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Langmuir 2002, 18, 7916-7922
NMR Investigation on Melaleuca alternifolia Essential Oil Dispersed in the Monoolein Aqueous System: Phase Behavior and Dynamics Francesca Caboi,† Sergio Murgia,† Maura Monduzzi,*,† and Paolo Lazzari‡ Dipartimento Scienze Chimiche, CSGI Universita` di Cagliari, S.S. 554 Bivio Sestu, 09042 Monserrato (CA), Italy, and ATLANTIS S.p.A., Piazza del Carmine 22, 09124 Cagliari, Italy Received May 27, 2002. In Final Form: July 19, 2002 The antiseptic, antibacterial, and antifungal properties of the essential oil of Melaleuca alternifolia, commonly called tea tree oil (TTO), are well-known. This mixture of monoterpenes, sesquiterpenes, and their alcohols is utilized in many therapeutic and cosmetic applications, directly as essential oil or in different kinds of formulations. The terpinen-4-ol, which is present in the commercial essential oil samples in the minimum amount of 30 wt %, is recognized as the main active agent of TTO. Considering the good performance of lipid-based liquid crystalline phases as drug delivery systems, the monoolein/water system has been assessed for both terpinen-4-ol and TTO formulations. The phase behavior of the ternary systems has been investigated by means of optical microscopy and 2H NMR quadrupolar splitting. 13C NMR spinlattice relaxation times and self-diffusion coefficients (measured by 13C NMR PGSE technique) of the lipid matrix and the active agent have been determined to give insights on the dynamic and localization of the molecules in the different microstructures.
Introduction The healing properties of the Melaleuca alternifolia leaves have been well-known by Australian Aborigenes for several thousand years. In the 1920s, European colonists discovered the antiseptic, antibacterial, and antifungal effects of the essential oil obtained by steam distillation of the leaves. Therapeutic applications in the treatment of diabetic ulcers, vaginal infections, gingivitis, and pyorrhoea were found.1 However, the use of M. alternifolia essential oil was drastically reduced in the first half of the 1940s as a consequence of the advent of antibiotics.2 In recent years, the steam-distilled oil from freshly harvested leaves and terminal branchlets of M. alternifolia, commonly known as tea tree oil (TTO), has been rediscovered especially for its antimicrobial and antiseptic activities for the treatment of disorders and diseases of skin, nails, and mucous membranes. The antibacterial activity of the essential oil has been scientifically verified by in vitro assessment against various bacteria and in some cases also through the study of clinical cases.1-6 M. alternifolia essential oil consists of a complex mixture of approximately 100 components, mainly monoterpenes, sesquiterpenes, and their alcohols. However, up to 90 wt % of the whole oil is constituted by less than 10 components, including terpinen-4-ol (Tp), 1,8-cineole, R-terpineol, terpinolene, and R- and γ-terpinene.1 The concentration of each component varies depending on the * Corresponding author. E-mail:
[email protected]; phone: (39) 070 6754385; fax: (39) 070 6754388. † Universita ` di Cagliari. ‡ ATLANTIS S.p.A. (1) Carson, C. F.; Riley, T. V.; Cookson, B. D. J. Hospital Infect. 1998, 40, 175. (2) Saller, R.; Berger, T.; Reigchling, J. Phytomedicine 1998, 5, 489. (3) Hammer, K. A.; Carson, C. F.; Riley, T. V. J. Appl. Microbiol. 1999, 86, 985. (4) Nenoff, P.; Haustein, U.-F.; Brandt, W. Skin Pharmacol. 1996, 9, 388. (5) Carson, C. F.; Riley, T. V. J. Appl. Bacteriol. 1995, 78, 264. (6) Tong, M. M.; Altman, P. M.; Barnetson, R. S. C. Australas J. Dermatol. 1992, 33, 145.
