Antimicrobial and Anti-Inflammatory Activity of Five Taxandria Fragrans
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Microbiol Immunol 2008; 52: 522–530 doi:10.1111/j.1348-0421.2008.00070.x ORIGINAL ARTICLE Antimicrobial and anti-inflammatory activity of five Taxandria fragrans oils in vitro Katherine A. Hammer1, Christine F. Carson1, Janet A. Dunstan2,JasmineHale2, Heidi Lehmann2, Christopher J. Robinson3, Susan L. Prescott3 and Thomas V. Riley1,4 1Discipline of Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, and 2School of Paediatrics and Child Health, The University of Western Australia, Crawley, 3Greening Australia Western Australia, Albany, and 4Division of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Nedlands, Australia ABSTRACT The antimicrobial activity of five samples of Taxandr ia fragrans essential oil was evaluated against a range of Gram-positive (n = 26) and Gram-negative bacteria (n = 39) and yeasts (n = 10). The majority of organisms were inhibited and/or killed at concentrations ranging from 0.06–4.0% v/v. Geometric means of MIC were lowest for oil Z (0.77% v/v), followed by oils X (0.86%), C (1.12%), A (1.23%) and B (1.24%). Despite differences in susceptibility data between oils, oils A and X did not differ when tested at 2% v/v in a time kill assay against Staphylococcus aureus. Cytotoxicity assays using peripheral blood mononuclear cells demonstrated that T. fragrans oil was cytotoxic at 0.004% v/v but not at 0.002%. Exposure to one or more of the oils at concentrations of ≤0.002% v/v resulted in a dose responsive reduction in the production of proinflammatory cytokines IL-6 and TNF-α, regulatory cytokine IL-10, Th1 cytokine IFN- γ and Th2 cytokines IL-5 and IL-13 by PHA stimulated mononuclear cells. Oil B inhibited the production of all cytokines except IL-10, oil X inhibited TNF-α, IL-6 and IL-10, oil A inhibited TNF-α and IL-6, oil C inhibited IL-5 and IL-6 and oil Z inhibited IL-13 only. IL-6 production was significantly inhibited by the mostoils(A,B,CandX),followedbyTNF-α (oils A, B and X). In conclusion, T. fragrans oil showed both antimicrobial and anti-inflammatory activity in vitro, however, the clinical relevance of this remains to be determined. Key words Agonis, myrtaceae, myrtenol, terpenes. Taxandr ia fragrans (J.R. Wheeler & N.G. Marchant) J.R. the Myrtaceae family of plants, which contains approxi- Wheeler & N.G. Marchant, comb. nov., formerly known as mately 130 genera. Of these genera, several are well known Agonis fragrans (1, 2), is native to the south-west corner of for having essential oil-producing species. A few examples Western Australia. This region of Australia is internation- of these are eucalyptus oil from various Eucalyptus species, ally recognized for its biodiversity, particularly in relation tea tree from Melaleuca alternifolia (5), clove from Syzy- to flowering plants (3, 4). The genus Taxandr ia belongs to gium aromaticum (6), bay from Pimenta racemosa,kanuka Correspondence Katherine A. Hammer, Discipline of Microbiology and Immunology (M502), School of Biomedical, Biomolecular and Chemical Sciences, Uni- versity of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia. Tel: +61 8 9346 4730; fax: +61 8 9346 2912; email: [email protected] Received 28 March 2008; revised 20 June 2008; accepted 25 June 2008. List of Abbreviations: ATCC, American type culture collection; C. albicans, Candida albicans; cfu, colony forming units; CNS, coagulase-negative staphylococci; dpm, disintegrations per minute; E. faecalis, Enterococcus faecalis; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; K. pneumoniae, Klebsiella pneumoniae; LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; MIC90, minimum inhibitory concentration for 90% of isolates; MLC, minimum lethal concentration; MLC90, minimum lethal concentration for 90% of isolates; NCTC, National collection of type cultures; P. aeruginosa, Pseudomonas aeruginosa; PBS-Tw, phosphate buffered saline supplemented with 0.001% Tween 80; PHA, phytohaemagglutinen; S. aureus, Staphylococcus aureus; S. marcescens, Serratia marcescens; T. fragrans, Taxandria fragrans; Th, T-helper; TNF-α, tumor necrosis factor alpha 522 c 2008 The Societies and Blackwell Publishing Asia Pty Ltd Bioactivity of Taxandria fragrans oil from Kunzea ericoides and manuka from Leptospermum Table 1 Major components (%) of five samples of T. fragrans essential scoparium (7). These oils are produced commercially and oil† are marketed for a variety of uses, including as medicinal T. fragrans oil composition (%) and cleaning agents. Their uses may be largely attributed to their antimicrobial activity against a wide range of bacteria Component A B C X Z and yeasts (8) and for some to their anti-inflammatory ac- 1,8-Cineole 28.3 29.8 34.2 31.7 34.1 tivity in vitro (9) and in vivo (10). Given that myrtaceous α-Pinene 28.0 25.1 24.2 20.9 13.7 plants have