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 »  Abstract
 »  Introduction
 »  Materials and Me...
 »  Results
 »  Discussion
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SHORT COMMUNICATION
Year : 2010  |  Volume : 42  |  Issue : 2  |  Page : 105-107
 

In vitro antibacterial evaluation of Anabaena sp. against several clinically significant microflora and HPTLC analysis of its active crude extracts


1 Department of Botany and Microbiology, Gurukul Kangri University, Hardwar, Uttarakhand - 249 404, India
2 Homoeopathic Pharmacopoeia Laboratory, Ghaziabad - 201 001, India

Date of Submission27-Jun-2008
Date of Decision01-Dec-2008
Date of Acceptance20-Apr-2010
Date of Web Publication23-Jun-2010

Correspondence Address:
Abhishek Chauhan
Department of Botany and Microbiology, Gurukul Kangri University, Hardwar, Uttarakhand - 249 404
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7613.64490

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 » Abstract 

The present study was conducted to evaluate the possible antibacterial activity of Anabaena extracts. Anabaena was isolated from a natural source and cultured in vitro. after suitable growth, cyanobacterial culture was harvested using different solvents. Extracts, thus prepared, were evaluated for their antibacterial potential by agar-well diffusion assay against bacterial species of clinical significance. MIC values were determined further to check the concentration ranges for significant inhibition. HPTLC analysis was done to separate the components of active crude extract in an attempt to identify the bio-active chemical entity. Methanol extract exhibited more potent activity than that of hexane and ethyl acetate extracts. No inhibitory effect was found against Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae. Staphylococcus aureus required about 256 μg/ml of the crude methanol extract for effective inhibition. HPTLC evaluation at λ 254 nm was performed for the separation of a complex mixture of the methanol extract. The results provide evidence that Anabaena sp. extracts might indeed be potential sources of new antibacterial agents.


Keywords: Blue-green algae, drug-resistance, biologically active substances, bacterial species


How to cite this article:
Chauhan A, Chauhan G, Gupta PC, Goyal P, Kaushik P. In vitro antibacterial evaluation of Anabaena sp. against several clinically significant microflora and HPTLC analysis of its active crude extracts. Indian J Pharmacol 2010;42:105-7

How to cite this URL:
Chauhan A, Chauhan G, Gupta PC, Goyal P, Kaushik P. In vitro antibacterial evaluation of Anabaena sp. against several clinically significant microflora and HPTLC analysis of its active crude extracts. Indian J Pharmacol [serial online] 2010 [cited 2019 Oct 20];42:105-7. Available from: http://www.ijp-online.com/text.asp?2010/42/2/105/64490



 » Introduction Top


Cyanobacteria (also known as blue-green algae due to the presence of certain pigments) are among the oldest phototrophic procaryotic organisms. The search for cyanobacteria with antimicrobial activity has gained importance in recent years due to growing worldwide concern about an alarming increase in the emergence of antibiotic resistance and further increase in the rate of infection by these antibiotic-resistant microorganisms. Various strains of cyanobacteria are known to produce intracellular and extracellular metabolites with diverse biological activities such as antibacterial and antifungal. [1],[2],[3] The aim of study reported here was to investigate the antibacterial activity of various organic extracts and spent medium of Anabaena sp. against the bacteria of clinical significance.


 » Materials and Methods Top


Isolation and Culturing of Cyanobacterium

Cyanobacterial mats collected from Naini-Lake, Nainital, India, were crushed with sterilized glass beads and diluted in presterilized double distilled water and were spread on solidified agar plates containing BG-11 medium and incubated at a light intensity of 3500 lux until visible colonies were appeared. Isolated cyanobacterium was identified by Prof. P. Kaushik at the Department of Botany and Microbiology, Gurukul Kangri University, Hardwar, India, as Anabaena species on the basis of its characteristic features. Scanning electron microscopy of isolated species was done for further confirmation on the basis of its morphological characteristics [Figure 1].

Preparation of Extracts

At the stationary phase of growth (25 days), Anabaena culture was dried in a hot air oven at 60°C for 1 h. The dried biomass was extracted with the solvents viz. methanol, ethyl acetate, hexane, and dichloromethane (Qualigens, AR grade). The extracts were centrifuged at 4000 rpm for 10 min, and were further concentrated in vacuum under reduced pressure and dried in a desiccator. Dried extracts were stored in labeled sterile screw capped bottles in a refrigerator till further use.

Test Microorganisms

The bacterial cultures selected for the present study were Staphylococcus aureus Scientific Name Search  MTCC-740, Bacillus subtilis Scientific Name Search  MTCC-736,  Bacillus cereus Scientific Name Search C-430,  Bacillus pumilus Scientific Name Search CC-1607,  Escherichia More Details coli MTCC-739,  Pseudomonas aeruginosa Scientific Name Search CC-741,  Salmonella More Details typhi MTCC-733, and Klebsiella pneumoniae Scientific Name Search  MTCC-139. All organisms were maintained on specific media slants at 4°C and revived prior to use. An inoculum size of 10 6 CFU/ml of bacteria, compared with 0.5 McFarland turbidity standards, was used.

