Ascaricidal activity of Rhoicissus tridentata root-tuber ethanolic and water extracts

A S Nalule, J M Mbaria*, J W Kimenju** and D Olila

Department of Wildlife and Aquatic Animal Resource, College of Veterinary Medicine, Animal Resource and Bio-security,
Makerere University, P. O. Box 7062, Kampala, Uganda
snalule@vetmed.mak.ac.ug   or   snalule@gmail.com
* Department of Public Health, Pharmacology and Toxicology, University of Nairobi, P. O. Box 29053 00625 Nairobi, Kenya
** Department of Plant Protection and Crop Science, University of Nairobi, P. O. Box 29053 00625 Nairobi, Kenya

Abstract

This study was conducted to determine in vitro ascaricidal activity of ethanolic and water extract of root tuber Rhoicissus tridentata against adult nematodes. Adult worm motility inhibition assay was conducted using Ascaris suum model. Ethanolic and water root tuber Rhoicissus tridentata extracts were used in serial dilutions including 4, 8, 16, 32 and 64mg/ml and; 8,16, 32, 64 and 128mg/ml respectively, parallel to Albendazole and Goodwins’ controls in three replicates. Ten adult Ascaris suum were added to each concentration and controls and incubated at 37⁰C for 48hours. Standard phytochemical analysis methods were used to determine the secondary plant metabolites in the extracts.

A significant motility inhibition in all dose levels that was dose-dependent was observed (F _{(5, 53)} = 4.14, p =0.005; R² = 0.90). There was however, no significant interaction between methods of extraction and the dose effect on motility inhibition in R. tridentata (F _{(10, 53)} =1.02, p =0.450). The ethanolic and water extracts maximum response did not significantly differ (p=0.082) although their median effective doses were 12.3 and 23.5mg/ml respectively. R. tridentata extracts have immense in vitro ascaricidal potential supporting its use in ethno-veterinary medicine although anthelmintic potency of plant extracts depend on solvent used for extraction. There is however, need to determine in vivo ascaricidal activity and safety for sustainable utilization of this medicinal plant.

Key words: Ascaris suum, dose-dependent, median effective dose, motility inhibition

Introduction

Helminthes infections are wide-spread across the world affecting both livestock and humans. They are a major cause of reduced productivity in livestock leading to enormous economic losses particularly by resource poor farmers in extensive grazing systems calling for continued search for sustainable and inexpensive control strategies. The prevalence of helminthosis is attributed to increased drugs costs, unavailability of drugs especially in developing countries and the development of drug resistance to commercially available anthelmintics (Behnke et al 2008). Resistance to anthelmintics has become a major problem in veterinary medicine, and threatens agricultural income and animal welfare (Wolstenholme et al 2004) yet is likely to increase under changing environment in the face of climate change (Weaver et al 2010). It is well documented that parasites undergo evolution to adapt to opportunities presented by climate change or anthelmintic use or undoubtedly as a manifestation of ‘survival of the fittest’ (Sargison et al 2007; Davey et al 2009). Despite the high helminthes prevalence in Ugandan drylands (Ocaido et al 2009) and their related effects, helminthes control remain a neglect area in veterinary extension services particularly in extensive systems leading to continued search for inexpensive control remedies worldwide (Waller and Thamsborg 2004). Exploitation of medicine plants provides alternative disease control options that are culturally acceptable; ecologically and environmentally sound (Nalule et al 2011).

Rhoicissus tridentata (L.f.) Wild & R.B. Drummond or wild grape (Vitaceae) is a common shrub, widely used in drylands of Uganda. The family Vitaceae consists of approximately 14 genera and approximately 900 species primarily distributed in tropical regions in Asia, Africa, Australia, the neotropics, and the Pacific islands, with a few genera in temperate regions (Soejima and Wen 2006; Wen et al 2007; Iju 2009). However, R. tridentata is one of the 15 genera exhibiting strict regional distribution (Iju 2009). According to the site: http://www.gwannon.com/species/Rhoicissus-tridentata; the species is widely distributed in Ethiopia, Tanzania, Kenya, South Africa, Botswana, Malawi, Zambia, Mozambique, Swaziland, Uganda, Namibia, Democratic Republic of the Congo, Burundi, Rwanda and Angola. The species is a native of coastal dunes and forest margins throughout South to north-eastern Africa.

