Antivenin plants used for treatment of snakebites in Uganda: ethnobotanical reports and pharmacological evidences

Snakebite envenomation is a serious public health concern in rural areas of Uganda. Snakebites are poorly documented in Uganda because most occur in rural settings where traditional therapists end up being the first-line defense for treatment. Ethnobotanical surveys in Uganda have reported that some plants are used to antagonize the activity of various snake venoms. This review was sought to identify antivenin plants in Uganda and some pharmacological evidence supporting their use. A literature survey done in multidisciplinary databases revealed that 77 plant species belonging to 65 genera and 42 families are used for the treatment of snakebites in Uganda. The majority of these species belong to family Fabaceae (31%), Euphorbiaceae (14%), Asteraceae (12%), Amaryllidaceae (10%) and Solanaceae (10%). The main growth habit of the species is shrubs (41%), trees (33%) and herbs (18%). Antivenin extracts are usually prepared from roots (54%) and leaves (23%) through decoctions, infusions, powders, and juices, and are administered orally (67%) or applied topically (17%). The most frequently encountered species were Allium cepa, Carica papaya, Securidaca longipedunculata, Harrisonia abyssinica, and Nicotiana tabacum. Species with global reports of tested antivenom activity included Allium cepa, Allium sativum, Basella alba, Capparis tomentosa, Carica papaya, Cassia occidentalis, Jatropa carcus, Vernonia cinereal, Bidens pilosa, Hoslundia opposita, Maytensus senegalensis, Securinega virosa, and Solanum incanum. There is need to identify and evaluate the antivenom compounds in the claimed plants.


Introduction
Snake envenoming is a global health problem and a justification for morbimortality and various socio-economic losses. A recent conservative global estimate points that about 5.5 million snakebite cases are encountered every year causing about 2 million deaths [1,2]. Up to 500,000 of these cases are reported in Africa [3][4][5]. In 2002, 108 cases of snakebites were reported in Gulu Regional Hospital (Uganda) though none of the victims died [6]. About 151 cases were reported in neighboring Kenya in 1994 with 19% of these from venomous snakes [7].
A recent study [8] in 118 health facilities throughout Uganda revealed that only 4% of the facilities stocked antivenin sera, thus most victims rarely seek medical care when bitten by snakes. A retrospective part of this study showed that in 140 surveyed facilities, 593 snakebite cases were recorded within six months with bites reported in the rainy seasons from April 2018 to June 2018 and then October 2018 to December 2018 [8]. Thus, fatalities in rural areas are due to lack of antidotes within the 24 h recommended [6,9,10] and antisera administration problems [11,12].
Snakes are taxonomically carnivorous vertebrates of class Reptilia, order Squamata, sub-order Serpentes and families: Colubridae, Boidae, Elapidae, Pythonidae, Viperidae that characteristically kill their prey by constriction The use of plants in addressing medical challenges have been witnessed since antiquity and is regaining shape in the modern era due to their safety, effectiveness, cultural preferences, inexpensiveness, abundance, and availability. In Uganda, more than 230 species of angiosperms belonging to about 168 genera and 69 families are being utilized for treatment of erectile dysfunction, malnutrition, sickle cell anemia, hernia, venereal diseases (syphilis, HIV, and gonorrhoea), post-partum hemorrhage, snakebites, cancer, menorrhagia, threatened abortion, skin diseases, jaundice, and cough . This study compiled information on antivenin plants reported in different districts of Uganda and presented some experimental evidence supporting their use in antivenom therapy.

