Antioxidant Properties and Phenolic Composition of Propolis from Diverse

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1 Food Sci. Technol. Res., 19 (2), , 2013 Antioxidant Properties and Phenolic Composition of Propolis from Diverse Geographic Regions in Korea Su Jin Choi 1, Kohsuke Shimomura 2, Shigenori Kumazawa 2 and Mok-Ryeon Ahn 1* 1 Department of Food Science and Nutrition, Dong-A University, 840 Hadan 2-dong, Saha-gu, Busan , Korea 2 Department of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka , Japan Received September 6, 2012; Accepted November 6, 2012 Propolis is a resinous substance collected by honeybees from various plant sources. In this study, we examined the antioxidant properties of propolis from diverse geographic regions of Korea including Jeju Island. Ethanol extracts of propolis (EEP) were prepared and evaluated for their antioxidant properties by β-carotene bleaching, 1,1-diphenyl-2-picrylhydrazyl free radical-scavenging, 2,2 -azinobis-(3- ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization, and ferric reducing/antioxidant power assays. Furthermore, the major constituents in EEP were identified by HPLC analysis with a photodiode array and mass spectrometric detection, and each component was quantitatively analyzed. All propolis samples except that from Hongcheon, Iksan, Aewol and Pyoseon had relatively strong antioxidant activity accompanied by high total polyphenol contents. Propolis with strong antioxidant activity contained large amounts of antioxidative compounds, such as caffeic acid, kaempferol, phenethyl caffeate and galangin. On the other hand, propolis from Jeju Island had weak antioxidant activities and had only a few compounds in propolis from other regions. Keywords: propolis, antioxidant properties, phenolics, PDA, Korea Introduction Propolis, a natural resinous substance with a complex composition, is collected by honeybees from various sprouts, plant exudates, buds and leaves and is modified in their hives. Propolis has been used in the folk medicine since the primordial times of humanity, having acquired popularity Egyptians Arabs, Greeks, and many other civilizations (Abd El Hary and Hegazi, 2002). It has been reported to various biological activities, such as antibacterial, antiviral and antifungal (Kujumgiev et al., 1999), antihepatotoxic (Banskota et al., 2001a), antioxidant (Kumazawa et al., 2004), antitumoral (Russo et al., 2004), anti-diabetic, and antihypertensive (Ahuja and Ahuja, 2011) properties. In recent decades, propolis has gained prevalent as a health drink and also has been used extensively in food and beverages, and is thought to improve human health and to prevent diseases such as inflammation, heart disease, and even cancer (Banskota et al., 2001b). *To whom correspondence should be addressed. mokuren@dau.ac.kr Propolis usually contains a variety of chemical compounds such as polyphenols (flavonoids, phenolic acid and their esters), terpenoids, steroids and amino acids, but its composition varies qualitatively and quantitatively with the geographical region and botanical origin (Marcucci, 1995). Some of the observed biological activities might be attributable to identified chemical compositions, in particular its high polyphenol contents. Thus, propolis samples of the world, Europe, Asia and South America, have different chemical compositions (Kalogeropoulos et al., 2009; Ahn et al., 2007; Laskar et al., 2010; Silva et al., 2011). European propolis, such as Europe, North America, and non-tropic regions of Asia, contains the typical poplar bud phenolics (many flavonoids and phenolic acid esters) (Bankova, 2005; Bankova et al., 2002). Poplar trees, mainly the black poplar Populus nigra, are common only in the temperate zone and they cannot grow in tropical and subtropical regions. Propolis from tropical origins has a different chemical composition from that of poplar type propolis. In contrast, the major components of Brazilian propolis are terpenoids and prenylated derivatives of p-coumaric acids and that the source plant is