Antibacterial Testing

Agar well diffusion assay


In vitro evaluation of antibacterial activity of different crude extracts of Anabaena sp. was done using the agar well diffusion assay. [4]  Petri dish More Detailses (90 mm diameter) were poured with Mueller Hinton Agar media (HI-Media) and allowed to solidify to make a base layer. A seed layer was prepared by inoculating 100 μl of each test organism's suspension in 25 ml Mueller Hinton Agar. After complete solidification of media, wells (of 6 mm in diameter) were made in the agar medium with the help of a sterile steel borer. 100 μl of each extract (stock solution 10 mg/ml) was added aseptically in respective wells. All the plates were incubated at 37±1ΊC for 24 h. At the end of the period, the inhibition zone formed in the medium was measured in millimeters.

Microdilution assay

MIC values were determined for the sensitive bacteria to the methanol extract using a two-fold serial microdilution method. [5] The concentrations of the extract used in experiment ranged from 10 μg/ml to 512 μg/ml. The methanol extract was added to sterile microtiter plate containing Cation-Adjusted Mueller-Hinton Broth (CAMHB, Hi-Media). In each plate, 1 Χ 10 6 CFU/ml bacterial suspensions were added. The MIC value was taken as the lowest concentration of the extract in wells of the microtiter plate that showed no turbidity after 24 h of the incubation at 37°C. The turbidity of the wells in the microtiter plate was interpreted as visible growth of the microorganisms. Experiments were carried out in duplicate.

HPTLC analysis

HPTLC analysis was performed on a silica gel F- 254 (E-Merck grade) pre-coated aluminum plate. A band of 7 mm was applied at a distance of 10 mm from the bottom of the plate. The methanol extract (10 μl) was applied on a chromatoplate (CAMAG Linomat-5) and run in the solvent system (chloroform and methanol, 7:3).The plate was developed up to 8 cm in a twin trough chamber previously equilibrated with mobile phase for 20 min. Densitometric evaluation of the plate was performed at l 254 nm.


 » Results Top


The results shown in [Table 1] indicate that the methanol extract revealed a prominent effect on various bacterial species. However, the hexane and ethyl acetate extract exhibited a moderate effect and DCM and spent medium did not exhibit any antibacterial activity. As shown in [Table 2], MIC (minimum inhibitory concentrations) for the methanol extract was found to be >512 μg/ml for Bacillus pumilus and Bacillus cereus. On the other hand, Bacillus subtilis and Escherichia coli was inhibited at the dose of 512 μg/ml of the crude extract. While Staphylococcus aureus required about 256 μg/ml. Results of HPTLC analysis revealed that the methanol extract contained a mixture of different component that were eluted at R F = 0.19, R F = 0.30, R F = 0.33, R F = 0.42, R F = 0.54, R F = 0.66, R F = 0.73 as shown in [Figure 2]a and b.


 » Discussion Top


There is a clear indication that Anabaena sp. extracted in different solvents has significant antibacterial effects on both Gram-positive and Gram-negative bacterial species. The current scenario of antibiotics is very threatening due to significant emergence of resistance among bacterial pathogens against available antibiotics. [6] The present study focused to the discovery of novel chemicals that can lead to the development of pharmaceuticals of cyanobacterial origin. The combination of a chromatographic technique with serial dilution in tubes was capable of identifying the active components of methanol extract responsible for this activity. This manifests the importance of using the correct combination of chemical screening and biological analysis.

 
 » References Top

1.Kreitlow S, Mundt S, Lindequist U. Cyanobacteria-a potential source of new biologically active substances. J Biotechnol 1999;70:61-3.  Back to cited text no. 1  [PUBMED]    
2.Falch BS, Konig GM, Wright AD, Sticher O, Angerhofer CK, Pezzuto JM et al Biological activities of cyanobacteria: Evaluation of extracts and pure compounds. Planta Med 1995;61:321-8.  Back to cited text no. 2      
3.Mundt S, Kreitlow S, Nowotny A, Effmert U. Biological and pharmacological investigation of selected cyanobacteria. Int J Hyg Environ Health 2001;203:327-34.  Back to cited text no. 3  [PUBMED]    
4.Perez C, Pauli M, Bazerque P. An antibiotic assay by agar-well diffusion method. Acta Biol Med Exp 1990;15:113-5.  Back to cited text no. 4      
5.NCCLS-National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard M7-A4, Wayne, Pa;1997.  Back to cited text no. 5      
6.Kaushik P, Chauhan A, Chauhan G, Goyal P. Evaluation of Nostoc commune for potential antibacterial activity and UV-HPLC analysis of methanol extract. Int J Microbiol 2008;5:1-5.  Back to cited text no. 6      


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]

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