Chemical constituents from the twigs and leaves of Trichilia sinensis and their biological activities

Article abstract of DOI:10.1016/j.phytol.2018.11.020

Phytochemical investigation on the twigs and leaves of Trichilia sinensis led to the isolation of two previously undescribed limonoids (i.e., trichiliasinenoids D and E, 1 and 2), two previously undescribed phenolic acids (3 and 4), and one previously undescribed natural phenolic acid dimer (5), together with 11 known compounds (6-16). Their structures were elucidated by extensive spectroscopic analysis (IR, UV, HRESIMS, 1D and 2D NMR) and chemical techniques. The potential anti-inflammatory activities of all the compounds were evaluated in lipopolysaccharide-stimulated RAW 264.7 cells. Among these isolates, compounds 1, 2, 6, and 11-13 expressed weak NO inhibition. The antibacterial activities of all the compounds against bacteria were also tested in vitro. Compound 16 exhibited moderate antibacterial activities against Escherichia Coli.

Full text of DOI:10.1016/j.phytol.2018.11.020

Phytochemistry Letters 29 (2019) 142–147
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Chemical constituents from the twigs and leaves of Trichilia sinensis and their
biological activities
a,b,1
a,b
a,b,1
a,b
, Shang-Gao Liao , Li-She Gan , Lin Yang , Jian-Neng Yaob,e,
c
a,b
d
a
Dong-Hua Cao
, Peng Sun
e,f
a
Zong-Yi Zhang , Jin-Feng Li , Xiao-Ling Zheng , Yi-Dian Xiao , Chun-Fen Xiao ,
a
a
a,⁎
Ping Zhang , Hua-Bin Hu , You-Kai Xu
a
CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, 666303, PR
China
b
c
d
e
University of Chinese Academy of Sciences, Beijing, 100049, PR China
State Key Laboratory of Functions and Applications of Medicinal Plants & School of Pharmacy, Guizhou Medical University, Guizhou, 550025, PR China
Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, PR China
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China
f
School of Chemical Science and Technology, Key Laboratory of Medicinal Chemistry for Nature Resource, Ministry of Education, Yunnan University, Kunming, 650091,
PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Phytochemical investigation on the twigs and leaves of Trichilia sinensis led to the isolation of two previously
undescribed limonoids (i.e., trichiliasinenoids D and E, 1 and 2), two previously undescribed phenolic acids (3
and 4), and one previously undescribed natural phenolic acid dimer (5), together with 11 known compounds (6-
Trichilia sinensis
Meliaceae
Mexicanolide-type limonoid
Phenolic acid
1
6). Their structures were elucidated by extensive spectroscopic analysis (IR, UV, HRESIMS, 1D and 2D NMR)
and chemical techniques. The potential anti-inflammatory activities of all the compounds were evaluated in
lipopolysaccharide-stimulated RAW 264.7 cells. Among these isolates, compounds 1, 2, 6, and 11-13 expressed
weak NO inhibition. The antibacterial activities of all the compounds against bacteria were also tested in vitro.
Compound 16 exhibited moderate antibacterial activities against Escherichia Coli.
Anti-inflammatory activity
Antibacterial activity
1
. Introduction
limonoids. Limonoids from this plant were reported to possess anti-
inflammatory, cytotoxic, and acetylcholinesterase (AChE) inhibitory
Trichilia (Meliaceae) is a genus of perennial herbs that were mainly
activities (Cao et al., 2017; Liu et al., 2017, 2016; Xu et al., 2013). In
our previous study, three novel limonoids with an unprecedented C-29-
C-7 connecting carbon skeleton were isolated from the twigs and leaves
of T. sinensis collected from Xishuangbanna, China (Cao et al., 2017). As
a continuing phytochemical investigation on this plant, two previously
undescribed limonoids (i.e., trichiliasinenoids D and E, 1 and 2), two
previously undescribed phenolic acids (3 and 4), and one previously
undescribed natural phenolic acid dimer (5), together with 11 known
compounds (6–16) were isolated (Fig. 1). These compounds were
evaluated for their anti-inflammatory activities via the inhibition of li-
popolysaccharide (LPS)-induced NO production in RAW264.7 cells and
cytotoxicities toward RAW264.7 cells. Furthermore, the antibacterial
activities of these compounds were tested against Gram-positive bac-
teria (Staphylococcus aureus and Candida albicans) and Gram-negative
bacteria (Escherichia coli and Pseudomonas aeruginosa). Herein, we
distributed throughout the tropical America and Africa, India,
Indochina, and the Malay Peninsula. There are approximately 86 spe-
cies in this genus, of which two species and one variant grow in China
(
Chen et al., 1997). The genus Trichilia is well known for producing
different types of limonoids with a wide range of biological activities
(
e.g. antifeedant, antibacterial, anticancer, and anti-inflammatory ac-
tivities) (Tan and Luo, 2011; Zhang and Xu, 2017). Trichilia sinensis
Bentv, a shrub native to southern China and Vietnam, has been used in
folk medicine to treat diseases including abdominal pain caused by
Ascaris lumbricoides, chronic osteomyelitis, scabies, and eczema
(
Editorial Committee of Chinese Materia Medica and The
Administration Bureau of Traditional Chinese Medicine, 2000). Pre-
vious phytochemical investigations on T. sinensis indicated that the
plant was a rich source of mexicanolide-type and phragmalin-type
⁎
Corresponding author.
