Structural determination and DPPH radical-scavenging activity of two acylated flavonoid tetraglycosides in oolong tea (Camellia sinensis)

Article abstract of DOI:10.1248/cpb.56.851

Two major acylated flavonoid tetraglycosides were isolated from the methanol extract of oolong tea. Their structures were elucidated by spectroscopic methods as quercetin 3-O-[2^G-(E)-coumaroyl-3 ^G-O-β-D-glucosyl-3^R-O-β-D-g

Full text of DOI:10.1248/cpb.56.851

June 2008
Notes
Chem. Pharm. Bull. 56(6) 851—853 (2008)
851
Structural Determination and DPPH Radical-Scavenging Activity of Two
Acylated Flavonoid Tetraglycosides in Oolong Tea (Camellia sinensis)
,d
Viola Szu-Yuan LEE,a Chiy-Rong CHEN,b Yun-Wen LIAO,c Jason Tze-Cheng TZEN ,a and Chi-I CHANG
,
*
*
a Graduate Institute of Biotechnology, National Chung Hsing University; Taichung 40227, Taiwan: b Department of
Biological Science and Technology, Meiho Institute of Technology; Pingtung 91201, Taiwan: c Department of Food
Science, National Pingtung University of Science and Technology; and d Graduate Institute of Biotechnology, National
Pingtung University of Science and Technology; Pingtung 91201, Taiwan.
Received December 9, 2007; accepted February 27, 2008; published online March 21, 2008
Two major acylated flavonoid tetraglycosides were isolated from the methanol extract of oolong tea. Their
structures were elucidated by spectroscopic methods as quercetin 3-O-[2G-(E)-coumaroyl-3G-O-b-D-glucosyl-3R-
O-b-D-glucosylrutinoside] (1) and kaempferol 3-O-[2G-(E)-coumaroyl-3G-O-b-D-glucosyl-3R-O-b-D-glucosylruti-
noside] (2). Compounds 1 and 2 exhibited scavenging activity against DPPH radical with EC50 values of 30.5 and
487.2 mM, respectively.
Key words Camellia sinensis; oolong tea; quercetin; kaempferol; 1,1-diphenyl-2-picrylhydrazyl
Tea is a beverage widely consumed in the world for over scribe the extraction, purification, and structural elucidation
several thousand years. Various teas as well as their ac- of the two relatively abundant acylated flavonoid tetraglyco-
tive ingredients, such as catechins and polyphenols, have sides, 1 and 2, from the methanol extract of oolong tea. Scav-
been demonstrated to possess antioxidant and biological ac- enging activities against 1,1-diphenyl-2-picrylhydrazyl
tivities.1) They are mainly classified into green tea (unfer- (DPPH) radical of these two compounds were evaluated.
mented), oolong tea (partially fermented), and black tea
Compound 1 was isolated as a light yellow amorphous
(fully fermented) according to the degree of fermentation solid. The molecular formula of 1 was established as
during their preparations, where the term ‘fermentation’ C48H56O28 by the positive HR-FAB-MS showing a quasi-
refers to natural browning reactions induced by oxidative en- molecular ion peak at m/z 1081.3032 [MꢀH]ꢀ, 13C-NMR
zymes in the cells of tea leaves.2) Oolong tea possessing a spectrum, and DEPT spectrum indicating 21 degrees of un-
taste and color somewhere between green and black teas is saturation. The IR spectrum of 1 displayed absorptions
the most popular tea in Taiwan, and different process condi- for hydroxyl (3383 cmꢁ1), conjugated ester (1703 cmꢁ1),
tions have been practiced to generate variable products of conjugated ketone (1659 cmꢁ1), and benzene ring (1606,
this tea by local manufacturers.