2 212 Baccharis dracunculifolia (Marcucci and Bankova, 1999). Due to the differences in their chemical compositions, the biological activities of propolis from different origins are also different. Many methods have been used to evaluate the antioxidant properties of various substances in vitro. The ranking of the antioxidant activity of experimental samples may vary with the analysis methods because the mechanism for each analysis is different. Therefore, one analysis method can not accurately evaluate the total antioxidant activity. The inhibition of β-carotene bleaching in a coupled oxidation with linoleic acid is a well-known methodology used for evaluating the antioxidant activity. β-carotene is discolored easily by the oxidation of linoleic acid, because its double bonds are sensitive to oxidation (Singh et al., 2002). The 1,1-diphenyl- 2-picrylhydrazyl (DPPH) and the 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods measure the ability of antioxidants to scavenge the free radicals, but the free radicals are produced in different way. DPPH is a free radical stable at room temperature and accepts an electron or hydrogen radical to become stable diamagnetic molecule. The ABTS radical cation is engendered by the oxidant of ABTS with potassium persulfate and is reduced in the presence of such a hydrogen-donating antioxidant. The ferric reducing/antioxidant power (FRAP) assay takes advantage of electron-transfer reaction. The principle of this method is based on the reduction of a ferric-tripyridyl-triazine complex to its ferrous, colored form in the presence of antioxidants (Benzie and Strain, 1996). The biological activity and chemical constituent of propolis have been studied extensively in many countries, but only a few reports can be found on data concerning the geographical region of Korean propolis. Previously, we first reported the antioxidant activity and constituents of six propolis samples collected on Korea (Ahn et al., 2004). Although some compositions and functional properties of Korean propolis have been investigated, there is no detailed information of that. In addition, six samples of Korean propolis are not enough to accurately determine and that there are no reports on the antioxidant activity and the detailed quantitative analysis of the phenolic compounds of propolis from Jeju Island. Jeju Island is the southern-most part of the Korea, the region is in the subtropical regions and has moderate climate. Kumazawa et al. (2006) studied that propolis from Jeju Island has constituents not present in that from other regions. Moreover, three compounds were isolated and identified from the propolis collected on Jeju Island; however, the other components in it have remained unknown. In this study, we investigated the in vitro antioxidant activity of the ethanol extracts of propolis (EEP) from all pro- S.J. Choi et al. vincial regions (Gyeonggi, Gangwon, Chungbuk, Chungnam, Jeonbuk, Jeonnam, Gyeongbuk, Gyeongnam and Jeju Island) in Korea and analyzed the individual components in EEP. We performed four different systems: 1) the inhibition of linoleic acid oxidation by β-carotene bleaching, 2) the free radicalscavenging activity on DPPH, 3) the scavenging activity on ABTS radical cation and 4) FRAP assays. Furthermore, we identified and quantitatively analyzed several compounds in EEP by high-performance liquid chromatography (HPLC) analysis with a photodiode array (PDA) and mass spectrometric (MS) detection and quantitatively analyzed each component. Materials and Methods Materials Caffeic acid (a), p-coumaric acid (b), ferulic acid (c), α-tocopherol (VE), butylated hydroxytoluene (BHT), quercetin, ABTS, β-carotene, linoleic acid, DPPH, 2,4,6-tripyridyl-s-triazine (TPTZ) and ascorbic acid were purchased from Sigma (St. Louis, MO, USA). 3,4-Dimethoxycinnamic acid (d), pinobanksin 5-methyl ether (e), pinobanksin (f), cinnamylideneacetic acid (i), pinocembrin (j), benzyl caffeate (k), pinobanksin 3-acetate (l), cinnamyl caffeate (p), pinostrobin (q), and tectochrysin (r) were isolated from the ethanol extract of Uruguayan propolis (Kumazawa et al., 2002). Kaempferol (g), apigenin (h), chrysin (m) and galangin (o) were purchased from Extra Synthese (Genay, France). Phenethyl caffeate (n) were purchased from Bachem (Bubendorf, Switzerland). Aluminium chloride, iron chloride, gallic acid and acetic acid were purchased from Junsei Chemical (Tokyo, Japan). Tween 40 was purchased from Yakuri Pure Chemical (Osaka, Japan). Propolis samples were collected as the crude materials by beekeepers from diverse geographic regions in Korea. Figure 1 shows the collection sites of each sample. The crude propolis materials (100 mg each) were extracted with ethanol (3 ml) at room temperature for 24 h. The ethanol suspension was separated