Tea catechins and flavonoids from the leaves of Camellia sinensis inhibit yeast alcohol dehydrogenase

Article abstract of DOI:10.1016/j.bmc.2012.02.002

Four new quercetin acylglycosides, designated camelliquercetisides A-D, quercetin 3-O-[α-l-arabinopyranosyl(1→3)][2-O'-(E)-p-coumaroyl] [β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→6)] -β-d-glucoside (17), quercetin 3-O-[2-O'-(E)-p-coumaroyl][β-d- glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→6)] -β-d-glucoside (18), quercetin 3-O-[α-l-arabinopyranosyl(1→3)] [2-O'-(E)-p-coumaroyl][α-l-rhamnopyranosyl(1→6)]-β-d-glucoside (19), and quercetin 3-O-[2-O'-(E)-p-coumaroyl][α-l- rhamnopyranosyl(1→6)]-β-d-glucoside (20), together with caffeine and known catechins, and flavonoids (1-16) were isolated from the leaves of Camellia sinensis. Their structures were determined by spectroscopic (1D and 2D NMR, IR, and HR-TOF-MS) and chemical methods. The catechins and flavonoidal glycosides exhibited yeast alcohol dehydrogenase (ADH) inhibitory activities in the range of IC₅₀ 8.0-70.3 μM, and radical scavenging activities in the range of IC₅₀ 1.5-43.8 μM, measured by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.

Full text of DOI:10.1016/j.bmc.2012.02.002

Bioorganic & Medicinal Chemistry 20 (2012) 2376–2381
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tea catechins and flavonoids from the leaves of Camellia sinensis inhibit
yeast alcohol dehydrogenase
Md. Maniruzzaman Manir a, Jeong Kee Kim b, Byeong-Gon Lee b, Surk-Sik Moon a,
⇑
a Department of Chemistry, Kongju National University, Gongju 314-701, Republic of Korea
b Research Institute, AmorePacific R&D Center, Yongin 446-729, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new quercetin acylglycosides, designated camelliquercetisides A–D, quercetin 3-O-[
a
-
L
-arabinopyr
-rhamnopyranosyl(1?6)]-b- -gluco-
-glucopyranosyl(1?3)- -rhamnopyranosyl(1?6)]-b-
-arabinopyranosyl(1?3)][2-O00-(E)-p-coumaroyl][
-rhamnopyrano
-glucoside (19), and quercetin 3-O-[2-O00-(E)-p-coumaroyl][
-rhamnopyranosyl(1?6)]-
-glucoside (20), together with caffeine and known catechins, and flavonoids (1–16) were isolated from
a n o s y l (1?3)][2-O00-ðEÞ-p-coumaroyl][b-
D
-glucopyranosyl(1?3)-
a-L
D
Received 21 December 2011
Revised 1 February 2012
Accepted 1 February 2012
Available online 9 February 2012
side (17), quercetin 3-O-[2-O00-(E)-p-coumaroyl][b-
D
a-L
D
-glucoside (18), quercetin 3-O-[
syl (1?6)]-b-
b-
a-L
a-L
D
a-L
D
Keywords:
Green tea
the leaves of Camellia sinensis. Their structures were determined by spectroscopic (1D and 2D NMR, IR,
and HR-TOF-MS) and chemical methods. The catechins and flavonoidal glycosides exhibited yeast alcohol
dehydrogenase (ADH) inhibitory activities in the range of IC50 8.0–70.3 lM, and radical scavenging activ-
Camellia sinensis
Theaceae
Quercetin glycosides
Camelliquercetisides
Alcohol dehydrogenase (ADH)
ities in the range of IC50 1.5–43.8
l
M, measured by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
research has been performed on the chemical ingredients of green
tea that interact with the alcohol metabolizing enzymes ADH and
ALDH. Recently, we have reported isolation and characterization of
triterpenoidal saponins from the green tea root, which showed
strong inhibitory effects against ADH.18 In an ongoing search for bio-
logically active compounds from medicinal plants,19,20 we have
found that the extract of tea leaves strongly inhibits ADH. Here, we
report the isolation and characterization of four new acylated quer-
cetin glycosides, named camelliquercetisides A–D (17–20), along
with 16 known isolates from the tea leaves. Their ADH-inhibitory
and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activ-
ities are reported.
