An antibacterial composition and uses thereof

Abstract

The invention relates to the identification of the Populus ciliata tree bark as a natural antioxidant and with potential antimicrobial activities. Crude methanol bark extract and its fractions (ethyl acetate, n-butanol, chloroform and n-hexane) were screened for antioxidant analysis and antimicrobial activities against gram positive (B. subtilis, S. aureus) and gram negative (E. coli, P. aeruginosa and K. pneumoniae) bacteria. Results of DPPH, ABTS and FRAP assays revealed the highest antioxidant potential of n-butanol and ethyl acetate fractions with lower IC50 value as compared to standard ascorbic acid, crude methanol extract and others fractions. Total phenolic content and total flavonoids content were also observed to be higher in n-butanol fraction (317.83±2.95 mg / g gallic acid and 271.26±0.89 mg / g quercetin, respectively) followed by ethyl acetate fraction, than crude methanol extract, chloroform and hexane fractions of bark indicating the highest antioxidant potential of P. ciliata bark extracts. Antibacterial assay also showed higher zone of inhibition of n-butanol and ethyl acetate fractions of P. ciliata bark against gram positive and gram negative bacteria, later on supported by the lower MIC value (62.5µg / ml) of both fractions. Present invention concluded P. ciliata tree bark as higher antioxidant and antibacterial potential agent whose n-butanol and ethyl acetate fraction was observed to be more effective than crude extract alone. This work further includes the extraction and identification of compounds with higher biological activities (antioxidant and antifungal activities) which would be further helpful in decreasing the pressure of pharmaceutical companies on wild herbaceous medicinal plants.

Description

FIELD OF INVENTIONThis invention relates to antibacterial composition prepared from methanolic extract of bark of P. ciliate.BACKGROUND OF INVENTIONMedicinal plants have been used as source of molecules of therapeutic potential for a long time and now represent an important source for the identification of novel drug leads. Approximately 80% of the total world populations rely upon traditional medicines, whereas the remaining 20% population is also utilizing the benefit of natural products directly or indirectly. Although every plant has some medicinal value, but among all, most exploited ones are herbaceous medicinal plants while some of them are shrubs and trees. The extensive or massive use of medicinal plants in pure form or in various formulations has created a danger to the existence of plant and they are entering into the state of getting threatened or endangered. The pressure on these herbaceous medicinal plants can be overcome by utilization of easy and fast growing trees with higher medicinal value. Literature showed that the medicinal values in the plants were due to the presence of secondary metabolites particularly phenolic compounds. So the plants / trees with higher phenolic content can be explored in this field.Populus ciliata Wall ex. Royle, also known as Himalayan Poplar is the multipurpose native tree of the Himalaya. The plant has a fast growth rate, easy to grow, i.e. can be propagated by cutting and can be grown easily in less nutritive soil or on unstable hilly areas. The plant has also been reported in bio-remediation purpose. The plant is mostly used by the local people as fodder or fire wood purpose, but the medicinal properties of its bark have also been reported in literature. P. ciliata is rich in phenolic content like Caffeic acid; 4- Coumaric acid; Ferulic acid, etc. therefore, can be explored in the area of drug discovery. Previous work on this plant was only concerned with the sex identification and genetic variation among its different populations. Populus tremuloides, P. nigra, P. ussuriensis etc. are some species of Populus which have been screened for antioxidant and antibacterial potential. No prior work has been reported in the literature where bark of P. ciliata has been used for screening of various biological activities. Since, this tree has higher amount of phenolic content, therefore it can be explored for identification and isolation of antioxidant and antibacterial compounds and further helpful in the development of effective drug system for the cure of various diseases.Natural plant based product for new drug discovery is very useful for human kind and this science is as old as our