Ethnomedicinal Information on Plants Used for the Treatment of Bone Fractures, Wounds, and Sprains in the Northern Region of the Republic of Benin

Medicinal plants are frequently used in African countries due to their importance in the treatment of various conditions. In the northern Republic of Benin, traditional healers are recognized as specialists in the treatment of fractures, wounds, and sprains. The present study was conducted to document the practices (diagnosis and materials) and traditional knowledge accumulated by healers in this region on their area of specialty. In addition, literature-based research was performed to support the usage of the most cited plants. Sixty traditional healers identified as “reference persons” from Atakora and Donga departments in the northern Republic of Benin, who specialized in the treatment of fractures, wounds, and sprains, were interviewed in their communities through a semi-structured questionnaire. Information about the practice, age of the healers, medicinal plants used in this treatment, methods of preparation, and administration were collected. Samples of the plant species were also collected, identified, and stored in the national herbarium at the University of Abomey-Calavi, the Republic of Benin. The study enabled the identification of thirty-four (34) species belonging to twenty-three (23) families. Ochna rhizomatosa and Ochna schweinfurthiana (21%) were the most quoted plants among the species, followed by Chasmanthera dependens (12.1%), Piliostigma thonningii (11.3%), and Combretum sericeum (8.1%). These plants were reported to strengthen bones, reduce inflammation, relieve pain, and promote healing in the northern part of the Republic of Benin. Besides their ability to treat fractures, wounds, and sprains, they are also used for multiple purposes in the West African subregions. According to the available literature, some of the plants will need to be investigated for their phytoconstituents and pharmacological activity to validate their ethnobotanical uses. These results confirm the need for documenting traditional knowledge since it represents an opportunity for exploring plant species with potentially good pharmacological effects, which have been barely investigated. Plants identified may constitute a significant source of bioactive compounds in the treatment of various ailments such as skin inflammation and musculoskeletal disorders. They can be further explored to justify their use in traditional Beninese medicine.


Introduction
In sub-Saharan African countries, herbal medicine practice constitutes an important source of medicinal compounds essential in the health care system of the local population [1,2]. Although modern medicine has made signifcant progress, the local population remains committed to the use of folk medicine. Te reliance of the rural community on herbal medicine is still high in countries like the Republic of Benin [3]. Traditional medicine is based on knowledge, beliefs, and practices developed, preserved, and shared over generations [4]. Economic, social, and cultural factors have concurred in the preservation of these practices [5]. In Beninese, Ethiopian, and Indian traditional cultures, herbal medicine is used in the treatment of bone fractures [6][7][8], suggesting that phytochemicals may be promoted as a candidate therapy. Tey will contribute to shortening the healing period and enhancing the healing quality. A fracture can be described as a partial or entire separation in the continuity of the bone. For its repair, four overlapping stages such as hematoma formation, infammation, repair, and remodeling are included [9]. In the case of an open fracture, the skin is injured, and this may lead to a microbial infection [10]. Te practice of using herbal medicine to treat bone fractures may have been preserved due to diferent conditions including restricted access of the population to modern medicine and the unavailability of modern health facilities [11]. Terefore, rural dwellers in the Republic of Benin are still constrained to refer to traditional medicines for their common daily ailments. Tus, it is expected that substantial knowledge of the use of plants to treat bone fractures has been gathered in areas where plant use is widespread [12]. In the northern region of the Republic of Benin, traditional healers are known to be specialized in the healing of bone fractures, wounds, and sprains [13]. Tey are the main stakeholders in the transmission of the practice. Te use of plants in the management of bone fractures, wounds, and sprains in the north area of the Republic of Benin is barely documented [13], which justifes the interest in collecting data of this knowledge through an ethnomedicinal study. Documenting this indigenous knowledge is crucial to the conservation and utilization of biological resources and the identifcation of bioactive compounds with therapeutic relevance [7]. Te aim of the present study was to collect and document knowledge regarding the medicinal plants used by the local traditional healers in the treatment of bone fractures, wounds, and sprains; their usage, methods of preparation, and administration in the northern departments of the Republic of Benin (Atakora and Donga). In addition, a literature-based study was performed to provide an overview of the bioactive compounds and pharmacological activities of the most cited species, which support their usage in folk medicine as well as the ethnobotanical uses and phytoconstituents of identifed species.