by centrifugation, and the supernatant was concentrated under reduced pressure to give EEP. EEP was stored under dry conditions at 4 until analyzed. Total polyphenol and flavonoid contents Total polyphenol contents in EEP were determined according to the Folin- Ciocalteu colorimetric method (Singleton et al., 1999). EEP solution (0.5 ml) was mixed with 0.5 ml of the 10% Folin Ciocalteu reagent (Kanto Chemicals, Tokyo, Japan) and 0.5 ml of 10% Na 2 CO 3, and the absorbance was measured at 760 nm after 1 h of incubation at room temperature. EEP samples were evaluated at the final concentration of 20 μg/ ml. Total polyphenol contents were expressed as mg/g of gallic acid equivalents. Contents of flavonoid in EEP were determined according

3 Antioxidant Properties and Phenolic Composition of Korean Propolis Fig. 1. Collection sites of Korean propolis. P1, Uijeongbu; P2, Ansan; P3, Hongcheon; P4, Youngwol; P5, Chungju; P6, Cheongju; P7, Cheonan; P8, Daejeon; P9, P10, Iksan; P11, Jangseong; P12, Kwangju; P13, Sangju; P14, Gumi; P15, Changnyeong; P16, Uiryeong; P17, Aewol; P18, Pyoseon. to the method of Woisky and Salatino (1998), with minor modifications. To 0.5 ml of EEP solution was added 0.5 ml of 2% AlCl 3 -ethanol solution. After 1 h at room temperature, the absorbance was measured at 420 nm. EEP samples were evaluated at a final concentration of 20 μg/ml. Total flavonoid contents were calculated as quercetin equivalents (mg/g) from a calibration curve. Antioxidant activity on linoleic acid oxidation This experiment was carried out according to the method of Emmons et al. (1999), with some modifications. β-carotene (3 mg) was dissolved in 30 ml of chloroform, and 3 ml was added to 40 mg of linoleic acid and 400 mg of Tween 40. Chloroform was removed under a stream of nitrogen gas. Then distilled water (100 ml) was added, and the solution was mixed well. Aliquots (3 ml) of the β-carotene/linoleic acid emulsion were mixed with 50 μl of EEP solution and incubated in a water bath at 50. Oxidation of the emulsion was monitored spectrometrically by measuring absorbance at 470 nm over a 60 min period. The control sample contained 50 μl of solvent in place of the extract. The antioxidant activity is expressed as percent inhibition relative to the control after a 60 min incubation using the equation AA = (DR C DR S ) / DR C 100 where AA is the antioxidant activity, DR C is the degradation rate of the control (= ln(a/b)/60), DR S is the degradation rate in the presence of the sample (= ln(a/b)/60), a is the initial absorbance at time 0, and b is the absorbance at 60 min. EEP samples were evaluated at a final concentration of 10 μg/ ml, and BHT and VE at 1 μg/ml were used as the reference samples. Free radical-scavenging activity on DPPH The reaction mixture contained 1.5 ml of ethanol, 0.5 mm DPPH and test samples. After a 1 h incubation at room temperature in the dark, the absorbance was recorded at 517 nm. Control solution contained only ethanol and DPPH. Results were expressed as percentage decrease with respect to control values (Chen and Ho, 1995). EEP samples were evaluated at a final concentration of 20 μg/ml, and BHT and VE at the same concentration were used as the reference samples. Scavenging activity of ABTS radical cation The ABTS radical cation (ABTS + )-scavenging activity was measured according to the method described by Fellegrini et al. (1999), with some modifications. ABTS was dissolved in water to a 7 mm concentration. The ABTS radical cation was produced by reacting the ABTS stock solution with 140 mm potassium persulfate (final concentration) in the dark at room temperature for h to allow the completion of radical generation. This solution was then diluted with ethanol so that its absorbance was adjusted to 0.70 ± 0.02 at 734 nm. To determine the scavenging activity, 990 μl of diluted ABTS + solution were added to 10 μl of EEP solution, and the absorbance was measured at 734 nm 2.5 min after the initial mixing, using ethanol as the blank. The percentage inhibition was calculated by the equation % inhibition = (A C A S ) / A C where A C is the absorbance of the control and A S is the absorbance of the samples. BHT and VE at the 1/10 concentration of EEP samples were used as the reference samples. Ferric reducing/antioxidant power (FRAP) assay The FRAP assay was measured according to the method described by Benzie and Strain (1996). The FRAP reagent contained 2.5 ml of a 10 mm 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mm HCl plus 2.5 ml of 20 mm FeCl 3 6H 2 O and 25 ml of 0.3 M acetate buffer, ph 3.6. Aliquots of 100 μl of test sample were mixed with 3 ml FRAP reagent and the absorbance of the reaction mixture at 593 nm was measured spectrophotometrically after incubation at room temperature for 3 min. The FRAP activity was calculated by the equation FA = (A C / A A ) 100 where FA is the FRAP activity, A C is the absorbance of the samples, A A is the absorbance of the ascorbic acid, 100 is the concentration of the ascorbic acid (100 μg/ml). BHT and VE at the same concentration were used as the reference

