Antinociceptive effects of the extracts of xylopia parviflora bark and its alkaloidal components in experimental animals

Article abstract of DOI:10.1007/s11418-009-0356-2

In the present study, we attempted to elucidate the antinociceptive activity of Xylopia parviflora bark using the acetic acid-induced writhing test, hot plate test, and formalin test in mice. The MeOH extract (100 and 200 mg/kg, administered intraperitoneally (i.p.)) had an antinociceptive effect demonstrated by its inhibitory effects on writhing number induced by acetic acid. Three alkaloidal fractions exhibited significant antinociceptive effects in three animal models; the chloroform-soluble fraction, including secondary and tertiary alkaloids, exhibited the strongest effect. This result supported its use in folk medicine as an analgesic agent. We tested the main alkaloids of these fractions for their antinociceptive effects to clarify the active components. (+)-Corytuberine (6.3 and 12.5 mg/kg, i.p.) showed very strong activity, had a significant antinociceptive effect in the acetic acid-induced writhing test (with 49.4 and 98.9% reduction of writhes), in the hot plate test, and in the formalin test (with 55.4 and 90.6% inhibition during the first phase, and 73.9 and 99.9% during the second phase, respectively). (+)-Glaucine (12.5 and 25 mg/kg, i.p.) showed strong activity in three animalmodels, too. The activity of these compounds was also observed following oral administration in the acetic acidinduced writhing test. The Japanese Society of Pharmacognosy and Springer 2009.

Full text of DOI:10.1007/s11418-009-0356-2

J Nat Med (2010) 64:9–15
DOI 10.1007/s11418-009-0356-2
ORIGINAL PAPER
Antinociceptive effects of the extracts of Xylopia parviflora bark
and its alkaloidal components in experimental animals
Yumi Nishiyama Æ Masataka Moriyasu Æ Momoyo Ichimaru Æ
Kinuko Iwasa Æ Atsushi Kato Æ Simon G. Mathenge Æ
Patrick B. Chalo Mutiso Æ Francis D. Juma
Received: 1 May 2009 / Accepted: 30 July 2009 / Published online: 3 September 2009
Ó The Japanese Society of Pharmacognosy and Springer 2009
Abstract In the present study, we attempted to elucidate
the antinociceptive activity of Xylopia parviflora bark
using the acetic acid-induced writhing test, hot plate test,
and formalin test in mice. The MeOH extract (100 and
models, too. The activity of these compounds was also
observed following oral administration in the acetic acid-
induced writhing test.
2
00 mg/kg, administered intraperitoneally (i.p.)) had an
Keywords Xylopia parviflora Á Annonaceae Á
Antinociceptive effect Á Isoquinoline alkaloids
antinociceptive effect demonstrated by its inhibitory effects
on writhing number induced by acetic acid. Three alka-
loidal fractions exhibited significant antinociceptive effects
in three animal models; the chloroform-soluble fraction,
including secondary and tertiary alkaloids, exhibited the
strongest effect. This result supported its use in folk med-
icine as an analgesic agent. We tested the main alkaloids of
these fractions for their antinociceptive effects to clarify
the active components. (?)-Corytuberine (6.3 and
Introduction
Xylopia parviflora Spruce (Annonaceae) is a tall tree dis-
tributed in east Africa and widely used in folk medicine.
The Nyamwezi and coastal peoples take a decoction of the
root for stomach disorders. It is also used by Nyamwezi
women for barrenness. Other medicinal uses include
insertion of root pieces into nostrils for headache relief, and
its bark is also used for analgesic and antispasmodic pur-
poses (Chalo Mutiso, private communication). In our pre-
vious study, we reported that a large number of
isoquinoline alkaloids, especially benzylisoquinoline and
aporphine-type, were identified as the constituents of this
plant [1, 2]. Some medicinal plants rich in similar alkaloids
are often used as an analgesic, just like this plant [3–6]. We
hypothesized that the analgesic effect of this plant was
caused by these alkaloidal components. To pursue the
active principles of X. parviflora, the antinociceptive effect
of its methanolic extract was first determined according to
acetic acid-writhing, hot plate, and formalin tests. We
further investigated the activities of the alkaloidal and non-
alkaloidal fractions from the extract using similar animal
model tests, and found that the alkaloidal fractions showed
a significant effect. To clarify the active components, we
tested the activity of the main alkaloids in these fractions
including corytuberine and glaucine, which were reported
to have antinociceptive activity [7].
