Anthocyanin chemical structure. Flavonoids. Different aglycones found in nature. ound. Backgro. Each Aglycone has a characteristic color and spectra

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1 Anthocynins H + Science of Foods for Helth CH Anthocynin Pigments: Stbility, Avilbility, nd Biotrnsformtion in the Gstrointestinl Trct M. Mónic Giusti Deprtment of Food Science & Technology hio Stte University, Columbus, USA Bsics bout nthocynins Vlue of nthocynins Bckgro ound Nturl pigments (blue, purple, red) Potent ntioxidnts (- times > vitmin E) 7 found in nture (Andersen nd Jordheim, ) Structure nd color chnge with ph Aglycone R H + R Acyltion H Glycosyltion Bckgro ound Reserch helth benefits Prevention of crdiovsculr diseses (Dy et l. 997) Relief of oxidtive stress (Rmirez-Tortos et l. ) Anti-cncer (Reen et l. ; Hrris et l. ) Prevention of obesity (Kwon et l. 7; Tsud ) Food industry Nturl colornt Added vlue ingredient Good imge Anthocynin chemicl structure Different glycones found in nture R Flvonoids R Ech Aglycone hs chrcteristic color nd spectr H + R C C C H + R nthocynin C-C-C skeleton Aglycon R R λ mx (nm) visible / color nthocynin Pelrgonidin H H 9 nm / ornge Cynidin H nm / ornge-red Peonidin Me H nm / ornge-red Delphinidin nm / bluish-red Petunidin Me nm / bluish-red Mlvidin Me Me nm / bluish-red

2 H H C H C In nture, nthocynins re lwys glycosylted nd cn be cylted Anthocynin sources H + H + CH CH H + H C CH - cid with cids ttched to the sugrs H Aliphtic cid (mlonic cid) H H Cinnmic cid (p-coumric cid) Berries: Simple pigments ther Sources: Complex cylted pigments HPLC nthocynin profiles Compring different nthocynin pigment profiles nm A B C 9 9,7 9,,, 7 Bilberry Grpe 7 9 Anthocynin profiles cn vry lot mong plnts simple profile one glycone (cy) different sugrs Bilberry different glycones different sugrs Grpe ne sugr: glucose different glycones cyltion Del--glu Del --Glu Cy--Gl D el--ar Cy y--gl Cy--glu Cy--Glu Cy--Arb Pet--glu Peo--glu Cy--Ar Pet--Gl Cy- --Glu Del- --Gl Pet--r Ml --gl Pet- --glu Ml--glu Cy--xyl Peo o--glu Ml--Arb Ml l--glu Grpe Bilberry Acylted nthocynins Minutes minutes Science of Foods for Helth There is evidence tht nthocynins my contribute to the protective effect of fruits nd vegetbles Anthocynins cn be used s food colornts of dded vlue

3 Anthocynins re poorly bsorbed Anthocynins re widely distributed in nture nd in foods High dily intke (+ mg/dy) HWEVER levels of nthocynins found in plsm re very low Less thn % of the dietry intke is bsorbed So cn compounds tht re not even bsorbed hve ny impct on helth? Science of Foods for Helth In vitro tests: Anthocynins nd colon cncer protection ti Cells lining the intestine cn be ffected by compounds entering the blood s well s by compounds tht re not bsorbed due to direct contct. Wht is colorectl cncer? Who does colorectl cncer ffect? Epithelil cell lyer of the colon, rectum, nd ppendix Five stges Symptoms Unexplined weight loss Ftigue Anemi Chnge in bowel hbits Liver metstsis Anthocynins in direct contct with the GI trct Third most prevlent cncer in the Western World 7% lifetime risk of developing Risk fctors Age Heredity Diet Smoking Alcohol Physicl inctivity Americn Cncer Society Americn Cncer Society Anthocynin extrct preprtion HT-9 Cell tretment Plnt Mteril Acetone/Chloroform Extrction HT-9 cells seeded t, cells/ml -hrs C Semi-purifiction Frctionted w/ Ethyl Acette Rotoevported Sponified w/ K Rotoevported HT-9 cells treted with nthocynin extrcts -hrs SRB Assy Lyophilized Lyophilized Acylted Anthocynin Sponified Anthocynins Plte 9 nm

4 HT-9 Cell tretment procedure Percent inhibition of HT-9 Cells Blnk Control Anthocynin Tretment Concentrtions Unused % Growth Inhibition = (T trt T ) x / (T ctr T ) A B C D E F G H ug ug ug.ug ug ug ug.ug B C Acylted Sponified Anthocynins Anthocynins Where: T is time zero T trt is the bsorbnce with tretment t 7 hrs T ctr is the bsorbnce with out tretment t 7 hrs GI : mount of extrct required to inhibit % growth Inhibitory effects of frctiontions of chokeberry on HT-9 colon cells Frctiontion of nthocynins Bckground Info ormtion on (% control) Growth Inhibitio ARE : Anthocynin-rich extrct Dose (ug/ml) Zho et l., Different solvents used to seprte nthocynin frction from other phenolics frction Very high efficiency nd low cost More thn 9% pure Experimentl design The GI of nthocynin-rich extrcts from different nturl sources Purple corn Bilberry Purple crrot Grpe Elderberry Red rdish ARE: nthocynin-rich extrct ACN: nthocynin frction PF: other phenols frction SPE ARE In vitro model: Semi-purifiction HT9 cell line Frctiontion (C SPE) SRB test GI PF ACN Bckground Info ormtion /ml) GI (ug/. Subset I.. Subset II. 7. Subset III. 7.7 Jing et l.,

