Lipase inhibitory and LDL anti-oxidative triterpenes from Abies sibirica

Article abstract of DOI:10.1016/j.phytochem.2012.11.017

A methanol extract of Abies sibirica Ladeb, a Mongolian medicinal plant, had an inhibitory effect on both lipase activity in mouse plasma and LDL anti-oxidative activity, which are preventative factors for arteriosclerosis. The extract was fractionated by silica gel column chromatography and its active constituents were sought. From lipid soluble fractions, 20 terpenoids including seven hitherto unknown triterpenes were isolated. The latter triterpenes had either a γ-lactone ring with a lactol or a derivative thereof. Their chemical structures were determined by spectroscopic methods. The lipase inhibitory activity and LDL anti-oxidative activity of these compounds were evaluated. Some constituents (either lipase inhibitory or LDL anti-oxidative activities) had moderate inhibitory activities.

Full text of DOI:10.1016/j.phytochem.2012.11.017

Phytochemistry 86 (2013) 168–175
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Lipase inhibitory and LDL anti-oxidative triterpenes from Abies sibirica
a
a
b
Mizuho Handa a, Toshihiro Murata a, , Kyoko Kobayashi , Erdenechimeg Selenge , Toshio Miyase ,
⇑
Javzan Batkhuu c, Fumihiko Yoshizaki a
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
c School of Biology and Biotechnology, National University of Mongolia, POB 617, Ulaanbaatar 46A, Mongolia
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 Abies sibirica Ladeb, a Mongolian medicinal plant, had an inhibitory effect on both
lipase activity in mouse plasma and LDL anti-oxidative activity, which are preventative factors for
arteriosclerosis. The extract was fractionated by silica gel column chromatography and its active
constituents were sought. From lipid soluble fractions, 20 terpenoids including seven hitherto unknown
Received 16 February 2012
Received in revised form 31 August 2012
Available online 20 December 2012
triterpenes were isolated. The latter triterpenes had either a c-lactone ring with a lactol or a derivative
Keywords:
Abies sibirica
Pinaceae
Lipase inhibitor
LDL anti-oxidative activity
Triterpene
thereof. Their chemical structures were determined by spectroscopic methods. The lipase inhibitory
activity and LDL anti-oxidative activity of these compounds were evaluated. Some constituents (either
lipase inhibitory or LDL anti-oxidative activities) had moderate inhibitory activities.
Ó 2012 Elsevier Ltd. All rights reserved.
c
-Lactone ring
1. Introduction
A previous study on the constituents of this plant mainly resulted
in isolation of triterpenes (Yang et al., 2008).
Risk factors for cardiovascular disease have important meaning
for the prevention and treatment of life style-related diseases, and
our focus is on lipids and lipoproteins in bloods. Ingested triacyl-
glycerols (TGs) and TGs in the bloodstream are hydrolyzed and
resynthesized by lipases. For example, gastric and pancreatic li-
pases are enzymes which digest TGs to free fatty acids. After intes-
tinal absorption, they are resynthesized into TGs by lipases in
serums. Inhibitors and activators of these enzymes are expected
to be key factors regulating lipid metabolism. Lipase inhibitors
are considered to prevent digestion and intestinal absorption of
fatty acids from dietary fats. Actually, lipase inhibitors such as orli-
stat have been used to treat of obesity, and extracts of various
plants and their constituents were reported as anti-obesity agents
based on their lipase inhibitory activity (Tucci et al., 2010; Yamada
et al., 2010). Low-density lipoprotein (LDL) has important roles in
the maintenance of the body. However, oxidized LDL is a factor
for arteriosclerosis. So, LDL anti-oxidative activity is a target in
the treatment of cardiovascular diseases. Abies sibirica Ladeb is a
medicinal plant in Mongolia, used to treat tumors and diarrhea
(Boldsaikhan, 2004). Consequently, their constituents and biologi-
cal activities have been studied extensively (Ou-Yang et al., 2011;
Xia et al., 2012). A. sibirica is a coniferous plant of the family Pina-
ceae distributed in Eurasia including Russia and Northern Europe.
In the present study, seven new triterpenes were isolated to-
gether with eleven known triterpenes, a known sesquiterpene,
and a known monoterpene. Four of the new triterpenes had a
c-lactone ring which is typical of the genus Abies. The other three
had a carboxylic acid, and their absolute configuration at the
connected carbon was determined using phenylglycine methyl es-
ter (PGME). Lipase inhibitory activity using the 2,3-dimercapto-1-
propanol tributyrate (BALB) method and LDL anti-oxidant activity,
both preventative factors for arteriosclerosis, of these constituents
and the extract of A. sibirica were examined.
2. Results and discussion
A MeOH extract of A. sibirica had an inhibitory effect on lipase
activity in isolated mouse plasma in vitro (IC50, 0.39 mg/mL). The
extract was dissolved in water and extracted with diethyl ether.
The latter extract was found to exhibit an inhibitory effect (73.3%
inhibition, 0.3 mg/mL), whereas the aqueous extract had low activ-
ity (1.6–26% inhibition, 0.3 mg/mL). The diethyl ether layer extract
was purified on a silica gel column, and yielded 9 fractions (frs. 1A-
1I, 5.7–89.7% inhibition, 0.3 mg/mL). Fr. 1B (89.7% inhibition,
0.3 mg/mL) was further fractionated using both an ODS column
and HPLC, in accordance with the guide to monitoring inhibitory
activity. Similarly, the diethyl ether layer obtained from another
⇑
MeOH extract was passed through
(CHCl3-MeOH) to yield 7 fractions (frs. 2A-2G). Frs. 2D-2F were
a column of silica gel
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 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.11.017

M. Handa et al. / Phytochemistry 86 (2013) 168–175
169
21
30
22
20
17
23
24
25
27
12
8
27
11
9
13
19
O
O
O
O
O
O
16
18
15
14
26
OMe
OMe
OH
1
4
10 H
2
3
OH
HO
OH
H
H
5
7
6
HO
HO
H
H
H
1
2
3
29
28
OH
O
OH
O
OH
O
O
O
O
27
H
O
O
HO
H
H
H
6
4
5
OH
O
O
O
H
7
Fig. 1. Structures of 1–7.
