Skin Penetration of Terpenes from Essential Oils and Topical Vehicles

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Materials
• Skin absorption studies
• The quantitative analysis of terpenes
• Data analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

The purpose of this study was to evaluate the in vitro cutaneous penetration of five terpenes - linalool, linalyl acetate, terpinen-4-ol, citronellol and α-pinene - applied in pure essential oils or in dermatological formulations (o/w emulsion, oily solution or hydrogel) containing 0.75 % w/w of the essential oils. Different skin absorption was observed depending on the type of the vehicle and terpenes’ log P values. Cutaneous accumulation of terpenes is several times higher when they are applied in pure essential oils than in topical vehicles. Penetration of terpinen-4-ol to the skin was better from an oily solution (∼90 μg/cm²) than from an emulsion (˜60 μg/cm²). No penetration of linalyl acetate from topical vehicles into viable skin was observed, but also for this terpene penetration to the upper layers of the stratum corneum was 2-times higher when an oily solution was used. In contrast, the cutaneous absorption of linalool was the same from both vehicles (50 - 60 μg/cm²). The skin penetration of α-pinene was not traceable when it was applied in an oily solution. Only a small amount (∼5 μg/cm²) of this terpene was determined in viable skin after application as a hydrogel. Citronellol applied in a hydrogel penetrated into all skin layers in a total amount of 25 μg/cm², while no penetration into viable skin layers after application of an oily solution was noted. Only citronellol permeated into the acceptor medium.

Abbreviations

C:citronellol

L:linalool

LA:linalyl acetate

αP:α-pinene

T:terpinen-4-ol

SC:stratum corneum

SC I:outer fraction of stratum corneum

SC II:middle fraction of stratum corneum

SC III:inner fraction of stratum corneum

ED:epidermis with dermis

Introduction

Terpenes, alone or as essential oils, are components of several hundred medicinal preparations, the majority being used as cutaneous topical products. These are primarily solutions, ointments, emulsions and gels. Some of these products are also intended for inhalation after spreading on the skin of the chest. In drug formulations, terpenes appear not only as drugs themselves, but are often present in mixtures with other active compounds. Terpenes are also present in many cosmetic preparations and toiletries. Percutaneous permeation of terpenes through the intact skin has been proven [1]. As small, unionised and lipophilic molecules, they readily overcome biological barriers [1], [2]. In experimental dermatology, terpenes are widely used as penetration enhancers - excipients which reversibly disturb the structure of the stratum corneum (SC) leading to increased permeability of the skin for terpenes alone and for other substances.

The quantitative evaluation of the percutaneous penetration of terpenes is an important issue, for predicting their activity as penetration enhancers or understanding the mechanism of their action in the skin. Unfortunately, only few publications concern the quantitative determination of terpenes penetrated to the skin [3], [4], [5], [6]. The absorption of these compounds is most often demonstrated indirectly, by enhancement of the permeation of other drugs through skin treated with terpenes [3], [7], [8], [9]. In our previous studies, skin penetration of pure terpenes as well as from a transdermal matrix-type system was determined [5], [6]. The present study has been performed to investigate the skin penetration of terpenes after application of essential oils and after application of essential oils incorporated in dermatological vehicles: o/w emulsion, oily solution or hydrogel. A tape-stripping method was chosen to obtain data about the in vitro process of skin penetration.

Materials and Methods

Materials

Lavender, tea tree, geranium and juniper essential oils were a gift from Pollena-Aroma Fragrances & Flavours (Warsaw, Poland). Their composition with respect to the investigated terpenes, namely terpinen-4-ol (T), linalool (L), linalyl acetate (LA), citronellol (C) and α-pinene (αP) is shown in Table [1], and the structural formulas of the investigated terpenes together with their log P values are given in Fig. [1]. The lavender and tea tree essential oils were incorporated into dermatological vehicles: o/w emulsion or grape seeds oil. The geranium and juniper essential oils were added to grape seeds oil or hydrogel. The o/w emulsion consists of (% w/w): water, mineral oil (10.0), isohexadecane (3.0), C_{12 - 20} alkyl glucoside and C_{14 - 22} alkyl alcohol (together 2.0), decyl oleate (0.5) and polysorbate 80 (0.1) as surfactants; in turn the hydrogel consists of water, carbomer (0.5), triethanolamine (0.75) and ethanol (<1.0) necessary for dissolving the essential oils.

