Plant Derived Therapeutics for the treatment Metabolic
Syndrome
Plant Derived Therapeutics for the treatment Metabolic
SyndromeWilliam T. Cefalu, M.D.
Pennington Biomedical Research Center
Changing Nutrition/Health ParadigmChanging Nutrition/Health Paradigm
What food is required for me? Nutritional deficiency:
Iron - Anemia Iodine – Thyroid goiter Vitamin D - Rickets
Identifying essential nutrients required for promoting growth and sustaining life.
Vitamins Essential minerals – Calcium Essential amino acids
1920s-1980s1920s-1980s
Changing Nutrition/Health ParadigmChanging Nutrition/Health Paradigm
What does food do to me? Nutritional excess and imbalances
Calories - obesity Fiber – Colon cancer
Identifying nutrients and components that contribute to premature death
Saturated fat Cholesterol Sodium
1950s-1990s1950s-1990s
Seven Countries Study: CHD Events areCorrelated with Saturated Fat
Seven Countries Study: CHD Events areCorrelated with Saturated Fat
0 5 10 15 20
% Calories from Saturated Fat
0
1
2
3
4
5
CHD
De
ath
s an
d M
I/10
0 R = 0.84
V
MC
DG
SW
B
Z
UN
E
K
Keys, 1970
Changing Nutrition/Health ParadigmChanging Nutrition/Health Paradigm
What does food do for me? Nutritional optimization of quality of life Identification of physiological active
components to prevent or delay premature onset of chronic disease
Phytochemicals Pre/Probiotics Fiber
1990s-Today!1990s-Today!
Identification of Phytochemicals
Phytochemicals as Functional ComponentsPhytochemicals as Functional Components
Individual compounds in plants that have evolved in part as protective mechanisms against environmental insult
Phytochemicals with biological activity have had great utility as pharmaceuticals and pest-management agents.
Very few of these potentially active compounds have been examined thoroughly.
Functional FoodsFunctional Foods DefinitionDefinition
Generic description of foods, that when ingested, provide demonstrated physiological health benefits beyond simple nutritional value
Expanded Definition Similar in appearance to conventional foods;
Are consumed as part of a usual diet
How to Be a Functional FoodHow to Be a Functional Food
A natural food product can be engineered to become a functional food by 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 SourcesPhytochemicals and Reported Sources
PolyphenolsPolyphenols
Epicatechin Epigallocatechin Epigallocatechin gallate
Source: Green tea; grapes, red wine
Benefits: Anti-cancer; CHD protective
Function: Inhibit chemical carcinogenesis and tumor formation; inhibit cancer
cell growth; antioxidant; reduces free radical/oxidative damage
IsoflavonesIsoflavones
Genistein Daidzein
Source: Soybean, flaxseed
Benefits: Relieves menopausal symptoms; prevents osteoporosis; anti-cancer; CHD protective
Function: Estrogen-like activity; inhibit growth of breast cancer cells; stimulate Ca absorption; lower cholesterol levels
Genistein
PhytosterolsPhytosterols
-sitosterol Campesterol -sitostanol
Source: Plant oils
Benefits: CHD protective
Function: Inhibit cholesterol absorption
-sitosterol
CarotenoidsCarotenoids
Lycopene -carotene -cryptoxanthin Lutein
Source: Tomatoes, carrots, yams, cantaloupe, spinach, sweet potatoes; citrus fruits
Benefits: Anti-cancer; CHD protective
Function: Antioxidant; free radical scavenger; induction of cell-cell communication and
growth control; inhibit tumor growth
-carotene
n3-Fatty Acidsn3-Fatty Acids
-linoleic acid Docosahexaenoic acid Eicosapentaenoic acid
Source: Flaxseed oil, fish oil
Benefits: CHD protective; anti-cancer; anti-inflammatory
Function: Lower triglycerides; inhibit platelet aggregation; affect eicosanoid
production
DHA
FlavonoidsFlavonoids
Quercetin Apigenin Luteolin Myricetin
Source: Citrus fruits; vegetables
Benefits: Anti-cancer; CHD protective
