Bioactive constituents of Chinese natural medicines. VII.1 Inhibitors of degranulation in RBL-2H3 cells and absolute stereostructures of three new diarylheptanoid glycosides from the bark of Myrica rubra

Article abstract of DOI:10.1248/cpb.50.208

Three new diarylheptanoid glycosides, named (+)-S-myricanol 5-O-β-D-glucopyranoside, myricanene A 5-O-α-L- arabinofuranosyl(1→6)-β-D-glucopyranoside, and myricanene B 5-O-α-L-arabinofuranosyl(1→6)-β-D-glucopyranoside, were isolated from the bark of Chines

Full text of DOI:10.1248/cpb.50.208

208
Chem. Pharm. Bull. 50(2) 208—215 (2002)
Vol. 50, No. 2
Bioactive Constituents of Chinese Natural Medicines. VII.1) Inhibitors of
Degranulation in RBL-2H3 Cells and Absolute Stereostructures of Three
New Diarylheptanoid Glycosides from the Bark of Myrica rubra
Hisashi MATSUDA, Toshio MORIKAWA, Jing TAO, Kazuho UEDA, and Masayuki YOSHIKAWA*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received August 20, 2001; accepted October 23, 2001
Three new diarylheptanoid glycosides, named (؉)-S-myricanol 5-O-b-D-glucopyranoside, myricanene A 5-
O-a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside, and myricanene B 5-O-a-L-arabinofuranosyl(1→6)-b-D-
glucopyranoside, were isolated from the bark of Chinese Myrica rubra, together with twenty known compounds.
The absolute stereostructures of the new diarylheptanoid glycosides were elucidated on the basis of chemical and
physicochemical evidence, including the application of the modified Mosher’s method. The inhibitory effects of
isolated constituents on the release of b-hexosaminidase from RBL-2H3 cells were examined, and several diaryl-
heptanoids, myricanol, (؉)-S-myricanol, myricanone, and myricanenes A and B, and a flavonol, myricetin, were
found to show the inhibitory activity.
Key words Myrica rubra; degranulation inhibitor; RBL-2H3 cell; (ϩ)-S-myricanol 5-O-b-D-glucopyranoside; myricanene A
5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside; myricanene B 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside
The Myricaceae plant Myrica rubra SIEB. et ZUCC. is 50% aqueous ethanolic extract and some constituents inhib-
widely distributed in China, Taiwan, Japan and Korea. The ited melanin biosynthesis10) and showed anti-androgenic ac-
bark of M. rubra (Myricae cortex) has been used locally as tivity.11)
an astringent, antidote, and antidiarrheic in Japanese folk
In the course of characterization studies on the bioactive
medicine, and has also been used externally for burns and constituents of Chinese natural medicines,1,12) we have iso-
skin diseases in Chinese traditional medicine. Previously, lated three new diarylheptanoid glycosides, termed (ϩ)-S-
several flavonoids,2) tannins,2) triterpenes,3) and diarylhep- myricanol 5-O-b-D-glucopyranoside (1), myricanene A 5-O-
tanoids4—8) were isolated from the bark of M. rubra. Pharma- a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside (2), and
cological studies of this natural medicine have reported that myricanene B 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopy-
its methanolic extract showed protective effects on CCl4Ϫ and ranoside (3), together with twenty known compounds from
a-naphthylisothiocyanate-induced liver injury,9) whereas the the bark of M. rubra. This paper deals with the absolute
Chart 1
To whom correspondence should be addressed. e-mail: shoyaku@mb.kyoto-phu.ac.jp
© 2002 Pharmaceutical Society of Japan

February 2002
209
Chart 2
with twenty known compounds, myricanol (4,4,5) 0.73%),
myricanol glucoside (5,4) 0.20%), myricanone (6,4,5) 0.036%),
ursolic acid (7,13) 0.027%), acetylursolic acid (8,13) 0.0021%),
ursonic acid (9,13) 0.0067%), rhoiptelenol (10,14) 0.0036%),
taraxerol (11,3) 0.014%), myricadiol (12,3) 0.020%), maslinic
acid (13,6,15) ϭcrategolic acid, 0.00083%), alphitolic acid
(14,6) 0.0014%), myricitrin (15,2,10) 5.59%), myricetin (16,2,10)
0.027%), quercitrin (17,16) 0.0028%), 1817) (0.0059%), (Ϫ)-
epigallocatechin (19,18) 0.0066%), (Ϫ)-epigallocatechin 3-O-
gallate (20,18) 0.043%), 3,5-dimethoxy-4-hydroxyphenol 1-O-
b-D-(6Ј-O-galloyl)glucopyranoside (21,19) 0.012%), b-sitos-
terol20) (0.0036%), and gallic acid20) (0.026%).
stereostructure elucidation of three new diarylheptanoid gly-
cosides (1—3). In addition, we describe the inhibitory effect
of components isolated from the bark of M. rubra on an im-
mediate allergic reaction by monitoring the release of b-hex-
osaminidase from rat basophilic leukemia (RBL-2H3) cells.
Isolation of Chemical Constituents from the Dried
Bark of M. rubra The methanolic extract of the bark of M.
rubra (cultivated in Guang Dong province, China) was sus-
pended in MeOH and filtered by Kiriyama funnel to give a
soluble fraction and insoluble residue, which was recrystal-
lized with aqueous MeOH to furnish myricitrin (15, 4.6%
from natural medicine). The soluble fraction was partitioned
in an n-BuOH–H2O (1 : 1) mixture to give an n-BuOH-solu-
ble fraction and H2O-soluble fraction. The n-BuOH-soluble
fraction was subjected to ordinary- and reversed-phase silica
gel column chromatography, and finally HPLC, to furnish
(ϩ)-S-myricanol 5-O-b-D-glucopyranoside (1, 0.074%), myri-
canene A 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyra-
noside (2, 0.0032%), and myricanene B 5-O-a-L-arabinofu-
ranosyl(1→6)-b-D-glucopyranoside (3, 0.0029%), together
b
Absolute Stereostructure of (؉)-S-Myricanol 5-O- -D-
Glucopyranoside (1) (ϩ)-S-Myricanol 5-O-b-D-glucopy-
ranoside (1) was isolated as a white powder with positive op-
tical rotation ([a]D22 ϩ81.3°, EtOH). The positive- and nega-
tive-ion fast atom bombardment (FAB)-MS of 1 showed qua-
simolecular ion peaks at m/z 543 (MϩNa)ϩ and 519 (MϪ
H)Ϫ respectively, in addition to a fragment ion peak at m/z
357 (MϪC6H11O5)Ϫ. The molecular formula C27H36O10 of 1

210
Vol. 50, No. 2
Fig. 1. Absolute Stereostructure of (ϩ)-S-Myricanol 5-O-b-D-Glucopyranoside (1)
Table 1. 13C-NMR Data for (ϩ)-S-Myricanol 5-O-b-D-Glucopyranoside
(1), Myricanene A 5-O-a-L-Arabinofuranosyl(1→6)-b-D-glucopyranoside
(2), and Myricanene B 5-O-a-L-Arabinofuranosyl(1→6)-b-D-glucopyra-
noside (3) and Their Aglycons (1a, 2a, 3a)
was determined from quasimolecular ion peaks and by high-
resolution MS measurement. In the UV spectrum of 1, ab-
sorption maxima were observed at 216 (log e 4.5), 256 (4.2),
and 299 (3.7) nm, which were suggestive of a biphenyl type
diarylheptanoid structure.8) The IR spectrum of 1 showed ab-
sorption bands at 3568, 1598, 1560, 1508, and 1073 cmϪ1,
ascribable to hydroxyl, benzene ring, and ether functions. On
acid hydrolysis with 5% aqueous sulfuric acid (H2SO4)–1,4-
dioxane (1 : 1, v/v), 1 liberated D-glucose, which was identi-
fied by GLC analysis of the trimethylsilyl thiazolidine deriva-
1a)
2a)
3a)
1ab)
2ab)
3ab)
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
Glc-1
2
126.4
129.6
149.0
146.3
149.6
130.5
27.1
26.6
23.4
40.5
67.7
126.2
129.0
148.6
145.7
149.5
132.1
30.8
25.2
31.4
134.5
132.5
35.6
126.1
129.3
148.6
146.3
150.3
131.6
27.1
25.4
30.1
32.6
135.9
130.2
34.6
132.1
129.4
116.4
153.0
138.4
130.0
61.3
123.1
122.7
147.8
138.7
145.9
124.7
34.8
23.0
25.5
25.8
68.7
124.7
123.9
145.5
138.3
146.9
124.6
29.3
25.0
31.4
134.2
132.8
35.2
123.8
123.3
145.5
138.5
147.5
124.3
25.6
24.7
29.9
32.3
135.3
129.9
34.3
132.0
128.7
116.0
151.6
136.4
129.4
61.5
1
tive.21) The H-NMR (C5D5N) and 13C-NMR (Table 1) spec-
tra of 1 showed signals assignable to two methoxyl groups [d
3.96, 4.10 (both s, 20, 21-H3)], a methine bearing a hydroxyl
group [d 4.34 (m, 11-H)], a D-glucopyranosyl moiety [d 5.75
(d, Jϭ7.0 Hz, 1Ј-H)], and four aromatic protons [d 7.12 (s,
19-H), 7.18 (d, Jϭ8.0 Hz, 16-H), 7.19 (dd, Jϭ2.0, 8.0 Hz,
15-H), 7.44 (br s, 18-H)], together with six methylenes (7, 8,
9, 10, 12, 13-H2), which were very similar to those of myri-
canol glucoside (5).
