Journal of Ethnopharmacology 158 (2014) 255–263
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep
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: ahensel@uni-muenster.de (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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C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263
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).
C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263
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
257
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
261
C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263
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
C. Agyare et al. / Journal of Ethnopharmacology 158 (2014) 255–263
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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