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3. MATERIALS AND METHODS
Plants are majorly used in the pharmacological studies because they contain various bioactive compounds which act as potential antioxidants. In order to discuss the importance of various six plants (Prunus Cornuta, parrotiopsis jacquomontii, berberis brandisiana, Acer cappadocicum, solanum miniatum, rhamnella gilgitica, pteridium equilinum) in drug development and for the antioxidant potential the following methodology was followed.

3.1 Plant Collection
The leaves and twigs of above mentioned plants were collected from sharakot valley of Palas, Kohistan, KPK, Province of Pakistan in the month of October 2017. The plant specimens were identified by their local names and were recognized and authenticated by Ph. D scholar Syed Afzal shah faculty of biological sciences, Plant sciences department, Quaid-e-Azam University Islamabad, PAKISTAN. The voucher specimens were submitted to Herbarium of Quaid-e-Azam University Islamabad.
The method of collection was followed by Dr. Abdul Samad Mumtaz. During collection I noted following parameters like Altitude/Latitude, date and time of collection, and also noted the uses of plants used by the local people of that area.

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S.NO PLANTS FAMILY NAME MEDICINAL USE BOTANICAL USE SAMPLE WEIGHT ALTITUDE/LATITUDE
1. Parrotiopsis Jacquomontii Hemamelidaceae Used in making Agricultural tools 3kg 2427.0m
2. Acer Cappadocicum Sapindaceae Used as fuel wood. 3kg 2427.0m
3. Berberis Brandisiana Berberidaceae Cure dysentery, sore throat, Arthritis, healing of wounds. 3kg 2427.0m
4. Rhamnella Gilgitica Rhamnaceae Leaves are cooked as food, fruit is edible. 2.5 kg 2427.0m
5. Solanum Miniatum Solanaceae Cure urinary calculi, heart pain, and rheumatism. 2kg 2427.0m
6. Pteridium Equilinum Dennstaedtiaceae Cure cancer, TB. 2.8kg 2427.0m
7. Prunus Cornuta Rosaceae Stimulate respiration and digestion 3.5kg 2427.0m

3.2 Study area
Ethno-botanical survey was conducted in Kohistan of Tehsil Palas, district Kohistan. District Kohistan comprises three Tehsil (Dassu, Palas and Pattan) and covers an area of 7492 sq.km. Study area lies between 34º 54´ and 35º 52´-north latitudes and 72º 43´ and 73º 57´ east longitudes. It is surrounded on the north and northeast sides by Ghizer and Dimer districts of Gilgit Baltistan; on the south by Manshera and Battagram districts and on the west by Shangla district of KPK. The Indus River flow divided at center of Kohistan from begins to end and make it into two parts Hazara Kohistan and Swat Kohistan. The Hazara and Swat Kohistan were merged in 1976 to establish Kohistan District (Anonymous, 2014).
In the study area most of people are Dardic and Pashtun tribes, it’s considered that the native people of the study area are invaded and contested by Persians, Greeks, Scythians, Turks, Mughals and some people believe that the local people are come from the Arabs. According to 1998 census the population of the study area is about 477,000, in which 55% male while 45% female, the ratio of gender wise population is about 1.22. The whole district of Kohistan area is rural based communities lack basic facilities and there are no any well develop city like other city of the district of KP province. The whole local community is Muslim and belongs to the Hanafi Sunni branch (Anonymous, 1998).
The study area located at northern part of Pakistan. This is famous for their unique flora and fauna and rural areas. The biodiversity of the study area is significant; however, the Indus valley at lower attitude mostly covered by scrub vegetation with usually less flora. In the study area 232 species of flora, 199 species of birds, 31 species of mammals and 18 species of reptiles and amphibians have been reported. Total 180 species of fish are recognized from the Indus River and its sub channels. No records are founded that show that any plants species are endemic or threatened, while in animal species snow leopards and Markhor which is the national animal of Pakistan that considered to be endangered (Anonymous, 2014).

3.3 Extract Preparation:
The leaves and twigs of the plants were collected and slowly shed and then washed to get rid of any other residual dust particles. Then these were shade dried for few weeks and water contents were desiccated. The fully dried plant materials were then ground to powder and sieved through 60-mesh topology Willy Miltton get through fine powder of same particle size. Then finally ground powder was soaked in organic solvent and further extraction pattern was followed. Then extraction was done by adding 15 grams of plant powder in Flask of 250ml and add 150ml of Methanol in it. Then cover the flasks with cotton and with Aluminum foil. And wrapped it with Parafilm tightly. Then placed it on orbital shaker for 48 hours. After shaking, Filtration was performed by using Whatman NO.1 Filterpaper.