In Uganda, R. tridentata is used in treatment of a number of ailments of man and livestock, helminthosis inclusive (Nalule et al 2011). For instance, the Pokots of Kenya use the root tuber to treat malaria of which Gakunju et al. (1995) demonstrated an in vitro activity of the water plant extract. Anti bacterial and anti protozoal activity (Naidoo et al 2005), ant-inflammatory and anti-microbial effects (Hamza et al., 2006; Opoku et al., 2007) have been demonstrated. McGaw and Eloff (2008) reported the root tubers are used in treatment of heart-water, red water, internal parasites and general ailments. Opoku et al (2007) demonstrated a hepatoprotective effects of R. tridentata. Despite several reports on traditional uses and biological activities, experimental reports on anthelmintic activity of the root tuber of this plant are scarce. In addition, different reports have indicated that the anthelmintic properties of plants may dependent on solvent used for extraction of active ingredients (Malu et al 2009). This study was undertaken to determine in vitro ascaricidal activity of ethanolic and water root tubers’ extracts of R. tridentata for claimed anthelmintic potential using Ascaris suum model. It was anticipated that the result of this study would have potential to help poor resource users in Ugandan drylands to improve their agricultural productivity, increase their food nutrition and income security and better manage their natural resources.

Materials and methods

Plant collection and identification

The plant root tubers were collected from Nakasongola District and identified by a plant taxonomist in the herbarium of department of Botany, Makerere University, Kampala, Uganda where a voucher specimen was deposited under accession number: 39701.

Determination of community adopted dosage and preparation of test materials

The dosage, preparation and route of administration adopted by the livestock farmers was obtained from thirty two (n=32) individuals’ collections out of the 198 farmers (N=198) interviewed during field exploration that was conducted between January and March 2010. Each individual’s plant material collected was first washed off soil then weighed while flesh, followed by chopping into smaller parts. The chopped materials were dried under the shade at room temperature for ten days and individually reweighed after drying. The weights were recorded and analysed to obtain the mean weight and standard deviation (n=32). The roots were then pulverized into powder and stored in amber plastic containers till further processing was done.

Extraction of active ingredients and determination of extraction efficiency

Two hundred fifty grams (250g) of the freshly dried powdered root tuber was macerated in 2000ml of 70% ethanol for 72hours with intermittent shaking in duplicates. Filtration through cotton wool was done to remove coarse particles (residues) and finely through filter paper 12.5mm (Whatman®, No.1). The filtrate was concentrated on Rota-vapor under reduced pressure at 40⁰C. The concentrated extracts were later dried on weighed kidney dishes to a constant weight at 50⁰C. The above procedures were repeated with water as solvent. The dried extracts were packed into universal bottles and kept at 4⁰C until needed for bioassays.

Collection and maintenance of worms

Adult Ascaris suum worms were collected from small intestines of pigs obtained from Wambizi slaughter house in Kampala, Uganda. Immediately after slaughter, adult worms were collected and transported in flask containing Goodwin’s solution to the pharmacology laboratory. The worms were selected and washed in warm water at 37⁰C and maintained in Goodwin’s solution at 37⁰C.

Preparation of Goodwin’s physiological solution

Goodwin’s physiological solution was prepared according to Lamson and Brown (1936) and Donahue et al. (1981). The solution was made by dissolving 0.20g calcium chloride, 5g glucose, 0.10 magnesium chloride, 0.2g potassium chloride, 0.15g sodium bicarbonate, 8g sodium chloride and 0.5g sodium hydrogen phosphate, all quantities in 1 liter of distilled water. Calcium chloride was added later after dissolving other salts to discourage precipitation of other salts. The solution was pre warmed to 37⁰C before putting in the warms.

