Assessment of the Anthelmintic Efficacy of an Aqueous Crude Extract of Vernonia amygdalina.

Abstract

The anthelmintic efficacy of an aqueous crude extract of the leaves of Vernonia amygdalina. Del. (Compositae) was studied in 16 Nigerian puppies. They were divided into four groups of four puppies per group. The group A animals were uninfected and hence were untreated, whereas those in groups B, C, and D were naturally infected with helminths. Group B animals were untreated whereas group C animals were treated with a proprietary anthelmintic (Mebendazole). Group D animals were treated with aqueous crude extracts of V. amygdalina. using the oral route. Two weeks after treatment, blood and fecal samples were collected to evaluate the hematologic values and fecal egg counts, respectively. Before the onset of this study, fecal egg counts showed that ascarids (Toxocara canis.) and hookworm (Ancylostoma caninum.) were present in the experimental animals. The results of this study showed that the aqueous crude extracts of V. amygdalina. after its administration into local dogs produced significant changes (p < 0.05) in PCV, RBC, Hb concentration, and TWBC. The fecal egg counts also showed remarkable and significant reduction in the levels of the identified helminths. The effects of the plant extracts were broad spectrum in action. Because the aqueous crude extract of V. amygdalina. significantly reduced the fecal egg count of the helminths, it could be a potential source of a new lead anthelmintic agent.

Keywords:

• Anthelmintic
• helminths
• hematology
• Mebendazole
• V. amygdalina

Introduction

The use of herbal products for medicinal benefit has played an important role in nearly every culture on earth, and knowledge about plants, herbs, and spices, and their respective and collective roles in promoting health is modest (Ballatine et al., 1999; Izevbigie, 2003). Herbs have been used as food and medicinal purposes for centuries by ancient peoples in Africa, Asia, Europe, and the Americas (Wargovich et al., 2001). The use of medicinal herbs has, however, increased over the past few years, and research interest has focused on various herbs that possess hypolipidemic, antiplatelet, antitumor, or immune-stimulating properties that may be useful adjuncts in helping reduce the risk of cardiovascular disease and cancer (Craig, 1999).

Parasitic diseases remain a major constraint to livestock productivity across all ecological zones and production systems in Africa, and gastrointestinal nematodes remain of major economic consequence in domesticated livestock throughout the world (Prichard 1994; Alawa et al., 2003). In recent times, there has been an increasing interest in ethnomedical and ethnoveterinary practices across the world, especially as they relate to the use of medicinal plants in treating various ailments. In the developed world, this move is in response to the production of animals free from industrial chemical inputs (Gasbarre et al., 2001) and the need to discover new therapeutic substances of natural origin with possibly low toxicity to man and animals (Guarrera, 1999). In African livestock, this is borne out of the economics of affordability (Schillhorn van Veen, 1997; Ketzis, 1999; Alawa et al., 2003).

The importance of helminthosis can be underscored by the fact of the clinical signs associated with it. These include lethargy, dullness, inappentence, loss of general body condition, rough or starry hair coat, loss of weight, pallor of visible mucous membrane, depression, anemia, protein losing enteropathy leading to hypoproteinemia, gastroenteritis, and so forth (Thompson, 1988; Okewole & Oduye, 2001).

In this study, close attention is paid to Vernonia amygdalina. Del. (Compositae) because it has been listed by several authors as a plant being used as an anthelmintic by local livestock farmers in Nigeria (Ibrahim et al., 1984; Kudi & Myint, 1999; Abdu & Faya, 2000; Abdu et al., 2000; Alawa et al., 2003). Vernonia amygdalina., also known as bitter leaf, is a plant that is widely used in Africa to treat a multiplicity of diseases including schistosomiasis, amebic dysentery, and gastrointestinal problems (Huffman et al., 1996; Masaba, 2000). Although any part of the plant can be used, there seems to be a preference for the leaves, which are quite bitter in taste, the bitterness being taken as a sign of potency (Masaba, 2000).