Green tea from the plant Camellia sinensis has a long history of use
as a social, medicinal, and health drink.1,2 The medicinal effects of
green tea are mainly attributed to caffeine, theanine, catechins,
and polyphenols.3–6 These compounds have been shown to exhibit
antibacterial, anticarcinogenic, and antioxidant properties and play
a protective role against cognitive deficits and coronary heart
disease.7–10 A crude extract of green tea and its main components,
catechins and caffeine, have been found to reduce the concentration
of ethanol and acetaldehyde in the blood and liver of ICR mice.11
Consumed ethanol is mostly metabolized to acetaldehyde by NAD-
dependent alcohol dehydrogenase (ADH) and further to acetic acid
by NAD-dependent aldehyde dehydrogenase (ALDH), mostly in the
liver (Fig. 1). Accumulation of acetaldehyde in the body is considered
to be the main cause of hangovers, which are characterized by
unpleasant symptoms such as headache, nausea, vomiting, thirst,
dizziness, vertigo, and other decreased sensory abilities, after the
heavy drinking of alcohol.12,13 The intermediate acetaldehyde is
potently toxic, mutagenic, and carcinogenic. It is believedto be a ma-
jor factor in the development of alcohol-associated cancers, liver and
pancreatic diseases, as well as coronary heart disease.14–17 Oxida-
tion of ethanol to acetaldehyde is thought to be a convenient and
useful means of preventing alcoholic disorders. However, little
2. Results and discussion
The extract of green tea leaves (Camellia sinensis) was sus-
pended in H2O and partitioned successively with n-hexane and
n-BuOH. The n-BuOH layer was subjected to multiple chromato-
graphic purification steps including reversed-phase C18 flash col-
umn chromatography and preparative C18 HPLC to produce four
new (17–20) and sixteen known compounds (1–16) (Fig. 2).
Compounds 1–16 were identified by comparison of their spec-
troscopic data with those reported in the literature, as (ꢀ)-
epigallocatechin (EGC) (1),21 caffeine (2), (+)-catechin (3),21
(ꢀ)-epigallocatechin gallate (EGCG) (4),21 (ꢀ)-epicatechin gallate
(ECG) (5),21 myricetin 3-O-b- -galactopyranoside (6),22 myricetin
D
⇑
Corresponding author. Tel.: +82 41 850 8495; fax: +82 41 850 8479.
E-mail address: ssmoon@kongju.ac.kr (S.-S. Moon).
3-O-b-
D
-glucopyranoside (7),23 (ꢀ)-epicatechin 3-O-(30-O-methyl)
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.002

M. M. Manir et al. / Bioorg. Med. Chem. 20 (2012) 2376–2381
2377
ADH
ALDH
NAD+
NADH
O
C
H
C
H
H
C
H3C
OH
H3C
OH
H3C
O
NAD+
NADH
Figure 1. Metabolic pathway for alcohol: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; NAD+, nicotinamide adenine dinucleotide; and NADH, a reduced
form of NAD+.
gallate (8),24 (ꢀ)-epiafzelechin gallate (9),25 (ꢀ)-epigallocatechin
3-O-(Z)-p-coumaroate (10),26 (ꢀ)-epigallocatechin 3-O-(E)-p-coumar-
br s) for the A ring; dH 7.67 (1H, d, J = 16.0 Hz), 7.44 (2H, d,
J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), and 6.39 (1H, d, J = 16.0 Hz) for a
p-coumaric acid moiety; and dH 5.56 (1H, d, J = 8.0 Hz), 4.61 (1H,
br s), 4.45 (1H, d, J = 7.6 Hz), and 4.33 (1H, d, J = 6.8 Hz) for the ano-
meric protons of four carbohydrate units. Additionally, the corre-
sponding anomeric carbons of the carbohydrates were observed at
d 101.0, 102.3, 105.9, and 105.3 in the 13C NMR spectra, assigned
withassistance from the HSQC experiment. Acid hydrolysis of 17 fol-
lowed by TLC analysis identified glucose, rhamnose, and arabinose
as the carbohydrate moieties. Their absolute configurations were
oate (11),27 quercetin 3-O-b-
D
-glucopyranosyl(1?3)-
a
-
L
-rhamnopyr
-gluco-
-galactopyranoside
-glucopyranosyl(1?3)- -rhamnopyr-
-galactopyranoside (14),28 kaempferol 3-rutinoside
(15),29 and kaempferol 3-O-b- -galactopyranoside (16).22
anosyl(1?6)-b-
pyranosyl(1?3)-
(13),28 kaempferol 3-O-b-
anosyl(1?6)-b-
D
-glucopyranoside (12),28 quercetin 3-O-b-
D
a-L
-rhamnopyranosyl(1?6)-b-
D
D
a-L
D
D
Compounds 17–20 were obtained as pale brown solids. The
