Transcript Functional Foods - China Medical University, Taiwan

Plant Derived Therapeutics
for the treatment Metabolic
Syndrome
William T. Cefalu, M.D.
Pennington Biomedical Research Center
Changing Nutrition/Health Paradigm
1920s-1980s
s
What food is required for me?
ä
Nutritional deficiency:
ä
ä
ä
ä
Iron - Anemia
Iodine – Thyroid goiter
Vitamin D - Rickets
Identifying essential nutrients required for
promoting growth and sustaining life.
ä
ä
ä
Vitamins
Essential minerals – Calcium
Essential amino acids
Changing Nutrition/Health Paradigm
1950s-1990s
s
What does food do to me?
ä
Nutritional excess and imbalances
ä
ä
ä
Calories - obesity
Fiber – Colon cancer
Identifying nutrients and components that
contribute to premature death
ä
ä
ä
Saturated fat
Cholesterol
Sodium
CHD Deaths and MI/100
Seven Countries Study: CHD Events are
Correlated with Saturated Fat
5
R = 0.84
E
4
N
U
3
2
C
W
S
M
G
D
1
B
Z
V
0
0
5
K
10
15
20
% Calories from Saturated Fat
Keys, 1970
Changing Nutrition/Health Paradigm
1990s-Today!
s
What does food do for me?
ä
ä
Nutritional optimization of quality of life
Identification of physiological active
components to prevent or delay premature
onset of chronic disease
ä
ä
Phytochemicals
Pre/Probiotics
Fiber
Identification of Phytochemicals
ä
Phytochemicals as Functional Components
s
Individual compounds in plants that have evolved
in part as protective mechanisms against
environmental insult
s
Phytochemicals with biological activity have had
great utility as pharmaceuticals and pestmanagement agents.
s
Very few of these potentially active compounds
have been examined thoroughly.
Functional Foods
s
Definition
ä
s
Generic description of foods, that when
ingested, provide demonstrated physiological
health benefits beyond simple nutritional value
Expanded Definition
ä
Similar in appearance to conventional foods;
Are consumed as part of a usual diet
How to Be a Functional Food
s
A natural food product can be engineered to
become a functional food by
ä
ä
ä
ä
Increasing specific components (Phytochemicals)
to reach a concentration more likely to express
health benefits
Adding components not normally present but
having a beneficial effect
Replacing a component that is excessive and
harmful with one having a beneficial effect
Improving the bioavailability of components
having desired health benefits
Phytochemicals and Reported Sources
Polyphenols
ä
ä
ä
Epicatechin
Epigallocatechin
Epigallocatechin gallate
s
Source:
Green tea; grapes, red wine
s
Benefits: Anti-cancer; CHD protective
s
Function: Inhibit chemical carcinogenesis and
tumor formation; inhibit cancer cell
growth; antioxidant; reduces free
radical/oxidative damage
Isoflavones
ä
ä
Genistein
Daidzein
Genistein
s
Source:
Soybean, flaxseed
s
Benefits: Relieves menopausal symptoms; prevents
osteoporosis; anti-cancer; CHD protective
s
Function: Estrogen-like activity; inhibit growth of
breast cancer cells; stimulate Ca
absorption; lower cholesterol levels
Phytosterols
ä
ä
ä
-sitosterol
Campesterol
-sitostanol
s
Source:
Plant oils
s
Benefits: CHD protective
s
Function: Inhibit cholesterol absorption
-sitosterol
Carotenoids
ä
ä
ä
ä
Lycopene
-carotene
-cryptoxanthin
Lutein
-carotene
s
Source:
Tomatoes, carrots, yams, cantaloupe,
spinach, sweet potatoes; citrus fruits
s
Benefits: Anti-cancer; CHD protective
s
Function: Antioxidant; free radical scavenger;
induction of cell-cell communication and
growth control; inhibit tumor growth
n3-Fatty Acids
ä
ä
ä
-linoleic acid
