INTRODUCTION

Microorganisms are present in the air that we breathe, the food that we eat and the water we drink and medicines we use unless specific measures are adopted to exclude them (Aulton, 2002). Although, some types of pharmaceutical products for example ophthalmic and injectable preparations are sterilized by physical methods (autoclaving for 20 min at 15 pounds pressure and 121°C, dry heat at 180°C or bacterial filtration) many of them also require an antimicrobial preservative to maintain their aseptic condition throughout storage and use. Other types of preparations those are not sterilized during their preparation but are particularly susceptible to microbial growth. Natures of their ingredients are protected by the addition of antimicrobial preservative. Preparations that provide excellent growth media for microbes are most aqueous preparations such as syrups (Allen et al., 2005).

Syrups are concentrated solutions of a sugar such as sucrose in water or other aqueous liquids. Flavored and medicated syrups are the preferred dosage form of choice for both children and adults because they contain no or very little alcohol. Patients frequently use multidose syrups and most of the syrups are sold over the counter since the active ingredients are usually not potent, for this reason manufacturers add preservatives to prevent accidental contamination to opened bottles. Preservatives are widely employed in the cosmetics and pharmaceutical industries as well as in a variety of other manufacturing industries (Hugo and Russells, 2005). The preservative system includes both ingredients with known antimicrobial activity and ingredients formula that may contribute directly or indirectly to antimicrobial activity (Spiegeleer et al., 2006). Antimicrobial preservatives work by reducing the numbers of organisms and inhibiting the growth of microorganisms that may be introduced during repeated use accidently (Rosenthall et al., 2006). The amount of a preservative varies with the proportion of water available for growth, the nature and inherent preservative activity of some formulative materials and the capability of the preservative itself. Among the preservatives commonly used in syrups with the usually effective concentrations are benzoic acid 0.1-0.2%, sodium benzoate 0.1-0.2% and various combinations of methylparabens, propylparabens and butylparabens. Frequently alcohols are used in syrups to dissolve alcohol-soluble ingredients but it could act as preservative if is concentration exceeds 15% (Aulton, 2002). The activity of pure preservative must be evaluated using appropriate in vitro test, in addition to that other sort of challenge test involving final product is required to be done for determination of the preservative efficacy (Hugo and Russells, 2005). The final product is deliberately inoculated with a suitable microorganisms which may be fungal e.g., Candida or bacterial e.g., Staphylococcus aureus, Escherichia coli and Pseudomonas aeroginosa. (Hugo and Russells, 2005). The level of contamination is then monitored at several time intervals (Hugo and Russells, 2005; Spiegeleer et al., 2006; Rosenthall et al., 2006; Cremieux et al., 2005; Souza and Ohara, 2003).

The aim of this study is to evaluate different preservatives (efficacy of preservatives) in different cough and expectorant syrups manufactured by different pharmaceutical companies.

MATERIAL AND METHODS

The methodology of this study was familiar to that followed by Wachowski et al. (1999) and Crowther et al. (1996). The experimental study has been done during 3 months, April-June 2009. It was conducted at the Department of Biotechnology, Faculty of Science at Philadelphia University. Overnight cultures of S. aureus (ATCC 259231), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) and C. albicans (ATCC 14053) (gifted by Dr. A. Shehabi, faculty of medicine, Jordan University) were diluted to a density of 0.5 McFarland units with 0.9% sterile nonbacteriostatic saline using spectrophotometer (Cecil, England). Each organism solution was further diluted 1:50 with sterile 0.9% saline. Aliquots of 0.2 mL of each diluted organism were then added to sterile sealed culture vials containing 20 mL of the following solutions: three vials of syrup (A) A (glycerol and propylene glycol); three vials of syrup (B) (propylene glycol and glycerin); three vials of syrup (C) (glycerin, propylene glycol and butyl paraben); three vials of syrup D (methyl paraben and probylparaben) and three vials of 0.9% nonbacteriostatic saline as a control. Each organism solution was vortexed before addition to the 20 mL vials. After the organisms were added, each vial was vortexed for 1 min and subplated to three plates of Trypticase Soy Agar (TSA). Vials were subplated out at 0, 3, 6, 12, 24 and 48 h intervals for a total of nine plates per solution per sampling period and stored at 20°C between samplings. The plates were then incubated at 37°C for 24 h. Each plated medium was read and numbers of Colony Forming Units (CFUs) were counted and recorded using colony counter (Galaxy 230, USA). For each microorganism, the number of CFUs per plate was averaged for each studied period. Data are presented as the mean of nine replicate assays. A probability of p = 0.05 was taken to indicate statistical significance.