Phytochemistry 130 (2016) 291e300
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Chemical constituents from Melicope pteleifolia leaves
Ngoc Hieu Nguyen a, Thi Kim Quy Ha a, Sangho Choi b, Sangmi Eum b, Chul Ho Lee b,
Tran The Bach c, Vu Tien Chinh c, Won Keun Oh a, *
a
Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 151-742,
Republic of Korea
b
International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Republic of Korea
c
Department of Botany, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, 10307, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 November 2015
Received in revised form
16 June 2016
Accepted 23 June 2016
Available online 30 June 2016
Five acetophenones bearing spiroketal-hexofuranoside rings, one di-C-glycosidic acetophenone and two
benzopyrans, along with 16 known compounds were isolated from the leaves of Melicope pteleifolia.
Structures of all the isolates were elucidated using extensive spectroscopic methods, including 1D, 2DNMR and HRESIMS. All the isolates were also evaluated for their neuraminidase inhibitory activities
against H1N1, H9N2, wild-type H1N1 and oseltamivir-resistant H1N1 (H274Y mutation) virus strains. Of
the isolates, tamarixetin 3-robinobioside was found to exhibit the strongest enzymatic inhibition (IC50
24.93 ± 3.46, 23.19 ± 5.41, 26.67 ± 5.16 and 40.16 ± 4.50 mM, respectively). Selected candidates,
kaempferol 3-robinobioside, kaempferol 3-O-b-D-glucopyranosyl (1 / 2)-a-D-xylopyranoside and tamarixetin 3-robinobioside, also showed moderate reductions in H1N1-induced cytopathic effects on MDCK
cells.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
Melicope pteleifolia
Rutaceae
Spiroketal
Neuraminidase
H1N1
1. Introduction
Polyphenol-bearing spiroketal-hexofuranoside rings are a rare
group of natural products. There have been limited reports on their
occurrence in nature, including pinnatifins C and D (Zhang and Xu,
2001), pinnatifinosides A-D (Zhang and Xu, 2001), aquilarinoside A
(Qi et al., 2009) and upuborneols A and B (Ito et al., 2012). It is
believed that spiroketal compounds are biosynthesized from their
C-glycoside precursors through dehydrogenation-reductive cyclization reactions, followed by isomerization (Ito et al., 2012), which
leads to diversity in the absolute configurations of the spiroketal
moieties. However, currently known natural spiroketals were isolated as only minor constituents from plants. Additionally, the absolute configurations of those compounds have not been fully
assigned. Recently, there have been advances in the synthetic
procedures for generating spiroketals, including the semi-synthesis
of catechin-C-spiro-glycosides through Maillard reactions with
catechin, glycine and identified sugars (D-glucose, D-xylose or Dgalactose) (Zhang et al., 2014), suggesting that the absolute configurations of spiroketal moieties were closely related to their
* Corresponding author.
E-mail address: wkoh1@snu.ac.kr (W.K. Oh).
http://dx.doi.org/10.1016/j.phytochem.2016.06.011
0031-9422/© 2016 Elsevier Ltd. All rights reserved.
original sugars. To date, spiroketals have become a point of interest
for synthetic chemists due to their unique structural features,
limited natural availability, and potential anti-inflammatory activities (Furuta et al., 2004; Nakatsuka et al., 2004; Sato et al., 2008).
Melicope pteleifolia (Champ. ex Benth) T.G. Hartley (www.
theplantlist.org) (synonym Evodia lepta (Spreng.) Merr.), is a
medium-sized tree that belongs to the Rutaceae family, which is
widely distributed throughout Vietnam and China. In Vietnamese
traditional medicine, the extract prepared from M. pteleifolia, under
the local name of “Ba chac”, is empirically used for the treatment of
high fevers, colds and various inflammatory conditions. Previous
phytochemical investigations established that the main constituents of this plant were benzopyrans (Guo-Lin et al., 1997;
Kamperdick et al., 1997), furoquinoline alkaloids (Sichaem et al.,
2014) and glycosidic compounds (Zhang et al., 2012). It was also
found that the methanol extract from its leaves and twigs exhibited
anti-inflammatory activities through either 5-lipoxygenase (5-LOX)
inhibition (Shaari et al., 2011) or interactions with Syk/Src targets
(Yoon et al., 2013), which are ethnopharmacologically relevant for
its traditional usage. Ongoing chemical investigations herein
established the major occurrence of rare spiroketal constituents in
M. pteleifolia leaves, contributing to the chemical diversity of the
overall natural products library, and providing a rich source of
spiroketals for further pharmacological studies.
292
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
Influenza, commonly called flu, is a respiratory disease caused
by RNA viruses of the Orthomyxoviridae family. They are classified
into three types A, B and C; among them, type A is of clinical significance due to its association to respiratory complications, and
hospitalization, as well as pandemic mortality. Until now, besides
annual vaccination, another preventive means is using antiviral
agents, specifically neuraminidase inhibitors (zanamivir, oseltamivir and peramivir). Influenza neuraminidase (NA) is an antigenic
glycoprotein, which locates on the surface of influenza virions. NA
plays its important role in the release of viral progeny from infected
cells by cleaving the sialic acid residues (N-acetyl-neuraminic acid)
from the surface glycoproteins (Grienke et al., 2012). Additionally,
NA is also believed to relate to the mobility of influenza virus within
the respiratory tract mucous (Xie et al., 2011a). Due to the key roles
in viral replication, spread and pathogenesis, NA is considered a
promising druggable target in the fight against influenza (Xie et al.,
2011a).
In this paper, purification and structural determination of five
new spiroketals (1e5), one new phloroacetophenone (11), and two
new benzopyrans (13 and 14), along with 16 known compounds
(6e10, 12 and 15e24) from the methanol extract of M. pteleifolia
leaves are reported (Fig. 1). All compounds were also examined for
their inhibitory activities against various neuraminidases from
H1N1, H9N2, wild-type H1N1, and oseltamivir-resistant H1N1
(H274Y mutation) strains. To confirm the viral inhibition in cells,
selected candidates were also examined for their abilities to reduce
cytopathic effects (CPE) on H1N1-infected MDCK cells.
