Ethnoveterinary practises of medicinal plants used for the treatment of different cattle diseases: A case study in East Khasi Hill district of Meghalaya, North East India

Introduction For generations, the inhabitants of Meghalaya have relied on medicinal plants to maintain the health of their livestock and treat various illnesses that may afflict their animals. Due to the lack of survey for use and documentation, these plants have never been undertaken. Therefore, it is imperative to explore the diversity, utilization, and phytochemical profile of these plants and quantitatively analyse the data to identify important medicinal plants. By doing so, we can better understand the potential of these plants for developing novel drugs. Methods Frequent field trips were made for the collection of ethnoveterinary data of medicinal plants from local animal-keepers, traditional healers (THs) and inhabitants of different age groups. This information was gathered through semi-structured interviews, individual discussions, direct field-use observation, and questionnaires. A total of 52 informants (35 females and 17 males) were interviewed from seven rural villages and the information obtained from them were quantitatively analysed using the informant consensus factor (ICF), and fidelity level (FL). Additionally, for each documented plant, available published literature was extensively surveyed to identify the presence of bioactive chemical compounds responsible for their therapeutic effects. Results During the present study, a total 96 plants, distributed into 87 genera and 43 families were identified and recorded for their use in ethnoveterinary practices against more than 25 diseases. Out of the recorded plant species, the Fabaceae family was found to be the most dominant with seven species, followed by Poaceae and Lamiaceae with six species each, and Moraceae with five species. The leaves (50.00%) and seeds (12.50%) were the most frequently used plant parts, while the paste (30 species) was the common mode of application. Aegle marmelos Correa exhibited a fidelity level (FL) of 100% for indigestion, while Tagetes erecta L. had a fidelity level of 94.11% for wound treatment, making them the most promising candidates for further study. The highest FIC value of 1.00 was recorded for the treatment of neurological disorder (1.00), followed by foot and mouth disease (FIC 0.91), which depicted that some species were frequently utilized to treat multiple livestock ailments. Conclusion The study presents trustworthy information about medicinal plants and their associated indigenous ethnoveterinary knowledge. It has been scientifically proven that these plants contain bioactive compounds responsible for their therapeutic properties. However, this knowledge is in danger of being lost due to factors like socioeconomic changes, environmental and technological alterations, and lack of interest from younger generations. Therefore, it is essential to document this empirical folklore knowledge systematically and take measures to protect and conserve it.

& 92 • 09″ E Longitude. It is located in the extreme northeastern region of the country, along with the international border with Bangladesh ( Fig. 1). The altitude ranges from 150-2000 m asl, having the highest being Shillong peak point (1966 m). The climate of the study area ranges from temperate in the plateau region to the warmer tropical and sub-tropical pockets in the Northern and Southern regions. The area has highly diverse community forests as compared to the other districts of the state and they are protecting their forest by themselves.

Identification of informants
The present study attempts to survey medicinal plants used in ethnoveterinary practices. Information about their uses was collected from seven rural (hilly) village areas: Iew Pynursla, Wahlyngkhat, Umkor, Pynursla, Laitlyting, Lait Mynrieng, and Urksew Wahpathaw (Table 1). Prior to conducting the fieldwork, a meeting was arranged with the village heads to discuss the objectives and  methodology of the research. In every village, resource persons or traditional healers were identified to provide ethnoveterinary information. A total of 52 practitioners aged between 35 and 60 years were identified for the survey. Periodic field surveys were carried out at the study site between November 2018 and March 2020 to collect plants and associated ethnoveterinary information.

Data collection
Information about the use of plants was collected from 52 selected informants (35 female and 17 male) having traditional knowledge on folk veterinary medicines by following questionnaire survey, semi-structured interviews, individual discussions, and direct field-use observation, living in the investigated area. The informants were selected through purposive sampling based on their experience and expertise in ethnoveterinary practices. The questions were designed to elicit information on: (1) the personal characterization of participants; (2) the types of medicinal plants used in ethnoveterinary practices; and (3) the mode of preparation and administration, and the effectiveness of the treatments. A field data sheet has been prepared to record the details of plants with ethnoveterinary uses from the informants. Information viz., local name of the plant, part used for curing, method of preparation, any other plants/agents used as ingredients, modes of administration, dosage were recorded for each collected taxon. Participants interviewed in this study have given their full consent to participate and affirm that the information they have provided is true, accurate, and complete. The information was cross-checked by the information provided by the other tribal practitioners during the earlier visits.

