Antidiabetogenic oligostilbenoids and 3-ethyl-4-phenyl-3,4- dihydroisocoumarins from the bark of Shorea roxburghii

Article abstract of DOI:10.1016/j.bmc.2011.11.067

A methanol extract of the bark of Shorea roxburghii (Dipterocarpaceae) was found to inhibit plasma glucose elevation in sucrose-loaded mice. From the extract, three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, 1′S-dihydrophayomphenol A₂ (1) an

Full text of DOI:10.1016/j.bmc.2011.11.067

Bioorganic & Medicinal Chemistry 20 (2012) 832–840
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Bioorganic & Medicinal Chemistry
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Antidiabetogenic oligostilbenoids and 3-ethyl-4-phenyl-3,4-
dihydroisocoumarins from the bark of Shorea roxburghii
Toshio Morikawa a, Saowanee Chaipech a, Hisashi Matsuda b, Makoto Hamao b, Yohei Umeda b,
Hiroki Sato b, Haruka Tamura b, Haruka Kon’i b, Kiyofumi Ninomiya a, Masayuki Yoshikawa a,b
,
Yutana Pongpiriyadacha c, Takao Hayakawa a, Osamu Muraoka a,
⇑
a Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan
b Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
c Faculty of Science and Technology, Rajamangala University of Technology Srivijaya, Thungsong, Nakhonsithammarat 80110, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
A methanol extract of the bark of Shorea roxburghii (Dipterocarpaceae) was found to inhibit plasma glucose
elevation in sucrose-loaded mice. From the extract, three new 3-ethyl-4-phenyl-3,4-dihydroisocouma-
rins, 10S-dihydrophayomphenol A2 (1) and phayomphenols B1 (2) and B2 (3), were isolated together with
24 known compounds including 20 stilbenoids and oligostilbenoids. The structures of 1–3 were deter-
mined on the basis of their spectroscopic properties as well as of chemical evidences. Among the isolates,
Received 2 September 2011
Revised 28 November 2011
Accepted 29 November 2011
Available online 13 December 2011
(ꢀ)-hopeaphenol (6), hemsleyanol D (8), (+)- -viniferin (15), and (ꢀ)-balanocarpol (18) showed inhibitory
a
Keywords:
Antidiabetogenic activity
Oligostilbenoid
Shorea roxburghii
Phayomphenol
activity against plasma glucose elevation in sucrose-loaded rats at doses of 100–200 mg/kg, p.o. To clarify
the mode of action of the antihyperglycemic property, effects of these oligostilbenoids on gastric emptying
in mice, those on glucose uptake in isolated intestinal tissues as well as inhibitory activities against rat
intestinal
a
-glucosidase and rat lens aldose reductase were examined.
Ó 2011 Elsevier Ltd. All rights reserved.
3-Ethyl-4-phenyl-3,4-dihydroisocoumarin
a-Glucosidase inhibitor
1. Introduction
(3) have been isolated as minor constituents. This paper deals with
the structural elucidation of these new dihydroisocoumarins (1–3)
as well as inhibitory effects of the principal oligostilbenoid constit-
uents on increase in plasma glucose levels in sucrose-loaded mice.
To clarify the mechanism of action of the antidiabetogenic activity
observed, effects of these oligostilbenoids on gastric emptying in
mice, those on glucose uptake in isolated intestinal tissues as well
Shorea roxburghii G. DON (Dipterocarpaceae) is widely distrib-
uted in Thailand and its neighboring countries such as Cambodia,
India, Laos, Malaysia, Myanmar, and Vietnam, etc. The bark of
S. roxburghii (‘Phayom’ in Thailand) has been used as an astringent
or a preservative for traditional beverages in Thailand. In Indian folk
medicine, they have been used for treatments of dysentery, diarrhea,
and cholera, etc.1 In the course of our exploratory studies on bioac-
tive constituents in Thai natural medicines,2–13 we have reported
isolation and structural elucidation of dihydroisocoumarins, stilbe-
noids, and oligostilbenoids from a methanol extract of the bark of S.
roxburghii.2 We also revealed that the methanol extract and several
oligostilbenoid constituents showed antihyperlipidemic effect in
olive oil-loaded mice and pancreatic lipase inhibitory activity.2 As
a continuing study, we conducted further evaluations of the extract
and/or constituents, and found that the methanol extract showed
inhibitory activity against increase in plasma glucose levels in
sucrose-loaded mice. By the intensive fractionalization of the ex-
tract, three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins named
10S-dihydrophayomphenol A2 (1) and phayomphenols B1 (2) and B2
as their inhibitory activities against rat intestinal
and rat lens aldose reductase were also examined.
a-glucosidase
2. Results and discussion
2.1. Effect of methanol extract of the bark of S. roxburghii on
plasma glucose elevation in sucrose-loaded mice
The bark of S. roxburghii (collected in Phatthalung Province,
Thailand) was extracted with methanol under reflux to yield a meth-
anol extract (15.6% from the dried bark). The methanol extract was
subjected to Diaion HP-20 column chromatography (H2O ? MeOH)
to give H2O- and MeOH-eluted fractions (3.2% and 10.6%, respec-
tively). As shown in Table 1, the methanol extract and its MeOH-
eluted fraction showed inhibitory effect against increase in plasma
glucose levels in sucrose-loaded mice at a dose of 500 mg/kg, p.o.
⇑
Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6729 3577.
E-mail address: muraoka@phar.kindai.ac.jp (O. Muraoka).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.067

T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
833
Table 1
Inhibitory effects of the methanolic extract from the bark of S. roxburghii and its methanol- and H2O-eluted fractions on plasma glucose levels in sucrose-loaded mice
Treatment
Dose (mg/kg, p.o.)
n
Plasma glucose (mg/dL)a
0.5 h
1.0 h
2.0 h
Normal
Control
MeOH extract
—
—
4
6
4
4
4
4
4
4
4
4
121.4 14.7c
225.1 12.5
207.6 22.6
188.1 17.9
154.4 9.8c
198.0 13.9
159.9 17.2
137.3 4.4c
176.7 12.6
230.6 34.3
124.9 7.2c
229.9 16.2
213.4 19.2
180.0 23.2
179.3 12.1
185.8 14.4
172.7 6.1
106.5 3.9b
145.2 7.2
156.0 7.4
146.9 12.6
167.6 6.9
152.5 3.4
152.8 3.4
162.3 8.0
142.7 14.4
142.3 14.6
125
250
500
125
250
500
250
500
MeOH-eluted fraction
H2O-eluted fraction
160.5 14.4a
178.6 14.0
210.0 20.9
Normal
Control
Acarbose
—
—
10
20
6
9
6
6
124.8 7.3c
218.7 4.0
143.0 5.4c
208.9 6.8
183.8 3.8b
185.4 8.1b
131.8 6.4c
163.7 3.7
151.5 6.3
152.8 3.8
162.4 11.7c
153.8 10.2c
a
b
c
Each value represents the mean S.E.M.
