Structures of new flavonoids and benzofuran-type stilbene and degranulation inhibitors of rat basophilic leukemia cells from the Brazilian herbal medicine Cissus sicyoides

Article abstract of DOI:10.1248/cpb.57.1089

Three new flavonoid glycosides (cissosides I, II, and III) and a new benzofuran-type stilbene (cissusin) were isolated from the methanolic extract of the aerial parts of Cissus sicyoides cultivated in Brazil. Their structures were elucidated on the basis

Full text of DOI:10.1248/cpb.57.1089

October 2009
Chem. Pharm. Bull. 57(10) 1089—1095 (2009)
1089
Structures of New Flavonoids and Benzofuran-Type Stilbene and
Degranulation Inhibitors of Rat Basophilic Leukemia Cells from the
Brazilian Herbal Medicine Cissus sicyoides
Fengming XU, Hisashi MATSUDA, Hiroki HATA, Kaoru SUGAWARA, Seikou NAKAMURA, and
Masayuki YOSHIKAWA*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received May 29, 2009; accepted June 22, 2009; published online July 15, 2009
Three new flavonoid glycosides (cissosides I, II, and III) and a new benzofuran-type stilbene (cissusin) were
isolated from the methanolic extract of the aerial parts of Cissus sicyoides cultivated in Brazil. Their structures
were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the isolated
constituents on the release of b-hexosaminidase as a marker of degranulation in rat basophilic leukemia (RBL-
2H3) cells were examined. Cissusin, flavonols (kaempferol, quercetin), flavones (7,3ꢀ,4ꢀ-trihydroxyflavone, lanceo-
latin B), pterocarpanes (homopterocarpin), chalcones (isoliquiritigenin, E-7-O-methylpongamol), and tryptan-
thrin markedly inhibited the release of b-hexosaminidase.
Key words Cissus sicyoides; flavonoid; benzofuran-type stilbene; cissoside; cissusin; degranulation inhibitor
Cissus sicyoides (Vitaceae) (Brazilian name: Insulina) is a a-L-rhamnopyranoside (12, 0.0010%),11) kaempferol-3-
herbaceous plant widely distributed in Brazil. Plant infusions O-a-L-arabinopyranosyl-7-O-a-L-rhamnopyranoside
(13,
are used in Brazilian folk medicine to treat diabetes mellitus, 0.0019%),12) kaempferol-3-O-a-L-arabinofuranosyl-7-O-a-L-
while the leaves are used externally against rheumatism and rhamnopyranoside (14, 0.0041%),8) quercetin (15,
wounds.1) Bioactive studies indicated that the water extract 0.00077%),9) quercetin-3-O-a-L-rhamnopyranoside (16,
of C. sicyoides (500 mg/kg body weight) improved hyper- 0.00045%),9)
quercetin-3-O-b-D-glucopyranoside
(17,
glycemia after the oral administration of maltose to KK-Ay 0.00017%),9) quercetin-3-O-b-D-galactopyranoside (18,
mice.2) Previous phytochemical investigations of this plant 0.000092%),9) quercetin-3-O-a-L-arabinopyranoside (19,
yielded coumarins, flavonoids, and anthocyanins.1,3) In the 0.00051%),9) quercetin-3-O-a-L-arabinofuranoside (20,
course of our characterization studies of traditional Brazilian 0.00057%),9) rutin (21, 0.017%),13) quercetin-3-O-a-L-rham-
medicines,4—7) we systematically investigated the chemical nopyranosyl-7-O-a-L-rhamnopyranoside (22, 0.00043%),11)
constituents of the methanolic extract from the aerial parts of two flavones 7,3ꢀ,4ꢀ-trihydroxyflavone (23, 0.00039%),14)
C. sicyoides, which resulted in the isolation of three new and lanceolatin B (24, 0.00094%)15); three pterocarpanes,
flavonoid glycosides, cissosides I (1), II (2), and III (3), and medicarpin (25, 0.00011%),16) homopterocarpin (26,
the new benzofuran-type stilbene cissusin (4), together with 0.00018%),16) and melilotocarpan D (27, 0.00043%)17); three
39 known constituents. This paper deals with the isolation chalcones, isoliquiritigenin (28, 0.00075%),18) pongamol
and structural elucidation of four new constituents (1—4), (29, 0.0018%),19) and E-7-O-methylpongamol (30, 0.0017%)19)
and the inhibitory effects of both the extracts and isolated benzofuran-type stilbene, 2-(2ꢀ,4ꢀ-dimethoxyphenyl)-5,6-
compounds on the release of b-hexosaminidase from RBL- methylenedioxybenzofuran (31, 0.00016%)20); a lignan, (ꢁ)-
2H3 cells.
;
a
pinoresinol (32, 0.00068%)21); an indole quinazoline alka-
The methanolic extract from the dried aerial parts of C. loid, tryptanthrin (33, 0.0024%)22); a chromone, 7-hydroxy-
sicyoides (cultivated in Brazil) was partitioned into a mixture chromone (0.000085%)23); seven phenolics, syringaldehyde
of ethyl acetate (EtOAc) and water to furnish the EtOAc-sol- (0.00011%),24) 4-hydroxybenzoic acid (0.00028%),25) vanillic
uble fraction and an aqueous layer. The aqueous layer was acid (0.00078%),25) 4-hydroxycinnamic acid (0.00017%),26)
extracted with n-butanol (n-BuOH) to give n-BuOH- and gallic acid (0.0069%),27) methyl gallate (0.00034%),28)
H2O-soluble fractions. The EtOAc- and n-BuOH-soluble and glucosyl salicylate (0.00084%)29); an indole, indican
fractions were subjected to silica gel and octadecyl silica (0.0037%)30); and
(ODS) column chromatography and finally to HPLC to fur- (0.0030%).31)
a catechin, epicatechin 3-O-gallate
nish three new flavonoid glycosides, cissoside I (1, 0.0018%
Cissoside I (1) was obtained as a yellow powder with neg-
from natural medicine), cissoside II (2, 0.00029%), and ative optical rotation ([a]D27 ꢂ176.8°). In the positive-ion
cissoside III (3, 0.00038%); a new benzofuran-type stil- FAB-MS of 1, a quasimolecular ion peak was observed at
bene, cissusin (4, 0.00054%); 18 flavonols, kaempferol m/z 629 (MꢁNa)ꢁ, and high-resolution (HR) positive-ion
(5, 0.0011%),8) kaempferol-3-O-a-L-rhamnopyranoside FAB-MS analysis revealed the formula of 1 to be C28H30O15.
(6,
0.00023%),8)
kaempferol-3-O-b-D-glucopyranoside The IR spectrum of 1 suggested the presence of hydroxyl
(7, 0.00029%),9) kaempferol-3-O-a-L-arabinofuranoside (3415 cmꢂ1), acetyl carbonyl (1720 cmꢂ1), and g-pyrone
(8, 0.00024%),8) kaempferol-7-O-a-L-rhamnopyranoside (1655 cmꢂ1) groups, and an aromatic ring (1601, 1493 cmꢂ1).
