Veterinary Parasitology 117 (2003) 43–49
Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. extracts on Haemonchus contortus L.M. Assis a , C.M.L. Bevilaqua a,∗ , S.M. Morais a , L.S. Vieira b , C.T.C. Costa a , J.A.L. Souza a a
Faculdade de Veterinária, Universidade Estadual do Ceará, Av. Paranjana 1700, Fortaleza, Ceara 60740-000, Brazil b EMBRAPA, CNPC, Brazil
Received 25 October 2002; received in revised form 15 June 2003; accepted 31 July 2003
Abstract The rapid development of anthelmintic resistance, associated with the high cost of the available anthelmintic drugs, had limited the success of gastrointestinal nematodiasis control in sheep and goats and thus awakened interest in the study of medicinal plants as alternative sources of anthelmintics. Spigelia anthelmia extracts obtained with hexane, chloroform, ethyl acetate or methanol, were tested on Haemonchus contortus eggs and larvae via egg hatch and larval development tests. The extracts were evaluated at five concentrations: 3.1, 6.2, 12.5, 25.0 and 50.0 mg ml−1 . At 50.0 mg ml−1 , the ethyl acetate extract inhibited 100% of the egg hatching and 81.2% of the larval development. In a similar way the methanolic extract inhibited 97.4% of the egg hatching and 84.4% of larval development. These results suggest that utilization of S. anthelmia extracts may be useful in the control of sheep and goats gastrointestinal nematodes. © 2003 Elsevier B.V. All rights reserved. Keywords: Spigelia anthelmia; Haemonchus contortus; In vitro; Egg; Larvae
1. Introduction Sheep and goats breeding are the more developed activity in semi-arid northeastern Brazil for its capacity of resistance to adverse conditions. The main interest of small ruminant exploitation is the production of meat for feeding rural low-income populations. However, gastrointestinal nematode parasitism is pointed out as the main retrain factor of this activity. An epidemiological investigation carried out in this region showed that Haemonchus ∗ Corresponding author. Tel.: +55-85-299-2753; fax: +55-85-299-2740. E-mail address:
[email protected] (C.M.L. Bevilaqua).
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.07.021
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contortus represents 80% of sheep and goats worm burden (Arosemena et al., 1999). The control of gastrointestinal nematodes of small ruminants is performed almost exclusively with anthelmintics. However, the rapid development of resistance to these drugs among nematodes, associated with high cost, food residues and environmental pollution have awakened interest in medicinal plants as an alternative source of anthelmintic drugs (Herd, 1996; Vieira et al., 1999; Melo et al., 2003). Spigelia anthelmia Linn. (Logoniaceae), a native plant from Asia and tropical America is used against gastrointestinal helminths in the folk medicine of Brazil, Panama and Costa Rica (Achenbach et al., 1995; Braga, 2001). Preliminary reports about aqueous extract of this plant related a promising anthelmintic activity (Batista et al., 1999). The aim of the present study was to evaluate the in vitro effects of S. anthelmia extracts prepared with hexane, chloroform, ethyl acetate and methanol on eggs and larvae of the abomasal nematode of sheep and goats, H. contortus. 2. Materials and methods 2.1. Plant extracts preparation Dried aerial parts of S. anthelmia (2 kg) were mixed with ethanol (5 l) and, left in contact for 1 week at room temperature (28 ◦ C). After this period, the solvent was evaporated and the residue (350 g) was mixed with the same amount of silica gel. This mixture was submitted to a vacuum chromatographic column, being eluted with the organic solvents, hexane, chloroform, ethyl acetate and methanol. After evaporation of solvents, the obtained extracts were tested for anthelmintic activity. An initial solution was prepared with the hexane and chloroform extracts (5 g) with 3 ml dimethylsulfoxide to improve solubility in water (100 ml solution). For the ethyl acetate extract (5 g) Tween 80 (3 ml) was used to make a 100 ml aqueous solution. Aliquots of the start solutions (50 mg ml−1 ) were taken to obtain the following concentrations: 3.1, 6.2, 12.5, 25.0 and 50.0 mg ml−1 . 2.2. H. contortus egg recovery 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 (Le Jambre and Royal, 1977) were mixed with tepid water and filtered through 590, 149, 101 and 30 m aperture sieves. The eggs retained on the 30 m sieve were collected. 2.3. Egg hatch test The in vitro egg hatching test was based on the method described by Coles et al. (1992). A suspension of 0.2 ml was distributed in a 24-flat-bottomed microtitre plate containing approximately 100 fresh eggs/well and mixed with the same volume of plant extract. The control plates contained the diluent water and DMSO or Tween 80, depending on the extract, and the egg solution. The eggs were incubated in this mixture for 48 h at room temperature.