E-mail address: xyk@xtbg.ac.cn (Y.-K. Xu).
1
Contributed equally to this work.
https://doi.org/10.1016/j.phytol.2018.11.020
Received 29 August 2018; Received in revised form 16 November 2018; Accepted 27 November 2018
Available online 05 December 2018
1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

D.-H. Cao et al.
Phytochemistry Letters 29 (2019) 142–147
Fig. 1. The chemical structures of compounds 1–5.
report the isolation, structural elucidation, and anti-inflammatory and
antibacterial activities of the sixteen compounds (1–16) from the twigs
and leaves of T. sinensis.
Table 1
1
H and 13C NMR data of compounds 1 and 2.
position
1
2
2
. Results and discussion
δ
H
(multi, J in Hz)
δ
C
δ
H
(multi, J in Hz)
δ
C
1
2
214.9
47.9
213.6
49.3
Compound 1 was isolated as a white, amorphous powder. Its mo-
3.35, dd (9.1, 6.0)
5.54, d (9.1)
3.54, dd (8.6, 6.0)
5.27, d (8.6)
lecular formula
C
33
H
33NO10 was established by HRESIMS (m/z
3
73.3
70.6
+
6
26.2004 [M + Na] , calcd as 626.1997), corresponding to 18 degrees
4
47.4
47.4
5
4.38, s
45.3
4.11, s
44.6
of unsaturation. The IR spectrum indicated the presence of ester/ketone
−
1
−1
13
6
172.2
169.7
126.8
51.7
172.6
168.2
140.3
55.9
(
1722 cm ) and olefinic (1625 cm ) groups. The C NMR (Table 1)
7
as well as DEPT and HSQC spectra of 1 displayed 33 carbon resonances
8
as four methyls (including one O-methyl), four methylenes, 13 me-
9
2.21, dd (12.2, 5.1)
2.35, dd (11.6, 5.0)
2
3
thines (including seven sp and three oxygenated sp ), and 12 qua-
10
51.3
48.8
1
1
1
1
1α
1β
2α
2β
2.11, m
1.86, m
1.52, m
1.25, m
17.1
2.11, m
2.02, m
1.85, m
1.73, m
21.5
ternary carbons (including five carbonyl ones). A β-substituted furan
ring (δ
H
6.46, br s; 7.44, br s, and 7.55, br s; δ 110.1, 120.3, 141.6, and
C
29.6
34.7
1
1
1
43.3), a nicotinoyl (δ
.7 Hz; 8.89, dd, J = 4.8, 1.7 Hz; 9.27, d, J = 1.7 Hz; δ
37.1, 151.7, and 155.5), a keto carbonyl (δ 214.9), and four ester/
H
7.52, dd, J = 8.2, 4.8 Hz; 8.23, dt, J = 8.2,
C
123.8, 124.0,
13
38.6
134.8
33.2
37.6
45.8
29.3
1
1
1
1
4
2.27, d (6.7)
C
5α
5β
6
3.13, d (18.4)
3.13, d (18.4)
2.81, dd (18.9, 6.7)
2.71, d (18.9)
lactone carbonyls (δ
C
163.9, 168.8, 169.7, and 172.2) were evident
from the NMR data (Table 1). The above observations suggested that 1
168.8
81.2
167.0
76.6
was a mexicanolide-type limonoid (Xu et al., 2013). Its NMR data
17
18
19
20
5.42, s
1.00, s
1.47, s
5.21, s
1.07, s
1.41, s
showed great similarity to those of trichiliasinenoid A (Cao et al.,
17.1
21.7
1
15.4
15.2
2
017), suggesting their close structures. Detailed analysis of the 1D ( H
1
3
1
1
120.3
141.6
110.1
143.3
23.3
162.2
103.7
122.7
169.1
22.7
and C NMR) and 2D NMR data ( H– H COSY, HSQC, and HMBC)
2
2
1
2
7.55, br s
6.46, br s
7.44, br s
1.32, s
5.73, s
6.28, s
(
Fig. 2a) indicated that both compounds shared the same skeleton, and
the major differences were the presence of a C-8-C-14 double bond
23
2
2
3
3
8
1.28, s
instead of a C-8-C-30 one in 1. The conclusion was supported by the
9
4.53, s
81.3
4.52, s
81.9
significant downfield shift for C-8 and C-30 (Δδ -15.2 and -88.8, re-
C
0α
0β
2.64, d (15.5)
2.26, dd (15.5, 6.0)
3.30, s
32.2
5.26, d (6.0)
122.6
spectively) and upfield shift for C-14 (Δδ 89.4) as determined by the
C
HMBC correlations from H-30 (δ
H
2.64) to C-8 and C-14.
