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Crystallization studies of epigallocatechin gallate

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Crystallization studies of epigallocatechin gallate
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Kesani, Sheshanka
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EGCG
Flavonoids
Green tea
Pharmaceutical crystal forms
Supramolecular chemistry
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bibliography   ( marcgt )
theses   ( marcgt )
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ABSTRACT: Flavonoids are a long and well known class of natural products. Their potential health benefits can be attributed to their antioxidant activity, and modulation of cell signaling pathways. Green tea one of the most widely consumed beverages, consist of flavonoids such as catechins and tannins. Epigallocatechin gallate (EGCG) the major catechin of green tea exhibits multiple health benefits due to its antioxidant nature. The radical scavenging activity of EGCG is attributed to its structure. Therefore, a study on molecular features of EGCG would provide valuable information on structural modifications, which may change the physiochemical properties such as bioavailability and solubility. Although flavonoids are abundant and commercially available they are difficult to purify and crystallize. In this respect, crystallizing EGCG was challenging. By exploring different techniques EGCG was crystallized. Here in this study one new form of EGCG and two solvates, acetonitrile and nitrobenzene, have been synthesized and structurally characterized by differential scanning calorimetry (DSC), infrared (IR) and powder X-ray diffraction (PXRD). The crystal structures were solved by single-crystal X-ray diffraction and a detailed description of synthesis and about the supramolecular synthons that exist in these crystal forms are given.
Thesis:
Thesis (M.S.)--University of South Florida, 2007.
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by Sheshanka Kesani.
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Crystallization Studies of Epigallocatechin Gallate by Sheshanka Kesani A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Michael J. Zaworotko, Ph.D. Roland Douglas Shytle, Ph.D. Abdul Malik, Ph.D. Date of Approval: July 2, 2007 Keywords: Flavonoids, EGCG, Green t ea, Pharmaceutical crystal forms, Supramolecular chemistry. Copyright 2007 Sheshanka Kesani

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Dedication To Sadguru Shiva nanda Murthy Garu and my Family

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Acknowledgements I would like to thank my advisor, Professor Michael J. Zaworotko, for the opportunity to conduct research under his supervision, and for his advice and gui dance throughout the Graduate Program. I would also like to thank Dr Malik and Dr. Shytle, my committee members, for their helpful comments and encouragements. I would like to acknowledge all members of my research group as well as Faculty and Staff of the Chemistry Department of University of South Florida, for their friendly accommodation. I would like to express my deepest thanks to my closest family and friends who constantly supported me throughout the years of studies.

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i TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES ix ABSTRACT x 1. INTRODUCTION 1 1.1 Flavonoids 1 1.1.1 History 1 1.1.2 Structure 2 1.1.3 Classification of Flavonoids 3 1.1.4 Occurrence and distribution of Flavonoids 5 1.1.5 Extraction and Purification 5 1.1.6 Uses 6 1.2 Green Tea 7 1.2.1 History 7 1.2.2 Green tea production and trade 8 1.2.3 Extraction and purification of green tea. 9 1.2.4 Bioactivity 10 1.3 Crystal engineering 13 1.3.1 Introduction 13 1.3.2 Pharmaceutical Crystal Forms 14

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ii 2. CSD STATISTICS 21 2.1 Cambridge Structural Database (CSD) 21 2.2 Supramolecular chemistry 21 2.3 Supramolecular synthons 22 2.4 CSD Statistics for Polyphenols 23 3. EXPERIMENTAL DATA 29 3.1 Infrared Spectroscopy (IR) 29 3.2 Differential Scanning Calorimetry (DSC) 29 3.3 Powder X-ray Diffraction (PXRD) 29 3.4 Single Crystal X-Ray Crystallography 30 3.5 Form I 31 3.5.1. Physical properties 31 3.5.2 IR of Form I 31 3.5.3 DSC of Form I 32 3.5.4 Powder X-ray Diffract ion of EGCG form I 33 3.6 Form II 34 3.6.1 Synthesis 34 3.6.2 Physical properties 34 3.6.3 IR of Form II 34 3.6.4 DSC of Form II 35 3.6.5 TGA of Form II 35 3.6.6 Powder X-ray Diffraction of form II 36

