s ay on i s su ace (e.g., UV p o ec ion, p ebio ic) o
deli e ed in o deepe skin laye s (e.g., AHR ecep o s imula ion, collagen syn hesis,
in lamma o y diseases ea men ) [
3
–
8
].The pe mea ion o ac i e subs ances h ough he
s a um co neum gene ally depends on he in e ac ion be ween he skin, ac i e compound
and di e en o mula ion componen s, and ha in e dependence p o ides possibili y o
Pha maceu ics 2024,16, 1209. h ps://doi.o g/10.3390/pha maceu ics16091209 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2024,16, 1209 2 o 13
con olled o sus ained d ug elease which is p e e ed o opical, cu aneous and sys emic
deli e y [1,3,8].
Fo he labo a o y-scale e alua ion o ac i e ing edien s’ skin pe mea ion and elease
om opical o mula ions, F anz di usion cell expe imen s a e being usually used wi h di -
e en memb anes [
7
]. When ca ie sys ems a e examined o d ug elease, cellulose ace a e
memb ane wi h la ge po e diame e , which does no ep esen a di usion
ba ie [9,10]
, is
commonly applied, while o cu aneous and ansde mal deli e y s udies, animal models
a e p e e ed [
11
,
12
]. Recen ly, syn he ic S a -M
®
memb ane was p o en as an adequa e
eplacemen o biological skin models since, besides ha ing no e hical issues, i gi es an
ad an age o su ace uni o mi y and cons an cha ac e is ics, hus p o iding he highes
possible ep oducibili y and eliabili y o o mula ion and ac i e ing edien s sc eening
da a [13,14].
Blackcu an is a be y ui ich in phenolic compounds, pa icula ly an hocyanins [
15
],
which a e known o hei an ioxidan ac i i y and pho o-p o ec i e ole agains pho o-
inhibi o y and pho o-oxida i e damage caused by high ligh [
16
]. Polyphenols occu in
ee o ms wi hin he be ies bu also a e bound o he cell wall, which complica es hei
ex ac ion h ough adi ional sol en -based ex ac ion. In o de o o e come his p oblem,
he use o ca bohyd a e-hyd olysing enzymes o b eak down he cell wall can signi ican ly
imp o e polyphenol yield in a sho e ime. Fu he mo e, enzyme-assis ed ex ac ion is
conduc ed unde mild condi ions, wi hou he use o o ganic sol en s, making i a mo e
ad an ageous op ion han sol en -based ex ac ion [
15
,
17
]. We ha e p e iously p o en
ha blackcu an ex ac possesses he po en ial o egula e skin mic obio a composi ion by
posi i ely in luencing S aphylococcus epide midis (skin commensal) and S aphylococcus au eus
(oppo unis ic pa hogen) balance, whe eas his p ebio ic e ec is e en mo e p onounced
when enzyma ically de i ed ex ac is applied [
17
]. The e o e, ou goal was o cha ac e ize
blackcu an ex ac ob ained in an enzyme-assis ed p ocess in e ms o o al polyphenol
con en and di e en polyphenol classes’ ( la onoid, la onol, an hocyanin, phenol acid,
hyd olysable annin and condensed annins) abundance and o co ela e hem wi h an ioxi-
dan ac i i y o ex ac s de e mined by se e al
in i o
me hods (DPPH, ABTS, CUPRAC
and FRAP). Fu he mo e, ex ac was inco po a ed in se e al cosme ic o mula ions in
o de o e alua e hem as an hocyanin ca ie sys ems o opical, cu aneous and/o ans-
de mal deli e y by pe o ming di usion expe imen s wi h cellulose ace a e and S a -M®
memb ane. Ob ained esul s we e modelled in o de o de e mine a elease mechanism o
each indi idual an hocyanin compound and calcula e hei e ec i e di usion coe icien s.
2. Ma e ials and Me hods
2.1. Ma e ials
Lyophilised blackcu an (LBC), a ie y Ben Ne is, was supplied by D eno ac d.o.o.,
A ilje, Se bia. The used blackcu an s ha e pe ec ly sphe ical be ies wi h a diame e o
~1 cm and a deep pu ple colou coming om hei ull ipeness. Fo he ex ac ion p ocess,
be ies we e g ound in a mill (Re sch MM 400, Re sch GmbH, Haan, Ge many) o a powde .
Analy ical g ade chemicals, including sodium ace a e ihyd a e, sodium chlo ide,
sodium ca bona e, aluminium chlo ide, i on(III) chlo ide hexahyd a e and i on(II) sulpha e,
we e ob ained om Cen ohem (S a a Pazo a, Se bia). HPLC-g ade sol en s (me hanol and
wa e ), i luo ace ic acid (TFA), 2,4,6- is(2-py idyl)-s- iazine, ca eic acid and T olox we e
pu chased om Sigma-Ald ich (Schnelldo , Ge many). An hocyanin HPLC s anda ds,
delphinidin 3-O- u inoside chlo ide, delphinidin 3-O-glucoside chlo ide, cyanidin 3-O-
u inoside chlo ide and cyanidin 3-O-glucoside chlo ide we e pu chased om Ca l Ro h
(Ka ls uhe, Ge many). Gallic acid and que ce in we e ob ained om Me ck (Da ms ad ,
Ge many). Folin–Ciocal eu phenol eagen was ob ained om Ca lo E ba (A ese, I aly).
