Identification, Enhancement and Concentration of Phenolic Compounds in Muscadine Grapes

MISSING IMAGE

Material Information

Title:
Identification, Enhancement and Concentration of Phenolic Compounds in Muscadine Grapes
Physical Description:
1 online resource (148 p.)
Language:
english
Creator:
Sandhu, Amandeep Kaur
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Gu, Liwei
Committee Members:
Yang, Weihua Wade
Sims, Charles A
Gray, Dennis J
Brecht, Jeffrey K

Subjects

Subjects / Keywords:
aba -- adsorption -- anthocyanins -- antioxidants -- concentration -- desorption -- dpph -- enhancement -- flavonols -- florida -- grapes -- hplc-dad-esi-ms -- identification -- kaempferol -- kinetics -- meja -- muscadine -- myricetin -- orac -- phenolics -- phytochemicals -- pomace -- quercetin -- resin -- solvents -- spectrometer -- tannins -- thermodynamics
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre:
Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The Muscadine grape (Vitis rotundifolia) is native to the southeastern United States and possesses a unique phytochemical profile. The phytochemical composition of grapes affects the quality of grapes, such as color and flavor, as well as their health promoting benefits. Therefore, the first objective of this project was to identify various phytochemicals in seeds, skin and pulp of Florida grown muscadine grapes. The second objective was to enhance the phytochemical content by application of pre and post-harvest plant growth regulators abscisic acid (ABA) and methyl jasmonate (MeJA), respectively. Finally, we developed a method to obtain concentrated phytochemical extract from muscadine pomace using resin adsorption technology. High-performance liquid chromatography equipped with diode array (HPLC-DAD) and electrospray ionization mass spectrometric detection (ESI-MSn) was used to identify the phenolic compounds in seeds, skin, and pulp of Noble grapes. On average, 87.1, 11.3, and 1.6% of phenolic compounds were present in seeds, skin, and pulp, respectively. A total of 88 phenolic compounds of diverse structures were tentatively identified in Noble, which included 17 in pulp, 28 in skin, and 43 in seeds. Seventeen compounds were identified for the first time in muscadine grapes. ABA treatment enhanced the antioxidant capacity by 38 and 18% in Noble variety at first and second sampling, respectively. A significant increase in individual anthocyanins was observed in treated Noble grapes at both sampling times. However, increase in the content of ellagic acid and flavonols was observed at first sampling only. No effects of ABA treatment were seen in Alachua grapes. Our results indicate that exogenous application of ABA enhances the antioxidant capacity, anthocyanins and phenolic content of muscadine grapes but these effects may vary depending upon the cultivar. Post harvest MeJA treatment didn’t show any effect on the phenolic compounds in muscadine grapes. The adsorption/desorption characteristics of anthocyanins from muscadine pomace were evaluated on five Amberlite resins (FPX-66, XAD-7HP, XAD-16N, XAD-1180 and XAD-761). FPX-66 and XAD-16N showed highest adsorption and desorption capacities, and ratios. Dynamic testing was done on a column packed with FPX-66 which resulted in a concentrated pomace extract that contained 13% (w/w) anthocyanins with no detectable sugars.
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 Amandeep Kaur Sandhu.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Gu, Liwei.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-11-30

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045280:00001


This item is only available as the following downloads:


Full Text

PAGE 1

1 IDENTIFICATION, ENHANCEMENT AND CONCENTRATION OF PHENOLIC COMPOUNDS IN MUSCADINE GRAPES By AMANDEEP K. SANDHU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

PAGE 2

2 2013 Amandeep K. Sandhu

PAGE 3

3 To my parents and in laws for encouraging me at every step; my husband for his unconditional love and support and my precious son

PAGE 4

4 ACKNOWLEDGMENTS I would like to express my deepest gratitude to my advisor Dr. Liwei Gu for giving me the opportunity to pursue PhD d egree in his lab Dr. Gu has taught me how to critically evaluate my own data and how to get to the root of any underlying problem in research. He has served as a role model of perseverance and I thank him for his support and advice during this endeavor His willingness to support new ideas and confidence has molded me into an independent researcher. I am thankful to Dr. Charles Sims for helping me with juice and wine making. I want to express my gratitude to Dr. Dennis Gray for helping with ABA experimen ts in his vineyard at Apopka. I would also like to thank Dr. Jeffrey Brecht for his excellent suggestions with MeJA study, providing resources for that study and for critically reviewing my dissertation. A special thanks to Dr. Weihua Yang for his insightf ul comments and questions that have been valuable in improving my experiments It would not have been possible to achieve this honorable milestone without the assistance from my fellow lab mates who provided hours of discussion and interaction when needed I would like to thank all the present and past members of the Gu lab for helping me with various experiments, coursework and for making it a great place to work: Wei Wang, Zheng Li, Haiyan Liu, Hanwei Liu, Keqin Ou, Timothy Buran, Sara Marshall, Bo Zhao, Kyle Song, Dr. Tao Zou and Dr. Vishnupriya Gourineni. I will always cherish the chats, humors and lab potlucks. I acknowledge all the assistance provided to me by the University of Florida, College of Agriculture and Life Sciences and the Department Food Science and Human Nutrition. I would like to thank Carmen Graham, Bridget Stokes, Parker Hall Sheila, Marianne Mangone, and Julie Barber, for their help and services in our department.

PAGE 5

5 I would like to allude to the tremendous support and advice I have gott en from my parents, Kuldip and Gurmeet without whom this would not have been possible. I would also like to extend my heartfelt gratitude to my in laws and siblings for their encoura gement and support. Most of all, I would like to thank my husband Milap, w ho provided me the strength to succeed, encouraged and guided me when required, and helped me stay focused. A special thanks to my precious son Armaan, who was born during my PhD program for being the sunshine of my life Finally, I would like to thank the Almighty for showering me with all the blessings.

PAGE 6

6 TABLE OF CONTENTS p age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 Muscadine Grapes ................................ ................................ ................................ .. 17 Phytochemicals i n Muscadine Grapes ................................ ................................ .... 17 Antimicrobial Activity ................................ ................................ ............................... 18 Health Benefits ................................ ................................ ................................ ....... 18 Research Objectives ................................ ................................ ............................... 20 2 ANTIOXIDAN T CAPACITY AND PHENOLIC COMPOSITION IN SEEDS, SKIN AND PULP OF FLORIDA GROWN MUSCADINE GRAPES ................................ .. 21 Background ................................ ................................ ................................ ............. 21 Materials and Methods ................................ ................................ ............................ 22 Chemicals ................................ ................................ ................................ ......... 22 Sample Preparation ................................ ................................ .......................... 23 HPLC DAD ESI MS n Analysis ................................ ................................ .......... 24 Folin Ciocalteu Assay ................................ ................................ ....................... 24 Oxygen Radical Absorbance Capacity (ORAC FL ) Assay ................................ .. 25 Statistical Analyses ................................ ................................ .......................... 25 Results and Discussion ................................ ................................ ........................... 26 Total Phenolic Content and Antioxidant Capacity ................................ ............. 26 Phenolic Identification on HPLC DAD ESI MS n ................................ ............... 27 Summary ................................ ................................ ................................ ................ 37 3 EFFECTS OF EXOGENOUS ABSCISIC ACID ON ANTIOXIDANT CAPACITIES, ANTHOCYANINS AND FLAVONOL CONTENTS OF MUSCAD INE GRAPE SKINS ................................ ................................ ................. 53 Background ................................ ................................ ................................ ............. 53 Materials and Methods ................................ ................................ ............................ 55 Chemicals ................................ ................................ ................................ ......... 55 ABA Treatment ................................ ................................ ................................ 55

PAGE 7

7 Fruit Weight, pH and Total Soluble Solids ................................ ........................ 56 Extraction and Sample Preparation ................................ ................................ .. 56 Folin Ciocalteu Assay ................................ ................................ ....................... 57 Ox ygen Radical Absorbance Capacity (ORAC) ................................ ............... 57 DPPH Assay ................................ ................................ ................................ ..... 57 HPLC Analysis of Phenolic Compounds ................................ .......................... 58 Anthocyanins ................................ ................................ ............................. 58 Ellagic acid and flavonols ................................ ................................ ........... 58 Statistical and Multivariate Analysis ................................ ................................ 59 Results ................................ ................................ ................................ .................... 59 Fruit Weight, pH and Tot al Soluble Solids ................................ ........................ 59 Total Phenolic Content and Antioxidant Capacities ................................ .......... 60 HPLC Analysis of Anthocyanins and Other Phenolic Compounds ................... 61 Principal Component Analysis (PCA) ................................ ............................... 62 Discussion ................................ ................................ ................................ .............. 63 Summary ................................ ................................ ................................ ................ 66 4 COMPARISON OF DIFFERENT SOLVENTS FOR THE EXTRACTION OF PHENOLIC COMPOUNDS FROM NOBLE JUICE AND WINE POMACE .............. 74 Background ................................ ................................ ................................ ............. 74 Materials and Methods ................................ ................................ ............................ 75 Chemicals ................................ ................................ ................................ ......... 75 Muscadine Juice and Wine Pomace Preparation ................................ ............. 75 Extracts Preparation ................................ ................................ ......................... 76 Total Anthocyanin Assay ................................ ................................ .................. 7 6 Folin Ciocalteu Assay ................................ ................................ ....................... 77 Oxygen Radical Absorbance Capacity (ORAC) ................................ ............... 77 HPLC Analysis of Phenolic Compounds ................................ .......................... 78 Anthocyanins ................................ ................................ ............................. 78 Ellagic acid and flavonols ................................ ................................ ........... 78 Statistical Analysis ................................ ................................ ............................ 79 Results and Discussion ................................ ................................ ........................... 79 Summary ................................ ................................ ................................ ................ 81 5 ADSORPTION/DESORPTION CHARACTERISTICS AND SEPARATION OF PHYTOCHEMICALS FROM MUSCADINE POMACE USING MACROPOROUS ADSORBENT RESINS ................................ ................................ ........................... 86 Background ................................ ................................ ................................ ............. 86 Materials and Methods ................................ ................................ ............................ 88 Chemicals ................................ ................................ ................................ ......... 88 Muscadine Juice and Wine Pomace Preparation ................................ ............. 88 Preparation of Juice and Wine Pomace Water Extracts ................................ ... 89 Characterization and Phytochemical Analysis of Extracts ................................ 89 Pretreatment of Macroporous Resins ................................ ............................... 91 Static Adsorption and Desorption Tests for Screening of Resins ..................... 92

PAGE 8

8 Adsorption Kinetics ................................ ................................ ........................... 93 Adsorption Isotherms ................................ ................................ ....................... 94 Dynamic Adso rption and Desorption Tests ................................ ...................... 95 Statistical Analysis ................................ ................................ ............................ 95 Results and Discussion ................................ ................................ ........................... 95 Screening of the Resins ................................ ................................ ................... 95 Total anthocyanin content ................................ ................................ .......... 96 Total phenolic content ................................ ................................ ................ 98 Adsorption Kinetics ................................ ................................ ......................... 100 Adsorption Isotherms ................................ ................................ ..................... 101 Dynamic Br eakthrough Curve on FPX 66 Resin ................................ ............ 102 Dynamic Desorption Curve on FPX 66 Resin ................................ ................ 102 Characterization of Concentrated Extracts from Muscadine Juice Pomace ... 103 Summary ................................ ................................ ................................ .............. 104 6 EFFECT OF METHYL JASMO NATE TREATMENT ON POSTHARVEST QUALITY OF MUSCADINE GRAPES AT DIFFERENT STORAGE TEMPERATURES ................................ ................................ ................................ 119 Background ................................ ................................ ................................ ........... 119 Materials and Methods ................................ ................................ .......................... 120 Chemicals ................................ ................................ ................................ ....... 120 MeJA Treatment ................................ ................................ ............................. 121 Decay Assessment ................................ ................................ ......................... 122 pH and Total Soluble Solids ................................ ................................ ........... 122 Extraction and Sample Preparation ................................ ................................ 122 Total Anthocyanin Assay ................................ ................................ ................ 122 Folin Ciocalteu Assay ................................ ................................ ..................... 123 Oxygen Radical Absorbance Capacity (ORAC) ................................ ............. 123 DPPH Assay ................................ ................................ ................................ ... 124 Statistical Analysis ................................ ................................ .......................... 124 Results and Discussion ................................ ................................ ......................... 124 CO 2 and O 2 Analysis ................................ ................................ ...................... 124 Decay Assessment ................................ ................................ ......................... 125 pH and Total Soluble Solids ................................ ................................ ........... 125 Total Anthocyanin Content ................................ ................................ ............. 125 Total Phenolic Content and Antioxidant Capacities ................................ ........ 126 Summary ................................ ................................ ................................ .............. 127 7 CONCLUSIONS ................................ ................................ ................................ ... 134 LIST OF REFERENCES ................................ ................................ ............................. 136 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 148

PAGE 9

9 LIS T OF TABLES Table page 2 1 Total phenolic content and antioxidant capacity of seeds, skin and pulp of different cultivars of muscadine grapes. ................................ ............................. 39 2 2 Retention time and mass spectrometric data of phenolic compounds in muscadine grape pulp (cv. Noble) determined by HPLC ESI MS n ..................... 40 2 3 Retention time and mass spectrometric data of phenolic compounds in muscadine grape skin (cv. Noble) determined by HPLC ESI MS n ..................... 41 2 4 Retention time and mass spectrometric data of phenolic compounds in muscadine grape seed (cv. Noble) determined by HPLC ESI MS n .................... 44 3 1 Average berry weight, total soluble solids ( Brix) and pH of juice from control and ABA treated muscadine grapes. ................................ ................................ .. 68 3 2 Total phenolic content and antioxidant capacities (ORAC and DPPH) of control and ABA treated muscadine grape skins. ................................ ............... 69 3 3 Anthocyanin content of control and ABA treated muscadine grape skins. .......... 70 3 4 Ellagic acid, myricetin, quercetin and kaempferol content of control and ABA treated muscadine grape skins. ................................ ................................ .......... 71 4 1 Total phenolics, antioxidants and total anthocyanins extracted from Noble juice and wine pomace using different solvents. ................................ ................. 83 4 2 Anthocyanins extracted from Noble juice and wine pomace using different solvents. ................................ ................................ ................................ ............. 84 4 3 Ellagic acid, myricetin, quercetin and kaempferol extracted from Noble juice and wine pomace using different solvents. ................................ ......................... 85 5 1 Physical characteristics of adsorbent resins. ................................ .................... 105 5 2 Kinetic parameters of muscadine juice and wine pomace on FPX 66, XAD 16N and XAD 1180 resins at room temperature (25C) based on total anthocyanins. ................................ ................................ ................................ ... 106 5 3 Kinetic parameters of muscadine juice and wine pomace on FPX 66, XAD 16N and XAD 1180 resins at room temperature (25C) based on total phenolics. ................................ ................................ ................................ ......... 107

PAGE 10

10 5 4 Langmuir and Freundlich parameters for the adsorption of muscadine juice pomace on FPX 66, XAD 16N and XAD 1180 resins based on total anthocyanins. ................................ ................................ ................................ ... 108 5 5 Comparison of phytochemical and sugar content of muscadine juice pomace water extract with concentrated extract. ................................ ........................... 109 6 1 ANOVA for dependent variables for MeJA treatment, storage time, temperature and their interactions for Noble and Alachua grapes. ................... 128 6 2 CO 2 and O 2 levels in the headspace of the containers after MeJA treatment of Noble and Alachua grapes. ................................ ................................ .......... 129 6 3 Effect of MeJA treatment on decay of Noble and Alachua grapes after 2 weeks of storage at 5 and 20C. ................................ ................................ ....... 130 6 4 pH and total soluble solids (Brix) of Noble and Alachua juice from control and MeJA treated muscadine grapes. ................................ .............................. 131 6 5 Total anthocyanin, total phenolic and antioxidant capacities (ORAC and DPPH) of control and MeJA treated Noble grape skins. ................................ ... 132 6 6 Total anthocyanin, total phenolic and antioxidant capacities (ORAC and DPPH) of control and MeJA treated Alachua grape skins. ............................... 133

PAGE 11

11 LIST OF FIGURES Figure page 2 1 Proposed structures of phenolic compounds in muscadine grapes. ................... 47 2 2 Negative ion electrospray product ion mass spectra (MS 2 and MS 3 ) of phenolic compounds identified for the first time in musc adine (cv. Noble) pulp, skin and seeds. ................................ ................................ .......................... 48 2 3 Negative ion electrospray product ion mass spectra (MS 2 and MS 3 ) of phenolic compounds identified for the first time in muscadine (cv. Noble) seeds. ................................ ................................ ................................ ................. 49 2 4 HPLC DAD chromatograms of muscadine (cv. Noble) pulp .............................. 50 2 5 HPLC DAD chromatograms of muscadine (cv. Noble) skin. .............................. 51 2 6 HPLC DAD chromatograms of muscadine (cv. Noble) seed. ............................. 52 3 1 Score plots of principal component analysis of control and ABA treated muscadine grapes: A. Noble B. Alachua. ................................ ........................... 72 3 2 Loading plot of principal component analysis of muscadine grapes (cv. Noble) based on principal component 1 and 2 ................................ ................... 73 5 1 Static adsorption results based on total anthocyanin content on different resins in juice pomace ................................ ................................ ...................... 110 5 2 Static adsorption results based on total anthocyanin content on different resins in wine pomace. ................................ ................................ ..................... 111 5 3 Static adsorption results based on total phenolic content on different resins in juice pomace. ................................ ................................ ................................ ... 112 5 4 Static adsorption results based on total phenolic content on different resins in wine pomace. ................................ ................................ ................................ ... 113 5 5 Adsorption kinetic curves for juice pomace on FPX 66, XAD 16N and XAD 1180 ................................ ................................ ................................ ................. 114 5 6 Adsorption kinetic curves for wine pomace on FPX 66, XAD 16N and XAD 1180.. ................................ ................................ ................................ ............... 115 5 7 Adsorption isotherms for juice pomace based on t otal anthocyanin content on FPX 66, XAD 16N and XAD 1180. ................................ ................................ ... 116 5 8 Dynamic breakthrough curves of total anthocyanins from muscadine juice pomace on column packed with FPX 66 resin at different flow rates. .............. 117

PAGE 12

12 5 9 Dynamic desorption curves of total anthocyanins from muscadine juice pomace on column packed with FPX 66 resin at different flow rates.. ............. 118

PAGE 13

13 LIST OF ABBREVIATIONS ABA Abscisic acid AAPH azobis(2 amidin opropane) ANOVA Analysis of variance BV Bed volume CO 2 Carbon dioxide DAD Diode array detector DPPH 2,2 diphenyl 1 picrylhydrazyl g Gram g Relative centrifugal force h Hour (s) HPLC High performance liquid chromatography Kg Kilogram L Liter m/z Mass to c harge ratio MS Mass spectrometer MeJA Methyl jasmonate g Microgram L Microliter mol Micromole mL Milliliter mm Millimeter min Minute (s) nm Nanometer nM Nanomole

PAGE 14

14 ORAC Oxygen radical absorbance capacity O 2 Oxygen psi Pounds per square inch PCA Principal component analysis PC1 Principal component 1 PC2 Principal component 2 rpm Revolutions per minute s Second (s) Trolox 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid TSS Total soluble solids UV Ultraviolet Vis Visible v Volume w Weight

PAGE 15

15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IDENTIFICATION, ENHANCEMENT AND CONCENTRATION OF PHENOLIC COMPOUNDS IN MUSCADINE GRAPES By Amandeep K. Sandhu M ay 2013 Chair: Liwei Gu Major: Food Science and Human Nutrition The Muscadine grape ( Vitis rotundifolia ) is native to the southeastern United States and possesses a unique phytochemical profile. The phytochemical composition of grapes affects the quality of grapes, such as color and flavor, as well as their health promoting benefits. Therefore, the first objective of this project was to i dentify various phytochemicals in seeds, skin and pulp of Florida grown muscadine grapes. The second objective was to enhance the phytoc hemical content by application of pre and post harvest plant growth regulators abscisic acid (ABA) and methyl jasmonate (MeJA), respectively. Finally, we developed a method to obtain concentrated phytochemical extract from muscadine pomace using resin adsorption technology. High performance liquid chromatography equipped with diode array (HPLC DAD) and electrospray ionizati on mass spectrometric detection (ESI MS n ) was used to identify the phenolic compounds in seeds, skin, and pulp of Noble grapes. On average, 87.1, 11.3, and 1.6% of phenolic compounds were present in seeds, skin, and pulp, respectively. A total of 88 phenol ic compounds of diverse structures were tentatively identified in Noble which included 17 in pulp, 28 in skin, and 43 in seeds. Seventeen compounds were identified for the first time in muscadine grapes.

PAGE 16

16 ABA treatment enhanced the a ntioxidant capacity by 38 and 18% in Noble variety at first and second sampling, respectively. A significant increase in individual anthocyanins was observed in treated Noble grapes at both sampling times. However, increase in the content of ellagic acid and flavonols was observed at first sampling only. No effects of ABA treatment were seen in Alachua grapes. Our results indicate that exogenous application of ABA enhances the antioxidant capacity, anthocyanins and phenolic content of muscadine grapes but these effects may vary depending upon the compounds in muscadine grape s The adsorption/desorption characteristics of anthocyanins from muscadine pomace were evaluated on five Amberlite resins (FPX 66, XAD 7HP, XAD 16N, XAD 1180 and XAD 761). FPX 66 and XAD 16N showed highest adsorption and des orption capacities, and ratios. Dynamic testing was done on a column packed with FPX 66 which resulted in a concentrated pomace extract that contained 13 % (w/w) anthocyan ins with no detectable sugars.

PAGE 17

17 CHAPTER 1 INTRODUCTION Muscadine Grapes The Muscadine grape ( Vitis rotundifolia ) is indigenous to the southeastern United States. They differ from other grapes ( Viti s vinefera ) both in morphology and composition. They are either light skinned (green or bronze) or dark skinned (red to almost black) 1 2 and are 2 .5 3 8 c m in diameter with thick, tough skin which protects them from heat, UV radiation, humidity, insects and fungi. They grow in tight small clusters of 3 to 10 berries in contrast to other grape species which grow in bunches. Muscadine grapes are resistant to P d isease, which prevents the commercial production of Vitis vinifera grapes 3 The disease resistance of muscadine grapes has made them uniquely fit for production in the southeastern United States in comparison to traditional bunch grapes. There are more than 300 muscadine cultivars and among them Carlos, Noble, and Magnolia are of commercial importance. Muscadine grapes are consumed as fresh fruit or processed into wine, juice, jam or jelly. Muscadine wines are gaining popularity as consumers begin to appreciate their unique fruity bouquet and the positive healt h effects. Phytochemicals in Muscadine Grapes Phytochemicals originate as a result of secondary plant metabolism and are essential for reproduction, stability and growth processes of plants. They are recognized not only for their health promoting benefits but also for their contribution to color and flavor in different fruits and vegetables. They are categori z ed into different classes depending upon their structures varying from simple phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid) to comple x polyphenols (hydroly s able and condensed

PAGE 18

18 tannins). Several classes of phytochemicals are found in grapes however, structures and contents of these phytochemicals differ in the grapes of different variety or genotype. Muscadine grapes possess unique phytoc hemical composition that differentiates them from other Vitis species such as presence of ellagic acid and its derivatives, and anthocyanin 3, 5 diglucosides 4 5 Other phytochemicals found in muscadine are flavanols (catechin and epicatechin), condensed tannins (oligomeric flavanols), flavonols (myricetin, quercetin and kaempferol), resveratrol and gallic acid. About 80% and 18% of phytochemicals are located in the muscadine seeds and skin, respectively. The pulp contains very low amount of phytochemicals 6 Antimicrobial Activity The high phenolic content not only confers disease resistance in muscadine grapes but also provide antimicrobial activity. Several studies have investigated the effect of muscadine gra pe components and extracts on pathogenicity. Many of these studies worked with gram negative microorganisms including Eschericia coli O157:H7, Enterobacter sakazakii Cronobacter sakazakii Salmonella enteriditis and Listeria monocytogenes pathogens that are watched closely by the food industry. The results from these studies suggest that muscadine grapes and extracts could be used as a natural preservative in beverages and other products 7 9 Health Benefits In addition to their disease resistance and antimicrobial activity, muscadine grapes have potential to positively affect a wide variety of health issues including muscadine wine showed that moderate consumption of muscadine wine attenuates cognitive deterioration in Tg2576 mice by interfering with the oligomerization of amyloid

PAGE 19

19 molecular responsible for initiating a cascade of cellular events resulting in cognitive decline. Thus, it can be used for the pre 10 I nflammation plays a pivotal role in various immunopathophysiological conditions. Muscadine skin, seed, and combination skin/seed extracts e xhibit significant topical anti inflammatory properties in mice models 11 Similarly, another study reported the anti inflammatory properties of muscadine s kin powder both in vitro and in vivo with possible mechanisms including the inhibition of cytokine and superoxide release 12 Cancer is a rapidly growing health problem that is the biggest challenge to researchers and medical professionals. Muscadine grapes, wines and extracts have shown promising results in preventing various types of cancers. For example, cell culture studies have suggested that polyphenols from muscadine grapes were effective in in hibiting cancer cell viability and inducing apoptosis in a liver cell model (i.e., HepG2 cells) and a human colon cell (i.e., Caco 2 cells), and that the greatest anticancer activities were attributed to muscadine fractions containing ellagic acid and anth ocyanins, respectively 13 14 Muscadine grape skin extract inhibited tumor cell growth in prostate cancer cell lines and exhibited high rates of apoptosis by targeting phosphatidylinositol 3 kinase Akt and mitogen activated protein kinase survival pathways 4 Extracts from red muscadine wines induced cell death in MOLT 4 human leukemia cells 15 Obesity is a multifactorial condition posing major health problems worldwide that leads to various other metabolic complications. Muscadine grape and wine phytochemicals prevented obes ity associated metabolic complications in C57BL/6J mice 16 Several studies have suggested the anti diabetic activity of muscadine grapes. A recent study reported that anthocyanins from

PAGE 20

20 glucosidase and pancrea tic lipase in vitro the enzymes implicated in diabetes 17 Extracts of muscadine grape and seeds have also prevented f ormation of advanced glycation end products in vitro which have been associated with diabetic complications 18 A human study conducted by Banini et. al., showed that nightly supplementation of 150 m L of muscadine wine and dealcoholized wine both comparably moderated blood glucose, glycated hemoglobin, and insulin in participants with and without Type II diabetes 19 Research Objectives This research was conduct ed to get the comprehensive knowledge of phenolic compounds in muscadine grapes and to investigate the effect of pre and post harvest growth regulators on the ir content In addition, a method was developed to extract and concentrate phytochemicals from mu scadine pomace using resin adsorption technology. The specific objectives were: 1. To evaluate antioxidant capacity, phenolic content and perform an extensive identification of the phenolic compounds in seed, skin and pulp of Florida grown muscadine grapes us ing HPLC DAD ESI MS n 2. To investigate the effects of exogenous abscisic acid on antioxidant capacities, anthocyanins, and flavonol contents of muscadine grape skins. 3. To compare different solvents for the extraction of phenolic compounds from Noble juice and wine pomace. 4. To study adsorption/desorption characteristics and separation of phenolic compounds from muscadine pomace using macroporous adsorbent resins. 5. To investigate the effects of methyl jasmonate on postharvest quality of muscadine grapes at differe nt storage temperatures

