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Evaluation of antibacterial properties of celtis australis against S. aureus and P.

aureginosa

INTRODUCTION
There is a continuous and urgent need to discover new antimicrobial compound with divers and novel mechanism of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases. Another big concern is the development of resistance to the antibiotics in current clinical use. In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. (Dowzicky and park,2008). The bacterial infection diseases causes problem for humankind beyond historical age. The researcher to find antimicrobial medicine have been launched for over 50 year(Rudrappa and bais,2008).However, we discovered many anti-biotic drug,we still facing multidrug resistance bacteria(Dowzicky and park,2008,Saonuam et al,2008;Tilloston et al.,2008) . There are the report about the adverse effect of antibiotic treatment in children (Khotaei et al.,2008) and adults (Lin et al.,2009).Furthermore reported about the decreasing susceptibility in pathogenic bacteria (Dowzicky and park,2008;Saonuam et al.,2008) . Commonly Drug resistance to human pathogenic bacteria has been reported all over the world (Piddok and wise,1989; Singh et al.,1992;Mulligen al.,1993;Davis,1994;Robin et al.,1998). However, the situation is alarming in developing as well as developed countries due to indiscriminate use of antibiotics. The drug resistance bacteria and fungal pathogens have further complicated the treatment of infectious diseases in immunocompromised, AIDS and cancer patient (Rinaldi,1991;Diamond,1993).In the present scenario of emergence of multiple drug resistance to human pathogenic organism, has necessitated a search for new antimicrobial substances from other sources including plants. Therefore, the antimicrobial research was become interesting to support the information et

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


for development of the anti -infection diseases remedy especially the development of folkloric medicine which has been used locally before (Nascimento et al.,2000; Tongson et al .,2005). Nature has been a source of medicinal Plant for thousands of years and since the beginning of man . In Nigeria ,almost all plants are medicinal and the application of medicinal plants especially in traditional medicine is a currently well acknowledge and established as a viable profession(Kafaru,1994).Traditionally medicinal plants are used to produce variety of compounds of known therapeutic properties (Lyengar ,1985;Chopra et al ,1992;Harborne and Baxter,1995). The use of medicinal plants as a source for relief from illness can be traced back over five millennia to written documents of the early civilization in China ,India and the Neareast, but it is doubtness an art as old as mankind . In present day Iraq used plants such as holyback, these plants are still widely used in ethnomedicine around the world (Thomson et al .,1978,Stockwell et al .,1988). Antimicrobial properties of medicinal plants are being increasingly reported from differents parts of the word (Grosvenor et al .,saxena and Sharma,1999). Medicinal plants of India have been found of immense global importance in treatment because of adverse effect of synthetic drug had created varied type of complicated diseases.(Govindrajan et al.,2005). The substances that can either inhibit the growth of pathogens or kill them and have no or least toxicity to host cell are considered candidates for developing new antimicrobial drugs. so far, more than 100,000 biologically active secondary plant compound have been isolated from higher plants ,with most of these diverse structures falling in to four main chemical classes, the phenolics (phenol, flavonoids, quinines, tannins and linins), terpenoids(monoterpenes ,saponins ),sulphur compounds (disulphide and acetynic thiophenes)and

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


nitrogen compound (alkaloids, amines ,non-proteins, amino acid and cyanogenic glycosides). Antimicrobial activities of plants have been evaluated by earlier workers(At Delaimy and Ali,1970;Watanab,1974;Tansey and Appleton,1975;Thinel and dehia,1976;et al.,1977) Higher and aromatic plants have been used traditionally in folk medicine as well as to extend the shelf life of foods, showing inhibition against bacteria ,fungi ,and yeast(Hulin et al.,1998). There are about 47,000 plants species in india out which 7,500 plants species are medicinal value .only 800 plants species are used in preparation of herbal drugs. Plants produces many substances for self-defence against microbial infection and deterioration.these phytochemicals possess potential significant therapeutic application against human pathogens such as bacteria and fungi(Perez,2003). Medicinal plants represent a rich source of antimicrobial agent (Mahesh and Satis,2008) Due to a rapid increase in the rate of infections, antibiotic resistance in microorganisms and due to side effects of synthetic antibiotics, medicinal plants are gaining popularity over these drugs.( Mahesh, B S, 2008 ). Celtis australis vern. Kharik belonging to family Ulmaceae is a deciduous tree distributed to montane and submontane Himalaya . The paste obtained from the bark of C. australis is effective remedy for bone fracture and also applied on pimples, contusions, sprains and joint pains . Previously, betulin-3,3-di-Omethylellagic acid, gallic acid and quebrachilol were reported from the bark whereas acacetin 7-O-glucoside, isovitexin and cytisoside were isolated from leaves of the plant . Recently, we have isolated a novel sulphonated phenolic celtisanin from the fruits of this plant . This is the first chemical report together with antimicrobial activity on fruits of this plant.

