Anthelmintic activity of Phytolacca dodecandra and Vernonia amygdalina leaf extracts in naturally infected small East African goats

A S Nalule, C N Karue*, E Katunguka-Rwakishaya

Department of Wildlife Animal Resource management, School of Veterinary Medicine, Makerere University,
P.O. Box 7062, Kampala, Uganda
snalule@vetmed.mak.ac.ug   /   snalule@gmail.com
* Department of Land Resource Management and Agriculture Technology, University of Nairobi,
P. O. Box 29053 00625 Nairobi, Kenya.

Abstract

Livestock contribute in a major way to household food security and income.  However livestock productivity of free ranging stock is undermined by helminthes infections despite infection is the most neglected area of veterinary extension service. The objective of this study was to determine the anthelmintic efficacy of aqueous crude extract of Phytolacca dodecandra and Vernonia amygdalina against naturally acquired mixed parasites infections in goats as alternative control strategies. The efficacy of single dose extract of P. dodecandra and V. amygdalina with negative and positive controls were evaluated in 6-9 months old goats, naturally infected with nematodes parasites over a 28 day period using faecal egg count reduction test (FECRT). Modified McMaster egg counting technique was used for quantification of nematode eggs on individual faecal samples collected once-weekly for four weeks of the experimental period. Culture were done to isolate and identify the infecting parasites using modified Baerman technique.

The P. dodecandra, V. amygdalina crude extracts and Albendazole achieved a maximum percentage mean epg per gram (EPG) output reduction of 57%, 66% and 99.8% respectively by 21 days post treatment. Both P. dodecandra and V. amygdalina crude extracts significantly reduced the worm egg production in goats compared (p< 0.001) with the negative control but the two plants were not significantly different in the effect (p>0.05).  The two plants’ extracts effects also significantly differed with the positive control, albendazole (p < 0.001). The study revealed that the goats were parasitized by Oesoghagostomum, Haemonchus, Nematodirus, Strongyloides, Cooperia, Trichostrongylus, Bunostomum, Tricuris and Strongylus sp. The current study showed conclusively that P. dodecandra and V.amygdalina plant extracts possess anthelmintic activity thus providing support for their traditional use in veterinary practices. More detailed studies on these plant species involving changing dose rates on a specific parasite and different bioassay should be done.  

Key words: efficacy, helminthes, medicinal plants, Ugandan cattle corridor

Introduction

The economic and social impact of helminthes in sub-Saharan Africa cannot be underestimated with the effect ranging from stunted growth, reduced weight gain, diarrhoea, respiratory problems, reduced productivity and death in livestock especially in the tropics and developing countries (Mcleod 1995; Grade´ et al 2008; Charlier et al 2009). This undermines the economic status of those that derive livelihoods from livestock based industries. Development of anthelmintic resistance, the problem of drug residues, deficient livestock extension services and the high cost of conventional anthelmintics, has led to the evaluation of medicinal plants as an alternative source of anthelmintics for treatment of livestock against helminthes. In humans helminthes cause innumerable suffering such as gastroenteritis, anemia, stunted growth, blindness and lameness worldwide. Consequently, control of helminthosis has received major focus in biomedical research and veterinary extension where synthetic drugs have been developed and used (Sebuguzi 2000). Conversely, many parasites have developed resistance to these drugs (Wolstenholme et al 2004; Behnke et al 2008) while others have had serious side effects (Siddiqui and Hussein 1992) in addition to being costly for the poor countries thus necessitating the search for alternative sustainable control strategies. This has led to increasing demand for herbal medicines worldwide in the recent past.

In Uganda, helminthes infections are one of the major health conditions affecting humans and livestock and of which herbalists have confident in treating and continued to claim effective        (Nalule et al 2011). The search for plants with anthelmintic activity against the gastrointestinal nematodes of livestock has emerged from ethno-veterinary surveys and previous agro-pastoral community claims of efficacy of these plants (Katunguka-Rwakishaya et al 2004; Nalule et al 2011). With these features, P. dodecandra (Phytolaccaceae) and V. amygdalina (Asteraceae) appeared to be some of the good candidate to evaluate as anthelmintics against nematode parasites of small ruminants.  P. dodecandra and V. amygdalina are frequently used in treating gastro-intestinal parasites of both humans and livestock (Nalule et al 2011) although there is limited scientific evidence to this claim.

