An ethnopharmacological survey and in vitro confirmationof the ethnopharmacological use of medicinal plants as anthelminticremedies in the Ashanti region, in the central part of Ghana

Christian Agyare a,1, Verena Spiegler c,1, Herbert Sarkodie a, Alex Asase b, Eva Liebau d,Andreas Hensel c,n

a Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghanab Department of Botany, University of Ghana, Legon, Ghanac University of Münster, Institute for Pharmaceutical Biology and Phytochemistry,Corrensstraße 48, D-48149 Münster, Germanyd Institute for Zoophysiology, Schlossplatz 8, D-48143 Münster, Germany

a r t i c l e i n f o

Article history:Received 31 March 2014Received in revised form15 October 2014Accepted 17 October 2014Available online 29 October 2014

Keywords:AnthelminticCaenorhabditis elegansCombretum mucronatumPaullinia pinnataPhyllanthus urinaria

a b s t r a c t

Ethnopharmacological relevance: Infections with helminths are still a big problem in many parts of theworld. The majority of the people in West Africa treat such infections with medicinal plants related to thelocal traditional medicine. The present study aims at identifying medicinal plants traditionally used forworm infections in the Ashanti region, Ghana. In vitro screening of selected extracts from plants on whichscientific knowledge is limited was to be performed.Materials and methods: Validated questionnaires were administered to 50 traditional healers in theAshanti region, Ghana. Interviews and structured conversations were used to obtain relevant informa-tion. Quantitative and qualitative evaluation was performed additionally to structured cross-referencingof the data using SciFinders data base. Selected plant species were used for in vitro testing onanthelmintic activity against the free-living model nematode Caenorhabditis elegans.Results: 35 plant species were recorded for the use in humans and 6 for the use in animals. Plant materialmost frequently used were the seeds from Carica papaya, mentioned by nearly all healers. Theplausibility of most plants used for treatment of infections with helminths was given in most cases bydocumentation of potential anthelmintic activity in recent scientific literature. 9 species from plants notor scarcely described in literature for this indication were investigated on in vitro activity. Ahydroethanolic (1:1) extract of Combretum mucronatum was most active with a survival rate ofnematodes of 89% at 0.1 mg/mL and 58% at 1 mg/mL respectively (levamisole 16%). Extracts of Paulliniapinnata and Phyllanthus urinaria were also assessed to exhibit a minor (85% and 89% respectively at 1 mg/mL), but still significant activity.Conclusion: Traditional use of anthelmintic plants from Ghana can be well rationalized by cross-referencing with published literature and phytochemical/pharmacological plausibility.The in vitroinvestigations of extracts from Combretum mucronatum, Paullinia pinnata and Phyllanthus urinariaexhibited significant effects against nematodes. The anthelmintic activity of these plants should beinvestigated in detail for pinpointing the respective lead structures responsible for the activity.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Infections with parasitic nematodes, including intestinal helm-inths and filarial worms, are among the most widespread diseasesworldwide with a global prevalence of more than 2 billion (Hotez

et al., 2008) Thereof intestinal or also called soil-transmitted hel-minths (STH), are the major agents. The World Health OrganizationWHO estimates the number of people suffering from STH infectionsto approximately 1.5 billion, which accounts for almost a quarterof the world's population (WHO, 2013a) While soil-transmitted

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.10.0290378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: DMSO, dimethyl sulfoxide; GNATH, Ghana National Association of Traditional Healers; MDA, mass drug administration; NC, negative control; NGM,nematode growth medium; PC, positive control; STH, soil-transmitted helminthes; WHO, World Health Organization

n Corresponding author. Tel.: þ49 251 8333380; fax: þ49 251 8338341.E-mail address: ahensel@uni-muenster.de (A. Hensel).1 Contributed equally to this work.

Journal of Ethnopharmacology 158 (2014) 255–263

helminthiases in humans play a minor role in Europe, they occurthroughout the tropical and subtropical regions of Sub-Saharan Africa,the Americas and Asia, imposing a great burden mainly on inhabi-tants of developing areas (Hotez et al., 2008; Lustigman et al., 2012).

Intestinal helminthiases are most commonly caused by round-worms (Ascaris lumbricoides), whipworms (Trichuris trichiura) andhookworms (Ancylostoma duodenale and Necator americanus)(Lustigman et al., 2012; WHO, 2013a) although coinfestations withmore than one parasite are rather normal (Bethony et al., 2006).

