Effects of Vernonia amygdalina methanol leaf extract and fractions on Ascaridia galli in experimentally infected birds with regard to its pathological effect

Helminthiasis, usually a chronic problem in poultry, is the major cause of economic losses in the poultry industry. This study was undertaken with the aim of evaluating the in vitro and in vivo anthelmintic effects of Vernonia amygdalina methanol leaf extract and fractions on Ascaridia galli in experimentally infected birds. Standard protocols for extraction, phytochemical screening, isolation and infection of parasite and histological examination were followed. At 50 mg/ml albendazole (ALB) caused the highest inhibition of embryonation (97.3%) followed by crude methanol extract (CME) (94.5%), ethyl acetate fraction (EAF) (81.3%) and butanol fraction (BF) (80.3%). The lowest faecal egg counts (FEC) and highest FEC at 21 days post-treatment were observed in the group treated with albendazole (99.2%) and CME at 800 mg/kg (96.9%). Deparasitization at 200 mg/kg caused by ALB was (93.8%), while at 200, 400 and 800 mg/kg, CME was (77.5%, 87.5% and 91.86%), BF was (75.51%, 79.61% and 85.73%) and EAF was (73.48%, 75.5% and 81.63%), respectively. At post-mortem, broiler chickens infected with A. galli and treated with CME, BF and EAF at 200 mg/kg had desquamation of the villi while at 400 and 800 mg/kg treatments, the intestines were intact. The extract and fractions of V. amygdalina were found to possess anthelmintic activity against A. galli due to the inhibition of embryonation of eggs and the reduction in faecal egg count; further research is required to fully ascertain their mechanism of action of the extract.


Background
Poultry production is an important means of providing high-quality protein for human consumption and contributes about 30% of all animal protein consumed globally (FAO 2010). Poultry reared in rural scavenging system face various hindrances among which helminthiasis plays a vital role (Fakae and Paul-Abiade 2003). Studies conducted in different parts of the world indicated that the proportion of chicken infection with gastrointestinal helminths is high; therefore, helminths are considered to be an important cause of ill health and reduction in poultry productivity (Ajala et al. 2007;Ogbaje et al. 2012;Idika et al. 2016;Ola-Fadunsi et al. 2019). Helminthiasis is a chronic problem in the poultry industry and accounts for economic losses (Newbold et al. 2017). Ascaridia galli is the largest, most prevalent and one of the most pathogenic nematodes of birds. Infection may cause reduction in growth rate, weight loss, sometimes serious illness, economic losses and occasionally, mortality (Yousfi et al. 2013;Naphade 2014;Silva et al. 2015;Sahu and Sinha 2016). There are reports of emerging resistance to some anthelmintics (Coles et al. 1998) due to the use, overuse and misuse of the drugs in the treatment and prevention of parasitic infections (Douglass et al. 2015;Lawal et al. 2015). Ethno-veterinary medicine (EVM) as an alternative for controlling both internal and external parasites in livestock production systems involves the use of indigenous beliefs, knowledge, skills, methods and practices pertaining to the health care of animals (Mathius-Mundy and McCorkle 2004). V. amygdalina commonly called bitter leaf (English) due to its bitter taste was named after an English Botanist William Vernon (Alara et al. 2017). In Nigeria, it is known as 'Ewuro' in Yoruba, 'Etidot' in Ibibio, 'Onuigbo' in Igbo, 'Ityuna' in Tiv, 'Oriwo' in Edo and 'Shuwaka' in Hausa (Farombi and Owoeye 2011). So, the aim of the study was to evaluate the anthelmintic and histopathological efficacy of the methanol leaf extract and fractions of V. amygdalina against experimental A. galli infection in broilers and assess the histopathological lesions in the extracts-treated birds. The traditional use of the plant as an anthelmintic necessitated this study.

Ethical clearance
Ethical approval for the use of animal for this study was approved by the Ahmadu Bello University, Committee on Animal Use and Care (ABUCAUC/2021/042), ABU Zaria.

Experimental location
This study was carried out in the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University (ABU) Zaria.