The V. amygdalina Del (Asteraceae) commonly called bitter leaf is a shrub of 2-5 m tall sometimes a tree to 10 m that grows in secondary scrub, forest edges, thickets and invades cultivated areas (Katende et al 1995; Yeap et al 2010). Many herbalists use aqueous extracts of bitter leaf as treatment for emesis, nausea, diabetes, loss of appetite-induced abrosia, dysentery and other gastrointestinal tract problems (Iwu, 1993).  In Uganda, the plant is traditionally for medicine, nutriceutical and construction purposes (Tabuti 2009) as well as for increasing uterine contraction during child birth in human (Kamatenesi and Oryema-Oringa 2007). Bitter leaf is a widely used medicinal plant in Africa for treatment of various ailments of man and animals including treatment for emesis, nausea, diabetes, loss of appetite-induced abrosia, dysentery and other gastrointestinal tract problems (Iwu, 1993; Bizimana 1994; Farombi and Owoeye 2011). In West and East Africa, V. amygdalina is used in treatment of constipation, hepatitis, malaria fever, gastro-intestinal parasites (Engel 2007; EL-Kamali 2009; Ademola and Eloff 2011; Nalule et al 2011; Farombi and Owoeye 2011), urinary tract inflammations and as a purgative and as soup vegetable (Yeap et al 2010). Both aqueous and alcoholic extracts of the stem, bark, roots and leaves are used as a purgative, antimalarial and for treatment of eczema (Kupcham 1971). In western Kenya, the leaf concoction is used to treat local chicken of Ascaridia galli (Siamba et al 2007). In Tanzania, the local people use the plant for treatment of malaria fever, stomache, schistosomiasis, amoebic dysentery, and other intestinal parasites (Huffman and Seifu 1989). It has also been reported to significantly reduce glucose levels in diabetic patients (Nwanjo 2005; Asuquo et al 2010). Huffman, 2003 reported the wild chimpanzees use the bitter leaf to relieve stomach pain probably involving internal parasites. Anti-malarial and anthelminthic properties (Abosi and Raseroka 2003); anti-tumour properties (Izevbige et al 2004; Sweeney et al. 2005; Song et al. 2005; Opata and Izevbigie 2006) and hepato-protective activity (Ojiako and Nwanjo 2006; Arhoghro et al 2009) and anti-bacterial (Hamil et al 2003) have been demonstrated. The roots of V. amygdalina have been used for treatment of gingivitis and toothache (Elujoba et al 2005). It is also documented that V. amygdalina provide anti-oxidant benefits (Erasto et al 2007) and enhance the immune system through many cytokines regulation (Sweeney et al 2005) while antibacterial activity has been documented in Côte d’Ivoire (Bolou et al 2011).

The P. dodecandra L’Herit belonging to the family, Phytolaccaceae and commonly called endod or soap berry is found in secondary scrub, thickets, forest edges and disturbed areas in forests (Katende et al 1995; Esser et al 2003). The plant is native to sub-Saharan Africa and Madagascar (Schmelzer et al 2008). The plant is used for various medicinal purposes (Ndamba et al 1989; Nalule et al 2011). In central, East Africa and Madagascar, the leaves are used to treat various ailments in humans and animals. The leaves, roots and fruits and seeds are used as purgatives, anthelmintics, laxatives, emetics and diuretics and are also used in the treatment of diarrhea, abdominal pains, edema, and intestinal problems in animals and humans (Bizimana 1994: Schmelzer et al 2008; Nalule et al 2011). In Ethiopia the plant leaves are used in treatment of Malaria (Mesfin et al 2009). The leaf juices are used in treatment of wounds, skin diseases like ringworms, scabies while young leaves are used to induce abortions (Schmelzer et al 2008). In Ethiopia and Zimbabwe, unripe fruits are used to control bilhazia transmitting snails (Schmelzer et al. 2008). The roots and leaves are particularly poisonous.  Juice from the leaves or roots can cause abortion and can kill sperms or germ cells in addition to killing mosquito larvae in ponds and snails (Ndamba and Chandiwana 1986; Treyvaud et al 2000).  The fruits are widely used as soap (Esser et al 2003). According to Katende et al (1995), Phytolacca sp is “a very poisonous” plant, both to people and grazing animals. In Rwanda, the plant is used in treatment of emesis, otitis and pneumonia (EL-Kamali 2009). The berries of a related plant, Phytolacca icosandra have traditionally been used as soap for washing cotton clothes in addition to having molluscicidal, spermicidal and haemolytic properties (Treyvaud et al 2000). The leaves and roots are used in treatment of scabies, ringworm, dandruff, itching, headache, rheumatism, skin irritations, stomach pain and intestinal roundworms (Fonnegra and Jimenez 2007).