OH
OH
O
OH
O
O
O
O
13
O
H
H
30
O
HO
HO
O
H
10
H
H
8
9
OH
O
OH
O
OH
O
O
HO
O
O
H
H
O
O
11
H
H
H
H
13
12
15
O
O
HO
O
O
O
OH
OH
OH
H
H
H
HO
HO
O
16
H
H
14
OH
O
O
O
O
OH
H
H
O
HO
HO
17
H
18
20
19
COOH
Fig. 2. Structures of 8–20.
separated further using an ODS column and HPLC. Major compo-
nents in the active fractions were terpenoids such as 1–20, includ-
ing seven new triterpenes (1–7, Fig. 1).
These results suggested that some triterpenes from A. sibirica
including 10 and 11 have the potential to affect lipid metabolism
as a lipase inhibitor.
In the inhibitory assay with mouse plasma, compounds 10 and
11 showed concentration-dependent inhibition and IC50 values of
0.51 and 0.27 mM, respectively (Table 1). Although IC50 values
were not estimated for their small yields, compounds 6, 9, 12,
and 15 showed inhibitory activity (24.9–63.4%, Table 1) at
0.1 mg/mL. Orlistat (IC50: 0.09 mM) was used as a positive control.
The methanol extract of A. sibirica also had LDL anti-oxidative
activity (Table 1). Five constituents (5, 6, 8–10) were found to have
moderate activity (IC50: 39.42–198.62 lM, Table 1).
The compounds 8–20 (Fig. 2) were identified based on spectro-
scopic data as (5R,20R)-23-hydroxy-8(14?13R)-abeo-17,13-frie-
do-3-oxolanosta-8,14(30),24-triene-26,23-olide (8) (Ou-Yang et al.,

170
M. Handa et al. / Phytochemistry 86 (2013) 168–175
2011), (3
a
,9b,17
a
)-3,23,23-trihydroxy-17-methyl-
c
-lactone-18-
signals (H-27) instead of the exomethylene signals of 1. In the
HMBC spectrum, the methoxy protons and the singlet methyl pro-
tons were long-range coupled with the oxygenated carbon (C-25).
Moreover, the H-24 methylene protons were correlated with C-23,
25, 26, and 27. Another methylene proton (H-22) correlated with
C-20, 21, 23, and 24. These results established that the side-chain
of 2 was as shown in Fig. 1. Absolute stereochemistry at C-25
was determined by the PGME method (Yabuuchi and Kusumi,
2000). Compounds 2a and 2b were (S)- and (R)-PGME amides of
2, and 1H NMR values (2a–2b) suggested that C-25 has the R-con-
figuration (Fig. 3). The NOE experiment showed that the C-3 hydro-
nordammara-7,12,24-trien-26-oic acid (9) (Hasegawa et al.,
1987b; Gao et al., 2008), 7,14,24-mariesatrien-26,23-olide-3
a,
23-diol (10) (Gao et al., 2008), 23-hydroxy-3-oxo-9b-lanosta-
7,24-dien-26,23-olide (11) (Raldugin et al., 1989), 23-hydroxy-
3-oxolanosta-8,24-dien-26,23-olide (12) (Grishko et al., 1998),
3,4-seco-4(28),6,8(14),24-mariesatetraene-26,23-olide-23-hydro-
xy-3-oic acid (13) (Gao et al., 2008), 23-oxo-mariesiic acid A (14)
(Hasegawa et al., 1987b), 23-oxo-mariesiic acid B (15) (Hasegawa
et al., 1987b), (23R,24Z)3-oxo-9b-lanosta-7,24-dien-23-hydroxy-
27-oic acid (16) (Yang et al., 2010), 13,17-friedo-3a-hydroxy-
9b-lanosta-7,12,25(27)-trien-23-oxo-26-oic acid (17) (Yang et al.,
2010), isopseudolarifuroic acid B (18) (Yang and Yue, 2001), dehy-
droabietic acid (19) (Shitara et al., 2007), and bornyl acetate (20)
(Mino et al., 2007).
The novel triterpenes 1–7 were isolated as amorphous powders
with the 1H and 13C NMR spectroscopic data (measured in CDCl3 at
30 °C) shown in Table 2 and the Section 4.
xy group had the a-configuration (H-3 correlated with H-28, 29).
H-7 correlated with H-15 and H-27 correlated with H-24 in the
NOE spectra, supporting this conclusion.
For 3, the 1H and 13C NMR spectra were similar to those of 15
and 17 (Hasegawa et al., 1987b; Yang et al., 2010) except for the
signals of the side-chain. In the HMBC spectrum, the methyl proton
at d 1.17 (3H, s, H-30) was correlated with olefinic carbon reso-
nances at d 145.9 (C-8) and 156.1 (C-13). These results suggested
that the A–D ring moiety was 7,12-dien-3-ol. Signals of the side-
chain in the spectra were similar to those of 2. In the HMBC spec-
trum, the methoxy proton resonance (d 3.30, 3H, s) and the singlet
methyl proton signal (d 1.45, s, H-27) were long-range coupled
with an oxygenated carbon (d 77.7, C-25) as in the case of 2. Com-
pound 3 was an isomer of 2 as shown in Fig. 1.