Collection, identification and preparation of V. amygdalina leaves
Fresh V. amygdalina leaves and flowers were collected from the Botanical Garden of Ahmadu Bello University, Zaria, between the months of October and November 2019. The Sample was taken to the Herbarium of the Department of Botany, ABU Zaria and compared with voucher specimens kept at the Herbarium and a voucher number (ABU07425) corresponding to that specimen was allocated. The leaves were then dried under the shade at room temperature (Abdalla et al. 2012). The dried leaves were made into powder using mortar and pestle. The powdered leaf material was weighed, poured into a polythene bag and kept at room temperature.

Extraction and solvent partitioning
The extraction of the V. amygdalina leaves was carried out at the Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, ABU Zaria according to the method described by (Handa et al. 2008). Briefly, about 3.5 kg of the powdered leaves of V. amygdalina was extracted with 10.5 L of absolute methanol by maceration for 48 h and filtered. The procedure was repeated three times to exhaustively extract the constituents of the plant materials. Thereafter, the extract was filtered. The filtrates were pooled together and solvent was evaporated in vacuo at 40 °C using a rotary evaporator to obtain the crude methanol extract (CME). The CME was divided into two portions: One portion for the partitioning while the other portion was kept for the treatment during in vivo studies. One hundred and fifty grams (150 g) of the solid crude methanol extract (CME) was dissolved in 1.5 L of distilled water to form an aqueous extract with 1.5L of Butanol added and exhaustively mixed together. The suspension was sieved through a filter paper to obtain the Butanol fraction (BF). 1.5 L of ethyl acetate was added to the aqueous extract and exhaustively mixed together, sieved through a filter paper to obtain the Ethyl acetate fraction (EAF). Both fractions were concentrated to a semi gel-like material by allowing the solvent to evaporate at room temperature. The process was done for hexane (HF) fractions also.

Phytochemical screening
The phytochemical screening of the plant extract and fractions were carried out in the Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, ABU Zaria. Phytochemical test was carried out on the crude methanol extract, hexane, butanol, ethyl acetate fractions according to the methods described by Trease and Evans (1996).

Molisch's test
Three drops of Molisch's reagent were added to about 0.1 g of each crude extract and the fractions (hexane, ethyl acetate and butanol), each in a test tube, followed by addition of concentrated sulphuric and proper mixing. The mixtures were allowed to stand for two minutes. The formation of a reddish colour ring at the interface indicated the presence of carbohydrate.

Test for flavonoids Shinoda test (Stephen et al. 2021)
One hundred microgram of the extract and fractions were each dissolved in 2 ml of methanol and pieces of magnesium chips were added, followed by addition of 3 drops of concentrated hydrochloric acid. The formation of a pink, orange or red to purple colouration indicated the presence of flavonoids.

Sodium hydroxide test (Stephen et al. 2021)
Two drops of 10% sodium hydroxide solution were added to 0.1 g of the extracts (CME) and each of the fractions (hexane, ethylacetate, butanol), Appearance of yellow colouration indicated the presence of flavonoids.

Test for saponin
Frothing test (Stephen et al. 2021) Ten ml of distilled water was added to 0.1 g of the extract and each fraction (hexane, ethylacetate, butanol), with continuous shaking for 30 s, following which the solution was allowed to stand for 5 min. The formation of a persistent froth indicated the presence of saponins.

Salkowskis test
One hundred micrograms of the extract and fractions (hexane, ethylacetate, butanol) were each dissolved in 2 ml of chloroform and 3 drops of concentrated sulphuric acid (Salkowskis reagent) added at the side of the test tube. The appearance of a reddish colouration at the interface indicated the presence of terpenoids.

Liebermann-Burchard test
To 0.1 g of the extract (CME) and fractions (hexane, ethylacetate, butanol), in a test tube, was added to equal volume of acetic anhydride, and mixed together gently; 1 ml of concentrated sulphuric acid was then added to the test tube. The appearance of blue-green colour at the upper layer and a reddish, pink or purple colour at the junction of the two layers within one hour indicated the presence of triterpene.

Test for alkaloids (Stephen et al. 2021) Dragendroff's test
One microgram of the extract and fractions (hexane, ethylacetate, butanol) were dissolved in 2 ml of 1% hydrochloric acid with vigorous stirring in a water bath. The mixture was filtered and few drops of Dragendroff 's reagent added. The appearance of rose red precipitate indicated the presence of alkaloids.

