BIOAVAILABILITY AND CATABOLISM OF CRANBERRY PROCYANIDINS By KEQIN OU 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 2014
2014 Keqin Ou
To my family and friends
4 ACKNOWLEDGMENTS I would like to express my deepest gratitude to my advisor Dr. Liwei Gu for his guidance, patience, and providing me with a supportive environment for doing research. His enthusiasm in scientific research , immense knowledge, and perseverance served as a rol e model . His willingness to encourage new ideas and to trust students on problem solving has molded me into an independent researcher. I would like to thank Dr. Susan S. Percival for her generous help on offering me facilities to perform the cell culture study. I am thankful to Dr. Keith R. Schneider for his critical review of microbiological experiments and providing resources for this experiment. I want to express my gratitude to Dr. Donald J. Huber for guiding me to broaden my knowledge on phytochemical s. A special thanks to Dr. Maurice R. Marshall for his insightful comments and questions that have been valuable in improving my experiments. I would also like to thank Dr. Paul Sarnoski for his constructive suggestion and providing the instrument for meta bolites analysis. It has been my pleasure to work with my fellow labmates, the present and past members of the Gu lab. They are Wei Wang, Amandeep K. Sandhu, Haiyan Liu, Zheng Li, Hanwei Liu, Timothy Buran, Dr. Tao Zou, Bo Zhao, Sara Marshall, Kaijie Song, Brittany Hubbard, Yun Cai, Weixin Wang and Dr. Vishnupriya Gourineni. We discussed together, shared our ideas, and helped each other. Those made my years at University of Florida the mos t memorable period in my life. 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,
5 Marianne Ma ngone, Rhonda Herring and Julie Barber, for their help and services in our department. I would also like to thank China Agricultural University for providing me the platform to apply for the government scholarship. I acknowledge the financial support of Ch ina Scholarship Council for my doctoral program. I would like to allude to the tremendous support and advice I have gotten from my parents, Shulie Ou and Binglan Li, without whom this would not have been possible. I would like to give my heartfelt gratitude to my in laws for their encouragement and support. Even though we are thousands of miles away, you were always there whenever I needed you. I would also like to thank my cousin, Cun Shen for his unfailing support. Most of all, I would like to thank my l oving husband Huaping Xiao , who provided continuous encouragement and gave me strength to succeed.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 A REVIEW: ABSORPTION AND METABOLISM OF PROCYANIDINS .................. 14 Cranberries and Procyanidins ................................................................................. 14 Health Benefits ....................................................................................................... 15 Absorption of Procyanidins ..................................................................................... 16 Microbial Catabolism of Procyanidins ..................................................................... 20 Research Objectives ............................................................................................... 25 2 TRANSPORT OF CRANBERRY A TYPE PROCYANIDIN DIMERS, TRIMERS, AND TETRAMERS ACROSS MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS ................................................................................. 27 Background ............................................................................................................. 27 Materials and Methods ............................................................................................ 28 Chemicals and Materials .................................................................................. 28 Purification of Cranberry Procyanidins ............................................................. 28 Cell Culture ....................................................................................................... 29 Transport Experiments ..................................................................................... 30 Solid Phas e Extraction ..................................................................................... 31 HPLCMSn Analysis .......................................................................................... 31 Determination of Procyanidin Contents, Apparent Permeability Coefficient and Transport Ratio ...................................................................................... 32 Statistical analysis ............................................................................................ 32 Results and Discussion ........................................................................................... 33 Composition of Procyanidin Fractions Purified from Cranberries ..................... 33 Transport of Cranberry A type Procyanidins on Caco2 Cells .......................... 35 Summary ................................................................................................................ 39 3 DEPOLYMERIZATION OPITIMIZATION OF CRANBERRY PROCYANIDINS AND TRANSPORT OF RESULTANT OLIGOMERS ON MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS ............................................. 48
7 Background ............................................................................................................. 48 Materials and Methods ............................................................................................ 49 Chemicals and Materials .................................................................................. 49 Preparation of Partially Purified Procyanidin Polymers (PP) ............................ 49 Depolymerization of Cranberry Procyanidins. .................................................. 50 HPLCESI MSn Analysis of Procyanidins ......................................................... 51 Transport Experiments ..................................................................................... 52 Optimization of Depolymerization Conditions ................................................... 52 Statistical Analysis ............................................................................................ 53 Results and Discussion ........................................................................................... 53 HPLCMSn Analysis of Procyanidins Before and After Depolymerization ......... 53 Transport of Depolymerized Cranberry Procyanidins on Caco2 Cell Monolayers .................................................................................................... 56 Optimization of Depolymerization ..................................................................... 57 Summary ................................................................................................................ 59 4 MICROBIAL CATABOLISM OF PROCYANIDINS BY HUMAN GUT MICROBIOTA ......................................................................................................... 67 Background ............................................................................................................. 67 Materials and Methods ............................................................................................ 68 Chemicals and Materials .................................................................................. 68 Extracti on and Purification of Procyanidins from Apples and Cranberries ........ 69 HPLCMSn Analysis of Procyanidins ................................................................ 70 In vitro Fermentation of Procyanidins with Human Fecal Microbiota ................ 70 Sample Extraction and Derivatization for GC MS Analysis .............................. 71 GC MS Analysis ............................................................................................... 72 Statistical Analysis ............................................................................................ 72 Results and Discussion ........................................................................................... 72 Composition of Partially Purified Apple and Cranberry Procyanidins ............... 72 Identification of Microbial Metabolites ............................................................... 73 Quantitation of Microbial Metabolites ............................................................... 75 Summary ................................................................................................................ 79 5 CONCLUSIONS ..................................................................................................... 88 LIST OF REFERENCES ............................................................................................... 90 BIOGRAPHICAL SKETCH ............................................................................................ 99
8 LIST OF TABLES Table page 2 1 type and B type procyanidin oligomers in purified procyanidin fractions ........................................................................................... 40 2 2 Transport ratio of A type procyanidin oligomers on Caco2 cells ........................ 41 3 1 Recovery rate and transport ratio of oligomers from depolymerized cranberry procyanidin polymer ........................................................................................... 61 4 1 Procyanidin content in the partially purified apple and cranberry procyanidins .. 80 4 2 Retention time, molecular weight, and major ions of identified microbial metabolites and an internal standard present in fermentation broth. Peak numbers match those in Figure 42. ................................................................... 81 4 3 Mass recoveries from microbial catabolites of epicatechin, catechin, procyanidin B2, A2, partially purified apple and cranberry procyanidins over 24 h . ................................................................................................................... 82
9 LIST OF FIGURES Figure page 1 1 Structure of ( ) epicatechin, (+) catechin, B type procyanidin dimer B2 and A type procyanidin A2 ............................................................................................ 26 2 1 HPLC chromatogram of procyanidins in fraction I and fraction II. ....................... 42 2 2 HPLC chromatogram of solutions from transport experiments. .......................... 43 2 3 Product ion spectrum of peak X1 ........................................................................ 44 2 4 Postulated fragmentation pathway of pr ocyanidin A2. . ....................................... 45 2 5 HPLC chromatogram of solutions from transport experiments. .......................... 46 2 6 Product ion spectrum of detected peaks. A) peak X2 and B) product ion peak X3. ...................................................................................................................... 47 3 1 Chromatograms of cranberry procyanidin polymers before and after depolymerization. ............................................................................................... 62 3 2 HPLC chromatogram of oligomers from depolymerization without added epicatechin ......................................................................................................... 63 3 3 HPLC chromatogram of oligomers from depolymeri zation with added epicatechin. ........................................................................................................ 64 3 4 3D graphic surfaces of the effect of variables (temperature, time, acid concentration) on the yield of oligomers in the depolymerization without added epicatechin .............................................................................................. 65 3 5 3D graphic surfaces of the effect of variables (temperature, time, acid concentration, and epicatechin/polymer ratio) on the yield of oligmers in depolymerization with added epicatechin ........................................................... 66 4 1 HPLC chromatograms of partially purified procyanidins using florescent detection.. ........................................................................................................... 83 4 2 Gas chromatograms of microbial metabolites in ferments with substrate epicatechin ......................................................................................................... 84 4 3 Degradation curve of epicatechin, catechin, procyanidin B2, proc yanidin A2, partially purified apple and cranberry procyanidins. ............................................ 85 4 4 Formation of microbial metabolites with 12 carbon(s) on the side chain. .......... 86 4 5 Formation of microbial metabolites with 35 carbons on the side chain.. ............ 87
10 LIST OF ABBREVIATIONS ANOVA Analysis of variance ATCC American Type Culture Collection BSTFA N, O Bistrifluoroacetamide Cat Catechin CO2 Carbon dioxide cm Centimeter DP Degree of polymerization d d (s) EI Electron impact epi Epicatechin g Gram g Relative centrifugal force HBSS Hanksâ€™ balanced salt solution HCl Hydrochloric acid HPLC High performance liquid chromatography HRF Heterocyclic ring fission h Hour (s) Kg Kilogram L Liter MS Mass spectr ometer m/z Mass to charge ratio min Minute (s) mL Milliliter mM Millimole
11 mm Millimeter nM Nanomole nm Nanometer O2 Oxygen Papp Apparent permeability coefficients PP Partially purified procyanidin polymers psi Pounds per square inch QM Quinone methide RDA Retro Diels Alder rpm Revolutions per minute s Second (s) TEER Transepithelial electric resistance TMCS Trimethylchlorosilane UV Ultraviolet g Microgram L Microliter mol Micromole Vis Visible v Volume w Weight
12 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 BIOAVAILABILITY AND CATABOLISM OF CRANBERRY PROCYANIDINS By Keqin Ou August 2014 Chair: Liwei Gu Major: Food Science The absorption, depolymerization and microbial catabolism of cranberry procyanidins were investigated in this study. In th e first objective, p rocyanidins were extracted from cranberries and purified using chromatographic methods. Fraction I ( contained predominantly A type procyanidin dimer A2) and f raction II ( contained primarily A type trimers and tetramers, with B type trimers, A type pentamers, and A type hexamers being minor components ) were added onto the apical side of the Caco2 cell me mbranes. Data indicated that procyanidin dimer A2 in fraction I and A type trimers and tetramers in fraction II traversed Caco2 cell monolayers with transport ratio of 0.6%, 0.4%, and 0.2%, respectively. In the second objective, cranberry procyanidin pol ymers were depolymeriz ed with or without added epicatechin and yield ed 644 g and 202 g oligomers (monomer through tetramers) per mg partially purified polymers (PP), respectively . O ligomers yielded from both methods transported through Caco2 cell monolayer albeit absorption rates were low. With the aid of response surface methodology, the optimum depolymerization conditions were determined to be 60 oC , 0.1 M HCl in m ethanol and 3
13 h without added epicatechin. The predicted maximum yield was 364 g oligomers per mg partially purified cranberry procyanidins . The optimum depolymerization condition with added epicatechin shared the same temperature, acid concentration and reaction time in addition to an epicatechin/PP mass ratio of 2.19 . Its predicted maxima oligomer yield was 1089 g/mg. In the third objective, ( ) e picatechin, (+) catechin, procyanidin B2, procyanidin A2 , partially purified apple procyanidins, and partially purified cranberry procyanidins were fermented with human gut microbiota anaerobically at a concentration equivalent to 0.5 mM epicatechin. C ommon metabolites of the six substrates were benzoic acid, phenylacetic acid, 2 ( 3 â€™ hydroxypenyl ) acetic acid, 2 ( 4 â€™ hydroxyphenyl ) acetic acid, phenylpropionic acid, 3 ( 3 â€™ hydroxyphenyl ) propionic acid and hydroxyphenylvaleric acid. 5 ( 3 â€™, 4 â€™ Dihydroxyphenyl ) valerolactone, 5 ( 3 â€™ hydroxyphenyl ) valerolactone compounds were identified as microbial metabolites of epicatechin, catechin , procyanidin B2, and apple procyanidins but not from the procyanidin A2 or cranberry procyanidin ferments. 2 ( 3 â€™ ,4 â€™ Dihydroxyphenyl ) acetic acid was found in the fermented broth of procyanidin B2 , A2 , apple and cranberry procyanidins but not in epicatechin or catechin metabolites. T he mass recoveries of microbial metabolites range from 20 % to 57% for the six substrates a fter 24 h of fermentation .
14 CHAPTER 1 A REVIEW: ABSORPTION AND METABOLISM OF PROCYANIDINS Cranberr ies and Procyanidins Cranberry, known scientifically as Vaccinium macrocarpon Ait ., is a native fruit in North America. A pproximately 8 x 108 lb of cranberries are harvested in the United States per year. The major areas of cranberry production include Wisconsin, Massachusetts, New Jersey, Oregon and Washington. Wi sconsin is the leading producer, which accounts for over half of U.S. production. Massachusetts is t he second largest U.S. producer. Fresh cranberries have a bitter and tart taste. They are too tangy to be eaten plain and raw. O nly a small portion of the production is sold fresh to consumers. The majority of them (> 96% ) are processed into juices, dried cranberr y sauce , and sauces for consumption. Cranberries contain over 80% water , 10% carbohydrates and a complex mixture of organic acids, vitamin C, flavonoids, anthoc yanins, catechin, and triterpenoids ( Guay, 2009; Raz, Chazan, & Dan, 2004) . Among these compounds, procyanidins are thought to be the ma jor bio active constitutes in cranberry . Procyanidins, also known as con densed tannins, are oligomers or polymers of flavan3 ols [e.g. ( ) epicatechin or (+) catechin] linked through interflavan bonds. B type procyanidins contain an additional ether bond between (DP). Procyanidins with a degree of polymerization of 1, 2, 3 or 4 are called monomers, dimers, trimers, or tetramers, respectively. P rocyanidins with DP 24 are defined a s oligomers, DP >4 as polymers, and DP >10 as high polymers, respectively. The flavan3 ol units at the end of the procyanidins are terminal units. All flavan3 ols above the
15 terminal unit are extension units. Flavan3 ol has two aromatic rings (A & B) and a heterocyclic ring C. The structure of epicatechin, catechin, B type procyanidin dimer, and A type procyanidin dimer are shown in Figure 11. The most ubiquitous procyanidins in foods are the B type procyanidins . A type procyanidins are less common in nature. Over 50% of procyanidins in cranberries are A type ( Gu, Kelm, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . Health B enefits of Procyanidins Numerous studies have reported a variety of physiological activities for p rocyanidins. For example, procyanidins were more effective than resveratrol or ascorbic acid in scavenging free radicals ( Maldonado, RiveroCruz, Mata, & PedrazaChaverr, 2005) . A recent study suggested cranberry procyanidins may help to prevent lung cancer by inducing rapid cancer cell apoptosis and growth arrest ( Kresty, Howell, & Baird, 2011) . Cranber ry procyanidins may also serve as a chemoprevention agent against esophageal cancer by inducing apoptosis and inhibiting proliferation ( Kresty, Howell, & Baird, 2008) . Procyanidins from peanut skin decreased the production of inflammatory cytokines, tumor necrosis factor 6 in cultured human monocytic THP 1 cells in response to lipopolysaccharide ( Tatsuno, Jinno, Arima, Kawabata, Hasegawa, Yahagi, et al., 2011) . Administering grape seed procyanidins reduced lung inflammation and decr eased IL 4, IL 5, and IL 13 expression in a mouse model of acute or chronic asthma ( Lee, Kwon, Bang, Lee, Park, Moon, et al., 2012) . Procyanidins from c ocoa were found to inhibit growth of human breast cancer and colonic cancer cells ( Carnsecchi, Schneider, Lazarus, Coehlo, Goss, & Raul, 2002; Ramljak, Romanczyk, Metheny Bar low, Thompson, Knezevic, Galperin, et al., 2005) .
16 Abso r p tion of P rocyanidins Procyanidins in a solid food matrix are not available for absorption ( SauraCalixto, Serrano, & Goi, 2007 ) . Only solubilized procyanidins in the aqueous phase are bioaccessible for the enterocyte surface of the small intestine. No transporters have been identified for procyanidins. Pro cyanidins are absorbed through passive diffusion. P rocyanidins are not likely to pass the lipid bilayer via the transcellular pathway due to their large number of hydrophilic hydroxyl groups . P aracellular diffusion was thought to be a preferential absorpti on mechanism ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . The absorption and metabolism of flavan3 ol monomer s was investigated extensively. Both human and animal studies indicated that (+) catechin and ( ) epicatechin were rapidly absorbed from the upper portion of the small intestine. A maximum level of (+) catechin at 76.7 nmol/L was detected in humans at 1.4 h after intake of 121 mol (+) catechin in dealcoholized red wine ( Bell, Donovan, Wong, Waterhouse, German, Walzem, et al., 2000) . A peak plasma ( ) epicatechin level of 260 nmol/L was achieved within 2 h in humans after the consumption of 557 mg of procyanidins containing 137 mg of ( ) epicatechin fro m a procyanidinrich chocolate ( Rein, Lotito, Holt, Keen, Schmitz, & Fraga, 2000) . Upon absorption, (epi)catechin undergoes extensive phase II metabolism in the intestine and liver to form g lucuronidated, s ulfated, and/or methylated conjugates. These metabolites are present in blood and tissues. Major conjugates of ( ) epicatechin in human plasma , bile, and urine were ( ) O sulfonate and ( ) O glucuronide after ingestion of 50 mg of ( ) epicatechin by volunteer s ( Romanov Michailidis, Viton, Fumeaux, Lvques, Actis Goretta, Rein, et al., 2012) .
