Antibacterial abietane-type diterpenoid, taxodone from Metasequoia glyptostroboides Miki ex Hu 533 Brief communication Antibacterial abietane-type diterpenoid, taxodone from Metasequoia glyptostroboides Miki ex Hu VIVEK K BAJPAI and SUN CHUL KANG* Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea *Corresponding author (Fax, +82-53-850-6559; Email, [email protected]) In an attempt to isolate bioactive constituents, ethyl acetate cone extract of Metasequoia glyptostroboides was subjected to a column chromatographic analysis that resulted in isolation of an abietane-type diterpenoid, taxodone. Its structure was elucidated by spectroscopic means. Further, taxodone showed potential antibacterial effect as diameters of zones of inhibition against foodborne pathogenic bacteria, such as Listeria monocytogenes ATCC 19166, Salmonella typhimurium KCTC 2515, S. enteritidis KCTC 2021, Escherichia coli ATCC 8739, E. coli O157:H7 ATCC 43888, Enterobacter aerogenes KCTC 2190, Staphylococcus aureus ATCC 6538 and S. aureus KCTC 1916, were found in the range of 9.4 to 14.2 mm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of taxodone against the employed bacterial pathogens were found in the range of 250 to 1000 and 250 to <2000 μg/ml, respectively. Also the compound had a strong antibacterial effect on the viable counts of the tested bacteria. These fi ndings indicate that the bioactive compound taxodone present in M. glyptostroboides could be used as an antibacterial agent in food industry to inhibit the growth of certain important foodborne pathogens. [Bajpai V K and Kang S C 2010 Antibacterial abietane-type diterpenoid, taxodone from Metasequoia glyptostroboides Miki ex Hu; J. Biosci. 35 533–538] DOI 10.1007/s12038-010-0061-z 1. Introduction reported to have various biological activities (Gao and Han 1997; Singh and Singh 2003). Plants contain a great number of secondary metabolites, Foodborne pathogenic bacteria cause several foodborne many of which display biological activity as “natural diseases (Tauxe 2002). The indiscriminate and excessive products” with a role in plant defense against bacteria, fungi use of a wide range of chemical bactericides has led to and other microorganisms. There has been a growing interest extended environmental pollution and the production of in new and effective antibacterial substances from natural resistant pathogen populations. During the last few years, sources like plants to reduce cases of bacterial diseases. In research on plant-based antibacterial agents has produced a many cases, antibacterial compounds from plants, herbs and diverse range of products with novel modes of action, which spices in low concentrations have been used effectively in are expected to have a signifi cant impact on the control of the food industry. Biologically active secondary metabolites foodborne diseases in coming decade. Therefore, the demand of spices, herbs and medicinal plants, due to their diverse for plant based antimicrobials as novel bactericide sources range of antibacterial activities, are becoming increasingly for controlling foodborne pathogens is rapidly increasing important in food industry as potential food preservatives (Ulubelen et al. 2000; Kubo et al. 2004; Giang et al. 2006). to control foodborne pathogenic bacteria. Several terpenoid Some synthetic chemicals have been used to control compounds have been isolated from many plant species and microbial growth and reduce the incidence of foodborne Keywords. Antibacterial activity; diterpenoid; foodborne pathogens; Metasequoia glyptostroboides; taxodone Abbreviations used: DMSO, dimethylsulphoxide; KFDA, Korea Food and Drug Administration; LB, Luria–Bertani; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration http://www.ias.ac.in/jbiosci J. Biosci. 35(4), December 2010, 533–538, © IndianJ. Biosci. Academy 35(4), of December Sciences 2010 533 534 Vivek K Bajpai and Sun Chul Kang illness. Although these synthetic preservatives are effective, Korea Food and Drug Administration (KFDA), and were they might be detrimental to human health. Consumers are maintained on Luria–Bertani (LB) agar medium at 4°C. concerned about the safety of food products that contain artifi cial preservatives. Recently, the ban of chemical 2.3 Plant material additives by some consumers has driven the food industry and food research towards the search for natural antibacterial The cones of M. glyptostroboides were collected from the compounds from plant origin. Many natural compounds local area of Pohang, Republic of Korea, in November and (e.g. terpenoids) found in dietary plants possess antibacterial December 2008, and identifi ed by the morphological features activities against foodborne pathogens (Dellar et al. 1996; and the database present in the library at the Department Singh and Singh 2003; Aiyelaagbe et al. 2007). of Biotechnology. A voucher specimen (DUB-0038) was Metasequoia glyptostroboides Miki ex Hu