ag onomy
A icle
The Elucida ion o To al Polyphenols, Indi idual Phenolic
Compounds, An ioxidan Ac i i y o Th ee Unde u ilized F ui
Species—Black C owbe y, Honeybe y, Eu opean C anbe y
wi h Thei Accumula ion
Tünde Ju íko á1, Jiˇ íMlˇcek 2,*, Š e an Balla 1, Monika Ond ášo á2, Libo Dokoupil 3, Jiˇ íSocho 4,
L’uba ˇ
Du išo á5, Pa ol Eliáš J . 5, Anna Adámko á2, Mojmí Ba oˇn 4and Sezai E cisli 6


Ci a ion: Ju íko á, T.; Mlˇcek, J.; Balla,
Š.; Ond ášo á, M.; Dokoupil, L.;
Socho , J.; ˇ
Du išo á, L.; Eliáš, P., J .;
Adámko á, A.; Ba oˇn, M.; e al. The
Elucida ion o To al Polyphenols,
Indi idual Phenolic Compounds,
An ioxidan Ac i i y o Th ee
Unde u ilized F ui Species—Black
C owbe y, Honeybe y, Eu opean
C anbe y wi h Thei Accumula ion.
Ag onomy 2021,11, 73.
h ps://doi.o g/10.3390/
ag onomy11010073
Recei ed: 23 No embe 2020
Accep ed: 29 Decembe 2020
Published: 31 Decembe 2020
Publishe ’s No e: MDPI s ays neu-
al wi h ega d o ju isdic ional clai-
ms in published maps and ins i u io-
nal a ilia ions.
Copy igh : © 2020 by he au ho s. Li-
censee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and con-
di 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/).
1
Ins i u e o Teache T aining, Facul y o Cen al Eu opean S udies, Cons an ine he Philosophe Uni e si y in
Ni a, D ažo ská4, 949 74 Ni a, Slo akia; [email p o ec ed] (T.J.); [email p o ec ed] (Š.B.)
2Depa men o Food Analysis and Chemis y, Facul y o Technology, Tomas Ba a Uni e si y in Zlín,
Va ecko a 275, 760 01 Zlín, Czech Republic; [email p o ec ed] (M.O.); [email p o ec ed] (A.A.)
3
Depa men o B eeding and P opaga ion o Ho icul u al Plan s, Facul y o Ho icul u e, Mendel Uni e si y
in B no, Val ická337, 691 44 Lednice, Czech Republic; libo [email p o ec ed]
4Depa men o Vi icul u e and Enology, Facul y o Ho icul u e, Mendel Uni e si y in B no, Val ická337,
691 44 Lednice, Czech Republic; [email p o ec ed] (J.S.); mojmi [email p o ec ed] (M.B.)
5Depa men o En i onmen and Biology, Facul y o Ag obiology and Food Resou ces, Slo ak Uni e si y o
Ag icul u e in Ni a, A. Hlinku 2, 949 76 Ni a, Slo akia; [email p o ec ed] (L.ˇ
D.);
[email p o ec ed] (P.E.J.)
6Depa men o Ho icul u e, Ag icul u al Facul y, A a u k Uni e si y, 25240 E zu um, Tu key;
[email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +420-576-033-030
Abs ac :
To al polyphenols con en (TPC), an ioxidan ac i i y (AA) and polyphenolic spec um
assay o h ee unde u ilized ui species—black c owbe y (Empe um nig um), honeybe y (Lonice a
kam scha ica) and Eu opean c anbe y (Vaccinium oxycoccos) is he aim o he p esen wo k. TPC
and AA assay was pe o med by spec opho ome y and “indi idual phenolic compounds” we e
es ablished by high-pe o mance liquid ch oma og aphy. The esul s showed ha TPC anged
om 1.61
±
0.16 (Eu opean c anbe y) up o 5.65
±
0.01 (Black cowbe y) gallic acid mg/g esh
weigh . The highes alue o DPPH (2,2-diphenyl-1-pic ylhyd azyl) ee adical sca enging assay
was de e mined in black c owbe y ui (7.43
±
0.34 mmol TROLOX/g esh weigh o ui ).
In all samples, he mos p e alen phenolic acid was e ulic acid wi h he highes con en in he
samples o black c owbe y (77.73
±
3.99
µ
g/g FW o ui ), black c owbe y and honeybe y we e
p esen by he highes le el o gallic acid (21.82
±
1.53; 15.07
±
2.45
µ
g/g FW o ui ). Honeybe y
ep esen ed a aluable sou ce o que ce in (12.18
±
7.88
µ
g/g FW o ui ), whe eas Eu opean
c anbe y and honeybe y a e a sou ce o u in (28.48
±
0.83 and 27.99
±
1.78
µ
g/g FW o ui ,
espec i ely). The esul s o s a is ical analyses p o ed signi ican di e ences be ween cowbe y
and Eu opean c anbe y in TPC con en and in AA among assayed species. In he same way,
s a is ically signi ican di e ences we e con i med in phenolic acids among he assayed species
excep o Eu opean c anbe y-honeybe y (in ans p-couma ic, p o oca equic and cinnamic acid)
and honeybe y-cowbe y (in cinnamic acid con en ). The ca echin con en is signi ican ly in luenced
by species (p
≤
0.05), on he o he hand he species has no in luence on he es e a ol con en
(p> 0.05). Each o he s udied species can be conside ed as aluable sou ce o o al polyphenols and
indi idual phenolic compounds. Polyphenolic compounds we e mos equen ly accumula ed in he
acuole as well as in he ou e laye s o pe ica p.
