Determination antioxidant activity and metal accumulation capacity of wild bilberry (Vaccinium myrtillus L., Ericaceae) from Montenegro

Crnoj Gori ima u obilju, i tradicionalno se koristi u ishrani ljudi ali i za pripremu ekstrakata i tinktura u tretmanu dijabetesa. S obzirom da borovnica sadrži različite koncentracije makroelemenata i mikroelemenata, koji su važni kao esencijalni elementi u tragovima, ili su potencijalno toksični, zavisno od koncentracije, posebna pažnja je usmjerena na sadržaj 16 elemenata, uključujući makroelemenata (Al, Ca, Fe, K i Mg) i mikroelemente (As, Ba, Cd, Co, Cu, Cr, Mn, Ni, Pb, Sr i Zn) u korijenu, stablu, listu i plodu divlje borovnice i zemljišnim supstratima kako bi se procijenila biodostupnost i migracija elemenata iz zemljišnih supstrata u korijen i određene nadzemne dijelove biljke. Što se tiče toksičnih metala As nije detektovan u borovnici. Borovnica je pokazala i vrlo mali stepen akumulacije Pb u svim dijelovima biljke što je od izuzetne važnosti, s obzirom da je BCR sekvencijalna ekstrakcija potvrdila izuzetnu mobilnost a samim tim i biodostupnost Pb na ispitivanom zemljišnom supstratu. U slučaju Cd nešto veći biokoncentracioni faktor se javlja kod korijena biljke, mada je njegova vrijednost bila ispod jedinice. Ipak, vrijednost translacionog faktora ukazuje, što je takođe vrlo važno, da se ovaj toksični mikroelement u značajnijoj mjeri transportuje do stabla nego plodova i listova borovnice. Dobijeni translokacioni podaci vezani za ispitivane toksične metale, pored biokoncentracionih vrijednosti pokazali su da se plod i listovi borovnice mogu bezbjedno primjenjivati kako u ishrani stanovništva tako i u tradicionalnoj medicini. Visoka bioakumulaciona vrijednost u plodu borovnice zabilježena je samo za K. Ono što je interesantno je da ovaj makroelement jedini i pokazuje značajan stepen translokacije prema plodu borovnice. Sa druge strane, Ca, Mg, K, Fe, Al, Sr, Ba, Mn, Cu i Zn se u vrlo značajnoj mjeri transportuju od korijena do listova, dok su Ca, Mg, K, Sr, Ba, Mn i Zn takođe pokazali veliku sposobnost akumuliraja u listu borovnice. Dnevni doprinos Cr, Mn i Cu iz jedne porcije ispitivanih borovnica je vrlo značajan, s obzirom na preporučene vrijednosti dnevne potrebe esencijalnih elemenata, prema direktivi Evropske unije. S druge strane, konzumiranjem 100 g ispitivane borovnice unijeti toksični metali čini samo mali procenat od maksimalno prihvatljivog na dnevnom nivou, što nam ukazuje da borovnica ne predstavlja toksikološki rizik po zdravlje konzumatora. Osnovni cilj ove teze bilo je i ispitivanje korelacije između hemijskog sastava i antioksidativne moći ekstrakata divlje borovnice (lista i ploda) dobijenih različitim ekstrakcionim postupcima: Soxhlet ekstrakcijom, postupkom maceracije i ekstrakcijom vrelom vodom. Iako su hemijski profil i antioksidativni kapacitet borovnice prilično ispitani, ovo je prvo istraživanja o njihovoj korelaciji u različitim ekstraktima lista i ploda borovnice. Uzimajući u obzir da se biljni vodeni infuzi najčešće koriste za pripremu tradicionalnih fitopreparata, ali uzimajući u obzir i činjenicu da industrijska proizvodnja obično koristi etanol kao rastvarač, koristili smo različite metode ekstrakcije i rastvarače različitih polarnosti za nalaženje pogodnih uslova za dobijanje ekstrakata bogatih polifenolima. Ovo istraživanje je potvrdilo da su ispitivani ekstrakti lista i ploda borovnice izuzetno bogati fenolnim jedinjenjima i da posjeduju visoku antioksidativnu aktivnost mjerenu dvjema spektrofotometrijskim metodama čiji rezultati su potvrđeni takođe i cikličnom voltametrijom. Naši ekstrakti pokazali su visok stepen korelacije između sadržaja fenola, tanina, flavonoida, antocijana, procijanidina, makroelemenata i mikroelemenata i njihove antioksidativne aktivnosti. U ovom istraživanju, potvrđen je i veliki uticaj primjenjenog postupka ekstrakcije na sadržaj fenolnih jedinjenja u ispitivanim ekstraktima. HPLC analizom u ispitivanim ekstraktima lista i ploda borovnice identifikovano je osamnaest fenolnih jedinjenja, u različitim količinama, koji su u literaturi prepoznati kao nosioci antidijabetskog potencijala. Najzastupljenija fenolna kiselina u ekstraktima bila je hlorogenska kiselina, praćena protokatehinskom kiselinom, dok su u značajnim količinama u ispitivanim ekstraktima zastupljeni i resveratrol, izokvercetin, kvercetin, hiperozid...The subject of our research