Mayer's test
To 0.1 g of the extract (CME) and fractions (hexane, ethylacetate, butanol), each in a test tube, 4 drops of Mayer's reagent was added. A cream precipitate indicated the presence of alkaloids.

Test for free anthraquinones (Stephen et al. 2021)
One hundred micrograms of extract (CME) and fractions (hexane, ethylacetate, butanol), each in a test, 5 ml of chloroform was added, followed by shaking for 5 min, filtration and addition of 5 ml of 10% ammonia solution and agitation. A bright pink colour in the upper aqueous layer indicated the presence of anthraquinones.

Test for tannins Ferric chloride test
Three drops of ferric chloride solution were added to 0.1 g each of the extract (CME) and fractions (hexane, ethylacetate, butanol), each in a test tube. Appearance of blue-black colouration indicated the presence of tannins.

Lead sub-acetate test
Four drops of lead sub-acetate solution were added to 0.1 g each of the extract and fractions in a test tube. The formation of a cream colour precipitate will indicate the presence of tannins.

Isolation, culture and recovery of infective A. galli eggs
Female Ascaridia galli were obtained from the intestine of chicken slaughtered at Sabon-Gari poultry slaughter slab in Zaria. The intestines were collected into a beaker, and transported to the Helminthology Laboratory, Department of Veterinary Parasitology and Entomology, Ahmadu Bello University, Zaria. Adult female Ascaridia galli worms were obtained from the infected birds according to standard parasitological techniques described by Stephen et al. (2021), Yusuf (2010) and Skallerup et al. (2005). The uteri of gravid female worms were dissected and the eggs recovered. The recovered eggs were washed with 0.5 M (KOH) solution and agitated gently for 30 min in order to dissolve the sticky albuminous layer and allow for uniform sampling. Thereafter centrifuged at 1500 × g for 3 min, with supernatant decanted and eggs washed with distilled water thrice by centrifuging. The supernatant decanted and the suspended eggs were transferred into Petri dishes to which embryonating fluid (0.1 M sulphuric acid) was added before incubating for 21 days at 37ºC (Das et al. 2010). The culture in test tubes was centrifuged and the eggs washed twice. The eggs were then pipetted onto the glass slide and examined under the microscope at × 10 for the evidence of embryonation.

In vitro evaluation of V. amygdalina extract and fractions for inhibition of A. galli embryonation
The methods described by Gill et al. (1995) and Coles et al. (2006)  eggs in 200 µl of water were pipetted into each of 96 wells of microtitre plate. The in vitro inhibitory activity of the extract and fractions of V. amygdalina were evaluated at five different concentrations of 3.15, 6.25, 12.5, 25, and 50 mg/ml. Also, Albendazole was used as standard (positive) control at the same concentration of 3.15, 6.25, 12.5, 25, and 50 mg/ml. All tests were performed in triplicates. Similarly, distilled water was employed as negative control. The plate was then incubated at 37ºC for 21 days (Coles et al. 1992). Thereafter, aliquots of 100 µl from each well were pipetted onto a clean glass slide, for examination at × 10 under a light microscope. The numbers of embryonated and unembryonated eggs were counted. The percent inhibition (PI) was estimated using the following formula (Coles et al.,1992): where P unembryonation Mean number of unembryonated eggs in each well. P total = Mean number of eggs in each well.

Determination of median lethal dose (LD 50 ) of V. amygdalina
The median lethal dose (LD 50 ) of CME, EAF and BF was determined, as described by Lorke (1983). The trial was carried out in two phases using a total of twelve birds for each extract and fraction. The LD 50 was calculated by taking the geometric mean of the lowest dose that produced mortality and the highest dose that did not produce death was assumed to be greater than or equal to ( ≥) 5000 mg/kg.

Experimental birds
A total of one hundred and fifty-day-old broiler chickens were purchased for this study, were housed and fed ad libitum (birds were fed without restriction). The birds were divided at random into 6 groups of (A, B, C, D, E and F) with birds in group A, B and C further divided into 3 subgroups of ten birds each and received 200, 400 and 800 mg/kg. Birds in groups D were treated with a single dose of albendazole (200 mg/kg), while group E birds were given distilled water and served as infected non-treated controls. Birds in groups F served as the non-infected non-treated controls, respectively.

