A Validated Spectrophotometric Method for Quantification of Prenylated Flavanones in Pacific Propolis from Taiwan

Transcription

1 Research Article Received: 19 February 2009; Revised: 1 September 2009; Accepted: 14 September 2009 Published online in Wiley Interscience: 25 ctober 2009 ( DI /pca.1176 A Validated Spectrophotometric Method for Quantification of Prenylated Flavanones in Pacific Propolis from Taiwan Milena Popova, a Chia-Nan Chen, a Pen-Yuan Chen, b Chung-Yang Huang b and Vassya Bankova a * ABSTRACT: Introduction Because of its chemical diversity, the only way to standardise propolis is to specify multiple standards for different propolis types according to the corresponding chemical profile. So far, this has been done only for European propolis. bjective To develop a rapid low-cost spectrophotometric procedure for quantification of bioactive prenylated flavanones in Taiwanese propolis. Methodology The proposed method quantifies the total flavanones on the basis of their absorption as coloured phenylhydrazones formed by interaction with 2,4-dinitrophenylhydrazine. The procedure was validated through model mixture of compounds representing the composition of Taiwanese propolis according to previous studies. The major flavanones of the propolis samples (propolins C, D, F and G) were quantified by HPLC. Antiradical activity against DPPH was also measured. The DNP (dinitrophenylhydrazine) spectrophotometric method is applied for the first time for quantification of prenylated flavanones. Results Spectophotometric procedure applicable to new type propolis (Macaranga type) was developed with recovery between 105 and 110% at the concentration range of mg/ml. Six propolis samples were analysed by spectrophotometry using the procedure developed and validated, and by HPLC as the results demonstrated satisfactory agreement. Neither the spectrophotometric data nor the values measured by HPLC showed significant correlation with the antiradical activity against DPPH. Conclusion The proposed spectrophotometric procedure is useful for routine analyses of Macaranga-type propolis, because of its simplicity, repeatability and acceptable accuracy. Its application to a number of commercial samples could be used as a basis for standardisation and quality control of Pacific propolis. Copyright 2009 John Wiley & Sons, Ltd. Keywords: Taiwanese propolis; prenylated flavanones; spectrophotometric quantification; validation 186 Introduction Propolis (bee glue) is a resinous material, collected by bees from plants and used in the hive as sticky building material and as major chemical defence against microorganisms. Because of its diverse biological activity antimicrobial, antiviral, antiinflammatory, antioxidant, antitumor, etc. (Banskota et al., 2000; Bankova, 2005) propolis has been used by humans for centuries and it is a popular remedy in the folk medicine of many nations, and a raw material for numerous over-the-counter preparations (Zhou et al., 2008). Its chemical composition depends strongly on the phytogeographic characteristics of the site of collection, because in different habitats bees often choose different plant species as propolis sources. As a result, the chemical composition of propolis is highly variable and this makes it a valuable source of diverse biologically active natural compounds of different structural types. However, this feature is a significant problem concerning propolis standardisation and quality control based on quantification of the active substances. Hence, it is impossible to formulate a universal standard for propolis, but it is possible to specify multiple standards for different propolis types according to their plant source and the corresponding chemical profile. Until now, this task has been implemented only for poplar-type propolis (Popova et al., 2007), which is the predominant chemical propolis type in the temperate zones in Europe, Asia and the Americas, even in New Zealand (Greenaway et al., 1987; Garcia-Viguera et al., 1993; Markham et al., 1996; Bankova et al., 2002; Usia et al., 2002). Tropical propolis, on the other hand, was * Correspondence to: V. Bankova, Institute of rganic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str. Bl. 9, 1113 Sofia, Bulgaria. bankova@orgchm.bas.bg a Institute of rganic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str. Bl. 9, 1113 Sofia, Bulgaria b NatureWise Biotech & Medicals Corporation, Taipei, Taiwan Copyright 2009 John Wiley & Sons, Ltd. Phytochem. Anal. 2010, 21,

