SCREENING OF ANTIMICROBIAL PROPERTIES OF PHENOL SCHIFF BASESHTML Full Text
SCREENING OF ANTIMICROBIAL PROPERTIES OF PHENOL SCHIFF BASES
N. A. Ghanwate*1, A. W. Raut 2 and N. N. Vidhale 3
Department of Microbiology 1, Sant Gadge Baba Amravati University, Amravati - 444602, Maharashtra, India.
Department of Chemistry 2, Department of Microbiology 3, Shri Shivaji Science College, Amravati - 444603, Maharashtra, India.
ABSTRACT: In their fight for supremacy and survival, microorganisms have been developing and devising various methods to inactivate the action of antibiotics. The numbers of drug resistant microorganisms with reduced susceptibility to various antibiotics are increasing. Therefore, new infection-fighting strategies are required to control microbial infections. The interaction between potent chemicals and living system contribute to the understanding of life processes and provide effective methods for treatment, prevention and diagnosis of many diseases. Phenol Schiff bases like chalcone imines and flavone imines were screened for their antimicrobial activity against a wide range of pathogenic bacteria and fungi. The in vitro activity of the compounds was evaluated by the Kirby-Bauer disc diffusion method and the MIC was determined for a few selected compounds. Most of the compounds showed significant antimicrobial activity. The compounds were found toxic to bacteria like Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, S. typhimurium, Shigella flexneri and Proteus species. Pseudomonas aeruginosa was found to be resistant to most of the compounds. It was noted that all the compounds were active against fungi like Cryptococcus neoformans, Candida albicans, Trichophyton mentagrophytes, Microsporum gypseum, Mucor and Rhizopus but inactive towards Aspergillus niger, Asp. flavus and Asp. fumigatus. It was interesting to note that the compounds were more fungi toxic than antibacterial. Flavone imines showed more antimicrobial activity than chalcone imines.
Phenol Schiff bases, Antimicrobial Activity, Chalcone imines, Flavone imines
INTRODUCTION: Schiff bases have attracted much more attention due to their biochemical and biological activities 1 such as antiviral 2, anticancer 3, 4, antimicrobial5-8 and antibacterial9,10. Anticancer Schiff bases have been synthesized by conden-sation of aniline with substituted benzaldehyde 11. Schiff bases like chalcone imines have been reported to exhibit antimicrobial properties 12, 13.
Preparation of 2’-hydroxy substituted 14-16 chalcone imine has been reported. Synthesis of 2'-hydroxy- 3'- Nitro- 5- chloro- 4- substituted- N-(substituted phenyl) chalcone imine have been reported 17. 2-Hydroxy chalcones and 2-hydroxy substituted chalcone are prepared by known methods 18.
Chalcone condenses with substituted aniline in ethanol in presence of 2, 3 drops of concentrated H2SO4 to give chalcone imine. Chalcone imine reacts with DMSO-I2-H2SO4 system to give flavone imine. DMSO-I2 with or without H2SO4 reagent has been used for oxidative cyclization of 2-hydroxy chalcone to flavones and dehydrogenation to flavonoids 19. Phenol Schiff bases with DMSO-I2-H2SO4 system give N-phenyl benzisoxazolines 20-22. Synthesis of 8-nitro-5-chloro-4-substituted-N-(substituted phenyl) flavone imine has been reported 23.
The synthesized Schiff bases were screened for their antimicrobial activity against bacteria like E. coli, Klebsiella pneumoniae, Salmonella typhi, Salmonella typhimurium, Shigella flexneri, Proteus sp., Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Aerobacter aerogenes, Staphylococus aureus and Bacillus sp. 24, 25 and fungal isolates like Cryptococus neoformans, Candida albicans, Trichophyton mentagrophytes, Microsporum gypseum, Mucor, Rhizopus, Aspergillus niger, Aspergillus flavus and Aspergillus fumigates 26 by disc diffusion method 27 by dissolving the compounds in methanol 28. Those Schiff bases with good antimicrobial activity were further evaluated for their minimum inhibitory concentration.
MATERIAL AND METHODS: 2'-Hydroxy-3'-bromo-5'-chloro-4-methoxy chalcone condenses with p-toludene in ethanol in presence of 2,3 drops of conc. H2SO4 gives 2'hydroxy-3'-bromo-5'-chloro-4-methoxy-N-(para tolyl) chalcone imine m.p. 146 °C, yield 72%.
The following compounds were synthesized by known methods 29-31.