17 An ear ly study suggested that procyanidin oligomers (trimers to hexamers) were depolymeri z ed into mixtures of ( ) epicatechin and dimers in simulated gastric fluid (pH 2.0, 37 oC ) ( Spencer, Chaudry, Pannala, Srai, Debnam, & RiceEvans, 2000) . These authors also detected ( ) epicatechin as a major metabolite after ex vivo perfusion of rat small intestines with procyanidin dimer B2 or B5 extracted from cocoa ( Spencer, Schroeter, Shenoy, Srai, Debnam, & RiceEvans, 2001) . A study in humans demonstrated that depolymerization did not occur and procyanidins were st able during gastric transit ( Rios, Bennett, Lazarus, Rmsy, Scalbert, & Williamson, 2002) . A later study confirmed that procyanidin dimers and trimers were highly stable under gastric and duodenal digestion conditions ( Serra, Maci, Romero, Valls, Blad, Arola, et al., 2010) . Oligomeric p rocyanidins in grape seed extract or s orghum were not depolymeri z ed in the gastrointestinal tract releasing monomer ic flavan 3 ols after ingestion by rats ( Gu, House, Rooney, & Prior, 2007; Tsang, Auger, Mullen, Bornet, Rouanet, Crozier, et al., 2005) . A recent study compared the plasma concentration of ( ) epicatechin in h uman blood and urine after volunteers were given ( ) epicatechin, cocoa procyanidin monomers [predominantly ( ) epicatechin] through decamers, or cocoa procyanidins dimers through decamers. It was found that all absorbed ( ) epicatechin in blood or urine w ere from ingested ( ) epicatechin. No ( ) epicatechin was derived from ingested oligomers and polymers ( Ottaviani, Kwik Uribe, Keen, & Schroeter, 2012) . One study detected procyanidin B2 and ( ) epicatechin in rat plasma and urine after administration of purified B2. It was suggested that a portion of the dimer was degraded into ( ) epicatechi n . C leavage of the interflavan bond likely occurred in the
18 large intestine by microbiota. Catabolism of purified procyanidin B2 with human fecal microbiota in a static in vitro culture model caused less than 10% of dimer s to be converted to ( ) epicatechin ( Stoupi, Willamson, Dry nan, Barron, & Clifford, 2010) . In contrast, scission of the interflavan bond was not observed in vivo in rats (Gu, House, Rooney, & Prior, 2007; Tsang, Auger, Mullen, Bornet, Rouanet, Crozier, et al., 2005) . It can be concluded from these studies t hat depolymerization of procyanidins to monomers is negligible in the gastrointestinal tract in vivo . Results from in vitro and in vivo models demonstrated that procyanidin oligomers with a degree of polymerization lower than 5 are absorbable. D prez et al observed that (+) catechin, procyanidin dimer and trim er had similar permeability coeffi cients to that of mannitol, a marker of paracellular transport , on human intestinal epithelial Caco2 monolayers. Permeability of procyanidin polymers with an average degree of polym erization of 6 were 10 times lower ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . A study employing in situ perfusion of rat small intestine with procyanidin B2 from grape seeds revealed that procyanidin B type dimer was absorbed from the small int estine but the absorption rate was only 510% of that of ( ) epicatechin ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . Spencer et al also demonstrated that procyanidin dimer B2 or B5 from cocoa transferred from the lumen of isolated rat small intestines to the serosal side of enterocytes but only to a very small extent (<1% of the total transferred flavanol like compounds ) ( Spencer, Schroeter, Shenoy, Srai, Debnam, & Rice Evans, 2001) . Shoji et al gave apple procyanidins to rat s at a dose of 1 g/kg and found that the concentration of ( ) epicatechin, procyanidin dimer B2, and trimer C1 in the plasma reached Cmax of 1.3 M, 0.4 M, and 0.14 M respectively using HPLC -
19 mass spectrometry . Dimer and trimer concentration peaked at 2 h after administration, whereas monomer concentration peaked 1 h after administration. Using a mass spectrometer and the Porter method, t hey also detected free nonconjugat ed procyanidin dimer s to tetr amer s in rat plasma after oral administration of purified oligomer s to rats at a dose of 1 g / kg ( Shoji, Masumoto, Moriichi, Akiyama, Kanda, Oh take, et al., 2006) . Pentamers were detected using the Porter method only (Shoji, Masumoto, Moriichi, Akiyama, Kanda, Ohtake, et al., 2006) , but these data should be interpreted cautiously because Porter method lacks the specificity and accuracy to detect procyanidins in b lood ( Gu, 2012) . The concentration of procyanidin B1 [epic atechin (4 8) catechin] in human serum was 10.6 nmol/L 2 h after intake of 2.0 g of grape seed extract ( Sano, Yamakoshi, Tokutake, Tobe, Kubota, & Kikuchi, 2003) . The Cmax of procyanidin dimer B2 and ( ) epicatechin in human plasma was 41 nmol/L and 5.9 mol/L, respectively, 2 h after consumption of 0.375 g cocoa/kg body weight . The plasma concentration of dimer was only 3% of ( ) epicatechi n ( Howell, Reed, Krueger, Winterbottom, Cunningham, & Leahy, 2005) . Serum concentration of ( ) epicatechin, procyanidin dimer s, and trimers reached Cmax of 2.5 nM, 0. 5 7 nM, and 0.1 0 nM, respectively, 1 h after a grape seed extract was gavage d to rats using 1 g/kg body weight . The absorption rate of dimers and trimers was calculated as 1.69% and 0.04%, respectivel y (Serra, Maci, Romero, Valls, Blad, Arola, et al., 2010) . Unlike extensive phase II metabolism on absorbed monomers, phase II metabolism on dimers appeared to be limited because glucuronidated or sulfated metabolites of dimers were not detected in biological fluids after intestinal perfusion in rat s ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . One study detected
20 methylated B type dimer and trimer, but not glucuronide forms, in rat plasma using a mass spectrometer. The amount of methylated metabolites was not determined but appeared to be low compared with that of intact oligomer s (Shoji, Masumoto, Moriichi, Akiyama, Kanda, Ohtake, et al., 2006) . The extent of phase II metabolism of oligomers remained unclear in humans. An in situ perfusion study showed that A type procyanidin dimer A1 [e picatechinepicatechin] and A2 were absorbed in the small intestine of rats with absorption rates higher than B type dimer . This was the first study that detected A type procyanidin in blood ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . A type trimers or tetramers have not been detected in biological fluids. F ood matrices m arkedly alter the oral absorption of pro cyanidins . A carbohydraterich diet significantly decreased the absorption of procyanidins in rats . Plasma concentration of dimers reached 0.57 nM in rats when a grape seed extract was gavaged (1 g/kg) without carbohydrate. It decreased to 0.12 nM when the same amount of extract was administered with carbohydrat e (Serra, Maci, Romero, Valls, Blad, Arola, et al., 2010) . Serafini et al . showed that milk markedly decreased the absorption ( ) epicatechin from c hocolat e ( Serafini, Bugianesi, Maiani, Valtuena, De Santis, & Crozier, 2003) . These findings suggested that protein may negatively impact the absorption of procyanidins; however, additional research is needed to confirm this. Microbial Catabolism of P rocyanidins After ingestion, a small amount of flavan3 ols or procyanidins oligomers are absorbed in the small intestine. The majority of them reach the colon. A previous study detected 3 (3â€™ hydroxyphenyl)propionic acid in the urine of rats fed a diet with (+) catechin, and first suggested t he formation of this metabolite could depend on the action
21 of intestinal microflor a ( Griffiths, 1962 ) . Their subsequent work confirmed this by comparing metabolites from rats fed a (+) catechin diet with and without added antibiotic s ( Griffiths, 1964 ) . There is resurgence in research on the microbial degradation of polyphenols due to the recognition of the importance of human microbiota in the metabolism of procyanidins and human health in general. The role of gut microflora in the catabolism of procyanidins was often explored using a static anaerobic incubation system in which procyanidins were fermented with freshly collected human colonic fecal bacteria. Using this system, D prez et al. showed that polymeric procyanidins from willow tree were completely degraded after 48 h of incubation. Major catabolites included 3(3â€™ hydroxyphenyl)propionic acid, 2 ( 4 â€™ hydroxyphenyl ) acetic acid, 3(4â€™ hydryoxphenyl)propionic acid, and 3phenylpropionic aci d ( Deprez, Brezillon, Rabot, Philippe, Mila, Lapierre, et al., 2000) . Procyanidin B2 was degraded by human fecal flora twice as fast as ( ) epicatechin. 5(2â€™,4â€™ dihydroxy) phenyl 2 ene valeric acid and 5(3â€™,4â€™ dihydroxyphenyl) valeric acid were tentatively identified after in vitro anaerobic incubation of procyanidin B2 with human fecal bacteria but not from ( ) epicatechin. They were likely unique metabolites of dimer s ( Stoupi, Willamson, Drynan, Barron, & Clifford, 201 0 ) . It should be noted that t h e ability of bacteria to catabolize procyanidi ns in the gut decreases with an increase of molecular size. The yield of phenolic acids in rat gut was 10% and 7% for monomers and dimers, whereas it decreased to 0.7% and 0.5% for trimers and polymers, respectivel y ( Gonthier, Cheynier, Donovan, Manach, Morand, Mila, et al., 2003) . These results suggested that bioavailability of procyanidins decrease drastically
22 with the increase of molecular size in the form of intact procyanidins or microbial metabolites. A type procyanidins have a rigid interflavan linkage due to an additional C2O7 covalent bond. This linkage is more stable than B type procyanidins ( Gu, Kelm, Hammerstone, Beecher, Holden, Haytowitz, et al., 2003) . Their unique prevention of urinary tract infection makes the investigation of their microbial catabolism of great importance. To date, only fragmentary information is available on the microbial catabolism of A type procyanidins. A recent study utilized a pig cecum model to investigate the metabolism of p rocyanidin A2 and cinnamtannin B1 (an A type trimer) . They found that 80% of A type dimers and 40% of cinnamtannin B1 were degraded within 8 h of incubation. Procyanidin A type tri mer exhibited a more complicated pattern of hydroxylated catabolites than procyanidin A2, which probably resulted from the larger and more complex structure of trimers . Both A type procyanidins show ed C ring cleavage on the terminal unit during deg radation. Further metabolism led to the generation of hydroxy or dihydroxy benzoic acids, phenylacetic acids, phenylpropionic acids , and phloroglucinol ( Engemann, Hubner, Rzeppa, & Humpf, 2012) . In contrast to human colon microbiota, no interflavan bond scission was observed in the pig cecum model . Few studies have investigated the activity of specific bacterial species on flavan3 ol catabolism. Human fecal organism Eubacterium oxidoreducens was able to insert oxygen to form a new hydrox yl group in the A ring , which facilitates A ring opening ( Stoupi, Williamson, Drynan, Barron, & Clifford, 2010) . Eggerthella lenta rK3 has the capability of cleaving the heterocyclic C epicatechin and (+) -
23 catechin giving rise to 1(3 â€™ ,4 â€™ dihydroxyphenyl) 3 (2 â€™â€™,4 â€™â€™,6 â€™â€™trihydroxyphenyl)propan2 o l ( Kutschera, Engst, Blaut, & Bra une, 2011) . Flavonifractor plautii can further convert 1 (3 â€™ ,4 â€™ dihydroxyphenyl) 3 (2 â€™â€™,4 â€™â€™,6 â€™â€™trihydroxyphenyl)propan2 ol to 5 (3 â€™ ,4 â€™ dihydroxyphenyl) valerolactone and 4hydroxy 5 (3 â€™ ,4 â€™ dihydroxyphenyl) valeric aci d ( Kutschera, Engst, Blaut, & Braune, 2011; Winter, Popoff, Grimont, & Bokkenheuser, 1991) . Two probiotics, Streptococcus thermophilus and Lactobacillus casei 01 were able to metabolize A type procyanidins from Litchi pericarp during their log phase of growth . Streptococcus thermophilus transformed procyanidin A2 to its isomer and Lactobacillus casei 01 decomposed avan3 ols into 2 ( 3 â€™ ,4 â€™ di hydroxyphenyl ) acetic acid, 3 ( 4 â€™ hydroxyphenyl ) propionic acid, m coumaric acid, and p coumaric aci d ( Chen, Yang, Wu, Lv, Xie, & Sun, 2013) . Aspergi llus fumigatus was able to grow on purified B type procyanidins from apples. Oxygenase from this fungus modified the terminal unit of procyanidin B2 to form a metabolite with a lactone moiet y ( Contreras Domnguez, Guyot, Marnet, Le Petit, PerraudGaime, Roussos, et al., 2006) . In vivo studies showed that m icrobial derived p henyl valerolactone and phenolic acids were the predominant metabolites of procyanidins in blood and urine. After humans were given cocoa procyanidins that contained dimers to decamers, urinary excretion (07 h after dosing) of 5 (3 â€™ ,4 â€™ dihydroxyphenyl) valerolactone was 30 mol in contrast to about 2 mol for flavan3 ol monomer s ( Ottaviani, Kwik Uribe, Keen, & Schroeter, 2012) . In humans who consumed procyanidinrich cocoa regularly, blood level of ( ) e picatechin and 5(3â€™ , 4â€™ dihydroxyphenyl) valerolactone were undetectable and 0.48 M, respectivel y (Urpi Sarda, Monagas, Khan, Llorach, LamuelaRavents, Juregui, et al., 2009) . Urinary excretion of p coumaric acid, vanillic acid, 3 -
24 h ydroxybenzoic acid, and ferulic acid increased over 2 fold in humans after consumption of 40 g of procyanidinrich cocoa powder (Urpi Sarda, Monagas, Khan, LamuelaRaventos, Santos Buelga, Sacanella, et al., 2009) . There was an increase in urinary excretion of 3(3â€™ hydroxyphenyl)propionic acid, 2(3â€™ hydroxyphenyl)acetic acid, vanillic acid (4 hydroxy 3 methoxybenzoic acid) , and m hydroxybenzoic acid after human volunteers consumed 439 mg procyanidins and 147 mg catechin monomer s ( Rios, Gonthier, Rmsy, Mila, Lapierre, Lazarus, et al., 2003) . M ajor microbial metabolites of procyanidins in rat serum were 3, 4 dihydroxybenzoic acid, vanillic aci d, and 2 ( 4 â€™ hydroxyphenyl ) acetic acid after animals were given sorghum bran ( Gu, House, Rooney, & Prior, 2007) . About 5080% ingested procyanidins were degraded in the gastrointestinal tract. Up to 11% of ingested procyanidins were excreted in 24 h urine in the form of phenolic acid s ( Gu, House, Rooney, & Prior, 2007) . The absorption of procyanidins in the form of phenolic acids was likely underestimated because it was impossible to quantify all the microbial metabolites derived from procyanidins. A recent study using 14C labeled procyanidin B2 showed that the absolute bioavai lability of dimer was about 82% using the value of total urinary excretion. After an oral dose, 58% of ingested procyanidins were excreted in 24h urine and additional 40% of total radioactivity was excreted in feces (096 h after administration) ( Stoupi, Williamson, Dryn an, Barron, & Clifford, 2010) . This study confirmed that a major portion of ingested procyanidins were degraded by gut microflora before absorption. Evidence from animal and human studies suggested that B type procyanidins are absorbable and can be degraded by human gut microflora; however, information on the
25 absorption, microbial conversion and subsequent metabolites of A type procyanidin is scarce . T his dissertation aimed to contribute to cranberry rese arch by pursuing the following objectives. Research Objectives This research has three specific objectives: 1. T o investigate the transport of A type procyanidins on human intestinal epithelial Caco 2 cells a s the first step to elucidate the bioavailability of A type cranberry procyanidins in humans. 2. To convert cranberry procyanidin polymers into oligomers and monomers using two depolymerization methods and to investigate the absorbability of the depolymerized procyanidins using Caco 2 cell model. 3. To identify the microbial metabolites of partially purified cranberry procyanidins derived from in vitro fecal fermentation and compare them with those from ( ) epicatechin, (+) catechin, procyanidin B2, procyanidin A2 and partially purified apple procyanidins.
26 Figure 11. Structure of ( ) epicatechin, (+) catechin, B type procyanidin dimer B2 and A type procyanidin A2 Epicatechin Catechin Procyanidin B2 Epicatechin (4 8) epicatechin Procyanidin A2 Epicatechin (4 8, 2 epicatechin Extension Unit Terminal Unit
27 CHAPTER 2 T RANSPORT OF CRANBERRY A TYPE PROCYANIDIN DIMERS, TRIMERS, AND TETRAMERS ACROSS MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS Background Cranberries have been shown to be effective in preventing urinary tract infections ( Howell, Botto, Combescure, Blanc Potard, Gausa, Matsumoto, et al., 2010; Su, Howell, & D'Souza, 2010) , dental caries ( Bonifait & Grenier, 2010; Koo, Duarte, Murata, ScottAnne, Gregoire, Watson, et al., 2010) , cardiovascular diseases ( Caton, Pothecary, Lees, Khan, Wood, Shoji, et al., 2010) , cancer s ( Kresty, Howell, & Baird, 2011; Singh, Singh, Kim, Satyan, Nussbaum, Torres, et al., 2009) , and ag e related disorder s ( Wilson, Singh, Vorsa, Goettl, Kittleson, Roe, et al., 2008) . These physiological functions are attributed in part to the procyanidins in cranberries. Procyanidins are oligomers and polymers that consist of (+) catechin or ( ) epicatechin as constituent units. The most ubiquitous procyanidins in foods are the B type procyanidins, in which (epi)catechin units are linked through the C4C8 or C4 C6 interflavan bonds. A type procyanidins are less common in nature. The (epi)catechin units in A type procyanidins are linked by an additional ether bond between C2O7. Over 50% of the procyanidins in cranberries are A type ( Gu, Kelm, Hammerstone, Beecher, Holden, Haytowitz, et al., 2004) . A type procyanidin oligomers isolated from cranberries were able to inhibit adherence of uropathogenic E. coli, whereas the B type procyanidins were not ( Foo, Lu, Howell, & Vorsa, 2000; Nowack & Schmitt, 2008) . These previous studies suggest A type procyanidins are the major bioactive components in cranberries. B type procyanidin dimers and trimers were absorbed and present in the blood of human subject s ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009; Baba, Osakabe,
28 Natsume, & Terao, 2002; Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . An in situ intestinal perfusion experiment in anesthetized rats revealed t hat A type procyanidin dimer A1 [ epicatechin(2 O 7, 4 â€“ 8) catechin] and A2 [ epicatechin(2 O 7, 4 â€“ 8) epicatechin] isolated from peanut skin were absorbed and their transport ratio were higher than that of dimer B2 [ epicatechin(4 â€“ 8) epicatechin] ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . H owever, A type procyanidins were not known to be absorbed by humans. As the first step to elucidate the bioavailability of A type cranberry procyanidins in humans, human intestinal epithelial Caco2 cells were employed to investigate the transport of A typ e procyanidins. M aterials and Methods Chemicals and M aterials Freezedried cranberries were provided by Ocean Spray Cranberries, Inc (Lakeville Middleboro, MA) . A type procyanidin dimer A2 (epicatechin(2 O 7, 4 8) epicatechin) was purchased from Chromadex Inc. (Irvine, CA). B type procyanidin dimer [ epicatechin(4 8) epicatec hin] was obtained from Indofine Chemical Company, Inc. (Hillsborough, NJ). A B type procyanidin standard that contained monomers to decamers was kindly provided by Mars Botanicals (Roc kville, MD). Amberlite FPX 66 resin was a product from Dow Company (Midland, M I ). Caco 2 cells originating from human colorectal carcinoma were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Solidphase extraction cartridges and other reagents were obtained from Fisher Scientific Co. (Pittsburg h , PA). Purification of C ranberry P rocyanidins Five hundred grams of spray dried cranberry powder was extracted with 4 L of methanol at room temperature for 48 h. Extracts obtained after vacuum filtration were
29 combined and concentrated under a partial vacuum using a rotary evaporator. The concentrated extract was resuspended in 100 mL of water and loaded onto a column (3.844 cm) packed with Amberlite FPX 66 resin. The column was eluted with 5 L deionized water to remove sugars followed by 2 L of methanol to yield cranberry phytochem ical powder ( ca. 28 g). Part of this powder (27 g) was suspended in 80 mL of DI water and loaded onto a column (28 cm, 5.8 cm i.d.) packed with Sephadex LH 20, which was soaked in 30% methanol for over 4 h before use. The column was eluted with 30% methanol (1.6 L), 60% methanol (1.2 L), 80% methanol (1.2 L), 100% methanol (1.2 L) and 70% acetone (1.2 L). Every 400 mL eluent was collected as a fraction. The third fraction of 80% methanol eluent was referred to as fraction I ( ca. 1.3 g) . T he 100% methanol el u ent was concentrated to yield 3 g of dry extract. It was suspended in 20 mL water and partitioned with 100 mL ethyl acetate three times. The ethyl acetate phases were combined and evaporated to yield 0.45 g of extract. This was referred to as fraction II . Cell C ulture Caco 2 cells between passages 2832 were used in these experiments. Cells were cultured in Dulbeccoâ€™s modified Eagleâ€™s medium (DMEM) with glucose (4.5 g/L), Lglutamine, and 20% fetal bovine serum as previously described ( Bose, Seetharam, Dahms, & Seetharam, 1997) . At 80 90% confluence, cells were seeded a t a level of 1 105 cells/cm2 in 6 well transwell plates (Corning Inc. Corning, NY) and cultured in an incubator at 37 oC with 5% CO2. The mediums were changed every 2 days for the first 7 days. After 8 days, the medium in apical chamber w as changed every day while the basolateral chamber medium w as changed every other day.
