Inhibition of Alpha-Amylase and Alpha-Glucosidase by Bioflavanoids: In Vitro and In Vivo Studies Anneke Tucker Dr. Me

1. Inhibition of Alpha-Amylase andAlpha-Glucosidase by Bioflavanoids: In Vitro and In Vivo Studies Anneke TuckerDr. Meltem MusaDr. Balz Frei HHMI Symposium Presentation September 23, 2010

2. -10.7% of people in the United States have diabetes Medical expenditures are 2.3 times higher with diabetes than without Diabetes causes more deaths/year than breast cancer and AIDS combined, and was the 7th leading cause of death in the United States in 2006 71000 amputations are performed each year, with the number increasing annually, because of diabetes related complications 1.6 million new cases of diabetes are diagnosed yearly There are over 23 million people in the United States with diabetes The total cost of diabetes per year is $218 billion dollars in the US -10.7% of people in the United States have diabetes Medical expenditures are 2.3 times higher with diabetes than without Diabetes causes more deaths/year than breast cancer and AIDS combined, and was the 7th leading cause of death in the United States in 2006 71000 amputations are performed each year, with the number increasing annually, because of diabetes related complications 1.6 million new cases of diabetes are diagnosed yearly There are over 23 million people in the United States with diabetes The total cost of diabetes per year is $218 billion dollars in the US

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 Medical Significance With diabetes, there is two different issues� either the body doesn�t produce enough insulin, which is typically a result of a genetic defect, or the body�s cells ignore the presence of insulin, which is the case with most Americans. This type of diabetes is called Type II diabetes, determined by having an elevated fasting blood glucose level of 126 mg/dL or higher. There are several precursors for �getting� diabetes, such as an obese body type, a diet high in fat and alcohol, genetic predisposition, high blood pressure, and leading a sedentary lifestyleWith diabetes, there is two different issues� either the body doesn�t produce enough insulin, which is typically a result of a genetic defect, or the body�s cells ignore the presence of insulin, which is the case with most Americans. This type of diabetes is called Type II diabetes, determined by having an elevated fasting blood glucose level of 126 mg/dL or higher. There are several precursors for �getting� diabetes, such as an obese body type, a diet high in fat and alcohol, genetic predisposition, high blood pressure, and leading a sedentary lifestyle

4. 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 (a-amylase and a-glucosidase) slow the absorption of carbohydrates. 4

5. Digestive Enzymes of Interest a-Amylase Digests starches and oligosaccharides Secreted by the pancreas, component of saliva Located in mouth and small intestine a- Glucosidase Digests starches and oligosaccharides Located in the epithelium of the small intestine Amylase is an oral and intestinal enzyme that digests long chain starches and ogliosaccharides, and is more prevalently active in the small intestine. Glucosidase also digests starches and ogliosaccharides, and is located strictly in the epithelium of the small intestine. (difference between insulin resistance and pompe disease� pompe is caused by a transversion mutation, that results in a non functioning lysosomal acid alpha-glucosidase enzyme� aka acid maltase, required for the degradation of a small percentage of cellular glycogen� results in an accumulation of structurally normal glycogen in lysosomes and cytoplasm� the usual metabolism of glucose and glycogen is still present and functioning� it�s simply and curiously only the glucosidase in the lysosome that can�t be broken down.Amylase is an oral and intestinal enzyme that digests long chain starches and ogliosaccharides, and is more prevalently active in the small intestine. Glucosidase also digests starches and ogliosaccharides, and is located strictly in the epithelium of the small intestine. (difference between insulin resistance and pompe disease� pompe is caused by a transversion mutation, that results in a non functioning lysosomal acid alpha-glucosidase enzyme� aka acid maltase, required for the degradation of a small percentage of cellular glycogen� results in an accumulation of structurally normal glycogen in lysosomes and cytoplasm� the usual metabolism of glucose and glycogen is still present and functioning� it�s simply and curiously only the glucosidase in the lysosome that can�t be broken down.

