Biosystematic and Pharmaceutical Analysis of Genus Ficus from Bhimber Azad Jammu and Kashmir, Pakistan

Abstract

The current research was conducted to explore Biosystematics and Pharmaceutical Analysis (BPA) of ten species of genus Ficus naturally occurring in District Bhimber of Azad Jammu and Kashmir (AJK), Pakistan. Fruits and other plant parts of these species have been used in traditional folklore therapeutics as to cure heart diseases, high blood pressure, constipation, fever, piles, leucorrhoea and energetic tonic. The selected taxa have also been playing pivotal role in ecosystem services in wild areas of AJK, Pakistan for indigenous communities. As Ficus species are important source of green medicines and other pharmaceutical products which belong to family Moraceae. Albeit these taxa have been classified according to traditional taxonomic principles but due to plasticity of floral parts, flower types and positon of occurrence has lead into many mis-interpretation of species of the genus, hence there is need to conduct comprehensive taxonomic research using different approaches for valid identification, demarcation and classification of the species. This is first attempt to determine biosystematic positon of species of Ficus occurring in District Bhimber of AJK, Pakistan by four different methods. In first part of research, Biosystematic analysis (BSA) of genus Ficus was conducted using taximetrics, histology, chemotaxonomy and molecular approaches. For BSA; in taximetric technique selected ten species viz: Ficus benghalensis, F. religiosa, F. sarmentosa, F. benjamina, F. semicordata, F. racemosa, F. auriculata, F. carica, F. elastica and F. palmata were morphologically surveyed and their sixteen distinctive phenetic characters were analyzed and coded in form of numerical values in matrix. The data was analyzed by PAST software version 2.17 and MVSP which divided the species into four clusters depicting linkage distance (LD) of 3.9. Ficus sarmentosa appeared as separate line with LD 3.8 depicting that it is morphologically different from other allied taxa of the genus. The species F. religiosa, F. benghalensis, F. benjamina and F. elastica belong to one sub-genus Urostigma. F. elastica and F. benghalensis depicted the closest relationship with LD 0.9. The leaf epidermal and stem histological data of the species depicted that Ficus sarmentosa delimited as one unique group with LD 2.6. The least LD (0.15) was found between F. elastica and F. benghalensis showing strong histological based affinity proving that these are genetically near relative. Other close-affinity was found in section sycomorus having LD 0.25. In chemotaxonomic analysis, Ficus sarmentosa showed maximum LD 4.6 delineating it as one unit proving it is one separate sub-genus. A close affinity was found among the members of subsection conosycea with LD 1.0. F. racemosa and F. sarmentosa depicted least LD value of 0.05 which proves that these are genetically closest members. In molecular analysis, in which DNA based taxonomic studies were conducted where Ficus sarmentosa was also present as most distant group of all ten species proving that it belongs to separate subgenus synoecia having LD 2.4. At subsection level close association was found among the F. benghalensis, F. elastica, F benjamina and F. elastica with LD of 1.4 which proved that these are genetically very close to each other and hence these all fall in subsection Urostigma. These all approaches prove that all the ten species can be identified and classified on basis of any one or more of the above techniques and which can be used to delimit it from its allied taxa. The molecular approach DNA barcoding was shown more reliable than others as it is least effected by climate impacts. These comprehensive taxonomic approaches will be useful for quality control (QC) of herbal drugs and allopathic medicines by authentic identification and demarcation. Prior to conduction of pharmaceutical analysis, preliminary research was conducted by using an ethnobotanical analysis (EBA) method. The EBA was recorded through open ended (OE) and close ended (CE) interviews of 100 indigenous people of age groups from 25 to 70 years old comprising of both genders. The ethnobotanical (EB) data was collected in field note book (FNB) and analyzed through microstatistical tools such as UV (used value), ICF (Informant census factor), and FL (fidelity level) which depicted that Ficus species of study area were prevalently used to cure different diseases by indigenous communities of the area. The analysis revealed that Ficus palmata, F. semicordata, F. carica and F. auriculata are commonly used as green/botanic drugs with UV of 0.06 and highest ICF values: 0.97, 0.95 and 0.95; respectively