BOSNlAN lOUlNAl Ol BASlC MlDlCAl SCllNClS 2008, 8 (3).

23+-238
&
Abstract
Anthocvanins are eective antioxidants but thev have also been proposed to have other bioloical activi-
ties independent ot their antioxidant capacities that produce health benets. lxamples rane trom inhibi-
tion ot cancer cell rovth in vitro, induction ot insulin production in isolated pancreatic cells, reduction
ot starch diestion throuh inhibition ot a-lucosidase activitv, suppression ot inammatorv responses as
vell as protection aainst ae-related declines in conitive behavior and neuronal dvstunction in the cen-
tral nervous svstem. Hovever, to achieve anv bioloical eect in a specic tissue or oran, anthocvanins
must be bioavailable, i.e. eectivelv absorbed trom the astrointestinal tract (GlT) into the circulation
and delivered to the appropriate location vithin the bodv. ln this studv, ve assess the stabilitv ot antho-
cvanins trom commercial Black currant (Ribes nigrum L.) uice usin an in vitro diestion procedure that
mimics the phvsiochemical and biochemical conditions encountered in the astrointestinal tract (GlT).
Te main obective ot this vork vas the evaluation ot stabilitv ot anthocvanins durin in vitro diestion
in astric and intestinal uid reardin vhether appropriate enzvme (pepsin or pancreatin) vas added
or not. Anthocvanins present in commercial black currant uice remain stable durin in vitro diestion in
gastric uid reardless vhether pepsin vas added into the medium or not. Also, thev remain stable dur-
in in vitro diestion in simulated intestinal uid vithout pancreatin. Te stabilitv studies ot anthocvanins
in the intestinal uid containin pancreatin indicated reduced stabilitv, vhich also mainlv contribute to
sliht reduction ot total anthocvanins content (-+,8) in commercial black currant uice.
llY WOlDS. anthocvanins, stabilitv, black currant, astrointestinal diestion
STABILITY OF
ANTHOCYANINS
FROM COMMERCIAL
BLACK CURRANT JUICE
UNDER SIMULATED
GASTROINTESTINAL
DIGESTION
Alija Uzunovi*, Edina Vrani
lnstitute tor Qualitv Control ot Medicines,
Titova ,, ;+ ooo Saraevo, Bosnia and Herzeovina
Department ot Pharmaceutical Technolov, lacultv ot Pharmacv,
Universitv ot Saraevo, ekalua ,o, ;+ ooo Saraevo, Bosnia and Herzeovina
Correspondin author
BOSNlAN lOUlNAl Ol BASlC MlDlCAl SCllNClS 2008, 8 (3). 233-238
AlllA UZUNOVl, lDlNA VlANl. STABlllTY Ol ANTHOCYANlNS llOM COMMllClAl BlACl
CUllANT lUlCl UNDll SlMUlATlD GASTlOlNTlSTlNAl DlGlSTlON
Introduction
Anthocvanins belon to the tlavonoid roup ot polv-
phenolic compounds and are responsible tor the red,
purple and blue hues present in plant orans such as
truits, overs, rains and leaves as vell as in products
made trom those sources (+). Pelaronidin, cvanidin, pe-
onidin, delphinidin, petunidin, and malvidin are the six
common anthocvanidins tound in nature. Teir struc-
tures mav be altered bv lvcosidic substitution (lucose,
alactose, rhamnose, xvlose, and arabinose) at the and
, positions in the A and C rins. Tev are industriallv
important natural tood colorins (.). Additional varia-
tions occur bv acvlation ot the suar roups vith acids.
Acetic acid, p-coumaric acid, caeic acid, malonic acid,
sinapic acid, terulic acid, oxalic acid, and succinic acid
are some ot the commonlv tound acvlatin roups (, |).