OMe-7
53.3
3.59, s
3.70, s
52.9
The relative configuration of 1 was determined by the ROESY data
OMe-21
58.5
1
2
3
4
′
′
′
′
163.9
123.8
151.7
166.2
127.0
141.9
14.9
(
Fig. 2b). The ROESY correlations of H-5/H-17, H-17/H-11β, H-17/H-
1
5β, H-15β/H-30β indicated that these protons were all β-oriented. The
9.27, d (1.7)
6.68, q (4.5)
1.82, d (4.5)
1.82, s
ROESY correlations of H-2/H-3, H-3/H-28, H-2/H-30α, H-30α/H-15α,
H-15α/H
3
-18, H
3
-28/H-9, H-9/H
3
-19, H-9/H-11α revealed the α-or-
5′
8.89, dd (4.8, 1.7)
7.52, dd (8.2, 4.8)
8.23, dt (8.2, 1.7)
155.5
124.0
137.1
12.2
6
7
′
′
ientation of the corresponding protons. Therefore, the structure of
compound 1 was finally established as depicted and was named tri-
chiliasinenoid D.
at 600 MHz ( H NMR, J in H) and 150 MHz (13C
1
Data were measured in CDCl
NMR).
3
Compound 2, obtained as a white amorphous powder, possessed a
molecular formula of C33
H O12 as deduced from the HRESIMS peak at
38
+
m/z 625.2275 ([M-H] , calcd for C33
H O12, 625.2291). The IR spec-
37
suggested a mexicanolide-type limonoid structure with a tigloyloxy
group (δ 1.82, s; 1.82, d, J = 4.5 Hz; 6.68, q, J = 4.5 Hz; δ 12.2, 14.9,
27.0, 141.9) for 2. Comparison of its NMR data with those of
trum implied the presence of γ-lactone and ester/ketone carbonyl
H
C
functionalities on the basis of the absorption bands at 1793 and
1
−
1
13
1
748 cm , respectively. Analysis of the 1H and C NMR data for 2
143

D.-H. Cao et al.
Phytochemistry Letters 29 (2019) 142–147
Fig. 2. Selected 1H- H COSY, HMBC, and ROESY correlations of compounds 1 and 2.
1
trichiliasinenoid B (Cao et al., 2017) suggested that, both compounds
shared similar structural features, the only difference being the re-
placement of the furan ring by a 21-methoxybutenolide moiety at C-17
169.7 was attached to C-1 by the HMBC correlations from H-2/H-6 to C-
1 and C-7. HMBC correlations from the remaining methoxyl (δ
H
3.89) to
C-5 and from H-6 to C-1 (δ
149.4), C-7 (δ 169.7), as well as from H-2 to C-1 (δ
147.1), C-4 (δ 141.7), C-6 (108.5), C-7 (δ
C
121.5), C-2 (110.2), C-4 (δ
C
C
141.7), C-5 (δ
C
C
in 2. This was verified by the HMBC correlations of H-17 (δ
H
5.21) with
122.7), and of OMe-21
103.7). The relative configuration of 2 was
established by a ROESY experiment (Fig.2d). The C-21 configuration in
C
C
121.5), C-3 (δ
C-20 (δ
C
162.2), C-21 (δ
C
C
103.7), and C-22 (δ
C
C
169.7) indicated the pre-
(
δ
H
3.70) with C-21 (δ
sence of a 3,4,5-tri-O-substituted benzoic acid fragment. Connection of
the two benzoyl groups via a C -O-C1′ ether linkage was deduced from
their chemical shifts (δ 137.7 for C-1′ and δ 147.1 for C-3) and HMBC
3
2
was not assigned by the available data. Thus, the structure of 2 was
C
C
established as drawn and was named trichiliasinenoid E.
