Identification of Dimethylmatairesinol as an Immunoglobulin E-suppressing Component of the Leaves of Cinnamomum camphora
ABSTRACT Immunoglobulin E (IgE) plays an important role in allergic diseases. In this study, we found that a methanol extract of leaves of the camphor tree Cinnamomum camphora (C. camphora) reduced the amount of IgE secreted by human myeloma U266 cells. When the methanol extract was fractionated by extraction with organic solvents, the ethyl acetate fraction showed the highest activity. The fraction was further separated into several subfractions by preparative TLC. We identified the component of one of the active subfractions as dimethylmatairesinol. Authentic dimethylmatairesinol exhibited similar ac-tivity. Thus, the extract of C. camphora and its com-ponents including dimethylmatairesinol have poten-tial as an anti-allergic agent.
Journal of Health Science, 57(2) 184–187 (2011)
Dimethylmatairesinol as an
Component of the Leaves of
Hiroki Tanabe,aRyuuta Fukutomi,b
Kensuke Yasui,cAkihiro Kaneko,c
Shinjiro Imai,bTsutomu Nakayama,a
and Mamoru Isemura∗, a
aGraduate School of Nutritional and Environmental Sciences,
University of Shizuoka, Shizuoka 422–8526, Japan,bNisshin
Seifun Group Inc., Fujimino 356–8511, Japan andcHealth
Care Research Center, Nisshin Pharma Inc., Fujimino 356–
(Received November 4, 2010; Accepted December 20, 2010;
Published online December 27, 2010)
Immunoglobulin E (IgE) plays an important role
in allergic diseases.In this study, we found that
a methanol extract of leaves of the camphor tree
Cinnamomum camphora (C. camphora) reduced the
amount of IgE secreted by human myeloma U266
cells. When the methanol extract was fractionated
by extraction with organic solvents, the ethyl acetate
fraction showed the highest activity.
was further separated into several subfractions by
preparative TLC. We identified the component of one
of the active subfractions as dimethylmatairesinol.
Authentic dimethylmatairesinol exhibited similar ac-
tivity. Thus, the extract of C. camphora and its com-
ponents including dimethylmatairesinol have poten-
tial as an anti-allergic agent.
The recent epidemic of allergies has heightened
interest in immunoglobulin E (IgE) which plays an
∗To whom correspondence should be addressed: Graduate
School of Nutritional and Environmental Sciences, University
of Shizuoka, Shizuoka 422–8526, Japan. Tel. & Fax: +81-54-
264-5824; E-mail: email@example.com
important role in the allergic response. It is impor-
tant to search for a new compound which reduces
excess production or secretion of IgE for patients
with allergic diseases.
The human myeloma cell line U266 produces
IgE and secretes it into the culture medium spon-
taneously.1–6)We have used these cells to evalu-
ate suppressive effect of IgE production or secre-
tion and examined methanol extracts from more
than 200 species of plant. As a result, we found
that a methanol extract from leaves of the camphor
tree Cinnamomum camphora (C. camphora Lau-
raceae) decreased the amount of IgE in the culture
medium of U266 cells dose-dependently. Although
C. camphora has long been prescribed in traditional
medicine for the treatment of inflammation-related
diseases,7)no report has described that an extract
from C. camphora suppresses IgE production or se-
cretion by U266 cells. In the present study, we iden-
tifieddimethylmatairesinol asone oftheactivecom-
ponents in the C. camphora extract which has a sup-
pressive effect on IgE production/secretion in U266
MATERIALS AND METHODS
Cells and Chemicals—— U266 cells were ob-
tained from Dainippon-Sumitomo Pharmaceuti-
cal Co. Ltd. (Osaka, Japan) and maintained in
RPMI1640 medium supplemented with 10% fetal
bovine serum, 50U/ml penicillin, 50µg/ml strep-
tomycin, and 2.5µg/ml amphotericin B at 37◦C
under a 5% CO2 atmosphere.
(MESACUP IgE Test) was purchased from Medi-
cal and Biological Laboratories Co. Ltd. (Nagoya,
Japan).Dimethylmatairesinol and matairesinol
were obtained from APIN Chemicals Ltd. (Oxon,
U.K.)and Cayman ChemicalCompany (Ann Arbor,
MI, U.S.A.), respectively.