Experimental infection of birds and in vivo anthelmintic screening of V. amygdalina leaf extract
The embryonated eggs of A. galli were suspended in water inside a beaker and thoroughly agitated. Two hundred microlitre (0.2 ml) of the suspension was pipetted PI = P unembryo × 100 P total and eggs in it were counted under light microscope. This was done three times and average of the eggs in it was taken. One hundred and twenty broilers were orally infected at three weeks of age with 1000 A. galli eggs contained in 0.5 ml of water per bird using an 18G needle attached to a 2 ml syringe. Patency was monitored by simple flotation method. Faecal drops on the wood shavings were collected from the different groups of birds starting from week three of infection and examined by the flotation method. Collection and examination of samples were done twice in a week until the infection was established using simple flotation protocol (Cringoli et al. 2010;Stephen et al. 2021). Briefly, a suspension of about 2 g of faeces in flotation fluid was filtered through layers of gauge (in a funnel) into a test tube until a convex meniscus was formed. A cover slip which had been allowed to stay on the meniscus for 3-5 min was carefully removed, placed on a clean glass slide for examination at × 10 objective of a light microscope. One hundred and twenty birds that were shedding A. galli in faeces were selected for the in vivo screening. The birds were divided at random into 6 groups (A, B, C, D, E and F). Groups A, B and C contain 30 birds each, while groups D and E have 10 birds each. Birds in groups A, B and C were further allocated randomly into three groups of 10 birds each (Groups A 1 , A 2 and A 3 ; B 1, B 2 and B 3 ; C 1, C 2 and C 3 ). Groups A 1 , A 2 and A 3 were treated with CME at 200,400 and 800 mg/kg, respectively. Similarly, birds in groups B 1, B 2 and B 3 were given EAF at 200, 400 and 800 mg/kg, respectively. Also, BF at 200, 400 and 800 mg/ kg was given to birds in groups C 1, C 2 and C 3 , respectively. Birds in groups D were treated with albendazole (200 mg/kg), while group E birds were given distilled water (5 mg/kg) and served as infected non-treated controls. Birds in groups F served as the non-infected nontreated controls, respectively. All the treatments were given orally for three consecutive days.

Faecal egg count
Fresh droppings were collected from birds at day 0 (for pre-treatment FEC) and on days 7-, 14-and 21-day posttreatment from each group of bird into different sample bottles and labelled accordingly (Das et al. 2010). Faecal samples were collected from each group of birds on Egg per gram of faeces (epg) and were determined using the modified McMaster technique (Cringoli et al. 2010). Briefly, 2 g of faeces from each group was homogenized in a 28 ml flotation solution of zinc sulphate of specific gravity of 1.28. The supernatant was sieved through a tea sieve into a bottle and the sediment discarded. The supernatant from each bottle was then dispensed into both chambers of the MaMaster slide using a transfer pipette, and then allowed to stand for 5 min. The MaMaster slide was then mounted onto a microscope and viewed to count the eggs at × 10 magnification. To obtain the egg per gram (epg), the total number of eggs in the two chambers was multiplied by 50, obtained thus: The anthelmintic effect of leaf extracts (CME) and fractions (BF, EAF) was determined by comparing the egg per gram (epg) of the treated group with that of those treated with distilled (untreated group) and Albendazole treated group. The percentage reduction in A. galli eggs in the treatment groups was calculated using the method by Kamrun et al. (2017).

Post-mortem worm count
Three weeks post-treatment, all the birds were deprived of feed but not water for a day to empty the gastrointestinal tract for easy worm count. Three birds were randomly selected from each group and then sacrificed by euthanization. Thereafter the intestines were removed, opened and checked for the presence of adult A. galli. The ascarids recovered were counted and preserved in 10% buffered formalin. The percentage deparasitization was calculated (Stephen et al. 2021).
where "N" mean number of worms found in untreated control birds. "n" mean number of worms found in treated birds.
Percentage deparasitization of 70 and above was considered significant (Stephen et al. 2021;Suleiman et al. 2005).

