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Inhibitory Effects of Brown Algae (Fucus Vesiculosus) Extracts and Its Constituent Phlorotannins on the Formation of Adv...

Permanent Link: http://ufdc.ufl.edu/UFE0043773/00001

Material Information

Title: Inhibitory Effects of Brown Algae (Fucus Vesiculosus) Extracts and Its Constituent Phlorotannins on the Formation of Advanced Glycation Endproducts
Physical Description: 1 online resource (70 p.)
Language: english
Creator: Liu, Haiyan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: advanced -- endproducts -- fucusvesiculosus -- glycation -- methylglyoxal -- phlorotannins
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: Advanced glycation endproducts (AGEs) are a group of complex and heterogeneous compounds generated during protein glycation. Accumulation of AGEs is associated with aging, diabetes, Alzheimer's disease, renal failure, etc. Reactive carbonyls produced during lipid peroxidation or sugar glycoxidation play an important role in protein glycation. Carbonyl compounds rapidly react with amino groups in biological molecules to form AGEs. Previous studies showed that polyphenols scavenged reactive carbonyls and had inhibitory effects on the formation of AGEs. Brown algae Fucus vesiculosus contains a wide range of bioactive phytochemicals including phlorotannins, which are the oligomers and polymers of phloroglucinol found exclusively in brown algae. We hypothesized that F. vesiculosus may inhibit the formation of AGEs by scavenging reactive carbonyl compounds. F. vesiculosus phlorotannins were extracted using 70% acetone. The resultant extract was fractionated into dichloromethane, ethyl acetate, butanol and water fractions using a liquid-liquid partition method. The ethyl acetate fraction was further fractionated into four subfractions (Ethyl-F1 to F4) using a Sephadex LH-20 column. The antioxidant capacities of F. vesiculosus acetone extract or fractions were evaluated. The inhibitory effects of F. vesiculosus extracts on the formation of AGEs were investigated in bovine serum albumin (BSA)-glucose model and BSA-methylglyoxal model. The capacity of F. vesiculosus extracts to scavenge methylglyoxal was examined. Phloroglucinol, the constituent unit of phlorotannins, was incubated with methylglyoxal and glyoxal at pH 7.4. Phloroglucinol-carbonyl adducts were detected, and their structures were tentatively identified using HPLC-ESI-MSn. Liquid-liquid partition of F. vesiculosus acetone extract caused phlorotannins to be concentrated in butanol and ethyl acetate fractions. HPLC-ESI-MSn analyses showed that Ethyl-F1 and F2 contained phlorotannin oligomers and polymers. Etnyl-F3 and F4 contained exclusively phlorotannin polymers. Phlorotannin trimers through pentamers were identified in Ethyl-F1. Phlorotannin pentamer, heptamer and octamers were identified in Ethyl F-2. F. vesiculosus acetone extract or fractions significantly displayed high capacities to scavenge free radicals. All the extracts inhibited the formation of AGEs mediated by glucose and methylglyoxal. The inhibitory effects increased in a concentration-dependent manner. The concentrations of F. vesiculosus extracts required to inhibit 50% of albumin glycation (EC50) in BSA-methylglyoxal assay were lower than those of aminoguanidine (a drug candidate for diabetic complication), except for F. vesiculosus acetone extract and dichloromethane fraction. In BSA-glucose assay, F. vesiculosus extracts inhibited glycation of bovine serum albumin more or as effectively as aminoguanidine, except for Ethyl-F3 and F4. Ethyl acetate fraction and its four subfractions scavenged more than 50% of methylglyoxal in two hours. The reaction between phloroglucinol with glyoxal or methylglyoxal led to the generation of adducts. The structures of phloroglucinol-carbonyl adducts were tentatively identified using HPLC-ESI-MSn. Our study showed that F. vesiculosus phlorotannins inhibited the formation of AGEs by scavenging reactive carbonyls. F. vesiculosus in diet may provide benefits in preventing carbonyl or AGE-related chronic diseases.
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 Haiyan Liu.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Gu, Liwei.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043773:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043773/00001

Material Information

Title: Inhibitory Effects of Brown Algae (Fucus Vesiculosus) Extracts and Its Constituent Phlorotannins on the Formation of Advanced Glycation Endproducts
Physical Description: 1 online resource (70 p.)
Language: english
Creator: Liu, Haiyan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: advanced -- endproducts -- fucusvesiculosus -- glycation -- methylglyoxal -- phlorotannins
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: Advanced glycation endproducts (AGEs) are a group of complex and heterogeneous compounds generated during protein glycation. Accumulation of AGEs is associated with aging, diabetes, Alzheimer's disease, renal failure, etc. Reactive carbonyls produced during lipid peroxidation or sugar glycoxidation play an important role in protein glycation. Carbonyl compounds rapidly react with amino groups in biological molecules to form AGEs. Previous studies showed that polyphenols scavenged reactive carbonyls and had inhibitory effects on the formation of AGEs. Brown algae Fucus vesiculosus contains a wide range of bioactive phytochemicals including phlorotannins, which are the oligomers and polymers of phloroglucinol found exclusively in brown algae. We hypothesized that F. vesiculosus may inhibit the formation of AGEs by scavenging reactive carbonyl compounds. F. vesiculosus phlorotannins were extracted using 70% acetone. The resultant extract was fractionated into dichloromethane, ethyl acetate, butanol and water fractions using a liquid-liquid partition method. The ethyl acetate fraction was further fractionated into four subfractions (Ethyl-F1 to F4) using a Sephadex LH-20 column. The antioxidant capacities of F. vesiculosus acetone extract or fractions were evaluated. The inhibitory effects of F. vesiculosus extracts on the formation of AGEs were investigated in bovine serum albumin (BSA)-glucose model and BSA-methylglyoxal model. The capacity of F. vesiculosus extracts to scavenge methylglyoxal was examined. Phloroglucinol, the constituent unit of phlorotannins, was incubated with methylglyoxal and glyoxal at pH 7.4. Phloroglucinol-carbonyl adducts were detected, and their structures were tentatively identified using HPLC-ESI-MSn. Liquid-liquid partition of F. vesiculosus acetone extract caused phlorotannins to be concentrated in butanol and ethyl acetate fractions. HPLC-ESI-MSn analyses showed that Ethyl-F1 and F2 contained phlorotannin oligomers and polymers. Etnyl-F3 and F4 contained exclusively phlorotannin polymers. Phlorotannin trimers through pentamers were identified in Ethyl-F1. Phlorotannin pentamer, heptamer and octamers were identified in Ethyl F-2. F. vesiculosus acetone extract or fractions significantly displayed high capacities to scavenge free radicals. All the extracts inhibited the formation of AGEs mediated by glucose and methylglyoxal. The inhibitory effects increased in a concentration-dependent manner. The concentrations of F. vesiculosus extracts required to inhibit 50% of albumin glycation (EC50) in BSA-methylglyoxal assay were lower than those of aminoguanidine (a drug candidate for diabetic complication), except for F. vesiculosus acetone extract and dichloromethane fraction. In BSA-glucose assay, F. vesiculosus extracts inhibited glycation of bovine serum albumin more or as effectively as aminoguanidine, except for Ethyl-F3 and F4. Ethyl acetate fraction and its four subfractions scavenged more than 50% of methylglyoxal in two hours. The reaction between phloroglucinol with glyoxal or methylglyoxal led to the generation of adducts. The structures of phloroglucinol-carbonyl adducts were tentatively identified using HPLC-ESI-MSn. Our study showed that F. vesiculosus phlorotannins inhibited the formation of AGEs by scavenging reactive carbonyls. F. vesiculosus in diet may provide benefits in preventing carbonyl or AGE-related chronic diseases.
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 Haiyan Liu.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Gu, Liwei.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043773:00001


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1 INHIBITORY EFFECTS OF BROWN ALGAE ( FUCUS VESICULOSUS ) EXTRACT S AND ITS CONS TITUENT PHLOROTANNINS ON THE FORMATION OF ADVANCED GLYCATION ENDPRODUCTS By HAIYAN LIU 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 2011

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2 2011 Haiyan Liu

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3 To my family and friends

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4 ACKNOWLEDGMENTS I want to express my gratitude to my major advisor, Dr. Liwei Gu, for his patience, continuous encouragement and mentorship. Without his guidance and support, this research could not be accomplished. I am grateful for my committee members, Dr. Maurice R. Marshall Dr. W. Steven Otwell, and Dr. E dward J. Phlips for their valuable time and suggestions. I cherished the friendship created with my lab group members Keqin Ou, Wei Wang, Hanwei Liu, Amandeep K. Sandhu, Zheng Li and Timothy Buran. They were always willing to offer helping hands The laughter we shared brought abundant joy and made our lives memorable. I also want to give my thanks to Sara Marshall, who kindly offered help to proofread the present thesis. M ost important ly I want to express my deepest gratitude to my parents for their constant love and great support of my education. Without their love, patience and unconditional support I would not be able to successfully accomplish my graduate studies.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 13 Background ............................................................................................................. 13 Marine Algae ........................................................................................................... 13 Health Benefits ................................................................................................. 13 Phytochemicals in Marine Algae ...................................................................... 14 Reactive Carbonyls ................................................................................................. 15 Exogenous Sources of Reactive Carbonyl Compounds ................................... 15 Formation of Reactive Carbonyl Compounds ................................................... 16 Advanced Glycation Endproducts (AGEs) .............................................................. 16 The Formation of AGEs .................................................................................... 16 Inhibi tion of AGE s Formation ............................................................................ 17 Research Objectives ............................................................................................... 18 2 EXTRACTION, FRACTIONATION AND HPLCESI MS IDENTIFICATION OF PHLOROTANNINS FROM FUCUS VESICULOSUS .............................................. 21 Background ............................................................................................................. 21 Materials and Methods ............................................................................................ 21 Chemicals and Materials .................................................................................. 21 Phlorotannin Extraction and Fractionation ........................................................ 21 Folin Ciocalteu Assay ....................................................................................... 22 Oxygen Radical Absorbance Capacity Assay .................................................. 23 Total Phlorotannin Content Assay .................................................................... 23 P hlorotannin Identification by HPLC DADESI MSn ......................................... 24 Data Expression and S tatistical Analysis .......................................................... 25 Results and Discussion ........................................................................................... 25 Total Phenolic Content ..................................................................................... 25 Oxygen Radical Absorbance Capacity ............................................................. 25 Total Phlorotannin Content ............................................................................... 26 Phlorotannin Identification by HPLCDADESI MSn ......................................... 27

