PHYTOTHERAPY RESEARCH

Phytother. Res. 29: 1956–1963 (2015)

Published online 14 October 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5490

Anti-inflammatory Activity of Constituents

Isolated from Aerial Part of Angelica
acutiloba Kitagawa

Takuhiro Uto,1† Nguyen Huu Tung,1,2† Risa Taniyama,1 Tosihide Miyanowaki,1

Osamu Morinaga1 and Yukihiro Shoyama1*
1
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch,
Sasebo, Nagasaki 859-3298, Japan
2
School of Medicine and Pharmacy, Vietnam National University, 144 Xuan Thuy St., Cau Giay, Hanoi, Vietnam

Recently, the resources of medicinal plants have been exhausting. The root of Angelica acutiloba is one of the
most important ingredients in Japanese Kampo medicine for the treatment of gynecological diseases. In our
search for alternative medicinal plant resources of the root of A. acutiloba, we found that its aerial part has
the anti-inflammatory potency as well as the root. Phytochemical investigation of the aerial part resulted in
the isolation of four compounds including a new dimeric phthalide, namely tokiaerialide (2), along with
Z-ligustilide (1), falcarindiol (3), and bergaptol (4). Next, we investigated the in vitro anti-inflammatory activity
of 1–4 in lipopolysaccharide-stimulated RAW264 macrophages. Among the isolated compounds, 1 exhibited the
most potent inhibition against lipopolysaccharide-induced production of prostaglandin E2, nitric oxide, and
pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α). Compounds 3 and 4 also inhibited
all inflammatory mediators, but their inhibitory abilities were weaker than those of 1. Furthermore, 1, 3, and
4 strongly also induced heme oxygenase-1. These results suggest that 1, 3, and 4 potentially exert
anti-inflammatory activity, and the aerial part of A. acutiloba may be considered to be a useful medicinal
resource for inflammatory diseases. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: Angelica acutiloba; aerial part; Z-ligustilide; tokiaerialide; anti-inflammation; macrophages.

In Japan, the root of Angelica acutiloba Kitagawa

INTRODUCTION
Traditional herbal medicines have recently attracted at-
tention, owing to the rising interest in their health bene-
fits. The consumption of medicinal plants is markedly
increasing with economic development, and the depletion
of medicinal plant resources is becoming recognized as a
serious problem. Recently, there has been interest in the
effective utilization of non-supplemented medicinal parts
of medicinal plants as an approach to solve this problem.
The root of Bupleurum falcatum L. (Umbelliferae) is one
of the important ingredients in Japanese Kampo medicine
(JKM) and traditional Chinese medicine, while its aerial
part is not utilized as medicinal material. Our recent study
demonstrated that phytochemical analysis of the aerial
part of B. falcatum resulted in the isolation of major
phenolic components together with low concentrations
of saikosaponins (Tung et al., 2015). Furthermore, some
isolated components showed anti-proliferative activity
against HL-60 and HepG2 cancer cell lines. These find-
ings support utilization of the aerial part of B. falcatum
as medicinal material, and other medicinal plants also
have possibilities to contain active components in non-
supplemented medicinal parts.
* Correspondence to: Yukihiro Shoyama, Department of Pharmacognosy,
Faculty of Pharmaceutical Sciences, Nagasaki International University,
2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan.
E-mail: shoyama@niu.ac.jp
†
These authors contributed equally to this work.
Copyright © 2015 John Wiley & Sons, Ltd.
(Umbelliferae) is known as Toki and listed in Japanese
Pharmacopeia. A. acutiloba root has been used in JKM
for the treatment of gynecological diseases like female
reproductive diseases (Tsuchida et al., 1987). In China,
the root of Angelica sinensis (Oliv.) Diels (Danggui in
Chinese) has been used in traditional Chinese medicine
for the same purpose as A. acutiloba (Chao and Lin,
2011). A. acutiloba and Angelica gigas Nakai are com-
monly used as the substitutes of A. sinensis in the mar-
ket of Southeast Asia because of the shortage of A.
sinensis (Lao et al., 2004).
