A lipase inhibitor monoterpene and monoterpene glycosides from Monarda punctata

Article abstract of DOI:10.1016/j.phytochem.2010.08.009

An 80% acetone extract of Monarda punctata showed an inhibitory effect on lipase activity in isolated mouse plasma in vitro and carvacrol was obtained as the active constituent. It had an IC₅₀ value of 4.07 mM in vitro and suppressed elevations in blood triacylglycerol levels in olive oil-loaded mice. Furthermore, from the whole plant, 22 compounds were isolated. Six monoterpene glycosides (3-8), a flavone glucuronide (9), and other known compounds were identified based on the results of spectroscopic analyses.

Full text of DOI:10.1016/j.phytochem.2010.08.009

Phytochemistry 71 (2010) 1884–1891
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
A lipase inhibitor monoterpene and monoterpene glycosides from Monarda punctata
a
b
a
Keiko Yamada a, Toshihiro Murata a, , Kyoko Kobayashi , Toshio Miyase , Fumihiko Yoshizaki
⇑
a Department of Pharmacognosy, Tohoku Pharmaceutical University, 4-1 Komatsushima 4-chome Aoba-ku, Sendai 981-8558, Japan
b School of Pharmaceutical Sciences, University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2010
Received in revised form 5 June 2010
An 80% acetone extract of Monarda punctata showed an inhibitory effect on lipase activity in isolated
mouse plasma in vitro and carvacrol was obtained as the active constituent. It had an IC50 value of
4.07 mM in vitro and suppressed elevations in blood triacylglycerol levels in olive oil-loaded mice. Fur-
thermore, from the whole plant, 22 compounds were isolated. Six monoterpene glycosides (3–8), a fla-
vone glucuronide (9), and other known compounds were identified based on the results of
spectroscopic analyses.
Keywords:
Monarda punctata
Lamiaceae
Ó 2010 Elsevier Ltd. All rights reserved.
Lipase inhibitor
Monoterpene
Carvacrol
Glycoside
1. Introduction
dionin and rhodiosin in Rhodiola rosea (Kobayashi et al., 2008a) and
myricitrin and myricetin in Myrica rubra (Kobayashi et al., 2008b).
Monarda punctata L. (Lamiaceae) is a traditional herbal medi-
cine of North American Indians used as a remedy for colds and a
treatment for nausea, vomiting, and rheumatic pains (Chevallier
and Nanba, 2000). Previous studies on the ingredients of M. punc-
tata have identified essential oils using gas chromatography (Scora,
1965).
In the present study, a major component of essential oil, carva-
crol (1), was obtained as a mouse lipase inhibitor, which depressed
the elevation of blood TG levels in olive oil-loaded mice, from M.
punctata. Additionally, six new monoterpene glycosides, monar-
dins A–F (3–8) and a new flavone glucuronide, keshonin (9), were
isolated from the plant together with 14 known compounds (2, 10–
22). We describe the structural elucidation of novel compounds
and inhibitory effects of several constituents on lipase in isolated
mouse plasma.
Lipase is an enzyme that hydrolyzes triacylglycerols (TGs). The
digestion and absorption of natural lipids begins with hydrolysis
by pancreatic lipase. The activity of this enzyme greatly affects
the metabolism of fat and the concentration of TG in blood (Mu
and Porsgaard, 2005). Recently, inhibitors of lipase and lipid
absorption have been isolated from natural sources with the aim
of preventing and treating metabolic syndrome. These inhibitors
include terpenoids (Bitou et al., 1999; Ninomiya et al., 2004; Jang
et al., 2008; Kim et al., 2009), triterpene glycosides (Zheng et al.,
2004; Xu et al., 2005; Yoshizumi et al., 2006; Morikawa et al.,
2009; Sugimoto et al., 2009), flavonoids (Kawaguchi et al., 1997;
Hatano et al., 1997; Won et al., 2007), catechins (Yoshikawa
et al., 2002; Nakai et al., 2005), stilbenes (Matsuda et al., 2009),
chitosan (Ostanina et al., 2007), and phenyl compounds
(Raghavendra and Prakash, 2002; Han et al., 2007). Most of these
inhibitory activities were examined using porcine pancreatic
lipase. On the other hand, we have reported inhibitory effects of
natural products on mouse lipase in vitro and in vivo, namely, rho-
2. Results and discussion
An 80% acetone extract of M. punctata had an inhibitory effect on
lipase activity in isolated mouse plasma in vitro (Table 1; IC50
,
2.0 mg/mL). The extract was dissolved in water and extracted with
diethyl ether. An essential oil fraction was obtained from the
diethyl ether layer and its extract was found to exhibit an inhibitory
effect (Table 1; IC50, 0.18 mg/mL). A major component of the essen-
tial oil fraction was identified as carvacrol (1) (Han et al., 2005). 1
showed a concentration-dependent inhibition and its IC50 value
was 4.07 mM (Table 1). This suggested that 1 has the potential to
affect lipase activity in the mouse alimentary canal. Then, we eval-
uated the suppressive effect of 1 on the increase in blood TG levels
in olive oil-loaded mice. The TG levels of control mice (olive oil
alone, 5 mL/kg, p.o.) gradually increased, but 1 (300 mg/kg, p.o.) sig-
nificantly suppressed the increase at 90, 120, and 180 min as shown
in Fig. 2. Orlistat (positive control, 10 mg/kg, p.o.) likewise
depressed the increase in the blood TG level. As the diethyl ether
⇑
Corresponding author. Tel./fax: +81 22 727 0220.
E-mail address: murata-t@tohoku-pharm.ac.jp (T. Murata).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.08.009

K. Yamada et al. / Phytochemistry 71 (2010) 1884–1891
1885
H-10) and 4.72 (1H, d, J = 7.5 Hz, H-100), and the 1H–1H COSY, 13C-
NMR and HMBC spectra showed the presence of two 6-malonated
b-D-glucopyranose moieties. The missing malonyl methylene pro-
Table 1
Inhibitory effects of extracts on lipase in
isolated plasma of mice.
ton and carbon signals in CD3OD were confirmed by 1H- and 13C-
NMR spectra in DMSO-d6 at dY 3.19, dY 3.22, dC 41.7, and dC 42.2.