correlations above (Li et al., 2008). Thus, compound 3 was established
and named 3-(2′,6′-dimethoxy-4′-(methoxycarbonyl)phenoxy)-4-hy-
droxy-5-methoxybenzoic acid.
Compound 3 was obtained as white amorphous power. Its HRESIMS
+
displayed a pseudomolecular ion peak [M+Na] at m/z 401.0843
(
calcd 401.0843), corresponding to the molecular formula of C18
H
18
O
9
.
Compound 4 was obtained as white amorphous powder and gave a
−
1
+
The IR absorptions showed the presence of hydroxy (3426 cm ),
sodiated molecular ion [M+Na] at m/z 563.1372 (calcd 563.1371) in
−
1
−1
carbonyl (1718 cm ), and aromatic (1502 and 1598 cm ) groups.
the HRESIMS, suggesting a molecular formula of C24
H
28
O
14 for 4. The
1
−1
The H NMR spectrum of 3 (Table 2) showed signals for a symmetric
IR spectrum showed the absorption bands of hydroxy (3426 cm ),
−1 −1
2
′,6′-dimethoxy-1′,4′-disubstituted benzene ring (δ
H
7.40, br s, 2 H;
carbonyl (1721 cm ), and aromatic (1599, 1501 cm ) groups. The
1
13
3
.79, s, 6 H) and an asymmetric 1,3,4,5- or 1,3,4,6-tetrasubstituted
6.74, br s; 7.28, br s), and two additional methoxy
H and C NMR spectra of 4 (Table 2) showed, except for signals for an
benzene ring (δ
H
extra hexosyl moiety (δ
H
3.26–3.78, 6 H and 5.28, 1 H; δ 62.6, 71.4,
C
1
3
groups [δ
Table 2) with ten aromatic carbon signals (three methines and seven
quaternary carbons) and four methoxy signals (δ 56.8, 56.8, 56.8, and
2.9) were in agreement with the above deduction. Besides, two car-
H
3.90 (3H), 3.89 (3H)]. The C NMR (with DEPT) spectrum
75.7, 77.8, 78.4, 104.8), distinct signals similar to those for compound
3. The noticeable differences were only observed for C-1 (Δ + ca
6 ppm), C-3 (Δ + ca 5 ppm), C-4 (Δ - ca 2 ppm), C-5 (Δ + ca 5 ppm) in
accordance with typical glycosylation shifts of such a phenyl group with
glycosylation occurred at C-4 (Miyase et al., 1988). HMBC correlation
between H-1′′ (δ 5.28, d, J = 7.5 Hz) and C-4 (δ 139.6) was further
verified the location of the glucosyl group at C-4. The hexose was de-
termined to be D-glucose by GC analysis of the hexose methyl ether
prepared from methylation of its hydolysate, while the β-configuration
of the glucose was established by the large coupling constant of the
anomeric proton (J = 7.5 Hz). Thus, the structure of 4 was established
(
C
5
1
3
boxyl/ester carbonyls (δ
C
169.7, 167.9) were also observed in the
C
NMR spectrum. Structural details of 3 were further established on the
basis of 2D NMR data (Fig. 3). The HMBC correlations from H-3′/H-5′ to
C-7′ (δ
C
167.9), C-4′ (δ
C
128.7), and C-1′ (δ 137.7), as well as from the
C
two symmetric methoxy groups to C-2′/C-6′ (δ
methoxy at δ 3.90 to C-7′ suggested the presence of a methyl 2′,6′-
dimethoxy-1′-O-substituted benzoate. The carboxyl resonance at δ
C
154.5) and from the
H
C
144

D.-H. Cao et al.
Phytochemistry Letters 29 (2019) 142–147
Table 2
7.60, d, J = 2 Hz, 2H; 7.68, d, J = 2 Hz, 2 H; 3.87, s, 6H; δ 56.5, 125.6,
C
1
H and 13C NMR spectroscopic data for compounds 3–5.
