INTRODUCTION:

Today more than 30,000 diseases are clinically described. Less than one third of these can be treated symptomatically and only a few can be cured. Consequently, there exists a strong interest in getting access to new therapeutic agents which is major driving force for advanced drug discovery strategies. The screening approach has been employed extensively in the search for microorganism capable of producing useful antibiotics. Endophytic actinomycetes have been defined as that can be isolated from the disinfected surfaces of plant tissues or that can be extracted from within the plant that do not cause visible harm to the host.

They can promote the growth of many field crops by producing plant growth-promoting substances and by fixing nitrogen from the atmosphere and potential sources of novel natural products for exploitation in medicine, agriculture and industry. It is noteworthy that, of the nearly 300,000 plant species that exist on the earth, each individual plant is host to one or more endophytes

There is a general call for new antibiotics, chemotherapeutic agents, and agrochemicals that are highly effective, possess low toxicity, and have a minor environmental impact. This search is driven by the development of resistance in infectious microorganisms (e.g., species of Staphylococcus, Mycobacterium and Streptococcus) to existing compounds and by the menacing presence of naturally resistant organisms. Most of the drug mainly produced by actinomycetes.

MATERIALS AND METHODS:

Collection of sample:

The different parts like leaves, twigs, fruits were collected from a species (Citrus aurantifolia) of the citrus plant were collected from different regions of India like Tamilnadu and Uttar Pradesh.

Surface sterilization:

Collected plant parts first rinsed with sterilized water then plant parts were kept in sterile beaker consists of 70% ethanol for 3 min. After that the ethanol sterilized plant parts were kept in beaker consist of Sodium hypochlorite (5%) with Tween 20 (0.1%) for 5 minutes. At last plant parts rinsed with sterilized water. Sterility checked and the surface sterilized plant parts were taken and crushed using sterile pestle and mortar and spread on the three of the media ( Starch casein agar, YMA media, Albumin media) consisting of Nystatin 50 µg/ ml and kept at 28⁰C for 3 weeks.

Name of Plants Consisting Endophytic Bacteria:

S.No.

NAME OF PLANTS

Agropyron elongatum (tall wheat grass)

Agropyron intermedium (intermediate wheat grass)

Allium porrum

Amarylis belladonna

Amorpha canescens (lead plant )

Andropogon gerardi (big blue stem)

Andropogon scoparius (little bluestem)

Baptisia leucantha (white false indigo)

Betula pendula

Bouteloua curtipendula(sideoats grama)

Bouteloua gracilis (blue grama)

Brassica sps.(mustard)

Callirhoe involucrate (purple poppy mallow)

Calathea sp.

Calluna vulgaris

Camellia japonica

Dicanthelium oligosathes (panicgrass)

Euphorbia podperae (leafy spurge)

Euphorbia sp.

Festuca rubra

Fragaria vesca

Glycine max (soyabean)

Triticum aestivum (wheat)

Vicia villosa (hairy vetch)

Isolation of actinomycetes producing antibacterial compound:

Isolated actinomycetes were streaked on solidified media at straight line and kept for incubation in incubator at 37⁰C for nearly about 4 days. After completion of 4 days sensitive microbes streak at angle of 90⁰ and incubated at 37⁰C for 24 hrs and observe zone of inhibition.

Identification of different actinomycetes:

Isolated actinomycetes were streaked on solidified ISP-media media in zigzag fashion and kept for incubation in incubator at 37⁰C for about 24 hrs, and Biochemical test and Gram’s staining also performed.

Media (plant parts)

Colour of Colony

Code

Starch Casein agar

(Citrus aurantifolia twigs)

Yellow Colony

Dark Yellow Colony

Milky White Colony

Red Colony

Creamy white Colony

CT1

CT2

CT3

CT4

CT5

Table no.1. Shows the actinomycetes isolated from Citrus aurantifolia twigs on the Starch Casein media.

Production of antibacterial compound using starch casein broth and L.B. broth:

The 3 strains of actinomycetes CT1, CT2, CT3, producing antibacterial compound were inoculated into L.B. broth and Starch casein broth and kept for incubation at 37⁰C for the production of antibacterial compound for 18 days.

Media(plant parts)

Color of Colony

Code

YMA media

(Citrus aurantifolia Twigs)

Brown Colony

Light White Colony

YMT1

YMT2

Table no.2.Shows the actinomycetes isolated from Citrus aurantifolia twigs on the YMA media.

Antibacterial activity of isolates checking through cylinder plate method:

After incubation of 7 days of fermentation of L.B.broth and Starch Casein Broth antibacterial activity was observed from 7^th day of fermentation by taking isolates and concentrated up to 10 times at 45⁰C by using cylinder plate method against some gram +ve and some gram –ve bacteria, this antibacterial activity checking procedure continued upto 18^th day.