1
2.5 mg/kg, i.p.) showed very strong activity, had a sig-
nificant antinociceptive effect in the acetic acid-induced
writhing test (with 49.4 and 98.9% reduction of writhes), in
the hot plate test, and in the formalin test (with 55.4 and
9
0.6% inhibition during the first phase, and 73.9 and 99.9%
during the second phase, respectively). (?)-Glaucine (12.5
and 25 mg/kg, i.p.) showed strong activity in three animal
AY .. KN ai st ho iyama (&) Á M. Moriyasu Á M. Ichimaru Á K. Iwasa Á
Department of Natural Medicinal Chemistry,
Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi,
Higashinada-ku, Kobe 658-8558, Japan
e-mail: nisiyama@kobepharma-u.ac.jp
S. G. Mathenge Á P. B. Chalo Mutiso
Department of Botany, University of Nairobi,
P.O. Box 30197, Nairobi, Kenya
F. D. Juma
Department of Clinical Pharmacology,
Kenyatta National Hospital, University of Nairobi,
P.O. Box 19676, Nairobi, Kenya
123

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J Nat Med (2010) 64:9–15
Materials and methods
an ion-pair extraction method using sodium perchlorate [8].
The insoluble part (NA-1) in 2.5% tartaric acid and the
soluble part (NA-2) in acidic diethyl ether were also
obtained as non-alkaloidal fractions (Fig. 1).
Plant material
The bark of Xylopia parviflora was collected in the Kwale
district of Kenya in 2000. This plant was identified and
authenticated by Mr. S. G. Mathenge and Mr. P. B. Chalo
Mutiso, two of the authors. Voucher specimens of this
plant were deposited both in the laboratory of Kobe
Pharmaceutical University (2000 No. 3) and the herbarium
of University of Nairobi (No. 00/3).
Preparation of alkaloidal components
The major alkaloidal component in BE was (?)-reticuline,
followed by considerable amounts of (?)-coclaurine and
(
?)-glaucine [2]. The main alkaloid of BC was (?)-
corytuberine [2]. The most abundant alkaloid in BD was
?)-magnoflorine, then (?)-tembetarine [1]. The three
(
Extraction, fractionation, and isolation
obtained alkaloidal fractions were subjected to an ion-pair
preparative HPLC system [8], and preparative TLC was
applied, as necessary, for the isolation of their main con-
stituents. The quantities of isolated compounds were not
sufficient for carrying out examinations using animal
models, because the amount of the plant materials avail-
able was limited. So, we isolated each compounds from
other available plants rich in these alkaloids. (?)-Reticu-
line, (?)-coclaurine, and (?)-magnoflorine were obtained
from the extract of Magnolia salicifolia [9], and (?)-
tembetarine was obtained from Zanthoxylum usambarense
The dried stem bark of X. parviflora (3 kg) was finely cut
and extracted with hot MeOH (3 L, refluxed for 5 h) three
times. The resulting MeOH extract was evaporated under
reduced pressure and the residue (165 g) was re-extracted
with 2.5% tartaric acid. The acidic solution was subjected
to conventional methods for the isolation of secondary and
tertiary alkaloids, and gave a diethyl ether-soluble fraction
(
(
BE fraction: 3830 mg) and a chloroform-soluble fraction
BC fraction: 260 mg). Furthermore, we obtained the
quaternary alkaloidal fraction (BD fraction: 1220 mg) by
[
10] by conventional extraction and isolation. (?)-Cory-
tuberine was derived from (?)-isocorydine by demethyl-
ation using 47% HBr. (?)-Glaucine was supplied by
Roth Co. Spectroscopic instruments, especially NMR and
MS data, were used to identify the structures of these
compounds (Fig. 2).
Xylopia parviflora bark
extracted with MeOH
evap.
MeOH extract
Animals
acidified (2.5% tartaric acid)
Four-week-old ddY male mice were purchased from Nihon
SLC Co. and adapted to constant conditions (temperature
aqueous layer
diethyl ether
residue Fraction NA-1
22 ± 2°C, light/dark cycle 12/12 h) for 1 week. The
experiments were conducted on mice weighing 25–35 g.
