An evidence based efficacy and safety assessment of the ethnobiologicals against poisonous and non-poisonous bites used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects.

INTRODUCTION: To explore the ethnobiological wisdom of the tribals of three western districts of West Bengal, India against poisonous and non-poisonous bites and stings, a quantitative approach was adopted. These age-old yet unexplored knowledge can be utilized in finding lead-molecules against poisonous and non-poisonous animal-bites. Further, an evidence-based approach is needed to assess the venom-neutralization ability of plants by experimental studies.
MATERIALS AND METHODS: During 2008-2009 and 2012-2017, 11 ethnomedicinal surveys were carried out to explore the use of medicinal flora and fauna via conducting open semi-structured interviews with 47 traditional healers (THs) or informants. The retrieved dataset was statistically evaluated using seven quantitative-indexes: use-value (UV), informants'-consensus-factor (ICF), fidelity-level (FL), relative-importance (RI), cultural importance-index (CI), index of agreement on remedies (IAR) and cultural agreement-index (CAI). Anti-phospholipaseA2 (PLA2) properties of selected plant extracts were also examined. In addition, the cytotoxicity and genotoxicity of the water extract of the plants showing high FL as well as significant PLA2 inhibitory potential were investigated using Allium cepa root tip assay.
RESULTS: A total of 41 traditional-formulations (TFs) containing 40 plant species (of 39 genera from 28 families) and 3 animal species were prescribed by the THs. Fabaceae exhibited most number of medicinal plants. Piper nigrum (1.78) and Apis cerana indica and Crossopriza lyoni (both 0.21) exhibited the highest UV among the plants and the animals respectively. Stinging of centipede and dog/cat/hyena bite displayed highest ICF (1.00 each). Among the plants, the maximum RI (0.91) and CI (4.98) values were observed for Aristolochia indica. IAR (1.00) was recorded maximum for Achyranthes aspera, Gloriosa superba, Lycopodium cernuum, Smilax zeylanica and Streblus asper. Maximum CAI value was noted for Piper nigrum (5.5096). Among the animals, Apis cerana indica (0.31) and Crossopriza lyoni (1.52) displayed the highest RI and CI values respectively. Crossopriza lyoni (0.99) and Apis cerana indica (1.3871) exhibited maximum IAR and CAI values respectively. Plants showing higher FL exhibited higher anti-PLA2 activity via selective inhibition of human-group PLA2. In addition, Allium cepa root tip assay has indicated the safety and/or toxicity of the plant parts prescribed by the THs. Root water extracts of Aristolochia indica and Gloriosa superba exhibited significant genotoxicity and cytotoxicity.
CONCLUSIONS: Three western districts of West Bengal is the natural abode for many tribal and non-tribal communities. A noteworthy correlation was established between the plants used against poisonous-bites and their anti-PLA2 activity. A few plant parts used by the THs also exhibited high toxicity. Such alternative medical practices serve as the only option in these underprivileged and backward areas during medical-exigencies.

Introduction

Snake envenomation is considered as a major problem worldwide especially in the tropical and subtropical countries including South East Asia [1-4]. There are more than 440,000 snake envenoming and 20,000 deaths each year [2]. Antivenom is considered as an effective treatment against snakebite; however snakebite-associated mortality remains significantly high due to unavailability, limited therapeutic efficacy and safety concerns of conventional antivenins [5]. India presents a very high number of snakebite incidences compared to other countries. Insufficient hospital-based reports indicated the total number of snakebite mortality to be ranging over 1,300 to 50,000 in India [6]. Besides snakebites, scorpion stings also cause severe venom-related injury [7]. Scorpion stings result in severe consequences in human which may lead to even mortality due to neurotoxins present in the venom [8]. Centipede bites have reportedly caused local pain, erythema and edema, nausea and vomiting, headache, lymphadenopathy, rhabdomyolysis, myocardial infarction, hypotension etc. in humans due to myocardial toxic effects of the venom and anaphylaxis [9, 10]. Bee stings are also known to cause heart blocks, syncope and cardiac arrest [11]. Even the stings of the members of Hymenoptera such as bees, hornets, and wasps can be fatal [12]. Animal bites are reported as a serious global health concern and are responsible for almost 1–2% of all visits to the hospital emergency. Most of the animal bites are imposed by dogs (80–90%) and cats (5–15%) and the most common complication following animal bite is wound infection [by Pasteurella multocida, Capnocytophaga canimorsus, Eikenella corrodens and Rhabdovirus (rabies only) etc.] which may lead to sepsis and death especially in the immunocompromised victims [13, 14].

There has always been a philosophical conflict between the mainstream Western medicine and the traditional and complementary medicine or TCM with their possible coexistence and prevalence in the global context [15]. World Health Organization (WHO) has advocated herbal medicines as a valid alternative therapy against many human ailments. According to the WHO, almost 80% of the world’s population rely on TCM for primary healthcare [16]. In India, other alternative medication strategies such as Ayurveda, Siddha, Unani, Tibbi and Homoeopathy have also been popularly used [17]. Purulia, one of the remote and backward districts of India is rich in aboriginals with their age old ethnomedicinal treatments. Remoteness from the nearby towns, unfavorable topography, sparse healthcare facilities and poverty have persuaded the aboriginals to use ethnomedicines especially during serious medical exigencies such as poisonous envenomations.

Phospholipase A2 (PLA2) enzymes, commonly present in the venoms of the snakes from the families Viperidae, Hydrophidae and Elaphidae are implicated to the venom-induced toxic effects [18-20]. Many natural compounds from plants, marine organisms, serum plasma etc. have been tested for their anti-PLA2 properties on snake venoms and/or isolated toxins [20]. Higher PLA2 inhibitory properties of the plant extracts indicate their possible snake venom neutralization ability. Earlier, in-vitro, in-vivo and in-silico studies performed on the anti-PLA2 properties of the natural compounds indicated the tremendous potential of the compounds as novel inhibitors of ophidian toxins [21-23].

Standardized Allium cepa root tip test is commonly used as a fast and reliable method to assess cytotoxicity and genotoxicity of plant extracts, isolated compounds, synthetic derivatives, nano-materials, environmental pollutants, pesticides, industrial effluents etc. mostly on the basis of mitotic anomalies and chromosomal aberrations studies [24-28]. Medicinal plants, although used for their therapeutic properties, also exhibit dose dependent toxic effects. Allium cepa root tip meristematic cells assay easily determines dose dependent cytotoxicity and genotoxicity of medicinal plant extracts and plant-derived compounds [29-32].

The primary aim of the present study is to enumerate the indigenous use of phyto- and zoo-therapeutics prescribed by the traditional healers (THs) practicing in the three westernmost districts of West Bengal, India, against poisonous and non-poisonous animal bites and insect stings. The present work is also intended to elucidate the preparations and applications of traditional formulations (TFs) and the statistical interpretation of the retrieved ethnobiological information. In the present study, anti-PLA2 properties of selected plant extracts were also evaluated against human group PLA2 and porcine group PLA2 to find out any selective inhibition of the said extracts on pro-inflammatory human group PLA2 without or minimally inhibiting the porcine group digestive PLA2. In addition, Allium cepa root tip meristematic cells were employed to assess the genotoxic and cytotoxic potential of selected antivenin plants showing higher fidelity level (FL) as well as superior PLA2 inhibitory potential.

Materials and methods

Ethics statement

The permission (ethnomedicinal survey, sanctioned in 2012) was granted by the West Bengal Biodiversity Board [OM No. 040/3/K(Bio)−1/2012], Govt. of West Bengal, India. West Bengal Biodiversity Board is a Govt. of the State of West Bengal who issues necessary permission for ethnobiological surveys. Earlier, preliminary information on the area and the people were gathered by an approval granted by West Bengal Biodiversity Board (1.1.2008–22.5.2009) [Memo No. 5k(bio)-2/2007]. This Govt. body has approved this work which compiles with the guidelines. In addition, written and verbal consent were taken from the local people during the survey which also compiles to the ethical guidelines as provided by the aforesaid Govt. body.

Selection of informants and data collection

During 2008–2009, initial surveys were conducted to explore the people and their practices in different villages of the three districts (Fig 1). Later, during 2012–2017, a total number of 11 ethnomedicinal surveys were conducted to explore the use of medicinal flora and fauna to treat poisonous and non-poisonous animal bites and insect stings. A total number of 47 THs (also known as the informants) were interviewed on the basis of their ethnomedicinal knowledge as well as their reputation and social acceptability as practitioners. A semi-structured questionnaire (S1 and S2 Figs) was supplied to each TH to explain the use (against particular ailments) and composition of each monoherbal or polyherbal ethnomedicinal preparations, their local names, plant/animal parts used, detection of disease symptoms, method of composing formulations, route of administration, addition of other ingredients etc. Before the start of the questionnaire session, prior consent was sought from the informants (S3 Fig). Besides, name, gender, age, aboriginal group etc. of the THs were also noted. Habit, habitat, taxonomic and identification features of the ethnobiologicals as well as date, time, season and site of collection were also documented. Herbarium sheets were prepared using medicinal plants preferably in their flowering phases and specific voucher numbers were assigned to each. Plants, plant parts and methods of preparations were also photographed for documentation. Literatures such as Prain (1903) [33] and Sanyal (1994) [34] were used to identify local flora and Tropicos plant database from Missouri Botanical Garden (www.tropicos.org) was consulted to verify names, synonyms and authors’ citations. Economic Botany Data Collections Standards (EBDCS) proposed by Cook (1995) [35] was used to present the use categories mentioned by the informants (Table 1). Herbarium specimens with respective voucher numbers were preserved at the Department of Zoology, Sidho-Kanho-Birsha University, Purulia, West Bengal. Ethnozoologicals were initially documented during the field visits and were subsequently identified by the first author of this article. Animal samples were photographed and common samples were preserved for future reference.