2 Quantification of Prenylated Flavanones in Pacific Propolis found to be much more diverse with respect to plant origin and chemical composition (Bankova et al., 2000). Recently, several reports have been published on the chemical composition and biological activity of propolis from Taiwan and kinawa (Chen et al., 2003; Kumazawa et al., 2004, 2007; Huang et al., 2007). They show that this propolis has different chemical composition and hence it belongs to another, as yet unknown, chemical type. Its main bioactive constituents are prenylated flavanones (propolins) originating from the resinous surface material on the fruits of the tropical tree Macaranga tanarius L. (Kumazawa et al., 2008). It is important to note that prenylated flavanones are known to possess antibacterial, antioxidant and antitumour activities (Shirataki et al., 2001; Huang et al., 2007; Kumazawa et al., 2007; Weng et al., 2007). This propolis could be regarded as a new type, Pacific (Macaranga) propolis. Because of the growing commercial interest in this propolis and in its remarkable biological activity, a reliable procedure for the quantification of its bioactive constituents is needed for the purposes of standardisation and quality control. The quantification of propolins in the Pacific propolis that are its antibacterial, antioxidant and cytotoxic principles has been performed by HPLC (Kumazawa et al., 2007; Chen et al., 2008). However, the HPLC method is limited to the quantitative determination of the major constituents. Moreover, recent data demonstrate that the quantification of propolis active principles as groups of compounds with close chemical structures by spectrophotometry correlates better with the biological activity, especially with antimicrobial and antioxidant action, than the quantification of individual constituents. In this article the development and validation of a rapid, easy-to-perform spectrophotometric method for measuring total flavanones in Pacific propolis is reported. The DNP (dinitrophenylhydrazine) spectrophotometric method is applied for the first time for quantification of prenylated flavanones. Experimental Propolis collection Six samples of Taiwanese propolis (TP) were collected from six different regions of Taiwan: 1, Taichung (Taiping); 2, Taichung (Wufeng); 3, Changhua; 4, Chiayi; 5, Tainan; and 6, Ping Tung. All samples were collected from May to July of 2008, using propolis collectors. The propolis sample from each location was gathered and kept at 4 C until processed. The colour of all samples was green. The propolis (1 g) was extracted with 95% ethyl alcohol (50 ml), sonicated for 3 h, and left to stand for 72 h at room temperature. The extracts were filtered through a MILLEX-GS 0.22 μm filter (Millipore, Malsheim, France) to remove impurities. The extraction of each sample was carried out in triplicate. From each of the three parallel extracts, 2 ml were evaporated in vacuo at 45 C until constant weight of the dry extract. The yield of the dry extract was determined and calculated as a percentage of the raw propolis weight (Table 1). Chemicals and reagents HPLC-grade methanol was purchased from Carlo Erba, Italy. Potassium hydroxide and 2,4-dinitrophenylhydrazine (DNP), both of analyticalreagent grade, were purchased from Merck, Darmstadt, Germany. Propolins C (1), D (2), F (3) and G (4) were previously isolated, purified and characterised as described in Chen et al. (2003) and Huang et al. (2007), and their purity was confirmed by HPLC. All other reagents were also of analytical grade. TLC confirmation of identity of Macaranga propolis TLC (thin-layer chromatography) test for confirmation of propolis origin by using of standard subtances was performed on silica gel (Alufolien Kieselgel Merck F 254 ) with mobile phase petroleum ether:ethyl acetate 7 : 3. For visualisation of the spots, UV light (366 nm) and spraying with vanillin sulfuric acid in methanol (5 : 95 w/v vanillin:methanol solution, freshly mixed with a 5 : 95 v/v sulfuric acid : methanol solution) and further heating at 100 C were used. As standard substances the propolins C, D F and G were used: propolin C (R f 0.48; colour at 366 nm, yellow; colour after spaying and heating, greenish-brown), propolin D (R f 0.56; colour at 366 nm, yellow; colour after spaying and heating, orangebrown), propolin F (R f 0.71; colour at 366 nm, yellow; colour after spraying and heating, orange-brown) and propolin G (R f 0.80; colour at 366 nm, yellow; colour after spraying and heating, orange-brown). Spectrophotometric procedure Instrumentation. Spectrophotometric measurements were performed on a Helios Gamma spectrophotometer (Thermo Electron Corporation, Whaltham, Massachusetts, USA) equipped with 1 cm quartz cells. Controls. Controls were prepared by replacing the respective sample with equivalent aliquot of 95% ethanol and were carried out through all stages of the procedure. Each assay was carried out in triplicate. Stock and working solutions. Stock standard solution of standard mixture of propolin C (1) and propolin D (2) in weight ratio 4 : 1 and total concentration of mg/ml was prepared by dissolving appropriate amounts of the compounds in methanol in 10 ml volumetric flask. A series of working standard solutions was prepared by appropriate dilution of the stock standard solution with methanol to give a concentration range of mg/ml of the standard mixture. Reference mixture. A stock reference mixture representing qualitatively and quantitatively the major constituents of Taiwanese propolis was prepared by dissolving propolin C (7.74 mg), propolin D (4.77 mg), propolin Table 1. Yield of ethanol dry extract, total flavanones and wax content of Taiwanese raw propolis samples Sample (origin) Dry extract yield (%) Total flavanones (% of raw propolis) Wax content (%) Taichung (Taiping) 77.5 ± ± ± 2 Taichung (Wufeng) 80.0 ± ± ± 0.9 Changhua 83.2 ± ± ± 0.3 Chiayi 68.5 ± ± ± 0.2 Tainan 84.0 ± ± ± 0.6 Ping Tung 71.0 ± ± ± Phytochem. Anal. 2010, 21, Copyright 2009 John Wiley & Sons, Ltd.

