2015

NPC Natural Product Communications Vol. 10

No. 3
Anti-inflammatory Compounds from Ampelopsis cantoniensis 383 - 385
Nguyen Van Thua,b, To Dao Cuonga,c, Tran Manh Hunga, Hoang Van Luongb, Mi Hee Wooa, Jae Su Choid,
Jeong-Hyung Leee, Jeong Ah Kimf* and Byung Sun Mina*
a
College of Pharmacy, Catholic University of Daegu, Gyeongsan 712–702, Korea
b
Vietnam Military Medical University, 160 Phung Hung, Ha Dong, Hanoi, Vietnam
c
Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet,
Caugiay, Hanoi, Vietnam
d
Faculty of Food Science and Biotechnology, Pukyung National University, Busan 608–737, Korea
e
College of Natural Sciences, Kangwon National University, Gangwon-Do 200-701, Korea
f
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu,
702-701 Korea

jkim6923@knu.ac.kr (J.A.Kim), bsmin@cu.ac.kr (B.S.Min)

Received: May 11th, 2014; Accepted: November 17th, 2014

Many natural products have been shown to have an inhibitory effect on nitric oxide (NO), and are used as chemotherapy agents for inflammation disease. The
current study was designed to evaluate the anti-inflammatory activity of chemical components from the leaves of Ampelopsis cantoniensis. Sixteen compounds
(1―16) were isolated and identified. Phloretin (5) and 5,7,3′,5′-tetrahydroxyflavanone (16) inhibited nitric oxide (NO) production with IC50 values of 5.2, and
18.5 μM, respectively. The inhibitory effect of compounds 5 and 16 were accompanied by dose-dependent decreases in LPS-induced nitric oxide synthase
(iNOS) in RAW 264.7 cells, respectively.This study investigated the significant anti-inflammatory properties of isolated compounds from the leaves of
A. cantoniensis for the first time. The findings demonstrate that A. cantoniensis could be used beneficially in the treatment of inflammation disease.

Keywords: Ampelopsis cantoniensis, Vitaceae, Anti-inflammatory activity, RAW264.7 cells.