Glacial ace ic acid, 96% ( / ) e hanol and hyd ochlo ic acid we e om Zo ka Pha ma (Šabac,
Se bia). Comme cial enzyme p epa a ion Viscozyme
®
L was ob ained om No ozymes
(Bags ae d, Denma k), while Rohapec
®
MC was a kind dona ion om AB Enzymes
(Da ms ad , Ge many). The o mula ions we e p epa ed using A is o ex
®
AVC polyme om
Pha maceu ics 2024,16, 1209 3 o 13
Cla ian In e na ional L d. (Basel, Swi ze land), gelling agen HeliogelTM om Lucas Meye
Cosme ics (Pa is, F ance), isop opyl my is a e and Lane e
®
O (ce ea yl alcohol) om BASF
(Ludwigsha en, Ge many), and Sabowax FL 65K (glyce ol–s ea a e, PEG-100 s ea a e) om
(Sabo, Le a e, I aly). The cellulose ace a e and S a
®
-M memb anes we e pu chased om
Wha man (Dassel, Ge many) and Me ck Millipo e (Bille ica, MA, USA), espec i ely.
2.2. Me hods
2.2.1. Enzyme-Assis ed Ex ac ion
Enzyme-assis ed ex ac ion o polyphenols om LBC was pe o med using a mix u e
o comme cial enzyme p epa a ions—Viscozyme
®
L and Rohapec
®
MC, in acco dance
wi h a p ocedu e p e iously op imized by ou esea ch g oup [
17
]. B ie ly, LBC and ace a e
bu e (pH 4.5) we e mixed a a 1:10 a io, and enzyme mix u e was added (Viscozyme
®
L and Rohapec
®
MC in a 2 o 1 a io wi h o e all concen a ion o 0.05 mL/g DM).
Ex ac ion ook place a 50
◦
C wi h shaking a 150 pm du ing 60 min, ollowed by enzyme
deac i a ion (hea ing a 100
◦
C o 5 min). All p epa ed ex ac s we e s o ed a
−
20
◦
C
un il u he use.
2.2.2. Ex ac s Composi ional Analysis
The o al polyphenol con en in he ex ac s was assessed using a modi ied Folin–
Ciocal eu me hod [
18
]. The esul s we e exp essed as millig ams o gallic acid equi alen s
(GAE) pe g am o d y ma e (DM) o he LBC. The o al la onoid con en in he ex ac s
was e alua ed using he aluminium chlo ide colo ime ic me hod [
19
]. The esul s we e
p esen ed as millig ams o que ce in equi alen s (QE) pe g am o d y ma e (DM) o
he LBC. The o al an hocyanin con en in ex ac s was ob ained by he pH di e en ial
me hod [
19
]. The inal esul s we e exp essed as millig ams o cyanidin-3-O-glucoside
equi alen s (cya-3-glu) pe g am o d y ma e (DM) o he LBC. The o al la onol con en
in he ex ac s was de e mined using he me hod epo ed p e iously [
20
]. The esul s we e
exp essed as millig ams o que ce in equi alen s (QE) pe g am o d y ma e (DM) o he
LBC. The o al phenolic acid con en in he ex ac was quan i ied using he me hod wi h
A now’s eagen [
21
]. The esul s we e exp essed as millig ams o ca eic acid (CAE) pe
g am o d y ma e (DM) o LBC. To al hyd olysable annins con en in he ex ac was
de e mined by he po assium ioda e solu ion me hod [
22
]. The o al amoun o annins in
he sample was exp essed in millig ams o gallic acid equi alen s (GAE) pe g am o d y
ma e (DM) o LBC. To al condensed annins con en in he ex ac was de e mined by
he n-bu anol- hyd ochlo ic acid assay [
23
]. The concen a ion o condensed annins was
calcula ed as millig ams o cyanidin equi alen s (CyE) pe g am o d y ma e (DM) o
LBC using mola ex inc ion coe icien (34,700 L/(mol
·
cm) and mola mass (287.28 g/mol)
o cyanidin.
2.2.3. An ioxidan Ac i i y De e mina ion
To assess he an ioxidan ac i i y o he ex ac s, se e al spec opho ome ic me hods
we e applied: e ic- educing an ioxidan powe (FRAP) me hod, 2,2-diphenyl-1-pic ylhyd azyl
(DPPH) adical sca enging ac i i y assay, 2,2-azino-bis-3-e hylbenzo hiazoline-6-sulphonic
acid (ABTS) adical sca enging assay and cup ic ion- educing an ioxidan capaci y (CUPRAC)
me hod.All me hods we e pe o med acco ding o p e iously desc ibed
p o ocols [18,24,25]
,
and he inal esul s we e exp essed as mic omoles o T olox equi alen (TE) pe g am o
d y ma e (DM) o LBC.
