Natural Product Research, Vol. 20, No.

3, March 2006, 253257

Acetogenins in Annona muricata L. (annonaceae)

leaves are potent molluscicides
J. DE S. LUNAy, J. M. DE CARVALHOz,
M. R. F. DE LIMAz, L. W. BIEBERy, EDSON DE S. BENTOz,
X. FRANCKx and A. E. G. SANTANA*z
yDepartamento de Qu mica Fundamental, Universidade Federal
de Pernambuco, 50.740-901, Recife-PE, Brazil
zDepartamento de Qu mica, Universidade Federal de Alagoas,
57072-970, Maceio-AL, Brazil
xLaboratoire de Pharmacognosie, BioCIS-CNRS (UMR 8076), Universite Paris Sud,
Faculte de Pharmacie, 92296 Chatenay-Malabry CEDEX, France
(Received 12 July 2004; in final form 2 October 2005)
An ethanolic extract of the leaves of Annona muricata was shown to be toxic to adult forms
of the snail Biomphalaria glabrata (LC50 9.32 mg mL
1) and to larvae of the brine shrimp
Artemia salina (LC50 0.49 mg mL
1). Activity-guided fractionation of the extract gave rise to
a sample with high molluscicidal activity that contained the acetogenins, annonacin (90%),
isoannonacin (6%) and goniothalamicin (4%).
Keywords: Annona muricata; Molluscicide; Larvicide; Annonacin; Isoannonacin;
Goniothalamicin

1. Introduction
Annona muricata L. has been used in folk medicine by virtue of its anti-diarrheic,
anti-diabetic, sedative, insecticidal and parasiticidal properties [13]. Various chemical
and biological studies have been carried out on different parts of the plant, and
anti-parasitic, anti-depressive, anti-herpes, leishmanicidal, molluscicidal and cytotoxic
activities have been reported [1,310].
Schistosomiasis is a human tropical disease caused by schistosomes, of the species
Schistosoma mansoni, S. japonicum and S. haematobium. The schistosomes first infect
and then mature in fresh water snails of the genus Biomphalaria (intermediate host)
before transferring to humans. In Brazil, B. glabrata is responsible for more than
95% of the transmission of schistosomiasis [11,12]. In this article we describe
the activity-guided isolation of molluscicidal compounds from an ethanolic extract
of the leaves of A. muricata, and elucidation of their structures by NMR.

*Corresponding author. Fax: 55 82 214 1700. Email: aegs@qui.ufal.br

Natural Product Research
ISSN 1478-6419 print: ISSN 1029-2349 online 2006 Taylor & Francis
http://www.tandf.co.uk/journals
DOI: 10.1080/14786410500161445