Extraction of antibacterial compound using different solvents:

Broth taken at the end of 15^th day and centrifuged at 10,000 rpm for 20 min to separate the mycelial biomass, the supernatant was obtained separated by filtration using Whatman filter paper. Certain solvents used for extraction of antibacterial compound like butanol, n-hexane, ethyl acetate, petroleum ether, chloroform, ethanol (1:1) ratio. Supernatant mixture was agitated for 45 min. with homogenizer and the solvent was separated from broth by separating funnel, Solvent present in the broth was separated by centrifugation at 5000 rpm for 15 min to remove traces of fermentive broth. All extracts obtained through this method were assayed for

antibacterial study against different microbes using respective solvents as control by agar well diffusion method.

Purification of antibacterial compound:

Purification of the compound was performed using TLC and Column chromatography using n-butanol: acetic acid: water (2:1:1) mobile phase or eluent.

Media

CT1

CT2

CT3

CT4

CT5

YMT1

YMT2

ISP2

ISP4

ISP5

ISP6

ISP7

Table.no. 3. Shows the growth on different types of actinomycetes strains on ISPmedia.

Identification of antibacterial compound:

The structure elucidation of the compound was performed by using UV, FT-IR and NMR.

RESULT AND DISCUSSION:

Actinomycetes isolate:

There were 7 types of the actinomycetes were isolated (given code) from different parts of the species of Citrus plant (Citrus aurantifolia) on the three of nutrient media given in table no.1-3 and out of 7 actinomycetes.

Isolation of the antibacterial Compound producing actinomycetes:

Total seven actinomycetes showed the production of antibacterial compound on Bennet agar and CT1 showing strong antibacterial activity was selected for further study.

Identification and biochemical test:

The growth ISP-media was shown in table no.4

S.No.

Name of

microorganisms

Zone of inhibition

(mm)

E. coli ATCC 8739

S. typhi ATCC 23564

S. aureus ATCC 29736

M. luteus ATCC11880

K. pneumoniae ATCC 10031

S. fecalis ATCC 8043

B. subtilis ATCC 6633

S. boydi ATCC 9207

P. mirabilis ATCC 2124

8.2 mm

7.3 mm

8.9 mm

0 mm

6.2 mm

0 mm

7.0 mm

0 mm

Table.no.4. Shows the zone of inhibition against different microbes by the antibacterial compound.

Production of antibacterial compound:

One actinomycetes isolated from Citrus aurantifolia twig (CT1) was selected for the production of antibacterial because that showing higher activity on the Bennet agar shown in figure no.1. After that CT1 shows the production of antibacterial activity in L.B. Broth media and antibacterial activity was checked through after concentration of broth at 45⁰C upto 10 times against some gram +ve and gram –ve microbes.

Extraction of antibacterial compound:

At last after extraction of the L.B. Broth (using petroleum ether showed good antibacterial activity) the brown gummy like substance obtained.

Graph no.1 Shows the λ Max of the antibacterial compound.

Fig no.1 Shows actinomycetes (CT1) strain showing strong antibacterial activity against some bacteria

Purification of antibacterial compound:

After Purification of the antibacterial compound using TLC and Column chromatography { n-butanol: acetic acid :water (2:1:1) mobile phase or eluent.},whitish brown powder was obtained which showed good antibacterial activity shown in table no. 4.

Fig no.2. Shows the I.R spectra of the antibacterial compound

Structural analysis of the antibacterial compound:

The structure elucidation of the compound was performed by using UV, FT-IR and NMR. The λ max of antibacterial compound was found 318 when taken in CHCl₃shown in graph no.1.The IR of the compound were shown in figure no. 2. The I.R. spectra show the presence of OH group, presence of aromatic ring and presence of NH₂ group. The N.M.R. spectra of the compound shows the proton attached to hetero in nature, proton attached to Benzene ring.

CONCLUSION:

The actinomycetes were isolated from citrus aurantifolia plant and the production of antibacterial compound was carried out by using L.B. broth which showed antibacterial activity against the some gram + and gram– bacteria (E.coli, S.typhi, S.aureus, B.subtilis, P.mirabilis, S.boydi) which have got resistance against some antibiotic so from our study it was concluded that antibacterial compound produced by endophytic actinomycetes isolated from citrus aurantifolia will be useful for future study.

ACKNOWLEDEMENT:

The authors are thankful to Prof. D Shankar, SASTRA University, Thanjavur to provide NMR facility.

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Received on 20.04.2008 Modified on 28.04.2008

Research J. Pharm. and Tech. 1(2): April-June. 2008;Page 112-115