Matrix metalloproteinase-2 inhibitors from Clinopodium chinense var. parviflorum

Article abstract of DOI:10.1021/np800781t

From the aerial parts of Clinopodium chinense var. parviflorum, nine new phenylpropanoids, clinopodic acids A-I (2-10), were isolated together with a known phenylpropanoid, rosmarinic acid (1). The structures of these new compounds were elucidated on the

Full text of DOI:10.1021/np800781t

J. Nat. Prod. 2009, 72, 1379–1384
1379
Matrix Metalloproteinase-2 Inhibitors from Clinopodium chinense var. parWiflorum
Toshihiro Murata,† Kenroh Sasaki,† Kumiko Sato,† Fumihiko Yoshizaki,† Haruna Yamada,‡ Hiromichi Mutoh,‡
Kaoru Umehara,‡ Toshio Miyase,*,‡ Tsutomu Warashina,§ Hiroaki Aoshima,⊥ Homare Tabata,| and Kouichi Matsubara|
Tohoku Pharmaceutical UniVersity, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan, School of Pharmaceutical Sciences, UniVersity of
Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan, Institute for EnVironmental Sciences, UniVersity of Shizuoka, 52-1, Yada,
Suruga-ku, Shizuoka 422-8526, Japan, Department of Pharmacy, Shizuoka Saiseikai General Hospital, 1-1-1 Oshika, Suruga-ku,
Shizuoka 422-8527, Japan, and Hokkaido Mitsui Chemicals, INC., 1-Toyonuma, Sunagawa-shi, Hokkaido 073-0138, Japan
ReceiVed December 9, 2008
From the aerial parts of Clinopodium chinense var. parViflorum, nine new phenylpropanoids, clinopodic acids A-I
(2-10), were isolated together with a known phenylpropanoid, rosmarinic acid (1). The structures of these new compounds
were elucidated on the basis of spectroscopic analysis. Clinopodic acid C (4) showed MMP-2 inhibitory activity (IC50
3.26 µM).
In the course of our research on the saponins from Clinopodium
species,1,2 we reported the structures of oleanane-type triterpene
saponins related to the antihepatotoxic3 saikosaponins from Cli-
nopodium chinense (Benth.) O. Kuntze var. parViflorum (Kudo)
Hara. (Labiatae). The ethanol extract of C. chinense Kuntze showed
antihyperglycemic effect.4 The water extract of C. Vulgare L.
showed strong antitumor activity in an in Vitro screening.5 In a
search for bioactive compounds from the water extract of C.
chinense var. parViflorum, we now report the isolation and the
structural elucidation of eight new phenylpropanoids, named
clinopodic acids A-I (2-10), and a known phenylpropanoid,
rosmarinic acid (1). Clinopodic acid C (4) showed MMP-2
inhibitory activity (IC50 3.26 µM).
the molecular formula C27H23O12. The 1H NMR spectrum showed
ABC-type aromatic protons at δ 6.81 (1H, d, J ) 8.0 Hz), 6.84
(1H, dd, J ) 8.0 and 2.0 Hz), and 6.96 (1H, d, J ) 2.0 Hz) and
AB-type aliphatic proton signals at δ 5.10 (1H, d, J ) 3.5 Hz) and
5.48 (1H, d, J ) 3.5 Hz) in addition to a set of proton signals due
to a rosmarinic acid moiety. Irradiating at the proton doublet at δ
5.48 showed ROEs at two aromatic protons at δ 6.84 and 6.96,
which were coupled with each other, and at an aliphatic proton
doublet at δ 5.10, which was coupled with a proton at δ 5.48. In
the HMBC spectrum, the aliphatic proton at δ 5.48 was long-range
coupled with a carbon at δ 146.5, which was assigned to C-4 of
the caffeoyl moiety because of the long-range coupling with the
aromatic proton at δ 7.24 (1H, d, J ) 8.0 Hz) due to H-6. These
NMR data suggested that this compound had a phenyl-benzodioxane
moiety.7 The CD spectrum showed a positive Cotton effect at 244
nm, suggesting that the absolute configuration of the benzylic carbon
was S.8 The C-8′ configuration was established as R from the
retention time of the amide with (S)-2-phenylglycine methyl ester,
which was synthesized after alkaline hydrolysis.8 From these data
the structure of 4 was determined as indicated.
Results and Discussion
Clinopodic acid A (2) was isolated as a colorless, amorphous
solid. Its molecular formula was established as C18H16O7 on the
basis of a pseudomolecular ion peak at m/z 345.0975 ([M + H]+).
1
The H NMR spectrum was similar to that of rosmarinic acid (1)
except for the presence of AA′BB-type aromatic proton signals at
δ 6.90 (2H, d, J ) 8.0 Hz) and 7.54 (2H, d, J ) 8.0 Hz). In the
ROE experiment, an ROE was observed at the aromatic proton (δ
7.54) on irradiation at an olefinic proton at δ 7.63 (d, J ) 15.5
Hz). The CD spectrum showed negative Cotton effects at 230 and
282 nm and positive Cotton effects at 300 and 329 nm and were
similar to those of 1. From these data, the structure of clinopodic
acid A was elucidated as 2. Compound 2 is assumed to be a
precursor of rosmarinic acid.6
Clinopodic acid B (3) was isolated as a colorless, amorphous
solid, and the 1H NMR spectrum of 3 was also similar to that of 1
except for the presence of a methoxy proton singlet at δ 3.83. In
the ROE experiment, ROEs were observed at the aromatic proton
[δ 6.97 (1H, d, J ) 2.0 Hz)] on irradiation at the O-methyl signal
and on irradiation at two methylene protons [δ 3.09 (dd, J ) 14.0
and 9.0 Hz); 3.19 (dd, J ) 14.0 and 4.0 Hz)]. Therefore the position
of the O-methyl group was decided to be C-3′. The absolute
configuration was determined from the dextrorotatory specific
rotation and the CD spectrum, which was similar to those of 1.
The HRFABMS spectrum of clinopodic acid C (4) showed a
pseudomolecular ion [M + H]+ at m/z 539.1218, consistent with
1
The H NMR spectrum of clinopodic acid D (5) was similar to
that of 4 except for the presence of an O-methyl singlet at δ 3.83
(3H, s). ROEs were observed at the aromatic proton signal at δ
6.88 (1H, brs) on irradiation at the O-methyl signal, and at the
O-methyl and the methine protons at δ 5.25 (1H, dd, J ) 8.0 and
4.5 Hz) on irradiation at the aromatic proton at δ 6.88. In the HMBC
spectrum, an oxygenated aromatic carbon signal at δ 148.8 was
long-range coupled with the O-methyl proton and the aromatic
proton at δ 6.88. The configuration of the benzodioxane moiety
was established as 7′′S, 8′′S from the CD data and that at C-8′ to
be R from the retention time of the amide derivative as in the case
of 4.