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iii 3.6.7 Picture of Single crystal 37 3.6.8 Single Crystal X-ray Crystallography Data 38 3.7 Form III 38 3.7.1 Synthesis 38 3.7.2 Physical properties 38 3.7.3 IR of Form III 39 3.7.4 DSC of Form III 39 3.7.5 TGA of Form III 40 3.7.6 Powder X-ray Diffraction of form III 40 3.7.7 Picture of single crystal 42 3.7.8 Single Crystal X-ray Crystallography Data 42 3.8. Form IV 43 3.8.1 Synthesis 43 3.8.2 Physical Properties 43 3.8.3 IR of Form IV 43 3.8.4 DSC form IV 44 3.8.5 TGA form IV 44 3.8.6 Powder X-ray Diffraction of form IV 45 3.8.7 Picture of Single Crystal 46 3.8.8 Single Crystal X-ray Crystallography Data 47 4. RESULTS AND DISCUSSION 48 4.1 Structure Description 48

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iv 4.1.1 Form II 48 4.1.2 Form III 53 4.1.3 Form IV 57 4.2 Conformational analysis 60 4.3 Discussion 62 5. CONCLUSION 66 REFERENCES 68

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v LIST OF FIGURES Figure 1.1: Skelet on of Flavonoid 2 Figure 1.2: Structural repres entation of Flavonoids 2 Figure 1.3: Chalcone 3 Figure 1.4: Skeleton of Fl avone 3 Figure 1.5: Quericetol 3 Figure 1.6: Skeleton of Fl avanone 3 Figure 1.7: Anthocyanins 3 Figure 1.8: Skeleton of Isoflavonoi d 3 Figure 1.9: Skeleton of Neoflavonoids 4 Figure 1.10 : Structure of EGCG 8 Figure 1.11: Structure of Catechins (68) 11 Figure 1.12: Structure of Catechin 12 Figure 1.13: Galloyl Group 12 Figure 1.14: Form I 19 Figure 1.15: Form II 18 Figure 1.16: Powder X-ray diffraction of form I and II 19 Figure 2.1 Examples of supr amolecular homosynthon (left) and supramolecular heterosynthon (right) 23

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vi Figure 2.3: Synthon I; OH---OH, Synthon II OH---CO, Syntho n IIIOH----O(ether) interactions, (R:C6H5OH) 24 Figure 2.4:Histograms of contacts for supr amolecular synthons I (OH---OH), II (OH---CO) and (OH----O) III 25 Figure 2.5: Synthon IV represents OH--CN and Synthon V represents OH---NO interaction 26 Figure 2.6 : Histograms of contacts for supramolecular synthons IV 26 Figure 3.1: IR of Form I 31 Figure 3.2: DSC of Form I 32 Figure 3.3: DSC of Form I from Literature 32 Figure 3.4: PXRD of Form I 33 Figure 3.5: PXRD of Form I from literature 33 Figure 3.6: IR of Form II 34 Figure 3.7: DSC of Form II 35 Figure 3.8: TGA of Form II 35 Figure 3.9: Experimental PXRD of Form II 36 Figure 3.10: Calculated PXD of Form II 36 Figure 3.11: Calculated Vs Experimental PXRD of Form II 37 Figure 3.12: Single crystal of Form II 37 Figure 3.13: IR of form III 39 Figure 3.14: DSC of Form III 39 Figure 3.15: TGA of Form III 40

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vii Figure 3.16: Experimental PXRD of Form III 40 Figure 3.17: Calculated PXRD of Form III 41 Figure 3.18: Calculated Vs Experimental PXRD of Form III 41 Figure 3.19: Single crystal of Form III 42 Figure 3.20: IR of Form IV 43 Figure 3.21: DSC of Form IV 44 Figure 3.22: Experimental PXRD of Form IV 45 Figure 3.23: Calculated PXRD of Form IV 45 Figure 3.24: Calculated Vs experimental PXRD of Form IV 46 Figure 3.25: Single crystal of Form IV 46 Figure 4.1: Structure of EGCG 48 Figure 4.2: Representation of OH----OH synthon in Form II 49 Figure 4.3: Representation of OH---O synthon in form II 50 Figure 4.4: Representation of OH---CO synthon in form II 51 Figure 4.5: Representation of OH---CN synthon in form II 51 Figure 4.6: Solvent entrapment in form II 52 Figure 4.7: Unit cell packing of form II 52 Figure 4.8: Representation of OH ----OH synthon in Form III 54 Figure 4.9: Representation of OH---CO synthon in Form III 54 Figure 4.10: Representation of Synthon OH---NO in Form III 55 Figure 4.11: Representation of Synthon OH---O in Form III 55 Figure 4.12: Solvent entrapment in form III 56