Function: Antioxidant; inhibit platelet aggregation; inhibit cancer cell growth and
proliferation; cytotoxic to cancer cells
Luteolin
The Promise of Plant TherapeuticsThe Promise of Plant Therapeutics
Better health through improved nutrition can:
Increase quality of lifeEnhance productivityReduce health-care costs - by preventing or delaying the onset of
chronic disease, i.e diabetes
- or improving metabolic factors related to the disease, i.e. glucose
Health claims need to Health claims need to be verified be verified
with carefully controlled studieswith carefully controlled studies
O
Type 2 diabetes
Years from Years from diagnosisdiagnosis
0 5-10 -5 10 15
Pre-diabetes
Onset Diagnosis
Insulin resistanceInsulin resistance
Post-Meal glucose
Fasting glucoseFasting glucose
““Nutraceutical (Botanical/Bioactive)Nutraceutical (Botanical/Bioactive)Strategies”Strategies”
““Pre-Diabetes”Pre-Diabetes”
Type 2 diabetes
Years from Years from diagnosisdiagnosis
0 5-10 -5 10 15
Pre-diabetes
Onset Diagnosis
Insulin secretion
Insulin “inefficiencyInsulin “inefficiency
Ramlo-Halsted BA, Edelman SV. Prim Care. 1999;26:771-789 Nathan DM. N Engl J Med. 2002;347:1342-1349
Post-Meal glucose
Fasting glucoseFasting glucose
Pancreas function
Progressive -Cell Failure
Natural History of Type 2 DiabetesNatural History of Type 2 Diabetes
Re
lati
ve
Ris
k o
f M
I o
r S
tro
ke
0
1
2
3
4
5
6
7
NondiabeticThroughout
2.4
>15 YrBefore Dx
10-14.9 YrBefore Dx
3.64
<10 YrBefore Dx
DiabeticThroughout
5.02
3.19
1.0
Hu FB, et al. Diabetes Care. 2002;25:1129-1134.
Non-Diabetic Diabetes
Cardiovascular Risk in Pre-diabetesCardiovascular Risk in Pre-diabetes
Pre-Diabetes
Type 2 diabetes
Years from Years from diagnosisdiagnosis
0 5-10 -5 10 15
Pre-diabetes
Onset Diagnosis
Insulin resistanceInsulin resistance
Ramlo-Halsted BA, Edelman SV. Prim Care. 1999;26:771-789 Nathan DM. N Engl J Med. 2002;347:1342-1349
Post-Meal glucose
Fasting glucoseFasting glucose
Natural History of Type 2 DiabetesNatural History of Type 2 Diabetes
““Nutraceutical (Botanical/Bioactive)Nutraceutical (Botanical/Bioactive)Strategies”Strategies”
Phytochemicals and Reported SourcesPhytochemicals 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 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
Baseline
Blueberry Group
Placebo (Control)
Screening
Insulin Sensitivity Test
1 2 3 4 5 6 7 8 9
End
End
Visits (Weeks)
*Randomized
*Started consuming smoothies
Hyperinsulinemic Euglycemic Clamp
“Gold Standard” for objectively assessing whole body insulin action
Nutritional Value (per 16oz) Bioactives PlaceboEnergy, kcal 239 234Carbohydrate, g 48.5 48.6 Fiber, g 4.2 4.3Protein, g 11.9 11.1Fat, g 0.08 0.08 Saturated Fat, g 0.05 0.05
Each subject consumed two 16oz smoothies per day
16 oz Smoothie (Bioactives) =
Nutritional Value of InterventionNutritional Value of Intervention
Placebo Smoothie
Stull AJ et al. J Nutr. 2010 Oct;140(10):1764-8.
The Ability of Insulin to Work in All ParticipantsFrom Beginning to End of Study
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Placebo (Control)Placebo (Control)GroupGroup
Bioactives Bioactives GroupGroup
Individual Response in Insulin Individual Response in Insulin SensitivitySensitivity
Technical Variability
Blueberry Bioactives Improved Insulin Sensitivity
∆ insulin Sensitivity = ability of insulin to work from week 0 to week 6
4-Fold4-Fold
Botanicals showing Promise to Botanicals showing Promise to Favorably modulate Carbohydrate Favorably modulate Carbohydrate
Metabolism Metabolism
Botanicals showing Promise to Botanicals showing Promise to Favorably modulate Carbohydrate Favorably modulate Carbohydrate
Metabolism Metabolism
• Bitter Melon ( Momordica charantia)
• Hoodia (Hoodia gordonii) • Artemisia dracunculas and related sp.