5 Inhibitory effects of frctiontions of chokeberry on HT-9 colon cells Combintion Index: Interction Between Anthocynins nd other Phenols Bckground Info ormtion on (% control) Growth Inhibitio ARE : Anthocynin-rich extrct AP: ACN+PF ACN: Anthocynins frction PF : ther phenolics frction Dose (ug/ml) Jing et l., It is used to determine if the compounds tested work more efficiently lone or combined. CI i = Source [ACN ] [PF ] + [ACN ] [PF ] Combintion Index GI GI GI Anthocynins + ther Phenols... Combintion Effect Additive Additive Additive Chou nd Tlly (9). ur cell line studies showed tht Wht questions should we sk? All AREs inhibited growth of HT9 colon cncer cells, with little effect on NCM nontumorogenic cells Dt suggests tht t the protective effect of specific nthocynins or extrcts my depend on the chemicl structure of nthocynins present Control ARE (ug/ml) Totl Cell No.(x ) Norml Mlik et l., Nutr Cncer. HT-9 Are these compounds ctully reching the tissue? And if so re these the ctul forms of the compounds tht rech the tissue (or hve they been trnsformed?) Are they present in concentrtions tht re high enough to mke ny impct? Are the effects observed relly due to the compounds of interest? r re there other potentilly bioctive compounds? How will the compounds impct cncer cells nd how will they impct norml cells? Will these results be reproduced in n in-vivo system? FST Fll Seminr Series M. Mónic Giusti Anthocynin bsorption nd chemoprevention in n niml model Animl models: Anthocynin bsorption nd chemoprotection Animl tril: F rts / tretment wk ARE tretments: g nthocynin/kg diet Week Week Week AM injection ( mg/kg body wt) Urine, feces Colon, Plsm

6 Anthocynins in Plsm ( nm) Anthocynin metbolites in urine. to μg/ml plsm Mjor peks detected Acylted nthocynin detected Anthocynin metbolites Bilberry Grpe Cy--Gl Cy--Gl Cy--Glu Cy--Arb Cy--Arb??? Cy--xyl Urine ARE Anthocynin methyltion vs glucuronidtion Methylted nthocynin were found in urine Cy--glu M+ 9 (9-7) H CH Methyltion Glucuronidtion H C + H CH CH + Pn--glu M+ ( -- ) Cy--glucuronide M+ ( -- 7) Cy--Gl Cy--Gl Cy--Glu Cy--Arb Cy--Arb Pd--Gl Pd--Ar Pd--G Glu Urine ~ % methylted pigments ARE Cy--xyl ur Animl Studies Show... Effect of AREs on Aberrnt Crypt Foci Presence of intct nthocynins nd metbolites in plsm nd urine, demonstrting bsorption Anthocynin chemicl structure my ffect nthocynins bsorption nd excretion. urine Cy--Gl Cy--Glu Pt--Glu Mv--Arb Cy--Gl Cy--Arb Cy--Glu Cy--Arb ARE diet Pd--Gl Pd--Ar Pd--Glu Cy--xyl Urine Plsm feces He et l., r Men ACF Number Anthocynin levels in feces correlted with inhibition of erly cncer lesions, suggesting unbsorbed nthocynins my be chemoprotective Control Bilberry Grpe * * * * * Smll (-) Middle(-) Lrge(>) Totl ACF Multiplicity Ll et l.. Nutr Cncer ()