Gross pathology
Three birds from each group were euthanized; the gastrointestinal tracts removed and observed for gross pathological lesions.

Histopathological processing and examination
Section of the small intestine was collected from each bird in all the groups for histopathological preparations and examination. The method described by Baker 0.3 ml (volume of the two chambers) = X eggs Therefore, 30 ml (final volume) = X × 30 eggs 0.3 2 g of faeces = 100X eggs Therefore, 1 g of faeces = 50X eggs et al. (2000) was used for processing for histology. The tissues were fixed in 10% buffered neutral formalin and after which they were dehydrated in ascending grades of alcohol, embedded in paraffin wax and then sectioned at 5 microns. The sections were mounted on clean greasefree slides, stained with haematoxylin and eosin (H and E) stain and were examined microscopically at different magnifications of × 40, × 100, × 200 and × 400.

Data analyses
Data obtained were expressed as mean ± standard error of mean (SEM) and subjected to two-way analysis of variance (ANOVA) using GraphPad Prism version 8.0.2 for windows to compare the anthelmintic effect of different extract and fractions of V. amygdalina and presented in tables and charts. Means in different groups were compared using Bonferroni's post hoc test. Values of P ≤ 0.05 were considered significant. Table 1 shows the feed composition that was giving to the chickens throughout the course of the experiments.

The yield of V. amygdalina powdered leaves extract
Three and half kilograms (3.5 kg) of the powdered leaves of V. amygdalina yielded 280 g (8%) of crude methanol extract (CME). One hundred and fifty grams (53.6%) of CME was serially partition with solvents of different polarities to yield 42 g (28%) of BF, 9 g (6%) of EAF (Table 2).

Phytochemical constituents of V. amygdalina
The CME contained alkaloids, flavonoids, saponins, tannins, steroids, anthraquinone, cardiac glycosides and carbohydrates. The aqueous extract contained all but 3 (flavonoids, steroids and cardiac glycosides) of those found in CME, while EAF lacked saponin, cardiac glycoside and carbohydrate. The BF contained all but 1 (flavonoids) of the constituents found in the other extracts (Table 3).

Acute toxicity studies
No signs of toxicity or death were observed following individual oral administration of crude methanol extract, ethylacetate and butanol fractions of V. amygdalina at doses between 10 and 5000 mg/kg. The LD 50 was therefore assumed to be ≥ 5000 mg/kg.

Inhibition of embryonation of A. galli eggs in vitro by extract and fractions of V. amygdalina
The results of inhibition of embryonation of A. galli eggs in vitro by extract and fractions of V. amygdalina are presented (Table 4). The mean inhibition of embryonation was significantly (P < 0.05) lower in the distilled water group (46.5 ± 1.5) compared to the group treated with albendazole (200 mg/ml), CME, EAF and BF fractions of V. amygdalina. There were significant (P < 0.05) differences in the inhibition of embryonation with increasing concentrations of the albendazole, extract and fractions. At 3.25 mg/ml concentration, there was no significant difference in the inhibition caused by EAF (125.5 ± 2.5) and BF (128.5 ± 0.5) and the inhibition caused by ALB (141 ± 2.0) was significantly higher compared to all the fractions and extract. Similar patterns were observed for the inhibition of embryonation at concentrations of 6.5 mg/ml, 12.5 mg/ml and 25 mg/ml. At concentration of 50 mg/ml, there was no statistically significant differences in the inhibition of embryonation by CME (189.0 ± 2.0) and ALB (194.5 ± 2.5), but these were significantly (P < 0.05) higher compared to those caused by EAF (162.5 ± 1.5), BF (160.5 ± 1.5).

