Ethno-Pharmacognosy and Diversity Encourage Conservation of Wild Ziziphus species Collected from KP, Pakistan

The genus Ziziphus consists of almost 100 species has been used in folk and alternative systems of treatment in order to combat different diseases such as; fever, diabetes, skin infections, antipyretic, antinociceptive, antioxidant, antilisterial and larvicidal [14]. Due to peculiar geographical region, Pakistan exhibits a great diversity of flora as represented by 7 genera along with 13 species for Ziziphus [5].

tropical and sub-tropical areas of the world and mostly found in all continent of the world and native of Afghanistan, Australia, North Africa, North India, Malaysia, and southern China and Pakistan Z. mauritiana is a dominates species/variety of wild vegetation in different arid and as well as desert while in great examples of drought and hard species of Ziziphus [17,18]. In Pakistan, it is widely distributed in three provinces i.e. KPK, (Banuu, Karak and Kohat) while district swat, Buner and Dir is found in only wild from, Punjab (Attock, Chakwal and Mianwali districts) and Sindh province (Karachi, Hyderabad and Nawabshah districts). In Pakistan, jujube is cultivated on an area about 5.425ha with an annual production of 28.000 tones [19].
There is lack of information regarding jujube cultivars in Pakistan. Indigenous cultivars are missing and there is little research work available on their botanical classification [18]. Fluctuation in yield and quality of jujube fruit is greatly affected by the soil properties, climatic factors and cultivar selection [20]. Fruit quality attributes largely depend upon cultivar to be selected. Previously, variations in fruit weight, juice content [21] and seed weight has been recorded in jujube cultivars [22].
Morpho-biochemical methods are used to screened best genotypes in large collected germplasm of different crop species [23][24][25][26][27][28][29][30]. Among these methods the SDS-PAGE method is effectively used to determinate the taxonomic and evolutionary difficulties of certain plant species [28]. The seed storage protein studies help in documentation and description of variability in crop varieties, cultivars and their wild species but also rich genetic variability and phylogeny association of the accessions. It is considered that variability in protein bands intricate the association among the assortment from various geographical regions [31]. SDS PAGE as a powerful tool that has been used in the solution of problems in the field of taxonomy and explains the origin and evolution of cultivated plants, including the fenugreek [32].
It provides maximum variability among different crop species and the level of polymorphism depends upon the plant species [33]. Haliem and Huqail [34] found 168 different polypeptides bands among diverse Fenugreek genotypes of Saudi Arab and Yemen through SDS-PAGE method. They recorded 26 different polymorphic bands characterized Brassica rapa sub-species brown sarson through this method and recorded 83.33% polymorphic protein bands. They also noted four different cluster groups for the twenty studied genotypes. Jan et al. [28], evaluated three different ecotypes of B. rapa through SDS-PAGE method and high level of variability were noted in protein bands size. Protein electrophoresis is considered a reliable, practical and reproducible method because seed storage proteins are the third hand copy of genomic DNA and largely independent of environmental fluctuations [34,35].
The main objectives of the current research studies on Ziziphus species (1) ethno medicinal uses of Ziziphus species in local peoples (2) to explore different aspects the genetic relationship of wild Ziziphus species (Z. oxyphylla and Z. mauritiana) genotypes based on different morphometric collected from different regions of district Swat KP, Pakistan (2) to study genetic diversity based on store seed protein (SDS-PAGE) of wild Ziziphus species of their genotypes (3) by the using of multivariate analysis, phylogenetic, PCA, for the checking of their relationship of wild Ziziphus species collected from different regions of distract Swat KP, Pakistan.

Samples collection and Ethno-pharmacognosy
Exploratory trips were arranged to different areas of KP, Pakistan years, 2017-2018 and total two Ziziphus species (50 genotypes) were identified and investigated for morphological characterization and SDS PAGE protein profiling. The plants (Ziziphus species) Z. oxyphyla and Z. mauritina specimen were store in the herbarium Department of Botany, Hazara University, Mansehra, KP, Pakistan; the specimens were recognized referring different Floras, viz., Hooker (1872-1897). Ethno medicinal data has been collected through Participatory Rural Appraisal (PRA), which is based on communication with indigenous people and direct communication in the field Martin [36]. The data have been noted through semi-structured interviews with people involved in the plants, organization [37]. A total of 130 residents have been interviewed during the field survey, information on uses of plants to cure various diseases of human being, parts used, of medicine have been collected. Based on the information obtained from the informants in the study area, all the reported diseases have been classified into 12 groups.
The different level of the similarity among information delivered by various informants was calculated by the Informants' Consensus Factor, ICF Trotter and Logan [38] by applying the following formula: Where, Nur = number of use reports from informants for a specific plant-use category; Nt = number of taxa or species that are used for that plant use category for all informants.
ICF Values range between 0 and 1, where '1' indicates the highest level of informant agreement. The fidelity level (FL), the percentage of informants claiming the use of a certain plant species for the same major purpose, was calculated for the most frequently reported diseases or ailments as: Where, Np = number of informants that claim a use of a plant species to treat a particular disease; N = number of informants that use the plants as a medicine to treat any given disease [37].

