An ethnobotanical survey of indigenous medicinal plants in Hafizabad district, Punjab-Pakistan

Present paper offers considerable information on traditional uses of medicinal plants by the inhabitants of Hafizabad district, Punjab-Pakistan. This is the first quantitative ethnobotanical study from the area comprising popularity level of medicinal plant species intendedby using relative popularity level (RPL) and rank order priority (ROP) indices.Ethnobotanical data were collected by interviewing 166 local informants and 35 traditional health practioners (THPs) from different localities of Hafizabad district. Demographic features of informants; life form, part used, methods of preparation, modes of application and ethnomedicinal uses were documented. Ethnobotanical data were analyzed using quantitative tools, i.e. Relative frequency citation (RFC), use value (UV), informant consensus factor (ICF) fidelity level (FL), RPL and ROP indices. A total of 85 species belonging to 71 genera and 34 families were documented along with ethnomedicinal uses. Solanum surattense, Withania somnifera, Cyperus rotundus, Solanum nigrum and Melia azedarach were the most utilized medicinal plant species with highest used value. The reported ailments were classified into 11 disease categories based on ICF values and highest number of plant species was reported to treat dermatological and gastrointestinal disorders. Withania somnifera and Ranunculus sceleratus with maximum FL (100%), were used against gastrointestinal and urinary disorders, respectively. The RPL and ROP values were calculated to recognize the folk medicinal plant wealth; six out of 32 plant species (19%) were found popular, based on citation by more than half of the maximum number of informant viz. 26. Consequently, the ROP value for these species was more than 75. The comparative assessment with reported literature revealed 15% resemblance and 6% variation to previous data;however79% uses of the reported species were recorded for the first time. The diversity of medicinal plant species and associated traditional knowledge is significant in primary health care system. Medicinal plant species with high RPL values should be screened for comprehensive phytochemical and pharmacological studies. This could be useful in novel drug discovery and to validate the ethomendicinal knowledge.


Data collection and identification of plant species
Semi structured interviews and group conversation with local inhabitants were conducted to gather information on medicinal uses of plant species from 201 informants including male, female respondents and traditional healers. Data on age, gender, educational status and linguistics of respondents were also gathered. Information regarding the local plant names, part (s) used, methods of preparation and application were documented. The medicinal plant species used by the local communities of the study area were authenticated using the international plant name index (http://www.ipni.org), the plant list (www.theplantlist.org) and GRIN taxonomy site (http://www.ars-grin.gov/cgi-bin/npgs/html/queries.pl), while that of families follow A.P.G. system [29]. The species entries were complemented along with data on taxonomic position (family), vernacular name, common name, flowering period, life form and folk medicinal uses. The life form was categorized into herbs, shrubs, grasses and trees (annual, biennial or perennial), according to the system proposed by Raunkiaer [30,31] and modified by Brown [32]. Collected plant species were identified by Prof. Dr. Rizwana Aleem Qureshi (Plant Taxonomist, Quaid-i-Azam University, Islamabad and by using Flora of West Pakistan [33] and Flora of Punjab [34]. The voucher specimens were submitted in Herbarium of Pakistan (ISL) Quaid-i-Azam University Islamabad.

Quantitative analysis
The ethnobotanical data was analyzed using different quantitative indices including Informant Consensus Factor (ICF), Use value (UV), Relative frequency citation (RFC), Fidelity level (FL), Relative popularity level (RPL), Rank order priority (ROP). Data were reported in proportions and percentages.
Informant consensus factor (ICF). ICF value describes informants' consensus on the medicinal plant consumption species, and evaluates variability in mode of utilization against reported diseases. Before calculating ICF value, ailments are broadly categorized into different categories [35]. The maximum ICF value i.e. close to 1 indicates that well known species are used by a large proportion of local communities due to their authenticity regarding diseases. However, low ICF index close to 0 specifies that the informants use this species randomly to treat reported diseases [35].
The ICF value was calculate using the formula as described earlier [36,37]: Where, "N ur " is the total number of use reports for each disease category and "N t " indicates the number of species used in said category. Use value (UV). Use value (UV) determines the relative importance on uses of plant species. It is calculated using the following formula as explained before [37,38]: Where, "UV" indicates use value of individual species, "U" is the number of uses recoded for that species and "N" represents the number of informants who reported that species. Relative frequency of citation (RFC). Relative frequency of citation(RFC)signifies the local importance of each species in a study area [39,40]. This index is determined by dividing the number of informants citing a useful species (FC) by total number of informants in the survey (N). RFC is calculated by the formula as described earlier [41]:

Fidelity level (FL).
FL is the percentage of informants who mentioned the uses of certain plant species to treat a particular ailment in a study area. The FL index is calculated using formula as reported previously [42,43]: Where, 'Np' is the number of informants that claimed a use of certain plant species for a particular disease and 'N' is the total number of informants citing the species for any disease. The maximum FL indicates the frequency and high use of the plant species for treating a particular ailment by the informants of the study area. Relative popularity level (RPL). RPL is the ratio between number of ailments treated by a particular plant species and the total number of informants for any disease. However, plant species with comparable FL may vary in their healing potential. A correction scale is therefore introduced, in which all the encountered plant species are divided into popular and unpopular groups. The relative popularity level (RPL) assumes a value 0 and 1.0, with '1' being complete popularity of a plant for major ailments and '0' no ailments treated by a plant species. When all plant species are frequently used to treat some major ailments, popularity index would be maximum (1.0); then decrease towards zero as the relative popularity of the species diverge away from popular side. For popular plant species, the RPL value is rationally selected to equal unity (i.e. equal to 1), while RPL value is less than 1 for unpopular plant species. The relative popularity level (RPL) of the plant species is calculated and designated as popular or unpopular. The RPL value may be determined for each specific plant in accordance with its exact position on graph [43,44].
Rank order priority (ROP). ROP is a correction factor, used for appropriate ranking of the plant species with different FL and RPL values. The ROP is derived from FL; by multiplying RPL and FL values as explained earlier [43,44].

Results and discussion
Demographic features of the informants A total of 201 local informants including 175 males and 26 females were interviewed. Based on demography these informants were categorized into different classes as given in Fig 2. In the present survey male participants were higher than females. The prevalence of male informants is due to the fact that females of the study area were reluctant in conversation with male strangers (the interviewers). The local informants were farmers, foresters or herdsmen, craftsmen, shopkeepers, teachers and housewives. The traditional health practioners (THPs) hold significant information on the medicinal uses of local plant species to treat various ailments. THPs were classified into five groups on the basis of their experience such as THPs having less than 2 years' experience (9), THPs with 2-5 years' experience (12), THPs of 5-10 years' experience (7), THPs of 10-20 years' experience (4) and 3 THPs have more than 20 years' experience. Among others, maximum informants having traditional knowledge regarding the use of medicinal plants were fall in secondary school education level or even below this and often spoke only Punjabi language. The maximum information was collected from the informants above 60 years age possess significant traditional knowledge whereas little information was shared by young respondents. Moreover, illiterate informants shared possess more information on the traditional use of medicinal plant species compared to educated respondents. This may be due to changing lifestyle, increase in the use of allopathic medicine and urbanization. Similar findings have also been reported from Bangladesh [45]and Turkey [9,46].