3 M. Popova et al. F (2.05 mg) and propolin G (3.35 mg) in 10 ml of methanol. The reference mixtures were used to validate the spectrophotometric method for determination of propolins tested in this work. Sample preparation. The extract was prepared as described above. After filtration it was diluted to 100 ml with 95% ethanol in a volumetric flask. The solution was then analysed to determine the total flavanone content. Concentration effect graphs were built and EC 50 values determined. EC 50 value is the effective concentration that could scavenge 50% of the DPPH radicals. Statistics. For comparison of mean values, ANVA and Student s t-test were used. For correlation analysis, Pearson correlation coefficients were applied. 188 Total flavanone content. The method described by Nagy and Grancai (1996) was used, based on the interaction of flavanones with 2,4-dinitrophenylhydrazine (DNP) in acidic media (sulfuric acid) to form coloured phenyl hydrazones. ne millilitre of test solution and 2 ml of DNP solution (1 g DNP in 2 ml 96% sulfuric acid, diluted to 100 ml with methanol in a volumetric flask) were heated at 50 C for 50 min in a water bath. After cooling to room temperature, the mixture was diluted to 10 ml with 10% K in methanol (w/v). ne millilitre of the resulting solution was added to 10 ml methanol and was diluted to 50 ml with methanol in a volumetric flask. Absorbance was measured at 486 nm. Each assay was carried out in triplicate. HPLC analysis Sample preparation. Extraction was performed as described above. After filtration the extract was evaporated to dryness under reduced pressure at 45 C to yield a brown gum (kept at 20 C until used), in order to prepare solutions with a concentration of 2 mg/ml. Powders of propolins C (1), D (2), F (3) and G (4) were dissolved in methanol at a concentration of 8 mm. All propolin concentrations were determined using linear calibration curves based on the peak area for each propolin. Each calibration curve contained five concentrations of propolins: 2, 1, 0.5, 0.25 and mm. sthol was used as an internal standard. Analytical conditions. The propolin profile in the TP extracts was analysed with an RP HPLC-UV. The conditions were as follows: RP column, Thermo (C 18, mm i.d., particle size, 5 μm); solvent system, methyl alcohol water 80 : 20 (isocratic elution); flow-rate, 1.0 ml/min; UV detection at 280 nm; injection volume, 10 μl; column temperature, 25 ± 2 C. Wax content. The wax content was determined according to the procedures described by Woisky and Salatino (1998). The powdered propolis sample (3 g) was treated with chloroform in a Soxhlet for 6 h. The extract was concentrated to dryness under reduced pressure and 120 ml of hot methanol was added to the residue. The mixture was boiled until there was a clear solution on top and a small oily residue on the bottom of the flask, which solidified upon cooling. The methanolic phase was filtered through filter paper, taking care to avoid the oily residue, and transferred, while still hot, to a weight 150 ml flask. The latter was cooled to 0 C and the content filtered through a weight filter paper. The flask and the residue were washed with 25 ml cold methanol. After drying in the air, the flask and the residue were conditioned in a desiccator. The analysis was performed in duplicate. 2,2-Diphenyl-2-picryhydrazyl (DPPH) free radical scavenging activity. The free radical scavenging capacities of TP extracts at various concentrations (5, 10, 20, 40 and 60 μg/ml) and caffeic acid phenethyl ester (CAPE), as a positive control for various concentrations (2, 4, 6, 8 and 10 μg/ml) were measured with 1.0 ml of 0.15 mm DPPH in methanol. The DPPH radical has a deep violet colour due to its unpaired electron, and its radical scavenging capability can be followed spectrophotometrically by an absorbance loss at 517 nm when the pale yellow nonradical form is produced. The mixtures were vigorously shaken and left to stand at room temperature for 30 min in the dark. Absorbance at 517 nm was measured vs methanol as a blank. The capability of scavenging DPPH radicals was then calculated by the following equation: scavenging effect % = [1 (A 517 of sample/a 517 of control)] 100% Results and Discussion The first step in the quantification of biologically active