Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes

Article abstract of DOI:10.1002/prp2.166

Kujin contains antiallergic compounds that inhibit upregulation of histamine H₁ receptor (H1R) and interleukin (IL)-4 gene expression. However, the underlying mechanism remains unknown. We sought to identify a Kujin-derived antiallergic compound and investigate its mechanism of action. The H1R and IL-4 mRNA levels were determined by real-time quantitative RT-PCR. To investigate the effects of maackiain in?vivo, toluene-2,4-diisocyanate (TDI)-sensitized rats were used as a nasal hypersensitivity animal model. We identified (?)-maackiain as the responsible component. Synthetic maackiain showed stereoselectivity for the suppression of IL-4 gene expression but not for H1R gene expression, suggesting distinct target proteins for transcriptional signaling. (?)-Maackiain inhibited of PKCδ translocation to the Golgi and phosphorylation of Tyr³¹¹ on PKCδ, which led to the suppression of H1R gene transcription. However, (?)-maackiain did not show any antioxidant activity or inhibition of PKCδ enzymatic activity per se. Pretreatment with maackiain alleviated nasal symptoms and suppressed TDI-induced upregulations of H1R and IL-4 gene expressions in TDI-sensitized rats. These data suggest that (?)-maackiain is a novel antiallergic compound that alleviates nasal symptoms in TDI-sensitized allergy model rats through the inhibition of H1R and IL-4 gene expression. The molecular mechanism underlying its suppressive effect for H1R gene expression is mediated by the inhibition of PKCδ activation.

Full text of DOI:10.1002/prp2.166

ORIGINAL ARTICLE
Maackiain is a novel antiallergic compound that suppresses
transcriptional upregulation of the histamine H1 receptor
and interleukin-4 genes
Hiroyuki Mizuguchi1, Yuki Nariai1, Shuhei Kato1, Tomohiro Nakano1, Tomoyo Kanayama1,
Yoshiki Kashiwada2, Hisao Nemoto3, Kazuyoshi Kawazoe4, Yoshihisa Takaishi2, Yoshiaki Kitamura5,
Noriaki Takeda5 & Hiroyuki Fukui6
1Department of Molecular Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, 1-78-1 Sho-machi, Tokushima
770-8505, Japan
2Department of Pharmacognosy, Institute of Biomedical Sciences, Tokushima University Graduate School, 1-78-1 Sho-machi, Tokushima
770-8505, Japan
3Department of Pharmaceutical Chemistry, Institute of Biomedical Sciences, Tokushima University Graduate School, 1-78-1 Sho-machi, Tokushima
770-8505, Japan
4Department of Clinical Pharmacy, Institute of Biomedical Sciences, Tokushima Tokushima University Graduate School, 3-18-15 Kuramoto,
Tokushima 770-8503, Japan
5Department of Otolaryngology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto, Tokushima
770-8503, Japan
6Department of Molecular Studies for Incurable Diseases, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15
Kuramoto, Tokushima 770-8503, Japan
Abstract
Keywords
Allergic disease sensitive gene, histamine H1
receptor gene, IL-4 gene, Kujin, maackiain,
PKCd
Kujin contains antiallergic compounds that inhibit upregulation of histamine
H1 receptor (H1R) and interleukin (IL)-4 gene expression. However, the under-
lying mechanism remains unknown. We sought to identify a Kujin-derived
antiallergic compound and investigate its mechanism of action. The H1R and
IL-4 mRNA levels were determined by real-time quantitative RT-PCR. To
investigate the effects of maackiain in vivo, toluene-2,4-diisocyanate (TDI)-sen-
sitized rats were used as a nasal hypersensitivity animal model. We identified
(ꢀ)-maackiain as the responsible component. Synthetic maackiain showed ste-
reoselectivity for the suppression of IL-4 gene expression but not for H1R gene
expression, suggesting distinct target proteins for transcriptional signaling. (ꢀ)-
Maackiain inhibited of PKCd translocation to the Golgi and phosphorylation of
Tyr311 on PKCd, which led to the suppression of H1R gene transcription. How-
ever, (ꢀ)-maackiain did not show any antioxidant activity or inhibition of
PKCd enzymatic activity per se. Pretreatment with maackiain alleviated nasal
symptoms and suppressed TDI-induced upregulations of H1R and IL-4 gene
expressions in TDI-sensitized rats. These data suggest that (ꢀ)-maackiain is a
novel antiallergic compound that alleviates nasal symptoms in TDI-sensitized
allergy model rats through the inhibition of H1R and IL-4 gene expression. The
molecular mechanism underlying its suppressive effect for H1R gene expression
is mediated by the inhibition of PKCd activation.
Correspondence
Hiroyuki Fukui, Department of Molecular
Studies for Incurable Diseases, Institute of
Biomedical Sciences, Tokushima University
Graduate School, 3-18-15 Kuramoto,
Tokushima 770-8503, Japan. Tel: +81-88-
634-6410; Fax: +88-633-7264;
E-mail: hfukui@tokushima-u.ac.jp
Present address
Yuki Nariai, Nakamura-ku, Nagoya, Aichi
Japan
Funding Information
This work was financially supported in part
by Grants-in-Aid for Research on Allergic
disease and Immunology from The Ministry
of Health Labor and Welfare of Japan
(10103185) to H.F, Grants-in-Aid for
Scientific Research (B) (26305004 to H.F.)
and Scientific Research (C) (15K07933 to
H.M.) from the Japan Society for the
Promotion of Science, and by a grant from
the Osaka Medical Research Foundation for
Incurable Diseases.
Abbreviations
DNP-BSA, 2,4-dinitrophenylated bovine serum albumin; DPPH, 2,2-diphenyl-1-
picrylhydrazyl; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled
receptors; H1R, histamine H1 receptor; IL, interleukin; MEK, mitogen-activated
protein kinase; PARP-1, poly(ADP-ribose) porymerase-1; PKCd, protein kinase
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and
no modifications or adaptations are made.
2015 | Vol. 3 | Iss. 5 | e00166
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Maackiain Suppresses H1R and IL-4 Gene Transcriptions
H. Mizuguchi et al.
Received: 26 May 2015; Revised: 10 June
C-d; PMA, phorbol-12-myristate-13-acetate; TDI, toluene-2,4-diisocyanate; TH1/
TH2, helper T cell type 1/2.
