Mark Lange, Ph. D.Market America

Director of Quality Control

The Science of Resveratrol

Important StatementThis presentation is on the latest research

in the areas of longevity, cancer, and heart disease.

Claims are not being made to treat disease or health conditions with dietary supplements.

This research is in its infancy; please refrain from drawing conclusions based on preliminary data.

Berry and Fruit Extracts to be Discussed

Resveravine® – whole grape extract containing resveratrol

BioVin® Advanced – grape skin extract containing OPCs, anthocyanins, and resveratrol

Vita Blue® – wild blueberry extract containing anthocyanins

Resveravine®

A natural extract from grape combining the synergistic biological effects of trans-resveratrol and -viniferin

A standardized extract with 20% total trans-resveratrol and -viniferin

The most concentrated source of Resveratrol

Resveratrol is naturally produced by plants as a defense system againstinfection and oxidative stress due to external attacks.

UV

Fungi

Copper

Ozone time

Resveratrol level

Sources of ResveratrolResveratrol is found in

Grapes (only in skin)WineGrape JuicePeanutsBlueberriesBilberriesCranberries

Benefits of Resveratrol Induces apoptosis

(programmed cell death) in unhealthy cells

Promotes normal nuclear factor kappa B and cyclooxygenase-2 activity

Promotes normal activity of the SIRT-1 gene (involved in calorie restriction, fat mobilization, mitochondrial function, longevity)

Promotes cardiovascular health Normal platelet activityNormal vasorelaxationAntioxidant protection

of LDL particles

Possibly Fights Obesity and Diabetes

Apoptosis

Apoptosis is programmed cell death. The lifespan of cells have genetic limitations. The apoptotic process safely disposes of old cells and cell fragments and does not trigger inflammation.

Microscope View of Apoptosis

A. Unfertilized embryonic cell receives a biochemical signal to die.

B. Cell starts to shrink as activated enzymes cut up the cytoskeleton and DNA in the nucleus.

C. Cell prepares itself to be engulfed by macrophages.

D. Cell fragments into blobs.

Apoptosis is a Normal Process

Rapid turnover of white blood cells in the bodyRemodeling of the developing fetus (fingers

and toes)Formation of connections (synapses) between

neurons in the brainMenstruation – sloughing of the uterine lining

Apoptosis in Cancer Cells

Unlike normal cells, cancer cells proliferate rapidly and lose the ability to respond to cell death signals and apoptosis. Resveratrol has been found to inhibit proliferation and induce apoptosis in a number of cancer cell lines.

Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 2004;24(5A):2783-2840.

Importance of Apoptosis

Defective cell cycle regulation may result in the propagation of mutations that contribute to the development of cancer. Resveratrol has been found to induce cell cycle arrest when added to cancer cells grown in culture.

Joe AK, Liu H, Suzui M, Vural ME, Xiao D, Weinstein IB. Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines. Clin Cancer Res. 2002;8(3):893-903.

Resveratrol Research

Resveratrol caused significant cytotoxicity and increased apoptosis in malignant hemorrhagic tumors.

Resveratrol blocks the multi-step process of carcinogenesis at various stages: suppressing tumor initiation, promotion and progression.

Resveratrol shown to delay development and reduce the metastasizing capacity of spontaneous mammary tumors in mice due to apoptosis.

NIH Resveratrol Study

National Institutes of Health is currently sponsoring a Phase I Study of Resveratrol in Patients With Surgically Removable Colorectal Cancer

Increased frequency of apoptosis in Increased frequency of apoptosis in two tumor cell lines induced by two tumor cell lines induced by polyphenolic extractspolyphenolic extracts

0

10

20

30

40

50

60

70

0,1 1 3 10µg/ml

Caco-2 control

resveratrol

resveravine

Poligonumcuspidatumextract

Cel

ls d

eath

rat

e (%

)

0

10

20

30

40

50

60

70

80

0,1 1 3 10µg/ml

U937

Cel

ls d

eath

ra

te (

%)

(Japanese knotweed)

Nuclear factor-kappa B

is a protein involved in cellular responses to stress (free radicals, UV light, and bacterial or viral attack).

NF-κB plays a key role in regulating the immune response to infection.

Incorrect regulation of NF-κB has been linked to cancer, inflammatory and viral infection.