The experimental protocols were conducted according to
Kobe Pharmaceutical University’s Guidelines for the Care
and Use of Laboratory Animals.
aqueous layer
basified with Na CO
diethyl ether layer
diethyl ether layer
Fraction NA-2
2
3
partitioned with diethyl ether
aqueous layer
CHCl3
Acetic acid-induced writhing test
Fraction BE
Fraction BC
Mice were randomly divided into several groups. Test
solution was intraperitoneally (i.p.) administrated 30 min
(
or orally at 60 min) before the experiment and further
% acetic acid–saline was injected intraperitoneally
0.1 mL/10 g). After the injection of acetic acid–saline
aqueous layer
CHCl layer
3
1
acidified with HClO4
(
NaClO (0.5M)
4
1
,2-dichloroethane
(5 min), each mouse was placed in a transparent cage,
and the frequency of writhing was counted for 15 min.
Aminopyrine was used as the positive control agent. The
antinociceptive activity was evaluated in terms of per-
centage inhibition of writhes.
aqueous layer
1,2-dichloroethane layer
Fraction BD
Fig. 1 Extraction and isolation of alkaloids from Xylopia parviflora
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J Nat Med (2010) 64:9–15
11
Fig. 2 Structures of main
alkaloidal constituents from
each fraction
Alkaloids from BE fraction
H3CO
H3CO
H3CO
HO
N
H
N
H
CH3
NH
H
HO
CH3
H CO
3
H CO
3
H3CO
HO
(+)-Reticuline
(+)-Glaucine
(+)-Coclaurine
OCH3
OH
Alkaloids from BD fraction
Alkaloid from BC fraction
H CO
3
H3CO
CH3
H3CO
N
CH3
CH3
HO
CH3
N
H
N
H
CH3
H
HO
HO
HO
HO
H3CO
H CO
3
H CO
3
OH
(+)-Corytuberine
(+)-Tembetarine
(+)-Magnoflorine
Hot plate test
using Dunnet’s multigroup test at the 1 and 5% level of
significance.
The hot plate test was used to measure latency by the
method of Bispo et al. [11] with small modifications. Mice
were divided into several groups. The temperature of the
plate (Model MK-350B, Muromachi Kikai Co., Japan) was
fixed at 53 ± 0.2°C. The reaction time (jumping, licking
one of the hind legs or one of the forelegs, tapping) was
measured 0, 30, and 60 min after intraperitoneal adminis-
tration of the test solution. When 40 s elapsed without the
animal presenting any of the above behaviors, it was
removed from the plate to avoid tissue damage.
Results
Acetic acid-induced writhing test
The effects of the MeOH extract and each fraction obtained
from the MeOH extract in the acetic acid-induced writhing
test in mice are shown in Table 1. Intraperitoneal admin-
istration of the MeOH extract (50, 100, and 200 mg/kg)
inhibited the number of writhes induced by acetic acid. All
alkaloidal fractions inhibited writhing, in particular frac-
tions BC and BD showed especially strong inhibition.
Small doses (12.5 mg/kg) of BC and BD fraction showed
42.1 and 46.1% inhibition, respectively.
Formalin test
The test was carried out as described by Hunskaar and Hole
[
12] with small modifications. Mice were injected with
0 lL of 2.5% formalin (in saline) subcutaneously into the
hind paw, and the duration of paw licking was determined
–5 min (first phase) and 15–30 min (second phase) after
2
The effects of the alkaloidal constituents of each frac-
tion are summarized in Table 2. (?)-Reticuline, which is
the major constituent of BE, did not inhibit writhing, but
(?)-coclaurine and (?)-glaucine (each 25 mg/kg, i.p.)
showed 44.8 and 81.0% inhibition, respectively. In par-
ticular, (?)-glaucine showed significant inhibition (59.8%)
even at 12.5 mg/kg. (?)-Corytuberine showed stronger
inhibition than (?)-glaucine, causing 49.4% inhibition at a
lower dose, 6.3 mg/kg. In the BD fraction, (?)-magnoflo-
rine did not show any inhibition at all, but (?)-tembetarine
showed 58.0% inhibition at 25 mg/kg.
0
formalin application. Test solution was given intraperito-
neally 30 min prior to formalin.
