In vivo anthelmintic potentials of Gongronema latifolium and Picralima nitida against gastrointestinal parasite (Heligmosomoides bakeri)

The fresh roots of G. latifolium and seeds of P. nitida were collected from Ogunmakin, Ogun State, Nigeria. The plant materials were identified and authenticated by plant taxonomists Mr. Nodza, G.I and Adeyemo, A. at the herbarium unit of University of Lagos, Nigeria and Forest Research Institute of Nigeria (FRIN) Ibadan, Nigeria respectively where the plant specimens were deposited with voucher numbers.

The plants materials were rapidly washed under running tap water and air-dried in the laboratory at room temperature. The dried plant materials were then crushed into coarse powder with plant grinder. Two hundred and fifty grams (200g) of each the resulting powder was soaked in 2000 ml 95 % ethanol stirred intermittently for 48 hours at room temperature and later filtered. Clean muslin cloth and Whatman filter paper 1 were used to filter to obtain a clear solution. The filtrate was evaporated to dryness over water bath at 45°C and the residue was stored for further use. The choice of the solvent was based on the mode of preparation mentioned by the herb sellers during questionnaire administration.

The quantitative phytochemical tests for plant secondary metabolites such as flavonoids, tannins, alkaloids, saponin and total polyphenol were evaluated. Flavonoid was determined by repeated extraction with aqueous methanol at room temperature, as described previously Boham and Kocipai (1974). Tannin determination was carried out by using Folin – Dennis titration method as described by Pearson and Cox (1974). Alkaloids and saponin were estimated according to the methods of Harborne (1973) and Bruner (1984) respectively. Total polyphenol content was determined according to Attard (2013).

The crude protein, ash, crude fat, carbohydrate and moisture content of the G. latifolium roots and P. nitida seeds were determined according to AOAC (2005).

A total of 74 Mus musculus BALB/c strain each weighing between 26 and 28g of similar age commercially obtained from Central Animal house, University of Ibadan, Ibadan, Nigeria was used for this study. The mice were free of pathogens and have not been used for any initial experiment.

The mice were kept in plastic cages which contained dried wood shavings as beddings at room temperature (28 ± 2°C), fed with growers’ mash (Top Feeds Limited, Ibadan) and clean water in the animal house in the Department of Zoology, Federal University of Agriculture, Abeokuta, Nigeria. The mice were acclimatized for two weeks before the commencement of the experiment and kept at 12 h light and dark cycle.

Thirty-nine BALB/c male mice weighing 26 – 28g were used to determine lethal dose (LD₅₀) of the extracts. The toxicity study was conducted in two phases. In phase one, eighteen mice divided into six groups of three mice were used for the two plant extracts. The mice received orally 10, 100 and 1000mg/kg body weight for each of the extracts (Lorke, 1983). Another three mice received distilled water. The animals were observed for signs of toxicity and mortality. Based on the results of phase one, another set of eighteen mice were randomized into six groups of three mice for the two extracts. The mice were given 1600, 2900 and 5000mg/kg body weight for each of the extracts. Immediately after dosing, the animals were observed for any behavioural changes, any sign of toxicity and mortality for critical 4hrs and thereafter, daily for 7 days.

The gastrointestinal nematode parasite, Heligmosomoides bakeri was obtained from the Department of Veterinary Parasitology and Entomology, Micheal Okpara University, Abia State where it was maintained in albino mice.

The culture was set according to the method of Fakae et al., (1994). Fresh faecal pellets were collected from the infected mice and broken up in distilled water in a coffee strainer. The supernatant was decanted while the pasty sediment was smeared on moistened filter paper placed in plain petri dishes. The cultures were left for 7 days in a refrigerator (4°C) to ensure optimal hatching of the H. bakeri eggs.

At the end of the culture period, a few drops of distilled water were sprinkled on and underneath the filter paper and left for 1 hour. The water obtained from the recovered infective larvae was pipetted into clean test tube, then allowed to settle under laboratory conditions and excess water was decanted using Pasteur pipette. The suspension was mixed thoroughly by gently shaking of the tube to ensure an even distribution of larvae. Thereafter, an aliquot of 0.2ml was drawn into a clean microscope slide for five times and larvae were counted under 4x objective lens.

Each worm free mouse was infected orally with 150 L₃ H. bakeri suspended in 0.2ml of distilled water (Ngogeh & Fakae, 2011). The larvae suspension was inoculated by properly restraining the mice and using blunted tip slightly curved 18gauge needle mounted on tuberculin (1 ml) syringe.

Thirty-five male BALB/c mice each weighing between 26g and 28g were randomly divided into seven groups consisting five mice each. Five animals were used per group in order to prevent loss of animals or data due to the infection. Groups 1 – 4 were infected but treated with graded doses (300 and 500mg/kg) of G. latifolium and P. nitida extracts orally for five consecutive days, group 5 was infected but treated with 10mg/kg albendazole once (positive control), group 6 was infected untreated (negative control) while group 7 was uninfected control group. At day 12 post treatment, mice in each group were euthanized by cervical dislocation. The in vivo anthelmintic studies were conducted three times for quality control.

