ORIGINAL PAPER

Inhibitory effect of Iboga-type indole alkaloidson capsaicin-induced contraction in isolated mouse rectum

Mee Wah Lo • Kenjiro Matsumoto •

Masumi Iwai • Kimihito Tashima • Mariko Kitajima •

Syunji Horie • Hiromitsu Takayama

Received: 13 July 2010 / Accepted: 17 September 2010 / Published online: 2 November 2010

� The Japanese Society of Pharmacognosy and Springer 2010

Abstract Voacanga africana (Apocynaceae) is used as an

anti-diarrheal medicine in West Africa. In the present study,

we investigated the effect of an extract of V. africana and its

constituents on smooth muscle contraction induced by cap-

saicin in mouse rectum, where transient receptor potential

vanilloid type 1 (TRPV1)-immunoreactive fibers are abun-

dant. Methanol and alkaloid extracts of the root bark of

V. africana were found to inhibit capsaicin-induced con-

traction in a dose-dependent manner (30–300 lg/ml). Major

constituents isolated from the alkaloid extract were then

studied for their effects on the capsaicin-induced contrac-

tion. The main active constituents were found to be Iboga-

type alkaloids, including voacangine (1), 3-oxovoacangine

(2), voacristine (3), and (7a)-voacangine hydroxyindolenine

(4). The voacangine concentration dependently (3–100 lM)

inhibited the capsaicin-induced contraction. The capsaicin-

induced contraction was almost completely inhibited by the

TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloro-

pyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC).

On the other hand, the Iboga-type alkaloids did not inhibit

the contractions induced by 3 lM acetylcholine and

300 lM nicotine. These results suggest that Iboga-type

alkaloids isolated from V. africana inhibit capsaicin-

induced contraction in the mouse rectum, possibly via the

inhibition of a TRPV1-mediated pathway. This inhibition

may be involved in the anti-diarrheal effect of V. africana.

Keywords Apocynaceae � TRPV1 � Iboga alkaloid �Voacangine � Colon � Diarrhea

Introduction

Transient receptor potential vanilloid type 1 (TRPV1) was

first identified by its responsiveness to a pungent compo-

nent of hot chili peppers, capsaicin, from the genus Cap-

sicum. Capsaicin activates TRPV1 channels on sensory

neurons, and leads to the activation of unmyelinated C

fibers and thinly myelinated Ad fibers [1, 2]. TRPV1 is a

polymodal nociceptor that is also activated by moderate

heat (C43�C), low pH (B5.9), and endogenous lipid sig-

naling molecules such as anandamide [3, 4]. In the gut,

TRPV1 participates in the regulation of gastrointestinal

motility, blood flow, secretion, mucosal homeostasis, and

nociception [5–7]. Activation of the afferent nerves in the

gastrointestinal tract provides a local efferent-like effect by

releasing neuropeptides, such as tachykinins and calcitonin

gene-related peptide (CGRP), that modulate intestinal

motility and is postulated to involve a TRPV1-mediated

pathway. As such, capsaicin has been utilized in

the investigation of gastrointestinal motility involving

TRPV1 [8].

Voacanga africana (Apocynaceae) is a tree widely dis-

tributed in West Africa and as far as the Congo and even

Tanzania. In Cote d’Ivoire, it is used as a decoction for

diarrhea, leprosy, generalized edema, convulsions in chil-

dren, and madness [9]. Practitioners of traditional medicine

in Cameroon also suggested that V. africana possesses

anti-ulcer properties [10], and this was confirmed by Tan

M. W. Lo � M. Iwai � M. Kitajima � H. Takayama (&)

Department of Biofunctional Molecular Chemistry,

Graduate School of Pharmaceutical Sciences, Chiba University,

1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

e-mail: takayama@p.chiba-u.ac.jp

M. W. Lo � K. Matsumoto (&) � K. Tashima � S. Horie

Laboratory of Pharmacology, Faculty of Pharmaceutical

Sciences, Josai International University, 1 Gumyo Togane,

Chiba 283-8555, Japan

e-mail: kenjiro@jiu.ac.jp

123

J Nat Med (2011) 65:157–165

DOI 10.1007/s11418-010-0478-6

et al. [11, 12], who showed the protective effects of

aqueous and methanol extracts of the bark of V. africana

on gastric ulcers induced by HCl/ethanol using rats. In a

study of some Congolese plant extracts used as an anti-

diarrheic in traditional medicine, a water extract and

extracts for alkaloids, flavonoids, tannins, steroids, and

terpenes from the root bark of V. africana showed anti-

amoebic activity [13].