Plant Material and Extraction—— Leaves of C.
camphora were collected in Shizuoka, Japan, in
August 2009. The washed leaves were dried at
60◦C for 24hr. Crushed dry leaves (20g) were ex-
tracted at 25◦C with absolute methanol (100ml) for
1 week with occasional shaking. The methanol ex-
tract thus obtained was filtered by filter paper and
the filtrate was dried under reduced pressure to yield
303mg. The extract (273mg) was then extracted
with n-hexane, ethyl acetate, n-butanol, and water
to yield 53.6, 46.8, 50.9, and 70.1mg of dry mate-
An IgE assay kit
C ?2011 The Pharmaceutical Society of Japan
rial, respectively. These materials were dissolved in
dimethyl sulfoxide for use.
TLC—— Samples dissolved in methanol were
spotted on TLC Plate Silica Gel 60 F254 (Merck
Ltd., Tokyo, Japan) and developed in chloroform-
ethyl acetate (95:5) or (9:1). Spots were visual-
ized by immersing in 5% ethanolic phosphomolyb-
dic acid,8)followed by heating. Preparative TLC
was performed similarly using a solvent mixture of
Enzyme-linked Immunosorbent Assay (ELISA)
of IgE—— U266 cells were plated in flat-bottomed
96-well plates at 3.5×104cells per well and treated
with test samples. After incubation for 4hr, the
supernatants were collected by centrifugation at
1400× g for 10min and IgE concentrations were
determined using the MESACUP IgE Test accord-
ing to the manufacturer’s instructions.
Cytotoxicity—— Cell viability was determined by
the Trypan blue dye exclusion test as reported pre-
Time-of-flight Mass Spectrometry (TOF-MS)
and NMR Analysis—— High-resolution TOF-
MS was performed using a Shimadzu sys-
tem (MALDI-TOFMS-AXIMA, Shimadzu, Kyoto,
Japan).1H NMR (400MHz),13C NMR (100MHz),
HH-correlation spectroscopy (COSY), heteronu-
clear single-quantum coherence, and heteronu-
clear multiple-bond correlation spectra (HMBC)
were recorded in CDCl3at room temperature with
a JEOL JNM-A400 system (JEOL Ltd., Tokyo,
Japan). Samples (3mg) were dissolved in 0.5ml of
chloroform-d (ISOTEC Ltd., Tokyo, Japan). Chem-
ical shifts were expressed in parts per million rela-
tive to tetramethylsilane as an internal standard.
Statistical Analysis—— Data were analyzed by
one-way analysis of variance (ANOVA), and post
hoc Tukey-Kramer tests. Differences were consid-
ered significant at p < 0.05. Data are expressed as
means±S.E. The statistical calculations were car-
ried out using Stat View 5.0 computer software
(SAS Institute, Tokyo, Japan).
RESULTS AND DISCUSSION
When U266 cells, human IgE-bearing B cells,
were incubated with the methanol extract from C.
camphora at a final concentration of 10, 30, and
120µg/ml, the results of ELISA showed that the
amount of IgE in the culture medium decreased
dose-dependently as compared with the control
Fig. 1. Effect of the Methanol Extract from C. camphora on
the IgE Concentration in the Culture Medium of U266
The IgE concentrations in the presence of the methanol extract at
different concentrations are expressed as the mean±S.E. from 3 deter-
minations. The data with different alphabetic letters are different with
statistical significance from each other.
(vehicle-treated cells, Fig.1). Theresults ofthe Try-
pan blue assay indicated that cell viability was more
than 90% in each case (data not shown). These re-
sults suggested that the methanol extract contained
some compound(s) which suppressed IgE produc-
tion/secretion in U266 cells and did not cause sig-
nificant cytotoxicity at least up to a concentration of
The methanol extract from C. camphora was
then fractionated by successive extraction with or-
ganic solvents and the effects of the different frac-
tions on U266 cells were examined by ELISA. The
amount of IgE in the culture medium of the cells
treated with the hexane, ethyl acetate, butanol, and
water fractions at 120µg/ml for 4hr was 50.5±2.2,
25.3±2.1, 71.5±3.2 and 77.7±1.2%, respectively,
of the control value (100±7.4%).
TLC of the ethyl acetate fraction, which exhib-
ited the strongest inhibitory activity, indicated that
several compounds were present (Fig.2A). We sep-
arated this fraction into 9 fractions by preparative
TLC (Fig.2B) and examined their effects on U266
cells (data not shown). We found that fraction 6 ex-
hibited the IgE-reducing activity and one of three
subfractions (6b, Fig.2B) contained the active sub-
stance (Fig.2C) which was named as compound 1.
The yield of compound 1 was about 1 mg from
100mg of the methanol extract from C. camphora.
