Isolation of α-glusosidase inhibitors from hyssop (Hyssopus officinalis)

Article abstract of DOI:10.1016/j.phytochem.2003.10.009

α-Glucosidase inhibitory activity was found in aqueous methanol extracts of dried hyssop (Hyssopus officinalis) leaves. Active principles against α-glucosidase, prepared from rat small intestine acetone powders, were isolated and characterized. The structures of these isolated compounds were determined to be (7S, 8S)-syringoylglycerol-9-O-(6′-O-cinnamoyl)- β-D-glucopyranoside and (7S, 8S)-syringoylglycerol 9-O-β-D- glucopyranoside by analysis of physical and spectroscopic data (FDMS, ¹H NMR, ¹³C NMR, HMQC, and HMBC experiments) together with chemical syntheses.

Full text of DOI:10.1016/j.phytochem.2003.10.009

Phytochemistry 65 (2004) 91–97
www.elsevier.com/locate/phytochem
Isolation of a-glusosidase inhibitors from hyssop
(Hyssopus officinalis)
Hideyuki Matsuuraa,*, Hiroyuki Miyazakia, Chikako Asakawaa, Midori Amanoa,
Teruhiko Yoshiharab, Junya Mizutanic
aNorthern Advancement Center for Science and Technology, Kita-21, Nishi-12, Sapporo, Hokkaido 001-0021, Japan
bDivision of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan
cPlant Ecochemicals Research Center, R&BP Center Bldg. 3F, E-310, 3-1-1, Megumino-kita, Eniwa, Hokkaido 061-1374, Japan
Received 23 June 2003; received in revised form 26 September 2003
Abstract
a-Glucosidase inhibitory activity was found in aqueous methanol extracts of dried hyssop (Hyssopus officinalis) leaves. Active
principles against a-glucosidase, prepared from rat small intestine acetone powders, were isolated and characterized. The structures
of these isolated compounds were determined to be (7S, 8S)-syringoylglycerol-9-O-(60-O-cinnamoyl)-b-d-glucopyranoside and (7S,
8S)-syringoylglycerol 9-O-b-d-glucopyranoside by analysis of physical and spectroscopic data (FDMS, 1H NMR, 13C NMR,
HMQC, and HMBC experiments) together with chemical syntheses.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Hyssopus officinalis; Labiatae; Hyssop; a-Glucosidase inhibitor; Diabetes; Syringoylglycerol
1. Introduction
analysis of the secondary metabolites synthesized by
plants would be a promising line of inquiry, since plants
are known to have many nutritional and health benefits.
We surveyed a number of herbs and edible wild plants
for a-glucosidase inhibitors, and reported the inhibitor
from balsam pear (Momordica charantia) and Grifola
frondosa (Matsuura et al., 2002). We report here the iso-
lation and characterization of a-glucosidase inhibitors
from hyssop.
Diabetes is a lifestyle-related disease known to trigger
many complications. Recently, the rapid increase in the
already large number of patients has become a major
health issue. The a-glucosidase complex in the small
intestine is involved in sugar absorption. Thus, inhibi-
tors of a-glucosidase would limit the absorption of
dietary carbohydrates and in turn suppress postprandial
hyperglycemia. Therefore, a-glucosidase inhibitors are
promising drug candidates in the treatment and pre-
vention of diabetes. Nojirimycin (Inouye et al., 1968;
Reese and Parrish, 1968) and acarbose (Truscheit et al.,
1981) are known to be powerful a-glucosidase inhibitors
derived from microorganisms. However, the potency of
these inhibitors precludes their use in humans without
careful medical consultation. In the search for
alternative a-glucosidase inhibitors, we speculated that
2. Results and discussion
Extracts were obtained in MeOH–H2O (7:3) of 13
varieties of herb; yarrow (Achillea millefolium), basil
(Ocimum basilicum), spearmint (Mentha spicata), sage
(Saliva officinalis), rocket (Eruca vesicaria), parsley
(Petroselinum crispum), watercress (Nasturtium offici-
nale), lemon balm (Melissa officinalis), chervil (Anthris-
cus cerefolium), oregano (Origanum vulgare), hyssop
(Hyssopus officinalis), chamomile (Matricaria chamo-
milla), and tansy (Tanacetum vulgare). Each extract was
divided into EtOAc and H2O soluble fractions. The
H2O and EtOAc soluble layers were examined for their
inhibitory activities. Both layers of oregano, tansy, and
* Corresponding author at present address: Division of Applied
Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo, Hokkaido 060-8589, Japan. Tel.: +81-11-706-2495; fax:
+81-11-706-2505.