The H-20 resonance at d 3.76 (1H, dd, J = 9.0, 7.5 Hz) and the C-20
(d 83.2) carbon signals were shifted downfield relative to the reso-
nances of 3. In the HMBC spectrum, the H-100 signal was long-range
coupled with C-20. Hence, the structure of 3 was formulated as
shown in Fig. 1.
Extract
Lipase inhibition (IC50)
80% Acetone
Water
Ether
Carvacrol
Orlistat
2.0 mg/mL
5.6 mg/mL
0.18 mg/mL
4.07 mM
0.09 mM
extract exhibited stronger activity, presence of other minor constit-
uents having strong inhibitory activities in the diethyl ether layer
are suggested.
The water layer, its extract having showed weak inhibition
(Table 1; IC50, 5.6 mg/mL), was passed through a column of HP-
20, a porous polymer gel. The 80% methanol eluate was separated
by preparative Yamazen cartridge column chromatography (YCCC)
and HPLC to afford nine monoterpene glycosides (2–8), a flavone
glucuronide (9), and other known compounds (10–22). The com-
pounds 2 and 10–22 were identified based on spectroscopic data
Monardin C (5) had the molecular formula C16H24O7 according
to HRFABMS (m/z 351.1412, calcd for C16H24O7Na, 351.1419). Its
1H- and 13C-NMR spectra were similar to those of 2 except for
the presence of a methylene group in 5 instead of a methyl group
in 2. The 1H-NMR spectrum showed oxymethylene proton signals
at d 4.53 (1H, d, J = 12.5 Hz, H-7) and d 4.81 (1H, d, J = 12.5 Hz, H-
7). These signals were long-range coupled with C-1, 2 and 6 in
the HMBC spectrum. They indicated that 5 possesses a –CH2OH
moiety instead of the methyl group of 2. Hence, the structure of
5 was formulated as depicted in Fig. 1.
as 2-methyl-5-(1-methylethyl)phenyl-b-
(Shimoda et al., 2006), apigenin 7-O-b- -glucoside (10) (Oyama
and Kondo, 2004), apigenin 7-O-(6-O-malonyl-b- -glucoside) (11)
(Svehlikova et al., 2004), apigenin 7-O-b- -rutinoside (12) (Wang
et al., 2003), apiin (13) (Yoshikawa et al., 2000), 600-O-malonylapiin
(14) (Yoshikawa et al., 2000), apigenin 7-O-b- -glucuronide (15)
(Flamini et al., 2001), luteolin 7-O-b- -glucuronide (16)
(Vanhoenacker et al., 2002), chrysoriol 7-O-b- -glucuronide (17)
(Stochmal et al., 2001), scutellarin B (18) (Kawasaki et al., 1988),
4-allyl-1-O-b- -glucopyranosyl-2-hydroxybenzene (19) (Norr and
D
-glucopyranoside (2)
The molecular formula C19H26O10 for monardin D (6) was de-
duced by HRFABMS (m/z 437.1431, calcd for C19H26O10Na,
D
D
437.1431). In the 1H- and 13C-NMR spectra of 6, the H-60 resonance
at d 4.26 (1H, dd, J = 12.0, 6.5 Hz) and 4.56 (1H, dd, J = 12.0, 2.0 Hz)
were shifted downfield relative to those of 5, and the two carbonyl
carbons at d 168.6 and 170.0 suggested the presence of a malonyl
moiety. In the HMBC spectrum, the H-60 signals were long-range
coupled with d 168.6 (C-70). Hence, 6 was the malonate derivative
of 5.
D
D
D
D
D
The 1H- and 13C-NMR spectra (Table 2) suggested that aglycone
moieties of monardin E (7) and monardin F (8) were p-menth-
3-ene-1,2-diol, similar to those of (1R,2R)-p-menth-3-en-1,2-diol 2-
Wagner, 1992), citrusin C (20) (Teng et al., 2005), rosmarinic acid
(21) (Jayasinghe et al., 2003), and eritrichin (22) (Fedoreyev
et al., 2005).
O-b-D-glucopyranoside (Kitajima et al., 2004). The molecular formu-
The novel monoterpene glycosides (3–8) were isolated as amor-
phous powders with the 1H- and 13C-NMR spectroscopic data
(measured in methanol-d4 at 30 °C) shown in Table 2.
Monardin A (3) was deduced to have the molecular formula
las C16H28O7 for 7 and C19H30O10 for 8 were determined (see
Section 4). For 7, the anomeric proton at d 4.71 (1H, d, J = 7.5 Hz,
H-10) and the coupling constants of the sugar protons in the
1H-NMR spectrum, 13C-NMR spectrum and sugar analysis suggested
C
19H26O9 based on HRFABMS (m/z 399.1649, calcd for C19H27O9,
the presence of an b-D-allopyranosyl unit. In the HMBC spectrum, the
399.1655). Its 1H- and 13C-NMR spectra were similar to those of
2 except for the presence of a 6-acylated glucopyranose moiety
in 3. The 1H-NMR spectrum showed ABX-type aromatic protons
at d 7.02 (1H, d, J = 9.0 Hz), 6.80 (1H, dd, J = 9.0, 2.0 Hz) and
6.96 (1H, br s). Two doublet methyl [d 1.22 (6H, d, J = 7.0 Hz),