35.7
27.8
27.0
39.5
33.6
33.5
131.0
130.5
117.1
153.1
135.2
130.2
61.2
61.7
105.7
75.7
132.7
128.9
116.1
153.2
137.0
130.2
61.2
61.5
105.6
75.5
130.7
129.9
116.9
151.5
133.1
129.4
61.4
133.2
128.7
116.0
151.5
135.5
130.0
61.7
The planar structure of 1, including the positions of two
methoxyl groups and a glycosidic linkage, which was the
same as that 5, was constructed on the basis of 1H–1H corre-
lation spectroscopy (H–H COSY) and heteronuclear multiple
bond correlation (HMBC) experiments. And the H–H COSY
experiment on 1 indicated the presence of two partial struc-
tures written in the bold line, whereas, in the HMBC experi-
ment, long-range correlations were observed between the
protons and carbons, as shown in Fig. 1. Enzymatic hydroly-
sis of 1 with b-glucosidase liberated (ϩ)-S-myricanol (1a).
61.8
105.8
75.7
61.4
61.5
61.4
3
78.3
78.2
78.4
4
71.5
71.8
72.0
5
78.4
76.8
76.9
6
62.5
68.1
68.3
Ara(f)-1
2
109.8
83.1
110.1
83.2
1
The proton and carbon signals in the H-NMR (CDCl3) and
13C-NMR (Table 1) spectra of 1a were superimporsable on
those of myricanol (4), while the optical rotation of 1a ([a]D22
ϩ37.3°, CHCl3) was the opposite to that of 4 ([a]D24 Ϫ48.3°,
CHCl3) [lit.4) ([a]D30 Ϫ62.9°, CHCl3)]. Finally, pyridinium
chlorochromate (PCC) oxidation of 1a furnished myricanone
(6). The above-mentioned evidence suggests that 1a is an
enantiomer of 4.
3
78.7
78.7
4
85.7
86.0
5
62.5
62.6
a) Measured in pyridine-d5; b) CDCl3 at 125 MHz. Glc: b-D-glucopyranosyl; Ara(f):
a-L-arabinofuranosyl.
as shown in Fig. 1. Thus, diazomethane methylation of 1a
In order to confirm the absolute stereostructure of 1a, we furnished the dimethylated-derivative (1b), which was sub-
carried out an application of the modified Mosher’s method,22) jected to the modified Mosher’s method for the 11-mono-(R)-

February 2002
211
and (S)-2-methoxy-2-trifluorophenylacetate (MTPA esters, 19-H), 7.41 (br s, 18-H)], together with five methylenes (7, 8,
1c, d). As shown in Fig. 1, the signals due to protons attached 9, 12, 13-H2). The proton and carbon signals in the 1H-NMR
to 12, 13, 15, 16, and 18-carbons in the 11-mono-(S)-MTPA (C5D5N) and 13C-NMR (Table 1) spectra of 3 were found to
ester (1d) were observed at lower field fields compared with be similar to those of 2, except for signals due to an olefin
those of the 11-mono-(R)-MTPA ester (1c) [Dd: positive], function. New aglycons, myricanenes A (2a) and B (3a), were
while signals due to protons of 7, 8, 9, 10-carbons in 1d were obtained by enzymatic hydrolysis of 2 and 3 with naring-
observed at higher fields compared with those of 1c [Dd: inase, respectively. On the other hand, dehydration of myri-
negative]. Consequently, the absolute stereostructure at the canol (4) with thionyl chloride (SOCl2) yielded 2a and 3a, so
11-positions of 1 and 1a have been determined to be S. Pre- that the structures of 2a and 3a were determined to be as
viously, a (Ϯ)-myricanol mixture was isolated from the bark shown in Fig. 2. The structure of the disaccharide moiety and
of Myrica cerifera, and its structure was established by crys- the position of the glycosidic linkage in 2 and 3 were eluci-
tal X-ray analysis.23) This paper is the first report for the (ϩ)- dated by HMBC experiment. Namely, long-range correla-
form of myricanol.
tions were observed between the 1Ј-proton of the glucopyra-
Stereostructures of Myricanene A 5-O-a-L-Arabinofu- nosyl moiety and the 5-carbon of the aglycon moiety, and be-
→
b
ranosyl(1 6)- -D-glucopyranoside (2) and Myricanene B tween the 1Љ-proton of the arabinofuranosyl moiety and the
→
b
5-O-a-L-Arabinofuranosyl(1 6)- -D-glucopyranoside (3) 6Ј-carbon of the glucopyranosyl moiety. Consequently, the
Myricanene A 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopy- stereostructures of 2 and 3 were elucidated as shown.