A
B
C
Figure 3
D
E
F
Bitter Melon and Glucose tolerance Bitter Melon and Glucose tolerance Bitter Melon and Glucose tolerance Bitter Melon and Glucose tolerance
IRS-2
IRS-1
-actin
Group LFD HFD BM
IR
GLUT4
PI 3K
Akt1
Akt2
Skeletal muscleIRS-1 p (Tyr612)
Akt1 p (ser473)
Insulin - + - + - +Group LFD HFD BM
IRS-1
Akt1
Akt2
Akt2 p (ser474)
Mouse # 1 2 3 4 8 9 7 11 13 15 16 18Insulin - + - + - + - + - + - +Group LFD HFD BM
0.0
0.5
1.0
1.5
2.0
2.5
LFD HFD BM
Basal
Insulin
Fo
ld o
fL
FD
a
t
ba
sa
l
**
**
*
###
Bitter Melon and Glucose tolerance Bitter Melon and Glucose tolerance Bitter Melon and Glucose tolerance Bitter Melon and Glucose tolerance
Figure 4.
A
B
Bitter Melon Modulates Intracellular Bitter Melon Modulates Intracellular lipids lipids
Bitter Melon Modulates Intracellular Bitter Melon Modulates Intracellular lipids lipids
Hoodia gordonii and Asclepias incarnata (Swamp milkweed) Belong to the
Same Plant Family
cellhealthmakeover.com abnativeplants.com
In collaboration with Analyticon
Natural Diversity of PregnanesIsolated from A. incarnata Extract
ikemagenin
An approach to the study of An approach to the study of Phytochemicals and Human Health Phytochemicals and Human Health
An approach to the study of An approach to the study of Phytochemicals and Human Health Phytochemicals and Human Health
Phytochemicals and Carbohydrate Phytochemicals and Carbohydrate Metabolism Metabolism
Phytochemicals and Carbohydrate Phytochemicals and Carbohydrate Metabolism Metabolism
Alcoholic Extract of Artemisia dracunculus L.Russian Tarragon (PMI 5011)
Ribnicky DM, et al. Am J Physiol Endocrinol Metab. 2007
• Agriculture SOPs must be developed for each crop (Germination; Plant nutrition Pest management)
Sourcing and Sourcing and Standardization of Phytochemicals
Sourcing and Sourcing and Standardization of Phytochemicals
Artemisia dracunculusArtemisia dracunculus in in Hydroponics at ARC GreenhousesHydroponics at ARC Greenhouses
• Agriculture SOPs must be developed for each crop (Germination; Plant nutrition Pest management)
• Optimization of activity and composition
Sourcing and Sourcing and Standardization of Phytochemicals
Sourcing and Sourcing and Standardization of Phytochemicals
Phytochemicals and Carbohydrate Phytochemicals and Carbohydrate Metabolism Metabolism
Phytochemicals and Carbohydrate Phytochemicals and Carbohydrate Metabolism Metabolism
Flowering Artemisia dracunculus
LC-MS Comparison of PMI-5011 Extracts Made LC-MS Comparison of PMI-5011 Extracts Made from Plants at Different Stages of Developmentfrom Plants at Different Stages of Development
Flowering Stage
Growth/ Non-flowering Stage
• 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)
Sourcing and Sourcing and Standardization of Phytochemicals
Sourcing and Sourcing and Standardization of Phytochemicals
5011
ADEX
ADEX
ADEX
Bioactivity GuidedFractionization
Identification and Isolation of PhytochemicalsIdentification and Isolation of Phytochemicals
BMI 29.5
0 4 6 8 12 hours Type 2 DM subjects:
BMI 31.1
BMI 36.8
PTP-1B
-actin
PTP-1B
-actin
PTP-1B
-actin
PTP1B Levels and Artermisa sp (5011) Human Skeletal MuscleHuman Skeletal Muscle
PTP1B Levels and Artermisa sp (5011) Human Skeletal MuscleHuman Skeletal Muscle
Time Course Studies: 5011
IRS-1
IRS-2
PTP 1B
IR
PI 3
-Actin
Fraction7 Contr 2 3 4 5 6 7 8 9 10 11 5011D
Akt-p
Insulin Signaling ParametersInsulin Signaling Parameters
Human Skeletal Muscle Culture5011 Fractions
Phosphatase
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.
