investigation of the flavonoids in croatian propolis by thin-layer chromatography

Journal of Planar Chromatography VOL. 17. MARCH/APRIL 2004 95

SummaryFlavonoids and phenolic acids with a variety of biological activity

are considered to be the main compounds in propolis – a natural

product produced by the honey bee. TLC can be used for rapid

screening of pharmacologically active components and to establish

the difference between different propolis samples. Our goal was to

optimize chromatographic conditions for separation of flavonoids

and phenolic acids and to apply the optimized method for analysis

of propolis samples from different geographic regions of Croatia.

For chromatographic analysis we used 20 cm × 20 cm glass-backed

TLC plates coated with 0.25 mm layers of silica gel 60 F254

. Ethano-

lic standard solutions (80%) of the flavonoids and phenolic acids

(10 μL) were applied to the plates. Chromatograms were developed

at room temperature by ascending development in previously satu-

rated vertical, flat-bottomed glass chambers with glass lids. Visual-

ization was performed in short- and long-wavelength UV light and

in long-wavelength UV light after spraying with different reagents.

After calculation of RF

values numerical taxonomy methods were

used to test the efficiency of 11 mobile phases and to optimize chro-

matographic conditions for separation of 19 standard solutions. We

established the most appropriate mobile phases

(chloroform–methanol–(98–100%) formic acid, 44.1 + 3 + 2.35, and

n-hexane–ethyl acetate–glacial acetic acid, 31 + 14 + 5) for separa-

tion of standards. The results obtained were used for analysis of

propolis samples. TLC was shown to be a highly suitable method

for rapid analysis of propolis samples. It can be used to establish

differences between the amounts of pharmacologically active com-

pounds in propolis from different geographic regions of Croatia.

1 Introduction

Propolis is made from the resinous material collected by honey-bees from a variety of plant sources. The resin is masticated,salivary enzymes are added, and the partially digested materialis mixed with beeswax and used in hive construction [1, 2]. Theexact composition of propolis depends on its origin. Its chemi-cal composition is rather complicated – over 150 componentshave been identified [2–4]. The main compounds are resins(which include flavonoids, phenolic acids, and esters), waxesand fatty acids, essential oils, pollen, other organic compounds(for example steroids, benzoic acids, vitamins, sugars), andminerals (14 trace minerals of which iron and zinc are the mostcommon). Propolis has been used for decades in folk medicinefor maintaining health, and many studies have confirmed itsantiseptic, antifungal, antibacterial, antiviral, anti-inflammato-ry, and antioxidant properties. The most important pharmaco-logically active constituents in propolis are flavonoids and phe-nolic acids; it has been suggested these are responsible for thebiological activity of the material [5–8]. Flavonoids are a groupof plant phytochemicals that cannot be biosynthesized byhumans. They are benzo-γ-pyrone derivatives which resemblecoumarin and are divided in six groups (flavanols, flavones,flavonols, flavanons, isoflavons, and anthocyanidins), depend-ing on the substituents on the main ring (Figure 1) [9]. Inpropolis flavonoids occur as aglycons and glycosides.

Investigation of the Flavonoids in Croatian Propolisby Thin-Layer Chromatography

Ivona Jasprica, Asja Smolčić-Bubalo, Ana Mornar, and Marica Medić-Šarić*

Key Words:

PropolisFlavonoidsPhenolic acidsNumerical taxonomyTLC

I. Jasprica, A. Smolèiæ-Bubalo, A. Mornar, and M. Mediæ-Šariæ, Department ofPharmaceutical Chemistry, Faculty of Pharmacy and Biochemistry, University ofZagreb, A. Kvoaèiæa 1, 10 000 Zagreb, Croatia.

This paper was presentedat the 5th Balaton Symposium

on High-Performance Separation Methods,Siófok, Hungary,

September 3–5, 2003

Figure 1

The basic structure of flavonoids.

DOI: 10.1556/JPC.17.2004.2.3

Current applications of propolis include over-the-counter prepa-rations for treatment of colds. Most of these are ethanolicextracts of propolis. With increasing use of these preparations itbecame important to know the precise composition of propolis.Different analytical methods can be used to establish the com-position of propolis samples, for example TLC [10–12], HPLC,and MS–MS [13]. Compared with other analytical methodsthin-layer chromatography is rapid and rather inexpensive andcould be used to screen propolis extracts for pharmacologicallyactive substances (flavonoids and phenolic acids) before use ofmore expensive and time-consuming methods.