Description of the study area
Uganda is a landlocked country straddling the equator in Eastern Africa [61]. It is flanked by Lake Victoria, Tanzania, and Rwanda to the south, Kenya to the East, South Sudan to the North and Democratic Republic of Congo to the West (Fig. 1). The climate experienced is equatorial moderated by relatively high altitudes with a mean annual temperature of 20.5°C. The country's population is estimated to be 35.92 million with 5 main ethnic families: Nilotics (Acholi, Alur, Padhola, Lulya, and Jonam), Bantu (Baganda, Banyankole, Batoro, Bagwere, Bakiga, Bakiga, Banyarwanda, Bakonjo, Banyoro, and Bakiga), Hamities (mainly constituted by the Bahima), the Nilo-Hamities (Teso, Karamojong, Kakwa, Sebei, Labwor, and Tepeth) and the Sudanics (Lugwara, Madi, and Lendu) [62]. Health care services are inadequate [63], and access to allopathic drugs is limited in rural areas due to their prohibitive cost, poor transport network, chronic poverty and the general belief in efficacy of traditional medicine than western medicine [64]. The key search words used were "snakebite," "vegetal," "traditional medicine," "ethnobotany," "alternative medicine," "ethnopharmacology," "antivenom," "antiophidian," "antitoxin," "snake antidotes," and "Uganda." The botanical names of the plants were vetted in botanical databases: the Plant List [71], International Plant Names Index (IPNI) [72], NCBI taxonomy browser [73], and Tropicos [74]. Where a given species was considered as distinct species in different reports, the nomenclature as per the botanical databases took precedence. The families, local names (Lango, Acholi, Ateso, Luganda, Lunyoro, Rukiga, and Lusoga), growth habit, part(s) used, conservation status, preparation and administration mode, status of antivenin activity investigation of the plants, and the districts where the plants were surveyed are reported (Table 1, Additional file 1). Pertaining to pharmacological reports, the snake venom studied, phytochemicals, and positive results obtained using plants identified by this study (or species from the same genus) are reported. In some cases, some activities of the plant extracts such as antioxidant and radical scavenging activities are reported as these are some of mechanisms by which snake venoms are countered.

Results and discussion
Only full-text articles in English, Lango, Acholi, Ateso, Luganda, Lunyoro, Rukiga, and Lusoga were considered. A total of 15 articles (13 in English, 1 in Luganda, and 1 in Lusoga) with information on antivenin plants were retrieved, but two of these did not meet inclusion criteria because one was not a full-text article while the other had only one botanically unidentified antivenin plant. Thus, the following reports of interest specifically on the subject of antivenin plants in Uganda were retrieved (Table 1).

Traditional concept of snakebites in Uganda
From the electronic survey of data, it is indubitable that the local communities in Uganda have different perceptions about snakebites. The beliefs appear to be clanrelated and include snakes "can protect" (among the Baganda) [18,75] or "are dangerous and connected to witchcraft" in most communities [8]. By comparison, the Luo of Kenya associate snakes with witchcraft [76].
Growth habit, parts used, preparation, and administration of antivenin preparations  used plant parts are roots (54%) and leaves (23%) followed by whole plant (4%), bark (4%), and tuber (4%) (Figs. 4 and 5). The regular use of roots and leaves in antivenin preparations is a characteristic feature of traditional antivenin therapy [17,81,86,99,100], no wonder some of these plants are named "snakeroot" in some rural communities [101]. Comparatively, embryonal plant parts such as fruits, seeds, buds, bulbs, and flowers which have reputation for accumulating certain compounds are less frequently used, concordant with reports from other countries [17,81]. Majority of the plants reported grow in the wild (82%), 14% are cultivated while 4% are semi-wild (occurs in the wild but can also be cultivated). The commonest mode of preparation is as decoctions and infusion. The plants are collected from fallow land, cultivated fields or home gardens when needed. Traditional medicine practitioners either collect herbal plants personally or hire collectors. All traditional medical practitioners cultivate some medicinal plants especially fast growing ones around their homes and shrines in order to have them within easy access when needed. The antidotes are administered orally (67%) or applied at the point of snakebite (17%). In this survey, it was noted that few plant species are used against snakebites simultaneously in different districts. This could probably be attributed to the abundant distribution of the analog active substances among species especially those of family Fabaceae. Some of the plants listed are also used for wading off or discouraging snakes from reaching human and livestock abodes. In most instances, the plants possess a strong smell that causes discomfort and disorientation to snakes when they slither over them. In exceptional cases as with tobacco, the plant (dried whole plant or leaves) are burnt to produce unpleasant odor that discourages snakes ( Table 2). The Lango of Northern Uganda burn bicycle, motorcycle, and vehicle tyres to discourage snakes.

