SKIN PROTECTION 111
Table 1: Protocol conditions. Cell model
Keratinocytes Fibroblasts LED-BL exposure (energy dose and time) Concentration of olive fruit extract 45 J/cm2 5 J/cm2 for 1.5 hours for 15 minutes
Figure 2: Queen Cleopatra used olive oil in her beauty routine. Today olive fruit helps skin and hair to counteract the signs of blue light exposure.
antioxidant defences, they have the ability to reduce apoptotic markers (cytoprotecting activity).9-12 Olea-HT 10 has been already tested for
UV protection on skin and hair and has effectively demonstrated to be a suitable ingredient to prevent the signs of photoageing.
The activity of Olea-HT 10 against blue
light effects was assessed in an in vitro model. The objective of the study was to investigate the protective effect of Olea-HT 10 on human keratinocytes (NCTC 2544) and fibroblasts treated with light-emitting, diode–generated blue light (LED-BL). We evaluated cell viability/proliferation, generation of reactive oxygen species, DNA damage through the quali- quantitative analysis of 8-OHdG (8-dihydro- 2-deoxyguanosine) and of a nuclear protein, PCNA (Proliferating Cell Nuclear Antigen). 8-OHdG is a pivotal biomarker of DNA damage. It is a modified DNA base product generated by ROS and photodynamic action (e.g. light irradiation). DNA damage and repair are also linked to the activation of PCNA. We, therefore, performed immunocytochemical staining and Western Blot analysis, using specific antibodies for 8-OHdG and PCNA. As regards the extracellular matrix (ECM)
LEB-BL-45 J/cm2 a
Control
Blue light control
10 25 50
0 0.2 0.4 0.6 * * * * 0.8 1 1.2 1.4 Cell viability (OD value at λ=550 nm) 1.6 (1.5 hours)
components, we have evaluated by Western Blot important factors of ECM turn-over such as collagen type I and MMP- 1 (matrix metalloproteinase-1) enzyme, responsible for collagen breakdown in skin. Additionally, we detected the MMP-12, a skin elastase implicated in the metabolism of elastic fibres and associated with the decrease in skin elasticity and, consequently, the formation of wrinkles in various types of tissues during inflammation, as well as during ageing. We demonstrated that Olea-HT 10 (now
referred to as ‘olive fruit extract’) is a promising photo-damage protective agent and, as a consequence, a key cosmetic ingredient to counteract the signs of blue light-induced premature ageing.
Experimental protocol We planned to irradiate cell cultures with a light-emitting diode-generated blue light (LED-BL) source at the distance of 10 cm (from the light tip to the cell monolayer), at different energy doses and LED-BL was set at a wavelength of 450 nm. The chosen energy dose of LED-BL was
different between the two cell models: 45 J/cm2 J/cm2
for 1.5 hours for keratinocytes and 5 for 15 min for fibroblasts. In fact,
preliminary results (data not shown) demonstrated that these cells seem to be more sensitive to blue light treatment. All the tests were performed on cells not exposed to LED-BL (control), on cells exposed to LED-BL and not treated with olive fruit extract (blue light control), on keratinocytes and fibroblasts exposed to LED-BL and treated with olive fruit extract. The tested concentrations of olive fruit
LED-BL-5 J/cm2 b
Control
Blue light control
10 25 50
100 0 0.2 0.4 0.6 0.8 1 * * * * * 1.2 1.4 Cell viability (OD value at λ=550 nm)
Figure 3: Cell viability was measured by MTT assay. *p< 0.05 vs. non-exposed control. Data are presented as mean ± standard error of the mean (SEM) of optical densities (OD) of two experiments performed in quadruplicate. – a) keratinocytes; b) fibroblasts.
April 2018 PERSONAL CARE EUROPE 1.6 (15 min) 10, 25 and 50 µg/ml 10, 25, 50 and 100 µg/ml
extract were 10, 25 and 50 μg/ml for keratinocytes and 10, 25, 50 and 100 μg/ml for fibroblasts.
Effect of olive fruit extract on cell viability Design
Cell viability was evaluated through the colorimetric assay MTT, a reliable technique for determining the number of viable cells in a given culture.
Keratinocytes and fibroblasts were pre-
treated with olive fruit extract at the different concentrations and exposed to LED-BL. After 24 hours cell viability assay was performed.
Results
Keratinocytes and fibroblasts, treated with LED-BL, showed a strong reduction of viability, 71.5% and 35.7% respectively, versus untreated control (Fig 3a, b). The pre-treatment of keratinocytes with 10, 25 and 50 μg/ml of olive fruit extract increases cell viability of about 59%, 78% and 150% in comparison with the blue light control, whereas in fibroblasts, the pre- treatment with olive fruit extract at the concentrations of 10, 25, 50 and 100 μg/ml, led to an increase that ranged from 9.63% to 23.70%. These results highlighted that olive fruit
extract protects LED-BL-damaged keratinocytes and fibroblasts.
Antioxidant activity of olive fruit extract - reactive oxygen species (ROS) assay Design
The level of intracellular ROS was assessed
Olea-HT 10
(µg/mL)
Olea-HT 10
(µg/mL)
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