Although not lethal, infections can cause symptoms rangingfrom abdominal pain and diarrhea to more severe impairmentssuch as anemia as well as growth and cognitive retardation inchildren due to blood loss and malnutrition (Hotez et al., 2008;WHO, 2013a) depending on the type of nematode and the wormburden. Since in most cases people affected by helminthiases facepoverty accompanied by poor sanitary conditions and limitedaccess to medication these diseases hinders them from socio-economic development, leading to a vicious circle (Lustigmanet al., 2012).

In the recent past considerable efforts have been made by theWHO to tackle the currently 17 neglected tropical diseases,including guidelines for preventive chemotherapy in helminthiasisand recommendations for treatment and donations of anthelmin-tic medicines (WHO, 2013a). The set up of mass drug administra-tion (MDA) programs to treat inhabitants in endemic areas withanthelmintics given annually or biannually has led to a significantsuccess in the reduction of morbidity. While the WHO (2013b)currently lists four anthelmintics, namely albendazole, mebenda-zole, levamisole and pyrantel pamoate as essential drugs in mostcases benzimidazoles, and in particular albendazole, is adminis-tered due to its low costs and broad spectrum of activity (forreview see Humphries et al. (2012)). Still, great differences in thesusceptibility among STH species and also in the over-all success ofthe anthelmintic treatment could be observed, which raises thequestion on the reliability of MDA, particularly with respect todevelopment of benzimidazole, levamisole and pyrantel resistanthelminth strains. Also adverse effects that occur in quantity duringdeworming of whole populations should be considered (for reviewsee Keiser and Utzinger (2008); Vercruysse et al. (2011) andHumphries et al. (2012); ). Finally, these programs focus on themost commonly occurring helminthiases, neglecting millions ofinfections with less prominent infectious agents, such as Strongy-loides, of which little is known about its epidemiology and thesuccess of MDA measures (for review see Olsen et al. (2009)).

Instead of conventional drugs, many African patients (almost70% of the people) rely on remedies of traditional healers andherbal practitioners (Agyare et al., 2009). Therefore, it is not sur-prising that plants or plant products have also been successfullyused in the treatment of filarial (Ndjonka et al., 2011, 2013) andintestinal parasites (Waterman et al., 2010; Koné et al., 2012) andare also a considerable resource for nematode control in livestockand agriculture (Chitwood, 2002).

Furthermore, Fabricant and Farnsworth (2001) not only under-line the importance to confirm the traditional use of herbalremedies by in vitro investigations, but also highlight the potentialof ethnopharmacological approaches towards the discovery ofnew lead compounds that are certainly awaited.

In this study we aim to identify herbal remedies that aretraditionally used to treat helminth infections in a defined area ofGhana and to confirm the efficacy of the most promising candidatesrevealed in the field study in a bioassay. By this approach we intendto support the ethnopharmacological use by in vitro data and toidentify previously undescribed herbal drugs for this indication thatcould be the base for further phytochemical and pharmacologicalresearch.

2. Material and methods

2.1. Study area and survey

The ethnopharmacological survey was performed in Ashantiregion in the central part of Ghana, located between 0.15–2.251Wand 5.50–7.461N (Fig. 1). The region shares boundaries with four ofthe 10 political regions of Ghana. The region covers a total landarea of 24,389 km2, representing 10.2% of the total land areaof Ghana.

The ethnopharmacological survey was carried out from October2012 to February 2013 in accordance with the national rights ofGhana and with acceptance and in close co-operation with GhanaNational Association of Traditional Healers (GNATH). A house tohouse visitation strategy was employed for interviewing the prac-tioners. All participants were informed about the survey and personalvisits were made to their facilities, centers and homes. In respect tothe local tradition, some gifts in cash or kinds were given. Interviewsand conversations were used to administer the questionnaires.Questionnaires were designed in English and administered to 50

Map of Ghana Districts in Ashanti region

Ashanti region

Fig. 1. Map of Ghana with Ashanti region (shaded) with detailed boundaries of all the districts (study area).

C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263256

traditional healers and herbal practitioners who nearly represent 50%of the GNATH membership in the region.

2.2. Plant material and chemicals

Plant material from Azadirachta indica A. Juss. (No. 297), Sennaalata (L.) Roxb. (No. 302), Combretum mucronatum Schumach. &Thonn. (No.305), Vernonia amygdalina Delile (No. 298), Bryophyl-lum pinnatum Lam. (Oken) (No. 303), Phyllanthus urinaria L. (No.304), Musa x paradisiaca L. (No. 299), Brachyachne obtusiflora(Benth.) C.E. Hubb. (No. 296) and Paullinia pinnata L. (No. 300)was catalogized in the plant archives and voucher specimen havebeen deposited in the herbarium of the Institute of PharmaceuticalBiology and Phytochemistry, University of Münster, Germany. Allplant materials were collected between November 2013 andJanuary 2014 from the Bosomtwi area and identified botanically.

