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Quantitative transdermal behavior of pellitorine from Anacyclus pyrethrum extract.

ABSTRACT

The plant Anacyclus pyrethrum (AP) consists of several N-alkylamides with pellitorine as main constituent. AP extracts are known to be biologically active and some products for topical administration containing AP plant extracts are already commercially available with functional cosmeceutical claims. However, no transdermal data for pellitorine are currently available. Therefore, our general goal was to investigate the local skin pharmacokinetics of the plant N-alkylamide pellitorine using a Franz diffusion cell setup. Two different forms were applied on human skin: purified pellitorine and the AP extract. Our study demonstrated that pellitorine is able to cross the stratum corneum and the subsequent skin layers. A significantly higher permeability coefficient was observed when the AP extract ([K.sub.p] = 2.3 x [10.sup.-4] cm/h) was administered, compared to purified pellitorine ([K.sub.p] = 1.1 x [10.sup.-4] cm/h). With the obtained pellitorine concentrations in the skin layers and the receptor fluid, it is concluded that local and systemic effects can be expected after topical application. Due to these findings and as a regulatory consequence, products containing reasonable concentrations of pellitorine are recommended to be classified as a medicinal product.

[c] 2014 Elsevier GmbH. All rights reserved.

Keywords:

Anacyclus pyrethrum

Pellitorine

Franz diffusion cells

Transdermal

Human skin

Introduction

Anacyclus pyrethrum DC (AP) is a plant belonging to the Asteracea family. Several N-alkylamides (NAAs) present in the AP extract have already been identified and reported. Thirteen NAAs were identified in an ethanolic AP root extract with HPLC-UV/ESI-MS: N-isobutylamides, N-methylisobutylamides, 4-hydroxyphenylethylamides and 2-phenylethylamides. The most abundant NAA in this AP extract is pellitorine (deca-2E,4E-dienoic acid isobutylamide) (Boonen et al., 2012a). The structure of pellitorine is given in Fig. 1.

Extracts of Anacyclus pyrethrum are ethnopharmacologically used to treat various diseases. Root extracts are known to have antimicrobial, local anaesthetical, anti-depressive, insecticidal, and saliva stimulating features and are used to treat epilepsy, paralysis, toothache and rheumatism (Annalakshmi et al., 2012; Boonen et al., 2012b). Hydro-alcoholic and chloroform extracts of AP have anticonvulsive effects (Zaidi et al., 2009; Pahuja et al., 2012). In addition, aqueous extracts are used as aphrodisiac to improve the libido of men (Sharma et al., 2009).

It was shown that the main biologically active AP constituents, N-alkylamides, inhibit the enzymes of cyclooxygenase (Muller-Jakic et al., 1994). This anti-inflammatory effect was confirmed in vitro with different AP extracts (Rimbau et al., 1996). In addition, pellitorine was demonstrated in vivo to have a strong tingling and saliva stimulating effect (Sharma et al., 2010).

Topical products containing Anacyclus pyrethrum extracts are already commercially available with functional, cosmeceutical claims. AP root extracts are used in lotions, creams, toners, gels, moisturisers and bath care products, marketed as a cosmetic not only for moisturizing the skin but also for stimulating cell regeneration to reduce discolouration.

In order to exert a biological effect after topical application, the active N-alkylamides must permeate through the stratum corneum into the viable skin layers for a local effect and penetrate through the skin for a systemic effect. Previous research from our group has shown that spilanthol, present in Spilanthes acmella extract, can permeate pig mucosa and human skin, with systemic effects very likely to be present (Boonen et al., 2010a,b). Until now, spilanthol (deca-2E,6Z,8E-trienoic acid isobutylamide), containing three unsaturated double bounds in the fatty acid chain (a triene), is the only plant N-alkylamide on which research was done to evaluate the permeation through human skin: no other plant N-alkylamides were investigated yet for transdermal purposes. In this study, the skin permeability of another N-alkylamide, with a different structure, was investigated: pellitorine, containing two double bounds (a diene). Pellitorine and spilanthol have the same isobutylamide group and both alkylamides possess a 2E double bound in the fatty acid chain, but pellitorine contains 2 double bounds instead of three and the second double bound is on C4, instead of C6 or C8 in case of spilanthol. Both purified pellitorine, as well as the ethanolic extract of Anacyclus pyrethrum were used in this study to investigate if there was a difference in penetration of pellitorine through human skin, as co-compounds in the plant extract could influence the skin penetration as well.