Resveratrol and NF-kB

Resveratrol was shown to be a potent inhibitor of NF-kB by blocking IkB kinase activityCancer Research. 60:3477-3483, 2000.

This study examined the anti-inflammatory effects of resveratrol on human airway epithelial cells, resveratrol inhibited NF-kB Am J Physiol Lung Cell Mol Physiol, 2004 .

Resveratrol has been shown to promote normal NF-kB activity in several studies.Cancer Research. 60:3477-3483, 2000.Polish Journal of Pharmacology. 53(6):557-69, 2001.

Resveratrol Inflammation Research

Cyclooxygenase (COX) – Blocking this enzyme impedes the production of the chemical messengers (prostaglandins) that cause pain and swelling from inflammation.

Suppression of COX-2 expression by blocking the

activation of MAPKs and AP-1 may represent possible molecular mechanisms responsible for previously reported anti-tumor promoting effects of resveratrol on mouse skin carcinogenesis.Biofactors. 21(1-4):33-39, 2004.

Study concluded in addition to other effects on gene expression, resveratrol also directly inhibited the activity of COX-2. J Biol Chem. 273(34):21875-21882, 1998.

Non-Steroidal Anti-Inflammatory Drugs

Commonly prescribed for rheumatoid arthritis and osteoarthritis.

Sept. 30, 2004: Vioxx recalled due to sudden heart attacks, strokes, and blood clots.

Resveratrol for Cardiovascular Support

Healthy Platelet Activity

Study reported a dose-dependent inhibition of platelet aggregation (blood clotting) by resveratrol.Clin Chim Acta. 235:207-219, 1995.

Rodent study demonstrated that resveratrol protects the heart and improves heart functions after diminished blood flow, affecting developed pressure and arterial flow.Journal of Cardiovascular Pharmacology. 35(2):263-268,

2000.

Shown to inhibit platelet aggregation both in vitro and in vivoInternational Journal of Molecular Medicine. 9:77-9, 2002.

Resveratrol Inhibits Platelet Aggregation

Platelet aggregation is one of the first steps in the formation of a blood clot that can occlude a coronary or cerebral artery, resulting in myocardial infarction or stroke. Resveratrol has been found to inhibit platelet aggregation in vitro.

Blood Cells Mol Dis. 2000;26(2):144-150. Clin Chim Acta. 1995;235(2):207-219.

Resveratrol stimulates eNOS

eNOS is an enzyme that catalyzes the formation of nitric oxide (NO) by vascular endothelial cells. NO is needed to maintain arterial relaxation (vasodilation), and impaired NO-dependent vasodilation is associated with increased risk of cardiovascular disease. Resveratrol has been found to stimulate eNOS activity in cultured endothelial cells.

Circulation. 2002;106(13):1652-1658.

0

20

40

60

80

100

ControlControl

E-NOS E-NOS InhibitoInhibito

r (L-r (L-NNA)NNA)

Endothelium Endothelium RemovedRemoved

Endothelium Endothelium IntactIntact

Art

eri

al

Art

eri

al R

ela

xati

on

(%

)R

ela

xati

on

(%

)

1 55

10

10

50 100

Red Wine Extract Concentration Red Wine Extract Concentration (μM)(μM)

A study of mechanisms involved in vasodilation by Resveratrol

Li etal. A Study of Mechanisms Involved in Vasodilation Induced by Resveratrol in Isolated Porcine Coronary Artery. Physiol. Res. 55:365-72, 2006.

1 5 50

100

0

20

40

60

80

100

Cardiovascular Support Antioxidant protection of LDL particles

Resveratrol was shown to protect LDL by scavenging free radicals and chelating copper Fremont L et al. Antioxidant activity of resveratrol and alcohol-free

wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. 64(26):2511-21, 1999.

Belguendouz, L., et al. Resveratrol inhibits metal ion-dependent and independent peroxidation of porcine low-density lipoproteins. Biochem Pharmacol. 53(9):1347-1355, 1997.

Low-density Lipoprotein Particle (LDL)

Resveravine protects LDL from oxidation Resveravine protects LDL from oxidation better than synthetic resveratrol, catechin better than synthetic resveratrol, catechin and vitamin Eand vitamin E

0

100

200

300

400

500Tlag (min)

control

vitamin E

catechin

resveratrol

resveravine

Caloric RestrictionCaloric restriction is reducing caloric intake

without malnutrition.