2.3. Preparation of plant extracts

A standard protocol for the preparation of the extracts used20 g of the powdered plant material, which was extracted in200 mL of ethanol/water (50:50 v/v) by Ultra-Turraxs (IKA,Staufen, Germany) for 10 min at 9500 rpm under ice cooling. Fortechnical reasons (strong floating during the extraction andinsufficient sedimentation during centrifugation) the standardextraction protocol had to be changed for the preparation of theextracts from the leaves of Brachyachne obtusiflora and the root ofMusa x paradisiaca: these plant materials were extracted withdifferent plant-solvent ratios (Brachyachne obtusiflora 15 g in350 mL solventand Musa x paradisiaca 7 g in 200 mL solvent. Theextracts were centrifuged (3000g, 10 min) and the supernatantwas filtered (595 qualitative filter paper, S&S Dassel, Germany) toobtain a clear solution. The supernatant was concentrated byvacuum evaporation not exceeding 40 1C, lyophilized and thepowder was stored under vacuum at room temperature.

2.4. Anthelmintic assay

2.4.1. Monoxenic and axenic maintenance of Caenorhabditis elegansCultures of Caenorhabditis elegans wildtype (N2 Bristol strain)

were maintained monoxenically as described by Stiernagle (2006)at 20 1C on petridishes containing Nematode Growth Medium(NGM; 1.5 g NaCl, 1.25 g peptone from casein, 8.5 g Agar-agar(Roth, Karlsruhe, Germany), 500 mL 1 M CaCl2, 500 mL 1 M MgSO4,

500 mL Cholesterol (Calbiochem / Merck, Schwalbach, Germany)5 mg/mL in ethanol and 12.5 mL 1 M KH2PO4 / K2HPO4 per 500 mLH2O) supplemented with 800 mL of Escherichia coli OP50 strain as afood source (Brenner, 1974).

Age synchronous cultures were obtained by the alkalinebleaching method described by Ndjonka et al. (2011): worms wererinsed from the petridish with M9 buffer solution (3 g KH2PO4, 6 gNa2HPO4, 5 g NaCl, 0.25 g MgSO4�7H2O in 1 L of water) (Brenner,1974), centrifuged at 2000g for 1 min and treated with alkalinesolution (600 mL sodium hypochlorite solution, (Sigma-Aldrich,Steinheim, Germany), 100 mL 10 M sodium hydroxide solution,1.300 mL H2O) for 7 min after the supernatant was removed.While worms and bacteria are dissolved, the eggs withstand thebleaching procedure and after three washing steps with M9 buffersolution they can be seeded onto plates or into liquid medium.

For the in vitro assay, synchronous cultures of Caenorhabditiselegans were initiated in axenic liquid medium to avoid interferingeffects of the bacteria with the test substances.

The liquid mediumwas composed of 3.0 g Bacto™ yeast extract(Becton-Dickinson, Heidelberg, Germany), 3.0 g soy peptone fromcasein (Sigma-Adrich, Steinheim, Germany), 1.0 g dextrose, and0.25 mL cholesterol solution (5 mg / 1.0 mL in ethanol) in 100 mL

of H2O and autoclaved. To start the axenic culture, synchronousworm cultures were seeded into the axenic medium supplemen-ted with 0.05% hemoglobin (stock solution: 5% (w/v) of bovinehemoglobin (Sigma-Adrich, Steinheim, Germany) in 0.1 M KOH,autoclaved for 10 min) (Lenaerts et al., 2008) and 0.1% (v/v)penicillin/streptomycin solution (10,000 U / 10,000 mg/mL; Gibco/ Invitrogen, Darmstadt, Germany) (Ndjonka et al., 2011).

2.4.2. In vitro screeningStock solutions of 50 mg/mL of the dried plant extracts were

prepared with M9 buffer solution using Dimethyl sulfoxide(DMSO) as a solubilizer and centrifuged at 2000g for 1 min. Thefinal concentration of DMSO did not exceed 1% (v/v). Aliquots ofthe stock solution were added to a 24-well microtiter platecontaining filter-sterilized “enriched” M9 buffer solution (50 mL20% (w/v) dextrose solution, 500 mL of a solution from cholesterol5 mg/mL in ethanol, 500 mL 1 M CaCl2, 500 mL 1 M MgSO4, 12.5 mL1 M KH2PO4/K2HPO4 and 500 mL penicillin/streptomycin solution(10,000 U/10,000 mg/mL) in 500 mL M9 buffer solution) to a finalvolume of 500 mL per well resulting in final concentrations of0.1 mg/mL and 1 mg/mL. Each extract was tested in 4 replicatesper treatment and each experiment was independently performedin triplicate. A solution of levamisole hydrochloride (40 mM)served as a positive control, DMSO 1% (v/v) was used as a negativecontrol.