1514 cmꢁ1) functionalities. The UV absorption maxima at
1
In the production of oolong tea, young green shoots are 260, 271, and 354 nm, as well as the H-NMR signals for a
freshly harvested and allowed to wither under sunlight for a 5,7-dihydroxylated pattern of ring A [(dH 6.13 (1H, br d,
few hours prior to undergoing the semi-fermentation process, Jꢂ1.6 Hz, H-6); 6.32 (1H, br d, Jꢂ1.6 Hz, H-8)], and a set of
in which tea leaves are oxidized, pan fired at ca. 200 °C, ABX coupling pattern of ring C [(dH 7.59 (1H, d, Jꢂ1.6 Hz,
rolled to form ball shape, and then dried in a specialized H-2ꢃ); 6.88 (1H, d, Jꢂ8.4 Hz, H-5ꢃ); 7.53 (1H, dd, Jꢂ1.6,
oven at various desired temperatures. The reaction time for 8.4 Hz, H-6ꢃ)] suggested that the aglycone of 1 was
1
the contact between phenolic compounds and oxidative en- quercetin.13) The H-NMR spectrum of 1 (Table 1) also
zymes is empirically controlled by experts during this semi- showed the resonances for a (E)-coumaroyl group [dH 7.45
fermentation process, and the final fermentation degree of (2H, d, Jꢂ8.8 Hz, H-2ꢄ, 6ꢄ); 6.79 (2H, d, Jꢂ8.8 Hz, H-3ꢄ,
oolong tea ranges at 20—80% depending on the demand of 5ꢄ); 7.67 (1H, d, Jꢂ16.0 Hz, H-7ꢄ); 6.41 (1H, d, Jꢂ16.0 Hz,
customers. Experientially, fermentation (oxidation) and dry- H-8ꢄ)], four anomeric protons [dH 5.54 (1H, d, Jꢂ8.0 Hz, H-
ing in the specialized oven are two key steps for the quality 1 of Glc-I); 4.43 (1H, d, Jꢂ7.6 Hz, H-1 of Glc-II); 4.44 (1H,
control of oolong tea preparation. Therefore unique com- br d, Jꢂ7.6 Hz, H-1 of Glc-III); 4.62 (1H, br s, H-1 of Rha)],
pounds are possibly generated in various preparations of oo- and other sugar protons [dH 3.22—4.01; 5.21]. The sugar
long tea manufactured by different process conditions.3—5)
Many kinds of compounds have been identified in various
teas, such as flavan-3-ols, flavonoids, gallic acid, quinic es-
ters of caffeic, coumaric and gallic acid, theaflavins, thearu-
bigins, and alkaloids.6—11) Although oolong tea is becoming
increasingly more popular in the world, there is much less in-
vestigation of the infusions of different oolong tea products
in comparison with the vigorous studies that have been con-
ducted on the active ingredients found in green and black
teas. Recently, six novel acylated flavonol glycosides were
identified in the infusion of oolong tea, and their chemical
structures were approximately predicted by electrospray ion-
ization tandem mass spectrometry.12) In this report, we de-
∗ To whom correspondence should be addressed. e-mail: changchii@mail.npust.edu.tw; TCTZEN@dragon.nchu.edu.tw © 2008 Pharmaceutical Society of Japan

852
Vol. 56, No. 6
Table 1. 1H- and 13C-NMR Data for 1 and 2 (400, 100 MHz in CD3OD)
moiety of 1 was proposed to comprise one internal glucose
[dH 5.54 (H-1 of Glc-I); dC 100.9, 74.4, 84.6, 70.0, 76.5,
68.4], one internal rhamnose [4.62 (H-1 of Rha-I); dC 102.2,
71.2, 82.9, 72.4, 69.4, 17.9], and two terminal glucoses {[dH
4.43 (H-1 of Glc-II); dC 104.5, 74.6, 77.3, 71.2, 77.9, 62.4]
and [dH 4.44 (H-1 of Glc-III); dC 105.4, 75.3, 77.3, 70.7,
77.5, 62.0]} by the HOHAHA, HMQC experiments, and di-
rect comparisons of the 1H- and 13C-NMR data with those of
