Dott.ssa Emanuela EspositoDott.ssa Emanuela EspositoDipartimento di Scienze Biologiche ed AmbientaliDipartimento di Scienze Biologiche ed Ambientali

UniversitUniversit àà degli Studi di Messinadegli Studi di Messina

Gli alimenti nella prevenzione di malattie croniche e tumorali:

casi studio Bologna, 21 Febbraio

Free radicals attack all your vital cellular structures, such as cell membranes and stimulate processes that

have been linked to accelerated cellular aging. An antioxidant’s job is to neutralize the free radical cells thus protecting the cells in our body from accelerated

aging.

Free Radicals

A free radical is defined as any atom or molecule possessing unpaired electrons

The biologically relevant free radicals derived from oxygen are:

• superoxide anion (O2-.)

•Hydrogen peroxide (H2O2.)

•hydroxyl radical (HO.)

•nitric oxide (NO.).

Production of reactive nitrogen species (RNS)Production of reactive nitrogen species (RNS)

NO���� O2��������+ ONOO����

�

HONOO

Peroxynitrousacid

Hydroxylradical

��

Peroxynitrite

�

H+

NO2••••

O2 O2�������� H2O2

����OH

NO���� ONOO���� HONOOL-ARG

+

iNOS

����OH

�Lipid peroxidation�DNA damage �PARP activation�Nitrotyrosine modification

�Apparent Hydroxyl Radical Production by Peroxynitrite :

Implications for endothelial Injury from Nitric Oxi de and Superoxide. Beckman et al., PNAS USA87:1620-1624, 1990

Frequent targets of ROSFrequent targets of ROS

OO22••••••••--

HH22OO22

HOClHOCl••••••••OOHH

gutgut

heart & heart & vesselsvessels

airwaysairways

brain &brain &nervesnerves

Pathological conditions that may have a free radical component and sites of ROS actions

Atherosclerosis

Smoking,air pollutants

&drug induced

reactions

intracellular extracellularIntracellular &extracellular

Hypo-, hyper-oxygenation

& Reperfusion

after ischemia CataractogenesisImmune

reactions

Parkinsonism

Diabetes

Iron, drug& chemicaltoxicity

Chemical cancerogenesisRadiation injury

Ageing & senile dementia

FREE RADICALS

Cancer

Inflammatoryreactions

ROS are tightly controlled resulting in a physiological balance between their

production and elimination

ROS are tightly controlled resulting in a physiological balance between their

production and elimination

c-cytosolic, m-mitochondrial, p-peroxisomal

ROS:ROS:OO22

•−•−•−•−•−•−•−•−, H, H22OO2 2 , , 11OO2 2 ••••••••OH, HOClOH, HOCl

Enzymes:Enzymes: SOD (SOD (c, mc, m) , ) , GPX (GPX (c, mc, m), CAT (), CAT (c, pc, p))

NonNon--enzyme antioxidants:enzyme antioxidants:vitaminesvitamines (E,A,C), thiols, (E,A,C), thiols, phenols, ceruloplasmin, phenols, ceruloplasmin, transferrin, uric acid,transferrin, uric acid,

albumin, etc.albumin, etc.

ROS:ROS:OO22

•−•−•−•−•−•−•−•−, H, H22OO2 2 , , 11OO2 2 ••••••••OH, HOClOH, HOCl

Enzymes:Enzymes: SOD (SOD (c, mc, m) , ) , GPX (GPX (c, mc, m), CAT (), CAT (c, pc, p))

NonNon--enzyme antioxidants:enzyme antioxidants:vitaminesvitamines (E,A,C), thiols, (E,A,C), thiols, phenols, ceruloplasmin, phenols, ceruloplasmin, transferrin, uric acid,transferrin, uric acid,

albumin, etc.albumin, etc.

ROS:ROS:OO22

•−•−•−•−•−•−•−•−, H, H22OO2 2 , , 11OO2 2 ••••••••OH, HOClOH, HOCl

Enzymes:Enzymes: SOD (SOD (c, mc, m) , ) , GPX (GPX (c, mc, m), CAT (), CAT (c, pc, p))

NonNon--enzyme antioxidants:enzyme antioxidants:vitaminesvitamines (E,A,C), thiols, (E,A,C), thiols, phenols, ceruloplasmin, phenols, ceruloplasmin, transferrin, uric acid,transferrin, uric acid,

albumin, etc.albumin, etc.