commercial oil sample, although for the major 14 components the composition range is determined by ISO 4730, the international standard for TTO. In particular, TTO samples should be characterized by a maximum of 15 wt % of 1,8-cineole and a minimum of 30 wt % of Tp to reduce tissue irritation and to ensure antimicrobial activity in the skin and mucous membrane treatments. Actually, Tp is considered the main antibacterial component of TTO, although R-terpineol has been described as having a considerable antimicrobial activity as well;5 on the contrary, 1,8-cineole is reported as irritating the skin.2 It is generally believed that 1,8-cineole produces tissue irritation in concentrations above 10%. Thus, a content lower than 7 wt % is preferred in TTO samples for cosmetic applications. In addition, Hausen et al. have demonstrated the presence of sensitizing agents more active than 1,8cineole in TTO because of oxidation processes.7 The authors have pointed out the necessity to keep M. alternifolia essential oil in plugged brown bottles to avoid the photooxidation of R-terpinene to p-cymene and ascaridol (endoperoxide) and the oxidation of Tp to p-cymene and 1,2,4-trihydroxy menthane, being that these products of degradation reactions are very allergenic agents. At present, pure TTO is sold in bottles or as pharmaceutical and cosmetic commercial formulations for acne treatments, mouthwashes, shampoos, soaps, deodorants, and hand and body lotions. In a recent patent, TTO has been used as an antimicrobial agent together with coenzyme Q10, a gum tissue regeneration agent, for treating and preventing periodontal disease.8 Both the active compounds were dissolved in oil. Though the necessary concentration for the effective topical treatment is still undefined, prescriptions using 5-10% TTO seem to be sufficient in most cases. Despite this wide use of the essential oil of M. alternifolia in commercial applications, a rational study of the properties and characteristics of its formulates is still at (7) Hausen, B. M.; Reichling, J.; Harkenthal, M. Am. J. Contact Dermatitis 1999, 10, 68. (8) Bozzacco, C. U.S. Patent 5,908,613.
10.1021/la0259959 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/13/2002
NMR Investigation on Melaleuca alternifolia
the beginning. In addition, new formulations based on components that display high skin compatibility are well suited, particularly if characterized by a high shelf life and controlled delivery of the active principles. Here we propose the possibility of incorporating the TTO in liquid crystalline (lc) devices to improve its availability and delivery. For this purpose, monoacylglycerol or monoolein (MO) (a polar lipid commonly used as a food emulsifier) offers a valid alternative way of drug administration.9,10 The interest devoted to the study of MO as a matrix for sustained drug release is due to different advantages. It is biocompatible and fully biodegradable by the action of lipase. The MO/water (W) lc phases (especially cubic phases) show a great flexibility in solubilizing molecules of different polarity and size and possess protective properties toward proteins and vitamins (e.g., K and A).11-15 Two types of bicontinuous cubic phases (the gyroid (CG) and the diamond (CD), with space groups Ia3d and Pn3m, respectively) have been identified in the MO/W binary phase diagram.16 The CD can coexist with excess water. Moreover it shows a good bioadhesiveness to the oral mucosa, giving the possibility to administer the precursor lamellar (LR) or micellar (L2) phases that evolve to the final cubic phase in the presence of an aqueous environment, like the mouth.17 The MO/H2O phase diagram has been thoroughly investigated by Hyde and co-workers.16 In an earlier work, we have studied the phase diagrams and the stability of cubic phases of different MO/H2O/additive systems.15 Some of the hydrophobic additives promoted the formation of hexagonal phases, while an evolution with time (from a cubic to a hexagonal microstructure, essentially attributed to MO hydrolysis products) has been observed for both binary and ternary samples. The analysis of 13C spinlattice relaxation rates, measured for some MO carbons in binary and ternary systems, showed that the local order and dynamics of the lipophilic environment were not significantly affected by the presence of additives or by hydrolysis products.18 The present work focuses on the study of the solubilization of the pure active agent Tp and of the essential oil of M. alternifolia (TTO) in the MO/D2O (D) system. The phase behavior of the MO/D/Tp and MO/D/TTO ternary systems (5 Tp wt % and 5-15 TTO wt %) was investigated by NMR 2H quadrupolar splitting and optical microscopy. Measurements of 13C NMR spin-lattice relaxation times (T1) for Tp and MO, along with the values of self-diffusion coefficients of Tp and MO in L2 samples by 13C NMR PGSE technique, were performed to get information on the localization and dynamics of the active agent in the lipid matrix. (9) Engstro¨m, S.; Larsson, K.; Lindman, B. Proc. Int. Symp. Controlled Release Bioact. Mater. 1988, 15, 105. (10) Ganem-Quintanar, A.; Quintanar-Guerrero, D.; Burri, P. Drug Dev. Ind. Pharm. 2000, 26, 809. (11) Ericsson, B.; Larsson, K.; Fontell, K. Biochim. Biophys. Acta 1983, 729, 23. (12) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyte`, Z.; Monduzzi, M.; Larsson, K. Langmuir 1997, 13, 5476. (13) Ericsson, B.; Ericsson, P. O.; Lofroth, J. E.; Engstrom, S. Am. Chem. Soc. Symp. Ser. 1991, 469, 251. (14) Razumas, V.; Larsson, K.; Miezes, Y.; Nylander, T. J. Phys. Chem. 1996, 100, 11766. (15) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids 2001, 109, 47. (16) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213. (17) Nielsen, L. S.; Schubert, L.; Hansen, J. Eur. J. Pharm. Sci. 1988, 6, 231. (18) Murgia, S.; Caboi, F.; Monduzzi, M. Chem. Phys. Lipids 2001, 110, 11.