is a deciduous deposited in the herbarium of College of Engineering, conifer of the redwood family of Cupressaceae. It is only Department of Biotechnology, Daegu University, Republic living species in the genus Metasequoia propagated and of Korea. distributed in many parts of eastern Asia and North America as well as Europe. Previously we reported various biological properties of organic extracts and essential oils from M. 2.4 Extraction and isolation glyptostroboides such as antibacterial (Bajpai et al. 2007a; Bajpai and Kang 2009b), antioxidant/antibacterial (Bajpai Dried cones of M. glyptostroboides (2 kg) were milled et al. 2009a), antidermatophytic (Bajpai et al. 2009c) and into powder and then extracted with ethyl acetate at room antifungal (Bajpai et al. 2007b) activities. However, there is temperature for 12 days. The extract was evaporated no report available in the literature on the biologically active under reduced pressure using a rotary evaporator (EYELA constituents of M. glyptostroboides and their antibacterial N1000). The dried ethyl acetate extract (7 g) was subjected properties against foodborne pathogenic bacteria. to column chromatography over silica gel (230–400 mesh, In this study, we report for the fi rst time an abietane-type Merck, Germany) and was eluted with hexane-ethyl acetate- diterpenoid, taxodone from the ethyl acetate cone extract methanol solvent system to give 20 fractions. Of the fractions of Metasequoia glyptostroboides and evaluated its effi cacy obtained, the fraction-12 was further purifi ed by preparative as a potential antibacterial agent against some important TLC over silica gel GF254 by using hexane-ethyl acetate (2: foodborne pathogens. 1) as a mobile phase to give one compound (96 mg). 2. Materials and methods 2.5 Taxodone 2.1 General The purifi ed compound was crystallized from n-hexane- methanol to yield yellow round crystals (96 mg), mp 176– 1 Melting points were recorded on a Gallenkamp apparatus. 177°C; H NMR (pyridine-d5, 250 MHz) δ: 5.30 (1H, m, W1/2 Optical rotations were measured using a JASCO DIP-1000 = 23), 3.45 (1H, d, J = 2.5 Hz), 3.19 (1H, br s), 7.00 (1H, (Tokyo, Japan) automatic digital polarimeter. IR spectra septet), 2.51 (1H, s, 11-OH), 8.78 (9H), 8.83 (3H), 8.90 (3H); 13 (KBr) and UV spectra (MeOH) were obtained from Shimadzu C NMR (pyridine-d5, 250 MHz) δ: 181.6 (C-1), 149.3 (C- DR-8001 IR and LKB 4053 UV spectrophotometer, 2), 143.3 (C-3), 141.3 (C-4), 135.7 (C-5), 130.3 (C-6), 126.2 respectively. 1D and 2D NMR spectra were measured in (C-7), 77.5 (C-8), 77.0 (C-9), 76.4 (C-10), 69.9 (C-11), 57.8 (C-12), 43.1 (C-13), 40.6 (C-14), 36.6 (C-15), 34.1 (C-16), CDCl3 on a Brucker ARX 250 MHz instrument with TMS as an internal standard. Purity of the compounds was checked 26.6 (C-17), 21.3 (C-18), 20.7 (C-19), 18.7 (C-20). by TLC and HPLC. 2.6 Antibacterial activity assay 2.2 Microorganisms Standard agar diffusion method was used for antibacterial Microorganisms as foodborne pathogenic bacteria such assay (Murray et al. 1995). Petri plates were prepared by as Listeria monocytogenes ATCC 19166, Salmonella pouring 20 ml of LB medium and allowed to solidify. typhimurium KCTC 2515, S. enteritidis KCTC 2021, Plates were dried, and 100 μl of standardized inoculum Escherichia coli ATCC 8739, E. coli O157:H7 ATCC 43888, containing 107 CFU/ml of bacterial suspension was poured Enterobacter aerogenes KCTC 2190, Staphylococcus and uniformly spread. The inoculum was allowed to dry aureus ATCC 6538 and S. aureus KCTC 1916 were used in for 5 min. A Whatman No. 1 sterile fi lter paper disc (6 mm this study. The bacterial pathogens were obtained from the diameter) was impregnated with 50 μg/disc of the compound J. Biosci. 35(4), December 2010 Antibacterial abietane-type diterpenoid, taxodone from Metasequoia glyptostroboides Miki ex Hu 535 on the agar surface in the lowest concentration of sample was defi ned as the minimum bactericidal concentration (MBC). Each assay in this experiment was replicated three times. 2.8 Effect of taxodone on viable count of the tested bacteria Active cultures for viable count assay were prepared in LB broth medium (Bajpai et al. 2009a). For each strain, 1 ml of active stock solution was transferred to 4 ml of LB broth. All treated cultures were kept at 37°C for 2 h. The cultures were then centrifuged at 10000 rpm for 10 min. The pellets were retained and re-suspended with 1 ml of phosphate-buffered saline. For viable counts, each of the tubes containing re- suspended bacterial suspension (approximately 107 CFU/ Figure 1. Chemical structure of taxodone isolated from the ethyl ml) of L. monocytogens ATCC 19166, E. coli ATCC 8739, acetate cone extract of Metasequoia glyptostroboides. S. aureus ATCC 6538 and E. aerogenes KCTC 2190 was inoculated with 250 μg/ml concentration of the compound in 10 ml LB broth and kept at 37°C. Samples for viable cell taxodone. The compound was dissolved in 5% DMSO.