La biodisponibilità dei composti antiossidanti. La capacità antiossidante totale (TAC)

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1 La biodisponibilità dei composti antiossidanti e La capacità antiossidante totale (TAC)

Study on the bioavailability of green and

UV - Ion Trap Mass Spectrometry Daniele Del

2 Study on the bioavailability of green and black tea antioxidant compounds using HPLC - UV - Ion Trap Mass Spectrometry Daniele Del Rio Human Nutrition Unit Department of Public Health University of Parma Plant Products and Human Nutrition Group Division of Biochemistry and Molecular Biology Institute of Biomedical and Life Sciences University of Glasgow

Mortality rates for CHD in Europe 400 0 50 100 150 200 250 300 350 400 350 300 250 200 150 100 50 0 SCO FIN IRE UK TZE NOR DAN SWE HOL POL GER BEL AUS BUL ITA ROM YUG SWI GRE POR SPA FRA

3 Mortality rates for CHD in Europe SCO FIN IRE UK TZE NOR DAN SWE HOL POL GER BEL AUS BUL ITA ROM YUG SWI GRE POR SPA FRA Age-standardized Age-standardized death death rates rates per per in in men men aged aged (Source: (Source: James, James, WPT, WPT, Healthy Healthy Nutrition Nutrition 1988) 1988)

5 Green Black

6 Mass Spectrometry in brief

7 One step ahead: The Ion Trap

10 Green and Black tea antioxidant compounds: identification and quantification using HPLC UV Ion Trap Mass Spectrometry

11 Principle flavan-3-ols in tea leaves HO O OH OH OH OH HO O OH OH OH OH HO O OH OH OH OH OH (-)-Epicatechin (+)-Catechin (+)-Gallocatechin HO O OH OH OH HO OH O O OH OH O HO OH O OH OH OH O O OH OH (-)-Epigallocatechin HO OH OH (-)-Epicatechin gallate HO OH OH (-)-Epigallocatechin gallate

12 MS/MS identification RT: SM: 7G NL: 7.77E5 nm= PDA green tea uau Time (min)

13 green tea #751 RT: AV: 1 NL: 8.21E6 T: - c ESI Full ms [ ] Relative Abundance m/z

14 green tea #752 RT: AV: 1 NL: 3.19E6 T: - c d Full ms @35.00 [ ] Epicatechin-3-O-gallate 65 Relative Abundance m/z

15 4-25% CH 3 CN Green tea Black tea (120 min)

16 10-30% CH 3 CN Green tea Black tea (120 min)

18 Length of fermentation period (min) Compound Gallic acid 6.0 ± ± ± ± ± 7.5 Theogallin 122 ± ± ± ± ± 0.8 (-)-Gallocatechin 383 ± ± ± 0.0 n.d. n.d. (-)-Epigallocatechin 1565 ± ± ± ± ± 0.8 (-)-Epigallocatechin gallate 1255.± ± ± ± ± 0.0 (+)-Catechin 270 ± ± ± ± ± 0.1 (-)-Epicatechin 738 ± ± ± ± ± 0.2 (-)-Epicatechin gallate 361 ± ± ± ± ± Caffeoylquinic acid 60 ± ± ± ± ± Caffeoylquinic acid 231 ± ± ± ± ± p-Coumaroylquinic acid 160 ± ± ± ± ± 0.2 Quercetin-rhamnosylgalatoside 28 ± ± ± ± ± 0.3 Quercetin-3-rutinoside 126 ± ± ± ± ± 0.5 Quercetin-rhamnose-hexose-rhamnose 5.2 ± ± ± ± ± 0.0 Quercetin-3-galactoside 57 ± ± ± ± ± 0.2 Quercetin-3-glucoside 122 ± ± ± ± ± 0.4 Kaempferol-rhamnose-hexose-rhamnose 99 ± ± ± ± ± 1.7 Kaempferol-galatoside/rutinoside 539 ± ± ± ± ± 5.9 Quercetin-arabinoside 3.1 ± ± ± ± ± 0.1 Kaempferol-3-glucoside 581 ± ± ± ± ± 1.9 Kaempferol-arabinoside n.q. n.q. n.q. n.q. n.q. Quercetin-rhamnose-hexose-rhamnose n.q. n.q. n.q. n.q. n.q. Unknown quercetin conjugate 11 ± ± ± ± ± 0.2 Unknown quercetin conjugate 17 ± ± ± ± ± 0.6 Unknown kaempferol conjugate 28 ± ± ± ± ± 0.5 Unknown quercetin conjugate n.q. n.q. n.q. n.q. n.q. Unknown kaempferol conjugate n.q. n.q. n.q. n.q. n.q. Theaflavin n.d. 200 ± ± ± ± 0.2 Theaflavin-3-gallate n.d. 147 ± ± ± ± 0.6 Theaflavin-3'-gallate n.d. 118 ± ± ± ± 0.8 Theaflavin-3,3'-digallate n.d. 148.± ± ± ± 0.1