H-9, 10] and methine [d 2.84 (1H, m), H-8] protons showed the
presence of a (CH3)2–CH– spin system. These signals and a singlet
methyl proton [d 2.22 (3H, s)] suggested the presence of a carva-
crol moiety as an aglycone. The anomeric proton at d 4.86 (1H,
overlapped, H-10), 1H–1H COSY and 13C-NMR spectra showed
the presence of a 6-acylated glucopyranosyl moiety. The sugar
H-10 signal was long-range coupled with d 86.6 (C-2) and d 4.08 (1H,
overlapped, H-2) was long-range coupled with d 102.3 (C-10). The rel-
ative configuration was determined from the 1H-NMR and NOE spec-
tra recorded in pyridine-d5 (Fig. 3 and Section 4), and 13C-NMR
spectra recorded in CDCl3 (Table 3). In the NOE spectrum, a methyl
proton at d 1.48 (3H, s, H-7) was correlated with a C-5ax proton at d
2.00 (1H, overlapped), a C-6 eq proton at d 1.85–1.98 (overlapped),
and other methyl protons at d 0.87 (3H, d, J = 6.6 Hz, H-10) and 0.88
(3H, d, J = 6.6 Hz, H-9), which showed the methyl was axial. An oxy-
genated methine proton at d 4.47 (br s, H-2) was correlated with a
C-6ax proton at d 1.85–1.94 (overlapped) in the NOE spectrum. The
decoupling 1H-NMR spectra on irradiation around d 2.00–2.07
showed a couplingconstant between H-2and H-3ofJ = 1.5 Hz, which
suggested a trans-diol conformation (Suga et al., 1968; Kitajima et al.,
2004). Moreover, 1H- and 13C-NMR (CDCl3, Table 3) signals of the
aglycone moiety were partly superimposable with those of a
(1R,2R)-3-p-menth-3-ene-1,2-diol (Abraham et al., 1986; Jefford
et al., 1989). Hence, 7 was rel-(1R,2R)-p-menth-3-ene-1,2-diol 1-O-
analysis showed that it was
anomeric configuration of the
D
-glucose (Tanaka et al., 2007). The
-glucosyl moiety was determined
D
to be b from the chemical shifts in the 13C-NMR spectrum (Kasai
et al., 1979). The H-60 resonances at d 4.25 (1H, dd, J = 12.0,
6.0 Hz) and 4.54 (1H, dd, J = 12.0, 2.0 Hz) were shifted downfield
relative to those of 2. The two carbonyl carbons at d 168.6 and
170.0, the methylene carbon at d 41.9, and the corresponding
methylene proton at d 3.35 (s) suggested the presence of a
malonyl moiety. In the HMBC spectrum, the H-60 signals were
long-range coupled with d 168.6 (C-70), and the H-10 signal was
long-range coupled with d 157.0 (C-2). Hence, the structure of 3
was determined as shown in Fig. 1.
b-D-allopyranoside. Although the absolute configuration of 7 was
not determined, it was deduced by application of the 13C-NMR gly-
cosidation shifts rule of a secondary alcohol (Seo et al., 1978). The
paper indicated that the method can be extended to all glycopyrano-
sides including b-
group at C-20 of a sugar moiety. The glycosidation shift for the ano-
meric carbon (C-10) [
d = d (alcoholic glycoside) ꢀ d (methylglyco-
side)] was
ꢀ0.7 ppm (CD3OD, Li et al., 2003), and the
glycosidation shifts for the aglycone moiety [ d = d (alcoholic glyco-
side) ꢀ d (alcohol)] were shown in Fig. 3 (pyridine-d5) and Table 3
D-allopyranoside having an equatorial hydroxyl
Monardin B (4) was deduced to have the molecular formula
C
28H38O17 by HRFABMS (m/z 647.2166, calcd for C28H39O17
,
D
647.2187). Its 1H- and 13C-NMR spectra were similar to those of
3 except for the presence of another 6-acylated glucopyranose
moiety in 3. The anomeric protons at d 5.07 (1H, overlapped,
Dd
D

Table 2
NMR spectroscopic data (CD3OD) for compounds 3–8.
Position
3
4
5
6
7
8
(J in Hz)
HMBC
(H–C)
(J in Hz)
HMBC
(H–C)
(J in Hz)
HMBC
(H–C)
(J in Hz)
HMBC
(H–C)
(J in Hz)
HMBC
(H–C)
(J in Hz)
HMBC
(H–C)
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
1
2
126.6
157.0
126.0
156.7
129.7
157.4
129.8
157.2
72.3
86.6
72.4
87.7
4.08,
Overlapped
5.40, br s
1, 3, 4,
10
4.04, m
3, 10
3
6.96, br s
115.2 2, 4, 5, 8 6.92, d (1.5)
114.0
2, 4, 5, 8 7.11, d (1.0)
115.7 2, 4, 5,
7.06, d (2.0) 115.8 2, 5, 8
121.4 1, 5, 8
5.39, br s
121.4
1, 5, 8
8
4
5
149.0
149.2
121.1
151.7
121.8 1, 3, 6,
151.6
121.8 3, 6, 8
146.5
146.8
25.9
6.80, dd
(9.0, 2.0)
121.4 1, 3, 6, 8 6.79, dd (7.5,
1, 3, 6, 8 6.91, dd (7.5,
1.0)
2, 4, 5, 7 7.24, d (7.5)
6.93, dd
(8.0, 2.0)
2.07, m
25.9
35.4
21.0
1, 4, 6, 8 2.07, m
1, 4, 5, 7 1.69, m
6
1.5)
8
6
7
7.02, d (9.0) 131.5 1, 4, 5, 7 7.03, d (7.5)
131.6
16.4
130.1 2, 4, 5,
7
60.9