Significantly different from the control, p <0.05.
Significantly different from the control, p <0.01.
2.2. Chemical constituents from the bark of S. roxburghii
nol (7, 0.53%), hemsleyanol D (8, 0.30%), (ꢀ)-ampelopsin H (9,
0.015%), vaticanols B (10, 0.031%), C (11, 0.032%), A (12, 0.28%), E
In the preceding paper,2 we reported the isolation and structure
elucidation of two new dihydroisocoumarins, phayomphenols A1
(4) and A2 (5), relatively as the major constituents, the yield of
which from the dried bark being 0.29% and 0.11%, respectively.
By the intensive fractionation of the MeOH-eluted fraction in this
study, three related compounds, 10S-dihydrophayomphenol A2 (1,
0.0061%) and phayomphenols B1 (2, 0.0005%) and B2 (3, 0.0006%)
were isolated as new constituents. Twenty kinds of stilbenoids
and oligostilbenoids, (ꢀ)-hopeaphenol (6, 0.63%), (+)-isohopeaphe-
(13, 0.30%), and G (14, 0.042%), (+)-a-viniferin (15, 0.10%), paucifl-
orol A (16, 0.014%), hopeafuran (17, 0.012%), (ꢀ)-balanocarpol (18,
0.070%), (ꢀ)-ampelopsin A (19, 0.012%), malibatols A (20, 0.0029%)
and B (21, 0.0007%), (+)-parviflorol (22, 0.0029%), trans-resveratrol
10-C-b-
glucopyranoside (24, 0.0081%), and piceid (25, 0.0098%), and a fla-
vonol glycoside, quercetin 3-O- -rhamnopyranoside (0.0050%),
D-glucopyranoside (23, 0.90%), cis-resveratrol 10-C-b-D-
a-L
and a megastigmane glycoside, (6S,9S)-roseoside (0.0017%), were
also isolated from the extracts of this plant (Chart 1).
Chart 1. Constituents from the bark of S. roxburghii.

834
T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
2.3. Structures of 10S-dihydrophayomphenol A2 (1) and phayom-
phenols B1 (2) and B2 (3)
Table 3
13C NMR (125 MHz) data on 10S-dihydrophayomphenol A2 (1) and 1a
Position
1a
1b
1ab
dC
10S-Dihydrophayomphenol A2 (1) was obtained as a white pow-
dC
dC
der with positive optical rotation (½a D25
ꢁ
+79.5 in MeOH). Its IR spec-
1
3
165.2
90.3
166.9
90.7
166.4
90.3
38.3
158.7
105.6
161.6
104.5
127.9
123.0
70.1
trum showed absorption bands at 3390, 1698, 1615, 1514, 1474,
1362, and 1258 cmꢀ1 ascribable to hydroxyls, lactone carbonyl
functions, and an aromatic ring. The positive-ion FABMS spectrum
of 1 showed a quasimolecular ion peak at m/z 339 (M+Na)+, and
the molecular formula was determined as C17H16O6, a two hydro-
gen homolog to compound 5, by high-resolution positive-ion FAB-
MS measurement. The 1H and 13C NMR spectral properties of 114
(Tables 2 and 3) were quite similar to those of 5 (Tables 4 and 5),
except for signals due to 10 and 20 positions. Instead of a signal
due to the carbonyl carbon [(dc 205.4 in CD3OD)] observed in the
13C NMR spectrum of 5, a signal of methine carbon bearing an oxy-
gen appeared at dc 69.9 (in CD3OD) in that of 1. In the 1H NMR
spectrum of 1 (pyridine-d5, Tables 2 and 3), an additional methine
signal appeared at d 4.43 (1H, dq, J = 6.2, 6.6 Hz, H-10), and thus the
signal of the adjacent methyl appeared as a doublet at d 1.52 (3H, d,
J = 6.6 Hz, H3-20). Therefore, the structure of 1 was speculated to be
the reduced product of 5 at C-10 position. Actually oxidation of 1
with chromium trioxide (CrO3) in pyridine afforded phayomphenol
A2 (5). The CD spectrum of the oxidized product was completely in
accord with that of 5, thus the absolute stereochemistry at C-3 and
C-4 being approved to be S and S, respectively.
4
5
6
7
8
9
38.0
37.9
157.0
109.1
159.0
107.4
128.6
119.7
69.5
156.8
109.3
158.8
107.4
128.0
119.7
69.9
10
10
20
20.8
20.0
20.0
100
134.2
129.5
116.5
157.8
134.2
129.6
116.3
157.3
135.3
129.5
115.0
160.1
55.7
200,600
300,500
400
5-OCH3
7-OCH3
400-OCH3
56.1
56.4
a
Measured in pyridine-d5.
Measured in CD3OD.
b
Table 4
1H NMR (CD3OD) data on phayomphenols B1 (2), B2 (3), A1 (4), and A2 (5)
Position 2a
dH (J Hz)
3a
4b
dH (J Hz)
5b
dH (J Hz)
The absolute configuration of the C-10 position in 1 was eluci-
dated by the application of the modified Mosher’s method.15
Namely, 5,7,400-trimethyl ester (1a), which was obtained from 1
upon reaction with trimethylsilyldiazomethane (TMSCHN2), was
derived to 10-(R)-MTPA ester (1b) by treatment with (R)-2-meth-
oxy-2-trifluoromethylphenylacetic acid [(R)-MTPA] in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCꢂHCl) and
4-dimethylaminopyridine (4-DMAP). On the other hand, 10-(S)-
MTPA ester (1c) was obtained from 1a using (S)-MTPA under the
same conditions. As shown in Figure 2, signals due to the protons
at C-3 and C-4 in 1c were observed at lower field compared with
dH (J Hz)
3
4
6
8
20
200
300
500
600
5.11 (d, 3.9)
4.54 (d, 3.9)
6.57 (d, 2.2)
7.04 (d, 2.2)
1.88 (3H, s)
6.45 (d, 2.2)
6.62 (d, 8.2)
6.62 (d, 8.2)
5.27 (d, 1.2)
4.74 (br s)
5.14 (d, 4.0) 5.28 (d, 1.5)
4.61 (d, 4.0) 4.80 (br s)
6.57 (d, 2.3) 6.55 (d, 2.3)
7.04 (d, 2.3) 6.99 (d, 2.3)
1.86 (3H, s) 2.33 (3H, s)
6.82 (d, 8.6) 6.96 (d, 8.6)
6.64 (d, 8.6) 6.71 (d, 8.6)
6.64 (d, 8.6) 6.71 (d, 8.6)
6.55 (d, 2.2)
6.98 (d, 2.2)
2.22 (3H, s)
6.54 (d, 2.2)
6.70 (d, 8.2)
6.70 (d, 8.2)
6.33 (dd, 2.2, 8.2) 6.51 (dd, 2.2, 8.2) 6.82 (d, 8.6) 6.96 (d, 8.6)
a
Measured at 700 MHz.