E-mail address: matsuura@chem.agr.hokudai.ac.jp
(H. Matsuura).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2003.10.009

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H. Matsuura et al. / Phytochemistry 65 (2004) 91–97
hyssop extract showed inhibitory activities (data not
shown). Air-dried leaves of hyssop (1 kg) were soaked in
MeOH–H2O (7:3). The extract was partitioned with
EtOAc and H2O. The H2O and EtOAc soluble layers
were purified by a series of various chromatography
techniques to obtain (7S, 8S)-syringoylglycerol 9-O-b-d-
glucopyranoside (1, 565 mg) and (7S, 8S)-syr-
ingoylglycerol 9-O-(60-O-cinnamoyl)-b-d-glucopyrano-
side (2, 90 mg), respectively (Fig. 1). Compound 1 was
obtained as a colorless residue with ions at m/z 405
[MꢀH]ꢀ (negative, matrix:TEA) and 429 [M+Na]+
(positive, matrix:glycerol) by FAB-MS spectrometry.
The molecular formula of heptaacetyl derivative (3),
which was derived from 1, was determined to be
C31H40O18 by HR-FD–MS spectrometry. Thus, the
rotation, [ꢀ]2D3 ꢀ17.7ꢁ (c=0.5, MeOH), agreed well with
previously reported data (Kijima et al., 1997), and
therefore, the planar structure of 1 was determined to be
as in Fig. 1. The analysis of 1H NMR, 13C NMR,
DEPT, HMQC, and HMBC spectral data gave the total
1H and 13C assignments for 1, as in Tables 1 and 2,
respectively.
In order to ascertain the absolute configurations of
C-7 and 8, compound 1 was converted according
to Scheme 1 to afford (7S, 8S)-4-O-methylsyrin-
goylglycerol (5), with a specific rotation of [ꢀ]2D3 ꢀ21.1ꢁ
(c=0.59, MeOH). Independently, (7R, 8R)-4-O-methyl-
syringoylglycerol (8) was synthesized according to
Scheme 2 using 3, 4, 5-trimethoxycinnamic acid (6) as a
starting material. Its chirality of (7R, 8R) was intro-
duced by a reaction of the catalytic asymmetric dihy-
dration developed by Kolb et al. (1994) using the
molecular formula of 1 was determined to be
C17H26O11. Spectroscopic data including the specific
1
reagent of AD-mix-b, and the total assignment of H
1
for 8 was performed by a H–1H COSY experiment.
1
The H NMR, IR, and EI-MS spectra of (7R, 8R)-4-O-
methylsyringoylglycerol (8) agreed well with those of 5,
but the direction of its specific rotation, [ꢀ]2D3 ꢀ17.0ꢁ
(c=0.50, MeOH), was opposite. Therefore, the absolute
configurations of C-7 and -8 of 1 were determined to be
(7S) and (8S), respectively. Although the expected [ꢀ]D23
of 8 should be close to ꢀ21.1ꢁ, the observed value was
due to incomplete enantio-selectivity of catalytic asym-
metric dihydroxylation conducted by our group.
Fig. 1. a-Glucosidase inhibitors isolated from hyssop (H. officinalis).