Phytochemical analyses of the roots of A. acutiloba
and A. sinensis have shown that volatile alkylphthalide
derivatives are principal components and Z-ligustilide
is the main component among the phthalides along with
several dimers (Tsuchida et al., 1987; Chao and Lin,
2011; Deng et al., 2006). Because the root of A. acutiloba
is the most important herbal medicine in JKM, several
biological activities have been elucidated. A. acutiloba
root extract relieves insulin resistance induced by high
sugar diet in rats (Liu et al., 2011a) and improves
advanced glycation end-product-mediated renal injury
in a diabetic model rat (Liu et al., 2011b). The polysac-
charide fraction from the root of A. acutiloba showed a
potent anti-tumor activity (Yamada et al., 1990). In the
field of inflammation, it has been reported that the
water extract of A. acutiloba root inhibited adjuvant ar-
thritis (Cho et al., 1982) and mast cell-derived allergic
reactions (Lee et al., 2012). Moreover, Z-ligustilide has
Received 01 May 2015
Revised 17 August 2015
Accepted 21 September 2015
 ANTI-INFLAMMATORY CONSTITUENTS OF AERIAL PART OF A. ACUTILOBA
an anti-asthma activity (Tao et al., 1984). However,
because the aerial part of A. acutiloba is not used as
an ingredient in JKM, phytochemical investigation of
its aerial part and its anti-inflammatory activity has not
been studied previously.
Inflammation involves multiple events regulated by
activation of inflammatory or immune cells. In the in-
flammatory process, activated macrophages play a key
role in inflammatory diseases by producing inflamma-
tory mediators, such as prostaglandin E2 (PGE2), nitric
oxide (NO), and pro-inflammatory cytokines [interleu-
kin-6 (IL-6) and tumor necrosis factor-α (TNF-α)]
(Fujiwara and Kobayashi, 2005; Berenbaum, 2000).
Overproduction of inflammatory mediators in macro-
phages has been implicated in several diseases, includ-
ing rheumatoid arthritis (Davignon et al., 2013),
atherosclerosis (Lind, 2003), asthma (Naik and Wala,
2013), chronic hepatitis (Tilg et al., 1992), and pulmo-
nary fibrosis (Coker and Laurent, 1998). Therefore,
inhibition of the production of these inflammatory me-
diators may thus prevent or suppress a variety of inflam-
matory diseases.
Heme oxygenases (HO) catalyze the rate-limiting step
in heme degradation, resulting in the formation of carbon
monoxide, biliverdin, and ferrous iron (Maines, 1988).
Several lines of evidence have shown that HO-1, an in-
ducible isoforms of HO, acts as a cytoprotective factor
against inflammatory responses and oxidative insults
through the antioxidant activity of biliverdin and its me-
tabolite, bilirubin, as well as the anti-inflammatory action
of carbon monoxide (Otterbein and Choi, 2000). There-
fore, the induction of HO-1 is beneficial in response to
stressful situations like inflammation.
Our pilot study on A. acutiloba showed that the aerial
part extract suppressed PGE2 and NO production with
similar potency to the root extract in lipopolysaccharide
(LPS)-stimulated murine macrophage-like RAW264
cells, and the phytochemical profile by TLC and HPLC
fingerprinting revealed the occurrence of the major
compound, Z-ligustilide, at a relatively high concentra-
tion in the aerial part rather than the root. In this study,
we showed the phytochemical analyses of the aerial part
of A. acutiloba. Moreover, we investigated the effects of
the isolated compounds on inflammatory mediators and
induction of HO-1. Our findings may open the possibil-
ity of the aerial part of A. acutiloba as a resource instead
of the root.
MATERIALS AND METHODS
General procedures. Optical rotations were measured
with a DIP-360 digital polarimeter (JASCO, Easton,
USA). NMR spectra were recorded on a JEOL ECX
400 FT-NMR spectrometer (JEOL, Tokyo, Japan) at
20 °C using Jeol’s standard pulse program, with
tetramethylsilane as the internal standard, and chemical
shift values were expressed in δ (ppm). HR-ESI-
TOFMS experiments employed a JEOL AccuTOFTM
LC 1100 mass spectrometer (JEOL, Tokyo, Japan).