Despite the various studies and reports revealing interesting results on traditional uses of these plants, there is insufficient in-vivo studies on these plants claimed to treat helminthes infections and neither have they been licensed as medicines. Yet communities continue to claim anthelmintic efficacy even though animals continue to suffer from helminthes infections and related complications.  Therefore, the current study was undertaken to determine the in vivo anthelmintic activity of the water extracts of P. dodecandra and V. amygdalina on naturally acquired gastrointestinal nematodes in the small East African goats using FECRT. This could contribute to the knowledge base of materia medica for sustainable animal health management and the well-being of people whose livelihoods are livestock based industries.

Materials and methods

Collection and preparation of extracts

The study plants P. dodecandra (Photo 1) and V. amygdalina (Photo 2) were collected from Nakasongola district, in the mid Ugandan cattle corridor.  Mature green P. dodecandra (weighing approximately 500 grams) were carefully picked from the plants per goat.  The plant crude extract was prepared as reported by agro-pastoral community by boiling in 3L of water for one hour to remain with about 1.5L and the extract was then cooled. Concurrently mature leaves of V. amygdalina weighing approximately 0.5kg were also harvested from the plants per goat.  The leaf crude extract were obtained by pounding the fresh leaves in a mortar. The paste was added to one litre of distilled water and thoroughly mixed before straining through cotton wool. A frequently used anthelmintic, albendazole (albendazole bolus 250mg) one of the benzimidazole compounds was purchased from the Veterinary drug shop in Kampala, Uganda.

Photo 1. Leaves, flowers and fruits of Phytolacca dodecandra
Photo 2. The leaves and flowers of Vernonia amygdalina

Collection of fecal material and quantification of egg output

The freshly passed feces were collected directly from the rectum per animal using rubber glove. Each collection was put in a container labeled with the number of the animal and the date of collection. Thereafter, the fresh samples were kept refrigerated to avoid hatching till the counting exercise.  Modified McMaster egg counting technique was used for quantification of nematode eggs (MAFF 1986; Bondarenkoet al 2009) from individual faecal samples.

Animal infection

The 6-9 months old Small East African goats purchased from Ugandan cattle corridor were allowed to graze around School of Veterinary Medicine, Makerere University for six weeks so as to acquire natural infection of worms during which no treatment was administered to any goat.  During this pre-treatment period, the build-up of worms was monitored by collecting faecal samples on a weekly basis to monitor the level of infection and the trend of epg of faeces of goats during free grazing.  By the end of six weeks of monitoring period, the worm burden as reflected by epg had increased to above threshold infection level of 500 epg as recommended by Blood et al (1994) for treatment.

Experimental goats during the 4 weeks period

The twenty small East African goats were randomly distributed to four treatment groups (I-IV) of five animals each following natural infection with parasites.  Animals in each group were separately confined in standard goat pens adjacent to each other in the animal house in School of Veterinary Medicine, Makerere University. Each animal was fed on elephant grass and banana peels in addition to supplementation with cotton seed cake, salt lick and water ad libtum.  The elephant grass was tied up to hang to avoid faecal contamination and assimilate browsing.  The peels and cake were also always put into raised feeding troughs to avoid faecal contamination.  Each animal cubical contained a feeding and drinking trough.  The ecto-parasites were controlled using acaricide, Tactic.  The pens for the goats on experiment (walls and floor) were regularly scrubbed and disinfected using Lysol 12%. 