1
The H, 13C NMR and MS data of clinopodic acid E (6) were
similar to those of 4. In the CD spectrum, 6 showed a negative
Cotton effect at 231 nm, suggesting a 7′′R absolute configuration.
The NMR spectra of clinopodic acids F (7) and G (8) were
similar to those of clinopodic acid D (5) and had one and two
O-methyl groups, respectively.
The 1H NMR spectrum of clinopodic acid F (7) showed an
O-methyl signal at δ 3.84 (3H, s), which was located at C-3′ from
the ROE data.
1
The H NMR spectrum of clinopodic acid G (8) showed two
* To whom correspondence should be addressed. Tel and Fax: 81-54-
264-5661. E-mail: miyase@ys7.u-shizuoka-ken.ac.jp.
† Tohoku Pharmaceutical University.
O-methyl signals at δ 3.83 and 3.84 (each 3H, s) located at C-3′′
and C-3′, respectively. These locations were confirmed by ROE
and HMBC spectra.
The HRFABMS spectrum of clinopodic acid H (9) showed a
molecular ion peak at m/z 566.1435 (calcd for C29H26O12 566.1424).
‡ School of Pharmaceutical Sciences, University of Shizuoka.
§ Institute for Environmental Sciences, University of Shizuoka.
⊥ Department of Pharmacy, Shizuoka Saiseikai General Hospital.
| Hokkaido Mitsui Chemicals.
10.1021/np800781t CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/03/2009

1380 Journal of Natural Products, 2009, Vol. 72, No. 8
Murata et al.
1
102 mass spectrometer in the negative mode using a m-nitrobenzyl
alcohol matrix. Preparative HPLC was performed on a JASCO 800
instrument.
Plant Material. C. chinense var. parViflorum was harvested on
September 2006 from our medicinal botanical garden. The plant was
authenticated by Prof. A. Ueno, School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka, Japan. A voucher specimen
(200609030) has been deposited at the Herbarium of the University of
Shizuoka.
The H NMR spectrum showed two O-methyl signals at δ 3.80
and 3.82 and two aliphatic doublet proton signals at δ 4.57 (1H, d,
J ) 5.0 Hz) and 6.02 (1H, d, J ) 5.0 Hz) coupling with each other
in the H-H COSY spectrum. These protons were assigned to the
aliphatic proton of a dihydrobenzofuran moiety. The coupling
constant of these protons suggested trans fusion.9 The positive
Cotton effect at 253 nm in the CD spectrum of 9 suggested a 7′′S
absolute configuration.10 The location of two O-methyl groups was
decided to be C-3′ and C-3′′ by an ROE experiment. Alkaline
hydrolysis gave a dextrorotary optically active 3-(3-methoxy-4-
hydroxyphenyl)-2-hydroxypropanoic acid similar to (2R)-3-(3,4-
dihydroxyphenyl)-2-hydroxypropanoic acid from rosmarinic acid
(1) and 8-epibechnic acid.9
The absolute configuration of the 3-(3,4-dihydroxyphenyl)-2-
hydroxypropanoic acid moiety in compound 6 and the 3-(3-
methoxy-4-hydroxyphenyl)-2-hydroxypropanoic acid moiety in
compounds 5 and 7-9 were decided to be both R in the same way
as for 4. Compound 9 has the same structure as lithospermic acid,11
but their NMR data are not identical.
Materials. p-Aminophenylmercuric acetate was purchased from
Sigma (St. Louis, MO). The reagents used for SDS-PAGE were
purchased from Bio-Rad Co. Ltd. (Hercules, CA).
Extraction and Isolation. Powdered aerial parts of C. chinense var.
parViflorum (1.13 kg) were extracted with hot H2O. The H2O extract
was dissolved in H2O, and the aqueous solution was passed through a
porous polymer gel column (Mithubishi Diaion HP-20, 9 × 45 cm).
The adsorbed material was eluted with 30% MeOH (5 L), 50% MeOH
(5 L), and MeOH (8 L) to give 30% MeOH eluate (26.7 g), 50% MeOH
eluate (16.9 g), and MeOH eluate (19.3 g). Some of the 30% MeOH eluate
(3.0 g) was subjected to HPLC [column, YMC ODS, 5 × 100 cm; solvent,
H2O-CH3CN (80:20) f (78: 22) linear gradient; flow rate, 45 mL/
min; detection, UV 320 nm] to afford 12 fractions. Fractions 4-12
were subjected further to preparative HPLC to give compounds 1 (152
mg), 2 (3 mg), 4 (20 mg), 6 (15 mg), 7 (8 mg), and 10 (13 mg). The
50% MeOH eluate (16.9 g) was subjected to a silica gel column [Fuji
Silysia PSQ-100B, 7 × 85 cm; solvent, CHCl3-MeOH-H2O (80:18:
2) f + MeOH gradient] to afford eight fractions. Fraction 2 (2.3 g)
was subjected to HPLC [column, YMC ODS, 5 × 100 cm; solvent,
H2O-CH3CN (80:20) f (70:30) linear gradient; flow rate, 45 mL/
min; detection, UV 320 nm] to afford 10 fractions. Fractions 5-10
were subjected further to preparative HPLC to give compounds 2 (20
mg), 3 (24 mg), 5 (19 mg), 7 (57 mg), 8 (13 mg), and 9 (228 mg).
