Udio flavonoida u 20 uzoraka propolisa sakupljenih iz košnica s 10 različitih lokaliteta kontinentalne Hrvatske određen je spektrofotometrijskim postupcima. Flavoni i flavonoli prezentirani su kao ekvivalentni kvercetina i određeni uporabom aluminijevog klorida, a flavononi su prezentirani kao ekvivalent naringenina i određeni uporabom 2,4-dinitrofenilhidrazina. Količina flavona i flavonola je vrlo slična u većini ispitivanih uzoraka i kreće se između 2,0 i 2,3%, osim jednog uzorka s koncentracijom od 1,3% i jednog uzorka u kojem flavone i flavonole nije bilo moguće otkriti. Količina flavonona u uzorcima propolisa je vrlo varijabilna. 55% uzoraka sadrži flavanone između 15 i 24%, a 45% uzoraka sadži flavanone u koncentraciji između 4 i 14%. Ukupan udio flavonoida u uzorcima sirovog propolisa kreće se između 5 i 26%. U većini uzoraka (75%) dokazan je ukupan udio flavonoida između 15 i 26%. Visoke razlike u koncentraciji flavanona utjecat će na biološku aktivnost preparata s propolisom.Spectrometric analyses of flavonoids in twenty propolis samples collected from ten different geographic localities in northern Croatia using two complementary methods, are reported. Flavones and flavonols were determined using aluminum chloride and expressed as quercetine equivalent while flavanones were determined using 2,4-dinitrophenylhydrazine and expressed as naringenin. Contents of flavones and flavonols were similar for most samples and ranged from 2 to 2.3%, except for one sample with a concentration of 1.3% and one sample in which it was not possible to detect flavones and flavonols. The content of flavanones in propolis samples is very variable. 55% of samples contain flavanons between 15 and 24% and 45% of samples between 4 and 14%. Total levels of flavonoids in raw propolis samples ranged between 5 and 26%; for the majority of samples (75%), the total level of flavonoids ranged between 15 and 25.9%. The high variability of flavanone concentration will affect biological activity of propolis preparations

IVAN KOSALEC

MARINA BAKMAZ

SANDA VLADIMIR-KNEZEVIC

STJEPAN PEPELJNJAK

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Propolis is a sticky, gummy, resinous substance collected by honeybees (Apis mel-lifica L.) from various plant sources. Bees collect propolis to seal holes in the hives,smooth out the internal walls and protect the entrance against intruders. The main plantsources of resin from cracks in trees and leaf buds in the northern hemisphere are poplar(Populus spp.), birch (Betula spp.), etc. (1).Raw propolis is composed of approximately 50% resin (polyphenolic fraction), 30%wax, 10% essential oils, 5% pollen and 5% various organic and inorganic compounds (1,2). Chemical composition of propolis is very complex. More than 200 compounds havebeen identified (1–3). Its biological activity depends on compounds from the polyphe-65Acta Pharm. 54 (2004) 65–72 Short communicationQuantitative analysis of the flavonoids in rawpropolis from northern Croatia*IVAN KOSALEC1**MARINA BAKMAZ2STJEPAN PEPELJNJAK1SANDA VLADIMIR-KNE@EVI]31 Institute of MicrobiologyFaculty of Pharmacy and BiochemistryUniversity of ZagrebHR-10000 Zagreb, Croatia2 Analytical LaboratoryPharmacy »Zagreb«HR-10412 Rakov Potok, Croatia3 Institute of PharmacognosyFaculty of Pharmacy and BiochemistryUniversity of ZagrebHR-10000 Zagreb, CroatiaReceived October 29, 2003Accepted January 12, 2004Spectrometric analyses of flavonoids in twenty propolissamples, collected from ten different geographic localitiesin northern Croatia using two complementary methods,are reported. Flavones and flavonols were determinedusing aluminum chloride and expressed as quercetineequivalent while flavanones were determined using 2,4--dinitrophenylhydrazine and expressed as naringenin.Contents of flavones and flavonols were similar for mostsamples and ranged from 2 to 2.3%, except for one sam-ple with a concentration of 1.3% and one sample inwhich it was not possible to detect flavones and flavo-nols. The content of flavanones in propolis samples isvery variable. 