127.5, 122.0, 112.2, 148.7, 150.2, 170.0) were easily recognized by the
1
13
H and C NMR spectra (Table 2). 2D NMR experiments were further
position
3
4
5
conducted to figure out the structural details. The carboxyl resonance at
170.0 was attached to C-3 by the HMBC correlation from H-2 and H-4
to C-7. The HMBC correlations of H-4/C-2, C-5, C-6, C-7 and the ROSEY
δ
H
(multi, J
δ
C
δ
H
(multi, J
δ
C
δ
H
(multi, J
δ
C
in Hz)
in Hz)
in Hz)
correlation of H-4/OCH -5 indicated the methoxy group was attached
3
1
2
3
4
5
6
7
121.5
127.5
109.4 7.68, d (2.0) 127.5
152.0 122.0
125.6
6.74,br s
110.2 6.74, d (2.0)
to C-5 (Fig.3). The hydroxy group was assigned to C-6 by the HMBC
147.1
correlation from H-2 to C-1, C-4, C-6, C-7, as well as from H-4 to C-2, C-
141.7
139.6 7.60, d (2.0) 112.2
5
, C-6, C-7 (Fig.3). Two 5-methoxy-6-hydroxyl-1-substituted benzoic
149.5
154.5
109.0
169.0
136.6
148.7
150.2
170.0
125.6
acid fragments were thus established. Connection of the two subunits
via a C -C1′ linkage was deduced from their chemical shifts (δ 125.6 for
7.28, br s
108.5 7.34, d (2.0)
169.7
1
C
1
2
3
4
5
6
7
′
′
′
′
′
′
′
137.7
C-1 and C-1′) and HMBC correlations above (Li et al., 2008). Compound
154.5
154.4 7.68, d (2.0) 127.5
5
was therefore established as depicted and was named 6,6′-dihydroxy-
7.40, br s
7.40, br s
107.8 7.43, br s
128.7
107.9
122.0
5
,5′-dimethoxy-[1,1′-biphenyl]-3,3′-dicarboxylic acid.
129.0 7.60, d (2.0) 112.2
In addition to the five previously undescribed compounds (1-5),
107.8 7.43, br s
154.5
107.9
154.4
167.8
56.8
148.7
150.2
170.0
eleven known compounds named β-hydroxypropiovanillone (6)
(Karonen et al., 2004), kaempferol-3-O-rutinoside (7) (Feng et al.,
167.9
OMe-2′, 6′ 3.79, s
56.8
56.8
3.81, s
3.91, s
2
007), kaempferol 3-O-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside
OMe-5
OMe-5′
OMe-7′
3.89, s
3.90, s
57.1
3.87, s
3.87, s
56.5
56.5
(
8) (Szewczyk et al., 2014), Kaempferol 3-O-glucoside (9) (Senatore
et al., 1999), meliavosin (10) (Rogers et al., 1998), stigmast-4-ene-
3β,6β-diol (11) (Qu et al., 2009), 7-ketositosterol (12) (Byung et al.,
2007), lyoniside (13) (Zheng et al., 2011), 3,4,6-trimethoxy phenyl-O-
D-glucoside (14) (Kimura et al., 1984), 1-hexadecanoyl propan-2,3-diol
52.9
3.92, s
53.0
104.8
75.7
77.8
71.4
78.4
62.6
1
2
3
4
5
6
′′
′′
′′
′′
′′
′′
5.28, d (7.5)
3.53, m
3.42, m
3.42, m
3.26, m
3.68, dd
(
15) (Misra and Siddiqi, 2000), and 3,5-dimethoxy-4-hydroxy-benzal-
dehyde (16) (Yuan et al., 2016) were also obtained and structurally
determined by comparing their spectroscopic data with those in the
literature. All the compounds were isolated from the plant for the first
time.
(
12.0, 5.0)
.78, dd
12.0, 2.9)
3
(
OD at 500 MHz ( H NMR) and 125 MHz (13C
1
All the isolates were evaluated for their inhibitory effects on nitric
oxide (NO) production stimulated by LPS in RAW264.7 cells using L-
NMMA (NG-monomethyl L-arginine) as the reference compound with
an IC50 value of 35.5 μM. The cytotoxicity of the tested samples were
assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at the con-
centrations (≤100 μM) showing no cytotoxicity. Among the isolates,
compounds 1, 2, 6, and 10-13 exhibited weak NO inhibition and the
rest compounds were inactive (IC50 ≥100 μM) (Table 3). All the iso-
lated compounds were also evaluated for their in vitro antibacterial
activities against Gram-positive bacteria (Staphylococcus aureus and
Candida albicans) and Gram-negative bacteria (Escherichia coli and
Pseudomonas aeruginosa) using a microbroth dilution method for the
determination of the minimum inhibitory concentrations (MICs) (Sup-
porting Information, Table S1). Compound 16 showed moderate in-
hibitory activity against Escherichia coli.
