ABSTRACT TANNINS AS ANTI-INFLAMMATORY AGENTS By Melanie Diane Jeffers The potential of dietary and herbal polyphenols to act as beneficial antioxidants or anti-inflammatory agents, or as harmful pro-oxidants, is a topic of intense current interest. The first half of this research involved using SDS-PAGE gels to elucidate tannin-protein complexation. The second half of this study attempts to evaluate the anti- inflammatory or pro-inflammatory activity of the tannin pentagalloyl glucose within an ex vivo model system using peripheral blood mononuclear cells from rabbit. The oxidative burst from cells stimulated with phorbol ester was monitored with cytochrome c. The results provide preliminary data that supports the hypothesis that tannins such as pentagalloyl glucose minimize the inflammatory response. TANNINS AS ANTI-INFLAMMTORY AGENTS A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Masters of Science Department of Chemistry and Biochemistry By Melanie Diane Jeffers Miami University Oxford, Ohio 2006 Advisor _______________________ Dr. Ann E. Hagerman Reader ________________________ Dr. John W. Hawes Reader _________________________ Dr. Gary A. Lorigan Reader _________________________ Dr. Kevin W. Kittredge Table of Contents Page List of Figures iv Abbreviations vi 1. Introduction: Tannins as Anti-Inflammatory Agents 1.1. Tannins 1 1.1.1. What is a tannin 1 1.1.2. Human exposure to tannins 2 1.1.3. Tannin-protein complexes 3 1.1.4. Anti-nutritional effects of dietary tannins 4 1.2. Tannins and Reactive Oxygen Species 4 1.2.1. Tannins and the inflammatory system 6 1.2.2. Inflammatory diseases 7 1.2.3. Inflammatory bowel diseases 8 1.3. Hypothesis 10 1.4. Chapter 1 References 12 2. Characterization of soluble protein-tannin complexes using Ribonuclease A and Pentagalloyl glucose (β-1,2,3,4,6-Pentagalloyl-O-D-Glucopyranose) 2.1. Introduction 18 2.2. Material and Methods 19 2.3. Results and Discussion 20 2.4. Chapter 2 References 41 3. Analytical methods for monitoring the inflammatory the response 3.1. Introduction 43 3.1.1. Superoxide as one of the first signaling molecules 43 3.1.2. Rationale for using cytochrome c 44 3.2. Methods 45 3.2.1. Reagents 45 3.2.2. Validation 45 3.3. Results and Discussion 45 3.4. Chapter 3 References 51 ii 4. Investigation of tannins as anti-inflammatory agents 4.1. Introduction 53 4.1.1. Peripheral blood mononuclear cells (PBMCs) 53 4.2. Methods 55 4.2.1. Reagents 55 4.2.2. Isolation of peripheral blood mononuclear cells (PBMC) 56 4.2.3. Incubation and priming of PBMCs 57 4.2.4. PMA stimulation of PBMCs 57 4.2.5. Superoxide production from PBMCs 57 4.2.6. PGG interference with cytochrome c 57 4.2.7. Lucigenin as a probe for superoxide 58 4.3. Results 58 4.4. Discussion 60 4.5. Chapter 4 References 70 iii List of Figures 1. Chapter 1. Figures Page 1.1. Structures of representative tannins. 11 2. Chapter 2. Figures 2.1. Optimal pH for PGG-RNase complex formation. 26 2.2. Optimal RNase:PGG ratio for complex formation. 27 2.3. Effect of excess PGG on complex formation. 28 2.4. SDS PAGE to detect PGG-RNase complexes. 29 2.5. Higher percent acrylamide SDS PAGE to better resolve PGG-RNase complexes. 30 2.6. Gradient SDS-PAGE to detect PGG-RNase complexes. 31 2.7. Effect of dialysis on PGG-RNase complexes. 32 2.8. Effect of β-ME on PGG-RNase complexes. 33 2.9. Order of addition of reagents. 34 2.10. Effect of urea on PGG-RNase complex formation. 35 2.11. Proteolysis with trypsin. 36 2.12. Proteolysis with subtilisin and trypsin. 37 2.13. Proteolysis and Tris/Tricine gels. 38 2.14. Proteolysis with 14C radiolabelled PGG using subtilisin. 39 2.15. Periodate reaction with RNase. 40 3. Chapter 3. Figures 3.1. Cytochrome c spectrum. 48 3.2. Cytochrome c reduction by stimulated monocyte cells. 49 3.3. Relationship between reduced cytochrome c and slope of the spectral peak. 50 4. Chapter 4. Figures 4.1. Superoxide production from rabbit PBMCs in the presence of cytochrome c. 63 4.2. Superoxide production represented as slope of the spectral data. 64 4.3. Superoxide production from cells exposed to tannin. 65 iv 4.4. PGG reduces cytochrome c. 66 4.5. Superoxide production in PGG-treated cells. 67 4.6. Superoxide production from rabbit PBMCs probed with lucigenin. 68 4.7. Superoxide production from cells exposed to tannin using lucigenin as a probe. 