Micronutrients of!Citrus fruits :composition, bioavailability and

!metabolism benefits.

Marie Josèphe Amiot-CarlinUMR INRA 1260/ Université de la Méditerranée

Campus Santé la TimoneMarseille, France

Citrus and Health" meeting, Valencia,

2010 March 8-10

Low energyLow energy

Minerals

Organicacids

Antioxidants

Vitamins

Phytosterols,carotenoids,

polyphenols,

sulfur compounds…

Sugars

terpenes

Fibres

Fruit and vegetables : a complex assemblywith low energy and a large diversity of

phytochemicals

Composition in micronutrients and changes

Bioavailability of micronutrients?

Bioactivities and metabolic consequences

Further research

Micronutrients : substances that are essential in tinyamounts for the proper growth and metabolism of a livingorganism. They must be obtained in general from outsidesources.

!Deficiency diseases

!Increased needs for specific populations

Vitamins, Minerals and Trace elements

Recommended dietary allowances

Citrus fruits are rich in Vitamin C, in calcium and in fibers

Vitamins (mg)

Vitamine C (ascorbic acid) 53

Provitamin A (carotene) 0.12

Vitamin B1 (thiamin) 0.09

Vitamin B2 (riboflavin) 0.04

Vitamin B3 ou PP (nicotinamide) 0.28

Vitamin B5 (pantothenic acid ) 0.30

Vitamin B6 (pyridoxin) 0.06

Vitamin B9 (folic acid) 0.03

Vitamin E (tocopherols) 0.24

Components (g)Carbohydrates 9Proteins 1Lipids 0.2Water 86Fibers 1.8

Minerals (mg)P 16Ca 40Mg 10

Graphic build on the data food questionnaires of a representative French sub-population of536 men & 665 women - 18 à 75 year-old (INCA study -Afssa- coordinator: J.L Volatier)

Increase of fruit and vegetables intake contributesto reach RDA (fibres and micronutrients)

!"cryptoxanthinHO

Retinolor retinol ester for OH =FFA

O

O

OHOH

CH2OH

HOCH

Vitamin C

Vitamins A and C

CH3(CH2)12COO

!"cryptoxanthin esters

20-25 %Lemon (Meyer)

93-96 %Mandarin (Hansen)

82-88 %Orange (Shamouti)

BCX-Esters

Place of Citrus in food profiling

Nicole

Darmon

Some micronutrients are non-essentialUsually called phytochemicalsFlavonoids, Carotenoids, Terpenoids…

No recommended dietary allowances

!None deficiency

!Low biological effect

!Chronic age-related conditions

Changes in micronutrient level from plant to human?Changes in micronutrient level from plant to human?

Genetic selection /production practicesF

ood m

atri

x /

other

mea

l co

mpo

nent

s

Processing/culinary practices

Individ

ual characteristics

Plant content

Bioavailable content

IngestionIngestion

AbsorptionAbsorption

Factors influencing micronutrient contents in plants

*****Maturitystage

**(increase when less N)

**(increase when less N)Fertilization

*****Light

***total polyphenols9-337 mg/100 gHesperetin9-41 mg/100 g

***40-80 mg/100 g

***B-cryptoxanthinorange116-168 mg/100 gMandarin560 mg/100 g

Variety

FlavonoidsVitamin CCarotenoids

Amiot-Carlin MJ 2003 Quality of plant-derived products. Forum Nutr 56:318-321

Data axis 1 and axis 2 (97% )

Salustiana

HamlinMaltaise

Shamouti

Sanguinelli

Valencia

Pera

Mandarin

Clementine

-2

-1,5

-1

-0,5

0

0,5

1

1,5

2

2,5

-6 -4 -2 0 2 4

-- axis 1 (75% ) -->

-- a

xis

2 (

21

% )

-->

Correlation : axis 1 and axis 2

(97%)