Keywo ds:
Empe um nig um;Lonice a kam cha ica aVaccinium oxycoccus; ui s; o al phenolic con en ;
an ioxidan ac i i y
Ag onomy 2021,11, 73. h ps://doi.o g/10.3390/ag onomy11010073 h ps://www.mdpi.com/jou nal/ag onomy
Ag onomy 2021,11, 73 2 o 13
1. In oduc ion
A lo o species o less—known and unde u ilized be y c op in he e i o y o Slo-
akia (black mulbe ies, co nelian che ies, blackbe ies, black ho n, owanbe ies) and
honeybe y (Lonice a kam scha ica Se as . Poja k) con ain nu ien s wi h many non-nu ien
bioac i e compounds (phy ochemicals) possess a high an ioxidan ac i i y [
1
]. Di e en
s udies indica ed ha especially polyphenols (phenolic acids, la onoids, s ilbenes) as he
majo g oup o seconda y me aboli es p esen ed a key ole in be ies in he apy and p e-
en ion o ch onic diseases as well [
2
]. Excep o he de e mina ion o he majo bioac i e
compounds i has been e y impo an o s udy he accumula ion o hese compounds o
u he u iliza ion o ui in p ocessing.
Black c owbe y includes wo o ms—Empe um nig um L. (c owbe y o black c ow-
be y) and i s e aploid subspecies E. nig um ssp. he maph odi um. C owbe y (Em-
pe um nig um L.) ep esen ed less u ilized ne e heless widely dis ibu ed wild be ies
in no he n a eas [
3
] wi h he high con en o phenolic compounds. An hocyanins and
la onols a e he mos abundan phenolic compounds o ui s [
4
]. Que ce in, kaemp e ol,
my ice in oge he wi h ca echins p esen he majo la onols in ui [
5
–
7
]. The analyses o
phenolic acids con i med he p esence o e ulic and gallic acid [
6
]. The s udy o Ju iko a
e al. (2019) ied o connec he s udy o ui ana omy o black c owbe y (Empe um
nig um) wi h de ec ion and quan i ica ion o main non-an hocyanin polyphenolic com-
pounds. The expe imen esul s p o ed he highes accumula ion pigmen s—an hocyanins
oge he wi h o he polyphenols in ou e laye s in ma u ed ui . The men ioned polyphe-
nolic compounds in cells we e iden i ica ed as u in, que ce in and ca echins, es e a ol,
couma ic, p-couma ic, ca eic, e ulic acids, gale, anilic, sy inge, cinnamic and ca eic
acids [
6
]. The polyphenols we e accumula ed in ui . The an i-in lamma o y e ec o black
c owbe y has been widely used o ea men o cys i is, neph i is, and e h i is [
5
,
7
] and
as an e ec i e an ibac e ial and an i ungal emedy [
7
–
9
] oge he wi h an i-adhe ence [
10
]
and an i-swa ming ac i i y and an iangiogenic ac i i y [
11
]. Un o una ely, despi e all
heal h bene i s hese species ha e been neglec ed [12].
Vaccinium oxycoccos plan s include o ms such as he li le-lea c anbe y, V. oxycoccos
. mic ophylla, syn. = Oxycoccus mic oca pus (Tu cz.), and he la ge -lea o m V. oxycoccos
L. subsp. palus is = Oxycoccus quad ipe alus (Tu cz.). I occu s in o es a eas in Eu ope,
Asia, and No h Ame ica [
13
]. Vaccinium sp. a e one o he iches sou ces o an ioxidan s,
such as an hocyanins, among ui s and ege ables [
14
]. The majo i y o esea ch pape s
and s udies ha e ocused only on “la ge c anbe y” o “Ame ican c anbe y” (Vaccinium
mac oca pon Ai s), while he s udies dealing wi h Eu opean c anbe y ha e occu ed only
a ely (V. oxycoccos) [
13
]. Eu opean c anbe y, in he same way as la ge c anbe y ui s
ep esen s a la ge numbe o bioac i e compounds, especially polyphenolic compounds
(mainly phenolic acids, la onols, an hocyanins, and p oan hocyanidins). O e all, hese
be y species a e alued o he high polyphenol con en mainly esponsible o high an iox-
idan ac i i y [
8
,
15
,
16
]. The an ioxidan ac i i y was co ela ed wi h he con en o phenolic
compounds, la onoids and p oan hocyanidins [
17
]. Kona ska e al. (2015) s udied he
ana omy o ma u ed ui . Phenolic compounds (an hocyanins and annins) we e p esen ed
mainly in he epica p [
18
]. Mo eo e , he be ies display an i–in lamma o y p ope ies
u ilized in he p e en ion and ea men o ca dio ascula diseases and cance [
13
]. Ex-
ac s om na u al c anbe y juices migh be used o sa e and e icien supp ession o o al
pa hogenic bac e ial species [
19
] and can be u ilized in he p e en ion agains u ina y ac
in ec ions [20].