Ethnomedicinal Survey and Selection of Participants.
Te ethnomedicinal survey was conducted between February 2017 and January 2019 in agreement with the executive committee of the Federation of National Associations of Traditional Medicine Actors of Benin (FANAMETRAB), Republic of Benin. Tis study was authorized by the Research Ethics Committee of the Interregional University of Industrial Engineering, Biotechnology, and Applied Sciences under the number B001331455. As this work focuses on bone fractures, wounds, and sprains, traditional healers who specialize in the treatment of bone fractures and associated complications and are willing to share their practical knowledge were considered. Selection of the healers was based on the existing register of herbalists specialized in this area of treatment by the representatives of healers' association. Te local population was of great help in locating them, especially in remote and hard-to-reach areas. Healers, who had specialized in the treatment of fractures, wounds, and sprains, were interviewed in almost every community. Participants were visited at their homes or workplaces and informed about the questionnaires. Informed consent of healers was obtained before administering the questionnaire. Tis process allowed the healers to feel valued and comfortable in their working environment. It also enabled us to collect relevant information about materials used for the treatment of patients. Te questionnaire was designed in French and conducted either in French or the local language with the help of a translator or resident able to speak the local language. A pretesting of the semi-structured oral questionnaire with two healers in Natitingou was performed, and the questionnaire readjusted for an efcient collection of data. Sixty (60) healers specialized in the treatment of fractures, and associated complications have been interviewed. Te full survey is available in the supplement. For dedicating time to the study and in respect of their tradition, a token was given to each healer. After the questionnaires and interviews were completed, healers were asked to provide samples of the plant materials mentioned. Samples were collected, labelled, and kept as voucher specimens for identifcation. Voucher specimens (see Table 1) of all plants were identifed and deposited at the National Herbarium of the University of Abomey-Calavi, where each was given a defned ID-number.

Data Collection and Analysis.
Questionnaires and interviews were used to collect biographical data of the respondents and details on their knowledge of the disease conditions (fractures, wounds, and sprains), years of experience, learning of practice, method of diagnosis, recipe, storage, and therapeutic usage of plants and plant parts used, their vernacular names, and the duration of treatment. Medicinal plants were rated based on how often the informants cited a particular plant. Data obtained from the ethnobotanical survey of medicinal plants used in the treatment of fractures, wounds, and sprains were recorded in 2 Evidence-Based Complementary and Alternative Medicine Microsoft Excel spreadsheet software version 2016 and analyzed using descriptive statistical tools to determine frequency of citation (FC), informant consensus factor (F ic ), percentages, and frequencies of quantitative data.

Frequency of Citation.
Te frequency of citation (FC) of a species corresponds to the ratio between the number of respondents (n) who cited the species and the total number of respondents (N) and is calculated as follows [14]: Te informant consensus factor (F ic ) varies from 0 to 1. Values are low (close to 0) if plants are selected randomly or if no exchange of information about plants has occurred between informants. It approaches one (1) when there is a well-defned selection criterion in the community and/or if information is exchanged between informants.

Literature Validation of the Data. Plant species identifed
were confrmed using the Plant List (https://www.theplantlist. org/) for the accepted scientifc names. Considering the probability of having the most cited plants according to our study reported in the scientifc literature for treatment of fractures, wounds, and sprains, a cross-referencing was performed by entering their botanical names in combination with the search term "fracture, wound, or sprain" into the PubMed, Google Scholar, and ScienceDirect database. Databases were also used to look for available published data regarding the ethnobotanical uses and phytoconstituents of the plant species mentioned by the healers and reported in Table 2.

Geographical Distribution of Respondents.
Te geographical coordinates of each healer were recorded to have an insight into the geographical distribution in these departments (see Figure 2). Participants were well distributed in most of the communities, thus depicting the widespread dissemination of traditional medicine practiced by healers in rural areas.