7. Acarbose(Precose, Bayer Pharmaceuticals) Obtained from the fermentation processes of a microorganism, Actinoplanesutahensis Reduces postprandial hyperglycemia Oligosaccharide that competitively and reversibly inhibits the activity of a-glucosidase and a-amylase Issues: Cost Side effects, including gastrointestinal disturbances (stomach distress, diarrhea, bloating, etc.) and nausea Enteric: of or pertaining to the enteron Affects liver because it is metabolized in the liverEnteric: of or pertaining to the enteron Affects liver because it is metabolized in the liver

8. Negative effects Dumping syndrome Gas Bloating CostNegative effects Dumping syndrome Gas Bloating Cost

9. A Natural Solution: Bioflavonoid A class of plant secondary metabolites Primary function is to provide protection to plants against biological attacks Are known for their biological activities Bioflavoniods act like an antibody for the plant; a field that has more exposure to insects or harsher environmental conditions will contain more bioflavonoids than a field that is otherwise. Not all carrots are created equal!Bioflavoniods act like an antibody for the plant; a field that has more exposure to insects or harsher environmental conditions will contain more bioflavonoids than a field that is otherwise. Not all carrots are created equal!

10. 10 Flavonoids inhibit a-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. Catechins 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 Found most abundantly in green tea leaves!!! Berries, chocolate, red wine, applesFound most abundantly in green tea leaves!!! Berries, chocolate, red wine, apples

12. Hypothesis Inhibition of a-amylase and a-glucosidase by specific flavonoids during the digestion of starches helps control post-prandial hyperglycemia and ultimately Type II Diabetes Mellitus. Type II Diabetes and metabolic syndrome go hand in hand!Type II Diabetes and metabolic syndrome go hand in hand!

13. a-Amylase Method 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 IC50 calculation When the enzyme acts upon the glycosidic bonds, it releases a �quench�, which allows it to fluoresce. When the enzyme acts upon the glycosidic bonds, it releases a �quench�, which allows it to fluoresce.

14. Calculating the IC50 IC50= the concentration of the inhibit or when the enzyme has been reduced to 50% of it�s original activity Check FDAIC50= the concentration of the inhibit or when the enzyme has been reduced to 50% of it�s original activity Check FDA

15. Positive Control: AcarboseCalculating the Activity -graph of incidence of fluorescence over a period of time, compared to the standard, which is zero inhibition of amylase activity -incidence of fluorescence measured at differing concentrations of inhibitor - The top diamond line represents uninhibited enzymatic activity. The other lines represent various compound concentrations. As the level of fluorescence goes down, that indicates to us a decrease in the activity of the enzyme -graph of incidence of fluorescence over a period of time, compared to the standard, which is zero inhibition of amylase activity -incidence of fluorescence measured at differing concentrations of inhibitor - The top diamond line represents uninhibited enzymatic activity. The other lines represent various compound concentrations. As the level of fluorescence goes down, that indicates to us a decrease in the activity of the enzyme

16. a-Amylase Data Catechin and EC are two monomers found in GSE, but there are oligomers such as dimers and trimers as well. (They may be responsible or play a role in the inhibition, but we have not yet determined the SAR)Catechin and EC are two monomers found in GSE, but there are oligomers such as dimers and trimers as well. (They may be responsible or play a role in the inhibition, but we have not yet determined the SAR)

17. a-Amylase Data Catechin and EC are two monomers found in GSE, but there are oligomers such as dimers and trimers as well. (They may be responsible or play a role in the inhibition, but we have not yet determined the SAR)Catechin and EC are two monomers found in GSE, but there are oligomers such as dimers and trimers as well. (They may be responsible or play a role in the inhibition, but we have not yet determined the SAR)

18. a-Amylase Data

19. a-amylase Data

20. a-Amylase Summary Two separate graphs� Smaller bars means more efficient inhibitor!!Two separate graphs� Smaller bars means more efficient inhibitor!!

22. Positive Control: AcarboseCalculating the IC50 Instead of reading activity over a period of time, we instead read the various levels of amylase activity that decrease with increasing concentration Instead of reading activity over a period of time, we instead read the various levels of amylase activity that decrease with increasing concentration

23. a-glucosidase Data Show GCG smaller concentration graph!!!Show GCG smaller concentration graph!!!

