Anti-inflammatory isocoumarins from the bark of Fraxinus chinensis subsp. rhynchophylla

Article abstract of DOI:10.1080/14786419.2020.1715401

A new isocoumarin (1) named fraxicoumarin was isolated from the bark of Fraxinus chinensis subsp. rhynchophylla along with three known compounds (2–4). The structure of the new compound was established by extensive spectroscopic studies and chemical evidence. The anti-inflammatory effects of the isolated compounds (1–4) on lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells were evaluated in?vitro. Of the compounds tested, compounds 1 and 3 inhibited LPS-induced nitric oxide (NO) production in RAW 264.7 cells. Consistent with these findings, they also suppressed LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein level in RAW 264.7 cells.

Full text of DOI:10.1080/14786419.2020.1715401

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Anti-inflammatory isocoumarins from the bark of
Fraxinus chinensis subsp. rhynchophylla
Beom Zoo Lee, Chan-Sik Kim, Soon-Kyu Jeong, Sulhae Lee, Ik Soo Lee &
KwangWon Hong
To cite this article: Beom Zoo Lee, Chan-Sik Kim, Soon-Kyu Jeong, Sulhae Lee, Ik Soo Lee &
KwangWon Hong (2020): Anti-inflammatory isocoumarins from the bark of Fraxinusꢀchinensis
subsp. rhynchophylla, Natural Product Research, DOI: 10.1080/14786419.2020.1715401
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1715401
Anti-inflammatory isocoumarins from the bark of
Fraxinus chinensis subsp. rhynchophylla
a,b
c
b
b
c
Beom Zoo Lee , Chan-Sik Kim , Soon-Kyu Jeong , Sulhae Lee , Ik Soo Lee and
a
KwangWon Hong
a
Department of Food Science and Biotechnology, College of Life Science and Biotechnology,
b
Dongguk University, Goyang, Republic of Korea; Chemland Co., Ltd., Gunpo, Republic of Korea;
c
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea
ABSTRACT
ARTICLE HISTORY
A new isocoumarin (1) named fraxicoumarin was isolated from
the bark of Fraxinus chinensis subsp. rhynchophylla along with
three known compounds (2–4). The structure of the new com-
pound was established by extensive spectroscopic studies and
chemical evidence. The anti-inflammatory effects of the isolated
compounds (1–4) on lipopolysaccharide (LPS)-induced RAW 264.7
macrophage cells were evaluated in vitro. Of the compounds
tested, compounds 1 and 3 inhibited LPS-induced nitric oxide
Received 18 October 2019
Accepted 4 January 2020
KEYWORDS
Fraxinus chinensis subsp.
rhynchophylla; isocoumarins;
anti-inflammation; nitric
oxide; inducible nitric oxide
synthase; cyclooxygenase-2
(NO) production in RAW 264.7 cells. Consistent with these
findings, they also suppressed LPS-induced expression of indu-
cible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)
at the protein level in RAW 264.7 cells.
1. Introduction
The genus Fraxinus comprises approximately 60 species worldwide. Fraxinus chinensis
subsp. rhynchophylla (Hance) A.E.Murray (Oleaceae) is a deciduous tree that grows
widely in Korea and China. The bark of this plant has been used as an antibacterial,
analgesic, and anti-inflammatory agent, and is known as the traditional Chinese herbal
drug ‘Qinpi’ in the Chinese Pharmacopoeia. It also has diuretic, anticoagulant, and
CONTACT Ik Soo Lee
knifer48@kiom.re.kr; KwangWon Hong
hkwon@dongguk.edu
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1715401.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
B. Z. LEE ET AL.
anti-allergic properties (Kim et al. 1999). Numerous bioactive substances have been
isolated from Fraxinus species, including coumarins, lignans, flavonoids, secoiridoid
glucosides, and simple phenolic compounds, and their pharmacological activities have
been verified in vitro (Kwon and Kim 1996; Kostova and Iossifova 2007; Si et al. 2008,
2009; Ahn et al. 2013; Hadroug et al. 2019). Further phytochemical studies of
F. chinensis subsp. rhynchophylla bark to search for other anti-inflammatory compounds
resulted in the isolation of a new isocoumarin (1) and three known compounds (2–4).
