Antimicrobial Activity of Essential Oil from Nelumbo nucifera Gaertn. Pollen

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Life International Journal of Pharmacology ISSN 1811-7775 Life science alert ansinet Asian Network for Scientific Information

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International Journal of Pharmacology 5 (1): 98-100. 2009 ISSN 1811-7775 2009 Asian Network for Scientific Information Antimicrobial Activity of Essential Oil from Nelumbo nucifera Gaertn. Pollen Chaiyasit Sittiwet Biomedical Research Unit, Department of Chemistry, Faculty of Science, Mahasarakham University, Khamreung, Kantharawichai, Mahasarakham 44150, Thailand Abstract: The Nelumbo nucifera Gaertn. pollen essential oil was extracted by using vapor distillation. Antimicrobial activity of Nelumbo mucifera Gaertn. pollen essential oil has been investigated using agar diffusion susceptibility test and broth macro-dilution. The essential oil were tested against both gram positive (S. aureus ATCC 25923, S. epidermidis ATCC 12228, M. luteus ATCC 9341, B. subtillis ATCC 6633 and 1. plantarum ATCC 14917) and gram negative (F. coli ATCC25922, S. typhimurium ATCC 14028. K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315, Ps. aeruginosa ATCC 9721) bacteria using agar diffusion susceptibility test. Significant zone of inhibition were observed for S. typhimurium ATCC14028 and E. coli ATCC25922). The MICS and MBCs are 10-10 and 20-80 ml L', respectively. Nelumbo mucifera pollen essential oil show inhibitory effect on growth of food born pathogen bacteria in low concentration which indicated the possibility of used as food preservation additive. Key words: Nelumbo nucifera, food borne pathogen, anti-microbial, Nymphaceae INTRODUCTION The Nelumbo mucifera Gacitn. is an aquatic plant belonging to the family Nymphaceae (Gaertn) which almost all parts are edible (La-ongsri et al., 2008). The leaves, root and the embryonic stage of the plant have reported to contain alkaloids such as such as roemerine, nuciferine. normiciferine, nelumboside. anonaine, 5-methoxy-5-hydroxyaporphine, liensinine and asimilobine (Kulkarni and Juvekar, 2008). It has been reported to have anti-stress (Kulkarni and Juvekar, 2008), anti-obesity (Ono et al., 2006) and anti-oxidant (Rai et al., 2006), hepatoprotective (Sohn et al., 2003), anti-diabetic activity (Mukherjee et al., 1997a), anti-inflammatory (Mukherjee et al., 1997b), antipyretic (Mukherjee et al., 1996) activities. The anti-malaria and antifungal activity of N. mucifera extract have also been reported (Agnihotri et al., 2008). The leaves have been indicated as part of the plant. responsible for its anti-malarial activity (Li and Xu, 2008). In many Asian countries N. nucifera part was found in the recipe of food and folklore such as root and seed (La-ongsri et al., 2008). However, the N. nucifera pollen essential oil anti-microbial activity has never been reported. The present study deals with the antimicrobial activity of the essential oil extracted from N. nucifera. MATERIALS AND METHODS Sample collection identification and extraction: The N. nucifera was collected on May 2008 from Ayuthaya Province. Thailand The plan was identified by Department. of Biology: Faculty of Science. Mahasarakham University, Thailand. Ten gram of dried flowers were hydro-distilled in 500 mL water and repeat 3 times. The yield of vapor distillation was 0.5-1% of dried weight of dried plant's flowers. In this study used pooled of batches of the same condition of distillation throughout the studies. Microbial cultures: Laboratory isolates of the pure culture of gram positive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus subtillis ATCC 6633, Lactobacillus plantarum ATCC 14917) and gram