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
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
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