2. Results and discussion
Compound 1 was isolated as a brownish gum. Its molecular
formula was determined as C20H26O13 from the positive-charged
peak [MþH]þ at m/z 475.1433 (calcd for C20H27O13, 475.1446) of
its HRESIMS spectrum, corresponding to eight degrees of unsaturation. Its IR spectrum showed absorption bands for hydroxy (3696
and 3351 cm 1) and carbonyl (1628 cm 1) functionalities. The 13C
NMR and HSQC spectra showed a total of seven quaternary carbon
signals in the range of dC 100e170 ppm (dC 164.8, 163.4, 156.2, 120.4,
105.1, 102.4 and 101.1), suggesting the existence of one fully
substituted benzene ring with three oxygenated aromatic carbons
and a characteristic oxygen-bearing carbon. An acetyl group was
also observed by the appearance of a downfield methyl singlet at dH
2.61 (3H, s, H3-8) and a ketone signal at dC 203.0 (s). The attachment
of this acetyl group to C-1 was determined by the HMBC correlation
from H3-8 (dH 2.61) to C-1 (dC 105.1). In addition to the appearance
of an acetophenone moiety, the existence of two hexose sugars was
evident in this structure based on the twelve remaining carbon
signals. Because there were no observations of anomeric protons or
carbons of an O-glycosidic sugar in the NMR spectra, it was
reasonable to believe that two sugars would be linked to an acetophenone moiety by a C-glycosidic bond. This suggestion was also
supported by spectroscopic comparisons with previous literature
reports (Sato et al., 2004). The position of the C-glycosidic sugar
was assigned to C-5 based on HMBC correlations of H-100 (dH 4.36)
and C-4/5/6 (dC 163.4, 101.1 and 164.8, respectively). However, the
remaining sugar residue appeared to be different from typical Cglycosidic sugars due to unusual carbon signals at dC 120.4 (q) and
31.9 (t) and through the occurrence of diastereotopic methylene
protons at dH 2.79 (d, J ¼ 16.5 Hz) and 3.36 (d, J ¼ 16.5 Hz). These
observations implied the existence of a special hexofuranose moiety. Because six out of the eight hydrogen deficiency indices were
accounted for in one benzene ring, one acetyl group and one glucopyranose, the two remaining indices were attributed to a tworing system. All of these deductions suggested the existence of a
spiro(benzofuran-[2H]furan) ring. Moreover, by NMR data
comparison, this sugar appeared to closely resemble pinnatifinoside A (Zhang and Xu, 2001) and aquilarinoside A (Qi et al., 2009),
which possess spiroketal moieties. The HMBC spectrum provided
evidence on the presence of a special type of spiroketal ring in
compound 1, which was supported by the correlations of H-10 (dH
3.36 and 2.79) to C-2/3/4 (dC 156.2, 102.4 and 163.4, respectively) of
the acetophenone moiety and to C-20 /30 (dC 120.4 and 80.9,
respectively) of the hexofuranoside moieties. These correlations
also allowed deduction of the position of the spiroketal residue
within the whole structure (Fig. 2).
The C-glycosidic sugar moiety was assigned as a b-D-glucose by
ROESY cross-peaks of H-100 /H-300 , 500 and H-200 /H-400 , 600 and by
comparing the spectroscopic data with that of 3,5-di-C-b-D-glucopyranosyl phloroacetophenone (Sato et al., 2004). The b-orientation of the sugar was also confirmed by the 3JHH of the anomeric
proton being 9.6 Hz (Agrawal, 1992). The relative configuration of
the spiroketal ring of compound 1 was assigned based on ROESY
correlations of H-30 (dH 4.05)/H-50 (dH 3.84), 30 -OH (dH 5.74)/H-40 (dH
3.76), 40 -OH (dH 5.33)/H-30 (dH 4.05) and H-40 (dH 3.76)/H-10 (dH
2.79). The relative configuration was also simulated by observing a
3D model (CambridgeSoft Corporation, PerkinElmer, Inc.) with
reasonable spatial proximity between two protons (less than 4 Å).
Although there was one compound previously described in the
€s et al.,
literature with the same relative configuration as 1 (Bla
2004), its absolute configuration has not been determined yet.
The absolute configuration of 1 was assigned by comparing its
spectra with previous spiroketal compounds that were synthesized
from known starting materials (i.e., catechin, glycine and specific
sugars) through a Maillard-Catechin reaction (Zhang et al., 2014).
Interestingly, the 13C NMR data of 1 were in good agreement with
compounds synthesized from D-glucose, indicating the absolute
stereochemistry of the spiroketal moiety of 1 as (20 R, 30 S, 40 S, 50 R),
also suggesting its origin from b-D-glucopyranose. Therefore, the
structure of compound 1 was elucidated and accordingly named
melicospiroketal A.
Compounds 2e5 shared the same molecular formula as 1,
C20H26O13, which was established from HRESIMS data. Moreover,
the NMR patterns of these compounds also resembled those of 1,
suggesting that they all possessed the same backbone but had
different spiroketal ring stereochemistry (Tables 1 and 2). Hence,
the determination of the absolute configuration of melicospiroketal
A played a key role in the structural elucidation of other spiroketal
compounds.
The ROESY spectrum of melicospiroketal B (2) showed crosspeaks of H-30 (dH 4.10)/H-40 (dH 3.81), H-50 (dH 3.90) and H-40 (dH
3.81)/H-50 (dH 3.90), indicating that these three adjacent protons
were on the same side of the molecular plane. Furthermore, H-30
and H-50 were shown to correlate with H3-8 (dH 2.55), suggesting
the same relative configuration as that of a-D-tagatofuranose (Ito
et al., 2012). Moreover, the 13C NMR data of the spiroketal moiety
of 2 were found to be identical to those of upuborneol B (Ito et al.,
2012), whose spiroketal moiety originated from b-D-galactopyranose in the biosynthetic pathway of the Upuna borneensis.
Collectively, the absolute configuration of spiroketal residue of 2
was established as (20 R, 30 S, 40 R, 50 R).
The relative configuration of melicospiroketal C (3) was characterized as follows: ROESY experimental data showed correlations
of H-30 (dH 3.76)/H-40 (dH 4.05) and 30 -OH (dH 5.56), 40 -OH (dH 5.06)/
H-50 (dH 3.91), suggesting that H-30 and H-40 were co-facially oriented, but positioned opposite to H-50 . Cross-peaks of 30 -OH (dH
5.56)/H-10 (dH 3.37), and H-50 (dH 3.91)/H-10 (dH 2.85) revealed the
relative configuration of 3. Noticeably, the 13C NMR data of the
spiroketal ring of 3 closely resembled those of pinnatifinoside C
(Zhang and Xu, 2001), which is a flavone-bearing spiroketal ring
originating from b-D-allopyranose (Sato et al., 2004, 2008). Based
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
293
Fig. 1. Structures of compounds isolated from Melicope pteleifolia leaves.
on all these observations, the absolute stereochemistry of the spiroketal moiety of 3 was proposed to be (20 S, 30 R, 40 S, 50 R).
Compounds 4 and 5 were determined to possess the same
relative configurations through ROESY spectroscopic analysis. The
ROESY spectrum of 4 showed correlations of 40 -OH (dH 5.44)/H30 (dH 3.86) and H-50 (dH 3.74), H-30 (dH 3.86)/H-50 (dH 3.74), and H-50
(dH 3.74)/H-10 (dH 2.98). In the case of compound 5, correlations
were found for H-30 (dH 3.89)/H-50 (dH 3.77), H-30 (dH 3.89)/H-10 (dH
3.13) and H-40 (dH 3.94)/H3-8 (dH 2.57). However, the differences in
their NMR data suggested that the two compounds had different
absolute stereochemistry (Table 1). The 3D simulated models
showed noticeable differences between the two configurations.
While the distance between H-40 and H3-8 in model A was over 5 Å,
the distance of model B was approximately 3 Å, indicating that only
model B could show a ROESY correlation between H-40 and H3-8
(Fig. 3). Moreover, the 13C NMR data of 5 was shown to be identical
to those of upuborneol A (Ito et al., 2012), which was determined to
be an acetophenone bearing spiroketal ring originating from b-Dglucopyranose. Based on all these observations, the absolute
configuration of the spiroketal residue of 4 was determined to be
(20 R, 30 R, 40 R, 50 S), while that of 5 was determined to be (20 S, 30 S, 40 S,
50 R). These two new compounds were named as melicospiroketal D
and E, respectively, which were isolated the first time from nature.