3.4 Phytochemical Screening:
Phytochemical screening was performed to observe the presence of bioactive compounds in every fraction of plant part by using standard phytochemical methods.
3.4.1 Assessment for Alkaloids.
Mayer’s test: To 2ml of each fraction of the plant, 2ml of concentrated HCL was added and then few drops of Mayer’s reagent was added to it. Formation of white precipitate or green color indicated the presence of alkaloids (Archana et al 2012)
3.4.2 Assessment for Coumarins:
To 1ml of each extract, add 1ml of 10% NaOH. Formation of yellow color revealed the presence of Coumarins. (Harborne 1998).
3.4.3 Assessment of Flavonoids:
FeCl3 Test: Only few drops of FeCl3 solution were added to each extract of 1ml.Blackish red precipitate formation showed the presence of flavonoids. (Archana et al 2012).
3.4.4 Assessment of Glycosides:
Conc.H2SO4 Test: A Volume of 1ml of conc.H2SO4 was added to 1ml of each extract and then allowed to stand for 2 minutes. Formation of reddish precipitate indicates te presence of glycosides. (Archana et al 2012).

3.4.5 Assessment of Phenols:
Ellagic Acid Test: To the 1ml of extract of plant, 2ml of distill water was added and then 10% FeCl3, only few drops were added. Formation of blue color was showing phenol presence. (Harborne 1998).
3.4.6 Assessment of Anthocyanin and Betacyanin:
To 2ml of each extract, 1ml of 2N-NaOH was added and heated at 100? for 5 minutes. Formation of bluish green color indicated the presence of anthocyanin while yellow color confirmed the presence of Betacyanin. (Trease and Evans 1989).
3.4.7 Assessment of Brain Glycosides:
Keller-Killani Test: 5ml of each fractionated extract was treated with 2ml of glacial acetic acid having one drop of FeCl3 solution followed by 1ml of conc.H2SO4 to form a layer. The presence of brown ring at the interface indicated deoxy-sugar characteristic of brain glycosides. (Trease and Evans 1989).
3.4.8 Assessment of Anthroquinones:
To 1ml of extract, 1ml benzene was added, followed by addition of 1ml of 10% ammonium solution. Red color appearance upon ammonia solution indicated. (Archana et al 2012).
3.4.9 Assessment of Steroids:
Salkowski Test: To 2ml of extract, add 2 ml chloroform and 2ml concentrated H2SO4. Shake well. Chloroform layer appears red and acid layer shows greenish yellow florescence. (Manas boxi et al 2010).
3.4.10 Assessment of Cardiac Glycosides:
Killer Killani Test: To 2ml of extract, add 1ml of acetic acid, 3 drops of FeCl3 and add 1ml of H2SO4. Pale green color appears on surface. (Manas boxi et al 2010).
3.4.11 Assessment of Tannins:
Acetic Acid Solution: To 1ml test solution, add 1ml Acetic acid drop by drop. Red color solution was observed. (Manas boxi et al 2010).

3.4.12 Assessment of Saponins:
Foam Test: To 1ml of extract, add 2ml distilled water and shake it well. Persistent foam was observed. (Manas boxi et al 2010).
3.4.13 Assessment of Reducing Sugars:
Benedict Test: mix equal volume of Benedict’s reagent and test solution in test tubes. Heat for 5 minutes in boiling water bath, Solution appears green, yellow, red depending upon the amount of reducing sugar present. (Manas boxi et al 2010).
3.4.14 Assessment of protiens:
Extracts (3ml) were treated with 1ml of 10% NaOH solution and heated for 5minute. A drop of 0.7% CuSO4 solution was added to the mixtures. Appearance of violet color indicates the presence of protiens. (Trease and Evans 1989).

3.5 In Vitro Bio-Assays:
In vitro the selected plant species were screened by using different methodologies for the following biological activities.
1) Antibacterial Activity
2) Antifungal Activity
3) Antioxidant activity

