1

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 et

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

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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 nitrogen compound (alkaloids, amines ,non-

proteins, amino acid and cyanogenic glycosides).

3

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

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-O-methylellagic 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.

OBJECTIVE OF THE STUDY

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

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.

REVIEW OF LITERATURE

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

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 protozoan’s (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)

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

GENERAL INTRODUCTION OF TEST PATHOGENS

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)

Scientific classification

Kingdom:

Bacteria

Phylum:

Proteobacteria

Class:Gamma Proteobacteria

Order:Pseudomonadales

Family:Pseudomonadaceae

Genus: Pseudomonas

Species:

Pseudomonas aeruginosa

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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 Non–spore 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 yellow-green 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)

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

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.

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

9

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

plant pathogens, notably all of the other members of the P. syringae subgroup, but P. syringae is the most widespread and best studied.

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 aureusDomain: 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

Gram-positive 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

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

production of an enterotoxin, and pathogenicity is also associated with

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 first-

line therapy is most commonly a penicillinase-resistant β-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

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

high risk of 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): 178–82.

GENERAL INTRODUCTION OF PLANT TAKEN DURING THE

COURSE OF STUDY

Celtis Australis

Chinese Hackberry (C. sinensis) leaves and fruit

Scientific classification

Kingdom: Plantae

(unranked): Angiosperms

(unranked): Eudicots

(unranked): Rosids

Order: Rosales

Family: ulmaceae

Genus: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 (23-79) to the unrelated Ziziphus lotus. (KEELER, HARRIET L. 1900)

Description

Celtis species are generally medium-sized trees, reaching 10–25 m (33–82 ft) tall, rarely up to 40 m (130 ft) tall. The leaves are alternate, simple, 3–15 cm (1.2–5.9 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 6–10 mm (0.24–0.39 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 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.

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

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 contains C. 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.

MATERIAL AND METHOD

Material required

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

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. Apparatus Company

1 Autoclave Scientific equipment

2 Hot air oven Scientific equipment

3 Electronic balance Sartorius

4 Laminar air flow Zenith

5 Incubator Toshiba

6 Refrigerator Sanyo

7 Sterile cotton & swab

tubes

Hi -media

8 Micropipette Torson

9 Glassware 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.

METHODS

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

Table:2 Preparation of Media (Nutrient Broth) for revival of Bacterial

culture.

Nutrient Broth with following composition was used for Bacterial

culture.

S. No. Composition Amount (1000ml)

1 Beef Extract 1.5g

2 Yeast Extract 1.5g

3 Peptone 5.0g

4 NaCl 5.0g

5 Distilled water 1000ml

6 pH 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) .

Preparation of Media (Muller Hinton) for revival of Bacterial culture.

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

Table:3 Muller Hinton media with following compound was used for

Bacterial culture.

S.No. Composition Amount 1000ml

1 Beef infusion 300g

2 Casamino acid 17.5g

3 Starch 1.5g

4 Agar 17.0g

5 Distilled 1000ml

6 pH 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 the condenser and then solvent come back to R.B.F. with

compound of leaf. Extract containing volatile solvent, were concentrated

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

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 4°C.

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

140°C 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 37°C 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.

RESULTS

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

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.

Table-:4 Antibiogram of isolated Pseudomonas aeruginosa :

Determinatio

21

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

S No. Antibiotics Symbol

n of Zone of

inhibition in

(mm)Result

1

Chloramphenico

l

C

8 intermediate

2

Ampicillin

A

No zone Resistant

3

Tetracycline

T

6 Intermediat

e

4

Gentamicin

G

10 sensitive

5

Co-trimoxazole

Co

8 intermediate

6

Cefriaxone

Ci

7 intermediate

7

Cefuroxime

Cu

6 intermediate

8

Ciprofloxine

Cf

15 sensitive

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

Table-:5 Antibiogram of isolated Staphylococcus aureus.

Determinatio

n of Zone of

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

S No. Antibiotics Symbol

inhibition in

(mm)Result

1

Chloramphenico

l

C

10 Sensitive

2

Ampicillin

A

No zone Resistant

3

Tetracycline

T

12 sensitive

4

Gentamicin

G

14 sensitive

5

Co-trimoxazole

Co

16 sensitive

6

Cefriaxone

Ci

8 intermediate

7

Cefuroxime

Cu

6 intermediate

8

Ciprofloxine

Cf

13 sensitive

24

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

ANTIMICROBIAL PROPERTIES OF CELTIS AUSTRALIS

25

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

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.

