Bioactive constituents of Cirsium japonicum var. australe

Article abstract of DOI:10.1021/np500233t

Cirsium japonicum var. australe, used as a folk medicine in Taiwan, has been employed traditionally in the treatment of diabetes and inflammatory symptoms. Bioactivity-guided fractionation of its ethanolic extract, utilizing centrifugal partition chromatography monitored by DPPH-TLC analysis, led to the isolation of three new acetylenic phenylacrylic acid esters (1-3) and two new polyacetylenes (4 and 5), together with seven known compounds (6-12). The structures of 1-5 were elucidated by spectroscopic methods including 1D and 2D NMR techniques. The absolute configurations of 4 and 7 were determined utilizing Mosher's method and ECD/CD experiments. The DPPH scavenging activity of the constituents isolated from the C. japonicum var. australe ethanolic extract was evaluated. The potential antidiabetic activity of some of the isolates was evaluated using in vitro cellular glucose uptake and oil red staining assays.

Full text of DOI:10.1021/np500233t

Article
pubs.acs.org/jnp
Bioactive Constituents of Cirsium japonicum var. australe
Wan-Chun Lai,†,‡,# Yang-Chang Wu,§,⊥,∥,# Balazs Danko,†,‡ Yuan-Bin Cheng,† Tusty-Jiuan Hsieh,†
́
́
Chi-Ting Hsieh,† Yu-Chi Tsai,† Mohamed El-Shazly,†,∇ Ana Martins,△,□ Judit Hohmann,‡
Attila Hunyadi,*,‡ and Fang-Rong Chang*,†,○
†Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
‡Institute of Pharmacognosy, University of Szeged, H-6720 Szeged, Hungary
§School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan
⊥Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan
∥Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan
∇Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Organization of
African Unity Street, Abassia, Cairo 11566, Egypt
△Department of Medical Microbiology and Immunobiology, University of Szeged, H-6720 Szeged, Hungary
□Unidade de Parasitologia e Microbiologia Med
1349-008 Lisbon, Portugal
́
ica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,
○Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
S
* Supporting Information
ABSTRACT: Cirsium japonicum var. australe, used as a folk
medicine in Taiwan, has been employed traditionally in the
treatment of diabetes and inflammatory symptoms. Bioactivity-
guided fractionation of its ethanolic extract, utilizing centrifugal
partition chromatography monitored by DPPH-TLC analysis,
led to the isolation of three new acetylenic phenylacrylic acid
esters (1−3) and two new polyacetylenes (4 and 5), together
with seven known compounds (6−12). The structures of 1−5
were elucidated by spectroscopic methods including 1D and
2D NMR techniques. The absolute configurations of 4 and 7
were determined utilizing Mosher’s method and ECD/CD
experiments. The DPPH scavenging activity of the constitu-
ents isolated from the C. japonicum var. australe ethanolic extract was evaluated. The potential antidiabetic activity of some of the
isolates was evaluated using in vitro cellular glucose uptake and oil red staining assays.
of three new acetylenic phenylacrylic acid esters (1−3), two
new polyacetylenes (4 and 5), and seven known compounds
(6−12). Compounds 1−3 are esters of a polyacetylene unit
and a phenylacrylic acid. The structures of the new compounds
were established by means of spectroscopic data interpretation.
The absolute configurations of 4 and 7 were determined by CD
and by Mosher’s experiments. The isolated compounds were
evaluated for their antidiabetic and antioxidant activities.
Cirsium japonicum DC. var. australe Kitam. (Asteraceae) is
known as “Formosan thistle”.1,2 It is native to Taiwan, mainland
China, Vietnam, and Australia and is used as a folk medicine for
treating diabetes and symptoms associated with inflamma-
tion.1,3 Despite its ethnopharmacological significance, no
chemical investigation on C. japonicum var. australe is available
in the scientific literature. However, this plant is frequently
mixed up with Cirsium japonicum DC. (“Japanese thistle”),
which has been utilized as a traditional Chinese medicine for
the treatment of hypertension, traumatic hemorrhage, and
inflammation.4 Previous biological studies have indicated that
flavones from C. japonicum enhanced the differentiation of
adipocytes and their glucose uptake activity.5−7
RESULTS AND DISCUSSION
■
The fresh material of the whole plant of C. japonicum var.
australe was extracted with 90% aqueous EtOH to yield a crude
extract, which was then subjected to partition with solvents of
different polarities. Among them, the EtOH extract exhibited
The absence of prior phytochemical studies on C. japonicum
var. australe encouraged the present phytochemical inves-
tigation to reveal its secondary metabolites and then to
determine their biological activities. Bioactivity-guided fractio-
nation of the whole plant ethanolic extract led to the isolation
Received: March 12, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
a
1
Table 1. H NMR and 13C NMR Data for 1−3
1
2
3
position
δC mult.
δH (J in Hz)
δC mult.
δH (J in Hz)
7.03, d (1.7)
δC mult.
δH (J in Hz)
1
127.1, C
109.5, CH
146.9, C
148.1, C
114.8, CH
123.3, CH
145.1, CH
115.5, CH
167.3, C
63.1, CH2
27.8, CH2
16.4, CH2
75.9, C
127.0, C
126.9, C
109.5, CH
146.9, C
148.3, C
114.9, CH
123.4, CH
145.7, CH
114.8, CH
166.7, C
63.8, CH2
139.5, CH
112.3, CH
75.9, C
2
7.03, d (1.8)
109.5, CH
146.9, C
7.02, d (1.3)
3
4
148.3, C
5
6.92, d (8.2)
114.9, CH
123.4, CH
145.8, CH
114.9, CH
166.7, C
6.92, d (8.2)
6.92, d (8.2)
6
7.08, dd (8.2, 1.8)
7.61, d (15.9)
6.28, d (15.9)
7.09, dd (8.2, 1.7)
7.64, d (15.9)
6.30, d (15.9)
7.07, dd (8.2, 1.3)
7.63, d (15.9)
6.29, d (15.9)
7
8
9
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
O-CH3
4.28, t (6.2)
1.93 m
63.7, CH2
139.8, CH
112.1, CH
79.1, C
4.77, dd (5.7, 1.6)
6.37, dt (15.9, 5.7)
5.90, d (15.9)
4.74, dd (5.8, 1.5)
6.33, m
2.42, tt (7.0, 1.0)
5.82, d (15.9)
66.2, C
75.5, C
72.5, C
65.4. C
64.9, C
65.2, C
78.1, C
79.5, C
85.1, C
21.3, CH2
21.9, CH2
13.6, CH3
56.1, CH3
2.22, tt (7.0, 1.1)
1.53, m
109.2, CH
143.4, CH
16.7, CH3
56.1, CH3
5.57, d (10.9)
6.17, dq (10.9, 6.9)
1.93, dd (6.9, 1.6)
3.94, s
21.9, CH2
21.7, CH2
13.6, CH3
56.1, CH3
2.30, t (7.0)
1.57, h (7.3)
1.00, t (7.4)
3.92, s
0.98, t (7.4)
3.93, s
a
Compound 1 was measured at 600 MHz; compounds 2 and 3 were measured at 400 MHz. For all compounds NMR spectra were obtained in
CDCl3; J values (Hz) are given in parentheses. Assignments were made based on the analysis of H−1H COSY, HSQC, and HMBC data.