for cure of heart diseases, high blood pressure and constipation. The highest fidelity level was shown for fever, piles and leucorrhoea with 90%, 70% and 70% values, respectively. Thus, EB studies proved good indicator for drug discovery and these species having high EB statistical indices were subjected to pharmacological analysis (PCA).In PCA, different parameters like antibacterial activity (ABA), antifungal activity (AFA) anti-diabetic activities (ADA), anti-lipidemic activity (ALA), antioxidant activity (AOA) and enhancer of uretic potential (DP) were conducted. The leaf, stem and fruit parts were powdered and extracted in four solvents using petroleum ether (PE), chloroform (Chl), methanol (MeOH) and Aqueous (Aq). For some activities ethanolic (EtOH) solvent was employed. The methanol proved to be the best solvent followed by the aqueous, chloroform and P.E. Highest extraction yield was found for leaf parts of the taxa in MeOH solvent with 5.95mg/g. For antibacterial analysis (ABA) three bacterial strains: Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli, and for antimycotic studies (AMS) three fungal species viz; Fusarium solani, Aspergillus flavus and Candida albicans were tested against all Ficus species’ extracts using Agar Well Diffusion (AWD) method and Micro Dilution (MD) method. Extracts of plant Ficus racemosa were extracted in four solvents and it was found that during ABA highest zone of inhibition (ZI) was found for MeOH extraction fraction. It showed maximum value of ZI 19.8±0.35 against Staphylococcus aureus with activity index (AI) of 0.94 while least impact was seen for Pseudomonas aeruginosa with ZI of 17.1±0.21 and AI of 0.80. Highest MIC was seen for aqueous extract with 49.6±3.4 and MBC was found 96.8±0.2 against S. aureus while in fungal analysis highest MIC was 58.3±0.4 and MFC was 116.4±0.2. Extracts of Ficus auriculata in MeOH solvent depicted highest ZI values against S. aureus with 18.3±0.5 and AI of 0.94. While for antimycotic studies (AMS), methanolic extract depicted highest ZI (19.8±0.5) and AI 1.2 against fungus C. albicans. The aqueous extract of the leaf depicted highest MIC (56.1±0.5) and MBC value of 97.8±0.3 against bacterium S. aureus while its aqueous extract showed highest values of MIC (56.1±0.8) and MFC (116.2 ± 0.13) against Aspergillus flavus. MeOH extract of Ficus palmata depicted very significant zone of inhibition (ZI) with 17.8±0.12 and AI of 0.84 against S. aureus while these were good near to control/standard values of 19.3± 0.2. For antimycotic analysis, the best activity of ZI and AI was found against fungus C. albicans having 18.1±0.8 and 1.21, respectively. The aqueous extract of the plant depicted highest MIC value with 46.4 ± 0.6 and MBC (95.8±0.50) against bacterium E. coli. In antifungal activity, highest MIC value (58.7±0.8) and MFC with 117.1±10 was seen against F. solani. The MeOH extract of Ficus religiosa showed least activity against bacterium P. aeruginosa having ZI with 15.3±1.0 and AI 0.90 while it had very prominent control activity for S. aureus with ZI of 17.2± 0.5 and AI of 0.84. For antifungal analysis, methanolic fraction depicted highest ZI (18.7±1.4) was found against A. flavus and AI of 1.06. For MIC and MBC analysis, it was found that highest values were found against S. aureus with 49.8±0.7 and 96.9±0.1. In antimycotic analysis, methanolic extract showed the best activity with 60.3±1.1 and 117±0.40 against the F. solani fungus. For extracts of Ficus benghalensis, it was found that water extract depicted the least ZI activity (6.5±0.1) with AI (0.26) against S. aureus while MeOH extract showed highest ZI 20.5±0.10 and AI of 1.19 against fungus Aspergillus flavus. The water extract of the plant showed highest MBC with 99.8±0.95 for S. aureus and MFC 119.7±0.81 for A. flavus. For plant Ficus carica, out of different extracts MeOH extract depicted highest ZI (20.5±0.55) with 0.93 AI against P. aeruginosa bacterium. The fungus research proved that MeOH extract was found the best against C. albicans with ZI of 21.8±0.95 and AI having 1.24 values. For MBC analysis, it was found that highest MBC (105.6±0.09) was found for P. aeruginosa and the best MFC was found for F. solani having 122.2±0.40 values. The methanolic extract of Ficus sarmentosa depicted highest ZI 19.7±0.55 with AI of 1.38 against E. coli and highest antifungal activity with ZI of 23.1±0.6 and AI was 1.18 for C. albicans. For MBC highest reading was found for aqueous extract against S. aureus while the best MFC was found for chloroform extract against F. solani with 115.8±0.5. For Ficus semicordata, the leaf extract in solvent MeOH showed the maximum ZI 19.5±0.8 with AI of 0.92 against S. aureus bacterium while P. aeruginosa was most resistant