negative bacteria (Escherichia coli ATCC25922. Salmonella typhimurium ATCC 14028, Klebsiella pneumonia ATCC 10031, Pseudomonas aeruginosa ATCC9721, Proteus vulgaris ATCC 13315) were obtained from the Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand. Antimicrobial sensitivity test Agar disc diffusion test: The disc diffusion method used. as described in the standard guideline technique (Lorian 1996). All test bacteria were cultured overnight on Tryplic Soy Agar (TSA) slant at 37 C. Bacteria were washed from surface agar slant with sterile normal saline solution (0.9% NaCl) then adjusted to match turbidity of standard Mcfarland No. 0.5 before used as starter 98

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Int. J. Pharmacol., 5 (1): 98-100, 2009 solution. Twenty milliliter of Mueller Hinton agar (MHA) was put in cultivation plates and leave until the agar become rigid. The starter solution was swabbed on agar surface by using swab collon. N. mucifera essential oil was dissolved in olive oil (1.25, 2.5 and 5% v/v) and put in sterile stainless steel cylinders (6 mm internal diameter and 10 mm height) were placed on the inoculated agar surface. The various concentrations of plant extract solution were filled in the cylinders (300 μL cylinder After pre-diffusion at room temperature for 1 hour, the plates were incubated at 37 °C for 19 h. The NSS (normal saline solution) filled in the cylinder was used as control and 10 mg L gentamicin sulphate (Sigma Chemical Co., St. Louis, USA) solution was used as standard in same cultivation plate. MICs and MBCs determination using agar dilution and broth macro dilution methods: MICs of crude water extract of N. nucifera essential oil were determined by agar dilution method (Merck) (Lorian, 1996) while MBCs were determined by broth macro-dilution method were (Lorian, 1996). Gentamicin sulphate (Sigma Chemical Co.. St. Louis, USA) was used as a reference antibiotic. Inoculates were prepared in the same medium at density adjusted to 0.5 McFarland turbidity standard (10³ colonyforming units (cfu mL ') and two fold dilution for the broth macro-dilution procedure. The inoculated tube were incubated at 37°C and the MICs were recorded aller 24 h of incubation. The MIC was defined as the lowest concentration of ME or gentamicin sulphate at which the microorganism lested did not showed visible growth while MBC was defined as the minimuni bactericidal concentration with negative subcultures on agar medium. Values were means of triplicate. RESULTS AND DISCUSSION The control growth of food home pathogen bacteria such as S. typhimurium, E. coli, S. aureus, Ps. aeruginosa and B. subtilis are common in the tropical countries especially in country where poultry products are produced (Van Dijk et al., 2007). The present study has investigated anti-microbial activity of N. nucifera which used in food and folklore. Furthermore, N. mucifera has almost every part is edible which also mean less toxicity. Therefore incorporation of N. nucifera in to food as food bom protection consider less toxic. In this study antimicrobial activity of N. nucifera pollen essential oil has been test against various bacteria include food born pathogen bacteria. The anti-malarial and antifungal activity of N. nucifera extract has been reported (Agnihotri et al.. 2008). In earlier study indicated that the leaves of N. nucifera may responsible for antibacterial activity (Li and Xu, 2008). However, the pollen of N. mucifera has never been investigated even if used as the ingredient of food and some folkloric medicine. In this study the essential oil of N. nucifera were tested against various bacteria to screen the possibility to use it as food preservation additive. The N. nucifera pollen essential