Regulation of Wheal and Flare by Tea Tree Oil: Complementary Human and Rodent Studies
2004; Elsevier BV; Volume: 123; Issue: 4 Linguagem: Inglês
10.1111/j.0022-202x.2004.23407.x
ISSN1523-1747
AutoresZeinab G. Khalil, Annette Pearce, Narmatha Satkunanathan, Emma Storer, John J. Finlay‐Jones, Prue H. Hart,
Tópico(s)Essential Oils and Antimicrobial Activity
ResumoWhen applied 20 min after injection of histamine into human forearm skin, tea tree oil (TTO) reduces the developing cutaneous vascular response. In this study, the effect of TTO on inflammatory microvascular changes was dissected at the base of an experimental blister on rat skin. 1,8-Cineole, representing 2% of TTO, reduced vascular changes induced by sensory neuropeptides released when the distal portion of a cut sciatic nerve was electrically stimulated. The pre-terminal modulatory effect of 1,8-Cineole was confirmed in tests in sensory-denervated rats. Terpinen-4-ol (approximately 40% TTO) reduced substance P-induced microvascular changes and protein extravasation by a direct nitric oxide-mediated effect on the microvasculature, without sensory nerve involvement. α-Terpineol (3% of TTO) regulated both pre- and post-sensory nerve terminals. In human skin, terpinen-4-ol applied 10 min after histamine injection, but not α-terpineol or 1,8-cineole, regulated the developing wheal and flare suggesting that the histamine-induced responses in humans (at the dose used in this study, 50 μL of 330 μM histamine) are in large part determined by histamine directly affecting the vasculature via post-terminal-mediated events. The underlying strength of these studies is the use of a well-established rat physiologic model to differentiate the mechanism of regulation of microvascular changes by modulatory agents. When applied 20 min after injection of histamine into human forearm skin, tea tree oil (TTO) reduces the developing cutaneous vascular response. In this study, the effect of TTO on inflammatory microvascular changes was dissected at the base of an experimental blister on rat skin. 1,8-Cineole, representing 2% of TTO, reduced vascular changes induced by sensory neuropeptides released when the distal portion of a cut sciatic nerve was electrically stimulated. The pre-terminal modulatory effect of 1,8-Cineole was confirmed in tests in sensory-denervated rats. Terpinen-4-ol (approximately 40% TTO) reduced substance P-induced microvascular changes and protein extravasation by a direct nitric oxide-mediated effect on the microvasculature, without sensory nerve involvement. α-Terpineol (3% of TTO) regulated both pre- and post-sensory nerve terminals. In human skin, terpinen-4-ol applied 10 min after histamine injection, but not α-terpineol or 1,8-cineole, regulated the developing wheal and flare suggesting that the histamine-induced responses in humans (at the dose used in this study, 50 μL of 330 μM histamine) are in large part determined by histamine directly affecting the vasculature via post-terminal-mediated events. The underlying strength of these studies is the use of a well-established rat physiologic model to differentiate the mechanism of regulation of microvascular changes by modulatory agents. NG-nitro-L-arginine tea tree oil Tea tree oil (TTO) is the essential oil steam-distilled from Melaleuca alternifolia, an Australian native plant. There are now many reports of the susceptibility of a range of bacteria, yeasts, and fungi to the antimicrobial properties of TTO that support the increasing popularity of TTO as an antimicrobial agent for the treatment of conditions such as tinea pedis and acne (Bassett et al., 1990Bassett I.B. Pannowitz D.L. Barnetson R.St.C. A comparative study of tea tree oil versus benzoylperoxide in the treatment of acne.Med J Aust. 1990; 153: 455-458PubMed Google Scholar;Tong et al., 1992Tong M.M. Altman P.M. Barnetson R.St.C. Tea tree oil in the treatment of tinea pedis.Australas J Dermatol. 1992; 33: 145-149Crossref PubMed Scopus (84) Google Scholar;Carson and Riley, 1994Carson C.F. Riley T.V. The antimicrobial activity of tea tree oil.Med J Aust. 1994; 160: 236PubMed Google Scholar;Nenoff et al., 1996Nenoff P. Haustein U.F. Brandt W. Antifungal activity of the essential oil of Melaleuca alternifolia (tea tree oil) against pathogenic fungi in vitro.Skin Pharmacol. 1996; 9: 366-394Crossref PubMed Scopus (105) Google Scholar;Concha et al., 1998Concha J.M. Moore L.S. Holloway W.J. Antifungal activity of Melaleuca alternifolia (tea tree) oil against various pathogenic organisms.Podiatr Med Assoc. 1998; 88: 489-492Crossref PubMed Scopus (31) Google Scholar). There have also been reports of TTO having anti-inflammatory properties. In the mouse, TTO reduced the edema but not the influx of inflammatory cells associated with the efferent phase of a contact hypersensitivity response but was without effect on an irritant response or the edema associated with exposure to UVB radiation (Brand et al., 2002aBrand C. Grimbaldeston M.A. Gamble J.R. Drew J. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces the swelling associated with the efferent phase of a contact hypersensitivity response.Inflamm Res. 2002; 51: 236-244Crossref PubMed Scopus (47) Google Scholar). Furthermore, TTO reduced immediate type hypersensitivities in the mouse, i.e., topical application of TTO significantly suppressed histamine-induced ear swelling in murine ears (Brand et al., 2002bBrand C. Townley S.L. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced oedema in murine ears.Inflamm Res. 2002; 51: 283-289Crossref PubMed Scopus (54) Google Scholar). Topically applied TTO also reduced the wheal induced by intradermal injection of histamine in human skin (Koh et al., 2002Koh K.J. Pearce A.L. Marshman G. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced skin inflammation.Br J Dermatol. 2002; 147: 1212-1217https://doi.org/10.1046/j.1365-2133.2002.05034.xCrossref PubMed Scopus (115) Google Scholar). In that study, 21 volunteers were injected intradermally in each forearm with histamine. After 20 min, TTO was applied topically to one forearm, leaving the other as a control. Flare and wheal diameters, and double skin fold thickness were measured every 10 min for an hour. Mean wheal volume, but not flare area, significantly decreased 10 min after TTO application (Koh et al., 2002Koh K.J. Pearce A.L. Marshman G. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced skin inflammation.Br J Dermatol. 2002; 147: 1212-1217https://doi.org/10.1046/j.1365-2133.2002.05034.xCrossref PubMed Scopus (115) Google Scholar). It was important to determine the mechanism by which TTO reduced histamine-induced skin inflammation and to characterize the component of TTO responsible. For the TTO used, gas chromatography/mass spectrometry identified terpinen-4-ol (42% of TTO), α-terpineol (3%), and 1,8-cineole (2%) as the main water-soluble components of TTO (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar). These are the components of TTO that would penetrate into the dermis and regulate both the vasculature and peripheral nervous systems. In this study, we investigated the regulatory properties of TTO, as well as terpinen-4-ol, α-terpineol, and 1,8-cineole, on peripheral inflammatory responses in rat skin. Histamine receptors have been identified on both endothelial cells and sensory nerves (Greaves and Wall, 1996Greaves M.W. Wall P.D. Pathophysiology of itching.Lancet. 1996; 348: 938-940https://doi.org/10.1016/S0140-6736(96)04328-0Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar;Clough et al., 1998Clough G.F. Bennett A.R. Church M.K. Effects of H1 antagonists on the cutaneous vascular response to histamine and bradykinin: A study using scanning laser Doppler imaging.Br J Dermatol. 1998; 138: 806-814Crossref PubMed Scopus (70) Google Scholar;Clough, 1999Clough G. Experimental models of skin inflammation.Clin Exp Allergy. 1999; 29: 105-108Crossref PubMed Scopus (7) Google Scholar) and it has been documented that histamine causes vasodilation and an increase in microvascular permeability leading to increased tissue perfusion and plasma extravasation (wheal and flare responses) by direct effects on the former cells or indirect effects on the activity of sensory nerves. To determine how TTO was regulatory, we employed a well-validated rat physiological model to differentiate between regulation of pre- and post-sensory nerve terminal events. This involved assessing the inflammatory response at the base of blisters raised on the rat hind footpad after superfusing inflammatory mediators over the blister base or electrically stimulating the distal portion of a cut sciatic nerve to release sensory neuropeptides (Khalil et al., 1994Khalil Z. Ralevic V. Bassirat M. Dusting G.J. Helme R.D. Effects of ageing on sensory nerve function in rat skin.Brain Res. 1994; 641: 265-272https://doi.org/10.1016/0006-8993(94)90153-8Crossref PubMed Scopus (56) Google Scholar;Khalil, 1999Khalil Z. Sensory peptides: Effects on ageing and wound healing.in: Moore P.K. Brain S.D. Pain and Neurogenic Inflammation. Life Science. Birkhauser Verlag, Basel1999: 275Crossref Google Scholar). These responses were also studied in sensory-denervated rats (Lembeck and Holzer, 1979Lembeck F. Holzer P. Substance P as a neurogenic mediator of antidromic vasodilatation and neurogenic plasma extravasation.Naunyn-Schmiedeberg's Arch Pharmacol. 1979; 310: 175-183Crossref PubMed Scopus (1006) Google Scholar). Finally, the components of TTO that modulate histamine-induced wheal and flare in human skin were characterized and their mechanism of action interpreted in the light of investigations of the activity of TTO on vascular changes in the rat hind footpad. As in models previously described (Khalil et al., 1994Khalil Z. Ralevic V. Bassirat M. Dusting G.J. Helme R.D. Effects of ageing on sensory nerve function in rat skin.Brain Res. 1994; 641: 265-272https://doi.org/10.1016/0006-8993(94)90153-8Crossref PubMed Scopus (56) Google Scholar), selective electrical stimulation of C fibers by antidromic stimulation of the sciatic nerve caused the release of neuropeptides and an increase in the microvascular blood flow at the base of an experimentally induced blister on a rat hind footpad. It is generally accepted that the regulation of the vasodilator response to electrical stimulation primarily reflects control of pre-terminal events with an additional post-terminal component (Khalil et al., 2001Khalil