2015; Accepted: 11 June 2015
Pharma Res Per, 3(5), 2015, e00166,
doi: 10.1002/prp2.166
doi: 10.1002/prp2.166
toward a pronounced TH2 profile (Holgate 1993). TH2
cytokines, including IL-4 and IL-5, are also upregulated
Introduction
Pollinosis, a seasonal allergic rhinitis caused by hypersen-
sitivity to tree or grass pollens, affects more than 36 mil-
lion people in the U.S. and about 16% of the Japanese
population (Nathan et al. 1997; Baba et al. 2009). Anti-
histamines are a widely employed first-line treatment for
nasal symptoms of pollinosis. These drugs were thought
to antagonize histamine and prevent it from binding to
the histamine H1 receptor (H1R). However, nowadays, it
is considered that antihistamines act as inverse agonists
and antagonize histamine by blocking its binding with the
H1R and suppress constitutive H1R activity (Simons
2004; Mizuguchi et al. 2012a). Recently, we reported that
the H1R gene expression strongly correlated with the
severity of allergic symptoms in toluene-2,4-diisocyanate
(TDI)-sensitized allergy model rats and patients with pol-
linosis (Mizuguchi et al. 2008, 2010). Compounds that
suppress upregulation of H1R gene expression can allevi-
ate allergy symptoms (Matsushita et al. 2008; Dev et al.
2009; Shahriar et al. 2009; Nurul et al. 2011; Hattori et al.
2013). These findings strongly suggest that H1R signaling
is very important for the development of pollinosis and
that drugs designed for the suppression of H1R signaling
will be effective for allergic diseases. Previously, we
reported that histamine and phorbol-12-myristate-13-ace-
tate (PMA) stimulation increased H1R at both mRNA
and protein levels by the activation of H1R in HeLa cells
endogenously expressing H1R (Das et al. 2007). Hista-
mine- and PMA-induced upregulation of H1R gene
expression was suppressed by rottlerin, a protein kinase
C-d (PKCd)-selective inhibitor, indicating that upregula-
tion of H1R gene expression is PKCd dependent. Further
studies showed that both histamine- and PMA-induced
upregulation of H1R gene expression involved common
downstream mediators of PKCd signaling. Recently, we
investigated the molecular mechanism of PMA-induced
upregulation of H1R gene expression in HeLa cells and
demonstrated that the PKCd/extracellular signal-regulated
kinase (ERK)/poly(ADP-ribose) polymerase-1 (PARP-1)
signaling pathway was involved in PMA-induced upregu-
lation of HIR gene expression in HeLa cells (Mizuguchi
et al. 2011).
in TDI-sensitized rats and patients with pollinosis
(Shahriar et al. 2009; Kitamura et al. 2012). We
reported that the expression levels of these cytokines
strongly correlated with that of H1R (Mizuguchi et al.
2008; Shahriar et al. 2009), suggesting the existence of
cross talk between H1R signaling and TH2 cytokine sig-
naling. Consistent with this observation, we found that
intranasal application of IL-4 and histamine increased
H1R and IL-4 gene expression, respectively (Shahriar
et al. 2009).
Kujin is the dried root of Sophorae flavescens AITON
of the Leguminosae family. This Chinese herb has been
used extensively in the treatment of allergic diseases and
many other pathological conditions for many years in
Asian countries. Phytochemical studies have shown that
it contains quinolizidine, alkaloids, flavonoids, and trit-
erpenoids (Chen et al. 2004; Li and Wang 2004; Piao
et al. 2006; Ling et al. 2007). Kujin and its active compo-
nents have been reported to possess many biologically
relevant properties, exerting anti-inflammatory (Kim
et al. 2002), antiasthmatic (Hoang et al. 2007), antitumor
(Sun et al. 2007), and antimicrobial (Kuroyanagi et al.
1999) effects. In the previous study, we showed that
Kujin extract inhibited upregulation of H1R and IL-4
gene expression in TDI-sensitized rats (Dev et al. 2009).
However, so far, little work has been done to justify the
usefulness of Kujin extract or to elucidate the mechanism
behind its effects.
In the present study, we identified (ꢀ)-maackiain as
the primary ingredient responsible for the antiallergic
action of Kujin. Our data revealed that (ꢀ)-maackiain
inhibits the activation of PKCd, a key enzyme in H1R sig-
naling, resulting in the inhibition of PKCd translocation
to the Golgi, thereby causing suppression of H1R gene
transcription. Treatment with maackiain alleviated nasal
symptoms and suppressed TDI-induced upregulation of
H1R and IL-4 gene expression in TDI-sensitized rats. Our
data suggest that (ꢀ)-maackiain is a novel antiallergic
compound that alleviates nasal symptoms in TDI-sensi-
tized allergy model rats through the inhibition of H1R
and IL-4 gene expression. The molecular mechanism
underlying its suppressive effect for H1R gene expression
is mediated by the inhibition of PKCd activation.
Allergic reactions are also characterized by a disrup-
tion of the helper T cell type 1/2 (TH1/TH2) balance
2015 | Vol. 3 | Iss. 5 | e00166
Page 2
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

H. Mizuguchi et al.
Maackiain Suppresses H1R and IL-4 Gene Transcriptions
The synthesis of (ꢁ)-3-benzylmaackiain was performed as
follows. To a solution of 7-benzyloxy-2H-1-benzopyran
(300.0 mg, 1.26 mmol, 1.0 equiv), 2-bromo-4,5-methylene
dioxyphenyl (1.093 g, 5.03 mmol, 3.0 equiv) in anhydrous
DMF (5 mL), suspended with potassium acetate (1.300 g,
13.2 mmol, 9.0 equiv) was added PdCl2(PhCN)2 (49.0 mg,
0.128 mmol, 0.1 equiv) in one portion. The resulting sus-
pension was stirred at 40°C. After 24 h, addition of
PdCl2(PhCN)2 (49.0 mg, 0.128 mmol, 0.1 equiv) was car-
ried out to the resulting reaction mixture, and the same
procedure was repeated after 48 h and 72 h, respectively
(total amount of used PdCl2(PhCN)2 was 196.0 mg).
Finally, the resulting mixture was stirred for additional
24 h (totally, 96 h). The resulting mixture was diluted with
ethyl acetate (50 mL), and filtered through Florisil. The fil-
trate was washed with brine, dried over sodium sulfate,
and concentrated in vacuo. The residue was purified by sil-
ica gel 60N column chromatography (dichloromethane:
hexane = 1:1) to afford (ꢁ)-3-benzylmaackiain (77.1 mg,
0.206 mmol, 16% yield) as a colorless oil. m.p. 146–147°C;
IR (KBr): 3446, 2924, 1619, 1541, 1506, 1474, 1434, 1377,
Materials and Methods
Isolation of (ꢀ)-maackiain from Kujin extract
Kujin extract was prepared as reported previously (Dev
et al. 2009). In briefly, 60 g of Kujin (“Kojima Kujin M,”
Lot 902607; Kojima Kampo, Osaka, Japan) was added to
1 L of distilled water, boiled for 1.5 h, and filtered twice to
remove insoluble materials. The extract was then concen-
trated and used for this study. The yield of freeze-dried
extract was 20% (w/w) with respect to the dried root. To
separate the crude extract into acidic, alkaline, and neutral
fractions, the pH of the Kujin extracts (30 mg/mL) was first
adjusted to three using 1 mol/L HCl, and then extracted
three times with ethyl acetate (EtOAc) to recover the acidic
fraction. Subsequently, the pH of the water-soluble fraction
was adjusted to 10 with 1 mol/L NaOH and was then
extracted with EtOAc to recover the alkaline fraction.
