Anthelmintic potential of three plants used in Nigerian ethnoveterinary medicine

Abstract

Context: The leaves of Irvingia gabonensis Baill. Ex Lanen (Irvingiaceae), Ficus exasperata Vahl (Moraceae), and Vernonia amygdalina Delile (Asteraceae) are folklorically used in treating worm infestation in Eastern Nigeria. The anthelmintic potential of the ethanol extracts of the leaves of I. gabonensis, F. exasperata, and V. amygdalina was investigated.

Materials: Acute toxicity tests were done in mice using 10, 100, and 1000 mg/kg/bw of extracts. In vitro larval assays of Heligmosomoides bakeri larvae at various extract concentrations (125, 250, and 500 mg/kg) were done. Mice experimentally infected with H. bakeri were treated with F. exasperata extract (200, 400, 800 mg/kg).

Results: At concentrations of 500, 250, and 125 mg/ml F. exasperata caused 100% larval mortality. V. amygdalina extract caused 71.43, 57.14, and 57.14% larval deaths while I. gabonensis extract caused 71.43, 57.14, and 42.9% larval deaths at the same concentrations. There was no significant difference in the fecal egg output, packed cell volumes and body weights of the F. exasperata treated mice when compared with the infected untreated group.

Discussion and conclusion: Leaf extracts of F. exasperata, V. amygdalina, and I. gabonensis exhibited varying degrees of larvicidal activities on the infective stage larvae of H. bakeri in vitro whereas F. exasperata showed no activity on the parasites in vivo.

Keywords::

• Irvingia gabonensis
• Ficus exasperata
• Vernonia amygdalina
• Heligmosomoides bakeri
• ethnomedicine

Introduction

During informal oral interviews of folks about plants used in treating worm infestation in man in rural areas in Eastern Nigeria mention was made of the use of the leaves of Irvingia gabonensis Baill. ex Lanen (Irvingiaceae), Ficus exasperata Vahl (Moraceae), and Vernonia amygdalina Delile (Asteraceae). I. gabonensis is locally called “ogbono” (Igbo). F. exasperata is “asisa” (Igbo) while V. amygdalina is known as “onugbu” (Igbo). The young tender leaves are macerated in water and drunk. These infusions are claimed to be potent in treating cases of helminthosis.

V. amygdalina has been reported as being used by wild chimpanzees for self-deparasitization (Jisaka et al., 1992). According to Iwu (1993) it is used to treat a variety of illnesses. The leaves are reputed to be an effective remedy for gastrointestinal disorders, fevers and abortion (Lawal et al., 2010). I. gabonensis seeds have been found to reduce blood lipids and body weight in treated obese subjects (Ngondi et al., 2005). In a report by Krief et al. (2005) the leaves of F. exasperata are used for the treatment of edema, healing abscess, and ulcer. The leaves of F. exasperata are also traditionally employed in the control of lice by placing them in the bird shed (Fajimi & Taiwo, 2004).

Other plants with anthelmintic properties include Annona senegalensis Pers. (Alawa et al., 2003) Ocimum gratissimum Linn. (Njoku & Asuzu, 1998) Nauclea latifolia Sm. (Fakae et al., 2000), and Piliostigma thonningii Schum. (Asuzu & Onu, 1994). Plants have been sources of medicines over the years. In fact, ~80% of all medicines are related to natural products (Harvey, 2008). Traditional veterinary practices are used in most developing countries and being an integral part of the peoples’ culture, their use is not likely to change to a significant degree in years to come.

Anthelmintics are drugs that are effective against any one or all the three groups of gastrointestinal parasites called cestodes (tapeworms), trematodes (flukes), and nematodes (roundworms). They are also defined as agents that kill, destroy and expel worms from the gastrointestinal tract. An anthelmintic can either be a vermicide or a vermifuge. The former destroys worms without necessarily expelling them whereas the latter is any substance that expels worms from the bowel.

The major impediment to the use of synthetic anthelmintics in livestock production is the growing rate of parasite resistance. The earliest reports of anthelmintic resistance involved parasites of sheep such as Haemonchus contortus and the benzimidazole group of anthelmintics (Borgsteede et al., 1996). Overdependence on drugs, frequent treatments and misuse of chemical-based anthelmintics has led to the development of anthelmintic resistance, particularly in sheep and goats worldwide.

