Laxative effect of agarwood leaves and its mechanism

Article abstract of DOI:10.1271/bbb.70361

We investigated the laxative activity of an extract of agarwood leaves from Aquilaria sinensis. The laxative activity was measured in mice by counting the stool frequency and stool weight, and the drugs were orally administered. An acetone extract of agar

Full text of DOI:10.1271/bbb.70361

Biosci. Biotechnol. Biochem., 72 (2), 335–345, 2008
Laxative Effect of Agarwood Leaves and Its Mechanism
y
Hideaki HARA,1; Yasuaki ISE,2 Nobutaka MORIMOTO,1 Masamitsu SHIMAZAWA,1
2
Koji ICHIHASHI,2 Masayoshi OHYAMA,2 and Munekazu IINUMA
1Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University,
Gifu 502-8585, Japan
2Department of Bioactive Molecules, Pharmacognosy, Gifu Pharmaceutical University, Gifu 502-8585, Japan
Received June 7, 2007; Accepted October 23, 2007; Online Publication, February 7, 2008
[doi:10.1271/bbb.70361]
We investigated the laxative activity of an extract of
agarwood leaves from Aquilaria sinensis. The laxative
activity was measured in mice by counting the stool
frequency and stool weight, and the drugs were orally
administered. An acetone extract of agarwood leaves
and senna (a representative laxative drug) both increas-
ed the stool frequency and weight, but a methanol
extract did not. The laxative effect of the acetone extract
was milder than that of the anthraquinoid laxative,
senna, and the former did not induce diarrhea as a
severe side effect. We identified the main constituent
contributing to the laxative effect of the acetone extract
as genkwanin 5-O-ꢀ-primeveroside (compound 4).
Compound 4 strengthened the spontaneous motility
and induced contraction in the ileum. This ileal
contraction induced by compound 4 was inhibited by
atropine, but not by azasetron, suggesting that the effect
of compound 4 was mediated by acetylcholine receptors,
and not by serotonin. The laxative mechanism for
compound 4 may in part involve stimulation of intesti-
nal motility via acetylcholine receptors.
genkwa, Thymelaeaceae), and pharbitis seed (Pharbitis
nil, Convolvulaceae). However, their purgative activ-
ities are also too powerful, and they are not now in
common use as laxatives. Against this background, we
searched for other naturally occurring laxatives. We
describe here a new laxative source, namely, agarwood
leaves (Aquilaria sinensis, Thymelaeceae; ‘‘Jinko’’ in
Japanese). Agarwood is well known as incense in the
Oriental region, and has also been used as a sedative,
analgesic, and digestive in traditional medicine. Char-
acteristic sesquiterpenes4–6) and chromone deriva-
tives7–9) have been isolated from agarwood, and some
of these sesquiterpenes have sedative and analgesic
effects.10,11) Although thorough phytochemical research
has been carried out on the trunk and resin of agarwood,
little is known about the pharmacological effect of
agarwood leaves. The purpose of the present study was
to elucidate this effect. We first evaluated the laxative
activities of an acetone extract of agarwood leaves
(EAL) and methanol EAL, then identified the major
laxative constituents of EAL, and finally investigated its
mechanism of action.
Key words: laxative effect; agarwood leaf; Aquilaria
sinensis
Materials and Methods
Constipation is a life-style problem and may increase
during aging. It can be a chronic condition for which
patients may need to take laxatives in the long term.
Nowadays, 20 to 30% of people over the age of 60 use
laxatives more than once a week,1) the major laxatives
used being anthraquinoids based on a crude extract of
senna (Cassia angustifolia, C. acutifolia, Legumino-
ceae) or rhubarb (Rheum palmatum, R. tanguticum, etc.,
Polygonaceae).2) However, the powerful purgative
activity of anthraquinoids makes them unsuitable for
regular use, and even periodic use of these laxatives can
induce pseudomelanosis coli, a risk factor for colorectal
neoplasma.3) Other crude purgative drugs include rose
fruit (Rosa multiflora, Rosaceae), genka (Daphne
Materials. Agarwood (Aquilaria sinensis) and senna
leaves were purchased from Kaya Co., Ltd. (Osaka,
Japan) and Matsuura Yakugyo Co., Ltd. (Aichi, Japan),
respectively. Acetylcholine chloride, serotonin, atropine
sulfate, azasetron hydrochloride, and dimethyl sulfoxide
were purchased from Wako Pure Chemical Co., Ltd.
(Osaka, Japan).
Extraction and isolation procedures. The process of
extraction and isolation is shown in Fig. 1. Ground
agarwood leaves (1.5 kg) were successively extracted
with acetone (8-liter) four times, and then six times with
methanol for two days. Evaporation of the solvents in
vacuo gave acetone (70 g) and methanol (150 g) extracts
y
To whom correspondence should be addressed. Tel/Fax: +81-58-237-8596; E-mail: hidehara@gifu-pu.ac.jp
Abbreviations: EAL, extract of agarwood leaf

336
H. HARA et al.
Aquillaria sinensis dried leaves ( 1.5 kg )
Extracted at r.t.