In Eu ope he edible honeysuckle has ep esen ed a new aluable species o en i on-
men al condi ions and i s cul i a ion has an inc easing endency.
The edible honeysuckle species belong o he old Linnean species Lonice a cae ulea L.
I is possible o classi y he ound geno ypes o Lonice a cae ulea in o wo geog aphical
subspecies: L. cae ulea subsp. kam scha ica (Poja k.) Plekhano a, and ano he subspecies,
L. cae ulea L. subsp. ocho ensis
Ag onomy 2021,11, 73 3 o 13
Smekal., Holubec e S obodo a, subsp. no a, has dis ibu ion in Sakhalin and he
adjacen Ku il Islands (Holubec e al., 2018) [21].
The edible honeysuckle has been used as a be y c op o di ec consump ion and
medicinal c op in he e i o y o Russia, Japan, and China. Nowadays, i has been no iced
he inc easing popula i y o Lonice a sp. (Lonice a cae ulea, Lonice a kam scha ica) in Eu ope,
USA and Ko ea [
22
]. The p edominan g oup o bioac i e compounds in he edible hon-
eysuckle ui was c ea ed by polyphenols (mainly la onoids—6 an hocyanins oge he
wi h 9 la onols, mainly que ce in and u in) [
22
,
23
] and 15 i idoids [
24
]. Acco ding o
ˇ
Du išo áe al. (2020) he an hocyanins we e localized in he skin and ou e laye s o ui
exoca p, polyphenols we e accumula ed also in acuoles [25].
The use o he blue honeysuckle ui s in he die as a po en ial sou ce o bioac i e com-
pounds wi h heal h-p omo ing p ope ies can be ex emely bene icial o consume s [
21
].
The polyphenols linked wi h i idoids o edible honeysuckle possess an i-in lamma o y,
an imic obial (agains six bac e ia and six ungi [
26
], an icance e ec [
27
]. The p edomi-
nan an hocyanin cyanidin-3-O-glucoside in haskap (Lonice a cae ulea L.) be y ex ac s can
a enua e he ca cinogen-induced DNA damage in no mal lung epi helial cells
in i o
[
28
].
Fang e al. [
29
] also poin ed o he hepa op o ec i e, an i-alle gic, an i- umo , and im-
munomodula o y e ec s o be ies. G ea po en ial bene i s o his nu i ious ood a e i s
abili y o minimize he nega i e e ec s o UV adia ion, diabe es melli us and neu odegen-
e a i e diseases and ca diop o ec i e ac i i y [30].
The majo g oup o polyphenols p esen ed in assayed species ep esen pigmen s—
an hocyanins. The pigmen s causing he colo o he ui s signi ican ly a ec s hei
ansmission by animals [
31
]. Un o una ely, he majo i y o s udies deals wi h well-known
ui species. In many plan species, he pigmen s a e localized mos ly in he skin o he
ui . The densi y o ed pigmen s in cells o he ou e laye o he ui skin ed apple
“Fuji” is esponsible o he colo o he ui s [
32
]. The cells wi h colo ed acuoles we e
obse ed in he epide mal and subepide mal cells in he ma u ing ui o Empe um nig um.
Kona ska no iced ha The he phenolic compounds localized mainly in he epide mal and
hypode mal cells in he ma u e ui s o P unus domes ica cul i a s [
33
]. The an hocyanins
in he ma u e be ies o Vi is ini e a DeChaunacwe e localized in a shallow subepide mal
laye , while hey we e no de ec ed in he epide mis [
34
]. The accumula ion o bioac i e
compounds has been impo an o he u he u iliza ion o he ui .
The assayed h ee be y c op a e alued o high polyphenols con en mainly con-
ibu ed o an ioxidan ac i i y. So his in es iga ion e alua ed and compa ed he o al
polyphenols con en (TPC), an ioxidan ac i i y o he ui , and he p o ile o he selec ed
phenolic compounds p esen ed in h ee unde u ilized be y c ops—black cowbe y (Em-
pe um nig um), Eu opean c anbe y (Vaccinium oxycoccos)—widely g own and collec ed in
na u e, we e s udied. Honeybe y (Lonice a kam scha ica) widely occu ed in Kam scha ka
was collec ed om a Bo anical ga den in Ni a. Selec ed, in e es ing polyphenols a e
included. The p esen s udy o e s he compa a i e s udy o h ee unde u ilized ui
species despi e hei high nu i ional alue. The assayed species ha e no been e alua ed
in espec o TPC and p edominan polyphenolic compounds oge he ye . Excep o he
de e mina ion o polyphenols con en , he s udy aims a de ec ing he accumula ion o
polyphenols.
2. Ma e ials and Me hods
The ui sample o Empe um nig um L. o igina ed om he Na u e Rese e Rudné
(NW Slo akia). The Na u e Rese e Rudné ep esen ed he adminis a i e a ea o Suchá
Ho a in he alley O a skáko lina a an al i ude o 750 m a. s. l. Today i is only a
agmen (2.06 ha) o one o he mos aluable aised bogs in Slo akia, his a ea cons i u ed
an su ace o abou 100 ha. The ese e was launched in 1984 [
35
]. Acco ding o he
phy ogeog aphical di ision o Slo akia, i is included in he e i o y o he Wes e n
Ca pa hian egion (Ca pa icum occiden ale) and he dis ic ZápadnéBeskydy M s [36].