Column chromatography was performed on silica gel
60 (230–400 mesh; Nacalai Tesque Inc., Kyoto, Japan).
TLC was performed on Kieselgel 60 F254 (Merck,
Darmstadt, Germany) plates. Spots were visualized by
Copyright © 2015 John Wiley & Sons, Ltd.
1957
spraying with 1% Ce(SO4)2–10% aqueous H2SO4 solution,
followed by heating.
Chemicals. Lipopolysaccharide (Escherichia coli sero-
type 055:B5) was purchased from Sigma Chemical Co.
(St. Louis, MO, USA). An enzyme-linked immunosor-
bent assay (ELISA) kit for PGE2 was purchased from
Cayman (Ann Arbor, MI, USA). ELISA kits for IL-6
and TNF-α were purchased from Diaclone (Besançon,
France). Antibodies against HO-1 and β-actin were
obtained from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). All other chemicals were obtained from
Wako Pure Chemical Industries (Osaka, Japan).
Plant material. A. acutiloba were collected at the Insti-
tute of Genkai medicinal plant cultivation (Genkai
town, Saga prefecture, Japan) in 2013 and authenticated
by one of the authors (Y. S.). A voucher specimen (Code
AA201301) has been deposited in the Department of
Pharmacognosy, Faculty of Pharmaceutical Sciences,
Nagasaki International University.
Preparation of the MeOH extracts of aerial and root
parts for the assay of anti-inflammatory activity. The
dried aerial or root parts (100 mg) of A. acutiloba were
extracted with MeOH (1.2 mL) under sonication three
times at 40 °C individually and then filtered and evapo-
rated with N2 gas at 60 °C.
Extraction and isolation of compounds 1-4. The pulver-
ized air-dried aerial part (79.0 g) was extracted with
MeOH (1.0 L × 3 times) at 40 °C with sonication. The
combined extracts were concentrated to a dark brown
residue (15.3 g). The obtained crude extract was
partitioned with a solvent mixture of hexane–MeOH–
H2O (200 mL:200 mL:20 mL, v/v/v) to obtain a hexane
layer (2.60 g after removing the solvent) and MeOH–
H2O layer, which was then evaporated and further
partitioned between EtOAc and water (200 mL:200 mL)
to obtain an EtOAc residue (1.40 g). The hexane extract
(2.60 g) was loaded onto a silica gel column
(30 × 340 mm) and eluted with hexane–EtOAc (8:1, v/v)
to yield compound 1 (120 mg). The EtOAc extract was
chromatographed on a silica gel column (30 × 300 mm)
and eluted with a gradient of hexane–EtOAc (8:1 → 2:1,
v/v) to afford additional compound 1 (60 mg) and five
fractions (fractions 1–5). Fraction 2 (25 mg) was subjected
to preparative TLC (200 × 200 × 0.25 mm) with a mobile
phase of hexane–EtOAc (6:1, v/v) to yield compound 2
(8.0 mg). Finally, fraction 5 (145 mg) was chrom-
atographed on a silica gel column (20 × 400 mm) with
hexane–EtOAc (5:2, v/v) to afford compounds 3
(15.0 mg) and 4 (8.0 mg). Compound 1, 3, and 4 were
identified as Z-ligustilide (Mitsuhashi et al., 1963;
Tsuchida et al., 1987), falcarindiol (Tanaka et al., 1977),
and bergaptol (Caceres et al., 2001; Girennavar et al.,
2007) by the comparisons of spectral data, respectively.