Other ethnomedicinal uses and toxicity of the reported antivenin plants
Almost all the plants recapitulated in this review are employed for the treatment of various ailments. For example, Bidens pilosa L. has been reported to be useful in the treatment of more than 40 disorders including inflammation, immunological disorders, digestive disorders, infectious diseases, cancer, metabolic syndrome, and wounds among others [103][104][105][106]. Albizia coriaria (Welw. ex) Oliver is used in the management of syphilis, postpartum haemorrhage, sore throats, menorrhagia, threatened abortion, skin diseases, jaundice, cough, sore eyes, and as a general tonic [35]. Such plants tend to be used in different communities for treating snakebites and can be a justification of their pharmacological efficacy [107].
On the other hand, some of the antivenin plants cited exhibit marked toxicity. A striking example is Jatropha carcus L. leaf and latex which contain a purgative oil (irritant curcanoleic acid and croton oil), curcin (toxalbumin), and diterpene of tigliane skeleton classified as phorbol esters [108]. Curcin has protein translation inhibitory (N-glycosidase) activity whereas phorbol esters are amphiphillic molecules that can bind phospholipid membrane receptors [109]. This observation explains why some antivenin preparations in Uganda are applied topically or ingested in small amounts. Fortuitously, topical application is a better approach for reducing the local action of venoms at the bitten site.

Knowledge dynamics of antivenin plants in Uganda
Knowledge of traditional medicine and medicinal plants are usually acquired and passed on orally from the elders to the young [34]. This is comparable to reports from other African countries [17,78]. Knowledge is gained through trainings, divine call, and in some instances, the plant to be used can be asked for from the dead [42,59]. Because of civilization, efforts to pass on traditional medical knowledge to children is impeded by lack of interest and the fact that most children spend their youthful years in school [17,34,60]. Most Ugandans know that their current social conditions such as poverty, sleeping in mud houses and activities such as cultivation, hunting, and herding cattle increase their chances of getting bitten by a snake. Snakebites are always taken as exigencies with economic implications due to the expenses involved in transporting the victims for treatment, the care needed, enforced borrowing, amputation of necrosed legs, and arms as well as loss of time [8].

Treatment of snakebites
Treatment of snakebites in Uganda involves various procedures that vary from culture to culture and religion to religion, for example, Pentecostal Assemblies of God (PAG) believe prayers can treat snakebites. Use of tourniquets to tie the injured part above the affected area to prevent the venom from spreading to heart, the lungs, kidney, and other delicate parts of the body has been prescribed as a supportive first aid in Northern Uganda [6]. This is usually done at five-minute intervals to avoid the weakening of the local tissues. Among the Baganda (Central Uganda), the use of black stones (carbonized absorptive animal bone) and Haemanthus multiflorus bulb have been reported (Fig. 6) [10]. A black stone is placed on incisions made around  the bitten area until it sticks. It is administered to reassured victims and left for 20-30 minutes for it to "suck out" the poison. The stone is reported to be 30% effective and can be reused if boiled in hot water after use and can be used alongside other medical treatments [10]. For Haemanthus multiflorus, the bulb is chewed by the victim or it is crushed and put on the bite. In Northern Uganda, the use of 500 Uganda shilling copper coins and black stones have been reported [6]. The copper coins are placed on the bite until it gets stuck and it is left to fall off on its own. In some communities like Lango of Northern Uganda, antivenin therapy involves oral administration of egg yolk and albumin similar to the therapy reported among the Luo of Kenya [17]. Overall, traditional antivenin therapy in Uganda involves administration of plant preparations to the victims [35].