Ag onomy 2021,11, 73 4 o 13
The ui samples o Vaccinium oxycoccos L. we e collec ed in h ee locali ies o pea -
bog habi a s si ua ed in NW Slo akia: he Klinské ašelinisko Na u e Rese e, he Rudné
Na u e Rese e and he Medzi Bo mi Na u e Rese e. Acco ding o he phy ogeog aphical
di ision o Slo akia, hey a e included in he e i o y o he Wes e n Ca pa hian egion
(Ca pa icum occiden ale). The Klin Na u e Rese e and he RudnéNa u e Rese e belong
o he dis ic o ZápadnéBeskydy M s, while he Medzi Bo mi Na u e Rese e is si ua ed
in he Ta y M s dis ic , he ZápadnéTa y M s subdis ic [36].
The samples o honeybe y (Lonice a kam scha ica (Se as .) Poja k) widely g own in
Kam scha ka we e ob ained om he Bo anical ga den o Slo ak Uni e si y o Ag icul u e
in Ni a.
F ui s we e ha es ed when ipe and eady o consump ion om i e sh ubs o each
cul i a unde s udy in he cou se om June o July. Twen y andomly chosen ui s om
each sh ubs we e used o chemical analyses (i.e., al oge he 100 pe each cul i a ). Fo
ana omical obse a ions 30 ui s o 4 sh ubs o a o al o 120 ui s we e used We choose
only heal hy be ies wi hou damage. F esh ha es ed samples we e homogenized and
lyophilized by Alpha 1-4 LSC (Ma in Ch is Ge ie ocknungsanlagen GmbH, Os e ode
am Ha z, Ge many) a
−
55
◦
C and 0.120 mba o 48 h. Fu he , lyophilized samples we e
subjec ed o he analysis.
2.1. Pho ome ic Quan i ica ion o To al Polyphenols
The quan i ica ion o o al phenolic con en (TPC) o ui was de e mined by he Folin-
Ciocal eu Assay [
37
,
38
]. 1 mL o ex ac was added o 50 mL olume ic lask wi h 20 mL
o deionized wa e . One millili e o Folin-Ciocal eu’s phenol eagen , (Sigma-Ald ich,
S Louis, MO, USA) was added o he mix u e and shaken. To al o 5 mL o 20% Na
2
CO
3
(Sigma Ald ich, S Louis, MO, USA) was added a e h ee minu es and he sample was
mixed. Deionized wa e was added o an o e all olume o 50 mL. A e incuba ion o
30 min a oom empe a u e, he abso bance a 765 nm was de e mined by an UV-Mini
1240 spec opho ome e (Shimadzu, Kyo o, Japan). To exp ess he esul s, uni millig ams
o gallic acid (GAE) (Sigma Ald ich, S Louis, MO, USA) pe g ams o esh weigh o ui
we e used. All samples we e analyzed in h ee epe i ions.
2.2. De ec ion o Indi idual Phenolic Compounds
The concen a ion o he p edominan phenolic compounds in samples we e de-
e mined by Dionex Ul iMa e 3000 High-pe o mance Liquid Ch oma og aphy sys em
(Dionex Co po a ion, Sunny ale, CA, USA) wi h a diode-a ay de ec o (DAD) and Kine-
ex column C-18 (150
×
4.6; 2.6
µ
m) (Phenomenex, To ance, CA, USA) acco ding o he
me hod by Sy aˇ o áe al. (2020) and Sumczynski e al. (2017) [
39
,
40
]. Indi idual phenolic
compounds we e iden i ied using hei e en ion imes and quan i ied by he me hod o
s anda d addi ion. Da a signals we e p ocessed by LC Ch omeleonTM 7.2 Ch oma og aphy
Da a Sys em (Dionex Co po a ion, Sunny ale, CA, USA). A e age alues o measu emen s
we e p esen ed in h ee epe i ions. The con en o indi idual phenolic compounds in he
ui was exp essed as µg/g o esh weigh o ui .
2.3. An ioxidan Ac i i y o F ui
The an ioxidan ac i i y assay by DPPH (2,2-diphenyl-1-pic ylhyd azyl) (Sigma Ald ich,
S Louis, MO, USA) was conduc ed acco ding o p e iously desc ibed me hods [
41
–
43
]
wi h some modi ica ions. S ock solu ions we e p epa ed by dissol ing DPPH (24 mg) in
me hanol (100 mL) (Sigma Ald ich, S Louis, MO, USA), and hen s o ed a
−
20
◦
C un il
needed. Wo king solu ions we e p epa ed by mixing o he s ock solu ion (10 mL) wi h
me hanol (45 mL) o ob ain an abso bance o 1.1
±
0.02 uni s a 515 nm using LIBRA S6
Spec opho ome e (Bioch om L d., Camb idge, UK). F ui s ex ac s (150
µ
L) we e exposed o
he eac ion wi h he DPPH solu ion (2.850
µ
L) o 1 h in he da k. Following his p ocedu e,
abso bance was measu ed again a 515 nm. An ioxidan ac i i y was calcula ed as a dec ease
o he abso bance alue using he o mula: (%) = (A
0−
A
1
/A
0
)
×
100%, whe e A
0
is he
Ag onomy 2021,11, 73 5 o 13
abso bance o he blank (wi hou he sample), and A
1
is he abso bance o he mix u e
con aining he sample. The abso bance esul s we e con e ed using a s anda d calib a ion
cu e and exp essed as TROLOX equi alen s (AA) [
24
]. The analyses o ui ex ac s we e
p o ided in h ee epe i ions.