Docosahexaenoic acid
Eicosapentaenoic acid
DHA
s
Source:
Flaxseed oil, fish oil
s
Benefits: CHD protective; anti-cancer; antiinflammatory
s
Function: Lower triglycerides; inhibit platelet
aggregation; affect eicosanoid production
Flavonoids
ä
ä
ä
ä
Quercetin
Apigenin
Luteolin
Myricetin
Luteolin
s
Source:
Citrus fruits; vegetables
s
Benefits: Anti-cancer; CHD protective
s
Function: Antioxidant; inhibit platelet aggregation;
inhibit cancer cell growth and
proliferation; cytotoxic to cancer cells
The Promise of Plant Therapeutics
s
Better health through improved nutrition
can:
Increase quality of life
ä Enhance productivity
ä Reduce health-care costs
ä
- by preventing or delaying the onset of
Health claims need to
be verified
with carefully controlled studies
chronic disease, i.e diabetes
- or improving metabolic factors related to the
disease, i.e. glucose
O Years from
-10
diagnosis
-5
Onset
Insulin resistance
0
5
10
Diagnosis
“Nutraceutical (Botanical/Bioactive)
Strategies”
Post-Meal glucose
Fasting glucose
Pre-diabetes
Type 2 diabetes
15
Natural History of Type 2 Diabetes
Progressive -Cell Failure
Years from
diagnosis
-10
Pancreas function
0
-5
Onset
Insulin “inefficiency
Insulin secretion
5
10
15
Diagnosis
“Pre-Diabetes”
Post-Meal glucose
Fasting glucose
Pre-diabetes
Type 2 diabetes
Ramlo-Halsted BA, Edelman SV. Prim Care. 1999;26:771-789 Nathan DM. N Engl J Med. 2002;347:1342-1349
Cardiovascular Risk in Pre-diabetes
Non-Diabetic
Diabetes
Relative Risk of MI or Stroke
7
5.02
6
3.64
5
3.19
4
2.4
3
2
1.0
1
0
Nondiabetic
Throughout
>15 Yr
Before Dx
10-14.9 Yr
Before Dx
Pre-Diabetes
Hu FB, et al. Diabetes Care. 2002;25:1129-1134.
<10 Yr
Before Dx
Diabetic
Throughout
Natural History of Type 2 Diabetes
Years from
diagnosis
-10
-5
Onset
0
5
10
15
Diagnosis
Insulin resistance
“Nutraceutical (Botanical/Bioactive)
Strategies”
Post-Meal glucose
Fasting glucose
Pre-diabetes
Type 2 diabetes
Ramlo-Halsted BA, Edelman SV. Prim Care. 1999;26:771-789 Nathan DM. N Engl J Med. 2002;347:1342-1349
Phytochemicals and Reported Sources
Blueberries as a Therapy for
Type 2 Diabetes
• Literature Review
– Blueberries are a folk remedy in Canada for
treatment of diabetes (Martineau LC et al., Phytomedicine, 2006)
– Blueberries have been found to:
• Reduce blood glucose concentrations in rats and
humans (Abidov M et al, 2006 ; DeFuria J et al, 2009)
• Increase glucose uptake in muscle and fat cells
(Tri Vuong et al, 2006)
• Protect against obesity in rats (DeFuria J et al, 2009)
PBRC Blueberry Research Study
• Primary Objective
– To evaluate the effect of blueberry bioactives
on improving a pathophysiologic parameter
contributing to the development of type 2
diabetes in humans
• Hypothesis
– Increased consumption of blueberry
bioactives will result in an increase in whole
body insulin action, i.e. insulin sensitivity, in
pre-diabetic individuals
Study Design
Insulin Sensitivity Test
Blueberry
End
Screening
Group
Hyperinsulinemic
Euglycemic
Baseline
Clamp
*Randomized
Placebo
End
*Started consuming
“Gold
Standard” for
objectively
(Control)
smoothies
assessing
whole
body
1 2
3
4
5 insulin
6
7 action
8
9
Visits (Weeks)
Nutritional Value of Intervention
Nutritional Value (per 16oz)
Energy, kcal
Carbohydrate, g
Fiber, g
Protein, g
Fat, g
Saturated Fat, g
Bioactives
239
48.5
4.2
11.9
0.08
0.05
Placebo
234
48.6
4.3
11.1
0.08
0.05
Each subject consumed two 16oz smoothies per day
16 oz
Smoothie
(Bioactives)
=
Stull AJ et al. J Nutr. 2010 Oct;140(10):1764-8.