IIA: 2- HYDROXY- 3- BROMO- 5- CHLORO- 1- (α-PARAMETHYL PHENYL-IMINO) ETHYL BENZENE
IIB: 7- BROMO- 5- CHLORO- 3- METHYL- 2- (PARA TOLYL) BENZISOXAZOLINE
TABLE 1: THE FOLLOWING CHALCONE IMINES WERE SYNTHESIZED
|Sr. No.||Compound No.||R1||R2||R3||R4||m.p. (°C)||Yield (%)|
Substituted chalcone imines were dissolved in DMSO (40ml) and conc. H2SO4, 2, 3 drops was added. The mixture was refluxed for 10 min. It was then cooled and little catalytic amount of iodine was added. The reaction mixture was again heated for 1 hour in water bath then cooled and diluted with cold water. The resulting solid mass was treated with water 10% Sodium thiosulphate solution to remove iodine and again by water and crystallized from alcohol acetic acid mixture to get flavone imine.
TABLE 2: THE FOLLOWING FLAVONE IMINES WERE SYNTHESIZED
|Sr. No.||Compound No.||R1||R2||R3||R4||m.p. (°C)||Yield (%)|
The compounds were tested in vitro for their antimicrobial activity by disc diffusion method.
Bacterial and Fungal Culture: Twelve pure bacterial cultures and eight pure fungal isolates were selected for the study. The cultures were procured from the Department of Microbiology, Government Medical College, Nagpur. The culture were confirmed by the conventional techniques 24-26 and maintained by sub culturing on semisolid nutrient agar and stored at 4-8 °C. NCTC strains of S. aureus, E. coli and Pseudomonas aeruginosa were used as control strains.
TABLE 3: MICROORGANISMS
|Gram Negative||Gram Positive||Yeast||Yeast like Fungi||Moulds|
|Escherichia coli NCTC 10418||Staphylococcus aureus NCTC 6751||Cryptococcus neoformans ATCC 14116||Candida albicans ATCC 10231||Trichophyton mentagrophytes ATCC 9533|
|Salmonella typhi||Bacillus species||-||-||Microsporum gypseum|
|Salmonella thyphimurium ATCC 51812||-||-||-||Mucor|
|Shigella flexneri ATCC 9199||-||-||-||Aspergillus niger ATCC 16404|
|Klebsiella pneumoniae ATCC 10031||-||-||-||Aspergillus flavus|
|Aerobacter aerogenes||-||-||-||Aspergillus fumigatus|
|Proteus mirabilis ATCC 12453||-||-||-||-|
|Proteus vulgaris ATCC 33420||-||-||-||-|
|Pseudomonas NCTC 10662||-||-||-||-|
Antibiotic Susceptibility Testing: The compounds were tested in vitro for their antimicrobial activity by Kirby Bauer disc diffusion method 21.
Preparation of Discs: Wet discs were used 6.25 mm diameter discs were punched from No. 1 Whatman filter paper and dispensed in batches of 100 in screw-capped bottles and sterilized by dry heat at 140 °C for 60 minutes. The selected compounds were dissolved in methanol 22 and their solutions were prepared such that 1 ml contains 100 times the amount of antibiotic required in the disc. 1ml of this solution was added to each bottle of 100 discs and as the whole of this volume was absorbed, it was assumed that each disc will contain approximately 0.01ml of the solution.
Culture Medium: The culture medium used was the HI-media India (make) Nutrient Agar for bacterial cultures and Sabouraud Dextrose Agar for fungal isolates.
Preparation of Inoculum: A small loopful (about 0.01 ml) of an overnight broth culture (about 10g bacteria/ml) was added to 5ml sterile isotonic saline to a concentration of 105-106 bacteria per ml.
Test Procedure: The culture plates were dried in the incubator with the lid ajar until its surface was free from moisture. 2ml of the inoculum suspension was transferred to the plate with a sterile Pasteur pipette and the plate was rotated to wet the whole of its surface. The excess fluid was removed with the pipette and the plates were dried in the incubator for 30 minutes. The compound discs were applied (2cm apart) to the surface of the plate with sterile fine pointed forceps and gently pressed to ensure full contact with the medium.
Immediately the plates were transferred to the incubator and incubated at 37 °C for 24 hours for bacteria and at 28 °C for 72 hours for fungi.