Table 2
13C NMR (67.5 MHz, CD3OD) assignments of 1 and 2
Table 1
1H NMR (270 MHz, CD3OD) assignments of 1 and 2
1
2
1
2
C-1
C-2
133.6
105.3
148.9
135.9
148.9
105.3
75.5
133.5
105.1
148.8
135.7
148.8
105.1
75.3
H-2
H-6
H-7
6.62 (1H, s)
6.62 (1H. s)
4.54 (1H, d,
J=6.2 Hz)
6.62 (1H, s)
6.62 (1H, s)
4.59 (1H, d,
J=6.5 Hz)
C-3
C-4
9
=
;
C-5
C-6
H-8
H-9a
H-9b
3.73 (1H, m)
3.63 (1H, m)
3.47 (1H, dd,
J=10.0, 3.5 Hz)
3.73–3.20 (3H, m)
C-7
C-8
76.0
72.0
75.9
72.1
H-10
H-20
H-30
H-40
H-50
H-60a
4.15 (1H, d, J=7.6 Hz)
4.20 (1H, d, J=7.6 Hz)
3.73–3.20 (4H, m)
C-9
9
>
9
C-10
C-20
C-30
C-40
C-500
C-600
C-100
C-200
C-300
C-4
104.6
71.7
75.2
104.9
71.8
75.1
>
9
8
8
>
=
>
=
>
>
>
>
>
>
3.30–3.10 (4H, m)
3.73 (1H, m)
=
<
<
>
>
>
;
>
;
77.9
78.0
75.2
77.7
>
>
>
>
;
>
:
>
:
4.46 (1H, dd,
J=13.0, 2.1 Hz)
4.23 (1H, dd,
62.8
64.9
H-60b
3.55 (1H, dd,
J=11.6, 5.4 Hz)
168.2
118.4
146.4
135.5
129.2
129.9
131.4
129.9
129.2
56.7
J 13.0, 6.2 Hz)
6.43 (1H, d, J=16.1 Hz)
7.58 (1H, d, J=16.1 Hz)
7.49 (1H, m)
H300
C-500
C-600
C-700
C-800
C-900
H500
H600
7.33 (1H, m)
7.33 (1H, m)
H-700
H-800
H-900
Ph–OCH3
7.33 (1H, m)
7.49 (1H, m)
3.73 (6H, s)
Ph–OCH3
56.9
3.77 (6H, s)

H. Matsuura et al. / Phytochemistry 65 (2004) 91–97
93
Compound 2 was obtained as a colorless residue and
gave a molecular ion at m/z 536 [M]+ by FD–MS spec-
trometry. Compound 2 was treated with a solution of
pyridine and acetic anhydride to afford 9, whose
molecular formula was determined to be C38H44O18 (m/
z 788.2508 [M]+) by HR-FD–MS spectrometry. Thus,
the molecular formula of 2 was determined to be
C26H32O12. Compounds 1 and 2 showed similar features
The inhibitory activities of 1 and 2 were compared with
1-deoxynojirimycin (11) (Yoshikuni, 1988) (Fig. 3). Com-
pounds 1 and 2 exhibited 53 and 54% inhibitory activity
at the concentration of 3 ꢂ 10ꢀ3 M, but 1-deoxy-
nojirimycin (11) exhibited 58% inhibitory activity at 3 ꢂ
10ꢀ7 M. Compared with the activity of 1-deoxynojiri-
mycin (11), those of 1 and 2 were very low. However,
the activity of 1-deoxynojirimycin (11) is too potent to
administer without medical supervision. On the other
hand, hyssop has been consumed as a food material for
a long time, thus it has a proven safety record. If the
plant is regularly incorporated into the diet, it could
compensate for the low a-glucosidase inhibitory activity.
1
in their H and 13C NMR spectra (Tables 1 and 2), and
the acetyl-derivatives also exhibited similar tendencies in
1H and 13C NMR spectra. The 13C NMR resonance of ꢁ
168.2 of 2 suggested that an ester moiety existed in the
structure. As shown in Scheme 3, alkaline hydrolysis of
2 afforded 1 and 10. Compound 10 was identified as
1
cinnamic acid since spectral data of EI-MS, H and 13C
NMR of 10 agreed closely with those of an authentic
specimen of cinnamic acid (data not shown). This result
revealed that compound 2 was composed of 1 and cin-
namic acid in its ester form, and the partial structures
were connected by HMBC correlation to construct the
1
structure of 2 (Fig. 2). The analysis of H NMR, 13C
NMR, DEPT, HMQC, and HMBC spectral data gave
1
the total H and 13C assignments for 2, as in Tables 1
and 2, respectively.