26

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 given

on plate

Cons./ml Zone of

inhibition

(in mm)

Celtis

australi

s

Leaves

Aque

ous

T 200 mg/ml 8

T1 100 mg/ml 7.5

T2 50 mg/ml 6.5

T3 25 mg/ml No zone

T4 12.5mg/ml No zone

T5 6.25mg/ml No zone

Methan

ol

T 200mg/ml 9

T1 100mg/ml 8.5

T2 50mg/ml 7.5

T3 25mg/ml 6

T4 12.5mg/ml No zone

T5 6.25mg/ml No zone

Cephotaxime Drug 30mcg 15

27

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

Table-:7 Zone of inhibition (in mm) of different leaves fractions of celtis australis

against Staphylococcus aureus.

28

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

Plant Fractions Code given

on plate

Cons./ml Zone of

inhibition

(in mm)

Celtis

australi

s

Leaves

Aque

ous

T 200 mg/ml 8.5

T1 100 mg/ml 7.5

T2 50 mg/ml 6.5

T3 25 mg/ml No zone

T4 12.5mg/ml No zone

T5 6.25mg/ml No zone

Methan

ol

T 200mg/ml 10.5

T1 100mg/ml 9

T2 50mg/ml 8.5

T3 25mg/ml 7

T4 12.5mg/ml No zone

T5 6.25mg/ml No zone

Cephotaxime Drug 30mcg 15

29

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

SUMMARY AND CONCLUSSION

30

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

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).

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

31

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

(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).

BIBLIOGRAPHY

32

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

1. A. Y. Itah and J. P. Essien, Growth Profile and

Hydrocarbonoclastic Potential of Microorganisms Isolated from

Tarballs in the Bight of Bonny, Nigeria, World Journal of Microbiology

and Biotechnology, Volume 21, Numbers.

2. At-Deleimy K.S. & Ali S.H. 1970. Antibacterial action of vegetable

extract on the growth of pathogenic bacteria J. sci. Food. Agric.21:110-

111.

3. Balcht, Aldona & Smith, Raymond (1994). Pseudomonas

Aeruginosa: Infections and Treatment. Informa Health Care. pp. 83–

84. ISBN 0-8247-9210-6

4. Bayer AS, Bolger AF, Taubert KA, et al. 1998. "Diagnosis and

management of infective endocarditis and its complications". Circulation

98 (25): 2936–48.

5. BROWER, ANDREW V.Z. (2006): Problems with DNA barcodes for species delimitation:

‘ten species’ of Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae).

Systematics and Biodiversity 4(2): 127–132.

6. Chambers HF 2001. "The changing epidemiology of

Staphylococcus aureus?". Emerg Infect Dis 7 (2): 178–82.

7. Chambers HF 2001. "The changing epidemiology of Staphylococcus

aureus?". Emerg Infect Dis 7 (2): 178–82.

Chopra R.N.,Nayer S.l., Chopra I.C.1992 Glossary of Indian

Medical plants,3rd Edn.CSIR,New Delhi,7-246

8. Cimolai. 2008. MRSA and the environment: implications for

comprehensive control measures. European journal of clinical

microbiology & infectious diseases : official publication of the

European Society of Clinical Microbiology vol. 27 (7) pp. 481-93.

9. Clauditz A, Resch A, Wieland KP, Peschel A, Götz F 2006.

"Staphyloxanthin plays a role in the fitness of Staphylococcus

aureus and its ability to cope with oxidative stress". Infection and

immunity 74 (8): 4950–3. doi:10.1128/IAI.00204-06.

33

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

10. Cooper M, Tavankar GR, Williams HD (2003). "Regulation of

expression of the cyanide-insensitive terminal oxidase in

Pseudomonas aeruginosa". Microbiology 149 s(Pt 5): 1275–1284

11. Cosgrove SE, Vigliani GA, Campion M, et al. 2009. "Initial

low‐dose gentamicin for Staphylococcus aureus bacteremia and

endocarditis is nephrotoxic". Clin Infect Dis 48 (6): 713–721.

