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Journal of Food and Drug Analysis, Vol. 10, No. 3, 2002, Pages 178-182
Estimation of Total Flavonoid Content in Propolis by Two Complementary Colorimetric Methods
CHIA-CHI CHANG1, MING-HUA YANG2*, HWEI-MEI WEN1 AND JIING-CHUAN CHERN1
Department of Food Science, National Pingtung University of Science and Technology (NPUST), 1 Hseuh-Fu Road, Neipu Hsiang, Pingtung Hsien 912, Taiwan, Republic of China
Department of Food and Nutrition, Hung-Kuang Institute of Technology,
34 Chung-Chi Road, Shalu, Taichung Hsien 433, Taiwan, Republic of China
(Received: January 25, 2002; Accepted: May 16, 2002)
Flavonoids, with various biological activities, are considered as key compounds in propolis. In this study, quantitative determinations of flavonoids in propolis were conducted by two complementary colorimetric methods, aluminum chloride method and 2,4-dinitrophenylhydrazine method. Results suggested that the sum of flavonoid contents determined by the above two individual methods may represent the real content of total flavonoids. In this work, six raw propolis samples were investigated and the total contents of flavonoids ranged from 10.38 ± 0.14% to 24.91 ± 0.53%. As for the 12 commercial propolis products examined, the levels of total flavonoids in tinctures were all below 7% and those in powdery products varied from 2.97 ± 0.05% to 22.73 ± 0.72%. Key words: Propolis, flavonoids, flavones, flavonols, flavanones, flavanonols, quantitative determination, colorimetric method, aluminum chloride reaction, 2,4-dinitrophenylhydrazine reaction
Propolis, the material used by bees to protect their hives, is a glue-like substance composed of plant resins, bee waxes and pollens. Since various biological activities of propolis such as antibacterial, antiviral, anti-inflammatory and anaesthetic properties were found(1-3), it is used as a health food. The chemical composition of propolis is quite complicated and over 150 components have been identified(2,4). Among these compounds flavonoids were suggested to be responsible for the biological activities (5,6) . Therefore, the content of flavonoids is considered as an important index for evaluating propolis quality. The analysis of flavonoids in propolis has been done by colorimetric methods(7-10), thin layer chromatography(9,11), gas chromatography(12,13), gas chromatography-mass spectrometry(14-16) and high performance liquid chromatography(7,15,17,18). Although chromatographic techniques in combination with absorption spectrum analysis and mass spectrometry provide definitive information for identification and quantification of flavonoids in propolis, these methods usually require advanced instruments, various authentic standards and are time-consuming. On the other hand, colorimetric methods targeting flavonoids of similar structures are convenient and appropriate for routine analyses. However, none of the colorimetric methods can detect all kinds of flavonoids. For instance, within four major groups of flavonoids in propolis, only flavones and flavonols were found to complex stably with aluminum chloride(19), while
* Author for correspondence. Tel: 886-4-26318652 ext. 5032 Fax: 886-4-26319176; Email: email@example.com
flavanones and flavanonols reacted better with 2,4-dinitrophenylhydrazine(8). Considering the need of criteria for both consumers and government agencies to evaluate numerous commercial propolis products, we propose to determine the content of total flavonoids in propolis complementarily by aluminum chloride and 2,4-dinitrophenylhydrazine reactions. In this work, six raw propolis samples and 12 commercial products were investigated to compare the differences of results from each colorimetric analysis.
