Chemical components from the seeds of: Catalpa bungei and their inhibitions of soluble epoxide hydrolase, cholinesterase and nuclear factor kappa B activities

Article abstract of DOI:10.1039/c6ra04207d

Two new chlorinated iridoids, named bungosides A (1) and B (2), were isolated from the seeds of Catalpa bungei (family Bignoniaceae). Their structures were elucidated on the basis of NMR analysis. Twenty known compounds (3-22) were also characterized, including in one case (6-O-p-hydroxybenzoylglutinoside, 3) the assignment of the absolute configuration by employing electronic circular dichroism (CD) and time-dependent density functional theory (TDDFT) calculations. Compounds 1-3 were unusual cage-like iridoids with an oxygen bridge between C-3 and C-10. Among the isolates, ursolic acid lactone (14) was the most active soluble epoxide hydrolase (sEH) inhibitor with an IC₅₀ 19.1 ± 0.8 μM. In addition, d-pinoresinol (19) and ladanein (21) displayed selectively inhibitory effects on butyrylcholinesterase (BChE) with IC₅₀ of 31.5 ± 0.4 μM and 33.0 ± 2.3 μM, respectively, but not acetylcholinesterase (AChE) activity, and only compound 18 suppressed NF-κB activity in HepG2 cells (IC₅₀ 21.53 ± 2.37 μM).

Full text of DOI:10.1039/c6ra04207d

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PAPER
Chemical components from the seeds of Catalpa
bungei and their inhibitions of soluble epoxide
hydrolase, cholinesterase and nuclear factor kappa
B activities†
Cite this: RSC Adv., 2016, 6, 40706
Hao-Yu Tang,a Meng-Meng Bai,a Jun-Mian Tian,a Gennaro Pescitelli,b Trpimir Ivsic,bc
ˇ ´
a
Xiao-Hua Huang, Hyunwoo Lee,d Ya Nan Son,d Jang Hoon Kim,d Young Ho Kimd
*
a
*
and Jin-Ming Gao
Two new chlorinated iridoids, named bungosides A (1) and B (2), were isolated from the seeds of Catalpa
bungei (family Bignoniaceae). Their structures were elucidated on the basis of NMR analysis. Twenty
known compounds (3–22) were also characterized, including in one case (6-O-p-
hydroxybenzoylglutinoside, 3) the assignment of the absolute configuration by employing electronic
circular dichroism (CD) and time-dependent density functional theory (TDDFT) calculations. Compounds
1–3 were unusual cage-like iridoids with an oxygen bridge between C-3 and C-10. Among the isolates,
ursolic acid lactone (14) was the most active soluble epoxide hydrolase (sEH) inhibitor with an IC50
19.1 ꢀ 0.8 mM. In addition, D-pinoresinol (19) and ladanein (21) displayed selectively inhibitory effects on
butyrylcholinesterase (BChE) with IC50 of 31.5 ꢀ 0.4 mM and 33.0 ꢀ 2.3 mM, respectively, but not
acetylcholinesterase (AChE) activity, and only compound 18 suppressed NF-kB activity in HepG2 cells
(IC50 21.53 ꢀ 2.37 mM).
Received 16th February 2016
Accepted 18th April 2016
DOI: 10.1039/c6ra04207d
www.rsc.org/advances
becomes an effective pathway to treat cardiovascular disease.
Alzheimer's disease (AD) is one of the most common neurode-
Introduction
generative diseases in elderly population. Reduced synthesis of
the neurotransmitter acetylcholine is thought to be a main
cause of AD.3 Therefore, cholinesterase inhibitors became an
important cognitive enhancing treatment based on the facts
that they could increase the content of acetylcholine at cholin-
ergic synapses. Both acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BChE) participate in hydrolyzing brain
acetylcholine in different manners.4 AChE and BChE have thus
been the main targets to prevent AD.
Inammation contributes to the pathogenesis of cancer,
cardiovascular diseases, and type-2 diabetes mellitus.5 The
nuclear factor kappaB (NF-kB) is a key regulator of many pro-
inammatory pathways.5 NF-kB normally resides in the cyto-
plasm, where it is retained by association with the endogenous
inhibitor of kappa B (IkB) protein.6 However, when activated by
inammatory stimuli such as tumor necrosis factor alpha (TNF-
a), injury, or other stress-related stimuli, NF-kB is released,
translocates to the nucleus and binds to DNA resulting in the
transcription of proinammatory target genes.7 Therefore,
inhibitors targeting NF-kB signaling are considered as potential
candidates for both prevention and therapy of inammation.
The Catalpa plants of Bignoniaceae family include about 13
species in the world and are mainly distributed in America and
East Asia. Previous phytochemical investigation indicated that
Soluble epoxide hydrolase (sEH) is an important bifunctional
enzyme with potent biological activities on vascular and renal
systems. It can hydrolyze epoxyeicosatrienoic acids (EETs),
metabolites of arachidonic acid generated by epoxygenase CYP
enzymes, into dihydroxyeicosatrienoic acids (DHETs).1 The
former produce signicant biological effects in vascular and
renal systems while the latter don't show much benecial
function. EETs are endothelium-derived hyperpolarizing factors
(EDHFs) which act as regulators of vascular function.2 The
cardiovascular effects of EETs include vasodilation, anti-
migratory actions on vascular smooth muscle cells and anti-
inammatory action.1 Therefore, the inhibition of sEH
aShaanxi Key Laboratory of Natural Products & Chemical Biology, College of Science,
Northwest A&F University, Yangling 712100, P. R. China. E-mail: jinminggao@
nwsuaf.edu.cn; x.h.huang@163.com; Tel: +86-29-87092515
b
`
Dipartimento di Chimica e Chimica Industriale, Universita di Pisa, via Moruzzi 3, I-
56124 Pisa, Italy
c
ˇ
´
Division of Organic Chemistry and Biochemistry, Rudꢀer Boskovic Institute, 10000
Zagreb, Croatia
dCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of
Korea
† Electronic supplementary information (ESI) available: HR-ESI-MS, NMR, and IR
spectra of compounds 1–3, bioactive data of compounds. See DOI:
10.1039/c6ra04207d
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Catalpa plants contain iridoids and naphthoquinones as the tertiary hydroxyl group (dC 79.9), one oxymethylene (dH 4.09 and
main constituents with various pharmacological properties 3.72, dC 62.1), an aromatic unit, and a b-glucopyranosyl moiety.