Skin absorption studies

With permission of the Ethics Committee of the Medical University of Gdansk, human skin was obtained from the thorax region of three cadavers (40 - 50-year-old Caucasian women). Before the experiment, the skin was kept frozen at -20 °C. The penetration studies were carried out in flow-through Teflon® diffusion cells (Crown Glass Company, USA) with a diffusion area 0.65 cm². An acceptor medium, isotonic pH 7.3 phosphate buffer (10 mL) preserved with 0.005 % sodium azide (Fluka, Buchs, Switzerland), was recirculated beneath the skin with a constant rate of 10 mL/h, protected against evaporation. The system was maintained at 37 ± 0.5 °C. In the reservoir of the acceptor fluid, 5 mL of methylene chloride were in contact with the buffer thereby allowing achieving sink conditions. The two-phase acceptor system was described in detail earlier [5].

Following the 24 h equilibration, the essential oils or essential oils incorporated in the dermatological vehicles were applied onto the skin surface in the quantity of 200 mg and left for 8 h, while the donor compartment was occluded with Parafilm® (Sigma-Aldrich, Steinheim, Germany). After that time, the penetrants were removed, and the skin was rinsed very briefly with methanol. The SC layers were separated by a tape-stripping method, using 21 fragments of an adhesive tape (3M Medica Pharma, St. Paul, USA). Fragments of the tape with adhering SC were combined into three groups, 7 samples each, representing 3 fractions of the SC: outer (SC I), middle (SC II) and inner (SC III). Each fraction, as well as the remaining epidermis with dermis (ED), was extracted with 5.0 mL of methanol (HPLC grade, P.O.Ch. Gliwice, Poland) by shaking for 24 h at room temperature in tightly closed vials. The methanol extracts were collected into chromatographic vials (Chromacol, Trumbull, USA) and stored at 4 °C prior to analysis. The acceptor medium was collected for analysis as well. The absorption experiments were repeated in quadruplicate.

The quantitative analysis of terpenes

The terpenes were analysed by GC (ATI Unicam 610 series apparatus with the flame-ionisation detector, Unicam Chromatography, Cambridge, UK). The chromatographic conditions were as follows: the detector temperature was 250 °C; the initial oven temperature was 100 °C for 10 min, increasing gradually at 5 °C/min up to 200 °C; the Chromosorb 80/100 glass column 2.1 m × 6 mm × 4 mm was filled with 10 % of non-polar stationary phase OV-101. The peak areas were measured by an integrator. Concentrations of terpenes in the samples were calculated on the basis of the experimentally obtained standard curves. The linearity of the method was demonstrated, and the detection limit was 0.5 μg/mL for all terpenes.

Data analysis

Log P values of terpenes were calculated using ACD/LogP (Advanced Chemistry Development, Toronto, Canada) software. The amounts of terpenes extracted from the skin were expressed per 1 cm² area. Statistical analysis was performed using one-way analysis of variance (ANOVA) and differences were considered significant at P < 0.05.

Results and Discussion

The SC is the main barrier for the percutaneous permeation of xenobiotics. The octanol-water partition coefficient, usually presented as a logarithm (log P), is a parameter that provides basic information on the ability of a molecule to permeate the lipophilic barrier of the SC, as well as on the ability to partition between the SC and either hydrophilic or lipophilic vehicle. The log P values of three out of the five investigated terpenes, namely of L, T and C, are in the range between 2 and 4 (Fig. [1]), regarded as optimum for percutaneous penetration.

The investigated terpenes were applied in the essential oils or the essential oils were introduced to the dermatological vehicles. In the grape seed oil, essential oils were completely dissolved, while in the o/w emulsion they were present in several forms - dissolved in the oily internal phase, emulsified with the surfactants and forming an internal oily phase, as well as dissolved in surfactant micelles. The concentration of the investigated terpenes in the essential oils was in the range of 28 - 41 %. The concentration of the essential oils in the vehicles was approximately 0.75 % (w/w), which corresponded to concentrations of terpenes in the range of 0.21 - 0.31 % (Table [1]). The final concentrations of the investigated terpenes in preparations were more than 100-fold lower than in the pure essential oils (Table [1]). The in vitro penetration experiment was carried out for 8 h, which is longer than the in vivo residence time of dermatological preparations on the skin (usually up to 6 h), but equilibrium of absorption was guaranteed [6].

The amounts of L, LA and T penetrated to the skin layers are presented in Fig. [2] , and amounts of C and αP are shown in Fig. [3]. A gradient of concentrations in the SC layers can be demonstrated for terpenes applied in essential oils. When terpenes were applied in pure essential oils, their total content in the skin was greater than after application in the dermatological vehicles. However, more than 100-fold higher concentrations of terpenes in the essential oils than in the prepared formulations resulted only in 10 - 30-times larger accumulation. It is a result of the capacity of the skin being exceeded, thus proportionality to the penetrant concentration could not be observed.

