3,4-seco-Dammarane Triterpenoid Saponins with Anti-Inflammatory Activity Isolated from the Leaves of Cyclocarya paliurus

Article abstract of DOI:10.1021/acs.jafc.9b06898

Cyclocarya paliurus is commonly used for the prevention and treatment of hypertension, diabetes, and inflammation in South China. Although research on the anti-inflammatory effects of C. paliurus leaves has been reported, no active anti-inflammatory compounds have been identified. In the present study, RAW 264.7 cells were used to establish a bioactivity-guided identification model to verify the inhibitory effects of C. paliurus leaves on inflammation and identify the anti-inflammatory constituents. The active fraction was isolated to yield 18 dammarane triterpenoid saponins, including 11 new 3,4-seco-dammarane triterpenoid saponins (1-11), the structures of which were identified on the basis of analyses of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) and comparison with literature data. Compounds 7, 8, 10, and 11 showed strong inhibition on nitric oxide (NO) productions, with IC₅₀ values ranging from 8.23 to 11.23 μM. These four compounds significantly decreased the secretion of tumor necrosis factor-alpha (TNF-α), prostaglandin E₂ (PGE₂), and interleukin 6 (IL-6) in lipopolysaccharide-activated RAW 264.7 cells. Furthermore, compound 7 decreased the expression of the proteins cyclooxygenase-2 (COX-2), inducible nitric-oxide synthase (iNOS), and nuclear factor kappa-B (NF-κB/p65). In addition, the structure-activity relationships of the isolates were investigated. The results suggest that 3,4-seco-dammarane triterpenoid saponins may be used as potential anti-inflammatory drugs and warrant further investigation.

Full text of DOI:10.1021/acs.jafc.9b06898

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Bioactive Constituents, Metabolites, and Functions
3,4-Seco-Dammarane Triterpenoid Saponins with Anti-inflammatory
Activity Isolated from the Leaves of Cyclocarya paliurus
Wei Liu, Shengping Deng, Dexiong Zhou, Yan Huang, Chenguo Li, Lili Hao,
Gaorong Zhang, Shanshan Su, Xia Xu, Rui-Yun Yang, Jun Li, and Xishan Huang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06898 • Publication Date (Web): 22 Jan 2020
Downloaded from pubs.acs.org on January 26, 2020
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3,4-Seco-Dammarane Triterpenoid Saponins with Anti-inflammatory
Activity Isolated from the Leaves of Cyclocarya paliurus
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Wei Liu, Shengping Deng, Dexiong Zhou, Yan Huang, Chenguo Li, Lili Hao, Gaorong Zhang,
Shanshan Su, Xia Xu, Ruiyun Yang, Jun Li *, Xishan Huang*
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004,
China
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ABSTRACT: Cyclocarya paliurusis commonly used for the prevention and treatment
of hypertension, diabetes, and inflammation in South China. Although research on the
anti-inflammatory effects of C. paliurus leaves has been reported, no active
anti-inflammatory compounds have been identified. In the present study, RAW 264.7
cells were used to establish a bioactivity-guided identification model to verify the
inhibitory effects of C. paliurus leaves on inflammation and identify the
anti-inflammatory constituents. The active fraction was isolated to yield eighteen
dammarane triterpenoid saponins, including eleven new 3,4-seco-dammarane
triterpenoid saponins (1-11), the structures of which were identified on the basis of
analyses of nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry
(MS) and comparison with literature data. Compounds 7, 8, 10, and 11 showed strong
inhibition on nitric oxide (NO) productions, with IC50 values ranging from 8.23 μM to
11.23 μM. These four compounds significantly decreased the secretion of tumor
necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2) and interleukin 6 (IL-6) in
lipopolysaccharide (LPS)-activated RAW 264.7 cells. Furthermore, compound 7
decreased the expression of the proteins cyclooxygenase-2 (COX-2), inducible
nitric-oxide synthase (iNOS), and nuclear factor kappa-B (NF-κB/p65). In addition,
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the structure-activity relationships of the isolates were investigated. The results
suggest that 3,4-seco-dammarane triterpenoid saponins may be used as potential
anti-inflammatory drugs and warrant further investigation.
INTRODUCTION
Cyclocarya paliurus (Batal.) Iljinskaja, a Chinese indigenous plant, is the sole
species in the genus Cyclocarya Iljinskaja of the Juglandaceae family. The leaves of
C. paliurus are used not only as drug formulations in traditional Chinese medicine to
treat hyperlipidemia, hypertension, diabetes mellitus, chronic inflammatory diseases
and as an antioxidant but also as a famous “sweet tea”, with a very sweet taste, in folk
medicine in China to prevent and treat hypertension, diabetes, and inflammation.1-6
The leaves of C. paliurus were approved as a new raw food material by The National
Health Commission of the People’s Republic of China in 2013.7 Currently, C.
paliurus is an important and valuable crop in the functional food market. A variety of
triterpenoid glycosides, flavonoids, and other compounds have been isolated from the
leaves of this plant.8 Among its constituents, 3,4-seco-dammarane triterpenoids are
considered characteristic taxonomic indicators of this plant and the primary active and
functional constituents in C. paliurus. To date, 20 seco-dammarane triterpenoid
glycosides, which are cleaved in plants by hydroxylation or methyl esterification at
C-3 and C-4 followed by glycosylation at C-12, have been reported.9-14 Some of them
have a sweet taste and are used as functional food ingredients. For example,
cyclocariosides I-II are approximately 50 to 100 times sweeter than 2% sucrose15-16
and are used as sweeteners and antitumor agents.17 Although research on the
anti-inflammatory effects of an extract of C. paliurus has been reported,18 no active
anti-inflammatory compound has been reported. To explore potential bioactive
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compounds from the leaves of C. paliurus, a bioactivity-guided investigation of
anti-inflammatory compounds was carried out. The active fraction was isolated to
yield eleven new 3,4-seco-dammarane triterpenoid saponins (1−11) and seven known
dammarane triterpenoid saponins (12−18) (Figure 1). The inhibition of NO by the
extracts and compounds were evaluated. Furthermore, the effects of the
anti-inflammatory compounds on the levels of inflammatory cytokines TNF-α, PGE2
and IL-6 and the expression of proteins iNOS, COX-2 and NF-κB/p65 were tested
using LPS-activated RAW 264.7 macrophage cells. We describe herein the separation
and structural identification of seco-dammarane triterpenoid saponins, their
anti-inflammatory effects, and their structure-activity relationships.
MATERIALS AND METHODS
Instruments. The detailed experimental instruments are seen in the Support
Information (Text S1).
Chemicals. Silica gel (Qingdao Marine Chemical Co., Ltd., China), Sephadex
LH-20 gel (Amersham Pharmacia Biotech AB, Sweden), and RP-C18 (Merck,
Germany) were used for various separations. D-glucose, L-glucose, L-arabinose,
D-arabinose, D-quinovose, and L-quinovose were used as the sugar standards
(Sigma-Aldrich, Munich, Germany).
Plant materials. The leaves (20.0 kg) of C. paliurus were bought from Xiushui
county, Jiangxi Province, China in July 2017 and identified by Associate Professor
Qiang Xie (Guangxi Normal University). A specimen (No. ID-20170705) was
preserved in the State Key Laboratory of Medicinal Resources of Guangxi Normal
University.