Under pathological conditionthe physiological balance is lost

Under pathological conditionthe physiological balance is lost

ROS:ROS:OO22

•−•−•−•−•−•−•−•−, H, H22OO2 2 ,, 11OO22, , ••••••••OH, HOClOH, HOCl

Enzymes:Enzymes: SOD,GPX,CATSOD,GPX,CATNonNon--enzyme antioxidants:enzyme antioxidants:vitaminesvitamines (E,A,C), (E,A,C), thiolsthiols, , uric acid, ceruloplasmin, uric acid, ceruloplasmin,

transferrin, phenols, transferrin, phenols, albumin, etc.albumin, etc.

What are antioxidants?

Antioxidants come in different shapes and sizes: they are nutrients (vitamins and minerals) or enzymes.

These tiny superheroes counteract the negative effects of oxidation in animal tissue.

How do anti-oxidants work?

The free radical tug-of-war for electrons damages cells, proteins and DNA. If Wright's antioxidant vitamins are developed, they may be able to shorten the battle time between the two molecules. He is targeting Vitamin E since this is where he has detected a weakness in the body

RO•, ·O2-

HO· HOO·

H2O2,

ROO•, 1O2,

AntioxidantsRO•, •O2

-

HO•, HOO•,H2O2

ROO• , 1O2

Antioxidants preventor treat

�Cancer

�Diabetes

�Heart disease

�High blood pressure

Prevention of disease shown in populations having a high consumption of fruits and vegetables

(A) An antioxidant-enriched diet maintains healthy arteries that release vasodilators and fibrinolytic compounds. (B) An antioxidant-inadequate diet results in free radical proliferation and LDL oxidati on. Macrophages engulf oxidized LDL, die and form foam cells in the arterial intima. (C) Increased LDL oxidation and foam cell proliferation forms plaque and blocks the formation of fibrinolytic compounds and vasodilators. (D) A clinical event occurs when a 20-30% lumen-obstructing plaque ruptures. The plaque rupture causes immediate release of clot-forming and vasoconstriction compounds. Lumen obstructions lead to myocardial infarction or death. (LDL=low-density lipoprotein)

A B C D

*

Superfoods

Sure, everyone knows that an apple is a better snack than potato chips, but do you know that a

daily handful of walnuts or a bowl of blueberries can actually improve your well-

being and longevity?

Free Radicals

Vegetal pigments

Vitamins

Micronutrients and Enzymes

Polyphenol, bioflavonoid

Vitamin C, Vitamin E, Betacarothen

Polyphenol, bioflavonoid

Selenium, Cu, Zn, Glutathione, Melatonin, Uric Acid

Antioxidants in Diet

H2NNH

NH COONa

COOH

O

O

S

S

COOH O

O

H2N COONaNHNH

Glutathione

FeTMPyP

Mn-Salens

MnTBAP

Tempol

M40401

Melatonin

M40403

Evidence That Antioxidant Flavonoids in Tea and Cocoa Are Beneficial for

Cardiovascular Health

By

Penny M. Kris-Etherton and Carl L. Keen

Current Opinion in Lipidology 2002, 13:41-49

Anthocyanins

Evaluation of antioxidant and antinflammatory activity in vivo and in vitro sperimental model of pulmonary

damage in rat

Antioxidant vitamin C

Food sources:

�Citrus fruit

�Tomatoes

�Peppers

�Broccoli

�Melons

�Strawberries

Antioxidant vitamin E

Food sources:

�Oil (60%)

�Fruits and veggies, esp.dark green leafy (10%)

�Grains

�Nuts and seeds

Antioxidant-beta carotene

Food sources:

�Carrots

�Sweet potatoes

�Oranges

�Cantaloupe

�Tomatoes

�Dark green leafy vegetables

*

To drink moderatly and regularly attenuates stroke risk

Recently on Nature review it was published that wine has beneficial effect on safety. Prof. R.Corder, of London school of

Medicine, showed that polyphenols, bioflavonoid and proantocyanins can have beneficial effect on arteries and veins,

stopping endotelin-1 production.

Polyphenols, are substances so powerfull that a moderatly assumption, can be very efficacy.

