Influence of injection volume and solvent strength on spilantholchromatography using RP fused-core amide stationary phase

For high-throughput purposes, newly developed Fused-Core HPLC stationary phases (HALO® columns) have attracted the interest of the chromatographic community. Due to their small particle size (2.7 μm) and unique particle technology with 0.5 μm porous shell fused to a solid core particle (1.7 µm diameter), these columns create fast and high performance separations. For our skin research, in vitro FDC experiments are performed, resulting in large sample numbers of low concentrated bio-active in physiologic receptor media (e.g. PBS). Spilanthol, present in Spilanthes acmella as most prominent N-alkylamide and having promising transdermal activity [1, 2], could be analyzed in reversed-phase mode on the HALO® amide stationary phase with methanol/H2O (70/30, V/V) acified with 1% formic acid as mobile phase. The influence of the injection volume and the sample solvent composition on six chromatographic characteristics characteristics (retention time, area, height, theoretical plate, symmetry factor and limit of detection) was investigated. Applying different injection volumes (2–100 µl), the chromatographic responses were obtained from a Spilanthes extract, dissolved in a purely aqueous (PBS) as well as methanol-based (70/30, V/V) sample solvent. Our results show that the chromatographic characteristics are highly dependent on the injection volume and solvent strength

New N-alkylamides from Anacyclus pyrethrum

The roots of Anacyclus pyrethrum DC (Asteraceae) are frequently used in traditional medicine e.g. as aphrodisiac [1]. Depending on the extraction method and solvent, different yields of N-alkylamide constituents can be found, possibly resulting in alterations in biological effects and toxicity. Therefore, analytical profiling of the bio-active N-alkylamides in these plant preparations is an inevitable quality parameter, with liquid chromatography-electrospray mass spectrometry (HPLC/ESI-MS) as recommended technique for comprehensive analysis of alkylamides in plant extracts [2-4].An N-alkylamide profiling from an ethanolic Anacyclus pyrethrum root extract was performed using a gradient reversed phase HPLC/ESI-MS method on an embedded polar column. MS1 and MS2 fragmentation data were used for identification purposes, while UV was used for quantification. Thirteen N-alkylamides (five N-isobutylamides, three N-methyl isobutylamides, four tyramides and one 2-phenylethylamide) were detected. Five of are novel compounds, which have never been identified in Anacyclus pyrethrum or other plants: Acknowledgements: Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) (no. 091257) and the All India Council for Technical Education, New Delhi, India. References: 1. Sharma, V., Thakur, M., Chauhan, N., Dixit, V. 2010 Planta Med 76:1214-1214. 2. Sharma, V., Boonen, J., Chauhan, N., Thakur, M. De Spiegeleer, B., Dixit, V. 2011 Phytomedicine, In press. 3. Kartal, M., Kan, Y., Gulpinar, A. R. 2007 Planta Med 73:253. 4. Boonen, J., Baert, B., Burvenich, C., Blondeel, P., De Saeger, S., De Spiegeleer, B. 2010 J Pharmaceut Biomed 53:243-249

Transdermal penetration enhancing effect of the N-alkylamide spilanthol

The dermal penetration of compounds may be influenced by other compounds when mixtures are presented to the skin. Plant extracts, often used in cosmeceuticals, are complex mixtures of wanted bio-actives as well as undesirable impurities like nanoparticles and mycotoxines. A major question is if plant bio-actives (like spilanthol) can significantly alter the dermal penetration of other compounds which can be actives (like testosterone) or impurities (like mycotoxins). If so, the qualification assessment of the product quality needs to include this influence within the Quality-by-Design strategy. Therefore, the concentration-dependent penetration promoting effect of spilanthol was investigated on the three CART transdermal model compounds i.e. caffeine, ibuprofen and testosterone [1]. It was shown that spilanthol has a compound and concentration dependent penetration enhancing effect. No significant penetration enhancing effect for ibuprofen has been observed. However, with increasing spilanthol concentration (from 0 up to 1%, m/V), the permeability of caffeine increases, resulting in an enhancing ratio (ER) of 4.60 ± 0.49 (mean ± SEM, n=4). For testosterone, a maximal penetration enhancing concentration of 0.5% spilanthol was found (ER = 4.13 ± 0.44 (mean ± SEM, n=3)). Our findings with these model compounds are also confirmed with mycotoxins [2]. In conclusion, the existence of a significant mutual influence of compounds towards skin penetration should always be considered as part of the functional quality evaluation or in topical product development. References [1] B. Baert, E. Deconinck, M. Van Gele, M. Slodicka, P. Stoppie, S. Bode, G. Slegers, Y. Vander Heyden, J. Lambert, J. Beetens, B. De Spiegeleer. Transdermal penetration behaviour of drugs: CART-clustering, QSPR and selection of model compounds, 2007, Bioorganic & medicinal chemistry, 15(22): 6943-6955. [2] B. De Spiegeleer, J. Boonen, L. Veryser, L. Taevernier, S.V. Malysheva, J. Diana Di Mavungu, S. De Saeger, N. Roche, P. Blondeel. Skin penetration enhancing properties of the plant N-alkylamide spilanthol, 2012, manuscript in preparation

Analytical profiling, modeling and transdermal/transmucosal characteristics of bioactive N-alkylamides

N-alkylamides are a group of bioactive molecules found in several plants. Extracts and formulations derived from these plants are not only used orally, but are applied on the skin and buccal mucosa as well. However, there is no specific information available about the intrinsic local pharmacokinetics of N-alkylamides after topical application, questioning the role of this mode of administration. Therefore, we investigated the transdermal and transmucosal behaviour of spilanthol, a prominent N-alkylamide, in a commercial Spilanthes extract, two mouth gels and different propylene glycol (PG)/H20 solutions

