Parasitol Res (2012) 111:325–330 DOI 10.1007/s00436-012-2843-8
ORIGINAL PAPER
In vitro and in vivo efficacy of aqueous extract of Caryocar brasiliense Camb. to control gastrointestinal nematodes in sheep Flávia A. Nogueira & Leydiana D. Fonseca & Rayana B. da Silva & Adriano V. de Paiva Ferreira & Patrícia S. Nery & Luciana C. Geraseev & Eduardo R. Duarte
Received: 20 April 2011 / Accepted: 24 January 2012 / Published online: 11 February 2012 # Springer-Verlag 2012
Abstract A major problem faced in sheep rearing has been the rapid acquisition of anthelminthic-resistant populations of gastrointestinal nematodes. In the search for alternatives, aqueous extract of the peel of Caryocar brasiliense was evaluated for larval development inhibition, egg-hatching inhibition, and fecal nematode egg count reduction in sheep. For in vivo analysis, the doses were calculated according to a 10% lethal dose derived from acute toxicity tests in mice, and the efficacy was evaluated for two periods following oral administration of the extract. Egg-hatching inhibition at concentrations of 15 and 7.5 mg/ml was significantly higher than observed in negative controls with distilled water. For larval development inhibition, all concentrations showed anthelminthic activity significantly higher than controls and were not significantly different from ivermectin treatment. The LC90 of larval development inhibition was 53.19 mg/ml. In vivo analysis for first and second weeks after treatment found 32.2% and 33% anthelminthic efficacy, respectively.
Introduction Sheep are bred by production methods ranging from family farms to larger rural companies. Helminthiasis represents F. A. Nogueira : L. D. Fonseca : R. B. da Silva : A. V. de Paiva Ferreira : P. S. Nery : L. C. Geraseev : E. R. Duarte (*) Instituto de Ciências Agrárias, Universidade Federal de Minas Gerais, Av Universitária 1000, Bairro Universitário, Montes Claros, Minas Gerais 39400-006, Brazil e-mail:
[email protected]
the major health problem for these animals, and both sexes at all age levels may be intensely affected, not only reducing weight gain and reproductive capacity but also milk, wool, and hide production (Bizimenyera et al. 2006; Pires et al. 2000). Gastrointestinal nematode (GIN) control has relied on the frequent use of synthetic anthelminthic drugs. However, a significant decrease in their efficacy has been observed in the major sheep-producing regions due to insufficient technological training or lack of adequate information on treatment protocols and correct use (Vieira and Cavalcante 1999). Multiresistant parasite strains have been found on several farms (Molento and Prichard 2001; Taylor et al. 2009). The possibility of anthelminthic residues in the environment and in animals reared for consumption (Hammond et al. 1997), and the spread of multiresistant strains demand research on alternatives for the control of GIN. The utilization of plants containing secondary compounds such as condensed tannins may expand the organic alternatives to controlling GINs (Athanasiadou et al. 2007; Kahn and Diaz-Hernandez 2000). Caryocar brasiliense Camb. is a native tree in the Brazilian Cerrado and widely distributed in tropical areas of Latin America. The fruits, known as “pequi,” are used for both human consumption and therapeutic purposes. Recent studies have demonstrated fungicidal and molluscicidal action (Bezerra et al. 2002; Motter et al. 2004; Passos et al. 2002), as well as antileishmanial, bactericidal, and antioxidant activity (Paula-Junior et al. 2006). The endocarp is a relevant food of the Cerrado, and its commercialization is an important source of family income (Chévez-Pozo 1997). One way of adding value to this plant species would be to use other parts, such as the peel, exocarp, and mesocarp that
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are not used commercially and represent approximately 80% of the total fruit weight (Vera et al. 2005). Pequi peel is a residue widely available in Cerrado, and our recent study assessed its potential as forage for ruminants (Ribeiro et al. 2011). The aim of this study was to evaluate the anthelminthic efficacy in vitro and in vivo of an aqueous extract of the peel of C. brasiliense on gastrointestinal nematodes in sheep.