The HRFABMS spectrum of clinopodic acid I (10) showed a
pseudomolecular ion peak at m/z 717.1476 (calcd for C36H29O16
717.1455). The 1H NMR spectrum was similar to that of 9 except
for signals of an additional 3-(3,4-dihydroxyphenyl)-2-hydroxypro-
panoic acid moiety. In the ROE spectra, ROEs were observed at
two aromatic protons at δ 6.57 (1H, dd, J ) 8.0 and 2.0 Hz) and
6.79 (1H, d, J ) 2.0 Hz) on irradiation at an aliphatic proton at δ
5.33 (1H, dd, J ) 8.0 and 4.5 Hz), at three protons at δ 6.74 (1H,
dd, J ) 8.0 and 2.0 Hz) and 6.90 (1H, d, J ) 2.0 Hz) on irradiation
at δ 5.78 (1H, d, J ) 5.0 Hz), at four protons at δ 3.01 (1H, dd, J
) 14.5 and 7.0 Hz), 3.10 (1H, dd, J ) 14.5 and 4.5 Hz), 4.53 (1H,
d, J ) 5.0 Hz), and 6.70 (1H, d, J ) 8.0 Hz) on irradiation at δ
5.28 (1H, dd, J ) 7.0 and 4.5 Hz), at two protons at δ 3.01 and
5.28 on irradiation at δ 6.70, and at two aromatic protons at δ 6.94
(1H, dd, J ) 8.0 and 2.0 Hz) and 7.07 (1H, d, J ) 2.0 Hz) on
irradiation at δ 7.53 (1H, d, J ) 16.0 Hz). The absolute
configurations of the two 3-(3,4-dihydroxyphenyl)-2-hydroxypro-
Rosmarinic acid (1): colorless, amorphous solid; [R]23 +37.2 (c
D
2.92, MeOH); CD (c 0.030, MeOH) λmax nm ([θ]) 218 (-2.40 × 103),
232 (-9.36 × 103), 280 (-2.88 × 103), 298 (+4.80 × 103), 320 (+3.12
× 103).
Clinopodic acid A (2): colorless, amorphous solid; [R]23D +83.8 (c
1.60, MeOH); UV (MeOH) λmax (log ꢀ) 230 (4.84), 279 (4.23), 308
(4.01), 319 (4.02); CD (c 0.050, MeOH) λ max nm ([θ]) 230 (-7.91 ×
103), 250 (+5.50 × 103), 282 (-3.44 × 103), 300 (+6.88 × 103), 325
1
(+4.47 × 103); H and 13C NMR, Table 1; HRFABMS m/z 345.0975
1
[M + H]+ (calcd for C18H17O7, 345.0974), 367.0783 [M + Na]+
(C18H16O7Na, 367.0794).
panoic acid moieties were established as R, from the H NMR
chemical shifts of H-7′ and H-7′′ (Table 2) of the amides with (S)-
phenylglycine methyl ester and (R)-phenylglycine methyl ester.8
Clinopodic acid B (3): colorless, amorphous solid; [R]23D +79.3 (c
1.91, MeOH); UV (MeOH) λ
(log ꢀ) 220sh (4.19), 232sh (4.13),
max
Matrix metalloproteinase-2 (MMP-2) inhibitory activity was
measured for these compounds. Rosmarinic acid (1), clinopodic
acid C (4), and lithospermic acid12 showed inhibitory activity, (IC50
27.2, 3.26, and 10.2 µM, respectively). Series of MMPs play an
important part in the regulation of both cell-cell and cell-extra-
cellular matrix interactions. Especially, the activation of MMP-2
contributes to proteolytic activity and the degrading of denatured
collagens, gelatins, and extracellular matrix.13 This can be related
to the phenomena of tissue regeneration and/or various diseases
such as cancer invasion or metastasis, arthritis, and blood vessel
impairment.13,14 Moreover, the relationship between antioxidants
and MMP inhibitory activity of rosmarinic acid (1) had been
reported.15 Thus, some MMP-2 inhibitors observed in this paper
would play a role not only in ameliorating the diseases described
above but also in a possible cosmetic for skin aging.16
288 (4.07), 326 (4.17); CD (c 0.050, MeOH) λ max nm ([θ]) 230 (-7.33
× 103), 250 (+6.43 × 103), 275 (-2.99 × 103), 295 (+5.54 × 103),
325 (+4.19 × 103); 1H and 13C NMR, Table 1; HRFABMS m/z
375.1077 [M + H]+ (calcd for C19H19O8, 375.1080), 397.0903 [M +
Na]+ (C19H18O8Na, 397.0899).
Clinopodic acid C (4): colorless, amorphous solid; [R]23D -14.0 (c
1.10, MeOH); UV (MeOH) λmax (log ꢀ) 221sh (4.13), 233sh (4.01),
289 (3.96), 326 (3.95); CD (c 0.050, MeOH) λmax nm ([θ]) 214 (-2.15
× 104), 229 (+7.09 × 103), 244 (+1.88 × 104), 294 (-1.26 × 104),
1
306 (-9.39 × 103), 322 (-1.21 × 104); H and 13C NMR, Table 1;
HRFABMS m/z 539.1218 [M + H]+ (calcd for C27H23O12, 539.1190).
Clinopodic acid D (5): colorless, amorphous solid; [R]23D -56.5 (c
0.46, MeOH); UV (MeOH) λmax (log ꢀ) 223sh (4.46), 233sh (4.38),
288 (4.29), 324 (4.31); CD (c 0.050. MeOH) λmax nm ([θ]) 213 (-2.83
× 104), 227 (+1.35 × 103), 233 (+1.24 × 104), 244 (+2.45 × 104),
1
300 (-1.30 × 104), 306 (-1.10 × 104), 321 (-1.57 × 104); H and
13C NMR, Table 1; HRFABMS m/z 575.1127 [M + Na]+ (calcd for
C28H24O12Na, 575.1165).
Experimental Section
Clinopodic acid E (6): colorless, amorphous solid; [R]23 +4.4 (c
D
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP-1000 digital polarimeter. UV spectra were measured
in MeOH on a Hitachi U-2010 spectrophotometer. CD spectra were
0.46, MeOH); UV (MeOH) λmax (log ꢀ) 223sh (4.11), 234sh (3.98),
288 (3.91), 326 (3.81); CD (c 0.052, MeOH) λmax nm ([θ]) 231 (-1.08
× 104), 250 (-1.45 × 103), 282 (-6.21 × 103), 320 (+5.38 × 103);
1H and 13C NMR, Table 1; HRFABMS m/z 539.1167 [M + H]+ (calcd
for C27H23O12, 539.1190).