Finally, pH of the water-soluble fraction was neutralized
with 10% (w/v) citric acid and was designated the neutral
fraction. After removal of the solvent, each fraction was
tested for its ability to suppress the IgE-stimulated upregula-
tion of IL-4 gene expression in RBL-2H3 cells. Activity was
found in the acidic fraction. The acidic fraction was sub-
jected to chromatography on a silica gel column (Silica gel
60N; Kanto Kagaku, Tokyo, Japan). Elution of the column
with CHCl3:MeOH (9:1) yielded 21 fractions. TLC analysis
showed a common spot in two active fractions (fraction #1
and #2). These fractions were combined and rechromatog-
raphy was performed on a silica gel column eluted with hex-
ane:EtOAc (2:1). Fractions containing the common spot,
which was identified using TLC analysis, were combined
and their ability to suppress IL-4 gene expression in RBL-
2H3 cells was confirmed. After rechromatography on a silica
gel column using benzene:EtOAc (20:1), the active fraction
was further purified with a Mightysil RP18 GP HPLC col-
umn (Kanto Kagaku) using 65% methanol. Finally, the pure
compound was obtained by crystallization in methanol,
rechromatography on the HPLC column using 55% metha-
1329, 1269, 1146, 1038, 939, 828, 779 cmꢀ1 1H NMR
;
(400 MHz; CDCl3): d 7.48–7.31 (m, 6H), 6.77–6.70 (m,
2H), 6.57 (d, J = 2.4, 1H), 6.45 (s, 1H), 5.93 (d, J = 10.8,
1H), 5.93 (d, J = 10.8, 1H), 5.51 (d, J = 6.8, 1H), 5.08 (s,
2H), 4.25 (dd, J = 11.2, 5.2, 1H), 3.68 (dd, J = 11.2, 11.2,
1H), 3.50 (ddd, J = 11.2, 6.8, 5.2, 1H); 13C NMR
(100 MHz, CDCl3): d 160.2 (C), 156.5 (C), 154.2 (C),
148.1 (C), 141.7 (C), 136.7 (C), 131.7 (CH), 128.6
(CH 9 2), 128.0 (CH), 127.4 (CH 9 2), 117.9 (C), 112.7
(C), 109.8 (CH), 104.7 (CH), 102.7 (CH), 101.2 (CH2),
93.8 (CH), 78.4 (CH), 70.0 (CH2), 66.5 (CH2), 40.2 (CH);
HRESIMS: m/z calcd for C23H18O5Na [M + Na]+:
397.1052, found: 397.1054. Preparation of (ꢁ)-maackiain
[(ꢁ)-hydroxy-8,9-methylenedioxypterocarpan] was carried
out from (ꢁ)-3-benzylmaackiain according to the previ-
ously reported method (Breytenbach et al. 1980; To}kꢀes
et al. 1999). Separation of (ꢀ)-maackiain from (+)-ma-
ackiain was performed using a Chiral Pack IC column
(0.46 ID 9 25 cm, Daicel Co, Osaka, Japan) using hexane/
methanol [80:20 (v/v)] as a mobile phase. The retention
times of (ꢀ)-maackiain and (+)-maackiain were 73.68 min
and 99.60 min, respectively.
nol, and recrystallization in methanol. H NMR and 13C
1
NMR spectra of the purified maackiain in CDCl3 were
recorded using a Bruker AV400N instrument (Hitachi
High-Tech, Tokyo, Japan) with tetramethylsilane as internal
standard. MS spectra were obtained with an LCT PREMIER
system (Waters/Micromass, Milford, MA). IR spectra were
recorded on FT/IR-6000 (JASCO, Tokyo, Japan). Optical
rotations of purified and synthesized maackiain were
measured using a CD-J600 spectropolarimeter (JASCO).
Animal studies
Six-week-old male Brown Norway rats weighing 200–
250 g (Japan SLC, Hamamatsu, Japan) were used for the
present study. Rats were allowed free access to water and
food and kept in a room maintained at 25 ꢁ 2°C and
55 ꢁ 10% humidity with a 12-h light/dark cycle. Sensiti-
zation with TDI was performed by the method described
by Dev et al. (2009; Fig. 1). In brief, 10 lL of a 10%
Synthesis of maackiain
Bis(benzonitrile)palladium(II) dichloride [PdCl2(PhCN)2]
was prepared as reported previously (Doyle et al. 1960).
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2015 | Vol. 3 | Iss. 5 | e00166
Page 3

Maackiain Suppresses H1R and IL-4 Gene Transcriptions
H. Mizuguchi et al.
5 days
2 days
5 days
9 days
Control
Ethyl acetate
TDI
5 days
5 days
2 days
2 days
5 days
5 days
9 days
9 days
TDI
Water
( )-maackiain
TDI +
( )-maackiain
Figure 1. Experimental protocol for animal studies. Rats were sensitized with TDI for 2 weeks. The control group was sensitized with only ethyl
acetate. (ꢁ)-Maackiain (5–20 mg/kg) was administered once a day for 3 weeks. (ꢁ)-Maackiain was administered 1 h before TDI application when
rats were sensitized with TDI. TDI, toluene-2,4-diisocyanate.
solution of TDI in ethyl acetate (EtOAc; Wako Chemical,
Tokyo, Japan) was applied bilaterally on the nasal vesti-
bule of each rat once a day for five consecutive days. This
sensitization procedure was then repeated after a 2-day
interval. Nine days after the second sensitization, 10 lL
of 10% TDI solution was again applied to the nasal vesti-
bule to provoke nasal symptoms. The control group was
sensitized and provoked with 10 lL of EtOAc using the
same procedure. Synthetic (ꢁ)-maackiain (5–20 mg/kg)
was administered orally once a day for 3 weeks and nasal
symptoms were measured during the 10 min period just
after TDI provocation. Symptoms included the number
of sneezes and the nasal score, which included the extent
of watery rhinorrhea, swelling, and redness, measured on
a scale ranging from 0 to 3 (Table 1). All experimental
procedures were performed in accordance with the
guidelines of the Animal Research Committee of The
Tokushima University.
