Jente Boonen

Human skin penetration of selected model mycotoxins

Dermal exposure data for mycotoxins are very scarce and fragmentary, despite their widespread skin contact and hazard toxicity. In this study, the transdermal kinetics of aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1), citrinin (CIT), zearalenone (ZEA) and T-2 toxin (T-2) were quantitatively evaluated, using human skin in an in vitro Franz diffusion cell set-up. All mycotoxins penetrated through the skin, except for FB1, which showed concentrations in the receptor fluid below the LoD, resulting in a K(p)<3.24×10(-6)cm/h. OTA showed the highest permeation (K(p)=8.20×10(-4)cm/h), followed by CIT (K(p)=4.67×10(-4)cm/h). AFB1 and ZEA showed lower permeability rates (K(p)=2.11 and 2.33×10(-4)cm/h, respectively). T-2 was found to have the lowest permeability (K(p)=6.07×10(-5)cm/h). From literature-based mycotoxin-concentrations, dermal contact surface, exposure time and apparent K(p)s obtained in this study, the daily dermal exposure (DDE) in two industrial and one residential scenario was estimated. Dermal exposure to the DNA-reactive genotoxic carcinogenic AFB1 can lead to a health risk for agricultural workers which are exposed to a mycotoxin contaminated solution in a worst case situation. For all the other investigated mycotoxins, no significant health risk is calculated after dermal contact in neither agricultural nor residential environments.

Alkamid database: chemistry, occurrence and functionality of plant N-alkylamides

ETHNOPHARMACOLOGICAL RELEVANCE N-Alkylamides (NAAs) are a promising group of bioactive compounds, which are anticipated to act as important lead compounds for plant protection and biocidal products, functional food, cosmeceuticals and drugs in the next decennia. These molecules, currently found in more than 25 plant families and with a wide structural diversity, exert a variety of biological-pharmacological effects and are of high ethnopharmacological importance. However, information is scattered in literature, with different, often unstandardized, pharmacological methodologies being used. Therefore, a comprehensive NAA database (acronym: Alkamid) was constructed to collect the available structural and functional NAA data, linked to their occurrence in plants (family, tribe, species, genus). MATERIALS AND METHODS For loading information in the database, literature data was gathered over the period 1950-2010, by using several search engines. In order to represent the collected information about NAAs, the plants in which they occur and the functionalities for which they have been examined, a relational database is constructed and implemented on a MySQL back-end. RESULTS The database is supported by describing the NAA plant-, functional- and chemical-space. The chemical space includes a NAA classification, according to their fatty acid and amine structures. CONCLUSIONS The Alkamid database (publicly available on the website http://alkamid.ugent.be/) is not only a central information point, but can also function as a useful tool to prioritize the NAA choice in the evaluation of their functionality, to perform data mining leading to quantitative structure-property relationships (QSPRs), functionality comparisons, clustering, plant biochemistry and taxonomic evaluations.

LC-MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behaviour of its main bio-active spilanthol

N-Alkylamides are a promising group of naturally occurring bio-actives, with evidence for immune stimulating properties, which find applications i.a. in buccal preparations. In Spilanthes extracts, these properties are mainly ascribed to the most abundant N-isobutylamide, spilanthol. Yet, other N-alkylamides present in these extracts may contribute to this effect, as well as to its potential toxicity and physiologic interactions. Therefore, N-alkylamide profiling of an ethanolic Spilanthes extract was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS(1) and MS(2) fragmentation data were used for identification purposes. Moreover, the transmucosal behaviour of spilanthol, formulated in this ethanolic extract and in two commercially available oral gels, was evaluated using porcine buccal mucosa in a Franz diffusion cell experimental set-up. A high-throughput HPLC-UV method was used for the quantification of spilanthol in the receptor phase. Fundamental permeation characteristics of spilanthol in a solvent-independent way (100% aqueous dose solution) were obtained using different propylene glycol/water ratios. Eight N-isobutylamides, two 2-methylbutylamides and one 2-phenylethylamide were detected, with spilanthol as most abundant N-alkylamide (88.8%). Up till now, two of these N-isobutylamides were not yet described in Spilanthes extracts. We demonstrated for the first time that spilanthol permeates the buccal mucosa. Depending on the formulation, a more pronounced local or systemic effect is achieved, which is important for the regulatory product classification. The purely aqueous permeation coefficient K(p,aq) (+/-SEM) was found to be 11.3 (+/-0.403)10(-3)cm/h.

Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts

AIM OF THE STUDY N-Alkylamides are a large group of bioactive molecules found in several plants from the genera Echinacea, Xanthoxylum and Spilanthes. Extracts and formulations derived from these plants are not only orally used, but also applied on the skin as well. However, there is currently no specific information available about the intrinsic local pharmacokinetics of N-alkylamides after topical application on human skin, questioning the role of this mode of administration. The present study investigates the transdermal behaviour of spilanthol, a prominent N-alkylamide. MATERIALS AND METHODS Two pharmaceutically accepted dose solutions (ethanol and propylene glycol based aqueous donor vehicles), combined with three different receptor fluids (PBS, PBS+0.5% HPbetaCD, EtOH/H(2)O (30:70, v/v)), were applied on split-thickness human skin in a Franz diffusion cell (FDC) system. Fundamental permeation characteristics of spilanthol in a solvent-independent way (100% aqueous dose solution) were also obtained using an extrapolation approach with different organic solvent/H(2)O ratios. RESULTS AND CONCLUSIONS We demonstrated for the first time that spilanthol permeates the skin. The following aqueous-extrapolated primary transdermal parameters were obtained (mean+/-SEM): K(p,aq)=3.31 (+/-0.29)x10(-3)cm/h, D(m,aq)=1.86 (+/-0.09)x10(-4)cm(2)/h and K(m,aq)=7.28 (+/-1.59)x10(-1). Partitioning (K(m)) was strongly dependent on the donor solution composition, while diffusion (D(m)) was mainly influenced by the receptor fluid composition.

Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats

According to Indian Systems of Medicine, Spilanthes acmella (L.) Murr. (Family - Asteraceae), is considered effective in the treatment of sexual deficiencies especially due to ageing. In the present study, characterization of ethanolic extracts of the Spilanthes acmella flower and its effect on general mating pattern, penile erection and serum hormone levels of normal male Wistar albino rats were investigated and compared with sildenafil citrate. In vitro nitric oxide release was also investigated in human corpus cavernosum cell line. As N-alkylamides are a promising group, their profiling was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS(1) and MS(2) fragmentation data were used for identification purposes. For assessment of sexual behavior, animals were divided into five groups of eight male rats. The extracts (50, 100 and 150mg/kgbodyweight/day) and sildenafil citrate (5mg/kgbodyweight/day) (positive control) were administered orally for 28 days. The behavioral and sexual parameters were observed at days 0, 15, 28 and after a lapse of 7 and 14 days of discontinuance of drug treatment. Five N-isobutylamides, one 2-methylbutylamide and one 2-phenylethylamide were identified. The orally administered extract had a dose dependent positive effect on mounting frequency, intromission frequency and ejaculation frequency and the most significant effects (p<0.05) were observed at 150mg/kg treatment, even after a lapse of 7 and 14 days of discontinuance of drug treatment. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. With 150mg/kg of ethanolic extract the values for FSH, LH and testosterone were 3.10±0.25mlU/ml, 6.87±0.18mlU/ml and 3.72±0.12ng/ml, respectively. In vitro nitric oxide release was 21.7±2.9μM, which was significantly higher compared to the control group (p<0.01). Sildenafil citrate exhibited also a significant effect on NO release, but no effect on hormone levels of rats was observed. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in vitro and in vivo. N-Alkylamides might attribute to the improved sexual potential. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent.

A New Discriminative Criterion for the Development of Franz Diffusion Tests for Transdermal Pharmaceuticals

PURPOSE In vitro skin/membrane permeation profiling of topical pharmaceuticals is an important overall quality attribute in the evaluation of product consistency and it is also used for IVIVR (in vitro - in vivo relationship) purposes in product development and change control. Franz diffusion cell (FDC) experiments are emerging as a generally accepted methodology in this field, where the choice of operational conditions requires a data-supported justification towards the discriminating power of the test. A response function is therefore proposed to objectively quantify the discriminating power. METHODS We evaluated the usefulness of the proposed response function by studying one of the operational conditions, i.e. the influence of receptor medium composition, on the FDC in vitro penetration behaviour of the model compound testosterone formulated in four different topical preparations, using both artificial membranes and dermatomed human skin. RESULTS From the obtained cumulative amount of testosterone in the receptor fluid versus time curves, the permeability coefficient Kp of testosterone from each formulation was calculated. The evaluation of the discriminating power of the different media was performed using our new objective response function based upon an equal spread criterion of normalised Kp values. CONCLUSION We demonstrated significant differences in discriminating power between the different media used, with the overall best results obtained with hydroxypropyl-beta-cyclodextrine (HPBCD) containing media. The proposed new criterion was found to be useful for the rational design of an in vitro diffusion test for transdermal pharmaceuticals.

Skin penetration enhancing properties of the plant N-alkylamide spilanthol.