7.23, d (8.0) 130.0 2, 4, 5, 7 1.68, m
35.5
21.1
4, 5, 7
1, 2, 6
2.22, s
16.1
35.1
1, 2, 6
2.22, s
1, 2, 6
4.53, d (12.5)
4.81, d (12.5)
2.88, m
1, 2, 6
4.52, d
(12.5)
4.73, d
(12.5)
61.0
35.3
1, 2, 6
1, 2, 6
1.14, s
1, 2, 6
1.10, s
1, 2, 6
8
2.84, m
3, 4, 5,
9, 10
4, 8, 10
4, 8, 9
2.84, m
35.3
3, 4, 5,
9, 10
4, 8, 10
4, 8, 9
2
35.4
3, 4, 5,
9, 10
4, 8, 10
4, 8, 9
2
2.89, m
3, 4, 5,
9, 10
4, 8, 10
4, 8, 9
2.22, m
35.5
3, 4, 5,
9, 10
4, 8, 10
4, 8, 9
2
2.22, m
35.5
3, 5, 9,
10
4, 8, 10
4, 8, 9
2
9
10
10
1.22, d (7.0) 24.5
1.22, d (7.0) 24.5
1.22, d (6.5)
1.22, d (6.5)
5.07, d (7.5)
24.6
24.7
100.7
1.24, d (6.5)
1.24, d (6.5)
4.71, d (7.5)
24.4
24.4
103.7
1.24, d (7.0) 24.5
1.24, d (7.0) 24.5
4.84, d (7.5) 103.7 2, 30
1.02, d (6.5) 21.7
1.02, d (6.5) 21.8
4.71, d (7.5) 102.3
1.02, d (7.0) 21.7
1.02, d (7.0) 21.7
4.73, d (8.0) 102.4
4.86,
103.1 2, 200
Overlapped
3.49,
Overlapped
3.49,
Overlapped
3.38,
Overlapped
3.63, m
20
30
40
50
60
74.9
77.9
71.5
75.3
65.6
3.76, dd (7.5,
9.0)
83.2
77.9
71.3
75.2
3.49,
75.2
78.1
69.2
78.4
3.51, dd
(7.5, 9.0)
3.47, dd
(9.0, 9.0)
3.38, dd
(9.0, 9.0)
3.65, m
75.0
77.7
71.5
75.5
65.6
3.37, dd
(7.5, 3.0)
4.06, dd
(3.0, 3.0)
3.47, dd
(9.5, 3.0)
3.75, m
72.2
73.1
69.2
75.6
63.2
3.39, dd
(8.0, 3.5)
4.07, m
72.2
73.1
69.2
75.7
66.3
Overlapped
3.40–3.50,
Overlapped
3.40–3.50,
Overlapped
3.40–3.50,
Overlapped
3.74, dd (12.0, 62.7
5.0)
3.90, dd (12.0,
2.0)
3.66–3.73,
Overlapped
3.66–3.73,
Overlapped
3.44, dd (9.0,
9.0)
4.24, dd (11.5, 65.6
6.5)
4.54, dd (11.5,
2.0)
40, 50
30, 60
60
60
60
70
70
3.50, dd
(9.5, 3.0)
3.99, m
4.25, dd
(12.0, 6.0)
4.54, dd
70
4.26, dd
(12.0, 6.5)
4.56, dd
70
3.62, dd
(11.5, 6.5)
3.87, dd
4.17, dd
(12.0, 7.5)
4.56, dd
70
70
70
(12.0, 2.0)
(12.0, 2.0)
(11.5, 2.0)
(12.0, 2.5)
70
80
168.6
41.9
168.6
Missing
168.6
41.9
168.3
Missing
3.35 (s)
70, 90
Missing
3.38,
Missing
Overlapped
90
170.0
170.1
170.0
170.2
90-OMe
100
4.72, d (7.5)
3.27,
105.3
76.1
20
200
Overlapped
3.35–3.41,
Overlapped
3.35–3.41,
Overlapped
3.35–3.41,
Overlapped
4.14,
300
400
500
600
77.7
71.2
75.5
65.3
600
600
700
Overlapped
700
800
900
168.6
Missing
170.1
Missing

K. Yamada et al. / Phytochemistry 71 (2010) 1884–1891
1887
9
8
3
6'
R2O
RO
O
O
1
1
O
O
HO
1 Carvacrol
1'
1'
OH
OH
7
7
OH
OH
OH
OR1
OH
R1
H
R2
H
R
H
2
3
5
O
O
O
O
H
6
9'
7'
9'
7'
HO
HO
HO
O
O
O
O
HO 9''
7''
6''
O
4
O
OH
1''
HO
OH
10'''
5'''
3'''
OH
9'''
3
1'''
RO
O
O
1 OH
3'
O
OH
7
O
HO
1'
6''
8
O
O
O
1'
HO
HO OH
OH
1''
10
R
H
O
3
5
7
HO
OH
OH
O
O
8
9'
7'
HO
9
Fig. 1. Structures of compounds 1–9.
Blood TG level
(mg/dL)
H
H
CH
H
3
-0.9*
HO
OH
H
OH
H
CH
3
+0.7*
+0.4*
H
O
O
H
-0.2*
-0.1*
CH
3
-1.0*
H
O
H
+12.5*
H
HO
H
H
OH
: key HMBC correlations
: key NOE correlations
H
OH
Fig. 3. Key HMBC and NOE correlations for the relative configuration of 7 in
*
pyridine-d5 and ‘‘Sterically Hindered Case II” for 7 in glycosidation shifts rules The
glycosidation shifts in pyridine-d5 (Dd in ppm).
Table 3
13C-NMR Spectroscopic Data (CDCl3) for p-Menth-3-ene-1,2-diols, 7, and 7a.
Position
cysa,b
dC
transb,c
dC
7 (7-transb,c
dC
)
7ad (7a-transb,c
)
dC
Time after oral administration
Fig. 2. Variation in blood triacylglycerols levels in olive oil-loaded mouse plasma
after the oral administration of carvacrol. d, olive oil alone (5 mL/kg, p.o.); N,
orlistat (10 mg/kg, p.o.) and olive oil (5 mL/kg, p.o.); 4, carvacrol (300 mg/kg, p.o.)
and olive oil (5 mL/kg, p.o.). All points represent the mean for four mice per group.
Vertical lines show the standard error of the mean. Significantly different from the
1
2
3
4
5
6
7
8
9
10
70.1
71.8
119.7
147.8
24.4
32.4
24.4
34.5
21.4
21.3
72.7
74.8
121.0
145.0
25.0
34.0
21.3
34.0
21.2
20.1
71.7 (ꢀ1.0)
71.0 (ꢀ1.7)
89.0 (14.2)
119.7 (ꢀ1.3)
146.2 (1.2)
24.9 (ꢀ0.1)
33.9 (ꢀ0.1)
20.5 (ꢀ0.8)
34.1 (0.1)
87.0 (12.2)
119.8 (ꢀ1.2)
146.2 (1.2)
24.9 (ꢀ0.1)
34.1 (0.1)
20.5 (ꢀ0.8)
34.2 (0.2)
21.5 (0.3)
21.2 (1.1)
*
**
control, p < 0.05 and p < 0.01.
21.4 (0.2)
21.1 (1.0)
(7a: tetra-O-acetylglycoside, CDCl3). The NMR data of (1R,2R)-3-p-
menth-3-ene-1,2-diol recorded in pyridine-d5 and in CDCl3 were
cited from the literatures (Kitajima et al., 2004; Abraham et al.,
a
b
c
(1R,2S)-3-p-menth-3-ene-1,2-diol.