19 Study on the bioavailability of green and black tea antioxidant compounds in ileostomy volunteers

20 Ileum

21 Possible routes for consumed dietary phenolics in humans Dietary phenolics Tissues Small intestine Liver bile Kidney Colon Faeces Urine

22 Protocol Two different teas studied (green tea and black tea). Volunteers will be asked to follow a low flavonoid diet for 2 days. Advice will be given on what to avoid during the course of the study. Volunteers will be asked to abstain from alcohol for the duration of the study. On day 3, a sample of ileostomy fluid, blood and urine will be collected. Volunteers will then be asked to drink 300 ml (1 mug) ) of tea. All ileostomy fluid excreted will be collected, divided into 3 samples: 0-2 hr after drinking tea 2-55 hr after drinking tea 5-24 hr after drinking tea Blood samples will be collected 0, 1, 2, 3, 4, 5 and 24 hr after drinking tea Urine samples will be collected before the study and for the subsequent 5 h

24 Low Flavonoid Diet The following foods must be avoided during the course of the study : o Tea and herbal tea o Fruit Juice o Alcohol (especially red wine) o Tomatoes o Onions o Red pepper o Broccoli o Lollo rosso lettuce o Strawberries o Raspberries o Chocolate o Coffee Minimise the intake of other fruits and vegetables as much as possible.

25 MS/MS identification ileal fluid p1 gt 2-5h 2 #1008 RT: AV: 1 NL: 6.07E5 T: - c ESI Full ms [ ] MS 65 Relative Abundance m/z

26 ileal fluid p1 gt 2-5h 2 #1009 RT: AV: 1 NL: 1.64E5 T: - c d Full ms @35.00 [ ] MS 2 Relative Abundance m/z

27 ileal fluid p1 gt 2-5h 2 #799 RT: AV: 1 NL: 2.01E6 T: - c d Full ms @35.00 [ ] Relative Abundance MS 3 Epicatechin-3-O-gallate + sulphate group , m/z

32 What happens in plasma?

33 Ileostomy volunteer RT: uau SM: 7G NL: 9.53E5 nm= PDA TEA PLASMA VOL 2 GT 1HR BILL EXTR FS Relative Abundance NL: 1.30E5 m/z= MS TEA PLASMA VOL 2 GT 1HR BILL EXTR FS Relative Abundance NL: 8.64E4 m/z= MS TEA PLASMA VOL GT 1HR BILL EXTR FS Time (min)

34 Healthy volunteer RT: uau SM: 7G NL: 7.81E5 nm= PDA Alex Plasma 1Hr + IS Relative Abundance Relative Abundance Time (min) NL: 2.08E5 m/z= MS Alex Plasma 1Hr + IS NL: 3.05E5 m/z= MS Alex Plasma 1Hr + IS

38 Hypotesis: EGCG and ECG are absorbed in the large intestine.

39 Anaerobic fermentation of flavanols from tea and ileal fluid: Samples (fermenting substrate): Green Tea, Black Tea, Ileal fluid after Green Tea consumption, Ileal fluid after Black Tea consumption. Fermenting microorganisms: faecal microflora (from( a stool sample). Times: : 6 and 24 hours.

40 Green Tea after 24h colonic-like like fermentation RT: SM: 7G uau Relative Abundance Relative Abundance EGCG ECG Time (min) NL: 1.10E6 nm= PDA ferm sample ddr8 NL: 1.99E5 m/z= MS ferm sample ddr8 NL: 2.18E4 m/z= MS ferm sample ddr8

41 RT: SM: 7G uau Relative Abundance Green Tea related Ileal Fluid after 24h colonic- like fermentation EGCG NL: 2.31E5 nm= PDA ferm sample ddr4 NL: 1.98E5 m/z= MS ferm sample ddr4 Relative Abundance ECG Time (min) NL: 3.14E5 m/z= MS ferm sample ddr4