2.4. S a is ical Analysis
Each expe imen was pe o med in h ee epe i ions. Fo s a is ical analyses he Ads a
so wa e (Ve sion 1.25) (T iloBy e S a is ical so wa e, s. .o., Pa dubice, CZ) was used and
esul s we e exp essed as means alue ±s anda d de ia ions.
Fo e i ica ion o signi icance o di e ences among samples, one-way analyses o
a iance we e u ilized (
α
= 0.05). This s a is ical analysis was pe o med wi h p og am
STATISTICA CZ e sion 12 (TIBCO So wa e Inc., Palo Al o, CA, USA), and was also
used o nex da a analysis. A Shapi o–Wilk no mali y es (
α
= 0.05) was applied on all
measu ed da a g oups as he i s es . Homogenei y o a iances was es ed using he
Le ene’s es o homogenei y o a iances (
α
= 0.05). As nex es , he pa ame ic ANOVA
es (
α
= 0.05) was pe o med. Compa ison o s a is ical di e ences be ween g oups o da a
in indi idual g oups was pe o med using pa ame ic pos -hoc LSD es (α= 0.05).
Resul s we e e alua ed s a is ically using he so wa e STATISTICA CZ e sion 12
(TIBCO So wa e Inc., Palo Al o, CA, USA). Co ela ion was pe o med using co ela ion
ma ices wi h calcula ion o , p- alues, and N’s. Pea son co ela ion coe icien s ( ) we e
calcula ed as well wi h le el α= 0.05.
2.5. Ana omical C oss—Sec ion o Ma u ed F ui
The ma u e ui s we e sec ioned and subsequen ly ixed in FAA ixa i e ( o maldehyde-
e hanol-ace ic acid). Pa a in embedding was pe o med acco ding o Pazou ko á[
44
].
The pe ica p o he ui s was cu a he hickness 8–10
µ
m using he o a y mic o ome CUT
4055 Mic oTec (mic oTec Labo ge ä e GmbH, Walldo , Ge many). The slides we e coun-
e s ained wi h sa anin and as -g een [
45
]. The mic oscopic sec ions we e obse ed by
ligh mic oscope Olympus BX 41 (Olympus Co po a ion, Tokyo, Japan) and pho og aphed
wi h a came a Olympus E-520 (Olympus Co po a ion, Tokyo, Japan).
3. Resul s and Discussion
Chemical analyses ocused on he de e mina ion o o al polyphenols con en (TPC)
and an ioxidan ac i i y (AA) oge he wi h p edominan polyphenolic compounds (HPLC
me hod) o less u ilized ui —Empe um nig um,Lonice a kam scha ica and Vaccinium oxy-
coccos. The esul s a e summa ized in Table 1.
Table 1.
The esul s o analyses o o al polyphenols con en (TPC) (gallic acid mg/g esh weigh o
ui ) and an ioxidan ac i i y (mmol TROLOX/g esh weigh o ui ) o Empe um nig um,Lonice a
kam scha ica and Vaccinium oxycoccus ui s.
Pa ame e . Eu opean C anbe y Honeybe y Black Cowbe y
o al polyphenols con en
(TPC) (gallic acid mg/g
esh weigh o ui )
1.61 ±0.16 ‡3.11 ±1.67 5.65 ±0.01 ‡
an ioxidan ac i i y o ui
(mmol TROLOX g/ esh
weigh o ui )
1.62 ±0.03 *,‡ 3.02 ±0.05 *,† 7.43 ±0.34 †,‡
Legend: *
—
s a is ical signi icance be ween ui species Eu opean c anbe y and honeybe y,
†
—s a is ical
signi icance be ween ui species honeybe y and black cowbe y,
‡
—s a is ical signi icance be ween ui
species Eu opean c anbe y and black cowbe y.