1
the most potent biological activity. Hence, this extract was
selected for further bioactivity-guided isolation.
purification. Five new polyacetylenes (1−5) and seven known
compounds, 8-(hept-1-enyl)-1′,1′-dimethyl-8,9-dioxolan-9-yl)-
octa-11,13-diyn-1-ol (6),8 ciryneol C (7),9 heptadeca-1-ene-
11,13-diyne-8,9,10-triol (8),9 ciryneol H (9),10 ethyl caffeate
(10),11 apigenin (11),12 and scopoletin (12),13 were isolated.
Compound 1 was obtained as a pale yellow, amorphous
solid. It exhibited a molecular ion peak [M + Na]+ at m/z
349.1413 in the HRESIMS, corresponding to the molecular
formula C20H22O4. The IR spectrum showed a hydroxy group
(3406 cm−1), an ester carbonyl (1700 cm−1), a conjugated
acetylene (2200 cm−1), and an aromatic (1590 and 1509 cm−1)
absorption band. The UV absorption maxima at 237, 281, 296,
The fractionation was initiated with polyamide column
chromatography using gradient elution with different solvent
mixtures possessing a broad polarity range. Elution started with
10% aqueous MeOH, which was increased gradually to 100%
MeOH, and then CH2Cl2 was added to the solvent system at
increasing percentages to reach 100% CH2Cl2, resulting in the
separation of nine pooled fractions. The active fractions were
subjected to different chromatographic techniques such as
centrifugal partition chromatography, column chromatography
on silica, C18, and Sephadex LH-20 as well as HPLC for final
B
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and 317 nm suggested the presence of a polyacetylene moiety
and a conjugated phenyl group.14,15
In the H NMR spectrum of 1, signals for an ABX system
109.5), C-6 (δC 123.3), and C-9 (δC 167.3), H-5 (δH 6.92) to
C-1 (δC 127.1) and C-3 (δC 146.9), H-8 (δH 6.28) to C-1 (δC
127.1), and H-2 (δH 7.03) to C-4 (δC 148.1) and C-6 (δC
123.3) revealed the presence of a 3,4-substituted phenylacrylic
ester moiety (Figure 1). The methoxy group (δH 3.93) showed
an HMBC correlation to C-3 (δC 146.9), which along with the
NOESY correlation between the methoxy proton and H-2 (δH
7.03) suggested the presence of a 4-hydroxy-3-methoxyphenyl
acrylic ester moiety. The HMBC correlations between H-1′ (δH
4.28) and the ester carbonyl C-9 (δC 167.3) confirmed that the
deca-4,6-diyne moiety is linked to the 4-hydroxy-3-methox-
yphenyl acrylic ester (Figure 1). The structure of 1 was
elucidated as (E)-deca-4,6-diynyl 3-(4-hydroxy-3-
methoxyphenyl)acrylate and was given the trivial name
cirsiumyne A.
1
(δH 6.92, d, J = 8.2 Hz; δH 7.03, d, J = 1.8 Hz; δH 7.08, dd, J =
8.2, 1.8 Hz) of a phenyl moiety, a trans-double bond (δH 7.61,
d, J = 15.9 Hz; δH 6.28, d, J = 15.9 Hz), a methoxy group (δH
3.93, 3H, s), a terminal methyl (δH 0.98, 3H, t, J = 7.4 Hz), an
oxymethylene (δH 4.28, 2H, t, J = 6.2 Hz), and four methylenes
(δH 2.42, 2.22, 1.93 and 1.53) were observed (Table 1). The
13C and DEPT spectra suggested the presence of two methyls,
five methylenes, five methines, and eight quaternary carbons
including four C−C triple-bond carbons (δC 78.1, 75.9, 66.2,
and 65.4) (Table 1). On the basis of the information from the
HSQC and COSY correlations, a trimethylene unit (δH 4.28 for
H-1′, δH 1.93 for H-2′, and δH 2.42 for H-3′) and a propyl
group (δH 2.22 for H-8′, δH 1.50 for H-9′, δH 0.98 for H-10′)
were suggested (Figure 1). In the HMBC spectrum, the
Compound 2 (cirsiumyne B) was isolated as a pale yellow,
amorphous solid, and it was assigned the molecular formula
C20H18O4 from the HRESIMS at m/z 345.1102 [M + Na]+
(calcd for C20H18O4Na, 345.1103). The IR spectrum indicated
the presence of a hydroxy group (3406 cm−1), a conjugated
acetylene (2200 cm−1), an ester carbonyl (1709 cm−1), and
aromatic (1590 and 1514 cm−1) units. The UV absorption
maxima at 238 and 316 nm suggested the presence of a phenyl
1
moiety possibly conjugated to a carbonyl group. The H and
Figure 1. Key COSY (H−H) and HMBC (H→C) correlations of 1.
13C NMR spectra of 2 were similar to those of 1 (Table 1).
Instead of the four methylenes (δH 1.53, 1.93, 2.22, and 2.42) in
1, the 1D NMR spectra of 2 suggested the presence of a cis-
disubstituted double bond [δH 5.57 (d, J = 10.9 Hz) and δH
6.17 (dq, J = 10.9, 6.9 Hz)] and an additional trans-
disubstituted double bond [δH 6.37 (dt, J = 15.9, 5.7 Hz)
and δH 5.90 (d, J = 15.9 Hz)] (Table 1). The COSY
correlations of H-8′ (δH 2.22) to C-6′ (δC 65.4) and C-7′ (δC
78.1) and of H-3′ (δH 2.42) to C-4′ (δC 75.9) and C-5′ (δC
66.2) indicated that a diyne unit (C-4′, C-5′, C-6′, and C-7′)
may be placed between the trimethylene unit and the propyl
group, forming a deca-4,6-diynyl moiety in 1 (Figure 1).