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6 3 INHIBITORY EFFECT S OF FUCUS VESICULOSUS ON THE FORMATION OF ADVANCED GLYCATION EndPRODUCTS ........................................................... 43 Background ............................................................................................................. 43 Materials and Methods ............................................................................................ 43 Chemicals and Materials .................................................................................. 43 B o vine Serum Albumin (BSA) Glucose Assay ................................................. 43 BSAMethylglyoxal Assay ................................................................................. 44 Methylglyoxal Scavenging Assay ..................................................................... 44 Data Expression and S tatistical Analysis .......................................................... 45 Results .................................................................................................................... 45 Anti Glycation Effects in BSA Glucose Assay .................................................. 45 Anti Glycation Effects in BSA Methylglyoxal Assay .......................................... 46 Methylglyoxal Scavenging Capacity ................................................................. 46 Discussion .............................................................................................................. 47 4 HPLCESI MS IDENTIFICATION OF PHLOROGLUCINOL CARBONYL ADDUCTS .............................................................................................................. 53 Background ............................................................................................................. 53 Materials and Methods ............................................................................................ 53 Chemic als ......................................................................................................... 53 Phloroglucinol Glyoxal/Methylglyoxal Reaction and Adduct Identification ........ 53 Results .................................................................................................................... 54 Discussion .............................................................................................................. 56 5 CONCLUSION ........................................................................................................ 61 LIST OF REFERENCES ............................................................................................... 62 BIOGRAPHICAL SKETCH ............................................................................................ 70

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7 LIST OF TABLES Table page 2 1 Retention times and mass spectrometric data of phlorotannins in F. vesiculosus determined by HPLC ESI MSn. ....................................................... 32 3 1 EC50 of F. vesic ulosus extracts inhibiting protein glycation in two assays. ......... 49

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8 LIST OF FIGURES Figure page 1 1 Structures of phlorotannins from F. vesiculosus. ................................................ 19 1 2 Structures of reactive carbonyl compou nds. ....................................................... 19 1 3 Structures of advance glycation endproducts (AGEs). ....................................... 20 2 1 Extraction and fractionation of phlorotannins from F. vesiculosus ..................... 33 2 2 Total phenolic content of F. vesiculosus extracts determined by FolinCiocalteu assay. ................................................................................................. 34 2 3 Antioxidant capacities of F. vesiculosus extracts determined by oxygen radical absorbance c apacity assay ..................................................................... 35 2 4 Total phlorotannin content of F. vesiculosus extracts determined by DMBA assay. Results are means standard deviation of triplicate assay. Bars with diff erent letters were significantly different (P < 0.05) from each other. .............. 36 2 5 HPLCDAD chromatograms of F. vesiculosus fractions .................................... 37 2 6 MS and MS2 spectra of phlorotannin peaks in Ethyl F1. .................................... 39 2 7 MS and MS2 spectra of phlorotannin peaks in Ethyl F2. .................................... 40 2 8 The proposed structures and fragmentation of phlorotannins in Ethyl F1 and Ethyl F2. ............................................................................................................. 41 3 1 Inhibitory effects of F. vesiculosus extracts on the formation of AGEs in BSA glucose assay.. ................................................................................................... 50 3 2 Inhibitory effects of F. vesiculosus extracts on the formation of AGEs in BSA methylglyoxal assay. .......................................................................................... 51 3 3 The capacity of F. vesiculosus extracts to scavenge methylglyoxal .................. 52 4 1 HPLCDAD chromatograms of the phloroglucinol after incubation for two hours ................................................................................................................. 58 4 2 MS and MS2 spectra of phloroglucinol glyoxal/methyglyoxal adducts peaks ...... 59 4 3 The proposed structures of phloroglucinol glyoxal/methylglyoxal adducts and their product ions ............................................................................................... 60

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9 LIST OF ABBREVIATION S AGE s Advanced Glycation Endproducts BSA Bovine serum a lbumin DAD Diode array detector HPLC High performance liquid chromatogram M Molar Mg Milligram Min Minute(s) mL Milliliter mM Millimolar m/z Mass to charge ration ORAC Oxygen radical absorbance capacity Trolox 6 Hydroxy2, 5, 7, 8 tetramethylchroman2 carboxylic acid g Microgram L Microliter

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10 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 INHIBITORY EFFECTS OF BROWN ALGAE ( FUCUS VESICULOSUS ) EXTRACTS AND ITS CONSTITUENT PHLOROTANNINS ON THE FORMATION OF ADVANCED GLYCATION ENDPRODUCTS By Haiyan Liu December 2011 Chair: Liwei Gu Major: Food Science and Human Nutrition Advanced glycation endproducts (AGEs) are a group of complex and heterogeneous compounds generated during protein glycation. Accumulation of AGEs is associated with aging, diabetes Alzheimers disease, renal failure etc. Reactive carbonyls produced during lipid peroxidation or sugar glycoxidation play a n important role in protein glycation. Carbonyl compounds rapidly react with amino groups in biological molecules to form AGEs. Previous studies showed that polyphenols scavenged reactive car bonyls and had inhibitory effects on the formation of AGEs. Brown algae F ucus vesiculosus contains a wide range of bioactive phytochemicals including phlorotannins, which are the oligomers and polymers of phloroglucinol found exclusively in brown algae. W e hypothesized that F. vesiculosus m ay inhibit the formation of AGE s by scaveng ing reactive carbonyl compounds. F. vesiculosus phlorotannins were extract ed using 70% acetone. The resultant extract was fractionated into dichloromethane ethyl acetate, butanol and water f ractions using a liquid liquid partition method. The ethyl acetate fraction was further fractionated into four subfractions ( E thyl F1 to F4) using a Sephadex LH 20 column. The antioxidant

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11 capacities of F. vesiculosus acetone extract or fractions were evaluated. The inhibitory effects of F. vesiculosus extracts on the formation of AGEs were investigated in bovine serum albumin (BSA) glucose model and BSA methylglyoxal model The capacity of F. vesiculosus extracts to scavenge methylglyoxal was examined. Phloroglucinol the constituent unit of phlorotannins, was incubated with methylglyoxal and glyoxal at pH 7. 4. Phloroglucinol carbonyl adducts were detected and their structures were tentatively identified using HPLC ESI MSn. Liquid liquid partition of F. vesiculosus acetone extract caused phlorotannins to be concentrated in butanol and ethyl acetate fractions. HPLC ESI MSn analyses showed that Ethyl F1 and F2 contained phlorotannin oligomers and polymers. Etnyl F3 and F4 contained exclusively phlorotannin polymers. Phlorotannin trimers through pentamers were identified in Ethyl F1. Phlorotannin pentamer, heptamer and octamers were identified in Ethyl F 2. F. vesiculosus acetone extract or fractions significantly displayed high capacities to scavenge free radicals. All the extracts inhibited the formation of AGEs mediated by glucose and methylglyoxal. The inhibitory effects increased in a concentrationdependent manner The concentrations of F. vesiculosus extracts required to inhibit 50% of albumin glycation (EC50) in B SAmethylglyoxal assay were lower than th ose of aminoguanidine ( a drug candidate for diabetic complication) except for F. vesiculosus acetone extract and dichloromethane fraction. In BSAglucose assay F. vesicu losus extract s inhibited glycation of bovine serum albumin more or as effectively as aminoguanidine, except for Ethyl F3 and F4. Eth yl acetate fraction and its f our subfractions scavenged more than 50% of methylglyoxal in two hours The reaction

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12 between p h loroglucinol with glyoxal or methylglyoxal led to the generation of adducts. The structures of phloroglucinol carbonyl adducts were tentatively identified using HPLCESI MSn. Our study showed that F. vesiculosus phlorotannins inhibited the formation of AGEs by scaveng ing reactive carbonyls. F. vesiculosus in diet may provide benefit s in preventing carbonyl or AGE related chronic diseases.

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13 CHAPTER 1 INTRODUCTION Background The a dvance glycation end products (AGEs) are a class of compounds with brown, fluorescent, or cross linked characteristics Accumulation of AGEs is associated with aging, diabetes Alzheimers disease, renal failure and many other chronic diseases Reactive carbonyls such as glyoxal and methylglyoxal are formed during glycoxidation. They play a pivotal role in the formation of AGEs. Reactive carbonyls cause p rotein dysfunctions and tissue damage leading to pathological consequences such as inflammation and apoptosis that contribute to the progression of diseases (1). Marine Algae Marine algae, classified as Laminariales (brown), Chlorophyta (green) and Rhodophyta (red), have a long history of use in the Asian diet and are considered underexploited resources ( 2 3 ) They are known to contain a wide range of bioactive natural substances with diverse health benefits. Health Benefits Previous epidemiological studies have demons trated that marine algae provided protective effects against mammary ( 4 5 ) intestinal ( 6 7 ) and skin carcinogenesis ( 8 9 ) Rodents fed algae exhibited suppression of tumor initiation and mutagenic inhibition in the colon and skin ( 7, 9 ) Red and brown algae extracts have been shown to have inhibitory effects on breast and colon cancer induction ( 10) Anti inflammatory and proliferative ac tivities by a variety of red algal and kelp ex tracts w ere also documented ( 11) Brown algae w as effective in reducing blood cholesterol and lowering blood pressure, as well as preventing arteriolosclerosis ( 1213) Blood pressure control,

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14 constipation prevention and improvement of various gastroenteric disorders have been attributed to the polysaccharide alginic acid in the algae ( 14) Moreover, a number of studies have sho w n that marine algae have potent antioxidant activities ( 1519) Phytochemicals i n Marine Algae Marine algae contain a variety of phenolic phytochemicals. Flavonols and flavonol glycosides have been identified in the methanol extracts of red and green algae ( 20) Phlorotannins are oligomers and p olymers of phloroglucinol that exist exclusively in brown algae ( 2122) They have been identifi ed from several brown algal families such as Alariaceae, Fucaceae and Sargassaceae ( 23) Three types of phlorotannins including fucols, fucophlorethols and phlorethols were isolated and identified in brown algae F. vesi culosus Fucols contain phloroglucinol units which are linked by aryl aryl bonds. Phloroglucinol units in fucophlorethols are connected with ether and aryl aryl bonds, and only ether bonds are present in phlorethols ( 24) The structures of F. vesiculosus phlorotannins are illustrated in Figure 1 1. Three fucophlorethols isolated form F. vesiculosus scavenged free radical s and inhibited cytochrome P450 enzyme ( 24) A polyhydroxylated fucophlorethols from F. vesiculosus showed antibacterial effects ( 25 ) Phlorofucofuroeckol B isolated from a brow algae E. arborea had anti allergic effects ( 26) Phloroglucinol derivatives such as dioxinodehydroeckol, eckol, eckstolonol, phlorofucofuroeckol A, dieckol, triphlorethol B, 2 phloroeckol and 7phloroeckol were isolated from E. stolonifera ( 27 28) These compounds possessed antibacterial, antitumor, nitric oxide inhibitory and reverse transcriptase inhibition activities ( 2932) Oligomeric phlorotannins including eckol, phlorofucofuroeckol A, dieckol and 8, 8 bieckol were identified in the Japanese Laminariaceous brown algae Eisenia bicyclis Setchell and Ecklonia kurome. These