7 The GI of Anthocynin-rich Extrcts from Different Nturl Sources Anthocynin impct on GIT L) GI (ug/m. Subset I.. Subset II. 7. Subset III ~µg/g in Feces (GI trct) Biovilbilityil <.µg/ml in plsm Bckgro ound Importnce Improve lumen condition Protect epithelil cells Dose dependnt effects Stbility In vivo evidence is currently scrce Anthocynin-rich diets vs. rt colon cncer development control chokeberry bilberry grpe Colonic cell prolifertion index Lrge ACF multiplicity ND Fecl nthocynin (nmol/g) Fecl moisture content (%) Fecl bile cids (μmol/g) Colon cncer development (Source: Ll et l. ) Anthocynins in colonic lumen Jing et l., Experimentl Design Anthocynins Profile Chnge Anthocynins i Blck rspberry nthocynin-rich extrcts, nd lyophilized were used in feeding mle Fischer rts ( wks of ge) per group Single dose of ± mg nthocynins in.% citric cid solution by stomch tube fter hr fsting Tretments Control (.% citric cid) min min min min Bldder urine Gstric nd smll intestinl contents Gstric nd smll intestinl tissues HPLC-PDA-MS Anthocynins i Absorbnce (t nm) Cy glycone stndrd f Cy--glu Pek Are% o. Blck rspberry extrct n=.b Gstric content n= Smll intestinl content Stomch Intestine contents contents Cynidin glycon ws found in the gstric nd intestinl contents. Attributed to cid hydrolysis in the stomch nd β-glucosidse ctivity in the smll intestine membrne bound (?) Reltive proportion of Cy--glu decresed in the smll intestine Anthocynins in Stomch nd Smll Intestine Anthocynins in Stomch nd Smll Intestine Anthocynins i Anthocynins in the gstric content linerly decresed with t½ of min. Anthocynins in the smll intestinl content reched mximum t min before decresing. Anthocynins recovered from gstric nd smll intestinl lumen ccounted for 7-79% of the dministered dose between nd min. Anthocynin Recovered (mg cy--glu eq.) Stomch Intestine Combined GIT content b b b b b b bc d b 9 Anthocynins i Anthocynins in the smll intestinl tissue lso reched mximum t min before decresing. red (mg cy--glu eq.) The smll intestine tissue took up 7.% of the. dministered nthocynins, very high compred to bsorption into the plsm. levels.. Totl Anthocynins Recover.. b b b b b bc Stomch Intestine Combined b b d A) GIT content 9 Intestine B) GIT tissue 9 7

8 Bldder Urine Anthocynins Anthocynin-protein Complex in Stomch Tissue Anthocynins i Are% Pek A 9. n= Cy--glu% in urine. n= 7.. Urine nthocynin profile closely reflected tht in the lumen of bsorption sites n= n= Are% Pek A Cy--glu% in GIT. n=.b n= Gstric Intestinl content content Anthocynins Stomch tissue extrcts exhibited red color tht decresed over time. However, free nthocynins were not detected by HPLC Spectrl dt obtined by ltering the ph of the solution confirmed the presence of nthocynins, suggesting binding to other compounds, possibly protein trnsporter for nthocynins (Pssmonti, S. et l. ). Absorbnce (AU) Bound nthocynins ph Bound nthocynins ph. Free nthocynins ph Wvelength (nm) 7 Enzyme ctivity Some concluding remrks IT Enzymes The Effect of GI n (µm) Concentrtio LPH substrte specificity on the nthocynin glucosides in blueberry Dp--glu Cy--glu Pt--glu Pn--glu Mv--glu 9 Cy--gly H + Dp--gly H + Mv--gly H + Gly Gly CH Gly CH Pn--gly H + Pt--gly H + CH Gly CH Gly Results obtined in in-vitro nd niml experiments were in greement, suggesting protective effect of nthocynins ginst colon cncer. Anthocynins were reltively stble in the gstric nd smll intestinl lumens of fsted rts in contrst to reports from some in vitro studies. A significnt portion of nthocynins ws tken up into the GIT tissues, but neither extensively bsorbed or retined. This suggests tht nthocynins my hve in-situ protective effects in this tissue. The chemicl structure of the nthocynin molecule will impct its stbility to the conditions of the GIT, nd therefore, its chnces to interct with the GIT lining tissues. Anthocynins could be incorported into foods s food colornts with dded vlue Some concluding remrks In the news Anthocynins re bundnt in nture, nd could be incorported into foods s food colornts with dded vlue Not lwys we require bsorption of compounds in order to exert protective ction The ctivity it of phytochemicls h in the body will be ffected by other components present in the food nd compounds present. Some compounds cn ct synergisticlly, in n dditive wy or cn inhibit ech other s ction. August, 7

9 Wht do you think??? Acknowledgements Do you think nthocynins cn provide protection ginst colon cncer? Could compound tht is not bsorbed into the plsm hve n impct on helth? How nd or why could the chemicl structure ffect the bioctivity of the compounds? Wht re some importnt questions to sk when working with bioctive components using in-vitro experiments? How much cn we conclude from n niml model test? Wht would be some dvntges nd disdvntges of using n in-vitro test vs n niml test vs clinicl tril? Collbortors: SU:Joshu Bomser, Mrk Fill, Steven Schwrtz, Lur Kresty. MD: Bern Mgnuson UNALM: D. Cmpos, F. Sls, F. Luden, V. Norieg, I. Betlleluz, B. Htt, R. Chirinos. Chin: P. Jing Students nd Reserch Assistnts Pu Jing, Jin He, Tylor Wllce, Kristin Ketley, Lucy Zho, Minnie Mlik, Geet Ll, Qingguo Tin. Artemis Interntionl, Inc / Polyphenolics, Inc. / GlobeNturl Interntionl / Agricomseeds Reserch supported by USDA-NRI competitive grnts nd ARDC 9