To obtain optimum and reproducible results it is necessary tooptimize chromatographic conditions. Optimization can beachieved by different methods [14–17]. We used numerical tax-onomy methods [18], applying the computer search programKT1 [19] to test and compare the efficacy of eleven mobilephases used for TLC of fifteen standard solutions of flavonoidsand four standard solutions of phenolic acids [20]. The resultsobtained (the most appropriate mobile phases for chromatogra-phy of standards) were then used for analysis of fourteen sam-ples of propolis from different regions of Croatia.

The term ‘numerical taxonomy’ denotes the grouping bynumerical methods of taxonomic units (groups of any nature orrank) on the basis of their character states. These methodsrequire conversion of information about taxonomic entities intonumerical quantities (flavonoids and phenolic acids representthe taxonomic entities and their numerical characteristics aregiven by their RF values for a particular mobile phase). Estima-tion of resemblance is the most important and fundamental stepin numerical taxonomy and classification is generally based ona matrix of resemblance. In this example resemblance of RF val-ues is analyzed to find the mobile phase in which the resem-blance is the lowest and separation of the analyzed compoundsis optimum. It is important to choose an adequate number ofcompounds to establish a manageable taxonomic group (thelowest ranking taxonomic group employed in a study is calledan operational taxonomic unit – OTU). To estimate the resem-blance between pairs of OTU, data are arranged in the form ofan N × t matrix, where t columns represent the t OTU to be clas-sified on the basis of resemblance and N rows are N unit char-acters (properties). Similarity between two OTU is usually esti-mated by means of a similarity coefficient which quantifies theresemblance between the elements in the two columns of thedata matrix representing the character states of two OTU com-pared. If any two OTU have identical character, the distancebetween them is considered to be zero (they are positioned inthe same spot in space). Distance is considered to be the com-plement of similarity [14]. The distance dj,k between OTU j andk is equal to:

dj,k= (1)

and the mean taxonomic distance is:

Δj,k = (dj,k2/N)1/2 (2)

For thin-layer chromatography the mobile phases are the OTUand RF values are the identification characteristics. The comput-er search program KT1 uses different approaches to calculatethe amount of information and the similarity coefficients. TheShannon equation describes the average information content(entropy) [16]:

I(X) = H(X) = –Σ(nk/n)ld(nk/n) (bit) (3)

TLC of Flavonoids in Croatian Propolis

96 VOL. 17. MARCH/APRIL 2004 Journal of Planar Chromatography

Table 1

Origin of Croatian propolis.

Sample Geographic origin

1 Zabok – Pregrada, North Croatia

2 Pelješac, South Dalmatian* coast

3 Braè, Vis, Central Dalmatian islands

4 Gorski Kotar, Central Croatia

5 Split, Central Dalmatian coast

6 Dalmatinska Zagora – behind Klis, Hinterland of CentralDalmatian coast

7 Metkoviæ, The river Neretva delta, South Dalmatia

8 Kaštela, Central Dalmatian coast

9 Kamešnica, Hinterland of Central Dalmatian coast –border with Hercegovina

10 Imotski, Hinterland of Central Dalmatian coast

11 Imotska krajina, Hinterland of Central Dalmatian coast –border with Hercegovina

12 Trogir, Central Dalmatian coast

13 Šolta, Central Dalmatian islands

14 Dalmatinska Zagora – all, Hinterland of Central Dalmatiancoast

*Dalmatia – Southern part of Croatian coast

Table 2

The mobile phases studied.

No Solvents Proportions (v/v)

1 Toluene–ethyl acetate–(98–100%) 36 + 12 + 5formic acid

2 Cyclohexane–ethyl acetate–(98–100%) 30 + 15 + 5formic acid

3 Toluene–ethyl acetate–glacial acetic acid 36 + 12 + 5

4 Cyclohexane–ethyl acetate–glacial acetic 31 + 14 + 5acid

5 n-Hexane–ethyl acetate–(98–100%) formic 31 + 14 + 5acid

6 Toluene–acetone–(98–100%) formic acid 38 + 10 + 5

7 n-Hexane–ethyl acetate–glacial acetic acid 31 + 14 + 5

8 Petroleum ether (40–70°C)–ethyl 30 + 15 + 5acetate–(98–100%) formic acid

9 Carbon tetrachloride–acetone–(98–100%) 35 + 10 + 5formic acid

10 n-Hexane–ethylacetate–glacial acetic acid 30 + 20 + 1.5

11 Chloroform–methanol–(98–100%) 44.1 + 3 + 2.35formic acid

This equation represents the distribution of RF values intogroups, with error factor E (usually E = 0.05 or less), in RF units,assuming nk RF values in the kth group. The similarity of com-pounds can be described by the discriminating power (DP),value T and the formation of clusters. DP for one or more chro-matographic systems represents the probability of separating

two randomly selected standards by use of one (or at least one)chromatographic system:

DPk = 1 – 2M/[N(N – 1)] (4)

where k is the number of the chromatographic system, N thenumber of compounds analyzed, and M the total number ofmatching pairs. It is important that differences between identifi-cation values (RF) do not exceed the given error factor E. Thevalue T represents the average number of similar substances forthese chromatographic systems [21]:

T = 1 + (N – 1)(1 – DPk) (5)

Chromatographic systems of high similarity can be groupedinto clusters (sets of OTU that meet one or more of criteriaimposed by a particular cluster definition). In this study clusterformation was carried out by a weighted pair group methodusing the arithmetic average [18] and was followed by con-struction of a dendrogram [22].

2 Experimental

As mentioned above, most propolis preparations available com-mercially are extracts in ethanol. We used 80% ethanol toextract flavonoids and phenolic acids from 14 propolis samples[23] (the geographic origins of the propolis are listed inTable 1).



Table 3

Input data – RF values of phenolic acid and flavonoid standards.

Standard Mobile phase: Fluorescence under long-1 2 3 4 5 6 7 8 9 10 11 wavelength UV lighta)

Phenolic acids

o-Coumaric acid 0.55 0.38 0.51 0.51 0.37 0.48 0.73 0.75 0.28 0.47 0.34 Yellow–orange

p-Coumaric acid 0.55 0.36 0.51 0.49 0.34 0.47 0.69 0.75 0.22 0.42 0.34 Dark blue

Caffeic acid 0.38 0.26 0.30 0.33 0.22 0.34 0.43 0.62 0.23 0.25 0.22 Intensive yellow

Ferulic acid 0.56 0.32 0.49 0.49 0.28 0.45 0.63 0.70 0.28 0.41 0.51 Dark blue

Flavonoids

Flavanone 0.67 0.40 0.62 0.65 0.38 0.62 0.75 0.76 0.85 0.80 0.91 –

Naringenin 0.54 0.37 0.58 0.44 0.24 0.44 0.52 0.73 0.35 0.38 0.16 Orange

Flavone 0.88 0.62 0.92 0.86 0.66 0.85 0.91 0.92 0.69 0.55 0.81 –

3-Hydroxyflavone 0.77 0.51 0.80 0.76 0.56 0.66 0.82 0.83 0.70 0.55 0.85 White–yellow

6-Hydroxyflavone 0.67 0.39 0.61 0.62 0.36 0.56 0.75 0.80 0.51 0.56 0.46 White

6’-Hydroxyflavone 0.52 0.32 0.46 0.51 0.28 0.48 0.56 0.73 0.38 0.34 0.47 Light pink

7-Hydroxyflavone 0.46 0.30 0.42 0.42 0.26 0.46 0.47 0.70 0.36 0.29 0.28 Intensive pink

3,6-Dihydroxyflavone 0.54 0.36 0.51 0.52 0.34 0.46 0.56 0.72 0.37 0.43 0.49 Light blue

3,7-Dihydroxyflavone 0.54 0.36 0.50 0.48 0.33 0.47 0.54 0.70 0.44 0.39 0.32 Light blue

Morin 0.23 0.16 0.14 0.14 0.13 0.23 0.13 0.32 0.14 0.00 0.04 Yellow

Chrysin 0.62 0.38 0.60 0.53 0.36 0.56 0.68 0.74 0.41 0.51 0.58 Dark orange

Quercetin 0.39 0.27 0.27 0.28 0.22 0.35 0.30 0.60 0.20 0.00 0.14 Light orange

Galangin 0.65 0.44 0.64 0.57 0.37 0.60 0.72 0.85 0.59 0.53 0.45 Orange

Apigenin 0.44 0.33 0.47 0.33 0.21 0.37 0.39 0.67 0.31 0.26 0.11 Light pink

Kaempferol 0.51 0.37 0.50 0.39 0.23 0.40 0.47 0.77 0.36 0.29 0.10 Yellow

a)After spraying with 1% methanolic diphenylboryloxyethylamine and 5% ethanolic poly(ethylene glycol) 4000 or 1% ethanolic AlCl3

Table 4

DP and I output data for error factor E = 0.03 for each mobile phase.