Preparation of extracts and isolation of triterpenoid saponins. The fresh, dried
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leaves (20.0 kg) of C. paliurus were extracted with 75% ethanol (EtOH, 4 × 3 h), and
the solvent was evaporated under vacuum to obtain an extract. The crude extract (2.0
kg) was suspended in distilled water and partitioned with polyethylene (PE), ethyl
acetate (EtOAc) and n-butanol (n-BuOH) to obtain a PE extract (480 g), an EtOAc
extract (450 g) and an n-BuOH extract (600 g). The anti-inflammatory effects were
analyzed by measuring the inhibition of NO production in LPS-activated RAW 264.7
cells.
The active EtOAc fraction was loaded to a chromatography column (CC) on silica
gel eluted with CH2Cl2-MeOH mixtures (from 1:0 to 0:1) to yield eight fractions (Fr.
A to Fr. H). The anti-inflammatory fraction D (45 g) was fractionated using an MCI
gel CC eluting with MeOH-H2O (from 3:7 to 1:0) to obtain Fr. D-1 to Fr. D-4. Fr. D-3
(10 g) was further separated using RP-C18 CC eluting with gradients of MeOH-H2O
(from 6:10 to 1:0) to afford Fr. D-3-1 to Fr. D-3-4. Fr. D-3-2 (0.8 g) was loaded to
Sephadex LH-20 eluting with gradients of H2O-MeOH (from 1:0 to 0:1), and further
separated by RP-C18 semi-preparative HPLC to furnish compounds 1 (28.0 mg), 2
(15.4 mg), and 3 (7.1 mg). Fr. D-3-3 (3.2 g) was applied to Sephadex LH-20 CC and
eluted with MeOH and then chromatographed with semi-preparative RP-C18 HPLC to
afford compounds 4 (28.0 mg), 5 (15.2 mg), and 13 (30.3 mg). Fr. D-3-4 (4.2 g) was
chromatographed by silica gel and eluted with gradients of CH2Cl2-MeOH (from 1:0
to 0:1) to create Fr. D-3-4-1 to Fr. D-3-4-5. Fr. D-3-4-2 was subjected to Sephadex
LH-20 CC and RP-C18 HPLC to get compounds 6 (28.2 mg), 7 (15.2 mg), 11 (11.4
mg), and 12 (1.0 mg). In addition, Fr. D-3-4-3 was isolated by Sephadex LH-20 CC
and RP-C18 HPLC to afford compounds 8 (28.2 mg), 9 (15.0 mg), and 10 (19.7 mg).
The anti-inflammatory Fr. E (80 g) was subjected to MCI gel eluting with gradients of
MeOH-H2O (from 6:4 to 1:0) to yield Fr. E-1 to Fr. E-4. Active Fr. E-3 (23 g) was
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further chromatographed by RP-C18 CC eluted with gradients of MeOH-H2O (from
6:10 to 1:0) to afford Fr. E-3-1 to Fr. E-3-4. Fr. E-3-2 was subjected to Sephadex
LH-20 CC and semi-preparative RP-C18 HPLC to afford compounds 14 (28.3 mg), 15
(15.4 mg), 16 (12.1 mg), 17 (12.0 mg), and 18 (12.3 mg).
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Cyclocarioside R (1): The various chromatographic isolation and purification gave
[훼]2D5
28.0 mg of white, amorphous powder;
+ 38.0 (c 0.16, MeOH); negative
−
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HRESIMS m/z calculated for C36H60ClO10 [M + Cl] , 687.3859, found: 687.3875;
1H- (500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 1 and 3).
Cyclocarioside S (2): The various chromatographic isolation and purification gave
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15.4 mg of white, amorphous powder;
+ 28.6 (c 0.12, MeOH); positive
[훼]D
+
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HRESIMS m/z calculated for C36H60O10Na [M + Na] , 675.4073, found: 675.4084;
1H- (500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 1 and 3).
Cyclocarioside T (3): The various chromatographic isolation and purification gave
[훼]2D5
7.1 mg of white, amorphous powder;
+ 10.6 (c 0.19, MeOH); negative
−
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HRESIMS m/z calculated for C37H62ClO10 [M + Cl] , 701.4142, found: 701.4032;
1H- (500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 1 and 3).
Cyclocarioside U (4): The various chromatographic isolation and purification gave
[훼]2D5
28.0 mg of white, amorphous powder;
+ 27.6 (c 0.14, MeOH); positive
+
1
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HRESIMS m/z calculated for C36H45O4 [M + Na] , 641.4069, found: 641.4029; H-
(500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 1 and 3).
Cyclocarioside V (5): The various chromatographic isolation and purification gave
[훼]2D5
15.2 mg of white, amorphous powder;
+ 4.8 (c 0.14, MeOH); negative
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HRESIMS m/z calculated for C37H60ClO8 [M + Cl] −, 667.3960, found: 667.3977; 1H-
(500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 1 and 3).
Cyclocarioside W (6): The various chromatographic isolation and purification gave
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[훼]2D5
+ 11.3 (c 0.14, MeOH); positive
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28.2 mg of white, amorphous powder;
+
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HRESIMS m/z calculated for C37H60O9Na [M + Na] , 671.4136, found: 671.4135;
1H- (500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 1 and 3).
Cyclocarioside X (7): The various chromatographic isolation and purification gave
[훼]2D5
15.2 mg of white, amorphous powder;
+ 24.8 (c 0.07, MeOH); negative
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HRESIMS m/z calculated for C37H62ClO9 [M + Cl] −, 685.4074, found: 685.4082; 1H-
(500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 2 and 3).
Cyclocarioside Y (8): The various chromatographic isolation and purification gave
[훼]2D5
28.2 mg of white, amorphous powder;
+ 1.5 (c 0.14, MeOH); positive
+
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HRESIMS m/z calculated for C38H64O9Na [M + Na] , 687.4491, found: 687.4448;
1H- (500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 2 and 3).
Cyclocarioside Z1 (9): The various chromatographic isolation and purification gave
[훼]2D5
15.0 mg of white, amorphous powder;
+ 2.9 (c 0.19, MeOH); positive
+
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HRESIMS m/z calculated for C37H62O9Na [M + Na] , 673.4279, found: 673.4292;
1H- (500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 2 and 3).
Cyclocarioside Z2 (10): The various chromatographic isolation and purification
[훼]2D5
gave 19.7 mg of white, amorphous powder;
+ 34.0 (c 0.10, MeOH); negative
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HRESIMS m/z calculated for C37H62ClO9 [M + Cl] −, 685.4072, found: 685.4082; 1H-
(500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 2 and 3).
Cyclocarioside Z3 (11): The various chromatographic isolation and purification
[훼]2D5
gave 11.4 mg of white, amorphous powder;
+ 38.9 (c 0.14, MeOH); negative
−
1
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HRESIMS m/z calculated for C38H64ClO9 [M + Cl] , 699.422, found: 699.4239; H-
(500 MHz) and 13C-NMR (125 MHz) data (pyridine-d5, Tables 2 and 3).
Determination of the absolute configurations of the sugars. Each new
compound was dissolved in 1 M HCl and stirred at 80 °C for 2 hours. The solution of
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the reactant was then extracted with EtOAc 3 times. The H2O layers were evaporated
under vacuum. The presence of L-arabinose in the aqueous layers of compounds 1, 2,
4, 7, and 10 were detected by Sigel TLC (H2O-MeOH-CH2Cl2, 1:8:12) with standard
sample. D-quinovose was confirmed as the sugar moiety of compounds 3, 5, 8, 9 and
11 by Sigel TLC (H2O-MeOH-CH2Cl2, 1:8:12) with standard sample, and D-glucose
was identified as the sugar moiety of compound 6 by Sigel TLC
(H2O-MeOH-CH2Cl2, 1:8:12) with standard sample.