ranoside (2) and myricanene B 5-O-a-L-arabinofuranosyl
Inhibitory Effect of Constituents from M. rubra on the
(1→6)-b-D-glucopyranoside (3) were isolated as white pow- Release of b-Hexosaminidase from RBL-2H3 Cells Hist-
ders with negative optical rotations (2: [a]D22 Ϫ0.6°; 3: [a]D22 amine, which is released from mast cells stimulated by an
Ϫ10.8° in EtOH). In the positive- and negative-ion FAB-MS antigen or a degranulation inducer, is usually determined as a
of 2 and 3, common quasimolecular ion peaks were observed degranulation marker in in vitro experiments on immediate
at m/z 657 (MϩNa)ϩ and m/z 633 (MϪH)Ϫ, together with allergic reactions. b-Hexosaminidase is also stored in secre-
a fragment ion peak at m/z 339 (MϪC11H19O9)Ϫ, and the tory granules of mast cells, and is also released concomi-
molecular formula C32H42O13 was determined by high-reso- tantly with histamine when mast cells are immunologically
lution MS measurement. The UV spectra (EtOH) of 2 and 3 activated.24) Therefore, it is generally accepted that b-hex-
showed absorption maxima [nm (log e): 2, 215 (4.2), 257 osaminidase is a degranulation marker of mast cells. Previ-
(3.9), 300 (3.6); 3, 214 (4.5), 253 (4.1), 297 (3.7)] suggestive ously, we reported the isolation and structural elucidation
of a biphenyl type diarylheptanoid moiety. The IR spectra of of various antiallergic constituents from natural medicines,
2 and 3 showed absorption bands due to hydroxyl, olefin, such as Hydrangea macrophylla var. thunbergii,25) Hovenia
benzene ring, and ether functions (2: 3651, 1659, 1610, dulcis,26) Rhodiola quadrifida,27) R. sacra,28) Alisma orien-
1502, 1059 cmϪ1; 3: 3657, 1651, 1593, 1504, 1059 cmϪ1). tale,29) Kochia scoparia,30) Corchorus olitorius,31) Phaseolus
On acid hydrolysis with 5% aqueous H2SO4–1,4-dioxane vulgaris,32) Zizyphus jujuba var. spinosa,33) and Benincasa
(1 : 1, v/v), 2 and 3 liberated D-glucose and L-arabinose, hispida.34) In our continuous search for antiallergic principles
which were identified by GLC analysis of their trimethylsilyl from natural sources, we examined the effects of constituents
1
thiazolizine derivatives.21) The H-NMR (C5D5N) and 13C- from the bark of M. rubra on the release of b-hexos-
NMR (Table 1) spectra of 2 showed signals assignable to two aminidase induced by dinitrophenylated bovine serum al-
methoxyl groups [d 3.97, 4.12 (both s, 20, 21-H3)], an E- bumine (DNP-BSA) from RBL-2H3 cells sensitized with
conformed olefin function [d 5.22 (dt, Jϭ15.5, 6.0 Hz, 10- anti-DNP IgE. As shown in Table 2, biphenyl type diarylhep-
H), 5.62 (dt, Jϭ15.5, 8.5 Hz, 11-H)], a L-arabinofuranosyl tanoids, (ϩ)-S-myricanol (1a, IC50ϭ28 mM), myricanenes A
moiety [d 5.58 (br s, 1Љ-H)], a D-glucopyranosyl moiety [d (2a, 98 mM) and B (3a, ca. 100 mM), myricanol (4, 63 mM),
5.69 (d, Jϭ7.3 Hz, 1Ј-H)], and four aromatic protons [d 7.13 myricanone (6, 46 mM), and a flavonoid, myricetin (16,
(dd, Jϭ2.0, 8.0 Hz, 15-H), 7.14 (d, Jϭ8.0 Hz, 16-H), 7.21 (s, 23 mM), showed strong activity. On the other hand, biphenyl
Fig. 2. Chemical Correlations of 2 and 3 with Myricanol (4)

212
Vol. 50, No. 2
Table 2. Inhibitory Effects of Constituents from the Bark of M. rubra on the Release of b-Hexosaminidase from RBL-2H3 Cells
Inhibition (%)
IC50
Sample
(mM)
0 mM
10 mM
30 mM
100 mM
(ϩ)-S-Myricanol 5-O-b-D-
glucopyranoside (1)
(ϩ)-S-Myricanol (1a)
0.0Ϯ3.7
0.0Ϯ2.0
Ϫ3.6Ϯ5.9
17.0Ϯ1.6**
(Ϫ1.1)
Ϫ5.8Ϯ4.0
54.0Ϯ1.9**
(4.5)
Ϫ13.1Ϯ3.6
96.1Ϯ0.5**
(7.4)
—
28
Myricanene A 5-O-a-L-arabinofuranosyl(1→6)-
b-D-glucopyranoside (2)
Myricanene A (2a)
0.0Ϯ3.5
0.0Ϯ3.4
0.1Ϯ3.4
16.4Ϯ5.1
(Ϫ1.5)
Ϫ0.4Ϯ3.3
29.5Ϯ4.0**
(2.5)
7.9Ϯ1.4
50.6Ϯ3.9**
(3.5)
—
98
Myricanene B 5-O-a-L-arabinofuranosyl(1→6)-
b-D-glucopyranoside (3)
Myricanene B (3a)
0.0Ϯ2.7
0.0Ϯ2.4
Ϫ0.2Ϯ4.5
13.6Ϯ2.1
(1.2)
4.4Ϯ2.8
25.2Ϯ3.5**
(4.2)
Ϫ5.2Ϯ2.8
44.1Ϯ2.9**
(5.1)
—
—
Myricanol (4)
0.0Ϯ3.0
13.4Ϯ2.7
(Ϫ1.1)
28.6Ϯ3.1**
(4.9)
73.5Ϯ1.3**
(4.1)
63
Myricanol glucoside (5)
Myricanone (6)
0.0Ϯ3.4
0.0Ϯ3.3
1.6Ϯ3.2
10.4Ϯ2.7
(0.2)
6.3Ϯ4.9
39.4Ϯ3.4**
(4.3)
6.7Ϯ5.5
85.3Ϯ0.7**
(7.3)
—
46
Ursonic acid (9)
Rhoiptelenol (10)
Taraxerol (11)
Myricadiol (12)
Myricitrin (15)
Myricetin (16)
0.0Ϯ2.5
0.0Ϯ2.6
0.0Ϯ1.7
0.0Ϯ1.4
0.0Ϯ1.9
0.0Ϯ5.6
4.4Ϯ2.4
Ϫ1.6Ϯ4.8
Ϫ3.3Ϯ3.4
Ϫ1.1Ϯ3.7
3.7Ϯ1.4
3.9Ϯ5.1
(Ϫ3.2)
13.5Ϯ2.7**
Ϫ3.5Ϯ4.0
Ϫ0.5Ϯ3.1
Ϫ3.7Ϯ1.1
12.1Ϯ1.4
74.2Ϯ0.7**
(1.2)
20.2Ϯ1.3**
Ϫ8.0Ϯ1.1
Ϫ3.0Ϯ0.9
Ϫ12.0Ϯ1.9
12.6Ϯ4.2
99.6Ϯ0.2**
(Ϫ4.8)
—
—
—
—
—
23
18
0.0Ϯ3.8
0.0Ϯ2.9
0.0Ϯ4.1
2.5Ϯ3.4
2.1Ϯ5.0
Ϫ3.7Ϯ3.9
Ϫ3.3Ϯ1.9
12.1Ϯ1.4
1.6Ϯ3.3
39.8Ϯ1.4**
12.6Ϯ4.2
0.8Ϯ1.6
—
—
—
(Ϫ)-Epigallocatechin (19)
(Ϫ)-Epigallocatechin 3-O-gallate (20)
3,5-Dimethoxy-4-hydroxy-phenol 1-O-b-D-(6Ј-O-galloyl)-
glucopyranoside (21)
Gallic acid
Curcumin
0.0Ϯ2.4
0.0Ϯ2.1
0.0Ϯ1.8
7.0Ϯ3.0
Ϫ4.5Ϯ2.2
2.2Ϯ2.0
(2.6)
6.1Ϯ3.6
Ϫ3.3Ϯ3.5
14.2Ϯ1.6**
(4.1)
Ϫ4.7Ϯ2.3
Ϫ6.5Ϯ1.3
62.6Ϯ1.0**
(6.9)
—
—
82
Each value represents the meanϮS.E.M. (nϭ4). Significantly different from the control, ** pϽ0.01. Values in parentheses indicate enzyme inhibition (%) against b-hex-
osaminidase.
type diarylheptanoid glycosides (1—3, 5), flavonoid glyco-
sides (15, 18), and catechins (19, 20) exhibited weak inhibi-
tion. In addition, the effects of test compounds on b-hex-
osaminidase activity were examined to clarify whether their
effects were due to the inhibition of enzyme activity or of de-
chromatography (HPTLC), pre-coated TLC plates with Silica gel RP-18
WF254S (Merck, 0.25 mm); detection was achieved by spraying with 1%
Ce(SO4)2–10% aqueous H2SO4 followed by heating.