Mobile phase DP I (bit)

1 0.8538 3.221

2 0.7836 2.735

3 0.8655 3.076

4 0.8947 3.616

5 0.8187 2.860

6 0.8480 3.011

7 0.9298 3.682

8 0.7895 3.050

9 0.9240 3.511

10 0.9181 3.471

11 0.9415 3.827

Chromatography was performed on 20 cm × 20 cm plates pre-coated with 0.25 mm silica gel 60 F254 (Merck, Darmstadt, Ger-many). Before use plates were cleaned with methanol and acti-vated for 30 min at 105°C. Each standard (purchased from theDepartment of Pharmacognosy, Faculty of Pharmacy and Bio-chemistry, University of Zagreb; 1 mg) was dissolved in 80%ethanol (10 mL) and these solutions (10 μL) were applied to theplate, as bands, by means of a 25-μL Hamilton microsyringe.

To find the most appropriate mobile phases for the separationwe used numerical taxonomy methods. Eleven different sol-vents (all analytical grade; Kemika, Zagreb, Croatia) wereselected and combined according to their polarity to give themobile phases listed in Table 2. After establishing optimumconditions for the separation of standards, propolis sampleswere analyzed.

Chromatograms were developed at room temperature (24 ±1°C) to a distance of approximately 16 cm from the origin byascending development in previously saturated (saturation time30 min) vertical flat-bottom glass chambers (20 cm × 20 cm ×10 cm) with glass lids (Camag, Switzerland). The average

development time was 1 h 45 min. Visualization was performedunder UV illumination with short-wavelength UV light (λ = 254nm), long-wavelength UV light (λ = 366 nm) after sprayingwith 1% methanolic diphenylboryloxyethylamine and 5%ethanolic poly(ethylene glycol) 4000 [24], and long-wavelengthUV light (λ = 366 nm) after spraying with 1% ethanolic AlCl3

[25]. A list of the standards used, with their RF values, is givenin Table 3.

3 Results and Discussion

The efficiency of the eleven mobile phases in TLC separation ofstandards of compounds that might be present in propolis wastested. RF values of the separated standards are listed in Table 3and Table 4 gives discriminating power (DP) and informationcontent (I) output data for each chromatographic system with anerror factor E = 0.03. Output data for combined systems (two



Table 5

DP and T output data for combined mobile phases (K = 2, E = 0.03 and K = 3, E = 0.05).

Combination Mobile E = 0.03 Combination Mobile E = 0.05sequence phases (K = 2) DP T sequence phases (K = 3) DP T

1 10, 11 0.9942 1.105 1 3, 9, 10 1.0000 1.000

2 9, 11 0.9942 1.105 2 2, 9, 10 1.0000 1.000

3 9, 10 0.9883 1.211 3 9, 10, 11 0.9942 1.105

4 7, 11 0.9883 1.211 4 8, 10, 11 0.9942 1.105

5 7, 10 0.9883 1.211 5 8, 9, 11 0.9942 1.105

6 7, 9 0.9883 1.211 6 8, 9, 10 0.9942 1.105

7 4, 7 0.9883 1.211 7 7, 10, 11 0.9942 1.105

8 3, 9 0.9883 1.211 8 7, 9, 11 0.9942 1.105

9 2, 10 0.9883 1.211 9 6, 9, 11 0.9942 1.105

10 8, 11 0.9825 1.316 10 6, 9, 10 0.9942 1.105

Table 6

Formation of clusters.

Cluster Mobile phase Mobile phase Distance Distance [%]

1 4 6 0.0461 15.9

2 1 3 0.0545 18.8

3 2 4 0.0621 21.4

4 1 3 0.0687 23.6

5 1 3 0.1032 35.5

6 4 5 0.1111 38.2

7 4 5 0.1397 48.1

8 2 4 0.1646 56.6

9 1 3 0.1945 66.9

10 1 2 0.2906 100.0

Figure 2

Dendrogram for 11 TLC mobile phases.

mobile phases, K = 2, E = 0.03 and three mobile phases K = 3,E = 0.05) are presented in Table 5. Selection of the most appro-priate mobile phase is based on the highest values of DP and I.As is apparent from Table 4, the best mobile phase is number 11,chloroform–methanol–(98–100%) formic acid, 44.1 + 3 + 2.35

(DP = 0.9415, I = 3.827) followed by mobile phase number 7,n-hexane–ethyl acetate–glacial acetic acid, 31 + 14 + 5 (DP =0.9298, I = 3.682). For combination of two or three mobilephases the highest value of DP (ideally DP = 1) and the small-est value of T (ideally T = 1) is attributed to the most appropri-



Table 7

Analysis of propolis samples.