Z. Merhi M. Livett B.G. Differential involvement of conotoxin-sensitive mechanisms in neurogenic vasodilatation responses—Effects of age.J Gerontol Biol Sci. 2001; 8: B1-B8Google Scholar). When an unfractionated preparation of the water-soluble components of TTO (0.125%) was perfused for 10 min prior to, during, and for 20 min subsequent to the 1 min electrical stimulation, there was a significant reduction in the area under the response curve (Figure 1). A significant reduction (approximately 50%) in the microvascular blood flow was also measured after perfusion with 1,8-cineole (0.0025%, i.e., 2% of 0.125%) or α-terpineol (0.0038%, i.e., 3% of 0.125%), but not after perfusion with terpinen-4-ol (0.053%, i.e., 42% of 0.125%). Perfusion of substance P over the base of an experimentally induced blister causes a vasodilator response. It is known that the vasodilatation response to substance P is the outcome of a combined post-terminal component with a significant pre-terminal component (Khalil and Helme, 1989Khalil Z. Helme R.D. Sequence of events in substance P mediated plasma extravasation in rat skin.Brain Res. 1989; 500: 256-262https://doi.org/10.1016/0006-8993(89)90321-1Crossref PubMed Scopus (38) Google Scholar). When perfused for 10 min prior to, and 30 min together with substance P, an unfractionated preparation of the water-soluble components of TTO (0.125%) significantly reduced the substance P-induced increase in microvascular flow by approximately 30% (Figure 2a). α-Terpineol (0.0038%), but not 1,8-cineole (0.0025%), or terpinen-4-ol (0.053%), significantly suppressed substance P-induced increases in microvascular flow (Figure 2a). A significant inhibitory effect of terpinen-4-ol was detected only at a concentration of 0.2% (Figure 2b). Regulation of substance P-induced plasma extravasation was also investigated by measurement of protein in the perfusates every 10 min during the 30 min perfusion with substance P, and then twice at 10 min intervals during the post-stimulation period of perfusion with Ringer's solution. As the plasma extravasation response to substance P is the outcome of only a post-terminal effect on neurokinin-1 receptors on post-capillary venules (Khalil and Helme, 1989Khalil Z. Helme R.D. Sequence of events in substance P mediated plasma extravasation in rat skin.Brain Res. 1989; 500: 256-262https://doi.org/10.1016/0006-8993(89)90321-1Crossref PubMed Scopus (38) Google Scholar), regulators of this response must be modulating a vascular response unrelated to sensory nerve involvement. An unfractionated preparation of the water-soluble components of TTO (0.125%), terpinen-4-ol (0.053%), and α-terpineol (0.0038%), but not 1,8-cineole (0.0025%), significantly reduced substance P-induced plasma extravasation. Significant inhibition was detected in the last 10 min of perfusion with substance P and in the perfusates after substance P was removed (Figure 3). To further clarify whether (a) the regulatory effects of an unfractionated preparation of the water-soluble components of TTO (0.125%), terpinen-4-ol (0.053%), and α-terpineol (0.0038%) on microvascular changes were principally mediated by direct action on endothelial cells, and (b) the regulatory effect of 1,8-cineole (0.0025%) on microvascular changes was principally indirect and was by an effect on sensory nerves, blisters were induced in sensory-denervated rats and substance P-induced vasodilation was examined. In sensory-denervated rats, perfusion with an unfractionated preparation of the water-soluble components of TTO (0.125%) significantly suppressed the smaller substance P-induced increases in microvascular blood flow by 25% (Figure 4a) and also suppressed plasma extravasation (Figure 5). A significant reduction in both parameters of inflammation was also detected for terpinen-4-ol (0.053%) and α-terpineol (0.0038%), but not 1,8-cineole (0.0025%) (Figure 4a and Figure 5). The effect of terpinen-4-ol was concentration-dependent with 90% inhibition of the substance P-induced microvascular blood flow at 0.2% (Figure 4b).Figure 5Effect of 1,8-cineole, terpinen-4-ol, and α-terpineol, as well as an unfractionated preparation of the water-soluble components of tea tree oil (TTO), on substance P-induced protein extravasation in sensory-denervated rats. Protein levels in the perfusate were measured every 10 min during perfusion with substance P (SP1, SP2, SP3), as well as during two 10 min periods of perfusion with buffer (R2, R3). Plasma extravasation was measured for six to eight capsaicin-pre-treated rats per group. The test reagents were perfused at concentrations of 0.0025%, 0.053%, 0.0038%, and 0.125%. An asterisk denotes significant difference from control at that time point. Mean±SEM.View Large Image Figure ViewerDownload (PPT) Nitric oxide (NO) production constitutes the main mechanism by which substance P induces vasodilatation at a post-terminal level (Ralevic et al., 1995Ralevic V. Khalil Z. Helme R.D. Dusting G.J. Role of nitric oxide in the actions of substance P and other mediators of inflammation in rat skin microvasculature.Eur J Pharmacol. 