254

J. De S. Luna et al.

2. Results and discussion

At the present time, the strongest molluscicidal activity (LC50 0.12 mg mL
1) has been
reported from Euphorbia milii (latex) [13], although a number of other species have
also shown very high activities, namely, Anacardium occidentale (cashew nut shell)
LC90 0.35 mg mL
1 [14], Pithecolobium multiflorum (stem bark) LC90 4.9 mg mL
1 [15],
Euphorbia cotinifolia (leaves) LC90 3.4 mg mL
1 [16] and Phytolacca dodecandra LC50
2.57 mg mL
1 [17]. The molluscicidal activity, characterized by LC90 73.17 mg mL
1,
LC50 9.32 mg mL
1 and LC10 1.19 mg mL
1, of the ethanolic extract of the leaves of
A. muricata is somewhat lower than those of the most active species listed above.
However, the activity compares favourably with those of extracts from seeds of
A. crassiflora and A. glabra (LC50 values 13.21 and 17.02 mg mL
1, respectively [4]),
from bark of Acioa barteri and A. rudatisii (LC100 values 100 mg mL
1 [18]), from latex
of Euphorbia tirucalli (LC90 85.0 mg mL
1 [19]), from roots of Jatropha elliptica (LC90
44.48 mg mL
1 [20]) and from leaves of Renealmia exaltata (LC50 28.03 mg mL
1 [20]).
Furthermore, the ethanolic extract from leaves of A. muricata showed very high toxicity,
characterized by LC90 2.32 mg mL
1, LC50 0.49 mg mL
1 and LC10 0.10 mg mL
1,
towards brine shrimp larvae. In 1993, WHO recommended that plants, which yielded
extracts having molluscicidal activities characterized by LC50 values <40 mg mL
1,
would be of value for direct use in the control of schistosomiasis [21].
Activity-guided fractionation of the ethanolic extract of leaves of A. muricata was
problematic because of the presence of chlorophyll. In order to remove chlorophyll
and to reduce the complexity of the initial ethanolic preparation from leaves of
A. muricata, the crude extract was submitted to a filtration step over activated charcoal
using a procedure previously described for the isolation of iridoids [22,23]. The initial
fractions so obtained (F1F5) were submitted to bioassay (table 1). The main
molluscicidal activity resided in F3 and F4, whilst F5 presented an intense green
colour associated with the presence of chlorophyll. F4 (161.53 g) was selected for
further fractionation by column chromatography (CC) to yield fractions F4.1F4.5
(table 1). Fraction F4.4 was submitted to CC over silica gel, activated charcoal

Table 1. Molluscicidal activity and cytotoxicity of fractions obtained following

chromatographic separation of an ethanolic extract of A. muricata.
Measured mortality (%)
Fraction/Controla
F1, F2
F3
F4
F5
F4.1
F4.2, F4.3
F4.4
F4.5
Niclosamide (3 mg mL
1)
Cupric carbonate (50 mg mL
1)
Thymol
Dechlorinated water/DMSO (0.1%)
a

B. glabrata assay

A. salina assay

0
100
100
80
20
100
100
40
100
100

0
74
100
100
0
100
86
70

100
0

Activity determined at a concentration of 10 mg mL
1 except where otherwise indicated.

Acetogenins in A. muricata L. (annonaceae) leaves

255

and LH-20 Sephadex to give rise to a sample which was highly active in the
molluscicide bioassay and homogeneous by TLC. This sample was subjected to
high resolution EIMS and the measured molecular mass was 596.4652 indicating
a molecular composition of C35H64O7 (596.4654). The IR spectra showed absorption
bands (cm
1) at 3426 (OH), 2971, 2850 (CH), 1743 (CO) and 1073 (CO).
Analysis of the 13C-NMR and the 90 and 135 DEPT spectra revealed the presence
of two quaternary carbon atoms (one being a carbonyl), eight methine, two methyl
and 23 methylene carbons. The chemical shifts indicated the presence of an , -unsaturated- -lactone ring, a hydroxyl group in position 4 and a tetrahydrofuran ring
flanked by two hydroxyl groups with a threo-trans-threo stereochemistry that are characteristic of an acetogenin according to the literature [9,24].
OH

OH

32

H3C

10

( )n

( )m

( )5

O
OH

OH

33

34

35CH3

(1) Annonacin (m = 11, n = 3)

OH

(3) Goniothalamicin (m = 13, n = 5)

OH
O

H3C
( ) 11

( )5
CH3

( )3
OH

O
O
O

(2) Isoannonacin

The sample was subjected to HPLC-MS analysis and, following comparison of the
MS obtained with those of reference compounds, was found to consist of annonacin
(1; 90%) together with isoannonacin (2; 6%; stereochemistry undetermined) and
goniothalamicin (3; 4%), all of which have been previously found in the leaves and
seeds of A. muricata [2527]. The sample showed significant toxicity (100% mortality
at 10 ppm) to B. glabrata.
The present results show for the first time (to the best of the authors knowledge) that
acetogenins possess potent molluscicidal activity, and also indicate the potential use
of leaf material of A. muricata in the control of the snail B. glabrata. Since this plant
is already cultivated in Brazil, leaf material is readily available on a large scale, and
the material itself, or crude extracts therefrom, could have immediate application
as an efficient biocontrol agent.