Adult worm motility inhibition assay

Fifty four (54) 250ML conical flasks were divided into three major groups (ethanol, water and albendazole) of 18 flasks each to cater the five serial dilutions of crude extracts and negative control or blank and albendazole, all in three replicates. To the blank flasks, 100ml of Goodwin’s physiological solution was used in negative control. To group one (ethanolic extract flasks), 100ml of ethanolic extract at concentrations; 4, 8, 16, 32 and 64mg/ml were added. To group two (water extract), 100ml of water extract at concentrations 8, 16, 32, 64 and 128mg/ml were added. In a parallel set up (group three) flasks, 100ml of Albendazole (Valbazen®) 10% (KELA, Belgium) at 6.25, 12.5, 25.0, 50.0 and 100.0mg/ml were added. All groups were prepared in three replicates. The first dose of extracts were calculated to represent half of the community dose and based on extraction efficiency. Stock solutions were prepared by dissolving weighed amount of crude extract in 5ml of Dimethyl sulfoxide (DMSO) and then made to the highest concentration (mg/ml) required with Goodwins’ physiological solution. One hundred milliliter (100) of reconstituted R. tridentata root tuber extracts was used to ensure full submerge of the heavy parasites. Ten average size motile adult worms were randomly distributed into each of the flasks above and the flasks were incubated at 37⁰C for 48hours.

Worm motility assessment

In preliminary experiments, a Criteria used for assessing the effects of crude plant extracts on the motility of adult Ascaris suum was developed and combined the procedures described by Kotze et al (2004); Paolini et al (2004) and Marie-Magdeleine et al (2009). The worm motility was assessed at 24hours and 48hours post treatment. After 24 and 48 hours of incubation post extract administration in an incubator at 37⁰C, the worms were gently removed from the text treatment and re-suspended in Goodwins’ physiological solution at the same temperature for 30 minutes for possible recovery of parasite motility. The worms were assessed for death and/or paralysis. A worm was considered to be motile if it moved in a sinusoidal motion when stimulated by water at 40-50°C. Similarly it was considered paralyzed if on stimulating it by water at 50⁰C only part of the body responded either by raising the head and whether some parts showed autolysis and change of colour to pale white. Motility was also assessed by pressing the worm with an index finger, or using water at 50 - 60⁰C to differentiate dead from paralysed. The percent worm immotile or dead was calculated as the number of dead worms divided by the total number of worms per flask multiplied by 100.

Data analysis and determination of ED₅₀

The bioassay data was analyzed by the Generalized Linear Model procedures for regression and determination of median effective dose (ED₅₀) using the Graph Pad Prism version 5.01 software (Inc San Diego, CA USA). Prism was used to determine the means of percent motility inhibition and the ED₅₀ with confidence intervals (95% CI). Prism was also used generate the dose-response curves and to fit the non linear regression equations. Two-way analysis of mean variance was carried out in GenStat Release 13.2 (PC/Windows7) followed by Bonferroni post hoc t-test. P- value = 0.05 was used for significance level.

Results

Community based extract preparation and adopted dosage

The mean dry weight (farmers’ adopted dosage) of root tuber used was 493 ± 22g (n=32). The root tubers are harvested, washed in clean water, chopped and pounded in a traditional mortar before they are boiled in two liters of water. The boiling takes on average 30 minutes. The young animals including pigs, goats and cattle are drenched with 0.5 liter of the filtrate cooled extract while the adult animals are administered one liter of the same extract.

The ethanol and water extraction yield and efficiency of root tuber R. tridentate

The water and ethanol mean extraction efficiency and percent yield per 250g of Rhoicissus tridentata root tuber plant material used are given in table 1. The water extracts of R. tridentata yielded higher in water than ethanol extracts. Analysis of yields by the ethanol and water solvents revealed a significant difference in yield (p <0.05).