1995; 284: 231-239https://doi.org/10.1016/0014-2999(95)00321-BCrossref PubMed Scopus (31) Google Scholar). NG-Nitro-L-arginine (L-NAME), an inhibitor or NO biosynthesis, at the submaximal concentration of 100 μM inhibited the vascular response to substance P by 44% (Figure 6). Although terpinen-4-ol at 0.2% inhibited the vascular response to substance P by 30%, when combined with L-NAME, the overall inhibition (40%) was not additive (Figure 6). We have previously reported that when applied 20 min after histamine injection, TTO (100%) reduced the developing wheal, but not flare response (Koh et al., 2002Koh K.J. Pearce A.L. Marshman G. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced skin inflammation.Br J Dermatol. 2002; 147: 1212-1217https://doi.org/10.1046/j.1365-2133.2002.05034.xCrossref PubMed Scopus (115) Google Scholar). But when TTO (100%) was applied at both 10 and 20 min after histamine injection, TTO significantly reduced both the flare and wheal response (Figure 7). The effect of terpinen-4-ol (100%), 1,8-cineole (100%), and α-terpineol (100%) was also examined with application (25 μL) of the TTO components at both 10 and 20 min after histamine injection. The components were tested undiluted to prevent an interfering effect of a diluent. No adverse effect was measured in response to the undiluted chemicals. Terpinen-4-ol (Figure 8), but not 1,8-cineole or α-terpineol (data not shown), significantly reduced the histamine-induced wheal and flare response. The intensity of the erythema induced by histamine injection was also measured using a fiberoptic tissue spectrum analyzer (Sumitomo Electrics, Tokyo, Japan); the erythema index was determined by subtracting the mean reading of normal skin (adjacent to the reaction site) from the mean reading of the reaction site. Terpinen-4-ol, but not 1,8-cineole, or α-terpineol, also reduced the histamine-induced erythema index (data not shown).Figure 8Effect of terpinen-4-ol on histamine-induced (A) flare and (B) wheal in 10 volunteers. In (A), the mean flare area and (B) the mean wheal volume for the control (triangles) and study arms (squares) with increasing time after histamine injection are shown. Terpinen-4-ol was applied 10 and 20 min after histamine administration. Mean±SEM. An asterisk indicates a significant difference between control and terpinen-4-ol-treated arms at that time point.View Large Image Figure ViewerDownload (PPT) This study illustrates the ability of complementary rodent and human studies to determine the mode of action of immunoregulatory preparations on skin inflammation. We had previously shown that topical TTO could reduce skin inflammation experimentally induced by histamine. The study of microvascular changes in a blister base suggested that TTO was regulating both the sensory-nerve-associated and endothelial components of vasodilation. Further, the pre-terminal effect was due to 1,8-cineole and α-terpineol that comprise approximately 2% and 3%, respectively, of TTO. The post-terminal effects of TTO on microvascular flow were due to the actions of terpinen-4-ol (approximately 42% of TTO) and α-terpineol. Having determined the mode of action of the TTO components in this well-validated rat model that parallels the microvascular changes that take place in human skin, the component of TTO responsible for the immunoregulatory effects of TTO on human skin was examined. Terpinen-4-ol, but not 1,8-cineole or α-terpineol, significantly reduced both the wheal and flare following histamine injection. Thus, under conditions similar to those used in our human study, the effects of TTO on human skin involve regulation of post-terminal events, without involvement of sensory nerves. This study suggested that the histamine-induced responses in human skin >10 min after administration and at the dose used in this study (50 μL of 330 μM histamine) are in large part determined by histamine effects on endothelial cells (i.e., via post-terminal mediated events). This is supported by evidence that histamine is only able to activate sensory nerve terminals at much higher concentrations at the mM range (Khalil and Helme, 1989Khalil Z. Helme R.D. Sequence of events in substance P mediated plasma extravasation in rat skin.Brain Res. 1989; 500: 256-262https://doi.org/10.1016/0006-8993(89)90321-1Crossref PubMed Scopus (38) Google Scholar). Solubility tests of TTO in water in which the aqueous layer is filtered through filter paper, extracted with ether, and assessed by gas chromatography/mass spectrometry, demonstrated that the three main water-soluble components of TTO are terpinen-4-ol, α-terpineol, and 1,8-cineole (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar). It has however, been shown that the water-soluble components of TTO can be separated without difficulty by their ability to remain in serum-free tissue culture medium although the cell-toxic oil-soluble components adhere to the side of polystyrene plastic tubes (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar). The use of this simple separation procedure for identification of an active fraction was validated when terpinen-4-ol, α-terpineol, and 1,8-cineole were tested as pure substances. In the rat experiments, the plastic-non-adherent, water-soluble components (in this study called an unfractionated preparation of the