An alternative control strategy that is being considered is the use of forages or plants that are rich in condensed tannins (proanthocyanidins) since they were found to improve performance of parasitized sheep (Niezen et al., 1998). In the developed world, this is in response to the demand for production of animals free from industrial chemical inputs (Gasbarre et al., 2001) and the need to discover new therapeutic substances of natural origin with low toxicity for man and animals (Guarrera, 1999). The use of plants for deworming livestock in Africa is chiefly borne out of the economics of affordability (Schillhorn van Veen, 1997).

This study was designed to investigate the anthelmintic potentials of these three plants—I. gabonensis, F. exasperata, and V. amygdalina which are used in Nigerian ethnomedicine.

Materials and methods

Plant collection and extraction

I. gabonensis (with voucher number Intercedd/967) was collected from Enugu-Ezike in Igbo-Eze South local Government area of Enugu State in January, 2008. F. exasperata leaves (with voucher number Intercedd/705) were collected from Kogi State while V. amygdalina leaves (with voucher number Intercedd/968) were obtained from the Nsukka campus of the University of Nigeria. The plant materials are very common in this locality and were identified by Mr. A. O. Ozioko of International Centre for Ethnomedicine and Drug Development (Intercedd) Nsukka where voucher specimens were deposited. The leaves were dried in a shade and ground into fine powder using a laboratory mill. Each plant was extracted by maceration in 80% ethanol for 96 h. The extracts were filtered, concentrated and stored in a refrigerator until use.

Parasites

Heligmosomoides bakeri infective larvae (L₃) were obtained from the Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka. They were freshly harvested and preserved in a refrigerator at 4°C until use.

Acute toxicity test

Thirty-six-week old albino mice weighing an average of 20 g were used for the experiment. Mice were randomly divided into three groups of three animals each (Lorke, 1983) for each extract. Three mice served as a common untreated control group for all three extracts. They were dosed intraperitoneally with graded doses (10, 100, and 1000 mg/kg) of each extract. The animals were monitored for 24 h for signs of toxicity. This experimental design was approved by the Faculty board of Veterinary Medicine, University of Nigeria, Nsukka. Laboratory animals were maintained in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (DHHS, NIH Publication No. 85-23, 1985).

In vitro larval assay

The L₃ of H. bakeri were used for the in vitro anthelmintic assay according to the method adapted from Chiejina and Fakae (1984). Various concentrations (125, 250, and 500 mg/ml) of each extract were prepared. Larval suspensions containing an average of seven larvae were delivered into each well of a microtitre plate. Then extracts were added according to their concentrations. Each concentration was replicated three times. Pyrantel embonate was the standard drug given to the positive control group. The negative control wells contained neither extract nor drugs. The content of each well was examined after 12 and 24 h, respectively, using both light and stereo microscopes. Larvae were classified as “alive” if moving or “dead” if no observable motion took place after 5 min. The percentage of dead larvae was calculated.

In vivo anthelmintic assay

In vivo anthelmintic assay was done according to the method of Fakae et al. (1994). Thirty-six male albino mice aged between 8–10 weeks and of an average weight of 30 g were used for the study. They were randomly divided into six groups of six animals each. Groups 1–5 were orally infected with about 200 L₃ of H. bakeri. Group 6 was the uninfected control group. All the mice were fed ad libitum They were fed with pelleted grower’s mash containing 14.5% crude protein (Vital Feeds, Grand Cereals, & Oil Mills Ltd. Jos, Plateau State, Nigeria) and had free access to drinking water. On establishment of infection, the mice in groups 1, 2, and 3 were treated with graded doses (800, 400, and 200 mg/kg) of F. exasperata extract per os for five consecutive days. Group 4 was treated with pyrantel embonate (19 mg/kg/day) for three consecutive days while group 5 remained as the untreated control.

Fecal egg counts

Fecal egg counts (FEC) were monitored using a modified McMaster technique (Fakae et al., 1994). Briefly, 1 g of feces was crushed and put into a coffee strainer for sieving. The filtrate was collected in a 50 ml beaker. The suspension was then adequately stirred and a Pasteur pipette was used to rapidly fill the two chambers of the Mc Master slide. This was then mounted under the light microscope and allowed for 5 min to settle. All eggs within the square on each of the chambers were counted at ×10 objective. The number of eggs per gram (EPG) was expressed as follows

where N = Total number of eggs counted in the two chambers.