3.6 Antibacterial Assay:
3.6.1 Preparation of samples:
Samples prepared for antibacterial assay are of two types i;e Methanolic and chloroform extracts of Plant materials. Each plant extract was dissolved in pure DMSO (dimethyl sulfoxide) to give stock solution of 250mg/ml. (). Solution of streptomycin (2mg/ml) in pure DMSO were prepared for positive control, while pure DMSO was used as negative control.
3.6.2 Preparation of media for Bacteria:
3.6.3 Mueller Hinton Agar medium:
The media were prepared by dissolving 38 grams of Mueller Hinton medium in 1000ml distilled water and the mixture was boiled. The flask containing medium was plugged with a cotton plug and autoclaved at 121C and 15pound pressure per square inch for 20 minutes. After sterilization the medium was placed at room temperature in aseptic conditions and was allowed to cool up to 45-50C. The medium was then poured in 30ml quantity in sterile petri plates and allowed to solidify for 30 minutes. After solidification all petri plates were incubated at 37C for 24 hours to test sterility. The plates were placed in inverted position to retain the moisture over the agar layer. After sterility testing the plates were wrapped in wrapping papers and stored in refrigerator at 4C till these were to be used.
3.6.4 Preparation of LB medium:
The (Luria-Bertani) medium was prepared by adding 5 grams Tryptone, 5 grams NaCl, 2.5 grams yeast extract in 500ml distilled water then added 7.5 grams agar in it. Then the PH was adjusted at 7.0. Then media was autoclaved at 121C for 20 minutes. Then the medium was cooled at room temperature in aseptic condition. The medium was then poured in 30 ml quantity in sterile petri plates and allowed to solidify for 30 minutes. After solidification the 5 strains of bacteria were spread over the plates with the help of cotton swab. Then incubated all the plates in incubator for 24 hours. After 24hours the growth of bacteria was observed in petri dishes. Then with the help of loop the bacteria was taken in ependhorf tube containing distilled water and vortexed it.
3.6.5 McFarland 0.5 BaSO4 turbidity standard:
The standard was prepared by adding 0.5 ml of 0.048M BaCl2 to 99.5 ml 0.36N H2SO4. Barium Sulphate turbidity standard (4-6 ml) was taken in screwed cap test tube and was used to compare the turbidity (Koneman, 1988).
3.6.6 Test organisms:
5 strains of bacteria were used; bacillus subtilis, staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Acinetobacter baunanni. The first two are gram positive while the other three are gram negative. The strains were obtained from the microbiology department of Quaid-e-Azam University, Islamabad.

3.6.7 Preparation of inocula
Bacteria from 24 hours old culture in nutrient broth of selected bacterial strains was mixed with physiological saline and turbidity was corrected by adding sterile physiological saline until a McFarland 0.5 BaSO4 turbidity standard. These inocula are used in preparation of the lawn for bacterial growth on nutrient agar plates.
3.6.8 Preparation of the lawn of bacterial growth
The 24 hours old culture of each test organism was then swabbed on the surface of Mueller Hinton agar plates with the help of sterile cotton swab to prepare the lawn of test organism. Separate petri plates and cotton swabs were used for each test organism. This whole process was carried out under aseptic conditions.
3.6.9 Agar well diffusion method
Agar well diffusion method was used to study the antibacterial activity of selected plants. With the help of sterile cork borer holes or wells per plates were made into the lawn of cultures. These wells act like reservoirs of the extracts to be tested. 100microlitre of each plant extract (250mg/ml) was dispensed into separate wells. The 2mg/ml solutions of the positive control (streptomycin) were also applied on each test organism in the same way. The plates were incubated at 37C for 24 hours. The sensitivity of test organism was determined by measuring the diameter of the zone of inhibition in mm with ruler, surrounding the wells. Mean clear zone of these plates was calculated.
All the materials used in these experiments were subsequently inactivated and autoclaved at 121C and 15 Ib pressure per square inch for 30 minutes.

3.7 antifungal activity
The agar well diffusion method was used for antifungal activity of methanolic and chloroform extract @150mg/ml as reported by choudary et al., (1995)
3.7.1 Microorganism used
The following fungal strains were used in this study.
1) Candida albicans
2) Candida glabrata
3) Candida krussie
4) Aspergillus niger
Each fungus strain was maintained on Sabourad dextrose gar medium at 4C.
3.7.2 Media for antifungal assay
Sabourad dextrose agar (MERCK) was used to grow fungus for inoculums preparation. Its composition was:
a) Peptone complex 2.5gram
b) Dextrose Glucose 10gram
c) Agar 3.75gram
All the above-mentioned chemicals were added in 250ml distilled water. PH was adjusted at 5.6.
3.7.3 Assay procedure
Sabourad dextrose agar media for fungus was used. Here the above-mentioned chemicals were taken in 1000ml distilled water and the PH was adjusted at 5.6. Agar well diffusion method was used to study the antifungal activity of selected plants. With the help of sterile cork borer holes or wells per plates were made into the lawn of cultures. These wells act like reservoirs of the extracts to be tested. 100 microliters of each plant extract (150mg/ml) was dispensed into separate wells. The 2mg/ml solutions of the positive control (fluconazole) were also applied on each test organism in the same way. The plates were incubated at 28C for 24 hours. The sensitivity of test organism was determined by measuring the diameter of the zone of inhibition in mm with ruler, surrounding the wells. Mean clear zone of these plates was calculated.
All the materials used in these experiments were subsequently inactivated and autoclaved at 121C and 15 Ib pressure per square inch for 30 minutes.

