support of the American people through the United States Agency for Internatonal Development (USAID). The contents are the responsibility of Texas A&M University and Udayana University as the USAID Tropical Plant Curriculum Project partners and do not necessarily reect the views of USAID or the United States Government. 1
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INHIBITORY ACTIVITY OF LEMONGRASS ESSENTIAL OIL AGAINST Eschericia Coli, Staphylococcus Aureus, AND Vibrio Cholera Nyoman Semadi Antara*, Dwi Ayu Kirani Paramita, Anak Agung Duwipayana, Ida Bagus Wayan Gunam Laboratory of Bioindustry,
Department of Agroindustrial Technology, Faculty of Agricultural Technology, Udayana University, Kampus Bukit Jimbaran, Bali. *e-mail: ns_antara@yahoo.com ABSTRACT The lemongrass essential oil (LEO) extracted from the leaf of lemongrass (Cymbopogon citratus) was tested for its inhibitory effects against three pathogenic bacteria (Escherichia coli, Staphylococcus aureus, and Vibrio cholerae). This research used a Completely Randomized Design with 5 treatments of LEO concentration in 1% tween 80, namely 1%, 2%, 3%, 4%, 5% (v/v). By using of GC-MS to determine LEO chemical composition, the LEO was composed of several compounds which citral, a monoterpene compound, was the dominant compound. This LEO could inhibit significantly the growth of E. coli, S. aureus and V. cholerae. The most sensitive of the bacteria against the LEO was Vibrio cholerae. In addition, the minimum inhibitory concentration (MIC) of LEO was 1.0%, 0.8%, and 0.6% respectively against E. coli, S. aureus, and V. cholera. Keyword : inhibitory activity, lemongrass essential oil, Escherichia coli, Staphylococcus aureus, Vibrio cholera Abstrak Minyak atsiri daun sereh (MADS) yang diekstrak dari daun sereh dapur (Cymbopogon citratus) diuji daya hambatnya terhadap tiga bakteri pathogen (Escherichia coli, Staphylococcus aureus, dan Vibrio cholera). Penelitian ini dirancang menggunakan Rancangan Acak Lengkap dengan 5 perlakuan konsentrasi minyak atsiri daun sereh dapur di dalam 1% tween 80, yaitu 1%, 2%, 3%, 4%, 5% (v/v). Komposisi kimia MADS yang dianalisis dengan GC-MS terdiri dari beberapa senyawa dengan citral, senyawa monoterpene, merupakan senyawa yang paling dominan. MADS ini dapat menghambat secara nyata pertumbuhan E. coli, S. aureus, dan V. cholera. Konsentrasi penghambatan minimal (KPM) MADS terhadap E. coli, S. aureus, dan V. cholera adalah berturut-turut 1,0%, 0,8%, dan 0,6%. Kata kunci: daya hambat, minyak atsiri daun sereh, Escherichia coli, Staphylococcus aureus, Vibrio cholerae.