Materials and methods

Chemicals and reagents

Ultrapure water ([H.sub.2]O) of 18.2M[ohm] cm quality was produced by an Arium 611 purification system (Sartorius, Gottingen, Germany). Absolute ethanol (EtOH, 99.8% V/V) and acetic acid were purchased from Sigma-Aldrich (Bornem, Belgium). 0.01 M phosphate buffered saline (PBS) was purchased from Sigma-Aldrich as well and prepared according to the instructions of the supplier. HPLC gradient grade methanol (MeOH) and acetonitrile (ACN) came from Fisher Scientific (Erembodegem, Belgium), while formic acid (FA) was bought from Riedel-de Haen (Seelze-Hannover, Germany). Denaturated ethanol (up to 5% ether) came from Chem Lab (Zedelgem, Belgium).

Analytical characterisation of the Anacyclus pyrethrum extract

The Anacyclus pyrethrum root extract was prepared as previously described (Boonen et al., 2012a). For the analytical characterisation of the extract, the extract was dissolved in ethanol, vortexed, sonificated for 2 h and centrifuged for 15 min at 3220 x g at room temperature. A 30:70 [H.sub.2]O/EtOH (V/V) solution was prepared, centrifuged again and the supernatant was filtered using a 0.45 [micro]m nylon HPLC filter (Whatman). 25 [micro]l of this solution was injected on a Grace Prevail C18 (250 x 4.6 mm, 5 [micro]m) column using a Waters HPLC equipped with a Waters 2487 Dual Absorbance detector set at 258 nm. A gradient with a flow rate of 1.0 ml/min, was used as follows: t = 0 min: 80:20 A:B (V/V), t= 0-150 min: 10:90 A:B (V/V), t= 150-151 min: 80:10 A:B (V/V), t= 151-166 min: 80:20 A:B (V/V), t= 166 min: 80:20 A:B (V/V) (with A = 1% acetic acid in [H.sub.2]O and B = ACN). The identity of the mean peak in the AP extract was determined using HPLC-MS, based upon the precursor ion m/z and the fragmentation pattern. The HPLC-MS analysis was done on a HPLC system which consisted of a Spectra System SN4000 interface, a Spectra System SCM1000 degasser, a Spectra System P1000XR pump, a Spectra System AS3000 autosampler, and a Finnigan LCQ Classic ion trap mass spectrometer in positive ion mode (all Thermo, San Jose, CA, USA) equipped with a SPD-10A UV-vis detector (Shimadzu, Kyoto, Japan) and Xcalibur 2.0 software (Thermo) for data acquisition. The extract, dissolved in 50:50 ACN:[H.sub.2]O (V/V), was injected into the LC-MS apparatus and the MS method according to Boonen et al. (2012a) was used.

Purification of pellitorine

Pellitorine was isolated and purified from the Anacyclus pyrethrum extract by means of semi-preparative HPLC as follows. The Anacyclus pyrethrum root extract was dissolved in acetonitrile, vortexed and sonificated for 2h and centrifuged for 15 min at 3220 x g at room temperature. A 1:1 dilution of the supernatant was prepared with [H.sub.2]O. The solution was centrifuged again and the supernatant was filtered using a HPLC filter. 1.0 ml of the solution was injected on a Vydac C18 monomeric semi-preparative column (Grace, 250 mm x 10 mm, 5 [micro]m) using a Waters HPLC equipped with a Waters 2487 Dual Absorbance detector. The sample compartment and column temperature were maintained at room temperature. An isocratic elution mode was used with as mobile phase 50:50 A:B (V/V) (A: 0.1% FA in [H.sub.2]O and B: 0.1% FA in MeOH). A flow rate of 6.0 ml/min was used and UV detection was performed at 258 nm. Fractions between 50 and 57 min retention time on the semi-preparative HPLC were collected and evaporated to dryness using a rotavapor (Buchi rotavapor R-200 with Buchi heating batch B-490). The obtained dried fractions were dissolved in methanol, combined and evaporated to dryness under nitrogen.