TRPV1 is of particular interest based on its role in

gastrointestinal disorders. For instance, the upregulation

of TRPV1 is implicated in rectal hypersensitivity, fecal

urgency [14], irritable bowel syndrome [15], and

inflammatory bowel disease [16]. Therefore, it would

appear that TRPV1 antagonists are of value for the

treatment of a range of gastrointestinal disorders in

inhibiting gastrointestinal hyperalgesia related to activa-

tion (sensitization) or the upregulation of TRPV1 [17,

18]. While searching for plant extracts with TRPV1

antagonistic properties, we hypothesized that the under-

lying mechanisms of the anti-diarrheal properties of

V. africana may involve a TRPV1-mediated pathway. In

the present study, we investigated whether an extract of

the root bark of V. africana inhibits TRPV1 activities by

examining the effects of the extract on capsaicin-induced

contraction in rectum isolated from healthy mice.

Capsaicin is used to activate TRPV1 channels because

they do not always function under physiological condi-

tions. We also report the effects of alkaloids isolated

from V. africana on capsaicin-induced contraction in

order to identify the constituents responsible for the

effect.

Materials and methods

Animals

Male ddY-strain mice (Japan SLC, Hamamatsu, Japan)

7–8 weeks of age were used. Animals were housed under

controlled environmental conditions (temperature at

24 ± 2�C and light between 7:00 a.m. and 7:00 p.m.) and

had free access to food and water. Animals were fasted

overnight with free access to water before each tension

measurement experiment. Animal experiments were per-

formed in compliance with the ‘‘Guiding Principles for the

Care and Use of Laboratory Animals’’ approved by the

Japanese Pharmacological Society, and the guidelines

approved by the Institutional Animal Care and Use Com-

mittee of Josai International University. The number of

animals used was kept to the minimum necessary for a

meaningful interpretation of the data. Animals were

euthanized by cervical dislocation before the isolation of

tissues.

Drugs

The drugs used in this study were acetylcholine chloride,

capsaicin, nicotine tartrate, and atropine sulfate (Wako Pure

Chemical Industries, Tokyo, Japan), N-(4-tertiarybutyl-

phenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-

carbox-amide (BCTC) (Biomol International, Boechout,

Belgium), and hexamethonium chloride (Sigma-Aldrich, St.

Louis, MO, USA). Capsaicin was dissolved in 96% ethanol

and the other drugs were dissolved in distilled water. The

vehicles used had no pharmacological effects on the tonus of

preparations or capsaicin-induced contraction.

Plant material

The root bark of V. africana was provided by Dr. Ruri

Kikura-Hanajiri and Dr. Yukihiro Goda from the National

Institute of Health Sciences, Tokyo, Japan.

Extraction and isolation

The dried powder of the root bark (601 g) was extracted

exhaustively with methanol, and after removal of the sol-

vent in vacuo, yielded 70.12 g of crude methanol extract.

The crude methanol extract was then dissolved in AcOEt

and extracted with 1 N HCl. The aqueous layer obtained

was basified to pH 8 with Na2CO3, followed by partitioning

with chloroform. The chloroform fraction was then dried in

vacuo to give a crude alkaloid extract (15.45 g). The crude

alkaloid extract was chromatographed on silica gel [Silica

gel 60 (70-230 mesh, Merck KGaA, Darmstadt, Germany),

MeOH–CHCl3 ? AcOEt–hexane] to give voacangine (1)

(782.5 mg; yield, 5.07% of the crude alkaloid extract),

3-oxovoacangine (2) (21.7 mg; yield, 0.14% of the crude

alkaloid extract), voacristine (3) (239.8 mg; yield, 1.55%

of the crude alkaloid extract), (7a)-voacangine hydrox-

yindolenine (4) (371.2 mg; yield, 2.40% of the crude

alkaloid extract), tetrahydroalstonine (7) (8.4 mg; yield,

0.05% of the crude alkaloid extract), voacamine (8)