Ascaricidal effect of ethanolic and water extracts of R. tridentata on Ascaris suum

In vitro adult motility inhibition assay revealed the two extracts exhibited motility inhibition. The ascaricidal single dose effect of R. tridentata extract increased with increasing concentration of the extract as shown in Table 2. The highest concentration of 64 and 128mg/ml for ethanol and water extracts gave maximum mean percent ascaricidal activity by 48 hours as 80.0 ± 10.0 and 90.0 ± 0.6% respectively compared with negative and positive controls. The median effective doses (ED₅₀) of the plant extracts and positive control are given in table 3. However, the community dose concentration of 8 and 16mg/ml for ethanol and water extracts inhibited motility of the worms by 36. 7 ± 6. 7% and 43.3 ± 6.7% respectively 48 hours post treatment.

Irrespective of solvent used for extraction of bioactive ingredients, a significant adult worm motility inhibition (AWMI) in all dose levels of ethanolic and water R.tridentata root tuber extracts that was dose-dependent was observed (F _{(5, 53)} = 4.14, p =0.005; R² = 0.90) compared with the untreated worms. However, the ethanolic and water extracts maximum response did not show significant difference when R. tridentata root tuber extracts were compared with albendazole (p=0.082). Nevertheless, water extract was more potent than ethanol extract as indicated by their median effective doses in Table 3. The decreasing order of potency was albendazole, ethanolic extracts and water extract as illustrated by the shift of the graphs to the left of the albendazole (Fig.1). There was no significant interaction observed between methods of extraction and the dose effect on motility inhibition in R. tridentata (F _{(10, 53)} =1.02, p =0.450).

Table 3: The median effective doses (ED50) of the ethanol, water extract and albendazole
Extract	Maximum  motility inhibition %	Median effective dose (ED50)mg/ml	95% CI of ED50  mg/ml
Ethanol	80	12.3	6.7 – 22.7
Water	90	23.5	11.5 – 48.3
Albendazole	100	15.1	6.95 - 32.9

Figure 1.  Dose-response curves of adult Ascaris suum motility inhibition by ethanol and water crude extracts of Rhoicissus tridentata 48 hours post treatment. Nonlinear regression curves of treatments are defined as; Percentage motility inhibition (Y) is = A(1,2,3) + C/(1 + EXP(-B*(X - M))). Where; Y(1,2,3) are proportions of worm motility inhibited by ethanol, water extracts and albendazole. A is Y-value when X=0; C is the top – bottom of each curve i.e X=0 and X= maximum; B is a rate constant expressed as reciprocal of X; M is random error and X is the dose of treatment (ethanolic, water and albendazole). A1, A2, A3 are parameter estimates for ethanol extracts, water extracts and albendazole respectively given as 1.84, -5.05 and 8.52 respectively and C = 90.6, B = 1.85 with s.e of 0.243, and M = 2.64 with s.e of 0.077, X = treatment doses (level 1 to level 6) for ethanol, water and albendazole. Percentage variance accounted for 90.0 with a standard error of observations estimated at 11.2. The error bars show the standard error of the percent worm motility inhibition.

Discussion

This study as assessed through in vitro adult worm motility inhibition assay showed that R. tridentata used in ethno-veterinary medicine could be of value in the treatment of livestock helminthosis in the Ugandan cattle corridor. The motility decreased with increasing extract concentration and incubation period although with the low rate of change of worm response at bottom and peak concentrations. Paralysis of worms was very evident in treated groups that progressed till death. The paralysis could probably be linked to the action of sitosterol, sitosterolln and proanthocyanidins reported to be present in this plants (Brookes and Katsoulis 2006). The worms also probably died of starvation since they became paralyzed and fail to feed. Schoenian (2008) held similar observations that anthelmintic drugs kill worms either by starving them to death or causing paralysis since worms have no means of storing energy that force them to feed continuously to meet their metabolic requirements. Interfering with worm feeding for 24 hours or less is sufficient to kill most adult parasites. Plant metabolites like tannins bind to glycoprotein on the cuticle of the parasite and disturb the physiological functions like motility, feed absorption and reproduction as reported by Hoste et al (2006); Githiori et al (2006) and Brunet et al (2008). Aiyegoro et al (2008) observed that the concentrations of plant extracts cause protein leakages in the test organisms leading to disruption of cell membrane.