Measured at 500 MHz.
b
those of 1b [
10 in 1c was observed at higher field compared with that of 1b
d: negative]. Thus, the absolute configuration at C-10 of 1a was
Dd: positive], while the signal due to the proton at C-
[D
hydroxyls, a carbonyl, and a lactone carbonyl functions, and an aro-
matic ring. The molecular formula determined by means of high-
resolution EIMS was C17H14O7, a one oxygen homolog of 4. Instead
of ortho-coupled A2B2-type aromatic protons observed in the 1H
NMR spectrum of 4 [d 6.64, 6.82 (2H each, both d, J = 8.6 Hz, H-
300,500, 200,600)], ortho- and meta-coupled ABC-type aromatic protons
determined to be S. Consequently, the absolute stereostructure of
1 was elucidated to be as shown. 1H–1H COSY, HMQC, HMBC, and
NOESY spectral properties also supported the depicted structure.
Phayomphenol B1 (2), ½a D25
ꢁ
+162.9 (in MeOH), was obtained as a
white powder. Its IR spectrum showed absorption bands at 3400,
1717, 1698, 1617, 1362, 1250, and 1125 cmꢀ1 ascribable to
Table 2
1H NMR (500 MHz) data on 10S-dihydrophayomphenol A2 (1) and 1a–1c
Position
1a
1b
1ab
1bb
1cb
dH (J Hz)
dH (J Hz)
dH (J Hz)
dH (J Hz)
dH (J Hz)
3
4
5.01 (br d, ca. 6)
5.10 (br s)
4.39 (dd, 1.2, 6.9)
4.39 (br s)
4.42 (dd, 1.2, 5.8)
4.43 (br s)
4.58 (dd, 1.2, 6.9)
4.38 (br s)
4.64 (dd, 1.2, 8.0)
4.47 (br s)
6
7.11 (d, 2.4)
7.42 (d, 2.4)
4.43 (dq, 6.2, 6.6)
1.52 (3H, d, 6.6)
7.46 (2H, d, 8.6)
7.09 (2H, d, 8.6)
6.58 (d, 2.3)
7.01 (d, 2.3)
3.79 (dq, 6.9, 6.6)
1.19 (3H, d, 6.6)
6.89 (2H, d, 8.6)
6.68 (2H, d, 8.6)
6.81 (d, 2.3)
7.21 (d, 2.3)
3.82 (m)
1.17 (3H, d, 6.6)
6.96 (2H, d, 8.6)
6.81 (2H, d, 8.6)
3.72 (3H, s)
6.68 (d, 2.3)
7.22 (d, 2.3)
5.29 (m)
1.45 (3H, d, 6.6)
6.91 (2H, d, 8.6)
6.80 (2H, d, 8.6)
3.68 (3H, s)
3.89 (3H, s)
3.73 (3H, s)
6.86 (d, 2.3)
7.22 (d, 2.3)
5.25 (m)
1.31 (3H, d, 6.3)
6.95 (2H, d, 8.6)
6.82 (2H, d, 8.6)
3.77 (3H, s)
3.90 (3H, s)
3.74 (3H, s)
8
10
20
200,600
300,500
5-OCH3
7-OCH3
400-OCH3
10-OMTPA
3.86 (3H, s)
3.73 (3H, s)
3.50 (3H, s)
3.50 (3H, s)
7.36–7.41 (3H, m)
7.45–7.47 (2H, m)
7.39–7.42 (3H, m)
7.47–7.48 (2H, m)
a
Measured in pyridine-d5.
Measured in CD3OD.
b

T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
835
[d 6.33 (1H, dd, J = 2.2, 8.2 Hz, H-600), 6.45 (1H, d, J = 2.2 Hz, H-200),
6.62 (1H, d, J = 8.2 Hz, H-500)] were detected in the 1H NMR spec-
trum of 2. As a HMBC cross-peak was observed between the signals
due to C-4 (dc 40.5) and a signal at d 6.45, which was assigned to
H-200, the position of the newly introduced OH group was deduced
to be at C-300. The 1H–1H COSY, HMBC and NOESY (Fig. 1) spectra
supported well the structure of 2.
Table 5
13C NMR (CD3OD) data on phayomphenols B1 (2), B2 (3), A1 (4), and A2 (5)
Position
2a
3a
4b
5b
dC
dC
dC
dC
1
3
4
5
6
7
8
9
10
10
20
100
200
300
400
500
600
166.9
86.9
166.7
89.3
166.9
86.7
166.7
89.1
38.5
40.5
38.7
40.2
156.5
109.8
159.2
107.6
126.7
121.7
207.8
27.7
156.8
109.2
159.3
107.5
127.8
117.6
205.4
26.0
156.4
109.7
159.1
107.5
126.9
121.6
207.6
27.6
156.7
109.2
159.3
107.5
127.8
117.5
205.4
26.0
132.4
129.8
116.3
157.6
116.3
129.8
Phayomphenol B2 (3) was also isolated as a white powder with
positive optical rotation (½a D25
ꢁ
+212.1 in MeOH). The molecular
formula, C17H14O7, obtained by the positive-ion high-resolution
FABMS measurement, was found to be the same as that of 2. The
1H and 13C NMR spectroscopic properties of 3 (CD3OD, Tables 4
and 5) were quite similar to those of 2, except for the signal due
to the methyl group [d 2.22 (3H, s, H3-20)]. The position of the new-
ly introduced OH was determined in the same manner as was ap-
plied in the case for 2, the ortho- and meta-coupled ABC-type
aromatic protons [d 6.51 (1H, dd, J = 2.2, 8.2 Hz, H-600), 6.54 (1H,
d, J = 2.2 Hz, H-200), 6.70 (1H, d, J = 8.2 Hz, H-500)] and a cross-peak
between the signal due to C-4 (dc 38.7) and the signal at d 6.54
being observed in the 1H NMR and HMBC spectra, respectively.