Moreover, the HMBC correlations of H-7 (δH 7.61) to C-2 (δC
a
1
Table 2. H NMR Chemical Shifts (δ) of Compounds 4−7
4
5
6
7
position
1
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
5.00, ddt (17.0, 1.8, 1.7)
4.94, ddt (10.2, 2.2, 1.7)
5.80, ddt (17.0, 10.2, 6.8)
2.06, 2H, m
4.99, ddt (17.0, 1.7, 1.6)
4.93, ddt (10.2, 1.7, 1.3)
5.80, ddt (17.0, 10.2, 6.8)
2.05, 2H, m
4.99, ddt (17.0, 1.2, 1.1)
4.92, ddt (10.3, 2.4, 1.2)
5.82, ddt (17.0, 10.1, 6.7)
2.07, 2H, m
5.00, ddt (17.0, 1.7, 1.6)
4.94, ddt (10.2, 2.3, 1.6)
5.80, ddt (17.0, 10.2, 6.7)
2
3
2.05, 2H, m
b
4a
1.40, m
1.42, m
1.42, m
1.42−1.38,
m
4b
1.35, m
1.35, m
5
1.40, 2H, m
1.42, 2H, m
1.42, 2H, m
1.44, 2H, m
b
b
b
6a
1.75−1.90, 2H, m
1.62, m
1.38−1.44, 2H, m
1.38−1.48, 2H, m
6b
1.56, m
7
1.83, 2H, m
1.63, 2H, m
1.73, 2H, m
1.81, 2H, m
8
4.30, ddd (8.9, 5.0, 4.2)
3.74, dd (5.5, 4.2)
4.59, d (5.5)
4.07, td (8.0, 3.8)
3.81, dd (7.9, 3.8)
4.61, dd (7.9, 4.3)
4.00, td (8.1, 3.4)
3.67, ddd (7.6, 3.4, 5.0)
4.41, d (5.0)
4.28, ddd (10.2, 4.9, 4.0)
3.71, dd, br d (6.2, 6.0, 4.0)
4.51, d (6.0)
9
10
11
12
13
14
15
5.53, dq (10.9, 1.7)
6.20, dq (10.9, 6.9)
1.93, 3H, dd (6.9, 1.7)
5.52, dq (10.8. 1.7)
6.20, dq (10.8, 6.9)
1.92, 3H, dd (6.9, 1.7)
2.28, 2H, td (6.9, 0.7)
1.55, 2H, m
2.26, 2H, td (7.0, 0.8)
1.55, 2H, m
16
17
1.00, 3H, t (7.4)
0.99, 3H, t (7.4)
1′
CH3-1′a
CH3-1′b
1.43, 3H, s
1.42, 3H, s
1.43, 3H, s
1.42, 3H, s
a
Compounds 4, 6, and 7 were measured at 400 MHz; compound 5 was measured at 600 MHz. For all compounds NMR spectra were obtained in
CDCl3; J values (Hz) are given in parentheses. Assignments were made based on the analysis of H−1H COSY, HSQC, and HMBC data.
1
b
Multiplicity patterns are unclear due to signal overlapping.
C
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a
Table 3. 13C NMR Chemical Shifts (δ) of Compounds 4−7
correlations between the methylene protons adjacent to the
allylic and ester functionalities at δH 4.77 (H-1′) and the trans-
disubstituted olefinic proton at δH 6.37 (H-2′) and between the
terminal methyl proton (δH 1.93, dd, J = 6.9, 1.6 Hz, H-10′)
and the cis-disubstituted olefinic group (δH 6.17, dq, J = 10.9,
6.9 Hz, H-9′) indicated the location of the two double bonds.
The above data, along with the HMBC correlations of H-2′/C-
4′, H-3′/C-5′, and H-10′/C-7′, suggested that 2 contains a
diendiynyl moiety between C-1′ and C-10′. Moreover, the 1D
and 2D NMR spectra revealed that this compound also
possesses the same 4-hydroxy-3-methoxyphenyl acrylic ester
moiety as in 1. The HMBC cross-peak between H-1′ (δH 4.77)
and the ester carbonyl C-9 (δC 166.7) was used to deduce the
structure of 2, and it was identified as (E)-((2E,8Z)-deca-2,8-
dien-4,6-diynyl) 3-(4-hydroxy-3-methoxyphenyl)acrylate.
4
5
6
7
position
δC, mult.
δC, mult.
δC, mult.
δC, mult.
1
114.6, CH2
139.0, CH
33.8, CH2
28.8, CH2
28.6, CH2
26.4, CH2
34.6, CH2
64.6, CH
75.7, CH
64.8, CH
80.0, C
114.4, CH2
139.2, CH
34.0, CH2
25.4, CH2
33.8, CH2
26.0, CH2
33.8, CH2
77.4, CH
82.7, CH
63.4, CH
87.2, C
114.8, CH2
140.1, CH
34.8, CH2
29.9, CH2
30.1, CH2
26.9, CH2
35.1, CH2
79.4, CH
84.4, CH
64.0, CH
75.2, C
114.6, CH2
139.0, CH
33.8, CH2
28.5, CH2
28.8, CH2
26.3, CH2
34.6, CH2
64.5, CH
75.8, CH
64.6, CH
72.9, C
2
3
4
5
6
7
8
9
10
11
12
71.9, C
70.8, C
71.5, C
72.2, C
Compound 3 was isolated as a pale yellow, amorphous solid
and was assigned the molecular formula C20H20O4, as
evidenced by the HRESIMS at m/z 347.1257 [M + Na]+
(calcd for C20H20O4Na, 347.1259), suggesting one additional
degree of unsaturation when compared to 1. The UV
absorptions at 269, 286, and 319 nm in combination with an
IR band at 2229 cm−1 suggested the presence of a
polyacetylene functionality and a conjugated phenyl
group.14,15 The IR absorptions at 3511 (hydroxy group),
2229 (conjugated acetylene), 1704 cm−1 (ester carbonyl), and
1590/1514 cm−1 (aromatic hydrocarbons) of 3 were similar to
13
76.5, C
67.7, C
65.5, C
64.4, C
14
77.4, C
72.7, C
82.0, C
82.5, C
15
108.7, CH
144.2, CH
16.8, CH3
108.9, CH
144.0, CH
16.6, CH3
109.0, C
21.7, CH2
22.8, CH2
13.7, CH3
110.3, C
21.4, CH2
21.7, CH2
13.6, CH3
16
17
1′
CH3-1′a
CH3-1′b
29.9, CH3
27.7, CH3
27.9, CH3
27.2, CH3
a
Compounds 4, 6, and 7 were measured at 100 MHz; compound 5
was measured at 150 MHz. For all compounds NMR spectra were
obtained in CDCl3. Multiplicity was obtained from the DEPT
spectrum. Assignments were based on HSQC and HMBC NMR
spectra.
1
those of 1 and 2. The H and 13C NMR spectroscopic data
revealed the presence of a similar acrylate moiety to that
present in 1 and 2 (Table 1). In addition, the 1D and 2D NMR
spectra demonstrated that 3 possesses the same 4-hydroxy-3-
methoxyphenyl acrylic ester moiety as in 1. In comparison with
the seven methylenes in 7, the 1D NMR spectra of 4 revealed
the presence of five methylenes (δC 33.8, 28.8, 28.6, 26.4, and
34.6) and an additional cis-disubstituted double bond [δH 5.53
(dq, J = 10.9, 1.7 Hz) and δH 6.20 (dq, J = 10.9, 6.9 Hz)]. The
chemical shift of C-8 in 4 was shifted upfield to δC 64.6, and its
proton was shifted downfield to δH 4.30, suggesting that the
chlorine atom is attached to C-8.8,9 The COSY correlations of
the methine protons revealed the spin system of the 8-
chloropropane-9,10-diol (Figure 2). The HMBC correlations of
1
the H NMR data of 1, the only difference between these two
compounds was in the presence of an additional trans-olefinic
group [δH 5.82 (d, J = 15.9 Hz) and δH 6.33 (m)] in 3, in
agreement with the observation of the one additional degree of
unsaturation. The COSY correlation from the trans-olefinic
proton (H-2′) to the oxymethylene proton (δH 4.74, H-1′),
along with the HMBC correlations of H-1′ (δH 4.74) to C-3′
(δC 146.9), H-3′ (δH 5.82) to C-5′ (δC 72.5), and H-8′ (δH
2.30) to C-6′ (δC 65.2) and C-7′ (δC 85.1), revealed an endiyne
moiety between C-1′ and C-8′. Therefore, compound 3
(cirsiumyne C) was determined as (E)-[(E)-deca-2-en-4,6-
diynyl] 3-(4-hydroxy-3-methoxyphenyl)acrylate.