Compound 1 was deduced to have the molecular formula
C
30H44O4 based on HREIMS (m/z 468.3243, calcd for C30H44O4,
468.3241). The 13C NMR spectrum of its A–D ring was similar to
that of 7,14,22Z,24-mariesatetraen-26,23-olide-3- -ol (Gao et al.,
a
2008). Typical signals in the 1H NMR spectrum, five singlet methyl
proton resonances (d 0.85, 0.86, 0.94, 0.97, 0.99), a doublet methyl
proton signal at d 0.85 (3H, d, J = 7.0 Hz, H-21), and four olefinic
proton resonances at d 5.18 (1H, dd, J = 3.0, 1.5 Hz, H-15), 5.57
(1H, dd, J = 6.0, 2.0 Hz, H-7), 5.75 (1H, br s, H-27), and 6.45 (1H,
br s, H-27), were detected. These singlet methyl proton signals
were assigned to the methyls in the A–D ring (Table 1) based on
analysis of 2D-NMR spectra. In the HMBC spectrum, the H-7 proton
was long-range coupled with d 38.0 (C-5), 53.0 (C-9), and 152.9
(C-14), and the H-15 proton was long-range coupled with d 45.0
(C-16), 50.5 (C-17), 51.7 (C-13), and 136.7 (C-8). Moreover, two
methyl proton resonances at d 0.94 and 0.99 (H-29 and 28) corre-
lated with an oxygenated carbon C-3 (d 76.8), a quaternary carbon
C-4 (d 37.2), and a methine carbon C-5. These results established
that the A–D ring moiety was a 7,14-dien-3-ol. Signals of the
side-chain were very similar to those of methyl isofirmanoate
(Hasegawa et al., 1987a) and 17. Two carbonyl carbons at d 170.4
(carboxyl, C-26) and 207.2 (ketone, C-23) were correlated with
H-24 methylene proton resonances at d 3.34 (1H, d, J = 17.0 Hz)
and 3.43 (1H, d, J = 17.0 Hz), the terminal olefinic methylene pro-
tons [H-27, d 5.75 (br s) and 6.45 (br s)] were correlated with C-
26, and the H-21 methyl proton signal was correlated with C-17
(d 50.5), C-20 (d 33.6), and C-22 (d 46.7), in the HMBC spectrum.
The NOE experiment showed that the hydroxy group at C-3 has
Compounds 4–7 have the same side-chain moiety, a c-lactone
ring with a lactol as shown in Fig. 1. Compounds 8–13 also have
the side chain moiety. Some signals in the 1H NMR spectra of these
compounds showed broad weak peaks or divided peaks. Moreover,
more resonances were observed in the 13C NMR spectra than ex-
pected from the MS and analyses. These spectra were attributed
to tautomeric mixtures from the
c-lactone moiety (Gao et al.,
2008). Main signals are listed in Table. 1 and minor tautomeric
peaks are shown in Section 4. The chemical structure was deter-
mined using the main signals.
The molecular formula C30H42O4 for 4 and 5 was determined by
HREIMS (4: m/z 466.3087, 5: m/z 466.3076, calcd for C30H42O4,
466.3084).
For 4, the resonances of the A–D ring in the 13C NMR spectrum
were almost superimposable onto those of methyl 3,23-dioxo-
mariesiate A (Hasegawa et al., 1987b), except for the side chain sig-
nals. A carbonyl carbon resonance at d 216.9 was assigned to C-3
by HMBC correlations with H-1, 2, 28, and 29. An olefinic proton
at d 5.61 (1H, br s, H-7) and H-28, 29 were correlated with C-5 (d
45.0) in the HMBC spectrum, and H-7 was correlated with H-15
(d 5.22, 1H, br s) in the NOE spectrum. These results suggested that
the A–D ring moiety of 4 was the same as that of methyl 3,23-
dioxo-mariesiates A. Signals of 1H, 13C NMR spectra for the
side-chain moiety were almost superimposable onto those of
the
a-configuration (H-3 correlated with H-28 and -29) (Gao
et al., 2008). These results suggested that the structure of 1 was
as shown in Fig. 1. H-7 was correlated with H-15 and H-24 corre-
lated with H-27 correlated with each other in the NOE spectra sup-
porting this conclusion.
7,14,24-mariesatrien-26,23-olide-3a,23-diol (Gao et al., 2008).
Oxygenated carbon (d 106.3, C-23), olefinic carbon (d 147.9 and
131.6, C-24 and 25), carbonyl carbon (d 171.7, C-26), and methyl
proton (d 1.92, s, H-27) resonances were typical for a c-lactone ring
The 1H NMR, 13C NMR, and MS spectra of 2 and 3 resembled
each other. The molecular formula C31H48O5 for 2 and 3 was
determined by HRFABMS (2, m/z 523.3390; 3, 523.3412, calcd for
C
31H48O5Na, 523.3400).
For 2, the 1H and 13C NMR spectra were similar to those of 1 ex-
+0.07
+0.07
+0.08
+0.10
+0.04
−0.01
cept for the signals of the side-chain. The 2D-NMR and NOE spectra
showed that the A–D ring was the same as that of 1. A singlet
+0.07
CO-PGME
−0.10
O
C
0
O
O
methyl (d 1.47, 3H, H-27),
a doublet methyl (d 0.83, 3H,
+0.08
+0.04
OMe
NH
0
H3C
−δ
C
CH3
CH2
J = 7.0 Hz, H-21), a methoxy (d 3.32, 3H, s), and two sets of methy-
lene proton [d 2.19 (1H, overlapped, H-22), 2.46 (1H, d, J = 16.5 Hz,
H-22), 2.93 (1H, d, J = 16.5 Hz, H-24), and 2.98 (d, J = 16.5 Hz, H-
24)] resonances were observed in the 1H NMR spectrum. A car-
boxyl carbon (d 174.9, C-26) and a ketone (d 207.5, C-23) signal
were detected in the 13C NMR spectrum. In the spectra, 2 had an
oxygenated quaternary carbon (d 77.9, C-25) signal and the methyl
H
+δ
+0.03
OMe
HO
H
COOMe
0
0
Δδ = δ (S) − δ (R)
Fig. 3. Differences in chemical shifts of 1H NMR (2a–2b) used to the determine
absolute stereochemistry of C-25 for 2.

M. Handa et al. / Phytochemistry 86 (2013) 168–175
171
Table 1
singlet proton resonance in the NOE spectrum. The singlet proton
signal at d 1.09 (s, H-19) correlated with C-1 (d 30.1), C-9
(d 149.6), and C-10 (d 40.1). These results showed that C-9–11
was a double bond. In the NOE spectrum, the H-3 (d 3.44, m) cor-
related with H-28 (d 0.99, s) and H-29 (d 0.90, s), suggesting that
Lipase inhibitory activity and LDL anti-oxidative activity of methanol extract and
constituents of Abies sibirica.
