Medicinal plants species used by herbalists in the treatment of snakebite envenomation in Uganda

Background There are high mortality and morbidity rates due to poisonous snakebites globally with sub-Saharan Africa having some of the highest cases. However, traditional medicine practitioners (TMP) have been treating snakebites in Uganda for long despite the fact that few studies have been conducted to document such vital and rich indigenous traditional knowledge before it is lost. This study aimed to document the medicinal plant species used by experienced TMP in treating snakebite envenomation in selected post-conflict parts of Uganda. An ethnopharmacological survey was conducted in Kitgum, Serere, Kaberamaido and Kaabong districts in Uganda. Twenty-seven TMP with expertise in treating snakebites were purposively identified using the snowball technique and interviewed using semi-structured questionnaires. Data were analysed using simple descriptive statistics. Results Sixty plant species from 28 families were documented with high consensus among the isolated indigenous Ik tribe of Kaabong district. Most of the plant species used were from the Asteraceae and Fabaceae families with eight species each. The genus Echinops was the most well-represented with three species. The most commonly used plant species were of citation were Steganotaenia araliaceae (16), Microglossa pyrifolia (Lam.), Gladiolus dalenii Van Geel (13), Aframomum mildbraedii Loes. (11), Jasminum schimperi Vatke and Cyathula uncinulata (Schrad) Schinz (10) and Crinum macowanii Baker and Cyphostemma cyphopetalum (Fresen.) Desc. ex Wild & R.B. Drumm (10). S. araliaceae which was mentioned by all the TMP in the Ik community was used for first aid. Most of the plant species were harvested from the wild (68.75%) and were herbs (65.0%) followed by trees (23.3%). The most commonly used plant parts were roots (42.6%) and leaves (25.0%). Thirteen different methods of preparation and administration were used. Most of the medicines were administered orally (61.2%) and topically (37.6%). The commonest methods of oral application were cold water infusions (32.5%) and decoctions (21.7%). Conclusions TMP widely use several medicinal plant species for treating snakebite envenomation in the selected post-conflict regions of Uganda


Background
Worldwide, more than five million people suffer snakebite envenomation leading to 25,000-125,000 deaths, while an estimated 400,000 people are left with permanent disabilities [1]. The World Health Organization (WHO) has classified snakebites as one of the most neglected tropical diseases (NTD) in terms of incidence, severity, and clinical characteristics. This has served as a basis for the advocacy for snakebite envenomation [2]. The burden of snakebite envenomation was eventually recognized in June 2017 and then enlisted as a NTD category A by WHO [2,3].
Snakebite envenoming primarily affects residents of rural communities in Africa, Asia, Latin America and New Guinea and possess a serious health challenge [4]. It is an occupational, environmental and domestic health hazard that exacerbates the already impoverished state of these communities [1]. Venoms consist of mainly toxic modified saliva of poisonous snakes. They are complex mixtures of enzymes, proteins, non-proteins and metalloproteinases [5]. The most important venom components that cause serious clinical effects are pro-coagulant enzymes, cytolytic or necrotic toxins, haemolytic and myolytic phospholipases A2, pre-synaptic and post-synaptic neurotoxins and haemorrhagins [6]. Broadly, there are two types of toxins, namely, neurotoxins, which attack the central nervous system, and haemotoxins, which target the circulatory system and kill victims very first. Snakes with neurotoxic venom include cobras, mambas, sea snakes, kraits and coral snakes [5]. Snakes with haemotoxic venom include rattlesnakes, copper head and cottonmouths [7]. Snake venoms can be neutralized by antibodies obtained after immunizing domestic animals with them. This led to the production of anti-venom called antisera. A major drawback of serum therapy is its prohibitive cost and chance that victims are often some distance away from medical care when bitten [8]. The search for novel venom inhibitors from natural products is therefore relevant because of their potential to complement serum therapy in neutralizing mainly the local damages of envenomation. Plant extracts constitute an excellent alternative with a range of antivenom activities [7].
Africans have traditionally been treating poisonous snakebites using herbs [9][10][11][12]. For instance, of the 147 patients bitten by snakes seen between November 1995 and October 1996, 90% of them used herbs in KwaZulu-Natal, South Africa [13]. In Kenya, 32 medicinal plants have been documented for the treatment of snakebites [14,15]. In central Uganda, 36 plant species were documented for treating snakebites [10]. A total of 25 plants were documented for the treatment of snakebites in eastern Uganda, Bulamogi County, Uganda [11]. Five other medicinal plant species were documented for treatment of snakebites in the Northern sector of Kibale National Park in western Uganda [16].
The current population of Uganda is over 45.3 million [17]. More than 80% of Ugandans are involved in agriculture and live in rural areas [18]. This makes them highly vulnerable to snakebite envenomation coupled with lack of access to antisera in health facilities. There is a widespread use of medicinal plants for the treatment of snakebites in Uganda. However, there are no statistics available on snakebite envenomation and treatment. Additionally, ethnopharmacological surveys of plants used for the treatment of snakebites have not been done in many parts of Uganda. This study aimed to document the plant species used in the treatment of snakebite envenomation in the Acholi, Teso and Karamoja sub-regions of Uganda. These are post-conflict regions and were affected during the war led by Joseph Kony's Lord's resistance army (LRA) rebel outfit. The LRA war begun in 1986 and lasted over 18 years [19]. Anecdotal evidence points to a high prevalence of snakebite envenomation experienced by returnees during the postconflict resettlement in northern Uganda. This is because as many as 2 million people who had fled the fighting were forced into internally displaced people's camps in north of the country [20].