To confirm the absolute configuration of spiroketal moiety, compound 4 was esterified using p-bromobenzoyl chloride with DMAP
to synthesize the p-bromobenzoylated derivative for X-ray crystallography experiment (Niwa et al., 1991). However, the obtained
product was actually a mixture of many esters due to the presence
of total 9 hydroxy groups in the structure of 4, leading to insufficient material for purification and crystallization procedures.
Compound 11 was isolated as a colorless gum and had the
molecular formula of C29H34O16, which was established from the
294
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
Fig. 2. Key HMBC correlations (H/C) of new compounds (1e5, 11, 13 and 14).
sodium adduct ion peak at m/z 661.1731 [MþNa]þ (calcd for
C29H34O16Na 661.1739) in its HRESIMS spectrum. The diagnostic
signals for a trans-p-coumaric acid moiety were observed in its 1H
and 13C NMR spectra, including two overlapping ortho-aromaticproton signals at dH 6.78 (2H, d, J ¼ 8.5 Hz) and 7.55 (2H, d,
J ¼ 8.5 Hz), two trans-olefinic protons at dH 6.42 (1H, d, J ¼ 16.0 Hz)
and 7.55 (1H, d, J ¼ 16.0 Hz), and an ester carbon at dC 166.6 (q).
Those characteristic signals were also confirmed by comparing
spectroscopic data with those that were previously reported
(Konishi and Shoji, 1981). The remaining carbon signals were
similar to those of compound 6 (Sato et al., 2004), which indicated
the presence of a di-C-b-D-glucosidic acetophenone. The key HMBC
correlations of 11 confirmed that it had the same backbone as
compound 6, except for the presence of an additional trans-pcoumaric acid moiety (Fig. 2). The linkage of the p-coumaroyl group
was assigned to be connected to the C-6 position of the glucose
moiety based on key HMBC correlations from H-600 (dH 4.23, dd,
J ¼ 6.0, 12.0 Hz and dH 4.44, d, J ¼ 12.0 Hz) to C-9 of the p-coumaric
acid moiety (dC 166.6). The presence and linkage of the p-coumaric
acid residue to the C-6 of glucose were also supported by
comparing spectroscopic data published in previous papers (Du
et al., 2010; Hirobe et al., 1997). Thus, the structure of 11 was
deduced as 5-C-b-D-glucopyranosyl-3-C-(6-O-trans-p-coumaroyl)b-D-glucopyranoside phloroacetophenone and was determined to
be a new compound.
Compound 13 was isolated as a yellow amorphous powder, with
a molecular formula of C15H18O5, which was established based on
the positive ion peak [MþH]þ at m/z 279.1220 (calcd for C15H19O5
279.1227) of its HRESIMS spectrum and 13C NMR data. An IR
spectrum of 13 showed absorption bands at 3781 and 1730 cm 1,
which corresponded to hydroxy and carbonyl groups, respectively.
The 1H and 13C NMR data showed typical patterns of a chromenetype compound (Kamperdick et al., 1997) and exhibited peaks
resembling those of leptonol (Li and Zhu, 1998). However, the
arrangement of functional groups in the aromatic ring of 13
appeared to be different from those of reported compounds. Two
methoxy groups were attached to C-5 and C-8, which was proven
by HMBC correlations of H3-5 (dH 3.78)/C-5 (dC 132.9) and H3-8 (dH
3.72)/C-8 (dC 154.2). Furthermore, an acetyl group was determined
to be present on C-7 based on the HMBC correlation of H3-7 (dH
295
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
Table 1
13
C NMR spectroscopic data for compounds 1e5 and 11 (d in ppm).
Position
1a
1
105.1
2
156.2
3
102.4
4
163.4
5
101.1
6
164.8
7
203.0
8
32.7
10
31.9
120.4
20
30
80.9
0
4
76.2
0
83.5
5
60
60.9
Glucose moiety
1
73.5
2
70.5
3
78.8
4
70.5
5
81.3
6
61.6
Coumaric acid moiety
1
2, 5
3, 6
4
7
8
9
a
b
2b
3b
4b
5b
11a
100.7
160.1
103.1
159.4
105.2
162.5
201.6
31.0
31.8
121.2
81.0
77.0
84.7
61.3
105.2
163.0
102.1
163.0
101.1
164.4
202.9
32.7
31.7
120.6
74.4
71.3
84.6
63.1
105.1
156.4
102.3
163.8
101.1
164.4
203.1
32.8
33.5
116.8
79.6
73.9
83.6
63.4
100.4
160.4
102.6
158.8
104.8
162.4
201.6
31.0
33.1
117.1
79.5
73.4
83.4
62.7
104.9
162.4
103.4
162.2
103.7
162.1
202.9
32.7
74.7
71.6
78.4
69.9
81.1
63.8
74.0
71.4
78.6
70.1
81.2
60.9
73.3
71.0
78.6
70.4
81.1
61.5
73.5
71.2
78.7
69.9
80.8
61.0
73.9
71.3
78.5
69.9
81.1
60.8
75.0
72.6
78.0
69.1
81.1
59.9
125.0
130.4
115.8
159.9
145.0
113.9
166.6
125 MHz.
150 MHz. All samples were measured in DMSO-d6.
which was deduced from the protonated ion peak [MþH]þ at m/z
279.1236 (calcd for C15H19O5 279.1227). Their similar NMR patterns
suggested that these two compounds shared the same backbone
and functional groups. However, the order of the groups on the
benzene moiety appeared to be different from that of 13. Two
methoxyl groups were assigned to be on C-5 and C-7 based on
HMBC correlations of H3-5 (dH 3.71)/C-5 (dC 145.6) and H3-7 (dH
3.89)/C-7 (dC 144.1). The acetyl group protons (dH 2.51) were shown
to correlate with C-6 (dC 123.0), establishing its location. The HMBC
correlations of the hydroxyl peak at dH 5.24 with C-7 (dC 144.1), C-8
(dC 134.4), and C-9 (dC 141.9) further supported the positioning of all
the functional groups (Fig. 2). Thus, the structure of 14 was determined as 6-acetyl-8-hydroxy-5,7-dimethoxy-2,2-dimethyl-2H-lbenzopyran.