Compound
Lipase inhibitory activity
LDL anti-oxidative activity
a
b
%
IC50 (mM)
%
IC50 (lM)
the C-3 hydroxy group has an a-configuration. Thus, the structure
of 6 was determined as shown in Fig. 1.
1
2
NO
NO
4
5
6
7
8
9
10
11
12
13
14
15
16
19
20
NO
NO
43.0 1.5
NO
NO
24.9 3.4
65.6 2.0
75.6 0.9
63.4 5.2
NO
NO
32.0 2.2
NO
The molecular formula C30H42O4 for 7 was determined by HRE-
IMS (m/z 466.3070, calcd for C30H42O4, 466.3084), and 7 is an iso-
mer of 8. The 1H and 13C NMR spectra of 8 were similar to those of
(24Z)-8(14–13)-abeo-17,13-friedolanosta-8,14(30),24-triene-3,23-
dion-26-oic acids (Raldugin et al., 1992) except for signals of the
side chain moiety. For 7, resonances of the side chain were typical
25.2 4.9
60.3 5.0
79.0 0.6
198.62
55.32
64.11
81.3 0.7
92.2 2.1
NO
49.14
39.42
0.51
0.27
ND
of a c-lactone ring the same as in 4–6. Its NMR spectra were similar
to those of 8 except for resonances of the D-ring. Compound 7 has a
vinylic methyl group (d 1.46, s, H-30) and an olefinic proton (d 5.23,
br s, H-15) instead of the C14–30 exomethylene group of 8. In the
HMBC spectrum, the H-30 signal correlated with a quaternary car-
bon resonance at d 71.1 (C-13), two olefinic carbon signals at d
144.7 (C-14) and 123.0 (C-15), and a methylene carbon resonance
at d 40.5. These results suggested that C14–15 was a double bond
in 7, instead of C14–30 in 8, as shown in Fig. 1.
NO
NO
NO
methanol ext.
orlistat
probucol
39.9 1.0c
0.39 mg/mL
0.09
64.1 5.9d
0.02 mg/mL
10.53
Concentration of test samples in reaction solution, a0.1 mg/mL, b0.05 mg/mL,
c0.3 mg/mL, d0.025 mg/mL. Each value represents the mean (n = 3). Data are
expressed as means standard error (SE). NO, not observed. ND, not determined.
The A–D ring skeletons of 1–7 were considered to be the results
of biosynthesis of the lanostane skeleton by enzymic dehydrogena-
tion of H-17 (Hasegawa et al., 1987a,b; Kuroyanagi et al., 2000).
The configurations at C-17, C-20 (1–5, 7) and C-13, C-20 (6) were
supposed to be as shown in Fig. 1.
having a lactol structure (Gao et al., 2008; Li et al., 2009). In the
HMBC spectrum, an olefinic proton at d 6.86 (br s, H-24) was
long-range coupled with C-23, 25–27. In the NOE spectrum, a
methyl proton (H-27) was correlated with the olefinic proton (H-
24). A doublet methyl proton (d 0.96, 3H, J = 7.0 Hz, H-21) and cor-
related carbon signals [d 51.7 (C-17), 33.3 (C-20), 41.2 (C-22)] in
the HMBC spectrum were resonances of the side-chain moiety.
These results suggested that the side-chain moiety was an ali-
phatic chain with a lactone ring as shown in Fig. 1.
3. Conclusion
From the leaves of A. sibirica, seven new triterpenes (1–7) and
13 known terpenoids (8–20) were isolated. Compounds 4–7 have
a c-lactone ring with a lactol. Compounds 10 and 11 had IC50 val-
ues of 0.51 and 0.27 mM respectively for the inhibition of lipase
activity in isolated mouse plasma in vitro using the BALB method.
Some other triterpenes (6, 9, 12, and 15) also showed inhibitory
activity. Five of these triterpenes (5, 6, 8–10) were LDL oxidation
inhibitors.
In the 1H and 13C NMR spectra of 5, signals for the A–D ring, four
olefinic carbon resonances, and only one olefinic proton at d 5.33
(br s, H-15) were observed. Signals for the A and D ring and side-
chain moieties were similar to those of 4, and the 2D-NMR spectra
suggested the same partial structure as 4. In the HMBC spectrum,
H-15 was long-range coupled with the olefinic carbon at d 123.8
(C-8), and H-19 (d 1.12, s) correlated with another olefinic carbon
at d 140.1. These correlations suggested that C-8,9 was a double
bond. Thus, the chemical structures of 4 and 5 were determined
as shown in Fig. 1.
4. Experimental
4.1. General
Optical rotations were measured on a Jasco P-2300 polarimeter.
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 (measured in
CDCl3). Inverse-detected heteronuclear correlations were measured
Compound 6 was deduced to have the molecular formula
C
30H44O4 by HREIMS (m/z 468.3237, calcd for C30H44O4,
468.3241). In the 1H and 13C NMR spectra, signals of the side chain
showed that 6 has a -lactone ring the same as 4 and 5. The molec-
c
1
ular formula suggested that 6 was an isomer of 9 and 10. However,
the olefinic and singlet protons of the A–D ring in 6 differed from
those in 9 and 10. A singlet proton resonance at d 0.88 (s, H-18)
was long-range coupled with carbons at d 30.8 (C-12), 51.6 (C-
13), 46.8 (C-14), and 157.1 (C-17). Another singlet proton signal
at d 0.82 (s, H-30) was long-range coupled with C-13, 14, and 15
(d 40.8). So these methyl groups face each other across the C-13
and 14 of the C and D rings. A doublet methyl proton signal of
the side-chain at d 1.10 (d, J = 7.0 Hz, H-21) and an olefinic proton
signal at d 5.44 (m, H-16) correlated with C-17 in the HMBC spec-
trum, and H-16 correlated with H-15 (d 1.70–2.30, overlapped) in
the NOE spectrum. These correlations suggested that C16–17 of
the D-ring is a double bond. Another olefinic proton resonance at
d 5.33 was assigned to H-11, because it was long-range coupled
with C-10, 12, 13 in the HMBC spectrum, and correlated with H-
12 proton signals at d 1.50–1.90 (overlapped) and a H-19 methyl
using HMQC (optimized for JC–H = 145 Hz) and HMBC (optimized
n
for JC–H = 8 Hz) pulse sequences with a pulsed field gradient.