PAGE 21

21 CHAPTER 2 ANTIOXIDANT CAPACITY AND PHENOLIC C OMPOSITION IN SEEDS, SKIN AND PULP OF FLORIDA GROWN MUSCADINE GRAPES Background Phenolic compounds are a class of phytochemicals that play an important role in the nutritional an d sensory properties of various fruits and vegetables. They are categorized into different classes depending upon their structures varying from simple phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid) to complex polyphenols (hydrolysable and co ndensed tannins) 20 21 Phenolic compounds have been linked to many positive health benefits including the protective effects against certain diseases such as cancer and cardiovascular diseases 22 24 The protective effect of phenolic compounds has been attributed in part to their antioxidant capacity 25 26 Muscadine grapes are comm only grown in the southeastern United States and are well adapted to warm, humid climates, which are unsuitable for the growth of other grapes ( Vitis vinifera ). They are either light skinned (green or bronze) or dark skinned (red to almost black) 1 2, 27 and are 2 5 3 8 c m in diameter with thick, tough skin which protects them from heat, UV radiation, humidity, insects and fungi. They grow in tight small clusters of 3 to 10 berries and are marketed in fresh and processed forms such as jui ce, wine and jam. Muscadine grapes contain a large variety of antioxidant phytochemicals. They are reported to contain hydroxybenzoic acids, ellagic acid in free and conjugated form, resveratrol and flavonoids including anthocyanins, quercetin, myricetin and kaempferol 1, 5, 28 Cell culture studies have suggested that p olyphenols from muscadine grapes can inhibit proliferation of colon cancer cells and induce apoptosis in them 13 14 However, the phytochemical profiles of muscadine grapes have been documented by only a few

PAGE 22

22 studies. The phenolic compounds in muscadine grapes have been quantified after acid hydrolysis of the samples, which limits their actual structural identification 27 Two other studies identified the phenolic compounds in the skin of muscadine grapes 5, 28 however, specific information on the identification of phenolic co mpounds in seed and pulp of muscadine grapes is lacking. High performance liquid chromatography coupled with diode array detector and a mass spectrometry (HPLC DAD MS n ) provides a powerful tool for phytochemical analysis in crude plant extracts. It provide s useful structural information and allows for tentative compound identification when standard reference compounds are unavailable and when peaks have similar retention times and UV absorption spectra 29 30 The aim of our study was to evalu ate the antioxidant capacity and total phenolic content in seed, skin and pulp of eight cultivars of Florida grown muscadine grapes In addition, an extensive identification of the p henolic compounds in seed, skin and pulp of Noble variety was done using a simple and rapid high performance liquid chromatography and mass spectrometry (HPLC MS n ) technique. Seventeen different phenolic compounds were identified for the first time in musc adine grapes. The comprehensive knowledge of phenolic compounds in seed, skin and pulp of muscadine grapes can contribute to a better understanding of their influence on the quality of muscadine products especially wine and juice. Materials and Methods Ch emic als Ellagic acid, (+) catechin, ( ) epicatechin, quercetin 3 O glucoside, ( ) catechin gallate, ( ) gallocatechin and ( ) epigallocatechin were purchased from Sigma Aldrich (St. Louis, MO). Quercetin 3 O rhamnoside was purchased from Indofine ( Hillsborough, NJ). ( ) Epicatechin gallate and trans resveratrol 3 O glucoside were

PAGE 23

23 azobis(2 amidinopropane)) was a product of Wako Chemic als inc. (Bellwood, RI). Gallic acid, 6 Hydroxy 2,5,7,8 tetramethy lchroman 2 carboxylic acid (Trolox), HPLC grade methanol, acetic acid, formic acid, Folin Ciocalteu reagent, Flourescein, 96 well black plate with clear bottom wells and a lid, and sodium carbonate were purchased from Fischer Scientific Co. (Pittsburg, PA) Sample Preparation Eight cultivars of muscadine grapes, four bronze (Doreen, Fry, Carlos and Triumph) and four black (Southland, Magoon, Alachua and Noble) were obtained from a local vineyard in central Florida. The grapes were manually separated into s eed, skin and pulp within 2 hours of harvest and the separated portions were kept at 20C until further analysis. The seed, skin and pulp of grapes were freeze dried and ground to a fine powder using Waring kitchen blender. One gram of freeze dried pulp a nd 0.5 g of seed or skin were weighed in 20 mL screw capped glass tubes. The weighed samples were extracted with 10 mL of acetone: water: acetic acid (acetone/ H 2 O /acetic acid; 70:29.7:0.3, v/v) solvent. The extraction tubes were vortexed for 30 s and son icated for 5 min, then kept at room temperature for 20 min an d sonicated for another 5 min. The tubes were centrifuged at 3000 rpm for 10 min and supernatant collected in separate glass tubes. For identification of phenolic compounds, the same extraction p rocedure was followed. The collected supernatant was evaporated in a SpeedVac Concentrator (Thermo scientific ISS110, Waltham, MA) under reduced pressure at 25 C to remove the solvent. The solids obtained after evaporati on were dissolved in 5 mL of 70 % ac i dified (1 % formic acid) methanol and sonicated for 5 min to resuspend the solid

PAGE 24

24 residue. The samples were filtered through 0.45 m filter prior to injection (20 L) to the HPLC system. All the prepared samples were kept at 20 C until analysis. HPLC DAD ES I MS n An alysis Chromatographic analyses were performed on an Agilent 1200 series HPLC (Agilent, Palo Alto, CA) equipped with an autosampler/injector and diode array detector. A Zorbax Stablebond Analytical SB C18 column (4.6 250 mm, 5 m, Agilent Techno logies, Rising Sun, MD) was used for separation. Elution was performed using mobile phase A (0.5% formic acid aqueous solution) and mobile phase B (methanol). UV/Vis spectra were scanned from 220 to 600 nm on diode array detector with detection wavelengths at 280, 360 and 520 nm. The flow rate was 1 mL/min and the linear gradient used was: 0 2 min 5% B; 2 10 min 5 20% B; 10 15 min 20 30% B; 15 20 min 30 35% B; 60 65 min 80 85% B; 65 70 min 85 5% B followed by 5 min of re equilibration of the column for the next run. Electrospray mass spectrometry was performed with a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Column effluent was monitored in positive and negative ion mode of the MS in an alternative man ner during the same run. Other ex perimental conditions on the mass spectrometer were as follows: nebulizer, 45 psi; dry gas, 1 1.0 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 spe ctrometer was operated in Auto MS 3 mode. MS 2 was used to capture and fragment the most abundant ion in full scan mass spectra and MS 3 was used to fragment the most abundant ion in MS 2 Folin Ciocalteu Assay The acetone/ H 2 O /acetic acid extracts were diluted to appropriate concentration for analysis. Total phenolic content was determined by using modified method of

PAGE 25

25 Singleton and Rossi 31 The extracts were mixed with diluted Folin Ciocalteu reagent and 15% sodium carbonate. Absorption at 765 nm was measured in a microplate reader (SPECTRAmax 190, Sunnyvale, CA) after incubation for 30 minutes at room tempera ture. The results were expressed as milligrams of gallic acid equivalents per gram of fresh weight (mg of GAE/g) using a standard curve generated with 100 600 mg/L of gallic acid. Oxygen Radical Absorbance Capacity (ORAC FL ) Assay ORAC assay for extracted samples was carried out on a Spectra XMS Gemini plate reader (Molecular Devices, Sunnyvale, CA). Briefly, 50 L of standard and samples were added to the designated wells of 96 well black plate. This was followed by addition of 100 L of Flourescein (20 nM ). Mixture was incubated at 37C for 7 minutes before the addition of 50 L AAPH. Fluorescence was monitored using 485 nm (excitation) and 530 nm (emission) at 1 min interval for 40 minutes. Trolox was used to generate a standard curve. The results of the antioxidant capacity of extracts were expressed as micromoles of Trolox equivalents (TE) per gram fresh weight (mol of TE/g). Statistical Analyses One way analyses of variance (ANOVA) with Tukey HSD pair wise comparison of the means were performed using JMP software (Version 7.0, SAS Institute Inc. Cary, NC). Total phenolic and ORAC values are expressed as mean standard deviation of three independent observations. Data points from two samples were omitted as outliers based on the Q test 32 and the results from those values are expressed as dup licates. A

PAGE 26

26 Results and Discussion The phytochemical constituents of the muscadine grape ( Vitis rotundifolia ) differ from V itis v inifera in many aspects. The presence of ellagic acid in muscadine grapes i s unique and is found in the form of free ellagic acid, ellagic acid glycosides, and ellagitannins 33 Another unique feature is that the anthocyanins are present as 3, 5 diglucosides (as opposed to 3 glucosides in Vitis vinifera ) of delphini din, cyanidin, petunidin, peonidin, pelargonidin and malvidin in nonacylated forms. No condensed tannins were identified in muscadine skin as opposed to Vinifera skins 34 Red muscadine wines are more susceptible to browning and loss of color after a geing. This may be due to slow association of anthocyanin diglucosides and tannins present in muscadine wines 35 Total Phenolic Content and Antioxidant Capacity Table 2 1 shows the total phenolic content and antioxidan t capacity in eight cultivars of Florida grown muscadine grapes. Based on fresh weight, the total phenolic content was highest in seeds (27.0 81.2 GAE mg/g), followed by skin (4.3 10.2 GAE mg/g) and pulp (0.3 1.2 GAE mg/g). Among the seeds, the total pheno lic content was highest in Alachua and lowest in Magoon variety of muscadine grapes. The skin of Carlos variety had highest total phenolic content compared to skin of other varieties. Accordingly, the antioxidant capacity, based on fresh weight was also hi ghest in seeds (276.6 4.6 On average the phenolic content in seeds, skin and pulp was 87.1%, 11.3% and 1.6% of that in whole grapes, respectively. Similarly, the average antioxidant capacity among all the grape cultivars was 93.9% in seeds, 5.6% in skin and 0.5% in pulp. The

PAGE 27

27 correlation coefficient (r) between the total phenolic content and antioxidant capacity in seeds, skin and pulp of eight cultivars of muscadines was 0.87. Phenolic Identification on HPLC DAD ESI MS n Identification of phenolic compounds was done in Noble variety of muscadine grapes The HPLC DAD chromatograms of pulp, skin and seed were recorded at 280, 360 and 520 nm (Figures 2 4 to 2 6 ). Most of the phenolic compounds can be detected at 280 nm. Ellagic acid derivatives and flavonols have maximum absorption at 360 nm. Anthocyanins were detected at 520 nm in the skin. Anthocyanins we re also detected in the pulp, however, it could be due to migration of these pigments from skin to the pulp during the separation of fruit into its parts. Mass Spectrometer (MS) was operated in both positive and negative ionization modes in the same HPLC r un. Anthocyanins have inherent positive charge so they have maximum sensitivity in the positive modes of MS, however, for most other flavonoids the highest sensitivity was obtained in negative ionization mode. Most flavonoids are present in nature as glyco sides and other conjugates 36 37 Identification of sugar moiety attached to phenolic compounds was based on fragmentation data from MS and previous literature reports 5 Phenolic compounds in the pulp, skin and seed were identified based on mass spectrum data, chromatography of pure standards and UV/Vis spectra on diode array detector. Seventeen phenolic compounds are reported for the first time in Noble pulp that include caffeic acid hexoside, hydrolysable tannins, mostly gallotannins, epicatechin, epicatechin gallate, ellagic acid and its conjugates, flavonol glycosides and isomeric forms of resveratrol glucoside (Table 2 2). In Noble skin, twenty eight phenolic compounds are reported and among those eight compounds were identified for the first time (Table 2 3). These compounds were caffeic acid hexoside,

PAGE 28

28 hexahydroxydiphenoic glucose (HHDP glucose), monogalloyl glucose, ellagic acid hexoside, kaempferol rutinoside and h exosides of myricetin, quercetin, and kaempferol. No condensed tannins were identified in the skin. Forty three different phenolic compounds identified in Noble seed are outlined in Table 2 4. No previous study on muscadine grapes has identified these comp ounds. Among the various phenolic compounds in seed, hydrolysable tannins were most prominent. Condensed tannins and flavan 3 ols, ellagic acid conjugates, quercetin rhamnoside and caffeic acid hexoside were also identified in the N oble seed. Hydroxycinna mic acid derivatives: Compound 1 (Tables 2 2, 2 3 and 2 4, Figure 2 2) had m/z 377 [M + Cl ] ion, which indicates a chloride adduct that fragmented to yield m/z 341 [M H] as the most intense ion in MS 2 Compound with m/z 341 further dissociated to give ion at m/z 179 by losing a hexose sugar and was tentatively identified as caffeic acid hexoside. Similar MS fragmentation data was observed in previous studies 38 39 Hydroxybenzoic acid: The identification of gallic acid (Compound 8, Table 2 4) was confirmed by same retention time and MS data of pure standard which gave m/z 169 [M H] ion that dissociated to form m/z 125 via loss of CO 2 Gallic acid has been previously identified in muscadine grapes 5 Hydrolysable t annins: Hydrolysable tannins are categorized into gallotannins and ellagitannins. Gallotannins consist of a glucose molecule in which hydroxyl groups are partly or completely s ubstituted with galloyl groups and ellagitannins are esters of hexahydroxydiphenoyl (HHDP) group consisting of polyol core (glucose or quinic acid). Additionally, galloyl residues may be attached to glucose core via m depside bonds 40

PAGE 29

29 42 Based on MS data the main fragmentation pattern from gallotannins involved the loss of one or more galloyl groups (152 atomic mass unit, amu ) and/or gallic acid (170 amu ) from the deprotonated molecule [M H] However, the fragment ation pattern of ellagitannins was less clear than gallotannins as ellagitannins display enormous structural variability because of different linkages of HHDP residues with glucose molecule and their strong tendency to form C C and C O C linkages 41, 43 The presence of HHDP moiety was confirmed from MS data by the presence of an ion at m/z 301 from the deprotonated molecule [M H] as reported in previous studies with fruit an d plant material 5, 38, 44 49 The presence of a compound with same molecular weight at different retention times illustrated isomeric forms of that compound. Different isomeric forms of hyd rolysable tannins were observed and have been reported previously in Eucalyptus 45 Compound 2 (Table 2 4) and Compound 3 (Table 2 3) had a m/z 481 [M H] which fragmented to ga ve an intense product ion at m/z 301 [M H 162] by losing one glucose unit (Figure 2 2). Based on the fragmentation pattern and literature data 45 these compounds were tentati vely identified as isomers of HHDP glucose. Compounds 7, 10 and 16 (Table 2 4) had identical m/z 483 [M H] ions which fragmented to form ions at m/z 331 [M H 152] and m/z 169 [M H 162] after sequential removal of galloyl group and glucosyl group. They were tentatively identified as isomers of digalloyl glucose based on fragmentation data and previous literature reports 45, 47, 49 51 Compound 6 (Table s 2 2 and 2 3, Figure 2 2) gave identical [M H] ions at m/z 331, which yielded deprotonated gallic acid residue ( m/z 169) owing to loss of glucose unit [M H 162] The gallic acid anion decarboxylates to form fragment at m/z 125 (169

PAGE 30

30 44, loss of CO 2 ) This compound was tentatively identified as monogalloyl glucose 51 52 Compound 4 (Table 2 2), Compound 11 (Table 2 3) and Compounds 5, 9 and 19 (Table 2 4) eluted at different time but gave identical [M H] and product ions as compound 6. These compounds are tentatively identified as isomers of monogalloyl glucose, where gallic acid is attached to different hydroxyl group of the glucose. Compound 13 (Table s 2 2 and 2 4, Figure 2 3) was tentatively identified as monogalloyl diglucose wit h m/z 493 [M H] ion fragmenting to yield ions at m/z 331 and m/z 169 after sequential removal of two glucosyl groups (162 amu ). Similar observations were also reported in longan seeds 52 Compound 22 (Table 2 3) had [M H] ion at m/ z 625 which dissociated to give intense MS 2 ion at m/z 463 [M H 162] indicating loss of glucose unit. The major ion in MS 3 was at m/z 301 [M H 162 162] suggesting loss of another glucose unit. Based on fragmentation data this compound was tentatively identified as HHDP diglucoside 5 Com pound 12 (Table 2 4) had [M H] ion at m/z 633 and major MS 2 fragment at m/z 481, indicating presence of HHDP glucose by losing galloyl unit [M H 152] and minor fragment at m/z 301. Loss of galloyl unit suggested that galloyl units were not directly linked to glucose core but were attached via m depside bond, thus the compound was identified as Galloyl HHDP glucose 44, 49 However, compound 15 (Table 2 3) and compounds 14, 17, 20, 23, 28 and 30 (Table 2 4) gave identical deprotonated ions at m/z 633 [M H] but the intense MS 2 fragment was at m/z 301 [M H 331] instead of m/z 481 indicating loss of galloylglucose. The fragmentation data suggests that galloyl unit was directly linked to glucose core, thus the compounds were tentatively identified as isomeric forms of HHDP galloyl glucose. Compounds 21, 24, 31 (Figure 2

PAGE 31

31 3), 37, 42 and 45 (Table 2 4) were tentatively identified as isomeric forms of HHDP digalloyl glucose. The deprotonated molecule with m/z 785 [M H] fragmented to give a major ion at m/z 633 [M H 152] indicating loss of galloyl group and minor ions at m/z 483 [M H 301] and m/z at 301 [M H 483] showing the presence of HHDP moiety and loss of digalloyl glucose 5, 47, 49 Compounds 25, 26 (Figure 2 3), 32, 35, 41 and 43 (Table 2 4) had [M H] at m/z 635. The major ions in MS 2 were at m/z 483 [M H 152] 465 [M H 170] and 423 [M H 212] indicat ing loss of galloyl group, gallic acid and loss of another galloyl group along with cross ring fragmentation of glucose 53 respectively. However, MS 3 yielded fragments at m/z 331 [M H 152 152] 313 [M H 152 152 18] 271 [M H 212 152] and 169 indicating successive loss of galloyl groups. Some other minor fragments were also observed in MS 3 The different retention time and fragmentation pattern suggests presence of isomeric forms of the given molecule. These compounds were tentatively identified as isomers of trigalloyl glucose 45, 47, 49 50 Compounds 47, 51 and 52 (Table 2 3) were tentatively identified as ellagitannins yielding deprotonated ions at m/z at 813, 831 and 817. The presence of HHDP was supported by the formation of m/z 301. However, structural elucidation of these compounds was not done due to lack of complete fragmentation data. Ellagitannins have been previously identified in muscadine grapes 5 Compounds 40, 48 (Figure 2 3) and 49 (Table 2 4) were tentatively identified as isomers of tetragalloyl glucose which dissociated to give identical m/z at 787 [M H] The fragmentation of deprotonated ion in MS 1 and MS 2 yielded ions at m/z 635 [M H 152] 617 [M H 152 18] 483 [M H 152 152] 465 [M H 152 152

PAGE 32

32 18] 313 [M H 152 152 18 152] and 169 [M H 152 152 18 152 144] indicating consecutive losses of galloyl groups and water molecules, and finally loss of glucose fr om dehydrated galloyl glucose molecule to give deprotonated gallic acid. These findings were confirmed by previous literature reports 45, 47, 49 Compounds 57 (Figure 2 3) and 58 (Table 2 4) gave identical m/z at 939 [M H] dissociating to yield ions at m/z 787 [M H 152] 769 [M H 152 18] 635 [M H 152 18 134] 617 [M H 152 152 18] 483 [M H 152 152 18 152] and 465 [M H 152 152 18 152 18] suggesting loss of galloyl groups and water molecules. Due to lack of complete structural elucidation by fragmentation data in MS 1 and MS 2 these compounds were tentatively identified as isomers of pentagalloyl glucose 49 Compound 61 (Table 2 4, Figure 2 3) was tentatively identified as hexagalloyl glucose 49 with m/z 1091 [M H] that fragment ed to give 939 [M H 152] and 787 [M H 152 152] in MS 2 indicating loss of galloyl groups and presence of pentagalloyl and tetragalloyl glucose residues. The MS 3 fragments were at m/ z 787 [M H 152 152] 769 [M H 152 152 18] and 6 17 [M H 152 152 18 152] indicating loss of galloyl groups and water molecules. Similar results were reported by Soong et al 52 Anthocyanins: Six different anthocyanins were identified in Noble grape skin (Compounds 27, 29, 33 38, 39 and 44 (Table 2 3). The anthocyanins coeluted and represented only three peaks in the chromatogram but they had different retention times. So the peak numbers were marked according to the retention times of individual anthocyanins (Figure 2 5). Al though, previous studies have identified and quantified the anthocyanins in muscadine grapes 2, 54 most of the identification and quantification was

PAGE 33

33 done after hydrolysis 2, 28 which does not justify the structure of anthocyanin diglucosides. In the present study, fragmentation pattern of anthocyanins from MS 2 and MS 3 is provided. Similar fragmentation pattern was observed for all anthocyanins indicating l oss of glucose residues and formation of aglycone. Anthocyanins in muscadine grapes have been reported to exist in 3, 5 diglucoside forms 54 56 Compound 27 with m/z 627 [M] + fragmented to two product ions in MS 2 at m/z 465 [M 162] + and 303 [M 162 162] + corresponding to delphinidin glucoside and delphindin, respectively. So, this compound was tentativel y identified as delphinidin 3, 5 diglucoside. Compound 29 had m/z at 611 [M] + that fragmented to yield two product ions at m/z 449 [M 162] + and 287 (cyanidin) [M 162 162] + indicating the compound to be cyanidin 3,5 diglucoside. Compound 33 was tentat ively identified as petunidin 3, 5 diglucoside. The molecular ion at m/z 641 [M] + fragmented to yield two product ions in MS 2 at m/z 479 [M 162] + and 317 (petunidin) [M 162 162] + indicating two glucose molecules attached to petunidin. Compounds 38 an d 39 coeluted having the same retention time but the mass spectrum of the peaks suggested two molecular ions at m/z 595 [M] + and 625 [M] + The MS 2 spectrum of molecular ion at m/z 595 (compound 38) fragmented into two product ions at m/z 433 [M 162] + and 271 [M 162 162] + which corresponded to pelargonidin glucoside and pelargonidin, respectively. Compound 38 was tentatively identified as pelargonidin 3, 5 diglucoside. Similarly, the molecular ion at m/z 625 (compound 39) had two product ions at m /z 463 [M 162] + and 301 [M 162 162] + which corresponded to peonidin glucoside and peonidin, respectively. This compound was tentatively identified as peonidin 3, 5 diglucoside. Compound 44 had molecular ion at m/z 655 [M] + and fragment ions at m/z 4 93 [M 162] + and 331 [M

PAGE 34

34 162 162] + Based on mass fragmentation this compound was tentatively identified as malvidin 3, 5 diglucoside. We were able to confirm the presence of pelargonidin 3, 5 diglucoside in muscadine grapes as reported in a previous s tudy 2 Flavan 3 ols and condensed t annins: The condensed tannins were identified only in Noble seed compared to skin and pulp. Both galloylated and non galloylated flavan 3 ols and condensed tannins were identified (Tables 2 2 to 2 4). Compound 18 (Table 2 3) had deprotonated ion at m/z 305 [M H] and was identified as gallocatechin. Its identity was confirmed by the same retention time as standard and formation of MS 2 fragment ions at m/z 285, 263, 219, 179, 165 and 125 5, 57 Comp ound 34 (Table 2 4, Figure 2 3) with [M H] ion at m/z 729 generated main MS 2 fragment ion at m/z 577 [M H 152] and MS 3 fragment ion at m/z 289 [M H 152 288] corresponding to the loss of galloyl group and (epi)catechin gallate moiety, respectively. Based on mass spectral data this compound was tentatively identified as galloyl procyanidin dimer 57 58 Compound 36 (Table 2 4) was tentatively identified as procyanidin dimer with its m/z 577 [M H] dissociating to yield ions at m/z 425 [M H 152] and 289 [M H 288] in MS 2 indicating characteristic loss of 152 amu due to Retro Diels Alder (RDA) fission 57 and loss of (epi)catechin molecule, respectively. Mass spectral data from MS 3 showed further dissociation of flavanol rings. Compound 46 (Tables 2 2 and 2 4) with identical [M H] ion at m/z 289 gene rated the major MS 2 ions at m/z 245 (loss of CO 2 ) and minor ions at m/z 205 (cleavage of A ring of flavan 3 ol), 137 (RDA fission). The major ion in MS 3 was at m/z 203 (cleavage of A ring of flavan 3 ol). Compared with the standard, this compound was ident ified as epicatechin 30, 38, 44 Compound 53 (Tables 2 2 and 2 4, Figure 2 3) dissociated at m/z 441 [M H] to yield product ions in MS 2 at

PAGE 35

35 m/z 289, 169 and 125 corresponding to the deprotonated ion of (epi)catechin and gallic acid, and decarboxylated gallic acid, respectively. The major fragment ion in MS 3 was at m/z 245 indicating decarboxylation of epicatechin and minor fragments at m/z 205 and 137 suggesting characteristic fragmentation pattern of (epi)catechin 44, 57 Based on the same retention time with standard and mass spectral data this compound was identified as epicatechin gallate. Compound 60 (Table 2 4, Figure 2 3) gave [M H] ion at m/z 593, which yielded major ions at m/z 441 [M H 152] in MS 2 spectra and m/z 289 [M H 152 152] in MS 3 spectra, corresponding to loss of successive galloyl groups from (epi)catechin. Based on the mass spectral data this compound was tentatively identified as (epi)cat echin digallate 59 Ellagic acid and conjugates: Ellagic acid had been identified and quantified in muscadine grapes in the previous studies 5, 27 In this study we identified ellagic acid hexoside in N oble pulp and skin for the first time (Tables 2 2 and 2 3). Compound 55 (Tables 2 2 and 2 3, Figure 2 2) had identical [M H] ion at m/z 463 wh ich yielded major ion at m/z 301 [M H 162] and minor ions at m/z 284, 257, 229 characteristic of ellagic acid fragmentation 30, 44 The loss of 162 am u corresponded to the loss of hexose sugar and based on the fragmentation pattern this compound was tentatively identified as ellagic acid hexoside 47, 60 Compound 63 (Tables 2 2, 2 3 and 2 4) was tentatively identified as ellagic acid xyloside with its [M H] ion at m/z 433 dissociating to form major ions at m/z 301 via loss of xylose (132 amu ) and minor ions at m/z 284, 257 and 185 indicating presence of ellagic acid 5, 48, 52 The MS spectra of compound 65 (Tables 2 2 and 2 3) gave identical [M H] ion at m/z 447 which dissociated to yield major ion at m/z 301 [M H 146] and minor ions at 257, 229 and 185 corresponding

