Biol. Pharm. Bull. 32(7) 1244—1250 (2009)
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Vol. 32, No. 7
Antihypertensive Effects of Flavonoids Isolated from Brazilian Green Propolis in Spontaneously Hypertensive Rats Hiroe MARUYAMA,a Yoshiki SUMITOU,a Takashi SAKAMOTO,a Yoko ARAKI,a and Hideaki HARA*,b a
Nagaragawa Research Center, API Co., Ltd.; 692–3 Nagarayamasaki, Gifu 502–0071, Japan: and b Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University; 5–6–1 Mitahora-higashi, Gifu 502–8585, Japan. Received November 27, 2008; accepted May 9, 2009; published online May 11, 2009 Propolis, a honeybee product, has become popular as a food and alternative medicine. Its constituents have been shown to exert pharmacological effects, such as anticancer, antimicrobial, and anti-inflammatory effects. The present study was performed to investigate whether Brazilian green propolis exerts antihypertensive effects in spontaneously hypertensive rats (SHR) and which constituents are involved in its effects. Brazilian green propolis was extracted with ethanol and subjected to LH-20 column chromatography eluted with ethanol. The ethanol-eluted fractions at 10 mg/kg were administered orally to SHR for 14 d. Significant decreases in blood pressure were observed in fractions 6 and 7. The active constituents were purified and identified to be four flavonoids: dihydrokaempferide and isosakuranetin in fraction 6 and betuletol and kaempferide in fraction 7. These flavonoids at 10 mg/kg were administered orally to SHR for 28 d, and as a result, isosakuranetin, dihydrokaempferide and betuletol produced significant decrease in blood pressure, especially marked were the effects observed in the group that received isosakuranetin. Brazilian green propolis, fractions 6 and 7, and the 4 active constituents relaxed isolated SHR aorta in a concentration-dependent manner. Therefore, these finding suggest that the vasodilating action may be partly involved in the mechanism of antihypertensive effect. Hence, the ethanol extract of Brazilian green propolis and its main constituents may be useful for prevention of hypertension. Key words
Brazilian green propolis; antihypertensive effect; flavonoid; spontaneously hypertensive rat
Propolis is a resinous substance collected by honeybees from various plant sources. It has been suggested that propolis is used as a sealant to block unnecessary openings in the hive and to avoid invasion by fungi and bacteria. Propolis is used in folk medicines in many regions of the world and has been reported to have various biological activities, such as antibacterial,1) antiviral,2) anti-inflammatory,3) and anticancer4,5) properties. The composition of propolis depends primarily on the vegetation of the area from where it was collected and secondarily upon the solvents used for its extraction. In Japan, Brazilian green propolis has recently come to be used as a health food, which is of sufficiently good quality to provide experimental reproducibility. The ethanol extract of propolis is the major form used in health food. Antihypertensive effects of various foods and natural products have been reported. These have various activities such as inhibitory activity against angiotensin converting enzyme,6) vasodilatory action, or inhibitory effects on release of noradrenaline from sympathetic nerves.7) Hypertensive patients show increased levels of plasma superoxide and hydrogen peroxide.8) Moreover, in vessels from spontaneously hypertensive rats (SHR), a valuable animal model of essential hypertension in humans, enhanced endothelial superoxide anion production has been described and these effects are related to impairment of endothelium-dependent relaxation.9,10) Recently the antihypertensive effects of 25% ethanol extract of Brazilian green propolis were reported in SHR11,12) and the active constituent was shown to be caffeoylquinic acid.12) However, little information is available about the effects of absolute ethanol extract of Brazilian green propolis and its main constituents on hypertension in SHR. Generally, propolis is known to yield different constituents depending on the liquid used for extraction and the concentration. Therefore, the present study was performed to evaluate the ∗ To whom correspondence should be addressed.
