Abstract
Based on the results of ethnomedical and ethnoveterinary surveys that we had carried out in northern Côte d'Ivoire during 1999–2001, 79 plant species belonging to 36 families were selected for in vitro. anthelmintic screening. The activity of ethanol crude extracts was investigated using the nematode Haemonchus contortus. Rudolphi (1803) as the test species. Of 115 extracts tested, 50 showed larvicidal activity and two were ovicidal. The most active came from Sclerocarya birrea. (A. Rich) Hochst followed by Lannea kerstingii. Engl. & K. Krause, Aframomum alboviolaceum. (Ridely) K. Schum., Pericopsis laxiflora. (Benth) van Meeuwen, Pseudocedrela kotschyi. (Schweinf.) Harms, Securidaca longepedunculata. Fres., Alchornea cordifolia. (Schum. & Thonn.) Müll. Arg., Anthostema senegalense. A. Juss, Ficus vallis-choudae. Del., Ampelocissus grantii. (Lour) Merrill, Vitellaria paradoxa. C. F. Gaertn, and Hibiscus asper. Hook f. The results supported the traditional uses of some of the tested plants in the treatment of intestinal helminthosis. As Haemonchus contortus. is a pathogenic nematode in small ruminants, the active plant species could be a potential source of new lead anthelmintic agents.
Introduction
Livestock are important in supporting the livelihoods of poor farmers, consumers, traders, and laborers throughout the developing world. The enhancement of livestock-production systems will have an important impact in order to favor a sustainable development and to improve the conditions of the poor population (FAO, Citation2002). In the Côte d'Ivoire as well as in many West African countries, the dominant systems of livestock production are traditional pastoral and sedentary village husbandry. In these conditions gastrointestinal nematode infections are of considerable economic importance, causing clinical disease with mortalities but more importantly by causing chronic production losses as a result of reduced weight gain, weight loss, and reduced milk production (FAO, Citation1992). Among the helminths encountered in the Côte d'Ivoire, the parasites of the genus Haemonchus. represent the dominant species in cattle and small ruminants (Komoin-Oka et al., Citation1999Citation2000; Achi et al., Citation2003aCitationb). Under field conditions in the savanna in northern Côte d'Ivoire, Haemonchus contortus. Rudolphi (1803) was found in 96.5% and 97.6% of the sheep and goat populations studied, respectively, and the proportion of this species within the total number of worms identified in these hosts was 96.1% for sheep and 89.4% for goats (Achi et al., Citation2003c). This gastrointestinal nematode is one of the most common and widespread pathogenic parasites, with a largest clinical and economic impact on sheep and goat production (FAO, Citation1992).
An efficient therapy of worm infections remains a problem in many breeding areas in Côte d'Ivoire. Hardly any control of parasitic gastroenteritis infections is practiced in most traditional production systems (Baker, Citation1995; Chiejina, Citation1995). Modern veterinary cure is inefficient because of the relatively high cost of anthelmintics, the scarcity of modern anthelmintic drugs, and the inaccessibility of many villages as well as the nomadic and transhumant tradition. Herbal treatments are therefore often used as a potential alternative, and people know a great deal about medicinal plants in the breeding areas such as the Ferkessédougou region in the north of Côte d'Ivoire. At first, an ethnobotanical survey was carried out in this area in 1999–2001 on plants used in traditional medicine to treat intestinal worm infections and associated discomforts.
The biological activity of medicinal plants against helminths is poorly investigated, and a corresponding literature about anthelmintic activity of medicinal plants from the Côte d'Ivoire is scarce (Engoué, Citation1986; N'goran, Citation1986; Diehl, Citation1998; Okpekon et al., Citation2004).
As a single, inexpensive, and available alternative, traditional medicine is often used in the form of herbal treatment. Studies on medicinal plants used for animal health could contribute to increase livestock production in Côte d'Ivoire. In this paper, we report the in. vitro. anthelmintic activity of 79 of the 137 plants collected in a 1999–2001 survey.