oil showed anti-microbial activity against gram negative S. typhimurium ATCC14028 and E. coli ATCC 25922 (Table 1). The MICs are 10 and 40 and MBCs are 20 and 80 ml L (Table 2). In conclusion N. mucifera pollen essential oil show inhibitory effect on growth of food born pathogen bacteria in low concentration which indicated the possibility of used as food preservation additive. Table 1: Diameter of zone of inhibition of bacterial organisms of N. nucifera pollen essential oil against various bacteria Inhibition zone diameter (in) Bacteria Gram N. accifera (5% v/v) N. nucifera (2.5% v/v) N. nucifera (1.25% v/v) S. aureus ATCC 25923 + 012 JIZ [LZ Gentamicin sulphate (10 μg mL-¹) 21.0±1.00 S epidermidis ATCC 12228 + [12 JIZ [L 19.8±1.73 Mateus ATCC 9341 [12 [L 19.0±0.61 B. subtilis ATCC 6633 [12 SIZ [L 18.7±1.47 L. plantarum ATCC 14917 + [12 JIZ [L 22.3±1.85 E. coli ATCC 25922 19.3 JIZ [L 20.6±0.88 K preumoniae ATCC 10031 [12 16.4±1.11 S typhimurium ATCC 14028 17.5 12.5 [LZ 16.5±1.10 Ps. aeruginosa ATCC 9721 [12 JIZ [LE 17.9±1.61 P. vulgaris ATCC13315 [12 JIZ 20.7±0.41 Data are mean SD (n = 3); nz: No inhibition zone Table 2: The MICs and MBCs of N. nucifera pollen essential oil against various bacteria N nucifera pollen cssential oil Bacteria MIC (ml L¹) E. coli ATCC 25922 40 Styphimurian ATCC 14028 10 ud: Not Determine 99 Gentamicin sulphate MBC (ml L-) 80 20 MIC (μg mL-¹) <0.5 <0.5 MBC (ug mL-¹) nd nd

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Int. J. Pharmacol., 5 (1): 98-100, 2009 ACKNOWLEDGMENT Author would like to express the appreciation to partially support from faculty of Science, Mahasarakham University, Thailand. REFERENCES Agnihotri, V.K., H.N. EISohly, S.I. Khan, M.R. Jacob and V.C. Joshi, 2008. Constituents of Nelumbo mucifera leaves and their antimalarial and antifungal activity. Phytochem. Lell. 1: 89-93. Kulkarni MP. and A.R. Juvekar, 2008. Attenuation of acute and chronic restraint stress-induced perturbations in experimental animals Nelumbo nucifera Gaertn. Indian. J. Pharmacol. Sci., 70: 327-332. by La-ongsri, W., C. Trisonthi and H. Balslev, 2008. Management and use of Nelumbo mucifera Gaertn. in Thai wetlands. Wellands Ecol. Mariage. (In Press). Li, M. and Z. Xu, 2008. Quercetin in a lotus leaves extract may be responsible for antibacterial activity. Arch. Pharm. Res., 31: 640-644. Lorian, V., 1996. Antibiotics in Laboratory Medicine. 4th Edn., Williams and Wilkins, Baltimore, London, ISBN: 97807817-19831. Mukherjee, P.K., J. Das, K. Saha, S.N. Giri, M. Pal and B.P. Saha, 1996. Antipyretic activity of Nelumbo mucifera rhizome extract. Indian. J. Exp. Biol., 34: 275-276. Mukherjee, P.K., K. Saha, M. Pal and B.P. Saha, 1997a. Effect of Nelumbo mucifera rhizome extract on blood sugar level in rats. J. Ethnopharmacol., 58: 207-213. Mukherjee, P.K., K. Saha, J. Das, M. Pal and B.P. Saha, 1997b. Studies on anti-inflammatory activity of rhizomes of Nelumbo nucifera. Planta. Med., 63: 369-369. Ono, Y., L. Hattori, Y. Fukaya, S. Imai and Y. Ohizumi, 2006. Anti-obesity effect of Nelumbo nucifera leaves extracts in mice and rats. J. Ethnopharmacol.. 106: 238-2141. Rai, S., A. Wahile, K. Mukherjee, B.P. Saha and P.K. Mukherjee, 2006. Antioxidant activity of Nelumbo nucifera (sacred lotus) seeds. J. Ethnopharmacol., 104: 322-327. Sohn, D.II., Y.C. Kim, S.II. Oh, E.J. Park, X. Li and B.H. Lee, 2003. Hepatoprotective and free radical scavenging effects of Nelumbo mucifera. Phytomedicine, 10: 165-169. Van Dijk, A., E.J. Veldhuizen, S.I. Kalkholve J.L. Tjeerdsma-Van Bokhoven, R.A. Romiin and H.P. Haagman, 2007. The beta-defensin gallinacin-6 is expressed in the chicken digestive track and has antimicrobial activity against food-borne pathogens. Antimicrob Agents Chemother, 51: 912-922. 100