Fig. 2. The important HMBC correlations of compound 2.
Scheme 1. (a) b-Glucosidase/0.1 N AcONH4–AcOH buffer (pH 5.6), 37 ꢁC (67%) (b) CH2N2/MeOH, 0 ꢁC (35%).
Scheme 2. (a) CH2N2/MeOH at 0 ꢁC, (b) LAH/THF at 0 ꢁC ( 28%), (c) AD-mix-b at 4 ꢁC (9%).
Scheme 3. (a) 1% NaOH/EtOH, room temp. (76% for 1, 74% for 10).

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H. Matsuura et al. / Phytochemistry 65 (2004) 91–97
In our search for a-glucosidase inhibitors from edible
mass spectra (FD–MS) and electron impact mass spec-
tra (EI-MS) were on Jeol JMS-O1SG-2 and JMS-DX-
300 mass spectrometers, respectively. NMR spectra
were recorded on a Bruker AM-500 (1H at 500 MHz)
and Jeol JNM-EX 270 FTNMR system ( 1H at 270
MHz; 13C at 67.5 MHz). In the 1H NMR spectra,
chemical shifts are reported using TMS as an internal
standard. In the 13C NMR spectra, chemical shifts are
reported as ꢁ (ppm) values relative to the carbon signals
(ꢁ 49.0) and (ꢁ 77.0) of CD3OD and CDCl3, respec-
tively. IR spectra were on Perkin Elmer System 2000
FT-IR spectrometer.
plants, compound 1 was rediscovered from hyssop (H.
officinalis) and we determined its absolute configuration.
In addition, a new related compound 2 was identified. It
is hoped that these preliminary experiments will provide
the basis for further examination of the suitability of
hyssop as a medicinal supplement that contributes
toward the treatment and prevention of diabetes.
3. Experimental
3.1. General
Medium pressure liquid chromatography (MPLC):
EFC-1000 pump (Tokyo Rikakiki, stroke length, 50;
stroke rate, 70), Lobar LiChroprep RP-18 (Merck, 440
ꢂ 37 mm), UV-absorbance (A254nm); flash column
chromatography (FCC): EFC-1000 pump (Tokyo
Rikakiki), Wakogel (Wako Pure chem., C-200); Sepha-
dex LH-20 (Amersham Pharmacia Biotech); Diaion
HP-20 (Mitsubishi Chem.); TLC, Merck silica gel 60
F254 (Art 5554); HPLC : Hitachi liquid chromatograph
L-7000 series system.
Fast atom bombardment mass spectra (FAB-MS)
were obtained on a Jeol HX-100, and field desorption
3.2. Plant material
Yarrow (A. millefolium) grew wild, and basil (O.
basilicum), spearmint (M. spicata), sage (S. officinalis),
rocket (E. vesicaria), parsley (P. crispum), watercress (N.
officinale), lemon balm (M. officinalis), and chervil (A.
cerefolium) were harvested from the experimental field
of the Plant Ecochemicals Research Center. Air-dried
aerial parts of oregano (O. vulgare), hyssop (H. offici-
nalis), chamomile (M. chamomilla), tansy (T. vulgare)
were purchased from Koyu-Seikatsu, Inc. (Kitami,
Hokkaido, Japan).
3.3. Chemicals
1-Deoxynojirimycin (11) and glucose B test were pur-
chased from Wako pure chemical industries, Ltd. Rat
intestinal acetone powder, 3, 4, 5-trimethoxycinnamic
acid, and AD-mix-b were purchased from Sigma
Aldrich Japan Co., Tokyo.
3.4. Assay for rat intestinal ꢀ-glucosidase inhibitory
activity
The a-glucosidase inhibitory activity was measured
according to the described method (Toda et al., 2001).
A crude enzyme solution was prepared from rat intest-
inal acetone powder. Before the amount of glucose
derived from sucrose in the reaction mixture was mea-
sured, the reaction mixture was passed through an alu-
mina column (1.6 g, ICN Alumina B, ICN Biomedicals
GmbH) in order not to hinder the glucose B test by
phenolic compounds.
