Evaluation of anthelmintic activity of Nigerian ethnoveterinary plants; Cassia occidentalis Guiera senegalensis

Gastrointestinal nematode infections pose a major threat to small ruminant production. is a helminth parasite that feeds on blood of small ruminant animals and causes anaemia, anorexia, loss of condition, and eventual death of the host animal. The parasite is particularly found in tropical and subtropical regions worldwide [1]. In Nigeria and most developing countries, parasitic helminths cause large scale loss in animal productivity and pose a great threat to livestock development. The climate in Nigeria is humid and favours the development of helminth parasites [2]. Control of gastrointestinal nematodes, particularly in ruminants over the past 3 decades has been carried out almost entirely using conventional anthelmintic drugs. Most of these anthelmintics are derived from synthetic or semi-synthetic sources. These compounds are mainly considered to be the only effective way of controlling helminthosis in livestock. [3]. Some of the important drawbacks associated with the use of modern anthelmintics include resistance developed by the parasites to the drugs, which is a serious problem for livestock and a potentially growing problem for human helminth infections [4]. A good number of helminth parasites of domestic animals have the capacity to develop resistance to anthelmintics. Resistance to anthelmintics in sheep and goats is rapidly increasing, especially in warm and humid climatic regions, this could be as a result of increased rate of dosing with anthelmintics and also the adoption of common management, nutritional, and therapeutic strategies [5]. In addition, anthelmintic substances have been reported to cause considerable toxicity to humans and livestock, posing serious threat to human health [6]. In Africa, the rising cost of veterinary drugs and services has made it difficult for resource-poor farmers to access modern veterinary care including treatment of gastrointestinal helminth parasites in livestock [7]. Other issues of concern, such as drug residues in animal products and environment have limited the use of modern anthelmintic in veterinary practice [8, 9]. ( ) is a flowering plant belonging to the family Caesalpinaceae [10]. The entire parts of Haemonchus contortus


Introduction
Gastrointestinal nematode infections pose a major threat to small ruminant production.
is a helminth parasite that feeds on blood of small ruminant animals and causes anaemia, anorexia, loss of condition, and eventual death of the host animal. The parasite is particularly found in tropical and subtropical regions worldwide [1]. In Nigeria and most developing countries, parasitic helminths cause large scale loss in animal productivity and pose a great threat to livestock development. The climate in Nigeria is humid and favours the development of helminth parasites [2].
Control of gastrointestinal nematodes, particularly in ruminants over the past 3 decades has been carried out almost entirely using conventional anthelmintic drugs. Most of these anthelmintics are derived from synthetic or semi-synthetic sources. These compounds are mainly considered to be the only effective way of controlling helminthosis in livestock. [3]. Some of the important drawbacks associated with the use of modern anthelmintics include resistance developed by the parasites to the drugs, which is a serious problem for livestock and a potentially growing problem for human helminth infections [4]. A good number of helminth parasites of domestic animals have the capacity to develop resistance to anthelmintics. Resistance to anthelmintics in sheep and goats is rapidly increasing, especially in warm and humid climatic regions, this could be as a result of increased rate of dosing with anthelmintics and also the adoption of common management, nutritional, and therapeutic strategies [5]. In addition, anthelmintic substances have been reported to cause considerable toxicity to humans and livestock, posing serious threat to human health [6]. In Africa, the rising cost of veterinary drugs and services has made it difficult for resource-poor farmers to access modern veterinary care including treatment of gastrointestinal helminth parasites in livestock [7]. Other issues of concern, such as drug residues in animal products and environment have limited the use of modern anthelmintic in veterinary practice [8,9].
( ) is a flowering plant belonging to the family Caesalpinaceae [10]. The entire parts of : This study was designed to evaluate the anthelmintic activity of the stem-barks of and which are used traditionally to treat gastrointestinal helminth infections in goat.
: Phytochemical analysis using standard techniques was used to detect secondary metabolites contained in the plants.
anthelmintic activity of the crude methanol extracts of the plants was determined using egg hatch inhibition test (EHIT) and larval development inhibition assay (LDIA).
: The extracts of and inhibited hatching of eggs and larval development of in a concentration-dependent manner. At concentrations of 0.1, 1, 10 and 100 mg/ml, the extract of produced significant (p < 0.05) inhibition of egg hatching of eggs when compared with the untreated (distilled water) control group.
inhibited, by up to 86%, the larval development of . Similarly, the extract of at concentrations of 10 and 100 mg/ml produced significant (p < 0.05) inhibition of egg hatching of eggs and inhibited larval development by up to 85% of the parasite.
(EC = 4.23 mg/ml) was found to be more efficacious than (EC = 88.24 mg/ml) against eggs. However, (EC = 0.0012 mg/ml) was more effective than (EC = 0.11 mg/ml) against the larvae of .
: The crude methanol extracts of and possess anthelmintic activity against that requires detailed pharmacological and toxicological trials to justify their use in clinical veterinary practice. : , , medicinal plants. ', respectively. All the plant parts are used for medicinal purposes. The leaf has a bitter taste and is widely acknowledged as a 'cure-all' medicine. The leaf part has been shown to have gastroprotective effect when tested experimetally against ethanol, water immersion stress and aspirin-induced gastric ulcer in rats [15].