Extraction and Isolation The dried bark of Myrica rubra (5.0 kg, culti-
vated in Guang Dong, China and purchased from MAE CHU Co., Ltd.,
Nara, Japan) was finely cut and extracted three times with MeOH at room
granulation. As a result, these active constituents (1a, 2a, 3a, temperature for 1 d. Evaporation of the solvent under reduced pressure pro-
vided a MeOH extract (1600 g). The MeOH extract (1400 g) was suspended
in a small amount of MeOH, and the suspension was filtered to give a solu-
ble fraction (1200 g) and an insoluble fraction, which was recrystallized with
aqueous MeOH to furnish myricitrin (15, 200 g). The soluble fraction
4, 6, 16) did not affect the enzyme activity of b-hexos-
aminidase. Previously, a diarylheptanoid, curcumin, and a
few flavonoids such as myricetin were reported to inhibit the
degranulation,35) but this is the first report of the degranula-
(722 g) was partitioned in an n-BuOH–H2O (1 : 1) mixture to give n-BuOH-
and H2O-soluble fractions (380 g and 342 g), respectively. The n-BuOH-sol-
uble fraction (50 g) was subjected to ordinary-phase silica gel column chro-
matography [1.5 kg, CHCl3–MeOH (10 : 1→5 : 1)→CHCl3–MeOH–H2O (6 :
4 : 1)→MeOH] to give seven fractions {Fr. 1 (5.5 g), Fr. 2 (1.0 g), Fr. 3 (4.8
g), Fr. 4 (1.2 g), Fr. 5 [ϭmyricitrin (15, 19.4 g)], Fr. 6 (1.4 g), Fr. 7 (16.7 g)}.
Fraction 1 (4.4 g) was subjected to reversed-phase silica gel column chro-
matography [130 g, MeOH–H2O (60 : 40)→MeOH] to furnish two fractions
[Fr. 1-1 (2.4 g), Fr. 1-2 (2.0 g)]. Fraction 1-1 (2.4 g) was purified by ordinary-
phase silica gel column chromatography [70 g, n-hexane–AcOEt (4 : 1→
3 : 1)] to give myricanol (4, 2.0 g) and myricanone (6, 100 mg). Fraction 1-2
(2.0 g) was subjected to reversed-phase silica gel column chromatography
[60 g, MeOH–H2O (70 : 30→80 : 20→90 : 10)→MeOH] to furnish seven
fractions [Fr. 1-2-1 (145 mg), Fr. 1-2-2 (93 mg), Fr. 1-2-3 (388 mg), Fr. 1-2-4
(424 mg), Fr. 1-2-5 (100 mg), Fr. 1-2-6 (234 mg), Fr. 1-2-7 (616 mg)]. Frac-
tion 1-2-3 (388 mg) was purified by HPLC [detection RI, YMC-Pack ODS-
A (250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–H2O (90 : 10)] to give ursolic
acid (7, 76 mg) and ursonic acid (9, 19 mg). Fraction 1-2-4 (424 mg) was re-
crystallized with MeOH to furnish myricadiol (12, 57 mg) and a triterpene
mixture, which was further purified by HPLC [detection RI, YMC-Pack
tion inhibitory activity of biphenyl type diarylheptanoids.
Experimental
The following instruments were used to obtain physical data: specific ro-
tations, Horiba SEPA-300 digital polarimeter (lϭ5 cm); UV spectra, Shi-
madzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrome-
ter; electron impact (EI)-MS and high-resolution MS, JEOL JMS-GCMATE
mass spectrometer; FAB-MS and high-resolution MS, JEOL JMS-SX 102A
mass spectrometer; 1H-NMR spectra, JNM-LA500 (500 MHz) spectrometer;
13C-NMR spectra, JNM-LA500 (125 MHz) spectrometer with tetramethylsi-
lane as an internal standard; HPLC detector, Shimadzu RID-6A refractive
index detector and Shimadzu SPD-10A UV-VIS detector.
The following experimental conditions were used for chromatography: or-
dinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji
Silysia Chemical, Ltd., 150—350 mesh); reversed-phase silica gel column
chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd.,
100—200 mesh); TLC, pre-coated TLC plates with Silica gel 60F254
(Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck,
0.25 mm) (reversed phase); reversed-phase high performance thin-layer

February 2002
213
ODS-A (250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–H2O (90 : 10)] to give
acetylursolic acid (8, 6 mg). Fraction 1-2-6 (334 mg) was subjected to ordi-
nary-phase silica gel column chromatography [10 g, n-hexane–AcOEt (25 :
1→5 : 1)→CHCl3] to give rhoiptelenol (10, 10 mg), taraxerol (11, 46 mg),
and b-sitosterol (10 mg). Fraction 2 (1.0 g) was subjected to reversed-phase
silica gel column chromatography [30 g, MeOH–H2O (25 : 75→40 : 60→
60 : 40→90 : 10→95 : 5)→MeOH] to give six fractions [Fr. 2-1 (141 mg), Fr.
2-2 (114 mg), Fr. 2-3 (113 mg), Fr. 2-4 (56 mg), Fr. 2-5 (150 mg), Fr. 2-6
(426 mg)]. Fraction 2-5 (150 mg) was subjected to ordinary-phase silica gel
column chromatography [5 g, n-hexane–AcOEt (2 : 1)] and finally to HPLC
[detection RI, YMC-Pack ODS-A (250ϫ20 mm i.d., YMC Co., Ltd.),
MeOH–H2O (85 : 15)] to give maslinic acid (13, ϭcrategolic acid, 3 mg) and
alphitolic acid (14, 5 mg). Fraction 3 (4.8 g) was subjected to reversed-phase
silica gel column chromatography [140 g, MeOH–H2O (50 : 50→70 : 30)→
MeOH] to give six fractions [Fr. 3-1 (371 mg), Fr. 3-2 (184 mg), Fr. 3-3
(1.4 g), Fr. 3-4 (2.5 g), Fr. 3-5 (105 mg), Fr. 3-6 (240 mg)]. Fraction 3-1
(371 mg) was purified by HPLC [detection RI, YMC-Pack ODS-A
(250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–H2O (25 : 75 and 5 : 95)] to give
(Ϫ)-epigallocatechin (19, 23 mg) and gallic acid (90 mg). Fraction 3-3
(400 mg) was purified by HPLC [detection RI, YMC-Pack ODS-A (250ϫ
20 mm i.d., YMC Co., Ltd.), MeOH–H2O (50 : 50)] to give (ϩ)-S-myricanol
5-O-b-D-glucopyranoside (1, 74 mg) and myricanol glucoside (5, 201 mg).
Fraction 3-5 (105 mg) was purified by HPLC [detection RI, YMC-Pack
ODS-A (250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–H2O (50 : 50)] to give
UV [EtOH, nm, (log e)]: 214 (4.5), 253 (4.1), 297 (3.7). IR (KBr): 3657,
1
2910, 1651, 1593, 1504 1495, 1059 cmϪ1. H-NMR (C5D5N) d: 1.65, 1.75
(1H each, both m, 9-H2), 1.91 (2H, m, 8-H2), 2.09, 2.18 (1H each, both m,
10-H2), 3.00, 3.39 (1H each, both m, 7-H2), 3.30 (2H, m, 13-H2), 3.84, 4.22
(each 3H, both s, 20 and 21-H3), 5.59 (1H, br s, 1Љ-H), 5.65 (1H, d,
Jϭ6.5 Hz, 1Ј-H), 5.69 (1H, dt, Jϭ15.5, 7.5 Hz, 12-H), 5.89 (1H, dt, Jϭ15.5,
7.5 Hz, 11-H), 7.10 (1H, s, 19-H), 7.15 (1H, d, Jϭ8.5 Hz, 16-H), 7.19
(1H, dd, Jϭ1.5, 8.5 Hz, 15-H), 7.75 (1H, br s, 18-H). 13C-NMR (C5D5N)
dC: given in Table 1. Positive-ion FAB-MS m/z: 1291 (2MϩNa)ϩ, 657
(MϩNa)ϩ. Negative-ion FAB-MS m/z: 633 (MϪH)Ϫ, 339 (MϪC11H19O9)Ϫ.
