Journal of Ethnopharmacology 158 (2014) 255–263

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An ethnopharmacological survey and in vitro confirmation of the ethnopharmacological use of medicinal plants as anthelmintic remedies 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, Ghana Department of Botany, University of Ghana, Legon, Ghana University of Münster, Institute for Pharmaceutical Biology and Phytochemistry,Corrensstraße 48, D-48149 Münster, Germany d Institute for Zoophysiology, Schlossplatz 8, D-48143 Münster, Germany b c

art ic l e i nf o

a b s t r a c t

Article history: Received 31 March 2014 Received in revised form 15 October 2014 Accepted 17 October 2014 Available online 29 October 2014

Ethnopharmacological relevance: Infections with helminths are still a big problem in many parts of the world. The majority of the people in West Africa treat such infections with medicinal plants related to the local traditional medicine. The present study aims at identifying medicinal plants traditionally used for worm infections in the Ashanti region, Ghana. In vitro screening of selected extracts from plants on which scientific knowledge is limited was to be performed. Materials and methods: Validated questionnaires were administered to 50 traditional healers in the Ashanti region, Ghana. Interviews and structured conversations were used to obtain relevant information. Quantitative and qualitative evaluation was performed additionally to structured cross-referencing of the data using SciFinders data base. Selected plant species were used for in vitro testing on anthelmintic 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 material most frequently used were the seeds from Carica papaya, mentioned by nearly all healers. The plausibility of most plants used for treatment of infections with helminths was given in most cases by documentation of potential anthelmintic activity in recent scientific literature. 9 species from plants not or scarcely described in literature for this indication were investigated on in vitro activity. A hydroethanolic (1:1) extract of Combretum mucronatum was most active with a survival rate of nematodes of 89% at 0.1 mg/mL and 58% at 1 mg/mL respectively (levamisole 16%). Extracts of Paullinia pinnata 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 crossreferencing with published literature and phytochemical/pharmacological plausibility.The in vitro investigations of extracts from Combretum mucronatum, Paullinia pinnata and Phyllanthus urinaria exhibited significant effects against nematodes. The anthelmintic activity of these plants should be investigated in detail for pinpointing the respective lead structures responsible for the activity. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Anthelmintic Caenorhabditis elegans Combretum mucronatum Paullinia pinnata Phyllanthus urinaria

1. Introduction Infections with parasitic nematodes, including intestinal helminths and filarial worms, are among the most widespread diseases worldwide with a global prevalence of more than 2 billion (Hotez

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

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.: þ 49 251 8333380; fax: þ 49 251 8338341. E-mail address: [email protected] (A. Hensel). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2014.10.029 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

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helminthiases in humans play a minor role in Europe, they occur throughout the tropical and subtropical regions of Sub-Saharan Africa, the Americas and Asia, imposing a great burden mainly on inhabitants of developing areas (Hotez et al., 2008; Lustigman et al., 2012). Intestinal helminthiases are most commonly caused by roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura) and hookworms (Ancylostoma duodenale and Necator americanus) (Lustigman et al., 2012; WHO, 2013a) although coinfestations with more than one parasite are rather normal (Bethony et al., 2006). Although not lethal, infections can cause symptoms ranging from abdominal pain and diarrhea to more severe impairments such as anemia as well as growth and cognitive retardation in children due to blood loss and malnutrition (Hotez et al., 2008; WHO, 2013a) depending on the type of nematode and the worm burden. Since in most cases people affected by helminthiases face poverty accompanied by poor sanitary conditions and limited access to medication these diseases hinders them from socioeconomic development, leading to a vicious circle (Lustigman et al., 2012). In the recent past considerable efforts have been made by the WHO to tackle the currently 17 neglected tropical diseases, including guidelines for preventive chemotherapy in helminthiasis and recommendations for treatment and donations of anthelmintic medicines (WHO, 2013a). The set up of mass drug administration (MDA) programs to treat inhabitants in endemic areas with anthelmintics given annually or biannually has led to a significant success in the reduction of morbidity. While the WHO (2013b) currently lists four anthelmintics, namely albendazole, mebendazole, levamisole and pyrantel pamoate as essential drugs in most cases benzimidazoles, and in particular albendazole, is administered due to its low costs and broad spectrum of activity (for review see Humphries et al. (2012)). Still, great differences in the susceptibility among STH species and also in the over-all success of the anthelmintic treatment could be observed, which raises the question on the reliability of MDA, particularly with respect to development of benzimidazole, levamisole and pyrantel resistant helminth strains. Also adverse effects that occur in quantity during deworming of whole populations should be considered (for review see Keiser and Utzinger (2008); Vercruysse et al. (2011) and Humphries et al. (2012); ). Finally, these programs focus on the most commonly occurring helminthiases, neglecting millions of infections with less prominent infectious agents, such as Strongyloides, of which little is known about its epidemiology and the success of MDA measures (for review see Olsen et al. (2009)).