compounds in propolis is to determine the chemical type of the samples (Bankova and Marcucci, 2000). Based on the present knowledge of the chemical composition of Macaranga tanarius fruit exudates (Kumazawa et al., 2008) and Pacific propolis samples, propolins C (1), D (2), F (3) and G (4) were chosen as taxonomic markers. TLC tests of all six samples confirmed the presence of these specific taxonomic markers as major constituents. Moreover, the observed biological activity of Pacific propolis (antibacterial, antitumour, antioxidant) has been attributed exclusively to prenylated flavanones (Huang et al., 2007; Kumazawa et al., 2007; Weng et al., 2007). For this reason the measurement of the total flavanones will be a specific and suitable procedure for the evaluation of the amount of bioactive constituents in the samples. For quantification of flavanones the spectrophotometric method by Das Deutsche Arzneibuch 9 (1986), as modified by Nagy and Grancai (1996) for propolis, was used. At first pinocembrin was used as a calibration standard, as for poplar propolis (Popova et al., 2004), but the recovery was only 16%. bviously, the prenyl side chain influenced the reaction of propolins with DNP. To be as close as possible to the real structure of the compounds measured, the use of a calibration standard of propolin C (1), one of the main components of Taiwanese propolis, was decided. That led to a recovery of 130%. With propolin D (2) as calibration standard, 56% recovery was obtained. Considering these results, a mixture of two propolins appeared to be a useful alternative. Preliminary experiments showed that the mixture propolin C (1) propolin D (2) in proportion 4 : 1 provided acceptable recovery, so this mixture was used as the calibration standard. Linearity of the spectrophotometric method A calibration graph was constructed at five concentration levels in the corresponding range. Three independent determinations were performed at each concentration (n = 3). The calibration equation was: y = x (y absorbance, x concentration). Regression analysis was employed to determine the linearity of the calibration graph, R 2 = The calibration graph was linear in the range mg/ml. Precision of the spectrophotometric method Precision was evaluated as repeatability and intermediate precision. For the precision measurements, the reference mixture described in the Experimental was used. The reference mixture contains the four major constituents of Pacific propolis, considering previous results on the composition of this (Macaranga) propolis type (Huang et al., 2007; Chen et al., 2008; Kumazawa et al., 2008). The repeatability was expressed as the relative standard deviation by replicate (n = 6) measurements of the reference Copyright 2009 John Wiley & Sons, Ltd. Phytochem. Anal. 2010, 21,

4 Quantification of Prenylated Flavanones in Pacific Propolis mixture. The results are presented in Table 2. The obtained results were considered satisfactory since the RSD did not exceed 6.3%. The intermediate (inter-batch) precision of the method was determined on the bases of the reference mixture with 12 solutions, six prepared on day I and six prepared on day II, by two different analysts. The obtained results were considered satisfactory since the RSD was 8.2%. The analysis of variances demonstrated that there were no statistically significant differences between the inter-batch results obtained by the two analysts on two different days. Accuracy The accuracy of the method was established by measuring the reference mixture at three different concentration levels. The results displayed in Table 2 show that the used spectrophotometric method led to recovery of total propolins between 105 and 110% at the concentration range mg/ml. Analysis of the propolis solution and verification of the results by HPLC The spectrophotometric procedure was applied to six Taiwanese propolis samples from different regions in Taiwan. Independently, the same samples were subjected to HPLC analysis and the four major propolins (C, D, F and G) were quantified (Table 3). The propolis samples displayed similar chemical composition and HPLC chromatogram of one of them (Taichung-Taiping) is presented in Fig. 1. It was assumed that as only four propolins were determined by HPLC, which were the