4 214 samples. Results were expressed as micrograms per milliliter of ascorbic acid equivalent. HPLC analysis with PDA and MS detection To identify and determine the constituents in EEP, we used HPLC with PDA and MS detection. EEP samples were dissolved in ethanol (10 mg/ml) and filtered with a 0.45 μm filter (German Sciences, Tokyo, Japan). The HPLC-PDA system used was a Jacso system control program HSS-1500 (Tokyo, Japan) equipped with Jasco-Borwin chromatography data station, pump PU-1580, autosampler AS-1559, column oven CO-1565, and PDA system MD-1510 for monitoring at all wavelengths from 195 to 650 nm. For the column, Capcell Pak UG-120 (Shiseido Tokyo, Japan) C18 column ( mm i.d., 3 μm) was used. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was 20 80% B (0 110 min) at a flow rate of 0.65 ml/min. The injection volume for the extract was 10 μl. Another set of extracts dissolved in methanol was injected into the HPLC- ESIMS/MS system to obtain more information about the structure of each compound. A portion of the filtrate (5 μl) was subjected to SI-1 HPLC system (Shiseido, Tokyo, Japan) on a Capcell Pak UG-120 C18 column (250 2 mm i.d., 5 μm). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was 20 80% B (0 110 min) at a flow rate of 0.2 ml/min. The elution of extracts was monitored at 270 nm and then introduced into the ion-trap electrospray S.J. Choi et al. mass spectrometer equipped with ESI (LCQ, Thermo Fisher Scientific K.K., San Jose, CA, USA). The operating parameters were as follows: source voltages, 5 kv; ES capillary voltage, 10 V; capillary temperature, 260. All MS data were acquired in negative ionization. Quantification of each compound in EEP was performed using the HPLC-PDA described above. The stock standard solution of each compound (a r) was prepared as follows: an accurately weighed amount of pure compound (2 5 mg) was placed into a vial and adequate volume of methanol was added. The external calibration curve was generated using five data points covering the concentration ranges. Injections were performed in triplicate for each concentration level. The calibration curve was obtained by plotting the peak area of the compound at each level versus the concentration of the sample. The limits of detection (LOD) and quantification (LOQ) were determined at a signal-to-noise ratio of 3 and 10, respectively. The reproducibility of the determinations was confirmed by analyzing five technical replicates of a standard solution. Results and Discussion Total polyphenol and flavonoid contents of propolis Propolis is generally available as capsules made from ethanol extracts in many countries. We therefore examined the ethanol extracts from propolis samples obtained from diverse geographic regions in Korea as shown in Table 1. All propolis samples, except that from Hongcheon (P3), Aewol (P17) Table 1. Collection sites, province, total polyphenol, and flavonoid contents of EEP. Propolis Collection site Province Total polyphenol a (mg/g of EEP) Flavonoid b (mg/g of EEP) P1 Uijeongbu Gyeonggi ± ± 2.6 P2 Ansan Gyeonggi ± ± 4.2 P3 Hongcheon Gangwon ± ± 4.7 P4 Youngwol Gangwon ± ± 4.2 P5 Chungju Chungbuk ± ± 1.3 P6 Cheongju Chungbuk ± ± 2.9 P7 Cheonan Chungnam ± ± 2.9 P8 Daejeon Chungnam ± ± 3.2 P9 Iksan Jeonbuk ± ± 1.7 P10 Iksan Jeonbuk ± ± 3.2 P11 Jangseong Jeonnam ± ± 0.0 P12 Kwangju Jeonnam ± ± 0.4 P13 Sangju Gyeongbuk ± ± 1.7 P14 Gumi Gyeongbuk ± ± 1.9 P15 Changnyeong Gyeongnam ± ± 2.2 P16 Uiryeong Gyeongnam ± ± 3.5 P17 Aewol Jeju Island ± ± 2.4 P18 Pyoseon Jeju Isalnd ± ± 6.9 a Total polyphenol contents were determined by the Folin-Ciocalteu method. Each value is the mean ± standard deviation. b Flavonoid contents were determined by AlCl 3 coloration. Each value is the mean ± standard deviation.