Culture for larvae identification

At the end of the monitoring period, the faecal samples were collected to culture for isolation of larvae and later their identification. On each sampling day composite faecal cultures were

made for each treatment group. Cultures were incubated for 10 days at 27⁰C. Third stage

larvae were recovered from the cultures by the Baerman technique and identified  according to Thienpont  et al (1979); MAFF (1986) and Hansen and Perry (1990).

Administration of crude extract

The four treatment groups were randomly assigned treatment. Group 1 received no treatment (negative control), Group II received P. dodecandra crude extract, group III received V. amygdalina extract while group IV received albendazole (positive control). Groups II and IV received single doses while Group III received two doses according to cattle corridor’s agro-pastoralists dosage, table 1 below. The faecal samples were then collected at day- zero (pre-treatment), 7 days, 14 days, 21 days and 28 days post treatment. Faecal egg counts were performed on individual faecal samples and expressed as eggs per gram faeces (EPG).

Determination of crude extract efficacy

The FECRT was employed to provide an estimate of anthelmintic efficacy by comparing worm egg counts from goats before and after treatment (Coles et al 1992; Cabaret and Berrag 2004) with modification on data collection period. Data was recorded over a period of 28 days.  In addition to the general control, the zero-day epg of each group acted as a control for that particular group.  FECRT’s were undertaken with samples being collected on day 0, 7, 14, 21 (day of treatment) and 28. The percent epg reduction was calculated in reference to zero day (pre-treatment) epg level on weekly basis.  This was done to standardize the infection level of all groups to a hundred percent.  The percent faecal egg count reduction (FECR) post treatment was calculated on geometric means of EPG based on the differences in egg counts between treated and control groups using a method described by Coles et al (1992) criteria as below:

% Reduction = 100 (1-X_t /X_c )

where X are the arithmetic means, t- is the treated group count at end of third week (21 days) post treatment and c is the group fecal egg count at zero day (control).  Twenty one days post treatment was considered because after that period the egg output started rising again.

Data analysis

The anthelmintic efficacy was determined by comparing the parasites egg population means in treated groups and untreated groups of animals and zero day fecal egg collection record. T- tests and ANOVA were used to determine whether differences between treated and untreated groups over time schedules. The data was analyzed using SAS software computer programme and Excel to generate tables and graphs drawn.

Results

In vivo anthelmintic activity of plant crude extracts and the positive control

 The mean epg output of goats treated with P. dodecandra, V. amygdalina crude extracts and Albendazole and untreated control are presented in tables 2, 3 and table 4. The mean epg trends of control group steadily rose from day-zero to throughout the experimental period  table 5. Notably P. dodecandra, V. amygdalina crude extracts and Albendazole achieved a maximum percentage mean epg output reduction of 57%, 66% and 99.8% respectively by 21 days post treatment.  However, the egg output of the untreated controls had risen by 65% of pre-treatment egg output by end of the experiment (table 5).

The results on the fecal egg count from goats 28days post treatment with P. dodecandra crude extracts at 500ml/ml and V. amygdalina at 1500ml compared to values obtained with albendazole at 250mg bolus and negative control indicated the two plants extracts significantly reduced the worm egg production in goats (p = 0.001) compared with the negative control.  The results also showed that the plant extracts were less effective when compared with albendazole. There was however, no significant difference in effect when the two plant extracts were compared (p= 0.49). The study also showed that the trend of V. amygdalina in controlling the worms egg output closely followed that of albendazole but significantly different (p = 0.004). P. dodecandra also trailed V. amygalina trend but highly differed from albendazole (p=0.0008).

Notably there was a reverse in the trend of the treated animals insinuated by the percent epg retention of the 28^th day being higher than that of the 21st day post treatment illustrated in Figure 1.