Map of Ghana

Instead of conventional drugs, many African patients (almost 70% of the people) rely on remedies of traditional healers and herbal practitioners (Agyare et al., 2009). Therefore, it is not surprising that plants or plant products have also been successfully used in the treatment of filarial (Ndjonka et al., 2011, 2013) and intestinal parasites (Waterman et al., 2010; Koné et al., 2012) and are also a considerable resource for nematode control in livestock and agriculture (Chitwood, 2002). Furthermore, Fabricant and Farnsworth (2001) not only underline the importance to confirm the traditional use of herbal remedies by in vitro investigations, but also highlight the potential of ethnopharmacological approaches towards the discovery of new lead compounds that are certainly awaited. In this study we aim to identify herbal remedies that are traditionally used to treat helminth infections in a defined area of Ghana and to confirm the efficacy of the most promising candidates revealed in the field study in a bioassay. By this approach we intend to support the ethnopharmacological use by in vitro data and to identify previously undescribed herbal drugs for this indication that could be the base for further phytochemical and pharmacological research.

2. Material and methods 2.1. Study area and survey The ethnopharmacological survey was performed in Ashanti region in the central part of Ghana, located between 0.15–2.251W and 5.50–7.461N (Fig. 1). The region shares boundaries with four of the 10 political regions of Ghana. The region covers a total land area of 24,389 km2, representing 10.2% of the total land area of Ghana. The ethnopharmacological survey was carried out from October 2012 to February 2013 in accordance with the national rights of Ghana and with acceptance and in close co-operation with Ghana National Association of Traditional Healers (GNATH). A house to house visitation strategy was employed for interviewing the practioners. All participants were informed about the survey and personal visits were made to their facilities, centers and homes. In respect to the local tradition, some gifts in cash or kinds were given. Interviews and conversations were used to administer the questionnaires. Questionnaires were designed in English and administered to 50