Analytical characterisation of purified pellitorine

The identity of the purified pellitorine was confirmed using HPLC-MS. The equipment is already described in a previous section. A prevail RP C18 column (Grace, 250 x 4.6 mm, 5 [micro]m) with a suitable guard column was used. The sample compartment was kept constant at 20[degrees]C, while the column temperature was maintained at 30[degrees]C. The purified pellitorine was dissolved in 50:50 ACN:[H.sub.2]O (V/V), 25 [micro]l was injected and the flow rate was set to 1.0 ml/min. A gradient was used as follows: t = 0 min: 60:40 A:B (V/V), t= 0-5 min: 50:50 A:B (V/V), t= 5-35 min: 40:60 A:B (V/V), t= 35-40 min: 10:90 A:B (V/V), t = 40-41 min: 60:40 A:B (V/V) and t=41-46 min: 60:40 A:B (V/V) (with A = 1% acetic acid in [H.sub.2]O and B=ACN). ESI was conducted with a capillary voltage of 3 V. Nitrogen was used as sheath and auxiliary gas. The temperature of the heated capillary was set at 275[degrees]C. MS-MS spectra were obtained by collision-induced dissociation (CID) of the parent m/z, with the relative collision energy set to 35%. Identification was based on the parent m/z values and fragmentation ions.

The purity of purified pellitorine was determined using HPL-CUV. 25 [micro]l of purified pellitorine, dissolved in 30:70 [H.sub.2]O:EtOH (V/V) was injected on a Prevail C18 column (Grace, 250 x 4.6 mm, 5 [micro]m) with guard column. HPLC analysis was performed on a Waters Alliance 2695 HPLC equipped with a Waters 2998 Photo Diode Array detector. The same gradient method was used as previously described above for the characterisation of the extract. The reporting threshold was set on 0.10%.

Preparations of Franz diffusion dose solutions

Dose solutions of the Anacyclus pyrethrum extract and the purified pellitorine were prepared in 30:70 [H.sub.2]O:EtOH (V/V). In these dose solutions, the experimentally determined pellitorine concentration was 816 [micro]g/ml in the case of the purified pellitorine (purity factor: 0.93) and 316 [micro]g/ml in case of the Anacyclus pyrethrum extract. The purified pellitorine concentration was determined using ultraviolet-visible spectrophotometry (UV-vis) (Ultrospec 4000 Pharmacia Biotech), using [E.sup.1%.sub.1cm] = 1330 (Merck Index, 2001), while the concentration in the AP extract was determined using the purified pellitorine as reference standard. Negative controls were included in the study as well (dose solutions without NAA).

In vitro skin permeation study

Static Franz diffusion cells (Logan Instruments Corp., New Jersey, USA) with a receptor compartment of 5 ml and an available diffusion area of 0.64 [cm.sup.2] were used to determine the skin permeation of pellitorine in the different formulations. Human skin was used and the analyses were done in fourfold for the purified pellitorine and in sextuplicate for the Anacyclus pyrethrum extract, using a randomised blocked design. The skin samples were obtained from aesthetic body contouring surgery of three healthy female patients (40 years old [+ or -] 10, mean [+ or -] SD), supplied by the Department of Plastic and Reconstructive Surgery of the University Hospital (Ghent, Belgium). Confidentiality procedures with informed consent were applied. Immediately after the surgical procedure, the skin was cleaned with 0.01 M PBS pH 7.4, the subcutaneous fat was removed and the skin was subsequently stored at -20[degrees]C for not longer than 6 months. Just before the start of the FDC experiments, the full-thickness skin was thawed, mounted on a template and sliced to obtain split-thickness human skin, using an electrical powered dermatome. An actual skin thickness of 295 [+ or -] 15.5 [micro]m (mean [+ or -] SE, n = 20), 220 [+ or -] 9.35 [micro]m (mean [+ or -] SE, n = 30) and 235 [+ or -] 9.09 [micro]m (mean [+ or -] SE, n = 23) was experimentally determined with a micrometer (Mitutoyo, Tokyo, Japan) from the different patients. The receptor chambers were filled with 0.01 M PBS. The skin samples were visually inspected for skin damage and were mounted on the FDC between the donor and the receptor chambers, with the epidermis side upwards ensuring that no air was present under the skin. A Teflon coated magnetic stirring bar (400 rpm) allowed that the receptor fluid was continuously mixed. Skin integrity was controlled by measuring the skin impedance using an automatic micro-processor controlled Tinsley LCR Impedance Bridge (Croydon, U.K.). Skin pieces displaying an impedance value below 10 k[ohm] were discarded and replaced by a new skin piece (De Spiegeleer et al., 2008). 500 [micro]l of the dose solutions were applied on the skin surface with a micropipette. The donor chamber was covered with parafilm to prevent evaporation of the dose formulations. During the FDC experiment, the temperature of the receptor compartment was kept constant at 32[degrees]C by a water jacket. 200 [micro]l samples of receptor fluid were taken at regular time intervals (0 h, 1 h, 2h, 4h, 8h, 12 h, 18h, 21 h, 24 h) from the sample port and were immediately replaced by 200 [micro]l fresh receptor fluid. This was taken into account for the calculation of the cumulative permeated concentrations. Immediately after the last sample had been drawn, the remaining dose formulation was removed from the skin surfaces using a cotton swab. The epidermis and dermis were separated with forceps and pellitorine was extracted from the skin layers with ethanol. In addition, the ratio [C.sup.24h.sub.epidermis]/[C.sup.24h.sub.vehiculum] was obtained. The concentration of pellitorine in the (epi)dermis was obtained by dividing the amount of extracted pellitorine (experimentally determined) by the volume of the (epi)dermis (thickness of (epi)dermis (cm) x skin surface (0.64 [cm.sup.2])). The thickness of the epidermis was assumed to be 50 [micro]m, and the thickness of the dermis was the total measured skin thickness minus the thickness of the epidermis. The concentration of pellitorine in the remaining dose solution after 24 h was calculated by dividing the amount of pellitorine in the dose solution left after 24h (experimentally determined) by the applied volume of the dose solution, i.e. 500 [micro]l. A linear relationship of the individual cumulative amount of pellitorine versus time was observed, confirming steady-state conditions. Sink conditions were confirmed by the data.