Demographic Characteristics of Surveyed Traditional
Healers. Te interview enabled the collection of sociodemographic information concerning the age, gender, educational level, ethnic group, and religion of the respondents who specialized in the treatment of fractures, sprains, and wounds. Healers were in the 21-90 age range, with 35% between (31-50) years and (51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70) years and 25% between (71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90) years. In addition, only 3% of healers were represented in the 21-30 age range. Te gender distribution indicated that 90% of the practitioners were male. Te sociodemographic characteristics of the traditional healers are presented in Table 1. Te age distribution demonstrates that elders were more involved in this practice than young people, which was also revealed in studies carried out in Togo and Ethiopia [96,97]. According to the results, women in this practice were in the minority, which confrms the fndings of Upadhya et al. [8]. Te number of patients visiting the healers depended on the season. During the rainy season, the poor and slippery conditions of the roads increased the number of accidents related to motorcycle use in the northern area of the Republic of Benin [98,99]. According to the results, the level of education is such that 27% of the healers are educated, out of which 22% have attended primary school, 5% secondary school, and 73% have no education. Te level of education of the healers in these areas is comparable to healers in the northern region of Ghana and the Republic of Benin [100,101]. Healers belong to various ethnic groups like Waama, Ditammari, Bariba, Yiende, and Natimba in Atakora and Dindi, Yôme, Ani, Koura, Peulh, and Kotocoli in Donga. As far as religion is concerned, 23% of the healers were Christian, 25% Muslim,

Practical Knowledge of Healers in the Treatment of Bone
Fractures, Wounds, and Sprains. Te knowledge of treating fractures and its complications is, in most cases, passed on through generations. Te skills obtained by practicing with a family member who has the experience or knowledge is complemented by one's own observations. Te general process for treating fractures, wounds, and sprains can be described in three signifcant steps. Initially, an examination is made by the healer to identify the type of bone fracture or sprain. Te next step of massage is performed by preparing an infusion or a decoction of plants that is applied to the injured part, followed by the next step of applying either the seed or the bark of the roots of plants in a powdered, incinerated, or chewed paste form. Regarding open fractures, all healers unanimously agreed that the wound must be healed before a treatment can be given. Tis report follows hospital practices where minimizing the risk of infection is a priority [10]. Sprains, open and closed fractures, and wounds can be treated by 97% of the healers while only 3% focus on the treatment of fractures. Tirty-one percent (31%) of the healers claim the ability to treat these conditions without the help of modern medicine. In contrast, the association with modern medicine such as radiography, the use of analgesics (e.g., paracetamol), sewing of the open wound before treating it, and collaboration with health workers is done by 69% of the healers. Tis, combined with modern medicine, is a proof that traditional medicine sometimes relies on modern medicine to ascertain a diagnosis [8]. Te recovery time is variable, depending on the severity of the fracture. Two weeks are needed for a child to recover and 1-3 months for an adult. Te application of the treatment is made once or twice a day, depending on the severity of the case. Te materials needed in traditional medicine include mats, banana bark, and bandages, which are comparable to plaster casts used in modern medicine for immobilizing an injured part [103].