Materials and Methods
Plant selection
Seventy-nine plant species belonging to 36 families were selected for in vitro. anthelmintic screening on the basis of a questionnaire study carried out during 1999–2001 in the Ferkessédougou region. This area is located in northern Côte d'Ivoire about 585 km from Abidjan. The traditional use of the selected plants is shown in . Breeders as well as healers for traditional treatment of intestinal worm infections and discomforts associate use as individual applications rather than as mixtures for most of these plant species. The plant species were identified by a botanist (Henri Tere, CSRS). Voucher specimens are preserved in the herbarium of the Centre Suisse de Recherches Scientifiques (CSRS) in Adiopodoumé, Côte d'Ivoire.
Table 1.. Traditional uses of medicinal plants from the Ferkessédougou region selected for anthelmintic screening.
Preparation of crude extracts
After collection, plant parts were dried at ambient temperature. For the laboratory analysis, they were dried in an air-conditioned room (18°C) and then pounded by hand. Crude plant extracts were then prepared by extracting the plant powders in a 10-fold excess of 90% ethanol, with mechanical stirring, during 14 h at room temperature (25°C). The ethanol was completely evaporated at 40°C, and the extracts were lyophilized and stored at 4°C until the in vitro. screening.
Isolation of eggs
Sheep were experimentally infected with 3000–6000 third-stage larvae (L3) of a local isolate of Haemonchus contortus.. Three weeks after infection, fecal egg counts were made to determine the number of eggs produced by the sheep. A number of 3000 eggs per gram feces (EPG) were considered as a reasonable level to undertake anthelmintic tests. Feces were collected in the morning and triturated during 5–10 min to obtain a soft paste that was diluted with tap water and filtered through a coarse gauze. The filtrate was passed through a sieve with 32-µm pore size using an abundant water flow. The retained solid part was collected, solubilized in a saturated sugar solution (1:3) to obtain 200 to 250 ml of suspension, and centrifuged at 1500 rpm/min during 10 min. The supernatant was passed through two sieves composed of, first, a 50-µm, followed by a 32-µm meshwork superimposed. The eggs retained on the second sieve were recovered using water. The suspension was adjusted with water to obtain a concentration of 80 eggs per 20 µl.
Anthelmintic tests
The anthelmintic assays were performed using the method previously described by Diehl (Citation1998). Lyophilized plant extracts (30 mg) were suspended in 1 ml of DMSO and diluted with demineralized water to final concentrations from 1.7 to 0.0013 mg/ml in wells of flat-bottomed microtitration plates. Agar (140 µl) (45–50°C) containing 2% of amphotericin B was added to the wells, and 80 eggs were transferred onto the agar. The microtitration plates were kept in a humid atmosphere (90%) during 6 days at 27°C. In order to monitor the normal development of the larvae, wells containing eggs in water only were used. The number of unhatched eggs and the number of larvae were counted; the developmental stages of larvae and their mobility were also noted. The larvicidal (LC100) or ovicidal (OC100) concentration was defined as the lowest concentration that was still able to block completely the normal larval development. The tests were repeated three times with all extracts that showed some anthelmintic effect. Ivermectin and fenbendazole, commercial anthelmintics, were used as standard controls (from 0.283 to 1.67 × 10−4 mg/ml).
Results
Of 115 crude extracts tested, 52 (45.22%) showed some activity against Haemonchus contortus.. When considering LC100 values, the most active extracts containing larvicide potency (0.0963–0.00546 mg/ml) were obtained from 15 plant species belonging to 13 families. These plants were Sclerocarya birrea. followed by Lannea kerstingii, Aframomum alboviolaceum, Pericopsis laxiflora., Pseudocedrela kotschyi., Securidaca longepedunculata, Anogeissus leiocarpus., Alchornea cordifolia., Annona senegalensis., Anthostema senegalense., Piliostigma thonningii., Ficus vallis-choudae., Ampelocissus grantii., Vitellaria paradoxa., and Hibiscus asper. (). Only 2 extracts exhibited ovicidal activity with OC100 value of 0.849 mg/ml. These came from Hoslundia opposita. and Cochlospermum planchonii. ().
Table 2.. Values of larvicidal or ovicidal concentrations (mg/ml) of active plants and anthelmintics.
It was observed that a great number of active extracts came from plant species belonging to botanical families such as Euphorbiaceae, Fabaceae, Moraceae, Rubiaceae, and Anacardiaceae ().
