ANTIMICROBIAL EFFICACY OF THE METHYLPARABEN AND BENZOATE SODIUM AGAINST SELECTED STANDARD MICROORGANISMS, CLINICAL AND ENVIRONMENTAL ISOLATES IN VITRO

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1 ANTIMICROBIAL EFFICACY OF THE METHYLPARABEN AND BENZOATE SODIUM AGAINST SELECTED STANDARD MICROORGANISMS, CLINICAL AND ENVIRONMENTAL ISOLATES IN VITRO Seyedeh Zahra Mirsonbol 1, *Khosro Issazadeh 1, Mohammad Reza Majid Khoshkholgh Pahlaviani 2 and Naeem Momeni 1 1 Department of Microbiology, College of Basic Sciences, Lahijan Branch, Islamic Azad University, Lahijan, Iran 2 Department of Biotechnology, College of Basic Sciences, Lahijan Branch, Islamic Azad University, Lahijan, Iran *Author for Correspondence ABSTRACT Pharmaceutical preparations are particularly susceptible to microbial growth because of the nature of their ingredients. Such preparations are protected by the addition of anti-microbial agents in the formulation to destroy and inhibit the growth of those microorganisms that may contaminate the product during manufacture or use. Amongst the most commonly used preservative in the conservation of liquid pharmaceutical preparation are sodium benzoate and Methyl hydroxy benzoate (Methylparaben). ρ- Hydroxybenzoic esters (parabens) have been used as preservative for over 70 years. Parabens are the most commonly used since they have synergistic effects. In this study, standard, clinical and environmental strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger as test microorganisms were used. The efficacy of Methylparaben and Sodium benzoate as preservatives in Dextromethorphan syrup and Phenylephrine nasal drop respectively demonstrated first by inoculating the products with proper cultures of test microorganisms and second by challenging prepare dilutions of above-mentioned preservatives with microorganism specified. Then, their growth inhibitory power was determined in terms of minimum inhibitory concentration (MIC) and OD level. Furthermore, the synergistic effects of two preservatives were measured against test microorganisms. Finally, statistical analysis of P value at 0.05 was carried out. The results showed that the antibacterial activity of Methylparaben was stronger than Sodium benzoate and the assessment of synergistic effects indicated that a combination of two preservatives may extend the spectrum of preservative system with potentiated antibacterial / antifungal properties rather than when each used alone. Environmental strains of Aspergillus niger were the most sensitive microorganisms against preservatives. Keywords: Methylparaben, Benzoate Sodium, Antimicrobial Properties, Environmental, Standard, Microorganism INTRODUCTION Preservatives have been commonly used as additives in pharmaceutical products, cosmetics and foods. Preservatives are mainly effective in controlling molds, inhibiting yeast growth and protecting against bacterial proliferation. Their antibacterial and antifungal properties make them an integral part of the product formulation (Chawki et al., 2009). Examination of the literature strongly indicates that preservative activity should be biocidal rather than biostatic and also emphasizes the necessity of establishing standards for preservative activity, such standards to include in requirements for the rate of kill or time to be able to sterilize specified standard infections from a product and a specification for the capacity of the preservative to deal with successive infections of the product. The chemical availability of a preservative in a product may alter during storage as a result of absorption by the package, in-use infection, or change in storage temperature and the microbiological significance of the loss may be estimated from a knowledge of the concentration exponent and/ or temperature coefficient of the preservative (Bean, 1972). Pharmaceutical preparations which need an aqueous vehicle such as syrups Copyright 2014 Centre for Info Bio Technology (CIBTech) 363