Grape Nuts Extract

Used like as:•Antioxidant•Allergy, asthma•Vein fragility, arteriosus disfunction

Mechanism of Action:•Antioxidant•Stabilitazion of 1-antitrypsine•Inibition of inflammation mediator release (Hystamine and PGS)•Inibition of platelet aggregation

Precaution of use:•People with evidence of present or past emorragie, alterations of hemostatic components•Anticoagulator and antiaggregator drugs

General Warming:•Caution with children under 2 years•Caution with pregnant or lactating women

*

Antioxidant restricted diet increases oxidative stress during acute exhaustive exercise.

Watson TA, Callister R, Taylor R, Sibbritt D, MacDonald-Wicks LK, Garg ML.Asia Pac J Clin Nutr. 2003;12 Nutrition and Dietetics, University of Newcastle

Antioxidant on exhaustive exercise

Exercise – Antioxidant manipulation

Diet

- Little research

- Antioxidant deficiency

reduces exercise capacity

• Supplementation

- Mixed findings

- Reduced oxidative stress markers

- No impact on performance

Watson TA et al., Asia Pac J Clin Nutr. 12 2003

Acute disease: Carrageenan-induced

pleurisy (CAR)

Chronic disease: Type II collagen-induced

arthritis (CIA)

The major constituent of the leaves and unprocessed olive drupes

of “Olea europaea” is oleuropein and the majority of polyphenols found in olive oil or table olives are derived from its hydrolysis.

Olive oil is an integral ingredient

of the traditional Mediterranean

diet and several studies attribute

many of the healthy advantages of

this diet to olive oil's unique

characteristics.

• Car + Saline • Car + Ole Aglycone (40µg/Kg)• CAR + Ole Aglycone (100µg/Kg)• Sham + Saline • Sham + Ole Aglycone

Ole aglycone30min 4h

sacrificeCAR

Mice were anaesthetized and subjected to a skin incision at the level of

the left sixth intercostals space. The underlying muscle was dissected

and saline or saline containing 2% λ-carrageenan (0.1 ml) was injected into the pleural cavity.

Experimental design – “CAR” Model

HISTOLOGICAL EVALUATIONHISTOLOGICAL EVALUATION

SHAM CAR CAR+OLE AGLYCONE

012345678

SHAM CAR

His

tolo

gic

al s

core

Vehicleole aglycone (40ug/kg)ole aglycone (100ug/kg)

TNF-αααα, TNF-αααα,

0102030405060708090

Sham CAR

TN

F- αα αα

(ng

/ml) Vehicle

ole aglycone

0

20

40

60

80

100

120

140

Sham CAR

IL-1

ββ ββ(p

g/m

l)

Vehicleole aglycone

IL -1ββββ

NO LEVELS NO LEVELS

0

20

40

60

80

100

120

Sham CAR

Nitr

ite/ N

itrat

e es

suda

te

leve

ls (n

mol

/ml)

Vehicleole aglycone

MPO ACTIVITYMPO ACTIVITY

0

200

400

600

800

1000

1200

Sham CAR

Vehicleole aglycone

NITROTYROSINE EXPRESSIONNITROTYROSINE EXPRESSION

SHAM CAR+OLE AGLYCONECAR

SHAM CAR+OLE AGLYCONECAR

PAR EXPRESSIONPAR EXPRESSION

0

50

100

150

200

250

300

SHAM CAR

Lung

mal

ondi

alde

hyde

act

ivity

( µµ µµ

M/ 1

00 m

g w

et ti

ssue

)

Vehicleole aglycone

ICAM -1 (A), P-SELECTIN (B) EXPRESSIONICAM -1 (A), P-SELECTIN (B) EXPRESSION

A

SHAM

SHAM

CAR

CAR

CAR+OLE AGLYCONE

CAR+OLE AGLYCONE

B

Experimental design – “CIA” Model

• CIA Control • CIA + Ole Aglycone (20µg/Kg)• CIA + Ole Aglycone (40µg/Kg)• Sham Control • Sham + Ole Aglycone

DAYS

21

2°(CII)

1 25

ole aglycone i.p.

daily35

On day 1, CIA was induced in mice by an intradermal injection of 100 μl of

an emulsion containing 100 μg of bovine type II collagen (CII) and

complete Freund's adjuvant (CFA) at the base of the tail.