molecular formula of 17 was determined to be C47H54O27 based on
its HR-TOF-MS (m/z 1051.3065 [M+H]+, positive ESI mode). The 1H
NMR and 1H–1H COSY data of 17 suggested the presence of querce-
tin, trans-4-hydroxycinnamic [(E)-p-coumaric] acid, and four mono-
carbohydrate moieties: dH 7.59 (1H, d, J = 2.0 Hz), 7.54 (1H, dd,
J = 8.4, 2.0 Hz), and 6.87 (1H, d, J = 8.4 Hz) for a 3,4-disubstitution
on the B ring of the quercetin unit; dH 6.33 (1H, br s) and 6.13 (1H,
determined to be D-, L-, and L-, respectively, from HPLC analysis of
the chiral-derivatized sample of the hydrolysate using (R)- or (S)-
(ꢀ)-1-phenylethylamine.30,31 The HMBC correlations established
the glycosidic linkages as follows: dH 5.56 (H-1, Glc1) to dC 134.9
(C-3 of quercetin unit), dH 4.61 (H-1, Rha) to dC 68.5 (C-6, Glc1), dH
4.45 (H-1, Glc2) to dC 83.1 (C-3, Rha), dH 4.33 (H-1, Ara) to dC 84.5
OH
OH
O
N
OH
OH
OH
N
N
HO
O
HO
O
O
N
OH
OR
OH
OH
3
2
1
4
(EGC) R = H
(EGCG) R = Galloyl
10
11
R = (Z)-p-coumaroyl
R = (E)-p-coumaroyl
OH
OH
OH
OH
OH
HO
O
HO
O
O
HO
O
O
OH
OH
OH
OH
OH
O
O
OH
OR
O
OH
OH
OH
OR
5 (ECG) R = H
6
R = Gal
9
8
7 R = Glc
R = CH3
OH
OH
HO
O
O
OH
OH
OH
O
OH
HO
O
HO
O
O
OH
O
Rha
HO
HO
O
OR
O
OR
O
Glc
R1
O
OH
O
OH
O
R2
O
OH
14
15
16
12
13
R = Glc(1 3)Rha(1 6)Glc
R = Rha(1 6)Glc
R = Gal
R = Glc(1 3)Rha(1 6)Glc
R = Glc(1 3)Rha(1 6)Gal
17 R1 = α-L-Ara R2 = β-D-Glc
18 R1 = H
R2 = β-D-Glc
1
R = -L-Ara R2 = H
19
20
α
R1 = H
R2 = H
Figure 2. Chemical structures of isolates 1–20 from the leaves of C. sinensis.

2378
M. M. Manir et al. / Bioorg. Med. Chem. 20 (2012) 2376–2381
(C-3, Glc1), dH 3.87 (H-3, Glc1) to dC 105.3 (C-1, Ara), dH 3.61 (H-3,
Rha) to dC 105.9 (C-1, Glc2), and dH 3.88 and 3.52 (H-6, Glc1) to dC
102.3 (C-1, Rha). 3-O-glycosylation at the quercetin moiety was fur-
It is notable none of the isolates showed any significant ALDH-
inhibitory or activating activities (data not shown), unlike disulfi-
ram. The isolates were also tested for radical scavenging activities
using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Table 2).
The new isolates 17–20 exhibited moderately good radical-scav-
ther evidenced by the upfield shift of C-3 (
D
d 2.1 ppm) and the
d 2.2 ppm),
downfield shift of both C-2 ( d 11.9 ppm) and C-4 (
D
D
compared to those of unsubstituted quercetin.32 The attachment
of a p-coumaroyl group to C-2 of the Glc1 moiety was established
from the HMBC correlation [H-2 (d 5.22, Glc1) to the carbonyl carbon
(d 168.6, coumaroyl group)]. It is notable that the H-2 of Glc1 was
downfield-shifted compared to that of Glc2 due to the ester linkage.
The configurations of the two glucopyranosyl, rhamnopyranosyl,
enging activities in the IC50 range of 19.8–29.2
lM, while tocoph-
erol (Vit. E) showed an IC50 of 11.4 M. The DPPH-radical
l
scavenging activities of the isolates do not seem to positively cor-
relate with the ADH activities. The yeast ADH used in this study is a
metallodehydrogenase containing stoichiometric amounts of zinc and
having a high specificity towards ethanol.33,35–37 The ADH-inhibitory
effects of the isolates may be related to their metal-chelating ability as
in zinc-chelating pyrazoles and o-phenanthroline.38,39 Interestingly,
phytophenols in whiskey were reported to depress alcohol
metabolism through ADH inhibition.40 Green tea and tea products
are proven to be rich in catechins and flavonoidal polyphenols by
numerous quantifying techniques.41,42
In the present study, we have demonstrated that catechins and
flavonoids including four new quercetin glycosides isolated from
tea leaves showed strong yeast ADH-inhibitory activities as well
as DPPH-radical scavenging activities. Although yeast ADH may
not behave like human ADHs and oval or colonic bacterial
ADHs,33,37,43 consumption of green tea or tea extract products
could help prevent alcohol metabolism and the release of toxic
acetaldehyde (or even detrimental formaldehyde, a metabolite of
methanol), alleviating alcohol hangovers and reducing a risk of
developing alcohol-related health disorders.
