Ethnopharmacological Survey of Medicinal Plants Used by Traditional Healers and Indigenous People in Chittagong Hill Tracts, Bangladesh, for the Treatment of Snakebite

Snakebites are common in tropical countries like Bangladesh where most snakebite victims dwell in rural areas. Among the management options after snakebite in Bangladesh, snake charmers (Ozha in Bengali language) are the first contact following a snakebite for more than 80% of the victims and they are treated mostly with the help of some medicinal plants. Our aim of the study is to compile plants used for the treatment of snakebite occurrence in Bangladesh. The field survey was carried out in a period of almost 3 years. Fieldwork was undertaken in Chittagong Hill Tracts, Bangladesh, including Chittagong, Rangamati, Bandarban, and Khagrachari. Open-ended and semistructured questionnaire was used to interview a total of 110 people including traditional healers and local people. A total of 116 plant species of 48 families were listed. Leaves were the most cited plant part used against snake venom. Most of the reported species were herb in nature and paste mostly used externally is the mode of preparation. The survey represents the preliminary information of certain medicinal plants having neutralizing effects against snake venoms, though further phytochemical investigation, validation, and clinical trials should be conducted before using these plants as an alternative to popular antivenom.


Introduction
Snakebite, caused by a bite from a snake, is an accidental injury, which results in puncture wounds inflicted by the animal's fangs and sometimes causes envenomation. Snakes are carnivorous vertebrates of the class Reptilia, order Squamata, and suborder Serpentes. Snakes usually kill their prey with constriction rather than venom, though venomous snakes can be found on every continent except Antarctica [1]. 15% of the almost 3000 known species of snakes are venomous [2][3][4] and, in South Asia, four species were thought to be responsible for causing almost all the deadly cases of venomous snakebites which are referred to as the "big four, " which include the Indian cobra (Naja naja), the common krait (Bungarus caeruleus), Russell's viper (Daboia russelii), and the saw-scaled or carpet viper (Echis carinatus). However, other venomous snakes may also be found in this area [5][6][7] and thus represent a major cause of morbidity and mortality to humans [8][9][10][11]. Exact numbers on the global prevalence of snakebites and the percentage of severe or fatal cases are largely unknown [12]. However, at least 421,000-1,841,000 envenoming and 20,000-94,000 deaths occur worldwide each year due to snakebite [1]. According to Williams et al. [13] these events surpass the number of deaths from tropical diseases such as hemorrhagic fever, dengue, cholera, leishmaniasis, and the Chagas disease.
Incidence of snakebites in Bangladesh is very high like other tropical countries of Southeast Asia [1]. Here most snakebite victims dwelling in rural areas are farmers, fishermen, and hunters [7][8][9] and also there are a high number of Chittagong Hill Tracts C h it ta go n g H il l T ra ct s B a n d a r b a n C h i t t a g o n g R a n g a m a t i K h a g r a c h a r i snakebite occurrences that happened at their homes as most of the snakes are nocturnal animals and poor people have the practice of sleeping on the floor [7]. An epidemiological study estimated about 8000 snakebites per year with 22% mortality which has been identified to be one of the highest in the world [10,14]. Nonetheless, there are approximately 80 species of snakes found in Bangladesh; among them only few are venomous. These are cobra, krait, Russell's viper, sawscaled viper, green snakes, and sea snakes. However, most of the bites are reported by nonvenomous snakes and even as many as 40% bites inflicted by venomous snakes do not produce signs of envenoming [15].
Antivenom is the only therapeutic agent against snake venom available throughout the world. These antivenoms have highly effective neutralizing systemic effects but show some limitations in the inhibition of the local disorders [16,17] and also a chief drawback of serum therapy is its excessive cost and likelihood that victims are often at some distance away from availability of modern treatment when bitten as antivenom treatment should be sought as soon as possible for their potential efficacy. Moreover, there is a crisis in the quality and supply of antivenom serum in the rural areas where most incidences of snakebites occurred [18]. These problems could be subsided by using traditional plant based treatment since approximately 700 plant species are known to possess potential antivenom [19][20][21][22].
Ethnopharmacological survey is important for the conservation and utilization of biological resources [23] since of the 422,000 flowering plants found globally [24] more than 50,000 are used for medicinal purposes [25] and these plants contribute to 33% of drugs produced worldwide [26]. To date approximately more than 6,000 species of indigenous and naturalized plants have been identified out of which more than one thousand contain medicinally useful chemical substances [27,28]. Due to this rich diversity apparently more than 80% of the Bangladeshi use alternative (Ayurveda, Siddha, Unani, and Homeopathy) medicines for their healthcare and herbs constitute a major source of these alternative systems of medicine [29,30].
Several ethnobotanical investigations have been carried out at different parts of the world to explore the herbal treatment against snakebite [16,[31][32][33][34][35]. But there are very few ethnobotanical surveys carried out in Bangladesh to explore the medicinal plants used here in the treatment of snakebite. The present study was conducted in order to document the traditional knowledge of the medicinal plants used by the traditional healers of Bangladesh for treating against snakebite.