Anthocvanins are eective antioxidants (., ,) but thev
have also been proposed to have other bioloical ac-
tivities that are independent ot their antioxidative ca-
pacities and produce health benets. lxamples rane
trom inhibition ot cancer cell rovth in vitro (6), in-
duction ot insulin production in isolated pancreatic
cells (;), reduction ot starch diestion throuh inhi-
bition ot a-lucosidase activitv (8), suppression ot in-
ammatorv responses (,) as vell as protection aainst
ae-related declines in conitive behavior and neu-
ronal dvstunction in the central nervous svstem (+o).
Hovever, to achieve anv bioloical eect in a specic tis-
sue or oran, anthocvanins must be bioavailable, i.e. et-
tectivelv absorbed trom the astrointestinal tract (GlT)
into the circulation and delivered to the appropriate
location vithin the bodv. Oral intake ot anthocvanin-
rich truits, extracts or pure anthocvanins has proved
to have benecial eects in preventin or suppressin
ailments in vivo (++, +.). Studies ot oral administration
ot anthocvanins have conrmed increased antioxidant
status ot the serum (++, +, +|) but this is usuallv ac-
companied bv verv lov uptake ot anthocvanins into
the serum (+ ot dose) (+,, +6, +;)) and correspond-
inlv lov levels ot urinarv excretion as intact or conu-
ated torms. Te apparent lov bioavailabilitv ot antho-
cvanins seems to cast doubt on their abilitv to exert
their proposed benecial eects throuhout the bodv.
Assessment ot true bioavailabilitv ot anv class
ot phvt ochemi cal s requi res dat a concern-
in their absorption, metabolism, tissue and or-
an distribution and excretion (+8). Such stud-
ies carried out in animals or human subects are
complex, expensive and raise moral and ethical questions.
The relative stabilitv ot the anthocvanins un-
der GlT conditions is crucial tor the bioavailabil-
itv ot these compounds, as it vill determine the
pool size tor vhatever active mechanisms are pres-
ent in the stomach (+,) or the small intestine (.o).
ln this studv, ve assess the stabilitv ot anthocvanins
trom Black currant (Ribes nigrum L.) uice usin an
in vitro diestion procedure that mimics the phvs-
iochemical and biochemical conditions encoun-
tered in the astrointestinal tract (GlT). The main
obective ot this vork vas the evaluation ot stabil-
itv ot anthocvanins durin in vitro diestion in as-
tric and intestinal tluid reardin vhether appropri-
ate enzvme (pepsin or pancreatin) vas added or not.
Materials and Methods
Samples
luice sample, that vas not blend ot ditterent
truits, and that represented an arrav ot antho-
cvanin containin products available at a local
market vas purchased, brouht to the labora-
torv, aliquoted and stored at -.ooC until analvzed.
Chemicals and Reagents
Te used reaents vere all ot analvtical rade, unless
othervise stated. Glacial acetic acid (,8), orthophos-
phoric acid (8,) and sodium chloride vere provided
bv Carlo lrba, monobasic potassium phosphate, con-
centrated hvdrochloric acid (;) and sodium hv-
droxide vere provided bv Merck. Acetonitrile vas
HPlC rade (Sima-Aldrich), pepsin trom ho stom-
ach (lluka), pancreatin trom porcine pancreas (Sima).
In vitro digestion procedure
The di l ut ed bl ack currant ui ce vas sub-
ected to successive in vitro astric and pan-
creati c di esti on, t ol l ovi n the procedure.