MATERIALS AND METHODS
I. Materials Six raw propolis samples named Taiwan-1, Taiwan-2, Taiwan-3, Brazil-1, China-1 and China-2 were provided by Miaoli District Agricultural Improvement Station (Miaoli, Taiwan). Twelve commercial propolis products were obtained from local retailers and the description is shown in Table 1. All samples were stored at room temperature until analysis. Fifteen flavonoid standards including chrysin (catalog number C-3018), apigenin (A-3145), luteolin (L-9283), rutin (R-5143), morin (M-4008), quercetin (Q-0125), myricetin (M-6760), kaempferol (K-0133), quercitrin (Q3001), galangin (06829HS), naringin (N-1376), (±)-naringenin (N-5893), hesperetin (H-4125), daidzein (D-7802) and genistein (G-6776) were purchased from Sigma-Aldrich (St. Louis, MO) with product numbers in parentheses. The systematic names of 15 flavonoid standards are listed in Table 2. All reagents used were of analytical grade. 2,4-
7-dihydroxyflavone 4’. the supernatant was combined with previous supernatant and adjusted to 25 mL with 80% ethanol. No.5. III. Ten milligrams of quercetin was dissolved in 80% ethanol and then diluted to 25.1 to 1 g (accurately weighed to 0.4’. the absorbance of the reaction mixture was measured at 415 nm with a Shimadzu UV-160A spectrophotometer (Kyoto. the wavelength scans of the complexes of 15 standards with aluminum chloride showed that . II.7-pentahydroxyflavone-3-Lrhamnopyranoside 3. Liquid propolis products were directly diluted with 80% ethanol to the concen- RESULTS AND DISCUSSION I. Japan).4-dinitrophenylhydrazine reagent and 2 mL of methanol at 50˚C for 50 min. 0.5 mL) were separately mixed with 1. Vol. 1000 and 2000 µg/mL.5. In addition.4-Dinitrophenylhydrazine Colorimetric Method The current method was modified from the procedure described by Nagy and Grancai(8).7-trihydroxyflavanone 3’. One milliliter of each of the diluted standard solutions was separately reacted with 2 mL of 1% 2. In preliminary experiments. After cooling to room temperature.1 mL of 10% aluminum chloride.179 Journal of Food and Drug Analysis.5.0001 g) of raw propolis was extracted with 25 mL of 95% ethanol under 200 rpm shaking for 24 hr. Aluminum Chloride Colorimetric Method The principle of aluminum chloride colorimetric method is that aluminum chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols.7-dihydroxyisoflavone 4’.5 mL of 95% ethanol.000 x g for 10 min to remove the precipitate. the reaction mixture was mixed with 5 mL of 1% potassium hydroxide in 70% methanol and incubated at room temperature for 2 min.7-pentahydroxyflavone-3-rutinoside 2’. 0.0001 g) was first dissolved with 10 mL of 80% ethanol. The supernatant was collected and adjusted to 25 mL.5.7-hexahydroxyflavone 3. After centrifugation at 1. 3. 10.5.4’.5’.5. Then. (±)-Naringenin was used as the reference standard. the supernatant was collected and the precipitate was then extracted with 5 mL of 80% ethanol twice. the filtrate was adjusted to 25 mL with 80% ethanol and stored in an amber bottle. After filtration.1 mL of 1M potassium acetate and 2. Extraction of Flavonoids from Propolis About 1 g (accurately weighed to 0.5. 0.5. The absorbance of the supernatant was measured at 495 nm.3.7-trihydroxyflavone 4’. abbreviated 2.7-trihydroxy-4’-methoxyflavanone 4’.3’. mixed with 5 mL of methanol and centrifuged at 1. Quercetin was used to make the calibration curve. The ethanol extracts of propolis and 15 flavonoid standard solutions (1000 ppm) were similarly reacted with 2.4-dinitrophenylhydrazine for determination of flavonoid content as described above. 1 mL of the mixture was taken.3’. Finally. 50 and 100 µg/mL.7-trihydroxyflavanone-7-rhamnoglucoside 4’.8 mL of distilled water. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank.7-tetrahydroxyflavone 3.5. Dinitrophenylhydrazine reagent. Twenty milligrams of (±)-naringenin was dissolved in methanol and then diluted to 500.3’.5. Similarly.7-pentahydroxyflavone 3.4’.7-trihydroxyisoflavone trations appropriate for colorimetric analysis. The description of 12 commercial propolis products used in the present study Sample Form Color Country of Production A powder light brown Brazil B powder light brown Brazil C powder light brown Brazil D powder light brown Brazil E liquid yellowish brown Brazil F liquid dark brown Brazil G liquid dark brown England H liquid dark green Australia I liquid reddish brown New Zealand J liquid light yellowish green Brazil K liquid yellowish brown Brazil L liquid yellowish green Brazil Table 2.5. aluminum chloride forms acid labile complexes with the orthodihydroxyl groups in the A.4’.or B-ring of flavonoids(19).7-pentahydroxyflavone 3. Systematic present study Flavonoids Flavones chrysin apigenin luteolin Flavonols rutin morin quercetin myricetin kaempferol quercitrin galangin Flavanones naringin (±)-naringenin hesperetin Isoflavones daidzein genistein names of the 15 flavonoid standards used in the Systematic name 5.5.4-dinitrophenylhydrazine in 2 mL of 96% sulfuric acid and then diluting to 100 mL with methanol. was prepared by dissolving 1 g of 2. Estimation of Total Flavonoid Content (I) Aluminum Chloride Colorimetric Method The aluminum chloride colorimetric method was modified from the procedure reported by Woisky and Salatino(10).4-D.000 x g for 10 min.7 -trihydroxyflavone 3’.5 mL of ethanol extracts or 15 flavonoid standard solutions (100 ppm) were reacted with aluminum chloride for determination of flavonoid content as described above.4’. 2002 Table 1. For commercial propolis products in solid form.4’. The diluted standard solutions (0. After incubation at room temperature for 30 min.4’.5.7 -tetrahydroxyflavone 3. (II) 2.3’. 0.