water-soluble components of TTO) were examined at a concentration of TTO of 0.125%. This concentration was found in studies with human monocytes and neutrophils to be immunoregulatory but not toxic (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar;Brand et al., 2001Brand C. Ferrante A. Prager R.H. Riley T.V. Carson C.F. Finlay-Jones J.J. Hart P.H. The water-soluble components of the essential oil of Melaleuca alternifolia (tea tree oil) suppress the production of superoxide by human monocytes, but not neutrophils, activated in vitro.Inflamm Res. 2001; 50: 213-219Crossref PubMed Scopus (96) Google Scholar). As it is recommended that a 100% solution of TTO is applied to skin, this concentration represents approximately one-thousandth of that used and is a level less than the water solubility of TTO (1.6 g per liter) (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar). The ability of TTO components to reach the epidermal and dermal tissues and enter the systemic circulation has not been measured. TTO contains several components known to enhance skin penetration of other compounds, e.g., 1,8-cineole (Obata et al., 1991Obata Y. Takayama K. Machida Y. Nagai T. Combined effect of cyclic monoterpenes and ethanol on percutaneous absorption of diclofenac sodium.Drug Des Delivery. 1991; 8: 137-144Google Scholar), limonene (Okabe et al., 1990Okabe H. Obata Y. Takayama K. Nagai T. Percutaneous absorption enhancing effect and skin irritation of monocyclic monoterpenes.Drug Des Delivery. 1990; 6: 229-238Google Scholar), terpinen-4-ol (Magnusson et al., 1997Magnusson B.M. Runn P. Koskinen L.O.D. Terpene-enhanced transdermal permeation of water and ethanol in human epidermis.Acta Dermatol Venereol. 1997; 77: 264-267PubMed Google Scholar), and α-terpineol (Magnusson et al., 1997Magnusson B.M. Runn P. Koskinen L.O.D. Terpene-enhanced transdermal permeation of water and ethanol in human epidermis.Acta Dermatol Venereol. 1997; 77: 264-267PubMed Google Scholar). To test the activity of terpinen-4-ol, α-terpineol, and 1,8-cineole in isolation, calculations were made according to their concentration in whole TTO and therefore their approximate level in TTO solutions of 0.125%. For testing on human skin, pure compounds were used undiluted. No control oil was included in this study. Application of liquid paraffin has been used previously on human skin as a control for TTO and was without effect on the histamine-induced wheal and flare (Koh et al., 2002Koh K.J. Pearce A.L. Marshman G. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced skin inflammation.Br J Dermatol. 2002; 147: 1212-1217https://doi.org/10.1046/j.1365-2133.2002.05034.xCrossref PubMed Scopus (115) Google Scholar). In this study, the non-reactive components of TTO provided a control for the activity of TTO and terpinen-4-ol on histamine-induced responses in human skin. This study highlights the complexity of bioactivity of natural plant oils. The three main water-soluble components of TTO had different, but readily dissected, modulatory effects on the vascular responses in an experimentally induced blister. Terpinen-4-ol, the main water-soluble component of TTO, was without effect on sensory nerves but instead modulated vasodilation and plasma extravasation. In a recent report, another property of terpinen-4-ol was identified; it induced vascular smooth muscle relaxation in the deoxycorticosterone–acetate–salt hypertensive rat, without any effect on sympathetic nervous system activity (Lahlou et al., 2003Lahlou S. Interaminense L.F.L. Leal-Cardoso J.H. Duarte G.P. Antihypertensive effects of the essential oil of Alpinia zerumbet and it main constituent, terpinen-4-ol, in DOCA-salt hypertensive conscious rats.Fundam Clin Pharmacol. 2003; 17: 323-330https://doi.org/10.1046/j.1472-8206.2003.00150.xCrossref PubMed Scopus (95) Google Scholar). In contrast, in our skin models of inflammation in rat and human, the inhibitory effects of terpinen-4-ol were attributed to modulation of a post-terminal endothelium-mediated vasodilatation. In our animal experiments, a small vasodilatory response to terpinen-4-ol was observed upon its perfusion over the blister base; however, this effect was subsequently masked by its inhibitory effect on the inflammatory response to substance P. The inhibitory effect of terpinen-4-ol on the inflammatory response was then confirmed by the data demonstrating its inhibitory effect on histamine-induced inflammation in human skin. The possibility that terpinen-4-ol has an inhibitory effect on the vasodilatation response when applied locally in an inflammatory environment (this study), as well as a smooth muscle relaxant effect when applied systemically in a hypertensive environment (Lahlou et al., 2003Lahlou S. Interaminense L.F.L. Leal-Cardoso J.H. Duarte G.P. Antihypertensive effects of the essential oil of Alpinia zerumbet and it main constituent, terpinen-4-ol, in DOCA-salt hypertensive conscious rats.Fundam Clin Pharmacol. 