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Antiglycation Effects and Reactive Carbonyl Trapping Capacity of Berry and Grape Phytochemicals

Permanent Link: http://ufdc.ufl.edu/UFE0042631/00001

Material Information

Title: Antiglycation Effects and Reactive Carbonyl Trapping Capacity of Berry and Grape Phytochemicals
Physical Description: 1 online resource (129 p.)
Language: english
Creator: Wang, Wei
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: ages, antiglycation, berries, carbonyls, phytochemicals
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxidation of carbohydrates and proteins in foods and human body generates reactive carbonyls, which include glyoxal, methylglyoxal, malondialdehyde and acrolein. Reactive carbonyls react with proteins to form advanced glycation end-products (AGEs) and cause aging, diabetic complications and other chronic diseases. Previous studies suggested that plant extracts and certain phenolic compounds were able to inhibit AGE formation or directly scavenge reactive carbonyl compounds. Berries and Muscadine grapes are known as good sources of antioxidants and phenolic compounds. Therefore, we hypothesized that they may have the inhibitory effects on AGE formation and direct carbonyl trapping capacities. Phytochemicals were extracted and purified on adsorption resin to remove sugars. Sugar-free phytochemicals purified from blueberries were fractionated into five fractions using a Sephadex LH-20 column. The resultant extracts and fractions were tested in three AGE generation models that simulated physiological conditions. The model systems included bovine serum albumin (BSA)-fructose, BSA-methylglyoxal and arginine-methylglyoxal models. AGEs were detected using florescence. The capacity of sugar-free phytochemicals to scavenge reactive carbonyl was tested on methylglyoxal. Catechin, epicatechin, quercetin, chlorogenic acid and resveratrol were used to react with glyoxal, methylglyoxal, malondialdehyde and acrolein. Reaction kinetics were evaluated and phytochemical-carbonyl adducts were tentatively identified using HPLC-ESI-MSn. Results showed that sugar-free phytochemicals from berries and grapes, and blueberry phytochemical subfractions, inhibited AGE generation in BSA-fructose, BSA-methylglyoxal and arginine-methylglyoxal models by 22-88%. Berry and grape phytochemicals and blueberry subfractions scavenged methylglyoxal by 47-80% within six hours. Pure phytochemicals (catechin, epicatechin, quercetin and resveratrol) reacted with glyoxla, methylglyoxal, malondialdehyde and acrolein and formed various phytochemical-carbonyl adducts. Results in this study indicated that phytochemicals from berries and grapes are effective AGE inhibitory agents that are useful to alleviate AGE-related chronic diseases. Carbonyl scavenging and adduct formation was one of the mechanisms for berry phytochemicals to inhibit AGE formation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Wei Wang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Gu, Liwei.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042631:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042631/00001

Material Information

Title: Antiglycation Effects and Reactive Carbonyl Trapping Capacity of Berry and Grape Phytochemicals
Physical Description: 1 online resource (129 p.)
Language: english
Creator: Wang, Wei
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: ages, antiglycation, berries, carbonyls, phytochemicals
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxidation of carbohydrates and proteins in foods and human body generates reactive carbonyls, which include glyoxal, methylglyoxal, malondialdehyde and acrolein. Reactive carbonyls react with proteins to form advanced glycation end-products (AGEs) and cause aging, diabetic complications and other chronic diseases. Previous studies suggested that plant extracts and certain phenolic compounds were able to inhibit AGE formation or directly scavenge reactive carbonyl compounds. Berries and Muscadine grapes are known as good sources of antioxidants and phenolic compounds. Therefore, we hypothesized that they may have the inhibitory effects on AGE formation and direct carbonyl trapping capacities. Phytochemicals were extracted and purified on adsorption resin to remove sugars. Sugar-free phytochemicals purified from blueberries were fractionated into five fractions using a Sephadex LH-20 column. The resultant extracts and fractions were tested in three AGE generation models that simulated physiological conditions. The model systems included bovine serum albumin (BSA)-fructose, BSA-methylglyoxal and arginine-methylglyoxal models. AGEs were detected using florescence. The capacity of sugar-free phytochemicals to scavenge reactive carbonyl was tested on methylglyoxal. Catechin, epicatechin, quercetin, chlorogenic acid and resveratrol were used to react with glyoxal, methylglyoxal, malondialdehyde and acrolein. Reaction kinetics were evaluated and phytochemical-carbonyl adducts were tentatively identified using HPLC-ESI-MSn. Results showed that sugar-free phytochemicals from berries and grapes, and blueberry phytochemical subfractions, inhibited AGE generation in BSA-fructose, BSA-methylglyoxal and arginine-methylglyoxal models by 22-88%. Berry and grape phytochemicals and blueberry subfractions scavenged methylglyoxal by 47-80% within six hours. Pure phytochemicals (catechin, epicatechin, quercetin and resveratrol) reacted with glyoxla, methylglyoxal, malondialdehyde and acrolein and formed various phytochemical-carbonyl adducts. Results in this study indicated that phytochemicals from berries and grapes are effective AGE inhibitory agents that are useful to alleviate AGE-related chronic diseases. Carbonyl scavenging and adduct formation was one of the mechanisms for berry phytochemicals to inhibit AGE formation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Wei Wang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Gu, Liwei.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042631:00001


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1 ANTIGLYCATION EFFECTS AND REACTIVE CARBONYL TRAPP ING CAPACITY OF BERRY AND GRAPE PHYTOCHEMICALS By WEI WANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Wei Wang

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3 To my family and friends

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4 ACKNOWLEDGMENTS I want to express my sincere appreciation to my major advisor, Dr. Liwei Gu, for his patience, advice and mentorshi p. Without his guidance and support, this research could not be accomplished. I am grateful for my committee memb ers, Dr. Susan S. Percival, Dr. Maurice R. Marshall and Dr. Donald J. Huber, for their valuable time and suggestions. I would like to especiall y thank Dr. Susan S. Percival for her mentorship and generosity in providing the instrument for fluorescent intensity measurement in this study. Further I extend my appreciation to Dr. Cecilia Do Nascimento Nunes and Yavuz Yagiz for their assistance with sugar analysis for berry extracts. I cherished the friendship and time spent with my lab group members: Amandeep K. Sandhu, Haiyan Liu, Hanwei Liu, Keqin Ou, Timothy Buran Sara M. Marshall and Zheng Li. They were always willing to offer helping hands and emotional support. The laughter and foods we shared brought abundant joy and ma de our everyday life memorable. Last and most important, I express my deepest gratitude to my parents for their constant love and commitment to my education. They we re my firs t teachers and ga ve me many good lessons. They always encourage d me to pursue my dream, and this is why I earn ed this degree on the other side of the earth from my hometown I would also like to extend my gratitude to my beloved husband who is always p rou d of eve ry single achievement I obtain Because of his love, patience and unconditional support, I gained the strength to complete this program.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 TABLE OF CONTENTS ................................ ................................ ................................ .. 5 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 17 Introduction ................................ ................................ ................................ ............. 17 Reactive Carbonyl Compounds ................................ ................................ .............. 17 Sources of Reactive Carbonyl Compounds ................................ ...................... 18 Reactivity of Reactive Carbonyl Compounds ................................ ................... 19 Advanced Glycation End products (AGEs) ................................ ............................. 20 Berries and Muscadine Grape ................................ ................................ ................ 22 Health Related Benefits ................................ ................................ .................... 22 Phytochemicals in Berries and Muscadine Grapes ................................ .......... 22 Reactions Between Phytochemicals and Reactive Carbonyls ................................ 23 Research Objectives ................................ ................................ ............................... 23 2 FLUORESCENT AGES PRODUCTION FROM IN VITRO INCUBATION .............. 25 Materials and Methods ................................ ................................ ............................ 25 Chemicals ................................ ................................ ................................ ......... 25 Model System for Fluorescent AGEs Formation ................................ .............. 25 Emission and Excitation Spectra of Fluorescent AGEs ................................ .... 26 Fluorescent Intensity Measurement ................................ ................................ 26 Data Expression and Grafting ................................ ................................ .......... 27 Results ................................ ................................ ................................ .................... 27 Fluorescent AGEs Formation ................................ ................................ ........... 27 Discussion ................................ ................................ ................................ .............. 29 3 ANTIGLYCATION EFFECT OF BERRY AND GRAPE PHYTOCHEMICALS ......... 34 Materials and Methods ................................ ................................ ............................ 34 Chemicals and Materials ................................ ................................ .................. 34

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6 Extraction and Purification of Sugar free Phytochemicals ................................ 35 Analysis of Sugars in Phytochemical Extracts ................................ .................. 36 Folin Ciocalteu Assay ................................ ................................ ....................... 36 Oxygen Radical Absorbance Capacity ................................ ............................. 37 Total Anthocyanin Content ................................ ................................ ............... 37 Total P rocyanidin Content ................................ ................................ ................ 38 Antiglycation Effects in BSA Fructose Model ................................ ................... 38 Antiglycation Effects in BSA Methylglyoxal Model ................................ ............ 39 Antiglycation Effects in Arginine methylglyoxal Model ................................ ...... 39 Direct Methylglyoxal Trapping ................................ ................................ .......... 40 Data Expression, Grafting and Statistics ................................ .......................... 41 Results ................................ ................................ ................................ .................... 41 Phytochemical Extraction and Purification ................................ ........................ 41 Folin Ciocalteu Assay ................................ ................................ ....................... 42 Oxygen Radical Absorbance Capacity ................................ ............................. 42 Total Anth ocyanin Content ................................ ................................ ............... 42 Total Procyanidin Content ................................ ................................ ................ 43 Anti glycation Effects in Selected Models ................................ ......................... 43 Direct Methylglyoxal Trapping ................................ ................................ .......... 44 Discussion ................................ ................................ ................................ .............. 44 4 FRACTIONATION OF BLUEBERRY PHYTOCHEMICALS, COMPOUN D IDENTIFICATION AND ANTIGLYCATION EFFECTS ................................ ............ 54 Materials and Methods ................................ ................................ ............................ 54 Chemicals and Materials ................................ ................................ .................. 54 Fractionation of Sugar free Blueberry Phytochemicals ................................ .... 54 Phytochemical Identification on HPLC ESI MS n ................................ ............... 55 Total Phenolic Contents, Antioxidant Capacity, Total Anthocyanin Content and Total Procyanidin Content ................................ ................................ ...... 55 Anti glycation Effect in Selected Models ................................ .......................... 56 Direct Methylglyoxal Trapping Assay ................................ ............................... 56 Data Expression, Grafting and Statistics ................................ .......................... 56 Results ................................ ................................ ................................ .................... 57 Phytochemical Fractionation ................................ ................................ ............ 57 Phytochemical Identification on HPLC ESI MS n ................................ ............... 57 Total Phenolic Content ................................ ................................ ..................... 59 Antioxidant Capacity ................................ ................................ ......................... 60 Total Anthocyanin Content ................................ ................................ ............... 60 Total Procyanidin Content ................................ ................................ ................ 60 Anti glycation Effect in Selected Models ................................ .......................... 61 Direct Methylglyoxal Trappi ng ................................ ................................ .......... 62 Discussion ................................ ................................ ................................ .............. 62 5 REACTIONS BETWEEN PHYTOCHEMICALS AND REACTIVE CARBONYL SPECIES ................................ ................................ ................................ ................ 75

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7 Materials and Methods ................................ ................................ ............................ 75 Chemicals ................................ ................................ ................................ ......... 75 Preparation of Malondialdehyde ................................ ................................ ....... 75 Preparation of Phytochemical and Carbonyl Solutions ................................ ..... 76 Preparation of Derivatization Reagents ................................ ............................ 76 Catechin carbonyl reaction study ................................ ............................... 77 Epicatechin carbonyl reaction and chlorogenic acid carbonyl reaction studies ................................ ................................ ................................ .... 79 Resveratrol carbonyl reaction and quercetin carbonyl reaction studies ..... 79 Data Calculation, Grafting and Statistics ................................ .......................... 80 Results ................................ ................................ ................................ .................... 81 Reaction Kinetics between Phenolics and Carbonyl Compounds .................... 81 Adduct Identification ................................ ................................ ......................... 83 Catechin carbonyl adducts ................................ ................................ ......... 83 Epicatechin carbonyl adducts ................................ ................................ .... 85 Quercetin carbonyl adducts ................................ ................................ ....... 87 Resveratrol carbonyl adducts ................................ ................................ .... 87 Discussion ................................ ................................ ................................ .............. 88 6 CONCLUSION ................................ ................................ ................................ ...... 119 LIST OF REFERENCES ................................ ................................ ............................. 120 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 129

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8 LIST OF TABLES Table page 2 1 Wavelength of maxima excitation and emission of fluorescent AGEs in various model systems. ................................ ................................ ...................... 31 3 1 Weight of frozen berries, crude extracts, and sugar free extra cts. ..................... 46 3 2 Sugar concentrations in crude and sugar free extracts. ................................ ..... 46 3 3 Total phenolic, antioxidant capacity, total anthocyani ns and total procyanidins in sugar free extracts. ................................ ................................ ......................... 46 4 1 Weights of blueberry extracts and fractions. ................................ ....................... 64 4 2 Tentatively identif ied compounds in blueberry fractions. ................................ .... 64 4 3 Total phenolic, antioxidant capacity, total anthocyanins and total procyanidins in blueberry extract and fractions. ................................ ................................ ....... 65 5 1 Empirical degradation half time of phytochemicals in different incubation mixtures. ................................ ................................ ................................ ............. 90 5 2 Empirical degradation half time of reactive carbonyl compound s in different incubation mixtures. ................................ ................................ ............................ 90 5 3 Phytochemical carbonyl adduct identification. ................................ .................. 100

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9 LIST OF FIGURES Figure page 1 1 Structures of reactive carbonyls. ................................ ................................ ........ 24 1 2 Structures of selected advanced glycation end products (AGEs). ...................... 24 2 1 Excitation (A) and emission (B) spectra of BSA fructose model. ........................ 31 2 2 Fluorescent AGE generation in bovine serum albumin (BSA) monosaccharide incubation models at 37 C. ................................ .................... 32 2 3 Fluorescent AGE generation in bovine serum albumin (BSA)/ reactive carbonyl incubation models at 37 C. ................................ ................................ 32 2 4 Fluore scent AGE generation in amino acids monosaccharide incubation models at 37 C. ................................ ................................ ................................ 33 2 5 Fluorescent AGE generation in amino acid reactive carbonyl incubation models at 37 C. ................................ ................................ ................................ 33 3 1 Extraction and purification of sugar free phytochemicals from berries and noble muscadine grapes. ................................ ................................ ................... 47 3 2 HPLC chromatogram of sugars in (A ) cranberry crude extract and (B) cranberry sugar free extract. ................................ ................................ .............. 48 3 3 Anti glycation effects of berry extracts in bovine serum albumin fructose model. Bars represent mean standard deviation o f triplicate tests. ................... 49 3 4 Anti glycation effects of berry extracts in bovine serum albumin methylglyoxal model. Bars represent mean standard deviation of triplicate tests. ................... 50 3 5 Anti glycation effects of berry extracts in arginine methylglyoxal model. Bars represent mean standard deviation of triplicate tests. ................................ ....... 51 3 6 Chromatogram of methylglyoxal (A), methylglyoxal after reaction with blueberry sugar free extract (B) and methylglyoxal after reaction with aminoguanidine (C). ................................ ................................ ........................... 52 3 7 The percentage of remain ing methylglyoxal after reacting with berry and grape extracts for 0.5, 1, 2, 4, 6h. ................................ ................................ ....... 53 4 1 Fractionation flow chart of blueberry sugar free phytochemicals. ....................... 66 4 2 HPLC chromatograms of blueberry fraction I at 280 nm (A) and 520 nm (B). .... 67

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10 4 3 HPLC chromatograms of blueberry fraction II at 280 nm (A) and 520 nm (B). ... 67 4 4 HPLC chromatograms of blueberry fraction III at 280 nm (A), 360 nm (B) and 520 nm (C). ................................ ................................ ................................ ......... 68 4 5 HPLC chromatograms of blueber ry fraction IV at 280 nm (A) and 520 nm (B). .. 68 4 6 HPLC chromatograms of blueberry fraction V at 280 nm (A) and 520 nm (B). ... 69 4 7 Anti glycation effects of blueberry extract and fractions in bovine serum albumin fructose model. ................................ ................................ ..................... 70 4 8 Anti glycation effects of blueberry extract and fractions in bovine serum albumin m ethylglyoxal model. ................................ ................................ ............ 71 4 9 Anti glycation effects of blueberry extract and fractions in arginine methylglyoxal model. ................................ ................................ .......................... 72 4 10 Ch romatogram of methylglyoxal (A), methylglyoxal after reaction with blueberry fraction III (B) and methylglyoxal after reaction with aminoguanidine (C). ................................ ................................ ........................... 73 4 11 The percentage of remaining meth ylglyoxal after been incubated with blueberry extract and fractions for 0, 0.5, 1, 2, 4, 6h. ................................ ......... 74 5 1 Percentage of remaining catechin (CAT) during 37 C incubation with phosphate buffer (blank) glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). ................................ ..................... 91 5 2 Percentage of remaining epicatechin (EPI) during 37 C incubation with phosphate buffer (blank), glyoxal ( GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). ................................ ..................... 92 5 3 Percentage of remaining chlorogenic acid (CGA) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), m ethylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). ................................ ..................... 93 5 4 Percentage of remaining quercetin (QUE) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). ................................ ..................... 94 5 5 Percentage of remaining resveratrol (RES) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malo ndialdehyde (MDA) and acrolein (ACR). ................................ ..................... 95 5 6 Percentage of remaining glyoxal (GO) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin (EPI), quercetin (QUE), res veratrol (RES) and chlorogenic acid (CGA). ................................ ...... 96