kaempferol and quercetin were all widely found in propolis(7). (±)-naringenin. we chose (±)-naringenin with one more hydroxyl group at the 4’ position to make the calibration curve. The absorbance readings of flavonoid standards reacted with 2. we also found that the extra complexes formed between aluminum chloride and the adjacent hydroxyl groups in B ring not only influenced the absorption spectra but also increased the absorbanc at 415 nm.001 0. 2.000 0.291 ± 0. the absorbance readings of the complexes formed by flavone and flavonol compounds were much higher than those formed by flavanones and isoflavones as shown in Table 3. factors such as the absorption maxima and absorbance at 415 nm were considered.000 ± 0.000 ± 0.000 Quercitrin 0. However.007 0. therefore.000 ± 0.001 Flavonols Rutin 0.e. 2002 the complexes formed by flavonols with C-3 and C-5 hydroxyl groups. flavonols and isoflavones with the C2-C3 double bond could not react with 2. (±)naringenin and hesperetin.000 0.016 ± 0.000 Kaempferol 0.427 ± 0. Similarly. to react with aluminum chloride and 2. Therefore.000 Genistein 0. Therefore.000 0.000 ± 0. Therefore. kaempferol.005 (±)Naringenin 0.004 0.002 Hesperetin 0. i. Generally speaking. while rutin. Among the 15 flavonoid standards investigated. Comparing the data of chrysin and apigenin with those of other flavones and flavonols shown in Table 3. Pinocembrin was the most abundant flavanone found in various propolis samples(7) and used as the reference compound in quantitative determination of flavonoid content of propolis by Nagy and Grancai(8). To select a standard for calibration.4-dinitrophenylhydrazine reaction.000 0. we found that flavones. 385 and 415 nm. II.004 0.000 0. quercetin.4-dinitrophenylhydrazine colorimetric methods Flavonoidsa AlCl3 2.4-dinitrophenylhydrazine separately. The coefficients of determination (R2) obtained were all above 0. naringin. the wavelength 415 nm was chosen for absorbance measurement. quercetin. The absorbance of 15 flavonoid standards determined by aluminum chloride and 2. as well as those with extra ortho-dihydroxyl groups.005 0.4- dinitrophenylhydrazones. Results showed that these isoflavone compounds did not form hydrazone product with 2.037 ± 0. showed maximum absorbance at 495 nm (data not shown). instead of pinocembrin.000 ± 0. daidzein and genistein. while the hydrazones of all flavanone standards. quercitrin and galangin belong to flavonols.000 Quercetin 0. Quercetin which gave the second high absorbance reading among the 15 standards (Table 3) was used as the reference compound by Woisky and Salatino(10). had maximum absorbance at 415-440 nm (data not shown).024 ± 0. myricetin. morin and kaempferol.4-Dinitrophenylhydrazine Colorimetric Method The principle of this method is that 2. we observed the λmax of the complexes of chrysin and apigenin having only the C-5 hydroxyl group and that of luteolin having the C-5. we used two isoflavones. III. such as rutin. such as galangin.113 ± 0. 3. . However. Interestingly. As expected.4-D Abs (415 nm)b Abs (495 nm)b Flavones Chrysin 0. the absorbance at 415 nm was too low to make meaningful contribution to total absorbance.000 ± 0.000 a: The concentrations of each authentic standard are 100 ppm in aluminum chloride reaction and 1000ppm in 2.000 Flavanones Naringin 0.99.240 ± 0.000 Morin 0.016 ± 0.000 ± 0.4-dinitrophenylhydrazine reaction. we could not obtain commercial pinocembrin. there was no commercial flavanonol available.001 0. 