Placebo
Smoothie
The Ability
of Insulin
to Work inin
AllInsulin
Participants
Individual
Response
From Beginning to End of Study
Sensitivity
100
90
% ∆ Insulin Sensitivity
80
70
Bioactives
Group
Placebo (Control)
Group
60
50
40
30
20
10
0
-10
-20
-30
Bioactives
Group
Placebo
Technical Variability
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
25 11
26 12
27 13
28 14
29 30
1 17
2 18
3 19
4 20
5 21
6 22
7 23
8 24
9 10
15 31
16 32
17
Subject Numbers
Blueberry Bioactives Improved Insulin
Sensitivity
%∆ Insulin Sensitivity
25
22.2
4-Fold
20
15
10
4.9
5
0
Blueberry
Placebo
∆ insulin Sensitivity = ability of insulin to work from week 0 to week 6
Botanicals showing Promise to
Favorably modulate Carbohydrate
Metabolism
• Bitter
Melon ( Momordica charantia)
• Hoodia (Hoodia gordonii)
• Artemisia dracunculas and related sp.
Figure 3
Glucose (mg/dl)
A
LFD
250
HFD
Glucose (mg/dl)
Bitter Melon and Glucose tolerance
BM
200
D
**
#
150
100
50
HFD
BM
***
#
300
***
250
***
#
200
#
150
100
0
6
12
0
Weeks
LFD
2.5
HFD
BM
E
***
2.0
*
1.5
#
1.0
0.5
0.0
0
20
40
60
80
100
120
Time (min)
Glucose (mg/dl)
Insulin (ng/ml)
***
350
0
B
IPGTT
400
6
12
***
200
***
150
HFD
BM
LFD
IPITT
***
***
***
#
100
#
50
0
Weeks
0
20
40
60
80
100
1.5
LFD
HFD
BM
F
***
1.0
###
*
***
0.5
0.0
0
6
Weeks
12
(pmol/ml)
C
HOMA-IR
Time (min)
15000
12000
9000
6000
3000
0
Plasma leptin concentration in mice
***
##
***
LFD
HFD
Groups
BM
120
Bitter Melon and Glucose tolerance
Skeletal muscle
IRS-1 p (Tyr612)
IRS-1
IRS-2
IRS-1
IR 
Akt1 p (s er473)
PI 3K
Akt1
Akt1
Akt2 p (s er474)
Akt2
GLUT4
Akt2
Insulin
Group
-actin
Group
LFD
Mouse #
Insulin
Group
HFD
1
-
2
3
+
LFD
4
+
-
BM
8
-
9
7
+
HFD
11
+
13
-
15
+
16
BM
18
+
Basal
Fold of LFD at basal
2.5
**
###
Insulin
2.0
*
1.5
**
1.0
0.5
0.0
LFD
HFD
BM
+
LFD
-
+
HFD
-
+
BM
Bitter Melon Modulates Intracellular
A
lipids
B
Short/medium chain acyl carnitine in muscle tissues
*
25
***
HFD
BM
20
*
#
15
***
10
5
###
##
#
0
C3
Acyl carnitine (pmol/mg protein)
LFD
C4
C4-DC
#
Acyl carnitine (pmol/mg protein)
Figure 4.
C6
C8:1
C12-OH
Long chain acyl carnitine in muscle tissues
30
LFD
25
HFD
BM
20
15
###
10
5
0
***
***
###
C14:2
C14:1
C14-OH
##
C16
C18-OH
Hoodia gordonii and Asclepias incarnata (Swamp
milkweed) Belong to the
Same Plant Family
cellhealthmakeover.com
abnativeplants.com
Natural Diversity of Pregnanes
Isolated from A. incarnata Extract
12-hydroxy (lineolon)
12-cinnamoyl
ikemagenin
12-benzoyl
12-acetyl
In collaboration with Analyticon
12-tigloyl
12-nicotinoyl
An approach to the study of
Phytochemicals and Human Health
Phytochemicals and Carbohydrate
Metabolism
Alcoholic Extract of Artemisia dracunculus L.
