Anthelmintic acetogenin from Annona squamosa L. Seeds

CHEMICAL SCIENCES

Anthelmintic acetogenin from Annona squamosa L. Seeds

Marta M.C. Souza^I; Claudia M.L. Bevilaqua^I; Selene M. Morais^II; Cícero T.C. Costa^I; Ana R.A. Silva^II; Raimundo Braz-Filho^III

^ILaboratório de Doenças Parasitárias, Universidade Estadual do Ceará, Av. Parajana 1700, 60740-000 Fortaleza, CE, Brasil

^IICurso de Química, Universidade Estadual do Ceará, Av. Parajana 1700, 60740-000 Fortaleza, CE, Brasil

^IIISetor de Química de Produtos Naturais, Universidade Estadual do Norte Fluminense, 28013-600 Campos, RJ, Brasil

^{Correspondence to}

ABSTRACT

Annona squamosa seeds extracts showed anthelmintic activity against Haemonchus contortus, the main nematode of sheep and goat in Northeastern Brazil. A compound 1 was isolated from ethyl acetate extract and inhibited the egg hatching of H. contortus at 25 mg ml^-1. The structure of 1 was determined as a C₃₇ trihydroxy adjacent bistetrahydrofuran acetogenin based on spectroscopic analysis.

Key words:Haemonchus contortus, nematodes, Annonacea, phytotherapy.

RESUMO

Extratos das sementes de Annona squamosa demonstraram atividade anti-helmíntica contra Haemonchus contortus, o principal nematódeo de ovinos e caprinos no Nordeste do Brasil. O composto 1 foi isolado do extrato acetato de etila e inibiu a eclosão dos ovos de H. contortus a 25 mg ml^-1. A estrutura de 1 foi determinada como uma acetogenina C₃₇ tri-hidroxi bis-tetrahydrofurano adjacente, baseando-se nos dados espectrais.

Palavras-chave:Haemonchus contortus, nematódeos, anonácea, fitoterapia.

INTRODUCTION

In Northeastern Brazil, gastrointestinal parasitism ofsheep and goats is one of the leading causes of mortality, producing high economic losses (Pinheiro et al. 2000). Among gastrointestinal nematodes, Haemonchus contortus is the most frequent and pathogenic, being responsible for the high mortality rate in young animals during the rainy season (Menezes et al. 1992, Arosemena et al. 1999). Trying to reduce these losses, synthetic anthelmintics are routinely used, often indiscriminately, causing reduced efficacy besides environmental pollution and food residues (Waller et al. 1995, Herd 1995).To decrease these negative impacts, the phytotherapy is an alternative tool that has been studied by many researches nowadays. The study of plants from Annonacea family has demonstrated the presence of active substances with parasiticidal effects (Duret et al. 1998). Annona squamosa seed powder is popularly used against insects in Northeast of Brazil (Braga 2001). In the search for new anthelmintic agents to be used in the control of goat nematodes, the anthelmintic activity of extracts and isolated compounds of A. squamosa seeds were evaluated on egg hatching of H. contortus.

MATERIALS AND METHODS

PREPARATION OF A. squamosa EXTRACTS

The seeds of A. squamosa were obtained by manual extraction from fruits bought in the central market of Fortaleza. The extracts were prepared by the methodology performed by Nonfon (1990) as follows: A. squamosa seeds (2 kg) were triturated to a powder that was mixed with methanol/water solution (90:10, 3 l) and left in contact for seven days. This mixture was filtered and an aliquot of this initial solution (300 ml) was evaporated to dryness for bioassay analysis. The aqueous-methanol solution was evaporated under reduced pressure using a rotatory evaporator. Methanol was eliminated and the remaining aqueous solution was transferred to a separatory funnel and washed up four times (4 × 30 ml) with ethyl acetate that was evaporated to obtain the correspondent extract (15.0 g). The aqueous layer was dried in a water bath to obtain the aqueous extract (44.0 g).

ISOLATION OF ACETOGENIN 1

The Ethyl acetate extract (EtOAc) (15 g) was submitted to a silica gel (0.063-0.2 mm) being eluted with chloroform, ethyl acetate and methanol in mixtures of increasing polarity. Fifty-two (10 ml) fractions were obtained and compared by TLC using silica gel kieselgel 60 from Merck, visualized by exposure to I₂ vapor. Compound 1 (302 mg) was isolated by elution the column with 100% ethyl acetate.

PREPARATION OF ACETYL DERIVATIVE (1a)

The acetyl derivative was obtained by reaction of compound 1 (100 mg) with acetic anhydride (3 ml) and pyridine (1 ml) mixture at room temperature for 24 hours. Then a hydrochloric acid solution (5%) was added(10 ml). The reactional mixture was transferred to a separatory funnel and extracted with chloroform (3 × 5 ml). The organic layer, containing the acetyl derivative was washed with water (5 × 5 ml), dried with sodium sulfate and the solvent was evaporated leaving 1a (80 mg) (Furniss et al. 1989).