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


OBJECTIVE OF THE STUDY Considering the therapeutic potentials of celtis australis Following objectives are scheduled for experimentation : Determination of antimicrobial activity of celtis australis leaf extract (aquous& Methanol) against Staphylococcus aureus . Determination of antimicrobial activity of celtis australis leaf extract(aquous& methanol) against Pseudomonas Aureginosa.

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


REVIEW OF LITERATURE The present literature deals with the antimicrobial activity of various medicinal plants , general introduction of test pathogen and description of celtis australis.

ANTIMICROBIAL ACTIVITY OF MEDICINAL PLANT An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans (Merriam-Webster., 2009-05-02). The discovery, development, and clinical use of antibiotics during the 20th century have decreased substantially the mortality from bacterial infections. The antibiotic era began with the pneumatic application of nitroglycerine drugs, followed by a golden period of discovery from about 1945 to 1970, when a number of structurally diverse, highly effective agents were discovered and developed. However, since 1980 the introduction of new antimicrobial agents for clinical use has declined, in part because of the enormous expense of developing and testing new drugs. Paralleled to this there has been an alarming increase in bacterial resistance to existing agents. (Levy SB, 1994)

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa GENERAL INTRODUCTION OF TEST PATHOGENS Pseudomonas aeruginosa
Scientific classification Kingdom: Bacteria Phylum: Proteobacteria Class: Order: Family: Genus: Species: Gamma Proteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, the versatility enables the organism to infect damaged tissues or people with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs, such as the lungs, the urinary tract, and kidneys, the results can be fatal (Balcht, Aldona & Smith, Raymond 1994) Because it thrives on most surfaces, this bacterium is also found on and in medical equipment, including catheters, causing cross-infections in hospitals and clinics. It is implicated in hot-tub rash. It is also able to decompose hydrocarbons and has been used to break down tarballs and oil from oil spills.( A. Y. Itah and J. P. Essien)

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Characteristics Members of the genus display the following defining characteristics.( Cornelis P (editor). (2008).

Rod shaped Gram-negative One or more polar flagella, providing motility Aerobic Nonspore forming positive catalase test positive oxidase test.

Other characteristics which tend to be associated with Pseudomonas species (with some exceptions) include secretion of pyoverdine, a fluorescent yellowgreen siderophore under iron-limiting conditions. Certain Pseudomonas species may also produce additional types of siderophore, such as pyocyanin by Pseudomonas aeruginosa. (Fine MJ, Smith MA, Carson CA et al. (1996)and thioquinolobactin by Pseudomonas fluorescens,. (Diekema DJ, Pfaller MA, Jones RN et al. (1999) Pseudomonas species also typically give a positive result to the oxidase test, the absence of gas formation from glucose, glucose is oxidised in oxidation/fermentation test using Hugh and Leifson O/F test, beta hemolytic (on blood agar), indole negative, methyl red negative, Voges Proskauer test negative, and citrate positive. The members of the genus demonstrate a great deal of metabolic diversity, and consequently are able to colonise a wide range of niches. King EO, Ward MK, Raney DE (1954).Their ease of culture in vitro and availability of an increasing number of Pseudomonas strain genome sequences has made the genus an excellent focus for scientific research; the best studied species include P. aeruginosa in its role as an opportunistic human pathogen, the plant pathogen P. syringae, the soil bacterium P. putida, and the plant growth promoting P. fluorescens. Antibiotic resistance Being Gram-negative bacteria, most Pseudomonas spp. are naturally resistant to penicillin and the majority of related beta-lactam antibiotics, but a number are sensitive to piperacillin, imipenem, ticarcillin, tobramycin, or ciprofloxacin.(University of Chicago Medical Center (2009-04-14) This ability to thrive in harsh conditions is a result of their hardy cell wall that contains porins. Their resistance to most antibiotics is attributed to efflux pumps, which pump out some antibiotics before the antibiotics are able to act.