One to two microliters of the liquid medium, containingapproximately 10–20 worms (L4 larvae or young adults) weretransferred into each well and the plate was incubated at 20 1C.After 72 h mortality was assessed by counting the number of deadworms under a dissecting microscope: worms that were immotileand completely straight were counted as dead if they did notrespond when hit with an eyelash. The percentage of dead wormswas calculated as the number of dead worms in relation to thetotal number of worms per well.

2.4.3. Statistical analysisData obtained from the in vitro assay were analyzed using

GraphPad Prisms Ver. 3 (GraphPad Software, Inc., La Jolla, USA).Mean values of mortality rates were compared by a one-wayANOVA test followed by a Dunnett Multiple Comparison Test versusthe negative control. po0.05 was considered to be significant andpo0.01 to be highly significant.

3. Results and discussion

3.1. Field study and survey on anthelmintic plants

An ethnopharmacological field study on the use of medicinalplants for the management of helminthiasis was carried out indifferent districts of Ashanti region, Ghana, as indicated in Fig. 1.During the personalized and structured interviews differentiationwas made for plant species used for treatment of human hel-minthiasis and worm infections in animals. 50 professional healerswere recruited with the help of the Department of Herbal Medicineand GNATH. 72% were men and 28% were women. This genderrelation is in good accordance with a recent field study in Ghana ontraditional healers (Agyare et al., 2009). Professional healers weremainly (4 90%) older than 40 years: 2% were between 21 and 30years, 8% between 31 and 40 years, 58% between 41 and 60 yearsand 28% were older than 61 years. This clearly shows that tradi-tional healing is mostly related to older, more experienced practi-tioners. It was also interesting to find that most respondents hadhigher education: 10% hold a university degree, 14% completedsenior high school, 42% passed middle and junior high schooleducation, 30% primary school and 4% were illiterate. Considering

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the duration of practice of the healers, only 22% had less than 11years of experience. 54% worked for 11–20 years in this profession,18% for 21–40 years and 6% had more than 41 years of practicalexperience. In principle, these biodata indicate that the subject oftraditional healers in Ashanti region in Ghana is dominated by men,with a good formal school or university education, but also with along time of training on the job. When asking the respondents on amore detailed classification of their job, 54% classified themselves as“traditional healer”, 40% as “herbal practitioner”, and 6% as “herbaland divine healer”. Out of the 50 practioners, a majority of 48% hasthe traditional medicine as the sole way for regular income, 26%had an additional work or income as farmers, 10% as traders, 6% aspreachers, 6% as vocation and 4% as farmers and traders.

The study indicated that 84% of all practioners had no problemsin the collection of the plants. 80% of the practitioners identifiedtheir herbal raw material from long years of experience with therespective plants. Almost 16% of the respondents used otherfeatures and characteristics and 2% identified plants from theirspecific habitat.

All interview partners exclusively use plant based remedies forthe management of worm infections and do not add chemicallysynthesized antihelmintic drugs to the preparations.

3.2. Anthelmintic plants against human worm infections

During the field study 35 plant species from 28 genera, belong-ing to 25 families were recorded as remedies for treating hel-minthiasis in humans (Table 1).

Most recorded species (5 species/15%) were from the plantfamily Euphorbiaceae, followed by Rutaceae (4 species/12%). Theseplants are mostly applied by oral intake of a decoction. Also the oraluse of powdered material, suspended in water, juices or porridgeseems to be common. Some healers prefer enemas for the treat-ment of intestinal worms (Table 1). For skin associated-wormstopical and oral use of the plant extracts is described (Table 1).

Quantitative evaluation (Table 3) of the data from the mostcommonly used medicinal plant for helminth infections revealedCarica papaya (74% of all healers used this plant) as the “top hit”with the seeds being grinded and mixed with liquid and takenorally 2–3 times daily for 3 days. 16% of the healers used thedecoctions of the leaves from Senna alata mostly against intestinalworms, but in single cases also for Onchoceriasis. The use of leavedecoctions from Vernonia amygdalina was named with 14% againstintestinal worms, especially pinworms. 10% of the interview part-ners used leaves from Azadirachta indica against different kinds ofworms, the leaves from Combretum mucronatum, a plant speciesmonographed in the Ghana Herbal Pharmacopoeia for the treat-ment of infections with worms, and the leaves from Ananascomosus. Other plants listed by the healers did not have a verywide distribution within the healer community (Table 3).