such as antifungal, cytotoxic, antitumor, and antiplasmodial These assignments were further conrmed by the HMBC
activities.8–11 Catalpa bungei C. A. Mey is widely distributed in correlations forming the cyclopentanopyrane ring skeleton, a p-
China, and the fruits of the tree have been used in folk medicine hydroxybenzoyl and a b-glucopyranosyl residue. The 1H NMR
for edema, nephritis, cystitis, eczema, leprosy and gastric spectrum (Table 1) showed the presence of a doublet signal at
cancer.12 However, few secondary metabolites from the seeds of dH 5.70 (J ¼ 2.1 Hz, 1H) that is a characteristic signal for H-1 of
this plant have been reported.
an iridoid.35 The 13C signals at dC 98.9, 74.2, 78.1, 71.6, 78.1, and
In continuation of our investigation on the bioactive 62.7 in the 13C NMR spectrum were characteristic for a b-D-
constituents from traditional Chinese medicines in the Qinba glucopyranosyl residue. The b-conguration for the glucose was
Mountains,13–16 we carried out chemical investigations on the supported from the large coupling constant (J ¼ 8.0 Hz) for the
seeds of C. bungei and evaluated their sEH, AChE and BChE anomeric doublet signal at dH 4.73 (H-10). HMBC correlations of
inhibitory activities. In this study, two new compounds (1, 2), dH 5.70 (H-1) and dC 98.9 (C-10), and dH 4.73 (H-10) and dC 93.1 (C-
along with 20 known natural compounds that belong to various 1) indicated that the glucopyranosyl unit was attached to C-1 of
groups of iridoids, triterpenoids, phenylethanoid glycosides, the aglycone. The chemical shi of C-7 at dC 70.4 indicated that
and lignans, were isolated from the seeds of C. bungei. Their the Cl atom was located at C-7.36,37 Detailed interpretation of the
inhibitions of sEH, AChE and BChE were also assessed. Herein, 1H and 13C NMR spectral data (Table 1) indicated that 3
we report the isolation and structural elucidation of the new contains a p-hydroxybenzoyl group [dH 7.92 (d, J ¼ 8.8 Hz, H-
compounds (1, 2), as well as enzymatic inhibitory activity of 200,600), 6.87 (d, J ¼ 8.8 Hz, H-300,500); dC 168.6 (C-700), 164.3 (C-400),
these compounds. The present study could lay a foundation for 121.4 (C-100)]. The attachment of the p-hydroxybenzoyl unit on
the medicinal development of C. bungei.
C-6 (dC 87.9) of the aglycone was established from the HMBC
correlation between H-6 at dH 5.13 and the ester carbonyl
carbon (dC 168.6) of the p-hydroxybenzoyl group (Fig. 2).
The relative stereochemistry at C-1, C-5, and C-9 was deter-
Results and discussion
The EtOAc-soluble fraction of a 75% EtOH extract of seeds of mined to be the same as found in other iridoid glucosides based
C. bungei, was chromatographed by means of different chro- on the small coupling of J1,9 (2.1 Hz) and the large coupling of
matographic techniques using normal-phase silica gel, J5,9 (9.8 Hz). The conguration of the 8-OH was conrmed to be
reversed-phase C18 gel, Sephadex LH-20 and RP-HPLC to afford b from the C-9 chemical shi (dC 48.3), which is a characteristic
three new (1 and 2) and 20 known compounds (Fig. 1).
of assignment of the conguration of the 8-OH.38 This
The identication of the known compounds, des-p-hydroxy- b-conguration causes the deshielding of C-9 in comparison to
benzoyl kisasagenol B (4),11 rehmaglutin C (5),17 des-p-hydroxy- its a counterpart in the following pairs of C-8 isomers, 10-des-
benzoyl-3-deoxycatalpin (6),18 6-O-(p-hydroxybenzoyl)-D-glucopyr- cinnamoylglobularimin and 10-des-cinnamoylglobularinin,39
anose (7),19 6-O-[(E)-feruloyl]-D-glucopyranose (8),20 6-O-p-cou- and gardenoside and monotropein methyl ester.38 The cong-
maroyl-D-glucopyranose (9),21 ursolic acid (10),22 rotundic acid uration of the H-3 proton was determined to be b on the basis of
(11),23 arjunic acid (12),24 2a,3a,19a-trihydroxy-12-oleanen-28-oic the formation of the intramolecular acetal of C-3 with the C-10
acid (13),25 ursolic acid lactone (14),26 martynoside (15),27 verbas- OH.40 As a result, compound 3 was elucidated as a rigid tricyclic
coside (kusaginin or acteoside, 16),28 balanophonin (17),29 9a- iridoid. Similar iridoids possessing a cage-like skeleton have
hydroxysesamin (18),30 D-pinoresinol (19),31 9-methoxy-a-lapa- been isolated from Picrorhiza kurroa,41 Catalpa bignonioides,42
chone (20),32 ladanein (21),33 and p-hydroxybenzoic acid (22),34 was and Rehmannia glutinosa43 and also obtained through bromi-
conrmed by comparing their physical and spectroscopic data nation and dehydration of the catalposide extracted from the
with those reported in the literature. The known compounds roots of Picrorhiza kurroa.44
included various types of iridoids, triterpenoids, phenylethanoid
Compound 3 is a derivative of glutinoside, which was iso-
glycosides, and lignans. To the best of our knowledge, lated previously from the dried root of Rehmannia glutinosa.45 It
compounds 1–22 (except 3) were isolated from C. bungei for the had not been isolated previously as a pure natural form but as
rst time, of which 7–9, 11–14, and 17–19, in particular the a semi-synthetic hexaacetate, obtained through acetylation of
occurrence of ursane and oleanane triterpenes, were rst isolated crude extract of Catalpa species.11 Here, the NMR data of 3 are
from the genus of Catalpa.
