WORLD JOURNAL OF EXPERIMENTAL BIOSCIENCES
Vol. 3, No. 2: 150-154 (2015). ISSN: 2313-3937
Research article
Inhibitory effect of menthol extracted from Mentha rubra
on
Methicillin-Resistant
Staphylococcus
aureus Haider S. Kadhem1*, Harith J. F. Al-Mathkhury1
ABSTRACT Menthol is an organic compound (essential oil) made synthetically or obtained from peppermint or other mint oils. Menthol has chemical structure C10H16 and it is enriched in isoprene structure (terpens). It was extracted by Soxhlet from Mentha rubra and confirmed by Fourier transform infrared spectroscopy (FTIR) and characterized by High-performance liquid chromatography (HPLC) from other substances, which extracted from Mint. In vitro studies of menthol showed that it has inhibitory effect on Staphylococcus aureus, through menthol has lipophilic character and work as organic solvent for lipids which effected by essential oils. The minimum inhibitory concentrations (MIC) values of menthol MIC was evaluated, our study showed that the diameters of inhibition zones ranged from 15-28 mm in disc diffusion method when the MIC was 25 μg/ml. Keywords: FTIR, Menthol, MIC, MRSA.
Citation: Kadhem HS, Al-Mathkhury HJF. (2015) Inhibitory effect of menthol extracted from Mentha rubra on Methicillin-Resistant Staphylococcus aureus. World J Exp Biosci 3: 150-154. Received December 5, 2015; Accepted December 22, 2015; Published December 30, 2015.
INTRODUCTION Plants and their essential oils are potentially useful sources of antimicrobial compounds. Numerous studies have been published on the antimicrobial activities of plant compounds against many different types of microbes, including food-borne pathogens [1]. The main constituents of essential oils – mono- and sesquiterpenes including carbohydrates, phenols, alcohols,
Mint is widely used in food, cosmetics and medicines. It has been proven helpful in symptomatic relief of the common cold. It may also decrease symptoms of irritable bowel syndrome and decrease digestive symptoms such as dyspepsia and nausea, although more research is needed. It is used topically as an analgesic and to treat headaches [1].
*Correspondence:
[email protected]. Department of Medical Laboratory Techniques, Al-Esraa University College, Baghdad, Iraq. Full list of author information is available at the end of the article.
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Copyright: © 2015, Kadhem HS & Al-Mathkhury HJF. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any site, provided the original author and source are credited.
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Kadhem HS, Al-Mathkhury HJF. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 150-154. ethers, aldehydes and ketones are responsible for the biological activity of aromatic and medicinal plants as well as for their fragrance [2]. Menthol from mint essential oil (40-90%) is an ingredient of many cosmetics and some perfumes. Menthol and mint essential oil are also much used in medicine as a component of many drugs. Infections due to bacterial species also remain a serious clinical problem. Emerging resistance of bacterial species is seriously decreasing the number of effective antimicrobials. Because of increasing pressure of consumers and legal authorities, the food industry has tended to reduce the use of chemical preservatives in their products to either completely nil or to adopt more natural alternatives for the maintenance or extension of product shelf life [3]. Methicillin - resistant Staphylococcus aureus (MRSA) is a nosocomial pathogenic bacterium that is resistant to some kinds of antibiotics. It is resistant to a family of antibiotics related to penicillin that includes antibiotics called methicillin and oxacillin, and is often resistant to many other antibiotics as well [4]. Recently, cases of MRSA have been documented in healthy communitydwelling persons without established risk factors for MRSA acquisition [5]. Many studies were characterized MRSA isolates from individual hospitals or countries and identified strains that appear to be well adapted into hospital environment, They are established in several hospitals within a country, or have spread internationally (epidemic MRSA, EMRSA) [6]. The presence of the mecA gene recognizes MRSA; however, some studies do not test this gene, and define MRSA by antibiotic susceptibility testing. Caution must be used when using susceptibility testing as the criterion for MRSA, as some testing methods can overestimate methicillin resistance. The aim of this study was testing the antimicrobial activity of aromatic substance (Mentol) which was extracted from Mint against MRSA by susceptibility test.
MATERIALS AND METHODS Plant M. rubra The leaves of M. rubra were bought from market then washed by distilled water and left in air for drying then grinned by hands. Soxhlet extractor was used for extraction which supplied with 300 gm of fine ground leaves and 1.5 L hexane as organic solvent, the soxhlet operated for 8 h at 60°C. The solvent was removed by rotary vacuum evaporator. Then ethylene glycol was used for preparing different concentrations of menthol. Chemical analysis menthol was separated on Fast Liquid Chromatographic (FLC) column under the optimum condition.