(9, 0.00065%),10) kaempferol-3-O-a-L-rhamnopyranosyl- Its UV spectrum in methanol showed absorption maxima at
7-O-a-L-rhamnopyranoside (10, 0.010%),8) kaempferol- 266 nm (log e 4.32) (band II) and 347 nm (4.21) (band I),
3-O-b-D-glucopyranosyl-7-O-a-L-rhamnopyranoside
1
(11, suggesting the presence of a flavonol skeleton. The H- and
0.00039%),10)
kaempferol-3-O-b-D-galactopyranosyl-7-O- 13C-NMR (pyridine-d5, Table 1) spectra of 1, which were as-
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp

1090
Vol. 57, No. 10
Chart 1. Structures of Principal Constituents Isolated from the Aerial Parts of C. sicyoides
Table 1. 13C-NMR Data for 1—4, 14 and 31
1a)
14a)
2a)
3a)
4b)
31b)
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-1ꢀ
C-2ꢀ
C-3ꢀ
C-4ꢀ
C-5ꢀ
C-6ꢀ
C-1ꢃ
C-2ꢃ
C-3ꢃ
C-4ꢃ
C-5ꢃ
C-1ꢅ
C-2ꢅ
C-3ꢅ
C-4ꢅ
C-5ꢅ
C-6ꢅ
CH3CO
158.6
135.0
179.1
162.3
100.4
162.8
94.9
157.0
106.9
121.8
131.8
116.5
161.9
116.5
131.8
110.0
83.7
79.4
84.2
64.9
100.0
71.6
72.4
73.6
71.5
18.7
158.3
135.1
179.3
162.3
100.4
162.8
94.9
157.0
106.9
121.8
131.8
116.5
161.9
116.5
131.8
110.0
83.7
78.9
88.6
62.5
100.0
71.6
72.4
73.6
71.5
18.7
158.5
135.1
179.3
162.3
100.3
162.8
94.8
157.0
106.9
122.2
116.6
147.5
150.9
117.1
122.7
109.9
83.6
79.0
88.6
62.4
100.0
71.6
72.4
73.6
71.5
18.7
158.8
135.1
179.2
162.3
100.3
162.8
94.8
157.0
106.9
122.2
116.6
147.5
150.9
117.1
122.7
109.9
83.6
79.5
84.2
64.8
100.0
71.6
72.4
73.6
71.5
18.7
C-2
C-3
C-3a
C-4
C-5
C-6
C-7
C-7a
152.1
104.7
99.3
145.6
144.2
93.2
152.1
104.8
99.3
145.6
144.2
93.2
123.2
148.8
123.2
148.8
C-1ꢀ
C-2ꢀ
C-3ꢀ
C-4ꢀ
C-5ꢀ
C-6ꢀ
OCH2O
OCH3
112.9
157.4
99.3
156.6
107.5
127.5
101.1
55.5
113.0
157.2
98.8
160.5
104.8
127.3
101.1
55.5ꢄ2
20.6
20.6
170.5
170.5
a) Measured in pyridine-d5 at 150 MHz. b) Measured in CDCl3 at 150 MHz.
signed by various NMR experiments (distortionless enhance- ple quantum correlation (HMQC), and heteronuclear multi-
ment by polarization transfer (DEPT), double quantum filter ple bond connectivtiy (HMBC)), showed signals assignable
correlation spectroscopy (DQF COSY), heteronuclear multi- to a kaempferol part {two meta-coupled aromatic protons

October 2009
1091
Fig. 1. Significant H–H COSY, HMBC, and NOE Correlations for 1—4
[d 6.78, 6.96 (1H each, both d, Jꢆ2.1 Hz, H-6, -8)], four 2 were clarified in HMBC experiments (Fig. 1). Thus long-
ortho-coupled aromatic protons [d 7.33, 8.32 (2H each, range correlations were observed between H-1ꢃ [6.53 (1H,
both d, Jꢆ8.6 Hz, H-3ꢀ, -5ꢀ, -2ꢀ, -6ꢀ)]}, an arabinofuranosyl br s)] and C-3 (dC 135.1) as well as between H-1ꢅ [6.22 (1H,
moiety {d [4.42 (1H, dd, Jꢆ6.8, 12.4 Hz), 4.62 (1H, dd, br s)] and the carbon-7 (dC 162.8). On the basis of this
Jꢆ3.4, 12.4 Hz) H2-5ꢀ], 4.64 (1H, m, H-3ꢃ), 4.66 (1H, m, H- evidence, the structure of cissoside II was determined to
4ꢃ), 5.25 (1H, br d, Jꢆ2.8 Hz, H-2ꢃ), 6.49 (1H, br s, H-1ꢃ)}, a be quercetin 3-O-a-L-arabinofuranosyl-7-O-a-L-rhamnopy-
rhamnopyranosyl moiety [1.68 (3H, d, Jꢆ6.1 Hz, H3-6ꢅ), ranoside (2).
4.31 (1H m, H-5ꢅ), 4.41 (1H dd, Jꢆ8.9, 9.6 Hz, H-4ꢅ), 4.67
Cissoside III (3) was also obtained as a yellow powder. Its
(1H, dd, Jꢆ3.4, 9.6 Hz, H-3ꢅ), 4.73 (1H, br s, H-2ꢅ), 6.26 positive-ion FAB-MS and HR-FAB-MS analysis revealed the
(1H, br s, H-1ꢅ)], and an acetyl group [d 1.88 (3H, s, –CH3)]. formula of 3 to be C28H30O16. The proton and carbon signals
1
The position of the acetyl group in 1 was confirmed in an in the H- and 13C-NMR (pyridine-d5, Table 1) spectra of 3
HMBC experiment (Fig. 1), in which a long-range correla- were very similar to those of 2, except for the signals due to
tion was observed between the proton-5ꢃ [4.42 (1H, dd, an additional acetyl group [(dC 20.6, 170.5), (dH 1.89, s)].
Jꢆ6.8, 12.4 Hz), 4.62 (1H, dd, Jꢆ3.4, 12.4 Hz) H2-5ꢀ] and Comparison of the 13C-NMR spectra (Table 1) of 3 with
the acetyl carbonyl carbon (dC 170.5). In addition, the those of 2 revealed an acylation shift around the C-5ꢃ posi-
alkaline hydrolysis of 1 with 0.5% sodium methoxide tion of the a-L-arabinofuranosyl moiety. This was also con-
(NaOMe)–MeOH at room temperature furnished kaempferol firmed in an HMBC experiment, as shown in Fig. 1. Further-
3-O-a-L-arabinofuranosyl-7-O-a-L-rhamnopyranoside (14).8) more, the alkaline hydrolysis of 3 with 0.5% NaOMe–MeOH
These findings led us to formulate the structure of cissoside I at room temperature furnished cissoside II (2). On the basis
to be kaempferol 3-O-a-L-(5ꢃ-O-acetyl)-arabinofuranosyl-7- of the above-mentioned evidence, the structure of cissoside
O-a-L-rhamnopyranoside (1).