antihypertensive effects of Brazilian green propolis extracted with ethanol in SHR and to clarify the antihypertensive effects of its main constituents. MATERIALS AND METHODS Materials Brazilian green propolis was collected in Minas Gerais state, Brazil. The main botanical source was Baccharis dracunculifolia DC. Propolis was extracted with 95% ethanol, and stirred for 16 h at room temperature. The insoluble part was removed by filtration, and adjusted to about 20% solid was designed EEP-B20 (Api Co., Ltd., Gifu, Japan). Isosakuranetin and kaempferide were purchased from Funakoshi, Ltd. (Tokyo, Japan). Dihydrokaempferide and betuletol were purified as follows. Amlodipine was purchased from Pfizer (Norvasc® OD Tablets; Tokyo, Japan). Fractionated Propolis Ethanol-extracted propolis (EEPB20; Api Co., Ltd.) was applied to Sephadex™ LH-20 (Amersham Biosciences, Piscataway, NJ, U.S.A.) column chromatography (f 62 mm 270 mm) eluted with ethanol. Ten fractions (each 200 ml) were collected and their composition monitored by TLC (solvents: CHCl3–MeOH 9 : 1 or CHCl3 only) and those with similar TLC profiles were combined into 8 major fractions denoted as Fr. 1 to Fr. 8 (Fig. 1). Each fraction was collected and dried. Isolation Fraction 6 was further purified by repeated silica gel column chromatography and eluted with CHCl3–MeOH (gradually replaced with MeOH 0—5%), finally yielding purified compounds 1 and 2. Fraction 7 was further purified using the silica gel column chromatography CHCl3–MeOH system (gradually replaced with MeOH 1— 5%) yielding purified compounds 3 and 4. Identification of Flavonoids Flavonoids were identified by NMR (Varian, MERCURY plus 300 MHz; Varian Tech-
e-mail: hidehara@gifu-pu.ac.jp
© 2009 Pharmaceutical Society of Japan
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Fig. 1. Flowchart of the Isolation Process for the Ethanol Extract of Propolis Ethanol extract of propolis was subjected to Sephadex TM LH-20 column chromatography and eluted with ethanol. Isolation of flavonoids from the ethanol extract of propolis: LH-20 column chromatography fractions were further purified by repeated silica gel column chromatography, finally yielding purified 4 compounds.
nologies Japan Ltd., Osaka, Japan) spectroscopic methods in dimethyl sulfoxide (DMSO)-d6 and wherever possible by chromatographic TLC and high-performance liquid chromatography (HPLC) comparisons with authentic markers. The chemical shift values are reported in ppm (d ), and the coupling constants (J) are reported in Hz. An HPLC system equipped with a model 2695 and 996-Photodiodearray detector was used (Waters, Osaka, Japan). Chromatography was performed at 40 °C using a CAPCELLPAK ACR column (Shiseido, Tokyo, Japan). Elution was performed in stepwise gradient mode. A: H2O containing 1% acetic acid. B: CH3CN containing 1% acetic acid [B concentration 20→80% (0→60 min, 80% hold (60—70 min)] at a flow rate of 1.0 ml/min. Isolation of Four Flavonoids for Animal Experiments Ethanol extract of propolis (EEP-B20; Api Co., Ltd.) was applied to a column of LH-20 gel, and eluted with ethanol four times. Fractions 6 and 7 were concentrated under reduced pressure to afford a yellow material (10 g). A portion of the 10 g extract was pre-adsorbed onto silica gel, applied to a silica gel column, and eluted with CHCl3–MeOH (1% increments of MeOH), and finally with MeOH. Thirteen fractions were collected and their compositions were monitored by TLC (solvent: CHCl3 : MeOH 1 : 1 or CHCl3 only) or HPLC. The purity was confirmed by HPLC, and solutions of 90% purity or greater were used in the experiments. Animals and Measurement of Blood Pressure Male SHR and male Wistar Kyoto (WKY) rats (11—12 weeks old, body weight 260—310 g) were purchased from Hoshino Laboratory Animals (Saitama, Japan). SHR and WKY rats were housed individually in steel cages in a room kept at 23 °C with a 12 h light–dark cycle (lights on 8:00—20:00), and fed a laboratory diet (CE-II, CLEA Japan Inc., Tokyo,