was investigation of different extracts of bilberry, (Vaccinium myrtillus L, Ericaceae), a plant widespread in Montenegro, traditionally used in the treatment of diabetes, but also recognised as valuable nutraceutical. As bilberry is a source of macroelements and microelements in different concentrations, one of the aims of this investigation was to determine the concentrations of 16 elements including macro (Al, Ca, Fe, K and Mg) and microelements (As, Ba, Cd, Co, Cu, Cr, Mn, Ni, Pb, Sr and Zn) in roots, stems, leaves and fruits of wild bilberry and corresponding soil sample in order to investigate element mobility and availability in soil and the manner of translocation of elements taken up from the soil to the roots and the above-ground parts of the plant. Depending on the concentration, the abovementioned elements might be present in traces, representing the essentuials metals necessary for regular metabolic process, but being present in greater quantities they can be potentially toxic. Regarding toxic metals, As was not detected in bilberry. Pb had also low bio-concentration factor in all the parts of bilberry, which is extremely important, because the obtained results of applied BCR sequential extraction procedure revealed that Pb could be considered as the most easily mobilized metal in investigated soil samples. In the case of Cd, a slightly higher bioconcentration factor occurs at the roots of the plant, although its value was below one. However, the value of the translation factor indicates, which is also very important, that this toxic microelement is translocated to the stems mostly but not to leaves or fruits. The obtained translocation data related to the researched toxic metals, in addition to bio-concentration values, shows that the fruits and leaves of bilberry may safely be used in both human nutrition and traditional medicine. The high bioconcentration value in the fruits was only found for K. Interestingly, this is the only element that has showed a significant level of translocation to the fruits. On the other hand, Ca, Mg, K, Fe, Al, Sr, Ba, Mn, Cu and Zn were mostly translocated from roots to leaves, while Ca, Mg, K, Sr, Ba, Mn and Zn also exhibited a high capability to be accumulated in leaves. The daily contribution of Cr, Mn and Cu from one portion of the examined bilberry was very significant, considering the recommended daily allowance values of these essential elements, in accordance to the Directive of European Union. On the other hand, by consuming 100 g of tested bilberry, the intake of toxic elements was only a small percentage of the maximum tolerable daily intake, which indicates that bilberry possessed no toxicological risk to the consumer’s health. Taking into account the abovementioned, the additional aim of this study was to investigate the correlation between chemical profile and antioxidant activity of wild bilberry extracts (leaves and fruits), obtained by different extraction procedures, namely Soxhlet extraction, maceration and infusion. Although bilberry have been described elsewhere in terms of their chemical profile and/or their antioxidant capacities, this the first research focusing on their correlation in different types of bilberry leaves and fruits extracts. Althoughthe water herbal infusions are most often used for preparation of traditional phytopreparations, the industrial scale production usually employes ethanol as a solvent, different extraction methods and solvents with different polarity were used to establish the suitable conditions for obtaining the extracts rich in polyphenolic composition. Our results revealed that all investigated bilberry leaves and fruits extracts were rich in phenolic compounds and demonstrated good antioxidant activity measured by using two spectrophotometric methods, the results verified with the cyclic voltammetry tests, as well. Our extracts showed a high degree of correlation between phenols, tannins, flavonoids, anthocyanins, procyanidins, macro and microelements content and their antioxidant activity. This research also enabled to determine the effect of applied extraction procedure on the phenolic content in examined extracts. Using a HPLC method, we confirmed the presence of eighteen individual phenolic compounds in different amounts in the leaves and fruits extracts, known from the up to date literature data to possess antidiabetic potential..