Fig. 3. a-Glucosidase inhibitory activities of 1 and 2 (a) 1-deoxy-
nojirimysin showed its inhibitory activities of 21 and 58% at 1 ꢂ 10ꢀ8
ꢀ7
M and 3 ꢂ 10
M, respectively; (b) bars represent the meansꢃS.D.
of three measurements.

H. Matsuura et al. / Phytochemistry 65 (2004) 91–97
95
3.5. Extraction and separation
were combined and concentrated in vacuo. The resul-
tant black oil was subjected to Sephadex LH-20 (250 g)
with solvent MeOH–CHCl3 (7:3) to afford active frac-
tion C, which was applied to FCC using MeOH–CHCl3
(1:9) to afford active fraction D. Further purification of
fraction D by MPLC (MeOH–H2O–HOAc 50:50:0.1)
followed by another purification by HPLC (YMC,
YMC-Pack ODS-AM, 300 ꢂ 10 mm, MeOH–H2O–
AcOH 40:60:0.1, flow rate: 2.0 ml/min, A254nm) to afford
(7S, 8S)-syringoylglycerol 9-O-(60-O-cinnamoyl)-b-d-
glucopyranoside (2) (90 mg).
Air-dried leaves of hyssop (1 kg, H. officinalis) were
soaked in MeOH–H2O (7:3). After filtration using filter
paper (No. 1, Toyo), the filtrate was concentrated in
vacuo to about 1.5 l. The resulting extract was parti-
tioned between EtOAc (1 l ꢂ 3) and H2O to afford H2O
soluble and EtOAc soluble layers.
3.5.1. Purification (7S, 8S)-syringoylglycerol 9-O-(60-
O-cinnamoyl)-ꢂ-d-glucopyranoside (1)
The H2O soluble layer was concentrated under
reduced pressure to about 500 ml. To the concentrated
extract was added a solution of EtOH (2 l), and the
mixture was allowed to stand at ꢀ 25 C for 48 h. The
3.5.4. (7S, 8S)-Syringoylglycerol 9-O-(60-O-cinnamoyl)-
ꢂ-d-glucopyranoside (2)
Colorless oil; [ꢀ]D23 ꢀ 31.5ꢁ (c=1.0, MeOH); FD–MS
ꢁ
1
supernatant and precipitation were separated with filter
paper (No. 1, Toyo). The solution of the supernatant
was concentrated in vacuo, and the resultant oil was
subjected to Diaion HP-20 (500 g). The resins were
successively washed by H2O (2.5 l), MeOH–H2O (7:3) (4
l), and MeOH (2 l). The eluent of MeOH–H2O (7:3),
that exhibited a-glucosidase inhibitory activity, was
applied to Sephadex LH-20 (MeOH–H2O 7:3) to yield
an active fraction A, which was subjected to MPLC
(MeOH–H2O–HOAc 50:50:0.1) to give an active frac-
tion B. Further purification of fraction B by HPLC (GL
Sciences Inc., Inertsil ODS, 20 ꢂ 250 mm, MeOH–
H2O–HOAc 15:85:0.1, flow rate: 2 ml/min, A254nm) fol-
lowed by another purification by HPLC (Kanto chemi-
cal Co. Inc., Mightysil RP-18, 4.6 ꢂ 250 mm, CH3CN–
H2O–HOAc 8:92:0.1, flow rate: 1 ml/min, A254nm) affor-
ded (7S, 8S)-syringoylglycerol 9-O-(60-O-cinnamoyl)-b-
d-glucopyranoside (1) (565 mg)
m/z (rel. int.): 536 [M]+ (100); H NMR (270 MHz,
CD3OD):Table 1; 13C NMR (67.5 MHz, CD3OD):
Table 2.
3.6. Preparation of heptaacetylderivative (3)
To a stirred mixture of 1 in pyridine was added a
solution of acetic anhydride, and the reaction mixture
was further stirred for 24 h at room temperature. The
usual work-up was employed followed by the purifica-
tion of the extract to afford 3 quantitatively.
