Assorbimento e biodisponibilità di biofenoli in sistemi biologici

C. Filesi

Centro Nazionale per la Qualità degli Alimenti e per i Rischi Alimentari

Biophenols: FOCUS OF MUCH CURRENT NUTRITIONAL

AND THERAPEUTIC INTEREST

CARDIOPROTECTIONRole for flavonoid-rich dietary components in

reduction in risk of cardiovascular disease• NEUROPROTECTIONAnthocyanin-rich fruit associated with protection

against age-related decline in cognitive functionCHEMOPREVENTION

Absorption and Bioavailability

In vivo studies: humans or animal models

Or

In vitro studies: tissue or cell cultures

The major olive oil phenolic compounds

WHAT’S HAPPENING IN VIVO?

MAJOR METABOLIZING ENZYMES:small intestine / liver / colon

• Hydrolases

• Esterases

• Cytochrome P450s

OTHERS:• Glutathione-S transferases• Quinone reductases

• Glucosidases

• UDP-glucuronosyltransferases

• Catechol-O-methyl transferases

• Sulfotransferases

Hydroxyl groups confer upon the moleculehydrophilicity thus reducing the solubility in lipid

bilayer

Methoxy groups introduce unfavorable steric effectsbut increase the lipophilicity and membrane

partitioning

Absorption and Biotransformation of Dietary Flavonoids In Vivo

Monomericunits

OligomericFlavonoids

Stomach

Small Intestine

jejunum

ileum

Phase I and IImetabolism

Colon

Liver

Phenolic acids

glucuronides

glucuronides Kidney

Urine

O-methylated

SulphatesPortalvein

Furthermetabolism

Renal excretionof glucuronides

Oligomerscleaved cells

SKIN AND BRAIN

Gut microfloraFlavonoid

C. Rice-Evans

•glucuronidation•sulphation

•methylation• oxidation

•cleavage

OHO

OHOH

OHOH

POTENTIAL MOLECULAR SITES OF METABOLIC MODIFICATION

Influence of conjugation and metabolism on structural parameters

governing biological propertiesconsequently their physiologic

behaviour is differentfrom that of the native compounds

STRUCTURAL CHANGES ON ABSORPTION

Reduction potentials of resulting conjugates

Cellular access and partition coefficients

Intracellular/extracellular metabolism and structural modifications

Methods to obtain conjugates:

a) isolation from plants

b) isolation from blood after consumption of phenolic precursors

c) chemical synthesis

d) enzymatic synthesis

e) microbiological transformation

In vivo and in vitro studies on the absorption and bioavailability of biophenols contained in Mediterranean diet components associated

to a decreased risk of cardiovascular diseases.

(Grant 0F18 ISS/NIH)

The study is aimed to evaluate the effects of extra virgin olive oil or extra virgin olive oil extract in dyslipidemicpatients by evaluating some parameters indicative of

oxidative stress and inflammation.

Moreover it will be determined the concentration of the major biophenols and metabolites in plasma and urine

of treated subjects.

The absorption and metabolism will be also evaluated in suitable cellular in vitro models using the same

extract or individual purified components.

Experimental design

24 mildhyperlipidemicpatients (Total Chol <= 270; BMI >26, <30)Without anyother riskfactors, no drugtherapy, ecc.

polyphenols) Ol Supplement

+EVOO

Placebo+

EVOOPlacebo+

EVOOControlleddiet (low

ive oil

Supplement+

Olive oil

Supplement+

EVOO

2 weeks 4weeks 4 weeks 4 weeks

T1 T2 T3 T4

Blood and urine will be collected at the defined time

Plasma

•Total antioxidant capacity (chemiluminescenze)

•Circulating oxidized LDL (ELISA)

•Tocopherols and carotenoids

•Inflammatory cytokines (IL6, IL8, IL1β)

•Cytokine mRNA in PBMC

•Major biophenol

Urine

•Isoprostanes

•Major biophenols and metabolites

In vitro study

The in vitro study will be performed on differentiated Caco-2 cells, considered a suitable model to evaluate intestinal absorption.

Caco-2 cells, derived from a human colon adenocarcinoma, spontaneously undergoes full differentiation with enterocyte-like features both structurally

and functionally, becoming a suitable model to study the effects, absorption and metabolism of nutrient components.

Cell differentiation will be obtained seeding cells in polyester membrane chambers inserted in wells. At confluence, the cells start to differentiate

forming a tight impermeable monolayer, which prevents passive diffusion of components from apical to basolateral compartment allowing us to identify

• the intestinal absorption; • the transport pathways involved in the uptake; • the possible intracellular compartimentalization of biophenols;• the metabolic fate and the principal metabolites produced.

Cellular uptakePreliminary experiments were performed in undifferentiated Caco-2 cells incubated with (

•protocatechuic acid

•hydroxytyrosol

•tyrosol

•oleuropein

HPLC analysis

Phenolic compounds were extracted from cell monolayer by methanol and analyzed by reversed phase HPLC :

•C18 column T 28°C

•Detector UV\VIS DAD set at 280nm.

•Gradient elution: solution A (3% acetic acid in water)

solution B (50% methanol, 50% acetonitrile)

Flow rate 1.0 ml/min

790 ± 17034 ± 6Tyrosol

65 ± 7590 ± 28Oleuropein

9.8 ± 0.784 ± 11Protocatechuic acid

n.d.1.41± 0.4Hydroxytyrosol

ng/mg protein 2h 18h