Morphological analysis
In the current work qualitative and quantitative characterizations were carried out of the collected samples, Qualitative traits were recorded on the general visualization (phenotypic observations). Ten qualitative traits i.e. Tree vigor, Leaf type, Leaf shape, Leaf colure, Tomentose, stem color, Spines, fruit color, leaf Agricultural Research & Technology: Open Access Journal margin, fruit shape and quantitative characters which were measured with the help of vernier caliper for the measurement of plant height(feet), Branching, Leaf length(mm), Leaf width(mm), Leaf thickness(mm), Petiole length(mm), Inter node length(cm), Stem diameter(inches), fruit weight, fruit diameter (mm) and fruit length (mm).

Protein extraction and their preparation
Total seed protein (SDS-PAGE), a single mature and uncontaminated seed of Z. oxyphylla and Z. muritiana was selected for the analyses of total protein form each genotype collected from different regions of Swat KP, Pakistan. The seed was ground through the pestle and mortar and for the extraction of total protein and were add 400μl protein extraction buffer composition as (0.5M Tris-HCL pH 8.0, 0.2% SDS, 5M Urea, 1% B-mercaptoethanol) to 0.1g of seed powder into 1.5mL eppendr of tube and then vertex for three (3), mints thoroughly to homogenize the powder and solution.
Bromo-Phenol Blue (BPB) solution was added to the protein extraction buffer as tracking dye to monitor the movement of protein in the gel, then samples were centrifuge for 40 mints at 14,000rpm at 10 0 C and follow the standard electrophoretic method [4,39]. After centrifugation samples the curd protein were recovered as a clear supernatant on the top of the tube. Then were transferred into a new tube and were store at 120 0 C until electrophoresis. After that for the checking of make 12% polyacrylamide gel the separation gel were making form the mixing of (3.0M Tris-HCl pH9.0, 0.4% SDS) and 4.5% stacking gel (0.4M Tris-HCl pH8.0, 0.4% SDS), Electrode buffer (0.025 M Tris, 129 M Glycine, 0.125% SDS) was add into the top of the gel plate and select total volume as 8μl of the protein extraction mixture were loaded into each well of the gel with the help of micropipette and add molecular markers and finally run 100V until the blue color were passed through the bottom of the gel plates, the gel were stained by the staining solution containing 0.2% BPB dissolved in 10% glacial acetic acid, 40% methanol and water in the ratio of 10:40:50. Gels were destained in a solution consisting 5% acetic acid and 20% methanol for 15 minutes [40].

Data analysis
The current data was recorded from the design gel (destined) on the basis of absences and presences of total seed protein gel bands, 1 is denoted for the presence and 0, for the absence of the loci were arranged in Microsoft excel 2010, and this 0, 1 data were analyzed for cluster analysis and PCA (Principles Component Analysis) was performed by PCord 5.0, SSPS and Statistics.

Ethno-pharmacognosy
The current research work on two wild Ziziphus species Z. oxyphylla and Z. mauritina collected from different regions of KP, Pakistan, we have been in listed for control of diseases categories as12 and for each Ziziphus species , scientific name, family , local name, illnesses to be treated, and parts used for the different diseases were noted in (Table 1). Consumption of plant parts as medicine among the informants shows disparities. Fruits are mostly used part for majority, followed by roots, leaves and bark, this was the first time of the study area threat to the species is marginal as seeds are the leading plant part used for medicinal purposes. It was supposed that the collection of part of plant as medicinal part from the wild were not manageable. According to residents, this type of activity is done by the collectors related to illegal activity of medicinal plants. Ziziphus species mostly Z. oxyphylla is vulnerable to this type of activity in the study region. ICF values were established to know the settlement among the informants of Swat valley for usage of plants to cure certain illness groups. The ICF values ranges from 0.992 to 0.124 with an average value of 0.440. Jaundice has the highest ICF value 0.992 with 130 use-reports for 2 plant species.  munication among people in various areas. To discover conventionally significant medicinal species in the society, Fidelity Level (FL) of plants has been predicted based on use reports which have been cited by 50 or more informants for being used against a given disorder and the examination demonstrated that the highest FL value found in Z. oxyphylla followed by Z. mauritina respectively. The least FL value was found in the case of Z. oxyphylla. FIC and FL studies presented that the most commonly used species in the study area are Z. oxyphylla (ICF = 0.992) with 130 use-reports and FL value (100%). When choosing the most ideal plant species for each ailment category, we took the high-fidelity Level (%) in each category of ailment due their high biological compounds which were used for the control of various numbers of diseases (Table 2).