scripture. The extraction of natural compounds in pure form as the2 standardized extract provides unlimited opportunities for new drug discovery because of their unmatched chemical diversity. Secondary metabolites present in plants have a role in defence (protection from harmful UV light, abiotic factors, pest and diseases, etc.), in pollination and also act as signalling molecules.The production of natural drug has now acquired various limitations and one of the major limitations is the extensive use of medicinal plants for extraction of bioactive compounds resulted into loss of these medicinal important plants. Due to this, many of the pharmaceutical industries have shifted their interest toward the discovery of synthetic compounds. But these new chemically synthesized drugs have not shown the satisfactory results and do not fulfil the expectations; therefore their number is now declining into the market. All these circumstances have now strengthened the interest of researchers in natural product-based discovery. The discussed limitation can be overcome by the use of fast, easily growing and vegetative reproducing trees as they can be sustainably utilized for compounds extraction purpose. https: / / iopscience.iop.org / article / 10.1088 / 2053-1591 / ab19c8; Muhammad Hafeez , Rabia Arshad, Muhammad Usman Hameed, Bilal Akram, Muhammad Naeem Ahmed, Syed Ayaz Kazmi, Ikhtiar Ahmad and Shaukat Ali disclose utilization of the aqueous extract of Populus ciliata (Wall. ex Royle) leaves to synthesize zinc oxide nanoparticles (ZnO-NPs) at mild temperature. The nanoparticles formation, their dispersion, shape and size were confirmed by various characterization techniques i.e. Fourier Transform Infra-Red (FT-IR) spectroscopy, Scanning Electron Microscopy (SEM), Tunneling Electron Microscopy (TEM), x-ray diffraction (XRD) and Energy Dispersive x-ray (EDX). In addition to this, the FTIR analysis indicated the presence of important functional groups. The as synthesized NPs have been used for antibacterial studies against human pathogenic bacteria; E. coli, P. aeruginosa, K. pneumoniae, S. aureus and S. pyogene. An increase in the zone of inhibition was observed with increasing ZnO-NPs, concentration.IN2007DE01591A; Ajay Kumar Thakur, Mr. Rajesh Kumar Shandil, Mr. Dr Dinesh Kumar Srivastava disclose a reproducible high efficiency plant regeneration protocol through direct organogenesis after Agrobacterium tumefaciens - mediated npt-II and gus genes transfer in male plant of Himalayan poplar (Populus ciliata Wall) comprising culturing disarmed Agrobacterium tumefaciens LBA 4404 strain containing ß-glucuronidase, gusgene in a binary vector pBI121 alongwith a kanamycin resistance gene npt II for overnight for example at 28°C, centrifuging for 10 minutes at 5000 rpm, resuspending the pellet in MS liquid medium to get the concentration of 108 cells / ml, cocultivating 0.5 cm X 0.5 cm size leaf explants in the bacterial suspension for 15-20 seconds and inoculating on the respective medium containing BAP (1.5-2.0 mg / l)+ IAA (0.1-3 0.5 mg / 1) and kinetin (0.5-2.0 mg / 1) + IAA (0.1-0.5 mg / 1), preferably 0.5 mg / 1 kinetin,0.2 mg / 1 IAA for 24, 48 and 72 hrs, preferably 48 hrs and then transferring to the same media containing kanamycin 50 mg / 1, cefotaxime 500 mg / 1 for 14-21 days followed by transferring elongated shoots to the rooting media with various concentration of different auxins IAA, NAA, IBA, 2, 4 D .05-. 20 mg / 1 preferably 0.10 mg / 1 IAA for 10-15 days.None of the prior art indicate above either alone or in combination with one another disclose what the present invention has disclosed. The objective of the present invention is to screen the crude extracts of Populus ciliata bark for different biological activities (antioxidant and antimicrobial).SUMMARY OF INVENTIONHimalayan Poplar also called Populus ciliata Wall. ex Royle (family Salicaceae) is one of the native, fast growing, medicinally important forest trees of temperate Himalaya who has been reported as a valuable source for therapeutic agents. It has many ecological, economical and medicinal qualities. Not much prior work has been done of P. ciliata regarding the screening of crude extracts for biological activities. Although various Populus species have been screened for the isolation of biologically active phenolic glycosides, Literature shows that P. ciliata is rich in phenolic compounds (Caffeic acid, 4-coumaric acid, Ferulic acid), phenolic glycosides (populin, salicin), tannins, etc. Moreover, phenolic glycosides have already been proven with biological properties like antimicrobial, antioxidant, anti-inflammatory, etc. Ethno medicinally, the bark of the plant has been reported in the purification of blood, treatment of rheumatism and fatigue and also in the relieving of menstrual cramps pain. Traditionally, local people mixed the paste of the bark with cow dung ash and applied for the treatment of swelling. Therefore, the extraction of the natural compounds as expected from Himalaya poplar will be an innovative study to explore new series of compounds with enhanced biological potential. The first step for the isolation of bioactive compounds from the plant bark is the screening of crude extracts for different biological activities. Therefore, the present work was focused on the screening of various fractions of methanolic extract of P. ciliata bark for antioxidant and biological activities.BRIEF DESCRIPTION OF DRAWINGS:Figure 1: Radial scavenging activity for DPPH assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF-hexane fraction, CF- chloroform fraction, AA- ascorbic acid).4 Figure 2: Radial scavenging activity for ABTS assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF-hexane fraction, CF- chloroform fraction, AA- ascorbic acid).Figure 3: FRAP activity in terms of Fe (II) equivalents in mM in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF- hexane fraction, CF- chloroform fraction, AA- ascorbic acid).Figure 4: Quantification of total phenolic content in crude methanolic extract of bark and their fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF-hexane fraction, CF- chloroform fraction)Figure 5: Quantification of total flavonoids content in crude methanolic extract of bark and their fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF-hexane fraction, CF- chloroform fraction)Figure 6: Antibacterial activity in different concentration of crude methanolic extract and its various fractions against E. coli (1, A-E), K. pneumoniae (2, A-E), B. subtilis (3, A-E), S. aureus (4, A-E) and P. aeruginosa (5, A-E). Crude extract (1-5A), butanol fraction (1-5B), ethyl acetate fraction (1-5C), chloroform fraction (1-5D) and hexane fraction (1-5E); where a-200µg / ml, b-400µg / ml, c-600µg / ml, d-800µg / ml, e- antibiotic Ampicillin (100µg / ml) Table 1: Table 1: Radial scavenging activity for DPPH assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF- hexane fraction, CF- chloroform fraction, AA- ascorbic acid)Table 2: Radial scavenging activity for ABTS assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF- hexane fraction, CF- chloroform fraction, AA- ascorbic acid)Table 3: Fe (II) equivalents in mM for FRAP assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF-hexane fraction, CF- chloroform fraction, AA- ascorbic acid)Table 4: Comparative antioxidant potential in terms of IC50 value for DPPH, ABTS and FRAP assay in crude methanolic extract and its various fractions of crude methanolic extract (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF- hexane fraction, CF- chloroform fraction, AA- ascorbic acid)Table 5: Inhibition zone (mm) in different concentration of crude methanolic extract and its various fractions against E. coli, K. pneumoniae, P. aeruginosa, B. subtilis, S. aureus (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF- hexane fraction, CF- chloroform fraction)5 Table 6: MIC values of crude methanolic extract and its various fractions against E. coli, K. pneumoniae, P. aeruginosa, B. subtilis, S. aureus (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF- hexane fraction, CF- chloroform fraction.DETAILED DESCRIPTION OF INVENTION Chemicals and mediaMethanol Hi-AR (Himedia), n-Hexane 80% AR, Chloroform Hi-AR (Himedia), Ethyl acetate Hi-AR (Himedia), n-butanol A.R. (Himedia) Nutrient agar (Himedia), potato dextrose agar (Himedia), DMSO (Solvent) (Himedia), Ampicillin (Himedia), 2,2 Diphenyl-1-picryl hydrazyl radical (Sigma – Aldrich), Ammonium persulphate (Himedia), 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Himedia), Sodium acetate ACS, 2,4,6-Tripyridyl-S-Triazine CDH, Ferric chloride (Loba), Ferrous sulphate (Loba) and Resazurin Dye (Himedia), Aluminium chloride (Loba), Folin-Ciocalteu reagent (Himedia), Gallic acid extra pure (Loba), Sodium carbonate (Himedia), Quercetin hydrate (Loba), Sodium nitrate (Loba), Sodium hydroxide (Loba).Instruments: Bacterial Incubator (Radical), Laminar air flow