Reading Results: After incubation degree of sensitivity to compounds was determined by measuring the clear visible areas of growth free zones (zones of inhibition) produced by diffusion of compounds in the media. Zones of inhibition measured (including 6.25 mm of disc diameter) by calipers were reported in mm as shown in Table 4 and 5.
TABLE 4: BACTERIA SHOWING SENSITIVITY TO THE COMPOUNDS (SENSITIVITY MEASURED AS ZONE OF INHIBITION IN mm)
|Compd.||Escherichia coli||Klebisellan pneumoniae||Salmonella typhi||Salmonella typhimurium||Shigella flexneri||Proteus spp.||Proteus merabilis||Proteus vulgaris||Pseudomonas aeruginosa||Aerobacter aerogenes||Staphylococcus aureus||Bacillus spp.|
TABLE 5: FUNGI SHOWING SENSITIVITY TO THE COMPOUNDS (SENSITIVITY MEASURED IN mm)
|Compd.||Cryptococcus neoformans||Candida albicans||Trichophyton mentagrophytes||Microsporum gypseum||Mucor||Rhizopus||Aspergillus niger||Aspergillus flavus||Aspergillus fumigatus|
Evaluation of Minimum Inhibitory Concentration (MIC) of a Few Selected Compounds: After screening of 14 compounds, 3 compounds with good antimicrobial activity were selected for evaluation of their MIC values. The following compounds were selected-
- IIa: 2-Hydroxy-3-bromo- 5- chloro-1- (α-para methyl phenyl imino) ethyl benzene.
- IIIi: 2-Hydroxy-5-chloro-4-methoxy- N- (ortho nitro phenyl) chalcone imine.
- IVf: 6-Chloro-4-methoxy-4-ortho nitro phenyl imino flavone.
The MIC was determined by disc diffusion method 21 against the different bacteria stated in Table 1.
Disc with Different Concentration of Compounds: Stock solutions of the selected 3 compounds were prepared in methanol, each with a concentration of 10,000 µg/ml. Different concentrations were prepared from the stock solution. One ml from each concentration was added to 100 sterile discs. The final concentration per disc was 100, 80, 60, 40, 20 and 10µg. Culture medium, inoculum, test procedure and reading of results were carried out as described above.
Determination of MIC: The MIC of the compounds for a bacterial isolate was estimated from measurement of the zone of inhibition in the disc test by reference to a standard graph which related the MIC to the zone diameter for the control strain of known degree of sensitivity. Discs containing different amounts of the compound were tested under carefully standardized conditions against the following control strains.
- Escherichia coli NCTC 10418.
- Pseudomonas aeruginosa NCTC 10662.
- Staphylococcus aureus NCTC 6571.
Initially the minimum concentration of E. coli NCTC 10418, Pseudomonas aeruginosa NCTC 10662 and Staphylococcus aureus NCTC 6571 was determined towards the 3 selected compounds (IIa, IIIi and IVf). MIC of the compounds was determined by incorporating the compound in the nutrient agar in the concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140µg/ml. The inoculum of the control strain was prepared as in case of disc diffusion method and 1-2ml of the inoculum was applied on the agar surface. The plates were incubated at 37 °C for 18-24 hours and results read. The lowest concentration of the compound at which there was no visible growth was taken as the minimum inhibitory concentration.
Later the test micro-organisms were tested under the same conditions. The concentration of compound required for the inhibition of growth (MIC) was calculated from the measurement of the diameter of the zone of inhibition by reference to the standard graph.
The degree of sensitivity of the test bacteria was calculated as follows.
MIC of =
|Amount of antibiotic per disc required to inhibit the test bacterium at a given size of zone||
x MIC of
|Test bacteria||Amount of antibiotic per disc required to inhibit the control strain at the same size of zone||control strain|
TABLE 6: MIC OF IIa, IIIi AND IVf AGAINST THE CONTROL STRAINS (µg/ml)
|Compound||E. coli NCTC 10418||Pseudomonas aeruginosa NCTC 10662||Staphylococcus aureus NCTC 6571|
TABLE 7: MIC (µg/ml) OF COMPOUNDS IIa, IIIi AND IVf FOR DIFFERENT TEST BACTERIA
|S. no||Test Bacteria||IIa||IIIi||IVf|
FIG. 1: MIC OF COMPOUNDS IIa, IIIiAND IVf FOR DIFFERENT TEST BACTERIA
DISCUSSION: From the results it is clear that flavone imines showed greater antimicrobial activity than chalcone imines. Antibacterial activity of chalcone imines is highest against Salmonella typhimurium, Proteus vulgaris and Shigella flexneri, moderate against E. coli, Klebsiella, Proteus mirabilis and Aerobacter. It is inactive against S. aureus. Synthesis and antibacterial activity of 2-hydroxy-5-bromo-N-(substituted phenyl) chalcone imine was also reported by Shubhangi et al., against S. aureus, E. coli, Pseudomonas aeruginosa and S. typhimurium 32. Synthesis, characterization and study of antimicrobial activity of 2- hydroxy 3-nitro-5-methyl-n-(substituted phenyl) chalcone imines was also reported by Borul and Agarkar in 2015 33 and Xianwen et al., in 2014 13. While the flavone imines are very active against S. aureus, Bacillus and Aerobacter though moderate to others.