In order to investigate whether these species are already knownplants for helminthiasis, cross-referencing was made to standardliterature by using SciFinders data base (Table 1). From the 35plants found in our survey only a minor amount is described inscientific literature for treating helminthiasis, with in many caseslow impact and very preliminary results. In addition, the surveyperformed within this study revealed 7 plants not described at alluntil now for anthelmintic activity (Brachyachne obtusiflora, Bryo-phyllum pinnatum, Citrus sinensis, Citrus limon, Euphorbia pulcher-rima, Gossypium arboreum and Mansonia altissima).

3.3. Anthelmintic plants against worm infections in animals

Despite the fact that worm infections of animals are in generala big problem in the daily live of rural people and farmers, not somany of the healers treat animal worm infections. This might be

due to the fact that animal treatment is rather delegated toveterinary healers, but this hypothesis has not been investigatedin our survey. From the 50 healers interviewed only 13 treatedanimals. Only six plant species were recorded as remedies fortreating helminthiasis in animals (Table 2). Again the seeds fromCarica papaya seemed to be the herbal material of the first choicefor treating animal helminth infections, while the other fivespecies named for this indication seem to be not very widely usedor well-known in this field.

In order to investigate whether these species are already knownplants for the treatment of helminthiasis, cross-referencing wasmade to standard literature (Table 3). From the six plants found inthis survey four are described in scientific literature for treatinghelminthiasis (Table 2). Two plants, namely Brachyachne obtusifloraand Mansonia altissima, have not been described at all until now forthis disease.

3.4. In vitro investigation of anthelmintic activity of plant extractsagainst. Caenorhabditis elegans

For a detailed functional investigation, nine plants were cho-sen (Table 3), either because of their frequency in the survey(leaf extracts of Azadirachta indica, Senna alata, Combretum mucro-natum and Vernonia amygdalina) or because little to nothing isdescribed about their constituents or their anthelmintic activity inpublished literature (leaves of Brachyachne obtusiflora, shoots ofBryophyllum pinnatum and Phyllanthus urinaria and the roots ofPaullinia pinnata and Musa x paradisiaca.). To our knowledge, withthe exception of Azadirachta indica, these plants have not beentested on Caenorhabditis elegans before. The leaves of Euphorbiapulcherrima are known to be moderately toxic causing gastro-intestinal disorders. Although we cannot be sure that the sameagents are responsible for the anthelmintic activity on the one sideand the toxicity on the other, this plant did not seem attractive forfurther investigations. The other species (Citrus, Gossypium) havealready been subject of extensive studies, since they are wellknown plants and frequently used for various purposes. Althoughmentioned by far most frequently, the seeds of Carica papaya werenot included in the bioassay, since the activity of this drug hasbeen well confirmed and bioactive compounds have been char-acterized (Kermanshai et al., 2001). The leaves of Ananas comosuswere not further investigated due to availability problems. Also,this drug has been tested in vitro before (Hordegen et al., 2003;Githiori et al., 2004) and did not exhibit any activity in either ofthe experiments.

Ethanol–water extracts were prepared from the respectiveparts of these plants and assayed in vitro for anthelmintic activityagainst the free-living nematode Caenorhabditis elegans usinglevamisole as a positive control. Due to genetic and morphologicalsimilarities Caenorhabditis elegans shares with other nematodes, itis a useful model for parasitic nematodes showing the advantageof easy and low cost laboratory maintenance (Bürglin et al., 1998;Katiki et al., 2011).

3.4.1. Plants with significant activity against Caenorhabditis elegansOut of the nine extracts tested, three showed significant

anthelmintic effects compared to the negative control (Fig. 2).The extract of Combretum mucronatum was most active with asurvival rate of 89.2% at 0.1 mg/mL and 58.1% at 1 mg/mL followedby Paullinia pinnata (85.2%) and Phyllanthus urinaria (89.2%) at1 mg/mL respectively. As expected, the positive control levamisolecaused the lowest survival rate of 16%.

There has only been a single report on confirmed nematocidaleffects for each of these three plants: a hydro-ethanolic extract(90%) of the leaves of Combretum mucronatum was found to be

C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263258

Table 1Medicinal plants used for the management of helminthiasis in humans in the Ashanti region, Ghana. Potential anthelmintic activity is cross-referenced to publishedliterature.

Plant family/Scientificname/Local name(Asante-Twi )

Type of worm infection Part of plant used Formulations Cross-reference

AmaranthaceaeAmaranthus spinosus

L. NkasεenkasεeHookworm, pinworm Leaves, whole plant,

seeds. Often incombination with lemon,ginger

Decoction as enema Chaudhary et al. (2012)

AmaryllidaceaeAllium sativum L.Gyene kankan

Pinworm Clove/ Bulb Decoction Singh et al. (2009); Ayaz et al. (2008);Kumar et al. (2005); Jeyathilakan et al.(2012); Ahmed et al. (2013

AsteraceaeAgeratum conyzoidesL. Guakuro

Intestinal worms Leaves Decoction de Melo et al. (2011); Nakajima et al.(2004; Okunade (2002).