Enzymatic Hydrolysis of 1—3 A solution of 1 (21.0 mg, 0.040 mmol)
in 0.2 M acetate buffer (pH 4.4, 3.0 ml) was treated with b-glucosidase
(40 mg, from almond, Oriental Yeast Co., Ltd.), and the solution was stirred
at 38 °C for 2 d. After treatment of the reaction mixture with EtOH, the mix-
ture was evaporated to dryness under reduced pressure and the residue was
purified by ordinary-phase silica gel column chromatography [1.0 g, CHCl3–
MeOH–H2O (7 : 3 : 1, lower layer)] to give (ϩ)-S-myricanol (1a, 14.4 mg,
99%).
Through a similar procedure, a solution of 2 or 3 (3.0 mg each, 0.005
mmol) in 0.2 M acetate buffer (pH 4.0, 2.0 ml) was treated with naringinase
(10 mg, from Penicillium decumbens, Sigma Co., Ltd.), and the solution was
stirred at 40 °C for 4 d, respectively. After treatment of the reaction mixture
with EtOH, the mixture was evaporated to dryness under reduced pressure,
and the residue was purified by ordinary-phase silica gel column chromatog-
raphy [500 mg, n-hexane–AcOEt (3 : 1)] to give myricanenes A (2a, 1.0 mg,
62%) or B (3a, 1.0 mg, 62%).
myricanene
A 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside (2,
11 mg) and myricanene B 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyra-
noside (3, 10 mg). Fraction 4 (1.2 g) was subjected to reversed-phase silica
gel column chromatography [36 g, MeOH–H2O (20 : 80→50 : 50→70 : 30)→
MeOH] to furnish five fractions [Fr. 4-1 (65 mg), Fr. 4-2 (507 mg), Fr. 4-3
(100 mg), Fr. 4-4 (253 mg), Fr. 4-5 (275 mg)]. Fraction 4-2 (114 mg) was pu-
rified by HPLC [detection RI, YMC-Pack ODS-A (250ϫ20 mm i.d., YMC
Co., Ltd.), MeOH–H2O (35 : 65)] to give (Ϫ)-epigallocatechin 3-O-gallate
(20, 30 mg) and 3,5-dimethoxy-4-hydroxyphenol 1-O-b-D-(6Ј-O-galloyl)glu-
copyranoside (21, 9 mg). Fraction 4-3 (100 mg) was purified by HPLC [de-
tection RI, YMC-Pack ODS-A (250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–
H2O (30 : 70)] to give (Ϫ)-epigallocatechin 3-O-gallate (20, 15 mg). Fraction
4-4 (253 mg) was purified by HPLC [detection RI, YMC-Pack ODS-A
(250ϫ20 mm i.d., YMC Co., Ltd.), CH3CN–H2O (35 : 65)] to give quercitrin
(17, 9 mg) and 18 (20 mg). Fraction 6 (1.4 g) was subjected to reversed-
phase silica gel column chromatography [45 g, MeOH–H2O (30 : 70→50 :
50→70 : 30)→MeOH] and finally to HPLC [detection RI, YMC-Pack ODS-
A (250ϫ20 mm i.d., YMC Co., Ltd.), CH3CN–H2O (25 : 75)] to give
myricetin (16, 98 mg). The known compounds (4—21) were identified by
comparison of their physical data ([a]D, IR, 1H-NMR, 13C-NMR) with
reported values.2—6,10,13—19)
(ϩ)-S-Myricanol (1a): A white powder, [a]D22 ϩ37.3° (cϭ0.2, CHCl3).
High-resolution EI-MS: Calcd for C21H26O5 (Mϩ): 358.1780. Found:
358.1774. UV [EtOH, nm, (log e)]: 214 (4.2), 261 (3.7), 298 (3.5). IR (KBr):
1
3400, 2932, 1598, 1560, 1500, 1450 cmϪ1. H-NMR (CDCl3) d: 1.54, 1.91
(1H each, both m, 10-H2), 1.56, 1.70 (1H each, both m, 9-H2), 1.68, 2.34
(1H each, both m, 12-H2), 1.92 (2H, m, 8-H2), 2.55, 2.79 (1H each, both m,
7-H2), 2.92 (2H, m, 13-H2), 3.88, 4.00 (each 3H, both s, 20, 21-H3), 4.09
(1H, m, 11-H), 6.89 (1H, s, 19-H), 6.91 (1H, d, Jϭ8.5 Hz, 16-H), 7.09 (1H,
dd, Jϭ2.5, 8.5 Hz, 15-H), 7.18 (1H, br s, 18-H). 13C-NMR (CDCl3) dC:
given in Table 1. EI-MS m/z (%): 358 (Mϩ, 100), 340 (MϩϪH2O, 14).
Myricanene A (2a): A white powder. High-resolution EI-MS: Calcd for
C21H24O4 (Mϩ): 340.1674. Found: 340.1668. UV [EtOH, nm, (log e)]: 214
(4.0), 259 (3.4), 297 (3.3). IR (KBr): 3362, 2926, 1655, 1610, 1506,
1
1456 cmϪ1. H-NMR (CDCl3) d: 1.93 (2H, m, 8-H2), 2.37 (2H, m, 12-H2),
2.42 (2H, m, 9-H2), 2.72 (2H, m, 13-H2), 2.77 (2H, m, 7-H2), 3.86, 4.00
(each 3H, both s, 20, 21-H3), 5.39 (1H, dt, Jϭ15.5, 5.5 Hz, 10-H), 5.59 (1H,
dt, Jϭ15.5, 6.5 Hz, 11-H), 6.83 (1H, d, Jϭ8.0 Hz, 16-H), 7.02 (1H, dd,
Jϭ2.5, 8.0 Hz, 15-H), 7.07 (1H, s, 19-H), 7.36 (1H, d, Jϭ2.5 Hz, 18-H). 13C-
NMR (CDCl3) dC: given in Table 1. EI-MS m/z (%): 340 (Mϩ, 100), 325
(MϩϪCH3, 14).
(ϩ)-S-Myricanol 5-O-b-D-glucopyranoside (1): A white powder, [a]D22
ϩ81.3° (cϭ0.2, EtOH). High-resolution positive-ion FAB-MS: Calcd for
C27H36O10Na (MϩNa)ϩ: 543.2206. Found: 543.2218. UV [EtOH, nm,
(log e)]: 216 (4.5), 256 (4.2), 299 (3.7). IR (KBr): 3568, 2932, 1598, 1560,
Myricanene B (3a): A white powder. High-resolution EI-MS: Calcd for
C21H24O4 (Mϩ): 340.1674. Found: 340.1683. UV [EtOH, nm, (log e)]: 215
(4.1), 253 (3.7), 301 (3.4). IR (KBr): 3566, 2926, 1655, 1633, 1506,
1
1508, 1450, 1073 cmϪ1. H-NMR (C5D5N) d: 1.52, 1.79 (1H each, both m,
1456 cmϪ1 1H-NMR (CDCl3) d: 1.72 (2H, m, 9-H2), 1.94 (2H, m, 8-H2),
.
9-H2), 1.82, 1.91 (1H each, both m, 8-H2), 1.88, 1.93 (1H each, both m, 10-
H2), 1.97, 2.44 (1H each, both m, 12-H2), 2.99, 3.40 (1H each, both m, 7-
H2), 3.01, 3.33 (1H each, both m, 13-H2), 3.96, 4.10 (each 3H, both s, 20,
21-H3), 4.34 (1H, m, 11-H), 5.75 (1H, d, Jϭ7.0 Hz, 1Ј-H), 7.12 (1H, s, 19-
H), 7.18 (1H, d, Jϭ8.0 Hz, 16-H), 7.19 (1H, dd, Jϭ2.0, 8.0 Hz, 15-H), 7.44
(1H, br s, 18-H). 13C-NMR (C5D5N) dC: given in Table 1. Positive-ion FAB-
MS m/z: 543 (MϩNa)ϩ. Negative-ion FAB-MS m/z: 519 (MϪH)Ϫ, 357
(MϪC6H11O5)Ϫ.