The well-known fact that Pseudomonas and S. aureus show multiple drug resistance is also noted in this study. All fungi are sensitive to almost all the compound except the Aspergillus species which are found to be sensitive only to IVc and IVf. Here also the flavone imines were more active than the chalcone imines. Cytotoxic and antimicrobial activity of some synthetic flavones were studied and reported by Sohel et al., against selected bacterial and fungal strain 34. Antifungal activity of chalcone imines is highest against the dermatophytes and candida while flavone is more active against dermatophytes, mucor, Rhizopus and Aspergillus.
It was seen that the presence of -OCH3 group invariably increased the antimicrobial activity of these compounds. It is also found that compounds containing bromo and nitro group together are more active than simple one.
CONCLUSION: All the compounds except III d and IV d were found to exhibit strong antibacterial and antifungal properties. It was also concluded that flavones imines (compounds IV a and IV f) were more active than chalcone imines. As compared to other compounds, II a, III I and IV f showed the highest antimicrobial activity.
ACKNOWLEDGEMENT: The authors greatly acknowledge the Microbiology Department of Government Medical College, Nagpur, for providing the bacterial and fungal cultures for the present investigation.
CONFLICT OF INTEREST: The authors have no conflict of interest.
- Arulmurugan1 S, Helen PK and Venkatraman BR: Biological activities of schiff base and its complexes: A review. Rasayan Journal of Chemistry 2010; 3: 385-410.
- Das A, Trousdale MD, Ren S and Lien EJ: Inhibition of herpes simplex virus type 1 and adenovirus type 5 by heterocyclic Schiff bases of aminohydroxyguanidine tosylate. Antiviral Res 1999; 44: 201-208.
- Sengupta J: Indian J. App. Chem 1964; 2: 29.
- Modi JD, Sabnis SS and Deliwala EV: Journal of Molecular Chemistry1970; 13: 935.
- Ahmad Sabry Abu-Khadra1, Rabie SF, El-Dine A and Abdel-Hady M: Synthesis, Characterization and Antimicrobial Activity of Schiff Base (E)-N-(4-(2-Hydroxybenzylideneamino) Phenylsulfonyl) Acetamide Metal Complexes. American Journal of Analytical Chemistry 2016; 7: 233-245.
- Sikarwar P, Tomar S and Singh AP: Synthesis, Spectral Characterization and Antimicrobial Activity of Schiff Bases and their Mixed Ligand Metal Complexes of Co (II), Ni (II), Cu (II) and Zn (II). American Journal of Chemistry 2016, 6(5): 119-125.
- da Silva CM, da Silva DL, Modolo LV, Rosemeire BA, de Resende MA, Martins CVB and Aˆ ngelo de Fa´ tima: Schiff bases: A short review of their antimicrobial activities. Journal of Advanced Research 2011; 2: 1–8.
- Ashraf MA, Mahmood K and Wajid A: Synthesis, Characterization and Biological Activity of Schiff Bases. International Conference on Chemistry and Chemical Process IPCBEE 2011; 10.
- Debasis M: Antibacterial studies of the polymeric phenolic Schiff bases containing aminothiazole moiety. International Journal of Research in Pharmaceutical and Nano Sciences 2014; 3(3): 215 – 221.
- Demir MA, Özkalp B and Mercimek B: Synthesis and antibacterial activity of Schiff base derivatives. International Journal of Drug Development and Research 2010; 2(1): 102-107.
- Popp FD: Synthesis of potential anticancer agents. II. Some Schiff bases. Journal of Organic Chemistry 1960; 26: 1566.
- Raut AW, Doshi AG and Raghuwanshi PD: Oriental Journal of Chemistry 1998; 14(2): 337-338.