Taxonomic classification
In total 85 medicinal plant species belonging to 71 genera and 34 families were documented ( Table 1). All reported species were angiosperms, which include: monocotyledonous and dicotyledonous groups (10.6 and 89.4%, respectively). Asteraceae and Poaceae were the most dominant families (with 9 species each), followed by Fabaceae (8 species), Moraceae and Euphorbiaceae (6 species each), Chenopodiaceae (5 species), Malvaceae and Solanceae (4 species each), Amaranthaceae (3 species) and Meliaceae, Myrtaceae, Oleaceae, Polygonaceae and Rhamnaceae (2 species each), while other families were represented by one species only (S1 Fig) The utilization of medicinal plant species belonging to Asteraceae and Poaceae families was in agreement with ethnomedicinal flora reported from other parts of Pakistan and in other areas of the world [23,[47][48][49]. This may be due to their wide distribution of plant species belonging to Asteraceae [50] and Poaceae [51] and their traditional uses known by the indigenous communities living in different parts of the world.  The life forms of the reported species are mentioned in Table 1. The herbaceous flora constitutes highest contribution (44%) of the reported plant species, which is comparable to Mahmood et al. [2], who reported 54% contribution of wild herbs in medicinal plants used by the local communities of Gujranwala district, Pakistan. The herbaceous species comprise perennial, biennial and annuals (56.5, 1.2 and 27.1%, respectfully).Furthermore, wild and cultivate trees contribute (17 and 10%, respectively), wild grass (8%), wild and cultivate shrubs (9 and 7%, respectively), cultivated herbs (4%) and cultivated grasses (1%) in descending order (S2 Fig). These findings were in consistence with previous reports [19,49,[52][53][54][55][56]. The frequent use of herbs by indigenous communities may be due to their accessibility and high efficacy in the treatment of diseases compared to other life form [57][58][59][60].It is well-known that the medicinal plants having perennial nature require prolonged period of growth i.e. about 6-8 years depending on the type of plant species. Therefore, the perennial life cycle is more prominent in medicinal plant species than annual [22,[61][62][63].

Plant part(s)used, mode of preparation and application
Even though all plant parts are significant in the treatment of different ailments; nevertheless in the present study as shown in Fig 3, leaves were the most commonly utilized plant part with38% application in traditional medicinal recipes, followed by whole plant (14%), root (10%), stem (8%), fruit, seed, flower (7% each), bark (5%), shoot and rhizome (2% each). Bradacs et al. [64] and Leto et al. [65]also reported leaves as commonly utilized plant part in herbal medicine used by the inhabitants of islands and Italy. It has been reported that the use of leaves is better for the survival of medicinal plants collected by herbalists compared to the collection of whole plant, roots and stem, which may cause severe threat to local flora [66].Even though some plant species such as Calotropis procera, Croton sparsiflorus, Datura innoxia, Euphobia spp., Lantana camara, Solanum spp. and Ranunculus sceleratus are consider as poisonous; however used to treat various ailments by local inhabitants. It has been reported that plant species with potent bioactive compounds are often characterized as poisonous and medicinal as well, and a beneficial or an adverse result may depends on method of drug preparation and utilization [67]. It was observed that inhabitants of the study area use above mentioned species in modest quantity, therefore no toxic effect was mentioned by respondents.
Local inhabitants of the study area use different methods i.e. decoction, extract, juice, powder, paste, infusion, poultice, tea, and ash etc. to prepare a recipe for the treatment of various ailments (Fig 3). Decoction was the most common method of drug preparation (121 applications), followed by powder, juice, paste, extract and infusion (76, 62, 45, 34 and 32 applications, respectively). Whereas, milky latex and oil were used in 12 applications each, poultice in 10 applications, cooked food, tea and ash with 5 applications each and plant sap with 2 applications. Such a wide array of preparation methods has also been reported previously from different parts of Pakistan [2,23,68,69] and in other countries [49,[70][71][72][73][74]. The traditional healers of the study area also use more than two plant species along with other ingredients i.e. milk, honey, egg, butter, salt, sugar and water etc. The widespread use of decoction and powder in the study area is comparable to Mahmood et al. [2], Ahmad et al. [68] and Boudjelal et al. [75] who reported decoction as a most commonly utilized method of preparation followed by powder. Decoction is used as one of the major practices to prepare drug in traditional healing system, because it is easy to make by mixing with tea, water or soup [76,77].While making decoction, plant material is boiled in water until volume of the water reduced to one-fourth of its original volume [69], whereas crude extract is obtained by squeezing or crushing the plant parts [78].
As far as mode of administration is concerned, around 69% medications were taken orally (S3 Fig), followed by administered topically (24%), as eye drops and gargle (2% each), and as toothbrush, inhale and anal application (1% each). These findings were comparable to earlier reports [45,79,80]. Moreover, some herbal preparations were used for bathing, sniff and as eardrops. The medicinal plant species used as sniff are burnt to inhale fumes. Likewise, some plant parts were just crushed and smelled. Similar modes of applications were reported in Gujranwala [2]. The leaf and bark of some plant species are boiled and decoction is used to take bath against body pain. However, the trend of herbal bath is declining due to changing life style and now days only the leaves of Zizyphus mauritiana are used to bath the dead bodies. For topical use, direct application of paste, poultice or medicated oil are common, which are Ethnobotany of Punjab-Pakistan mostly used to treat skin infections, cuts, wounds, scorpion bites, rheumatism headache and body pain.