Packed cell volume and body weight

Packed cell volume (PCV) was assessed by the method of Coles (1968). Briefly, blood was collected from the tail veins of the mice into heparinized capillary tubes. One end of the tube was sealed using plasticin. The tubes were stood on a capillary tube rack and centrifuged with a microhematocrit centrifuge at 5000 revolutions/min for 5 min. The PCV was then read using a hematocrit reader. The mice were weighed weekly using a weighing balance.

Statistical analysis

Data are presented as mean ± SEM. All data were subjected to statistical analysis using one-way analysis of variance to determine significant difference between the means. Differences were considered significant at p < 0.05.

Results

All the plant extracts were chocolate brown and pasty in consistency. The I. gabonensis extract was darkest in color and the oiliest. During the acute toxicity test of I. gabonensis, deaths were recorded at doses above 500 mg/kg. In the V. amygdalina dosed mice, deaths occurred at doses above 100 mg/kg but in the F. exasperata dosed animals, deaths occurred at doses above 1000 mg/kg.

The result of the in vitro anthelmintic assay is shown in Table 1. F. exasperata extract and pyrantel embonate had 100% larvicidal activity against H. bakeri L₃ at all the tested concentrations. V. amygdalina extract caused 71.43, 57.14, and 57.14% larval deaths at concentrations of 500, 250, and 125 mg/ml, respectively. I. gabonensis extract caused 71.43, 57.14, and 42.9% larval deaths at concentrations of 500, 250, and 125 mg/ml, respectively.

Table 1.  The percentage mortalities of H. bakeri L3 larvae at different concentrations of plant extracts in vitro.

Plants/drug	Percentage of mortality
	Concentrations (mg/ml)
	500	250	125
Ficus exasperata	100	100	100
Irvingia gabonensis	71.43	57.14	42.9
Vernonia amygdalina	71.43	57.14	57.14
Pyrantel embonate	100	100	100

The effect of graded doses of F. exasperata extract on the mean fecal egg count (MFEC) of H. bakeri infected mice is shown in Table 2. The MFEC rose steadily among the infected groups from day 12 to 18 postinfection (PI). Following treatment on day 19, the MFEC of the pyrantel embonate treated group was from day 20 to 28 PI significantly (p < 0.05) lower than those of all the infected groups. By day 28 PI, MFEC of the pyrantel embonate treated group became zero and remained so until the end of the experiment. There was no significant difference between the MFEC of the F. exasperata extract-treated groups and the infected untreated group.

Table 2.  The effect of graded doses of F. exasperata extract on the mean fecal egg count (MFEC) of H. bakeri infected mice ± SD.

Days	Group 1	Group 2	Group 3	Group 4	Group 5
	800 mg/kg	400 mg/kg	200 mg/kg	Pyrantel embonate 19 mg/kg	Infected untreated
D12	2450.00 ± 970.05	2600.00 ± 784.22	2920.00 ± 586.00	2233.33 ± 409.61	1788.75 ± 744.65
D14	6680.00 ± 1307.54^ab	8320.00 ± 348.07^b	8830.00 ± 2068.43^b	3283.33 ± 341.97^a	3900.00 ± 398.43^a
D16	12410.00 ± 3746.34^a	10240.00 ± 1116.62^ab	10030.00 ± 1122.90^ab	6466.67 ± 1201.85^ab	3925.00 ± 867.35^b
D18	14000.00 ± 3053.52	10560.00 ± 840.60	13260.00 ± 1677.38	8833.33 ± 1695.42	7875.00 ± 2984.23
D20	15560.00 ± 2905.44^a	13000.00 ± 2437.42^ab	18180.00 ± 2472.35^a	5946.67 ± 3020.85^b	10225.00 ± 2587.59^ab
D22	22080.00 ± 3781.32^a	21930.00 ± 4918.98^a	41280.00 ± 6065.01^b	27.00 ± 12.10 ^c	17225 ± 2709.67^a
D24	19560.00 ± 1320.83^a	20960.00 ± 1518.75^a	30600.00 ± 1351.67^b	17.33 ± 13.38^c	16675.00 ± 2560.40^a
D26	20300.00 ± 5844.66^a	19820.00 ± 2820.89^a	32380.00 ± 5472.60^a	7.33 ± 5.46^b	20700.00 ± 8086.82
D28	12000.00 ± 2213.59^a	16200.00 ± 4083.00^a	22200.00 ± 5938.00^a	0.00 ± 0.00^b	12975.00 ± 2521.11^a
D30	28720.00 ± 4292.48^a	17280.00 ± 4083.80^ab	29880.00 ± 8525.22^a	0.00 ± 0.00^b	16350.00 ± 7077.00^ab
D32	19380.00 ± 4096.75^a	23680.00 ± 5423.23^a	27580.00 ± 8262.35^a	0.00 ± 0.00^b	15725.00 ± 4663.94^ab