Macrophages play an important role in inflammatory disease related
to the overproduction of pro-inflammatory cytokines, including
interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF)-α, and
inflammatory mediators, including reactive oxygen species (ROS),
prostaglandin E2 (PGE2) and nitric oxide (NO) [1a]. NO is
produced by inducible nitric oxide synthase (iNOS) in
macrophages, hepatocytes, and renal cells, under the stimulation of
lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-α),
interleukin-1, and interferon-gamma [1b]. After exposure to
stimulants such as LPS from Gram-negative bacteria or lipoteichoic
acid from Gram-positive bacteria, iNOS can be induced to trigger
several disadvantageous cellular responses, including inflammation
[1c]. Therefore, the NO production induced by LPS through iNOS
can reflect the degree of inflammation, and a change in the NO
level through the inhibition of iNOS enzyme activity or iNOS
induction provides a means of assessing the effects of agents on the
inflammatory process.
Ampelopsis cantoniensis (Hook. & Arn.) K.Koch (Vitaceae) is
distributed in China, Taiwan, India, Japan, and Vietnam. This is a
wild plant that has been used as a traditional herbal medicine to
treat inflammatory diseases, such as rheumatic-arthritis, hepatitis,
dermatitis, and gastritis [2a]. The methanol extract of A.
cantoniensis induces apoptosis in human promyelocytic leukemia
HL-60 cells [2b], inhibits the proliferation of murine leukemia
wehi-3 cells, and promotes a range of immune responses in vivo
[2c]. This plant was reported to be an herbal medicine rich in
flavonoids, such as ampelopsin and myricetin [2d]. Ampelopsin,
which was purified from A. cantoniensis, can protect sensitive cells
against HIV-1 infection, reduce HIV-1 antigen P24 expression, and
have protective effects on oxidant stress-induced apoptosis in MT-4
cells, a CD4 T lymphocyte cell line [2e]. In Vietnam, this plant has
been used as a tea with anti-stress, analgesic, anti-bacterial, and
anti-gastritis properties [2f]. To study further the inhibitory effect of
LPS-induced NO production in macrophage RAW264.7 cells,
fractionation of the EtOAc-soluble fraction resulted in the isolation
of sixteen compounds (1−16). This study describes their isolation
and characterization, as well as the evaluation of their inhibitory
effects on LPS-induced NO production in macrophage RAW264.7
cells.
The MeOH extract of the dried leaves of A. cantoniensis was
partitioned into n-hexane-, CHCl3-, EtOAc-, and n-BuOH-soluble
fractions, and a H2O layer. Chromatographic purification of
the EtOAc-soluble fraction led to the isolation of sixteen
compounds (1−16). The isolated compounds were identified
as dihydromyricetin (1), myricetin (2), myricitrin (3), quercitrin
(4), phloretin (5), phlorizin (6), 5,7-dihydroxychromone (7),
3,5,7–trihydroxychromone (8), alstilbin (9), aromadendrin (10),
methyl gallate (11), 3,4-dihydro-2-(3′,4′,5′-trihydroxyphenyl)-2H-
chromene-4,5,7-triol (12), arbutin (13), 2-(3,4-dihydroxy-
phenyl)ethyl-O-β-D-glucopyranoside (14), gallic acid (15), and
3′,5′,5,7-tetrahydroxyflavanone (16) by comparing their
physicochemical and spectroscopic data with those reported in the
literature [2g-2k].
In murine RAW 264.7 macrophages, LPS stimulation alone can
induce iNOS transcription and protein synthesis and subsequent NO
production [3a]. Therefore, this cell system is an excellent model
for evaluating topical agents and screening potential inhibitors of
the pathways that induce iNOS and NO production. Due to the rapid
half-life of NO in vivo, we used nitrite production as a biomarker of
NO production in LPS-stimulated RAW 264.7 cells [3b]. The
amount of NO production was determined by the amount of nitrite,
384 Natural Product Communications Vol. 10 (3) 2015
a stable metabolite of NO. To assess the effect of these compounds
on the LPS-induced production of NO in RAW264.7 cells, a cell
culture medium was harvested, with the production of nitrite
measured using the Griess reaction. NO production was monitored
in RAW264.7 cells stimulated by LPS either in the presence or
absence of all isolated compounds for 24 h. LPS (1 μg/mL) evoked
a 6.5-fold increase in nitrite production compared with the naive
control. This induction was inhibited in a dose-dependent manner
by treatment with celastrol, a positive control agent, with an IC50 -
value of 1.0 μM [3c] (Table 1). Of the isolated compounds,
phloretin (5) and 3′,5′,5,7-tetrahydroxyflavanone (16) exhibited
inhibitory potency with IC50 values of 5.2, and 18.5 μM,
respectively, whereas the others were inactive. An examination of
the cytotoxicity of phloretin (5) and 3′,5′,5,7-tetrahydroxyflavanone
(16) in RAW264.7 macrophages by usingMTT assay indicated that
both compounds did not affect the viability of RAW264.7cells,even
at 50 μM (data not shown).
Table 1: NO production inhibitory activity of isolated compounds from A.
cantoniensis.
Compds IC50, μMa Compds
1 > 50 10
2 > 50 11
3 > 50 12
4 > 50 13
5 5.2 ± 0.4 14
6 > 50 15
7 > 50 16
8 > 50 Celastrolb)
9 > 50
a
b
50% inhibition concentrations are expressed as the mean±S.E.M. of triple experiments;
Positive control.
Among the tested compounds, 5 and 16 were most effective on the
LPS-induced production of NO in RAW 264.7 cells. Therefore,
Western blot was performed to determine the inhibitory effects of
these compounds on the modulation of iNOS protein expression