Cissoside II (2) was obtained as a yellow powder with neg- arabinofuranosyl-7-O-a-L-rhamnopyranoside (3).
ative optical rotation ([a]D26 ꢂ181.5°). In the positive-ion
Cissusin (4) was isolated as a colorless powder. The mo-
III was established to be quercetin 3-O-a-L-(5ꢃ-O-acetyl)-
FAB-MS of 2, a quasimolecular ion peak was observed at lecular formula of 4 was established to be C16H12O5 by HR-
m/z 603 (MꢁNa)ꢁ, and HR-FAB-MS analysis established its EI-MS measurement. The IR spectrum of 4 suggested the
formula to be C26H28O15. Its IR spectrum showed absorption presence of a hydroxyl group (3433 cmꢂ1), an aromatic ring
bands at 3400, 1655, 1609, and 1509 cmꢂ1 ascribable to hy- (1623, 1614, 1592, 1501 cmꢂ1), and a methylenedioxy group
droxyl and carbonyl functions and aromatic rings. The UV (1036, 945 cmꢂ1). Its UV spectrum showed absorption max-
spectrum of 2 in methanol showed absorption maxima at ima at 330 nm (log e 4.38) and 345 nm (4.39), suggesting the
258 nm (log e 4.54) (band II) and 349 nm (3.93) (band I), presence of a 2-arylbenzofuran structure.20) The 1H- and 13C-
suggesting the presence of a flavonol skeleton. The acid hy- NMR (CDCl3, Table 1) spectra of 4 showed signals assigna-
drolysis of 2 with 1 M of hydrochloric acid (HCl)–1,4-diox- ble to one methoxyl group [d 3.93 (3H, s, OCH3-2ꢀ)], one
ane (1 : 1, v/v) liberated quercetin, L-arabinose, and L-rham- methylenedioxy group [d 5.97 (2H, s, OCH2O-5, 6)], three
nose, which were identified with HPLC analysis using a re- ortho- and meta-coupled aromatic protons [d 6.52 (1H, d,
1
fractive index or optical rotation detector.32,33) The H- and Jꢆ2.1 Hz, H-3ꢀ), 6.54 (1H, dd, Jꢆ2.1, 8.9 Hz, H-5ꢀ), 7.81
13C-NMR (pyridine-d5, Table 1) spectra of 2 showed signals (1H, d, Jꢆ8.9 Hz, H-6ꢀ)], three single aromatic protons [d
assignable to a quercetin part {two meta-coupled aromatic 6.92, 6.98, 7.06 (1H each, all s, H-4, 7, 3). The structure of 4
1
protons [d 6.75, 6.84 (1H each, both d, Jꢆ2.0 Hz, H-6, -8)], was confirmed in H–1H correlation spectroscopy (COSY)
three ortho- and meta-coupled aromatic protons [d 7.37 (1H, and HMBC experiments. As shown in Fig. 1, the former in-
d, Jꢆ8.2 Hz, H-5ꢀ), 7.99 (1H, dd, Jꢆ2.0, 8.2 Hz, H-6ꢀ), 8.19 dicated the presence of one partial structure shown by the
(1H, d, Jꢆ2.0 Hz, H-2ꢀ)]}, an a-L-arabinofuranosyl moiety bold line, while long-range correlations in the HMBC experi-
{d [4.10 (1H, dd, Jꢆ4.1, 11.7 Hz), 4.17 (1H, dd, Jꢆ2.7, ment on 4 were observed between the following proton and
11.7 Hz), H2-5ꢃ], 4.75 (1H, m, H-4ꢃ), 4.91 (1H, m, H-3ꢃ), carbon pairs: H-3 and C-3a, -4, -7a, 1ꢀ; H-4 and C-3, -5, -6,
5.20 (1H, br d, Jꢆ2.8 Hz, H-2ꢃ), 6.53 (1H, br s, H-1ꢃ)}, and -7a; OCH2O-5, -6 and C-5, 6; H-7 and C-3a, -5, -6; OCH3-2ꢀ
an a-L-rhamnopyranosyl moiety [1.68 (3H, d, Jꢆ6.2 Hz, H3- and C-2ꢀ; H-3ꢀ and C-1ꢀ, -4ꢀ, -5ꢀ; H-5ꢀ and C-1ꢀ, -3ꢀ; H-6ꢀ
6ꢅ), 4.30 (1H m, H-5ꢅ), 4.42 (1H dd, Jꢆ8.9, 9.6 Hz, H-4ꢅ), and C-2, -2ꢀ, -4ꢀ. The position of a methoxyl group was also
4.66 (1H, dd, Jꢆ2.8, 9.6 Hz, H-3ꢅ), 4.71 (1H, br s, H-2ꢅ), supported by the observation of a nuclear Overhauser effect
6.22 (1H, br s, H-1ꢅ)]. The linkages of the sugar moieties in (NOE) at H-3ꢀ upon irradiation of OCH3-2ꢀ. Furthermore,

1092
Vol. 57, No. 10
Table 2. Inhibitory Effects of Constituents (4, 5, 15, 23—33) from C. sicyoides on the Release of b-Hexosaminidase by RBL-2H3 Cells
Inhibition (%)
Inhibition (%) of
b-hexosaminidase
at 100 mM
0 mM
3 mM
10 mM
30 mM
100 mM
IC50 (mM)
Cissusin (4)
0.0ꢇ3.5
0.0ꢇ2.9
0.0ꢇ8.6
0.0ꢇ6.1
0.0ꢇ6.3
0.0ꢇ4.1
0.0ꢇ3.5
0.0ꢇ6.0
0.0ꢇ1.4
0.0ꢇ1.6
0.0ꢇ3.7
0.0ꢇ4.1
0.0ꢇ2.6
0.0ꢇ3.5
—
—
12.0ꢇ4.5
30.8ꢇ5.1**
78.8ꢇ1.2**
30.4ꢇ3.5**
52.7ꢇ4.5**
—
16.4ꢇ4.5**
—
28.1ꢇ2.8**
—
24.1ꢇ6.1**
79.6ꢇ2.5**
87.3ꢇ0.7**
71.8ꢇ2.1**
55.8ꢇ3.9**
—
28.1ꢇ1.8**
—
54.5ꢇ3.2**
—
51.6ꢇ2.7**
93.2ꢇ0.6**
90.1ꢇ0.4**
90.8ꢇ2.0**
69.0ꢇ3.6**
34.4ꢇ4.9**
74.7ꢇ0.1**
16.9ꢇ5.8
91.3ꢇ0.3**
36.7ꢇ2.6**
81.1ꢇ2.4**
25.7ꢇ3.6**
ꢂ11.3ꢇ3.5
89.4ꢇ0.7**
95
15
3.3
2.0
3.6
4.8
5.2
4.3
—
1.5
—
5.3
—
Kaempferol (5)
Quercetin (15)a)
46.0ꢇ3.4**
1.6ꢇ5.2
20.7ꢇ4.7*
7,3ꢀ,4ꢀ-Trihydroxyflavone (23)
Lanceolatin B (24)
Medicarpin (25)
Homopterocarpin (26)
Melilotocarpan D (27)
Isoliquiritigenin (28)
Pongamol (29)
E-7-O-methylpongamol (30)
31
(ꢁ)-Pinoresinol (32)
Tryptanthrin (33)
17
ca. 11
ꢈ100
54
—
24
ꢈ100
29
ꢈ100
—
—
—
—
15.4ꢇ2.2**
—
—
—
—
—
8.6ꢇ3.0
—
—
51.5ꢇ1.4**
—
ꢂ5.5
—
—
—
19.4ꢇ4.0**
85.6ꢇ1.7**
ca. 19
ꢂ1.1
Inhibition (%)
Inhibition (%) of
b-hexosaminidase
at 300 mM
0 mM
30 mM
100 mM
300 mM
IC50 (mM)
Ketotifen fumarate
Tranilast
0.0ꢇ5.2
0.0ꢇ6.3
10.8ꢇ5.4
25.4ꢇ4.3**
30.6ꢇ4.8**
44.5ꢇ4.4**
68.9ꢇ2.4
75.3ꢇ0.2**
176
112
7.3
7.1
Each value represents meanꢇS.E.M. (nꢆ4). Significantly different from the control, ∗ pꢉ0.05, ∗∗ pꢉ0.01. a) Inhibition at 1 mM was 6.7ꢇ7.6%.