2.2.4. Fo mula ion P epa a ion
Hyd ogel was p epa ed as he 2% (w/ ) wa e solu ion o comme cial polyme
A is o lex
®
AVC and mixed using an o e head s i e (Heidolph Hei-TORQUE Value
100, Schwabach, Ge many) a 800 pm o 5 min un il gel o ma ion occu ed. To make an
oil-in-wa e (O/W) c eam gel, 3% ( / ) o Heliogel
™
gelling agen and 10% ( / ) isop opyl
my is a e we e mixed a 500 pm o 2 min. Then, 87% ( / ) wa e was added, and he
Pha maceu ics 2024,16, 1209 4 o 13
mix u e was s i ed a 1000 pm o 5 min un il a c eam gel was o med. Fo p epa a ion
o he oil-in-wa e (O/W) emulsion, i s ly, 84% ( / ) wa e and a mix u e o 5% (w/ )
emulsi ie con aining PEG-100 s ea a e and glyce ol–s ea a e (Sabowax FL 65, Sabo), 1%
(w/ ) ce ea yl alcohol (Lane e-o, Bas ), and 10% ( / ) isop opyl my is a e we e hea ed
sepa a ely o 75
◦
C in a wa e ba h (Memme , Schwabach, Ge many). A e eaching
he desi ed empe a u e and mel ing he lipid componen s, wa e and he mix u e we e
combined, and s i ed a 500 pm o 1 min, ollowed by s i ing a 1000 pm o 2 min, and
hen a 700 pm o 3 min. A e he empe a u e o he emulsion d opped o 50
◦
C, i was
s i ed a 1100 pm o 1 min o achie e be e dispe sion o oil pa icles in he aqueous
phase. Blackcu an ex ac was added o all o mula ions o he inal concen a ion o
1% ( / ).
2.2.5. Release S udies
Expe imen s we e pe o med using F anz di usion cell (20 mL olume and 25 mm o i-
ice diame e , Pe meGea , Inc., Helle own, PA, USA) and wo memb ane ypes—cellulose
ace a e memb ane and ansde mal es di usion model, S a -M
®
memb ane (Me ck KgaA,
Da ms ad , Ge many). The p epa ed o mula ion, weighing 2 g
±
5%, was placed in he
dono compa men . The ecep o compa men was illed wi h he 0.01 M phospha e
bu e saline (PBS) pH 7.4, which was con inuously s i ed wi h a small magne ic ba
(750 pm) and he mos a ed (25
◦
C). Be o e applica ion, he cellulose ace a e memb ane
was p e-soaked in ecep o medium o 30 min, whe eas he S a -M
®
memb ane did no
necessi a e p e-hyd a ion. A he p ede ined ime, he samples (0.5 mL) we e collec ed om
he ecep o chambe and immedia ely eplaced wi h equi alen olume o esh ecep o
medium. The amoun o an hocyanins ha c ossed he cellulose ace a e and S a -M
®
mem-
b ane was quan i ied by he RP-HPLC-UV me hod. Addi ionally, he S a -M
®
memb ane
was subjec ed o e hanol ex ac ion o de e mine i polyphenol molecules emained in he
memb ane laye s a e he ansde mal di usion s udy. The memb ane was cu in o small
pieces (~1 mm
2
) and mixed wi h 10 mL o 50% ( / ) e hanol o 24 h a 25
◦
C wi h cons an
s i ing a 150 pm. The esul ing sol en ex ac was hen concen a ed using a o a y
e apo a o (Ro a apo R-210, Buchi, Flawil, Swi ze land) and analysed by HPLC.
2.2.6. RP-HPLC-UV Analysis
Quan i a i e analyses o samples and an hocyanin s anda ds we e pe o med by a
Dionex Ul ima e 3000 The mo Scien i ic (Wal ham, MA, USA) high-pe o mance liquid
ch oma og aphy (HPLC) sys em and a e e se-phase column (ZORBAX Eclipse Plus
C18, 4.6, 150 mm, 5
µ
m) by a me hod p e iously applied o blackcu an an hocyanins
sepa a ion [
17
]. We applied g adien elu ion wi h sol en (A) H
2
O:TFA = 100:0.1% and
sol en (B) MeOH:TFA = 100:0.1% in he ollowing way: 0–5 min isoc a ic 5% B, 5–45 min
g adien om 5% o 50% B, hen 45–55 min isoc a ic 50% B, 55–55.1 min g adien om
50% o 5% B, 55.1–60 min isoc a ic 5% B. Eluen low a e o 1 mL/min was used, and he
column was kep a 45
◦
C. The injec ion olume o he samples was 50
µ
L. De ec ion o
an hocyanins om analysed ex ac s and comme cial s anda ds was ca ied ou by a UV
de ec o a 520 nm. An hocyanin concen a ions we e calcula ed based on s anda d cu es
ob ained wi h comme cial an hocyanin s anda ds. These cu es we e made by injec ing
he same olume (50
µ
L) o a se ially dilu ed s ock solu ion o known conce a ion in o
HPLC and plo ing peak a ea agains concen a ion o each an hocyanin. The slopes o
s anda d cu es we e 0.00125 mmol/L o delphinidin-3-glucoside, 0.00128 mmol/L o
delphinidin-3- u inoside, 0.00157 mmol/L o cyanidin-3-glucoside and 0.00160 mmol/L
o cyanidin-3- u inoside. The e en ion imes we e 24.7 min o delphinidin-3-glucoside,
25.9 min o delphinidin-3- u inoside, 27.0 min o cyanidin-3-glucoside and 28.4 min o
cyanidin-3- u inoside.