Fig 1

Map of the study area (maps not to scale) (Map was created using the editor tools of the ArcGIS 10.3.1 software.).

Table 1

Traditional Formulations (TFs) used against poisonous and non-poisonous animal bites and insect stings.

Disease/ Disorder1 1: Scorpion sting (20090400)
symptoms: pain and burning sensation around stinging area and full organ
Traditional Formulations (TFs)	Composition3	Modes of preparation and methods of administration2
TF1	Bisalya karani (Barleria lupulina) bark: 50 g	Bark is grinded to make a paste; the paste is used as ointment (1070000) on the wound.
TF2	Khakra change/Bandarlej (Setaria glauca) roots: 10 g	Roots are grinded to make a paste; the paste is applied on the wound.
	Tar genda (Tridax procumbens) roots: 10 g
	Bhin Kambal (Premna herbacea) roots: 10 g
TF3	Rasun (Allium sativum) bulbs: 5 in no.	Roots and Rasun bulbs are grinded to make a paste; the paste is applied on the wound with a pinch of salt.
	Nirbisi (Cyperus kyllingia) root: 20 g
	Common salt (NaCl): a pinch
TF4	Seowra (Streblus asper) leaves: 4–5 in no.	Leaves are taken and rubbed by hand to extract juice and the juice is given on the stinging area.
TF5	Alkushi (Mucuna pruriens) seeds: 2–3 in no.	A powder is made from the seeds; the powder is applied over the stinging areas.
TF6	Harin singha (Euphorbia tirucalli) latex: 3–4 drops	Applied on the stinging area.
TF7	Gave verenda (Jatropha gossypifolia) bark	Bark paste is directly applied on the affected area.
TF8	Scorpion (Buthus sp.)	Internal material/ digestive system of scorpion by which the person was attacked is to rub on stinging area.
TF9	Ol (Amorphophallus paeoniifolius) stem	The stem is heated over the flame and the affected area is covered with this heated stem.
TF10	Kend (Diospyros melanoxylon) bark	Bark juice is applied on the stinging area.
TF11	Kalojira (Nigella sativa) seeds	Grinded seeds are applied on the stinging area.
TF12	Liyaya ful/ Bishlanguli (Gloriosa superba) roots	Fresh root is rubbed on the affected area.
TF13	Chatpati ghash (Ruellia tuberosa) leaves: 10 g	Paste is applied on the affected area.
	Tarnd mula (Premna herbacea) roots: 10 g
TF14	Narkol (Cocos nucifera) oil: 5 to 10 ml	Both the ingredients are mixed well; the mixture is used as an ointment on the stinging area.
	Naphthalene: 1 ball
TF15	Sada dhurba ghash (Cynodon dactylon) leaves: 10 g	Both the ingredients are crushed to make a paste; the paste with kalo saban is applied on the stinging area.
	Boka benri (Ipomoea carnea) leaves: 10 g
	Kapor kachar kalo saban (local black soap for washing clothes): 5 g
Disease/ Disorder 2: Stinging of honey bee or wasp or hornet (20090200 and 20090300)
Symptoms: Swelling of affected area and severe burning sensation
TF16	Hetal (Crateva adansonii) bark	Bark of Hetal is grinded to make paste. Bark paste is applied on the wound.
TF17	Lal Tulsi (Ocimum sanctum) leaves: 50 g	Leaf paste is smeared over the body to keep away bees.
TF18	Narkol (Cocos nucifera) oil: 5 to 10 ml	Both the ingredients are mixed well. Mixture is used like ointment on stinging area.
	Naphthalene: 1 ball
TF19	Common salt (NaCl): ½ teaspoonful	Application of a lotion (1050000) prepared from the salt and the kerosene oil just after stinging is helpful to relieve pain.
	Kerosene oil: 1 teaspoonful
TF20	Petrol: 5–10 ml	After removing the sting, cotton is soaked with petrol and it is rubbed on the stinging area until the burning sensation is gone. Then juice of Piyaj is then applied on the affected area.
	Cotton/cotton cloth: 1 in no.
	Piyaj (Allium cepa) bulb: 50 g
TF21	Liyaya ful/Bishlanguli (Gloriosa superba) roots	Fresh root is rubbed on the affected area.
TF22	Baichi (Flacourtia indica) stem and bark: 10 g	A paste of stem and bark is applied on the wound.
TF23	Rangani/Shiyal kanta (Argemone mexicana) seeds: 10 g	The seeds are crushed well to make a paste and the paste is applied on the stinging area.
Disease/ Disorder 3: Stinging of centipede
TF24	Ol (Amorphophallus paeoniifolius) stem	The stem is heated over the flame and the affected area is covered with this heated stem.
Disease/ Disorder 4: Dog/cat/hyena bite (12020000)
TF25	Chitchiti (Achyranthes aspera) roots: 20 g	Root paste is applied on the wound and molasses is taken orally.
	Molasses: 20 g
TF26	Crossopriza lyoni (Blackwall, 1867) (tailed cellar spiders): 1–2 in no.	Ingredients are properly mixed and taken orally to reduce the effect of the poison.
	Chanchhi (The deposits at the bottom of a pot after the milk is boiled): 50 g
TF27	Tentul (Tamarindus indica) tender twigs	Freshly taken twigs are heated over the flame and to cover the wound.
Disease/ Disorder 5: Snake bite (20090600)
TF28	Bisalya karani (Barleria lupulina) bark: 50 g	Bark is grinded to make a paste. The paste is used as ointment at the wound.
TF29	Mrita sanjiboni (Lycopodium cernuum) leaves: 50 g	All the ingredients are grinded to make a paste; the paste is applied on the wound.
	Asthi sancharini (Scindapsus officinalis) leaves: 50 g
	Bisalya karani (Barleria lupulina) leaves: 50 g
TF30	Chotopard (Cissampelos pareira) roots: 10 g	Root is grinded to make a paste; the paste is fed to the victim to prevent the spread of the poison.
TF31	Bamun hati/Ram datum (Smilax zeylanica) roots: 1 inch	Root of Ram datum and Iswarmul along with Rabing seeds are grinded to form a paste; the paste is applied on the wound.
	Iswarmul (Aristolochia indica) roots: 1 inch
		If patient becomes senseless, powder of dry leaves and roots of Kanch mala is given to the victim as snuff.
	Black pepper or Rabing (Piper nigrum) seeds: 2½ in no.
	Kanch mala (Abrus precatorius) dry leaves: 5 in no.
	Kanch mala roots: 2 g
TF32	Dusatin lata (Gloriosa superba) bulb	Dusatin lata is purified with bovine urine for seven days. The purified Dusatin lata is taken with dust of black pepper.
	Black pepper or Rabing (Piper nigrum) seeds
	Bovine urine
TF33	White Kuch fall/Kukurmala (Abrus precatorius) roots: 2 ½ g	Roots of Kuch fall and black pepper are grinded together and taken orally.
	Black pepper or Rabing (Piper nigrum) seeds: 10 in no.
TF34	Ishwarmul (Aristolochia indica) roots: 1½ inch	Paste is made and given to the patient orally.
	Rangani (Solanum surattense) roots: 1½ inch
	Bagh nakhi (Martynia annua) roots: 1½ inch
	Piyal (Buchanania lanzan) bark: 1½ inch
	Black pepper or Golki/Rabing (Piper nigrum) seeds: 12 in no.
TF35	Ishwarmul (Aristolochia indica) bark and roots: 5 g	Juice of bark and root is drunk twice daily.
TF36	Barachadar (Rauvolfia canescens) roots: 10 g	10 ml of root juice is prescribed orally and is also applied on the wound.
TF37	Anantamul/Analsing (Hemidesmus indicus) roots: 10 g	Root paste is applied on the wound.
	Apis cerana indica Fabricius (Indian honeybee) fresh honey from hives: 50 ml
TF38	Ishermul/Bhedi-Janete (Aristolochia indica) roots	Root paste with a paste of ten peppers is given as antidote.
	Black pepper or Golki/Rabing (Piper nigrum) seeds: 10 in no.
TF39	Chotopar/Chotkipar/Tijumala (Cissampelos pareira) roots	Root paste with a paste of ten peppers is given as antidote.
	Black pepper or Golki/ Rabing (Piper nigrum) seeds: 10 in no.
TF40	Mahadevjata/Ishwarjata (Uraria picta) leaves	Leaf paste is given as an antidote
TF 41	Swet akanda (Calotropis gigantea) leaves	A paste is prepared with all the ingredients and is offered to the patient to drink.
	Bishaynandi (Glossogyne bidens) whole plant
	Krishnajata (Nardostachys jatamansi) roots

1, 2 The numbers used in parenthesis after the diseases/disorders and methods of administration are according to the recommendations given by Cook, 1995 as Economic botany data collection standard (EBDCS), plant parts, body parts and processes, disorders/effects, medicinal applications and non-vertebrate organisms (Master lists of states for Level 3 descriptors) (Economic Botany Data Standard; https://www.kew.org/tdwguses/rptMasterListMain.htm).

3In composition, in no. used after the numbers stands for in number i.e. the number of that plant part used.