treatment of 4 with trimethylsilyldiazomethane (TMSCHN2)
yielded 31.20) Consequently, the structure of 4 was deter- 5 and 23 showed less activity than 15 and luteolin
mined to be as shown. (IC50ꢆ2.9 mM, data not shown) indicating that the 3ꢀ,4ꢀ-dihy-
With regard to structural requirements for the activity,
Inhibitory Effects of the Constituents of C. sicyoides droxyl group at the B-ring and the 5-hydroxyl group are im-
on the Release of b-Hexosaminidase by RBL-2H3 Cells portant for the greater activity, in agreement with our previ-
Histamine, which is released from mast cells upon stimula- ous results.37) The 4ꢀ-hydroxyl group of the benzofuran-type
tion with an antigen or a degranulation inducer, is usually stilbene 4 is suggested to be important for the activity, since
used as a degranulation marker in in vitro experiments on im- the methylated compound 31 showed less activity than 4.
mediate allergic reactions. b-Hexosaminidase is also stored Among the pterocarpans, 26 showed stronger activity than 25
in the secretory granules of mast cells and is released con- and 27, suggesting that the 7-methoxy group is important,
comitantly with histamine when mast cells are immunologi- but the 8- and/or 3ꢀ-hydroxyl groups reduced the activity.
cally activated.34,35) Therefore it is generally accepted that b- The detailed structural requirements for the activity should
hexosaminidase is a degranulation marker of mast cells.
As a part of our characterization studies of bioactive com-
be studied further.
In conclusion, three new flavonoid glycosides, cissosides I
ponents from natural medicines, we previously reported vari- (1), II (2), and III (3), and a new benzofuran-type stilbene,
ous inhibitors of the release of b-hexosaminidase including cissusin (4), were isolated from the methanolic extract of the
flavonoids and lignans.36—38) The glycosides (6—14, 16—22) aerial parts of C. sicyoides cultivated in Brazil. Their struc-
were not subjected to the examination, since the glycoside tures were elucidated on the basis of chemical and physico-
moiety of flavonoids markedly reduces their activity in this in chemical evidence. The inhibitory effects of the isolated con-
vitro bioassay method.37) As a continuing study of the anti- stituents on the release of b-hexosaminidase as a marker of
allergic constituents of natural medicines,39—48) the effects of degranulation in RBL-2H3 cells were examined. We found
the principal constituents of C. sicyoides lacking a glycoside that cissusin (4), flavonols [kaempferol (5), quercetin (15)],
moiety on the release of b-hexosaminidase in RBL-2H3 flavones [7,3ꢀ,4ꢀ-trihydroxyflavone (23), lanceolatin B (24)],
cells were examined. As shown in Table 2, cissusin (4, a pterocarpane [homopterocarpin (26)], chalcones [isoliquir-
IC50ꢆ95 mM), kaempferol (5, 15 mM), quercetin (15, 3.3 mM), itigenin (28), E-7-O-methylpongamol (30)], and tryptanthrin
7,3ꢀ,4ꢀ-trihydroxyflavone (23, 17 mM), tryptanthrin (33, ca. (33) markedly inhibited the release of b-hexosaminidase, and
19 mM), lanceolatin B (24, ca. 11 mM), homopterocarpin (26, several structural requirements of the active compounds for
54 mM), isoliquiritigenin (28, 20 mM), and E-7-O-methylpong- the inhibition were suggested.
amol (30, 29 mM) inhibited the release of b-hexosaminidase
Experimental
without inhibiting its enzyme activity [inhibition (%): ca.
The following instruments were used to obtain physical data: specific ro-
tations, Horiba SEPA-300 digital polarimeter (lꢆ5 cm); UV spectra, Shi-
madzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrome-
ter; EI-MS, CI-MS, and high-resolution CI-MS, JEOL JMS-GCMATE mass
5.3% at 100 mM] and their activities were more potent than
those of the antiallergic compounds tranilast (112 mM) and
ketotifen fumarate (176 mM).

October 2009
1093
spectrometer; FAB-MS and high-resolution MS, JEOL JMS-SX 102A mass [MeOH–H2O (50 : 50, v/v)] to give kaempferol (5, 32.4 mg, 0.0011%),
spectrometer; 1H-NMR spectra, JNM-LA500 (500 MHz) and JNM-ECA600 quercetin (15, 22.5 mg, 0.00077%), kaempferol-3-O-b-
-glucopyrano-
(600 MHz) spectrometers; 13C-NMR spectra, JNM-LA500 (125 MHz) and side (7, 8.6 mg, 0.00029%), kaempferol-3-O-a-L-rhamnopyranoside (6,
JNM-ECA600 (150 MHz) spectrometers with tetramethylsilane as an inter- 6.6 mg, 0.00023%), kaempferol-7-O-a-L-rhamnopyranoside (9, 19.0 mg,
D
nal standard; and HPLC detector, Shimadzu RID-6A refractive index and 0.00065%), and 7,3ꢀ,4ꢀ-trihydroxyflavone (23, 11.3 mg, 0.00039%). Fr. 4
SPD-10Avp UV–VIS detectors. For HPLC, COSMOSIL 5C18-PAQ (5.16 g) was subjected to reversed-phase silica gel column chromatography
250ꢄ4.6 mm i.d. and 250ꢄ20 mm i.d. columns were used for analytical and [150 g, MeOH–H2O (10 : 90→30 : 70→50 : 50→75 : 25, v/v)→MeOH] to
preparative purposes, respectively.
give 12 fractions [Fr. 4-1 (170 mg), Fr. 4-2 (287 mg), Fr. 4-3 (318 mg), Fr. 4-
The following experimental conditions were used for chromatography: or- 4 (107 mg), Fr. 4-5 (556 mg), Fr. 4-6 (254 mg), Fr. 4-7 (906 mg), Fr. 4-8
dinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji (540 mg), Fr. 4-9 (606 mg), Fr. 4-10 (140 mg), Fr. 4-11 (110 mg), and Fr. 4-
Silysia Chemical, Ltd., Aichi, Japan, 150—350 mesh); reversed-phase silica 12 (824 mg)]. Fr. 4-2 (287 mg) was separated using HPLC [MeOH–H2O
gel column chromatography, Chromatorex ODS DM1020T (Fuji Silysia (10 : 90, v/v)] to give gallic acid (202.8 mg, 0.0069%). Fr. 4-3 (318 mg) was
Chemical, 100—200 mesh); TLC, precoated TLC plates with Silica gel
separated using HPLC [MeOH–H2O (15 : 85, v/v)] to give indican (34.7 mg,
60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S 0.0012%). Fr. 4-5 (556 mg) was separated using HPLC [MeOH–H2O
(Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC (30 : 70, v/v)] to give epicatechin 3-O-gallate (60.4 mg, 0.0021%). Fr. 4-7
plates with Silica gel RP-18 WF254S (Merck, 0.25 mm); and detection was (906 mg) and Fr. 4-8 (540 mg) were separated using HPLC [MeOH–H2O
achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4, followed by (40 : 60, v/v)] to give cissoside I (1, 51.8 mg, 0.0018%), cissoside II (2,
heating.