cultured to 70% confluency in 6-well dishes were serum-
starved for 24 h and then treated with reagents 24 h
before PMA stimulation. After a 3-h treatment with PMA,
the cells were harvested with 700 lL of RNAiso Plus (Ta-
kara Bio Inc. Kyoto, Japan), mixed with 140 lL of chloro-
form, and centrifuged at 17,400g for 15 min at 4°C. The
aqueous phase was collected, and RNA was precipitated by
the addition of isopropyl alcohol. After centrifugation at
17,400g for 15 min at 4°C, the resulting RNA pellet was
washed with ice-cold 70% ethanol. Total RNA was resus-
pended in 10 lL of diethylpyrocarbonate-treated water,
and 5 lg of each RNA sample was used for the reverse
transcription reaction. For determination of IL-4 mRNA,
RBL-2H3 cells were used instead of HeLa cells. RBL-2H3
cells were cultured at 37°C under a humidified 5% CO2,
95% air atmosphere in a MEM containing 10% fetal calf
serum and 100 IU/mL penicillin (Sigma–Aldrich) and
50 lg/mL streptomycin (Sigma–Aldrich, St, Louis, MO,
USA). RBL-2H3 cells cultured 70% confluency in 6-well
dished were treated with antidinitrophenol (DNP) IgE
(SPE-7, 100 ng/mL; Sigma-Aldrich , St. Louis, MO, USA)
18 h before DNP-HSA (100 ng/mL) stimulation. After 2-h
stimulation with DNP-HSA, the cells were harvested and
total RNA was prepared. Rat nasal mucosa samples were
collected in RNAlater (Applied Biosystems, Foster City,
CA) 4 h after provocation. Nasal mucosa samples were
homogenized using a Polytron (Model PT-K; Kinematica
AG, Littau/Luzern, Switzerland) in 10 volumes of ice-cold
RNAiso Plus Reagent. The homogenates were mixed with
chloroform and centrifuged at 17,400g for 15 min at 4°C.
The aqueous phase containing RNA was transferred to a
new tube, and the RNA was precipitated by the addition
of isopropanol and centrifugation at 17,400g for 15 min at
4°C. The RNA samples were reverse-transcribed to cDNA
using a High Capacity cDNA Reverse Transcription kit
(Applied Biosystems). TaqMan primers and the probe
were designed using Primer Express (Applied Biosystems).
Real-time quantitative RT-PCR
HeLa cells were cultured at 37°C under a humidified 5%
CO2, 95% air atmosphere in a minimal essential medium-
a containing 8% fetal calf serum, and 1% antibiotics–an-
timycotics (Invitrogen, Carlsbad, CA, USA). HeLa cells
Table 1. Criteria for grading the severity of TDI-induced nasal
responses in rats.
Score
Nasal
response
0
1
2
3
Watery
(ꢀ) At nostril Between 1 and 3 Drops of discharge
rhinorrhea
from nose
Swelling and (ꢀ) Slightly
redness swollen
Between 1 and 3 Strong swelling
and redness
TDI, toluene-2,4-diisocyanate.
2015 | Vol. 3 | Iss. 5 | e00166
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ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

H. Mizuguchi et al.
Maackiain Suppresses H1R and IL-4 Gene Transcriptions
Table 2. Nucleotide sequences for primers and probes used in this
stimulation with PMA. The cells were harvested in TBS
containing proteinase inhibitors (Complete Mini, Sigma-
Aldrich, St. Louis, MO, USA) and phosphatase inhibitors
(Phos STOP, Sigma-Aldrich, St. Louis, MO, USA), and
whole cell extracts were prepared by sonication. For the
immunoblot analysis, 30 lg of each protein sample was
separated on a 10% SDS-PAGE gel and then transferred
onto a nitrocellulose membrane (Bio-Rad, Richmond,
CA, USA). The membrane was briefly rinsed in TBS con-
taining 0.1% Tween 20 (TBS-T) and then incubated for
1 h at room temperature in TBS-T containing 5% skim
milk (Difco, BD Japan, Tokyo, Japan) or 3% BSA (for
detecting phosphoproteins; Sigma). The membrane was
then incubated with a primary antibody [PKCd (C-20),
Santa Cruz Biotechnology (Santa Cruz Biotechnology,
Santa Cruz, CA, USA); phospho-PKCd (Tyr311) and
b-actin, Cell Signaling (Cell Signaling Technology Japan,
Tokyo, Japan)] overnight at 4°C. Goat anti-rabbit IgG
(H ꢁ L)-HRP conjugate (Bio-Rad) was used as the sec-
ondary antibody, and proteins were visualized with an
Immobilon Western Chemiluminescent HRP substrate
(Merk Millipore Billerica, MA, USA).
study.
Primer/probe name
Sequence
Human H1R mRNA
Sense primer
Antisense primer
Probe
50-CAGAGGATCAGATGTTAGGTGATAGC-30
50-AGCGGAGCCTCTTCCAAGTAA-30
FAM-CTTCTCTCGAACGGACTCAGATACCACC-
TAMRA
Rat H1R mRNA
Sense primer
Antisense primer
Probe
50-TATGTGTCCGGGCTGCACT-30
50-CGCCATGATAAAACCCAACTG-30
FAM-CCGAGAGCGGAAGGCAGCCA-TAMRA
Rat IL-4 mRNA
Sense primer
Antisense primer
Probe
50-CAGGGTGCTTCGCAAATTTTAC-30
50-CACCGAGAACCCCAGACTTG-30
FAM-CCCACGTGATGTACCTCCGTGCTTG-
TAMRA
H1R, histamine H1 receptor.
Real-time PCR was conducted using a GeneAmp 7300
sequence detection system (Applied Biosystems). The
sequences of the primers and TaqMan probe are listed in
Table 2. To standardize the starting material, the human
GAPDH gene and Rodent GAPDH Control Reagents
(VICTM Probe; Applied Biosystems) were used, and data
were expressed as the ratio of H1R mRNA to GAPDH
mRNA.
Subcellular localization of PKCd
To determine the subcellular localization of PKCd, HeLa
cells were plated onto 35-mm glass-bottomed dishes
(Asahi Techno Glass, Chiba, Japan). HeLa cells were
serum-starved for 24 h. The cells were then stimulated
with 100 nmol/L PMA for 5 min. The cells were treated
with the (ꢀ)-maackiain (30 lmol/L) for 24 h before
PMA stimulation. After stimulation, the cells were washed
once with PBS and fixed with ice-cold methanol, and the
PBS was then replaced. The subcellular localization of
the PKCd was determined with anti-PKCd antibody as
the primary antibody and Cy3-conjugated donkey anti-
rabbit IgG as the secondary antibody (Jackson Immuno-
Research Laboratories, West Grove, PA, USA) using a
confocal laser microscope (LSM510; Carl Zeiss, Oberko-
chen, Germany). Localization of the Golgi was deter-
mined with anti-58K Golgi marker Protein antibody (as
the primary antibody, Abcam, Tokyo, Japan) and
DyLight488-conjugated donkey anti-rabbit IgG (as the
secondary antibody, Jackson ImmunoResearch).