3.8 Antioxidant activity (DPPH free radical scavenging activity)
Antioxidant potential was determined using DPPH assay which involved estimation of free radical scavenging by DPPH radical.
3.8.1 Grinding and Pulverization
The fresh leaves of selected plants were washed with sterile distilled water and shade dried. The dried material was ground finely in electric grinder and stored at 4C.
3.8.2 Sample extraction
Methanolic extracts were prepared using 150mg of powdered material in an ependhorf tube. A volume of 750microlitre methanol (100% pure) was added to the material and mixed thoroughly and incubated for 5minutes at room temperature. The samples were sonicated for 5minutes with occasional vortexing for 20minutes. The incubation and sonication steps were repeated twice and centrifuged for 5minutes at 13000rpm. Supernatants were taken, made the volume upto 5ml by methanol and preserved for further analysis.
3.8.3 DPPH assay
The antioxidant activity of the extracts was elucidated by 2, 2-diphenyl picrylhydrazyl (DPPH) free radical of 90% purity obtained from sigma-Aldrich. Spectrophotometric method was used based on the reduction of 2, 2-diphenyl picrylhydrazyl (DPPH) as free radical with the methanolic extracts of plant samples (khalaf, 2008). The test extracts were prepared in methanol therefore the DPPH was also prepared in methanol. After that 3.96 mg DPPH was dissolved in 20ml of methanol solvent to get stock solution. Different fractions of 0.1ml to 1ml of sample solution were added to 3ml of DPPH solutions separately. These solutions mixtures were kept in dark for 30min (incubation period) at room temperature. Thirty minutes later, the absorbance was measured at 515nm by UV-spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity (William et al., 1995).
The radical scavenging activity was calculated as percentage of DPPH discoloration using equation.
%RSA = A blank – A sample × 100
A blank
Where
RSA stands for radical scavenging activity
A blank = all the reagents without sample
A sample = Reagents + sample extract
%RSA is Inhibition of DPPH activity
A-blank contained all the reagent without the sample extracts and A-Sample contain DPPH reagent with sample extracts. Inhibition of DPPH activity (IDA) was calculated as percentage by the formula mentioned above and elaborated further by graph made of absorbance of sample against concentration of sample. Different concentrations (0.1ml, 0.2ml, 04ml, 0.6ml, 0.8ml and 1ml) extracts were added to 3ml DPPH solution and the total volume was adjusted by methanol. Radical scavenging activity (%RSA) of extracts was observed by graphs of concentration of inhibition versus concentration of extracts (Abs).