Faecal egg counts (FEC) were monitored on day 16 post infection using a modified McMaster technique (Fakae et al., 1994). Briefly, 0.1g of fresh faecal pellet from each mouse was homogenised in 1.4ml of saturated sodium chloride solution and faecal suspension was filtered through a tea strainer. The filtrate was collected into a test tube and then adequately stirred. Pasteur pipette was used to withdraw the filtrate and rapidly used to fill the two chambers of the McMaster 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 calculated using formula described by Zajac and Conboy (2012) in equation (1). Eggs per gram=Number of eggs counted×TV/F {{Eggs}}\;{{per}}\;{{gram}} = {{\left\{ {{{Number}}\;{{of}}\;{{eggs}}\;{{counted}} \times \left( {\frac{T}{V}} \right)} \right\}} \mathord{\left/ {\vphantom {{\left\{ {{{Number}}\;{{of}}\;{{eggs}}\;{{counted}} \times \left( {\frac{T}{V}} \right)} \right\}} F}} \right.} F} Where T = total volume of faeces/flotation solution mixture, V = volume of aliquot examined in slide, and F = grams of faeces used. The anthelmintic effects of each of the plant extracts were determined by comparing the EPG of the treated groups with that of those treated with distilled water (untreated control) using the formula described by Suleman et al. (2005) in equation 2 % ChemosuppressionDeparasitisation=N−nN×100 {\raise0.7ex\hbox{${\% \;{{Chemosuppression}}}$} \!\mathord{\left/ {\vphantom {{\% \;{{Chemosuppression}}} {{{Deparasitisation}}}}}\right.}\!\lower0.7ex\hbox{${{{Deparasitisation}}}$}} = {{N - n} \over N} \times 100

On day 27 post infection, all the mice were deprived of food but not water for 24 hours so as to empty the gastrointestinal tract to make worm counting easier. Mice were euthanized by cervical dislocation. Modified method of Ngongeh (2013) was used for worm count. Summarily, the entire length of the small intestine of each mouse was removed and opened by cutting it longitudinally with a pair of surgical scissors. The adult worms were recovered individually by suspending each intestine with fine threads, then dipping into normal saline and incubating at room temperature (28 ± 2°C) for 20 hours to enable the migration of the worms into the solution. At the end of incubation, the suspensions were centrifuged at 250 rpm (11 g) for 2 minutes. The supernatant was discarded and the sediment containing the worms was viewed using the magnifying lens where the reddish worms were identified, counted and recorded.

The blood samples obtained from retro-orbital sinus of the mice were collected without anticoagulant to obtain non-haemolysed serum used for the biochemical analysis. Cholesterol was determined by enzymatic method (Allain et al., 1974), glucose was estimated by glucose oxidase method (Trinder, 1969), total protein and albumin were estimated by Biuret and bromocresol green method (Doumas et al., 1971), triglyceride concentration was determined enzymatically by glycerol-3-phosphate oxidase-phenol+aminophenazone method (Jacobs & VanDemark, 1960; Schettler & Nussel, 1975). High density lipoprotein cholesterol (HDL-C) was determined by precipitation methods of Friedewald, Levy and Frieddrickson 1972; Gordon et al., 1977 using test kits by Agappe Diagnostics Limited. LDL-C was calculated using Friedewald formula: LDLc=TC−HDLc+TG5 {LDL_c} = TC - \left( {{HDL_c} + {{TG} \over 5}} \right)

Mice were euthanized by cervical dislocation, intestines of the mice that received highest dose (500mg/kg) of G. latifolium and P. nitida, albendazole and untreated were removed, washed with saline and fixed in 10 % buffered neutral formalin for 24 hours. Subsequently, the tissue was subjected to histological techniques, including dehydration in ascending series of alcohols, clearing in Xylol and embedding in paraffin. Then, 5μm-thick sections were obtained in a manual rotation microtome and stained with Haematoxylin and Eosin (HE).

Data obtained were subjected to Shapiro-Wilk test for normality and was analysed using one-way analysis of variance (ANOVA) to compare the means across the groups using Statistical Package for Social Science (SPSS) version 20. The results were expressed as Mean ± SE and values of p<0.05 were considered significant. Independent sample t-test was used to analyse proximate composition and phytochemical constituents by comparing the means of G. latifolium and P. nitida.

The ethical conditions governing the use laboratory animals were maintained in accordance with the recommendations in the Guide for Animal Research: Reporting of In Vivo Experiments (ARRIVE) and the experimental protocol was approved by College of Veterinary Medicine Research Ethics Committee (CREC), Federal University of Agriculture, Abeokuta, Nigeria with approval reference number FUNAAB/COLVET/CREC/005/19.

In acute toxicity study, the roots extract of G. latifolium and seeds extract of P. nitida up to 5000mg/kg neither produced mortality nor any sign of toxicity. Hence, the oral LD₅₀ of G. latifolium and P. nitida was estimated to be greater than 5000mg/kg body weight (Table 1).