TlST +. An aliquot ot uice vas diluted vith hvdrochlo-
ric acid solution, supplemented vith sodium chloride
and adusted to pH +,. (+oo ml ot uice - |oo ml hvdro-
chloric acid solution at pH +,. - NaCl), chromatorams
vere analvzed betore and atter dissolution procedure (+
hour at ;oC, paddle method, stirrin speed +oo rpm)
BOSNlAN lOUlNAl Ol BASlC MlDlCAl SCllNClS 2008, 8 (3). 236-238
AlllA UZUNOVl, lDlNA VlANl. STABlllTY Ol ANTHOCYANlNS llOM COMMllClAl BlACl
CUllANT lUlCl UNDll SlMUlATlD GASTlOlNTlSTlNAl DlGlSTlON
lST .. An aliquot ot uice vas diluted vith hvdro-
chloric acid solution, supplemented vith sodium
chloride and pepsin and adusted to pH +,. (+oo ml
ot uice - |oo ml ot hvdrochloric acid solution at
pH +,. - NaCl- pepsin), chromatorams vere ana-
lvzed betore and atter dissolution procedure (+ hour
at ;oC, paddle method, stirrin speed +oo rpm)
TlST . An aliquot ot uice vas diluted vith phos-
phate butter at pH ;,, (+oo ml ot uice - |oo ml ot
phosphate butter at pH ;,,), chromatorams vere
analvzed betore and atter dissolution procedure (.
hours at ;oC, paddle method, stirrin speed +oo rpm)
TlST |. An aliquot ot uice vas diluted vith phos-
phate butter at pH ;,, and pancreatin (+oo ml
ot uice - |oo ml ot phosphate butter at pH ;,, -
pancreatin), chromatorams vere analvzed be-
tore and atter dissolution procedure (. hours
at ;oC, paddle method, stirrin speed +oo rpm)
Te tests vere pertormed usin USP apparatus ., Van
lel Vl ;o+o dissolution tester (Van lel, Carv). Te dis-
solution apparatus vas maintained at ;C throuhout
the experiment. Test samples vere ltered usin a o,|,
m nvlon svrine lters tor HPlC (Cronus). All tests
vere pertormed in triplicate. Prior to use, the dissolution
media and uice samples vere also equilibrated at ;C.
Samples vere dravn at the tollovin points. at the
beinnin (immediatelv thereatter stirrin vas
started) and at the end ot test procedure (atter one
hour tor testin in astric conditions vithvithout
pepsin added and atter tvo hours tor testin in in-
testinal conditions vithvithout pancreatin added).
HPLC analysis
Chromatoraphic analvsis (radient HPlC analvsis)
ot anthocvanins vas done accordin to the method ot
lee at all. (,) usin a Zorbax StableBond-C+8 column
(.,omm|,6 mm, m). Te analvsis vas pertormed on
an HPlC svstem composed ot a pump, inection valve,
autosampler and variable (DAD) vavelenth detector
(Shimadzu). Mobile phase A vas +oo acetonitrile, vhile
mobile phase B consisted ot + phosphoric acid and +o
acetic acid (lacial) (vv) in deionized vater. Te mate-
rial used as a reterence sample vas native uice prepared
trom tresh black currant truits usin pressure procedure.
Te proram vas as tollovs. o-., min linear radient
trom ,8 to 8o B, ., o min linear radient trom 8o
to 6o B, o . min linear radient trom 6o to ,8 B
and .-; min linear radient ,8 B. Te ov rate vas
+,o mlmin, the inection volume ., l, the column tem-
perature |o
o
C and the detection vavelenth ,.o nm.
Wavelenth selection vas in accordance vith maxi-
mum absorption tor anthocvanins. Media vas se-
lected vith the intention to determine possible in-
tluence ot astric and intestinal tluids on stabilitv ot
anthocvanins in acidicbasic conditions in presence
absence ot diestive enzvmes (pepsin and pancreatin).
BOSNlAN lOUlNAl Ol BASlC MlDlCAl SCllNClS 2008, 8 (3). 23-238
AlllA UZUNOVl, lDlNA VlANl. STABlllTY Ol ANTHOCYANlNS llOM COMMllClAl BlACl
CUllANT lUlCl UNDll SlMUlATlD GASTlOlNTlSTlNAl DlGlSTlON
Results and Discussion
Stabilitv ot anthocvanins in commercial black cur-
rant uice vas analvzed on the basis ot tour charac-
teristic peaks vhich vere observed in native black
currant uice chromatoram (liure +.). The native
uice vas prepared trom tresh black currants usin
pressure procedure. This chromatoram vas used
as tinerprint chromatoram ot black currant uice.
The same peaks vere observed in chromatorams
ot the analvzed commercial uice, vhich conve-
nientlv served as identitv contirmation as vell.
Tot al ant hocvani ns cont ent and percent -
ae chanes duri n i n vi t ro di est i on ot
black currant uice are presented in Table +.
Te analvsis ot content ot tour basic anthocvanin compo-
nents in commercial black currant uice in simulated as-
tric uid vithout pepsin indicated sliht increase vhich
is in accordance vith previouslv published data (.+).
Decreased content vas noted tor anthocvanin com-
ponents . and | (-.,8o and -,,+,) atter in vitro
diestion in intestinal tluid containin pancreatin.
When the intestinal tluid vithout pancreatin vas
used, increased content ot anthocvanin components
. and | (,,8 and o,+.) could be determined. The
ettective content reduction ot anthocvanin compo-
nents . and | is -6,;8 and -,,o, respectivelv, and
does not represent siniticant reduction as it vas
perceived in some previouslv published studies (.+).
Neative level ot anthocvanin components content
(T| - T) vas perceived tor components + and
(-,,+ and -6,;8) in intestinal tluid containin pan-
creatin, reardless ot the tact that it ve consider them
individuallv, in media at pH ;,, vith vithout enzvme
addition, the content ot these components increased.
TABll 1. Total anthocvanins content and percentae chanes durin in vitro diestion ot black currant uice
Averae area ot the
peak 1 (n3)
Averae area ot the
peak 2 (n3)
Averae area ot the
peak 3 (n3)
Averae area ot the
peak + (n3)
Total (1-2-3-+)
letention time
(minutes)
6,1 ,2 8,9 10,2
TlST 1
at the beginning of the
procedure
112923 31+80 38830 32028 99363
at the end of the
procedure
113016 339208 +0038 339088 103331
betore atter () 1,83 +,3 3,11 3,69 +,00

