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Arch Pharm Res Vol 30, No 2, 146-150, 2007
http://apr.psk.or.kr

Flavonoids from the Flower of Rhododendron yedoense var.

Poukhanense and Their Antioxidant Activities
Sung Je Jung, Dong-Hyun Kim, Yoon-Hee Hong, Jin-Hee Lee, Hyo-Nam Song1, Young-Deok Rho, and
Nam-ln Baek
Graduate School of Biotechnology & Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701,
Korea and 1Department of Oriental Medical Food and Nutrition, Semyung University, Jecheon 390-711, Korea

(Received June 7, 2006)

Antioxidant flavonoids have been isolated from the flower of Rhododendron yedoense vat.
poukhanense. One new flavonoid and three known flavonoids, quercetin-5-O-//-D-glucopyrano-
side (1), quercetin (3), and quercitrin (4), were isolated from the butanol and ethyl acetate
extracts of the plant. The new flavonoid was identified as myricitrin-5-methyl ether (2). The isola-
tion of these flavonoids from this plant, for the first time, is a valuable finding. The flavonoids were
evaluated for their antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH),
TBARS (thiobarbituric acid reactive substance) and superoxide anion radical (02) in the xan-
thine/xanthine oxidase assay system. In the DPPH scavenging assay, the IC50 values were
4.5+0.48 p.M for compound 2 and 9.7+0.29 p.M for compound 3, which showed an antioxidant
activity approximately 1.5-2 times higher than the antioxidant activity of ~z-tocopherol (9.8+0.94
#M). Additionally, the antioxidant activities of myricitrin-5-methyl ether (2) (ICso = 1.7+0.22 pM)
and quercetrin (4) (IC~o = 1.9+0.63 #M) were higher than that of L-ascorbic acid (IC50= 7.4+0,63
l.tM) when evaluated using a TBARS assay. Compound 2 showed high activity in both the inhibi-
tion of xanthine oxidase (1.1+0.21 mM) and in the activation of superoxide scavenging.
Key words: Rhododendron yedoense, Ericaceae, Antioxidant activity, DPPH scavenging
assay, TBARS assay, Xanthine oxidase inhibition assay, Superoxide anion radical scavenging
assay, Myricitrin-5-methyl ether

INTRODUCTION cardiotonic phlegm (Kim, 1996).

Free radicals, formed by various environmental chemicals
Rhododendron yedoense var. poukhanense (Ericaceae) as well as by the endogenous metabolism of the plant,
grows widely on the sunny-side of mountains in all can cause oxidative damage to DNA, lipids and proteins.
regions of Korea. The height of the stem is 1-2 meters This damage results in the failure of cellular functions,
and the calyx gives off a sticky secretion. As soon as the which can cause tumors, inflammation, shock, atheroscle-
flower of Rhododendron mucronulatum Turcz. has fallen, rosis, diabetes, and ischemia (Jin et al., 1998). Several
the flower of Rhododendron yedoense var. poukhanense natural agents, such as cancer chemopreventive agents,
blooms between April and May (Lee, 1996). Although the anti-inflammatory agents, anti-diabetic and ischemic agents,
fower of the plant has been known to show toxicity in exhibit antioxidant activities through their ability to scavenge
cases of ingestion, there were no research reports on the oxygen radicals, including singlet oxygen, peroxy radicals,
chemical component, the toxicology or the physiology of superoxide, and hydroxy radicals (Ito et al., 1999; Wei et
the plant. R. mucronulatum Turcz., the most well-known al., 1993). To screen antioxidants from natural products,
plant of the Rododendron species in Korea. It has been an in vitro assay was performed using the free radical
reported to contain flavonoids, phenolic acids, etc., and to scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl
have pharmacological activity, such as the discharge of radical (DPPH) (Blois, 1958), thiobarbituric acid reactive
substance (TBARS) (Yuan et al., 2004) and the superoxide
Correspondence to: Nam-ln Baek, Graduate School of Biotech- anion radical (02) in the xanthine/xanthine oxidase assay
nology, KyungHee University, Seochun-Ri 1, Kiheung-Eup, Suwon system (Noro et al. 1983). In this report, we describe the
449-701, Korea chemical components of the butanol and ethyl acetate
Tel: 82-31-201-2661, Fax: 82-31-201-2157
E-mail: nibaek@khu.ac.kr extracts from Rhododendron yedoense var. poukhanense

