Broad-spectrum disinfectant composition containing a synergistic combination of cinnamon oil and citric acid

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1 117 e rc EORC International Journal of Essential Oil Therapeutics Broad-spectrum disinfectant composition containing a synergistic combination of cinnamon oil and citric acid N. Baiju, S. M. Modak * Department of Surgery, College of Physicians and Surgeons of Columbia University, C, 630 West 168th Street, New York, NY 10032, USA Abstract Most surface and skin cleansers contain the antimicrobial triclosan and are widely used to reduce the transmission of pathogens in households and healthcare settings. However, triclosan-containing products have been reported to produce resistant microbes. In this study, botanicals such as essential oils and fruit acids have been investigated for their disinfectant activity. The antimicrobial efficacy of low concentrations of essential oils (0.25% to 0.50%) in combination with fruit acids (citric and lactic acids) showed synergistic activity in vitro. All the essential oils exhibited synergistic activity with 1% of citric acid when tested against Staphylococcus aureus ( log reduction from the control). Cinnamon, basil and citronella oil showed superior synergistic activity against all the bacteria tested, which included E. coli, Methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa ( log reduction from the control). Most importantly, only the cinnamon oil-citric acid combination (Cin-Cit) was highly effective against Candida albicans ( log reduction). Cin-Cit was incorporated in a liquid soap base (Cin-Cit soap), and the rapid antimicrobial efficacy (post-30-second contact) was evaluated and compared with that of a commercial hand soap containing triclosan by the tube-dilution method. The results showed that the antimicrobial activity of Cin-Cit soap was much higher (5.09 to 6.10 log reduction from the control) than that of soap containing triclosan (0.27 to 0.75 log reduction). Cin-Cit may provide rapid and broad spectrum disinfectant action when used in skin and surface cleansers. Key words: synergistic combination, essential oils, cinnamon oil, citric acid, organisms, surface cleanser, soap, triclosan. Introduction The major cause of microbial contamination and disease transmission in healthcare workers and in the general public is primarily contact with contaminated skin and hard surfaces. To address this problem, cleaning products such as surface cleansers, laundry detergents and skin and hand cleansers containing antibacterial compounds and disinfectants have been formulated and used. The main antibacterial agent used in these products is triclosan. Widespread use of triclosan-containing products can produce resistant microbes, resulting in the propagation of resistant microbial variants in the environment [1-4]. The recent trend in the use of environment-friendly * Corresponding author. address: smm4@columbia.edu Essential Oil Resource Consultants. All rights reserved. products has led to the increased use of natural products to treat diseases. The antiseptic properties of natural products such as aromatic and medicinal plants and their extracts have been known since ancient times. The essential oils from many of these plants have been characterized [5], and they have been found to possess antimicrobial, antiviral, and anti-inflammatory properties [6-9]. In recent years, there has been a significant increase in the use of plant products as preservatives in food and to treat disease [10, 11]. However, there have been reports of toxicity and contact dermatitis with use of essential oils at the effective concentrations [12]. Previously, we showed that the essential oils and their ingredients (sesquiterpenoids) exhibit synergistic action with quaternary ammonium compounds (QAC) [13], which are commonly used as preservatives. In order to make use of the beneficial effect of essential oils

2 118 International Journal of Essential Oil Therapeutics (2007) 1, with reduced risk of deleterious effects, we investigated the antimicrobial efficacy of low concentrations of essential oils in combination with other plant products, such as fruit acids. In this study, the synergistic effect of essential oils and fruit acids against various pathogens was evaluated. Furthermore, the utility of these botanicals as a natural antimicrobial-disinfectant composition for use in skin cleansers was also investigated. Materials Microorganisms Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 15442) and Candida albicans (ATCC 11651) were obtained from the American Type Culture Collection (ATCC). The clinical isolate used in this study, Methicillin-resistant Staphylococcus aureus (MRSA), was obtained from the New York Presbyterian Medical Center, NY. Essential oils and fruit acids The essential oils of basil (Ocimum sanctum), cinnamon bark and leaf (Cinnamomum zeylanicum), clove (Syzygium aromaticum), lavender (Lavandula officinalis) and lemon (Citrus limonum) were obtained from Vaishali Industries, India. Tea tree (Melaleuca alternifolia), citronella (Cymbopogon nardus), and orange (Citrus aurantium) oils were obtained from Herbal Remedies, USA. Lactic and citric acids were obtained from Purac and J.T. Baker Chemical Co., respectively. Disinfectant composition The disinfectant composition consisted of 6.94% cinnamon oil, 13.8% citric acid and 2.8% orange oil (for fragrance) dissolved in 76.4% SDA40-B alcohol (Cin-Cit). Soap containing Cin-Cit A soap base containing thickening agents, surfactants, and lathering agents was prepared. Cin-Cit was incorporated at 7% concentration into this base. Triclosan soap Soft soap containing 0.15% triclosan (TC) (Soft soap TC) was purchased from a local drugstore. Dilution fluid with neutralizer (DFN) Ten ml of Tween 80 and 3 ml of lecithin were added to 987 ml of deionized water containing 34 g KH 2 PO 4. The ph was adjusted to 7.2 prior to autoclaving at 121 C. Methods Evaluation of the synergistic antimicrobial effect of basil oil and organic acids against S. aureus Various concentrations of basil oil, organic acids and combinations were prepared in water containing 10% SDA40-B alcohol. With the exception of citric acid, all ingredients were added by volume. Nine ml of the solutions listed above were dispensed in sterile culture tubes in triplicate, and 0.1 ml of 1x10 7 cfu/ml of S. aureus culture was added to each of the tubes and mixed by vortexing. The tubes were incubated at room temperature for 5 minutes and then 0.9 ml of the dilution fluid with neutralizer (DFN) were added to each tube, mixed well, and serial dilutions made with DFN. The dilutions in 0.5 ml volumes were plated on trypticase soy agar (TSA) plates. As a control, water containing 10% SDA40-B alcohol was used and processed similarly. The plates were incubated at 37 C for hours, and the colony counts were determined. Evaluation of the synergistic antimicrobial effect of various essential oils with citric acid against S. aureus The various essential oils at 0.25%, such as tea tree, clove, lavender, lemon, orange citronella, pine, cinnamon leaf, and cinnamon bark either alone, or in combination with 1% citric acid, were dissolved in water containing 10% SDA40-B alcohol. These solutions in 0.9 ml volumes were dispensed in sterile culture tubes in triplicate. The tests were carried out as described above. Efficacy of combination of citric acid with basil, cinnamon, and citronella oils against different organisms. In this experiment, S. aureus, E. coli, P. aeruginosa, MRSA and C. albicans were used as test organisms; 0.25% of each of the essential oils (such as basil, cinnamon, and citronella oils) and 1% citric acid were dissolved in water containing 10% SDA40-B alcohol. These solutions at 0.9 ml were dispensed in sterile culture tubes in triplicate and the tests were carried out as described before. Evaluation of the antimicrobial activity of hand soap containing Cin-Cit The rapid antimicrobial efficacy of soaps was measured using a method previously published [13] with the following exception: a lower concentration of bacterial culture was used in this test. In short, a mixture of 0.1 ml containing 10 7 CFU/ml of different bacterial cultures and 0.1 ml of bovine serum were placed in a sterile culture tube. Cin-Cit soap (0.8 ml) was added to the tube and vortexed for 30 seconds. Nine ml of DFN was added to the tube to neutralize the activity of the soap; the tube was then vortexed and serially diluted with DFN. The diluted solutions were plated on trypticase soy agar plates in 0.5 ml volumes, incubated at 37 C for h and the colony counts determined. Soft soap TC was also tested as described above. Phosphate-buffered saline containing the culture was used as the control. Results Evaluation of the synergistic antimicrobial effect of basil oil and organic acids against S. aureus Table 1 shows the synergistic activity of basil oil and organic acids against S. aureus. The log reduction from control bacterial counts for 1.0% basil oil alone was 2.7, while acetic acid, citric acid, and lactic acid at the same concentration had log reductions of 0.2, 0.1, and 2.3, respectively. The combination of 1% basil oil and 1% of the acids showed higher log reductions ( ). Higher synergistic activity was seen with the combination of basil oil and citric acid. Evaluation of the synergistic antimicrobial effect of various essential oils with citric acids against S. aureus Since citric acid showed superior synergistic activity with basil oil, other essential oils such as cinnamon, citronella, orange, lavender, tea tree, pine, and lemon were tested to determine their synergistic activity with citric acid.