Pha maceu ics 2024,16, 1209 5 o 13
2.2.7. Calcula ion o E ec i e Di usion Coe icien s
The expe imen ally ob ained esul s o he ini ial elease ime ( i s 6 h) in his s udy
we e modelled using he Ko smeye –Peppas equa ion:
M
M∞=k· n(1)
whe e M
is an amoun o bioac i e compound eleased a ime , M
∞
is an amoun o
d ug elease a e in ini e ime, k is a kine ic cons an and nis an exponen deno ing he
cha ac e is ic o he elease mechanism o he sys em. In case o he hin ilm, n= 0.5
signi ies Fickian di usion, n= 1.0 ep esen s elaxa ion o he polyme ic chain, and he
0.5 < n< 1.0
sugges s anomalous o non-Fickian di usion, indica ing a combined e ec o
bo h Fickian di usion and polyme elaxa ion [
26
]. Addi ionally, i he e is a delay (
lag
)
be o e he di usion s a s, he modi ied o m o Equa ion (1) was employed [27,28].
M
M∞=k· − lagn(2)
E ec i e di usion coe icien s (De ) we e inally calcula ed as ollows [27,28]:
De =π·δ2·k2
4(3)
whe e δ ep esen s he sum o memb ane and o mula ion hicknesses.
The quali y o model p edic ions wi h espec o he expe imen al alues was e alua ed
using mean pe cen age e o (MPE):
MPE(%)=1
n
n
∑
i=1
M exp
i−M mod
i
M exp
i
·100 (4)
whe e nis he o al numbe o measu emen s o each componen being analysed, M
iexp
is he amoun o he bioac i e componen as obse ed in he expe imen and M
imod
is
amoun o bioac i e componen calcula ed h ough he model [29].
2.2.8. S a is ical Analysis
All expe imen s we e epea ed wice, and he esul s o expe imen s we e p o ided as
mean ±s anda d de ia ion.
3. Resul s and Discussion
3.1. Polyphenol Con en and An ioxidan Ac i i y o Blackcu an Ex ac
The majo classes o polyphenols ound in blackcu an s include la onoids, such as
an hocyanins and la onols, as well as phenolic acids and annins. The combina ion o
hese an ioxidan compounds in blackcu an s con ibu es o hei nume ous heal h bene i s
and makes hem a aluable componen in unc ional oods and bioac i e cosme ics [
30
].
To assess he polyphenol con en and an ioxidan p ope ies o enzyma ically de i ed
blackcu an ex ac , a ious spec opho ome ic assays we e employed.
The polyphenolic composi ion o blackcu an ex ac ob ained wi h Viscozyme
®
L
and Rohapec
®
MC, enzyme p epa a ions wi h high celluloly ic and pec inoly ic ac i i y,
a e p esen ed in Table 1. The o al polyphenol con en was 26.9 mg GAE/g DM, which
is abou 3 imes highe compa ed o he polyphenol con en o ex ac ob ained by con-
en ional ex ac ion p ocess (9.35 mg GAE/DM [
17
]) and ul asound-assis ed ex ac ion
(
10.23 mg GAE/g DM [31]
). To al con en s o majo polyphenol classes, la onoids and
phenolic acids, we e 3.98 mg QE/g DM and 3.85 mg CAE/g DM, espec i ely. I is known
ha p edominan la onoids in blackcu an s a e an hocyanins, which con ibu e o hei
ib an pu ple colou and a e conside ed o be pa icula ly po en an ioxidan s [
30
]. Thei
Pha maceu ics 2024,16, 1209 6 o 13
o al con en in his ex ac eached 2.21 mg Cy3GE/g DM. Fla onols, ano he impo an
subclass o la onoids, we e p esen in ex ac a a concen a ion o 2.57 mg QE/g DM,
which is signi ican ly highe compa ed o he p e iously epo ed la onol con en in black-
cu an juice (1.31 mg QE/g DM [
32
]). Bo h classes o annins, hyd olysable and condensed,
we e de ec ed in he ex ac in concen a ions o 12.5 mg GAE/g DM and 0.15 mg CyE/g
DM, espec i ely. This dual p esence o hyd olysable and condensed annins con ibu es
o he di e se and bene icial polyphenolic p o ile o blackcu an s, showcasing po en ial
an ioxidan and heal h-p omo ing p ope ies [33].
Table 1. Polyphenol composi ion and an ioxidan ac i i y o blackcu an ex ac .
Polyphenol Composi ion
Polyphenols
(mg GAE/g DM)
Fla onoids
(mg QE/g DM)
An hocyanins
(mg Cy3GE/g
DM)
Fla onols
(mg QE/g DM)
Phenol Acids
(mg CAE/g DM)
Hyd olysable
Tannins
(mg GAE/g DM)
Condensed
Tannins
(mg CyE/g DM)
26.9 ±1.45 3.98 ±0.24 2.21 ±0.08 2.57 ±0.13 3.85 ±0.27 12.5 ±0.42 0.15 ±0.01
An ioxidan ac i i y
FRAP
(µmol TE/g DM)
ABTS
(µmol TE/g DM)
CUPPRAC
(µmol TE/g DM)
DPPH
(µmol TE/g DM)
122.6 ±7.05 208.2 ±5.34 338.4 ±16.64 103.9 ±5.19
The esul s we e p o ided as mean ±s anda d de ia ion. The expe imen s we e conduc ed in duplica e.
To e alua e blackcu an polyphenol’s abili y o neu alise ee adicals and ac as
agen s in sa egua ding cells om oxida i e ha m, ou di e en me hods we e applied.