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15 compounds exhibited inhibitory effects on phospholipid peroxidation and radical scavenging activities (33) The 7 phloro eckol 6,6 bieckol l of phloroglucinol derivatives and fucodiphloroethol G from Ecklonia cava showed antioxidant properties ( 34) Phlorofucofuroeckol A from brown algae had inhibitory effects on the formation of AGEs mediated by glucose ( 35) Besides phlorotannins, brown algae also contain carotenoid pigments such as carotene, astaxanthin and fucoxanthin ( 23 ) Carotenoids ( 36) protect cells and tissues from damaging effects of free radicals and singlet oxygen ( 37) Fucoxanthin is a unique marine carotenoid that possess e s excellent antioxidant ( 38 39) anti inflammatory ( 40) and anti obesity effects ( 41) Fucoxanthinol and halocynthiaxanthin are two metabolites of fucoxanthin in marine organisms ( 4243) Fucoxanthin could be hydrolyzed to fucoxanthinol during absorption by Caco2 human and mouse intestine cells ( 44 ) Reactive Carbonyls Reactive carbonyl compounds generated endogenously from sugar glycoxidation and lipid peroxidation rapidly react with amino groups in biological molecules to form advanced glycation end products (AGEs) and advanced lipid p eroxidation end products (ALEs) ( 45) Th eir accumulat ion in vivo is associated with aging diabetes Alzheimer s, renal failure and other chronic disease ( 46 47 ) Exogenous Sources of Reactive Carbonyl Compounds The structures of reactive carbonyl compounds are depicted in Figure 12. Reactive carbonyls including methylglyoxal and glyoxal are found in various foods includi ng sugar sweetened beverages, high lipids content foods, and thermal ly processed products. Methylglyoxal and glyoxal were detected in cookies ranging from 3.7 to 81.4 mg/kg, and 4.8 to 26.0 mg/kg, respectively ( 48) In toast ed bread,

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16 methylglyoxal and glyoxal content rang ed from 0.5 to 2.5 mg/kg ( 49) B everages such as carbonated soft drinks are important exogenous sources of dicarbonyls due to the high fructose corn syrup content ( 50) Other drinks such as coffee, wine, and beer were also found to contain carbonyl compounds ( 51 ) Dicarbonyl compounds found in commercial soybean, olive and corn oil products were attribut ed to lipid peroxidation during manufacturing and storage ( 52) Soy sauce and brewed coffee contained the highest amount of methylglyoxal in food products and beverages ( 53) Formation of Reactive Carbonyl Compounds Carbonyl compound are generated under oxidati ve conditions in vivo and in vitro Lipid peroxidation produces a number of reactive carbonyl compounds. For example, oxidation of polyunsaturated fatty acid leads to the formation of unsaturated hydroxyalkenals, such as 4hydroxynonenal and 4hydroxyhexenal ( 45) Methylglyoxal and glyxoal can also be generated from sugar glycoxidation( 45) During protein glycation, degradation of Schiff bases and oxidation of Amadori products produce reactive carbonyls such as methylglyoxal, glyoxal, and 3 deoxyglucosone ( 54) Besides the nonenzymatic pathway, reactive carbonyls can be generated via enzymatic catalyzed metabolism The catabolism of lipids and amino acid produces methylglyoxal ( 55) Methylglyoxal can also be generated from ketone bodies by cytochrome P450 enzymes (56) Enzymatic oxidation of theronine by myeloperoxidase produces acrolein (57) Advanced Glycation Endproducts (AGEs) The Formation of AGEs Advanced glycation endproducts (AGEs) are a group of complex and heterogeneous compounds generated during protein glycation ( 58) Protein glycation

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17 has three stages : the initial stage, intermediate stage, and the late stage ( 54) In the initial stage, reducing sugar s such as glucose react with the primary amino groups of a lysine residue to form a Schiff base, which is reversible. N substituted glycosylamine is formed from the cyclization of the Schiff base ( 59) This unstable N substituted glycosylamine undergo Amadori rearrangement to generate relatively stable Amadori products ( 60) Such reactions can be catalyzed by either iminium ion or transition metal ions ( 54) On the other hand, the unstable Schiff base may degrade to generate carbonyls including glyxoal and glycoaldehyde via the Namiki pathway ( 61) In the intermediate stage, Amadori products decompose to generate the dicarbonyls such as 3 deoxyglucosone. In addition to the degradation of Schiff base and oxidation of Amadori products dicarbonyls such as glyxoal, methylglyoxal and 3deoxyglucosone can also be generated from sugar glycoxidation ( 45) T he lat e stage of protein glycation is mediated primarily by these dicarbonyls Glyoxal reacts with proteins to form several AGEs including (carboxymethyl)lysine, (carboxymethyl )arginine, glyoxal lysine dimer, argininelysine glyoxal crosslink, and pentosidine ( 54) Methylglyoxal react s with amino group of lysine residue in protei ns to form (carboxymethyl)lysine, N(carboxyethyl)lysine ( 6263) and methylglyox al lysine dimer ( 64) 3 deoxyglucosone modifies lysine residue in proteins t o generate AGEs such as labile pyrraline and imidazolone ( 6566) The structure of pentosidine, pyrraline and (carboxymethyl)lysine are illustrated in Figure 1 3 Inhibition of AGE s Formation Studies have shown that the formation of AGEs is a major pathogenic factor in diabetes ( 67 ) advance d aging ( 68) Alzheimers disease ( 68 ) renal failure ( 69) and other chronic diseases The current approach to prevent these conditions is to use

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18 antiglycation agents. Antiglycation agents suppress the formation of AGEs by either scavenging reactive carbonyls or alleviating oxidative stress ( 54) A minoguanidine, metformin and pyridoxamine are antigl ycation drug candidates that inhibit AGE inhibition by scavenging reactive dicarbonyls ( 70) P yridoxamine also prevent s the conversion of Amadori product to (carboxymethyl) lysine ( 71) But the application of these drugs in clinical trial was not successful due to toxicity It was re ported that phenolic compounds from plants had antiglycation effects. Polyphenols from cinnamon bark and mung bean inhibited pr otein glycation and the formation of AGEs (72 73) C atechin epicatechin and epigallocatechin from green tea had inhibitory effects on the protein glycation (74 75) It was found that these phenolic compouds scavenged reactive carbonyls Research Objectives T he investigation of carbonyl scavenging agents and AGEs inhibitor s provides a novel approach to prevent diabetic complications and other pathological diseases We hypothesized that phytochemicals from edible marine algae are able to inhibit the formation of AGEs by scavenging reactive carbony ls. There were three research objectives in this thesis. 1. To extract, fractionate and identify phlorotannins from F vesiculosus 2. To investigate the antiglycation effects and carbonyl scavenging activities of F. vesiculosus extracts. 3. To identify phlorogluci nol carbonyl adducts formed as a result of carbonyl scavenging

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19 Figure 1 1 Structures of phlorotannins from F. vesiculosus Figure 1 2 Structures of reactive carbonyl compounds O O H O H O H OH OH O H O H OH OH O H OH Phloroglucinol Fucophlorethol A OH O H O H O H OH OH O H O H OH O OH O H OH O H OH O O H O H OH O H OH OH O H OH O H O H OH OH O H O H Tetrafucol A Trifucodiphlorethol A O O O O O OH O O O OH O glyoxal methylglyoxal malondialdehyde acrolein 4-Hydroxy-2-nonenal (4-HNE) 4-Hydroxy-2-hexenal (4-HHE)

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20 Figure 1 3 Structures of advance glycation endproducts (AGEs). C H2 CO OH N H ( C H2)4 N H2 CO OH N HOH2C ( C H2)4 N H2 C HO CO OH N+ N N H N H ( H2C )3 N H2 ( C H2)4 N H2 HO OC CO OH N-carboxylmethyl lysine pyrraline pentosidine

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21 CHAPTER 2 EXTRACTION, FRACTIONATION AND HPLCESI MS IDENTIFICATION OF PHLOROTANNINS FROM FUCUS VESICULOSUS Background Phlorotannins are oligomers and polymers of phloroglucinol that exist exclusively in brown algae. The objective of this chapter was to extract phlorotannins from brown algae F. vesiculosus and tentatively identify these compounds using HPLC ESI MSn. Materials and Methods Chemicals and Materials Dry F. vesiculosus was obtained from Maine Seaweed Co. (Steuben, Maine). Phloroglucinol, N, N dimethylformamide, and 2 4 dimethoxybenzaldehyde were products from Acros Organics (Morris Plains, NJ). Sephadex LH 20 was obtained from Sigma Aldrich (St. Louis, MO). HPLC grade ancetonitrile and other organic solvent s were purchased from Fisher Scientific Co. (Pittsburg, PA). AAPH (2 2 azotis(2amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI) 6 Hydroxy 2, 5, 7, 8 tetramethylchroman2 carboxylic acid (Trolox) was purchased from SigmaAldrich (St. Louis, MO). Phlorotannin Extraction and Fractionation The phlorotannin extraction and fractionation procedure is depicted in Figure 21 Three hundred grams of F. vesiculosus materials was ground into fine pow der using a blender and extracted with 1200 mL acetone: water: acetic acid (70:29.7:0.3, v/v/v). The mixture was sonicated in a water bath sonicater (FS30, Fisher Scientific) for 30 min utes and then kept at room temperature for two hours. The extraction was repeated once. Extracts obtained after vacuum filtration were combined and concentrated under partial vacuum using a rotary evapor ator F ifty four grams of crude extract was obtained. Part

PAGE 22

22 of this extract (30 g) was suspended in 100 mL of water and parti tioned with 100 mL dichloromethane three times in a separation funnel. The dichloromethane phases were combined and evaporated to yield 4.934 g extract. The aqueous phase was then partitioned with ethyl acetate three times (100 mL each) before it was partitioned with butanol an additional three times (100 mL each) The ethyl acetate and butanol phases were evaporated to yield 4.946 g and 4.983 g extracts, respectively. The aqueous phase was dried in a rotary evaporator to y ield the water fraction (2.787 g). An extra sample ethyl acetate fraction (5.231 g) was obtained by repeating the previous fractionation steps. Part of the e thyl acetate fraction (7.622 g) was dissolved in 60% methanol and loaded onto a Sephadex LH 20 col umn (5.828 cm). The column was eluted with 60% methanol (3 L), 80% methanol (2 L), 90% methanol (2.5 L), 100% methanol (2 L) and 70% acetone (4 L). Ethyl acetate subfraction I (Ethyl F1, 0.823 g) was obtained by combining 60% and 80% methanol eluent. Ethy l F2 (0.834 g) was obtained by combining 90% and 100% methanol eluent. Ethyl F3 (3.492 g) and Ethyl F4 (0.669 g) were from 70% acetone eluent FolinCiocalteu Assay The total phenolic contents of F. vesiculosus extracts were determined by FolinCiocalteu assay with modification ( 76) F. vesiculosus extracts was dissolved in methanol and diluted to the appropriate concentration for analysis F. vesiculosus extracts (100 L) were mixed with Folin Ciocalteu reagent (1 ml, 0.2 N) and sodium carbonate (1 ml, 15%) Absorption at 765 nm was measured in a microplate reader (SPECTRAmax 190 Molecular Devices, Sunnyvale, CA ) after incubation for 30 min at room temperature. Phlorogl ucinol solutions with concentrations rang ing from 100 400 mg/L were used to generate a standard curve. The results were expressed as