reported for the rst time. The structure of this compound was
Compound 3 was obtained as a white powder. The molecular conrmed by detailed analysis of the 2D-NMR data (HSQC,
formula was established as C22H27ClO12 by HR-ESI-MS at m/z HMBC, 1H–1H COSY, and NOESY spectra) (Fig. 2). On the basis
541.1081 [M + Na]+, indicating nine degrees of unsaturation. Its of the above analysis, the structure of 3 was determined to be
IR spectrum indicated the presence of hydroxy (3400 cmꢁ1), 6-O-p-hydroxybenzoylglutinoside. In order to further determine
conjugated carbonyl ester (1693 cmꢁ1), and aromatic (1607 and the structure of 3, we prepare its hexaacetate (3a), whose NMR
1514 cmꢁ1) functionalities in the molecule. The 13C NMR and data are in good agreement with published data.11 Since the
DEPT data (Table 1) exhibited 22 signals, including three absolute conguration of the hexaacetate of 3 was not directly
methylenes, een methines, and four quaternary carbons. The determined in the original paper,11 we decided to assign it
1H and 13C NMR spectroscopic data suggested the presence of independently by means of electronic circular dichroism (CD)
two acetal groups (dH 5.70, dC 93.1 and dH 5.36, dC 95.9), one spectroscopy. The experimental UV absorption and CD
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Fig. 1 Structures of isolated compounds 1–22 from C. bungei.
spectrum of (ꢁ)-3 in acetonitrile are reported in Fig. 3. The UV based on a Monte Carlo conformational search followed by
consists of two main bands mainly associated with the elec- geometry optimizations with DFT method. The resulting set of
tronic transitions of the p-hydroxy benzoate chromophore. conformations was composed by two main families, one devoid
Correspondingly, the CD spectrum shows three major bands of any intramolecular hydrogen bond, and one showing
above 220 nm and a positive tail at shorter wavelengths. To consistently an intramolecular hydrogen bond between glucose
simulate the CD spectrum by TDDFT calculations,46 we rst 60-OH and the ester carbonyl group. Such a hydrogen bond leads
generated
a
set of structures reproducing low-energy to the formation of a 14-membered cycle which is quite dis-
conformers in solution, using a well-established procedure favored by entropic factors, and it is likely to be a computational
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Table 1 NMR spectroscopic data of compounds 1–3 (in MeOH-d4)
1
2
3
No.
dC
dH (multi. J in Hz)
dC
dH (multi. J in Hz)
dC
dH (multi. J in Hz)
1
3
4
93.1 d 5.70 d, 2.1
95.9 d 5.35 d, 3.0
34.4 t 2.49 dd, 13.7, 8.3; 2.13 dd, 13.7, 3.0 34.4 t 2.43 dd, 13.6, 8.5; 2.07 dd, 13.6, 2.9 34.4 t 2.49 dd, 13.5, 8.5; 2.16 dd, 13.5, 3.0
93.1 d 5.64 d, 2.0
95.9 d 5.30 d, 2.9
93.1 d 5.70 d, 2.1
95.9 d 5.36 d, 3.0
5
6
7
8
34.8 d 2.40, m
87.7 d 5.05 dd, 8.3, 2.7
70.4 d 4.46 d, 8.3
79.9 s
34.7 d 2.34 m
87.7 d 4.99 dd, 8.3, 2.7
70.4 d 4.41 d, 8.1 Hz
79.9 s
34.7 d 2.43, m
87.9 d 5.13 dd, 8.3, 2.7
70.4 d 4.54 dd, 8.3, 0.9
79.9 s
9
48.3 d 2.65 d, 9.9
62.1 t 4.08 d, 11.9; 3.72 d, 11.9
99.0 d 4.73 d, 8.0
74.7 d 3.19 dd, 9.1, 8.0
78.2 d 3.41, m
71.6 d 3.31, m
78.1 d 3.31, m
62.7 t 3.70, m; 3.90, m
127.6 s
111.9 s 7.25 d, 1.8
149.4 s
48.3 d 2.59 d, 9.9
62.1 t 4.02 d, 11.8; 3.65 m
98.9 d 4.67 d, 8.3
74.7 d 3.14 t, 8.3
78.1 d 3.35, m
48.3 d 2.67 d, 9.8
62.1 t 4.09 dd, 12.2, 0.8; 3.72 d, 12.2
98.9 d 4.73 d, 8.0
74.2 d 3.18 dd, 9.0, 8.0
78.1 d 3.40, m
10
10
20
30
40
500
600
100
200
300
400
5
71.6 d 3.26, m
78.1 d 3.28, m
71.6 d 3.31, m
78.1 d 3.30, m
62.7 t 3.85, m; 3.65, m
127.0 s
131.3 d 7.46 d, 8.6
116.9 d 6.79 d, 8.6
161.5 s
116.9d 6.79 d, 8.6
131.3 d 7.46 d, 8.6
147.2 d 7.62 d, 15.8
114.5 d 6.36 d, 15.9
168.9 s
62.7 t 3.70, m; 3.91, m
121.4 s
132.9 d 7.92 d, 8.8
116.4 d 6.87 d, 8.8
164.3 s
116.4 d 6.87 d, 8.8
132.9 d 7.92 d, 8.8
168.6 s
150.8 s
116.5 d 6.85 d, 8.2
124.3 d 7.13 dd, 8.2, 1.8
147.5 d 7.68 d, 15.9
114.9 d 6.45 d, 15.9
168.8 s
600
700
800
900
OCH3 56.5 q 3.93, s
Fig. 2 Selected HMBC, COSY and NOESY correlations for 1–3.
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measured for (ꢁ)-3. Therefore, we may assign 6-O-p-hydroxy-
benzoyl glutinoside the absolute conguration (ꢁ)-(1S,3R,5R,6S,
7R,8S,9S). Not surprisingly, this is the same conguration re-
ported for the analogs of glutinoside,45 6-O-p-hydroxybenzoyl
asystasioside E11 and piscroside A,48 all of which are ultimately
related to rehmaglutin D.49
Compound 1 was obtained as a white powder. The molecular
formula C25H31ClO13 was established by HR-ESI-MS at m/z
597.1343 [M + Na]+, requiring 10 degrees of unsaturation. Its IR
spectrum indicated the presence of hydroxy (3393 cmꢁ1) and
a,b-unsaturated ester carbonyl (1697 cmꢁ1) groups, and an
aromatic moiety (1598, 1516 cmꢁ1) in the molecule. The 13C
NMR and DEPT data (Table 1) showed the presence of one
methyl, three methylenes, sixteen methines, and ve quaternary
carbons. The presence of a sugar moiety was evidenced by a set
1
of signals observed in the H NMR spectrum between dH 4.73
and 3.19, and signals observed in the 13C NMR spectrum
(Table 1) at dC 62.7, 71.6, 74.7, 78.1, 78.2, and 99.0. The coupling
constant of the anomeric proton at dH 4.73 (d, J ¼ 8.0 Hz, H-10)
indicated the b-conguration of the glucopyranose. Acid
hydrolysis afforded D-glucose based on GC analysis.