Total polyphenol, flavonoid and tannin content were significantly higher (p<0.05) in P. nitida than G. latifolium. However, saponin was most abundant followed by alkaloid in G. latifolium root while alkaloid was most abundant in P. nitida seeds (Table 2). Proximate analysis revealed that the root of G. latifolium contained significantly higher (p<0.05) carbohydrate (41.90 ± 0.02 %) and ash content (11.70±0.35 %) when compared with seeds of P. nitida. Higher crude protein and moisture were recorded for P. nitida seeds (Table 2). Calcium, Magnesium, Zinc, Copper, Iron and Manganese were significantly (p< 0.05) higher in G. latifolium than P. nitida. However, Potassium and Phosphorus were significantly (p< 0.05) higher in the seeds of P. nitida. Calcium is the dominant mineral in the roots of G. latifolium while Potassium is the dominant in P. nitida seeds (Table 2).

The faecal egg count of H. bakeri was significantly higher (p<0.05) in untreated mice throughout the experiment than mice treated with G. latifolium (Fig. 1a). Meanwhile, the faecal egg count of H. bakeri reduced in mice treated with albendazole when compared with the extract of G. latifolium. Although a decline was recorded in the faecal egg output of H. bakeri in mice that received varying doses of G. latifolium, however the decline was not as steep as that of mice that were treated with albendazole. Furthermore, the extract of G. latifolium at 300mg/kg reduced the faecal egg count of H. bakeri till day 8. However, on day 9 the faecal egg count increased till day 11 (Fig. 1a). There was reduction in the faecal egg count of H. bakeri in mice treated with extract and albendazole throughout the experiment (Fig. 1b). The faecal egg output of H. bakeri was significantly (p<0.05) lower from day 1 till day 7 in mice treated with extract of P. nitida at 500mg/kg when compared with other treatment groups. However, from day 8, there was no significant (p>0.05) difference in the faecal egg count of mice treated with extract of P. nitida at 500mg/kg and albendazole (Fig. 1b).

Fig. 1.

There was significant (p<0.05) difference in the percentage chemo suppression among all the treatment groups. Mice treated with P. nitida extract recorded higher level of chemo suppression (p<0.05) when compared with G. latifolium at the same doses (Table 3). Extract of P. nitida at 500mg/kg produced higher chemo suppression from day 1 till day 7 post treatment when compared to albendazole. On the other hand, albendazole gave higher chemo suppression (p>0.05) from day 8 till day 11 post treatment compared to P. nitida (Table 3). There was dose dependency in chemo suppression of mice treated with P. nitida throughout the experiment. At 300 and 500mg/kg body weight, extracts of P. nitida produced highest chemo suppression of 84.91 % and 92.45 % respectively while G. latifolium gave 43.54 % and 65.82 % respectively (p<0.05). The extract of G. latifolium at 300 and 500mg/kg recorded highest chemo suppression at day 8 and 10 post treatment respectively. However, extract of P. nitida recorded highest chemo suppression at day 11 at 300 and 500mg/kg (Table 3).

A significant reduction (p<0.05) was recorded in the worm burden of mice treated with different doses of the plant extracts when compared with the untreated mice. The reduction in worm burden was dose dependent (Table 4). However, mice treated with P. nitida at 500 mg/kg body weight had the lowest worm burden followed by mice treated with albendazole. The deparasitization rate produced by different doses of G. latifolium was significantly lower than that of P. nitida and albendazole. There was no significant (p>0.05) difference in the worm burden of mice treated with extract of P. nitida and albendazole (Table 4).

The level of glucose increased significantly (p<0.05) in infected mice treated with extract of G. latifolium than infected mice treated with P. nitida and the untreated. However, extract of PN reduced the glucose level of infected mice when compared with untreated and uninfected treated mice (Table 5). On the other hand, cholesterol level was significantly (p<0.05) higher in all infected mice compared with uninfected mice. The protein content was lower in mice treated with P. nitida compared to mice treated with G. latifolium. Meanwhile, there was no significant difference (p >0 05) in protein content of both untreated and infected untreated. Higher albumin level was recorded in uninfected untreated mice when compared to infected but treated with plant extract (Table 5). Generally, triglyceride and HDL levels were significantly (p<0.05) higher in infected mice when compared with uninfected mice. However, the levels of triglyceride and HDL increased in both G. latifolium and P. nitida as the doses increased (Table 5). Furthermore, the LDL was in significantly reduced in infected but treated mice when compared to untreated and uninfected untreated mice (Table 5).

Several villi atrophy and intra-luminal parasites were observed in the intestinal segments of mice that did not receive treatment (Fig. 2). However, there was moderate villi atrophy and cryptal hyperplasia in intestine of mice that received 500mg/kg G. latifolium when compared to the untreated. Furthermore, no cryptal hyperplasia was observed in the intestine of mice that received 500mg/kg P. nitida (Fig. 2).

Fig. 2.