A new iridoid and effect on the rat aortic vascular smooth muscle cell proliferation of isolated compounds from Buddleja officinalis

Article abstract of DOI:10.1016/j.bmcl.2011.03.078

A new iridoid, named methylscutelloside (1) together with 19 known compounds belonging to the iridoids (2-4), monoterpenoids (5), flavonoids (6-8), triterpenoids (9-14), and phenylethanoids (15-20) were isolated from the flowers of Buddleja officinalis. Their chemical structures were elucidated on the basis of physicochemical properties, and by spectroscopic methods including 1D, 2D NMR, and MS. All isolated compounds were tested in vitro for their effects on the proliferation of rat aortic vascular smooth muscle cells (VSMCs). Among them, iridoids were the main active components and showed significant inhibitory effects on PDGF-BB-induced proliferation in rat aortic VSMCs. 2011 Elsevier Ltd. All rights reserved.

Full text of DOI:10.1016/j.bmcl.2011.03.078

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3462–3466
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new iridoid and effect on the rat aortic vascular smooth muscle cell
proliferation of isolated compounds from Buddleja officinalis
Bui Huu Tai a,b, Nguyen Xuan Nhiem a,c, Tran Hong Quang c, Nguyen Thi Thanh Ngan a, Nguyen Huu Tung a,
Yohan Kim a, Jung-Jin Lee a, Chang-Seon Myung a,d, Nguyen Manh Cuong b, Young Ho Kim a,
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Natural Products Chemistry, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam
c Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam
d Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A new iridoid, named methylscutelloside (1) together with 19 known compounds belonging to the irid-
oids (2–4), monoterpenoids (5), flavonoids (6–8), triterpenoids (9–14), and phenylethanoids (15–20)
were isolated from the flowers of Buddleja officinalis. Their chemical structures were elucidated on the
basis of physicochemical properties, and by spectroscopic methods including 1D, 2D NMR, and MS. All
isolated compounds were tested in vitro for their effects on the proliferation of rat aortic vascular smooth
muscle cells (VSMCs). Among them, iridoids were the main active components and showed significant
inhibitory effects on PDGF-BB-induced proliferation in rat aortic VSMCs.
Received 24 December 2010
Revised 4 March 2011
Accepted 23 March 2011
Available online 30 March 2011
Keywords:
Methylscutelloside
Vascular smooth muscles cells
Platelet derived growth factor
Buddleja officinalis
Ó 2011 Elsevier Ltd. All rights reserved.
Buddleja officinalis Maxim. (Buddlejaceae), a shrub tree, is culti-
vated widely throughout the temperate regions of the Americas,
Africa and Asia. Phytochemical studies on Buddleja species have
led to the isolation of flavonoids,1 triterpenoids,2 and phenyletha-
noids3 which are the main active components for the treatment
of stroke, headache, neurological disorders, conjunctival conges-
tion, clustered nebulae, removal of heat, replenishment of the liver,
and clearing of corneal opacity in several folk medicine reme-
dies.4,5 Recent studies showed that, the water extract of B.
officinalis inhibited high glucose-induced matrix metalloproteinase
activity,6 vascular inflammation7,8 in human umbilical vein endo-
thelial cells (HUVECs), and suppressed high glucose-induced
atherosclerotic processes through inhibition of p38 mitogen-acti-
vated protein kinases (MAPK), JNK phosphorylation, nuclear fac-
growth factor beta polypeptide (PDGF-BB) signal pathway are also
discussed.
Dried flowers of B. officinalis were ultrasonically extracted with
methanol. The crude extract was then successively partitioned
with n-hexane, dichloromethane, ethyl acetate, and n-butanol.
