Research Article - (2012) Volume 2, Issue 5
1Institute of Pharmaceutical Sciences and Research Center, Bhagwant University Ajmer Rajasthan
2College of Pharmacy Agra, Nandha College of Pharmacy, Erode, Tamilnadu, India.
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. Actinomyctes is a potential producer of many 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 It is noteworthy that, of the nearly 300,000 plant species that exist on the earth, In our study we have isolated the endophytic actinomycetes from citrus fruit. The total 5 actinomycetes were isolated from citrus fruits (Citrus aurantifolia) using Starch Casein agar, Out of 5 actinomycetes strains, 2 actinomycetes showing antibacterial activity were recovered using Bennet agar media. The production of antibacterial compound was performed using L.B. Broth. Out of 2 actinomycetes strain only one show (CT1) shows the strong antibacterial activity and fermentation carried out using L.B. Broth for 15 days. After fermentation, extraction of the supernatant was carried out using solvent petroleum ether. The Antibacterial compound was recovered using TLC and Column Chromatography. The Rf value of the compound was found to be (0.6152 ). The structural study of the extracted compound was carried out by UV-spectroscopy, FT-IR. The antibacterial compound was effective against E. coli, S. typhi, K. pneumoniae, S. aureus bacteria some of them got resistance against some antibiotic drug.
Endophytic actinomycetes, Citrus aurantifolia, Antibacterial activity, Structure elucidation.
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.
Collection of sample
Citrus aurantifolia fruits were collected from different regions of India
Surface sterilization:
Collected fruits 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.
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 90° and incubated at 37°C 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 37°C for about 24 hrs, and Biochemical test and Gram’s staining also performed.
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 37°C for the production of antibacterial compound for 18 days.
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 7th day of fermentation by taking isolates and concentrated up to 10 times at 45°C by using cylinder plate method against some gram +ve and some gram –ve bacteria, this antibacterial activity checking procedure continued upto 18th day.
Extraction of antibacterial compound using different solvents:
Broth taken at the end of 15th 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.
Identification of antibacterial compound
The structure elucidation of the compound was performed by using UV, FT-IR.
Actinomycetes isolates
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.
Table no.1. The actinomycetes isolated from Citrus aurantifolia twigs on the Starch Casein media.
Table no.2. The actinomycetes isolated from Citrus aurantifolia twigs on the YMA media.
Table.no. 3. The growth on different types of actinomycetes strains on ISPmedia.
Isolation of the antibacterial Compound producing actinomycetes
Total 7 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
Table.no.4. 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 45°C upto 10 times against some gram +ve and gram –ve microbes.
Figure no.1: Actinomycetes (CT1) strain showing strong antibacterial activity against some bacteria.
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.
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.
Structural analysis of the antibacterial compound
The structure elucidation of the compound was performed by using UV, FT-IR . The λ max of antibacterial compound was found 318 when taken in CHCl3 shown in graph no.1.The IR of the compound were shown in figure no. 2. The I.R. spectra shows the presence of OH group, presence of aromatic ring and presence of NH2 group.
Graph no.1 λ Max of the antibacterial compound.
Figure no.2. I.R spectra of the antibacterial compound.
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
The authors are thankful to Prof. M. Jagdisharan, Nandha College of Pharmacy to provide FTIR facility.