Standard Propolis sample:1 2 3 4 5 6 7 8 9 10 11 12 13 14

o-Coumaric acid + + + – + – + + + – + + + +

p-Coumaric acid – + – – + + – + + – – – + –

Caffeic acid + + + + + + + + + + + + + +

Ferulic acid – – – – – – – – – – – – – –

Flavanone – – – – – – – – – – – – – –

Naringenin – – – – – – – – – – – + – –

Flavone – – – – – – – – + – – – – –

3-Hydroxyflavone – – – – – – – – – – – – + –

6-Hydroxyflavone + – – – – – – – – – – – + +

6’-Hydroxyflavone – – – – – – – + + + + + – +

7-Hydroxyflavone – – – – – – – – – – – – + –

3,6-Dihydroxyflavone – – – – – – – – – – – – – –

3,7-Dihydroxyflavone + – – – – – – – – – – – – –

Morin – – – – – – – – – – – + – –

Chrysin + + + – – + + + + + + + + +

Quercetin – – – + – – – – + + + + + +

Galangin + – + + + + + + + + + + + +

Apigenin + + + + + + + + + + + + + +

Kaempferol – + – + – – – – + – – – – –

Figure 3

Results from separation with mobile phase number 7; visualization under short-wavelength UV light: 1. caffeic acid; 2. ferulic acid; 3. quercetin; 4. propolis sam-ple number 9; 5. o-coumaric acid; 6. p-coumaric acid; 7. morin; 8. chrysin.

Figure 4

Results from separation with mobile phase number 7; visualization under short-wavelength UV light: 1. 3-hydroxyflavone; 2. 3,6-dihydroxyflavone; 3. 3,7-dihy-droxyflavone; 4. propolis sample number 9; 5. 7-hydroxyflavone; 6. flavone;7. flavanone; 8. 6-hydroxyflavone.

ate combination. For a series of two mobile phases (E = 0.03)the most suitable combinations were combinations of mobilephases 10 and 11 (DP = 0.9942, T = 1.105) and mobile phases 9and 11 (with the same DP and T values). The most suitable com-binations of three mobile phases (E = 0.05) were shown to becombinations of mobile phases 3, 9, and 10 and 2, 9, and 10with DP = 1 and T = 1. Cluster formation (Table 6), graphical-ly presented as a dendrogram in Figure 2, shows that for this

analysis most suitable mobile phases are 11 (chosen from clus-ter 1) and 7 (chosen from cluster 2).Mobile phases 11 and 7 were found to be the most appropriatefor propolis analysis. Results obtained after chromatographicanalysis of 14 propolis samples are presented in Table 7. Of the19 standards available we managed to identify 9 or fewer com-pounds in each sample. Figures 3–8 show results obtained frompropolis sample 9 (from Kamešnica) analyzed under short-



Figure 5

Results from separation with mobile phase number 7; visualization under short-wavelength UV light: 1. 6’-hydroxyflavone; 2. galangin; 3. naringenin; 4. propolissample number 9; 5. apigenin; 6. kaempferol.

Figure 6

Results from separation with mobile phase number 7; visualization under long-wavelength UV light after spraying with 1% ethanolic AlCl3: 1. caffeic acid; 2. fer-ulic acid; 3. quercetin; 4. propolis sample number 9; 5. o-coumaric acid; 6. p-coumaric acid; 7. morin; 8. chrysin.

Figure 7

Results from separation with mobile phase number 7; visualization under long-wavelength UV light after spraying with 1% ethanolic AlCl3: 1. 3-hydroxyflavone;2. 3,6-dihydroxyflavone; 3. 3,7-dihydroxyflavone; 4. propolis sample number 9;5. 7-hydroxyflavone; 6. flavone; 7. flavanone; 8. 6-hydroxyflavone.