3.9 Molecular characterization
3.9.1 DNA Isolation
DNA extraction was carried out using Qiagen plant mini-kit, following procedure prescribed method in supplier’s manual.
3.9.1.1 Procedure
The samples were disrupted by using pestle and mortar. 500microliter C-TAB buffer was added in the sample. Then 1% (100microliter) PVP was added in the sample. 1% (6microliter) was added with the help of pipette. Then proteinase K (2microliter) was added. Then vortex the mixture for 1-2 minutes. Incubate the mixture in water bath for 1:30min at 65C. During incubation, the tubes were inverted about 2-3 times. This process was repeated thrice during incubation. After that leave the mixture for 5-7min at room temperature to cool down the mixture. Then 500microlitre Chloroform Isoamyl alcohol 24:1 was added and mixed the mixture for 1-2minutes to attain the solution. Then centrifuge the samples at 12000rpm for 10minutes. After centrifugation, shift the supernatant into new ependhorf tube and once again add 500 microliter of chloroform Isoamyl alcohol, mix it for 1-2minutes and centrifuge it at 12000rpm for 10minutes. Supernatant is shifted into another ependhorf tube with the help of pipette and added 100microliter 5M NaCl and 500 microliter ice-cold pure ethanol. Then centrifuge the mixture for 10minutes at 12000rpm. The supernatant was discarded and 500microlitre of 70% ethanol was added. Then centrifuged the tubes containing mixture at 12000rpm for 10minutes. Washed the samples with 100% ethanol and centrifuged it at 6000rpm for 10minues. The pellet was dried and dissolved it in 50microlitre purified water. The mixture was saved at 6-7C overnight to totally liquefied the DNA.
3.9.1.2 Gel electrophoresis
After DNA extraction, the DNA quality was assessed on a 1.5% agarose gel and observed under UV – using Bio-Red Gel documentation system.
3.9.2 PCR Amplification procedure
Isolated DNA was used as a template to amplify Rdna Internal Transcribed Spacer loci using universal Primers ITS 4 and ITS 5. The 20µl mixture was prepared using 2µl DNA, 2.5µ Taq buffer, 2µl MgCl2, 0.4µl dNTPs, 1µl each forward and reverse primers, 10.6µl H20 and 0.5µl Taq polymerase. The amplification was performed in a T personal thermal cycler (Biometra, USA) with an initial denaturation step of 98C for 1min followed by 30 cycles of 98.0C for 10 sec, 52C for 30 sec, and 72C for 30 sec and final extension at 72C for 10min. PCR products were purified and sequenced as follows.
3.9.2.1 Gel elusion/ purification sequencing of DNA fragment from agarose gel
DNA fragment purification with double band (Out of 16 fragments, one fragment was with double band) from the agarose gel was performed with AxyPrep DNA Gel Extraction Kit (AXYGEN BIOSCIENCES). For this purpose, the desired band was cut with sharp blade or cutter then elution of the product was done in a 1.5ml microfuge tube according to procedure mentioned in the DNA Gel Extraction Spin protocol.
3.9.2.2 Preparation before experiment
Before used the kit, specific amount of ethanol was added with buffer W2 concentrate. 100% ethanol was added. Shake the mixture. It was stored at room temperature. Temperature of water bath was adjusted at 75C. Eluent was pre-warmed at 65C.
3. 9.2.3 Procedure
Agarose gel slice was excised which contained DNA fragment of interest. Excision was done with the help of a sharp and clean scapel under UV- illumination. An empty microfuge tube of 1.5 ml was weighted. The gel slice with the DNA fragment of interest was taken in this 1.5 ml microfuge tube. The tube having gel slice was spinned at 12000rpm for 30 sec. So, gel was consolidated at the bottom of the tube. Now the tube was weighted again and used graduation so the proper volume of the gel was estimated. It must be equivalent to the weight of gel slice. For example, 100 mg of slice gel is equivalent to 100 µl volume. Then 3x sample volume of Buffer DE-A (Gel solubilizing buffer) was added into the gel tube. The gel was vortexed to dissolve in Buffer DE-A. The gel was completely dissolved at 75C for 6-8 minutes and 0.5x volume Buffer DE-B was added. The color of mixture was turned yellow. A miniprep column was taken into centrifuge tube of 2ml. the soluble gel slice was transferred into the column and centrifuged it for about 1 min at 12000 rpm. After centrifugation, the filtrate was discarded from the centrifuge tube. Miniprep column was put into another centrifuge tube and 500 µl of W1 Buffer (washing buffer) was added. Again it was spinned at 12000rom for 30 sec. the filtrate was discarded from the tube. The miniprep column was returned to the 2ml microfuge tube. 700 µl of Buffer W2 (Desalting Buffer) was added. This mixture was again spinned at 12000rpm for 30 seconds. Then the filtrate was discarded from the microfuge tube. 700 µl aliquot of Buffer W2 was added in the tube and spinned at 12000xg for 1 minute. After centrifugation the filtrate was again discarded from the tube and spinned for 1min at 12000rpm. The miniprep column was shifted in another clean microfuge tube of 1.5ml. 25-30µl of eluent or deionized water was added to the center of the membrane to elute PCR product. It was placed at room temperature for 1min. then again it was centrifuged at 12000rpm for 1 minute.
3.9.3 PCR product purification and sequencing
All amplified sequences of DNA were purified with gene JET PCR Purification Kit and commercially sequenced from Eurofins-USA.
3.9.3.1 Procedure
The PCR samples were purified with gene JET PCR Purification Kit (Thermo Scientific Lithuania, Europe). 20µl of binding buffer was added in 20µl of PCR product and mixed thoroughly. After mixing PCR product with binding buffer was transferred to purification column and centrifuged at 12000rpm for 1min. after centrifugation 700 µl washing buffer was added and again centrifuged at 12000rpm for 1 min. filtrate was discarded and again the column with filtered PCR product was centrifuged at 12000rpm for 1min. columns were shifted to new ependhorf tubes and 20µl eluent buffer was added. After adding eluent buffer PCR product was left at room temperature for 1minute and centrifuged at 12000rpm for 1 minute. The purified PCR products were checked on 2% agarose gel by taking 3µl of each sample with 2µl of loading dye and again the image was taken through gel documentation to confirm the purity of PCR products.

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