Analyses of ethnobiological data

Use Value (UV)

Use value (UV) is a quantitative analysis applied to enumerate the relative importance of local ethnobiologicals [36]. The equation is: (where U denotes the number of citations per ethnobiological and n denotes the informant numbers who were interviewed for a given ethnobiological)

Informant Consensus Factor (ICF)

The informant consensus factor (ICF) was calculated to evaluate user variability of ethnobiologicals [37]. The ICF is estimated by the following equation: (where nur is the used citation number in each category and nt is the number of ethnobiologicals reported)

Fidelity Level (FL)

The fidelity level (FL) is determined as the percentage of informants reporting the application of specific ethnobiological for similar purpose [38]. It was calculated as: (where Np is the informant numbers separately reporting an application of an ethnobiological to treat a specific disease whereas N is the total number of informants citing the use of an ethnobiological to treat any specific ailment)

Relative Importance (RI)

The relative importance (RI) of an ethnobiological depicts the most used ethnobiologicals with the most notable number of ethnomedicinal applications [39, 40]. The RI is represented by the equation: (where PP is the total number of curative properties ascribed to an ethnobiological divided by the highest number of similar activities ascribed to the widely used ethnobiologicals. AC represents the number of ailment categories reported to be cured by a specific ethnobiological divided by the highest number of ailment categories treated by the most prolifically used ethnobiologicals).

Cultural Importance index (CI)

The cultural importance index (CI) is calculated by the total number of informants citing the use of each ethnobiological [41]. The CI is represented by: (where UR represents the use report, N represents the gross number of informants whereas from u1 to uNC represent UR of each use-category [41, 42]).

Index of Agreement on Remedies (IAR)

The index of agreement on remedies (IAR) depicts the significance of each ethnobotanicals [43]. IAR is calculated as: (where nr is the summation of the reports on the use of an ethnobiological and na is the number of ailment categories against which the ethnobiological is administered [44]).

Cultural Agreement Index (CAI)

The cultural agreement index (CAI) [45] is calculated as: (where CII represents the cultural importance index and IAR represents the index of agreement on remedies)

Enzymes and reagents

Human group PLA2 and porcine group PLA2 were used as PLA2. Lecithin (egg yolk phosphatidylcholine), red phenol, sodium taurodeoxycholate (NaTDC) and ethylmethanesulfonate (EMS) were procured from Sigma, India. A UV-VIS spectrophotometer was used for the measurements using the visible spectra.

PLA2 inhibitory activity

PLA2 inhibitory activity of medicinal plants were tested only with the plants showing high FL. A protocol by De Aranjo and Radvany (1987) was followed to assess the PLA2 inhibitory activity of the plant extracts [46]. In this study, the inhibitory effects of the water extracts were assessed on the pro-inflammatory human group PLA2. As a control set, porcine group PLA2 was used. The substrate was composed of egg yolk phosphatidylcholine or lecithin (3.5 mM) in a mixture containing NaTDC (3 mM), NaCl (100 mM), CaCl2 (10 mM) and red phenol (0.055 mM) employed as colorimetric indicator dissolved in H2O (100 ml). The pH of the reaction mixture was calibrated to 7.6. The two PLA2 were solubilized in acetonitrile (10%) at 0.02 and 0.002 μg/μl concentrations respectively. Each plant extract (10 ml) was incubated with PLA2 solution (10 μl) at room temperature for 20 min. Following this, PLA2 substrate (1 ml) was added, and following hydrolysis in next 5 min, the OD was read at 558 nm. The inhibition percentage was determined by comparing with the control (absence of plant extract) and the IC50 values were obtained from the curve.

Allium cepa root tip meristem genotoxicity test

A protocol by Aşkin Çelik and Aslantürk (2010) with minor modifications was employed to assess the genotoxicity and cytotoxicity of medicinal plants with high FL as well as superior human-group PLA2 inhibitory properties [47]. Tap water was used as negative control whereas ethyl methanesulfonate (EMS, 2 X 10−2 M) was employed as positive control. After 24 h exposure to the control sets and the aqueous extract (2.5, 5 and 10 mg/ml) of the plants, Allium cepa root tips were cut from the bulbs to fix them in ethanol: glacial acetic acid: ethanol (1:3 v/v) overnight at 4 ˚C. Following fixation, the roots were put in 70% (v/v) aqueous ethanol and were stored in a fridge. The root tips were then hydrolyzed using hydrochloric acid (HCl) (1N) for 3 min and were subsequently squashed on microscope slides with 45% acetic acid after staining with aceto-orcein [2% (w/v)]. Slides were visualized under a Carl Zeiss compound microscope with 40X10 magnification (n = 5/set). The cytotoxic and genotoxic features visualized were i. mitotic-index (MI) which is the ratio of the total number of mitotically dividing cells and the total number of cells present in a microscopic field expressed in percentage; ii. micronuclei (MNC) formation during interphase cells per 1000 cells (‰MNC) and iii. chromosomal anomalies viz. chromosomal fragments, anaphase bridges, multipolarity, laggard chromosomes and ghost cells.

Results

Demographic profile of the THs

Among the 47 THs interviewed, 41 were men and 6 were women aged between 32 to 85 years. Thirty three male THs were having primary or parallel professions like agriculture, animal husbandry or other services whereas the women were all housewives. Remaining eight male THs practiced traditional medicine as their primary profession. District-wise, among 47 THs, 33 represented Purulia and 8 and 6 THs represented Bankura and West Midnapore districts respectively. Most THs have been practicing traditional medicine for years which they acquired from their ancestry.

Traditional therapeutics using ethnobiologicals

A total number of 41 TFs were prescribed by the THs against poisonous and non-poisonous animal bites and insect stings (Table 1). They have reported 40 plant species (of 39 genera from 28 families) and 3 animal species (of 3 genera from 3 families) as direct ingredients in TFs against 5 poisonous and non-poisonous animal bites and insect stings. Among plant families, Fabaceae exhibited most number of medicinal plants (4 species) (Fig 2). A total number of 3 animal species were reported (Table 1), parts of which were used as direct ingredients or as additives in TFs. Salt, soap, kerosene oil, petrol, molasses, black pepper, milk, bovine urine and honey were added as additives, as taste enhancers and/or to enhance the efficacy of formulations applied topically. The plant and animal species with their local names, families and part(s) used are presented in Table 2 indicating their use in respective TFs and against the particular type of bite. Plant voucher numbers and habit types are also tabulated in Table 2. Herbs (20 records) exhibited the most common plant habit (Fig 3) whereas roots (21 uses) were reported as the most commonly used plant part (Fig 4).

Fig 2

Distribution of plant families.

Table 2

Details of plants and animals as ingredients in Traditional Formulations (TFs) against poisonous and non-poisonous animal bites and insect stings.