ETHNOPHARMACOLOGICAL RELEVANCE Plants are often used for skin diseases in different ethnopharmacological systems. Local and systemic effects of topically applied compounds can be significantly increased by plant constituents having skin penetration enhancers. MATERIALS AND METHODS In this study, we examined the proposed penetration enhancing properties of spilanthol, an N-alkylamide abundantly present in several Asteraceae plants like Spilanthes acmella L., on three model drugs (caffeine, testosterone and ibuprofen). Moreover, as plants are frequently contaminated with toxic environmental substances, the mutual influence on the transdermal behavior between spilanthol and six model mycotoxins (aflatoxin B1, ochratoxin A, fumonisin B1, citrinin, zearalenone, T-2 toxin) was investigated. RESULTS Spilanthol exhibits component and concentration dependent penetration enhancing effects. No significant penetration enhancing effect for ibuprofen has been observed, but with increasing spilanthol concentration (from 0 up to 1w/V%), the permeability of caffeine increased, resulting in an enhancing ratio (ER) of 4.60. For testosterone, a maximal penetration enhancing concentration of 0.5% spilanthol was found (ER=4.13). Next to its beneficial applicability to increase local as well as systemic pharmacological effects of dermally co-administrated drug, this N-alkylamide negatively influences human health risk if spilanthol containing formulations are polluted with mycotoxins: the presence of spilanthol (0.3w/V%) induced a significant increase of permeability coefficient Kp of five investigated mycotoxins, with ER values ranging between 1.57 and 6.37. On the other hand, mycotoxins themselves do not significantly influence the transdermal behavior of spilanthol. CONCLUSIONS The existence of a significant mutual influence of compounds towards skin penetration should always be considered during the development or as part of the functional quality evaluation of topical products.

Vancomycin release from poly(d,l-lactic acid) spray-coated hydroxyapatite fibers

The influence of the poly(D,L-lactic acid) (PDLLA) coating thickness on the in vitro vancomycin release from a hydroxyapatite (HA) carrier was studied. Microporous HA fibers with a porosity of 51 v% and an average pore diameter of 1.0 μm were fabricated by a diffusion-induced phase separation technique. They were loaded with 38 mg vancomycin hydrochloride (VH)/gHA, and their cylindrical shape enabled the application of the spray coating technique for the deposition of uniform PDLLA coating thicknesses, varying from 6.5 μm to 28 μm. The resulting in vitro VH release varied from a complete release within 14 days for 6.5 μm coatings to a release of 23% after 28 days for 28 μm coatings. It was clear that the VH release rate from a HA fiber can be adjusted by varying the PDLLA coating thickness. Microbiological tests of these fibers against a methicillin-resistant Staphylococcus aureus (MRSA) isolate pointed to the importance of the initial burst release and confirmed that the released antibiotics had the potential to interfere with S. aureus biofilm formation.

Androgenic and spermatogenic activity of alkylamide-rich ethanol solution extract of Anacyclus pyrethrum DC.

Anacyclus pyrethrum (A. pyrethrum) has been used as Vajikaran Rasayana (aphrodisiac) in traditional Indian ayurvedic medicine to treat male sexual dysfunction, including infertility. Aphrodisiac activity may be due to an increase in the production or effect of androgens, so this study sought to evaluate the androgenic and spermatogenic potential of the alkylamide‐rich ethanol solution extract. Male Wistar strain rats weighing between 150 and 180 g were completely randomized divided into five groups. The ethanol solution extract of A. pyrethrum was administered to groups of rats in 50, 100, and 150 mg/kg doses for a period of 28 days, and the action was compared with control and testosterone‐treated rats. Thirteen N‐alkylamides were detected in the extract by using HPLC/UV/electrospray ionization mass spectrometry method. Extract administration at all the doses produced significant increase in body weight, sperm count, motility, and viability along with serum testosterone, luteinizing hormone, and follicle‐stimulating hormone concentrations. Histoarchitecture of testis revealed increased spermatogenic activities. Seminal fructose content was also significantly increased after 28 days of treatment. Our results suggest that the ethanol solution extract of the roots of A. pyrethrum has androgenic potential and may improve male fertility by enhancing spermatogenesis. Copyright

Improved positive electrospray ionization of patulin by adduct formation: usefulness in liquid chromatography-tandem mass spectrometry multi-mycotoxin analysis.

Several sensitive methods have been developed for patulin determination; however, mass spectrometric (MS) detection of this toxin in the positive electrospray ionization (ESI(+)) mode is not straightforward. Furthermore, the combined determination of patulin with other mycotoxins in one single run has not been reported yet. The present paper demonstrates the formation and use of a methanol adduct of patulin in ESI(+). A study of the fragmentation pathway confirmed the authenticity of the patulin adduct, while the use of ion trap and high resolution Orbitrap mass spectrometry allowed reliable assignment of the patulin fragment ions. Exploiting the formation of the methanol adduct, patulin has been successfully included in a single run multi-mycotoxin liquid chromatography tandem mass spectrometric (LC-MS/MS) method in support of ex vivo-in vitro biomedical studies.