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After this time a Lugol iodine solution was added to stop the eggs from hatching. All the eggs and first-stage larvae (L1) in each plate were counted. There were five replicates for each concentration and control. Data are expressed as percentage of unhatched eggs. 2.4. Larval development test The procedures used were a modification of the technique described by Hubert and Kerbouef (1992). 170 l aliquots of a suspension with about 100 eggs/well, and 50 l of filtrate obtained by fecal washing during egg recovering were distributed to wells of a 24-well flat-bottomed microtiter plate. This suspension was supplemented with 20 l of the nutritive medium described by Hubert and Kerboeuf (1984). 10 l of amphotericine B solution (Fungizone® Squibb) at 5 g/ml was added to the egg suspension to avoid fungal development. The plates were incubated at room temperature. After 48 h, 250 l of the extract or diluent (control) were added. Seven days after, one drop of Lugol’s iodine solution was added. At this time the parasites were counted by separating the larvae into two classes, third-stage larvae (L3) and other developmental stages larvae (L1 and L2). There were five replicates for each concentration and control. Data are expressed as percentage of L1 + L2. 2.5. Statistical analysis Data obtained in the egg and larval development tests were transformed by the formula: log(x+1), subjected to variance analysis and Duncan’s test to compare the mean percentages of egg hatch and larval development inhibition among the concentrations of each extract and the control and among different extracts of S. anthelmia, using a statistical probability of 5% as the criterion for significance. 3. Results The methanol and ethyl acetate extracts inhibited (P < 0.05) hatching of a higher number of eggs than controls in all concentrations tested. The methanol extract at 25.0 and 50.0 mg ml−1 concentrations obtained greater inhibition of egg hatch than the controls. The chloroform extract did not obtain values statistically different from the controls. Ethyl acetate and methanol extracts were more effective against egg hatch compared to the hexane and chloroform extracts at 50.0 mg ml−1 (Table 1). At all concentrations of the ethyl acetate extract the percentage of larvae that failed to develop was higher than the controls. At 25.0 and 50.0 mg ml−1 methanol extract and only at 50.0 mg ml−1 of hexane extract, the percentage of larval development inhibition was higher (P < 0.05) than the control. The inhibition of larval development in the presence of the chloroform extract was not different from the control. Table 2 shows the effect of S. anthelmia extracts on H. contortus larvae development. Unlike other extracts, the ethyl acetate extract inhibited larval development at the two lowest concentrations tested. The ethyl acetate and methanol extracts at 50.0 mg ml−1 concentration inhibited development of a higher number of larvae than the hexane and
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Table 1 Mean inhibition percentage±S.D. of H. contortus egg hatch with different concentrations of S. anthelmia extractsa Concentration (mg ml−1 ) 3.1 6.2 12.5 25.0 50.0 Control
Extract Hexane
Chloroform
Ethyl acetate
Methanol
28.0 ± 16.8 aA 32.1 ± 26.8 aA 42.4 ± 29.8 aA 48.9 ± 23.3 aA 46.5 ± 27.3 aB 9.3 ± 8.7 bA
15.0 ± 20.2 aB 16.1 ± 13.1 abA 31.0 ± 20.6 abA 53.4 ± 29.2 aA 46.7 ± 29.4 aB 19.8 ± 6.4 abA
14.5 ± 16.8 bA 13.8 ± 6.4 bA 20.9 ± 11.2 bA 83.8 ± 28.4 aA 100.0 ± 0.0 aA 4.6 ± 4.6 cA
22.8 ± 11.4 bcA 35.8 ± 31.7 bcA 35.0 ± 20.6 bcA 59.3 ± 29.3 abA 97.4 ± 5.9 aA 19.7 ± 20.2 cA
a Small letters compare means in the columns and capital letters in the lines. Different letters indicate significantly different values (P < 0.05).