5 Antioxidant Properties and Phenolic Composition of Korean Propolis and Pyoseon (P18), were dark yellowish-orange in color. The color of propolis from Hongcheon (P3), Aewol (P17) and Pyoseon (P18) were brown reddish-orange, dull yelloworange and brown purplish-red, respectively. Table 1 shows the collection sites, total polyphenol and flavonoid contents of EEP. The amount of total polyphenol and flavonoid contents in Korean propolis varied widely, ranging from to mg/g of EEP and from 36.9 to mg/g of EEP, respectively, and it had more than those from Uruguayan propolis (Silva et al., 2011). EEP from Chungju (P5), Cheongju (P6), Cheonan (P7) and Changnyeong (P15) showed higher total polyphenol and flavonoid contents than those from other regions. However, the amount of total polyphenol and flavonoid contents in EEP from Aewol (P17) and Pyoseon (P18) were much lower than those from any other region. We previously reported that the polyphenol content of propolis from various geographic origins in the world. The polyphenol content of EEP from Argentina, Bulgaria, China, Hungary, Japan and Uruguay was ~ mg/g of EEP (Kumazawa et al., 2010; Ahn et al., 2007; Hamasaka et al., 2004). Therefore, Korean propolis samples contain an equivalent level of polyphenol. Propolis contains a wide variety of phenolic compounds, mainly flavonoids. The determination of total polyphenol and flavonoid contents are important in various food materials. It has been suggested that flavonoids and other phenolic constituents play a preventive role in the development of cancer and heart disease (Banskota et al., 2001b). The Folin- Ciocalteu method and the AlCl 3 coloration are widely used to determine the total polyphenol and flavonoid contents, respectively (Laskar et al., 2010; Silva et al., 2011). These physicochemical methods are useful for evaluating various propolis samples because propolis contains many kinds of phenolics. Effects of propolis on linoleic acid oxidation The antioxidant activity of EEP samples was evaluated using the β-carotene-linoleic acid system, and results are shown in Fig. 2. Most of the EEP samples had high antioxidant activity, over 40%. EEP from Chungju (P5), Cheonan (P7) and Changnyeong (P15) had a stronger antioxidant activity than those from other regions. The contents of both total polyphenol and flavonoid were high in EEP from these samples. However, EEP from Hongcheon (P3), Iksan (P10), Aewol (P17) and Pyoseon (P18), in which total polyphenol and flavonoid contents were small (Table 1), exhibited weak antioxidant activity. Hamasaka et al. (2004) previously reported that antioxidant activity of EEP from Japan was 20 60%. EEP from various regions of Korea had activities similar to Japanese propolis. Figure 3 shows the relation between antioxidant activity of various EEP and total polyphenol con- tents. The antioxidant activity of various EEP had a positive correlation with total polyphenol contents (R 2 = 0.656). DPPH free radical-scavenging activity of propolis The DPPH free radical-scavenging activity of various EEP samples is shown in Fig. 4. DPPH free radical-scavenging activity assay system is a widely used for quickly evaluating the antioxidant activity of various samples. Most of the EEP samples showed DPPH free radical-scavenging activity. P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 BHT VE Antioxidant activity (%) Antioxidant activity (%) R² = Contents of polyphenol (mg/g of EEP) 215 Fig. 2. Antioxidant activity of EEP (P1 P18) of Korean propolis from various geographic origins in the β-carotene linoleic acid system. Means and standard deviations are indicated (n = 3). Fig. 3. Relation between antioxidant activity of EEP and total polyphenol contents.