2 and powders for oral suspensions require safeguards from microbial contamination, which may affect product stability or infect the consumers. This is accomplished by the addition of anti-microbial agents in the formulation to destroy and inhibits the growth of those organisms that may contaminate the product during manufacture or use. Bacterial contamination of the products through consumers use has resulted in presence of mixed and harmful microbial flora in product (Kamble, 2011). The inhibitory effect of preservatives varies, depending on the concentration and type of preservative, ph of the medium and the species of microorganism (Restaino and Komatsu, 1981). Among the most commonly used preservatives in the conservation of liquid pharmaceutical preparations are sodium benzoate, potassium sorbate, and methyl hydroxybenzoate methyl paraben (Chawki, 2009). Therefore, preservatives are being added to the preparation to prolong their shelf life by preventing the microbial attack (Akpan et al., 2007; Chaudhary et al., 2008). The antimicrobial and antifungal properties of fatty acids have been studied extensively and the development and use of safe antimicrobial preservatives in pharmaceutical preparations continue to be of great interest to the pharmaceutical industry (Chaudhary et al., 2008). Sodium benzoate is the sodium salt of benzoic acid. One gram of the salt is soluble in 2 ml of water. A typical aqueous solution will be slightly alkaline and typically has a sweetish astringent taste. Sodium benzoate is a slightly hygroscopic, white, odorless or nearly odorless product. Sodium benzoate is a preservative that is widely used in the food industry. It is used as an antifungal agent to conserve margarine, fresh juices, and sweets (Restaino and Komatsu, 1981). Hydroxybenzoate (parabens) are alky esters of ρ-hydroxybenzoic acid with antibacterial and antifungal properties. While the antimicrobial activity increases with increasing alkyl chain length of the ester group, the aqueous solubility decreases, making the use of shorter chain esters more common because of their high solubility in water. The activity may also be improved by combining two hydroxybenzoates with short alkyl chains. Methylparaben (C 8 H 8 O 3, molecular mass g mol -1 ) is used alone or in combination with other parabens in some preparations, as they act as synergists (Restaino and Komatsu, 1981). MATERIALS AND METHODS First, under aseptic conditions, each of the above listed microorganisms, standard (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404),clinical and environmental strains, were become active and after being assured of their purification, subcultured and stored in the freezer at C for subsequent tests. Effects of Challenging Dextromethorphan Syrup and Phenylephrine Nasal Drop with Microbial Suspensions Fresh culture of each test microorganisms was diluted to a density of 0.5 Mc Farland units with 0.9% sterile saline. Each microorganism solution was further diluted 1:50 with sterile 0/9% saline. Then, 10 ml liquid of Dextromethorphan syrup and Phenylephrine nasal drop were challenged with 0.1ml of each prepared microbial suspensions. Inoculated test products are incubated at 37 0 C for 48 h and sampled to determined microorganism concentration at zero, 3/6/12/24 and 48 hour intervals. To ensure of growth or no growth of microorganisms, 1 ml of both contaminated products were plated in Nutrient Agar for bacteria and Sabouraud Dextrose Agar for fungi. Determination of Microbial Inhibitory Concentration using Methylparaben and Sodium Benzoate At this stage, dilutions of preservatives were prepared. While distilled water was used as solvent for Sodium benzoate, Methylparaben was dissolved in alcohol. To avoid the effects of alcohol on microorganism during test process, an equal volume of a polar solvent, Then, 0.1 ml of microbial suspension with a turbidity equivalent to 0.5 Mc Farland units was added to the preservatives dilution prepared. Inoculated dilution was incubated at 37 0 C for 24h for bacteria and 48 to 72h at room temperature ( C) for fungi. Afterward, optical density of those dilutions that showed no growth was read using a spectrophotometer at wavelength of 600nm. Finally MIC relevant to each of dilution was calculated statistical analysis P value=0.05. To assess the synergistic effects of preservatives, equal Copyright 2014 Centre for Info Bio Technology (CIBTech) 364