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Japan). Water was freely available. Systolic blood pressure and heart rate were measured by the tail-cuff method with a Softron BP system (BP-98A; Softron, Tokyo, Japan) after warming the animals in a chamber maintained at 39 °C for 5 min. At least six determinations were made in every session of systolic blood pressure measurements and the mean of six values was taken as the systolic blood pressure level.12) Repeated Oral Administration of Propolis Fractions in SHR After acclimation of SHR in the environment described above, animals (16 weeks old) with systolic blood pressure over 200 mmHg were used in the experiments. Using the starting blood pressure and body weight, animals were divided into 9 groups (control group, and Fr. 1 to Fr. 8). Each fraction was suspended in a 0.5% solution of gum Arabic and administered orally at 10:00 a.m. each day for 14 d at a dose of 10 mg/kg/10 ml. The control group was administered the same volume of gum Arabic solution. Systolic blood pressure and heart rate were measured at 0, 7, and 14 d before the administration. Repeated Oral Administration of Flavonoid in SHR Using the starting blood pressure and body weight, animals (13 weeks old) were divided into 5 groups (control group, and dihydrokaempferide-, isosakuranetin-, kaempferide-, and betuletol-treated groups) and 2 groups (control group, and amlodipine-treated groups). Each flavonoid was suspended in a 1% solution of gum Arabic (vehicle) and administered orally at 10:00 am each day for 28 d at a dose of 10 mg/kg/10 ml. Amlodipine, calcium channel antagonist used as a positive control at a dose of 3 mg/kg/10 ml, was administered orally in the same method. The control group was administered the same volume of gum Arabic solution. Systolic blood pressure and heart rate were measured at 0, 7, 14, 21, 28 d before the administration and measured at 7, 14, 21, 28 d 2 h after the administration. Repeated Oral Administration of Isosakuranetin and Amlodipine in WKY Using the starting blood pressure and body weight, animals (13 weeks old) were divided into 3 groups (control group, isosakuranetin-, and amlodipinetreated groups). Isosakuranetin (10 mg/kg/10 ml) and amlodipine (3 mg/kg/10 ml) were administered orally at 10:00 a.m. each day for 28 d and the measurement of systolic blood pressure and heart rate was performed as described above. Relaxation in the Aorta Isolated from SHR Male SHR weighting 250—300 g were killed by cervical dislocation and exsanguination. The main branch of the superior mesenteric artery was excised and cut into helical strips (2—3 mm in width and 20—30 mm in length) as described previously.13) Arterial strips were then suspended between two stainless wire hooks in a 10 ml organ bath. The upper wire was connected to a force-displacement transducer and the lower wire was fixed to the bottom of the organ bath. The organ bath was filled with Krebs solution (mM: NaCl, 119; KCl, 4.7; NaHCO3, 25; CaCl2, 2.5; MgCl2, 1; KH2PO4, 1.2; glucose, 11; and ascorbic acid, 0.3). The bath was continuously oxygenated with a mixture of 95% O2 and 5% CO2, and maintained at 37 °C to give a pH of approximately 7.4. The strips were placed under an optimal resting tension of 0.5 g, which had been determined by length–tension relationship experiments. The tissues were allowed to equilibrate for 90 min, during which time the bath solution was replaced with pre-
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warmed and oxygenated Krebs solution every 20 min. The resting tension was readjusted to 1.0 g when necessary. The endothelial layer was mechanically removed by gently rubbing the intimal surface of the vessel. The absence of endothelium was confirmed by the absence of relaxing effects of acetylcholine (1 m M). The relaxing effects of propolis, flavonoid fraction mixed Fr. 6 with Fr. 7, and four flavonoids on the contraction induced by norepinephrine (10 7 M) were studied in the aorta without endothelium. Values for 50% inhibitory concentration (IC50) were obtained by linear regression analysis. Data Analysis The results are expressed as means and standard deviations (S.D.). The significance of the differences in systolic blood pressure and heart rate at each time point after repeated administration was analyzed by Dunnett’s multiple-range test. The comparison of the control and amlodipine-treated group in SHR was evaluated by Student’s t-test.
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Fig. 2. Effects of Repeated Oral Administration of Fractions 6 and 7 on Systolic Blood Pressure in SHR Fractions 6 and 7 were administered to SHR for 14 d (10 mg/kg). Fractions 6 and 7: fractionated ethanol extract of propolis on LH-20 column chromatography eluted with ethanol. Differences in systolic blood pressure compared with the control group were examined for statistical significance. ∗∗ p 0.01 vs. control (Dunnett’s multiple-range test). Each point with a vertical bar represents the mean S.D., n 4.