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11 5 7 Percentage of remaining methylglyoxal (MGO) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin (EPI), querceti n (QUE), resveratrol (RES) and chlorogenic acid (CGA). ................................ ...... 97 5 8 Percentage of remaining malondialdehyde (MDA) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin ( EPI), quercetin (QUE), resveratrol (RES) and chlorogenic acid (CGA). ................................ ...... 98 5 9 Percentage of remaining acrolein (ACR) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicat echin (EPI), quercetin (QUE), resveratrol (RES) and chlorogenic acid (CGA). ................................ ...... 99 5 10 HPLC FLD (Ex=231nm, Em=320nm) chromatogram of the reaction products in the incubation of catechin with phosp hate buffer (A), catechin with glyoxal (B), catechin with methylglyoxal (C), catechin with malondialdehyde (D) and catechin with acrolein (E). ................................ ................................ ................ 101 5 11 MS and MS 2 spectra of the precursor ions of m/z =619[M H] (A), m/z =347[M H] (B) and m/z =638[M] (C) in the incubation of catechin and glyoxal in negative electrospray mode. ................................ ................................ ............. 102 5 12 The proposed structures of reaction adducts forme d by catechin and glyoxal. 103 5 13 MS and MS 2 spectra of the precursor ions of m/z =433[M H] (A) and m/z =361[M H] (B) in the incubation of catechin and methylglyoxal in negative electrospray mode. ................................ ................................ ............. 104 5 14 The proposed structures of reaction adducts formed by catechin and methylglyoxal. ................................ ................................ ................................ ... 104 5 15 MS and MS 2 spectra of the precursor ions of m/z =433[M H] (A) and m/z =361[M H] (B) in the incubation of catechin and malondialdehyde in negative electrospray mode. ................................ ................................ ............. 105 5 16 The proposed structures of reaction add ucts formed by catechin and malondialdehyde. ................................ ................................ ............................. 105 5 17 MS and MS 2 spectra of the precursor ion of m/z =345[M H] in the incubation of catechin and acrolein in negative electrospray mode. ................................ .. 106 5 18 The proposed structure of reaction adduct formed by catechin and acrolein. .. 106 5 19 HPLC FLD (Ex=231nm, Em=320nm) chromatogram of the reaction products in the incubation of epicatechin with phosphate buffer (A), epicatechin with glyoxal (B), epicatechin with methylglyoxal (C), epicatechin with malondialdehyde (D) and epicatechin with acrolein (E). ................................ ... 107

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12 5 20 MS and MS 2 spectra of the precursor ions of m/z =347[M H] (A), m/z =619[M H] (B) and m/z =677[M H] (C) in the incubation of epicatechin and glyoxal in negative electrospray mode. ................................ ................................ ............. 108 5 21 The proposed structures of reaction adducts formed by epicatechin and glyoxal. ................................ ................................ ................................ ............. 109 5 22 MS and MS 2 spectra of the precursor ions of m/z =361 [M H] and m/z 433 [M H] in the incubation of epicatechin and methylglyoxal in negative electrospray mode. ................................ ................................ ........................... 110 5 23 The proposed structures of reaction adducts formed by epicatechin and methylglyoxal. ................................ ................................ ................................ ... 110 5 24 MS and MS 2 spectra of the precursor ion of m/z =433[M H] in the incubation of epicatechin and malondialdehyde in negative electrospray mode. ............... 111 5 25 The proposed structure of reaction adduct formed by epicatechin and malondialdehyde. ................................ ................................ ............................. 111 5 26 MS and MS 2 spectra of the precursor ions of m/z =345[M H] in the incubat ion of epicatechin and acrolein in negative electrospray mode. ............................. 112 5 27 The proposed structures of reaction adducts formed by epicatechin and acrolein. ................................ ................................ ................................ ............ 112 5 28 HPLC DAD (360nm) chromatogram of the reaction products in the incubation of quercetin with phosphate buffer (A), quercetin with glyoxal (B), quercetin with methylglyoxal (C) and quercetin with acrolein (D). ................................ .... 113 5 29 MS and MS 2 spectra of the precursor ions of m/z =341 [M H] (A, quercetin and glyoxal), m/z =373 [M H] (B, quercetin and methylglyoxal) and m/z =357 [M H] (C, quercetin and acrolein) in the incubation s of quercetin and reactive carbonyl compounds in negative electrospray mode. ................................ ....... 114 5 30 The proposed structures of reaction adducts formed by quercetin with glyoxal, methylglyoxal, malondialdeh yde and acrolein. ................................ .... 115 5 31 HPLC FLD (Ex=330nm, Em=374nm) chromatogram of the reaction products in the incubation of resveratrol with phosphate buffer (A), resveratrol with glyoxal (B), resveratr ol with methylglyoxal (C), resveratrol with malondialdehyde (D) and resveratrol with acrolein (E). ................................ .... 116 5 32 MS and MS 2 spectra of the precursor ions of m/z =285 [M H] (A, resveratrol and glyox al), m/z =299 [M H] (B, resveratrol and methylglyoxal), m/z =299 [M H] (C, resveratrol and malondialdehyde), m/z =283 [M H] and m/z =339 [M H] (D and E, resveratrol and acrolein) in the incubation of resveratrol and reactive carbonyl compounds in negative electrospray mode. ......................... 117

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13 5 33 The proposed structures of reaction adducts formed by resveratrol with glyoxal, methylglyoxal, malondialdehyde and acrolein. ................................ .... 118

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14 LIST OF ABBREVIATION S g Microgram L Microliter M Micromolar AGE Advanced Glycation E nd products ANOVA Analysis of Variance BSA Bovine Serum Albumin DAD Diode array detector DMSO Dimethyl sulfoxide FLD Fluorescent detector Fluorescent AGEs Fluorescent A dvanced Glycation End products. H Hour(s) HPLC High performance liquid chromatogram M Mole/Liter M g Milligram min Minute (s) mL Milliliter mM Millimolar m/z Mass to charge ratio nm Nanometer ORAC Oxygen radical absorbance capacity Trolox 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid

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15 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANTIGLYCATION EFFECTS AND REACTIVE CARBO NYL TRAPP ING CAPACITY OF BERRY AND GRAPE PHYTOCHEMICALS By Wei Wang December 2010 Chair: Liwei Gu Major: Food Science and Human Nutrition Oxidation of carbohydrates and proteins in foods and human body generate s reactive carbonyls, which include glyoxa l, methylglyoxal, malondialdehyde and acrolein. Reactive carbonyls react with proteins to form a dvanced glycation end produ cts (AGEs) and cause aging diabetic complications and other chronic diseases P revious studies suggested that plant extracts and cer tain phenolic compounds we re able to inhibit AGE format ion or directly scavenge reactive carbonyl compounds Berries and Mus cadine grape s are known as good sources of antioxidants and phenolic compounds. Therefore, w e hypothesized that they may have the in hibito ry effects on AGE formation and direct carbonyl trapping capacities. Phytochemicals were extracted and purified on adsorption resin to remove sugar s Sugar free phytochemicals purified from blueberries were fractionated into five fractions using a Se phadex LH 20 column. The resultant extracts and fractions were tested in three AGE generation models that simulate d physiological conditions. The model systems included bovine serum albumin (BSA) fructose, BSA methylglyoxal a nd arginine methylglyoxal model s AGEs were detected using florescence. The capacity of sugar free phytochemicals to scavenge reactive carbonyl was tested on methylglyoxal.

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16 Catechin, epicatechin, quercetin, chlorogenic acid and resveratrol were used to react with glyoxal, methylglyoxal, malondialdehyde and acrolein. Reaction kinetics were evaluated and phytochemical carbonyl adducts were tentatively identified using HPLC ESI MS n R esults showed that sugar free phytochemicals from berries and grape s and blueberry phytochemical subfractio ns, inhibited AGE generation in BSA fructose, BSA methylglyoxal and arginine methylglyoxal models by 22 8 8 % Berry and grape phytochemicals and blueberry subfractions scavenged methylglyoxal by 4 7 80 % within six hours. Pure phytochemicals ( catechin, epicat echin, quercetin and resveratrol) reacted with glyoxla, methylglyoxal, malondialdehyde and acrolein and form ed various phytochemical carbonyl adducts. Results in this study indicated that phytochemicals from berries and grapes are effective AGE inhibit ory agents that are useful to alleviate AGE related chronic disease s Carbonyl sca venging and adduct formation was one of the mechanisms for berry phytochemicals to inhibit AGE formation.

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17 CHAPTER 1 BACKGROUND Introduction Reactive carbonyl compound s, also cal led reactive carbonyl species, have been studi ed for decades due to their negative impacts o n human health. Methods have been developed to detect their presence in various foods and in the environment. M ost of the research has focus ed on their chemical cha racteristics their endogenous generation and reaction s with biological macro molecules The capacity of carbonyls to modif y proteins and gener at e advanced glycation end products (AGEs) is a major caus e of various disease s A new trend in research is to dis cover food source compounds that are able to scavenge reactive carbonyl specie s and prevent the production of AGEs Based on literature study, we hypothesized that phytochemicals from edible berries and muscadine grape s are able to scavenge reactive carbon yl species by adduct formation, thus reducing the gener ation of AGEs Reactive Carbonyl Compound s Reactive carbonyl compounds are a group of compounds that contain one or more carbonyl groups. The most reactive carbonyls are dicarbonyls and u nsaturated aldehydes. Dicarbon yl compound s contain two carbonyl groups in each molecule, such as glyoxal, methylglyoxal and malondialdehyde. unsaturated aldehydes contain a carbon carbon double bond in conjugat ion with an aldehyde group, such as a crolein and 4 hydroxynonenal. R eactive carbonyl compounds react with proteins to ge nerat e AGEs which cause various chronic diseases.

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18 Sources of Reactive Carbonyl C ompounds Reactive carbonyls are absorbed from foods and the environment or they are generat ed endogenously. In the environment, small amount s of acetaldehyde, acrolein, glyoxal and methylglyoxal exist ( 1 ) Higher a mount s of acrolein, methylglyoxal and glyoxal were detected in the air from urban area s than from rural area s ( 1 ) Acrolein and methylglyoxal were the major constituents in cooking fume s and cigarette smoke ( 2 ) Studies show that reactive carbonyls fro m the air are absorbed into the blood stream by inhalation ( 3 4 ) Reactive carbonyls are also absorbed from diet. These compounds were found in various foods, especially foods which have high lipid content and were processed at a high temperature ( 5 ) Acrolein, methylglyoxal and glyoxal ha ve been found in heated foods, such as baked meats. Methylglyoxal was also detected in many sugar containing food s i ncluding soy sauce and carbonated soft drinks ( 5 6 ) The mechanisms of the formation of reactive carbonyls are si milar under physiological condition or during food process ing Reactive carbonyls are produced by oxidation in vivo and in vitro Under physiological condition, they are produced via enzymatic metabolism In the first mechanism, carbohydrate s lipid s and amino acids, which are abundantly present throughout the body and in foods, are the precursors of reactive carbonyls. Glycoaldehyde, methylglyoxal and glyoxal are generated from carbohydrate or ascorbate metabolism or from their autoxidation ( 1, 7 ) Lipids, especially polyunsaturated fatty acids, produce malondialdehyde, acrolein and 4 hydroxynonenal through degradation, oxidation and cleavage reactions ( 8 10 ) Reactive carb onyls are also produced as intermediates of amino acid metabolism ( 10 11 )

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19 The oxidation of protein glyca tion products is an other source of reactive carbonyl compounds Protein glycation, known as the Maillard reaction when it occurs in vitro is the non enzymatic reaction between reducing sugars and proteins, eventually generating products with browning, flu orescence and cross linking characteristics. During the reaction, reducing sugars, for instance glucose, react with amino groups of proteins, producing Schiff bases. Schiff bases undergo Am adori rearrangement to produce relatively stable compound s called A madori product s At low pH or oxidative conditions, the degradation of Schiff bases or Amadori products generate s reactive carbonyls, such as methylglyoxal ( 1, 12 15 ) The se reactive carbonyls continue to attack ma cromolecules at a much fas ter speed than do reducing sugars Additionally, r eactive carbonyls are generated during enzymatic catalyzed metabolism under physiological conditions. For example, w hen polyamine oxidase catalyzes the oxidation of spermine to for m 3 aminopropanal, acrolein is automatically generated from aminopropanal ( 16 17 ) Similarly, acrolein is generated when myeloperoxidase catalyzes oxidation of threonine ( 18 ) Methylglyoxal can be formed from ketone bodies, catalyzed by cytochrome P450 enzyme s ( 19 ) Reactivity of Reactive Carbonyl C ompounds Most carbonyls produced from macronutrient oxidation and Amadori product s degradation are highly reactive. The most reactive carbonyls are dicarbonyls (glyoxal, methylglyoxal and malondialdehyde) and unsaturated aldehydes (acrolein and 4 hydroxynonenal) ( Figure 1 1 ) ( 8 ) T he carbonyl groups of these compounds are electrophilic readily attack ing the amino groups of amino acids in proteins. The reaction rate between reactive carbonyl compounds and proteins is much faster than that between sugars and proteins. For example, glyoxal and methylglyoxal react 20 times

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20 faster than sugar s to form protein adducts ( 20 ) unsaturated aldehydes contain a C2=C3 unsaturated double bond linked to the C1 aldehyde group. Therefore, the C3 carb on is a strong electrophile which can undergo Michael addition to the nucleophilic groups on proteins, DNAs and lipids ( 11, 13, 21 ) These reactions toward macromolecules cause structural and functional change s of proteins, DNAs and lipids. Reactive carbonyls cause various adverse biological effects mainly through the formation of AGEs Advanced Glycation End products (AGEs) Advanced glycation end products are a group of compounds with diverse molecular stru ctures and biological functions. They are primarily formed from reactive carbonyls and proteins ( 12, 22 ) and ha ve various characteristics including browning, fluorescence and cross linking. When similar reactions occur in food, some of the AGEs are known as Maillard reaction products ( 23 ) Generally AGEs are formed from the reaction between sugars and proteins, followed by the oxidation of Amadori products. P entosidine and N (carboxylmethyl) lysine are such examples. They can also be generated from the cross linking between reactive carbonyls and proteins. Reactive carbonyls target the amino, guanidinum and sulphydryl functional groups of intracellular and ext racellular proteins. Reactions between reactive carbonyl compounds and amino group on lysine or the guanidinium group on arginine results in AGEs such as N (carboxymethyl) lysine, pyrraline, methylglyoxal derived lysine dimer and glyoxal derived lysine dimer ( 12 ) Structures of N carbonxylmethyl lysine, pyrraline and pentosidine are show n in figure 1 2. Since reactive carbonyls are able to modify protein structures, the corresponding protein functions can be changed. Reactive carbonyls are able to react with pr oteins

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21 with various functions, causing deactivation of membrane transporters, enzymes, signaling co mponents, transcription factors, eventually causing protein degradation or cytotoxic effects ( 24 26 ) For example, methylglyoxal inhibit s mitochondrial respiration and glycolysis by inactivating membrane ATPase and glyceraldehydes 3 phosphate dehydrogenase ( 14 ) Reactive carbonyls reduce the intracellular level of glutathione a nd lead to increased oxidative stress ( 27 ) The formation of AGEs is accelerate d under oxidative condition s In the presence of transition metal ions, sugars undergo au to o xidation to form hydrogen peroxide and keto aldehydes which speed up the formation of AGEs ( 28 ) Once formed, AGEs are more susceptible to degradation and proteolysis when compared to the original proteins. A ccumulated AGE s have negative bi ological effects causing various cellular process disruptions. Studies have show n that AGEs are the causing factor of many pathological conditions, including diabetic complication s disease, cataract s aging and other chronic diseases ( 1, 9, 13, 29 30 ) AGEs contribute to diabetic complications through a series of pathological changes, such as increasing atherogenicity of low density lipoprotein, increasing membrane permeability and dec reasing insulin binding to its receptors ( 11, 31 32 ) AGE s also act as li gands and bind to the AGE receptors on the cell membrane to in duce signal cascades, causing in appropriate cellular activities and gene expres sions ( 12 ) Advanced or intermediate glycation products induce fre e radical production in vitro and in vivo ( 33 34 ) Therefore, the formation of AGEs can trigger an oxidative environment in which more AGEs are generated