30 Transport E xperiments The transport experiment was conducted when the transepithelial electrical resistance (TEER) of the C aco 2, typically a t 17 days post seeding. Briefly, growth medium was removed from the transwells by aspiration, and the upper and lower chambers were rinsed with Hank â€™ s Balance Salt Solution (pH 7.2 7.4 ). Then a 1.5 mL and 2.6 mL Hanksâ€™ balanced salt solution (HBSS, pH 7.27.4) were added in to the apical and basolateral chambers, respectively. After 30 min of preincubation, the HBSS w as aspirated. Fraction I or procyanidin dimer A2 standard was dissolved in 1.5 mL HBSS (contained 0.5% DMSO to dissolve procyanidins ) to a con centration of 1 mM A type procyanidin dimer. A solution of fraction II with 0.4 mM A type procyanidin trimers and 0.3 mM tetramers was used to mimic the expected concentration of procyanidins occurring in the gut ( Deprez, Mila, Huneau, Tome, & Scalbert , 2001) . These solutions were added to the apical chambers . Meanwhile, 2.6 mL HBSS with 0.5% DMSO was added to the basolateral chambers. Transport was carried out at 37 oC for 2 h as previously described ( Brand, van der Wel, Rein, Barron, Williamson, van Bladeren, et al., 2008) . At the end of uptake, 2.6 mL solutions in the basolateral chamber and 1.5 mL solutions in the apical chambers were collected. E ach of the apical or basolateral chamber s w ere washed with 1.0 mL or 2.4 mL of phosphatebuffered saline (pH 7.4). Rinsing solutions were added into the HBSS. Transport experiment s w ere carried out on 3 ind ividual wells for each sample. Wells with a TEER value below 300 2 at the end of transport w ere discarded to ensure the integrity of monolayer s ( Manna, Galletti, Maisto, Cucciolla, D'Angelo, & Zappia, 2000) .
31 Solid P hase E xtraction Procyanidins w ere extracted from HBSS using C18 solidphase extraction cartridges immediately after the transport experiments. The cartridges were washed with 3 mL of methanol and equilibrated with 3 mL of water. After HBSS solution from apical or basolateral chambers w as loaded, the cartridges were washed with 3 mL of water. Procyanidins w ere recovered with 3 mL of 100% methanol. The methanol eluent was dried in a SpeedVac concentrator (Fisher Scientific, Pittsburgh, PA). Dried samples were dissolved in methanol for HPLC MSn analyses. HPLCMSn A nalysis Chromatographic analyses were performed on an Agilent 1200 HPLC system ( Palo Alto, CA ) equipped with a binary pump, an autosampler, a fluorescence detector, and a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA) . Separation of procyanidins w as carried out on a 250 mm x 4.6 mm i. d., 5 m, Develosil Diol 100 column, with a 4 mm x 3 mm i.d. guard column (Phenomenex, Torrance, CA) at a column temperature of 35 oC . The binary mobile phase consisted of (A) a cetonitrile/acetic acid (98:2, v/v) and (B) methanol/water/acetic acid (95:3: 2, v/v/v). The 76 min gradient was as follows: 012 min, 7% B isocratic; 1260 min, 737.6% B linear; 6063 min, 37.6100% B linear, 6370 min 100% B isocratic; 70 76 min 1007% B linear; followed by 5 min of re equilibration of the column before the next run. Excitation and emission of the fluorescent detector were set at 231 and 320 nm, respectively. Electrospray ionization at negative mode was performed using nebulizer 50 psi, drying gas 10 L/min, drying temperature 350 oC , and capillary 4000 V. Mass spectra were recorded from m/z 150 to 2000 . The most abundant ion in full scan was isolated, and its product ion spectra were recorded.
32 Determination of P rocyanidin C ontents, A pparent P ermeability Coefficient and T ransport R atio ( ) Epicatechin, and B type and A type procyanidin dimers were quantitated using external standards. A type procyanidin trimers and tetramers were quantified using B type procyanidins standard from Mars Botanicals (Rockville, MD) . Apparent permeability coefficients (Papp, cm/s), transport ratio and recovery rates were calculated using the following equations. Papp= (dQ/dt)x(1/AC0) ( 2 1 ) where dQ/dt is the permeability rate (g/s), C0 is the initial concentration in the donor chamber (g/mL), and A is the surface area of the filter (cm2), which is 4.67 cm2 in this study. Transport ratio= (Procyanidin t ransported)/(Total procyanidins )100% ( 2 2 ) Recovery rate= [Procyanidin (transported+remaining )]/(Total procyanidins)100% ( 2 3 ) â€˜Procyanidin transportedâ€™ represented the amount of procyanidins on the basolateral side of the transwell after transport. Total procyanidins w ere the total amount of procyanidins added on the apical side of the transwell at the beginning of the experiment. Procyanidins (transported+remaining ) were the sum of procyanidins that traversed across Caco 2 membranes and those that remained in the apical side after transport. Statistical A nalysis All data were expressed as the mean standard deviation. Oneway analyses of variance (ANOVA) with Tukey Kramer HSD pair wise comparison of the means were performed using JMP software (Version 9 .0, SAS Institute Inc., Cary, NC). A difference of p
33 R esults and Discussion Composition of P rocyanidin Fractions P urified from C ranberries Cranberry methanol extracts contained sugars and phytochemicals such as anthocyanins, flavonols and oligomeric and polymeric procyanidins ( Fuleki & Francis, 1968; Prior, Lazarus, Cao, Muccitelli, & Hammerstone, 2001; Yan, Murphy, Hammond, Vinson, & Neto, 2002) . Amberlite resin FPX66 was used to remove sugars from the cranberry extracts. A Sephadex LH 20 column was employed to separate anthoc yanins and flavanols from procyanidins. Anthocyanins and flavanols bind to Sephadex LH 20 with lower affinity, and thus were eluted earlier. Procyanidin oligomers and polymers bind to Sephadex LH 20 with stronger affinities and were eluted by methanol or aceton e (Gu, Kelm, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . Two fractions were obtained from a Sephadex LH 20 column and their HPLC chromatograms are illustrated in Figure 2 1 . The chromatogram of fraction I showed a major peak (2A) at 8.2 min and a minor peak at 17.418.2 min. Peak 2A was identified as procyanidin A2 because it had the same retention time and mass spectra as procyanidin A2 standard. This peak showed [M H]m/z 575. Its product ion at m/z 285 was due to quinone methide (QM) cleavage of A type interflavan bonds. Product ion at m/z 423 resulted from the retroDiels Alder (RDA) fission of the heterocyclic ring (Table 2 1). The minor peaks with retention time of 1 7.4 18.2 min were identified as A type procyanidin trimers according to [M H]m/z 863. The diagnostic ion at m/z 575 derived from QM indicated that these trimers had a connection sequence of (epi)cat (epi)cat A (epi)cat . The (epi)cat denotes catechin or epicatechin since they could not be distinguished by mass spectrometer. â€“ A â€“ represented an A type linkage. The A type interflavan linkage
34 in the trimer was between the middle unit and the terminal units. A p roduct ion of m/z 711 was due to retroDiels Alder (RDA) fission. Ion m/z 287 was derived from QM cleavage of B type interflavan linkage between the upper and middle units. Ion m/z 693 was generated from RDA cleavag e and a loss of water (Table 2 1). The content of procyanidin dimers and trimers in fraction I were 44.1% and 7.6% (w/w), respectively. Fraction II contained oligomers with degree of polymerization between 3 and 6. Peak 3A with retention time of 17. 4 min s howed [M H]m/z 863 and product ions similar to peak 3A in fraction I. They were deduced to be the same compound. Peak 3A ' at 21.6 min had a [M H]m/z 863 and similar product ion spectra to that of peak 3A. This peak was also an A type trimer with connec tion sequence of (epi)cat (epi)cat A (epi)cat. Foo et al. ( Foo, Lu, Howell, & Vorsa, 2000) isolated two A type procyanidin trimers from cranberries with this same connection sequence. They were epicatechin(4 6) epicatechin(4 8, 2 O 7) epicatechin and epicatechin(4 8) epicatechin(4 8, 2 O 7) epicatechin. Peak 3A and 3A ' were likely these two trimers. Two peaks of A type tetramers 4A ( 28.7 min) and 4A ' (31.6 min) were identified. Peak 4A gave r ise to p roduct ions m/z 863 and 575 due to QM cleavage of interflavan linkages, indicating a connection sequence of (epi)cat (epi)cat (epi)cat A (epi)cat. This connection sequence was consistent with the absence of product ion m/z 573. Product ions m/z 861 and 573 derived from QM cleavage suggested peak 4A ' ha d a connection sequence of (epi)cat A (epi)cat (epi)cat (epi)cat . Peak 5A ([M H]m/z 1439.2) was identified as an A type pentamer. Its product ions at m/z 575, 863, 1149 and 1151 due to QM cleavage of interflavan bonds, and m/z 861 from QM cleavage in A type interflavan bond indicated this pentamer had a structure of (epi)cat (epi)cat (epi)cat A -
35 (epi)cat (epi)cat . Peak 6A ( M H]m/z 1725.4 w as identified as a hexamer with two A type interflavan linkages . We were not able to identify its connection sequence due to a large number of isomers and complexity of the mass spectra data. The content of procyanidin trimers, tetramers, pentamers, and hexamers in fraction II were 15.2%, 11.7%, 7.2%, and 5.3% (w/w), respectively. Transport of C ranberry A type P rocyanidins on Caco2 Cells Chromatogram of fraction I dissolved in HBSS is shown in Figure 2 2A. This solution was added into the apical side of Caco2 cells membranes. After 2 h, the HBSS solution from the basolateral side of the membrane was analyzed. Its chromatogram is depicted in Figure 2 2B . No peak was observed in the control sample where no procyanidins were added (Figure 2 2C). A peak X1 with retention time 7. 5 min gave rise to m/z [M H]575 w ith product ion at m/z 285 and 423 (Figure 2 3). This peak was identified as procyanidin dimer A2. The structure of A2 and its fragmentation pathway is depicted in Figure 2 4 . The HBSS solution of fraction II, with the chromatogram shown in Figure 2 5 A, w as applied on the apical side of differentiated Caco2 cells monolayer. After 2 h, the HBSS solution from the basolateral side showed a chromatogram (Figure 2 5 B) that was similar to Figure 2 5 A. No peak of procyanidins existed in the control (Figure 2 5 C) . A peak (retention time 16.5 min, referred as X2) showed m/z [M H]863 with product ion m/z [M H]575 and 693 (Figure 2 6 A). This peak was the same as peak 3A in fraction II. It was an A type trimer with a connection sequence of (epi)cat (epi)cat A (epi)cat. Peak X3 (retention time 28.0 min) showed m/z [M H]1151 with product ion m/z [M H]575 and 693 (Figure 2 6B ). This peak was the same as peak 4A in fraction II. It was an A -
36 type tetramer with a connection sequence of (epi)cat (epi)cat (epi) cat A (epi)cat . Identifications of other procyanidins in media of the basolateral side w as inconclusive. These results indicated that the A type procyanidin dimers, trimers and tetramers can traverse through Caco 2 cells monolayer. These obs ervations were consistent with the findings of Appeldoorn et al . ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) who found procyanidin dimers A1 and A2 were absorbable from the small intestine of rats during an in situ perfusion. However, they did not detect A type trimers in rat blood, likely due to very low concentrations of A type trimers in their testing samples ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . Deprez et al. also showed that B type procyanidin dimers, trimers, and tetramers transported through Caco2 me mbranes ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . The Papp values, recovery rates and transport ratios of A type procyanidin dimers, trimers, and tetramers on this Caco2 cell model are shown in Table 2 2 . R ecovery rates ranged from 93% for cr anberry A type tetramers to 104% for procyanidin A2 standard. Generally, the transport ratios were lower than 5%. This could be explained by Lipinskiâ€™s â€œRule of Fiveâ€, which suggests that compounds with five or more hydrogen bond donors, or ten or more hydrogen bond acceptor s, or molecular weight s greater than 500 Da have poor bioavailability due to their large apparent siz e ( Lipinski, Lombardo, Dominy, & Feeney, 1997) . The transport ratio of A type procyanidin dimer in fraction I was 0.6%, which was significantly lower than that of pure dimer standard A2, 4.8% ( ). Its permeability was ~ 8 times lower than that of the reported B3 trimer ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . The HPLC chromatogram suggested the existence of other
37 phytochemicals than procyanidin A type dimer in this fraction. Other phytochemicals in fraction I could possibly interfere with transport of procyanidin dimer, thus decreasing the amount of dimer transported. Another possible reason for the discrepancy in transport ratio as well as Papp values is that procyanidins with high polymerization are astringent and may complex the membrane proteins and strengthen intercellular tight junctions ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . This was confirmed by a lower transepithelial electrical resistance value of Caco2 monolayers in procyanidin A2 standard transport experiments than those for fraction I (data not shown). A type procyanidin dimer demonstrated a significantly higher transport ratio than B type dimer (3.0%). This was in agreement with an early study which showed that A1 and A2 dimers were better absorbed than dimer B2 in the rat small intestine ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . For the procyanidin A2 standard, its concentration in the basolateral side of Caco2 cell monolayer after transport was about 14 g/ mL. This concentration is likely enough to exert antibacterial activity because an early study reported inhibition of E. coli adherence by cranberry procyanidins over a concentration range of 5 to 75 g/m L ( Gupta, Chou, Howell, Wobbe, Grady, & Stapleton, 2007) . The permeability for A type trimer was almost 10 times less than that o f C2 trimer and the A type tetramer showed similar permeability with B type polymer with a degree of polymerization of six ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . The transport ratio of trimer and tetramer w ere 0.4% and 0.2% respectively, which were significantly lower than that of dimer s in fraction I (p 0.05). The transport ratio decreased with an increase in molecular weight. Similar observations have been made by other researchers. For example, dimer s w ere absorbed at a much lower efficac y than
38 monomer s ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009; Holt, Lazarus, Sullards, Zhu, Schramm, Hammerstone, et al., 2002) . Permeability of a procyanidin polymer with an average polymerization degree of 6 (molecular weight 1,740) on Caco2 monolayers was approximately 10 times lower than (+) catechin ( Ap peldoorn, Vincken, Gruppen, & Hollman, 2009) . However, lower permeability of epicatechin than dimer and trimer standard has been observed in both a previous and current study ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . This may be due to an epicatechin specific efflux pump localized on the apical m embrane ( Vaidyanathan & Walle, 2001) . In this study, a Papp value of 0.7x106 cm / s was observed for epicatechin. This value is si milar to the 0.9x106 cm/s reported in a previous study ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001 ) . The transport ratio of epicatechin (1.6%) was much lower than the values of 11.6% and 55.9% observed in jejunum and ileum using isolated rat smal l intestine perfusion model ( Kuhnle, Spencer, Schroeter, Shenoy, Debnam, Srai, et al., 2000) . The different models employed may be the major reason for the distinct results. The result on permeability of B type dimer was consistent with a previous study on Caco 2 cells ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) . This study showed cranberry A type procyanidin dimers, trimers, and tetramers traverse d across Caco 2 cell monolayers with a very low transport ratio. It suggested that these oligomers are absorbable in humans but their bioavailability is likely very low. This in part can explain the failure of detecting them in human blood. Appeldoorn et al . ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) reported about 0.11% of A type procyanidin dimers were absorbed after in situ intestinal perfusion in anesthetized rats for 30 min. The transport mechanism of procyanidin oligomers was likely permeation or
39 diffusion, since no transporters have been identif ied that can transport epicatechin into intestinal epithelial cells. Absorbed epicatechin undergo extensive phase II metabolism in the intestine, such as methylation, glucuronidation, and sulfation ( Li, Me ng, Winnik, Lee, Lu, Sheng, et al., 2001) . We did not detect any conjugates of A type procyanidin dimers, trimers, or tetramers. This observation was consistent with that of Appeldoorn et al . who found procyanidin dimers were not conjugated or methylate d in rats ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009) . Another explanation could be that the levels of conjugates were below the detection limit of the HPLC MSn method. Summary This study found that A type procyanidins dimers, trimer s and tetramers from cranberr ie s can be absorbed across Caco2 cells although the transport ratios were strikingly low. It was suggest ed that A type procyanidins dimers, trimer s and tetramers are bioavailable in humans after cranberry consumption.
40 Table 2 1. type and B type p rocyanidin o ligomers in p urified p rocyanidin f ractions Fraction Rt (min) Peak label Connection sequence [M H] m/z Product ions I 8.2 2A epicatechin(2 O 7, 4â€“ 8) epicatechin* 575 285(QM of A type linkage) , 423RDA,449HRF, 539(2 H2O loss) 16.5 3A (epi)cat (epi)cat A (epi)cat 863 423RDA, 449HRF, 575QM, 693(RDA, H2O loss), 711RDA, 845(H2O loss) II 16.5 3A (epi)cat (epi)cat A (epi)cat 863 287QM, 575QM, 693(RDA, H2O loss), 711RDA, 845(H2O loss) 21.6 3A' (epi)cat (epi)cat A (epi)cat 863 287QM, 575QM, 693(RDA, H 2 O loss), 711RDA 24.2 3B (epi)cat (epi)cat (epi)cat 865 575QM, 577QM, 695(RDA, H2O loss), 713RDA, 739HRF 28. 0 4A (epi)cat (epi)cat (epi)cat A (epi)cat 1151 411HRF, 575QM, 863QM, 981(RDA, H2O loss) 31.6 4A' (epi)cat A (epi)cat (epi)cat (epi)cat 1151 573QM, 691(RDA, H2O loss), 861QM, 981(RDA, H2O loss) 36.0 5A (epi)cat (epi)cat (epi)cat A (epi)cat (epi)cat 1439 575QM, 693(RDA, H2O loss), 861QM, 863QM, 1149QM, 1151QM 39.8 6A ND 1725 573QM, 691(RDA, H2O loss), 711RDA, 861QM, 1151QM, 1435QM Symbol (epi)cat stands for cat echin or epicatechin . RDA, retro Diels Alder cleavage; HRF, heterocyclic ring fission; QM, quinone methide cleavage. A denotes A . * id entified by comparing with standard. ND, unable to be determined.