RESULTS Effects of Repeated Oral Administration of Fractions 6 and 7 in SHR Repeated oral administration was performed using fractions of the ethanol extract. Only Fr. 6 and Fr. 7 produced significant decreases in blood pressure (Fig. 2). Measurements before the administration indicated significantly lower values in the groups that received Fr. 6 and Fr. 7 compared with the control group (p 0.01 on day 14). Other fractions were not decreased significantly, and throughout the period of administration, there were no differences between groups as regards heart rate and body weight of animals (data not shown). Isolation and Identification of Flavonoids in the Active Fractions The results of the present study indicated that Fr. 6 and Fr. 7 obtained by LH-20 column chromatography exhibited antihypertensive effects in SHR. We isolated the active components in the ethanol extract of propolis (EEP-B20) fractionated by LH-20 column chromatography eluted in ethanol four times, and then obtained the corresponding fraction (10 g). These active fractions including Fr. 6 and Fr. 7 were subjected to silica gel column chromatography eluted gradually with CHCl3 and methanol. Thirteen fractions were collected and their compositions were monitored by TLC (solvent: CHCl3 : MeOH 1 : 1 or CHCl3 only) or HPLC. For final purification, each was recrystallized in methanol, yielding compound 1 (0.92 g), compound 2 (0.1 g), compound 3 (1.09 g), and compound 4 (0.77 g), respectively. The purity of these flavonoids was confirmed to be 90% or more by HPLC analysis. The NMR data of compounds 1 to 4 are as follows: Compound 1: Dihydrokaempferide: 1H-NMR (DMSO-d6) d : 3.78 (3H, s, –OMe), 4.60 (1H, d, J 11.0 Hz), 6.24 (1H, d, J 2.0 Hz, H-6), 5.11 (1H, d, J 11.4 Hz, H-2), 5.86 (1H, br s, H-6), 5.91 (1H, br s, H-8), 6.97 (2H, d, J 8.8 Hz, H-3 , 5 ), 7.44 (2H, d, J 8.8 Hz, H-2 , 6 ), 11.80(1H, br s, –OH). Compound 2: Isosakuranetin: 1H-NMR (DMSO-d6) d : 2.72 (1H, dd, J 17.0, 3.0 Hz, Ha-3), 3.27 (1H, dd, J 17.0,13.0 Hz, Hb-3), 3.77 (3H, s, –OMe), 5.50 (1H, dd, J 13.0, 3.0 Hz, H-2), 5.88 (1H, d, J 2.0 Hz, H-6), 5.89 (1H, d, J 8.5 Hz, H-3 , 5 ), 7.44 (2H, d, J 8.5 Hz, H-2 , 6 ), 12.14 (1H, s, –OH).
Fig. 3. HPLC Profiles of the Components of Fractions 6 and 7 from Propolis Extract Elution was performed in stepwise gradient mode. A: H2O containing 1% acetic acid. B: CH3CN containing 1% acetic acid [B conc. 20→80% (0→60 min), 80% hold (60—70 min)] at a flow rate of 1.0 ml/min.
Compound 3: Kaempferide: 1H-NMR (DMSO-d6) d : 3.85 (3H, s, –OMe), 6.20 (1H br s, H-6), 6.45 (1H, br s, H-8), 7.11 (2H, d, J 8.9 Hz, H-3 , 5 ), 8.14 (2H, d, J 8.9 Hz, H-2 , 6 ), 12.44 (1H, br s, –OH). Compound 4: Betuletol: 1H-NMR (DMSO-d6) d : 3.77 (3H, s, 6-OMe), 3.85 (3H, s, 4 -OMe), 6.57 (1H, s, H-8), 7.11 (2H, d, J 8.9 Hz, H-3 ,5 ), 8.14 (2H, d, J 8.9 Hz, H2 ,6 ), 12.52 (1H, br s, –OH). These compounds were identified as dihydrokaempferide and isosakuranetin in Fr. 6 and kaempferide and betuletol in Fr. 7 by HPLC (Fig. 3) and NMR, which showed that the
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Fig. 4. Chemical Structures of 4 Compounds in Fractions 6 and 7 from the Ethanol Extract of Propolis Compound 1: dihydrokaempferide. Compound 2: isosakuranetin. Compound 3: kaempferide. Compound 4: betuletol.
