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

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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. 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.
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
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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. 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..

Nội dung

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: 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. Keywords: absolute configuration, circular dichroism, NMR, X-ray analysis. Classification number: 2.2 Introduction the applicability of these approaches in souNatural rproducts are an important determining the absolutebconfiguration determination of absolute configuration the purified constituents were examined the structure elucidation of chiral natural for their anti-inflammatory activity. products, especially those with complex structures. The available methods significant inhibition of superoxide derivatization, analytical chemistry, anion generation and elastase release. X-ray crystallography for t crystalline to be used as anti-inflammatory agents. chiroptical approaches [1]. Among these, X-ray crystallography probably remains the most powerful and effective General experimental procedures approach. However, the complete structure elucidation of new compound with a JASCO P-2000 digital polarimeter involve many different spectroscopic in a i0.5 dm cell. The UV spectra-were spectrophotometer while the IR spectra were measured with a Shimadzu FTIR The purpose of this review is to use Prestige-21 spectrometer. The ECD several examples, representing different spectra were recorded on a JASCO J-720 classes of natural products, to illustrate spectrometer. The 1H- and 13C-NMR spectra were measured using Bruker 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 SPD-M20A diode array detector at 250 nm, a Purospher STAR RP-8e c (5 μm, 250×4.6 mm), Cosmosil 5C18 ARII (250×4.6 mm i.d. Nacalai Tesque Inc.), 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 at room temperature, the leukocyte-rich 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 Physical sciences | Chemistry rich plasma was transferred on top of a 20 ml Ficoll solution (1.077 g/ml) and spun down at 400 g for 40 min at 20°C. The granulocyte/erythrocyte pellets were resuspended in ice-cold 0.2% NaCl to lyse the erythrocytes. After 30 s, the same volume of 1.6% NaCl solution was added to reconstitute the 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). 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 activated by 100 nM FMLP and 0.5 μg/ ml cytochalasin B while the changes in absorbance at 405 nm were continuously monitored to assay the elastase release. The results were expressed as the percentage of elastase release in the FMLP/CB-activated, drug-free control system [2]. 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 Fig. 1. The lactam compounds 1-15. (C-14), 45.3 (C-5), 43.5 (C-13), 41.8 (C-2’), 41.1 (C-22), 37.1 (C-10), 36.8 (C-4), 35.4 (C-15), 30.7 (C-20), 30.5 (C-1), 30.1 (C-12), 28.0 (C-30), 27.6 (C-28), 26.5 (C-6), 23.1 (C-2), 21.7(C-29), 20.2 (C-11), 19.4 (C-21), 18.8 (C-19), 17.9 (C-7), 16.5 (C-18), 10.8 (C-31), 8.5 (C-27); ESIMS m/z 621 ([M+K]+, 60), 605 ([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. 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 considered [3]. Therefore, the absolute 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 absolute configuration. Consequently, 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 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 Physical sciences | Chemistry 1 2 3 4 O HO N OCH3 5 6 8 9 Fig. 3. Selected NOESY (↔) correlations for compounds 1-6, 8, and 9. 