Districts in Ashanti region

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

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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), Senna alata (L.) Roxb. (No. 302), Combretum mucronatum Schumach. & Thonn. (No.305), Vernonia amygdalina Delile (No. 298), Bryophyllum 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 have been deposited in the herbarium of the Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Germany. All plant materials were collected between November 2013 and January 2014 from the Bosomtwi area and identified botanically. 2.3. Preparation of plant extracts A standard protocol for the preparation of the extracts used 20 g of the powdered plant material, which was extracted in 200 mL of ethanol/water (50:50 v/v) by Ultra-Turraxs (IKA, Staufen, Germany) for 10 min at 9500 rpm under ice cooling. For technical reasons (strong floating during the extraction and insufficient sedimentation during centrifugation) the standard extraction protocol had to be changed for the preparation of the extracts from the leaves of Brachyachne obtusiflora and the root of Musa x paradisiaca: these plant materials were extracted with different plant-solvent ratios (Brachyachne obtusiflora 15 g in 350 mL solventand Musa x paradisiaca 7 g in 200 mL solvent. The extracts were centrifuged (3000g, 10 min) and the supernatant was filtered (595 qualitative filter paper, S&S Dassel, Germany) to obtain a clear solution. The supernatant was concentrated by vacuum evaporation not exceeding 40 1C, lyophilized and the powder was stored under vacuum at room temperature. 2.4. Anthelmintic assay 2.4.1. Monoxenic and axenic maintenance of Caenorhabditis elegans Cultures 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 mL H2O) supplemented with 800 mL of Escherichia coli OP50 strain as a food source (Brenner, 1974). Age synchronous cultures were obtained by the alkaline bleaching method described by Ndjonka et al. (2011): worms were rinsed from the petridish with M9 buffer solution (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 0.25 g MgSO4  7H2O in 1 L of water) (Brenner, 1974), centrifuged at 2000g for 1 min and treated with alkaline solution (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 the bleaching procedure and after three washing steps with M9 buffer solution they can be seeded onto plates or into liquid medium. For the in vitro assay, synchronous cultures of Caenorhabditis elegans were initiated in axenic liquid medium to avoid interfering effects of the bacteria with the test substances. The liquid medium was composed of 3.0 g Bacto™ yeast extract (Becton-Dickinson, Heidelberg, Germany), 3.0 g soy peptone from casein (Sigma-Adrich, Steinheim, Germany), 1.0 g dextrose, and 0.25 mL cholesterol solution (5 mg / 1.0 mL in ethanol) in 100 mL

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of H2O and autoclaved. To start the axenic culture, synchronous worm cultures were seeded into the axenic medium supplemented with 0.05% hemoglobin (stock solution: 5% (w/v) of bovine hemoglobin (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 screening Stock 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. The final concentration of DMSO did not exceed 1% (v/v). Aliquots of the stock solution were added to a 24-well microtiter plate containing filter-sterilized “enriched” M9 buffer solution (50 mL 20% (w/v) dextrose solution, 500 mL of a solution from cholesterol 5 mg/mL in ethanol, 500 mL 1 M CaCl2, 500 mL 1 M MgSO4, 12.5 mL 1 M KH2PO4/K2HPO4 and 500 mL penicillin/streptomycin solution (10,000 U/10,000 mg/mL) in 500 mL M9 buffer solution) to a final volume of 500 mL per well resulting in final concentrations of 0.1 mg/mL and 1 mg/mL. Each extract was tested in 4 replicates per treatment and each experiment was independently performed in triplicate. A solution of levamisole hydrochloride (40 mM) served as a positive control, DMSO 1% (v/v) was used as a negative control. One to two microliters of the liquid medium, containing approximately 10–20 worms (L4 larvae or young adults) were transferred into each well and the plate was incubated at 20 1C. After 72 h mortality was assessed by counting the number of dead worms under a dissecting microscope: worms that were immotile and completely straight were counted as dead if they did not respond when hit with an eyelash. The percentage of dead worms was calculated as the number of dead worms in relation to the total number of worms per well. 2.4.3. Statistical analysis Data 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-way ANOVA test followed by a Dunnett Multiple Comparison Test versus the negative control. po0.05 was considered to be significant and po0.01 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 medicinal plants for the management of helminthiasis was carried out in different districts of Ashanti region, Ghana, as indicated in Fig. 1. During the personalized and structured interviews differentiation was made for plant species used for treatment of human helminthiasis and worm infections in animals. 50 professional healers were recruited with the help of the Department of Herbal Medicine and GNATH. 72% were men and 28% were women. This gender relation is in good accordance with a recent field study in Ghana on traditional healers (Agyare et al., 2009). Professional healers were mainly (4 90%) older than 40 years: 2% were between 21 and 30 years, 8% between 31 and 40 years, 58% between 41 and 60 years and 28% were older than 61 years. This clearly shows that traditional healing is mostly related to older, more experienced practitioners. It was also interesting to find that most respondents had higher education: 10% hold a university degree, 14% completed senior high school, 42% passed middle and junior high school education, 30% primary school and 4% were illiterate. Considering