(Figure 1). RAW264.7 cells were stimulated with 1 μg/mL of LPS
for 24h in the presence of increasing concentrations of compounds 5
and 16(1–30 µg/mL). Compounds 5 and 16 reduced the LPS-
induced iNOS expression in a dose-dependent manner, but did not
change α-tubulin expression (Figure 1).
Figure 1: Inhibition of LPS-induced iNOS expression in RAW 264.7 cells by
compounds 5 and 16.
Macrophages are centrally involved in acute and chronic
inflammatory responses. LPS-stimulated macrophages induce the
expression of iNOS protein to produce NO. NO assisted the
activation of macrophages through signal transduction, thereby
enhancing the ability of macrophages to kill microorganisms [4a].
On the other hand, the overproduction of NO by macrophages
caused an immune hypersensitivity reaction and tissue or cell
injury. Min et al. showed that a chalcone compound isolated from
Caesalpinia sappan had a potent inhibitory activity of NO
production [4b]. In this study, compounds 5 and 16, which belong
to the chalcone and flavanone classes of flavonoids [4c], exhibited
the strongest nitrite inhibitory production. The glycosylation of
phloretin to phlorizin resulted in a loss of nitrite inhibitory
production in RAW264.7 murine macrophages. This is in
accordance with a previous study, which reported that after
pretreatment with various concentrations of either phloretin or
phlorizin of RAW264.7 cells, phloretin inhibited the levels of NO,
Thu et al.
PGE2, IL-6, TNF-α, iNOS and COX-2 significantly, whereas
phlorizin did not suppress the inflammatory response in LPS-
stimulated RAW264.7 cells [4d]. Although NO might also regulate
physiological processes in chondrocytes, including collagen type X
expression and alkaline phosphatase activity, several reports have
shown that osteoarthritis (OA) chondrocytes overexpress iNOS and
that the resulting excess NO production by OA chondrocytes is a
contributing factor to OA cartilage degradative processes [4e]. In
this study, we demonstrated that compounds 5 and 16 inhibit the
production of NO in LPS-stimulated macrophages, suggesting that
these compounds could suppress LPS-induced iNOS expression at
the transcription level.
Experimental
Plant material: Ampelopsis cantoniensis (Vitaceae) was collected
in May, 2010 in Cao Bang province, in northernVietnam. That plant
sample was authenticated by Professor Vu Xuan Phuong, and the
voucher specimen has been deposited at the Herbarium of the
Institute of Ecology and Biological Resources, Vietnam Academy
IC50, μMa
of Science and Technology.
> 50
> 50 Extraction and isolation: The dried leaves of A. cantoniensis (2.0
> 50
> 50
kg) were extracted with MeOH (3 ×6 L) at room temperature. After
> 50 evaporation of the solvent under reduced pressure, the crude MeOH
> 50 extract (368.0 g) was suspended in distilled water (0.5 L) and
18.5±1.0
1.0 ± 0.1 partitioned with n-hexane, CHCl3, EtOAc, and n-BuOH,
successively. By bio-guided fractionation, the EtOAc soluble
fraction (41.1 g), which had shown the strongest inhibition of NO
production, was subjected to CC over silica gel (CHCl3-acetone, 5:1
→ 0:1) to yield 10 fractions (E1~ E10), respectively. Fraction E3
was fractionated by silica gel CC (CHCl3-acetone, 10:1 → 0:1) to
yield 2 sub-fractions (E3.1 ~ E3.2). Sub-fraction E3.1 was further
purified by silica gel CC (CHCl3-MeOH-H2O, 15:3:1→6:4:1)
resulting in the isolation of compounds 11 (9.2 mg) and 12 (7.8
mg). Sub-fraction E3.2 was purified by MPLC [using an Ultra pack,
ODS-S-50B column (26 × 300 mm); MeOH-H2O, 1:4 → 1:0]
resulting in the isolation of compounds 7 (10.8 mg), 8 (11.0 mg)
and 9 (8.7 mg). Fraction E7 was fractionated by silica gel CC
(CHCl3-acetone, 6:1 → 0:1) to yield 2 sub-fractions (E7.1~E7.2)
and compound 1 (200.6 mg). Sub-fraction E7.1 was purified by
semi-preparative HPLC [using a gradient solvent system of MeOH-
H2O (1:3→2:3); flow rate 5 mL/min; for 90 min; UV detection at
210 nm; an YMC-Pack ODS-A, 250 × 20 mm column] resulted in
the isolation of compounds 2 (20.7 mg, tR = 34.4 min) and 15 (11.5
mg, tR = 39.4 min). Sub-fraction E7.2 was also purified by MPLC
[using an Ultra pack, ODS-S-50B column (26 × 300 mm); ACN-
H2O, 1:2→1:1] resulting in the isolation of compound 10 (8.3 mg).
Fraction E9 was fractionated by silica gel CC (CHCl3-acetone,
6:1→0:1) to yield 3 sub-fractions (E9.1~ E9.3). Sub-fraction E9.1
was purified by MPLC [using an Ultra pack, ODS-S-50A column
(11 × 300 mm); MeOH-H2O, 2:3→3:1] resulting in the isolation of
compounds 5 (15.2 mg) and 6 (10.7 mg). Sub-fraction E9.3 was
also purified by semi-preparative HPLC [using an isocratic solvent
system of 35% MeOH; flow rate 5 mL/min; for 90 min; UV
detection at 210 nm; an YMC-Pack ODS-A, 250 × 20 mm column]
resulting in the isolation of compounds 13 (15.2 mg, tR = 38.7 min)
and 16 (11.4 mg, tR = 41.4 min). Sub-fraction E9.2 was also purified
by semi-preparative HPLC [using a gradient solvent system of
MeOH-H2O (7:13→1:1); flow rate 5 mL/min; for 90 min; UV
detection at 210 nm; an YMC-Pack ODS-A, 250 × 20 mm column]
resulting in the isolation of compounds 4 (10.3 mg, tR = 36.3 min), 3
(8.1 mg, tR = 40.2 min), and 14 (9.7 mg, tR = 42.4 min).
Determination of NO production and cell viability assay: The level
of NO production was determined by measuring the amount of
Chemical components from the leaves of Ampelopsis cantoniensis Natural Product Communications Vol. 10 (3) 2015 385