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22 Berries and Muscadine Grape Berries such as blueberries, strawberries, r aspberries, blackberries and cranberries are popular fruits throughout the United States. Muscadine grapes are cultivated and consumed in the southwestern United States. They are known to contain a wide variety of antioxidant phytochemicals with diverse he alth benefits Health Related Benefits E pidemiological studies suggest a phytochemical rich diet with fruits and vegetables decrease s the incidence of chronic diseases. Most edible berries have high antioxidant capacities which are considered important f actors for health. They are effective in stimulating apoptosis and inhibiting the proliferation of cancer cells both in vivo and in vitro ( 35 37 ) Blueberr ies inhibited the development of hemangioendothelioma ( 38 ) and reduced DNA damage and lipid peroxidation in vivo ( 39 40 ) Phenolic compounds from berry extracts inhibited the growth of food borne bacteria and gastrointestinal path ogens, including Escherichia coli Salmonella enteric and Staphylococcus aureus (41) Clinical research has also show n that c ranberry effectively inhibit s urinary tract infection s ( 42 ) Phytochemicals in B erries and Muscadine Grape s Berries and grapes contain phenolic phytochemicals of various structures M ajor phenolic compounds in berries are anthocyanins, hydrolysable and condensed tannins, flavonoids, phenolic acids and stilbenoids Flavonoids in berries include anthocyanins, flavonols and flavan 3 ols. Anthocyanins give the red, blue and purple color to berries. Oligomers and polymers of proanthocyanidins we re found in blueberries strawberr ies cranberr ies blackberr ies, and raspberr ies ( 43 ) E llagictannins were found in strawberries blackberries raspberries and Muscadine grapes ( 44 48 )

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23 Reactions B etween P hytochemical s and Reactive Carbonyl s Because r eactive carbonyl compounds react with proteins to generate AGEs causing diseases and aging drugs have been designed to scavenge reactive carbonyls and reduce the production of AGEs Several synthetic compounds with carbo nyl trapping capacity have been tested to prevent and treat diabetic complications. Th e se compounds include aminoguanidine, OPB 9195 [( ) 2 isopropyli denehydrazono 4 oxo thiazolidin 5 yl acetanilide] and penicillamine. However, clinical applications of thes e medications were not successful due to high dose requirement and hepatotoxicity in diabetic patients ( 49 ) Previous research suggested that phenolic compounds in foods can scavenge reactive carbonyls and inhibit AGE generatio n. For instance, c atechin, epicatechin, epigallocatechin, epigallocatechin 3 digallate from black tea were found to inhibit different stages of protein glycation ( 6, 50 ) Proanthocyanidin monomer and oligomers from cinnamon bark directly trap ped methylglyoxal by forming adducts ( 51 ) Phloretin and other phenoli c compounds effectively quenched unsaturated aldehydes ( 52 ) Carbonyl phytochemical adducts have been isolated and identified for catechin, epigallocactechin 3 gllate, ploretin and resveratrol ( 52 56 ) Research Objectives 1. To evaluate fluorescent AGE generation in different model systems 2. To extract sugar free phytochemicals from berries and muscadine grapes; and to investigate the ir capacities of inhibiting AGE generation and scavenging reactive carbonyls. 3. To fractionate blueberry phytochemicals and investigate the ir carbonyl scavenging capacities and antiglycation effects.

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24 4. To investigate reaction kinetics between pure phytochemicals and reactive carbonyls and tentatively ide ntify phytochemical carbonyl adducts on HPLC ESI MS n Figure 1 1. Structure s of reactive carbonyls Figure 1 2. Structure s of selected advanced glycation end products (AGEs)

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25 CHAPTER 2 FLUORESCENT AGES PRO DUCTION FROM IN VITRO INCUBATION Stable model systems are needed to study the inhibitory effects of phytochemicals on AGE production. An ideal model system provides a good simulation of physiological conditions and produces a sufficient amount of AGEs that can be easily detected. Several A GE generation model systems have b een used in previous research The performances of these model systems have not been evaluated. The objecti ve of this chapter wa s to evaluate fluorescent AGE s generation in different model systems and identify appropriate models for future studies Materials and Methods Chemical s Glyoxal (40% solution in water) was a product from Acros Organics ( Morris Plains, NJ ). Methylglyoxal ( 40% aqueous solution ) was purchased from MP Biomedicals, LLC ( Solon, OH). Bovine serum albumin (BSA) glucose, fructose, ribose, galactose, ascorbic acid, arginine, lysine sodium azide monobasic and dibasic sodium phosphate and 96 well plates with clear bottom wells were purchased from Fi s her Scientific Co. (Pit tsburg, PA) Model System for F luorescent AGEs F ormation BSA monosaccharide model : BSA (2 0 mg/ml 1 ml ) was mixed separately with 1ml of glucose, fructose, ribose, galactose or ascorbic acid ( 1.0 M). The mixtures were incubated at 37 C with s odium azide (0.02%, w/v) serving as an aseptic agent. BSA Carbonyl model : BSA ( 2 0 mg/ml 1ml ) w as mixed individually with 1 ml of glyoxal or methylglyoxal ( 4 0 mM) The mixtures were incubated at 37 C with 0.02% (w/v) of sodium azide.

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26 Amino acid monosacchride model : lysine and arginine ( 4 0 mM 1ml ) were mixed separately with 1 ml of glucose, fructose or ribose ( 1.0 M). The mixtures were incubated at 37 C with 0.02% (w/v) of sodium azide. Amino acid carbonyl model : methylglyoxal and arginine ( 40 mM each 1ml each ), gly oxal and lysine ( 4 0 mM each 1ml each ) were mixed and incubated. The mixtures were incubated at 37 C with 0.02% (w/v) of sodium azide. All reagents were dissolved in 50 mM phosphate buffer ( pH 7.4) Incubation m ixtures were placed in 10 ml s crew capped gl ass tubes and were kept in an incubator at 37 C ( Model 304, Lab Line Instrument, Melrose Park, IL ) Individual r eactants in phosphate buffer were i ncubated in the same condition s as the blank controls Incubations were conducted in triplicate s Emission and Excitation Spectra of Fluorescent AGEs After incubation for three days, incubation media (20 l) was injected into a n Agilent 1200 fluorescence detector ( Agilent Technologies, Palo Alto, CA ) without column separation. Emission spectra were obtained usi ng 280 nm as the excitation wavelength. Excitation spectra were obtained using 420 nm as the emission wavelength. Spectra were recorded using ChemStation software (Version B 01.03, Agilent Technologies, Palo Alto, CA). Fluorescent Intensity Measurement Ev ery 24 h, a 200 l aliquot from each incubation media were transferred into a 96 well plate with clear flat bottom to measure fluorescen t intensity on a microplate reader (Spectra XMS Gemini Molecular Device, Sunnyvale, CA ). F luorescent intensity

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27 was rec orded and plotted against incubation time until the fluorescent reading reache d a plateau. Data Expression and Grafting Data are expressed as mean standard deviation for triplicate tests. Data grafting wa s done with SigmaPlot (Version 11.0 Systat Softwar e Inc, San Jose, CA). Results Fluorescent AGEs Formation Excitation and emission spectra of BSA fructose system are shown in Figure 2 1. Wavelength of maxima e xcitation and emission for different incubation model s are listed in Table 2 1. The m ajority of t he incubation model s gave maxima excita tion at about 340 nm Wavelength of maxima emission fell in a range of 380 nm to 420 nm. Wavelength of maxima excitation and emission for BSA solution were 270nm and 400 nm, respectively. After BSA was incubated with carbonyl sources, its wavelength of maxima excitation and emission shifted, suggesting structural modification of BSA and generation of AGEs. This is in agreement with a previous study, which showed the maxima excitation and emission of BSA changed to 325 nm and 382 nm respectively, after incubating with methylglyoxal at 37 C for six days. Modification of BSA by reactive carbonyl compounds in cludes irreversible and reversible modifications, targeting mainly arginine, lysine and cysteine residues. For inst ance, methylglyoxal modifies BSA irreversibly mainly with arginine residues, and reversibly binds to BSA with arginine, lysine and cysteine residues in the ratio 110:51:0.6. Complex protein carbonyl adducts, such as imidazolone, contribute to fluorescence ( 57 ) Fluorescent intensities increased with incubation time in most model systems. Among BSA monosaccharide models (Figure 2 1) the BSA ribose mixture showed the

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28 most remarkable increase in fluorescent signal However it had hi gher standard deviation s than other BSA monosaccaride models. Fluorescent intensit ies in BSA fructose and BSA galactose systems increased gradually with incubation time and reached maxima value within a week. Data from these two systems showed lower standa rd deviation s suggesting these two systems are more stable. The increase of fluorescent intensities from BSA glucose and BSA ascorbic acid models w ere not as prominent as the other three models. The fluorescent intensity generated in the BSA fructose mode l was much higher than that in the BSA glucose model This is in agreement with the work by Lee et al ( 25 ) The BSA fructose model was chosen for anti glycati on experiment in future studies When BSA was incubated with glyoxal or methylglyoxal (Figure 2 3 ) the fluorescent intensity increased similarly to that in the BSA monosaccharide models. The fluorescent intensity was much less in the BSA methylglyoxal m odel than in the BSA glyoxal model after one day of incubation. However, since the reading s in the BSA methylglyoxal model increased more rapidly there was no significant difference between the two models after six day s of incubation. The BSA methylgly oxal model was chosen to represent protein and reactive carbonyl compound reactions. Models of amino acids and monosaccharide s (fructose/ ribose/ glucose) (Figure 2 4 ) were designed to produce pentosidine, a specific advanced glycation end product. However the fluorescent intensi ty generated from these models showed a lot of fluctuation It suggested that fluorescent detection may not be suitable to monitor AGE generation in amino acid monosaccharide models T herefore no model was chosen from this categor y.

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29 In the case of amino acid reactive carbonyl compound models (Figure 2 5 ) the fluorescent reading increased dramatically in the arginine methylglyoxal model with a small standard deviation during the incubation period. But the fluorescent reading wa s only slightly decreased in the lysine glyoxal model. Thus the arginine methylglyoxal model was selected to represent reactions between amino acids and reactive carbonyl compounds. Discussion Reactive carbonyl compounds originated from reducing sugars react with proteins to ge nerat e advanced glycation end products More than t hirty AGEs have been identified. T hey were generated from carbonyl compounds and proteins of different structures and modification sites ( 58 ) F luorescent detection is a convenient way to monitor AGE generation ( 14, 59 60 ) However, this method is not able to detect AGEs that do not fluoresce such as N carbonylmethyl lysine ( 11 ) BSA fructose, BSA methylglyoxal and arginine methylglyoxal w ere selec ted for further experime nts, represent ing the reactions between protein s and sugar, protein s and carbonyls and amino acid s and carbonyl s Methylglyoxal was reported as a potent agent for AGE generation. It modified proteins reversibly or irreversibly ( 57 ) by targeting the side chains of arginine ( 14 ) at a much faster rate than reducing sugars P hosphate buffer saline (50 mM) was used as the matrix because the protein modification by carbon yls required a sufficient ionic strength ( 14, 57 ) T he concentration of phosphate buffer used for this experiment was slightly higher than the physiological condition and was sufficient to maintain protein glycatio n. However further increase of the concentration of phosphate buffer to 250 and 500 mM did not shorten the incubation period ( 14, 57, 61 ) In the BSA fructose model, fluorescent reading reached a plateau

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30 after six days It suggested the modification sites on BSA, such as side chains of arginines, lysines and cysteines were saturated. In conclusion, BSA fructose, BSA methylglyoxal and arginine methylglyoxal models were stable systems in generating fluorescent AGE s These model systems are suitable to test the antiglycation effect of berries in future studies

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31 Table 2 1. Wavelength of maxima e xcitation and emission of fluorescent AGEs in various model systems. Incubation s Excitation (nm) Emission (nm) BSA glucose 280 410 BSA fructose 340 420 BSA galactose 340 410 BSA ribose 340 420 BSA ascorbic acid 280 305 BSA glyoxal 360 430 BSA methylglyoxal 280 375 Arginine lysine glucose 320 410 Arginine lysine fructose 322 425 Arginine lysine ribose 340 380 Arginine methylglyoxal 340 380 Lysine glyoxal 350 420 Figure 2 1. E xcitation (A) and emission (B) s pectr a of BSA fructose mod el

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32 Figure 2 2 Fluorescent AGE generatio n in bovine serum albumin (BSA) monosaccharide inc ubation models at 37 C. Data are mean standard deviation for three independent tests. Figure 2 3 Fluorescent AGE generation in bovine serum albumin (BSA)/ reactive carbonyl incubation models at 37 C. Data are mean standard deviation for three independent tests.

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33 Figure 2 4 Fluorescent AGE generation in amino acids monosaccharide incubation models at 37 C. Data are mean standard deviation for three independent tests. Figure 2 5 Fluorescent AGE generation in amino acid reactive carbonyl incubation models at 37 C. Data are mean standard deviation for three independent tests.

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34 CHAPTER 3 ANTIGLYCATION EFFECT OF BERRY AND GRAPE P HYTOCHEMICA LS Over production of carbonyl s cause s AGEs production and other adverse biological effects that are collectively called carbonyl stress. Carbonyl stress can be alleviated by using carbonyl scavengers. Berries and grapes are known as rich sources of antiox idant s and phenolic phytochemicals ( 44, 48, 62 63 ) Eating berries has been shown to reduce oxidative stress in animals and in human s ( 38 40, 64 65 ) However, it is not kno wn if berries and grapes can reduce carbonyl stress. The objective of this chapter was to investigate the carbonyl trapping capacities and inhibitory effects on AGE generation by berry and grape extracts. Materials and Methods Chemicals and M aterials S tra wberr ies cranberr ies and raspberries were purchased from local supermarkets. Southern high bush blueberries, blackberr ies and noble muscadine grape s were obtained fro m Straughn farm (Waldo, Florida), Coggins Farms (Plan City, ard (Florahome, Florida), respectively. azotis(2 amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI). 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid (Trolox) (+) catechin and ( ) epicatechin were purchased from Sigma A ldrich (St. Louis, MO). Aminoguanidin was a product from Acros Organics ( Morris Plains, NJ ). Amberlite XAD 7 resin was a product of Rohm and Haas Co. (Philadelphia, PA) HPLC grade m ethanol and other c hemicals were purchased from Fis her Scientific Co. (Pit tsburg, PA).

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35 E xtraction and P urificati on of Sugar f ree P hytochemicals The phytochemicals extraction and purification procedure is depicted in a flow chart in Figure 3 1. Frozen berries (blueberr ies strawberr ies cranberr ies raspberr ies and blackberr ies ) and muscadine grape s (Noble cultivar) were thawed under room temperature. F ruits (200 g) were blend ed with methanol (200 ml with 1% formic acid) in a kitchen blender The mixture was sonicated in a water bath sonicater ( FS30, Fisher Scientific ) for 10 min, then kept at room temperature for 20 min and filtered through Whatman no.4 filter paper s The extraction was repeated once on the remaining solid and the two methanol extracts were combined. The extract s w ere transferred to weighted contai ners and solvent was removed using a SpeedVac c oncentrator (Thermo scientific ISS110, Waltham, MA) at 25 C The dried crude extracts were stored at 4 C Amberlite XAD 7 resin was used to remove sugar s from the extracts R esin was suspended in 80% methanol and packed into a glass column. R esin (70g) in the column was cleaned by the following process: washed by 300 ml of methanol, 800 ml of distilled water, 300 ml of 0. 2% sodium hydroxide followed by distilled water until elute was neutral, then 300 ml of methanol followed by distilled water until all methanol was removed. After cleaning, the resin was read y for sample loading. Dried berry crude extract was re dissolved in 20 ml of distilled water containing 1% formic acid and loaded to an Amberlite XAD 7 resin column After sample loading elution was halted for 10 min to facilitate the a d sorption of phyto chemicals on resin beads. Sugars in the crude extrac ts were removed by elution with 600 ml of acidified (1% formic acid) water P hytochemicals absorbed on resin were recovered b y 250 ml of

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36 methanol (80%, with 1% formic acid) Elutes from resin column were dried on SpeedVac concentrator, and t he w eights of the dried phytochemicals were recorded. Analysis of Sugars in Phytochemical E xtracts S ugar analysis was conducted using a Hitachi HPLC system with a refractive index detector and a Shodex SP0810 column ( 300 mm 8 mm, Shodex, Colorado springs, CO) with a SP G guard column (2 mm x 4 mm). An isocratic solvent delivery of water was run at 1.0 mL/min. Crude extracts (from raspberries and cranberr ies ) and purified s ugar free extracts ( from blueberries, strawb erries, cranberries raspberr ies blackberries and noble grape s ) were dissolved in distilled water to a concentration of 15 mg/ml. Sample injection volume was 5 L. S ucrose, glucose, fructose, lactose and maltodextrin were used as standards. Each sample wa s analyzed in duplicate s Folin Ciocalteu A ssay The total phenolic contents of berry and grape sugar free extracts were determined by Folin Ciocalteu assay which was modified from a published method ( 66 ) S ugar free phytochemical extracts were dissolved in phosphate buffer (50 mM, pH 7.4) with 5% dimethyl sulfoxide (DMSO) at a concentration of 0.75 mg/ml. One hundred microliter of each was mixed with Folin Ciocalteu reagent (1 ml, 0.2 N) and sodium carbonate (1 ml, 15%) Gallic acid solutions with concentrations rang ing from 100 600 mg/L were used to generate a standard curve. Absor ption at 765 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA) after 30 min of incubation at room temperature. Results of total phenolics were expressed as milligram s gallic acid equivalents per milligram of sugar free extract (mg GAE/mg).