Study Area.
The study was conducted in four districts in Chittagong Hill Tracts ( Figure 1) in Bangladesh which is located in South Asia and bordered by India and Myanmar and by the Bay of Bengal to the south (latitudes 20 ∘ and 27 ∘ N and longitudes 88 ∘ and 93 ∘ E) with population over 162 million having 35 smaller groups of indigenous people. The vegetation type of the study area falls under tropical evergreen and semievergreen forests. More than 3 million people live in this

Plants Using in Treatment of Snakebite and Other Relevant
Information. 116 plant species belonging to 48 plant families have been identified as being used in the treatment of snakebite by traditional healers in Bangladesh. The largest number of species was noted from the family Fabaceae (10 species), followed by Apocynaceae (8 species), Caesalpiniaceae (7 species), and Euphorbiaceae (6 species) ( Figure 2). Leaves (43%) were the most frequently used plant parts, followed by roots (27%) and roots stem (9.4%) ( Figure 3). The major mode of preparation is paste (69.3%) followed by juice (21%) and powder (11.23%). Preparations were made with water, honey, wine, lime water, and milk as solvent. The mode of administration was oral (31.9%), topical (56.03%), and oral and topical (12.07%) ( Figure 4). 32% of the reported species were herb which was followed by tree (23.3%) and climber (9.5%). Most of the plants are wild (70%) and some are cultivated (18%), whereas others are both cultivated and wild (  Table 3.

Discussion
Fabaceae is the most dominant family in the current investigation. This is perhaps because of worldwide prevalence of the species from this family [112,113]. Leaves were the major plant parts used solely or mixed with other parts in the treatment of snakebite. Ease of collection of leaves is the prime reason compared to roots, flowers, and fruits [114][115][116].
On the other hand, herbs and trees were the most common habit of the reported plants which might be attributed to the huge number of trees or herbaceous plants naturally abundant in this hilly area [117].   It was very common that blend of different adjuvant including other plant parts was used for the preparation of medication to counteract snake venom. Several researchers also reported this kind of polyherbal treatment [118][119][120][121]. The frequent use of multiple plant remedies might be illustrated by the phenomenon of synergistic actions where two or more plants produce an effect greater than the sum of their individual effects [122]. This is particularly true in case of medicinal plant treatment, since each medicinal plant contains numerous pharmacologically active compounds [118].  Among the management options after snakebite, snake charmers (Ozha in Bengali language) were the first contact following a snakebite for more than 80% of the victims in these areas [10]. We also noticed that the field of "snakes and snakebite" has a mythological fragrance in the mind of people living here. The Ozha not only depends on herbal remedies but also recites mantras (magical/mystical words) to enthrall people. There are also potentially harmful approaches reported few of which are making multiple incisions around the bite site, incorrect application techniques in Evidence-Based Complementary and Alternative Medicine 5          Paste of leaf, fruit, and seed is used on the infected area 2/3 times daily for 2/3 days. Juice of leaf, fruit, and seed is also taken by grinding with milk and sugar. 2 Abroma augusta Linn. f. Root juice is used after maceration. 3 Abrus precatorius L. Seed powder is mixed with Andrographis paniculata seed powder to consume with lemon juice.
3-4 pieces of fresh root are crushed and squeezed; the extract is taken 3-5 times a day for 1 day.
5 Acalypha indica L. Whole plant is made into paste, and the paste thus obtained is divided into 4-5 equal parts; each part is given at 6-hour intervals as an antidote. 6 Achyranthes aspera L.
Fresh leaves extract of about 2 teaspoonfuls is given 4-6 times a day. 7 Acorus calamus L. Fresh rhizome is made into paste. The paste is given with a glass of lukewarm water twice a day for 3 successive days. 8 Aegle marmelos (L.) Corr. Serr.
Whole plants, in tender condition, are made into paste. The paste thus obtained is divided into two equal halves, one half mixed with rice beer applied for cleaning the biting site and the other half again divided into 6 equal portions, and each part is given at an interval of 4-6 hours of time for one day. 9 Albizia procera (Roxb.) Benth.
Fresh root of about one inch long, collected from the plants which are yet not flowered, is given once as an antidote of snakebite.
10 Allium cepa L. Two teaspoonfuls of bulb juice of the plant mixed with mustard oil and administered to expel poison by vomiting.