Botanical/Zoological binomials	Voucher No.1	Family	Vernacular names	Habit	Part(s) used2	Used in TFs3	Ailment/disorder treated4	UV	RI	CI	IAR	CAI
Abrus precatorius L.	PB001	Fabaceae	Kuch fall/ Kukurmala/Kanch Mala	climber	root (12040000)	31, 33	5	0.22	0.31	1.43	0.98	1.4014
Achyranthes aspera L.	PB002	Amaranthaceae	Chitchiti	herb	root	25	4	0.23	0.34	1.54	1.00	1.54
Argemone mexicana L.	PB036	Papavaveraceae	Rangani/ Shiyal kanta	herb	seed (11000000)	23	2	0.24	0.39	1.59	0.99	1.5741
Allium cepa L.	PB003	Amaryllidaceae	Piyaj	herb	bulb (12020000)	20	2	0.27	0.37	1.57	0.98	1.5386
Allium sativum L.	PB004	Amaryllidaceae	Rasun	herb	bulb	3	1	0.31	0.37	2.66	0.98	2.6068
Amorphophallus paeoniifolius (Dennst.) Nicolson	PB005	Araceae	Ol	herb	stem (6000000)	9, 24	1, 3	0.48	0.55	3.71	0.99	3.6729
Apis cerana indica Fabricus (8031100)	-	Apidae	Moumachhi	-	honey	37	5	0.21	0.31	1.43	0.97	1.3871
Aristolochia indica L.	PB006	Aristolochiaceae	Iswarmul/Ishermul/Bhedi-Janete	herb	root, bark (7000000)	31, 34, 35, 38	5	1.59	0.91	4.98	0.99	4.9302
Barleria lupulina Lindl.	PB007	Acanthaceae	Bisalya karani	herb	bark	1, 28, 29	1, 5	1.38	0.89	3.87	0.98	3.7926
Buchanania lanzan Spreng.	PB008	Anacardiaceae	Piyal	tree	bark	34	5	0.20	0.26	1.55	0.98	1.519
Buthus sp. Leach, 1815 (8010100)	-	Buthidae	Kankra bichha	-	digestive system (6000000)	8	1	0.19	0.25	1.44	0.97	1.3968
Cissampelos pareira L.	PB009	Menispermaceae	Chokipar/Tijumala/ Chotopard	climber	root	30, 39	5	1.11	0.79	3.65	0.98	3.577
Cocos nucifera L.	PB010	Arecaceae	Narcol	tree	oil	14, 18	1, 2	0.98	0.77	3.60	0.98	3.528
Crateva adansonii DC.	PB011	Capparaceae	Hetal	tree	bark	16	2	0.20	0.30	1.49	0.97	1.4453
Crossopriza lyoni (Blackwall, 1867) (8010200)	-	Pholcidae	Makadsa	-	whole body (11040000)	26	4	0.21	0.28	1.52	0.99	1.5048
Cynodon dactylon (L.) Pers.	PB037	Poaceae	Sada dhurba ghash	herb	leaf (8000000)	15	1	0.20	0.29	1.49	0.97	1.4453
Cyperus kyllingia Endl.	PB012	Cyperaceae	Nirbishi	herb	root	3	1	0.21	0.30	1.52	0.99	1.5048
Diospyros melanoxylon Roxb.	PB013	Ebenaceae	Kend	tree	bark	10	1	0.23	0.29	1.57	0.97	1.5229
Euphorbia tirucalli L.	PB014	Euphorbiaceae	Harin singha	tree	latex (13020000)	6	1	0.24	0.32	1.57	0.99	1.5543
Flacourtia indica (Burm. f.) Merr.	PB015	Salicaceae	Baichi	tree	stem, bark	22	2	0.22	0.32	1.53	0.98	1.4994
Gloriosa superba L.	PB016	Colchicaceae	Liyaya ful/Bishlanguli/Dusatin lata	herb	root	12, 21, 32	1, 2, 5	1.25	0.88	3.43	1.00	3.43
Glossogyne bidens (Retz.) Alston	PB039	Asteraceae	Bishaynandi	herb	whole plant (2000000)	41	5	0.21	0.28	1.47	0.99	1.4553
Hemidesmus indicus (L.) R. Br. ex Schult.	PB017	Apocynaceae	Anantamul/Analsing	herb	root	37	5	0.29	0.37	1.59	0.98	1.5582
Ipomoea carnea Jacq.	PB038	Convolvulaceae	Boka benri	shrub	leaf	15	1	0.28	0.38	1.55	0.98	1.519
Jatropha gossypifolia L.	PB018	Euphorbiaceae	Gave verenda	shrub	bark	7	1	0.22	0.33	1.49	0.97	1.4453
Lycopodium cernuum L.	PB019	Lycopodiaceae	Mrita sanjibani	herb	leaf	29	5	0.23	0.33	1.50	1.00	1.5
Martynia annua L.	PB020	Martyniaceae	Bagh nakhi	herb	root	34	5	0.23	0.29	1.51	0.98	1.4798
Mucuna pruriens Scop.	PB021	Fabaceae	Alkushi	shrub	seed	5	1	0.31	0.28	1.77	0.97	1.7169
Nardostachys jatamansi (D. Don) DC.	PB040	Caprifoliaceae	Krishnajata	herb	root	41	5	0.21	0.29	1.41	0.98	1.3818
Nigella sativa L.	PB022	Ranunculaceae	Kalojira	herb	seed	11	1	0.29	0.35	1.75	0.99	1.7325
Ocimum sanctum L.	PB023	Lamiaceae	Lal tulsi	herb	leaf	17	2	0.30	0.34	1.86	0.99	1.8414
Piper nigrum L.	PB024	Piperaceae	Rabing/Golki	vine	seed	31, 32, 33, 34, 38, 39	5	1.78	0.80	5.68	0.97	5.5096
Premna herbacea Roxb.	PB025	Lamiaceae	Tarnd mula	herb	root	2	1	0.25	0.33	1.49	0.98	1.4602
Rauvolfia canescens L.	PB026	Apocynaceae	Barachadar	shrub	root	36	5	0.25	0.31	1.54	0.98	1.5092
Ruella tuberosa L.	PB027	Acanthaceae	Chatpati ghash	herb	leaf	13	1	0.24	0.30	1.65	0.99	1.6335
Scindapsus officinalis (Roxb.) Schott	PB028	Araceae	Asthi sancharini	climber	leaf	29	5	0.20	0.29	1.49	0.97	1.4453
Setaria glauca (L.) P. Beauv.	PB029	Poaceae	Khakra change/Bandarlej	herb	root	2	1	0.21	0.29	1.50	0.98	1.47
Smilax zeylanica L.	PB030	Smilacaceae	Bamunhati/Ramdatun	shrub	root	31	5	0.25	0.29	1.55	1.00	1.55
Solanum surattense Burm. f.	PB031	Solanaceae	Rangani	herb	root	34	5	0.19	0.25	1.46	0.99	1.4454
Streblus asper Lour.	PB032	Moraceae	Seowra	tree	leaf	4	1	0.22	0.31	1.49	1.00	1.49
Tamarindus indica L.	PB033	Fabaceae	Tentul	tree	twig	27	4	0.27	0.31	1.60	0.97	1.552
Tridax procumbens L.	PB034	Asteraceae	Tar genda	herb	root	2	1	0.26	0.32	1.58	0.98	1.5484
Uraria picta (Jacq.) Desv.	PB035	Fabaceae	Mahadevjata/Ishwarjata	herb	leaf	40	5	0.22	0.32	1.49	0.99	1.4751

1Voucher no. indicates the herbarium sheet number deposited for future reference.

2The numbers used in parenthesis after the parts used are according to the recommendations given by Cook, 1995 as Economic botany data collection standard (EBDCS), plant parts, body parts and processes, disorders/effects, medicinal applications and non-vertebrate organisms (Master lists of states for Level 3 descriptors) (Economic Botany Data Standard; https://www.kew.org/tdwguses/rptMasterListMain.htm).

3Used in TFs indicates the plants used in traditional formulations given in Table 1. UV: use value; RI: Relative importance; CI: Cultural importance index; IAR: Index of agreement on remedies; CAI: cultural agreement index.

4The numbers use in this column are in accordance to the numbers used for different disease/ disorders in Table 1.

Fig 3

Distribution of plant habit types.

Fig 4

Distribution of plant and animal parts used by the THs.

Preparations and applications of TFs

Paste or ointment (23 uses) was recorded as the most used form of preparations (Fig 5) and topical administration was the most common mode of application (Fig 6). Among the animal parts, whole body, digestive system and honey were used. In most of the cases, plant and/or animal parts were ground to powder to make an ointment and were applied topically. In a few cases, the plant parts were heated over the flame and the affected area was covered with this heated part. TF19 was found to be applied as a lotion prepared from the salt and the kerosene oil and was applied on the stinging area to relieve pain just after an insect-sting. Most of the preparations were used either orally or topically; however, TF36 was used both orally as well as topically on the wound. In another interesting study, in TF8, the internal material/ digestive system of scorpion (Buthus sp.) by which the person was stung was rubbed on the stinging area. In TF26, Crossopriza lyoni (tailed cellar spiders) was used with plant part and milk and was taken orally to reduce the effect of the poison.

Fig 5

Distribution of drug preparations used by the THs.

Fig 6

Distribution of modes of drug administration prescribed by the THs.

Quantitative ethnobiology: UV, ICF, FL, RI, CI, IAR and CAI

Piper nigrum (1.78) and Apis cerana indica and Crossopriza lyoni (both 0.21) exhibited the highest UV among the plants and the animals respectively (Table 2). Stinging of centipede and dog/cat/hyena bite displayed highest ICF (ICF = 1.00 each) (Table 3). The ethnobotanicals/ethnozoologicals exhibiting high (70–100%), moderate (50–70%) and low (<50%) FL are presented in Table 4.

Table 3

Sting/bite category and corresponding Informant Consensus Factor (ICF) depicted from the interviews with the THs.

Sting/bite category	nur = number of use citations in each category	nt = number of species used against a particular ailment by all informants	Informant consensus factor (ICF) nur−nt/nur−1
Scorpion sting	28	22	0.22
Stinging of honey bee or wasp or hornet	16	9	0.47
Stinging of centipede	4	1	1
Dog/cat/hyena bite	7	1	1
Snake bite	30	25	0.17

Table 4

Fidelity Level (FL) of the ethnobiologicals used depicted from the interviews with the THs.

Disease/ailment category	FL (%) category	Most favored plant/animal used against particular bite/sting
Scorpion sting	High FL (70–100%)	Allium sativum
		Barleria lupulina
		Cynodon dactylon
		Cyperus kyllingia
		Gloriosa superba
		Ipomoea carnea
		Mucuna pruriens
		Nigella sativa
		Premna herbacea
		Setaria glauca
	Moderate FL (50–70%)	Diospyros melanoxylon
		Euphorbia tirucalli
		Jatropha gossypifolia
		Streblus asper
	Low FL (<50%)	Amorphophallus paeoniifolius
		Buthus sp.
		Cocos nucifera
		Ruellia tuberosa
Stinging of honey bee or wasp or hornet	High FL (70–100%)	Allium cepa
		Cocos nucifera
		Ocimum sanctum
	Moderate FL (50–70%)	Argemone Mexicana
		Flacourtia indica
		Gloriosa superba
	Low FL (<50%)	Crateva adansonii
Stinging of centipede	Moderate FL (50–70%)	Amorphophallus paeoniifolius
Dog/cat/hyena bite	Moderate FL (50–70%)	Achyranthes aspera
		Crossopriza lyoni
		Tamarindus indica
Snake bite	High FL (70–100%)	Abrus precatorius
		Apis cerana indica
		Aristolochia indica
		Cissampelos pareira
		Gloriosa superba
		Hemidesmus indicus
		Piper nigrum
		Rauvolfia canescens
		Smilax zeylanica
	Moderate FL (50–70%)	Barleria lupulina
		Buchanania lanzan
		Glossogyne bidens
		Martynia annua
		Nardostachys jatamansi
		Scindapsus officinalis
	Low FL (<50%)	Calotropis gigantea
		Lycopodium cernuum
		Solanum surattense
		Uraria picta

FL: fidelity level.

Other quantitative indices viz. RI, CI, IAR and CAI were also calculated for the plants and animals used as ethnomedicine (Table 2). Among the plants, maximum RI value (0.91) as well as CI value (4.98) were observed for Aristolochia indica. IAR which was calculated on the basis of importance of each species, was recorded maximum (1.00) for 5 plant species such as Achyranthes aspera, Gloriosa superba, Lycopodium cernuum, Smilax zeylanica and Streblus asper. Maximum CAI value was noted for the plant species Piper nigrum (5.5096). Among the animals, Apis cerana indica (0.31) displayed the highest RI value and Crossopriza lyoni (1.52) exhibited maximum CI value. Crossopriza lyoni also exhibited maximum IAR value (0.99) whereas Apis cerana indica (1.3871) was recorded for the maximum CAI value.