Compound 2: pale-yellow oil; ½α25 D -21° (c 0.15,
CHCl3); ESI-MS m/z (% base peak): 381 (39), 191
(100); HR-ESI-MS m/z: 381.2079 [M + H]+ (calcd for
C24H29O4, 381.2065); 1H-NMR (CDCl3, 400 MHz) δ:
Phytother. Res. 29: 1956–1963 (2015)
1958 T. UTO ET AL.
6.20 (ddd, J = 10.4, 6.4, 2.0 Hz, H-6′), 6.15 (dt, J = 9.6,
2.0 Hz, H-7), 5.97 (dt, J = 9.6, 4.4 Hz, H-6), 5.80
(brd, J = 10.4 Hz, H-7′), 4.86 (t, J = 7.6 Hz, H-8′), 2.78
(t, J = 8.0 Hz, H-8), 2.75 (m, H-4a), 2.60 (m, H-4b), 2.36
(m, H-5), 2.18 (dt, J = 14.0, 6.8 Hz, H-9′), 1.86-1.91
(m, H-5′), 1.66 (m, H-4′a), 1.58 (m, H-4′b), 1.25 (m, H-
9), 1.10–1.20 (overlapped, H-10,10′), 0.92 (t, J = 7.2 Hz,
H-11′), 0.89 (t, J = 6.8 Hz, H-11), and 13C-NMR (CDCl3,
100 MHz); δ: 172.8 (C-1′), 169.4 (C-1), 158.4 (C-3a), 148.8
(C-3′), 134.2 (C-6′), 130.4 (C-6), 126.1 (C-7a), 120.8
(C-7′), 116.6 (C-7), 107.8 (C-8′), 93.5 (C-3), 59.1 (C-7′a),
50.4 (C-3′a), 45.5 (C-8), 30.7 (C-4′), 29.7 (C-9), 27.4
(C-9′), 24.5 (C-5′), 22.5 (C-5), 21.7 (C-4), 20.7 (C-10),
20.6 (C-10′), 14.1 (C-11), and 13.7 (C-11′).
All compounds (1–4) were ≥95% pure (HPLC) and
were dissolved in DMSO at the concentration 100 mM
as the stock solution, which were stored at 20 °C until
their anti-inflammatory analysis.
Anti-inflammatory evaluation
Cell culture. The murine macrophage-like cell line,
RAW264 (RCB No. 0535), was obtained from the RIKEN
BioResource Center Cell Bank and cultured at 37 °C in a
5% CO2 atmosphere in Dulbecco’s modified Eagle’s me-
dium (DMEM) containing 10% FBS. To stimulate the cells,
the medium was replaced with fresh DMEM, and then LPS
was added in the presence or absence of compounds to the
indicated concentrations. Jolkinolide B (Uto et al., 2012)
was used as a positive control to assess the test validity of
anti-inflammatory activity of isolated compounds.
Measurement of PGE2 and cytokines. RAW264 cells
were plated at 4 × 105 cells/well in 24-well plates and
then treated with or without the compounds before ex-
posure to 50 ng/mL LPS. After 12 h of incubation, the
supernatant of the culture medium was collected. The
amounts of PGE2, IL-6, and TNF-α in the culture super-
natants were measured by ELISA according to the man-
ufacturer’s manual.
Measurement of NO. RAW264 cells were plated at
4 × 105 cells/well in 24-well plates and treated with or
without the compounds before exposure to 50 ng/mL
LPS. After 12 h of incubation, the level of nitrite (an indi-
cator of NO synthesis) was measured using the Griess re-
action. Briefly, the cell culture medium was mixed with
an equal volume of Griess reagent (1% sulfanilamide in
0.1% naphthylethylenediamine dihydrochloride and 5%
phosphoric acid in water). The absorbance was measured
with a microplate reader at 540 nm.
Western blotting analysis. RAW264 cells were plated at
1 × 106 cells/well in 6-cm dish and treated with or without
the compounds before exposure to 50 ng/mL LPS. After
12 h of incubation, the harvested cells were lysed, and
the supernatants were boiled for 5 min. Equal amounts
of lysate protein were run on 10% SDS-PAGE and elec-
trophoretically transferred to PVDF membrane. After
blotting, the membrane was incubated with specific pri-
mary antibody overnight at 4 °C and further incubated
for 1 h with HRP-conjugated secondary antibody. Bound
antibodies were detected by ECL Plus Western Blotting
Detection System with a LAS4000mini (GE Healthcare,
Princeton, NJ, USA).