Morphological characterization
In the current work qualitative and quantitative characterizations were carried out of the collected samples, Qualitative traits were noted on the general visualization (phenotypic observations). qualitative traits i.e. Leaf pubescent, Leaf shape, Leaf col-or, fruit color, Seed shape and quantitative characters which were measured with the help of vernier caliper for the measurement of plant height (feet), Branching, Leaf length (mm), Leaf width (mm), Leaf thickness (mm), Petiole length (mm), Inter node length (cm), Stem diameter (inches), fruit weight, fruit diameter (mm) and fruit length (mm). By using the Pearson correlation coefficient, the result for the association coefficient among the various traits for the two species of Ziziphus (Z. oxyphylla and Z. muritiana) was performed (Table 3  and   *. Correlation is significant at the 0.05 level (2-tailed). The double data matrix of 50 genotypes based on morphology was analyzed for the construction of phylogenetic tree to represents the similarity of various species or genera and the two species of the Ziziphus were investigated for similarities and the phylogenetic tree was constructed. The phylogenetic tree divided the two species in three groups R1. RII and RII (Figure 1). R1 and RII consisted of total genotypes of Z. oxyphylla. While the RII was into composed of all genotypes of Z. muritiana. The similarity indexes were performed for all the genotype of 2 species that was 23.529% for Z. muritiana and Z. oxyphylla (Table 5).

Discussion
Many of the local communities in the regions depends on the consumption of medicinal plants to use for the control/use against different number of diseases, However, the vanishing of these plant species is steadily reported chiefly due to fluctuations in the environment, land degradation and unsustainable use of these plants; moreover, the expansion of invasive species has donated a lot to their disappearance [41]. Conservation of medicinal plant genetic diversity has freshly created a lot of attention in the tropics as a result of many years of misconduct, adverse environment as well as socio-economic changes. Population genetic theory expects that the reduction in the genetic diversity limits a species ability to keep pace with the changing selection pressure [42]. Plant species mainly the medicinal plants rely on the existing genetic diversity for constancy and survival under the ever-fluctuating environments [43]. Understanding medicinal plants species population genetic structure is vital for their conservation, planning and justifiable organization [44].

Agricultural Research & Technology: Open Access Journal
Therefore, a common goal line of conservation is to preserve genetic diversity in "red listed" species, which is crucial for longterm survival and evolutionary response to the altering environment [45]. One main implication of this method, from the viewpoint of conservation genetics, is that it could help us set sampling intervals of areas within populations to optimize the genetic diversity in collections from local populations of rare, endangered, or endemic plant species [46]. For the purpose of conservation of plant species, most of the investigations deal only with determination of genetic diversity in individual populations [40].
The purpose of this study was to recognize the phylogenetic relationship, genetic diversity, genetic structure, and a core collection of Ziziphus species. Now, we explain our consequences with respect to genetic diversity and the causes of the genetic idleness. The current position of genetic structure is briefly debated. Moreover, we further clarify the competence of the plan used to build the core collection. Genetic redundancy is a significant issue in plant genetic resource management. The identification of duplicates is important in germplasm repositories, particularly when considering the construction of core collections [40].
Various apparatuses are now presented for documentation of required differences in the genotypes, including morphological / phenotypic, biochemical and molecular markers [40]. Though morphological description is the principal step in the description and alliance of crops genotypes, but these are highly subjective by the environment [40]. The double data matrix of 50 genotypes based on morphology was analyzed for the construction of phylogenetic tree to represents the similarity of various species or genera and the two species of the Ziziphus were investigated for similarities and the phylogenetic tree was constructed (Figure 1). The phylogenetic tree divided the two species in three groups R1, RII and RII (Figure 2). R1 and RII consisted of total genotypes of Z. oxyphylla. While the RII was into composed of all genotypes of Z. muritiana. The similarity indexes for all the genotype of 2 species was 23.529% for Z. muritiana and Z. oxyphylla [47].
The two plant species under the genus Ziziphus study exposed that no two plants have similar protein banding patterns which demonstrates the presence of genetic diversity between these species. The presence of common bands/locus (L-1, 6, 7, 8) among these two Ziziphus species suggests their close genetic similarity and common ancestry [40]. Also, [40] accredited the appearance of a common locus/band in all individual in a population to the fact that the gene coding for the enzyme or protein does not differ. Due to High inter-species locus contribution toward genetic disagreement SDS-PAGE could be a reliable technique for identification of these two species, while intra-specie locus contribution toward genetic diversity was high in genotypes of Ziziphus oxyphylla (41.66%) as compare to Z. muritiana (25%).

Conclusion
Best of our current study was first time to investigated that the seed protein and ethno medicinal uses of wild Ziziphus species collected from different regions of KP, Pakistan, the genetic pool of dissimilarity within genotypes and Z. oxyphylla and Z. mauritiana as well, is due to selection as well as for the crops/plants species improvement and their conservation for the better studies of genetic diversity and its distribution in the wild Ziziphus species of the studies needed duet to their conservation and will be help greatly to labeling what to conserve as well as where to conserve and will be and will enhance our information and understanding of the taxonomy, origin and evolution of wild Ziziphus species (Z. oxyphylla and Z. mauritiana) respectively