Reported value (Abraham et al., 1986).
(1R,2R)-3-p-menth-3-ene-1,2-diol.
Tetra-O-acetylated 7.
1986), respectively. Shift values [
(C-2), and
d ꢀ0.2 ppm (C-3) in pyridine-d5 and
(C-1), d + 14.2 ppm (C-2), and
D
d ꢀ1.0 ppm (C-1),
D
D
d + 12.5 ppm
d ꢀ1.7 ppm
D
d
D
D
d ꢀ1.3 ppm (C-3) inCDCl3 (7a)]sug-
gested that ‘‘Sterically Hindered Case II” discussed in the paper
(Seo et al., 1978) was applicable to 7 and 7a, which shows that C-2
glycoside had not been established except for iridoid glycosides
(Tagawa and Murai, 1997). Therefore, it was not possible to confirm
the absolute stereochemistry of 7 from only the shifts rule. The
is S configuration. However, the
Dd (C-3) values were larger than
those for C-1, and parameters of shifts rule for this allyl alcohol type

1888
K. Yamada et al. / Phytochemistry 71 (2010) 1884–1891
4. Experimental
OH
4.1. General
O
Optical rotations were measured on a Jasco P-2300 polarimeter.
OH
O
UV spectra were recorded on a Shimadzu MPS-2450. 1H-NMR
(400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Jeol
JNM-AL400 FT-NMR spectrometer, and chemical shifts were given
as d values with TMS as an internal standard at 30 °C. Inverse-de-
tected heteronuclear correlations were measured using HMQC
HO
HO
O
O
O
O
OH
: key HMBC corrations
: key NOE corrations
OH
OH
1
n
(optimized for JC–H = 145 Hz) and HMBC (optimized for JC–
H = 8 Hz) pulse sequences with a pulsed field gradient. HRFABMS
data were obtained on a Jeol JMS700 mass spectrometer, using a
m-nitrobenzyl alcohol or a glycerol matrix. YCCC and HPLC were
performed on a Jasco 2089 with UV at 210 nm, using the following
columns: Ultra Pack ODS-SM-50C-M [Yamazen, 37 ꢁ 100 mm; mo-
bile phase, MeOH–0.2%TFA in H2O (35:65) ? (65:35)], ODS-100V
[Tosoh, 20 ꢁ 250 mm; mobile phase, CH3CN–0.2%TFA in H2O
(20:80) ? (35:65)], Cosmosil 5C18–AR II column [Nacalai Tesque,
20 ꢁ 250 mm; mobile phase, CH3CN–0.2%TFA in H2O (25:75)], Cap-
cell-Pak Ph [Shiseido, 20 ꢁ 250 mm; solvent, CH3CN–H2O (20:80)
or (25:75)] and Mightysil RP-18 GP [Kanto Chemical, 10 ꢁ 250
mm; solvent, acetonitrile–water (25:75)]. Olive oil, 5,50-dithio-
bis(2-nitrobenzoic acid), and phenylmethylsulfonyl fluoride were
purchased from Nacalai Tesque Inc. (Kyoto, Japan), while 2,3-di-
mer-capto-1-propanol tributyrate was purchased from Aldrich
Chemical Co. (Milwaukee, WI). Orlistat was obtained from Roshe
Products Ltd. (Auckland, NZ). Carvacrol was purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan).
Fig. 4. Key HMBC and NOE correlations for 9 in DMSO-d6.
1H- and 13C-NMR spectra of 8 were similar to those of 7 except for the
presence of a 6-acylated allopyranose moiety in 8 instead of an allo-
pyranose in 7. The H-60 resonance at d 4.17 (1H, dd, J = 12.0, 7.5 Hz)
and 4.56 (1H, dd, J = 12.0, 2.5 Hz) were shifted downfield relative to
those of 7. The two carbonyl carbons at d 168.3 and 170.2 suggested
the presence of a malonyl moiety. The missing malonyl methylene
proton and carbon signals in CD3OD were confirmed by 1H- and
13C-NMR spectra in DMSO-d6 at dH 3.51 and dC 40.7. In the HMBC
spectrum, the H-60 signals were long-range coupled with d 168.3
(C-70). Hence, 8 was a malonyl derivative of 7.
Keshonin (9) was an amorphous powder with 1H- and 13C-NMR
spectroscopic data (measured in DMSO-d6, 30 °C) assigned as
shown in the Section 4. On the basis of HRFABMS, the molecular
formula C31H30O13 was determined (m/z 611.1757, calcd for
C
31H31O13, 611.1764). The 1H- and 13C-NMR spectra of 9 showed
a flavone glycoside moiety and a monoterpenoid moiety. The aro-
matic carbons d 94.4 (C-8), 99.4 (C-6), 103.5 (C-3), 105.3 (C-10),
115.9 (C-20), 117.1 (C-50), 121.4 (C-10), 122.5 (C-60), 146.2 (C-30),
151.9 (C-40), 156.7 (C-9), 161.1 (C-4), 162.4 (C-7), and 163.7 (C-2)
and the carbonyl carbon (d 181.7) of a flavone moiety were partly
superimposable with those of a 7,30-substituted luteoline skeleton
(Lu and Foo, 2000). The anomeric proton at d 5.14 (1H, d, J = 6.5 Hz,
H-100), and 1H–1H COSY and 13C-NMR spectra showed the presence
of glucuronic acid unit. The sugar analysis (Tanaka et al., 2007) and
4.2. Plant material
M. punctata L. was collected in July 2008 in Sendai City, Japan.
The young plants were purchased from and identified by Tamaga-
wa engei, Nagano, Japan. A voucher specimen is deposited at the
herbarium of Tohoku Pharmaceutical University, No. 20080716.
the coupling constant of the anomeric proton indicated a b-D-glu-
4.3. Extraction and isolation
curonic acid. In the HMBC spectrum (Fig. 4), the H-100 signal was
long-range coupled with d 162.4 (C-7). Accordingly, the flavonegly-
Powdered whole plants (640 g) of M. punctata were extracted
with acetone–H2O (80:20) (7 L) twice at 50 °C for 3 h. The extract
(75.6 g) was suspended in H2O (1.5 L), and extracted with Et2O
(3 ꢁ 1 L). The ether extract (17.5 g) was suspended in EtOH–H2O
(8:2) (1.5 L), and extracted with hexane (3 ꢁ 1 L). The hexane layer
extract (7.0 g) was passed through a silica gel column (Wakogel
C-200, Wako Pure Chemical Industries Ltd., Osaka, Japan, 30 g)
yielding 14 fractions, one of which, eluted with CHCl3-MeOH
(99:1) was an essential oil fraction whose major component was
1 (3.4 g). The H2O layer extract (50.5 g) was a red-brown syrup.