Copyright © 2015 John Wiley & Sons, Ltd.
RNA isolation and RT-PCR analysis. RAW264 cells
were plated at 1 × 106 cells/well in 6-cm dish and treated
with or without the compounds before exposure to
50 ng/mL LPS. After 6 h of incubation, total RNA was
extracted using Trizol Reagent (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s instructions.
From each sample, 1 μg of total RNA was used to synthe-
size cDNA using PrimeScript first-strand cDNA Synthesis
Kit (Takara Bio, Shiga, Japan). Then, PCR analyses were
performed on aliquots of cDNA preparations to detect
HO-1 and GAPDH using TaKaRa Ex Taq (Takara Bio).
After initial denaturation for 2 min at 94 °C, the PCR
primers used in this study are as follows: HO-1, sense 5′-
CTGTGTAACCTCTGCTGTTCC-3′ and antisense 5′-
CCACACTACCTGAGTCTACC-3′; GAPDH, sense
5′-GACCCCTTCATTGACCTCAAC-3′ and antisense
5′-CATACCAGGAAATGAGCTTG-3′. PCR products
were separated on 2 % agarose gels and photographed
under UV excitation after ethidium bromide staining.
Statistical analyses. The experimental results are pre-
sented as mean ± SE. Each experiment was repeated at
least three times. Data were compared using Student’s
t test, and P values less than 0.05 were considered to
be significant.
RESULTS
Effects of extracts of aerial and root parts of A.
acutiloba on LPS-induced PGE2 and NO production
To assess the anti-inflammatory potency of the aerial
part of A. acutiloba, we compared the effect of MeOH
extracts of its aerial and root parts on LPS-induced
PGE2 and NO production in RAW264 cells. As shown
in Fig. 1A, the aerial part extract significantly attenu-
ated LPS-induced PGE2 with similar potency to the root
extract. Moreover, the inhibitory potency of NO pro-
duction by the aerial part extract was stronger than that
by root extract (Fig. 1B). These results indicate that the
aerial part of A. acutiloba might contain the anti-
inflammatory compounds as well as the root. The cyto-
toxicity of the extracts of aerial and root parts to cells
was assessed using MTT assay, and the cell viability
was not affected in the concentration range tested
(Supporting Information Fig. S1). Thus, the inhibitory
effects were not attributable to cytotoxic effects.
Isolation and structural elucidation of the compounds
The MeOH extract of the aerial part of A. acutiloba
was partitioned with hexane–MeOH–water (10:10:1,
v/v/v) and then between ethyl acetate and water,
followed by column chromatography to yield a new
phthalide dimer (2) along with Z-ligustilide (1) (Chen
et al., 2007), falcarindiol (3) having anti-nociceptive
(Tanaka et al., 1977) and anti-tumor activities (Jin et al.,
2012), and bergaptol (4) having radical scavenging activity
(Girennavar et al., 2007; Fig. 2).
Z-Ligustilide (1) was known to be one of the major
compounds of the A. acutiloba root (Tsuchida et al.,
1987; Miyazawa et al., 2004; Fukuda et al., 2009). We
Phytother. Res. 29: 1956–1963 (2015)
 ANTI-INFLAMMATORY CONSTITUENTS OF AERIAL PART OF A. ACUTILOBA 1959

Figure 1. Effects of extracts of aerial and root parts of A. acutiloba on LPS-induced PGE2 (A) and NO (B) production of RAW264 cells.
RAW264 cells were treated with the indicated concentrations of the extracts of aerial and root parts for 30 min before incubation with
50 ng/mL LPS for 12 h. The concentrations of PGE2 and nitrite were determined as described in the Materials and Methods section. Data
are expressed as the means ± SE of three independent experiments. *p < 0.05 vs. LPS alone.

