Patent Application
20080027009
1) Determination ex vivo of the Effect of Surfactants on the Trypsin Activity in the Human Epidermis
For standardizing the skin of the test subjects, the subjects were requested to use only a mild (with 3-10% myrystyl sulfate instead of lauryl ether sulfate) when washing for two weeks. After this preconditioning, the forearms were in each case divided into two test areas. The test areas were treated for three days in succession in each case 3 times daily with 1 ml of washing product (examples 1, 2, 3, 4, 5 or 6) or a tap water control for 45 s per area. After the treatment, the test area was rinsed off with tap water for 30 s and dried using a disposable paper towel. On the 1st and 2nd day, the areas were treated three times (morning, midday and afternoon), on the 3rd day they were treated twice (morning and midday). Up to three washing products were tested against one another and against water.
2) Extraction of the Skin Biopsy and Measurement of the SCTE Activity
On the 4th day, SC samples were stripped from the areas by means of a microscope slide coated with sugar solution. Later on, the corneocytes were detached from the microscope slide with PBS buffer and the specific SCTE activity was determined.
3) Stratum Corneum Tryptic Enzyme (SCTE) Activity Assay
100 μl of human skin extract were incubated for 24 h with 150 μl of N-t-BOC-Phe-Ser-Arg-7-amido-4-methylcoumarin (33 μM in PBS; Sigma, St Louis, USA) at 37° C. The SCTE-specific release of fluorescent 7-amino-4-methylcoumarin was ascertained using a fluorescence plate reader (filter ex=360 nm+40, em=460 nm+40 nm, CytoFluor 4000, PerSeptive Biosystems, Framingham, USA).
4) Measurement of the Protein Concentration
In order to calculate the specific trypsin activity of the extracts, the protein content was determined by means of the ninhydrin method following alkaline hydrolysis. The corneocyte solutions were evaporated to dryness and the proteins were hydrolyzed for 5 h at 150° C. with 2 ml of sodium hydroxide solution (6M). The solution was neutralized with 2 ml of hydrochloric acid (6M) and 1 ml of sodium propionic acid buffer (3.35 M, pH 5.5) was added. 50 μl of the lysate were then diluted with 450 μl of double-distilled water and incubated for 20 min at 70° C. with 25 μl of formic acid (0.4% (v/v)) and 500 μl of ninhydrin solution (2% (w/v) ninhydrin in 3.35 M sodium propionic acid buffer with 50% (v/v) ethylene glycol monomethyl ether (Sigma, St Louis, USA)). After cooling, 5 ml of ethanol (50% (v/v) in double-distilled water) were added. The absorption was measured at a wavelength of 570 nm using a spectrophotometer (UVICON 942, Kontron, Milan, Italy) and the corresponding protein concentration was calculated.
5) Shower Gels
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/51392 | 3/24/2005 | WO | 00 | 5/14/2007 |