Langmuir, Vol. 18, No. 21, 2002 7917 Table 1. Main Components of Tea Tree Oil Sample (Australian Bodycare)a compound
% by wt
compound
% by wt
R-thujene R-pinene β-pinene myrcene R-phellandrene R-terpinene p-cymene 1,8-cineole
0.8 2.6 0.7 0.8 0.4 8.5 3.8 3.7
d-limonene γ-terpinene terpinolene terpinen-4-ol R-terpineol aromadendrene viridiflorene δ-cadinene
1.0 20.8 3.1 41.2 3.0 1.3 1.2 1.3
a GC: Thermoquest TRACE 2000; GC column: Chrompack CPSIL 5 CB-MS, 15 m, 0.25 mm i.d.(0.25 µm); injector temperature: 250 °C; detector temperature (FID): 300 °C; gas flow rate: 1 mL/ min; split: 10:1; temperature program: 40 °C for 4 min, 4 °C/min up to 120 °C, and 10 °C/min up to 300 °C.
Experimental Section Materials. MO (RYLO MG 90-glycerol monooleate; 98.1 wt % monoglyceride) has been kindly provided by Danisco Ingredients, Brabrand, Denmark. The fatty acid composition of GMO, as here verified through 13C NMR spectra, was 92% oleic acid, 6% linoleic acid, and 2% saturated fatty acids. The TTO sample was obtained by Australian Bodycare; Tp was purchased from Fluka. According to the GC method conditions by Hausen et al.,7 TTO characterization (GC-FID) showed the correspondence of the sample with the ISO 4730,19 being the composition of the main mono- and sesquiterpenic constituents of the oil as reported in Table 1 (identification by standard substances), while components occurring with less than 0.4 wt % are not listed. Sample Preparation. Samples were prepared by weighing the components into glass tubes (L ≈ 0.5 cm) that were centrifuged, frozen for 12 h, and flame-sealed. They were homogenized by repeated cycles of heating at 39 °C and centrifuging back and forth at 3000 rpm at 25 °C. The samples were stored at 25 °C in the dark; the tubes were covered with a black paper pocket to prevent the well-known photooxidation process of TTO with the consequent modification of the composition.7 Since the phase diagrams were mainly defined by the NMR 2H quadrupolar splitting technique, all samples were prepared in D2O. The one-phase regions of the cubic phase have been easily identified for their complete transparency, high viscosity, and optical isotropy, while the birefringent samples were analyzed by optical microscopy and NMR 2H quadrupolar splitting. Optical Microscopy. The anisotropic lc phases were observed by optical microscopy (Zeiss 200) in polarized light at 25 °C. The obtained patterns were compared with the typical textures of other surfactants.20 Nuclear Magnetic Resonance (NMR). 2H and 13C NMR measurements were performed by a Bruker Avance 300 MHz (7.05 T) spectrometer at the operating frequencies of 46.072 and 75.468 MHz, respectively, at 25 °C. A standard variabletemperature control unit (with an accuracy of (0.5 °C) was used. The spectrometer is equipped with a Bruker field gradient probe that can reach field gradients of 300 G/cm. 2H NMR spectra were periodically recorded, without lock, to verify the achievement of equilibrium and to characterize the lc microstructure in mono- or multiphase samples. Nuclei with a spin quantum number I g1 (such as 14N or 2H) have an electric quadrupolar moment that can interact with nonzero net electric field gradients giving multiple resonance of 2I peaks, separated by the splitting
∆νq ≈ (3/m)PbχSb where m ) 4 and 8 for the LR and hexagonal phase, respectively. Pb is the fraction of the observed nucleus in the bound state, χ is the quadrupolar coupling constant, and Sb ) 1/2(3 cos2 ϑD 1) is the order parameter related to the average time orientation (ϑD) of the nucleus with respect to the lipid chain axis. The inner and outer peaks correspond to the nuclei orientations at 90° and (19) International Standard I. ISO 4730. International Organization for Standardisation: Geneva, Switzerland, 1996. (20) Rosevear, F. B. J. Am. Chem. Soc. 1954, 31, 628.