HRFABMS and HREIMS data were obtained on a Jeol JMS700 mass
spectrometer, using either a m-nitrobenzyl alcohol or a glycerol ma-
trix. Preparative Yamazen Cartridge column chromatography (CC)
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; mobile phase, CH3CN–0.2%CF3CO2H (80:20)],
Cosmosil 5C18–AR II [Nacalai Tesque, 20 ꢀ 250 mm; mobile phase,
CH3CN–H2O (80:20) or (85:15) or (90:10)], Cosmosil 5PE-MS [Naca-
lai Tesque, 20 ꢀ 250 mm; solvent, CH3CN–0.2%CF3CO2H (70:30) or
MeOH–H2O (90:10)], Develosil C30-UG-5 [Nomura Chemical,
20 ꢀ 250 mm; solvent, CH3CN–0.2%CF3CO2H (85:15) or (90:10) or
(95:5)] and Mightysil RP-18 GP [Kanto Chemical, 10 ꢀ 250 mm; sol-
vent, CH3CN–0.2%CF3CO2H (85:15)]. 5,50-Dithio-bis(2-nitrobenzoic
acid) and phenylmethylsulfonyl fluoride were purchased from

172
M. Handa et al. / Phytochemistry 86 (2013) 168–175
Nacalai Tesque Inc. (Kyoto, Japan), while 2,3-dimer-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).
4.7. 23-Hydroxy-3-oxomariesia-7,14,24-trien-26,23-olide (4)
Colorless amorphous powder; ½a D21
ꢁ
+35.1 (c 1.99, MeOH); HREIMS
m/z 466.3087 [M]+ (calcd for C30H42O4, 466.3084); for main peaks of
1H NMR and 13C NMR (CDCl3) spectroscopic data, see Table 2;
Observable peaks of another minor tautomer in 1H-NMR spectrum
(CDCl3) d 6.88 (1H, br s, H-27); Observable minor peaks of 13C NMR
spectrum (CDCl3) d 120.0 (C-7), 33.4 (C-20), 17.5 (C-21), 40.8
(C-22), 106.7 (C-23), 147.7 (C-24), 131.5 (C-25), 171.6 (C-26).
4.2. Plant material
A. sibirica was collected in July 2008 in Mongolia. A voucher
specimen has been deposited at the herbarium of Tohoku Pharma-
ceutical University, No. 20100112. The plant was identified by Dr.
C. Sanchir, Institute of Botany, Mongolian Academy of Sciences.
4.8. 23-Hydroxy-3-oxomariesia-8(9),14,24-trien-26,23-olide (5)
Colorless amorphous powder; ½a D21
ꢂ2.5 (c 1.14, MeOH); HRE-
ꢁ
4.3. Extraction and isolation
IMS m/z 466.3076 [M]+ (calcd for C30H42O4, 466.3084); UV (MeOH)
kmax nm (loge
): 249 (4.88); for 1H NMR and 13C NMR (CDCl3) spec-
Dried leaves (150 g) of A. sibirica were extracted with MeOH (3 L
2ꢀ) at room temperature for a month’s duration. The combined ex-
tract (37.6 g) was suspended in H2O (1.5 L), and extracted with
Et2O (1.0 L 3ꢀ). The resulting Et2O extract (12.3 g) was passed
through a silica gel column [Wakogel C-200, Wako Pure Chemical
Industries Ltd., Osaka, Japan, 30 g; solvent, CHCl3, CHCl3–MeOH
(99:1), (98:2), and MeOH] yielding 9 fractions (frs. 1A-1I) and 20
(1.08 g). Fr. 1B [CHCl3–MeOH (99:1) (2.54 g)] was applied to a re-
versed-phase column using ODS (Cosmosil 140C18-OPN, Nacalai
Tesque, Osaka, Japan, 150 g) and eluted with MeOH–water (8:2),
MeOH, and EtOAc, respectively. The MeOH–H2O (8:2) fraction
(1.8 g) was next subjected to preparative HPLC, yielding com-
pounds 11 (11.7 mg), 12 (16.1 mg), and 19 (38.0 mg). Subse-
quently, dried leaves (650 g) of A. sibirica were extracted with
MeOH (7 L 2ꢀ) at room temperature for a month’s duration. The
resulting extract (164.9 g) was suspended in H2O (1.5 L), and ex-
tracted with Et2O (1.0 L 3ꢀ). The Et2O extract (68.9 g) was passed
through a silica gel column [Wakogel C-200, Wako Pure Chemical
Industries Ltd., Osaka, Japan, 200 g; solvent, CHCl3–MeOH
(100:0) ? (0:100) gradient] yielding 7 fractions (frs. 2A–2G). Frs.