It was dissolved again in H2O, and the aqueous solution was passed
through a porous polymer gel column (Mitsubishi Diaion HP-20,
Mitsubishi Chemical Corporation, Tokyo, Japan, 60 ꢁ 300 mm)
and eluted with H2O, MeOH–H2O (80:20) and MeOH. The MeOH–
H2O (80:20) eluate (10.2 g) was subjected to on a reversed-phase
CC using ODS (Cosmosil 140C18-OPN, Nacalai Tesque, Osaka, Japan,
150 g) and eluted with 20%, 30%, 40%, 50%, 60%, 80% MeOH in H2O,
and MeOH (fractions 1A–1G). Fraction 1B (2.19 g) was subjected to
YCCC and HPLC, yielding compounds 5 (6.8 mg), 6 (10.0 mg), 7
(15.8 mg), 12 (1.0 mg), 13 (297.1 mg), 16 (22.1 mg), 18 (31.0 mg),
19 (1.0 mg), 20 (2.0 mg) and 21 (12.4 mg). Fraction 1C (1.54 g)
coside unit was 30-O-substituted luteoline 7-b-
D-glucuronic acid.
The 1H- and 13C-NMR spectra of 9 suggested the presence of a
5000-O-substituted thymoquinol moiety as compared to zataro-
side-A or B (Ali et al., 1999). In the NOE spectrum (Fig. 4), the H-
9000 and -10000 methyl signals (d 1.16, 3H, d, J = 7.0 Hz; d 1.18, 3H,
d, J = 7.0 Hz) were correlated with the H-20 signal (d 7.24, 1H, d,
J = 2.0 Hz). Hence, the structure of 9 was formulated as shown in
Fig. 1.
The mouse lipase inhibitory activities of monoterpene glyco-
sides (2–4, 6 and 7) were evaluated but none of the compounds
showed significant activity. However, it is expected that their
hydrolysis influences lipase activity in the alimentary canal of mice
after conversion to aglycons by enterobacilli.
3. Conclusion
From whole plant of M. punctata, a lipase inhibitor: carvacrol (1),
six new monoterpene glycosides (3–9) and a new flavone glycoside
(9) were isolated. 1 had an IC50 value of 4.07 mM for lipase in
isolated mouse plasma in vitro and suppressed the postprandial ele-
vation of blood TG concentrations in mice in vivo. Compounds 2–4
were glucosides of carvacrol, whereas 7 and 8 were monoterpene
allopyranosides. The sugar moieties of 3, 4, 6, and 8 were malonated,
and 9 was a complex of the flavone glucuronide and the monoter-
pene moiety.
was subjected to YCCC and HPLC, yielding compounds
2
(10.5 mg), 4 (4.9 mg), 8 (4.3 mg), 10 (29.4 mg), 11 (19.9 mg), 14
(33.3 mg), 15 (38.3 mg), 17 (4.6 mg) and 22 (18.9 mg). Fraction
1D (750 mg) was subjected to YCCC and HPLC, yielding compounds
3 (13.4 mg) and 9 (2.8 mg).

K. Yamada et al. / Phytochemistry 71 (2010) 1884–1891
1889
4.4. Compound 1
4.11. Compound 8
Pale yellow oil; EI-MS m/z 150; 1H-NMR (CDCl3) d 6.71 (1H, d,
J = 7.5 Hz), 6.70 (1H, dd, J = 7.5, 1.0 Hz), 6.62 (1H, br s), 5.40 (1H,
br s), 2.77 (1H, sep, J = 7.0 Hz) 2.19 (3H, s), 1.18 (6H, d,
J = 7.0 Hz); 13C-NMR (CDCl3) d 153.5, 148.3, 130.8, 121.1, 118.7,
113.1, 33.6, 23.9, 15.3.
Colorless amorphous powder; ½a D22
ꢂ
ꢀ21.7 (c 0.23, MeOH);
HRFABMS m/z 441.1726 [M+Na]+ (calcd for C19H30O10Na,
441.1736); for 1H-NMR and 13C-NMR spectroscopic data, see Table
2. 1H-NMR signal of methylene (DMSO-d6) d 3.51; 13C-NMR signal
of methylene (DMSO-d6) d 40.7.
4.5. Compound 3
4.12. Compound 9
Colorless amorphous powder; ½a D22
ꢂ
ꢀ16.1 (c 0.41, MeOH);
Colorless amorphous powder; ½a D24
ꢀ6.9 (c 0.29, MeOH); UV
ꢂ
HRFABMS m/z 399.1649 [M+H]+ (calcd for C19H27O9, 399.1655);
(MeOH) kmax nm (log e): 202 (4.92), 253 (4.40), 269 (4.39), 345
for 1H-NMR and 13C-NMR (CD3OD) spectroscopic data, see Table 2.