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Cell viability. RAW 264.7 cell viability was performed using the previously
described MTT method.19 The detailed procedures are seen in the Support
Information (Text S2).
NO production assay. NO production was detected according to a previous
report.20 The detailed procedures are seen in the Support Information (Text S3).
Enzyme-linked immunosorbent assay (ELISA). The expression of
proinflammatory cytokines (TNF-α, PGE2, and IL-6) was tested using the previously
described method.21 The detailed procedures are seen in the Support Information
(Text S4).
Western blotting analysis. The expressions of proteins iNOS, NF-κB/p65, and
COX-2 were assayed by Western blotting analysis according to standard procedures
published previously.19 The detailed procedures are seen in the Support Information
(Text S5).
Statistical analysis. All data were performed by Student’s test. The results are
displayed as the mean ± standard deviation (SD) for three independent experiments.
Differences were considered statistically significant at p < 0.05.
RESULTS AND DISCUSSION
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Structural elucidation of the 3,4-seco-dammarane triterpenoid saponins.
Cyclocarioside R (1) yielded a negative-ion peak [M + Cl] − at m/z 687.3859 (calcd.
for C36H60ClO10, 687.3875) in its HRESIMS spectrum (Figure S1), which
demonstrated a molecular formula, C36H60O10, in light of its 1D NMR (pyridine-d5)
1
and HRESIMS data. The H NMR data (pyridine-d5, Table 1) of 1 exhibited six
methyl signals at δH 0.96 (s, 30-CH3), 1.02 (s, 18-CH3), 1.24 (s, 19-CH3), 1.58 (s,
21-CH3), 2.06 (s, 27-CH3), and 1.80 (s, 29-CH3); a methoxy group at δH 3.51 (s,
3-OMe); two terminal methylene olefinic protons at δH 5.33 (br s, H-26a), 5.15 (br s,
H-26b), 4.87 (br s, H-28a), and 4.95 (br s, H-28b); and a glycosyl anomeric proton at
13
δH 4.80 (d, JH-1'/H-2' = 7.4 Hz, H-1'). The C-NMR (pyridine-d5, Table 3) and the
distortionless enhancement by polarization transfer (DEPT) (Figure S4) spectra
displayed the existence of a carboxyl group (–C=O) at δC 176.3 and two terminal
double bonds (–C=CH2) at δC 148.9 (C-4), 114.3 (C-28) and δC 147.8 (C-25), 113.5
1
(C-26). The H-1H correlation spectroscopy (COSY) (Figures 2 and S6) of 1
demonstrated the presence of four fragments (H-1/H-2, H-5/H-6/H-7, H-11/H-12/
H-13/H-17/H-16/H-15, H-22/H-23/H-24). In its HMBC spectrum (Figures 2 and S7),
a key correlation from H-3-OMe (δH 3.51, s) to C-3 (δC 176.3) suggested that the –
OCH3 connected to C-3. The cross peaks from H-28 (δH 4.95, 4.87, br s) to C-5 (δC
52.5) and C-29 (δC 24.2, s) and from H-26 (δH 5.33, 5.15, br s) to C-24 (δC 80.9) and
C-27 (δC 19.1) indicated the two terminal double bonds (–C=CH2) linked to C-4 and
C-25, respectively. The HMBC correlations from H-21-CH3 (δH 1.58, s) to C-17 (δC
52.3) confirmed the side chain linked to C-17. The HMBC correlations from the H-1′
(δH 4.80, d, anomeric proton) to C-12 (δC 75.7) suggested that the arabinopyranose
was linked to C-12. Its diagnostic ROESY correlations of H-5/H-9/H-12/H-17/
H-CH3-21/ H-23/H-CH3-30 suggested that H-5, H-9, H-12, H-17, CH3-21, H-23, and
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CH3-30 are α-oriented (Figures 3 and S8). The β-orientations of H-13, H-CH3-18, and
H-CH3-19 were then verified by the ROESY correlations of H-13/H-CH3-18
/H-CH3-19. In addition, the pentose unit was assigned as α-L-arabinopyranosyl based
on its 1H-and 13C-NMR data (pyridine-d5, Tables 1 and 3) and comparison with other
3,4-seco-dammarane triterpenoid saponin from this plant11-13 as well as further
hydrolysis of 1 with 1M HCl and Sigel TLC with standard L-arabinose. The coupling
constant of JH-1'/H-2' = 7.4 Hz of the anomeric proton suggested that the L-arabinose
was also α-oriented. Analyses of the 1D and 2D-NMR data of 1 showed high
similarity to those of cyclocarioside K,14 suggesting that 1 includes the basic
3,4-seco-dammarane triterpenoid skeleton, differing from cyclocarioside K only in the
signals of the side chain. The 20-, 23- and 24-OH substituents were confirmed by the
1H-1H COSY correlations of H-22/H-23/H-24 along with the key HMBC correlations
from H-21-CH3 to C-20 (δC 76.2) and C-22 (δC 41.9) and from H-23 to C-20 (δC
76.2), C-24 (δC 80.9) and C-25 (δC 147.8) (Figure 2). The above information indicated
that 1 was a characteristic 3,4-seco-dammarane saponin.
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The molecular formula of cyclocarioside S (2) was deduced to be C36H60O10 based
+
on its sodium adduct molecular ion peak [M + Na] at m/z 675.4073 (calcd. for
C36H60O10Na, 675.4084) in its HRESIMS (Figure S9) spectrum, which has the same
formula as that of 1. Compound 2 had almost identical NMR (Tables 1 and 3) data to
that of 1, as revealed by comparisons of their MS and NMR signals. Small differences
1
existed in the H- and 13C-NMR (pyridine-d5) data of positions 22, 23, and 24
between 2 and 1. The 1H NMR signals of H-22, 23, and 24 shifted from δH 1.90, 2.17,
4.54, and 4.40 in 1 to δH 2.12, 2.29, 4.61, and 4.57 in 2, respectively (Table 1).
13
Moreover, the C NMR signals of C-22, 23, and 24 shifted from δC 41.9, 71.8, and
80.9 in 1 to δC 40.1, 71.6, and 79.9 in 2, respectively (Table 3). Furthermore,
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compounds 1 and 2 exhibited two peaks with different retention times in the HPLC
chromatogram (Figure S17), indicating that 1 and 2 are epimers. The results indicate
that 2 is also a 3,4-seco-dammarane triterpenoid saponin.
To assess the relative configurations of positions 23 and 24 in their side chains of 1
1
and 2, their ROESY correlations and H-1H coupling constant values were focused.
The ROESY correlation of H-23/H-CH3-21 indicated that H-23 and H-CH3-21 are
α-oriented (Figures 3 and S8). Moreover, the ROESY spectrum of 2 (Figures 3 and
S16) showed a correlation between H-24/H-CH3-21, indicating that H-CH3-21 and
H-24 are α-oriented. Furthermore, piscidinol A22 and 24-epi-piscidinol A,23 which
possessed the same eight-carbon side chains as 1 and 2 with syn and anti orietations
between hydrogens at positions 23 and 24, revealed coupling constants of J ≈ 0 Hz
and 8.0 Hz, and their structures were identified by X-ray analysis. Correspondingly,
combretanone A,24 alisol E25 and 23,24,25-trihydroxycycloartan-3-one,26 which have
syn and anti orietations between hydrogens at positions 23 and 24, exhibited coupling
constants of J ≈ 0 Hz and 6.0 Hz, respectively. The similar coupling constants of
respective J = 6.4 Hz and J = 5.8 Hz of 1 and 2 between hydrogens at positions 23
and 24 suggested that both hydrogens linked to positions 23 and 24 were in
anti-configurations. Thus, on the basis of their ROESY correlations and coupling
constants between hydrogens at positions 23 and 24, the absolute configurations of
carbons at positions 23 and 24 of 1 and 2 were determined as 23R, 24S, and 23S, 24R,
respectively. Compounds 1 and 2 were named cyclocarioside R and cyclocarioside S,
respectively (Figure 1).