Using various types of column chromatography, a new iridoid
named methylscutelloside (1) together with 19 known compounds
6-O-vanilloyl ajugol (2),10 methylcatalpol (3),11,12 buddlejoside A4
(4),11 betulalbusides A (5),13,14 apigenin (6),3 isorhoifolin (7),3 lina-
rin (8),3 b-phenylethoxy-b- -glucopyranoside (9),3 salidroside
D
(10),3 bioside (11),3 acteoside (12),3 poliumoside (13),3 echinaco-
side (14),3 buddlejasaponin III (15),15,16 buddlejasaponin IV
(16),15 buddlejasaponin I (17),17,18 buddlejasaponin Ia (18),19,20
buddlejasaponin IVa (19),15 and mimengoside B (20)21,22 were iso-
lated.3,23 Their chemical structures (see Fig. 1) were elucidated
base on the results of 1D, 2D NMR, MS data, as well as by compar-
ison with compounds reported in the literatures. Compound 1 was
obtained from n-butanol extract as a pale yellow amorphous pow-
tor-jB (NF-jB) and matrix metalloproteinase (MMP) signal
pathways in human aortic smooth muscle cells.9 Previously, flavo-
noids and phenylethanoids from B. officinalis were reported to act
as anti-oxidative stress inhibitors against peroxynitrite.3 For con-
tinuous investigation on the chemical components and bioactivi-
ties of B. officininalis, we report the isolation and structure
elucidation of a new iridoid and 19 known compounds. In addition,
their inhibitory effects on rat aortic vascular smooth muscle cell
(VSMC) proliferation through the homodimer platelet-derived
der, ½a 2D2
= ꢁ7.6 (c 0.31, MeOH). Its molecular formula was deter-
ꢀ
mined to be C16H26O11 based on the quasi-molecular ion peak
observed at m/z 395 [M+H]+ in positive mode, electrospray ioniza-
tion mass spectrometry (ESI-MS) and m/z 395.1561 [M+H]+ (calcd
C
16H27O11 for 395.1553) in high resolution ESI-MS (HR-ESI-MS)
analysis. The 13C NMR and Distortionless Enhancement by Polari-
zation Transfer (DEPT) spectral data of 1 showed 16 carbons
including one methoxy carbon, three methylene carbons, 11 ter-
tiary carbons, and one quaternary carbon. Among them, one
⇑
Corresponding author. Tel.: +82 42 821 5933; fax: +82 42 823 6566.
E-mail address: yhk@cnu.ac.kr (Y.H. Kim).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.078

B. H. Tai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3462–3466
3463
ValO
R2O
HO
H
H
MeO
H
6
3
5
10
HO
O
1
O
O
9
O
HO
O
HO
8
HO
H
H
OGlc
H
OGlc
OR1
OGlc
R1
Glc
Glc
R2
2
1
5
3
4
CH3
X
OR1
O
R2O
O
O
OH
OH
Me
OH
R1
O
O
R2O
R2
R1
R2
Glc
Xyl(1-4)Glc
Rham(1-4)Glc
OR1
6
7
8
H
H
CH3
H
15
16
17
Glc
Glc
Glc
OH
Rham(1-6)Glc
Rham(1-6)Glc
OR5
R1
O
R4O
R3O
O
R2
MeO
OH
OH
R1
H
H
R2
H
OH
R3
H
H
Rham
Rham Caf
Rham Caf Rham
Rham Caf Glc
R4
H
H
R5
H
H
H
H
R1
9
10
OH
R3O
O
O
R1
R2
R3
Glc
11 OH OH
12 OH OH
13 OH OH
14 OH OH
H
18
19
20
OH Glc
Glc
OH Glc
OR2
H
Rham(1-4)Glc
Rham(1-4)Glc
OH
MeO
HO
Me
O
O
MeO
HO
O
HO
O
O
OH
O
O
Caf
Val
X
Me
Figure 1. Chemical structures of isolated compounds.
HMBC
a
O
H
4
HO
HO
5
3
O
O
6
9
H
7
1
8
O
OH
10
b
O
HO
HO
OH
Figure 2. Selected HMBC and ROESY correlations for 1.