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viii Figure 4.13: Unit cell packing of Form III 56 Figure 4.14: Representation of Form IV structure 57 Figure 4.15: Representation of OH---OH synthon in Form IV 58 Figure 4.16: Representation of OHO synthon in Form IV 58 Figure 4.17: Representation of OHCO synthon in FormIV 59 Figure 4.18: Unit cell packing of Form IV 60 Figure 4.19: Representation of different conformations of EGCG (116) 61 Figure 4.20: LC-MS analysis of 90% pure EGCG 62 Figure 4.21: LC-MS analysis of Form II 63 Figure 4.22: LC-MS analysis of Form III 63 Figure 4.23: LC-MS analysis of of Form IV 64

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ix LIST OF TABLES Table 2.1: Frequency of occurence of synthon I in phenols and polyphenols 27 Table 2.2: Frequency of occurrence of synthons from II to V in phenols and polyphenols 27 Table 2.2.3: Distance ranges within which different synthons exists 28 Table 4.1 : Representing the components eluted at different retention times 64

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x ABSTRACT Flavonoids are a long and well kno wn class of natural products. Their potential health benefits can be attributed to their antioxidant activity, a nd modulation of cell signaling pathways. Green tea one of the most widely consumed beverages, consist of flavonoids such as catechins and tannins. Epigallocatechin gallate (E GCG) the major catechin of green tea exhibits multiple health benefits due to its antioxidant nature. The radical scavenging activity of EGCG is attributed to its structure. Therefore, a study on molecular features of EGCG would provi de valuable information on structural modifications, which may change the physiochem ical properties such as bioavailability and solubility. Although flavonoids are abundant and co mmercially available they are difficult to purify and crystallize. In this respect, crystallizing EGCG was challenging. By exploring different techniques EGCG was crystallized. Here in this study one new form of EGCG and two solvates, acetonitrile and nitrobenzene, have been synthesized and structurally characterized by differential scanning calorim etry (DSC), infrared (IR) and powder X-ray diffraction (PXRD). The crystal structures were solv ed by single-crystal X-ray diffraction and a detailed descri ption of synthesis and about the supramolecular synthons that exist in these crystal forms are given.

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1 1. INTRODUCTION 1.1 Flavonoids 1.1.1 History Flavonoid compounds, which are ubiquitous in nature, occupy a prominent position among the plant phenols. Though flavonoids are present in other parts of plants, many flavonoids are easily recognized as flower pi gments in most angiospermic plants. 3 Flavonoids are distributed in higher plants, bacteria, alg ae, fungi, and animals. The earliest suggestion that substances existed in nature that would even tually be recognized as flavonoids can be traced back to the 17t h century. In 1682 Nehemiah Grew discussed the differences between the sol ubility properties of pigments, which can be recognized as the starting point for naturally occurring compounds. 1 Flavonoids are responsible for coloration of the plants. Autumn coloration of leaves has attracted the attention of many workers for years. Anthocyanin pigments have played a major role in the history of naturally occurring compounds. The term flavone first appeared in a paper by Kostanecki and Tambor (1895). 1 In 1893 De Larie and Tiemann obtained the first isoflavone from the rhizomes of Iris Florentina. The first flavanone reco gnized as naturally occurring was butrin 1 and the first isoflavanoid discovered structurally is Prunetin described by Finnamore (1910). 1

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St.Von Kostanecki first established the basic structure of the flavones and synthesized a number of natural compounds. 2 In a continuation to these st udies, Herzig and A.G Perkin determined the positions of attachment of sugar residues in the glycosides. 2 Out of all the flavonoids, anthocyanins are the first com pounds which were studied in detail. This is due to their wide abundance in nature as a coloring compound in plants. 1.1.2 Structure Flavonoids are based on a 15 carbon atom skeleton. A simple way to describe the flavonoid skeleton is, two phenyl rings connected by three carbonbridges. It can also be represented as C 6 C 3 C 6 Figure 1.1: Skeleton of Flavonoid The chemical structure of the flavonoids are based on a C 15 skeleton with a chromane ring bearing a second aromatic ring B in position 2,3 or 4. 3 Figure 1.2: Structural representation of Flavonoids 2