Mean faecal egg counts of broiler chickens infected with A. galli and treated with extract and fractions of V. amygdalina
In all the birds infected with A. galli and treated with the different doses of extract and fractions (200,400 and 800 mg/kg) no significant differences (P > 0.05) in epg were observed. In infected chickens administered distilled water (DW), the change in FEC was not statistically significant (P > 0.05) throughout the period. At 7 days post-treatment, the decrease in FEC was significant (P < 0.05) in chickens administered CME (200 mg/kg), BF (200 mg/kg) and EAF (200 mg/kg). The decrease in FEC in chickens administered ALB (217 ± 117) and 800 mg/ kg of CME (400 ± 153) showed no statistically significant (P > 0.05) difference at 7 days post-treatment. At 14 days post-treatment, no significant difference was observed in the decrease in FEC in chickens administered 200 mg/kg of CME, BF (200, 400 and 800 mg/kg) and EAF (400 and 800 mg/kg). At 21 days post-treatment, there was significant decreases in FEC in chickens administered albendazole (17 ± 17) and 800 mg/kg of CME (67 ± 33) compared to chickens administered 200 and 400 mg/kg of CME, all dosages of BF and EAF (Table 6).

Percentage faecal egg count reduction of A. galli in broiler chickens treated with extract and fractions of V. amygdalina
Albendazole, extract and fractions of V. amygdalina of all caused dose and time-dependent decreased in percentage faecal egg count reduction (FECR). Crude methanol extract (CME) caused the highest FECR followed by Butanol fraction (BF  (Table 7).

Mean worm counts and percentage deparasitization of A. galli by extract and fractions of V. amygdalina
The mean count of A. galli was significantly (P < 0.05) higher in chickens administered distilled water  Table 6 Mean faecal egg counts of broiler chickens infected with Ascaridia galli and treated with extract and fractions of Vernonia amygdalina Values with the different superscript alphabets in the same row differ significantly at P < 0.05. Means preceded by different superscript letters differ significantly (P < 0.05) from one another: a and b (between DW and ALB, Extract and fractions), c and d (between ALB and the fractions), e and f (between the extract) DW, distilled water; ALB, albendazole; CME, crude methanol extracts; HF, hexane fraction; BF, butanol fraction; EAF, ethyl acetate fraction (16.0 ± 1.0) compared to chickens in all other treatment groups (Table 8). The lowest mean count of (1.0 ± 0.0) recorded in the ALB-treated was not significantly different (P < 0.05) compared to the count of (1.3 ± 0.9) in chickens administered 800 mg/kg of CME. However, the mean worm count of the ALB-treated chickens was significantly (P < 0.05) different compared to the other treatment groups. No significant differences existed in the mean worm counts of chickens administered CME (200 and 400 mg/ml), BF (200, 400 and 800 mg/ml) and EAF (200, 400 and 800 mg/ml). There was significant percentage deparasitization (> 70%) of A. galli by extract and fractions of V. amygdalina, with the highest recorded in chickens administered 800 mg/ml (91.86%) and 400 mg/ml (87.75%) of CME and least in chickens administered 200 mg/ml (73.5%) of EAF. Figure 1A-L shows the histopathological changes in the various groups of broiler chickens used in this study.

Post-mortem examination of broiler chickens infected with A. galli and treated with extract and fractions of V. amygdalina
The histopathological findings revealed that in broiler chickens that were not infected and not treated, villi and mucosa glands were intact; while in broiler chickens that were infected and not treated, desquamation, necrosis of villi and mucosal gland occurred; in broiler chickens infected and treated with albendazole the villi and mucosa gland were intact. In those treated with the extract and the fractions, with increase in concentration, the necrosis of villi and mucosa gland was greatly reduced.