Liquid chromatography of the FDC samples

Two methods were used, i.e. HPLC-UV and UHPLC-UV. The HPLC-UV method was applied for the FDC experiments with the purified pellitorine. However, this method required too long run times when applied on AP extract samples due to the presence of other compounds which had to be eliminated from the column before a next run. Hence, an adapted UHPLC method was applied on the FDC samples of the extract.

The HPLC-UV method used a Waters Alliance 2695 separation module and a dual absorbance detector 2487, equipped with Empower 2 software (Waters, Millford, USA) were part of the HPLC apparatus. 25 [micro]l of each sample was injected on a Symmetry C18 column (75 mm x 4.6 mm, 3.5 [micro]m) (Waters, Milford, USA) with an appropriate guard column. The sample compartment was kept constant at 20[degrees]C, while the column temperature was maintained at 30[degrees]C. A degassed isocratic mobile phase of 0.1% FA in 30:70 [H.sub.2]O:MeOH (V/V) was used at a flow rate of 1.5 ml/min was used. The run time was 5 min and UV detection was performed at 258 nm with peak areas used for quantification. An analytical validation was performed and the LoD and LoQ defined as the concentrations equivalent to a signal to noise value of 3 and 10, respectively, were determined to be 6.92 ng/ml and 23.06 ng/ml. Linearity, with determination coefficient [R.sup.2] = 1.000, is assured in a working range of 23.06 ng/mL (LoQ) up to 204 [micro]g/ml.

The UHPLC-UV method used a Waters Acquity UPLC with a Waters Photo Diode Array (PDA) detector, equipped with Empower software (Waters, Millford, USA) were part of the HPLC apparatus. 2 [micro]l of each sample was injected on a Acquity UPLC C18 column (50 mm x 2.1 mm, 1.7 [micro]m) (Waters, Milford, USA) with an appropriate guard column. The sample compartment was kept constant at 5[degrees]C, while the column temperature was maintained at 30[degrees]C. A gradient was used (t = 0-1.8 min: 100% A, t= 1.8-2.3 min: 100% B. t = 2.3-3.55 min: 100% B, t= 3.55-4.05 min: 100% A, t = 4.05-6.55 min: 100% A with A: 0.1% FA in 30:70 (V/V) [H.sub.2]O:MeOH and B: 0.1% FA in MeOH) at a flow rate of 0.5 ml/min was used. UV detection was performed at 258 nm with peak areas used for quantification. An analytical validation was performed and the LoD and LoQ defined as the concentrations equivalent to a signal to noise value of 3 and 10, respectively, were determined to be 2.97 ng/ml and 9.91 ng/ml. Linearity, with determination coefficient [R.sup.2] = 1.000, is assured in a working range of 9.91 ng/ml (LoQ) up to 204 [micro]g/ml.