Different superscripts (^ab) in a row indicates significant difference between the means at the level of probability: p ≤ 0.05.

The effect of F. exasperata extract on mean packed cell volume (MPCV) is shown in Table 3. The MPCV of the uninfected mice was significantly (p < 0.05) higher than those of the extract-treated mice from day 14 till the end of the study. From day 21 PI until the end of the experiment, there was no significant difference between the MPCV of the extract-treated groups and that of the infected untreated group. There was also no significant difference in the MPCV of the pyrantel embonate treated and uninfected controls from day 21 to the end of the experiment.

Table 3.  The effect of graded doses of F. exasperata extract on the mean packed cell volume (MPCV) of H. bakeri infected mice ± SD.

Days	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
	800 mg/kg	400 mg/kg	200 mg/kg	Pyrantel embonate 19 mg/kg	Infected untreated	Uninfected group
D0	54.80 ± 1.36	58.80 ± 2.50	55.60 ± 1.54	54.67 ± 2.58	59.50 ± 1.94	57.40 ± 1.33
D7	52.00 ± 0.84^a	56.20 ± 2.15^ab	53.4 ± 1.54^ab	53.00 ± 1.53^ab	57.75 ± 1.75^b	57.60 ± 0.98^b
D14	50.8 ± 1.66^a	52.40 ± 1.21^ab	50.20 ± 1.59^a	52.33 ± 1.67^ab	55.50 ± 0.96^bc	57.40 ± 1.33^c
D21	49.80 ± 1.80^a	50.80 ± 1.02^a	50.60 ± 1.33^a	52.33 ± 1.20^ab	54.00 ± 0.91^ab	56.40 ± 1.57^b
D28	49.80 ± 1.43^a	50.40 ± 0.93^a	51.60 ± 1.72^a	56.00 ± 2.00^b	54.00 ± 0.91^ab	57.60 ± 1.03^b
D35	48.8 ± 1.32^a	51.20 ± 1.20^a	50.40 ± 1.63^a	56.67 ± 1.33^b	53.00 ± 1.08^ab	57.60 ± 1.86^b

Different superscripts (^a–c) in a row indicates significant difference between the means at the level of probability: p ≤ 0.05.

The effect of F. exasperata extract on mean body weight (MBW) of the mice is shown in Table 4. By day 21 PI, the MBW of the uninfected mice was significantly (p < 0.05) higher than those of the 800 mg/kg treated, the pyrantel embonate treated and the infected untreated mice groups. From day 28 to 35 PI until the end of the experiment, there was no significant difference in MBW of all the experimental groups.

Table 4.  The effect of graded doses of F. exasperata extract on the mean body weight (MBW) of H. bakeri infected mice ± SD.

Days	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
	800 mg/kg	400 mg/kg	200 mg/kg	Pyrantel embonate 19 mg/kg	Infected untreated	Uninfected
D0	30.60 ± 1.21	31.60 ± 1.29	32.00 ± 1.14	31.00 ± 1.00	30.25 ± 0.63	31.20 ± 1.36
D7	28.80 ± 1.16	29.40 ± 1.40	29.40 ± 0.68	29.33 ± 0.88	27.75 ± 1.03	30.00 ± 0.95
D14	27.60 ± 0.81^ab	27.80 ± 1.02^ab	28.20 ± 0.66^ab	28.67 ± 0.67^b	25.50 ± 1.32^a	29.20 ± 0.73^b
D21	28.80 ± 0.37^a	29.60 ± 0.81^ab	29.60 ± 0.24^ab	28.67 ± 1.33^a	28.00 ± 0.82^a	31.40 ± 0.60^b
D28	27.80 ± 0.66	27.40 ± 1.08	28.40 ± 0.51	28.00 ± 1.15	29.00 ± 1.08	28.80 ± 1.03
D35	27.40 ± 0.87	28.00 ± 0.63	28.40 ± 0.68	27.67 ± 0.33	27.75 ± 1.03	29.00 ± 0.63