0

1°(CII)

HISTOLOGICAL EVALUATIONHISTOLOGICAL EVALUATION

0

1

2

3

4

Sham CIA

His

tolo

gic

al d

am

ag

e s

core

VEHICLE

OLE AGLYCONE (40ug/kg)

CIASHAM

CIA+OLE AGLYCONE

0

2

4

6

8

10

12

14

0 21 26 28 32 35

Pa

w S

core

Time (days)

CIA+VEHICLECIA+OLE AGLYCONE (40ug/kg)CIA+OLE AGLYCONE (20ug/kg)

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0 21 26 28 32 35

Foo

t in

cre

ase

(ml)

Time (days)

CIA+VEHICLE

CIA+OLE AGLYCONE (40ug/kg)

CIA+OLE AGLYCONE (20ug/kg)

18

18,5

19

19,5

20

20,5

21

21,5

22

22,5

0 21 26 28 32 35

Bo

dy

We

igh

t (g

r)

Time (days)

CIA+VEHICLE

CIA+OLE AGLYCONE (40 ug/kg)

CIA+OLE AGLYCONE (20 ug/kg)

0

20

40

60

80

100

0 21 26 28 32 35

Art

hriti

c m

ice

(%)

Time (days)

CIA + VEHICLECIA + OLE AGLYCONE (40 ug/kg)CIA + OLE AGLYCONE (20 ug/kg)

** ** * *

**

**

°°°°

°°°°

°°°° °°°°

°°°°°°°°

°°°° °°°°

* *

**

°°°°°°°°

°°°°

°°°°

°°°°

°°°°°°°°

°°°°

*

**

*

°°°°

°°°°

°°°°

°°°°

°°°°

°°°°

°°°°

°°°°

°°°°

°°°° °°°° °°°° °°°°

°°°° °°°° °°°°

COX-2 EXPRESSION COX-2 EXPRESSION

CIA

CIA+OLE AGLYCONE

CIA

CIA+OLE AGLYCONE

iNOS EXPRESSION iNOS EXPRESSION

0

10

20

30

40

50

60

SHAM CIA

TNF-

αα ααle

vels

(pg/

ml)

VehicleOLE AGLYCONE (40ug/kg)OLE AGLYCONE (20ug/kg)

CYTOKINES LEVELS CYTOKINES LEVELS

0

10

20

30

40

50

SHAM CIA

IL-1

ββ ββ(p

g/m

l)

VehicleOLE AGLYCONE(40ug/kg)OLE AGLYCONE (20ug/kg)

°°°°

°°°°

*

*°°°°

°°°°

CIA CIA+OLE AGLYCONE

NITROTYROSINE EXPRESSIONNITROTYROSINE EXPRESSION

CIA CIA+OLE AGLYCONE

PAR EXPRESSIONPAR EXPRESSION

Why almond skins???

The polyphenols localized in almond skins include a variety flavonols, flavanones and simple phenolic acids which have a role in reducing risk factors against chronic inflammatory

diseases and ageing disorders

Chronic disease: Colitis

Experimental design- colitis induction

4 days

DNBS 15’

(observation time)

Start treatment

0

Sacrifice

Histological and immunoistochemical

studies

1. Sham + Vehicle group: saline was administered daily orally.2. Sham + NS powder (30 mg/kg): was administered daily orally. 3. DNBS+Vehicle: administration of DNBS. 4. DNBS + NS powder (30 mg/kg): administered daily starting from 3 h after the administration of DNBS.

3 hr

Effect of NS powder treatment on clinical expression of DNBS-induced colitis-macroscopic damage score and body weight changs

Effect of NS powder treatment on colon injury and histological score

Effect of NS powder treatment on colon evels of cytokine and MPO activity and lipid peroxidation

Effect of NS powder treatment on immunohistochemical localization of TNFαααα, ICAM -1 and P-selectin

Effect of NS powder treatment on immunohistochemical localization of iNOS, pJNK and FAS-L in the colon

Effect of NS powder treatment on iNOS and pJNK

Effect of NS powder treatment on NF-kBp65 and IkB-αααα

Conclusion

The data presented in these studies demonstrate that oleuropein aglycone and natural almond skin reduce the development of acute and chronic inflammation. Moreover, future studies using different models are needed in order to better clarify the possible use of oleuropein aglycone and natural almond skin powder for the treatment of inflammatory diseases in patients.