12.Curran JP, Al-Salihi FL 1980. "Neonatal staphylococcal scalded skin

syndrome: massive outbreak due to an unusual phage type". Pediatrics

66 (2): 285–90.

13. Davis j.,1994 inactivation of antibiotic & the dissemination of

resistance gene . science 264:375-382.

14.Diamond R.D.,1993. The growing problem of mycoses in patients

infected with HIV. Review of infectious diseases.13:480-486.

15. Dowzicky M. J. and Park C.H. 2008. Update on antimicrobial

susceptibility rates among gram-negative and gram-positve

organism in United states: result from the tigecycline evaluation

and surveillance trial (TEST)2005 to 2007. Clin. Ther.,30, 2040-

2050.

16. Farrukh A., & Ahamad I.2003. Board spectrum antibacterial and

antifungal properties of certain traditionally used Indian medicinal plant.

World J. Microbiol. Biotechnol. 19:653-657.

17. Ghule B.V.,Gghante M.H., soaji A.N. & yeole P.G.

2006 .hypolipidemic & antihyperlipidemic effects of lagenaria siuraria

fruit extracts .Indian J. exp.biol. 44 (11):905-909.

18.Govindarajan R.,Vijayakumar M. Singh, M. Rao. C.L.V.

Ashirwaikar A.K.S. pushpangadan P. 2005.Antiulcers and

antimicrobial activity of some anogeissum latifolia, J.

Ethanopharmacology.101:57-61.

19.Grosvenor P.W., Spriono A. and Gray D.O. 1995. Medicinal plant

from Riau province, Sumatra, Indonesia. Part 2, Antibacterial and

antifungal activity. J. of Ethanopharmacology.45.97-111.

34

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

20. Harborne S.B. and Baxter H.1995. Phytochemical

dictionary.A handook of bioactive compound from plants.

21. HÉBERT, PAUL D.N.; PENTON, ERIN H.; BURNS, JOHN M.; JANZEN, DANIEL H. &

HALLWACHS, WINNIE (2004): Ten species in one: DNA barcoding reveals cryptic species

in the semitropical skipper butterfly Astraptes fulgerator. PNAS 101(41):

22.Hulin V., Mathat A.G.,Mafart P. and Dufosse L. 1998. Lee

proprieties antimicrobiennes des hiles essentielles et composes daromes.

Sci. Aliments,18:563-582.

23. Kafaru E.,1994.immense help from nature’ workshop.Elika help

services Ltd.academic press Plc. Lagos Njgeria.pp.1-27

24. KEELER, HARRIET L. (1900): Our Native Trees and How to Identify Them. Charles

Scriber's Sons, New York

25. Khotaei G.T.,Fattahi F., Purpak Z.,Moinfar Z., Aghaee

F.M., Gholami K. and Moin M. 2008. Adverse reaction to

antibiotics in hospitalized Iranian children. J. Microbiol. Immunel.

Infect.,41: 160-164.

26. Kluytmans J, van Belkum A, Verbrugh H 1997. "Nasal

carriage of Staphylococcus aureus : epidemiology, underlying

mechanisms, and associated risks". Clin. Microbiol. Rev. 10 (3):

505–20

27. Korzeniowski O, Sande MA 1982. "Combination antimicrobial

therapy for Staphylococcus aureus endocarditis in patients addicted

to parenteral drugs and in nonaddicts: a prospective study". Ann

Intern Med 97 (4): 496–503.

28. Len R.Y. Nusuzzaman F. & shah S.N. 2009.Incidence & impact of

adverse effect to antibiotics in hospitalized adults with pneumonia J.

hosp.med 4:E7-E15.

29. lglewski BH (1996). Pseudomonas. In: Baron's Medical

Microbiology (Baron S et al., eds.) (4th ed.). Univ of Texas Medical

Branch.

35

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

30.Liu GY, Essex A, Buchanan JT, Datta V, Hoffman HM, Bastian JF,

Fierer J, Nizet V 2005. "Staphylococcus aureus golden pigment impairs

neutrophil killing and promotes virulence through its antioxidant activity".

J Exp Med 202 (2): 209–15.

31. Lyengar M.A.1985.study of crude drug,2nd Edn. College of

pharmaceutical sciences,Manipal.13-78.

32.Mahesh B. & Satish S. 2008. Antimicrobial activity of some important

medicinal plant against plants and human pathogens. world J. of

agricultural science 4: 839-843.