Vernonia amygdalinaDelile, Awonwene

Pinworm, intestinalworms

Leaves Decoction Ademola and Eloff (2011a, 2011b);Iwalokun (2008); Rwangabo et al.(1986)

BromeliaceaeAnanas comosus (L.)

Merr. AborɔbεPinworm, intestinalworms

Leaves Mostly poultice, together with ginger;decoction

Kataki (2010); Hordegen et al. (2003)

CannabaceaeTrema orientalis (L.)Blume. Sesea

Hookworm, roundworm Leaves, bark Decoction McGaw et al., 2000

CaricaceaeCarica papaya L.

BorɔferεHookworm, pinworm,nematodes, lymphaticfilariasis, guinea worm

Mostly seeds; rarelyleaves

Grinded seed, applied often together withhoney, sugar solution, porridge. 10–20 mgtwice daily. Leaves as decoction

Okeniyi et al. (2007); Kermanshai et al.(2001)

CombretaceaeCombretummucronatumSchumach. & Thonn.

Hwerεmoo

Guinea worm, hookworm,pinworm

Leaves Decoction Koné et al. (2012)

CrassulaceaeBryophyllumpinnatum (Lam.)Oken, Tameawu

Guinea worm Aerial herbal material Poultice as a paste. For abdominal pain asdecoction.

CurcubitaceaeMomordica charantiaL. Nyanya

Pinworm, internal andexternal worms

Leaves Poultice with water or lemon Beloin et al. (2005); Grover and Yadav(2004)

EuphorbiaceaeAlchornea cordifolia(Schumach. & Thonn.)Müll.Arg. Gyama

Roundworm Leaves Decoction Koné et al. (2005); Okpekon et al.(2004)

Euphorbiapulcherrima Willd.exKlotzsch, Adankomilk

Intestinal worm Leaves Decoction

Jatropha curcas L.Nkrangyedua

Guinea worm Root, leaves Root: poultice together with salt; leaves:decoction

Monteiro et al. (2011); Rug and Ruppel(2000)

Mallotus oppositifolius(Geiseler) Müll.Arg.Nyanyanfrowa

Pinworm Leaves Poultice with lemon, enema Okpekon et al. (2004)

Phyllanthus urinaria L.Bɔwomaguwakyi

Skin-related worms Leaves Poultice, lemon added for topical use Nguyen et al. (2009)

FabaceaeAlbizia lebbeck (L.)Benth.

Tapeworm Bark Decoction Kumar et al. (2007)

Senna alata (L.) Roxb.

ƆsempεHookworm, roundworm,Nematodes

Leaves Decoction; for topical use (Onchoceriasis)poultice with shea butter

Kundu and Lyndem, 2013); Kundu et al.(2012)

Senna occidentalis (L.)

Link MmɔfraborɔdeεTapeworm Leaves, seeds Decoction Kundu & Lyndem, 2013); Eguale et al.

(2011).; Ademola and Eloff (2011a,2011b)

LamiaceaeOcimum gratissimumL. Nunum

Intestinal worms Leaves Decoction Fakae et al. (2000)

MalvaceaePinworm, roundworm Leaves Poultice with lemon

C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263 259

strongly active against the nematode Trichuris muris (minimumlethal concentration 10 mg/mL in vitro and a worm burden reduc-tion of 85.3% in vivo) and moderately active against Schistosomamansoni (Koné et al., 2012).

Activity of methanolic extracts from the leaves and roots ofPaullinia pinnata against the free- living nematode Rhabditis pseu-doelongata (EC50¼2.5 mg/mL) was reported by Okpekon et al. (2004).

Nguyen et al. (2009) tested a methanolic extract of the leaves ofPhyllanthus urinaria against the plant parasite Bursaphelenchusxylophilus at a concentration of 10 mg/mL, but despite a significant

reduction in the motility of the worms, no lethal effects could beobserved.

