1. © Mibelle Biochemistry, Switzerland 2007 Detoxophane Activating the skin‘s detoxification system

2. © Mibelle Biochemistry, Switzerland 2007 Mom was right: „Eat your vegetables, they are good for you!“ Phytonutrients are plant chemicals reported to exert a specific effect on health or on amelioration of diseases Health benefits of vegetables: Lower risk for certain cancers, stroke, and high blood pressure

3. © Mibelle Biochemistry, Switzerland 2007 The Vegetables of the Mustard Family Broccoli sprouts are now extreme popular as food supplements in the USA Vegetables of the mustard (Brassicaceae) family have been shown to be extremely healthy They contain a high concentration of sulforaphane Members of the Brassicaceae vegetables are broccoli, cauliflower, radishe, water and garden cress

4. © Mibelle Biochemistry, Switzerland 2007 Garden Cress Sprouts: The Source for a New Cosmetic Ingredient Garden cress is a suitable form for sulforaphane for cosmetics Fresh sprouts are homogenized and extracted with water The extract is purified, concentrated and encapsulated into liposomes

5. © Mibelle Biochemistry, Switzerland 2007 Composition Lepidium Sativum Sprout Extract Glycerin Lecithin Phenoxyethanol Aqua/Water

6. © Mibelle Biochemistry, Switzerland 2007 Isothiocyanates: Molecular Structures In the plant, isothiocyanates are present as glucose derivatives, called glucosinolates. When the plant cells are broken, the enzyme myrosinase is liberated. This thioglucosidase transforms glucosinolates into free glucose and isothiocyanates. Sulforaphane

7. © Mibelle Biochemistry, Switzerland 2007 Sulforaphane: Mechanism of Action Sulforaphane activates the body‘s detoxification system composed of phase I and phase II enzymes: Phase I reactions increase the reactivity of hydrophobic molecules by addition of functional polar groups (->reactive oxygen species). If these reactive molecules are not further metabolized by phase II conjugation, they may cause damage to DNA and induce mutations. Phase II reactions are either conjugation reactions of phase I products with small hydrophilic substances to form water-soluble products that can be excreted or they are antioxidant enzyme reactions that inhibit strong oxidant formation.

8. © Mibelle Biochemistry, Switzerland 2007

9. Detoxophane is More than an Antioxidant UV Toxin AGE DNA damage Lipidperoxides ROS Antioxidant Detoxophane

10. © Mibelle Biochemistry, Switzerland 2007 Activation of Gen Expression with the Antioxidant Response Element (ARE) The antioxidant response element ARE is a regulatory promoter sequence of genes coding for antioxidant systems and phase II detoxification enzymes The transcription factor Nrf2 is the critical regulator of ARE- dependent gen expression Under normal conditions Nrf2 is blocked by the repressor Keap1 Sulforaphane was found to disrupt the Nrf2/ Keap1 complex

11. © Mibelle Biochemistry, Switzerland 2007

12. Detoxophane: The Next Generation Antioxidant Stimulates the expression of antioxidants and detoxification enzymes in the skin to protect against Environmental pollutants (cigarette smoke, exhaust fumes, smog, endotoxins, heavy metals) Reactive oxygen species (extrinsic and intrinsic) DNA damage and thus premature skin aging

13. © Mibelle Biochemistry, Switzerland 2007 Modulation of Enzyme Expression Method Human keratinocytes were grown to 80% confluence in a standard growth medium. Then cells were incubated during 24 hours with different concentrations of Detoxophane. Cells were harvested and total RNA extracted. Real time quantitative PCR was used to analyze gene expression Phase I- Cytochrome P450 isoenzyme 2E1 (CYP2E1) Phase II- NADPH: quinone reductase 1 (NQO1) - Heme oxygenase 1 (HO-1) - Thioredoxin reductase 1 (TrxR1)

14. © Mibelle Biochemistry, Switzerland 2007 Modulation of Enzyme Expression Enzyme expression rel. to control (%) Concentration of Detoxophane0.05%0.2% Phase I enzyme Cytochrome P450 isoenzyme 2E1-40-38 Phase II enzyme NADPH:quinone reductase 175214 Heme oxygenase 12124182 Thioredoxin reductase 11842316 S-131

15. © Mibelle Biochemistry, Switzerland 2007 Protection Against Oxidative Stressors Method Growth of human keratinocytes to 80% confluence in a standard growth medium Pre-treatment for 24 hours with 0.05% Detoxophane Incubation for 4 hours with increasing concentrations of toxins: - 4-hydroxynonenal  HNE - tert-butyl hydroperoxide  t-BH Analysis of cell viability with MTT assay

16. © Mibelle Biochemistry, Switzerland 2007 Protection Against Tert-Butyl Hydroperoxide S-148

17. © Mibelle Biochemistry, Switzerland 2007 Detoxophane exerted an impressive protection against t-BH. At 0.05% Detoxophane, the IC 50 value increased from 258 microM of the control to 646 microM. Detoxophane exerted a moderate protection against HNE. At 0.05% Detoxophane, the IC 50 value increased from 76 microM of the control to 90 microM. Protection Against Tert-Butyl Hydroperoxide

18. © Mibelle Biochemistry, Switzerland 2007 Summary Vegetables of the mustard family, such as broccoli and garden cress sprouts, are known for their remarkable health benefits The phytonutrient sulforaphane has been identified as the active compound Garden cress sprouts have been used to develop Detoxophane as cosmetic sulforaphane preparation

19. © Mibelle Biochemistry, Switzerland 2007 Claims Detoxophane… Activates the detoxification system of skin cells Protects skin against pollutants Prevents visible signs of skin aging Protects against city pollution Prevents DNA damage Reveals a healthy looking skin

20. © Mibelle Biochemistry, Switzerland 2007 Application Anti-Ageing Formulations Detox City Creams Sun Protection Formulations Hand Creams Protective Foundations

21. © Mibelle Biochemistry, Switzerland 2007 Film