Statistical analysis
Results were expressed as the mean ± SEM. Results
were analyzed using one-way analysis of variance
(
ANOVA). Significant differences was further analyzed
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Table 1 Effect of each fraction on acetic acid-induced writhing in
mice
Table 3 Effect of each fraction on acetic acid-induced writhing in
mice
Group
Dose (mg/kg, i.p.) Writhing number Inhibition (%)
Group
Dose
mg/kg, p.o.)
Writhing
number
Inhibition
(%)
(
Control
–
22.8 ± 2.0
0
Control
–
23.7 ± 1.7
0
MeOH ext.
200
3.8 ± 1.3**
4.0 ± 0.8**
10.1 ± 2.3**
83.3
82.5
55.7
(?)-Glaucine
50
8.1 ± 1.9**
11.2 ± 1.5**
7.8 ± 2.0**
13.5 ± 2.2**
13.8 ± 2.2**
65.9
52.7
67.1
43.0
41.8
1
00
2
5
5
0
(?)-Corytuberine
25
Alkaloidal fractions
1
2.5
BE
BC
50
50
9.2 ± 2.3**
1.3 ± 1.3**
7.6 ± 2.1**
13.2 ± 2.0*
3.3 ± 1.4**
12.3 ± 3.5**
59.6
94.3
66.7
42.1
85.5
46.1
Aminopyrine
50
2
1
5
Writhing number is expressed as the mean ± SE of 9–10 mice
Statistically significant difference compared with controls: *p \ 0.05,
2.5
*
*p \ 0.01
BD
25
2.5
Non-alkaloidal fractions
1
strong inhibition (52.7, 65.9%) and (43.0, 67.1%), respec-
tively (Table 3).
NA-1
50
7.3 ± 2.3**
15.2 ± 2.1
68.0
33.3
47.4
64.9
2
5
NA-2
50
12.0 ± 2.4**
8.0 ± 1.9**
Hot plate test
Aminopyrine 25
Results obtained in the hot plate test for the MeOH extract
and each fraction are shown in Table 4. The MeOH extract
Writhing number is expressed as the mean ± SE of 7–10 mice
Statistically significant difference compared with controls: *p \ 0.05,
(200 mg/kg) and two non-alkaloidal fractions (each 50 mg/
*
*p \ 0.01
kg) did not prolong latency at all. In the alkaloidal frac-
tions, only the BC fraction showed significant extension of
latency time by the dosage of 50 mg/kg at 30 and 60 min
later compared with both controls and 0-min values.
Table 5 shows the results for alkaloidal constituents.
Table 2 Effect of each alkaloidal constituents on acetic acid-induced
writhing in mice
Group
Dose
mg/kg, i.p.)
Writhing
number
Inhibition
(%)
(
(
?)-Glaucine and (?)-corytuberine caused significant
Control
–
17.4 ± 1.9
0
extension; (?)-corytuberine especially showed significant
extension at 6.3 mg/kg after 30 min compared with 0-min
values.
Constituents of BE fraction
(
(
(
?)-Coclaurine
?)-Reticuline
?)-Glaucine
25
25
25
9.6 ± 1.2*
13.8 ± 1.6
3.3 ± 1.3**
7.0 ± 2.6**
12.8 ± 1.2
44.8
20.7
81.0
59.8
26.4
Formalin test
1
2.5
.3
6
In the formalin test, the MeOH extract at applied doses did
not significantly decrease the licking time compared with
control. The BC fraction (25 and 50 mg/kg) obtained from
the MeOH extract, however, exhibited very strong effect in
both the first and second phases. The BD fraction also
showed a significant effect in both phases (Table 6).
Concerning the effect of alkaloidal constituents, (?)-
glaucine and (?)-corytuberine exhibited a significant an-
tinociceptive effect, but (?)-magnoflorine and (?)-tembe-
tarine, the main components of BD, did not show activity at
all even though BD showed a significant effect (Table 7).