11
Andrographis paniculata (Burm. f.) Wall. ex Nees (1) Fresh root and leaves are mixed in a ratio of 2 : 3 and the whole mixture is made into paste with a little water. The paste thus obtained is divided into 12-16 equal parts (based on the condition of the patient) and each part is given at regular intervals of 1-2 hours for 2-3 days.
(2) Root paste along with honey in equal parts is given 6-8 times a day as an antidote of snake venom. 12 Annona squamosa L. Incision of snakebite is washed with the juice of plants. 13 Aristolochia indica L.
(1) Fresh root extract mixed with equal amount of root extract of Rauvolfia serpentina is given 4-6 times a day.
(2) Root paste along with honey in equal parts is given 6-8 times a day. 14

Asparagus racemosus
Willd. Leaf extract is applied on the bitten area.

15
Bacopa monnieri (L.) Pennell Dried plant (except the root portion) powder, about 1 teaspoonful, is given with a cup of warm goat milk or black tea 2-3 times of day as an antidote of snakebite.
16 Baliospermum montanum L. Paste is prepared with leaf and applied externally twice a day for 4/5 days. 17 Begonia barbata C.B. Clarke Paste is prepared with stem and leaves and applied once a day for 2/3 days. 18 Bombax ceiba L.
Fresh young shoots (3-5 pieces) are made into paste with black peppers seed (Piper nigrum) and a pinch of camphor (Karpur); the paste thus obtained is given after mixing with a spoonful of honey as an antidote of snake venom.

Stem bark (fresh or dried) about 20 g is made into paste with zinger (rhizome of Zingiber officinale).
The whole paste thus obtained is divided into four equal parts. Each of these 4 parts is given 4 times a day.

20
Byttneria pilosa Roxb. Paste of stem and leaf is applied twice a day until the area is cured.

21
Cajanus cajan (L.) Huth. Paste is made with seed powder of the plant and the juice of leaf of Senna tora. It is then applied twice a day for 2/3 days.

22
Calotropis gigantea (L.) Ait. f. Fresh root with milk of cow is ground to a fine paste and taken as an antidote for snakebite.
About three drops of latex are put on the snake-bitten area and pressed downwards to bleed; root extract is given two cups a day; flower powder is mixed with black pepper and taken. 24

Calycopteris floribunda
Lamk. Root juice is used in infected area.

25
Capparis zeylanica L. Dried fruits with seeds are made into dust; this dust is given as 1 teaspoonful with a glass of lukewarm water as a snake venom antidote.
14 Evidence-Based Complementary and Alternative Medicine 27 Cassia occidentalis L. 20-30 gm of root (fresh or dried) is made into paste with 3-4 pieces of "garlic" (Allium sativum) and a little "gur" (Jaggery); the whole mixture thus obtained is given as an antidote to snakebite. 28 Cassia sophera L. Root (fresh or dried) of about 20 gm is made into paste with 5-7 pieces of black peepers (seeds of Piper nigrum) and the paste is given as an antidote. 29

Catharanthus roseus D. Don
Leaf is grinded after maceration.

30
Cissampelos pareira L. Root paste with 10 g long pepper is prescribed once daily for 5 days.

31
Cissus adnata Roxb. Leaf paste is applied on infected place.

32
Cissus javana DC. Paste is made with leaf and stem, mixing with lime, and applied externally on the biting place tying a piece of cloth for 3/4 days.