the literature.13—15) The monosaccharides obtained by acid
hydrolysis of the saponin mixture were identified as D-glu-
cose and L-rhamnose by TLC and by measurement of their
specific rotation after purification.16) The b-configuration for
1
2
dC
dH
dC
dH
Aglycone
2
3
4
5
6
7
8
158.8
134.9
178.9
162.8
99.8
159.0
134.8
179.0
162.8
99.9
6.13 br d (1.6)
6.32 br d (1.6)
6.15 br d (2.0)
6.34 br d (2.0)
165.5
94.9
165.6
95.0
9
10
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
158.2
105.8
123.2
117.5
145.9
149.6
116.2
123.5
158.3
105.8
122.8
132.3
116.3
161.2
116.3
132.3
3
the three D-glucoses was determined from their large JH1,H2
coupling constants (Jꢂ7.6—8.0 Hz), while the a-configura-
tion for the L-rhamnose was judged from the broad singlet
signal of anomeric proton.17,18) The internal glucose was con-
nected to C-3 of aglycone, which was deduced from the
HMBC correlation between the anomeric proton of Glc-I (dH
5.54) and C-3 of aglycone (dC 134.9). In turn, the HMBC
correlations between the anomeric proton of Rha (dH
4.62)/C-6 of Glc-I (dC 68.4), the anomeric proton of Glc-II
(dH 4.43)/C-3 of Glc-I (dC 84.6), and the anomeric proton of
Glc-III (dH 4.44)/C-3 of Rha (dC 82.9) suggested that the
connection order of sugar moiety at the C-3 of aglycone was
Glc-I (6→1)[(3→1) Glc-II] Rham (3→1) Glc-III. Further-
more, the coumaroyl unit was located at the C-2 position of
Glc-I, which was also assured by the observation of the
HMBC relationship between H-2 of Glc-I (dH 5.21) and C-9ꢄ
(dC 168.8). The assignment structure of 1 closely resembles
the known acylated flavonoid tetraglycoside, quercetin 3-O-
(2G-p-coumaroyl-3G-O-b-L-arabinosyl-3R-O-b-D-glucosyl-
rutinoside), which was isolated from oolong tea by Mihara
7.59 d (1.6)
7.96 d (8.8)
6.90 d (8.8)
6.88 d (8.4)
7.53 dd (1.6, 8.4)
6.90 d (8.8)
7.96 d (8.8)
Glc-I
1
2
3
4
5
6
100.9
74.4
84.6
70.0
76.5
68.4
5.54 d (8.0)
5.21 t (9.8)
3.94 t (8.8)
3.56 m
3.57 m
3.56 m, 3.89 m
100.8
74.4
84.5
70.1
76.6
68.4
5.54 d (8.0)
5.20 t (9.2)
3.92 t (8.4)
3.50 m
3.54 m
3.51 m, 3.88 m
Glc-II
1
2
3
4
5
6
104.5
74.6
77.3
71.2
77.9
62.4
4.43 d (7.6)
3.22 t (8.4)
3.33 m
3.30 m
3.33 m
104.7
74.7
77.4
71.2
78.0
62.4
4.43 d (8.0)
3.20 t (8.0)
3.30 m
3.30 m
3.32 m
3.62 m, 3.86 m
3.60 m, 3.86 m
Glc-III
1
2
3
4
5
6
105.4
75.3
77.3
70.7
77.5
62.0
4.44 br d (7.6)
3.29 m
3.46 m
3.41 m
3.27 m
105.5
75.3
77.4
70.7
77.5
62.0
4.41 br d (7.2)
3.28 m
3.42 m
3.40 m
3.28 m
1
et al.19) By comparison of H- and 13C-NMR data of 1 with
those of quercetin 3-O-(2G-p-coumaroyl-3G-O-b-L-arabi-
nosyl-3R-O-b-D-glucosylrutinoside), the only difference was
that the C-3 substituent of Glc-I of rutin was an arabinosyl in
the known flavonoid tetraglycoside instead of a glucosyl in 1.
Hence compound 1 was characterized as quercetin 3-O-[2G-
(E)-coumaroyl-3G-O-b-D-glucosyl-3R-O-b-D-glucosylruti-
3.71 m, 3.76 m
3.72 m, 3.75 m
Rha
1
2
3
4
5
6
102.2
71.2
82.9
72.4
69.4
17.9
4.62 br s
4.01 br s
3.64 m
3.46 m
3.52 m
102.2
71.3
83.0
72.4
69.4
18.0
4.60 br s
4.00 br s
3.60 m
3.48 m
3.49 m
1
noside]. Complete H- and 13C-NMR chemical shifts were
1
established by H–1H COSY, HMQC, HMBC, and NOESY
1.11 d (6.4)
1.11 d (6.4)
spectra.
