In Vitro Assessment of Antibacterial Activity of lamatecara Preservatives Abstract: The objective of the current research was to evaluate the efficacy of different preservatives of paracetamol syrup against five bacterial clinical isolates including gram positive (Staphylococci aureus,streptococcus and Bacillus subtilis), gram negative (E. coli, ) and Candida albicans (opportunistic pathogen) using agar diffusion methods. Three different preservatives benzoic acid, sodium benzoate and ascorbic acid were used. The results revealed that 0.3% Benzoic acid and Sodium benzoate were found to be active against, S. aureus, Bacillus subtilis, Streptococcus and E. Coli with inhibition zones ranging between 10 mm to 15mm followed by 0.2% concentration (10 mm to 12mm). Ascorbic acid was active against E.coli (15mm ) and was partially inhibitory against Streptococcus sp. (6 mm). None of the preservatives had any effect on Candida albicans. The preservative's minimum inhibitory concentration ranging from 4 to 0.002 % (v/v) was determined. Key words: Paracetamol, antibacterial activity, chemical preservative, zone of inhibition. Introduction: Paracetamol (acetaminophen), is mostly used as an antipyretic & analgesic agent. It is one of the main medication in cold and flu. It is also used in combination with other NSAIDS in the treatment of severe pain in pregnant women (4).Paracetamol syrups are readily accepted by all ages. The USP states that syrups may contain preservatives to prevent microbial contamination (5).High sugar pediatric syrups have been formulated to ensure efficiency, palatabilty microbial stability, & acceptability to infants. The formulation of such products has necessitated the incorporation of wide variety of ingredients in a complex physicochemical state,thus offering considering potential
for microbial extensive growth in the products. Some of the formulation ingredients in pediatric syrups include sweetening agent,suspending, thickening agents & preservatives to prevent spoilage (1). Different types of preservatives could be added, one of the most common is the antimicrobial. Adding different antimicrobial preservatives to the formulations is to prevent contamination by microorganisms that may be introduced during their production as well as to limit microbial contamination during normal conditions of storage and use, particularly for multidose containers (2). Thus, the main objective of this research was the assessment of the preserving efficacy of paracetamol syrup to five strains. Materials and Methods: Strains: Five strains ( Staphylococci aureus, Streptococcus sp., Bacillus subtilis,e. coli and Candida albicans) were clinical isolates at Jazan General Hospital. Samples were cultured on nutrient agar and incubated at 37 C for 24h for all strains except for Candida albicans at 28 C.
Preparation of Paracetamol: Ingredients Quantity for 100 ml Quantity for 100 ml Paracetamol 2.5 % 2.5 G Propyleneglycol 20% 20.0mL Flavoured syrup 70 % 70 ml Water up to 100mL 100mL Procedure Heat propyleneglycol at 50 C and add Paracetamol in it. Stir the solution for 30 minutes. Transparent solution will be obtained. Weigh accurately sucrose. Add sucrose in hot water 65 C under continuous stirring till it dissolved.after that transfer flavoured syrup to a beaker, while stirring in a homogeniser add the paracetamol solution. Add water until it reaches 100 ml then transfer to a clean container (British Pharmacopoiea). Screening for antimicrobial activity using agar well diffusion method: 100 µl of each organism were inoculated on Muller Hinton agar plates. A sterile cork borer (5mm) was used to make ditches in each plate 100µl volume of paracetamol with different chemical preservative(0.1, 0.2, & 0.3%concentration) was dispensed into each ditch. The prepared plates were kept at 25 o C for ten minutes allowing the preservative diffusion. The Muller Hinton plates were incubated at 37 C for a day and then checked for antibacterial activity by determining the zone of inhibition surrounding the well, expressed in millimeters.
MIC and MBC determination MIC stands for minimum inhibitory concentration, is the least concentration of an antimicrobial that will inhibit the visible microorganism's growth after overnight incubation. MBC stands for minimum bactericidal concentration, is the least concentration of antimicrobial that will prevent the growth of a microorganism after the subculture on a media free from preservatives.. MIC and MBC of benzoic acid were determined by macrodilution agar method. Macrodilution agar method: In a macrodilution agar method, two fold serial dilution of paracetamol syrup containing the preservative was prepared in 8 sterile distilled water tubes.the concentrations in percentage were ranging from 4-.002 % (v/v). 5mm diameter sterile cork borer was used to make wells in a presolidified Muller Hinton agar and 100µl volume of each dilution was added asceptically into the wells seeded with the standardized inoculum.100µl distilled water introduced into the well in place of the preservative was used as a control. All the test plates were incubated at 37 C and checked for growth after a day. The lowest concentration of the preservative showing a clear zone of inhibition was considered as MIC.