129.1
117.3
146.2
145.8
116.2
121.7
133.2
115.9
146.4
145.5
116.5
120.1
128.3
131.2
116.1
157.9
116.1
131.2
a
Measured at 175 MHz.
Measured at 125 MHz.
b
Figure 1. 1H–1H COSY, HMBC, and NOESY correlations of 1–3.
Figure 2. Determination of absolute stereochemistry of 1. Reagents and conditions: (a) CrO3/pyridine, rt, 8 h, 29%. (b) TMSCHN2/MeOH, rt, 8 h, quant. (c) (R)- or (S)-MTPA,
EDCꢂHCl,4-DMAP/CH2Cl2, , 15 h, 1b: 43%, 1c: 66%.
D

836
T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
Finally, the absolute stereochemistry of 2 and 3 were confirmed
by their CD spectra, which showed a negative (
ꢀ0.14 at
296 nm) and a positive Cotton effect ( +4.37 at 294 nm), respec-
Next, the mode of action for the antihyperglycemic effects of
D
e
the stilbenoids was studied. Plasma glucose levels are known to
be regulated by many factors such as transport of sugar in the
digestive tract, the secretion and release of hormones, and absorp-
tion of glucose through membranes of the small intestine.29 Previ-
ously, we reported that inhibition of gastric emptying markedly
inhibited plasma glucose and triglyceride elevations in sucrose-
and olive oil-loaded mice or rats, respectively.3,30–37 Thus, effects
of the oligostilbenes (6–8, 15, and 18) on the gastric emptying rate
were examined. As shown in Table 7, compounds 6, 7, and 18 sig-
nificantly suppressed the gastric emptying in mice at a dose of
200 mg/kg, p.o., and 15 also tended to suppress gastric emptying
(p = 0.063) at the same dose. These findings suggest that these oli-
gostilbenoids inhibited the plasma glucose elevation in orally su-
crose-loaded mice possibly in part by suppressing the transfer of
sucrose from the stomach to the small intestine.
D
e
tively. According to published CD data for 3,4-dihydroisocouma-
rins, these Cotton effects were assignable to n ? p⁄ transition of
the lactone carbonyl group of dihydroisocoumarin chromo-
phore.2,16,17 This indicated that the 3-acetyl group in 3 was in
a-ax-
ial orientation, and accordingly the absolute configuration at C-3 of
3 was concluded to be S.18–23 In the same manner, the absolute
configuration at C-3 of 2 was confirmed to be R. These results indi-
cated that the 3,4-dihydroxyphenyl moieties at C-4 in 2 and 3 were
both in b-axial orientation, and their absolute configurations were
also concluded to be S. On the basis of the foregoing evidences, the
absolute stereostructures of 2 and 3 were confirmed as (3R,4S)-
and (3S,4S)-3-acetyl-5,7-dihydroxy-4-(3,4-dihydroxyphenyl)-3,4-
dihydroisocoumarin, respectively.
Next, effects of these constituents on glucose uptake in rat small
2.4. Antihyperglycemic effects of the chemical constituents
intestinal tissues38,39 and also those on inhibitory activities28,31,40–42
from the bark of S. roxburghii
against rat small intestinal a-glucosidases, maltase and sucrase,
were examined. As shown in Table 8, principal oligostilbenoids
(6–8, 15, and 18) did not inhibit glucose uptake at a concentration
of 1 mM. On the other hand, most of the oligostilbenoids (6–13,
With respect to the principal constituents of S. roxburghii,
phayomphenols A1 (4) and A2 (5), (ꢀ)-hopeaphenol (6), (+)-isoho-
peaphenol (7), hemsleyanol D (8), (+)-
a-viniferin (15), (ꢀ)-balano-
15, 17, 20, and 21) moderately inhibited the
as shown in Table 9. These results suggest that the intestinal
a-glucosidase inhibitory activity is also involved in the inhibitory
a-glucosidase activities
carpol (18), trans-resveratrol 10-C-b-D-glucopyranoside (23), and
resveratrol (25a),24–27 effects on increase in plasma glucose levels
in sucrose-loaded mice were examined. As shown in Table 6, oligo-
stilbenoids, 6, 8, 15, and 18 showed significant inhibitory activity
against increase in plasma glucose levels at a dose of 100 or
200 mg/kg, p.o., and at 200 mg/kg, p.o. (p = 0.070), 7 tended to inhi-
bit the elevation. The inhibitory effects of 8 and 15 were stronger
than those of 6, 7, and 18, while a stilbene monomer (25a) and its
glycoside (23) showed weak effects. In this experiment, an intestinal
effects of the oligostilbenoids (6–8 and 15, but not 18) against blood
glucose elevation in sucrose-loaded mice.
Finally, inhibitory effects of these constituents including minor
contents on rat lens aldose reductase were examined. Aldose
reductase is known to be a key enzyme which catalyzes the
reduction of glucose to sorbitol in the polyol processing pathway.
Sorbitol dose not readily diffuse across cell membranes, and the
intracellular accumulation of sorbitol has been implicated in the
chronic complications of diabetes such as cataracts.27,43 As shown
in Table 9, several dihydroisocoumarins (2–5) and stilbenoids (6,
a-glucosidase inhibitor, acarbose, was employed as a positive
control, which showed reasonable inhibition as was reported in
the literature (Table 1).28
Table 6
Inhibitory effects of the constituents from tfhe bark of S. roxburghii on plasma glucose levels in sucrose-loaded mice
Treatment
Dose (mg/kg, p.o.)
