23,600,000 Diabetics 7 th Leading Cause of Death Estimated $218 Billion 5.2X higher medical expenditures 71,000 amputations 1.6 million.

Inhibition of Alpha-Amylase andAlpha-Glucosidase by Bioflavanoids:

In Vitro and In Vivo Studies

Anneke TuckerDr. Meltem Musa

Dr. Balz Frei

HHMI Symposium Presentation September 23, 2010

The Problem

TYPE II DIABETES MELLITUS

www.diabetes.org

23,600,000 Diabetics

7th Leading Cause of Death

Estimated $218 Billion

5.2X higher medical expenditures

71,000 amputations

1.6 million new cases per year

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

6.2 MILLION PEOPLE ARE UNDIAGNOSED

54 MILLION PEOPLE ARE

PRE-DIABETIC

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

Digestive Enzymes of Interest

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

OH

OH

O

CH2OH

Lumen of the Small Intestine

Epithelial Brush Border of the Small Intestine

Enzyme

OH

OH

O

CH2OH

OH

OH

O

CH2OH

OH

OH

O

CH2OH

Acarbose (Precose, Bayer Pharmaceuticals)

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

Lumen of the Small Intestine

Epithelial Brush Border of the Small Intestine

Enzyme

Inhibitor: Acarbose

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

Flavone backbone structure

α- Amylase Inhibition AssayCommon structural features of flavonoids to inhibit α-amylase

CatechinEpicatechin

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

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

Catechin

Hypothesis

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.

α-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 http://www.cryst.bbk.ac.uk/pps97/assignments/projects/

gazerro/amylase.gif

Calculating the IC50

IC50

The IC50 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

0 2 4 6 8 10 12 14 160

10

20

30

40

50

60

Amylase Activity in Fluorescence

0

Time (min)

Rela

tive F

luore

scence

0 10 20 30 40 50 60 700

5

10

15

20

25

30

35

40

200

100

50

25

0

Dose-dependent Inhibition by Acarbose

Time (min)

Rela

tive F

Luore

scence

Positive Control: AcarboseCalculating the Activity

μM

μM

μM

μM

μM

Concentration of Inhibitor

Slopes converted to percentage activity

α-Amylase Data

IC50= 9.44 ± 0.70 μg/mlIC50= 7.37 ± 0.26 μg/ml

Catechin Epicatechin

α-amylase DataIC50= 7.37 ± 0.26 μg/ml

α-a

myla

se a

cti

vit

y (

%)

IC50= 44.62 ± 0.52 μg/ml

IC50= 356.73 ± 31.60 μg/ml

α-a

myla

se a

cti

vit

y (

%)

α-Amylase Summary

Acarb

ose

Gra

peseed E

xtra

ct

Gre

en Tea E

xtra

ct

White

Tea E

xtra

ct0

10

20

30

40

50

7.37 9.44

44.62

IC50

μg

/mL

Epigallo

cate

chin

Gal

late

Epicate

chin

Galla

te

Catech

in

Epicate

chin

0

10

20

30

40 34.0031.00 32.00

35.00

Maximum Inhibition (%)

300

320

340

360 356.73

α-glucosidase Method

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 IC50

http://www.rcsb.org/pdb/explore/explore.do?structureId=2ZE0

Positive Control: AcarboseCalculating the IC50

0 200 400 600 800 1000 12000

10

20

30

40

50

60

70

80

90

100

110

Acarbose

Acarbose Concentration (μg/mL)

Enzym

e A

cti

vit

y (

%)

IC50= 102.67 ± 3.77 μg/ml

0 1 2 3 4 5 60

20

40

60

80

100

120

Green Tea Extract

μg/mL

Enzym

e A

cti

vit

y (

%)

0 2 4 6 8 10 120

50

100

150

200

250

White Tea Extract

μg/mL

Enzym

e A

cti

vit

y (

%)

0 2 4 6 8 10 120

50

100

150

200

250

Grape Seed Extract

μg/mL

Enzym

e A

cti

vit

y (

%)

α-glucosidase Data

0 200 400 600 800 1000 12000

20

40

60

80

100

120

Acarbose

μg/mL

Enzym

e A

cti

vit

y (

%)

α-Glucosidase Summary

Acarb

ose

Gre

en Tea E

xtra

ct

White

Tea E

xtra

ct

Gra

peseed E

xtra

ct

Epicate

chin

Galla

te

EGCG

0.01.02.03.04.05.06.07.08.0

102.67

0.64

7.17

1.101.87

0.11

IC50

μg

/mL

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-à-vis carbohydrates/starch

› Blood collection time points

0 15 30 45 60 75 9060

70

80

90

100

110

120

130

Blood Glucose Test n=1

Time (min)

Blo

od

Glu

cose

(m

g/d

L)

Preliminary Data: Grape Seed Extract

Peak Area (AUC)= 21.9

With Inhibitor

Peak Area (AUC)= 21.7

Carbohydrate Only

Future plansα-Glucosidase and α-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-prandial glycemia and insulinemia

Pancreatic Lipase (fat absorption):› Initial studies (in vitro) and optimization in Fall/Winter› Preliminary in vivo studies (time permitting)

Acknowledgements 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

Morin

Baicalein Scutellarein

Myricetin

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

Metabolic

Syndrome3 of 5 symptoms