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In certain flavonoids the C ring is e ither in an open form or replaced by a five membered ring (eg: aurones). 1.1.3 Classification of Flavonoids Figure 1.3: Chalcone Figure 1.4: Skeleton of Flavone Figure 1.5: Quericetol Figure 1.6: Skeleton of Flavanone Figure 1.7: Anthocyanins Figure 1.8: Skeleton of Isoflavonoid 3

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Figure 1.9: Skeleton of Neoflavonoids Flavonoids are classified according to the substitution pattern of the C-ring. The oxidation state of the heterocyclic ring and th e position of the B ring are also considered in classifying flavonoids. There ar e 6 major sub groups of flavonoids. 3 Chalcones, (Fig: 1.3) the first sub cl ass of flavonoids contain three carbons bridge in open form. 5 Flavones (Fig: 1.4) are based on th e backbone of 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one). Generally, f ound in herbaceous families like Labiatae, Umbellifereae, and Compositae etc. 3, 4 Flavanols (Fig: 1.5) contain a hydroxyl group at C-3 position (Eg: Quericetol). In flavanone (Fig: 1.5) the hydroxyl group at C-3 position is absent. Anthocyanins (Fig: 1.6) are considered as major cl ass of pigments. Isoflavanoid (Fig: 1.7) are isomeric to flavones by virtue of their having the B-ring attached to position-3 rather than position-2 as in flavones. 1, 3 Neoflavonoids (Fig1.8) contain B-ring in 4 th position. Derived from the 4-phenylcoumarine (4-phenyl-1, 2-benzopyrone) structure. 3, 6 4

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5 1.1.4 Occurrence and distribution of Flavonoids Although flavonoids are widely distribut ed in nature and in foods, they lack uniform distribution throughout the plant kingdom. Flavonoid compounds occur in all parts of the higher plants: roots, stems, leaves, flowers, pollen, fruit, seeds, wood and bark. Ferns contain many flavonoid compounds of th e types found in flowering plants. 2 Different food items contain varying concentrations of flavonoids. Teas, fruits, and dark chocolates contain moderate to high concentrations of flavonoids. Vegetabl es like broccoli and some fruit juices like cranberry and orange provide low levels of flavonoid content. Black and oolong tea contents have high contents of derived tannins. 7 1.1.5 Extraction and Purification Different parameters effect the extraction of the flavonoids from plant materials; their chemical nature, storage time and c onditions, and the presence of interfering substances. The solubility of the flavonoids depend upon the polarity of the solvent used, degree of polymerization, inte raction with other food cons tituents and formation of insoluble complexes. There is no uniform or completely satisfactory procedure that is suitable for extraction of all flavonoids or spec ific type of flavonoids in plant materials. The most commonly used solvents in extrac tion of these compounds are methanol, ethyl acetate, acetone, and water, their combin ations are also used. Propanol and dimethylformamide, are also used but to a le sser extent. Extraction period usually varies from 1min to 24 hrs.

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6 Extraction of polyphenols from food constituen ts also depends on sample-solvent ratio. The Flavonoid extracts can be partia lly purified, using ion exchange resins. Extracts can also be purified and fr actioned by using solid phase extraction or solid phase microextraction (SPME), and column chroma tography. Different columns are used for extraction of flavonoids, like C-18 column, Sephadex column etc. Recently counter current chromatography (CCC) has been used as an alternative to liquid chromatography for fractionation of various flavonoids. High speed centrifugal countercurrent chromatography is used in the separation of flavonoids like tea catechins, anthocyanins, theaflavins, etc. 8 Quantification of flavonoids is carrie d out by different spectroscopic techniques. Gas Chromatography (GC) and high performance liquid chromatography (HPLC) are widely used in separation and quantitation of flavonoids Structure elucidation is carried out by a combination of GC or HPLC with mass spec trometry and also various other relevant techniques. 8 1.1.6 Uses Due to their beneficial health effects, flavonoids have garnered more interest in the past 2 decades. It was initially hypothesized that pharmacological effects of flavonoids would be related to their antioxidant activity, but the available evidence from cell culture experiments suggested that modulation of cell signaling pathway by flavonoids was attributed to their most of th e biological effects. Studies in cell cultures have suggested that flavonoids may affect the chroni c disease by inhibiting the kinases. 115