n
Plasma glucose (mg/dL)a
1.0 h
0.5 h
2.0 h
Normal
Control
Phayomphenol A1 (4)
—
—
6
7
7
7
6
7
7
10
6
7
6
7
6
7
6
7
6
7
126.6 5.2c
220.5 16.9
189.3 10.1
184.1 15.5
231.2 22.6
177.0 10.4
115.7 9.4c
232.9 11.8
191.6 13.9
158.3 5.6c
182.5 19.1
179.7 8.2
139.8 5.6c
221.4 9.1
199.5 10.1
178.1 11.0b
226.7 19.8
197.3 6.0
125.2 10.3c
229.0 9.6
194.9 5.9
187.9 6.1
204.9 7.3
209.4 11.0
211.8 24.3
201.7 24.9
219.8 6.1
184.2 7.7
210.6 12.0
192.0 10.8
116.0 3.2
160.8 3.5
159.2 5.9
166.2 7.0
179.4 12.9
166.9 8.0
129.9 10.2c
172.0 11.3
160.6 7.3
179.4 4.4
191.6 10.8
189.1 +6.8
174.1 16.6
177.4 15.2
173.3 9.1
167.2 6.9
157.4 5.1
156.4 9.7
100
200
100
200
—
Phayomphenol A2 (5)
Normal
Control
(ꢀ)-Hopeaphenol (6)
—
100
200
100
200
100
200
100
200
100
200
(+)-Isohopeaphenol (7)
Hemsleyanol D (8)
173.5 16.8b
142.4 22.7c
151.2 21.2c
153.5 19.2c
227.9 12.6
169.2 14.5c
(+)-a-Viniferin (15)
(ꢀ)-Balanocarpol (18)
Normal
Control
—
—
100
200
8
8
8
8
119.4 4.8c
226.5 17.3
184.3 17.8
188.7 10.1
131.9 2.7c
200.9 4.3
183.4 10.0
202.3 12.4
114.7 3.9c
148.8 6.3
140.9 6.0
147.9 3.1
trans-Resveratrol 10-C-Glc (23)
Normal
Control
Resveratrol (25a)
—
—
100
200
5
10
7
137.5 7.2c
230.9 15.7
218.3 12.2
205.6 8.0
142.1 11.0c
203.0 9.3
210.9 14.0
192.7 7.7
135.9 8.1c
174.5 7.1
181.4 4.5
158.7 7.5
6
a
b
c
Each value represents the mean S.E.M.
Significantly different from the control, p <0.05.
Significantly different from the control, p <0.01.

T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
837
Table 7
Inhibitory effects of the constituents from the bark of S. roxburghii on gastric emptying in CMC-Na-loaded mice
Treatment
Dose (mg/kg, p.o.)
n
Gastric emptying (%)a
Inhibition (%)
Control
—
9
8
8
4
8
7
82.0 3.1
58.2 2.7b
64.7 5.1b
74.9 6.5
69.4 3.4
61.7 2.3b
—
(ꢀ)-Hopeaphenol (6)
(+)-Isohopeaphenol (7)
Hemsleyanol D (8)
200
200
200
200
200
29.0
21.1
8.7
15.4
24.8
(+)-a-Viniferin (15)
(ꢀ)-Balanocarpol (18)
a
Each value represents the mean S.E.M.
Significantly different from the control, p <0.01.
b
Table 8
Inhibitory effects of the constituents from the bark of S. roxburghii on glucose uptake in rats intestinal tissues
Conc. (mM)
n
Glucose uptakea (dpm/100 mg tissue)
Inhibition (%)
Control
Phrolizin
(ꢀ)-Hopeaphenol (6)
(+)-Isohopeaphenol (7)
Control
—
1
1
1
—
1
1
1
1
6
6
6
6
10
5
5
5
5
3172 419
1606 103b
2830 259
3038 240
3992 370
1642 128b
3595 311
3564 254
4088 396
0.0
100.0
21.8
8.5
0.0
Phrolizin
Hemsleyanol D (8)
100.0
16.9
18.2
ꢀ4.1
(+)-a-Viniferin (15)
(ꢀ)-Balanocarpol (18)
a
Each value represents the mean S.E.M.
Significantly different from the control, p <0.01.
b
Table 9
Inhibitory effects of the methanolic extract from the bark of S. roxburghii and its methanol- and H2O-eluted fractions and the constituents on enzyme activities of
a-glucosidases,
and aldose reductase activities
a
a
a
-Glucosidase IC50
(
lg/mL)
Aldose reductase IC50
(
lg/mL)
Maltase
Sucrase
MeOH extract
150
128
19.6
MeOH-eluted fraction
H2O-eluted fraction
235
183
17.9
>400 (4.8)c
>400 (6.5)c
>400 (0.9)c
a
a
a-Glucosidase IC50
(l
M)
Aldose reductase IC50
(
lM)
Maltase
>400 (31.4)c
>400 (20.9)c
>400 (40.4)c
>400 (4.0)c
>400 (19.6)c
338
Sucrase
>400 (30.3)c
ca. 400
211
10S-Dihydrophayomphenol A2 (1)
Phayomphenol B1 (2)
Phayomphenol B2 (3)
Phayomphenol A1 (4)
Phayomphenol A2 (5)
(ꢀ)-Hopeaphenol (6)
(+)-Isohopeaphenol (7)
Hemsleyanol D (8)
>100 (18.8)b
32.5
26.6
39.8
47.7
>400 (6.4)c
>400 (3.7)c
195
69.0
216
266
90.0
218
>100 (34.4)b
29.4
(ꢀ)-Ampelopsin H (9)
178
233
97.1
294
50.2
30.0
Vaticanol B (10)
Vaticanol C (11)
140
218
342
94.2
148
88.9
21.2
23.7
30.8
Vaticanol A (12)31
Vaticanol E (13)31
Vaticanol G (14)31
>400 (32.2)c
172
>400 (37.8)c
234
>100 (46.7)b
7.8
(+)-a-Viniferin (15)
Pauciflorol A (16)
Hopeafuran (17)
(ꢀ)-Balanocarpol (18)
(ꢀ)-Ampelopsin A (19)
Malibatol A (20)
>400 (44.0)c
142c
55.7
105
29.5
6.9
30.0
68.8
>400 (29.4)c
> 400 (–6.3)
362
>400 (36.6)c
>400 (0.4)c
231
35.6
Malibatol B (21)
262
143
10.0
(+)-Parviflorol (22)
trans-Resveratrol 10-C-Glc (23)
cis-Resveratrol 10-C-Glc (24)
Piceid (25)
>400 (6.8)c
>400 (20.1)c
>400 (36.6)c
>400 (38.2)c
>400 (36.9)c
2.0
>400 (-0.8)c
>400 (14.7)c
>400 (26.8)c
>400 (48.0)c
>400 (35.7)c
1.7
>100 (43.7)b
>100 (30.3)b
>100 (39.2)b
ca. 100
25.027
Resveratrol (25a)
Acarbose31
Epalrestat31
0.072
a
Each value represents the mean of 2–4 experiments.
Values in parentheses present inhibition % at 100 or 400
b,c
lM.