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Figure 1. Phase diagrams at 25 °C of (a) MO/D and (b) MO/ D/Tp (Tp: 5 wt %) systems. 0°. Each sample was left for 30 min in the NMR probe to ensure thermal equilibrium and alignment in the magnetic field before recording the spectra at 25 °C. The error in the quadrupolar splitting measurements was around 7%. The 13C NMR T1 relaxation times were measured in the L2 phases by the standard inversion recovery sequence (PD-180τ-90-AC) by acquiring the partially relaxed spectra at 15-20 different τ values.22 The T1 relaxation times were obtained by the three-parameter nonlinear fitting:
Figure 2. Phase diagrams at 25 °C of MO/D/TTO systems.
I(τ) ) A - B exp(-τT1-1) The error on the fitting is always less than 1%, while the reproducibility of the T1 values is within (3%. Self-diffusion measurements were performed using the stimulated echo sequence of the FT PGSE technique, as previously described.21 The experiments were carried out by varying the gradient strength (G) while keeping the gradient pulse length (δ) and the pulse intervals (∆) constant. Self-diffusion coefficients were calculated by means of a two-parameter nonlinear fit of the echo intensity decay measured at 18-20 different G values. The echo intensity decay as the value of G is increased is given by
Figure 3. Optical micrograph texture of the hexagonal phase in a MO/D/TTO sample (77/15/8 wt %).
I(δ) ) I(0) exp[-(γGδ)2D(∆ - δ/3)] where D is the self-diffusion coefficient, I(0) is the echo intensity in the absence of any gradient, and γ is the magnetogyric ratio of the observed nucleus. The error on the fitting is always less than 1%, while the reproducibility (as judged by repeated measurements) is estimated to be smaller than (5%. To avoid a selective enhancement of the 13C experiments peak intensities because of NOE effects in the 1H-decoupled spectra, an inverse-gated 1H-decoupling sequence was used. All samples were prepared in duplicate, and all measurements were carried out in triplicate.
Results and Discussion Phase Behavior. The study of the solubilization of a third component in the lipid aqueous system cannot leave out of consideration the binary system phase behavior. Depending on the purity of MO or on the substitution of H2O with D2O, the MO/H2O binary phase diagram can present small shifts in the phase boundaries. For this reason, we have analyzed the MO/D phase diagram at 25 °C (Figure 1a). From now on “water” should be read as “deuterated water”. Compared with the MO/W phase diagrams reported by Hyde et al.16 and Briggs et al.,23 here the LR region does not extend beyond a water content of 7 wt %, while the bicontinuous cubic (V2) region forms at about 12 up to 42 wt % of water. Tp is the major accountable component for the antibacterial and antiseptic activity of TTO, and its individual effect on the MO/D phase behavior has been investigated (21) Stilbs, P. P. Prog. Nucl. Magn. Reson. Spectrosc. 1987, 19, 1. (22) Abragam, A. Principles of Nuclear Magnetism; Oxford University Press: Oxford, 1961. (23) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II Fr. 1996, 6, 723.