and arabinopyranosyl units were establishedas b, b, a, and a, respec-
tively, from their respective coupling constants (JH1–H2 8.0, 7.6, ꢁ0,
6.8 Hz). Thus, the structure of 17 was determined to be quercetin
3-O-[
copyranosyl(1?3)-
designated camelliquercetiside A.
a
-
L
-arabinopyranosyl(1?3)][2-O00-(E)-p-coumaroyl][b-
D
-glu-
a-L-rhamnopyranosyl(1?6)]-b-D-glucoside,
Compound 18 showed similar 1H and 13C NMR spectral patterns
to 17 except that the arabinopyranosyl unit was not observed. The
molecular formula was determined to be C42H46O23 from HR-TOF-
MS analysis ([M+H]+ m/z 919.2536). The configurations of the two
glucopyranosyl units and the rhamnopyranosyl unit were estab-
lished as b, b, and
constants (JH1–H2 8.0, 7.6, and ꢁ0 Hz). Thus, the structure of 18
was established as quercetin 3-O-[2-O00-(E)-p-coumaroyl][b-
glucopyranosyl(1?3)- -rhamnopyranosyl(1?6)]-b- -glucoside,
a, respectively, from their respective coupling
D
-
a
-
L
D
camelliquercetiside B. The presence of the carbohydrate units was
confirmed from the analysis of acid hydrolysate and their connec-
tivity was confirmed from the interpretation of HMBC spectral data.
3. Experimental
Compound 19, with
a molecular formula of C41H44O22
determined by the HR-TOF-MS analysis, exhibited similar NMR
spectroscopic patterns compared to 17. However, the terminal glu-
copyranosyl moiety in 17 was not observed in 19. The linkage of the
arabinopyranosyl unit to the glucopyranosyl unit was confirmed
from the HMBC [H-3 (d 3.85, Glc) to C-1 (d 105.3, Ara) and H-1
(Ara) to C-3 (Glc)] and the ROESY correlations [NOE between H-3
(d 3.85, Glc) and H-1 (d 4.32, Ara)]. The a-configuration of the ara-
binopyranosyl unit was confirmed from the coupling constant
(J = 6.8 Hz) of H-1 at d 4.32. Thus, the structure of 19 was
3.1. General experimental procedures
Melting points were measured on a Fisher melting point appa-
ratus and are uncorrected. Optical rotations were measured on a
Perkin Elmer 341-LC polarimeter. IR spectra were recorded on a
Perkin Elmer BX FT-IR spectrometer. NMR spectra were recorded
on a Varian Mercury 400 spectrometer with standard pulse se-
quences operating at 400 MHz for 1H NMR and 100 MHz for 13C
NMR. Chemical shifts, measured in ppm, were referenced to sol-
vent peaks (dH 3.31 and dC 49.15 for CD3OD). HR-TOF-MS spectra
were recorded in the positive ESI mode on a Waters LCT Premier
mass spectrometer coupled with a Waters AQUITY HPLC. Flash col-
determined to be quercetin 3-O-[
a
-
L
-arabinopyranosyl(1?3)]
[2-O00-(E)-p-coumaroyl][
a-L-rhamnopyranosyl(1?6)]-b- -glucoside,
D
camelliquercetiside C.
Compound 20 showed two anomeric proton signals at d 5.52
(J = 8.0 Hz) and 4.56 (br s) with the respective carbon signals at d
100.9 and 102.3, which indicated the presence of two carbohydrate
moieties. The molecular formula was determined to be C36H36O18
from the HR-TOF-MS analysis. Acid hydrolysis and chiral derivati-
umn chromatography was conducted on a C18 column (40–63 lm,
90 id ꢂ 180 mm, Merck). Medium-pressure liquid chromatography
(MPLC) was conducted on reversed-phase C18 silica gel (Cosmosil
40 C18 Prep, 40 id ꢂ 320 mm) at 5 mL/min. Preparative C18 HPLC
was performed on a Waters 600 model system equipped with a
photodiode array UV detector 996 using a Varian Dynamax HPLC
column (Microsorb 100-5, C18, 21.4 id ꢂ 250 mm) at 7 mL/min
with a UV detection at 220 or 270 nm. HPLC analyses of the (S)-
(ꢀ)-1-phenylethylamine derivatives of the hydrolysate of flavonoi-
dal glycosides and standard monosaccharides were performed on a
zation identified the two monosaccharides as
-rhamnose. From the spectroscopic data (COSY, HSQC, and HMBC
spectra) (Table 1), compound 20 was characterized as quercetin
3-O-[2-O00-(E)-p-coumaroyl][
-rhamnopyranosyl(1?6)]-b- -glu-
coside, camelliquercetiside D.
