2. www.diabetes.org 23,600,000 Diabetics 7 th Leading Cause of Death Estimated $218 Billion 5.2X higher medical expenditures 71,000 amputations 1.6 million new cases per year

3.  Type II Diabetes is most common form › Fasting blood glucose >126 mg/dL (NIH) › Excessive glucose levels in the blood causes health problems  Two different causes › The body does not produce enough insulin › The body’s cells become resistant to insulin  Increasing risk › Obesity and lack of physical activity › Genetics › High fat diet and alcohol intake › High blood pressure 6.2 MILLION PEOPLE ARE UNDIAGNOSED 54 MILLION PEOPLE ARE PRE-DIABETIC 54 MILLION PEOPLE ARE PRE-DIABETIC

4. Drug Therapies for Diabetes Mellitus  Biguanides such as Metformin (Glucophage) decrease the production of glucose in the liver.  Sulfonylureas (Amaryl, DiaBeta, Glucotrol, Micronase) and Meglitinides (Prandin, Starlix) stimulate the pancreas to release more insulin.  Thiazolidinediones (Actos, Avandia) increase the sensitivity of fat and muscle cells to insulin.  Inhibitors of digestive enzymes (α-amylase and α- glucosidase) slow the absorption of carbohydrates. 4

5.  α-Amylase › Digests starches and oligosaccharides › Secreted by the pancreas, component of saliva › Located in mouth and small intestine  α- Glucosidase › Digests starches and oligosaccharides › Located in the epithelium of the small intestine

6. OH O CH 2 OH Lumen of the Small Intestine Epithelial Brush Border of the Small Intestine Enzyme OH O CH 2 OH OH O CH 2 OH OH O CH 2 OH

7.  Obtained from the fermentation processes of a microorganism, Actinoplanes utahensis  Reduces postprandial hyperglycemia › Oligosaccharide that competitively and reversibly inhibits the activity of α-glucosidase and α-amylase  Issues: › Cost › Side effects, including gastrointestinal disturbances (stomach distress, diarrhea, bloating, etc.) and nausea

8. Lumen of the Small Intestine Epithelial Brush Border of the Small Intestine Enzyme Inhibitor: Acarbose

9.  A class of plant secondary metabolites  Primary function is to provide protection to plants against biological attacks  Are known for their biological activities Flavone backbone structure

10. Amylase Inhibition Assay α- Amylase Inhibition Assay Common structural features of flavonoids to inhibit α-amylase Catechin Epicatechin EGC 10 C R6 R7 R4΄ R5΄  Flavonoids inhibit α-amylase only if they contain (Lo Piparo et al., 2008):  4-oxo-flavonoid nucleus as C-ring  C2-C3 double bound conjugated to the 4-keto group  Specific pattern of OH groups:  R6 or R7 of the A-ring  R4’ or R5’ of the B- ring

11.  Belong to a subgroup of bioflavonoids called “Flavan-3-ols”  Chosen because of structural components  Monomers can form dimers or trimers that are structurally similar to acarbose  Found in grape seed and tea extracts, for example Catechin

12. Inhibition of α-amylase and α-glucosidase by specific flavonoids during the digestion of starches helps control post-prandial hyperglycemia and ultimately Type II Diabetes Mellitus.

13.  Incubate enzyme with test compound  Add derivatized starch (provided in an assay kit)  Read fluorescence in 96- well plate reader at Ex/Em 485/530 nm every 1 minute for 15 minutes  Determine inhibition through IC 50 calculation http://www.cryst.bbk.ac.uk/pps97/assignments/projects/gaz erro/amylase.gif

14. IC 50 The IC 50 value is defined as the concentration of inhibitor required to inhibit 50% of the α-amylase activity… …which is a measure of the inhibitor’s relative effectiveness

15. μMμM μMμM μMμM μMμM μMμM Concentration of Inhibitor Slopes converted to percentage activity

16. IC 50 = 9.44 ± 0.70 μg/ml IC 50 = 7.37 ± 0.26 μg/ml

17. IC 50 = 9.44 ± 0.70 μg/ml IC 50 = 7.37 ± 0.26 μg/ml Catechin Epicatechin

18. IC 50 = 7.37 ± 0.26 μg/ml α-amylase activity (%) IC 50 = 44.62 ± 0.52 μg/ml Epigallocatechin Gallate

19. IC 50 = 7.37 ± 0.26 μg/ml α-amylase activity (%) IC 50 = 44.62 ± 0.52 μg/ml IC 50 = 356.73 ± 31.60 μg/ml α-amylase activity (%)

20. 356.73

21.  Incubate enzyme with test compound  Add substrate p- nitrophenyl ß-O-D- glucopyranoside (pNPG) and incubate  Read absorbance in 96-well plate reader at 405 nm  Determine IC 50 http://www.rcsb.org/pdb/explore/explore.do?structureId=2ZE0

22. IC 50 = 102.67 ± 3.77 μg/ml

25.  Preliminary experiments for a human trial  Variables: › Amount of carbohydrate (starch) challenge › Nature and dose of test compound(s) › Time point of administration of test compound(s) vis-à-vis carbohydrates/starch › Blood collection time points

26. Peak Area (AUC)= 21.9 With Inhibitor Peak Area (AUC)= 21.7 Carbohydrate Only

27. α-Glucosidase and α-Amylase:  In Vitro Studies › Confirm in vitro results (n=3, statistical evaluation) › Vary inhibitor concentration to determine K i for select bioflavonoids/extracts  Human Studies › Optimize conditions  Carbohydrate dose  Supplement (mixture) to test  Dose of supplement  Administration time › Conduct trial, measure post-prandial glycemia and insulinemia Pancreatic Lipase (fat absorption): › Initial studies (in vitro) and optimization in Fall/Winter › Preliminary in vivo studies (time permitting)

28.  Dr. Meltem Yilmazer-Musa  Dr. Balz Frei  Mary Garrard  Dr. Dan Sudakin  Dr. Alex Michels  Linus Pauling Institute  Howard Hughes Medical Institute  Undergraduate Research and Innovation, Scholarship and Creativity  USANA › http://en.wikipedia.org › http://www.webmd.com/ › www.diabetes.org › Peters, A. and Votey, R. “Diabetes Mellitus, Type 2- A Review”. WebMD. http://emedicine.medscape.com/article/766143. Accessed 8/19/2010 › www.nih.gov

30. Morin Baicalein Scutellarein Myricetin

31. American Heart Association Waist circumference greater than 40 in (men) or 35 inches (women) Elevated blood pressure, equal to or greater than 130/85 Elevated fasting glucose, greater than 100 mg/dL Reduced HDL, less than 40 gm/dL (men), and 50 mg/dL (women) Elevated blood triglycerides, above 150 mg/dL