The (3R,9R)-3-hydroxy-7,8-dihydro-β-ionol disaccharide glycoside is an aroma precursor in tea leaves

Article abstract of DOI:10.1016/S0031-9422(00)00361-7

The disaccharide glycoside, (3R,9R)-3-hydroxy-7,8-dihydro-β-ionyl 6-O-β-D-apiofuranosyl-β-D-glucopyranoside was isolated as an aroma precursor from the leaves of Camellia sinensis var. sinensis cv. Yabukita. Its stereochemistry was elucidated on the basis of spectral data and chemical synthesis.

Full text of DOI:10.1016/S0031-9422(00)00361-7

Phytochemistry 56 (2001) 819±825
www.elsevier.com/locate/phytochem
The (3R,9R)-3-hydroxy-7,8-dihydro-b-ionol disaccharide
glycoside is an aroma precursor in tea leaves
Seung-Jin Ma a, Naoharu Watanabe b, Akihito Yagi b, Kanzo Sakata a,*
aInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
bDepartment of AppliedBiological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8017, Japan
Received 2 December 1999; received in revised form 3 August 2000
Abstract
The disaccharide glycoside, (3R,9R)-3-hydroxy-7,8-dihydro-b-ionyl 6-O-b-d-apiofuranosyl-b-d-glucopyranoside was isolated as
an aroma precursor from the leaves of Camellia sinensis var. sinensis cv. Yabukita. Its stereochemistry was elucidated on the basis
of spectral data and chemical synthesis. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Camellia sinensis; Theaceae; Glycosidic aroma precursor; Disaccharide glycoside; (3R,9R)-3-Hydroxy-7,8-dihydro-b-ionyl 6-O-b-d-apio-
furanosyl-b-d-glucopyranoside; C13-Norisoprenoid
1. Introduction
process (Takeo, 1981; Fischer et al., 1987; Morita et al.,
1994, Moon et al., 1994). In the course of our study on
the molecular basis of aroma formation during tea pro-
cessing, we have isolated and identi®ed glycosidic aroma
precursors such as b-primeverosides (6-O-b-d-xylopyr-
anosyl-b-d-glucopyranosides) of linalool, geraniol, 2-
phenylethanol, benzyl alcohol, etc., 6-O-b-d-apiofur-
anosyl-b-d-glucopyranosides of methyl salicylate and of
linalool oxides II and III and a b-d-glucopyranoside of
(Z)-3-hexenol from fresh leaves of oolong tea cultivars
(C. sinensis var. sinensis cv. Shuixian and Maoxie) (Guo
et al., 1993, 1994; Moon et al., 1994; Ogawa et al.,
1995). We also puri®ed a b-primeverosidase which is
considered to be a speci®c glycosidase to hydrolyze the
disaccharide glycosidic aroma precursors (Guo et al.,
1996; Ogawa et al., 1997; Ijima et al., 1998).
In the course of these studies, 3-hydroxy-7,8-dihydro-
b-ionol, a C13-norisoprenoid, was found to be generated
from some glycosidic fractions from tea leaves (C.
sinensis var. sinensis cv. Yabukita) when treated with a
crude enzyme prepared from the fresh tea leaves.
Because 3-hydroxy-7,8-dihydro-b-ionol has never been
reported as a tea aroma, it was searched among volatile
¯avor compounds from green, oolong and black tea by
the Simultaneous Distillation and Extraction (SDE)
method (Schultz et al., 1977; Berderich et al., 1992) and
found only from a black tea made from C. sinensis var.
assamica.
Thirteen-carbon (C13-) norisoprenoids are well known
as important aroma constituents of grape juices and
wines (Strauss et al., 1987; Winterhalter et al., 1990) and
recent studies have revealed that the volatile nor-
isoprenoids are derived from glycosidic precursor forms
(Skouroumounis and Winterhalter, 1994). A number of
these compounds have been detected in grapes, wine
(Strauss et al., 1987; Winterhalter et al., 1990), quince
fruit (Guldner and Winterhalter, 1991; Winterhalter et
al., 1991), tobacco (Baumes et al., 1994) and Osmanthus
absolute (Kaiser and Lamparsky, 1978). However, the
absolute con®guration of a limited number have been
determined with both isomers having 9R (Andersson
and Lundgren, 1988; Winterhalter et al., 1991; Pabst et
al., 1992; Matsuda et al., 1997) and 9S (Miyase et al.,
1989; Pabst et al., 1992; Baumes et al., 1994) con®gura-
tions being reported. Most ionone-type glycosides on
the other hand, have a 9R-con®guration.