Informant consensus factor (ICF)
To calculate ICF, the reported ailments were first classified into 11 different disease categories on the basis of their use reports (Table 2).Among three major disease categories, dermatological disorders were dominated with 111 use-reports, followed by glandular complaints and respiratory diseases (76 and 52 use-reports, respectively) as mentioned in S4 Fig. Around  79.1% plant species were used to treat dermatological ailments, followed by gastrointestinal track (GIT) disorders, glandular complaints, respiratory diseases, ENEM diseases, cardiovascular disorders, urinary problems, muscle and skeletal disorders, sexual diseases and nervous disorders (76.7, 62.8, 47.7, 33.7, 25.6, 24.4, 23.3, 22.1 and 14%, respectively). These findings signify that dermatological and GIT disorders are prevalent in the study area. Similar findings have already been reported [53,64,65,[81][82][83][84][85][86]. However, Kadir et al. [45] and Singh et al. [57] described more number of species to treat gastrointestinal diseases compared to dermatological ailments. The ICF value of different disease categories was ranged from0 (nervous disorder) to 0.39 (GIT diseases) The average ICF value for all categories was 0.16, which was similar to previous studies carried out in Pakistan [23,48].

Relative frequency of citation (RFC) and use value (UV)
The RFC and UV indices are applied to select potential plant species for further pharmacological study and recommendation in drug development. The relative frequency citation (RFC) index authenticates the frequency of citation of a medicinal plant species used for various ailments. The RFC of the reported species ranged from 3to 17% (Table 1). However, on average, the RFC was9%. The highest RFC was calculated for Solanum surattense (0.17), Eclipta alba (0.15) and Triticum aestivum (0.15).The positions of these plant species correspond to the fact that they were reported by maximum number of informants, therefore having high frequency of citation (FC).
The use value (UV) index demonstrates the relative importance of plant species and families for a population [87]. In the present investigation, the UV of the reported medicinal plant Ethnobotany of Punjab-Pakistan species varied from 0.14 to 0.86 (Table 1). The highest UV was calculated for Solanum surattense and Withania somnifera (UV = 0.86, for each), Cyperus rotundus (UV = 0.84) and Solanum nigrum (UV = 0.83). These findings demonstrate the extensive use of above mentioned species in the treatment of various ailments by local inhabitants/healers and the consciousness of indigenous peoples, which makes such medicinal plants, the first choice to treat a disease. The lowest UV of Anethum graveolens, Brassica rapa and Chrozophora tinctoria may be due to less accessibility and minimum ethnobotanical uses. The results of UV in the present study were comparable with previously reported from Gujranwala, Pakistan [2].It has been reported that S. surattense exhibits hepato-protective [88], anti-asthmatic [89],antioxidant, anthelmintic, antimicrobial [90], wound healing [91], diuretic [92] and antipyretic [93] properties.Devi et al. [94]evaluated antibacterial, anti-fungal and antitumor properties of W. somnifera while its root has been used since long time for both sexes and even during pregnancy [95]. Another medicinal plant, C. rotundus has been studied against skin disease [96]. Recently, anticancer activity of S. nigrum has been reported by Lai et al. [97]. These findings confirm high RFC and UV of these plant species in the study area.

Relative popularity level (RPL)
Our 201 informants cited 85 plant species for 11 different disease categories. Of them, 32 species as given in Table 3, received more consideration by informants; therefore included for further discussion. The correlation between number of informants citing a particular plant species and the number of application used is given in S5 Fig This is the first baseline study on the indigenous knowledge of local peoples regarding the use of popular plant species for a particular ailment. These findings were in consistent with previous studies on the status of medicinal plants among Bedouins of Negev district [43] and medicinal plant survey in Palestinian area [44]. In these studies Alhagimaurorum (urinary disorder) and Tamarixaphylla (eye problem) were reported as unpopular plant species due to low RPL.