Discussion and Conclusions
The aim of this study was to evaluate the anthelmintic potency of plants used in traditional medicine in Côte d'Ivoire for the treatment of intestinal helminthosis. Some of the plants used in Côte d'Ivoire to heal intestinal worm infections do contain ovicidal or larvicidal activity against Haemonchus contortus., a gastrointestinal nematode responsible for important losses in small ruminant production. Most of these tested plant species are used in ethnoveterinary medicine for the treatment of gastroenteritis infections in the Ferkessédougou region or in other areas of the Côte d'Ivoire and Africa (Aké Assi, Citation1992; Bizimana, Citation1994). This report ascertains the value of some of the plants used in Ivorian traditional medicine that could be of great interest to the development of new anthelmintic agents for the treatment of animal gastrointestinal helminthosis. To a certain extent, one could expect also an action against intestinal helminths in man, as most of these active plants are also traditionally used in Africa for human intestinal helminthiasis and associated discomforts. For example, some of the most actives plants such as Annona senegalensis. (Abbiw, Citation1990; Aké Assi & Guinko, Citation1991; Lejoly et al., Citation1994, Neuwinger, Citation1996; Koné et al., Citation2002), Anogeissus leiocarpus. (Abbiw, Citation1990; Aké Assi & Guinko, Citation1991; Koné et al., Citation2002; Okpekon et al., Citation2004), Lannea kerstingii. (Abbiw, Citation1990; Arbonnier, Citation2002), Sclerocarya birrea. (Abbiw, Citation1990; Lejoly et al., Citation1994; Iwu, Citation1993), Securidaca longepedunculata. (Iwu, Citation1993; Lejoly et al., Citation1994), and Pseudocedrela kotschyi. (Abbiw, Citation1990) are widely used in African traditional medicine against intestinal worm infections. For other plant species, such as Vitellaria paradoxa. and Pericopsis laxiflora., the traditional use for symptomatic treatment (diarrhea, dysentery, and abdominal pain) had switched to anthelmintic properties.
Previous studies reported the anthelmintic activity of Securidaca longepedunculata. (Ousoumanou et al., Citation1991; Diehl, Citation1998), Anogeissus leiocarpus., Alchornea cordifolia., and Bridelia ferruginea. (Okpekon et al., Citation2004). Kaurenoic acid isolated from Annona senegalensis. and protoanthocynidin from Piliostigma thonningii. displayed some activity against Haemonchus contortus. (Neuwinger, Citation1996; Fakae et al., Citation2000). The current study confirmed the anthelmintic activity previously found in these plant species.
Some botanical families, such as Euphorbiaceae, Fabaceae, and Moraceae, are interesting when considering the number of active plant species. These families could be targeted in the search for novel intestinal worm therapeutics. Anthelmintic properties were previously described for species of these families. For the Moraceae, it was shown that the anthelmintic activity of the latex was due to a proteolytic fraction, called ficin (de Amorin et al., Citation1999). The pharmacological studies are advanced on some species like Ficus glabrata. (Moraceae) that are well-known in the Amazonian area for its action against intestinal worms. Preclinical trials resulted in the recommendation of doses for the administration of this plant extract (Hansson et al., Citation1986). Euphorbiaceae are also a promising family in displaying anthelmintic activity. The three Euphorbiaceae species tested by Opkekon et al. (Citation2004) exhibited an anthelmintic activity. From Picrodendron baccatum. (Euphorbiaceae), picrodendrin P, a terpene, was isolated as the compound responsible for the nematocidal activity. This substance was ninefold more efficient than the kainic acid and santonin (Watanabe et al., Citation1999). All these could be corroborated by the antiparasitic activity previously found in some plant species of this family. For example, Euphorbia hirta. had been shown to be effective in vitro. and in vivo. against the protozoan parasite Entamoeba. (Sofowora, Citation2002).
This study provides evidence that some of the plants used in Côte d'Ivoire for traditional treatment of intestinal helminthiasis could be leads for new veterinary anthelmintic agents. Phytochemical investigations are underway on three plants in order to identify the active principle(s) responsible for anthelmintic activity.
Acknowledgments
We are thankful to our sponsors: the Centre Suisse de Recherches Scientifiques in Côte-d'Ivoire (CSRS) and the International Foundation for Science (IFS, grant B/3177). Thank are also due to all the breeders and healers we met in the Ferkessédougou region.
Related Research Data
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