부산대학교 도서관

Spectrophotometric procedures for the rapid characterization of propolis have been performed on propolis samples from different regions of Croatia. In order to determine the major groups of bioactive compounds in propolis, the following optimised and validated spectrophotometric methods were carried out: the Folin-Ciocalteu method for the content of total phenolics (TPs) and two distinct methods for the content of total flavonoids: aluminium chloride ([AlCl.sub.3]) complexation method for total flavones/flavonols (TFFs) and the 2.4-dinitrophenylhydrazine (2.4-DNPH) method for total flavanones/dihydroflavonols (TFDs). Validation parameters, including linearity, sensitivity, range, accuracy, limit of detection, limit of quantification, precision and robustness were implemented. The following polyphenol standards were used for the validation procedure: gallic acid (GA), pinocembrin (PC), galangin (GN), quercetin (QC) and a mixture of PC and GN. Validated methods were applied to analyse six samples of raw propolis from Croatian continental and Adriatic regions. The high qualitative/quantitative variability of the TP, TFF and TFD content was observed. Although the method of extraction (ultrasonic-assisted extraction or microwave assisted extraction) showed a non-significant effect on extraction yield (P>0.05) and the polyphenolic concentrations obtained of each sample in general, ultrasonic extraction was found to be more selective. Furthermore, the calibration compound used for constructing the calibration curve highly influenced the final concentrations of TPs and TFFs. The study showed good linearity, accuracy, repeatability, intermediate precision, LOD and LOQ for all three spectrophotometric methods, considering that these analyses are the basis for further research into the individual polyphenolic compounds in the propolis samples covered by this research. Keywords: raw propolis; polyphenols; ultrasonic-assisted extraction; microwave-assisted extraction; spectrophotometric determination; validation U ovom su radu provedene spektrofotometrijske metode za brzu analizu uzoraka propolisa iz razlicitih podrucja Republike Hrvatske. S ciljem odredivanja glavnih skupina bioaktivnih spojeva u propolisu, optimizirane i validirane su tri spektrofotometrijske metode: Folin-Ciocalteuova metoda za odredivanje ukupnih fenola i dvije razlicite metode za sadržaj ukupnih flavonoida - metoda kompleksiranja s aluminijevim kloridom za ukupne flavone/flavonole te 2,4-DNPH metoda za ukupne flavanone/dihidroflavonole. Validacijom su odredeni sljedeci parametri: linearnost, osjetljivost, raspon, tocnost, granica detekcije, granica kvantifikacije, preciznost i robusnost. Za provedbu validacijskog protokola korišteni su standardi polifenola, ukljucujuci galnu kiselinu (GA), pinocembrin (PC), galangin (GN), kvercetin (QC) te smjesu pinocembrina i galangina (PC-GN). Validiranim metodama analizirano je šest uzoraka sirovog propolisa iz kontinentalne i jadranske regije Hrvatske. Primijecena je velika raznolikost u sadržaju ukupnih fenola, ukupnih flavona/flavonola te ukupnih flavanona/dihidroflavonola medu uzorcima iz dviju hrvatskih regija. Iako metode ekstrakcije (ultrazvucna ekstrakcija ili ekstrakcija potpomognuta mikrovalovima) nisu pokazale statisticki znakovitu razliku u obliku prinosa ekstrakcije (P > 0,05), te gledajuci koncentracije polifenola cjelokupno, ultrazvucna je ekstrakcija bila selektivnija. Nadalje, velik utjecaj na konacne koncentracije ukupnih fenola i ukupnih flavona/flavonola imao je standard korišten za izradu kalibracijske krivulje. Dobivena je dobra linearnost, tocnost, ponovljivost, srednja preciznost, granica detekcije i granica odredivanja u svim trima spektrofotometrijskim metodama te su ovi rezultati temelj za daljnja istraživanja pojedinacnih polifenolnih spojeva u uzorcima propolisa. Kljucne rijeci: sirovi propolis; polifenoli; ultrazvucna ekstrakcija; ekstrakcija potpomognuta mikrovalovima; spektrofotometrijsko odredivanje; validacija
Introduction Propolis, a bee product, continues to fascinate many researchers around the world. Regardless of its complex and diverse composition, the increasingly frequent application of propolis in veterinary medicine could [...]