33
Clitoria ternatea L. Root powder mixed with milk is taken orally immediately after snakebite.

34
Cycas pectinata Griff. Paste of flower is applied thrice a day for 2/3 days.

35
Cyperus rotundus L. Bulb powder mixed with cow butter to treat snakebite.
Juice is prepared with shoots and mixing with shoots of Peperomia pellucida and 2 spoonfuls are taken thrice a day for 4/5 days. 37

Emblica officinalis
Gaertn. Stem infusion is given orally as an antidote. 38 Entada rheedii Spreng. Paste is prepared with leaf and applied externally once a day for 4/5 days.

39
Erythrina variegata L. Stamen and root bark are mixed in a ratio of 1 : 3 and then they are made into paste. This paste is applied in both ways externally and internally to reduce the swelling, pain of snakebite.

40
Ficus racemosa L. A few drops of its decoction are put into the nostrils, resulting into vomiting and relief; bark paste is applied over the injury.

41
Gmelina arborea L. Inner portion of fresh root (after peeling off the bark) about 20 g is made into paste and this paste is given with a spoonful of honey as an antidote of snakebite.

42
Hedyotis scandens Roxb. Paste is prepared with leaf and stem and applied in warmed condition externally twice a day for 3/4 days.

43
Hemidesmus indicus (L.) R. Br. Root paste is applied as an antidote to snakebite.

Holarrhena antidysenterica (Heyne ex Roth.) Conessi
The roots were rubbed on a stone with a few drops of water and the paste obtained is given internally and applied externally in snakebite.

45
Holarrhena pubescens (Bach.-Ham.) Wall. Seed paste is applied locally as antidote and also for reducing the swelling and pain of snakebite.

46
Homalomena aromatica (Roxb. ex Sims) Schott. Paste of rhizomes is applied until the area is cured.

47
Hyptis suaveolens (L.) Poit. Juice is extracted from leaf and applied externally twice a day for 3 days.
Fresh roots (about 100 g) are crushed and squeezed; the aqueous extract thus obtained is given 10-12 times a day as an antidote.

49
Ixora cuneifolia Roxb. Bark is grinded with water and the paste applied on the biting area twice a day for 4/5 days. 50
Fresh leaves extract is given 20-30 mL at every 1-hour interval for up to 18 hours of snakebite as an antidote.

51
Lantana camara L. Decoction of roots, flowers, and stems is prescribed.

52
Leucas aspera (Willd.) Link. Leaves with pepper and garlic are chewed and spit into the nostrils. 53

Melastoma malabathricum L.
Juice is prepared with leaf and applied externally twice a day for 3/4 days.

54
Melochia corchorifolia L. Leaf paste is applied on infected place.

55
Mirabilis jalapa L. Leaf juice is extracted and 2 spoonfuls are taken twice a day for 2/3 days. Also it is applied topically twice a day in infected areas. 57

Morinda persicifolia
Ham. Paste is prepared with leaf and applied externally twice a day for 3/4 days.

58
Mussaenda roxburghii Hook. f. Paste of leaf is applied on the infected place with tying a piece of cloth.

59
Peliosanthes teta Andr. Paste is prepared with root tuber and applied externally once a day for 2 days.

60
Peperomia pellucida (L.) HBK Juice is prepared with shoots and mixing with shoots of Desmodium triflorum and 2 spoonfuls are taken thrice a day for 4/5 days.

61
Polygonum chinense L. Paste is prepared with leaf and applied externally once a day for 2 days. 62 Pouzolzia indica Gaud. Paste of leaves is applied twice a day for 2/3 days.

63
Premna esculenta Roxb. Paste is prepared with leaf and applied externally twice a day for 3/4 days.

64
Rauvolfia serpentina (L.) Benth. ex Kurz Roots and leaf buds are crushed with milk and made into a paste and used internally and externally on the affected area; rhizome and root decoction is given orally.