146
Antioxidant Flavonoids of Rhododendron yedoense 147

flowers, and their antioxidant activities determined using Quercetin-5-O-~D-glucopyranoside (1)

various assay systems. Yellow powder (MeOH-H20); m.p. 263-265~ pos. FAB/
MS: 487 [M+Na]+; IR,, (film, cm 1) 3204, 1623, 1602; 1H-
MATERIALS A N D M E T H O D S NMR (400 MHz, pyridine-d~, 5) 8.46 (1H, d, J=2.0 Hz, H-
2'), 8.08 (1H, dd, J=8.4, 2.0 Hz, H-6'), 7.25 (1H, d, J=8.4
Plant materials Hz, H-5'), 6.67 (1H, d, J=2.0 Hz, H-8), 6.62 (1H, d, J=2.0,
The flowers of Rhododendron yedoense var. poukha- H-6), 6.09 (1H, d, J=7.6 Hz, H-I"); 13C-NMR (100 MHz,
nense were collected at Kyung Hee University in Suwon, pyridine-ds, ~)179.0 (C-4), 166.1 (C-7), 162.9 (C-5), 158.0
Korea, in May 2002 and were identified by Prof. Dae- (C-9), 157.7 (C-2), 151.0 (C-4), 146.9 (C-3'), 136.0 (C-3),
Keun Kim, Woosuk University, Jeonju. A voucher specimen 123.0 (C-1'), 122.5 (C-6'), 118.1 (C-5'), 116.5 (C-2'), 105.6
(KHU02214) was reserved at the Laboratory of Natural (C-10), 105.4 (C-1"), 100.1 (C-6), 94.8 (C-8), 78.0 (C-5"),
Products Chemistry, KyungHee University, Suwon, Korea. 75.8 (C-3"), 73.7 (C-2"), 70.1 (C-4"), 62.2 (C-6").

Instrumentation Myricitrin-5-methyl ether (2)