2003; 17: 323-330https://doi.org/10.1046/j.1472-8206.2003.00150.xCrossref PubMed Scopus (95) Google Scholar), is an intriguing possibility that needs further investigation. In our study, 1,8-cineole acted directly on sensory nerves, highlighting its potential anesthetic properties. This supports a study of oral feeding of 1,8-cineole to rats and mice resulting in decreased chemical nociception induced by intraplantal formalin or intraperitoneal acetic acid (Santos and Rao, 2000Santos F.A. Rao V.S.N. Antiinflammatory and antinociceptive effects of 1,8-cineole a terpenoid oxide present in many plant essential oils.Phytother Res. 2000; 14: 240-244https://doi.org/10.1002/1099-1573(200006)14:4 3.0.CO;2-XCrossref PubMed Scopus (357) Google Scholar). The third water-soluble component of TTO, α-terpineol, had both anti-edema and anesthetic properties with both a direct and indirect effect on the microvascular responses of the blister base. As terpinen-4-ol, the main water-soluble component of TTO was the most active component in regulating the wheal and flare response in human skin; its anti-inflammatory potential and possible mechanism of action were further investigated. In sensory-intact animals (where the response to substance P is mainly pre-terminal with some post-terminal component), terpinen-4-ol reduced microvascular blood flow by 10%, 22%, and 30% (at 0.05%, 0.1%, and 0.2% concentration, respectively). In sensory-denervated animals (where the response to substance P is mainly post-terminal), terpinen-4-ol inhibited the response by 35%, 55%, and 90%, respectively. These data together with the ability of terpinen-4-ol to inhibit the plasma extravasation response to substance P (a post-terminal mediated event) and its inability to inhibit the vascular response to sensory nerve stimulation (mainly a pre-terminal-mediated event) further support our argument that terpinen-4-ol is mainly acting via a post-terminal-mediated mechanism. NO is involved in the vascular response to substance P (Ralevic et al., 1995Ralevic V. Khalil Z. Helme R.D. Dusting G.J. Role of nitric oxide in the actions of substance P and other mediators of inflammation in rat skin microvasculature.Eur J Pharmacol. 1995; 284: 231-239https://doi.org/10.1016/0014-2999(95)00321-BCrossref PubMed Scopus (31) Google Scholar). There was no additive effect between L-NAME and terpinen-4-ol in reducing substance P-induced microvascular blood flow and this suggests that terpinen-4-ol could be acting via a mechanism that involves modulating the ability of substance P to release endothelial NO. The involvement of other mechanisms in the anti-inflammatory actions of terpinen-4-ol, however cannot be excluded. The study of physiologic models in rats contributes to an understanding of the bioactivity of TTO in other models of skin inflammation. In studies of histamine-induced edema in murine ears, terpinen-4-ol was equivalent in potency to TTO in suppressing the swelling and thus, could be interpreted as TTO acting on components of the vasculature, and not on sensory nerves (Brand et al., 2002bBrand C. Townley S.L. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced oedema in murine ears.Inflamm Res. 2002; 51: 283-289Crossref PubMed Scopus (54) Google Scholar). This was supported by the unaltered ability of TTO to reduce histamine-induced ear swelling in sensory neuropeptide-depleted mice (Brand et al., 2002bBrand C. Townley S.L. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced oedema in murine ears.Inflamm Res. 2002; 51: 283-289Crossref PubMed Scopus (54) Google Scholar). In contrast, TTO, as well as terpinen-4-ol and α-terpineol, could reduce the edema associated with the efferent phase of a contact hypersensitivity response suggesting some sensory nerve control of this response (Brand et al., 2002aBrand C. Grimbaldeston M.A. Gamble J.R. Drew J. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces the swelling associated with the efferent phase of a contact hypersensitivity response.Inflamm Res. 2002; 51: 236-244Crossref PubMed Scopus (47) Google Scholar). In summary, terpinen-4-ol is the active component of TTO in the regulation of histamine-induced wheal and flare in human skin. The underlying strength of these studies is, however, the use of a well-established rat physiologic model to dissect the mechanism of regulation of microvascular changes by modulatory agents. TTO was provided by Novasel Australia Pty Ltd (Mudgeeraba, Queensland, Australia) and fulfilled the criteria of the Australian Standard (ISO-4370, 1996 International Organisation for Standardisation, Geneva, Switzerland). Gas chromatographic analysis of the TTO used in this study by Wollongbar Agricultural Institute, Wollongbar, Australia, showed the following proportions: terpinen-4-ol, 41.6%; γ-terpinene, 21.5%; α-terpinene, 10.0%; terpinolene, 3.5%; α-terpineol, 3.1%; α-pinene, 2.4%; 1,8-cineole, 2.0%; p-cymene, 1.8%; aromadendrene, 1.1%; δ-cadinene, 1.0%; limonene, 0.9%; ledene, 0.9%; globulol, 0.5%; sabinene, 0.4%; and viridiflorol, 0.2%. TTO was kept in 10 mL aliquots in brown glass bottles to minimize oxidation and discarded after 1 mo. An unfractionated preparation of the water-soluble components of TTO was obtained as previously described (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar;Brand et al., 2001Brand C. Ferrante A. Prager R.H. Riley T.V. Carson C.F. Finlay-Jones J.J. Hart P.H. The water-soluble components of the essential oil of Melaleuca alternifolia (tea tree oil) suppress the production of superoxide by human monocytes, but not neutrophils, activated in vitro.Inflamm Res. 2001; 50: 213-219Crossref PubMed Scopus (96) Google Scholar). Briefly, TTO preparations of 1.25% (vol/vol) were prepared in polystyrene plastic tubes in serum-free culture medium, mixed well, and left to stand for 10 min. Gas chromatography/mass spectrometry confirmed that the water-insoluble components adhered to the side of the plastic tubes, whereas the water-soluble components remained in the culture medium (Hart et al., 2000Hart P.H. Brand C. Carson C.F. Riley T.V. Prager R.H. Finlay-Jones J.J. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes.Inflamm Res. 2000; 49: 619-626https://doi.org/10.1007/s000110050639Crossref PubMed Scopus (262) Google Scholar). For individual study, terpinen-4-ol and α-terpineol were obtained from Fluka (Buchs, Switzerland) and 1,8-cineole from Sigma Chemical Co. (St Louis, Missouri). Outbred male Sprague–Dawley rats (3 mo of age) were used. Anesthesia was induced with sodium pentobarbitone (60 mg per kg, i.p.) and maintained by supplementary injections (15 mg per kg, when needed). All experiments were performed according to the ethical guidelines of the National Health and Medical Research Council of Australia. A blister of 0.25 cm2 was induced on the hind footpad of the anesthetized rat by applying a vacuum pressure of -40 kPa to the glabrous skin for approximately 30 min, using a metal suction cap heated to 40°C by an attached heating element (Khalil and Helme, 1989Khalil Z. Helme R.D. Sequence of events in substance P mediated plasma extravasation in rat skin.Brain Res. 1989; 500: 256-262https://doi.org/10.1016/0006-8993(89)90321-1Crossref PubMed Scopus (38) Google Scholar;Khalil et al., 1994Khalil Z. Ralevic V. Bassirat M. Dusting G.J. Helme R.D. Effects of ageing on sensory nerve function in rat skin.Brain Res. 1994; 641: 265-272https://doi.org/10.1016/0006-8993(94)90153-8Crossref PubMed Scopus (56) Google Scholar;Khalil, 1999Khalil Z. Sensory peptides: Effects on ageing and wound healing.in: Moore P.K. Brain S.D. Pain and Neurogenic Inflammation. Life Science. Birkhauser Verlag, Basel1999: 275Crossref Google Scholar). When a blister was established, the surface epithelium was removed and a perspex chamber with inlet and outlet ports was fixed over the blister base. Perfusion of the drugs over the blister was maintained at 4 mL per h by a peristaltic pump (Microperpex S, LKB, Bromma, Sweden). Both perfusion temperature and body temperature were kept at 37°C. The experimental protocol consisted of an initial 20 min equilibration with Ringer's solution to establish a stable baseline. Sodium nitroprusside, a direct smooth muscle vasodilator, was perfused at 100 μM for 10 min. The latter is used to control for the variability in smooth muscle reactivity between rats. This was followed by perfusion of Ringer's solution to re-stabilize the baseline. An unfractionated preparation of the water-soluble components of TTO, or purified preparations of 1,8-cineole, α-terpineol, or terpinen-4-ol, was prepared in Ringer's solution, then perfused at concentrations of 0.125%, 0.0025% (equivalent to 2% of 0.125%), 0.0038% (equivalent to 3% of 0.125%), and 0.053% (equivalent to 42% of 0.125%), respectively. Concentrations of terpinen-4-ol of 0.1% and 0.2% were also tested, as was L-NAME (100 μM in Ringer's solution, Sigma) with or without terpinen-4-ol. The compounds were perfused for 10 min prior to and for 30 min together with substance P (1 μM). Finally, there was a 20 min post-stimulation period with perfusion of Ringer's solution. Immediately after blister induction but prior to removal of the epidermis, the hair was trimmed at the right midthigh region and a small incision was made in the skin (Khalil et al., 2001Khalil Z. Merhi M. Livett B.G. Differential involvement of conotoxin-sensitive mechanisms in neurogenic vasodilatation responses—Effects of age.J Gerontol Biol Sci. 2001; 8: B1-B8Google Scholar). The sciatic nerve was carefully exposed using blunt-end dissection. It was then cleared and mobilized from the surrounding connective tissue, and cut as proximally as possible. The distal portion of the cut nerve was placed over bipolar platinum electrodes and immersed in a paraffin oil pool formed using the skin flaps of the wound. The paraffin oil was pre-heated to 37°C. The electrodes were fixed in such a position that electrical leakage to adjacent nerve structures was minimized. The blister and the perfusions were as described above, except that the TTO compounds were perfused for 10 min prior to, during and for 20 min subsequent to electrical stimulation whereby the distal portion of the sciatic nerve was stimulated with a Grass S48 stimulator (Grass Instrument Company, Quinay, Massachusetts) using parameters of 20 V, 5 Hz, and 2 ms square waves, for 1 min duration. Neonatal rats were pre-treated on the second day of life with a single subcutaneous injection of 50 mg per kg capsaicin. Blisters were induced and peptides perfused in these rats at the age of 3 mo. Efficacy of this treatment to permanently destroy the majority of sensory nerve fibers was confirmed as previously