5S, and 6S (Fig. 2). Hence, compound 5 was characterized as lansamide-6. A positive Cotton effect at 223 nm in the ECD spectrum (Fig. 4) suggested a 3S absolute configuration. The absolute configuration was established as 3S, 4S, and 5S, while the structure of lansamide-7 (6) was characterized as shown. Based on these results and the single-crystal X-ray diffraction analyses using Cu Kα radiation (Fig. 2), the structure of lansamide-8 (7) was identified as shown. The crystals of compound 7 were orthorhombic and belonged to the space group, Pbca. As shown in the ORTEPdrawing (Fig. 2), the X-ray analysis revealed that compound 7 was a racemic mixture presumably originating from the reaction between pyridine-2,3,6-trione and acetone. From the spectroscopic analysis and the single-crystal X-ray diffraction data (Fig. 2), the absolute configuration was confirmed by the Flack parameter 0.0(2) and defined as 3S, 4S, 5R, and 6S. The structures of compounds 9 and 10 were confirmed by the HRESIMS data and single-crystal X-ray diffraction analysis (Fig. 2). These structures have been reported as synthetic products, but they were isolated from their natural sources for the first time. Compounds 12 and 13 were identified as (-)-clausenamide and (-)-neoclausenamide through the 1H and 13C NMR [1], the positive Cotton effect in the ECD spectrum [at 230 and 229 nm] (Fig. 4), single-crystal X-ray diffraction analysis (Fig. 2), and its negative specific rotation [-148.5 (c 0.8, MeOH) and -71.8 (c 1.8, MeOH)]. Compounds 14 and 15 were reported as racemates in a previous study [5], but the negative specific rotation [-107.8 (c 1.4, MeOH) and -117.1 (c 0.7, MeOH)] and a high-amplitude Cotton effect (Fig. 4) confirmed that they were pure enantiomers. Their structures were confirmed by the positive Cotton effects in their ECD spectra [at 230 and 231 nm] (Fig. 4) and single-crystal, X-ray diffraction analyses (Fig. 2). Some relationships between the ECD spectra and the absolute configurations could be found from the above results. In the ECD spectra, δ-lactams 4, 14, and 15, with 3S, 4S, and 5R absolute configurations, exhibited negative and positive Cotton effects near 210 and 230 nm, respectively. Compound 5 and 6, possessing 3S, 4S, and 5S absolute configurations, displayed ECD spectra with a positive Cotton effect at 220 nm. For the γ-lactam group, compounds 1, 12, and 13, with 3S and 4R stereochemistry, exhibited similar ECD spectra. However, the absolute configurations of compound 12 at C-5 and C-6 were different from those of compounds 1 and 13. This implied that the absolute configuration of C-5 and C-6 had little contribution to the ECD spectra. In contrast, compounds 3 and 9 possessed 3S and 4S absolute configurations and showed different ECD spectra, as compared to those of compounds 1, 12, and 13. This indicated that the C-4 phenyl group may have a significant influence on the Cotton effect near 230 nm. Furthermore, a comparison of the ECD spectra of compounds 3 and 9 showed that the absolute configuration at C-5 may influence the wavelength of the Cotton effect. In the other joint-study, air-dried and powdered fruiting bodies of H. apiaria were extracted with methanol and the combined extracts were concentrated under reduced pressure to produce a deep brown syrup. The crude extract was suspended in water and partitioned with ethyl acetate to afford ethyl acetate and water-soluble fractions. Purification of the ethyl acetate fraction by a conventional combination of column chromatographies yielded four new triterpenoids (16-19) and hexatenuin A [6]. Compound 16 was obtained as an optically active white powder, with [α]25D +57 (c 0.6, MeOH). The positive-mode HRESIMS of compound 16 showed a pseudo-molecular ion peak at m/z 605.3451 ([M+Na]+, calcd for C35H50O7Na, 605.3454), corresponding to the molecular formula of C35H50O7 with 11 indices of hydrogen deficiency (IHD). The UV spectrum of compound 16 exhibited an absorption maxima at 262 nm, compatible with an α,β-unsaturated carbonyl chromophore [7]. The IR absorption bands at 2946, 1759, and 1693 cm-1 suggested the presence of aliphatic