Original Article 77

ANTIMICROBIAL ACTIVITIES OF ESSENTIAL OIL FROM ETLINGERA PUNICEA Sarin Tadtong1, Piyapan Wannakhot1, Wanrawin Poolsawat1, Sirivan Athikomkulchai1,∗ and Nijsiri Ruangrungsi2,3

1Faculty of Pharmacy, Srinakharinwirot University, Nakhonnayok 26120, 2Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand

ABSTRACT: The purpose of this study is to determine the chemical compositions and the antimicrobial activities of the essential oil from rhizome of Etlingera punicea (Roxb.) R.M. Smith. The rhizome of is used as spice and ingredient in noodle and curry in Thailand. Essential oil from rhizome of the plant was extracted by hydrodistillation and analyzed by GC/MS. The major composition of the essential oil is methyl chavicol. This essential oil possesses fungicidal activity against Candida albicans ATCC 10231 and shows bacteriostatic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella albany DMST 10641, whilst no activity was found on gram negative bacteria Pseudomonas aeruginosa ATCC 27853 by agar disc diffusion assay. The minimal inhibitory concentration (MIC) against Candida albicans ATCC 10231 was further determined by broth microdilution assay which was 45%v/v. Keywords: Etlingera punicea, antimicrobial, volatile oil, methyl chavicol

INTRODUCTION: is a good clevenger apparatus. The volatile oil was collected º resource of spices. It is a large family comprising and stored at 2-4 C until being used. of more than 200 species. The large genera found Essential oil composition analysis in Thailand are Curcuma, Hedychium, Zingiber, The chemical analysis was done by gas Alpinia, Kaempferia and Etlingera. Etlingera chromatography mass spectrometry (GC/MS) on punicea (Roxb.) R.M. Smith is also in the family a Finnigan Trace GC ultra (Thermo Electron 1) Zingiberaceae . The rhizome is the source of Corporation, USA) with quadrupole mass spice, the ingredient in noodle and curry which spectrometer. The column was ZB-5 fused silica are commonly used in Chantaburi province, linked methyl silicon capillary column (30 m. x 2) Thailand . The objectives of this study are to 0.22 mm. i.d.; 0.25 μM); oven temperature determine the chemical compositions and programming was 50-250°C at 7°C/min; injector evaluate the antimicrobial activities of essential oil and detector temperature were 250 and 280°C, from the rhizome of E. punicea. respectively; sample volume injected was 1 μl; MATERIALS AND METHODS: split ratio was 100:1; and the carrier gas was He (2 ml/min). Plant materials Compounds were identified by comparing the The of E. punicea were collected from Kovats gas chromatographic retention indices of Chantaburi Province, Thailand during July- the peaks on the HP-5MS column with literature August, 2008. The plant was identified by one of values, computer matching using the Masslynx us (NR.). The voucher specimen (SWU 131910) database, and comparison of the fragmentation was deposited in the herbarium at the Faculty of patterns of the mass spectra with those reported Pharmacy, Srinakharinwirot University, Nakhon- in the literature3-4). nayok Province, Thailand. Antimicrobial assay Extraction of essential oil Test organisms Plant materials were cut into small pieces. The bacterial strains used were the gram They were extracted by hydrodistillation with positive bacteria, Staphylococcus aureus ATCC

∗To whom correspondence should be addressed. E-mail: [email protected], Tel.+66 2664 1000 ext. 1700, Fax.+66 3739 5096 J Health Res 2009, 23(2):77-79 78 Original Article

25923, the gram negative bacteria, Escherichia Tryptic soy broth or Saboraud’s dextrose broth coli ATCC 25922, Pseudomonas aeruginosa ATCC and then diluted to make the stock solutions at 27853, and Salmonella albany DMST 10641. The concentration of 90% v/v, 80% v/v, 70% v/v, fungus used was Candida albicans ATCC 10231. 60% v/v, and 50% v/v, respectively. MIC values of All bacteria were cultured in Tryptic soy broth the essential oil against the test microorganisms at 37°C for 24 h prior to use while the fungus was were evaluated based on a microdilution cultured on Saboraud’s dextrose agar at 37°C for method5). 24 h then suspended in Saboraud’s dextrose In brief, the 96-well plates were prepared by broth prior to use. applying 95 μl of Tryptic soy broth or Saboraud’s Saboraud’s dextrose broth was prepared by dextrose broth and 5 μl of the inocula into each filtering the suspension of Saboraud’s dextrose well. A hundred μl of each stock solution of the agar in water through cotton for removing the essential oil was added into the wells to make the agar before sterile. test concentrations at 45% v/v, 40% v/v, 35% v/v, 30% v/v, and 25% v/v, respectively. The well Chemicals containing 200 μl of the medium without the Tryptic soy broth, Muller-Hinton agar, and essential oil and inocula was used as negative Sabouraud dextrose agar were purchased from control. Neomycin (1 mg/ml), amoxicillin (1 Himedia Laboratories Limited (). Amoxicillin, mg/ml), and clotrimazole (1 mg/ml), were used as neomycin, and clotrimazole were purchased from positive control. The plate was incubated at 37°C Sigma Chemical CO (USA). Dimethylsulfoxide for 24 h. Microbial growth was determined by (DMSO) was purchased from Sigma-Aldrich absorbance at 600 nm using the microplate (USA). reader (Anthos, Zenith 200rt). Three independent Agar disc diffusion method experiments were performed and each experiment The antimicrobial assay was performed by the was run in triplicate. agar disc diffusion method5). In brief, microbial RESULTS AND DISCUSSION: The GC/MS suspension containing 108 CFU/ml of bacteria or results in Table 1 showed that the major fungus was swabbed and spread on Muller- compounds in the volatile oil from the rhizomes of Hinton agar or Sabouraud dextrose agar, E. punicea was a phenolic compound derivative respectively. The essential oil (20 μl) was applied namely methyl chavicol (estragole or estragol)6) on paper disc (6 mm in diameter) and placed on (Fig 1). the inoculated agar. Neomycin (20 μg/disc), amoxicillin (20 μg/disc), and clotrimazole (20 μg/disc), were used as positive control standards to determine the sensitivity of gram negative bacteria, gram positive bacteria, and fungus, H CO respectively. The inoculated plates were incubated 3 at 37°C for 24 h. The antimicrobial activity was Figure 1 Chemical structure of methyl chavicol evaluated by measuring the diameter of the zone of inhibition against the test microorganisms. The antimicrobial assay was performed by the Microdilution method agar disc diffusion method. The result was The minimal inhibitory concentration (MIC) obtained by measuring the zone of inhibition values were determined only for the microbial diameter (cm). strains, being sensitive to the essential oil in disc The oil revealed bacteriostatic activity against diffusion assay. Microbial suspension containing gram positive bacteria Staphylococcus aureus 108 CFU/ml of bacteria or fungus was adjusted to ATCC 25923 (0.87 cm), gram negative bacteria McFarland No. 0.5 standard turbidity. The Escherichia coli ATCC 25922 (0.78 cm), essential oil was dissolved in 10% DMSO in Salmonella albany DMST 10641 (0.82 cm), while