Methanol ext. ( 150 g )
Acetone ext. ( 68 g )
SiO2 CC
( CHCl3:MeOH=5:1 , gradient)
Filtration
( CHCl3:MeOH=1:1 )
Compound 1
( 3:1 )
( MeOH )
( 2.45 g )
Filtrate ( 65 g )
SiO2 CC
Fr. 5 ( 12 g )
Fr. 6 ( 75 g )
Diaion HP-20
(H2O→EtOH, gradient)
( CHCl3:MeOH=10:1 , gradient )
EtOH Fr.( 15 g )
( 3:1 )
( 5:1 )
DMS CC
( 20% MeOH)
Decantation
( CHCl3:MeOH=5:1 )
Fr. 3 ( 4 g )
Fr. 4 ( 6 g )
Fr. 2 ( 4.5 g )
Recrystallized
( MeOH:H2O=9:1 )
Sephadex LH-20
( 50% MeOH)
SiO2 CC
( Benzene:MeOH:H2O
=8:2:0.1 )
Sephadex LH-20
( MeOH )
Frs. 2-13 ( 1.5 g )
Recrystallized
( MeOH:H2O=9:1 )
Sephadex LH-20
( 90% MeOH )
Compound 2
Compound 4
( 0.63 g )
Compound 4
( 0.33 g )
Compound 7
( 1.5 g )
( 0.72 g )
Fig. 1. Flowchart of the Isolation Process for the Acetone and MeOH Extracts of Leaves of Aquillaria sinensis.
which were used in the laxative assay and for identi-
fication of the constituents. Ground senna leaves (0.5 kg)
were extracted with 50% aqueous ethanol (3-liter) for
one day. Evaporation of the solvent in vacuo gave an
aqueous ethanol extract (32.4 g) which was used in the
laxative assay.
The acetone extract of agarwood leaves was suspend-
ed in CHCl3-methanol (1:1), and the insoluble part was
recrystallized from methanol to yield compound 1
(2.45 g). The filtrate was subjected to chromatography
on silica gel eluted with a CHCl3-methanol solvent
system (10:1–1:1, linear gradient) to prepare six crude
fractions. The 5:1 crude fraction was separated in a
Sephadex LH-20 column eluted with methanol to give
compound 2 (1.5 g). The polar crude fraction (3:1) was
separated by chromatography in a silica gel column
eluted with a benzene-methanol-H2O solvent system
(8:2:0.1), and then recrystallized from methanol-H2O
(9:1) to give compound 4 (genkwanin 5-O-ꢀ-primevero-
side) (0.63 g).
The methanol extract of agarwood leaves was sub-
jected to chromatography in a silica gel column eluted
with a CHCl3-methanol solvent system (5:1–1:1, finally
methanol, linear gradient) to prepare six crude fractions.
The 2:1 crude fraction was purified by decantation with
CHCl3-methanol (5:1), and then recrystallized with
methanol-H2O (9:1) to yield compound 4 (0.33 g). The
crude methanol fraction was separated in a Diaion HP-
20 column (H2O-ethanol, linear gradient), DMS column
(20% methanol, linear gradient) and Sephadex LH-20
column (50% and 90% methanol) to give compound 7
(0.72 g).
Animals. The experimental design and all procedures
were in accordance with the Animal Care Guidelines
issued by the Animal Experimental Committee of Gifu
Pharmaceutical University. Male ddY mice (7–10 weeks
old), 20–35 g body weight, were purchased from Japan
SLC (Hamamatsu, Japan). Adult JW rabbits and adult
Hartley guinea pigs were purchased from Kitayama
Labes Co. (Nagano, Japan) and Nippon SLC Co.
(Shizuoka, Japan), respectively. The animals were
housed at a controlled room temperature (24.5–
25.0 ꢀC) with a 12/12 h light/dark cycle. Animal food
pellets and tap water were provided ad libitum.
Frequency and weight of stools. Test samples were
dissolved or suspended in distilled water and adminis-
tered orally at 0.1 ml/10 g of body weight to mice by an
oral tube (FG6202, Fuchigami Kikai Co., Kyoto, Japan).
The frequency and weight of the stools were measured
as the frequency and total stool wet weight produced by
each mouse during four consecutive 2-h periods (0–2 h,
2–4 h, 4–6 h, and 6–8 h) after the administration of a test
sample or distilled water (control). Each mouse was
observed in a cage (11 cm height, 17.5 cm width, and
11 cm depth).
Diarrhea frequency. The diarrhea frequency was
determined by counting the number of episodes of
diarrhea in each mouse during the four 2-h periods (0–
2 h, 2–4 h, 4–6 h, and 6–8 h) after the administration of a
test sample or distilled water (control). The criterium for
diarrhea was whether ordinary or non-ordinary stools
were produced. An ordinary stool means in pellet form,

Laxative Effect of Agarwood Leaves
337
brown color, and rugby ball shape. A non-ordinary stool
means loose, muddy, and watery. The judgment was
made by a masked observer (Y.I.).
Major constituents of EAL and their spectral data
The structures of the major constituents of EAL are
shown in Fig. 4. Two new benzophenone derivatives
[iriflophenone 2-O-ꢁ-rhamnoside (compound 2) and
iriflophenone 3, 5-C-ꢀ-diglucoside (compound 7)] and
two known compounds [mangiferin (compound 1) and
genkwanin 5-O-ꢀ-primeveroside (compound 4)] were
isolated, together with three minor compounds [gen-
kwanin 5-O-ꢀ-glucoside (compound 3), genkwanin
(compound 5), and genkwanin 40-methyl ether 5-O-ꢀ-
primeveroside (compound 6)].