Proven method for extending life span (30-50%) and postponing age-related functional decline and age-related disease.

Resveratrol Research in ExtendingLife Span

Restricting calorie intake extends the life spans of organisms ranging from yeast cells to lab animals.

Sirtuins enhance cell survival during times of stress, especially lack of food.

Sirtuins are regulators of aging in virtually all living organisms.

Resveratrol activates sirtuin activity without the extreme difficulty of starving oneself!

Sirtuins Help Cells Survive

Sirtuins help rejuvenate old or dying cells by beefing up their DNA repair process and stimulating production of protective antioxidants.

If a cell is at a point of deciding whether to live or die, Sirtuins push toward the survival mode by making the cell fix itself.

Studies Backing Resveratrol’s Activation of Sirtuins

Tests on Nothobranchius furzeri, a short-lived seasonal fishResveratrol extended the maximum life span

by 59% and extended the median lifespan by 56%Study also showed resveratrol supplementation

improved swimming performance and delayed motor and cognitive age-related declineTrends in Pharmacological Sciences 26, 2005. Current Biology, 2006.

Obese mice supplemented with resveratrol lived an average of 15% longer than obese mice not given resveratrolNature, 2006.

Studies Backing Resveratrol’s Activation of Sirtuins

Saccharomyces cerevisiae (yeast), resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. Howitz, K. T., et al. Small molecule activators

of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003.

Drosophila melanogaster, resveratrol identified as being beneficial and shown to extend lifespan under normal laboratory conditions Bauer, J. H., et al. An accelerated assay for

the identification of lifespan-extending interventions in Drosophila melanogaster. Proc Natl Acad Sci U S A. 101:12980-12985, 2004.

Resveratrol is a Superior Antioxidant

Oxidative stress is implicated in numerous disease processes

A lead resveratrol investigator, Dr. Milos Sovak commented, “There is no question that resveratrol is one of the best free-radical scavengers and that it has many effects whose ramifications might affect not only longevity but also general health.”

Resveratrol as an Antioxidant

In vitro studies revealed that red wine extract is a potent antioxidant as evidenced by its ability to scavenge peroxyl radical in vitro.

Sato M et al. Myocardial protection with red wine extract. J Cardiovasc Pharmacol.35(2):263-8, 2000.

Olas et al demonstrated that resveratrol inhibits the production of several different reactive oxygen species (O2

.-, H2O2, singlet oxygen and organic radicals in pig blood platelets

Olas B et al. Resveratrol may reduce oxidative stress induced by platinum compounds in human plasma, blood platelets and lymphocytes. Anticancer Drugs. 16(6):659-65, 2005.

Protection against oxidative stress

Influence of three related polyphenols on the oxidative stress of lymphocytes infected by the

Epstein-Barr virus (EBV)

Per

oxi

dat

ion

rat

e (%

)

Resveratrol Might Fight Obesity

June 16th. Google: “Martin Wabitsch endo08 webcast”

Human fat cells exposed to resveratrol or not exposed (control).

Number of fat cells in control doubled. Number of fat cells exposed to resveratrol decreased by 50%.

Resveratrol Might Fight Obesity

In addition, exposure to resveratrol also reduced the secretion of substances called interleukin 6 and 8, which may be linked to the development of diabetes and clogged arteries, both thought to be obesity-related problems.

Safety of ResveratrolSafety of Resveratrol

Data in humans have revealed that resveratrol is safe to ingest.Aggarwal B et al. Role of resveratrol in

prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 24(5A):2783-840, 2004.

Human trials on Resveravine®

Single oral doses of 1, 2.5 and 5 grams were given to 29 volunteers, and no serious adverse events were reported.

Possible Herb-Drug Interactions

Resveratrol has been found to inhibit human platelet aggregation in vitro. Theoretically, high intakes of resveratrol (e.g., from supplements) could increase the risk of bleeding when taken with anticoagulant drugs, such as warfarin (Coumadin), and antiplatelet drugs, such as clopidogrel (Plavix), dipyridamole (Persantine), non-steroidal anti-inflamatory drugs (NSAIDs), aspirin and others.