major components in the samples (over 90% of the total UV-absorbing components), the comparison between the two sets of results would be acceptable and informative. The comparison of these results is presented in Table 4. In general, the results obtained by the two methods demonstrated a satisfactory agreement, as proven by Student s t-test and ANVA. In most cases the values for propolins determined by HPLC were lower than those obtained by the spectrophotometric procedure. The reason, presumably, was that some minor components of the corresponding type (e.g. propolins A, B, E, etc.) were not quantified by HPLC. No Pearson correlation between the two sets of results was observed. In view of the two sets of results, obtained by the spectrophotometric method and by HPLC, it was of interest to check the correlation between the composition (quantity) determined by each method and the antiradical activity of the samples against DPPH radicals (Table 4). The statistics showed low correlation coefficients between propolins content and EC 50, for both sets of results, (r 2 = 0.429, for the results obtained by spectrophotometry, and r 2 = for those measured by HPLC). This result suggests that the spectrophotometric method, which is fast and easy to perform, is appropriate for quality control of Macaranga type propolis samples with respect to quantification of its antioxidant compounds. ther constituents of Taiwanese propolis The experiments performed indicated that propolins constituted from 43 to 66% of the weight of the raw propolis. The wax content of the six samples analysed was also determined and the results are presented in Table 1. The wax content was between 5 and 21%, which is similar to values obtained for samples from different regions of the world and it is below 25%, the upper limit for wax content in propolis, proposed by the International Honey Commission (Bogdanov and Gallmann, 2008). A statistically significant negative correlation was observed between the Table 2. Validation of the spectrophotometric quantification of total flavanones in Taiwanese propolis (using a reference mixture): accuracy and immediate precision Added reference mixture (mg/ml) Accuracy Precision (repeatability) Found (mg/ml) Recovery (%) SD a (mg/ml) RSD b (%) N c a Standard deviation. b Relative standard deviation. c Number of replicates. Table 3. Amount of propolins C, D, F and G by HPLC in Taiwanese propolis extract Propolis sample Propolin C (1) Propolin D (2) Propolin F (3) Propolin G (4) Propolins C + D + F + G Taichung (Taiping) ± ± ± ± Taichung (Wufeng) ± ± ± ± Changhua ± ± ± ± Chiayi ± ± ± ± Tainan ± ± ± ± Ping Tung ± ± ± ± Phytochem. Anal. 2010, 21, Copyright 2009 John Wiley & Sons, Ltd.

5 M. Popova et al. H Propolin F (3) H H Propolin G (4) Propolin C (1) H Propolin D (2) Figure 1. HPLC chromatogram of an extract of propolis from Taichung (Taiping). Peak 1, osthol (internal standard); peak 2, propolin D (2); peak 3, propolin F (3); peak 4, propolin C (1); peak 5, propolin G (4). For chromatographic conditions see Experimental. Table 4. Quantification of the main biologically active compounds in Taiwanese propolis and antiradical activity of the extracts Sample Total flavanones in extract (spectrophotometric) (% of dry extract) Propolins C + D + F + G in extract (HPLC) (% of dry extract) EC 50 DPPH (μg/ml, dry extract) Taichung (Taiping) ± 0.2 Taichung (Wufeng) ± 0.5 Changhua ± 0.1 Chiayi ± 0.1 Tainan ± 0.1 Ping Tung ± 0.2 CAPE 5.2 ± wax content and the percentage of total flavanones (r 2 = 0.893, p = 0.004). Until now, propolins and waxes were the only known constituents of Pacific propolis. GC-MS analysis of the propolis extracts after silylation (data not shown) revealed that the prenylated flavanones were the only detected phenolic compounds in the samples analysed. The other types of compounds present were sugars: mono and disaccharides (common for propolis samples of any plant origin), and some triterpenes. That is why, despite some drawbacks, the proposed spectrophotometric procedure seems to be very useful for routine analyses, because of its simplicity, good repeatability and acceptable accuracy. The application of the validated procedure for analysis of a significant number of commercial samples of Pacific propolis and further study of the correlation between the total propolin concentration and biological activity are needed. This approach will lead to determination of flavanone quantities of Macaranga (Pacific) type propolis for the purposes of its standardisation and Copyright 2009 John Wiley & Sons, Ltd. Phytochem. Anal. 2010, 21,