Vernonia amygdalina. has been suggested as a plant used by wild chimpanzees and gorillas for self-deparasitization, and study has found two classes of bitter compounds in this plant: a novel sigmastane-type steroid glucoside (Ohigashi et al., 1991; Jisaka et al., 1992a; Jisaka et al., 1992b; Jisaka et al., 1993) and four known sesquiterpene lactones, vernodalin, vernolide, hydroxyvernolide, and vernodalol (Jisaka et al., 1993). Almost all the pharmacological activity observed for this plant has been attributed to these bioactive compounds (Alawa et al., 2003).

This study is aimed at exploring the anthelmintic activity of the plant extract in puppies and comparing this with mebendazole (Janssen-Cilag, USA), a proprietary anthelmintic, which belongs to the benzimidazole group of anthelmintics.

Materials and Methods

Preparation of the plant extract

Vernonia amygdalina. leaves were collected freshly from the backyard and washed with clean water to remove dirt. The plant was identified and authenticated at the herbarium of the Department of Botany and Microbiology, University of Ibadan, Nigeria, and the voucher specimen was deposited there.

The leaves were weighed (75 g) and cut into small bits and then dissolved in hot water and allowed to stand overnight. The supernatant was filtered through sterile filter papers into a conical flask as the study extract. One milliliter of the filtrate is expected to contain 0.5 g (i.e., 500 mg/mL).

Proprietary anthelmintic (mebendazole)

The daily dosage of mebendazole for therapeutic use in Nigerian dogs is 50 mg/kg body weight in divided doses for 3 consecutive days (Liu & Weller, 1993; Gann et al., 1994). For an animal weighing 8 kg, such will receive 400 mg of the active ingredient per day. This amounted to 4 tablets per day as each tablet of mebendazole contains 100 mg of active ingredients.

Animal grouping and treatment

Twelve of the 16 dogs used in this study were procured from the local market in Ibadan, Nigeria. They consisted of 8 males and 4 females that were kept in concrete-floored, clean, separate cages in the kennels of the Faculty of Veterinary Medicine, University of Ibadan, where adequate water and feed were supplied. The kennel was cleaned and fumigated before the animals were put there. These animals were naturally infected with helminths. The remaining 4 animals (2 males and 2 females) were uninfected and untreated. The average weight was 7 kg and the average age was 7 months.

They were divided into four groups of four puppies per group. The group A animals were uninfected and hence were untreated, whereas those in groups B, C, and D were naturally infected with helminths. Group B animals were untreated whereas group C animals were treated with proprietary anthelmintic (mebendazole). The group D animals were treated with aqueous crude extracts of V. amygdalina. using the oral route. Both the drug and the extracts were administered for 3 consecutive days.

Hematologic and parasitologic analysis

The fecal sample of each dog was collected in labeled, sterile universal bottles for identification of the type of helminths present using floatation techniques. The blood samples were also collected from each animal into labeled EDTA bottles for hematologic analysis. After 2 weeks, the animals were weighed and the average weight was 7 kg. The average age was 7 months. After the initial stabilization, the baseline hematologic and coprologic evaluations were made.

The floatation method, which involves the use of salted (NaCl) water, was used to determine the helminths present in the fecal samples, and the modified McMaster egg-counting technique was used for nematode counts. In determining fluke count, the modified McMaster egg-counting technique as used for nematode counts was employed except that saturated zinc sulpfhate solution was used for estimation of fluke egg counts.

Blood samples were collected from the cephalic vein of each animal using 5-ml syringes and 25-gauge needles and were put in appropriately labeled EDTA bottles. Estimation of hemoglobin (Hb) concentration was by Sahli's method. The erythrocytes and leukocytes were counted manually using Neubau's hemaocytometer. Packed cell volume (PCV) was determined by conventional method (i.e., microhematocrit method). Total white blood cell (TWBC) counts were also determined.

Statistical analysis

Where necessary, results were subjected to the Student's t.-test. ANOVA test was also employed to compare group results. Results were considered significant at p < 0.05 (Prasad, 2003).