Penetration enhancing effect of phytoceramides

Ceramides are essential components in the stratum corneum barrier function. Different classes of ceramides are present in human skin, differing in the nature of sphingosine and acyl moieties with respect to chain length, degree of saturation and the presence of an OH group [1]. Ceramides with a saturated sphingosine base containing a hydroxyl function at C4 are known as phytoceramides. A few studies demonstrated the penetration enhancing properties of ceramides [2-5], however, systematic studies using phytoceramides are lacking. This led us to assess the penetration enhancing effect of phytosphingosine and a series of nine phytoceramides via transdermal experiments using in vitro Franz diffusion cells. As transdermal model compounds, testosterone, caffeine and ibuprofen were tested in a 50:50 (V/V) EtOH:H2O dose formulation [6]. Results showed that the penetration enhancing effect of the phytoceramides depends on the used model compound. Selected phytoceramides exhibited a penetration enhancing ratio of more than two. References [1] Janůšová, B.; Zbytovská, J.; Lorenc, P.; Vavrysová, H.; Palát K.; Hrabálek, A.; Vávrová, K. (2011). Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochimica et Biophysica, 1811, 129–137. [2] Vávrová, K.; Hrabálek, A.; Dolezal, P.; Holas, T.; Zbytovská, J. (2003). L-serine and glycine based ceramide analogues as transdermal permeation enhancers: polar head size and hydrogen bonding. Bioorganic & medicinal chemistry letters, 13, 2351-2353. [3] Vávrová, K.; Zbytovská, J.; Hrabálek, A. (2005). Amphiphilic transdermal permeation enhancers: structure-activity relationships. Current Medicinal Chemistry, 12, 2273-2291. [4] Novotý, J.; Janůšová, B.; Novotý, M.; Hrabálek, A.; Vávrová, K. (2009). Short-chain ceramides decrease skin barrier properties. Skin pharmacology and Physiology, 22, 22-30. [5] Sinko, B.; Kökösi, J.; Avdeef, A.; Takács-Novák, K. (2009). A PAMPA study of the permeability-enhancing effect of new ceramide analogues. Chemistry & Biodiversity, 6, 1867-1874. [6] Baert, B.; Deconinck, E.; Van Gele, M.; Slodicka, M.; Stoppie, P.; Bode, S.; Slegers, G.; Vander Heyden, Y.; Lambert, J.; Beetens, J.; De Spiegeleer, B. (2007). Transdermal penetration behaviour of drugs: CART-clustering, QSPR and selection of model compounds. Bioorganic & medicinal chemistry, 15(22), 6943-6955

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 aging. 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. N-alkylamides are a promising group of naturally occurring bio-actives in Spilanthes spp. Therefore, N-alkylamide profiling of ethanol extract of Spilanthes acmella flowers 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. MS1 and MS2 fragmentation data were used for identification purposes. The extracts (50, 100 and 150 mg/kg body weight/day) and sildenafil citrate (5mg/kg) were administered orally for 28 days. The behavioral parameters were observed at day 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 tentatively identified. The orally administered extract had a dose dependent effect on mounting frequency, intromission frequency and ejaculation frequency. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in-vitro and in-vivo. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent

LC-MS N-alkylamide profiling of an ethanolic Anacyclus pyrethrum root extract

An N-alkylamide profiling from an ethanolic Anacyclus pyrethrum DC. (Asteraceae) root extract was performed using a gradient reversed phase high performance liquid chromatography/UV/electrospray-ionization ion-trap mass spectrometry (HPLC/UV/ESI-MS) method on an embedded polar column. MS1 and MS2 32 fragmentation data were used for identification purposes, while UV was used for quantification. Thirteen N-alkylamides (five N-isobutylamides, three N-methyl isobutylamides, four tyramides and one 2-phenylethylamide) were detected. Six of them, identified as undeca-2E,4E-diene-8,10-diynoic acid isobutylamide, undeca-2E,4E-diene-8,10-diynoic acid N-methyl isobutylamide, tetradeca-2E,4E-diene-8,10-diynoic acid tyramide, deca-2E,4E-dienoic acid N-methyl isobutylamide, tetradeca-2E,4E,XE/Z-trienoic acid tyramide and tetradeca-2E,4E,XE/Z,YE/Z-tetraenoic isobutylamide, are novel compounds which have never been reported before in Anacyclus pyrethrum

Transdermal evaluation of caffeine in different formulations and excipients

Background: The stratum corneum(SC) forms adifficultphysical barrier fordrugs to pass through the skin. Several strategieswere developed to overcome this barrier.Optimization of topical drug formulations by selected excipients may facilitate the penetration of drugs through the SC into the viable skin cells and ultimately into the systemic circulation. Methods: Here, both the influence of two formulations (a classic carbomer-based gel and a novel Pluronic® lecithin organo gel (PLO gel)) and selected excipients (ethanol, propylene glycol, diethylene glycol monoethyl ether, isopropyl myristate (IPM), and water) with or without the penetration enhancer miconazole nitrate on the transdermal penetration characteristics of caffeine were determined using an in vitro Franz diffusion cell setup. Results: Higher fluxes were observed for the carbomer-based gel compared to the PLO gel. Among the commonly used excipients, IPM showed the best penetration enhancing properties, while the presence of the penetration enhancer miconazole nitrate did not significantly alter the apparent skin permeation characteristics for caffeine. Conclusion: The high ethanol percentage in the carbomer-based gel could explain the results as supported by our excipient data.Moreover, IPMcould play a beneficial role in topical formulations as this excipient was responsible for a significant increase in the amount of caffeine penetrated through the skin. No overall statistical significant effect of the presence of miconazole nitrate as a penetration enhancer was observed