PAGE 36

36 to loss of rhamnose and characteristic fragmentation pattern of ellagic acid. So this compound was tentatively identified as ellagic acid rhamnoside. Compound 67 (Tables 2 2, 2 3 and 2 4) was identified as free ellagic acid based on characteristic fragmentation pattern, standard and previous literature data 5, 30, 38, 44, 52, 60 Flavonols: The flavonols previously identified in muscadine grapes were glycosides of quercetin, kaempferol and myricetin 5 In this study m yricetin hexoside, kaempferol hexoside, quercetin glucoside and kaempferol rutinoside were identified for the first time (Tables 2 2 and 2 3). Compound 54 (Tables 2 2 and 2 3, Figure 2 2) had i dentical [M H] ion at m/z 593 which fragmented to produce product ions at m/z 447 [M H 146] and at m/z 285 [M H 146 162] indicating loss of rhamnosyl grou p and hexosyl rhamnosyl group, respectively. The fragment ion at m/z 285 corresponded to aglycone of kaempferol, therefore this compound was tentatively identified as kaempferol rutinoside 57 Compound 59 (Table 2 3, Figure 2 2) with deprotonated ion at m/z 479 [M H] gave product ions at m/z 317 [M H 162] and minor ions at m/z 271, 179 and 151, suggesting loss of hexose and producing aglyco ne myricetin. On the basis of the mass spectral data and previously published data 57, 61 this compound was tentatively identified as myricetin hexoside. Compound 62 (Table s 2 2 and 2 3) was tentatively identified as myricetin rhamnoside (464 amu ) based on MS data that produced major fragment of aglycone myricetin ( m /z 317) by losing rhamnose (146 amu ) 5, 39 Compound 64 (Table 2 3, Figure 2 2) had [M H] ion at m/z 463, dissociating to yield fragment ions at m/z 301 [M H 162] and m/z 271, 179 and 151, a characteristic of quercetin fragmentation. This compound was identified as quercetin 3 O glucoside based on mass spectral d ata and standard 30, 61 62 Compound 68 (Tables 2 2, 2 3 and

PAGE 37

37 2 4) was identified as quercetin 3 O rhamnoside based on standard and mass fragmentation which produced m/z 447 [M H] and dissociated to give ions at m/z 301 [M H 146] and m/z 179, 151, corresponding to loss of rhamnose and fragmentation of quercetin, respectively 5, 52, 62 Compound 69 (Tables 2 2 and 2 3, Figure 2 2) gave deproton ated ion at m/z 447 which further fragmented to produce major ion at m/z 285 [M H 162] and minor ions at m/z 255, 227, the compound was tentatively kaempferol hexoside 44, 62 Compound 70 (Tables 2 2 and 2 3) had [M H] ion at m/z 431, which fragmented to yield major ion at m/z 285 [M H 146] in MS 2 Based on mass spectral data and previous study on muscadine grapes 5 this compound was tentatively identified as kaemp ferol rhamnoside. Stilbenes: Compound 50 and 56 (Table 2 2) had [M H] ion at m/z 425, due to formation of chloride adduct. The ion at m/z 425 [M+Cl ] dissociated to yield two product ions one at m/z 389 [M H] and other at m/z 227 [M H 162] corresponding to loss of chloride ion and glucose, respectively. Similarly, Compound 66 (Table 2 3) had [M H] at m/z 389. The MS 2 spectrum of the deprotonated ion at m/z 389 produced product ion at m/z 227 [M H 162] resulting from loss of glucose u nit. Based on mass spectral data and standard, compound 56 was identified as trans resveratrol 3 O glucoside. The other two compounds (compound 50 and 66) having the same mass spectral data were tentatively identified as isomeric forms of resveratrol glu coside 58, 63 Summary Our results indicate that muscadine seeds have high phenolic content and antioxidant capacity compared to skin and pulp. The high antioxidant capacity and total phenolic content of the muscadine seeds makes them potentially significant source of

PAGE 38

38 compounds wit h nutraceutical properties. Additionally, it was confirmed from the results that HPLC ESI MS n operated under both positive and negative ionization, is a valuable tool for the identification of wide array of known phenolic compounds as well as for the preliminary identification of novel compounds. This method allows simultaneous identification of various phenolic compounds (phenolic acids, anthocyanins, flavonols, flavan 3 ols and condensed tannins, hydrolysable tannins, and stilbenes) under similar chr omatographic conditions. The prominent class of phenolic compounds in Noble skin and pulp belong to flavonols compared to seeds in which the majority of compounds belong to hydrolysable and condensed tannins category. The phenolic compounds from the skin a nd seed get extracted into the wine and juice, and are important quality components that contribute to the color and taste of these products. Thus, the structural elucidation of phenolic compounds in muscadine grapes could provide a better understanding of color and flavor changes occurring in muscadine wine and juice upon storage.

PAGE 39

39 Table 2 1. Total p henolic content and a ntioxidant c apacity of s eeds, s kin and p ulp of d ifferent c ultivars of m uscadine g rapes. Cultivar Total Phenolic content (GAE mg/g ) Antioxidant capacity (ORAC, mol TE/g) Seeds Skin Pulp Seeds Skin Pulp Bronze Doreen 45.1 5.3 b 4.5 0.4 cd 1.0 0.0 b 797.9 29.8 c 26.0 5.3 d 4.0 0.3 ab Fry 68.8 5.9 a 4.7 0.3 cd 0.9 0.0 bc 1538.4 41.8 a # 37.9 0.3 bcd 4.0 0.3 ab Carlos 37.4 2.0 bc 10.2 0.6 a 0.8 0.0 c 499.6 8.2 de 43.1 0.6 b 2.4 0.3 c Triumph 40.0 7.7 bc 4.3 0.5 d 0.3 0.0 d 530.8 39.8 d 27.1 3.2 cd 2.3 0.4 c Black Southland 44.4 2.7 b 6.2 0.4 bc 1.2 0.1 a 313.9 2.5 f # 43.9 0.2 b 3.6 0.2 b Magoon 27.0 2.2 c 5.9 0.6 bcd 0.9 0.0 bc 432.2 16.5 e 37.9 1.0 bc 3.4 0.3 b Alachua 81.2 5.8 a 6.1 0.7 bc 0.9 0.0 bc 1105.4 8.8 b 42.1 1.8 b 3.3 0.3 b Noble 36.6 5.6 bc 7.5 0.8 b 0.9 0.0 bc 276.6 18.3 f 77.5 8.1 a 4.6 0.1 a Results are mean standard deviation of three determinations on fresh weight basis. Values with # are in duplicates due to rejection of data points based on Q test. Different superscripts in each column indicate the significant differences in the mean at

PAGE 40

40 Table 2 2. Retention t ime and m ass s pectrometric d ata of p henolic c ompounds in m uscadine g rape p ulp (cv. Noble) determined by HPLC ESI MS n Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compou nd Hydroxycinnamic acid derivatives 1 3.1 342 377 [M+Cl ] [341+36] 341 215 179, 161 113, 101 Caffeic acid hexoside* Hydrolysable tannins 4 5.7 332 331 [M H] 313, 271, 169 125 Monogalloyl glucose* 6 7.2 332 331 [M H] 271, 211, 169 125 Monogalloyl glucose 13 9.4 494 493 [M H] 456, 377, 331 169, 157 Monogalloyl diglucose* Flavan 3 ols 46 20.4 290 289 [M H] 245 227, 205, 179, 137, 109 203 188, 161, 123 Epicatechin# 53 24.2 442 441 [M H] 289 169, 125 245 230, 203, 179, 107 Epicatechin gallate*# Ellagic acid and conjugates 55 25.6 464 463 [M H] 301 284, 257, 229, 217 Ellagic acid hexoside* 63 31.7 434 433 [M H] 301 284 257 Ellagic acid xyloside 65 32.9 448 447 [M H] 301 299, 257 300, 185 Ellagic acid rhamnoside 67 34.0 302 301 [M H] 262, 257 Ellagic acid# Flavonols 54 25.4 594 593 [M H] 535, 447, 285 Kaempferol rutinoside* 62 30.5 464 463 [M H] 405, 317 316, 271, 179 287 271, 215, 179,126 Myricetin rhamnoside 68 35.6 448 447 [M H] 437, 376, 344, 329, 321, 301 271, 255, 228, 191, 179, 167, 151 271, 179 Quercetin 3 O 69 36.0 448 447 [M H] 327, 285 257, 179, 134 Kaempferol hexoside* 70 39.6 432 431 [M H] 285 255, 214, 179, 163 Kaempferol rhamnoside Stilbenes 50 22.9 390 425 [M+Cl ] [389+36] 389 227 Resveratrol glucoside 56 25.7 390 425 [M+Cl ] [389+36] 389 227 Trans resveratrol 3 O glucoside# a Ions in boldface indicate the most intense product ion (100% relative intensity). Compounds with were identified for the first time in muscadine grapes. The compounds with # were identified using pure standards. All other compounds were tentatively iden tified based on mass data.

PAGE 41

41 Table 2 3. Retention t ime and m ass s pectrometric d ata of p henolic c ompounds in m uscadine g rape s kin (cv. Noble) determined by HPLC ESI MS n Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compound Hydroxycinnamic acid derivatives 1 3.1 342 377 [M+Cl ] [341+36] 341 215, 179 179 161, 143, 131, 125, 101 Caffeic acid hexoside* Hydrolysable tannins 3 4.2 482 481 [M H] 421, 301 258, 201, 185 175 HHDP glucose* 6 7.3 332 331 [M H] 271, 211, 169 125 125 Monogalloyl glucose* 11 8.8 332 331 [M H] 169 125 125 Monogalloyl glucose 15 9.6 634 633 [M H] 613, 481, 301 275, 250, 230, 178 300, 257 HHDP galloyl glucose 22 12.9 626 625 [M H] 623, 481, 463 320, 301, 239, 193 355, 319, 301 275, 257, 239, 215, 193, 175,164, 147 HHDP diglucoside 47 21.0 814 813 [M H] 781, 763 753, 745, 735, 725, 511, 301, 257 Ellagitannin 51 23.5 832 831 [M H] 813 795, 787, 769, 752, 741, 723, 707, 697, 680, 664, 611, 578, 451, 365, 301, 291, 254 Ellagitannin 52 23.8 818 817 [M H] 773 755, 729, 712, 701, 685, 673, 667, 655, 655, 647, 621, 617, 541, 503, 371, 237 729 712, 701, 685, 655, 617, 577, 531, 465, 407, 301, 237 Ellagitannin Flavan 3 ols 18 11.8 306 305 [M H] 285, 263, 247, 219 198, 179, 165, 151, 137, 125 Gallocatechin#

PAGE 42

42 Table 2 3. contd Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compound Anthocyanins 27 14.7 627 627 [M] + 465, 303 285, 257 229, 149 Delphinidin 3,5 diglucoside 29 16.1 611 611 [M] + 449, 287 269, 241, 213, 189 167, 149, 137, 109 Cyanidin 3,5 diglucoside 33 17.1 641 641 [M] + 479, 317 302 274, 218 Petunidin 3,5 diglucoside 38 18.2 595 595 [M] + 433, 271 225 215, 197, 187, 169, 141, 131, 121 Pelargonidin 3,5 diglucoside 39 18.2 625 625 [M] + 463, 301 286 Peonidin 3,5 diglucoside 44 20.0 655 655 [M] + 493, 331 315 299, 287, 270, 243, 179 Malvidin 3,5 diglucoside Ellagic acid and conjugates 55 25.6 464 463 [M H] 301 300 284, 257, 157 Ellagic acid hexoside* 63 31.8 434 433 [M H] 301 300, 257, 245 229, 188, 145 Ellagic acid xyloside 65 32.9 448 447 [M H] 301 300, 257, 229, 216 160 Ellagic acid rhamnoside 67 34.0 302 301 [M H] 284, 257 229, 201, 173 Ellagic acid# Flavonols 54 25.4 594 593 [M H] 534, 431, 333, 285 211 Kaempferol rutinoside* 59 28.2 480 479 [M H] 359, 317 270, 179 287, 271 259, 227, 179, 151, 125, 109 Myricetin hexoside* 62 30.4 464 463 [M H] 317 271, 179 287, 271, 242, 193, 179 151, 137 Myricetin rhamnoside 64 32.4 464 463 [M H] 301 151 299, 271, 255, 230, 212, 179 151, 121 Quercetin 3 O glucoside*# 68 35.6 448 447 [M H] 301 271, 255, 226, 193, 179, 151 Quercetin 3 O rhamnoside#

PAGE 43

43 Table 2 3. contd Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compound 69 36.0 448 447 [M H] 285 255, 227 267, 255 239, 227, 199, 169, 135 Kaempferol hexoside* 70 39.6 432 431 [M H] 285 267, 255 241, 229, 213, 195, 187, 174 Kaempferol rhamnoside Stilbenes 66 33.3 390 389 [M H] 227 185 157, 143 R es veratrol glucoside a Ions in boldface indicate the most intense product ion (100% relative intensity). Compounds with were identified for the first time in muscadine grapes. The compounds with # were identified using pure standards. All other compounds were tentatively iden tified based on mass data

PAGE 44

44 Table 2 4. Retention t ime and m ass s pectrometric d ata of p henolic c ompounds in m uscadine g rape s eed (cv. Noble) determined by HPLC ESI MS n Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compound Hydroxycinnamic acid derivatives 1 3.0 342 377 [M+Cl ] [341+36] 341 215, 179, 161 179 143, 131, 119, 113 Caffeic acid hexoside* Hydroxybenzoic acid 8 7.8 170 169 [M H] 125 Gallic acid# Hydrolysable tannins 2 3.6 482 481 [M H] 301 169 147 HHDP glucose* 5 6.7 332 331 [M H] 271, 241, 211, 169 125 125 Monogalloyl glucose* 7 7.4 484 483 [M H] 331 313, 169 Digalloylglucose 9 8.3 332 331 [M H] 271, 169 125 125 Monogalloyl glucose 10 8.6 484 483 [M H] 331 211, 169 169 Digalloylglucose 12 8.8 634 633 [M H] 481 301 185 Galloyl HHDP glucose 13 9.2 494 493 [M H] 331 169 169 Monogalloyl diglucose* 14 9.2 634 633 [M H] 593, 481, 301 284, 229 HHDP galloyl glucose 16 11.2 484 483 [M H] 331 313, 169 271, 241, 211, 169 Digalloylglucose 17 11.2 634 633 [M H] 613, 481, 301 185 257, 185 HHDP galloyl glucose 19 12.1 332 331 [M H] 169 125 125 Monogalloyl glucose 20 12.7 634 633 [M H] 615, 481, 421, 301 229, 185 300, 257, 201, 187 HHDP galloyl glucose 21 12.7 786 785 [M H] 748, 633 615, 483, 331, 301, 275 HHDP digalloyl glucose* 23 13.2 634 633 [M H] 613, 572, 483, 301 275, 257, 228, 201 257 HHDP galloyl glucose 24 13.2 786 785 M H] 765, 633 301, 275, 231, 223 HHDP digalloyl glucose 25 13.7 636 635 [M H] 613, 483 331, 211 313 207, 169 Trigalloyl glucose* 26 14.2 636 635 [M H] 614, 483 301, 229 331, 169 Trigalloylglucose 28 14.8 634 633 [M H] 613, 566, 483, 301 284, 257, 229, 185 HHDP galloyl glucose

PAGE 45

45 Table 2 4. contd Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compound 30 16.2 634 633 [M H] 465, 301 257 229 HHDP galloyl glucose 31 16.2 786 785 [M H] 633 543, 483, 301 482, 301 275 HHDP digalloyl glucose 32 16.5 636 635 [M H] 597, 483 465, 420, 313, 193 424, 331, 313, 169 Trigalloylglucose 35 17.3 636 635 [M H] 599, 483 423, 406, 332, 235, 194 405, 331 313, 271, 211, 169 Trigalloylglucose 37 18.1 786 785 [M H] 768, 633 615, 596, 465, 419, 301, 285 313, 301 275, 214 HHDP digalloyl glucose 40 19.4 788 787 [M H] 635 483 617 483, 424, 313, 211, 169 Tetragalloyl glucose* 41 19.4 636 635 [M H] 617, 545, 483, 465 314, 213 313, 249, 169 Trigalloylglucose 42 19.8 786 785 [M H] 765, 633 483, 423, 301, 276, 241 301 284, 275 HHDP digalloyl glucose 43 19.8 636 635 [M H] 617, 483, 466, 423 405, 271, 211, 193 271 251, 235, 211, 193, 179, 169 Trigalloylglucose 45 20.4 786 785 [M H] 765, 633 615, 482, 393, 301 483, 447, 301 187 HHDP digalloyl glucose 48 22.5 788 787 [M H] 635, 617 573, 403, 325 573, 529, 465 404, 313, 211, 197 Tetragalloyl glucose 49 23.1 788 787 [M H] 635 617, 483, 465, 447 617, 483 465, 423, 357, 331, 313, 271, 253, 235, 212, 193 Tetragalloyl glucose 57 26.2 940 939 [M H] 787, 769 617 725, 617 601, 573, 465, 431, 387, 295, 260 Pentagalloyl glucose* 58 27.3 940 939 [M H] 787 769, 635 617, 483, 465, 447, 277 Pentagalloyl glucose 61 29.6 1092 1091[M H] 939 787 787, 769 617 Hexagalloyl glucose*

PAGE 46

46 Table 2 4. contd Compound number Retention time t R (min) Molecular weight MS 1 (m/z) MS 2 (m/z) a MS 3 (m/z) a Identified compound Flavan 3 ols and Condensed Tannins 34 17.3 730 729 [M H] 641, 577 407, 299, 211 411, 289 Galloyl procyanidin dimer* 36 17.6 578 577 [M H] 559, 515, 425 407, 289, 228, 161 407 299, 257 Procyanidin dimer 46 20.4 290 289 [M H] 245 205, 188, 179, 165, 137, 126, 110 227, 203 191, 161, 123 Epicatechin# 53 24.2 442 441 [M H] 332, 289 169, 125 271, 245 203, 165, 143 Epicatechin gallate*# 60 28.6 594 593 [M H] 441 321, 289, 169 397, 332, 289 169 (Epi)catechin digallate* Ellagic acid and conjugates 63 31.6 434 433 [M H] 301 300 259, 228, 213, 201, 185 Ellagic acid xyloside 67 34.0 302 301 [M H] 284, 257 229, 185 Ellagic acid# Flavonols 68 35.6 448 447 [M H] 301 273, 151 271, 255, 179 164, 151, 121, 107 Quercetin 3 O rhamnoside# a Ions in boldface indicate the most intense product ion (100% relative intensity). Compounds with were identified for the first time in muscadine grapes. The compounds with # were identified using pure standards. All other compounds were tentatively iden tified based on mass data

PAGE 47

47 Figure 2 1 Proposed structures of phenolic compounds in muscadine grapes.

PAGE 48

48 Figure 2 2. Negative ion electrospray product ion mass spectra (MS 2 and MS 3 ) of phenolic compounds identified for the first time in muscadine (cv. Noble) pulp, skin and seeds. The numbers in bold in each product ion spectra corresponds to the compound number in tables.

PAGE 49

49 Figure 2 3. Negative ion electrospray product ion mass spectra (MS 2 and MS 3 ) of phenolic compounds identified for the first time in muscadine (cv. Noble) seeds. The numbers in bold in each product ion spectra corresponds to the compound number in tables. C ompound numbers with also identified in pulp.

PAGE 50

50 Figure 2 4. HPLC DAD chromatograms of muscadine (cv. Noble) p ulp: (A) 280 nm and (B) 360 nm. Peak with label U was unidentified. Peak numbers correspond to compound numbers in table 2 2.

PAGE 51

51 Figure 2 5 HPLC DAD chromatograms of muscadine (cv. Noble) skin: (A) 280 nm; (B) 360 nm and (C) 520 nm. Peak having two numbe rs indicate coeluting compound s Peak numbers correspond to compound numbers in table 2 3.

PAGE 52

52 Figure 2 6 HPLC DAD chromatograms of muscadine (cv. Noble) seed: (A) 280 nm and (B) 360 nm. Peaks with label U were unidentified and p eaks having two numbers indicate coeluting compounds Peak numbers correspond to compound numbers in table 2 4.

PAGE 53

53 CHAPTER 3 EFFECTS OF EXOGENOUS ABSCISIC ACID ON ANTIOXIDANT CAPACITIES, ANTHOCYANINS AND FLAVONOL CONTENTS OF MUSCADINE GRAPE SKINS Background Phenolic compound s are secondary metabolites produced by plants as a defense mechanism against various biotic and abiotic stresses. They are categorized into different classes depending upon their structures varying from simple phenolic acids (hydroxybenzoic acid and hydro xycinnamic acid) to complex polyphenols (hydroly s able and condensed tannins) 20 21 Phenolic compounds play an important role in color and sensory characteristics of fruits and vegetables. The protective effects of these compounds against various chronic diseases such as cancer and cardiovascular diseases are well recognized among researchers and health conscious consumers 22 23 Muscadine grapes ( Vitis rotundifolia ) are commonly grown in the southeastern United States and are well adapted to warm, humid climates, which are un suitable for the growth of Vitis vinifera They are either light skinned (green or bronze) or dark skinned (red to almost black) and are 2.5 3.8 cm in diameter with thick skin. They grow in tight small clusters of 3 to 10 berries and are marketed in fresh and processed forms such as juice, wine, and jam. Muscadine grapes possess unique phytochemical composition that differentiates them from other Vitis species such as presence of ellagic acid and its derivatives, and anthocyanin 3, 5 diglucosides 5, 28 In addition, they also contain hydroxybenzoic acid s, resveratrol, quercetin, myricetin, and kaempferol 1, 27 Cell culture studies have suggested that polyphenols from muscadine grapes can inhibit proliferation of colon cancer cells and induce apoptosis 13 14 The concentration of phenolic compounds in fruits and vegetables is regulated by genetic, environmental, physiological and chemical factors such as temperature, light,

PAGE 54

54 rainfall, soil, chemicals and plan t growth regulators. Various agronomic strategies such as alteration of environmental conditions, water management, grafting of plants, application of elicitors, stimulating agents and plant activators have been employed to enhance the biosynthesis of phen olic compounds in fruits and vegetables 64 One such strategy is the pre harvest application of abscisic acid, a plant growth regulator involved in various physiological processes including color development 65 Endogenous abscisic acid (ABA) plays an important role in plant growth. For example, ABA promotes seed maturation and germination, and serves as a signaling compound when plants are under stresses such as drought, high salinity, cold and microbial infections 66 I n addition, ABA also participates in the initiation of ripening and related changes in grape development 67 69 It has been demonstrated that exogenous application of ABA increases the anthocyanins in grape skins 70 72 and can improve the color and q uality of the grapes 65, 73 Lacampagne e t al., 74 reported that ABA regulates enzymes involved in tannin biosynthesis and thus elevates tannin content of green grapes at veraison. These studies suggest that ABA plays a significant role in triggering the flavonoid biosynthetic pathway. Most studies investigating the effect of exogenous application of ABA have been conducted in Vitis vinifera grapes and their hybrids. However, the effects of ABA in muscadine grapes ( Vitis rotundifolia ) have not been investigated. In this study, we hypothesized that exogenous ABA treatment will affect the anthocyanin accumulation, phenolic content and composition of red muscadi ne grapes.