All data were measured in CD
3
NMR).
Table 3
Effect of compounds 1–16 on LPS-induced NO production in RAW264.7 cellsa,b.
Compound
IC50/μM
CI (95%, n = 3)
1
93.8
[91.4, 95.9]
[87.4, 89.8]
–
1
1
Fig. 3. Selected H- H COSY, HMBC, and ROESY correlations of compounds
2
88.3
3
–5.
3-5
6
> 100
92.5
[91.8, 92.9]
–
7
9
-8
> 100
> 100
73.9
and
named
3-(2′,6′-dimethoxy-4′-(methoxycarbonyl)phenoxy)-5-
–
methoxy-4-O-β-D-glucopyranosylbenzoic acid.
10
[73.4, 74.3]
[92.3, 93.1]
[76.2, 76.8]
[92.6, 94.1]
–
1
1
92.7
Compound 5 was obtained as white amorphous power. The
+
12
76.5
HRESIMS displayed a pseudomolecular ion at m/z 357.0572 [M+Na]
1
1
3
93.1
(
calced 357.0581), consistent with a molecular formula of C16
H
14 8
O .
4-16
> 100
35.5
c
The IR absorption bands indicated the presence of hydroxy
L-NMMA
[32.7, 38.1]
−
1
−1
(
3508 cm ), conjugated carboxyl (1692 cm ), and aromatic (1491
−1
a
b
c
Results are expressed as IC50 values in μM.
Compounds with IC50 > 100μM are not shown.
Positive control.
and 1599 cm ) groups. Taking into consideration the molecular for-
mula, two identical methoxy 1,5,6-trisubstituted benzoyloxy groups (δ
H
145

D.-H. Cao et al.
Phytochemistry Letters 29 (2019) 142–147
3
. Experimental
phase preparative thin-layer chromatography using CHCl
) afforded 3 (8 mg), 4 (11 mg), 5 (5 mg), and 14 (15 mg), respectively.
Fr. E (5.2 g) was chromatographed on an ODS (2 cm × 40 cm) column
3
/acetone (10:
1
3.1. General experimental procedures
eluted with a MeOH/H O gradient (30:70, 40:60, 50:50, 60:40, 70:30,
2
Optical rotations were obtained with a JASCO P-1020 polarimeter.
80:20, 90:10, v/v, each 1 L) to yield seven sub-fractions, E-1∼E-7. Sub-
fraction E-5 (134 mg) was subjected to semi-preparative HPLC
UV spectra were measured with a Shimadzu UV-2401 A instrument. IR
spectra (KBr) were determined on a Bruker Tensor-27 infrared spec-
trometer. 1D and 2D NMR spectra were recorded on Bruker DRX-500
and Bruker Avance III 600 spectrometers with TMS as an internal
standard. ESIMS and HRESIMS were recorded on an AutoSpec Premier
P776 instrument. Semi-preparative HPLC was performed on a Waters
(10 mm × 300 mm, MeCN/H O, 30: 70, v/v, 3 mL/min) to give com-
2
pounds 7 (15 mg), 8 (7 mg), 9 (41 mg), and 13 (23 mg). Sub-fraction E-
6 (80 mg) was purified by normal-phase preparative thin-layer chro-
matography (CHCl /MeOH, 10: 1, v/v) to give 6 (5 mg). All of the
3
compounds met the criteria of ≥ 95% purity, as determined by NMR
analysis.