24. a-Glucosidase Summary

25. Human Studies 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-�-vis carbohydrates/starch Blood collection time points

26. Preliminary Data: Grape Seed Extract

27. Future plans a-Glucosidase and a-Amylase: In Vitro Studies Confirm in vitro results (n=3, statistical evaluation) Vary inhibitor concentration to determine Ki for select bioflavonoids/extracts Human Studies Optimize conditions Carbohydrate dose Supplement (mixture) to test Dose of supplement Administration time Conduct trial, measure post-prandialglycemia and insulinemia Pancreatic Lipase (fat absorption): Initial studies (in vitro) and optimization in Fall/Winter Preliminary in vivo studies (time permitting) -triplicate experiments will be completed as the summer progresses, of each of the compounds, successful and otherwise, in vitro -milimolar concentrations will be expanded to find a more specific IC50 value for each compound -we have found several compounds that have given promising data, and human assay studies are expected to be completed by the end of the summer -are optimizing the conditions for the glucosidase and lipase experiments, and expect to begin screening compounds within the next several weeks -triplicate experiments will be completed as the summer progresses, of each of the compounds, successful and otherwise, in vitro -milimolar concentrations will be expanded to find a more specific IC50 value for each compound -we have found several compounds that have given promising data, and human assay studies are expected to be completed by the end of the summer -are optimizing the conditions for the glucosidase and lipase experiments, and expect to begin screening compounds within the next several weeks

28. Acknowledgements Dr. MeltemYilmazer-Musa Dr. BalzFrei 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

29. 200g CHO 60-60-80= 800 calories 48% 150g P 40-50-60= 600 36% 65-70 g fat= 585 16% 415 total= 2085 calories 1500 calories= 720 CHO, 180g 540 P, 135g 240 F, 27g200g CHO 60-60-80= 800 calories 48% 150g P 40-50-60= 600 36% 65-70 g fat= 585 16% 415 total= 2085 calories 1500 calories= 720 CHO, 180g 540 P, 135g 240 F, 27g

30. Scutellarein: blue skullcap, a mint native to North America�mild sedative and sleep promoter, used for incense Baicalein: baikal skullcap, used in traditional chinese medicine as one of 50 fundamental herbs, active ingredient in a Japanese herbal supplement, believed to enhance liver health�studies are underway to support this claim Morin: horse-apple from Oklahoma, Texas and Arkansas�if eaten, can cause vomiting (although not poisonous, but makes great firewood) Myricetin: red wineScutellarein: blue skullcap, a mint native to North America�mild sedative and sleep promoter, used for incense Baicalein: baikal skullcap, used in traditional chinese medicine as one of 50 fundamental herbs, active ingredient in a Japanese herbal supplement, believed to enhance liver health�studies are underway to support this claim Morin: horse-apple from Oklahoma, Texas and Arkansas�if eaten, can cause vomiting (although not poisonous, but makes great firewood) Myricetin: red wine

31. Elevated waist circumference: Men � Equal to or greater than 40�inches (102�cm) Women � Equal to or greater than 35�inches (88�cm) Elevated triglycerides: Equal to or greater than 150�mg/dL Reduced HDL (�good�) cholesterol: Men � Less than 40�mg/dL Women � Less than 50�mg/dL Elevated blood pressure: Equal to or greater than 130/85�mm Hg or use of medication for hypertension Elevated fasting glucose: Equal to or greater than 100�mg/dL (5.6�mmol/L) or use of medication for hyperglycemia Having any three is defined as having the metabolic syndrome Elevated waist circumference: Men � Equal to or greater than 40�inches (102�cm) Women � Equal to or greater than 35�inches (88�cm) Elevated triglycerides: Equal to or greater than 150�mg/dL Reduced HDL (�good�) cholesterol: Men � Less than 40�mg/dL Women � Less than 50�mg/dL Elevated blood pressure: Equal to or greater than 130/85�mm Hg or use of medication for hypertension Elevated fasting glucose: Equal to or greater than 100�mg/dL (5.6�mmol/L) or use of medication for hyperglycemia Having any three is defined as having the metabolic syndrome