- Xianwen F, Bingqin Y, Zhao C, Pengfei Z and Meipan Y: Synthesis and antimicrobial activity of novel chalcone Derivatives. Res Chem Intermed 2014; 40: 1715–1725.
- Raut AW, Doshi AG and Raghuwanshi PD: Synthesis of some new Phenolic Azo Schiff Bases- Part I. Asian Journal of Chemistry 2000; 12(2): 619.
- Deshmukh AY, Raghuwanshi PD and Doshi AG: Synthesis of 4'-Nitro-N-(Substituted Phenyl) Chalcone Imine and 4'-Chloro-N (Substituted Phenyl) Chalcone Imine. Oriental Journal Chemistry 2002; 18(1): 101-104.
- Rajput ND: Ph.D. Thesis, "Reaction of para chloro-meta-cresol in the synthesis of O2 and N2 containing heterocycles". Amravati University, Amravati 2002; 128- 131.
- Rathi SR and Doshi AG: Synthesis of N-Substituted Phenyl Benzisoxazolines. Oriental Journal of Chemistry 2006; 22(1): 177-180.
- Doshi AG and Ghiya BJ: Improved synthesis of chalcones using pulverized potassium hydroxide and dimethyl-formamide. Current Science 1986; 55(10): 502-503.
- Doshi AG, Soni PA and Ghiya BJ: Oxidation of 2'-hydroxychalcones. Indian Journal of Chemistry 1986; 25B: 759.
- Lokhande PD and Ghiya BJ: Oxidation of Dihydroflavones. Journal of Indian Chemical Society 1991; 68: 412-413.
- Kadu VB and Doshi AG: Oriental Journal of Chemistry 1997; 13(3): 277-280.
- Raut AW, Doshi AG and Raguwanshi PB: Synthesis and Antimicrobial activity of chalcone imine and its cyclization by using DMSO-I~ 2-H~ 2SO~ 4 system. Oriental Journal of Chemistry 1998; 14(2): 363-364.
- Rathi SR: Ph.D. Thesis, S.G.B. Amravati University 2005.
- Banerjee MM: Essentials of Medical Microbiology, 1999; 1-2.
- Ananthanarayan R and Hayram Panikar CE: Text Book of Microbiology, Orient Longman, Fifth Edition 2002.
- Konemann EW and Roberts GD: Practical Laboratory Mycology, Third Edition 2001.
- Robert C, Duguid JP, Marimicon BP and Swain RHA: Medical Microbiology, Churchill living Store, Edinburgh, London, New York, Twelth Edition 1995; 11.
- Donald CG and William AR: In assay methods of Antibiotic in lab manual medical encyclopedia Inc. 1955.
- Ghanwate NA: Microbial and Biological effects of Phenol Schiff’s Bases. Ph.D. Thesis, S.G.B. Amravati University 2004.
- Ghanwate NA, Raut AW and Doshi AG: Synthesis and antimicrobial properties of flavone imines. Oriental Journal of Chemistry 2008; 24(2): 721-724.
- Ghanwate NA, Raut AW and Doshi AG: Synthesis and antimicrobial activity of chalcone imines. Oriental Journal of Chemistry 2008; 24(1): 733-736.
- Patil S, Utale P, Gholse S, Pande S and Thakur S: Synthesis, characterization and antimicrobial activity of 2-hydroxy-5-bromo-N-(substituted phenyl) chalcone imine. Asian Journal of Biochemical and Pharmaceutical Res. 2013; 2(3): 114-122.
- Borul SB and Agarkar SV: Synthesis, characterization and study of antimicrobial activity of 2- hydroxy 3-nitro-5-methyl-N-(substituted phenyl) chalcone imines. International Journal of Pharmaceutical Sciences and Research 2015; 6(8): 3424-3427.
- Sohel M, Sayed A and Azizul I: Cytotoxic and antimicrobial activities of some synthetic flavones. Indian Journal of Chemistry 2006; 45B: 1478-1486.
How to cite this article:
Ghanwate NA, Raut AW and Vidhale NN: Screening of antimicrobial properties of phenol schiff bases. Int J Pharm Sci Res 2017; 8(12): 5161-67.doi: 10.13040/IJPSR.0975-8232.8(12).5161-67.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
N. A. Ghanwate*, A. W. Raut and N. N. Vidhale
Department of Microbiology, Sant Gadge Baba Amravati University, Maharashtra, India.
12 April, 2017
17 June, 2017
29 June, 2017
01 December, 2017