Assorbimento e biodisponibilità di biofenoli in sistemi...
Transcript of Assorbimento e biodisponibilità di biofenoli in sistemi...
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Assorbimento e biodisponibilità di biofenoli in sistemi biologici
C. Filesi
Centro Nazionale per la Qualità degli Alimenti e per i Rischi Alimentari
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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
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Absorption and Bioavailability
In vivo studies: humans or animal models
Or
In vitro studies: tissue or cell cultures
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The major olive oil phenolic compounds
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WHAT’S HAPPENING IN VIVO?
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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
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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
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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
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•glucuronidation•sulphation
•methylation• oxidation
•cleavage
OHO
OHOH
OHOH
POTENTIAL MOLECULAR SITES OF METABOLIC MODIFICATION
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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
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Reduction potentials of resulting conjugates
Cellular access and partition coefficients
Intracellular/extracellular metabolism and structural modifications
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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
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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)
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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.
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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
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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
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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.
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Cellular uptakePreliminary experiments were performed in undifferentiated Caco-2 cells incubated with (
•protocatechuic acid
•hydroxytyrosol
•tyrosol
•oleuropein
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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
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790 ± 17034 ± 6Tyrosol
65 ± 7590 ± 28Oleuropein
9.8 ± 0.784 ± 11Protocatechuic acid
n.d.1.41± 0.4Hydroxytyrosol
ng/mg protein 2h 18h