Coumaroyl
Compound 2 was isolated as a light yellow amorphous
solid. Its HR-FAB-MS spectrum showed a quasi-molecular
ion peak at m/z 1065.3080 [MꢀH]ꢀ, which corresponded
to the molecular formula C48H56O27, indicating 21 degrees
of unsaturation. The IR spectrum of 2 indicated signals
for hydroxyl (3383 cmꢁ1), conjugated ester (1698 cmꢁ1),
conjugated ketone (1654 cmꢁ1), and phenyl group (1601,
1518 cmꢁ1) functionalities. The UV spectrum showed
1ꢄ
127.3
131.4
116.7
161.0
147.4
115.1
168.8
127.3
131.4
116.8
161.1
147.3
115.1
168.7
2ꢄ, 6ꢄ
3ꢄ, 5ꢄ
4ꢄ
7ꢄ
8ꢄ
7.45 d (8.8)
6.79 d (8.8)
7.45 d (8.8)
6.80 d (8.8)
7.67 d (16.0)
6.41 d (16.0)
7.67 d (15.6)
6.41 d (15.6)
9ꢄ
absorption bands at 267 and 355 nm characteristic of The 1H- and 13C-NMR data of 1 and 2 were essentially iden-
1
kaempferol.15) The H-NMR spectrum of 2 (Table 1) also tical in the carbohydrate region, revealing that the two com-
showed the resonances for a kempferol moiety [dH 6.15 (1H, pounds exhibited the same sugar compositions and linkages.
br d, Jꢂ2.0 Hz, H-6); 6.34 (1H, br d, Jꢂ2.0 Hz, H-8); 7.96 The molecular weight of 2 was 16 mass units less than that of
(2H, d, Jꢂ8.0 Hz, H-2ꢃ, 6ꢃ); 6.90 (2H, d, Jꢂ8.8 Hz, H-3ꢃ, 1, which supported the conjecture that the aglycone of 2 was
5ꢃ)], a (E)-coumaroyl group [dH 7.45 (2H, d, Jꢂ8.8 Hz, kaempferol instead of quercetin in 1. Accordingly, compound
H-2ꢄ, 6ꢄ); 6.80 (2H, d, Jꢂ8.8 Hz, H-3ꢄ, 5ꢄ); 7.67 (1H, d, 2 was elucidated as kaempferol 3-O-[2G-(E)-coumaroyl-3G-
Jꢂ15.6 Hz, H-7ꢄ); 6.41 (1H, d, Jꢂ15.6 Hz, H-8ꢄ)], and four O-b-D-glucosyl-3R-O-b-D-glucosylrutinoside].
anomeric protons [dH 5.54 (1H, d, Jꢂ8.0 Hz, H-1 of Glc-I);
Compounds 1 and 2 were evaluated for their antioxidant
4.43 (1H, d, Jꢂ8.0 Hz, H-1 of Glc-II); 4.41 (1H, br d, activities through scavenging activity assay against DPPH
Jꢂ7.2 Hz, H-1 of Glc-III); 4.60 (1H, br s, H-1 of Rha-I)]. radical with BHT (2,6-di-tert-butyl-4-methylphenol) and

June 2008
853
quercetin as positive controls (EC50ꢂ56.8, 7.2 mM).20) Com-
pound 1 showed moderate scavenging activity with an EC50
value of 30.5 mM, while compound 2 exhibited weaker scav-
enging activity with an EC50 value of 487.2 mM.
with EtOAc, the aqueous layer was repeatedly evaporated with MeOH
under vacuum to remove the solvent completely. Purification of the sugar
mixture was achieved by preparative silica TLC (developing solvent
CH2Cl2–MeOH–H2O, 15 : 5 : 1) to yield L-rhamnose (5.1 mg, [a]D25 9.5°,
cꢂ0.5, MeOH) and D-glucose (11.3 mg, [a]D25 38.1°; cꢂ0.6, MeOH), re-
spectively.16)
Determination of Scavenging Activity against DPPH Radical Scav-
enging activity against DPPH radical was determined by a modified method
according to Shimada et al.20) DPPH radical was prepared in methanol as a
400 mM solution. DPPH solution (150 ml) was added to 50 ml of different
tested samples in methanol (final concentration was 10—1000 mM) in each
well of a 96-well flat-bottom EIA microtitration plate. The mixture was
shaken vigorously and kept in the dark for 90 min. The absorbance was
measured on a microplate spectrophotometer (SpectraMax Plus384, Molec-
ular Devices, CA, U.S.A.) at 517 nm against methanol without DPPH as the
blank reference. The percent DPPH scavenging effect was calculated using
the following equation:
Experimental
General Experimental Procedures Optical rotations were measured by
Optical Activity AA-10R automatic spectropolarimeter. UV spectra were
measured in MeOH on a Shimadzu UV-1700 spectrophotometer. IR spectra
were recorded on a Bruker vector 22 FT-IR spectrometer. NMR spectra were
recorded in CD3OD at room temperature on a Varian Mercury plus 400
NMR spectrometer, and the solvent resonance was used as internal shift ref-
erence (TMS as standard). The 2D NMR spectra were recorded using stan-
dard pulse sequences. FAB-MS and HR-FAB-MS spectra were recorded on
a Finnigan/Thermo Quest MAT 95XL spectrometer. TLC was performed
using silica gel 60 F254 plates (Merck). Diaion HP-20 (Mitsubishi) and
Sephadex LH-20 (Pharmacia biotech) were used for column chromatogra-
phy. HPLC was performed on a Hitachi L-7000 chromatograph with a
Thermo Betasil C-18 (5 mm) column (250ꢅ10 mm).