Plant Material The aerial parts of C. sicyoides cultivated in Brazil were
obtained in January 2005 via Tamura Pharmaceutical Co., Ltd. (Wakayama,
Japan). A voucher specimen (2005.01, Brazil-02) is on file in our laboratory.
Extraction and Isolation The aerial parts of C. sicyoides (4.5 kg) were
8.5 mg, 0.00029%), cissoside III (3, 11.2 mg, 0.00038%), kaempferol-3-O-
a-L-rhamnopyranosyl-7-O-a-L-rhamnopyranoside (10, 51.7 mg, 0.0018%),
and kaempferol-3-O-a-L-arabinofuranosyl-7-O-a-L-rhamnopyranoside (14,
120.5 mg, 0.0041%). Fr. 4-9 (606 mg) was separated using HPLC
[MeOH–H2O (60 : 40, v/v)] to give tryptanthrin (33, 42.1 mg, 0.0015%). Fr.
powdered and extracted three times with methanol (MeOH) under reflux for 5 (4.12 g) was subjected to reversed-phase silica gel column chromatography
3 h. Evaporation of the solvent under reduced pressure gave the methanolic [120 g, MeOH–H2O (15 : 85→30 : 70→45 : 55→70 : 30, v/v)→MeOH] to
extract (199.7 g, 4.4%). The MeOH extract (174.7 g) was partitioned into an give 10 fractions [Fr. 5-1 (384 mg), Fr. 5-2 (270 mg), Fr. 5-3 (170 mg), Fr. 5-
EtOAc–H2O (1 : 1, v/v) mixture, and removal of the solvent in vacuo yielded 4 (233 mg), Fr. 5-5 (278 mg), Fr. 5-6 (960 mg), Fr. 5-7 (280 mg), Fr. 5-8
an EtOAc-soluble fraction (59.3 g, 1.5%) and an aqueous phase. The aque- (326 mg), Fr. 5-9 (335 mg), and Fr. 5-10 (540 mg)]. Fr. 5-2 (270 mg) was
ous phase was further extracted with n-BuOH to give an n-BuOH-soluble separated using HPLC [MeOH–H2O (30 : 70, v/v)] to give epicatechin 3-O-
fraction (39.4 g, 1.0%) and a water-soluble fraction (76.0 g, 1.9%).
gallate (26.4 mg, 0.00090%). Fr. 5-5 (278 mg) was separated using HPLC
[CH3CN–H2O (15 : 85, v/v)] to give quercetin-3-O-a-L-rhamnopyranoside
(16, 13.2 mg, 0.00045%), quercetin-3-O-b-D-glucopyranoside (17, 5.0 mg,
0.00017%), quercetin-3-O-b-D-galactopyranoside (18, 2.7 mg, 0.000092%),
The EtOAc-soluble fraction (44.3 g) was subjected to ordinary-phase sil-
ica gel column chromatography [1.4 kg, n-hexane–EtOAc (10 : 1→5 : 1→
2 : 1→1 : 1→1 : 2, v/v)→CHCl3–MeOH–H2O (10 : 3 : 1, lower layer→7 : 3 : 1,
lower layer→6 : 4 : 1, v/v/v)→MeOH] to give six fractions [Fr. 1 (9.27 g), Fr. and rutin (21, 8.6 mg, 0.00029%). Fr. 5-6 (960 mg) was separated using
2 (7.48 g), Fr. 3 (11.14 g), Fr. 4 (5.16 g), Fr. 5 (4.12 g), and Fr. 6 (6.28 g)]. Fr. HPLC [MeOH–H2O (45 : 55, v/v)] to give kaempferol-3-O-a-L-rhamnopyra-
1 (9.27 g) was subjected to reversed-phase silica gel column chromatography
nosyl-7-O-a-L-rhamnopyranoside (10, 129.9 mg, 0.0044%).
The n-BuOH-soluble fraction (27.7 g) was subjected to ordinary-phase
[270 g, MeOH–H2O (30 : 70→45 : 55→60 : 40→75 : 25, v/v)→MeOH] to
give seven fractions [Fr. 1-1 (110 mg), Fr. 1-2 (151 mg), Fr. 1-3 (658 mg), Fr. silica gel column chromatography [800 g, CHCl3–MeOH–H2O (10 : 3 : 1,
1-4 (1922 mg), Fr. 1-5 (751 mg), Fr. 1-6 (814 mg), and Fr. 1-7 (3884 mg)]. lower layer→7 : 3 : 1, lower layer→6 : 4 : 1, v/v/v)→MeOH] to give seven
Fr. 1-2 (151 mg) and Fr. 1-3 (658 mg) were purified using HPLC fractions [Fr. 1 (0.35 g), Fr. 2 (1.69 g), Fr. 3 (4.33 g), Fr. 4 (3.46 g), Fr. 5
[CH3CN–H2O (60 : 40, v/v)] to give pongamol (29, 52.1 mg, 0.0018%). Fr. 2 (6.40 g), Fr. 6 (4.84 g), and Fr. 7 (2.45 g)]. Fr. 3 (4.33 g) was subjected to re-
(7.48 g) was subjected to reversed-phase silica gel column chromatography versed-phase silica gel column chromatography [120 g, MeOH–H2O
[220 g, MeOH–H2O (20 : 80→30 : 70→45 : 55→60 : 40→75 : 25, v/v)→ (10 : 90→30 : 70→45 : 55→75 : 25, v/v)→MeOH]] to give eight fractions
MeOH] to give 11 fractions [Fr. 2-1 (330 mg), Fr. 2-2 (473 mg), Fr. 2-3 [Fr. 3-1 (165 mg), Fr. 3-2 (603 mg), Fr. 3-3 (407 mg), Fr. 3-4 (445 mg), Fr.
(318 mg), Fr. 2-4 (270 mg), Fr. 2-5 (310 mg), Fr. 2-6 (240 mg), Fr. 2-7 3-5 (359 mg), Fr. 3-6 (575 mg), Fr. 3-7 (406 mg), and Fr. 3-8 (740 mg)]. Fr.
(200 mg), Fr. 2-8 (256 mg), Fr. 2-9 (335 mg), Fr. 2-10 (658 mg), and Fr. 2-11 3-2 (603 mg) was separated by HPLC [MeOH–H2O (10 : 90, v/v)] to give
(2860 mg)]. Fr. 2-1 (330 mg) was separated using HPLC [CH3CN–H2O glucosyl salicylate (23.3 mg, 0.00084%) and indican (70 mg, 0.0025%).