Plant collection and identification
The plants mentioned by the informants were collected and identified with the help of taxonomic experts, available published literature [27][28][29][30], botanical keys, online efloras (Flora of China, India, Nepal, Pakistan, etc.), consulting the Herbaria of Department of Botany, NEHU, Shillong and verified using World Flora Online (http://www.worldfloraonline.org/database). The voucher specimens were then catalogued and preserved in the herbarium of the Department of Botany, NEHU for future references.

Data analysis and statistical evaluation
Data collected from the field through interviews of the informant were tabulated in Microsoft Excel spreadsheets and analysed using two quantitative indices: informant consensus (FIC) and fidelity level (FL). Informant consensus factor (FIC) was used to find the level of homogeneity among the information provided by different informants. The informant's consensus factor (FIC) [31] for the use of plants in the treatment of different ailments of cattle was calculated by using the following formula:  Where Nur is the number of use reports from informants for a particular plant use category and Nt is the number of species that are used for that plant use category for all informants. FIC values range between 0 and 1, where '1' indicates the highest level of informant consent. The Fidelity level (FL %) enumerates the significance of a species for a given purpose, was calculated by using the following formula:        where, Np = number of informants who cited the species for the particular use; N = total number of informants that mentioned the plant for any use.

Medicinal efficacy
To determine the efficacy of documented medicinal plants used in ethnoveterinary practices, a literature survey was conducted to identify the presence of bioactive chemical compounds responsible for their therapeutic effects. Pharmacological activities of identified compounds were searched using various scientific databases, such as Google Scholar, Science Direct, PubMed, SciFinder, Web of Science, ACS Publications, and Wiley Online library by using different keywords. Therapeutic potential of the major bioactive compounds was also done by reviewing the literature that investigated the pharmacological activities of the compounds, including their antidiarrheal, antimicrobial, anti-inflammatory, antioxidant, and other bioactivities. The chemical structures of the important phytochemical compounds were drawn using ChemDraw Pro software and confirmed by PubChem (https://pubchem.ncbi.nlm.nih. gov/) database.

Population structure of the informants
Data on ethnoveterinary practices were gathered from a diverse group of informants with varying ethnic and educational backgrounds, through a series of 52 systematic interviews. The majority of whom were analphabetic, and were middle-aged or older men (35-60 years). Out of the 52 participants, 35 were female and 17 were male. They represented various occupations, with small-scale farmers and traditional healers being the most commonly represented.

Documentation of traditional ethnoveterinary knowledge
A total of 96 plant species of different plant groups (i.e., 54 herbs, 27 trees, 10 shrubs, 3 climbers, 1 fern and 1 parasite) belonging to 43 families have been documented for the treatment of more than 20 categories of ailments (Fig. 4). For each species botanical name, voucher specimen number, local name, family, part(s) used, ailments to be treated, mode of use and mode of application was recorded and arranged family-wise in alphabetical order ( Table 2). Majority of them are collected from wild (78%), while some species (21%) are in cultivation, either in gardens or fields (Image 1). Among all the plant families recorded, the most species-rich were the Fabaceae accounted 7 species, followed by Poaceae and Lamiaceae (6 spp. each) and Moraceae (5 spp.), whereas Solanaceae, Rutaceae, Euphorbiaceae, Apiaceae and Amaranthaceae contributed 4 species each (Fig. 6). Five families (Acanthaceae, Amaryllidaceae, Asteraceae, Rosaceae and Zingiberaceae) represented 3 species each and the remaining 29 reported families were constituted by only one or two species. Leaves (50.00%) were found to be the most frequently utilized plant parts even solely or mixed with other parts for ethnoveterinary usage followed by seed (12.50%), fruit (10,42%) and whole plant, while bulb (3.13%) found to be the less utilized plant parts (Fig. 5). During the treatment of illnesses, several methods of medicinal preparations were used. The most regularly used mode of preparation was paste, which accounted the highest (31.25%) followed by direct feeding (23.96%), decoction (19.79%), infusion (15.63%) juice and powder (7.29% each) while oil (1.04%) was reported in only one species (Fig. 3). In the present survey, it was recorded that the majority of preparations were taken orally (57.29%) to treat cattle ailment followed by topical routes (26.04%) N.B: FL = Fidelity Level, SF = is the number of informants who independently cited the importance of a species for treating a particular disease, and TF = total number of citations.   Indigestion, foot and mouth disease, fever, ecto and endo parasites, deworming, skin infection, dysuria and conjunctivitis were the most frequently reported health problems (Table 2). From all the ailments occurring in the district, a total of twenty diseases were categorized.