(4.42); HRFABMS m/z 611.1757 [M+H]+ (calcd for C31H31O13
,
611.1764); 1H-NMR (DMSO-d6) d 6.70 (1H, s, H-3), 6.45 (1H, d,
J = 2.0 Hz, H-6), 6.62 (1H, d, J = 2.0 Hz, H-8), 7.24 (1H, d,
J = 2.0 Hz, H-20), 7.07 (1H, d, J = 8.5 Hz, H-50), 7.68 (1H, dd, J = 8.5,
2.0 Hz, H-60), 5.14 (1H, d, J = 6.5 Hz, H-100), 3.2–3.4 (3H, overlapped,
H-200, 300 and 400), 3.89 (1H, d, J = 8.0 Hz, H-500), 6.80 (1H, s, H-3000),
6.52 (1H, s, H-6000), 2.03 (3H, s, H-7000), 3.12 (1H, m, H-8000), 1.16
(3H, d, J = 7.0 Hz, H-9000), 1.18 (3H, d, J = 7.0 Hz, H-10000); 13C-NMR
(DMSO-d6) d 163.7 (C-2), 103.5 (C-3), 181.7 (C-4), 161.1 (C-5),
99.4 (C-6), 162.4 (C-7), 94.4 (C-8), 156.7 (C-9), 105.3 (C-10),
121.4 (C-10), 115.9 (C-20), 146.2 (C-30), 151.9 (C-40), 117.1 (C-50),
122.5 (C-60), 99.4 (C-100), 72.7 (C-200), 75.8 (C-300), 71.3 (C-400), 75.0
(C-500), 170.1 (C-600), 122.5 (C-1000), 151.9 (C-2000), 112.6 (C-3000),
136.8 (C-4000), 144.8 (C-5000), 120.2 (C-6000), 15.6 (C-7000), 26.1 (C-
8000), 23.1 (C-9000), 22.9 (C-10000).
4.6. Compound 4
Colorless amorphous powder; ½a D22
ꢀ18.5 (c 0.43, MeOH);
ꢂ
HRFABMS m/z 647.2166 [M+H]+ (calcd for C28H39O17, 647.2187);
for 1H-NMR and 13C-NMR (CD3OD) spectroscopic data, see Table 2.
1H-NMR signals of methylene (DMSO-d6) d 3.19, 3.22; 13C-NMR
signals of methylene (DMSO-d6) d 41.7, 42.2.
4.7. Compound 5
Colorless amorphous powder;
½
a 2D5
ꢂ
ꢀ4.9 (c 0.90, MeOH);
HRFABMS m/z 351.1412 [M+Na]+ (calcd for C16H24O7Na,
351.1419); for 1H-NMR and 13C-NMR (CD3OD) spectroscopic data,
see Table 2.
4.13. Tetra-O-acetylation of sugar moiety of 7
4.8. Compound 6
Compound 7 (5.0 mg) was dissolved in pyridine (1 mL) and stir-
Colorless amorphous powder;
½
a 2D5
ꢂ
ꢀ6.7 (c 0.95, MeOH);
red with Ac2O (200
(5.5 mg).
lL) for 5 h at 90 °C to yield compound 7a
HRFABMS m/z 437.1431 [M+Na]+ (calcd for C19H26O10Na,
437.1423); for 1H-NMR and 13C-NMR (CD3OD) spectroscopic data,
see Table 2.
4.14. Sugar identification
4.9. Compound 7
Each compound [3 (1.3 mg), 4 (0.5 mg), 5 (0.9 mg), 6 (1.0 mg), 7
(2.3 mg), 8 (1.4 mg), and 9 (0.3 mg)] was refluxed with 7% HCl
(1 mL) for 2 h at 60 °C. After cooling, the reaction mixture was
passed through an Amberlite IRA400 column, and the eluate was
concentrated. The residue was dissolved in pyridine (0.5 mL) and
Colorless amorphous powder; ½a D25
ꢀ26.0 (c 0.12, MeOH);
ꢂ
HRFABMS m/z 333.1907 [M+H]+ (calcd for C16H29O7, 333.1914);
for 1H-NMR and 13C-NMR (CD3OD) spectroscopic data, see Table 2.
1H-NMR (pyridine-d5) d 4.47 (1H, br s, H-2), 5.57 (1H, br s, H-3),
2.00 (overlapped, H-5ax), 2.05 (1H, m, H-5 eq), 1.85–1.94 (over-
lapped, H-6ax), 1.85–1.98 (overlapped, H-6 eq), 1.48 (3H, s, H-7),
2.00 (overlapped, H-8), 0.88 (3H, d, J = 6.6 Hz, H-9), 0.87 (3H, d,
J = 6.6 Hz, H-10), 5.05 (1H, d, J = 8.4 Hz, H-10), 4.07 (1H, dd, J = 8.4,
3.0 Hz, H-20), 4.76 (1H, dd, J = 3.0, 3.0 Hz, H-30), 4.16 (1H, dd,
J = 9.6, 3.0 Hz, H-40), 4.58 (1H, m, H-50), 4.30 (1H, dd, J = 11.4,
7.2 Hz, H-60), 4.61 (1H, dd, J = 11.4, 2.4 Hz, H-60); 13C-NMR (pyri-
dine-d5) d 70.8 (C-1), 87.3 (C-2), 121.7 (C-3), 145.0 (C-4), 25.4 (C-
5), 35.3 (C-6), 21.8 (C-7), 34.3 (C-8), 21.2 (C-9), 21.5 (C-10), 103.3
(C-10), 72.2 (C-20), 73.1 (C-30), 69.6 (C-40), 76.3 (C-50), 63.1 (C-60).
1H-NMR (CDCl3) d 5.25 (1Y, s), 4.73 (1H, br d, J = 6.5 Hz), 4.24
(1H, br s), 4.11 (1H, s), 3.45–4.00 (overlapped), 2.22 (1H, m), 2.04
(2H, br s), 1.71 (2H, m), 1.13 (3H, s), 1.00 (6H, d, J = 6.5 Hz); 13C-
NMR (CDCl3), d 101.3, 74.3, 71.0, 70.8, 68.1, 62.5, and Table 3.
stirred with
then o-tolyl isothiocyanate (20
L
-cysteine methyl ester (5 mg) for 1.0 h at 60 °C, and
L) was added to the mixture and
l
heated to 60 °C for 1.0 h. The reaction mixture was analyzed by
HPLC and detected at 250 nm. Analytical HPLC was performed on
a Cosmosil 5C18–AR II column (4.6 x 250 mm) at 20 °C using
CH3CN–0.2%TFA in H2O (25:75) as mobile phase. Peaks were de-
tected with a Tosoh UV8010 detector.
was identified as the sugar moieties of 3–6 by comparing retention
times with those of authentic samples of -glucose (tR 17.0 min)
and -glucose (tR 15.5 min). -Glucuronic acid (tR 16.8 min) was
identified as the sugar moiety of 9 by comparing retention times
with those of authentic samples of -glucuronic acid (tR
16.8 min) and -glucuronic acid (using -cysteine methyl ester
and -glucuronic acid, tR 16.1 min) (Tanaka et al., 2007). The
-derivative from -allose (tR 34.9 min) was identified as the sugar
moieties of 7 and 8 by comparing retention times with that of the
authentic sample of -allose (tR 34.9 min). After the reaction of
-allose, -cysteine methyl ester and o-tolyl isothiocyanate as
above, -thiohydantoin derivative (tR 24.5 min) was mainly
obtained instead of the expected -allose derivative.