These me hods we e based on single-elec on ans e eac ions (CUPRAC and FRAP) as
well as mixed elec on and hyd ogen a om ans e (ABTS and DPPH) [
34
]. The an iox-
idan ac i i y o blackcu an ex ac ob ained h ough enzyme-assis ed ex ac ion was
338.4 µmol TE/g DM
de e mined wi h he CUPRAC me hod, whe eas he FRAP me hod
yielded an an ioxidan capaci y o 122.6
µ
mol TE/g DM. This esul indica es an inc eased
an ioxidan ac i i y compa ed o esul s o s udy using ul asound-assis ed ex ac ion o
blackcu an be ies, whe e he an ioxidan ac i i y measu ed by he FRAP me hod was
36.5 µmol TE/g DM [31].
Acco ding o he ABTS me hod, he an ioxidan ac i i y o blackcu an ex ac in
he p esen s udy was 208.2
µ
mol TE/g DM, while he alue ob ained h ough he DPPH
me hod was 103.9
µ
mol TE/g DM. Fo example, he an ioxidan ac i i y o blackcu an
be y cul i a s p e iously de e mined by he ABTS me hod was up o
54 µmol/TE g DM [35]
,
indica ing ha enzyme-assis ed ex ac ion no only enabled inc eased ex ac ion e iciency
by eleasing polyphenols apped in cell wall s uc u es bu u he enhanced he ex ac an-
ioxidan ac i i y. By neu alizing di e en ee adicals, polyphenolic compounds p esen
in enzyma ically de i ed blackcu an ex ac showed e sa ile an ioxidan ac i i y. This
unde sco es hei po en ial as e ec i e agen s in mi iga ing damage caused by oxida i e
s ess and p omo ing o e all cellula well-being by bo h o al and opical adminis a ion.
De e mina ion o Blackcu an An hocyanins’ Composi ion
Since an hocyanins a e polyphenols o pa icula in e es o cosme ic applica ion, due
o hei high de ma ologic ac i i y and signi ican ole in imp o ing he appea ance o
he skin, hei composi ion in enzyma ically ob ained blackcu an ex ac was analysed
in mo e de ails [
36
]. Fo ha pu pose, e e se-phase HPLC-UV analysis was pe o med,
and he cha ac e is ic HPLC p o ile o he ex ac , eco ded a 520 nm, is p esen ed in
Figu e 1. Based on comme cial s anda ds, ou majo an hocyanin compounds we e
iden i ied, delphinidin-3-glucoside (del-3-glu), delphinidin-3- u inoside (del-3- u ), and
cyanidin-3-glucoside (cya-3-glu) and cyanidin-3- u inoside (cya-3- u ), a concen a ions o
1.34 mmol/L, 3.43 mmol/L, 0.92 mmol/L and 3.25 mmol/L, espec i ely. As p e iously
epo ed, blackcu an ex ac an hocyanin composi ion s ongly depends on he ex ac ion
me hod used and plan cul i a s [
35
,
37
], which de e mines he sha e o each molecule in
he ex ac . In o de o e alua e he possibili y o opical, cu aneous and/o ansde mal
deli e y o iden i ied an hocyanins, enzyma ically ob ained ex ac o de ined an hocyanin
Pha maceu ics 2024,16, 1209 7 o 13
composi ion was inco po a ed in di e en cosme ic o mula ions, and hei pe mea ion
h ough wo di e en memb anes was moni o ed.
Pha maceu ics 2024, 16, x FOR PEER REVIEW 7 o 14
u e 1. Based on comme cial s anda ds, ou majo an hocyanin compounds we e iden i-
ied, delphinidin-3-glucoside (del-3-glu), delphinidin-3- u inoside (del-3- u ), and cya-
nidin-3-glucoside (cya-3-glu) and cyanidin-3- u inoside (cya-3- u ), a concen a ions o
1.34 mmol/L, 3.43 mmol/L, 0.92 mmol/L and 3.25 mmol/L, espec i ely. As p e iously e-
po ed, blackcu an ex ac an hocyanin composi ion s ongly depends on he ex ac ion
me hod used and plan cul i a s [35,37], which de e mines he sha e o each molecule in
he ex ac . In o de o e alua e he possibili y o opical, cu aneous and/o ansde mal
deli e y o iden i ied an hocyanins, enzyma ically ob ained ex ac o de ined an hocya-
nin composi ion was inco po a ed in di e en cosme ic o mula ions, and hei pe mea-
ion h ough wo di e en memb anes was moni o ed.
Figu e 1. Cha ac e is ic ch oma og am o he an hocyanins om blackcu an ex ac : (1)—del-
phinidin-3-glucoside, (2)—delphinidin-3- u inoside, (3)—cyanidin-3-glucoside and (4)—cyanidin-
3- u inoside (cya-3-glu), eco ded a 520 nm.
3.2. Release S udy
An hocyanin- ich blackcu an ex ac a a concen a ion o 1% ( /w) was inco po-
a ed in o h ee di e en cosme ic o mula ions (hyd ogel, oil-in-wa e c eam gel and oil-
in-wa e emulsion), and he di usion p o iles o ou an hocyanins ac oss cellulose ace a e
memb ane we e de e mined. Cellulose ace a e memb ane was applied o assess he pe -
o mance o di e en opical o mula ions o be used as ca ie o polyphenol compounds
[10]. The mola concen a ions o an hocyanins in he ecep o compa men which di -
used om es ed cosme ic o mula ions h ough cellulose ace a e memb ane as well as
hei cumula i e elease ( he p opo ion o each di used an hocyanin) p o iles a e p e-
sen ed in Figu es 2–4. Rep esen a i e o e lapped ch oma og ams o an hocyanins di u-
sion o e ime a e p o ided in he Appendix A as Figu e A1.