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the duration of practice of the healers, only 22% had less than 11 years of experience. 54% worked for 11–20 years in this profession, 18% for 21–40 years and 6% had more than 41 years of practical experience. In principle, these biodata indicate that the subject of traditional healers in Ashanti region in Ghana is dominated by men, with a good formal school or university education, but also with a long time of training on the job. When asking the respondents on a more detailed classification of their job, 54% classified themselves as “traditional healer”, 40% as “herbal practitioner”, and 6% as “herbal and divine healer”. Out of the 50 practioners, a majority of 48% has the traditional medicine as the sole way for regular income, 26% had an additional work or income as farmers, 10% as traders, 6% as preachers, 6% as vocation and 4% as farmers and traders. The study indicated that 84% of all practioners had no problems in the collection of the plants. 80% of the practitioners identified their herbal raw material from long years of experience with the respective plants. Almost 16% of the respondents used other features and characteristics and 2% identified plants from their specific habitat. All interview partners exclusively use plant based remedies for the management of worm infections and do not add chemically synthesized antihelmintic drugs to the preparations. 3.2. Anthelmintic plants against human worm infections During the field study 35 plant species from 28 genera, belonging to 25 families were recorded as remedies for treating helminthiasis in humans (Table 1). Most recorded species (5 species/15%) were from the plant family Euphorbiaceae, followed by Rutaceae (4 species/12%). These plants are mostly applied by oral intake of a decoction. Also the oral use of powdered material, suspended in water, juices or porridge seems to be common. Some healers prefer enemas for the treatment of intestinal worms (Table 1). For skin associated-worms topical and oral use of the plant extracts is described (Table 1). Quantitative evaluation (Table 3) of the data from the most commonly used medicinal plant for helminth infections revealed Carica papaya (74% of all healers used this plant) as the “top hit” with the seeds being grinded and mixed with liquid and taken orally 2–3 times daily for 3 days. 16% of the healers used the decoctions of the leaves from Senna alata mostly against intestinal worms, but in single cases also for Onchoceriasis. The use of leave decoctions from Vernonia amygdalina was named with 14% against intestinal worms, especially pinworms. 10% of the interview partners used leaves from Azadirachta indica against different kinds of worms, the leaves from Combretum mucronatum, a plant species monographed in the Ghana Herbal Pharmacopoeia for the treatment of infections with worms, and the leaves from Ananas comosus. Other plants listed by the healers did not have a very wide distribution within the healer community (Table 3). In order to investigate whether these species are already known plants for helminthiasis, cross-referencing was made to standard literature by using SciFinders data base (Table 1). From the 35 plants found in our survey only a minor amount is described in scientific literature for treating helminthiasis, with in many cases low impact and very preliminary results. In addition, the survey performed within this study revealed 7 plants not described at all until now for anthelmintic activity (Brachyachne obtusiflora, Bryophyllum pinnatum, Citrus sinensis, Citrus limon, Euphorbia pulcherrima, Gossypium arboreum and Mansonia altissima). 3.3. Anthelmintic plants against worm infections in animals Despite the fact that worm infections of animals are in general a big problem in the daily live of rural people and farmers, not so many of the healers treat animal worm infections. This might be

