Ethnobotanical Survey and Cercaricidal Activity Screening of Medicinal Plants Used for Schistosomiasis Treatment in Atwima-Nwabiagya District, Ashanti Region, Ghana

This study focused on documenting and evaluating the cercaricidal activity of medicinal plants used for schistosomiasis treatment in an endemic area in Ghana. Through semistructured questionnaires, personal interviews with herbalists in communities surrounding the Barekese dam in the Atwima-Nwabiagya district, where the disease is endemic, were carried out. Thirty medicinal plants distributed in 19 families were reported to be used for schistosomiasis treatment in the survey. Information on the plants, including scientific names, common names, families, and the used plant part were recorded. The families Apocynaceae and Euphorbiaceae recorded the highest number of plants (14% each), followed by Asteraceae (10%), Loranthaceae (7%), and Rubiaceae (7%). In vitro cercaricidal activity of methanol extracts of nine out of the thirty plants was performed by exposing human Schistosoma mansoni cercariae obtained from Biomphalaria pfeifferi to various concentrations of extracts over a duration of 240 minutes. All the plants tested demonstrated time- and concentration-dependent cercaricidal activity. With lethality being set at <1000 μg/mL, the cercaricidal activity in order of decreasing potency was as follows: Withania somnifera (LC50 = 1.29) > Balanites aegyptiaca (LC50 = 7.1) > Xylia evansii (LC50 = 11.14) > Jathropha multifida (LC50 = 12.9) > Justicia flava (LC50 = 22.9) > Anopyxis klaineana (LC50 = 182.81) > Ximenia americana (LC50 = 194.98) > Loranthus lecardii (LC50 = 223.87) > Bridelia tenufolia (LC50 = 309.03) > Zanthoxylium zanthoxyloides (LC50 = 851.94). Phytochemicals, including alkaloids, tannins, triterpenes, saponins, phytosterols, and flavonoids were identified in the plants. The result of this study gives scientific credence to the traditional use of these plants in the treatment of schistosomiasis and proves that the rich botanical knowledge of medicinal plants provides an incredible starting point for the discovery of new anti-schistosomal drugs for the local population.