Cassia occidentalis
Due to the problems arising from the use of conventional anthelmintics highlighted above, there is the need to find alternative therapeutic agents for the treatment and control of helminth infections. One practical way of developing cheaper and effective anthelmintics is to study indigenous herbal remedies that are used as anthelmintics [16]. There have been many reports indicating the effectiveness of plant products against helminth infections in animals mainly from Africa and Asia [17,18,19]. This will suggest a high chance of discovering compounds with anthelmintic effect in some of such traditional medicinal plants.
The aim of this study was to evaluate the effect of the stem-barks of and against gastrointestinal nematode under condition.
Fresh stem-barks of and were collected around Zaria, Nigeria in the month of July, 2012. The plants were identified and authenticated in the Herbarium, Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria and voucher specimen numbers of 4751 and 1823 for and , respectively, were deposited. The stem-barks of the plants were individually chopped into smaller pieces, air-dried and pulverized into powder by pounding in a mortar using a pestle. Similarly, the powder of (230 g) and (150 g) were individually cold-extracted exhaustively in a separating funnel by using methanol and , subsequently concentrated at 55°C to solid darkbrown and brown extracts for and , respectively [20]. The extracts were kept in a refrigerator at 4°C until required.
Two grams of each extract were dissolved in 20 ml of distilled water to produce a stock solution of 100 mg/ml. Thereafter, 10-fold serial dilutions were made. The solutions were used for the anthelmintic tests. The abomasums of 5 goats naturally infected with were obtained from Zaria abattoir in Nigeria. Both ends of the collected abomasums were ligated and immediately taken to the Veterinary Helminthology laboratory of the Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria for the recovery, quantification and identification of using the procedure described by Hansen and Perry [21]. The abomasums were opened along their greater curvature and their contents washed into a bucket, a total volume of 2 litres was obtained. Mature female adult were picked, crushed and sieved to obtain the eggs. About 40 ml of the filtrate was collected and the concentration of eggs was estimated in 200 µl of the solution. By dilution and concentration, about 100 eggs in 200 µl of the filtrate were obtained. The eggs were viewed under a light microscope at × 40 magnification.
The egg hatch assay was done as described by Coles [22]. Approximately, 100 eggs in 200 µl of distilled water were pipetted into each well of a 48-well microtitre plate. To each of the test wells, 200 µl of each plant extract was added to a final volume of 400 µl per well. The plant extracts were tested at concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 mg/ml. Similarly, albendazole (200µl) (standard anthelmintic drug) at concentration of 0.25 mg/ml was used as a positive control, while distilled water (200µl) was used as a non-treated control. Each test was done in three replicates. The plate was incubated in a humidified incubator at 37 ºC for 48 h. Thereafter, a drop of Lugol's iodine solution was added to each well to stop further hatching. All unhatched eggs and L larvae in each well were counted. The percentage inhibition of eggs hatching was calculated.

Materials and Methods
Plant collection, identification and processing: Phytochemical screening: Recovery and preparation of eggs: Egg hatch inhibition assay (EHIA):