due to the fact that animal treatment is rather delegated to veterinary healers, but this hypothesis has not been investigated in our survey. From the 50 healers interviewed only 13 treated animals. Only six plant species were recorded as remedies for treating helminthiasis in animals (Table 2). Again the seeds from Carica papaya seemed to be the herbal material of the first choice for treating animal helminth infections, while the other five species named for this indication seem to be not very widely used or well-known in this field. In order to investigate whether these species are already known plants for the treatment of helminthiasis, cross-referencing was made to standard literature (Table 3). From the six plants found in this survey four are described in scientific literature for treating helminthiasis (Table 2). Two plants, namely Brachyachne obtusiflora and Mansonia altissima, have not been described at all until now for this disease. 3.4. In vitro investigation of anthelmintic activity of plant extracts against. Caenorhabditis elegans For a detailed functional investigation, nine plants were chosen (Table 3), either because of their frequency in the survey (leaf extracts of Azadirachta indica, Senna alata, Combretum mucronatum and Vernonia amygdalina) or because little to nothing is described about their constituents or their anthelmintic activity in published literature (leaves of Brachyachne obtusiflora, shoots of Bryophyllum pinnatum and Phyllanthus urinaria and the roots of Paullinia pinnata and Musa x paradisiaca.). To our knowledge, with the exception of Azadirachta indica, these plants have not been tested on Caenorhabditis elegans before. The leaves of Euphorbia pulcherrima are known to be moderately toxic causing gastrointestinal disorders. Although we cannot be sure that the same agents are responsible for the anthelmintic activity on the one side and the toxicity on the other, this plant did not seem attractive for further investigations. The other species (Citrus, Gossypium) have already been subject of extensive studies, since they are well known plants and frequently used for various purposes. Although mentioned by far most frequently, the seeds of Carica papaya were not included in the bioassay, since the activity of this drug has been well confirmed and bioactive compounds have been characterized (Kermanshai et al., 2001). The leaves of Ananas comosus were 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 of the experiments. Ethanol–water extracts were prepared from the respective parts of these plants and assayed in vitro for anthelmintic activity against the free-living nematode Caenorhabditis elegans using levamisole as a positive control. Due to genetic and morphological similarities Caenorhabditis elegans shares with other nematodes, it is a useful model for parasitic nematodes showing the advantage of easy and low cost laboratory maintenance (Bürglin et al., 1998; Katiki et al., 2011). 3.4.1. Plants with significant activity against Caenorhabditis elegans Out 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 a survival rate of 89.2% at 0.1 mg/mL and 58.1% at 1 mg/mL followed by Paullinia pinnata (85.2%) and Phyllanthus urinaria (89.2%) at 1 mg/mL respectively. As expected, the positive control levamisole caused the lowest survival rate of 16%. There has only been a single report on confirmed nematocidal effects for each of these three plants: a hydro-ethanolic extract (90%) of the leaves of Combretum mucronatum was found to be

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Table 1 Medicinal plants used for the management of helminthiasis in humans in the Ashanti region, Ghana. Potential anthelmintic activity is cross-referenced to published literature. Plant family/Scientific name/Local name (Asante-Twi ) Amaranthaceae Amaranthus spinosus

Type of worm infection

Part of plant used

Formulations

Cross-reference

Hookworm, pinworm

Leaves, whole plant, seeds. Often in combination with lemon, ginger

Decoction as enema

Chaudhary et al. (2012)

Pinworm

Clove/ Bulb

Decoction

Singh et al. (2009); Ayaz et al. (2008); Kumar et al. (2005); Jeyathilakan et al. (2012); Ahmed et al. (2013

Intestinal worms

Leaves

Decoction

Pinworm, intestinal worms

Leaves

Decoction

de Melo et al. (2011); Nakajima et al. (2004; Okunade (2002). Ademola and Eloff (2011a, 2011b); Iwalokun (2008); Rwangabo et al. (1986)

Pinworm, intestinal worms

Leaves

Mostly poultice, together with ginger; decoction

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

Hookworm, roundworm

Leaves, bark

Decoction

McGaw et al., 2000

Hookworm, pinworm, nematodes, lymphatic filariasis, guinea worm

Mostly seeds; rarely leaves

Grinded seed, applied often together with honey, sugar solution, porridge. 10–20 mg twice daily. Leaves as decoction

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

Guinea worm, hookworm, Leaves pinworm

Decoction

Koné et al. (2012)

Guinea worm

Aerial herbal material

Poultice as a paste. For abdominal pain as decoction.