Introduction
Schistosomiasis is the second most endemic parasitic worm disease caused by species of dioecious blood flukes of the genus Schistosoma, including Schistosoma guineensis, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma japonicum, Schistosoma mansoni, and Schistosoma mekongi [1]. More than 200 million individuals in tropical and subtropical regions of the world are affected by this neglected tropical disease; especially in deprived communities, which lack access to safe drinking water and adequate sanitation [2]. High prevalence rates have been recorded in Africa, the Caribbean, Latin America, and Asia with over 90% of cases reported in sub-Saharan Africa [3].
Considerable efforts to eradicate schistosomiasis from endemic areas through repeated mass preventive chemotherapy, improvement of water, sanitation, and hygiene, snail control, behavioural changes, and environmental management have not been entirely successful [4]. This is mainly due to the high cost involved in treatment campaigns (including basic health education, drugs, and logistics) and the inequity of access to preventive chemotherapy [5,6]. Moreover, praziquantel, which is currently the only drug of choice for treatment, has been faced with the challenge of reduced drug sensitivity to juvenile parasites, and the suspected emergence of drug-resistant strains [7][8][9]. While emphasizing the need to eliminate schistosomiasis by 2030, the World Health Organization (WHO) proposed the development of new intervention tools and alternative drugs to praziquantel [10].
In Ghana, schistosomiasis is present in all sixteen regions, mostly in rural and some rural-urban communities [11]. Even though mass drug administration began in 2008 and has continued, the disease is still highly endemic with a nationwide prevalence rate of~23% reported in 2015 and a focal prevalence of >50% in the country's capital [12,13]. Between 2017 and 2021, the population in need of preventive chemotherapy for schistosomiasis in Ghana increased from 10.1 to 11.5 million with only 24.1% receiving preventive chemotherapy [14]. High disease prevalence has been reported from communities around the Densu River in Greater Accra and Eastern regions [13,15], towns surrounding the Barekese and Owabi dams in the Ashanti region [16], along the Tono irrigation dam in Northern Ghana [17], and along the volta basin in the Volta region [18]. The construction of dams, lack of potable water, and dependence of the riparian communities on river sources are some reasons for this high prevalence in these districts.
As is the case in most African countries, many people resort to the use of traditional herbal remedies for the treatment of schistosomiasis in Ghana. This is mainly due to their affordability, accessibility, efficacy, and perceived safety over orthodox medicines [19,20]. As usual, the efficacy claims for most of these herbal remedies are based on past experiences without any scientific proof. In recognizing the importance of medicinal plants in the health care system of developing countries, the WHO encouraged the inclusion of herbal medicines with sufficient data on efficacy and safety into mainstream clinical practice [21]. Given this, the 21st century witnessed several research reports from the African Continent, focusing on the validation of the efficacy of traditionally used anti-schistosomal plants [21][22][23][24][25]. The artemisinin derivatives, artesunate and artemether from Artemesia annua, and myrrh from Commiphora momor are examples of plant-based products that are currently registered for the treatment of schistosomiasis in some African countries [26,27].
The Ghanaian flora is an invaluable source of new medicinal agents for the local population [28]. Unfortunately, the significance of these medicinal plants in the bioeconomy has so far not been realized. Traditional medical approaches to the treatment of schistosomiasis in Ghana are not well documented, and only a few studies exist on the antischistosomal activities of Ghanaian medicinal plants [21,29,30]. This research was thus undertaken to document herbal remedies used to treat schistosomiasis in selected communities surrounding the Barekese dam in the Atwima-Nwabiagya district, where the disease was reported to be endemic [16]. Consequently, knowledge of such herbal medicines, followed by biological activity testing, could lead to the identification of potential anti-schistosomal remedies for the future development of affordable community-based remedies for incorporation into primary health care. 2.3. Study Area. The present study was carried out in the 'Atwima-Nwabiagya' district (latitude 6°40 ′ 0 ″ N and longitude 1°49 ′ 0 ″ W) in the Ashanti region of Ghana ( Figure 1). It is 1 of the 21 political and administrative districts in the Ashanti region [31]. According to the 2010 Population and Housing Census in Ghana [63], the district covers an estimated area of 2,411 km 2 and an average height of about 77 m above sea level. The population of the district is about 149,025 with the majority (68.5%) of the population living in rural localities (2010 Population and Housing Census, Ghana) [63]. The surface area of the district is mainly drained by the 'Offin', 'Owabi', and 'Tano' rivers. There are, however, several streams in the district. These include 'Kobi' and 'Dwehen'. 'Owabi' and 'Barekese' are the two major dams have been constructed across the 'Owabi' and the 'Offin' rivers, respectively (2010 Population and Housing Census, Ghana) [63]. The major occupation in the district is wholesale and retail trade, cocoa farming, forestry, and fishing [32]. The Atwima-Nwabiagya district was selected for this survey based on the previously reported prevalence of urinary schistosomiasis in the area [16].

Materials and Methods
2.4. Sampling Procedure and Data Collection Methods. The ethnobotanical survey was carried out in August 2020 in Marbang, Adankwame, Ayensua fufuo, Ayensua kokoo, Barekese, Barekuma, and Akyena, which are all rural to urban-rural settlements through which tributaries of the Offin River are found. Eleven herbalists in the community were identified with assistance from community members and interviewed one on one using open-ended semistructured pretested questionnaires,  [34]. Briefly, fresh water host snails of S. mansoni parasites were scouted for and collected from their natural habitat in River Densu at Tomefa near Weija in the Ga South Municipal District, Greater Accra Region of Ghana. Harvesting was done by examining different sites of the river where they normally reside, thus, water, plants, and muddy areas that are rich in decaying organic matter, rocks, and stones. With the aid of forceps and an artist's brush, the fresh water snails were collected into aquarium plastic bowls partially filled with fresh 3 Journal of Parasitology Research water to mimic their natural habitat. The collected snails were first washed with fresh water to remove debris and snail excreta with an aquarium net/sieve, then subsequently washed with aged water (tap water that has been kept in a container and covered for 14 days). They were kept in beakers half-filled with aged water and transported in wellventilated aquarium plastic bowls (measuring 27 cm × 12 cm) to the Parasitology Laboratory at Noguchi Memorial Institute for Medical Research (NMIMR), Accra, where the experiment was performed.