Fidelity level (FL)
The fidelity level (FL) of the 32 most important plant species ranged from 14.3 to 100% (Table 3). In general, the high FL of a species indicates the prevalence of a specific disease in an area and the utilization of plant species by the inhabitants to treat it [23,98]. Withania somnifera and Ranunculus sceleratus depicted 100% FL against stomach and urinary disorders, respectively. The fidelity levels calculated for Solanum surattense, Triticum aestivum, Melia azedarach, Solanum nigrum and Calotropis procera to treat kidney stones, late puberty, malarial fever, cardio-vascular diseases, and wound healing were 84.6, 78.6, 78.6, 76.9, 76.9%, respectively. The plant species with100% FL for instance W. somnifera have also been reported as chemo-preventive against stomach and skin carcinogenesis [99]. In another study, the R. sceleratus was reported as antiurolithiatic agent to treat urinary disorder [100].

Rank order priority (ROP)
The Rank order priority (ROP) index is used to rank appropriately the plant species with different FL values. The resultant RPL values given in S5 Fig were used as correction factor to adjust the FL values. The ROP values are thus obtained are given in Table 3. Of the 32 species, only eight species attained ROP above 50.This is probably due to decreasing popularity of herbal medicines among the local communities of the study area. Based on ROP value  [43] and in Palestinian area [44] were comparable to present findings. However, Alhagim aurorum (ROP = 31) and Tamarix aphylla (ROP = 15), were used to relieve urinary system disorder and eye disease, respectively by the inhabitants of the study area.

Novelty and future impact
Present study is the first document on ethnobotanical uses of 85 medicinal plant species used by the inhabitants of Hafizabad district, Punjab-Pakistan. The current ethnomedicinal uses of reported plant species were compared previous studies conducted in Pakistan and other areas [21,26,66,80,[101][102][103][104][105] as shown in Table 1, to find the novelty index. Approximately,15% medicinal uses of reported species were similar, whereas 6% were dissimilar to previous reports. However, 79% medicinal uses were new in the present study. Moreover, medicinal uses of plant species reported in neighboring areas i.e. from Layyah district, Punjab province, Pakistan [21] showed more resemblance compared to those documented in other areas. The data collected from the study area reveal considerable difference in plant parts used, mode of herbal preparation and its utilization as reported from other regions. Some of the newly documented medicinal uses and relevant plant species include: Anethum graveolens (indigestion), Chrozophora tinctoria (stomachache), Cirsium arvense (to heal wounds), Euphorbia prostrate (diarrhea and dysentery), Ficus benjamina (stomach disorder), Jasminum officinale (fever), Kochia indica (toothache), Malvastrum tricuspidatum (sores & wounds), Morus nigra (asthma), Parthenium hysterophorus (constipation), Prosopis juliflora (kidney stones), Pterospermum acerifolium (bleeding piles), Ranunculussceleratus (asthma), Setaria glauca (skin disease), Sorghum halepense (indigestion), Trifolium resupinatum(sore throat) and Veronica polita (indigestion). The plant species with new medicinal uses and high RPL value could be studied further to screen bioactive compounds and their pharmacological activities to introduce novel drugs.

Conclusion
Present survey revealed that a number of medicinal plant species are used by indigenous people of the study area to treat various ailments. The indigenous community still relies on traditional medicine although; the modern health-care services are available, which indicates the significance of plant based traditional recipes. Our findings provide baseline data to establish a connection between the traditional health practioners and scientific communities, which could be substantial in novel drug discovery. Furthermore, ethnobotanical data is of significant value for conservation managers and policy makers for sustainable management of medicinal plant species, which are under threat due to over exploitation. The high RPL value of medicinal plant species is an indication of their preference by local inhabitants to treat particular ailments. Therefore such popular plant species could be further analyzed for bioactive constituents, in vivo/in vitro biological activities, which may leads to new and potential drugs.  Table 3. (TIF)