6
00 pump system with a 2996 photodiode array detector by using a
-5
YMC-Pack ODS-A column (300 × 10 mm, S μm). Silica gel (200 − 300
mesh, Qingdao Marine Chemical Factory, Qingdao, China), Sephadex
LH-20 gel (40 − 70 μm, Amersham Pharmacia Biotech AB, Uppsala,
Sweden), C18-reversed phase silica gel (250 mesh, Merck) and MCI gel
3.3.1. Trichiliasinenoid D (1)
2
5.7
White power; [α]
D
-24.9 (c 0.06, MeOH); UV (MeOH) λmax (log ε):
O), 2927, 1722,
203 (2.97), 261 (2.20) nm; IR (KBr) νmax: 3428 (H
2
−1
(
CHP20/P120, 75 − 150 μm, high-porous polymer, Mitsubishi
1625, 1385, 1275, 1029, 743 and 582 cm ; HRESIMS m/z 626.2004
+
1
13
Chemical Corporation, Tokyo, Japan) were used for column chroma-
tography (CC). Pre-coated silica gel GF254 plates (Qingdao Haiyang
Chemical Co. Ltd) were used for analytical TLC. All solvents used for CC
were of analytical grade (Shanghai Chemical Reagents Co. Ltd), and all
solvents used for HPLC were of spectral grade.
[M+Na] (calcd for C33
H
33NO10Na, 626.1997); H and C NMR data,
see Table 1.
3.3.2. Trichiliasinenoid E (2)
2
5.7
White power; [α]
D
-6.0 (c 0. 15, MeOH); UV (MeOH) λmax (log ε):
O), 2924, 1793, 1748, 1631,
2
03 (3.17) nm; IR (KBr) νmax: 3429 (H
2
−1
3.2. Plant material
1440, 1384, 1259, 1212, 1118, 1053, 995 and 583 cm ; HRESIMS m/z
+
1
13
6
25.2275 [M−H] (calcd for C33
H O12, 625.2291); H and C NMR
37
The twigs and leaves of T. sinensis were collected from
data, see Table 1.
Xishuangbanna Tropical Botanical Garden (XTBG), Chinese Academy of
Science (CAS), Mengla Country, Yunnan Province, People’s Republic of
China in May 2017, and they were identified by one of the authors
3.3.3. 3-(2′,6′-dimethoxy-4′-(methoxycarbonyl)phenoxy)-4-hydroxy-5-
methoxybenzoic acid (3)
(
Chun-Fen Xiao). A voucher specimen (No. HITBC-028935) is deposited
White power; UV (MeOH) λmax (log ε): 211 (4.05), 257 (3.53), 287
(3.14) nm; IR (KBr) νmax: 3426, 2924, 1718, 1598, 1502, 1384, 1217,
in the herbarium at XTBG.
−
1
+
1
126 and 766 cm ; HRESIMS m/z 401.0843 [M + Na] (calcd for
1 13
3.3. Extraction and isolation
C
18
H
18
O Na, 401.0843); H and C NMR data, see Table 2.
9
The dried and powered twigs and leaves of T. sinensis (5.0 kg) were
3.3.4. 3-(2′,6′-dimethoxy-4′-(methoxycarbonyl)phenoxy)-5-methoxy-4-O-
percolated with 95% aqueous EtOH (40 L) three times (for seven days
each time) at room temperature. Removal of the solvent from the
combined extracts in vacuo afforded a crude residue (274 g), which was
β-D-glucopyranosylbenzoic acid (4)
2
5.7
White power; [α]
D
-2.4 (c 0. 1, MeOH); UV (MeOH) λmax (log ε):
211 (4.05), 257 (3.53), 287 (3.14) nm; IR (KBr) νmax: 3426, 2942, 1721,
−1
then suspended in distilled H
2
O and successively partitioned with
1599, 1501, 1418, 1340, 1217, 1086 and 764 cm ; HRESIMS m/z
+
1
13
EtOAc and n-BuOH. The EtOAc-soluble fraction (145 g) was separated
563.1372 [M+Na] (calcd for C24
H
28
O
14Na, 563.1371); H and
C
over a MCI gel column (8 cm × 100 cm) chromatography and eluted
NMR data, see Table 2.
with MeOH-H O (20:80, 40:60, 60:40, 80:20, 100:0, v/v, each 8 L) to
2
give five fractions. The fourth fraction (68 g) was chromatographed on
3.3.5. 6,6′-dihydroxy-5,5′-dimethoxy-[1,1′-biphenyl]-3,3′-dicarboxylic
acid (5)
a silica gel column (6 cm × 70 cm, 200–300 mesh) with gradient mix-
tures of CHCl
3
-MeOH (100:0, 50: 1, 20:1, 10:1, 5: 1, 2:1, 1: 1, v/v, each
White power; UV (MeOH) λmax (log ε): 211 (4.05), 257 (3.53), 287
(3.14) nm; IR (KBr) νmax: 3508, 3422, 2943, 1692, 1599, 1491, 1410,
4
8
2
L) elution to yield seven fractions, Frs. A–G (6.5, 4.2, 20.4, 12.7, 5.2,
.6 and 3.2 g, respectively). Fr. B (4.2 g) was subjected to Sephadex LH-
−
1
+
1269, 1039 and 769 cm ; HRESIMS m/z 357.0572 [M+Na] (calcd
0 (2 cm × 100 cm) eluted with CHCl
3
-MeOH (1: 1, v/v) to give six
for C16
H
14
O
8
Na, 357.0581); 1H and C NMR data, see Table 2.