first. Figure 1b reports the MO/D/Tp (Tp: 5 wt %) phase diagram. A reverse L2, LR, and V2 and reverse hexagonal (H2) regions are found with increasing the water content. It should be noticed that 5 wt % of Tp (at low water content) does not change the interfacial curvature of the lipid system and that the L2, LR, and V2 phases (as in the binary system) are observed, although with differences in the phase boundaries. Particularly, the V2 phase is seen to occur up to 18 water wt % only. The increase of water content evidently allows rearrangements of Tp within the lipid bilayer that induce a negative curvature of the polar-apolar interface, and a H2 microstructure is observed up to a water content of about 32 wt %. Further increase of water content produces only biphasic systems. The MO/D/TTO ternary phase diagrams (with 5, 8, 12, and 15 wt % of TTO) are reported in Figure 2. A L2 phase, common to all the phase diagrams, forms in the 3-7 wt % range of water content. Increasing the water content causes the phase behavior to differentiate, depending on the solubilized amount of TTO. The cubic microstructure is found only in the system with the lowest TTO content (5 wt %) and extends up to about 17 water wt %, as in the MO/D/Tp system. Conversely, the LR microstructure that forms in the binary and in the ternary phase diagram with Tp is no longer favored when the essential oil is solubilized in the lipid system. A lc H2 phase was ascertained (by means of optical microscopy and NMR quadrupolar splitting) in all the analyzed systems, either in monophasic (with 5 or 8 TTO wt %) or in two-phase regions (with 12 or 15 TTO wt %). Figure 3 reports an example of an optical micrograph obtained for the hexagonal sample with composition MO/
NMR Investigation on Melaleuca alternifolia
D/TTO ) 77/15/8 (wt %). The phase boundaries move toward lower water content as the amount of essential oil increases. At the highest amounts of TTO, namely, 12 and 15 wt %, the hexagonal region (apparently monophasic by visual inspection and microscopic analysis) indeed coexists with a different anisotropic lc phase, as demonstrated by the presence of two NMR 2H quadrupolar splittings (see next paragraph). Increasing the water content, a two-phase region (namely, hexagonal plus water) is formed at all analyzed TTO percentages. The inferior limit of this region shifts toward a lower water content as the TTO amount increases. This is obvious since the total lipid content (MO + TTO) is also increasing. At high water content and in the systems with 12 and 15 TTO wt %, three-phase systems are observed because of a phase separation of both an excess of water and an excess of TTO oil. The effect of TTO in the aqueous lipid system varies depending on the amount of the solubilized oil and on the water content. At a water content around 5 wt %, a L2 phase is found independently of the TTO content. In the range of 8-16 wt % water content, only 5 wt % of TTO can be entrapped into a V2 cubic microstructure. At high water content, TTO has the same effect as that found for hydrophobic molecules with very different structures (such as vitamin K1) such as a promotion of a reverse curvature and formation of a H2 phase.12 By comparing the MO/D, MO/D/Tp, and MO/D/TTO (5 wt % TTO) phase diagrams, the most relevant effect is that the solubilization of pure Tp only allows the formation of the LR and V2 phases. On the contrary, the typical bilayers of the LR phase cannot form when TTO is present. This is presumably due to the presence of hydrophobic and sterically hindered minor components, which allows only an average zero curvature of the interface, typical of a V2 phase. NMR Quadrupolar Splitting. 2H NMR of deuterated water is an indispensable tool for characterizing surfactant system phase diagrams.24,25 The 2H quadrupolar splitting measurements were here performed to ascertain the single and multiphase samples and the type of microstructure in the birifrengent systems. Our samples, which had enough time to equilibrate during homogenization and storage, contain four types of deuterium atoms: the deuterium of water, the two different exchangeable OH groups of MO glycerol moiety, and the exchangeable OH of the Tp molecule. Theoretically, four different quadrupolar splittings corresponding to the nonequivalent 2H atoms would be expected unless fast deuterium exchange occurs. This seems to be the case for the hydroxyl group of the glycerol moiety of MO and Tp. Concluding, the only observed signals (in binary and ternary systems) are the splittings due to the D molecules bound to the lipid polar head and the isotropic signal of free water. Figure 4 reports the inner ∆νq (in general better resolved as compared to the outer) versus the lipid (surfactant)/ water molar ratio (S/D) (Tp and TTO are not included in the surfactant calculation) for MO/D binary and MO/D/ TTO, Tp ternary systems. The 2H splitting (measured in binary samples with a water content around 5 wt %) has been taken as the reference splitting of the LR phase (∆νq ) 1700 Hz, Figure 5a). Most of the quadrupolar splittings (24) Wennestro¨m, H.; Lindblom, G.; Lindman, B. Chem. Scr. 1974, 6, 97. (25) Khan, A.; Fontell, K.; Lindblom, G.; Lindman, B. J. Phys. Chem. 1982, 86, 4266.
