Diversity of flora used for the cure of equine diseases in selected peri-urban areas of Punjab, Pakistan

Background Plants have widely been used and documented for their therapeutic potential in many parts of the world. There are, however, few reports on the use of plants for the treatment of diseases of equines. To this end, participatory epidemiology and rapid rural appraisal techniques were used to document the plants having pharmacotherapeutic significance against different ailments of equines in selected population of Punjab, Pakistan. Methods A survey was conducted to interview a total of 450 respondents (150 from each of the districts of Faisalabad, Lahore and Sargodha of Pakistan) to collect information about disease recognition of the equines and their treatment on a well − structured questionnaire. A total of 60 plants belonging to 40 families were documented. An inventory was developed depicting detailed information of plants used in treatment of different conditions of equines. Results The top ten species of plants used were: Allium cepa, Zingiber officinale, Vernonia anthelmintica, Capsicum annum, Brassica campestris, Trachyspermum ammi, Anethum graveolens, Picrorhiza kurroa, Azadirachta indica, and Citrullus colocynthis. Seeds were the most frequently used (n = 16/60) parts, followed by leaves (n = 12/60) and fruits (n = 11/60) of plants. Based on the combination of different parts of plants used in different ratios and variation in their dose or mode of preparation led to a large number of recipes/remedies against wounds, lameness, bronchitis, colic, anorexia, dermatitis, weakness, parasitism (internal & external), fever, heat stress, urine retention, swelling, toxemia, and indigestion. Conclusions This study generated lot of data on phytomedicinal approach for the treatment of ailments in the equines in some selected areas. It would, therefore, be imperative to expand similar studies in other parts of Pakistan and elsewhere. Moreover, use of the documented plants may be validated employing standard scientific procedures, which may have their application in the drug discovery/development by the pharmaceutical industry.


Background
Equines (horses, donkeys and mules) are playing key roles in providing an economical draught power to resource-poor countries like Pakistan. Equines suffer from a variety of health conditions that not only hamper optimum performance, but also cause huge losses due to mortality [1]. Parasitism has been reported as the major health issue of equines in Punjab, Pakistan followed by wound, bacterial infections, lameness, bronchitis, dermatitis, and colic [2]. In addition to allopathic/ modern medicine, there is extensive use of traditional herbs for the treatment of different diseases in equines all over the world [3]. Ethnobotany has revealed that the indigenous knowledge of a community is a key player in the identification of medicinal plants which have been tested through generations in the human history [4]. Traditional medicine and bio-prospecting [5] may often lead to the development of a new herbal product based on their use by significant numbers of people over the extended periods of time [6]. The plant-based medicines have particularly been found promising as anti-parasitics, stomachics, and in treatment of various respiratory ailments [7][8][9][10][11][12]. So far, only a handful of investigations are reported on the use of plants for different ailments/conditions; however, inventories of plants for phyotherapy in the food animals are extensively reported. The present study was, therefore, carried out to document the plants being used in traditional veterinary practices for equines in some selected peri-urban areas of Punjab (Pakistan) where equines are frequently used for different purposes.

Study districts
Three districts of the central Punjab; Faisalabad, Lahore and Sargodha were included in the present survey. District Lahore is the capital of Punjab (second largest city of Pakistan after Karachi), while district Faisalabad is the hub of textiles (third largest city) of the country. District Sargodha is comparatively smaller city and considered as an agricultural trade center with various industries. The equine population of the three districts has been estimated as 24628 horses, 174994 donkeys and 7849 mules [13]. The use of equines in the three selected industrial districts of Punjab is frequent because it is the cheapest source for carriage of industrial raw materials and products from and to the market [2]. Figure 1 shows physical map of Punjab province and the three study districts.

Selection of respondents
A small scaled rapid rural appraisal (RRA), an exploratory phase [14] was conducted in metropolitan Faisalabad, Punjab, Pakistan for the purpose of collecting an initial data from candidates who could participate in the second (surveillance) phase of the project.
Of the total 1000 registered farmers (who submitted their willingness to participate in the survey), 450 were selected as key respondents for this study. One hundred and fifty respondents represented each of the three districts of Faisalabad, Lahore and Sargodha. Selection of respondents within each district was done using proportional allocation and map grid methods in order to collect information from the selected districts. The selected respondents belonged to 9 sites each of districts Faisalabad and Sargodha, and 6 sites of Lahore (Table 1).