PAGE 55

55 Materials and M ethods Chemicals azobis(2 amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI). Gallic acid, 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid (Trolox), HPLC grade methanol, acetic acid, formic acid, hydrochloric acid, Folin Ciocalteau reag ent, Flourescein, Tween 20, and sodium carbonate were purchased from Fischer Scientific Co. (Pittsburg, PA). Standards of the 3 O glucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin (six mixed anthocyanin standard, HPLC grade), were purchased from Polyphenols Laboratories (Sandnes, Norway). Ellagic acid, myricetin, quercetin and kaempferol were obtained from Sigma Aldrich (St. Louis, MO). S abscisic acid was received as a gift from Valent BioSciences Corporation (Libertyv ille, IL). ABA T reatment ABA treatments were conducted in 2009 on two cultivars of muscadine grapes, Alachua and Noble. Alachua is a red table grape with large berry size, whereas Noble is a red wine gr ape with much smaller berries. ABA treatment on Alach ua grapes was carried out in an experimental vineyard at the Mid Florida Research and Education Center, Apopka, Florida. For Noble grapes, ABA treatment was carried out in an experimental vineyard at the Center for Viticulture and Small Fruit Research, Flo rida A&M University, Tallahassee, Florida. The grapevines of the same age, size, and growth conditions were selected and assigned to receive one of the two treatments: control (water only) and 300 ppm of ABA. Tween 20 was used as a wetting agent in both th e control and 300 ppm ABA solutions a t a concentration of 315 L/L. The s pray aimed directly on the grapes with a hand held sprayer until run off. For Noble variety 9

PAGE 56

56 vines were used each for ABA treatment and control, however for Alachua 13 vines were use d for control and 12 vines for ABA treatment. ABA application dates were chosen to avoid rainfall and wind. The f irst ABA spray was done at veraison (the onset of ri pening 20% grapes with red color ) on both the varieties. The application dates were July 2 7th for Alachua and August 8th for Noble. The second spray treatment was done 13 and 8 days post veraison (60 % of grapes developed red color) for Alachua and Noble, respectively. Two hours before the second spray, 60 grapes were randomly sampled from each vine. Ripe grapes were harvested for second sampling i. e. 10 and 8 days after the second spray for Noble and Alachua, respectively. Fruit W eight, pH and T otal S oluble S olids The grapes were weighed after each sampling and average berry weight was record ed. During separation, juice was collected, filtered, and analyzed for pH and total soluble solids ( Brix). pH was measured using a pH meter and total soluble solids ( Brix) were measured using a bench top refractometer (Leica Abbe Mark 11, Fisher Scientific, Pittsburg, PA). Extraction and Sample P reparation The skin of the grapes was manually separated from seeds and pulp, and freeze dried. Subsequently, it was ground into a fine powder using a Waring kitchen blender and extracted (1 g) with 15 mL of methanol/water/acetic acid (85:15:0.5; v/v) in glass tubes. The samples were then vortexed for 30 s, sonicated for 5 min and kept in the dark at room temperature for 20 min The tubes were then centrifuged at 1317 g for 10 min and the supernatant was removed. The samples were extracted again with 10 mL of methanol/water/acetic acid using the same procedure. The supernatants from two

PAGE 57

57 extractions were pooled and transferred in to a 25 mL volumetric flask. Methanol/water/acetic acid was added to make up the final volume to 25 mL. Folin Ciocalteu Assay The extracts were diluted to appropriate concentration for analysis. The total phenolic content was determined as reported previ ously 6 .The results were expressed as milligrams of gallic acid equ ivalents per gram of fresh grape skins (mg of GAE/ g). Oxygen Radical Absorbance Capacity (ORAC) The ORAC assay for extracted samples was conducted on a Spectra XMS sa mples were added to the designated wells of a 96 well black plate. This was followed The mixture was incubated at 37 C for excitation and 530 nm emissions at 1 min intervals for 40 min. Trolox was used to generate a standard curve. The antioxidant capacities of extracts were expressed as DPPH Assay The DPPH scavenging activities of samples were measured using a previously published method 75 In summary, DPPH stock solution was prepared by dissolving 20 mg of DPPH in 10 0 mL methanol and stored at 20 C prior to use. DPPH working solution was freshly prepared by mixing 2.8 mL DPPH stock sol ution and 7.2 mL methanol. Absorbance at 515 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA). Diluted extracts (50 L) were added to 950 L DPPH working solution and incubated for 60 min in the dark at room tempera ture. Trolox solutions from 100 to 1000 M were added to DPPH working

PAGE 58

58 solution as standards. Results of the DPPH scavenging activity of grape skin extracts were expressed as micromoles of Trolox equivalents (TE) per gram of fresh grape skins HPLC Analysis of Phenolic C ompounds Chromatographic analysis was performed on an Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector and a fluorescent detector (Agilent Technologies, Palo Al to, CA). Reversed phase chromatography was used for the quantification of anthocyanins, ellagic acid and flavonols. An Agilent Zorbax Stablebond SB particle size, Agilent Technologies, Palo Alto, CA) was used for separatio n of phenolic compounds. Anthocyanins The extracts (1 mL) were filtered through 0.45 m filter units and 5 L injected directly without any purification. Elution was performed using mobile phase A (5% formic acid aqueous solution) and mobile phase B (meth anol). The flow rate was 1 10 min, 5 15% B; 10 25 min, 15 25% B; 25 30 min, 25 30% B; 30 45 min, 30% B; 45 47 min, 30 70% B; 47 50 min, 70 5% B; followed by 5 min of re equilibration of the column befor e the next run. UV vis spectra were scanned from 220 to 600 nm on a diode array detector and the detection wavelength for the anthocyanins was 520 nm. Ellagic a cid and f lavonols Acid hydrolysis of the samples was done before HPLC analysis. Extract (5 mL) was concentrated in a Speed Vac Concentrator (Thermo scientific ISS110, Waltham, M A) under reduced pressure at 25 C to remove the solvent. The solids were dissolved

PAGE 59

59 in 5 mL of methanol (50%) containing 1.2 N HCl and sonicated for 5 min. Hydrolysis was con ducted in a precision water bath (Thermoscientific Waltham, MA) for 80 min at 90 C and aglycones were separated and quantified using HPLC. The binary mobile phase consisted of (A) 0.5% aqueous formic acid solution and (B) acetonitrile. The flow rate was 1 mL/min and 25 min gradient was used. The gradient is described as follows: 30% B; 5 10 min, 30 40% B; 10 20 min, 40 50% B; 20 23 min, 50 70% B; 23 25 min, 70 10% B; followed by 5 min of re equilibration of the column before the next run. The c olumn t emperature was maintained at 30 C. UV vis spectra were scanned from 220 to 600 nm on a diode array detector and the detection wavelength for the ellagic acid and flavonols was 360 nm. Statistical and Multivariate Analysis Data were expressed as mea test was performed for the comparison of the means between control and treated groups, and paired comparison T test was done between the same treatment group at two different sampling times using JMP software (Version 8. 0, SAS Institute Inc., Cary, NC). P values less than 0.05 were considered statistically significant. Principal component analysis (PCA) was done to examine the grouping of samples, outliers and to visualize the relative distribution of the control and trea ted samples. PCA was done with the JMP software using the content of individual anthocyanins, ellagic acid and flavonols as variables without data transformation or normalization Results Fruit W eight, pH and T otal S oluble S olids ABA treatment had no effect on the average berry weight in both cultivars. Sampling time also had no effect on the average berry weight within ABA treated Noble,

PAGE 60

60 however, a significant decrease was observed in weight of control group of Noble grapes at sec ond sampling (Table 3 1). In case of Alachua, sampling time significantly decreased the average berry weight in control and increased the weight in ABA treated grapes Application of ABA did not affect the fruit juice composition of either cultivar No sig nificant differences were observed in soluble solid content ( Brix) and pH of the control and ABA treated juice. Similar observations were reported on Vitis vinifera 65, 71, 73 There was a significant increase in the soluble solids content and pH of control and ABA treated Alachua juice at second sampling. A significant increase in the soluble solid content of ABA treated Noble juice was also observed at second sampling (Table 3 1). Total P henolic C ontent and A ntioxidant C apacities A significant increase in the antioxidant capacities determined by ORAC and DPPH assays in ABA treated Noble was observed at both sampling times, ho wever, effect of ABA treatment on total phenolic content of Noble was observed at first sampling only (Table 3 2). Antioxidant capacity (ORAC) was enhanced by 38% and 18% in treated Noble samples compared to controls at first and second sampling, respectiv ely. Antioxidant capacity determined by DPPH assay also showed a similar effect, however the values were lower than ORAC assay. This is likely caused by the differences in the reaction mechanism of these two methods. ORAC assay applies a competitive reacti on scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds, whereas DPPH assay measures the capacity of an antioxidant in the reduction of an oxidant, which changes color when r educed 76 Overall, the total phenolic content was 30% higher in treated samples than controls after the first sampling. No effects of ABA

PAGE 61

61 treatment w ere seen on the Alachua. A time dependent significant increase in total phenolic content and antioxidant capacities in control group of Noble was also observed. There was a significant increase in the total phenolic content and antioxidant capacities of co ntrol and ABA treated Alachua at second sampling (Table 3 2). HPLC A nalysis of A nthocyanins and O ther P henolic C ompounds The effect of ABA treatment on the individual anthocyanins (delphinidin cyanidin petunidin, peonidin, and malvidin 3, 5 diglucoside) is reported in Table 3 3. ABA treated Noble showed an increase in the concentration of individual anthocyanins throughout the ripening period. The content of delphinidin, cyanidin, and peonidin 3, 5 diglucoside was 50% higher in ABA treated grapes compare d to controls at first sampling. Among the anthocyanins, cyanidin 3, 5 diglucos ide showed highest increase (63 %) in ABA treated grapes compared to controls at first sampling. The concentration remained constant at second sampling. Over all, total anthocyani ns were 51% and 39 % higher in ABA treated Noble compared to controls at first and second sampling, respectively. ABA treatment did not affect the content of individual anthocyanins in Alachua at the first sampling; however the level of peonidin 3, 5 diglu coside was increased by 20% in ABA treated grapes compared to controls at second sampling. Time related increase in the content of anthocyanins of control and ABA treated grapes was observed in both cultivars, however, this effect was more pronounced in Al achua. ABA treated Noble grapes had higher concentrations of ellagic acid and flavonols (myricetin, quercetin and kaempferol) compared to controls at first sampling (Table 3 4). Ellagic acid content was enhanced by 47% in treated samples compared to contr ols. Similarly the levels of myricetin, quercetin and kaempferol were enhanced by 54%, 45% and 48%, respectively, in treated Noble grapes compared to controls. Even

PAGE 62

62 though the effect of ABA treatment on content of ellagic acid and flavonols was not statist ically significant at the second sampling, the trend showed an increase in their content. A comparison of control groups from first and second sampling in Noble showed a significant increase in the ellagic acid and kaempferol content. No effect of ABA trea tment was seen on the ellagic acid and flavonol content of Alachua grapes. However, the effect of sampling time showed a significant increase in the levels of ellagic acid, myricetin and quercetin in both control and ABA treated Alachua. Principal Compone nt Analysis (PCA) The score plots of the first two principal components (PC1 and PC2) for the control and ABA treated muscadine grapes are shown in Figure 3 1. Generally, samples with similar characteristics cluster but samples with different characterist ics segregate on score plots. The first three components accounted for 92% of variance in the control and ABA treated Noble samples, where PC1 explains 60%, PC2 26% and PC3 7% of the variance, respectively. Grouping of control samples on left side of the p lot and ABA treated samples on the right side was observed (Figure 3 are from the same grapevines but sampled at two different times A change in position of these samples on the s core plot showed the effect of time on the phenolic compounds As the ripening progresses the phenolic content of grapes changes which is depicted in the labeled samples. Contrary to Noble, the score plots of Alachua grapes showed no segregation between th e control and ABA treated samples (Figure 3 1B). The contribution of individual phenolic compounds to overall variance in Noble grapes was visualized on the loading plot of PCA. Anthocyanins and flavonols appeared as two clusters on the loading plot. Ellag ic acid influenced the variance of Noble grapes similar to flavonols (Figure 3 2).

PAGE 63

63 Discussion Exogenous ABA has been investigated as a novel strategy to improve the quality of grapes. A few studies have examined the effects of ABA on anthocyanin metaboli sm and maturity parameters in Vitis vinifera grapes and hybrids 65, 70 71, 73, 77 However, the effects of exogenous ABA o n muscadine grapes ( Vitis rotundifolia ) have not been reported. In this study, we investigated the effect of ABA on enhancement of anthocyanins and other phenolic compounds in muscadine grape skin. The concentration of ABA chosen for treatment was based on our previous experiment on lettuce 78 Similar concentration of ABA has been applied on cabernet sauvignon grapes 79 No effect of sampling time was seen on berry weight and pH in Noble, which indicates that grapes reached their m aximum weight and acidity before the second spray. However, a significant increase in the total soluble solids content of treated Noble was observed with time, suggesting that ABA may positively affect sugar biosynthesis in ripening grapes. Grape berry wei ght decreased in control Alachua and increased in treated Alachua grapes at two different sampling times. Increase in total soluble solids and pH was observed in Alachua juice at two sampling times. These results suggest that the physiological changes, suc h as berry expansion and sugar accumulation, occur much faster in Noble than Alachua. The weight of control Noble and Alachua grapes decreased at second sampling. Although the reason for the weight loss is not known, we speculate that it may have been caus ed by dehydration of the grapes at ripening. PCA illustrated the patterns found in the data and revealed the relationships between variables. The score plot of Noble showed clustering of control and ABA treated samples with some overlapping. The overlapp ing of the clusters could be due to large variation in the samples. PC1 variable appears to be strongly associated with the

PAGE 64

64 grouping of control and ABA treated samples and also with the progression of ripening. In contrast, control and ABA treated Alachua samples were randomly scattered on the score plot without any grouping, suggesting no effect of ABA treatment. The loading plot provides a relationship among the variables and how much each variable contributes to each PC. Variables plotted in the same dir ection from the center are positively correlated, while those on opposite sides are negatively correlated. Loading plot of Noble showed positive correlation in variables 1, 2, 3, 4 and 5 (delphinidin, cyanidin, petunidin, peonidin, and malvidin 3, 5 digluc oside) through PC1. Variables 6, 7, 8 and 9 (ellagic acid, myricetin, quercetin and kaempferol) were positively correlated and contributed equally to PC1 and PC2. Simultaneous interpretation of score and loading plots reveal that samples on right side of t he score plot correspond to the direction of variables (anthocyanins, flavonols and ellagic acid) in the loading plot, suggesting higher values of these variables in the respective samples. This could be explained in terms of ABA treatment effect, as group ing of ABA treated samples, which have higher content of phenolic compounds, is observed on the right side of the score plot. ABA treatment stimulated the accumulation of anthocyanins and total phenolics, as well as enhanced the antioxidant capacities in N oble. This effect paralleled the increase in individual anthocyanins, flavonols and ellagic acid in ABA treated grapes compared to controls. Based on these observations it could be stated that ABA triggers the secondary metabolism of grapes including antho cyanin biosynthesis. Anthocyanins, flavonols and ellagic acid are products of secondary metabolism in plants and generated by common phenylpropanoid pathway 80 Although these three phen olic compounds share a common biosynthetic pathway, specific enzymes are involved in

PAGE 65

65 their synthesis, which may respond differently to exogenous ABA. Anthocyanins are synthesized from phenylalanine through an anthocyanin biosynthetic pathway regulated by e nzyme activities 81 and gene expression 82 A few examples of anthocyanin biosynthe tic pathway genes are phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3 hydroxylase ( F3H), dihydroflavonol 4 reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose: flavonoid 3 O glucosyltran sferase (UFGT) The key enzyme involved in the biosynthesis of flavonols is flavonol synthase (FLS). Ellagic acid is an upstream product of phenylpropanoid pathway and is generated by the hydrolysis of ellagitannins 83 Previous studies on grapes have reported positive impact of exogenous ABA on color development and anthocyanin biosynthesis 65, 70 73 The improved accumulation was correlated with an induced expression of a U FGT gene coding for an enzyme specific to the anthocyanin pathway and other genes coding upstream located enzymes such as P AL, CHI, CHS, DFR, F3H and LDOX 70 71 ABA treatment enhanced the content of flavonols and ellagic acid at first sampling but there was little effect at ripening. Fujita et al., 84 reported an increase in the transcript levels of two flavonol synthase genes and enhancement of quercetin content in the skin of Merlot grapes after veraison. Second application of ABA di d not change the levels of flavonols and ellagic acid however increase in the levels of anthocyanins was observed. One possibility could be that accumulation of flavonols occurs in the early stages of development and at ripening but accumulation of anthoc yanins begins at ripening and continues until grapes are fully ripe 84

PAGE 66

66 The increase in antioxidant capacities determin ed by ORAC and DPPH assays were consistent with the increase in total phenolic content and total anthocyanins of treated N oble grapes. However, total phenolic content in ABA treated grapes was similar to control at second sampling. This was probably caused by the differences in the chemistry and scope of compounds that were measured using these methods. Contrary to the results in Noble, no effect of ABA treatment was seen in Alachua. This may be due to variation in genetic make up of two cultivars, thus le adi ng to different ABA responses. The two vineyards used for these trials had similar weather, viticultural practices, and soil conditions. Minor differences in the environmental conditions were less likely to be the major factors that caused different ABA responses in two cultivars. The mechanism by which ABA gets adsorbed into the grape is not well understood. There are at least two possible hypotheses: either sprayed ABA penetrates through the skin, accumulates inside the grape and enhances the generati on of phenolic compounds and ripening related changes or exogenous ABA acts as a signaling agent that triggers the synthesis of endogenous ABA 69 As small grapes have larger skin sur face for the same weight, we speculate that they will adsorb ABA more efficiently than larger grapes and hence be more susceptible to ABA treatment. This may, in part, explain the different effects of ABA on Alachua and Noble grapes. Summary In concl usion, exogenous ABA enhance d the antioxidant capacity, anthocyanins and phenolic content of muscadine grapes but these effects varied depending upon the cultivar and were possibly influenced by other environmental factors since the grape varieties were gr own in different locations ABA is ubiquitous in nature and synthesized

PAGE 67

67 by all higher plants. Once production of S ABA for agricultural use becomes cost effective, this plant growth regulator will be a promising tool for the enhancement of phenolic compoun ds in fruits and vegetables. ABA applied at critical stages of grape development offers opportunities to increase the content of key phytochemicals without affecting the yield. The grapes with enhanced phytochemicals could attract health conscious consumer s and also increase the marketability of fresh fruits.

PAGE 68

68 Table 3 1. Average berry weight, total soluble solids ( Brix) and pH of juice from control and ABA treated muscadine grapes. Variety Sampling time Treatment Average berry weight (g) Total soluble solids ( Brix) pH Noble First sampling Control (n=9) 3.81 0.58 9.17 0.59 3.12 0.10 300 ppm ABA (n=9) 3.25 0.58 8.89 0.72 3.10 0.07 Second sampling Control (n=9) 3.09 0.19 a 9.44 1.64 3.05 0.09 300 ppm ABA (n=9) 2.96 0.28 9.86 1.26 a 3.10 0.08 Alachua First sampling Control (n=13) 7.16 0.44 10.81 1.31 3.24 0.06 300 ppm ABA (n=12) 7.17 0.63 10.76 1.48 3.24 0.05 Second sampling Control (n=13) 7.09 0.66 a 15.40 0.72 a 3.31 0.06 a 300 ppm ABA (n=12) 7.48 0.90 a 15.53 0.57 a 3.38 0.09 a Results are mean standard deviation on fresh weight basis. a indicates significant differences (p < 0.05, Paired comparison t test) between corresponding values during first sampling.

PAGE 69

69 Table 3 2. Total phenolic content and antioxidant capacities (ORAC and DPPH) of c ontrol and ABA treated muscadine grape skins. Variety Sampling time Treatment Total phenolics (GAE mg/g) ORAC DPPH Noble First sampling Control (n=9) 3.72 0.56 54.68 8.43 20.49 5.14 300 ppm ABA (n=9) 4.83 1.04 b 75.61 13.34 b 25.15 4.52 b Second sampling Control (n=9) 4.44 0.73 a 67.97 12.01 a 21.71 2.90 300 ppm ABA (n=9) 5.04 0.66 79.94 9.00 b 24.64 2.01 b Alachua First sampling Control (n=13) 3.76 0.64 55.27 10.52 21.51 4.67 300 ppm ABA (n=12) 3.59 0.94 52.02 12.51 21.43 4.52 Second sampling Control (n=13) 4.52 0.52 a 65.38 8.73 a 26.60 3.40 a 300 ppm ABA (n=12) 4.84 0.77 a 72.86 13.22 a 25.07 3.29 a Results are mean standard deviation on fresh weight basis. a indicates significant differences ( p < 0.05, Paired comparison t test) between corresponding values during first sampling. b Values are significantly different than control ( p test).

PAGE 70

70 Table 3 3. Anthocyanin content of control and ABA treated muscadine grape skins. Variety Sampling time Treatment Delphinidin 3, 5 diglucoside (mg/g) Cyanidin 3, 5 diglucoside (mg/g) Petunidin 3, 5 diglucoside (mg/g) Peonidin 3, 5 diglucoside (mg/g) Malvidin 3, 5 diglucoside (mg/g) Total Anthocyanins (mg/g) Noble First sampling Control (n=9) 0.41 0.17 0.08 0.03 0.17 0.05 0.10 0.02 0.06 0.02 0.82 0.27 300 ppm ABA (n=9) 0.63 0.21 b 0.13 0.06 b 0.24 0.07 b 0.16 0.05 b 0.08 0.02 b 1.24 0.40 b Second sampling Control (n=9) 0.47 0.13 0.08 0.02 0.21 0.05 a 0.13 0.02 a 0.08 0.02 a 0.97 0.23 a 300 ppm ABA (n=9) 0.65 0.15 b 0.13 0.05 b 0.27 0.06 b 0.19 0.05 b 0.11 0.03 b a 1.35 0.32 b Alachua First sampling Control (n=13) 0.92 0.20 0.07 0.02 0.18 0.04 0.04 0.01 0.04 0.01 1.25 0.28 300 ppm ABA (n=12) 0.82 0.30 0.06 0.03 0.16 0.05 0.04 0.01 0.05 0.01 1.13 0.40 Second sampling Control (n=13) 1.27 0.14 a 0.10 0.02 a 0.24 0.03 a 0.05 0.01 a 0.05 0.01 a 1.71 0.20 a 300 ppm ABA (n=12) 1.29 0.22 a 0.12 0.03 a 0.25 0.04 a 0.06 0.02 a b 0.06 0.01 a 1.78 0.32 a Results are mean standard deviation on fresh weight basis. a indicates significant differences ( p < 0.05, Paired comparison t test) between corresponding values during first sampling. b Values are significantly different than control ( p < test).

PAGE 71

71 Table 3 4. Ellagic acid, myricetin, quercetin and kaempferol content of control and ABA treated muscadine grape skins. Variety Sampling time Treatment Ellagic acid (mg/g) Myricetin (mg/g) Quercetin (mg/g) Kaempferol (mg/g) Noble First sampling Control (n=9) 2.67 0.73 0.28 0.07 0.23 0.08 0.11 0.03 300 ppm ABA (n=9) 3.92 0.87 b 0.44 0.10 b 0.34 0.06 b 0.16 0.04 b Second sampling Control (n=9) 3.46 1.32 a 0.36 0.10 0.27 0.08 0.14 0.05 a 300 ppm ABA (n=9) 4.31 1.31 0.38 0.08 0.34 0.08 0.18 0.05 Alachua First sampling Control (n=13) 1.56 0.28 0.21 0.05 0.19 0.05 0.08 0.02 300 ppm ABA (n=12) 1.64 0.36 0.21 0.06 0.20 0.06 0.09 0.02 Second sampling Control (n=13) 2.01 0.24 a 0.28 0.05 a 0.25 0.05 a 0.09 0.01 300 ppm ABA (n=12) 2.00 0.44 a 0.28 0.09 a 0.26 0.07 a 0.10 0.03 Results are mean standard deviation on fresh weight basis. a indicates significant differences ( p < 0.05, Paired comparison t test) between corresponding values during first sampling. b Values are significantly different than control ( p test).

PAGE 72

72 Figure 3 1. Score plots of princip al component analysis of control and ABA treated muscadine grapes: A. Noble B. Alachua in Noble score plot refer to control and ABA treated grapes and are the samples from the same grapevine s (control and treated) at two different sampling times.

PAGE 73

73 Figure 3 2. Loading plot of principal component analysis of muscadine grapes (cv. Noble) based on principal component 1 and 2: 1. delphinidin 3, 5 diglucoside, 2. cyanidin 3, 5 diglucoside, 3. petunidin 3, 5 diglucoside, 4. peonidin 3, 5 diglucoside, 5. malvidin 3, 5 diglucoside, 6. ellagic acid, 7. myricetin, 8. quercetin, 9. kaempferol

PAGE 74

74 CHAPTER 4 COMPARISON OF DIFFERENT SOLVENTS FOR THE EXTRACTION OF PHENOLIC COMPOUNDS FROM NOBLE JUICE AND WINE POMACE Background Muscadines ( Vitis rotundifolia ) are an integral part of agriculture in the They have a high content of phytochemicals primarily tannins, flavonols, anthocyanins, and resveratrol, th at are concentrated in the seeds and skins 6 Muscadine grapes are mainly processed into juice and wine, thus generating considerable amounts of pomace as a waste. Pomace accounts for 40% of the grape by fresh weight and consists of seeds, skin, pulp and residual solids. Because grape skins and seeds are the predominant c onstituents in the pomace, this biomass is a rich source of antioxidants. These antixodants may provide protective effects against chronic diseases such as cancer, diabetes and cardiovascular diseases 22 There has been a considerable interest in utilization of phenolic compoun ds from grape pomace into value added products. Extraction is the first step in obtaining the phenolic compounds from the plant matrix in a highly concentrated form. Different extraction solvents and procedures are used for recovering the phenolic compounds in plant materials 85 Solid/liquid extraction is a very efficient technique, but its effectiveness depends on several factors such as raw material composition, conditioning, solvent polarity (and phenolic compounds polarity), time, temperature and solid/solvent ratio during the extraction 86 Anot her important factor that should be considered is the cost of sample preparation. Aqueous organic solvents are most commonly used for the sample preparation. These solvents can be toxic and the extracts cannot be utilized in food industry. In this study a simple

PAGE 75

75 method using distilled water as a solvent instead of organic solvents was used for the extraction of anthocyanins and other phenolic compounds from muscadine pomace. The objectives of this research were to 1) compare the extraction ability of common ly used solvents (methanol, acetone and distilled water) in terms of phenolic content, antioxidant capacity and anthocyanin content and 2) quantify the phenolic compounds in different extracts of muscadine pomace. Materials and M ethods Chemicals Gallic acid, HPLC grade ethanol, acetonitrile, methanol, formic acid, hydrochloric acid, Folin Ciocalteau reagent, sodium hydroxide and sodium carbonate were purchased from Fischer Scientific Co. (Pittsburg, PA). Standards of the 3 O glucosides of pelargonidi n, cyanidin, peonidin, delphinidin, petunidin, and malvidin (six mixed anthocyanin standard, HPLC grade), were purchased from Polyphenols Laboratories (Sandnes, Norway). Ellagic acid, myricetin, quercetin, kaempferol and cyanidin 3 rutinoside were obtained from Sigma Aldrich (St. Louis, MO). Muscadine Juice and Wine Pomace Preparation Noble grapes were purchased from a local vineyard in central Florida. Grapes were crushed using a manual grape crusher and juice was extracted by hot pressing technique in w hich the crushed grapes were heated for 30 mins at 60C. After heating the grapes were pressed by using a stainless steel vertical bladder press to obtain the juice pomace. For wine making, the grapes were crushed using a manual grape crusher inoculated w ith yeast, and kept for fermentation at room temperature for 7 days. The fermented must was pressed using a stainless steel vertical bladder press to obtain the wine pomace Pomace was ground to a fine paste using a mill (Robot Coupe

PAGE 76

76 USA, Inc. Jackson, Mississippi), packaged into gallon z iploc bags and were kept in the freezer ( 20C) until used for extraction. Extracts Preparation Three different solvent systems i.e. 1) methanol:acetic acid (99.7:0.3 v/v); 2) acetone:acetic acid (99. 7:0.3 v/v); 3) acidified hot water (1 % formic acid in 25 mL and 50 mL volume) at three different temperatures a) 40C, b) 60C and c) 90C were used in this study Fresh pomace (10 g) was extracted twice with each solvent system using a method described by Wu et al 87 The first extraction was done with 15 mL of each solvent, than samples were vortexed for 30 s followed by sonication for 5 min. The samples were kept at room temperature for 20 min in darkness, being vortexed and sonicated again before centrifugation. The tubes were centrifuged at 1317 g for 10 min and the supernatant removed. The samples were extracted one more time with 10 mL of solvent using the same procedure, and the supernatants were pooled. The combined supernatant was transferred into a 25 mL volumetric flask, and final vol ume was made up to 25 mL using the respective solvents. In case of hot water apart from similar extraction to other solvents, the volume of water was doubled (50 mL) to make up for the lower extraction efficiency of water The extracts were used for chemic al and HPLC analysis. Total Anthocyanin Assay Total anthocyanin content in pomace extracts was measured using the pH differential spectrophotometric method described by Giusti and Wrolstad 88 The extracts were dissolved in 0.025 mol/L potassium chloride buffer, pH 1.0 and 0.4 mol/L sodium acetate buffer, pH 4.5 with pre determined dilution factor. Absorbance at 520 and 700 nm was measured on a DU 730 Life Science UV/vis spectrophotometer

PAGE 77

77 (Bec kman Coulter, Fullerton, CA) after 30 min of incubation at room temperature. The absorbance (A) of the diluted sample was then calculated using (A 520 A 700 ) pH 1.0 (A 520 A 700 ) pH 4.5 The monomeric anthocyanin concentration in the original sample was ca lculated in cyanidin 3, 5 diglucoside equivalents according to this formula: (A MW diglucoside is used, the molar gram to milligram and the A was absorbance. Results for total anthocyanin content were expressed as milligram cyanidin 3, 5 diglucoside equivalent per gram of fresh pomace (mg cyanidin 3, 5 diglucoside/g). Folin Ciocalteu Assay The extracts were diluted to appropriate concentration for analysis. The total phenolic content was determined as reported previously 6 .The results were expressed as milligrams of gallic acid equivalents per gram of fresh pomace (mg of GAE/ g). Oxygen Radical Absorbance Capacity (ORAC) The ORAC assay for extracted samples was conducted on a Spec tra XMS samples were added to the designated wells of a 96 well black plate. This was followed The mixture was incubate d at 37 C for excitation and 530 nm emissions at 1 min intervals for 40 min. Trolox was used to generate a standard curve. The antioxidant capacities of extracts were expre ssed as