Constituents of BC fraction
?)-Corytuberine 12.5
(
0.2 ± 0.2**
8.8 ± 1.7**
13.4 ± 1.9
98.9
49.4
23.0
6
.3
.2
3
Constituents of BD fraction
(
(
?)-Magnoflorine
?)-Tembetarine
25
25
17.3 ± 2.7
7.3 ± 2.1**
17.0 ± 2.0
7.3 ± 1.6**
0.6
58.0
2.3
1
2.5
Aminopyrine
25
58.0
Writhing number is expressed as the mean ± SE of 8–10 mice
Statistically significant difference compared with controls: *p \ 0.05,
*
*p \ 0.01
Discussion
In addition, to confirm whether these compounds were
effective not only by the i.p. administration but also orally
Table 8 summarizes the results obtained in this study. The
MeOH extract of X. parviflora did not show an obvious
antinociceptive effect because it still contained many
(
(
p.o.), (?)-glaucine (25, 50 mg/kg) and (?)-corytuberine
12.5, 25 mg/kg) were examined again. These showed
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13
Table 4 Effect of each fraction on heat-induced pain in mice (hot plate test)
Group
Dose
(
Latency (s)
min
mg/kg, i.p.)
0
30 min
60 min
Control
–
13.38 ± 0.79
13.05 ± 1.16
13.44 ± 0.92
12.28 ± 1.49
13.85 ± 0.96
12.95 ± 1.54
MeOH ext.
200
Alkaloidal fractions
#
BE
BC
50
50
13.16 ± 0.72
12.49 ± 0.65
12.41 ± 1.08
12.92 ± 0.91
17.78 ± 2.34
16.33 ± 1.28
#
#
##
28.28 ± 3.59**
15.31 ± 1.65
16.34 ± 1.90
27.33 ± 3.40**
13.37 ± 1.48
12.53 ± 1.03
2
5
BD
25
Non-alkaloidal fractions
NA-1
50
50
50
12.07 ± 0.93
13.51 ± 0.99
12.74 ± 0.94
13.56 ± 1.17
11.86 ± 1.04
12.88 ± 1.17
11.72 ± 1.03
12.14 ± 1.25
NA-2
#
Aminopyrine
16.59 ± 1.69
Latency is expressed as the mean ± SE of 9–10 mice
Statistically significant difference compared with controls: *p \ 0.05, **p \ 0.01
#
##
Statistically significant difference compared with 0-min values: p \ 0.05, p \ 0.01
Table 5 Effect of each alkaloidal constituents on heat-induced pain in mice (hot plate test)
Group
Dose
mg/kg, i.p.)
Latency (s)
min
(
0
30 min
60 min
Control
–
12.58 ± 0.58
12.70 ± 0.58
12.82 ± 1.28
Constituents of BE fraction
#
(
?)-Coclaurine
50
11.35 ± 0.59
13.03 ± 0.71
11.67 ± 0.60
12.33 ± 0.99
12.33 ± 0.90
14.40 ± 1.05
11.91 ± 0.65
14.16 ± 0.61
12.94 ± 0.99
12.92 ± 1.25
12.22 ± 0.72
19.56 ± 2.76*
12.21 ± 1.35
2
5
#
(
(
?)-Reticuline
?)-Glaucine
50
50
#
#
##
##
#
32.58 ± 2.95**
#
18.82 ± 2.14
#
2
5
Constituents of BC fraction
?)-Corytuberine
(
12.5
13.46 ± 0.66
13.21 ± 0.64
26.78 ± 2.81**
#
16.02 ± 0.99
19.48 ± 1.90*
12.61 ± 0.90
6
.3
Constituents of BD fraction
?)-Tembetarine
#
(
50
12.85 ± 0.69
13.08 ± 0.79
12.74 ± 0.94
17.36 ± 1.35
15.21 ± 1.31
14.35 ± 0.99
12.14 ± 1.25
2
5
14.73 ± 1.19
#
Aminopyrine
50
16.59 ± 1.69
Latency is expressed as the mean ± SE of 10 mice
Statistically significant difference compared with controls: *p \ 0.05, **p \ 0.01
#
##
###
Statistically significant difference compared with 0-min values: p \ 0.05, p \ 0.01, p \ 0.001
components. The alkaloidal fractions from this extract,
however, showed remarkable antinociceptive effect in
three animal models. The BC fraction exhibited the
strongest effect, and (?)-corytuberine, which was the main
component of BC, also showed very strong antinociceptive
activity. The (?)-corytuberine content of BC amounted to
about 25%, so (?)-corytuberine will likely bear the activity
of the BC fraction. The diethyl ether-soluble fraction (BE)
also showed a significant effect compared with the control,
but was somewhat weaker than BC. (?)-Reticuline, (?)-
coclaurine, and (?)-glaucine, which were the main com-
ponents of BE, were tested in the present study. (?)-Ret-
iculine and (?)-coclaurine, both benzylisoquinoline-type
alkaloids, showed no activities, whereas (?)-glaucine,
which is an aporphine-type alkaloid, showed a strong
effect. Therefore, we considered that it would not be ben-
zylisoquinoline-type but aporphine-type alkaloids that
show the analgesic effect. The content of (?)-glaucine in
BE was smaller than other two compounds and many
minor aporphine-type alkaloids were found in BE; so, in
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Table 6 Effect of each fraction on formalin-induced pain in mice (formalin test)
Group
Dose (mg/kg, i.p.)