with less ZI of 10.2±0.2 having AI of 0.45. The aqueous extract of the plant showed maximum MBC (101.5±0.8) and the best MFC was recorded against F. solani with value of 121.5±0.6. In antimicrobial analysis, in extracts of Ficus elastica, it was found that MeOH extract depicted the highest value of ZI (13.3±0.1) against bacterium S. aureus followed by 12.1±0.7 ZI value for P. aeruginosa. For antimycotic activity, the best ZI (19.3±0.4) was recorded for C. albicans with AI 1.66. Aqueous extract of plant showed the highest MBC with 91.9±1.2 against S. aureus and the best MFC value viz: 112 ±1.2 was found for the A. flavus species. For Ficus benjamina plant, in antibacterial analysis it was found that MeOH extract depicted the highest ZI was 111.5 ±0.15 with AI 0.74 against E. coli bacterium and while the best MBC was recorded for methanolic fraction with value of 80.6 ±0.7. For antimycotic research, it was found that the highest ZI (21.5 ±0.5) was found for water extract against F. solani fungus and its best MFC was recorded for 111.7 ±0.5 for the P.E. fraction against fungus A. flavus. For antidiabetic analysis (ADA), MeOH leaf extract of ten species were testified against the model organism (rabbits) for 5, 10 and 15 days using positive and negative control. It was found that Ficus palmata decreased the blood serum sugar with 125 ±2.6, followed by antidiabetic potential of F. racemosa with 134 ±3.5 value and 135 ±2.5 for F. carica as compared with positive control value of 325 ± 4.2. The antilipidemic analysis (ALA) was determined for the five species of Ficus selcedted on basis of EBA and it was found that leaf MeOH extract of Ficus religiosa depicted the best antilipidemic potential (with dose of 500mg/Kg on model organisms/rabbits) as compared with control (Fenofirbrate) with dose of 65mg/Kg in which total cholesterol (TC) was reduced from 100.38 ± 3.5 (Day-0) to 85.66±1.5 (Day-28). Total glycerides (TGs) were also considerably lowered by these Ficus green medicines. Similar good results were prevalently obtained for other extracts of species Ficus sarmentosa, F. semicordata and F. auriculata showed good hypolipidemic activity and could be potential mean of use as drug development and discovery. Diuretic potential (DP) was determined for ten taxa of Ficus and; its two EtOH and MeOH extracts were prepared and applied for treatment on the model organisms/rabbits. It was found that all plants depicted good ADA but out of these F. benghalensis MeOH extract produced urine volume (UV) of 2.2 ±0.74; conc. of Na (3.1±1.2), K (2.1±1.2) and Cl (3.31±1.2) as compared with negative control (NC) (5.2±0.91), Na (5.65±1.4), K (2.13±1.0), Cl (3.57±4.1) and positive control (PC) with UV (1.4±0.31), Na (3.5±0.5), K (1.98±1.24) and Cl (2.98±1.2). Ficus religiosa extracts of depicted good ADA and it was found that UV was 2.2±0.7 for EtOH and 1.45±1.24 for MeOH as compared with NC 5.1±0.82 and PC 1.4±0.41. The concentration of other electrolytes in EtOH solvent was Na (3.5±2.0), K (2.2±1.14), Cl (3.05±1.24) and in MeOH it showed Na (3.1±1.2), K (2.10±1.04) and Cl (3.31±1.25). All other plant testified plants extracts also showed good uretic activity in model organisms. The antioxidant activity (AOA) was determined by using DPPH (1, 1diphenyl 2-picrylhyorazyl) method; in which MeOH extract with conc. of 250 mL of Ficus benghalensis and F. racemosa depicted highest AOA values of activity 92.4±1.2 and 91.80±0.07 among all the tested taxa and it was near to control used 95.2±0.92. For EtOH extract highest values (95.42± 0.11) was found for Ficus benghalensis and F. racemosa ethanolic fraction also showed 2nd high value with 91.81± 0.05 that were coincided with control used, proving their prevalent AOA potential. The current findings on ten wild species Ficus of occurring wild in District Bhimber Azad Jammu and Kashmir (Pakistan) might be useful for preparation of optimized herbal drugs or green drugs, already being indigenous part of traditional cultural folklore treatments. The fruits of these plants can be used as food phytonyms or phytomedicines in form of food medicines which are commonly used by rural and mountainous areas inhabitants. Furthermore, these findings obtained through EBA and PCA can be proved as way forward for allopathic drug discovery or drug development which will be milestone towards for pharmaceutical industries to produce novel drugs against the resistant gaining microbes against the known antibiotics/drugs. These research outcomes will culminate into alternative medicine development by local healers and practioners as indigenous pharmacopeia and ultimately novel allopathic drugs can be developed through comprehensive pharmaceutical research and by commercial procedures of the industries.