Material and methods Aqueous extract preparation Pequi peels were obtained at the municipal market of Montes Claros city, North Minas Gerais State of Brazil. The peels, exocarps, and mesocarps were carefully inspected, and those with gross lesions or damage were discarded. The material was sliced and dried to constant weight in a forced air circulating drier at 40±2°C, ground, and refrigerated at 4°C until use. The dry peels were rehydrated in a distilled water bath at 60°C for 60 min, then hot-filtered through a gauze funnel. The aqueous extract was again dried in an oven with forced air at 40°C, and subsamples were transformed to dry matter (dm) at 105°C. The main secondary metabolites present in the extracts were identified using the colorimetric method described by Matos (1997). Tannins was tested using lead acetate, copper acetate, and lead acetate with glacial acetic acid reactions, phenols by ferric chloride test, flavonoids by Shinoda, ferric chloride, and sodium hydroxide tests. Steroids and terpenoids were shown by Lieberman–Burchard reaction; alkaloids using Dragendorff, Mayer, and Bourchard reagents; and saponins using foam test (Nery et al. 2010). In vitro anthelminthic tests Egg-hatching inhibition The aqueous extract was standardized at 15 mg/ml (dm) and diluted in sterile distilled water at concentrations of 7.5, 3.75, and 1.87 mg/ml immediately after dissolution. These extracts were used in egg-hatching inhibition (EHI) tests with four replicates (Coles et al. 1992). Flotation, sedimentation, and filtration techniques in saturated NaCl solution were conducted to obtain nematode eggs from feces of two Santa Inés ewes infected only with Haemonchus contortus and with an average fecal egg count (FEC) of >3,000 g−1, determined using the modified McMaster technique (Gordon and Whitlock 1939). Experimental mixtures contained: 300 μl fecal suspension with an average of 600 fresh eggs, and 300 μl of the extract, or a positive control solution with albendazole (10 mg ml−1); or a negative control with sterile distilled water. The samples were
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homogenized and incubated in a BOD incubator at 28°C for 48 h. Subsequently, 15 μl Lugol’s solution was added to each tube, which was then stored at 4°C for subsequent counting of unembryonated eggs, embryonated eggs, and first-stage larvae (L1, Coles et al. 1992). The number of L1 relative to the initial number of eggs was determined for each sample and subjected to variance analysis. Probit regression was employed to analyze the data using the statistical package Saeg 9.1 (2007) and to determine the extract concentrations sufficient to inhibit 50% (LC50) and 90% (LC90) of egg hatching. The formula given below was used to determine the EHI effectiveness: % effectiveness ¼ 100 ð1 L1=initial egg numberÞ
Larval development inhibition test A 200-mg ml−1 aqueous extract was diluted with sterile distilled water at 160, 100, 60, and 40 mg ml−1. Five naturally infected Santa Inês crossbred lambs, aged 6–8 months and producing >500 FEC, were selected. Eggs were suspended in saturated sodium chloride solution and counted in a McMaster chamber (Ueno and Gonçalves 1998). Quantitative coproculture adapted test was performed (Borges 2003; Nery et al. 2010). Unpreserved feces collected directly from the rectal ampulla of each selected animal were transported to the laboratory, where they were mixed and divided into 2-g samples distributed in clean disposable plastic cups. Two milliliters of each extract or 2 ml of ivermectin solution (Ranger LA, Vallée, Montes Claros, MG, Brazil) at a concentration of 16 μg ml−1 (positive control), or 2 ml sterile distilled water (negative control) was added to the feces. On day 7 of the culture, the nematode larvae were collected in a test tube and held at ~4°C before counting. In order to identify the genus, duplicate slides were prepared with Lugol’s iodine, and the Keith (1953) key was used for characterization at ×40 objective magnification. The number of L3 was divided by two to give the number of L3 per gram of feces (LPGF). The formula below, adapted from Borges (2003), was used to determine the percent reduction in larva numbers per gram of feces: % efficacy ¼ 100 ð1 LPGF of the treated group=LPGF of the untreated groupÞ
The data were log transformed, log (x+1) and submitted to variance analysis. The means were compared through the Scott–Knott test, the 5% probability calculated, and LC90 was determined by probit analysis using the statistical package Saeg 9.1 (2007).
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In vivo test in lambs Anthelminthic efficacy was determined by FEC reduction test according to the methodology described by Coles et al. (1992). The analyses were conducted in 36 four- to six-month-old Santa Inês lambs of 20 kg bw average (range 18–25 kg) of mixed sexes. Prior to the beginning of the trial, all lambs were administered with albendazole (10 mg kg−1 bw) and levamisole (15 mg kg−1 of bw) orally to ensure that they were worm free. The animals were held in an individual sheepfold and fed twice daily on 800 g of sorghum silage and 600 g of concentrate, according to the age category requirements, with water ad libitum. Experimental procedures were carried out in accordance with the Animal Experience Ethical Committee of Minas Gerais Federal University (CETEA—UFMG) and approved by this committee, under protocol number 042/2008. The 36 lambs, having zero FEC, were infected with 1,500 L3 from ewes naturally contaminated in pastures. Twentyone days postinfection, lambs were separated according to FEC, weight, and sex to obtain three homogeneous groups. Experiments were conducted in the morning, after 12 h fasting. A group of untreated animals served as the negative control, and a group orally administered with levamisole (15 mg kg−1 bw) represented the positive control. The extract doses were calculated to not exceed the LD10 observed in acute toxicity tests in mice established in our preliminary study (Almeida et al. 2010). The pequi aqueous extract was orally administered by esophageal probe at 2 gkg−1 bw (dm). The doses were calculated for each lamb and standardized at 300 ml of final volume in sterile distilled water. Animals were monitored for clinical signs. Initial FEC value was recorded, based on the mean values of 3 days prior to initiation of treatment. Subsequently, mean FEC was determined for two periods of efficacy analysis, days 5, 6, and 7 posttreatment and days 12, 13, and 14 posttreatment. A pool of feces from each group was sampled 7 days posttreatment, and eggs cultured for larvae genus identification (Ueno and Gonçalves 1998).
The FEC data were transformed to log10 (x+1), submitted to variance analysis and the means compared by the Scott– Knott test with a 5% significance level. A formula adapted from Coles et al. (1992) was employed to determine the percentage of FEC reduction: % efficacy ¼ 100 ð1 FEC mean of treated group=mean FEC of untreated groupÞ
Results The qualitative phytochemical analysis showed that the pequi extracts contained cachectic and condensed tannins, flavonoids, saponins, xanthones, catechins, steroids, and simple phenols. In the egg-hatching inhibition test, the 15and 7.5-mg/ml concentrations showed anthelminthic efficacy corresponding to 98.7% and 91.8%, respectively. For these concentrations, the L1 means were significantly lower than treatment with distilled water or albendazole (Table 1, P