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Pseudomonas aeruginosa is a highly relevant opportunistic human pathogen. One of the most worrying characteristics of P. aeruginosa is its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes (e.g. mexAB-oprM, mexXY, etc., and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develops acquired resistance either by mutation in chromosomally-encoded genes, or by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown phenotypic resistance associated to the emergence of small-colony-variants may be important in the response of P. aeruginosa populations to antibiotic treatment.( Worlitzsch D, Tarran R, Ulrich M et al. (2002).

Pathogenicity
Animal pathogens Infectious species include P. aeruginosa, P. oryzihabitans, and P. plecoglossicida. P. aeruginosa flourishes in hospital environments, and is a particular problem in this environment since it is the second most common infection in hospitalized patients(nosocomial infections). This pathogenesis may in part be due to the proteins secreted by P. aeruginosa. The bacterium possesses a wide range of secretion systems, which export numerous proteins relevant to the pathogenesis of clinical strains.( Rahme LG, Tan MW, Le et al. (1997) Plant pathogens P. syringae is a prolific plant pathogen. It exists as over 50 different pathovars, many of which demonstrate a high degree of host plant specificity. There are numerous other Pseudomonas species that can act as plant pathogens, notably all of the other members of the P. syringae subgroup, but P. syringae is the most widespread and best studied.

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Although not strictly a plant pathogen, P. tolaasii can be a major agricultural problem, as it can cause bacterial blotch of cultivated mushrooms. Similarly, P. agarici can cause drippy gill in cultivated mushrooms.( Mahajan-Miklos S, Tan MW, Rahme LG, Ausubel FM 1999)

Staphylococcus aureus
Domain: Bacteria Kingdom: Eubacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species: aureus Binomialname:Staphylococcusaureus Scanning electron micrograph of S. aureus, 20,000 times enlargement, Staphylococcus aureus meaning the "golden grape-cluster berry," and also known as "golden staph" and Oro staphira, is a facultative anaerobic Grampositive coccal bacterium. It is frequently part of the skin flora found in the nose and on skin, and in this manner about 20% of the human population are long-term carriers of S. aureus.( Kluytmans J,et.al. 1997). S. aureus is the most common species of staphylococci to cause Staph infections. One of the reasons for this is a carotenoid pigment staphyloxanthin that is responsible for the characteristic golden colour of S. aureus colonies. This pigment acts as a virulence factor, with an antioxidant action that helps the microbe evade death by reactive oxygen species used by the host immune system.( Clauditz A, et.al. 2006 ,Liu GY,et.al.2000).

Yellow colonies of S. aureus on a blood agar plate, note regions of clearing around colonies caused by lysis of red cells in the agar (beta hemolysis) Strains are responsible for food poisoning through the production of an enterotoxin, and pathogenicity is also associated with

Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


coagulase positivity. S. aureus may occur as a commensal on skin; it also occurs in the nose frequently (in about a third of the population).( Whitt, et.al. 2002) and the throat less commonly. The occurrence of S. aureus under these circumstances does not always indicate infection and, therefore, does not always require treatment (indeed, treatment may be ineffective and recolonisation may occur). It can survive on domesticated animals, such as dogs, cats, and horses, and can cause bumblefoot in chickens. It can survive for hours to weeks, or even months, on dry environmental surfaces, depending on strain.( Cimolai et.al.2008) It can host phages, such as Panton-Valentine leukocidin, that increase its virulence. S. aureus can infect other tissues when barriers have been breached (e.g., skin or mucosal lining). This leads to furuncles and carbuncles (a collection of furuncles). In infants, S. aureus infection can cause a severe disease - staphylococcal scalded skin syndrome (SSSS). (Curran JP, et.al.1980). S. aureus reproduces asexually. It starts this process by reproducing its DNA. The membrane stretches out and separates the DNA molecules. The cells form a hollow space that eventually divides into two new cells. The new cell wall does not fully separate from the existing cell wall, which is why the cells are observed in clusters. This cell will eventually reproduce, and cells will attach to it.( Staphylococcus aureus: Reproduction". palexander13.webs.com.) The treatment of choice for S. aureus infection is penicillin; in most countries, though, penicillin resistance is extremely common, and firstline therapy is most commonly a penicillinase-resistant -lactam antibiotic (for example, oxacillin or flucloxacillin). Combination therapy with gentamicin may be used to treat serious infections, such as endocarditis. (Korzeniowski O,et.al. 1982). (BayerAS, Bolger AF, Taubert KA, et al. 1998). but its use is controversial because of the high risk of