838
T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
8–13, 15–21, and 25a) moderately inhibited the activity of rat lens
aldose reductase.
combined extracts under reduced pressure provided a MeOH extract
(575.7 g, 15.6%). An aliquot (525.7 g) was subjected to Diaion HP-20
CC (3.0 kg, H2O ? MeOH, twice) to give H2O-eluted (107.0 g, 3.2%)
and MeOH-eluted (358.5 g, 10.6%) fractions. An aliquot (180.0 g) of
the MeOH-eluted fraction was subjected to normal-phase silica gel
CC [3.0 kg, CHCl3–MeOH–H2O (10:3:0.4 ? 7:3:0.5 ? 6:4:1, v/v/
v) ? MeOH] to give eight fractions [Fr. 1 (2.82 g), Fr. 2 (8.20 g), Fr.
3 (9.20 g), Fr. 4 (66.38 g), Fr. 5 (18.29 g), Fr. 6 (20.11 g), Fr. 7
(25.93 g), and Fr. 8 (31.25 g)]. The fraction 2 (8.20 g) was subjected
to reversed-phase silica gel CC [250 g, MeOH–H2O (20:80 ?
40:60 ? 50:50, v/v) ? MeOH] to afford seven fractions [Fr. 2-1
(4.8 mg), Fr. 2-2 (41.1 mg), Fr. 2-3 (55.3 mg), Fr. 2-4 (774.3 mg), Fr.
2-5 (1256.3 mg), Fr. 2-6 (5540.5 mg), and Fr. 2-7 (1081.9 mg)] as
reported previously.2 The fraction 2-4 (500.4 mg) was purified by
HPLC [Cosmosil 5C18-MS-II, MeOH–1% aqueous AcOH (20:80, v/v)]
to give 10S-dihydrophayomphenol A2 (1, 22.5 mg, 0.0021%) and
phayomphenols B1 (2, 5.1 mg, 0.0005%) and B2 (3, 6.7 mg, 0.0006%)
together with phayomphenols A1 (4, 6.4 mg, 0.0006%) and A2 (5,
442.6 mg, 0.040%). The fraction 2–5(500.7 mg) was purified by HPLC
[Cosmosil 5C18-MS-II, MeOH–1% aqueous AcOH (30:70, v/v)] to give
1 (9.9 mg, 0.0015%) together with 4 (123.0 mg, 0.018%) and 5
(165.4 mg, 0.025%).
In conclusion, the methanol extract of the bark of S. roxburghii
was found to inhibit plasma glucose elevation in sucrose-loaded
mice. Three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, 10S-
dihydrophayomphenol A2 (1) and phayomphenols B1 (2) and B2
(3), were isolated together with 24 known compounds including
20 stilbenoids and oligostilbenoids. Among the isolates, (ꢀ)-hopea-
phenol (6), hemsleyanol D (8), (+)-
a
-viniferin (15), and (ꢀ)-balano-
carpol (18) significantly inhibited plasma glucose elevation in
sucrose-loaded mice at doses of 100–200 mg/kg, p.o., and (+)-isoho-
peaphenol (7) tended to inhibit it at a dose of 200 mg/kg, p.o. As the
possible mechanism of action responsible for the antihyperglyce-
mic effect of the extract, following multiple modes were recursively
induced on the basis of the observed activities of the constituents;
(1) inhibition of gastric emptying: constituents 6, 7, and 18 signifi-
cantly suppressed the gastric emptying, and 15 also acted moder-
ately, (2)
a-glucosidase inhibitory activity: constituents 6–8 and
15 moderately inhibited enzyme activities of rat small intestinal su-
crose, (3) aldose reductase inhibitory activity: constituents 2–6, 8–
13, 15–21, and 25a moderately inhibited the rat lens aldose reduc-
tase. Inhibition of glucose uptake was excluded as a mode of the ac-
tion because major constituents 6–8, 15, and 18 did not inhibit
glucose uptake in rat small intestinal tissues.
3.3.1. 10S-Dihydrophayomphenol A2 (1)
A white powder, ½a D25
ꢁ
+79.5 (c 0.17, MeOH). Positive-ion FABMS:
m/z 339 (M+Na)+. High-resolution positive-ion FABMS: Calcd for
C
3. Experimental
3.1. General
17H16O6Na (M+Na)+: 339.0845. Found: 339.0849. CD [MeOH, nm
)]: 228 (ꢀ22.20), 265 (+2.86), 330 (+1.25). UV [MeOH, nm
(De
(loge)]: 261 (3.72), 327 (3.45). IR (KBr): 3390, 1698, 1615, 1514,
1474, 1362, 1258, 1223, 1123, 1017 cmꢀ1. 1H NMR (500 MHz, pyr-
idine-d5 and CD3OD) d: given in Table 2. 13C NMR (125 MHz, pyri-
dine-d5 and CD3OD) dc: given in Table 3.
The following instruments were used to obtain spectral and
physical data: specific rotations, Horiba SEPA-300 digital polarime-
ter (l = 5 cm); CD spectra, JASCO J-720WI spectrometer; UV spectra,
Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100
spectrometer; 1H NMR spectra, JEOL JNM-ECA700 (700 MHz),
JNM-ECA500 (500 MHz), and JNM-ECS400 (400 MHz) spectrome-
ters; 13C NMR spectra, JEOL JNM-ECA700 (175 MHz), JNM-ECA500
(125 MHz), and JNM-ECS400 (100 MHz) spectrometers with tetra-
methylsilane as an internal standard; EIMS and HREIMS, JEOL
JMS-GCMATE mass spectrometer; FABMS and HRFABMS, JEOL
JMS-SX 102A mass spectrometer; HPLC detector, Shimadzu SPD-
10A UV–VIS detectors; HPLC column, Cosmosil 5C18-MS-II and
3.3.2. Phayomphenol B1 (2)
A white powder, ½a D25
ꢁ
+162.9 (c 0.10, MeOH). EIMS m/z (%): 330
(M+, 11), 43 (100). High-resolution EIMS: Calcd for C17H14O7 (M+):
330.0740. Found: 330.0733. CD [MeOH, nm (
(+2.56), 296 (ꢀ0.14). UV [MeOH, nm (log )]: 262 (3.53), 328 (3.30).
1H NMR
D
e
)]: 228 (ꢀ6.79), 267
e
IR (KBr): 3400, 1717, 1698, 1617, 1362, 1250, 1125 cmꢀ1
.
(700 MHz, CD3OD) d: given in Table 2. 13C NMR (175 MHz, CD3OD)
dc: given in Table 3.
p
NAP (250 ꢃ 4.6 mm i.d. and 250 ꢃ 20 mm i.d. for analytical and
3.3.3. Phayomphenol B2 (3)
preparative purposes, respectively).