First phase
Second phase
Licking time (s)
Inhibition (%)
Licking time (s)
Inhibition (%)
Control
–
73.0 ± 6.9
54.9 ± 4.9
63.8 ± 5.0
0
123.6 ± 12.4
75.6 ± 12.6
86.2 ± 11.6
0
MeOH ext.
200
24.8
12.6
38.8
30.2
1
00
Alkaloidal fractions
BE
BC
50
50
51.2 ± 5.4
29.9
81.4
54.0
43.3
65.0 ± 13.4*
6.9 ± 6.8**
47.4
94.4
73.0
58.6
13.6 ± 4.0**
33.6 ± 3.9**
41.4 ± 5.6**
2
5
33.4 ± 8.8**
51.2 ± 16.3**
BD
25
Non-alkaloidal fractions
NA-1
50
50
25
72.9 ± 6.3
76.8 ± 9.7
59.1 ± 6.2
0.1
-5.2
19
65.3 ± 14.5*
126.5 ± 21.3
45.9 ± 13.5**
47.2
-2.3
62.9
NA-2
Aminopyrine
Licking time is expressed as the mean ± SE of 10 mice
Statistically significant difference compared with controls: *p \ 0.05, **p \ 0.01
Table 7 Effect of each alkaloidal constituents on formalin-induced pain in mice (formalin test)
Group
Dose (mg/kg, i.p.)
First phase
Second phase
Licking time (s)
Inhibition (%)
0
Licking time (s)
Inhibition (%)
0
Control
–
72.6 ± 6.9
149.4 ± 19.5
Constituents of BE fraction
(
(
(
?)-Coclaurine
?)-Reticuline
?)-Glaucine
25
25
25
64.7 ± 9.6
69.0 ± 4.8
8.0 ± 1.8**
24.9 ± 4.9**
59.5 ± 5.9
10.9
5.0
97.1 ± 16.9
130.4 ± 12.8
8.7 ± 4.8**
35.0
12.7
94.2
62.7
29.3
89.0
65.7
18.0
1
2.5
.3
55.7 ± 12.6**
105.6 ± 16.8
6
Constituents of BC fraction
?)-Corytuberine
(
12.5
6.8 ± 4.2**
32.4 ± 4.3**
53.4 ± 5.9
90.6
55.4
26.4
0.2 ± 0.2**
39.0 ± 6.1**
86.2 ± 22.4*
99.9
73.9
42.3
6
.3
.2
3
Constituents of BD fraction
(
(
?)-Magnoflorine
?)-Tembetarine
25
25
25
77.4 ± 5.0
75.8 ± 8.8
57.7 ± 5.9
-6.6
-4.4
20.6
131.6 ± 19.0
136.2 ± 15.7
46.7 ± 13.3**
11.9
8.8
Aminopyrine
68.7
Licking time is expressed as the mean ± SE of 9–10 mice
Statistically significant difference compared with controls: *p \ 0.05, **p \ 0.01
addition to (?)-glaucine, other aporphine-type alkaloids in
BE may contribute to the activities. In the examination of
the quaternary alkaloidal fraction (BD), a dose of 50 mg/kg
was often fatal due to its toxicity, so 25 mg/kg was adop-
ted. As a result, BD showed a significant effect on acetic
acid-induced writhing and in the formalin test, but the
activity of the two main components (?)-magnoflorine and
showed very strong activity. Secondly, the toxicities of BD
influenced the result of the animal experiment. At present
we believe that the latter was more plausible. Since (?)-
magnoflorine, an N-methyl derivative of the strongly active
(?)-corytuberine, had no activity, we suppose that the
presence of this type of quaternary group rendered the
molecule ineffectual.