3 volumes of Methylparaben and Sodium benzoate were combined. Then, 10-1 to dilutions of this combination were prepared. Each dilution was challenged with 0.1ml of microbial suspension. Inoculated dilutions with bacterial suspensions were incubated at 37 0 C for 24h while, those with fungal suspensions were placed at room temperature for 48-72h. In order to investigate rate of microbial growth, both Nutrient Agar (for bacteria) and Sabouraud Dextrose Agar (for fungi) were used. Optical density of inoculated dilution with no growth was determined. RESULTS AND DISCUSSION Results After incubating 10ml of mentioned drugs with 0.1ml of microbial suspensions, it was observed that none of the studied bacteria and fungi grew and when the bacteria were subcultured on Nutrient Agar, spore forming Gram-positive bacteria were found but no growth observed in Sabouraud Dextrose Agar. According to tables 1,2 and 3, it can be concluded that the least and most resistance to synergistic effects of Methylparaben and Sodium benzoate belong to A. niger and C. albicans respectively. The Results of Microbial Growth Inhibition by Sodium Benzoate Concentration Table 1: Concentration of microbial growth inhibition by sodium benzoate and their OD Microorganisms Microbial Growth OD Inhibition(mg/ml) S. aureus ATCC /052 E. coli ATCC /072 P. aeruginosa ATCC /065 A. niger ATCC /165 C. albicans ATCC /076 S. aureus (Clinical) /171 E. coli(clinical) /087 P. aeruginosa (Clinical) /094 S. aureus (Environmental) /046 E. coli (Environmental) /050 A. niger (Environmental) /0106 Table 2: Concentrations of microbial growth inhibition by methyl paraben and their OD Microorganisms Microbial Growth Inhibition(mg/ml) OD S. aureus ATCC /05 E. coli ATCC /03 P. aeruginosa ATCC /021 A. niger ATCC /14 C. albicans ATCC /056 S. aureus (Clinical) /12 E. coli (Clinical) /105 P. aeruginosa (Clinical) /021 S. aureus (Environmental) /237 E. coli (Environmental) /141 A. niger (Environmental) /05 Copyright 2014 Centre for Info Bio Technology (CIBTech) 365

4 Table 3: Concentrations of microbial growth inhibition by synergistic effects of methyl paraben and sodium benzoate and their OD Microorganisms Microbial Growth OD Inhibition(mg/ml) S. aureus ATCC /142 E. coli ATCC /089 P. aeruginosa ATCC /061 A. niger ATCC /241 C. albicans ATCC /028 S. aureus (Clinical) /079 E. coli (Clinical) /055 P. aeruginosa (Clinical) /109 S. aureus (Environmental) /169 E. coli (Environmental) /121 A. niger (Environmental) /061 Discussion Synergistic effects that anti-microbial preservatives in this case increased and easier than when each is used alone can lead to the growth of microorganisms. The antimicrobial effect of the environment on the strain increases. The standard strains of microorganisms most sensitive and most resistant microorganism Aspergillus niger synergistic preservative is also a Candida albicans. High degree of water availability in pharmaceutical products may give rise to their contamination by microorganisms which may cause spoilage of the product along with loss of therapeutic properties and if they are pathogenic serious infections can arise (Chaudhary et al., 2008; Judge et al., 2008). Protective material not only reduces the number of microorganisms due to strict observance of the manufacturing processes used. With this in mind the specificities of each antimicrobial substances protective substances show a protective effect, so all of these antimicrobial substances are toxic. The safety of patients must keep their concentration on products with antimicrobial properties is reduced to a minimum. It is empirically clear that Sodium benzoate exerted high antimicrobial effects on the growth, survival and aflatoxin production ability of A. niger, A. flavus and A. fumigatus (The United State Pharmacopeia, 1995). Syrups are concentrated solutions of sugar such as sucrose in water or other aqueous liquid. They have unusual opportunities as vehicles in extemporaneous compounding and are readily accepted by both children and adults. Because they contain no or very little alcohol, they are vehicles of choice from many of the drugs that are prescribed by pediatricians (Khalil et al., 2011; Parfitt, 1999). All of tested preservatives exerted an antimicrobial effect, antifungal action requiring higher concentrations of the preservatives than antibacterial action (Stanojevic et al., 2009). The results showed that sodium benzoate on growth and aflatoxin production lasting impression is produced by the fungus, increases with increasing concentration and duration of the preservative effect on the fungus antifungal effect is greater (Ogiehhor and Ikenebomeh, 2004). The effectiveness of use of preservatives is often dictated by limitations in their action or by sensitivity of the microorganisms themselves (Stickler and Tomas, 1982). The presence of methyl paraben and sodium benzoate preservative in pharmaceutical preparations exceeded, leading to skin reactions such as itching and hives are (Chawki et al., 2009; Beringer et al., 2006). Determination of preservatives in medicines to ensure quality and consumer safety is important (Chawki et al., 2009; Maybe et al., 2002). The