3.6.1. Heptaacetylderivative (3)
FD–MS m/z (rel. int.) 700 [M]+ (100); HR-FD–MS
m/z: 700.2190 [M]+ (calc. For C31H40O18: 700.2214); IR
1
ꢃmax (NaCl) cmꢀ1: 2944, 1755, 1222, 1040; H NMR
(270 MHz, CD3OD): ꢁ 6.62 (2H, s), 5.92 (1H, d, J= 8.1
Hz), 5.25 (1H, m), 5.22 (1H, t, J= 9.2, H-30), 5.06 (1H,
t, J= 9.7 Hz, H-40), 5.01 (1H, dd, J= 9.7, 7.8 Hz, H-20),
4.52 (1H, d, J= 7.7 Hz, H-10), 4.24 (1H, dd, J= 12.2,
4.7, H-60a), 4.06 (1H, dd, J= 12.2, 2.4, H-60b), 3.83 (3H,
s), 3.66 (3H, m), 2.32 (3H, s), 2.15 (3H, s), 2.09 (3H, s),
2.07 (3H, s), 2.06 (3H, s), 2.02 (3H, s).
3.5.2. (7S, 8S)-Syringoylglycerol 9-O-(60-O-cinnamoyl)
-ꢂ-d-glucopyranoside (1)
Colorless oil; [ꢀ]D23 ꢀ17.7ꢁ (c=0.5, MeOH); FAB-MS
(positive, matrix: glycerol) m/z (rel. int.): 429 [M+Na]+
(100); FAB-MS (negative, matrix: TEA) m/z (rel. int.): 405
1
[MꢀH]ꢀ (10.8); H NMR (270 MHz, CD3OD): Table 1;
3.7. Preparation of hexaacetylderivative (9)
13C NMR (67.5 MHz, CD3OD): Table 2.
To a stirred mixture of 2 in pyridine was added a
solution of acetic anhydride, and the reaction mixture
was further stirred for 24 h at room temperature. The
usual work-up was employed followed by the purifica-
tion of the extract to afford 9 quantitatively.
3.5.3. Purification of (7S, 8S)-syringoylglycerol 9-O-
(60-O-cinnamoyl)-ꢂ-d-glucopyranoside (2)
The EtOAc-soluble layer was concentrated under
reduced pressure, and the resultant black oil was sub-
jected to Diaion HP-20 (500 g). The resins were succes-
sively washed by MeOH–H2O (1:1) (3 l), MeOH–H2O
(7:3) (3 l), and MeOH (3 l). The eluate of MeOH–H2O
(1:1), that showed the inhibitory activities, was applied
to a silica gel (Wakogel C-200, Wako Pure Chem., 350
g) CC. The column was successively eluted with CHCl3
(2 l), MeOH–CHCl3 (5:95) (1 l), MeOH–CHCl3 (1:9) (1
l), MeOH–CHCl3 (3:7) (2 l), and MeOH–CHCl3 (4:6) (1
l). The eluate of MeOH–CHCl3 (1:9) and MeOH–
CHCl3 (3:7), that exhibited the inhibitory activities,
3.7.1. Hexaacetylderivative (9)
FD–MS m/z (rel. int.): 788 [M]+ (100), 494 (40); HR-
FD–MS m/z: 788.2508 [M]+ (calc. For C38H44O18:
788.2527); IR ꢃmax (NaCl) cmꢀ1: 2944, 1756, 1219, 1038;
1H NMR (270 MHz, CDCl3): ꢁ 7.65 (1H, d, J= 16.0
Hz), 7.50 (2H, m), 7.35 (3H, complex), 6.59 (2H, s), 6.40
(1H, d, J= 16.0 Hz), 5.90 (1H, d, J= 7.9 Hz), 5.24 (1H,
m), 5.22 (1H, br.t, J= 9.4 Hz), 5.07 (1H, br.t, J= 10.0
Hz), 4.92 (1H, dd, J= 9.5, 7.8 Hz), 4.52 (1H, d, J= 7.6

96
H. Matsuura et al. / Phytochemistry 65 (2004) 91–97
Hz), 4.26 (2H, m), 3.78 (6H, s), 3.74–3.56 (3H, com-
plex), 2.26 (3H, s), 2.12 (3H, s), 2.04 (3H, s), 2.02 (3H,
s), 2.00 (3H, s), 1.99 (3H, s). 13C NMR (67.5 MHz,
CDCl3): ꢁ 170.0, 169.8, 169.4, 169.2, 168.3, 166.2, 152.1,
145.6, 134.1, 134.0, 130.3, 128.7, 128.1, 117.0, 103.9,
101.0, 74.0, 73.6, 72.6, 71.9, 71.2, 68.6, 67.2, 62.0, 56.2,
21.0, 20.9, 20.7, 20.6, 20.5.