Based on concen a ion p o iles o molecules eleased om hyd ogel shown in Figu e
2a, i is ob ious ha he amoun o a hocyanidin-3- u inosides (cya-3- u and del-3- u )
was signi ican ly highe o e ime compa ed o an hocyanidin-3-glucosides (cya-3-glu
and del-3-glu).The concen a ion o cya-3- u was consis en ly highe han del-3- u , wi h
he g ea es ise occu ing wi hin he ini ial 2 h o he expe imen , a e which i displayed
a somewha slowe di usion a e. Since highe ini ial amoun s o del-3- u we e p esen
in he dono compa men compa ed o cya-3- u , i can be assumed ha di e ences in
hei molecula s uc u es a e in luencing hei ansmemb ane di usion. On he o he
hand, he de ec ed concen a ions o cya-3-glu and del-3-glu we e e y simila o e he
obse ed pe iod. A e 24 h o di usion, del-3-glu, del-3- u , cya-3- u and cya-3-glu mol-
ecules eached concen a ions o 0.255 µmol/L, 1.226 µmol/L, 0.340 µmol/L and 1.389
µmol/L, espec i ely, in he ecep o compa men . When compa ing he cumula i e e-
leases (Figu e 2b), i can be obse ed ha he elease o bo h cyanidin molecules we e e y
close and cons an ly highe han he elease o bo h delphinidins molecules. On he o he
Figu e 1. Cha ac e is ic ch oma og am o he an hocyanins om blackcu an ex ac : (1)—delphinidin-
3-glucoside, (2)—delphinidin-3- u inoside, (3)—cyanidin-3-glucoside and (4)—cyanidin-3- u inoside
(cya-3-glu), eco ded a 520 nm.
3.2. Release S udy
An hocyanin- ich blackcu an ex ac a a concen a ion o 1% ( /w) was inco po-
a ed in o h ee di e en cosme ic o mula ions (hyd ogel, oil-in-wa e c eam gel and
oil-in-wa e emulsion), and he di usion p o iles o ou an hocyanins ac oss cellulose
ace a e memb ane we e de e mined. Cellulose ace a e memb ane was applied o assess
he pe o mance o di e en opical o mula ions o be used as ca ie o polyphenol
compounds [
10
]. The mola concen a ions o an hocyanins in he ecep o compa men
which di used om es ed cosme ic o mula ions h ough cellulose ace a e memb ane
as well as hei cumula i e elease ( he p opo ion o each di used an hocyanin) p o iles
a e p esen ed in Figu es 2–4. Rep esen a i e o e lapped ch oma og ams o an hocyanins
di usion o e ime a e p o ided in he Appendix Aas Figu e A1.
Pha maceu ics 2024, 16, x FOR PEER REVIEW 8 o 14
hand, del-3-glu and del-3- u had simila p o iles only in he i s 6 h o di usion, ollowed
by signi ican ly as e elease o del-3- u . The p opo ion o di used cyanidin molecules
du ing he i s 6 h was 7.26–9.47%, while he p opo ion o bo h delphinidin molecules
was 1.88–3.27%, indica ing slowe di usion o delphinidin glycosides compa ed o cya-
nidin glycosides. No ably, a e 24 h o di usion, he p opo ion o cya-3- u emained he
highes a 39.22%. The e was no signi ican di e ence in he p opo ions o cya-3-glu and
del-3- u wi h cumula i e amoun s o 32.78% and 33.81%, espec i ely, while only 17.51%
o he ini ial del-3-glu amoun di used du ing he obse ed ime, indica ing sus ained
elease om A is o lex® AVC-based hyd ogel.
(a)
(b)
Figu e 2. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he hyd ogel.
Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e he s and-
a d de ia ions.
The elease kine ic o blackcu an an hocyanins om he second es ed o mula ion,
o/w c eam gel, which is shown in Figu e 3a, e ealed ha cya-3- u was mos abundan in
he ecep o solu ion (0.330 µmol/L), ollowed by del-3- u (0.135 µmol/L), cya-3-glu (0.060
µmol/L) and del-3-glu (0.024 µmol/L). Howe e , he p opo ion o di used cya-3- u du -
ing 24 h (Figu e 3b) was no ably educed, almos 4.3 imes lowe han ha obse ed om
he hyd ogel (9.15%), while he cumula i e amoun o he o he h ee an hocyanins was
e en lowe —5.7, 9.3 and 10.2 imes o cya-3-glu, del-3- u and del-3-glu, espec i ely.
(a)
(b)
Figu e 2. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he hyd ogel.
Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e he
s anda d de ia ions.
Pha maceu ics 2024,16, 1209 8 o 13
Pha maceu ics 2024, 16, x FOR PEER REVIEW 8 o 14
hand, del-3-glu and del-3- u had simila p o iles only in he i s 6 h o di usion, ollowed
by signi ican ly as e elease o del-3- u . The p opo ion o di used cyanidin molecules
du ing he i s 6 h was 7.26–9.47%, while he p opo ion o bo h delphinidin molecules
was 1.88–3.27%, indica ing slowe di usion o delphinidin glycosides compa ed o cya-
nidin glycosides. No ably, a e 24 h o di usion, he p opo ion o cya-3- u emained he
highes a 39.22%. The e was no signi ican di e ence in he p opo ions o cya-3-glu and
del-3- u wi h cumula i e amoun s o 32.78% and 33.81%, espec i ely, while only 17.51%
o he ini ial del-3-glu amoun di used du ing he obse ed ime, indica ing sus ained
elease om A is o lex® AVC-based hyd ogel.