Compound 4 was isolated as a colorless oil. Its HRESIMS
showed a sodiated molecular ion peak at m/z 317.1282 [M +
Na]+ (calcd for C17H23ClO2Na, 317.1284), which, together
with the 37Cl isotope at m/z 319.1268 [M + 2 + Na ]+ (calcd
for C17H23ClO2Na, 319.1255), revealed the presence of a
chlorine atom. The IR spectrum indicated the presence of a
hydroxy group (3397 cm−1), conjugated acetylene (2229
cm−1), and terminal vinyl (1638 and 909 cm−1) absorptions.
The UV absorption maxima at 255, 269, and 285 nm suggested
that compound 4 contains an ene-diynyl moiety.14,16 In the 1H
NMR spectrum, a terminal methyl resonance at δH 1.93 (dd, J
= 6.9, 1.7 Hz, H-17), a terminal vinyl at δH 5.00 (ddt, J = 17.0,
1.8, 1.7 Hz, H-1a), 4.94 (ddt, J = 10.2, 2.2, 1.7 Hz, H-1b), and
5.80 (ddt, J = 17.0, 10.2, 6.8 Hz, H-2), and three downfield
shifted methine protons (δH 4.30, 3.74, and 4.59) were
observed. The 13C, DEPT, and HSQC spectra indicated the
presence of two C−C triple bonds (δC 80.0, 71.9, 76.5, and
Figure 2. Key COSY (H−H) and HMBC (H→C) correlations of 4.
H-9/C-7, C-8, C-10, and C-11 and of H-10/C-11 confirmed
that the chlorine atom is connected to C-8 (Figure 2). On the
basis of the COSY correlations of H-1a (δH 5.00) and H-1b (δH
4.94)/H-2 (δH 5.80), H-16 (δH 6.20)/H-17 (δH 1.93), and H-
16 (δH 6.20)/H-15 (δH 5.53), a terminal vinyl and a propenyl
group were assigned at both ends of the molecule of 4 (Figure
2). The above data, together with the HMBC correlations of H-
3/C-1, C-2, C-4 and C-5, H-7/C-5 and C-9, H-9/C-8 and C-
11, H-10/C-11, and H-17/C-13, C-14, C-15 and C-16,
suggested that the ene-diyne unit may be placed between C-
10 and C-17 (Figure 2). Thus, the planar structure of 4 was
assigned as (Z)-8-chloroheptadeca-1,15-diene-11,13-diyne-
9,10-diol.
9
1
77.4), similar to those present in 7. The H and 13C NMR
spectra of 4 were comparable to those of 7 (Tables 2 and 3), a
compound previously isolated from C. japonicum.9 Instead of
On comparing the coupling constants and NOE correlations
of 4 and its analogue 7, it was confirmed that the relative
configurations of these compounds are identical. Furthermore,
D
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the positive Cotton effect at 220 nm and the negative Cotton
effects around 240−270 nm suggested similar absolute
configurations for 4 and 7 (Figures S1 and S2, Supporting
Information). Unfortunately, the yield of compound 4 was
extremely small. Accordingly, 7 was selected for processing with
the modified Mosher’s method to reveal the absolute
configuration of the two analogues.19,20 Treatment of 7 with
(R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride
[(R)-MTPA-Cl] and (S)-(+)-α-methoxy-α-(trifluoromethyl)-
phenylacetyl chloride [(S)-MTPA-Cl] in deuterated pyridine
afforded the (S)- and (R)-bis-MTPA ester derivatives (7s and
7r).19,20 The ΔS−R values were calculated, and the absolute
configuration of 7 was found to be 9R and 10R (Figure S5,
Supporting Information).21,22 In addition, the absolute
configuration of 7 was confirmed by electronic circular
dichroism (ECD) calculation and circular dichroism (CD)
analysis. The experimental CD spectrum of 7 exhibited a
positive Cotton effect at 220 nm and two negative Cotton
effects around 240−270 nm (Figure S2, Supporting Informa-
tion), which was in agreement with the calculated ECD
spectrum of 7 with an 8S, 9R, 10R configuration (Figures S2
and S4, Supporting Information). Thus, the absolute
configuration of 7 was determined as 8S, 9R, and 10R.
Therefore, compound 4 (cirsiumyne D) was proposed as
(8S,9R,10R,Z)-8-chloroheptadeca-1,15-diene-11,13-diyne-9,10-
diol.
Compound 5 was obtained as a colorless oil, and it was
assigned the molecular formula C20H28O3 from the HRESIMS,
[M + Na]+ ion peak at m/z 339.1933 (calcd for C20H28O3Na,
339.1936), indicating seven degrees of unsaturation. The IR
spectrum demonstrated the presence of a hydroxy group (3425
cm−1), a conjugated acetylene (2229 cm−1), and terminal vinyl
(1642 and 904 cm−1) functionalities. UV absorptions at 254,
268, and 284 nm were consistent with the presence of an ene-
diyne moiety.16 In the 1D NMR spectra, three methyls [δH 1.92
(H-17), δH 1.43 (H-2′), and δH 1.42 (H-3′)], a cis-disubstituted
double bond [δH 5.52 (dq, J = 10.8, 1.7 Hz, H-15) and δH 6.20
(dq, J = 10.8, 6.9 Hz, H-16)], a terminal double bond [δH 4.99
(ddt, J = 17.0, 1.7, 1.6 Hz, H-1a), δH 4.93 (ddt, J = 10.2, 1.7, 1.3
Hz, H-1b), and δH 5.80 (ddt, J = 17.0, 10.2, 6.8 Hz, H-2)], and
three oxymethine carbons (δC 77.4 for C-8, δC 82.7 for C-9,
and δC 63.4 for C-10) were observed (Table 3), suggesting a
similar skeleton to that of 6.8 On comparing their mass and 1D
NMR spectra, compound 5 showed one more degree of
unsaturation than 6, which was in agreement with the presence
of an additional cis-olefinic group (Tables 2 and 3). The COSY
correlation between one of the cis-olefinic protons at δH 6.20
and the terminal methyl at δH 1.93 (H-17) revealed the
presence of a propenyl group. The HMBC correlations of the
methylene proton δH 2.05 (H-3) to the terminal vinyl group δC
114.4 and δC 139.2 (C-1 and C-2) as well as the methyl protons
δH 1.43 and 1.42 (H-2′ and H-3′) to δC 109.0 (C-1′) were
consistent with the presence of propene and an acetonide
moiety as found in 6.8 The EIMS fragment peaks at m/z 89 and
m/z 65 correlated with the presence of a hepta-5-ene-1,3-diyne
fragment (Figures S7 and S8, Supporting Information), which
suggested that the diyne unit may be placed at C-11 to C-14
and is connected to the propenyl group (C-15 to C-17). In
addition, ion peaks at m/z 197 and m/z 119 confirmed that 5
possesses 4-(hept-6-enyl)-2,2-dimethyl-1,3-dioxolane and octa-
6-ene-2,4-diyn-1-ol moieties (Figures S7 and S8, Supporting
Information).