horizontal (Radical) model-RS-58-06, Vertical autoclave-RAV-40, Grinder, UV-visible Spectrophotometer 2205 (Systronics), Refrigerator, Hot air oven, Soxhlet apparatus, Separating funnel.Bacteria used: Two Gram's positive bacteria (Staphylococcus aureus and Bacillus subtilis) and three Gram's negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa)Techniques:In vitro antioxidant assays- DPPH radical scavenging assay, ABTS radical scavengingassay, FRAP assay.For phenolic and flavonoids content determination- Folin- Ciocalteau method andAluminium chloride methodFor Antibacterial assay- Disc diffusion method, Resazurin based Microtiter DilutionAssay (RMDA)2. Methods:2.1 Plant material collection4 kg bark was collected from P. ciliata tree from district Shimla near Sankat Mochan Temple in the month of June. The sampling site is located on 31° 06' N / 77° 13' E, northern6 India. The average elevation is 1975 m above mean sea level. The collected bark was dried in the oven at 40ºC for 15 days, crushed in a grinder and coarse powder was stored in airtight bottles for further use.2.2 Extract preparation50 gm dried coarse powder material of bark was extracted with 500 ml methanol (used as primary solvent) for 72 h in a Soxhlet apparatus. The extract was made solvent free by distillation process under reduced pressure and the resulting semisolid residue was vacuum dried using rotary flash evaporator. The crude extract was collected, weighted and stored in a refrigerator.2.3 Fractions of the methanolic extract of bark of P. ciliata5g crude extract was dissolved in 100 ml triple distilled water to make the mother solution which was partitioned off successively by four solvents (n-hexane, chloroform, ethyl acetate and n-butanol) in order of increasing polarity by using separating funnel. Equal volume of mother solution and solvent was used for fractions. All the four fractions of each plant extract were dried by evaporating respective solvent using rotary evaporator. All crude extracts were weighted and stored at 4ºC in airtight containers till further analysis.2.4 Biological potential screening of crude methanolic extract and fractions of P. ciliatabark2.4.1 Antioxidant assay2.4.1.1 DPPH assayThe free radical scavenging activity of bark methanol extract and their fractions against 2,2 Diphenyl-1-picryl hydrazyl radical was determined by via spectrophotometer at 517 nm. The activity was measured by preparing different concentration (20 µg / ml, 40 µg / ml, 60 µg / ml, 80 µg / ml, 100 µg / ml) of the sample in methanol. To the 100 μl of extract 900 μl of methanol solution of DPPH (0.004%) was added. The reaction mixture was allowed to react at room temperature for 30 minutes. Methanol served as the blank and DPPH in methanol, without the extracts, served as the positive control. After 30 minutes of incubation, the discoloration of the purple color was measured at 517 nm in a spectrophotometer. The radial scavenging activity was calculated as follows:7 Where, A (control) is the absorption of the DPPH and A (sample) is the absorption of the plant extract and the graph was plotted against radial scavenging activity at different concentration. IC 50 value (µg extract / ml) is the inhibitory concentration at which DPPH radicals was scavenged by 50 %. Ascorbic acid was as standard for used for comparison.2.4.1.2 ABTS assayThe ABTS radial scavenging activity of bark methanolic extract and their fractions was measured by preparing crude extract solutions of different concentration in methanol. 7 mM of ABTS was mixed with 2.45 mM ammonium persulphate and the mixture was allowed to stand in dark at room temperature for 12-16 hours before use. The activity was measured by preparing different concentration (20µg / ml, 40 µg / ml, 60µg / ml, 80 µg / ml, 100 µg / ml) of the sample in methanol. To the 100μl of extract 900 μl of diluted ABTS was added. Methanol served as control. The absorbance was read at 734 nm and the percentage inhibition was calculated by following the equation:Where, A (control) is the absorption of the DPPH and A (sample) is the absorption of the plant extract and the graph was plotted against radial scavenging activity at different concentration. IC 50 value (µg ex1tract / ml) is the inhibitory concentration at which DPPH radicals was scavenged by 50 %. Ascorbic acid was as standard for used for comparison.2.4.1.3 FRAP (ferric reducing antioxidant power) assayThe FRAP reagent was generated by mixing 300 mM sodium acetate buffer (pH 3.6), 10.0 mM (tripyridyl triazine) TPTZ solution and 20.0 mM FeCl3.6H2O solution in a ratio of 10:1:1 in volume. Samples at different concentrations (20µg / ml, 40 µg / ml, 60µg / ml, 80 µg / ml, 100 µg / ml) were prepared and then added to 3 ml of FRAP reagent. The reaction mixtures were incubated at 37°C for 30 minutes. The increase in absorbance at 593 nm was8 measured. A fresh working solution of FeSO4 (1mM) was used for calibration. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as µM FeSO4 equivalents per gram of sample (DW).2.5 Quantification of phenolic and flavonoids in crude methanolic extract and itsvarious fractions2.5.1 Folin- Ciocalteau method: The amount of total phenolics in extracts was determined with the Folin- Ciocalteau regent. Gallic acid was used as a standard and the total phenolics were expressed as mg / g gallic acid equivalents (GAE). 0.5 ml of plant sample (crude extract) was introduced into test tube and mixed with 2.5 ml of a 10 fold dilute Folin-Ciocalteau reagent and 2 ml of 7.5% sodium carbonate. The tube was covered with aluminum foil and allowed to stand for 30 minutes at room temperature before and the absorbance was at read at 760 nm spectrometrically. The method was repeated with all the fractions. Results were recorded in triplicates.2.5.1 Aluminum chloride method: In this method Quercetin was used as standard and flavonoid contents were measured as quercetin equivalent. 1ml of crude extract solution was taken into test tube, containing 5 ml of distill water. 0.3 ml of 5% NaNO2 was added to the test tube. After 5 minutes, 0.3 ml 10% AlCl3 was added to the mixture. At the 6 minutes 2 ml of 1M NaOH was added and final volume was made up to 10 ml with distilled water. The absorbance was noted at 510 nm using UV-visible spectrophotometer. The method was repeated with all the fractions. Results were recorded in triplicates.2.6 Antibacterial assay2.6.1 Microbial strainsThe six bacteria (two Gram's positive, viz. Staphylococcus aureus and Bacillus subtilis and three Gram's negative bacteria, viz. Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa) were used to study antibacterial activity of methanolic extract of bark and its fractions were purchased from School of Biotechnology, Shoolini University, Solan.9 2.6.2 Disc diffusion methodThe antimicrobial activity of methanolic crude extract and its various fractions (hexane, chloroform, ethyl acetate and n-butanol) was evaluated by disc diffusion method. 100 µl of bacterial culture (108 colony forming units (CFU) was uniformly spread on the surface of the nutrient agar plates using sterile cotton swabs. Stock solution of each extract was prepared by dissolving 20 mg of each extract into 1 ml of dimethyl sulfoxide (DMSO). The different concentration (200µg / ml, 400µg / ml, 600µg / ml, 800µg / ml) of extract was tested using 6 mm sterilized filter paper discs. The plates were allowed to stand at 4ºC for 2 hours for incubation with test microbial agents and then placed in 37ºC for 24 hours. After 24 h of incubation the zone of inhibition was measured using a Hi Antibiotic Zone scale-C (Himedia Biosciences, Mumbai (India). Each antimicrobial assay was performed in triplicates. Standard ampicillin served as positive controls for antimicrobial activity. DMSO (solvent) was used as solvent control. The similar assay was repeated for each fraction.2.6.3. Resazurin based Microtiter Dilution Assay (RMDA)96 well microtitre plates (Tarson) were used for Resazurin based Microtitre Dilution Assay. The first row of microtiter plate was filled with 100 µl of test materials (20mg in DMSO). All the wells of microtitre plates were filled with 100 µl of nutrient broth. Two-fold serial dilution (throughout the column) was achieved by starting transferring 100 µl test material from first row to the subsequent wells in the next row of the same column and so that each well has 100 µl of test material in serially descending concentrations. Finally, a volume of 10 µl was taken from bacterial suspension and then added to each well to achieve a final concentration of 5×106 CFU / ml. To avoid the dehydration of bacterial culture, each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Each micro titre plate had a set of different controls: (a) a row with dilution of Ampicillin (10mg / ml) (b) a row with broth except bacterial solution (c) five column with all test solutions except bacterial solution (d) two column with positive control (Ampicillin, 10mg / ml) and contain all solutions (e) two columns with negative control (DMSO) and contain all solutions. The plates were incubated in temperature controlled incubator at 37° C for 24 h. 10 µl of resazurin solution as indicator was added in each well. The colour change in the well was then observed visually. The lowest concentration of plant leaf extract at which colour change occurred was recorded as the MIC value.2.8 Data analysis: Data was analysed by taking mean and standard deviation.10 Results:5 g crude plant extract was produced from 50 g dried coarse powder material of bark whereas crude extracts obtained in different fractions were 640 mg in n-hexane fraction, 765 mg in chloroform extract, 900 mg in ethyl acetate and 1.2 g in n-butanol fraction.3.1 Antioxidants assayIn the present study, different antioxidant assays (DPPH, ABTS, FRAP) were performed to check antioxidant potential of crude methanolic extract and their fraction.In DPPH assay, results showed highest antioxidant potential in n-butanol and ethyl acetate fraction (IC50-1.73 µg / ml and 1.46µg / ml, respectively) as compared to that of ascorbic acid (IC50-1.86 µg / ml) and crude methanolic extract of bark (IC50-2.16µg / ml). The least antioxidant potential for DPPH assay was observed in hexane fraction of bark (IC50-2.89 µg / ml) (Table 1&4; Figure 1).In ABTS assay, maximum antioxidants were in n-butanol and ethyl acetate fraction (IC50-1.57 µg / ml and 1.66 µg / ml, respectively) as compared to that of ascorbic acid (IC50-2.10 µg / ml) and crude methanolic extract of bark (IC50-2.16 µg / ml). The least antioxidants for ABTS was observed in hexane fraction of bark (IC50-2.37 µg / ml) (Table 2&4; Figure 2). In FRAP assay, result showed the IC50 values was found to be lowest in n-butanolic fraction (IC50-3.94 µM Fe (II) equivalents) indicating its more antioxidant activity as compared to that of standard ascorbic acid (IC50-4.23 µM Fe (II) equivalents) and higher IC50 was observed from chloroform fraction [6.79µM Fe (II) equivalents] (Table 3&4; Figure 3).3.2 Quantification of total phenols and flavonoidsTotal phenolic content was calculated from the standard curve of gallic acid using equation: y = 0.236x + 0.9758 while total flavonoid content was calculated using standard curve of quercitin using equation: y = 0.1982x + 1.0226. The result of total phenolic content was shown in Fig. 4. The phenolic content was found to be more in n-butanol and ethyl acetate fraction as compared to that of crude extract of bark. The order of total phenolic content in bark extract was highest in n-butanol fraction (317.83± 2.95 mg / g gallic acid) and ethyl acetate fraction (291.820 ± 3.22 mg / g gallic acid) than crude extract (210.36 ± 2.44 mg / g gallic acid). Whereas it was minimum in chloroform fraction (97.99 ± 1.70 mg / g gallic acid) and hexane fraction (54.88 ± 1.36 mg / g gallic acid).Similar trend was observed in the total flavonoid content of bark extract. It was observed highest in n-butanolic fraction (271.26 ± 0.89 mg / g quercetin) followed by ethyl acetate11 fraction (230.54 ± 1.28 mg / g quercetin), crude extract (203.20± 1.15 mg / g quercetin), chloroform fraction (105.72 ± 2.27 mg / g quercetin) and hexane fraction (74.94 ± 2.01 mg / g quercetin) (Fig. 5).3.3 Antibacterial assayTo observe the antibacterial potential of crude extract and their fractions, four concentrations were used i.e., 200µg / ml, 400µg / ml, 600µg / ml, 800µg / ml from the stock solution (20 mg in 100% DMSO). Results showed that as the concentration increased from 200µg / ml to 800µg / ml the antibacterial activity (zone of inhibition) was also increases. Therefore, 800µg / ml concentration was used for antibacterial activity against all tested bactera. Among all the fractions used, n-butanol and ethyl acetate fraction of bark exhibited more antimicrobial activity as compared to other fractions and crude methanolic extract against all pathogenic strains (Table 3; Figure 6).Against E. coli, the zone of inhibition diameters of butanol fraction was highest (18±0.54mm) and lowest (10.68±0.40 mm) in chloroform fraction at whereas against K.pneumoniae zone of inhibition was highest (17.66±0.58 mm) for ethyl acetate fraction andlowest (11.44±0.57 mm) in hexane fraction. In B. subtilis, zone of inhibition was maximumand similar (17.33±0.44 mm and 17.00±0.40) for both butanol and ethyl acetate fractionsand lowest (11.66±0.44 mm) in hexane fraction whereas in S. aureus, zone