Antioxidant activity of (ꢀ)-maackiain
The antioxidant capacity of purified (ꢀ)-maackiain was
determined using the DPPH (1,1-diphenyl-2-pic-
rylhydrazyl) radical scavenging activity assay previously
described by Jung et al. (Jung et al. 2005). L-Ascorbic acid
was used as a positive control. In brief, various concentra-
tions of (ꢀ)-maackiain or L-ascorbic acid in methanol
were incubated with 150 lmol/L DPPH for 30 min at
room temperature. Absorbance at 520 nm derived from
the DPPH radical was measured. DPPH radical scaveng-
ing activity was calculated as follows:
DPPH radical scavenging activity ð%Þ
¼ C ꢀ ðSt ꢀ SbÞ=C ꢂ 100;
where, St represents absorbance of sample at 520 nm; Sb,
absorbance of blank (methanol solution) at 520 nm; and
C, absorbance of control (DPPH in methanol only) at
520 nm.
PKCd kinase assay
The PKCd kinase assay was performed using the PKCd
kinase enzyme system (Promega, Madison, WI, USA)
and ADP-Glo kinase assay kit (Promega), according to
the manufacturer’s instructions. In brief, recombinant
PKCd (3 ng) and substrates (50 lmol/L ATP and
0.2 mg/mL CREBtide) were incubated with or without
Immunoblot analysis
HeLa cells were serum-starved for 24 h and stimulated
with 100 nmol/L of PMA for 10 min in 100-mm dishes.
Cells were pretreated with (ꢀ)-maackiain for 24 h before
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2015 | Vol. 3 | Iss. 5 | e00166
Page 5

Maackiain Suppresses H1R and IL-4 Gene Transcriptions
H. Mizuguchi et al.
various concentrations of (ꢀ)-maackiain or stauro-
sporine, a PKC inhibitor, for 20 min at 25°C. The
reaction was stopped by the addition of the ADP-Glo
Reagent solution, and the luminescence derived from
the ADP formed was measured using an Infinite M200
microplate reader (Tecan Japan, Kanagawa, Japan).
fraction on the IgE-stimulated upregulation of IL-4 gene
expression in RBL-2H3 cells. Kujin extract was partitioned
into acidic, basic, and neutral fractions (Fig. 2B), and the
active (acidic) fraction was further purified using silica gel
column chromatography (Fig. 2C). Pure compound was
obtained by rechromatography using silica gel, followed by
reversed-phase HPLC and crystallization (Fig. 2D). The
purified compound was identified as maackiain by struc-
ture determination using a combination of several 2D
NMR spectroscopic techniques and mass spectrometry.
Determination of the specific rotation of the purified ma-
Statistical analysis
The results are shown as mean ꢁ SEM. The data from
the experiments using TDI-sensitized rats are shown as
mean ꢁ SD. Statistical analyses were performed using
unpaired t-tests or ANOVA with Dunnett’s multiple com-
parison test using the GraphPad Prism software (Graph-
Pad Software Inc., La Jolla, CA). P < 0.05 was considered
statistically significant.
ackiain ([a]16 = ꢀ176.4°) revealed that (ꢀ)-maackiain
D
(Fig. 2D) is the active compound. Purified (ꢀ)-maackiain
suppressed upregulation of H1R and IL-4 gene expression
(Fig. 2E).
Effect of synthetic maackiain on
upregulation of H1R and IL-4 gene
expression in cultured cells and nasal
symptoms in TDI-sensitized rats
Results
Identification of (ꢀ)-maackiain as an anti-
allergic compound in Kujin
The synthesis of racemic maackiain has already been
reported using HgCl2 as a catalyst (Breytenbach et al.
1980). To eliminate the possibility of Hg contamination in
the final product, we established a new method for the syn-
thesis of (ꢀ)-maackiain that does not use HgCl2. In our
method, the Heck reaction was used for the preparation of
(ꢁ)-3-Benzyloxy-8,9-methylenedioxypterocarpan [(ꢁ)-3-
Benzyloxymaackiain] from 2-Bromo-4,5-methylenedioxy-
phenol and 7-Benzyloxy-2H-1-benzopyran. After racemic
maackiain synthesis (Fig. 3), we separated (ꢀ)-maackiain
from (+)-maackiain using a chiral column (Fig. 4A) and
investigated the effect of these compounds on the stimuli-
We have previously reported that Kujin extract alleviates
nasal symptoms and suppresses TDI-induced upregulation
of H1R and IL-4 gene expression in the nasal mucosa of
TDI-sensitized rats (Dev et al. 2009). To identify the active
compounds responsible for this effect, we investigated the
effect of Kujin extract on stimuli-induced upregulation of
H1R and IL-4 gene expression in cell systems. Kujin extract
inhibited stimuli-induced H1R (Nurul et al. 2011) and IL-
4 mRNA elevations (Fig. 2A). Therefore, we tried to purify
the active compounds from Kujin extract by partitioning
the fractions and assessing the suppressive activity of each
Figure 2. Identification of (ꢀ)-maackiain as an antiallergic compound in Kujin. (A) Effect of Kujin extract on IgE/antigen-stimulated IL-4 mRNA
expression in RBL-2H3 cells. RBL-2H3 cells were treated with 100 ng/mL anti-DNP IgE for 18 h and then stimulated with 100 ng/mL DNP-HSA for
2 h. Kujin extract was treated 24 h before DNP-HSA stimulation. After stimulation, total RNA was isolated, and the IL-4 mRNA levels were
determined by real-time quantitative RT-PCR. Data are presented as the mean ꢁ SEM (n = 3). **, P < 0.01 versus control; ##, P < 0.01 versus
DNP-BSA; one-way ANOVA and Dunnett’s multiple comparison test. (B) Effect of the fractions resulting from pH partition on IgE/antigen-
stimulated upregulation of IL-4 mRNA expression in RBL-2H3 cells. Acid fraction (a), alkaline fraction (b), and neutral fraction (c) were treated
24 h before DNP-HSA stimulation. Data are presented as the mean ꢁ SEM (n = 3). **, P < 0.01 versus DNP-HSA; one-way ANOVA and
Dunnett’s multiple comparison test. (C) Effect of the fractions obtained from silica gel column chromatography on IgE/antigen-stimulated
upregulation of IL-4 mRNA expression in RBL-2H3 cells (a), Fractions (20 lg/mL for F1, F3, F7, and F9; 50 lg/mL for F2) were treated 24 h before
DNP-HSA stimulation. Data are presented as the mean ꢁ SEM (n = 3). **, P < 0.01 and *, P < 0.05 versus DNP-HSA; unpaired t-tests. (b), TLC
analysis of the active fractions indicates existence of the common compound in F1 and F2 (arrow). (D) Elution profile from HPLC of the purified
active compound (a), and the structure of (ꢀ)-maackiain (b). (E) Effect of purified (ꢀ)-maackiain on PMA-induced H1R mRNA elevation in HeLa
cells (a) and IgE/antigen-stimulated IL-4 mRNA elevation in RBL-2H3 cells (b). (a), HeLa cells were serum-starved for 24 h, and stimulated with
100 nmol/L PMA for 3 h. Purified (ꢀ)-maackiain was treated 24 h before PMA stimulation. After stimulation, total RNA was isolated, and the
H1R mRNA levels were determined by real-time quantitative RT-PCR. In (b), RBL-2H3 cells were treated with 100 ng/mL anti-DNP-HSA IgE for
18 h and then stimulated with 100 ng/mL DNP-HSA for 2 h. Purified (ꢀ)-maackiain was treated 24 h before DNP-HSA stimulation. After
stimulation, total RNA was isolated, and the IL-4 mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the
mean ꢁ SEM (n = 3). **, P < 0.05 versus PMA or DNP-HSA; one-way ANOVA and Dunnett’s multiple comparison test. H1R, histamine H1
receptor; PMA, phorbol-12-myristate-13-acetate.