1
measured on a JASCO J-20A spectrometer. H (400 MHz) and 13C
NMR (100 MHz) spectra were recorded on a JEOL JNM R-400 FT-
NMR spectrometer, and chemical shifts are given as δ values with
TMS as an internal standard at 35 °C. Inverse-detected heteronuclear
correlations were measured using HMQC (optimized for 1JC-H ) 145
Hz) and HMBC (optimized for nJC-H ) 8 Hz) pulse sequences with a
pulse field gradient. HRFABMS data were obtained on a JEOL JMS
700 mass spectrometer in the positive mode and on a JEOL JMS SX
Clinopodic acid F (7): colorless, amorphous solid; [R]23D +38.4 (c
0.35, MeOH); UV (MeOH) λmax (log ꢀ) 222sh (4.36), 233sh (4.28),
288 (4.22), 323 (4.21); CD (c 0.050, MeOH) λmax nm ([θ]) 218 (+1.41
× 104), 238 (-7.29 × 103), 259 (-1.32 × 103), 275 (-3.75 × 103),
1
296 (-1.02 × 104), 315 (+8.39 × 103); H and 13C NMR, Table 1;
HRFABMS m/z 551.1198 [M - H]- (calcd for C28H23O12, 551.1189).

Matrix Metalloproteinase-2 Inhibitors from Clinopodium
Journal of Natural Products, 2009, Vol. 72, No. 8 1381
1
Clinopodic acid G (8): colorless, amorphous solid; [R]23 +59.8
(+1.25 × 104); H and 13C NMR, Table 1; HRFABMS m/z 566.1435
D
[M]+ (calcd for C29H26O12, 566.1424), 567.1500 [M + H]+ (calcd for
C29H27O12, 567.1503), 589.1313 [M + Na]+ (calcd for C29H26O12Na,
589.1322).
(c 0.31, MeOH); UV (MeOH) λmax (log ꢀ) 224sh (4.43), 233sh (4.38),
288 (4.28), 325 (4.28); CD (c 0.050, MeOH) λmax nm ([θ]) 223 (+1.52
× 104), 237 (-5.66 × 103), 273 (-3.17 × 103), 293 (+1.11 × 104),
320 (+1.02 × 104); 1H and 13C NMR, Table 1; HRFABMS m/z
565.1340 [M + H]+ (calcd for C29H25O12, 565.1346).
23
Clinopodic acid I (10): colorless, amorphous solid; [R]D -76.6
(c 0.55, MeOH); UV (MeOH) λmax (log ꢀ) 231sh (4.35), 289 (4.21),
Clinopodic acid H (9): colorless, amorphous solid; [R]23 +131.0
D
331 (4.17); CD (c 0.050, MeOH) λmax nm ([θ]) 225 (-3.52 × 104),
(c 2.59, MeOH); UV (MeOH) λmax (log ꢀ) 229 (4.53), 254 (4.42), 288
(4.35), 310 (4.39), 333 (4.33); CD (c 0.050, MeOH) λmax nm ([θ]) 212
(-3.85 × 104), 217 (-1.81 × 104), 226 (+1.87 × 104), 234 (+9.62 ×
103), 253 (+5.60 × 104), 280 (-6.79 × 103), 295 (+6.79 × 104), 334
248 (+4.30 × 103), 293 (-3.04 × 104), 328 (-8.04 × 103); H and
1
13C NMR, Table 1; HRFABMS m/z 717.1476 [M - H]- (calcd for
C36H29O16, 717.1455).
Table 1. NMR Spectroscopic Data (400 MHz) of Compounds 2-10
2a
3a
4a
HMBC
ROE
HMBC
ROE
(H to H)
HMBC
ROE
(H to H)
position
δH (J in Hz)
7.54, d (8.0)
δC
(H to C) (H to H)
δH (J in Hz)
7.16, d (2.0)
δC
127.5
(H to C)
δH (J in Hz)
7.33, d (2.0)
δC
(H to C)
1
2
3
4
126.9
116.7
131.1
146.2
129.2
4
1
115.3 4, 7
148.1
117.4 3, 4
143.7
6.90, d (8.0)
148.9
146.5
5
6
7
8
6.90, d (8.0)
7.54, d (8.0)
7.63, d (15.5)
6.36, d (15.5)
131.1 1, 7
116.7
146.2 3, 5, 9
6.87, d (8.0)
116.4 4, 7
122.6 4′, 7
146.5 1, 2, 6, 9
114.9 1, 9
166.8
6.97, d (8.0)
118.4
123.5
145.7 1, 2, 6, 9
116.7 1, 7, 9
166.6
3
4
7.03, dd (8.0, 2.0)
7.58, d (16.0)
6.30, d (16.0)
7.24, dd (8.0, 2.0)
7.63, d (16.0)
6.45, d (16.0)
2, 6
2, 6
2′, 6′
115.0
166.9
1
9
OMe
1′
2′
3′
4′
5′
6′
7′
3.83, s
56.3 3′
128.9
113.9 1′, 4′
146.5
146.4
115.6 4′
122.9 4′
2′
129.3
117.3 4′
145.7
129.2
117.3 4′
145.7
6.85, d (2.0)
6.97, d (2.0)
6.87, d (2.0)
144.7
144.8
6.75, d (8.0)
116.0 1′, 3′
121.7 4′
37.5 2′, 6′, 9′
2′, 6′, 9′
73.8
6.76, d (8.0)
6.76, d (8.0)
6.69, dd (8.0, 2.0)
115.9 3′
121.7 4′
37.5 1′, 2′, 6′
1′, 2′, 6′
73.8 1′
171.0
6.68, dd (8.0, 2.0)
3.04, dd (13.5, 8.0)
3.14, dd (13.5, 4.0)
5.24, dd (8.0, 4.0)
6.79, dd (8.0, 2.0)
3.09, dd (14.0, 9.0)
3.19, dd (14.0, 4.0)