2D–2F [CHCl3–MeOH (99:1)–(9:1) (26.8 g)] were applied to a re-
versed-phase column using ODS (Cosmosil 140C18-OPN, Nacalai
Tesque, Osaka, Japan, 150 g) and eluted with MeOH–H2O (8:2),
MeOH, and EtOAc. The MeOH–H2O (8:2) fraction (13.9 g) was sub-
jected to preparative HPLC, yielding compounds 1 (7.7 mg), 2
(7.5 mg), 3 (3.1 mg), 4 (37.9 mg), 5 (21.2 mg), 6 (14.6 mg), 7
(11.1 mg), 8 (1.7 mg), 9 (22.0 mg), 10 (88.1 mg), 11 (170.2 mg),
12 (33.0 mg), 13 (50.1 mg), 14 (41.0 mg), 15 (16.8 mg), 16
(27.9 mg), 17 (1.6 mg), and 18 (0.3 mg), repectively.
troscopic data, see Table 2; Observable peaks of another minor tau-
tomer in 1H NMR spectrum (CDCl3) d 0.86 (3H, s, H-18), 0.97 (3H, d,
J = 7.0 Hz, H-21), 6.90 (1H, m, H-24), 1.94 (3H, s, H-27); Observable
minor peaks of 13C NMR spectrum (CDCl3) d 35.0 (C-2), 29.8 (C-10),
45.1 (C-16), 50.8 (C-17), 17.3 (C-21), 40.8 (C-22), 106.7 (C-23),
147.6 (C-24), 131.7 (C-25), 171.5 (C-26), 26.6 (C-28).
4.9. 3a,23-Dihydroxylanosta-9(11),16,24-trien-26,23-olide (6)
Colorless amorphous powder; ½a D21
ꢁ
+19.4 (c 0.72, MeOH); HRE-
IMS m/z 468.3237 [M]+ (calcd for C30H44O4, 468.3241); UV (MeOH)
kmax nm (loge
): 246 (4.70); for 1H NMR and 13C NMR (CDCl3) spec-
troscopic data, see Table 2; Observable peaks of another minor tau-
tomer in 1H NMR spectrum (CDCl3) d 1.33 (1H, m, H-5), 5.31 (1H,
m, H-16), 0.71 (3H, s, H-18), 1.08 (3H, s, H-19), 1.11 (3H, d,
J = 7.0 Hz, H-21), 1.88 (3H, d, J = 1.5 Hz, H-27), 0.81 (3H, s, H-30);
Observable minor peaks of 13C NMR spectrum (CDCl3) d 31.3
(C-12), 51.2 (C-13), 46.9 (C-14), 120.5 (C-16), 156.3 (C-17), 19.7
(C-18), 27.8 (C-20), 22.8 (C-21), 43.5 (C-22), 147.1 (C-24), 131.5
(C-25), 10.5 (C-27).
4.10. 23-Hydroxy-8(14?13)-abeo-17,13-fried-3-oxolanosta-
8,14(15),24-triene-26,23- olide (7)
Colorless amorphous powder; [
a
] ½a 2D1
ꢂ64.4 (c 2.21, MeOH);
ꢁ
HREIMS m/z 466.3070 [M]+ (calcd for C30H42O4, 466.3084); UV
(MeOH) kmax nm (loge
): 246 (4.66); for 1H NMR and 13C NMR
(CDCl3) spectroscopic data, see Table 2; Observable peaks of an-
other minor tautomer in 1H NMR spectrum (CDCl3) d 0.93 (3H, s,
H-18), 6.84 (1H, br s, H-24); Observable minor peaks of 13C NMR
spectrum (CDCl3) d 144.6 (C-14), 50.7 (C-17), 22.1 (C-18), 37.6
(C-20), 20.9 (C-21), 41.6 (C-22), 107.7 (C-23), 148.5 (C-24), 131.1
(C-25), 172.8 (C-26).
4.4. 3a-Hydroxymariesia-7,14,25(27)-trien-23-oxo-26-oic acid (1)
Colorless amorphous powder; ½a D23
ꢁ
+30.7 (c 0.56, MeOH); HRE-
IMS m/z 468.3243 [M]+ (calcd for C30H44O4, 468.3241); for 1H NMR
and 13C NMR (CDCl3) spectroscopic data, see Table 2.
4.11. (S)- and (R)- PGME amides of 2
4.5. (25R)-3
oic acid (2)
a
-Hydroxy-25-methoxy-23-oxo-mariesia-7,14-dien-26-
To 2 (2.0 mg each) in N,N-dimethylformamide (0.5 mL) was
added either (S)-PGME or (R)-PGME (5.0 mg). Benzotriazotriaz-
ole-1-yl-oxy-tris-pyrrolidinophoniumhexafluorophosphate (10 mg),
1-hydroxybenzotriazole (4.0 mg) and N-methylmorpholine
Colorless amorphous powder; ½a D22
ꢁ
+20.0 (c 0.66, MeOH);
HRFABMS m/z 523.3390 [M+Na]+ (calcd for C31H48O5Na,
523.3400); for 1H NMR and 13C NMR (CDCl3) spectroscopic data,
see Table 2.
(30 lL) were then added. The mixtures were stirred for 15 h at
room temperature, and subjected to HPLC [columns, Mightysil
RP18GP, 10 ꢀ 250 mm; solvents CH3CN–0.2%CF3CO2H (90:10);
detector, UV 210 nm], yielding the (S)-amide of 2 (0.7 mg) and
(R)-PGME amide of 2 (0.7 mg), respectively.
4.6. (25R)-3a-Hydroxy-25-methoxy-23-oxo-mariesia-7,12-dien-26-
oic acid (3)
4.12. (S)-PGME amide of 2 (2a)
Colorless amorphous powder; ½a D21
ꢂ103.1 (c 0.32, MeOH);
ꢁ
HRFABMS m/z 523.3412 [M+Na]+ (calcd for C31H48O5Na,
Colorless amorphous powder; FAB-MS m/z 648 [M+H]+, 670
[M+Na]+, 1H NMR (400 MHz, CDCl3) d 5.57 (1H, m, H-7), 5.18
(1H, m, H-15), 0.87 (3H, s, H-18), 0.97 (3H, s, H-19), 2.42 (1H, m,
523.3400); UV (MeOH) kmax nm (log
e
): 245 (4.75); for 1H NMR
and 13C NMR (CDCl3) spectroscopic data, see Table 2.

Table 2
NMR spectroscopic data (400 MHz) for compounds 1–7, 9.