The structures of the known compounds (6e10, 12 and 15e24)
were identified by means of spectroscopic analyses (ESIMS, 1H and
13
C NMR) and by comparing the spectroscopic data with literature
values of the following compounds: 3,5-di-C-b-D-glucopyranosyl
phloroacetophenone (6) (Sato et al., 2004), kaempferol 3robinobioside (7) (Brasseur and Angenot, 1986), kaempferol 3-Ob-D-glucopyranosyl (1 / 2)-a-D-xylopyranoside (8) (Fiorentino
et al., 2009), icariside B5 (9) (Matsunami et al., 2010), tamarixetin
3-robinobioside (10) (Barrero et al., 1998), leptonol (12) (Li and Zhu,
1998), 8-acetyl-7-hydroxy-5,6-dimethoxy-2,2-dimethyl-2H-l-benzopyran (15) (Kamperdick et al., 1997), evodione (16) (Kamperdick
et al., 1997), leptene A (17) (Guo-Lin et al., 1997), isoevodionol (18)
(Li and Zhu, 1999), 6-acetyl-7-hydroxy-5-methoxy-2,2-dimethyl2H-chromene (19) (Donnelly et al., 1995), 6-(1-methoxyethyl)5,7,8-trimethoxy-2,2-dimethyl-2H-l-benzopyran (20) (Kamperdick
Table 2
1
H NMR spectroscopic data for compounds 1e5 and 11 (d in ppm, mult., J in Hz).
Position
1a
2b
3b
4b
5b
11a
8
10
2.61, s
3.36, d (16.5)
2.79, d (16.5)
2.55, s
3.34, d (16.2)
2.79, d (16.2)
2.61, s
3.37, d (16.2)
2.85, d (16.2)
2.61, s
3.11, d (16.2)
2.98, d (16.2)
2.57, s
3.13, d, (16.2)
2.96, d, (16.2)
2.56, s
4.71, d (10.0)
4.05,
3.76,
3.84,
3.49,
3.57,
t (5.0)
m
m
m
d (11.5)
4.10,
3.81,
3.90,
3.59,
3.44,
3.76,
4.05,
3.91,
3.60,
3.45,
m
m
dt (3.0, 7.2)
m
m
3.86,
3.85,
3.74,
3.55,
3.46,
d (8.4)
dd (7.2, 8.4)
dt (3.0, 7.2)
d (12.0)
m
3.89,
3.94,
3.77,
3.59,
3.45,
4.36,
3.88,
3.18,
3.09,
3.14,
3.66,
3.38,
d (10.0)
m
m
m
m
d (11.5)
m
4.61, d (9.6)
3.72, m
3.20, m
3.19, m
3.21, m
3.64, d (10.2)
3.49, dd (3.6, 10.2)
13.57, s
4.34,
3.74,
3.17,
3.07,
3.12,
3.66,
3.38,
d (9.6)
d(9.6)
t (8.4)
m
m
d (11.4)
m
4.38,
3.79,
3.19,
3.17,
3.14,
3.61,
3.46,
d (10.2)
m
m
m
m
d (12.0)
m
4.59, d (9.6)
3.70, m
3.20, m
3.32, m
3.18, m
3.63, d (10.8)
3.49, m
13.60, s
20
30
40
50
60
Glucose moiety
1
2
3
4
5
6
6-OH
30 -OH
5.74, d (5.0)
5.33, d (5.0)
40 -OH
60 -OH
Coumaric acid moiety
2,5
3,6
7
8
a
b
d (3.6)
dd (3.6,
dd (3.6,
dd (3.6,
dd (7.2,
6.0)
6.0)
12.0)
12.0)
5.56, d (4.2)
5.06, d (6.0)
4.72, brt
brd (8.4)
t (8.4)
dt (2.4, 6.6)
d (10.2)
m
3.65,
3.53,
3.27,
3.27,
4.44,
4.23,
m
m
m
m
d (12.0)
dd (6.0, 12.0)
4.73,
3.46,
3.25,
3.33,
3.27,
3.62,
d (10.0)
m
m
m
m
m
7.55,
6.78,
7.55,
6.42,
d
d
d
d
5.10, d (5.4)
5.44, brs
(8.5)
(8.5)
(16.0)
(16.0)
500 MHz.
600 MHz. All samples were measured in DMSO-d6.
2.62)/C-7 (dC 109.1). HMBC correlations from 6-OH (dH 13.51) to C5/6/7 also strongly supported the positions of all the functional
groups (Fig. 2). Therefore, the structure of 13 was elucidated as 7acetyl-6-hydroxy-5,8-dimethoxy-2,2-dimethyl-2H-l-benzopyran.
Compound 14 had the same molecular formula as 13, C15H18O5,
et al., 1997), acronyculatin G (21) (Kozaki et al., 2014), 6-(lhydroxyethyl)-5,7,8-trimethoxy-2,2-dimethyl-2H-1-benzopyran
(22) (Kamperdick et al., 1997), 3,5,40 -trihydroxy-8,30 -dimethoxy-7(3-methylbut-2-enyloxy)flavone (23) (Sultana et al., 1999), and
3,5,30 -trihydroxy-8,40 -dimethoxy-7-(3-methylbut-2-enyloxy)
296
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
Fig. 3. Key ROESY correlations of compounds 4 and 5 (models simulated by Chem3D); (a) model A (compound 4); (b) model B (compound 5).
flavone (24) (Xie et al., 2011b).
To provide scientific evidence regarding the traditional use of
M. pteleifolia leaves to treat fevers and colds, all the isolates were
screened for their enzymatic inhibitory activities against neuraminidase (NA) of the H1N1 influenza virus. The screening data
indicated that flavonoids 7, 8, 10, 23 and 24 could be used to test for
their NA inhibitory activities, while the remaining compounds
showed little to no inhibition up to concentrations of 400 mM
(Supplementary data). The potential candidates were then examined in further detail against NAs from four different viral strains,
including H1N1, H9N2 (avian influenza virus), novel H1N1 (wild
type) and oseltamivir-resistant novel H1N1 (H274Y mutation). Of
the five tested flavonoids, compound 10 exhibited the strongest
inhibitory activity with an IC50 ranging from 23.19 ± 5.41 to
40.61 ± 4.50 mM, while compounds 7 and 8 only showed moderate
inhibition (IC50 values from 84.52 ± 10.51 to 129.83 ± 12.53 mM). As
shown in Table 4, while the inhibitory effects of the positive control
(oseltamivir) dropped remarkably against the H1N1 mutant strain
(oseltamivir-resistant, H274Y) (IC50 dropped from 41.42 ± 3.94 nM
Table 3
1
H and 13C NMR spectroscopic data for compounds 13 and 14.
Position
13a
2
3
4
5
6
7
8
9
10
11
12
2-Me2
5-OMe
7-OMe
8-OMe
6-OH
8-OH
77.7
128.3
116.5
132.9
158.7
109.1
154.2
152.9
107.3
203.5
31.2
28.1
60.7
dC
63.2
14a
dH
5.57, d (10.2)
6.44, d (10.2)
2.62, s
1.44, s
3.78, s
dC
77.8
129.5
116.7
145.6
123.0
144.1
134.4
141.9
111.1
201.5
32.7
28.1
64.1
61.9
dH
5.61, d (10.2)
6.50, d (10.2)
3. Conclusions
Phytochemical investigations of M. pteleifolia leaves led to the
isolation of five new spiroketals (1e5), one new acetophenone (11),
and two new benzopyrans (13, 14), along with 16 known compounds (6e10, 12 and 15e24). Our study established the major
occurrence of spiroketal-type compounds from the methanol
extract of M. pteleifolia leaves. All isolates were also screened for
their neuraminidase inhibitory activities. Of the isolates, compound
10 was shown to have the strongest inhibitory effects against
several types of neuraminidase enzymes (IC50 values ranging from
23.19 ± 5.41 to 40.16 ± 4.50 mM). Furthermore, a cytopathic effect
(CPE) assay demonstrated that compounds 7, 8 and 10 also reduced
H1N1-induced cytopathic effects in MDCK cells.