Isolated Compounds ALR2 PTP-1B PEPCK
4,5-di-O-caffeoylquinic acid * ◊ ○ A - -
Davidigenin * ◊ † ‡ A - -
A - A
2′,4′-dihydroxy- 4- methoxydihydrochalcone * ◊ ○ † ‡ §
A A A
2,4-dihydroxy- 4- methoxydihydrochalcone * ◊ ○ † ‡
- A -
- A -
6-demethoxycapillarisin * ◊ ○
Sakuranetin ▪ ◊ ○
* - confirmed with NMR
◊ - new compound to A . dracunculus
○ - activity reported for the first timeA - active† - dihydrochalcone‡ - new compound to genus Artemisia§ - first report as a constituent of plants
▪ - flavonoids
ALR2- Aldose reductase
PTP-1B - Protein tyrosine phosphatase - 1B
PEPCK - Phosphoenolpyruvate carboxykinase
Bioactives Isolated from Bioactives Isolated from Artemisia dracunculus Artemisia dracunculus and PMI-5011 by Activity-Guided Fractionation and PMI-5011 by Activity-Guided Fractionation Bioactives Isolated from Bioactives Isolated from Artemisia dracunculus Artemisia dracunculus and PMI-5011 by Activity-Guided Fractionation and PMI-5011 by Activity-Guided Fractionation
**2′, 4′–dihydroxy-4-methoxydihydrochalcone
0
50
100
150
200
250
Labrasol 50 mg/kgchalcone
150 mg/kgchalcone
300 mg/kgchalcone
metformin 300mg/kg
blo
od
glu
co
se
(m
g/d
l)
0 hr
6 hr
*
*
***
All treatments provided with 66% LabrasolAll treatments provided with 66% Labrasol
The Pure Active DMC-2* from Artermisia is Validated in vivo with Comparable Activity to Metformin
The Pure Active DMC-2* from Artermisia is Validated in vivo with Comparable Activity to Metformin
• 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 Sourcing and Standardization of Phytochemicals
Sourcing and 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
Sourcing and Sourcing and Standardization of Phytochemicals
Sourcing and Sourcing and Standardization of Phytochemicals
-20ºC freezer
22ºC open container
22ºC desiccator
37ºC open oven
Stability of the ExtractStability of the Extract
PMI-5011 is stable under various
storage conditions
Stability was investigated over
a period of 7 months by a
validated HPLC method
HPLC-Chromatograms of PMI-5011, stored under different conditions
Sourcing and Sourcing and Standardization of Phytochemicals
Sourcing and Sourcing and Standardization of Phytochemicals
Assuring Supply of materials for researchAssuring Supply of materials for research
• in vitro studies
• Toxicology
• in vivo preclinical
• clinical
Animals Animals ReceivedReceived
50115011
PlaceboPlacebo
InterventionIntervention
Animals: Animals: KK-KK-AAyy
InterventionIntervention:: 5011 Total Extract vs control conditions5011 Total Extract vs control conditionsEndpoint:Endpoint: Insulin and Glucose levels, Muscle signalingInsulin and Glucose levels, Muscle signaling
BaselineBaseline
• Insulin Stimulation/Muscle Insulin SignalingInsulin Stimulation/Muscle Insulin Signaling• Serial Insulin/GlucoseSerial Insulin/Glucose
BaselineBaseline
Study Design: In vivo MechanismStudy Design: In vivo MechanismStudy Design: In vivo MechanismStudy Design: In vivo Mechanism
Ribnicky DM, et al. Phytomedicine. 2006 Sep;13(8):550-7.
Body Weight/Food IntakeBody Weight/Food IntakeBody Weight/Food IntakeBody Weight/Food Intake
Plasma insulin
0 2 4 6 80
5
10
15
20
Control
5011
Weeks of Study
Insu
lin (
pg
/ml)
Mean + SEM, * P<0.05, ** P<0.01.
