Physical sciences | Chemistry
Using NMR, X-ray, and CD analysis in the study
on natural products obtained from Vietnamese plant
and fungi in terms of pharmaceutical product development
Dinh Thang Tran1*, Cong Dung Vo1, Ngoc Tuan Nguyen1, Manh Dung Doan2, Yang-Chang Wu3, Tian-Shung Wu4
1Faculty of Chemistry, Vinh University, Vietnam
2Faculty of Chemistry, Hue University of sciences - Hue University, Vietnam
3School of Pharmacy, College of Pharmacy, China Medical University, Taiwan
4School of Pharmacy, National Cheng Kung University, Taiwan
Received 8 June 2017; accepted 7 November 2017
Abstract:
spectra
were
measured
using
Bruker
NMR, X-ray analysis, and CD methods are powerful techniques for the study
of absolute configuration of bioactive compounds from natural resources. This
study presents the results of a joint-study between Vietnam and Taiwan on the
bioactive compounds obtained from Vietnamese plants and fungi. Among the
tested compounds, hexatenuin A displayed the most significant inhibition of
superoxide anion generation and elastase release. These triterpenoids may be
used as potential anti-inflammatory agents.
AMX-400 and AV500 spectrometers
with TMS as the internal reference, while
the chemical shifts were expressed in δ
(ppm). The ESIMS and HRESIMS were
collected on a Bruker Daltonics APEX II
30e spectrometer. HPLC was performed
on a Shimadzu LC-10ATVP (Japan)
system, equipped with a Shimadzu
Keywords: absolute configuration, circular dichroism, NMR, X-ray analysis.
SPD-M20A diode array detector at 250
nm, a Purospher STAR RP-8e c (5 μm,
Classification number: 2.2
250×4.6
mm),
Cosmosil
5C18
ARII
(250×4.6 mm i.d. Nacalai Tesque Inc.),
Introduction
Natural products are an important
source for drug discovery. The
determination of absolute configuration
is one of the most challenging tasks in
the structure elucidation of chiral natural
products, especially those with complex
structures. The available methods
include NMR spectroscopy/chiral
derivatization, analytical chemistry,
X-ray crystallography for crystalline
compounds, chemical synthesis, and
chiroptical approaches [1]. Among
these, X-ray crystallography probably
remains the most powerful and effective
approach. However, the complete
structure elucidation of new compound
may require considerable effort and
involve many different spectroscopic
and, sometimes, computational
techniques.
The purpose of this review is to use
the applicability of these approaches in
determining the absolute configuration
of natural products obtained from
Vietnamese plants and fungi. Moreover,
the purified constituents were examined
for their anti-inflammatory activity.
Among the tested compounds,
hexatenuin A displayed the most
significant inhibition of superoxide
anion generation and elastase release.
These triterpenoids may have potential
to be used as anti-inflammatory agents.
Experimental
General experimental procedures
The optical rotations were measured
with a JASCO P-2000 digital polarimeter
in a 0.5 dm cell. The UV spectra were
obtained with a Hitachi UV-3210
spectrophotometer while the IR spectra
were measured with a Shimadzu FTIR
Prestige-21 spectrometer. The ECD
and Astec Cellulose DMP (150×4.6
mm i.d. 5 μm) columns. The X-ray
diffraction experiments were performed
on a Bruker D8 Venture with a Photon
100 CMOS detector system equipped
with a Cu Incoatec IμS microfocus
source (λ = 1.54178 Å).
Preparation of human neutrophils
Neutrophils were isolated by a
standard method of dextran
sedimentation, prior to their
centrifugation in a Ficoll Hypaque
gradient and hypotonic lysis of
erythrocytes. Blood was drawn from
healthy human donors (20-30 years
old) by venipuncture into heparin-
coated Vacutainer tubes, using a
protocol approved by the institutional
review board at Chang Gung Memorial
Hospital [2]. The blood samples were
mixed gently with an equal volume
of 3% dextran solution. After the
sedimentation of the red cells for 30 min
several examples, representing different
spectra were recorded on a JASCO J-720
at room temperature, the leukocyte-rich
classes of natural products, to illustrate
spectrometer.