The relative stereochemistry of 5 could be proposed from the
NOESY spectrum and the 1H NMR coupling constants of H-8,
H-9, and H-10 (Table 2). The NOESY correlations of H-9/H-
8/H-10/CH3-1, H-7/CH3-1/H-10, and H-8/H-9/H-10
showed that H-8 and H-9 have a syn/erythro relationship
(Figure 3). Moreover, the NOESY spectrum showed a
Figure 3. Newman projection of the C-8−C-9 center illustrating the
key NOE correlations indicating the stereochemistry of 5 and 6.
correlation from H-8 to H-10, but no cross-peak between H-
10 and CH3-1, which suggested that H-8 and H-10 are syn
positioned (Figure 3). Comparing the NOESY spectroscopic
data of 5 and 6 confirmed that the relative configurations of
these compounds are the same. Moreover, the CD spectrum of
5 showed two positive Cotton effects at 220 and 240 nm and
three negative Cotton effects at 246, 260, and 275 nm (Figures
S5 and S6, Supporting Information), which were similar to
those of 6, suggesting a similar absolute configuration. Again,
the isolated amount of 5 was insufficient to determine its
absolute configuration, so its analogue 6 was subjected to the
modified Mosher’s method.19,20 Unfortunately, the data were
not clear and precluded the determination of the C-10 absolute
configuration (the values of ΔδS−R were negative at C-9 and
positive at C-8) (Figure S40, Supporting Information). Thus,
the structure of 5 (cirsiumyne E) was identified as shown.
The potential antidiabetic effect of the compounds isolated
was determined by the percentage of glucose consumption in
3T3-L1 adipocytes.17 Certain compounds were isolated in
minor quantities not sufficient for biological screening, and thus
only compounds 8, 10, and 11 were selected for the evaluation
of their in vitro hypoglycemic activity. The crude extract and its
EtOH layer promoted glucose uptake activity in 3T3-L1
adipocytes (Figure 4). Previously, the antidiabetic activity of
Cirsium was attributed to its flavonoid content.5−7 However,
only two pure flavones from C. japonicum, pectolinarin and 5,7-
dihydroxy-4′,6-dimethoxyflavone, were investigated for their
potential antidiabetic activity, and both showed PPARγ
activation.6 Polyacetylenes from this genus have never been
evaluated for their antidiabetic activity. According to the results
obtained, heptadeca-1-ene-11,13-diyne-8,9,10-triol (8) and
ethyl caffeate (10) showed significant in vitro hypoglycemic
activity, while apigenin (11) decreased the glucose uptake of
3T3-L1 adipocytes (Figure 4). The 3T3-L1 adipocytes were
stained with Oil Red O after coculturing with the test samples
for 7 days. Compound 8 increased glucose consumption in
adipocytes without any promotion of lipid accumulation
(Figure 5). Furthermore, the major compound (10) showed
significant DPPH and ABTS+ scavenging activities with EC50
values of 0.18 and 0.19 μg/mL, respectively.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with JASCO DIP-370 and P-1020 digital polarimeters. UV
■
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SCPC-250 instrument from Armen Instrument (Armen, Saint-Ave,
France) with a 250 mL column volume. For the preparative reversed-
phase HPLC, a Phenomenex Gemini-NX C18 (110A, 5 μm, 250 × 10.0
mm; Phenomenex Inc., Torrance, CA, USA), a Zorbax Eclipse XDB-
C8 (5 μm, 250 × 9.4 mm; Agilent Technologies Inc., Santa Clara, CA,
USA), or a Merck Hibar Purospher STAR C18 (5 μm, 250 × 10.0 mm)
semipreparative column (Merck KGaA, Darmstadt, Germany) was
used, and HPLC equipment consisted of two JASCO PU-2080 HPLC
pumps connected to a JASCO MD-2010 Plus multiwavelength
detector (JASCO Inc., Tokyo, Japan). The instrumentation for
normal-phase HPLC (NP-HPLC) consisted of dual Shimadzu LC-
10AT pumps and a Shimadzu SPD-10A UV−vis detector (Shimadzu
Inc., Kyoto, Japan), and a Phenomenex Luna CN (5 μm, 250 × 10.0
mm) semipreparative column (Phenomenex Inc., Torrance, CA, USA)
was used.
Plant Material. The fresh plant of C. japonicum var. australe was
collected from Puli, Nantou County, Taiwan, in November 2011. The
material was identified by Dr. Ming-Hong Yen, and a voucher
specimen (voucher number: Cirsium 01-1) was deposited in the
Graduate Institute of Natural Products, Kaohsiung Medical University,
Kaohsiung, Taiwan.
Extraction and Isolation. The fresh whole plants of C. japonicum
var. australe (12.0 kg) were extracted with 90% aqueous EtOH (20 L ×
4) at room temperature and then concentrated at 37 °C under reduced
pressure. The crude extract (320.0 g) was partitioned with 10%
aqueous MeOH (2 L × 3) and EtOAc (2 L × 3) to yield an aqueous
layer (233.8 g), an insoluble portion (8.0 g), and an EtOAc layer (77.4
g). The EtOAc layer was then partitioned between n-hexane (2 L) and
90% aqueous EtOH (2 L × 3) to provide an n-hexane layer (38.2 g)
and an EtOH layer (30.9 g). Among the layers obtained, the EtOH
layer exhibited the most potent potential hypoglycemic, DPPH
scavenging, and xanthine oxidase inhibitory activities and was selected
for further bioactivity-guided fractionation.
Figure 4. Effects of C. japonicum var. australe extracts and pure
compounds on the glucose consumption of 3T3-L1 adipocytes. 3T3-
L1 cells were treated with CJVA (C. japonicum var. australe ethanolic
extract, 50 μg/mL), CJVA-E (ethanolic layer from CJVA, 50 μg/mL),
CJVA-H (n-hexane layer from CJVA, 50 μg/mL), CJVA-W (water
layer from CJVA, 50 μg/mL), CJVA-Bu (butanol layer from CJVA, 50
μg/mL), heptadeca-1-ene-11,13-diyne-8,9,10-triol (8) (50, 25, 12.5
μg/mL), ethyl caffeate (10) (50 μg/mL), and apigenin (11) 50 μg/
mL. DMSO (0.1%, v/v) was used as the control. Insulin (3.2 × 10−7
M) and pioglitazone (PIO, 50 μg/mL) were selected as the positive
controls. The data are expressed as mean SEM of four independent
experiments. **: p < 0.01, ***: p < 0.001 compared to the control.
The glucose consumption data showed no significant difference
between 8 (25 and 50 μg/mL) and insulin. This means that 8 showed
equal hypoglycemic activity to insulin.