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7 Flavonoids have a wide range of beneficial health effects such as, reducing the risk of cancer, 116,117 anti-inflammatory, 118 reducing the risk of developing diabetes mellitus, 119 infertility, 120 anticholesterolemia, 121 antiatherosclerosis, 121 antiulcer, ability to inhibit human platelet aggregation, 123 reducing the skin wrinkling, a nd also in reducing the risk of cardiovascular diseases. 124 Flavonoids are most widely used as dietary supplements. 1.2 Green Tea 1.2.1 History Green tea one of the most widely consumed beverages in th e world is an aqueous infusion of dried leaves of Camellia Sinensis. The birth place of green tea is in Asia. Tea plants grow only in warm climates. Alt hough abundant foilage is produced by the tea plant, only the two leaves and the buds at each young shoot are picked for tea. The type of tea produced depends on the way in which leaves are processed: green, oolong, black. Green tea is the least processe d tea; it is made by steaming the harvested leaves, rolling them and then spreading them out to dry until they become crispy (14). The chemical contents of green tea include; flavonoids such as catechins and tannins, free amino acids, caffeine, ascorbic acid, saponins, and unsat urated fatty acids. 15, 16 Catechins constitute a major component of green tea, which include Epicatechin (EC), Epigallocatechin (EGC), Epicatechin gallate (ECG), Epigallocatechin gallate (EGCG). These compounds constitute 30% of the chemical composition of green tea. 12, 13 The most active component of green tea is EGCG, which constitut es 9-13% weight of green tea. It possesses a much greater antioxidant activ ity than the other catechins, and plays an

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important role in the prevention of cancer and cardiovascular diseases. 17 A literature Search thr ough Scifinder reveals that there are more than 8000 citations related to chemistry, bioactivity, pr oduction and potential h ealth benefits of green tea. Out of 8000, 4000 references are re lated to EGCG and ot her natural products of green tea. 18 Figure 1.10 : Structure of EGCG 1.2.2 Green tea production and trade World wide annual production of tea is estimated around 1 million tonnes and 70 percent of it is black tea and remaining is green tea. The worlds major producers of green tea are China, Japan, Korea, Taiwan, and Indonesia. China accounts for 65 percent of world production and 85 percents of world exports of green tea, where as Japan accounts for 20 percent of world production and five percent of world exports. United States of America and Canada are the major e xport destinations of gr een tea. It is also exported to Germany, United Kingdom, and Saudi Arabia. 11 8

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9 1.2.3 Extraction and purification of green tea. Various methods are used in extracti ng and purifying green tea. Most of these methods are patented. As EGCG is the principa l active ingredient of green tea, most of the methods in extracting green tea are concentrated on extracting the pure EGCG. In most of the methods, polyphenols of green t ea are extracted by so lvent extraction, using different solvents like water, ethanol, diethyl ether, ethyl acetate, acetone etc. Purification of green tea is performed by us ing chromatographic techniques. Different types of columns are used in the separati on of polyphenols, like Sephadex LH 20 column, 19 Silica gel column, 20 C-18 attached silica monolith microcolumns, 24 and Lignocellulose column. 25 Different lengths and particle size of HPLC columns are used for separation of catechins. 21 Weakly basic anion exchange resins are also used in the separation of polyphenols. 22 Polyphenols of green tea are also separated by Reverse phase liquid chromatography, and High sp eed counter current chromatography. 26, 27 During the purification process the time and te mperature at which brewing is performed, is also considered (higher th e brewing time higher the concen tration of the catechins in green tea). 70 C is recognized as the optimum te mperature for achieving the maximum concentration of polyphenols. 23 EGCG is also synthesized chemica lly, by the transformation of retero-chalcones into 1, 3-diarylpropene which are then subjected to asymmetric dihydroxylation. The resulting diarylpropane-1, 2-diols serv e as Chirons for essentially enantiopure flavan-3-ols. 28-30

