23,600,000 Diabetics 7 th Leading Cause of Death Estimated $218 Billion 5.2X higher medical...
-
Upload
isis-whitney -
Category
Documents
-
view
218 -
download
0
Transcript of 23,600,000 Diabetics 7 th Leading Cause of Death Estimated $218 Billion 5.2X higher medical...
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