Pinworm, internal and external worms

Leaves

Poultice with water or lemon

Beloin et al. (2005); Grover and Yadav (2004)

Roundworm

Leaves

Decoction

Koné et al. (2005); Okpekon et al. (2004)

Intestinal worm

Leaves

Decoction

Guinea worm

Root, leaves

Pinworm

Leaves

Root: poultice together with salt; leaves: decoction Poultice with lemon, enema

Monteiro et al. (2011); Rug and Ruppel (2000) Okpekon et al. (2004)

Skin-related worms

Leaves

Poultice, lemon added for topical use

Nguyen et al. (2009)

Tapeworm

Bark

Decoction

Kumar et al. (2007)

Hookworm, roundworm, Nematodes

Leaves

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

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

Tapeworm

Leaves, seeds

Decoction

Kundu & Lyndem, 2013); Eguale et al. (2011).; Ademola and Eloff (2011a, 2011b)

Intestinal worms

Leaves

Decoction

Fakae et al. (2000)

Pinworm, roundworm

Leaves

Poultice with lemon

L. Nkasεenkasεe

Amaryllidaceae Allium sativum L. Gyene kankan Asteraceae Ageratum conyzoides L. Guakuro Vernonia amygdalina Delile, Awonwene Bromeliaceae Ananas comosus (L.) Merr. Aborɔbε Cannabaceae Trema orientalis (L.) Blume. Sesea Caricaceae Carica papaya L. Borɔferε Combretaceae Combretum mucronatum Schumach. & Thonn. Hwerεmoo Crassulaceae Bryophyllum pinnatum (Lam.) Oken, Tameawu Curcubitaceae Momordica charantia L. Nyanya Euphorbiaceae Alchornea cordifolia (Schumach. & Thonn.) Müll.Arg. Gyama Euphorbia pulcherrima Willd.ex Klotzsch, Adanko milk Jatropha curcas L. Nkrangyedua Mallotus oppositifolius (Geiseler) Müll.Arg. Nyanyanfrowa Phyllanthus urinaria L. Bɔwomaguwakyi Fabaceae Albizia lebbeck (L.) Benth. Senna alata (L.) Roxb. Ɔsempε Senna occidentalis (L.) Link Mmɔfraborɔdeε Lamiaceae Ocimum gratissimum L. Nunum Malvaceae

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Table 1 (continued ) Type of worm infection

Part of plant used

Formulations

Threadworm

Stem Bark

Decoction

Nematodes, urogenitalrelated worms Hookworm, liver flukes, guinea worm

Leaves, rarely stem bark

Decoction; Poultice as enema

Stem bark

Decoction; poultice for topical use

Leaves, stem bark

Poultice, mix with alcohol

Faiyaz et al. (2012); Nweze Nwakaego et al. (2013)

Roundworms, pinworms

Seeds, leaves

Poultice with honey or sugar solution for seeds; decoction for leaves

Abubakar et al. (2002)

Unripe exocarp: pinworms; roots: guinea worm

Unripe exocarp, roots

Roots for topical use.

Hussain et al. (2011)

Leaves

Poultice, lemon added for topical use

Nguyen et al. (2009)

Intestinal worms

Leaves

Poultice

Nematodes

Root

Decoction

Fakae et al. (2000)

Pinworms, intestinal worms

Seeds

Seeds are chewed after meal

Taur et al. (2009)

External and internal worms

Juice, fruits

Juice mixed with warm water

Skin related

Bark

Decoction

Intestinal worms

Fruits

For eating with meals; fry and grind

Balde et al. (1989)

Sapotaceae Vitellaria paradoxa C. F.Gaertn. Krenkun

Guinea worm, tapeworm

Stem bark

Decoction, 10–20 mL 3  per day

Koné et al. (2005)

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

Intestinal worms

Leaves

Poultice with water

Maphosa and Masika (2012); Maphosa et al. (2010).