2.8.3.
Maintenance of Host Snails. The transported snails were maintained in the Parasitology Laboratory at NMIMR under standard laboratory conditions at a temperature of 28°C. The snails were fed with fresh lettuce and left to acclimatize to the laboratory conditions overnight. The water in the aquarium was changed every day until the work was completed.
2.8.4. Exposure of Host Snails and Cercariae Shedding. Cercariae shedding was accomplished following the method by Obare et al., and the method description partly reproduces their wording [35]. Briefly, single snails were individually placed in each well of a 24-well microtiter plate containing 1 mL of distilled water with the aid of forceps. The plates were covered to prevent snails from crawling out and were  [36,37]. Hence, praziquantel was not employed as a positive control in cercaricidal activity screening. The plant Balanites aegyptiaca is reported to exhibit significant cercaricidal activity against S. mansoni cercariae [38][39][40] and has been employed as an internal control to evaluate the adequacy of experimental conditions in many studies evaluating the cercaricidal activity of plants [29,41]. Hence B. aegyptiaca extracts, prepared at the same concentrations of test samples, were employed as an internal control, and distilled water was used as the negative control in this study. The cercaricidal activity was performed according to the method described by Tekwu et al., and the method description partly reproduces their wording [42].
2.9. Statistical Analysis. All data were calculated as the mean ± standard error of the mean (SEM). Statistical analysis was carried out using the Graphpad ® Prism Version 7.0 (Graphpad Software, San Diego, CA, USA) for Windows. Time-course curve of the percentage mortality of the plant extracts against time was plotted. The LC 50 (i.e., the concentration at which 50% of the cercariae died was determined by plotting a nonlinear regression curve (log concentration of inhibitor vs. % mortality).

Ethnobotanical Studies
3.1.1. Socio-Demographic Details of Respondents. Eleven traditional medicine practitioners, who were identified by community members in the study area were interviewed during 5 Journal of Parasitology Research the survey. All traditional healers interviewed belonged to the Akan tribe. The inclusion criterion for the survey was that the practitioner used herbal medicine. They comprised 77% men and 23% women aged between 31 and 70 years. A majority of respondents (61%) had practiced for more than 15 years.

Knowledge and Perception of Healers about Schistosomiasis.
All interviewed herbalists reported having heard of and knew urinary schistosomiasis, which they locally referred to as 'gyonso mogya', literally meaning 'bloody urine' in the Akan language, alluding to haematuria, which was the most common symptom of the disease presentation. Meanwhile, none of the respondents knew about any other form of the disease when asked about intestinal schistosomiasis. They all responded affirmatively to treating at least one patient with 'bloody urine' in the past one year.
Even though the herbalists interviewed reported treating only urinary schistosomiasis (or its major symptom-haematuria), interactions with community members within the same locality, indicated intestinal symptoms, such as bloody stool, diarrhoea, and abdominal pain (unpublished results). From the responses from herbalists and community members, it could be inferred that intestinal schistosomiasis may not be uncommon in the area visited. Its symptoms may have been misconceived for other gastrointestinal tract infections. It was expected that nearby water bodies could be a source of the intermediate hosts for both S. haematobium and S. mansonii. However, only few B. pfeifferi were obtained from nearby river sources. This led to the selection of this species for the study.
Despite the appreciable level of awareness of the disease in the area, correct knowledge about the specific cause and modes of transmission was not adequate among respondents. In response to a multiple-choice question on the cause of schistosomiasis, more than half of the herbalists mentioned 'infected water' (68.8%) and 'some types of worms' (77.9%) as causes of the disease, whereas about 38% associated the disease with personal hygiene. The most frequently mentioned mode of transmission related to contact with infected water (72.1%) or 'bite' from a fresh water snail (32.2%), even though some respondents still believed that sex with infected persons (3.8%) or sharing toilet facilities with infected persons (15.5%) could also be a transmission route for the disease. Knowledge of the symptoms of schistosomiasis varied, with most of the respondents listing blood in urine, painful urination, and other non-specific symptoms, such as fever, vomiting, dizziness, and loss of appetite.
The least concentration of the extract that caused 50% cercariae mortality (LC 50 ) was determined from a plot of % mortality against log concentration for all extracts. From this analysis, the standard control, B. aegyptiaca had an LC 50 of 7.1 with a minimal lethal concentration (MLC) of 31.25 μg/mL, achieving 100% mortality at 120 minutes. W. somnifera exhibited a much more potent cercaricidal activity with an LC 50 of 1.29 and an MLC of 31.25 μg/mL, achieving 100% mortality by the 120th minute. This was followed by X. evansii (LC 50 = 11.14) and J. multifida (LC 50 = 12.90), both having an MLC of 31.25 μg/mL, achieving 100% mortality by the 120th minute. Among all extracts tested, B. tenufolia and Z. zanthoxyloides exhibited the least cercaricidal activity having LC 50 values of 309.03 and 851.94, respectively, with MLCs of 1,000 μg/mL for both extracts (Tables 3  and 4).