Here, we described the isolation and structural elucidation of these compounds and
characterized their anti-inflammatory effects by measuring nitric oxide (NO) production
and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein
expression in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages.
2. Results and discussion
The ethanol extract of F. chinensis subsp. rhynchophylla bark, which significantly inhib-
ited LPS-induced NO production in RAW 264.7 cells (IC50 ¼ 15.2 mg/mL), was subjected
to a series of chromatographic separation steps guided by NO production inhibitory
activity, leading to the isolation of four compounds (1–4) (Figure 1). Comparing their
physicochemical and spectral data with reported data, three known compounds
were identified as brevifolin (2) (Nawwar et al. 1994), brevifolin carboxylic acid (3)
(Jiang et al. 2008), and methyl brevifolin carboxylic acid (4) (Camacho-Luis et al. 2008).
Compound 1 was obtained as an amorphous yellowish powder, and had a
þ
molecular ion peak at m/z 629.0774 [M þ Na] under HRESIMS, corresponding to the
molecular formula C H O . The IR spectrum of 1 included absorption bands for
2
6 22 17
ꢀ
1
ꢀ1
hydroxy (3254 cm ), conjugated carbonyl (1665 cm ), and aromatic (1603 and
510 cm ) functional groups. Its UV absorption maxima (225, 278, and 355 nm)
ꢀ
1
1
further supported the presence of conjugated carbonyl and aromatic ring systems.
1
The H-NMR spectrum included characteristic peaks for a galloyl group at d 7.10 (2H,
H
s) (Kaunda and Zhang 2019) and sugar proton signals. In addition, there was an
aromatic singlet at dH 7.33 and aliphatic ABX-type signals [dH 4.58 (1H, dd, J ¼ 7.5,
1
.8 Hz) and a pair of methylene at d 3.03 (1H, dd, J ¼ 18.8, 7.7 Hz) and 2.60 (1H, dd,
H
J ¼ 18.8, 1.8 Hz)] attributable to a brevifolin carboxylic acid moiety (Jiang et al. 2008).
Figure 1. Chemical structures of compounds 1–4.

NATURAL PRODUCT RESEARCH
3
1
3
The C-NMR spectrum showed that 1 contained 26 carbons, 13 of which were
0
00
000
000
000
000
assigned to galloyl [d 168.6 (C-7 ), 146.9 (C-3 , 5 ), 140.4 (C-4 ), 121.8 (C-1 ), and
C
000 000 00 00 00 00
10.9 (C-2 , 6 )] and glucosyl [d 91.9 (C-1 ), 75.8 (C-2 ), 72.8 (C-4 ), 72.7 (C-3 ), 71.2
C
C-5 ), and 66.0 (C-6 )] moieties. The remaining 13 carbon signals indicated the pres-
1
00 00
(
ence of an isocoumarin skeleton, consisting of one methylene, one methine, eight
quaternary, and three carbonyl carbons. Of these, the two most upfield signals at dC
3
8.9 and 43.0 were assigned to the aliphatic methylenic C-3 and methinic C-2, respect-
ively, whereas the three most downfield signals at d 163.0, 175.0, and 195.9 were
C
0
attributed to the carbonyl C-7 , C-6, and C-4, respectively. Assignment of the other
eight carbons was aided by comparing the chemical shifts with those of the corre-
sponding carbon signals of brevifolin carboxylic acid (3). In addition, the J value
(3.5 Hz) of the anomeric proton at dH 5.32 indicated that the glucose moiety was
attached in the a-configuration (Roslund et al. 2008). Complete assignment of the
chemical shifts of 1 and its substitution pattern was made using various 2 D-NMR tech-
1
1
niques, including H- H COSY, HMQC, and HMBC. The HMBC correlations between H-
0
0
00
(d 4.35 and 4.50) and C-6 (d 175.0), and between H-2 (d 4.71) and the carbonyl
H C H
6