Results: Table 1. Efficacy assessment of five strains concentrations : to different preservatives Preservative % Preservative Concentration S.aureus B.subtilis Strepto- coccus sp. E.coli Candida albicans Benzoic acid 0.1 ++ ++ + ++ +++ 0.2 ++ + + + +++ 0.3 + ++ + + +++ Soduim benzoate 0.1 ++ ++ + + +++ 0.2 + + + + +++ 0.3 + + + + +++ Ascorbic acid 0.1 + ++ ++ + +++ 0.2 + ++ + + +++ 0.3 + ++ + ++ +++ (resistant: +++, intermediate: ++, susceptible: +)
Benzoic acid Sodium benzoate Ascorbic acid Staphylococcus aureus Benzoic acid Sodium benzoate Ascorbic acid Bacillus subtilis Benzoic acid Sodium benzoate Ascorbic acid Streptococcus sp. Benzoic acid Sodium benzoate Ascorbic acid Escherichia coli Fig1: Shows the preservatives inhibitory effect as zone of inhibition on the growth of gram-positive and gram-negative organisms.
Table 1 and Fig1 show the inhibitory effect as well as zone of inhibition of the different preservatives on the growth of gram-positive and gram-negative organisms. All preservatives studied showed an inhibitory effect on Staphylococcus aureus, Bacillus subtilis, Streptococcus sp., E. coli however Candida albicans appeared resistant towards all preservatives tested. Fig 2 : Effect of paracetamol syrup containing different concentration of benzoic acid on different microorganisms. Fig 3: Effect of paracetamol syrup containing different concentration of sodium benzoate preservative on different microorganisms.
Fig 4: Effect of paracetamol syrup containing different concentration of ascorbic acid preservative on different microorganisms. Regarding the previous figures conclusions were drawn that Streptococcus sp. had the best responds towards benzoic acid, while E.coli, S.aureus and Bacillus subtilis had approximately similar susceptibility.moreover, E.coli shows the most susceptibility towards paracetamol syrup regarding both sodium benzoate and ascorbic acid preservatives followed by Streptococcus sp. Fig 5: MIC and MBC of benzoic acid chemical preservative.
The in vitro MIC of benzoic acid that showed the best antimicrobial activity of all the tested preservatives, was tested against the four chosen bacteria using macrodilution agar plate method (Fig5). The MIC values of benzoic acid ranged from 1 to 4 % (v/v) against Staphylococcus aureus,bacillus subtilis, Streptococcus sp.and E.coli. In the same sense E. coli was found to be the most sensitive which survived only up to 1% concentration of the benzoic acid. MBC equaled the MIC of benzoic acid. Discussion: The use of weak organic acids as preservatives have been well reported for years, to control microbial growth, improve sensory attributes and extend the shelf life (3). The outcome of this study reveals that all preservatives studied demonstrated an inhibitory effect on Staphylococcus aureus, Bacillus subtilis, Streptococcus sp., E. coli however Candida albicans appeared resistant towards all preservatives tested (Table1&Fig1).Added that Streptococcus sp. and E.coli were the most sensitive organisms towards all preservatives ( Fig 2,3&4). As regard minimum inhibitory concentration E. coli was found to be the most sensitive which survived only up to 1% concentration of the benzoic acid. Conclusion: From the research work done it was concluded that benzoic acid,sodium benzoate and ascorbic acid can be used as antimicrobials against Gram positive and Gram negative. Furthermore increasing the concentration of preservative had no effect on the antimicrobial activity.
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Acknowledgement: First and foremost, we would like to thank our supervisor of this project, Prof Hanaa Elshafei, Mrs.Walaa eldakroury and Miss Farah Fakhrul Islam for their valuable guidance and advice. The authors would like to express their deepest gratitude and cordial appreciation to Dr.Mohamed El-Safhi Dean of the College of Pharmacy, Jazan University for his continous encouragement and generous attitude. The authors would also like to thank to Dr B.B.Barik head of the pharmaceutics department.