The
1H-
and
13C-NMR
plasma was collected,. The leukocyte-
*Corresponding author: Email: thangtd@vinhuni.edu.vn
14
Vietnam Journal of Science,
Technology and Engineering
December 2017 Vol.59 Number 4
D
3
max
max
3
3 3
3
3 3
3 3
3
3 3
3
2 3
3
Physical sciences | Chemistry
rich plasma was transferred on top of a
activated by 100 nM FMLP and 0.5 μg/
(C-14), 45.3 (C-5), 43.5 (C-13), 41.8 (C-
20 ml Ficoll solution (1.077 g/ml) and
ml cytochalasin B while the changes in
2’), 41.1 (C-22), 37.1 (C-10), 36.8 (C-
spun down at 400 g for 40 min at 20°C.
absorbance at 405 nm were continuously
4), 35.4 (C-15), 30.7 (C-20), 30.5 (C-1),
The
granulocyte/erythrocyte
pellets
monitored to assay the elastase release.
30.1 (C-12), 28.0 (C-30), 27.6 (C-28),
were
resuspended
in
ice-cold
0.2%
The
results
were
expressed
as
the
26.5 (C-6), 23.1 (C-2), 21.7(C-29), 20.2
NaCl
to
lyse
the
erythrocytes. After
percentage
of
elastase
release
in
the
(C-11), 19.4 (C-21), 18.8 (C-19), 17.9
30 s, the same volume of 1.6% NaCl
FMLP/CB-activated,
drug-free
control
(C-7), 16.5 (C-18), 10.8 (C-31), 8.5 (C-
solution was added to reconstitute the
system [2].
27); ESIMS m/z 621 ([M+K]+, 60), 605
isotonic condition. Purified neutrophils
were pelleted and then resuspended in
a calcium (Ca2+)- free Hank’s balanced
salt solution (HBSS) buffer at pH 7.4
and maintained at 4°C before use [2].
Measurement of superoxide anion
generation
The assay of the superoxide anion
generation was based on the SOD-
inhibitable reduction of ferricytochrome
c [2]. Briefly, after supplementation
with 0.5 mg/ml ferricytochrome c and
1 mM Ca2+, the neutrophils (6×105
cells/ml) were equilibrated at 37°C
for 2 min and incubated with drugs
or an equal volume of vehicle (0.1%
DMSO, negative control) for 5 min.
The cells were activated with 100 nM
FMLP during the preincubation of 1
μg/ml cytochalasin B (FMLP/CB) for
3 min. Changes in the absorbance, with
a reduction in ferricytochrome c at
550 nm, were continuously monitored
in a double-beam, six-cell positioner
spectrophotometer with constant
stirring (Hitachi U-3010, Tokyo, Japan).
Hexagonin A (16): white powder
(CHCl ); mp 184-185°C; [α]25 +57
(c 0.6, MeOH); UV (MeOH) λ (log
ε) 262 (2.65) nm; IR (neat) n 2946,
1759, 1693, 1455, 1376, 1256, 1219,
1156 cm-1; 1H-NMR (500 MHz, CDCl )
(d ppm): 4.71 (1H, br s, H-3), 4.32 (1H,
ddd, J = 11.5, 11.5, 5.0 Hz, H-16), 3.72
(3H, s, CH -4’), 3.40 (2H, s, CH -2’),
2.27 (1H, dd, J = 14.0, 11.5 Hz, H-15),
2.18 (1H, m, H-20), 2.05 (2H, m, H-6,
-11), 1.89 (1H, m, H-2), 1.84 (1H, m,
H-12), 1.71 (1H, m, H-2), 1.60 (3H, m,
H-7, -12, -22), 1.49 (3H, m, H-1, -7, -22),
1.41 (3H, m, H-1, -5, -17), 1.20 (1H, dd,
J = 14.0, 5.0 Hz, H-15), 1.94 (3H, d, J
= 0.5 Hz, CH -31), 1.81 (3H, d, J = 0.5
Hz, CH -27), 1.08 (3H, s, CH -30), 1.00
(3H, s, CH -19), 0.93 (3H, s, CH -29),
0.95 (3H, d, J = 6.5 Hz, CH -21), 0.88
(3H, s, CH -28), 0.68 (3H, s, CH -18);
13C-NMR (125 MHz, CDCl ) (d ppm):
172.2 (C-26), 165.9 (C-1’), 167.2 (C-3’),
157.4 (C-24), 135.1 (C-9), 133.8 (C-8),
125.2 (C-25), 108.2 (C-23), 79.8 (C-16),
79.6 (C-3), 54.6 (C-17), 52.3 (C-4’), 48.6
([M+Na]+, 26), 521 (33), 505 (100), 483
(48); HRESIMS m/z 605.3451 ([M +
Na]+, calcd for C35H50O7Na, 605.3454).
Results and discussions
A joint-study between Vietnam and
Taiwan on bioactive compounds from
the Vietnamese plant, Clausena lansium
Skeels (Rutaceae), was conducted.