Pinworm, intestinal worms

Rhizoms Fruits

Enema

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

Plant family/Scientific name/Local name (Asante-Twi ) Gossypium arboreum L. Asaawa Mansonia altissima A. Chev. Oprono Meliaceae Azadirachta indica A. Juss. Dua gyene Khaya senegalensis (Desv.) A.Juss. Kuntunkuri

Moraceae Ficus exasperata Vahl, Schistosomes Nyankyerεnee Moringaceae Moringa oleifera Lam. Arzantuga (mole) Musaceae Musa x paradisiaca L. Apem

Phyllantaceae Phyllanthus urinaria L. Skin-related worms Bɔwomaguwakyi Poaceae Brachyachne obtusiflora (Benth.) C. E. Hubb. Abirekyire

Cross-reference

Atawodi and Atawodi (2009); Iqbal et al. (2010); Hordegen et al. (2003) Ndjonka et al. (2011); Ademola et al. (2004)

abɔdwesε Rubiaceae Sarcocephalus latifolius (Sm.) E.A. Bruce, Kankanu or Owentin Rutaceae Citrus aurantiifolia (Christm.) Swingle, Ankaa Citrus limon (L.) Osbeck, Ankaatwareε Citrus sinensis (L.) Osbeck, Akutu Harrisonia abyssinica Oliv. Sinamme

Zingiberaceae Zingiber officinale Roscoe, Akekaduro

strongly active against the nematode Trichuris muris (minimum lethal concentration 10 mg/mL in vitro and a worm burden reduction of 85.3% in vivo) and moderately active against Schistosoma mansoni (Koné et al., 2012). Activity of methanolic extracts from the leaves and roots of Paullinia pinnata against the free- living nematode Rhabditis pseudoelongata (EC50 ¼2.5 mg/mL) was reported by Okpekon et al. (2004). Nguyen et al. (2009) tested a methanolic extract of the leaves of Phyllanthus urinaria against the plant parasite Bursaphelenchus xylophilus at a concentration of 10 mg/mL, but despite a significant

reduction in the motility of the worms, no lethal effects could be observed. The efficacy of the screened plant material appears weak at first sight when regarding the survival rates and screening at higher concentrations might have revealed a greater number of active plants. On the other hand, unselective effects by randomly high doses of extracts were meant to be avoided by choosing these concentrations. Also the relatively low sensivity of Caenorhabditis elegans to drug treatment in general (Hu et al. 2013) should be considered, e.g. there is a 10-fold difference in the level of

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261

Table 2 Medicinal plants used for the management of helminthiasis in animals in the Ashanti region, Ghana. Documented antihelmintic activity is cross-referenced to published literature. Plant Family/Scientific name/Local name (Asante-Twi )

Type of worm infection

Part of plant used

Formulations

Cross-reference, remarks

Intestinal worms for dogs, pigs

Mostly seeds; rarely leaves, fruits

Grinded seeds or whole fruits in food; leaves as poultice.

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

Leaves

Decoction

Koné et al. (2005); Okpekon et al. (2004)

Threadworm

Stem bark

Decoction

Intestinal worms

Leaves

Wash with salty water and allow to chew

Rubiaceae Morinda lucida Benth. Ɔkonkroma

Pinworm

Stem bark

Decoction

Hounzangbe-Adote et al. (2005a, 2005b); Hounzangbe-Adote et al.

Sapindaceae Paullinia pinnata L. Toa ntini

Hookworm

Root

Decoction

Okpekon et al. (2004)

Caricaceae Carica papaya L. Borɔferɛ

Euphorbiaceae Alchornea cordifolia (Schum. & Thonn.) Roundworm Müll.Arg. Malvaceae Mansonia altissima A. Chev. Oprono Poaceae Brachyachne obtusiflora (Benth.) C.E. Hubb. Abirekyire abɔdwesε

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

Plant species

Plant species used for animal treatment Frequency

Carica papaya 37 Senna alata 8 Vernonia amygdalina 7 Azadirachta indica 5 Combretum mucronatum 5 Ananas comosus 5 Ageratum conyzoides 3 Jatropha curcas 3 Moringa oleifera 3 Zingiber officinale 3 Amaranthus spinosus 2 Citrus aurantiifolia, Citrus limon Each 2 Khaya senegalensis 2 Momordica charantia 2 Musa x paradisiaca 2 Single 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.

levamisole experimentally used for Caenorhabditis elegans compared to the therapeutical use against parasitic nematodes.