(10 : 90, v/v)] to give 4-hydroxybenzoic acid (8.2 mg, 0.00028%), vanillic Fr. 3-6 (575 mg) was separated using HPLC [CH3CN–H2O (20 : 80, v/v)]
acid (23.0 mg, 0.00078%), and methyl gallate (10.0 mg, 0.00034%). Fr.
2-2 (473 mg) was purified using HPLC [CH3CN–H2O (15 : 85, v/v)] to
give 7-hydroxychromone (2.5 mg, 0.000085%), syringaldehyde (3.2 mg,
to give kaempferol-3-O-a-L-rhamnopyranosyl-7-O-a-L-rhamnopyranoside
(10, 88.6 mg, 0.0032%) and kaempferol-3-O-a-L-arabinopyranosyl-7-O-a-
L-rhamnopyranoside (13, 52.0 mg, 0.0019%). Fr. 4 (3.46 g) was subjected
0.00011%), and 4-hydroxycinnamic acid (5.0 mg, 0.00017%). Fr. 2-4 to reversed-phase silica gel column chromatography [100 g, MeOH–H2O
(270 mg) was separated using HPLC [CH3CN–H2O (25 : 75, v/v)] to give (20 : 80→30 : 70→45 : 55→60 : 40, v/v)→MeOH]] to give six fractions [Fr.
melilotocarpan
D (27, 12.7 mg, 0.00043%) and (ꢁ)-pinoresinol (32, 4-1 (430 mg), Fr. 4-2 (646 mg), Fr. 4-3 (737 mg), Fr. 4-4 (264 mg), Fr. 4-5
20.0 mg, 0.00068%). Fr. 2-5 (310 mg) was purified using HPLC
[MeOH–H2O (50 : 50, v/v)] to give medicarpin (25, 3.1 mg, 0.00011%) and
homopterocarpin (26, 5.2 mg, 0.00018%). Fr. 2-6 (240 mg) was separated
using HPLC [MeOH–H2O (55 : 45, v/v)] to give isoliquiritigenin (28,
11.5 mg, 0.00039%) and E-7-O-methylpongamol (30, 10.0 mg, 0.00034%).
Fr. 2-8 (256 mg) was separated using HPLC [MeOH–H2O (65 : 35, v/v)] to
give tryptanthrin (33, 27.1 mg, 0.00091%), isoliquiritigenin (28, 10.7 mg,
0.00036%), and E-7-O-methylpongamol (30, 23.1 mg, 0.00078%). Fr. 2-9
(103 mg), and Fr. 4-6 (730 mg)]. Fr. 4-3 (737 mg) was separated using HPLC
[CH3CN–H2O (15 : 85, v/v)] to give kaempferol-3-O-b-D-glucopyranosyl-7-
O-a-L-rhamnopyranoside (11, 10.7 mg, 0.00039%), kaempferol-3-O-b-D-
galactopyranosyl-7-O-a-L-rhamnopyranoside (12, 28.0 mg, 0.0010%), rutin
(21, 91.2 mg, 0.0033%), and quercetin-3-O-a-L-rhamnopyranosyl-7-O-a-L-
rhamnopyranoside (22, 12.0 mg, 0.00043%). Fr. 4-4 (264 mg) was separated
using HPLC [CH3CN–H2O (20 : 80, v/v)] to give kaempferol-3-O-a-L-
rhamnopyranosyl-7-O-a-L-rhamnopyranoside (10, 22.0 mg, 0.00079%). Fr.
(335 mg) was separated using HPLC [CH3CN–H2O (40 : 60, v/v)] to give 5 (6.40 g) was subjected to reversed-phase silica gel column chromatography
cissusin (4, 15.8 mg, 0.00054%), lanceolatin B (24, 27.7 mg, 0.00094%), [180 g, MeOH–H2O (30 : 70→50 : 50→75 : 25, v/v)→MeOH]] to give seven
E-7-O-methylpongamol (30, 17.5 mg, 0.00059%), and 2-(2ꢀ,4ꢀ- fractions [Fr. 5-1 (1250 mg), Fr. 5-2 (508 mg), Fr. 5-3 (422 mg), Fr. 5-4
dimethoxyphenyl)-5,6-methylenedioxybenzofuran (31, 4.6 mg, 0.00016%). (700 mg), Fr. 5-5 (859 mg), Fr. 5-6 (623 mg), and Fr. 5-7 (1377 mg)]. Fr. 5-3
Fr. 3 (11.14 g) was subjected to reversed-phase silica gel column chromatog- (422 mg) and Fr. 5-4 (700 mg) were separated using HPLC [CH3CN–H2O
raphy [330 g, MeOH–H2O (20 : 80→30 : 70→45 : 55→70 : 30, v/v)→MeOH]
(15 : 85, v/v)] to give rutin (21, 357.8 mg, 0.013%).
The known compounds were identified by comparison of their physical
to give nine fractions [Fr. 3-1 (448 mg), Fr. 3-2 (603 mg), Fr. 3-3 (574 mg),
Fr. 3-4 (492 mg), Fr. 3-5 (1064 mg), Fr. 3-6 (1430 mg), Fr. 3-7 (772 mg), Fr. data ([a]D, IR, 1H-, 13C-NMR, MS) with reported values8—15,17—29,31) or
3-8 (1335 mg), and Fr. 3-9 (2645 mg)]. Fr. 3-5 (1064 mg) was separated those of authentic samples.16,30)
using HPLC [MeOH–H2O (40 : 60, v/v)] to give kaempferol-3-O-a-L-arabi-
nofuranoside (8, 7.0 mg, 0.00024%), quercetin-3-O-a-L-arabinopyranoside
(19, 15.0 mg, 0.00051%), and quercetin-3-O-a-L-arabinofuranoside (20,
16.7 mg, 0.00057%). Fr. 3-6 (1430 mg) was separated using HPLC
Cissoside I (1): A yellow powder, [a]D27 ꢂ176.8° (cꢆ0.70, MeOH). UV
lmax (MeOH) nm (log e): 266 (4.32), 347 (4.21). IR (KBr) cmꢂ1: 3415,
1720, 1655, 1601, 1493, 1375, 1208, 1179, 1120, 1063. 1H-NMR (600 MHz,
pyridine-d5) d: 6.78, 6.96 (1H each, both d, Jꢆ2.1 Hz, H-6, 8), 7.33, 8.32

1094
Vol. 57, No. 10
(Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation [Shodex
OR-2 (Showa Denko Co., Ltd., Tokyo, Japan)]; mobile phase, CH3CN–H2O
(85 : 15, v/v); flow rate, 0.7 ml/min; and column temperature, room tempera-
ture. Identification of L-rhamnose (i) and L-arabinose (ii) from 2 present in
the aqueous layer was carried out by comparison of their retention times and
optical rotations with those of authentic samples. tR: (i) 8.7 min (negative op-
tical rotation); (ii) 11.4 min (positive optical rotation).
Methylation of 4 with TMSCHN2 A solution of 4 (3.4 mg) in MeOH
(1 ml) was treated with trimethylsilyldiazomethane (TMSCHN2) (10% in
hexane, 0.2 ml), and the mixture was stirred at room temperature for 3 h. Re-
moval of the solvent under reduced pressure gave a residue, which was puri-
fied using silica gel column chromatography [0.8 g, hexane–EtOAc (30 : 1,
v/v)] to give 31 (2.8 mg, 78.5%).