Results
Twenty-seven TMP were purposively selected and interviewed. Twenty-five were women and the rest were men. The average age of the respondents was 54.7 years and ranged from 36 to 95 years. The majority of the respondents (80%) were illiterate, with only 20% having attained primary education and were all peasant farmers (Table 1).      Sixty plant species from 28 families and fifty-one genera were documented ( Table 2). The plant families with most species were Asteraceae (8), Fabaceae (7), Asparagaceae and Amaranthaceae with 4 species each and Euphorbiaceae, Meliaceae and Solanaceae with 3 species each (Fig. 1). The genus with the most plant species was Echinops (3). This was followed by Annona, Chlorophytum spp., Eucalyptus and Solanum with two species each ( Table 2).

Medicinal plant preparation and administration
The methods of preparation and administration were grouped into thirteen categories. Most of the herbal medicines were prepared for oral administration (62.5%). The rest were administered topically (32.5%) with the exception of inhalation of smoke from burnt plant material (1.2%). The commonest methods of oral application were cold water infusions (31.8%), decoctions (21.2%) and chewing or squeezing juice from the plant material and drinking it (5.9%) (Fig. 4). The commonest topical methods of application were poultices (9.4%) and direct application of powders to the bitten site or juice (8.2%), followed by the application of powder to bite area after making small cuts with a razor blade (4.7%). Some of the plant species were used as snake repellents (3.6) and one specie was used for making an eyewash (1.2%) for cases of ocular envenomation by spitting cobras. One herbalist reported burning the plant material and making the patient inhale the smoke in cases where they were unconscious (1.2%). However, the herbalists were not aware of any specific modes of action of the medicinal plant species they used with the exception of a few species that acted as emetics ( Table 2). In the Ik community in Kaabong district, herdsmen, farmers and hunters usually moved with small quantities of G. dalenii powder as a quick remedy in case they were being bitten by a poisonous snake. In case of a snakebite, small cuts are made at the site and the powder applied. Generally, the consensus among the TMP was high in the relatively closed and isolated Ik community. Additionally, the medicinal plant species used by the Ik were generally unique to them and not used by the other communities in Kitgum, Kaberamaido and Serere districts. These included G. dalenii, E. longifolius, Cyathula uncinulata and Steganotaenia araliaceae.

