Rat small intestine muscle relaxation alkaloids from Orixa japonica leaves

Article abstract of DOI:10.1248/bpb.24.100

Two quinoline alkaloids, japonine (1) and eduline (2) were isolated from the methanol extract of the leaves of Orixa japonica as relaxants against rat small intestine muscle. The activity of these alkaloids was comparable to that of the typical muscle rel

Full text of DOI:10.1248/bpb.24.100

100
Notes
Biol. Pharm. Bull. 24(1) 100—102 (2001)
Vol. 24, No. 1
Rat Small Intestine Muscle Relaxation Alkaloids from Orixa japonica
Leaves
Shinji FUNAYAMA, ,a Rioko TANAKA,a Yasuhiro KUMEKAWA,a Toshiro NOSHITA,a Tetsuya MORI,a
*
b
Tadashi KASHIWAGURA,a and Kiyoshi MURATA
Department of Bioscience and Biotechnology, Aomori University,a 2–3–1 Kohbata, Aomori 030–0943, Japan and Aoba
Kasei Co., Ltd.,b 4–19–1 Ake-Dori, Sendai 981–3206, Japan. Received June 8, 2000; accepted September 23, 2000
Two quinoline alkaloids, japonine (1) and eduline (2) were isolated from the methanol extract of the leaves
of Orixa japonica as relaxants against rat small intestine muscle. The activity of these alkaloids was comparable
to that of the typical muscle relaxant papaverine (4). This is also the first report of the isolation of eduline (2)
from O. japonica.
Key words Orixa japonica; quinoline alkaloid; japonine; eduline; rat small intestine; relaxant effect
Orixa japonica (Rutaceae) is a shrub widely distributed in MATERIALS AND METHODS
Japan, Korea and China. The stems and leaves of this plant
were formerly used in Japan as an insecticide for livestock,
and this plant is regarded as one of the materials of the crude recorded on a JASCO A-100S spectrometer in KBr discs.
drug “Johzan.” UV spectra were taken on a Hitachi U-3200 spectrophotome-
General Experimental Procedures IR spectra were
In connection with our interest in naturally occurring bio- ter. Mass spectra and high-resolution EI-MS were taken on
logically active compounds of crude drug origin, two quino- JEOL JMX DX-303 and/or AX-500 mass spectrometers
1
line alkaloids, japonine (1) and eduline (2) were isolated using a direct inlet system. H-NMR (300 MHz) and 13C-
from the extract of the leaves of O. japonica, and it was NMR (75.5 MHz) spectra were measured by Hitachi R-3000
found that alkaloids 1 and 2 showed strong relaxant activity and Varian Gemini 2000 spectrometers with tetramethylsi-
against the small intestine muscle. This report is concerned lane as an internal standard, and chemical shifts were
with the isolation, identification and biological characteriza- recorded in d units. Wakogel C-200 (Wako Pure Chemical
tion of 1 and 2.1)
Ind., Ltd.) and ICN Alumina N, Akt. I (ICN Biomedicals)
To date, the following 20 quinoline alkaloids were isolated were used for column chromatography. DC-Fertigplatten
from various parts of O. japonica, from the stems, (ϩ)-orix- Kieselgel 60 F254 (0.25 mm thick, Merck) was used for
ine (3),2,3) O-methylbalfourodinium salt, orixinone,4) (Ϫ)- preparative TLC. DC-Alufolien Kieselgel 60 F254 (0.2 mm
preorixine,5,6) pteleprenine, isopteleprenine, N-demethyllu- thick, Merck) was used for TLC analyses.
nidonine, N-methylflindersine, 8-methoxy-N-methylflinder-
Plant Material Orixa japonica was collected near
sine, (ϩ)-isoptelefolidine,7) N-methylatanine, 8-O-methylgly- Sendai, Japan, in May, 1991 and cut and dried prior to ex-
cosolone, 4-O-methylhydroxyluninium chloride8) and (ϩ)-3Ј- traction. The plant was identified by one of us (S.F.), and a
O-acetylisopteleflorine,9) from the roots, (ϩ)-orixine,3) noror- voucher specimen is deposited in the herbarium of the De-
ixine,10) evolitorine,10) kokusagine2) and skimmianine,3) from partment of Bioscience and Biotechnology, Aomori Univer-
the leaves, evoxine11) and japonine,12) and from the fruits, sity.