Figure 1. Trend lines of faecal egg output of goats treated with 500ml of P. dodecandra, 1500ml of V. amygdalina and 250mg of albendazole in a single dose compared with the untreated controls. The mean egg output of treated animals steadily reduced till end of third week, and thereafter started increasing while the mean egg output in untreated animals steadily increased.

Parasites isolated in the goats

The culture results revealed presence of nine nematode species; Oesoghagostomum sp, Haemonchus sp, Nematodirus sp, Strongyloides sp, Cooperia sp, Trichostrongylus sp, Bunostomum sp, Tricuris sp and Strongylus sp as illustrated in table 6.

Discussion

The study revealed that the herbal preparation at the dose adopted by the agro-pastoral community inhibited production of eggs by gastrointestinal nematode parasites confirming the farmers’ use of P. dodecandra and V. amygdalina as anthelmintics, and their indigenous knowledge of phytotherapy.  However the worms resumed producing the eggs about four weeks later. It was also evident that the dose used by the community despite the anthelmintic potential of the plants was low that probably explains the low efficacy of plant extracts compared with the conventional drug observed. The low dosage may also lead to resistance of parasites. The anthelmintic activity of V. amygdalina was in agreement with observation by Adedapo et al (2007) who reported the anthelmintic efficacy of the aqueous crude extract on Toxocara canis (ascarids) and Ancylostoma caninum (hookworm). The anthelmintic activity of the crude extracts could be attributed to secondary plant metabolites that could be present. The reduction in egg production probably indicates death or paralysis of parasites while the re-emergency of eggs could be attributed to the maturation of the young stages of the parasites. This probably implies the extracts were effective in mature parasites but not young stages.  In the absence of intervention with the plant extract the epg would have risen to show trends as the control group and possibly causing death.  A repeat of the treatment after every three weeks would probably continue to control the buildup of the worms. It was also implicative that in the absence of intervention with P. dodecandra and V. amygdalina the epg would have risen from the initial level of infestation to a level that would possibly have caused production losses or even deaths of the animals. Because agro-pastoralist in the cattle corridor normally uses their local knowledge of phytotherapy, they guard against this state of affairs.

A study by Ademola and Eloff (2011) using Haemonchus contortus eggs and different solvent fractions and Wasswa and Olila (2006) using adult Ascaris suum parasites showed V.amygdalina demonstrated the anthelmintic activity, although Alawa et al (2003) were unable to demonstrate a significant anthlemintic activity at a concentration up to 11.2mg/ml of this plant using egg hatch assay. In addition, Ademola and Eloff (2011) study showed that efficacy varies with solvent used to extract active ingredients and the development stage of parasite which also agrees by the findings by Hernández-Villegas et al (2011). The P. dodecandra effects on fecal egg count closely agrees with findings of the Hernández-Villegas et al (2011) on a related plants species although these authors used different solvents and bioassays.  The authors using three solvents to extract  P. icosandra demonstrated the ethanolic extract of this plant inhibited 55.4% larval migration at 2mg/mL, and the dichloromethane extract showed 67.1% inhibition of migration at 3mg/mL while ethanol extract inhibited egg hatching by 72.6% by the lowest concentration tested (0.15 mg/mL.  However, there was no activity on larval migration inhibition (LMI) with n-hexane extract at any concentration explored in spite, both the ethanolic and dichloromethane extracts inhibited egg hatching by >90% at higher concentration (Hernández-Villegas et al 2011). A study by Alawa et al (2010) using aqueous extract of V. amygdalina at a rate of 1.1g/kg body weight in cattle caused faecal egg output per gram (EPG) reduction of 59.5%. A study by Siamba et al 2007 showed V amygdalina aqueous extract was able to inhibit larval migration of Ascaris galli by 63.7% in vitro assay and reduced egg production by infested chicken by 76.9%. The difference in efficacies could be attributable to source of the plant, maturity at harvesting, species of parasites and animal targeted and the dose administered.