3464
B. H. Tai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3462–3466
Table 1
acid hydrolysis and gas chromatography (GC) analysis, followed
The 1H and 13C NMR data for 1 in CD3OD
by comparisons of retention times with authentic samples.24 Next,
the stereostructure of 1 was elucidated by careful examination of
its 1D and 2D NMR spectra (Table 1). The broad singlet appearance
of H-1 and the cross-peak between H-1(dH 5.60) and H-9 (dH 2.47)
on COSY spectra confirmed the b-orientation of the H-9, since H-1
a
b
Position
dH (mult, J in Hz)
dC
1
3
4
5.60 (br s)
5.23 (br s)
93.3
95.7
35.3
1.63 (dd, 13.8, 2.4)
2.43 (m)
2.37 (t, 8.4)
3.68 (dd, 1.8, 7.8)
4.06 (d, 7.8)
—
2.47 (br d, 8.4)
3.57 (d, 11.4)
3.95 (d, 11.4)
is known to be
a-oriented in naturally occurring iridoid glyco-
5
6
7
8
9
10
33.3
95.0
72.4
80.2
48.1
62.1
sides.25,26 The stereochemistry of the ring fusion was cis, b-ori-
ented which was also confirmed by the large coupling constant
(J = 8.4 Hz) between H-9 (dH 2.47) and H-5 (dH 2.37). The ROESY
(Fig. 2) spectra showed a correlation signal between H-7 and H-9
indicating the b-configuration of H-7, thus confirming the
a-con-
figuration of the hydroxyl group attached to C-7. Moreover, the
1-O-Glc
10
20
30
40
50
60
4.66 (d, 7.8)
3.15 (dd, 8.1, 7.8)
3.26 (m)
3.26 (m)
3.34 (m)
3.62 (dd, 13.8, 3.0)
3.83 (d, 13.8)
3.38 (s)
98.9
74.7
78.1
71.6
78.1
62.7
ROESY correlation between H-10b (dH 3.57) and H-1 (dH 5.60) also
confirmed the
a-orientation of C-10 attached to C-8. The absence
of olefinic proton signals in the 1H NMR spectra indicated that no
double bond existed between C-3 and C-4 as described for com-
mon iridoids.25 In addition, the long range correlations between
proton H-3 (dH 5.23) and carbon C-10 (dC 62.1), and between pro-
ton H-10 (dH 3.95, 3.57) and C-3 (dC 95.7) indicated an ether link-
6-OMe
57.9
Assignments were done by HMQC, HMBC, 1H–1H COSY, and ROESY experiments.
age between C-3 and C-10 and the
a
-configuration of C-3. Finally,
a
the strong movement of the chemical shift (
D
dC-6 9.0) at C-6 to the
Measure at 600 MHz.
b
downfield of 1 (dC-6 95.0) in comparison with scutelloside27 (dC-6
86.0) indicated a methoxy group attached to C-6. These differences
were similar to those of methylcatalpol (3, dC-6 88.6) and catalpol28
Measure at 150 MHz.
(dC-6 79.6;
DdC-6 9.0). Furthermore, the long range correlation sig-
anomeric carbon observed at dC 98.9, four oxygenated methine car-
bons at dC 71.6–78.1, and one oxygenated methylene carbon at dC
62.7 were assigned to the sugar moiety. One methyl carbon at dC
57.9 ppm was assigned to the methoxy group and the nine remain-
ing carbons belonged to the aglycone moiety. These data suggested
that 1 had nine carbons in a catalpol-like iridoid structure linked to
a b-glucopyranosyl unit at C-1, which was also in agreement with
the correlations observed between anomeric proton H-10 (dH 4.66)
and C-1 (dC 93.3), and between proton H-1 (dH 5.60) and anomeric
carbon C-10 (dC 98.9) in the HMBC spectra (see Fig. 2). The coupling
constant of the anomeric proton was 7.8 Hz in doublet multiplicity
in the 1H NMR spectra which confirmed the b-configuration of glu-
nals between methoxy proton (dH 3.38, 6-OMe) and C-6 (dC 95.0),
H-6 (dH 3.68) and methoxy carbon (dC 57.9, 6-OMe) in HMBC spec-
tra also confirmed the methoxy group attached to C-6. The strereo-
chemistry of the methoxy group at C-6, however, was determined
to be the b-configuration due to the trans-orientation between pro-
tons H-6 and H-7 observed in the 1H NMR spectra with a coupling
constant of 7.8 Hz, and the ROESY correlation between H-6 (dH
3.68) and H-4a (dH 1.63). Based on the above data, compound 1
was determined to be a new compound named methylscutelloside
(Fig. 1). In a preliminary assay of atherosclerosis treatment using B.
officinalis, the isolated compounds were screened for their effects
on rat aortic VSMCs proliferation.29–31 The results showed that
cose. The presence of a
D
-glucose unit was further confirmed by
250
200
150
100
50
0
-50
-100
+
+
+
+
+
+
+
PDGF-BB (25 ng/mL)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Compound (50 µM)
Figure 3. Effects of isolated compounds on PDGF-BB-induced proliferation in rat aortic VSMCs. AG1295 (1
anti-proliferative agents. Data are expressed as Mean SD (n = 4, P <0.05 vs stimulus control).