kokusaginine and skimmianine.13)
Measurement of Rat Jejunum Smooth Muscle Relax-
This is the first isolation of eduline (2) from Orixa japon- ant Activity The jejunum was isolated from 48 h-starved
ica.
male rats of the Wistar strain, weighing 180—220 g. The re-
laxant activity was measured as described previously.14)
Thus, 2 cm length at both ends of the isolated intestine were
tied to the bottle edge with silk thread. The small intestine
was immersed in 20 ml of a solution consisting of (in mM)
149.1 Naϩ, 2.7 Kϩ, 1.1 Mg2ϩ, 1.8 Ca2ϩ, 143.1 ClϪ, 11.9
HCO3Ϫ, 0.4 H2PO4Ϫ and 5.6 glucose (pH 7.4Ϯ0.1) in a bath.
The solution was stirred by bubbling it with air. The edge of
the intestine was hooked to a strain transducer by a wire, and
the signal from the transducer was amplified and recorded
(Nihon Kohden isometric transducer TB-651T and isometric
amplifier EF-601G). All experiments were done at 37 °C.
Isolation of Japonine (1) and Eduline (2) The dried
leaves of O. japonica (3.8 kg) were extracted three times
(each 24 h) with n-hexane and MeOH at r.t., successively.
After filtration, the solvent was evaporated under reduced
pressure to afford n-hexane (59 g) and MeOH extracts
(1065 g).
The MeOH extract was suspended in H2O (2.25 l) and the
To whom correspondence should be addressed. e-mail: funayama@aomori-u.ac.jp
© 2001 Pharmaceutical Society of Japan

January 2001
101
7.49 (1H, d, Jϭ9 Hz, H-8), 7.51—7.59 (3H, m, arom.), 7.97
(1H, d, Jϭ3 Hz, H-5).
Fraction II-3 was chromatographed over Wakogel using n-
hexane–EtOAc (1 : 1) as a solvent to give Fr. III-1 (114 mg),
Fr. III-2 (150 mg), and Fr. III-3 (105 mg). Fraction III-2 was
further purified by column chromatography over Wakogel
using CHCl3 as a solvent to give eduline (2, 72.6 mg,
0.0098% yield) as a colorless powder. Rfϭ0.15 (benzene/
EtOAc (1 : 1)). UV lmax (MeOH) nm (emax): 254 (28700),
337 (9600), 350 (9600). IR (KBr) cmϪ1: 3450, 1595, 1560,
1482, 1309, 1251, 1228, 1033. EIMS m/z: 265 (Mϩ,
C17H15NO2, base), 250, 235, 222, 207, 194, 148. HREIMS
1
m/z: 265.1134 (Mϩ); Calcd for C17H15NO2; 265.1183. H-
NMR (CDCl3, R-3000) d: 3.62 (3H, s, N–CH3), 3.96 (3H, s,
C6–OCH3), 6.30 (1H, s, H-3), 7.34 (1H, dd, Jϭ3, 9 Hz, H-7),
7.38—7.49 (2H, m, arom.), 7.48—7.53 (3H, m, arom.), 7.52
(1H, d, Jϭ9 Hz, H-8), 7.92 (1H, d, Jϭ3 Hz, H-5). 13C-NMR
(CDCl3, R-3000) d: 37.3, 55.8, 105.7, 111.6, 117.7, 122.8,
127.9, 128.6, 128.7, 129.5, 135.8, 136.5, 153.8, 156.2, 176.8.
Synthesis of Eduline (2) To a stirred solution of diiso-
propylamine (1.0 g, 10.0 mmol) in THF (30 ml), was added
dropwise a 1.56 M solution of n-BuLi in hexane (6.4 ml,
10.0 mmol) at Ϫ30 °C under argon. The mixture was stirred
for 30 min. The solution was cooled to Ϫ70 °C, acetophe-
none (300 mg, 2.0 mmol) was added to the solution, and the
mixture was stirred for 1 h at Ϫ70 °C. The suspension of 6-
methoxy-1-methylisatoic anhydride15) (210 mg, 1.0 mmol)
was then added to the solution, and the mixture was stirred
for 1.5 h at Ϫ65 °C. The temperature of the reaction mixture
was then gradually raised to room temperature, the mixture
was stirred for 12 h, and the reaction was quenched with
AcOH. The reaction mixture was extracted with CH2Cl2, the
extract being washed with water and brine, and then dried
over CaCl2. Evaporation of the extract and purification of the
residue by recrystallization gave eduline (1, 103.5 mg, 0.4
Fig. 1. Rat Jejunum Smooth Muscle Relaxant Activity
Experimental procedures were described in MATERIALS AND METHODS Typical
responses of eduline (a) (2, 1ϫ10Ϫ5 M) and papaverine (b) (4, 1ϫ10Ϫ5 M) are presented.