Knowledge acquisition and transfer
Most herbalists acquired their knowledge on the use of medicinal plants for snakebite management from their parents and grandparents (80%) other relatives (12%). Two unique cases (8%) were registered. The first case involved the observation of self-medication in snakes by one herbalist. The said herbalist acquired the knowledge on the use of Microglossa pyrifolia for treatment of snakebites by observing a snake wounded in a fight with another snake. The injured snake reportedly recovered from its injuries after ingesting the leaves of M. pyrifolia. In the second instance, another herbalist who mainly

Type of snakebites treated
Cyathula uncinulata, Astripomoe amalvacea, Kalanchoe sp. and Hoslundia opposita were specifically mentioned as being used for treating puff adder bites. Euphorbia hypericifolia was also used for treating scorpion and spider stings. Microglossa pyrifolia was used for treating all types of snakebites except the puff adder bites. Bryophyllum delagoense and Steganotaenia araliaceae were used for treating cobra bites. In addition, S. araliaceae was used as first aid for all snakebites among the Ik only. Most of the plant species used were harvested from the wild (68.75%), whereas 24.24% were domesticated and 6.25% were both domesticated and wild. Thirty-seven (61.6%) of the documented plant species did not have any previous references about use in snakebite treatment literature. However, some of the plant species are from genera with many well-known plant species used for snakebite such as Solanum, Annona, Echinops, Euphorbia and Indigofera

Unidentified medicinal plant species used
An additional nine plant species were mentioned by the herbalists for treating snakebite envenomation in Kitgum district (Table 3). However, we were not able to collect voucher specimen for these species for identification for several reasons including wildfires that had destroyed some of their habitats, drought, insecurity near the  Uganda Sudan border and difficulty in locating some of the species because they were naturally rare.

Discussion
Even though most herbs were used singly, some of the herbalists prepared polyherbal formulations for use. One of the TMP disclosed his formula consisting of Pseudocedrela kotschyi, Gardenia ternifolia, Zanthoxylum chalybeum, Indigofera arrecta and Capsicum frutescens. The herbalists used different herbal formulations to treat their patients. These differences can be attributed to some variations in local flora of the regions, culture, training and the circumstances under which the patients presented. The most frequently cited plant species in this study were selected for farther evaluation of their antivenom potentials in vivo and their phytochemical composition. These experiments are on-going and the findings will be published in due course. The sustainable use of the plant resources raises concerns since most of them are harvested from the wild. To worsen matters, the roots of these plant species are the most widely used parts. Harvesting of roots is destructive to the plant species and is a threat to both the trade of the herbalists and the survival of the plant species. It is encouraging however to note that some herbalists had made attempts to domesticate some of the plant species they used by growing them in their backyards. Although we reported that most of the plant species recorded in this study do not have any previous documentation for use in snakebite treatment literature, some unique cases stand out. We recorded for the first time the use of Opilia amentacea in the treatment of snakebite envenomation in Uganda. Interestingly, the same species is used in India for treating snakebite envenomation [21]. However, O. amentacea was only reported by a single renowned traditional healer commonly known as "Dr. Snake". The use of O. amentacea was associated with the doctrine of signatures (DoS) or similarities. The selection of plant species for the treatment of particular conditions because of their resemblance to particular body organs is not a new concept. This DoS or similarities attributes the therapeutic properties of some plants to particular morphological characters or features they possess, i.e. "like treats like" [36]. This particular herbalist began using this plant because of the scale-like and dotted appearance of its bark and its creeping habit. This is the first report on the doctrine of signatures with O. amentacea with reference to snakebite. According to Bennett [37], the doctrine of signatures is found throughout the world and has had a long history of use. He further argues that considering the DoS from the classical morphological perspective has rarely led to the discovery of medicinal plants and the approach is therefore unproductive and largely untestable. The DoS cannot therefore be considered scientific [36,37], although parts of its utility lie in facilitating the process of understanding the subjective, psychological, and spiritual dimensions of nature [38].
Another interesting observation was the routine use of prayers during healing. One particular healer was observed to always begin his plant collection routine in the bush with prayers. He prayed to God beseeching him to give the plant species their healing power before he begun harvesting. He professed the catholic faith and attributed his success to his God. This particular healer had a medicinal plant garden and a special treatment room/hut in which he treated his patients. He got official recognition with a certificate from the ministry of culture in Uganda as early as 1986. The citation of prayers by herbalists who profess Christianity during healing rituals has previously been reported in parts of Uganda such as the west [39]. Prayers form an integral part of the belief system and are believed to make the treatment successful.
The transfer of traditional knowledge is by word of mouth. The TMP identify and train particular children on the identification, preparation and administration of the herbs. We report a unique case of self-medication in snakes. This provides an insight into the potential antivenom properties of Microglossa pyrifolia. Although previous studies have reported cases of self-medication especially in primates such as chimpanzees [40,41], we have not come across previous reports of selfmedication in snakes. However, according to Shurkin [42], some lizards are believed to survive venomous snake bites by eating roots of particular plants. It is therefore not farfetched to consider self-medication in snakes.
According to the in-charge of Timu health centre II in Kaabong district, there were relatively many reports of snake bites in the Ik community, but there were few cases reporting to the health facility. Even those who reported to the health centre came several days after being bitten by snakes for supportive treatment after initially managing the snakebites with herbs. The health centre also did not have any antisera for treatment of snake bites.