4. Experimental
4.1. General experimental procedures
2.51,
1.47,
3.71,
3.89,
s
s
s
s
3.72, s
13.51, s
All samples were measured in CDCl3.
a
600 MHz for proton NMR and 150 MHz for carbon NMR.
to 7.23 ± 0.73 mM), compound 10 still retained its inhibition effects
and had an IC50 of 40.61 ± 4.50 mM. These data suggest that the
methoxy substitution in the B-ring of flavonoids (7, 8 and 10) may
contribute to enzymatic inhibition while oxyprenylation in the Aring (23 and 24) may reduce inhibitory activity.
To confirm the viral inhibition effects of the selected candidates,
a cytopathic effect (CPE) assay was employed using two positive
controls, oseltamivir and ribavirin. As shown in Fig. 4, compounds 7,
8 and 10 were shown to reduce cytopathic effects on H1N1-infected
MDCK cells, while compounds 23 and 24 did not show clear activities due to their cytotoxicities at high concentrations. Of all the
selected candidates, compound 10 still exhibited the strongest
inhibitory activity against the H1N1 virus in the CPE assay.
5.24, brs
Nuclear magnetic resonance (NMR) spectra were obtained on
AVANCE-500, AVANCE-600 (Bruker, Germany) and JNM-ECA-600
(Jeol, USA) spectrometers at the National Center for InterUniversity Research Facilities (NCIRF) and College of Pharmacy,
Seoul National University, Korea. HRESIMS data were measured
with an Agilent Q-TOF MS 1260 Infinity (Agilent Technologies, Inc.,
Santa Clara, California, USA) spectrometer; circular dichroism (CD)
spectra were obtained from an Applied Photophysics Ltd.,
297
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
Table 4
Inhibitory effects (IC50) of compounds 7, 8, 10, 23 and 24 on neuraminidase activity.
Compound
H1N1 (mM)
H9N2 (mM)
H1N1 (WT) (mM)
H274Y (mM)
7
8
10
23
24
Oseltamivir
91.63 ± 7.32
107.22 ± 7.45
24.93 ± 3.46
173.07 ± 21.33
160.28 ± 26.32
99.91 ± 16.76 nM
88.04
84.52
23.19
NT
NT
14.16
94.41
95.48
26.67
NT
NT
41.42
129.83 ± 12.53
123.40 ± 17.25
40.61 ± 4.50
NT
NT
7.23 ± 0.73
± 5.55
± 10.51
± 5.41
± 0.79 nM
± 10.32
± 9.24
± 5.16
± 3.94 nM
NT: Not tested.
Fig. 4. Antiviral activities of compounds 7, 8, 10, 23 and 24 against the H1N1 A/PR/8/34 virus in a viral cytopathic effect reduction assay using MDCK cells. Data are presented as the
mean ± SD (n ¼ 3), *p < 0.05, **p < 0.01, and ***p < 0.001 for infected cells with the H1N1 virus control, #p < 0.05, ##p < 0.01 and ###p < 0.001 for uninfected cells with H1N1 virus
control (one-way analysis of variance followed by a two-tailed Student’s t-test).
Chirascan-plus CD spectrometer (Surrey, UK); infrared (IR) spectroscopic data were obtained on a JASCO FT-IR-4200 (USA); optical
rotations were measured on a JASCO P-2000 (USA) polarimeter
with a 10-mm path length cell at the College of Pharmacy, Seoul
National University, Korea. Column chromatography (CC) was performed using various adsorbents, including normal-phase silica gel
(63e220 mm particle size), reversed-phase C-18 silica gel
(40e63 mm particle size) purchased from Merck (Darmstadt, Germany) and Sephadex LH-20 (Sigma-Aldrich Corp., St. Louis, Missouri, USA) resin. Thin-layer chromatography (TLC) was carried out
on silica gel 60 F254 and RP-18 F254 plates purchased from Merck
(Darmstadt, Germany). Preparative HPLC was conducted on a Gilson system using an Optima Pak C18 column (10 250 mm, 10 mm
particle size, RS Tech, Seoul, Korea) and UV detector at wavelengths
of 205 and 254 nm. All solvents used for extraction and isolation
were of analytical quality. All 3D models of the spiroketal compounds were simulated using Chem3D Pro 14.0.0.117 software in
MM2 minimizing energy mode (CambridgeSoft Corporation, PerkinElmer, Inc.).
4.2. Plant materials
The leaves of Melicope pteleifolia (Champ. Ex Benth) T.G. Hartley
(Rutaceae) were collected from Ngoc Ha herbal distract, Hanoi,
Vietnam in March 2014. The sample was botanically identified by
Dr. Tran The Bach at the Institute of Ecology and Biological Resources, Hanoi, Vietnam. A voucher specimen (KRIBB 010471) is
deposited in the herbarium of the Korea Research Institute of
Bioscience and Biotechnology.
4.3. Extraction and isolation
The dried leaves of M. pteleifolia (1.0 kg) were extracted using
MeOH (5 L 3 times 4 hours at room temperature) with ultrasonic assistance. The combined extracts were filtered and evaporated under reduced pressure to yield a green residue (110.0 g). The
crude extract was suspended in distilled H2O (2 L) and successively
partitioned with EtOAc and n-BuOH. The n-BuOH portion (22.5 g)
was initially fractionated by normal-phase silica gel CC
(10 25 cm; 63e220 mm particle size) eluted with a gradient
solvent system of EtOAc and MeOH (ratio from 15:1 to 0:1) to
obtain 7 smaller fractions (F1-7). Fraction F3 (2.2 g) was then
applied to a reversed-phase silica gel CC (2 40 cm; 40e63 mm
particle size) eluted with MeOH:H2O (1:1, v/v) to obtain 6 subfractions (F3.1e3.6). Further separation of F3.1 (200 mg) by HPLC
(Gilson system using an Optima Pak C18 column (10 250 mm,
10 mm particle size, mobile phase MeOH: H2O containing 0.1%
HCO2H, 0e20 min: isocratic 20% MeOH in H2O, flow rate 2 mL/min;
UV detector wavelengths at 205 and 254 nm) resulted in isolation
of compound 2 (tR ¼ 16 min, 69.3 mg). Meanwhile, compounds 7
(tR ¼ 23 min, 8.5 mg) and 8 (tR ¼ 24 min, 2.5 mg) were isolated from
fraction F3.5 (100 mg) using the HPLC system (0e25min: isocratic
20% MeCN in H2O). Fraction 3.6 (80 mg) was also purified using
HPLC (0e20 min: 22% MeCN in H2O) to yield compound 10
(tR ¼ 17 min, 5.0 mg). Next, fraction F4 (0.6 g) was subjected to a
reversed-phase CC to separate out the polar fractions (F4.1, 100 mg).
F4.1 (100 mg) was then purified HPLC (0e25min: isocratic 8% MeCN
in H2O) to yield compound 5 (tR ¼ 14 min, 4.5 mg). The polar
portion of fraction F5 (300 mg) was successively purified by a RPC18 CC and HPLC (0e30 min: isocratic 15% MeCN in H2O) and
298
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
resulted in the purification of compound 9 (tR ¼ 21 min, 10.5 mg).