Spontaneous movement of the isolated ileum. The
rabbits and guinea pigs were killed by exsanguination
under deep anesthesia (pentobarbital and diethyl ether,
respectively). Segments of the rabbit ileum were
suspended at 1 g tension in an organ bath containing
Tyrode’s solution (137 mm NaCl, 5 mm KCl, 2.5 mm
.
CaCl2 2H2O, 0.1 mm MgCl2-6H2O, 0.3 mm NaHPO4-
2H2O, and 5.6 mm glucose; pH 7.4). Spontaneous
movement was monitored by a recorder (U-228; Pantos
Co., Kyoto, Japan) via an isotonic transducer (EG-650H;
Nihon Kohden, Tokyo, Japan). Segments of the guinea
pig ileum were suspended at 0.5 g tension in an organ
bath containing Tyrode’s solution, and recordings were
made as just described. Each test drug was cumulatively
added to the bath. Various receptor agonists and
antagonists (acetylcholine chloride, serotonin, atropine
sulfate, and azasetron hydrochloride) were also sepa-
rately added to the bath.
Mangiferin (compound 1): a pale yellow powder;
negative ion HR-FAB-MS m/z: 421.0779 [M-H]ꢂ
1
(calcd. 421.0771 for C19H17O11); H-NMR (DMSO-d6)
ꢂ: 3.14 (1H, dd, J ¼ 8:2, 7.8 Hz, glc-H-4), 3.22 (1H, dd,
J ¼ 8:2, 5.6 Hz, glc-H-5), 3.22 (1H, dd, J ¼ 9:0, 7.8 Hz,
glc-H-3), 3.34 (1H, dd, J ¼ 11:2, 5.6 Hz, glc-H-6), 3.67
(1H, d, J ¼ 11:2 Hz, glc-H-6), 4.03 (1H, dd, J ¼ 9:8,
9.0 Hz, glc-H-2), 4.58 (1H, d, J ¼ 9:8 Hz, glc-H-1), 6.36
(1H, s, H-4), 6.85 (1H, s, H-5), 7.37 (1H, s, H-8), 9.68
(1H, s, C-7-OH), 10.51 (1H, s, C-3-OH), 10.58 (1H, s,
C-6-OH), 13.77 (1H, s, C-1-OH); 13C-NMR (DMSO-d6)
ꢂ: 61.5 (glc-6), 70.2 (glc-4), 70.6 (glc-2), 73.1 (glc-1),
79.0 (glc-3), 81.6 (glc-5), 93.3 (C-4), 101.3 (C-9a),
102.6 (C-5), 107.6 (C-2), 108.1 (C-8), 111.7 (C-8a),
143.7 (C-7), 150.8 (C-10a), 154.0 (C-6), 156.2 (C-4a),
161.8 (C-1), 163.8 (C-3), 179.1 (C-9).
Statistical analysis. Data are presented as the mean ꢁ
S.E.M. Statistical comparisons were made with one-way
ANOVA followed by Dunnet’s multiple-comparison
test, paired t-test, or Student’s t-test (StatView software
version 5.0; SAS Institute, Cary, NC, USA). A value of
p < 0:05 is considered to indicate statistical signifi-
cance.
Iriflophenone 2-O-ꢁ-rhamnoside (compound 2): a
pale yellow powder; negative ion HR-FAB-MS m/z:
391.1019 [M-H]ꢂ (calcd. 391.1029 for C19H19O9);
EIMS m/z (rel. int.): 392 (M+, 4), 246 (M-rhamnose,
1
Results
96), 245 (100), 153 (54), 121 (28), 118 (6); H-NMR
(methanol-d4) ꢂ: 1.19 (3H, d, J ¼ 6:4 Hz, rha-H-6), 3.10
(1H, dd, J ¼ 9:6, 3.0 Hz, rha-H-3), 3.29 (1H, dd, J ¼
9:6, 3.6 Hz, rha-H-4), 3.41 (1H, dd, J ¼ 3:0, 0.8 Hz, rha-
H-2), 3.44 (1H, dd, J ¼ 6:2, 3.6 Hz, rha-H-5), 5.22 (1H,
d, J ¼ 0:8 Hz, rha-H-1), 6.07 (1H, d, J ¼ 2:0 Hz, H-5),
6.30 (1H, d, J ¼ 2:0 Hz, H-3), 6.81 (2H, d, J ¼ 8:6 Hz,
H-30,50), 7.61 (2H, d, J ¼ 8:6 Hz, H-20,60); 13C-NMR
(methanol-d4) ꢂ: 17.9 (rha-6), 70.7 (rha-5), 71.4 (rha-2),
71.7 (rha-3), 73.5 (rha-4), 100.2 (rha-1), 95.5 (C-3), 97.9
(C-5), 109.2 (C-1), 115.9 (C-30,50), 132.6 (C-10), 132.8
(C-20,60), 158.3 (C-6), 158.4 (C-2), 163.0 (C-4,40), 197.6
(C-7).