Use of ethnobotanicals: Toxicity aspects and conservation status

The use of the whole body of tailed cellar spider Crossopriza lyoni and the digestive system of the scorpion Buthus sp. may be implicated to possible toxicity and adverse effects in the recipients of oral or topical mode of administration. However, no such toxicity was reported by the THs. One of the prime conservation strategies adopted by the tribal people was to protect the plants by worshipping them in sacred groves. The authors found that plants such as Tamarindus indica, Cissampelos pareira, Streblus asper, Calotropis gigantea, Abrus precatorius, Ocimum sanctum, Achyranthes aspera and Aristolochia indica etc. were conserved in sacred groves.

Determination of PLA2 inhibition

The objective here was to find out plant extracts showing selective inhibition against the pro-inflammatory human group PLA2 without or minimally suppressing the porcine group digestive PLA2. Initially, three extracts [water, methanol and chloroform-ethanol (1:1)] exhibited promising outcome regarding PLA2 inhibition. However, only water extract was analyzed further since most of the ethnomedicines were prepared using water. The water extract of Aristolochia indica roots demonstrated the most significant inhibition of the enzyme human PLA2 with an IC50 value of 0.73 mg/ml. Following Aristolochia indica, water extracts of other plant parts showing promising PLA2 inhibition were Mucuna pruriens seeds (IC50 value = 0.79 mg/ml), Allium cepa bulbs, Gloriosa superba roots (both with IC50 value = 0.81 mg/ml), Hemidesmus indicus roots (IC50 value = 0.83 mg/ml), Nardostachys jatamansi roots (IC50 value = 0.88 mg/ml), Rauvolfia canescens roots (IC50 value = 0.90 mg/ml) and Piper nigrum seeds (IC50 value = 0.97 mg/ml) (Table 5). These findings indicated a selective inhibition of the extracts against the two PLA2.

Table 5

PLA2 inhibitory activities of the water extracts of the ethnobotanicals with high FL.

Ethnobotanicals and parts	IC50 values (mg/ml) on human group PLA2	IC50 values (mg/ml) on porcine group PLA2	Inhibition specificity (IC50 porcine group PLA2/IC50 human group PLA2)
Abrus precatorius L. root	1.76	>5	>2.84
Achyranthes aspera L. root	1.89	>5	>2.64
Argemone mexicana L. seed	1.67	>5	>2.99
Allium cepa L. bulb	0.81	3.2	3.95
Allium sativum L. bulb	1.98	>5	>2.52
Amorphophallus paeoniifolius (Dennst.) Nicolson stem	2.20	>5	>2.27
Aristolochia indica L. root	0.73	3.1	4.24
Barleria lupulina Lindl. bark	2.34	>5	>2.13
Buchanania lanzan Spreng. bark	3.34	>5	>1.49
Cissampelos pareira L. root	3.76	>5	>1.33
Cocos nucifera L. oil	3.67	>5	>1.36
Crateva adansonii DC. bark	2.89	>5	>1.73
Cynodon dactylon (L.) Pers. leaf	2.67	>5	>1.87
Cyperus kyllingia Endl. root	3.45	>5	>1.45
Diospyros melanoxylon Roxb. bark	4.54	>5	>1.10
Euphorbia tirucalli L. latex	3.09	>5	>1.62
Flacourtia indica (Burm. f.) Merr. stem	4.12	>5	>1.21
Gloriosa superba L. root	0.81	3.3	4.07
Glossogyne bidens (Retz.) Alston whole plant	2.98	>5	1.67
Hemidesmus indicus (L.) R. Br. ex Schult. root	0.83	3.4	4.09
Ipomoea carnea Jacq. leaf	3.67	>5	>1.36
Jatropha gossypifolia L. bark	3.09	>5	>1.61
Lycopodium cernuum L. leaf	4.86	>5	>1.03
Martynia annua L. root	4.71	>5	>1.06
Mucuna pruriens Scop. seed	0.79	3.2	4.05
Nardostachys jatamansi (D. Don) DC. root	0.88	2.9	3.29
Nigella sativa L. seed	3.22	>5	>1.55
Ocimum sanctum L. leaf	3.33	>5	>1.50
Piper nigrum L. seed	0.97	3.6	3.71
Premna herbacea Roxb. root	4.60	>5	>1.08
Rauvolfia canescens L. root	0.90	>5	>5.55
Ruella tuberosa L. leaf	4.23	>5	>1.18
Scindapsus officinalis (Roxb.) Schott leaf	3.49	>5	>1.43
Setaria glauca (L.) P. Beauv. root	3.32	>5	>1.50
Smilax zeylanica L.	3.48	>5	>1.43
Solanum surattense Burm. f. root	4.04	>5	>1.23
Streblus asper Lour. root	3.19	>5	>1.56
Tamarindus indica L. leaf	4.04	>5	>1.23
Tridax procumbens L. twig	4.54	>5	>1.10
Uraria picta (Jacq.) Desv. leaf	2.78	>5	>1.79

IC50: The half maximal inhibitory concentration; PLA2: phospholipases A2.

Assessment of cytotoxicity and genotoxicity by Allium cepa root tip assay

Aqueous extracts of Allium cepa bulb, Hemidesmus indicus root, Nardostachys jatamansi root and Piper nigrum seed did not affect MI significantly since they demonstrated almost similar results as shown by the negative control. Mucuna pruriens seed and Rauvolfia canescens root water extracts exhibited slight inhibition of MI whereas the roots of Aristolochia indica and Gloriosa superba exhibited significant inhibition of MI especially at higher concentrations. Although the positive control EMS (2 × 10−2 M) showed maximum inhibition of MI (%) (1.58), Aristolochia indica root extracts at 10 mg/ml concentration showed potent antimitotic activity in Allium cepa root meristems with an MI (%) of 1.77. In addition, with increasing concentration of the water extracts of the used plant parts, a gradual decline in the MI was noted which was statistically comparable with both the controls (Fig 7). A few plant part extracts, at higher concentration caused chromosomal aberrations such as chromosome breaks, anaphase bridges, multipolarity, laggard chromosomes and ghost cells. However, the chromosomal aberrations were most profound in root tips treated with the positive control EMS. Genotoxicity of the root extracts was also recorded in the interphase MNC formation which was expressed as per 1000 cells (‰MNC). The root water extracts of Aristolochia indica and Gloriosa superba exhibited maximum genotoxic and cytotoxic potential which was comparable to the EMS. MNC formation was also found to be the highest when root tip cells were treated with the root water extracts of Aristolochia indica and Gloriosa superba (Table 6).

Fig 7

Mitotic Index (MI) in the root meristematic cells of Allium cepa in control and treatment concentrations of water extract of ethnobotanicals with high Fidelity Level (FL).

Table 6

Chromosomal aberrations and micronuclei formation in the root meristematic cells of Allium cepa in control and treatment concentrations of water extract of ethnobotanicals showing high FL and high PLA2 inhibition.

Treatment groups	Concentrations	Chromosomal fragments ±SD	Multipolarity ±SD	Anaphase bridge ±SD	Laggard chromosome ±SD	Ghost cells ±SD	MNC (‰)±SD
negative control (tap water)	-	-	-	-	-	-	-
positive control (EMS)	2 × 10−2 M	-	9.61±4.49	4.02±3.56	3.39±2.45	13.90±3.32	0.48±0.19
Allium cepa L. bulb, water extract	2.5 mg/ml	-	-	-	-	-	-
	5 mg/ml	-	-	-	-	-	-
	10 mg/ml	-	-	-	-	-	-
Aristolochia indica L. root, water extract	2.5 mg/ml	6.65±1.19	7.85±2.21	1.91±2.22	2.46±3.33	10.59±2.87	0.54±0.23
	5 mg/ml	5.76±0.46	7.04±1.09	2.08±1.87	1.50±2.09	11.38±3.90	0.49±0.22
	10 mg/ml	6.09±0.44	6.28±2.24	2.23±1.89	1.70±3.45	11.10±4.05	0.44±0.45
Gloriosa superba L. root, water extract	2.5 mg/ml	4.66±0.37	6.84±1.76	2.66±0.71	1.02±0.67	12.76±3.67	0.39±0.17
	5 mg/ml	5.54±1.45	5.50±0.89	1.89±0.98	2.81±0.98	12.06±4.56	0.59±0.14
	10 mg/ml	4.81±0.67	6.93±0.69	1.94±0.91	1.19±0.20	10.90±2.68	0.60±0.09
Hemidesmus indicus (L.) R. Br. ex Schult. root, water extract	2.5 mg/ml	1.67±0.76	-	-	-	1.27±0.22	-
	5 mg/ml	2.02±0.34	-	-	-	2.32±1.48	-
	10 mg/ml	1.88±1.73	-	-	0.56±2.831.75	2.01±1.09	-
Mucuna pruriens Scop. seed, water extract	2.5 mg/ml	2.02±1.77	4.77±0.73	0.67±2.56	0.98±1.79	7.70±2.67	-
	5 mg/ml	1.72±0.80	5.07±0.66	1.04±1.29	0.68±1.20	8.93±2.90	0.12±0.08
	10 mg/ml	1.90±0.47	4.17±1.39	1.87±1.62	1.10±1.11	8.88±1.96	0.11±0.17
Nardostachys jatamansi (D. Don) DC. root, water extract	2.5 mg/ml	1.05±0.33	2.21±1.98	-	-	2.76±2.69	-
	5 mg/ml	0.66±0.21	1.65±1.54	-	-	2.90±2.34	-
	10 mg/ml	1.14±0.20	2.94±2.02	-	-	1.98±2.93	-
Piper nigrum L. seed, water extract	2.5 mg/ml	-	-	-	-	-	-
	5 mg/ml	-	-	-	-	-	-
	10 mg/ml	-	-	-	-	-	-
Rauvolfia canescens L. root, water extract	2.5 mg/ml	3.45±0.22	5.09±1.45	1.56±1.98	1.92±2.34	9.09±2.82	0.29±0.18
	5 mg/ml	4.98±0.29	4.96±0.46	2.20±2.74	2.02±2.67	11.10±2.78	0.32±0.23
	10 mg/ml	4.02±33	5.01±0.48	1.85±2.89	2.22±3.03	10.63±3.22	0.17±0.25