Fraction F6 (2.1 g) was first fractionated by reversed-phase CC, and
the polar fraction (F6.1, 200 mg) was then subjected to an HPLC
(0e30 min: isocratic 8% MeCN in H2O), which resulted in the
isolation of compounds 3 (tR ¼ 29 min, 10.3 mg), 1 (tR ¼ 32 min,
43.2 mg) and 4 (tR ¼ 34 min, 27.2 mg). Finally, the major fraction
(F7, 2.5 g) was applied to reversed-phase CC to divide into smaller
subfractions (F7.1e7.3). A part of F7.1 (1.6 g) was purified with a
Sephadex LH-20 CC (2 40 cm) and was eluted with MeOH: H2O
(1:1, v/v) to yield compound 6 (13.0 mg). F7.3 (30 mg) was applied
to an HPLC system (0e25 min: isocratic 24% MeCN in H2O) to obtain
compound 11 (tR ¼ 24 min, 3.5 mg).
The EtOAc portion (55.5 g) was initially separated by normalphase CC (10 25 cm; 63e220 mm particle size) using a stepwise
gradient solvent mixture of n-hexane/EtOAc (from 30:1 to 0:1), to
obtain 9 fractions (E1-9). The non-polar fraction E1 (4.3 g) was
fractionated again using normal-phase silica gel CC (5 40 cm;
40e63 mm particle size) with an n-hexane/EtOAc system (50:1) as
eluent to afford 3 subfractions (E1.1-3). Fraction E1.1 (270 mg) was
applied to HPLC (0e30 min: isocratic 80% MeOH in H2O) to yield
compounds 19 (tR ¼ 21 min, 16.5 mg), 15 (tR ¼ 23 min, 2.9 mg) and
12 (tR ¼ 25 min, 35.1 mg). While compound 18 (tR ¼ 16 min, 8.9 mg)
was purified from fraction E1.2 (67 mg) using HPLC with an isocratic 87% MeOH in H2O solvent system (0e20 min), compounds 17
(3.1 mg) and 21 (4.0 mg) were isolated from fraction E1.3 (200 mg)
with an RP-C18 column using isocratic 85% MeOH in H2O as eluent.
Fraction E2 (2.8 g) was applied to a reversed-phase column
(2 40 cm; 40e63 mm particle size) and was eluted with 75%
MeOH in H2O to yield compounds 16 (138.2 mg), 20 (14.6 mg) and
13 (98.2 mg). Chromatographic purification of E5 (0.4 g) by an ODS
column (2 40 cm; 40e63 mm particle size), eluting with MeOH:
H2O (3:1, v/v) yielded compound 14 (8.2 mg). Next, fraction E8
(1.2 g) was subjected into an RP-C18 column (2 40 cm; 40e63 mm
particle size) to obtain compound 22 (64.3 mg). Fine yellow crystals
appeared from E9 (2.2 g) which were filtered and washed with nhexane to obtain compound 23 (250 mg). The mother liquor was
then dried and applied to an HPLC system with appropriate conditions (0e30 min: isocratic 80% MeOH in H2O) to yield compound
24 (10.0 mg).
4.3.1. Melicospiroketal A (1)
Brownish gum; ½a20
D þ78.7 (c 0.3, MeOH); IR (KBr) nmax 3696,
3351, 1627, 1445, 1371, 1313 cm 1; UV lmax (MeOH) (log ε) (nm) 232
(3.48), 288 (3.49); CD (c 0.5, MeOH) 225 ( 8.30), 284 (þ3.00), 328
( 1.49); See Tables 1 and 2 for 1H NMR (500 MHz) and 13C NMR
(125 MHz) spectroscopic data; HRESIMS [MþH]þ m/z 475.1433
(calcd for C20H27O13 475.1446).
4.3.2. Melicospiroketal B (2)
Brownish gum; ½a20
D þ27.6 (c 0.3, MeOH); IR (KBr) nmax 3364,
2929, 1629, 1445, 1369, 1303 cm 1; UV lmax (MeOH) (log ε) (nm)
240 (3.48), 264 (3.49), 335 (3.37); CD (c 0.5, MeOH) 276 ( 0.50),
299 (þ2.63), 315 (þ1.65); See Tables 1 and 2 for 1H NMR (600 MHz)
and 13C NMR spectroscopic data; HRESIMS [MþH]þ m/z 475.1450
(calcd for C20H27O13 475.1446).
4.3.3. Melicospiroketal C (3)
Brownish gum; ½a20
D -25.4 (c 0.3, MeOH); IR (KBr) nmax 3379,
2935, 1627, 1444, 1371, 1315 cm 1; UV lmax (MeOH) (log ε) (nm) 229
(3.37), 288 (3.36); CD (c 0.5, MeOH) 221 ( 6.30), 248 (þ0.05), 286
( 4.4); See Tables 1 and 2 for 1H NMR (600 MHz) and 13C NMR
(150 MHz) spectroscopic data; HRESIMS [MþH]þ m/z 475.1440
(calcd for C20H27O13 475.1446).
4.3.4. Melicospiroketal D (4)
Brownish gum; ½a20
D -20.2 (c 0.3, MeOH); IR (KBr) nmax 3365,
2925, 1629, 1446, 1372, 1321 cm 1; UV lmax (MeOH) (log ε) (nm)
230 (3.22), 288 (3.19); CD (c 0.5, MeOH) 261 ( 0.56), 282 ( 1.13),
330 ( 0.50); See Tables 1 and 2 for 1H NMR (600 MHz) and 13C NMR
(150 MHz) spectroscopic data; HRESIMS [MþH]þ m/z 475.1442
(calcd for C20H27O13 475.1446).
4.3.5. Melicospiroketal E (5)
Brownish gum; ½a20
D þ7.6 (c 0.3, MeOH); IR (KBr) nmax 3370,
2938, 1626, 1440, 1369, 1294 cm 1; UV lmax (MeOH) (log ε) (nm)
230 (2.98), 284 (2.92); CD (c 0.5, MeOH) 215 ( 2.79), 228 ( 4.79),
284 (þ2.93); See Tables 1 and 2 for 1H NMR (600 MHz) and 13C NMR
(150 MHz) spectroscopic data; HRESIMS [MþH]þ m/z 475.1447
(calcd for C20H27O13 475.1446).
4.3.6. 5-C-b-D-glucopyranosyl-3-C-(6-O-trans-p-coumaroyl)-b-Dglucopyranoside phloroacetophenone (11)
Colorless gum; ½a20
D -5.5 (c 0.3, MeOH); IR (KBr) nmax 3703, 1691,
1615, 1518, 1274, 1042, 1024 cm 1; UV lmax (MeOH) (log ε) (nm) 230
(3.18), 290 (3.18); See Tables 1 and 2 for 1H NMR (500 MHz) and 13C
NMR (125 MHz) spectroscopic data; HRESIMS [MþNa]þ m/z
661.1731 (calcd for C29H34O16Na 661.1739).