Figure 8

Results from separation with mobile phase number 7; visualization under long-wavelength UV light after spraying with 1% ethanolic AlCl3: 1. 6’-hydroxyflavone;2. galangin; 3. naringenin; 4. propolis sample number 9; 5. apigenin; 6.kaempferol.

wavelength UV light (Figures 3–5) and under long-wavelengthUV light after spraying with 1% ethanolic AlCl3 (Figures 6–8).A Camag Reprostar 3 was used to document the chromato-grams.

As is shown in Table 7, propolis samples collected from differ-ent parts of Croatia were of different composition.

4 Conclusion

The numerical taxonomic procedures used in this study to selectthe most efficient chromatographic systems for TLC separationof the components of propolis enabled rational classificationand selection of mobile phases. Analysis of propolis samplesusing the most suitable mobile phases resulted in good separa-tion of known and unknown components. This study confirmedour expectation that TLC is a suitable analytical method forrapid screening of pharmacologically active substances(flavonoids and phenolic acids) in propolis extracts. In furtherwork we will use these results to investigate two-dimensionalTLC of propolis samples.

References

[1] E.L. Ghisalberti, Bee World 60 (1979) 59–84.

[2] M.C. Marcucci, Apidologie 26 (1995) 83–99.

[3] W. Greenway, T. Scaysbrook, and F.R. Whatley, Bee World 71

(1990) 107–118.

[4] V.S. Bankova, S.L. de Castro, and M.C. Marucci, Apidologie 31

(2000) 3–15.

[5] A. Kujumgiev, I. Tsvetkova, Y. Serkedjieva, V. Bankova, R. Chris-tov, and S. Popov, J. Ethnopharmacol. 64 (1999) 235–240.

[6] K. Bosio, C. Avanzini, A. D’Avolio, O. Ozino, and D. Savoia, Lett.Appl. Microbiol. 31 (2000) 174–177.

[7] S. Castaldo and F. Capasso, Fitoterapia 73 (2002) S1–S6.

[8] A. Russo, R. Longo, and A. Vanella, Fitoterapia 73 (2002)S21–S29.

[9] B.H. Havsteen, Pharmacol. Ther. 96 (2002) 67–202.

[10] E.M. Schneidewind, H. Kala, B. Linzer, and J. Metzner, Pharmazie30(12) (1975) 803.

[11] M. Vanhaelen and R. Vanhaelen-Fastré, J. Pharm. Belg. 34(5)(1979) 253–259.

[12] J. Metzner, H. Bekemeier, M. Paintz, and R. Schneidewind, Phar-mazie 34 (1979) 97–102.

[13] P.G. Pietta, C. Gardana, and A. M. Pietta, Fitoterapia 73 Suppl. 1(2002) S7–S20.

[14] S. Nyiredy, J. Chromatogr. Sci. 40 (2002) 553–563.

[15] A. Pelander, J. Summanen, T. Yrjönen, H. Haario, I. Ojanperä,and H. Vuorela, J. Planar Chromatogr. 12 (1999) 365–372.

[16] K. Morita, S. Koike, and T. Aishima, J. Planar Chromatogr. 11

(1998) 94–99.

[17] C. Cimpoiu, T. Hodiºan, and H. Naºcu, J. Planar Chromatogr. 10

(1997) 195–199.

[18] P.H.A. Sneath and R.R. Sokal, Numerical Taxonomy, W. H. Free-man, San Francisco, CA, 1973.

[19] M. Mediæ-Šariæ, S. Šariæ, and D. Maysinger, Acta Pharm. Jugosl.39 (1989) 1–16.

[20] Ž. Maleš, M. Mediæ-Šariæ, and D. Kuštrak, Acta Pharm. 44 (1994)183–191.

[21] A.C. Moffat, K.W. Smalldon, and C. Brown, J. Chromatogr. 90

(1974) 1.

[22] M. Mediæ-Šariæ, Ž. Maleš, G. Staniæ, and S. Šariæ, Croat. Chem.Acta 69 (1996) 1265–1274.

[23] Y.K. Park and M. Ikegaki, Biosci. Biotechnol. Biochem. 62 (1998)2230–2232.

[24] Ž. Maleš and M. Mediæ-Šariæ, J. Planar. Chromatogr. 12 (1999)345–349.

[25] T.G. Gage, C.D. Douglas, and S.H. Wender, Anal. Chem. 23

(1951) 1582.

Ms received: September 5, 2003Accepted by SN: March 30, 2004



investigation of the flavonoids in croatian propolis by thin-layer chromatography

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