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Introduction Essential oil produced from plant extraction process is composed of volatile secondary metabolites. Essential oils are found in all parts of the plant, such as flowers, fruit, leaves, stems, and roots. Although essential oils are spread in all parts of the plant, but generally concentrated in the leaves, bark, or fruit. Essential oils from different parts of the plant have a different chemical composition (Hili et al., 1997). Lemongrass (Cymbopogon citratus) concentrates their essential oil content in the leaves. Lemongrass essential oil (LEO) contained in the leaves has a different chemical composition compared to that extracted from the stem (Putra et al., 2012). Plants have a natural defense system. The content of natural bioactive compounds such as the compounds of terpenoids, alkaloids, and phenolic compounds that are generally used for self-defense of plants against invading microorganisms (Shukla et al., 2013). In addition, such compounds are also easily decomposed, environmentally friendly, and does not leave harmful residues (Bishop and Thornton, 1997). These characteristics will be helpful to explore and develop food bio-preservative recognized as safe. Among plant products, essential oils received great attention because of its high antimicrobial activity. Essential oils can be used as natural food additives and commercially produced through encapsulation technology (Burt, 2004; Arana-Sanchez et al., 2010). Many studies have been done related to the antibacterial and antifungal activity of essential oils. Former studies indicate a higher antibacterial effect of essential oils against Gram-positive than Gram-negative bacteria (Lang and Buchbauer, 2012). Garlic extract has bactericidal efficacy against Helicobacter pylori (Cellini et al., 1996). Some pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella typhi are also sensitive to garlic extract (Arora and Kaur, 1999). Garlic extract also showed anti-Candida activity. Extract of garlic and clove has efficacy as bacteriostatic and bacteriocidal against E. coli O157 and Sal. enteric serovar Enteritidis at concentrations between 0.25 to 1.0% (Leuschner and Zamparini, 2002). Oregano and thyme hydrosol also shown to kill pathogenic bacteria such as E. coli, S. aureus, and Yersinia enterocolitica (Sadi, 2003). In addition to bacterial pathogens, essential oils are also effective to inhibit spoilage bacteria. 3
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Essential oil of lemongrass could inhibit the growth of genus Aspergillus (Matasyoh et al., 2010; Ella et al., 2013) and detected as antifungal within wider spectrum (Mishra and Dubey, 1994). The oil was also found effective against several pathogenic bacteria, so it would be helpful in the treatment of infections caused by multidrug resistant organisms. Several essential oils showed efficacy to eradicate biofilm-forming bacteria with higher efficiency than using standard antimicrobial treatment (Kavanaugh and Ribbeck, 2012). In present study we investigated and confirmed the inhibition activity of essential oil extracted from lemongrass leaf against three important pathogenic bacteria related to food safety. The study also determined the minimum inhibitory concentration (MIC) of LEO to inhibit E. coli, Staph. aureus, and Vibrio cholera.
Materials and Methods Materials The main material of the study was LEO obtained from the extraction of lemongrass leaves Pelaga village origin, Regency of Badung, Bali, Indonesia. Bacterial cultures of E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, Vibrio cholerae were obtained from the Laboratory of Microbiology, Faculty of Medicine, University of Udayana. Another necessary reagents were an emulsifier tween 80, media Nutrient Agar (CM Oxoid 3), Nutrient Broth (CM Oxoid 1), Thiosulfate Citrate Bile-salt Sucrose Agar (TCBSA) (Oxoid CM 333), Eosin Methylen Blue Agar (EMBA) (Oxoid CM 69), NaCl (JT. Baker) obtained in UPT. Integrated Bioscien and Biotechnology Laboratory, University of Udayana.
Design of Experiment This research was a laboratory experiment designed by completely randomized design (CRD). Five treatment concentrations of LEO in 1% tween 80 experimented in this study are 1%, 2%, 3%, 4%, and 5% (v / v). Each treatment was tested against three pathogenic bacteria namely E. coli, S. aureus, and V. cholerae. Each experiment be repeated 3 times, so that the research was conducted in 45 experimental units. The data were analyzed with analysis of variance and followed by Duncan's test (Steel and Torrie, 1993). When the LEO concentration of 1% still has the inhibitory activity then do the treatment by 4