lM), a PDGF-BB receptor inhibitor, was used as a positive control of

B. H. Tai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3462–3466
3465
120
80
40
0
*
*
*
**
**
**
**
**
**
**
**
**
**
**
+ + + -
Comp. 8
+
PDGF-BB (25 ng/mL)
+ + + - + + + - + + + - + + + -
+
+ + + - + + + -
AG1295
Comp. 1
Comp. 2
Comp. 3
Comp. 4 Comp. 5
Comp. 7
Concentration (µM)
Figure 4. Effects of selected compounds on PDGF-BB-induced proliferation in rat aortic VSMCs. AG1295 (1
lM), a PDGF-BB receptor inhibitor, was used as a positive control
of anti-proliferative agents. Data are expressed as Mean SD (n = 4, ⁄P <0.05, ⁄⁄P <0.01 vs stimulus control).
8. Lee, Y. J.; Kyoung, M. M.; Hwang, S. M.; Yoon, J. J.; Lee, S. M.; Seo, K. S.; Kim, J. S.;
Kang, D. G.; Lee, H. S. Am. J. Chin. Med. 2010, 38, 585.
9. Lee, Y. J.; Kim, J. S.; Kang, D. G.; Lee, H. S. Exp. Biol. Med. 2010, 235, 247.
10. Nishimura, H.; Sasaki, H.; Morota, T.; Chin, M.; Mitsuhashi, H. Phytochemistry
1989, 28, 2705.
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606.
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Takeda, Y. Chem. Pharm. Bull. 2004, 52, 1086.
14. Calis, I.; Yuruker, A.; Ruegger, H.; Wright, A. D.; Sticher, O. Helv. Chim. Acta
1993, 76, 2563.
15. Yamamoto, A.; Miyase, T.; Ueno, A.; Maeda, T. Chem. Pharm. Bull. 1991, 39,
2764.
16. Pistelli, L.; Bilia, A. R.; Marsili, A.; De-Tommasi, N.; Manunta, A. J. Nat. Prod.
1993, 56, 240.
17. Liu, Z.; Li, D.; Owen, N. L.; Grant, D. M.; Cates, R. G.; Jia, Z. Nat. Prod. Lett. 1995, 6,
157.
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2002, 16, 161.
19. Ebata, N.; Nakajima, K.; Hayashi, K.; Okada, M.; Maruno, M. Phytochemistry
1996, 41, 895.
20. Yamamoto, A.; Miyase, T.; Ueno, A.; Maeda, T. Chem. Pharm. Bull. 1993, 41,
1780.
21. Ding, N.; Yahara, S.; Nohara, T. Chem. Pharm. Bull. 1992, 40, 780.
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most of compounds significantly inhibited VSMCs proliferation at a
concentration of 50 M (Fig. 3). Of these, compounds 1–5, 7 and 8
l
showed the potent inhibitory effects, while compounds 9–14 did
not show inhibitory effects on VSMCs proliferation after treatment
with 25 ng/mL homodimer platelet-derived growth factor beta
polypeptide (PDGF-BB). Compounds 6, 15–20 showed the pattern
of cytotoxic effects. Based on the above results, compounds 1–5,
7 and 8 were then further tested for concentration-dependent ef-
fects on VSMCs (0–50
centration of 50 M (Fig. 4). Compound 1 significantly decreased
cells proliferation at 10, 30 and 50 M, with inhibition of 39.1%,
lM) as they showed no cytotoxicity at con-
l
l
42.0% and 79.6%, respectively. Compounds 7 and 8 showed moder-
ate inhibition by 22.6% and 21.2%, respectively, at a concentration
of 30
compounds showed significantly inhibition effects on the prolifer-
ation of VSMCs at a concentration of 10 M. These results clearly
lM. With the exception of compounds 1 and 3, none of these
l
indicated that the iridoids from B. officinalis have potent inhibitory
effects on rat aortic VSMC proliferation and may be useful for the
treatment of atherosclerosis.