41 Table 2 2. Transport r atio of A type p rocyanidin o ligomers on Caco2 cells Sample added to apical side before transport (g) Basolateral side after transport (g) Apical side after transport (g) P app x10 6(cm/s) Transport ratio (%) Recovery rate (%) A type dimer in f raction I 624.9712.75 3.660.03 615.4416.21 0.260.01 A 0.60.0 A 99.10.6 A type trimer in f raction II 476.3220.17 1.750.24 476.961.31 0.160.03 B 0.40.1 B 100.50.3 A type tetramer in f raction II 529.96 34.59 1.23 0.039 493.31 14.52 0.11 0.01 C 0.2 0.0 C 93.3 2.7 ( ) Epicatechin 383.801.11 5.980.06 375.331.54 0.700.01 c 1.60.0 c 99.40.1 Procyanidin A2 744.0625.63 35.430.22 736.0630.23 2.110.05 a 4.80.1 a 103.77.6 Procyanidin B2 946.292.84 28.170.94 905.8219.36 1.330.04 b 3.00.1 b 98.71.9 Data were expressed as the mean standard deviation (n=3). Different letters in the same column indicate significant differences in means at p 0.05. Values with the same case were compared to each other.
42 Figure 21. HPLC chromatogram of procyanidins in fraction I and fraction II. Peak 2A, 3A, 4A, 5A, and 6A were A type dimers, trimers, tetramers, pentamers, and hexamers, respectively. Peak 3B was B type trimers. Fraction I2A 3 A LU 0 200 400 600 800 1000 1200 1400 min 0 10 20 30 40 50 60 70 0 20 40 60 80 100 120 140 160 Fraction II3 A 3A' 3B 4A 4A ' 5A 6A
43 Figure 22. HPLC chromatogram of solutions from transport experiments. A) HPLC chromatogram of fraction I in apical side at the beginning of transport experiment, peak 2A was A type dimer; B) HPLC chromatogram of H BSS from basolateral sides of C aco 2 monolayer after 2 h, and C) chromatogram of control solution in the basolateral side. Peak X1 was detected in the HBSS solution from basolateral side. LU 200 400 A 10 20 30 40 50 B min 0 10 20 30 40 50 60 70 2 4 6 8 10 12 14 CX12A
44 A. Figure 2 3 . Product ion spectrum of peak X1 288.6 284.6 422.7 448.8 538.8 (2H2O loss) MS2(575.0), 7.5min 0 500 1000 1500 2000 200 300 400 500 600 700 m/z Intens .
45 Figure 2 4 . P ostulated fragmentation pathway of procyanidin A2. RDA, retroDiels Alder cleavage; HRF, heterocyclic ring fission; QM, quinone methide cleavage. RDA QM HRF m/z 575 m/z 423 m/z 285 m/z 289 m/z 449 O H O H O H O H O H O O H O O O H O H O H O O O H O H O O H O H O H O O H O H O H O H O H O O H O O O H O H O H O H O H O H O H O H O O H O O +
46 Figure 25 . HPLC chromatogram of solutions from transport experiments. A) HPLC chromatogram of fraction II in apical side at the beginning of transport experiment, Peak 3A and 4A were A type trimers and tetramers, B) HPLC chromatogram of HBSS from basolateral sides of C aco 2 monolayer after 2 h, and C) chromatogram of control solution in the basolateral side. Peak X2 and X3 were detected in the HBSS solution from basolateral side. 14 min 10 20 30 40 50 60 70 2 4 6 8 10 12 C LU 10 20 30 40 A 5 10 15 20 25 B X2 X3 03A 4A
47 Figure 2 6 . Product ion spectrum of detected peaks. A) peak X2 and B) product ion peak X3. 574.8 692.9 845.8 MS2(863.3),16.5 min 0 200 400 600 Intens . 200 400 600 800 1000 m/z 800 711.2A B 860.9 998.7 1093.3 0 50 100 150 200 600 700 800 900 1000 1100 m/z MS2(1151.0), 28.0 min 574.6 981.0
48 CHAPTER 3 DEPOLYMERIZATION OPITIMIZATION OF CRANBERRY PROCYANIDINS AND TRANSPORT OF RESULTANT OLIGOMERS ON MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS Background Procyanidins in cranberries consist of oligomers and polymers of different sizes (Gu, Kelm, Hammerstone, Beecher, Holden, Haytowitz, et al., 2003) . Oligomers refer to procyanidin monomers through tetramers. Polymers include pentamers and above. A type oligomeric procyanidins from cranberries were known to inhibit the adherence of uropathogenic E. coli whereas B type were not ( Foo, Lu, Howell, & Vorsa, 2000; Shoji, Masumoto, Moriichi, Kobori, Kanda, Shinmoto, et al., 2005; Sugiyama, Akazome, Shoji, Yamaguchi, Yasue, Kanda, et al., 2007) . This study and reports from other researchers show that procyanidins dimer s through tetramers were absorbable in vitr o ( Zumdick, Deters, & Hensel, 2012) and in viv o (Baba, Osakabe, Natsume, & Terao, 2002; Stoupi, Williamson, Viton, Barron, King, Brown, et al., 2010) , whereas larger procyanidins are not absorbable. In cranberry, however, these absorbable oligomers only account for 15% of total procyanidins, while the rest are polymer s (Gu, Kelm, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . Such composition limits both the bioavailability and bioactivities of procyanidins in cranberries. Several methods were explored to conver t polymers into absorbable oligomers and monomers. E xtrusion of sorghum increased the levels of procyanidin oligomers with degree of polymerization (DP) 4 while decreas ing 6 ( Gu, House, Rooney, & Prior, 2008) . Extrusion also increased procyanidin monomer and dimers in blueberry pomac e ( Khanal, Howard, Brownmiller, & Prior, 2009) . T he improvement of procyanidin bioavailability through extrusion was verified in weanling
49 pig s ( Gu, House, Rooney, & Prior, 2008) . A recent study applied flavan3 ols as ch ain breakers to depolymerize procyanidins under a mild conditio n ( Liu, Zou, Gao, & Gu, 2013) . In this method , H+ catalyzes the cleavage of the interflavan bonds to form carbocation at C4. A nucleophilic addition between carbocations and added flavan3 ols resulted in the generation of new oligomers. T he chemical structure of newly generated oligomer after depolymerization was similar to that of naturally occurred procyanidins . However, it was not known if these oligomers were absorbable. Because bioactivities of procyanidins largely depend on their bioavailability , it is important to investigate their absorption. Therefore, the objective of this study is to investigate the transport of depolymerized cranberry procyanidins on Caco2 cell monolayers. If they can be absorbed, the depolymerization process will be optimized using r esponse s urface m ethodology. Materials and M ethods Chemicals and M aterials Freezedried cranberries were provided by Ocean Spray Cranberries, Inc (Lakeville Middleboro, MA) . ( ) E picatechin and Sephadex LH 20 were purchased from Sigma Chemical Co. (St. Louis, MO). Amberlite FPX 66 resin was a product from Dow Company (Midland, Michigan). Caco2 cells originating from hum an colorectal carcinoma were obtained from the American Type Culture Collection (Manassas, VA). Other reagents were obtained from Fisher Scientific Co. (Pittsburg h , PA). Preparation of P artially P urified P rocyanidin P olymers (PP) Five hundred grams of dri ed cranberry powder was extracted in 4 L of methanol at room temperature for 48 h. Extracts obtained after vacuum filtration were combined and concentrated under partial vacuum using a rotary evaporator. The concentrated
50 extract was resuspended in 100 mL of water and loaded onto a column (3.844 cm) pac ked with Amberlite FPX 66 resin. The column was eluted with 5 L deionized water to remove sugars followed by 2 L of methanol to yield cranberry phyt ochemical powder (about 28 g). Part of this powder (27 g) was suspended in 80 mL of 30% meth a n o l and loaded onto a column (28 cm, 5.8 cm i.d.) packed with Sephadex LH 20, which was soaked in 30% methanol over 4 h before use. The column was eluted with 30% methanol (1.6 L), 60% methanol (1.2 L), 80% methanol (1.2 L), 100% methanol (1.2 L) and 70% acetone (1.2 L). Every 400 mL eluent was collected as a fraction. The second fraction of 70% acetone elution was dried and stored for depolymerization experiments. The profile of procyanidins in this extract was analyzed using HPLC MS. Depolymerization of C ranberry P rocyanidins D epolymerization was carried out with or without added epicatechin . In experiments without added epicatechin, cranberry procyanidin extract w as dissolved in methanol to hav e a stock solution with a concentration of 10 mg/mL. The stock solution was then mixed with an equal volume of 2 M methanolic HCl . In the depolymerization with added epicatechin, procyanidin extract and ( ) epicatechin were dissolved in methanol to achieve a concentration of 20 mg/mL , respectively. After wards , equal volume of extract and ( ) epicatechin w as mixed with 2 volumes of 2 M methanolic HCl. The final concentration of cranberry procyanidin extract was 5 mg/m L . Depolymerization w as carried out in a 60 oC water bath for 60 min for both methods . The reaction was stopped by adjusting the pH of mixture to 5 with 1 M NaHCO3. The solutions were dried in a SpeedVac concentrator at 45 oC . T he dried sample was dissolved in water and partitioned with ethyl acetate 3 times. The ethyl acetate fraction was collected and dried in a SpeedVac concentrator .
51 HPLCESI MSn A nalysis of P rocyanidins Chromatographic analyses were performed on an Agilent 1200 HPLC system ( Palo Alto, CA ) equipped with a binary pump, an autosampler, a fluorescence detector, and a high capacity (HCT) ion trap mass spectrometer (Bruker Daltonics, Billerica, MA) . Separation was carried out on a Luna Silica (2) column (250 4.6 mm , size , P henomenex, Torrance, CA) at a column te mperature of 37 oC . The binary mobile phase consisted of (A) methylene chloride/methanol/acetic acid/water (82:14:2:2, v/v/v/v) and (B) methanol/acetic acid/ water (96:2:2, v/v/v). The 70 min gradient was as followed by 5 min of column reequilibration before the next injection . Excitation and emission of the fluorescent detector were set at 231 and 320 nm, respectivel y ( Robbins, Leonczak, Johnson, Li, Kwik Uribe, Prior, et al., 2009) . Electrospray ionization at negative mode was performed using nebulizer 50 psi, drying gas 10 L/min, and drying temperature 350 oC , capillary 4000 V. Mass spectra were recorded in a range of m/z 150 to 2200. The most abundant ion in full scan was isolated, and its production ion spectra were recorded. A calibration curve was generated using ( ) epicatechin standard. P rocyanidins were estimat ed using relative response factor s calculated according to Robbins et al . ( Robbins, Leonczak, Li, Johnson, Collins, Kwik Uribe, et al., 2012) . The relative response factor of nonamers was employed to estimate the amount of polymers . The yield of depolymerization was defined as the amount of oligomers produced per mg of partially purified polymers.
52 Transport E xperiments Transport experiment was carried out following the procedures described in C hapter 2. The concentration of depolymerized samples in Hankâ€™s Balanced Salt Solution (0.5% DMSO) was equivalent to 2 mM epicatechin. Optimization of D epolymerization C onditions A central composite experimental design was utilized to study the response pattern and determine optimum conditions . In depolymerization without added epicatechin, variables included time ( from 20 to 180 min) , temperature ( from 25 to 60 oC ) , and acid conce ntration (from 0.1 2 M HCl in methanol) . While in the method with added epicatechin, an additional factor of epicatechin/polymer ratio (w/w) (from 0 to 3) was taken into consideration. A total of 20 experiments for depolymerization without added epicatechi n and 30 for depolymerization with added epicatechin were conducted according to the design. Duplicates were performed for each run. Six replicates at the center of the design were used to allow the pure error square sum estimation. The condition at center point was 100 min, 42.5 oC , 1.05 M HCl in methanol for depolymerization without added epicatechin. The center point for depolymerization with added epicatechin had the same time, tempera ture, HCl concentration while the epicatechin/polymer ratio was 1.5. Experiments were randomized to maximize the effects of unexplained variability in the responses obtained due to external factors. Experimental data was analyzed using JMP software (Version 10.0, SAS Institute Inc., Cary, NC) . Q uadratic polynomial regression models were proposed to predict the Y variableyield of oligomers. The model proposed for the response of Y fitted equation was as follows: Y1=b0+b1X1+b2X2+b3X3+b11X1 2+b22X2 2+b33X3 2+b12X1X2+b13X1X3+b23X2X3 ( 3 1 )
53 Y2=b0+b1X1+b2X2+b3X3+b11X1 2+b22X2 2+b33X3 2+ b44X4 2+b12X1X2+b13X1X3+b14X1X4+b23X2X3+ b24X2X4+b34X3X4 ( 3 2 ) where Y1 and Y2 are the response (yield of oligomers) for depolymerization with out and with added epicatechin, respectively. The coefficients of the polynomial were represented by b0 (constant term); b1, b2, b3 and b4 (linear effects); b11, b22, b33, and b44 (quadratic effects); and b12, b13, b14, b23, b24, and b34 (interaction effects) and (random error). The fitted polynomial equations were expressed in 3D response surface s. The proportion of variance explained by the polynomial models obtained was given by the multiple coefficients (R2) of determination. The significance of each coefficient was also determined. JMP software was used to fit response surface, to optimize depolymerization process. The predicted optimum yield of oligomers was verified by the experiment s using the selected optimum values of the variables. Statistical A nalysis All data were expressed as the mean standard deviation. Oneway analyses of variance ( ANOVA) with Tukey Kramer HSD pair wise comparison of means were performed using JMP software (Version 10.0, SAS Institute Inc., Cary, NC). A difference of p Results and D iscussion HPLCMSn A nalysis of P rocyanidins B efore and A fter D epolymerization In the present study, procyanidin polymers were defined as those with degree of polymerization 5 . P olymers account for over 85% of the total procyanidins i n cranberr ie s (Gu, Kelm, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . Sugars in cranberry extracts were removed using Amberlite resin FPX66. Sephadex LH 20 was used as an adsorbent for pu rification of procyanidin polymers . The
54 adsorption affinity between Sephadex LH 20 and procyanidins increases with molecular size s (Gu, Kel m, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . The HPLC chromatogram of partially purified procyanidin polymers before depolymerization was shown in Figure 3 1A . Polymers with DP 5 10 eluted from 32 min to 52 min and those with DP > 10 elute as a single peak at 56 min. Insignificant amount of oligomers were also detected from 15 to 20 min. This result was in agreement with a previous study ( Gu, 2012) . The content of procyanidin polymers (510 accounted for 83.3%. During depolymerization reactions, procyanidins were cleaved by HCl into carbocation unit ( from extension units) and the flavan3 ol unit ( from terminal units). B type interflavan bonds cleave under mild acidic conditions, whereas A type interflavan bonds remained stable ( Thompson, Jacques, Haslam, & Tanner, 1972) . When epicatechin was added as a nucleophile, it added on to carbocation to form new oligomers. Without added epicatechin, new oligomers were those flavan3 ols released from terminal units. The HPLC chromatograms of the depolymerized samples are shown in Figure 3 1B & 3 1C. For both methods, the large peak of procyanidin polymers with DP>10 disappeared while new peaks appeared at retention time s from 10 to 30 min. The peak elut ing at 10 .6 min gave rise to m/z 289 [M H]and it was consistent with epicatechin and/or catechin , which cannot be separated using a normal phase HPLC . Peak 15.7 min showed m/z 575 [M H]-. It was identified as procyanidin A type dimer as it generated m/ z 423 due to retroDiels Alder fission of the heterocyclic ring and m/z 285 from quinone methide cleavage of the A type linkage. The peak with retention time of
55 18.2 min gave m/z 577 [m H]and was identified as B type dimer according to its product ions of 425, 475 and 287. Other peaks were identified as A type trimer s at 23.1 min, B type trimer s at 24.1 min and A type tetramers at 2 7.9 min, respectively. After depolymerization without added epicatechin (Figure 1B), the peak of A type dimer was hig her than that of B type dimer. The quantitation data showed that the yield of A type dimer was 68 .2 g oligomers per mg PP, which wa s about 4.5 fold of B type dimer (14 .9 g oligomers per mg PP). A fter depolymerization with added epicatechin, the yield of A type dimer ( 139. 2 g oligomers per mg PP) was lower than that of B type dimer ( 21 8.96 g oligomers per mg PP ) . The yield of total oligomers from depolymerization with added epicatechin was 643.9 g oligomers per mg PP, which wa s much higher than without added epicatechin (201.8 g oligomers per mg PP). There were two possible explanations for this observation. The A type linkage remains stable during depolymerization (Gu, K elm, Hammerstone, Beecher, Holden, Haytowitz, et al., 2003) . A previous study reported that 46.1% of the terminal units in the polymeric procyanidins in cranberry were A type dimers (Gu, Kelm, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . In the absence of added epicatechin, t he A type terminal unit s were released as A type procyanidin oligomers. The carbocation from ex tension units undergo structural rearrangement to form anthocyanidins. Therefore more A type oligomers were yielded than B type during depolymerization without added epicatechin. When epicatechin was added as a nucleophile, it reacted with the carbocations to form procyanidin oligomers. Because ex tension units are linked mostly by B type bonds, most extension units were turned into B type oligomers. This explained the higher yield of B type procyanidins than A type during depolymerization
56 with added epicatechin. The depolymerization without added epicatechin appears to be a good way to produce A type oligomers. This approach is desirable if a higher percentage of A type oligomers are needed in the final product for the prevention of urinary tract infection. Depolymerization with added epicatechin had a higher depolymerization efficiency and yield. This method is desirable when the total yield is the priority. Transport of D epolymerized C ranberry P rocyanidins on Caco2 C ell M onolayers After depolymerization, oligomers obtained from depolymerization with or without added epicatechin were dissolved in HBSS and added into the apical side of Caco2 cell monolayers (Figure 3 2A & 3 3A). After 2 h, the HBSS solution from the basolateral side of transwells was analy zed. Its chromatogram is depicted in Figure 3 2B & 3 3B. No peak was observed in the control sample where no procyanidins were added (Figure 3 2C & 3 3C). The peak with retention time of 10.5 min gave rise to m/z [M H]289 and was identified as epicatechin or catechin. Peak 15 .5 min showed m/z 575 [M H]and was fragmented into similar product ions of 423 and 285. This peak was identified as an A type dimer. Other peaks were identified as B type dimer (18.4 mi n) and A type t rimer (23 min). These results indicated that the oligomers (DP<4) from depolymerization can transport through Caco 2 cell monolayers. The natural procyanidin in cranberry consist s exclusive ly of epicatechin as the extension units. Only less than 10% catechin was found in the terminal units (Gu, Kelm, Hammerstone, Beecher, Cunningham, Vannozzi, et al., 2002) . Therefore, we chose epicatechin instead of catechin as the nucleophile in this study, so that newly generated oligomers may have similar structures to natural procyanidins.