PAGE 23

23 milligrams of phloroglucinol equivalents per gram of F. vesiculosus extracts (mg of PG /g ). Oxygen Radical Absorbance Capacit y Assay The oxygen radical absorbance capacity (ORAC) assay was used with modification ( 77) F. vesiculosus extracts were dissolved in phosphate buffer (50 mM, pH 7.4) Fifty microliter s of each extract were mixed with fluorescein solution (100 l, 20 nM) in a 96well black microplate. Mixture were incubated at 37 C for 10 min before the addition of 2,2 azobis(2amidinopropane) dihydrochloride (AAPH, 50 l, 0.14 M). Fluorescence was measured using 485 nm excitation and 530 nm emi ssion (Spectra XMS Gemini, Molecular Device, Sunnyvale, CA) Readings were taken at 1 min intervals for 40 min. 6Hydroxy2, 5, 7, 8 tetramethylchroman2 carboxylic acid (Trolox) w as used to generate a standard curve. The antioxidant capacities of extracts were expressed as mol Trolox equivalents (TE) per gram of F. vesiculosus extracts ( TE/g). Total Phlorotannin Content Assay T otal phlorotannins were quantified using 2 4 dimethoxybenzaldehyde ( DMBA ) assay ( 78) The 2 4 dimethoxybenzaldehyde (2.0 g/100 mL glacial acetic acid) and HCl (16.0 mL concentrated HC l in 100 mL gla cial acetic acid) w ere used as stock solutions. A w orking reagent was prepared by mixing equal volumes of these two solutions prior to use. A s tandard curve was prepared by making a phloroglucinol solution with concentrations of 0 mg/ mL 0.1 mg/ mL 0.25 mg/ mL 0.5 mg/ mL 1.0 mg/ mL and 1.25 mg/ mL Ten l of phloroglucinol solution w ere mixed with 10 l of N N dimethylformamide and 2.5 mL of working reagent. The mixtures were incubated at 30 oC for 60 min. After incubation, the absorbance was measured at 494 nm. Similarly 10

PAGE 24

24 l of F. vesiculosus ac e tone extract or fractions w ere mixed with 10 l N, N dimethylformamide and 2.5 mL working reagent. After incubating at 30 oC for 60 min, the absorbance was measured at 515 nm The results were expressed as milligrams of phloroglucinol eq uivalent per gram of F. vesiculosus extracts (mg of PG /g ). P hlorotannin Identification by HPLCDAD ESI MSn Chromatographic analyses were performed on an Agilent 1200 series HPLC system (Agilent, Palo Alto, CA) equipped with an autosampler/injector a binary pump, a column compartment, and a diode array detector. A Zorbax Stablebond Analytical SB C18 column (4.6 for separation. Mobile phases consisted of A (0.1% formic acid aqueous solution) and B m with a recording wav elength of 2 8 0 nm. The flow rate was 1 mL /min T he linear gradient was as follows : 10% B from 0 to 5 min, 10 to 26% B from 5 to 15 min, 26 to 30% B from 15 to 30 min, 30 to 44% B from 30 to 32 min, 44 to 60% B from 32 to 42 min, and 60 to 10% B from 42 to 45 min. Electrospray mass spectrometry was performed on a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Column effluent was monitored in the positive and negative ion mode of the instrument in an alternative manner during the same run. Other experimental conditions for the mass spectrometer were as follows: nebulizer, 45 psi; dry gas, 10.0 L/min; dry temperature, 350 C; ion trap, scan from m/z 100 to 2200; smart parameter setting (SPS), compound stability, 50%; trap drive level, 60%. The mass spectrometer was operated in Auto MS2 mode. MS2 was used to capture and fragment the most abundant ion in full scan mass spectra

PAGE 25

25 Data Expression and S tatistical Analysis One way ANOVA with Tukey Kramer HSD test were done using JMP software as signi ficant. All data were expressed as the mean standard deviation. Results and Discussion Total Phenolic Content The total phenolic content of F. vesiculosus extracts were determined using Folin Ciocalteu with modification ( 76) The r esults of the Folin Ciocalteu assay indicated that all the F. vesiculosus extracts contained various amounts of phenolic compounds, with a range from 24.9 to 594.5 mg phloroglucinol equivalents per gram extract (Figure 2 2 ). Ethyl F3 had the highest phenolic content ( 594.2 0.7 mg phloroglucinol/g extract ) among all the extracts. The total phenolic content of ethyl acetate fraction was higher than that of Ethyl F1 and F2, but was lower than that of Ethyl F 3 and F 4 Ethyl F1 showed comparable amounts of polyphenols as butanol and acetone extracts, and higher phenolic content than that of dichloromethane fraction. The phenolic content of water fractions was the lowest. Oxygen Radical Absorbance Capacity All the F. vesiculosus extracts showed high antioxidant capacity, with values higher than 539.7 mol Trolox e quivale nts per gram extract Ethyl acetate fraction and its four subfractions showed significantly higher antioxidant capacity than F. vesiculosus acetone extract (Figure 23 ). Ethyl F2 and Ethyl F3 had higher antioxidant capacities (6578.6 321.6, and 6288 171.4 mol T E/g extract ) than Ethyl F1 and Ethyl F4. The antioxidant capacities of four subfractions are comparable to th ose of ethyl acetate fractions. Dichloromethane and water fractions showed the lowest ORAC values. The

PAGE 26

26 antioxidant activities of F. ve siculosus extracts may be attributed to phenolic compounds, especially phlorotannin content Ethyl acetate fraction and its subfractions, which are concentrated with phlorotannins, showed significantly higher ORAC values than other extracts. This is in agr eement with previous research that showed phlorotannis scavenged oxygen species ( 7980) The antioxidant activity of phlorotannins from brown algae was associated with their unique molecular skeleton ( 81) The phenol rings of phlorotannins acted as electron traps to scavenge peroxy, superoxideanions and hydroxyl radicals ( 23) Phlorotannins from brown algae have eight interconnected rings, and exhibit greater free radi cal scavenging activity than other polyphenols like green tea catechin, which only has three to four rings ( 82) Total Phlorotannin Content The total phlorotannin content of F. vesiculosus extracts were determined using DMBA assay (78) The results were expressed as milligram phloroglucinol equivalent per gram dry weight. The 70% acetone was used for extraction because it was shown as the most effective solvent to extract phlorotannins from brown algae ( 83) Phytochemicals in the crude acetone extract was fractionated according to polarity using liquid liquid extraction. Dichloromethane was used to obtain lipid compounds from the extract. Both ethyl acetate and butanol were used to concentrated phlorotannins. As a result, ethyl acetate fraction showed the highest concentration phlorotannins. Phlorotannin content was also high in butanol fraction (Figure 24 ). Dichloromethane and water fractions had lower phlorotannins contents than the original extract. All four ethyl acetate subfractions had significantly higher phlorotannin content than original F. vesiculosus extract. Ethyl F1 showed the highest phlorotannin content (133.81 2.74

PAGE 27

27 mg ph loroglucinol /g extract). The phlorotannin content in Ethyl F3 was comparable to that of ethyl acetate fraction, but was higher than Ethyl F2 and Ethyl F4. After solvent partition, about 10%, 45%, 29.7%, and 1.6% of total phlorotannins were distributed in the dichloromethane, ethyl acetate, butanol, and water fractions, respectively A fter subfractionation of ethyl acetate fraction on Sephadex column, about 5.6 3.3 19.6 and 3.3% of initial phlorotannins were distributed in Ethyl F1, Ethyl F2, Ethyl F3, and Ethyl F4, respectively. Ph lorotannin Identification by HPLCDAD ESI MSn F. vesiculosus butanol and ethyl acetate fractions showed similar HPLC DAD chromatograms They both had a predominant peak at 35 min and many smaller peak s between 5 and 20 min (Figure 2 5 A, B ). The intensities of phlorotannins peaks in butanol fractions were much lower than those in ethyl acetate fraction, which was consistent with their phlorotannin contents. Ethyl F1 showed group peaks at 5 12 min and a smaller peak at 35 min (Figure 2 5 C ) Etn yl F2 showed a profile similar to ethyl acetate fraction, except for absence of a peak between 5 and 10 min ( Figure 25 D ) A single pea k at 35 min was observed for Ethyl F3 and F4 ( Figure 25 E F ) Mass spectrometry indicated that phlorotannin trimers through octamers including a group of structural isomers, eluted between 5 to 15 min Seven peaks of phlorotannin oligomers in Ethyl F1 and additional 5 peaks in Ethyl F2 were determined using a mass spectrometer ( Figure 2 5 G H ) Only o ne or two possible structures of each group of structural isomers were proposed since MS data was insufficient to determine the exact structure of each peak. The i dentification of peak eluted at 35 min was inclusive on a mass spectrometer. However this peak was deduced to be polymeric phlorotannins for the following reasons. First, the separation mode of tannins on Sephadex LH 20 was adsorption

PAGE 28

28 instead of gel permeation. Phlorotannins of smaller molecular size bind with Sephadex LH 20 with lower af finity, and thus elute earl ier than phlorotannins of larger molecular sizes. This was consistent with the chromatographic analysis and mass spectrometric data. Secondly, the peak at 35 min showed a typical UV vis spectrum that was characteristic for phlorotannins. A similar observation was made by Pent et a l on the HPLC analysis of procyanidins ( 84) In this previous study, procyanidin dimers through tetramers were separated on a reversedphase column. Polymers (pentamers and beyond ) eluted as a single peak at t he end of chromatogram. Th e mass spectra of tentatively identified phlorotannin peaks are shown in Figure 2 6 and 2 7 Structures of the phlorotannins isomers are depicted in F igure 28 The ESI MS (positive mode) of peak 1 (retention time 2.8 min) showed pseudomolecular ions at m/z 375 [M+H] +, indicating the compound was phlorotannin trimer. The compound gave rise to fragments at m/z 232 [M+H 18125] +, suggesting the loss of one phloroglucinol ring. The fragment at m/z 125 was phloroglucinol. The structure of one possible isomer is illustrated in Figure 2 8 The peak 2 (retention t ime 5.9 min) and 3 (retention t ime 7.0 min) gave m/z 499 [M+H]+, suggesting these two compounds were isomers of phlorotannin tetramer. Peak 2 produced fragment ions at m/z 356 [M+H 18125]+, 355 [M+H 18126]+, 358 [M+H 12516]+, 232 [M+H 18125124]+, 250 [M+H 125124]+. The fragments at m/z 356 and 232 were yielded after losing one phloroglucinol ring and hexahydroxybiphenyl, respectively. Peak 3 gave rise to the fragments at m/z 374 [M+H 125]+, 356 [M+H 18125]+, 232 [M+H 18125 124]+, 234[M+H 12512416]+. On the basis of the fragmentation pattern and published data (25) the structure of one isomer is shown in