Russian Tarragon (PMI 5011)
Ribnicky DM, et al. Am J Physiol Endocrinol Metab. 2007
Sourcing and Standardization of
Phytochemicals
• Agriculture SOPs must be developed for each crop
(Germination; Plant nutrition Pest management)
Artemisia dracunculus in
Hydroponics at ARC Greenhouses
Sourcing and Standardization of
Phytochemicals
• Agriculture SOPs must be developed for each crop
(Germination; Plant nutrition Pest management)
• Optimization of activity and composition
Phytochemicals and Carbohydrate
Metabolism
Flowering Artemisia dracunculus
LC-MS Comparison of PMI-5011 Extracts Made
from Plants at Different Stages of Development
Growth/
Non-flowering Stage
Flowering Stage
Sourcing and Standardization of
Phytochemicals
• Agriculture SOPs must be developed for each crop
(Germination; Plant nutrition Pest management)
• Optimization of activity and composition
• Identification of the active components for
standardization (Bioactivity guided Fractionization)
Identification and Isolation of Phytochemicals
ADEX
ADEX
5011
ADEX
Bioactivity Guided
Fractionization
PTP1B Levels and Artermisa sp (5011)
Human Skeletal Muscle
Time Course Studies: 5011
Type 2 DM subjects: 0
BMI 29.5
BMI 31.1
4
6
8
12
hours
PTP-1B
-actin
PTP-1B
-actin
BMI 36.8
PTP-1B
-actin
Insulin Signaling Parameters
Human Skeletal Muscle Culture
5011 Fractions
IRS-1
IRS-2
IR 
PI 3
Akt-p
Phosphatase
PTP 1B
-Actin
Fraction7 Contr 2
3
4
5
6
7
8
9
Wang ZQ et al. Metabolism. 2008 Jul;57(7 Suppl 1):S58-64.
Ribnicky DM et al. Am J Clin Nutr. 2008;87(2):472S-5S.
10
11 5011D
Bioactives Isolated from Artemisia dracunculus
and PMI-5011 by Activity-Guided Fractionation
Isolated Compounds
4,5-di-O-caffeoylquinic acid * ◊ ○
Davidigenin * ◊
† ‡
6-demethoxycapillarisin * ◊ ○
2′,4′-dihydroxy- 4methoxydihydrochalcone * ◊ ○
- confirmed with NMR
*
◊ - new compound to A . dracunculus
▪ - flavonoids
○ - activity reported for the first time
A - active
† - dihydrochalcone
‡ - new compound to genus Artemisia
§ - first report as a constituent of plants
PTP-1B
PEPCK
A
-
-
A
-
-
A
-
A
A
A
A
-
A
-
-
A
-
† ‡ §
2,4-dihydroxy- 4methoxydihydrochalcone * ◊ ○ †
Sakuranetin ▪ ◊ ○
ALR2
‡
ALR2- Aldose reductase
PTP-1B - Protein tyrosine phosphatase - 1B
PEPCK - Phosphoenolpyruvate carboxykinase
The Pure Active DMC-2* from Artermisia is Validated
in vivo with Comparable Activity to Metformin
250
*2′, 4′–dihydroxy-4-methoxydihydrochalcone
*
blood glucose (mg/dl)
200
*
**
150
*
0 hr
6 hr
100
50
0
Labrasol
50 mg/kg
chalcone
150 mg/kg
chalcone
300 mg/kg
chalcone
metformin 300
mg/kg
All treatments provided with 66% Labrasol
Sourcing and Standardization of
Phytochemicals
• Agriculture SOPs must be developed for each crop
(Germination; Plant nutrition Pest management)
• Optimization of activity and composition
• Identification of the active components for
standardization (Bioactivity Guided Fractionization)
• Standardized for Active Components
Sourcing and Standardization of
Phytochemicals
• Agriculture SOPs must be developed for each crop
(Germination; Plant nutrition Pest management)
• Optimization of activity and composition
• Identification of the active components for
standardization (Fingerprinting)
• Standardized for Active Components
• Stability Assessed
Stability of the Extract
-20ºC freezer
22ºC open container
22ºC desiccator
PMI-5011 is stable under
various
storage conditions
Stability was investigated
a period of 7 months by
validated HPLC method
37ºC open oven
HPLC-Chromatograms of PMI-5011,
stored under different conditions
Sourcing and Standardization of
Phytochemicals
Assuring Supply of materials for research
•
•
•
•
in vitro studies
Toxicology
in vivo preclinical
clinical
Study Design: In vivo Mechanism
Animals: KK-Ay
Intervention: 5011 Total Extract vs control conditions
Endpoint: Insulin and Glucose levels, Muscle signaling
Baseline
Animals
Received
Baseline
Intervention
5011
Placebo
• Insulin Stimulation/Muscle Insulin Signaling
• Serial Insulin/Glucose
Body Weight/Food Intake
Ribnicky DM, et al. Phytomedicine. 2006 Sep;13(8):550-7.