It is interesting to compare the accumulation in the lipophilic SC and in the hydrophilic ED: for the more lipophilic LA, SC/ED ratio is 3 : 1, for L is 1 : 1 and for the least lipophilic T it is 1 : 1.6 (Fig. [2]). These ratios for L and LA are different from the ratios 1 : 2.8 and 1 : 1.5, respectively, calculated when these terpenes were applied as neat substances [6]. This indicates that terpenes in the essential oils penetrate to the viable skin slower than pure substances. The other important difference is that, in spite of smaller penetration to the ED, accumulation of LA in the SC is 4-times larger when it is applied in lavender oil than after application of a pure compound [6]. Since L can act as a percutaneous penetration enhancer [8], its presence in the lavender oil promotes penetration of LA to the SC.

When LA was applied in the form of an emulsion or oily solution, its penetration was less, and the compound was detected only in the SC I (Fig. [2]). This confirms that viable skin, being hydrophilic, is a strong barrier against penetration of compounds with log P above 4. The amount of LA in the SC I was about 2-times larger when the oily solution was applied. In the case of L, a similar concentration profile was observed, irrespective of the type of vehicle. Both vehicles enabled the terpene to penetrate to the ED and, interestingly, the amounts of L determined in all SC layers were similar, without a concentration gradient.

A completely different profile was demonstrated for T: this terpene penetrated better when applied in the oily solution, but for both vehicles a large gradient in the SC existed, because the terpene was not detected in the SC II and SC III (Fig. [2]). This indicates that a good partitioning to the hydrophilic ED occurs, which can be explained with a relatively good solubility of T in water.

L, LA and T applied in essential oils penetrate easily to the SC and ED, although skin accumulation of the more lipophilic LA is smaller than observed for L and T. Due to a high affinity of terpenes for the skin tissue, no terpenes were detected in the receiver solution. For the more lipophilic LA and for the least lipophilic T, better accumulation in the SC was observed after application of the oily solution than following application of the o/w emulsion, which indicates that the oily vehicle is a good carrier for the terpenes to this layer, irrespective of the unfavourable (due to a very good solubility of terpenes in the oil) SC/vehicle partition coefficient.

Fig. [3] A presents the amounts of C and αP absorbed into skin layers after 8 h application of the essential oils. The absorption was similar for both terpenes. However, better accumulation of αP in deeper layers of the SC resulted in the lack of a concentration gradient in the SC, which was observed for the less lipophilic C. In comparison to other investigated terpenes, skin accumulation of αP and C was relatively small, comparable however to that determined for LA. The similar penetration of αP and LA can be explained by similar lipophilicity, but low absorption of C is in contrast to the very high penetration (1300 μg/cm²) of L, an acyclic terpene with a similar log P value.

When C is applied in 100-fold lower concentrations, in hydrogel with geranium oil, its accumulation in the skin is only 10-times smaller than after penetration from pure essential oil (Fig. [3]). However, the ratio of the amounts in the SC and ED is similar for both applications, i. e., between 1 : 1.3 and 1 : 1.5. This ratio indicates that C cumulates in larger amounts in hydrophilic skin layers when applied as pure terpene, because in that case the ratio 1 : 3 was found [6]. After application of geranium oil dissolved in grape seed oil, almost similar amounts of C were found in the SC as after application of hydrogel. However, no C was found in the ED. This can be explained by a very good partitioning of C to the acceptor medium and fast ”washing out” of the terpene from the ED. C is the only terpene under investigation which penetrated to the acceptor fluid under the experimental conditions. On the basis of the concentration of C in the acceptor medium after 8 h, the percutaneous permeation of C was calculated: 13.3 ± 2.5 μg/cm², 5.5 ± 0.8 μg/cm² and 7.9 ± 1.0 μg/cm² for pure geranium oil, hydrogel and oily solution, respectively.

Unexpected results were found for αP applied in dermatological formulations: no absorption was observed when an oily solution was applied and only a very small amount was detected in the ED after application of hydrogel - the penetration of highly lipophilic αP to the hydrated tissue is very poor. When the terpene is applied in a very small concentration (less than 0.5 %), its accumulation in the skin is below the limit of detection (less than 3 μg/cm²).