Langmuir, Vol. 18, No. 21, 2002 7919
Figure 4. Inner quadrupolar splittings (∆νq) vs surfactant/ water (S/D) molar ratio in MO/D binary and MO/D/TTO, Tp ternary samples.
of the birefringent phases of the ternary systems are in the range of 500-800 Hz, roughly corresponding to half of ∆νq measured for the LR phase. For this reason, they have been easily attributed to a hexagonal microstructure, as also suggested by the optical microscopy observation. Figure 5b-d shows examples of the hexagonal quadrupolar splittings, alone or coexisting respectively with an isotropic signal or with another anisotropic phase. This mesophase, characterized by a ∆νq of about 100 Hz, is found for few multiphase samples with high TTO content and can be assigned to a gel phase. The small ∆νq value is due to the low amount of bound water. The isotropic signal that appears in some samples together with the hexagonal splitting may be due either to some free water or to a cubic phase since the stiff appearance of these samples (because of the presence of the H2 phase) prevents the isotropic phase definition. Increasing the water content, free water separates at the bottom of the tubes, and this occurrence supports the assignment of the isotropic signal to free water. 13 C NMR Relaxation. NMR T1 is a useful parameter when studying the organization and dynamics of surfactant systems. Dipolar spin-lattice relaxation is the major mechanism for spin-1/2-nuclei such as 13C nuclei. In this case, the fluctuating magnetic field (source of relaxation) arises from the magnetic moment of the nearby nuclei with nonzero spin. Consequently, bound protons (characterized by the highest magnetic moment) are mainly responsible for relaxation of 13C nuclei. To gain information on the localization and dynamic of the Tp molecules in the lipid matrix, we measured 13C NMR T1 of Tp and MO in the L2 phases. Figure 6 reports the 1H-decoupled 13C NMR spectra of Tp in CDCl3 with the peak attributions referred to the Tp molecule structure and of a L2 sample (MO/D/Tp: 92.5/2.5/5 wt %) with the peak attributions referred to the MO molecule structure. We chose the L2 microstructure as it presents the highest resolution of the NMR spectra together with a quick preparation and an easy handling. Table 2 shows the effect of Tp on the MO/D system. It reports the results obtained for MO carbons in MO/D/Tp samples having 0, 5, and 10 wt % of Tp, while keeping constant the water content. The percent variations of the T1 values (∆1 and ∆2 in Table 2) are also shown. These experiments were carried out at 40 °C to ensure larger phase boundaries to the L2 phase region, since it was verified that the higher temperature does not alter the microstructure of the system. This choice was dictated by the appearance (at 25 °C) of small crystals in the L2 phase
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Figure 5. Examples of 2H NMR quadrupolar splittings in binary and ternary systems. (a) LR phase (MO/D: 95/5 wt %), (b) hexagonal phase (MO/D/TTO: 76/16/8 wt %), (c) hexagonal phase and water (MO/D/TTO: 54.1/40.9/5 wt %), and (d) hexagonal and gel phases (MO/D/TTO: 73.6/11.4/15 wt %).
Figure 6. 1H-decoupled 13C NMR spectra. (a) Tp in CDCl3 and peaks attributions referred to the molecule structure, and (b) L2 sample (MO/D/Tp: 92.5/2.5/5 wt %) and MO peak attributions referred to the molecule structure. Tp carbons are indicated with an asterisk. Table 2. Effect of Tp Additiona T1 (s) T1 (s) MO T1 (s) groups Tp ) 0 wt % Tp ) 5 wt % Tp ) 10 wt % G1 G2 G3 C1 C2 C3 C9, 10 C16 C17 C18
0.291 0.479 0.333 3.270 0.415 0.470 0.681 1.182 1.718 3.361
0.240 0.410 0.278 2.880 0.372 0.438 0.667 1.132 1.654 3.290
0.236 0.405 0.273 2.845 0.368 0.433 0.674 1.139 1.661 3.299
∆1b
∆2c
-17.5 -14.2 -16.5 -11.9 -10.4 -6.8 -2.1 -4.2 -3.7 -2.1
-1.7 -1.2 -1.8 -1.2 -1.1 -1.1 1.0 0.6 0.4 0.3
a NMR spin-lattice relaxation time (T ) of MO carbons and 1 percent variations measured at 40 °C in L2 of MO systems having a constant water content (2.5 wt %) and different Tp percentages (0, 5, 10 wt %). b ∆1 ) [T1(5%) - T1(0%)/T1(0%)] × 100). c ∆2 ) [T1(10%) - T1(5%)/T1(5%)] × 100).