yielded numerous essential oils with useful Linalool 11.9 10.4 9.2 3.3 14.7 medicinal properties, the likelihood of previously unin- α-Terpineol 5.9 5.2 5.9 5.9 5.6 vestigated myrtaceous plants containing ‘new’ essential Myrtenol 3.4 1.7 4.1 5.5 4.5 oils with similarly useful properties is high. Terpinen-4-ol 2.9 3.8 3.5 3.9 3.2 ρ-Cymene 2.5 2.4 2.6 2.8 2.5 The composition of the essential oil extracted from Myrcene 2.1 1.8 1.6 2.6 2.7 T.fragrans has recently been characterized (11). The major γ-Terpinene 2.1 2.4 2.0 2.0 1.9 components of the high-cineole variant were 1,8-cineole, β-Pinene 1.9 1.8 1.6 2.1 1.4 pinene and linalool, whilst for the less common low- Sesquiterpene alcohols‡ nd nd 0.2 3.8 2.0 cineole variant they were pinene, linalool and myrtenol Total 89.0 84.4 89.1 84.5 86.3 (11). Commercial production of T. fragrans oil began in † Data provided by the Wollongbar Agricultural Institute, Wollongbar, the 1990s with the establishment of plantations in the NSW, Australia. south-west of Western Australia. The oil, sold as Frago- ‡Includes α, β and γ-eudesmol, eudesma-5-en-11-ol, bicyclogerma- nia oil (The Paperbark Company, Harvey, WA, Australia), crene, humulene, humulene oxide, caryophyllene, aromadendrene, is marketed internationally for the treatment of minor cis-calamanene, cadina-1,4-diene, epiglobulol, globulol, spathulenol, ailments and inclusion in cosmetic products. Although, iso-spathulenol, viridiflorol and rosifoliol. based on the strong aroma released from crushed leaves, nd, not detected. it could be assumed that the T. fragrans plant was used in traditional medicine, such use of this particular species The antimicrobial activity of sample Y was examined has not been documented. To date the biological activ- against four organisms only. ity of T. fragrans oil has not been evaluated. The aim of this work was therefore to evaluate the antimicrobial and Evaluation of antimicrobial activity anti-inflammatory activity of T. fragrans oil, using several different oil samples. The investigation of these pharma- Clinical isolates and reference strains (n = 75) were ob- cological properties may give an indication of the potential tained from the Division of Microbiology and Infectious usefulness of the oil as a medicinal agent. Diseases at PathWest Laboratory Medicine WA, Nedlands, and the Microbiology and Immunology Discipline of the MATERIALS AND METHODS University of Western Australia, Crawley, Western Aus- tralia. The organisms were selected to represent a broad T. fragrans oils range of Gram-positive and -negative bacteria as well as yeasts, and also to represent both commonly encountered Five T. fragrans oils were selected from a collection of oils human pathogens and commensal human skin flora. Ref- produced by steam distillation as part of a larger survey on erence isolates (n = 14) were Acinetobacter baumannii the composition of T. fragrans oils. This survey was con- ATCC 15308, Candida albicans ATCC 10231, C. albicans ducted at Wollongbar Agricultural Institute (Wollongbar, ATCC 90028, Enterococcus faecalis ATCC 29212, E. faecalis NSW, Australia) and the specific details of plant source NCTC 8213, Escherichia coli NCTC 10418, Micrococcus and locations, oil extraction methods and compositional luteus ATCC 9341, Pseudomonas aeruginosa NCTC 10662, analyses are described in detail elsewhere (11). The five Serratia marcescens NCTC 1377, Staphylococcus aureus oils, designated A, B, C, X and Z were representative of NCTC 6571, S. aureus NCTC 7121, Staphylococcus epider- the range of compositions seen in initial gas chromatog- midis ATCC 12228, Staphylococcus warneri NCTC 7291 raphy analyses, were described as high cineole variants and Staphylococcus sp. ATCC 27626. Reference strains and and were each derived from a single provenance (11). The clinical isolates were routinely cultured on blood agar in- major components of oils A, B, C, X and Z are shown in cubated aerobically at 37◦C for 24–36 hr. Table 1. Later work at the Wollongbar Agricultural Insti- The broth microdilution methods recommended by the tute revealed a low cineole variant of T. fragrans oil des- Clinical and Laboratory Standards Institute (12, 13) were ignated Y, the major components of which were linalool used to determine the susceptibility of microorganisms to (25.3%), α-pinene (21.5%) and myrtenol (20.0%) (11). T. fragrans oils. Briefly, a series of doubling dilutions of c 2008 The Societies and Blackwell Publishing Asia Pty Ltd 523 K. A. Hammer et al. each oil (from 4.0 to 0.002% v/v) was made in a 96-well Mononuclear cells were isolated from peripheral blood microtitre tray in the relevant growth medium. Overnight collected from 13 healthy volunteers using Lymphoprep cultures of test organisms were adjusted using a colorime- (Nycomed Pharma, Oslo, Norway) gradient centrifuga- ter to 0.5 McFarland for bacteria, which corresponds to tion (16).