6 Quantification of Prenylated Flavanones in Pacific Propolis quality control. It is important to note that the procedure could only be applied to Macaranga samples and is not relevant to any other propolis type. Acknowledgements V.B. and M.P. are thankful to the National Science Fund (Bulgaria), for the partial support offered by contract TKX-1609, and for the postdoctoral fellowship of M.P (grant PD-3/2007). This work was also supported by Council of Agriculture Executive Yuan of Taiwan (97AS ST-aA). References Bankova V Recent trends and important developments in propolis research. Evid Based Complem Altern Med 2: Bankova V, Marcucci MC Standardization of propolis: present status and perspectives. Bee World 81: Bankova VS, de Castro SL, Marcucci MC Propolis: recent advances in chemistry and plant origin. Apidologie 31: Bankova V, Popova M, Bogdanov S, Sabatini AG Chemical composition of European propolis: Expected and unexpected results. Z Naturforsch C Biosci 57: Banskota AH; Tezuka Y, Kadota S Recent progress in pharmacological research of propolis. Phytother Res 15: Bogdanov S, Gallmann P Authenticity of honey and other bee products: state of the art. ALP Sci 520: Chen CN, Wu CL, Shy HS, Lin JK Cytotoxic prenylflavanones from Taiwanese propolis. J Nat Prod 66: Chen YW, Wu SW, Ho KK, Lin S-B, Huang CY, Chen CN Characterisation of Taiwanese propolis collected from different locations and seasons. J Sci Food Agric 88: Das Deutsche Arzneibuch Kommentar 3: Garcia-Viguera C, Ferreres F, Tomas-Barberan FA Study of Canadian propolis by GC-MS and HPLC. Z Naturforsch C Biosci 48: Greenaway W, Scaysbrook T, Whatley FR The analysis of bud exudates of Populus euramericana, and of propolis, by gaschromatography mass-spectrometry. Proc R Soc Lond Ser B Biol Sci 232: Huang WJ, Huang CH, Wu CL, Lin JK, Chen YW, Lin CL, Chuang SE, Huang CY, Chen CN Propolin G, a prenylflavanone, isolated from Taiwanese propolis, induces caspase-dependent apoptosis in brain cancer cells. J Agric Food Chem 55: Kumazawa S, Goto H, Hamasaka T, Fukumoto S, Fujimoto T, Nakayama T A new prenylated flavonoid from propolis collected in kinawa, Japan. Biosci Biotechnol Biochem 68: Kumazawa S, Ueda R, Hamasaka T, Fukumoto S, Fujimoto T, Nakayama T Antioxidant prenylated flavonoids from propolis collected in kinawa, Japan. J Agric Food Chem 55: Kumazawa S, Nakamura J, Murase M, Miyagawa M, Ahn MR, Fukumoto S Plant origin of kinawan propolis: honeybee behavior observation and phytochemical analysis. Naturwissenschaft 95: Markham KR, Mitchell KA, Wilkins AL, Daldy JA, Lu YR HPLC and GC-MS identification of the major organic constituents in New Zealand propolis. Phytochemistry 42: Nagy M, Grancai D Colorimetric determination of flavanones in propolis. Pharmazie 51: Popova M, Bankova V, Butovska D, Petkov V, Nikolova-Damyanova B., Sabatini AG, Marcazzan GL, Bogdanov S Validated methods for the quantification of biologically active constituents of poplar-type propolis. Phytochem Anal 15: Popova MP, Bankova VS, Bogdanov S, Tsvetkova I, Naydenski C, Marcazzan GL, Sabatini AG Chemical characteristics of poplar type propolis of different geographic origin. Apidologie 38: Shirataki Y, Motohashi N, Tani S, Sakagami H, Satoh K, Nakashima H, Mahapatra SK, Ganguly K, Dastidar SG, Chakrabarty AN In vitro biological activity of prenylflavanones. Anticancer Res 21: Usia T, Banskota AH, Tezuka Y, Midorikawa K, Matsushige K, Kadota S Constituents of chinese propolis and their antiproliferative activities. J Nat Prod 65: Weng MS, Liao CH, Chen CN, Wu CL, Lin JK Propolin H from taiwanese propolis induces G1 arrest in human lung carcinoma cells. J Agric Food Chem 55: Woisky RG, Salatino A Analysis of propolis: some parameters and procedures for chemical quality control. J Apicult Res 37: Zhou JH, Li Y, Zhao J, Xue XF, Wu LM, Chen F Geographical traceability of propolis by high-performance liquid-chromatography fingerprints. Food Chem 108: Phytochem. Anal. 2010, 21, Copyright 2009 John Wiley & Sons, Ltd.