(2H each, both d, Jꢆ8.6 Hz, H-3ꢀ, -5ꢀ, -2ꢀ, -6ꢀ), [H-arabinose part: 1.88 (3H,
s, CH3CO–), 4.42 (1H dd, Jꢆ6.8, 12.4 Hz, Ha-5ꢃ), 4.62 (1H dd, Jꢆ3.4,
12.4 Hz, Hb-5ꢃ), 4.64 (1H, m, H-3ꢃ), 4.66 (1H, m, H-4ꢃ), 5.25 (1H, br d,
Jꢆ2.8 Hz, H-2ꢃ), 6.49 (1H, br s, H-1ꢃ)], [H-rhamnose part: 1.68 (3H, d,
Jꢆ6.1 Hz, H3-6ꢅ), 4.31 (1H m, H-5ꢅ), 4.41 (1H dd, Jꢆ8.9, 9.6 Hz, H-4ꢅ),
4.67 (1H, dd, Jꢆ3.4, 9.6 Hz, H-3ꢅ), 4.73 (1H, br s, H-2ꢅ), 6.26 (1H, br s,
H-1ꢅ)]. 13C-NMR (150 MHz, pyridine-d5) dC: see Table 1. Positive-ion
FAB-MS m/z: 629 (MꢁNa)ꢁ. HR-FAB-MS m/z: 629.1476 [Calcd for
C28H30O15Na (MꢁNa)ꢁ: 629.1482].
Cissoside II (2): A yellow powder, [a]D26 ꢂ181.5° (cꢆ0.50, MeOH). UV
lmax (MeOH) nm (log e): 258 (4.54), 349 (3.93). IR (KBr) cmꢂ1: 3400,
1655, 1609, 1509, 1460, 1364, 1200, 1169, 1115, 1065. 1H-NMR (600 MHz,
pyridine-d5) d: 6.75, 6.84 (1H each, both d, Jꢆ2.0 Hz, H-6, -8), 7.37 (1H, d,
Jꢆ8.2 Hz, H-5ꢀ), 7.99 (1H, dd, Jꢆ2.0, 8.2 Hz, H-6ꢀ), 8.19 (1H, d, Jꢆ2.0 Hz,
H-2ꢀ), [H-arabinose part: 4.10 (1H dd, Jꢆ4.1, 11.7 Hz, Ha-5ꢃ), 4.17 (1H dd,
Jꢆ2.7, 11.7 Hz, Hb-5ꢃ), 4.75 (1H, m, H-4ꢃ), 4.91 (1H, m, H-3ꢃ), 5.20 (1H
br d, Jꢆ2.8 Hz, H-2ꢃ), 6.53 (1H, br s, H-1ꢃ)], [H-rhamnose part: 1.68 (3H, d,
Jꢆ6.2 Hz, H3-6ꢅ), 4.30 (1H m, H-5ꢅ), 4.42 (1H dd, Jꢆ8.9, 9.6 Hz, H-4ꢅ),
4.66 (1H, dd, Jꢆ2.8, 9.6 Hz, H-3ꢅ), 4.71 (1H, br s, H-2ꢅ), 6.22 (1H, br s,
H-1ꢅ)]. 13C-NMR (150 MHz, pyridine-d5) dC: see Table 1. Positive-ion
FAB-MS m/z: 603 (MꢁNa)ꢁ. HR-FAB-MS m/z: 603.1322 [Calcd for
C26H28O15Na (MꢁNa)ꢁ: 603.1326].
2-(2ꢀ,4ꢀ-Dimethoxyphenyl)-5,6-methylenedioxybenzofuran (31): A color-
less powder. 1H-NMR (600 MHz, CDCl3) d: 3.86, 3.96 (3H each, both s,
OCH3-4ꢀ, -2ꢀ), 5.98 (2H, s, OCH2O-5, 6), 6.55 (1H, d, Jꢆ2.1 Hz, H-3ꢀ), 6.59
(1H, dd, Jꢆ2.1, 8.6 Hz, H-5ꢀ), 6.93 (1H, s, H-4), 7.00 (1H, s, H-7), 7.09
(1H, s, H-3), 7.88 (1H, d, Jꢆ8.6 Hz, H-6ꢀ). 13C-NMR (150 MHz, CDCl3) dC:
see Table 1. EI-MS m/z: 298 (Mꢁ) (100). HR-EI-MS m/z: 298.0836 (Calcd
for C17H14O5: 298.0841). The physical data agreed with published data.20)
Inhibitory Effects on the Release of b-Hexosaminidase by RBL-2H3
Cells The inhibitory effects of the test samples on the release of b-hex-
osaminidase from RBL-2H3 cells (Cell No. JCRB0023, obtained from
Health Science Research Resources Bank, Osaka, Japan) were evaluated
using the method reported previously37—48) with some modifications. Briefly,
RBL-2H3 cells were dispensed into 48-well plates at a concentration of
4ꢄ104 cells/well using Eagle’s minimum essential medium (MEM, Sigma)
containing fetal calf serum (10%), penicillin (100 units/ml), streptomycin
(100 mg/ml), and 0.45 mg/ml of anti-dinitrophenyl (DNP) IgE. The mixture
was incubated overnight at 37 °C in 5% CO2 for sensitization of the cells.
Then, the cells were washed twice with 200 ml of Siraganian buffer [NaCl
119 mM, KCl 5 mM, MgCl2 0.4 mM, piperazine-N,Nꢀ-bis(2-ethanesulfonic
acid) (PIPES) 25 mM, and NaOH 40 mM, pH 7.2], and incubated in 80 ml of
Siraganian buffer [glucose 5.6 mM, CaCl2 1 mM, and 0.1% bovine serum al-
bumin (BSA) were added] for an additional 10 min at 37 °C. Aliquots
(10 ml) of test sample solution were added to each well and incubated for
10 min, followed by the addition of 10 ml of antigen (dinitrophenylated
bovine serum albumin (DNP-BSA), final concentration 10 mg/ml) at 37 °C
for 10 min to stimulate the cells to evoke allergic reactions (degranulation).
The reaction was stopped by cooling in an ice bath for 10 min. The super-
natant (40 ml) was transferred into a 96-well microplate and incubated with
40 ml of substrate (p-nitrophenyl-N-acetyl-b-D-glucosaminide 1 mM) in cit-
rate buffer 0.1 M (pH 4.5) at 37 °C for 2 h. The reaction was stopped by
adding 200 ml of stop solution (Na2CO3/NaHCO3 0.1 M, pH 10.0). The ab-
sorbance was measured using 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 0.1%).