Summarising the results of the studies, it can be concluded that a hydrogel enables good skin absorption of terpenes which can be explained by good partitioning of lipophilic molecules between the vehicle and the lipophilic SC [10], [11]. The skin penetration of terpenes is increasing in the following order, when they are applied in pure essential oils: C = LA = αP << L < T. For oily solutions the order is as follows: αP < C < LA < L < T. The above presented ranking of terpenes with respect to their ability to penetrate into the skin demonstrates that lipophilicity of the terpene is the factor influencing the kinetics of skin absorption. Penetration of highly lipophilic compounds (log P > 4) is low, while less lipophilic compounds penetrate into the skin very easily. This is not true, however, for C when it was applied in the presence of other terpenes present in geranium oil, since its penetration was similar to that observed for very lipophilic compounds. This is in contrast to its penetration as a pure terpene, because in that case the cutaneous absorption was in the range observed for L [6].

Acknowledgements

This study was supported by grant No 2 P05F 003 26 from State Committee for Scientific Research, Poland.

References

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• 11 Yener G, Gonullu U, Uner M, Degim T, Araman A. Effect of vehicles and penetration enhancers on the in vitro percutaneous absorption of celecoxib through human skin.  Pharmazie. 2003;  58 330-3
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Dr. Krzysztof Cal

Department of Pharmaceutical Technology

Medical University of Gdansk

Hallera 107

80-416 Gdansk

Poland

Phone: +48-58-349-3185

Fax: +48-58-349-3190

Email: kcal@wp.pl

References

• 1 Jager W, Buchbauer G, Jirovetz L, Fritzer M. Percutaneous absorption of lavender oil from a massage oil.  J Soc Cosmet Chem. 1992;  43 49-54
  PubMedSearch in Google Scholar
• 2 Buchbauer G, Jager W, Jirovetz L, Ilmberger J, Dietrich H. Therapeutic properties of essential oils and fragrances.  ACS Symp Ser. 1993;  525 159-65
  PubMedSearch in Google Scholar
• 3 Okabe H, Takayama K, Nagai T. Percutaneous absorption of ketoprofen from acrylic gel patches containing d-limonene and ethanol as absorption enhancers.  Chem Pharm Bull. 1992;  40 1906-10
  PubMedSearch in Google Scholar
• 4 Sugibayashi K, Kobayashi D, Nakagaki E, Hatanaka T, Inoue N, Kusumi S. et al . Differences in enhancing effect of l-menthol, ethanol and their combination between hairless rat and human skin.  Int J Pharm. 1995;  113 189-97
  CrossrefPubMedSearch in Google Scholar
• 5 Cal K, Janicki S, Sznitowska M. In vitro studies on penetration of terpenes from matrix-type transdermal systems through human skin.  Int J Pharm. 2001;  224 81-8
  CrossrefPubMedSearch in Google Scholar
• 6 Cal K, Sznitowska M. Cutaneous absorption and elimination of three acyclic terpenes - in vitro studies.  J Control Release. 2003;  93 369-76
  CrossrefPubMedSearch in Google Scholar
• 7 Bach M, Lippold B C. Percutaneous penetration enhancement and its quantification.  Eur J Pharm Biopharm. 1998;  46 1-13
  PubMedSearch in Google Scholar
• 8 Vaddi H K, Ho P C, Chan S Y. Terpenes in propylene glycol as skin-penetration enhancers: permeation and partition of haloperidol, Fourier transform infrared spectroscopy, and differential scanning calorimetry.  J Pharm Sci. 2002;  91 1639-51
  CrossrefPubMedSearch in Google Scholar
• 9 Songkro S, Rades T, Becket G. The effect of p-menthane monoterpenes and related compounds on the percutaneous absorption of propranolol hydrochloride across newborn pig skin. I. In vitro skin permeation and retention studies.  STP Pharm Sci. 2003;  13 349-57
  PubMedSearch in Google Scholar
• 10 Smith E, Surber C. The absolute fundamentals of transdermal permeation (drug delivery for dummies). In: Gabard B, Elsner P, Surber C, Treffel P, editors Dermatopharmacology of topical preparations. Berlin; Springer 1999: pp 23-35
  Search in Google Scholar
• 11 Yener G, Gonullu U, Uner M, Degim T, Araman A. Effect of vehicles and penetration enhancers on the in vitro percutaneous absorption of celecoxib through human skin.  Pharmazie. 2003;  58 330-3
  PubMedSearch in Google Scholar

Dr. Krzysztof Cal

Department of Pharmaceutical Technology

Medical University of Gdansk

Hallera 107

80-416 Gdansk

Poland

Phone: +48-58-349-3185

Fax: +48-58-349-3190

Email: kcal@wp.pl