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


damage to the kidneys.( Cosgrove SE, Vigliani GA, Campion M, et al. 2009). Antibiotic resistance in S. aureus was uncommon when penicillin was first introduced in 1943. Indeed, the original petri dish on which Alexander Fleming of Imperial College London observed the antibacterial activity of the Penicillium fungus was growing a culture of S. aureus. By 1950, 40% of hospital S. aureus isolates were penicillin-resistant; and, by 1960, this had risen to 80%.( Chambers HF (2001). "The changing epidemiology of Staphylococcus aureus?". Emerg Infect Dis 7 (2): 17882.

GENERAL INTRODUCTION OF PLANT TAKEN DURING THE COURSE OF STUDY


Celtis Australis

Chinese Hackberry (C. sinensis) leaves and fruit Scientific classification Kingdom: (unranked): (unranked): (unranked): Order: Family: Genus: Plantae Angiosperms Eudicots Rosids Rosales ulmaceae Celtis

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

Celtis (Hackberry) is a genus of about 60-70 species of deciduous trees widespread in warm temperate regions of the Northern Hemisphere, in southern Europe, southern and eastern Asia, and southern and central North America, south to central Africa, and northern and central South America. The genus is present in the fossil record at least since the Miocene of Europe. (BROWER, ANDREW V.Z. 2006) Previously included either in the elm family (Ulmaceae) or a separate family, Celtidaceae, the APG II system places Celtis in the hemp family (Cannabaceae). The generic name originated in Latin and was applied by Pliny the Elder (2379) to the unrelated Ziziphus lotus. (KEELER, HARRIET L. 1900)

Description Celtis species are generally medium-sized trees, reaching 1025 m (3382 ft) tall, rarely up to 40 m (130 ft) tall. The leaves are alternate, simple, 315 cm (1.25.9 in) long, ovate-acuminate, and evenly serrated margins. Small monoecious flowers appear in early spring while the leaves are still developing. Male flowers are longer and fuzzy. Female flowers are greenish and more rounded. The fruit is a small drupe 610 mm (0.240.39 in) in diameter, edible in many species, with a dryish but sweet, sugary consistency, reminiscent of a date. Formerly placed here

Trema cannabina Lour. (as C. amboinensis Willd.) Trema lamarckiana (Schult.) Blume (as C. lamarckiana Schult.) Trema orientalis (L.) Blume (as C. guineensis Schumach. or C. orientalis L.) Trema tomentosa (Roxb.) H.Hara (as C. aspera Brongn. or C. tomentosa Roxb.) ( HALLWACHS, WINNIE ,2004)

Uses and ecology Several species are grown as ornamental trees, valued for their drought tolerance. They possess the most bending tolerance of all species of wood. They are a regular feature of arboretums and botanical gardens, particularly in

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


North America. Chinese Hackberry (C. sinensis) is suited for bonsai culture, while a magnificent specimen in Daegu-myeon is one of the natural monuments of South Korea. Some, including Common Hackberry (C. occidentalis) and C. brasiliensis, are honey plants and pollen source for honeybees of lesser importance. The berries are often eaten locally. The Korean tea gamro cha sinensis leaves. Pathogens The plant pathogenic basidiomycete fungus Perenniporia celtis was first described from a Celtis hostplant. Some species of Celtis are threatened by habitat destruction. contains C.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


MATERIAL AND METHOD
Material required Plant sample Plant material i.e. leaves of celtis australis were collected from kashmir region. Leaves were washed and dried for one weak in shady place. Fine powdered of leaves was prepared by grinding leaves in mixer grinder. Apparatus used: Table :1 list of equipments used for study. S.N. 1 2 3 4 5 6 7 8 9 Apparatus Autoclave Hot air oven Electronic balance Laminar air flow Incubator Refrigerator Sterile cotton & swab tubes Micropipette Glassware Company Scientific equipment Scientific equipment Sartorius Zenith Toshiba Sanyo Hi -media Torson Borosil

Soxhlet Assembly, clevanger, Rotatory evaporator, Distillation assembly and other glassware of general use.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