PAGE 29

29 Figure 2 8 (2 ) The compou nd with m/z 499 has been isolated from F. vesiculosus previously ( 25) Peak s 4, 5, 9 and 10 had m/z 747 [M+H]+, suggesting that these compounds were isomers of phlorotannin hexamer. Peak 4 (retention time 8.4 min) yield ed f ragment ions at m/z 585, 586, 570, 462, 446, 338, and 332. Ion at m/ z 586 [M+H 36125] + indicated the loss of two water molecules and a phloroglucinol ring. The f ragment at m/z 570 [M+H 3612516] + suggested the loss of two water molecules along with a phloroglucinol ring and an ether bond. The ions at m/z 462 [M H 36125124]+,and 446 [M+H 3612512416]+, were deduced to be the fragments after loss of two phloroglucinol rings along with two water molecules and two phloroglucinol rings along with a ether bond and two water molecules, respectively. The ion at m/ z 338 [M+H 36125124 124]+, was generated after losing three phloroglucinol rings along with two water molecules. The compound lost three phloroglucinol rings along wit h one ether bond to yield fragment at m/z 322 [M+H 125124124 16]+. Peak 5 (retention time 10.3 min) showed a major ion in MS2 at m/z 711 [M+H 36]+. This compound gave rise to fragments at m/z 586 [M+H 36125]+, 570 [M+H 36125 16]+, 446 [M+H 36125 16125]+, suggesting the loss of a phloroglucinol ring and two water molecules, the loss of a phloroglucinol ring along with the ether bond and two water molecules, the loss of two phloroglucinol r ings along with the ether bond and two water molecules, respectively Peak s 9 (retention time 13.1 min) and 10 (retention time 13.8 min) had similar fragment patterns. The major fragment ions in MS2 were at m/z 622 [M+H 125]+, 462 [M+H 36125124]+,356 [M+H 18125124 124]+,338 [M+H 36125 124 124]+,

PAGE 30

30 suggesting the loss of a phloroglucinol ring the loss of two phloroglucinol rings with two water molecules, the loss of three phloroglucinol rings with one water molecule, and the loss of thre e phloroglucinol rings with two water molecules. Since MS data is insufficient to identify the exact structure of each peak, the structures of two possible isomers were given in Figure 2 8 The fifth structure illustrated in Figure 28 has been identified in F. vesiculosus according to previous research ( 24 ) Peak s 6 and 7 had the same [M+H]+ ion at m/z 623, suggesting that these compounds are the isomers of phlorotannin pentamer. Peak 6 (retention time 10.7 min) generated fragment s at m/z 480 [M+H 18125] + after losing a phloroglucinol ring and one water molecule. The fragment at m/z 464[M+H 1812516]+, was due to the loss of a phloroglucinol ring along with an ether bond and one water molecule. The compound gave the fragment ions at m/z 356 [M+H 18125124]+, 340 [M+H 18125124 16]+, suggesting the loss of two phloroglucinol rings and one water molecule, and the loss of two phloroglucinol rings along with an ether bond an one water molecule. Peak 7 (retention time 10.7 min) had the fragment ions in MS2 as 480 [M+H 18125] +, 356[M+H 18125 124 ] +, and 340[M+H 18125 12416 ] +. Based on the fragmentation patter n, the structure of one possible isomer was illustrated in Figure 28 Peak 8 (retention time 12.6 min) had a [M+H]+ ion at m/z 871, suggesting it is phlorotannin heptamer. The major fragments in MS2 included m/z 710 [M+H 36125]+, 586[M+H 36125 124]+, 462 [M+H 36125124 124]+. The fragment was produced after losing one phloroglucinol ring along with two water molecules, two phloroglucinol rings and two water molecule, and three phloroglucinol rings with two water molecules

PAGE 31

31 respectively. A possible str ucture of the phlorotannin heptamer was illustrated in Figure 2 8 Peak s 11 (retention time 14.4 min) and 12 (retention time 15.1) gave m/z 995 [M+H] +, suggesting the se compound s were phlorotannin octamer. Mass spectrometric data of peak 12 showed major ions in MS2 at m/z 977 [M+H 18]+, 834 [M+H 36125]+, 710 [M+H 36125124]+,694 [M+H 36125124 16]+, 586 [M+ H 36125 124 124]+, 462 [M+H 36125124124 124]+,338 [M+H 36125 124124 124 124]+. The fragments were generated after losing one water molecule, o ne phloroglucinol ring with two water molecules two phloroglucinol rings along with two water molecules two phloroglucinol rings along with one ether bond and two water molecules three phloroglucinol rings and two water molecules four phloroglucinol ri ngs and two water molecules five phloroglucinol rings with two water molecules, respectively. Peak 11 showed a similar fragment pattern. The structure of one possible isomer is illustrated in Figure 2 8 In conclusion, F. vesiculosus acetone extract was fractioned into dichloromethane, ethyl acetate, butanol and water fractions using liquidliquid extraction. All the extracts showed high phenolic content and antioxidant capacities. The ethyl acetate fraction was found to be enriched w ith phlorotannins. This fraction was separated into four subfractions on a Sephadex LH 20 column. The E thyl F1 and F2 contain a mixture of oligomers and polymers while the E thyl F3 and F4 contained exclusively polymers Phlorotannin trimer, teramer, panta mer and hexamer were found in Ethyl F1. H examer, heptamer and octamer were found in Ethyl F2.

PAGE 32

32 Table 2 1. Retention times and mass spectrometric data of phlorotannins in F. vesiculosus determined by HPLC ESI MSn. a Compounds followed by an asterisk were isolated from F. vesiculosus previously Peaks Phlorotannin compound R etention time ( min) M olecular weight MS1 ( m/z ) MS2 ( m/z ) 1* Trimer 2.8 374 375[M+H] + 357,232,231 2* Tetramer 5.9 498 499[M+H] + 481,358,356,355,250,232 3* Tetramer 7.1 498 499[M H] 374,373,358,357,356, 355,234,233,232,231 4* Hexamer 8.4 746 747[M+H] + 729,586,585,570,462, 461,462,446,337,338,322 5* Hexamer 10.3 746 747[M+H] + 711,586,585,570,462, 446,445 9* Hexamer 13.1 746 747[M+H] + 729,622,462,356,338,231 10* Hexamer 13.8 746 747[M+H] + 729, 622, 621,462, 356,338 6 Pentamer 10.7 622 623[M+H] + 605,480,464,356, 340,231,179 7 Pentamer 11.6 622 623[M+H] + 605,480,479,356,340,231 8 Heptamer 12.6 870 871[M+H] + 853,710, 586,462 11 Octamer 14.4 994 995[M+H] + 977,834,710,694,586,462,338 12 Octamer 15.1 994 995[M+H] + 977,834,710,586,462,338

PAGE 33

33 Figure 21. Extraction and fractionation of phlorotannins from F. vesiculosus. Dichloromethane fraction Eethyl F1 Dry F. vesiculosus powder F. vesiculosus acetone extract extract with 70% acetone water Ethyl acetate fraction Butonal fraction Water fraction Ethyl F2 Ethyl F3 Ethyl F4Dispersed in water Sephadex LH 20 6080% MeOH 90100% MeOH 70% Acetone 70% Acetone

PAGE 34

34 Figure 2 2 Total phenolic content of F. vesiculosus extracts determined by FolinCiocalteu assay. Results are means standard deviation of duplicate assay. Bars with different letters were significantly different (P < 0.05) from each other. 0 100 200 300 400 500 600 f c e g e d a b e Phloroglucinolequivalents (mg/g)

PAGE 35

35 Figure 2 3 Antioxidant capacities of F. vesiculosus extracts determined by oxygen radical absorbance capacity assay. Results are means standard deviation of duplicate assay. Bars with different letters were significantly different (P < 0.05) from each other. 0 1000 2000 3000 4000 5000 6000 7000 8000 Trolox equivalents (mol/g) d e a, b c, d e b a a b, c

PAGE 36

36 Figure 2 4 Total phlorotannin content of F. vesiculosus extracts determi ned by DMBA assay. Results are means standard deviation of triplicate assay. Bars with different letters were significantly different (P < 0.05) from each other. 0.00 40.00 80.00 120.00 160.00 e f b d g a d b cPhloroglucinol Equivalent (mg/g )

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37 Fi gure 25 HPLCDAD chromatogram s of F. vesiculosus fractions. A) B utanol fraction. B) E thyl acetate fraction. C) Ethyl F1. D) Ethyl F2. E) Ethyl F3. F) Ethyl F4. G) Magnified chromatogram of Ethyl F1. H) Magnified chromatogram of Ethyl F2. Peaks marked with numbers were tentatively identified using HPLC ESI MSn. min 10 20 30 40 mAU 0 100 200 300 min 10 20 30 40 mAU 0 200 400 600 B Ethyl acetate fraction min 10 20 30 40 mAU 0 100 200 300 C Ethyl -F1 D Ethyl -F2 min 10 20 30 40 mAU 0 200 400 600 800 1000 E Ethyl -F3 min 10 20 30 40 mAU 0 200 400 600 800 1000 1200 F Ethyl-F4 Polymer Polymer Polymer Polymer Polymer min 10 20 30 40 mAU 0 20 40 60 80 100 120 Polymer A Butanol fraction0

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38 Fi gure 25 Continued. min 0 10 20 mAU 0 100 200 300 G Ethyl -F1 4 2 3 1 5 6 7 0 10 20 mAU 25 0 25 50 75 100 8 9 10 11 12 minH Ethyl -F2

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39 Figure 2 6 MS and MS2 spectra of phlorotannin peaks in Ethyl F1. Number 17 on the figures match those in the chromatogram of Ethyl F1 in Figure 25 G 126.4 163.8 231.4 269.6 357.0 +MS2(375.3), 2.8min #136 0.0 0.2 0.4 0.6 0.8 100 200 300 400 500 600 700 800 m/z x1040.01 136.6 189.5 268.3 314.3 499.2 623.1 747.0 375.2 +MS, 2.8min #135 0.2 0.4 0.6 0.8 x105Intens 165.2 480.9 598.3 734.8 354.9 +MS2(499.4), 5.9min #299 0.0 0.5 1.0 1.5 100 200 300 400 500 600 700 m/z 355.6 232.0 521.1 645.0 746.9 499.2 +MS, 5.9min #298 1 2 3 4 5 Intens x1052x106 499.1 +MS, 7.1min #363 0.2 0.4 0.6 0.8 Intens 165.3 231.8 411.0 480.9 355.0 +MS2(499.1), 7.1min #364 0 2 4 6 100 200 300 400 500 600 700 800 900 m/z 373.8 355.8 x106 826.8 747.1 +MS, 8.4min #432 0.2 0.4 0.6 0.8 x106Intens 338.2 462.1 585.1 729.0 +MS2(747.1), 8.4min #433 0.0 0.2 0.4 0.6 0.8 1.0 100 200 300 400 500 600 700 800 900 m/z x105446.14 179.0 355.8 480.1 605.0 710.0 231.1 +MS2(623.1), 10.7min #558 0 2000 4000 6000 8000 100 200 300 400 500 600 700 800 900 m/z x105 702.9 747.0 623.1 +MS, 10.7min #557 1 2 3 4 5 Intens .6340.2 570.0 446.0 586.1 711.0 +MS2(747.1), 10.3min #538 0 2 4 6 100 200 300 400 500 600 700 800 900 m/z x1045 703.0 826.8 747.1 +MS, 10.3min #537 0 1 2 3 4 Intens x1053 356.0 479.8 604.9 231.1 +MS2(623.1), 11.6min #609 0 1 2 3 100 200 300 400 500 600 700 m/z 340.0 x1057 623.1 +MS, 11.6min #608 2 4 6 x105Intens