Cyclocarioside T (3) yielded a negative-ion peak [M + Cl] − at m/z 701.4142 (calcd.
for C37H62ClO10, 701.4132) in its HRESIMS spectrum (Figure S18), which
demonstrated a molecular formula, C37H62O10, in light of its 1D NMR (pyridine-d5)
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and HRESIMS data. The H-, C-NMR data (pyridine-d5, Tables 1 and 3) of 3 were
similar to those of compound 1, indicating that 3 also possesses a similar
3,4-seco-dammarane triterpenoid skeleton as 1, differing only in the presence of a
β-D-quinovopyranosyl unit at position 12 and the absence of an α-L-arabinopyranosyl
unit. The sugar unit was identified as a β-D-quinovopyranosyl moiety based on the
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1H-, C-NMR data (pyridine-d5, Tables 1 and 3), the hydrolysis of 3 with 1M HCl
and Sigel TLC with standard sample. In its HMBC spectrum (Figures 2 and S24), the
methyl singlets H-6'-CH3 (δH 1.59, d, J = 5.6 Hz) correlated with an oxymethine
carbon resonance at C-5' (δC 73.3), indicating that H-6'-CH3 was connected to C-5',
and H-1' (δH 4.88, d, J H-1'/H-2' = 7.9 Hz, a glycosyl anomeric proton) was correlated
with C-12 (δC 76.3, an oxymethine carbon resonance), suggesting the sugar group was
connected to C-12. In the ROESY spectrum of 1, the correlation of H-CH3-21/H-23
indicated that H-CH3-21 and H-23 are α-oriented (Figures 3 and S25). Moreover, the
coupling constant of J = 6.2 Hz of 3 suggested that H-23 and H-24 were in
anti-configurations with respect to their configurations in 1, and the absolute
configurations of carbons at positions 23 and 24 of 3 was assigned as 23R, 24S. Thus,
3 was determined and named cyclocarioside T (Figure 1).
+
Cyclocarioside U (4) yielded a positive-ion peak [M + Na] at m/z 641.4069
(calcd. for C36H58O8Na, 641.4029) in its HRESIMS spectrum (Figure S26), which
demonstrated a molecular formula, C36H58O8, in light of its 1D NMR (pyridine-d5)
1
and HRESIMS data. Its H-NMR data (pyridine-d5, Table 1) exhibited six methyl
signals at δH 0.95 (s, 30-CH3), 1.05 (s, 18-CH3), 1.25 (s, 19-CH3), 1.49 (s, 21-CH3),
1.89 (s, 27-CH3), and 0.95 (s, 29-CH3); a methoxy signal at δH 3.52 (s, 3-OMe); three
olefinic protons including two terminal double bond protons at δH 6.15 (m, H-23),
6.45 (d, J = 15.7 Hz, H-24); δH 5.07 (br s, H-26a), 4.98 (br s, H-26b); and δH 4.87 (br
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s, H-28a), 4.95 (br s, H-28b); and a glycosyl anomeric proton at δH 4.82 (d, JH-1'/H-2' =
13
7.4 Hz, H-1'). Its spectra of C-NMR (pyridine-d5, Table 3) and DEPT (Figure S29)
indicated the presence of a carboxyl unit at δC 176.3 (C-3) and three double bond
signals at δC 148.9 (C-4), 114.2 (C-28); δC 128.7 (C-23), 135.9 (C-24); and δC 143.1
(C-25), 115.4 (C-26). The 1H-, 13C-NMR data (pyridine-d5) for 4 displayed a number
of similarities to those of 1 but differed in the signals of the eight-carbon side chain,
indicating that 4 also possesses a 3,4-seco-dammarane triterpenoid skeleton. Its 1H-1H
COSY (Figures 2 and S31) correlations of H-22 (δH 2.58, 2.71, m)/H-23 (δH 6.15,
m)/H-24 (δH 6.45, d, J = 15.7 Hz) suggested the existence of a –CH2CH=CH–
fragment. The HMBC (Figures 2 and S32) correlations of H-23 (δH 6.15, m) with
C-20 (δC 74.9) and H-24 (δH 6.45, d, J = 15.7 Hz) with C-26 (δC 115.4) and C-27 (δC
19.3) indicated the location of the additional double bond is at positions 23 and 24. In
1
addition, the sugar unit was identified as α-L-arabinopyranosyl based on the H-,
13C-NMR (pyridine-d5, Tables 1 and 3) data, the hydrolysis of 4 with 1M HCl and
Sigel TLC with L-arabinose, as well as comparison with those of 1. The L-arabinose
is linked to C-12 by the HMBC correlation from H-1′ (anomeric proton) to C-12.
Therefore, the structure of 4 was identified and assigned as cyclocarioside U (Figure
1).
The molecular formula of cyclocarioside V (5), C37H60O8, was determined by 1D
−
NMR (pyridine-d5) data and HRESIMS m/z 667.3960 [M + Cl]
(calcd. for
C37H60ClO8, 667.3977) (Figure S34), which is 14 Da more than that of 4. The 1D
NMR (pyridine-d5, Tables 1 and 3) signals of 5 closely resembled those of 4; they
differed in the existence of a –CH3 at δH 1.55 (d, J = 5.6 Hz). Comparisons of the 1D
NMR, MS data between 5 and 4 revealed a pronounced difference in the signals for
the sugar moiety and the presence of a methyl hexacarbonose in 5, indicating that 5
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also possesses a 3,4-seco-dammarane triterpenoid skeleton. The hydrolysis of 5 with
1M HCl and Sigel TLC with standard sample attributed the sugar in 5 as a
quinovopyranose. The arabinopyranose linked to position 12 in 4 was replaced by a
quinovopyranose in 5. The coupling constant of JH-1'/H-2' = 7.3 Hz of the anomeric
proton, suggested the quinovopyranose was -oriented. In the HMBC spectrum, the
correlation of H-6'-CH3 (δH 1.55, d, J = 5.6 Hz) with C-5' (δC 73.2) indicated 6'-CH3
connected to C-5'. The HMBC correlation (Figures 2 and S40) of H-1' (anomeric
proton) with C-12 unambiguously confirmed that the sugar unit was situated at C-12.
Thus, the structure of 5 was deduced and given the common name cyclocarioside V
(Figure 1).
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−
Cyclocarioside W (6) yielded a negative-ion peak [M + Cl] at m/z 671.4136
(calcd. for C37H60ClO10, 671.4135) in its HRESIMS spectrum (Figure S42), which
demonstrated a molecular formula, C37H60O10, in light of its 1D NMR (pyridine-d5)
and HRESIMS data. The 1D NMR (pyridine-d5, Tables 1 and 3) spectra of 6 were
closely similar to those of 5 except for the absence of the –CH3 peaks [δH 1.55 (3H, d,
5.9 Hz); δC 18.9] observed in 5 and the existence of alternative –CH2OH singlets [δH
4.38, 4.52 (2H, m); δC 63.9] in 6, suggesting that 6 is also a 3,4-seco-dammarane
triterpenoid. In the HMBC spectrum (Figures 2 and S48), the correlation of 6'-CH2OH
(δH 4.38, 4.52, m) with C-5' (δC 72.8) indicated that the –CH2OH was linked to C-5'.