6 216 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 BHT VE DPPH free radical-scavenging activity (%) Fig. 4. DPPH radical-scavenging activity of EEP (P1 P18) of Korean propolis from various geographic origins. Means and standard deviations are indicated (n = 3). DPPH free radical-scavenging activity (%) R² = Contents of polyphenol (mg/g of EEP) Fig. 5. Relation between DPPH radical-scavenging activity of EEP and total polyphenol contents. As shown in Fig. 4, the EEP samples from Chungju (P5), Cheongju (P6), Cheonan (P7) and Changnyeong (P15) had strong DPPH free radical-scavenging activities, over 80%. Especially, the DPPH free radical-scavenging activity of the EEP samples from Cheongju (P6) and Changnyeong (P15) was higher than that of VE used as the positive control. These EEP samples had high total polyphenol and flavonoid contents (Table 1). EEP samples from Chungju (P5), Cheon- S.J. Choi et al. gju (P6), Cheonan (P7) and Changnyeong (P15) showed strong antioxidant activity, also in the assay system using the discoloration of β-carotene (Fig. 2). EEP from Hongcheon (P3), Iksan (P10) and Aewol (P17), which had weak antioxidant activities in the assay system using the discoloration of β-carotene, exhibited weak DPPH free radical-scavenging activity. EEP from Pyoseon (P18) was observed to have extremely weak DPPH free radical-scavenging activity. The DPPH free radical-scavenging activity shown in Fig. 4 seemed to correlate with the antioxidant activity shown in Fig. 2. The relationship between DPPH free radical-scavenging activity of various EEP and total polyphenol contents was examined, and a positive correlation between them was observed (R 2 = 0.760) (Fig. 5). Effect of propolis on ABTS radical cation All EEP samples showed ABTS radical cation-scavenging activity (Fig. 6). The ABTS radical cation decolorization assay is a spectrophotometric method widely used for the assessment of antioxidant activity of various substances. The ABTS radical cation-scavenging activity of all EEP samples except EEP from Pyoseon (P18) was higher than that of BHT used as the positive control. Furthermore, the percentage inhibition of ABTS radical cation for all EEP samples, except that Pyoseon (P18), was over 40%. EEP samples from Pyoseon (P18) showed weak antioxidant activity, also, in the assay system using the discoloration of β-carotene (Fig. 2) and DPPH free radical scavenging activity (Fig. 4). The ABTS radical cation-scavenging activity shown in Fig. 6 seemed to correlate with the antioxidant activity shown in Fig. 2. The relation between ABTS radical cationscavenging activity of EEP samples and total polyphenol contents is plotted in Fig. 7, and a positive correlation between them was observed (R 2 = 0.830). According to the results obtained, antioxidant activity in the ABTS assay was higher than those in the DPPH assay. The difference between the results of two antioxidant activities assay might be because of the fact that DPPH radical reacts only with lipophilic antioxidants while ABTS radical reacts with both hydrophilic and lipophilic antioxidants (Prior et al., 2005). However, more detailed qualitative and quantitative analyses of the compounds with antioxidant activity will be necessary to fully elucidate the antioxidant activity of propolis. Effect of propolis on FRAP assay The FRAP activity of various EEP samples is shown in Fig. 8. The FRAP activity for all EEP samples was higher than that of BHT used as the positive control. The EEP from Chungju (P5), Cheongju (P6), Cheonan (P7) and Changnyeong (P15) had strong FRAP activities of over 250 μg/ml. EEP samples from Iksan (P10), Aewol (P17) and Pyoseon (P18) showed weak the FRAP activity, also, DPPH free radical-scavenging activity (Fig.

7 Antioxidant Properties and Phenolic Composition of Korean Propolis P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 BHT VE ABTS radical cation-scavenging activity (%) Fig. 6. ABTS radical cation-scavenging activity of EEP (P1 P18) of Korean propolis from various geographic origins. Means and standard deviations are indicated (n = 3). ABTS radical cation-scavenging activity (%) R² = Contents of polyphenol (mg/g of EEP) Fig. 7. Relation between ABTS radical cation-scavenging activity of EEP and total polyphenol contents. 4) and the ABTS radical cation-scavenging activity (Fig. 6). The FRAP activity shown in Fig. 8 seemed to correlate with the antioxidant activity shown in Fig. 2. The relation between FRAP activity of EEP and total polyphenol contents is plotted in Fig. 9, and correlation was significant (R 2 = 0.751). The propolis with high antioxidant activity also had high FRAP activity. There are two categories of antioxidants-a synthetic and a natural. Synthetic antioxidants are compounds with phenolic structures of various degrees of alkyl substitution, whereas natural antioxidants can be phenolic compounds, nitrogen compounds, or carotenoids as well as ascorbic acid (Velioglu et al., 1998). Although synthetic antioxidants such as butylated hydroxyanisole (BHA), BHT, propyl gallate, and tert-butylhydroquinone are widely used in the food industry, there are some arguments about their safety. The development and utilization of more effective antioxidants P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 BHT VE Ascorbic acid equivalent (µg/ml) Fig. 8. FRAP activity of EEP (P1 P18) of Korean propolis from various geographic origins. Means and standard deviations are indicated (n = 3). Ascorbic acid equivalent (μg/ml) R² = Contents of polyphenol (mg/g of EEP) 217 Fig. 9. Relation between FRAP activity of EEP and total polyphenol contents.