55% of samples contained flavanones be-tween 15 and 24% and 45% of samples between 4 and14%. Total levels of flavonoids in raw propolis samplesranged between 5 and 26%; for the majority of samples(75%), the total level of flavonoids ranged between 15and 25.9%. The high variability of flavanone concentra-tion will affect the biological activity of propolis prepa-rations.Keywords: propolis, flavonoids, flavones, flavonols, fla-vanones, Croatia* A part of this work was presented at the 8th Croatian Biological Congress, Zagreb, September 27 – October 2, 2003.** Correspondence; e-mail: ivan.kosalec@zg.htnet.hrnolic fraction, mainly flavonoids, followed by aromatic acids, phenolic acid esters, tri-terpenes, lignans, etc. (1–5). These groups of compounds are reported to have bacteri-cidal (6–8), fungicidal (9, 10), antiviral (11), antiprotozoal (8), antioxidant (12), anti-in-flammatory (13) and immunomodulatory (14) activities.Flavonoids, one of the main groups of phenolic compounds in propolis, are the keycompounds for estimation of propolis quality. Flavonoids in propolis are aglycones(without the sugar component). These lipophilic flavonoids are chemically divided intosubgroups of flavones, flavanones, flavonols, dihydroflavonols and chalcones (15, 16).The concentration of flavonoids in propolis depends on the geographic origin and eco-system (plant sources) (17). It is known that flavonoid concentration will affect the bio-logical activity of propolis (11).Because of the phytogeographic dependence of the flavonoid content in raw pro-polis, our aim was to determine the concentration of flavonoids (flavones, flavonols andflavanones) in raw propolis samples collected from ten different localities in continentalCroatia.EXPERIMENTALReagents and solventsEthanol, methanol, potassium acetate and potassium hydroxide used were of ana-lytical grade and were purchased from Kemika (Croatia) and aluminum chloride and2,4-dinitrophenylydrazine used were of analytical grade purchased fromMerck (Germany).InstrumentsThe measurements were carried out using a PU 8625 UV-Visible spectrophotometerdiode-array (Philips, The Netherlands).66I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.Fig. 1. Localities for collectingpropolis samples.Propolis samples and extractsTwenty samples of raw propolis were collected by scraping it off hive frames inSeptember 2002. Geographic locations of hives: \akovo (45° 18’ N, 18° 24’ E), Virovitica(45° 50’ N, 17° 23’ E), ^akovec (46° 23’ N, 16° 26’ E), ^ulinec (45° 49’ N, 16° 00’ E), Osijek(45° 33’ N, 18° 42’ E), Slatina (45° 42’ N, 17° 42’ E), Sisak (45° 29’ N, 16° 22’ E), Gorjani(45° 24’ N, 18° 23’ E), Donji Miholjac (45° 36’ N, 14° 39’ E) (Fig. 1). Samples from one lo-cality were taken from different bee-keepers.The collected propolis samples were kept desiccated in the dark until analysis. Theappearance, form, color and smell of collected raw propolis samples are described in Ta-ble I.Propolis extract (PEE) was prepared as follows. One gram of raw propolis was ex-tracted with 25 mL of 95% ethanol (V/V) for 24 h at 37 °C and the filtrate was adjusted to25 mL with 80% ethanol (V/V).Determination of flavones and flavonolsFlavones and flavonols in propolis were expressed as quercetine equivalent. Quer-cetine (Sigma, Germany) was used to make the calibration curve standard solutions of12.5, 25.0, 50.0, 80.0 and 100.0 g mL–1 in 80% ethanol (V/V). The standard solutions orPEE (0.5 mL) were mixed with 1.5 mL 95% ethanol (V/V), 0.1 mL 10% aluminum chlo-ride (m/V), 0.1 mL of 1 mol L–1 potassium acetate and 2.8 mL water. The volume of 10%(m/V) aluminum chloride was substituted by the same volume of distilled water inblank. After incubation at room temperature for 30 minutes, the absorbance of the reac-tion mixture was measured at 415 nm. The coefficient of determination was r2 = 0.998.Determination of flavanonesFlavanones in propolis were expressed as ()-naringenin equivalent. ()-Naringenin(Sigma, Germany) was