Conclusion
TMP widely use several medicinal plant species for treating snakebite envenomation in the post-conflict sub-regions of Acholi, Teso and Karamoja in Uganda. There is a high consensus by herbalists in the Ik community on different plant species used. Most of the plant species are harvested from the wild, prepared as infusions and used orally. The knowledge of medicinal plant use is transmitted orally.

Study design and setting
An ethnopharmacological study was conducted in the districts of Soroti in Mukura/Asuret sub-counties (1.7229°N, 33.5280°E), Serere in n Bugondo, Okulonyo & Kyere sub-county (4994°N, 33.5490°E), Kaberamaido, Anyara sub-county (1.6963°N, 33.2139°E) in the Teso sub-region, Kitgum, Namukora and Orom sub-counties (3.3397°N, 33.1689°E, Acholi subregion) and Kaabong, Timu sub-county (3.5126°N, 33.9750°E, Karamoja sub-region) (Fig. 5). The data were collected between August and October 2017 using interviews with semi-structured questionnaires These areas have tropical and savanna type vegetation [43]. The study areas were selected because they are recovering from protracted LRA war; they are remote. Additionally, these areas have limited access to modern health facilities with antisera and have been reported to have frequent snakebites [44,45].

Characteristics of participants
Traditional medicine practitioners or herbalists with expertise in treating patients bitten by snakes were purposively selected and identified using the snowball technique [46]. In each of the study areas, there are associations of general traditional healers. Each of these associations has specialists such as traditional birth attendants, bone setters and those that treat snakebites within its ranks. Through these networks, we were able to get referrals to the specific snakebite treatment experts.

Plant collection and identification
Voucher specimens of the plant species mentioned in the study were collected using standard procedures [47] and taken to Makerere University herbarium for identification. The scientific names of the plant species were identified based on the plant list [48]. Plant families were verified using the angiosperm phylogeny group IV [49].

Data analysis
The data were analysed using simple descriptive statistics in Microsoft Excel 2019. traditional medicine practitioners from the Ik community in Kaabong, Kaberamaido, Kitgum and Serere districts; the local leadership of the respective districts for their support in conducting this survey; research assistants Benjamin Okello, Emmanuel Onekanono, Lazarus Aryon, Anthony Oluka Eridu and Ronald Onen; and survey guides and language translators Denis Omongin, Evalyne Auma Opio, Alex Okwel from Timu Health centre II.
Authors' contributions OFD conceptualized the study and wrote the protocol under the guidance of BR, NJ and AG. OFD and AG conducted the ethnopharmacological survey, analysed the data and drafted the manuscript under the supervision of BR and NJ. All authors read and approved the final manuscript.

Funding
This study was funded in part by the Swedish International Development Cooperation Agency 353 (Sida) and Makerere University under Sida contribution No: 51180060.
Availability of data and materials Supporting data to this article is publicly available in the Mendeley data repository: Data, V2, https://doi.org/10.17632/g788hgn5t2.2 Ethics approval and consent to participate Ethical approval for the study was obtained from Gulu University research ethic committee (GUREC-003-20) prior to the study and the Uganda National Council of Science & Technology (UNCST, No. SS 5207). Permission was also obtained from the local authorities to conduct the study in the respective sites. Written prior informed consent was obtained from each of the participants before every interview.

Consent for publication
All participants referred to in this study gave their consent for publication

Competing interests
All the authors declare that they have no competing interests Author details