2015 | Vol. 3 | Iss. 5 | e00166
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ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

H. Mizuguchi et al.
Maackiain Suppresses H1R and IL-4 Gene Transcriptions
(A) 16
**
12
8
##
##
4
0
Control DNP-HSA 3.0
2.1
1.2
DNP-HSA + extract
(mg/mL)
(B) (a)
(b)
(c)
24
20
16
12
8
25
40
Alkaline fraction
Acidic fraction
Neutral fraction
20
15
10
5
30
20
10
0
**
**
**
4
0
0
1
5
2
10
20
100
DNP-HSA
+ fraction (µg/mL)
500
DNP-HSA
+ fraction (µg/mL)
DNP-HSA
+ fraction (µg/mL)
(C) (a)
(b)
18
(D) (a)
*
(b)
15
12
9
O
HO
H
*
H
O
O
6
Injection
**
3
O
0
Control
DNP-HSA
F1
F2
F3
F7
F9
F1 F2
DNP-HSA + fraction
HPLC after recrystallization
Structure of (–)-maackiain
(E) (a)
(b)
30
25
20
15
10
5
7
6
5
4
3
2
1
0
**
**
**
**
**
0
Control
DNP-HSA
50
150
Control
PMA
10
20
PMA + purified
(–)-maackiain (µmol/L)
30
DNP-HSA + purified
(–)-maackiain (µmol/L)
induced upregulation of H1R and IL-4 gene expression. As
shown in Figure 4B, both (+)- and (ꢀ)-maackiain sup-
pressed H1R mRNA elevation, whereas only (ꢀ)-maackiain
inhibited IL-4 mRNA elevation, suggesting distinct target
molecules for the H1R and IL-4 signaling pathways. Next,
we investigated the effect of synthetic maackiain on nasal
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2015 | Vol. 3 | Iss. 5 | e00166
Page 7

Maackiain Suppresses H1R and IL-4 Gene Transcriptions
H. Mizuguchi et al.
NBS
CH Cl
2
2
Br
O
O
O
O
1~2 min
HO
HO
63% yield
H
SO
4
Benzyl chloride
NaI
2
Acrylonitrile
Triton B
NaOH
CH COOH
3
O
K
CO
2
3
H
2
acetone
HO
OH
HO
O
HO
O
13 h
8 h
18 h
NC
O
43% yield
31% yield
NaBH
p-toluenesulfonic acid
toluene
4
ethanol
rt
O
O
O
O
O
O
O
3 h
2 h
OH
56% yield
96% yield
93% yield
PdCl (PhCN)
2
2
KoAc
DMF
HO
O
Br
O
H -Pd/C
2
O
O
O
O
H
+
H
4 days
O
3 h
HO
H
O
O
H
O
O
O
O
86.3% yield
16.4% yield
Figure 3. Synthetic pathway of (ꢁ)-maackiain.
symptoms and upregulation of allergic disease-sensitive
gene expression in TDI-sensitized rats. Application of TDI
causes nasal symptoms such as sneezing and watery rhinor-
rhea. Repeated application of TDI also increased H1R and
IL-4 mRNA expression in the nasal mucosa of TDI-sensi-
tized rats (Mizuguchi et al. 2008). Oral treatment with
racemic maackiain (5–20 mg/kg) for 3 weeks alleviated
nasal symptoms and suppressed upregulation of H1R and
IL-4 gene expression in TDI-sensitized rats (Fig. 4C).
Molecular mechanism of suppressive effect
of (ꢀ)-maackiain on H1R gene expression in
HeLa cells
Pharmacological activity of many flavonoids has been
explained by their antioxidant activities. Therefore, we
investigated whether the antiallergic effect of (ꢀ)-maacki-
ain is due to its antioxidant activity using the 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) radical scavenging assay,
Figure 4. Characterization of synthesized (ꢀ)-maackiain. (A) Separation of (ꢀ)-maackiain from (+)-maackiain by Chiral column chromatography.
(B) Effect of synthesized (ꢀ)-maackiain and (+)-maackiain on PMA-induced upregulation of H1R mRNA expression in HeLa cells (a) and on IgE/
antigen-stimulated upregulation of IL-4 mRNA expression in RBL-2H3 cells (b) (a), HeLa cells were serum-starved for 24 h, and stimulated with
100 nmol/L PMA for 3 h. Synthesized (ꢀ)- or (+)-maackiain was treated 24 h before PMA stimulation. After stimulation, total RNA was isolated,
and the H1R mRNA levels were determined by real-time quantitative RT-PCR. (b), RBL-2H3 cells were treated with 100 ng/mL anti-DNP IgE for
18 h and then stimulated with 100 ng/mL DNP-HSA for 2 h. Synthesized (ꢀ)- or (+)-maackiain was treated 24 h before DNP-HSA stimulation.
After stimulation, total RNA was isolated, and the IL-4 mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the
mean ꢁ SEM (n = 3). **, P < 0.01 versus PMA or DNP-HSA; one-way ANOVA and Dunnett’s multiple comparison test. (C) Effect of (ꢁ)-
maackiain on TDI-induced nasal symptoms and up-regulations of H1R and IL-4 gene expressions in the nasal mucosa of TDI-sensitized rats. Rats
were sensitized and provoked as described in Materials and Methods. (a), The number of sneezes were counted for 10 min just after TDI-
provocation. (b), Degrees of swelling and redness and watery rhinorrhea were scores according to the criteria listed in Table 1 on a scale ranging
from 0 to 3. (c, d), Rats were sacrificed 4 h after TDI provocation and nasal mucosa was collected. Total RNA was isolated from the nasal mucosa
as described in Materials and Methods, and H1R and IL-4 mRNAs were determined by real-time quantitative RT-PCR. Data are presented as the
mean ꢁ SD (n = 3). **, P < 0.01 and *, P < 0.05 versus TDI; one-way ANOVA and Dunnett’s multiple comparison test. H1R, histamine H1
receptor; H1R, histamine H1 receptor, PMA, phorbol-12-myristate-13-acetate; TDI, toluene-2,4-diisocyanate.