Methicillin Resistant aureus (MRSA)
agar a selective and differential medium for S. aureus containing 7% NaCl, bile salt, then incubated at 37°C for 48 h. S. aureus isolates were identified based on the standard biochemical and microbiological methods such as: golden pigments of S. aureus when cultured on Milk Agar, clotting of the oxalated or citrated blood by S. aureus (Coagulase test) and production of bubbles when adding few drops of hydrogen peroxide on the grown S. aureus isolates on nutrient agar. Methicillin resistance was detected by cefoxitin disc diffusion test. All isolates were subjected to cefoxitin disc diffusion test using a 30 µg disc. A 0.5 Mc Farland standard suspension of the isolate was made and lawn culture done on Mueller Hinton agar (MHA) plate. Plates were incubated at 37°C for 18 h and inhibition zone diameters were measured. An inhibition zone diameter of ≤ 19 mm was reported as methicillin resistant and ≥20 mm was considered as oxacillin sensitive [7].
Antimicrobial activity of menthol After extraction of menthol and MRSA isolation, the sensitivity of MRSA toward menthol as well as the minimum inhibitory concentration (MIC) were determined.
Disc diffusion method (Kirby-Bauer test) Filter paper no.3 discs were prepared and immersed in menthol extract. Thereafter, they were placed on MHA agar (streaked previously with MRSA cultured) as 3 discs for each plate [8].
Minimum inhibitory menthol (MIC)
concentration
of
MIC is read as the lowest concentration of antibiotic which inhibited bacterial growth. The purpose of MIC is to know the minimum concentration of menthol that inhibit the pathogenic bacteria under test with minimal side effects on human, MIC determination was accomplished using ethylene glycol as a diluents. 0.5 ml ethylene glycol was added into six test tubes. Thereafter, 0.5 ml of menthol extract was added to the first tube and then an aliquot of 0.5 ml was transferred from the first tube to the subsequent tube. Three discs per dilution were prepared and placed on MHA seeded with MRSA (equal to McFarland standard tube no. 0.5). All plates were incubated at 37°C for 18 h then the inhibition zone diameter was estimated for each dilution [9].
RESULTS AND DISCUSSION The plant (M. rubra) has many essential oils with lipophilic effects. The essential oils are a hydrophobic liquid containing volatile aroma compounds of plants. Every essential oil has one main chemical component. This main chemical component determines the overall qualities (properties and best uses) of that essential oil. These main chemical components are grouped into
Staphylococcus
Ten specimens were collected from acne pimples by swabbing. All specimens were streaked on mannitol salt
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Kadhem HS, Al-Mathkhury HJF. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 150-154. several major classifications recognized by aromathecm-1) ascribed to aliphatic methyl group (3006.8) cm -1 rapy professionals and every essential oil on the market ascribed to aromatic methyl group (1745.5) cm -1 that should fall into one of these categories [10]. FTIR attributed to (C-O) bond and 1652.9 cm -1 corresponding spectrum confirmed the material isolated from M. rubra to aromatic (c=c) group, all of that confirmed the leaf oil as menthol (Fig. 1). Significant peaks were found acceptable purity of the isolated material. at 3469.7 cm-1 corresponding to hydroxyl group (2677.0 Moreover, menthol was characterized using the HPLC
Fig 1. FTIR spectra of menthol isolated from M. rubra leaves.
Menthol is an organic compound (essential oil) made synthetically or obtained from peppermint or other mint oils. The Menthol has the chemical structure C10H16 and menthol is enriched in isoprene structure (terpens) [10]. Ethylene glycol is an organic compound widely used
system (Fig. 2) and identified by comparing its retention time (tR) and UV spectrum with that of the standard compound. The retention time 7.09 min and UV spectra of the isolated compound on HPLC were identical to that of standard menthol (7.304).
Fig 2. HPLC chromatogram of standard menthol and menthol isolated from M. rubra leaves.
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Kadhem HS, Al-Mathkhury HJF. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 150-154. as automotive antifreeze. Menthol was isolated from the cell contents can then occur [12]. Although, a certain extracted essential oil, and then detected on TLC plates amount of leakage from bacterial cells may be tolerable in comparison with standard menthol. A purple zone with without loss of viability, extensive cell contents loss or a retention factor (Rf) value of 0.62 was identified as the exit of critical molecules and ions will lead to death menthol in comparison with standard menthol that had [13]. It has also been reported that Gram-negative the same Rf value. The FTIR spectrum confirmed the strains are less susceptible to essential oils due to the material isolated from which confirm the purity of the presence of an outer membrane surrounding the cell isolated material. Moreover, menthol was characterized wall which restricts diffusion of hydrophobic compounds using the HPLC system and identified by comparing its through its lipopolysaccharide covering. To characterize retention time (tR) and UV spectrum with that of the the mechanisms underlying these effects, menthol was standard compound. The retention time 7–8 min and UV tested for their membrane-damaging activity by spectra of the isolated compound on HPLC were determining the release of CF entrapped in the completely identical to that of standard menthol [10]. intraliposomal space [13]. The mentholol analysis was studied by GC and GC-MS. Table 2- Inhibition zones diameter of Minimum inhibitory This study allowed identification of 34 composed concentrations of menthol against MRSA. representing 97.2% of the totality of the components of the oil of M. longifolia. The oil is particularly rich in Isolate Inhibition zones diameter (mm) menthol (32.51%), menthone (20.71%), pulegone 1 21 22 22 (17.76%), 1,8-cineole (5.61%), terpineol-4 (4.87%) and 2 21 22 21 piperitone (2.16%). The major part of the identified 3 22 27 23 volatile compounds is oxygenated monoterpenes 4 18 15 18 (83.3%) followed by hydrocarbons and ether.