TlST 2
at the beginning of the
procedure
113831 311689 38+69 323221 989229
at the end of the
procedure
120308 33931 +2060 3+261+ 10++699
betore atter () 3,83 3,++ 9,33 3,33 3,61

T2
-
T1
+,00 0,1 6,22 1,66 1,61

TlST 3
at the beginning of the
procedure
8033 398+86 269 260831 63089
at the end of the
procedure
9332+ +1+336 31+22 261133 800236
betore atter () 19,36 3,98 13,+3 0,12 +,60

TlST +
at the beginning of the
procedure
830 +2+86+ 30+13 2+183 81033
at the end of the
procedure
9609 +1292 32+10 23996,00 802038
betore atter () 10,++ -2,80 6,36 -3,19 -1,83

T+
-
T3
-9,13 -6,8 -6,89 -3,30 -6,+3
BOSNlAN lOUlNAl Ol BASlC MlDlCAl SCllNClS 2008, 8 (3). 238-238
AlllA UZUNOVl, lDlNA VlANl. STABlllTY Ol ANTHOCYANlNS llOM COMMllClAl BlACl
CUllANT lUlCl UNDll SlMUlATlD GASTlOlNTlSTlNAl DlGlSTlON
Conclusion
Anthocvanins present in commercial black currant uice remain stable durin in vitro diestion in gastric uid re-
ardless vhether pepsin vas added into medium or not,
Anthocvanins present in commercial black currant uice remain stable durin in vitro diestion in simulated intesti-
nal uid vithout pancreatin,
Te stabilitv studies ot anthocvanins in the intestinal uid containin pancreatin indicated reduced stabilitv, primar-
ilv ot anthocvanin components . and |, vhich also mainlv contribute to sliht reduction in total anthocvanins con-
tent (-+,8) in commercial black currant uice.
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