carbon (d 168.6) of the galloyl group (Figure S1), indicated that the glucose moiety
C
was attached to C-6 of the brevifolin carboxylic acid structure, and the galloyl moiety
0
0
was linked to C-2 of glucose. Acid hydrolysis yielded D-glucose as a sugar residue,
which was identified by GC analysis of the acid hydrolysate, with gallic acid (1a) and
brevifolin carboxylic acid (1b) as aglycones. The phenylglycine methyl ester (PGME)
method (Nagai and Kusumi 1995) was used to determine the absolute configuration
1
of the chiral carboxyl group at C-2 of 1 based on the H-NMR data of the (S)- and
(
R)-PGME amides of 1b (Figure S2). A negative Dd (d ꢀd ) was observed for H-3, and
S
R
0
a positive value was observed for H-3 , indicating the S configuration of the secondary
carboxyl group at C-2 of 1b (Figure S3). Therefore, the structure of 1 was elucidated
as 2-O-galloyl 6-O-(S)-brevifolincarboxyl-a-D-glucopyranoside, and the compound was
named fraxicoumarin.
The ability of 1–4 to inhibit NO production in LPS-induced RAW 264.7 macrophages
was examined. In a toxicity test, these compounds did not affect cell viability at con-
centrations up to 20 lM (data not shown). Therefore, RAW 264.7 cells were treated
with these compounds at concentrations of 1–20 lM. Of the compounds tested, com-
pounds 1 and 3 inhibited LPS-induced NO production in a dose-dependent manner
(Figure S4). Treatment with 20 lM 1 and 3 decreased the LPS-induced NO production
by about 68% and 61%, respectively, compared with the LPS-treated control, which
G
was comparable to that of L-N -monomethyl arginine citrate (L-NMMA; 66% inhibition
at 20 lM). Because iNOS and COX-2 are crucial enzymes for NO production (Salvemini
et al. 2003), we examined the protein expression of iNOS and COX-2 in LPS-induced
RAW 264.7 cells by Western blotting. LPS strongly increased the expression of iNOS
and COX-2 protein, and pretreatment with 1 and 3 inhibited the LPS-induced iNOS
and COX-2 protein expression in a dose-dependent manner (Figure S4).
This study sought to identify effective anti-inflammatory agents from the bark of F.
chinensis subsp. rhynchophylla, and resulted in the isolation of four isocoumarins (1–4),
including one new compound (1). Of these isolates, compounds 1 and 3 dose-
dependently inhibited LPS-induced NO production in RAW 264.7 cells. Consistent with

4
B. Z. LEE ET AL.
this finding, compounds 1 and 3 also suppressed LPS-induced iNOS and COX-2 pro-
tein expression in RAW 264.7 cells. Further studies are needed to understand the spe-
cific mechanism(s) involved in the anti-inflammatory effects of these compounds.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured using a JASCO P-2000 digital polarimeter. UV spectra
were recorded using a JASCO V-550 UV/VIS spectrometer. IR spectra were recorded on
1
13
a JASCO 100 IR spectrometer. H- (400 MHz) and C-NMR (100 MHz) spectra were
obtained using a Bruker DRX-400 spectrometer with tetramethylsilane as an internal
1
1
standard. 2D-NMR experiments ( H- H COSY, HMQC and HMBC) were performed on a
Bruker Avance 500 NMR spectrometer. HRESIMS was performed using a Shimadzu
LCMS-IT-TOF spectrometer. Column chromatography was performed using Diaion HP-
20 (Supelco) and YMC-gel ODS-A (12 nm, S-75 mm, YMC). TLC was performed on pre-
coated silica gel 60 F254 (0.25 mm; Merck) and RP-18 F254s plates (0.25 mm; Merck).