5.26, dd (9.0, 4.0)
37.7 1′, 2′, 6′, 8′, 9′ 2′, 6′, 8′ 3.05, dd (13.5, 8.0)
1′, 2′, 6′, 8′ 9′ 2′, 6′, 8′ 3.14, dd (13.5, 4.0)
73.8 1′, 9, 9′
171.3
2′, 6′
2′, 6′
8′
9′
2′, 6′, 7′ 5.25, dd (8.0, 4.0)
171.3
OMe
1′′
2′′
3′′
4′′
5′′
6′′
7′′
8′′
9′′
127.8
115.0 4′′
145.8
146.3
116.0 3′′
119.8 4′′, 7′′
76.0 1′′, 2′′, 4, 6′′ 2′′, 6′′, 8′′
75.7 1′′, 3
168.0
6.96, d (2.0)
6.81, d (8.0)
6.84, dd (8.0, 2.0)
5.48, d (3.5)
5.10, d (3.5)
5b
6a
7a
HMBC
ROE
HMBC
(H to C)
ROE
(H to H)
HMBC
(H to C)
ROE
position
δH (J in Hz)
δC
(H to C)
(H to H)
δH (J in Hz)
7.29, d (2.0)
δC
δH (J in Hz)
7.29, d (2.0)
δC
(H to H)
1
2
3
4
5
6
129.4
129.3
129.2
117.3
143.6
146.2
118.5
7.27, d (2.0)
117.6 3, 4, 6
144.2
7, 8
117.4 3, 4
143.6
6
7, 8
147.0
146.2
118.6
6.93, d (8.5)
118.6 1, 3, 4
7.15, dd (8.5, 2.0) 124.0 2, 4
6.96, d (8.0)
7.24, dd (8.0, 2.0) 123.4
3
4
6.96, d (8.0)
7.22, dd (8.0, 2.0) 123.4
1
2
7
8
9
7.62, d (16.0)
6.39, d (16.0)
146.7 2, 6, 9
116.4 1, 7, 9
168.0
7.62, d (15.5)
6.44, d (15.5)
116.8 1, 2, 6
2, 6
7.63, d (15.0)
6.46, d (15.0)
116.7 1, 9
145.7 1, 2, 6, 9
166.6
2, 6
145.6
166.6
1
OMe
1′
2′
3′
4′
5′
6′
7′
129.3
114.3 1′, 3′, 4′, 7′
148.8
146.6
116.2 3′, 4′
123.1 1′, 2′, 7′
129.3
117.4 3′, 4′
145.6
144.9
116.0 1′, 3′
128.8
113.9 1′, 6′, 7′
148.1
146.3
115.6 1′
6.88, brs
OMe, 9′
6.86, d (2.0)
6.75, d (8.0)
6.97, brs
6.74, overlapped
6.73, overlapped
6.76, d (8.0)
6.68, dd (8.0, 2.0) 121.7 4′
3.04, dd (13.5, 8.0) 37.5
3.14, dd (13.5, 4.0)
6.80, dd (8.0, 2.0) 122.9 1′, 7′
3.10, dd (14.0, 8.0) 37.7 1′, 2′, 6′, 8′
3.09, dd (14.5, 8.0) 38.1 1′, 2′, 6′, 8′
2′, 6′
2′, 6′
3.17, dd (14.5, 4.5)
5.25, dd (8.0, 4.5)
1′, 2′, 6′, 8′
3.20, dd (14.0, 3.5)
5.27, dd (8.0, 3.5)
1′, 2′, 6′, 8′
8′
9′
74.7 1′, 9, 9′
173.4
56.5 3′
128.3
115.4 3′′, 4′′, 6′′, 7′′ 7′′, 8′′
146.2
146.8
116.1 1′′
120.1 7′′
76.9 1′′, 2′′, 4,
2′, 6′
2′
5.24, dd (8.0, 4.0)
6.97, brs
73.8 1′, 7′, 9, 9′ 2′, 6′
171.0
73.8 1′, 9, 9′
171.0
56.3
128.4
115.3 1′′, 6′′, 7′′
145.9
146.5
116.1 1′′, 7′′
120.0 1′′, 2′′, 7′′
76.6 1′′, 2′′, 4,
OMe 3.83, s
1′′
3.84, s
2′
128.5
115.3 4′′
146.0
146.6
116.1 3′′
120.1
2′′
3′′
4′′
5′′
6′′
7′′
6.83, d (1.5)
6.97, brs
OMe, 8′
6.70, d (8.0)
6.71, overlapped
5.45, d (3.0)
6.84, brs
6.84, brs
6.84, brs
6.84, brs
2′′, 6′′, 8′′ 5.31, d (4.5)
7′′, 8′′
2′′, 6′′
2′′, 6′′, 8′′ 5.32, d (5.0)
76.7
6′′, 8′′
6′′, 8′′
8′′
9′′
4.95, d (3.0)
76.5 1′′, 3, 7′′, 9′′ 7′′
170.5
4.99, d (5.0)
76.6
168.9
3
4.98, d (4.5)
76.6 1′′, 3, 7′′, 9′′ 2′′, 6′′
168.8

1382 Journal of Natural Products, 2009, Vol. 72, No. 8
Murata et al.
Table 1. Continued
8a
9a
10a
HMBC
ROE
HMBC
ROE
HMBC
(H to C)
ROE
position
δH (J in Hz)
7.29, d (2.0)
δC
(H to C)
(H to H)
δH (J in Hz)
δC
(H to C)
(H to H)
δH (J in Hz)
7.07, d (2.0)
δC
127.5
(H to H)
1
2
3
4
5
6
129.3
124.4
127.3
148.4
144.6
118.2
117.3 1, 3, 4
143.6
115.4 3, 4, 6
145.6
146.4
148.8
6.98, d (8.0)
7.23, dd
(8.0, 2.0)
7.62, d (16.0)
6.45, d (16.0)
118.6 1, 3
6.89, d (8.5)
7.27,
d (8.5)
7.85, d (16.0)
6.39, d (16.0)
1
6
6.88, d (8.0)
6.94, dd
(8.0, 2.0)
7.53, d (16.0)
6.12, d (16.0)
116.5 1, 4
122.8
123.4
4
121.5 2, 8′′
5, 7, 8
2, 4
2, 6
7
8
9
145.6 1, 2, 6, 9
116.8 1, 9
166.6
2, 6
143.2 1, 2, 6, 8, 9
116.6 1, 7, 9
166.6
6, 8′′
146.9 1, 2, 6, 9
114.6 1, 9
166.9
1′
2′
128.9
113.9 6′, 8′
128.8
113.9 6′, 7′
126.1
125.6
6.98, d (2.0)
6.92, d (2.0)
OMe, 7′, 7′,
8′
3′
4′
5′
6′
7′
148.1
146.2
115.7 1′, 3′
148.1
146.4
115.7 1′
148.6
141.2
117.2 1′, 3′
123.6 2′, 4′, 7′
6.76, d (8.0)
6.75, d (8.0)
6.75, d (8.0)
6.70, d (8.0)
6.80, dd (8.0, 2.0) 122.9 2′, 4′, 7′