Informant consensus factor
Informant consensus factor (Fic) values were determined to know the uniformity among the information provided by different informants. The Fic values ranged from 0.50 to 1.00 with an average value of 0.74 (Fig. 2). The highest Fic value of 1.00 with 4 usereports for 1 species were obtained for neurological disorder followed by foot & mouth diseases (Fic = 0.91; 5 species and 47 usereports) and deworming (Fic = 0.90; 4 species and 30 use-reports). Centella asiatica (L.) Urb., Melia azedarach L. and Zanthoxylum armatum Roxb. were the species that accounted for the highest consensus for neurological disorder, foot & mouth diseases and deworming. The lowest agreement between the informants was observed in respiratory diseases (Fic = 0.50; 4 species and 7 usereports) followed by fever (Fic = 0.52; 14 species and 28 use-reports) and skin infection (Fic = 0.60; 18 species and 44 usereports). Skin infection had the lowest Fic (0.60) after respiratory and fever but this ailment category ranked first in the number of taxa (18) and fifth in the number of use reports (44) attributed to this category.

Fidelity level
Plants frequently used for curing the major categories of ailment as claimed by different informants were analysed by using fidelity level (FL %) based on use reports. The fidelity level (FL %) values of species that were declared to be used by informants against the corresponding ailment are depicted in Table 4. The values ranged from 42.85% to 100% with an average value of 71.95%. After the data analysis, the FL value with 100% was found in Aegle marmelos Correa, followed by Tagetes erecta L.

Documentation of bioactive compounds
The collected ethnoveterinary plants possessed a number of pharmacological properties and produces a wide range of bioactive chemical compounds through their secondary metabolism. Available published literature (viz., Google Scholar (www.scholar.google. co.in), Web of Science (http://thomson reuters.com), PubMed (www.ncbi.nlm.nih.gov/pubmed), CAB direct (www.cabdirect.org), SciFinder (www.cas.org/products/scifinder) and Dictionary of Natural Products (www.dnp. chemnetbase.com) revealed that all the taxa used for the treatment of various cattle diseases contain a wide range of valuable chemical compounds (Table 3). These compounds are reported to have beneficial effects on the prevention of various diseases, depending on which species are used and how much quantity is taken.