ice bath was added excess CH2N2, and the reaction
ꢁ
mixture was further stirred at 0 C for 1 h. The volatile
components were removed under reduced pressure to
give a residue. The residue was reduced using LAH (182
mg) in THF (50 ml) to give 7 (250 mg, 28%)
3.10.1. 3-(30, 40, 50-Trimethoxyphenyl) prop-2-en-1-ol
(7)
3.8. Preparation of (7S, 8S)-syringoylglycerol (4)
IR ꢃmax (NaCl) cmꢀ1: 3405, 2940, 1584, 1507, 1240,
1127, 1008; EI-MS m/z (rel. int): 224 (100), 195 (29), 182
1
To a stirred mixture of 2 (146 mg, 0.27 mmol) in 3 ml
of 0.1 N AcONH4–HOAc buffer (pH 5.6) was added b-
glucosidase (5 mg), and the mixture was shaken (100
(57), 181 (59), 83 (28); H NMR (270 MHz, CD3OD): ꢁ
6.61 (2H, s), 6.53 (1H, d, J= 15.9 Hz), 6.28 (1H, dt, J=
15.9, 5.4 Hz), 4.32 (1H, d, J= 5.4 Hz), 3.87 (6H, s), 3.84
(3H, s).
ꢁ
rpm) at 37 C for 24 h. The mixture was put directly
onto the cartridge column of Bond Elut C18. The col-
umn was washed with H2O (3 ml ꢂ 3) and then with
MeOH–H2O (7:3) (3 ml ꢂ 3). The volatile components
of the MeOH–H2O (7:3) eluents were removed to give 4
(45 mg, 0.18 mmol, 67%).
3.11. Preparation of (7R, 8R)-4-O-methylsyringoyl-
glycerol (8)
To a stirred mixture of CH3SO2NH2 (64 mg, 0.67
mmol) and AD-mix-b (929 mg) in a solution of t-
BuOH–H2O (1:1) (3 ml) was added a solution of 7 (150
mg, 0.67 mmol) in a solution of t-BuOH–H2O (1:1) (7
3.8.1. (7S, 8S)-Syringoylglycerol (4)
FD–MS m/z (rel. int.) 244 [M]+ (100); IR ꢃmax (NaCl)
cmꢀ1: 3360, 2940, 1614, 1519, 1463, 1428, 1216, 1113,
ꢁ
ml). The reaction mixture was stirred at 4 C for 24 h.
1
750; H NMR (270 MHz, CD3OD): ꢁ 6.70 (2H, s), 4.55
To the reaction mixture was added sodium sulfite (640
mg, 5.1 mmol), and the mixture further stirred for 1 h at
room temp. The reaction mixture was poured into a
mixture of EtOAc (300 ml) and H2O (150 ml). The
EtOAc layer was washed with satd. aq. NaCl (50 ml ꢂ
2) and dried over Na2SO4. The volatile components of
the EtOAc layer was removed under reduced pressure to
give a residue, which was purified by preparative TLC
(MeOH–CHCl3 9:41) to afford 8 (15 mg, 0.058 mmol,
9%).
(1H, d, J= 5.9 Hz), 3.86 (3H, s), 3,69 (1H, m), 3.50 (1H,
dd, J= 11.2, 3.7 Hz), 3.30 (1H, dd, J= 11.2, 6.2 Hz);
13C NMR (67.5 MHz, CD3OD): ꢁ 148.9, 135.8, 133.9,
105.0, 77.5, 75.5, 64.2, 56.7.