2015 | Vol. 3 | Iss. 5 | e00166
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ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

H. Mizuguchi et al.
Maackiain Suppresses H1R and IL-4 Gene Transcriptions
(–)-maackiain
(+)-maackiain
(A)
Injection
Time (min)
(a)
(b)
(B)
5
4
3
2
1
0
15
12
9
6
3
**
**
**
**
0
PMA
(–)-maackiain
(µmol/L)
–
–
+
–
+
+
+
–
+
–
DNP-HSA
–
–
+
–
+
+
+
–
+
–
(–)-maackiain
(µmol/L)
10
30
40
80
(+)-maackiain
(µmol/L)
(+)-maackiain
(µmol/L)
10
30
–
–
–
–
40
80
–
–
–
–
(a)
(C)
100
(b)
4
3
2
1
0
80
60
40
20
0
*
*
*
**
10
20
Control
5
TDI
20
Control TDI
5
10
TDI + maackiain (mg/kg)
TDI + maackiain (mg/kg)
(c)
3
2.5
2
(d)
5
4
3
2
1
0
*
1.5
1
*
*
0.5
0
5
TDI
Control
10
20
Control
5
10
20
TDI
TDI + maackiain (mg/kg)
TDI + maackiain (mg/kg)
with L-ascorbic acid as a positive control. (ꢀ)-Maackiain
at concentrations up to 200 lmol/L showed no DPPH
scavenging activity per se (Fig. 5A). This result suggests
that the mechanism of the suppressive effect of maackiain
on nasal symptoms and H1R and IL-4 gene expressions
was not due an antioxidant activity but proceeded
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2015 | Vol. 3 | Iss. 5 | e00166
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Maackiain Suppresses H1R and IL-4 Gene Transcriptions
H. Mizuguchi et al.
Maackiain (µmol/L)
40 20
(A)
100
(B)
Control
PMA
(–)-maackiain
80
60
40
20
0
L-ascorbic acid
311
P-Y
PKC
PKC
-actin
0
50
100
150
200
Concentration (µmol/L)
(C)
(D)
120
merge
58K Golgi protein
PKC
100
80
60
40
20
0
Control
PMA
(–)-maackiain
Staurosporine
PMA
–10
–9
–8
–7
–6
–5
–4
Log [compound (M)]
Figure 5. Antiallergic activity of (ꢀ)-maackiain is not due the antioxidant activity. (A) DPPH radical scavenging assay. Various concentrations of
(ꢀ)-maackiain (●) or L-ascorbic acid (■) in methanol were incubated with 150 lmol/L DPPH for 30 min at room temperature. Absorbance at
520 nm derived from the DPPH radical was measured. DPPH radical scavenging activity was calculated as described in Materials and Methods. (B)
Effect of (ꢀ)-maackiain on the PMA-induced phosphorylation of Tyr311 on PKCd. HeLa cells were serum-starved for 24 h and treated with
100 nmol/L PMA for 10 min. (ꢀ)-Maackiain was treated 24 h before stimulation with PMA. After stimulation, total cell lysate was prepared and
subjected to immunoblot analysis. (C) Effect of (ꢀ)-maackiain on translocation of PKCd in response to PMA stimulation. HeLa cells were serum-
starved for 24 h and treated with 100 nmol/L PMA for 5 min. The cells were treated with 30 lmol/L (ꢀ)-maackiain 24 h before PMA stimulation.
The subcellular localization of PKCd was determined using a confocal laser microscope. Scale bars = 20 lm. (D) Effect of (ꢀ)-maackiain on PKCd
kinase enzymatic activity. Recombinant PKCd (3 ng) and substrate (50 lmol/L ATP and 0.2 mg/mL CREBtide) were incubated with or without
various concentrations of (ꢀ)-maackiain (●) or staurosporine (■) for 20 min at 25°C. The reaction was stopped by the addition of the ADP-Glo
Reagent solution, and the luminescence derived from the ADP formed was measured using an Infinite M200 microplate reader. DPPH, 2,2-
diphenyl-1-picrylhydrazyl; PMA, phorbol-12-myristate-13-acetate.
through an unknown mechanism. Previously, we demon-
strated that the PKCd/ERK/PARP-1 pathway is involved
in the histamine- and PMA-induced upregulation of H1R
expression level in HeLa cells (Mizuguchi et al. 2011)
and that the translocation of PKCd to the Golgi and
phosphorylation of Tyr311 on PKCd are crucial steps in
this signaling pathway. Therefore, we investigated the
effect of (ꢀ)-maackiain on PMA-induced translocation
and phosphorylation of PKCd. Pretreatment with (ꢀ)-
maackiain inhibited PMA-induced phosphorylation of
Tyr311 on PKCd, and its translocation to the Golgi
(Fig. 5B and C). However, (ꢀ)-maackiain did not inhibit
PKCd kinase activity (Fig. 5D). These data suggest that
inhibition of PKCd activation and not the inhibition of
2015 | Vol. 3 | Iss. 5 | e00166
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British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

H. Mizuguchi et al.
Maackiain Suppresses H1R and IL-4 Gene Transcriptions
PKCd enzymatic activity is the mechanism of (ꢀ)-ma-
H1R gene expression is highly correlated with the
severity of allergic symptoms, and compounds that sup-
press H1R gene expression alleviate allergic symptoms
(Das et al. 2007; Mizuguchi et al. 2011; Kitamura et al.
2012; Hattori et al. 2013). TH2 cytokines are also sug-
gested to play important roles in the pathogenesis of
allergic inflammation (Holgate 1993). We have demon-
strated the cross talk between H1R signaling and TH2
cytokine signaling in patients with pollinosis (Mizuguchi
et al. 2008; Shahriar et al. 2009) and have shown that
suppression of H1R signaling could inhibit TH2 cytokine
signaling. Therefore, we consider suppression of H1R sig-
naling to be crucial for the treatment of allergic diseases.
Recently, we have reported that PKCd/ERK/PARP-1 path-
way is involved in the PMA-induced upregulation of H1R
expression level in HeLa cells (Mizuguchi et al. 2011).
And the translocation of PKCd to the Golgi and phos-
phorylation of Tyr311 on PKCd are crucial steps in this
signaling pathway. Pretreatment with (ꢀ)-maackiain
inhibited PMA-induced phosphorylation of Tyr311 on
PKCd, and its translocation to the Golgi (Fig. 5B and C).
However, (ꢀ)-maackiain did not inhibit PKCd kinase
activity (Fig. 5D). These data suggest that the inhibition
of PKCd activation is the mechanism of (ꢀ)-maackiain
action for suppression of H1R gene expression. We iden-
tified additional antiallergic compounds, including epigal-
locatechin-3-O-gallate (EGCG) and quercetin that
suppress the upregulation of H1R gene expression in
HeLa cells and TDI-sensitized rats (Matsushita et al.
2008; Hattori et al. 2013). These compounds also inhibit
the phosphorylation of Tyr311 on PKCd (Mizuguchi et al.