As i has been shown in Table 1, he ui o black c owbe y displayed he highes
le el o o al polyphenols con en (5.65
±
0.01mg gallic acid/g) ha co esponded wi h
he highes alue o an ioxidan ac i i y (7.43
±
0.34 mmol TROLOX g/ esh weigh

Ag onomy 2021,11, 73 6 o 13
o ui ). In his s udy he co ela ion was de e mined = 0.9994 (p
≤
0.05) in black
cowbe y. The o al polyphenol con en (TPC) o black c owbe y ui has been e alua ed
as 4.3
±
0.09 mg GAE/g FW (gallic acid equi alen ) in esh ui [
7
]. I can be gi en by
di e en soil, clima ic condi ions o na u al locali y o ui occu ence [
6
,
46
]. A compa a i e
s udy by Dudonne e al. on he polyphenol composi ion o wel e na i e Canadian be ies:
Saska oon be y, alpine bea be y, chokebe y, black c owbe y, honeysuckle, chokeche y,
cloudbe y, elde be y, lowbush bluebe y, alpine bluebe y, lingonbe y, and highbush
c anbe y p o ed he highes alue o polyphenols as 4.54
±
3.77 mg GAE/g FW o he
clack cowbe y ui [
47
]. The signi ican ly lowe alue o TPC in Lonice a kam scha ica ui
eaching up 11.1 mg GAE/g FW was e alua ed in he s udies o Ba czak e al. (2007) unde
he condi ions o Canada [
48
]. In he esea ch o Senica e al. he highes o al phenolic
con en in 4 cul i a s o Lonice a kam scha ica was epo ed in he condi ions o und a, wi h
2.68 mg/g [49].
In honeybe y he co ela ion ( = 0.9983; p
≤
0.05) was de e mined, bu in case o
Eu opean c anbe y he co ela ion was no con i med ( = 0.6973; p> 0.05). The e o e,
his pape is in acco dance wi h he esea ch pape s [
4
,
21
,
25
], ha poin ed o he ac
ha polyphenols ep esen he majo g oup o seconda y me aboli es o black c owbe y
ui and hey mos ly con ibu ed o he an ioxidan ac i i y [
3
,
6
,
7
,
12
]. The TPC con en
oge he wi h an ioxidan ac i i y o ui is in acco dance wi h he p e ious s udies
o Ju iko a e al. [
50
] and ha e been signi ican ly in luenced by he o igin o be ies
samples [51–54].
S a is ical e alua ion o he di e ences in p edominan la onoids, o al polyphenol
con en (TPC) and an ioxidan ac i i y (AA) among Eu opean c anbe y, honeybe y and
black cowbe y is showed in Table 1. S a is ically signi ican di e ences we e ound in o al
an ioxidan ac i i y among all assayed species (ANOVA; F (2, N = 9) = 695.15; p
≤
0.01).
In case o o al polyphenol con en , a s a is ically signi ican di e ence was calcula ed by
ANOVA (F (2, N = 9) = 13.34; p
≤
0.01). LSD pos -hoc es de e mined ha a s a is ically
signi ican di e ence was calcula ed only be ween Eu opean c anbe y and black cowbe y
(p≤0.05).
The second pa o ou esea ch aimed a he de e mina ion o he selec ed polypheno-
lic compounds in he ui o Eu opean c anbe y, black cowbe y and honeybe y (phenolic
acids-gallic, anillic, ca eic, sy ingic, ans—p-couma ic, cinnamic and p o oca equic acids,
la onoids— o al con en o ca echin, que ce in, u in and es e a ol, espec i ely).
Fla onoids o ui Eu opean c anbe y, honeybe y (especially ca echin and u in)
ep esen ed he p edominan g oup o polyphenols ha ha e been in acco dance wi h
esea ch s udies o [51,52,55,56]. Resul s o chemical analyses a e summed up in Table 2.
The con en o que ce in in ho icul u al c ops did no each 10 mg/kg excep o
apple and onion [
53
], which was p o ed in ou esea ch. An excep ion was eco ded
in honeybe y samples, he que ce in con en eached 12.18
±
7.88
µ
g/g. In he same
way [
50
,
51
,
57
] eco ded, in less known ui species, he que ce in con en g ea e han
10 mg/kg was only in samples o Lonice a kam scha ica. Acco ding o [
20
] he edible honey-
suckle be ies eached up o he high le el o la onoids (2 g pe 100 g o d y ui weigh )
ha ep esen he simila concen a ion abundance in some bluebe ies. Resea ch wo ks
o [
24
,
58
–
60
] con i med, ha que ce in ep esen ed he p e ailed polyphenolic compound
in edible honeysuckle ui [
61
] ound ou , ha he samples o Eu opean c anbe y ui
con ained (5.15
µ
g/g o FW) que ce in bu esul s o assayed sampled poin ed o lowe
con en 1.49
±
0.19
µ
g/g [
16
,
62
] de e mined in di e en Polish cul i a s o Eu opean
c anbe y he que ce in con en om 0.52 o 1.54
µ
g/g. Ano he impo an la onoid ep e-
sen ed by ca echins [
55
,
58
,
63
] ha was p o ed in case o samples o Eu opean c anbe y
(206.0
±
3.85
µ
g/g) and honeybe y (38.88
±
3.78
µ
g/g). Simila ly high alues o u in
28.48
±
0.83 and 27.99
±
1.78
µ
g/g we e es ablished in he samples o Eu opean c anbe y
and honeybe y.
Ag onomy 2021,11, 73 7 o 13
Table 2.
E alua ion o he mos ly abundan polyphenolic compounds (
µ
g/g esh weigh o ui ) o
less known ui species (Eu opean c anbe y, honeybe y and black cowbe y).