D-Glucose (tR 17.0 min)
D
L
D
D
L
D
D
D
D
4.10. Compound 7a
D
L
L
1H-NMR (CDCl3) d 5.68 (1Y, br s), 5.10 (1H, s), 4.92 (3H,
overlapped), 4.30 (1H, br d, J = 12.0), 4.18 (1H, dd, J = 9.5, 7.5),
4.05–4.10 (2H, overlapped), 2.21 (1H, m), 2.15 (3H, s), 2.11 (3H,
s), 2.06 (3H, s), 2.06 (2H, overlapped), 2.01 (3H, s), 1.73 (1H, m),
1.68 (1H, m), 1.10 (3H, s), 1.00 (3H, d, J = 7.0 Hz), 1.00 (3H, d,
J = 7.0 Hz); 13C-NMR (CDCl3), d 170.7, 169.7, 169.1, 169.0, 100.2,
70.4, 69.2, 68.6, 66.7, 62.7, 20.7, 20.7, 20.7, 20.6, and Table 3.
L
L
4.14.1. D-Derivative from D-allose
Colorless amorphous powder; ½a D22
ꢂ
+67.8 (c 0.23, MeOH); FAB-
): 240 (5.18); 1H-
MS m/z 447 [M+H]+; UV (MeOH) kmax nm (log
e
NMR (400 MHz, CD3OD) d 5.89 (1H, br d, H-2), 5.86 (1H, t,

1890
K. Yamada et al. / Phytochemistry 71 (2010) 1884–1891
J = 8.0 Hz, H-4), 3.45 (2H, d, J = 8.0 Hz, H-5), 4.05 (1H, dd, J = 8.0,
3.5 Hz, H-6), 4.00 (1H, dd, J = 7.5, 3.5 Hz, H-7), 3.96 (1H, dd,
J = 7.5, 7.5 Hz, H-8), 3.82 (1H, m, H-9), 3.65 (1H, dd, J = 11.5,
6.0 Hz, H-10), 3.75 (1H, dd, J = 11.5, 3.5 Hz, H-10), 7.12–7.23 (4H,
overlapped, H-30-H-60), 2.26 (3H, s, 20-CH3), 3.87 (3H, s, OCH3);
13C-NMR (100 MHz, CD3OD) d 68.5 (C-2), 69.2 (C-4), 32.5 (C-5),
79.6 (C-6), 73.9 (C-7), 73.1 (C-8), 74.9 (C-9), 64.2 (C-10), 184.7
(C@S), 127.3, 127.8, 129.2, 131.5, 136.2, 140.1 (C-10-C-60), 18.5
(20-CH3), 174.2 (C = O), 53.4 (OMe).
References
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with 1-p-menthene skeleton. Appl. Microbiol. Biotechnol. 24, 24–30.
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cymene derivatives from Zataria multiflora. Phytochemistry 52, 685–688.
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Chevallier, A., Nanba, T., 2000. Sekai Yakuyou Syokubutu Hyakka Jiten, Original
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Fedoreyev, S.A., Veselova, M.V., Krivoschekova, O.E., Mischenko, N.P., Denisenko,
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Zhuravlev, Y.N., 2005. Caffeic acid metabolites from Eritrichium sericeum cell
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Flamini, G., Antognoli, E., Morelli, I., 2001. Two flavonoids and other compounds
from the aerial parts of Centaurea bracteata from Italy. Phytochemistry 57, 559–
564.
Han, X., Armstrong, D.W., 2005. Using geminal dicationic lonic liquids as solvents
for high-temperature organic reactions. Org. Lett. 7, 4205–4208.
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effects of compounds isolated from fruit of Juglans mandshurica on pancreatic
lipase. J. Nat. Med. 61, 184–186.
Hatano, T., Yamashita, A., Hashimoto, T., Ito, H., Kubo, N., Yoshiyama, M., Shimura,
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4.14.2.
L
-Thiohydantoin derivative from
L
-allose
+151.4 (c 0.42, MeOH); FAB-
): 240 (5.03), 276
Colorless amorphous powder; ½a D22
ꢂ
MS m/z 415 [M+H]+; UV (MeOH) kmax nm (log
e
(5.11); 1H-NMR (400 MHz, pyridine-d5) d 7.12 (1H, d, J = 2.0 Hz,
H-2), 5.50 (1H, t, J = 8.5 Hz, H-4), 3.38 (1H, dd, J = 10.5, 8.5 Hz, H-
5), 3.74 (1H, dd, J = 10.5, 8.5 Hz, H-5), 5.31 (1H, br d, J = 8.0 Hz,
H-6), 4.68 (1H, m, H-7), 4.75–4.80 (1H, overlapped, H-8), 4.75–
4.80 (1H, overlapped, H-9), 4.38 (1H, dd, J = 10.5, 5.5 Hz, H-10),
4.52 (1H, dd, J = 10.5, 3.0 Hz, H-10), 7.20–7.32 (4H, overlapped,
H-30-H-60), 2.22 (3H, s, 20-CH3); 13C-NMR (100 MHz, pyridine-d5)
d 69.7 (C-2), 67.8 (C-4), 32.3 (C-5), 75.3 (C-6), 74.7 (C-7), 74.1 (C-
8), 75.6 (C-9), 64.8 (C-10), 185.1 (C@S), 127.3, 129.6, 129.9,
131.3, 133.6, 137.4 (C-10-C-60), 17.6 (20-CH3), 172.4 (C@O).
Jayasinghe, C., Gotoh, N., Aoki, T., Wada, S., 2003. Phenolics composition and
antioxidant activity of sweet basil (Ocimum basilicum L.). J. Agric. Food Chem.
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4.15. Animals
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