Discussion
Meghalaya a part of the Indo Burma biological hotspot, sustains a diverse array of traditional knowledge which spreads across food, medicine, fuel, architecture and agro-forestry. Inhabitants of Meghalaya have a close association with nature as they are used plants for medicine in curing numerous ailments, due to its dominance of ethnic people, who are reliant on forest products since time eternal [128,129]. The region has vast pharmaceutical and commercial potentials as the knowledge of plants were inbuilt in their deep-rooted culture, which is being transferred from generation to generation. Hence, an attempt has been made in the present study to document the ethnoveterinary medicinal knowledge, focusing primarily to find out the gap and scope in further ethnobotanical research in the region for searching novel bioactive compounds. The present study reveals that plants and their byproducts are the most regular and easily available medicine used by traditional herbal healers and animal keepers on various ailments of their livestock [130][131][132]. The current findings are aligned with the various studies conducted in other parts of India on various ethnic groups [14,[19][20][21][22]24,26]. Plants like Zingiber officinal, Azadiracca indica, Allium sativum, Acorus calamus, Curcuma longa, Andrographis paniculata, etc., used by Khasi people for the treatment of the livestock were similarly found to be used by other ethnic people of the country [133][134][135][136][137][138], which signifies the recognition of these species as medicinal importance for noble drug findings [134,139]. The paste and powder are the most common mode of application of herbal medicines taken either orally or externally as seen in the present study have a similar finding cited in the literature as well [14,138,140,141]. Diseases recorded like foot and mouth disease, diarrhoea, bloating, etc. in the present study are also frequent among cattle in various states of India [23,[142][143][144][145]. In the present study, the species Centella asiatica, Melia azedarach and Zanthoxylum armatum were commonly used for the treatment of neurological disorders, foot & mouth diseases and deworming, resulting in the highest Fic value [146]. The plants with the highest Fic are because of their systematic selection and information procedures provided by informants [131]. The species with the highest FL of 100% were Aegle marmelos, Tagetes erecta, Melia azedarach, Camellia sinensis and for indigestion, wound treatment, foot and mouth diseases and fever [131,147,148]. So, we recommend these species for further phytochemical and pharmacological studies for the development of new, cheap, effective, and eco-friendly herbal formulations for healthcare management [149][150][151]. Priority should be given to the plants with the highest consensus factor and fidelity level, which could potentially guide the search for new pharmaceutical and commercial products of universal interest.

Scientific validation of medicinal plants
The medicinal plants with the highest consensus factor and fidelity level were compared with previously reported studies for biological activities and bioactive constituents responsible for their therapeutic properties. These species are Aegle marmelos Correa, Tagetes erecta L., Melia azedarach L., Centella asiatica (L.) Urb., Zanthoxylum armatum Roxb. and Ocimum basilicum L.
In support of our study, it has been scientifically proven that Aegle marmelos treats Indigestion and also possesses antidiarrheal, antimicrobial and antiviral properties [34]. The bioactive compounds viz., marmelosin, lupeol and aegeline [ Fig. 7(a-c)], isolated from Aegle marmelos have been reported to be effective against the several bacterial strains (Bacillus spp., Klebsiella aerogenes, Pseudomonas vulgaris, Vibrio cholerae, Escherichia coli, Shigella spp., etc.) and showed significant inhibitory action against castor oil induced diarrhoea [152]. This study also indicates that β-caryophyllene and quercetagetin [ Fig. 8(a and b)] are a very effective bioactive constituent for the treatment of wound healing, isolated from Tagetes erecta [148,153]. β-caryophyllene is a ligand of the cannabinoid receptor 2 (CB2) and on its activation it has the capability to improve wound healing by decreasing inflammation. It also improves re-epithelialization due to enhanced cell proliferation and cell migration [153]. This study also showed that quercetagetin is a very effective in wound healing process in I/R lesions by suppressing MAPK pathway, decreases immune cell infiltration and pro-inflammatory cytokines production [148]. Leaves of Melia azedarach used traditionally for foot and mouth diseases has been mostly found to contain meliacine, limonoic acid, β-sitosterol and rutin [ Fig. 9(a-c)]. The limonoids of Melia azeradach was found to inhibit herpes simplex virus [154] and are cytotoxic against different cancer cell lines [155]. It also indicates that meliacine is significantly suppress the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells by inhibiting vacuolar acidification = Datura metal L. [156]. Similarly, β-sitosterol and rutin are involved in the curative properties for inflammation, viral damage, ulcer and immune system booster, by controlling the production of inflammatory cytokines [157,158]. This study also indicates that rutin is an effective constituent against avian influenza strain H5N1 using plaque inhibition assay in the Madin-Darby canine kidney [158]. Centella asiatica is another scientifically proven ethnomedicinal plant to have positive effect on diseases of the nervous system by reducing inflammatory factors, ROS production and nerve cell apoptosis, repairing abnormal expression of mitochondrial-related proteins, and improving the survival rate of neural cells [159]. A summary of the related literature showed that the bioactive compounds (asiatic acid, asiaticoside and madecassic acid) isolated from Centella asiatica extract [ Fig. 10(a-c)] had positive effects against neurological diseases, generally through the mitogen-activated protein kinase (MAPK) signalling pathway by increasing the brain-derived neurotrophic factor (BDNF) contents [160]. The signalling (p38 MAPK and PI3K/Akt/mTOR) pathway can control various events in Alzheimer's and Parkinson's disease, viz., neuroinflammation, tau phosphorylation, and synaptic dysfunction [161,162]. The leaves and bark of Zanthoxylum armatum containing a major compound of limonene, linalool and cinnamic acid [ Fig. 11(a-c)], which shows the highest toxic activity against endoparasites. This study also specifies that limonene is a very effective against Leishmania species (L. major, L. braziliensis and L. chagasi) with 50% inhibitory concentrations of 252.0 ± 49.0 and 147.0 ± 46.0 μM, respectively [163]. Similarly, the compounds like linalool, β-ocimene, limonene, α-terpinolene, cinnamone and germacrene isolated from Z. armatum leaves exhibits strong DPPH radical scavenging activity (IC50 = 27 μg/mL) relative to activity of ascorbic acid (IC50 = 15.0 μg/mL) [124]. The present study also confirms that Ocimum basilicum contains several bioactive constituents like phenols (eugenol), flavonoids (luteolin and eriodictyol) and a polyphenol (rosmarinic acid), which are scientifically studied to have therapeutic properties for venomous bites [ Fig. 12(a-d)]. These active compounds isolated from the leaf extract of O. basilicum may be the potential mechanism contributing to treat insect stings, snake bites and skin infections [164]. Rosmarinic acid extracted from O. basilicum has proved the anti-inflammatory, antioxidant, and neuroprotective activities [165]. Literature also showed that rosmarinic acid has anti-inflammatory properties, stimulates differentiation of cell by controlling the extracellular signal regulating kinase (ERK1/2) signaling pathway and improves cholinergic activities in rat PC12 cells [166]. Rosmarinic acid also prevent Aβ-induced memory loss, inhabits apoptosis and decrease hyperphosphorylation of tau protein [166,167].