(1,441.4 mg; yield, 9.33% of the crude alkaloid extract),

and subsessiline (9) (65.3 mg; yield, 0.42% of the crude

alkaloid extract) (Fig. 1). Constituents were identified by

the comparison of their physical data—[a]D, [1H]-nuclear

magnetic resonance (NMR), [13C]-NMR, and electron

ionization mass spectroscopy (EIMS)—with authentic

constituents. Two Iboga-type alkaloids, coronaridine (5)

and isovoacangine (6) isolated from Tabernaemontana

subglobosa [19], were also used for biological evaluation

(Fig. 1). The constituents were first dissolved in 100%

dimethylsulfoxide (DMSO) (Wako Pure Chemical Indus-

tries) to yield 30-mM solutions and subsequently diluted

with distilled water. The final concentration of DMSO was

less than 0.01%, which did not affect the smooth muscle

158 J Nat Med (2011) 65:157–165

123

contractions in response to 10 lM acetylcholine (data not

shown). The vehicles used had no pharmacological effects

on the resting tonus or agonist-induced contraction.

Cryosection preparation

for immunohistochemical study

The immunohistochemical procedures were performed as

described by our previous reports [20, 21]. Segments of

mouse rectum and distal, transverse, and proximal colon

were removed, fixed by immersion in fresh 4% parafor-

maldehyde in 0.1 M phosphate buffer for 2 h, washed three

times with phosphate-buffered saline (PBS), and cryopro-

tected overnight in 0.1 M phosphate buffer containing 20%

sucrose. The next day, the tissues were frozen in OCT

mounting medium (Sakura Finetek, Tokyo, Japan) and

sectioned on a cryostat (Leica Instruments, Nussloch,

Germany) at a thickness of 40 lm. The transverse sections

were thaw-mounted onto SuperFrost Plus slides (Matsun-

ami Glass, Osaka, Japan).

Immunohistochemical study

Immunohistochemical study was performed according to

the methods described by Horie et al. [20], Matsumoto

et al. [21], and Watanabe et al. [22]. Prior to staining,

the slide-mounted sections were successively incubated

in 10% normal donkey serum containing 0.2% Triton

X-100 and 0.1% sodium azide in PBS for 1 h, followed by

PBS containing 0.3% hydrogen peroxide for 30 min to

quench endogenous peroxidase activity, and then washed

three times for 10 min each with PBS. In addition, the

avidin and biotin sites of the sections were successively

blocked using an avidin–biotin blocking kit (Vector Labo-

ratories, Peterborough, UK), and the sections were then

washed three times in PBS again. Subsequently, the sec-

tions were incubated in rabbit polyclonal anti-TRPV1

antibody (1:60,000; mouse TRPV1 C-terminus; Neuromics,

Minneapolis, MN, USA) for 40 h at room temperature.

After three washes in PBS, the sections were incubated with

biotinylated donkey anti-rabbit immunoglobulin G (1:400;

Jackson ImmunoResearch Laboratories, West Grove, PA,

USA) for 90 min at room temperature. After three further

washes, the sections were incubated in streptavidin biotin–

peroxidase complex (1:5; Vectastain Elite ABC kit, Vector

Laboratories) for 1 h at room temperature, followed by

fluorescein isothiocyanate (FITC) tyramide (1:75; TSA kit,

PerkinElmer Life Sciences, Boston, MA, USA). In control

experiments, the TRPV1 antibody was omitted from the

staining procedures to verify the specificity of the staining.

No immunolabeling was observed in these controls (data

not shown).

Microscopy and image analysis

The sections were viewed at 109 magnification (Zeiss

Plan-NeoFluar) via an inverted fluorescence microscope

(Axioskop 2 plus, Zeiss, Gottingen, Germany) equipped

with a filter for the detection of fluorescein (FITC,

488 nm). Images were acquired via a charge-coupled

device camera (AxioCam MRm, Zeiss), stored in a per-

sonal computer, and analyzed with Zeiss imaging software

(AxioVision LE version 4.6).