Naturally synthesized by the plant, it is involved in the defense system against various fungi such as Botrytis cinerea . It is naturally present in the grapes and wine

Like Resveratrol, -Viniferin has also very interesting biological properties:

• anti-inflammatory • antioxidant effects

As mentioned, Resveravine also provides -Viniferin, which is a dimer of Resveratrol :

BioVinBioVin® Advanced AdvancedBioVin Advanced is a French red wine

extract, offering a full 5% of pure resveratrol

Made exclusively from French red wine grapes, naturally high in resveratrol

Contains red wine polyphenols and trans-resveratrol

BioVin® Advanced vs. Wine

Amount of Resveratrol in 200mg BioVin®

Advanced

Amount required to be equivalent to 200 mg BioVin® Advanced

Red Wine White Wine

10 mg10 glasses

(1500 ml)40 glasses

(6000 ml)

Benefits of BioVin® Advanced

Due to the synergistic blend of OPCs and resveratrol,

BioVin Advanced offers numerous health advantages

Cardiovascular healthImproves blood circulationStrengthens blood vesselsDecreases platelet aggregation

Cellular health (as described earlier for Resveratrol)Induces apoptosis, involvement in SIRT-1 activity, involvement

in nuclear factor-kappa B and cyclooxygenase-2 activityVision healthExcellent antioxidant capabilitiesSkin health

VitaBlue™ Wild Blueberry Extract

Through a unique proprietary process, VitaBlue™ extract has concentrated the phytochemicals of the blueberry into an extract of unmatched purity, anthocyanin content and antioxidant potency.

Only the finest, ripest North American wild

blueberries qualify to be used in this patented extract.

12.5% Anthocyanins, 40% Polyphenolics, 6500 ORAC units, including pterostilbene.

ORAC of Selected Foods 2007 U.S. Department of Agriculture

Cranberry 9584 Blueberry 6552 Plum 6259 Blackberry 5347 Raspberry 4882 Strawberry 3577 Cherry 3365 Apple 3082 Pear 2941

Benefits of Blueberries

Blueberries have been shown to be beneficial in several applications

Provides 30 anthocyanins and pterostilbene Powerful antioxidant Inhibits cellular proliferation and induces

apoptosis (programmed cell death in unhealthy cells)

Promotes cardiovascular health Contains COX-2 inhibitors Promotes healthy vision

Blueberries and Cell Health

The following review summarizes current knowledge on the various molecular evidences of cancer chemoprevention by anthocyanins including 1) antioxidant activity 2) the molecular mechanisms involved in anti-carcinogenesis 3) the molecular mechanisms involved in the induction of apoptosis in tumor cells.

Hou D et al. Potential mechanisms of cancer chemoprevention by anthocyanins. Curr Mol Med. 3(2):149-159, 2003.

In this study, blueberry exhibited potential anti-carcinogenic activity as evaluated by in vitro screening tests.Bomser, J., et al. In vitro anticancer activity of fruit extracts from

Vaccinium species. Planta Medica. 62(3):212-216, 1996.

Hypoxia-Inducible Factor (HIF) – Why Antioxidants May Help to Prevent

Cancer

NORMAL CONDITIONS

OXYGEN

Hypoxia OXYGEN

Nat Rev Cancer. 2003 Oct;3(10):721-32

HIF Helps Cells Survive, But Free Radicals Must Be Present

OXYGENHypoxia HIF

FREE RADICALS"HIF-1 helps an oxygen- starved cell convert sugar to energy without using oxygen and also initiates the construction of new blood vessels to bring in a fresh oxygen supply."

Cancer Cells Consume All The Oxygen Around Them

OXYGENHypoxia

OXYGEN

But HIF Can’t Help the Cancer Cells Survive When Antioxidants Have Neutralized All The

Free Radicals.

OXYGENHypoxia

HIF

FREE RADICALS

Antioxidants

And the Cancer Cells Die….

OXYGENHypoxia

Antioxidants

Resveratrol

Blueberries

Grape Extract

Heart, Antioxidants

Eyes

Longevity, Obesity?, Diabetes?

Apoptosis, Inflammation

Skin

Cells

Conclusions about Berry Extracts and Cell Health

The active chemicals in selected berries have shown promise in maintaining cardiovascular, immune, and visual health, as well as lengthening life span and using fat stores more efficiently in experimental animal models.

The active chemicals are more than potent antioxidants – they interact with key enzymes and signaling pathways in cells.

Research is ongoing. More to come….