3.09, dd (14.0, 8.5) 37.7 1′, 2′, 6′, 8′, 9′ 2′, 6′
6.79, dd (8.0, 2.0) 123.0 2′, 7′
3.08, dd (14.5, 8.5) 37.7 1′, 2′, 6′, 8′, 9′
7′, 8′
3.01, dd (14.5, 7.0) 34.2 1′, 2′, 6′, 9′
3.20, dd (14.0, 4.0)
5.27, dd (8.5, 4.0)
1′, 2′, 6′, 8′, 9′ 2′, 6′
73.9 1′, 7′, 9, 9′
3.16, dd (14.5, 4.0)
5.24, dd (8.5, 4.0)
1′, 2′, 6′, 8′, 9′
73.9 1′, 9, 9′
3.10, dd (14.5, 4.5)
5.28, dd (7.0, 4.5)
1′, 2′, 6′, 9′
8′
9′
73.1 1′, 7′, 9, 9′
6′, 7′,
7′, 8′′
171.1
56.4 3′
128.1
111.9 1′′, 3′′, 6′′, 7′′
148.5
148.1
171.1
56.3 3′
132.9
110.4 1′′, 6′′
148.5
147.8
170.8
OMe 3.84, s
1′′
2′
3.80, s
1′
134.3
113.4 6′′, 7′′
146.0
145.8
116.3 1′′
2′′
3′′
4′′
5′′
6′′
7′′
7.13, d (2.0)
7.09, d (2.0)
OMe, 7′′, 8′′ 6.90, d (2.0)
6.83, d (8.0)
115.6 1′′, 3′′, 4′′, 6′′
6.84, d (8.0)
115.9 1′′, 7′′
6.82, d (8.0)
6.95, dd (8.0, 2.0) 121.3 2′′, 4′′
6.91, dd (8.0, 2.0) 119.4 2′′
6.74, dd (8.0, 2.0) 118.3 2′′, 7′′
5.35, d (5.0)
76.8 1′′, 2′′, 4, 8′′ 2′′, 6′′
6.02, d (5.0)
88.3 1′′, 2, 2′′, 3, 6′′, 2′′, 6′′
5.78, d (5.0)
87.8 1′′, 2′, 2′′, 3′,
2′′, 6′′
8′′, 9′′
6′′, 9′′
8′′
9′′
5.05, d (5.0)
76.7 3, 7′′, 9′′
168.9
56.3 3′′
4.57, d (5.0)
3.82, s
56.7 1, 1′′, 2, 3, 7′′, 9′′
172.8
56.4 3′′
4.53, d (5.0)
57.3 1′′, 2′, 3′, 9′′
172.3
OMe 3.83, s
2′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
128.7
117.2 6′′′, 7′′′
145.9
144.8
116.1 1′′′
6.79, d (2.0)
6.72, d (8.0)
6.57, dd (8.0, 2.0) 121.9 2′′′, 4′′′, 7′′′
3.02, dd (14.5, 8.0) 37.1 1′′′, 2′′′, 6′′′, 9′′′
3.15, dd (14.5, 4.5)
5.33, dd (8.0, 4.5)
1′′′, 2′′′, 6′′′, 9′′′
74.6 7′′′, 9′′, 9′′′
8′′′
9′′′
2′′′, 6′′′,
8′′
170.5
a In acetone-d6. b In methanol-d4.
(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid (12 mg) as a colorless,
Table 2. NMR Data (400 MHz, Acetone-d6) for (S)- and
(R)-PGME Amide of Clinopodic Acid I (10)
1
amorphous solid: [R]23 +13.9 (c 2.54, acetone); H NMR (acetone-
d6) δ 2.78 (1H, dd, J )D14.0, 7.5 Hz), 2.97 (1H, dd, J ) 14.0, 4.5 Hz),
4.31 (1H, dd, J ) 7.5, 4.5 Hz), 6.60 (1H, dd, J ) 8.0, 2.0 Hz), 6.71
(1H, d, J ) 8.0 Hz), 6.79 (1H, d, J ) 2.0 Hz).
δ(S)-PGME amide
- (R)-PGME amide
(S)-PGME amide
(R)-PGME amide
H-7′ and H-7′′ δH (J in Hz) H-7′ and H-7′′ δH (J in Hz)
Condensation of the Acid Moiety of Rosmarinic Acid (1) with
Phenylglycine Methyl Ester. The acid moiety (5 mg) and (S)-
phenylglycine methyl ester (PGME) (10 mg) were dissolved in dry
DMF (0.5 mL), and to the solution were added benzotriazol-1-yl-oxy-
tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP) (20 mg),
1-hydroxybenzotriazole (HOBT) (7 mg), and N-methylmorpholine (30
µL). The reaction mixture was stirred overnight. EtOAc (2 mL) was
added, and resulting diluted solution was successively washed with
dilute HCl, saturated NaHCO3, and saturated NaCl solutions. The
EtOAc layer was separated by HPLC [column, TOSOH TSKgel ODS-
80Ts, 0.46 × 25 cm; H2O-CH3CN (80:20) + 0.05% TFA; flow rate,
1 mL/min; detector, UV 280 nm] to give (S)-amide (0.5 mg) as a
colorless, amorphous solid, tR 25.5 min; 1H NMR (acetone-d6) δ 2.682
(1H, dd, J ) 14.0, 7.5 Hz), 2.992 (1H, dd, J ) 14.0, 3.5 Hz), 4.234
(1H, dd, J ) 7.5, 3.5 Hz), 5.52 (1H, m), 6.58 (1H, dd, J ) 8.0, 2.0
Hz), 6.70 (1H, d, J ) 8.0 Hz), 6.76 (1H, d, J ) 2.0 Hz), 7.38 (5H, m).
By the same method, (R)-amide (0.5 mg) was obtained as a colorless,
amorphous solid, tR 27.4 min; 1H NMR (acetone-d6) δ 2.688 (1H, dd,
J ) 14.0, 8.0 Hz), 2.952 (1H, dd, J ) 14.0, 3.5 Hz), 4.274 (1H, dd, J
) 7.5, 3.5 Hz), 5.50 (1H, m), 6.64 (1H, dd, J ) 8.0, 2.0 Hz), 6.67
(1H, d, J ) 8.0 Hz), 6.74 (1H, d, J ) 2.0 Hz), 7.27-7.38 (5H, m).