PAGE 78

78 HPLC Analysis of Phenolic C ompounds Chromatographic analysis was performed on an Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartm ent, a diode array detector and a fluorescent detector (Agilent Technologies, Palo Alto, CA). Reversed phase chromatography was used for the quantification of anthocyanins, ellagic acid and flavonols. An Agilent Zorbax Stablebond SB C18 column (250 mm 4. particle size, Agilent Technologies, Palo Alto, CA) was used for separation of phenolic compounds. Anthocyanins The extracts (1 mL) were filtered through 0.45 m filter units and 5 L injected directly without any purification. Elution was perf ormed using mobile phase A (5% formic acid aqueous solution) and mobile phase B (methanol). The flow rate was 1 10 min, 5 15% B; 10 25 min, 15 25% B; 25 30 min, 25 30% B; 30 45 min, 30% B; 45 47 min, 30 70% B; 47 50 min, 70 5% B; followed by 5 min of re equilibration of the column before the next run. UV vis spectra were scanned from 220 to 600 nm on a diode array detector and the detection wavelength for the anthocyanins was 520 nm. Ellagic a cid and f l avonols Acid hydrolysis of the samples was done before HPLC analysis. Extract (5 mL) was concentrated in a Speed Vac Concentrator (Thermo scientific ISS110, Waltham, M A) under reduced pressure at 25 C to remove the solvent. The solids were dissolved in 5 mL of methanol (50%) containing 1.2 N HCl and sonicated for 5 min. Hydrolysis was conducted in a precision water bath (Thermoscientific Waltham, MA) for 80 min at 90 C and aglycones were separated and quantified using HPLC. The binary mobile

PAGE 79

79 phase consist ed of (A) 0.5% aqueous formic acid solution and (B) acetonitrile. The flow rate was 1 mL/min and 25 min gradient was used. The gradient is described as follows: 30% B; 5 10 min, 30 40% B; 10 20 min, 40 50% B; 20 23 min, 50 70% B; 23 25 min, 70 10% B; followed by 5 min of re equilibration of the column before the next run. The column t emperature was maintained at 30 C. UV vis spectra were scanned from 220 to 600 nm on a diode array detector and the detection wavelength for the ellagic acid and fl avonols was 360 nm. Statistical Analysis One way analyses of variance (ANOVA) with Tukey HSD pairwise comparison of the means were performed using JMP (version 7.0, SAS Institute Inc., Cary, NC). Data are expressed as means standard deviation of three independent observations. A p Results and Discussion The extraction efficiency of phytochemicals depends upon the extraction solvents and conditions used. Therefore, the aim of an extraction process is to provide the extracts with maximum yield and the highest quality of the compounds of interest. Table 4 1 summarizes total phenolics, total anthocyanins and antioxidants extracted from juice and wine pomace using different solvents Acetone extracted the highest amount of t otal phenolic s followed by methanol extract ion in both juice and wine pomace. Water at a volume two times of acetone or methanol extracted more phenolics from pomace Me thanol extracted the highest amount of antioxidants from juice pomace. However, in wine pomace hot water (60C, 50 mL) yielded the highest amount of antioxidants followed by methanol. The highest amount of total anthocyanin content was obtained in hot water at 90C (50 mL), followed by methanol and acetone in both juice and wine

PAGE 80

80 pomace. Hot water extracts (25 mL) at different temperatures yielded the lowest amount of total anthocyanin s in both juice and wine pomace. T he amount of extracted individual anthocyanins was highest in methanol and hot water (50 mL) at different temperatures in juice pomace Acetone and hot water extracts (25 mL) contained the l east amount of individual anthocyanins (Table 4 2). However, for wine pomace the highest amount of individual anthocyanins were extracted in methanol followed by hot water (50 mL) and acet one. Hot water extracts (50 mL) gave the highest yield for cyanidin and peonidin 3, 5 diglucoside. Ellagic acid, myricetin, quercetin and kaempferol were found to be highest in methanol and acetone extracts of both juice and wine pomace (Table 4 3). An ext raction solvent is generally selected according to the purpose of extraction, polarity of the interested components, polarity of undesirable components, overall cost, and safety 89 To conduct various analyses, o rganic solvents such as alcohols and acetone, with different levels of water, have been widely used for the extraction of phenolic components from plant materials. Water is a universal solvent which is cheap and safe to be used in food industry Very few studies have explored the use of water for extraction of phenolic compounds. In this study we compared hot w ater extraction of anthocyanins from muscadine pomace with organic solvents. The solubility of phenolics is governed by the ir chemical structures, as well as the polarity of the solvents used. Phenolic compounds in p lant materials vary from simple to high ly polymerized substances in different quantities. Therefore, it is difficult to find a universal extraction solvent suitable for extraction of all plant phenolics. Acetone extracts gave much higher yields of total phenolic compounds than methanol and wate r extracts, which can mainly be attributed to its ability to dissolve both the hydrophilic and

PAGE 81

81 lipophilic components from plant materials. Muscadine pomace contains mixture of polar and non polar components such as anthocyanins, flavonols, ellagic acid and conjugates, and flavan 3 ols (proanthocyanidins). Total anthocyanins and antioxidant capacity was lower in acetone extracts compared to methanol; therefore it can be assumed that these extracts were rich in proanthocyanidins and other non polar compounds. A study conducted on extraction of phenolic compounds from barley showed that aqueous acetone was a better mixture for extracting phenolic compounds (which had an overall non polar character) than aqueous methanol 90 The high antioxidant capacity for methanol extract in case of juice pomace can be explained by its ability to penetrate the cell wall thus releasing more phenolic compounds. Hot water extracts (90C, 50 mL) gave the highest value for total anthocya nins. The solubility of anthocyanins was highest in methanol, a little lower in acetone and the lowest in hot water. Similar results were reported in a study on grape skins 91 .However, doubling the volume of hot water resulted in increased extraction efficiency for anthocyanins. Therefore, hot water was as effective as acidified methanol and acetone in extracting anthocyanins from grape pomace. Extraction is the fi r st step before phenolic com p ounds can be concentrated The extraction yield and antioxidant activity of the extracts highly depend on the solvent polarity, which determines both quantitatively and qualitatively the extracted phenolic compounds. Knowing the extraction ability of various solvents will help to select a particular solvent for the extraction of compounds of interest. Summary In this study, we found hot water to be the most suitable solvent for the extraction of anthocyanins. Organic solvents work best for the extraction of ellagic acid and

PAGE 82

82 flavo nols. The advantages of using water as a solvent outweigh the use of organic solvents as it is non toxic and inexpensive source. In addition, it fits better for the industry scale production because it significantly reduces the overall production cost.

PAGE 83

83 Table 4 1. Total p henolic s a ntioxidant s and t otal a nthocyanin s extracted from Noble j uice and w ine p omace using different solvents Extraction Solvent Total Phenolic s extracted (GAE mg/g) Antioxidant capacity (ORAC, mol TE/g) Total Anthocyanin s extracted (mg/g) Juice Pomace Methanol:acetic acid (99.7:0.3 v/v) 5.69 0.43 b 119.94 19.20 a 1.33 0.07 ab Acetone:acetic acid (99.7:0.3 v/v) 9.32 0.69 a 91.56 4.59 b 1.30 0.04 b Extraction with 25 mL water Hot water 40C 3.86 0.11 de 71.66 7.71 bc 1.05 0.04 c Hot water 60C 3.56 0.11 e 87.72 8.57 b 1.12 0.02 c Hot water 90C 4.05 0.17 cde 75.71 11.11 bc 1.09 0.03 c Extraction in 50 mL water Hot water 40C 4.48 0.09 cd 61.10 2.07 c 1.43 0.05 a Hot water 60C 4.78 0.28 c 57.03 9.25 c 1.38 0.04 ab Hot water 90C 4.60 0.10 cd 61.18 6.07 c 1.43 0.01 a Wine Pomace Methanol:acetic acid (99.7:0.3 v/v) 5.76 0.20 b 84.90 10.78 ab 0.93 0.00 a Acetone:acetic acid (99.7:0.3 v/v) 8.66 0.32 a 73.08 5.24 bc 0.89 0.04 a b Extraction in 25 mL water Hot water 40C 2.47 0.08 d 61.25 4.80 cd 0.58 0.02 c Hot water 60C 2.71 0.27 d 49.93 6.04 d 0.61 0.04 c Hot water 90C 2.71 0.12 d 74.24 1.81 bc 0.64 0.01 c Extraction in 50 mL water Hot water 40C 3.80 0.15 c 68.82 4.91 bcd 0.89 0.03 ab Hot water 60C 3.98 0.08 c 96.94 7.49 a 0.83 0.00 b Hot water 90C 3.98 0.11 c 83.32 14.10 abc 0.90 0.01 a Results are mean standard deviation of three determinations on fresh weight basis. Different

PAGE 84

84 Table 4 2. Anthocyanin s extracted from Noble juice and wine pomace using different solvents Extraction Solvent Delphinidin 3, 5 diglucoside (mg/g) Cyanidin 3, 5 diglucoside (mg/g) Petunidin 3, 5 diglucoside (mg/g) Peonidin 3, 5 diglucoside (mg/g) Malvidin 3, 5 diglucoside (mg/g) Total Anthocyanins (mg/g) Juice Pomace Methanol:acetic acid (99.7:0.3 v/v) 0.26 0.01 a 0.07 0.00 a 0.14 0.00 a 0.09 0.02 a 0.06 0.00 a 0.71 0.01 a Acetone:acetic acid (99.7:0.3 v/v) 0.22 0.01 b 0.06 0.00 a 0.13 0.00 b 0.14 0.00 a 0.07 0.02 a 0.61 0.02 b Extraction with 25 mL water Hot water 40C 0. 22 0.01 b 0.06 0.00 a 0.12 0.00 b 0.11 0.00 b 0.07 0.00 a 0.59 0.01 b Hot water 60C 0.23 0.01 b 0.06 0.00 a 0.13 0.00 b 0.11 0.00 b 0.07 0.00 a 0.60 0.02 b Hot water 90C 0.23 0.01 b 0.06 0.00 a 0.13 0.00 b 0.11 0.00 b 0.06 0.00 a 0.59 0.01 b Extraction with 50 mL water Hot water 40C 0.28 0.00 a 0.07 0.00 a 0.16 0.00 a 0.14 0.00 a 0.09 0.00 a 0.73 0.01 a Hot water 60C 0.27 0.00 a 0.07 0.00 a 0.16 0.00 a 0.14 0.00 a 0.09 0.00 a 0.72 0.01 a Hot water 90C 0.28 0.00 a 0.07 0.00 a 0.16 0.00 a 0.14 0.00 a 0.09 0.00 a 0.74 0.01 a Wine Pomace Methanol:acetic acid (99.7:0.3 v/v) 0.12 0.00 a 0.05 0.00 a 0.11 0.00 a 0.12 0.00 a 0.07 0.00 a 0.47 0.01 a Acetone:acetic acid (99.7:0.3 v/v) 0.10 0.00 b 0.05 0.00 ab 0.09 0.00 b 0.12 0.00 a 0.05 0.00 b 0.40 0.00 b E xtraction with 25 mL water Hot water 40C 0.06 0.00 f 0.04 0.00 b 0.06 0.00 d 0.07 0.00 b 0.04 0.00 b 0.27 0.01 c ot water 60C 0.06 0.00 f 0.04 0.00 b 0.07 0.00 cd 0.08 0.00 b 0.05 0.00 b 0.29 0.03 c Hot water 90C 0.07 0.00 e 0.04 0.00 b 0.07 0.00 d 0.08 0.00 b 0.04 0.00 b 0.30 0.00 c E xtraction with 50 mL water Hot water 40C 0.09 0.00 cd 0.05 0.00 a 0.09 0.00 b 0.11 0.00 a 0.06 0.00 a 0.41 0.01 b Hot water 60C 0.08 0.00 d 0.05 0.00 a 0.09 0.00 b 0.11 0.00 a 0.07 0.00 a 0.41 0.01 b Hot water 90C 0.09 0.01 bc 0.05 0.00 a 0.09 0.00 b 0.11 0.00 a 0.07 0.00 a 0.42 0.02 b Results are mean standard deviation of three determinations on fresh weight basis. Different superscripts in each column indicate the significant

PAGE 85

85 Table 4 3. Ellagic acid, myricetin, quercetin and kaempferol extracted from Noble juice and wine pomace using different solvents Extraction Solvent Ellagic acid (mg/g) Myricetin (mg/g) Quercetin (mg/g) Kaempferol (mg/g) Juice Pomace Methanol:acetic acid (99.7:0.3 v/v) 2.43 0.05 a 0.31 0.01 a 0.15 0.00 a 0.07 0.00 a Acetone:acetic acid (99.7:0.3 v/v) 2.39 0.10 a 0.30 0.01 a 0.14 0.01 a 0.07 0.00 a Extraction with 25 mL water Hot water 40C 1.15 0.22 b 0.18 0.01 bc 0.08 0.00 bc 0.04 0.00 bc Hot water 60C 1.27 0.14 b 0.18 0.01 bc 0.09 0.00 bc 0.04 0.00 bc Hot water 90C 0.95 0.31 b 0.15 0.03 c 0.07 0.02 c 0.03 0.01 c Extraction with 50 mL water Hot water 40C 1.18 0.20 b 0.20 0.02 b 0.10 0.01 b 0.04 0.00 b Hot water 60C 1.15 0.21 b 0.19 0.01 b 0.09 0.00 b 0.04 0.00 bc Hot water 90C 0.93 0.07 b 0.18 0.00 bc 0.09 0.00 bc 0.04 0.00 bc Wine Pomace Methanol:acetic acid (99.7:0.3 v/v) 0.12 0.00 a 0.05 0.00 a 0.11 0.00 a 0.12 0.00 a Acetone:acetic acid (99.7:0.3 v/v) 0.10 0.00 ab 0.05 0.00 a 0.09 0.00 a 0.12 0.00 b Extraction with 25 mL water Hot water 40C 0.06 0.00 b 0.04 0.00 b 0.06 0.00 b 0.07 0.00 c Hot water 60C 0.06 0.00 b 0.04 0.00 b 0.07 0.00 b 0.08 0.00 c Hot water 90C 0.07 0.00 b 0.04 0.00 b 0.07 0.00 b 0.08 0.00 c Extraction with 50 mL water Hot water 40C 0.09 0.00 b 0.05 0.00 b 0.09 0.00 b 0.11 0.00 c Hot water 60C 0.08 0.00 b 0.05 0.00 b 0.09 0.00 b 0.11 0.00 c Hot water 90C 0.09 0.01 b 0.05 0.00 b 0.09 0.00 b 0.11 0.00 c Results are mean standard deviation of three determinations on fresh weight basis. Different superscripts in each column indicate the significant diff

PAGE 86

86 CHAPTER 5 ADSORPTION/DESORPTION CHARACTERISTICS AND SEPARATION OF PHYTOCHEMICALS FROM MUSCADINE POMACE USING MACROPOROUS ADSORBENT RESINS Background Muscadine grapes are native to the southeastern United States. They contain a wide variety of antioxidants and possess a unique phytochemical profile. They are reported to contain anthocyanins and other polyphenols including ellagic acid, quercetin, myrice tin and kaempferol 1, 5, 28 Cell culture studies have suggested th at polyphenols from muscadine grapes can inhibit proliferation of colon cancer cells and induce apoptosis 13 14 The anthocyanins from muscadine grapes are also reported to gluco sidase and pancreatic lipase, the two major enzymes involved in diabetes 17 During juice and wine processing 40% of t he fruit is lost as pomace which consists of seeds, skin, pulp and residual solids. Since p henolic compounds in muscadine grapes are concentrated in seeds and skin 6 considerable quanti ties are still left in pomace. Therefore, muscadine pomace is an attractive source of bioactive phytochemicals including anthocyanins, ell agic acid, and flavonols 92 These bioactive compounds, also known as nutraceuticals possess high antioxidant capacity and may provide protective effects against chronic diseases such as cancer, diabetes and cardiovascular diseases 22, 24 There is an increasing demand by the grape indust ry for the extraction of these phenolic compounds from the pomace and utilization for the production of functional foods. Muscadine pomace is currently utilized as compost or animal feed. Utilization of pomace to extract the phenolic compounds will increas e the economic value of the muscadine grapes and wines.

PAGE 87

87 Several extraction and separation methods are used for the enrichment of phenolic compounds from the plant based materials such as liquid liquid extraction, membrane filtration, ion exchange and chro matography. However, these methods have several disadvantages; for example, they are time consuming, laborious, expensive with poor recovery, and not suitable for large scale industrial production. Alternatively, great progress has been made in recent year s to separate these compounds from the plant materials with macroporous resins. The resins are polar or non polar polymers having characteristics of good selectivity, different surface properties, high mechanical strength and fast adsorption speed 93 The advantages of using macroporous resins outweigh other methods of separation and enrichment as they are relatively low cost, easy to use and regenerate, have high efficiency and are suitable for the industrial scale up. Moreover, some of these resins meet food grade standards of Food and Drug Administration; therefore, the extracted compounds can be used as food ingredients or dietary supplements. Adsorption by macroporous resins has been successfully applied f or the recovery of various phenolic compounds such as hesperidin from citrus peels during citrus processing 94 flavonoids from mulberry leaves 95 lycopene from tomato skins 96 vitexin and isovitexin from pigeon pea 97 and polyphenols from apple juice 98 100 No studies have been done to investigate the use of resins for the separation and enrichment of phenolic compounds from muscadine pomace. The objectives of this study were to investigate the adsorption/desorption behaviors of anthocyanins and other phenolic compounds from muscadine pomace extracts on different amberlite resins, and optimizing the conditions for the selection of one resin for conducting the dynamic adsorption and desorption.

PAGE 88

88 Materials and M ethods Chemicals Gallic acid, HPLC grade ethanol, acetonitrile, methanol, formic acid, hydrochloric acid, Folin Ciocalteau reagent, sodium hyd roxide, sodium carbonate, sucrose, glucose and fructose were purchased from Fischer Scientific Co. (Pittsburg, PA). Standards of the 3 O glucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin (six mixed anthocyanin standard HPLC grade), were purchased from Polyphenols Laboratories (Sandnes, Norway). Ellagic acid, myricetin, quercetin, kaempferol, cyanidin 3 rutinoside, (+) catechin and ( ) epicatechin were obtained from Sigma Aldrich (St. Louis, MO). Resins FPX 66, XAD 16N, XAD 1180, XAD 7HP, and XAD 761 were products of Rohm and Haas Co. (Philadelphia, PA). Muscadine Juice and Wine Pomace Preparation Noble grapes were purchased from a local vineyard in central Florida. Muscadine juice is normally processed by hot or cold pressing. For this study g rapes were crushed using a manual grape crusher and juice was extracted by hot pressing technique in which the crushed grapes were heated for 30 mins at 60C. After heating the grapes were pressed by using a stainless steel vertic al bladder press to obtain the juice pomace. For wine making, the grapes were crushed using a manual grape crusher, inoculated with yeast, and kept for fermentation at room temperature for 7 days.The fermented must was pressed using a stainless steel verti cal bladder press to obtain the wine pomace. Pomace was ground to a fine paste using a mill (Robot Coupe USA, Inc. Jackson, Mississippi), packaged into gallon bags and were kept in the freezer ( 20C) until used for extraction.

PAGE 89

89 Preparation of Juice and Wine Pomace Water Extracts Pomace was extracted twice with acidified hot water (1% formic acid) at 90C. For the first extraction, pomace (200 g) was mixed with 500 mL of acidified hot water (1% formic acid) at 90C, sonicated for 15 min and kept in the d ark at room temperature for 1 h. The samples were sonicated again for 15 min before filtration using muslin cloth. The residue from the muslin cloth was extracted with 400 mL of acidified hot water (1% formic acid) using the same procedure. The filtered ex tracts from the two extractions were pooled and transferred into a 1 L volumetric flask. Acidified water (1% formic acid) was added to make up the final volume to 1 L. The final extract was filtered using Whatmann No. 4 filter paper. Characterization and P hytochemical A nalysis of E xtracts The extracts were diluted to appropriate concentration for analysis. Total anthocyanin content in pomace extracts and concentrated extracts obtained after resin adsorption/desorption processes was measured using the pH di fferential spectrophotometric method described by Giusti and Wrolstad 88 The extracts were dissolved in 0.025 mol/L potassium chloride buffer, pH 1.0 and 0.4 mol/L sodium acetate buffer, pH 4.5 with pre determined dilution factor. Absorbance at 520 and 700 nm was measured on a DU 730 Life Science UV/vis spectrophotometer (Beckman Coulter, Fullerton, CA) after 30 min of incubation at room temperature. The absorbance (A) of the diluted samp le was then calculated using (A 520 A 70 0 ) pH 1.0 (A 520 A 700 ) pH 4.5 The monomeric anthocyanin concentration in the original sample was calculated in cyanidin 3, 5 diglucoside equivalents according to this formula: (A MW DF 1000) / MW (611) of cyanidin 3, 5 was 30,175; the DF was dilution factor; 1000 is the factor to convert gram to milligram

PAGE 90

90 and the A was absorbance. Results for total anthocyanin content were expressed as milligram cyan idin 3, 5 diglucoside equivalent per gram of fresh pomace (mg cyanidin 3, 5 diglucoside/g). The total phenolic content was determined as reported previously 6 The results were expressed as milligrams of gallic acid equivalents per gram of fresh pomace (mg of GAE/ g). HPLC a nalysis of anthocyanins, other phenolic phytoch emicals, and sugars was performed on an Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector, fluorescent detector and a refractive index detector (Agilent Technologies, Palo Alto, CA). Reversed phase chromatography was used for the quantification of anthocyanins, ellagic acid, flavonols, catechin and epicatechin. An Agilent Zorbax Stablebond SB C18 column (250 mm 4.6 mm, 5 m particle size, Agilent Technologies, Palo Alto, CA) was used for the separation of phenolic compounds. For anthocyanin analysis, the extracts (1 mL) were filtered through 0.45 M filter units and 5 L injected directly without any purification. Elution was performed using mobile phase A (5% formic acid aqueous solution) an d mobile phase B (methanol). The flow rate was 1 mL/min with the gradient as follows: 10 min, 5 15% B; 10 25 min, 15 25% B; 25 30 min, 25 30% B; 30 45 min, 30% B; 45 47 min, 30 70% B; 47 50 min, 70 5% B; followed by 5 min of re equilibrati on of the column before the next run. UV vis spectra were scanned from 220 to 600 nm on a diode array detector and the detection wavelength for the anthocyanins was 520 nm. The individual anthocyanins were quantified using standards. The analysis of ellagi c acid, flavonols, catechin and epicatechin was done on HPLC after acid hydrolysis of the samples. The solids were dissolved in 5 mL of methanol (50%)

PAGE 91

91 containing 1.2 N HCl and sonicated for 5 min. Hydrolysis was conducted in a precision water bath (Thermos cientific, Waltham, MA) for 80 min at 90C and aglycones were separated and quantified using HPLC. The binary mobile phase consisted of (A) 0.5% aqueous formic acid solution and (B) acetonitrile. The flow rate was 1 mL/min and 25 min gradient was used. The 30% B; 5 10 min, 30 40% B; 10 20 min, 40 50% B; 20 23 min, 50 70% B; 23 25 min, 70 10% B; followed by 5 min of re equilibration of the column before the next run. The column temperature was maintained at 30C. The detection wavelength for the ellagic acid and flavonols was 260 and 360 nm, respectively. Catechin and epicatechin were quantified using fluorescent detection. Excitation and emission of the fluorescent detector were set at 230 and 321 nm, respectivel y. Sugar analysis was conducted using a Restek ultra ami 4.6 mm). Acetonitrile: water (65:35 v/v) was used as the mobile phase at a constant flow rate of 1.0 mL/min. The column temperature was maintained at 30C and a 5 L of sample was injected. The optical unit temperature was set at 35C and the refractive index detector signal was monitored in positive polarity. The run time for each sample was 15 min followed by 5 min post time before the next run. Calibration curves were constructe d using pure standards of glucose and fructose. Pretreatment of Macroporous Resins The physical characteristics of five different resins used in this study are summarized in Table 5 1. Resins were soaked in ethanol overnight and then treated with 2 bed vo lumes (BV) of 4% HCl and 5% NaOH solutions to remove salts and other impurities trapped inside the pores due to synthesis process. After acid and base wash,

PAGE 92

92 resins were neutralized with distilled water. To determine the moisture content of resins, three sa mples of each kind of pretreated resin were weighed and dried at 60C for 24 h. Static Adsorption and Desorption Tests for Screening of Resins The static adsorption and desorption experiments were performed as follows: 1 g resin (wet weight) was introduce d into a 125 mL Erlenmeyer flask. Then, 50 mL of juice/wine pomace water extract was added to each flask. A control sample was employed to monitor any change in the initial concentration values, to exclude effects on measured absorbance. The flasks were th en shaken (120 rpm) for 24 h at room temperature (25C) in a shaking water bath. After adsorption, resins were filtered and washed with 50 mL of distilled water. For desorption 70 and 95 % acidified (1 % formic acid) ethanol solutions were tested. Fifty mi lliliters of ethanol water solution was added to the flasks containing the adsorbate laden resins. The flasks were kept for shaking (120 rpm) for 24 h at room temperature (25C) in a shaking water bath. The concentrations of anthocyanins and total phenolic s in the liquid phase after adsorption and desorption, were then analyzed by respective assays. Candidate resins were selected in terms of their adsorption capacities, adsorption and desorption ratios and recovery (%).The following equations were used to q uantify the capacities of adsorption and desorption as well as their ratios. Adsorption evaluation ( 5 1 ) ( 5 2 ) Where Q e is adsorption capacity at adsorption equilibrium (mg/g resin), E is the adsorption ratio (%), C 0 and C e are initial and equilibrium concentrations (mg/L) of

PAGE 93

93 solute in the solution, respectively, V 0 the volume of initial sample solution (mL), and m is the weight of dry resin (g). Desorption evaluation ( 5 3 ) ( 5 4 ) Where, D is the desorption ratio (%), C d concentration of the solute in desorption solution (mg/L), V d volume of desorption solution ( mL) and R is the recovery (%). C 0 C e and V 0 are the same as described above. Adsorption Kinetics The adsorption kinetics was studied on FPX 66, XAD 16N and XAD 1180 resins The test for adsorption kinetics on the selected resins were conducted by adding 50 m L water extracts of juice/wine pomace with 1g (wet weight) resin, and then shaking (120 rpm) for 8 h at room temperature (25C). Aliquot (1 mL) of sample solution was tak en at equal time intervals until the equilibrium was reached and analyzed for total anthocyanins and total phenolic content. Two mathematical models were applied to simulate the uptake of phenolic compounds on the selected resins with time i. e., pseudo fi rst order (Eq. 5 5) 101 and pseudo second kinetic model (Eq. 5 6) 102 ( 5 5 ) ( 5 6 ) Where, Q e and Q t (mg/g) are the amount of phenolic compounds adsorbed per gram of resin at equilibrium and anytime t (min), respectively and k a (min ) is the equilibrium rate constant. k b (g/mg min) is the second order model rate constant.

