Journal of Alternative Complementary & Integrative Medicine Category: Medicine Type: Case Report
In Vitro Evaluation of Anthelmintic Efficacy of Some Plant Species Possessing Proteinases and/or Other Nitrogenous Compounds in Small Ruminantsq
- Sylvester W Fomum1, Ignatius V Nsahlai2*
- 1 Department Of Animal And Poultry Science, College Of Agriculture, Engineering And Sciences, Scottsville, South Africa
- 2 Department Of Animal And Poultry Science, College Of Agriculture, Engineering And Sciences, 127 Rabie Saunders Building, SAEES, Private Bag X01, Scottsville 3209, PMB Campus Of UKZN, South Africa
*Corresponding Author:Ignatius V Nsahlai
Department Of Animal And Poultry Science, College Of Agriculture, Engineering And Sciences, 127 Rabie Saunders Building, SAEES, Private Bag X01, Scottsville 3209, PMB Campus Of UKZN, South Africa
Received Date: Jul 13, 2017 Accepted Date: Sep 18, 2017 Published Date: Sep 29, 2017
Animal species affected (P = 0.0004) anthelmintic efficacy. Similarly, concentration affected (P=0.0002) anthelmintic efficacy. Additionally, interaction between animal species and concentration also affected (P = 0.0015) anthelmintic efficacy. Animal species, concentration and their interaction are crucial to retaining consistent in vitro efficacy of selected plant species. None of these observations could be explained by alkaloid, flavonoid or tannin content.
Developed and developing regions of the worlds have both expressed renewed interest in this field as a result of intense selection for resistance by most pathogenic bacteria and gastrointestinal helminth parasites of livestock [7-11] against antibiotics and chemical anthelmintics. On a global scale, most of these communities are not sufficiently equipped with the technical and managerial capacity to deter re-infection and optimize the efficacy of these plant remedies.
Phytochemical control of gastrointestinal nematodes presents an important area of research, given its historical and traditional background, and incidental application in most communities around the world . They have the potential of being sustainable in addition to their environmental friendly nature [12,13]. Plant-based remedies are also credited with being non-resistible compared to chemical ones, and do not lodge chemical residues in animal products and the environment [14-16]. They also possess fewer or no side effects and contraindications relative to chemical anthelmintics . Additionally, they have benefits such as low cost, availability and acceptability, which are not prohibitive to their use relative to conventional chemical anthelmintics . Consequently, they are suitable for organic farming of livestock. Authors acknowledged the paucity of work relating to number of plants analysed for their biological activities and toxicity in addition to other related studies in view of scientific global validation .
Plant species containing proteinases and other protein/nitrogen related compounds make an important contribution to helminth parasite management and control in livestock . Five plant species; Allium cepa, Ananas comosus, Bidens pilosa, Carica papaya and Ricinus communis, some of which contain proteinases and others nitrogenous compounds were evaluated for their anthelmintic activity at three different concentrations in vitro on mixed nematode L3 larval species. Besides the study of A. comosus, these plants have been used locally without much evidence to relieve animals from gastrointestinal nematodes . So it would be interesting to establish a link between plant metabolites and anthelmintic efficacy. Anthelmintic efficacy measures the degree to which each extract can kill gastrointestinal nematodes and larvae, thus reducing the chance of re-infection and disease incidence. The objective of study was to evaluate the anthelmintic efficacy of five plants species at different concentration for sheep and goats.
MATERIALS AND METHODS
Plant collection and identification
Tannin determination was done following HCl-Butanol proanthocyanidin assay as leucocyanidin equivalent and absorbance’s read using Beckman DU®640 Spectrophotometer at visible wavelength of light 550 nm [31,32].
Flavonoid content of each milled sample was determined following . Into 100 ml of 80 % aqueous methanol was added to 10 g DM of milled material in 250 ml sterile beakers. The content was allowed to stand for 10 hours at room temperature, while being shaken intermittently after each hour with a magnetic stirring bar over a magnetic rotor without heat. The solution was filtered individually through Whatman No 42 filter paper. The filtrate of each milled sample was transferred into pre-weighed 250 ml conical flask and evaporated to dryness over a water bath set at 80ºC. Flasks and flavonoid contents were allowed to cool and subsequently placed in desiccators for one hour. Each of them was weighed and the weight of the sterilized conical flask deducted from that of flask and flavonoid. Flavonoid (the difference) was computed as a percentage of the initial sample weight.