Calculation of skin permeation parameters

The cumulative amounts of pellitorine (in pg) permeated through human skin were plotted as a function of time (in hours). For the calculations of the transdermal parameters, the individual graphs were used. Steady-state flux ([J.sub.ss], [micro]g/([cm.sup.2] h)) was calculated from the slope of the linear portion of the cumulative amount versus time curve divided by 0.64 to correct for the exposed skin area ([cm.sup.2]). The lag time (h) was obtained by setting y = 0 in the individual linear regression equation. The [Q.sub.1d] is the cumulative quantity, expressed as % of the effective dose applied, obtained after 1 day. The indicated parameters are the secondary parameters, i.e. directly obtained from and dependent on the experimental data. The primary parameters, i.e. derived from the experimentally obtained secondary parameters and independent of some operational experimental conditions, are calculated in accordance with ECETOC, CEFIC (ECETOC, 1993): the steady-state permeability coefficient ([K.sub.p], cm/h) was calculated as follows:

[K.sub.p] = [J.sub.ss]/[C.sub.d]

where [C.sub.d] ([micro]g/ml) is the concentration of pellitorine in the dose formulation. Furthermore, the apparent diffusion ([D.sub.m], [cm.sup.2]/h) and partition ([K.sub.m]) coefficients were determined using the following equations:

[D.sub.m] = [d.sup.2]/6 x [t.sub.lag]

[K.sub.m] = [K.sub.p] x d/[D.sub.m]

where d and [t.sub.lag] are the measured skin thickness (cm) and the lag time (h). respectively. Furthermore, the steady-state plasma concentration after topical application of pellitorine (ng/ml) was calculated using the following formula:

[C.sup.top.sub.pl,ss] = A x [K.sub.p] x [C.sub.d]/Cl

in which Cl is the plasma clearance (l/h), A is the skin surface ([cm.sup.2]) and [C.sub.d] the concentration in the dose solution applied on the skin.

Results

Identification and purity of pellitorine

The purity of the Anacyclus pyrethrum extract and the purified pellitorine was determined by HPLC-UV. Fig. 2 shows the HPLC-UV chromatogram of the used extract. The main peak was pellitorine (deca-2E,4E-dienoic acid isobutylamide or [C.sub.14][H.sub.25]NO). identified by the molecular ion m/z signal observed in [MS.sup.1] (precursor ion m/z = 224) and complemented by the CID fragmentation data in [MS.sup.2]. The product ions (m/z) were 74, 83, 123, 133, 151 and 168, respectively: with fragment losses of 150,141,101,91, 73 and 56, respectively. Its normalised concentration, based on [UV.sub.258nm] peak area, was 32.0%. The other peaks observed are other N-alkylamides, e.g. anacycline (tetradeca-2E,4E-diene-8,10-diynoic acid isobutylamide).

The isolated pellitorine had a normalised concentration of 93.3%, with only one additional N-alkylamide peak observed at 67.0 min (Fig. 3). [MS.sup.1] and [MS.sup.2] data confirmed the identity of both peaks. The minor peak observed at 67.0 min with a precursor ion of 336 m/z ([C.sub.22][H.sub.25]N[O.sub.2]) resulting in product ions (m/z) of 121, 129, 171, 199 and 216 with fragment losses of 215, 207, 165, 137 and 120, respectively, was identified as tetradeca-2E,4E-diene-8,10-diynoic acid 4-OH phenylethylamide.

Franz diffusion cell transdermal results

Our results showed that pellitorine was able to diffuse through human skin when applied in an EtOH:[H.sub.2]O solution. All individual runs confirmed the sink and steady-state conditions. A linear relationship of the individual cumulative amounts versus time was observed between time points 8 and 24 h ([R.sup.2] not less than 0.96). The mean percentage of pellitorine of the applied dose solution permeated through the skin versus time, is visually presented in Fig. 4 for both the purified pellitorine and pellitorine in the Anacyclus pyrethrum extract.

During the first hours of the FDC experiment, the percentage of pellitorine permeated through the skin was very similar for both formulations up to 8h. From then on (8-24 h), the percentage of pellitorine penetrating the skin was higher for pellitorine in the extract and an increase in difference between the two formulations was seen toward the end of the experiment (24 h). The curve of the AP extract exhibited a higher slope than the slope of purified pellitorine.