Antivenin activity of plants and pharmacological evidence
Pharmacological studies have revealed that some plants used in traditional medicine are able to antagonize the activity of various crude venoms and purified toxins [110][111][112]. Antigen-antibody interaction is the proposed mechanism through which the activity of venoms is countered by antivenins. Reported mechanisms of venom inactivation include precipitation or inactivation of the toxic venom proteins [113], inactivation, or enzyme inhibition [114], chelation [115], adjuvant action [116], antioxidant activity or a synergistic interaction of these mechanisms. Enzyme inhibition and protein precipitation are by far the most conventionally accepted mechanisms [117]. To start with, plant metabolites such as flavonoids, polyphenols, saponins, tannins, terpenoids, xanthenes, quinonoids, steroids, and alkaloids have been reported to snuggly bind to toxic proteins of snake venoms, thereby offsetting their deleterious effects. Another explained scientific possibility is the competitive blocking of the target receptors [118]. For example, atropine (an alkaloid reported in family Solanaceae) is reported to inhibit the activity of green and dark mamba (Drendroaspis angusticeps and D. polylepsis) venoms by blocking cholinergic nerve terminals usually attacked by the venoms. Aristolochic acid I (8-methoxy-6-nitro-phenanthro(3,4-d)1,3-dioxole 5-carboxylic acid), an alkaloid present in Aristolochia species acts in the same way.
Another mechanism of snake venom inactivation involves inhibition of the active enzymes such as phospholipase A 2 , metalloproteases, and hyaluronidases by polyphenolic compounds such as tannins. In this scenario, the metabolites interact with the venom enzymes by non-specific binding proteins [119] through hydrogen bonding with hydroxyl groups in the protein molecules generating chemically stable complexes [120]. For example, in a study experimented with aristolochic acid I and PLA 2 isolated from Viper russelli venom, molecular interactions between the two were reported to be between their hydroxyl groups which formed two hydrogen bonds with Granulocyte Marker Monoclonal Antibody (His48) and myotoxins I (Asp49) of the venom [121]. Aristolochic acid I is also an inhibitor of hyaluronidase of Naja naja venom [122]. Other examples of these are outlined in Table 3. Chelation on the other hand is reported to be effective for antivenin plant extracts with molecules (compounds) capable of binding to divalent metal ions necessary for some enzymatic activities. For the cause that chemical coordination of metal ions is indispensable for normal hydrolytic activities of phospholipases and metalloproteases, secondary metabolites capable of disrupting the enzyme-metal ion bondage inhibits enzymatic progression [166]. In antioxidation mechanism, plant metabolites (flavonoids, terpenoids, tannins, polyphenols, vitamins A, C, E, and minerals such as selenium) prevent, stop or reduce oxidative damage due to phospholipase A 2 activity by selectively binding to the active sites or modifying the conserved residues that are inevitable for phospholipase A 2 catalytic action [119].
The efficacy of plant extracts in antivenom action tends to be related to the solvent used for the extraction of the bioactive compounds. A study [152] reported that  The antioxidant activity by DPPH was 35.50 ± 0.02%, by phosphomolybdate assay was 41.22 ± 0.17 mg/kg ascorbic acid equivalent, and the reducing power increased with increase in concentration up to a maximum at 800 μg/ml in alloxanized male mice (aqueous extracts).

Leaves Water, ethanol
Hepatoprotective against carbon tetrachloride induced hepatotoxicity in mice.
Saponins, cardiac glycosides, alkaloids, phenolic acids, chlorogenic acid, flavonoids and coumarin compounds [137][138][139][140] Carissa  methanolic extracts of Jatropa curcas L. were more effective than the aqueous and chloroform fractions in inhibiting phospholipase A 2 activity. The authors attributed this to the possible presence of divalent ions (Calcium (II), Strontium (II), and Barium (II) ions) or quercetin-like compounds which are reported to augment the activity of phospholipase A 2 through induction of conformational changes in its substrate-binding sites [167,168]. Table 3 summarizes some of the solvents employed by studies done on antivenom activity of some plants reported in this survey. It is worth noting that methanol appears to be the solvent of choice probably because of its ability to dissolve both polar and nonpolar compounds [169,170]. Testing for the efficacy of plants as antivenins has been perfected using mice as the test specimens. Experimentally, the extracts are tested against the lethal dose of the venom that causes death of 50% of the subjects (LD 50 ). Tests are done either in vivo or in vitro on specific toxic activities of venoms. So far, the inhibitory activity of most extracts has been tested against phospholipase A 2 , one of the toxic constituents of snake venoms [111].

Conclusions and recommendations
Uganda has over 125 districts hence less than 1% of the country have been surveyed for antivenin plants. The inventory of plants utilized by Ugandan communities present considerable potential for the treatment of snake envenomation. The present review therefore opens the lead for isolation and elucidation of the chemical structures of the antivenom compounds from the claimed plants that could be harnessed in combined therapy with commercial antiserum. There is a need for concerted efforts by scholars, traditional healers, local authorities, and the state to address the ongoing African snakebite crisis and meet World Health Organizations' great interest in documenting the various medicinal plants utilized by different tribes worldwide.
Additional file 1. Family, local name, botanical name, growth habit, conservation status, part used, method of preparation and route of administration of antivenin plants used in different districts of Uganda. Authors' contributions TO, SK, and OB designed the study. AO, TO, SS, and KMK performed the literature search. TO, AO, TO, KMK, and OB analyzed the collected data. TO, SK, TO, SS, and OB verified the plant names in botanical databases, Lusoga, Lango, Luganda, and Acholi, respectively. TO, SK, AO, TO, and OB wrote the first draft of the manuscript. All authors revised and approved the final manuscript.

Funding
This research received no external funding.

Availability of data and materials
This is a review article and no raw experimental data was collected. All data generated or analyzed during this study are included in this published article.
Ethics approval and consent to participate Not applicable