The HMBC correlation of H-1' (δH 4.94, anomeric proton) with C-12 (δC 76.6)
indicated the sugar was connected to C-12. The hydrolysis of 6 with HCl yielded
D-glucose as identified by Sigel TLC analysis. The coupling constant of JH-1'/H-2' = 7.3
Hz of the glucosyl anomeric proton indicated the β-orientation for 6. Consequently,
the structure of 6 was deduced and given the common name cyclocarioside W (Figure
1).
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Cyclocarioside X (7) yielded a positive-ion peak [M + Na] at m/z 673.427 in its
HRESIMS spectrum (Figure S50), which demonstrated a molecular formula,
C37H62O9, in light of its 1D NMR (pyridine-d5) and HRESIMS data. The 1D NMR
spectra (pyridine-d5, Tables 2 and 3) of 7 were closely analogous to those of 4 except
in the signals of their side chains, indicating that 7 also possesses a
3,4-seco-dammarane triterpenoid skeleton. There were signals from a methyl group
(δH 1.34, s; δC 26.4, 26-CH3), a quaternary carbon (δC 74.6, C-25) and an additional
methoxy group (δH 3.24, s; δC 50.7, 25-OCH3) in 7, replacing the terminal olefin at δH
4.95 and 5.01, and at δC 115.4 and 143.1 observed in 4. The HMBC (Figures 2 and
S56) correlations from δH 3.24 (25-OCH3) and δH 1.34 (CH3-26/27) to δC 74.6 (C-25)
confirmed that the methoxy group and gem-dimethyl were linked to position C-25.
The hydrolysis of 7 with 1M HCl yielded L-arabinose. The coupling constant of
JH-1'/H-2' = 7.4 Hz of the sugar anomeric proton indicated the α-orientation for the
arabinosyl group. Accordingly, the structure of 7 was established and given the
common name cyclocarioside X (Figure 1).
Cyclocarioside Y (8) was revealed to have the molecular formula C38H64O9 by the
NMR data (1H, 13C, pyridine-d5) and its positive-ion peak [M + Na] + at m/z 687.4491
(calcd. for C38H64O9Na, 687.4448) (Figure S58) in the HRESIMS spectrum, which is
14 Da more than that of 7. The NMR data (1H, 13C, pyridine-d5, Tables 2 and 3) of 8
were closely analogous to those of 7; they differed in the existence of a methyl group
signals H-6'-CH3 (δH 1.59, d, 6.0 Hz; δC 18.9) in 8, indicating that 8 is also a
3,4-seco-dammarane triterpenoid saponin. In the HMBC spectrum, the correlation of
H-6'-CH3 (δH 1.59, d, J = 6.0 Hz) with C-5' (δC 73.2) indicated that H-6'-CH3 is linked
to C-5'. The HMBC correlation (Figures 2 and S40) of H-1' (δH 4.94) with C-12 (δC
76.3) indicated the linkage of the sugar unit to the 3,4-seco-dammarane triterpenoid
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skeleton was at C-12. The hydrolysis with 1M HCl yielded quinovopyranose. The
coupling constant of JH-1'/H-2' = 7.2 Hz suggested the quinovopyranose was β-oriented.
Thus, the structure of 8 was elucidated and given a trivial name cyclocarioside Y
(Figure 1).
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Cyclocarioside Z1 (9) was found to have the formula C37H62O9 via the NMR data
(1H, 13C, pyridine-d5) and its negative-ion peak [M + Cl] − at m/z 685.4074 (calcd. for
C37H62ClO9, 685.4082) (Figure S66) in the HRESIMS, which is 14 Da less than that
of 8. The NMR data (1H, 13C, pyridine-d5, Tables 2 and 3) of 9 showed that the signals
were analogous to those of 8 except for the absence of an –OCH3 signals (δH 3.23/δC
50.7) in 9, suggesting that 9 is also a 3,4-seco-dammarane triterpenoid saponin. The
NMR data (1H, 13C, pyridine-d5, Tables 2 and 3, Figures 2 and S72) confirmed that an
–OCH3 at position C-25 in 9 was substituted with a –OH in 8. The sugar unit was
confirmed by hydrolysis to be quinovopyranose. The coupling constant of JH-1'/H-2'
=
7.2 Hz demonstrated that the quinovopyranose was β-oriented. Thus, the structure of 9
was identified and given the common name cyclocarioside Z1 (Figure 1).
Cyclocarioside Z2 (10) has the same molecular formula of C37H62O9 as 7, as
evidenced by the NMR (1H, 13C, pyridine-d5) data and its negative-ion peak [M + Cl]
−
at m/z 685.4072 (calcd. for C37H62ClO9, 685.4082) (Figure S74) in the HRESIMS
1
spectrum. The H- and 13C-NMR data (pyridine-d5, Tables 2 and 3) of 10 were
analogous to those of 7 except for the positions of the double bond and the –OH of the
side chain, indicating that 10 also possesses a 3,4-seco-dammarane skeleton. In its
1H-1H COSY spectrum (Figures 2 and S79), the correlations of H-22/H-23/H-24
suggested that there is a –CH2CH(O)CH=C fragment. In its HMBC spectrum (Figures
2 and S80), the correlations of H-23-OCH3 (δH 3.24, s) with C-23 (δC 76.3) and H-24
(δH 5.22, d, J = 6.0 Hz) with C-25 (δC 135.9)/C-26 (δC 18.7)/C-27 (δC 26.2) indicated
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that the methoxy group and the double bond were located at C-23 and C-24/C-25,
respectively. The hydrolysis of 10 yielded L-arabinose. The α-configuration for the
sugar was confirmed by its coupling constant of 7.4 Hz between H-1′ and H-2′. These
findings indicated that 10 possessed a skeleton of 3,4-seco-dammarane triterpenoid.
Cyclocarioside Z3 (11) has the molecular formula C38H64O9 (Figure S82) by the
NMR (1H, 13C, pyridine-d5) data and its negative-ion peak [M + Cl] – at m/z 699.4222
(calcd. for C38H64ClO9, 699.4239) in the HRESIMS spectrum, which is 14 Da more
than that of 10. The NMR (1H, 13C, pyridine-d5, Tables 2 and 3) spectra of 11
suggested that its basic signals were analogous to those of 10 except for the existence
of a β-D-quinovopyranosyl unit at C-12 and the absence of an α-L-arabinopyranosyl
moiety at C-12. The sugar unit was determined to be a β-D-quinovopyranosyl moiety
based on the hydrolysis of 11 with 1M HCl and Sigel TLC with D-quinovose and the
13
NMR (1H, C, pyridine-d5, Tables 2 and 3) data. The HMBC (Figures 2 and S88)
correlation of H-6'-CH3 (δH 1.58) with C-5' (δC 73.2) suggested H-6'-CH3 linked to
C-5'. The HMBC correlation of H-1' (δH 4.94, anomeric proton) with C-12 (δC 76.2)
indicated that the linkage of the sugar unit to the 3,4-seco-dammarane skeleton was at
C-12. The results revealed that 11 was also a 3,4-seco-dammarane triterpenoid
saponin.