D-glucose and
L
a-
L
D
ADH activities of isolates 1–20 were evaluated using the yeast
ADH enzyme.33 These isolates showed strong inhibitory effects
Varian Chromsep HPLC column (Microsorb 100-5, C18
, 4.6
id ꢂ 250 mm) with a 0.8 mL/min gradient elution of 30 to 46%
aqueous MeCN for 60 min at 220 nm. TLC was performed on pre-
coated silica gel plates (Kieselgel 60, F254, Merck). Spots were de-
tected by staining with a solution of p-anisaldehyde-sulfuric acid
in methanol, followed by heating. Optical density for a 96-well
microplate was measured on a Tecan Sunrise microplate reader
at 340 and 517 nm for the ADH and DPPH assays, respectively.
(R)-1-Phenylethylamine, (S)-(ꢀ)-1-phenylethylamine, NaBH3CN,
ADH (alcohol dehydrogenase from Saccharomyces cerevisiae, EC
1.1.1.1, >90% protein, 331 units/mg), b-NAD+ (b-nicotinamide ade-
nine dinucleotide, oxidized form), ethanol (99.5%), and tetraethyl-
on ADH in the IC50 range of 8.0–70.3 lM, except caffeine 2,
whereas disulfiram, an alcohol aversive drug, known to be an
excellent inhibitor of both ADH and ALDH,34 inhibited ADH at an
IC50 of 247.9 lM (Table 2). Isolate 11 (EGC coumarate) was found
to be the strongest and appeared to be more effective than 1
(EGC), which is likely due to an additional p-hydroxycinnamoyl
group. The enhanced effect of an additional p-hydroxycinnamoyl
on the inhibition of ADH was also observed in 18, as compared
to 1. Attachment of an arabinopyranosyl group at the C-3 position
of Glc did not seem to show a considerable effect (17 vs 18; 19 vs
20). All flavonoidal glycosides, including newly isolated quercetin
glycosides 17–20, were more or less comparable to tea catechins.

M. M. Manir et al. / Bioorg. Med. Chem. 20 (2012) 2376–2381
2379
Table 1
1H and 13C NMR spectroscopic data of compounds 17–20 in CD3ODa
Position
17
18
19
20
dc
dH (J in Hz)
dc
dH (J in Hz)
dc
dH (J in Hz)
dc
dH (J in Hz)
Quercetin
2
3
4
5
158.8
134.9
178.8
162.9
99.8
—
—
—
—
158.7
134.9
178.9
162.9
99.8
—
—
—
—
158.7
134.8
178.8
162.9
99.8
—
—
—
—
158.7
134.9
178.9
162.9
99.8
—
—
—
—
6
6.13, br s
6.15, d (2.0)
6.14, d (2.0)
6.15, d (1.6)
7
8
165.5
94.8
—
165.5
94.8
165.6
94.8
—
165.5
94.8
—
6.33, br s
6.34, d (1.6)
6.33, d (2.0)
6.33, d (1.6)
9
158.3
105.5
123.2
117.5
145.8
149.5
116.2
123.4
—
—
—
158.2
105.6
123.2
117.5
145.7
149.5
116.1
123.5
—
—
—
158.2
105.8
123.1
117.4
145.8
149.6
116.1
123.5
—
—
—
158.2
105.8
123.2
117.4
145.8
149.5
116.1
123.5
—
—
—
10
10
20
30
40
50
60
7.59, d (2.0)
—
—
6.87, d (8.4)
7.54, dd (8.4, 2.0)
7.59, d (2.0)
—
—
6.87, d (8.4)
7.55, d (8.4, 1.6)
7.57, d (2.0)
—
—
6.87, d (8.4)
7.55, d (8.4, 2.0)
7.57, br s
—
—
6.86, d (8.0)
7.55, dd (8.4, 2.0)
p-coumaric acid
1
127.3
131.3
116.7
161.1
115.1
147.2
168.6
—
127.2
131.2
116.8
161.1
115.2
146.9
168.5
—
127.3
131.3
116.8
161.1
115.1
147.2
168.6
—
127.2
131.2
116.8
161.1
115.2
146.9
168.5
—
2, 6
3, 5
4
a
b
7.44, d (8.4)
6.79, d (8.4)
—
6.39, d (16.0)
7.67, d (16.0)
—
7.44, d (8.4)
6.80, d (8.8)
—
6.37, d (16.0)
7.68, d (16.0)
—
7.44, d (8.8)
6.80, d (8.4)
—
6.38, d (16.0)
7.66, d (16.0)
—
7.44, d (8.4)
6.80, d (8.4)
—
6.38, d (16.0)
7.67, d (16.0)
—
C@O
Glc1
1
2
3
4
101.0
74.6
84.5
70.2
76.8
68.5
5.56, d (8.0)
5.22, t (8.6)
3.87
3.50
3.54, m
101.0
75.8