Conclusion
The present study provides trustworthy information on the indigenous ethnoveterinary knowledge of plants. This knowledge is as senescent as human civilization and is transmitted orally from generation to generation. Consequently, it was found that only the older generation is responsible for knowing about native plants and their uses. This knowledge is at risk of being lost due to urbanization, societal changes, and the lack of interest in the uses of plants for curing domestic animals among the younger generation. Even the anthologies of these medicinal plants are unscientific viz., uprooting, scraping bark, etc. which reduces their chances of proliferation.
To overcome these problems, there is an urgent need for collective efforts from taxonomists, ethnobotanists, and pharmacologists to collect, document and conserve this precious folklore knowledge related to the utilization of medicinal and other wild plants. This knowledge is essential for future generations to properly cure their domestic animals through scientific implementation. The inhabitants of the study area need to be aware of sustainable collection, domestication (for personal or trade use), and conservation. This will improve their socio-economic conditions and reduce pressure on natural resources. Lastly, the documented plants require further scientific analysis (phytochemical and pharmacological screening) for their effective utilization for medicinal purposes. In the future, this knowledge could potentially guide the search for developing new pharmacological products.

Author contribution statement
Nazir Ahmad Bhat: Conceived and designed the experiments; Performed the experiments; Analysed and interpreted the data; Contributed materials, analysis tools or data; Wrote the paper. Licha Jeri, Dolly Karmakar: Performed the experiments; Contributed materials, analysis tools or data; Wrote the paper. Puranjoy Mipun, Nilofer sheikh, Chester John Nongkynrih: Performed the experiments; Contributed materials, analysis tools or data. Pankaj Bharali, Yogendra Kumar: Designed the experiments; Analysed and interpreted the data.

Additional information
The plant specimens were deposited and preserved in the herbarium of Botany Department, North-Eastern Hill University, Shillong, Meghalaya for future references.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. grateful to the Head, Department of Botany, NEHU Shillong, for providing necessary facilities and Botanical Survey of India (Eastern Circle, Shillong) to consult the herbarium.