Polifenoli del vino e prevenzione di patologie cronico-degenerative

P. Morazzoni, W. Cabri Indena R&D

Corporate Twitter: @IndenaSpA

New England Journal of Medicine 366, 2333 (2012)

TOP 10 CAUSES OF DEATH (1900 vs 2010)

The role of diet, even at epigenetic level, in contributing to

modulate chronic-degenerative pathologies has been

established in a number of epidemiological studies.

In this contest, the role of secondary (and even primary)

metabolites contained in edible plants seems to be pivotal

in human homeostasis and in the modulation of the risk of

pathological conditions including cardiovascular diseases

and cancer.

ROLE OF DIET AND EDIBLE PLANTS

Epigenetic regulations consist of potentially reversible

changes in DNA methylation, histone modifications,

alteration in microRNA (miRNA) expression, without any

change in DNA sequence.

Several phytonutrients are demonstrated to modulate these

epigenetic regulators inducing heritable changes in gene

expression (i.e. those codifying for pivotal inflammation-

regulating factors, such as NF-kB …..).

S. Reuter et al., Genes Nutr. 6, 93 (2011)

EPIGENETIC REGULATIONS AND ROLE OF

DIETARY PHYTONUTRIENTS

R&D Indena focus is to identify phytonutrients provided

with a solid scientific background and to develop these

ingredients by optimizing their biological benefits for

improving quality of life.

INDENA MISSION IN CONSUMER HEALTH

IS THERE A REASONABLE BASIS

IN ORDER TO INTEGRATE PHYTONUTRIENTS

(AS SUPPLEMENTS OR FUNCTIONALIZED FOOD)

IN THE MODERN DIET?

EDIBLE PLANTS ARE TODAY USUALLY CHARACTERIZED BY A LOWER CONTENT OF PHYTONUTRIENTS

Agricultural selection has reduced the contents of phytochemicals in edible plants which have been selected for factors like appearance, succulence and yields (by means of pesticides).

INTENSIVE AGRICULTURE IS A WORLDWIDE NEED

Productivity

Hectares dedicated to agriculture

Journal of the American College of Nutrition 2004, 23, 669

25% less proteins

MODERN DIET IS MARKEDLY REDUCED IN DIVERSITY

•Only 1% of the total 300,000 higher plants has been historically utilized for food. Roughly 150 species are commercially cultivated.

•Caloric intake is derived mostly from 4 plant species (wheat, rice, soy, corn). These plants represent the basis upon which the world’s population is fed.

The concept of

optimized nutrition

arises from the

convergence of

information from

basic biomedical

science with

epidemiological data.

HIGH QUALITY INGREDIENTS

FULFILLING OF HEALTH CLAIM CRITERIA

COMPATIBILITY WITH FOOD PREPARATION PROCESSES

THE PROCESS OF

OPTIMIZING NUTRITION

CAN BE ENVISAGED ALSO AS A

CONSTANT SUPPLY OF SELECTED PHYTONUTRIENTS

(MOSTLY FROM EDIBLE PLANTS)

CHARACTERIZED BY WELL ESTABLISHED BIOLOGICAL PROPERTIES

DIETARY SUPPLEMENTS FUNCTIONALIZED FOOD

INDENA STRATEGIC APPROACHES FOR THE DEVELOPMENT OF EDIBLE PLANT-DERIVED PRODUCTS

•Rediscovery of plants from Eastern and Western area

Grape, artichoke, lupin….

•Targeted combinations

Combination for appetite control, cholesterol lowering formulation….

•Optimization of extract “characteristics” (bioavailability, standardization, new formulation)

Phaseolus vulgaris, curcumin….

INDENA MAJOR AREA IN HEALTH NUTRITION

CARDIOVASCULAR HEALTH

Metabolic disorders

GASTROENTEROLOGY

Functional dyspepsia

IBS/IBD

MENTAL HEALTH

Cognitive decline

EXAMPLES OF INDENA EDIBLE PLANTS DERIVED STANDARDIZED EXTRACTS

LEUCOSELECT® Vitis vinifera L.

OLEASELECT®

Olea europaea L.

GREENSELECT® Camellia sinensis (L.) O. Kuntze

MIRTOSELECT® Vaccinium myrtillus L.