2.21 (2H, m, 10-H2), 2.65 (2H, m, 7-H2), 3.31 (2H, m, 13-H2), 3.89, 4.00
(each 3H, both s, 20, 21-H3), 5.73 (1H, dt, Jϭ15.5, 6.8 Hz, 12-H), 5.90 (1H,
dt, Jϭ15.5, 7.0 Hz, 11-H), 6.84 (1H, d, Jϭ8.0 Hz, 16-H), 6.96 (1H, s, 19-H),
7.09 (1H, dd, Jϭ2.5, 8.0 Hz, 15-H), 7.63 (1H, d, Jϭ2.5 Hz, 18-H). 13C-NMR
(CDCl3) dC: given in Table 1. EI-MS m/z (%): 340 (Mϩ, 100), 325
(MϩϪCH3, 9).
PCC Oxidation of 1a A solution of 1a (4.0 mg, 0.011 mmol) in CH2Cl2
(1.0 ml) was treated with pyridinium chlorochromate (PCC, 5.0 mg, 0.023
mmol), and the whole mixture was stirred at room temperature for 30 min
under N2 atmosphere. The reaction mixture was poured into ice-water and
the whole was extracted with AcOEt. The AcOEt extract was washed with
saturated aqueous NaHCO3 and brine, then dried over MgSO4 powder and
filtered. Removal of the solvent from the filtrate under reduced pressure fur-
nished a residue, which was then purified by silica gel column chromatogra-
phy [500 mg, n-hexane–AcOEt (4 : 1)] to give 6 (3.0 mg, 75%).
Myricanene A 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside (2):
A white powder, [a]D22 Ϫ0.6° (cϭ0.1, EtOH). High-resolution positive-ion
FAB-MS: Calcd for C32H42O13Na (MϩNa)ϩ: 657.2523. Found: 657.2531.
UV [EtOH, nm, (log e)]: 215 (4.2), 257 (3.9), 300 (3.6). IR (KBr): 3651,
1
2907, 1659, 1610, 1502, 1495, 1059 cmϪ1. H-NMR (C5D5N) d: 1.93 (2H,
m, 8-H2), 2.27 (2H, m, 12-H2), 2.44 (2H, m, 9-H2), 2.65 (2H, m, 13-H2),
3.15, 3.45 (1H each, both m, 7-H2), 3.97, 4.12 (each 3H, both s, 20, 21-H3),
5.22 (1H, dt, Jϭ15.5, 6.0 Hz, 10-H), 5.58 (1H, br s, 1Љ-H), 5.62 (1H, dt,
Jϭ15.5, 8.5 Hz, 11-H), 5.69 (1H, d, Jϭ7.3 Hz, 1Ј-H), 7.13 (1H, dd, Jϭ2.0,
8.0 Hz, 15-H), 7.14 (1H, d, Jϭ8.0 Hz, 16-H), 7.21 (1H, s, 19-H), 7.41 (1H,
br s, 18-H). 13C-NMR (C5D5N) dC: given in Table 1. Positive-ion FAB-MS
m/z: 1291 (2MϩNa)ϩ, 657 (MϩNa)ϩ. Negative-ion FAB-MS m/z: 633
(MϪH)Ϫ, 339 (MϪC11H19O9)Ϫ.
Diazomethane Methylation of 1a A solution of 1a (4.0 mg, 0.011
mmol) in MeOH (1.0 ml) was treated with CH2N2·Et2O (ca. 5 ml), and the
whole mixture was stirred at room temperature for 30 min. Removal of the
solvent under reduced pressure gave 1b (4.3 mg, quant.).
1b: A white powder. High-resolution EI-MS: Calcd for C23H30O5 (Mϩ):
386.2093. Found: 386.2104. 1H-NMR (CDCl3) d: 1.54, 1.72 (1H each, both
m, 9-H2), 1.56, 1.92 (1H each, both m, 10-H2), 1.67, 2.32 (1H each, both m,
12-H2), 1.92 (2H, m, 8-H2), 2.54, 2.81 (1H each, both m, 7-H2), 2.92 (2H, m,
13-H2), 3.89, 3.92, 3.97, 4.00 (all 3H, s, –OCH3), 4.10 (1H, m, 11-H), 6.92
Myricanene B 5-O-a-L-arabinofuranosyl(1→6)-b-D-glucopyranoside (3):
A white powder, [a]D22 Ϫ10.8° (cϭ0.1, EtOH). High-resolution positive-ion
FAB-MS: Calcd for C32H42O13Na (MϩNa)ϩ: 657.2523. Found: 657.2518.

214
Vol. 50, No. 2
(1H, d, Jϭ8.5 Hz, 16-H), 6.89 (1H, s, 19-H), 7.09 (1H, dd, Jϭ2.5, 8.0 Hz, (5.6 mM glucose, 1 mM CaCl2, 0.1% BSA were added) for an
15-H), 7.19 (1H, br s, 18-H). EI-MS m/z (%): 386 (Mϩ, 19), 372 (MϩϪCH3,
additional 10 min at 37 °C. Then, 20 ml of test sample solu-
100), 358 (MϩϪC2H6, 44).
tion was added to each well and incubated for 10 min, fol-
Preparation of the (R)-MTPA Ester (1c) and (S)-MTPA Ester (1d)
from 1b A solution of 1b (1.0 mg, 0.029 mmol) in CH2Cl2 (1.0 ml) was
treated with (R)-2-methoxy-2-trifluoromethylphenylacetic acid [(R)-MTPA]
lowed by the addition of 20 ml of antigen (DNP-BSA, final
concentration was 10 mg/ml) at 37 °C for 10 min to stimulate
(6.6 mg, 0.028 mmol) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC·HCl, 5.5 mg, 0.028 mmol) and 4-dimethylaminopyri-
dine (4-DMAP, 2.0 mg, 0.016 mmol), and the mixture was heated under re-
flux for 6 h. After cooling, it was poured into ice-water and the whole was
extracted with AcOEt. The AcOEt extract was successively washed with 5%
aqueous HCl, saturated aqueous NaHCO3 and brine, then dried over MgSO4
powder and filtered. Removal of the solvent from the filtrate under reduced
pressure furnished a residue which was purified by silica gel column chro-
matography [500 mg, n-hexane–AcOEt (2 : 1)] to give 1c (1.0 mg, 69%).
Through a similar procedure, 1d (1.0 mg, 69%) was prepared from 1b (1.0
mg, 0.029 mmol) using (S)-2-methoxy-2-trifluoromethylphenylacetic acid
[(S)-MTPA] (6.6 mg, 0.028 mmol), EDC·HCl (5.5 mg, 0.028 mmol) and 4-
DMAP (2.0 mg, 0.016 mmol).
the cells to evoke allergic reactions (degranulation). The re-
action was stopped by cooling in an ice bath for 10 min. Su-
pernatant (50 ml) was transferred into a 96-well microplate
and incubated with 50 ml of substrate (1 mM p-nitrophenyl-N-
acetyl-b-D-glucosaminide) in 0.1 M citrate buffer (pH 4.5) at
37 °C for 1 h. The reaction was stopped by adding 200 ml of
stop solution (0.1 M Na2CO3/NaHCO3, pH 10.0). The ab-
sorbance was measured by a microplate reader at 405 nm. The
test sample was dissolved in dimethylsulfoxide (DMSO), and
the solution was added to siraganian buffer (final DMSO
concentration was 0.1%).