3.9. Preparation of (7S, 8S)-4-O-methylsyringoyl-
glycerol (5)
To a stirred mixture of 4 (16 mg, 0.065 mmol) in
MeOH (10 ml) cooled with an ice bath was added excess
CH2N2. The reaction mixture was further stirred for 1 h
3.11.1. (7R, 8R)-4-O-Methylsyringoylglycerol (8)
[ꢀ]2D3 ꢀ17.0ꢁ (c=0.50, MeOH); HR-EI-MS m/z:
258.1115 [M]+ (calc. for C12H18O6: 258.1103).
ꢁ
at 0 C. The volatile components were removed under
reduced pressure to give a colorless residue, which was
purified using preparative TLC (MeOH–CHCl3 1:9) to
afford 5 (5.9 mg, 0.022 mmol, 35%).
3.12. Alkaline hydrolysis of 2
3.9.1. (7S, 8S)-4-O-Methylsyringoylglycerol (5)
[ꢀ]2D3 21.1ꢁ (c=0.59, MeOH) ; EI-MS m/z (rel. int): 258
(34), 198 (12), 197 (100), 196 (34), 169 (41), 154 (17), 138
(20); HR-EI-MS m/z: 258.1096 [M]+ (calc. for C12H18O6:
258.1103); IR ꢃmax (NaCl) cmꢀ1: 3383, 2940, 1593, 1506,
1463, 1422, 1328, 1235, 1125, 1005, 754; 1H NMR
(CD3OD, 500 MHz); ꢁ 6.71 (2H, s, H-20 and -60), 4.59 (1H,
d, J=2.9 Hz, H-3), 3.84 (6H, s, C-30-OMe and C-50-OMe),
3.74 (3H, s, C-40-OMe), 3.67 (1H, m, H-2), 3.54 (1H, dd,
J=6.1, 2.4 Hz, H-1a), 3.40 (1H, dd, J=6.1, 3.3 Hz, H-1b).
To a stirred solution of 1% NaOH/EtOH (25 ml) was
added a solution of 2 (25 mg, 0.046 mmol) in MeOH (10
ml), and the reaction mixture was stirred for 5 h. The
reaction mixture was treated with the resins of amberlite
120 B (Organo, 150 g). The resins was washed with
MeOH (50 ml). The combined eluents were con-
centrated under reduced pressure, and the resultant
residue was dissolved in 1 ml of MeOH–H2O (1:9) and
put onto the cartridge column of Bond Elut C18. T he
column was washed with MeOH–H2O (1:9) (3 ml ꢂ 2)
and then with MeOH–H2O (4:1) (3 ml ꢂ 2). The eluate
of MeOH–H2O (1:9) was concentrated under reduced
pressure to give 1 (14 mg, 0.035 mmol, 76%), and the
eluate of MeOH–H2O (4:1) to afford 10 (50 mg, 0.034
mmol, 74%).
3.10. Preparation of (2E) 3-(30, 40, 50-trimethoxyphenyl)
prop-2-en-1-ol (7)
To a stirred mixture of 3, 4, 5-trimethoxycinnamic
acid (6, 952 mg, 4 mmol) in MeOH (20 ml) cooled with

H. Matsuura et al. / Phytochemistry 65 (2004) 91–97
97
Matsuura, H., Asakawa, C., Kurimoto, M., Mizutani, J., 2002. a-
Glucosidase inhibitor from the seeds of balsam pear (Momordica
charantia) and the fruit of Grifola frondosa. Biosci. Biotechnol. Bio-
chem. 66, 1576–1578.
Acknowledgements
The authors thank Mr. Kenji Watanabe and Dr. Eri
Fukusi for FAB-mass, FD-mass, EI-mass, and 2D
NMR measurements.
Reese, E.T., Parrish, F.W., 1968. Nojirimycin and D-glucono-1, 5-
lactone as inhibitors of carbohydrases. Carbohydr. Res. 18, 381–
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Toda, M., Kawabata, J., Kasai, T., 2001. Inhibitory effects of ellagi-
and gallotannins on rat intestinal a-glucosidase complexes. Biosci.
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