Discussion
The high incidence of schistosomiasis in some Ghanaian communities, coupled with the unavailability and reduced efficacy of praziquantel, the drug of choice, has prompted the exploration of other sources of new or alternative treatment options for the disease. In several African countries, including Ghana, medicinal plants have proven to be an invaluable source of healing agents for the treatment of a plethora of diseases. This rich botanical knowledge provides an incredible starting point for the discovery of new drugs for the local population. In this study, medicinal plants used in the treatment of schistosomiasis in selected endemic   Journal of Parasitology Research communities around the Barekese Lake in the Atwima-Nwabiagya District of Ghana were recorded in an ethnobotanical survey. Furthermore, the cercaricidal activity of selected plants was investigated against the S. mansoni cercariae in an in vitro assay. From the ethnobotanical survey, thirty medicinal plants belonging to 19 families were listed by herbalists to be used by them in the management of schistosomiasis. According to the practitioners, formulations comprised of one to three plants dispensed as dried powdered herbs are to be prepared as decoctions for oral administration for at most 2 weeks. The efficacy claims were based on the number of people who experienced reduction or relief from symptoms as a result of using the herbal formulations. The use of medicinal plants for the treatment of schistosomiasis is a common practice in several African countries. Previous ethnopharmacological studies make mention of the use of different plants in the treatment of schistosomiasis in South Africa [44], Zimbabwe [45], Mali [46], Kenya [25], Morocco [47], and Ivory Coast [48]. Some of the plant species listed in the ethnobotanical survey conducted had been previously reported in other studies for cercaricidal and anti-schistosomal activity. In a study by Acheampong et al., Azazdirachta indica and Nauclea latifolia exhibited a very potent cercaricidal effect on S. mansoni cercariae, and further significantly reduced worm burden in schistosomiasis-infected mice [29]. In another study, solvent fractions and erythroivorensin, a flavanone isolated from the roots of Erythrophluem ivorense, displayed a remarkable cercaricidal effect against S. haematobium cercariae [30]. The stems and roots of D. crassifolium together with its triterpenoids and sterol isolates also demonstrated significant cercaricidal activity against S. haematobium [43]. In a recent study, the hydroethanolic and alkaloidal extracts from   11 Journal of Parasitology Research the stem bark of H. floribunda also showed potent cercaricidal activity against S. haematobium cercariae [41]. Others include preparations from Khaya senegalensis, V. amygdalina, A. indica, A. barbadensis, and T. officinale, which exhibited varying inhibitory effects against newly transformed schistomula [21]. These scientific reports, justifying the traditional claims of these plants in the treatment of schistosomiasis, confirm the important relationship between therapeutic agents derived from plants and their ethnomedicinal applications.
Nine out of the thirty listed medicinal plants were tested for cercaricidal activity against S. mansoni. All plants tested demonstrated time-and concentration-dependent cercaricidal activity against S. mansoni cercariae at varying levels. This was congruent with previous studies on the cercaricidal effects of plant extracts, which also showed a time and concentration relationship for various extracts against Schistosoma parasites [42]. Probit analysis of all plants' activities showed a strong positive correlation between plant concentration and mortality. As established earlier, B. aegyptiaca, used as the standard control, was reported to exhibit very significant activity against Schistosoma cercariae [29]. The cercaricidal activity of the plants screened in order of decreasing potency (LC 50 ) was as follows: W. somnifera showed the highest cercaricidal activity against S. mansonii cercariae with an LC 50 of 1.29. Similar to B. aegyptiaca, W. somnifera had a MLC of 31.25 μg/mL at 120 minutes. W. somnifera is widely used in folk medicine in the treatment of several diseases. Its medicinal properties have been mainly