8 218 of natural origin are desired, because BHA and BHT have suspected of being responsible for liver damage and carcinogenesis (Gülçin et al., 2002). As mentioned above, according to most of the EEP samples have antioxidant activities more than BHT used as positive control. Thus, Korean propolis has enough values to quality as natural antioxidants. HPLC analysis of propolis We identified the major components in EEP samples, by HPLC analysis by PDA and MS detection. Figure 10 shows the structures of the main compounds from propolis. Previously, Kumazawa et al. (2002) isolated and identified 33 compounds, which are 18 flavonoids, 4 aromatic carboxylic acids, and, 11 phenolic acid esters from Uruguayan propolis. Concerning the com- S.J. Choi et al. pounds that could not be obtained from commercial sources, we used those isolated from Uruguayan propolis as authentic compounds to identify each component. Figure 11 shows the HPLC chromatograms of EEP samples P1 P18. To identify each peak (a r), UV spectra and the selected ion monitoring of MS spectra of all peaks were compared with those of authentic samples. Fujimoto et al. (2001) classified the propolis into two groups, namely, Brazilian-type (Baccharis-type) and European-type (poplartype), according to the difference of their components. Especially, European-type propolis is rich in flavonoids and is collected, not only in Europe but also China and other countries. The present study revealed that Korean propolis were Fig. 10. Structures of the compounds identified from Korean propolis. a, caffeic acid; b, p-coumaric acid; c, ferulic acid; d, 3,4-dimethoxycinnamic acid; e, pinobanksin 5-methyl ether; f, pinobanksin; g, kaempferol; h, apigenin; i, cinnamylideneacetic acid; j, pinocembrin; k, benzyl caffeate; l, pinobanksin 3-acetate; m, chrysin; n, phenethy caffeate; o, galangin; p, cinnamyl caffeate; q, pinostrobin; r, tectochrysin.

9 Antioxidant Properties and Phenolic Composition of Korean Propolis 219 Fig. 11. HPLC chromatograms of EEP (P1 P18) of Korean propolis from various geographic origins. similar to poplar-type propolis. The quantitative analysis of the identified peaks was carried out using calibration curves for standard compounds. A calibration curve for each compound was constructed and tested for linearity. Good linearity (r 2 > 0.998) was observed for the relationship of each compound peak areas and concentrations over the tested range. The LOD and LOQ values were in the range 1.6 ~ 4.8 μg/ml and 2.6 ~ 8.1 μg/ml, re-

10 220 spectively (Table 2). The results indicate that the HPLC-PDA method was sufficiently sensitive for the determination of phenolic acids and flavonoids in propolis samples. The precision of the HPLC system was tested by performing intra- and inter-day multiple injections of a standard solution containing pure standards of phenolic acids and flavonoids identified Table 2. Sensitivity parameters for the constituents used as propolis standards (μg/ml). Compound LOD a LOQ b Caffeic acid (a) p-coumaric acid (b) Ferulic acid (c) ,4-Dimethoxycinnamic acid (d) Pinobanksin 5-methyl ether (e) Pinobanksin (f) Kaempferol (g) Apigenin (h) Cinnamylidenacetic acid (i) Pinocembrin (j) Benzyl caffeate (k) Pinobanksin 3-acetate (l) Chrysin (m) Phenethyl caffeate (n) Galangin (o) Cinnamyl caffeate (p) Pinostrobin (q) Tectochrysin (r) a LOD: limit of detection. b LOQ: limit of quantification. S.J. Choi et al. in this study. The recovery and precision of the method were satisfactory for quantitative analysis and good reproducibility of the results has been achieved. The results of the quantitative analysis of all EEP samples are shown in Table 3. Values are expressed as means of triplicate analyses for each sample. The EEP samples from Chungju (P5), Cheongju (P6), Cheonan (P7) and Changnyeong (P15) had strong antioxidant activities as mentioned above. Furthermore, these EEP samples contained a large amount of antioxidative compounds, such as caffeic acid (a), kaempferol (g), galangin (o) and cinnamyl caffeate (p). We previously reported that phenolics such as caffeic acid (a), kaempferol (g), phenethyl caffeate (n), galangin (o) and