Detailed interpretation of the 1H and 13C NMR spectral data
(Table 1) indicated that the chemical structure of compound 1
was very similar to those of 6-O-p-hydroxybenzoylglutinoside (3),
except for the presence of an (E)-p-feruloyl group [dH 7.25 (d, J ¼
1.8 Hz, H-200), 7.13 (dd, J ¼ 8.2, 1.9 Hz, H-600), 6.85 (d, J ¼ 8.2 Hz,
H-500), 7.68 (d, J ¼ 15.9 Hz, H-700), 6.45 (d, J ¼ 15.9 Hz, H-800); dC
168.8 (C-900), 127.6 (C-100)] in 1. The location of the feruloyl group
at C-6 (dC 87.7) was supported by the correlation of H-6 (dH 5.05,
dd, J ¼ 8.3, 2.7 Hz) to the ester carbonyl at C-900 (dC 168.8) in the
HMBC spectrum (Fig. 2). The HMBC correlations of the H-1
acetal signal at dH 5.70 (d, J ¼ 2.1 Hz) to the C-10 anomeric
carbon at dC 99.0 and the C-3 acetal carbon at dC 95.9 indicated
that the glucose residue was attached to C-1 of the aglycon,
which was further linked to C-3 through an ether linkage. In
addition, on the basis of the downeld shi of C-10 (dC 62.1)
Fig. 3 Experimental UV absorption and CD spectra of compound
(ꢁ)-3 compared with TDDFT-calculated spectra at B3LYP/TZVP level
on B3LYP/6-31G(d) geometries for (1S,3R,5R,6S,7R,8S,9S)-3, as
Boltzmann average at 300 K for all structures with population >1%.
Experimental spectra: concentration 0.54 mM in acetonitrile; cell
path-length 0.05 cm. Calculated spectra: Gaussian band-shape with
0.2 eV exponential half-width; scaled by a factor 3; no shift applied.
Vertical bars represent computed rotational strengths. Inset: lowest-
energy DFT conformer of compound (1S,3R,5R,6S,7R,8S,9S)-3
showing no 60-OH/O]C intramolecular hydrogen bond.
artifact.47 In fact, the NOESY spectrum of 3 did not show any and HMBC correlation from H2-10 (dH 4.08, 3.72) to C-3 (dC
cross-peak expected for such a cyclic structure, e.g. between 95.9), the acetal carbon C-3 was determined to be linked to C-10
H-200 and CH2-60. Therefore, we neglected these hydrogen- through an oxo bridge. The methine carbon at dC 70.4 was
bonded conformers from our set; however we also checked assigned to C-7, attached to one chlorine atom,36,37 which was
that the inclusion of hydrogen-bonded conformers in TDDFT conrmed from the HMBC correlation of H2-10 to C-7 and from
calculations would not affect the nal assignment (results not HR-ESI-MS data.
shown). As a consequence, we obtained a restricted set of low-
The relative conguration of 1 was conrmed by a NOESY
energy conformers including 9 structures with relative ener- experiment (Fig. 2) as well as from biosynthetic considerations.
gies within 1.8 kcal molꢁ1 and populations >1% at room According to molecular modeling of this compound, the oxo
temperature; the lowest-energy conformer is shown in the inset bridge from C-3 to C-10 could only be a-oriented, and the 8-OH
in Fig. 3. All 9 conformers share the same conformation of the and H-9 could only be b-oriented. In the NOESY spectrum, the
polycyclic skeleton, which seems to be very rigid, and also the correlations between H-7 and H-5b, H-7 and H-9b, and H-6 and
same conformation of the p-hydroxy benzoate group, dening H-1a indicated that H-7 and H-6 were in b- and a-orientation,
a dihedral angle (O])C–O–C-6–H-6 around ꢁ40ꢂ. The 9 struc- respectively, thereby establishing the stereochemistry of the Cl
tures differ only in the ip of 400-OH and in the rotamerism of and the feruloyl group as a and b, respectively. In addition, the
the glucose OH groups. Accordingly, the CD spectra calculated chemical-shi as well as coupling-constant values of the iridoid
with TDDFT method at B3LYP/TZVP level for the whole moiety of 1 (Table 1) were very similar to those reported for
conformational set were similar to each other. The Boltzmann- compound 3, suggesting that 1 possesses the same congura-
weighted average of the spectra is shown in Fig. 3. The CD tion (ꢁ)-(1S,3R,5R,6S,7R,8S,9S) as 3. The structure of this
spectrum calculated for enantiomer (1S,3R,5R,6S,7R,8S,9S)-3 is compound was conrmed by detailed analysis of the 2D-NMR
in very good agreement with the experimental CD spectrum data including its HSQC, HMBC, 1H–1H COSY, and NOESY
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spectra (Fig. S5–S8 and Table S1 in the ESI†). Consequently, the over 50% on sEH. Their IC50 values were determined in the
structure of 1 was determined to be 6-O-feruloylglutinoside, following work (Table 2). Among the seven compounds tested,
named bungoside A.