To determine the relative configurations of position 23 of 10 and 11, their ROESY
13
spectra and comparisons of the NMR (1H, C, pyridine-d5) data with two known
epimers which possess the same side chain were focused. In the ROESY spectra of 10
and 11 (Figures 3, S81 and S89), the correlations of H-23/H-21-CH3 indicated that
13
H-23 in each compound was α-oriented. In addition, the C-NMR data at δC 127.4
(C-24), δC 135.9 (C-25) of 10 and at δC 127.5 (C-24), δC 136.4 (C-25) of 11 more
closely resembled those at δC 128.4 (C-24), δC 135.6 (C-25) of
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23β-cycloarta-24-ene-3β,23-diol than those at δC 129.1 (C-24), δC 133.8 (C-25) of
23α-cycloarta-24-ene-3β,23-diol.27 From the above analyses, the methoxy groups in
position 23 of the side chains of 10 and 11 were deduced to be -oriented.
Correspondingly, the absolute configurations of position 23 in 10 and 11 were
unambiguously confirmed to be R. Compounds 10 and 11 were designated
cyclocarioside Z2 and cyclocarioside Z3, respectively (Figure 1).
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By comparisons of the NMR data (1H, C) to those reported in the literatures, the
known isolates were identified as cyclocariol H (12),10 cyclocarioside O (13),28
cyclocarioside P (14),28 cyclocarioside J (15),29 cyclocarioside I (16),16 cyclocarioside
H (17),11 and cyclocarioside K (18).12
The extracts and the compounds suppress the release of NO in LPS-activated
RAW 264.7 cells. As shown in Figure 4, relative to the PE, EtOAc, n-Butanol, and
H2O extracts, the EtOAc fraction of the 75% EtOH-H2O extract revealed strong
inhibition to the release of NO in LPS-activated RAW 264.7 cells, comparable to the
inhibition achieved by the positive control dexamethasone. Thus, the
anti-inflammatory compounds were further isolated from the EtOAc fraction.
The cell viability assay showed that these compounds did not exhibit any
cytotoxicity, and the survival rates of RAW 264.7 cells were over 90% at a
concentration of 40 μM of each compound. Studies have shown after RAW 264.7
cells are treated with LPS, large amounts of NO, TNF-α, PGE2 and IL-6 can be
released, which result in inflammation.30-32
The inhibitory activities of 1-18 against LPS-activated NO release in RAW 264.7
cells are shown in Table 4. From the results, it was deduced that compounds 2, 4, 6-8,
10-13, and 16-17 suppressed the release of NO, with IC50 values ranging from 8.23
μM to 34.57 μM. Compounds 7, 8, 10, and 11 showed strong inhibition to the
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production of NO with IC50 values of 8.23 μM, 9.41 μM, 9.36 μM, and 11.23 μM,
respectively, comparable to the inhibition achieved by the positive control
dexamethasone (IC50: 8.34 μM). Compounds 1, 3, 5, 9, 14-15, and 18 showed weak
inhibition of the release of NO, with IC50 values above 40 μM.
A comparison of the structures of these dammarane triterpenoid saponins with their
activities revealed that the methoxy group in the side chain seemed to be related to
high potency against LPS-activated NO release in RAW 264.7 cells. For example,
among the compounds, compounds 7, 8, 10, and 11, with methoxy groups at positions
25 or 23, achieved the most effective inhibition of NO release. Moreover, the
dammarane triterpenoid saponins (1-6, 9, and 12-18), without methoxy groups on the
side chain, achieved reduced or no inhibition of the release of NO. Furthermore, by
comparing the inhibitory activities against NO release with their structures, it was
deduced that the kind of sugar group was important in influencing the inhibitory effect
on NO release. Compounds 2, 4, 7, and 10, with an arabinopyranosyl moiety at
position 12, exhibited stronger activity against the release of NO than their
corresponding compounds 3, 5, 8, and 11, with a quinovopyranosyl moiety at position
12. Similarly, compounds 13, with two arabinopyranosyl moieties at positions 3 and
12, revealed stronger inhibitory activities against the release of NO than their
corresponding compounds 14, with an arabinopyranosyl unit and a quinovopyranosyl
unit at positions 3 and 12, respectively. Furthermore, the inhibitory activities against
the release of NO were related to the absolute configurations of the side chain at
positions 23 and 24. For example, compound 2 possesses different absolute
configurations of the side chain at positions 23 and 24 than compounds 1 and 3 and
showed higher activity than both 1 and 3. The inhibitory activity against the release of
NO was also found to be related to the substitution positions of the double bond and –
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OCH3 in the side chain. For example, the substitution positions of the double bond
and –OCH3 in the side chains of 7 and 8 differ from those of 10 and 11 and showed
different activity. Comparison of the structures of 4 and 13 revealed that the inhibitory
activity against NO release appears largely unrelated to the presence of cleavage in
positions 2 and 3. In addition, comparison of the structures of 13 and 16 indicated that
the inhibitory activity is related to the existence of a ring in the side chain. For
example, compound 13, without an oxygen-substituted five-member ring in the side
chain, revealed weaker inhibitory effects on NO release than did compound 16, which
has a ring. In summary, the inhibitory effects of dammarane triterpenoid saponins
1-18 on the release of NO depend on the substitution of the –OCH3 on the side chain,
the kind of sugar, the absolute configuration of the side chain, the substitution
positions of the double bond and –OCH3, the cleavage in positions 2 and 3, and the
presence of a ring in the side chain.
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Active compounds reduce the expression of TNF-α, PGE2 and IL-6 in
LPS-activated RAW 264.7 cells. To explore the inhibition to inflammation of active
compounds 7, 8, 10, and 11, three crucial proinflammatory mediators, TNF-α, PGE2
and IL-6, were investigated whether these compounds are able to decrease these
proinflammatory molecules stimulated by LPS. The levels of cytokines TNF-α, PGE2,
and IL-6 markedly increased after LPS treatment. However, the overexpression of
cytokines TNF-α, PGE2, and IL-6 was reduced by treatment with 7, 8, 10, and 11
(Figures 5, 6, and 7). Generally, the results suggested that these isolates can inhibit the
expression of cytokines TNF-α, PGE2, and IL-6 in a concentration-dependent manner
in LPS-activated RAW 264.7 cells. Notably, among the inhibitory effects of the four
isolates on the expression levels of cytokines TNF-α, PGE2, and IL-6, those of
compound 7 were the most significant. The inhibitory activity of 7 was almost
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equivalent to that of the positive control, dexamethasone.
Compound 7 inhibits the levels of proteins COX-2, iNOS, and NF-κB/p65 in
LPS-activated RAW 264.7 cells. NF-κB is a critical transcription factor regulating
proinflammatory cytokine expression during inflammation.33-36 Moreover, proteins
iNOS and COX-2 are important proinflammatory mediators that contribute to
inflammation.37-40 To detect the effects of active compound 7 on protein and gene
expression, the levels of COX-2, iNOS, and NF-κB/p65 in LPS-activated cells were
measured. Activated by LPS, RAW 264.7 cells exhibited markedly increased levels of
the proteins COX-2, iNOS, and NF-κB/p65 relative to the levels of control cells
(Figure 8). After treatment with compound 7, the levels of proteins COX-2, iNOS,
and NF-κB/p65 in LPS-activated cells were markedly decreased. Compound 7
inhibited the expression levels of proteins COX-2, iNOS, and NF-κB/p65 in
LPS-activated RAW 264.7 cells in a dose-dependent manner.