C-H, lactonic carbonyl, and carbon-carbon double bond functionalities. The 1H NMR spectrum of compound 16 displayed five methyl singlets at δ 0.68 (3H, CH3-18), 0.88 December 2017 • Vol.59 Number 4 Vietnam Journal of Science, Technology and Engineering 17 Physical sciences | Chemistry Fig. 4. ECD spectra of compounds 1-6 and 8-15. (3H, CH3-28), 0.93 (3H, CH3-29), 1.00 (3H, CH3-19), and 1.08 (3H, CH3-30), respectively. In addition, one doublet methyl group at δ 0.95 (3H, J = 6.5 Hz, CH3-21) suggested the presence of the lanostane skeleton. Two vinyl methyl signals at δ 1.81 (3H, d, J = 0.5 Hz, CH3-27) and 1.94 (3H, d, J = 0.5 Hz, CH3-31), along with the 13C NMR signals at δ 8.5 (C-27), 10.8 (C-31), 108.2 (C-23), 125.2 (C-25), 157.4 (C-24), and 172.2 (C-26), indicated a γ-lactone ring cyclized between C-23 and C-26. This was verified by the HMBC correlations from CH3-31 to C-23, -24, and -25 as well as from CH3-27 to C-24, -25, and -26, respectively. In the downfield region of the 13C NMR spectrum, there were two oxygenated methines at δ 79.6 (C-3) and 79.8 (C-16), one set of tetra-substituted double bonds at δ 133.8 (C-8) and 135.1 (C-9), and two ester carbonyl carbons at δ 165.9 (C-1′) and 167.2 (C-3′). The location of the tetra-substituted CH3-19 and C-9 and between CH3-30 and C-8. The HMBC cross-peaks from H-16 (δ 4.32, 1H, ddd, J = 11.5, 11.5, 5.0 Hz) to C-20 (δ 30.7), from H-3 (δ 4.71, 1H, br s) to C-29 (δ 21.7), C-1 (δ 30.5), C-5 (δ 45.3), C-1′; from CH2-2′ (δ 3.40, 2H, s) to C-1′ and C-3′; and from CH3-4′ (δ 3.72, 3H, s) to C-3′ evidenced that the C-16 had been oxygenated while the C-3 had been acetylated by the carbomethoxyacetyloxy group. The elucidations provided above constructed the chemical skeleton of 1 with 10 IHDs. The last IHD was afforded by the cyclization between C-16 and C-23 through the ether linkage with a spiro structure. These spectra evidenced that compound 16 was very similar to the reported compound hexatenuin A [8], with the only difference being that compound 16 was the methyl derivative of hexatenuin A. The coupling constants of H-3 (br s) and H-16 (11.5, 11.5, 5.0 Hz) indicated their orientations to be further established as β and β, according to the NOESY analysis and comparison of the spectral data of compound 16 and hexatenuin A [8]. The successive two-dimensional spectral experiments, including COSY, NOESY, HMQC, and HMBC accomplished the assignments of all the proton and carbon signals of compound 16, and therefore its chemical structure was established as shown in Fig. 5 and named trivially as hexagonin A. Compounds 17-19 were all obtained as optically active white powder, displaying similar UV spectra and IR absorption bands as those of compound 16. Moreover, the proton resonances for the eight methyl groups, characteristic of the triterpenoid basic skeleton, were all observed in their 1H NMR spectra. These data indicated that compounds 16-19 were structurally similar compounds (Fig. 6). double bond at C-8/C-9 was determined equatorial and axial. The stereochemical The purified triterpenoids, which by the 3J-HMBC correlations between configurations of H-3 and H-16 were were isolated in sufficient quantity, 18 Vietnam Journal of Science, Technology and Engineering December 2017 • Vol.59 Number 4 Physical sciences | Chemistry were examined for their inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB (Table 1). Among the examined constituents, hexatenuin A displayed the most significant inhibition of superoxide anion generation and elastase release, with IC values of 1.9±0.2 and 4.3±1.4 μM, as compared to the reference compound LY294002,12 with IC values of 0.4±0.02 and 1.5±0.3 μM for superoxide anion generation and elastase release, respectively. In addition, the following structure- (B) Fig. 5. Significant HMBC (A) and NOESY (B) correlations of compound 16. activity relationships could be deduced from the bioactivity data. Hexagonins B (17) and D (19), which possess the basic