of the binary MO/D system, which perturbed the T1 measurements. In addition, it was ascertained that adding 10 wt % of Tp (at 40 °C and at a low water content) does not change the L2 microstructure. The T1 values of MO carbons in the binary system range between 0.3 and 3.4 s and are comparable to T1 profiles
reported in our previous work.18 The lowest T1 values were found for the carbons of the glycerol region, and the highest were found for the methyl terminal group. In that work, we showed that the presence of a low amount of different types of additives did not affect the T1 values of the MO significantly. Here more detailed investigation was carried out, and T1 values of MO carbons were seen to decrease when Tp was present in the system. This effect is significant particularly on the carbons of the polar head region (composed of the glycerol backbone and the C1-C2 carbons of MO). It becomes less important for the other carbons of the MO chain (see ∆1 values in Table 2). A comparison between the data of the ternary samples (with a Tp content of 5 and 10 wt %, respectively) shows a small decrease of T1 again in the polar head of the molecule, although the percent differences are now less significant (cf. ∆2 values in Table 2). Table 3 shows the effect of the water content on the T1 values of MO (Table 3, Section a) and Tp (Table 3, Section b) carbons, measured at 25 °C in MO/D/Tp ternary samples having a constant Tp content of 10 wt % and 4 and 9 wt % of D. As expected, with increasing the water content, the variations of T1 for the MO carbons are larger within
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Langmuir, Vol. 18, No. 21, 2002 7921
Table 3. Effect of Water Contenta
Table 4. D Measured by
Section a MO groups
T1 (s) D ) 4 wt %
T1 (s) D ) 9 wt %
∆3b
G1 G2 G3 C1 C2 C3 C9,10 C16 C17 C18
0.169 0.292 0.187 2.220 0.277 0.331 0.447 0.783 1.112 2.337
0.207 0.352 0.232 2.630 0.311 0.360 0.479 0.842 1.201 2.497
22.5 20.5 24.1 18.5 12.3 8.8 7.2 7.5 8.0 6.8
Section b Tp groups
T1 (s) D ) 4 wt %
T1 (s) D ) 9 wt %
∆4c
C1 C2 C3 C4 C5 C7 C8 C9, 10
1.472 0.314 0.177 2.241 0.173 0.902 0.308 0.649
1.732 0.364 0.201 2.682 0.197 0.978 0.351 0.708
17.7 15.9 13.6 19.7 13.9 8.4 14.0 9.1
a NMR T and percent variations of MO Section a and Tp Section 1 b carbons measured at 25 °C in MO/D/Tp L2 phase. Samples have a constant Tp content (10 wt %) and different water percentages (4 and 9 wt %). b ∆3 ) [T1(9%) - T1(4%)/T1(4%)] × 100. c ∆4 ) [T1(9%) - T1(4%)/T1(4%)] × 100.
the polar head than along the hydrophobic tail (see ∆3 values in Table 3, Section a). The changes observed for Tp carbons, with increasing the water content, are similar to those of the MO polar head, with the exception of the C7-C9,10 carbons (see ∆4 values in Table 3, Section b). All these findings are consistent with a model in which the Tp molecules are mainly located close to the polar head of MO. Although the Tp molecular structure is not properly that of a highly polar organic molecule, it is evident that the OH group confers a fair polar character. However, the fact that a further addition of 5 wt % of Tp to the ternary system with 5 wt % of Tp (Table 2) does not produce the same effect as the addition of 5 wt % of Tp to the binary system suggests that the limits imposed by the MO-water interfacial curvature prevent a further location of Tp in the proximity of the polar head region. Consequently, part of the Tp molecules, because of their oil character, can intercalate among the apolar tails of MO. In addition, solubilization in the aqueous phase cannot be excluded. This is also consistent with a minimum solubilization of Tp in the aqueous phase, as ascertained by Brand et al.26To strengthen these findings, D measurements were then performed on the ternary MO/D/ Tp-TTO. 13C NMR PGSE D Measurements. D, especially when measured in colloidal systems, provide detailed and easily interpreted information on the microstructure and molecular organization. In particular, the NMR PGSE technique gives the possibility to determine the individual translation mobility of all the species present in the system. The most common nucleus selected for the PGSE technique is 1H, which offers a fast answer despite of a low resolution of the spectrum when multicomponent systems are examined. On the contrary, 13C (though the experiments are time-consuming due to the low natural abundance) offers a larger chemical shift range. This gives (26) Brand, C.; Ferrante, A.; Prager, R. H.; Riley, T. V.; Carson, C. F.; Finlay-Jones, J. J.; Hart, P. H. Inflammation Res. 2001, 50, 213.