used to make the calibration curve (standard solution of 0.25,0.30, 0.50, 1.00 and 2.00 mg mL–1 in methanol). One mL of standard solution or PEE wasseparately mixed with 2 mL of 1% 2,4-dinitrophenylhydrazine (m/V) and 2 mL of metha-nol at 50 °C for 50 min. After cooling at room temperature, the solution was mixed with5 mL of 1% potassium hydroxide (m/V) in 70% ethanol (V/V). Then, 1 mL of the mixturewas taken and centrifuged at 1000xg for 10 min and the supernatant was filteredthrough Whatman No. 1 filter paper. The filtrate was adjusted to 25 mL. The absorbanceof the filtrate was measured at 495 nm. The coefficient of determination was r2 = 0.996.Statistical analysisThe results were expressed as mean  SD obtained upon three independent analy-ses. Statistical significance of the differences was evaluated using Student’s t-test forsamples from \akovo and Virovitica.67I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.68I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.TableI.PhysicalcharacteristicsandflavonoidcontentinrawpropolissamplesfromcontinentalCroatiaSampleNo.LocalityAppearanceandformColourandsmellFlavonoidsFlavonesandflavonolsa,cxSDdFlavanonesb,cxSDdEasternpart2\akovoWaxyBrown,notaromatic2.20.32.20.120.70.1A16.24.3G6Dry,waxyLightbrown,notaromatic2.20.115.10.2A,B7WaxyLightbrown,notaromatic2.30.413.80.2A,B,C9WaxyLightbrown,aromatic2.20.410.30.1A,B,C,D10SolidDarkbrown,notaromatic2.20.120.60.1B,C,D13SolidLightbrown,notaromatic2.20.220.30.1B,C,D17WaxyDarkbrown,aromatic2.30.212.40.1A,B,C,D15GorjaniWaxy,solidBrown,aromatic2.20.223.80.11OsijekGummy,waxyLightbrown,aromatic2.10.122.20.116DonjiMiholjacWaxyBrown,aromatic2.30.113.10.1Westernpart4ViroviticaWaxy,solidLightbrown,notaromatic2.20.32.10.119.40.2E15.13.2I11VerysolidLightbrown,notaromatic2.00.411.60.1EF12Solid,waxyLightbrown,notaromatic2.10.114.40.1EF5^akovecDryDarkbrown,notaromatic2.30.220.60.114SolidLightbrown,notaromatic2.30.417.80.118^ulinecWaxyBrown,aromatic2.30.217.90.119PowderedYellowish-brown,aromatic1.30.33.90.38SisakSolidDarkbrown,notaromatic2.00.213.10.23SlatinaDry,solidBrown,aromatic2.10.217.80.220^abarSolidYellowish-brown,notaro-maticND10.60.1aExpressedasquercetinequivalent.bExpressedasnaringeninequivalent.cMeanSDofdatafromthesamelocality.dGrandmeanSD(n=3).ND–Notdetected.A–F–Eachlatterdenotessignificantdifferences(p<0.001)betweentherespectivesamples.G–Significantdifference(p<0.05).RESULTS AND DISCUSSIONTwenty samples of raw propolis were collected from hives situated on 10 differentlocations in continental Croatia. The samples studied varied in consistency, colour andsmell (Table I). Geographic position of the hives (eastern and western parts of continen-tal Croatia) did not influence the physical characteristics of propolis. Appearance andform did not influence the colour and smell of propolis samples. Only one propolis sam-ple, powdered (No. 19) differed markedly from other samples. Samples of propolis fromdifferent hives but from the same locality differed to the same extent as samples fromdifferent localities. For example, four samples from \akovo did not differ in the appear-ance and form of wax (samples 2, 7, 9, and 17) but they differed in colour and smell.Samples from the same locality with a solid form (samples 10 and 13) have a differentcolor but the same (not aromatic) smell.Flavonoids were determined by two independent colorimetric methods, one for de-terimination of flavones and flavonols and the other for determination of flavanones, asreported by Chang et al. (18). The results are shown in Table I.In the seven propolis samples from \akovo, the level of flavones and flavonols var-ied from 2.2 to 2.3% (average 2.2%), the content of flavanones varied between 10.3 and20.7% (average 16.2%). The level of total flavonoids in \akovo propolis samples variedbetween 12.4 and 20.7%.Three propolis samples from Virovitica contained between 2.0 and 2.2% (average2.1%) of flavones and flavonols, and between 11.6 and 19.4% (average 15.1%) of flava-nones. The level of total flavonoids