cinnamyl caffeate (p) exhibited strong radical-scavenging activity (Ahn et al., 2009; Kumazawa et al., 2004). On the other hand, constituents of propolis from Aewol (P17) and Pyoseon (P18) were apparently different from those from other regions and contain a few compounds shown in Fig. 11. EEP from Jeju Island, Aewol (P17) and Pyoseon (P18), had relatively weak antioxidant activity, which was consistent with the low total polyphenol and flavonoid contents, as mentioned above. Eighteen compounds were identified from 18 Korean propolis and the quantitative values of each compound were determined in the present study. The characteristic compounds of poplar-derived propolis are pinocembrin (j), chrysin (m), galangin (o) and tectochrysin (r) (Fujimoto et al., 2001). These compounds were detected from the EEP samples from regions other than Jeju Island that the main Table 3. Content of the constiuents in EEP samples. Content a (mg/g of EEP) P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 Caffeic acid (a) p-coumaric acid (b) Ferulic acid (c) ,4-Dimethoxycinnamic acid (d) Pinobanksin 5-methyl ether (e) Pinobanksin (f) Kaempferol (g) Apigenin (h) Cinnamylideneacetic acid (i) Pinocembrin (j) Benzyl caffeate (k) Pinobanksin 3-acetate (l) Chrysin (m) Phenethyl caffeate (n) Galangin (o) Cinnamyl caffeate (p) Pinostrobin (q) Tectochrysin (r) : not detected. a Each value is the mean of triplicate analyses for each sample.

11 Antioxidant Properties and Phenolic Composition of Korean Propolis 221 plant source of this propolis is poplar. All Korean propolis samples, except that from Jeju Island, had relatively strong antioxidant activity accompanied by high total polyphenol contents. Propolis with strong antioxidant activity contained large amounts of antioxidative compounds, such as caffeic acid (a), kaempferol (g), phenethyl caffeate (n) and galangin (o). Laskar et al. (2010) reported that two flavonoids, pinocembrin (j) and galangin (o) were isolated from Indian propolis; among which (o) showed high DPPH radicalscavenging activity. Furthermore, phenethyl caffeate (n) and cinnamyl caffeate (p) possessed strong scavenging effects toward DPPH radical and their scavenging strengths were nearly equal to that of VE and stronger than that of ascorbic acid (Banskota et al., 2001a). Banskota et al. (2001a, 2001b) reported that the antioxidative activity of propolis is due to its phenolic components, which also possess antitumor and antihepatotoxic activities. In our previous report, we also found a correlation between antioxidant activity and antiangiogenic activity in various components from propolis (Ahn et al., 2009). The components from propolis such as kampferol (g), phenethyl caffeate (n), galangin (o) and quercetin not only had strong antioxidant activity but also antiangiogenic activity. Thus Korean propolis may have the potential to be developed into pharmaceutical drugs for the treatment of various free radical related diseases. Conclusions In this study, we investigated the antioxidant properties of propolis samples from diverse geographic regions in Korea. Major polyphenols were identified in propolis and quantitatively analysed. All Korean propolis samples had relatively similar antioxidant properties and compounds, except those from Aewol and Pyoseon, which are located in southern Korea, distant from the other sample regions. The results show that propolis from Korea contains a many amount of phenolic antioxidants to counteract the damaging effects of free radicals and can effectively prevent the oxidative stressrelated diseases. In contrast, EEP from Aewol and Pyoseon had singular HPLC chromatogram pattern from other that. Propolis samples from that have differences in their chemical compounds, due to Jeju Island is in a subtropical or tropical region and abundant in forests. To our knowledge, this is the first paper describing the diversity of Korean propolis from diverse geographic regions including Jeju Island. Therefore, this result will offer a meaningful reference of Korean propolis because propolis from all provincial regions is compared. 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