2012b; Hattori et al. 2013). These findings suggest that
inhibition of PKCd activation could be a novel therapeu-
tic strategy for allergic diseases. So far, we have no direct
evidence that activation of PKCd upregulates H1R gene
expression in allergic nasal mucosa and suppression of its
activation is target to maackiain treatment. However, as
described above, pretreatment with compounds that inhi-
bit PKCd activation suppressed nasal symptoms. These
findings support the possibility that the activation of
PKCd in the nasal mucosa is also involved in the nasal
symptoms in allergy model rats. Recently, it is demon-
strated that H1R immunoreactivity was observed in epi-
thelial cells and vascular endothelial cells in human nasal
mucosa (Shirasaki et al. 2012). H1R mRNA was increased
in human nasal epithelial cells after stimulation with die-
sel exhaust particles that are known to cause chronic air-
way diseases, and histamine stimulation induced
production of granulocyte macrophage-colony-stimulating
factor (GM- CSF) and IL-8 in human nasal epithelial cells
(Terada et al. 1999). In addition, involvement of H1R-
PKC-ERK signaling pathway in eliciting GM- CSF and
IL-8 production from bronchial epithelial cells stimulated
ackiain action.
Discussion
In the present study, we showed that (ꢀ)-maackiain is
the prominent component responsible for the antiallergic
activity of Kujin. Maackiain is a pterocarpan that is
widely distributed in leguminous plants (Shibata and
Nishikawa 1963). Bioactivities of maackiain, including
anticancer and antimicrobial effects, have been reported
(Honda and Tabata 1982; Aratanechemuge et al. 2004).
(ꢀ)-Maackiain also inhibits aryl hydrocarbon hydroxy-
lase (Gelboin et al. 1981). However, the antiallergic activ-
ity of maackiain had not yet been reported. We
demonstrated that pretreatment with synthetic racemic
maackiain for 3 weeks significantly suppressed TDI-
induced nasal symptoms and upregulation of H1R and
IL-4 gene expression in TDI-sensitized rats (Fig. 4C). To
our knowledge, this is the first report to show the antial-
lergic activity of maackiain. As shown in Figure 4C,
5 mg/kg of racemic maackiain treatment showed nearly
maximal alleviation of sneezing and nasal scores
although suppression of H1R and IL-4 gene expressions
were suppressed moderately. We previously reported that
treatment with histamine up-regulated IL-4 and IL-5
gene expression and IL-4 treatment stimulated H1R gene
expression in normal (i.e., non-TDI-sensitized) rats
(Shahriar et al. 2009). We also reported that the expres-
sion of H1R gene is strongly correlated with those of IL-
5 (Kitamura et al. 2012), IL-9, IL-13, and CD40L (H.
Mizuguchi, H. Ogishi, and H. Fukui, unpubl. data).
These data suggest the existence of the cross talk between
the H1R signaling and IL-4 signaling. This is perhaps the
reason why strong suppression of nasal symptoms was
observed even if suppression of mRNA expression in
each protein is moderate.
Pharmacological activity of many flavonoids has been
explained by their antioxidant activities. However, (ꢀ)
-maackiain did not show any DPPH scavenging activity
per se (Fig. 5A). Furthermore, studies using synthetic
(ꢀ)-maackiain and (+)-maackiain also revealed that ma-
ackiain showed stereoselectivity for IL-4 gene suppression
but not for H1R gene expression (Fig. 4B), suggesting the
existence of distinct target proteins for these transcrip-
tional signaling. We did not obtained clear dose-depen-
dency of maackain, especially experiments with HeLa
cells. Over 30 lmol/L of (ꢀ)-maackiain showed cell tox-
icity in HeLa cells. On the other hand, (ꢀ)-maackiain
did not show any cell toxicity up to 150 lmol/L in RBL-
2H3 cells. This cell-dependent toxicity failed to use suit-
able concentration of (ꢀ)-maackiain to obtain the clear
dose-dependency.
ª 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2015 | Vol. 3 | Iss. 5 | e00166
Page 11

Maackiain Suppresses H1R and IL-4 Gene Transcriptions
H. Mizuguchi et al.
trifolirhizin (maackiain glycoside) isolated from sanzukon
by histamine was reported (Matsubara et al. 2006).
Involvement of PKCd was also reported by Masaki et al.
(2011). These findings suggest that epithelial cells are one
of the candidates responsible for histamine-induced
upregulation of H1R gene expression. As HeLa cells are
the cell line derived from cervical cancer cells arising in
epithelial cells, we believe that HeLa cells can be the
model to analyze molecular mechanism of histamine-
induced upregulation of H1R gene expression in the nasal
mucosa although HeLa cells are not representative for
typical target cells involved in allergic reactions. Further-
more, very recently, we found that Heat shock protein 90
(Hsp90) is one of the target proteins for (ꢀ)-maackiain
and disruption of the interaction between PKCd and
Hsp90 is the mechanism of suppression of PKCd activa-
tion by (ꢀ)-maackiain (Y. Nariai, H. Mizuguchi, T. Oga-
sawara, H. Nagai, Y. Sasaki, Y. Okamoto, Y. Yoshimura,
Y. Kitamura, H. Nemoto, N. Takeda, and H. Fukui, un-
publ. data). This finding combined with the data that
(ꢀ)-maaackiain does not inhibit PKCd enzymatic activity
suggests that enzymatic activity of PKCd is not directly
related to H1R gene upregulation.
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Das AK, Yoshimura S, Mishima R, Fujimoto K, Mizuguchi
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Dev S, Mizuguchi H, Das AK, Maeyama K, Horinaga S, Kato
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In summary, we have shown that (ꢀ)-maackiain is a
novel antiallergic compound that alleviates nasal symptoms
in TDI-sensitized allergy model rats through the inhibition
of H1R and IL-4 gene expression. The molecular mecha-
nism underlying its suppressive effect for the upregulation
of H1R gene expression is the inhibition of PKCd activa-
tion. Our data also suggest that PKCd is a novel target pro-
tein for allergic diseases and compounds that suppress
PKCd activation may improve allergic symptoms or pre-
vent development of allergic diseases, contributing to their
clinical application for allergic diseases.
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Author Contributions
Hoang BX, Shaw DG, Levine S, Hoang C, Pham P (2007).
New approach in asthma treatment using excitatory
modulator. Phytother Res 21: 554–557.
Mizuguchi designed the project and wrote the manu-
script. Nariai, Kato, Nakano, and Kanayama performed
the experimental work. Nariai, Kashiwada, Nemoto,
Kawazoe, and Kitamura analyzed the data and partici-
pated in the data interpretation. Takaishi and Takeda
supervised the research and wrote the manuscript. Fukui
conceived the project, supervised the research, and wrote
the manuscript.
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Disclosure
Kim DW, Chi YS, Son KH, Chang HW, Kim JS, Kang SS,
et al. (2002). Effects of sophoraflavanone G, a prenylated
flavonoid from Sophora flavescens, on cyclooxygenase-2 and
in vivo inflammatory response. Arch Pharm Res 25: 329–335.
The authors declare no financial conflicts of interest.
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