1. PARALLEL SESSION A Auditorium Descartes Selected communications Chairs: Glaucia Maria Pastore - UNICAMP- Campinas - Brazil Peter Hollman - Wageningen University - The Netherlands Multiple-approach studies to assess anthocyanin gastric absorption – potential role of GLUT1 Iva Fernandes - University of Porto – Portugal

2. 7 th International Conference on Polyphenols and Health Multiple-approach studies to assess anthocyanin gastric absorption: potential role of GLUT1 Iva Fernandes

3. Consumption in Europe: ♂ : 19.8 (Netherland) – 64.9 mg/d (Italy) ♀ : 18.4 (Spain) – 44.1 mg/d (Italy) (Heinonen 2007) (Zamora-Ros, Knaze et al. 2011) Anthocyanins consumption and human health Anthocyanins Change and improve human health! Diabetes Obesity Cardiovascular

4. Anthocyanins in food and pH equilibrium forms Cólon Food Stomach Small Intestine pH 1-5pH 5-7.4pH 7.4-8

5.  Low concentration in biological samples  Matrix complexity (e. g. EtOH)  Structural diversity  Metabolic disorders (e. g. Obesity)  Cleanup and concentration process  Lack of standards  Analytical methods  pH equilibrium forms  Low concentration in biological samples  Matrix complexity (e. g. EtOH)  Structural diversity  Metabolic disorders (e. g. Obesity)  Cleanup and concentration process  Lack of standards  Analytical methods  pH equilibrium forms (Manach, Williamson et al. 2005) Detection of anthocyanins in human samples Overlooked factors

6. Small intestine Stomach Enterohepatic recirculation Microbiota Fecal Excretion Kidneys Bladder metabolites/catabolites absorption Colon Urinary Excrection Liver Systemic circulation Eyes Heart Brain Adipose tissue Tissue/organs Methyl Sulphate Glucuronil Glucose Anthocyanins Transporter? Absorption and metabolism

7. Human trial Multiple approach studies... In vitro / theoretical studies

8. Human gastric barrier model: MKN-28 cell line Tight junctions Hanks medium pH 5.0 Policarbonate membrane Transwell insert Apical side Basolateral side S TOMACH MKN-28 CELL LINE 12-well transwell, 0.4-µm pore size, 7 days, TEER > 150 Ω.cm 2 HPLC analysis 30’ 60’ 120’ 180’ (Fernandes, de Freitas et al. 2012)

9. Anthocyanins transport in MKN-28 cell model Red Wine 0 % alc/vol Red Wine 4 % alc/vol Red Wine 40 mM glucose Glucose affects wine anthocyanins absorption patterns. ~ > Individual anthocyanins have similar absorption efficiencies. Wine anthocyanins are absorbed at gastric level, independent on ethanol. (Fernandes, de Freitas et al. 2012; Oliveira, Fernandes et al. 2015)

10. Apical pH effect Higher transport efficiency at lower pH values 1 < pH < 5 6.5 < pH < 8 (Oliveira, Fernandes et al. 2015) Equilibrium form and possible transporters

11. qRT-PCR - transporters expression in MKN-28 Apical side Basolateral side MKN-28 cell line GLUT-1 (Oliveira, Fernandes et al. 2015)

12. Molecular docking studies: anthocyanins/GLUT1 – extracellular and intracellular sides anthocyanins (ring B or glucose) – stable complexes with GLUT1 Docking studies – anthocyanin/GLUT1 Gastric barrier GLUT1 pH < 5.0 (Oliveira, Fernandes et al. 2015)

13. Molecular docking studies: Asn280, Asn403, Asn407, Trp380, Gln284 in transporter core Ongoing dynamic studies: transport from extracellular to cytoplasmatic side

14. Apical side Basolateral side MKN-28_SLC2A1 cell line GLUT-1 Stable transfection of GLUT1 in MKN-28 cell line

15. ConclusionGastric transport of anthocyanins Negative feedback after anthocyanins treatment (3h): GLUT1 levels similar to MKN-28 cell line Slightly higher transport efficiency of Dp3glc in transfected cell line, without significance Passive diffussion/paracellular transport with similar levels in both cell lines, at 4ºC Active transport! 4ºC

16. Multiple approach studies... Human trial In vitro / theoretical studies

17. Blackberry juice Anthocyanins ~ 650 mg Overnight fasting [Anthocyanins] juice = 2.92 ± 0.66 mg/mL Human Trial 8 volunteers BMI≤25

18. 0.010.020.030.040.050.060.0 Time [min] 120 min 60 min 30 min 0 min 15 min 0.0 Abs (520 nm) Plasma samples – HPLC analysis

19. C max (Cy3glc) located within the 2 h, at ~60 min C max (Conjugates) > 2h Conjugates have significantly higher plasmatic levels Different letters are significantly different Plasmatic levels of Cy3glc and conjugates

20. In vitro gastric absorption of anthocyanins In vitro gastric absorption of anthocyanins across MKN-28 and MKN-28_GLUT1 cells, at similar levels Theoretical gastric transporter Putative involvement of glucose transporter 1 in anthocyanin gastric absorption mechanism Human absorption and metabolism Detection of anthocyanins and their phase II conjugates in human plasma samples GLUT-1 Phase II enzymes Ongoing studies… Silencing GLUT1/GLUT3 expression with small interfering RNA! First steps…

21. Research team Iva Fernandes, Ph.D Natércia Brás, Ph.D. Helder Oliveira, M.Sc. Prof. Nuno Mateus Claudia Marques, M.Sc. Prof. Victor de Freitas Prof. Conceição Calhau Ana Faria, Ph.D.