Compound was assigned
14 showed the most active inhibitory activity on sEH with an
molecular formula of IC50 value of 19.1 ꢀ 0.8 mM; other compounds exhibited weak
2
a
C
24H29ClO12, as established by the HR-ESI-MS at m/z 567.1243 activity with IC50 values of 30–50 mM. In addition, kinetic curve
[M + Na]+. The IR, UV, and NMR spectroscopic data (Table 1) of 2 of 14 on sEH showed it inhibits sEH by a mixed mode with
closely resembled those of 1, except that the C-6 substituent inhibition constant Ki value 7.9 ꢀ 1.7 mM (Fig. 4). Compounds
group in 2 is a (E)-p-coumaroyl group [dH 7.46 (2H, d, J ¼ 8.6 Hz, 19 and 21 were found to display satisfactory inhibitory effects on
H-200,600), 6.79 (2H, d, J ¼ 8.6 Hz, H-300,500), 7.62 (1H, d, J ¼ 15.9 AChE and BChE. Then only components 19 and 21 were tested
Hz, H-700), 6.36 (1H, d, J ¼ 15.9 Hz, H-800); dC 168.9 (C-900), 127.0 their IC50 values on the two enzymes, and the results are
(C-100)] instead of an (E)-feruloyl group. This was supported by observed in Table 2. Compounds 19 and 21 displayed selectively
the correlation of H-6 at dH 4.99 (dd, J ¼ 8.3, 2.7 Hz) to the ester moderate inhibitory effects on butyrylcholinesterase (BChE)
carbonyl at C-900 (dC 168.9) in the HMBC spectrum. The structure with IC50 values of 31.5 ꢀ 0.4 mM and 33.0 ꢀ 2.3 mM, respec-
of this compound was conrmed by detailed analysis of the 2D- tively, but not acetylcholinesterase (AChE) activity. Moreover,
NMR data including its HSQC, HMBC, 1H–1H COSY, and kinetic curve of 19 and 21 on BChE (Table 2) showed they
NOESY spectra (Fig. S14–S17 and Table S2 in the ESI†). ROESY inhibited this enzyme by a mixed mode with Ki values of 7.8 ꢀ
correlations suggested the same relative conguration as 1 and 0.3 and 11.3 ꢀ 2.9 mM, respectively (Fig. 5). To the best of our
3. Similarly, compound 2 was suggested to possess the same knowledge, this is the rst report on 14 as sEH inhibitor as well
absolute conguration (ꢁ)-(1S,3R,5R,6S,7R,8S,9S) due to the as on the inhibition of 21 on BChE.
same sign of specic rotation as 3 and 1 as well as to their same
Furthermore, compounds 1–22 (except 9) were assessed for
biogenetic relationships. Consequently, the structure of 2 was their effects on TNFa-induced NF-kB transcriptional activity in
established to be (ꢁ)-(1S,3R,5R,6S,7R,8S,9S)-6-O-(E)-p-coumar- human hepatocarcinoma (HepG2) cells using an NF-kB-
oylglutinoside, named bungoside B.
luciferase assay. As shown in Table 2, among the tested
The compounds 1–22 (except 9) were tested for their inhib- compounds, only compound 18 signicantly inhibited TNFa-
itory effects on soluble epoxide hydrolase,16 acetylcholinesterase induced NF-kB transcriptional activity with IC50 values of 21.53
(AChE) and butyrylcholinesterase (BChE) at 100 mM (Table S4†). ꢀ 2.37 mM, while others were weak little or inactive. Apigenin
Compounds 1, 8, 10, 14, 15, 17 and 20 showed inhibitory rate (IC50 1.64 mM) was used as a positive control. To date, there is no
Table 2 sEH, AChE, BChE and NF-kB inhibitory activities of compounds
sEH inhibitory activity
AChE inhibitory activity
BChE inhibitory activity
NF-kB inhibitory activity
IC50c,d (mM)
compda
IC50a,c (mM)
IC50c,d (mM)
IC50c,d (mM)
1
2
50.4 ꢀ 2.0
NTe
NT
NT
NT
ND
>100
NT
3
4
NT
NT
NT
NT
NT
NT
52.78 ꢀ 2.26
>100
5
NT
NT
NT
ND
6
NT
NT
NT
ND
7
NT
NT
NT
ND
8
9
44.6 ꢀ 3.4
NT
NT
NT
—
>100
—
—
10
11
36.6 ꢀ 7.7
NT
NT
NT
NT
33.93 ꢀ 2.53
NT
>100
12
13
14
15
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
96.4 ꢀ 2.35
98.0 ꢀ 2.36
32.88 ꢀ 2.46
46.91 ꢀ 2.74
>100
19.1 ꢀ 0.8
30.6 ꢀ 2.0
16
NT
NT
NT
17
18
19
20
21
22
53.8 ꢀ 1.2
NT
NT
NT
NT
NT
NT
ND
NT
21.53 ꢀ 2.37
NT
31.59 ꢀ 0.44
>100
37.0 ꢀ 1.4
NT
31.64 ꢀ 2.20
52.4 ꢀ 2.25
>100
—
—
NT
86.36 ꢀ 0.93
33.09 ꢀ 2.34
NT
NT
NT
AUDAb
Tacrineb
Apigeninb
10.9 ꢀ 1.2 nM
—
—
—
—
207.84 ꢀ 4.29 nM
7.46 ꢀ 0.86 nM
—
—
1.64 ꢀ 0.19
a
All compounds were examined in a set of experiments three times. b Positive control. c Statistical signicance was determined by one-way analysis
d
of variance followed by Dunnett's multiple comparison test, P < 0.05 versus control. Data were expressed as mean ꢀ SD of at least three
e
experiments performed in triplicate. NT not tested.
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Fig. 4 Lineweaver–Burk plot for the sEH inhibition of compound 14 (A). Dixon plot of sEH inhibition by 14 (B).
Fig. 5 Lineweaver–Burk plot for BChE inhibition of compounds 19 (A) and 21 (B); Dixon plot of BChE inhibition by 19 (C) and 21 (D).
literature on the anti-inammatory effects of compound 18. B (2), as well as 19 compounds, were isolated from the seeds of
These results led us to conclude that ursolic acid lactone (14) and Catalpa bungei. Among the isolates, ursolic acid lactone (14)
9a-hydroxysesamin (18) from the seeds of Catalpa bungei exhibit showed a potential inhibitory activity against soluble epoxide
signicant anti-inammatory effects by inhibition of sEH enzyme hydrolase (sEH), and 9a-hydroxysesamin (18) signicantly
and TNFa-induced NF-kB activation, and that D-pinoresinol (19) inhibited TNFa-induced NF-kB transcriptional activity.
and ladanein (21) inhibit BChE. These results also provide Furthermore, D-pinoresinol (19) and ladanein (21) displayed
scientic support for the use of Catalpa bungei in the prevention marked inhibitory effects on BChE. Our observations provide
of cancer, inammatory and neurodegenerative diseases.
scientic support for the use of Catalpa bungei in the
In conclusion, three cage-like chlorinated iridoid gluco- prevention of cancer, inammatory and neurodegenerative
sides (1–3) including two new bungoside A (1) and bungoside diseases.