Position
1
2
3
4 (main)
dH (J in Hz)
dc
HMBC (H to
C)
NOE (H to dh (J in Hz)
H)
dc
HMBC (H to
C)
NOE (H to dh (J in Hz)
H)
dc
HMBC (H to NOE (H to dh (J in Hz)
dc
HMBC (H to NOE (H to
C)
H)
C)
H)
1
2
3
0.95a
2.04, m
1.63, m
1.98a
28.8
25.3
76.8
0.94a
1.98a
28.7
25.3
1.02, m
1.96a
29.3
25.3
77.2
1.60a
35.5
35.0
2, 3, 5, 9
2
1.82a
3,19
1.62a
19
1.63, m
1, 3
1, 5
19
2.29, m
2.74, m
1, 3
1,19
2.00a
3.47, br s
1.94a
3.47, t (3.0)
1, 2, 5, 28,
29
28,29
76.4a 4, 5, 28, 29
28,29
3.47, t (3.0)
2,28,29
216.9
4
5
37.2
38.0
37.2
36.8
38.1
47.3
45.0
1.56, dd
(11.5,5.0)
1.88–2.01a
5.57, dd
(6.0,2.0)
1.55, dd
(11.5,5.0)
1.85–2.05a
5.57, br d
(5.0)
38.0
23.0
9
1.45a
1.52a
1, 4, 6, 10
5, 7, 8
6
7
23.1
1.94a
5.63, m
23.2
118.8
5, 7
5,7
6
1.90–2.10a
5.61, br s
23.9
120.1 5, 6, 9
5,7
6,15
120.7 5, 6, 9, 14
6,15
120.8 5, 6, 8, 9, 14 6,15
8
9
10
11
136.7
136.7
145.9
50.9
34.8
28.1
136.8
1.42
53.0
34.8
25.4
7, 8, 14
1.41a
53.0
34.8
25.2
8, 11, 14
9
1.41, m
5.12a
52.4
34.6
25.1
7, 8, 11
1.42a
1.41a
1.80a
1.45a
1.80, m
2.24a
5.45, dd
(8.5,2.0)
1.36a
1.65a
1.50a
1.80–1.90a
1.44a
12
33.7
33.5
122.4 11, 14, 17
33.1
1.82a
1.82a
2.05a
13
14
15
51.7
152.9
51.7
152.8
156.1
50.0
37.0
51.5
152.3
115.5
5.18, dd
(3.0,1.5)
1.91, dd
(16.0,3.0)
2.20, br d
(16.0)
115.0 8, 13, 14, 16, 7,16
17
45.0
5.18, brs
1.89a
115.0 8, 13, 14, 16, 7,16,18
1.40–1.47a
1.40–1.47a
1.90a
5.22, br s
1.90a
7,16
17
16
14, 15
45.0
38.3
45.0
2.20a
2.17, br d
(15.5)
17
18
50.5
19.3
50.0
19.3
46.3
24.7
51.7
16.7
0.88, s
13, 16, 17,
20
0.87, s
16, 20
15,16,20
20,22
0.91
13, 16, 17
1, 5, 9, 10
12,16
0.88, s
13, 16, 17
1, 5, 9, 10
19
20
21
22
0.97, s
2.46, m
0.85, d (7.0)
2.53, br d
(16.5)
22.3
33.6
16.6
46.7
0.97, s
22.7
33.9
16.6
47.6
1, 9, 10
0.94, s
22.1
38.4
15.7
47.2
1.13, s
21.7
33.3
17.4
41.2
2
2.42a
2.20a
1.55–1.70a
0.96, d (7.0)
2.04, m
17, 20, 22
17, 20, 21,
23
0.83, d (7.0)
17, 20, 22
20, 21, 23,
24
0.87, d (6.0)
17, 20, 22
20, 21, 23
17, 20, 22
20, 23, 24
24
2.19a
2.18a
2.29 dd
2.46, d (16.5)
2.76, d (13.5)
(16.5,11.0)
23
24
207.2
46.0 22, 23, 25, 26,
27
207.5
49.5
208.0
49.0
106.3
147.9 23, 25, 26,
27
3.34, d (17.0)
3.43, d (17.0)
27
24
2.93, d (16.5)
2.98, d (16.5)
23, 25, 26,
27
27
2.88–2.92
2.98–3.02
23, 25, 26,
27
6.86, br s
1.92, s
22,27
24
25
26
27
133.8
170.4
130.7 24, 25, 26
77.9
174.9
21.3
77.7
174.3
21.4
131.6
171.7
10.4
5.75, br s
6.45, br s
0.99, s
0.94, s
0.86, s
1.47, s
24, 25, 26
24,OMe
1.45
24, 25, 26
OMe
23, 25, 26
28
29
30
28.2
23.0
17.1
3, 4, 5, 29
3, 4, 5, 28
12, 13, 14,
17
3,5
3
0.99, s
0.94, s
0.85, s
28.2
23.1
17.1
3, 4, 5, 29
3, 4, 5, 28
13,17
3,5
3
0.98, s
0.93, s
1.17
28.2
23.0
25.9
3, 4, 5, 29
3, 4, 5, 28
8, 13, 14, 15
1.10, s
1.10, s
0.88, s
25.5
22.6
19.0
3, 4, 5, 29
3, 4, 5, 28
12, 13, 17
5
2
OMe
3.32, s
51.6
25
3.30-
51.5
25
(continued on next page)

Table 2 (continued)
Position 5 (main)
6 (main)
7 (main)
9 (main)
dH (J in Hz)
dC
HMBC (H to C)
NOE (H to
H)
dH (J in Hz) dC
HMBC (H to
C)
NOE (H to
H)
dH (J in Hz) dC
HMBC (H to
C)
NOE (H to
H)
dH (J in Hz)
dC
HMBC (H to
C)
1
2
1.60a
34.9
33.4
1.30–2.40a
30.1
1.60a
36.2
1.96a
29.3
24.3
10, 19
1.90a
1.98a
2.45a
2.57, m
1, 3
1.70, m
2.03, m
3.44, br s
25.7
1, 3
1
2.56, m
34.6
1, 3, 10
1
1.63a
1.96a
3
4
5
6
217.8
47.3
51.1
22.8
76.3
37.9
46.7
22.0
28,29
219.3
47.4
51.3
3.47, br s
77.2
37.0
37.9
22.9
1, 4, 29
1.68, m
4, 6, 7, 10, 28, 29
8, 9
1.36, m
1.62a
4
1.48a
2.00–2.20a
1.30–1.50a
5, 7
30
1.45–1.80a 20.3
1.42a
5, 7, 8
5, 7, 8
1.96a
7
1.60–1.70a
19.5
2.20–2.50a
2.38, m
28.0
1.73a–
2.00a
2.00a
26.2
5.63, br s
118.7 5, 9
8
9
10
11
123.8
140.1
37.7
40.0
149.6
40.1
137.5
144.1
36.0
145.7
50.6
34.8
28.4
1.70a
34.4
8, 9
5.33, m
114.0 10, 12, 13
12
15
2.00a
26.2
2.15–2.30a
5.57, m
2.45a
12