69 v Abbreviations 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) bovine serum albumin (BSA) cyclooxygenase (COX-2) dimethyl sulfoxide (DMSO) ethylenediamine-tetraacetic acid (EDTA) epigallocatechin gallate (EGCG) gastrointestinal (GI) Hank’s balanced salt solution (HBSS) helper T-cells (Th) hydrogen peroxide (H2O2) hydroxyl radical (OH•) hypochlorous acid (HOCl) inducible nitric oxide synthase (iNOS) inflammatory bowel disease (IBD) interferons (IFN) interleukins (IL) lipopolysaccharide (LPS) β-mercaptoethanol (β-ME) nitric oxide radical (NO•) nitroblue tetrazolium (NBT) β-1,2,3,4,6-pentagalloyl-O-D-glucopyranose (PGG) peripheral blood mononuclear cells (PBMC) phorbol 12-mysristate 13-actetate (PMA) potassium superoxide (KO2) procyanidin (PC) prostaglandin E2 (PGE2) reactive oxygen species (ROS) ribonuclease A (RNase) sodium dithionite (Na2S2O4) vi sodium dodecyl sulfate (SDS) sodium periodate (NaIO4) •- superoxide anion (O2 ) superoxide dismutase (SOD) tumor necrosis factors (TNF) vii 1. Introduction: Tannins as Anti-Inflammatory Agents 1.1. Tannins 1.1.1. What is a tannin? Tannins are a complex family of naturally occurring polyphenols that most characteristically precipitate gelatin and other proteins (1). Other phenolic compounds also have the ability to bind with protein, but do not form the insoluble, cross-linked complexes typically formed by tannin-protein mixtures (2). Tannins were originally used in the process of tanning or conversion of animal hides into leather by precipitating the proteins, such as collagen, found in the skin of the animal (3). Their name is derived from the Celtic word for oak, a traditional source for tannin-containing extracts for preparing leather (4). Tannins are secondary metabolites produced by plants presumably to provide protection from disease and mammalian herbivores. These polymeric polyphenols, which have molecular weights ranging from 0.5 to 22 kD, have unique chemical and biological activities (5). In addition to binding protein, tannins are also potent metal ion chelators, tightly binding essential metals such as iron and calcium (6, 7). Polymeric polyphenols are also potent radical scavengers and antioxidants (8) and thus have been targeted as good candidates for therapeutic agents against diseases associated with oxidative stress. There are two classes of tannins that are of biological significance to humans; condensed and hydrolyzable tannins. Condensed tannins, or proanthocyanidins, can all be degraded to the pigments known as anthocyanidins. The flavanoid backbones of the condensed tannins are derived from phenylalanine and polyketide biosynthesis (9). The majority of condensed tannins are produced from the flavan-3-ols (+)-catechin (2,3-trans) and (-)-epicatechin (2,3-cis) (10). For example, procyanidin (PC) is a polymer of catechin and epicatechin subunits (Fig.1.1). Hydrolyzable tannins maintain a core polyol, usually glucose, with galloyl groups derived from shikimic acid esterified to the core (9). These tannins can polymerize through oxidative coupling of the galloyl groups resulting in complex structures known as ellagitannins. Pentagalloyl glucose (β-1,2,3,4,6- pentagalloyl-O-D-glucopyranose) or PGG, is one of the simplest gallotannins (Fig.1.1). 1 It consists of a glucose core with five galloyl groups linked to the five alcoholic positions on the pyranose ring by ester bonds (4). PGG is the polyphenol used in the experiments described in this thesis. 1.1.2. Human exposure to tannins Tannins, both condensed and hydrolyzable, are widely dispersed in the plant kingdom, with some plants containing as much as several grams of tannin per kilogram of tissue (2). Fruits such as blueberries and strawberries are some of the most commonly consumed tannin-containing foods in the American diet. Beverages such as red wine, cranberry juice, and apple juice contain approximately 750, 248, and 48 mg tannin per liter, respectively (2). It is estimated that tannin makes up somewhere between 37-56% of green tea constituents and that as much as 10% of black tea solids are made up of unoxidized catechins (11). Consumption of tannin-containing food products varies from region to region. Legumes and beans, staples in many countries, contain large quantities of condensed tannins (12, 13). Sorghum, which contains between 0.13-7.2% condensed tannin, is a common food staple in Asia, Africa, and countries in the Near and Middle East (14). The North American diet, which is relatively poor in grains, legumes and fruits, contains much less tannin. It has been reported that tannin consumption in India ranges from 1.5- 2.5 g/day, depending on the region, and about 1g/day within the USA (2). Tannins are important components of medicinal plants widely used in many cultures, particularly in Asia. For instance, shigyaku-san, used for gastritis and peptic ulcers in Japan, has the root of Paeonia among its many components. The outer skin of this root is also used to treat disorders of the cardiovascular system, such as high blood pressure. PGG is thought to be the biologically significant portion of that root (15). Polyphenolic compounds, when applied topically, may aid in wound healing, burns and inflammation by creating an impenetrable layer with protein or polysaccharide and therefore natural healing can occur underneath (5). Other plants reported to have medicinal properties, and that contain polyphenolic compounds include; Arctostaphylos uva-ursi, used as a diuretic for kidney disorders; Geranium maculatum, used as an anti- inflammatory agent and astringent; and Rubus idaeus or raspberry leaves, used for disorders of the stomach in children (5).