65
Senna hirsuta (L.) Irwin & Barneby Paste of leaf is applied topically on the biting place.

66
Senna tora (L.) Roxb. Paste is prepared with leaf and applied once a day for 2/3 days.

67
Sida cordifolia L. Leaf juice is applied to cure snakebite. 68 Sida rhombifolia L. Paste is prepared with leaf and stem and applied topically once a day to infected areas. Also juice of extracted leaf and stem is 2 spoonfuls which are taken four times a day for 4/5 days.
Root juice is mixed with 250 mL water and 100 mL mustard oil. First, ammonium chloride is rubbed on the snake-bitten area and then the mixture of root juice, water, and oil is given orally. Otherwise, 1 handful of fruit is boiled in 1/2 litre of water. The fruits are then squeezed to get the juice, which is orally given to the snake-bitten person to vomit out the poison.
To treat snakebite, spoonful powder with honey is consumed thrice a day after every two hours.
Stem bark powder (about 10 g) is made into paste with a teaspoon full of honey and 5-7 pieces of black pepper (Piper nigrum); this paste is given with a glass of lukewarm goat milk as an antidote to snake venom.

73
Trewia nudiflora L. Paste of leaf is applied topically on the biting place.
Root juice is prepared after maceration and 1 spoonful is taken twice a day until the area is cured.
Handful of leaves is crushed in urine of snake-bitten person and 2-3 drops of extract are passed through the nostrils.

