ACTIVES 2.5 2.0 1.5 1.0 0.5 0 DMSO PGFE Figure 1: MMP-1 gene expression in primary dermal fibroblasts. In order to obtain a product with
precisely these effects, there was a need for the development of a combination of several components into one carrier system. To promote collagen synthesis the stable ascorbic acid derivative sodium ascorbyl phosphate (SAP), already known from literature and used in cosmetics, was chosen.9,10
In order to find a new
component that reduces MMP-1 synthesis, a screening was carried out on several plant extracts based on a literature research. In this connection, the effects of the chosen extracts on MMP-1 expression in UVA irradiated primary dermal fibroblasts was investigated. At the end of this series of tests, a pomegranate flower extract (PGFE) showed the desired MMP-1 expression-reducing properties. The aim of this study was to demonstrate
that the two active components, sodium ascorbyl phosphate and pomegranate flower extract, could be combined into an effective and safe active ingredient for anti-ageing purposes. Collagen synthesis should be increased by SAP and the breakdown of collagen reduced by the pomegranate flower extract. In this way the balance between collagen synthesis and collagen breakdown should be shifted in favour of collagen synthesis. Wrinkles already present should be reduced and the development of new wrinkles prevented.
Not irradiated ** ** *p0.01-0.05; ** p<0.01; *** p<0.001
Not irradiated Irradiated
120 100 80 60 40 20 0
*** *p0.01-0.05; ** p<0.01; *** p<0.001 *** ***
Blank
Blank+UV Figure 2: UVA cytotoxicity assay.
Material and methods MMP-1 gene expression analysis (real-time RT-PCR)
Selected plant extracts were examined in a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) screening for MMP-1 gene expression-inhibiting properties. Primary dermal fibroblasts were cultivated in 6-well culture plates and treated for 24 hours with 0.1% DMSO (negative control) or extract. In addition, one group was irradiated with 75 mJ/cm2 UVA light. Twenty-four hours after irradiation of the cells the mRNA was extracted and the relative MMP-1 gene expression determined using real time RT-PCR.
MMP-1 synthesis (ELISA) In order to also confirm the results obtained in the RT-PCR tests at the protein level, collagen production was investigated in normal human dermal fibroblasts (NHDF, Bioalternatives, France). NHDF were cultivated up to senescence (passage 17 (P17)) and then treated with the test substance in 96-well plates for 72 hours. Then the quantity of secreted MMP-1 in supernatant was determined using an enzyme-linked immunosorbent assay (ELISA). A senescent NHDF culture (P17) treated with 10 ng/mL transforming growth factor beta (TGF-β) and an untreated non- senescent NHDF culture (passage 7 (P7))
Irradiated
were used as positive controls. The MMP-1 production of an untreated P17 culture was used as a reference.
UVA cytotoxicity assay Normal human dermal fibroblasts (NHDF, PromoCell, Germany) were grown to confluence in 96-well plates and Petri dishes with Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS) content. Subsequently the DMEM with 10% FCS content was replaced by DMEM with 1.25% FCS content. In addition, 0.1% pomegranate flower extract was added to the medium of selected cultures and then incubated for 24 hours. Half of the cultures were irradiated with UVA. To do this, a UV crosslinker (Vilber Lourmat, Eberhardzell Germany) equipped with UVA lamps (T-8.L UV tube 8W – 365 nm) was used. For irradiation, the culture medium was suctioned off and the cells were covered with PBS. After irradiation with 12.5 mJ/cm2
UVA light, the PBS was
aspirated off and fresh culture medium was added accordingly with or without the addition of 0.1% pomegranate flower extract. The non-irradiated cells were also treated with PBS and covered with fresh culture medium. Cell irradiation was carried out three times. Between each irradiation there was a 24 hour incubation time in each case. For evaluation, images of the
Irradiated +0.1% PGFE
PGFE
PGFE+UV
Figure 3: Light microscopic images of NHDF in the UVA cytotoxicity assay. 46 PERSONAL CARE March 2012
Rel. MMP-1 mRNA expression
Viability (%)
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