Linear regression on the linear sections of the individual curves, relating the cumulative amounts of pellitorine versus time, was performed to calculate the skin parameters. The values of the transdermal parameters, calculated on the individual linear trend lines, are given in Table 1.

The difference in the apparent primary skin parameter [K.sub.p] between the two formulations was significant at the 0.05 significance level using an independent t-test (p < 0.05). The steady-state permeability coefficient of pellitorine in the Anacyclus pyrethrum extract was thus significantly higher than the [K.sub.p] of the purified pellitorine.

After separating the epidermis from the dermis at the end of the FDC experiment (24 h), the remaining amounts of pellitorine present in the applied dose solution, in the epidermis and in the dermis were determined. Fig. 5 presents an overview of the pellitorine concentrations in the different compartments (dose solution, epidermis, dermis, receptor fluid) obtained under our experimental conditions, applying dose solutions of 816 [micro]g/ml pellitorine (as purified form) and 316 [micro]g/ml pellitorine (as AP root extract).

Concentrations of pellitorine in the epidermis and dermis ranged between 282-730 [micro]g/ml and 50-56 [micro]g/ml, respectively. Pellitorine in the purified preparation showed a decreasing concentration gradient from the dose solution over the epidermis and dermis to the receptor fluid, while pellitorine in the plant extract gave a different concentration distribution, with much higher concentrations in the epidermis ('reservoir effect'), compared not only to the dose solution, but also to the purified formulation.

Discussion

Our findings demonstrate for the first time that the plant diene N-alkylamide pellitorine from an Anacyclus pyrethrum extract permeates the skin. This corroborates well with a previous study from our research group, demonstrating the skin permeability of the triene N-alkylamide spilanthol (Boonen et al., 2010a). Spilanthol and pellitorine only differ in the place and the number of unsaturated bonds in the fatty acid chain. Spilanthol, having an extra double bond in the fatty acid chain, is slightly more hydrophilic than pellitorine. This is reflected in the log P values: spilanthol has a slightly lower log P value (3.39) than pellitorine (3.65) (calculated with Hyperchem professional 8.0 software). Both these logP values are acceptable values for skin penetrators, with the optimal target logP value currently proposed ranging between 1 and 3 (Brain and Chilcott, 2008). Due to its slightly more optimal logP value, spilanthol is expected to penetrate the skin more. Indeed, spilanthol has higher [K.sub.p] values (purified: 1.66 x [10.sup.-4] cm/h and in extract: 4.29 x [10.sup.-4] cm/h) compared to the [K.sub.p]'s of pellitorine (purified: 1.10 x [10.sup.-4] cm/h and in extract: 2.25 x [10.sup.-4] cm/h).

In this study, two different forms were tested: purified pellitorine and pellitorine in Anacyclus pyrethrum extract. Upon comparison of the apparent permeability coefficients of pellitorine between the two formulations, a significant difference between the [K.sub.p]'s of the purified pellitorine versus the AP extract was observed: a two times higher [K.sub.p] was obtained for pellitorine in the extract. As a result of this, the percentage of pellitorine that had penetrated through the skin after 24 h was significantly higher when the extract was used as dose solution. This can only be due to the other compounds present in the extract, which improved the penetration of pellitorine. The AP extract does indeed contain other N-alkylamides (Fig. 2) (Boonen et al., 2012a,b), which can themselves function as skin penetration enhancers. This corroborates well with a previous study, where the NAA spilanthol was demonstrated to act as a compound dependent skin penetration enhancer (De Spiegeleer et al., 2013). The results of the current study with the pellitorine extract suggest that also other NAAs can exert a penetration enhancing effect. Furthermore, based upon these results, a similar study was undertaken comparing the [K.sub.p]'s of purified spilanthol versus the [K.sub.p] of spilanthol in Spilanthes acmella extract. A 2.6 times higher [K.sub.p] (4.29 x [10.sup.-4] cm/h) was obtained for the extract compared to purified spilanthol (1.66 x [10.sup.-4] cm/h), confirming and generalizing our findings.