described (Khalil et al., 1994Khalil Z. Ralevic V. Bassirat M. Dusting G.J. Helme R.D. Effects of ageing on sensory nerve function in rat skin.Brain Res. 1994; 641: 265-272https://doi.org/10.1016/0006-8993(94)90153-8Crossref PubMed Scopus (56) Google Scholar;Khalil, 1999Khalil Z. Sensory peptides: Effects on ageing and wound healing.in: Moore P.K. Brain S.D. Pain and Neurogenic Inflammation. Life Science. Birkhauser Verlag, Basel1999: 275Crossref Google Scholar). A laser Doppler flowmeter probe (Periflux, PF2B, Perimed, Sweden) was positioned vertically over the exposed blister in the hind paw via the perspex chamber (Khalil et al., 2000Khalil Z. Georgiou G.M. Ogedegbe H. Cone R.E. Simpson F. Little C.H. Immunological and in-vivo neurological studies on a benzoic acid-specific T-cell-derived antigen-binding molecule from the serum of a toluene-sensitive patient.Arch Environ Health. 2000; 55: 304-318Crossref PubMed Scopus (7) Google Scholar). The flux output of the laser Doppler monitor is a function of the concentration and the velocity of the red blood cells moving in the tissue penetrated by the laser light. The changes in relative blood flow (as determined by changes in red cell flux) were continuously displayed on a chart recorder. Raw data were evaluated by calculating the area under the response curve (cm2). All measurements were made relative to a stable baseline obtained prior to drug perfusion. The baselines did not differ between control and capsaicin-pre-treated rats or control and any other acute treatment groups. The perfusate was collected during the 30 min perfusion with substance P in three 10 min intervals (SP1, SP2, SP3) and during two 10 min intervals post substance P perfusion (R1, R2). Plasma extravasation was determined by assaying the protein content of the perfusate using the Bradford method (Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein, utilising the principle of protein-dye binding.Anal Biochem. 1976; 72: 248Crossref PubMed Scopus (205559) Google Scholar). Forty healthy people in total (some on more than one occasion) were tested for the regulatory effects of TTO (n=18), terpinen-4-ol (n=10), 1,8-cineole (n=10), or α-terpineol (n=10). Seventy percent (28 of 40) had previously used a TTO product on their skin. Participants had no severe generalized skin conditions such as eczema or psoriasis, atopy (eczema, hayfever, or asthma), or previous skin or systemic sensitivity to TTO and had no severe allergic reactions in the past. The participants were not on systemic immunosuppressant therapy and had not taken oral anti-histamines or topical corticosteroids in the preceding 2 wk. This study was approved by the Clinical Investigation Committee of Flinders Medical Centre, Adelaide, Australia. Histamine (50 μL of 100 μg per mL solution (330 μM)) was injected intradermally into the inner forearm skin (approximately midway along the volar aspect) of both arms and the resulting wheal and flare measured at 10 min intervals for 60 min (Koh et al., 2002Koh K.J. Pearce A.L. Marshman G. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced skin inflammation.Br J Dermatol. 2002; 147: 1212-1217https://doi.org/10.1046/j.1365-2133.2002.05034.xCrossref PubMed Scopus (115) Google Scholar). After 10 min and again after 20 min, 25 μL of undiluted TTO, terpinen-4-ol, α-terpineol, or 1,8-cineole were applied topically with a pipette to cover the flare and wheal on the experimental arm. Study arms (TTO or components) and control arms were assigned in an alternating fashion from subject to subject. In this way, each subject acted as his or her own control. Wheal and flare diameters (cm) were measured with calipers (Mitutoyo Corp., Tokyo, Japan). Wheal skin double thickness (mm) was measured by lightly pinching the skin and measuring with a spring-loaded gauge (Mitutoyo). Flare area (cm2) and wheal volume (μL) were calculated as previously described (Marshman et al., 1996Marshman G. Burton J.L. Archer C.B. Comparison of the actions of kallidin and bradykinin in the skin of normal and psoriatic subjects.Clin Exp Dermatol. 1996; 21: 112-115https://doi.org/10.1046/j.1365-2230.1996.d01-199.xCrossref PubMed Google Scholar;Koh et al., 2002Koh K.J. Pearce A.L. Marshman G. Finlay-Jones J.J. Hart P.H. Tea tree oil reduces histamine-induced skin inflammation.Br J Dermatol. 2002; 147: 1212-1217https://doi.org/10.1046/j.1365-2133.2002.05034.xCrossref PubMed Scopus (115) Google Scholar). Vasodilator responses in rat skin were measured as the area under the response curve (cm2) using a digital planimeter (Tamaya, Ohta-Ku, Japan). Results are expressed as mean±SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post hoc test. Sodium nitroprusside responses were used as a covariate in the analysis. For comparison of wheal and flare responses, the readings for control and test arms for a single time point were compared by a paired Student's t test. Significance was set at p<0.05. We are grateful to Novasel Australia Pty Ltd for support of this project and to all the subjects who volunteered to participate. Dr A. Pearce was supported by a scholarship from the Flinders Foundation, Adelaide, Australia.
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