Larval development inhibition assay:
Haemonchus contortus 1 done in three replicates. At the end of the sixth day after incubation, the different larval stages of the parasites (L , L and L ) from each well were recovered and counted under a light microscope at × 40 magnifi-cation.
The percent (%) inhibition of egg hatching and larval development was calculated by using the formula described by Coles [16]. Percent inhibition = 100 (1-P /P ) where P = number of eggs hatched (or larval forms) in the case of egg hatch test (EHT) assay, or the number of hatched larvae that developed into infective (L ) in the larval development inhibition assay (LDIA). P = number of hatched eggs or developed larvae in the distilled water treated control wells.
All values were transformed into means ± S.E.M. Comparisons between groups of data were conducted using Student's -test.
values of < 0.05 were considered significant. The concentrations of the plant extract and the controls were transformed into natural logarithm and plotted against the percentage inhibition using Microsoft Excel, 2003 software. The straight line graph obtained from the best line of fit was used to calculate the ED as a measure of the effectiveness of the plant extracts.
The percentage yields of the extracts after concentration were 18.5% and 23.3% for and , respectively.
The methanol extracts of . and .
The extracts of and inhibited hatching of eggs of in a concentration-dependent manner ( Figure-1 and 2). At concentrations of 0.1, 1, 10 and 100 mg/ml, the extract of produced significant (p<0.05) inhibition of egg hatching of eggs when compared with the group treated with distilled water. However, at the concentrations of 0.01 and 0.001 mg/ml the extract showed no significant anthelmintic effect when compared with distilled water (untreated control). Similarly, the extract of at concentrations of 10 and 100 mg/ml produced significant (p < 0.05) inhibition of egg hatching of eggs when compared with the group treated with distilled water. However, at the concentrations of 0.001 and 0.01 mg/ml the extract showed no significant difference when compared with the untreated control group. Equation of the dose-response curves of the extracts of and were y = 4.94x + 42.88 and y = 4.11x + 31.58, respectively. The correlations of the dose-response curves were 98% and 78 % for and , respectively. The concentration that is effective in producing 50% anthelmintic effect also know as effective concentration 50 (ED ) of the extracts of and against hatching of eggs deduced from the dose-response curves were, therefore, 4.23 mg/kg and 88.24 mg/kg, respectively.   The problem of anthelmintic resistance, toxicity and the increasing concern over the presence of drug residues in animal products has led to a renewal of interest in the use of plant-developed drugs, in the form of extracts containing mixture of different plant secondary compounds [8]. The utilization of plants for the treatment of diseases of human and animal origin continues to rise although with few studies demonstrating proof of these effects [24]. Some plant extracts that were found to exhibit anthelmintic activity are [25], [2], [18], [26], [27] and and [28] The extracts of and were evaluated for anthelmintic activity in this study because reports had shown their traditional use as anthelmintic agents [10,29].
The safety and efficacy of and as traditional medicinal herbs have been demonstrated [30]. Scientific evaluation is, however, required for the demonstration of the safety and efficacy potentials of medicinal plants [31]. The present study evaluated the possible inhibition of eggs and larvae by the methanol extracts of and . good test screen for studies because of its longer survival rate in distilled water. Different stages of the parasite and that of other similar had been used for anthelmintic screening by other workers [32][33][34][35].
Both the extracts of and inhibited hatching and development of eggs and larvae in concen-tration-dependent fashions. The main advantages of using assays to test for the anti-parasitic properties of plant and plant extracts are the low costs and rapid turnover which allow large scale screening of plants [36].Also due to high cost of tests, tests have been used for initial screening of plant extracts for their anthelmintic activity [37]. Albendazole showed higher anthelmintic action than any of the extracts. The superior anthelmintic action of albendazole could perhaps be attributed to its purity when compared with the extracts that have many compounds contained in it. Perhaps if the active compound(s) is isolated from the extract it may have comparable anthelmintic action when compared with albendazole.
The extracts appeared to be having more effect against the larvae of than the eggs. This could be attributed to egg shell resistance. The egg shell comprises 3 layers; an external lipoprotein layer, a middle chitinous protein layer and an inner lipid layer. The middle and inner layers are resistant to salts and chemicals and also protect the eggs from desiccation, strong acids and bases, oxidants, reductive agents, detergents and proteolytic compounds [38].
Secondary chemical metabolites like saponins, tannins, phenolic glycones, flavonoids, triterpenes and steroids were demonstrated in the extracts of and . In addition, alkaloids were also present in the extract of . The observed anthelmintic effect of the extracts could be attributed to one or more of these secondary metabolites contained in the extract. Monodesmoside saponins destabilize membranes and increase cell permeability in helminth parasites by combining with membraneassociated sterols [39,40]. Monomers of condensed tannins are reported to have the capacity to bind to protein and prevent the exsheathment of nematode third stage larvae [41]. Another possible anthelmintic effect of tannins is binding to free proteins in the gastrointestinal tract of host animals [8] or glycoproteins on the cuticle of the parasite [42]   Guiera senegalensis in vitro H. contortus of condensed tannins from an extract of has been reported [8]. Substituted phenols such as disophenol, niclofolan and nitroxynil are established anthelmintics which act by uncoupling the mitochondrial reaction involved in electron transportassociated event of ATP generation by helminths. This causes exhaustion and death of helminths [3]. These compounds may act singly or in synergy to produce the observed anthelmintic effect. The extract of was shown to have strong antioxidant effect attributed to the tannins and alkaloids [43]. This could be responsible for enhancing the anthelmintic action of the extract. The antioxidant effect of against DPPH radical has been demonstrated [44]. The various proposed mechanisms of action of antioxidants are: direct radical scavenging, inhibition of enzymes (such as NO-synthase, xanthine oxidase, cyclooxygenase and lipoxygenase), iron chelating, and direct inhibition of lipid peroxidation [45].
The difference of and studies with regards to drug action is greatly due to differences in the physiology of the host animals and of the mechanisms that inactivate exogenous substances and the toxic effects of plant preparations within the animal host [7]. Hence an appropriate study that would involve toxicological and residual effect is required before a conclusion is made on the usefulness of as an effective anthelmintic.
The findings from this study suggest that the crude methanol extracts of the stem-bark of and contain constituents with anthelmintic properties. The results obtained, therefore, suggest that the extracts of and are good candidates for developing potentially useful and effective drugs for the control of in livestock. However, further studies on the active principles and the development of quality assurance protocol involving the use of reference substance of plant origin are required.
MMS, AS and EJI designed and performed the experiment. MMS and AMT analysed the data. MM, MMS and MT had given technical guidance during the experiment, drafted and revised the manuscript. All authors read and approved the final manuscript.
The authors are thankful to all technical staff in the Departments of Veterinary Pharmacology and Toxicology, Physiology and Parasitology and Entomology for their assistance. The authors are thankful to the authorities of Ahmadu Bello University, Nigeria for providing funds and facilities for this research.
The authors declare that they have no competing interests.