triterpenoid skeleton without the malonyl substitution at C-3, did not show any anti-inflammatory bioactivity. Comparatively, hexagonin A (16), with its triterpenoid skeleton and malonyl and methyl ester functions, also failed to exhibit significant activity. Hexatenuin A, which had the triterpenoid skeleton as well as a free malonic acid group, displayed the most significant inhibitory effects in the bioactivity examination. Consequently, the free malonic acid function was important for anti- Fig. 6. Chemical structures of all the purified compounds. Table 1. Inhibitory effects of purified samples from H. apiaria on superoxide Anion generation and elastase release by human neutrophils, in response to N-Formyl-Lmethionyl-phenylalanine/Cytochalasin B (FMLP/CB). inflammatory activity. From the above data, it was concluded that the purified triterpenoids of H. apiaria are new potential leads for anti-inflammatory drug development and the starting fungus can be used as a health food with a possible and known mechanism of action. Compound 16 17 18 19 hexatenuin A LY294002 c IC50 (μM)a Superoxide anion generation >10 >10 >10 6.0±1.0*** 1.9±0.2*** 0.4±0.02*** Elastase release - b - b - b >10 4.3±1.4*** 1.5±0.3*** Therefore, it is not surprising that intrinsic anti-inflammatory properties demonstrated in vitro with H. apiaria can be transferred in vivo after mushroom consumption as food or nutraceutical food. This study has identified the ability for food processing to anti-inflammatory. The process extraction for H. apiaria identified a five-step process that would address certain critical aspects in the design and development of functional aconcentration necessary for 50% inhibition. results are presented as mean ± SD (n = 3-4). ***p < 0.001 compared with the control value. bIncreasing effects were observed. cA phosphatidylinositol-3-kinase inhibitor was used as a positive control for superoxide anion generation and elastase release. food (Fig. 7). Conclusions A total of 15 lactams were isolated December 2017 • Vol.59 Number 4 Vietnam Journal of Science, Technology and Engineering 19 Physical sciences | Chemistry Org. Chem., 14(16), pp.1678-1697. [2] S.c. Yang, P.J. chung, c.m. Ho, c.Y. Kuo, m.F. Hung, Y.T. Huang, W.Y. chang, Y.W. chang, K.H. chan, T.l. Hwang (2013), “Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1”, J. Immunol., 190(12), pp.6511-6519. [3] D.Y. Shen, T.N. Nguyen, S.J. Wu, Y.J. Shiao, H.Y. Hung, P.c. Kuo, D.H. Kuo, T.D. Thang, T.S. Wu (2015), “γ- and δ-lactams from the leaves of clausena lansium”, Journal of Natural Products, 78(11), pp.2521-2530. [4] T. Konno, H. meguro, K. Tuzimura (1975), “circular dichroism of γ-lactams and their sign determinating factors”, Tetrahedron Lett., 16, pp.1305-1308. [5] Z.Q. Feng, X.Z. li, G.J. Zheng, l. Huang (2009), “Synthesis and activity in enhancing Fig. 7. The process of extraction for H. apiaria. long-term potentiation (lTP) of clausenamide stereoisomers”, Bioorg. Med. Chem. Lett., from the methanolic extract of C. lansium. This research work enabled the determination of the absolute configuration of these classes of compounds using MS, NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses with Cu Kα radiation. In the other study, a chemical investigation of the fruiting bodies of H. apiaria resulted in the identification of five compounds, hexagonins A-D (16-19) and hexatenuin A. 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 the potential to be used as anti-inflammatory agents. This study has identified abilities from food processing to anti-inflammatory. The process extraction for H. apiaria identified a five-step process that would address certain critical aspects in the design and development of functional food. REFERENCES [1] X.c. li, D. Ferreira, Y. Ding (2010), “Determination of absolute configuration of natural products: theoretical calculation of electronic circular dichroism as a tool”, Curr.

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