AUTOFLUORESCENCE VISUALIZATION OF PHENOLIC AUTOFLUORESCENCE VISUALIZATION OF PHENOLIC
COMPOUND UPTAKE BY MICROSCOPIC AND SPECTROSCOPIC COMPOUND UPTAKE BY MICROSCOPIC AND SPECTROSCOPIC
TECHNIQUES IN CACO
TECHNIQUES IN CACO - - 2 CELL LINE MODEL 2 CELL LINE MODEL
C. PETCHLERT
C. PETCHLERT
1,41,4, C. DHUIQUE , C. DHUIQUE- -MAYER MAYER
22, B. CAPORICCIO , B. CAPORICCIO
11and and L.P.B. BIDEL L.P.B. BIDEL
331
1UMR : PrUMR : Préévention des Malnutritions et des Pathologies Associvention des Malnutritions et des Pathologies Associéées, ED SPes, ED SP--SA, UM1, UM2, Montpellier, France.SA, UM1, UM2, Montpellier, France.
2
2UMR UMR QualisudQualisud, Centre International de Recherche Agronomique pour le D, Centre International de Recherche Agronomique pour le Dééveloppement (CIRAD), Montpellier, France. veloppement (CIRAD), Montpellier, France.
3
3UMR : DiversitéUMR : Diversitéet Adaptation des Plantes Cultivéet Adaptation des Plantes Cultivées, UM II, Montpellier, France.es, UM II, Montpellier, France.
4
4Department of Biochemistry, Faculty of Science, Department of Biochemistry, Faculty of Science, BuraphaBuraphaUniversity, ChonburiUniversity, Chonburi, Thailand., Thailand.
Objective Use the natural fluorescence of certain Use the natural fluorescence of certain phenolic phenolic compounds : compounds : quercetin quercetin and and hesperidin hesperidin to characterize and investigate their localization and uptake by
to characterize and investigate their localization and uptake by human intestinal epithelial cells human intestinal epithelial cells
Quercetin
Quercetin HesperidinHesperidin (Hesperetin
(Hesperetin--77--rhamnoglucoside)rhamnoglucoside)
Cell Culture : Caco-2 cell lines were seeded on cover glass in 4-well plate.
This culturing method was specially adapted for the confocal microscopy.
450500 550600650 700 Emission wavelength (nm) 0
50 100 150 200 250 Intensity
ROI 1 ROI 2
450 500 550 600 650 700 Emission wavelength (nm) 0
50 100 150 200 250 Intensity
ROI 1 ROI 2 ROI 3
Emission spectra filtrated through the optical pathway of quercetin(left) and hesperidin(right) detected by LSM510 META, Zeiss.
(Images by LSM Image Browser 4.2 and
(Images by LSM Image Browser 4.2 and ImarisImaris6.2 after taken them with LSM510 META, Zeiss6.2 after taken them with LSM510 META, Zeiss)) GG
A, B, C
A, B, CQuercetin(blue) in different level of localization after 10 min of incubation.
D, E, F
D, E, FHesperidin (green) in different level of localization after 90 min treatment.
GGOrthogonal section display of hesperidin after 90 min of incubation.
It absolutely appears at the basal side of the cells
Actin
Actin(red) is presented in all images.
Absorption ratios of 375/650nm and 320/650nm were performed to investigate the uptake of quercetin (left) and hesperidin (right), respectively. Both figures show different polyphenol assimilative rate via human intestinal Caco-2 cell lines : the quercetin is remarkably reduced faster than the hesperidin at similar concentration (250 µµµµM) before all of them approach to constant rate at the final stage.
However, either quercetin or hesperidin clearly display in the exponential manner for the uptake through enterocytes .
The passage of The passage of quercetinquercetinoccurred rapidly with occurred rapidly with transcellulartranscellular mechanism whereas the passage of
mechanism whereas the passage of hesperidinhesperidinwas observed with was observed with transcellular
transcellularas well at the first stage, then became progressively as well at the first stage, then became progressively in
in paracellularparacellularmechanism after incubation time of 90 min.mechanism after incubation time of 90 min.
According to the UV-According to the UV-visible spectroscopy, we have also seen visible spectroscopy, we have also seen the absorptive manner of
the absorptive manner of quercetinquercetin (aglycone(aglycone flavonoidflavonoid) and ) and hesperidin
hesperidin(glycosylated(glycosylatedflavonoidflavonoid) that correlated to the results ) that correlated to the results of
of confocalconfocal microscopy. The microscopy. The quercetinquercetin can be assimilated can be assimilated exponentially by human intestinal epithelial cells faster than t exponentially by human intestinal epithelial cells faster than the he hesperidin
hesperidin..
Caco-2 cells confluency(phase-contrast inverted microscope-left) and natural fluorescence of quercetin in Caco-2 cells (epifluorescence microscope-right).
A. Quercetin 10min on apical side along Z-axis A.A. QuercetinQuercetin10min on 10min on apical
apical side along Zside along Z--axisaxis
B. Quercetin 10min in media along Z-axis B. QuercetinB.Quercetin10min in10min in
media mediaalong Z-along Z-axisaxis
C. Quercetin 10min on basal side along Z-axis C. QuercetinC.Quercetin10min on10min on basal
basalside along Zside along Z--axisaxis
D. Hesperidin 90min on apical side along Z-axis D.D. HesperidinHesperidin90min on 90min on
apical
apicalside along Z-side along Z-axisaxis
E. Hesperidin 90min in media along Z-axis E.E. HesperidinHesperidin90min in 90min in
media mediaalong Zalong Z--axisaxis
F. Hesperidin 90min on basal side along Z-axis F.F. HesperidinHesperidin90min on 90min on
basal side basal side along Zalong Z--axisaxis
ICPH 2009 ICPH 2009
4
4ththInternational Conference on International Conference on PolyphenolsPolyphenolsand and Health
Health
Harrogate International Centre, Yorkshire, UK: 7
Harrogate International Centre, Yorkshire, UK: 7--1111ththDecember 2009December 2009
Quercetin Quercetin
Quercetin Quercetin Caco Caco--22
Caco Caco--22
Base value of cells