Results

Fecal egg count

The study showed that the animals in groups B, C, and D were heavily infested with hookworms (Ancylostoma caninum.) and ascarids (Toxocara canis.). Administration of drug and extracts produced significant reduction in the worm burden in group C and D animals. ANOVA tests showed that when all the identified helminths were compared between groups and within groups, the differences were significant at p < 0.05 (Table 1).

Table 1.. Fecal egg counts of dogs in the drug trial.

Parameters	Baseline	After 2 weeks^a.
Ancylostoma caninum. (eggs/gram feces)	0 (Group A)	0
	4000 ± 10.8 (Group B)	4200 ± 21.2
	2225 ± 21.3 (Group C)	450 ± 12.3
	6200 ± 10.2 (Group D)	3250 ± 8.5
Toxocara canis. (eggs/gram feces)	0 (Group A)	37.5 ± 2.1
	3850 ± 10.7 (Group B)	4500 ± 12.5
	2300 ± 14.2 (Group C)	150 ± 2.2
	3300 ± 12.5 (Group D)	275 ± 7.6

Results expressed as means ± SEM. Note that animals in group A were not infected hence did not any receive any treatment.

^a.Two weeks after administration of drug/plant extracts.

Hematologic reports

The baseline hematologic results showed that the PCV mean values for groups A, B, C, and D were 40.2 ± 4.0%, 20.8 ± 1.7%, 20.4 ± 2.3%, and 25.0 ± 1.9%, respectively. After treatment, this parameter experienced significant increase. For hemoglobin, baseline values for groups A, B, C, and D were 12.0 ± 2.0 g/L, 6.5 ± 1.5 g/L, 6.9 ± 1.1 g/L, and 8.2 ± 1.5 g/L, respectively. After treatment, this parameter also experienced significant increase. In the case of the red blood cell (RBC), the mean values for groups A, B, C, and D before treatment were 5.5 ± 1.2 × 10^12/L, 3.6 ± 0.4 × 10^12/L, 3.7 ± 0.9 × 10^12/L, and 4.6 ± 0.5 × 10^12/L, respectively. Like the other two parameters, RBC also experienced significant increase after treatment. In the case of TWBC, this parameter experienced decreasing level with drug and extracts administration. ANOVA tests showed that when all the hematologic parameters were compared between groups and within groups, the differences were significant at p < 0.05 (Table 2).

Table 2.. Hemogram of the experimental dogs in the drug trial.

Parameters	Baseline	After 2 weeks^a.
PCV (%)	40.2 ± 4.0 (Group A)	39.8 ± 2.6
	20.8 ± 1.7 (Group B)	17.9 ± 1.5
	20.4 ± 2.3 (Group C)	29.7 ± 2.6
	25 ± 1.9 (Group D)	27.5 ± 2.6
Hemoglobin concentration (mg/dL)	12.0 ± 2 (Group A)	12.4 ± 2.1
	6.5 ± 1.5 (Group B)	6.0 ± 1.2
	6.9 ± 1.1 (Group C)	11.2 ± 1.2
	8.2 ± 1.5 (Group D)	9.1 ± 1.3
RBC (× 10^6/µL)	5.5 ± 1.2 (Group A)	5.6 ± 1.0
	3.6 ± 0.4 (Group B)	3.0 ± 0.9
	3.7 ± 0.9 (Group C)	5.5 ± 0.6
	4.6 ± 0.5 (Group D)	5.3 ± 0.3
TWBC (× 10^3/µL)	13.2 ± 4.6 (Group A)	13.5 ± 3.2
	13.1 ± 4.6 (Group B)	13.5 ± 1.9
	18.6 ± 2.1 (Group C)	12.9 ± 1.4
	16.5 ± 1.8 (Group D)	10.2 ± 0.6

Results expressed as means ± SEM. Note that animals in group A were not infected and hence did not receive any treatment.