PAGE 40

40 Figure 2 7 MS and MS2 spectra of phlorotannin peaks in Ethyl F 2 Number 8 12 on the figures match those in the chromatogram of Ethyl F2 in Figure 25 H 433.5 462.3 586.1 681.1 710.1 853.0 +MS2(871.1), 12.7min #660 0 2 4 6 300 400 500 600 700 800 900 1000 m/z x104 827.0 950.9 994.8 871.1 +MS, 12.6min #659 0 1 2 3 4 5 x105Intens .8 9 747.1 +MS, 13.1min #684 0 2 4 6 8 x105Intens 230.9 356.1 462.0 622.0 728.9 +MS2(747.1), 13.1min #686 0 1000 2000 3000 4000 5000 100 200 300 400 500 600 700 800 m/z 338.0 231.0 268.9 355.9 462.0 602.9 728.9 +MS2(747.1), 13.9min #725 0.0 0.2 0.4 0.6 0.8 1.0 100 200 300 400 500 600 700 800 m/z 622 338.2 x104 826.9 884.9 747.1 +MS, 13.9min #724 0.2 0.4 0.6 0.8 x106Intens .10 337.8 462.1 515.0 586.0 710.0 834.0 976.9 +MS2(995.0), 14.4min #755 0 2 4 6 x104 300 400 500 600 700 800 900 1000 m/z 694.0 747.1 1074.8 995.0 +MS, 14.4min #754 0.2 0.4 0.6 0.8 x106Intens .11 337.8 391.0 462.0 516.0 586.1 694.0 834.2 976.9 +MS2(995.0), 15.1min #795 0 1 2 3 4 5 300 400 500 600 700 800 900 1000 m/z 710.2 x104 893.1 1074.8 995.0 +MS, 15.1min #794 2 4 6 x106Intens .12

PAGE 41

41 Figure 2 8 The proposed structures and fragmentation of phlorotannins in Ethyl F1 and Ethyl F2. Numbers on the figures m atch those in the chromatograms and mass spectra O O O H O H O H O H O H O H O H O H O H O H 373 357 249 233 MW=498 m/z=499 [M+H]+ O H O H O H O H O H O H O H O H O O O H O H O H O H O O H O H O H 461 445 337 321 569 585 (loss of 2 water molecule) MW=746 m/z=747 [M+H]+ O H O H O H O H O H O H O H O H O H O O H O H O H O H O H O O H O H 585 585 569 569 MW=746 m/z=747 [M+H]+ O O H O H O H O H O H O H O H O H 231 125 MW=374 m/z=375 [M+H]+1 2 4 5

PAGE 42

42 Figure 2 8 Continued. O H O H O H O H O H O H O H O H O O O H O H O H O H O H 479 463 355 339 MW=623 m/z=624 [M+H]+ O O H O H O H O H O O H O H O H O H O H O H O H O H O O H O H O H O H O H O H 461 585 709 (loss of 2 water molecule) MW=870 m/z=871 [M+H]+ O O H O H O H O H O O H O H O O O H O H O H O H O H O H O H O H O H O H O H O H O H O 461 585 693 709 ( loss of 2 water molecules ) 833 337 MW=994 m/z=993 [M+H]+6 8 12

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43 CHAPTER 3 INHIBITORY EFFECT S OF FUCUS VESICULOSUS ON THE FORMATION OF ADVANCED GLYCATION E NDPRODUCTS Background Reactive carbonyls accelerate the for mation of advance glycation endproducts (AGEs). Scavenging reactive carbonyls is an effective approac h to inhibit protein glycation. F. vesiculosus acetone extract and its fractions showed high phlorotannin content. However, it is not known whether F. vesiculosus extract s are effective in inhibiting protein glycation The objective of this chapter was to investigate the inhibitory effects of F. vesiculosus extract s on the formation of AGEs as well as their capacities to scavenge reactive carbonyls. Materials and Methods Chemicals and Materials Aminoguanidine, o phenylenediamine were products from Acros Organics (Morris Plains, NJ). Methylglyoxal (40% aqueous solution) and glucose wa s obtained from MP Biomedicals, LLC (Solon, OH). Bovine serum albumin (BSA), sodium azide, monobasic and dibasic sodium phosphate, 96 well plates with clear bottom wells, acetone and other organic solvent were purchased from Fisher Scientific Co. (Pittsburg, PA) B ovine Serum Albumin (BSA) Glucose Assay This assay evaluates protein glycation mediated by glucose. Bovine serum albumin and glucose were dissolved in phosphate buffer ( 100 mM pH 7.4) to a concentration of 100 mg/mL and 1.6 M, respectively F. vesiculosus acetone extract or fractions were dissolved in the same phosphate buffer. One mL of the BSA solution was mixed with 1 mL of glucose solution and 1 mL of F. vesiculosus extracts The mixtures

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44 were incubated at 37 oC Sodium azide (0.2 g/L) was used as an aseptic agent Phosphate buffer was used as a blank. Aminoguanidine and phloroglucinol were used as positive controls. After seven days of incubation, the fluorescence of samples was measured using an excitation of 330 nm and an emission of 410 nm respectively. The % inhibition of AGEs formation= [1 (fluorescence of t he test group/fluorescence of t he control group)] x 100%. BSA Methylglyoxal Assay This assay evaluates the middle stage of protein glycation. BSA and methylglyoxal were dissolved in phosphate buffer (100 mM, pH 7.4) to concentrations of 20 mg/ mL and 60mM, respectively. F. vesiculosus acetone extract or fractions were dissolved in the same phosphate buffer. One mL of the BSA solution was mixed with 1 mL of methylglyoxal solution and 1 mL F. vesiculosus extracts The mixture was incubated at 37 oC Sodium azide ( 0.2 g/L ) was used as an aseptic agent Phosphate buffer was used as a blank. Aminoguanidine and phloroglucinol were used as positive controls After seven days of incubation, the fluorescence of samples was measured using an excitation of 3 4 0 nm and an emission of 420 nm, respectively. The % inhibition of AGEs formation= [1 (fluorescence of the test group/fluorescence of the control group)] x 100%. Methylglyoxal Scavenging Assay Methylglyoxal scavenging assay followed a published method with modifications (72) Methylglyoxal (5 mM), ophenylenediamine (derivatization agent, 20 mM) were freshly prepared in phosphate buffer (100 mM, pH 7.4). F. vesiculosus acetone extract or fractions and phloroglucinol were dissolved in the same buffer to a concentration of 1 mg/mL. Aminoguanidine (5 mM) was used as a positive control. Methylglyoxal solution

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45 (0.25 mL) was mixed with 0.25 mL of phosphate buffer (blank) or test samples The mixtures were incubated at 37 oC for 0, 5, 10, 20, 40, 60, and 120 min, respectively. After incubation, 0.125 mL of o phenylenediamine was added to each test solution. The mixtures were kept at room temperature for 30 min for derivatization to complete. The m ixture after derivatization was injected for HPLC analysis (Agilent Technologies, Palo Alto, CA) Compound s eparation was carried out on a Z orbax SB C18 column (4.6250 mm, 5 m, Agilent Technologies, Palo Alto, CA). Mobile phases were composed of 0.1% formic acid in water (phase A) and methanol (phase B). The flow rate was set at 1 mL /min and the injection volume was 15 l. The linear gradient for elution was: 03 min, 5 50% B; 316 min, 50 50% B; 1617 min, 50 90% B; 1719 min, 90 90% B; 1919.5 min, 905% B; followed by 1 min of reequilibration. Methylglyoxal reacted with ophenylenediamine to form 1 methylquinoxaline, which eluted at 12.9 min using detection wavelength of 315 nm. Data Expression and S tatistical Analysis Half inhibition concentrations (EC50) were determined using Probit analysis function of SPSS software (Version 13, SPSS Inc., Chicago, IL.) One way ANOVA with Tukey Kramer HSD test w as done using JMP software (Version 8.0, SAS Institute Inc., Cary, NC) ficant. All data were expressed as the mean standard deviation. Results Anti Glycation Effects in BSA Glucose Assay F. vesiculosus acetone extract and its fractions significantly inhibited protein glycation mediated by glucose, and the antiglycation effects increased with concentration (Figure 3 1 ). EC50 was defined as t he concentrations of F. vesiculosus

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46 extracts required to inhibit 50% of BSA glycation. Ethyl acetate fraction was a more potent antiglycation agent than the acetone extract and other fractions. Dichloromethane fraction had much lower phlorotannin content than butanol fraction, yet it showed similar antiglycation activities. Ethyl F1 and F2 were similar to phloroglucinol Ethyl F3 and F4 had the much lower activity compar ed to all other fractions except that of water fraction. Anti Glycation Effects in BSA Methylglyoxal Assay F. vesiculosus acetone e xtract and its fractions inhibited the formation of fluorescent AGEs mediated by methylglyoxal. The inhibitory effects increased in a concentrat ion dependent manner (Figure 3 2 ). The ethyl acetate fraction had lower EC50 (0.169 mg/mL), therefore was more effective than other F. vesiculosus fractions. Subsequently, antiglycation activities of four subtractions showed similar antiglycation effects to ethyl acetate fracti on and aminoguanidine (Table 3 1 ). Phloroglucinol the constituent unit of phlorotannins, appeared to be the most effect ive antiglycation agent with an EC50 value of 0.058 mg/ mL Dichloromethane and water fraction had the least antiglycation activities, which was consistent with their low phlorotannin co ntent s. M ethylglyoxal Scavenging Capacity Methylglyoxal content decreased significantly after incubation with F. vesiculosus extract and fractions for 120 min (Figure 3 3 ). After incubating with butanol fraction, ethyl acetate fraction and its four subfra ctions, l ess than 50% of methylglyoxal remained at 120 min Aminoguanidine and phloroglucinol scavenged 82.6 % and 77.2% of methylglyoxal after 120 min of incubation. Ethyl acetate fraction and its four subfractions showed the strongest capacity to scavenge methylglyoxal, followed by butanol fraction, acetone extract, dichloromethane fraction and water fraction.