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10 1.2.4 Bioactivity Various diseases like cardiovascular diseases and cancer are considered to be caused by the activity of oxygen radicals; hence th ey are expected to be prevented by antioxidative compounds. It is well recognized that tea catechins (+) ca techin (+C), (-)epicatechin (-EC), (-) epigallocatechin (-EGC ), (-) epigallocatechin (-EGC), (-) Epigallocatechin gallate show potent antioxidant activity. 73, 74 Epidemiological studies have shown that the intake of tea catechins decrease the risk of coronary heart disease, stroke and cancer. 75-77 Hence analysis of radical scavenging activity of the phenolic compounds present in different types of teas (green tea, oolong tea, black tea) has gained in importance. Different techniques such as the oxygen electrode method, 69 High performance liquid chromatography, 70 Electron spin resonance spectrometry, 71 Nuclear magnetic resonance 72 etc are used to analyze the scav enging activity of catechins on free radicals. As mentioned earlier, ep igallocatechin gallate (EGCG), the most abundant catechin in tea catechins, is shown to have the most effective s cavenging activity among the tea catechins. 67 The ortho trihydroxy group in the B ring and the galloyl moiety attached at 3 rd position contribute to the strong scavenging activity of Epigallocatechin gallate. 68

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Figure 1.11: Structure of Catechins (68) To elucidate the molecular mechanism underlying the radical scavenging and antioxidant activities of antioxidants, (+)catechin was reacted with 1, 1diphenyl-2picrylhydrazyl (DPPH; stable free radical) as a model reaction. Th e reaction mixture is analyzed by C13 NMR. Two new carbonyl peak s were detected in the spectra, and the disappearance of characteristic peaks of the B ring was also observed, suggesting that two hydroxyl groups in the Bring are more impor tant in radical scavenging activity among the 4 phenolic hydroxyl groups of catechin and the B ring was changed to a quinone structure. Signals ascribable to A and C ring remained unchanged. 72 11

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12 AC B Figure 1.12: Structure of Catechin Experimental results have shown that the galloyl moiety in epigallocatechin gallate is more important in radical scavenging activ ity than the hydroxyl groups on the B ring. 72 Figure 1.13: Galloyl Group Therefore catechins are useful in the prevention of di seases caused by oxygen radicals. Consumption of green tea can decrea se the cholesterol ab sorption, can decrease the body weight by interfering with sympathoadren al system, inhibit the low density lipid

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13 oxidation (LDL), antithrombotic activities and decrease the systolic and diastolic blood pressure. 79 Catechins may ameliorate the impairments or injuries caused by intracellular active oxygen (Senile disorders), and might b ecome useful for pr otecting human from Alzheimers disease. 78 EGCG, the most abundant catechin of gr een tea catechins, has se veral health benefits. However the beneficial effects of EGCG on human diseases are inconclusive, therefore epidemiological studies on prot ective action of green tea and EGCG yet to be explored 1.3 Crystal engineering 1.3.1 Introduction Crystal engineering the design of solids 62 is target oriented and property directed synthesis of molecular crystals. 80 Traditionally, crystal engineering focuses on the relationship between the struct ure and properties of solids. Currently this concept is expanded into diverse areas such as material chemistry, supramolecular chemistry, molecular recognition and biology. 81 The term Crystal engineering was introduced by Pepinsky (1955) in the context of crystallization of organic i ons with metal complexes. 63 Schmidt applied the concept of crystal engineering in th e context of solid state photodimerisation reactions. 64 Modern Crystal engineering can be de scribed as an interdiscip linary subject, owing to its implications for organic, inorganic, metal organic, theoretical and materials chemistry. 65 A more useful definition for crystal engineer ing is provided by Desiraju, who illustrates

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14 crystal engineering as a fiel d with broader discipline des igning crystals with desired properties. Definition Crystal engineering is the understanding of intermolecular interactions in the context of crystal packing and in the ut ilization of such understanding in the design of new solids with desired physical and chemical properties. 62 Gautam Desiraju. The concept of crystal engineering has been successfully exploited in the synthesis of porous solids 82 and ion exchange materials. 83 Crystal engineering strategies are applied in the design of pharmaceuticals, 38 photographic materials, 84 and in novel optical, electronic and magnetic materi als. 1.3.2 Pharmaceutical Crystal Forms The majority of the drug substances are solid dosage forms, most of which contain active ingredient in a crystallin e state. The inherent stability of crystalline materials and the well established impact of crystallizati on process on purification and isolation have made the chemists and the engineers to de liver the pharmaceutically active compounds in crystalline form. 31 The physical properties of the cr ystal such as purity, size and shape are determined by the operating conditions of crystallization process, in turn these properties determine the efficiency of the operations filtration, drying, formulating and also product effectiveness such as bioavail ability and shelf-life Drug dissolution and toxicity are affected by solid st ate phase and purity of the product, this leads to batch to