obtained 1 from both the hexane and EtOAc extracts of
the aerial part, and its content was 0.228 dry weight (dw)
%, although the other alkyl phthalides, including
butylidene phthalide, butylphthalide, senkyunolide A,
and tokinolide A were isolated from the A. acutiloba
root (Tsuchida et al., 1987; Kim et al., 2006). HPLC fin-
gerprint of the aerial and root parts showed that 1 is
one of the major compounds of the aerial part as well
as root (Supporting Information Fig. S2). In addition,
the minor compound falcarindiol (3) was also found in
the root part. Meanwhile, the coumarin component
bergaptol (4) with low yield was isolated for the first
time from A. acutiloba.
Compound 2 was obtained as pale yellow oil with
½α20
D -21° (c 0.15, CHCl3). Its molecular formula was
determined to be C24H28O4 based on the observed
molecular peak at m/z 381.2079 [M + H]+ (calcd for
C24H29O4, 381.2065) in the HR-ESI-TOF-MS spec-
trum. The 1H-NMR (Supporting Information Fig. S3)
and H–H COSY spectra displayed the presence of a butyl
side chain [δ 2.78 (t, J = 8.0 Hz, H-8), 1.25 (m, H-9), 1.10–
1.20 (overlapped, H-10), and 0.89 (t, J = 6.8 Hz, H-11)]
and butylidene side chain [δ 4.86 (t, J = 7.6 Hz, H-8′),
2.18 (dt, J = 14.0, 6.8 Hz, H-9′), 1.10–1.20 (overlapped,
H-10′), and 0.92 (t, J = 7.2 Hz, H-11′). In addition, four
olefinic protons accounting for two isolated double bonds
[δ 5.97 (dt, J = 9.6, 4.4 Hz, H-6), 6.15 (dt, J = 9.6, 2.0 Hz,
H-7, 6.20 (ddd, J = 10.4, 6.4, 2.0 Hz, H-6′), and 5.80 (br d,
J = 10.4 Hz, H-7′) were observed. The 13C-NMR
Copyright © 2015 John Wiley & Sons, Ltd.
spectrum (Supporting Information Fig. S4) associated with
the DEPT spectrum revealed 24 carbons including two
ester carbonyl signals [δ 169.4 (C-1) and 172.8 (C-1′)], six
olefinic carbons, three quaternary carbons, a methine,
eight methylenes, and two methyls. The 24 carbon signals
and their resonance pattern suggested that 2 might be a
phthalide dimer formed by two ligustilide units (Tsuchida
et al., 1987; Deng et al., 2006). The upfield shift of the
olefinic proton signal of the butylidene side chain [δ 4.86
(t, J = 7.6 Hz)] indicated that it was not affected by
deshielding of the neighboring lactone and indicated
the butylidene side chain in the ligustilide moiety has
a Z-geometry form (Tsuchida et al., 1987; Deng et al.,
2006; Chen et al., 2007). The 1H- and 13C-NMR spectra
data of 2 were found to be similar to those of
sinaspirolide (Deng et al., 2006) and neodiligustilide
(Chen et al., 2007), which further suggested the struc-
ture of 2 as a dimeric ligustilide forming a cyclobutane
ring. This hypothesis was confirmed by the presence
of the prominent fragment ion of the monomer m/z
191 in the ESI-MS spectrum (Supporting Information
Fig. S5). The interlinkage between the two monomers
was established by the HMBC correlations of H-8/C-3′,
H-8/C-4′, and H-7′/C-3, respectively, supporting the
carbon bonding of C-3 → C-7′a and C-8 → C-3′a to build
the fused cyclobutane ring. In addition, the HMBC
spectrum of 2 showed correlations of H-6/C-7a, H-7/
C-1, H-7/C-3a, H-8/C-3, H-8/C-3a, H-8/C-9, H-6′/C-7′a,
H-7′/C-1′, H-7′/C-3′a, and H-8′/C-3′a confirmed the
Phytother. Res. 29: 1956–1963 (2015)
1960
Figure 2. Structures of compounds 1–4.
partial structures of 2. Moreover, the relative configura-
tion of compound 2 was also established by the NOESY
spectrum. The cross-peaks of H-8/H-4 and H-8′/H-4′b
further confirmed the Z geometry of the two ligustilide
moieties (Chen et al., 2007), and the NOE correlations
of H-8/H-7′, H-8/H-4′a, H-7′/H-4′a, and H-7′/H-6′
established that these protons lie in the same plane
(Fig. 3). Based on the above evidences, 2 was deter-
mined to be a new 3-7′a, 8-3′a Z-ligustilide dimeric
phthalide and named as tokiaerialide.