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37 Oxygen R adical Absorbance C apacity The oxygen radical absorbance capacity (ORAC) as say used a published method with minor modification ( 67 ) All suga r free e xtracts were dissolved in phosphate buffer (50 mM, pH 7.4) with 5% dimethyl sulfoxide (DMSO) at a concentration of 15 g/ml. Fifty microliter s were mixed with fluorescein solution (100 l, 20 nM) in a 96 well microplate with clear flat bottom The m ixture s were incubated at 37 C for 10 min azobis(2 amidinopropane) dihydrochloride (AAPH 50 l, 0.14 M ). Fluorescence was measured on a fluorescent microplate reader (Spectra XMS Gemini, Molecular Device, Sunnyvale, CA) using 485 an d 530 nm as the excitation and emission wavelengths. Readings were taken at 1 min interval s for 40 min. 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid (Trolox) solutions with concentration of 3.125, 6.25, 12.5 and 25 M were used to generate a stan dard curve. The results of the antioxidant capacity were expressed as mol Trolox equivalents per milligram sugar free extract ( mol TE/mg). Total A nthocyanin C ontent Total anthocyanin content was measured by using a pH differential assay ( 68 ) Blueberry and blackberry extracts (5 mg/ml in methanol) were diluted 80 times in potassium chloride buffer (0.025 M, pH 1) and sodium acetate buffer (0.4 M pH 4.5), respectively. Other berry and grape extracts (5 mg/ml in methanol) were diluted 20 times in the same buffers Absorbance at 520 nm and 700 nm were measured on a Life Science UV/Vis spectrophotometer (DU 730, Beckman Coulter, Fullerton, CA) afte r 30 min of incubation at room temperature. Absorbance (A) was calculated using (A520 A700) pH 1.0 (A520 A700) pH 4.5 Total anthocyanin content ( g Cy G/ m g) was calculated using (A 449.2 80 1000) / (26900 1) for blueberry and blackberry

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38 extrac ts and (A 449.2 20 1000) / (26900 1) for other berry and grape extracts. Results of total anthocyanin content for berry and grape extracts were expressed as milligram cyanidin 3 glucoside equivalent per gram of dried berry and grape extracts ( g Cy G/ m g). Total P rocyanidin C ontent Total procyanidin in berry and grape extracts were determined using 4 dimethylaminocinnamaldehyde (DMAC) colorimetric method ( 69 ) Five milligram of berry and grape extracts were dissolved in 5 ml of acetone: water: acetic acid ( 70:29.5:0.5, v:v:v) then diluted to a proper concentration with ethanol. An aliquot of 70 l of diluted berry and grape extracts were mixed with DMAC solution (0.1%, 210 l) in a 96 well plate. Epicatechin solutions with concentrations ranging from 1 15 g /ml were used to generate a standard curve. Absorption at 640 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA) after 30 min of incubation in dark ness Results of total procyanidins were expressed as micrograms epica techin equivalents per mi lligram of sugar free extract ( g epicatechin /mg). Antiglycation E ffect s in BSA F ructose M odel F ructose ( 1 .5 M 1 ml ) was mixe d with the sugar free phytochemical extracts catechin or epicatechin (0. 1 5 mg/ml 1 ml ) in sodium phosph ate bu ffer ( 50 mM, pH 7.4 with 0.02% sodium azide ) in capped test tubes and incubated at 37 C for 2 h. BSA ( 3 0 mg/ml 1ml ) was added in each test tube and the mixtures were incubated in an incubator ( Model 304, Lab Line Instrument, Melrose Park, IL ) at 3 7 C for six days. BSA and f ructose with a phosphate buffer (1 ml) or aminoguanidin (3 0 mM 1ml ) were used for negative and positive control s Fluorescent AGE s from each incubation were monitored by taking the fluore scent reading of mixture s (200 l each) using a microplate

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39 reader (Spectra XMS Gemini, Molecular Device, Sunnyvale, CA) using 340 and 420 nm as the excitation and emission wavelengths. Samples, blank s and positive control s were prepared in triplicate Percentage of the AGE inhibition of extracts was calculated by the following equation: Antiglycation E ffects in BSA M ethylglyoxa l M odel Procedure of antiglycation effect in the BSA methylglyoxal model wa s similar to that of the BSA fructose model. M ethylglyoxal ( 6 0 mM 1 ml ) was incubated individually with phosphate buffer ( 1 ml, negative control ), phytochemical extracts ( 1 .5 mg/ml 1 ml ) catechin (1. 5 mg/ml, 1 ml), epicatechin (1.5 mg/ml, 1 ml) or aminoguanidin ( 3 0 mM 1 ml, positive control ) in sodium phosphate buffer ( 50 mM, pH 7.4, with 0.02 % sodium azide) at 37 C for 2 h. BSA ( 3 0 mg/ml 1 ml ) was added to each test tube and incubated at 37 C for six days. The f luorescent intensity was taken for each mixture (200 l) using 340 and 380 nm as the excitation and emission wavelengths Samples, blank s and positive control s were prepared in triplicate Percentage of the AGE inhibition of extracts was ca lculated by the following equation: Antiglycation Effects in Arginine methylglyoxal M odel M ethylgl yoxal ( 6 0 mM 1 ml ) was incubated with phosphate buffer ( 1 ml, negative control ), phytochemical extracts ( 0.7 5 mg/ml, 1 ml ) catechin (0.75 mg/ml, 1 ml), epicatechin (0.75 mg/ml, 1 ml) or aminoguanidin ( 10 mM 1 ml, positive control ) in 50 mM sodium phosph ate buffer (pH 7.4, with 0.02% sodium azide) at 37 C for 2 h.

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40 Arginine ( 6 0 mM 1ml ) was added to each test tube and incubated at 37 C for six days. Fluorescent signal was taken for each mixture (200 l) using 340 and 380 nm as the excitation and emission wavelengths Samples, blank s and positive control s were prepared in triplicate Percentage of the AGE inhibition of extracts was calculated by the following equation : Direct Methylglyoxal T rapping Test of the direct methylglyoxal trapping used a published method with minor modifications ( 51 ) Methylglyoxal (10 mM) and o phenylenediamine (derivatization agent, 50 mM) were freshly prepared in phosphate buffer (50 mM, pH 7.4). Berry and grape phytochemicals were diluted to 2.5 mg/ml. Catechin (5 mM) and aminoguanidine (10 mM) were used as a pure compound control and a positive control, respectively. Methylglyoxal solution (0.125 ml) was mixed with 0.125 ml of phosphate buffer ( negative control ) or berry phyt ochemicals and incubated at 37 C. After i ncubating for 0.5 h, 1 h, 2 h, 4 h and 6 h, o phenylenediamine (0.25 ml) was added into each sample and kept for 30 min for the derivatization reaction to complete. HPLC analysis of incubation media was performed o n an Agilent 1200 HPLC system (Agilent technologies, Palo Alto, CA) Compound separation was carried out in a zorbax stablebond analytical SB C18 column (4.6 250 mm, 5 m, Agilent Technologies, Palo Alto, CA ). Mobile phases were composed of 0.1% formic aci d in millipore water (mobile phase A) and pure methanol (mobile phase B). The flow rate was 1 ml/min and the injection volume was 15 l. The linear gradient for elution was: 0 3 min, 5 50% B; 3 16 min, 50 50% B; 16 17 min, 50 90% B; 17 19 min, 90 90% B; 19 19.5 min, 90 5% B;

PAGE 41

41 followed by 1 min of re equilibration of the column Methylglyoxal o phenylenediamine adduct, 1 methylquinoxaline was detected at 315 nm using a diode array detector. Its retention time was 12.9 min. The p eak area of 1 methylquinoxalin e in each sample was integrated. The p ercentage of remain ing methylglyoxal was calculated by the following equation: Data E xpression, Grafting and S tatistics Samp les were analyzed in triplicate and data was expressed as mean standard deviation unless otherwise noted One way analyses of variance with Tukey Kramer HSD pair wise comparison of the me ans were performed using JMP software (Version 8 s ignificant. Data grafting was done using SigmaPlot (Version 11.0, Systat Software Inc, San Jose, CA). Results Phytochemical E xtractio n and P urification F rozen berries and grapes approximately 200 g, were extracted. After extraction, filtration and solvent evaporation, about 20 g of crude extracts were obtained. The c rude extracts were sticky due to the high content of sugar s S ugar s we re not detected after crude extracts were purified on resin s. About one gram of sugar free extract s was obtained The weights of frozen fruits, crude extracts and sugar free extracts are listed on Table 3 1.

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42 Sugar concentration was measured in two crude e xtracts (raspberries and cranberries ) and in all the sugar free extracts. Sucrose, glucose and fructose were detected in raspberry crude extracts. Glucose and fructose were detected in cranberry crude extracts (Figure 3 2) For both crude extracts, they co ntained approximately 50 % of sugar s In contrast, no sugar was found in any o f the sugar free extracts (Table 3 2) which demonstrated that our sugar removal process was effective. Folin Ciocalteu Assay Sugar free berry and grape extract s contain ed more t han 0.38 g gallic acid equivalents per g extract ( Table 3 3 ) N oble grape s and blueberries had the highest total phenolic content (0.56 0.03 and 0.53 0.01 g gallic acid equivalents per g extract). The t otal phenolic content of strawberr ies cranberr ies and blackberr ies was significantly lower Raspberr ies had the lowest phenolic content, but did not significantly differ from the extracts of strawberr ies and cranberr ies Oxygen Radical Absorbance C apacity Based on ORAC results, the antioxidant capacities of berry and grape extracts ranged from 4.86 6.91 mol Trolox per mg extract ( Table 3 3 ). N oble grape s showed the highest antioxidant capacity value (6.91 0.08 mol Trolox/mg extract) However, unlike the result of the Folin Ciocalteu assay, blueberr ies sh owed the lowest antioxidant capacity value (4.86 0.05 mol Trolox/mg extract) in the ORAC assay. Total Anthocyanin C ontent Total anthocyanin content in berry and grape extracts ranged from 20.9 163.5 g Cyanidin 3 glucoside per mg extract ( Table 3 3 ). Blac kberry extract had the highest total anthocyanin content (163.5 2.19 g Cy G/mg) followed by blueberry extract

PAGE 43

43 (153.5 3.19 g Cy G/mg) Noble grape and raspberry extracts showed the lowest total anthocyanin content (23.6 and 20.9 g Cy G/mg, respectively) among all the extracts Total Procyanidin C ontent Total procyanidin content in berry and grape extracts ranged from 12.9 118.9 g epicatechin equivalent per mg extract ( Table 3 3 ). Noble grape extract had the highest total procyanidin content (118.9 5.44 g epicatechin equivalent /mg). Blueberry and raspberry extracts showed the lowest total procyanidin values (20.9 and 12.9 g epicatechin/mg, respectively) among all the extracts Anti glycation E ffect s in Selected M odel s Berry and grape e xtract s (0.05 mg/ ml) were able to inhibit formation of fluorescent AGEs in the BSA fructose model by over 60% in six days (Fig ure 3 3 ). Among sugar free berry or grape extracts, s trawberry extract showed the highest anti glycation effect (79.5%), and cranberry showed the l owest (60.1%). Results from the other extracts fell in between. Catechin and epicatechin inhibited fluorescent AGE generation by 81% and 72.5%, respectively. The positive control, aminoguanidine was able to inhibit fluorescent AGE generation by 95% In the BSA methylglyoxal model, antiglycation effects by extracts catechin and epicatechin (0.5 mg/ml) ranged from 34 % to 79% (Fig ure 3 4 ). Catechin showed the highest antiglycation effect (79%). Blueberry, cranberry, blackberry noble grape extracts and epicat echin were not significantly differe nt in terms of AGE inhibition in this assay They inhibited approximately 60% fluorescent AGE generation. Raspberry had the lowest antiglycation effect in this assay (34%) B erry and grape extracts (0.25 mg/ml) inhibit ed fluorescent AGE generation in the arginine methylglyoxal model over 45% (Figure 3 5 ) Blueberry blackberry and noble

PAGE 44

44 grape extract s had the highest inhibitory effect They inhibited 56.8%, 52.2% and 53.3% fluorescent AGE generation, respectively. In this assay, s trawberry extract had the lowest anti glycation effect (45.6%). Direct Methylglyoxal T rapping The direct methylglyoxal trapping curve was obtained by measuring the amount of methylglyoxal at various incubation period s after they reacted with the t ested samples. The chromatograms of methylglyoxal, methylglyoxal with blueberry sugar free extracts and methylglyoxal with aminoguanidine are show n in Figure 3 6 The direct methylglyoxal trapping curve of berry and grape extracts are presented in Figure 3 7 Result s showed that the raspberry extract was able to trap 46.9 % methylglyoxal within 6 h, whereas the other tested berries and grapes had higher capacities, trapping approximately 7 0 % methylglyoxal within 6 h. Discussion The results of this chapter showed that sugar free berry and grape extracts had high total phenol ic content, antioxidant capacit y and notable anti glycation effects. The antioxidant properties of many plant extracts ha ve been attributed to their phenolic content. Phenolic compounds play an important role in neutralizing free radicals, quenching singlet and triplet oxygen species and decomposing peroxides. It was known that a good correlation existed between the free radical scavenge activity and the inhibitory effect on AGE generatio n for pure phenolic compounds and phenolic rich plant extracts ( 70 71 ) O xidative stress was elevat ed under hyperglyc emic co nditions and free radicals accelerated the formation of AGEs ( 28 ) P henolic c ompounds inhibit AGE formation in part by functioning as free radical scavenger s In this study, phytochemical extracts with high total phenolic content and antioxidant capacities

PAGE 45

45 showed significant anti glycation effects This relation is in agreement wit h previous studies ( 72 75 ) I t wa s believed that direct trapping of reactive carbonyls wa s one of the major mechanisms for the inhibition in AGE formation. Result s from the direct methylglyoxal trapping assay showe d that sugar free berry and grape extracts drastically decreased the amount of methylglyoxal. Because methylglyoxal is an active intermediate for AGE formation, compounds from berry and grape extract s inhibit ed AGE generation by directly quenching reactive carbonyl compounds. A number of plant extracts showed anti glycation activity in vitro ( 51, 70, 73, 76 77 ) L eave s and stems of lowbush blueberries (Vaccinium angustifolium) in different seasons had anti glycation effects ( 78 ) However, anti glycation effects and carbonyl quenching activities of berries has not yet been studied. Result s in this study showed berry and grape extracts effectively prevented the formation of flu orescent AGEs and directly quenched methylglyoxal. I t is likely that a variety of compounds in berries and grapes act together to inhibit AGE formation. In conclusion, s ugar free phytochemicals from berries and grape s extracts inhibited AGE generation by 3 4 % 80% in BSA fructose, BSA methylglyoxal and arginine methylglyoxal models They scavenged 4 7 % 80% of the methylglyoxal within 6 h. Such effects were attributed to their high total phenolic contents and antioxidant capacities

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46 Table 3 1 Weight of froze n berries, crude extracts and sugar free extracts. Sample Name Frozen Berries (g) Crude Extracts (g) Sugar free Extracts (mg) Blueberry 200.8 26.0 839.6 Strawberry 200.8 19.1 728.8 Cranberry 200.0 20.5 1048.7 Blackberry 199.5 18.1 939.9 Raspber ry 200.1 14.2 496.5 Nobel grape 200.6 24.5 1318.9 Table 3 2. Sugar concentrati ons in crude and sugar free extracts Extracts Samples S ucrose ( % ) G lucose ( % ) F ructose ( % ) Total S ugar ( % ) Crude Extracts Raspberr ies 130. 2 160.4 240.04 530.9 Cran berr ies ND 4 0 0.0 150.0 550.0 Sugar free Extracts Blueberr ies ND ND ND ND Strawberr ies ND ND ND ND Raspberr ies ND ND ND ND Cranberr ies ND ND ND ND Blackberr ies ND ND ND ND Noble grape s ND ND ND ND Values are mean standard deviation of three tests. Table 3 3. Total phenolic, antioxidant capacity, total anthocyanins and total procyanidins in sugar free extracts. Extracts Total p henolics ( g GAE/ g) Antioxidant capacity ( mol Trolox/mg) Total anthocyanins ( g Cy G/mg) Total procyanidins ( g epicatechin/mg) Blueberries 0.50.0 a 4.9 0.0 e 153.5 3.2 b 20.9 0.2 de Strawberries 0.4 0.0 bc 5.1 0.1 d 43.2 0.0 d 27.7 1.7 de Cranberries 0.4 0.0 bc 5.6 0.0 c 51.9 0.2 c 47.1 4.7 c Raspberries 0.4 0.0 b 5.2 0.1 d 20.9 0.2 e 12.9 1.6 e B lackberries 0.4 0.0 c 6.0 0.1 b 163.5 2.2 a 57.9 3.0 b Noble grapes 0.6 0.0 a 6.9 0.1 a 23.6 0.1 e 118.9 5.4 a Values are mean standard deviation of three individual experiments for all the assays. Values in the same column with different letters denoted s ignificantly differences (P 0.05) from each other.