A white powder, ½a D25
ꢁ
+212.1 (c 0.08, MeOH). Positive-ion FAB-
The following experimental conditionswere used for chromatog-
raphy: normal-phase silica gel column chromatography (CC), silica
gel 60 N (Kanto Chemical Co., Ltd, 63–210 mesh, spherical, neutral);
reversed-phase silica gel CC, Diaion HP-20 (Nippon Rensui) and
Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd, 100–200
mesh); TLC, pre-coated TLC plates with silica gel 60F254 (Merck,
0.25 mm) (normal-phase) and silica gel RP-18 F254S (Merck,
0.25 mm) (reversed-phase); reversed-phase HPTLC, pre-coated TLC
plates with silica gel RP-18 WF254S (Merck, 0.25 mm), detection
was achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4, fol-
lowed by heating.
MS: m/z 353 (M+Na)+. High-resolution positive-ion FABMS: Calcd
for C17H14O7Na (M+Na)+: 353.0637. Found: 353.0630. CD [MeOH,
nm (
D
e
)]: 227 (ꢀ13.63), 270 (+3.83), 294 (+4.37). UV [MeOH, nm
(log
e
)]: 260 (3.52), 324 (3.28). IR (KBr): 3400, 1732, 1717, 1541,
1509, 1362, 1123 cmꢀ1
.
1H NMR (700 MHz, CD3OD) d: given in
Table 2. 13C NMR (175 MHz, CD3OD) dc: given in Table 3.
3.4. Chromium trioxide (CrO3) oxidation of 1
To a solution of 1 (3.5 mg) in pyridine (0.2 mL) was added CrO3
(6.6 mg)–pyridine (0.8 mL), and the mixture was stirred at 60 °C for
8 h. The reaction mixture was poured into saturated aqueous NaCl
and the resulting mixture was extracted with EtOAc. The extract
was washed with brine, and dried over anhydrous MgSO4. Removal
of the solvent under reduced pressure gave a pale yellow oil, which
was purified by HPLC [Cosmosil 5C18-MS-II, MeOH–1% aqueous
AcOH (70:30, v/v)] to furnish 5 (1.0 mg, 29%) as a white powder.
3.2. Plant material
The bark of S. roxburghii was collected in Phatthalung Province,
Thailand on September 2006. The plant material was identified by
one of the authors (Y.P.). A voucher specimen (2006.09. Raj-02) of
this plant is on file in our laboratory.
3.3. Extraction and isolation
3.5. Methylation of 1
Dried barks of S. roxburghii (3.7 kg) were finely cut and extracted
To a solution of 1 (8.5 mg) in MeOH (1.0 mL) was added trim-
three times with MeOH under reflux for 3 h. Evaporation of the
ethylsilyldiazomethane (TMSCHN2, 10% in hexane, ca. 0.5 mL).

T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
839
The mixture was stirred at room temperature for 8 h. Removal of
3.7.3. Effect on gastric emptying in mice
the solvent under reduced pressure gave 1a (9.6 mg, quant.).
The experiments were performed according to the method as
described in our previous reports.3,30–37 A solution of 1.5% CMC-
Na containing 0.05% phenol red as a marker was given intragastri-
cally (0.3 mL/mouse) to conscious mice. Thirty minutes latter, the
mice were killed by cervical dislocation. The abdominal cavity
was opened, the gastroesophageal junction and the pylorus were
elamped, and then the stomach was removed, weighed, placed in
10 mL of 0.1 M NaOH, and homogenized. The suspension was al-
lowed to settle for 1 h at room temperature, 1 mL of the superna-
tant was added to 0.1 mL of 20% (w/v) trichloroacetic acid, and
then centrifuged at 3000 rpm for 20 min. The supernatant
(0.1 mL) was mixed with 0.1 mL of 0.5 M NaOH, and the amount
of the phenol red was determined on the basis of the optical den-
sity (OD) at 560 nm using a microplate reader (SH-1000 Lab., Cor-
ona Electric Co., Ltd). A phenol red solution (0.3 mL) was used as
the standard (0% emptying). Test samples were given orally via a
metal orogastric tube 30 min prior to the administration of the test
meals. Gastric emptying rate (%) in the 30-min period was calcu-
lated according to the following equation:
3.5.1. Compound 1a
A white powder, ½a D25
ꢁ
+69.6 (c 0.10, MeOH). Positive-ion FABMS:
m/z 381 (M+Na)+. High-resolution positive-ion FABMS: Calcd for
C
20H22O6Na (M+Na)+: 381.1314. Found: 381.1309. UV [MeOH,
nm (log )]: 258 (3.67), 319 (3.45). IR (KBr): 3400, 1717, 1609,
e
1512, 1458, 1362, 1252, 1132, 1046 cmꢀ1 1H NMR (500 MHz,
.
CD3OD) d: given in Table 2. 13C NMR (125 MHz, CD3OD) dc: given
in Table 3.
3.6. Preparation of (R)-MTPA ester (1b) and (S)-MTPA ester (1c)
from 1a
A solution of 1a (1.9 mg) in CH2Cl2 (1.0 mL) was treated with
(R)-2-methoxy-2-trifluoromethylphenylacetic acid [(R)-MTPA,
6.5 mg] in the presence of 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDCꢂHCl, 5.6 mg) and 4-dimethylamino-
pyridine (4-DMAP, 2.0 mg), and the mixture was heated under
reflux for 15 h. The reaction mixture was poured into ice-water
and extracted with EtOAc. The extract was successively washed
with 5% aqueous HCl, saturated aqueous NaHCO3, and brine, then
dried over anhydrous MgSO4 and filtered. Removal of the solvent
from the filtrate under reduced pressure furnished a pale yellow
oil, which was purified by HPLC [Cosmosil 5C18-MS-II, MeOH–1%
aqueous AcOH (70:30, v/v)] to give 1b (1.3 mg, 43%). According
to the similar procedure, 1c (2.0 mg, 66%) was obtained from 1a
(1.9 mg) by using (S)-MTPA (6.5 mg), EDCꢂHCl (5.6 mg), and 4-
DMAP (2.0 mg).
