(Tea Tree Oil), Suppresses Inflammatory Mediator Production by Activated Human Monocytes
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© Birkhäuser Verlag, Basel, 2000 Inflamm. res. 49 (2000) 619–626 1023-3830/00/110619-08 $ 1.50+0.20/0 Inflammation Research Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes P. H. Hart1,C.Brand1,C.F.Carson2, T.V.Riley 2,R.H.Prager3 and J. J. Finlay-Jones1 1 Department of Microbiology and Infectious Diseases, School of Medicine, Flinders University, GPO Box 2100, Adelaide, 5001, Australia, Fax: 61882768658, e-mail: [email protected] 2 Department of Microbiology, University of Western Australia, Nedlands, Australia 6907 3 School of Chemistry, Physics and Earth Sciences, Flinders University, GPO Box 2100, Adelaide, 5001, Australia Received 1 January 2000; returned for revision 11 May 2000; accepted by R. O. Day 21 June 2000 Abstract. Objective and Design: To evaluate potential anti- tains over 100 components, the majority being monoterpene inflammatory properties of tea tree oil, the essential oil steam and sesquiterpene hydrocarbons and their alcohols. The anti- distilled from the Australian native plant, Melaleuca alterni- bacterial properties of tea tree oil have now been well docu- folia. mented, and there are susceptibility data on a wide range of Material and Methods: The ability of tea tree oil to reduce bacteria [1–9]. There is also considerable information iden- the production in vitro of tumour necrosis factor-a (TNFa), tifying the components of tea tree oil active against bacteria interleukin (IL)-1b, IL-8, IL-10 and prostaglandin E2 (PGE2) and yeasts [6]. Until recently there have only been anecdotal by lipopolysaccharide (LPS)-activated human peripheral claims about tea tree oil’s anti-inflammatory activity. The blood monocytes was examined. only specific report on anti-inflammatory properties of tea Results: Tea tree oil emulsified by sonication in a glass tube tree oil has been of an in vitro study published as an abstract into culture medium containing 10% fetal calf serum (FCS) [10] in which the addition of tea tree oil to lipopolysac- was toxic for monocytes at a concentration of 0.016% v/v. charide-primed neutrophils inhibited superoxide release by However, the water soluble components of tea tree oil at con- approximately 85%. The concentration of tea tree oil used centrations equivalent to 0.125% significantly suppressed was 0.05% v/v. LPS-induced production of TNFa, IL-1b and IL-10 (by ap- Several studies have investigated the anti-inflammatory proximately 50%) and PGE2 (by approximately 30%) after properties of compounds also found in tea tree oil in a rat car- 40 h. Gas chromatography/ mass spectrometry identified terpin- rageenan-induced hind paw oedema model [11–14]. These en-4-ol (42%), a-terpineol (3%) and 1,8-cineole (2%, respec- studies investigated the immunomodulatory effects of oils tively, of tea tree oil) as the water soluble components of tea tree from Bupleurum gibraltaricum, Bupleurum fruticescens, oil. When these components were examined individually, only Zingiber cassumunar and Salvia sclarea, respectively. terpinen-4-ol suppressed the production after 40 h of TNFa, Collectively, these studies concluded that a-pinene [11, 12], IL-1b, IL-8, IL-10 and PGE2 by LPS-activated monocytes. a-terpinene [13], terpinen-4-ol [13], a-terpineol [14] and Conclusion: The water-soluble components of tea tree oil can linalool [14] may have direct or indirect anti-inflammatory suppress pro-inflammatory mediator production by activated activity. The implications of these studies for the activity of human monocytes. tea tree oil are uncertain since the composition of the oils and the concentrations used were generally in excess of those Key words: Tea tree oil – Monocytes – Interleukin-1 – found in tea tree oil. However, they go some way to identify- Tumour necrosis factor-a – Prostaglandin E2 ing essential components of tea tree oil which possess anti- inflammatory activity. Whether there are synergistic or anta- gonistic interactions of several potential immunomodulators Introduction when they are in the same chemical mixture is not known. Some of the components of tea tree oil previously shown to Tea tree oil is the essential oil steam distilled from the Aus- have anti-bacterial properties, e.g. terpinen-4-ol and a-terpi- tralian native plant, Melaleuca alternifolia. Tea tree oil con- neol, are also anti-inflammatory in vivo [6]. In this study of the anti-inflammatory activity of tea tree Correspondence to: P. H. Hart oil in vitro, human peripheral blood monocytes were used as 620 P. H. Hart et al. Inflamm. res. a model for tissue macrophages. Upon activation with mole- ELISAs for TNFα, IL-1β, IL-8 and IL-10 cules such as LPS, these cells produce many mediators in- cluding the central mediators of inflammation, TNFa and Culture supernatants were stored at –20°C until used. TNFa, IL-8 and IL- IL-1b. Other important monocyte/macrophage-derived 10 were measured by sandwich ELISA using mAbs to human TNFa, IL- mediators of inflammation include IL-8, IL-10 and PGE . 