varied from 13.6 to 21.6%.Two samples from ^akovec contained equal shares of flavones and flavonols (2.3%)but different levels of flavanones (from 17.8 to 20.6%).Two samples from ^ulinec differed greatly in both flavones and flavonols (1.3 and2.3%), and flavanones (17.9 and 3.9%).Single samples from Osijek, Slatina, Sisak, Gorjani, Donji Miholjac and ^abar didnot differ in the content of flavones and flavonols (2.0 to 2.3%), but the level of flava-nones varied between 10.6 and 23.8%. The content of flavonoids in these samples variedfrom 10.6 to 25.9%.Determination of flavones, flavonols and flavanones in raw propolis samples sho-wed that 16 out of 20 samples (80%) contained more than 2% of flavones and flavonols(from 2.1 to 2.3%, average 2.2%). Only three samples contained less than 2% of flavonesand flavonols (from 1.3 to 2%, average 1.8%). We could not detect any flavones and fla-vonols in sample No. 20 from ^abar.The content of flavanones in the propolis samples studied varied greatly (4 to 24%,average 16%). Six propolis samples (30%) contained more than 20% of flavanones (from20.6 to 23.8%, average 21.4%) whereas the majority of the samples studied (13 from 20,or 65%) had 10 to 20% of flavanones. Only one sample (sample No. 19), which also had alow concentration of flavones and flavonols (1.3%), had a low concentration of flava-nones (3.9%).Comparison of the concentrations of flavones and flavonols in propolis samplesfrom the same locality and between different localities showed that there were no signi-69I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.ficant differences, except for sample No. 19 from ^ulinec and sample No. 20 from ^abar.However, significant differences (p < 0.001) of flavanone concentration were found with-in and between the localities of \akovo and Virovitica.With regard to the geographic position, samples from the eastern part of continentalCroatia and samples from the western part had comparable concentrations of flavones,flavonols and flavanones. The exceptions were the samples from the western part, na-mely from ^abar and one sample from ^ulinec, with low concentrations of flavonoids.The total level of flavonoids in the raw propolis samples studied varied between 5%and 26%, with an average of 19%. For the majority of propolis samples (75%), the totallevel of flavonoids was between 15 and 26%.Our results show uniformity of the concentrations of flavones and flavonols butgreat differences in flavanone concentrations. High variability of the flavonoid concen-tration led us to conclude that this will affect the biological activity.CONCLUSIONSOur results show very balanced concentrations of flavones and flavonols, but a highvariability of flavanones concentrations in the propolis samples collected in the conti-nental region of Croatia. Significant differences in the concentration of flavanones be-tween localities and within the same locality were observed. However, no influence ofthe geographic position of hives was found in the eastern and western parts of continen-tal Croatia.Differences in specific groups of flavonoids will affect biological activities and ourfuture research will be focused on investigating the influence of concentration of the fla-vones, flavonols and flavanones on the bactericidal activity, especially against Gram-po-sitive bacterial species.Our investigation has shown that application of two individual and complementarymethods (flavones and flavonols together with flavanones) makes a simple method forestimation of flavonoids as the key compounds for evaluating the quality of raw pro-polis. These spectrophotometric methods could be the first step for the estimation of rawpropolis quality. The same method might be applicable for the determination of flavo-noids in propolis preparations as well.REFERENCES1. G. A. Burdock, Review of the biological properties and toxicity of bee propolis (propolis), FoodChem. Toxicol. 