To hydrolyze compound 2, a solution of 2% H2SO4
(5 ml) containing compound 2 (3.0 mg) was refluxed for
2 h. After cooling the reactant, the precipitate was fil-
tered to give an aglycone (compound 2a). Compound 2a
(iriflophenone): 1H-NMR (acetone-d6) ꢂ: 5.97 (2H, s, H-
3,5), 6.85 (2H, d, J ¼ 8:4 Hz, H-30,50), 7.61 (2H, d,
J ¼ 8:4 Hz, H-20,60); 13C-NMR (acetone-d6) ꢂ: 95.9 (C-
3,5), 106.2 (C-1), 115.1 (C-30,50), 132.3 (C-20,60), 133.3
(C-10), 161.8 (C-40), 163.1 (C-2,6), 163.1 (C-2), 164.8
(C-4), 197.9 (C-7).
Laxative effects of acetone EAL and methanol EAL
To examine the laxative effects of EAL on the bowel
movement in mice, acetone EAL and methanol EAL
were orally administered (Fig. 2). Acetone EAL (at a
dose of 1000 mg/kg, p.o.) induced significant increases
(up to 2–3 times the control value) in stool frequency
and stool weight both 2–4 h and 6–8 h after its oral
administration (Fig. 2C and D). However, a lower dose
of acetone EAL (100 or 300 mg/kg, p.o.) and any
dose of methanol EAL (100, 300 or 1000 mg/kg, p.o.)
failed to induce a significant effect (Fig. 2A, B, E
and F).
Laxative effect of senna
To examine the laxative effect of senna leaves (used
as a positive control) on the bowel movement in mice,
the aqueous ethanol extract was orally administered
(Fig. 3). At a dose of 300 mg/kg, p.o., the senna extract
significantly increased the stool frequency and stool
weight both 2–4 h and 6–8 h after its administration
(Fig. 3A and B). At 1000 mg/kg, p.o., it significantly
increased (by approximately 10 times) both the stool
frequency and stool weight 2–4 h after its administration
(Fig. 3C and D), while it induced no changes at 30
mg/kg, p.o.
Genkwanin 5-O-ꢀ-primeveroside (compound 4): a
pale yellow powder; negative ion HR-FAB-MS m/z:
577.1547 [M-H]ꢂ (calcd. 577.1557 for C27H29O14);
EIMS m/z (rel. int.): 314 (16), 284 (M-primeverose,

338
H. HARA et al.
B
A
400
200
0
Control
20
10
0
Control
Acetone ext.
100 mg/kg
Acetone ext.
300 mg/kg
Acetone ext.
100 mg/kg
Acetone ext.
300 mg/kg
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
D
C
400
20
10
0
Control
Acetone ext.
1000 mg/kg
Control
Acetone ext.
1000 mg/kg
200
0
*
*
*
*
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
F
E
Control
Control
400
200
0
20
10
0
MeOH ext.
100 mg/kg
300 mg/kg
1000 mg/kg
MeOH ext.
100 mg/kg
300 mg/kg
1000 mg/kg
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
Fig. 2. Effects of EAL (the extract of agarwood leaves) on the Stool Frequency and Stool Weight in Mice.
The tool frequency (number) and weight (mg) were measured during four consecutive 2-h periods (0–2 h, 2–4 h, 4–6, and 6–8 h) after the oral
administration of acetone EAL or methanol EAL to mice. Data are shown as the mean ꢁ SE for 5–9 mice. ꢃ, p < 0:05 vs. Control at the same
time.
100), 255 (22), 241 (10), 166 (8), 118 (6); 1H-NMR
(DMSO-d6) ꢂ: [sugar moiety] 3.00 (1H, dd, J ¼ 8:1,
7.6 Hz, xyl-H-2), 3.03 (1H, dd, J ¼ 10:8, 5.5 Hz, xyl-H-
5), 3.10 (1H, dd, J ¼ 8:8, 8.7 Hz, xyl-H-3), 3.24 (1H, dd,
J ¼ 8:8, 5.5 Hz, xyl-H-4), 3.28 (1H, dd, J ¼ 9:6, 9.2 Hz,
glc-H-4), 3.34 (1H, dd, J ¼ 9:6, 8.8 Hz, glc-H-3), 3.39
(1H, dd, J ¼ 8:8, 7.6 Hz, glc-H-2), 3.56 (1H, dd,
J ¼ 9:2, 5.2 Hz, glc-H-5), 3.67 (1H, dd, J ¼ 10:6,
1.2 Hz, glc-H-6), 3.69 (1H, dd, J ¼ 10:8, 1.5 Hz, xyl-
H-5), 3.97 (1H, dd, J ¼ 10:6, 5.2 Hz, glc-H-6), 4.18 (1H,
d, J ¼ 7:6 Hz, xyl-H-1), 4.79 (1H, d, J ¼ 7:6 Hz, glc-H-
1), [aglycone moiety] 3.89 (3H, s, OMe), 6.69 (1H, s, H-
3), 6.85 (1H, d, J ¼ 2:4 Hz, H-6), 6.91 (2H, d,
J ¼ 8:8 Hz, H-30,50), 7.02 (1H, d, J ¼ 2:4 Hz, H-8),
7.91(2H, d, J ¼ 8:8 Hz, H-20,60), 10.31 (1H, s, C-40-
OH); 13C-NMR (DMSO-d6) ꢂ: [sugar moiety] 67.1 (xyl-
5), 68.8 (glc-6), 69.5 (glc-4), 71.1 (xyl-4), 73.5 (glc-2),
75.0 (xyl-3), 75.7 (glc-3,5), 75.9 (glc-5), 78.2 (xyl-3),
103.7 (glc-1), 106.0 (xyl-1), [aglycone moiety] 56.2
(OMe), 96.9 (C-8), 102.9 (C-6), 105.9 (C-3), 109.2 (C-
10), 115.9 (C-30,50), 121.2 (C-10), 128.1 (C-20,60), 158.1
(C-5), 158.5 (C-9), 160.9 (C-40), 161.4 (C-2), 163.6 (C-
7), 176.9 (C-4).