Participatory epidemiology and collection of data
A questionnaire containing a blend of open ended and closed (dichotomous and multiple choice) questions was prepared and refined through formal and informal testing [15]. For effective communication and data recording, a survey team was appointed; comprising of a veterinarian, professionals of The Brooke Hospital for Animals, and a community leader from the local village. Interviews, focused group discussions, and field visits were conducted with the respondents. The information about disease recognition of the equines and their treatments was collected using the well-structured questionnaire, open-ended interviews and guided-dialogue techniques. Focused group discussions were arranged to cross-check/ verify the information provided by the respondents to reach more accurate results. The respondents were asked to tell how they acquired the knowledge of phytotherapy related to the disease/condition recognition of equines. In addition, the direct observation approach was also followed as described by Etkins [16]. Thus, local names of plants, dose, parts of plant used, methods of preparation, and mode of administration were recorded. The farmers were asked to show the plant species described for the treatment of diseases/conditions for their taxonomic identification by the botanists at the Department of Botany, University of Agriculture, Faisalabad, Pakistan and the voucher specimens were preserved for record. The information collected was maintained in Microsoft Excel for further analyses and interpretation.

Respondents
Equine owners/traditional veterinary healers were well familiar with the signs and symptoms of the diseases/ conditions of equines in the study area and a majority of the botanical ingredients used in treatment were of indigenous origin. Therefore, farmers had these remedies available at their door step or at the most in the nearby grocery shops. The respondents were not trained by any authority about usage of plants for treatment of their animals. In fact, their knowledge was based on folk beliefs and previous practices based on hit and trial methods.

Diversity of plants used
A total of 60 plants were documented ( Table 2) for their use in different diseases/conditions of equines. Documented plants represented 40 families. Fabaceae was the largest represented family including five plants, followed by other families (Table 2). Composition, dosage, mode of preparation and administration, and frequency of usage of Traditional Veterinary Practices (TVPs) has been presented in Table 3. Plants and diversity of their usage in different diseases have been presented in Table 4. Thirty one species were used for the treatment of multiple diseases/conditions. Piyaz (Onion; Allium cepa), Adrak (Ginger; Zingiber officinale), Kali zeeri (Iron weed; Vernonia anthelmintica), Mirch (Chilli; Capsicum annum), Sarsoon (Rapeseed plany; Brassica campestris), Ajwain (Carom seeds; Trachyspermum ammi), Ajwain (Dill; Anethum graveolens), Kutka (Picrorhiza kurroa), Neem (Azadirachta indica), and Kor tumma (Bitter gourd; Citrullus colocynthis) were the top ten most frequently used plants as part of prescriptions in different diseases/conditions of equines.

Approaches adapted in plant usage
Seeds were the most frequently used (n = 16/60) part of plants as such or as their oils followed by leaves (n = 12/ 60) and fruit (n = 11/60). The other parts of plants used were: whole plant, rhizome, bark, branches, bulb, buds, flour, pepper corn, roots, etc ( Table 2). Prescriptions for treatment/control of different conditions of equines were based on single or multiple plants. Most of the recipes were prepared by mixing and grinding the ingredients. The powder was then made into physic balls as bolus to be given orally or decoctions were prepared for drenching the animals and/or used for topical application as washing, spraying, ointment, liniment, massage, etc. The prescriptions also differed in dose, method of preparation, and mode of administration of plants and/or materials other than plants; within and amongst the diseases/conditions. In some cases, frying, burning (to create smoke around animals), pouring, drinking, soaking before use, forced inhalation and steaming of ingredients were practiced.