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The solvent was evaporated under reduced pressure to obtain
a crude EtOH extract (1.042 g), which was suspended in H2O
and partitioned successively with petroleum ether (PE), EtOAc,
and n-BuOH. The EtOAc fraction (ca. 250 g) was chromato-
graphed on silica gel over CC eluting with EtOAc/MeOH (20 : 1,
15 : 1, 10 : 1, 7 : 1, 5 : 1) to give ve fractions 1–5. Fraction 4 (16
g) was subjected to silica gel CC using CHCl3/MeOH (10 : 1–
2 : 1) to give four fractions 4.1–4.4. Fraction 4.1 was fractionated
on Sephadex LH-20 with MeOH and then further puried by
a silica gel column using PE/acetone (7 : 1) to afford 10 (17 mg).
Fraction 4.2 was loaded to silica gel CC using CHCl3/MeOH
(5 : 1–2 : 1) as eluent to give four fractions (4.2.1–4.2.4). Fraction
4.2.2 was puried by RP-18 CC using MeOH/H2O (0 : 10–5 : 5) as
a solvent to give 4 (88 mg) and 15 (27 mg). Fraction 4.2.3 was
rechromatographed over RP-18 column (30% methanol) and
further puried by RP-HPLC using MeOH/H2O (25 : 75, ow
rate: 3 mL minꢁ1) to afford 1 (18.5 mg, tR ¼ 37.5 min) and 2 (17.1
mg, tR ¼ 42.5 min).
Experimental
General experimental procedures
Optical rotations were measured with a DKSH Automatic
A21101 polarimeter. UV spectra were obtained using a Thermo
Scientic Evolution 300 UV-VIS Spectrophotometer. 1H and 13
C
NMR spectra were recorded on 400 MHz and 500 MHz NMR
instruments (Bruker Daltonics Inc., Bremen, Germany).
Chemical shis were reported using solvent residual peak as the
internal standard. ESI-MS spectra were performed on a Thermo
Fisher LTQ Fleet instrument spectrometer (Thermo Fisher
Scientic Inc., MA, U.S.). Column chromatography (CC) was
performed on silica gel (90–150 mm) (Qingdao Marine Chemical
Inc., Qingdao, China), Sephadex LH-20 (40–70 mm) (Amersham
Pharmacia Biotech AB, Uppsala, Sweden), and Lichroprep RP-18
gel (40–63 mm; Merck, Darmstadt, Germany). GF254 plates
(Qingdao Marine Chemical Inc., Qingdao, China) were used for
thin-layer chromatography (TLC). Preparative TLC (PTLC) was
carried out on silica gel 60 GF254 (Qingdao Marine Chemical,
Ltd., Qingdao, China); semi-preparative HPLC (Waters Corp.,
Milford) was performed by a Thermo BDS Hypersil column (250
ꢃ 4.6 mm and 250 ꢃ 10 mm, 5 mm) (Thermo Fisher Scientic
Inc., MA).
Reagents. 3-Phenyl-cyano(6-methoxy-2-naphthalenyl)methyl
ester-2-oxiraneacetic acid (PHOME), 12-[[(tricyclo[3.3.1.13,7]
dec-1-ylamino)carbonyl]amino]-dodecanoic acid (AUDA), and
puried recombinant sEH were purchased from Cayman
(Cayman, Michigan). The other chemical reagents and standard
compounds were purchased from Sigma-Aldrich (St. Louis,
MO). AB-8 macroporous resin was purchased from the Chem-
ical Plant of Nankai University (Tianjin, China), which was
cross-linked polystyrene copolymer. All used chemicals (meth-
anol, chloroform, ethyl acetate, petroleum ether and n-butanol
etc.) were purchased from Kelong Chemical Engineering
(Kelong, Chengdu, China). HPLC methanol was purchased
from Tedia company (Tedia Inc., Faireld, Connecticut).
Ultrapure water was made in our lab.
Fraction 4.3 was separated by CC over RP-18 with 10–50%
MeOH. Subfraction eluted with 50% MeOH was puried by
silica gel CC eluting with CHCl3/MeOH (5 : 1), followed by semi-
preparative RP-HPLC using MeOH/H2O (40 : 60; ow rate ¼ 2
mL minꢁ1) to obtain 3 (30 mg, tR ¼ 18.5 min).
Fraction 4.4 was separated by a Sephadex LH-20 column and
then RP-18 CC. Its water fraction was puried on CC over silica
gel with CHCl3/MeOH (5 : 1) as eluent to give 5 (9 mg).
Fraction 1 was applied to a silica gel column by elution with
EtOAc/MeOH (20 : 1–5 : 1) to generate three fractions (1.1–1.3).
Fraction 1.1 was loaded to medium pressure liquid chroma-
tography over RP-C18 by gradient elution with MeOH/H2O (40–
100%) to furnish 6 fractions (1.1.1–1.1.6). Fraction 1.1.1 eluted
by 40% MeOH was puried by Sephadex LH-20 to give 22 (90
mg). Fraction 1.1.2 (eluted by 50% MeOH) was puried by
Sephadex LH-20 and then by CC over silica gel and eluted by
CHCl3/MeOH (10 : 1) to obtain 17 (14.5 mg). Fraction 1.1.3 was
separated successively by Sephadex LH-20, RP-18 CC (45%
MeOH) and then silica gel CC (CHCl3/MeOH 5 : 1) to give 20 (6.5
mg). Fraction 1.1.4 was submitted to Sephadex LH-20 to furnish
three fractions (1.1.4.1–1.1.4.3). Fraction 1.1.4.1 was puried by
CC on silica gel eluting with CHCl3/MeOH (7 : 1) to give 18 (20.0
mg). Fraction 1.1.4.2 was puried by CC on silica gel and further
Sephadex LH-20 to provide 19 (9.0 mg). Fraction 1.1.4.3 was
recrystallized to give 21 (20 mg) in MeOH. Fraction 1.1.5 (80%
MeOH) was separated successively by CC over Sephadex LH-20,
RP-18, and silica gel (CHCl3/MeOH 50 : 1) to produce 12 (11.6
mg) and 13 (6.7 mg). Fraction 1.1.6 was puried successively by
Sephadex LH-20, RP-18 (85% MeOH), and CC on silica gel
(CHCl3/MeOH 20 : 1) to afforded 14 (7.0 mg).
