ISSN 2319 – 2739
Int J Med Biosci. 2013; 2(1): 09 ‐ 15
International Journal of Medicine and Biosciences www.ijmbonline.com
Antimicrobial Activity of Cyanobacteria using In‐ Vitro and In Silico Analysis Vijayakumar Madhumathi and Subramaniyan Vijayakumar* *Department of Botany and Microbiology, A.V.V.M Sri Pushpam College (Autonomous),
Poondi‐613 503, Thanjavur. Received 10 November 2o12; accepted 24 December 2012; published online 31 January 2013
Research Article Microbiology Abstract Cyanobacteria have immense medicinal importance and are therefore used in the treatment of bacterial and fungal organism causing oral diseases. GC‐MS analysis of the ethanolic extract of the Oscillatoria laete‐virens and Lyngbya martensiana has resulted in the identification bioactive compound. The human oral cavity is a complex biological system in which members of a microbial community interact with themselves as well as with different host structures and components of Candida albicans and Streptococcus mutans. The pathogenesis of this dental infection is a multi‐factorial process that results in a serious degenerative disease of the Oral candidiasis. In the present study SAP (Secreted Aspartyl Proteinases in Virulence and Pathogenesis) is taken as a case study molecule to understand high reactive responses of various drugs administered for the oral candidiasis. The drugs are being compared with the SAP from Candida albicans and SpaP (cell surface antigen SpaP gene) from Streptococcus mutans. The SAP and SpaP interacted with formic acid, nerolidal, octanol and nonanoic acid using docking methods. Keywords: Cyanobacteria, bioactive compound, candidiasis, GC‐MS, docking
Introduction Cyanobacterial blooms have a wide range of social environmental and economic impacts. Cyanobacterial metabolites which are cytotoxic to other algae are of special interest. On the other hand, such compounds could provide helpful in combating algae blooms or in the development of environmentally friendly are tributilin free antofouling parts of ships. The knowledge about such compounds gives a better understanding of interaction between competing organisms of the same habitat. Furthermore, cyanobacterial biogenics, particularly cyotoxic compounds could provide leads for further development of new therapeutic agents for a variety of disease and for the development of new antibiotics. [1,2]. The Study was designed to investigate the Antimicrobial activity of cyanobacterial species Oscillatoria laete‐virens and Lyngbya martensiana. The study was extended to identify the individual components of their ethanolic extract by Gas chromatography (GC) coupled with Mass Spectrometry (MS). *Corresponding Author E‐mail:
[email protected] ©Copyright 2012, International Journal of Medicine and Biosciences All Rights Reserved
In oral cavity of human beings, Oral candidiasis diseases are of common occurrence. It is the inflammatory condition of the supporting tissues of the teeth caused by bacteria and fungi. The oral microbiota is a complex community of microorganisms. They function as multiple pathogens in the affected site. Oral disease, caused mainly by microbes, is characterized by inflammation, a strong immune response and loss of connective tissue attachment and bone disease is an infection of the supporting structures of the teeth. The oral diseases are treated with antibiotics and anti‐ therapeutics by the oral physicians. Even though, many antibiotics are administered, the nature of the response is different .To identify the suitable drugs against the pathogenic microorganisms, insilico analysis is carried out in the recent past. In the present study, Candida albicans and Streptococcus mutans have been selected for the insilico analysis to assess the suitable drug for the disease called oral candidiasis. In the present study SAP and SpaP molecules is taken as a case study molecule to understand high reactive responses of various drugs administered for the oral candidiasis. The drugs are being compared with the SAP and SpaP. The SAP and SpaP interacted with formic acid, nerolidal, octanol and nonanoic acid using docking
V. Madhumathi and S. Vijayakumar / Int J Med Biosci. 2013; 2(1): 09 ‐ 15 10 methods. The present application of computational biology and bioinformatics has the potential, not only of speeding up the drug discovery process but also of changing the drugs design. Materials and Methods Study Area Two Cyanobacterial strains were selected for screening of their antimicrobial activity Oscillatoria laete‐virens and Lyngbya martensiana from Samuthiram Lake Thanjavur, Tamilnadu, India. The algal species were maintained in BG11 medium at a temperature [3, 4, 5, 6, 7 &8] subcultured in BG11 medium [9] of 25 ± 1°C under continuous illumination (2500 lu for 60 μmol.m‐ 2s‐1) for 12 days. Cyanobacterial cells were collected by centrifugation in the exponential growth phase (after 12 days), washed with distilled water and the pellets were dried at room temperature. Chemical composition (GC‐MS analysis) The Gas chromatography mass spectrometry (GC‐ MS) analysis of the samples isolated from the ethanolic extract of such as Oscillatoria laete‐virens and Lyngbya martensiana was performed using a Shimzdu Mass Spectrometer‐2010 series system (AIRF, JNU, New Delhi) equipped with a AB inno‐wax column (60 m × 0.25 mm id, film thickness 0.25 μm). For GC‐MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as a carrier gas at a flow rate of 1.2 ml per minute. Injector and mass transfer line temperature were set at 270 and 280°C. The compounds were identified by comparing them with the standards, or the mass spectra were matched with the in built library (NIST/Wiley). Molecular Docking The various oral pathogenic microorganisms Candida albicans and Streptococcus mutans were selected for the study: In the above organisms, pathogenic protein, causing oral candidiasis, such as SAP and SpaP was selected for the present docking study for finding the best drug to be administered for the oral candidiasis. For the present protein‐ligand docking the following drugs were identified to act as ligands formic acid, nerolidal, octanol and nonanoic acid. The PDB (Protein Data Bank) is the single worldwide archive of Structural data of Biological macromolecules, established in Brookhaven National Laboratories (BNL) in 1971 6. It
contains Structural information of the macromolecules determined by X‐ray crystallographic, NMR methods etc. http:// www.ncbi. lm.nih. gov / PubMed. These specialized databases are funded by the National Institute of Dental and Craniofacial Research (NIDCR) within the National Institutes of Health, Bethesda Maryland (http://www.oralgen.lanl.gov/).