1c: 1H-NMR (CDCl3) d: 1.62, 1.74 (1H each, both m, 9-H2), 1.98 (2H, m,
12-H2), 2.01 (2H, m, 10-H2), 2.30 (2H, m, 8-H2), 2.36, 2.75 (1H each, both
m, 7-H2), 2.58, 2.86 (1H each, both m, 13-H2), 3.49, 3.57, 3.90, 3.92, 3.98
(all 3H, s, –OCH3), 5.35 (1H, m, 11-H), 6.79 (1H, dd, Jϭ1.8, 8.3 Hz, 15-H),
6.92 (1H, d, Jϭ8.3 Hz, 16-H), 7.00 (1H, s, 19-H), 7.13 (1H, br s, 18-H).
1d: 1H-NMR (CDCl3) d: 1.58, 1.71 (1H each, both m, 9-H2), 1.89 (2H, m,
10-H2), 2.05 (2H, m, 12-H2), 2.28 (2H, m, 8-H2), 2.32, 2.68 (1H each, both
m, 7-H2), 2.60, 2.89 (1H each, both m, 13-H2), 3.49, 3.51, 3.91, 3.92, 3.98
(all 3H, s, –OCH3), 5.35 (1H, m, 11-H), 6.93 (1H, d, Jϭ8.3 Hz, 16-H), 7.00
(1H, s, 19-H), 7.05 (1H, dd, Jϭ2.1, 8.3 Hz, 15-H), 7.16 (1H, br s, 18-H).
Dehydration of 4 A solution of 4 (50.0 mg, 0.014 mmol) in pryidine
(2.0 ml) was treated with SOCl2 (500 ml), and the mixture was stirred at 0 °C
for 1 h. The reaction mixture was poured into ice-water and the whole was
extracted with AcOEt. The AcOEt extract was washed with saturated aque-
ous NaHCO3 and brine, then dried over MgSO4 powder and filtrated. Re-
moval of the solvent from the filtrate under reduced pressure gave a residue.
The residue was purified by HPLC [detection UV (254 nm), YMC-Pack
For estimating the spontaneous release of b-hexos-
aminidase from cells, exactly the same procedure was fol-
lowed (Normal), but without adding antigen and IgE. Blank
absorbance of the test material was measured to eliminate in-
terference caused by the color of the test material itself. For
this, only test material and substrate were added without
adding cell extract (Blank). Thus, the inhibition % of the re-
lease of b-hexosaminidase by the test material was calculated
by the following equation.
TϪBϪN
inhibition (%)ϭ 1Ϫ
ϫ100
CϪN
Control (C): antigen-IgE response was evoked without test
ODS-A (250ϫ20 mm i.d., YMC Co., Ltd.), MeOH–H2O (80 : 20)] to give sample; Test (T): antigen-IgE response was evoked in the
myricanenes A (2a, 13.2 mg, 28%) and B (3a, 9.5 mg, 20%).
Acid Hydrolysis of 1—3 A solution of 1—3 (2 mg each) in 5% aqueous
H2SO4–1,4-dioxane (0.5 ml, 1 : 1, v/v) was heated under reflux for 1 h. After
presence of test sample; Blank (B): only test sample and sub-
strate were added; Normal (N): antigen-IgE response was not
evoked, test sample was not added.
In this condition, it was calculated that 40—50% of b-hex-
osaminidase was released from the cells in the control groups
by determination of the total b-hexosaminidase activity after
sonication of the cell suspension.
b-Hexosaminidase Inhibitory Activity The cell sus-
pension (5ϫ107 cells) in 6 ml of PBS was sonicated. The so-
lution was then centrifuged and the supernatant was diluted
with siraganian buffer and adjusted to equal the enzyme ac-
tivity of the degranulation test described above. The enzyme
solution (45 ml) and test sample solution (5 ml) were trans-
ferred into a 96-well microplate and incubated with 50 ml of
the substrate solution at 37 °C for 1 h. The reaction was
stopped by adding 200 ml of the stop solution. The ab-
sorbance was measured by a microplate reader at 405 nm.
Statistics Values were expressed as meansϮS.E.M.
One-way analysis of variance following Dunnett’s test was
used for statistical analysis.
cooling, the reaction mixture was neutralized with Amberlite IRA-400 (OHϪ
form), and the residue was removed by filtration. After removal of the sol-
vent from the filtrate under reduced pressure, the residue was transferred to a
Sep-Pak C18 cartridge with H2O and MeOH. The H2O eluate was concen-
trated and the residue was treated with L-cysteine methyl ester hydrochloride
(4 mg) in pyridine (0.5 ml) at 60 °C for 1 h. After reaction, the solution was
treated with N,O-bis(trimethylsilyl)trifluoroacetamide (0.2 ml) at 60 °C for
1 h. The supernatant was then subjected to GLC analysis to identify the de-
rivatives of D-glucose (i) from 1—3; and of L-arabinose (ii) from 2 and 3.
GLC conditions: column: Supeluco STBTM-1, 30 mϫ0.25 mm (i.d.) capil-
lary column; injector temperature: 230 °C; detector temperature: 230 °C;
column temperature: 230 °C; He flow rate: 15 ml/min; tR: i: 24.2 min, ii:
15.1 min.
Bioassay
b
Inhibitory Effect on the Release of -Hexosaminidase
from RBL-2H3 Cells Inhibitory effects of test samples on
the release of b-hexosaminidase from RBL-2H3 cells were
evaluated by the following procedure.36) RBL-2H3 cells were
grown in Minimum Essential Medium Eagle (MEM, Shigma
Co., Ltd.) containing fetal calf serum (10%), penicillin (100
units/ml), and streptomycin (100 mg/ml). Before the experi-
ment, cells were dispensed into 24-well plates at the concen-
tration of 2ϫ105 cells/well using a medium containing
0.45 mg/ml of anti-DNP IgE, and these were incubated
overnight at 37 °C in 5% CO2 for sensitization of the cells.
Then, cells were washed twice with 500 ml of siraganian
buffer [119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 25 mM
piperazine-N,NЈ-bis(2-ethanesulfonic acid) (PIPES), 40 mM
NaOH, pH 7.2] and incubated in 160 ml of siraganian buffer
References and Notes
1) Part VI: Matsuda H., Ohta T., Kawaguchi A., Yoshikawa M., Chem.
Pharm. Bull., 49, 69—72 (2001).
2) Nonaka G., Muta M., Nishioka I., Phytochemistry, 22, 237—241
(1983).
3) Sakurai N., Yaguchi Y., Inoue T., Phytochemistry, 26, 217—219
(1987).
4) Inoue T., Arai Y., Nagai M., Yakugaku Zasshi, 104, 37—41 (1984).
5) Takeda Y., Fujita T., Shingu T., Ogimi C., Chem. Pharm. Bull., 35,
2569—2573 (1987).
6) Yaguchi Y., Sakurai N., Nagai M., Inoue T., Chem. Pharm. Bull., 36,

February 2002
1419—1424 (1988).
7) Sakurai N., Yaguchi Y., Hirakawa T., Nagai M., Inoue T., Phytochem-
istry, 30, 3077—3079 (1991).
8) Inoue T., Yakugaku Zasshi, 113, 181—197 (1993).
9) Ohta S., Sakurai N., Kamogawa A., Yaguchi Y., Inoue T., Shinoda M.,
Yakugaku Zasshi, 112, 244—252 (1992).
215
23) Joshi B. S., Pelletier S. W., Newton M. G., Lee D., McGaughey G. B.,
Puar M. S., J. Nat. Prod., 59, 759—764 (1996).
24) a) Schwartz L. B., Lewis R. A., Seldin D., Austen K. F., J. Immunol.,
126, 1290—1294 (1981); b) Marquardt D. L., Wasserman S. I., ibid.,
131, 934—939 (1983).
25) a) Yoshikawa M., Uchida E., Chatani N., Murakami N., Yamahara J.,
Chem. Pharm. Bull., 40, 3121—3123 (1992); b) Yoshikawa M.,
Uchida E., Chatani N., Kobayashi H., Naitoh Y., Okuno Y., Matsuda
H., Yamahara J., Murakami N., ibid., 40, 3352—3354 (1992); c)
Yamahara J., Matsuda H., Shimoda H., Ishikawa H., Kawamori S.,
Wariishi N., Harada E., Murakami N., Yoshikawa M., Yakugaku
Zasshi, 114, 401—413 (1994); d) Yoshikawa M., Harada E., Naitoh Y.,
Inoue K., Matsuda H., Shimoda H., Yamahara J., Murakami N., Chem.