results demonstrate more effective antimicrobial action of preservatives when used in combination with other preservatives than when used individually, a finding that can contribute to more effective conservation of food (Stanojevic et al., 2009). Different types of preservatives could be added one of the most common is the antimicrobial preservatives which work by inhibiting the growth of microorganisms inadvertently introduced during manufacture or use (Khalil et al., 2011; Rosenthall et al., 2006; Elsawah et al., 1999). Preservatives levels were found to fall outside the typical allowed concentration range for 70% of the samples, with some exhibiting significant higher Copyright 2014 Centre for Info Bio Technology (CIBTech) 366

5 concentrations. The present study highlights the high amount of preservatives that may be found in some liquid pharmaceutical preparations. The reason behind such finding is unclear and could be due to poor quality control or to intentionally extend the shelf life of the products. Consequence to patient's health need to be evaluated, especially since most liquid pharmaceutical products are administered to the pediatric population (Chawki et al., 2009; Beringer et al., 2006). ACKNOWLEDGEMENT We are grateful to Islamic Azad University, Lhijan Branch Authorities, for their useful collaboration. REFERENCES Akpan UG, Mohammed AD and Aminu I (2007). Effect of preservative on the shelf life of yoghurt produced from Soya Beans Milk. Leonardo Electronic Journal of Practices and Technologies Bean HS (1972). Preservatives for Pharmaceuticals, Journal of the Society of Cosmetic Chemists Beringer PA Marderosian L et al., (2006). The Science and Practice of Pharmacy, 21 st edition (Lippicott Williams & Wilkins Philadelphia). Chaudhary J, Rajpal AK and Judge V et al., (2008). Preservative evaluation of caprylic acid derivatives in Aluminum Hydroxide gel-usp. Scientia Pharmaceutica Chawki B, Sarah AJ and Soula K (2009). Preservative in liquid pharmaceutical preparations, Department of pharmaceutical Sciences, School of pharmacy, Lebanese American University, Byblos, Lebanon 9(1,2). Elsawah MMA, Ashour EH and Shady TSM (1999). Characteristics of Streptomyces flavogriseus β- xylosidase and its use in Xylan hydrolysis. Pakistan Journal of Biological Sciences Judge V, Narasimhan B, Narang R et al., (2008). Preservative evaluation of novel 2, 4-Hexadienoic acid derivatives in Aluminum Hydroxide Gel-Usp. Scientia Pharmaceutica Kamble R, Singh S and Singh S (2011). Simultaneous Determination of preservatives (Methyl paraben and Propyl paraben) in sucralfate suspension using high performance liquid chromatography, E- Journal of Chemistry 8(1) Khalil R et al., (2011). Assessment of the preserving efficacy of the pharmaceutical Syrups to identified air-born microorganisms. Trends in Applied Sciences Research 6(2) Maybe Schatz M and Zeiger R (2002). Immediate hypersensitivity to Methyl paraben causing falsepositive results of local anesthetic skin testing or provocative dose testing, The Permanente Journal 6(4). Mincea M et al., (2009). Determination of Methyl paraben from cosmetic products by ultra performance liquid chromatography. Journal of the Serbian Chemical Society 74(6) Parfitt K (1999). Martindale, the Complete Drug Reference, 32 nd edition (Pharmaceutical press) London. Ogiehhor IS and Ikenebomeh MJ (2004). Antimicrobial effects of Sodium Benzoate on the growth, survival and Aflatoxin production potential of some species of Aspergillus ingarri during storage, Pakistan Journal of Nutrition 3(5) Restaino LK and Komatsu MJ (1981). Effects of acids on potassium sorbate inhibition on food-related microorganisms in culture media. Journal of Food Science Rosenthall RA et al., (2006). Evaluation of the preserving efficacy of lubricant eye drops with a novel preservative system. Journal of Ocular Pharmacology and Therapeutics Stanojevic DC et al., (2009). Antimicrobial effects of Sodium benzoate, Sodium nitrate and Potassium sorbate and Their synergistic action in vitro. Bulgarian Journal of Agricultural Science 15(4) Stickler DJ and Tomas B (1982). Intrinsic resistance to non antibiotic antibacterial agents. Principles and Practice of Disinfection, Preservation and Sterilization, edited by Russell AD, Hugo WB and Ayliffe GAJ (Blackwell scientific publications) Oxford The United State Pharmacopeia (1995). 23 the end National Formulary, 18 th edition, United state pharmacopeial Convention (68). Copyright 2014 Centre for Info Bio Technology (CIBTech) 367