Preparation of mouse rectum segments

and measurement of tension

The isolation and measurement of contraction of seg-

ments of the mouse rectum were performed according to

the methods described by Matsumoto et al. [21] and

Penuelas et al. [23]. Rectums were removed and placed

in Krebs–Henseleit solution (in mM: 112.08 NaCl, 5.90

KCl, 1.97 CaCl2, 1.18 MgCl2, 1.22 NaH2PO4, 25.00

NaHCO3, and 11.49 glucose) at pH 7.4. Each rectum

was cut into roughly 1.0-cm segments, set up under a

0.5-g load, and mounted longitudinally in a 10-ml organ

bath filled with Krebs–Henseleit solution. The bath was

maintained at 37�C and continuously bubbled with a

NH

N

MeO2C

HMeO

R2

R1

H

NN

MeO2C

HMeO

HO

NH

N

MeO2C

H

R

NH

N MeH

HMeO2C

NH

N

MeO2C

HMeO

NH

N

H

CO2Me

O

N N

OH

MeO

H

O

H

NH

N

OMeO2C

MeHH

H

1: R1 = H, R2 = H2

2: R1 = H, R2 = O

3: R1 = OH, R2 = H2

5: R = H6: R = OMe

4

7

98

Fig. 1 Chemical structures of voacangine (1), 3-oxovoacangine (2),

voacristine (3), (7a)-voacangine hydroxyindolenine (4), coronaridine

(5), isovoacangine (6), tetrahydroalstonine (7), voacamine (8), and

subsessiline (9)

J Nat Med (2011) 65:157–165 159

123

mixture of 95% O2 and 5% CO2. Contraction was recor-

ded using an isometric transducer (TD-112S, Nihon

Kohden, Tokyo, Japan), an isometric amplifier (JD-112S,

Nihon Kohden), and a PowerLab system (AD Instru-

ments, Castle Hill, Australia). Before performing the

experiments, the tissues were equilibrated for 15 min,

followed by three repeated additions of acetylcholine

(10 lM). Acetylcholine (10 lM) was added to the bath

to establish the integrity of the tissue at the end of the

experiment. Each response was expressed as a percent-

age of the maximum contraction induced by 10 lM

acetylcholine (% of acetylcholine contraction).

The biphasic contraction induced by capsaicin has been

previously reported in mouse rectum [21] and guinea pig

esophagus [24]. We also observed that capsaicin at 3 lM

induced a small, immediate, and transient contraction,

followed by a big, slowly developing, and long-lasting

contraction in mouse rectum (data not shown). In the

present study, effects of the plant extracts and their con-

stituents on the long-lasting contraction were evaluated.

Compound 1 at 30 lM slightly affected the resting tone,

and slightly potentiated spontaneous contractions of

smooth muscle in the rectum preparation. Application of

10 lM of 1, 2, 3, 4, 5, 6, 7, 8, and 9 did not affect the

resting tonus (data not shown). Therefore, we adopted the

10-lM concentration of the constituents shown in Figs. 4,

6, and 7.

In studies of the effects of the vehicle, BCTC, plant

extracts, and constituents on capsaicin-induced contrac-

tion, the mouse rectum segment was pre-incubated with

vehicle, BCTC, plant extracts, and constituents for

45 min. Then, capsaicin at 3 lM, a submaximal con-

centration for capsaicin-induced contraction [21], was

added to obtain contractions. The submaximal concen-

tration was determined by the concentration–response

curve.

In studies of the effects of the vehicle, atropine, and

constituents on acetylcholine-induced contraction, the

mouse rectum segment was pre-incubated with vehicle,

atropine, and constituents for 10 min. Then, acetylcholine at

3 lM, a submaximal concentration for acetylcholine-

induced contraction, was added to obtain contractions.

In studies of the effects of the vehicle, hexamethonium,

and constituents on nicotine-induced contraction, the

mouse segment was pre-incubated with vehicle, hexame-

thonium, and constituents for 10 min. Then, nicotine at

300 lM, a submaximal concentration for nicotine-induced

contraction, was added to obtain contractions. The drugs,

plant extracts, and constituents did not affect the baseline

tension. Each response was expressed as a percentage of

the maximum contraction induced by 10 lM acetylcholine

(% of acetylcholine contraction).