(a)
(b)
Figu e 2. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he hyd ogel.
Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e he s and-
a d de ia ions.
The elease kine ic o blackcu an an hocyanins om he second es ed o mula ion,
o/w c eam gel, which is shown in Figu e 3a, e ealed ha cya-3- u was mos abundan in
he ecep o solu ion (0.330 µmol/L), ollowed by del-3- u (0.135 µmol/L), cya-3-glu (0.060
µmol/L) and del-3-glu (0.024 µmol/L). Howe e , he p opo ion o di used cya-3- u du -
ing 24 h (Figu e 3b) was no ably educed, almos 4.3 imes lowe han ha obse ed om
he hyd ogel (9.15%), while he cumula i e amoun o he o he h ee an hocyanins was
e en lowe —5.7, 9.3 and 10.2 imes o cya-3-glu, del-3- u and del-3-glu, espec i ely.
(a)
(b)
Figu e 3. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he oil-in-wa e
gel c eam. Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e
he s anda d de ia ions.
Pha maceu ics 2024, 16, x FOR PEER REVIEW 9 o 14
Figu e 3. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he oil-in-wa e
gel c eam. Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e
he s anda d de ia ions.
When i comes o he di usion o an hocyanins om he o/w emulsion, he same
concen a ion end o hese compounds was obse ed (Figu e 4a) as wi h hyd ogel and
o/w c eam gel. Rega ding di used amoun s, 0.041 µmol/L, 0.181 µmol/L, 0.143 µmol/L,
0.577 µmol/L o del-3-glu, del-3- u , cya-3-glu and cya-3-glu we e de ec ed in he ecep o
luid, espec i ely. By analysing cumula i e amoun s o ou an hocyanins, i can be seen
(Figu e 4b) ha a e 24 h o pe mea ion, he p opo ions o del-3-glu, del-3- u , cya-3-glu
and cya-3- u we e 3.0%, 5.1%, 15.0% and 17.2%, espec i ely. These p opo ions we e
conside ably lowe compa ed o hose obse ed o hyd ogel, bu sligh ly highe han
hose ob ained o he o/w gel c eam, indica ing ha educ ion o de ec ed molecule con-
cen a ion in he ecep o solu ion could be asc ibed o o mula ion oil con en . Lipid com-
ponen s e iden ly had a nega i e e ec on he elease o he an hocyanins om he o/w
c eam gel as well as o/w emulsion, which is in line wi h o he s udies which showed ha
he highe sha e o he oil phase in he o mula ions led o he slowe elease o polyphe-
nols [38,39]. A second po en ial explana ion o he as e an hocyanins pe mea ion om
he hyd ogel compa ed o oil-based o mula ions is ha hese molecules ha e a suga
moie y in hei s uc u e, which gi es hem an amphiphilic cha ac e , making hem su -
ace-ac i e. Consequen ly, i is likely ha an hocyanins a e mo e s ongly a ached o he
oil–wa e in e ace, hinde ing hei elease [39].
(a)
(b)
Figu e 4. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he oil-in-wa e
emulsion. Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e
he s anda d de ia ions.
De e mina ion o Di usion Coe icien s
To comp ehensi ely desc ibe he elease p o iles o blackcu an an hocyanins om
hyd ogel, o/w c eam gel and o/w emulsion and ind ou he mechanism o elease as well
as kine ic cons an s, he di usion da a we e i ed o he Ko smeye –Peppas equa ion
(Equa ion (1)). This equa ion was sugges ed by se e al au ho s o in es iga ing he e-
lease o polyphenols om semisolid o mula ions [39–41]. E ec i e di usion coe icien s
(De ) and kine ic cons an s o del-3-glu, del-3- u , cya-3-glu and cya-3- u om blackcu -
an ex ac inco po a ed in h ee o mula ions ac oss he cellulose ace a e memb ane a e
p esen ed in Table 2.
Figu e 4. Concen a ion p o ile (a) and cumula i e elease (b) o an hocyanins om he oil-in-wa e
emulsion. Da a ep esen he mean alues o h ee independen expe imen s, and e o ba s indica e
he s anda d de ia ions.