In tea leaves, many kinds of alcohols such as geraniol,
linalool, etc., which contribute to the characteristic
¯oral tea aroma of oolong tea and black tea, have been
reported to be present as glycosides and liberated from
their glycosidic aroma precursors by the action of
endogenous enzymes during the tea manufacturing
* Corresponding author. Tel.: +81-774-38-3230; fax: 81-774-38-
3229.
E-mail address: ksakata@scl.kyoto-u.ac.jp (K. Sakata).
3-Hydroxy-7,8-dihydro-b-ionol has been isolated only
as the 3-glycosylated form from quince fruits and
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00361-7

820
S.-J. Ma et al. / Phytochemistry 56 (2001) 819±825
Prunus spinosa (Hump and Schreier, 1992; Winterhalter
and Schreier, 1994), but its absolute con®guration has
not been reported. This compound is the ®rst example
with a hydroxyl at C-9 from tea leaves (C. sinensis var.
sinensis), although several kinds of C13-norisoprenoid
compounds have been identi®ed in tea volatiles (Yama-
nishi, 1995). So we decided to determine the absolute
con®guration at C-9 of this compound, even though the
isolated amount was minute.
(ꢀ 3.50, 2H, s, H-500) as well as a cross peak between
methylene (ꢀ 3.50, H-500) and methine (ꢀ 3.82, 1H, d,
J=3.1 Hz, H-200) protons in the NOESY experiment.
The cross peaks between H-100 (ꢀ 4.92, 1H, d, J=3.1 Hz)
of the apiofuranose moiety and C-60 (ꢀ 68.5) of the glu-
copyranosyl moiety were clearly detected in the HMBC
spectrum. The apiofuranose was con®rmed to have a b-
1
con®guration by comparing the 13C and H NMR data
of the apiofuranose moiety of 1 with those of previously
reported papers (Moon et al., 1996; Oka et al., 1997).
Therefore, the disaccharide moiety of 1 was concluded
to be 6-O-b-d-apiofuranosyl-b-d-glucopyranoside.1
Thirteen carbon signals in the 13C NMR spectrum
accounted for the aglycone part of 1. The 1H N MR
spectrum showed the presence of two quaternary methyl
protons (ꢀ 0.88 and 0.90, 3H each, s, H-11 and 12), one
doublet methyl protons (ꢀ 1.07, 3H, d, J=6.1 Hz, H-10)
and one vinyl methyl protons (ꢀ 1.49, 3H, s, H-13),
indicating an ionone derivative. Two oxygen bearing
carbons (ꢀ 65.8, C-3 and ꢀ 76.4, C-9) and two sets of
down®eld-shifted methylene protons (ꢀ 1.89±1.95, 2H,
m, H-7 and ꢀ 1.37±1.43, 1.54±1.59, 1H each, m, H-8a,
This paper addresses with the isolation of the new
aroma precursor (1) of 3-hydroxy-7,8-dihydro-b-ionol
and the elucidation of its absolute stereochemistry.
2. Results and discussion
Table 1
13C NMR spectral data for compounds 1, 2, 5a and 5b in CD3OD
The glycosidic fractions obtained from hot water
extracts of tea leaves (cv. Yabukita) were subjected to
HPLC on an ODS column (gradient elution of 20±60%
C
1
5a
5b
1
40.0
49.8
65.8
40.5
33.5
32.7
33.5
32.6
2
MeCNin H O). The 25.5±29% MeCNeluate contain-
2
3
23.9
121.0
23.9
121.1
ing a glycoside (1) of 3-hydroxy-7,8-dihydro-b-ionol
was further puri®ed by repeated HPLC on an ODS
column (gradient elution of 10±50% MeCNin H 2O)
and then on Fluo®x column (eluted with 21% MeOH in
H2O) to give 1.1 mg of 1.
4
5
125.4
138.6
25.4
38.9
76.4
19.9
30.4
29.0
20.2
102.4
75.2
78.0
71.8
76.9
68.5
110.9
78.2
80.6
75.3
65.8
137.9
50.7
137.8
50.8
6
7
27.8 (27.9a)
27.7
38.0 (38.1a)
78.0 (78.1a)
21.8 (21.9a)
28.1
8
38.8 (38.9a)
9
76.1 (76.2a)
The molecular formula C24H42O11 of 1 was deter-
mined by high resolution FAB-MS analysis [m/z; found:
507.2798, calcd. for C24H43O11 ([M+H]+): 507.2806].