Statistical analysis

The data are presented as mean ± standard error of the

mean (SEM) for four to seven independent experiments.

The number in experiments (n) refers to the number of

experimental animals used. The statistical analyses were

performed by a Bonferroni multiple-comparison test for the

comparison of more than two groups. A P-value \ 0.05

was considered to be statistically significant.

Results

Localization of TRPV1 in isolated mouse rectum,

and distal, transverse, and proximal colon

We first investigated the localization and distribution of

TRPV1 in the mouse lower gastrointestinal tract using

immunohistochemistry. To increase the sensitivity of

detection, we used the fluorescein-conjugated tyramide

signal amplification (TSA) method to visualize the anti-

TRPV1 antibodies. In the rectum, numerous TRPV1-

immunoreactive nerve fibers were seen in the mucosa,

submucosal layers, and myenteric plexus. TRPV1-immu-

noreactive nerve fibers were also observed in the muscle

layer and around bundles of arterioles, venules, and lym-

phatic vessels in the submucosal layer of the rectum. In the

distal colon, the numbers of TRPV1-immunoreactive nerve

fibers were lower than in the rectum, and they were

observed in the mucosa, submucosal layer, and myenteric

plexus. In contrast, TRPV1-positive axons were hardly

observed in the transverse and proximal colon under the

conditions of the present experiments (Fig. 2). Because the

TRPV1-immunoreactive nerve fibers were most abundant

in the rectum in the isolated mouse lower gastrointestinal

tract, we used segments of mouse rectum in the measure-

ment of contractions.

Effects of extracts and their constituents

on capsaicin-induced contraction in isolated

mouse rectum

We investigated the effect of the methanol extract of

V. africana on capsaicin-induced contraction. The methanol

extract was found to reduce significantly the capsaicin-

induced contraction. The inhibitory effect of the methanol

extract occurred in a concentration-dependent manner

(30–300 lg/ml) (Fig. 3). We then further fractionated the

methanol extract to give a crude alkaloid extract. As shown

in Fig. 3, the alkaloid extract was found to exhibit more

potent inhibitory effects on the capsaicin-induced con-

traction than the methanol extract. The inhibitory effect of

160 J Nat Med (2011) 65:157–165

123

the alkaloid extract occurred in a concentration-dependent

manner (30–300 lg/ml).

Constituents isolated from the alkaloid extract of

V. africana by using silica gel chromatography were pre-

pared in order to examine their effects on capsaicin-

induced contraction (Fig. 4). Among them, the Iboga-type

alkaloids [voacangine (1), 3-oxovoacangine (2), voacristine

(3), and (7a)-voacangine hydroxyindolenine (4)] at 10 lM

were found to show potent inhibitions on capsaicin-induced

contraction to the same extent, whereas aspidosperma–

aspidosperma-type bisindole alkaloids [subsessiline (9)] at

10 lM moderately inhibited capsaicin-induced contrac-

tions. On the other hand, a heteroyohimbine-type alkaloid

[tetrahydroalstonine (7)] and an Iboga–vobasine-type

bisindole alkaloid [voacamine (8)] at 10 lM showed no

inhibitory effects on capsaicin-induced contractions. For

comparison, the effects of two other Iboga-type alkaloids,

coronaridine (5) and isovoacangine (6) isolated from

T. subglobosa, were examined. As a result, like constitu-

ents 1–4, coronaridine (5) and isovoacangine (6) were

found to show potent and significant inhibition on capsai-

cin-induced contractions at 10 lM.

To confirm the validity of this assay, the effect of the

TRPV1 antagonist BCTC was examined. BCTC (1 lM)

almost completely inhibited capsaicin-induced contractions

in the mouse rectum. The TRPV1 antagonist, BCTC, used

in this study has been well characterized in terms of the

potency and selectivity of its effects on TRPV1 channels

[25].