PAGE 94

94 Adsorption Isotherms The tests for adsorption isotherms on the FPX 66, XAD 16N and XAD 1180 resins were conducted at room temperature (25C), 30 and 35C for juice pomace extracts. Briefly, 50 mL water extracts of juice pomace at different initial concentrations were mixed with 1g (wet weight) resin, and then kept for shaking (120 rpm) for 8 h at selected temperatures. At equilibrium samples were analyzed for total anthocyanins and total phenolic content. Two standard theoretical models, i.e., Langmuir and Freundlich model, are used to describe the adsorption behavior between adsorbate and adsorbent 103 Equations describing Langmuir and Freundlich models are given below: ( 5 7 ) ( 5 8 ) Where, Q m (mg/g) is the maximum amount of adsorption, K L is the affinity constant in Langmuir model which can be calculated from the slope and intercept of the linear plot of C e /Q e versus C e respectively. K F and 1/ n are the constants as me asures of adsorption capacity and adsorption intensity in Freundlich model and can be determined from the intercept and slope of linear plot of ln Q e versus ln C e respectively. The essential characteristics of the Langmuir isotherm can be expressed by mea L dimensionless constant, called the separation factor or equilibrium parameter. R L can be calculated using the following equation: ( 5 9 ) R L values indicate the type of isotherm to be irreversible (R L =0), favorable (0 < R L <1), linear (R L =1), or unfavorable (R L >1) 104

PAGE 95

95 Dynamic Adsorption and Desorption Tests Dynamic adsorption and desorption tests were carried out on a glass column (22 mm 350 mm) packed with 28 g (wet weight) of the selected FPX 66 resin. The bed volume (BV) of resin was 30 mL and the packed length of resin bed was 19 cm. The adsorption process was performed by loading juice pomace water extract onto the pretreated resin filled in a glass column. Subsequently, the adsorbate laden column was washed first with 4 BV of distilled w ater, and then desorbed with 70 % acidified (1 % formic acid) ethanol solution. The effect of sample flow rate and ethanol solution flow rate on adsorption and desorption phenomenon were studied. After optimizing conditions for adsorption and desorption, the 70% ethanol eluent was collected and concentr ated to dryness in a Speed Vac Concentrator (Thermo scientific ISS110, Waltham, MA) under reduced pressure at 25C to remove the solvent. Statistical Analysis All data are expressed as mean standard deviation. Two way analyses of variance (2 way ANOVA) were performed for static desorption tests using Sigma Stat (version 11.0, Systat software Inc., Chicago, IL). One way analyses of variance (ANOVA) with Tukey HSD pairwise comparison of the means was performed using JMP software (version 8.0, SAS Institute Inc., Cary, NC) for other results. The mean values Results and Discussion Screening of the Resins Resins differ in chemical structure, polarity, particle size, porosity and surface area. The adsorption of a solute on an adsorbent is a complex process which involves the interactions among three components: the adsorbate (the solute), the adsorbent and

PAGE 96

96 t he solvent involving a physical action through V an der Waals force or hydrogen bonding. The adsorption and desorption properties of different resins were tested based on total phenolic and anthocyanin content in juice and wine pomace water extracts. Total a nthocyanin c ontent Juice pomace Adsorption capacity describes amount of anthocyanins/phenolic compounds adsorbed on 1 g of resin. Adsorption ratio is the percentage of anthocyanins/phenolic compounds adsorbed by resins from the aqueous extract. Adsorpt ion capacity was highest for FPX 66 and XAD 16N, followed by XAD 1180 and XAD 761 in juice pomace. Similarly, the highest adsorption ratio was observed for FPX 66 and XAD 16N, followed by XAD 1180. However, XAD 7HP showed the lowest adsorption capacity and ratio for juice pomace (Figure 5 1A). Recovery rate is the % of anthocyanins/phenolic compounds recovered from the resins by the desorbing solvent based on the initial concentration of the extract. The recovery (%) was highest in FPX 66 and XAD 16N, foll owed by XAD 1180 and 761 (Figure 5 1B). However, recovery (%) of anthocyanins with 95% ethanol was similar for FPX 66, XAD 1180 and 16N. The lowest recovery (%) was seen in XAD 7HP with both 70 and 95% ethanol. Desorption ratio is the % of anthocyanins/ph enolic compounds desorbed from the resins by the desorbing solvent. Significant differences were observed among resins in desorption ratio with 70% ethanol. The highest desorption ratio was observed in FPX 66 followed by XAD 16N using 70% ethanol as desorp tion solvent (Figure 5 1C). In contrast, the desorption ratio was similar for FPX 66, XAD 1180 and XAD 16N using 95% ethanol. Similar to recovery, XAD 7HP showed the lowest desorption ratio for

PAGE 97

97 anthocyanins with 70 and 95% ethanol. There was a statisticall y significant interaction between resins and % ethanol (p < 0.001). Wine pomace Adsorption capacity was highest for FPX 66 and XAD 16N, followed by XAD 1180 and XAD 761 in wine pomace as well. Highest adsorption capacity and ratio was observed in FPX 66, followed by XAD 16N and 1180 while the lowest adsorption capacity and ratio was observed in XAD 761 and 7HP (Figure 5 2A). The highest recovery (%) and desorption ratio was observed for XAD 16N and FPX 66, followed by XAD 761 and XAD 1180 using 70 and 95% ethanol while, XAD 7HP had lowest recovery (%) and desorption ratio. No signifi cant differences were observed in recovery (%) or desorption ratio in different resins by using 70 and 95% ethanol as desorption solution (Figures 5 2B and C). The adsorption and desorption of anthocyanins was highest on non polar resins (FPX 66, XAD 16N a nd 1180). The pore size of the porous adsorbents and the size of adsorbate molecules play an important role in the process of adsorption. If the pore diameter is too small it can restrict the diffusion of adsorbate molecules. On the other hand, if the pore diameter is too large the molecules adsorbed will be prone to desorption at the same time 105 In addition, the affinity between different polyphenols for the adsorption sites may vary, with some phenolics being preferentially selected over others. A nthocyanins are small molecules compared to other phenolic compounds present in pomace. They contain both polar hydroxy groups and non polar phenylallyl groups which could explain the differences in adsorption behaviors of resins with different polarity. E xcept for XAD 7HP, the amount of anthocyanins adsorbed seems to be proportional to the adsorbent surface area 94, 106 Resins showing high adsorption

PAGE 98

98 capacity had lower pore diameters which could be another factor for better ad sorption of anthocyanins on them. Desorption of anthocyanins was almost complete in case of FPX 66, XAD 16N and XAD 1180 resins. However, the desorption ratio was very low for polar resins i. e., XAD 761 and XAD 7HP, which might be due to strong interactio n between polar hydroxy groups of anthocyanins in the solute with the adsorbent material. The large surface area and ideal pore diameter for anthocyanins could be possible explanation for better adsorption and desorption characteristics of FPX 66, XAD 16N and XAD 1180 resins. Total phenolic content Juice pomace. Based on total phenolic content adsorption capacity was highest for XAD 7HP followed by FPX 66, XAD 16N and XAD 1180 for juice pomace. Among all the resins XAD 761 had the lowest adsorption capacit y and ratio (Figure 5 3A). The recovery (%) based on total phenolic content was found to be highest for FPX 66 resin followed by XAD 16N and XAD 7HP using 70% ethanol in juice pomace while, XAD 1180 and 761 gave lowest recovery (Figure 5 3B). However, with 95% ethanol the recovery (%) was similar for FPX 66, XAD 16N, XAD 7HP and XAD 1180. There was a statistically significant interaction between resins and % ethanol (p < 0.001). No significant differences were observed in desorption ratio in different resin s at both 70 and 95% ethanol as desorption solution in juice pomace (Figure 5 3C). Wine pomace Similar to juice pomace adsorption capacity was highest for XAD 7HP followed by FPX 66, XAD 16N and XAD 1180 while, XAD 761 had the lowest adsorption capacity and ratio (Figure 5 4A). The recovery (%) based on total phenolic content was found to be highest for FPX 66 resin followed by XAD 16N and XAD 7HP using 70% ethanol. XAD 1180 and 761 gave lowest recovery (%) with 70% ethanol. In

PAGE 99

99 contrast to juice pomace, r ecovery (%) based on total phenolic content in wine pomace was highest in XAD 1180, followed by FPX 66 and XAD 7HP using 95% ethanol. Resin XAD 761 and 16N had lowest recovery (%) (Figure 5 4B). Significant differences were observed in the desorption ratio among different resins with 70% ethanol. However, no differences were observed with 95% ethanol (Figure 5 4C). There was a statistically significant interaction between resins and % ethanol (p < 0.001) for recovery (%). Phenolics in pomace may vary from simple acids to complex flavonoids, hydrolysable tannins and condensed tannins. These compounds vary in their polarity depending upon their structure. XAD 7HP showed highest adsorption capacity for both juice and wine pomace followed by non polar resins (F PX 66, XAD 16N and XAD 1180). The adsorption of particular species can also depend upon its similarity to the more towards polar resins 106 XAD 7HP, which presented the best results in terms of the adsorptive capacity, is a non ionic aliphatic acrylic polymer (dipole moment = 1.80), wher eas the other adsorbents are made of styrene divinylbenzene, a hydrophobic polyaromatic polymer (dipole moment = 0.30), making them more efficient for adsorption of compounds of higher hydrophobicity 107 The adsorption of phenolics seems to be proportional to the surface area of the resins. Recovery (%) and desorption of phenolics was almost complete in both juice and wine pomace however, difference s were observed by using 70 or 95% ethanol. Based on the initial screening three resins (FPX 66, XAD 16N and XAD 1180) were selected for further testing of adsorption kinetics and thermodynamics.

PAGE 100

100 Adsorption Kinetics The experimental results of the adsorp tion kinetics of juice and wine pomace based on total anthocyanin and phenolic content on the three selected resins are shown in Figures 5 5 and 5 6. The adsorption of phenolic compounds from the aqueous solution on all the three resins increased quickly i n the first 90 mins and then increased slowly until adsorption equilibrium was reached at 490 mins in both the pomaces Different kinetic models are used to determine the rate of adsorption processes and mechanism of adsorption. The commonly used kinetic m odels are the pseudo first order and pseudo second order model 108 The pseudo first order is generally applicable over the initial stage of an adsorption process while pseudo second order model assumes that rate limiting step is chemisorptio n and predicts the behavior over the whole range of adsorption 109 The experimental data were fitted to these two models in order to determine which model best described the adsorption rate of phenolic compounds. Summary of the results are reported in Table 5 2. In the case of FPX 66 and XAD 16N resins, the higher correlation coefficients and agreement between experimental and calculated Qe values conclude that the pseudo first order model is a more favo rable fit for the adsorption of total anthocyanins from juice pomace extracts. However, for XAD 1180, pseudo second order seems to be a better fit than the pseudo first order model. For wine pomace pseudo second order was a better fit for all the three res ins based on total anthocyanins (Table 5 2). Except for XAD 16N in wine pomace, the high correlation coefficient (R 2 > 0.95) of pseudo second order model indicates that adsorption of phenolic compounds from muscadine pomace fit better by this model compare d to pseudo first order model (Table 5 3). Similar findings were reported by other studies 106, 110 Since, the results of wine pomace were comparable to juice

PAGE 101

1 01 pomace, so only juice pomace was selected for further testing with focus on anthocyanin content. Adsorption Isotherms The equilibrium adsorption isotherms of anthocyanins for juice pomace on thr ee selected resins were investigated at three temperatures (room temperature (25C), 30 and 35C) as shown in Figures 5 7 A, B and C. The Langmuir and Freundlich isotherms are two of the most commonly used models for describing adsorption isotherms. The La ngmuir model describes a monolayer adsorption with energetically identical sorption sites and without mutual interactions between the adsorbed molecules. The Freundlich model assumes adsorption to heterogeneous surfaces which is characterized by sorption s ites at different energies. This model can be used to describe the adsorption behavior of monomolecular layer as well as that of the multi molecular layer 109 The correlation coefficients for each model on three selected resins at different temperatures are listed in Table 5 4 For all three resins, the Langmuir model was considered as a better model for describing adsorption equilibrium due to higher correlation coefficients (R 2 = 0.9831 0.9994). The va lues of Q m (maximum adsorption capacity) increased in the order FPX 66>XAD 16N>XAD 1180. Our results indicate that the adsorption for anthocyanins on the selected resins is favorable and has R L value between 0 and 1 (Table 5 4 ). The Freundlich isotherm con stant n represents the strength of adsorption process and its value should be greater than 1 and less than 10 for favorable adsorption conditions 109 The n values obtained from Freundlich plots were gre ater than 1 for all the three resins indicating favorable adsorption conditions.

PAGE 102

102 Dynamic B reakthrough C urve on FPX 66 R esin In order to optimize the process of dynamic adsorption and desorption important factors such as feed volume, flow rate of feed and eluent, and volume of eluent were taken into consideration and tested. The process of adsorption involves diffusion of adsorbate, interaction between the adsorbate molecules and the resin including hydrogen bonding, simple stacking or hydrophobic interacti ons. Resins can easily adsorb the molecules due to their large surface area and a highly porous structure. When the adsorption reaches the breakthrough point (the point of maximum saturation and when the resin cannot hold the adsorbate molecules), the adso rption effect decreases and solute starts to leak from the resin. Thus, it is important to define the breakthrough point in order to calculate the resin quantity, processing volume of sample and proper sample flow rate. The best adsorption performance was observed at the slowest flow rate (2 BV/h) (Figure 5 8 ). The low flow rate allows more time for the adsorbate molecules to interact with the active sites of the resin at the expense of longer processing time. In contrast, a faster flow rate requires less t ime but has a negative impact on the adsorption capacity since breakthrough point reaches more quickly 106 The 5% ratio of exit to the inlet solute was defined as the break through point in this study. Based on the results and taking into consideration the processing time, flow rate of 4 BV/h was selected as the optimum for the adsorption with approximately 17 BV processing volume of the sample solution. Dynamic D esorption C urve on FPX 66 R esin These curves were based on the volume of desorption solvent used and the concentration of anthocyanins in the desorption solvent. A 70% acidified (1% formic acid) ethanol solution was used as a desorption solvent. It is important to select the

PAGE 103

103 proper flow rate for better desorption performance. As it can be seen from F igure 5 9 the best desorption performance was observed at slowest flow rate of 1 BV/h. There was no difference in the dynamic desorption at flow rates of 2 and 3 BV/h. Therefore, 2 BV/h was c hosen as proper flow rate for dynamic desorption on account of short working time. Approximately, 3 BV of desorption solution was used to completely desorb the anthocyanins from the resin. The yield of concentrated powder was 5.7 mg/g of fresh juice pomace The percent recovery of total anthocyanins was 73%, which was comparable with those in some previous studies 96, 111 Choice of organic solvent and poor stability of anthocyanins may also adversely affect the recovery 112 Characterization of C oncentrated E xtracts from M uscadi ne J uice P omace The content of individual phytochemicals in the concentrated extracts obtained after resin adsorption and desorption process is shown in Table 5 5 The values were compared with those in the initial water extract of muscadine juice pomace. The extracts were highly concentrated solutions of anthocyanins, ellagic aci d, flavonols, catechin and epi catechin without sugars 113 Resin adsorption increased the content of peonidin 3, 5 diglucoside by 38 times. Similarly, total anthocyanins and total phenolics were 38 and 42 times higher in the concentrated extract compared to the water extract. A study on muscadine pomace showed increase in the total phenolics and the anthocyanins by 25 times after resin adsorption/desorption process 114 Similarly, anthocyanins were increased 7 to 20 times from a by product of blood orange juice processing 113 The adsorption of phenolic phytochemicals on resin was not completely selective for anthocyanins. In addition to anthocyanins, the content of ellagic ac id, and total flavonols (myricetin, querceti n and kaempferol) in the concentrated extract also increased by 6

PAGE 104

104 and 5 times, respectively. Catechin and epicatechin were detected only in the concentrated extract but not in the initial water extract. Muscadine pomace water extract contained significant amount of glucose and fructose (Table 5 5 ). These sugars were completely removed by water wash during the resin adsorption process. Summary In summary, our results indicate that FPX 66 is the most suitable resin among selected commercial adsorbents for the recovery of anthocyanins from muscadine juice pomace. This is due to its high adsorption and desorption capacity, and greater affinity towards these phenolic compounds, with respect to the other tested resins under the same conditions. The optimization of resin adsorption process in the present study sets parameters for the development of pilot scale separation and concentration of anthocyanins from muscadine pomace. This extract could potentially find application as a natural colorant, dietary suppleme nt, antioxidant ingredient for functional foods, and/or as a raw material in the cosmetic and pharmaceutical industry preparations.

PAGE 105

105 Table 5 1. Physical c haracteristics of a dsorbent r esins. Resin Chemical nature Polarity Surface area (m 2 /g) Average pore diameter () XAD 7HP Aliphatic ester Polar 500 450 XAD 761 Phenol formaldehyde Polar 200 600 XAD 16N Polystyrene DVB* Non polar 800 150 XAD 1180 Polystyrene DVB* Non polar 700 400 FPX 66 Polystyrene DVB* Non polar 700 200 250 *DVB Divinyl benzene

PAGE 106

106 Table 5 2. Kinetic parameters of muscadine juice and wine pomace on FPX 66, XAD 16N and XAD 1180 resins at room temperature (25C) based on total anthocyanins. Resins Q e exp (mg/g) Pseudo first order Pseudo second order Q e cal (mg/g) K a (min 1 ) R 2 Q e cal (mg/g) K b (g/mg min) R 2 Juice Pomace FPX 66 27.12 25.27 0.0063 0.9883 31.55 2.96 10 4 0.9784 XAD 16N 25.32 23.23 0.0062 0.9907 29.50 4.06 10 4 0.9819 XAD 1180* 16.32 7.57 0.0051 0.8387 16.86 1.64 10 3 0.9920 Wine Pomace FPX 66 19.23 16.56 0.0067 0.9159 21.88 8.29 10 3 0.9740 XAD 16N 18.27 15.50 0.0065 0.9018 20.58 5.75 10 4 0.9731 XAD 1180 12.43 6.68 0.0055 0.6151 13.48 1.47 10 3 0.9836 exp experimental, cal calculated. Data are based on three replicates. based on two determinations

PAGE 107

107 Table 5 3 Kinetic parameters of muscadine juice and wine pomace on FPX 66, XAD 16N and XAD 1180 resins at room temperature (25C) based on total phenolics Resins Q e exp (mg/g) Pseudo first order Pseudo second order Q e cal (mg/g) K a (min 1 ) R 2 Q e cal (mg/g) K b (g/mg min) R 2 Juice Pomace FPX 66 155.83 187.67 0.0081 0.8745 196.08 5.38 10 5 0.9609 XAD 16N 144.88 176.69 0.0085 0.9045 178.57 4.06 10 5 0.9507 XAD 1180 118.17 113.11 0.0084 0.9587 133.33 1.02 10 4 0.9833 Wine Pomace FPX 66 109.13 109.76 0.0087 0.9667 129.87 4.39 10 5 0.9786 XAD 16N 103.71 110.10 0.0093 0.9832 123.46 8.48 10 5 0.9795 XAD 1180 85.29 75.31 0.0076 0.9486 96.15 1.34 10 4 0.9798 exp experimental, cal calculated. Data are based on three replicates.

PAGE 108

108 Table 5 4 Langmuir and Freundlich parameters for the adsorption of muscadine juice pomace on FPX 66, XAD 16N and XAD 1180 resins based on total anthocyanins. Temp (C) Langmuir Equation Freundlich Equation Q m (mg/g) K L (L/mg) R L R 2 n K F (L/mg) R 2 FPX 66 25 31.35 0.043 0.09 0.9974 1.94 2.65 0.9654 30 27.70 0.047 0.08 0.9994 1.97 2.49 0.9358 35 30.49 0.039 0.10 0.9979 1.76 2.04 0.9601 XAD 16N 25 30.22 0.035 0.10 0.9831 1.90 2.27 0.9627 30 26.81 0.047 0.08 0.9989 2.02 2.51 0.9543 35 27.86 0.040 0.09 0.9987 1.79 1.95 0.9555 XAD 1180 25 22.33 0.026 0.13 0.9915 1.94 1.52 0.9478 30 20.84 0.025 0.14 0.9981 1.88 1.29 0.9669 35 20.24 0.023 0.15 0.9987 1.74 1.03 0.9609 Data are based on three replicates.

PAGE 109

109 Table 5 5 Comparison of phytochemical and sugar content of muscadine juice pomace water extract with concentrated extract. Compounds Juice pomace water extract (mg/g dry weight) Concentrated extract (mg/g dry extract) Enrichment factor Anthocyanins Delphinidin 3,5 diglucoside 1.69 0.01 48.86 0.98 28.9 Cyanidin 3,5 diglucoside 0.44 0.00 12.77 0.23 29.1 Petunidin/Pelargonidin 3,5 diglucoside # 0.87 0.01 29.54 0.66 34.1 Peonidin 3,5 diglucoside 0.87 0.00 33.22 0.60 38.2 Malvidin 3,5 diglucoside 0.53 0.00 18.16 0.45 34.1 Total Anthocyanins* 3.41 0.20 130.04 2.73 38.1 Other phenolic compounds Ellagic acid 7.23 0.49 42.86 2.19 5.9 Myricetin 1.24 0.35 7.53 0.24 6.1 Quercetin 0.60 0.10 2.79 0.05 4.7 Kaempferol 0.30 0.04 1.33 0.00 4.4 Catechin ND 17.32 0.24 NA Epicatechin ND 10.56 0.29 NA Total Phenolic Content (mg GAE/g) 16.61 0.43 691.75 65.6 41.6 Sugars Fructose 165.66 4.06 ND NA Glucose 137.44 4.74 ND NA # Peaks coeluted on HPLC. Data are mean standard deviation for triplicate tests. determined by pH diffe rential method. ND Not Detected, NA Not Applicable

PAGE 110

110 Fig ure 5 1. Static adsorption results based on total anthocyanin content on different resins in juice pomace : A. Adsorption capacity and ratio Different upper case letters indicate significant differences of bars (p 0.05). Different lower case letters indicate significant differences of lines (p 0.05) B. Recovery rate (%) using 70 and 95% ethanol. indicate s significant differences (p 0.0 5) in the recovery (%). C. D esorption ratio (%) using 70 and 95% ethanol indicates significant differences (p 0.05) in the desorption ratio (%) Results are mean of three determinations.

PAGE 111

111 Figure 5 2. Static adsorption results based on total anthocyanin content on different resins in wine pomace: A. Adsorption capacity and ratio. Different upper case case Recovery rate (%) recovery (%). C. Desorption ratio (%) using 70 and 95% ethanol. indicates of three determinations

PAGE 112

112 Figure 5 3. Static adsorption results based on total phenolic content on different resins in juice pomace: A. Adsorption capacity and ratio. Different upper case letters ). Different lower case letters recovery (%). C. Desorption ratio (%) using 70 and 95% ethanol. ind icates of three determinations

PAGE 113

113 Figure 5 4. Static adsorption results based on total phenolic content on different resins in wine pomace: A. Adsorption capacity and ratio. Different upper case letters case letters 70 and 95% ethanol. indicates significant differences ( recovery (%). C. Desorption ratio (%) using 70 and 95% ethanol. indicates of three determinations

PAGE 114

114 Fig ure 5 5 Adsorption kinetic curves for juice pomac e A. Total anthocyanin content B. Total phenolic content on FPX 66, XAD 16N and XAD 1180 Results are mean of three determinations.

PAGE 115

115 Figure 5 6. Adsorption kinetic curves for wine pomace A. Total anthocyanin content B. Total phenolic content on FPX 66, XAD 16N and XAD 1180. Results are mean of three determinations.

PAGE 116

116 Fig ure 5 7 Adsorption isotherms for juice pomace based on total anthocyanin content on FPX 66, XAD 16N and XAD 1180 at A. Room temperature (25C) B. 30C C. 35C. Re sults are mean of three determinations.

PAGE 117

117 Figure 5 8. Dynamic b r eakthrough curves of total anthocyanins from muscadine juice pomace on column packed with FPX 66 resin at different flow rates. Results are mean of two determinations.

PAGE 118

118 Fig ure 5 9 Dynamic desorption curves of total anthocyanins from muscadine juice pomace on column packed with FPX 66 resin at different flow rates. Results are mean of two determinations.

PAGE 119

119 CHAPTER 6 EFFECT OF METHYL JASMONATE TREATMENT ON POSTHARVEST QUALITY O F MUSCADINE GRAPES AT DIFFERENT STORAGE TEMPERATURES Background Muscadine grapes harvested at optimum maturity should maintain their quality during the time required for storage and marketing. After harvest the major compositional changes occurring in g rapes are loss of sugars and organic acids. In addition, other oxidative changes can occur such as degradation of anthocyanins by enzymatic and non enzymatic oxidation resulting in dull or brownish colored product 115 Improper storage of fresh grapes can cause water loss and fruit softening, and favor development of decay 116 Muscadine grapes have a very short fresh storage life (2 to 3 weeks) compared to other V itis species which can be stored for 6 to 8 weeks 117 Extending the short storage life of muscadines would eliminate the major problem limiting fresh fruit marketing Moreover, antioxidant content is becoming an increasingly important parameter with respect to fruit and vegetable quality. Fruits and vegetables have been subjected to various post harvest treatments to prolong their shelf life and maintain secondar y metabolism and can affect the antioxidant s in them 118 Thus, such targeted treatments may be used to obtain fruits and vegetables enriched with phytochemicals for sale as fresh market products or used as raw materials for fun ctional foods and supplements 119 Postharvest elicitors can be divided into two categories: physical and chemical. The physical elicitors include low temperature, heat treatment, ultraviolet and gamma irradiation, and altered gas composition. Chemical elicitors are primarily plant hormones such as salicylic acid, jasmonic acid, methyl jasmonate (MeJA) and ethylene.