57 The recovery rate and transport ratio were calculated in Table 3 1. The recovery rates ranged from 93% to 104%. The transport ratio varied from 0.9% for A type dimer and 1.7% for A type trimer from depolymerization without added epicatechin. These values were comparable with the results in Chapter 2. Low absorption of procyanidins wer e also reported by Zumdick et al . using Caco2 cell model ( Zumdick, Deters, & Hensel, 2012) , Appeldoorn et al . using in situ perfusion ( Appeldoorn, Vincken, Gruppen, & Hollman, 2009 ) and Shoji et al . using rats (Shoji, Masumoto, Moriichi, Akiyama, Kanda, Ohtake, et al., 2006) . Factors that contribute to the low transport ratio included: 1) high molecular weight (Lipinski, Lombardo, Dominy, & Feeney, 2001; Shoji, Masumoto, Moriichi, Akiyama, Kanda, Ohtake, et al., 2006) ; 2) the formation of complexes with proteins ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001) ; and 3) efflux systems may pump out the polyphenols internalized into cells ( Zumdick, Deters, & Hensel, 2012) . No significant difference was observed in the transport ratio of oligomers from different depolymerized sample ( p >0.5). This suggested that structures of the oligomers from the two different depolymerization methods were similar. This was in agreement with mass spectr a data. Optimization of Depolymerization Central composite design in response surface methodology was applied to study three independent variables (time, temperature, and acid concentration) for depolymerization without added epicatechin and the four independent variables (time, temperature, acid concentration and epicatechin/PP ratio) for depolymerization with a dded epicatechin. ANOVA analysis was performed to check the adequacy of the suggested models and identify the significant factors. A secondorder polynomial model was obtained for the two depolymerization methods, respectively.
58 Y1= 140.55+32.34X1+75.27X22 0.82X3+30.59X1X213.56X1X319.71X1 2+42.74X2 2 ( 3 3 ) Y2 =542.79+43.94X1+64.03X2+291.54X467.25X1X3+124.29X3 2376.54X4 2 ( 3 4 ) where Y1 and Y2 are the experimental response--yield of oligomers ( g/mg PP) and X1, X2, X3, and X4 correspond to independent variables of time, temperature, acid concentration and epicatechin/ PP ratio, respectively. The analysis of variance (ANOVA) demonstrated that both models wer e suitable and had good predicta bility, as was evidenced by the very low probability of the F values (p<0.0001). R squared, which measures the modelâ€™s goodness of fit, was 0.93 and 0.87 for depolymerization without and with added epicatechin, respectively . The effects of the independent variables on yield was depicted using three dimensional (3D) response surface curves ( Wei, Liao, Zhang, Liu, & Jiang, 2009 ) . The response models were mapped against two experimental factors while the other independent parameters were fixed at zero (center value of the testing ranges). Figure 3 4A to 3 4C shows the 3D plot s of the response surface for the oligomer yield using depolymerization without added epicatechin. Figure 3 4A is the response surface plot showing the effect of time and temperature on the response at the fixed center values for acid concentration. The yield of oligomers increased as the time and temperature increases. However, the influence of time was not as significant as that of temperature. The response surface plot of acid concentration on the yield of oligomers is given in Figure 3 4B & 3 4C. The yie ld of oligomers decreased when the acid concentration increases. This result was not consistent with a previous study in which higher oligomer yield w as achieved in 1.0 M met hanolic HCl than in 0.1 M methanolic
59 HCl for a 20 min reaction at 40 oC ( Liu, Zou, Gao, & Gu, 2013) . This may be explained by the degradation of procyanidins at higher concentration of HCl and high temperature. Figure 3 5A to 3 5F shows the effects of independent variables on the yield of oligomers using depolymerization with added epicatehin. It was observed from the Figure 3 5A that the yield of oligomers increased as the time progress ed. There was a slight decrease in the yield when the temperature increased from 25 oC to 35 oC but it increased dramatically after the temperature went above 35 oC . With increasing ratio of epicatechin/PP, the yield of oligomers increased at first, but when the ratio reached a higher level around 2, the yield decreased (Figure 3 5C). Similar results between temperature and ratio, and acid concentration and ratio on the oligomer yield were observed (Figure 3 5D & 3 5E). Figure 3 5F indicated that the lower concentration of acid and higher temperature resulted in higher yield of oligomers . The optimum conditions of depolymerization and theoretical maximum values of yi eld were predicted using polynomial models . The forecasted optimum theoretical oligomer yield was 364 g/mg PP when depolymerized at 60 oC , 0.1 M HCl/ m ethanol for 3 h using depolymerization without added epicatechin, whereas the predicted yield was 1089 g/mg PP at the condition of 60 oC , 0.1 M HCl/ m ethanol, epicatechin: polymer (w/w) of 2.19 and reaction time of 3 h. The experiment al value of the oligomer yield was 324 26 and 1281 3 2 g/mg PP for without and with added epicatechin depolymerization under s uggested conditions, which matched well to the forecast ed values from the mathematical model. Summary In summary, cranberry procyanidin polymers were converted into oligomers using depolymerization with or without added epicatechin. Depolymerization without
60 added epicatechin generated higher proportion of A type oligomers. Depolymerization with added epica techin had higher yields of oligomers. Procyanidin oligomers from both processes transported through Caco2 cell monolayers. U sing response surface methodology, the depolymerization conditions were optimized and verified by experimental data.
61 Table 3 1. Recovery rate and transport ratio of oligomers from depolymerized cranberry procyanidin polymer Sample source Oligo mer Sample added to apical side before transport (g) Basolateral side after transport (g) Apical side after transport (g) Recovery rate (%) Transport ratio (%) Depolymerized without added epicatechin 2A 5657.2275.1 50.82.4 5242.0266.1 93.60.2 0.90.0c 2B 1441.763.1 21.92.6 1420.4163 99.97.1 1.50.1ab 3A 2541.7161.7 42.92.2 2605.5108 104.32.3 1.70.0a Depolymerized with added epicatechin 2A 9054.6367.8 105.617.1 8274.3164.2 92.61.8 1.20.1bc 2B 8142.2384.2 127.623.7 7451.51020.5 92.98.4 1.60.2ab 3A 6657.1366.9 102.39.5 6366.276.9 97.36.4 1.50.1ab Data were expressed as the mean standard deviation (n=3). Different letters in the same column indicate significant differences in means at p 0.05.
62 Figure 31. Chromatograms of cranberry procyanidin polymers before and after depolymerization. A) partially purified cranberry procyanidin polymer, B) after it was depolymerized without added epicatechin, and C) samples depolymerized with added epicatechin. 1, 2A, 3A, and 4A are epicatechin, A type procyanidin dimer, trimer, and tetramer, respectively. 2B and 3B denote B type dimer, trimer. LU 10 20 30 40 50 60 70 0 25 50 75 100 125 150 175 200 min 0 10 20 30 40 50 60 0 50 100 150 200 250 300 350 400 450 1 2A 2B 3A 4A 1 2A 2B 3A 4A 3B 3B Polymers
63 Figure 32. HPLC chromatogram of oligomers from depolymerization without added epicatechin A) in apical side at the begi nning of transport experiment; B) HBSS from basolateral side of C aco 2 monolayer after 2 h, and C) control so lution in the basolateral side min 0 10 20 30 40 50 60 5.5 6 6.5 7 7.5 8 8.5 C LU 0 50 100 150 200 A 3A 4A 2A 2B 6 8 10 12 14 16 18 B 2A 3A 2B
64 Figure 3 3 . HPLC chromatogram of oligomers from depolymer ization with added epicatechin. A) in apical side at the begi nning of transport experiment; B) HBSS from basolateral side of C aco 2 monolayer after 2 h, and C) control solution in the basolateral side min 0 10 20 30 40 50 60 5.5 6 6.5 7 7.5 8 8.5 C A 2A 2B 2B 3A LU 50 100 150 200 250 300 350 400 450 1 5 10 15 20 25 30 35 B 2A 2B 2B 3A 1
65 Figure 34. 3D graphic surfaces of the effect of variables (temperature, time, acid concentration) on the yield of oligomers in the depolymerization without added epicatechin A B C
66 Figure 35. 3D graphic surfaces of the effect of variables (temperature, time, acid concentration, and epicatechin/polymer ratio) on the yield of oligmers in depolymerization with added epicatechin D E A C B F
67 CHAPTER 4 M ICROBIAL CATABOLISM OF PROCYANIDINS BY HUMAN GUT MICROBIOTA Background Procyanidins are compounds present in fruits, berries, beans, nuts, cocoa and wine ( Santos Buelga & Scalbert, 2000) . Procyanidins have been reported to possess a variety of physiological activities such as antibacterial, antioxidant, antiviral, anticarcinogenic and anti inflammatory properties. Procyanidins are composed of ( ) epicatechin or (+) catechin. B bonds. Most common foods, such as apple, pear, cocoa, and blueberries, contain exclusively B ty pe procyanidins (Gu, Kelm, Hammers tone, Beecher, Holden, Haytowitz, et al., 2004) . A type procyanidins contain an additional ether linkage between the C2 of the upper unit and the oxygenbearing C7 of the lower unit. Cranberries are one of the few foods that contain significant amount s of A type procyanidins. A type procyanidin oligomers from cranberr ies were shown to inhibit adhesion of u ropathogenic P fimbriated E. coli bacteria wh ereas B type ha d no such activity ( Foo, Lu, Howell, & Vorsa, 2000; Howell, Reed, Krueger, Winterbottom, Cunningham, & Leahy, 2005) . In vitro experiments with Caco2 cells demonstrated poor permeability of procyanidins ( Deprez, Mila, Huneau, Tome, & Scalbert, 2001 ; Zumdick, Deters, & Hensel, 2012) . This study showed a low transport rate (<5%) of procyanidin monomer through tetramers for both A and B type s. Shoji et al . administrated apple procyanidins to rats using a dose of 1 g/kg . They found that the concentration of ( ) epicatechin, procyanidin dimer B2, and trimer C1 in the plasma reached Cmax of 1.3 M, 0.4 M, and 0.14 M , respectively (Shoji, Masumoto, Moriichi, Akiyama, Kanda, Ohtake, et al.,
68 2006) . A recent study utilizing pigs reported that 0.004% of ingested procyanidin trimer C1 and 0.019% of ingested procyanidin dimer B4 were excreted in 30 h urin e ( Rzeppa, Bittner, Dll, Dnicke, & Humpf, 2012) . T hese results indicated that 9095% of dietary procyanidins are not absorbed in the small intestine , but rather they reach the colon intact and are degraded into simple phenolic compounds and other metabolites by colon microbiota. These microbial metabolites are thought to be bioavailable (Rios, Gonthier, Rmsy, Mila, Lapierre, Lazarus, et al., 2003) and contribute to the health promoting properties of procyanidins in vivo ( Karlsson, Huss, Jenner, Halliwell, Bohlin, & Rafter, 2005; Unno, Tamemoto, Yayabe, & Kakuda, 2003) . S everal studies investigated the microbial catabolism of procyanidin monomers and B type dimer s. T he results were inconsistent due to the different composition of microbiota in different studies. The unique activity of A type procyanidins in preventing urinary tract infections makes the investigation of their microbial catabolism of great importance. However; there is a lack of information on the microbial catabolism of A type procyanidins. In this study, ( ) epicatechin, (+) catechin, procyanidin B2, A2, and partially purified procyanidins from apples and cranberr ies were incubated with human gut microbiota in a static anaerobic incubation system . Their catabolites were identifi ed and quantified to elucidate microbial catabolism of this type of compound. M aterials and Methods Chemicals and Materials A ll chemicals and reagents were obtained from Fisher Scientific Inc. ( Pittsburg h , PA, USA ) u nless otherwise stated. Freezedried cranberries were provided by Ocean Spray Cranberries, Inc . (Lakeville Middleboro, MA , USA ). () Epicatechin, (+) catechin and phenolic acids standards were purchased from SigmaAldrich (St. Louis,
69 M O , USA). A type procyanidin dimer A2 (epicatechin(2 O 7, 4 8) epicatechin) was purchased from Chromadex Inc. (Irvine, CA , USA ). B type procyanidin dimer B2 [epicatechin(4 8) epicatechin] was obtained from Indofine Chemical Company, Inc. (Hillsborough, NJ , USA ). 5 (2 â€™ hydroxyphenyl) valerolactone, 5 ( 3 â€™ hydrox yphenyl ) valerolactone, 5 ( 3 â€™ ,4 â€™ dihydroxyphenyl) valerolactone, and 5 ( 3 â€™ ,4 â€™ ,5 â€™ trihydroxyphenyl ) valerolactone were kindly provided by Dr. N . Nakajima (Toyama Prefectural University, Japan). Amberlite FPX 66 resin was a product from Dow Company (Midland, M I, USA ). Extraction and Purification of P rocyanidins from A pple s and C ranberr ies A total of 500 g Granny Smith apples were blended in 500 mL distilled water containing 2 g of sodium bisulf a te. The homogenates were put into 1500 mL boiling distilled water and boiled for 5 min. After cool ing down to room temperature, it was cen trifuged at 3346 x g for 10 min ( Xiao, Liu, Wu, Xie, Yang, & Sun, 2008) . For cranberries, 500 g of freezedried cranberry powder was extracted with 4 L of extraction solvent (acetone/water, 70:30, v/v) at room temperature (~25 C) for 48 h. Extracts obtained after vacuum filtration were combined and concentrated under a partial vacuum using a rotary evaporator. The concentrated extract was resuspended in 100 mL of water. Then the apple supernatant or cranberry extract solution wer e loaded separately onto a column (3.844 cm) packed with Amberlite FPX 66 resin. The column was eluted with 5 L deionized water to remove sugars followed by 2 L of methanol to yield apple or cranberry phytochemical extract . The se extracts w ere suspended in 80 mL of water and loaded onto a column (5.828 cm) packed with Sephadex LH 20, which had been soaked in 30% methanol for over 4 h before use. T he column was washed with 2.5 L of 30% methanol/water to remove anthocyanins and other flavonoids .
70 P rocyanidi ns were recovered from the column by eluting with 4 L of 70% (v/v) aqueous acetone. The eluents were evaporated to dryness under vacuum in a SpeedVac concentrator (Thermo Scientific Inc., Waltham, MA, USA) for further analys e s and experiments. HPLCMSn A n alysis of P rocyanidins Chromatographic analyses were performed on an Agilent 1200 series HPLC system ( Palo Alto, CA , USA ) equipped with a binary pump, an autosampler, a fluorescence detector, and a high capacity (HCT) ion trap mass spectrometer (Bruker Daltonics, Billerica, MA , USA ) . Separation and mass spectr a analysis was carried out following the method of Liu et al. ( Liu, Zou, Gao, & Gu, 2013) . Content of procyanidins was estimated using relative response factors calculated according to Robbins et al . (Robbins, Leonczak, Li, Johnson, Collins, Kwik Uribe, et al., 2012) . In vitro F ermentation of P rocyanidins with H uman F ecal M icrobiota A pool of human feces were obtained from four female volunteers who were 2050 years old, self claimed healthy, and had not taken antibiotics for at least 3 months. This research was approved by Institutional Review Boards at University of Florida (IRB #27 5 2012). The study was fully explained to the subjects and they gave their written informed consent . A carbonatedphosphate buffer (pH 5.5) , which was prepared according to Karppinen et al. ( Karppinen, Liukkonen, Aura, Forssell, & Poutanen, 2000) , was used for incubation. It was autoclaved at 121 C for 30 min and deoxygenated for 2 d under anaerobic conditions. Ten gram of freshly collected feces was added to 100 mL medium making a 10 % ( w/v ) fecal suspension . It was mixed well and filtered through sterilized two layer cheesecloth to remove large particles. The fecal suspension was left to equilibrate overnight under anaerobic conditions. Then, epicatechin, catechin ,
71 procyanidin B2, procyanidin A2 , partially purified apple or cranberry procyanidins were added into the suspension to achieve a concentration equivalent to 0.5 mM epicatechin, which was low enough to avoid microbial inhibition. For each substrate at each time point, there were five incubation flasks . The first flask was control 1 where procyanidins were added but no living flora was added. This was to subtract changes of procyanidins due to chemical degradation or transformation. The second flask was control 2 where living flora was added but no procyanidins were added. This was to subtract metabolites that were not from procyanidins. Flask 3, 4, and 5 contained living flora and a substrate in three replicat es. A control incubating procyanidins with heat inactivated flora was omitted since no matrix effect was observed in preliminary experiments. A series of fermentation s were conducted at 37 C under anaerobic condition for 0, 3, 6, 9, 12 and 24 h , respectiv ely. Sample Extraction and Derivatization for GC MS Analysis M icrobial metabolites were extracted from the fermentations by a method modified from a previous study (Deprez, Brezillon, Rabot, Philippe, Mila, Lapierre, et al., 2000) . order to keep the analytes in deionized form. Fifty microliter s was added as internal standard. The samples were extracted with 2 mL hexane twice to remove lipids. Then, 5 mL ethyl acetate was added to extract the metabolites. Samples were vortexed vigorously for 30 sec and centrifuged at 5229 x g for 10 min at room temperature. This extraction was repeated and the ethyl acetate layer was collected, combined and evaporated to dryness in a SpeedVac concentrator at 25 C . Derivatization was carried ou bistrifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) to the dried extracts and incubated at 50 C for 4
72 octane was added. The samples were then vortex mixed and analyzed on GC MS. GC MS A nalysis Derivatized samples were analyzed by gas chromatography coupled with a mass spectrometry detect ion (7 890 A gas chromatograph, 5975C mass specific detector, Agilent Technologies, Santa Clara, CA). The chromatographic separation took place on an HP5following temperature program: 50 oC was held for 1 min and then the temperature was raised 5 oC /min until 120 oC , which was held for 10 min and then increased by 20 C /min until 320 C . The final temperature was held for 4 min. One microliter was manually injected using the splitless mode at 320 oC . The mass spectrometer was used in the electron i mpact mode (EI) at 70 eV with a source temperature of 230 oC and a quadrupole temperature of 150 oC . Mass spectra were obtained in the full scan mode from m/z 50 to 650. Data were processed using MSD Chemstation software (Agilent, V ersion E.02.02.1431) . S tatistical Analysis All data were expressed as the mean standard deviation. Oneway analyses of HSD pairwise comparison of the means were performed using JMP software (Version 10 .0, SAS Institute Inc., Cary, NC). A difference of p 0.05 was considered significant. R esults and Discussion Composition of Partially Purified Apple and Cranberry Procyanidins The procyanidins purified from Granny Smith apples were exclusively B type (Figure 4 1A). Peaks 1, 2B, 3B, and 4B were identified as monomers, dimer, trimer, and
73 tetramers according to [M H]m/z 289, 577, 865, and 1153 mass spectra. B type pentamer and hexamer (peaks 5B and 6B) were identified by comparing with reported retention time (Gu, Kelm, Hammerstone, Beecher, Holden, Haytowitz, et al., 2003) . The total content of the procyanidins in apple extracts was 94% (Table 4 1). Among them, around 50% were dimers through tetramers. Monomers acc ounted for 2.36% (w/w) of the total procyanidins. This was because the majority of monomers were removed in the procyanidin purification steps. About 8% of purified apple procyanidins were high polymers. Procyanidins purified from cranberries were predo minantly A type (Figure 4 1B). Peaks 2A, 3A, and 4A were identified as A type dimer, trimer, and tetramers according to [M H]m/z 575, 863, and 1151 on mass spectra. The A type dimer appeared as a dominant peak . The total content of the procyanidins in cranberry extract was about 40%, which was much lower than that in apple extracts. This may be due to an underestimation of high polymers because they were a mixture of procyanidins with DP>10 . A pproximately half of the cranberry procyanidins were A type dimers to tetramers (Table 4 1) and p olymers accounted for the other half. Identification of M icrobial M etabolites The gas chromatograms of ferments with different substrates after 6 h are shown in Figure 4 2 . T he six substrates were degraded by human fecal microbiota to different extent s. The retention time and main ions with relative intensity of metabolites were listed in Table 4 2. Their identification was achieved by comparing their retention time and mass s pectra with reference standards. Compounds 1 6 appeared as the common microbial metabolites for epicatechin, catechin, procyanidin B2, A2 as well as apple and cranberry procyanidins. They were identified as benzoic acid, 2 phenylacetic acid, 3 -