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47 Figure 3 1. Extraction and purification of sugar free phytochemicals from berries and noble muscadine grape s

PAGE 48

48 Figure 3 2 HPLC chro matogram of sugar s in (A) cran berry crude extract and (B ) cran berry sugar free extract. The peak s represent (1) sucrose, (2 ) glucose and (3 ) fructose.

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49 Figure 3 3 Anti glycation effects of berry extracts in bovine serum albumin fructose model Bars represent mean standard deviation of triplicate tests. Bar s with different letters denoted significant differences at p 0.05. The concentrations of berry extracts catechin and epicatechin were 0.05 mg/ml. Aminoguanidin (10 mM) served as the positive control.

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50 Figure 3 4 Anti glycation effects of berry extracts in bovine serum album in methylglyoxal model. Bars represent mean standard deviation of triplicate tests. Bars with different letters denoted significant differences at p 0.05. The concentrations of berry extracts catechin and epicatechin were 0.5 mg/ml. Aminoguanidin (10 mM ) served as the positive control.

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51 Figure 3 5 Anti glycation effects of berry extracts in arginine methylglyoxal model. Bars represent mean standard deviation of triplicate tests. Bars with different letters denoted significant differences at p 0.05. The concentrations of berry extracts catechin and epicatechin were 0.25 mg/ml. Aminoguanidin (10 mM) served as the positive control.

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52 Figure 3 6 Chromatogram of methylglyoxal (A), methylglyoxal after reaction with blueberry sugar free extract ( B) and methylglyoxal after reaction with aminoguanidine (C ). Methylglyoxal was detected as 1 methylquinoxaline after derivatization using o phenylenediamine at 315 nm.

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53 Figure 3 7 The percentage of remaining methylgly oxal after reacting with berry and grape extracts for 0.5, 1, 2, 4, 6h. The concentrations of berry extracts and catechin were 2.5 mg/ml and 5 mM, respectively. Data points represent the mean percentage of remaining methylglyoxal with the standard deviatio n for two independent experiments.

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54 CHAPTER 4 FRACTIONATION OF BLUEBERRY PHYTOCH EMICALS COMPOUND IDENTIFICATION AND ANTIGLYCATION EF FECT S Sugar free phytochemicals extracted from berries and noble grapes effectively inhibited the formation of AGEs in th ree model systems. Such effects were attributed to t heir antioxidant and carbonyl scavenging capacities. Florida is a major producer of blueberries in the US. Most of the Florida blueberries are the early season high bush cultivars. They are harvested in l ate April and early May when blueberries from other part s of the US are not available ( 79 ) Blueberries have higher market values and per capita consumption compared to raspberries, blackberries, cr anberries and muscadine grapes ( 80 81 ) The objective of this chapter was to fractionate blueberry phytochemicals identify major phytochemicals in each fraction, and investigate the ir carbonyl scavenging capacities and antiglycation effects. Materials and Methods Chemicals and Materials Sephadex LH 20 was a product from Sigma Aldrich ( Saint Louis, MO ) Other chemicals and materials were purchased from different companies as described in Chapter 2 Chemicals and Chapter3 Chemicals and M aterials Fractionation of S ugar free Blueberry P hytochemicals Procedure for fractionation of sugar free blueberry ex tracts is depicted in Figure 4 1. Frozen b lueberr ies (200 g) were used to prepare sugar free phytochemicals using a procedure described in Chapter 3, Extraction and Purification of Sugar free P hytochemicals The resultant dried extract was homogenized in w ater (10 ml) and loaded into Sephadex LH20 (80 g ) through a column ( 2.855 cm, 500 ml ) Fractions I to V were collected by eluting the column with distilled water (500 ml ), methanol ( 20%, 700

PAGE 55

55 ml ), methanol ( 50%, 700 ml ), methanol ( 70%, 7 00 ml ) and acetone ( 80%, 500 ml ). All solvents used to wash the Sephadex LH 20 column were acidified with 1% formic acid. S olvents in all the fractions were removed and the remaining solids were weighed Phytochemical I dentification on HPLC ESI MS n Compound identification f or blue berry fractions were performed o n an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA) equipped with a diode array detector, a fluorescence detector and a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Compounds were separated on a Z orbax stablebond analytical SB C18 column (4.6 250 mm, 5 m, Agilent Technologies, Palo Alto, CA ). Millipore water with 0.5% formic acid and pure methanol was used as the mobile phase A and the mobile phase B for elution. Flow rate was 1 ml /min. The linear gradient for elution was: 0 2 min, 5% B; 2 10 min, 5 20% B; 10 15 min, 20 30% B; 15 20 min, 30 35% B ; 20 60 min, 35 80% B; 60 65 min, 80 85% B; 65 70 min, 85 5% B ; followed by 5 min re equilibration of the column Wavelengths were set at 2 80, 360 and 520 nm for diode array detect ion Excitation and emission of the fluorescent detector were set at 230 and 320 nm respectively Electro spray ionization at both negative and positive mode (alternative) at the same run was performed using nebuliz er 45 psi, drying gas 11 L/min and drying temperature 350 C. A f ull scan was obtained from m/z 100 to 2200. The most abundant ion in the full scan spectrum was isolated and the product ion spectra ( MS 2 ) were recorded. Total Phenolic C ontents Antioxida nt C apacity Total Anthocyanin Content and Total P rocyanidin Content Dried blueberry fractions were re dissolved in phosphate buffer ( 50 mM, pH 7.4) with 5% dimethyl sulfoxide (DMSO) to a concentration of 15 mg/ml. The blueberry

PAGE 56

56 fractions in the phosphate buffer were further diluted to appropriate concentration for Folin Ciocalteu assay ORAC assay total anthocyanin assay and total procyanidin assay whose procedu res were described in Materials and Methods in Chapter 3 Folin Ciocalteu Assay Antioxidant C a pacity Total Anthocyanin assay and Total procyanidin assay Anti glycation Effect in Selected M odels The anti glycation effects for five blueberry fractions were examined. A ll fractions were diluted to 0.1 5 mg/ml for BSA fructose assay, 1 .5 mg/ml for BS A methylglyoxal assay and 0. 7 5 mg/ml for arginine methylglyoxal assay. Other procedure s to determine anti glycation effect were described in Materials and Methods in Chapter 3 Anti glycation Effect in Selected M odels The p ercentage of inhibited fluoresce nt AGEs was calculated. Direct Methylglyoxal T rapp ing A ssay Blueberry fractions were diluted to 2.5 mg/ml for the direct methylglyoxal trapping assay. The procedures of this assay were described in Materials and Methods in Chapter 3 Direct Methylglyoxal T rapping A ssay Data E xpression, G rafting and S tatistics Samples were anal yzed in triplicate and data was expressed as mean standard deviation unless otherwise noted One way analyses of variance and Tukey Kramer HSD pair wise comparison of the means were performed using JMP software (Version 8 Data grafting was done with SigmaPlot (Version 11.0, Systat Software Inc, San Jose, CA).

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57 Results Phytochemical Fractionation W eights of blueberry e xtracts and fractions are shown in Table 4 1. F rozen blueberries (201.1 g) w ere used for the fractionation. The amount of crude extract and sugar free extract from blueberries were similar to the result s in Chapter 3 Extraction and Co lumn S eparation The water fraction (fraction I) yielded 272.1 mg while f raction II to fraction V weighed from 78.2 to 166.6 mg. Phytochemical I dentification on HPLC ESI MS n Phenolic compounds in five blueberry fractions were identified based on mass spe ctra, UV/Vis and fluorescent spectra. HPLC chromatogram s of fraction I to fraction V at 280 nm and 520 nm are show n in Figure 4 2 to Figure 4 6. Compounds identified from the different fractions were marked with numbers on the chromatograms and listed in T able 4 2. Fraction I and II contained phenolic acids. The major phenolic acid found in Fraction I and II was chlorogenic acid. Fraction II and III contained anthocyanin monoglycosides. F lavonols and proanthocyanidin monomers and dimers were identified in F raction IV Procyanidin oligomers (from monomer to tetramer) were identified in Fraction V Most of the anthocyanin monoglycosides were eluted with 20% methanol (Fraction II) and 50% methanol (Fraction III). However, a small amount of anthocyanin monoglyco sides were detect ed from Fraction I, IV and V. Anthocyanin diglycoside was not detected in any of the fractions. The m olecular ion at m/z 191 [M H] (compound 1) produced a fragment at m/z 111, which wa s tentatively identified as quinic acid ( 82 ) Compound 2 had m/z 481 [M H] which fragmented into m /z 301, 239 and 175. This compound was tentatively identified as hexahydroxydiphenoyl ( HHDP ) glucose ( 48 ) The MS spectrum for

PAGE 58

58 compound 3 showed m/z 169 [M H] and a fragment at m/z 125 [M H] which correspond s to gallic acid ( 48, 83 ) The m olecular ion at m/z 331 [M H] (compound 5) and its fragment at m/z 169 [M H 162] were consistent with a gallic acid hexoside C ompound 10 showed m/z 325 [M H] and fragment m/z 163 [M H 162] It was identified as a coumaric acid hexoside ( 62 ) Compound 11 had m/z 353 [M H] and fragment at m/z 191. It was identified as chlorogenic acid ( 83 ) Molecular ions at m/z 463 [M H] (compound 13) m/z 447 [M H] (compound 15) and m/z 433 [M H] (compound 18) produced fragments at m/z 301, 299 and 285 respectively They were identified as ellagic acid hexoside, ellagic acid rhamnoside and ellagic acid xyloside respectively ( 62 ) Compounds 6, 7, 8, 9, and 14 were identified as flavan 3 ols and procyanidin oligomers ( 44 ) Compounds 8 and compound 14 had m/z 289 [M H] and yield ed fragments at m/z 245 and m/z 187. They were identified as catechin and epicatechin. Compound 6 had a molecular ion at 577 [M H] and fragments at m/z 407 and 287, which was consistent with a procyanidin dimer. The molecul ar ion at m/z 865 [M H] (compound 7) produced fragments at m/z 847, 694 and 575. It was identified as a procyanidin trimer. Compound 9 had m/z 1153 [M H] and fragments at m/z 1027, 693 and 576 which was consistent with a procyanidin tetramer. C ompound 4 had m/z 305 [M H] and produced fragment ions at m/z 261, 247 and 179. This compound was tentatively identified as (epi)gallocatechin ( 48 ) Compound s 12, 16, 17, 19, 20, 21, 22, 23, and 24 were identified as anthocyanins based on UV absorbance ( max = 520 nm), their fragmentation patterns and previous studies ( 44, 83 ) Comp ound s 12 and 17 had m/z at 465 [M] + and 435 [M] + Both of them

PAGE 59

59 fragmented and yield ed an ion in the MS 2 at m/z 303 [M 162] + and [M 132] + They were tentatively identified as delphinidin hexoside and delphinidin xyloside. Compound 16 with m/z 449 [M] + and c ompound 20 with m/z 419 [M] + fragmented and produced ion with m/z 287 ([M 162] + and [M 132] + ). They were identified as cyanidin hexoside and cyanidin xyloside. M olecular ion s at m/z 479 [M] + (compound 19) and m/z 449 [M] + (compo und 21) fragmented to produc t ion s at m/z 317 ([M 162] + and [M 132] + ), which were consistent with petunidin hexoside and petunidin xyloside. C ompound 23 ( m/z 493 [M] + ) and compound 24 ( m/z 463 [M] + ) produced fragment ion at m/z 331 ([M 162] + and [M 132] + ), which correspond ed to malvi din hexoside and malvidin xyloside. Compound 22 had molecular ion at m/z 463 [M] + and fragment ion at m/z 301 [M 162] + It was tentatively identified as peonidin hexoside. Compound 25 to compound 31 were identified as flavonols ( 44, 62 ) Compound 25 ( m/z 479 [M H] ) and compound 26 ( m/z 463 [M H] ) produced a fragment at m/z 317 ([M H 162 ] and [M H 146 ] ), which corresponded to myricetin hexoside and myricetin rhamnoside ( 48 ) Compound 27 to 29 had molecular ions at m/z 463 [M H] 433 [M H] and 447 [M H] These three compounds yield ed fragments at m/z 301([M H 162] [M H 132] and [M H 146] ). T hey were identified as quercetin hexoside, quercetin xyloside and quercetin rhamnoside respectively Molecular ions m/z 447 [M H] (compound 30) and m/z 431 [M H] (compound 31) produced fragment at m/z 285 ([M H 162] and [M H 1 46] ). They were identified as kaempferol hexoside and kaempferol rhamnoside respectively ( 48 ) Total Phenolic C ontent The values of the total phenolic content of five blueberry fractions ( Table 4 3 ) were closed to that of blueberry sugar free extract. Fraction IV showed the highest value of

PAGE 60

60 total phenolic content (0.63 0.02 g gallic acid equivalents/ g dried fr actions) I t was significantly higher than that of the blueberry extract (p < 0.0001). Fraction V showed the lowest total phenolic content (0.31 0.01 g gallic acid equivalents/ g dried fractions). The phenolic content of fraction I and fraction V were sig nificantly lower than that of the blueberry extract (p < 0.0001). Fraction II and III had similar phenolic content to the original blueberry extract. Antioxidant Capacity Similar to the re sults of total phenolic content the antioxidant capacities of the f ive fractions ( Table 4 3 ) were close to that of the original blueberry extract. Fraction II and fraction IV showed the highest antioxidant capacities (8.20 1.02 and 8.30 0.37 mol Trolox equivalents/mg dried fraction). Fraction II, III and IV had significa ntly higher antioxidant capacities than the original extract (P=0.041 for fraction III and P < 0.0001 for fraction II and fraction IV). Fraction V showed the lowest antioxidant capacity (2.53 0.26 mol Trolox equivalents/mg dried fraction) and was signific antly lower than the original extract (p = 0.002). Total Anthocyanin Content Total anthocyanins of blueberry fractions ranged from 14.8 367.8 g Cyanidin 3 glucoside per mg dried fraction ( Table 4 3 ). Fraction I had the highest total anthocyanin value (367 .7 4.83 g Cy G/mg). It was the only fraction that had a significantly higher total anthocyanin value than blueberry extract. Fraction V showed the lowest total anthocyanin values (14.8 0.39 g Cy G/mg). Total Procyanidin Content Total procyanidins of blu eberry fractions ranged from 1.3 35.0 g epicatechin per mg dried fraction ( Table 4 3 ). Total procyanidins in fraction III, IV and V were

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61 significantly higher than blueberry extract. Fraction V had the highest total procyanidin value (35.0 1.21 g epicatec hin/mg). Fraction I showed the lowest total procyanidin values (1.3 0.08 g epicatechin/mg). Anti glycation Effect in Selected M odels Results of the BSA fructose model showed that all five fractions inhibited the generation of fluorescent AGEs in six days ( Figure 4 7 ) Fraction IV showed the highest anti glycation effect in this model with 88.7 0.6% of the fluorescent AGEs inhibited. Fraction I had the lowest anti glycation effect (58.5%) They both significantly differed from the original extract (p < 0.0 001 for fraction IV and p = 0.021 for fraction I). The anti glycation effect of fraction II, III and V fell in between and were not significantly different from the blueberry sugar free extract. In the BSA methylglyoxal model, blueberry extract inhibited m ore than 90% of the formation of fluorescent AGEs Five blueberry fractions were able to inhibit 27 % to 65% of the fluorescent AGE generation (Figure 4 8 ) Am ong these fractions, fraction III showed the highest anti glycation effect (64.9%), but it was not significantly different from the blueberry extract. Fraction V showed the lowest anti gl ycation effect (27.6%) among all the blueberry fractions In the arginine methylglyoxal model, all fractions inhibited more than 32 % of the fluorescent AGE generation (Figure 4 9 ) Fraction II III and IV showed 66.2, 62.2 and 69.4 % inhibition, respectively. They had significantly higher anti glycation effect s when compared to the original sugar free extract. Similar to the result in the BSA methylglyoxal model, fractio n V showed the lowest anti glycation effect (32.4%)