Zeaxanthin

Lutein

Violaxanthin

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

0,4

0,6

0,8

1

-1 -0,5 0 0,5 1

-- axis 1 (75% ) -->

ax

is2

(2

1%

) -

-> !-carotene

!-cryptoxanthin

C. DHUIQUE-MAYER,!C. CARIS-VEYRAT, P. OLLITRAULT, F. CURK & M. J. AMIOT

Journal of Agricultural and Food Chemistry, 2005

Varietal caracterisation of Citrus fruits in relationwith antioxidant micronutriment content

Cirad/Inra

1000 retinol equivalent (RE)/L

750 mg/L hesperidin

Vitamin C

FlavonoidsExcept

anthocyanins

Carotenoids

AlkaliAcidsOxidativeagents

LightT°Vitamins

Very sensitive sensitive

Factors affecting micronutrients during processing

Hypothetic changes of micronutrients

Cultivation

Storage

Processing

Packaging

Storage

ConsumerProcessing

Consumption

Composition in micronutrients and changes

Bioavailability of micronutrients?

Bioactivities and metabolic consequences

Further research

Food matrix

Release of Nutrients and Micronutrients

Proteins /carbohydrates /lipids/carotenoids/vitamins…

Lipophilic Micronutrients (micelles)

Intestine

Bioaccessibility

Absorption

Antioxidant micronutrients along the digestive tract

intestine colon

Vitamin C >>CarotenoidsPolyphenols

Vitamin C +++Carotenoids +Polyphenols +/-

Vitamin C low contentCarotenoids andpolyphenols >>

MetabolizationClivage ofprovitaminic Acarotenoids

Conjugaison ofphenolics

MetabolizationDegradation bymicroflora

Scavengers ofScavengers of

RNS and ROSRNS and ROS

OH°OH°

RRO°O° RROO22°°

HH22OO22

ScavengersScavengers

Inhibition ofInhibition ofLOX, COX2LOX, COX2

stomach

Halliwell et al, 2005, Am J Clin Nutr

Absorption

Our recent works support a protective and synergistic effect ofthe main antioxidants present in our dietat the pre-absorptive level

• Design : 16 experiences withVitamin C (100 mg), Vitamin E (12 mg),

Carotenoids (5 mg) (beta carotene, lycopene and lutein) Polyphenols (1g) (gallic acid, caffeic acid, flavanone and flavane)

• Physiological conditions

• Daily intake/micronutrient

What is the behaviour of micronutrients in upper digestivetract?

0

0,1

0,2

0,3

0,4

0,5Control beta -carotene

alone

with vitamin C

with polyphenols

with vitamin E

with vitamins C, Eand polyphenols

In general, stability of micronutrients is greatly improved whencombined

Results on carotenoids : beta-carotene is less stable with vitamin C andmore stable in stomach and duodenum when combined with phenoliccompounds

Inter-dependence of oxidative reactions

PP

PP•

ROO•

ROOH

!Car

!Car•

TO• AscO–

AscO–•TOHTOH

Synergistic effects of diverse micronutrients presentin fruits and vegetables

(bioaccessibility)

Two steps and two models to assess bioavailability

Cellular model:

Acellular model: In vitro digestion

Caco-2 (TC7 clone)

(intestinal uptake)

Enterocyte

Gastric step digestion

Ph 4, pepsin, 37°C, 30 min

Intestinal step digestion

Ph 6, Pancreatin-bile extract, 37°C, 30 min

Centrifugation-filtration (0.2"M)-dilution

Carotenoid-rich Micelles (2h30-incubation)

Basolateral medium

Apical medium

Differentiated Caco-2 cell monolayer

In vitro digestion/caco-2 cell culture model

· Mean of three determinations mg/200ml± SD; *significant difference (p < 0.05)

Free and esterified b-cryptoxanthin in native and digestive juices

!-cryptoxanthin in native juice

(mg/200ml)ｷ

!-cryptoxanthin in digested juice

Free Bcx BcxL BcxM Free Bcx BcxL BcxM

Orange 0.16ｱ 0.0 2

(18 %)