77
Vitis lanata Roxb. Paste is prepared with leaf and applied externally once a day for 3/4 days.

78
Willughbeia edulis Roxb. Latex is collected from stem and applied externally thrice a day for 2/3 days.
tourniquets (e.g., wrong pressure), and sucking blood orally from the multiple cuts which are practiced in an alarmingly high proportion of cases. The species with high FC values is a sign of their diverse and numerous medicinal activities and thus it offers further pharmacological, toxicological, and phytochemical analysis for the discovery of potential novel drugs.
Snake venom contains a complex mixture of enzymes, nonenzymatic proteins, carbohydrates, lipids, and other substances [123][124][125][126] most of which are extremely toxic. Snakebite envenoming has cytotoxic, hypotensive, neurotoxic, or anticoagulant effects [127]. Cytotoxic enzymes, phospholipases A 2 and metalloproteinases, activate proinflammatory mechanisms that result in edema, blister formation, and local tissue necrosis and facilitate the release of bradykinin, prostaglandin, cytokines, and sympathomimetic amines that cause the intense pain [128]. In addition, there are some venom toxins including aminopeptidases having the ability to alter the physiological function of the victims and ultimately causing systemic hypotension [126]. Many snake venoms have peptides that inhibit angiotensin-converting enzyme causing a slump in arterial blood pressure [129]. Moreover, some toxins such as safarotoxins and endothelins are potent vasoconstrictors of coronary arteries and might be 16 Evidence-Based Complementary and Alternative Medicine responsible for myocardial ischemia or cardiac arrhythmias [123]. Neurotoxins cause paralysis by affecting the neuromuscular transmission at either presynaptic or postsynaptic levels [130]. Presynaptic neurotoxins, also called b-neurotoxins, include taipoxin, paradoxin, trimucrotoxin, viperotoxin, Pseudocerastes, textilotoxin, and crotoxin [127] which are phospholipase A 2 complexes that inhibit the release of acetylcholine from the presynaptic terminal [131,132]. On the other hand, postsynaptic neurotoxins including irditoxin [127] called a-neurotoxins cause a reversible blockage of acetylcholine receptors [133][134][135]. Snake venom toxins may also interfere with blood coagulation and cause hemorrhages or thrombosis [125,127,136,137].
Elucidation of the mode of actions of 116 plants individually is beyond the scope of this study. Research suggests extract of different medicinal plants having antivenom activities such as reducing necrotic and hemorrhagic activity as well as preventing cardiac arrest and reversing the effect of paralysis of skeletal muscle caused by snake venom. Also they might inhibit phospholipase A 2 that causes degranulation of mast cell [138] and consequently they prevent release of platelet activating factors and histamine into circulation, preventing hypersensitive anaphylactic reaction [139].
Several studies have been conveyed in finding of active constituents in the plants used against snake venom. Among the 116 plants in this study, the phytochemical investigations are conducted in most of the plants though the compounds rational for antivenom properties are still unknown for most of them. Extensive phytochemical investigations on the plants mentioned in this study could be another mammoth task. Several plant constituents like flavonoids, quinonoid, xanthene, polyphenols, terpenoids lupeol, gymnemagenin, and pentacyclic triterpenes like oleanolic acid, ursolic, tannins, taraxasterol, amyrin, and so forth are found to be present in varying proportions in surveyed plants. These compounds have also been previously tested in vitro for possessing protein binding and enzyme inhibiting properties [140][141][142].
These literature studies revealed that the alkaloids (Eclipta prostrate, Rauvolfia serpentina, Strychnos nux-vomica, and Mimosa pudica), esters (Gloriosa superba), phenolic fraction (Hemidesmus indicus), terpenoids (Aristolochia indica, Andrographis paniculata), and flavonoids fraction (Tephrosia purpurea) neutralized the snake venom activities. Flavonoids have been shown to inhibit phospholipases A 2 , an important component of snake venoms [143]. The antivenom effects of wedelolactone, a coumestan isolated from the Eclipta prostrate, are well cited for antivenom activities [144]. 2-Hydroxy-4-methoxy benzoic acid, found in Hemidesmus indicus root extracts, was identified as a snake venom neutralizing factor which effectively neutralized viper venom induced lethal, hemorrhagic, coagulant, anticoagulant, and inflammatory activity [145]. This compound seems to act through free radical formation system [146] and is one of the mechanisms of venom inhibition. Caffeic acid is present in Strychnos nuxvomica, and the monomeric caffeic acid is a proven antidote against snake venoms when given as oral and parenteral administration [147]. Marmin in Aegle marmelos, a monoterpenoid substituted fernolin [148], has been mentioned as a remedy against snakebite. Piperine from Piper nigrum inhibits the adhesion of neutrophils to endothelial monolayers. Also it possesses inhibitory activities on prostaglandin and leukotrienes and thus possesses anti-inflammatory activity [149][150][151]. Quercetin is a potent inhibitor of lipoxygenase, and free quercetin and its glycosides rutin are present in Allium cepa skins [152]. The aristolochic acid content of Aristolochia indica contains a large number of proteins that cluster under native condition. It shows strong gelatinolytic, collagenase, nuclease, and peroxidase activities. It interacts with the components of snake venom and partially inhibits proteolytic and L-amino acid oxidase activities of the venom [12]. Active principle of Bauhinia forficata has thrombin-like enzyme that acts as potent inhibitor of clotting activity that otherwise causes persistent hemorrhage [153].
Most of the plants documented in this study are used for the treatment of versatility of disease. This trend is a possible indication of the tradition of THPs to develop local healing system through trials and errors for optimal treatment practices [154].
There are resemblances in comparative studies of these cited plants to other surveys regarding medicinal plants having antivenin characteristics (Table 4). Using the same plants in different areas by different cultures for the same purpose might be considered as a justification of their pharmacological efficacy [155].
12 of these cited plants had been found to possess possible toxic potentiality (Table 5). However, among those possibly the most toxic one is Abrus precatorius. It contains abrin, a serious toxic compound, which after penetrating the cells of the body inhibits cell protein synthesis. Human fatal dose of abrin is approximately 0.1-1 mg/kg. But toxins are released only if the seed is chewed and swallowed [91]. Another dangerous plant is Ageratum conyzoides which in ingestion can cause liver lesions and tumors [94,95]. There was a mass poisoning incident reported in Ethiopia as a result of contamination of grain with A. conyzoides [96]. In addition, epidemic dropsy and ocular toxicity have been reported by seed oil of Argemone mexicana [98][99][100][101] and latex of Calotropis procera [105], respectively; the rest are toxic only due to high doses of ingestion. However, a number of phytochemical investigations would be required to declare these plants as being toxic.

Conclusion
This survey represents the contribution of natural flora of Bangladesh to the global approach in the management of snakebite occurrences. The knowledge documented in this study possibly supports the development of novel plant based treatment. Further investigations should be carried on especially in order to ensure safe therapy concerning medicinal plants. Therefore, snake charmers should be trained on as a priority basis. Again, scarcity of supply of snake antivenin is a major factor which needs to be addressed by local production. And in that case these findings regarding herbal antidote would be useful in planning and formulating strategies and specific interventions to combat snakebite related health problems in Bangladesh.   Ficus racemosa L. Bark Tetracyclic triterpene derivatives Cause abnormality of liver and kidney [110] Lantana camara Leaf Triterpene acids Leaf extracts are cytotoxic [111]