PAGE 62

62 Direct Methylglyoxal T rapping The chromatograms of methylglyoxal, methylglyoxal with blueberry fraction III and methylglyoxal with aminoguanidine are shown in Figure 4 1 0 The amount of methylglyoxal de creased gradually when incubated with the blueberry fractions (Figure 4 1 1 ). All the fractions scavenged more than 45 % of the methylglyoxal within 6 h. Fraction I and Fraction III had the strongest capacity to reduce methylglyoxal (75.7 and 80.6, respectiv ely) while fraction II and fraction V were not as effective (48.5 and 50.4, respectively) Discussion The s eparation mechanism of phytochemicals on Sephadex LH 20 is primarily a normal phase partition with gel permeation also involved. P henolic acids are eluted with water, whereas most of the anthocyanin monoglycosides are eluted with 20% and 50% methanol. The total phenol ic content, antioxidant capacities as well as the antiglycation effects in selected models varied among the different fractions. Bluebe rry phytochemical fractions act ed either as antioxidants or scavengers of reactive carbonyl s. F lavonoids in blueberries are a class of plant phenolics with powerful antioxidant capacity The s tructures of flavonoids influence their radical scavenging activ ity. The radical scavenging activity of flavonoids is enhanced by the high degree of hydroxyl substitution ( 84 86 ) Flavonoid s may also inhibit AGE generat ed by chelating transi ti onal metal ions. A study from Urios et al. showed flavonoids prevent ed metal catalysed formation of hydroxyl radicals, thus inhibiting protein glycation ( 86 ) The f ive blueberry fractions contained different types of flavonoids. Fraction I contained phenolic acids, including chl orogenic acid, w ith a small amount of

PAGE 63

63 anthocyanin monoglycosides. The majority of compounds in fraction II were chlorogenic acid and anthocyanin monoglycosides. Fraction III contained large amount of anthocyanin monoglycosides and procyanidins oligomers (m onomer and dimer) Fraction IV was found to have flavonols and procyanidin oligomers (monomer to tetramer ) Fraction V contained a small amount of procyanidins Various phenolic compounds possess carbonyl trapping ability. Anthocyanins may ha ve potential c arbonyl quenching abilities. Anthocyanin s react with pyruvic acid and acetaldehyde to form pyranoanthocyanins during red wine aging, contributing to wine color, aroma and taste ( 87 89 ) Procyanidin monomer, catechi n and epicatechin were potent carbonyl scavengers ( 90 91 ) P rocyanidin B type dimer was shown to have methylglyoxal trapping ability as well as anti glycation property ( 51, 92 ) The d ifferent antioxidant capacity, anti glycation effects, and methylglyoxal scavenging ability in the five fractions may be explained by differences in phenolic composition. In conclusion, f ive fractions were obtained from a sugar free blueberry ex tract The m ajor compounds in those fractions were phenolic acids, procyanidins, anthocyanins and flavonols. Blueberry fractions inhibited 28% 88 % of AGE generation in BSA fructose, BSA methylglyoxal and arginine methylglyoxal models They scavenged 49 % 80 % of methylglyoxal in 6 h. Fraction II, fraction III and fraction IV showed higher AGE inhibitory effects, and methylglyoxal trapping capacities than the original blueberry extracts.

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64 Table 4 1 Weight s of blueberry extracts and fractions. Samples Weight Major p hytochemicals Frozen berries 201.1 g Crude Extract 25.3 g Sugar free Extract 925.3 mg Fraction I 272.2 mg Phenolic acids Fraction II 132.4 mg Chlorogenic acid, anthocyanins Fraction III 78.2 mg Procyanidins, anthocyanins Fraction IV 166.6 mg Procyanidins, flavonols Fraction V 106.4 mg Procyanidins Table 4 2 Tentatively identified c ompound s in blueberry fractions Compound number Retention time (min) MS ( m/z ) MS2 ( m/z ) MS3 ( m/z ) Identified compound Frations 1 5.7 191 [M H] 11 1 Quinic acid I 2 8.0 481 [M H] 301, 239, 175 HHDP glucose I 3 8.2 169 [M H] 125 Gallic acid V 4 11.8 305 [M H] 261, 247, 219, 179, 165 (Epi)gallocatechin V 5 12.9 331 [M H] 169, 125 125 Monogalloyl glucose I 6 14.6 577 [M H] 425, 407, 287 40 7 Procyanidin dimer III, IV, V 7 15.1 865 [M H] 847, 740, 694, 575 Procyanidin trimer V 8 16.7 289 [M H] 245, 203, 187 (Epi) catechin III, IV, V 9 17.0 1153 [M H] 1027, 983, 693, 576 Procyanidin tetramer V 1 0 18.7 325 [M H] 163, 263, 145 Coumari c acid 4 glucoside I 1 1 19.0 353 [M H] 191 Chlorogenic acid II 1 2 19.9 465 [M]+ 303 285, 257, 229, 151 Delphinidin hexoside I, II, III, IV, V 13 20.2 463 [M H] 301, 299, 284 256, 176, 125 Ellagic acid hexoside I, II, III, IV, V 14 21.4 289 [M H] 2 45, 187, 217 ( Epi ) catechin III,IV, V 15 21.6 447 [M H] 301, 299, 285 227 Ellagic acid rhamnoside I, II 16 21.7 449 [M]+ 287 241,231, 213, 157, 137, Cyanidin hexoside I, III 17 22.1 435 [M]+ 303 257, 229, 151, Delphinidin xyloside II, III, IV, V 18 22.4 433 [M H] 301, 299, 285 257 Ellagic acid xyloside I, II, III, V 19 22.7 479 [M]+ 317 302, 274, 261, 229 Petunidin hexoside I, II, IV, V

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65 Table 4 2. Continued. Compound number Retention time (min) MS ( m/z ) MS2 ( m/z ) MS3 ( m/z ) Identified compound Fra tions 20 23.8 419 [M]+ 287 213, 187, 149 Cyanidin xyloside II, III 21 24.7 449 [M]+ 317 302, 274, 261 Petunidin xylose I, II, III, IV, V 22 25.3 463 [M]+ 301 286 Peonidin hexoside II 23 25.5 493 [M]+ 331 315, 299, 287, 270, 243 Malvidin hexcoside I, II IV, V 24 27.4 463 [M]+ 331 316, 299, 288, 269 Malvidin xyloside I, II, III, IV, V 25 28.2 479 [M H] 317, 271, 179 287, 271, 179 Myricetin hexoside IV 26 30.7 463 [M H] 317, 271 271 Myricetin Rhamnoside IV 27 32.1 463 [M H] 301 271, 255, 179, 151 Q uercetin hexoside III, IV 28 33.9 433 [M H] 301 271, 255,215, 179, 107 Quercetin xyloside IV 29 35.6 447 [M H] 301 275, 257, 229, 165, 137 Quercetin Rhamnoside IV 30 36.2 447 [M H] 327, 285, 256 Kaempferol hexoside IV 31 39.8 431 [M H] 285, 255 K aempferol rhamnoside IV Table 4 3. Total phenolic, antioxidant capacity, total anthocyanins and total procyanidins in blueberry extract and fractions. Fractions Total phenolics ( g GAE/ g) Antioxidant capacity ( mol Trolox/mg) Total anthocyanins ( g Cy G/mg) Total procyanidins ( g epicatechin/mg) Blueberries 0.50.0 bc 4.9 0.0 c 153.5 3.2 b 20.9 0.2 c I 0.4 0.0 d 6.0 0.5 bc 367.8 4.8 a 1.3 0.1 d II 0.6 0.0 b 8.2 1.0 a 95.1 4.2 c 3.8 0.1 d III 0.5 0.0 c 6.4 0.5 b 68.2 2.2 d 29.1 3. 1 b IV 0.6 0.0 a 8.3 0.4 a 46.4 0.7 e 27.5 1.8 b V 0.3 0.0 e 2.5 0.3 d 14.8 0.4 f 35.0 1.2 a Values are mean standard deviation of three individual experiments for all the assays. Values in the same column with different letters denoted significan tly differences (P 0.05) from each other.

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66 Figure 4 1. Fractionation flow chart of blueberry sugar free phytochemicals

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67 Figure 4 2. HPLC chromatogram s of blueberry fraction I at 280 nm (A) and 520 nm (B). Peaks marked with numbers were tentatively id entified Mass spect ral data of identified compounds are liste d in Table 4 2. Figure 4 3. HPLC chromatogram s of blueberry fraction II at 280 nm (A) and 520 nm (B). Peaks marked with numbers were tentatively identified. Mass spectral data of identified compounds are listed i n Table 4 2.

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68 Figure 4 4. HPLC chromatogram s of blueberry fraction III at 280 nm (A), 360 nm (B) and 520 nm (C). Peaks marked with numbers were tentatively identified. Mass spectral data of identified compounds are listed in Table 4 2. Figure 4 5. HPLC chromatogram s of blueberry fraction IV at 280 nm (A) and 520 nm (B). Peaks marked with numbers were tentatively identified Mass spectral data of identified compounds are listed in Table 4 2.

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6 9 Figure 4 6. HPLC chromatogram s of blueberry fraction V at 280 nm (A) and 520 nm (B). Peaks marked with numbers were tentatively identified. Mass spectral data of identified compounds are listed in Table 4 2.

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70 Figure 4 7 Anti glycation effects of blueberry extract and fract ions in bovine serum albumin fructose model. The bars showed the mean percentage of fluorescent advanced glycation end product inhibition with their standard deviation for three independent experiments. Bars with different letters denoted significant diffe rences (p 0.05) from each other. The concentration of fractions catechin and epicatechin were 0.05 mg/ml. A minoguanidine (10 mM) served as the positive control.

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71 Figure 4 8 Anti glycation effects of blueberry extract and fra ctions in bovine serum albumin methylglyoxal model. The bars showed the mean percentage of fluorescent advanced glycation end product inhibition with their standard deviation for three independent experiments. Bars with different letters denoted significan t differences (p 0.05) from each other. The concentration of fractions, catechin and epicatechin were 0.5 mg/ml. Ami noguanidine (10 mM) s erved as the positive control.

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72 Figure 4 9 Anti glycation effects of blueberry extract and frac tions in arginine methylglyoxal model. The bars showed the mean percentage of fluorescent advanced glycation end product inhibition with their standard deviation for three independent experiments. Bars with different letters denoted significant differences (p 0.05) from each other The concentration of fractions, catechin and epicatechin were 0.25 mg /ml. Aminoguanidine (10 mM) served as the positive control.

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73 Figure 4 10 Chromatogram of methylglyoxal (A), methylglyoxal after reaction with blueberry fractio n III (B) and methylglyoxal after reaction with aminoguanidine (C ). Methylglyoxal was detected as 1 methylquinoxaline after derivatization using o phenylenediamine at 315 nm.

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74 Figure 4 11 The percentage of remaining m ethylglyoxal after been incubated with blueberry extract and fractions for 0, 0.5, 1, 2, 4, 6h The concentrations of blueberry fractions and catechin were 2.5 mg/ml and 5 mM, respectively. Scattered dots represented the mean percentage of remaining methylg lyoxal with the standard deviation for two independent experiments.

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75 CHAPTER 5 REACTIONS BETWEEN PH YTOCHEMICALS AND REA CTIVE CARBONYL SPECI ES Sugar free phytochemicals from berries and noble grapes i nhibit ed AGE formation part ly by scavenging re active carbonyls. Previous research demonstrated that a wide range of phytochemica ls exist in berries and grapes ( 62 63, 93 ) Flavan 3 ols, flavonols, anthocyani ns and chlorogenic acid were identified from fraction s of blueberr y extract using HPLC ESI MS n No research has been conduc t ed to systematically evaluate their scavenging capacity to different reactive carbonyls. The objective of this chapter wa s to investigate the reaction kinetics between pure phytochemica ls and reactive carbonyls and tentatively identify phytochemical carbonyl adducts on HPLC ESI MS n Materials and Methods Chemicals R esveratrol was a product from Quality Phytochemicals LLC. ( Edison, NJ ) (+) Catechin and ( ) epicatechin were purchased fro m Sigma Aldrich (St. Louis, MO). Glyoxal (40% solution in water) and quercetin (95% hydrate) were products from Acros Organics ( Morris Plains, NJ ). Methylglyoxal ( 40% aqueous solution ) and chlorogenic acid were purchased from MP Biomedicals, LLC ( Solon, O H). Acrolein (97% stabilized with hydroquinone) was purchased from Alfa Aesar (Ward Hill, MA). Other chemicals and materials were described in previous chapters. Preparation of Malondialdehyde Malondialdehyde was produced by hydrolysis of malonald ehyde bis (diethyl acetal or 1,1,3,3 tetraethoxypropane). Malonaldehyde bis (diethyl acetal, 166 l) was homogenized with hydrogen chloride (90 l, 1.0 M ) at 40 C for 2 min. Then distilled water (729 l) was added and the solution was incubated in 40 C water bath for 1 h for

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76 complete hydrolysis. After incubation, the pH of the solution was adjusted to 7.0 by adding NaOH (15 l, 6.0 M ). This solution was diluted by 10 5 times and the concentration of malondialdehyde was measured immediately on a spectrophotometer at MDA ( DUseries 700 UV/Vis Scanning Spectrophotometer, Beckman Coulter, Brea, CA ) ( 94 ) The concentration of malondialdehyde was calculated by the following equation: Preparation of P hytochemical and C arbonyl S olutions Solutions of phytochemicals and reactive carbonyl compounds were freshly prepared before the experiment. In the studies for catechin epicatechin and chlorog enic acid chemicals ( catechin, epicatechin, chlorogenic, glyoxal, methylglyoxal, malondialdehyde and acrolein ) were dissolved or diluted in a 50 mM phosphate buffer (pH 7.4) to a concentration of 10 mM In the studies for querce tin and resveratrol, these two phytochemicals and other reactive carbonyl c ompounds were dissolved or diluted in a DMSO: phosphate buffer (50:50, v/v) to a concentration of 10 mM. Preparation of Derivatization R eagent s The derivatization agent was o phenylenediamine for gly oxal and methylglyoxal and 2,4 d initrophenylhydrazine for acrolein. O phenylenediamine (50 mM) was prepare d fresh daily in phosphate buffer (50 mM, pH 7.4) 2,4 d initrophenylhydrazine solution (6 mM) was prepared weekly by dissolving 62.5 mg of 2,4 d initrophenylhyd razine crystal and hydrogen chloride (1.0 M 3 ml) in acetonitrile to a total volume of 50 ml Trichloroacetic acid buffer and thiobarbituric acid solution were prepared for the detection of malondialdehyde. T he t richloroacetic acid buffer (0.61 M)

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77 was pro duced by dissolving 10 g of trichloroacetic acid in 20 ml distilled water, followed by adding 80 ml of phosphate buffer (50 nM) containing 0.1% ethylenediaminetetraacetic acid (EDTA). T he t hiobarbituric acid solution (20 mM) was prepared by dissolving 1.44 1 g thiobarbituric acid in 500 ml distilled water. All solutions were sealed and kept at 4 C before use Catechin carbonyl r eaction s tudy Catechin (10 mM, 1 ml) was incubated with glyoxal, methylglyoxal, malondialdehyde, acrolein (10 mM, 1 ml) or phosphat e buffer (50 mM, pH 7.4 1 ml, control) separately for 0 48 h Carbonyls (10 mM, 1ml) were also incubated with phosphate buffer (50 mM, pH 7.4, 1 ml). I ncubation was conducted in duplicate A t 0, 0.5, 1, 2, 4, 6, 12, 24 and 48 h of incubation o phenylened iamine (50 mM, 200 l) was mixed with 200 l of catechin glyoxal or catechin methylglyoxal media to terminate the reaction. 2,4 Dinitrophenylhydrazine (6 mM, 300 l) was mixed with 50 l of catechin acrolein media to terminate the reaction. An aliquot of 2 00 l catechin control solution, catechin malondialdehyde media an d other catechin carbonyl media with derivatization agents were transferred into vial s for HPLC ESI MS n analysis. Detection of catechin and catechin carbonyl adducts were performed on an Agi lent 1200 HPLC system (Agilent Technologies, Palo Alto, CA) equipped with a diode array detector, a fluorescence detector and HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). A zorbax stable bond analytical SB C18 column (4.6 250 mm, 5 m, Agilent Technologies, Palo Alto, CA) was used for compound separation. Mobile phases were composed of 0.1% formic acid in Millipore water (mobile phase A) and pure methanol (mobile phase B). The linear gradient was: 0 5 min, 10 22.5% B; 5 15 min, 22.5 27. 5% B; 15 18 min, 27.5 70% B; 18 21 min, 70 90% B; 21 23 min, 90 10%