PAGE 120

120 Application of postharvest elicitors had been successfully used in fruits and vegetables with intent of improving their quality and phytochemical content 118 MeJA is a naturally occurring compound and plays an important role in plant growth and development, fruit ripening and responses to environmental stresses 120 Because MeJA is already classified by the U.S. Food and Drug Administration as a Generally Recognized As Safe (GRAS) substance, it has the potential for commercial applications in postharvest treatments for quality maintenance by reducing decay and enhancing the phenolic content. It has been reported that MeJA treatment could effectively suppress postharvest diseases of various fruits including sweet cherry 121 loquat 122 peach 123 and grapefruit 124 In addition, it has been reported that postharvest MeJA treatment maintained higher levels of bioactive compounds and enhanced antioxidant capacity in berry fruits including blackberries, raspberries, and strawberries 125 127 Postharvest treatment of Golden delicious apples with MeJA carotene accumulation 128 A study conducted on Chinese b ayberries showed that the postharvest application of MeJA can effectively reduce fruit decay and improve their antioxidant capacity 129 However, there is no published data on the effect of postharvest MeJA treatment on the changes in the quality and phenolic content of muscadine grapes upon storage. The objective of this research is to investigate the effect of postharvest MeJA treatment on muscadine grape quality and phytochemical composition. Materials and M ethods Chemicals azobis(2 amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI). Gallic acid, 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid

PAGE 121

121 (Trolox), HPLC grade methanol, acetic acid, formic acid, Folin Ciocalteau reagent, Flo urescein and sodium carbonate were purchased from Fischer Scientific Co. (Pittsburg, PA). Methyl jasmonate and 2,2 Diphenyl 1 picrylhydrazyl (DPPH) were obtained from Sigma Aldrich (St. Louis, MO). MeJA T reatment Noble and Alachua varieties of muscadine grapes were obtained at commercial maturity (bas ed on full color development, Brix above 1 1 and 14 for Noble and Alachua, respectively) from vineyards in Florida. Grapes were selected for uniform size, color and absence of defects and then randomly divided into three lots for each variety For each lot of Noble and Alachua variety 1.6 and 1.8 kg of grapes were weighed respectively and pla ced in 7 L airtight plastic container s for MeJA treatment. An appropriate amount of MeJA or water for control was spotted on a filter paper inside the containers and incubated at 20C for 24 hrs, allowing MeJA to evaporate. MeJA concentrations of 0, 10 and 100 mol/l were used 129 The concentrations of MeJA were chosen based on previous work on other fruits. Three replicates of each treatment were cond ucted. Before opening the containers the levels of CO 2 / O 2 were analyzed in each container using an O 2 and CO 2 meter (PBI Dansensor, Checkmate 9900, Ringsted, Denmark) The containers were ventilated in a fume hood and grapes from each variety were further divided into two lots of equal weight for storage at 5 and 20C for 7 days. Samples were taken at 3, 5 and 7 days during storage for quality parameter analysis. For analyzing fruit decay, 50 (Noble) and 100 (Alachua) grapes were taken f rom eac h lot and stored for 2 week s at 5 and 20 C

PAGE 122

122 D ecay Assessment Fruit decay was recorded after visually examining the 50 (Noble) and 100 (Alachua) grapes per replication for any defects such as mold growth, shrinkage or overripe grapes Grapes were monitored for two weeks at 5 and 20C storage temperatures and results were expressed as % decay. pH and T otal S oluble S olids pH of the juice was measured using a pH meter and total soluble solids (Brix) were measured using a bench top refractometer (Leica Abbe M ark 11, Fisher Scientific, Pittsburg, PA). Extraction and Sample P reparation The skin of the grapes was manually separated from seeds and pulp, and freeze dried. Subsequently, it was ground into a fine powder using a Waring kitchen blender and extracted (1 g) with 15 mL of methanol/water/acetic acid (85:15:0.5; v/v) in glass tubes. The samples were then vortexed for 30 s, sonicated for 5 min and kept in the dark at room temperature for 20 min. The tubes were then centrifuged at 1317 g for 10 min and the supernatant was removed. The samples were extracted again with 10 mL of methanol/water/acetic acid using the same procedure. The supernatants from two extractions were pooled and transferred into a 25 mL volumetric flask. Methanol/water /acetic acid was added to make up the final volume to exactly 25 mL. Total Anthocyanin Assay Total anthocyanin content in pomace extracts was measured using the pH differential spectrophotometric method described by Giusti and Wrolstad 88 The extracts were dissolved in 0.025 mol/L potassium chloride buffer, pH 1.0 and 0.4 mol/L sodium acetate buffer, pH 4.5 with pre determined dilution factor. Absorbance at 520

PAGE 123

123 and 700 nm was measured o n a DU 730 Life Science UV/vis spectrophotometer (Beckman Coulter, Fullerton, CA) after 30 min of incubation at room temperature. The absorbance (A) of the diluted sample was then calculated using (A 520 A 700 ) pH 1.0 (A 520 A 700 ) pH 4.5 The monomeric an thocyanin concentration in the original sample was calculated in cyanidin 3, 5 diglucoside equivalents according to this formula: (A MW diglucoside is used, the molar DF was dilution factor; 1000 is the factor to convert gram to milligram and the A was absorbance. Results for total anthocyanin content were expressed as milligram cyanidin 3, 5 diglucoside equivalent per gram of fresh grape skins (mg cyanidin 3, 5 digluco side/g). Folin Ciocalteu Assay The extracts were diluted to appropriate concentration for analysis. The total phenolic content was determined as reported previously 6 .The results were expressed as milligrams of gallic acid equivalents per gram of fresh grape skins (mg of GAE/ g). Oxygen Radical Absorbance Capacity (ORA C) The ORAC assay for extracted samples was conducted on a Spectra XMS samples were added to the designated wells of a 96 well black plate. This was followed by the add The mixture was incubated at 37 C for excitation and 530 nm emissions at 1 min intervals for 40 min. Trolox was used to generate a s tandard curve. The antioxidant capacities of extracts were expressed as

PAGE 124

124 DPPH Assay The DPPH scavenging activities of samples were measured using a previously published method 75 In summary, DPPH stock solution was prepared by dissolving 20 mg of DPPH in 10 0 mL methanol and stored at 20 C prior to use. DPPH working solution was freshly prepared by mixing 2.8 mL DPPH stock solution a nd 7.2 mL methanol. Absorbance at 515 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA). Diluted extracts (50 L) were added to 950 L DPPH working solution and incubated for 60 min in the dark at room temperature. T rolox solutions from 100 to 1000 M were added to DPPH working solution as standards. Results of the DPPH scavenging activity of grape skin extracts were expressed as micromoles of Trolox equivalents (TE) per gram of fresh grape skins Stati stical Analysis Three way analyses of variance (ANOVA) with Tukey HSD pairwise comparison of the means were performed using Sigmaplot (version 11.0, Systat software Inc., Chicago, IL). Data are expressed as means the standard deviation of three independ statistical results is shown in Table 6 1. Results and Discussion CO 2 and O 2 Analysis Table 6 2 shows the headspace CO 2 and O 2 concentration in the sealed containers after MeJA treatment of Noble and Alachua grapes. In Noble variety the % of CO 2 and O 2 were not affected by the MeJA treatment, however decrease in headspace O 2 and increase in CO 2 levels were observed in the MeJA treated Alachua grapes

PAGE 125

125 compared to control. This implies that rate of respiration was higher in MeJA treated Alachua grapes and modified atmosphere was created in the containers. D ecay Assessment There was no effect of the MeJA treatment on grape decay compared to control (Table 6 3 ). However, tempe rature had a significant effect (p<0.05) on decay with more number of grapes decayed at 20C compared to 5C. Similar effect of temperature was observed in strawberries 130 pH and Total S oluble S olids The effect of MeJA on pH and t otal soluble solids of the treated and control juice is shown in Table 6 4 pH of the juice from MeJA treated Noble and Alachua was significantly lower (p<0.001) compared to control at all the sampling times and storage temperatures (5 or 20C). This indic ates the amount of organic acids in the MeJA treated grapes were higher than control. The effect was more pronounced at 100 mol/L MeJA concentration. A number of studies showed increase in organic acid content of MeJA treated fruits 131 132 There was no effect of storage temperature on pH of the juice from control and MeJA treated grapes. Total soluble solids (TSS) were significantly higher (p<0.001) in juice from MeJA treated Alachua; however no effect of the treatment was observed in juic e from Noble variety. A study conducted on kiwifruit treated with MeJA showed increase in TSS 133 while another study on ras pberry showed no effect of MeJA on pH 115 A significant decrease in TSS of juice from control and MeJA treated Alachua and Noble grapes was observed with sampling time 115, 130 Total Anthocyanin C ontent There was no significant effect of the treatment on total anth ocyanin content of Noble and Alachua (Tables 6 5 and 6 6 ). The anthocyanin content was affected by

PAGE 126

126 storage time and temperature in both the varieties. In case of Noble grapes, the anthocyanin content increased 5 and 8% in control and 100 mol/L MeJA treate d samples, respectively after 7 days of storage at 5C. For the same storage time the anthocyanin content at 20C was increased by 26 and 33% in control and 100 mol/L MeJA treated samples, respectively. Similar effect of storage time and temperature was o bserved in Alachua variety. The increase in anthocynain content after storage at 5 or 20C might be due to ripening related changes going on during storage. In addition, the moisture loss and sugar metabolism during storage can also contribute to increase in anthocyanin content. The continuation of anthocyanin synthesis during postharvest storage has been report ed in strawberries, raspberries, highbush blueberries 134 and grapes 135 Total P henolic C ontent and A ntioxidant C apacities There was no significant difference in the phenolic content or antioxidant capacity (measured by ORAC or DPPH) in control and MeJA treated samples of both the varieties (Tables 6 5 ad 6 6 ). A significant increase in the phenolic content was observed in both varieties with storage time and temperature. The results were consistent with total anthocyanin con tent. Such an increase could be due to release of phenolic compounds from their complexes with other components like proteins and carbohydrates. Both storage time and temperature significantly increased the antioxidant capacity (ORAC and DPPH) in control and MeJA treate d Alachua grapes. However, only storage time affected the antioxidant capacity measured by ORAC in control or MeJA treated Noble grapes and there was no effect of storage time and temperature on the antioxidant capacity measured by DPPH assay. The inconsis tency

PAGE 127

127 in the antioxidant capacity results could be attributed to varietal differences or differences in chemistry and mechanisms involved in ORAC and DPPH assays. Summary Postharvest MeJA treatment did not affect the total anthocyanins, phenolic content a nd antioxidant capacity of muscadine grapes. However, there was a significant effect of storage time and temperature on the measured quality parameters. These observations contradicted other studies in which MeJA enhanced the phenolic compounds, anthocyani ns, antioxidant capacity, quality and postharvest life of Chinese bay berries 129 and raspberries 115 However, a study con ducted on strawberries showed no effect of postharvest MeJA on flavonol content 136 Several aspects such as different fruit s cultivars, and influence of other growth regulators such as ethylene and salicylic acid might affect the results. When applied on various fruits, MeJA stim ulates the production of phenylalanine ammonia lyase, a key enzyme of the phenylpropanoid pathway which is directly involved in the biosynthesis of phenolic compounds, including anthocyanins stilbenes, and flavonoids 137 The major effect of MeJA treatment on muscadine grapes appeared to be on pH and TSS which is in agreement with previous stud ies 131, 133 In conclusion, we saw no impact of MeJA treatment on decay, anthocyanins, phenolic content and antioxidant capacity of muscadine grapes.

PAGE 128

128 Table 6 1 ANOVA for dependent variables for MeJA treatment, storage time, temperature and their interactions for Noble and Alachua grapes. Variety Tests Time Temperature Treatment TimeTemperature # Noble pH NS NS TSS NS NS Total anthocyanins NS Total phenolics NS ORAC NS NS DPPH NS NS NS NS Alachua pH NS TSS NS Total anthocyanins NS NS Total phenolics NS NS ORAC NS DPPH NS NS represent significance and NS represents non significance at p 0.05. # Interactions with significant results are shown.

PAGE 129

129 Table 6 2 CO 2 and O 2 levels in the headspace of the containers after MeJA treatment of Noble and Alachua grapes. Variety MeJA concentration (mol/L) % Oxygen % Carbon dioxide Noble 0 13.00 0.75 a 7.33 0.50 a 10 12.70 0.30 a 7.57 0.21 a 100 13.10 0.14 a 7.30 0.14 a Alachua 0 14.87 0.59 a 6.57 0.64 b 10 12.93 0.38 b 8.73 0.40 a 100 12.90 0.40 b 8.73 0.57 a Results are mean standard deviation of three determi nations. Different superscripts for each variety

PAGE 130

130 Table 6 3 Effect of MeJA treatment on decay of Noble and Alachua grapes after 2 weeks of storage at 5 and 20C. Cultivar Storage Temperature (C) MeJA concentration (mol/L) % grape decay Noble 5 0 2.67 1.15 10 2.00 2.00 100 2.67 1.15 20 0 14.67 1.15 10 10.67 1.15 100 13.33 3.06 Alachua 5 0 1.67 0.58 10 4.00 2.65 100 5.00 2.00 20 0 22.00 3.46 10 20.33 5.86 100 25.67 5.03

PAGE 131

131 Table 6 4 pH and t otal soluble solids (Brix) of Noble and Alachua juice from control and MeJA treated muscadine grapes. Cultivar Storage Temperature (C) Days of Storage MeJA concentration (mol/L) pH Total soluble solids (Brix) Noble 5 3 0 3.24 0.04 11.20 0.52 10 3.24 0.01 11.33 0.23 100 3.23 0.03 11.73 0.23 5 0 3.27 0.03 11.53 0.55 10 3.27 0.02 11.67 0.25 100 3.22 0.02 11.53 0.32 7 0 3.60 0.02 11.70 0.17 10 3.54 0.02 11.27 0.49 100 3.50 0.04 11.13 0.06 20 3 0 3.26 0.04 12.07 0.21 10 3.26 0.02 11.83 0.68 100 3.19 0.08 11.93 0.29 5 0 3.24 0.02 11.27 0.32 10 3.25 0.04 11.47 0.25 100 3.28 0.05 11.75 0.07 7 0 3.53 0.02 11.33 0.21 10 3.55 0.06 11.40 0.36 100 3.51 0.03 11.23 0.12 Alachua 5 3 0 3.55 0.03 15.13 0.42 10 3.59 0.06 15.72 0.47 100 3.51 0.03 15.57 0.21 5 0 3.62 0.08 15.53 0.67 10 3.65 0.03 16.10 0.75 100 3.59 0.04 15.90 0.26 7 0 3.49 0.05 14.70 0.53 10 3.54 0.04 15.53 0.23 100 3.42 0.05 15.03 0.40 20 3 0 3.55 0.07 14.70 0.36 10 3.55 0.05 15.20 0.44 100 3.57 0.06 15.73 0.58 5 0 3.58 0.02 15.23 0.50 10 3.58 0.03 15.43 0.32 100 3.49 0.06 15.53 0.23 7 0 3.49 0.03 15.07 0.21 10 3.50 0.05 15.30 0.44 100 3.39 0.03 14.50 0.14

PAGE 132

132 Table 6 5 Total anthocyanin, total phenolic and antioxidant capacities (ORAC and DPPH) of control and MeJA treated Noble grape skins. Storage Temp (C) Days of Storage MeJA concentration (mol/L) Total anthocyanin content (mg/g) Total phenolic content (mg/g) DPPH ORAC 5 3 0 1.99 0.28 3.73 0.09 21.60 1.24 53.90 6.20 10 2.01 0.31 3.73 0.53 20.69 2.93 47.87 9.33 100 1.93 0.21 3.76 0.32 20.80 3.19 53.02 3.79 5 0 2.13 0.05 3.68 0.08 21.84 1.65 62.91 7.31 10 2.14 0.26 3.37 0.20 20.28 1.72 52.30 9.72 100 1.97 0.34 3.60 0.58 20.79 2.48 52.14 5.06 7 0 2.09 0.13 3.76 0.12 19.99 3.41 43.52 4.50 10 2.20 0.38 4.09 0.61 22.33 1.18 57.88 5.11 100 2.10 0.02 3.84 0.28 21.03 2.25 44.74 3.40 20 3 0 2.14 0.04 3.72 0.09 20.75 1.21 50.46 4.08 10 2.12 0.27 3.51 0.18 20.03 2.00 52.81 4.25 100 2.04 0.24 3.48 0.17 19.34 2.14 47.50 5.78 5 0 2.54 0.16 4.16 0.26 23.58 1.12 63.06 3.58 10 2.48 0.22 4.01 0.33 23.08 1.38 51.60 3.40 100 2.20 0.42 3.71 0.19 21.49 3.04 53.67 9.90 7 0 2.91 0.57 4.42 0.67 23.72 2.11 63.11 5.05 10 2.77 0.16 4.25 0.06 22.33 2.86 48.73 5.06 100 3.03 0.13 4.68 0.04 24.79 1.85 61.48 2.65

PAGE 133

133 Table 6 6 Total anthocyanin, total phenolic and antioxidant capacities (ORAC and DPPH) of control and MeJA treated Alachua grape skins. Storage Temp (C) Days of Storage MeJA concentration (mol/L) Total anthocyanin content (mg/g) Total phenolic content (mg/g) DPPH ORAC 5 3 0 2.70 0.57 3.73 0.20 19.05 4.77 67.70 4.60 10 2.75 0.87 3.42 0.84 18.79 2.23 53.06 8.44 100 2.41 0.92 3.56 1.10 18.62 5.90 68.08 10.24 5 0 3.83 0.17 6.42 0.50 22.53 1.18 71.65 5.90 10 3.70 0.35 5.27 0.24 22.54 0.79 64.63 4.85 100 3.21 0.35 4.81 0.37 20.14 0.26 65.43 5.35 7 0 3.36 0.75 4.36 0.83 22.92 2.76 55.41 3.12 10 3.74 0.27 5.22 0.81 23.41 4.10 64.11 2.38 100 2.82 0.70 4.02 0.79 19.80 4.77 50.86 7.68 20 3 0 3.22 0.23 4.02 0.16 22.16 3.97 64.02 2.35 10 2.81 0.14 4.10 0.54 22.47 1.75 73.86 11.34 100 2.70 0.46 3.57 0.18 20.08 2.55 55.57 2.41 5 0 3.63 0.10 5.98 1.22 24.40 0.56 49.68 2.37 10 4.09 0.70 5.88 0.33 23.06 2.20 71.13 7.81 100 3.95 0.66 5.49 0.43 23.94 0.61 83.37 13.91 7 0 3.72 0.27 5.48 0.03 22.93 2.81 67.78 4.57 10 4.21 0.44 5.49 0.36 26.09 3.20 42.59 1.17 100 3.78 0.52 5.99 0.52 25.05 3.22 59.93 14.60

PAGE 134

134 CHAPTER 7 CONCLUSIONS Muscadine seeds have high phenolic content and antioxidant capacity compared to skin and pulp. A total of 88 phenolic compounds of diverse structures were tentat ively identified in Noble variety including 17 compounds identified for the first time in muscadine grapes. The hig h antioxidant capacity and total phenolic content of the muscadine seeds make them a potentially significant source of compounds with nutraceutical properties. The structural elucidation of phenolic compounds in muscadine grapes could provide an improved u nderstanding of color and flavor changes occurring in muscadine wine and juice upon storage. ABA doe s not affect grape berry weight, pH or total soluble solids (Brix) of juice. E xogenous ABA enhance d the antioxidant capacity, anthocyanins and phenolic con tent of muscadine grapes but these effects varied depending upon the cultivar and possibly environmental factors. Further research may show that ABA applied at critical st ages of grape development offer opportunities to increase the content of key phytoch emicals without affecting the yield. The grapes with enhanced phytochemicals could attract health conscious consumers and also increase the marketability of fresh fruits. We found hot water to be the most suitable solvent for the extraction of anthocyanins from muscadine pomace. Organic solvents work ed best for the extraction of ellagic acid and flavonols. The advantages of using water as a solvent outweigh the use of organic solvents as it is non toxic and inexpensive. In addition water fits better for th e industry scale because it significantly reduces the overall production cost. Results from resin adsorption/desorption study indicate that FPX 66 is the most suitable resin among selected commercial adsorbents for the recovery of anthocyanins

PAGE 135

135 from muscadi ne juice pomace. Resin adsorption caused a tremendous increase in the content of anthocyanins, ellagic acid, and flavonols in the concentrated extract. Using FPX 66 resin, a concentrated pomace extract was produced that contained 13% (w/w) anthocyan ins wit h no detectable sugars. The optimization of the resin adsorption process in the present study sets parameters for the development of pilot scale separation and concentration of anthocyanins from muscadine pomace. This extract could potentially find applica tion as a natural colorant, dietary supplement, antioxidant ingredient for functional foods, and/or as a raw material in cosmetic and pharmaceutical industry preparations. Postharvest MeJA treatment did not affect the total anthocyanins, phenolic content and antioxidant capacity of muscadine grapes. However, significant effect s of time and temperature of storage o n measured quality parameters w ere observed. Contrary to the ABA study, the major effect of MeJA treatment appeared to be on pH and total soluble solids

PAGE 136

136 LIST OF REFERENCES 1. Ector, B. J.; Magee, J. B.; Hegwood, C. B.; Coign, M. J. Resveratrol Concentration in Muscadine Berries, Juice, Pomace, Purees, Seeds, and Wines. Am. J. Enol. Vitic. 1996, 47 57 62. 2. Lee, J. H.; Talcott, S. T. Ellagic acid and ellagitannins affect on sedimentation in muscadine juice and wine. J. Agric. Food Chem. 2002, 50 3971 3976. 3. Bouquet, A. Resistance to grape Fanleaf virus in muscadine grape inoculated wi th Xiphinema index. Plant Dis. 1981, 65 (10), 791 792. 4. Hudson, T. S.; Hartle, D. K.; Hursting, S. D.; Nunez, N. P.; Wang, T. T. Y.; Young, H. A.; Arany, P.; Green, J. E. Inhibition of Prostate Cancer Growth by Muscadine Grape Skin Extract and Resveratr ol through Distinct Mechanisms. Cancer Res 2007, 67 (17), 8396 8405. 5. Lee, J. H.; Johnson, J. V.; Talcott, S. T. Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC ESI MS. J. Agric. Food Chem. 2005, 53 6003 6 010. 6. Sandhu, A. K.; Gu, L. Antioxidant capacity, phenolic content, and profiling of phenolic compounds in the seeds, skin, and pulp of Vitis rotundifolia (Muscadine Grapes) as determined by HPLC DAD ESI MS n J. Agric. Food Chem. 2010, 58 4681 92. 7. Kim, T. J.; Silva, J. L.; Jung, Y. S. Antibacterial activity of fresh and processed red muscadine juice and the role of their polar compounds on Escherichia coli O157:H7. J App Microbio 2009, 107 (2), 533 539. 8. Kim, T. J.; Weng, W. L.; Silva, J. L.; Jung, Y. S.; Marshall, D. Identification of Natural Antimicrobial Substances in Red Muscadine Juice against Cronobacter sakazakii. J. Food Sci. 2010, 75 (3), M150 M154. 9. Park, Y. J.; Biswas, R.; Phillips, R. D.; Chen, J. Antibacterial Activities of Blue berry and Muscadine Phenolic Extracts. J. Food Sci. 2011, 76 (2), M101 M105. 10. Ho, L.; Chen, L. H.; Wang, J.; Zhao, W.; Talcott, S. T.; Ono, K.; Teplow, D.; Humala, N.; Cheng, A.; Percival, S. S.; Ferruzzi, M.; Janle, E.; Dickstein, D. L.; Pasinetti, G. M. Heterogeneity in Red Wine Polyphenolic Contents Differentially Influences Alzheimer's Disease type Neuropathology and Cognitive Deterioration. J Alzheimer's Dis 2009, 16 (1), 59 72. 11. Bralley, E. E.; Hargrove, J. L.; Greenspan, P.; Hartle, D. K. T opical anti inflammatory activities of vitis rotundifolia (muscadine grape) extracts in the tetradecanoylphorbol acetate model of ear inflammation. J. Med. Food 2007, 10 (4), 636 642.

PAGE 137

137 12. Greenspan, P.; Bauer, J. D.; Pollock, S. H.; Gangemi, J. D.; Mayer, E. P.; Ghaffar, A.; Hargrove, J. L.; Hartle, D. K. Antiinflammatory Properties of the Muscadine Grape ( Vitis rotundifolia ). J. Agric. Food Chem. 2005, 53 (22), 8481 8484. 13. Mertens Talcott, S. U.; Lee, J. H.; Percival, S. S.; Talcott, S. T. Induction of cell death in Caco 2 human colon carcinoma cells by ellagic acid rich fractions from muscadine grapes ( Vitis rotundifolia ). J. Agric. Food Chem. 2006, 54 5336 5343. 14. Yi, W.; Fischer, J.; Akoh, C. C. Study of anticancer activities of muscadine grape phenolics in vitro. J. Agric. Food Chem. 2005, 53 8804 8812. 15. Mertens Talcott, S. U.; Percival, S. S.; Talcott, S. T. Extracts from red muscadine and cabernet sauvignon wines induce cell death in MOLT 4 human leukemia cells. Food Chem. 2008, 108 (3), 824 832. 16. Gourineni, V.; Shay, N. F.; Chung, S.; Sandhu, A. K.; Gu, L. Muscadine Grape ( Vitis rotundifolia ) and Wine Phytochemicals Prevented Obesity Associated Metaboli c Complications in C57BL/6J Mice. J. Agric. Food Chem. 2012, 60 (31), 7674 7681. 17. You, Q.; Chen, F.; Wang, X.; Luo, P. G.; Jiang, Y. Inhibitory Effects of Muscadine Glucosidase and Pancreatic Lipase Activities. J. Agric. Food Chem. 20 11, 59 9506 9511. 18. Farrar, J. L.; Hartle, D. K.; Hargrove, J. L.; Greenspan, P. Inhibition of protein glycation by skins and seeds of the muscadine grape. BioFactors 2007, 30 (3), 193 200. 19. Banini, A. E.; Boyd, L. C.; Allen, J. C.; Allen, H. G.; S auls, D. L. Muscadine grape products intake, diet and blood constituents of non diabetic and type 2 diabetic subjects. Nutr 2006, 22 (11 12), 1137 1145. 20. Bravo, L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev 1998, 56 (11), 317 333. 21. Waterman, P. G.; Mole, S. Analysis of Phenolic Plant Metabolites Blackwell Scienctific Publications, (Chapter 1): Oxford, U. K., 1994; p 5 20. 22. Arts, I. C. W.; Hollman, P. C. H. Polyphenols and disease risk in epidemiologic studies1 4. Am. J. Clin. Nutr. 2005, 81 317S 325S. 23. Djousse, L.; Arnett, D. K.; Coon, H.; Province, M. A.; Moore, L. L.; Ellison, R. C. Fruit and vegetable consumption and LDL cholesterol: the National Heart, Lung, and Blood Institute Family Heart Study. Am. J. Clin. Nutr. 2004, 79 213 217.