The 13C NMR spectrum (Table 1) showed 11 carbon
signals ascribed to a sugar moiety including two
anomeric carbons (ꢀ 102.4 and 110.9) besides those of
an aglycone, 3-hydroxy-7,8-dihydro-b-ionol. The large
coupling constant of an anomeric proton (ꢀ 4.35, 1H, d,
J=7.9 Hz, H-10) and the sequential trans-1,2-diaxial
10
11
12
13
10
20
30
40
500
600
100
2
19.8 (19.9a)
28.1
28.0
24.0
102.0 (102.1a)
28.1
24.0
103.7 (103.8a)
75.2
78.2
71.8
77.8
62.9
±
75.3
78.2
71.8
77.8
62.9
±
relationship of H-10 to H-50 (J=7.9±9.2 Hz) in the H
1
±
±
NMR spectrum suggested the presence of a glucopyr-
anosyl moiety. The identi®cation of the remaining sugar
300
400
500
±
±
±
±
1
moiety was established by comprehensive H and 13C
±
±
NMR measurements including HSQC and HMBC.
Another anomeric proton (ꢀ 4.92, 1H, d, J=3.1 Hz, H-
100) having a small coupling constant and a signal cor-
responding to a quaternary carbon (ꢀ 80.6, C-300) indi-
cated a furanose moiety. This was determined as
apiofuranose based on the presence of AB quartet
methylene signals (ꢀ 3.71 and 3.85, 1H each, d, J=10.4
and 10.1 Hz, H-400a and -400b, respectively) and a singlet
a
1
Chemical shifts due to the epimer at C-6.
The absolute con®gurations of the sugar moieties were not deter-
mined, but we had determined the sugar moiety (d-glucose) of geranyl
b-primeveroside in a previous paper (Guo et al., 1993). Since only
d-apiose has been found in plant kingdom so far, both sugars were
deduced to be d-series.

S.-J. Ma et al. / Phytochemistry 56 (2001) 819±825
821
-8b) show that this aglycone is 3-hydroxy-7,8-dihydro-
b-ionol. The glycosidic linkage of the glucopyranosyl
moiety to C-9 position of the aglycone was con®rmed
by the cross-peak between an anomeric carbon signal (ꢀ
102.4, C-10) of the glucopyranose and H-9 (ꢀ 3.74±3.78,
1H, m) of the aglycone in HMBC spectrum.
The relative stereochemistry at C-3 in the aglycone
moiety of 1 was con®rmed as follows. In NOE experi-
ments, strong interactions were observed between H-11
and H-7 and between H-12 and H-3, which indicates the
hydroxyl group at C-3 is equatorial. This is the same
con®guration as those of previously reported 3-oxyge-
nated ionone derivatives, all of which have been repor-
ted having 3R-con®gurations (Miyase et al., 1988, 1989;
Matsushita et al., 1991; Guldner and Winterhalter,
1991; Baumes et al., 1994). Based on these results, the
absolute con®guration of C-3 was deduced to be R.
The absolute con®guration at the C-9 position with a
glycosyl substituent was determined using the model
compound, 7,8-dihydro-a-ionol (2), having a similar
structure to the aglycone moiety of 1 (Fig. 1), since the
isolated quantity of 1 was too small for further analysis.
A racemic mixture (6RS, 9RS) of 2 was reacted with
2,3,4,6-tetra-O-benzoyl-a-d-glucopyranosyl bromide (3)
to give the diastereoisomeric mixture of 4. The mixture
was separated on a silica gel column to a€ord two sets
[(6RS, 9R) and (6RS, 9 S)] of diastereomers (4a and 4b),
which gave diastereomixtures of a-ionyl glycosides (5a
and 5b), respectively, after deacylation with methanolic
ammonia. Seo et al. and Tagawa et al. reported the
absolute con®guration at C-9 could be determined
based on the glucosidation shift rule i.e. based on the C-
10 chemical shift di€erence between 9R- and 9 S-glyco-
sylated ionol epimers (Seo et al., 1978; Tagawa et al.,
1996). Actually, the C-10 carbon signals of 5a and 5b
were observed at ꢀ 19.8 (19.9*), 21.8 (21.9*) respectively
(Asterisks indicate chemical shifts of an epimer at C-6).