The EtOH layer (29.8 g) was subjected to polyamide column
chromatography (20.0 × 25.5 cm) under a gradient elution of 10%
aqueous MeOH to pure MeOH and then increasing ratios of CH2Cl2
in MeOH to 100% CH2Cl2 to yield nine fractions (Fr. 1−9). The
DPPH scavenging and xanthine oxidase inhibitory assays revealed the
most potent fractions to be Fr. 5 (2.8 g) and Fr. 6 (6.7 g). Accordingly,
Fr. 5 was eluted with CH2Cl2−MeOH (50:1 → 10:1) from silica gel to
obtain 10 subfractions (Fr. 5-1−5-10).
Fraction 5-4 (125.0 mg) was subjected to CPC using a solvent
system of n-hexane−EtOAc−MeOH−water (4:1:4:1) in the ascending
mode to give 12 subfractions (Fr. 5-4-1−5-4-12). Among them, Fr. 5-
4-4 (2.7 mg) was purified utilizing normal-phase HPLC (CN) (n-
hexane−CH2Cl2−MeOH, 100:10:1; flow rate 2.2 mL/min, UV 254
nm, 5 μm, 250 × 10.0 mm column) to obtain compounds 6 (1.2 mg)
and 5 (1.0 mg). Fraction 5-4-5 (1.3 mg) was purified with normal-
phase HPLC (CN) (flow rate 2.0 mL/min, UV 254 nm, 5 μm, 250 ×
10.0 mm column) in n-hexane−CH2Cl2−MeOH (130:10:1) to obtain
compound 6 (0.5 mg).
Fraction 5-5 (138.9 mg) was subjected to RPC eluting with n-
hexane−EtOAc−EtOH (200:10:0.5 → 1:2:0.1), and Fr. 5-5-4 (13.7
mg) was purified by reversed-phase HPLC (C18) (3 mL/min, PDA,
250 × 10.0 mm column) eluting with 66% aqueous ACN to give an
additional amount of compound 6 (5.5 mg). Fraction 5-5-18 (30.1
mg) was purified by reversed-phase HPLC (C8) (3 mL/min, PDA,
250 × 9.4 mm column) eluting with 43% aqueous MeOH to obtain
compound 10 (8.5 mg).
Fraction 5-7 was purified by CPC in n-hexane−EtOAc (1:1, 1 L)
and MeOH−H2O (1:1, 250 mL) to give compound 8 (94.0 mg) and
subfractions Fr. 5-7-1−5-7-14. Then, the pooled subfractions Fr. 5-7-7
and 5-7-8 (56.7 mg) were eluted from a Sephadex LH-20 column by
98% aqueous MeOH to obtain compound 10 (45.1 mg).
Fraction 6 (2.8 g) was chromatographed on a silica gel open column
with a gradient solvent system, from pure n-hexane to CH2Cl2−
MeOH (50:3), to yield subfraction Fr. 6-6 (548.5 mg) eluted with n-
hexane−CH2Cl2 (1:6). This fraction was loaded on a Sephadex LH-20
column and eluted with CH2Cl2−MeOH (3:1), to yield eight
subfractions (Fr. 6-6-1−6-6-8). Among them, Fr. 6-6-4 (245.8 mg)
Figure 5. Effect of the extracts and pure compound 8 from C.
japonicum var. australe on lipid accumulation in 3T3-L1 adipocytes.
3T3-L1 cells were treated with CJVA (20 μg/mL), CJVAE (20 μg/
mL), and compound 8 (20 μg/mL) for 7 days. Insulin (3.2 × 10−7 M)
and PIO (pioglitazone, 50 μg/mL) were taken as positive controls.
Cells were fixed and stained with Oil Red O dye. Images of the
representative cells were scanned and captured with a microscope
(40× magnification).
spectra were obtained using JASCO UV-530 ultraviolet spectropho-
tometers. IR spectra were obtained on a PerkinElmer FTIR-
spectrometer and Spectrum 2000 spectrophotometer. The CD spectra
were obtained using a JASCO J-810 spectrophotometer. NMR (400
and 600 MHz) spectra were obtained on a Varian Unity 400 MHz FT-
NMR, a JEOL JNM ECS 400 MHz FT-NMR, and a Varian Unity 600
MHz FT-NMR. ESIMS data were collected on a VG Biotech Quattro
5022 mass spectrometer. HRESIMS data were obtained on a Bruker
APEX II spectrometer (FT-ICR/MS, FTMS) (Bruker Daltonics Inc.,
Billerica, MA, USA). MN polyamide SC-9 gel was purchased from
ICN Biomedicals GmbH (Eschwege, Germany). Analytical and
preparative TLC was performed on silica gel 60 F254 plates (Merck
KGaA, Darmstadt, Germany), and spots were visualized by UV and by
spraying with vanillin sulfuric acid reagent followed by heating for 5
min. Rotational planar chromatography (RPC) was performed on a
Chromatotron apparatus (Harrison Research, Palo Alto, CA, USA).
Centrifugal partition chromatography (CPC) was performed on an
F
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was purified using silica gel and eluted with a gradient of n-hexane−
CH2Cl2 (2:3) to n-hexane−CH2Cl2−MeOH (10:30:1) to obtain
subfractions Fr. 6-6-4-5 (39.5 mg) and Fr. 6-6-4-6 (6.14 mg). Then,
normal-phase HPLC (CN) (2.2 mL/min, UV 254 nm, 5 μm, 250 ×
10.0 mm column) was utilized by eluting with n-hexane−CH2Cl2−
MeOH (40:10:1) to give pure compounds 7 (5.1 mg) and 4 (0.5 mg)
from subfraction 6-6-4-5. In addition, normal-phase HPLC eluting
with n-hexane−CH2Cl2−MeOH (100:10:1) gave the pure compounds
9 (1.2 mg) and 7 (1.0 mg) from fraction 6-6-4-6.
Fraction 6-6-6 (30.7 mg) was purified by normal-phase HPLC
(CN) (2.2 mL/min, UV 254 nm, 5 μm, 250 × 10.0 mm column),
eluting with n-hexane−CH2Cl2−MeOH (240:20:1), to give pure
compound 3 (1.9 mg). The residue (12.2 mg) was further purified by
reversed-phase HPLC (C18) (2 mL/min, PDA, 5 μm, 250 × 10.0 mm
column) and eluted with 72% aqueous MeOH to obtain compounds 1
(0.6 mg), 2 (2.7 mg), and 3 (2.1 mg).
Fraction 3 (1.3 g) was chromatographed by CPC in n-hexane−
EtOAc−MeOH−H2O (2:8:2:8, 1 L), and its subfraction Fr. 3-10 (45.6
mg) obtained from this separation was purified by reversed-phase
HPLC (C18) (2 mL/min, RI detector, 5 μm, 250 × 10.0 mm column),
eluting with 40% aqueous MeOH, to obtain compound 12 (2.3 mg).
Fraction 8 (1.0 g) was loaded on a C18 silica gel open column
eluting with 10% aqueous MeOH−100% MeOH. A subfraction
obtained, Fr. 8-2 (186.2 mg), was purified by RPC, eluting with a
gradient system of CH2Cl2−MeOH (50:1 → 6:1), to give compound
11 (2.3 mg).