4.3.7. 7-acetyl-6-hydroxy-5,8-dimethoxy-2,2-dimethyl-2H-1benzopyran (13)
Yellow amorphous powder, IR (KBr) nmax 3781, 2367, 2318, 1730,
1609, 1444, 1382, 1290 cm 1; UV lmax (MeOH) (log ε) (nm) 260
(3.45); See Table 3 for 1H NMR (600 MHz) and 13C NMR (150 MHz)
spectroscopic data; HRESIMS [MþH]þm/z 279.1220 (calcd for
C15H19O5 279.1227).
4.3.8. 6-acetyl-8-hydroxy-5,7-dimethoxy-2,2-dimethyl-2H-1benzopyran (14)
White amorphous powder, IR (KBr) nmax 2953, 2363, 2319, 1731,
1601, 1454, 1361, 1242 cm 1; UV lmax (MeOH) (log ε) (nm) 222
(2.52), 256 (2.27); See Table 3 for 1H NMR (600 MHz) and 13C NMR
(150 MHz) spectroscopic data; HRESIMS [MþH]þ m/z 279.1236
(calcd for C15H19O5 279.1227).
4.4. Cell culture and expression of neuraminidases
HEK293 cells (human embryonic kidney cells) and MDCK cells
(Madin-Darby canine kidney cells) were maintained in DMEM
(Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine
serum (FBS; Hyclone, Logan, UT, USA) at 37 C and 5% CO2. The
HEK293 cells were transfected with H1N1 (WT) plasmid or
oseltamivir-resistant novel H1N1 (H274Y) plasmids using the PEI
transfection reagent (Polyscience, Inc., Warrington, PA, USA) as
previously described (Nguyen et al., 2011). After transfection, the
cells were maintained in DMEM containing 5% FBS. After 48 h posttransfection, the medium was removed, and the cells were treated
with phosphate-buffered saline (PBS) supplemented with 0.02%
ethylene diamine tetraacetic acid (EDTA) and were then harvested.
4.5. Influenza H1N1 A/PR/8/34 and H9N2 A/Chicken/Korea/O1310/
2001 neuraminidase inhibition assays
The neuraminidase inhibition assay was performed as previously described with several modifications (Dao et al., 2010).
Briefly, influenza H1N1 or H9N2 viruses were injected into MDCK
cells in large-scale using DMEM containing trypsin (0.15 mg/mL)
and bovine serum albumin (5 mg/mL) (BSA; Sigma-Aldrich Co., St
Louis, MO, USA). To inactivate the ability for viral infection, formaldehyde was added to the viral suspension until the final
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
formaldehyde concentration was 0.1%. The neuraminidase activity
determined using 20 -(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (4-MU-NANA; Sigma-Aldrich Co., St Louis, MO, USA) as
the fluorescent substrate. Briefly, the assay was performed using
96-well plates containing viral suspensions (10 mL) and oseltamivir
phosphate (10 mL) (Hoffman-La Roche Ltd., Basel, Switzerland) or
test compounds diluted to corresponding concentrations with
enzyme buffer (32.5 mM 2-(N-morpholino)ethanesulfonic acid,
4 mM CaCl2, pH 6.5). After 30 min of incubation at 37 C, 30 mL of 4MU-NANA substrate (0.04 mM) was added to each well. The
enzymatic reactions were incubated at 37 C for 2 h and quenched
by treating the wells with a stop solution (150 mL) (25% EtOH in
H2O, 0.1 M glycine, pH 10.7). The fluorescence intensities of the
reactions were monitored using a fluorescence microplate reader
(Spectra Max GEMINI XPS, Molecular Devices, Sunnyvale, CA, USA)
with an excitation at 360 nm and emission at 440 nm. Halfmaximal inhibitory concentrations (IC50) were determined using
SigmaPlot 11.0 (SPCC Inc., Chicago, IL, USA).
4.6. Novel H1N1 (WT) and oseltamivir-resistant novel H1N1
(H274Y) neuraminidase inhibition assays
After post-transfection with H1N1 (WT) or H274Y plasmids, the
HEK293 cells were harvested with PBS containing 0.02% EDTA (pH
7.4) and were then washed with PBS at pH 7.4. Neuraminidase
suspensions were prepared by adding PBS (100 mL) containing
3.5 mM CaCl2 (pH 7.4) into the cells (approximately 2 106 cells)
and storing the samples at 80 C. The neuraminidase inhibition
assay was performed following a procedure similar to the one
described above.
4.7. Cytopathic effect (CPE) inhibition assay
MDCK cells were seeded into 96-well plates and incubated for 1
day. Then, the cells were washed twice with PBS and infected with
or without H1N1 A/PR/8/34 virus using DMEM containing trypsin
(0.15 mg/mL) and BSA (5 mg/mL). After 1 h of incubation, the medium was removed, the cells were washed with PBS, and the medium was replaced with new media containing several compounds
at different concentrations. After incubation for 3 days at 37 C
under a 5% CO2 atmosphere, the media were replaced with DMEM
and 2 mg/mL MTT (20 mL) was added to each well and incubated for
4 h at 37 C. Formazan crystals were dissolved in DMSO (100 mL),
added to each well, and the resulting absorbance for the wells were
measured at 550 nm using an absorbance microplate reader (VersaMax™, Randor, PA, USA).
4.8. Statistical analyses
The data were expressed as the means ± SD (n ¼ 3). Statistical
analyses were performed using Sigma Plot Statistical Analysis
software. Differences between the group means were determined
by a one-way analysis of variance followed by a two-tailed Student’s t-test. Statistical significance was accepted when at p < 0.05.
Acknowledgements
This work was supported in part by grants from the Korea
Bioactive Natural Material Bank (NRF-2012M3A9B8021570) and
from the Procurement and Development of Foreign Biological Resources (2012-K1A1A3307871) of the National Research Foundation of Korea (NRF), which is funded by the Korean government.
299
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2016.06.011.
References
Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31, 3307e3330.
~ oz-Dorado, M., Akssira, M., Sedqui, A., Mansour, I.,
Barrero, A.F., Haídour, A., Mun
1998. Polyacetylenes, terpenoids and flavonoids from Bupleurum spinosum.
Phytochemistry 48, 1237e1240.
€s, B., Zapp, J., Becker, H., 2004. ent-Clerodane diterpenes and other constituents
Bla
from the liverwort Adelanthus lindenbergianus (Lehm.) Mitt. Phytochemistry 65,
127e137.
Brasseur, T., Angenot, L., 1986. Flavonol glycosides from leaves of Strychnos variabilis.
Phytochemistry 25, 563e564.
Dao, T.-T., Tung, B.-T., Nguyen, P.-H., Thuong, P.-T., Yoo, S.-S., Kim, E.-H., Kim, S.-K.,
Oh, W.-K., 2010. C-methylated flavonoids from Cleistocalyx operculatus and their
inhibitory effects on novel influenza A (H1N1) neuraminidase. J. Nat. Prod. 73,
1636e1642.
Donnelly, D.M.X., Molloy, D.J., Reilly, J.P., Finet, J.-P., 1995. Aryllead-mediated synthesis of linear 3-arylpyranocoumarins: synthesis of robustin and robustic acid.