The difference in efficacies displayed by the previous studies and the current study may be due difference in animal species used, presence of resistant parasites, difference in parasite species and their development stages and the difference in the dose used in the different studies. A combination of more than one factor may also explain the observed activity against the parasites that were evaluated. The difference in efficacy of V. amygdalina may also be attributed to the solvent used, and dosage, source of the plant material and its maturity at harvest.  This difference is also a documented phenomenon in conventional anthelmintic drugs (Schoenian 2008).

The P. dodecandra and V. amygdalina extracts were 57% and 66 % effective respectively of the commercial anthelmintic and this could also be attributed to the effect of digestive enzymes on the extracts that could have made unavailable most of the active compounds. Similarly, it could have been due to lack of active ingredients compounds refinement and the dosage administered. The effect would probably have been much higher if the plant extracts were extracted using other solvents both polar and non polar or if the dose rates were increased.  However, it should be noted that refinement may render plant medicinal value inactive since plants impart their medicinal value through synergism, additive effect or antagonism to reduce toxicity to animal tissues.

The observed low anthelmintic activity of P. dodecandra when compared to V.amygalina may in part be due to difference in method of preparation of the concoction where by prolonged boiling was likely to have destroyed or weakened the active principles in P.dodecandra.  In addition, the difference in the dosage and active principles present in the two plants may have contributed to the difference in their efficacy. Although limited anthelmintic reports are available on P. dodecandra (Tuwangye and Olila 2006), the cercaricidal and miracidiacidal properties (Madhina and Shiff 1996; Birrie et al 1998), molluscicidal potency (Ndamba and Chandiwana 1988; Treyvaud et al 2000) of P.dodecandra have been documented.

Four weeks post treatment, the mean epg of the treated groups started rising.  This reverse may imply that the used dose rates was insufficient and the defeat to appreciate the relative resistance of the immature stages of most parasites in some instances by virtue of their inaccessibility while migrating through tissues as was suggested by Blood et al (1994). This rise may also probably be attributed to appearance of recently matured worms and presence of worm species with longer life histories that their maturity coincides with the time when the entire drug had been completely metabolized.  By the 21^st day the species that will have survived the first treatment will start laying eggs that would be detected as an increased epg on the 28th day.  This has an implication in the design of the treatment regimes against helminthosis in farm animals if the use of plant anthelmintics is to be promoted as provision of modern veterinary services to the disadvantaged pastoralists is poor. The epg is likely to exceed thresh hold (500 epg) for treatment in less than the expected time of repeating the treatment above. This period is too short to meet the recommendation by Blood et al (1994) for strategic treatment of two to four times a year.  These authors said it is necessary to repeat the treatment after several weeks to remove the recently matured worms that were in an immature, more resistant stage at the first treatment. This could be supported by the presence of worm genera; Oesophagostomum, Haemonchus, Trichostrongylus and Nematodirus in this study that have been reported to be resistant (McKenna 1995; Várady et al 2009) to most drugs and the resistance of these worms could be responsible for the epg rise observed. Likewise, it may be argued that after this period the worms may have got used to the weaning drug concentrations in the animal body that they no longer have an effect on their activity. 

Conclusion and recommendation

• The aqueous extracts from P. dodecandra and V. amygdalina showed a clear in vivo anthelmintic activity against mixed infection parasite egg production in goats with mixed infection.

  
  

• The success of these medicinal plants in reducing worm egg output confirms the plants traditional use in livestock disease management. Thus these plants could provide an alternative means of helminthes control whenever modern treatment is unavailable especially if dosage and dose are standardized and a better solvent for extraction are sought.  

  
  

• In view of these findings, it is proposed that further in vitro and in vivo anthelmintic potential studies of different plant parts and solvent extracts targeting specific worm species and development stages of parasites to determine sensitivity of specific worm species to these plants be carried out. Throughout, emphasis should be placed on determining the median effective concentration. In addition, the potential for toxicity need to be studied.

Acknowledgements

The authors are sincerely thankful to Pastoral Information Network Programme and Makerere University who funded this study.  An appreciation is also extended to the agro-pastoral community in the Ugandan cattle corridor who provided their indigenous knowledge that formed the basis of this study.

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Received 11 July 2011; Accepted 25 October 2011; Published 1 December 2011

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