Discussion

Study area and the aboriginals

The three western districts of the state of West Bengal, India are Purulia, Bankura and West Midnapore. This area is an extension of the Chota Nagpur Plateau (22°-25° 30'N and 83°47'-87° 50'E). Santhalis, Oraons, Mundas, Bhumijs, Birhors, Gonds, Kharias, Mal Pahariyas and Hos are the major tribal groups residing in these three districts. Austro-Asiatic and Dravidian languages are common among the tribal people. Natural tropical forests are prevalent in the westernmost district i.e. Purulia; however the forests have been constantly decreasing due to human exploitation. In this area, forests have supported the livelihood of its inhabitants via providing food, fodder, clothing, building materials, timber and so on. Undulated topography and extreme climate also counted for their overdependence on forest resources instead of opting for conventional agricultural practices. However the two districts Bankura and West Midnapore have cultivable lands and the presence of tribal groups is significantly lower in the two districts compared to Purulia. Over the past few years, irrigation and social forestry have improved the agricultural practices and productivity as well as forest coverage in the aforesaid areas. Once, Purulia was mentioned as the one of the 250 most backward districts (out of 640) of India, for which it received fund from the Backward Regions Grant und Program (BRGF) [48]. Non-availability of mainstream medication and remoteness of hospitals contribute to their over-reliance on age-old traditional medicines prescribed by THs. However, recent technological and communicational advancement alongside medical and backward development facilities have lifted the standard of living of the tribal populations. This elevation in social and economic conditions in the tribal populations subsequently declined the use of traditional medication that also led to the indifference and ignorance among the younger generations in taking up traditional healing as professions. Hence, the present documentation is also a timely representation of rapidly vanishing medicinal folklore of the local ethnic groups.

Earlier, a number of ethnobiological surveys were conducted globally to explore the use of plants and animals against poisonous bites in northwest Colombia [49], Ethiopia [50], Hainan Island, China [51] and so on. Moreover, plants used against snakebites in Santarém, western Pará, Brazil were validated scientifically against hemorrhagic activity caused by Bothrops jararaca venom [52]. Ethnobotanical use of antivenin plants used in Uganda were also evidenced by pharmacological analysis [53]. Neutralization of lethal, enzymatic and hemorrhagic effects of Bothrops atrox venom by medicinal plants from Colombia was documented in a series of studies [49, 54, 55].

Some plants viz. Hemidesmus indicus [56], Gloriosa superba [57], Tamarindus indica [58], Aristolochia indica [59-61] etc. depicted in the present study have been investigated previously in vitro and/or in vivo for evaluating the anti-venom properties of their extracts and phytochemicals. The present study also reveals a few animals or animal products used in the folkloric medicine in polyherbal formulations active against poisonous or non-poisonous bites. The authors have also noted the inseparable existence of magical beliefs and prescribed medicine. For example, in TF8, the scorpion envenomation victim is given the internal material/ digestive system of the same scorpion by which the person was stung.

In India, up to one million snake bites are recorded in a single year of which as many as 50,000 are recorded as initial death. In fact, roughly the number of people died of snakebites in India, is almost equal to the total deaths due to snakebite in the rest of the world. And yet most of these deaths could have been prevented if necessary medical care was taken. Till date, the snake anti-venom is the only treatment for poisonous snakebites, which is produced from the snake venom itself. Most of the snake bites are either dry bites or from non-poisonous snakes. Still death occurs. There are many instances where patients die only because of fear and psychological shock. THs provide at least first aid and mental support to both patient and his/her families, where there is no other option [62, 63].

Mode of administration of TFs against poisonous and non-poisonous bites seemed to have played a crucial role since few of the plant or animal materials would have caused toxicity if applied orally instead of topical application. Authors have noted that most of these medicines were applied on the bitten or stinging area as paste or ointment or lotion, which was obviously meant to relieve the pain instantly or to alleviate symptoms of poisonous bites at some later stages. Among the TFs, only TF26, TF30, TF32, TF33, TF35 and TF41 were taken orally. Rest of the TFs were applied topically. In case of TF31, if patient becomes senseless, powder of dry leaves and roots of Kanch mala is given to the victim as snuff. As a prophylactic measure, Lal Tulsi (Ocimum sanctum) leaves were smeared over the body to keep away bees. In TF25, Chitchiti (Achyranthes aspera) root paste and molasses were prescribed topically and orally respectively against dog/cat/hyena bite.

Quantitative ethnobiology

Relative importance of the plants and animals used in the ethnomedicines is indicated by UV and among the plants Piper nigrum exhibited maximum UV (1.78) because of its widespread use in TFs to attenuate bitter taste of the formulations. Aristolochia indica (1.59), Barleria lupulina (1.38) and Gloriosa superba (1.25) were recorded among the other botanicals with high UVs which reflects their widespread acceptance as ethnomedicine. Similarly, Aristolochia indica (0.91), Barleria lupulina (0.89) and Gloriosa superba (0.88) also exhibited high RI values. The highest CI and CAI values were displayed by Piper nigrum which were 5.68 and 5.95 respectively. Highest IAR (1.00) was exhibited by four plants such as Achyranthes aspera, Gloriosa superba, Lycopodium cernuum and Streblus asper. In comparison to the plants, very less number of animals were present in the TFs which is reflected in their low UVs. High/moderate/low FL is displayed by various plant or animal species since they have been differentially accepted by the THs in their formulations.

Use of plants and animals as drugs: Toxicity and conservation aspects

Despite being used since time immemorial, use of traditional medicine must consider safety and toxicity issues. Animal samples such as digestive system of scorpion, whole body of spider and animal excreta such as bovine urine may possess toxicity and pathogens causing adverse effects in the recipients. Topical application of naphthalene, kerosene and petrol is also needed to be assessed for toxicity and side effects. THs have reported Aristolochia indica as a potent anti-venom herb. However, it has also been reported to cause aristolochic acid nephropathy (AAN) in different parts of the globe which has led to the discontinuation of the drug in different herbal products containing aristolochic acid [64]. Among the ethnozoologicals, Apis cerana indica, Buthus sp. and Crossopriza lyoni have not yet been assessed by the International Union for Conservation of Nature and Natural Resources (IUCN) Red List [source: The IUCN Red List of Threatened SpeciesTM (http://www.iucnredlist.org/search))].

PLA2 inhibitory activity of the medicinal plants

Interestingly, most of the plants (Aristolochia indica, Mucuna pruriens, Allium cepa, Gloriosa superba, Hemidesmus indicus, Nardostachys jatamansi, Rauvolfia canescens and Piper nigrum) showing high FL (70–100%) exhibited high PLA2 inhibitory activity against human group PLA2 without or minimally inhibiting the digestive porcine group PLA2. Similar observations were noted while elucidating the anti-PLA2 activity of Aloe vera leaf skin extracts. Earlier, selective inhibition of pro-inflammatory PLA2 group IIA was attributed to the catechin tannins present in the water extract of Aloe vera. It was further suggested that the compounds present in the extracts exhibiting anti-PLA2 properties were different than those displaying antioxidant properties [65]. High PLA2 inhibitory potential of the plants indicates their possible effectiveness against snake venom. In exploration of alternative antivenin, especially in rural India where lack of conventional antivenin against poisonous snakebites causes a number of mortality and morbidity every year. Available literature also supports the traditional use of Aristolochia indica, Gloriosa superba, Hemidesmus indicus [66] and Nardostachys jatamansi [67] against snakebite. Snake venom neutralization ability of Hemidesmus indicus [55], Aristolochia indica [58] and Gloriosa superba [68] has also been assessed via in vitro and in vivo studies. A number of phytochemicals such as 2-hydroxy-4-methoxy benzoic acid and lupeol acetate from Hemidesmus indicus [69, 70] and aristolochic acid from Aristolochia indica [59, 60] also exhibited potent antivenin as well as anti-PLA2 activities.

Aristolochic acid [8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid], an uncompetitive inhibitor with a Ki of 9.9 × 10−4 M (with phosphatidylcholine as substrate), was found to interact with the major basic PLA2 from Vipera russelli venom. Administration of aristolochic acid inhibited edema-inducing activity of Vipera russelli PLA2. Suppression of edema-induction by aristolochic acid was manifested when it reached the site; however, it did not aid in recovery. Aristolochic acid also failed to suppress other pathological properties of PLA2 [60]. In another study, aristolochic acid inhibited human synovial fluid (HSF)-PLA2, porcine pancreatic PLA2, Naja naja PLA2, and human platelet derived PLA2 dose dependently with sensitivity of these PLA2s to aristolochic acid varied significantly: HSF-PLA2> Naja naja PLA2> human platelet PLA2> porcine pancreatic PLA2. In addition, it was indicated that inhibition of HSF-PLA2 was possibly mediated via direct interaction with the enzyme [70]. The compound 2-hydroxy-4-methoxy benzoic acid from Hemidesmus indicus exhibited adjuvant efficacy and antiserum potentiation in rabbits immunized with Vipera russellii venom demonstrating potent venom neutralization ability (lethal and hemorrhage) [69]. Hemidesmus indicus root extract-derived lupeol acetate remarkably neutralized Daboia russellii venom-induced edema, haemorrhage, defibrinogenation, PLA2 activity and lethality in male albino mice. Furthermore, Naja kaouthia venom-induced neurotoxicity, cardiotoxicity, respiratory modulations and lethality in the animals were also neutralized by the compound. In addition, venom-induced alterations in super oxide dismutase (SOD) activity and lipid peroxidation were also antagonized by lupeol acetate [70]. Therefore, ethnobiological use of a few medicinal plants reported in the present study is being supported by scientific literature describing their in vitro and in vivo efficacy as potent antivenin.

