ACTIVES
Petri dish cultures were taken using light microscopy and XTT cytotoxicity tests were performed on the 96-well plate cultures.
Product manufacture Pomegranate flower extract, sodium ascorbyl phosphate and maltodextrin were mixed and freeze-dried. The portion of pomegranate flower extract was selected based on its activity in the MMP-1 ELISA. In this way the product was standardised with respect to its effectiveness. The freeze-dried end product is called PGFE/SAP in the following.
Application formula For the in vivo tests on product safety and effectiveness, 1% and 5% respectively PGFE/SAP was introduced into a cream formulation (Table 1). The stability of the cream formulation was tested at room temperature and 40˚C for 6 months.
Tests concerning product safety In order to be able to guarantee the safety in use of PGFE/SAP and the cream formulation containing PGFE/SAP, several safety tests were carried out. PGFE/SAP was tested for cytotoxicity (MTT and XTT assays), phototoxicity (according to OECD guideline no. 432) and mutagenicity (Ames test according to OECD guideline no. 471). In addition, an eye irritation test (HET-CAM) was carried out and an allergy certificate in compliance with Directive 2003/15/EC Annex III issued. The cream formulation containing PGFE/SAP was investigated in two different skin irritation tests (SPT and RPT).
In vivo studies The effectiveness of PGFE/SAP was investigated in two different in vivo studies. Study 1 used a test panel of 20 female subjects with Caucasian skin type (phototype I-III). The female subjects, who were aged between 40 and 65 and
35 30 25 20 15 10 5 0
*p0.01-0.05; ** p<0.01; *** p<0.001 100 80 60 40 20 0 P17-NHDF P7-NHDF TGF-β PGFE/SAP SU PGFE/SAP L Figure 4: MMP-1 production in senescent dermal fibroblasts.
healthy, took part in the study on a voluntary basis without remuneration. A cream formulation with 1% PGFE/SAP content was applied twice daily for 56 days to the face and the inner side of the forearms. At three points in time (DO, D28, D56) skin thickness was determined by ultrasound and high-resolution facial photographs taken. In Study 2, a cream formulation with
1% PGFE/SAP content was tested on ten subjects (panel requirements according to Study 1). The cream formulation was applied to the face and tested for 56 days. Similarly to Study 1, facial photos were taken at measurement times D0, D28 and D56. In addition a skin profile was completed from the lateral corner of the eye (crow’s feet area).
Ultrasonography In Study 1, a Dermascan C ultrasound machine was used at a frequency of 20 MHz in order to determine the thickness of the upper layers of the skin. The measurements were taken on the inner side of the forearm. For evaluation the number of dark pixels was determined
*** 20 *p0.01-0.05; **
in the ultrasound images. A reduction in dark pixels was interpreted as an increase in skin thickness.
Profilometry (PRIMOS) The profilometric measurements in the framework of in vivo Study 2 were carried out using the PRIMOS Pico (GF Messtechnik) method in the area around the lateral corner of the eye (crow’s feet area). On measurement days D0, D28 and D56, three measurements were taken on each day. To calculate the skin profile and determine the depth of wrinkles, PRIMOS software V5.7, 2010 was used.
Visual evaluation (VisioFace Quick) In order to guarantee standardised photo documentation, a VisioFace Quick machine was used. The visual evaluation of treatment results was carried out by trained professional staff taking high-resolution images the face (10 MPixel).
Results Product safety Cytotoxicity To evaluate cytotoxicity two series of tests
p<0.01; *** p<0.001 ** 15 ** 10 ** PGFE *** **
*p0.01-0.05; ** *
p<0.01; ***
p<0.001 *
5
0 D0 D28 Figure 5: Increase in skin density on the volar forearm. D56 D0 D28
Figure 6: Reduction in wrinkle depth in the lateral corner of the eye (crow’s feet area).
March 2012 PERSONAL CARE 47 D56
Skin density increase (%)
MMP-1 production
Reduction of wrinkle depth (%)
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