0.25 ｱ 0. 03

(29 %)

0.47 ｱ 0.13

(53 % )

0.23 ｱ 0.02

(62 %)*

0.04 ｱ 0.004

(11 %)

0.1 ｱ 0.0 08

(27 %)

Mandarin 0.17 ｱ 0.06

(5 %)

1.28 ｱ 0.28

(39 %)

1.83 ｱ 0.32

(56 % )

0.32 ｱ 0.03

(20 % )*

0.50 ｱ 16

(31 %)

0.78 ｱ 0.28

(49 %)

Meyer

lemon

0.18ｱ 0. 03

(75 %)

0.03 ｱ 0.008

(11 %)

0.03 ｱ 0.0 06

(14 % )

0.19 ｱ 0.06

(86 %)*

0.01 ｱ .00 2

(5 %)

0.02 ｱ 0.003

(9 %)

Digestion liberates free form of beta-cryptoxanthin

Bioaccessibility of total b-cryptoxanthin (A) and b-carotene (B)for different Citrus juices

B

0

10

20

30

40

50

60

70

80

90

100

Mandarin Orange Meyer lemon

Bio

accessib

ilit

y

of !

-caro

ten

e (

%)

Mandarin Orange Meyer lemon

0

10

20

30

40

50

60

70

80

90

100

Bio

accessib

ilit

y

of !"

cry

pto

xan

thin

(%

)

A

Micellarization is an important factor for the bioavailability of carotenoids

Beta-cryptoxanthin is better transferred into micelles than beta-carotene ,than esters of beta-cryptoxanthin

Caco-2 (clone TC7) cell culture

Confluence after 7 days

21 days on transwells

Insert

Membrane 1 "m pore size Differentiated cell monolayers

Culturemedium

0

2

4

6

8

10

12

BC BCX BCXL BCXM

Caro

ten

oid

s u

pta

ke

eff

icie

ncy (

%)

0

10

20

30

40

50

60

70

80

90

pm

ol o

f caro

ten

oid

s / m

g c

ell

pro

tein

Uptake of carotenoids by Caco-2 cells

9.5

5.3

0.50.6

Beta-carotene is more absorbed than beta-cryptoxanthin,which is, as free form, more absorbed than the esters

Hesperidin and naringenin are poorly absorbed as the majority of phenoliccompounds and highly metabolized

(Manach et al., 2005, Human Nutrition Unit in Clermont_Ferrand, France )

Que

rcet

in

Hes

peri

din

Nar

ingi

n

Epic

atec

hin

Dai

dzei

n

Gen

iste

in

Caff

eic

acid

Feru

lic a

cid

EGCg

Proa

ntho

cyan

ins

Ant

hocy

anin

s

Chlo

roge

nic

Cate

chin

EGC

Gal

lic a

cid

0

10

20

30

40

50

60

(3) (3) (9)(9) (3) (2)

(3)(4) (3) (8)

(1) (3) (13)

(13)(3)

Single dose of 50 mg aglycone equivalentsUrinary recovery (% ingested dose)

(X) : number of studies

Polyphenol structures

Bioactivity of Citrus flavonones is different than directantioxidant mechanisms after absorption

Metabolites of hesperetin and naringenin

Composition in micronutrients and changes

Bioavailability of micronutrients?