Iriflophenone 3, 5-C-ꢀ-diglucoside (compound 7):
brown amorphous gum; negative ion HR-FAB-MS,
m/z: 570.1585 [M-H]ꢂ (calcd.: 570.1574 for C25H30-
O15); 1H-NMR (methanol-d4) ꢂ: [sugar moiety] 3.42
(2H, dd, J ¼ 12:8, 6.6 Hz, glc-H-5a,5b), 3.50 (2H, t, J ¼
8:6, glc-H-5a,5b), 3.52 (2H, t, J ¼ 8:6 Hz, glc-H-2a,2b),
3.68 (2H, dd, J ¼ 10:0, 8.6 Hz, glc-H-2a,2b), 4.92 (2H,
d, J ¼ 10:0 Hz, glc-H-1a,1b), [aglycone moiety] 6.78
(2H, d, J ¼ 8:8 Hz, H-30,50), 7.64 (2H, d, J ¼ 8:8 Hz, H-

Laxative Effect of Agarwood Leaves
339
A
B
Control
Senna ext.
30 mg/kg
100 mg/kg
Control
20
10
0
400
Senna ext.
30 mg/kg
100 mg/kg
300 mg/kg
200
0
300 mg/kg
*
*
*
*
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
C
D
Control
Senna ext.
1000 mg/kg
20
10
0
400
200
0
Control
*
Senna ext.
1000 mg/kg
*
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
Fig. 3. Effects of the Extract of Senna Leaves on the Stool Frequency and Stool Weight in Mice.
The stool frequency (number) and weight (mg) were measured during four consecutive 2-h periods (0–2 h, 2–4 h, 4–6, and 6–8 h) after the oral
administration to mice. Data are shown as the mean ꢁ SE for 5–13 mice. ꢃ, p < 0:05 vs. Control at the same time.
Fig. 4. Major Constituents Iisolated from Acetone EAL and Methanol EAL.
Two new benzophenone derivatives [iriflophenone 2-O-ꢁ-rhamnoside (compound 2) and iriflophenone 3, 5-C-ꢀ-diglucoside (compound 7)]
and two known compounds [mangiferin (compound 1) and genkwanin 5-O-ꢀ-primeveroside (compound 4)] were isolated.
20,60); 13C-NMR (methanol-d4) ꢂ: [sugar moiety] 61.9
(glc-6), 70.9 (glc-4), 74.1 (glc-2), 76.9 (glc-1), 79.1 (glc-
3), 82.5 (glc-5), [aglycone moiety] 105.3 (C-3,5), 115.6
(C-30,50), 132.6 (C-10), 132.7 (C-1), 133.0 (C-20,60),
158.9 (C-2,6), 160.1 (C-4), 162.9 (C-40), 198.6 (C-7).
Structural determination
1H- and 13C-NMR spectra were measured with a
JEOL AL-400 instrument operated at 400 MHz and
100 MHz in acetone-d6, methanol-d4, or DMSO-d6, with
tetramethylsilane (TMS) used as an internal standard.

340
H. HARA et al.
those of compound 2, suggesting that the aglycone of
compound 7 was an iriflophenone. In the 1H-NMR
spectrum, no aromatic protons based on the meta-
substituted benzene ring found in compound 2 were
apparent. In the 13C-NMR spectrum, 13 aromatic car-
bons were detected, and the carbons (C-10–C-60) were
superimposed, while the others (C-1–C-6) were shifted.
These results suggested that the aromatic ring was fully
substituted and had a symmetrical structure such as a 2,
4, 6-trihydroxybenzene ring (phloroglucinol unit). Mul-
tiplicity was revealed by the DEPT experiment, and
complete assignment was performed by HMBC and
COSY experiments. The 1H-NMR spectrum showed
a signal at ꢂ 4.92 (d, J ¼ 10:0 Hz) attributable to an
anomeric proton of a glucose moiety, and the carbon at ꢂ
76.9 was found to be C-1 of C-ꢀ-glucoside. The 13C-
NMR spectrum showed that the two glucosyl carbons
were completely superimposed, indicating that two C-ꢀ-
glucose moieties were symmetrically substituted at C-3
and C-5. Clear HMBC correlations were observed
between the anomeric proton of C-ꢀ-glucose and C-3,5
through J3 correlation, and C-4 through J4 correlation in
the aglycone moiety of iriflophenone (Fig. 5), this
confirming that the linkage positions of the C-ꢀ-glucose
moieties were at C-3 and C-5. The structure of
compound 7 was thus determined to be iriflophenone
3,5-C-ꢀ-diglucoside. The structure of genkwanin 5-O-ꢀ-
primeveroside (compound 4) was completely character-
ized after an analysis of the above-mentioned spectral
data together with 2D NMR experiments.