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lowering concentrations of LEO which will be tried on the concentration of 0.2%, 0.4%, 0.6%, and 0.8% in 1% tween 80 to determine the MIC. Determination of Chemical Composition of LEO Chemical composition of LEO was analyzed by using GC-MS (Agilent Technologies, USA). Gas chromatography was run on HP 5MS column with internal diameter and column length was 0.25 mm and 30 m. Helium was used as carrier gas at a flow rate of 1 ml/min. Injector temperature was set up at 250C. The oven temperature program began at 70C for 5 min and ramped to final temperature (270C) at 10C/min, the total run time was 25 min. Identification of compounds was done by using software Wiley 229, NIST 12, and NIST 62 Library. LEO Sterilization and Emulsion Preparation LEO was sterilized by filtering through 0.22 m filter (Millex-Gv) sterile. The LEO was then collected in dark bottle and subsequently stored in a refrigerator at 4C until used for experiments. LEO emulsion was made by mixing LEO into a 1% solution of tween 80 (v/v). And then the mixture was shaken until LEO emulsion uniform and stable. Preparation of the Bacteria Preparation of bacterial culture started with that stock cultures of bacteria E.coli cultured on EMBA, S. aureus cultured on agar MSA and V. cholerae on TCBSA and incubated at 37C for 24 hours. Furthermore, each culture was grown in agar slant for stock that will be used to test inhibition. A total of one loop of each bacterial culture was inoculated into 5 ml Nutrient Broth for E. coli and 5 ml Nutrient Broth containing 1.5% NaCl for S. aureus and V. cholerae, and then subsequent cultures were incubated at 37C for 8 hours. Further suspension of bacterial culture was used in the inhibitory activity assay. I nhibitory Activity Assay Inhibitory effect of LEO on the growth of indicator bacteria was carried by the agar diffusion method. The LEO was tested of its inhibitory activity against E. coli, S. aureus, and V. cholerae. The media used were Nutrient Agar, the steril 5
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agar medium was poured in petri dishes 2-3 mm thick (15 ml) and allowed to solidify. Indicator bacteria in Nutrient Broth were shaken using vortex to obtain a suspension of the bacteria. Then 200 l suspension of bacteria were spread on the surface of Nutrient Agar lawn by a bent glass rod and then allowed to dry for 30 minutes. Furthermore sterile paper discs with a diameter of 8 mm spotted by 50 l LEO of suitable concentration treatment, then placed paper discs on agar plates that have inoculated indicator bacteria suspension. Further, the agar plates were incubated for 24 hours at 37C. After 24 hours the plates were observed for the presence of a clear zone around the paper disc which is the inhibitory activity formation and the diameter of the inhibition areas in mm were measured using a caliper. Inhibition area is the average total diameter of inhibition reduced by paper disc diameter. Inhibition activity is positive if formed of at least 2 mm clear zone around the paper disc that showed inhibition of the growth of pathogenic bacteria test. Determination of Minimum I nhibitory Concentration In the process of determining the MIC, the medium used was sterile NA. Agar medium was poured in petri dishes 2-3 mm thick (15 ml) and allowed to solidify. Bacterial suspension of about 200 l was then inoculated and spreaded on agar lawn and then allowed to dry for 30 minutes. Furthermore sterile paper discs with a diameter of 8 mm to 50 mL spotted by LEO of 0.2%, 0.4%, 0.6%, and 0.8% and held for 5 minutes for dispersing LEO into the paper disc, then placed them on agar plates that have inoculated by bacterial suspension test. Further agar plates were incubated for 24 hours at 37C. After 24 hours the plates were observed in the presence of a clear zone around the paper disc. The MIC was determined by the minimal concentration of LEO showed inhibitory activity. Results and Discussion Chemical Composition of LEO Chemical composition of LEO determined by GC-MS showed that several components were detected as illustrated in Figure 1. By using the library three major compounds were identified as two isomers of citral and 3-carene, which citral were the dominant compounds contained in LEO (Table 1). This result of 6
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determination was in agreement with the obtained that the components of LEO were dominated by neral and geranial, which were two geometric isomers of aldehyde citral (Hanaa et al., 2012). Paranagama et al. (2003) confirmed by using thin layer chromatography (TLC), citral a and b are constituents LEO that has fungicidal activity.