J Health Res 2009, 23(2): 77-79 Original Article 79

exhibited fungicidal activity against Candida REFERENCES: albicans ATCC 10231 (1.18 cm) whilst no activity 1. Puangpen S. 1998. Thai Zingiberaceae: was found on gram negative bacteria Species diversity and their uses. The International conference on biodiversity and Pseudomonas aeruginosa ATCC 27853 (Table 2). bioresources: conservation and utilization. The essential oil was only further evaluated for Phuket, Thailand. Pure Appl Chem 70(11): 2111. the minimal inhibitory concentration (MIC) 2. Available from: http://intranet.m-culture.go. against Candida albicans ATCC 10231 due to its th/chanthaburi/herbs.html [Accessed on April antifungal activity. The result showed the MIC at 18, 2009] 45% v/v. 3. Adams RP. 1995. Identification of Essential Oil Components by Gas Chromatography-Mass The bacteriostatic and anitifungal activities of Spectrometry. Allured: Illinois. the essential oil from the rhizome of E. punicea 4. Davies NW. 1990. Gas chromatographic might be due to the presence of methyl chavicol retention indices of monoterpenes and as previously reported7-9). The possible sesquiterpenes on methyl silicone and Carbowax antimicrobial action of methyl chavicol might be 20M phases. J Chomatogr 503: 1-24. arrived from the complexation between the 5. Sokmen A, Gulluce M, Akpulat HA, Daferera D, Tepe B, Polissiou M, et al. 2004. The in vitro protein or other components of the cell membrane antimicrobial and antioxidant activities of the of the microbes and the phenolic components in essential oils and methanol extracts of endemic the essential oil as previous reported8). Thymus spathulifolius. Food Control 15: 627-634. Furthermore, methyl chavicol showed limited and 6. Available from: http://en.wikipedia.org/wiki/ weak antibacterial activity9) which was similar to Methyl_chavicol [Accessed on May 22, 2009] our report. According to its antifungal activity, the 7. Oxenham SK, Svoboda KP, and Walters DR. 2005. Antifungal Activity of the Essential Oil of volatile oil from this plant might be used as an Basil (Ocimum basilicum). J Phytopathol 153(3): active ingredient in antifungal formulations. 174-180. 8. Omidbeygi M, Barzegar M, Hamidi Z, and Naghdibadi H. 2007. Antifungal activity of thyme, Table 1 Chemical constituents of E. punicea volatile oil from hydrodistillation summer savory and clove essential oils against Aspergillus flavus in liquid medium and tomato Compounds %Area Retention Time Monoterpenes paste. Food Cont 18:1518-1523. α -Pinene 1.05 5.65 9. Bagamboula, CF, Uyttendaele M, and β -Pinene 0.95 6.85 Debevere J. 2004. Inhibitory effect of thyme and Sylvestrene 0.91 8.46 β -Ocimene (E) 0.10 9.10 basil essential oils, carvacrol, thymol, estragol, Sesquiterpenes linalool and p-cymene towards Shigella sonnei β -Bisabolene 0.10 27.74 and S. flexneri. Food Microbiol 21: 33-42. α -Copaene 0.19 22.40 β -Selinene 0.22 26.85 δ -Cadinene 0.65 28.34 Phenolic Methyl chavicol 95.73 15.07

Table 2 Antimicrobial activities of E. punicea volatile oil

Microorganisms Inhibition zone (cm) S. aureus ATCC 25923 0.87 E. coli ATCC 25922 0.78 P. aeruginosa ATCC 27853 - S. albany DMST 10641 0.82 C. albicans ATCC 10231 1.18

ACKNOWLEDGEMENTS: This work was supported by the grant from the Faculty of Pharmacy, Srinakharinwirot University.

J Health Res 2009, 23(2):77-79