Soxhlet assembly Soxhlet assembly is used for extraction of compound from leaf of plant: It is composed of RBF (Round Bottom flask), Soxhlet and condensor. Clevanger is used to extract essential oil from leaf. It is also composed of RBF (Round Bottom flask), clevanger and condensor. Rotatory evaporator is used to concentrate the extract of leaves and waste of essential oil which is called "Hydrosol". Chemical used: Petroleum ether (100%), Benzene, (100%), chloroform (100%) Benzene (100%). Bacterial strain: Bacterial type used for testing were Eschrichia coli, Bacillus cereus, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


METHODS Table:2 Preparation of Media (Nutrient Broth) for revival of Bacterial culture. Nutrient Broth with following composition was used for Bacterial culture. S. No. 1 2 3 4 5 6 Composition Beef Extract Yeast Extract Peptone NaCl Distilled water pH Amount (1000ml) 1.5g 1.5g 5.0g 5.0g 1000ml 7.0

Preparation of Bacterial Inoculum 5 test tube containing 10 ml of nutrient broth each was autoclaved at 15lbs (121OC) for 15 min. for inoculum of bacterial species with the help of sterilized inoculating loop , bacterial culture was transferred in 5 test tubes. Kept the tubes at 37OC in incubator for 24 hrs. The bacterial culture in broth was streaked on the nutrient agar plates. The media was autoclaved at 15 lbs (121OC) for 15 min.. The plates kept in incubator at 37OC for active growth of culture. The test organism maintained in the agar plates were used for inoculum.5 test tubes containing sterilized nutrient broth was inoculated with bacterial culture maintained in agar plate and tube kept in incubator at 37OC till the conc. of test organism matched with the MacFarland standard (9950 l, 1% H2SO4 + 50l BaCl2) .

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Preparation of Media (Muller Hinton) for revival of Bacterial culture. Table:3 Muller Hinton media with following compound was used for Bacterial culture.

S.No. 1 2 3 4 5 6

Composition Beef infusion Casamino acid Starch Agar Distilled pH

Amount 1000ml 300g 17.5g 1.5g 17.0g 1000ml 7.0

The media was autoclaved (121OC) for 15 min, at 15 lbs and agar plates were prepared for use in Disc Diffusion Method

Extract Preparation Methodology 25 gm of dry powder of leaves was added in 250 ml of different solvent and packed in soxhlet apparatus for extraction of respective soluble bioactive molecules from the plant. Soxhlet apparatus is combination of soxhlet; condenser and R.B.F. For extraction of compounds the RBF is heated on the heating mentle and evaporated solvent goes to soxhlet. Here it is cooled by the water moving in

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


the condenser and then solvent come back to R.B.F. with compound of leaf. Extract containing volatile solvent, were concentrated with the help of Rotary evaporator (Rota vapor) under reduce pressure, first of all vaccum compressor was switched on and extract was taken in a flask and it was set to adaptor and vaccum released nobe was closed, flask was dipped in water bath and the flask. Keep rotating with attached vaccum handle which lead to low temp condensing region were volatile from of solvent in condense to liquid and get collected with flask attached to bottom side. When sample was concentrated then rotor was switch off and vaccum was released to remove the flask. The concentrated extract was unloaded to sterilize collecting tube. Sample Preparation 200mg of extract was dissolve in 1ml of DMSO i.e., 200mg/ml stock solution was prepared and different dilution were prepared i.e., 50 mg/ml, 100 mg/ml and 200 mg/ml and kept in refrigerator at 4C. Screening of Antimicrobial Activity of Plant Extract To check the presence of Antimicrobial Substance, the antimicrobial susceptibility tests were preformed by standard disc diffusion method (Beghe, D.A.V., et al., 1991). Approximately 100 l of inoculum, which contain 105cells/ml were poured on agar plate. The culture was equally spread by a spreader under fully aseptic condition and was left for 5-10 min. Whatmann filter paper disc prepared and sterilized by dry heat at 140C in hot air oven for one or two hours, were used to determine Antimicrobial activity. The test compound (10l) was loaded onto the filter paper disc and allow to dry.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Already prepared antibiotics as standard (Chloramphenicol 10g/disc, Ampicillin 10g/disc, Levofloxacin/10g) positive control for testing of antimicrobial activity The disc were placed over the plates preceded with respective microorganism. The plates were kept inside the incubator at 37C for overnight. The antimicrobial Activity was determined by measuring the zone of inhibition around the disc. The zone of inhibition if any was observed and recorder in comparison to positive control.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