MADEGLUCYL® Syzygium cumini (L.) Skeels

BEANBLOCK®

Phaseolus vulgaris L.

PYCRINIL®

Cynara cardunculus L. subsp. Scolymus

PRO-GAMMA™ Lupinus albus L.

MERIVA® Curcuma longa L.

SOYSELECT®

Glycine max L.

ENOVITA® Vitis vinifera L.

orientalis

(from Jordan valley)

occidentalis

(from Nile valley and Western Europe)

pontica

(intermediate area)

sylvestris (Gmelin) Hegi

(South-West Asia, South-Central Europe)

sativa Hegi

(Many of these cultivated species are derived from the sylvestris subspecies)

Vitis vinifera L. (Vitaceae family)

Caspian Sea region (Asia minor)

EAST Turkey, Persia, India, China

(128 B.C.)

WEST North Africa, Egypt

(23th c. B.C.)

Greece (18th-16th c. B.C.)

Italy (9th-7th c. B.C.)

Gaul (6th c. B.C.)

Vitis vinifera L. CULTIVATIONS: THE HISTORICAL TRACK – OLD PERIOD

Mexico

USA

South America

(East Coast FAILED)

(West Coast SUCCESS)

Vitis vinifera L. CULTIVATIONS: THE HISTORICAL TRACK – NEW WORLD

South Africa

(16th c. A.D.)

Australia

New Zealand

Egypt (23th c. B.C.)

Wall paintings from the Kha'emwese's tomb of ancient Thebae: various steps of grape harvest.

Vitis vinifera L. CULTIVATIONS: THE HISTORICAL TRACK

Vitis vinifera L. THROUGH THE CENTURIES

Fruits and leaves (and their derivatives) have been utilized for

both edible and medicinal purposes by basically most of the

populations involved in the cultivation of grapevine.

Ancient Egyptians, the Hippocratics (5th-4th c. B.C.),

Theofrastus (4th c. B.C.), Dioscorides, Pliny (1st c. A.D.), Galen

(2nd c. A.D.) described the medicinal properties of grapes.

FRUITS

LEAVES

MAIN MEDICINAL PROPERTIES AND THERAPEUTICAL USES OF Vitis vinifera L. DERIVATIVES

Vitis vinifera L. SEEDS ARE THE PART OF THE PLANT WITH THE HIGHEST CONCENTRATION OF POLYPHENOLS

OLIGOMERIC PROANTHOCYANIDINS: ONE OF THE MOST CHARACTERIZING GROUPS OF POLYPHENOLS IN Vitis vinifera L.

Sharmila Shankar, Brahma N. Singh, and Rakesh K. Srivastava, in: Nutrition, Diet and Cancer, Chapter 10, Plant Polyphenols and Their Role in Cancer Prevention and Chemotherapy, p 209, S. Shankar and R.K. Srivastava (Eds.), Springer, 2012

Modified from:

OPC: THE PHARMACEUTICAL APPROACH…

The pharmaceutical utilization of OPC

(starting from the 1980s) has been mostly concerned

with a proprietary product (Endotelon) which contains

as active principle a highly standardized extract from

grape seeds.

… AND MODERN INSIGHTS INTO OPC HEALTH BENEFITS

•DIETARY SUPPLEMENTS (Mostly in the area of CVDs and stimulated by the

“French Paradox”* induced cascade of literature)

PREVENTION/RISK REDUCTION

•PHARMACEUTICAL PRODUCTS (Mostly concerned with vascular protection)

THERAPY

*S. Renaud and M. de Lorgeril, Lancet 339, 1523 (1992);

M. de Lorgeril et al., Cardiovas. Res. 54, 503 (2002).

THE RELEVANCE OF OPC AND RESVERATROL IN THE EPIDEMIOLOGICAL SCENARIO From “French Paradox” to “red wine paradox”?

Resveratrol in wine: 5 mg/L

One glass of 250 mL/day = 1.25 mg = 18 µg/kg

Oligomeric procyanidins in wine: 600 mg/L

One glass of 250 mL/day = 150 mg = 2.14 mg/kg

for a man of 70 kg b.w.