10.000 ± 0. The coefficients of determination (R2) obtained were all higher than 0.000 ± 0.000 Myricetin 0. although the presence of C-5 hydroxyl group in flavanones such as naringin. hesperetin and genistein helped these compounds complex with aluminum chloride.005 0.191 ± 0.4-dinitrophenylhydrazine reacts with ketones and aldehydes to form 2. the absorbance of apigenin after reaction with aluminum chloride was too low to be measured at concentrations lower than 100 ppm.358 ± 0. the aluminum chloride complexes of compounds with more functional groups absorbed stronger at 415 nm and showed the absorption maximum at longer wavelength (data not shown). Another flavone compound investigated. the λ max of the complexes formed by chrysin and apigenin which have only the C-5 hydroxyl and C-4 keto groups were at 395 and 385 nm.000 ± 0. which has the C-5 hydroxyl group and the ortho-dihydroxyl groups in B ring formed a complex that showed a strong absorption at 415 nm.180 Journal of Food and Drug Analysis.4-dinitrophenylhydrazine were shown in Table 3. To investigate more flavanoid compounds. C-3’ and C-4’ hydroxyl groups showed maximum absorbance at 395.471 ± 0. No. except for chrysin and apigenin.999. For instance. quercitrin and myricetin.000 Apigenin 0.032 ± 0.023 ± 0.000 ± 0.000 Luteolin 0. luteolin.4-dinitrophenylhydrazine.451 ± 0.016 ± 0. chrysin. Flavonoid Contents of Propolis Samples Flavonoid contents of six raw propolis samples deterTable 3.006 0. On the other hand.013 Isoflavones Daidzein 0.000 Galangin 0.4-dinitrophenylhydrazine. Vol.391 ± 0.167 ± 0. while only slight absorbance was obtained when genistein with the C-3 hydroxyl group complexed with aluminum chloride (Table 3). apigenin and luteolin belong to flavones. we used quercetin solutions at concentrations ranging from 0 to 100 ppm to build up the calibration curve.000 ± 0. b: Results were shown as mean ± SD (n=3).002 0.004 0. morin. respectively. Although apigenin. In compromise. the wavelength 495 nm was selected for all measurements in the 2.258 ± 0. respectively. in the absence of acid.
67 ± 0.05 17.03 4.23 D 1.43 ± 0.02 5. F and G.82 ± 0. The flavonoid contents of six raw propolis samples determined by aluminum chloride and 2.03 2.4-dinitrophenylhydrazine colorimetric method (Table 4). pinocembrin. L.02 18.34 ± 0.4-dinitrophenylhydrazine.26 ± 0.69 22. 10.06 G 1. there was tremendous variation in total flavonoid contents of commercial propolis products (Table 5).05 0.86 ± 0. modern technology of mass spectrometry facilitates the identification of suspicious peaks. while the three samples obtained from Taiwan contained higher levels of total flavonoids (20. 1995. Results showed that. Therefore.02 17.02% to 21. 2002 mined by aluminum chloride colorimetric method were generally lower than those determined by 2.19 Taiwan-2 3.29 ± 0.4-dinitrophenylhydrazine colorimetric methods Sample Flavonoid content (%)a AlCl3b 2.47 ± 0.76 ± 0. To avoid bias. the contents of flavanones were only 6-40% of total flavonoids in raw propolis and 4-9% in commercial propolis samples. . 3.67 ± 0.07 J 0.00 4. while another colorimetric method utilizing 2.73 ± 0.45 ± 0.29 ± 0.00 1.02 1. REFERENCES 1. Flavonoid contents (%) of tinctures = flavonoid (µg/mL) × dilution factor × 10-6 (g/µg) × 100. chrysin 7-methyl ether and galangin 7-methyl ether in eight tincture samples.35 ± 0.91 ± 0.17 21. flavonoid contents of commercial propolis samples.12 ± 0. Burdock. Uruguay and China by HPLC. A.34 %.95 ± 0.50 ± 0.01 1.02 5. Similarly.