Acetylcholinesterase (AChE) from electric eel (Type-VI-S, E.C.
3.1.1.7), acetylthiocholine iodide (ATChI), butyrylcholinesterase
(BChE) from equine serum (E.C. 3.1.1.8), butyrylthiocholine
iodide (BTChI), 5,50-dithiobis(2-nitrobenzoic acid) (DTNB),
tacrine were obtained from Sigma-Aldrich (St. Louis, MO).
Potassium phosphate were purchased from Amresco (K2HPO4)
and Daejung (KH2PO4).
Plant materials
The seeds of Catalpa bungei C. A. Mey were collected from
Lveyang County, Shaanxi Province of China on November 25,
2012. The plant was taxonomically identied by one of authors
(K. J. Z.). A voucher specimen (LXY-01239) has been deposited at
the Herbarium of College of Life Sciences, Northwest A&F
University, P. R. China.
Fraction 1.2 was fractionated successively by CC over silica
gel, Sephadex LH-20, and RP-18 (70% MeOH) and then by silica
gel CC (CHCl3/MeOH 25 : 1) to give 11 (9.3 mg). Fraction 3 was
separated by reduced pressure CC over RP-18 eluting by using
MeOH/H2O in a gradient elution (from 1 : 10 to 5 : 5) to yield six
fractions (3.1–3.6). Fraction 3.1 was separated by Sephadex LH-
20 and silica gel CC using CHCl3/MeOH (20 : 1) to give 6 (9 mg).
Extraction and isolation
The dried and powdered seeds (20 kg) of C. bungei were Fraction 3.2 was separated by Sephadex LH-20 to furnish two
extracted three times with 75% EtOH at room temperature. fractions (3.2.1–3.2.2). Fraction 3.2.1 was puried by semi-
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preparative RP-HPLC using MeOH/H2O (40 : 60; ow rate: 3 mL which L-cysteine methyl ester hydrochloride in pyridine (0.1 M,
minꢁ1) to give 9 (5.5 mg, tR ¼ 18.5 min) and 8 (13.5 mg, tR ¼ 19.3 0.5 mL) was added. Aer reacting at 60 C for 1 h, trimethylsi-
ꢂ
min). Compound 7 (15.1 mg) was isolated from fraction 3.2.2 by lylimidazole (0.05 mL) was added to the reaction mixture and
ꢂ
using silica gel CC eluted with CHCl3/MeOH (8.5 : 1), and kept at 4 C for another 8 h. The mixture was analysed by GC-
compound 16 (181 mg) was obtained from fraction 3.3 using MS. By comparison of the retention time of the authentic
Sephadex LH-20 and silica gel CC eluting with CHCl3/MeOH/ sample, the monosaccharide of compounds 1–3 was deter-
H2O (4 : 1 : 0.02).
mined to be D-glucose (tR ¼ 11.85 min) (tR ¼ 12.15 min of L-
The purity of every puried chemicals was more than 98% by glucose).
HPLC.
Bungoside A (1). White powder; [a]2D1 – 15.7 (c 0.17, MeOH);
UV (MeOH) lmax (log 3) 328 nm (4.28); IR (KBr) nmax 3393, 1697, Biological assays
1630, 1598, 1516 cmꢁ1; 1H and 13C NMR data, see Table 1. ESI-
sEH inhibition activity assay. The inhibition of sEH was
MS (positive) m/z 597.49 [M + Na]+; HR-ESI-MS m/z 597.1343 ([M
+ Na]+, calcd C25H31ClO13Na+, 597.1351).
assayed using a hydrolysis reaction of PHOME in the presence
of the sEH enzyme.16 Briey, 130 mL of sEH (62.5 ng mLꢁ1) in 25
mM bis–Tris buffer (including 0.1% bovine serum albumin, pH
7.0) and 20 mL of ligands diluted in MeOH were mixed in 96 well
plate. 50 mL of PHOME (40 mM) in buffer was added as substrate.
Aer starting the sEH reaction at 37 ꢂC, products were
measured using a Genios microplate reader (Tecan, Mannedorf,
Switzerland) during an hour at excitation and emission wave-
lengths of 320 and 465 nm, respectively. sEH inhibitory activity
for each sample was calculated as follows:
Bungoside B (2). White powder; [a]2D0 – 14.3 (c 0.12, MeOH);
UV (MeOH) lmax (log 3) 316 nm (4.23); IR (KBr) nmax 3383, 1690,
1631, 1604, 1514 cmꢁ1; 1H and 13C NMR data, see Table 1. ESI-
MS (positive) m/z 567.23 [M + Na]+; HR-ESI-MS m/z 567.1243 ([M
+ Na]+, calcd C24H29ClO12Na+, 567.1245).
6-O-p-Hydroxybenzoylglutinoside (3). White powder; [a]D21
–
19.5 (c 0.11, MeOH); UV (MeOH) lmax (log 3) 261 nm (4.37); IR
(KBr) nmax 3400, 1693, 1607, 1514 cmꢁ1; 1H and 13C NMR data,
see Table 1. ESI-MS (positive) m/z 541.38 [M + Na]+; HR-ESI-MS
m/z 541.1081 ([M + Na]+, calcd C22H27ClO12Na+, 541.1089).
sEH inhibitory activity (%) ¼ 100 ꢁ [(S60 ꢁ S0)/(C60 ꢁ C0)] ꢃ 100
where C60 and S60 were the uorescence of control and inhibitor
aer 60 min, S0 and C0 is the uorescence of control and
inhibitor at zero min.
Acetylation of 3
Compound 3 (3 mg) was treated with acetic anhydride (0.2 mL)
in dry pyridine (0.2 mL) at room temperature. The mixture was
stirred for 24 h, on workup to give a pure derivative 3a (ca. 3 mg).