Hex is an interactive protein docking and molecular superposition program, written by Dave Ritchie Molecular docking is a study of how two or more molecular structures, for example drug and enzyme or receptor of protein, fit together Molecular docking softwares are mainly used in drug research industry. The most interesting case is the protein‐ligand interaction, because of its applications in medicine. Ligand is a small molecule, which interacts with protein's binding sites. The Protein‐Ligand interaction plays a significant role in structural based drug designing. In our research work we have taken the SAP (Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis) and SpaP (spaP gene of Streptococcus mutans in dental plaque and its relationship with early childhood caries) receptor and the cyanobacterial bioactive compounds using as drugs against oral candidiasis. The receptor was docked to the above said drugs and the energy value obtained using the HEX docking software. Results The GC‐MS spectral results and comparison of results with library search successfully enabled the identification of the thirty compounds. The structures of all compounds were identified and detail given in (Table 1& Fig‐ 1, 2) Among the 30 compounds 8 compounds such very essential in order to bioactive compound with refered to molecular docking. The two organisms Candida albicans and Streptococcus mutans caused oral candidiasis, a disease encompassing a large of inflammatory and destructive conditions of the tissue surrounding the teeth. These bacteria after colonizing around the teeth in the gingival crevice , tissue damage has taken place by a combination of direct action of toxic bacterial products that include endotoxins and hydrolytic enzymes.
V. Madhumathi and S. Vijayakumar / Int J Med Biosci. 2013; 2(1): 09 ‐ 15 11 S. No 1 2. 3. 4. 5 6. 7 8 9 10 11 12 13 14 15 1 2. 3. 4. 5 6. 7 8 9 10 11 12 13 14 15
Compound name Oscillatoria laete‐virens Ethanoic acid Formic acid Octanal 3,7,11‐Trimethyl‐1,6,10‐dodecatrien‐3‐ol Bicyclo[10.1.0]tridecane Heptanol Nanonal Octadecanol 1,1‐Diethoxypropanal (Z)‐1,7‐Octadadiene 1‐Undecanol (CAS) 1‐Tetradecanol (CAS) 1‐Tridecene (CAS) Cyclohexyl 4‐cyanobutanoate 1‐Heptadecene (CAS) Lyngbya martensiana 1‐Heptanol Formic acid Ethaneloic acid Dihydrodiplodialide D3‐methyNitrate Cyclododecylhydroperoxide 1‐Dodecanol Octadecanol 2‐Octenal, (E)‐ (CAS) 6‐Iodo‐1‐heptene 1‐Nonadecanol Neopentyl 2‐oxobutanoate Heptane (CAS) 1‐Hexadecanol (CAS) NANONIC ACID
Retention time(min)
Area (%)
45.1 3.44 10.09 12.6 3.04 6.00 6.00 17.88 6.62 9.29 13.78 17.88 17.88 24.20 26.76
24.12 24.12 0.66 2.44 2.44 1.12 1.12 1.07 0.32 0.34 1.12 1.76 1.76 0.34 1.89
9.29 6.24 13.68 9.24 3.04 10.7 6.00 9.29 10.07 10.07 17.86 21.10 21.10 23.30 15.33
9.24 24.12 0.38 0.81 13.68 0.85 0.81 0.68 0.86 0.86 2.35 6.05 0.65 2.03 2.56
Table.1. GC‐MS Analysis of the components in ethanolic extracts of cyanobacteria
Fig.1. GC‐MS Chromatogaram of ethanolic extract of cyanobacteria Oscillatoria laete‐virens
V. Madhumathi and S. Vijayakumar / Int J Med Biosci. 2013; 2(1): 09 ‐ 15 12 Enzymes play an important role in disease making. Enzymes are the functional proteins, which is controlled by small molecules called ligands. These small molecules act as switches to turn on or off of the protein to function. The Protein‐Ligand interactions play a significant role in the identification of suitable drugs for suitable disease to the pathogens. Computational biology and bioinformatics have the potential not only for speeding up the drug discovery process, but also of changing the way drugs are designed. It involves variety of methods to identify novel compounds. One such method is docking of the drug molecule with receptor in the protein. Docking in process by which two molecules fit together in 3D space. To perform docking software packages of various kinds have been used. One such software packaged is here. In this investigation Hex is used for docking of drug molecules with the receptor molecules of SAP and SpaP. Molecular docking is like a “lock‐and‐key”, where one is interested in finding the correct relative orientation of the “key” which will open up the “lock”. In the present study the proteins SAP and SpaP is the lock and the ligand (Drugs) is the key.