13
sub-fractions (B-1∼B-6), according to their TLC profiles. Sub-fraction
B-4 was recrystallized to give 15 (30 mg). Fr. C (20.4 g) was further
separated by a silica gel column (6 cm × 70 cm, 200–300 mesh, pet-
roleum ether/acetone, from 50/1 to 1/1, v/v, each 1 L) to yield eight
fractions, Fr. C-1∼C-8. Sub-fraction C-3 (5 g) was purified by repeated
CC over Sephadex LH-20 (2 cm × 100 cm; MeOH) and semi-preparative
3.4. Determination of absolute configuration of sugar moieties
Compound 4 (3 mg) was separately dissolved in 1 N HCl and refl-
uxed for 6 h. After removal of HCl by evaporation and extraction with
CH
2
Cl
2
, the H O extract was again evaporated and dried in vacuo to
2
HPLC (10 mm × 300 mm, MeCN/H
2
O, 52:48, v/v, 3 mL/min) to yield 1
afford a residue that contained the monosaccharide. The residue was
dissolved in pyridine (1 mL), and 2 mg L-cysteine methyl ester hydro-
chloride was added to the solution. The mixture was kept at 60 °C for
(
6 mg), 2 (4 mg), and 16 (5 mg). Sub-fraction C-5 (6.5 g) was fractio-
nated by a reversed silica gel column (RP-18, 5 cm × 40 cm) eluted
with MeOH/H O (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10,
00:0, v/v, each 1 L) to give eight sub-fractions, C-5a∼C-5 h. Sub-
2
2 h and then the solvent was evaporated under a stream of N , to give a
2
1
residue. The residue was then trimethylsilylated via a reaction with N-
fraction C-5e (74 mg) was separated by semi-preparative HPLC
(trimethylsilyl) imidazole (0.2 mL) for 2 h. The mixture was partitioned
(
(
(
10 mm × 300 mm, MeCN/H
33 mg), 11 (19 mg), and 12 (16 mg). Fr. D (12.7 g) was subjected to CC
RP-18, 5 cm × 40 cm, MeOH/H O, 30:70, 50:50, 70:30, 90:10, and
2
O, 45: 55, v/v, 3 mL/min) to give 10
between n-hexane and H O (2 mL each), and the n-hexane extract was
2
analyzed by GC under the following conditions: L-chirasil-Val-column
(25 m × 0.25 mm, i.d.); detection, FID; detector temperature, 280 °C;
injection temperature, 250 °C; column temperature, 270 °C; and carrier,
2
1
00:0, v/v, each 1 L) to yield five fractions, Fr. D-1∼D-5. Fr. D-3 was
separated by CC over Sephadex LH-20 (2 cm × 100 cm) eluted with
MeOH to give four fractions, further purification of which by normal-
N gas, 250 kPa (Ma et al., 2018). The presence of D-glucose in the acid
2
hydrolysate of compound 4 was verified by comparison of the retention
146

D.-H. Cao et al.
Phytochemistry Letters 29 (2019) 142–147
times of their derivatives to those of corresponding control samples
prepared by the same procedure. The retention time of D-glucose was
online version, at doi:https://doi.org/10.1016/j.phytol.2018.11.020.
2
4.78 min.
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Tan, Q.G., Luo, X.D., 2011. Meliaceous limonoids: chemistry and biological activities.
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The authors declare no conflict of interest.
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This work was supported financially by Systematic Evaluation of
Ethnic Medicinal Plant Resources in Tropical Regions and Development
of Health Products for the Public (2017XTBG-F02), Conservation and
Application of National Strategic Tropical Plant Resources: Theory and
Practice (2017XTBG-F05) and the International Partnership Program of
Chinese Academy of Sciences (153631KYSB20160004), and the Central
Laboratory of XTBG for the technical support of this study.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
147