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78 B, followed by 2 min of re equilibration. The i njection volume was 5 l and flow rate was 1 ml/min. Peak areas of catechin in catechin carbonyl samples were integrated and compared to the peak area s of a catechin control. A m ass spectrometer with electro spray ionization interface was operated at both positive and negative modesusing nebulizer 50 psi, drying gas 10 L/min and drying temperature 300 C. Retention time for catechin was 12.4 min on a fluoresc ent chromatogram (excitation 231 nm, e mission 320 nm). Mass spectra were scanned from 50 to 800 m/z The most abundant ions in the full scan spectrum were isolated and their product ion spectra (MS 2 ) were recorded. Detection of glyoxal and me thylglyoxal were performed using the same HPLC system and column The flow rate was 1 ml/min and the injection volume was 15 l. The linear gradient for elution was: 0 3 min, 5 50% B; 3 16 min, 50 50% B; 16 17 min, 50 90% B; 17 19 min, 90 90% B; 19 19.5 mi n, 90 5% B; followed by 1 min of re equilibration. Glyoxal and methylglyoxal were detected in the form of quinoxaline and 1 methylquinoxaline at 315 nm at 11.7 min and 12.9 min respectively. The p eak areas of quinoxaline in the catechin glyoxal incubatio ns were integrated and compared with the peak area s in the glyoxal control. Peak areas of 1 methylquinoxaline in the catechin methylglyoxal incubations w ere integrated and compared with the peak area s in the methylglyoxal control. Detection of the remainin g acrolein was also completed by the same HPLC system. The mobile phases were 0.1% formic acid in Millipore water (mobile phase A) and pure acetonitrile (mobile phase B). An i socratic elution (1 ml/min) was used containing 40% A and 60% B. An aliquot of 15 l of catechin acrolein mixture with

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79 derivatization agent was injected. A crolein was detected in the form of acrolein 2,4 dinitrophenylhydrazine adduct at 372 nm at 13.8 min. The p eak areas of acrolein in the catechin acrolein incubations was integrated and compared with the peak areas in the acrolein control. Detection of the remaining malondialdehyde was modified from the method of thiobarbituric acid reactive substances (TBARS). An aliquot of 3 l of catechin malondialdehyde media or malondialdehyde co ntrol was mixed with trichloroacetic acid buffer (497 l) and thiobarbituric acid solution (500 l). A blank was prepared by adding distilled water instead of incubation media. Mixtures were heated in 80 C water bath for 1 h and cooled in ice. Mixtures we re diluted by half with distilled water. An aliquot of 200 l from each mixture were pipette d into a 96 well plate with clear flat bottom and the absorbance at 530 nm were recorded. Absorbance readings from the catechin malondialdehyde samples were compare d with the malondialdehyde control. Absorbance was measured at the linear range of the plate reader ( SPECTRAmax 190, Molecular Devices, Sunnyvale, CA ). Epi catechin carbonyl reaction and chlorogenic acid carbonyl reaction studies Procedures followed the C a techin carbonyl reaction study Epicatechin eluted at 19.4 min and was detected using a fluorescent detector (230 nm excitation and 320 nm emission). Peak of chlorogenic acid eluted at 15.0 min using 330 nm for detection Resveratrol carbonyl reaction and quercetin carbonyl reaction studies Incubation procedures were the same as Catechin carbonyl reaction study Detection of resveratrol, quercetin and their carbonyl adducts were performed on the same HPLC ESI MS n system. The mobile phases were composed of 0. 1% formic acid in Millipore water (mobile phase A) and pure acetonitrile (mobile phase B). The line ar

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80 gradient was: 0 10 min, 5 30 % B; 10 17 min, 30 40 % B; 17 20 min, 40 60 % B; 20 23 min, 60 5 % B, followed by 2 min of re equilibration. A r esveratrol peak appeared at 16.7 min on the fluorescent chromatogram ( 330 nm excitation /374 nm emission). Identification of quercetin was done at 3 6 0 nm by diode array detector. Detection of other carbonyl compounds, resveratrol carbonyl adducts and quercetin carbonyl a dducts followed the me thods described in Catechin carbonyl reaction study Data C alculation Grafting and S tatistics The p eak area of each phytochemical and carbonyl compound at different incubation time was integrated. The remaining percentage of phytoche mical or carbonyl compound was calculated by the following equation: The remaining percentage of malondialdehyde was calculated by the following equation: The remaining percentage of the phenolic compounds or carbonyls was plotted against incubation time. Samples were analyzed in duplicate and data was expressed as mean standard deviation. One way analyses of variance with Tukey Kramer HSD pair wise comparison of the means were performed using JMP software (Version 8 .0, SAS Institute Inc. Cary, NC) for m ean comparison at 24 h considered significant. Empirical d egradation half time for phytochemicals and reactive carbonyl compounds were calculated using Excel software (Version 2007, Microsoft Cooperation. Redmond, WA). Data grafting was done using Si gmaPlot (Version 11.0, Systat Software Inc, San Jose, CA).

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81 Results Reaction Kinetic s b etween P h enolic s and Carbonyl C ompounds Phenolic compounds were unstable in pH 7.4 buffer. After 48 hour incubation, 90% of catechin 63% of epicatechin and 44% of chloro genic acid remained. Quercetin and resveratorl were the least stable compounds among the five. They were not detected at 48 h The empirical degradation half time of the phytochemicals are listed i n Table 5 1. HPLC analysis with fluorescent detection show ed that catechin w as more susceptible to glyoxal and acrolein, than to methylglyoxal and malondialdehyde. The empirical degradation h alf times for catechin were 3.6 3. 6 7.0 and >48 h when reacted with glyoxal, acrolein, methylglyoxal and malondialdehyde, respectively (Table 5 1). At 48 h catechin was reduced to 1%, 12%, 27% and 72% of its original level by glyoxal, acrolein, methylglyoxal and malondialdehyde, respectively (Fig ure 5 1) Similar to the result found in catechin, epicatechin required 4.3 4. 6, 11.7 and >48 h for half reduction when reacted with glyoxal, acrolein, methylglyoxal and malondialdehyde, respectively (Table 5 1). Epicatechin was decreased to 1%, 12%, 24% and 68% of its original level after 48 h incubation with glyoxal, acrolein met hylglyoxal and malondialdehyde respectively (Fig ure 5 2) C hlorogenic acid was decreased by 75 % after reacting with glyoxal and methylglyoxal for 48 h The decrease of chlorogenic acid in the presence of glyoxal and methylglyoxal was more rapid than in t he control with an empirical degradation half time of 9.4 and 9. 2 h, respectively (Table 5 1). However, acrolein and malondialdehyde were similar to the buffer control in decreasing chlorogenic acid content (Fig ure 5 3)

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82 The decrease of q uercet in by meth ylglyoxal and glyox al was significantly faster than that in the control with an empirical degradation half time of 5. 5 and 6. 4 h (Table 5 1) A crolein slightly spe d up the degradation of quercetin. On the other hand malondialdehyde delayed the degradatio n of quercetin. At the end of the 48 h quercetin degraded completely with or without the presence of reactive carbonyl compounds (Fig ure 5 4) Resveratrol content decreased after incubation with glyoxal, methylglyoxal and acrolein It required 3. 8, 7.4 an d 7.4 h for half reduction when reacted with acrolein, glyoxal and methylglyoxal, respectively (Table 5 1). But the addition of malondialdehy de did not reduce the resveratro l amount compared to the control. After 48 h incubation, resveratrol degraded compl etely in all tested incubation systems including the controls (Fig ure 5 5) The empirical degradation half time for reactive carbonyl compounds are listed i n Table 5 2. Catechin, epicatechin, quercetin and resveratrol scavenged more than 90% of glyoxal in 48 hours (Figure 5 6) Glyoxal decreased by half a t 3.5 2.7, 3.6 and 4.3 h when reacted with catechin, epicatechin, quercetin and resveratrol, respectively (Table 5 2). Chlorogenic acid also significantly reduced the amount of glyoxal when compared to the control. Similar to the result s of glyoxal, methylglyoxal showed rapid reduction when incubated with phenolic compounds. It s empirical degradation half time was shorter than 1 h when reacted with catechin, epicatechin, quercetin and resveratrol. Epic atec hin was the most efficient agent for methylgly oxal scavenging, followed by catechin

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83 quercetin and resveratrol. C hlorogenic acid was less effective than resveratrol. (Fig ure 5 7). C atechin and epicatechin were the most efficient agents to scavenge malondia ldehyde followed by chlorogenic acid and quercetin. However, resveratrol did not significantly reduce the amount of malondialdehyde. (Fig ure 5 8) Although acrolein gradually decreased to 16% in the control solution at 48 h the addition of phenolic comp ounds efficiently scavenge d 70% of it in 6 h and more than 95% in 48 h compared to the control (Fig ure 5 9). Catechin and epicatechin were the most efficient agents at lowering the amount of acrolein in solution followed by quercetin and resveratrol. Chlo rogenic acid did not significantly lower the amount of acrolein. In general, the decrease of phenolic compound s occurred simultaneously with the decrease of carbonyl compound s. The p ercentage remaining decreased with reaction time. Adduct I dentification HP LC ESI MS n analysis was performed to identify adducts that were formed during phytochemical carbonyls incubation. Adducts were tentatively identified on the basis of [M H] and their product ion spectra. Identification was confirmed by comparing part of ad ducts to those previously identified in research. Phytochemical carbonyl adducts identified in this thesis are listed in Table 5 3 Catechin carbonyl a dducts Figure 5 10 show s the HPLC fluorescent chromatogram of catechin control (A), catechin with glyoxal (B), catechin with methylglyoxal (C), catechin with malondialdehyde (D) and catechin with acrolein (E) after six hours of incubatio n Three

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84 catechin glyoxal adducts were tentatively identified (Figure 5 10 B ) and their mass spectra are showed in Figure 5 11 The f irst adduct gave rise to m/z 619 [M H] and fragment ion s with m/z 468, 329, 289 and 245. This was consistent with an adduct that composed of two catechin molecules and a glyoxal molecule in between. The f ragment m/z 289 [M H] was catechin, which lost a CH 2 CHOH group in the benzofuran skeleton to yield m/z 245. This adduct los t a catechi n molecule to yield a f ragment m/z 329 [M catechin H] This compound was tentatively identified as a dicatechin monoglyoxal adduct. The s econd adduct gave [M H ] at m/z 347 [M H] and fragment ion s of m/z 329, 289, 245, and 205. It was identified as a monocatechin monoglyoxal adduct. The f ragments at m/z 289 were from catechin. Ions 245, 167 and 205 resulted from catechin fragmentation whic h has been reported pr eviously ( 95 ) The third adduct gave [ M H] m/z 63 7 and a product ion m/z 289 [M H] formed by catechin fragment. It was tentatively identified as a dicatechin monoglyoxal adduct. Figure 5 12 show s the proposed structures of catechin glyoxal adduct and their fragments. Reactive carbonyls atta ck ed the C 6 or C 8 positions on the A ring of catechin to form adducts ( 91 ) Because the positive carbonyl moieties on catechin cannot be confirmed by mass spectrometry, only one isomer of possible adducts are show n in this figure. Two catechin methylglyoxal adducts were ide ntified The ir mass spectra are show n in Figure 5 13 The first adduct yield ed m/z 361 [M H] and fragments at m/z 343, 289, 165 and 137. Ion m/z 343 [M H] was due to water elimination of m/z 361 [M H] Ion m/z 289 resulted from catechin. The cleavage of the C ring of catechin yield ed fragments with m/z 165 and 137 ( 96 ) T his compound was tentatively id entified as monocatechin monomethylglyoxal adduct. The second adduct with m/z 433 [M H] was

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85 tentative ly identified as a monocatechin dimethylglyoxal adduct. It yield ed m/z 415 due to water elimination, m/z 362 after losing a methylglyoxal m oiety and m/z 2 53 after the cleavage of the C ring of catechin Figure 5 14 show s the proposed structure s of catechin methyl glyoxal adduct and their fragments. Two catechin malondialdehyde adducts were identified and t he ir mass spectra are show n in Figure 5 15. An adduc t with m/z 361 [M H] was tentatively identified as a monocatechin monomalondialdehy de adduct. I t yield ed a catechin fragment at m/z 289 and m/z 327 due to water elimination The s econd adduct with m/z 433 [M H] was tentatively identified as a monocatechi n dimalondialdehyde adduct. It produced a fragment at m/z 326 [M] after losing a malondialdehyde m oiety Catechin ( m/z 289) fragment ed to generate m/z 163. Proposed structures of adducts and fragments are shown in Figure 5 16. O ne catechin acrolein adduct was identified and its MS/MS 2 spectra are shown in Figure 5 1 7. It yielded m/z 345 [M H] and a catechin fragment at m/z 289 Fragment s at m/z 245 and 215 were generated from catechin, which has been reporte d ( 97 ) Proposed structures of adduct and fragments are shown in Figure 5 18. Epicatechin carbonyl adducts Figure 5 19 shows the HPLC fluorescent chromatogr am of epicatechin control (A), epicatechin with glyoxal (B), methylglyoxal (C), malondialdeh yde (D) and acrolein (E) after six hours of incubation. Three epicatechin glyoxal adducts were identified and t heir mass spectra are shown in Figure 5 20. The first adduct gave m/z 347 [M H] and was tentatively identified as a monoepicatechin monoglyoxal adduct. The f ragment at m/z 329 was due to water elimination. Fragment s of m/z 289 and 245 were resulted from epicatechin. The second adduct with m/z 619 [M H] was tentatively identified as a

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86 diepicatechin monoglyoxal adduct Its fragmentation pattern was the same as the catechin glyoxal adduct. The third adduct yielded m/z 677 [M H] and was tentatively identified as a diepicatechin diglyoxal adduct. It produced fr agment at m/z 388 after los ing an epicatechin m oiety Fragment at m/z 347 was generated after los ing one glyoxal moiety and one epi catechin moiety The f ragment at m/z 289 [M H] was epicatechin Proposed structures of adducts and fragments are show n in Fi gure 5 21. Two epicatechin methylglyoxal adducts were tentatively identified Figure 5 22 show s their mass spectra. An adduct gave m/z 361 [M H] and was tentatively identified as a monoepicatechin monomethylglyoxal adduct. Its fragmentation pattern was th e same as the monocatechin monomethylglyoxal adduct. The second adduct yielded m/z 433 [M H] and was tentatively identified as a monoepicatechin dimethylglyoxal adduct. It yield ed an e picatechin fragment at m/z 289 and m /z 416 due to water elimination Th e f ragment at m/z 343 was yield ed after losing a methylglyoxal m oiety and a water molecule The f ragment m/z 181 was deduced to be a methylglyoxal linked with a phloroglucinol molecule after water elimination Proposed structures for adducts and fragments are show n in Figure 5 23. One epicatechin malondialdehyde adduct at m/z 433 [M H] was identified as a monoepicatechin dimalondialdehyde adduct. I ts mass spectra are show n in Figure 5 24. It produced a fragment at m/z 325 after losing a malondialdehyde moi ety and two water molecules Epicatechin ( m/z 289) fragmented to produce m/z 245. Figure 5 25 shows t he proposed structures of adduct and fragments A mono epicatechin mono acrolein adduct at m/z 345 [M H] was identified Figure 5 26 show s its MS and MS 2 sp ectra. It yield ed fragments at m/z 289 and 245, which

PAGE 87

87 resulted from epicatechin. A fragment at m/z 193 was due to the cleavage of C ring on the epicatechin skeleton (Figure 5 27). Quercetin carbonyl adducts Figure 5 28 show s the HPLC chromatogram of querce tin control (A), quercetin with glyoxal (B), methylglyoxal (C) and acrolein (D ). The mass spectra for mono quercetin mono glyoxal adduct ( m/z 341 [M H] ), mono quercetin mono methylglyoxal adduct ( m/z 373 [M H] ) and mono quercetin mono acrolein adduct ( m/z 356 [M H] ) are show n in Figure 5 29. They fragmented and produced the quercetin fragment at m/z of 301 after losing a glyoxal moiety, a methylglyoxal moiety and an acrolein moiety (Figure 5 30). Resveratrol carbonyl adducts Figure 5 31 show s the HPLC fluore scent chromatogram of resveratrol control (A), resveratrol with glyoxal (B), methylglyoxal (C), malondialdehyde (D) and acrolein (E). Adducts yielded m/z 285 [M H] m/z 299 [M H] m/z 299 [M H] m/z 283 [M H] (Figure 5 32) and were tentatively identifi ed as a monoresveratrol monoglyoxal adduct, a monoresveratrol monomethylglyoxal adduct, a monoresveratrol monomalondialdehyde adduct and a monoresveratrol monoacrolein adduct. They produced resveratrol fragment s at m/z 227 after losing a glyoxal, methylgly oxal, malondialdehyde or acrolein moiety An adduct at m/z 339 [M H] from resveratrol acrolein incubation was tentatively identified as a monoresveratrol diacrolein adduct, because it produce d a fragment at m/z 227 (resveratrol) and a fragment at m/z 283 (monoresveratrol monoacrolein) Figure 5 33 shows the proposed structures of adduct and fragments

PAGE 88

88 Discussion E merging research suggest s plant extracts with high phenolic contents effectively inhibit the generation of AGEs. Phenolic compounds were consid ered as major candidates for AGE inhibition due to their an tioxidant and chelating properties However, a limited number of studies have investigated their chemical reaction between phenolic compounds and reactive carbonyls. In this study, catechin epicat echin chlorogenic acid, quercetin and resveratrol were selected to represent various classes of phenolic compounds in berries and grapes. They were used to react with dicarbonyl compounds and unsaturated aldehydes. This was the first study that compare d the carbonyl scavenging capacity of selected phenolic compounds and the relative stability of phenolic compounds in the presence of carbonyls. Mass spectra data demonstrated that phenolic co mpounds scavenged reactive carbonyls by forming adducts of different structures Catechin and epicatechin were reported to react with glyoxal or methylglyoxal at the C 6 or C 8 position ( 91 ) Additional research showed their carbonyl trapping capacity was achieved under phys iological conditions. It was also suggested that catechin methylglyoxal adduct might undergo further metabolisms by enzymes in vivo ( 71 ) Our result, for the first time, showed that carbonyls can cross line two catechins or epicatechins to form dimers. tetrahydroxystilbene 2 O D glucoside and resveratrol, had been tested for their efficiency in trapping methylglyoxal. R esveratrol was less efficient than other stilbenes ( 56 ) In our study, adducts formed between resveratrol and glyoxal, malondialdehyde and acrolein w ere detected for the first time.