The C-10 carbon signal of the isolated glycoside (1) was
observed at ꢀ 19.9 (Table 1), indicating the con®guration
at C-9 is identical with that of 5a.
The absolute con®guration of C-9 of 5a was con-
®rmed by use of Mosher's Method (Dale et al., 1969;
Dale and Mosher, 1973). Chiral alcohol (2a) was pre-
pared by enzymatic hydrolysis of 5a, this being con-
verted into the R- and S-MTPA esters (6a and 6b).
From the Áꢀ values (Áꢀ=ꢀSꢀR), where protons on the
right-hand side of the MTPA plane must have positive
values, and those on the left-hand side must be negative
(Ohtani et al., 1991, 1995), were calculated. For the
MTPA derivatives (6a and 6b), the Áꢀ value for methyl
protons on C-10 was positive (ÁꢀH10=+0.08) and for
the methylene protons at C-8 and C-7 were negative
(ÁꢀH8=À0.08, ÁꢀH7=À0.06). The Áꢀ value for H-9,
however, was zero (ÁꢀH9=0), since it is located on the
MTPA plane (Fig. 2). These observations clearly show
that the absolute con®guration at C-9 of 2a is R and con-
sequently the C-9 stereochemistry of 1 is unambiguously
assigned as R.
Fig. 1. Synthesis of model compounds (6a and 6b) to determine the
absolute con®guration of C-9 of aglycone of 1. *Indicates C-10 che-
mical shift of 5a and 5b. (i) Hg(CN)2, dry MeCN, 36 h, rt; (ii) Silica gel
column chromatography (CH2Cl2); (iii) NH3 in MeOH, 24 h; (iv) b-
glucosidase, citrate bu€er (pH 4.4), 16 h, 38ꢀ; (v) (+) or (À)-MTPA
chloride, dry pyridine, dry CH2Cl2, 12 h, rt.
On the basis of the foregoing, the structure of 1 has
been concluded to be (3R,9R)-3-hydroxy-7,8-dihydro-b-
ionyl 6-O-b-d-apiofuranosyl-b-d-glucopyranoside.
3. Experimental
3.1. General
NMR spectra were recorded on JEOL JNM-AL 400
and JEOL LAMBDA 500 spectrometers, using either
CD3OD or D2O as solvent. Chemical shifts were given
in ꢀ values with TMS or TSP being employed as internal
Fig. 2. The conformation of MTPA esters (6a and 6b) with Áꢀ values
(shown in bracket, Áꢀ=ꢀsÀꢀr).

822
S.-J. Ma et al. / Phytochemistry 56 (2001) 819±825
standards. Mass spectra (EI, at 70 eV; FAB, using gly-
cerol or NBA as a matrix) were obtained with a JEOL
JMS 700 spectrometer. GC±MS analysis was carried
out with a JEOL JMS-DX 302 mass spectrometer
linked to a Hewlett±Packard 5890A gas chromatograph
(column, PEG-20 M, 0.25 mmÂ50 m; carrier gas, He at
1 ml minÀ1; temp. program, 60ꢀ to 230ꢀC at 3ꢀC minÀ1).
TLC was performed on silica gel plates (Kieselgel 60
MeOH (0, 10, 20, 40, 60 and 100% MeOH; 450 ml
each). The 10±100% MeOH fractions were combined
and loaded on an ODS column (YMC-GEL, ODS-A
60±200/60, 30Â480 mm, YMC Co. Ltd), which was
eluted with H2O±MeOH (10, 20, 30, 40, 60 and 80%
MeOH, 800 ml each). The 40% MeOH fraction from
which 3-hydroxy-7,8-dihydro-b-ionol was liberated
after enzymatic hydrolysis, was subjected to HPLC on
an ODS (YMC-Pack S-343 I-15 ODS column,
20Â250 mm, YMC Co. Ltd) with gradient elution of
F254, Merck) using CH2Cl2 for 2 and 4, CHCl3-MeOH
(5:1) for 5 and hexane-CHCl3 (1:3) for 6. They were
detected under UV 254 nm and by spraying with 0.2%
naphthoresorcinol in H2SO4-EtOH (1:19) (Touchstone,
1978).
20±60% MeCNin H O.
2
The 25.5±29% MeCNfractions containing a glyco-
side (1) were successfully puri®ed by repeated HPLC on
an ODS (YMC-PACKED S-5 120A ODS column,
20Â250 mm, YMC Co.; gradient elution of 10±50%
MeCNin H 2O) and Fluo®x (Fluo®x 120Ncolumn,
10Â150 mm, NEOS Co. Ltd; eluted with 21% MeOH in
H2O) column to give 1 (1.1 mg).