Several phthalide dimers have been isolated from A.
acutilaba roots and other members of the Umbelliferae
family such as A. sinensis, Cnidium officinale (Kobayashi
et al., 1987), and Ligusticum wallichii (Kaouadji et al.,
1986). Tokiaerialide (2) might be putatively composed
of two Z-ligustilide moieties attached via a cycloaddition
reaction reflecting anti-inflammatory activity of the A.
acutilaba aerial part.
Effect of the isolated compounds on PGE2, NO, IL-6,
and TNF-α production in LPS-stimulated RAW264
macrophages
To examine the anti-inflammatory effect of the isolated
compounds, we first measured the cytotoxicity of 1–4
against RAW264 cells using MTT assay (Supporting In-
formation Fig. S6). Cell viability was not altered by 1–4
Figure 3. Relative configuration of tokiaerialide (2) with key
NOESY correlations. This figure is available in colour online at
wileyonlinelibrary.com/journal/ptr.
Copyright © 2015 John Wiley & Sons, Ltd.
T. UTO ET AL.
below 25 μM. Therefore, cells might be treated with 1–4
at concentrations below 25 μM in the following
investigations of anti-inflammatory effect.
To investigate the effect of 1–4 on LPS-induced PGE2,
NO, IL-6, and TNF-α production, RAW264 cells were
treated with 1–4 at concentrations of <25 μM for 30 min
before exposure to 50 ng/mL LPS for 12 h. As shown in
Fig. 4A and B, the treatment of 1 between 3.125 and
25 μM strongly suppressed both LPS-induced PGE2
(IC50 = 1.05 μM) and NO production (IC50 = 8.45 μM).
Compounds 2, 3, and 4 also strongly reduced PGE2 (2,
IC50 = 15.61 μM; 3, IC50 = 7.78 μM; 4, IC50 = 7.18 μM)
and NO (2, IC50 > 25 μM; 3, IC50 = 11.24 μM; 4,
IC50 = 11.85 μM), but their inhibitory potency against
PGE2 and NO was weaker than that of 1. As shown in
Fig. 4C and D, 1 exhibited suppression of IL-6 and
TNF-α. On the other hand, compounds 3 and 4 weakly
reduced IL-6 and TNF-α. Compound 2 exhibited the
suppression of TNF-α at only 25 μM, but showed no
inhibition of IL-6 at any tested concentration. Overall,
the rank order of inhibitory potencies against inflamma-
tory mediators was 1 > 3 = 4 > 2. These results suggest
that 1 was the most potent inhibitor among the four
isolated compounds.
Effect of the isolated compounds on the induction of
HO-1 protein and mRNA expressions
Because HO-1 is known to be a novel protective gene
with potent anti-inflammatory effect, we examined
whether 1–4 enhance HO-1 protein expression in LPS-
stimulated RAW264 cells. As shown in Fig. 5, HO-1
protein and RNA levels were increased by the treat-
ment of 1–4. Among these compounds, compound 1 is
the strongest inducer of HO-1 expression at protein
and mRNA, and 3 and 4 also strongly increased HO-1
expression. However, the induction potency of 2 was
weaker than those of 1, 3, and 4. These data suggested
that 1, 3, and 4 are potent HO-1 inducer at the level of
protein synthesis.
DISCUSSION
In this study, we found that the aerial part of A. acutiloba
has anti-inflammatory potency similar to the root part.
