plant derived therapeutics for the treatment metabolic syndrome william t. cefalu, m.d. pennington...

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


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


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


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


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


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


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


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


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


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


Artemisia dracunculusArtemisia dracunculus in in Hydroponics at ARC GreenhousesHydroponics at ARC Greenhouses


• Optimization of activity and composition





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



• Identification of the active components for standardization (Bioactivity guided Fractionization)



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



• Identification of the active components for standardization (Bioactivity Guided Fractionization)

• Standardized for Active Components





• Identification of the active components for standardization (Fingerprinting)

• Standardized for Active Components

• Stability Assessed



-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



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


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)



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…..

plant derived therapeutics for the treatment metabolic syndrome william t. cefalu, m.d. pennington...

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