74 phenylpropionic acid, 2 ( 3 â€™ hydroxyphenyl ) acetic acid, 2 ( 4 â€™ hydroxyphenyl ) acetic acid , 3 ( 3 â€™ hydroxyphenyl ) propionic acid and hydroxyphenylvaleric acid. These metabolites have also been reported after the fermentation of red wine extracts ( Grun, van Dorsten, Jacobs, Le Belleguic, van Velzen, Bingham, et al., 2008) or grape seed polyphenols ( Sanchez Patan, Cueva, Monagas, Walton, Gibson, Martnlvarez, et al., 2012) in a colonic gut model. After female rats were fed containing sorghum bran for 50 days, the serum concentrations of 3,4dihydroxybenzoic acid, 3methox y 4 hydroxybenzoic acid, and 2 ( 4 â€™ hydroxyphenyl ) acetic acid increased. 2 ( 3 â€™ Methoxy 4 â€™ hydroxyphenyl ) acetic acid, 2 ( 3 â€™ hydroxyphenyl ) acetic acid, and 3 ( 3 â€™ hydroxyphenyl ) propionic acid were the dominate compounds in the urine( Gu, House, Rooney, & Prior, 2007 ) . A fter consum ing 439 mg procy anidins and 147 mg catechin monomers, an increase in urinary excretion of 3 ( 3 â€™ hydroxyphenyl ) propionic acid, 2 ( 3 â€™ hydroxyphenyl ) acetic acid, vanillic acid, and 3 hydroxybenzoic acid was observed in human volunteers (Rios, Gonthier, Rmsy, Mila, Lapierre, Lazarus, et al., 2003) . Peak 7 was only found in procyanidin B2, A2, apple and cranberry procyanidin samples and was not detected in epicatechin and catechin sample throughout the 24 h fermentation. This result was consistent with previous studies in which 2 ( 3 â€™ ,4 â€™ dihydroxy hphenyl ) acetic acid was observed as a metabolite of procyanidin B2 ( Appeldoorn, Vincken, Aura, Hollman, & Gruppen, 2009) while it was absent when pure monomers were used in the incu bation ( Aura, Mattila, SeppnenLaakso, Miettinen, Oksman ) . Compound s 9 11 were only found in epicatechin, catechin, procyanidi n B2 and apple ferments. Compound 9 was tentatively identified as hydroxyphenylvaleric acid according to National Institute of Standards and Technology
75 (NIST) library (Version 2.0 g, 2011) . However, the position of the hydroxyl group was not able to be con firmed due to a lack of commercial standard. Compounds 10 and 11 were identified as 5 ( 3 â€™ hydroxyphenyl ) valerolactone and 5 ( 3 â€™ ,4 â€™ dihydroxyphenyl ) valerolactone, respectively . They may be formed through the cleavage of A ring and the C ring opening. 5 ( 3 â€™ ,4 â€™ Dihydroxyphenyl ) valerolactone was regarded as a key metabolite in delineating the rate and extent of the microbial catabolism of flavan3 ols (Sanchez Patan, Cueva, Monagas, Walton, Gibson, Martnlvarez, et al., 2012) . 5 ( 3 â€™ Hydroxyphenyl ) valerolactone was probably a dehydroxylated product from 5 ( 3 â€™ ,4 â€™ dihydroxyphenyl ) valerolactone. These phenylvalerolactones were further degraded oxidation generated other small phenolic acids and their derivatives, such as phenylpropionic acid, phenylac e tic acid and benzoic acid. Compounds 1 2 and 13 appeared in epicatechin and catechin ferments at 3 and 6 h. Since these two compounds disappeared after 6 h, they might be intermediate metabolites of epicatechin and catechin. Based on their mass spectra and similar retention time to (epi)catechin, we tentatively identified peak 1 2 as 1 (hydroxyphenyl) 3 (2â€™â€™,4â€™â€™,6â€™â€™trihydroxyphenyl)propan2 ol and peak 13 as 1 (3â€™,4â€™ Dihydroxyphenyl) 3 (2â€™â€™,4â€™â€™,6â€™â€™trihydroxyphenyl)propan2 ol. NMR analysis of purified compounds is needed t o confirm their structures . Quantitation of M icrobial M etabolites Degradation of six substrates is shown in Figure 43 . Epicatechin and catechin were fully degraded in the first 6 h of incubation. Procyanidin B2 was broken down after 12 h. Procyanidin A2 was detected even after 24 h fermentation. Over 50% of
76 procyanidins remained in the samples of partially purified apple and cranberry procyanidins. The microbial metabolites of epicatechin, catechin, procyanidi n B2, procyanidin A2 , partially purified apple and cranberry procyanidins were quantitated using gas chromatography with syringic acid as an internal standard. Figure 44 shows formation of benzoic acid and phenylacetic acids. Figure 45 depicts the format ion of phenylpropionic acids, phenylvaleric acid, and hydroxyphenyl valerolactones . The major metabolites of epicatechin, catechin, and procyanidin B2 included benzoic acid, 2phenylacetic acid, 3phenylpropionic acid, 5(3â€™ hydroxyphenyl) valerolactone and 5(3â€™, 4â€™ dihydroxyphenyl) valerolactone. They accounted for over 70% of the total metabolites during fermentation. Microbial catabolism of procyanidin A2, partially purified apple procyanidins and partially purified cranberry procyanidins generat ed benzoic acid, 2phenylacetic acid, 3phenylpropionic acid as major metabolites, constituting over 50% of the total metabolites. 2 ( 3 â€™ Hydroxyphenyl ) acetic acid, 2 ( 4 â€™ hydroxyphenyl ) acetic acid and 3 ( 3 â€™ hydroxyphenyl ) propionic acid appeared to be minor metabolites . In epicatechin and catechin incubates, those three compounds account ed for around 10% of the total metabolites at each time point. This result was consistent with a previous report by Stoupi et al. except that we observed lower amount of 3 ( 3 â€™ hydroxyphenyl ) propionic acid and they did not detect benzoic acid( Stoupi, Willamson, Drynan, Barron, & Clifford, 2010) . One reason for the discrepancy could be the conversion of 3 ( 3 â€™ hydroxyphenyl ) propionic acid to benzoic acid. Another study administered 14C labelled 3 ( 3 â€™ hydroxyphenyl ) propionic acid to guinea pig and gave rise to labelled 3hydroxybenzoic acid ( Das & Griffiths, 1969 ) . Another possible reason
77 could be that part of 3 ( 3 â€™ hydroxyphenyl ) propionic acid was dehydroxylated into 3 phenylpropionic acid which were present at high content in the ferments. Although 2 ( 3 â€™ ,4 â€™ dihydroxyphenyl ) acetic aci d was detected as a unique metabolite of procyanidin B2 and A2, partially purified apple procyanidins, and partially purified cranberry procyanidins (Figure 4 4 E), its amount was small in those samples. This was different from the finding s of Appeldoorn et al., where 2 ( 3 â€™ ,4 â€™ dihydroxyphenyl ) acetic acid was observed as a primary metabolite with the highest amount. Inter individual differences, which have been observed in other studies (Sanchez Patan, Cueva, Monagas, Walton, Gibson, Martnlvarez, et al., 2012; Urpi Sa rda, Monagas, Khan, LamuelaRaventos, Santos Buelga, Sacanella, et al., 2009) , could be a major reason for the differences observed. Figure 4 5 show s that metabolites with 3 or 5 carbons on the side chain reached the peak concentration at about 12 h. Their concentration decrease d gradually afterwards. No phenylvalerolactone was detected throughout the fermentation time in the incubates of procyanidins A2 and partially purified cranberry procyanidins . H ydroxyphenylvaleric acid appeared at 9 h, which was 3 h later than B2, epicatechin and catechin. Compared to B2 or monomers, A t ype procyanidins are more stable due to an additional interflavan bond. Such rigid structure makes them more resistant to microbiota. They are less commonly found in the human diet. Therefore, gut flora may evolve limited capacity to degrade the m . Epicatechin and catechin not only had similar microbial metabolites but also exhibited similar patterns on the change in these metabolites . This could be explained by t heir similar chemical structure . Compared to the other 5 substrates, procyanidin B2
78 show ed relatively high concentration of most metabolites detected, such as benzoic acid, 2phenylacetic acid, 2(3â€™ hydroxyphenyl)acetic acid, 2(4â€™ hydroxyphenyl)acetic acid, 2(3â€™, 4â€™ dihydroxyphenyl)acetic acid, phenylpropionic acid, 3(3â€™ hydroxyphenyl)propionic acid and hydroxyphenylvaleric acid. Partially purified apple procyanidins and partially purified cranberry procyanidins generally produced the lowest amount of metabolites among the six substrates. The exceptional cases were cranberry procyanidins d emonstrated the highest amount of 2(3â€™ hydroxyphenyl)acetic acid and apple procyanidins demonstrated the highest amount of 3(3â€™ hydroxyphenyl)propionic acid during incubation. The mass recovery of metabolites was determined by dividing the total amount o f identified metabolites by the total procyanidins added to the fermented broth. After 24 h of fermentation, microbial metabolites accounted for 56.9%, 54. 2 % , 38. 3 %, 27. 7 %, 21.3%, and 20.0% of added epicatechin, catechin, procyanidin B2, procyanidin A2, partially purified apple procyanidins and partially purified cranberry procyanidins, respectively (Table 4 3). Procyanidin A2 had a much lower mass recovery of metabolites than pure procyanidin B2, epicatechin and catechin. The lower mass recovery for apple and cranberry procyanidins may be explained by the occurrence of procyanidins polymers. A recent study evaluated the ability of probiotic bacteria to convert procyanidins in vitro . The author found that 3 ( 4 â€™ hydroxyphenyl ) propionic acid derived from epica techin and A2 was much higher than that from Litchi pericarp oligomeric procyanidins, indicating that it was easier for Lactobacillus to transform the procyanidins with a lower degree of polymerization ( Chen, Yang, Wu, Lv, Xie, & Sun, 2013) . Another study using a pig cecum model showed similar phenomena i n which
79 only 40% of A type trim ers were degraded within 8 h of incubation compared to 80% of A type dimers ( Engemann, Hubner, Rzeppa, & Humpf, 2012) . These results and th ose of other stud ies supported the point that the ability of bacteria to catabolize procyanidins in the gut decreases with an increase of molecular size. Summary In summary , procyanidins, both B type and A type can be degraded by human gut microflora. C ommon metabolites include benzoic acid, 2 phenylacetic acid, 3 phenylpropionic acid, 2 ( 3 â€™ hydroxypenyl ) acetic acid, 2 ( 4 â€™ hydroxyphenyl ) acetic acid, 3 ( 3 â€™ hydroxyphenyl ) propionic acid and hydroxyphenylvaleric acid. Compared to B type, A type is more resistant to microbial catabolism. The accessibility of procyanidins to microbiota decreases when procyanidins with high degree of polymerization are present .
80 Table 4 1. P rocyanidin content in the partially purified apple and cranberry procyanidins Composition Content of procyanidin (w/w, %) Partially purified apple procyanidins Partially purified cranberry procyanidins Monomers 2.36 0.55 Dimers 18.2 8.52 (7.13a) Trimers 16.1 5.54 (3.89a) Tetramers 19.8 7.30b Pentamers 18.4 0.84b Hexamers 11.0 1.21b High polymers (DP>10) 8.0 16.1 Total 93.8 40.1 a Numbers in the parentheses represent the content of A type procyanidins. Numbers out of parenthesis are total procyanidins. b Tetramers through hexamers in partially purified cranberry procyanidins were predominantly A type according to mass spectr a data .
81 Table 4 2. Retention time, molecular weight, and major ions of identified microbial metabolites and an internal standard present in fermentation broth. Peak numbers match those in Figure 4 2. Peak number Retention time (min) Compounds Molecular weight Mass after derivatization Major ions (relative intensity) 1 8.397 Benzoic acid 122 194 194 (7), 179 (100), 135 (62), 105 (90), 77 (58), 51(13) 2 9.576 2 Phenylacetic acid 136 208 193 (13), 164 (20), 91 (21), 75 (35), 73 (100) 3 11.905 3 Phenylpropionic acid 150 222 222 (12), 207 (28), 104 (100), 75 (63) 4 14.892 2 (3â€™ Hydroxyphenyl)acetic acid 152 296 296 (25), 281 (25), 164 (44), 147 (30), 73 (100) 5 15.287 2 (4â€™ Hydroxyphenyl)acetic acid 152 296 296 (22), 281 (22), 252 (19), 179 (50), 164 (26), 73 (100) 8 16.613 3 (3â€™ Hydroxyphenyl)propionic acid 166 310 310 (35), 205 (100), 192 (70), 177 (327), 73 (40) 12 18.868 2 (3â€™,4â€™ Dihydroxyphenyl)acetic acid 182 384 384 (70), 267 (42), 237 (13), 179 (69), 73 (100) 13 20.464 Syringic acid (internal standard) 198 342 342 (42), 327 (59), 312 (38), 297 (40), 253 (29), 223 (19), 141 (25), 73 (100) 18 21.277 Hydroxyphenylvaleric acid 194 338 338 (19), 323(16), 248 (18), 233 (16), 206 (15), 180 (38), 147 (27), 75 (65), 73 (100) 14 21.775 5 (3â€™ Hydroxyphenyl) valerolactone 192 264 264 (34), 207 (21), 179 (20), 149 (20), 85 (100) 16 29.609 5 (3â€™,4â€™ Dihydroxyphenyl) valerolactone 208 352 352 (45), 267 (100), 205 (55), 179 (53), 73 (47) 40.731 ( ) Epicatechin 290 650 650 (7), 368 (80), 355 (27), 267 (88), 179 (15), 73 (100) 40.885 (+) Catechin 290 650 650 (10), 368 (82), 355 (25), 267 (70), 179 (13), 73 (100)
82 Table 4 3. Mass recoveries from micro b ial catabolites of epicatechin, catechin, procyanidin B2, A2, partially purified apple and cranberry procyanidins over 24 h* . Mass recoveries from identified catabolites (%) 0 h 3 h 6 h 9 h 12 h 24 h Epicatechin 7.90.7 a 14.22.8 ab 24.44.7 bc 28.11.2 b 52.82.1 a 56.98.0 a Catechin 0.00.0 c 10.20.1 bc 29.71.0 ab 47.80.9 a 43.61.4 b 54.26.0 a Procyanidin B2 0.00.0 c 10.61.0 bc 34.82.4 a 46.65.8 a 58.52.3 a 38.32.6 b Procyanidins A2 0.00.0 c 16. 01.0 a 17.62.7 cd 20.12.7 c 24.30.7 c 27.72.1 bc Partially purified apple procyanidins 4.90.5 b 8.60.7 c 16.41.4 d 20.11.4 c 22.64.7 c 21.31.3 c Partially purified cranberry procyanidins 0.00.0 c 9.51.3 c 11.82.6 d 15.61.2 c 19.03.7 c 20.05.8 c *Different letters in the same column indicate the significant differences at p
83 Figure 4 1 . HPLC chromatograms of partially purified procyanidins using florescent detection. A) apple and B) cranberry . 1, 2B, 3B, 4B, 5B, and 6B denote monomer, B type dimer, trimer, tetramer, pentamer, and hexamer, respectively. 2A, 3A, and 4A are A type dimer, trimer, and tetramer, respectively. Oligomers were identified using HPLC tandem mass spectrometry (MSn). Time (min) 0 10 20 30 40 50 60 0 50 100 150 200 250 300 (A) LU 2B 2B 3B 4B 5B 6B 1 Time (min) 0 10 20 30 40 50 60 LU 0 20 40 60 80 (B) 2A High polymers 2B 3A 3B 4A 1
84 Figure 42 . Gas chromatograms of microbial metabolites in ferments with substrate epicatechin A) catechin B), procyanidin B2 C) procyanidin A2 D) partial ly purified apple procyanidins E) and partially purified cranberry procyanidins F) after 6 h anaerobic incubation. Peak numbers ( 1 1 3 ) match those in Table 4 2.
85 Figure 43 . Degradation curve of epicatechin, catechin, procyanidin B2, procyanidin A2, partially purified apple and cranberry procyanidins. The amount detected at t= 0 h was expressed as 100%. Error bars represent the standard deviation of triplicates. Different letters at the same time indicate significant differences at p 0 20 40 60 80 100 120 0 3 6 9 12 24 Procyanidin percentage over initial value (%) Time (h) Epicatechin Catechin Procyanidin B2 Procyanidin A2 Partially purified apple procyanidins Partially purified cranberry procyanidins a a a a a a b ab b b b b d c e e c d b c b c c b
86 Figure 44 . Formation of microbial metabolites with 12 carbon(s) on the side chain. A) benzoic acid, B) p henylacetic acid, C) 2 (3â€™ hydroxyphenyl)acetic acid, D) 2(4â€™ hydroxyphenyl)acetic acid, E) 2 (3â€™, 4â€™ dihydroxyphenyl)acetic acid over 24 h in the microbial fermentation of epicatechin, catechin, procyanidin B2, procyanidin A2, partially purified apple pr ocyanidins and partially purified cranberry, corrected for the amount detected in the control samples. Error bars represent the standard deviation of triplicates. 0 20 40 60 80 100 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (A) Benzoic acid 0 10 20 30 40 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (B) 2 Phenylacetic acid 0 1 2 3 4 5 6 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (D) 2 (4 ' Hydroxyphenyl)acetic acid 0 1 2 3 4 5 6 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (E) 2 (3',4' Dihydroxyphenyl)acetic acid 0 5 10 15 20 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (C) 2 (3' Hydroxyphenyl)acetic acid
87 Figure 45 . Formation of microbial metabolites with 35 carbons on the side chai n. A) phenylpropionic acid, B) 3 (3â€™ hydroxyphenyl)propionic acid, C) hydroxyphenylvaleric acid, D) 5 (3â€™ h ydroxyphenyl) valerolactone, E) 5 (3â€™, 4â€™ hydroxyphenyl) valerolactone over 24 h in the microbial fermentation of epicatechin, catechin, procyani din B2, procyanidin A2, partially purified apple procyanidins and partially purified cranberry, corrected for the amount detected in the control samples. Error bars represent the standard deviation of triplicates. 0 20 40 60 80 100 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (A) 3 Phenylpropionic acid 0 10 20 30 40 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (B) 3 (3' Hydroxyphenyl)propionic acid 0 20 40 60 80 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (D) 5 (3 ' Hydroxyphenyl ) valerolactone 0 10 20 30 40 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (E) 5 (3',4' Dihydroxyphenyl) valerolactone 0 5 10 15 20 25 30 0 3 6 9 12 24 Amount of metabolites ( g/3 mL incubates) Time (h) (C) Hydroxyphenylvaleric acid
88 CHAPTER 5 CONCLUSIONS This dissertation increased our knowledge on the absorption, chemical conversion and microbial catabolism of cranberry procyanidins. A type procyanidins dimers, trimer s and tetramers from cranberr ies transport ed across Caco 2 cells although the transport r atios were low (Chapter 2) . It implie d that A type procyanidins dimmers through tetramers are bioavailable in humans after cranberry consumption. Future studies are needed to elucidate the absorption mechanism and identify possible transporter of procyanidins . The paracellular transport is the most probable absorption pathway; however this needs to be verified by experiments . In Chapter 3, t he nonabsorbable cranberry polymers were converted into absorbable oligomers by depolymerization under mild acid con dition with or without added epicatechin. O ligomers yielded from both methods transported through Caco2 cell monolayer with a comparable transport ratio to the natural oligomers . Reaction conditions affected both the yield and composition of oligomers . The depolymerization without added epicatechin method appear ed to be a good way to produce A type oligomers. This approach is desirable if a higher p ortion of A type oligomers is needed in the final product for the prevention of urinary tract infection. Depolymerization with added epicatechin had a higher depolymerization efficiency and yield. This method is desirable when the total yield is the priority. This study provided an effective and applicable m ethod for the industry to produce cranberry procyanidi n supplements with increased bioavailability and bioactivity. Procyanidin compositions were controllable by adjusting reaction conditions. Future studies were needed to determine the bioactivity
89 of newly generated cranberry oligomers, such as the preventi on effect of urinary tract infections, antioxidant capacity and anticancer properties . In Chapter 4, procyanidins, both B and A type s were catabolized by human gut microflora. Compared to B type, A type procyanidins were more resistant to microbial catabolism , which may result from their rigid structure. A total of eleven metabolites were identified from the ferments of ( ) epicatechin, (+) catechin, procyanidin B2, procyanidin A2, partially purified apple and cranberry procyanidins. Common metabolites inc lude d benzoic acid, phenylacetic acids, phenylpropionic acids and phenylvaleric acids with different number of hydroxyl groups on the benzene ring. Two phenylvalerolactones were identified. Limited numbers of metabolites were identified in this study due to the detection limits of GC MS methods and the complexity of human gut microbiota. Future studies are needed to identif y the strain s of bacteria which are responsible for the degradation of procyanidins. The present work used a pool of human fecal sa mple s which only showed the general trend on the microbial degradation of procyanidins. Microbial metabolites from individuals may be compared in future studies to determine the inter individual differences on the microbial catabolism patterns. Additional investigation s on the bioactivity and bioavailability of these microbial metabolites were also warranted.
