The elucidation of total polyphenols, individual phenolic compounds, antioxidant activity of three underutilized fruit species-black crowberry, honeyberry, european cranberry with their accumulation

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