3.2. Materials
3.2.1. Plant
Fresh tea leaves (Camellia sinensis var. sinensis cv.
Yabukita) were collected at the farm of the National
Research Institute of Vegetables, Ornamental Plants
and Tea at Kanaya, Shizuoka, Japan in May, 1997. The
leaves were steamed just after plucking and stored at
À70ꢀC before use.
3.6. (3R,9R)-3-Hydroxy-7,8-dihydro-ꢁ-ionyl 6-O-ꢁ-d-
apiofuranosyl-ꢁ-d-glucopyranoside (1)
HR-FABMS m/z; found: 507.2798, ꢀcalcd. for
C24H43O11 ([M+H]+): 507.2806; [a]2d7 À7.2 (MeOH, c
1
1.0); H NMR (500 MHz, D2O, TSP) ꢀ 0.88 (3H, s, H-
3.2.2. Chemical
12), 0.91 (3H, s, H-11), 1.07 (3H, d, J=6.1 Hz, H-10),
1.24 (1H, t, J=12.1 Hz, H-2a), 1.40 (1H, m, H-8a), 1.49
(3H, s, H-13), 1.54±1.59 (1H, m, overlapped with H-2b,
H-8b), 1.55 (1H, overlapped with H-8b, H-2b), 1.80
(1H, dd, J=4.7 and 11.6 Hz, H-4a), 1.89±1.95 (2H, m,
H-7), 2.10 (1H, dd, J=4.7 and 11.6 Hz, H-4b), 3.08
(1H, dd, J=7.9 and 9.2 Hz, H-20), 3.29 (1H, dd, J=8.2
and 9.2 Hz, H-40), 3.32 (1H, dd, J=8.2 and 9.2 Hz, H-
30), 3.37 (1H, m, H-50), 3.50 (2H, s, H-500), 3.54 (1H, dd,
J=6.6 and 11.6 Hz, H-60a), 3.71 (1H, d, J=10.4 Hz, H-
400a), 3.75 (1H, m, overlapped with H-3, H-9), 3.76±3.80
(1H, m, overlapped with H-9, H-3), 3.82 (1H, d,
J=3.1 Hz, H-200), 3.84 (1H, overlapped with H-200 and
400b, H-60b), 3.85 (1H, d, J=10.4 Hz, H-400b), 4.35 (1H,
d, J=7.9 Hz, H-10), 4.92 (1H, d, J=3.1 Hz, H-100); 13C-
NMR (125.7 MHz, CD3OD, TMS) Table 1.
The racemic mixture of 7,8-dihydro-a-ionol (2) was
kindly provided by Prof. Hideo Etoh (Faculty of
Agriculture, Shizuoka University).
3.3. Simultaneous distillation and extraction (SDE)
method
SDE was performed for 2 h using pentane-CH2Cl2
(2:1) as previously reported (Schultz et al., 1977). The
extracts were dried over anhydrous Na2SO4 and
concentrated under N2 stream for GC±MS analysis.
3.4. Detection of aroma precursor
To detect aroma precursors, enzymatic hydrolysis was
carried out as previously reported (Ogawa et al., 1995).
The characteristic signals in the 1H NMR spectrum,
corresponding to four methyl signals of the aglycone
moiety of 1, were used as a guide for the isolation.
3.7. Determination of the absolute con®guration in the
C-9 position of 1
3.7.1. Preparation of 4a and 4b by Koenigs±Knorr
Reaction (Sone andMisaki, 1986)
3.5. Extraction andisolation of aroma precursor
To a mixture of 2,3,4,6-tetra-O-benzoyl-a-d-gluco-
pyranosyl bromide (3, 0.96 g, 1.45 mmol), which was
prepared from d-(+)-glucose by a general method
(Fletcher and Hudson, 1947), Hg(CN)2 (0.3 g, 1.2 mmol)
and activated 4 A molecular sieves (4 g) in dry MeCN
(15 ml) was added to the racemic mixture (6RS, 9RS) of
2 (0.96 g, 1.45 mmol); The reaction mixture was then
allowed to stir at room temp. under argon for 36 h, after
which it was extracted with CH2Cl2 and washed with
Frozen tea leaves (3.8 kg) were extracted with hot
water (20 l, 10 lÂtwice) for 20 min and subjected to
active charcoal CC (85Â350 mm) eluted with H2O±
MeOH (0, 10, 20, 40, 60, 80 and 100% MeOH; 2 l each).