3. Experimental
3.1. Plant material
Leaves of A. muricata were collected in Alagoas State (Brazil) in August 2000 and were
authenticated by Cicero Barros (Instituto do Meio Ambiente [IMA], Alagoas State,
Brazil): voucher specimen number 8530 is deposited in the herbarium at IMA.

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J. De S. Luna et al.

3.2. Chromatographic and instrumental methods

Analytical TLC was carried out on silica gel 60 layers (Merck) eluted with CHCl3 and
CHCl3 : MeOH (9 : 1) and visualized by spraying with Kedde reagent followed by
heating at 100 C for 35 min. HPLC-MS were obtained using a Hewlett-Packard
model HP1100 chromatograph equipped with a Waters Microbondapak C18 column
(10  8 mm i.d.; 10 mm) and coupled to a Bruker Esquire LC mass spectrometer.
Chromatography was carried out with MeOH : H2O (85 : 15) as the mobile phase
delivered at a flow-rate of 1 mL min
1, and the injected sample size was 20 mg.
MS were measured using electrospray ionization (negative ion mode). IR spectra
were obtained using KBr discs in a Perkin-Elmer model FT-IR 1600 spectrometer.
One- and two-dimensional NMR spectra were measured with samples dissolved in
CDCl3 using Bruker models AMX 400 and AVANCE 400 and 600 spectrometers
(300K and TMS as the internal standard).
3.3. Extraction and isolation of the active components
Dried leaf material was ground in a laboratory mill to a moderately fine (2.0 mm
screen) powder. Dry powder (3.5 kg) was extracted with 95% ethanol (12 L)
at room temperature for five days. The crude extract (390 g) was solubilized in
acetone and incorporated onto activated charcoal (250 g). The resulting mixture
was dried, powdered and placed in a Buchner funnel (20 cm diameter) over a layer
(2.5 cm) of silica gel 60 (230400 mesh; Merck). Elution was performed stepwise
with 2.5 L each of H2O, EtOH, EtOAc, CHCl3, and mixtures thereof. The
fractions were combined on the basis of their similarity into five groups: F1 H2O
(109.29 g), F2 H2O : EtOH (7 : 3) (12.85 g), F3 H2O : EtOH (1 : 1) (23.57 g),
F4 [EtOH EtOH : EtOAc (1 : 1) EtOAc] (161.53 g) and F5 CHCl3 (15.19 g).
The whole of F4 was submitted to CC over silica gel 60 and fractions were evaporated
to dryness to provide residues F4.1 hexane (66.96 g), F4.2 [hexane : CHCl3 (8 : 2)]
(44.76 g), F4.3 [hexane : CHCl3 (1 : 1) CHCl3] (16.25 g), F4.4 EtOAc (16.26 g)
and F4.5 MeOH (12.14 g). The whole of F4.4 was submitted to CC over silica gel
[eluted with CHCl3 : MeOH (98 : 2)], filtered through activated charcoal [eluted with
EtOH and EtOH : EtOAc (1 : 1)], submitted to CC twice more over silica gel [eluted
with CHCl3 : MeOH (98 : 2)], and twice over LH-20 Sephadex (eluted with MeOH) to
yield a product (90 mg) that had a waxy nature. This product was subjected to bioassay
and also analysed by TLC, NMR and HPLC-MS.
3.4. Bioassays
Samples were assayed for activity against adult B. glabrata snails and against larvae
of A. salina according to methods already described [20].

Acknowledgements
This work was financed by the CNPq, CAPES and FAPEAL (Brazil). The authors
are grateful to Cicero Barros (Instituto do Meio Ambiente do Estado de Alagoas,
Maceio-AL, Brazil) for help in collection and identification of plant material.

Acetogenins in A. muricata L. (annonaceae) leaves

257

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