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47 Discussion The antiglycation activities of F. vesiculosus extracts were evaluated in selected models. In BAS g lucose assay, E thyl F1 and F2 were more effective than Ethyl F3 and F4 in preventing protein glycation, w hereas no differences were observed among these four subfractions in BSA MGO assay E th yl acetate fraction had high phlorotannin content which explained its significant inhibitory effects on the formation of AGEs. B utanol fraction had lower phlorotannins content than ethyl acetate fraction, which was consistent with lower antiglycation activity in the BSA MGO system. BSAglucose evaluates all stages of pr otein glycation, while BSA MGO assay assesses the protein glycation only in the middle stage. The data suggested that phlorotannins of lower molecular weights were more effective than phlorotannins of high molecular weight s in inhibiting protein glycation mediated by glucose. This was consistent with the potent antiglycation effects observed for phloroglucinol. On the other hand, molecular weight had little impact on methylglyoxal mediated protein glycation. Our results were consistent with a previous study where several phloroglucinol derivatives from brown algae were found effective in inhibiting the formation of AGEs in vitro ( 85) Methylglyoxal was stable in phosphate buffer. Its level was significantly decreased by F. vesiculosus extract s. Ethyl acetate fraction showed the highest methylglyoxal scavenging activity comparing to other fractions. Dichloromethane and water fractions sh owed the least activities. This data was consistent with their antiglycation activities. Interestingly, Ehtyl F1 to F4 showed similar methylglyoxal scavenging capacity, which explains their similarity in inhibiting methylglyoxal mediated protein glycation. Scavenging of reactive carbonyls appeared to be a major mechanism for algae extract to inhibit in protein glycation. Reactive carbonyls such as methylglyoxal, glyoxal

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48 and 3deoxyglucosone are formed from the degradation and oxidation of Amadori products in the mid dle stage of protein gl ycation (86) Alternatively, these key intermediates can be generated by the glucose glycoxidation and lipid peroxidation ( 45) Since methylglyoxal is an active intermediate of AGE formation, the inhibitory effect of F. vesicul osus phlorotannins was attributed in part to their abilities to scavenge reactive carbonyls. F. vesiculosus p hlorotannins had showed reactive oxygen species scavenging capacity It has been reported that the free radical scavenging activity of phenolic co mpounds and their inhibitory effects on the formation of AGEs are positively correlated in many plant extracts ( 87) Oxidative stress would accelerate the formation of AGEs. Phlorotannins may inhibit protein glycation by scavenging free radicals. In conclusion, F. vesiculosus acetone extract and its fractions inhibited the formation of AGEs in BSA glucose and BSA methylglyoxal models. Ethyl acetate fraction showed the highest antiglycation activities in BSA glucose, and BSA methylglyoxal models. Ethyl F1 and F2 were better antiglycation agents than F3 and F4 in BSAglucose assay. They appeared to equally active in BSA methylglyoxal assay. F. vesiculosus acetone extract and its fractions scavenged methylglyoxal, suggesting carbonyl scavenging was a major mechanism for protein glycation inhibition.

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49 Table 31 EC50 of F. vesiculosus extracts inhibiting protein glycation in two assays. F. vesiculosus extracts EC50 (mg/ mL ) BSAMGO assay BSAGlucose assay F. vesiculosus acetone extract 0.3930.0127 b 0.338 0.0146 c d Dichloromethane fraction 1.7760.0536 a 0.4890.0692 c Ethyl acetate fraction 0.1690.0050 d 0.2780.0186 d B utanol fraction 0.2370.0057 c 0.3860.0364 c d Water fraction > 6.0 >2 .0 Ethyl F1 0.1660.007 d 0.0450.001 e Ethyl F2 0.1660.012 d 0.0570.003 e Ethyl F3 0.1590.005 d 1.1570.046 b Ethyl F4 0.1620.012 d 1.5260.161 a Phloroglucinol 0.0580.0036 e 0.0680.0056 e Aminoguanidine 0.1970.0095 c, d 0.3100.0607 c d a Data are mean standard deviation of triplicate tests. b

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50 Figure 3 1 Inhibitory effects of F. vesiculosus extracts on the formation of AGEs in BSAglucose assay. Results are means standard deviation of triplicate assay. 0% 20% 40% 60% 80% 100% 0 0.5 1 1.5 2 2.5 Ethyl F1 Ethyl F2 Ethyl F3 Ethyl F4 Inhibition Percentage of AGEs Formation Concentration (mg/ml) B 0% 20% 40% 60% 80% 100% 0 0.2 0.4 0.6 0.8 1 Acetone extract Dichloromethane fraction Ethyl aceate fraction Butanol fraction Phloroglucinol Aminoguanidine Inhibition Percentage of AGEs Formation Concentration ( mg/ml) A

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51 Figure 3 2 Inhibitory effects of F. vesiculosus extracts on the formation of AGEs in BSAmethylglyoxal assay. Results are means standard deviation of triplicate assay 0% 20% 40% 60% 80% 100% 0 0.2 0.4 0.6 0.8 Ethyl F1 Ethyl F2 Ethyl F3 Ethyl F4 Concentration (mg/ml) Inhibition Percentage of AGEs Formation B 0% 20% 40% 60% 80% 100% 0 0.2 0.4 0.6 0.8 1 Acetone extract Dichloromethane fraction Ethyl acetate fraction Butanol fraction Phloroglucinol Aminoguanidine Inhibiton Percentage of AGEs FormationConcentration ( mg/ml) A

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52 Figure 3 3 The capacity of F. vesiculosus extracts to scavenge methylglyoxal. Methylglyoxal (5mM) was incubated with F. vesiculosus extracts ( 1.0 mg/ml). Aminoguanidine and phloroglucinol (5 mM) w ere used as positive control s. Results are means standard d eviation of duplicate assay. 0.0% 20.0% 40.0% 60.0% 80.0% 100.0% 0 20 40 60 80 100 120 Methylglyoxal +Buffer Ethyl F1 Ethyl F2 Ethyl F3 Ethyl F4 Incubation Time ( min) Percentage Remaining Methylglyoxal B 0.0% 20.0% 40.0% 60.0% 80.0% 100.0% 0 20 40 60 80 100 120 Methylglyoxal+Buff er Acetone extract Dichloromethane fraction Ethyl acetate fraction Butanol fraction Water fraction Phloroglucinol Aminoguanidine Percentage Remaining Methylglyoxal Incubation Time (min) A

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53 CHAPTER 4 HPLCESI MS IDENTIFICATION OF PHLOROGLUCINOL CARBONYL ADDUCTS Background F. vesiculosus extracts showed significant antiglycation activities S uch activities were attributed in part to phlorotannins and their abilities to scavenge reactive carbonyls. Phlorotannins are complex oligomers and polymers that consist of phloroglucinol. The objective of this c hapter was to use phloroglucinol as a model compound to explore the reaction mechanisms and tentatively identify phytochemical carbonyl adducts on HPLC ESI MSn. Materials and Method s Chemicals Phloroglucinol, glyoxal (40% wt solution) was obtanied from Acros Organics ( Morris Plains, NJ ). Methylglyoxal ( 40% aqueous solution) was purchased from MP Biomedicals, LLC ( Solon, OH) Other chemicals and materials were described in previous chapters. Phloroglucinol Glyoxal / Methylglyoxal Reaction and Adduct Identification The phloroglucinol carbonyls reaction was conducted in the phosphate buffer saline (pH 7.4). Phloroglucinol (10 mM) was incubated with methylglyoxal or glyoxal at the concentration of 1 mM at 37C. After two hours incubation, the adducts were analyzed using HPLC ESI MSn technique. An Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA) equipped with a diode array detector and HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA) was used for adduct identification. Compound separation w as carried out on a Zorbax SB Aqueous column (3.0250 mm, 5 m, Agilent Technologies, Palo Alto, CA). Mobile phases were composed of 0.1%

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54 formic acid in water (phase A) and 0.1% ancetonitrile (phase B). The linear gradient was: 0 20 min, 010% B; 20 22 min, 10 17% B; 22 37 min, 1730% B; 37 40 min, 3070% B; 4042min, 700% B, followed by 5 min of reequilibration. The flow rate was 0.25 mL/min. The detection wavelength on diode array detector was 270 nm. Electrospray ionization at both positive and negati ve modes was performed using nebulizer 50 psi, drying gas 10 L/min and drying temperature 300 oC. One precursor ion with the highest intensity was isolated and fragmented to obtain the product ion spectra of adducts product ion spectra of adducts. Results Phloroglucinol Glyoxal/Methylglyoxal Adducts Identification After phloroglucinol was incubated in phosphate buffer for two hours, no apparent degradation was observed (Figure 41A ). Its content decreased after incubating with glyoxal or methylglyoxal at m olar ratio of phloroglucinol : glyoxal/methylglyoxal =10:1 for two hours Three phloroglucinol glyoxal and two phloroglucinol methylglyoxal adducts were detected (Figure 4 1B,C ), and their mass spectra are shown in Figure 4 2 Proposed structures of adducts and fragments are illustrated in Figure 4 3 The first phloroglucinol glyoxal adduct eluted at 11.2 minutes and had m/z 183 [M H]-. It yielded a product ion at m/z 125 that was consistent with phloroglucinol moiety (Figure 4 2 A ). Fragment m/z 165 was due to water elimination from 183[M H]-. This adduct was tentatively identified as a monophloroglucinol monoglyoxal adduct. Its structure is depicted in Figure 4 3 A The second adduct at 16.5 min utes gave rise to m /z 349 [M H]and product ions at m/z 331, 291, 183, and 125 (Figure 4 2 B ). Fragment m/z 331 was due to water elimination from 349 [M H]-. F ragments with m/z 291 and 183 were produced after losing a glyoxal, a glyoxal moiety and one phloroglucinol moiety,

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55 respectively This adduct was tentatively identified as a diphloroglucinol di glyoxal adduct (Figure 4 3 B ). In this adduct, two phloroglucinol molecules were crossed linked by the aldehyde group of the glyoxal. A similar reaction had been reported for glyoxal and epicatechin ( 88 ) We postulated that the reaction started from protonation of glyoxal, which was att ac ked by one phloroglucinol through nucleophilic addition to form a glyoxal phloroglucinol intermediate. The intermediate continued to lose a water mo lecule to form a carbocation. The new carbocation then attacked another phloroglucinol to yield an ethyl linked adduct. The third glyoxal phloroglucinol adduct ( 21.4 minute s) showed m/z 291 [M H]( Figure 4 2 C ) This adduct lost one phloroglucinol moiety to yield a fragment at m/z 165 [M H]-. The fragment at m/z 125 [M H]was phloroglucinol. It was consistent with an adduct composed of two phloroglucinol molecules and one glyoxal molecule in between (Figure 4 3 C ). The first methylglyoxal phloroglucinol adduct eluted at 20.4 minutes and yield ed m/z 197 [M H]-. The fragment s at m/z 179 and 125 were due to water elimination and phloroglucinol, respectively ( Figure 4 2 D ) It was tentatively identified as monophloroglucinol monomethylglyoxal adduct ( Figure 4 3 D ) The second methylglyoxal phloroglucinol adduct eluded at 22.2 minutes and produced m/z 269 [M H]( Figure 4 2 E ) It lost one methylglyoxal moiety to generate a pr oduct ion at m/z 197. Fragment m/z 251 was due to water elimination from [M H]-, whic h continued to fragment to produce m/z 125. The structure of this adduct is illustrated in Figure 4 3 E. Glyoxal has two aldehyde groups. The methylglyoxal has a ketone group and an aldehyde group. In the phloroglucinol methylglyoxal reaction, the aldehyde group of methyl glyoxal molecules attacked phloroglucinol to form the adduct This occurred

