Mangifera indica activates key metabolic master switch enzymes The innovation for well-aging

Mangifera indica activates key metabolic master switch enzymes The innovation for well-aging Dr. S. Buchwald-Werner 6. May 2015 Vitafoods Europe Conference Healthy Aging Session

Consumer demand for healthy-aging products World s population is aging rapidly and life expectancy is increasing Aging consumers are the physically and mentally fittest in history Today all age-groups take personal responsibility for their health and are interested in products to reduce the signs of aging and the risk of metabolic diseases There is a 7 years gab between Life expectancy & healthy life expectancy In 2050 the proportion of people over 60 years is expected to increase from 605 million to 2 billion until 2050 There is a market for well-aging products Jane Fonda, 75 years, 2014 http://www.welt.de/lifestyle/article113599606/jane-fonda-fast-zu-perfekt-um-75-zu-sein.html Foto: Invision WHO 2012: Interesting facts about aging. http://www.who.int/ageing/about/facts/en/#; 18.08.2014O Euromonitor International Life expectancy 2013 2

Agenda 1. What happens during aging? 2. Emerging biological targets to develop ingredients for well-aging 3. Mangifera indica the natural well-aging ingredient for application in food supplements and foods; Ingredient introduction and scientific results for Key metabolic master switch enzyme: SIRT 1 Key metabolic master switch enzyme: AMPK Mitochondria and aging 3

1. What happens during aging? Biological aging is commonly defined as the successive accumulation of adverse effects in an organism with increasing age Our cells lose power because less power plants (Mitochondria) are produced and the remaining are less efficient Our cells lose direction because mistakes in DNA replication by oxidative stress and inflammation are adding up to first disturbances in our metabolism Enzymes and metabolic master switches decline, leading to an increase of blood lipids and body fat mass in combination with reduced muscle mass as well as elevated blood glucose and insulin resistance Jane Fonda, 75 years, 2014 http://www.welt.de/lifestyle/article113599606/jane-fonda-fast-zu-perfekt-um-75-zu-sein.html Foto: Invision 4

2. Emerging biological targets for well-aging Master switches, e.g. SIRT1, AMPK: Evolutionary conserved metabolic sensors that steer energy homeostasis by instructing the body to burn or not to burn energy and consequently to maintain essential body functions Mitochondria: working organelles that keep the cell full of energy The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy demand matches energy supply These master switches have homologs in all eukaryote; evolutionarily conserved - all the way from yeast to humans Effective mitochondrial biogenesis and turnover is critical for the maintenance of energy production, the prevention of oxidative stress and well-aging 6

3. Mangifera indica for well-aging 100% Mango fruit powder (Mangifera indica L.) Mangos are harvested at a special degree of ripeness from selected farmers to obtain high concentrations of secondary plant compounds which are important for the health benefits Several scientific studies have been carried out using the proprietary ingredient to investigate the effects of well-aging Nutritional benefits (fiber, vitamins and minerals) A high quality, sustainable natural ingredient Approved for use in dietary supplements & foods Careless is the first scientifically proven and IP protected ingredient that helps you care less about aging while delivering the great taste and pleasure of exotic mango fruits 8

3. Key metabolic master switch enzyme: SIRT 1 Sirtuin structure (MIT) 9

3. Key metabolic master switch enzyme: SIRT 1 Reduction of body fat Increased muscle mass NAD + Nicotinamid + ADP-ribose Balanced blood sugar & insulin SIRT1 Transcription/ Co-factors: e.g. PGC-1a FOXO LXR PPARg p53 Deacetylated targets Endothelial function DNA repair Cell protection Beta-cell protection Cholesterol homeostasis Boutant et al. Molecular Metabolism 2013 Li X Aca Biochim Biophys Sin 2013 Sirtuin structure (MIT) Hubbard ad Sinclair, Trends in Pharmacological Sciences (2014) 35: 10

3. Mangifera indica for well-aging Activation of SIRT 1 using human recombinant enzyme Enzyme deacetylation activity was compared, before and after treatment with a water extract based sample preparation of 4 different batches of Mangifera indica fruit powder and resveratrol as positive control. Optimal sample concentration of 500µg/ml was determined in a pre-test Two different test systems were used: 1. Fluorescence assay SIRT1 active recombinant human protein expressed in E.coli 2. Luminescence assay SIRT1 active recombinant human protein expressed in Sf9 cells 11

3. Mangifera indica for well-aging Activation of SIRT 1 using human recombinant enzyme - Results Fluorescence assay All Mangifera fruit powder batches activated SIRT1 by > 30% Resveratrol ~50% Luminescence assay All Mangifera fruit powder batches activated SIRT1 by > 40% Resveratrol ~95% Results show that Mangifera indica fruit powder is able to activate SIRT1 12

