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180 SKIN PROTECTION


mAU 900 800 700 600 500 400 300 200 100 0


0


*mAU 900 800 700 600 500 400 300 200 100 0


0 5 10


Figure 3: HPLC chromatogram comparison of SWE versus methanol extraction. l Gradient:


Time


45 47 49 50 55


A


095 595 50 0 0


95 95


B 5 5


50


100 100 5 5


Skin barrier default study To induce a skin barrier default, or atopic dermatitis, a mix of cytokines (IL-4, IL-13, IL-17E, IL-31, and TNFalpha at 15 ng/ml per cytokine) was supplemented to the medium from Day 6 up to Day 10. 25 or 50 µg/ml green tea active or reference (1 µM Tofacitnib) was supplemented to the medium from Day 6 up to Day 10. Characterisation and evaluation of the efficacy of the green tea active compared to the reference were performed using histological and biological analysis. A morphology evaluation (hemalun eosin coloration) and an immunofluorescence study on caspase-14 and filaggrin were conducted. TEWL and pH were also evaluated.


15 20 5 Epigallocatechin Gallaten (EGCG) Caffeine Gallocatechin Gallate (GCG) Catechin (C) Epicatechin (EC) Epigallocatechin (EGC)


Epicatechin Gallate (ECG) Catechin Gallate (CG)


Green Tea extracted via Methanol


10


15


20 Green Tea extracted via Methanol


UV stress study Tissues were pre-treated with Green Tea biomass (50 µg/ml) or by reference (vitamin E at 50 µg/ml) into the medium on Day 8. On Day 9, UVA + UVB, were introduced using Biosun equipment at 2MED. On Day 10 the tissues were evaluated. Characterisation and evaluation of the efficacy of green tea compared to vitamin E was performed using histological and biological analysis. A morphology evaluation (hemalun eosin coloration) on sunburn cells was conducted, as well as an immunofluorescence study on 8- hydroxydeoxyguanosine (8-OHdG) and cyclobutane pyrimidine dimer (CPD).


Results HPLC study Compared to the methanol extraction method, the green tea extract obtained via SWE has a more broad phytochemical composition with the presence of polyphenols, catechins, caffeine, and EGCG (Fig 3).


Skin barrier default study Compared to the normal morphology (see Fig 4), the atopic dermatitis model displays spongiosis, fluid buildup in the skin, and the


epidermis is not as cohesive as the normal morphology, which allows moisture to be lost and external aggressors to penetrate. With treatment of 50 µg/ml Green Tea, the spongiosis has decreased and the epidermis looks more cohesive resembling the normal morphology. Also in the skin barrier study, the atopic


dermatitis model decreased the level of Caspase-14 to 18%, whereas treatment with 25 & 50 µg/ml increased the level of Caspase-14 to 44% and 52%, respectively. The Green Tea active restores Caspase- 14 levels by 34% after skin barrier disruption, working as effectively as the positive control (Tofacitinib). In addition, the atopic dermatitis model decreased the level of filaggrin to 74%, whereas treatment with 25 & 50 µg/ml increased the level of filaggrin to 81% and 115%, respectively. The Green Tea active restores filaggrin levels by 41% after skin barrier disruption, completely normalising filaggrin levels, and working as effectively as the positive control (Tofacitnib). Furthermore, 50 µg/ml of Green Tea was able to normalise TEWL and pH after skin barrier disruption.


UV stress study Under normal conditions, sunburn cells are


Figure 4: Morphology of normal RHE, RHE under cytokines, and RHE with cytokines & green tea. PERSONAL CARE EUROPE April 2020


10.201


11.015 10.203


11.466


14.470 15.218 16.713 16.627


14.391 15.214


18.768 19.512


18.761


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