Bioactivities and metabolic consequences

Further research

Plasmatic concentrations of antioxidant micronutrients

73 à 2310,001 à 4 (according tostructures)

Hesperetin (1)

Flavonoids

426 (F)

2

!-cryptoxanthin (0,27)

Carotenoids

3317#-tocopherol

8260Vitamin C

Coefficient ofvariation %

Concentrations

micromol/L

Antioxidants

+ Endogenous antioxidants (uric acid, glutathione, albumin and bilirubin)

Micronutrients, especially non-essential micronutrients, probablyact as signalling molecules

Metabolic consequences

Sugiura et al, 2004Increase of beta-cryptoxanthin

Associated toHLD-cholesterol

non-smoking female+ Mandarinfruit

Gorinstein et al, 2006Diminution of LDL-cholesterol

and TG

with atheroslerosis+ Grapefruit

Obeses

withhypercholesteromia

Sujects

Weignt loss

Improvement ofInsulin sensitivity

Diminution of TG

Endpoint

Fujioka et al, 2008+ Grapefruit

Vinson & Jang, 2001+ Citrus juice

But notvitamin C alone

AuthorsIntake

Composition in micronutrients and changes

Bioavailability of micronutrients?

Bioactivities and metabolic consequences

Further research

NUTRI-OMIC

plasma, urines,

tissues

Data acquisition

RMN

LC/MS

GC/MS

« Data mining »:

Metabolites

Metabolic pathway

Statistics

-10

-5

0

5

10

-20 -10 0 10 20

t[2]

t[1]

OSCpl-la sans bruit.M5 (PLS-DA)

t[Comp. 1]/t[Comp. 2]

Colored according to classes in M5

R2X[1] = 0,359727 R2X[2] = 0,0860919 Ellipse: Hotelling T2 (0,95)

Class 1

Class 2

Class 3

Class 4

Class 5

Class 6

L11

L12

L13

L14

L15

L16 L17L18

L21

L23L25 L26

L28

L31

L32

L33

L34L35L36

L37

L38

L41

L42

L43L44

L45L46

L47

L48

L51

L52

L53 L54

L55

L56 L57

L58

L61L62

L63

L64

L65L66

L67

L68

AbsorptionMetabolismExcretion

Manach et al., 2009, Mol Nutr. Food Res.

N

N

O

O

O O

OH OH

OMeMeO

OOH

OH

OH

OH

OH

+

S

O

N

S

O S O

O

O

O

OHOH

OH

OH

OH

OMe

COOH

OH

COOH

OH

OH

OHOH

CO

OH

OH

OH

OH

OHOH

CO

O

OO

O

OHOH

OH

OH

OH

OH

CO

CO

O

O

O

OH

OH

OC

OH

OH

OHOH

OH

OH

OCCO

O

O

O

O

OH

OH

OH

OH

OH

OH

CO

OC

O O

CO

4

OO

OHOHOH

OH

O

OH

OHOH

O

OH

OH

OH

NH

CH3H

H

CH3

HH

HO

CH3

CH3

H

H

O

OH

OO

OH

OH

OH

OH

OH

OH

O

H

OH

OH

O

O

Specific signatures of food consumption

Food Metabolome

Biomarkers of consumption

+ + abondantabondant- abondant

OPC

358 ions after orange

juice consumption

477 ions after grapefruit

juice consumption

+ 214 ions

common

Urine

0-12h

LC:SM

Ions p

<0.0

5

Cluster analysis

Biomarkers of effects?

Global approachClaudine Manach, INRA UNH, Clermont

Hesperetin glucuronides

Hesperetin sulfate

Naringenine glucuronide

Stachydrine

Aucubin

Limonene epoxide

Dihydroperillic acid

Tetramethoxyflavone

Dihydroxypentamethoxyflavone

Dimethoxycoumarin glucuronide

Methyltyramine sulfate

Confertifolin

Myrcene diol

…

Orange

Naringenin glucuronides

Eriodictyol glucuronide

Bergaptol glucuronide

Stachydrine

Aucubin

Scopoletin

Demethylnobiletin

Dihydroxypentamethoxyflavone

Trimethoxycoumarin glucuronide

Myrcene Hydroxy Acid

Limonene epoxide

Nootkatone

Hydroxycarvone

…

Grapefruit

New identified biomarkers

Thank for attention

WHAT HEALTH!