CDCl3.19,20 (R)-MTPA ester 6: 1H NMR (400 MHz, CDCl3) δ 7.43−
7.37 (5H, m, aromatic H), 5.81 (1H, ddt, J = 17.2, 10.0, 6.7 Hz, H-2),
5.37 (1H, d, J = 7.1 Hz, H-10), 5.00, (1H, ddt, J = 17.2, 2.0, 1.7 Hz, H-
1a), 4.93 (1H, ddt, J = 10.0, 2.0, 1.7 Hz, H-1b), 3.97 (1H, m, H-8),
3.93 (1H, m, H-9), 3.58 (3H, OCH3), 2.27 (2H, t, J = 7.1 Hz, H-15),
2.03 (2H, q, J = 6.7 Hz, H-3), 1.69 (2H, m, H-7), 1.57 (2H, dq, J = 7.1,
7.4 Hz, H-16), 1.42−1.26 (6H, m, H-4, 5, 6), 1.40 (3H, s, H-2′), 1.34
1
(3H, s, H-3′), 0.99 (3H, t, J = 7.4 Hz, H-17). (S)-MTPA ester 6: H
NMR (400 MHz, CDCl3) δ 7.40−7.38 (5H, m, aromatic H), 5.78
(1H, ddt, J = 17.2, 10.0, 6.9 Hz, H-2), 5.72 (1H, d, J = 3.6 Hz, H-10),
4.99 (1H, m, H-1a), 4.94 (1H, m, H-1b), 4.99 (1H, m, H-8), 3.83 (1H,
dd, J = 3.8, 7.1 Hz, H-9), 3.52 (3H, OCH3), 2.26 (2H, t, J = 7.0 Hz, H-
15), 1.99 (2H, q, J = 7.0 Hz, H-3), 1.73 (2H, m, H-7), 1.57 (2H, dq, J
= 7.0, 7.4 Hz, H-16), 1.37−1.28 (6H, m, H-4, 5, 6), 1.33, (3H, s, H-
2′), 1.25, (3H, s, H-3′), 0.99 (3H, t, J = 7.4 Hz, H-17). (R)-Bis-MTPA
ester 7: 1H NMR (400 MHz, CDCl3) δ 7.44−7.34 (10H, m, aromatic
H), 5.80 (2H, m, H-2), 5.75 (1H, d, J = 6.8 Hz, H-10), 5.57 (1H, dd, J
= 6.8, 1.5 Hz, H-9), 5.01 (1H, m, H-1a), 4.94 (1H, m, H-1b), 4.00
(1H, m, H-8), 3.50 (OCH3), 3.55 (OCH3), 2.27 (2H, m, H-15), 1.99
(2H, m, H-3), 1.86 (2H, m, H-7), 1.57 (2H, m, H-16), 1.45−1.15 (6H,
m, H-4, 5, 6), 0.99 (3H, t, J = 7.4 Hz, H-17). (S)-Bis-MTPA ester 7:
1H NMR (400 MHz, CDCl3) δ 7.42−7.34 (10H, m, aromatic H), 5.88
(1H d, J = 8.0 Hz, H-10), 5.78 (2H, m, H-2), 5.50 (1H, dd, J = 8.0, 3.0
Hz, H-9), 5.00 (1H, m, H-1a), 4.94 (1H, m, H-1b), 3.56 (1H, m, H-8),
3.50 (3H, OCH3), 3.55 (3H, OCH3), 2.27 (2H, m, H-15), 2.01 (2H,
dd, J = 14.6, 6.8 Hz, H-3), 1.79 (1H, m, H-7), 1.57 (2H, m, H-16),
1.49−1.05 (6H, m, H-4, 5, 6), 0.98 (3H, t, J = 7.4 Hz, H-17).
ECD Calculation. Both 8R,9R,10R and 8S,9R,10R conformational
analysis of compounds 4 and 7 were conducted via Monte Carlo
searching with the MMFF94 molecular mechanics force field using
Molecular Operating Environment (MOE) software (Chemical
Computing Group, Montreal, Canada).23 The conformers were
optimized using DFT at the CAM-B3LYP/6-31G(d) level in the gas
phase with GAUSSIAN 09.23 The CAM-B3LYP/6-31G(d) harmonic
vibrational frequencies were computed to confirm their stability and to
provide their relative thermal free energy, which are used to evaluate
their equilibrium populations. The energies, rotational strengths, and
oscillator strengths of the 20 weakest electronic excitations of the
conformers were computed utilizing the TDDFT methodology at the
CAM-B3LYP/6-31G(d)/dichloromethane level in the gas phase.
Furthermore, the ECD spectra were then simulated using
GaussSum2.2.5 with a bandwidth σ of 0.20 eV. The corresponding
theoretical ECD spectra of (8R,9R,10R)-4 and (8R,9R,10R)-7 were
depicted by inverting those of (8S,9R,10R)-4 and (8S,9R,10R)-7. The
ECD spectra of the two stereostructures were compared with the CD
spectrum of 4 and 7 to determine their absolute configuration.
2-Deoxyglucose Uptake Assay. 3T3-L1 preadipocytes were
purchased from American Type Culture Collection (Manassas, VA,
USA) and cultured in Dulbecco’s minimal essential medium (DMEM)
supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin,
1% nonessential amino acid, and 10% calf serum (Hyclone, Logan,
UT, USA) in 5% CO2 at 37 °C.14 When the cell density reached 100%
confluence, 3T3-L1 preadipocytes were induced to differentiate by
treating the culture with 450 mg/dL glucose, 0.32 μM insulin, 0.5 mM
3-isobutyl-1-methylxanthine, and 1 μM dexamethasone.17 The
induction medium was removed, and the cells were maintained in
the culture medium with 450 mg/dL glucose in the presence or
absence of test samples and insulin after 2 days. Fresh medium and test
samples were treated every day, and 24 h glucose consumption was
measured by a Roche Cobas Integra 400 chemistry analyzer (Roche
Diagnostics, Taipei, Taiwan).17 The samples were dissolved in DMSO,
to which the medium was added to obtain the final concentration of
0.1% (v/v) of DMSO. Pioglitazone (50 mg/mL) was used as a positive
control, and DMSO was added to the blank control group.
Cirsiumyne A (1): pale yellow, amorphous solid; UV (CH2Cl2) λmax
(log ε) 237 (4.07), 281 (2.91), 296 (4.60), 317 (4.60) nm; IR (neat)
1
νmax 3406, 2918, 2200, 1700, 1590, 1509, 1152, 1028, 976 cm−1; H
NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see
Table 1; HRESIMS m/z 349.1413 [M + Na]+ (calcd for C20H22O4Na,
349.1416).
Cirsiumyne B (2): pale yellow, amorphous solid; UV (CH2Cl2) λmax
(log ε) 238 (4.57), 280 (2.61), 296 (3.80), 316 (3.94) nm; IR (neat)
1
νmax 3406, 2928, 2200, 1709, 1590, 1514, 1152, 1028, 976 cm−1; H
(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table
1; HRESIMS m/z 345.1102 [M + Na]+ (calcd for C20H18O4Na,
345.1103).
Cirsiumyne C (3): pale yellow, amorphous solid; UV (CH2Cl2) λmax
(log ε) 241 (3.01), 254 (2.63), 269 (3.25), 286 (3.76), 319 (3.11) nm;
IR (neat) νmax 3511, 3416, 2956, 2229, 1704, 1628, 1590, 1514, 1266,
1
1152, 1028, 976 cm−1; H (400 MHz, CDCl3) and 13C NMR (100
MHz, CDCl3) data, see Table 1; HRESIMS m/z 347.1257 [M + Na]+
(calcd for C20H20O4Na, 347.1259).