PAGE 89

89 Previous research found that plant extracts high in quercetin exhibited high inhibitory effects on the formation of AGEs ( 73 ) Our study confirmed that quercetin quenched carbonyls by forming adducts. Unlike catechin, no carbonyl cross linked quercetin dimers was identified. It was reported that chlorogenic acid from Ilex paraguariensis was the major anti glycation agent for BSA and histones ( 98 ) Another study indicated Ilex paraguariensis extracts had higher inhibitory effects on the formatio n of AGEs than green tea ( 99 ) A dducts formed between chlorogenic acid and carbonyl compounds were not detect ed in our study Except for antioxidant effects, mechanisms for chlorogenic acid inhibit ion AGE formation remain unclear. The A ring of flavonoids was suggested to be th e trapping site for reactive carbonyls, including dicarbonyls and unsaturated aldehydes. The A ring s of epigallocatechin 3 gallate, catechin, phloretin, phloridzin and naringenin were able to undergo nucleophilic substitution by reactive carbonyl compo unds ( 50, 52, 54, 90, 100 102 ) The c arbonyl group of both dicarbonyl compounds and unsaturated aldehydes can undergo electrophilic substitution to the A ring of flavonoids. In conclusion, p hytochemicals (cate chin, epicatechin, quercetin, resveratrol) rapidly reacted with carbonyls and resulted in the formation of phytochemical carbonyl adducts of different structures.

PAGE 90

90 Table 5 1. Empirical degradation half time of phytochemicals in different incubation mixt ures Time (h) Reactive carbonyl compounds Phytochemicals Control Glyoxal Methylglyoxal Malondialdehyde Acrolein Catechin -3. 7 7.0 >48 3.6 Epicatechin >48 4.3 11.7 >48 4. 7 Chlorogenic acid 41. 6 9.4 9. 2 >48 47.7 Quercetin 11.9 6. 4 5. 5 20. 9 6.9 Resveratrol 34.0 7.4 7.4 35.3 3. 8 Table 5 2 Empirical degradation half time of reactive carbonyl compounds in different incubation mixtures Time (h) Phytochemicals Carbonyls Control Catechin Epicatechin Chlorogenic acid Quercetin Resvera trol Glyoxal >48 3. 5 2.7 28. 2 3.6 4.3 Methylglyoxal >48 0.4 0.4 11. 1 0. 8 1.0 Malondialdehyde >48 28.9 24.5 30.1 >48 >48 Acrolein 9.5 0.6 0. 5 3.7 1. 5 1.5

PAGE 91

91 Figure 5 1. Percentage of remaining cat echin (CAT) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). Scattered dots represented the mean standard deviation of remaining catechin for two independent experiments. D ifferent letters denoted significant differences (p 0.05) from each other at 24 h

PAGE 92

92 Figure 5 2. Percentage of remaining epicatechin (EPI) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR) Scattered dots represented the mean standard deviation of remaining epicatechin for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 93

93 Figure 5 3. Per centage of remaining chlorogenic acid (CGA) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). Scattered dots represented the mean standard deviation of remaining chlorogenic acid for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 94

94 Figure 5 4. Percentage of remaining quercetin (QUE) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). Scattered dots represented the mean standard deviation of remaining quercetin for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 95

95 Figure 5 5. Percent age of remaining resveratrol (RES) during 37 C incubation with phosphate buffer (blank), glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and acrolein (ACR). Scattered dots represented the mean standard deviation of remaining resveratrol for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 96

96 Figure 5 6. Percentage of remaining glyoxal (GO) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin (EPI), quercetin (QUE), resveratrol (RES) and chloro genic acid (CGA). Scattered dots represented the mean standard deviation of remaining glyoxal for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 97

97 Figu re 5 7. Percentage of remaining methylglyoxal (MGO) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin (EPI), quercetin (QUE), resveratrol (RES) and chlorogenic acid (CGA). Scattered dots represented the mean standard devia tion of remaining methylglyoxal for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 98

98 Figure 5 8. Percentage of remaining malondialdehyde (MDA) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin (EPI), quercetin (QUE), resveratrol (RES) an d chlorogenic acid (CGA). Scattered dots represented the mean standard deviation of remaining malondialdehyde for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 99

99 Figure 5 9. Percentage of remaining acrolein (ACR) during 37 C incubation with phosphate buffer (blank), catechin (CAT), epicatechin (EPI), quercetin (QUE), resveratrol (RES) and chlorogenic acid (CGA). Scattered dots represented the mean st andard deviation of remaining acrolein for two independent experiments. Different letters denoted significant differences (p 0.05) from each other at 24 h.

PAGE 100

100 Table 5 3 P hytochemical carbonyl adduct identification RT (min) Molecular Weight MS1 MS2 Adduct Composition Catechin carbonyl systems 9.8 348 347 [M H] 329, 289, 245, 205, 167 Monocatechin + monoglyoxal 8.2 620 619 [M H] 468, 329, 289, 245 Dicatechin + monoglyoxal 9.5 638 638 [M] 289 Dicatechin + monoglyoxal 12.4 362 361 [M H] 343, 289, 245, 181 Monocatechin + monomethylglyoxal 20.2 434 433 [M H] 415, 361, 343, 289 Monocatechin + dimethylglyoxal 21.8 362 361 [M H] 327, 289 Monocatechin + monomalondialdehyde 20.4 434 433 [M H] 289, 163, 326 Monocatechin + dimalondialdehyde 20.1 346 345 [M H] 289, 245, 21 5, 16 1 Monocatechin + monoacrolein Epicatechin carbonyl systems 9.4 348 347 [M H] 329, 289, 245 Monoe picatechin + monoglyoxal 19.7 620 619 [M H] 329, 289, 206 Diepicatechin + monoglyoxal 20.0 6 78 67 7 [M H] 387, 347 289, 225 Diepicatechin + di glyoxal 12.9 362 361 [M H] 344, 289, 145 Monoepicatechin + monomethylglyoxal 20.6 434 433 [M H] 416, 343, 289 181 Monoepicatechin + dimethylglyoxal 21.1 434 433 [M H] 289, 245, Monoepicatechin + dimalondialdehyde 20.3 346 345 [M H] 315, 289, 245, 192 Monoepicatechin + monoacrolein C hlorogenic acid carbonyl systems ND ND ND ND ND Quercetin carbonyl sys tems 13.9 360 341 [M H H2O] 301, 249 Monoquercetin + monoglyoxal 12.6 374 373 [M H] 355, 301, 203 Monoquercetin + monomethylglyoxal 16.0 35 8 357 [M H ] 301, 265, 223 Monoquercetin + monoacrolein Resveratrol carbonyl systems 13.6 286 285 [M H] 267, 227, 161 Monoresveratrol + monoglyoxal 14.9 300 299 [M H] 227, 175 Monoresveratrol + monomethylglyoxal 16.0 300 299 [M H] 227 Monoresveratrol + monomalondialdehyde 18.0 284 283 [M H] 22 8 Monoresveratrol + monoacrolein 20.7 340 339 [M H] 228 283 Monoresveratrol + diacrolein

PAGE 101

101 Fig ure 5 10. HPLC FLD (Ex=231nm, Em=320nm) chromatogram of the reaction products in the incubation of catechi n with phosphate buffer (A), catechin with glyoxal (B), catechin with methylglyo xal (C), catechin with malondi al dehyde (D) and catechin with acrolein (E). Emerging peaks were reaction adducts and labeled with their molecular weight.

PAGE 102

102 Fig ure 5 11. MS and MS 2 spectra of the precursor ions of m/z = 619 [M H] (A), m/z = 347 [M H] (B) and m/z = 638 [M ] (C) in the incubation of catechin and glyoxal in negative electrospray mode

PAGE 103

103 Fig ure 5 12. The proposed structures of reaction adducts formed by catechin and glyoxal.

PAGE 104

104 Fig ure 5 13 MS and MS 2 spectra of the precursor ions of m/z = 433[M H] (A) and m/z = 361 [M H] (B) in the incubation of catechin and methylglyoxal in negative electrospray mode. Fig ure 5 14. The proposed structures of reaction adducts formed by catechin and methylglyoxal.

PAGE 105

105 Fig ure 5 15 MS and MS 2 spectra of the prec ursor ions of m/z = 433 [M H] (A ) and m/z = 361 [M H] ( B ) in the incubation of catechin and malondialdehyde in negative electrospray mode. Fig ure 5 16. The proposed structures of reaction adducts formed by catechin and malondialdehyde.

PAGE 106

106 Fig ure 5 17 MS and MS 2 spectra of the precursor ion of m/z = 345 [M H] in the incubation of catechin and acrolein in negative electrospray mode. Fig ure 5 18. The proposed structure of reaction adduct formed by catechin and acrolein.

PAGE 107

107 Fig ure 5 1 9 HPLC FLD (Ex=231nm, Em=320nm) chromatogram of the reaction products in the incubation of epi catechin with phosphate buffer (A) epicatechin with glyoxal (B), epicatechin with methylglyoxal (C), epicatechin with malondialdehyde (D) and epic atechin with acrolein (E ) Emerging peaks were reaction adducts and labeled with their molecular weight.

PAGE 108

108 Fig ure 5 20 MS and MS 2 spectra of the precursor ions of m/z = 347 [M H] (A), m/z = 619 [M H] ( B ) and m/z = 677 [M H] ( C ) in the incubation of epicatechin and glyoxal in negative electrospray mode.

PAGE 109

109 Fig ure 5 21. The proposed structures of reaction adducts formed by epicatechin and glyoxal.

PAGE 110

110 Fig ure 5 22 MS and MS 2 spectra of the precursor ions of m/z = 361 [M H] and m/z 433 [M H] in the incubation of epicatechin and methylglyoxal in negative electrospray mode. Fig ure 5 23. The proposed structures of reaction adducts formed by epicatechin and methylglyoxal.

PAGE 111

111 Fig ure 5 24 MS and MS 2 spectra of the precursor io n of m/z = 433[M H] in the incubation of epicatechin and malondialdehyde in negative electrospray mode. Fig ure 5 25. The proposed structure of reaction adduct formed by epicatechin and malondialdehyde.

PAGE 112

112 Fig ure 5 26 MS and MS 2 spectr a of the precursor ions of m/z = 345[M H] in the incubation of epicatechin and acrolein in negative electrospray mode. Fig ure 5 27. The proposed structures of reaction adducts formed by epicatechin and acrolein.

PAGE 113

113 Fig ure 5 28 HPLC D AD (360nm) chromatogram of the reaction products in the incubation of quercetin with phosphate buffer (A), quercetin with glyoxal (B), quercetin with methylglyoxal (C) and quercetin with acrolein ( D ) Emerging peaks were reaction adducts and labeled with t heir molecular weight.

PAGE 114

114 Fig ure 5 29 MS and MS 2 spectra of the precursor ions of m/z = 341 [M H] (A quercetin and glyoxal ) m/z = 373 [M H] (B quercetin and methylglyoxal ) and m/z = 357 [M H] (C quercetin and acrolein) in the incubations of quercetin and reactive carbonyl compounds in negative electrospray mode.

PAGE 115

115 Fig ure 5 30. The proposed structures of reaction adducts formed by quercetin with glyoxal, methylglyoxal, malondialdehyde and acrolein.

PAGE 116

116 Fig ure 5 31 HPLC FLD (Ex=330nm, Em =374nm) chromatogram of the reaction products in the incubation of resveratrol with phosphate buffer (A), resveratrol with glyoxal (B), resveratrol with methylglyoxal (C), resveratrol with malondialdehyde (D) an d resveratrol with acrolein (E). Emerging pea ks were reaction adducts and labeled with their molecular weight.

PAGE 117

117 Fig ure 5 32 MS and MS 2 spectra of the precursor ions of m/z = 285 [M H] (A, resveratrol and glyoxal), m/z = 299 [M H] (B, resveratrol and methylglyoxal), m/z = 299 [M H] (C, resveratrol and malondialdehyde) m/z =283 [M H] and m/z = 339 [M H] (D and E resveratrol and acrolein) in the incubation of resveratrol and reactive carbonyl compounds in negative electrospray mode.

PAGE 118

118 Fig ure 5 33. The proposed structures of reaction adducts formed by resveratrol with glyoxal, methylglyoxal, malondialdehyde and acrolein.

PAGE 119

119 CHAPTER 6 CONCLUSION In conclusion, su gar free phytochemicals extracted from berries and grapes effectively scavenged methylglyoxal. They had potent anti glycat ion capacities in BSA fructose, BSA methylglyoxal and arginine methylglyoxal assays. Phenolic compounds from berries and grapes scavenged reactive carbonyls by forming adducts of different structures.

PAGE 120

120 LIST OF REFERENCES 1. O'Brien, P J.; Siraki, A. G.; Shangari, N., Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit Rev Toxicol 2005 35, 609 62. 2. Kazutoshi, F.; Takayuki, S., Determination of toxic carbonyl compounds in cigarette smoke. Environmental Toxicology 2006 21, 47 54. 3. Andre, E.; Campi, B.; Materazzi, S.; Trevisani, M.; Amadesi, S.; Massi, D.; Creminon, C.; Vaksman, N.; Nassini, R.; Civelli, M.; Baraldi, P. G.; Poole, D. P.; Bunnett, N. W.; Geppetti, P.; Patacchini, R ., Cigarette smoke induced neurogenic inflammation is mediated by alpha,beta unsaturated aldehydes and the trpa1 receptor in rodents. J Clin Invest 2008 118, 2574 82. 4. Raij, L., Hypertension, endothelium, and cardiovascular risk factors. The American J ournal of Medicine 1991 90, S13 S18. 5. Nemet, I.; Varga Defterdarovic, L.; Turk, Z., Methylglyoxal in food and living organisms. Mol Nutr Food Res 2006 50, 1105 17. 6. Tan, D.; Wang, Y.; Lo, C. Y.; Sang, S.; Ho, C. T., Methylglyoxal: Its presence in b everages and potential scavengers. Ann N Y Acad Sci 2008 1126, 72 5. 7. dicarbonyl fragments from mono and disaccharides under caramelization and maillard reaction conditions. Zeitschrift fr Lebensmittelunters uchung und Forschung A 1998 207, 50. 8. Esterbauer, H.; Schaur, R. J.; Zollner, H., Chemistry and biochemistry of 4 hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991 11, 81 128. 9. Negre Salvayre, A.; Coatrieux, C.; Inguene au, C.; Salvayre, R., Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors. British Journal of Pharmacology 2008 153, 6 20. 10. Alarcon, R. A., Formation of acro lein from various amino acids and polyamines under degradation at 100c. Environmental Research 1976 12, 317 326. 11. Miyata, T.; Ishikawa, N.; de Strihou, C. v. Y., Carbonyl stress and diabetic complications. Clinical Chemistry & Laboratory Medicine 200 3 41, 1150 1158.

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129 BIOGRAPHICAL SKETCH Wei Wang was originally from Guangzhou, China. She degree in b iotechno logy from Su n Yat sen University in 2007. After that she entered a nutritional Ph D program at the Ohio State University in Columbus, Ohio Eager to explore her interest in food science, she transferr ed to the University of Florida and entered the Food Sci ence and Human Nutrition Department in 2008 under the supervision of Dr. Liwei Gu. While in graduate school Wei had one publication in the Journal of Food Chemistry. Further more she received the William L. and Agnes F. Brown Graduate Scholarship and Outstanding Academic Achievement Certificate from UF in 2010. Upon her completion of the m career in the food industry and is looking forward to any challenge that life will bring.