3. Key metabolic master switch enzyme: AMPK Steinberg GR, Kemp BE, AMPK in Health and Disease, Physiological Reviews (2009)89 : 3, 1025-1078 13

3. Key metabolic master switch enzyme: AMPK Improved hepatic fatty acid oxidation Cholesterol Triglyceride homeostasis AMPK AMP: ATP Kinases / Ca 2+ AMPK P Improved muscle fatty acid oxidation Transcription/ Co-factors, e.g. ACC COX PGC-1a GLUT1,4 mtor HMG-CoA Balanced blood sugar & insulin Improved muscle glucose uptake Steinberg GR, Kemp BE, AMPK in Health and Disease, Physiological Reviews (2009)89 : 3, 1025-1078 14

3. Mangifera indica for well-aging Activation of AMPK in human hepatic progenitor cell line (HepaRG) Enzyme activity was compared (phosphorylation) before and after treatment with a water extract based sample preparation of Mangifera indica fruit powder and resveratrol and metformin as positive control Cytotoxicity test was carried out Dose-dependency was tested (30µg/ml, 100µg/ml, 300µg/ml) Activity of 3 different batches was tested 15

3. Mangifera indica for well-aging Activation of AMPK in human hepatic progenitor cell line (HepaRG) Results: Mangifera fruit powder activated AMPK in a dose dependent manner, up to 100 % Resveratrol and Metformin ~100% All Mangifera fruit powder batches activated AMPK by > 50% Resveratrol ~82% Metformin ~130% Results show that Mangifera indica fruit powder is able to activate AMPK 16

3. Mitochondria and aging 17

3. Mitochondria and aging Mitochondria - processes Heat Production Thermogenesis Energy ATP Production Calcium storage ROS homeostasis cellular Signaling Hormones Apoptosis mt DNA 18

3. Mangifera indica for well-aging Mitochondrial biogenesis in liver and muscle cell lines Capacity to activate mitochondrial biogenesis before and after treatment with a water extract based sample preparation of Mangifera indica fruit powder Cytotoxicity test was carried out In murine muscle cell line (C2C12) dose-dependency was tested (200µg/ml, 400µg/ml); resveratrol as positive control In human hepatic cell line (HepG2) dose-dependency was tested (100µg/ml, 200µg/ml, 400µg/ml) 19

3. Mangifera indica for well-aging Mitochondrial biogenesis in liver and muscle cell lines - Results In murine muscle cell line (C2C12) Mangifera fruit powder enabled mitochondrial biogenesis > 7 % Resveratrol up to 17% In human hepatic cell line (HepG2) Mangifera fruit powder enabled mitochondrial biogenesis dose dependent > 15 % Results show that Mangifera indica fruit powder enabled mitochondrial biogenesis 20

3. Mangifera indica for well-aging Summary Reduction of body fat Increased muscle mass Well-aging Balanced blood sugar Oxidative stress resistance 21

3. Mangifera indica for well-aging R&D Outlook 1. Phytochemical investigation 2. In vitro study to investigate enos activation 3. In vivo animal study to investigate weight management - ongoing Body weight and Body fat Gene expression to confirm mode of action (SIRT, AMPK) 4. Human study Well-aging - ongoing Skin microcirculation acute study Vascular function acute study 5. Human study Weight management (to be defined based on animal study results) 6. Human study well-aging (to be defined based on 1 st human study results) 22

Vital Solutions - Company introduction Vital Solutions develops innovative condition specific natural ingredients for use in foods and dietary supplements, which serve consumer demands for health products and help to reduce health care costs Based on a market-oriented dynamic strong R&D platform, we focus on the development of natural ingredients which improve quality of human life by feeling good and aging well. Our research is focusing on: Anti-aging Brain health Digestive health & metabolism Over the past 5 years we have created an innovative product portfolio and a pipeline, capable of delivering sustainable sales growth. Market introduction of 4 innovative health care ingredients Set up of an innovative product pipeline of 4 ingredients Global distribution network Latest innovation: Ingredient for healthy-aging 23

Thank you for your attention! Meet us at Vitafoods Booth G51 Vital Solutions GmbH Dr. Sybille Buchwald-Werner Hausinger Straße 6 D-40764 Langenfeld Phone: +49 (0) 2173 10 98 202 E-Mail: sybille.buchwald-werner@vitalsolutions.biz All information given in this presentation reflects our current knowledge and interpretation of relevant legislations and legal practice. Vital Solutions expressly disclaims any responsibility for the suitability of this information for products and for any specific or particular purposes intended by the user. Our suggestions do not release Vital Solutions customers from evaluating the individual products as to their legal compliance. Vital Solutions does not assume any liability or risk involved in the use or marketing of its products as the conditions of use are beyond its control. The user of the products is solely responsible for compliance with all laws and regulations applying to the use of the products. 24