Different superscripts (^a,b) in a row indicates significant difference between the means at the level of probability: p ≤ 0.05.

Discussion and conclusion

From the results of the acute toxicity tests, F. exasperata extract had the highest safety margin followed by I. gabonensis and V. amygdalina extracts. F. exasperata extract also exhibited the highest larvicidal activity by killing all the H. bakeri larvae at the tested concentrations. V. amygdalina and I. gabonensis extracts showed some larvicidal activities too. A different result was obtained by Alawa et al. (2003) in an in vitro hatchability study where the effect of whole plant extracts of V. amygdalina was tested on H. contortus eggs. No significant reduction in hatchability was observed at the extract concentration of 11.2 mg/ml.

F. exasperata was unable to significantly reduce fecal egg counts of treated mice in the in vivo study. This result is similar to that obtained using Buchholzia coriacea seed extract by Nweze and Asuzu (2006). In that report, B. coriacea seed extract had in vitro larvicidal activities against Heligmosomoides polygyrus larvae but showed no effect on the adult worms and their eggs. These results show that an extract may exhibit an activity in vitro but not necessarily in vivo. The foregoing highlights one disadvantage of in vitro assays which is that conditions and concentrations are not always comparable to those in vivo and thus results can differ in the two assays (Athanasiadou et al., 2001). Also, considerable physiological differences are expected between conditions in vitro and in the predilection site of the parasite within their animal hosts that affect bioavailability of the active compounds (Githiori et al., 2006). In vitro tests are useful for initial screening of extracts and to establish biologically realistic drug concentrations for further animal testing.

Infection with H. bakeri was associated with reduction in PCV. From day 14 until the end of the experiment, the MPCV of the uninfected mice was significantly (p < 0.05) higher than those of the extract-treated groups. This finding could be attributed to the fact that H. bakeri is a heamatophagous nematode (Fakae et al., 1994). Treatment with the extract did not significantly increase the MPCV of the treated animals. This result could be due to the fact that the extract had no significant activity against the nematode parasites whose feeding was responsible for the drop in PCV.

By day 21, MBW gain of the uninfected mice was significantly (p < 0.05) higher than those of group 1 (800 mg/kg treated) pyrantel embonate treated and the infected untreated groups. By days 28 and 35, there was no significant difference in MBW of all the experimental groups. From the above result, parasitism caused loss of body weight in infected animals at the onset of infection but when the condition became chronic there was no significant difference observed between the infected and uninfected mice.

Preliminary phytochemistry of the leaf showed that it contains tannins, flavonoids, and saponins with no traces of alkaloids or anthraquinones (Ayinde et al., 2007). The presence of phenols was also detected in the ethanol extract of F. exasperata and is thought to be responsible for its observed antioxidant activity (Abotsi et al., 2010). F. exasperata has been shown to significantly increase motility and gastric emptying (Awe et al., 1999) owing to its content of a proteolytic enzyme, ficin (Evans, 2005). Increased gastric emptying and intestinal motility are possible causes of purgation which could lead to the expulsion of adult worms (Alawa et al., 2003). In this study, however, the ethanol extract of F. exasperata had no effect on the adult worms. This was evident in the inability of the extract to expel worms. F. exasperata extract exhibited a narrow spectrum of anthelmintic activity. The leaf extract of F. exasperata showed larvicidal activity on the infective stage larvae of H. bakeri in vitro.

Declaration of interest

The authors report no conflict of interest.

Notice of correction:

The version of this article published online on 16 November 2012 contained an error in the author list. Author name Lukas Ngongeh should have read Lucas A. Ngongeh. The error has been corrected for this version.