(
?)-tembetarine was very weak or not effective at all. We
Both (?)-corytuberine and (?)-glaucine, which were
already reported as having antinociceptive activities in the
hot plate test (52°C) and phenylquinone-induced writhing
considered that there were two possibilities from such
results. In the first, other minor alkaloids contained in BD
1
23

J Nat Med (2010) 64:9–15
15
Table 8 Summary of the results obtained in this study
Group
Acetic acid-writhing test
Hot plate test
i.p.
Formalin test (i.p.)
1st phase
i.p.
p.o.
2nd phase
MeOH ext.
BE fraction
?
–
–
–
?
?
–
?
(
(
(
?)-Coclaurine
?)-Reticuline
?)-Glaucine
??
–
?
–
–
?
–
–
???
???
????
???
–
???
???
???
????
–
???
???
???
????
??
–
BC fraction
?)-Corytuberine
BD fraction
???
(
????
????
??
–
(
(
?)-Magnoflorine
?)-Tembetarine
??
?
?
–
–
–
NA-1 fraction
–
?
NA-2 fraction
?
–
–
–
Aminopyrine (positive control)
??
??
?
–
??
test following subcutaneous administration [7], showed
significant effects again in this study. We examined these
two compounds in other animal models (acetic acid-
induced writhing test and formalin test) during this inves-
tigation and recognized the antinociceptive activity. The
formalin test can also be used to elucidate the mechanism
of pain and analgesia [13]. These compounds inhibited the
two phases equally in the formalin test and were centrally
acting drugs [14]. In addition, the analgesic activity of
these two compounds was recognized after oral adminis-
tration in the acetic acid-induced writhing test for the first
time.
4. Ichimaru M, Moriyasu M, Nishiyama Y, Kato A, Juma FD,
Nganga JN, Ogeto JO (1997) Studies on African medicinal
plants. Alkaloidal constituents of Uvaria acuminata and
U. lucida. Nat Med 51:272–274
5. Nishiyama Y, Moriyasu M, Sonoda M, Ichimaru M, Iwasa K,
Kato A, Mathenge SG, Juma FD (2000) Isoquinoline alkaloids
from Monodora junodii. Nat Med 54:42
6
. Nishiyama Y, Moriyasu M, Ichimaru M, Iwasa K, Kato A,
Mathenge SG, Chalo Mutiso PB, Juma FD (2000) Secondary and
tertiary isoquinoline alkaloids from Monodora junodii. Nat Med
54:338–341
7
. Zetler G (1988) Neuroleptic-like, anticonvulsant and antinoci-
ceptive effects of aporphine alkaloids: bulbocapnine, corytuber-
ine, boldine and glaucine. Arch Int Pharmacodyn Ther 296:255–
281
In conclusion, the results of the present study provide
support for the traditional use of X. parviflora as an anal-
gesic agent, and some of the active components and their
mechanism were clarified. A study of structure–activity
relationships of aporphine alkaloids and their detailed
mechanism is now progress in experimental animals to find
new analgesic medicines.
8. Moriyasu M, Ichimaru M, Nishiyama Y, Kato A (1993) The
acquisition of alkaloids from plant materials by a combination of
ion-pair extraction and preparative ion-pair HPLC using sodium
perchlorate. Bunseki Kagagu 42:659–665
9. Kikuchi T, Kadota S, Yanada K, Watanabe K, Yosizaki M,
Kimura M (1981) Studies on the constituents of crude drug
‘‘Shin-I’’ (dried buds of Magnolia salicifolia Maxim). Wakan-
yaku Shinpojumu [Kiroku] 13:101–104
1
1
1
0. Kato A, Moriyasu M, Ichimaru M, Nishiyama Y, Juma FD,
Nganga JN, Mathenge SG, Ogeto JO (1995) Examination of
alkaloidal constituents of Zanthoxylum usambarense by a com-
bination of ion-pair extraction and ion-pair chromatography using
sodium perchlorate. Phytochem Anal 6:89–95
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