A. Giacosa et al., Eur. J. Cancer Prev. 22, 90 (2013)

OPC: AN unicum AMONG POLYPHENOLS

• HIGH TROPISM FOR THE VASCULAR TISSUE

n 0 - 5

O

OH

OH

HO

OH

3

4

R

O

OH

OH

HO

OH

3

4

R

O

OH

OH

HO

OH

3

R

8

8

OLIGOMERIC PROCYANIDINS

• CAPACITY TO MODULATE ENDOTHELIAL

FUNCTIONAL RESPONSE

HIGH TROPISM FOR THE VASCULAR TISSUE

TISSUE DISTRIBUTION OF RADIOACTIVITY AFTER ORAL ADMINISTRATION OF 14C-LABELLED OPC IN RODENTS

Total blood

Muscle

Heart

Plasma

Lung

Adrenals

Spleen

Xiphoid cartilage

Skin

Kidney ( cortex )

Trachea

Liver

Duodenum

Aorta

6.310

6.885

7.459

8.677

10.424

11.370

12.822

17.504

17.864

22.534

24.427

34.056

42.579

49.205

±

±

±

±

±

±

±

±

±

±

±

±

±

±

1.671

1.301

1.588

2.203

2.220

3.217

3.141

4.113

4.216

6.399

5.740

9.024

10.346

11.513

Tissue dpm / g % radioactivity

( vs. blood )

1

1.09

1.18

1.37

1.65

1.80

2.03

2.77

2.83

3.57

3.87

5.40

6.75

7.80

J. Laparra et al.: Plantes médicinales et phytothérapie 11, 133 (1977)

OPC: DEMONSTRATED BASIC MECHANISMS REGULATING FUNCTIONAL ASPECTS IN MAINTAINING CARDIOVASCULAR HEALTH

• ACTIVATION AND UP-REGULATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE

• STIMULATION OF AN ENDOTHELIUM DEPENDENT RELAXATION OF BLOOD VESSELS

- M.B. Cishek et al., Clin. Sci. 97, 507 (1997)

- I. Edirisinghe et al., Clin. Sci. 114, 331 (2008)

- M. Ndiaye et al., Br. J. Pharmacol. 142, 1131 (2004); The FASEB Journal 19, 455 (2005)

O

OH

OH

HO

OH

3

4

R

O

OH

OH

HO

OH

3

4

R

O

OH

OH

HO

OH

3

R

8

8

OLIGOMERIC PROCYANIDINS

- M. Ndiaye et al., Br. J. Pharmacol. 142, 1131 (2004); The FASEB Journal 19, 455 (2005)

Schematic diagram showing the OPC-induced endothelium-dependent NO-mediated relaxation in the isolated porcine coronary artery

OPC: THE INDENA EXPERIENCE

IN ORDER TO CAPITALIZE

THIS IMPORTANT BACKGROUND WE STARTED YEARS AGO

WITH THE CHALLENGING TARGET TO

STANDARDIZED GRAPE SEEDS EXTRACTS

HIGHLY STANDARDIZED GRAPE SEEDS EXTRACTS: GENERAL MANUFACTURING PROCESS

ANALYSIS

OF THE

FINAL PRODUCT

ANALYSIS OF THE

FINAL PRODUCT

EXTRACTION PURIFICATION BY

SELECTIVE COUNTEREXTRACTION

CONCENTRATION

TO

AQUEOUS EXTRACT

FILTRATION

ATOMIZATION

MIXING / SIEVING

CONCENTRATION

TO

SOFT EXTRACT

DISSOLVE

IN WATER

GRAPE SEEDS

1) Extract GRAPE SEEDS, at room temperature, ....until exhaustion.

2) Concentrate the percolates in vacuum, under ....mild and controlled temperature conditions, ....until elimination of the extraction solvent.

3) Filter the aqueous concentrate obtained, to ....eliminate any precipitated material.

4) Purify the filtered solution by selective ....counterextraction.

5) Concentrate the purified product in vacuum, ....under mild and controlled temperature ....conditions, to yield a soft extract.

6) Dissolve the soft extract in water.

7) Atomize the aqueous solution obtained.

8) Mix the dried product and sieve it.

9) Send the final product for quality control analysis (HPLC-UV, HPLC-GPC) and packaging.