CHEMICAL ANALYSIS OF ACETOGENIN 1

Optical rotation was made in CHCl₃ (Perkin Elmer 341); IR spectra were recorded on Perkin Elmer FT-IR spectrum 100 spectrophotometer and the values are expressed in cm^-1. NMR spectra were recorded on a Brucker Avance DRX-500 spectrometer in CDCl₃. 70 eV EI-Mass spectra was obtained using a Hewlett-Packard 5971 GC/MS instrument employing the following conditions: column: Dimethylpolysiloxane DB-1 coated fused silica capillary column (30 m × 0.25 mm × 0.25 mm); carrier gas: He (1 ml/min); capillary injector operating at 250°C in the split mode (1:100); detector temperature: 200°C; column temperature: 35-180°Cat 4°C/min then 180-250°C at 10°C/min; mass spectra: electron impact. High-resolution mass spectra were obtained in an ultrOTOF_Q - ESI-TOF Mass Spectrometer, Bruker Daltonics, Billerica, MA, USA. Experiment conditions: Infusion bomb, Flux 300 µl/h, Mobile phase for solubilization: H₂O:MeOH (20:80). Detection mode was positive and negative for the sample. For internal calibration it was used a 10mg/ml NA-TFA solution and for external calibration a 10mM sodium formiate solution. Analysis conditions: End Plate: 4000 Volts, Capillary: 4500 Volts, Capillary Exit: 300 Volts, Skimmer 1: 50 Volts, Skimmer 2:25 Volts, Transfer: 90 µs, Collision Exit Gate: 80 µs. TLC analyses were performed on a 3-10 cm aluminum sheet precoated with silica gel 60-254 (Merck) (Solvent system: CHCl₃/EtOAc 3:7). SiO₂, 200-400 mesh (Merck) was used for column chromatography.

Compound 1 was isolated as a viscous oil: +11.57° (c=0.001, CHCl₃); UV (l_max, MeOH, nm): 225 (log e 3.19); IR (KBr) n_max 3418, 2927, 2855, 1748, 1652, 1463, 1319, 1118, 1068, 1028, 953, 877, 756, 666 cm^-1; ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) are showed in Table I. EIMS 70 eV m/z: 481(3), 341 (5), 327 (7), 281 (45), 253 (12), 155 (5), 111 (8), 85 (32), 71 (57), 57 (72), 43 (43). A mass spectral fragmentation of compound 1 in the EI mass spectrum is shown in Figure 1.

Compound 1a was obtained as a waxy solid: +15.57° (c=0.001, CHCl₃); UV (l_max, MeOH, nm): 225 (log e 2.61); IR (KBr) n_max 2928, 2855, 1748, 1653, 1465, 1436, 1317, 1250, 1120, 1070, 1026, 950, 876, 759, 668 cm^-1; ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz). EIMS 70 eV m/z: 481 (4), 341 (5), 281 (58), 253 (18), 155 (4), 141 (5), 137 (6), 125 (4), 111 (8), 97(17). C₃₇H₆₆O₇: 622.480306 (M+), 623.924945 (M+H), 645.906374 (M+Na).

ANTHELMINTIC BIOASSAY - in vitro EGG HATCH TEST

H. contortus eggs were recovered according to Hubert and Kerboeuf (1984). Briefly, 10 g of feces were collected directly from the rectum of sheep experimentally infected with H. contortus mixed with tepid water andfiltered through 590; 149; 101 and 30 µm aperturesieves. The eggs retained on the 30 µm sieve were collected.

The in vitro egg hatch test was carried out by the method of Coles et al. (1992). To perform this test, a volume of 500 µl, with 250 µl of the eggs solution (about 120 eggs) and 250 µL of extract solution were placed in 5 ml tubes. The extract solutions for the bioassay were prepared using the concentrations of the extracts tested were 25; 5; 1; 0.2; 0.04 mg ml^-1, the negative control was the diluent Tween 80 and the positive control thiabendazole (0.1 µg ml^-1). After incubation for 48 hours, a drop of Lugol was added. All larvae and eggs were counted using a microscope. Five replicates were performed for each concentration. Data were expressed as percentage of egg hatching inhibition. Statistical comparisons of the results from the egg hatching inhibition by the extracts on different concentrations were performed using Kruskal-Wallis test, with significance level of 5%. The effective concentration to inhibit half eggs hatching (CE₅₀) for each extract was calculated by the equation which showed the tendency line with minimum determination coefficient of 70%. The values of negative and positive controls are expressed as mean results.