Table 2 Mean inhibition percentage ± S.D. of H. contortus larval development with different extracts of S. anthelmiaa Concentration (mg ml−1 )
Hexane
Chloroform
Ethyl acetate
Methanol
3.1 6.2 12.5 25.0 50.0 Control
14.6 ± 21.6 bB 17.4 ± 3 bB 11.3 ± 12 abB 16.4 ± 4.1 abB 45.0 ± 26.7 aB 10.9 ± 17.2 bA
13.9 ± 20.4 aB 20.9 ± 28.1 aB 29.0 ± 37.4 aAB 17.2 ±18.6 aB 36.9 ± 24.3 aB 11.0 ± 13.3 aA
67.6 ± 28.8 bA 81.0 ± 18.5 bA 65.0 ± 36.5 bA 83.1 ± 19.3 bA 81.2 ± 17.8 bA 25.9 ± 16.4 aA
3.1 ± 3.9 cB 5.2 ± 4.0 cB 16.8 ± 24.5 bcB 36.1 ± 26.5 abAB 84.4 ± 14.3 aA 16.7 ± 27.9 cA
Extract
a Small letters compare means in the columns and capital letter in the lines. Different letters indicate significantly different values (P < 0.05).
chloroform extracts. There was no difference among the values obtained in the controls in either test. During the larval development test, disintegrated larvae were observed in cultures treated with the ethyl acetate and methanol extracts. These larvae were included in the L1 and L2 counts.
4. Discussion The tests performed in this study for screening the anthelmintic activity of S. anthelmia extracts were based on established methods, for evaluating the efficacy of anthelmintics. Several in vivo and in vitro techniques have been developed to detect anthelmintic resistance in nematodes (Craven et al., 1999). However, in vivo tests are not the best model to screen plants extracts with anthelmintic activity, since, these tests are time-consuming, expensive and present low precision and reproducibility due to interanimal variation and pharmacodynamics of the drug in the host (Lacey et al., 1990). Although the adult nematode is the major targets for the chemotherapy, gastrointestinal nematodes parasites cannot yet be raised in continuous culture (Geary et al., 1999). Thus,
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the lack of a culture system yielding adults of any nematode parasite prevents a preliminary study of the effect of medicinal plants on this stage. On the other hand, the in vitro tests using free living stages of parasitic nematodes offer a means of evaluating the anthelmintic activity of new plant compounds, as already reported by various authors (Robinson et al., 1990; Amorim et al., 1991, 1996, 1998; Asuzu and Njoku, 1996). These in vitro tests measure the effects of anthelmintics directly on physiological processes such as hatch, development and motility of the parasites (Varady and Corba, 1999). The high frequencies of egg hatch (80–93%) and larval development (74–89%) observed in the absence of the extracts showed that the different diluents, DMSO, and Tween 80, did not interfere with natural development of eggs and larvae. The results obtained with the ethyl acetate and methanol extracts at 50.0 mg ml−1 showed a high percentage egg hatch inhibition, varying from 100 to 97.4%, respectively. These results are in agreement with those obtained by Batista et al. (1999) who observed that 50% of eggs remained in the initial stages of development with 0.17 mg ml−1 concentration of S. anthelmia aqueous extract. Despite the fact that hexane extract gave some inhibition of egg hatch in all concentrations tested, it was not as efficient as the ethyl acetate and the methanol extracts. The egg hatch test is used to detect nematode resistance to benzimidazoles. It is believed that the anthelmintic acts to prevent egg embryonation. Higher levels of activity observed in the methanol and ethyl acetate extracts suggest that the anthelmintic component of S. anthelmia is a relatively polar compound. Gill et al. (1995) reported that the strongly hydrophobic nature of the avermectins prevents their penetration into nematode egg shells. The ethyl acetate extract was submitted for column chromatographic treatment and two compounds were isolated and characterized by spectral means as the alkaloid, spiganthine and a flavonoid, 3,7-dihydroxy-3 ,4 -dimethoxyflavone (Morais et al., 2002). The cardio active principle, spiganthine, isolated from S. anthelmia (Achenbach et al., 1995) has a chemical structure and biological function similar to ryanodine-type compounds, which cause a considerable delay in the development of contractions. Ryanodines were isolated from some plant species (Jefferies et al., 1992) and shown experimentally to reduce motility in the free living nematode Caenorhabditis elegans (Maryon et al., 1998). Ryanodine, after interaction with specific receptors called “ryanodine receptors” keeps the calcium channel in the sarcoplasmatic reticulum in the open state and induces tonic contractions in isolated skeletal muscle and tonic paralysis in mammals and worms (Kim et al., 1992; Hong, 2000). This type of compound is present in the S. anthelmia active extracts obtained with ethyl acetate and may be responsible for the inhibition of larval development. The investigation of chemical compounds from natural products is fundamentally important for the development of new anthelmintic drugs, especially in view of the vast worldwide flora. Based on the results presented in this work S. anthelmia offers an opportunity for a new compound. This plant may offer an alternative source for the control of gastrointestinal nematodes of sheep and goats. However, more detailed studies are needed to identify and evaluate the active components and the mechanisms of action of S. anthelmia extracts. Also, studies on toxicity, evaluation of the effects in vivo and the establishment of adequate doses for sheep, goats and cattle are needed.
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