Table 1.
Flavonoid Contents of Ethanol Extract of Propolis Compounds
Flavonoid contents from ethanol extract of propolis (solid) (%)
Dihydrokaempferide Isosakuranetin Kaempferide Betuletol
1.3 0.2 2.2 1.0
spectra were consistent with those reported previously.14—17) The structures of these four compounds were shown in Fig. 4. Contents of dihydrokaempferide and isosakuranetin in Fr. 6 and of kaempferide and betuletol in Fr. 7 were 10%, 3.1%, 36%, and 40%, respectively. The contents of these flavonoids in propolis extract were about 1.3%, 0.2%, 2.2%, and 1.0%, respectively (Table 1). The chemical structures of dihydrokaempferide, kaempferide, and isosakuranetin were A-ring 5,7-dihydroxyflavones (or flavonol) with B-ring 4 -methoxy groups. Betuletol had a 6,4 -methoxy group. Effects of Repeated Oral Administration of Flavonoids in SHR Measurements before the administration revealed that lower values in the groups that received flavonoid compared with the control group (Fig. 5A). The systolic blood pressure in SHR administered dihydrokaempferide at a dose of 10 mg/kg/d was significantly lower than that in the control group on days 21 (p 0.05) and 28 (p 0.01) of administration. Isosakuranetin significantly reduced systolic blood pressure in SHR as compared with the control group on days 14 (p 0.05), 21 and 28 (p 0.01). Systolic blood pressure was significantly lower in SHR treated with betuletol than in the controls on days 28 (p 0.05) of administration. No significant effect on the blood pressure was observed with kaempferide. Measurements conducted 2 h after administration showed that systolic blood pressure was significantly lower in the group that received isosakuranetin only (p 0.05 on day 14, 21 and 28, Fig. 5B). In contrast, amlodipine, calcium channel antagonist, significantly reduced systolic blood pressure before and 2 h after administration as compared with the control group on days 7 (p 0.05; before, p 0.01; 2 h after), 14, 21 and 28 (all p 0.01, Figs. 6A, B).
Fig. 5. Effects of Repeated Oral Administration of Flavonoids from Propolis on Systolic Blood Pressure in SHR Dihydrokaempferide, isosakuranetin, kaempferide, and betuletol were administered to SHR for 28 d (10 mg/kg). (A) Systolic blood pressure was measured before daily administration. (B) Systolic blood pressure was measured 2 h after daily administration. Differences in systolic blood pressure compared with the control group were examined for statistical significance. ∗ p 0.05, ∗∗ p 0.01 vs. control (Dunnett’s multiple-range test). Each point with a vertical bar represents the mean S.D., n 6—7.
As regards the heart rate, no differences were observed between the control group and the groups before administration of flavonoid (Fig. 7A), however, in the group which received betuletol and dihydrokaempferide, a significant increase of heart rate were observed at 2 h after administration on days 21 (p 0.05 and p 0.01, respectively, Fig. 7B). In the group given amlodipine, no differences in the change of heart rate was observed before administration, however, the heart rate showed significant increase 2 h after administration as compared with the control group on days 7 (p 0.01), 14 (p 0.05), 21 and 28 (p 0.01, Figs. 8A, B). Repeated Oral Administration of Isosakuranetin and Amlodipine in WKY In contrast to the SHR, no differences in the change of systolic blood pressure and heart rate of WKY were observed between the control group and the isosakuranetin-treated group before and 2 h after administration (Figs. 9A, B and Figs. 10A, B). Amlodipine significantly reduced systolic blood pressure 2 h after administration as compared with the control group on days 7, 14, 21 and 28 (all p 0.01, Fig. 9B). The heart rate of the group receiving amlodipine showed significant decrease before administration on days 14 (p<0.05, Fig. 10A) and showed significant increase 2 h after administration as compared with the control group on days 7, 14 and 28 (all p 0.01, Fig. 10B). Throughout the period of administration, there were no abnormal findings as regards the general condition of SHR and
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Fig. 6. Effect of Repeated Oral Administration of Amlodipine on Systolic Blood Pressure in SHR Amlodipine was administered to SHR for 28 d (3 mg/kg). (A) Systolic blood pressure was measured before daily administration. (B) Systolic blood pressure was measured 2 h after daily administration. Differences in systolic blood pressure compared with the control group were examined for statistical significance. ∗ p 0.05, ∗∗ p 0.01 vs. control (Student’s t-test). Each point with a vertical bar represents the mean S.D., n 6—7.