13
14
15
16
1.50–1.80a
29.9
47.9
148.1
116.9 8, 13, 16, 17
45.2
9, 11, 13, 17
1.50–1.90a
30.8
51.6
46.8
40.8
2.05–2.40a 29.2
123.0 9, 11, 17
71.1
144.7
156.6
50.2
36.8
38.4
5.33, br s
1.96, m
2.30, d
(16.0)
1.70–2.30a
5.44, m
5.23, br s
123.0 13, 16, 17, 30 30
1.30–1.70a
1.30–1.70a
14, 15, 17, 18
121.5 13, 15, 17
1.90–2.15a 40.5
17
18
19
20
21
50.7
17.1
18.7
33.1
17.5
157.1
50.8
46.8
0.78, s
13, 16, 17, 20
1, 9, 10
0.88, s
1.09, s
1.60a
19.9
21.1
27.7
23.3
12, 13, 14, 15 8,15
0.91, s
1.06, s
1.62a
21.9
19.5
37.2
20.6
13, 16, 17, 20 24
1, 5, 9, 10
23
0.93, s
25.8
22.1
37.9
18.0
13, 16, 17
1, 9, 10
1.12, s
1
1, 10
1,11
0.93, s
1.10a
1.30–1.70a
1.01, d (7.0)
1.00, d (7.0)
17, 20, 22
24
1.10, d
(7.0)
17, 20, 22
22
0.83, br s
17, 20, 22
15,24
17, 20, 22
22
2.17a
41.2
17, 20, 21, 23,
24
2.25–2.45a
43.0
1.60a
41.7
17, 20, 23
1.69, dd (14.0,
8.5)
2.44, br d (14.0)
40.5
17, 20, 21, 23
17, 20, 21, 23
1.85–2.10a
6.79, m
2.16a
23
24
25
26
27
106.2
147.7 23, 25, 26, 27
131.8
171.6
10.5
105.8
107.4
106.8
147.2 23, 25, 26
131.9
171.8
10.4
6.87, m
1.94, s
27
24
147.4 23, 25, 26, 27 27
131.3
171.8
6.86, br s
148.4 23, 25, 26, 27 18, 22, 27
131.2
172.9
6.71, s
1.89, s
24, 25, 26
1.90, d
(6.5)
10.4
24, 25, 26
16,24
1.92, s
10.3
24, 25, 26
24
24, 25, 26
28
29
30
1.11, s
1.08, s
0.86, s
26.7
21.3
15.7
3, 4, 5, 29
3, 4, 5, 28
12, 13, 14, 17
0.99, s
0.90, s
0.82, s
28.4
22.6
19.9
3, 4, 5, 29
3, 4, 5, 28
13, 14, 15
3,5
3
1.10, s
1.08, s
1.46, s
27.0
21.1
13.5
3, 5, 29
3, 5, 28
13, 14, 15
5
5
15
0.98, s
0.93, s
1.12, s
28.1
23.2
25.4
3, 4, 5, 29
3, 4, 5, 28
8, 14, 15
a Overlapped.
b Interchangeable.

M. Handa et al. / Phytochemistry 86 (2013) 168–175
175
H-20), 0.83 (3H, d, J = 7.0 Hz, H-21), 2.22 (1H, m, H-22), 2.46 (1H,
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0.84 (3H, s, H-30), 3.30 (3H, s, H-OMe).
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Colorless amorphous powder; FAB-MS m/z 648 [M+H]+, 670
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(1H, m, H-15), 0.79 (3H, s, H-18), 0.97 (3H, s, H-19), 2.36 (1H, m,
H-20), 0.76 (3H, d, J = 7.0 Hz, H-21), 2.14 (1H, m, H-22), 2.36 (over-
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enzyme in the absence of the test solution and B, that in the pres-
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activity of the H2O layer extract, ether layer extract, and fractions
obtained by column chromatography was 0.3 mg/mL.
4.15. Human LDL anti-oxidative activity
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The assay was carried out according to the method of Hirano
et al. (1997). The sample adjusted to each final reaction concentra-
tion in EtOH or DMSO was added to a 4.18 ꢀ 10ꢂ5 mM Human LDL
saline solution. After a 20 mM 2,2-azobis (4-methylvaleronitrile)
EtOH was added as an initiator, the change in absorbance of
234 nm was measured at 5 min intervals at 37 °C for 200 min.
LDL oxidation was shown as the reaction speed (DAbs/min) which
is the increase in absorbance from 100 min to 200 min after the
initiator was added. LDL oxidative inhibition was calculated using
the following equation; I (%) = (
min ꢀ 100, where Acon is the reaction speed without the test sam-
ple and Asam is the reaction speed with the sample. The increasing
D
Acon/min ꢂ
DAsam/min)/DAcon/
D
D
reaction speed of a blank (without LDL) was subtracted from each
reaction speed.
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Acknowledgments
We thank Mr. S. Sato and Mr. T. Matsuki of Tohoku Pharmaceu-
tical University, for assisting with the MS measurements and Ms. B.
Odonbyar of National University of Mongolia, for searching the liter-
ature on traditional medicines in Mongolia.