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99 BIOGRAPHICAL SKETCH Keqin Ou was born in Sichuan, China. She obtained her B. S. degree in f ood q uality and safety from China Agricultural University in 2007, and was admitted into a m asterâ€™s program in f ood science at the same university. She published 2 papers for her research project during her m aster â€™s studies . She joined the f ood science doctoral program at the Univer sity of Florida in the fall of 20 10 under the supervision of Dr. Liwei Gu . Upon her graduation in August 201 4, she had three publications for her Ph. D. She plans to go back to her home country and work as a research professional in food industry.
Depolymerizationofcranberryprocyanidinsusing(+)-catechin, ( )-epicatechin,and( )-epigallocatechingallateaschainbreakers HanweiLiua,TaoZoua,Jin-mingGaob,LiweiGua ,aFoodScienceandHumanNutritionDepartment,InstituteofFoodandAgriculturalSciences,UniversityofFlorida,Gainesville,FL32611,UnitedSta tesbNaturalProductsResourceResearchCenter,CollegeofScience,NorthwestA&FUniversity,Yangling,Shaanxi712100,ChinaarticleinfoArticlehistory: Received6September2012 Receivedinrevisedform15January2013 Accepted4March2013 Availableonline14March2013 Keywords: Cranberries Procyanidins DepolymerizationabstractProcyanidindimers,trimers,andtetramersareabsorbable,whereaslargeroligomersarenot.Procyanidinsincranberriesarebioactivecomponentsthathelppreventchronicdiseases;however,85%ofcranberryprocyanidinsarelargeoligomersorpolymerswithadegreeofpolymerizationabovefour.The objectiveofthisstudywastodepolymerizecranberryprocyanidins,particularlythepolymers,into absorbableoligomers.PartiallypuriÂ“edcranberryprocyanidins(PCP)wereobtainedusingchromatographicmethods.Theresultantextractcontainedpredominantpolymerswithadegreeofpolymerization aboveten(77.2%w/w).Theextractwasdepolymerized,using0.1or1MmethanolicHCl,with(+)-catechin,( )-epicatechin,or( )-epigallocatechingallate(EGCG)addedaschainbreakers.Depolymerization convertedpolymersintoA-typeandB-typedimers,trimersandtetramers.UseofEGCGasachainbreaker resultedinA-andB-typeoligomerswithEGCGasaterminalunit,indicatingthattheaddedÂ”avan-3-ol attachedtotheC4carbocationsfromprocyanidinsduringdepolymerization.TheyieldofB-typeoligomerswashigherthanthatofA-typeoligomers.TheyieldincreasedwhenhigheramountsofÂ”avan-3olswereusedfordepolymerization.EGCG,asachainbreaker,producedfewerprocyanidinoligomers thandidcatechinorepicatechin.Thisresearchprovidedapracticalapproachthatmayenhancethebioavailabilityandbioactivityofprocyanidinsincranberries. 2013ElsevierLtd.Allrightsreserved.1.Introduction Cranberries( Vacciniummacrocarpon )arenativetothenortheasternregionofNorthAmerica.TheirfruitscontainsigniÂ“cant amountsofprocyanidins( Guetal.,2002 ).Cranberrieshavetraditionallybeenusedforthetreatmentandpreventionofurinary tractinfections.Theireffectivenesswasdemonstratedbyarandomised,double-blindplacebo-controlledtrial( Avornetal., 1994 ).TheA-typeprocyanidintrimersfromcranberrieswere showntoinhibittheadherenceofuropathogenic E.coli ,suggesting thattheywerethebioactivecomponents( Foo,Lu,Howell,&Vorsa, 2000a,2000b ).Cranberryprocyanidinsalsohavepotentantioxidantcapacitiesandmayreducetheriskofcardiovasculardiseases andseveraltypesofcancers( Catonetal.,2010;Kresty,Howell,& Baird,2011;Maatta-Riihinen,Kahkonen,Torronen,&Heinonen, 2005 ). Procyanidindimers,trimers,andtetramerswereabsorbedand presentinbloodcirculation,whereasproanthocyanidinsofhigher molecularweightwerenot( Holtetal.,2002;Shojietal.,2006; Tsangetal.,2005 ).Additionally,Tomas-BarberÃ¡netal.(2007) provedthattheincreaseofmonomersofprocyanidinsshould enhancetheirbioavailability( Tomas-BarberÃ¡netal.,2007 ).Procyanidinsincranberriesconsistof15%ofmonomertotetramersand 85%ofpolymerswithadegreeofpolymerizationof5orlarger( Gu etal.,2002 ).Wefoundthatextrusiondepolymerizedprocyanidins andenhancedtheirabsorptioninweanlingpigs( Gu,House,Rooney,&Prior,2008 ).However,extrusionisaharshprocessingmethodthatcausesdrasticprocyanidindegradation.Depolymerization ofprocyanidinsbytoluene-a-thiol,phloroglucinol,orL-cysteine havebeenexplored( Fooetal.,2000a,2000b;Guetal.,2002;Torres&Selga,2003 ),butnoneofthesemethodsareapplicableinthe foodindustrybecausetheÂ“nalproductsarenolongerprocyanidins.Whiteetal.haverecentlydepolymerizedpolymericprocyanidinsfromcranberrypomacebyalkalinehydrolysis.However,only asmallpercentage(lessthan6%)ofthepolymerwasactuallyconverted,whiletheremainderwaslikelydegraded( White,Howard, &Prior,2010 ). Esatbeyoglu,Wray,andWinterhalter(2010) used (+)-catechinor( )-epicatechintodepolymerizeB-typeprocyanidinpolymersforthesemisynthesisofB-typedimers( Esatbeyoglu etal.,2010 ).Toourknowledge,itisnotknownifasimilarmethod canbeusedtodepolymerizeA-typeprocyanidins,suchasthose fromcranberries,orwhether( )-epigallocatechingalatecanfunctionasachainbreaker.Theobjectiveofthisstudywastodepolymerizecranberryprocyanidinpolymersintooligomers,using(+)catechin,( )-epicatechin,and( )-epigallocatechingalateaschain0308-8146/$-seefrontmatter 2013ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.003 Correspondingauthor.Tel.:+13523921991x210;fax:+13523929467. E-mailaddress: LGu@uÂ”.edu (L.Gu). FoodChemistry141(2013)488Â…494 Contentslistsavailableat SciVerseScienceDirectFoodChemistryjournalhomepage:www.elsevi er.com/locat e/foodchem
breakers.Itwasexpectedthatbothbioavailabilityandbioactivity ofprocyanidinscouldbeenhancedafterdepolymerization. 2.Materialsandmethods 2.1.Chemicalsandmaterials Freeze-driedcranberrypowderwasprovidedbyOceanSpray Cranberries,Inc.(Lakeville,MA).HPLC-gradeacetone,methylene chloride,methanol,hydrochloricacid,andaceticacidwereobtainedfromFisherScientiÂ“c(Pittsburg,PA).SephadexLH-20was purchasedfromSigmaChemicalCo.(St.Louis,MO).Amberlite FPX66resinwasaproductofRohmandHaasCo.(Philadelphia, PA).(+)-Catechin,( )-epicatechin,and( )-epigallocatechingallate werepurchasedfromSigmaChemicalCo.(St.Louis,MO).Aprocyanidinstandardthatcontainedmonomerthroughdecamerswas kindlyprovidedbyMarsInc.(Hackettstown,NJ).Thisstandard waspuriÂ“edfromunfermentedcocoaandcontainedB-typeprocyanidinsonly. 2.2.PreparationofpartiallypuriÂ“edcranberryprocyanidins(PCP) Onekilogrammeofcranberrypowderwasextractedwith4Lof methanol(acidiÂ“edwith0.5%aceticacid)atroomtemperaturefor 48h.ExtractsobtainedaftervacuumÂ“ltrationwerecombinedand concentratedat40 Cunderpartialvacuumusingarotaryevaporator.About742gofdrycrudeextractwereobtained.Partofthis crudeextract(150g)wassuspendedin150mlofwaterandpartitionedwithhexanetwice(500mleachtime)toremovelipidsubstances.Thewatersuspensionwaspartitionedwithethylacetate, threetimes.Afterwards,thewatersuspensionwasloadedontoa column(2.8 55cm)packedwithAmberliteFPX66resin.Thecolumnwaselutedwith3500mlof1%aceticacidinwatertoremove sugars,followedby1000mlof30%methanol/water,1000mlof 50%methanol/water,and1000mlofmethanolelution.Themethanolfractionwasdriedanddissolvedin30%methanolbeforeit wasloadedontoaSephadexLH-20column(5.8 28cm).Thecolumnwaselutedwith70%methanol(2.5L)toremoveanthocyaninsandÂ”avonols.Procyanidinswererecoveredusing70% acetone(1L)elution.About1.09gofpartiallypuriÂ“edcranberry procyanidins(PCP)wasyieldedafterthesesteps.Itspuritywas estimatedusingHPLC. 2.3.Depolymerizationofcranberryprocyanidins Depolymerizationwascarriedoutunderconditionsthatwere modiÂ“edfrompreviousresearch( Esatbeyoglu&Winterhalter, 2010;Esatbeyogluetal.,2010;Kohler,Wray,&Winterhalter, 2008 ).BrieÂ”y,10mgofPCPweredissolvedin0.1Mor1.0MmethanolicHCltoaconcentrationof10mg/ml;(+)-catechinand( )epicatechinweredissolvedinthesamemethanolicHCltohave concentrationsof4,10,20and30mg/ml.Theconcentrationsof ( )-epigallocatechingallatewere6.4,15.9,31.6and47.4mg/ml. The6.4mg/ml( )-epigallocatechingallateandthe4mg/ml;(+)catechinhadthesamemolarconcentrationof13.8mM.Afterdissolving,0.5mlofPCPsolutionwasmixedwithanequalvolumeof Â”avan-3-olsolutionsofdifferentconcentrations.Thisresultedin PCP-to-Â”avanolmassratioof5:2,5:5,5:10,and5:15forcatechin orepicatechin[collectivelycalled(epi)catechin].ThePCP-to-EGCG ratioswere5:3.2,5:7.9,5:15.8,and5:23.7.Thedepolymerization reactionwascarriedoutin0.1and1MmethanolicHClfor 20minat40 C.ThemixturesweresubjectedtoHPLCÂ…ESI-MSnanalysesimmediatelyafterdepolymerization. 2.4.HPLCÂ…ESI-MSnanalysisofprocyanidins ChromatographicanalyseswereperformedonanAgilent1200 HPLCsystem(PaloAlto,CA)equippedwithabinarypump,an autosampler,aÂ”uorescencedetector,andahighcapacity(HCT) iontrapmassspectrometer(BrukerDaltonics,Billerica,MA).SeparationwascarriedoutonaLunaSilica(2)column(250 4.6mm, 5lmparticlesize,Phenomenex,Torrance,CA)atacolumntemperatureof37 C.Thebinarymobilephaseconsistedof(A)methylene chloride/methanol/aceticacid/water(82:14:2:2,v/v/v/v)and(B) methanol/aceticacid/water(96:2:2,v/v/v).The70mingradient wasasfollows:0Â…20min,0.0Â…11.7%Blinear;20Â…50min,11.7Â… 25.6%Blinear;50Â…55min,25.6Â…87.8%Blinear;55Â…65min,87.8% Bisocratic;65Â…70min,87.8Â…0.0%Blinear;followedby5minof columnre-equilibrationbeforethenextinjection.Excitationand emissionoftheÂ”uorescentdetectorweresetat231and320nm, respectively( Robbinsetal.,2009 ).Electrosprayionisationatnegativemodewasperformedusingnebulizer50psi,dryinggas10l/ min,anddryingtemperature350 C,capillary4000V.Massspectra wererecordedinarangeof m / z 150Â…2200.Themostabundantion infullscanwasisolated,anditsproductionionspectrawere recorded. 2.5.Statisticalanalyses Depolymerizationexperimentswereconductedinduplicates. DatawereexpressedasthemeanÂ±standarddeviation.Two-tailed StudentÂs t -testswereperformedtocomparetheyieldofA-andBtypeoligomers,usingExcel(Version2007,Microsoft,Seattle,WA). One-wayANOVA,withTukeyÂ…KramerHSDpair-wisetests,was doneonJMP(Version8.0,SASInstituteInc.,Cary,NC).Adifference of p 6 0.05wasconsideredassigniÂ“cant. 3.Resultsanddiscussion 3.1.CharacterizationofpartiallypuriÂ“edcranberryprocyanidins Atwo-stepchromatographicmethodwasusedtoobtainthe partiallypuriÂ“edcranberryprocyanidins(PCP).Amberliteresin FPX66wasusedtoremovesugarsfromthecranberryextract. SephadexLH-20wasusedtoremoveanthocyanin,Â”avonols,and themajorityofsmallerprocyanidins.ThePCPwasanalysed,using normalphaseHPLC,whichwasabletoseparateprocyanidins accordingtodegreeofpolymerization.Procyanidinswithdegree ofpolymerizationover10weredeÂ“nedaspolymers( Guetal., 2002 ). Fig.1 AshowsthatPCPcontainedpredominantlypolymers thatelutedasalargepeakat56min( Guetal.,2002 ).Thecontent ofpolymersinpuriÂ“edcranberryprocyanidinswasestimatedtobe 77.2%(w/w),usinghexamersintheprocyanidinasstandard.Hexamerswereusedasexternalstandardbecausetheywerethelargest procyanidinsinwhichaccuratepeakintegrationcanbeobtained usingnormalphaseHPLC.SmallamountsofA-typeprocyanidindimer(0.3%w/w),trimer(0.5%w/w),andtetramer(0.9%w/w)were alsodetected.Thetotalamountoftheseoligomerswasabout2%of thepolymers. 3.2.HPLCÂ…MSnidentiÂ“cationofoligomersafterdepolymerizationin HClwithoutaddedÂ”avan-3-ols ItisknownthatprocyanidinscanbedepolymerizedbyconcentratedacidssuchasHCltoproduceanthocyanins.Thismethodhas beenusedtoestimateprocyanidincontentintheHCl/butanolassay( Porter,Hrstich,&Chan,1985 ).Insuchassay,H+catalyses thecleavageoftheinterÂ”avanbondsinprocyanidinstoformcarbocationsatC4(pathwayisdepictedin SupplementaryFig.S1 ).H.Liuetal./FoodChemistry141(2013)488Â…494 489
Thesecarbocationsundergostructuralrearrangementtoform anthocyanins.Becausethecarbocationscanonlybeformedon theextensionunitsofprocyanidins,aprocyanidinpolymerwith 4Â…8interÂ”avanconnectioncanproducemultipleanthocyanins andasingleÂ”avan-3-olfromaterminalunit.IntheHCl/butanolassay,procyanidinsweredepolymerizedin2.0MHClinaboiling waterbathfor50min( Porteretal.,1985 ).Thepresentstudyappliedmuchmilderconditions.Cranberryprocyanidinswerekept in0.1or1.0MmethanolicHClat40 Cfor20min.Mostofthe procyanidinpolymersremainedstableundertheseconditions. However,therewasanincreaseofpeaksfordimers,trimers,and tetramers( Fig.1 BandC).Wetheorisedthattheseoligomerswere releasedfromtheterminalunitsofsomepolymersafterincompletedepolymerization.Suchexplanationwasconsistentwith thelowdepolymerizationyield(6%)whenHClalonewasused. Procyanidinoligomershadthreecharacteristicfragmentation routes,whichhavebeendescribedas:quinonemethide(QM) cleavage,retro-DielsÂ…Alder(RDA)cleavage,andheterocyclicring Â“ssion(HRF)( Gu,Kelm,Hammerstone,Zhangetal.,2003 )(Pathwayisdepictedin SupplementaryFig.S2 ).Apeakelutedat 14.3mingaveriseto m / z 575[M H]anditwasidentiÂ“edasan A-typeprocyanidindimer( Fig.1 BandC).Itfragmentedtogenerate m / z 423,duetoretro-DielsÂ…Aldercleavage,and m / z 285,dueto quinonemethidecleavageoftheA-typelinkage( Table1 ).Thepeak at17.6minshowed m / z 577[M H]andproductionsthatwere characteristicofB-typedimers.FourpeakswereidentiÂ“edasAtypetrimersaccordingto m / z 863[M H]andproductionion spectra( Table1 ).Peaksat19.1,19.7,and21.3hadproductions m / z 737,711and575,respectively.Thediagnosticionat m / z 575 derivedfromQMcleavageoftheinterÂ”avanbondindicatedthat thistrimerhadaconnectionsequenceof(epi)cat-(epi)cat-A-(epi)cat.Peaksat19.1and19.7werealsopresentinPCPbeforedepolymerization( Fig.1 A).The(epi)catdenotescatechinorepicatechin sincetheycouldnotbedistinguishedbymassspectrometer. Fluorescent Intensity Fig.1. ChromatogramsofpartiallypuriÂ“edcranberryprocyanidins(PCP)in methanol(A)andaftertheyweredepolymerizedin0.1M(B)or1.0M(C) methanolicHCl.A2,A3,andA4areA-typeprocyanidindimer,trimer,andtetramer, respectively.B2denotesB-typedimer.Symbol(epi)catstandsforcatechinor epicatechin. Table1 IdentiÂ“cationofoligomersafterdepolymerizationofcranberryprocyanidinsinHClwithorwithoutaddedÂ”avan-3-ols.Flavan-3-olRT(min)Connectionsequence[M