Fig. 5. HMBC Correlations (!) and 1H-1H Correlations ($)
for Compounds 2 and 7.
Compound 2, iriflophenone 2-O-ꢁ-rhamnoside; compound 7,
Iriflophenone 3, 5-C-ꢀ-diglucoside.
The molecular formula C19H19O9 of compound 2 was
established from the negative ion HR FAB-MS and EI-
Laxative activity of the major compounds in EAL
As shown in Fig. 6, compound 2 (at 1000 mg/kg,
p.o.) significantly increased both the stool frequency and
stool weight 6–8 h after its administration (Fig. 6C and
D), but compounds 1 and 7 (at 1000 mg/kg, p.o.) had no
effects (Fig. 6A, B, E and F).
Compound 4 (at doses of 100–1000 mg/kg, p.o.) had
significant laxative activity (Fig. 7 C-H). At 1000 mg/
kg, p.o., it significantly increased both the stool
frequency and stool weight 2–4 h and 6–8 h after its
administration (Fig. 7G and H), whereas at 10 and
30 mg/kg, p.o. it had no significant laxative activity
(Fig. 7A and B). The smallest effective dose of
compound 4 was 100 mg/kg. The time-course charac-
teristics of the laxative effect of compound 4 were
similar to those of acetone EAL, suggesting that it was
one of the main laxative constituents of agarwood
leaves.
1
MS data, together with H- and 13C-NMR spectra. The
1H- and 13C-NMR spectra suggested the presence of a
rhamnose unit which was supported by an ion peak at
m/z 246 ([M-146]) in the EI-MS data. The 13C-NMR
spectrum displayed a signal at ꢂ 197.6 attributable to a
carbonyl group. The rest of the signals, corresponding to
twelve carbons, suggested the presence of two aromatic
benzene rings. In the 1H-NMR spectrum, the presence of
a para-substituted benzene ring was shown by proton
signals ꢂ 6.81 (1H, d, J ¼ 8:6 Hz) and 7.61 (1H, d, J ¼
8:6 Hz), in addition to a meta-substituted benzene ring
due to two doublets at ꢂ 6.07 (1H, d, J ¼ 2:0 Hz) and
6.30 (1H, d, J ¼ 2:0 Hz). The HMBC and COSY spectra
allowed complete assignment and identification of the
aglycone as 4, 20, 40, 60-tetrahydroxybenzophenone
(iriflophenone) (18, 19). The usual acidhydrolysis of
compound 2 gave iriflophenone. The presence of O-ꢁ-
rhamnose was confirmed by the characteristic carbon
signal of a methyl group at ꢂ 17.9 and by a proton signal
for an anomeric proton at ꢂ 5.22 (1H, d, J ¼ 0:8 Hz). Its
linkage position at C-2 was determined by HMBC
(Fig. 5). The structure of compound 2 was finally
concluded to be iriflophenone 2-O-ꢁ-rhamnoside.
Diarrhea frequency
Table 1 shows the diarrhea frequency for each 2-h
period. The senna extract at 300 mg/kg, p.o. (but not at
30 or 100 mg/kg) induced diarrhea. Methanol EAL and
acetone EAL (at 100–1000 mg/kg, p.o.) failed to induce
diarrhea. Likewise, the major constituents of EAL
(compounds 1, 2, 4 and 7) did not induce diarrhea,
even at 1000 mg/kg, p.o.
The molecular formula C25H30O15 of compound 7
was established from the negative ion HR FAB-MS
data. The 1H- and 13C-NMR spectra were similar to

Laxative Effect of Agarwood Leaves
341
A
B
300
15
10
Control
Control
Compound 1
1000 mg/kg
Compound 1
1000 mg/kg
200
100
0
5
0
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
C
D
Control
Compound 2
1000 mg/kg
300
200
100
0
15
10
Control
Compound 2
1000 mg/kg
5
0
*
*
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
E
F
300
200
100
0
15
10
Control
Compound 7
1000 mg/kg
Control
Compound 7
1000 mg/kg
5
0
0-2
2-4
4-6
6-8
0-2
2-4
4-6
6-8
Time after administration ( h )
Time after administration ( h )
Fig. 6. Effects of the Major Constituents (compounds 1, 2 and 7) of EAL on the Stool Frequency and Stool Weight in Mice.
The stool frequency (number) and weight (mg) were measured during four consecutive 2-h periods (0–2 h, 2–4 h, 4–6, and 6–8 h) after the oral
administration to mice. Data are shown as the mean ꢁ SE for 5–9 mice. ꢃ, p < 0:05 vs. Control at the same time.