The methanol extract from the dried
leaves of C. lansium was partitioned
between H O and CHCl . The
purification of the CHCl fraction by a
combination of column chromatographic
methods afforded eight new lactams,
including γ-lactams (1-3), δ-lactams
(4-7), and amide (8), along with seven
known lactams (9-15), which were
characterized from the leaves of C.
lansium (Fig. 1). Their structures were
elucidated using spectroscopic methods
[3] and the absolute configurations were
determined using electronic circular
dichroism (ECD) and single-crystal
X-ray diffraction analyses with Cu Kα
radiation.
Then calculations were based on the
differences in the reactions with and
without SOD (100 U/ml), divided by the
extinction coefficient for the reduction
of ferricytochrome c (ε = 21.1/mM/10
mm) [2].
Measurement of elastase release
The
degranulation
of
azurophilic
granules
was
determined
by
the
elastase release, as described previously
[2].
Experiments
were
performed
using
MeO-Suc-Ala-Ala-Pro-Val-p-
nitroanilide
as
the
elastase
substrate.
Briefly, after supplementation with MeO-
Suc-Ala-Ala-Pro-Val-p-nitroanilide (100
μM), the neutrophils (6×105 cells/ml)
were equilibrated at 37°C for 2 min and
incubated with drugs or an equal volume
of vehicle (0.1% DMSO, negative
control) for 5 min. The cells were
Fig. 1. The lactam compounds 1-15.
December 2017 Vol.59 Number 4
Vietnam Journal of Science,
Technology and Engineering
15
Physical sciences | Chemistry
The
ECD
sign
and
red
shift
of
the
Cotton
effect
were
shown
to
experimentally
determine
the
C-3
configuration as well as the sign and the
magnitude of the n → π* Cotton effect,
which are sensitive to the nature of the
C-3 substituent [4]. Therefore, the C-3
configuration of compound 1 with a
hydroxyl functionality was determined
as S, because it displayed a positive
Cotton effect near 230 nm. The absolute
configuration
of
compound
1
was
unambiguously defined, by a single-
crystal X-ray diffraction analysis with
Cu Kα radiation, as 3S, 4R, 5S, and 6R
(Fig. 2). Consequently, the structure of
the
6-O-methylneoclausenamide
(1)
was characterized, as shown in Fig.
1. The 2D structure of compound 2
was similar to compound 1, while the
relative
configuration
of
the
lactam
ring
was
assigned
as
being
similar
to compound 1, through the analysis
of their NOESY spectra (Fig. 3). In
addition,
the
absolute
configurations
at C-4, C-5, and C-6 were determined
by the single-crystal X-ray diffraction
pattern using the anomalous scattering
of Cu Kα radiation (Fig. 2). Therefore,
the
absolute
configuration
was
determined as 3S, 4R, 5S, and 6S. In
effect, the structure of 6-O-methyl-epi-
neoclausenamide (2) was assigned as
shown. The 2D structure of compound
3 was assigned to be identical to those
of compounds 1 and 2 by a comparison
of their UV, IR, MS, and NMR data
[2]. The ECD spectrum of compound
3
showed
a
low-amplitude
positive
Cotton effect near 236 nm. The ECD
spectrum of compound 12 showed a
high-amplitude positive Cotton effect
at 230 nm. Thus, the low-amplitude
positive Cotton effect at 238 nm in
Fig. 2. ORTEP drawings of compounds 1, 2, 5, 7, 8, and 10.
the ECD spectrum of compound 3
(Fig. 4) suggested 3S and 4S absolute
configurations [5]. By comparing
the specific rotation and absolute
configuration of compound 3 with the
16 stereoisomers of clausenamide,
the 3S, 4S, 5R, 6S and 3S, 4S, 5R,
6R configurations could be further
cisneoclausenamide (3) was established
as 3S, 4S, 5R, and 6R. The absolute
configuration of C-3 in compound 4 was
deduced by the ECD spectrum. In this
case, the ECD spectrum of compound
4 (Fig. 4) showed a positive Cotton
effect at 231 nm, which evidenced a 3S
4 was deduced as 3S, 4S, 5R, and 6R,
the structure of which was illustrated
as shown. To determine the absolute
configuration, compound 5 was
subjected to a single-crystal X-ray
diffraction analysis with Cu Kα radiation
(Fig. 2) which confirmed the structure
considered [3]. Therefore, the absolute
absolute
configuration.
Consequently,
unambiguously. Therefore, the absolute
configuration
of
6-O-methyl-epi-
the absolute configuration of compound
configuration was established as 3S, 4S,
16
Vietnam Journal of Science,
Technology and Engineering
December 2017 Vol.59 Number 4