Plant Species Cited and Teir Applications for the Treatment of Fractures, Wounds, and Sprains.
Te ethnomedical study resulted in the identifcation of thirty-four (34) species representing twenty-three (23) families that are used for the treatment of fractures, wounds, and sprains (Table 3). For each plant species, a recipe has been collected that is valid for treating fractures, wounds, and sprains. Plants species are used with a technicality that varies  Evidence-Based Complementary and Alternative Medicine 9 0.8 [112,113] Powdered bark of the roots of N. laevis + F. thonningii adventitious roots applied topically  [114] phytochemical study also shows the presence of a trifavonoid ester and bifavone derivatives, which were reported for the frst time in Ochna rhizomatosa leaves. A recent study allowed for the identifcation of three bifavones ((R)-rhizomatobifavonoid A-C) along with gerontoisofavone A, schweinfurthianone A and B, and calodenine B and investigated their inhibitory efect against HIV and malaria [73]. Regarding Ochna schweinfurthiana, it is used in traditional medicine to treat pain, infammation, skin infection, and arthritis [76,124]. During the evaluation of the antimalarial potential of Ochna schweinfurthiana roots, several bifavones were identifed, namely, calodenone, calodenine B, lophirone A, gerontoisofavone A, and 4‴methoxylophirone A, 4,4',4‴-trimethoxylophirone A [123]. A trimethoxy derivative of lophirone A has shown a potential antimicrobial efect [125]. In addition, other bifavones such as cupressufavone and robustafavone, together with epicatechin and 3-β-O-D-glucopyranosylβ-stigmasterol compounds, were isolated from the ethyl acetate extract of the stem bark of the plant [77]. Recently, hemerocallone, amentofavone, agathisfavone, lithospermoside, 6,7-dimethoxy-3′-4′-dimethoxyisofavone, and β-D-fructofuranosyl-α-D-glucopyranoside were isolated from the acqueous extract of the bark of Ochna schweinfurthiana [124].
Chasmanthera dependens cited 15 times, has been reported for the management of fractures and wounds in Nigeria [30]. Te species is known for its content of quaternary and tertiary phenolic alkaloid compounds [31]. It was shown to possess analgesic, anti-infammatory, and antimicrobial properties [30,126].
Combretum sericeum from the family Combretaceae, and 10 times cited has been barely investigated. Members of the genus Combretum have shown interesting properties and are widely used as medicinal plants [131]. C. sericeum is described as having several ethnopharmacological efects, e.g., against diarrhoea and gastrointestinal disorders [36,37], as well as antiplasmodial [132] and antimicrobial properties [133]. Despite the numerous features of this plant, only a few phytochemical studies have been performed. Tannins, terpenoids, saponins, various favonoids, and anthraquinones were detected in the plant [134]. As seen from the literature discussed here, all the plants contain interesting bioactive components such as favonoids and alkaloids. During bone fractures, wounds, and sprains, infammation plays a signifcant role. Te richness of these plants in various compounds may contribute to overcoming the infammation process [135,136]. Tis study demonstrates that medicinal plants used by traditional healers to treat fractures, wounds, and sprains may be potential candidates for pharmacological research focused on various conditions such as infammatory skin and musculoskeletal conditions. In addition, the literature review on the ethnobotanical uses and phytoconstituents of the identifed plant species revealed that Rourea coccinea, Ipomoea pyrophila, Combretum sericeum, and Apodostigma pallens require in-depth phytochemical studies. Finally, these investigations may lead to the development of reliable formulations of herbal medicines with proven clinical efcacy and confrmed safety.

Conclusion
Te north of the Republic of Benin constitutes an area where the knowledge for treating bone fractures, wounds, and sprains is advanced. Tis ethnomedicinal study resulted in the documentation of the traditional medicine practice and provided an overview of plant species, their medicinal use, and mode of application in the treatment of bone fractures, wounds, and sprains. Te recorded plants are administered as a decoction, infusion, or powder to strengthen bones, reduce infammation, relieve pain, and promote healing. Based on these results, pharmacological and clinical assessments of these natural remedies can be conducted to rationalize their ethnomedicinal use and enhance the promotion of these plants.

Data Availability
All data generated or analyzed during this study are included in this published article and its supplementary information fles.

Ethical Approval
Research Ethics Committee of the Interregional University of Industrial Engineering, Biotechnology and Applied Sciences under the number B001331455 has authorized this study. During the feld study, permission to conduct the study was also obtained from the executive committee of the Federation of National Associations of Traditional Medicine Actors of Benin (FANAMETRAB) to enable us to interact with their members.

Disclosure
Te author Cica Vissiennon is employed by Repha GmbH Biologische Arzneimittel. Cica Vissiennon and Karin Fester contributed evenly to this work, and share last authorship.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
NMCT performed data collection, evaluation, did feld work, and conceptualized the study; NMCT and EOB performed methodology and validated the study; VA provided resources; NMCT drafted the manuscript and prepared the fgures; NMCT, CV, KF, EOB, ZV, and VA contributed to the review and editing process. All authors have read and approved the fnal version of the manuscript.

Acknowledgments
Tis work was sponsored by the IRGIB-Africa University, which funded feldwork for data collection in local communities. Te frst author Codo Toafode Nonvignon Murielle received a mobility grant between Leipzig University (Germany) and IRGIB-Africa University (Benin) granted by the DAAD (German Academic Exchange Service); funding program/ID: 57299293.

Supplementary Materials
Supplementary material fle is entitled "Survey of the collection of data on plants used in the treatment of fractures, wounds, and sprains." (Supplementary Materials)