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IN VITRO ANTIMICROBIAL ACTIVITY OF SOME ESSENTIAL OILS AGAINST BACTERIAL PATHOGENS CAUSING SKIN DISEASES IN VAPOR PHASE

Objective: The objective of this study was to evaluate the antimicrobial activity of volatile oils from aromatic plants against pathogenic bacteria. Methods: Thai aromatic plants such as Pogostemon cablin (Blanco) Benth (Patchouli oil), Cymbopogon nardus Rendle (Citronella grass oil), Pelargonium roseum (Geranium oil), Syzygium aromaticum (L.) Merrill and Perry (clove oil), Cinnamomum spp.(cinnamon oil), and Cymbopogon citratus (DC.) Stapf. (lemongrass oil) were selected. Essential oils were obtained by water distillation and were stored at 4°C until use. Five human pathogenic bacteria were obtained from Thai traditional Medicine College, Rajamangala University of Technology, Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa. The antibacterial activity of volatile oils was determined by disc-diffusion assay. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each essential oil were determined. Results: Our study showed that 10% of essential oil from Cinnamomum spp. was the most potential against S. aureus, MRSA, and E. coli when assayed by disc-diffusion method with inhibition zones ranging from 37.66±0.57 to 45.33±1.15 mm and from 29.33±0.57 to 36.00±1.00 for lemongrass oil with MIC and MBC of 1.25%. Conclusion: From this study, it can be concluded that some essential oils have potential antibacterial activity. The present investigation provides support to the antibacterial properties of essential oils and will be applied to health-care product as aroma antibacterial products.


INTRODUCTION
Skin infectious disease is a disorder of exclusively the superficial layers of the skin, infected by organisms such as bacteria, viruses, fungi, and parasites [1]. The pathogens including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and drugresistant bacteria caused several skin human infections [2][3][4][5][6][7][8]. Thus, the global prevalence of infectious disease caused by bacteria is a major public health problem [9]. However, it is important to note that the effects of synthetic drugs can be highly unpredictable, making difficult to fully assess their physical impact. Hence, the natural compounds or essential oils from medicinal plant have an alternative to treat the antimicrobial agent. Medicinal plants have been used as primary treatment of skin disorders for centuries in Thailand Traditional Medicine, Chinese Traditional Medicine, and Indian Traditional Medicine.
Essential oils of medicinal plants present a great potential of application as natural antimicrobial agents [9]. They are mixtures of natural volatile molecule deriving from plant secondary metabolism which potent antibacterial, antifungal, antiviral, insecticidal and antioxidant activities. Generally, the biological activity of essential oil depends on their chemical structure, environmental, and agronomic conditions [10]. Thus, the objective of this research was to screen the antibacterial activity of volatile oil from medicinal plants in Thailand against skin human pathogenic bacteria. Stapf. (lemongrass oil) were selected. Briefly, the medicinal plant was completely immersed in water and heated to boiling, after which the essential oil was evaporated together with water vapor and finally collected after decantation. The distillate was isolated and dried in a Rotavapor to giving greenish-yellow oil. The oil was stored at 4°C until the antimicrobial screening [11].

Antibacterial activity of the essential oils Microorganisms
Antimicrobial activity determination was carried out against bacterial pathogens causing skin diseases, Staphylococcus epidermis, E. coli, methicillin-resistant S. aureus (MRSA), S. aureus, and P. aeruginosa.

Inocula
Inocula for the assays were prepared by incubation at 37°C for 18 h before use and were adjusted to McFarland scale of 0.5. Cell suspensions were finally diluted to 10 4 cfu/ml for being used in the activity assay.

Disc volatilization assay
This assay requires a culture agar plate inoculated with microbial suspension adjusted by McFarland No.0.5 containing 10 6 cfu/ml inserted down on top of a container (Fig. 1). A paper disc (6 mm) is deposited at the bottom of the container with 15 µl of essential oils. All plates inoculated and the disc should be sealed with parafilm to prevent the steam outlet and incubated at 37 C for 24 h, and the inhibition zone was measured in millilitre (mm) [12]. The experiments were done by tree replicates (Fig. 1).

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
To assess the MIC and MBC of essential oils, the serial two-flow dilution was carried out with the disc-diffusion assay. The MIC represented the lowest concentration showing an inhibition zone; the MBC was determined by subculturing which showed no bacterial growth on the agar plates after incubated at 37°C for 24 h in an incubator. The lowest concentration that did not show bacterial growth was defined as an MBC value. The experiment was conducted in triplicate, and the mean diameter of the zone of inhibition was recorded in millimeters (mm). Erythromycin was used as a positive control, and dimethyl sulfoxide (DMSO) solution was used as a negative one. All experiments were performed in triplicate. The results were represented by mean±standard deviation (SD) [13,14].

Statistical analysis
The results of the data experiments were expressed in mean±SD for groups (n=3).

Antibacterial activity of essential oils
Due to the absence of direct contact between the skin pathogenic bacteria and essential oils, the common skin pathogenic bacteria are listed in Table 1. The MIC was determined only for oils that presented positive on disc-diffusion assay. The results show a variable effect of essential oils on the bacterial pathogens. The essential oils from Cinnamomum spp. was great potential against MRSA, showing the lowest MIC values of 1.25% with clearing zone of 45.33±1.15 mm and MBC value of 1.25%. As it can be appeared in Table 1, the essential oil of cinnamon oil exhibited the growth of E. coli and S. aureus with MIC and MBC value of 1.25% and showed zone of inhibition of 20.00±0.00 and 37.66±0.57 mm, respectively. The essential oil with the lowest MIC and MBC was that of clove, with MIC and MBC of 5.0% against P. aeruginosa with halo zone of 44.33±1.15 mm ( Table 2). The MIC and MBC of clove oil at a concentration of 1.25% showed an inhibition zone of 33.66±1.52 mm with B. subtilis. However, as the same condition, it did not inhibit the growth of E. coli, S. aureus (MRSA) and S. aureus. The MIC and MBC of lemongrass oils at a concentration of 2.50% displayed the lowest concentration to inhibit the growth of MRSA, S. aureus, and B. subtilis with clearing zone of 36.00±1.00, 36.00±1.00 and 28.33±1.52 mm (Fig. 2). It also no antibacterial activity was observed in the controls as well as in the assays where only DMSO was used. Above 5.0%, patchouli oil, citronella oil, and geranium oil did not show any antibacterial effective against E. coli, S. aureus (MRSA), S. aureus, and P. aeruginosa. In contrary, the citronella oil showed potent inhibition against pathogens [13]. The vaporization of the inhibition zone of cinnamon oil was observed on NA with the difference various skin pathogens and concentration of essential oils. This result was similar of the previous reported, concerning the antioxidant and the antimicrobial activity [15][16][17][18][19][20], indicating that the different plants species have a variety of different kinds of molecules. Hence, differences in the composition of essential oils, in the interactions of their constituents, and in their extraction and purification operations affect their antimicrobial activity [21].

ACKNOWLEDGMENT
The authors extend their appreciation to the Thai Traditional Medicine College, RMUTT, for all facilities and financial supported.