3 119 When tested alone, the various essential oils at 0.25% concentration showed a log reduction ranging from from control counts. Table 1. Synergistic activity of basil oil in combination with organic acids against S. aureus. compounds % log 10 reduction* 1 basil basil basil acetic acid lactic acid citric acid basil + 1 lactic acid basil + 1 acetic acid basil + 1 citric acid basil + 1 citric acid basil citric acid basil + 1 citric acid basil citric acid 2.5 reduction from control bacterial counts that range from 1 x 10 6 to 5 x The log reductions of combinations of various essential oils at the 0.25 % concentration and citric acid at 1.0% ranged from (Table 2). Table 2. Synergistic activity of various essential oils with citric acid against S. aureus. compounds % log 10 reduction 1 citric acid cinnamon bark cinnamon bark + 1 citric acid cinnamon leaf cinnamon leaf + 1 citric acid citronella citronella + 1 citric acid orange orange + 1 citric acid lemon lemon + 1 citric acid lavender lavender + 1 citric acid clove clove + 1 citric acid tea tree tea tree + 1 citric acid pine pine + 1 citric acid 2.74 reduction from control bacterial counts that range from 1 x 10 6 to 5 x Almost all the essential oils tested showed synergistic activity with citric acid; however, cinnamon leaf oil and citronella oil showed superior synergistic activity (5.7 and 6.2 log reduction, respectively). Efficacy of the combination of citric acid with basil, cinnamon, and citronella oils against different microorganisms Basil, cinnamon, and citronella oils in combination with citric acid showed high antibacterial activity against S. aureus, E. coli, P. aeruginosa, and MRSA. Log reduction from control bacterial counts ranged from 4.3 to 6.5. However, only cinnamon oil showed high activity against C. albicans (4.5 and 5.4 log reduction). Citronella and basil oils exhibited log reductions of only 1 and 1.5, respectively, against C. albicans (Figure 1). Evaluation of a hand soap containing a synergistic combination of cinnamon leaf oil and citric acid The soap containing Cin-Cit was more effective against S. aureus, E. coli, P. aeruginosa, and MRSA (log reduction ) than Soft soap TC (log reduction ) (Table 3). Table 3. Evaluation of a hand soap containing a synergistic combination of cinnamon leaf oil and citric acid. bacteria Cin-Cit soap Soft soap TC S. aureus 5.11 ± ± 0.06 P. aeruginosa 6.10 ± ± 0.12 E. coli 5.09 ± ± 0.09 MRSA 5.71 ± ± 0.16 reduction from control bacterial counts that ranges from 1 x 10 6 to 5 x 10 6 for all the bacteria tested. Results are the average of six samples in each group and are expressed as mean ± SD. Discussion The beneficial effects of essential oils in humans have been well documented [14, 15]. Also, many studies have been published on the antimicrobial, antiviral and sporicidal activities of essential oils [16-19]. However, there are reports of allergic contact dermatitis from the topical use of essential oils and their ingredients [12]. In this study we found that low concentrations of essential oils in combination with other botanicals such as fruit acids (citric and lactic acids) exhibit synergistic antimicrobial efficacy. Essential oils at concentrations as low as 0.25% to 0.5% were found to exhibit synergistic activity in combination with 1% citric acid (Tables 1 and 2) when tested against S. aureus. The activity was much higher when citric acid was combined with cinnamon, basil or citronella oil. The latter combinations were also synergistically effective against E. coli, S. aureus, MRSA, and P. aeruginosa ( log reduction from control). However, only the cinnamon oil-citric acid combination was highly effective against C. albicans ( log reduction). The synergistic activity appears to result from the different modes of action of citric acid and the essential oils. The ingredients in the essential oils have been reported to disrupt the plasma membrane [20-22] with the change in ph. The synergistic effect of cinnamon oil and citric acid appears to result from the disruption of the plasma