The efficacy of the screened plant material appears weak at firstsight when regarding the survival rates and screening at higherconcentrations might have revealed a greater number of activeplants. On the other hand, unselective effects by randomly highdoses of extracts were meant to be avoided by choosing theseconcentrations. Also the relatively low sensivity of Caenorhabditiselegans to drug treatment in general (Hu et al. 2013) should beconsidered, e.g. there is a 10-fold difference in the level of

Table 1 (continued )

Plant family/Scientificname/Local name(Asante-Twi )

Type of worm infection Part of plant used Formulations Cross-reference

Gossypium arboreumL. AsaawaMansonia altissima A.Chev. Oprono

Threadworm Stem Bark Decoction

MeliaceaeAzadirachta indica A.Juss. Dua gyene

Nematodes, urogenital-related worms

Leaves, rarely stem bark Decoction; Poultice as enema Atawodi and Atawodi (2009); Iqbalet al. (2010); Hordegen et al. (2003)

Khaya senegalensis(Desv.) A.Juss.Kuntunkuri

Hookworm, liver flukes,guinea worm

Stem bark Decoction; poultice for topical use Ndjonka et al. (2011); Ademola et al.(2004)

MoraceaeFicus exasperata Vahl,

NyankyerεneeSchistosomes Leaves, stem bark Poultice, mix with alcohol Faiyaz et al. (2012); Nweze Nwakaego

et al. (2013)

MoringaceaeMoringa oleifera Lam.Arzantuga (mole)

Roundworms, pinworms Seeds, leaves Poultice with honey or sugar solution forseeds; decoction for leaves

Abubakar et al. (2002)

MusaceaeMusa x paradisiaca L.Apem

Unripe exocarp:pinworms; roots: guineaworm

Unripe exocarp, roots Roots for topical use. Hussain et al. (2011)

PhyllantaceaePhyllanthus urinaria L.Bɔwomaguwakyi

Skin-related worms Leaves Poultice, lemon added for topical use Nguyen et al. (2009)

PoaceaeBrachyachneobtusiflora (Benth.) C.E. Hubb. Abirekyire

abɔdwesε

Intestinal worms Leaves Poultice

RubiaceaeSarcocephaluslatifolius (Sm.) E.A.Bruce, Kankanu orOwentin

Nematodes Root Decoction Fakae et al. (2000)

RutaceaeCitrus aurantiifolia(Christm.) Swingle,Ankaa

Pinworms, intestinalworms

Seeds Seeds are chewed after meal Taur et al. (2009)

Citrus limon (L.)

Osbeck, AnkaatwareεExternal and internalworms

Juice, fruits Juice mixed with warm water

Citrus sinensis (L.)Osbeck, Akutu

Skin related Bark Decoction

Harrisonia abyssinicaOliv. Sinamme

Intestinal worms Fruits For eating with meals; fry and grind Balde et al. (1989)

SapotaceaeVitellaria paradoxa C.F.Gaertn. Krenkun

Guinea worm, tapeworm Stem bark Decoction, 10–20 mL 3 � per day Koné et al. (2005)

XanthorrhoeaceaeAloe vera (L.)Burm.f.Aloe vera

Intestinal worms Leaves Poultice with water Maphosa and Masika (2012); Maphosaet al. (2010).

ZingiberaceaeZingiber officinaleRoscoe, Akekaduro

Pinworm, intestinalworms

Rhizoms Fruits Enema Lin et al. (2010); Adewunmi et al.(1990); Mostafa et al. (2011)

C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263260

levamisole experimentally used for Caenorhabditis elegans com-pared to the therapeutical use against parasitic nematodes.

3.4.2. Plants showing little to no activityTo our surprise, the extracts of Azadirachta indica, Cassia alata

and Vernonia amygdalina had only little effects at 1 mg/mL (0.6%,0.6% and 1.7% mortality respectively), although being among theplants most commonly used against helminth infections in thissurvey.

Anthelmintic effects of Senna alata are mainly described fortapeworm infections (Kundu et al. 2012; 2013), but Ademola andEloff (2011a) report ovicidal and larvicidal effects in vitro againstthe nematode Haemonchus contortus.

Despite a widespread traditional use of various plant parts ofAzadirachta indica, in vitro and in vivo investigations lead to differentresults (reviewed by Atawodi and Atawodi (2009)). Akhtar (2000)

reports nematotoxic properties of aqueous leaf extracts of Azadir-achta indica, whereas Sharma et al. (2003) found an activity ofAzadirachtins A, B and H isolated from methanolic extracts ofdefatted seed kernels on Caenorhabditis elegans. However, theadministration of a methanolic extract and crude seed powder onlyhad minor effects in sheep infected with Trichostrongylus species(Iqbal et al., 2010).

A similar picture can be drawn for the leaf extract of Vernoniaamygdalina: butanol and methanol (35%) extracts were found to beactive against eggs and larvae of Haemonchus. contortus respectively(Ademola and Eloff, 2011b). On the other hand, aqueous extractscould not inhibit egg hatching of Haemonchus contortus eggs in vitro(Alawa et al., 2003). And despite leading to a significant wormburden reduction in puppies (Adedapo et al., 2007), it remainsunclear whether this effect can be addressed to an anthelminticactivity or rather to a faster expulsion of worms by an increase ofthe intestinal motility (Awe et al., 1999; Adedapo et al., 2007).