33. Mulligen M.E. murry –leisure K.A. ribner B.S. standiford H.C.,john

J.F.,karvick J.A. kaufman C.A.,Yu,V.L.,1993.methicillin resistant S. aureus

American J. of medicine 94:313-328.

34. Nair R. & Chanda S. 2007. Invitro antimicrobial activity of Psidium

guajava L. Leaf extracts against clinically important pathogenic microbial

strains. Braz J. Microbiol. 38:452-458.

35. Nascimento G.G.F., Locatelli J., Freitas P.C. and Silva

G.L.2000. Antibacterial activity of plant extracts and

phytochemicals on antibiotic resistant bacteria. Braz. J. Microbiol.

31: 247-256

36. Oyetayo F.L.,, oyetayo V.O. & Ajcwole V.2007. phytochemical

profile & antibacterial properties of the seed & leaf of the luffa

cylindrical.J. of pharmacology & Toxicology. 2(6):586-589.

37. Petrovie J., Stanojkovie A. Comic L. & Curcic 2004. Antibacterial

activity of Antibacterial activity of plant name Fitoteratina 75:737-739.

38. Piddock K.J.V., Wise R., 1989. Mechanisms of resistance to

quinolones and clinical perspective. J. of Antimicrobial

Chemotherapy 23: 475- 483.

39. Rinaldi M.G.,1991. Problems in the diagnosis of invasive

fungal diseases. Review of infectious diseases 13: 493-495.

40. Robin E.H.,Anil W.,Alexander M., Loeto M.,Kath K.,1998.

Nasopharyngeal carriage & antimicrobial resistance in isolates of

36

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

S.aureus & Heamophilus influenzae type b in children ,under 5 year of

age in Botswana. International J.of infectious diseases 1:18-25.

41. Rudrappa T. and Bais H.P. 2008. Curcumin a known phenolic

from Curcuma longa, attenuates the virulence of pseudomonas

aeruginosa PAO1 in whole plant and animal pathogenicity models. J.

Agric. Food Chem.,56: 1955-1962.

42. Saunuam P., Hiransuthikul N., saun kratay C., Malathum K. &

Danchaivijtra S. 2008 Risk factors for noscomial infections caused by

extended –spectrum a-lactamare producing E.coli or pneumonia in

Thailand.Asian biomed.,2:485-491.

43. Singh M.,chaudhary M.A., yadav J.N.S. & sanyal S.C.1992- the

spectrum of antibiotic resistance in human & veterinary isolates of E.coli

collected from 1984,1986 in northern Indian J. of antimicrobial

chemotherapy 29:159-168

44.Stockwell C.,1988. Nature’s pharmacy London, United Kingdom.

Century Hutchinson Ltd.

45.Tansey M.R. & Appletonl J.A. 1975. Inhibition of fungal growth by

garlic extract mycologia 67:409-413.

46.Thinnel T.S. Dahiya M.S. 1976. Inhibitory effect of essential oils of

some medicinal plants against soil inhibiting dematophytes. Indian

Drugs.14(11):17-18.

47. Thomson W.A.R.,1978. Medicines from the earth.

Maindendhead, U.K. McGraw-hill books.

48. Tillotson G.S., Draghi D.C., sahm D.F., Tomfohrde K.M.,del

fabro T. & gitch lay I.A. 2008. Susceptibility of infections in the united

state 2005-2007: laboratory-based surveillance study.J. antimicrobs

chemother.,62: 109-115.

49.Tongson C., Davidson P.M., Mahakarnchanakul W. & Vibulsresth

P. 2005. Antimicribial effect of Thai spices against Listereia

monocytogenes and Salmonella typhimurium DT 104. J. F ood

prot.,68:2054-2058.

37

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

50. Vlictink A.J. & Pieters L. 2005. Bio-guided Isolation of

pharmacologically active plant components, still a valuable strategy for

finding of new load compounds J. Ethanopharmacol.,100:57-60.

51.Watanab T. 1974. Garlic Therapy Japan Publiation Inc. Tokyo 66.

52. Worlitzsch D, Tarran R, Ulrich M et al. (2002). "Effects of

reduced mucus oxygen concentration in airway Pseudomonas

infections of cystic fibrosis patients". J. Clin. Invest. 109 (3): 317–

325