We studied the concentration-dependency of active

constituents. The amount of the most abundant constituent

isolated from the alkaloid extract, 1, allowed us to study its

dose–response effects. We studied the effects of 1 using

three concentrations, 3, 10, and 30 lM. The application of

1 reduced the capsaicin-induced contractions in a dose-

dependent manner (Fig. 5). A significant difference in the

number of contractions from the control was observed at

Fig. 2 Distribution of transient

receptor potential vanilloid

subtype 1 (TRPV1)

immunoreactivity in transverse

sections of rectum (a), and

distal (b), transverse (c), and

proximal colon (d). MUmucosa, CM circular muscle.

TRPV1-immunoreactive nerve

fibers are found in the mucosa

(arrows), submucosa layer

(double arrows), and myenteric

plexus (arrowheads). Scalebar = 100 lm

Fig. 3 Effects of methanol extract and alkaloid extract on capsaicin-

induced contraction in isolated mouse rectum. Isolated mouse rectum

was incubated with vehicle (V), 1 lM N-(4-tertiarybutylphenyl)-4-(3-

chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)

(B), methanol extract (30–300 lg/ml), or alkaloid extract (30–300

lg/ml) for 45 min. Then, 3 lM capsaicin was added to measure the

contractions. The data are expressed as a percentage of the maximal

contraction induced by 10 lM acetylcholine (ACh) (% of ACh

response). Each value represents the mean ± standard error of the

mean (SEM) of data obtained from five to seven mice. The asterisksindicate significant differences at *P \ 0.05 and **P \ 0.01 as

compared with the control (V) by a Bonferroni multiple-comparison test

J Nat Med (2011) 65:157–165 161

123

concentrations of 10 lM and higher. The concentration of

3 lM showed no significant inhibitory effect.

Effects of voacanga alkaloids on contractions induced

by the activation of muscarinic and nicotinic receptors

In order to clarify that 1, 2, 3, 4, 5, and 6 do not act on

cholinergic muscarinic and nicotinic receptors, we studied

the effects of the constituents on acetylcholine- and nico-

tine-induced contractions. The same concentration that

produced significant inhibition on capsaicin-induced con-

tractions was adopted in acetylcholine- and nicotine-

induced contractions. In this context, the constituents tested

at 10 lM did not inhibit acetylcholine-induced contraction,

and there was no significant difference from the control

experiment. We also evaluated the effect of a muscarinic

receptor antagonist, atropine, on the contractile responses.

Atropine at 1 lM almost completely inhibited the acetyl-

choline-induced contraction (Fig. 6). This suggests that the

constituents do not act on the cholinergic muscarinic

receptors under these conditions.

Moreover, the constituents at 10 lM also did not

inhibit contractions induced by nicotine. The effect of the

nicotinic blocker hexamethonium (100 lM) was also

evaluated, and it almost completely inhibited nicotine-

induced contractions in the rectum (Fig. 7). This suggests

Fig. 4 Effects of voacangine (1), 3-oxovoacangine (2), voacristine (3),

(7a)-voacangine hydroxyindolenine (4), coronaridine (5), isovoacan-

gine (6), tetrahydroalstonine (7), voacamine (8), and subsessiline (9) on

capsaicin-induced contraction in isolated mouse rectum. Isolated

mouse rectum was incubated with vehicle (V), 1 lM BCTC (B), or

10 lM constituents for 45 min. Then, 3 lM capsaicin was added to

measure the contractions. The data are expressed as a percentage of the

maximal contraction induced by 10 lM acetylcholine (ACh) (% of ACh

response). Each value represents the mean ± SEM of data obtained

from five to seven mice. The asterisks indicate significant differences at

*P \ 0.05 and **P \ 0.01 as compared with the control (V) by a

Bonferroni multiple-comparison test

Fig. 5 Effects of voacangine (1) on capsaicin-induced contraction in

isolated mouse rectum. Isolated mouse rectum was incubated with

vehicle (V), 1 lM BCTC (B), or voacangine (3–30 lM) for 45 min.