Plasma Insulin
Plasma insulin
Insulin (pg/ml)
20
**
*
*
15
Control
10
5011
5
0
0
2
4
6
Weeks of Study
Mean + SEM, * P<0.05, ** P<0.01.
8
Insulin Signaling Parameters
Skeletal Muscle
Control
5011
AS160
Akt-p
Akt-1
Akt-2
Glut
4
-Actin
Mice # 1
5011
-
2
-
3
-
9
+
10
+
11
+
Bench to Bedside
Animal Testing to Monitor
Safety and Effectiveness
Cells tested with
Botanical
Human Testing in
Several Phases
Clinical Trial Study Design
Subjects: Obese, Insulin Resistant, n = 28
Intervention: 5011 Total Extract vs Placebo
Endpoint: Insulin Sensitivity (Clamp Procedure)
Screening
Screening
Baseline
Baseline
Intervention
5011
Placebo
•Hyperinsulinemic Euglycemic Clamps
•Body Composition
Clinical Response
Insulin Sensitivity (mg/min/FFM)
Insulin Sensitivity
Baseline
End of Study
12
11
10
*
10
9
8
8
7
6
6
5
4
4
Pre
Post
Placebo
Pre
Post
5011
Placebo 5011
* P < .05
Phytochemical Characterization
Analysis of Compounds from PMI-5011 in
In Plasma by LC-MS-SIM
SIM for active compounds in PMI-5011
285
257
Standards
6-demethoxycapillarisin & davidigenin
271
285
Standards
sakuranetin &
2′, 4–dihydroxy-4′-methoxydihydrochalcone
SIM plasma analysis
SIM = Selected Ion Monitoring
Plasma Appearance of
Phytochemical
Abundance (X 107)
Time Course of Appearance
7
6
5
4
3
2
1
0
0
45
120
180
Minutes after Ingestion
240
Plasma Abundance
5011 Metabolites
Abundance (x 107)
Mean Levels over 4 hours
7
Ion 257: davidigenin
Ion 271 sakuranetin
Ion 285: chalcone
6
5
4
3
2
1
Undetectable
0
5011
Placebo
Potential Nutraceutical (Phytochemical)
Markets
1. Joint health
(n3-fatty acids, glucosamine, chondroitin sulfate)
2. Gastrointestinal health
(ginger, pepermint, fennel, prebiotics, probiotics)
3. Blood lipids
(n3-fatty acids, oat bran, phytosterols)
4. Bone density and skeletal health
(phytoestrogens, Ca, Zn)
5. Hormone replacement
(isoflavones, arginine, yohimbe)
Potential Nutraceutical (Phytochemical)
Markets
6. Body fat/Weight
(herbal phen-fen, Cr, garcinia cambogia)
7. Optimal vision
(lutein, zeaxanthin)
8. Stress and insomnia
(St. John’s Wort, tryptophan)
9. Breast and prostate health
(fruits, vegetables, saw palmetto)
10. Carbohydrate Metabolism
(Chalcones, Cinnamon polyphenols)
The most studied Phytochemical:
Wine
s
Over 40 prospective studies have documented an inverse
relationship between alcohol intake and heart disease
prevention
1.4
1.2
1
Relative 0.8
Risk 0.6
0.4
CVD
0.2
Non-CVD
0
0
<0.1
0.1 - 0.3
0.3 - 1
Drinks per Day
1.0 - 2.0
>2.0
Regarding Plant Therapeutics and Human Health…..
We Can’t Put Our Heads in the Sand any Longer !!!