Table 2Medicinal plants used for the management of helminthiasis in animals in the Ashanti region, Ghana. Documented antihelmintic activity is cross-referenced to publishedliterature.

Plant Family/Scientific name/Localname (Asante-Twi )

Type of worminfection

Part of plant used Formulations Cross-reference, remarks

CaricaceaeCarica papaya L. Borɔferɛ Intestinal worms

for dogs, pigsMostly seeds; rarelyleaves, fruits

Grinded seeds or whole fruits infood; leaves as poultice.

Okeniyi et al. (2007); Kermanshai et al.(2001)

EuphorbiaceaeAlchornea cordifolia (Schum. & Thonn.)Müll.Arg.

Roundworm Leaves Decoction Koné et al. (2005); Okpekon et al. (2004)

MalvaceaeMansonia altissima A. Chev. Oprono Threadworm Stem bark Decoction

PoaceaeBrachyachne obtusiflora (Benth.) C.E.Hubb. Abirekyire abɔdwesε

Intestinal worms Leaves Wash with salty water and allow tochew

RubiaceaeMorinda lucida Benth. Ɔkonkroma Pinworm Stem bark Decoction Hounzangbe-Adote et al. (2005a, 2005b);

Hounzangbe-Adote et al.

SapindaceaePaullinia pinnata L. Toa ntini Hookworm Root Decoction Okpekon et al. (2004)

Table 3Quantitative evaluation of plants used in the management of helminthiasis of humans and animals. Data are related to the information from 50 informants treating humansand 13 healers treating additionally animals. Species in bold were selected for further in vitro investigations.

Plant species used for human therapy Plant species used for animaltreatment

Plant species Frequency Plant species Frequency

Carica papaya 37 Carica papaya 6Senna alata 8 Alchornea cordifolia 1Vernonia amygdalina 7 Brachyachne obtusiflora 1Azadirachta indica 5 Morinda lucida 1Combretum mucronatum 5 Mansonia altissima 1Ananas comosus 5 Paullinia pinnata 1Ageratum conyzoides 3Jatropha curcas 3Moringa oleifera 3Zingiber officinale 3Amaranthus spinosus 2Citrus aurantiifolia, Citrus limon Each 2Khaya senegalensis 2Momordica charantia 2Musa x paradisiaca 2Single recommendation by healers: Albizia lebbeck, Alchornea cordifolia, Allium sativum, Aloe vera, Brachyachne obtusiflora,Bryophyllum pinnatum, Senna occidentalis, Citrus sinensis, Euphorbia pulcherrima, Ficus exasperata, Gossypium arboreum,Harrisonia abyssinica, Mallotus oppositifolius, Nauclea latifolia, Ocimum gratissimum, Phyllanthus urinaria, Trema orientalis,Vitellaria paradoxa.

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No significant mortality was found for Musa x paradisiaca(1.1% at 1 mg/mL) and no effect at all could be observed for theextracts of Brachyachne obtusiflora and Bryophyllum pinnatum.

The variations in the outcome of the in vitro tests could beexplained at least to some extent by the use of different solventsfor the extraction, but of course also by the test system.

It should be noted again that this result only refers to anactivity against the soil-living nematode Caenorhabditis elegans,and does not proof an inefficiency of the traditional treatment ofthe respective helminth diseases. Further investigations on differ-ent parasitic species would therefore be desirable where notconducted yet.

4. Conclusion

An ethnopharmacological survey among traditional healers inthe Ashanti region in Ghana revealed seven plants that have notbeen described previously for their anthelmintic properties. Out ofthe nine plants selected for in vitro screening, extracts fromCombretum mucronatum, Paullinia pinnata and Phyllanthus urinariashowed highly significant activity against Caenorhabditis elegans.These findings underline the value of ethnopharmacologicalapproaches for discovering new remedies and a validation of thetraditional use by providing in vitro confirmation of the reportedactivity. Phytochemical investigations to reveal the responsiblecompounds for the activity of the plant material are currentlyongoing.

Acknowledgments

We are grateful to Mr. John Ampomah of Ghana Herbariumand Department of Botany, University of Ghana, Accra, Ghana andMr. Eric Gyebi, Jachie, Ashanti region, Ghana for the collection ofthe plant materials for further investigations. The cooperationwith the traditional healers, herbalists and regional executivesand members of the Ghana National Association of TraditionalHealers (GNATH) in Ashanti region of Ghana in the survey isacknowledged.

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