PAGE 138

138 24. Kaur, C.; Kapoor, H. C. Antioxidants in fruits and vegetables The millenium health. Int. J. Food Sci.Tech. 2001, 36 703 725. 25 Bors, W.; Werner, H.; Michel, C.; Saran, M. Flavonoids as antioxidants: determination of radical scavenging efficiencies. Methods Enzymol. 1990, 186 343 355. 26. Hanasaki, Y.; Ogawa, S.; Fukui, S. The correlation between active oxygen scavenging and an tioxidative effects of flavonoids. Free Radical Biol. Med. 1994, 16 845 850. 27. Pastrana Bonilla, E.; Akoh, C. C.; Sellappan, S.; Krewer, G. Phenolic content and antioxidant capacity of muscadine grapes. J. Agric. Food Chem. 2003, 51 5497 5503. 28. Hu ang, Z.; Wang, B.; Williams, P.; Pace, R. D. Identification of anthocyanins in muscadine grapes with HPLC ESI MS. LWT Food Sci.Technol. 2009, 42 819 824. 29. Hakkinen, S. H.; Karenlampi, S. O.; Heinonen, I. M.; Mykkanen, H. M.; Torronen, A. R. Content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries. J. Agric. Food Chem. 1999, 47 2274 2279. 30. Seeram, N. P.; Lee, R.; Scheuller, H. S.; Heber, D. Identification of phenolic compounds in strawberries by liquid chromatography ele ctrospray ionization mass spectroscopy. Food Chem. 2006, 97 (1), 1 11. 31. Singleton, V. L.; Rossi, J. A., Jr. Colorimetry of Total Phenolics with Phosphomolybdic Phosphotungstic Acid Reagents. Am. J. Enol. Vitic. 1965, 16 144 158. 32. Smith, J. S. Evaluation of Analytical Data. In Food Analysis Third ed.; Nielsen, S. S., Ed. Plenum: New York 2003; pp 51 64. 33. Talcott, S. T.; Lee, J. H. Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. J. Agric. Food Chem. 2 002, 50 3186 3192. 34. Rodrguez Montealegre, R.; Romero Peces, R.; Chacn Vozmediano, J. L.; Martnez Gascuea, J.; Garca Romero, E. Phenolic compounds in skins and seeds of ten grape Vitis vinifera varieties grown in a warm climate. J. Food Comp. Anal y. 2006, 19 687 693. 35. Sims, C. A.; Morris, J. R. Effects of Acetaldehyde and Tannins on the Color and Chemical Age of Red Muscadine ( Vitis rotundifolia ) Wine. Am. J. Enol. Vitic. 1986, 37 163 165.

PAGE 139

139 36. Beecher, G. R. Overview of Dietary Flavonoids: Nomenclature, Occurrence and Intake. J. Nutr. 2003, 133 3248S 3254. 37. Peterson, J.; Dwyer, J. Flavonoids: Dietary occurrence and biochemical activity. Nutr. Res. 1998, 18 1995 2018. 38. Maatta Riihinen, K. R.; Kamal Eldin, A.; Torronen, A. R. Identification and Quantification of Phenolic Compounds in Berries of Fragaria and Rubus Species (Family Rosaceae). J. Agri. Food Chem. 2004, 52 6178 6187. 39. Regos, I.; Urbanella, A.; Treutter, D. Identification and quantification of phenolic compounds from the forage legume sainfoin ( Onobrychis viciifolia ). J. Agric. Food Chem. 2009, 57 5843 5852. 40. Haddock, E. A.; Gupta, R. K.; Al Shafi, S. M. K.; Layde n, K.; Haslam, E.; Magnolato, D. The metabolism of gallic acid and hexahydroxydiphenic acid in plants: biogenetic and molecular taxonomic considerations. Phytochem. 1982, 21 1049 1062. 41. Haslam, E. Gallic acid metabolism. In Plant Polyphenols, Vegetabl e Tannins Revisited Cambridge University Press: Cambridge, U. K., 1989; p 91. 42. Michael, N. C.; Augustin, S. Ellagitannins nature, occurrence and dietary burden. J. Sci. Food Agri. 2000, 80 1118 1125. 43. Khanbabaee, K.; van Ree, T. Tannins: classif ication and definition. Nat. Prod Rep. 2001, 18 641 649. 44. Aaby, K.; Ekeberg, D.; Skrede, G. Characterization of Phenolic Compounds in Strawberry ( Fragaria ananassa ) Fruits by Different HPLC Detectors and Contribution of Individual Compounds to Total Antioxidant Capacity. J. Agri. Food Chem. 2007, 55 4395 4406. 45. Barry, K. M.; Davies, N. W.; Mohammed, C. L. Identification of hydrolysable tannins in the reaction zone of Eucalyptus nitens wood by high performance liquid chromatography electrospray i onisation mass spectrometry. Phytochem. Anal. 2001, 12 120 127. 46. Hager, T. J.; Howard, L. R.; Liyanage, R.; Lay, J. O.; Prior, R. L. Ellagitannin composition of blackberry as determined by HPLC ESI MS and MALDI TOF MS. J. Agric. Food Chem. 2008, 56 6 61 669. 47. Hanhineva, K.; Rogachev, I.; Kokko, H.; Mintz Oron, S.; Venger, I.; Krenlampi, S.; Aharoni, A. Non targeted analysis of spatial metabolite composition in strawberry ( Fragaria ananassa ) flowers. Phytochem. 2008, 69 2463 2481.

PAGE 140

140 48. Mullen, W.; McGinn, J.; Lean, M. E. J.; MacLean, M. R.; Gardner, P.; Duthie, G. G.; Yokota, T.; Crozier, A. Ellagitannins, Flavonoids, and Other Phenolics in Red Raspberries and Their Contribution to Antioxidant Capacity and Vasorelaxation Properties. J. A gri. Food Chem. 2002, 50 5191 5196. 49. Salminen, J. P.; Ossipov, V.; Loponen, J.; Haukioja, E.; Pihlaja, K. Characterisation of hydrolysable tannins from leaves of Betula pubescens by high performance liquid chromatography mass spectrometry. J. Chromato gr. A. 1999, 864 283 291. 50. Meyers, K. J.; Swiecki, T. J.; Mitchell, A. E. Understanding the native Californian diet: Identification of condensed and hydrolyzable tannins in tanoak acorns ( Lithocarpus densiflorus ). J. Agric. Food Chem. 2006, 54 7686 7 691. 51. Wei, J.; Ying Feng, W.; Ri Li, G.; Hai Ming, S.; Cun Qin, J.; Peng Fei, T. Simultaneous analysis of multiple bioactive constituents in Rheum tanguticum Maxim. ex Balf. by high performance liquid chromatography coupled to tandem mass spectrometry. Rapid Commun. Mass Spectro. 2007, 21 2351 2360. 52. Soong, Y. Y.; Barlow, P. J. Isolation and structure elucidation of phenolic compounds from longan ( Dimocarpus longan Lour. ) seed by high performance liquid chromatography electrospray ionization mass s pectrometry. J. Chromatogr. A. 2005, 1085 270 277. 53. Bernard, Q.; Marie Christine, R. Evidence for linkage position determination in known feruloylated mono and disaccharides using electrospray ion trap mass spectrometry. J. Mass Spectro. 2004, 39 11 53 1160. 54. Flora, L. F. Influence of heat, cultivar and maturity on the anthocyanin 3,5 diglucosides of muscadine grapes. J. Food Sci. 1978, 43 1819 1821. 55. Goldy, R. G.; Ballinger, W. E.; Maness, E. P. Fruit anthocyanin content of some Euvitis Vitis rotundifolia hybrids. Hort. Sci. 1986, 111 955 960. 56. Lamikanra, O. Development of anthocyanin pigments in muscadine grapes Hort. Sci. 1988, 23 591 599. 57. Dou, J.; Lee, V. S.; Tzen, J. T.; Lee, M. R. Identification and comparison of phenolic compounds in the preparation of oolong tea manufactured by semifermentation and drying processes. J. Agric. Food Chem. 2007, 55 7462 7468. 58. Monagas, M.; Suarez, R.; Gomez Cordoves, C.; Bartolome, B. Simultaneous determination of nonanthocyanin phenol ic compounds in red wines by HPLC DAD/ESI MS. Am. J. Enol. Vitic. 2005, 56 139 147.

PAGE 141

141 59. Wang, D.; Lu, J.; Miao, A.; Xie, Z.; Yang, D. HPLC DAD ESI MS/MS analysis of polyphenols and purine alkaloids in leaves of 22 tea cultivars in China. J. Food Comp. A naly. 2008, 21 361 369. 60. Hukkanen, A. T.; Kokko, H. I.; Buchala, A. J.; McDougall, G. J.; Stewart, D.; Karenlampi, S. O.; Karjalainen, R. O. Benzothiadiazole Induces the Accumulation of Phenolics and Improves Resistance to Powdery Mildew in Strawberri es. J. Agric. Food Chem. 2007, 55 1862 1870. 61. Maatta, K. R.; Kamal Eldin, A.; Torronen, A. R. High Performance Liquid Chromatography (HPLC) Analysis of Phenolic Compounds in Berries with Diode Array and Electrospray Ionization Mass Spectrometric (MS) Detection: Ribes Species. J. Agric. Food Chem. 2003, 51 6736 6744. 62. Schieber, A.; Berardini, N.; Carle, R. Identification of flavonol and xanthone glycosides from mango ( Mangifera indica L. Cv. "Tommy Atkins") peels by high performance liquid chromatography electrospray ionization mass spectrometry. J. Agric. Food Chem. 2003, 51 5006 5011. 63. Francesca, B.; Franco, C.; Renata, J.; Mirco, M.; Alessandro, T. Analysis of some stilbenes in It alian wines by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21 2955 2964. 64. Treutter, D. Managing phenol contents in crop plants by phytochemical farming and breeding visions and constraints. Int. J. Mol. Sci. 2010, 11 807 857. 65. Cantn, C. M.; Fidelibus, M. W.; Crisosto, C. H. Application of abscisic acid (ABA) at veraison advanced red color development and mai ntained postharvest quality of Crimson Seedless' grapes. Postharv. Bio. Technol. 2007, 46 237 24 1. 66. Leung, J.; Giraudat, J. Abscisic Acid Signal Transduction. Annu. Rev. Plant Physiol. Plant Mol. Bio. 1998, 49 199 222. 67. Coombe, B. G.; Hale, C. R. The Hormone Content of Ripening Grape Berries and the Effects of Growth Substance Treatments. Pl ant Physiol. 1973, 51 629 634. 68. Deytieux Belleau, C.; Gagne, S.; L'Hyvernay, A.; Doneche, B.; Geny, L. Possible roles of both abscisic acid and indoleacetic acid in controlling grape berry ripening process. J. Int. Sci. Vigne Vin 2007, 41 141 148. 6 9. Wheeler, S.; Loveys, B.; Ford, C.; Davies, C. The relationship between the expression of abscisic acid biosynthesis genes, accumulation of abscisic acid and the promotion of Vitis vinifera L. berry ripening by abscisic acid. Austr. J. Grape Wine Res. 20 09, 15 195 204.

PAGE 142

142 70. Ban, T.; Ishimaru, M.; Kobayashi, S.; Shiozaki, S.; Goto Yamamoto, N.; Horiuchi, S. Abscisic acid and 2,4 dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in 'Kyoho' grape berries. J. Horti. S ci. Biotechnol. 2003, 78 586 589. 71. Jeong, S. T.; Goto Yamamoto, N.; Kobayashi, S.; Esaka, M. Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. Plant Sci 2004, 167 247 252. 72. Kataoka, I.; Sugiura, A.; Utsunomiya, N.; Tomana, T. Effect of abscisic acid and defoliation on anthocyanin accumulation in Kyoho grapes ( Vitis vinifera L. labruscana BAILEY). Vitis 1982, 21 325 332. 73. Peppi, M. C.; Fidelibus, M. W.; Dokoozlian, N. Abscisic acid application timing and concentration affect firmness, pigmentation, and color of 'flame seedless' grapes Hort. Sci. 2006, 41 1440 1445. 74. Lacampagne, S.; Gagn, S.; Gny, L. Involvement of Ab scisic Acid in Controlling the Proanthocyanidin Biosynthesis Pathway in Grape Skin: New Elements Regarding the Regulation of Tannin Composition and Leucoanthocyanidin Reductase (LAR) and Anthocyanidin Reductase (ANR) Activities and Expression. J. Plant Gro wth Regu. 2009, 29 81 90. 75. Brand Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 1995, 28 25 30. 76. Huang, D.; Ou, B.; Prior, R. L. The Chemistry behind Antioxidant Capacity Assays. J. Agric. Food Chem. 2005, 53 (6), 1841 1856. 77. Peppi, M. C.; Fidelibus, M. W. Effects of forchlorfenuron and abscisic acid on the quality of 'Flame Seedless' grapes. Hort Sci. 2008, 43 173 176. 78. Li, Z.; Zhao, X.; Sandhu, A. K.; Gu, L. Effects of Exogenous Abscisic Acid on Yield, Antioxidant Capacities, and Phytochemical Contents of Greenhouse Grown Lettuces. J. Agric. Food Chem. 2010, 58 6503 6509. 79. Balint, G.; Reynolds, A. G. Impact of Exogenous Abscisic Acid on Vine Physiology and Grape Composition of Cabernet Sauvignon. Am. J. Enol. Vitic. 2013, 64 (1), 74 87. 80. Vogt, T. Phenylpropanoid Biosynthesis. Mol. Plant 2010, 3 2 20. 81. Hrazdina, G.; Parsons, G. F.; Mattick, L. R. Physiological and Biochemical Events During Development and Maturation of Grape Berries. Am. J. Enol. Vitic. 1984, 35 220 227.

PAGE 143

143 82. Boss, P. K.; Davies, C.; Robinson, S. P. Analysis of the Expression of Anthocya nin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation. Plant Physiol. 1996, 111 1059 1066. 83. Crozier, A.; Clifford, M. N.; Ashihara, H. Phenols, Polyphenols and Tannins: An Overview. In Pl ant Secondary Metabolites: Occurrence, Structure and Role in the Human Diet Crozier, A.; Jaganath, I. B.; Clifford, M. N., Eds. Blackwell Publishing Ltd: UK, 2006; pp 1 24. 84. Fujita, A.; Goto Yamamoto, N.; Aramaki, I.; Hashizume, K. Organ Specific Tran scription of Putative Flavonol Synthase Genes of Grapevine and Effects of Plant Hormones and Shading on Flavonol Biosynthesis in Grape Berry Skins. Biosci., Biotechnol., Biochem. 2006, 70 632 638. 85. Naczk, M.; Shahidi, F. Extraction and analysis of phe nolics in food. J Chromatogr A 2004, 1054 (1 2), 95 111. 86. Spigno, G.; Tramelli, L.; De Faveri, D. M. Effects of extraction time, temperature and solvent on concentration and antioxidant activity of grape marc phenolics. J Food Eng 2007, 81 (1), 200 208. 87. Wu, X.; Gu, L.; Prior, R. L.; McKay, S. Characterization of Anthocyanins and Proanthocyanidins in Some Cultivars of Ribes, Aronia, and Sambucus and Their Antioxidant Capacity. J. Agric. Food Chem. 2004, 52 (26), 7846 7856. 88. Giusti, M. M.; Wr olstad, R. E. Unit F1.2: Anthocyanins. Characterization and measurement of anthocyanins by UV visible spectroscopy. In Current protocols in food analytical chemistry Wrolstad, R. E., Ed. John Wiley & Sons: New York, 2001; pp 1 13. 89. Yu, L.; Haley, S.; Perret, J.; Harris, M.; Wilson, J.; Qian, M. Free Radical Scavenging Properties of Wheat Extracts. J. Agric. Food Chem. 2002, 50 (6), 1619 1624. 90. Liu, Q.; Yao, H. Antioxidant activities of barley seeds extracts. Food Chem. 2007, 102 (3), 732 737. 91. Ju, Z. Y.; Howard, L. R. Effects of Solvent and Temperature on Pressurized Liquid Extraction of Anthocyanins and Total Phenolics from Dried Red Grape Skin. J. Agric. Food Chem. 2003, 51 (18), 5207 5213. 92. Cardona, J. A.; Lee, J. H.; Talcott, S. T. Color and Polyphenolic Stability in Extracts Produced from Muscadine Grape ( Vitis rotundifolia ) Pomace. J. Agric. Food Chem. 2009, 57 8421 8425.

PAGE 144

144 93. Wei min, Z. Chapter 2: Introduction to Natural Products Chemistry. In Extraction and Isolat ion of Natural Products Xu, R.; Yang, Y.; Wei min, Z., Eds. Science Press: Beijing, China, 2012; Vol. 1, pp 18 19. 94. Scordino, M.; Di Mauro, A.; Passerini, A.; Maccarone, E. Adsorption of Flavonoids on Resins: Hesperidin. J. Agric. Food Chem. 2003, 51 6998 7004. 95. Wang, J.; Wu, F. A.; Zhao, H.; Liu, L.; Wu, Q. S. Isolation of flavonoids from mulberry ( Morus alba L. ) leaves with macroporous resins. Afri. J. Biotechnol. 2008, 7 2147 2155. 96. Liu, Y.; Liu, J.; Chen, X.; Liu, Y.; Di, D. Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins. Food Chem. 2010, 123 1027 1034. 97. Fu, Y.; Zu, Y.; Liu, W.; Hou, C.; Chen, L.; Li, S.; Shi, X.; Tong, M. Preparative separation of vitexin and isovitexi n from pigeonpea extracts with macroporous resins. J. Chromatogr. A 2007, 1139 206 213. 98. Kammerer, D. R.; Carle, R.; Stanley, R. A.; Saleh, Z. S. Pilot Scale Resin Adsorption as a Means To Recover and Fractionate Apple Polyphenols. J. Agric. Food Chem. 2010, 58 6787 6796. 99. Kammerer, D. R.; Saleh, Z.; Carle, R.; Stanley, R. Adsorptive recovery of phenolic compounds from apple juice. Eur. Food Res. Technol. 2007, 224 605 613. 100. Saleh, Z. S.; Wibisono, R.; Lober, K. Recovery o f Polyphenolics from Apple Juice Utilizing Adsorbent Polymer Technology. Int. J. Food Eng. 2008, 4 (1), 1 20. 101. Lagergren, S. Zur Theorie der Sogenannten Adsorption Geloster Stoffe. Kungliga Svenska Vetenskapsakademiens: Handlingar, Germany 1898, 24 (4 ), 1 39. 102. Ho, Y. S.; McKay, G. Pseudo second order model for sorption processes. Process Biochem. 1999, 34 451 465. 103. Pompeu, D. R.; Moura, F. G.; Silva, E. M.; Rogez, H. Equilibria, Kinetics, and Mechanisms for the Adsorption of Four Classes of Phenolic Compounds onto Synthetic Resins. Sep. Sci. Technol. 2010, 45 700 709. 104. Webi, T. W.; Chakravort, R. K. Pore and solid diffusion models for fixed bed adsorbers. J. Am. Inst. Chem. Eng. 1974, 20 228 238. 105. Xu, Z.; Zhang, Q.; Chen, J.; Wang, L.; Anderson, G. K. Adsorption of napthalene derivatives on hypercrosslinked polymeric adsorbents. Chemosph. 1999, 38 2003 2011.

PAGE 145

145 106. Chang, X. L.; Wang, D.; Chen, B. Y.; Feng, Y. M.; Wen, S. H.; Zhan, P. Y. Adsorption a nd Desorption Properties of Macroporous Resins for Anthocyanins from the Calyx Extract of Roselle ( Hibiscus sabdariffa L.). J. Agric. Food Chem. 2012, 60 2368 2376. 107. Silva, E. M.; Pompeub, D. R.; Larondelle, Y.; Rogez, H. Optimisation of the adsorpti on of polyphenols from Inga edulis leaves on macroporous resins using an experimental design methodology. Sep Purifi Technol 2007, 53 274 280. 108. Rudzinski, W.; Plazinski, W. Kinetics of dyes adsorption at the solid solution interfaces: a theoretical description based on the two step kinetic model. Environ. Sci. Technol. 2008, 42 2470 2475. 109. Duran, C.; Ozdes, D.; Gundogdu, A.; Senturk, H. B. K inetics and Isotherm Analysis of Basic Dyes Adsorption onto Almond Shell ( Prunus dulcis ) as a Low Cost Adsorbent. J. Chem. Eng. Data 2011, 56 2136 2147. 110. Kumar, N. S.; Min, K. Removal of phenolic compounds from aqueous solutions by biosorption onto A cacia leucocephala bark powder: Equilibrium and kinetic studies. J. Chil. Chem. Soc. 2011, 56 539 545. 111. Jia, G.; Lu, X. Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins. J. Chromato gr. A 2008, 1193 136 141. 112. Kammerer, D.; Kljusuric, J. G.; Carle, R.; Schieber, A. Recovery of anthocyanins from grape pomace extracts ( Vitis vinifera L. cv. Cabernet Mitos) using a polymeric adsorber resin. Eur. Food Res. Technol. 2005, 220 431 437 113. Scordino, M.; Di Mauro, A.; Passerini, A.; Maccarone, E. Selective Recovery of Anthocyanins and Hydroxycinnamates from a Byproduct of Citrus Processing. J. Agric. Food Chem. 2005, 53 651 658. 114. Biswas, R. Development of technologies for the pr oduction of polyphenolic nutraceuticals from Muscadine grapes and Rabbiteye blueberries. PhD Dissertation 2007 1 328. 115. Ghasemnezhad, M.; Javaherdashti, M. Effect of Methyl jasmonate treatment on antioxidant capacity, internal quality and postharvest life of raspberry fruit. Caspian J. Env. Sci. 2008, 6 (1), 73 78. 116. Sabir, A.; Sabir, F. K. Postharvest treatments to preserve table grape quality during storage and approaches to find better ways alternative for SO 2 Adv Environ Bio 2009, 3 (3), 286 295.

PAGE 146

146 117. Morris, J. R. Handling and Marketing Of Muscadine Grapes. Fruit South 1980, 4 (2), 12 14. 118. Schreiner, M.; Huyskens Keil, S. Phytochemicals in Fruit and Vegetables: Health Promotion and Postharvest Elicitors Crit Rev Plant Sci 2 006, 25 (3), 267 278. 119. Cisneros Zevallos, L. The Use of Controlled Postharvest Abiotic Stresses as a Tool for Enhancing the Nutraceutical Content and Adding Value of Fresh Fruits and Vegetables. J. Food Sci. 2003, 68 (5), 1560 1565. 120. Creeman, R. A.; Mullet, J. E. Biosynthesis and action of jasmonate in plants. Annu. Rev. Plant Physiol. 1997, 48 355 381. 121. Yao, H. J.; Tian, S. P. Effects of pre and post harvest application of salicylic acid or methyl jasmonate on inducing disease resistance of sweet cherry fruit in storage. Postharvest Bio. Technol. 2005, 35 253 262. 122. Cao, S. F.; Zheng, Y. H.; Yang, Z. F.; Tang, S. S.; Jin, P.; Wang, K. T.; Wang, X. M. Effect of methyl jasmonate on the inhibition of Colletotrichum acutatum infection in loquat fruit and the possible mechanisms. Postharvest Bio. Technol. 2008, 49 301 307. 123. Yao, H. J.; Tian, S. P. Effects of a biocontrol agent and methyl jasmonate on postharvest diseases of peach fruit and the possible mechanisms involve d. J Appl Microbio 2005, 98 (4), 941 50. 124. Droby, S.; Porat, R.; Cohen, L.; Weiss, B.; Shapio, B.; Philosoph ; Hadas, S.; Meir, S. Suppressinig green mold decay in grapefruit with postharvest jasmonate application. J. Am. Soc. Hortic. Sci. 1999, 124 184 188. 125. Chanjirakul, K.; Wang, S. Y.; Wang, C. Y.; Siriphanich, J. Effect of natural volatile compounds on antioxidant capacity and antioxidant enzymes in raspberries. Postharvest Bio. Technol. 2006, 40 106 115. 126. Chanjirakul, K.; Wang, S. Y.; Wang, C. Y.; Siriphanich, J. Natural volatile treatments increase free radical scavenging capacity of strawberries and blackberries. J. Sci. Food Agric 2007, 87 1463 1472. 127. Wang, S. Y.; Bowman, L.; Ding, M. Methyl jasmonate enhances antioxidant acti vity and flavonoid content in blackberries ( Rubus sp.) and promotes antiproliferation of human cancer cells. Food Chem. 2008, 107 1261 1269. 128. Perez, A.; Sanz, C.; Richardson, D.; Olias, J. Methyl jasmonate promotes beta carotene synthesis and chlorop hyll degradation in Golden Delicious apple peel. J Plant Growth Regul 1993, 12 163 7.

PAGE 147

147 129. Wang, K.; Jin, P.; Cao, S.; Shang, H.; Yang, Z.; Zheng, Y. Methyl jasmonate reduces decay and enhances antioxidant capacity in Chinese bayberries. J Agric Food Chem 2009, 57 (13), 5809 15. 130. Ayala Zavala, J. F.; Wang, S. Y.; Wang, C. Y.; Gonzlez Aguilar, G. A. Effect of storage temperatures on antioxidant capacity and aroma compounds in strawberry fruit. LWT Food Sci Technol 2004, 37 (7), 687 695. 131. Gonzalez Aguilar, G.; Buta, J. G.; Wang, C. Y. Methyl jasmonate reduces chilling J. Sci. Food and Agri. 2001, 81 1244 1249. 132. Gonzlez Aguilar, G. A.; Buta, J. G.; Wang, C. Y. Methyl jasmonate and modified atmosphere packaging (MAP) reduce decay and maintain postharvest quality of Postharvest Bio. Technol. 2003, 28 (3), 361 370. 133. Wang, C. Y.; Buta, J. G. Maintaining quality of fresh cut kiwifruit with volatile c ompounds. Postharvest Bio. Technol. 2003, 28 (1), 181 186. 134. Kalt, W.; Forney, C. F.; Martin, A.; Prior, R. L. Antioxidant Capacity, Vitamin C, Phenolics, and Anthocyanins after Fresh Storage of Small Fruits. J. Agric. Food Chem. 1999, 47 (11), 4638 4644. 135. Cantos, E.; Garcia Viguera, C.; de Pascual Teresa, S.; Tomas Barberan, F. A. Effect of Postharvest Ultraviolet Irradiation on Resveratrol and Other Phenolics of Cv. Napoleon Table Grapes. J. Agric. Food Chem. 2000, 48 (10), 4606 461 2. 136. De la Pea Moreno, F.; Blanch, G. P.; Flores, G.; Ruiz del Castillo, M. L. Impact of postharvest methyl jasmonate treatment on the volatile composition and flavonol content of strawberries. J. Sci. Food Agri. 2010, 90 (6), 989 994. 137. Dixon, R. A.; Paiva, N. L. Stress lnduced Phenylpropanoid Metabolism. The Plant Cell 1995, 7 1085 1097.

PAGE 148

148 BIOGRAPHICAL SKETCH Aman Sandhu was born in Punjab, India. She attended St. Francis School, Tarn T aran and Khalsa College, Amritsar for her high school and senior secondary education, respectively. She obtained her B. S. degree in f ood s cience from Khalsa College, Amritsar in 2003 and M. S. degree in f ood s cience and t echnology from the Guru Nanak Dev University, India in 2005. She subsequently worked in f ood i ndustry as well as a college lecturer before joining the University of Florida. She joined the f ood s cience doctoral program at the University of Florida in the spring of 2009. She graduated in M ay 2013 with PhD in f ood s cience.