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15 batch uniformity and consistency. Therefor e, improved control of crystallization processes can achieve better cr ystal product quality, shorter pr ocess time and elimination of compromised batches. 32 Pharmaceutical crystals may be chir al or achiral and they may exist in different forms, salts, solvate or hydrate and polymor phs. The existence of a crystal in different crystalline phases results in polymorphs. 40 Solvates are Molecular complexes that have incorporated the crystallizing solvent molecule in their crystal lattice. 41 When a crystal lattice incorporates water, it is designated as a hydrate. Approximately one third of pharmaceutical substances are capable of forming crystalline hydrates. 42 Solvates and hydrates demonstrate different solubilities and dissolution rates when compared to their unsolvated counter parts. 43 The stability of the hydrates and solvates at different temperatures and different vapor pressure differ from their unsolvated forms. 44 These differences can influence the stability of the pharmaceutical substances under different storage conditions. The physical and chemical properties of a crystal depend upon the arrangement of the molecules in it and the physiochemical pr operties of the solid drug can affect its performance. Thus a study of the crystalline state can lead to an understanding of the drug properties which are important for preformulation and formulation. Different crystal packing changes the periodicity of the molecules which may in turn change physical properties of various crystal forms. 40, 45 Therefore, different solid forms can show different physical and chemical properties. Pharmaceutical drugs with different solid

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16 forms can lead to phase transformations dur ing processing and formulation, such as in theophylline, 46 carbamazepine, 47 Phenobarbital, 48 lactose 49 etc. The stability and bioavailability of the drug are affected by these phase changes. 50 Therefore, an understanding in relationship between the solid state properties and cr ystal structures can be utilized for optimizing fo rmulation strategies and in designing suitable stability protocols. 51, 52 Different solid forms of excipients, used in pharmaceutical formulations, can also affect the final p hysical form of a tablet. 53, 54 Opposite enantiomers of chiral drugs, show different pharmacological, toxilogical, pharmacodynamic and pharmacokinetic properties. This is due to the molecular environment, in which these solids exist. 55, 56 The presence of ions in pharmaceutical salts influences the physiochemical propert ies of the crystals, like dissolution rate, stability, solubility and hygroscopicity. 57 The structure of the crystal also affects mechanical properties, for example the intermolecular hydrogen bonds in theophylline m onohydrate results in higher mechanical strength and less brittle than th e anhydrous form of theophylline. 58 Similarly, the presence of water molecules in the monohydrat e of 4-hydroxy benzoic acid facilitates its plastic deformation. 59 Therefore, it is possible to pr edict the mechanical properties from its crystal structure. Different crystal forms of drug substa nces with different physiochemical properties can also affect scale up and transfer from laboratory quantities and procedure through pilot plant and full production. 60 The characterization of crystal forms plays a major role in quality control and regulatory processes. 61

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17 Physiochemical properties including mechan ical properties of crystalline drug are determined by its molecular arrangement, packing, conformation and intramolecular interactions in its lattice. These physiochemical prope rties affect the pharmaceutical properties of the drug product. In order to predict the properties of pharmaceutical crystals a thorough unde rstanding of the underlying crys tal structure is desirable. 40 1.3.3 Patent Relevance of Crystal Forms Patents are a mechanism for promoting resear ch for societys bene fit: the right to exclude others from practicing a patented i nvention affords an economic incentive to the inventor, while the limited term of the exclus ionary right ultimately delivers the invention into the public domain. 87 Andrew V. Trask Patent can be defined as A license conferring the sole right to manufacture, sell, or deal in a product or commodity; (now) spec. a license from a government conferring for a set period the sole right to make, use, or sell some process or invention. Oxford English Dictionary Criteria for obtaining a patent: A product or process must have novelty (not part of the s tate of the art), utility (must serve some worthwhile practical use), and must not be obvious (competent but without imagination). A product must be of human ingenuity, and should contain an enabling description. M.J. Zaworotko