In general, two processes have to be considered in skin permeation: (1) the partition of a compound from the dose solution (vehicle) into the SC and (2) the diffusion across the SC and subsequent layers. The first step is thus the partitioning of a compound into the SC, which can be quantified by a thermodynamic equilibrium constant between the concentration of the penetrant in the vehicle and the upper skin layer, i.e. the stratum corneum. Partitioning is important for skin absorption because it influences the amount of penetrant in the SC and subsequently this amount affects the thermodynamic gradient across the skin (Pugh and Chilcott, 2008).

The partition coefficient [K.sub.m] for pellitorine in the Anacyclus pyrethrum extract (0.265) was higher than the one of purified pellitorine (0.067). This can also be observed in the distribution graph, presented in Fig. 5, in which the epidermis contained the highest concentration of pellitorine when the dose solution was the AP extract: the ratio [C.sup.24h.sub.epidermis]/[C.sup.24h.sub.vehiculum] was 2.78 for the AP extract and 0.40 for purified pellitorine. The other N-alkylamides present in the extract will thus not only influence the pellitorine behavior in the dose solution, but will also alter the chemical properties of the SC and hence the partition coefficient [K.sub.m].

The second stage in skin absorption is the diffusion of the penetrant within the upper skin layers, which depends upon a thermodynamic gradient. Factors that can affect the diffusion are the size, the shape of the penetrant and stickiness of the penetrant to compounds present in the skin (hydrogen bonding) (Pugh and Chilcott, 2008). Furthermore, it is recognised that solvents, such as ethanol, as used in our study, can enhance solubility, influence drug permeation and alter skin barrier properties. Ethanol is a frequently used solvent in topically applied medicines and is known to also have a penetration enhancing effect at lower concentrations with a change in this property observed at concentrations above 70%. At these higher ethanol levels, there is i.a. a dehydration of human skin resulting in a decrease of permeability of compounds dissolved in high ethanol formulations (Williams and Barry, 2004; Watkinson et al., 2009; Boonen et al., 2010a). The co-compounds in the extract apparently do not significantly influence the diffusion in the viable skin layers and thus once in the skin, there is almost no difference between the [D.sub.m] of pellitorine in the two forms.

As there are already some topical products on the market containing pellitorine, it is important to determine the biological relevance of the obtained local skin kinetics data of pellitorine. Until now, there are no integrated pharmacokinetic/pharmacodynamic (PK/PD) models available to characterise the effects of pellitorine. Consequently, for example, the required plasma concentration of pellitorine for having systemic effects is not yet known. Therefore, this information is taken from another plant N-alkylamide, where PK/PD data are available. In the study of Guiotto et al. (2008), the apparent clearance of the N-alkylamide dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide from Echinacea purpurea was 1371/h and this value was also used for our calculations with pellitorine. With the obtained [K.sub.p] values in this study (1.1 x [10.sup.-4] for purified pellitorine and 2.3 x [10.sup.-4] for pellitorine in the extract) and under the assumptions that the exposed skin surface is 20 [cm.sup.2] and the concentration in the dose solution is 1 mg/ml, the steady-state plasma concentration of pellitorine after topical application can thus be calculated: the resulting steady-state plasma concentrations for the purified pellitorine and the pellitorine in the Anacyclus pyrethrum extract were 1.6 x [10.sup.-2] ng/ml and 3.2 x [10.sup.-2] ng/ml, respectively. When comparing these values with the steady-state plasma concentration of the Echinacea purpurea N-alkylamide (4.3 x [10.sup.-2] ng/ml) to have bioactivity, it is concluded that the pellitorine steady-state plasma concentrations are in the same order of magnitude as the bioactive Echinacea purpurea N-alkylamide, indicating pellitorine is able to elicit systemic effects after topical application (Guiotto et al., 2008). Furthermore, the epidermis and dermis concentration of pellitorine obtained with a concentration of pellitorine in the dose solution of 816 [micro]g/ml in the purified solution and 316 [micro]g/ml in the extract solution, were between 282-730 [micro]g/ml and 50-56 [micro]g/ml, respectively. Seen the high skin concentrations of pellitorine, local effects are certainly expected, e.g. immune-modulating activity. A topical formulation of pellitorine can thus be interesting for systemic and/or local functionalities.

This transdermal behavior, coupled to its pharmacodynamics properties, can have consequences for the regulatory classification of products containing plant-based N-alkylamides like pellitorine (Regulation (EC) No 1223/2009, Directive 2001/83/EC, Directive 2001/82/EC). This study clearly demonstrated that pellitorine was able to penetrate the skin and reach not only the viable cells of the skin in this study, but also the systemic circulation, recommending formulations containing reasonable concentrations of this plant N-alkylamide as a medicinal product.