76.3
71.0
77.6b
68.6
5.50, d (8.0)
5.04, t (8.6)
3.62, t (9.2)
3.40, dd (9.2, 8.8)
3.43, m
100.8
74.6
84.5
70.3
77.0
68.3
5.56, d (8.0)
5.21, dd (9.6, 8.0)
3.85, t (8.4)
3.48
3.49, m
3.87
100.9
75.8
76.3
71.7
77.4
68.4
5.52, d (8.0)
5.03, t (8.8)
3.61, t (9.0)
3.37, t (8.4)
3.40, m
5
6
3.88, dd (12.0, 2.4)
3.52
3.87, br d (10.8)
3.51
3.87, br d (10.0)
3.45
3.46
Glc2
1
2
3
4
105.9
75.6
4.45, d (7.6)
3.27, t (8.0)
3.42, t (9.0)
3.39, t (8.8)
3.26, m
105.9
75.5
77.3
71.7
77.6b
62.2
4.45, d (7.6)
3.28, t (8.4)
3.44, m
3.39, dd (9.2, 8.8)
3.26, m
77.6b
71.0
5
6
77.6b
62.2
3.74, dd (12.0, 2.0)
3.71, dd (12.0, 4.4)
3.76, dd (11.6, 2.0)
3.70, dd (11.6, 4.0)
Rha
1
2
3
4
102.3
71.3
83.1
72.6
69.56
18.2
4.61, br s
102.2
71.4
83.2
72.6
69.5
18.2
4.60, br s
3.97, br s
3.61, t (9.2
3.45
3.52, m
1.12, d (6.0)
102.4
72.2
4.57, br s
3.68, dd (3.2, 1.6)
3.54
3.27, t (9.6)
3.45, m
1.13, d (6.4)
102.3
72.2
72.3
74.0
69.8
18.1
4.56, br s
3.99, dd (3.2, 1.6)
3.61, dd (9.2, 3.2)
3.45, t (9.2)
3.51, m
3.66, dd (3.1, 1.7)
3.54, dd (10.0, 3.2)
3.26, t (9.2)
3.48, m
1.13, d (6.0)
72.3b
74.0c
69.9
5
6
1.13, d (6.0)
18.1
Ara
1
2
3
4
105.3
72.3
74.0
69.57
67.3
4.33, d (6.8)
3.53
3.47, dd (8.8, 3.6)
3.77, m
105.3
74.0c
4.32, d (6.8)
72.3b
3.47, dd (8.8, 3.6)
69.9
3.53
3.77
5
3.85
67.3
3.84, m
3.56, m
3.56
a
1H and 13C NMR measured at 400 and 100 MHz, respectively. The assignments are based on 1H–1H COSY, TOSCY, HSQC, HMBC, and ROESY experiments. Overlapped
signals are reported without designating multiplicity.
b,c
Overlapped within the same columns
thiuram disulfide (disulfiram, 97%) were purchased from Sigma–
Aldrich Chemical Co.
the Natural Product Chemistry Laboratory, Department of Chemis-
try, Kongju National University, Republic of Korea.
3.2. Plant material
3.3. Extraction and isolation
Green leaves of Camellia sinensis were freshly picked from a
20-year-old Yabukita cultivar in Jeju Island, a southern island of
the Republic of Korea, in May, 2005, and used for extraction after
immediate steaming and air-drying (also available in a local mar-
ket as Uksoocha). A voucher specimen (CS78) was deposited at
The leaves of Camellia sinensis (1.2 kg) were sequentially ex-
tracted with 80% aqueous MeOH (3 ꢂ 10 L, 1 day each time) and
CH2Cl2–MeOH (1:1, 10 L, 7 days) at room temperature. The com-
bined extracts were concentrated under reduced pressure. The
resulting dark green residue (585 g) was suspended in H2O and

2380
M. M. Manir et al. / Bioorg. Med. Chem. 20 (2012) 2376–2381
Table 2
Alcohol dehydrogenase (ADH) and radical scavenging (DPPH) activities of compounds 1–20
Compound
Activity (IC50
,
l
M)a
Compound
Activity (IC50, l
M)a
ADH
16.5 2.1
DPPH
ADH
19.7 1.5
DPPH
1
2
3
4
5
6
7
8
16.2 0.2
>100
12
13
14
15
16
17
18
19
7.7 0.4
30.1 0.2
>100
>100
>100
19.8 0.1
22.0 0.3
31.9 1.4
29.2 0.1
196.7 26.8
21.1 1.3
42.8 3.2
65.5 4.1
18.2 1.9
19.9 1.5
44.1 2.1
44.3 2.2
22.1 0.1
8.0 0.5
41.0 4.1
17.6 1.3
40.4 3.4
70.3 7.7
17.3 1.3
13.7 0.6
46.7 5.3
41.5 1.1
4.3 0.1
8.3 0.4
1.5 0.0
3.5 0.0
28.0 2.1
10.8 0.2
2.8 0.0
26.4 0.3
43.8 2.9
9
10
11
20
Disulfiramb
Tocopherolb
247.9 10.8
11.4 1.6
a
The experiments were performed in triplicate, and the average values are expressed with the standard errors of the mean.