RESULTS
ANTIBIOGRAM (ANTIBIOTIC DRUG SENSITIVITY) OF P.AERUGINOSA AND S.AUREUS
Staphylococcus aureus and P. aeruginosa isolated were assessed for the antibacterial drug susceptibility by using disc diffusion method .the results are given in table. Out of eight antibiotics Staphylococcus aureus isolate was found to be resistance for cefuroxime and ciprofloxacin and shown intermediate effect for gentamicin and cefriaxone with inhibition zone diameter ranging between 10-12 mm. Staphylococcus aureus. Strain was highly sensitive to chloramphenicol, ampicillin ,tetracycline and co-trimoxazole producing inhibition zone of 20mm.,18mm,16mm,and 15mm respectively. While of eight antibiotics, the P aeruginosa isolate was found to be resistant for chloramphenicol, ampicillin, tetracycline , cefuroxime and ciprofloxacin .Intermediates for co-trimoxazole with inhibition zone diameter ranging between 12mm p aeruginosa strain was sensitive gentamicin and cefriaxone with inhibition zone diameter ranging between 20mm and 15mm. It appeared that isolates have become resistance to some antibiotics because of constant exposure with these antibiotics.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

Table-:4 Antibiogram of isolated Pseudomonas aeruginosa : Determination of S No. Antibiotics Chloramphenicol 1 Ampicillin 2 Tetracycline 3 Gentamicin 4 Co-trimoxazole 5 Cefriaxone 6 Cefuroxime 7 Ciprofloxine 8 Cf Cu 15 sensitive Ci 6 intermediate Co 7 intermediate G 8 intermediate T 10 sensitive A 6 Intermediate C No zone Resistant Symbol Zone of in Result intermediate inhibition (mm) 8

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Table-:5 Antibiogram of isolated Staphylococcus aureus. Determination of S No. Antibiotics Chloramphenicol 1 Ampicillin 2 Tetracycline 3 Gentamicin 4 Co-trimoxazole 5 Cefriaxone 6 Cefuroxime 7 Ciprofloxine 8 Cf Cu 13 sensitive Ci 6 intermediate Co 8 intermediate G 16 sensitive T 14 sensitive A 12 sensitive C No zone Resistant Symbol Zone of in Result Sensitive inhibition (mm) 10

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa ANTIMICROBIAL PROPERTIES OF CELTIS AUSTRALIS
The study on antimicrobial properties of celtis australis of family of ulmaceae against various pathogen were conducted.The different leaf fraction of celtis australis at different concentration (200 mg/ml,100 mg/ml,50 mg/ml,25 mg/ml,12.5 mg/ml,6.25 mg/ml) were prepared and studied for the antimicrobial properties .The antibacterial activity of the plant was studied against two bacterial strain Staphylococcus aureus. and Pseudomonas aeruginosa. different culture media was used for performing experiment and maintenance of strain. Nutrient broth and nutrient agar were used for antibacterial study.The antimicrobial properties of the test pathogen were checked by different standard method. The antibacterial activity against both bacterial strain was studied by Disc diffusion method (Mukherjee et al.,1995) .

ANTIBACTERIAL PROPERTIES:
The data in table showing zone of inhibition for the both bacteria Staphylococcus aureus.and Pseudomonas aeruginosa due to the leaf fraction obtained from the celtis australis in comparison to control. This is clear from the table that leaf fraction of celtis australis plant showed inhibition against both the bacteria in more or less extent. It is also clear from the table that different leaf fraction obtained from celtis australis under study showed various degree of inhibition against both the bacteria Staphylococcus aureus.and Pseudomonas aeruginosa. We use both organic and inorganic solvent for the preparation of different leaf fraction of active compound from the celtis australis. The antibacterial activity of leaf fraction of celtis australis was assessed using the Disc diffusion method. By measuring the

diameter of growth inhibition zone with different concentration (200 mg/ml,100 mg/ml,50 mg/ml,25 mg/ml,12.5 mg/ml,6.25 mg/ml) .the result showed that the fraction possesses antibacterial activity against tested pathogen i.e. Staphylococcus aureus and Pseudomonas aeruginosa.