Gastric emptying rate ð%Þ
¼ ð1ꢀamount of the test sample=amount of the standardÞꢃ100
3.7.4. Effects on glucose uptake in rat small intestinal tissues
The experiments were performed according to the method as
described in our previous reports with a slight modification.38,39
Thus, small fragments (0.10–0.15 g) of everted rat intestine were
placed in 1 mL of modified Krebs–Henseleit solution, pH 7.4, with
14C-U-glucose (2 mM, 1 ꢃ 105 cpm/mL) with or without a test sam-
ple. Incubation was carried out at 30 °C for 6 min, then the pieces
were washed two times for 3–5 s with the medium containing
1 mM phlorizin without 14C-U-glucose, and placed on a filter paper
to absorb the water from the tissue. The tissue was then weighed
and dissolved using 2 M NaOH. After neutralization by 2 M HCl,
the radioactivity was examined. Each test sample was dissolved
in dimethyl sulfoxide (DMSO) and added to an incubation solution
(final DMSO concentration was 0.5%). Inhibitions of the uptake by
the vehicle only and 1 mM phrolizin were calculated to be 0% and
100%, respectively.
3.6.1. Compound 1b
1H NMR (500 MHz, CD3OD) d: given in Table 2.
3.6.2. Compound 1c
1H NMR (500 MHz, CD3OD) d: given in Table 2.
3.7. Bioassay
3.7.1. Animals
Male ddY mice were purchased from Kiwa Laboratory Animal
Co., Ltd, (Wakayama, Japan). The animals were housed at a con-
stant temperature of 23 2 °C and were fed a standard laboratory
chow (MF, Oriental Yeast Co., Ltd, Tokyo, Japan). The animals were
fasted for 20–24 h prior to the beginning of experiments, but were
allowed free access to a tap water. All of the experiments were per-
formed with conscious mice unless otherwise noted. The experi-
mental protocol was approved by the Experimental Animal
Research Committee of Kyoto Pharmaceutical University.
3.7.5. Effects on rat intestinal a-glucosidase
The experiments were performed according to the method as
described in our previous reports with a slight modification.18,27–
30
Thus, a rat small intestinal brush border membrane fraction
was prepared and its suspension in 0.1 M maleate buffer (pH 6.0)
was used to determine the small intestinal
a-glucosidase activity
of maltase and sucrase. A mixture of a substrate (maltose:
37 mM, sucrose: 37 mM), a test compound, and the enzyme in
0.1 M maleate buffer (pH 6.0, 0.1 mL) were incubated at 37 °C.
After 30 min of incubation, 0.4 mL of water was added to the test
tube, and the tube was immediately immersed in boiling water
for 2 min to stop the reaction and then cooled with water. The glu-
cose concentration was determined using the enzymatic method.
Each test sample was dissolved in DMSO and the measurements
were performed in duplicate, and IC50 values were determined
graphically. The concentration of each enzyme suspension was ad-
3.7.2. Effects on plasma glucose elevation in sucrose-loaded mice
The experiments were performed according to the method as
described in our previous reports with a slight modification.3,30,31
Thus, each test sample was administrated orally to the fasted male
ddY mice (body weight 24–27 g), and 20% (w/v) sucrose solution
(10 mL/kg, p.o.) was administrated 30 min thereafter. Blood sam-
ples (ca. 0.1 mL) were collected from the infraorbital venosus
plexus under ether anesthesia 0.5, 1, and 2 h after the oral admin-
istration of sucrose. The collected blood was immediately mixed
with heparin sodium (5 units/tube). After centrifugation of the
blood samples, the plasma glucose level was determined enzymat-
ically by the Glucose CII test Wako (Wako Pure Chemical Industries
justed to produce ca. 0.30 and 0.15
lmol/tube of D-glucose from
the substrate maltose and sucrose, respectively. The intestinal
glucosidase inhibitor acarbose was used as a reference compound.
a-
3.7.6. Effects on rat lens aldose reductase
Ltd, Osaka, Japan). An intestinal
a-glucosidase inhibitor acarbose
The experiments were performed according to the method as
described in our previous reports.27,43 Thus, the supernatant fluid
was used as a reference compound.

840
T. Morikawa et al. / Bioorg. Med. Chem. 20 (2012) 832–840
9. Matsuda, H.; Ninomiya, K.; Morikawa, T.; Yasuda, D.; Yamaguchi, I.; Yoshikawa,
M. Bioorg. Med. Chem. 2009, 17, 7313.
10. Morikawa, T.; Yamaguchi, I.; Matsuda, H.; Yoshikawa, M. Chem. Pharm. Bull.
2009, 57, 1292.
of a rat lens homogenate was used as a crude enzyme. The incuba-
tion mixture contained 135 mM phosphate buffer (pH 7.0),
100 mM Li2SO4, 0.03 mM NADPH, 1 mM DL-glyceraldehyde as a
11. Morikawa, T. Yakugaku Zasshi 2010, 130, 785.
substrate, and 100 lL of enzyme fraction, with 25 lL of sample
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solution, in a total volume of 0.5 mL. The reaction was initiated
by the addition of NADPH at 30 °C. After 30 min, the reaction
was stopped by the addition of 150 lL of 0.5 M HCl. Then, 0.5 mL
of 6 M NaOH containing 10 mM imidazole was added, and the
mixture was heated at 60 °C for 20 min to convert NADP into a
fluorescent product. The fluorescence intensity was measured
using a fluorescence microplate reader (FLUOstar OPTIMA, BMG
Labtechnologies) at an excitation wavelength of 360 nm and an
emission wavelength of 460 nm. Each test sample was dissolved
in DMSO and the measurements were performed in duplicate,
and IC50 values were determined graphically. The concentration
of enzyme suspension was adjusted to produce ca. 10 nmol/tube
of b-nicotinamide adenine dinucleotide phosphate (NADP). An al-
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3.8. Statistics
Values are expressed as means S.E.M. One-way analysis of
variance (ANOVA) followed by Dunnett’s test was used for statisti-
cal analysis. Probability (p) values less than 0.05 were considered
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Acknowledgments
28. Yoshikawa, M.; Morikawa, T.; Matsuda, H.; Tanabe, G.; Muraoka, O. Bioorg. Med.
Chem. 2002, 10, 1547.
O.M., T.M., and K.N. were supported by a Grant-in Aid for Scien-
tific Research from ‘High-tech Research Center’ Project for Private
Universities: matching fund subsidy from a Grant-in Aid for Scien-
tific Research from the Ministry of Education, Culture, Sports, Sci-
ence and Technology (MEXT) of Japan, 2007–2011 and also
supported by a Grant-in Aid for Scientific Research by Japan Society
for the Promotion of Science (JSPS). M.Y. and H.M. were supported
by the 21st COE program, Academic Frontier Project, and a Grant-
in Aid for Scientific Research from MEXT.
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