Alkaline Hydrolysis of Clinopodic Acids C (4) and E (6) and
Condensation with (S)-PGME. Each compound (1 mg) was dissolved
in 10% NaOH solution (1 mL), and the solution was stirred for 1 h at
3.007, 2H, dd (14.5, 7.5)
2.880, 1H, dd (14.5, 7.0)
2.932, 1H, dd (14.0, 8.0)
3.078, 1H, dd (14.0, 4.5)
3.108, 1H, dd (14.5, 5.5)
+
+
+
+
3.130, 1H, dd (14.5, 5.5)
3.151, 1H, dd (14.5, 4.5)
Table 3. MMP-2 Inhibitory Activity of Compounds 1-4, 6, 7,
and 9, Methyl Ester of 1, and Lithospermic Acid
compound
IC50 (µM)
compound
IC50 (µM)
1
27.2
6
7
9
>100
>100
>100
10.2
methyl ester of 1
>100
>100
>100
3.26
2
3
4
lithospermic acid
Alkaline Hydrolysis of Rosmarinic Acid (1). Rosmarinic acid (1)
(100 mg) was dissolved in 10% NaOH (5 mL), and the mixture was
stirred for 1 h at room temperature under N2. The reaction mixture
was passed through an Amberlite IR-120B column (2 × 10 cm) and
eluted with H2O (100 mL). The eluate was concentrated under reduced
pressure to give a brown residue (60 mg). The eluate was purified by
HPLC [column, Inertsil ODS-3, 3 × 50 cm; solvent, H2O-CH3CN
(93:7) + 0.1% TFA; flow rate, 9.5 mL/min; 280 nm) to give (2R)-3-

Matrix Metalloproteinase-2 Inhibitors from Clinopodium
Journal of Natural Products, 2009, Vol. 72, No. 8 1383
Chart 1
room temperature under N2. The reaction mixture was passed through
an Amberlite IR-120B column (15 × 40 mm) and eluted with H2O
(30 mL). The eluate was concentrated under reduced pressure to give
a brown residue (0.5 mg). The residue (0.5 mg) was treated the same
as above. The EtOAc layer was analyzed by HPLC [column, TOSOH
TSKgel ODS-80Ts, 0.46 × 25 cm; solvent, H2O-CH3CN (80:20) +
0.05% TFA; flow rate, 1 mL/min; 280 nm] to give amide peak, tR 25.5
min.
(S)-amide, tR 37.2 min, and (R)-amide, tR 38.8 min, in HPLC analysis
[column, TOSOH TSKgel ODS-80Ts, 0.46 × 25 cm; solvent,
H2O-CH3CN (77:23) + 0.05% TFA; flow rate, 1 mL/min; detector,
UV 280 nm].
Alkaline Hydrolysis of Clinopodic Acids D and F-H (5, 7-9)
and Condensation with (S)-PGME. Each compound (1 mg) was
treated as for the clinopodic acids C (4) and E (6) to give (S)-amide,
tR 37.2 min, in the HPLC analysis [column, TOSOH TSKgel ODS-
80Ts, 0.46 × 25 cm; solvent, H2O-CH3CN (77:23) + 0.05% TFA;
flow rate, 1 mL/min; detector, UV 280 nm].
Condensation of Clinopodic Acid I (10) with (S)-PGME. To a
stirred solution of a mixture of clinopodic acid I (10) (5.3 mg) and
(S)-PGME (12.8 mg) in DMF (700 µL) was successively added
benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate
(PyBOP, 11.1 mg), 1-hydroxybenzotriazole (HOBT, 7.4 mg), and
N-methylmorphorine (50 µL) at 0 °C. After the mixture was stirred at
room temperature overnight, EtOAc (20 mL) was added, and the
resulting diluted solution was successively washed with dilute HCl,
saturated NaHCO3, and saturated NaCl solutions. The EtOAc layer was
dried and concentrated to give a residue, which was purified by
Alkaline Hydrolysis of Clinopodic Acid H (9) and Condensation
with (S)- and (R)-PGME. Clinopodic acid H (9) (50 mg) was dissolved
in 10% NaOH (0.5 mL), and the mixture was stirred for 5 h at room
temperature under N2. The reaction mixture was passed through an
Amberlite IR-120B column (1 × 15 cm) and eluted with H2O (100
mL). The eluate was concentrated under reduced pressure to give a
brown residue (50 mg). The eluate was purified by HPLC (column,
Cosmosil 5C18-AR-II, 2 × 25 cm; solvent, H2O-CH3CN (85:15) +
0.05% TFA; flow rate, 6.5 mL/min; detector, UV 280 nm) to give (2R)-
3-(3-methoxy-4-hydroxyphenyl)-2-hydroxypropanoic acid (15 mg) as
a colorless, amorphous solid, [R]23D +25.8 (c 1.50, acetone); 1H NMR
(acetone-d6) δ 2.84 (1H, dd, J ) 14.0, 7.5 Hz), 3.02 (1H, dd, J ) 14.0,
4.5 Hz), 3.82 (3H, s), 4.34 (1H, dd, J ) 7.5, 4.5 Hz), 6.72 (2H, brs),
6.90 (1H, brs), and 4-(2-carboxyethenyl)-2-(3-methoxy-4-hydroxyphe-
nyl)-2,3-dihydro-7-hydroxy-3-benzofurancarboxylic acid (17 mg), as
a colorless, amorphous solid, [R]23D +173.9 (c 1.66, acetone); 1H NMR
(acetone-d6) δ 3.83 (3H, s), 4.56 (1H, d, J ) 5.5 Hz), 6.01 (1H, d, J )
5.5 Hz), 6.32 (1H, d, J ) 16.0 Hz), 6.83 (1H, d, J ) 8.0 Hz), 6.89
(1H, d, J ) 8.0 Hz), 6.90 (1H, brs), 6.91 (1H, dd, J ) 8.0, 2.0 Hz),
7.10 (1H, d, J ) 2.0 Hz), 7.25 (1H, d, J ) 8.0 Hz), 7.80 (1H, d, J )
16.0 Hz); 13C NMR (acetone-d6) δ 56.4, 56.7, 88.3, 110.4, 115.8, 117.6,
118.1, 119.5, 121.3, 124.7, 127.2, 132.9, 142.5, 144.3, 147.7, 148.4,
148.5, 167.9, 172.9.
preparative HPLC [column, YMC-ODS
1
×
25 cm; solvent,
H2O-CH3CN (72.5:37.5); flow rate, 3.5 mL/min; detector, UV 280
nm] to give (S)-PGME amide (2.3 mg): 1H NMR (acetone-d6) δ 3.007
(2H, dd, J ) 14.5, 7.5 Hz, H-7′ or H-7′′), 3.130 (1H, dd, J ) 14.5, 5.5
Hz, H-7′ or H-7′′), 3.151 (1H, dd, J ) 14.5, 4.5 Hz, H-7′ or H-7′′),
5.402 (1H, dd, J ) 7.5, 4.5 Hz, H-8′ or H-8′′), 5.422 (1H, dd, J ) 7.5,
5.5 Hz, H-8′ or H-8′′).
Condensation of Clinopodic Acid I (10) with (R)-PGME. Cli-
nopodic acid I (10) (5.4 mg) was treated with (R)-PGME (12.9 mg) by
the procedure described above to obtain the (R)-PGME amide (2.4 mg)
1
as a colorless, amorphous solid: H NMR (acetone-d6) δ 2.880 (1H,
(2R)-3-(3-Methoxy-4-hydroxyphenyl)-2-hydroxypropanoic acid (5
mg) was condensed with (S)-PGME and (R)-PGME by the same method
as for (2R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid and gave
dd, J ) 14.5, 7.0 Hz, H-7′ or H-7′′), 2.932 (1H, dd, J ) 14.0, 8.0 Hz,
H-7′ or H-7′′), 3.078 (1H, dd, J ) 14.0, 4.5 Hz, H-7′ or H-7′′), 3.108

1384 Journal of Natural Products, 2009, Vol. 72, No. 8
Murata et al.