WKY. There were also no differences between groups as regards the body weight of animals (data not shown). Relaxation Effect in the Aorta Isolated from SHR Propolis and the flavonoids isolated from it markedly relaxed contractions induced by norepinephrine (10 7 M) in the aorta without endothelium. The IC50 values for propolis and some flavonoids were 0.9—16.6 m g/ml (Table 2). The inhibitory effects of flavonoids on relaxed contraction were observed in the order of kaempferide isosakuranetin flavonoid fraction betuletol propolis dihydrokaempferide. DISCUSSION Propolis is usually extracted with ethanol or water, and these extracts have been used in folk medicine. The composition of propolis depends on the solvent used for its extraction. In addition, biological activities could be related to its chemical composition. Although 90—100% ethanol extract of Brazilian green propolis is commonly used as a health food, but little information is available regarding its biological activities and components. A recent study indicated that caffeoylquinic acid and its derivatives, which are among the components of the Brazilian green propolis extract, showed antihypertensive activities.12) However, the solvent used for extraction was 25% ethanol. In this study, the ethanol extract of propolis was fractionated, and repeated oral administration of these fractions was performed in SHR. Only Fr. 6 and Fr. 7 produced significant
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Fig. 7. Effects of Repeated Oral Administration of Flavonoids from Propolis on Heart Rate in SHR Four flavonoids were administered to SHR for 28 d at a dose of 10 mg/kg. (A) Heart rate was measured before daily administration. (B) Heart rate was measured 2 h after daily administration. Differences in heart rate compared with the control group were examined for statistical significance. ∗ p 0.05, ∗∗ p 0.01 vs. control (Dunnett’s multiple-range test). Each point with a vertical bar represents the mean S.D., n 6—7.
decreases in blood pressure. The active constituents were purified and identified to be four flavonoids such as dihydrokaempferide, kaempferide, isosakuranetin, and betuletol. Flavonoids are considered as an important part of the antioxidants in food. Dihydrokaempferide and betuletol isolated from Brazilian green propolis show anticancer activity in vitro.18) Kaempferide is known to have an antioxidative effect,19) and isosakuranetin is known to have antimicrobial activity.20) However, the antihypertensive effects of these flavonoids have not been clarified. Several previous studies have indicated various biological activities of flavonoids, including vasodilator effects in isolated aorta stimulated with noradrenaline or KCl.13,21) However, no antihypertensive activity was observed in SHR.13,21) Only a few reports have suggested that flavonoid shows an antihypertensive effect in SHR.22) In this study, effects of four active flavonoids (dihydrokaempferide, kaempferide, isosakuranetin, and betuletol) from Brazilian green propolis on blood pressure and heart rate were examined using SHR. As a result, isosakuranetin, dihydrokaempferide and betuletol produced significant decrease in blood pressure before daily administration, especially marked were the effects observed in the group that received isosakuranetin; the values obtained 2 h after administration were significantly lower on days 14, 21 and 28. In the present study, though dihydrokaempferide, kaempferide and betuletol were purified and administered at
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Fig. 8. Effect of Repeated Oral Administration of Amlodipine on Heart Rate in SHR Amlodipine was administered to SHR for 28 d (3 mg/kg). (A) Heart rate was measured before daily administration. (B) Heart rate was measured 2 h after daily administration. Differences in heart rate compared with the control group were examined for statistical significance. ∗ p 0.05, ∗∗ p 0.01 vs. control (Student’s t-test). Each point with a vertical bar represents the mean S.D., n 6—7.