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Table-:6 Zone of inhibition (in mm) of different leaves fractions of celtis australis against Pseudomonas aeruginosa :
Plant Fractions Code on plate given Cons./ml Zone inhibition (in mm) T T1 T2 T3 Aqueous T4 Celtis australis Leaves T T1 T2 T3 T4 Methanol T5 Cephotaxime Drug 6.25mg/ml 30mcg No zone 15 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 9 8.5 7.5 6 No zone T5 12.5mg/ml 6.25mg/ml No zone No zone 200 mg/ml 100 mg/ml 50 mg/ml 25 mg/ml 8 7.5 6.5 No zone of

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

Fig :Aqueous zone of inhibition of Pseudomonas aeruginosa

Fig :Methanol zone of inhibition of Pseudomonas aureginosa

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


Table-:7 Zone of inhibition (in mm) of different leaves fractions of celtis australis against Staphylococcus aureus.
Plant Fractions Code on plate given Cons./ml Zone inhibition (in mm) T T1 T2 T3 Aqueous T4 Celtis australis Leaves T T1 T2 T3 T4 Methanol T5 Cephotaxime Drug 6.25mg/ml 30mcg No zone 15 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 10.5 9 8.5 7 No zone T5 12.5mg/ml 6.25mg/ml No zone No zone 200 mg/ml 100 mg/ml 50 mg/ml 25 mg/ml 8.5 7.5 6.5 No zone of

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

Fig :Aqueous zone of inhibition of Pseudomonas aeruginosa

Fig: Methanol zone of inhibition of Pseudomonas aureginosa

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

SUMMARY AND CONCLUSSION


The aqueous and Methanol extract of celtis australis where study for the antibacterial activity against the two pathogens strains i.e. P.aeruginosa and S.aureus.The two microbial strain taken in the study have an intrinsic resistant to several antibiotics and are capable to acquire resistant during antibiotic therapy.Both extract of celtis australis shows inhibitory effect against both the test pathogens.the inhibitory effect of these extract of celtis australis against the test mircroorganism may be due to the presence of bioactive compound .among the two extract of celtis australis leaves Methanol shows MIC of about 25 mg/ml and aqueous shows MIC of about 50 mg/ml against S.aureus while in case of P.aeruginosa MIC of methanol 25 mg/ml while MIC of aqueous is 50 mg/ml. The some active substances were present in water extracts, but in low concentrations. Active substances were soluble in organic fraction solvents and therefore, not present in water extracts. Results also indicates that inhibitory effects of different fraction of plant against both the bacterial strains increased with an increases in concentration of different fractions .However degree of toxicity of different concentration of different fraction of plant may differ from one microorganism to another.The antibacterial activity of aqueous, and methanol extracts of celtis australis was reported against P.aeruginosa and S.aures (petronic et al.,2004). In most of the cases of root fraction showed comparatively higher degree of inhibition in regard to the leaf fractions.It may be because the leaf are rich in bioactive molecules which are known to show medicinal activity as well as exhibiting physiology and antimicrobial activities(Victinck and Pieter,2005). Celtis australis plant was active against gram negative and gram positive bacteria(Farrukh Aqil and Iqbal Ahmad,2003).

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa


This is evident from the result that the extract of plant studied.High inhibition against S.aureus is as compared to P.aeruginosa. According to (Nair and Chandra2007) in gram negative bacteria outer membrane acting as a barrier to many environmental substances including antibiotics(Burt,2004).It is not yet clear what are the mechanism of action of the antibacterial activity of the fraction; it is therefore difficult to speculate what could be the factor responsible for the differential response. It is however sufficed to mention for new that effect of the fraction is broad spectrum .Further study is therefore strongly indicated. It is important to investigate our plant kingdom, especially the world tropical reserves as an alternative for finding new and better drugs .it should therefore be essential to up this type of investigation to isolate and elucidate the active antimicrobial principles of this bioactive plant. It can be concluded that the plant fractions under study have great potential as antimicrobial compounds against microorganisms and they can be use in the treatment of infectious diseases caused by resistant micro organisms. Such screening of various natural organic compound and identification of active agent is the need of the hour because successful prediction of lead molecule and drug discovery will pay off late in drug development (Villsenor e al.,1995;Ghule et al.,2006;Oyetayo et al.,2007).

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Evaluation of antibacterial properties of celtis australis against S. aureus and P.aureginosa

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