Results of the culturing MRSA on Muller Hinton Agar as sensitivity test for menthol are summarized in table 1. Results of the minimum inhibitory concentration (MIC) of menthol to MRSA are presented in table 2. Table 1. Inhibitory effect of menthol on MRSA Isolates 1 2 3 4 5 6 7 8 9 10
Inhibitory zone diameter (mm) 13 15 9 8 9 9 9 8 8.5 9
In vitro studies of menthol showed that it has inhibitory effect on S. aureus, through menthol has lipophilic character and work as organic solvent for lipids which effected by essential oils. It has been shown that Grampositive bacteria more been effected by essential oils especially by menthol than Gram-negative bacteria. Such variation may due the presence of the hydrophilic outer membrane in Gram-negative bacteria, which prevent the penetration the hydrophobic essential oils and the cell wall thicker than the cell membrane in Gram-positive bacteria which lack to outer membrane and make it more susceptible to menthol than Gramnegative bacteria [11]. An important characteristic of essential oils and their components is their hydrophobicity, which enables them to partition in the lipids of bacterial cell membrane and mitochondria, disturbing the structures and rendering them more permeable as a result of which leakage of ions and other
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5 6 7 8 9 10
18 15 15 14 18 22
15 14 14 18 20 24
19 14 17 15 19 28
There are also morphological changes in MRSA after treatment by menthol, MRSA have a normal coccoid shape, After 24 h of bacteria treatment with menthol a hole can be seen on surface and cell wall. It has disappeared to reveal fine cytoplasm structure. It seems that coccoid bacteria are more resistant to menthol essential oil than rod bacteria. This resulted in membrane expansion, increased membrane fluidity and inhibition of a membrane-embedded enzyme [14] reported that the mode of action of essential oils is related to an impairment of a variety of enzyme systems, mainly involved in energy production and structural components synthesis. They also explain the mode of action through leakage of ions, ATP, nucleic acids and amino acids damage [15]. The MIC values which obtained from the researches of menthol against MRSA were varied, The highest MIC value (15.6 μg/ml) was observed against S. aureus. The MIC values the essential oil of M. longifolia against bacterial strains tested in microdilution assay. S. aureus (ATCC 25923) was (0.78 mg\ml) [16]. The essential oils activity against Gram-positive bacteria was less noteworthy with the lowest MIC values equal or more than 7.8 mg/ml, with exception for S. aureus ATCC 6533 that was more susceptible (2 mg/ml) [17]. Menthol diminishes α-hemolysin, Staphylococcal enterotoxins A and B (SEA and SEB), and shock syndrome toxin (TSST-1) levels in the culture supernatants. Among the extracellular proteins produced by S. aureus, αhemolysin is the key factor responsible for the hemolysis of rabbit erythrocytes, while Staphylococcal enterotoxins
Kadhem HS, Al-Mathkhury HJF. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 150-154. (SEs) and Toxic shock syndrome toxin (TSST-1) are the major toxins that can act as super antigens, inducing T cells to release Tumor necrosis factor (TNF-α). Accordingly, western blot analysis was used to determine whether the reduced hemolytic and TNF-α inducing activities of S. aureus culture fluids cultured with increasing concentrations of menthol were attributed to the decreased expression of these toxins. Treatment with increasing concentrations of menthol resulted in a dosedependent reduction in the production of α-hemolysin, SEA, SEB, and TSST-1. Exposure to 8 μg/ ml of menthol did not result in a significant decrease in the secretion of these toxins; however, at 64 μg/ml of menthol, no or little immunoreactive proteins could be observed in all tested strains. Our results showed that transcription from the agrA locus was significantly decreased when S. aureus was grown in the presence of 64 μg/ml of menthol [18]. Our results showed that the diameters of inhibition zones ranged from 15-28 mm in disc diffusion method, while the MIC was 25μg/ml. Our results was agreed with previous study [18] and disagreed with others [10, 16]. Conflict of interist The authors declare that they have no conflict of interests.
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Author affiliation: 1. Department of Medical Laboratory Techniques, Al-Esraa University College, Baghdad, Iraq. 2. Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq 2.
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