H]Productsions Without14.5(epi)cat-A-(epi)cat575557(H2Oloss),423RDA,285(QMofA-typelinkage) Flavanols17.6(epi)cat-(epi)cat577451HRF,425RDA,407(H2Oloss),287QM 19.1,19.7,21.3(epi)cat-(epi)cat-A-(epi)cat863737HRF,711RDA,575QM 22.2(epi)cat-A-(epi)cat-(epi)cat863711RDA,573QM,559(2RDA),451HRF,411HRF,289QM 27.2,28.7(epi)catÂ…(epi)catÂ…(epi)catÂ…AÂ…(epi)cat1151981(RDA,H2Oloss),863QM,739HRF,575QM,411HRF (+)-Catechin14.3(epi)cat-A-(epi)cat575557(H2Oloss),423RDA,285(QMofA-typelinkage) 17.5,19.4(epi)cat-(epi)cat577451HRF,425RDA,407(H2Oloss),287QM 21.3(epi)cat-(epi)cat-A-(epi)cat863737HRF,711RDA,575QM 22.1(epi)gallocat-(epi)cat593467HRF,425RDA,407(H2Oloss),289QM 22.8(epi)cat-A-(epi)cat-(epi)cat863711RDA,573QM,559(2RDA),451HRF,411HRF,289QM 23.4,24.1(epi)cat-(epi)cat-(epi)cat865739HRF,695(RDA,H2Oloss),577QM,287QM 27.3,28.3(epi)CatÂ…AÂ…(epi)CatÂ…(epi)CatÂ… (epi)Cat 1151981(RDA,H2Oloss),861QM,739HRF,573QM,411HRF ( )Epicatechin 14.6(epi)cat-A-(epi)cat575557H2Oloss,423RDA,285(QMofA-typelinkage) 17.7,19.2(epi)cat-(epi)cat577451HRF,425RDA,407H2Oloss,289QM 20.2(epi)gallocat-(epi)cat593467HRF,425RDA,407H2Oloss,289QM 21.5(epi)cat-(epi)cat-A-(epi)cat863711RDA,693(RDA,H2Oloss),575QM,423RDA 22.3(epi)cat-A-(epi)cat-(epi)cat863711RDA,573QM,559RDA,451HRF,411HRF,289QM 22.8,23.7(epi)cat-(epi)cat-(epi)cat865713RDA,695(RDA,H2Oloss),577QM,543(RDAfrom695) 27.4,28.0,28.9(epi)CatÂ…AÂ…(epi)CatÂ…(epi)CatÂ… (epi)Cat 1151981(RDA,H2Oloss),861QM,739HRF,573QM EGCG17.7(epi)cat-(epi)cat577451HRF,425RDA,289QM 19.2,19.9,21.5(epi)cat-(epi)cat-A-(epi)cat863845H2Oloss,711RDA,693(RDA,H2Oloss),575QM 22.9(epi)cat-EGCG745593gallateloss,457QM,423RDA-H2O 25.5(epi)gallocatechinÂ…EGCG761635HRF,609gallateloss,593RDA,575(RDA,H2Oloss),457QM,423RDA 26.3,26.7,27.4,28.9(epi)cat-(epi)cat-EGCG1033863gallateloss,745QM,457QM,423RDA 30.3Â…31.7(epi)cat-A-(epi)cat-(epi)cat-EGCG13191149gallateloss,745QM,861QM,573QM Symbol(epi)catstandsforcatechinorepicatechin.RDA,retro-DielsÂ…Aldercleavage;HRF,heterocyclicringÂ“ssion;QM,quinonemethidecleavage .Â…AÂ…denotesA-type interÂ”avanlinkage.EGCG,epigallocatechingallate. 490 H.Liuetal./FoodChemistry141(2013)488Â…494
Â…AÂ…representedanA-typelinkage.ThefourthA-typetrimer (22.2min)wasidentiÂ“edas(epi)cat-A-(epi)cat-(epi)cataccording toproduction m / z 573,duetoQMcleavage.TwopeaksofA-type tetramers(27.2and28.7min)wereidentiÂ“ed.Productions m / z 863and575,generatedafterQMcleavage,suggestthatthesetetramershadastructureof(epi)catÂ…(epi)catÂ…(epi)catÂ…A-(epi)cat. TheseA-typetetramersweredifferentfromthoseinPCPbefore depolymerization(24.2and26.7min, Fig.1 A),whichhadaconnectionsequenceof(epi)catÂ…A-(epi)catÂ…(epi)cat-(epi)catinaccordancewithmassspectra.ThissuggestedthattheyweretheÂÂnewÂÂ oligomersresultingfromdepolymerization. 3.3.HPLCÂ…MSnidentiÂ“cationofoligomersafterdepolymerizationin HClwithaddedÂ”avan-3-ols Depolymerizationofprocyanidins,usingPCP-to-catechinratio of5:10in0.1or1.0MmethanolicHCl,causedcompletedisappearanceofthepolymerpeaks.Thiswasaccompaniedbyappearanceof newpeaksat12Â…30min( Fig.2 ).Thelargepeakat10minwasdue totheadded(+)-catechin.TheHPLCchromatogramshowedfour dimerswith m / z [M H]575,577,577,and593( Fig.2 A).Peaks at17.5minand19.4minshowed m / z 577[M H]andproduct ionsthatwerecharacteristicofB-typedimers.The m / z [M H]593wasindicativeofaB-typedimerthatconsistedofan(epi)catechinandan(epi)gallocatehin.Ion m / z 289wasformedafterquinonemethide(QM)cleavageindicatedthatthisdimerhad (epi)gallocatechinasanextensionunitand(epi)catechinasaterminalunit. Thesecondpeakat22.8minshoweddiagnosticionsat m / z 573 and289fromQMcleavagethatwereconsistentwithanA-typetrimerwith(epi)cat-A-(epi)cat-(epi)catconnection.Threepeakswere identiÂ“edasB-typetrimersaccordingto m / z 865[M H]andtheir productions.TwopeaksofA-typetetramers(27.3and28.3min) wereidentiÂ“ed.Productions m / z 861and573generatedafter QMcleavage,suggestedthatthesetetramershadastructureof (epi)catÂ…AÂ…(epi)catÂ…(epi)catÂ…(epi)cat. Similarto(+)-catechin,depolymerizationofPCP,using( )-epicatechin,generatedbothA-andB-typeoligomers( Fig.3 ).IdentiÂ“edoligomerpeaksarelabelledonthechromatogramsandlisted in Table1 . Fluorescent Intensity Fig.2. ChromatogramofprocyanidinsafterPCPwasdepolymerizedusingPCP-tocatechinmassratioof5:10in0.1MmethanolicHCl(A)orin1.0MmethanolicHCl (B).A2,A3,andA4areA-typeprocyanidindimer,trimer,andtetramer,respectively. B2/593denotesB-typedimerwith m / z 593. Fluorescent Intensity Fig.3. ChromatogramofprocyanidinsafterPCPwasdepolymerizedusingPCP-toepicatechinmassratioof5:10in0.1MmethanolicHCl(A)orin1.0Mmethanolic HCl(B).A2,A3,andA4areA-typeprocyanidindimer,trimer,andtetramer, respectively.B2/593denotesB-typedimerwith m / z 593. Fluorescent Intensity Fig.4. ChromatogramofprocyanidinsafterPCPwasdepolymerizedusingPCP-toEGCGmassratioof5:15.8in0.1MmethanolicHCl(A)orin1.0MmethanolicHCl (B).B2/745,B3/1033,andA4/1319denoteB-typedimerswith m / z 745,B-type trimerswith m / z 1033,andA-typetetramerwith m / z 1319,respectively. H.Liuetal./FoodChemistry141(2013)488Â…494 491
Afterdepolymerization,usingepigallocatechingallate(EGCG) in0.1MmethanolicHCl,addedEGCGappearedasapeakat 13.2min( Fig.4 A).TheEGCGpeakwasmuchlowerinthedepolymerizationmixtureof1.0MmethanolicHCl.HClcanhydrolyze EGCGtogenerateepigallocatechin.However,epigallocatechin wasnotdetectableinthedepolymerizationmixtures.Thepeak at14.7min( m / z 519)wasnotepigallocatechinanditwasnotidentiÂ“ed.ItsuggestedthatthedecreaseofEGCGin1.0Mmethanolic HClwasmorelikelyduetodegradationthanhydrolysis.Apeak at22.9minshowed m / z 745[M H]( Table1 ).Fragment m / z 457,fromEGCGatterminalunitafterQMcleavage,suggestedthat thisdimerhadan(epi)catechinÂ…epigallocatechingallateconnection.The[M H]ionofadimerat25.5mingaveriseto m / z 761.Fragments m / z 635and593wereduetoHRFandRDA,respectively.Thefragmentof m / z 457,afterQMcleavage,indicatedthat thisdimerwas(epi)gallocatechinÂ…epigallocatechingallate.Three peaksshowed m / z 1033[M H].Thesetrimerslose(epi)catechin fromatopunitthroughQMcleavage,toyield m / z 745.Aproduct ion, m / z 457,resultingfromQMcleavages,indicatedthatEGCG layintheterminalunit.HencetheywereidentiÂ“edas(epi)catechin-(epi)catechinÂ…epigallocatechingallate.Aclusterofpeaksat 30.3Â…31.7mingave m / z 1319[M H].Fragment745wasformed from(epi)catechinÂ…epigallocatechingallateafterQMcleavage. The m / z 573,afterQMcleavage,suggestedthatthestructureof thetopunitwas(epi)catechin-A-(epi)catechin.Thereforethese clusterofpeakswereidentiÂ“edtobe(epi)catechin-A-(epi)catechin-(epi)catechinÂ…epigallocatechingallate.EGCGwasnotaconstituentunitincranberryprocyanidins( Guetal.,2002 ). Depolymerizationofcranberryprocyanidins,usingEGCG,generatedprocyanidinsthatcontainedEGCGasaterminalunit.This observationprovidedclearevidencethatEGCGattachedtothe C4carbocationformedfromprocyanidinsbyH+. 3.4.Yieldofoligomers OligomersgeneratedafterdepolymerizationwerequantiÂ“ed. YieldofoligomerswasdeÂ“nedastheratioofoligomersgenerated totheamountofpolymersusedfordepolymerization.When0.1or 1.0MHClwereusedalonetodepolymerizeprocyanidins,the yieldswere6%and8%,respectively.Adding(+)-catechin,( )-epicatechin,orEGCGaschainbreakerssigniÂ“cantlyincreasedthe yieldofoligomers( Fig.5 ). Inthe0.1MmethanolicHCl,anincreaseofPCP-to-catechinratiofrom5:2to5:10ledtoanincreaseofyieldfrom22%to61%.In PCPto Flavan-3-ol mass ratioYield percent (%) 0 20 40 60 80 100 A-type oligomers B-type oligomers Sum of A-and B-type oligomers A*ab*a b B a a a b 0 20 40 60 80 100 C a a a 5:05:25:55:105:15 5:05:25:55:105:15 D a a a a 5:05:3.25:7.95:15.85:23.7 -10 0 10 20 30 40 E b b b 5:05:3.25:7.95:15.85:23.7 F b b b c* * * * * * * * * * * * * * * Fig.5. YieldofprocyanidinoligomersafterPCPwasdepolymerizedwithcatechinin0.1M(A)and1.0M(B)methanolicHCl,withepicatechinin0.1M(C)and1.0M (D) methanolicHCl,andwithEGCGin0.1M(E)and1.0M(F)methanolicHCl.A-typeoligomersincludeddimer( m / z 575),trimer( m / z 863),andtetramer( m / z 1151).A-type oligomersafterdepolymerizationwithEGCGwereA-typetrimer( m / z 863)andtetramer( m / z 1319).B-typeoligomersincludeddimer( m / z 577)andtrimer( m / z 865).B-type oligomers,afterdepolymerizationwithEGCG,includeddimer( m / z 577),dimer( m / z 745,761),andtrimers( m / z 1033).Â‡SigniÂ“cantdifferencesbetweenyieldofA-typeandBtypeoligomers.YieldsofsumofA-andB-typeoligomerswithdifferentlettersatequivalentPCPtoÂ”avan-3-olmassratioweresigniÂ“cantlydifferen t. 492 H.Liuetal./FoodChemistry141(2013)488Â…494
the1.0MmethanolicHCl,anincreaseofPCP-to-catechinratio from5:2to5:10ledtoanincreaseofyieldfrom38%to88%.However,afurtherincreaseofthePCP-to-catechinratioto5:15caused adecreaseofoligomeryield.Thereasonforthisphenomenonwas unclear,butitwaslikelycausedbydegradationofpolymersor generationofbyproducts,suchasthechalcaneÂ”avan-3-oldimers (gambiriins)( Esatbeyoglu&Winterhalter,2010 ).Becausechalcane Â”avan-3-oldimers( m / z 579,[M H])werenotdetectedinthe depolymerizationmixturesinthisstudy,degradationwasspeculatedtobeamajorreason.Degradationofprocyanidinsinthiolysis hasbeenpreviouslyreported( Guetal.,2002 ).Incontrasttocatechin,adecreaseinoligomeryieldwasnotobservedatthehighest PCP-to-Â”avanolratioforepicatechinandEGCG.Inthesetwocases, yieldincreasedcontinuouslywhenhigheramountsofÂ”avan-3-ols wereaddedtothesystem.Thisobservationwasinaccordancewith apreviousreport( Esatbeyoglu&Winterhalter,2010 ). Incatechinorepicatechinsystems,oligomeryieldwashigherin 1.0MmethanolicHClthanin0.1MmethanolicHCl.Theopposite wasobservedwhenEGCGwasusedindepolymerization,although thedifferencewasnotsigniÂ“cantlydifferent,usingthe t -test.ItappearedthatthehigherconcentrationofHClfacilitatedthegenerationofcarboncationandincreasedtheoligomeryieldwhen catechinorepicatechinwereused.However,thehigherconcentrationofHClcausedEGCGdegradationandadecreaseinoligomer yield.TheyieldofoligomersinEGCGsystemwasmuchlowerthan thoseincatechinorepicatechinsystems.Thereweretwopossible reasonsforthisloweryield.First,themolecularsizeofEGCGwas muchlargerthancatechinorepicatechin.Thismaycreatesteric hindrancetoEGCGattackingprocyanidins.Second,epigallocatechinisknowntohavealowerÂ”uorescenceresponsethanepicatechin.IncorporationofEGCGintheprocyanidinmoleculecausesa decreaseofÂ”uorescencereading( Gu,Kelm,Hammerstone,Beecher,etal.,2003 ).Asaresult,theamountofnewprocyanidins mayhavebeenunderestimatedbyHPLCanalyses. TheyieldofB-typeoligomerswasmuchhigherthanthatofthe A-typeoligomersinallthedepolymerizationsystemsusingÂ”avan3-ols.TheB-typeprocyanindindimerwasthepredominantoligomerafterdepolymerization.Itwasexpectedthatdepolymerization ofprocyanidinwouldgeneratemoreB-typeprocyanidinsthanAtype.ThisisbecausethemajorityofA-typeprocyanidinoligomers incranberriescontainasingleA-typeinterÂ”avanlinkage.ForanAtypehexamerwithsingleA-typelinkageattheterminalunitand fourB-typelinkagesbetweenextensionunits,acompletedepolymerizationusingepicatechinwouldresultinfourB-typedimers andoneA-typedimer. Procyanidinpolymersincranberrieshadanaveragedegreeof polymerizationof15.3( Guetal.,2002 ).Theextensionunitsof thesepolymersconsistedexclusivelyofepicatechin.Onlyasmall amountofcatechin(7%)wasfoundintheterminalunits( Gu etal.,2002 ).Whencatechinorepicatechinwereusedasachain breaker,epicatechinwasincorporatedintothenewlygenerated oligomersasaterminalunit.Theseoligomerswereexpectedto havestructuressimilartothoseexistingnaturallyincranberries. EpicatechinappearedtobeanidealnucleophileforthedepolymerizationofcranberrypolymersamongthethreeÂ”avan-3-olsdueto itshigholigomeryield.NewA-andB-typeoligomerswereproducedwhen( )-epigallocatechingallatewasusedfor depolymerization. Procyanidinshavebeendepolymerizedusingphloroglucinolor toluene-a-thiol,bothofwhicharenucleophiles.TheC6andC8on theA-ringofÂ”avan-3-olsarenucleophilicbecausetheA-ringhasa 1,3,5-benzenetriolstructure,similartoaphloroglucinol.Hence, Â”avan-3-olscanattackthecarbocationatC4afteritwasformed bythecatalysisofstrongacid(H+).Asaresult,useofÂ”avan-3olstodepolymerizeprocyanidins,producedsmallerprocyanidin oligomersinsteadofanthocyanins.Furthermore,A-typeprocyanidinswerepreservedduringdepolymerizationusingÂ”avan-3-ols. Thesameobservationwasmadeinprocyanidinthiolysis,usingtoluene-a-thiol( Gu,Kelm,Hammerstone,Beecher,etal.,2003 ).Some ofthelargeprocyanidinsincranberriescontainedmorethanone setofA-typeinterÂ”avanlinkagesthatwereseparatedbyB-type linkages( Gu,Kelm,Hammerstone,Zhang,etal.,2003 ).Theselarge procyanidinsmaybeÂÂchoppedÂÂintoseveralsmallerA-typeprocyanidinoligomersbyÂ”avan-3-ols.Thisacidassisteddepolymerizationofpolymersgeneratedalargenumberofcompoundsthat wereidentiÂ“edasprocyanidins,usingtandemmassspectrometry. However,themassspectrometerlacksthecapacitytodistinguish catechinfromepicatechininprocyanidins.Theexactstructures, absorptionrate,andtoxicityofthesecompoundshavetobedeterminedbeforetheycanbeusedasfunctionalfoodingredients. 4.Conclusions Cranberryprocyanidinswereeffectivelydepolymerizedinto oligomericprocyanidinsunderacidicconditions,usingÂ”avan-3olsaschainbreakers.Thenewlyformedoligomersincludedimers, trimers,andtetramersofbothA-typeandB-type.DepolymerizationyieldedmoreB-typeprocyanidinsthanA-type.Becauseover 85%ofprocyanidinsincranberrieswerelargeoligomersorpolymersthatcannotbeabsorbed( Guetal.,2002 ),depolymerization ofcranberryprocyanidinswasexpectedtoincreasetheirbioavailabilityandbioactivities. AppendixA.Supplementarydata Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,at http://dx.doi.org/10.1016/j.foodchem.2013. 03.003 . ReferencesAvorn,J.,Monane,M.,Gurwitz,J.H.,Glynn,R.J.,Choodnovskiy,I.,&Lipsitz,L.A. 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