RESULTS AND DISCUSSION

Table I displays the ¹H and ¹³C-COSY 2D NMR (HMQC and HMBC) data of compound 1 and its acetyl derivative 1a. Compound 1 showed to be an acetogenin, a type of compound common in plants of Annonaceae family, with 37 carbons, three hydroxyl groups, an unsaturated lactone moiety (d 6.99, H-35; d 148.9, C-35; d 134.2, C-2; d 173.8, C-1) and two adjacent tetrahydrofuran(THF) rings (Figure 2). The adjacent bis-THF rings with flanking OH groups in 1 were indicated by ¹HNMR resonances at d 74.1 (C-15), 83.2 (C-16), 82.5 (C-19), 82.1 (C-20), 82.7 (C-23) and 71.4 (C-24). The relative stereochemistry of the bis-THF moiety of 1 was suggestedto be threo-trans-threo-trans-erythro by careful comparison of the ¹H and ¹³C NMR signals of 1 with stereo-chemically defined bis-THF acetogenins (Araya et al. 2002, Alali et al. 1999, Zhou et al. 2000, Hopp et al. 1998). The presence of three hydroxyl groups in the molecule was evidenced by an absorption band in the IR at 3418 cm^-1 and three singlets at d 2.08, 2.05, and 2.03 in the ¹H NMR spectra of the acetyl derivative 1a. The presence of two THF rings was suggested by signals in the ¹H NMR at d 3.43 (H-15), 3.84 (H-16, H-19, H-24) and 3.95 (H-20, H-23), which integrated constitutes 6 protons. A third hydroxyl group was evidenced by ¹H NMR signal at d 3.62 and a ¹³C NMR signal at d 71.7. Its position was predicted to be at C-5 based on the EIMS fragments ions at m/z 155, 111, 137, 111 and 97 (Figure 1) common in other C-5 hydroxylated acetogenins (Alali et al. 1999) and associated signals of ¹H-¹H COSY, HMQC and HMBC of the acetyl derivative 1a. In the bidimensional homonuclear ¹H-¹H COSY spectrum of 1a, the sequence of couplings between H-3 (t, d 2.32) and H-4 (m, d 1.55) and H-4 with H-5 (m, d 4.91) assures the presence of a hydroxyl group at C-5. The IR spectra of compound 1 showed as main peaks at 3418 cm^-1 for hydroxyl groups and at 1748 and 1652 cm^-1 for the C=O and the double bond of the lactone moiety respectively, that are compatible with data of similar acetogenins from seeds of A. squamosa (Rupprecht et al. 1990).

The percentage of egg hatching inhibition of H. contortus using MeOH/H₂O, aqueous, EtOAc extracts and compound 1 is shown in Table II.

The highest inhibition of egg hatching was obtained with the EtOAc extract, in the concentrations of 5 and 25 mg ml^-1, which did not demonstrate significant difference from thiabendazole, at 0.1 µg ml^-1. The aqueous extract showed an inhibition percentage higher than the negative control, Tween 80, at 25 mg ml^-1, but at 5 mg ml^-1 was not statistically different from the negative control. The EtOAc extract was submitted to a silica gel column chromatography and the main isolated compound 1 was evaluated for its anthelmintic activity. Compound 1 inhibited the egg hatching more than90% at 5 and 25 mg ml^-1 concentrations, similarly to the EtOAc extract. The concentration that is effective for killing half of larvae amount (CE₅₀) of the aqueous, methanol/water, ethyl acetate extracts and compound 1 was 10.02, 0.89, 0.76 and 0.78 mg ml^-1 respectively.

The extracts MeOH/H₂O, EtOAc and the acetogenin 1 at 5 mg ml^-1 concentration had similar results in the inhibition of egg hatching of H. contortus. This result could be explained by the fact that the extracts MeOH/H₂O and EtOAc possess several chemical compounds, and many of them could display ovicidal action. The total action of the extracts is a sum of the activities of their constituents (Rates 2001). A. squamosa aqueous extract did not show relevant activity, similarly to the aqueous extract of A. senegalensis bark in the H. contortus egg hatch inhibition test (Alawa et al. 2003). Probably these results are due to the low solubility of acetogenins in water.

The ovicidal effect obtained with ethyl acetate extract is lower than synthetic anthelmintics nevertheless it is important when compared with other plant extracts. Assis et al. (2003) reported 20% inhibition of H. contortus egg hatching with ethyl acetate extract of Spigelia anthelmia at 12.5 mg ml^-1 and the EtOAc extract of A. squamosa showed 99% egg hatch inhibition at 5 mg ml^-1. The effect of eight acetogenins, isolated from Uvaria hookeri and U. narum, against H. contortus adult specimens showed a negative relationship between the death time in minutes and acetogenins concentration (Padmaja et al. 1993). In conclusion, the seeds of A. squamosa represent an alternative natural source for anthelmintic compounds.

ACKNOWLEDGMENTS

This work received financial support from ConselhoNacional de Desenvolvimento Científico e Tecnológico(CNPq), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) and Projeto de Apoio ao Desenvolvimento de Tecnologias Agropecuárias para o Brasil/ Empresa Brasileira de Pesquisa Agropeduária (PRODETAB / EMBRAPA) government agencies. Authors are grateful to Centro de Ressonância Magnética Nuclear do Nordeste (CENAUREN), Universidade Federal do Ceará (UFC) for the NMR spectral data and to Dr. Alberto J. Cavalheiro from Chemistry Institute, Universidade Estadual Paulista (UNESP) for high resolution mass spectra.

Manuscript received on September 12, 2006; accepted for publication on November 12, 2007; contributed by RAIMUNDO BRAZ FILHO^*

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*

Member Academia Brasileira de Ciências

Publication Dates

• Publication in this collection
  14 May 2008
• Date of issue
  June 2008

History

• Received
  12 Sept 2006
• Accepted
  12 Nov 2007