Discussion
The anthelmintic activities of crude methanol leaf extract and fractions of V. amygdalina against A. galli were evaluated in this study. The yield of 8% obtained from the leaves of V. amygdalina was lower than the 14.35% reported by Okoduwa et al. (2017) following cold maceration of V. amygdalina leaves collected from a local farm at Samaru, Zaria. Danquah et al. (2012) also reported a higher yield of 16.0% after extraction of V. amygdalina leaves using water. Similarly, yields of 21%, 19% and 16% were obtained after extraction of the leaves of V. amygdalina using microwave-assisted, soxhlet and cold extraction methods, respectively (Okoduwa et al. 2018).
In similar studies, Sirama et al. (2015) reported yield of about 9% each when the root of V. amygdalina was extracted with acetone, methanol and water. Ekpo et al. (2007) obtained a yield of only 2% after extraction with ethanol. In this study, yield obtained after partitioning 150g of the CME was 28% (BF) which was the highest followed by EAF (6.00%). However, Okoduwa et al. (2017) reported lower yields by BF (2.39%) and EAF (1.73%) in a similar study. The phytochemical constituents present in V. amygdalina leaves in this study included alkaloids, flavonoids, saponins, tannins, steroids, cardiac glycosides, anthraquinones and carbohydrates. Report from other studies demonstrated the presence of these constituents in the extract and fractions of V. amygdalina leaves (Ekam et al. 2013;Zakaria et al. 2016;Ejiofor et al. 2017;Alara et al. 2019). In a similar study by Okoduwa et al. (2017), anthraquinones were not detected, while polyphenols   and triterpenes were present in the methanol leaf extract and fractions of V. amygdalina. Udochukwu et al. (2015) in addition, the presence of oxalate, phytate, phenols and cyanogenic glycosides in the aqueous and ethanolic leaf extracts of V. amygdalina. Also, the presence of phenolics and phlobatannins has been reported in the root and stem bark extracts of V. amygdalina collected from Lapai, Niger State (Audu et al. 2018). The differences in the yield of extracts and fractions phytochemical constituents could have resulted from differences in extraction method used, drying method, type and volume of solvent, season in which plant was obtained, part of plant used, soil characteristics, environmental factors and geographical locations (Ncube et al. 2012). The LD 50 of the extract and fractions V. amygdalina tested individually was ≥ 5000 mg/kg. Similarly, there were no signs of toxicity or death in following oral administration of the crude methanol extract, ethylacetate and butanol fractions. This was higher than the LD 50 of 1950 mg/kg reported by Sha'a et al. (2011) in rats and 2500.62 ± 5.24 mg/kg by Mebratu et al. (2013) in mice. Also, in a study by Ekpo et al. (2007), neither signs of toxicity nor mortality were observed in rats administered ethanol leaf extract of V. amygdalina at 100-1000 mg/kg. Hence, the increased LD 50 could probably be due to increased tolerance to the extract resulting from the larger size and weight of the chickens. The methanol leaf extract and fractions of V. amygdalina demonstrated anthelmintic activities in vitro and in vivo against A. galli in this study as evidenced by inhibition of embryonation, faecal egg count reduction, decreased mean worm counts and increased percentage deparasitization. The patterns of these anthelmintic activities were also observed to be concentration-dependent as evidenced by increased activities with increase in concentration of the extract and fractions in our study. At highest concentration (50 mg/ml) and dosage of (800 mg/ kg), CME produced the highest activities In vitro next to albendazole with no significant difference when compared to albendazole. Islam et al. (2008) reported that the inhibition of embryonation of A. galli eggs was increased with increase in concentrations of ethanol leaf extract of some indigenous plants in Bangladesh. Also, the increase in concentrations of aqueous and ethanol leaf extracts of V. amygdalina resulted in increased mortality of Heligmosomoides bakeri (Nweze et al. 2013) and increased inhibition of motility of Ascaris suum (Nalule et al. 2013) in vitro. Safitri et al. (2019) also reported that increasing concentration of V. amygdalina caused increased anthelmintic activity against A. galli worms in vitro. The enhanced activity with increasing concentrations could be due to saturation of target receptors by increased active ingredients resulting in hyperpolarization of parasite membranes (Lullman et al. 1993;Nalule et al. 2013). This hyperpolarization will in turn limit excitation and impulse transmission leading to flaccid paralysis of worm muscles.
The anthelmintic activity of V. amygdalina against A. galli in vitro and in vivo in this study could be due to the presence of secondary metabolites such as alkaloids, glycosides, saponin and tannin (Nalule et al. 2013). Though these metabolites are unstable, their biological activity is dependent on their structure, physical and chemical properties, and method of extraction (Waterman 1992;Alawa et al. 2003). The increased anthelmintic activity by CME could possibly be due to the presence of large amount of these metabolites compared to the fractions. Also, the findings of previous studies suggest that alkaloids, glycosides, saponins and tannins possess concentration-dependent anthelmintic activity either singly or in synergy (Kaufman et al. 1999;Githiori et al. 2006;Wynn and Fougere 2007;Islam et al. 2008). The nematicidal