Then, 3 lM capsaicin was added to measure the contractions. The data

are expressed as a percentage of the maximal contraction induced by

10 lM acetylcholine (ACh) (% of ACh response). Each value represents

the mean ± SEM of data obtained from five to seven mice. The asterisksindicate significant differences at *P \ 0.05 and **P \ 0.01 as

compared with the control (V) by a Bonferroni multiple-comparison test

Fig. 6 Effects of voacangine (1), 3-oxovoacangine (2), voacristine (3),

(7a)-voacangine hydroxyindolenine (4), coronaridine (5), and isovo-

acangine (6) on acetylcholine (ACh)-induced contraction in isolated

mouse rectum. Isolated mouse rectum was incubated with vehicle (V),

1 lM atropine (A), or 10 lM constituents for 10 min. Then, 3 lM

acetylcholine (ACh) was added to measure the contractions. The data are

expressed as a percentage of the maximal contraction induced by 10 lM

acetylcholine (% of ACh response). Each value represents the mean ±

SEM of data obtained from four to seven mice. The asterisks indicate

significant difference at **P \ 0.01 as compared with the control (V) by a

Bonferroni multiple-comparison test

162 J Nat Med (2011) 65:157–165

123

that the constituents do not act on the cholinergic nico-

tinic receptors.

Discussion

Inhibitory effects of crude extract and its constituents

Voacanga africana has been traditionally used as an anti-

diarrheal agent in West Africa. In the present study, we

attempted to study the working hypothesis that the anti-

diarrheal effect of V. africana involves mechanisms

underlying TRPV1 channels. Thus, we investigated the

effect of the methanol extract of V. africana on capsaicin-

induced contractions in isolated mouse rectum. The

methanol extract was found to reduce the capsaicin-

induced contraction significantly. We then further frac-

tionated the methanol extract to give a crude alkaloid

extract, and it was found that the alkaloid extract exhibits a

more potent inhibitory effect on capsaicin-induced con-

tractions than the methanol extract. These results suggested

the presence of active constituents in the alkaloid extract

and led us to further purify the alkaloid extract.

Constituents isolated from the alkaloid extract of

V. africana by using silica gel chromatography were

prepared in order to examine their effects on capsaicin-

induced contractions. Among them, the Iboga-type alkaloids,

which include 1, 2, 3, and 4, were found to show similar

potent and significant inhibitions on capsaicin-induced

contraction at 10 lM. Two Iboga-type alkaloids, 5 and 6

isolated from T. subglobosa, also showed similar and

potent and significant inhibitions. The potencies of their

inhibitory effects on capsaicin-induced contractions were

not affected by the different side chains of the Iboga-type

alkaloids. The aspidosperma–aspidosperma-type bisindole

alkaloid, 9, at 10 lM moderately inhibited capsaicin-

induced contractions. On the other hand, 7, a heteroyo-

himbine-type alkaloid, and 8, an Iboga–vobasine-type

bisindole alkaloid, at 10 lM had no inhibitory effects on

capsaicin-induced contraction. As such, the fundamental

ibogaine skeleton seems to act as an important key com-

ponent of the activity. To our knowledge, this is the first

report to show the TRPV1 antagonist-like activities of 1, 2,

3, 4, 5, and 6.

Selectivity of inhibitory effect

on capsaicin-induced contraction

The capsaicin-evoked response has been shown to involve

cholinergic participation, where acetylcholine is the final

transmitter [26–28]. Acetylcholine acts on muscarinic M3

receptors on smooth muscle cells, leading to the induction

of rectal contraction [6, 29]. Furthermore, capsaicin has

also been reported to exert nonspecific actions on nicotinic

receptors [30]. Nicotine acts on nicotinic receptors on the

parasympathetic postsynaptic nerve to elicit the release of

acetylcholine from the nerve ending, and then the released

acetylcholine induces smooth muscle contractions in the

rectum [6, 29].

In our study, the Iboga-type alkaloids that inhibited

capsaicin-induced contraction did not modify the acetyl-

choline-induced or nicotine-induced contractions. There-

fore, these results suggest that these constituents do not act

on muscarinic and nicotinic receptors.

This observation throws light on our speculation that the

inhibitory effects of the active constituents are attributable

to the inhibition of mechanisms underlying TRPV1 chan-

nels. However, we cannot exclude the possibility that the

constituents may have inhibitory effects on receptors for

other neurotransmitters mediating the capsaicin-induced

contraction. For instance, tachykinins (substance P and

neurokinin A) and CGRP are the most widely accepted

transmitters to mediate ‘‘local efferent’’ effects of capsai-

cin-sensitive nerves. We are currently studying the effects

of active constituents on substance P-, neurokinin A-, and

CGRP-mediated responses of the rectum.