42 Decrease of ECG and EGCG in Green Tea and related Ileal Fluid during fermentation Ileal EGCG Ileal ECG 80 Tea EGCG Tea ECG % hours

43 Conclusions: Antioxidant compounds in Green and Black tea are bioavailable, but the percentage of absorbtion and their in vivo transformation are markedly different among subjects. Apparently, most compounds are metabolised in the small intestine lumen or mucosa, glucuronidated or sulphated.. Some individuals has shown the ability to hydrolyse the sugar moisture to obtain the aglycone compound in the small intestine. The main flavanols remain mostly unmetabolised along the upper gastrointestinal tract. These results suggest that the main flavanols (EGCG and ECG) might be significantly absorbed in the large intestine, being undetectable in plasma of ileostomy volunteers and partially resistent to the attack of the colonic microflora.

44 Thanks to Human Nutrition Unit,, Parma Prof. Furio Brighenti Dr. Nicoletta Pellegrini Dr. Marta Bianchi Dr. Sara Salvatore Dr. Barbara Colombi All the students of the last years!! Human Nutrition at Glasgow Prof. Alan Crozier Dr. Bill Mullen Dr. Gina Borges Dr. Alex Degeneve Dr. Ctherine Tsang Dr. Alexander Laurentin Dr. Cyril Auger

49 Do flavan-3-ols from green tea reach the human brain? Relative Abundance A Plasma Blood cerebrospinal fluid barrier Relative Abundance Time (min) Time (min) B Cerebrospinal Fluid Relative Abundance C 305 m/z 0 m/z

50 La capacità antiossidante totale (TAC)

51 RED-OX NETWORK Recycling??? Unknown AA??? Synergism??? Vit. E Uric acidcoqvit. CCarotenoids Phenolics Thiols

52 Total Antioxidant Capacity O 2 - ONOO - OH ROO NO TA Antioxidant activity E C Redox interactions Stress Oxidizing Substrate Partitioning

53 TAC assays developed ( ) % of the total number of developed assays.

54 METHODS FOR MEASUREMENT F TOTAL ANTIOXIDANT CAPACITY OPD/H 2 O 2 (Nakamura 1987) ABTS+ (Rice Evans 1991) HRP/ H 2 O 2 (Whitehead 1982) AMVN (Metsa-Ketela 1992) Cu 2+ /cumene hydroperoxide (McKenna 1991) Superoxide (Ogasawara 1985) Dopa Oxidation (Cooper 1991) AAPH (De Lange 1989) Metal-chelating (Morel 1994) GENERATE RADICAL Fluorescence Inhibition ADD ANTIOXIDANT Chemiluminescence Absorbance change TBA-RS Oxygen Consumption OBSERVE ENDPOINT OBSERVE INHIBITION OF ENDPOINT

55 To TAC or not to TAC DPPH OH ECD DC TEAC FFRAP ORAC FluoLip TAC RP ABTS LP TRAP

56 Trolox Equivalent Antioxidant Capacity (TEAC ABTS)

57 TEAC - Sistema flow-injection B C D L E F A H G I

58 TEAC - Sistema flow-injection AU Minutes

59 Total Radical-trapping Antioxidant Parameter (TRAP)

60 Ferric Reducing Antioxidant Power (FRAP)

61 Oxygen Radical Absorbance Capacity Assay (ORAC).

TRAP increase (%) 8 0 6 0 4 0 2 0 0 Green tea Water 0 20

63 TRAP increase (%) Green tea Water Time (min) From: Serafini M et al. Lancet 1994;

64 T0 T2 T3 T6 Fresh MAP TAC increase (%) TAC increase (%)

65 Relative risk of gastric cancer in relation to TAC of consumed fruit and vegetables. OR TRAP (microm TE)

66 Effect of food association on plasma red-ox status FRAP (% increase over base-line value) *** DC + MK DC MC Time (hours) (-)-Epicatechin AUC (ng ml 1 h 1 ) a b c DC DC + MK MC Serafini et al. Nature, 2003

68 I fitoestrogeni

Bioavailability of dietary (poly)phenols following acute. ingestion of an enriched drink by ileostomists

SUPPLEMENTARY MATERIAL Bioavailability of dietary (poly)phenols following acute ingestion of an enriched drink by ileostomists Gina Borges a, Michael E.J. Lean b, Susan A. Roberts c, and Alan Crozier *a