(1H, dd, J ) 14.5, 5.5 Hz, H-7′ or H-7′′), 5.387 (1H, dd, J ) 7.0, 5.5
Hz, H-8′ or H-8′′), 5.427 (1H, dd, J ) 8.0, 4.5 Hz, H-8′ or H-8′′).
Preparation of Tissue Homogenate. Male Wistar rats age 9-10
weeks and weighing 200-220 g (Nihon SLC, Hamamatsu, Japan) were
housed in groups of two in stainless-steel cages with free access to
food (Japan CLEA; CE-2, Tokyo, Japan) and H2O. They were kept in
a room maintained at ambient temperature and humidity (25 ( 5 °C,
55 ( 5%) under a day/night regime (day 7:00-19:00 and night 19:
00-7:00). All animals were maintained in the laboratory for a minimum
of 1 week prior to the start of the experiment. Lung was collected from
nine naive rats by decapitation followed by immediately freezing in
liquid nitrogen and maintained at -80 °C until use. Animal treatment
and maintenance were conducted in accordance with the Guidelines
for Animal Experimentation of Tohoku Pharmaceutical University,
Japan.
58. The gel was then stained with Rapid Stain Coomassie Brilliant
Blue kit. The inhibitory activities are collected as the % of EDTA-
control value. Each experiment was performed independently in
triplicate, and the data are indicated as mean (n ) 3), followed by
calculated IC50 values (µM).
Quantification of Gelatinolytic Activity by SDS-PAGE. The SDS-
PAGE gel was digitized using a PC scanner (Epson ES-2200, Epson
Co. Ltd., Nagano, Japan) operating on the image acquisition and
analysis program L Process V2.0 and Image Gauge V4.0 (Fuji Photofilm
Co., Ltd., Tokyo, Japan).
References and Notes
(1) Yamamoto, A.; Miyase, T.; Ueno, A.; Maeda, T. Chem. Pharm. Bull.
1993, 41, 1270–1274.
(2) Yamamoto, A.; Suzuki, H.; Miyase, T.; Ueno, A.; Maeda, T.
Phytochemistry 1993, 34, 485–488.
(3) Abe, H.; Sakaguchi, M.; Yamada, M.; Arichi, S.; Odashima, S. Planta
Med. 1980, 40, 366–372.
(4) Tian, D. N.; Wu, F. H.; Ma, S. C.; Li, D.; Dai, Y. Zhongguo Zhong
Yao Za Zhi 2008, 33, 1313–1316.
Lung tissue was homogenized with five volumes of homogenate
medium composed of 50 mM Tris-HCl buffer, pH 7.5, at 4 °C followed
by centrifuge (9000g, 4 °C, 20 min) to obtain a supernatant fraction as
an enzyme source maintained as aliquots for repeated experiments at
-80 °C until use.
(5) Dzhambazov, B.; Daskalova, S.; Monteva, A.; Popov, N. Biol. Pharm.
Bull. 2002, 25, 499–504.
SDS-PAGE Gelatin Zymography. Substrate gelatin-gel zymogra-
phy was performed by nonreducible SDS-PAGE conditions with
modification to the literature.17 Briefly, a lung 9000g supernatant
fraction was activated by incubation at 37 °C for 1 h with 0.5 mM
APMA in a buffer containing 50 mM Tris-HCl, pH 7.5, and 50 mM
NaCl. The protein content was measured by the method of Bradford,18
and equal amounts (0.5 mg/mL) of protein were used. The effect of
acid derivatives on gelatinolytic activity was tested as follows. The
activated supernatant fraction was incubated with 10 µL of each acid
solution and 5 µL of Tris-HCl, pH 7.5, for 30 min at 37 °C. The final
concentration of each acid derivative was 5-100 µM prepared in
DMSO solution (5%). Thereafter, these were resuspended in laemmli
sample buffer with the exception of 2-mercaptoethanol and boiling
followed by separation with SDS-PAGE gel containing 1.0 mg/mL of
gelatin. EDTA at 10 mM was used as a control for inhibition. The
electrophoresis was carried out by loading onto 7.5% acrylamide/
bisacrylamide (29:1) separating gel and applied for SDS-PAGE
performed with a Mini-Protein III apparatus (Bio-Rad, Hercules, CA).
Electrophoresis was carried out with molecular mass maker of myosin
(200 kDa), ꢀ-galactosidase (116 kDa), and bovine serum albumin (66
kDa), at a constant voltage of 200 V for 40 min. Proteins were stained
by Rapid Stain Coomassie Brilliant Blue kit (Nacalai Tesque, Kyoto,
Japan). After electrophoresis, the gel was soaked in 0.25% Triton X-100
(30 min twice) at room temperature and rinsed in H2O following
incubation at 37 °C for 18 h in the incubation buffer containing 50
mM Tris-HCl (pH 7.6), 20 mM NaCl, 5 mM CaCl2, and 0.02% Brij-
(6) Petersen, M. Phytochemistry 1997, 45, 1165–1172.
(7) Kim, N.-C.; Graf, T. N.; Sparacino, C. M.; Wani, M. C.; Wall, M. E.
Org. Biomol. Chem. 2003, 1, 1684–1689.
(8) Satake, T.; Kamiya, K.; Saiki, Y.; Hama, T.; Fujimoto, Y.; Kitanaka,
S.; Kimura, Y.; Uzawa, J.; Endang, H.; Umar, M. Chem. Pharm. Bull.
1999, 47, 1444–1447.
(9) Tanaka, T.; Morimoto, S.; Nonaka, G.; Nishioka, I.; Yokozawa, T.;
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