Figure 1. GC-MS Chromatogram of essential oil of lemongrass leaf. Table 1. Bioactive compounds identified in essential oil of lemongrass leaves Peak No. Retention time (min) Compounds Molecular formula Relative conct. (%) Class of compound 6 11.61 Z-Citral C 10 H 16 O 32.93 monoterpene 7 11.78 3,7,7-trimethyl Bicyclo hept- 3-ene (3-Carene) C 10 H 16 3.82 monoterpene 8 12.09 E-Citral C 10 H 16 O 37,85 monoterpene
3-Carene 7
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I nhibitory Activity This study used sterile distilled water as a negative control and positive control used amoxicillin and tetracycline. The test results showed that the concentration of 30 g/100 ml amoxicillin with volume of 50 l was able to inhibit E. coli with diameter of 23.3 mm and 22.5 mm for S. aureus, and tetracycline concentration of 30 g/100 ml with a volume of 50 l was able to inhibit V. cholerae with diameter of 21.3 mm. Amoxicillin and tetracycline are a broad-spectrum antibiotic that inhibits bacterial Gram-positive and Gram-negative. The diameter of the resulting inhibition confirmed that E. coli, S. aureus, and V. cholerae were sensitive to amoxicillin and tetracycline. The results also showed that concentration of LEO highly significant affected the growth of E. coli, S. aureus and V. cholerae. The LEO inhibition activities against E. coli, S. aureus and V. cholerae are given in Table 2. Table 2. Inhibition diameter (mm) of LEO against E.coli, S. aureus, and V. cholerae LEO Conct. E. coli S. aureus V. cholera 1% 6,030,5 e 4,970,5 e 9,675,7 d 2% 7,030,5 d 5,970,5 d 10,675,7 c 3% 8,030,5 c 6,970,5 c 11,675,7 b 4% 9,030,5 b 7,970,5 b 12,835,7 a 5% 9,930,5 a 8,870,5 a 13,675,7 a Note: same letter behind the average value showed no significant difference (p<0.05)
All levels of treatment showed inhibition against test bacteria. LEO 5% concentration treatments resulted in inhibition diameter against E. coli, S. aureus, and V. cholerae was about of 9.93 mm, 8.87 mm, and 13.67 mm, respectiovely. LEO still gave inhibition at a concentration of 1% which was of 6.03 mm, 4.97 mm, and 9.67 mm, respectively. The results were consistent with those expressed by Eddy (2009) which states that the essential oil extracts from plants can suppress the growth of microorganisms. The higher concentration of the volatile oil content a higher of antimicrobial compounds, so the inhibition of the growth of the bacteria will become larger. Figure 1 shows the inhibitory effects of LEO against pathogenic bacteria E. coli, S. aureus, and V. cholerae.
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Figure 2. Inhibition of LEO of lemongrass against the growth of E. coli, S. aureus, and V. cholerae. A: 1% oil concentration; B: 5% oil concentration.
According Arswendiyumna (2010), compounds that have antimicrobial properties contained in lemongrass is compounds group of terpene which determined in the essential oil fraction. Derivative terpenes contained in the lemongrass oil are geranial ( citral) and neral (citral ). Inhibition of leaf oil of lemongrass against bacteria S. aureus is suspected as phenolic compounds, which are present in the essential oil of lemongrass leaves. The compounds contained in LEO inhibits the microbes by damaging the structure of peptidoglycan (protein) present in the cell wall and protein denaturation, which can damage cell walls or membranes and inactivate enzymes. In general, the inhibition activity can be caused by interference constituent compounds of the cell wall, increased permeability of the cell membrane that can lead to loss of the components of cells, inactivate enzymes, and the destruction or damage to the genetic material. Minimum inhibitory concentration (MIC) of LEO against E. col , S. aureus, and V. cholerae was of 1 %, 0.8 % and 0.6 %, respectively, with successive inhibition was equal to 6.03 mm , 4.77 mm and 3.70 mm (Table 2) . The results of this study indicated that V. cholerae was more sensitive than E. coli and S. aureus to the LEO. Figure 2 shows the inhibition activity of LEO against E. coli, S. aureus, and S. aureus E. coli V. cholerae A B 9
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V. cholerae at MIC by agar diffusion method . The results are consistent with those disclosed in Nursini (2005) that the natural antimicrobial compounds are highly effective in inhibiting or killing foodborne microbes, although it is used in relatively small concentrations. Generally, Gram- negative bacteria is inhibited by essential oils at lower concentrations than Gram-positive bacteria. Table 2. Minimum inhibitory concentration (MIC) of LEO against Escherichia coli, Staphylococcus aureus, and Vibrio cholerae. LEO Conct. Diameter Inhibition. (mm) E.coli (mm) S.aureus (mm) V.cholerae (mm) 0,2% 0 0 0 0,4% 0 0 0 0,6% 0 0 3,700,51c 0,8% 0 4,770,05 b 4,430,98 b 1% 6,030,05 a 4,970,05 a 9,670,57 a Keterangan : huruf yang sama dibelakang nilai rata-rata menunjukkan perbedaan tidaknyata (P > 0,05)
E Figure 2. Minimum inhibitory concentration (MIC) of LEO on the growth of E. coli, S. aureus, and V. cholerae.