The ligand molecule is the smaller one. In this study the drug molecules are the ligands. In the docking process the SAP and SpaP molecules is larger size while the ligand drug molecule is very smaller. The focus of molecular docking is to computationally simulate the molecular recognition process. The aim of molecular docking is to achieve an optimized conformation for both the protein and ligand and relative orientation between protein and ligand such that the free energy of the overall system is minimized. Docking results tabulated between the SAP and SpaP of Candida albicans and Streptococcus mutans with the drugs. Initially the SAP receptor of Candida albicans was docked with the drug molecule, formic acid. The e‐ value obtained was ‐64.91. Then the same drug molecule was docked with SpaP of Streptococcus mutans the e‐ value obtained – 50.57. Among the four drugs above energy values given, the drug nerolidal showed better energy value than that of other three drugs. The SAP receptor molecule of Candida albicans was docked with the above three drugs to identify the better drug. Among the four, nerolidal showed high energy value (‐ 80.37) than that of other three drugs. In the case of Streptococcus mutans, the e‐value was ‐56.57 (Table‐2& Fig‐3, 4).
Fig.2. GC‐MS Chromatogaram of ethanolic extract of cyanobacteria‐ Lyngbya martensiana
V. Madhumathi and S. Vijayakumar / Int J Med Biosci. 2013; 2(1): 09 ‐ 15 13 S.NO
E‐Value
Nerolidal Octonal
‐73.74 ‐78.99
Nanonic acid
‐80.37
Formic acid
‐55.63
nerolidal
‐ 56.57
Octonal
‐51.77
Nanonic acid
‐50.57
Streptococcus mutans
SAP
Bio active compound
‐ 64.91
2.
Candida albicans
Pathogenic enzyme
Formic acid
1.
Pathogenic Organism
SpaP
Table. 2. HEX e‐ Value of Bioactive pathogenic enzyme interaction
Fig. 3. Bioactive Pathogenic Enzyme Interaction. SAP – Nanonic acid interaction
Fig.4. SpaP – Nerolidal interaction
V. Madhumathi and S. Vijayakumar / Int J Med Biosci. 2013; 2(1): 09 ‐ 15 14 Discussion The antimicrobial activity of algal compounds extracted from algae depends upon the type of solvent used for extraction. The present study revealed that the use of organic solvents in the preparation of algal extracts provide more consistent antimicrobial activity. This observation clearly indicates that the polarity of antimicrobial compounds make them more readily extracted by organic solvents and using organic solvent does not negatively affect their bioactivity against antibacterial and antifungal species. Many investigations mentioned that the methanol extracts of Nostoc muscorum revealed antibacterial activity on Sclerotinia sclerotiorum. Also the methanolic extract of a blue green algae has been investigated for in vitro antimicrobial activity against Proteus vulgaris, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Aspergillus flavus and Rhizopus nigricans using agar cup diffusion method. The antimicrobial activity of methanolic extract of S. platensis was also explained due to the presence of γ‐ Linolenic acid and compound was also present in the methanol extract in the present study as observed by GC‐MS analysis. Previous publications reported that the compounds such as formic acid, nerolidal, octanol and nonanoic acid were found in both algae and plants show anticancer, antioxidant and antimicrobial activity [10, 11].
Docking plays an important role in the rational design of drugs. Therefore the molecular docking is biologically and pharmaceutically gains significance. A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. Docking is most innovative method used in the field of drug design. Most drugs are small organic molecules, and also docking is possible with these molecules. In the present study, docking analysis of Formic acid, nerolidal, Octonal and Nanonic acid was carried by HEX In this way the pharmacophoric part of the drug was partially identified. Acknowledgement The authors are thankful to the management of A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for providing them necessary facilities and support to carry out this work.
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