8 and IL-10 purchased as antibody pairs from PharMingen, San Diego, 2 CA. The antibody pair for measuring IL-1b was purchased from Endo- Together with other products of activated macrophages, gen, Woburn, MA. One antibody from each pair was purchased as a bio- these molecules can damage tissue or, in turn, activate other tinylated antibody. The assays were sensitive to levels of >40 pg/ml. cells to produce pro-inflammatory mediators. It was hypo- thesised that if anti-inflammatory, tea tree oil would reduce the production in vitro of TNFa, IL-1b, IL-8, IL-10 and Radioimmunoassay for PGE2 PGE2 by LPS-activated monocytes. The levels of PGE2 in the culture supernatants were determined by radioimmunoassay using competitive adsorption to dextran-coated charcoal (3H-PGE2, Amersham, Bucks, UK; PGE2 antiserum, Sigma). Materials and methods The assay was sensitive to levels of >30 pg/ml. Tea tree oil and its major components Solubility of tea tree oil in water Tea tree oil was kindly provided by Australian Plantations (Wyrallah, A sample of tea tree oil (500 mg) was rapidly stirred in water (50 ml) NSW, Australia). The levels of 15 major components of the batch of tea for 20 h at 20°C. The layers were separated and the aqueous layer fil- tree oil used in all experiments (97/03) was examined by Wollongbar tered through filter paper, then extracted with ether (3 ¥ 25 ml). The Agricultural Institute, Wollongbar, Australia, according to the inter- ether extracts were dried (Na2SO4), and the solvent carefully removed national standard for tea tree oil [15] and were as follows: terpinen-4- by distillation at 50°C in a closed system. The residue weighed 80 mg ol (41.6%), g-terpinene (21.5%), a-terpinene (10.0%), terpinolene giving a solubility of 1.6 g/l in water. The organic phase and the oil (3.5%), -terpineol (3.1%), -pinene (2.4%), 1,8-cineole (2.0%), p- a a adhering to the filter paper were extracted into ether, dried (Na2SO4) cymene (1.8%), aromadendrene (1.1%), d-cadinene (1.0%), limonene and evaporated as above to give 400 mg oil. Gas chromatographic (0.9%), ledene (0.9%), globulol (0.5%), sabinene (0.4%) and viridi- analysis as above showed the composition to be 84% terpinen-4-ol, 7% florol (0.2%) (Table 1). For individual study, terpinen-4-ol and a-ter- a-terpineol and 3% 1,8-cineole. pineol were obtained from Fluka, Buchs, Switzerland and 1,8-cineole For separation of tea tree oil preparations in culture medium into from Sigma Chemical Co., St Louis, MO. water and oil soluble components, very similar procedures were adop- ted. Immediately before separation, samples of 250 ml were vigorously shaken before separation of the aqueous and oil layers in a separating Monocyte isolation and culture funnel. The aqueous fraction was filtered through paper, extracted with ether (2 ¥ 25 ml), dried with sodium sulphate and the ether removed by distillation. The oily fraction, and the oil adhering to the filter paper, Human monocytes were isolated from peripheral blood as published was extracted in a similar manner. The residues from both fractions were [16, 17] to >93% purity by countercurrent centrifugal elutriation and analysed by gas chromatography/mass spectrometry (GC/MS) (Table 1). cultured in RPMI-1640 medium (Cytosystems, Castle Hill, Australia) supplemented with 13.3 mM NaHCO3 , 2 mM glutamine, 50 mM b- mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 nM 3-(N-morpholino)propanesulphonic acid with an osmolality of Expression of results 290 mmol/kg H2O (‘complete RPMI’). During isolation and subsequent culture of all cells, extreme care was taken to limit LPS contamination Cytokine measurements were performed on samples from triplicate cul- of buffers and culture fluids [16, 17]. tures and the LPS-induced level was normalised to 100%. The mean For measurement of regulation of cytokine production, freshly values from each set of replicates were used to determine the mean isolated monocytes were cultured at 106 cells/ml with tea tree oil or its + SEM for n donors. For comparison of responses by cell populations water-soluble components and, unless otherwise indicated, 1% fetal calf from a number of different donors, the Student’s paired t test was used. serum (FCS). LPS from Escherichia coli 0111:B4 (Sigma) was added A value of P < 0.05 was considered significant. to give a final concentration of 500 ng/ml. Triplicate cultures for each test variable were incubated at 37°C in 5% CO2. After 20 or 40 h, cultures were centrifuged and the supernatants harvested for measure- Results ment of TNFa, IL-1b, IL-8, IL-10 and PGE2.