Effects of compound 4 on the spontaneous motility in
an isolated rabbit ileum
pig ileum was antagonized by a pre-treatment with
atropine. The pretreatment with atropine, an acetylcho-
line-receptor antagonist, at 1 ꢄ 10ꢂ7 g/ml significantly
suppressed the contraction by compound 4. Com-
pound 4 at a concentration of 1 ꢄ 10ꢂ3 g/ml induced a
contraction in the guinea pig ileum, this contraction
being antagonized by atropine (Fig. 9). The contraction
by serotonin was antagonized by a pre-treatment with
azasetron. Compound 4 at a concentration of 1 ꢄ 10ꢂ3
g/ml induced a contraction. However, a pre-treatment
with azasetron, a 5-HT3 receptor antagonist, at 1 ꢄ
10ꢂ5 g/ml failed to suppress this contraction (Fig. 10).
Figure 8 shows a typical trace obtained from the
rabbit ileum after the application of compound 4 which
induced a contraction when applied at 1 ꢄ 10ꢂ3 g/ml.
The amplitudes of the contractions observed before the
application of compound 4 and after its application at
1:0 ꢄ 10ꢂ5, 1:0 ꢄ 10ꢂ4, and 1:0 ꢄ 10ꢂ3 g/ml were
3:72 ꢁ 0:11, 3:74 ꢁ 0:20, 3:76 ꢁ 0:23, and 4:66 ꢁ 0:35
cm, respectively (mean ꢁ SE, n ¼ 4) (equivalent to 100,
100, 101 and 125% of the ‘‘before’’ value). At 1:0 ꢄ
10ꢂ3 g/ml, compound 4 induced a significant contrac-
tion.
Discussion
Effect of a pretreatment with atropine or azasetron on
the contraction induced by compound 4 in an isolated
guinea pig ileum
In the present study, an oral administration of acetone
EAL, but not of methanol EAL, had a laxative effect on
mice, the effect induced by 1000 mg/kg, p.o. being
The contraction by acetylcholine in an isolated guinea

342
H. HARA et al.
B
A
15
300
Control
Compound 4
Control
Compound 4
10 mg/kg
10
200
100
0
10 mg/kg
Compound 4
Compound 4
30 mg/kg
30 mg/kg
5
0
0-2 2-4 4-6
6-8
0-2 2-4 4-6
6-8
Time after administration ( h )
Time after administration ( h )
D
C
300
15
Control
Control
Compound 4
100 mg/kg
Compound 4
100 mg/kg
10
200
100
0
5
0
*
0-2 2-4 4-6
6-8
0-2 2-4 4-6
6-8
Time after administration ( h )
Time after administration ( h )
F
E
Control
300
15
Control
Compound 4
300 mg/kg
Compound 4
300 mg/kg
10
200
100
0
5
0
*
*
0-2 2-4 4-6
6-8
0-2 2-4 4-6
6-8
GTime after administration ( h )
H
Time after administration ( h )
300
15
Control
Control
Compound 4
1000 mg/kg
Compound 4
1000 mg/kg
200
100
0
10
*
*
5
0
*
*
0-2 2-4 4-6
6-8
0-2 2-4 4-6
6-8
Time after administration ( h )
Time after administration ( h )
Fig. 7. Effects of Compound 4 (genkwanin 5-O-ꢀ-primeveroside) on the Stool Frequency and Stool Weight in Mice.
The stool frequency (number) and weight (mg) were measured during four consecutive 2-h periods (0–2 h, 2–4 h, 4–6, and 6–8 h) after the oral
administration to mice. Data are shown as the mean ꢁ SE for 5–10 mice. ꢃ, p < 0:05 vs. Control at the same time.
evident in two phases (2–4 h and 6–8 h after its
administration). This is the first report of EAL possess-
ing laxative activity in mice. An extract of senna, a
representative drug, exhibited laxative activity at 300
and 1000 mg/kg, p.o. The potency of acetone EAL at
1000 mg/kg, p.o. was the same as that of the senna
extract at 300 mg/kg, p.o., and the biphasic pattern of
the EAL effect was similar to that seen with the extract
of senna. The laxative effect of the senna extract at
1000 mg/kg, p.o. was intense but transient, being
evident only 2–4 h after its administration. These find-
ings indicate that, like senna, EAL had a laxative effect.
To identify the major laxative constituents of EAL,
we repeatedly separated and purified by chromatogra-
phy to isolate four compounds. These were two
new benzophenones [iriflophenone 2-O-ꢁ-rhamnoside
(Fig. 4, compound 2) and iriflophenone 3,5-C-ꢀ-diglu-
coside (compound 7)] and two known compounds
[mangiferin (compound 1) and genkwanin 5-O-ꢀ-pri-
meveroside (compound 4)]. Compound 4 (0.63 g) and
compound 2 (1.5 g) were purified from EAL (68 g), the
yield constant being approximately 1% and 2.2%,
respectively (Fig. 1). The rate of EAL and compound 4
was 100. On the other hand, the effective doses of EAL
and compound 4 to affect stool frequency and weight
were 1000 and 100 mg/kg, respectively. The rate was
10. This discrepancy may have been due to the differ-
ence in effect between a mixture (the extract) and a

Laxative Effect of Agarwood Leaves
Table 1. Diarrhea Frequency of EAL (extract of agarwood leaf),
343
Acetylcholine (1 × 107 g/ml)
Atropine (1 × 107 g/ml)
50% Ethanol Extract of Senna Leaf, and Major Components
(compounds 2 and 4) Isolated from EAL
Diarrea frequency (no.)