20,30,40,60,400,8-O-Hexaacetoyl-6-O-p-hydroxybenzoylglutinoside
(3a). White powder; C34H39ClO18; ESI-MS m/z 793.25 [M + Na]+;
Cholinesterase inhibition assay. The AChE and BChE
inhibitory activity measurements were performed according to
the literature protocol.50 In brief, 0.1 M potassium buffer (pH
7.4), 130 mL of 0.05 U mLꢁ1 AChE and BChE, 20 mL of tested
compound (1 mM) were mixed and pre-incubated for 30 min at
25 ꢂC. The reaction was then initiated with the addition of 25 mL
of 5 mM ATChI and BTChI, 25 mL of 1 mM DTNB. AChE and
BChE activity was determined spectrophotometrically through
measuring the change in ultraviolet absorbance of an assay
13C NMR (125 MHz, CDCl3)
d 170.8 (C–COCH3), 170.4
(C–COCH3), 167.0 (C–COCH3), 169.5 (C–COCH3), 169.21
(C–COCH3), 168.9 (C–COCH3), 165.7 (C-11), 154.8 (C-15), 131.6
(C-13,17), 127.0 (C-12), 121.9 (C-14, 16), 95.3 (C-10), 94.5 (C-1), 91.9
(C-3), 86.2 (C-6), 85.9 (C-8), 72.9 (C-30), 72.2 (C-50), 71.04 (C-20),
68.3 (C-40), 63.0 (C-7), 61.7 (C-60), 60.0 (C-10), 42.0 (C-9), 33.5 (C-5),
32.9 (C-4), 22.3 (C–COCH3), 21.3 (C–COCH3), 20.9 (C–COCH3),
20.8 (C–COCH3), 20.7 (C-2 ꢃ COCH3); 1H NMR (500 MHz, CDCl3)
d 8.08 (2H, d, J ¼ 8.7 Hz, H-13, 17), 7.20 (2H, d, J ¼ 8.7 Hz,
H-14,16), 5.58 (1H, d, J ¼ 2.0 Hz, H-1), 5.49 (1H, d, J ¼ 8.0 Hz,
H-7), 5.39 (1H, d, J ¼ 2.7 Hz, H-3), 5.22 (1H, ddd, J ¼ 9.4, 7.3, 2.2
Hz, 30), 5.17 (1H, dd, J ¼ 8.0, 2.8 Hz, H-6), 5.11 (1H, t, J ¼ 9.7 Hz,
H-40), 4.96 (2H, m, H-10,20), 4.31 (2H, m, H-60b,10a), 4.15 (1H, dd,
J ¼ 12.4, 2.3 Hz, H-60a), 3.90 (1H, d, J ¼ 12.5 Hz, H-10b), 3.73 (1H,
ddd, J ¼ 10.0, 4.1, 2.4 Hz, H-50), 3.60 (1H, br d, J ¼ 10.3 Hz, H-9),
2.40 (1H, m, H-5), 2.33 (3H, s, H–COCH3), 2.29 (1H, m, H-4b),
2.11, 2.10, 2.02, 2.01, 1.99 (each 3H, s, H–COCH3), 2.06 (1H, m,
H-4a).
ꢂ
solution at 405 nm over a period of 15 min at 37 C. Tacrine
(3.8–500 nM) was used as positive control.
NF-kB inhibitory activity assay.16 The luciferase vector was
rst transfected into human hepatocarcinoma HepG2 cells.
Aer a limited amount of time, the cells were lysed, and lucif-
erin, the substrate of luciferase, was introduced into the cellular
extract along with Mg2+ and an excess of ATP. Under these
conditions, luciferase enzymes expressed by the reporter vector
could catalyze the oxidative carboxylation of luciferin. Cells
were seeded at 2 ꢃ 105 cells per well in 12-well plates and grown.
Aer 24 h, cells were transfected with inducible NF-kB lucif-
erase reporter and constitutively expressing Renilla reporter.
Aer 24 h of transfection, medium was changed to assay
medium (Opti-MEM + 0.5% FBS + 0.1 mM NEAA + 1 mM sodium
pyruvate + 100 units per mL penicillin + 10 mg mLꢁ1 strepto-
Acid hydrolysis of 1–3 and determination of sugar
Compounds 1–3 (2.0 mg, each) was hydrolysed with 2 M HCl– mycin) and cells were pretreated for 1 h with either vehicle
dioxane (1 : 1, 1 mL) at 100 ꢂC for 2 h. The reaction mixture was (DMSO) and compounds, followed by 1 h of treatment with 10
partitioned between chloroform and H2O three times. The ng mLꢁ1 TNF-a for 20 h. Unstimulated cells were used as
aqueous layer was neutralized with 2 M Ag2CO3 and evaporated a negative control (ꢁ), apigenin was used as a positive control.
in vacuo. The residue was dissolved in pyridine (0.5 mL), to Dual luciferase assay was performed 48 h aer transfection, and
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promoter activity values are expressed as arbitrary units using 10 F. Dal Piaz, A. Vassallo, A. Temraz, R. Cotugno,
a Renilla reporter for internal normalization.
M. A. Belisario, G. Bifulco, M. G. Chini, C. Pisano, N. De
Tommasi and A. Braca, J. Med. Chem., 2013, 56, 1583–1595.
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Computational section
MMFF and DFT calculations were run with Spartan'14 (Wave-
function, Inc., Irvine CA, 2014), with standard parameters and
convergence criteria. TDDFT calculations were run with
Gaussian'09,51 with default grids and convergence criteria.
Conformational searches were run with the Monte Carlo algo-
rithm implemented in Spartan'14 using Merck molecular force
eld (MMFF). All structures thus obtained were optimized with
DFT method using B3LYP functional and 6-31G(d) basis set in
vacuo. TDDFT calculations were run using B3LYP functional
and TZVP basis set, including 24 excited states. On some
selected structures, other functionals (CAM-B3LYP, PBE0, M06,
M06-2X) and basis sets (augmented TZVP, ADZP) were also
tested, leading to consistent results. UV and CD spectra were
generated using the program SpecDis.52 by applying a Gaussian
band shape with 0.2 eV exponential half-width, from dipole-
length rotational strengths.
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Acknowledgements
This work was nancially supported by the National Forestry
Public Welfare Industry Research Project (201204603) and the
Program of Science & Technology of Shaanxi Province (2013SF2-
15) as well as by the Priority Research Center Program (2009-
0093815) through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Tech-
nology, Republic of Korea. T. I. gratefully acknowledges FP7-
REGPOT-2012-2013-1, grant agreement number 316289–
InnoMol.
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