The 60±100% MeOH fractions were treated with 20 g of
Polyclar SB-100 (Wako Chemical Co. Ltd, eluted with
H2O) to remove catechins, and then subjected to
Amberlite XAD-2 CC (40Â310 mm) eluted with H2O±

S.-J. Ma et al. / Phytochemistry 56 (2001) 819±825
823
1 M NaHCO3 soln. The organic layer was applied to
¯ash chromatography (eluted with CH2Cl2) to a€ord
two sets [(6RS, 9R) and (6RS, 9 S)] of diastereomers,
4a (Rf 0.47, CH2Cl2) and 4b (Rf 0.29, CH2Cl2) (100 mg
each, 42%).
with H-6, H-7), 1.54 (2H, m, H-8), 1.68 (3H, s, H-13),
1.96 (2H, m, H-3), 3.74 (1H, m, H-9), 5.30 (1H, m, H-4);
13C NMR (125.7 MHz, CDCl3, TMS) ꢀ 23.0 (C-10),
23.4 (C-3), 23.5 (C-13), 27.0 (C-7), 27.5 (C-12), 27.6 (C-
11), 31.6 (31.7*) (C-2), 32.6 (C-1), 39.8 (C-8), 49.3 (C-6),
68.7 (68.9*) (C-9), 120.3 (C-4) 136.4 (C-5) (Asterisks
indicate chemical shifts of epimer at C-6).
3.7.2. Preparation of 5a and 5b
Each diastereomer (4a and 4b, 100 mg each, 129 mmol)
was treated with 30 ml of the methanolic ammonia for
24 h at room temp. and puri®ed by ¯ash chromato-
graphy (CHCl3±MeOH, 9:1) to give 5a and 5b (30 mg
each, 90%), respectively. 5a: Rf 0.35, (CHCl3±MeOH,
5:1); HR-FABMS m/z; found: 359.2437, calcd. for
3.7.4. Preparation of (R)-MTPA ester of 7,8-dihydro-
ꢂ-ionol (6a)
To a solution of 7,8-dihydro-a-ionol (2a, 4.5 mg,
23 mmol) in pyridine (1.5 ml) and CH2Cl2 (1.5 ml) was
added (+)-MTPA chloride (10 ml, 52 mmol, Aldrich
Chemical Co.), and stirred at room temp. for 12 h (Dale
and Mosher, 1973). The reaction mixture was added
with ether, washed with cold diluted HCl, satd aq.
Na2CO3 and NaCl, respectively, and puri®ed by ¯ash
chromatography (hexane±CHCl3, 1:1) to a€ord the
desired (R)-(+)-MTPA ester (6a, 3 mg, 30%). 6a: Rf
0.60, (hexane±CHCl3, 1:3); HR-FABMS m/z; found:
1
C19H35O6 ([M+H]+): 359.2434; H NMR (500 MHz,
CD3OD, TMS) ꢀ 0.87 (3H, s, H-12), 0.93 (3H, s, H-11),
1.13 (1H, m, H-2a), 1.17 (3H, d, J=6.1 Hz, H-10), 1.44
(1H, overlapped with H-2b, H-6), 1.45 (1H, overlapped
with H-6 and H-7, H-2b), 1.48 (2H, overlapped with H-
2b, H-7), 1.53 (1H, m, H-8a), 1.62 (1H, m, H-8b), 1.68
(3H, s, H-13), 1.96 (2H, m, H-3), 3.14 (1H, dd, J=7.8
and 8.5 Hz, H-20), 3.24 (1H, m, H-50), 3.30 (1H, dd,
J=7.8 and 8.6 Hz, H-40), 3.34 (1H, dd, J=7.8 and
8.9 Hz, H-30), 3.67 (1H, dd, J=5.5 and 11.9 Hz, H-60a),