10 mg/kg, the effect on lowering blood pressure was not more potent than those of Fr. 6 and Fr. 7 tested on days 14. It might be speculated that the response on lowering blood pressure is different by SHR used different age, in Fr. 6 and Fr. 7 experiment, with systolic blood pressure over 200 mmHg established, and there may be some unknown materials decreasing blood pressure in Fr. 6 and Fr. 7, although further studies are needed to elucidate the active constituents which purified and identified. Four types of flavonoid have the flavonol, flavononol, and flavanon frame with a 4 -methoxyl group in the B ring. It was found that the chemical structures of flavonoids having the B-ring 3 ,4 -methoxy/hydroxyl groups were elevated for their inhitory activities of NO production23) and the flavonols having methylation of the 3-, 5-, or 4 -hydroxyl group enhanced the inhibitory activity of NO production.24) Jeong et al. have suggested that flavonoids with 3-OH group play a positive role in antioxidant activities.25) Thus, it is well known that there are differences in the anti-oxidative revitalization and anti-inflammatory actions according to the structure of the B ring of flavonoids. The observation that these four flavonoids from Brazilian green propolis have a 4 methoxyl group in the B ring is probably related to the antihypertensive activity. Brazilian green propolis, Fr. 6 and Fr. 7, and the 4 active
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Fig. 9. Effects of Repeated Oral Administration of Isosakuranetin and Amlodipine on Systolic Blood Pressure in WKY Isosakuranetin (10 mg/kg) and amlodipine (3 mg/kg) were administered to WKY for 28 d. (A) Systolic blood pressure was measured before daily administration. (B) Systolic blood pressure was measured 2 h after daily administration. Differences in systolic blood pressure compared with the control group were examined for statistical significance. ∗ p 0.05, ∗∗ p 0.01 vs. control (Dunnett’s multiple-range test). Each point with a vertical bar represents the mean S.D., n 6—7.
constituents relaxed the aorta isolated from SHR in a concentration-dependent manner. Its potency of four flavonoids was not necessarily corresponding to the antihypertensive effects in SHR. However, the vasodilating action may be partly involved in the mechanism of antihypertensive effect, though it is possible that the dissolubility and/or absorption of flavonoids in vivo might be different. In the present study, repeated oral administration of isosakuranetin and amlodipine, calcium channel antagonist, induced a progressive reduction of systolic blood pressure in SHR. Blood pressure at 2 h after administration was significantly lower in the groups that received isosakuranetin and amlodipine. However, while there was no change in heart rate in the group that received isosakuranetin, amlodipine produced significant increase 2 h after administration. In addition, while no differences in the change of systolic blood pressure and heart rate of WKY were observed between the control group and the isosakuranetin-treated group, amlodipine significantly reduced systolic blood pressure and increased heart rate 2 h after administration. Therefore, further studies are needed to clarify the possible mechanism for flavonoids, especially isosakuranetin, including calcium antagonistic vasorelaxing activity. In conclusion, the findings presented here indicate that
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ethanol extract of Brazilian green propolis and its main constituents would be beneficial for improving blood pressure as a functional food. REFERENCES
Fig. 10. Effects of Repeated Oral Administration of Isosakuranetin and Amlodipine on Heart Rate in WKY Isosakuranetin (10 mg/kg) and amlodipine (3 mg/kg) were administered to WKY for 28 d. (A) Heart rate was measured before daily administration. (B) Heart rate was measured 2 h after daily administration. Differences in heart rate compared with the control group were examined for statistical significance. ∗ p 0.05, ∗∗ p 0.01 vs. control (Dunnett’s multiple-range test). Each point with a vertical bar represents the mean S.D., n 6—7.
Table 2.
Relaxation Effect in the Aorta Isolated from SHR Compounds
IC50 (95% confidential interval) (m g/ml)
Dihydrokaempferide Isosakuranetin Kaempferide Betuletol Ethanol extract of propolis Flavonoid fraction
16.6 (10.4—22.7) 1.7 (1.2—2.5) 0.9 (0.2—1.6) 8.2 (5.8—10.6) 15.0 (10.5—22.0) 4.4 (3.4—5.4)
IC50 values (m g/ml) of different flavonoids to inhibit the contraction induced by 10 7 M norepinephrine. Data are means, n 3.