of inhibitionwas again similar and highest (17±0.84 mm and17±0.44) for butanol and ethyl acetatefractions and lowest (12.33±0.43) in hexane fraction. Against P. aeruginosa, zone ofinhibition was highest (16±0.70 mm) for butanol fraction and lowest (11.66±0.57) for hexane fractions. (Table 5; Figure 6).3.3.1 Microtiter Dilution AssayThe MIC values are shown in Table 3. MIC value of bark crude extract was 125 µg / mlagainst E. coli, K. pneumonia, B. subtilis, S. aureus and P. aeruginosa whereas forbutanol and ethyl acetate fraction MIC values were 62.5 µg / ml against all bacteria. The MIC values of bark chloroform fraction was 250 µg / ml against E. coli, K. pneumoniae, B. subtilis, S. aureus except P. aeruginosa where is was 125 µg / ml. Similarly, MIC values of bark hexane fraction was 250 µg / ml against all bacteria. MIC values of antibiotic (ampicillin) was observed 7.8 µg / ml against E. coli, K. pneumoniae, B. subtilis whereas for S. aureus and P. aeruginosa it was 3.9 µg / ml (Table 6).12 Table 1: Radial scavenging activity for DPPH assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, F£F-hexane fraction, CF- chloroform fraction, AA- ascorbic acid)Table 2: Radial scavenging activity for ABTS assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, F£F-hexane fraction. CF- chloroform fraction. AA- ascorbic acid") Table 3: Fe (II) equivalents in mM for FRAP assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, HF-hexane fraction, CF- chloroform fraction, AA- ascorbic acid) Table 4: IC50 value for DPPH, ABTS and FRAP assay in crude methanolic extract and its various fractions (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, F£F-hexane fraction, CF- chloroform fraction, AA- ascorbic acid)Table 5: Inhibition zone (mm) in different concentration of crude methanolic extract and its various fractions against E. coli, K. pneumoniae, B. subtilis, S. aureus, P. aeruginosa (CR-crude extract, BF- butanol fraction, EF- ethyl acetate fraction, F£F- hexane fraction, CF- chloroform fraction)17 Table 6: MIC values of crude methanolic extract and its various fractions against E. coli, K. pneumoniae, B. subtilis, S. aureus, P. aeruginosa. CR-crude extract, BF- Butanol fraction, EF-Ethyl acetate fraction, HF- Hexane fraction, CF- Chloroform fraction.

Claims

WE CLAIMS1. An antibacterial composition comprises of methanolic extract of bark of Populus. ciliate.

2. A composition as claimed in claim 1, wherein said composition shows antibacterial activity at the concetration of 800µg / ml.

3. The composition as claimed in claim 1, wherein said composition is active against bacteria (Staphylococcus aureus and Bacillus subtilis) and three Gram's negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa).

4. The composition as claimed in claim 1, wherein said composition is prepared by method comprising the steps of;a. Collecting 4 kg bark from P. ciliata tree from district Shimla 31° 06' N / 77° 13' E,northern India, dried in the oven at 40ºC for 15 days, crushed in a grinder and coarsepowder was stored in airtight bottles for further use.b. extracting 50 gm dried coarse powder material of bark with 500 ml methanol for 72 h in aSoxhlet apparatus;c. Dissolving 5g crude extract in 100 ml triple distilled water to make the mother solution whichis partitioned off successively by four solvents (n-hexane, chloroform, ethyl acetate and n-butanol) in order of increasing polarity by using separating funnel, drying all the four fractionsof each plant extract by evaporating respective solvent using rotary evaporator, weighing andstoring all crude extracts at 4ºC in airtight containers for further biological screening of extract.

5. The extract as claimed in claim 4, wherein the order of total phenolic content in bark extract was highest in n-butanol fraction (317.83± 2.95 mg / g gallic acid) and ethyl acetate fraction (291.820 ± 3.22 mg / g gallic acid) than crude extract (210.36 ± 2.44 mg / g gallic acid), minimum in chloroform fraction (97.99 ± 1.70 mg / g gallic acid) and hexane fraction (54.88 ± 1.36 mg / g gallic acid).

6. The extract as claimed in claim 4, wherein the total flavonoid content of bark extract is highest in n-butanolic fraction (271.26 ± 0.89 mg / g quercetin) followed by ethyl acetate fraction (230.54 ± 1.28 mg / g quercetin), crude extract (203.20± 1.15 mg / g quercetin), chloroform fraction (105.72 ± 2.27 mg / g quercetin) and hexane fraction (74.94 ± 2.01 mg / g quercetin). Dated this 1st May, 2019