nitrite present in cell culture supernatants, as described previously
[3c]. Briefly, the RAW264.7 cells (1×105cells/well) were stimulated
with or without 1 μg/mL of LPS (Sigma Chemical Co.; St. Louis,
MO, USA) for 24h in the presence or absence of the test
compounds (0.5−30 μM). The cell culture supernatant (100 μL) was
then reacted with 100 μL of Griess reagent (1% sulfanilamide in 5%
phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride
in distilled H2O). The absorbance at 540 nm was determined with a
microplate reader (Molecular Devices, Emax, Sunnyvale, CA,
USA), and the absorption coefficient was calibrated using a NaNO2
solution standard. Cell viability was measured with the MTT-based
colorimetric assay. For this experiment, celastrol was used as a
positive control [3c].
Immunoblotanalysis: Proteins were extracted from cells in ice-cold
lysis buffer (50mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM
EDTA, 1mM phenylmethyl sulfonyl fluoride, 1 μg/mL leupeptin,
1mM sodium vanadate, 150 mM NaCl). A 50 μg amount of protein
(for iNOS) per lane was separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride membrane (Millipore, Bedford, MA,
USA). The membrane was blocked with 5% skim milk and then
incubated with the corresponding antibody. The antibody for iNOS
was obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). The antibody for α-tubulin was obtained from Sigma. After
binding of an appropriate secondary antibody coupled to
horseradish peroxidase, proteins were visualized by enhanced
chemiluminescence according to the instructions of the
manufacturer (Amersham Pharmacia Biotec Buckinghamshire, UK)
[3c].
Statistical analysis: Values are expressed as mean ± S.E.M.
Acknowledgments - This research was supported by the Basic
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Science, ICT
& Future Planning (KRF-2012R1A1A2003547 and NRF-
2013R1A1A1008086). We are grateful to the Korean Basic Science
Institute (KBSI) for supplying the NMR spectra.

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