1) Drago L., Mombelli B., De Vecchi E., Fassina M. C., Tocalli L., Gismondo M. R., J. Chemother., 12, 390—395 (2000). Erratum in: J. Chemother., 13, 102 (2001). 2) Kujumgiev A., Tsvetkova I., Serkedjieva Y., Bankova V., Christov R., Popov S., J. Ethnopharmacol., 64, 235—240 (1999). 3) Wang L., Mineshita S., Ga I., Shigematsu T., Matsumoto T., Jpn. J. Clin. Pharmacol. Ther., 24, 223—224 (1993). 4) Scheller S., Krol W., Swiacik J., Owczarek S., Gabrys J., Shani J., Z. Naturforsch., 44, 1063—1065 (1989). 5) Grunberger D., Banerjee R., Eisinger K., Oltz E. M., Efros L., Caldwell M., Esterez V., Nakanishi K., Experientia, 44, 230—232 (1988). 6) Tokunaga K., Yoshida C., Suzuki K., Maruyama H., Futamura Y., Araki Y., Mishima S., Biol. Pharm. Bull., 27, 189—192 (2004). 7) Suzuki A., Kagawa D., Ochiai R., Tokimitsu I., Saito I., Hypertens. Res., 25, 99—107 (2002). 8) Lacy F., O’Connor D. T., Schmid-Schönbein G. W., J. Hypertens., 16, 291—303 (1998). 9) Kumar K. V., Das U. N., Free Radic. Res. Commun., 19, 59—66 (1993). 10) Jameson M., Dai F. X., Luscher T., Skopec J., Diederich A., Diederich D., Hypertension, 21, 280—288 (1993). 11) Kubota Y., Umegaki K., Kobayashi K., Tanaka N., Kagota S., Nakamura K., Kunitomo M., Shinozuka K., Clin. Exp. Pharmacol. Physiol., 31, S29—S30 (2004). 12) Mishima S., Yoshida C., Akino S., Sakamoto T., Biol. Pharm. Bull., 28, 1909—1914 (2005). 13) Duarte J., Perez Vizcaino F., Utrilla P., Jimenez J., Tamargo J., Zarzuelo A., Gen. Pharmacol., 24, 857—862 (1993). 14) Breitmair E., Voelter W., “Carbon-13 NMR Spectroscopy,” VCH Verlagsgesellschaft, Weinheim, 1987, pp. 450—456. 15) Manez S., Paya M., Terenico C., Villar A., Planta Med., 54, 187—188 (1988). 16) Agrawal P. K., Rastogi R. P., Heterocycles, 16, 2181—2236 (1981). 17) Ramachandran Nair A. G., Sivakumar R., Phytochemistry, 29, 1011— 1012 (1990). 18) Banskota A. H., Tezuka Y., Prasain J. K., Matsushige K., Saiki I., Kadota S., J. Nat. Prod., 61, 896—900 (1998). 19) He Z. W., Lu Z. P., Wu W. S., Zhu Y., Xibei Zhiwu Xuebao, 27, P1884—P1887 (2007). 20) Da Silva Filho A. A., de Sousa J. P., Soares S., Furtado N. A., Andrade e Silva M. L., Cunha W. R., Gregorio L. E., Nanayakkara N. P., Bastos J. K., Z. Naturforsch., 63, 40—46 (2008). 21) Morello S., Vellecco V., Alfieri A., Mascolo N., Cicala C., Life Sci., 78, 825—830 (2006). 22) Villar I. C., Jimenez R., Galisteo M., Garcia-Saura M. F., Zarzuelo A., Duarte J., Planta Med., 68, 847—850 (2002). 23) Kim S. J., Park H., Kim H. P., Arch. Pharm. Res., 27, 937—943 (2004). 24) Matsuda H., Morikawa T., Ando S., Toguchida I., Yoshikawa M., Bioorg. Med. Chem., 11, 1995—2000 (2003). 25) Jeong J. M., Choi C. H., Kang S. K., Lee I. H., Lee J. Y., Jung H., J. Pharm. Pharm. Sci., 10, 537—546 (2007).