Selec ed
Polyphenolic
Compounds o F ui
(µg/g FW o F ui )
Eu opean C anbe y Honeybe y Black Cowbe y
gallic acid 1.62 ±0.09 *,‡ 15.07 ±2.45 *,† 21.82 ±1.53 +,‡
ca echin 206.0 ±3.84 *,‡ 38.88 ±3.78 *,† 7.03 ±1.35 +,‡
anillic acid 0.77 ±0.04 *,‡ 2.34 ±0.76 *,† 6.40 ±0.24 +,‡
ca eic acid 1.02 ±0.10 *,‡ 5.98 ±1.67 *,† 0.66 ±0.09 +,‡
sy ingic acid 6.67 ±0.05 ‡ND 3.82 ±0.46 ‡
ans p-couma ic acid 4.98 ±1.84 ‡9.87 ±2.67 †0.44 ±0.13 +,‡
couma ic acid 0.50 ±0.03 * 6.4 ±2.56 *,† 1.66 ±0.24 †
e ulic acid 38.52 ±1.35 *,‡ 20.70 ±2.78 *,† 77.73 ±3.99 +,‡
u in 28.48 ±0.83 ‡27.99 ±1.78 †2.21 ±0.93 +,‡
p o oca equinic acid 2.04 ±0.02 ‡1.44 ±0.76 †0.12 ±0.03 +,‡
es e a ol 0.24 ±0.01 1.55 ±0.56 0.34 ±0.16
cinnamic acid 3.43 ±0.15 ‡4.44 ±3.34 0.59 ±0.11 ‡
que ce in 1.49 ±0.19 * 12.18 ±7.88 *,† 2.32 ±0.27 †
Legend: *—s a is ical signi icance be ween ui species Eu opean c anbe y and honeybe y,
†
—s a is ical
signi icance be ween ui species honeybe y and black cowbe y,
‡
—s a is ical signi icance be ween ui
species Eu opean c anbe y and black cowbe y.
A e y impo an g oup o less-known ui species is ep esen ed by de i a es o
hyd oxycinnamic acid ha was in acco dance wi h esea ch s udies [
64
–
66
]. Wi h espec
o phenolic acids, we can s a e ha e ulic acid p edomina ed (38.52
±
1.35, 20.70
±
2.78
and 77.73
±
3.99
µ
g/g, espec i ely) in he samples o Eu opean c anbe y, honeybe y
and black cowbe y. Simila ly, gallic acid p e ailed in he samples o black cowbe y
21.82 ±1.53 µg/g and honeybe y 15.07 ±2.45 µg/g.
In he same way Ogawa e al. [
63
] iden i ied ca eic and gallic acids as p e ailing
phenolic acids in black c owbe y samples.
Gallo anins ep esen ed he p edominan g oup o polyphenols in ana omical obse -
a ions o genus Lonice a kam scha ica and Vaccinium oxycoccos a e concen a ed in acuoles.
In he same way ca echins p edomina ed in polyphenolic compounds in ou analyses.
Howe e , anillic acid was p esen ed only in ace amoun in he samples o Eu opean
c anbe y 0.76
±
0.04
µ
g/g. The highes concen a ion o p-couma ic acid and ca eic
acid was achie ed in he samples o honeybe y (6.4
±
2.56 a 5.98
±
1.67
µ
g/g), ha is
in acco dance wi h esea ch wo k o [
67
]. The e y simila con en o p-couma ic and
co eic acid 4.08
µ
g/g, 1.4
µ
g/g was de e mined in esea ch wo k [
16
,
68
] wi h Eu opean
c anbe y ui . Acco ding o [
16
] he samples o di e en cul i a s o Eu opean c anbe y
showed highe alues o ca eic acid 3.6
±
0.13
µ
g/g and que ce in 2.52
±
0.22
µ
g/g in
compa ison wi h ou esul s. Di e ences can be caused by he in luence o locali y as i
has been con i med in he esea ch s udy o [
29
]. A he same ime he analyzed samples
o Eu opean c anbe y we e collec ed in na u e and he b eeding p ocess in case o Eu-
opean c anbe y species was ocused on he inc ease o bioac i e compounds including
polyphenols. The esul s o s a is ical e alua ion o di e ences in phenolic acids con en
(Table 2) p o ed a s a is ical signi icance di e ences in all case. Excep ions a e. in case o
di e ences in he con en o phenolic acids be ween Eu opean c anbe y and honeybe y
in ans p-couma ic, p o oca equic, and cinnamic acid con en whe e signi ican di e ences
we e no calcula ed. Likewise, signi ican di e ences be ween honeybe y and cowbe y in
cinnamic acid con en we e no obse ed.
The ca echin, one o he impo an la onoids, ha e s a is ically signi ican di e ences
among all assayed species (ANOVA; F (2, N = 9) = 74.53; p
≤
0.01). On he con a y, no
s a is ically signi ican di e ences in case o es e a ol was calcula ed be ween all assayed
species. In case o u in, a s a is ically signi ican di e ence was calcula ed by ANOVA
Ag onomy 2021,11, 73 8 o 13
(F (2, N = 9) = 430.40; p
≤
0.01), bu ollowing LSD pos -hoc es obse ed s a is ically
signi ican di e ence was only be ween Eu opean c anbe y and honeybe y (p
≤
0.05).
Nex , able showed he s a is ically signi ican di e ence in que ce in con en (ANOVA;
F (2, N = 9) = 5.12; p
≤
0.05) in case Eu opean c anbe y—honeybe y and honeybe y—
black cowbe y.