Discussion
There are several recent evidences of plant based treatment and control strategies from Pakistan, especially for parasitism [11,12]. This has been supported by repellent activity of Moringa oleifera [17], an indication to be used against ecto-parasites. Plants from different geographical regions have produced variable results [18] as the synthesis of secondary plant products can be affected by environmental/growing conditions. Steroid saponins show pharmacologic actions like antifungal, antibacterial, anti-inflammatory and hypocholesteremic influences (Wang et al., [19]). So, plants producing saponins and organosulfur compounds like those of genus Allium can be used in the conditions described above. Large number of qualified veterinarians also advocates the use of phytotherapy, other than preventive medication, but these practices are less organized in the form of scientific reports and are usually transferred orally as these have been developed by farmers, rather than by scientists in sophisticated laboratories. This situation is typical of a rural underdeveloped culture like that of pastoralists of Africa [20] and other parts of world having dependence on phytotherapy for their animals. Inadequate access to modern health care facilities due to cost-ineffectiveness, inherited beliefs, empirical evidence of efficacy, cultural acceptability and availability of botanicals at the farmers' doorstep are the main factors [21] that lead to dependence of livestock farmers on the phytotherapy. The indigenous knowledge and skills can contribute towards development of phytotherapy in less developed areas of the world [22,23]. Plants are considered to possess relatively higher bioactive secondary compounds, thus hold promise for drug discovery. Most of the plant-derived chemicals are secondary metabolites, of which at least 12,000 have been isolated; a number estimated to be less than 10% of the total [24]. Nok et al. [25,26] and Nok and Williams [27] have discussed the active principles as well as the mechanisms of action of some plant extracts that are used in phytotherapy.
Fruitful efforts have also been made previously to document TVPs in some parts of Punjab, Pakistan focusing livestock but not including equines [28][29][30][31][32]. Equine industry has the ready and largely uninformed access to herbal products. Therefore, at least to the extent of equines, herbals are more than traditional veterinary medicine. The researchers have, therefore, focused on documentation and validation of usage of plants based on the claims of traditional healers [33][34][35][36][37].
Results of the present study have revealed that equine owners and/or traditional veterinary healers have great wealth of indigenous knowledge based on their practices and experience, which is evident from the number of plants (n = 60) used for treatment of different diseases/ conditions of equines in Faisalabad, Sargodha and Lahore. Use of plants in multiple disorders indicates diversity of their pharmacological and toxicological impacts [38]. Plants have more than one mode of actions; therefore, provide broad spectrum activities in different diseases [39] due to diversity of phyto − chemicals. Twenty − four plants including Anethum graveolens, Bambusa bambos, Cascuta reflexa, Citrus limon, Cocus nucifera, Ficus religiosa, Geranium wallichianum, Grewia asiatica, Halorrhena pubescens, Lagenaria siceraria, Lepidium sativum, Mangifera indica, Medicago sativa, Myristica fragrans, Nigella sativa, Oryza sativa, Peganum harmala, Pennisetum glaucum, Picrorhiza kurroa, Piper betle, Prunus dulcis, Trifolium alexandrinum, Withania somnifera and Zea mays were found to be used for different indications in equines. As far as could be ascertained, there is no published literature on the use of plants for the treatment of ailments in equines. There are thousands (≈250,000) of species of plants naturally available [40], with a low proportionate exploited for medicinal purposes. Further, 5-5% of the higher plants have been investigated for their active constituents against a wide range of infectious and non-infectious diseases of humans and animals (Pieters and Vlietinck [41]). Plants are primary source of natural products used by traditional healers in 80% of the developing societies [42]. There was about 40% repetition in the ethno-botanical preparations (EBPs) documented in the present study and those documented by others for other species of animals [28][29][30][31][32]). It has been reported previously that different parts of the same plant (leaves, fruits, flowers, seeds, seed kernels, latex, stem, grains, bulbs, tuber, roots, basal rosette, bark, thallus, shoots, wood, buds, aerial parts, branches, etc.) and variety of solvents used for their extraction diversify their usage ( [43][44][45][46]). Variation in the doses and mode of preparation of remedies within and among different conditions has also been reported elsewhere [11,28,30,32,47]. The aspect of non-standardized doses in phytotherapy have been criticized, because of toxicity constraints, under dosing, and cost; however, cost can be reduced by proper standardization of doses [48,49]. The common adage that natural is synonymous with safe, has led to significant and widespread disease [50], and it is critical that those involved in equine husbandry and health care are aware of the potential dangers of herbal medicine. Therefore, researches on the standardization of doses regarding efficacy vs safety should be carried out.

Conclusions
The current research suggests that EBPs have a crucial role in animal health and production in the study area. The current study revealed a diverse range of plants which is in practice to treat the prevalent ailments in equine population of Punjab. The pitfalls of TVPs found in this study were related with the improper diagnosis of diseases (some cases), non-standardization of dosages, mode of preparation and administration, and lack of understanding regarding importance of value addition (e.g., validation) to the existing practices, adverse/overdosing effects and documentation of indigenous knowledge. For example, traditional healers were not aware of the minimum essentials of parasite biology and strategic worm control practices. Fundamental issues in phytotherapy; however, are the dose, efficacy and safety left to an educated guess or is completely ignored. However, a handsome volume of the indigenous knowledge has been documented for the first time in the region in relation to the treatment of equines which provides a baseline for future scientific investigations in phytochemistry. The promising candidates of plant origin can be isolated through modern chemistry protocols and authenticated for their medical value after in vivo and in vitro experimentations.