GRAPE SEEDS EXTRACTS PRODUCED BY INDENA

All these products are standardized dry extracts from grape seeds, and contain the purified fraction of characteristic grapes polyphenols. The products are manufactured in the same authorized industrial plant, by means of food grade solvents according to EU and US specifications. ENOVITA complies also with Japanese specifications, being produced with water and ethanol only.

ENOVITA

LEUCOSELECT

GRAPE SEED MONOMERS

OPC: THE INDENA EXPERIENCE

HIGHLY STANDARDIZED GRAPE SEEDS EXTRACTS

ALLOW

PRE-CLINICAL

AND

CONTROLLED CLINICAL INVESTIGATIONS

Comparative analytical data (according to USP)

WATER

(%)

MONOMERS

(%)

OPC

(%)

INSOLUBLE

RESIDUAL

(%)

ENOVITA 5.3 8.6 91.4 0.03 99

GRAPE SEED MONOMERS 3.3 21.4 82.3 0.50 103

OPC

MONOMERS

(%)

+

PRODUCT

GRAPE SEEDS EXTRACTS PRODUCED BY INDENA

OPC rich grape seeds extract is made exclusively with grape seeds from white wine production. Using only water as extraction solvent, ENOVITA is a food grade grape seed extract whose development has capitalized on Indena's 40 years experience in grape seed extract production. ENOVITA is produced according to Indena 30 Quality system and under HACCP conditions in a GMPs and ISO 14001 certified facility, ensuring full traceability from grape harvest to the finished product. Furthermore, ENOVITA is Halal and Kosher certified and its environmental friendly production process is designed to minimize the production of waste.

Stratification of total CV risk in categories of low, moderate, high and very high risk according to SBP and DBP and prevalence of RFs, asymptomatic OD, diabetes, CKD stage or symptomatic CVD.

NUMBER OF SUBJECTS: 119 TREATMENT GROUPS:

- Control (28 males + 19 females = 47) - ENOVITA 150 mg/day (17 males + 18 females = 35) - ENOVITA 300 mg/day (23 males + 14 females = 37) PERIOD OF TREATMENT: 16 weeks EVALUATION OF THE EFFECTS:

Blood pressure and blood flow at microcirculation level (foot skin) by Laser Doppler flowmetry; heart rate and plasma free radicals.

Mild hypertension: pre hypertension (120-139 / 80-89 mmHg) and Stage 1 hypertension (140-159 / 90-99 mmHg). All patients were undergoing Best Standard Management: weight control, exclusion of NaCl, caffeine, soft drinks and drinks containing caffeine; physical exercise 20 minutes/2 times/day.

ENOVITA

Registry study in mild hypertensive subjects (undergoing Best Standard Management)

Evidence-Based Complementary and Alternative Medicine, Volume 2013, Article ID 313142, http://dx.doi.org/10.1155/2013/313142

ENOVITA: effects on blood pressure and peripheral blood flow in mild hypertensive subjects

Evidence-Based Complementary and Alternative Medicine, Volume 2013, Article ID 313142, http://dx.doi.org/10.1155/2013/313142

ENOVITA: effects on heart rate and plasma free radicals in mild hypertensive subjects

Evidence-Based Complementary and Alternative Medicine, Volume 2013, Article ID 313142, http://dx.doi.org/10.1155/2013/313142

CONCLUSIONS

In healthy subjects bordering hypertension, the combination of the Best Standard

Management with ENOVITA (150-300 mg/day for at least 4 weeks) can

positively modulate blood pressure and blood flow at the level of

microcirculation.

This effect is paralleled by a significant reduction in heart rate.

A relevant reduction of plasma free radicals has also been observed.

ENOVITA has been well tolerated with a global positive compliance.

ENOVITA: registry study in mild hypertensive subjects (undergoing Best Standard Management)

ENOVITA OVERALL CONCLUSIONS

• OPC are the most characterizing polyphenols of Vitis vinifera L.

• Standardization of grape seeds extracts containing OPC is still a challenging task

• Highly standardized grape seeds extracts containing OPC are:

– clinically documented as venotropic agents – strongly effective in modulating endothelial functionality – promising co-adjuvant agents in reducing intermediate – risk factors (i.e. ENOVITA on blood pressure) in CVDs

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