3.83 (1H, m, overlapped with H-60b, H-9), 3.86 (1H,
overlapped with H-9, H-60b), 4.33 (1H, d, J=7.8 Hz, H-
10), 5.28 (1H, m, H-4); 13C NMR (125.7 MHz, CD3OD,
TMS) Table 1. 5b: Rf 0.35, (CHCl3±MeOH, 5:1); HR-
FABMS m/z; found: 359.2428, calcd. for C19H35O6
1
412.2218, calcd. for C23H31O3F3 (M+): 412.2225; H-
NMR (500 MHz, CDCl3, TMS) ꢀ 0.85 (3H, s, H-12),
0.87 (3H, s, H-11), 1.11 (1H, m, H-2a), 1.26 (3H, d,
J=6.1 Hz, H-10), 1.33 (1H, m, H-2b), 1.41 (1H,
overlapped with H-7, H-6), 1.42 (2H, overlapped with
H-6, H-7), 1.59 (1H, m, H-8a), 1.63 (3H, s, H-13), 1.72
(1H, m, H-8b), 1.94 (2H, m, H-3), 3.54 (3H, s, H-20-
OCH3), 5.09 (1H, m, H-9), 5.30 (1H, m, H-4), 7.38±7.40
(3H, m, H-Ph-300, -500 and 400), 7.52±7.54 (2H, H-Ph-200
and 600); 13C NMR (125.7 MHz, CDCl3, TMS) ꢀ 19.5
(C-10), 23.0 (C-3), 23.4 (C-13), 26.5 (26.6*) (C-7), 27.4
(C-12), 27.5 (C-11), 31.4 (31.6*) (C-2), 32.5 (C-1), 35.9
(36.2*) (C-8), 49.0 (49.1*) (C-6), 55.3 (C-20-OCH3), 74.6
1
([M+H]+): 359.2434; H NMR (500 MHz, CD3OD,
TMS) ꢀ 0.88 (3H, s, H-12), 0.94 (3H, s, H-11), 1.13 (1H,
m, H-2a), 1.23 (3H, d, J=6.1 Hz, H-10), 1.44 (1H,
overlapped with H-2b, H-6), 1.46 (1H, overlapped with
H-6 and H-8, H-2b), 1.48 (2H, overlapped with H-2b,
H-7), 1.53 (1H, m, H-8a), 1.63 (1H, m, H-8b), 1.69 (3H,
s, H-13), 1.96 (2H, m, H-3), 3.15 (1H, dd, J=7.7 and
8.5 Hz, H-20), 3.23 (1H, m, H-50), 3.28 (1H, dd, J=7.7
and 8.6 Hz, H-40), 3.34 (1H, dd, J=7.7 and 8.6 Hz, H-
30), 3.66 (1H, dd, J=5.5 and 11.9 Hz, H-60a), 3.76 (1H,
m, H-9), 3.86 (1H, dd, 0J=1.8 and 11.9 Hz H-60b), 4.32
(1H, d, J=7.7 Hz, H-1 ), 5.29 (1H, m, H-4); 13C-NMR
(125.7 MHz, CD3OD, TMS) Table 1.
(74.7*) (C-9), 84.4 (C-20, JCF=28.3 Hz), 120.5
2
(120.6*) (C-4), 123.4 [C-30 (CF3), JCF=289.2 Hz],
1
127.2 (C-Ph-200 and 600), 128.4 (C-Ph-300 and 500), 129.5
(C-Ph-400), 132.4 (C-Ph-100), 135.8 (136.0*) (C-5), 166.2
(C-10) (asterisks indicate chemical shifts of an epimer
at C-6).
3.7.5. Preparation of a (S)-MTPA ester of 7,8-dihydro-
ꢂ-ionol (6b)
3.7.3. Preparation of 2a from 5a
2a (4.5 mg, 23 mmol) was treated with (À)-MTPA
chloride (10 ml, 52 mmol, Aldrich Chemical Co.) in the
same way as preparation for 6a to give an (S)-(À)-
MTPA ester (6b, 3 mg, 30%). 6b: Rf 0.60, (hexane±
CHCl3, 1:3); HR-FABMS m/z; found: 412.2231, calcd.