Genotoxic and cytotoxic effects of plant extracts on Allium cepa root tip meristems

The plant parts used by the THs were extracted in water and the roots of Aristolochia indica and Gloriosa superba demonstrated significant antimitotic and genotoxic potential in Allim cepa root tip assay. Therefore, oral administration of such extracts are discouraged also keeping in mind the worldwide occurrence of aristolochic acid nephropathy (AAN) due to the consumption of Aristolochia preparations in traditional medicine and in health supplements [71]. Earlier, Allium cepa root tip meristems were used to assess the genotoxic and cytotoxic effects of Aristolochia birostris water and alcoholic extracts [72]. Hemidesmus indicus root extract was evaluated in cultured lymphocytes for its genotoxic and antigenotoxic effects [73]. Species of Gloriosa were also evaluated for their antimitotic effects on onion roots [74]. The present study evaluated the mutagenic and genotoxic properties of the plant extracts in a dose dependent manner and further suggested to take precautions before using few plant extracts in human.

Conclusions

The three western districts of the state of West Bengal are the natural dwelling place for many indigenous communities surviving the climatic and economic hardships and exercising their time tested medical practices mostly based upon the uses of ethnobiologicals. These traditional and alternative treatments serve as the only option in these underprivileged and geographically remote areas during medical-exigencies like snake envenomation. The present study depicts a quantitative ethnobiological analysis among the aboriginals from the area against poisonous and non-poisonous animal bites and insect stings. However, the efficacy of the antivenin ethnobiologicals are needed to be validated scientifically. In addition, bioactivity guided isolation of phyto-constituents may lead to the templates for synthesis of novel antivenins. In addition, plants with higher FL displayed superior anti-PLA2 properties via selective inhibition of human-group PLA2. In addition, Allium cepa root tip assay revealed significant genotoxic and cytotoxic properties of some plant extracts. Therefore, concomitant studies on toxicology and safety of the plant extracts are also needed for safe efficacious application of botanical-derived antivenin.

Interview data sheet.

(DOCX)

Click here for additional data file.

Specimen copy of an interview data sheet.

(DOCX)

Click here for additional data file.

A consent letter in Bengali language provided by one of the informants.

(DOCX)

Click here for additional data file.

(PPTX)

Click here for additional data file.

14 Jul 2020

PONE-D-20-17909

An evidenced based efficacy and safety assessment of the antivenom ethnobiologicals used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

PLOS ONE

Dear Dr. Malik,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The detailed reports of the reviewers are enclosed above. All questions deserve attention, especially the improvement of English language use and the discussion section's reformulation.

Please submit your revised manuscript by Aug 28 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Benito Soto-Blanco, DVM, MSc, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To ensure that you obtained ethics approval before your study began, please upload the ethics approval document issued by West Bengal Biodiversity Board approval (1.1.2008-22.5.2009) [Memo No. 5k(bio)-2/2007].

3. We note that [Figure(s) 1] in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1.    You may seek permission from the original copyright holder of Figure(s) [1] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2.    If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article “An evidenced based efficacy and safety assessment of the antivenom ethnobiologicals used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects.” presents a survey conducted with traditional healers from a specific region in India, aiming to catalogue traditional formulations used as treatments against animal bites and stings and assess their efficacy and safety using experimental assays with the reported ethnobotanicals. It is an interesting work that addresses an important matter because not only the traditional knowledge can drive further investigations on the pharmacological potential of the natural products, leading to new drugs, but it is also important to characterize possible toxic effects of such formulations in order to protect the population making use of them. Considering the neglected issue of envenoming and the scarcity of available treatments, and the fact that the populations living in poor and isolated districts are mostly affected by these problems, the presented article has merit.

I would recommend this work for publication in PLOSOne, but it must be submitted to a major revision, in order to attend the journal’s quality standards. First of all, the manuscript is to long. Information should be presented in a more concise and direct language, highlighting the main findings. For this, English language, although intelligible, must also be revised. Other issues are listed below:

� Title: the work title mentions “antivenom ethnobiologicals”, but dog/cat/hyena bites, which do not contain venom, are listed as ailments for the traditional formulations. Whether the title should be modified or these particular conditions must be taken out from the results.

� The interviewees are mentioned as “traditional healers” (TH) and “traditional medicine man” (TMM) along the text. This can cause confusion, so only one of the terms should be chosen.

� Material and Methods: in the “Study area and aboriginals” session, the authors make narrative description that, although interesting, should be better placed in the discussion session. The Material and Methods should deliminate the study subject in more direct form.

� The period of time when the survey was conducted differs between the ‘Abstract’ (2008-2009 and 2010-2017) and ‘Materials and Methods’. Which one is correct?

� Table 1 would be better placed in the ‘Results session’.

� In the Results session, it would be interesting to have the demographic profile of the interviewees (sex/age/profession/district).

� The second session in ‘Results’ is named ‘Preparations, application and dose-dependence of TFs’, but there is no mention of the dose-dependence in the results.

� Table 2 should me made horizontally, to increase readability and should be placed in the ‘Quantitative ethnobiology’ session. Also, in this table, what does “voucher number’ stands for? And the numbers that appear after the description of ‘parts used’? This information should be removed or explained in the table’s legend. Information on abbreviations is also lacking in table 5.

� As a suggestion, data presented in table 6 would be more clearly visualized as a Graph.

� Table 7 would also be better read horizontally.

� The Discussion session, unlike the other session, is rather succinct. I believe the other parts of the manuscript could be more concise and Discussion should be further explored, comparing the found information with other similar articles, performed in other regions.

� In line 419, authors state that ‘Most of the snake bites are either dry bites or from non-Poisonous snakes. Still death occurs. There are many instances where patients die only because of fear and psychological shock.’ This seemed odd to me. Are there any references to support this statement?

� In line 494 of the ‘Conclusion’, authors claim that ‘Various conservation strategies have also been described as adopted by the indigenous people in order to use plant resources sustainably.’. I did not find the description of these strategies throughout the text.

Reviewer #2: A broad review regarding the English language, punctuation and spacing should be made.

Several references are mentioned incorrectly during the text, such as seen on lines 102, 105 and 109, for example.

During introduction, authors stress that PLA2 enzymes are responsible for the inflammatory effects of snake bites, and that plant extracts detain some sort of PLA2 neutralizing capacity. However, activity of PLA2 of both human and porcine origin are also evaluated. These subgroups must be more clearly determined in methodology and results and discussion.

Authors should classify the different Authors also claim to evaluate genotoxicity and cytotoxicity, and that should be clear as an aim of the present study as well. The aim of the study is not clearly outlined by the authors.

On Table 1, TF4 is explained as masticated by hand, a term with dubious meaning.

Table 2 is impossible to read and contains some of the main results from the experiment that should be clearly available to the reader.

Table 7 also needs formatting.

Discussion is very superficial, mainly replicating results obtained. It should be rewritten, stressing what each plant extract detains from a biochemical or pharmacological level that corroborates with a lesser or more prominent effect regarding PLA2 neutralization.

Since no aim of the study was properly established, conclusion is also in need of revision.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Aug 2020

To ensure that you obtained ethics approval before your study began, please upload the ethics approval document issued by West Bengal Biodiversity Board approval (1.1.2008-22.5.2009) [Memo No. 5k(bio)-2/2007].

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. Thank you. The desired documents are attached. The document of project [Memo No. 5k(bio)-2/2007] carried out is approved by the Principal, Acchuram Memorial College, under Sidho Kanho Birsha University, the then institute of the first author. The same document also confirms the other projects carried out in this regard. The second document from West Bengal Biodiversity Board, a Govt. Body also confirms the same project [Memo No. 5k(bio)-2/2007] on “Preparation of people’s biodiversity register of Jhalda-Darda Gram Panchayat” carried out by the first author. This was issued by Sr. Research Officer, West Bengal Biodiversity Board. Authors having all these documents in original and can exhibit if needed. Permission Form or other proof of granted permissions as an "Other" file being submitted.

We note that [Figure(s) 1] in your submission contain [map/satellite] images which may be copyrighted. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: Map was created using the editor tools of the ArcGIS 10.3.1 software. It is mentioned now in the caption of Fig. 1.

Reviewer #1:

The article “An evidenced based efficacy and safety assessment of the antivenom ethnobiologicals used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects.” presents a survey conducted with traditional healers from a specific region in India, aiming to catalogue traditional formulations used as treatments against animal bites and stings and assess their efficacy and safety using experimental assays with the reported ethnobotanicals. It is an interesting work that addresses an important matter because not only the traditional knowledge can drive further investigations on the pharmacological potential of the natural products, leading to new drugs, but it is also important to characterize possible toxic effects of such formulations in order to protect the population making use of them. Considering the neglected issue of envenoming and the scarcity of available treatments, and the fact that the populations living in poor and isolated districts are mostly affected by these problems, the presented article has merit.

I would recommend this work for publication in PLOSOne, but it must be submitted to a major revision, in order to attend the journal’s quality standards. First of all, the manuscript is to long. Information should be presented in a more concise and direct language, highlighting the main findings. For this, English language, although intelligible, must also be revised. Thank you for your kind comments. Now after the revision, we have tried to use concise and direct language which has also been revised extensively.