Disulfiram: tetraethylthiuram disulfide; tocopherol: Vit. E.
b
extracted with n-hexane (6.4 L) and then with n-BuOH (4.8 L). A
portion (100 g) of the n-BuOH extract (360 g) was chromato-
graphed on a C18 flash column using a 10% stepwise gradient elu-
tion of increasing MeOH concentration in H2O (4 L each) to afford
eleven fractions. A portion (1.5 g) of the active 50% MeOH eluate
(5.7 g) was fractionated on a reversed-phase MPLC (30–60% MeOH)
1512, 1440, 1356, 1198, 1166 cmꢀ1 1H (CD3OD, 400 MHz) and
;
13C NMR (CD3OD, 100 MHz) data, Table 1; HR-TOF-MS (positive
ESI mode) m/z 919.2536 [M+H]+ (calcd for C42H46O23+H, 919.2508).
3.6. Camelliquercetiside C, quercetin 3-O-[
syl(1?3)][2-O00-(E)-p-coumaroyl][
-rhamnopyranosyl(1?6)]-
b- -glucoside (19)
a-L-arabinopyrano
a-L
to give ten subfractions (1–10). Subfractions
2 (53.0 mg), 3
D
(83.7 mg), 5 (330 mg), and 6 (85.7 mg) were purified to provide
compounds 1 (15.0 mg, tR 16.0 min), 2 (10.0 mg, tR 19.5 min), and
3 (8.3 mg, tR 20.6 min) from subfraction 2, compounds 4 (7.2 mg,
tR 26.7 min) and 5 (57.0 mg, tR 30.4 min) from subfraction 3, com-
pounds 6 (24.2 mg, tR 59.0 min), 7 (9.4 mg, tR 61.0 min), 8 (33.2 mg,
tR 63.7 min), 9 (9.9 mg, tR 65.0 min), 10 (14.8 mg, tR 68.0 min), and
11 (43.5 mg, tR 70.5 min) from subfraction 5, and compounds 12
(14.5 mg, tR 75.5 min) and 13 (17.0 mg, tR 77.7 min) from subfrac-
tion 6 by preparative C18 HPLC (0.05% trifluoroacetic acid-buffered
gradient elution of 10–15% aqueous MeCN for 20 min and 15–28%
aqueous MeCN for 80 min). Compounds 14 (73.4 mg, tR 26.0 min),
15 (4.83 mg, tR 29.5 min), and 16 (2.41 mg, tR 30.6 min) were ob-
tained from subfraction 7 (121.8 mg) by preparative C18 HPLC
(0.05% trifluoroacetic acid-buffered gradient elution of 22–28%
aqueous MeCN for 60 min). The 80% MeOH eluate (1.05 g, a deep
brown solid) from the flash column was fractionated by re-
versed-phase C18 MPLC (60–100% MeOH) to afford ten subfrac-
tions. Subfraction 5 (160 mg, a brown solid, 70% MeOH eluate)
was purified by preparative C18 HPLC (0.05% trifluoroacetic acid-
buffered gradient elution of 10–15% aqueous MeCN for 10 min
and 15–28% aqueous MeCN for 80 min) to afford compounds 17
(30.1 mg), 18 (15.4 mg), 19 (15.1 mg), and 20 (12.7 mg) with a tR
of 53.08, 56.6, 59.5 and 65.0 min, respectively.
A pale brown solid; mp 218–221 °C; ½a D20
ꢀ87.0 (c 0.1, MeOH);
ꢃ
UV (CH3OH) kmax (loge) 356 (4.56), 316 (4.45), 267 (4.33), 256
(4.31) nm; IR (neat) mmax 3352, 1734, 1647, 1602, 1514, 1440,
1356, 1195, 1166 cmꢀ1 1H (CD3OD, 400 MHz) and 13C NMR
;
(CD3OD, 100 MHz) data, Table 1; HR-TOF-MS (positive ESI mode)
m/z 889.2434 [M+H]+ (calcd for C41H44O22+H, 889.2402).