23. Dried flowers of B. officinalis Maxim. (2.0 kg) were extracted with methanol at
room temperature three times. After removal of the solvent under reduced
pressure, the crude extract (94.37 g) was dissolved in 1.0 L of H2O to form a
suspension that was successively partitioned with dichloromethane (CH2Cl2),
ethyl acetate (EtOAc), and n-butanol (BuOH) to give CH2Cl2 (10.77 g), EtOAc
(12.70 g), and BuOH (27.29 g) extracts, respectively. The BuOH extract was
then subjected to column chromatography using SiO2 (70–230 mesh), eluting
with Me2CO/CHCl3/H2O (3:1:0.2, v/v/v) to give five fractions (1a–e). Repeated
silica gel column chromatography of fraction 1a with CH2Cl2/MeOH/H2O
Acknowledgments
We would like to thank the Ministry of Science and Technology,
Vietnam, for financial support in form of the Vietnam-Korea Interna-
tional Collaboration Project (No. 30/823/2007/HD-NDT). This work
was supported partly by Priority Research Centers Program through
the National Research Foundation of Korea (NRF) funded by the Min-
istry of Education, Science and Technology (2009-0093815), Repub-
lic of Korea.
(4.5:1:0.1, v/v/v) and Me2CO/CHCl3/H2O (3.5:1:0.1) to give compounds
3
(13 mg) and 4 (18 mg). Fraction 1b was then subjected to a silica gel column
using an isocratic eluent of CHCl3/Me2CO/MeOH/H2O (3:2:1:0.1, v/v/v/v) and
further by YMC column with MeOH/H2O (1:2, v/v) afford compounds 1(9 mg),
2 (15 mg) and 5 (12 mg). Compounds 15 (14 mg), 16 (12 mg), and 19 (15 mg)
were isolated from fraction 1d using a silica gel column with CH2Cl2/MeOH/
H2O (4:1:0.1, v/v/v), further by YMC column and eluting with Me2CO/H2O (2:1,
v/v). Finally, fraction 1e was chromatographed using a YMC column with
MeOH/H2O (2:3, v/v) to afford five fractions (2a–e). Compounds 17 (90 mg), 18
(30 mg), and 20 (18 mg) were obtained from YMC gel column chromatography
of sub-fractions 2d and 2e. The purity of isolated compounds was more than
97% by high-performance liquid chromatography (HPLC) analysis.
References and notes
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24. Compound 1 (2.0 mg) was dissolved in 1.0 N HCl (dioxane/H2O, 1:1, v/v,
1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic solution
was neutralized with silver carbonate and the solvent thoroughly driven out
under N2 gas overnight. After extraction with ethyl acetate, the aqueous layer
was concentrated to dryness using N2 gas. The residue was dissolved in 0.1 mL
of dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine

3466
B. H. Tai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3462–3466
(0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at
Modified Eagle Medium (DMEM) containing 10% FBS for 48 h. The cells were
then cultured with serum-free medium containing various isolated compounds
for 24 h. Cells were then stimulated with 5% FBS for another 24 h. WST-1
reagent was added to each well at 22 h, and the plate was further incubated the
plate at 37 °C for 2 h. The optical density was read at 450 nm in an ELISA reader
(Molecular, USA). Cell proliferation was measured by direct counting. Initially,
the cells were seeded into 12-well culture plates at 4 ꢂ 104 cells/well and
cultured in DMEM containing 10% FBS at 37 °C for 24 h. When cells reached up
to 70% confluence, the medium was replaced with serum-free DMEM
60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added,
followed by heating at 60 °C for 1.5 h. The dried product was partitioned with
n-hexane and H2O (0.1 mL, each), and the organic layer was analyzed by gas
liquid chromatography (GC): Column: column SPB-1 (0.25 mm ꢂ 30 m);
detector FID, column temp 210 °C, injector temperature 270 °C, detector
temperature 300 °C, carrier gas He. The absolute configuration of the
monosaccharide was confirmed to be
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authentic sugar derivative samples prepared in the same manner ( -glucose
derivative tR 14.11 min, -glucose derivative tR 14.26 min).
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D
containing various concentrations (0–50 lM) of isolated compounds. After
L
24 h incubation, they were treated with 25 ng/ml PDGF-BB (Upstate
Biotechnology, NY, USA) and further incubated for another 24 h. The cells per
well were collected after treatment with trypsin-EDTA and then counted using
a hemocytometer.
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29. VSMC proliferation was assessed using the WST-1 (CCK-8) assay kit according
to the manufacturer’s instructions. In brief, cells were seeded at
a
concentration of 4 ꢂ 104 cells/well in 96-well plates and grown in Dulbecco’s