An epimeric mixture (5a; 30 mg, 84 mmol) was enzy-
matically hydrolyzed with b-glucosidase (30 mg, 120
units) from sweet almond (Oriental Yeast Co.) in
100 mM acetate bu€er (2 ml, pH 4.4) at 38ꢀ for 16 h
(Yoshikawa et al., 1998). The reaction mixture was
extracted with EtOAc, washed with satd aq. NaCl,
respectively, and puri®ed by ¯ash chromatography
(CHCl3±MeOH, 9:1) to give the aglycone, 7,8-dihydro-
a-ionol (2a, 9 mg, 55%). 2a: Rf 0.22 (CH2Cl2); 1H
NMR (500 MHz, CDCl3, TMS) ꢀ 0.87 (3H, s, H-12),
0.92 (3H, s, H-11), 1.13 (1H, m, H-2a), 1.19 (3H, d,
J=6.1 Hz, H-10), 1.30 (1H, m, H-2b), 1.43 (1H, over-
lapped with H-7 and H-8, H-6), 1.44 (2H, overlapped
1
for C23H31O3F3 (M+): 412.2225; H NMR (500 MHz,
CDCl3, TMS) ꢀ 0.80 (0.81*) (3H, s, H-12), 0.82(0.83*)
(3H, s, H-11), 1.08 (1H, m, H-2a), 1.29 (1H, m, H-2b)
1.34 (3H, d, J=6.1 Hz, H-10), 1.36 (2H, overlapped
with H-6, H-7), 1.38 (1H, overlapped with H-7, H-6),
1.51 (1H, m, H-8a), 1.57 (3H, s, H-13), 1.64 (1H, m, H-
8b), 1.92 (2H, m, H-3), 3.57 (3H, s, H-20-OCH3), 5.09
(1H,00m, H-9), 50.028 (1H, m, H-4), 7.37-7.39 (3H, m, H-
Ph-3 , -500 and 4 ), 7.52±7.54 (2H, H-Ph-200 and 600); 13C-

824
S.-J. Ma et al. / Phytochemistry 56 (2001) 819±825
NMR (125.7 MHz, CDCl3, TMS) ꢀ 19.7 (C-10), 23.0
(C-3), 23.3 (C-13), 26.3 (26.4*) (C-7), 27.3 (C-12), 27.5
(C-11), 31.4 (31.6*) (C-2), 32.5 (C-1), 35.9 (36.2*) (C-8),
49.0 (49.1*) (C-6), 55.4 (C-20-OCH3), 74.6 (74.7*) (C-9),
anoside: new C13 norisoprenoid glucoconjugates from sloe tree
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Kaiser, R., Lamparsky, D., 1978. Constituents of Osmanthus absolute.
Helv. Chim. Acta 61, 373±382.
84.4 (C-20, JCF=28.3 Hz), 120.5 (120.6*) (C-4), 123.4
2
[C-30 (CF3), JCF=289.2 Hz], 127.4 (C-Ph-200 and 600),
1
128.3 (C-Ph-300 and 500), 129.5 (C-Ph-400), 132.6 (C-Ph-
100), 135.8 (136.0*) (C-5), 166.2 (C-10) (asterisks indicate
chemical shifts of an epimer at C-6).
Matsuda, N., Isawa, K., Kikuchi, M., 1997. Megastigmane glycosides
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Miyase, T., Ueno, A., Takizawa, N., Kobayashi, H., Oguchi, H., 1988.
Studies on the glycosides of Epimedium grandi¯orum MORR. var.
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Acknowledgements
The authors thank the National Research Institute of
Vegetables, Ornamental Plants and Tea of Japan for
fresh tea leaves and Mr. M. Suzuki of Mitsui Norin Co.
for black tea samples. They are also grateful to Prof.
Hideo Etoh for his generous gift of compound 2.
Miyase, T., Ueno, A., Takizawa, N., Kobayashi, H., Oguchi, H., 1989.
Ionone and lignan glycosides from Epimedium diphyllum. Phy-
tochemistry 28, 3483±3485.
Moon, J.-H., Watanabe, N., Sakata, K., Yagi, A., Ina, K., Luo, S.,
1994. trans- and cis-Linalool 3,6-oxides 6-O-b-d-xylopyranosyl-b-d-
glucopyranosides isolated as aroma precursors from leaves for
oolong tea. Biosci. Biotechnol. Biochem. 58, 1742±1744.
Moon, J.-H., Watanabe, N., Ijima, Y., Yagi, A., Ina, K., Sakata, K.,
1996. cis- and trans-Linalool 3,7-oxides and methyl salicylate glycosides
and (Z)-3-hexenyl b-d-glucopyranoside as aroma precursors from tea
leaves for oolong tea. Biosci. Biotechnol. Biochem. 60, 1815±1819.
Morita, K., Wakabayashi, M., Kubota, K., Kobayashi, A., Herath,
N.L., 1994. Aglycone constituents in fresh tea leaves cultivated for
green and black tea. Biosci. Biotechnol. Biochem. 58, 687±690.
Ogawa, K., Moon, J.-H., Guo, W., Yagi, A., Watanabe, N., Sakata,
K., 1995. A study on tea aroma formation mechanism: alcoholic
aroma precursor amounts and glycosidase activity in parts of the tea
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