Spots were detected by UV light (254 nm) and spraying of 10% H SO followed by
2
4
heating. Preparative HPLC was performed on a Gilson TRILUTION system with a 321
pump and a UV/Vis 151 detector, and a GX-271 liquid handler (Gilson, Inc.), using a
YMC-pack Pro C18 (250 ꢁ 10 mm, i.d.) column.
3.2. Plant material
Bark of F. chinensis subsp. rhynchophylla was collected in Gagok-ri, Hwado-eup,
Namyangju-si, Gyeonggi-do, Korea, in April 2017, and identified by Prof. Ki Hwan Bae,
Chungnam National University, Republic of Korea. A voucher specimen (KIOM-321) has
been deposited in the Herbarium of the Korea Institute of Oriental Medicine, Republic
of Korea.
3
.3. Extraction and isolation
Air-dried F. chinensis subsp. rhynchophylla stem bark (500 g) was extracted using EtOH
three times, 10 L each time) at room temperature for 7 days, filtered, and concen-
trated to yield an ethanol extract (48 g). The extract (40 g) was subjected to Diaion HP-
0 column chromatography (50 ꢁ 10 cm) and eluted with a gradient solvent system
consisting of (A) MeOH and (B) water: 100% B (1 L), 80% B (1 L), 60% B (1 L), 40% B
1 L), 20% B (1 L), and 100% A (1 L). The resulting portions from column chromato-
graphic separation were combined into four fractions (A, 5.2 g; B, 4.2 g; C, 3.8 g; D,
.5 g) based on TLC results. In this bioassay-guided study, the most active column frac-
(
2
(
6
tion B was chromatographed on a YMC RP-18 column (50 ꢁ 6.5 cm) using a
MeOH–H O gradient solvent system (20:80!100:0) to yield three subfractions (B1–B3).
2
Fraction B1 was further chromatographed on a YMC RP-18 column (50 ꢁ 3.5 cm) and
eluted with a MeOH–H O gradient solvent system (30:70!80:20) to yield compounds
2
1
(65 mg) and 2 (53 mg). Fraction B2 was purified over a YMC RP-18 column
(
50 ꢁ 2.5 cm) and eluted with a MeOH–H O gradient solvent system (40:60!80:20) to
2

NATURAL PRODUCT RESEARCH
5
generate compound 3 (38 mg). Chromatography of fraction B3 on a YMC RP-18
column (50 ꢁ 2.5 cm) and elution with a MeOH–H O gradient solvent system
2
(40:60!90:10) yielded compound 4 (31 mg).
3
.3.1. Fraxicoumarin (1)
2
5
Amorphous yellowish powder; [a]D þ4.6 (c 0.1, MeOH); UV (MeOH) k
(log e) 225
max
(2.1), 278 (4.0), 355 (1.4) nm; IR (KBr) ꢀ
3254, 1699, 1665, 1603, 1510, 1447, 1391,
max
ꢀ
1
1
0
00
1307, 1199, 1090, 1029 cm ; H NMR (400 MHz, CD OD) d 7.33 (1H, s, H-3 ), 7.10 (2H,
3
H
0
00
000
00
s, H-2 , 6 ), 5.32 (1H, d, J ¼ 3.5 Hz, H-1 ), 4.71 (1H, dd, J ¼ 9.8, 3.5 Hz, H-2 ), 4.58 (1H,
0
0
dd, J ¼ 7.5, 1.8 Hz, H-2), 4.50 (1H, dd, J ¼ 12.1, 2.1 Hz, H-6 a), 4.35 (1H, dd, J ¼ 12.1,
0
0
00
00
5
.6 Hz, H-6 b), 4.08 (1H, m, H-5 ), 3.98 (1H, dd, J ¼ 9.8, 9.3 Hz, H-3 ), 3.35 (1H, dd,
00
J ¼ 9.7, 9.3 Hz, H-4 ), 3.03 (1H, dd, J ¼ 18.8, 7.7 Hz, H-3a), 2.60 (1H, dd, J ¼ 18.8, 1.8 Hz,
1