Phytochemical analysis of the aerial part resulted in the
isolation of four compounds including a new dimeric
phthalide, tokiaerialide (2). Furthermore, we demon-
strated that the aerial part contains a major marker
constituent Z-ligustilide (1) together with falcarindiol (3)
and bergaptol (4), and the concentrations of Z-ligustilide
in the aerial part is remarkably high (0.228 dw%) as
compared with that in roots (approximately 0.05 dw%;
Tsuchida et al., 1987; Miyazawa et al., 2004; Fukuda
et al., 2009). To our knowledge, this is the first report of
a chemical profile of the aerial part of A. acutiloba.
To further investigate the mechanism underlying the
anti-inflammatory effect of isolated compounds, we ex-
amined the effects of 1–4 on inflammatory mediators.
Our data showed that 1 exerted the most potent inhibi-
tion of LPS-induced production of PGE2, NO, IL-6, and
TNF-α. This evidence has a good agreement with previ-
ous result (Chung et al., 2012). Compounds 3 and 4 also
Phytother. Res. 29: 1956–1963 (2015)
 ANTI-INFLAMMATORY CONSTITUENTS OF AERIAL PART OF A. ACUTILOBA 1961

Figure 4. Effect of 1–4 on LPS-induced production of PGE2 (A), NO (B), IL-6 (C), and TNF-α (D) in RAW264 cells. RAW264 cells were treated
with the indicated concentrations of 1–4 for 30 min before incubation with 50 ng/mL LPS for 12 h. The concentrations of PGE2, nitrite, IL-6,
and TNF-α were determined as described in the Materials and Methods section. Data are expressed as the means ± SE of three independent
experiments. *p < 0.05 vs. LPS alone.

suppressed all inflammatory mediators, but their
inhibitory abilities were weaker than those of 1. Addi-
tionally, 1, 3, and 4 strongly induced HO-1 expression.
Based on these findings, 1, 3, and 4 may contribute to
the anti-inflammatory activity of the aerial part of A.
acutiloba.
Previously, almost all researches on herbal medicines
have been focusing on the chemical and biological
analyses of medicinal part defined by the Japanese
Pharmacopeia. However, it becomes important to
Copyright © 2015 John Wiley & Sons, Ltd.
consider the utilization of non-supplemented medicinal
parts for the resource protection of medicinal plants be-
cause it is well known that the resource of native medic-
inal plants like licorice has been exhausted (Marui et al.,
2014; Yamamoto and Tani, 2005). This is the reason why
we started to investigate the aerial part of medicinal
plants. Resource of ginsenosides, major bioactive com-
pounds in the root of Panax ginseng, has been supplied
from the leaves of American ginseng based on an inves-
tigation (Li et al., 1996). In our previous study, we
Phytother. Res. 29: 1956–1963 (2015)
1962 T. UTO ET AL.
Figure 5. Effect of 1–4 on HO-1 protein (A) and RNA expression (B). RAW264 cells were treated with the indicated concentrations of 1–4 for
30 min before incubation with 50 ng/ml LPS for 12 (A) or 6 h (B). Protein expression levels of HO-1 and β-actin were detected by Western
blotting analysis as described in the Materials and Methods section. RNA levels of HO-1 and GAPDH were analyzed by RT-PCR as described
in the Materials and Methods section. Data shown are representative of two independent experiments.
isolated major phenolic components together with
saikosaponins from the aerial part of B. falcatum, and
some isolated components suppressed the proliferation
of cancer cell lines (Tung et al., 2015). As a second trial,
we isolated four compounds including a major marker
constituent Z-ligustilide (1) and new compound
tokiaerialide (2) from the aerial part of A. Acutiloba
and clarify that 1, 3, and 4 suppress LPS-induced inflam-
matory mediators and induce HO-1 in this study. These
evidences may approach the development of not only a
potential resource in JKM prescription having anti-
inflammatory activities instead of A. acutiloba root but
also new usage of non-supplemented part of medicinal
herbs. From these findings, we suggest that other
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Phytother. Res. 29: 1956–1963 (2015)