J. Chem. Soc. Perkin Trans. 1, 2531e2534.
Du, Z.-Z., Yang, X.-W., Han, H., Cai, X.-H., Luo, X.-D., 2010. A new flavone C-glycoside
from Clematis rehderiana. Molecules 15, 672e679.
Fiorentino, A., Ricci, A., D’Abrosca, B., Golino, A., Izzo, A., Pascarella, M.T.,
Piccolella, S., Esposito, A., 2009. Kaempferol glycosides from Lobularia maritima
and their potential role in plant interactions. Chem. Biodivers. 6, 204e217.
Furuta, T., Kimura, T., Kondo, S., Mihara, H., Wakimoto, T., Nukaya, H., Tsuji, K.,
Tanaka, K., 2004. Concise total synthesis of flavone C-glycoside having potent
anti-inflammatory activity. Tetrahedron 60, 9375e9379.
Grienke, U., Schmidtke, M., von Grafenstein, S., Kirchmair, J., Liedl, K.R.,
Rollinger, J.M., 2012. Influenza neuraminidase: a druggable target for natural
products. Nat. Prod. Rep 29, 11e36.
Guo-Lin, L., Jia-Feng, Z., Chun-Qing, S., Da-Yuan, Z., 1997. Chromenes from Evodia
lepta. Phytochemistry 44, 1175e1177.
Hirobe, C., Qiao, Z.-S., Takeya, K., Itokawa, H., 1997. Cytotoxic flavonoids from Vitex
agnus-castus. Phytochemistry 46, 521e524.
Ito, T., Ito, H., Oyama, M., Tanaka, T., Murata, J., Darnaedi, D., Iinuma, M., 2012. Novel
isolation of acetophenone derivatives with spiroketal-hexosefuranoside in
Upuna borneensis. Phytochem. Lett. 5, 325e328.
Kamperdick, C., Van, N.H., Sung, T.V., Adam, G., 1997. Benzopyrans from Melicope
ptelefolia leaves. Phytochemistry 45, 1049e1056.
Konishi, T., Shoji, J., 1981. Studies on the constituents of Boschniakia rossica
FEDTSCH. et FLEROV. I. Isolation and structures of new phenylpropanoid glycosides, rossicasides B, C and D. Chem. Pharm. Bull. 29, 2807e2815.
Kozaki, S., Takenaka, Y., Mizushina, Y., Yamaura, T., Tanahashi, T., 2014. Three acetophenones from Acronychia pedunculata. J. Nat. Med. 68, 421e426.
Li, G.-L., Zhu, D.-Y., 1998. Two chromenes from Evodia lepta. Phytochemistry 48,
1051e1054.
Li, G.L., Zhu, D.Y., 1999. Two dichromenes from Evodia lepta. J. Asian Nat. Prod. Res. 1,
337e341.
Matsunami, K., Otsuka, H., Takeda, Y., Miyase, T., 2010. Reinvestigation of the absolute stereochemistry of megastigmane glucoside, icariside B(5). Chem.
Pharm. Bull. 58, 1399e1402.
Nakatsuka, T., Tomimori, Y., Fukuda, Y., Nukaya, H., 2004. First total synthesis of
structurally unique flavonoids and their strong anti-inflammatory effect. Bioorg. Med. Chem. Lett. 14, 3201e3203.
Nguyen, T.N.A., Dao, T.T., Tung, B.T., Choi, H., Kim, E., Park, J., Lim, S.-I.L., Oh, W.K.,
2011. Influenza A (H1N1) neuraminidase inhibitors from Vitis amurensis. Food
Chem. 124, 437e443.
Niwa, M., Sugino, H., Takashima, S., Sakai, T., Wu, Y.-C., Wu, T.-S., Kuoh, C.-S., 1991.
A new coumarin glucoside from Daphne arisanensis. Chem. Pharm. Bull. 39,
2422e2424.
Qi, J., Lu, J.J., Liu, J.H., Yu, B.Y., 2009. Flavonoid and a rare benzophenone glycoside
from the leaves of Aquilaria sinensis. Chem. Pharm. Bull. 57, 134e137.
Sato, S., Akiya, T., Suzuki, T., Onodera, J.-i, 2004. Environmentally friendly C-glycosylation of phloroacetophenone with unprotected d-glucose using scandium(III) trifluoromethanesulfonate in aqueous media: key compounds for the
syntheses of mono- and di-C-glucosylflavonoids. Carbohydr. Res. 339,
2611e2614.
Sato, S., Miura, M., Sekito, T., Kumazawa, T., 2008. Effective conversion of three
diacetyl-C-(b-D-Glycopyranosyl) phloroglucinols to spiroketal derivatives by
refluxing in water. J. Carbohydr. Chem. 27, 86e102.
Shaari, K., Suppaiah, V., Wai, L.K., Stanslas, J., Tejo, B.A., Israf, D.A., Abas, F., Ismail, I.S.,
Shuaib, N.H., Zareen, S., Lajis, N.H., 2011. Bioassay-guided identification of an
anti-inflammatory prenylated acylphloroglucinol from Melicope ptelefolia and
molecular insights into its interaction with 5-lipoxygenase. Bioorg. Med. Chem.
19, 6340e6347.
Sichaem, J., Jirasirichote, A., Sapasuntikul, K., Khumkratok, S., Sawasdee, P., Do, T.M.,
Tip-pyang, S., 2014. New furoquinoline alkaloids from the leaves of Evodia lepta.
Fitoterapia 92, 270e273.
300
N.H. Nguyen et al. / Phytochemistry 130 (2016) 291e300
Sultana, N., Hartley, T.G., Waterman, P.G., 1999. Two novel prenylated flavones from
the aerial parts of Melicope micrococca. Phytochemistry 50, 1249e1253.
Xie, Y., Gong, J., Li, M., Fang, H., Xu, W., 2011a. The medicinal potential of influenza
virus surface proteins: hemagglutinin and neuraminidase. Curr. Med. Chem. 18,
1050e1066.
Xie, Y., Liang, Y., Du, Q., Guo, L., 2011b. Study on the chemical constituents from
Melicope ptelefolia. Zhong Yao Cai 34, 386e388.
Yoon, J.Y., Jeong, H.Y., Kim, S.H., Kim, H.G., Nam, G., Kim, J.P., Yoon, D.H., Hwang, H.,
Kimc, T.W., Hong, S., Cho, J.Y., 2013. Methanol extract of Evodia lepta displays
Syk/Src-targeted anti-inflammatory activity. J. Ethnopharmacol. 148, 999e1007.
Zhang, L., Xia, Y., Peterson, D.G., 2014. Identification of bitter modulating Maillardcatechin reaction products. J. Agric. Food Chem. 62, 8470e8477.
Zhang, P.-C., Xu, S.-X., 2001. Flavonoid ketohexosefuranosides from the leaves of
Crataegus pinnatifida Bge. var. major N.E.Br. Phytochemistry 57, 1249e1253.
Zhang, Y., Yang, L.-J., Jiang, K., Tan, C.-H., Tan, J.-J., Yang, P.-M., Zhu, D.-Y., 2012.
Glycosidic constituents from the roots and rhizomes of Melicope pteleifolia.
Carbohydr. Res. 361, 114e119.