According to Baermann method for isolating surviving nematode larvae, each faecal culture was placed in a double cheese cloth, a porch loosely formed around and tide with a rubber band. All faecal cultures in cheese cloth porches were immersed into lurkwarm water-filled funnels, placed into holes, drilled into a table structure to hold them in place. Sufficient attention was taken to avoid porches from blocking migrating L3 larvae falling down the funnel stem, from where they were collected in fluid. The apparatus was left for 24 hours at room temperature and 15 ml of fluid collected in test tubes from the funnel stem. Tubes containing fluid were left to stand for 30 minutes to enable L3 larvae to settle at the bottom and avoiding agitation during collection, supernatant was drawn with a Pasteur pipette, a McMaster slide filled and mounted on a microscope. Samples were examined and larvae counted using 10 x magnification.
The experiment had two types of animals, five plant species and three extract concentrations; hence it can be described as a 2 x 5 x 3 factorial design. In each run, three plates were treated with each crude extract concentration of each plant species. Surviving L3 larvae were isolated, larval counts taken and mortality (based on a mean of three plates) calculated during each run. The study was re-run three times. Nematode mortality was calculated using Abbott’s formula, as follows :
Where n = number of larvae, T = treated and Co = control. Results are expressed as least square means ± standard error of least square means to serve as indices of dosed anthelmintic efficacy for each plant species.
?ijkl = µ + Ai + Sj + Ck + (A × S)ij + (A × C)ik + ( S × C )jk + (A × S × C )ijk + eijkl
Where, ?ijkl = individual observation; µ = overall mean; Ai = effect of animal species; Sj = effect of plant species; Ck = effect of concentration; (A × S)ij = interaction between animal and plant species effects; (A × C)ik = interaction between animal species and concentration effects; ( S × C )jk = interaction between plant species and concentration effects; (A × S × C)ijk = interaction among animal species, plant species and concentration effect; eijkl = the error term.
Anthelmintic efficacy measures the degree to which each extract can kill gastrointestinal nematodes. Both animal species differed (P = 0.0004) in anthelmintic efficacy, with relatively more radical changes in efficacy for goats than sheep (Figure 1). At parity, efficacies for goats were generally lower than those of sheep (Table 2; Figure 1). Concentration affected (P < 0.0001) anthelmintic efficacy, with various trends of increases in efficacy following increasing concentration (Figure 1). Interaction between animal species and concentration (P = 0.0015) also affected anthelmintic efficacy (Table 2; Figure 1). At the lowest concentration, there was a far lower efficacy for goats relative to sheep, which subsequently increased radically through the intermediate to the highest concentration for goats. Generally, mean efficacy for both animal species were between 66.4 ± 3.13 and 97.5 ± 1.95 % (Figure 2).
|Plant species||n||Alkaloid (gKg-1)||n||Tannin (gKg-1)||n||Flavonoids (gKg-1)|
|Allium cepa||2||5.7 ± 0.30B||6||4.7 ± 0.97A||2||550.4 ± 25.42A|
|Ananas comosus||2||47.5 ± 6.70A||6||4.4 ± 0.75A||2||133.5 ± 5.15B|
|Biden pilosa||2||39.5 ± 6.10A||6||5.9 ± 1.09A||2||163.5 ± 1.92B|
|Carica papaya||2||40.5 ± 6.10A||4||2.6 ± 0.76A||2||167.7 ± 12.38B|
|Ricinus communis||2||40.5 ± 6.10A||6||4.4 ± 1.56A||2||149.6 ± 10.27B|
|Allium c||53.7 ± 3.13||70.3 ± 2.69||77.7 ± 1.95||79.0 ± 3.13||91.4 ± 2.69||93.4 ± 1.95|
|Anana c||46.7 ± 3.13||62.6 ± 2.69||69.5 ± 1.95||96.2 ± 3.13||98.1 ± 2.69||99.4 ± 1.95|
|Bidens p||75.3 ± 3.13||83.3 ± 2.69||94.8 ± 1.95||90.8 ± 3.13||90.8 ± 2.69||97.2 ± 1.95|
|Carrica p||67.5 ± 3.13||82.2 ± 2.69||96.2 ± 1.95||96.1 ± 3.13||97.9 ± 2.69||98.7 ± 1.95|
|Ricinus c||37.3 ± 3.13||51.3 ± 2.69||83.2 ± 1.95||95.4 ± 3.13||98.4 ± 2.69||99.0 ± 1.95|
Allium c- Allium cepa; Ananas c- Ananas comosus; Bidens p - Bidens pilosa; Carica p- Carica papya; Ricinus c - Ricinus communis