13C
NMR PGSE at 25 °C × 1011 m2 s-1
MOD
sample MO/D/Tp, TTO
phase
100% Tp 100% TTO (Tp 41.2%) 92.5/2.5/5 (Tp) 87.5/2.5/10 (Tp) 80/15/5 (Tp) 79.6/4.8/15.6 (TTO)
oil oil L2 L2 V2 L2
0.74 0.94 1.25 1.08
× 1011 m2 s-1
TPD
15.10 71.20 2.75 2.94 3.14 4.13
the possibility to identify in the analyzed system the various molecules, even when those occur in low amount as compared to the others. 13C NMR PGSE measurements were performed at 25 °C on the L2 and cubic samples of the two ternary systems: MO/D/Tp and MO/D/TTO and, for comparison, on the pure Tp and on TTO. Table 4 reports the results obtained for MO and Tp. The rather different values of D of Tp (TpD) for pure and for 41 wt % of Tp in TTO are probably due to the different self-associations of pure Tp. The differences in D (found for the L2 and V2 samples) are mainly due to the different lipid/water ratio that is related to the dimension of the unit cell. The value of TpD in the aggregated phases is an order of magnitude lower than that in the essential oil or that in the pure component. This indicates a relevant decrease of the lateral molecular displacement of Tp when it is dispersed in the lipid matrix. Moreover, these values are of the same order of magnitude as that obtained for MO (10-11 m2 s-1), although slightly higher. This result can suggest again a partition of the Tp molecules between the interface and the oil domain. Indeed, if the observed TpD is due to the contribution of Tp located at the interface (Dinter) and in the oil domain (Dfree) according to the relation:
Dobs ) xinterTpDinter + (1 - xinter)TpDfree
Tp
it is evident that a high molar fraction of Tp molecules are located in the proximity of the interface, thus assuming a diffusion coefficient close to that found for MO carbons. The data found for MO are in agreement with the D measured in MO/W binary cubic samples by Eriksson and Lindblom by 1H PGSE. Those values ranged between 1.1 and 1.4 10-11 m2 s-1 depending on the water/lipid molar ratio.27 In a previous work, 1H PGSE measurements were performed on the cubic MO/W/vitamin K1 system.12 In that case also, the trends of DMO were similar to that of the third component DVK1, and both were dependent on the volume fraction of the lipid rather than on the amount of solubilized vitamin. On the contrary, the DVK1 value of the pure vitamin was lower as compared to the vitamin dissolved in molten MO or in the organized system, but this result was related to the ring stacking effects characteristic of the vitamin K1 molecular structure. Conclusion The potentiality of the MO/W system as a matrix for the solubilization of the essential oil of M. alternifolia and of the active agent Tp has been investigated at 25 °C. The study of the phase behavior of the two ternary systems (MO/D/Tp (5 wt %) and MO/D/TTO (TTO: 5,8,12,15 wt %)) shows that both the single active component and the complex oil can be solubilized in the aqueous lipid system, giving L2 and lc phases. These thermodynamically stable phases may be used as a delivery system for the therapeutic applications. (27) Eriksson, P. O.; Lindblom, G. Biophys. J. 1993, 64, 1993.
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In particular, Tp does not modify the interfacial curvature of the binary MO/W system at a low water content, giving L2, LR, and V2 phases. Increasing the water content, both Tp and TTO promote a negative curvature of the polar-apolar interface with the formation of the H2 phase. On the other hand, by increasing the Tp content along with the TTO, LR and V2 phases cannot form. Indeed, as demonstrated by 13C NMR T1 and D, only a limited amount of the Tp molecules are allowed to locate at the MO/W interface. Further addition of TTO modifies significantly the oil volume fraction since the essential oil
Caboi et al.
components intercalate among the MO chains, thus favoring at first a reverse curvature (H2 lc phase) and then a phase separation. Acknowledgment. Consorzio Sistemi Grande Interfase (CSGI, Italy), Italian MIUR, and Danisco (Denmark) are acknowledged for support. S.M. acknowledges MURST Law 488, Project 8, Cluster 08-B (Italy) for his 3-year position at Cagliari University. LA0259959