^a.Two weeks after administration of drug/plant extracts.

Discussion

This study has clearly shown that local puppies are heavily infested with worms except when treated with appropriate anthelmintics. The helminths infection manifested general clinical signs such as lethargy, dullness, inappentence, loss of general body condition, rough or starry hair coat, pallor of visible mucous membrane, loss of weight, and so fourth, necessitating the urgent need for treatment (Caroll & Grove, 1986; Thompson, 1988; Okewole & Oduye, 2001).

The V. amygdalina. extract administered in this study caused significant reduction in the worm burden of the puppies. It thus showed that the oral route of administration should be preferred with respect to this extract. This assertion has to do with the fact that earlier study on another plant extract using oral and parenteral routes showed that oral route is superior to the latter (Adedapo et al., 2005). It must be stressed however, that the effect of the proprietary anthelmintic is more pronounced on the worm than the effects of the extract of V. amygdalina..

The reduction of worm load observed with the extract of V. amygdalina. on group D animals may be attributed to the presence of some compounds that caused the contraction of the intestine leading to the expulsion of the worms from the system. Worms were actually seen on the floor of the kennel in the course of this study. This view was arrived at because a study showed that oral pretreatment with V. amygdalina. leaf methanol extract produced significant (p < 0.05) promotion of the motility of charcoal meal in mice. The study further showed that gastric emptying of stomach contents was also promoted. The study concluded, however, that the effects of the extract were not as pronounced as that of carbachol (Awe et al., 1999).

Alawa et al. (2003) in their in vitro. study of the extract of V. amygdalina. concluded that this plant is ineffective in inhibiting egg hatching at all concentrations used. The study postulated that as V. amygdalina. has been shown to significantly increase intestinal motility and gastric emptying, it may be that the ingestion of the plant material causes a purgative-like activity leading to the increased gastric emptying and intestinal motility. Purgation potentially causes the expulsion of adult worms that would be visible to the naked eye and which prompt local farmers to conclude that the plant has some efficacy as an anthelmintic. It may also need to be stated here that in another in vitro. study, it was stated that all of the sesquiterpene lactones present in this plant inhibited both the movement and egg-laying of schistosomes at a concentration of 200 µg/mL. At 20 µg/mL, vernodalin and vernolide inhibited both functions (Ohigashi et al., 1994).

Substituted phenols such as disophenol, niclofolan, and nitroxynil are established anthelmintics that act by uncoupling the mitochondrial reaction involved in electron transport–associated event from ATP generation. This is lethal to blood-sucking helminths (Behinke et al., 1991). Because it has been shown that lactones, vernodalin, and vernolide, inhibited both the movement and egg-laying capacity of schistosomes, it may well be that these compounds have uncoupling effects on the mitochondria reaction involved in electron transport–associated event to form ATP generation. These compounds present in the plant extract could have also caused reduction in worm load through this same mechanism that culminates in exhaustion and expulsion of worms. It thus showed that the efficacy of this plant as an anthelmintic against adult worms is established but may be combined with other agents that will act on other stages of the helminth growth and development.

The administration of the drug and extract brought about a remarkable improvement in the hematology of puppies in groups C and D because the worms that were responsible for reduction in the levels of these hematologic parameters have been removed to some extent. It is expected that through hemopoeisis, the parameters will begin to appreciate with time (Halton, 1974; Oyerinde, 1980; Omamegbe & Uche, 1985; Barriga & Omar, 1992). However, there will still be the need for further administration of hematinics and multivitamins in the course of deworming puppies.

The reduction of worm load by the extracts of V. amygdalina. in this study is a positive and welcomed development in our local helminths struggle because the plant V. amygdalina. is available year-round in Nigeria. The easy access to this plant and its availability might mean that the cost of medication would be drastically reduced. The plant extracts exhibited some high degree of broad-spectrum activity; this implies that total reliance on proprietary drugs, which in most cases are expensive, will be reduced. It also means that the risk of drug resistance could to some extent be avoided.