3.4.2. Plants showing little to no activity To 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 the plants most commonly used against helminth infections in this survey. Anthelmintic effects of Senna alata are mainly described for tapeworm infections (Kundu et al. 2012; 2013), but Ademola and Eloff (2011a) report ovicidal and larvicidal effects in vitro against the nematode Haemonchus contortus. Despite a widespread traditional use of various plant parts of Azadirachta indica, in vitro and in vivo investigations lead to different results (reviewed by Atawodi and Atawodi (2009)). Akhtar (2000)

Plant species

Frequency

Carica papaya Alchornea cordifolia Brachyachne obtusiflora Morinda lucida Mansonia altissima Paullinia pinnata

6 1 1 1 1 1

reports nematotoxic properties of aqueous leaf extracts of Azadirachta indica, whereas Sharma et al. (2003) found an activity of Azadirachtins A, B and H isolated from methanolic extracts of defatted seed kernels on Caenorhabditis elegans. However, the administration of a methanolic extract and crude seed powder only had minor effects in sheep infected with Trichostrongylus species (Iqbal et al., 2010). A similar picture can be drawn for the leaf extract of Vernonia amygdalina: butanol and methanol (35%) extracts were found to be active against eggs and larvae of Haemonchus. contortus respectively (Ademola and Eloff, 2011b). On the other hand, aqueous extracts could not inhibit egg hatching of Haemonchus contortus eggs in vitro (Alawa et al., 2003). And despite leading to a significant worm burden reduction in puppies (Adedapo et al., 2007), it remains unclear whether this effect can be addressed to an anthelmintic activity or rather to a faster expulsion of worms by an increase of the intestinal motility (Awe et al., 1999; Adedapo et al., 2007).

262

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Fig. 2. Influence of ethanol–water (50%) extracts on the survival of the free-living nematode Caenorhabditis elegans at 0.1 and 1 mg/mL after 72 h incubation time. Untreated Caenorhabditis elegans in medium containing 1% DMSO served as negative control (NC); levamisole HCl (40 mM) served as positive control (PC); **p o 0.01 compared to NC.

No significant mortality was found for Musa x paradisiaca (1.1% at 1 mg/mL) and no effect at all could be observed for the extracts of Brachyachne obtusiflora and Bryophyllum pinnatum. The variations in the outcome of the in vitro tests could be explained at least to some extent by the use of different solvents for the extraction, but of course also by the test system. It should be noted again that this result only refers to an activity against the soil-living nematode Caenorhabditis elegans, and does not proof an inefficiency of the traditional treatment of the respective helminth diseases. Further investigations on different parasitic species would therefore be desirable where not conducted yet.

4. Conclusion An ethnopharmacological survey among traditional healers in the Ashanti region in Ghana revealed seven plants that have not been described previously for their anthelmintic properties. Out of the nine plants selected for in vitro screening, extracts from Combretum mucronatum, Paullinia pinnata and Phyllanthus urinaria showed highly significant activity against Caenorhabditis elegans. These findings underline the value of ethnopharmacological approaches for discovering new remedies and a validation of the traditional use by providing in vitro confirmation of the reported activity. Phytochemical investigations to reveal the responsible compounds for the activity of the plant material are currently ongoing.

Acknowledgments We are grateful to Mr. John Ampomah of Ghana Herbarium and Department of Botany, University of Ghana, Accra, Ghana and Mr. Eric Gyebi, Jachie, Ashanti region, Ghana for the collection of the plant materials for further investigations. The cooperation with the traditional healers, herbalists and regional executives and members of the Ghana National Association of Traditional Healers (GNATH) in Ashanti region of Ghana in the survey is acknowledged.

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