(16) identified 10 major peaks in the HPLC chromatogram of a New Zealand propolis tincture by comparing the absorption spectra and using GC-mass spectrometry.79 ± 0. HPLC were greatly influenced by the selected authentic standards. the convenient colorimetric method utilizing aluminum chloride reaction to determine flavonoid contents was proved to be specific only for flavones and flavonols. They found that the total amounts of flavanones including pinobanksin. Propolis: a review. No.37 ± 0. M.02 1.4-dinitrophenylhydrazine method in addition to aluminum chloride method.02% to 7.82 ± 0.02 10. indicating that the quality of commercial products does require verification. biological properties and therapeutic activity.04 5.91%) (Table 4).01 K 1. CONCLUSIONS With various biological activities.30 ± 0.03 21.39 20.4dinitrophenylhydrazine reaction was specific for flavanones.00 0. Our results were consistent with their finding and indicated the importance of the quantitative determination by 2.16 a: All results were presented as mean ± SD (n=3). the amounts of flavonoids determined by Table 4.05 12.26 ± 0.48 ± 0. we conducted the quantitative determination of flavonoid contents in various propolis samples by 2.13 ± 0.10 L 1.05 2. Marcucci. we suggest both analyses be conducted so that the sum of the results may better represent the real content of total flavonoids.47 ± 0. Table 5. However.19 10.12 7.4-dinitrophenylhydrazine colorimetric methods Sample Flavonoid content (%)a AlCl3b 2.98 ± 0. flavonols and isoflavones investigated formed complexes only with aluminum chloride.00 ± 0.22 ± 0. Markham et al. G. while the latters ranged from 7. galangin. Since the flavones. the identification of flavonoid peaks in chromatograms may be incomplete.22 to 3.74 ± 0.181 Journal of Food and Drug Analysis.10 H 1. Ghisalberti.30 ± 0.06 10.73 ± 0.4-dinitrophenylhydrazine reaction (Table 5).72 C 3.14 China-1 5. The flavonoid contents of 12 commercial propolis products determined by aluminum chloride and 2.01 21.02 ± 0. According to Serra Bonvehi et al.60 –24.96 ± 0.24 a: Results were presented as mean ± SD (n=3). The formers ranged from 2. c: Flavonoid content (%) = naringenin equivalent (µg/mL) × total volume of ethanol extract (mL) ÷ sample weight (g) × dilution factor × 10-6 (g/µg) × 100. The results seem to be opposite to our general knowledge that the major flavonoid species of propolis from the temperate zone are flavones and flavonols.4-Dc Total Taiwan-1 2.82 ± 0. b: Flavonoid content (%) = quercetin equivalent (µg/mL) × total volume of ethanol extract (mL) ÷ sample weight (g) × dilution factor × 10-6 (g/µg) × 100. were found to be lower by aluminum chloride reaction than those obtained by 2. 1979.14 6.16 B 1.16 ± 0. However.47 ± 0. Food Chem.65 ± 0. except for tinctures E.16 4. Sometimes.19 %.01 2.12 ± 0.73 ± 0.84 ± 0. pinobanksin 3-acetate and pinocembrin 7-methyl ether were 2. Flavonoid content (%) of solid samples = flavonoid (µg/mL) × total volume of ethanol extract (mL) ÷ sample weight (g) × dilution factor × 10-6 (g/µg) × 100.4-Dc Total A 0.38 ± 0. c: Levels calculated as naringenin equivalents.02 ± 0. 1998.03 ± 0.07 China-2 7. Vol. among the six raw propolis samples.4-dinitrophenylhydrazine reaction.03 I 1. who analyzed 15 propolis samples from Brazil. Brazil propolis contained the lowest level of total flavonoids (10.97 ± 0. C. while flavanones strongly reacted only with 2.08 3. b: Levels calculated as quercetin equivalents.84 ± 0.60 ± 0. E. Bee World 60: 59-84. In fact.12 ± 0. 2.86 ± 0. (7). limited by the availability of authentic standards.05 E 1.49 ± 0.38 ± 0. 3. Toxicol. Review of the biological properties and toxicity of bee propolis (propolis).14%).07 ± 0.46 Brazil-1 3.12 ± 0. Fortunately. Apidologie 26: 83-99.58 ± 0. Propolis: chemical composition.00 0.02 F 1.17 6.95 ± 0.35 Taiwan-3 2.84 ± 0.02 1.14 times more than those of flavones and flavonols including chrysin.06 0.07 17.81 ± 0.34 24. the contents determined by the two methods were added up to evaluate the total content of flavonoids. flavonoids are as key candidate compounds for evaluating the quality of propolis products. 36: 347-363.82 ± 0.55 ± 0.
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