Conclusion. The C. paliurus is a famous edible medicinal plant in China, and its
potent anti-inflammatory effects have attracted increasing attention. However, before
the present study, its active anti-inflammatory compounds had not been identified. In
the present study, anti-inflammatory-guided isolation of the leaves of C. paliurus
identified eighteen dammarane triterpenoid glycosides, including eleven new
3,4-seco-dammarane triterpenoid glycosides (1-11). Bioassays demonstrated that
compounds 7-8 and 10-11 showed potent inhibitory effects on the expression of
cytokines NO, TNF-α, PGE2, and IL-6. In addition, compound 7 significantly
suppressed the expression levels of proteins COX-2, iNOS, and NF-κB/p65,
indicating that this compound produced anti-inflammatory effects through the
suppression of NF-κB signaling pathways. The structure-activity relationship analysis
suggested that the anti-inflammatory activities of dammarane triterpenoid saponins are
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correlated with the substitution of the methoxy group in the side chain, the kind of
sugar, and the absolute configuration of the side chain of the compounds. In summary,
C. paliurus contains abundant anti-inflammatory dammarane triterpenoid saponins,
and compound 7 is a promising natural anti-inflammatory drug that warrants further
investigation. These findings suggest that regular consumption of the leaves of C.
paliurus may help prevent the occurrence of inflammatory diseases.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
13
1
website at DOI: xxxxxx. 1H NMR, C NMR, DEPT, HSQC, H-1H COSY, HMBC,
ROESY and HRESIMS spectra for compounds 1−11 (PDF).
AUTHOR INFORMATION
Corresponding Authors
*E-mail: lijun9593@gxnu.edu.cn.
*E-mail: huangxishan13@foxmail.com.
ORCID
Jun Li: 0000-0003-2023-8609
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by National Natural Science Foundation of Guangxi
(21662004), the Province Natural Science Foundation of Guangxi (2018JJD140035
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and 2018JJA140613) and the Key Laboratory for the Chemistry and Molecular
Engineering of Medicinal Resources (CMEMR2019-A1).
REFERENCES
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553
(1) Jin, L.; Li, X. B.; Tian, D. Q.; Fang, X. P.; Yu, Yong-Ming M.; Zhu, H. Q.; Ge, Y. Y.; Ma,
G. Y.; Wang, W. Y.; Xiao, W. F. Antioxidant properties and color parameters of herbal teas in
China. Ind. Crop. Prod. 2016, 87, 198-209.
(2) Ning, Z. W.; Zhai, L. X.; Huang, T.; Peng, J.; Hu, D.; Xiao, H. T.; Wen, B.; Lin, C. Y.;
Zhao, L.; Bian, Z. X. Identification of α-glucosidase inhibitors from cyclocarya paliurus tea leaves
using UF-UPLC-Q/TOF-MS/MS and molecular docking. Food Funct. 2019, 10, 1893-1902.
(3) Xiao, H. T.; Wen, B.; Ning, Z. W.; Zhai, L. X.; Liao, C. H.; Lin, C. Y.; Mu, H. X.; Bian, Z.
X. Cyclocarya paliurus tea leaves enhances pancreatic β cell preservation through inhibition of
apoptosis. Sci. Rep. 2017, 7, 1-13.
(4) Xie, J. H.; Xie, M. Y.; Nie, S. P.; Shen, M. Y.; Wang, Y. X.; Li, C. Isolation, chemical
composition and antioxidant activities of a water-soluble polysaccharide from Cyclocarya
paliurus (Batal.) Iljinskaja. Food Chem. 2010, 119, 1626-1632.
(5) Wang, Q. Q.; Jiang, C. H.; Fang, S. Z.; Wang, J. H.; Ji, Y.; Shang, X. L.; Ni, Y. C.; Yin, Z.
Q.; Zhang, J. Antihyperglycemic, antihyperlipidemic and antioxidant effects of ethanol and
aqueous extracts of Cyclocarya paliurus leaves in type 2 diabetic rats. J. Ethnopharmacol. 2013,
150, 1119-1127.
(6) Zhang, J.; Shen, Q.; Lu, J. C.; Li, J. Y.; Liu, W. Y.; Yang, J. J.; Li, J.; Xiao, K. Phenolic
compounds from the leaves of Cyclocarya paliurus (Batal.) Ijinskaja and their inhibitory activity
against PTP1B. Food Chem. 2010, 119, 1491-1496.
(7) The National Health Commission of the people’s Republic of China. Eight new raw
materials for foods, such as Euglena gracilis. Bulletin of The National Health Commission of the
people’s Republic of China. 2013, 124, 4.
(8) Yang, H. M.; Yin, Z. Q.; Zhao, M. G.; Jiang, C. H.; Zhang, J.; Pan, K. Pentacyclic
triterpenoids from Cyclocarya paliurus and their antioxidant activities in FFA-induced HepG2
ACS Paragon Plus Environment

Page 23 of 36
Journal of Agricultural and Food Chemistry
554
steatosis cells. Phytochemistry 2018, 151, 119-127.
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
(9) Fang, Z. J.; Shen, S. N.; Wang, J. M.; Wu, Y. J.; Zhou, C. X.; Mo, J. X.; Lin, L. G.; Gan, L.
S. Triterpenoids from Cyclocarya paliurus that enhance glucose uptake in 3T3-L1 adipocytes.
Molecules 2019, 24, 187.
(10) Chen, Y. J.; Na, L.; Fan, J. L.; Zhao, J. P.; Hussain, N.; Jian, Y. Q.; Yuan, H. W.; Li, B.;
Liu, B.; Choudhary, M. I.; Khan, I.; Wang, W. Seco-dammarane triterpenoids from the leaves of
Cyclocarya paliurus. Phytochemistry 2018, 145, 85-92.
(11) Li, S.; Cui, B. S.; Liu, Q.; Tang, L.; Yang, Y. C.; Jin, X. J.; Shen, Z. F. New triterpenoids
from the leaves of Cyclocarya paliurus. Planta Med. 2012, 78, 290-296.
(12) Cui, B. S.; Li, S. New triterpenoids saponins from the leaves of Cyclocarya paliurus. Chin.
Chem. Lett. 2015, 26, 585-589.
(13) Xuan, T. Y.; Tan, J.; Sun, H. H.; Yang, C.; Lv, W. Y.; Zhang, J. H.; Zhang, K. Q.; Nie, Z.
Q.; Ye, Z. J.; He, X. A.; Zhu, G. Z.; Xu, K. P. Cyclocarioside O-Q, three novel seco-dammarane
triterpenoid glycosides from the leaves of Cyclocarya paliurus. Nat. Prod. Res. 2019.
Doi.org/10.1080/14786419.2019.1616722.
(14) Wu, Y.; Li, Y. Y.; Wu, X.; Gao, Z. Z.; Liu, C.; Zhu, M.; Song, Y.; Wang, D. Y.; Liu, J. G.;
Hu, Y. L. Chemical constituents from Cyclocarya paliurus (Batal.) Iljinsk. Biochem. Systemat.
Ecol. 2014, 57, 216-220.
(15) Shu, R. G.; Xu, C. R.; Li, L. N.; Yu, Z. L. Cyclocariosides II and III: two seco-dammarane
triterpenoid saponins from Cyclocarya paliurus. Planta Med. 1995, 61, 551-553.
(16) Kennelly, E. J.; Cai, L.; Long, L.; Shamon, L.; Zaw, K.; Zhou, B. N.; Pezzuto, J. M.;
Kinghorn, A. D. Novel highly sweet seco-dammarane glycosides from Pterocarya paliurus. J.
Agric. Food Chem. 1995, 43, 2602-2607.
(17) Zhu, K. N.; Jiang, C. H.; Tian, Y. S.; Xiao, N.; Wu, Z. F.; Ma, Y. L.; Lin, Z.; Fang, S. Z.;
Shang, X. L.; Liu, K. Two triterpenoids from Cyclocarya paliurus (Batal) Iljinsk (Juglandaceae)
promote glucose uptake in 3T3-L1 adipocytes: the relationship to AMPK activation.
