ACTIVES High density D0 D28 D56
Low density Figure 7: Ultrasonographic images of the volar forearm.
were carried out with different cell types. Up to 2.0% concentration levels PGFE/SAP showed no cytotoxic effect in an MTT assay on normal human dermal fibroblasts and in an XTT assay on NCTC2544 keratinocytes.
Phototoxicity In a study on phototoxicity conforming to OECD Guideline no. 432 on mouse embryo fibroblasts (3T3), PGFE/SAP was classified as ‘not phototoxic’.
Mutagenicity (AMES) The AMES reverse Mutations Assay on S. typhimurium and E. coli conform to OECD Guideline 471, could not demonstrate any mutagenicity caused by PGFE/SAP.
Eye irritation (HET CAM) In order to detect any possible eye- irritating potential of PGFE/SAP, tests were conducted on the chorioallantonic membrane of hens’ eggs (HET-CAM). PGFE/SAP was ‘practically non-irritating to the eyes’ at a concentration level of 1% and at 5% ‘slightly irritating to the eyes’.
Skin irritation (SPT/RPT) PGFE/SAP was worked into to a cream formulation at levels of 1% and 5% and classified in a single epicutaneous patch test (SPT) as ‘non-irritating to the skin’. In a repeated epicutaneous patch test (RPT) the cream formulation containing 1% PGFE/SAP caused no sensitisation of the skin. Likewise, application of a cream formulation containing 5% PGFE/SAP twice a day for 56 days caused no skin irritation or sensitisation.
Allergy research Based on an evaluation of allergy-releasing potential according to Directive 2003/15/EC Annex III, PGFE/SAP was classified as non-allergenic.
48 PERSONAL CARE March 2012
In vitro MMP-1 gene expression analysis (RT-PCR)
The influence of pomegranate flower extract on MMP-1 gene expression was investigated using real time RT-PCR, on irradiated and non-irradiated primary dermal fibroblasts. The results are shown in Figure 1. Primary dermal fibroblasts were treated
with 1 µL/mL DMSO and 7.11 µL/mL pomegranate flower extract (PGFE). Twenty- four hours after treatment one group was irradiated with 75 mJ/cm2
UVA light. The
mRNA was extracted 24 hours after irradiation. All results are shown as a multiple of the non-irradiated DMSO control group. The UVA irradiated cells of the control group treated with DMSO showed a 1.71-fold increase in the expression of MMP-1 mRNA. The MMP-1 mRNA expression in cells treated with PGFE was obviously reduced with and without UVA irradiation. Non-irradiated cells showed 0.42% and UV-irradiated cells showed 0.55% of normal MMP-1 mRNA expression. It should be pointed out that the PGFE treatment reduced not only the MMP-1 gene expression in irradiated cells (45% reduction) but also the MMP-1 gene expression in non-irradiated cells (58% reduction).
UV cytotoxicity The UV cytotoxicity test investigated the influence of pomegranate flower extract on the cell vitality of normal human dermal fibroblasts (NHDF) with and without UVA irradiation. The results are shown in Figure 2. For each series of tests the NHDF of
one group was treated with 0.1% PGFE. At the same time a control group was left untreated. Half of the cultures of each group were irradiated with UVA light (12.5 mJ/cm2
) three times. There was a 24-hour period between each irradiation.
Then an XTT cytotoxicity test was performed to determine cell vitality 24 hours after the last irradiation. Figure 2 shows the cell viability, calculated from cytotoxicity test results with the untreated non-irradiated control group set as 100%. After UVA irradiation, the untreated cells (Blank+UV) showed a vitality of 59% which corresponded to a vitality deficit of 41%. The cells treated with 0.1% pomegranate flower extract (PGFE+UV) showed clearly higher vitality (88%). The vitality deficit was therefore only 12%. The non-irradiated cells treated with 0.1% pomegranate flower extract (PGFE) showed an increased vitality value of 111%. Figure 3 shows light microscopy images of (a) non-irradiated, (b) irradiated and (c) irradiated cells treated with PGFE 0.1%, 24 hours after the last irradiation with UVA. The non-irradiated NHDF (a) showed a normal appearance. The irradiated cells without extract additive (b) showed severe UV damage (changed cell morphology with many dead cells in the medium). The irradiated culture treated with PGFE (c) demonstrated obviously less UV damage. The majority of cells still showed adhesion and were of a normal appearance. In comparison to culture (b) there were clearly fewer cells to be seen in the medium.
MMP-1 synthesis (ELISA) The ELISA was performed in order to demonstrate the MMP-1 inhibiting effect of both the PGFE component and the PGFE/SAP at the protein level and to prove that the PGFE extract as well as two variants of PGFE/SAP produced in different scale productions caused the same MMP-1 inhibiting effect in aged dermal fibroblasts. Figure 4 shows MMP-1 production
quantified by ELISA in senescent dermal fibroblasts. Aged fibroblasts (P17-NHDF) were treated for 72 hours with 10 ng/mL TGF-β, 0.3% PGFE/SAP SU (from Scale-Up methodology 150 kg), 0.3% PGFE/SAP L
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