3
000
0
H-3b); C NMR (100 MHz, CD OD) d 195.9 (C-4), 175.0 (C-6), 168.6 (C-7 ), 163.0 (C-7 ),
3
C
000
0
000
0
0
151.4 (C-4 ), 148.1 (C-5), 146.9 (C-3 , 5 ), 145.3 (C-6 ), 141.9 (C-5 ), 141.0 (C-1), 140.4
000 000 0 0 000 000 0 00
(C-4 ), 121.8 (C-1 ), 116.8 (C-2 ), 115.5 (C-1 ), 110.9 (C-2 , 6 ), 109.9 (C-3 ), 91.9 (C-1 ),
0
0
00
00
00
00
75.8 (C-2 ), 72.8 (C-4 ), 72.7 (C-3 ), 71.2 (C-5 ), 66.0 (C-6 ), 43.0 (C-2), 38.9 (C-3);
þ
HRESIMS m/z 629.0774 [M þ Na] (Calcd for C H O Na, 629.0779).
2
6 22 17
3.4. Acid hydrolysis and identification of sugar
Compound 1 (50mg) was dissolved in 10% aqueous HCl and refluxed for 3 h. On cool-
ing, the reaction mixture was extracted with EtOAc. After separating the organic layer,
the aqueous phase was neutralized with Na CO and concentrated. The organic layer
2
3
was evaporated under reduced pressure, and the residue (30 mg) was purified by pre-
parative RP-HPLC [Gilson TRILUTION system; YMC-pack Pro C18 (S-5 lm, 250 ꢁ 10 mm)
column; ACN/H O (20:80); UV detection, 280 nm; flow rate, 4.0 mL/min] to obtain com-
2
pounds 1a (8mg) and 1b (15mg). By comparing their spectral data with reported val-
ues, 1a and 1b were identified as gallic acid and brevifolin carboxylic acid, respectively.
A solution of the sugar residue of 1 in 1.5 mL pyridine was added to L-cysteine methyl
ꢂ
ester hydrochloride (1.5 mg) and kept at 60 C for 1 h. Trimethylsilylimidazole (1.5 mL)
ꢂ
was added to the mixture and kept at 60 C for 30min. The supernatant (4 mL) was
then subjected to GC analysis. GC conditions: GC-2010 (Shimadzu) instrument; detector,
FID; column, TC-1 capillary column (0.25mm ꢁ 30m; GL Sciences, Inc.); column tem-
ꢂ
ꢂ
perature, 230 C; programmed increase, 38 C/min; carrier gas, N (1 mL/min); injection
2
ꢂ
and detector temperature, 270 C. The sugar derivative obtained had a retention time
of 20.0min, identical with that of authentic D-glucose.
3
.5. Formation of (S)- and (R)-PGME amides
A solution of 1b (0.03 mmol) and (S)-PGME (0.05 mmol) in dry dimethylformamide
1 mL) was treated with benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate (0.05 mmol), hydroxybenzotriazole (0.05 mmol), and N-methylmorpholine
(
(
(
0.15 mmol). The reaction mixture was stirred at room temperature for 2 h. Water
1.0 mL) was added to the reaction mixture, which was then extracted with EtOAc. The
organic layer was evaporated under reduced pressure, and the residue (3 mg) was

6
B. Z. LEE ET AL.
purified by preparative RP-HPLC [Gilson TRILUTION system; YMC-pack Pro C18 (S-5 lm,
2
50 ꢁ 10mm) column; ACN/H O (20:80); UV detection, 280nm; flow rate, 4.0 mL/min] to
2
afford (S)-PGME amide 1c (1.8mg). Compound 1b (0.03 mmol) and (R)-PGME (0.05mmol)
were treated with the same procedure to afford (R)-PGME amide 1d (1.7 mg).