Other issues are listed below:

Title: the work title mentions “antivenom ethnobiologicals”, but dog/cat/hyena bites, which do not contain venom, are listed as ailments for the traditional formulations. Whether the title should be modified or these particular conditions must be taken out from the results. Thank you for your kind comments. It is corrected as suggested by you.

The interviewees are mentioned as “traditional healers” (TH) and “traditional medicine man” (TMM) along the text. This can cause confusion, so only one of the terms should be chosen. Thank you for your kind comments. It is corrected as suggested by you.

Material and Methods: in the “Study area and aboriginals” session, the authors make narrative description that, although interesting, should be better placed in the discussion session. The Material and Methods should deliminate the study subject in more direct form.

Thank you for your kind comments. It is corrected as suggested by you.

The period of time when the survey was conducted differs between the ‘Abstract’ (2008-2009 and 2010-2017) and ‘Materials and Methods’. Which one is correct?

Thank you for your kind comments. It is corrected as suggested by you.

Table 1 would be better placed in the ‘Results session’.

Thank you for your kind comments. It is corrected as suggested by you.

In the Results session, it would be interesting to have the demographic profile of the interviewees (sex/age/profession/district).

Thank you for your kind comments. It is corrected as suggested by you.

The second session in ‘Results’ is named ‘Preparations, application and dose-dependence of TFs’, but there is no mention of the dose-dependence in the results.

Thank you for your kind comments. It (dose dependence) is deleted as suggested by you.

Table 2 should me made horizontally, to increase readability and should be placed in the ‘Quantitative ethnobiology’ session.

Thank you for your kind comments. It is corrected as suggested by you.

Also, in this table, what does “voucher number’ stands for? And the numbers that appear after the description of ‘parts used’? This information should be removed or explained in the table’s legend.

Thank you for your kind comments. It is corrected as suggested by you. Voucher no. and the numbers that appear after the description of ‘parts used’ are explained in the footnote.

Information on abbreviations is also lacking in table 5.

Thank you for your kind comments. It is added as suggested by you.

As a suggestion, data presented in table 6 would be more clearly visualized as a Graph.

Thank you for your kind comments. It is corrected as suggested by you.Table 6 is replaced by

Table 7 would also be better read horizontally.

Thank you for your kind comments. It is corrected as suggested by you.

The Discussion session, unlike the other session, is rather succinct. I believe the other parts of the manuscript could be more concise and Discussion should be further explored, comparing the found information with other similar articles, performed in other regions.

Thank you for your kind comments. It is corrected as suggested by you.

Discussion part is further elaborated comparing the found information with other similar articles, performed in other regions.

In line 419, authors state that ‘Most of the snake bites are either dry bites or from non-Poisonous snakes. Still death occurs. There are many instances where patients die only because of fear and psychological shock.’ This seemed odd to me. Are there any references to support this statement?

Thank you for your kind comments. References are added to support this statement.

In line 494 of the ‘Conclusion’, authors claim that ‘Various conservation strategies have also been described as adopted by the indigenous people in order to use plant resources sustainably.’. I did not find the description of these strategies throughout the text.

Thank you for your kind comments. This part is deleted and rewritten.

Reviewer #2:

A broad review regarding the English language, punctuation and spacing should be made. Thank you for your kind comments. We have corrected the language with the best of our ability.

Several references are mentioned incorrectly during the text, such as seen on lines 102, 105 and 109,

Thank you for your kind comments. It is corrected as suggested by you.

During introduction, authors stress that PLA2 enzymes are responsible for the inflammatory effects of snake bites, and that plant extracts detain some sort of PLA2 neutralizing capacity. However, activity of PLA2 of both human and porcine origin are also evaluated. These subgroups must be more clearly determined in methodology and results and discussion.

Authors should classify the different Authors also claim to evaluate genotoxicity and cytotoxicity, and that should be clear as an aim of the present study as well.

Thank you for your kind comments. It is corrected as suggested by you. We have added portions in the methodology and result sections where the subgroups are more clearly determined. We have also added a number of references in the discussion to classify the different Authors also claim to evaluate genotoxicity and cytotoxicity.

The aim of the study is not clearly outlined by the authors.

Thank you for your kind comments. The aim of the study is now clearly added at the end of introduction section.

On Table 1, TF4 is explained as masticated by hand, a term with dubious meaning.

Thank you for your kind comments. It was corrected and replaced.

Table 2 is impossible to read and contains some of the main results from the experiment that should be clearly available to the reader.

Thank you for your kind comments. The table was reframed to be clearly available to the reader.

Table 7 also needs formatting.

Thank you for your kind comments. As suggested by the first reviewer we have replaced the data of Table 7 with Figure 7 presenting the same dataset.

Discussion is very superficial, mainly replicating results obtained. It should be rewritten, stressing what each plant extract detains from a biochemical or pharmacological level that corroborates with a lesser or more prominent effect regarding PLA2 neutralization.

Thank you for your kind comments. The discussion part is elaborated explaining the plant extract from a biochemical or pharmacological level with a number of newly added references which corroborates with a lesser or more prominent effect regarding PLA2 neutralization.

Since no aim of the study was properly established, conclusion is also in need of revision.

Thank you for your kind comments. We have rewritten the aim of the study and hence the conclusion is also rewritten accordingly.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

20 Oct 2020

PONE-D-20-17909R1

An evidence based efficacy and safety assessment of the ethnobiologicals against poisonous and non-poisonous bites used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

PLOS ONE

Dear Dr. Malik,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 04 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Benito Soto-Blanco, DVM, MSc, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although the manuscript has been consistently improved from the first submission, there are still some points that should be further addressed prior to publication:

--> In Table 1, there are still unexplained numbers, placed after "Disease/ Disorder" and after some words like "ointment

(1070000)". These numbers should be further explained in the legend or footnote. In addition, the units abbreviations described in the TF Composition (eg: Seowra (Streblus asper) leaves: 4-5 in no.) should also be described in the table's footnotes.

--> Likewise, for Table 2, numbers placed after "Parts used" (eg: root (12040000)), and "Ailment treated" are not explained neither in the table's legend nor in the footnote.

--> The session "Use of ethnobotanicals: toxicity, conservation status and Economic Botany Data Standard" is still a bit confusing, as different subjects are discussed in the same paragraph, without an evident connection between them.

--> I am still concerned about the statement in lines 428-429: "There are many instances where patients die only because of fear and psychological shock." I believe that, indeed, the stress caused by a snakebite can cause some harm but not be the responsible for the victim's death. I did not find support to this statement in the respective references. Therefore, I would recommend to rephrase the statement or to remove it from the discussion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Nov 2020

As needed Figures S1, S2 and S3 are included in this version of submission.

Although the manuscript has been consistently improved from the first submission, there are still some points that should be further addressed prior to publication:

Our answer: Thank you.

In Table 1, there are still unexplained numbers, placed after "Disease/ Disorder" and after some words like "ointment (1070000)". These numbers should be further explained in the legend or footnote.

In addition, the units abbreviations described in the TF Composition (eg: Seowra (Streblus asper) leaves: 4-5 in no.) should also be described in the table's footnotes.

Our answer: The numbers used in parenthesis after the diseases/disorders, parts and methods of administration in table 1 and 2 are according to the recommendations given by Cook, 1995 as Economic botany data collection standard (EBDCS), plant parts, body parts and processes, disorders/effects, medicinal applications and non-vertebrate organisms (Master lists of states for Level 3 descriptors) (Economic Botany Data Standard; https://www.kew.org/tdwguses/rptMasterListMain.htm). 3In composition, in no. used after the numbers stands for in number i.e. the number of that plant part used.

This information is added to the footnote in both the tables.

In addition, the units abbreviations described in the TF Composition (eg: Seowra (Streblus asper) leaves: 4-5 in no.) are also described in the table's footnotes.

Likewise, for Table 2, numbers placed after "Parts used" (eg: root (12040000)), and "Ailment treated" are not explained neither in the table's legend nor in the footnote.

Our answer: The numbers used in parenthesis after the diseases/disorders, parts and methods of administration in table 1 and 2 are according to the recommendations given by Cook, 1995 as Economic botany data collection standard (EBDCS), plant parts, body parts and processes, disorders/effects, medicinal applications and non-vertebrate organisms (Master lists of states for Level 3 descriptors) (Economic Botany Data Standard; https://www.kew.org/tdwguses/rptMasterListMain.htm). 3In composition, in no. used after the numbers stands for in number i.e. the number of that plant part used.

This information is added to the footnote in both the tables.

The session "Use of ethnobotanicals: toxicity, conservation status and Economic Botany Data Standard" is still a bit confusing, as different subjects are discussed in the same paragraph, without an evident connection between them.

Our answer: Yes, that is why we removed the irrelevant Economic Botany Data Standard from this section, explaining as footnotes in table 1 and 2 as suggested by the reviewers. This section of the result is now renamed as “Use of ethnobotanicals: toxicity, aspects and conservation status” in accordance to the section in the discussion “Use of plants and animals as drugs: toxicity and conservation aspects”

Submitted filename: Response to reviewers.docx

Click here for additional data file.

12 Nov 2020

An evidence based efficacy and safety assessment of the ethnobiologicals against poisonous and non-poisonous bites used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

PONE-D-20-17909R2

Dear Dr. Malik,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Benito Soto-Blanco, DVM, MSc, PhD

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

16 Nov 2020

PONE-D-20-17909R2

An evidence based efficacy and safety assessment of the ethnobiologicals against poisonous and non-poisonous bites used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

Dear Dr. Malik:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Benito Soto-Blanco

Academic Editor

PLOS ONE