Cirsiumyne D (4): colorless oil; [α]25 −18.5 (c 0.1, CH2Cl2); UV
D
(CH2Cl2) λmax (log ε) 242 (2.10) 255 (3.43), 269 (4.47), 285(3.51)
nm; IR (neat) νmax 3626, 3397, 2919, 2851, 2229, 1638, 1457, 1433,
1300, 1033, 995, 909, 719 cm−1; 1H (400 MHz, CDCl3) and 13C NMR
(100 MHz, CDCl3) data, see Tables 2 and 3; HRESIMS m/z 317.1282
[M + Na]+, 319.1268 [M + 2 + Na]+ (calcd for C17H23ClO2Na,
317.1284 and 319.1255).
Cirsiumyne E (5): colorless oil; [α]25D −30.0 (c 0.05, CH2Cl2); UV
(CH2Cl2) λmax (log ε) 242 (1.01), 254 (1.47), 268 (1.90), 284 (1.62)
nm; IR (neat) νmax 3425, 2918, 2851, 2315, 2354, 2229, 1728, 1642,
1
1452, 1371, 1095, 1042, 904, 871 cm−1; H (600 MHz, CDCl3) and
13C NMR (150 MHz, CDCl3) data, see Tables 2 and 3; HRESIMS m/
z 339.1933 [M + Na]+ (calcd for C20H28O3Na, 339.1936).
Preparation of (R)- and (S)-MTPA Esters (6r, 6s, 7r, 7s).
Compounds 6 and 7 (1.0 mg) were stored in separate NMR tubes and
dried under vacuum.19,20 Deuterated pyridine (0.6 mL) and (R)-
MTPA-Cl (10 μL) were added to the NMR tubes under a N2 gas
stream. The reaction NMR tubes were permitted to stand at room
temperature and monitored by 400 MHz NMR every hour. After 5 h,
1
the reaction was found to be completed, and the H NMR data was
obtained (400 MHz, in pyridine-d5). (S)-MTPA esters of 6 (6s) and 7
(7s) were obtained, and the 1H NMR data in CDCl3 (400 MHz) were
analyzed. Similar to 6s and 7s, (S)-MTPA-Cl (10 μL) and deuterated
pyridine (0.6 mL) were reacted at room temperature for 5 h, to afford
the (R)-MTPA ester derivatives (6r and 7r), in separate experiments.
The 1H NMR spectra were measured with 400 MHz NMR in
Oil Red O Staining. Fixed cells were washed with PBS buffer and
placed in 60% 2-propanol in PBS.18 Then, they were stained in freshly
diluted Oil Red O (Wako Pure Chemical, Tokyo, Japan) solution
[0.3% stock in 2-propanol−H2O (3:2)] for 15 min at 37 °C. DMSO
(0.1%, v/v) was used as the blank. Insulin (3.2 × 10−7 M) and PIO
(pioglitazone, 50 μg/mL) were taken as positive controls. The stained
G
dx.doi.org/10.1021/np500233t | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
lipid droplets were scanned and captured with a microscope (40×
magnification).
(12) Ha, T. J.; Lee, J. H.; Lee, M.-H.; Lee, B. W.; Kwon, H. S.; Park,
C.-H.; Shim, K.-B.; Kim, H.-T.; Baek, I.-Y.; Jang, D. S. Food Chem.
2012, 135, 1397−1403.
(13) Carpinella, M. C.; Ferrayoli, C. G.; Palacios, S. M. J. Agric. Food
Chem. 2005, 53, 2922−2927.
ASSOCIATED CONTENT
■
S
* Supporting Information
(14) Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. J. Nat. Prod.
1997, 60, 126−130.
(15) Hufnage, J. C.; Hofmann, T. J. Agric. Food Chem. 2008, 56,
1376−1386.
(16) Huang, H.-Q.; Zhang, X.; Shen, Y.-H.; Su, J.; Liu, X.-H.; Tian, J.-
M.; Lin, S.; Shan, L.; Zhang, W.-D. J. Nat. Prod. 2009, 72, 2153−2157.
(17) Hsieh, T.-J.; Tsai, Y.-H.; Liao, M.-C.; Du, Y.-C.; Lien, P.-J.; Sun,
C.-C.; Chang, F.-R.; Wu, Y.-C. Food Chem. Toxicol. 2012, 50, 779−
789.
(18) Choi, S.-S.; Cha, B.-Y.; Lee, Y.-S.; Yonezawa, T.; Teruya, T.;
Nagai, K.; Woo, J.-T. Life Sci. 2009, 84, 908−914.
(19) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092−4096.
(20) Takada, N.; Watanabe, M.; Yamada, A.; Suenaga, K.; Yamada,
K.; Ueda, K.; Uemura, D. J. Nat. Prod. 2001, 64, 356−359.
1D and 2D NMR spectra of compounds 1−5; GC-MS
spectrum of compound 5; CD spectra of compounds 4−7;
ECD spectra of compounds 4 and 7 are available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: +36-62-546-456. Fax: +36-62-545-704. E-mail: hunyadi.
a@pharm.u-szeged.hu (A. Hunyadi).
*Tel: +886-7-3121-101-2162. Fax: +886-7-3114-773. E-mail:
aaronfrc@kmu.edu.tw (F.-R. Chang).
Author Contributions
(21) Freire, F.; Seco, J. M.; Quinoa,
́
E.; Riguera, R. J. Org. Chem.
̃
#W.-C. Lai and Y.-C. Wu contributed equally to this study.
2005, 70, 3778−3790.
Notes
(22) Seco, J. M.; Quinoa,
́
E.; Riguera, R. Chem. Rev. 2012, 112,
̃
4603−4641.
(23) Liu, K.; Feng, J.; Young, S. S. J. Chem. Inf. Model. 2005, 45, 515−
522.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Science
Council, Taiwan, awarded to Y.-C.W. (NSC 103-2911-I-002-
303) and F.-R.C. (102-2911-I-037-501), and it was performed
within the framework of a bilateral mobility grant from the
National Science Council, Taiwan, and the Hungarian Academy
of Sciences (102-2911-I-037-501 and SNK-79/2013). We
gratefully acknowledge the support from the European Union
́
cofunded by the European Social Fund (TAMOP-4.2.2.A-11/
1/KONV-2012-0035), the Fundacao para a Ciencia e a
̧
̂
̃
Tecnologia, Portugal (PEsT-OE/SAU/UI0074/2011), and
the Center for Research Resources and Development
(CRRD), Kaohsiung Medical University, for technical support
and services in LC-MS, GC-MS, and NMR analysis. A.M.
acknowledges grant SFRH/BPD/81118/2011 from the FCT,
Portugal, supervised by Prof. Leonard Amaral and Prof. Joseph
Molnar. This work was also supported by the Cancer Center,
́
Kaohsiung Medical University Hospital, Kaohsiung Medical
University, Kaohsiung, Taiwan.
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