Phytomedicine 2015, 22, 837-846.
(18) Jiang, C. H.; Yao, N.; Wang, Q. Q.; Zhang, J. H.; Sun, Y.; Xiao, N.; Liu, K.; Huang, F.;
Fang, S. Z.; Shang, X. L.; Liu, B. L.; Ni, Y. C.; Yin, Z. Q.; Zhang, J. Cyclocarya paliurus extract
ACS Paragon Plus Environment

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Page 24 of 36
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
modulates adipokine expression and improves insulin sensitivity by inhibition of inflammation in
mice. J. Ethnopharmacol. 2014, 153, 344-351.
(19) Liu, S. H.; Lin, C. H.; Hung, S. K.; Chou, J. H.; Chi, C. W.; Fu, S. L. Fisetin inhibits
lipopolysaccharide-induced macrophage activation and dendritic cell maturation. J. Agric. Food
Chem. 2010, 58, 10831-10839.
(20) Shen, C. Y.; Zhang, T. T.; Zhang, W. L.; Jiang, J. G. Anti-inflammatory activities of
essential oil isolated from the calyx of Hibiscus sabdariffa L. Food Funct. 2016, 7, 4451-4459.
(21) Shen, C. Y.; Jiang, J. G.; Li, M. Q.; Zheng, C. Y.; Zhu, W. Structural characterization and
immunomodulatory activity of novel polysaccharides from Citrus aurantium Linn. variant amara
Engl. J. Funct. Foods 2017, 35, 352-362.
(22) McChesney, J. D.; Dou, J.; Sindelar, R. D.; Goins, D. K.; Walker, L. A.; Rogers, R. D.
Tirucallane-type triterpenoids: nmr and X-ray diffraction analyses of 24-epi-piscidinol A and
piscidinol A. J. Chem. Cryst. 1997, 27, 283-290.
(23) Kazufumi, T.; Takafumi, N.; Takashi, O.; Midori, A. A.; Takashi, K.; Thaworn, K.;
Masami I. Cycloartane triterpenes isolated from Combretum quadrangulare in a screening
program for death-receptor expression enhancing activity. J. Nat. Prod. 2011, 74, 249-255.
(24) Joycharat, N.; Greger, H.; Hofer, O.; Saifah, E. Flavaglines and triterpenoids from the
leaves of Aglaia forbesii. Phytochemistry 2008, 69, 206-211.
(25) Dirsch, V. M.; Stuppner, H.; Vollmar, A. M. The Griess assay: suitable for a bio-guided
fractionation of anti-inflammatory plant extracts. Planta Med. 1998, 64, 423-426.
(26) Yoshikawa, M.; Hatakeyama, S.; Tanaka, N.; Fukuda, Y.; Yamahara, J.; Murakami, N.
Crude drugs from aquatic plants. I. On the constituents of Alismatis Rhizoma. (1). Absolute stereo
structures of alisols E 23-acetate, F, and G, three new protostane-type triterpenes from Chinese
Alismatis Rhizoma. Chem. Pharm. Bull. 1993, 41, 1948-1954.
(27) Lago, J. H. G.; Brochini, C. B.; Roque, N. F. Terpenoids from Guarea guidonia.
Phytochemistry 2002, 60, 333-338.
(28) Wang, Y. R.; Cui, B. S.; Han, S. W.; Li, S. New dammarane triterpenoid saponins from the
leaves of Cyclocarya paliurus. J. Asian. Nat. Prod. Res. 2018, 20, 1019-1027.
(29) Liu, Y.; Zhang, M. Q.; Li, X. L.; Xu, T. H.; Xie, S. X.; Xu, Y. J.; Xu, D. M. Study on
ACS Paragon Plus Environment

Page 25 of 36
Journal of Agricultural and Food Chemistry
612
chemical constituents of Cyclocarya paliurus. J. Asian. Nat. Prod. Res. 2014, 16, 206-209.
(30) Coulter, J. A.; McCarthy, H. O.; Xiang, J.; Roedi, W.; Wagner, E.; Robson, T.; Hirst, D. G.
Nitric oxide-A novel therapeutic for cancer. Nitric Oxide 2008, 19, 192-198.
(31) Wu, Y. L.; Han F.; Luan S. S.; Ai R.; Zhang P.; Li H.; Chen L. X. Triterpenoids from
Ganoderma lucidum and their potential anti-inflammatory effects. J. Agric. Food Chem. 2019, 67,
5147-5158.
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
(32) Liu, Z. G.; Tang, L. G.; Zhu, H. P.; Xu, T. T.; Qiu, C. Y.; Zheng, S. Q.; Gu, Y. G.; Feng, J.
P.; Zhang, Y. L.; Liang, G. Design, synthesis, and structure–activity relationship study of novel
indole-2-carboxamide derivatives as anti-inflammatory agents for the treatment of Sepsis. J. Med.
Chem. 2016, 59, 4637-4650.
(33) Liu, Z. K.; Ng, C. F.; Shiu, H. T.; Wong, H. L.; Wong, C. W.; Li, K. K. A traditional
Chinese formula composed of Chuanxiong rhizoma and Gastrodiae Rhizoma (Da Chuanxiong
Formula) suppresses inflammatory response in LPS -induced RAW 264.7 cells through inhibition
of NF-κB pathway. J. Ethnopharmacol. 2017, 196, 20-28.
(34) Tak, P. P.; Firestein, G. S. NF-κB a key role in inflammatory diseases. J. Clin. Invest.
2001, 107, 7-11.
(35) Zhai, X. T.; Zhang, Z. Y.; Jiang, C. H.; Chen, J. Q.; Ye, J. Q.; Jia, X. B.; Yang, Y.; Ni, Q.;
Wang, S. X.; Song, J.; Zhu, F. X. Nauclea officinalis inhibits inflammation in LPS-mediated
RAW 264.7 macrophages by suppressing the NF-κB signaling pathway. J. Ethnopharmacol. 2016,
183, 159-160.
(36) Liu, Z. W.; Fan, Y. M.; Wang, Y.; Han, C.; Pan, Y.; Huang, H.; Ye, Y.; Luo, L.; Yin, Z.
M. Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in
macrophages and protects against endotoxic shock in mice. Febs. Lett. 2008, 582, 1643-1650.
(37) Moona, D. O.; Kima, M. O.; Lee, J. D.; Choi, Y. H.; Kim, G. Y. Rosmarinic acid sensitizes
cell death through suppression of TNF-α-induced NF-κB activation and ROS generation in human
leukemia U937 cells. Cancer Lett. 2010, 288, 183-191.
(38) Killeen, M. J.; Linder, M.; Pontoniere, P.; Crea, R. NF-κB signaling and chronic
inflammatory diseases: exploring the potential of natural products to drive new therapeutic
opportunities. Drug Discov. Today 2014, 19, 373-378.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 36
641
642
643
644
645
(39) Hayden, M. S.; Ghosh, S. Shared principles in NF-kappaB signaling. Cell 2008, 132,
344-362.
(40) Gupta, S. C.; Sundaram, C.; Reuter, S.; Aggarwa, B. B. Inhibiting NF-κB activation by
small molecules as a therapeutic strategy. Biochim. Biophys. Acta, Gene Regul. Mech. 2010, 1799,
775-787.
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Captions of Figures
Figure 1. Structures of compounds 1−18 isolated from the leaves of C. paliurus
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Figure 2. Key HMBC and COSY correlations of compounds 1−11
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Figure 3. ROESY correlations of compounds 1, 2, 4, 7, 10, and 11
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