2
5
(
S)-PGME amide (1c): Amorphous yellowish powder; [a] –8.5 (c 0.06, MeOH);
D
1
0
H-NMR (400 MHz, DMSO-d ) d 7.293 (1H, s, H-3 ), 4.367 (1H, dd, J ¼ 7.6, 1.8 Hz, H-2),
6
H
2
.976 (1H, dd, J ¼ 18.4, 7.6 Hz, H-3a), 2.491 (1H, dd, J ¼ 18.4, 1.8 Hz, H-3b).
2
5
1
(
R)-PGME amide (1d): Amorphous yellowish powder; [a] þ7.2 (c 0.05, MeOH); H-
D
0
NMR (400 MHz, DMSO-d ) d 7.287 (1H, s, H-3 ), 4.372 (1H, dd, J ¼ 7.6, 1.8 Hz, H-2),
6
H
3.001 (1H, dd, J ¼ 18.4, 7.6 Hz, H-3a), 2.504 (1H, dd, J ¼ 18.4, 1.8 Hz, H-3b).
3.6. Cell culture
RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC) and
maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher
Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher
ꢂ
Scientific) and 1% penicillin/streptomycin at 37 C in a humidified 5% CO incubator.
2
3.7. Cell proliferation
4
RAW 264.7 cells were plated in 96-well plates at a density of 1 ꢁ 10 cells/well and
incubated for 24 h. The medium was replaced with fresh medium containing the indicated
concentrations of samples followed by incubation for 24 h. The rate of cell proliferation
was measured by MTS assay (Promega) according to the manufacturer’s protocol.
3.8. Measurement of NO
4
RAW 264.7 cells were plated in 12-well plates at a density of 5 ꢁ 10 cells/well
and incubated for 24 h. The cells were stimulated with 1 mg/mL LPS for 24 h after
pretreatment with samples. The supernatants of the cell cultures were harvested and
NO production was determined by measuring the amount of nitrite using the Griess
reagent system (Promega). Briefly, 100 mL of cell culture medium was mixed
with 100 mL of Griess reagent (1% sulfanilamide and 0.1% naphthylenediamide
dihydrochloride in 5% H PO ) and incubated at room temperature for 10 min. The
3
4
absorbance was then measured at 550 nm using a microplate reader (Bio-Rad). Nitrite
G
levels in samples were obtained from a standard NaNO2 curve. L-N -monomethyl
arginine citrate (Abcam) was used as a positive control.
3.9. Western blot analysis
Cell lysates were prepared using 1 ꢁ Laemmli lysis buffer (2.4 M glycerol, 0.14 M
Tris-HCl, pH 6.8, 0.21 M sodium dodecyl sulfate, and 0.3 mM bromophenol blue), and
boiled for 10 min. The protein concentration was measured using a bicinchoninic acid
(BCA) protein assay kit (Pierce Biotechnology). Equal concentrations of protein (20 mg)
were separated by 4–20% SDS-PAGE and transferred onto polyvinylidene difluoride

NATURAL PRODUCT RESEARCH
7
membranes. After blocking, the membranes were incubated with antibodies against
b-actin, iNOS, and COX-2 (Cell Signaling Technology). The membranes were incubated
with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling
Technology) and developed using the enhanced chemiluminescence (ECL) detection
system (Amersham Biosciences). Protein activity was determined using an LAS-4000
image analyzer (Fujifilm) and Image Gauge software (Fujifilm).
3.10. Statistical analysis
The results are expressed as means ± standard deviation (SD) from three independent
experiments. Statistical significance was assessed using Student’s t-test with GraphPad
7.0 Prism software (GraphPad).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by
Oriental Medicine.
a grant (KSN1812080) from the Korea Institute of
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