Based on concen a ion p o iles o molecules eleased om hyd ogel shown in
Figu e 2a
,
i is ob ious ha he amoun o a hocyanidin-3- u inosides (cya-3- u and del-3- u ) was
signi ican ly highe o e ime compa ed o an hocyanidin-3-glucosides (cya-3-glu and
del-3-glu).The concen a ion o cya-3- u was consis en ly highe han del-3- u , wi h he
g ea es ise occu ing wi hin he ini ial 2 h o he expe imen , a e which i displayed a
somewha slowe di usion a e. Since highe ini ial amoun s o del-3- u we e p esen in
he dono compa men compa ed o cya-3- u , i can be assumed ha di e ences in hei
molecula s uc u es a e in luencing hei ansmemb ane di usion. On he o he hand,
he de ec ed concen a ions o cya-3-glu and del-3-glu we e e y simila o e he obse ed
pe iod. A e 24 h o di usion, del-3-glu, del-3- u , cya-3- u and cya-3-glu molecules
eached concen a ions o 0.255
µ
mol/L, 1.226
µ
mol/L, 0.340
µ
mol/L and 1.389
µ
mol/L,
espec i ely, in he ecep o compa men . When compa ing he cumula i e eleases
(Figu e 2b), i can be obse ed ha he elease o bo h cyanidin molecules we e e y close
Pha maceu ics 2024,16, 1209 9 o 13
and cons an ly highe han he elease o bo h delphinidins molecules. On he o he hand,
del-3-glu and del-3- u had simila p o iles only in he i s 6 h o di usion, ollowed by
signi ican ly as e elease o del-3- u . The p opo ion o di used cyanidin molecules
du ing he i s 6 h was 7.26–9.47%, while he p opo ion o bo h delphinidin molecules was
1.88–3.27%, indica ing slowe di usion o delphinidin glycosides compa ed o cyanidin
glycosides. No ably, a e 24 h o di usion, he p opo ion o cya-3- u emained he highes
a 39.22%. The e was no signi ican di e ence in he p opo ions o cya-3-glu and del-3- u
wi h cumula i e amoun s o 32.78% and 33.81%, espec i ely, while only 17.51% o he
ini ial del-3-glu amoun di used du ing he obse ed ime, indica ing sus ained elease
om A is o lex®AVC-based hyd ogel.
The elease kine ic o blackcu an an hocyanins om he second es ed o mula ion,
o/w c eam gel, which is shown in Figu e 3a, e ealed ha cya-3- u was mos abundan
in he ecep o solu ion (0.330
µ
mol/L), ollowed by del-3- u (0.135
µ
mol/L), cya-3-glu
(0.060
µ
mol/L) and del-3-glu (0.024
µ
mol/L). Howe e , he p opo ion o di used cya-3- u
du ing 24 h (Figu e 3b) was no ably educed, almos 4.3 imes lowe han ha obse ed
om he hyd ogel (9.15%), while he cumula i e amoun o he o he h ee an hocyanins
was e en lowe —5.7, 9.3 and 10.2 imes o cya-3-glu, del-3- u and del-3-glu, espec i ely.
When i comes o he di usion o an hocyanins om he o/w emulsion, he same
concen a ion end o hese compounds was obse ed (Figu e 4a) as wi h hyd ogel and
o/w c eam gel. Rega ding di used amoun s, 0.041
µ
mol/L, 0.181
µ
mol/L, 0.143
µ
mol/L,
0.577
µ
mol/L o del-3-glu, del-3- u , cya-3-glu and cya-3-glu we e de ec ed in he ecep o
luid, espec i ely. By analysing cumula i e amoun s o ou an hocyanins, i can be seen
(Figu e 4b) ha a e 24 h o pe mea ion, he p opo ions o del-3-glu, del-3- u , cya-3-glu
and cya-3- u we e 3.0%, 5.1%, 15.0% and 17.2%, espec i ely. These p opo ions we e
conside ably lowe compa ed o hose obse ed o hyd ogel, bu sligh ly highe han
hose ob ained o he o/w gel c eam, indica ing ha educ ion o de ec ed molecule
concen a ion in he ecep o solu ion could be asc ibed o o mula ion oil con en . Lipid
componen s e iden ly had a nega i e e ec on he elease o he an hocyanins om he
o/w c eam gel as well as o/w emulsion, which is in line wi h o he s udies which showed
ha he highe sha e o he oil phase in he o mula ions led o he slowe elease o
polyphenols [
38
,
39
]. A second po en ial explana ion o he as e an hocyanins pe mea ion
om he hyd ogel compa ed o oil-based o mula ions is ha hese molecules ha e a
suga moie y in hei s uc u e, which gi es hem an amphiphilic cha ac e , making hem
su ace-ac i e. Consequen ly, i is likely ha an hocyanins a e mo e s ongly a ached o
he oil–wa e in e ace, hinde ing hei elease [39].
De e mina ion o Di usion Coe icien s
To comp ehensi ely desc ibe he elease p o iles o blackcu an an hocyanins om
hyd ogel, o/w c eam gel and o/w emulsion and ind ou he mechanism o elease as
well as kine ic cons an s, he di usion da a we e i ed o he Ko smeye –Peppas equa ion
(
Equa ion (1)
). This equa ion was sugges ed by se e al au ho s o in es iga ing he e-
lease o polyphenols om semisolid o mula ions [
39
–
41
]. E ec i e di usion coe icien s
(D
e
) and kine ic cons an s o del-3-glu, del-3- u , cya-3-glu and cya-3- u om blackcu -
an ex ac inco po a ed in h ee o mula ions ac oss he cellulose ace a e memb ane a e
p esen ed in Table 2.
Based on he coe icien o de e mina ion (R
2
) and mean pe cen age e o (MPE), i
can be con i med ha he chosen elease model was sui able o desc ibing he elease
kine ic o blackcu an an hocyanins om es ed o mula ions. The ob ained alue o n
was 0.5 o all es ed o mula ions, sugges ing ha he elease mechanism o an hocyanins
om all o mula ions was o a Fickian ype, highly dependen on molecula di usion.
The lag phase, ime be o e molecules we e ound in he ecep o luid, was only de ec ed
o o mula ions con aining oil and o bo h delphinidin glycosides, while in o he cases,
di usion s a ed immedia ely a e expe imen s a . As can be seen om Table 2, kine ic
cons an and, consequen ly, di usion coe icien s showed ends which a e in line wi h