Conclusion

In this study, the N-alkylamide pellitorine was isolated from an ethanolic Anacyclus pyrethrum extract by means of semi-preparative HPLC. The purity was evaluated and was determined to be 93% using HPLC-UV. The transdermal behavior of pellitorine was investigated with Franz diffusion cells and pellitorine was applied on human skin in two different forms: as purified pellitorine and as Anacyclus pyrethrum extract. The purity of pellitorine in the extract was 32%. In both dose solutions, pellitorine was able to cross the stratum corneum and subsequently reach the viable epidermis and dermis. A higher permeability coefficient was observed for pellitorine when administered as extract (2.3 x [10.sup.-4] cm/h) than as purified pellitorine (1.1 x [10.sup.-4] cm/h). After 24 h, concentration profiles of pellitorine were obtained in the remaining dose solution, the epidermis, the dermis and the receptor fluid. An increased partitioning of pellitorine from the dose solution into the stratum corneum was observed when using the extract, which is also reflected by the higher partition coefficient (0.265) compared to the [K.sub.m] value of the purified pellitorine (0.067). Other N-alkylamides present in the Anacyclus pyrethrum extract thus contribute to the skin penetration of pellitorine. To conclude, due to the high systemic and skin concentrations of pellitorine, local dermal and systemic biological effects are expected, recommending that topically applied N-alkylamides like pellitorine are generally better classified as a medicinal product instead of a cosmetic.

Conflict of interest

No conflict to disclose.

Acknowledgements

The authors like to thank the Special Research Fund of Ghent University (BOF 01D23812 to Lien Taevernier) for their funding. The authors would like to thank Lieselot Minne for her technical assistance in the transdermal experiments and Dr. Vikas Sharma for his provision of the AP extract.

ARTICLE INFO

Article history:

Received 1 July 2014

Received in revised form 31 August 2014

Accepted 20 September 2014

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Lieselotte Veryser (a), Lien Taevernier (a), Nathalie Roche (b), Kathelijne Peremans (c), Christian Burvenich (c), Bart De Spiegeleer (a), *

(a) Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium

(b) Department of Plastic and Reconstructive Surgery, University Hospital Ghent, De Pintelaan 185, B-9000 Ghent, Belgium

(c) Department of Comparative Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

* Corresponding author. Tel.: +32 9 264 81 00; fax: +32 9 264 81 93.

E-mail address: Bart.DeSpiegeleer@UGent.be (B. De Spiegeleer).

http://dx.doi.org/10.1016/j.phymed.2014.08.015

Table 1 Transdermal parameters for pellitorine in different dose
solutions (mean [+ or -] SEM, with n=4 for purified pellitorine and
n=6 for pellitorine in AP extract).

Dose solution               Observed secondary parameters

                            [J.sub.ss] ([micro]g/[cm.sup.2] h)

Pellitorine                 0.089 [+ or -] 0.032
Pellitorine in AP extract   0.071 [+ or -] 0.008

Dose solution               Observed secondary parameters

                            [Q.sub.1d] (% of the applied dose solution)

Pellitorine                 0.314 [+ or -] 0.123
Pellitorine in AP extract   0.569 [+ or -] 0.065

Dose solution               Observed secondary parameters

                            [t.sub.lag] (h)

Pellitorine                 2.445 [+ or -] 0.674
Pellitorine in AP extract   5.050 [+ or -] 0.576

Dose solution               Apparent primary parameters

                            [K.sub.p] ([10.sup.-4] cm/h)

Pellitorine                 1.096 [+ or -] 0.397
Pellitorine in AP extract   2.253 [+ or -] 0.246

Dose solution               Apparent primary parameters

                            [D.sub.m] ([10.sup.-5] [cm.sup.2]/h)

Pellitorine                 5.479 [+ or -] 1.738
Pellitorine in AP extract   2.166 [+ or -] 0.207

Dose solution               Apparent primary parameters

                            [K.sub.m]

Pellitorine                 0.067 [+ or -] 0.030
Pellitorine in AP extract   0.265 [+ or -] 0.028
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Author:Veryser, Lieselotte; Taevernier, Lien; Roche, Nathalie; Peremans, Kathelijne; Burvenich, Christian;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUBL
Date:Dec 15, 2014
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