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56 because an aldehyde group has greater electrophilicity than a ketone group a nd it also has less steric hindrance during reaction ( 82 ) Similar observations had been ma de in methylglyoxal and epicatechin reactions ( 88 ) Discussion It is difficult to study the scavenging capacity of phlorotannins due to its structur al complexity. We hypothesized that the antiglycation activities of phlorotannins can be attributed to its constituent unit, the p hloroglucinol ( 1, 3, 5 trihydroxybenzene) Investigating the reaction between phloroglucinol and carbonyls will provid e insight about the functions of phlorotannins in carbonyl scavenging. T he electronegativity character of phloroglucinol leads to the nucleophilic addition reaction between phloroglucinol and the aldehyde group. One or two glyxoal molecules attached on t o one phloroglucinol molecule to form a monophloroglucinol monoglyoxal, or monophloroglucinol diglyoxal adduct. T wo phloroglucinol rings were cross linked by one glyoxal molecule. It has been reported that epicatechin, which includes one phloroglucinol ring, could react with glyoxal at the C 6 or C 8 position ( 89) The crosslinking of two epicatechin molecules by aldehydes has also been re ported. We speculated that the reaction began with protonation of glyoxal, which was then attacked by one phloroglucinol molecule through nucleophilic addition to form a monophloroglucinol monoglyoxal adduct. Then the monomer glyoxal phloroglucinol interm ediate lost one water molecule, leading to a new carbocation ( 9091) The new carbocation attacked another phloroglucinol molecule to generate an ethyl linked diphl oroglucinol monoglyoxal adduct. In the phloroglucinol methylglyoxal reaction, instead of ketone group, the aldehyde groups of methylglyoxal molecules attached on phloroglucinol molecule to form a

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57 monophloroglucinol mono methyl glyoxal, or monophloroglucinol di methylglyoxal adduct. One explanation is that the aldehyde group is more reactive than the ketone group due to its less steric hindrance. The carbon of ketone group is less partial positive charge than that of aldehyde group, which makes the carbonyl carbon as a weaker nucleus (82) methylglyoxal reacted with phloroglucinol to generate a series of stereoisomers. In conclusion, phloroglucinol rapidly reacted with methylglyoxal and glyoxal by forming adducts at pH 7.4. One to three glyoxal molecules attached on one or two phlor oglucinol molecules to form six phloroglucinol glyoxal compounds with different structures. Up to two methylglyoxal molecules attached on one phloroglucinol to generate two phloroglucinol methylglyoxal adducts. The r eaction between phloroglucinol and carbonyls provided insight in the carbonyl scavenging mechanism of phlorotannins.

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58 Figure 4 1. HPLC DAD chro matograms of the phloroglucinol after incubation for two hours. A) Phloroglucinol (10 mM) was incubated with phosphate buffer. B) Phloroglucinol (10 mM) was incubated with 1 mM glyoxal for two hours. C) Phloroglucinol (10 mM) was incubated with 1 mM methylglyoxal Peaks of identified adducts were labeled with their molecular weight. min 5 10 15 20 25 30 35 40 mAU 0 100 200 300 400 500 600 phloroglucinolA min 5 10 15 20 25 30 35 40 mAU 0 100 200 300 400 500 ( a) m/z=183 [M H](b) m/z=349 [M H](c) m/z=291 [M H]phloroglucinolB min 5 10 15 20 25 30 35 40 mAU 0 100 200 300 400 500 Phloroglucinol (d) m/z=197 [M H] (e) m/z=269[M H ] -C

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59 Figure 4 2. MS and MS2 spectra of phloroglucinol glyoxal/methyglyoxal adducts peaks Letter A E on the figures match those in the Figure 4 1 349.4 MS 0 1000 2000 3000 4000 Intens 125.0 164.9 182.9 204.8 222.8 290.9 330.8 MS2(349.4) 0 200 400 600 800 150 200 250 300 350 400 450 m/zB 183.0 MS 0.0 0.5 1.0 1.5 2.0 2.5 5 x10 Intens 125.1 164.9 MS2(183.0) 0 200 400 600 150 200 250 300 350 m/zA 125.2 178.8 MS2(196.9) 0 2000 4000 6000 8000 150 200 250 300 350 400 m/z 196.9 MS 0 2 4 6 4 x10 Intens DMS3(269.3>250.7) 125.0 232.7 0 200 400 600 800 150 200 250 300 350 400 m/z 1000 197.0 233.0 250.7 MS2(269.3) 0 1000 2000 3000 269.3 MS 0.00 0.50 1.00 4 x10 Intens E 290.9 MS 0 1 2 3 5 x10 Intens 165.0 MS2(290.9) 0 1 2 3 4 5 4 x10 150 200 250 300 350 400 m/zC 125.1 0 50 100 150 Intens 120 140 160 180 200 m/z 137.0 165.0

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60 Figure 4 3. The proposed structures of phloroglucinol glyoxal/methylglyoxal adducts and their product ions Letter A E on the figures match those in the Figure 41 and Figure 42. A B C D E Monophloroglucinol+Monoglyoxal MW=184 m/z=183 [M-H]m/z=125 [M-H]Diphloroglucinol+Diglyoxal MW=350 m/z=349 [M-H]m/z=291 [M-H]m/z=125 [M-H]m/z=183 [M-H]Diphloroglucinol+Monoglyoxal MW=292 m/z=291 [M-H]m/z=125 [M-H]m/z=165 [M-H]Monophloroglucinol+Monomethylglyoxal MW=198 m/z=197 [M-H]m/z=125 [M-H]Monophloroglucinol+Dimethylglyoxal MW=270 m/z=269 [M-H]m/z=197 [M-H]m/z=125 [M-H]O H O H O H O H O H O H O H C H3O O H O H O H O H C H3O O H C H3O O H O H O H O H O H O H O H C H3O O H O O H O H O H O H O H O H O H O H O H O O H O H O H O H O H O H O H O H O H O O H O H O H O H O O H O H O H O H O H O H O H O O O H O H O H O H O H O H O H O H O H O

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61 CHAPTER 5 CONCLUSION F. vesiculosus phytochemicals were extracted and fractioned into four fractions. Phlorotannins were concentrated in ethyl acetate and its subfractions. Ethyl F1 and F2 were found to contain a mixture of oligomers and polymers. Ethyl F3 and F4 had exclusively polymers All the F. vesiculosus extracts showed high antioxidant capacities antiglycation activities in BSAglucose and BSA methylglyoxal models and directly scavenged methylgl yoxal. The ethyl acetate fraction and its subfractions showed the h ighest antioxidant, antiglycation and reactive carbonyl scavenging activities. Phloroglucinol, the constitute unit of phlorotannins, rapidly reacted with reactive carbonyls by forming adducts, indicating that the ability of phlorotannins to react with carbonyls was the major mechanism for protein glycation inhibition

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62 LIST OF REFERENCE S 1. Petersen, D. R.; Doorn, J. A. Reactions of 4 hydroxynonenal with proteins and cellular targets. Free Radical Biology and Medicine. 2004, 37, 937945. 2. Yuan, Y. V.; Walsh, N. A. Antioxidant and antiproliferative activities of extracts from a variety of edible seaweeds. Food and Chemical Toxicology. 2006, 44, 11441150. 3. Jimnez Escrig, A.; Jimnez Jimnez, I.; Pulido, R.; SauraCalixto, F. Antioxidant activity of fresh and processed edible seaweeds. Journal of the Science of Food and Agriculture. 2001, 8 1, 530534. 4. Teas, J.; Harbison, M. L.; Gelman, R. S. Dietary Seaweed ( Laminaria) and Mammary Carcinogenesis in Rats. Cancer Res. 1984, 44, 27582761. 5. Yamamoto, I.; Maruyama, H.; Moriguchi, M. The effect of dietary seaweeds on 7,12dimethylbenz[a]anthraceneinduced mammary tumorigenesis in rats. Cancer Letters. 1987, 35, 109 118. 6. Yamamoto, I.; Maruyama, H. Effect of dietary seaweed preparations on 1,2dimethylhydrazineinduced intestinal carcinogenesis in rats. Cancer Letters. 1985, 26, 241251. 7. Lee, E. J.; Sung, M. K. Chemoprevention of azoxymethaneinduced rat colon carcinogenesis by seatangle, a fiber rich seaweed. Plant Foods for Human Nutrition (Formerly Qualitas Plantarum). 2003, 58, 1 8. 8. Yamamoto, I.; Maruyama, H.; Takahashi, M.; Komiyam a, K. The effect of dietary or intraperitoneally injected seaweed preparations on the growth of Sarcoma180 cells subcutaneously implanted into mice. Cancer Letters. 1986, 30, 125131. 9. HiqashiOkaj, K.; Otani, S.; Okai, Y. Potent suppressive effect of a Japanese edible seaweed, Enteromorpha prolifera (Sujiaonori) on initiation and promotion phases of chemically induced mouse skin tumorigenesis. Cancer Letters. 1999, 140, 2125. 10. Reddy, B. S.; Sharma, C.; Mathews, L. Effect of Japanese seaweed (Laminaria angustata) extracts on the mutagenicity of 7,12dimethylbenz[a]anthracene, a breast carcinogen, and of 3,2' dimethyl 4 aminobiphenyl, a colon and breast carcinogen. Mutation Resear ch/Fundamental and Molecular Mechanisms of Mutagenesis. 1984, 127, 113118. 11. Berge, J. P.; Debiton, E.; Dumay, J.; Durand, P.; Barthomeuf, C. In Vitro Anti inflammatory and Anti proliferative Activity of Sulfolipids from the Red Alga Porphyridium cruent um. Journal of Agricultural and Food Chemistry. 2002, 50, 62276232.

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70 BIOGRAPHICAL SKETCH Haiyan Liu was from Xi an, China. She received her bachelors degree in food safety and security from China Agricultural University in 2008 After that she was admitted as a master s student in the Food Science and Human Nutrition Department at the University of Florida. In graduate school, Haiyan presented her research at the IFT annual meeting in 2010. Her most recent research has resulted in two abstracts that were submitted to Experimental Biology and IFT annual meeting in 2011. Further, she received the William L. and Agnes F. Brown Graduate Scholarship from UF in 201 1 Upon her completion of the m asters degree in 2011, Haiyan plans to continue her study in the food science major and hopefully pursue the doctorate degree in the future.