Treatment
p.o.
mg/kg
n
0–2 h
2–4 h
4–6 h
6–8 h
Control
Agarwood
10
5
0/10ꢃ
0/5
0/10
0/5
0/5
0/9
0/10
0/5
0/5
0/9
0/10
0/5
0/5
0/9
100
300
5
0/5
0/9
1000
9
Control
Senna
10
10
10
12
6
0/10
0/10
0/10
0/12
0/6
0/10
0/10
0/10
7/12
6/6
0/10
0/10
0/10
7/12
4/6
0/10
0/10
0/10
4/12
0/6
30
100
300
1000
Control
Compound 2
10
5
0/10
0/5
0/5
0/9
0/10
0/5
0/5
0/9
0/10
0/5
0/5
0/9
0/10
0/5
0/5
0/9
1 × 10-3 g/ml
100
300
5
1000
9
Control
Compound 4
10
4
0/10
0/4
0/10
0/4
0/10
0/4
0/10
0/4
10
30
5
0/5
0/5
0/5
0/5
100
300
1000
10
10
8
0/10
0/10
0/8
0/10
0/10
0/8
0/10
0/10
0/8
0/10
0/10
0/8
1 × 10-3 g/ml
Control mice were administered with each vehicle. Diarrhea frequency was
measured as the total number in each 2 h. ꢃValues are presented as no. of
diarrhea incidents/no. tested.
Fig. 9. Effect of a Pretreatment with Atropine on the Contraction of
an Isolated Guinea Pig Ileum Induced by Compound 4 (genkwanin
5-O-ꢀ-primeveroside at 1:0 ꢄ 10ꢂ3 g/ml).
Segments of a guinea pig ileum were suspended at 0.5 g tension in
an organ bath containing Tyrode’s solution. Acetylcholine chloride
and atropine were also added to the bath.
laxative effects in this late phase. Meanwhile, com-
pounds 1 and 7 had no effects on bowel movement in
mice. Genkwanin, one of the flavones, has been reported
to have a variety of pharmacological effects such as
antioxidative,12) radical scavenging,13) and antimicrobial
activities.14) However, there are no reports of genkwanin
having a laxative effect.
0.5 cm
5 min
Fig. 8. Effect of Compound 4 (genkwanin 5-O-ꢀ-primeveroside) on
the Spontaneous Motility in an Isolated Rabbit Ileum.
Segments of the rabbit ileum were suspended at 1 g tension in an
organ bath containing Tyrode’s solution. Their spontaneous move-
ments were monitored on a recorder via an isotonic transducer.
The amplitude of contraction was observed before the application
of compound 4. Compound 4 induced significant contraction at
1:0 ꢄ 10ꢂ3 g/ml.
Senna is a traditional medicine containing anthraqui-
none derivatives, its effective constituents being senno-
sides A, B, C and D. Senna is a major and popular
laxative, and it has been reported to accelerate sponta-
neous ileal contractions to induce a purgative effect.15,16)
The mechanism involves the sennosides and their active
derivatives acting on the intestinal mucosal epithelium
and submucosal nerve bundles, thereby stimulating
prostaglandin (PG) synthesis and endogenous acetylcho-
line release, and consequently enhancing the colonic
smooth muscle contraction.17–19) Unfortunately, the
purgative activities of anthraquinoids (including senna)
are too powerful for them to be regularly used, and even
periodic use of these laxatives can induce pseudomela-
nosis coli, a risk factor for colorectal neoplasma.3)
Indeed, in the present study, the senna extract at 300 and
1000 mg/kg, p.o., had a strong laxative effect, and it
induced severe diarrhea at the same doses as those
single compound; for example, EAL contained such
compounds as 4 and 2 which each had a laxative effect.
Compound 4 at 100, 300, and 1000 mg/kg, p.o. had
the strongest laxative effects among these compounds
and was fast-acting (the laxative activity was first
evident 2–4 h after its administration). Furthermore, at
1000 mg/kg, p.o. it showed lasting laxative activity, (the
effect being still evident after 6–8 h). Its effect was
milder and longer lasting than that of the senna extract.
In contrast, compound 2 at 1000 mg/kg, p.o. displayed
slow-acting laxative activity, its effect only being
evident 6–8 h after the its administration). The metab-
olites of compound 2 may possibly have exerted

344
H. HARA et al.
Serotonin (1 × 10-6 g/ml)
Azasetron (1 × 10-5 g/ml)
indicate that compound 4 acted via a mechanism differ-
ent from that of senna.
In summary, EAL had a laxative effect on mice that
was milder than that of senna, and it did not produce
diarrhea as a severe side effect, unlike senna. Further-
more, we identified the main constituent contributing to
the laxative effect of EAL as genkwanin 5-O-ꢀ-
primeveroside (compound 4). Compound 4 may act as
a laxative partly by contracting the ileum via acetylcho-
line receptors.
Acknowledgments
The authors thank Shun-ichi Yasuda and Nihon
Bioresearch Inc. for his technical assistance. This work
was partially supported by grant-aid from the Japan
Science and Technology Agency (Innovation Plaza
Tokai, Feasibility Study).
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