Compound 1 was identified as dimethyl-
matairesinol (Fig.3) based on1H NMR spectrum,
Vol. 57 (2011)
Fig. 2. Fractionation of the Ethyl Acetate Fraction by TLC and
the Activity of the Subfractions
The ethyl acetate fraction (EA) was separated by preparative TLC
using a developing solvent of chloroform-ethyl acetate (95:5) to give
fractions 1–9 (A). The fraction 6 was further separated by preparative
TLCusing developing solventofchloroform-ethyl acetate (9:1)togive
6a, 6b, and 6c (B) and the culture medium IgE concentrations after
incubation for 4hr in the presence of these subfractions at 60µg/ml are
expressed relative to that for the control cells (370 IU/ml, 100%, C).
13C NMR spectrum, and TOF-MS.
(CDCl3, 400MHz) δ 2.50 (dd, J = 13.4, 8.0Hz,
H-7b), 2.51 (ddddd, J = 9.1, 8.2, 8.0, 7.3, 5.8Hz,
H-8), 2.60 (ddd, J = 9.1, 6.3, 5.6Hz, H-8?), 2.62
(dd, J = 13.4, 5.8Hz, H-7a), 2.91 (dd, J = 14.0,
5.6Hz, H-7?b), 2.95 (dd, J = 14.0, 6.3Hz, H-7?a),
3.83 (s, H-12), 3.84 (s, H-11), 3.86 (s, H-13), 3.87(s,
H-10), 3.89 (dd, J = 8.8, 8.2Hz, H-9b), 4.12 (dd,
J = 8.8, 7.3Hz, H-9a), 6.49 (d, J = 2.1Hz, H-2),
6.55 (dd, J = 8.1, 2.1Hz, H-6), 6.66 (dd, J = 8.3,
2.0Hz, H-6?), 6.69 (d, J = 2.0Hz, H-2?), 6.76 (d,
J = 8.3Hz, H-5?), 6.78 (d, J = 8.1Hz, H-5).
NMR (CDCl3, 100MHz) δ 34.49 (C-7?), 38.19 (C-
7), 41.07 (C-8), 46.57 (C-8?), 55.82 (C-12), 55.86
(C-11), 55.86 (C-13), 55.90 (C-10), 71.23 (C-9),
111.06 (C-5), 111.30 (C-5?), 111.81 (C-2), 112.33
(C-2?), 120.55 (C-6), 121.34 (C-6?), 130.18 (C-1?),
130.42 (C-1), 147.86 (C-3?), 147.94 (C-3), 149.02
Fig. 3. Chemical Structure of Compound 1 Identified as
A, COSY and HMBC correlations. B, Deduced chemical struc-
(C-4?), 149.04 (C-4), and 178.69 (C-9?). TOF-MS
(ESI+) m/z 387.1807 [39%, M+H]+[Calculated for
C22H27O6(M+H)+387.1808] and 405.1879 [88%,
M+H2O+H]+[Calculated for C22H29O7 (M+H)+
We then examined a commercially available
dimethylmatairesinol preparation for IgE-reducing
activity. The results showed that the IgE concentra-
tion in the culture medium of U266 cells decreased
dose-dependently (Fig.4). Cell viability was more
than 90% in each case. The concentrations higher
than 100µg/ml resulted in cytotoxicity, preventing
evaluations of IgE production. It should be noted
that authentic matairesinol had no IgE-suppressing
activity up to 100µg/ml.
Thus, we found that the methanol extract of
C. camphora showed activity to reduce the con-
centration of IgE in the culture medium of U266
cells. One of the active principles was identified as
dimethylmatairesinol. Many natural extracts have
been shown to suppress IgE production by U266
cells,2–6)but these studies failed to identify the ac-
tive principle. For example, green tea extract has
been shown to suppress IgE production in U266
cells, but the structure of the active component(s)
remains to be determined.6)Therefore, dimethyl-
Fig. 4. Effect of Authentic Dimethylmatairesinol on the IgE
Concentration in the Culture Medium of U266 Cells
The culture medium IgE concentrations in dimethylmatairesinol
at different concentrations are expressed as the mean±S.E. from 3 de-
terminations. The data with different alphabetic letters are different
with statistical significance from each other.
matairesinol represents one of the few natural prod-
ucts identified as having such activity.
The methanol extract of C. camphora seemed
to contain other active compounds, since the whole
activity of the methanol extract cannot be accounted
for by dimethylmatairesinol alone, judging from its
content and since the activity was also detected in
fractions 4, 5, and 7–9 in Fig.2 (data not shown).
Thus, the extract of C. camphora and its compo-
nents including dimethylmatairesinol have potential
as an anti-allergic agent. More studies are needed
to know if these fractions exhibit anti-allergic activ-
ities in vivo.
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