4 120 International Journal of Essential Oil Therapeutics (2007) 1, Figure 1. Efficacy of combination of citric acid with essential oils against different microorganisms. Log 10 reduction from control bacterial counts* E.coli P.aeruginosa MRSA C.albicans S.aureus 0.25 basil+ 1CA 0.25 cin bark + 1CA 0.25 citronella + 1CA 0.25 cin leaf +1CA reduction from control bacterial counts that range from 1 x 10 6 to 5 x 10 6 the exception being C. albicans that ranges from 1 x 10 5 to 5 x10 5. membrane and cellular functions induced by eugenol and cinnamaldehyde contained in the cinnamon oils [23], and influenced by the ph change caused by the citric acid. Citric acid has been reported to gain entry into the bacterial cell and lower the internal ph [24]. In the second part of the study, the rapid antibacterial activity of Cin Cit-containing hand soap was evaluated and compared with that of a commercial soap containing triclosan. Cin-Cit soap exhibited superior activity against all the bacteria tested ( log reduction) compared with the commercial soap containing triclosan ( log reduction) after a 30 second exposure (Table 3). Most people wash their hands for less than 30 seconds, and therefore washing with triclosan-containing soaps may be ineffective in disinfecting the hands. Furthermore, it has been suggested that the wide use of triclosan-containing soaps and other products can promote bacteria that are resistant to triclosan, cross-resistant to antibiotics, and thus alter a person s microflora [1]. Soaps containing Cin- Cit may show better disinfectant action even with 30- second hand washes. The synergistic combination of citric acid and essential oils, especially cinnamon oil, exhibit broad spectrum disinfectant activity. Use of cinnamon oil and citric acid in combination in skin and surface cleansers may provide rapid and broad-spectrum activity with a lower risk of emergence of resistant mutants and may be used as an alternative or adjuvant to triclosan in cleansing and other topical products. References Levy SB. Antibacterial household products: Cause for concern. Emerg Infect Dis. 2001;7(3): McMurry LM, Oethinger M, Levy SB. Triclosan targets lipid synthesis. Nature. 1998;(394): McMurry LM, Oethinger M, Levy SB. Over expression of mara, soxs or acrab produces resistance to Triclosan in Escherichia coli. FEMS Microbiol Lett. 1998;166: Suller MT, Russell AD. Triclosan and antibiotic resistance in Staphylococcus aureus. J Antimicrob Chemother. 2000;46:11-8. Dorman HJD., Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol ;88(2), Deans SG, Ritchie G. Antibacterial properties of plant essential oils. Int J Food Microbiol. 1987;5: Ultee A., Kets EPW, Smid EJ. Mechanism of action of carvacrol on the food borne pathogen Bacillus cereus. Appl Environ Microbiol. 1999;65: Karatzas AK, Kets EPW, Smid EJ, Bennik MHJ. The combined action of carvacrol and high hydrostatic pressure on Listeria monocytogenes Scott. J Appl Microbiol. 2001;90 (3): Marino M, Bersani C, Comi G. Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae. Int J Food Microbiol. 2001;67: Conner DE. In: Davidson PM, Branen AL, editors. Antimicrobials in food. New York: Marcel Dekker; Balandrin MF, Klocke JA, Wurtele ES, Bollinger WH. Natural plant chemicals; sources of industrial and medicinal materials. Science. 1985;228: Bleasel N, Tate B, Rademaker M. Allergic contact dermatitis following exposure to essential oils. Aust J Dermatol. 2002;43: Shintre MS, Gaonkar TA, Modak SM. Efficacy of an alcohol based healthcare hand rub containing synergistic combination of farnesol and benzethonium chloride. Int J Hyg Environ Health. 2006;209: Silva J, Abebe W, Sousa SM, Duarte VG, Machado MI, Matos FJ. Analgesic and anti-inflammatory effects of essential oils of Eucalyptus. J Ethnopharmacol. 2003;89(2-3): Hajhashemi V, Ghannadi A, Sharif B. Anti-inflammatory and analgesic properties of the leaf extracts and essential oil of Lavandula angustifolia Mill. J Ethnopharmacol. 2003;89(1): Garcia CC, Talarico L, Almeida N, Colombres S, Duschatzky C, Damonte EB. Virucidal activity of essential oils from aromatic plants of San Luis, Argentina. Phytother Res. 2003;17(9): Minami M, Kita M, Nakaya T, Yamamoto T, Kuriyama H, Imanishi J. The inhibitory effect of essential oils on Herpes simplex virus type-1 replication in vitro. Microbiol Immunol. 2003;47(9): Schuhmacher A, Reichling J, Schnitzler P. Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro. Phytomed. 2003;10(6-7): Valero M, Salmerion MC. Antibacterial activity of 11 essential oils against Bacillus cereus in tyndallized carrot broth. Int J Food Microbiol. 2003;85: Helander IM, Alakomi HL, Latva KK, Mattila ST, Pol I, Smid EJ, et al. Characterization of the action of selected essential

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