attributed to the presence of steroidal lactones called withanolides, which have shown antiparasitic, antibacterial, antiviral, molluscicidal, anthelminthic, and immune-modulating potencies [49,50]. In previous studies, formulations of W. somnifera leaves were found to be toxic to intermediate host snails of Schistosoma haematobium, that is, Biomphalaria alexandrina and Bulinus truncatus, through the destruction of their nucleic acids, proteins, and fatty acids, limiting their spread and reproduction rates [51]. Extracts from the leaves, fruits, and roots as well as isolated withanolides also demonstrated significant inhibitory effects against other human parasites including the leishmania parasite, Leishmania donovani [52,53] and filarial parasite, Brugia malayi [54]. The significant cercaricidal activity exhibited by W. somnifera in this study, together with its previously reported antiparasitic activity, connotes its potential as a novel source of the drug candidate for the treatment of schistosomiasis.
J. multifida exhibited remarkable cercaricidal activity with an LC 50 of 12.9 and MLC at 31.25 μg/mL within 120 minutes. Previous studies reported on the cercaricidal activity of some Jathropha species including Jathropha curcas and Jathropha elliptica against S. mansoni cercariae. In other reports, some species of the genus Jathropha, including Jathropha gossypiifolia, J. curcus, and J. elliptica showed highly toxic effects on the Schistosoma vector snails, Biomphalaria glabrata, B. truncatus, and Bulinus natalensis [55]. This implies that extracts and oils from Jathropha species may be useful as both vector control and treatment agents in schistosomiasis-endemic regions. Diterpenes and pyridine alkaloids identified in these species were responsible for the inhibition of oviposition in S. mansoni, causing external and internal damage to structures in adult worms [56,57]. The current result on the cercaricidal activity of J. multifida is congruent with these previous observations and suggests that Jathropha species, in general, may have a potential inhibitory effect on Schistosoma parasites and intermediate host snails. This is the first report on anti-schistosomal activity of the genus Xylia. X. evansii exhibited the second most potent cercaricidal activity with an LC 50 of 11.14 and MLC at 31.25 μg/mL within 120 minutes.
The cercaricidal activity demonstrated by the plants under study could be attributed to their secondary metabolites, such as flavonoids, saponins, and alkaloids [58]. In previous studies, the total alkaloids, saponins, and essential oils from Nigella sativa [59], isoflavonoids from Millettia thonningii [60], and alkaloids from J. elliptica [57] demonstrated significant cercaricidal activity against S. mansoni cercariae. Plant essential oils have also been reported to induce catalytic activity of acetylcholinesterase (AChE), which damages the cholinergic nervous system of cercariae. This interferes with cercariae motor activity leading to atypical movements, such as slow rotation and vibration as well as contortions [61]. These reports on cercaricidal activity of plant phytoconstituents, strongly support the effect of some of the plants under study in killing S. mansoni cercariae. According to Xiao et al. [62], cercaricidal activity usually results from an intensive disturbance in motor activity and lysis of cercarial tissues, followed by an extensive release of gland contents and separation of the tail from the body. Specifically, in this study, separation of cercariae head and tail as well as cercariae coiling was observed.

Conclusion
This study has highlighted the importance of medicinal plants in the treatment of schistosomiasis in Ghanaian communities. Furthermore, the remarkable cercaricidal activity demonstrated by some plant species provides scientific credence for their ethnomedicinal use and paves the way for further investigation into their anti-schistosomal activity.

Data Availability
The raw data/results from experiments used to arrive at the findings of this study are available from the corresponding author upon request. Previous reports that were used to support this study are cited at relevant places within the text as references.

Conflicts of Interest
The author(s) declare(s) that they have no conflicts of interest.