36 (1998) 347–363.2. V. S. Bankova., S. L de Castro and M. C. Marcucci, Propolis: recent advances in chemistry andplant origin, Apidologie 31 (2000) 3–15.3. E. Langner and H. Schilcher, Propolis – Qualität und Wirkungen von Propolis bzw. Propolis-zuberetungen, Dtsch. Apoth. Ztg. 37 (1999) 51–63.4. J. D. Fontana, M. Passos, M. H. R. dos Santos, C. K. Fontana, B. H. Oliveria, L. Schause, R. Pon-taralo, M. A. Barbirato, M. A. Ruggiero and F. M. Lanças, Profiling propolis flavonoids by means70I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.of micellar electrokinetic capillary chromatography, capillary gas chromatograpy and bacteri-cidal action, Chromatographia 52 (2000) 147–151.5. V. S. Bankova, R. S. Christov and A. D. Tejera, Lignans and other constituents of propolis fromthe Canary Islands, Phytochem. 49 (1998) 1411–1415.6. S. Pepeljnjak, I. Jal{enjak and D. Maysinger, Growth inhibition of Bacillus subtilis and composi-tion of various propolis extracts, Pharmazie 37 (1982) 864–865.7. S. Pepeljnjak, I. Jal{enjak and D. Maysinger, Flavonoid content in propolis extracts and growthinhibition of Bacillus subtilis, Pharmazie 40 (1985) 122–123.8. O. K. Mirzoeva, R. N. Grishanin and P. C. Calder, Antimicrobial action of propolis and some ofits components: the effects on growth, membrane potential and motility of bacteria, Microbiol.Res. 52 (1997) 239–246.9. S. Pepeljnjak and I. Jal{enjak, Usage of propolis extract for preserving food against microbiolo-gical contamination, Microbiol. Alim. Nutri. 2 (1984) 301–302.10. A. C. H. F. Sawaya, A. M. Palma, F. M. Caetano, M. C. Marcucci, I. B. da Silva Cunha, C. E. P.Araujo and M. T. Shimizu, Comparative study of in vitro methods used to analyse the activityof propolis extracts with different compositions against species of Candida, Lett. Appl. Microbiol.35 (2002) 203–207.11. A. Kujumgiev, I. 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Tew, B. A. G. Nguyen and G. Mathé, Polyphenols: do they play a role in theprevention of human pathologies?, Biomed. Pharmacother. 56 (2002) 200–207.17. M. I. Nieva Moreno, M. I. Isla, N. G. Cudmani, M. A. Vattuoene and A. R. Sampietro, Screeningof antibacterial activity of Amaicha del Valle (Tucumán, Argentina) propolis, J. Ethnopharmacol.68 (1999) 97–102.18. C. C. Chang, M. H. Yang, H. M. Wen and J. C. Chern, Estimation of total flavonoid content inpropolis by two complementary colorimetric methods, J. Food & Drug Analysis 10 (2002) 178–182.71I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.S A @ E T A KKvantitativna analiza flavonoida u sirovom propolisu sjeverne HrvatskeIVAN KOSALEC, MARINA BAKMAZ, SANDA VLADIMIR-KNE@EVI] i STJEPAN PEPELJNJAKUdio flavonoida u 20 uzoraka propolisa sakupljenih iz ko{nica s 10 razli~itih loka-liteta kontinentalne Hrvatske odre|en je spektrofotometrijskim postupcima. Flavoni iflavonoli prezentirani su kao ekvivalenti kvercetina i odre|eni uporabom aluminijevogklorida, a flavanoni su prezentirani kao ekvivalent naringenina i odre|eni uporabom2,4-dinitrofenilhidrazina. Koli~ina flavona i flavonola je vrlo sli~na u ve}ini ispitivanihuzoraka i kre}e se izme|u 2,0 i 2,3%, osim jednog uzorka s koncentracijom od 1,3% ijednog uzoraka u kojem flavone i flavonole nije bilo mogu}e otkriti. Koli~ina flavanonau uzorcima propolisa je vrlo varijabilna. 55% uzoraka sadr`i flavanone izme|u 15 i 24%,a 45% uzoraka sadr`i flavanone u koncentraciji izme|u 4 i 14%. Ukupan udio flavonoi-da u uzorcima sirovog propolisa kre}e se izme|u 5 i 26%. U ve}ini uzoraka (75%) doka-zan je udio ukupnih flavonoida izme|u 15 i 26%. Visoke razlike u koncentraciji flavano-na utjecat }e na biolo{ku aktivnost preparata s propolisom.Klju~ne rije~i: propolis, flavonoidi, flavoni, flavonoli, flavanoniFarmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu, ZagrebGradska ljekarna Zagreb, Rakov Potok, Zagreb72I. Kosalec et al.: Quantitative analysis of the flavonoids in raw propolis from northern Croatia, Acta Pharm. 54 (2004) 65–72.

Quantitative analysis of the flavonoids in raw propolis from northern Croatia

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