Conclusion Essential oil extracted from leaves of lemongrass (Cymbopogon citratus) could significantly inhibit the growth of E. coli, S. aureus and V. cholerae. Vibrio cholerae was more sensitive than E. coli and S. aureus. The minimum inhibitory concentration of the essential oils to the inhibition of V. cholerae, S. aureus, and E. coli was of 0.6%, 0.8%, and 1%, respectively. Further research is being carried out against spoilage bacteria and other pathogens and various types of fungi.
E. coli 1% S. aureus 0,8%, V. cholerae 0,6% 10
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Acknowledgement We are grateful thank to USAID TPC project for funding the experiment and coming to the national seminar of IAFT 2013. References Arana-Sanchez, A., Estarron-Espinosa, M. Obledo-Vazquez, E.N., Padilla- Camberos, E., Silva-Vazquez, R. and Lugo-Cervantes, E. 2010. Antimicrobial and antioxidant activities of Mexican oregano essential oils (Lippia graveolens H. B. K.) with different composition when microencapsulated in b-cyclodextrin. Letters in Applied Microbiology 50: 585590 Ardiansyah. 2007. Antimikroba dari Tumbuhan. (http://www.berita iptek.com/berita berita iptek). Diakses tanggal 7 Maret 2013. Arswendiyumna, R. 2010. Minyak Atsiri Dari Daun Dan Batang Tanaman Dua Spesies Genus Cymbopogon, Famili Gramineae Sebagai Insektisida Alami Dan Antibakteri. Prosiding Skripsi. Semester Genap. Fakultas MIPA. ITS, Surabaya. Bishop, C.D. & Thornton, I.B. (1997). Evaluation of the antifungal activity of the essential oils of Monarda citriodora var. citriodora and Melaleuca alternifolia on post harvest pathogens. Journal of Essential Oil Research, 9, 7782. Burt, S. 2004. Essential oils: their antibacterial properties and potential applications in foods a review. Int. J. Food Microbiol. 94: 223-253. Cellini, L., Di Campli, E., Masulli, M., Di Bartolomeo, S., and Allocati, N. 1996. Inhibition of Helicobacter pylori by garlic extract (Allium sativum). FEMS Immunology and Medical Microbiology 13: 273-277. Eddy, S. 2009. Daya Hambat Zat Anti Mikroba Ekstrak Daun Sambiloto (Andrographis paniculata (Burm.f) Ness) terhadap Pertumbuhan Jamur Candida albicans Secara In-Vitro.Vol 6 (1) : 9-15 Francisco V, Costa G, Fiqueirinha A, Marques C, Pereira P, Miquel Neves B. 2013. Anti. Inflamatory Activity of Cymbopogon citratus Leaves Infusion Via Proteasome and Nuclear factor - K B Pathway Inhibition : Contribution of Chlorogenic Acid. J. Ethnopharmacol. 148 (1) : 26-34 Guenther, E. 1987. The Essential Oils. Penerjemah S. Ketaren. Minyak Atsiri (Jilid I). UI-Press, Jakarta. Gupte, S. 1990. Mikrobiologi Dasar. Edisi Ketiga. Penerjemah Julius, E.S. Binarupa Aksara, Jakarta. Hanaa, A. R. M., Sallam, Y.I., El-Leithy, A.S., and Aly, S.E. 2012. Lemongrass (Cymbopogon citratus) essential oil as affected by drying methods. Annals of Agricultural Science. 57(2): 113116 11
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