The dose-response curve of eduline is also shown (c). Points and bars are meanϮS.D.
for three experiments. *Scale bar of relaxation tension.
Table 1. Effects of Japonine (1) and Eduline (2) Isolated from Orixa
japonica Leaves, Orixine (3) and Papaverine (4) on Relaxant Activity of Rat
Jejunum
Final Concentration
Relaxative Tension
(g)
Compounds
(mM)
Japonine (1)
Eduline (2)
Orixine (3)
5
0.12Ϯ0.03 (nϭ2)
0.17Ϯ0.05 (nϭ3)
0
1
10
10
10
mmol, 40%). The H-NMR data and Rf value of synthesized
eduline (1) were fully identical with those of the isolated ma-
terial.
(nϭ3)
Papaverine (4)
0.16Ϯ0.03 (nϭ10)
RESULTS AND DISCUSSION
suspension was extracted with CHCl3 (2 l) to give CHCl3 sol-
ubles (214 g). The aq. layer was further extracted with EtOAc
to give EtOAc solubles (5 g) and a residue (776 g). A part of
the CHCl3 soluble fraction (41.9 g) was chromatographed
over Wakogel and eluted with CH2Cl2, CH2Cl2/MeOH with
an increasing concentration of MeOH to give Fr. II (1.5 g:
this fraction was eluted with CH2Cl2–MeOH (20 : 1)) which
contained alkaloids 1 and 2, as well as the preceding Fr. I
(15.0 g) and following Fr. III (10.1 g). Fr. II (1.5 g) was fur-
ther chromatographed over silica gel and eluted with n-
hexane/EtOAc (1 : 1) to give Fr. II-1 (850 mg), Fr. II-2
(203 mg), and Fr. II-3 (371 mg).
Fraction II-2 was purified by p-TLC using benzene–EtOAc
(1 : 1) as a solvent to give japonine (1, 42.5 mg, 0.0057%
yield) as a colorless powder. Rfϭ0.17 (benzene/EtOAc
(1 : 1)). UV lmax (MeOH) nm (emax): 251 (34500), 341
(10700), 358 (10400). IR (KBr) cmϪ1: 3564, 3350, 1594,
1569, 1490, 1316, 1239, 1210, 1121, 1029. EIMS m/z 295
(Mϩ, C18H17NO3, base), 276, 264, 252, 235, 221, 207, 173,
140. 1H-NMR (CDCl3, GEMINI 2000) d: 3.53 (3H, s,
N–CH3), 3.66 (3H, s, C3–OCH3), 3.97 (3H, s, C6–OCH3),
7.32 (1H, dd, Jϭ3, 9 Hz, H-7), 7.35—7.41 (2H, m, arom.),
Isolation and Identification of Japonine (1) and Edu-
line (2) The MeOH of Orixa japonica extract was sus-
pended in H2O, and the suspension was extracted with CHCl3
and EtOAc, successively, to give a biologically active CHCl3
soluble fraction which was further chromatographed over sil-
ica gel and monitored by the biological activity test described
above to give japonine (1) and eduline (2).
Alkaloid 1 showed m/z 295 (Mϩ) in the EIMS, and in the
1H-NMR spectrum of this alkaloid, three aromatic signals at
d 7.32 (1H, dd, Jϭ3, 9 Hz, H-7), 7.49 (1H, d, Jϭ9 Hz, H-8)
and 7.97 (1H, d, Jϭ3 Hz, H-5) were coupled with each other,
and three O– and/or N–Me signals at d 3.53 (3H, s, N–CH3),
3.66 (3H, s, O–CH3) and 3.97 (3H, s, O–CH3) were observed,
together with five aromatic signals at d 7.35—7.41 (2H, m)
and 7.51—7.59 (3H, m), which was assigned to a C6H5-moi-
ety. These data suggested this alkaloid to be japonine (1) for-
merly isolated from this plant.12) The identification was ac-
complished by comparison of the spectral data of the isolate
with those reported.
