ANTI-POLLUTION 85


allowed database-assisted identification of secondary metabolites. Antioxidant power, a measure of antioxidant capacity and reactivity, were analysed by electron spin resonance spectroscopy. The overall antioxidant power is expressed as antioxidant units, where one unit corresponds to the activity of a 1 ppm solution of pure ascorbic acid as a benchmark.23


Results


Flavonoid HPTLC fingerprint analysis of the aqueous-alcoholic pre-material of the active ingredient revealed substantial amounts of flavonoids, which correlate with the antioxidative power. HPTLC also demonstrated excellent heat and storage stability. Heating to 80°C for 2 h or storage at 40°C for 1 month had no effect on the overall flavonoid pattern. Dereplication analysis identified flavonoids such as quercetin-, kaempferol-, and protogenkwanin-derivatives as well as phenylpropanoids of caffeoylquinic acid, or cichoric acid, as main constituents (Fig 3). In addition, the presence of isothiocyanates such as gluconasturtiin was confirmed by extracted ion chromatograms (not shown).


Particulate matter and pollutants impair skin cells Objective To show that particulate matter (PM) and


a 100 ** 75 ** 50 ** Quercetin derivate (6) 2.0 Flavonoid 1.5 (3) 1.0 0.5


Kaempferol derivate (2) (1)


Quercetin


Quercetin derivate (4)


Kaempferol derivate (5)


(9) Flavonoid Quercetin derivate (8) (10) Protogenkwanin 4-glucoside/ Gluconasturtiin (7)


0 4 5 6 7 Time (min)


Figure 3: The active ingredient combines a wealth of powerful metabolites. Dereplication analysis of the aqueous-alcoholic pre-material identified: (1) Tryptophan; (2) Kaempferol3-glucoside7-sophoroside; (3) 5-O- caffeoylquinic acid; (4) Quercetin 3,5-digalactoside; (5) Kaempferol-3-O-sophoriside; (6) Quercetin-3-O- rutinoside; (7) Protogenkwanin 4-glucoside/ Gluconasturtiin (an isothiocyanate); (8) Quercetin-3-0-(6‘‘- malonylglucoside); (9) 1,5-Di-O-caffeoylquinic acid; (10) Mesocichoric acid.


pollutants stress skin cells and reduce their metabolic activity and vitality.


Technique - MTT Assay Cultured, metabolically active human keratinocytes (HaCaT cells) transform the yellow dye MTT into blue crystals. The intensity of the blue stain correlates with the metabolic activity and the vitality of the cells. In the assay, cells were stressed for 48h with naturally occurring diesel particulate


b 100 *** 75 *** 50 *** *** 25 25


Activation of Nrf2 - enhancing Phase II detoxification Objective To show that the active ingredient enhances the Nrf2 signal pathway.


0 Unstressed 125 250 500 PM burden (μg/ml) 1000 0 Unstressed 0.16 0.63 2.5 BaP burden (μg/ml)


Figure 4: Particulate matter and pollutants reduce the vitality of skin cells. Human keratinocytes were incubated for 48 h with particulate matter (A; PM) and BaP (B), respectively. Metabolic activity and vitality decline with increasing PM and pollutant stress. N = 4; Mean ± SD; Student’s unpaired t-test versus unstressed; ** = p <0.01; *** = p <0.001.


5


Technique Nrf2 Transcription Factor Assay is an ELISA- based method to quantify the DNA binding activity of Nrf2 in nuclear extracts of primary human keratinocytes (NHEK). Increased binding activity signals increased expression of genes that encode for cyto-protective enzymes.


matter (PM 1650b) (Fig 4A) or benzo[a]pyrene (Fig 4B) - one of the most abundant polycyclic aromatic hydrocarbons (PAH).


Results


Particulate matter (PM), at a concentration as low as 125 μg/ml, reduced the metabolic activity and vitality of skin cells (Fig 4A). Toxic substances, adsorbed to PM, are considered to cause their toxicity3


and in


fact, BaP disrupted the metabolic activity and vitality of skin cells at concentrations as low as of 0.16 μg/ml (Fig 4B). The associated, stress-induced morphological changes of keratinocytes are presented in the light microscopic images in Figure 5.


8 9 10


Figure 5: Pollutants damage cultured human keratinocytes. Light microscopic images following 48 h of exposure to stressors. Unstressed keratinocytes showed a characteristic structure and monolayer organisation. Positive control cells, devitalized with detergents, shrank and became nodular or detached from the bottom of the culture dishes. These stress signals were also observed with cells that were treated with 500 μg/ml particulate matter (PM; note the particles in the image) or with 5 μg/ml BaP. Scale bars = 50 μm.


April 2018 PERSONAL CARE EUROPE


Metabolic Activity (%)


Metabolic Activity (%)


Intensity (mAU) x10-5


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