scavenging effect (%)ꢂ[1ꢁ(absorbance of sample/absorbance of
control)]ꢅ100
Plant Material The tea plant (Camellia sinensis L.) was cultivated in
Lugu village, Nantou County, Taiwan. Oolong tea was prepared by a local
manufacturer, Mr. Jim-Fang Huang, in November, 2006. A voucher speci-
men has been deposited in Graduate Institute of Biotechnology, National
Pingtung University of Science and Technology.
Acknowledgements This research was supported by grants from the
National Science Council of the Republic of China (NSC 94-2317-B-020-
001 and NSC 96-2317-B-020-003). We thank Mr. Jim-Fang Huang for his
kind gifts of oolong tea and Ms. Lih-Mei Sheu for the MS measurement in
the Instrumentation Center of the College of Science, National Chung Hsing
University.
Extraction and Isolation Oolong tea (500 g) was powdered and ex-
tracted three times with methanol (4 l) at room temperature (7 d each). The
methanol extract was evaporated in vacuo to generate a black residue, which
was suspended in H2O (500 ml) then partitioned sequentially using EtOAc
and n-BuOH (500 mlꢅ3). The n-BuOH fraction (45 g) was subjected to a
Diaion HP-20 column (6ꢅ55 cm), and eluted with mixtures of water and
methanol of reducing polarity as eluents. Eleven fractions were collected as
follows: fr. 1 [500 ml, water], fr. 2 [500 ml, water–methanol (9 : 1)], fr. 3
[1000 ml, water–methanol (8 : 2)], fr. 4 [1000 ml, water–methanol (7 : 3)], fr.
5 [1000 ml, water–methanol (6 : 4)], fr. 6 [1000 ml, water–methanol (5 : 5)],
fr. 7 [1000 ml, water–methanol (4 : 6)], fr. 8 [1000 ml, water–methanol
(3 : 7)], fr. 9 [1000 ml, water–methanol (2 : 8)], fr. 10 [(1000 ml, water–
methanol (1 : 9))], and fr. 11 (3000 ml, methanol). Fraction 8 (6.1 g) was
further chromatographed on a Sephadex LH-20 column (3ꢅ45 cm), eluted
with water–methanol (1 : 1 to 0 : 1) to afford nine fractions (each 500 ml),
fr. 8A—fr. 8I. Fr. 8D (105.2 mg) was further purified by preparative HPLC
using a Thermo Betasil C-18 column eluted with water–acetonitrile (9 : 1
to 1 : 1), 2 ml/min, to afford 1 (19.1 mg, tRꢂ19.7 min) and 2 (21.1 mg, tRꢂ
20.9 min), respectively.
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1
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Acid Hydrolysis of 1, 2 and the Saponin Mixture Compound 1 (5 mg)
was separately dissolved in 2 N CF3COOH (3 ml) and refluxed for 5 h. The
reaction mixture was diluted with water (3 ml) and extracted with EtOAc
(6 mlꢅ3). Through TLC comparison with authentic samples using
CH2Cl2–MeOH (9 : 1) as a developing system, trans-p-coumaric acid (Rf
0.40) and quercetin (Rf 0.70) were detected in the EtOAc layer.21) The aque-
ous layer was also identified by TLC on Si gel (developing solvent
CH2Cl2–MeOH–H2O, 10 : 5 : 1) as rhamnose (Rf 0.65) and glucose (Rf 0.45)
in comparison with authentic samples. Acid hydrolysis of compound 2 by
the same method for 1 led to decomposition of coumaric acid, kaempferol,
rhamnose, and glucose. A 100-mg aliquot of the crude saponin mixture was
dissolved in 2 N CF3COOH (10 ml) and refluxed for 5 h. After extraction
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