3.7. Camelliquercetiside D, quercetin 3-O-[2-O00-(E)-p-
coumaroyl][
a
-L
-rhamnopyranosyl(1?6)]-b-
D-glucoside (20)
A pale brown solid; mp 188–191 °C; ½a D20
ꢃ ꢀ101.54 (c 0.13,
MeOH); UV (CH3OH) kmax (loge) 354 (4.67), 315 (4.59), 268
(4.46), 257 (4.45) nm; IR (neat) mmax 3347, 1735, 1654, 1598,
1512, 1442, 1354, 1200, 1166 cmꢀ1 1H (CD3OD, 400 MHz) and
;
13C NMR (CD3OD, 400 MHz) data, Table 1; HR-TOF-MS (positive
ESI mode) m/z 757.2017 [M+H]+ (calcd for C36H36O18H, 757.1980).
3.8. Hydrolysis and identification of the sugar moieties in 17–20
Flavonoidal glycosides were hydrolyzed and analyzed according
to the literature precedent.18 Briefly, glycoside (17, 2 mg) was
heated in a 1 M HCl solution (2 mL, 1:1 dioxane–H2O) at 80 °C
for 2 h. The resulting mixture was concentrated and partitioned
between water (2 mL) and butanol (2 mL). The aqueous layer was
analyzed by TLC (70:27:3 CH2Cl2–MeOH–H2O) and was identified
to contain glucose (Rf 0.24), arabinose (Rf 0.32), and rhamnose (Rf
0.43), based on comparison with authentic samples. The aqueous
layer was reacted with (S)-(ꢀ)-1-phenylethylamine (2 mg) and
NaBH3CN (8 mg) followed by acetylation with acetic anhydride
(0.2 mL) and pyridine (0.2 mL). After drying, the resulting dark
brown residue was analyzed by HPLC. The carbohydrates were
3.4. Camelliquercetiside A, quercetin 3-O-[
anosyl(1?3)][2-O00-(E)-p-coumaroyl][b-
-glucopyranosyl(1?3)-
-rhamnopyranosyl(1?6)]-b- -glucoside (17)
a-L-arabinopyr
D
a-
L
D
A pale brown solid; mp 228–230 °C; ½a D20
ꢀ83.9 (c 0.13, MeOH);
ꢃ
1H (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table
1; HR-TOF-MS (positive ESI mode) m/z 1051.3065 [M+H]+ (calcd
for C47H54O27+H, 1051.2931).
3.5. Camelliquercetiside B, quercetin 3-O-[2-O00-(E)-p-couma
identified as
L-arabinose (tR, 28.1 min), D-glucose (tR 38.3 min),
and -rhamnose (tR 42.3 min) by comparing their retention times
L
royl][b-
D-glucopyranosyl(1?3)-a-L-rhamnopyranosyl(1?6)]-b-
with those of (S)-(ꢀ)-1-phenylethylamine derivatives of authentic
carbohydrates, while (R)-(+)-1-phenylethylamine derivatives of
authentic carbohydrates were detected at tR, 26.7 min, 35.5 min,
and 39.2 min, respectively. The same procedure was applied for
the identification of the sugar moieties in the other isolates.
D
-glucoside (18)
A pale brown solid; mp 220–222 °C; ½a D20
ꢀ92.65 (c 0.07,
ꢃ
MeOH); UV (CH3OH) kmax (loge) 355 (4.55), 316 (4.46), 267
(4.37), 257 (4.35) nm; IR (neat) mmax 3361, 1734, 1649, 1602,

M. M. Manir et al. / Bioorg. Med. Chem. 20 (2012) 2376–2381
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ADH activity was measured according to the literature prece-
dent.18,19 Briefly, stock solutions of samples in DMSO were serially
diluted with a phosphate buffer solution (PBS, 10 mM, pH 7.4). Eth-
anol (20
test solutions (20
and allowed to equilibrate for 5 min at 24 °C. After an ADH solution
(20 L, 1180 unit/L in PBS) was added, the mixture was incubated
l
L, 25% in PBS), b-NAD+ (20
l
L, 1.87 mM in PBS), and the
lL) were added to a 96-well plate (120 L, PBS)
l
l
at 24 °C for 2 h. The absorbance at 340 nm was measured at inter-
vals of 5 min for a period of 2 h to measure the amount of NADH
produced. The 50% inhibitory concentration (IC50, lg/mL) of the
sample was expressed as the sample concentration at which the
amount of NADH produced was reduced by 50%, compared to the
control (PBS) at 2 h. Tetraethylthiuram disulfide (disulfiram) was
used as the positive control, and the experiments were performed
in triplicate.
3.10. DPPH radical scavenging activity assay
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precedent.20 Briefly, the solutions of serially diluted samples
(20
(59
l
l
L) in MeOH were added to an EtOH solution (80
g/mL) in a 96-well plate. After incubation for 30 min at room
lL) of DPPH
temperature, the absorbance of the wells was measured at 517 nm
with a microplate reader. Radical scavenging activity was ex-
pressed in terms of IC50 (the concentration required for a 50% de-
crease in the absorbance of the DPPH control solution).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.002.
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