Cissoside III (3): A yellow powder, [a]D26 ꢂ187.9° (cꢆ0.70, MeOH). UV
lmax (MeOH) nm (log e): 257 (4.25), 350 (3.93). IR (KBr) cmꢂ1: 3397,
1717, 1655, 1603, 1509, 1370, 1206, 1177, 1115, 1061. 1H-NMR (600 MHz,
pyridine-d5) d: 6.75, 6.84 (1H each, both d, Jꢆ2.0 Hz, H-6, -8), 7.37 (1H, d,
Jꢆ8.2 Hz, H-5ꢀ), 7.99 (1H, dd, Jꢆ2.0, 8.2 Hz, H-6ꢀ), 8.19 (1H, d, Jꢆ2.0 Hz,
H-2ꢀ), [H-arabinose part: 1.89 (3H, s, CH3CO–), 4.42 (1H dd, Jꢆ6.9,
12.4 Hz, Ha-5ꢃ), 4.62 (1H dd, Jꢆ3.4, 12.4 Hz, Hb-5ꢃ), 4.65 (1H, m, H-3ꢃ),
4.75 (1H, m, H-4ꢃ), 5.22 (1H, br d, Jꢆ3.5 Hz, H-2ꢃ), 6.47 (1H, br s, H-1ꢃ)],
[H-rhamnose part: 1.68 (3H, d, Jꢆ6.2 Hz, H3-6ꢅ), 4.30 (1H m, H-5ꢅ), 4.41
(1H, dd, Jꢆ8.9, 9.6 Hz, H-4ꢅ), 4.66 (1H, dd, Jꢆ3.4, 9.6 Hz, H-3ꢅ), 4.71
(1H, br s, H-2ꢅ), 6.22 (1H, br s, H-1ꢅ)]. 13C-NMR (150 MHz, pyridine-d5)
dC: see Table 1. Positive-ion FAB-MS m/z: 645 (MꢁNa)ꢁ. HR-FAB-MS
m/z: 645.1429 [Calcd for C28H30O16Na (MꢁNa)ꢁ: 645.1432].
Cissusin (4): A colorless powder. UV lmax (MeOH) nm (log e): 212
(4.37), 282 (4.06), 330 (4.38), 345 (4.39). IR (KBr) cmꢂ1: 3433, 1623, 1614,
1
1592, 1501, 1458, 1321, 1202, 1173, 1036, 945, 845. H-NMR (600 MHz,
CDCl3) d: 3.93 (3H s, OCH3-2ꢀ), 5.97 (2H, s, OCH2O-5, -6), 6.52 (1H, d,
Jꢆ2.1 Hz, H-3ꢀ), 6.54 (1H, dd, Jꢆ2.1, 8.9 Hz, H-5ꢀ), 6.92 (1H, s, H-4), 6.98
(1H, s, H-7), 7.06 (1H, s, H-3), 7.81 (1H, d, Jꢆ8.9 Hz, H-6ꢀ). 13C-NMR
(150 MHz, CDCl3) dC: see Table 1. EI-MS m/z: 284 (Mꢁ) (100). HR-EI-MS
m/z: 284.0687 (Calcd for C16H12O5: 284.0685).
Deacylation of
1
and
3
A solution of 1 (5.2 mg) in 0.5%
NaOMe–MeOH (1.0 ml) was stirred at room temperature for 1 h. The reac-
tion mixture was neutralized with Dowex HCR-W2 (Hꢁ form), and the resin
was removed by filtration. Evaporation of the solvent under reduced pressure
furnished a residue, which was purified by silica gel chromatography [0.8 g,
CHCl3–MeOH–H2O (15 : 3 : 1, lower layer)] to give 14 (4.1 mg, 84.7%).
Using a similar procedure, 2 (3.3 mg, 78.6%) was obtained from 3 (4.5 mg).
Kaempferol-3-O-a-L-arabinofuranosyl-7-O-a-L-rhamnopyranoside (14):
A yellow powder, [a]D28 ꢂ170.6° (cꢆ0.80, MeOH). 1H-NMR (600 MHz,
pyridine-d5) d: 6.80, 6.98 (1H each, both d, Jꢆ2.1 Hz, H-6, -8), 7.33, 8.40
(2H each, both d, Jꢆ8.2 Hz, H-3ꢀ, -5ꢀ, -2ꢀ, -6ꢀ), [H-arabinose part: 4.09 (1H
dd, Jꢆ4.1, 11.7 Hz, Ha-5ꢃ), 4.15 (1H dd, Jꢆ2.7, 11.7 Hz, Hb-5ꢃ), 4.68 (1H,
m, H-4ꢃ), 4.92 (1H, m, H-3ꢃ), 5.26 (1H, br d, Jꢆ2.8 Hz, H-2ꢃ), 6.57 (1H,
br s, H-1ꢃ)], [H-rhamnose part: 1.67 (3H, d, Jꢆ6.2 Hz, H3-6ꢅ), 4.30 (1H m,
H-5ꢅ), 4.40 (1H dd, Jꢆ8.9, 9.6 Hz, H-4ꢅ), 4.67 (1H, dd-like, H-3ꢅ), 4.73
(1H, br s, H-2ꢅ), 6.27 (1H, br s, H-1ꢅ)]. 13C-NMR (150 MHz, pyridine-d5)
dC: see Table 1. Positive-ion FAB-MS m/z: 587 (MꢁNa)ꢁ. HR-FAB-MS
m/z: 587.1374 [Calcd for C26H28O14Na (MꢁNa)ꢁ: 587.1377]. The physical
data agreed with published data.8)
Acid Hydrolysis of 2 A solution of 2 (2.0 mg) in HCl–1,4-dioxane 1 M
(1 : 1, v/v, 1.0 ml) was heated under reflux for 2 h. After cooling, the reaction
mixture was poured into ice-water and neutralized with Amberlite IRA-400
(OHꢂ form), and the resin was removed by filtration. Then, the filtrate was
extracted with EtOAc. The EtOAc layer was subjected to HPLC analysis
under the following conditions: HPLC column, YMC-Pack ODS-A, (YMC,
4.6 mm i.d.ꢄ250 mm); detection, RID-6A refractive index; mobile phase,
CH3OH–H2O (50 : 50, v/v); and flow rate, 0.8 ml/min. Identification of
quercetin from 2 present in the EtOAc layer was carried out by comparison
of its retention time with that of an authentic sample. tR: 23.8 min. In addi-
tion, the aqueous layer was subjected to HPLC analysis under the following
conditions: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d.ꢄ250 mm
The percentage of inhibition of the release of b-hexosaminidase by the
test material was calculated using the following equation, and IC50 values
were determined graphically:
inhibition (%)ꢆ[1ꢂ(TꢂBꢂN)/(CꢂN)]ꢄ100
where C is the control with DNP-BSA (ꢁ), test sample (ꢂ); T is test with
DNP-BSA (ꢁ), test sample (ꢁ); B is blank with DNP-BSA (ꢂ), test sample
(ꢁ); and N is normal with DNP-BSA (ꢂ), test sample (ꢂ).
Under these conditions, it was calculated that 10—15% of b-hex-
osaminidase was released from the cells in the control groups by determina-
tion of the total b-hexosaminidase activity after treatment with 0.05% Triton
X-100.
To clarify whether the antiallergic effects of samples were due to the inhi-
bition of hexosaminidase release, but not a false positive due to the inhibi-
tion of b-hexosaminidase activity, the following assay was carried out. The
supernatant (36 ml) of the control group as an enzyme solution, substrate so-
lution (40 ml), and test sample solution (4 ml) were transferred into a 96-well
microplate and enzyme activity was examined as described above.
Statistical Analysis Values are expressed as meanꢇS.E.M. One-way
analysis of variance followed by Dunnett’s test was used for statistical analy-
sis.
Acknowledgments This research was supported by the 21st COE Pro-
gram, Academic Frontier Project, and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan.

October 2009
1095
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