Tachykinins and long-lasting contraction

induced by capsaicin

Tachykinins such as substance P and neurokinin A, neuro-

transmitters of enteric cholinergic neurones and extrinsic

afferent nerve fibers, have pathological implications for

secretory diarrhea [31, 32]. Substance P and neurokinin A

released from sensory nerves mainly stimulate NK1 and NK2

Fig. 7 Effects of voacangine (1), 3-oxovoacangine (2), voacristine

(3), (7a)-voacangine hydroxyindolenine (4), coronaridine (5), and

isovoacangine (6) on nicotine-induced contraction in isolated mouse

rectum. Isolated mouse rectum was incubated with vehicle (V),

100 lM hexamethonium (H), or 10 lM constituents for 10 min.

Then, 300 lM nicotine was added to measure the contractions. The

data are expressed as a percentage of the maximal contraction induced

by 10 lM acetylcholine (ACh) (% of ACh response). Each value

represents the mean ± SEM of data obtained from four to seven

mice. The asterisks indicate significant difference at **P \ 0.01 as

compared with the control (V) by a Bonferroni multiple-comparison

test

J Nat Med (2011) 65:157–165 163

123

receptors, respectively. The major final mediator is the

neurokinin A released from extrinsic sensory nerve endings.

Neurokinin A activates NK2 receptors on the smooth muscle

cells. The long-lasting contraction induced by capsaicin in

the mouse rectum may be attributed to the neurokin A reac-

tivity [33]. It has been reported that NK2 receptor antagonists

reduce the fecal output, fecal water content, and/or intestinal

hypermotility induced by Salmonella enteritidis endotoxin

[34], acetic acid enema [35], Escherichia coli toxin Sta, or

Clostridium difficile toxin [36]. Neurokinin A is responsible

for the hypermotility associated with diarrheic symptoms.

Therefore, in the present study, we evaluated the effects of

the plant extracts and their constituents on the long-lasting

contraction induced by capsaicin.

TRPV1 antagonists and functional

gastrointestinal diseases

Elevated numbers of TRPV1 channels are implicated in

various gastrointestinal diseases, such as rectal hypersensi-

tivity, fecal urgency [14], and irritable bowel syndrome [15].

Oral administration of a non-vanilloid, JNJ 10185734,

reduced diarrhea rates by as much as 70% [37]. We, there-

fore, speculate that the TRPV1 antagonist-like effects of the

Iboga-type alkaloids isolated in our study may play an

important role in the anti-diarrheic property of V. africana.

There is also accumulating evidence that TRPV1 antagonists

can be developed as novel treatments for gastrointestinal

diseases. For instance, the preclinical efficacy of a number of

TRPV1 antagonists involving capsazepine [38], BCTC [39],

AMG 9810 [40], and A-42519 [41] in models of inflamma-

tory and neuropathic pain in rodents has been documented.

Therefore, the isolated Iboga-type alkaloids may have ther-

apeutic potential as novel drug candidates for functional

gastrointestinal diseases.

Conclusion

In conclusion, our study showed that Iboga-type alkaloids

isolated from the alkaloid extract of Voacanga africana,

including voacangine (1), 3-oxovoacangine (2), voacristine

(3), and (7a)-voacangine hydroxyindolenine (4), exert a

potent inhibitory effect on capsaicin-induced contractions

in isolated mouse rectum, possibly through the blockage of

transient receptor potential vanilloid type 1 (TRPV1)

channels. These results suggest that their TRPV1 antago-

nist-like properties contribute to the anti-diarrheic activity

of V. africana, and may have potential for the treatment of

functional gastrointestinal diseases.

Acknowledgment We thank Dr. Ruri Kikura-Hanajiri and

Dr. Yukihiro Goda for the provision of the root bark of Voacanga

africana. This work was supported in part by Grants-in-Aid for Sci-

entific Research from the Ministry of Education, Science, Sports, and

Culture of Japan, and by the Uehara Memorial Foundation.

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