Alkaloid 2 showed a similar UV spectrum to that of 1. In

102
Vol. 24, No. 1
the HREIMS of 2, an ion at m/z 265.1134 (Mϩ) was ob-
served, and the molecular formula of this alkaloid was eluci-
dated as C17H15NO2 (Calcd 265.1183). In the 1H-NMR spec-
trum of alkaloid 2, three aromatic signals at d 7.34 (1H, dd,
Jϭ3, 9 Hz, H-7), 7.52 (1H, d, Jϭ9 Hz, H-8) and 7.92 (1H, d,
Jϭ3 Hz, H-5) were coupled with each other, and a set of five
aromatic signals at d 7.38—7.49 (2H, m, arom.) and 7.48—
7.53 (3H, m, arom.) attributed to a C6H5-moiety, as in the
case of alkaloid 1, were observed. On the other hand, only
two O– and/or N–Me signals at d 3.62 and 3.96 (each 3H, s),
were observed. Instead, one proton singlet at d 6.30 (1H, s)
was observed. From all of the data described above, this al-
kaloid was estimated to be attributed to eduline (2),16) and 2
was synthesized in 4 steps starting from 5-methoxy-2-ni-
kaloids 1 and 2 showed similar potency concerned with the
relaxant effect in rat jejunum smooth muscle, whereas orix-
ine (3) did not show such activity. This is the first report of
the rat small intestine relaxative activity of alkaloids 1 and 2,
and it is noteworthy that such activity of these compounds
(0.12Ϯ0.03 g and 0.17Ϯ0.05 g, respectively) were equal to
that of papaverine (4, 0.16Ϯ0.03 g) (Table 1).
Acknowledgements The authors thank Messrs. Hideki
Hayasaka and Keiji Ohba, Medicinal Plant Garden of To-
hoku University, for collecting the plant material, and the an-
alytical center of the Faculty of Pharmaceutical Sciences, To-
hoku University is acknowledged for the EI-MS data. We ap-
preciate Professor Geoffrey A. Cordell, Department of Medi-
cinal Chemistry and Pharmacognosy, College of Pharmacy,
University of Illinois at Chicago, for his help in reviewing
the manuscript prior to submission.
1
trobenzoic acid according to Coppola.15) The H-NMR spec-
troscopic features and Rf value of synthesized eduline (1)
were fully identical with those of the isolated material. Be-
cause d 3.96 (3H, s) was assigned to C6–O–CH3 in eduline
(2), it was elucidated that d 3.97 (3H, s) of japonine (1) was
assigned to the C6–O–CH3 and d 3.66 (3H, s) of 1 was as-
signed to C3–O–CH3, respectively. Eduline (2) was formerly
isolated from the seeds of Casimiroa edulis (Rutaceae),16)
whereas this is the first isolation of this compound from
Orixa japonica.
The relaxation activity of the rat jejunum smooth muscle
by the alkaloids extracted from Orixa japonica leaves was
examined in the bath solution.14) Thus, the addition of 5 mM
japonine (1) and 10 mM eduline (2) to the bath solution
caused relaxation of the intestine. No phasic relaxation effect
was observed; typical responses of eduline (2) and papaver-
ine (4) are shown in Figs. 1-a and 1-b. The first relaxation
was acute and then the response decreased progressively with
time. The maximal response was mostly observed around
3 min after the addition of alkaloids to the bath solution, and
the response was measured for about 5 min. The dose-re-
sponse curve of eduline (2) is also shown in Fig. 1-c.
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Table 1 shows the parameters of dose-response curves for
the relaxation induced by japonine (1) and eduline (2) ex-
tracted from Orixa japonica leaves, and orixine (3) isolated
from the stem of the same plant. As depicted in this table, al-
15) Coppola G. M., J. Heterocyclic Chem., 19, 723 (1982).
16) Dreyer D. L., J. Org. Chem., 33, 2577 (1968).