Given the same concentration, the anthelmintic efficacy for sheep was far higher than that of goats (Figure 1). This trend was suggestive of prior exposure to the same or similar active anthelmintic phytochemical by goats relative to sheep, in effect, creating some level of tolerance at low concentration. The most probable route of prior encounter is likely to have been through animal species diet, in which plant species or plant varieties possessing similar phytochemicals constituted some portion of their diet . Goats browse and graze, whereas untrained sheep graze, rendering tree parts such as shoots, leaves, growing parts of shrubs and forbes main constituents of goats’ diet . These forages contain plenty of plant secondary metabolites, some of which are implicated in control of parasitic nematodes and other microbial organisms [41,42]. Sheep on their part seldom encounter these plant secondary metabolites because their diet is predominantly grass in nature . Differences in anthelmintic efficacy were also observed in the treatment of infected lambs and mice with extracts of Albizia anthelmintica Brong, wherein, there was no anthelmintic activity observed against mice parasites . Though the outcome contrasted with that of the current study in which plant species possessing proteases and protein compounds exhibited anthelmintic activity against both goats and sheep in vitro, it highlights animal species differences vis a vis anthelmintic efficacy of the same plant species. How proteinases and related phytochemicals exercise their activity in helminth parasite control is worth brief exploration.
Concentration effect of plant crude extract was also observed to influence differences in anthelmintic efficacy in this study. Increase in concentration of plant species crude extract from lowest through the intermediate to the highest concentration for both animal species resulted to various changes in anthelmintic efficacy in conformation with dose-dependent activity of Ficus racemosa (Linn.) bark crude extract on adult earth worms . The primary anthelmintic bioactive phytochemical in the above crude extract is proteases in nature, and similar to that of some plant species in the current study . Cysteine proteinases from papaya latex administered to sheep infected with Haemonchus contortus and Trichostrongylus colubriformis, also exhibited dose dependent anthelmintic activity . Additionally, aqueous extracts of pineapple skin and bromelain exhibited dose-dependent in vitro inhibition on Haemonchus contortus egg-hatch and larval development in sheep . All of the former examples are corroborative of dose dependent anthelmintic activity of the same plant species and others in the current study. Variable anthelmintic efficacy emanating from different plant species possessing proteases such as pineapple, Kiwi fruits and pawpaw at the same and different concentration are in accord with results of the current study . Concentration or dose effect may also vary between in vitro and in vivo activity in consonance with with Luoga , as a result of animal host internal environment. There were differences between animal species and concentration.
Proteinases have a wide range of characteristics, some of which include tissue dissolution and remodelling . The same biochemical mode of action is used by parasites to penetrate host tissues in order to obtain nutrients, be it plug tissue feeding Strongyloides or other parasitic species that burrow and lodge in the subcutaneous walls of the gastrointestinal tract among others . These parasites protect themselves from host protease analogues which aid protein digestion and other biochemical processes by secreting proteases inhibitors . Proteases exert their activity in context by binding to protein or peptide substrate in their active site, in the process cleaving them, and retaining site specificity by amino acids on both sides of the cleavage . It is implicit that several active sites are therefore involved in dissolution of parasite protein or peptide, for them to exert anthelmintic activity. Extensive in vitro and in vivo cuticular damage was done to rodent intestinal nematode Heligmosomoid bakeri by cysteine proteases from Kiwi fruit (Actinidia deliciosa), latex of Carrica papya and stem and fruit bromelain of Ananas comosus, reaffirming tissue digesting trait of proteases . So a combination of proteases could even become more devastating on helminths.
cepa differed from the other four plants in alkaloids and flavonoids contents (Table 1), but A. cepa exerted similar anthelmintic efficacy (Figure 2) compared to A. comosus and R. communis at low and intermediate concentrations; delineation being obvious only for B. pilosa and C. papaya on the one hand and A. cepa. This lack of alignment between anthelmintic efficacy and these plant chemicals leaves proteases and/or other nitrogenous compounds, which were unable to separate and analyze needing verification, though some unknown biochemical constituents may be responsible.
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Citation:Fomum SW, Nsahlai IV (2017) in vitro Evaluation of Anthelmintic Efficacy of Some Plant Species Possessing Proteinases and/or Other Nitrogenous Compounds in Small Ruminants. J Altern Complement Integr Med 3: 038.
Copyright: © 2017 Sylvester W Fomum, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.