4. Ana omical Obse a ion o Ma u ed F ui s
The exoca p Eu opean c anbe y (Vaccinium oxycoccos) is buil o cells o polygonal
shape wi h s aigh walls. On he c oss-sec ion, hey we e ec angula in he ou line, sligh ly
elonga ed in he di ec ion o he ci cum e ence. One o wo laye s o cells unde he epide mis
had he cha ac e o a hypode m. The mesoca p was hick, leshly, c ea ed o big hin-walled
cells. The endoca p is o med by a single laye o cells wi h sligh ly wa y hin walls [
69
]. We
obse ed he highes accumula ion o phenolic subs ances in he ou e laye s o ipe ui s.
The s ikingly colo ed acuoles illed wi h globula bodies a e cha ac e is ic o epide mal cells.
La ge hypode mal cells con ained acuole wi h a numbe o small g anula deposi s. Some
cells o one laye unde he hypode mis had he same s uc u e, bu he acuoles con ained
signi ican ly smalle amoun s o he pigmen s. Smalle clus e s o colo ed g ains (g anules)
we e sca e ed in he deepe laye s o he pe ica p, oo (Figu e 1a). Simila a angemen
pigmen s bodies in ed apple (Malus domes ica c . ‘Fuji’) was s a ed by Bae and Kim [
32
].
They ound he highes densi y o ed pigmen was in cells o he ou e laye o he ui skin,
and g adually dec eased inwa d owa d he lesh. They s a ed ha elec on-dense ma e ial
accumula ed inside he cen al acuoles in skin likely co espond o phenolic compounds
and assume hey we e an hocyanins. The an hocyanins can o m cy oplasmic agg ega es ha
we e inco po a ed in o he acuole by mic oau ophagy [
70
]. An hocyanins a e s able in he
acuole, because he acuole solu ion is acid in mos plan issues [34].
The ui o mos edible Lonice a species a e cha ac e ized as be y-shaped mul iple
ui s, which de elops om o a y, calyx ube and b ac s [
71
]. Based on ou ana omical ob-
se a ions, we can conclude ha mainly b ac s su ounding he o a ies o ipe honeybe y
(Lonice a kam cha ica) ui s we e he si e o he highes accumula ion o phenolic subs ances.
The abaxial pa o he b ac s consis o se e al laye s o cells wi h clumps da k small g ains
co esponding o he pigmen . These clus e s we e ep esen ed mainly by inside cells o he
epide mis, in pa hypode mis and h ee subhypode mal laye s (Figu e 1b). The adaxial
pa o he b ac s also had a simila s uc u e. We also obse ed colo ed acuoles in he
epide mal cells o he o a ies. Addi ionally, da k pu ple colo ed acuoles we e p esen ed
in all men ioned laye s. As s a ed by Kona ska [
33
], acuoles wi h phenolic compounds in
he o m o he la ge b own-pu ple deposi s mainly in epide mal and hypode mal cells
as well as p esence o ib ous and g anula deposi s in pa enchyma o he P unus ui s
indica ed he ui ipening and senescence.
The ma u e ui o black c owbe y (Empe um nig um) is glab ous d upe. The shape
o he epide mal cells was polygonal, while he mesoca p cells we e adially elonga ed [
72
].
I has been ound ha he accumula ion o polyphenolic subs ances is concen a ed in
he ou e pa s o he de eloping pe ica p E. nig um [
10
]. We obse ed ha polyphenols
we e p esen in wo pigmen -bea ing cells laye s o exoca p o ipe E. nig um ui s. The
cells o he epide mis con ained a colo ed acuole wi h globula bodies illing he whole
acuole. Unlike epide mal cells, he hypode mis was cha ac e is ic by cells wi h small
colo ed bodies loca ed a ound he acuole as well as inside i (Figu e 1c). The shape o he
colo ed bodies was mos o en globula , howe e , he e we e obse ed also o al bodies.
Polyphenols compounds we e mos equen ly la onoid pigmen s mainly an hocyanins
ha accumula e in he la ge cen al acuole o he pe ica p o mos plan s. In he inside o
he acuole, an hocyanins can be ound uni o mly dis ibu ed o as pa o sub- acuola
bodies, he an hocyanic acuola inclusions (AVIs) [70,73].
Ag onomy 2021,11, 73 9 o 13
Ag onomy 2021, 11, x FOR PEER REVIEW 9 o 13
he acuole, an hocyanins can be ound uni o mly dis ibu ed o as pa o sub- acuola
bodies, he an hocyanic acuola inclusions (AVIs) [70,73].
Figu e 1. C oss sec ion o he ma u e ui s pe ica p: (a) Vaccinium oxycoccos (b) Lonice a kam cha ica
(c) Empe um nig um.
Figu e 1.
C oss sec ion o he ma u e ui s pe ica p: (
a
)Vaccinium oxycoccos (
b
)Lonice a kam cha ica
(c)Empe um nig um.
5. Conclusions
The esea ch a icle ocused on he compa ison o he less-known ui species and un-
de u ilized c op such as Eu opean c anbe y, black cowbe y, honeybe y. The men ioned