Pharm. Bull., 42, 2225—2230 (1994); e) Yamahara J., Matsuda H.,
Shimoda H., Wariishi N., Yagi N., Murakami N., Yoshikawa M., Folia
Pharmacol. Jpn., 105, 365—379 (1985); f) Yoshikawa M., Matsuda
H., Shimoda H., Shimada H., Harada E., Naitoh Y., Miki A., Yama-
hara J., Murakami N., Chem. Pharm. Bull., 44, 1440—1447 (1996); g)
Yoshikawa M., Shimada H., Yagi N., Murakami N., Shimoda H.,
Yamahara J., Matsuda H., ibid., 44, 1890—1898 (1996); h) Shimoda
H., Matsuda H., Yamahara J., Yoshikawa M., Biol. Pharm. Bull., 21,
809—813 (1998); i) Matsuda H., Shimoda H., Yoshikawa M., Bioorg.
Med. Chem., 7, 1445—1450 (1999); j) Matsuda H., Shimoda H.,
Yamahara J., Yoshikawa M., Biol. Pharm. Bull., 22, 870—872 (1999);
k) Matsuda H., Shimoda H., Kageura T., Yoshikawa M., ibid., 22,
925—931 (1999); l) Matsuda H., Shimoda H., Uemura T., Ueda T.,
Yamahara J., Yoshikawa M., Chem. Pharm. Bull., 47, 1753—1758
(1999).
10) Matsuda H., Higashino M., Chen W., Tosa H., Iinuma M., Kubo M.,
Biol. Pharm. Bull., 18, 1148—1150 (1995).
11) Matsuda H., Yamazaki M., Matsuo K., Asanuma Y., Kubo M., Biol.
Pharm. Bull., 24, 259—263 (2001).
12) a) Matsuda H., Kageura T., Morikawa T., Toguchida I., Harima S.,
Yoshikawa M., Bioorg. Med. Chem. Lett., 10, 323—327 (2000); b)
Yoshikawa M., Morikawa T., Toguchida I., Harima S., Matsuda H.,
Chem. Pharm. Bull., 48, 651—656 (2000); c) Matsuda H., Kageura T.,
Inoue Y., Morikawa T., Yoshikawa M., Tetrahedron, 56, 7763—7777
(2000); d) Matsuda H., Morikawa T., Toguchida I., Park J.-Y., Harima
S., Yoshikawa M., Bioorg. Med. Chem., 9, 41—50 (2001); e) Matsuda
H., Morikawa T., Ninomiya K., Yoshikawa M., ibid., 9, 909—916
(2001); f) Matsuda H., Kageura T., Oda M., Morikawa T., Sakamoto
Y., Yoshikawa M., Chem. Pharm. Bull., 49, 716—720 (2001); g) Mu-
rakami T., Kishi A., Sakurama T., Matsuda H., Yoshikawa M., Hetero-
cycles, 54, 957—966 (2001); h) Matsuda H., Morikawa T., Toguchida
I., Ninomiya K., Yoshikawa M., ibid., 55, 841—846 (2001); i) Kimura
I., Yoshikawa M., Kobayashi S., Sugihara Y., Suzuki M., Oominami
H., Murakami T., Matsuda H., Doiphode V. V., Bioorg. Med. Chem.
Lett., 11, 985—989 (2001); j) Matsuda H., Shimoda H., Yoshikawa
M., Bioorg. Med. Chem., 9, 1031—1035 (2001); k) Matsuda H., Shi-
moda H., Morikawa T., Yoshikawa M., Bioorg. Med. Chem. Lett., 11,
1839—1842 (2001); l) Kageura T., Matsuda H., Morikawa T., 26) Yoshikawa M., Murakami T., Ueda T., Matsuda H., Yamahara J., Mu-
Toguchida I., Harima S., Oda M., Yoshikawa M., Bioorg. Med. Chem.,
9, 1887—1893 (2001).
13) Ursolic acid (7), acetylursolic acid (8), and ursonic acid (9) were iden-
rakami N., Chem. Pharm. Bull., 44, 1736—1743 (1996).
27) Yoshikawa M., Shimada H., Shimoda H., Murakami N., Yamahara J.,
Matsuda H., Chem. Pharm. Bull., 44, 2086—2091 (1996).
tified by comparison of their methyl ester derivatives (diazomethane 28) Yoshikawa M., Shimada H., Horikawa S., Murakami T., Shimoda H.,
methylation) with reported values; Seo S., Tomita Y., Tori K., Tetrahe-
dron Lett., 1, 7—10 (1975).
Yamahara J., Matsuda H., Chem. Pharm. Bull., 45, 1498—1503
(1997).
14) Kitajima J., Arai M., Tanaka Y., Chem. Pharm. Bull., 42, 608—610
(1994).
29) Kubo M., Matsuda H., Tomohiro N., Yoshikawa M., Biol. Pharm.
Bull., 20, 511—516 (1997).
15) Maslinic acid (13, ϭcrategolic acid) was identified by comparison of 30) Matsuda H., Dai Y., Ido Y., Yoshikawa M., Kubo M., Biol. Pharm.
its methyl ester derivative (diazomethane methylation) with reported Bull., 20, 1165—1170 (1997).
values; Mallavarapu G. R., Rao S. B., Muralikrishna E., Rao G. S. R. 31) Yoshikawa M., Shimada H., Saka M., Yoshizumi S., Yamahara J., Ma-
S., Indian J. Chem., 19B, 713—714 (1980). tsuda H., Chem. Pharm. Bull., 45, 464—469 (1997).
16) Markham K. R., Ternai B., Stanley R., Geiger H., Marby T. J., Tetrahe- 32) Yoshikawa M., Shimada H., Komatsu H., Sakurama T., Nishida N.,
dron, 34, 1389—1397 (1978).
17) a) Fauran F., Feniou C., Mosser J., Prat G., Eur. J. Med. Chem.-Chim.
Yamahara J., Shimoda H., Matsuda H., Tani T., Chem. Pharm. Bull.,
45, 877—882 (1997).
Ther., 13, 503—508 (1978); b) Zapesochnaya G. G., Khim. Prir. 33) Yoshikawa M., Murakami T., Ikebata A., Wakao S., Murakami N.,
Soedin., 6, 695—709 (1982).
18) Nonaka G., Kawahara O., Nishioka I., Chem. Pharm. Bull., 31, 3906—
3914 (1983).
19) Ishimaru K., Nonaka G., Nishioka I., Phytochemistry, 26, 1147—1152
(1987).
20) b-Sitosterol and gallic acid were identified by comparison of their
physical data with those of commercially obtained reagents.
21) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 1843—1845
(1986).
Matsuda H., Yamahara J., Chem. Pharm. Bull., 45, 1186—1192
(1997).
34) Yoshizumi S., Murakami T., Kadoya M., Matsuda H., Yamahara J.,
Yoshikawa M., Yakugaku Zasshi, 118, 118—192 (1998).
35) a) Futagami Y., Yano S., Horie S., Tsuchiya S., Ikegami F., Sekine T.,
Yamamoto Y., Fujimori H., Takamoto K., Watanabe K., J. Trad. Med.,
13, 430—431 (1996); b) Cheong H., Ryu S. Y., Oak M. H., Cheon S.
H., Yoo G. S., Kim K. M., Arch. Pharm. Res., 21, 478—480 (1998).
36) Ho C., Choi E. J., Yoo G. S., Kim K. M., Ryu S. Y., Planta Med., 64,
577—578 (1998).
22) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem. Soc.,
113, 4092—4096 (1991).