*** *
Plasma InsulinPlasma InsulinPlasma InsulinPlasma Insulin
Akt-pAkt-p
Akt-1Akt-1
Akt-2Akt-2
AS160AS160
Mice # 1 2 3 9 10 11Mice # 1 2 3 9 10 115011 - - - + + +5011 - - - + + +
GlutGlut44
-Actin-Actin
Control 5011Control 5011
Insulin Signaling ParametersInsulin Signaling ParametersSkeletal MuscleSkeletal Muscle
Insulin Signaling ParametersInsulin Signaling ParametersSkeletal MuscleSkeletal Muscle
Bench to BedsideBench to Bedside
Cells tested with Cells tested with BotanicalBotanical
Human Testing in Human Testing in Several PhasesSeveral Phases
Animal Testing to MonitorAnimal Testing to MonitorSafety and EffectivenessSafety and Effectiveness
Screening Screening
Screening Screening
50115011
PlaceboPlacebo
InterventionIntervention
Subjects:Subjects: Obese, Insulin Resistant, n = 28 Obese, Insulin Resistant, n = 28InterventionIntervention:: 5011 Total Extract vs Placebo 5011 Total Extract vs PlaceboEndpoint:Endpoint: Insulin Sensitivity (Clamp Procedure) Insulin Sensitivity (Clamp Procedure)
•Hyperinsulinemic Euglycemic ClampsHyperinsulinemic Euglycemic Clamps•Body CompositionBody Composition
BaselineBaseline
BaselineBaseline
Clinical Trial Study DesignClinical Trial Study DesignClinical Trial Study DesignClinical Trial Study Design
4
5
6
7
8
9
10
11
12
Pre Post Pre Post
5011
4
6
8
10
5011Placebo
Insu
lin S
ensi
tivi
ty (
mg
/min
/FF
M)
Placebo
BaselineBaselineEnd of StudyEnd of Study
*
* P < .05
Clinical ResponseClinical ResponseInsulin SensitivityInsulin Sensitivity
Clinical ResponseClinical ResponseInsulin SensitivityInsulin Sensitivity
Analysis of Compounds from PMI-5011 in
SIM for active compounds in PMI-5011
Standards
6-demethoxycapillarisin & davidigenin
Standards sakuranetin &2′, 4–dihydroxy-4′-methoxydihydrochalcone
SIM plasma analysis
SIM = Selected Ion Monitoring
285257
271285
Phytochemical CharacterizationPhytochemical CharacterizationIn In Plasma by LC-MS-SIM
Phytochemical CharacterizationPhytochemical CharacterizationIn In Plasma by LC-MS-SIM
Time Course of Appearance
0
1
2
3
4
5
6
7
0 45 120 180 240
Minutes after Ingestion
Ab
un
dan
ce (
X 1
07)
Plasma Appearance of Plasma Appearance of PhytochemicalPhytochemical
Plasma Appearance of Plasma Appearance of PhytochemicalPhytochemical
Mean Levels over 4 hours
0
1
2
3
4
5
6
7
5011 Placebo
Ion 257: davidigenin Ion 271 sakuranetin Ion 285: chalcone
Undetectable
Ab
un
dan
ce (
x 10
7)
Plasma AbundancePlasma Abundance5011 Metabolites5011 Metabolites
Plasma AbundancePlasma Abundance5011 Metabolites5011 Metabolites
Potential Nutraceutical (Phytochemical) Markets
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)
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)
Potential Nutraceutical (Phytochemical) Markets
Potential Nutraceutical (Phytochemical) Markets
The most studied Phytochemical:Wine
The most studied Phytochemical:Wine
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 <0.1 0.1 - 0.3 0.3 - 1 1.0 - 2.0 >2.0
Drinks per Day
RelativeRisk
CVD
Non-CVD
Over 40 prospective studies have documented an inverse relationship between alcohol intake and heart disease
prevention
We Can’t Put Our Heads in the Sand any We Can’t Put Our Heads in the Sand any
Longer !!!Longer !!!
Regarding Plant Therapeutics and Human Health…..