Melting points were determined on a Fisher-John Yellow powder (MeOH-H20); m.p. 170-172~ [o~]D= -139 ~
apparatus and were left uncorrected (Fisher Scientific, (c=0.2, MeOH); El/MS m/z: 478, 332, 286, 213, 128,
Chicago, U.S.A.). Optical rotations were measured on a 73; HREIMS m/z 478.1119 [M] + (calcd for C22H22012 =
JASCO P-1010 digital polarimeter (JASCO, Tokyo, Japan). 478.1111); IR,, (film, cm -1) 3366, 2937, 1609, 1344, 1203;
El-MS and FAB/MS were recorded on a JEOL JMSAX 1H-NMR (400 MHz, CD3OD, 6) 6.89 (2H, s, H-2', 6'), 6.29
505-WA (JEOL, Tokyo, Japan). IR spectra were run on a (1H, s, H-8), 6.20 (1H, s, H-6), 5.25 (1H, s, H-I"), 3.74
Perkin Elmer Spectrum One FT-IR spectrometer (Perkin (3H, s, OMe), 0.90 (3H, d, J=5.6 Hz, H-6"); 13C-NMR (100
Elmer, Norwalk, USA). ~H-NMR (400 MHz) and 13C-NMR MHz, CD3OD, 5) 175.2 (C-4), 164.1 (C-7), 161.9 (C-5),
(100 MHz) spectra were taken on a Varian Unity Inova AS 159.7 (C-9), 156.6 (C-2), 146.2 (C-3', 5'), 137.9 (C-3),
400 FT-NMR spectrometer (Varian, California, U.S.A.). 137.0 (C-4'), 121.8 (C-1'), 109.4 (C-2', 6'), 108.2 (C-10),
103.0 (C-1"), 96.8 (C-6), 95.8 (C-8), 73.1 (C-4"), 71.9 (C-
Chemicals 3"), 71.7 (C-2", 5"), 56.3 (OMe), 17.5 (C-6").
The 1,1-diphenyl-2-picryl-hydrazyl (DPPH), linoleic acid,
2,6-dihydroxypurine (xanthine) and xanthine oxidase were Quercetin (3)
purchased from Sigma-Aldrich (St. Louis, U.S.A.). Yellow powder (H20); m.p. 311-313; El/MS re~z: 302,301,
274, 273, 245, 153, 137.109; IR,, (film, cm -1) 3350, 1685,
Isolation of flavonoids from the flower of Rhodo- 1615; 1H-NMR (400 MHz, pyridine-ds, 8) 8.62 (1H, d,
dendron yedoense var. Poukhanense J=2.2 Hz, H-2'), 8.12 (1H, dd, J=8.4, 2.2 Hz, H-6'), 7.40
The dried sample (7 kg) was extracted at room tem- (1H, d, J=8.4 Hz, H-5'), 6.77 (1H, d, J=2.0 Hz, H-8), 6.72
perature with 80% aq. MeOH (25 L x 2). The extracts were (1H, d, J=2.0 Hz, H-6); 13C-NMR (100 MHz, pyridine-ds, 5)
partitioned with water (2 L), EtOAc (2 L x 2) and n-BuOH 176.9 (C-4) 165.2 (C-7), 162.0 (C-5), 157.1 (C-9), 149.3
(2 L x 2). The n-BuOH extract (CJB, 20 g) was applied (C-4'), 147.4 (C-2), 146.8 (C-3'), 137.6 (C-3), 123.1 (C-1'),
during silica gel column (@5 x 15 cm) chromatography 120.7 (C-6'), 116.4 (C-2', 5'), 104.2 (C-10), 99.0 (C-6),
(c.c.), eluted with CHCI~:EtOH (15:1 --> 10:1) and CHCI3: 94.0 (C-8).
MeOH:H20 (9:3:1 --> 7:3:1, lower layer) for monitoring by
thin layer chromatography (TLC), in order to produce Quercetin-3-O-~-L-rham nopyranoside (4)
nineteen fractions (CJB1-CJB19). CJB9 (2 g)was purified Yellow powder (H20); m.p. 180-182~ pos. FAB/MS m/z:
by silica gel c.c. (@5.5 x 13 cm) using EtOAc:MeOH:H20 471 [M+Na]+; IR,, (film, cm -1) 3228, 1655, 1605; 1H-NMR
(18:3:1) as an eluting solution to ultimately produce (400 MHz, CD3OD, 8) 7.32 (1H, d, J=2.0 Hz, H-2'), 7.27
compound 1 (47 mg). CJB11 (4 g) was applied to the (1H, dd, J=8.4, 2.0 Hz, H-6'), 6.89 (1H, d, J=8.4 Hz, H-5'),
silica gel c.c. (@4.5 x 10 cm), eluted with EtOAc:MeOH: 6.30 (1H, d, J=1.6 Hz, H-8), 6.15 (1H, d, J=1.6, H-6), 5.34
H20 (12:3:1), to yield compound 2 (32 mg). In addition, (1H, d, J=2.0 Hz, H-I"); 13C-NMR (100 MHz, CD3OD, 8)
the EtOAc extract (CJE, 10 g) was applied to the silica gel 179.2 (C-4), 165.4 (C-7), 162.8 (C-5), 159.0 (C-9), 158.1
c.c. (7 x 18 cm), eluted with n-hexane:EtOAc (25:1 ~ 15:1) (C-2), 149.4 (C-4'), 146.0 (C-3'), 136.0 (C-3), 122.7 (C-1',
-->CHCI3:MeOH (10:1 ~ 8 : 1 -->6:1 -->3:1), to produce 6'), 116.8 (C-5'), 116.1 (C-2'), 105.7 (C-10), 103.3 (C-1"),
thirteen fractions (CJE1-CJE13). CJE10 (749 mg) was 99.6 (C-6), 94.6 (C-8), 73.1 (C-4"), 72.0 (C-3"), 71.9 (C-
applied to the silica gel c.c. (@4.4 x 13 cm) eluted with n- 2"), 71.8 (C-5"), 17.6 (C-6").
hexane:EtOAc:EtOH (5:4:1) to produce compound 3 (30
mg) and compound 4 (52 rag).