The relatively low ED₅₀ of crude extracts (25mg/ml) indicate the potential of the crude extract to control gastrointestinal nematodes. These findings confirm previous community claims that the plant is useful in the treatment of helminthosis (Nalule et al 2011). However, the community dosage of 16mg/ml yielded lower response than the maximum achieved by the highest dose in this study. This is a clear demonstration that the community under-dose the parasites. Repeated exposure to insufficient crude extract concentration could lead to worm resistance. This probably explains the continued reports of helminthosis and low livestock productivity despite community use of medicinal plants to contain the parasites. The lack of significant difference in the responses by the two extracts (80% and 90%) indicates the two solvents could be used interchangeably in extraction of active ingredients. However, ethanol has an added advantage of preservation and high potency thus reducing pressure on plant. This would not only reduce labor of repeated preparation but also promote species’ conservation.

It should be noted that although in vitro studies can be an invaluable tool in identifying a potential anthelmintic mechanism of this plant, such as inhibition of parasite motility, or structural destruction of the worm, variation in activity may occur when tested in an in vivo experiment. Despite the relatively higher percent motility inhibition caused by water extract, ethanolic extract was more potent as measured by the median effective dose (table 3). This could probably be attributed to the kind of bioactive substances extracted by the two solvents agreeing with reports by Harbone, (1973) that different solvents extract different compounds depending on type of substances and polarity. Ebrahimzadeh and Bahramian (2009) also noted differences in biological activity based on solvent used for extraction and the concentration of the extracts. A study by Ene et al (2008) of 15 ant-malarial plants using three solvents in mice revealed that different solvents had different efficacy.

Harbone (1973) recommended alcohol as a "good for all-purpose" solvent for preliminary extraction. Brookes and Katsoulis (2006), reported that R.tridentata has twenty novel compounds with health-promoting properties. Naidoo et al (2005) reported that the major chemical constituent as condensed tannins, proanthocyanldin monomers which probably account for the health properties although synergy by other compounds could have enhanced the activity. The role of condensed tannins in helminthes control has been demonstrated (Athnasiadou et al 2001; Cenci et al 2007; Brunet et al 2008). Chemically tannins are polyphenolic compounds (Muchuweti et al 2006) and synthetic phenolic anthelmintics like niclosamide and oxyclozanide are said to interfere with energy generation in helminthes parasites by uncoupling oxidative phosphorylation (Martin et al 2005). It is thus possible that tannins contained in the extracts of R. tridentata produced similar effects.

Conclusion

Three main conclusions can be drawn from our study.

• Firstly, R. tridentata root tuber extracts have immense ascaricidal potential supporting its use in ethno-veterinary medicine. Thus the ascaricidal efficacy of the plant makes it a potential candidate for a novel class of anthelmintic to provide a valuable alternative control of livestock helminthes to chemotherapy for resource poor agro-pastoralists to improve animal productivity in Ugandan drylands.

• Secondly, the anthelmintic potency of crude plant extracts varied with extraction solvent used as revealed by the median effective doses. The variability depending on the solvent used in extraction of active ingredient that in turn determine the chemical constituents and the complexity of the metabolite compounds interaction with parasite.

• Lastly, the study has revealed that the dosage adopted by the community fall far below the required quantity to produce beneficial helminthes control. This explains the persistent low productivity of livestock despite long term traditional use of this plant. However, there is need to conduct in vivo studies to determine the effect of digestive enzymes on this observed efficacy in addition to determining safety for sustainable utilization of this medicinal plant.

Acknowledgement

The authors are grateful to the Regional Universities Forum (RUFORUM), Association of African Universities (AAU), and Makerere University for funding this study. We are also grateful to the Nakasongola agro-pastoral community who provided information on which this study is based.

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Received 4 April 2012; Accepted 22 May 2012; Published 1 August 2012

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