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18 Newness of the drug product can be attributed not only for a new bioactive chemical form, but also for a different solid state form (polymorphic, amorphous or chiral), combination with other solids (carriers, co ating, excipients), a nd different delivery method or even for a different propor tion of the drug in this combination 85 are patented. Crystallization methods for a par ticular solid state form, new drugs, related manufacturing, and formulation aspects are protected by patents. Examples Co-crystallization process (patent number; WO0053283 Authors; Reuter Karl, Reuter Chemische Apparatebau) A process for preparation of urea complexe s of vitamin E and its esters (Patent number; IN182620 Authors; Bajaj Vikas; Madan Anil Kumar ) High-throughput formation, identification, and analysis of diverse solid-forms (patent number; 20030162226, Authors; Cima, Michael J.; Levinson, Douglas; Lemmo, Anthony V.; Galakatos, Nicholas; Putnam, David A.; ) Novel Conazole crystalline forms an d related processes, pharmaceutical compositions and methods (patent number; WO03101392, Remenar Julius; MacPhee Michael; Peterson Matthew Ly nn; Morissette Sherry L; Almarsson Orn ) Crystal forms of the chemical substances are patented on their powder X-ray diffraction pattern, because the powder pattern of a crysta lline form is recognized as a finger print of it. Chemical substances can exist in more than one crystal form; this phenomenon is referred to as polymorphism. As different polymorphs of the same drug can exhibit

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widely different solubility and bioavailab ility properties, polymorphism became an immediate concern for pharmaceutical industr ies during patent filing. A polymorph of a drug can be entitled to a differe nt patent protection, as it has a different legal entity. For example, the two polymorphs of Zantac (ranit idine hydrochloride) are patented by GlaxoWellcome. Ranitidine hydrochloride 86 Figure 1.14: Form I Figure 1.15: Form II Powder pattern for polymorphs of ranitidine Figure 1.16: Powder X-ray diffraction of form I and II 19

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20 As methods in generating the new patents guar antee the future financ ial security of the company, a complete screening and charac terization of single crystal forms and development of corresponding cr ystallization techniques should be carried out as early as possible.

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21 2. CSD STATISTICS 2.1 Cambridge Structural Database (CSD) Cambridge Crystallographic Data Centre (CCDC) was establishe d by Olga Kennard at Cambridge University in 1965. Cambridge structur al data base is the principal product of the CCDC. 33 The CSD comprises software for data base access, structure visualization, data analysis, and structural knowledge bases derived from the scientists world wide. It records X-ray and neutron diffraction data of organic and metal organic compounds. It also records bibliographic, chemical a nd crystallographic information of these compounds. Crystallographic information incl udes single crystal studies and powder diffraction studies. Crystal st ructure data arising from the publications in the open literature and from private communica tions are also included in CSD. CSD software includes ConQuest (search program), Mercury (visualiser), Vista (statistical analysis), and Pre quest (database creation) (33). 2.2 Supramolecular chemistry Supramolecular chemistry 90 which is also known as chemistry beyond the molecule 91 is based on the selective recognition of molecules which interact via non covalent forces to form well organized assemblies. During the 1960s and 1970s much of the supramolecular chemistry res earch, involved in host guest systems 91 for selective

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22 binding of small alkali metal cations by macrocyclic receptors. The fascination for molecular recognition phenomenon inspired chemists in further exploration of supramolecular systems in the context of w eak intermolecular interactions such as hydrogen bonds. 92-98 2.3 Supramolecular synthons The term synthon was introduced by Corey in 1967 (99). Corey defined synthon as; Structural units within the molecule s can be formed or assembled by known or conceivable synthetic operations. The term synthon is traditionally used in representing the key structural features in target molecule in organic synthesis. Crystal engineering has emerged around the idea of establishing and later utilizing the intermolecular interactio ns to govern the crystal packing with reasonable predictability. In this respect the term supramolecula r synthon was introduced by Desiraju in 1995. 34 It is defined as a structural unit within the supermolecule which can be formed and/or assembled by known or conceivable intermolecular interactions. Supramolecular synthons are also called as motifs 100 or patterns. 101 They can also be regarded as regions within a crystal structure where the rec ognition between constitu ent functional groups occurs. 102 Supramolecular synthons can be separated in to two distinct categories: Supramolecular homosynthon; which results from interaction of identical selfcomplementary functionalities 35 and supramolecular heteros ynthon which results from interaction of different but complementary functionalities 35 Examples of supramolecular homosynthons include carboxylic acid and amide dimmers, 103,104 while supramolecular

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heterosynthon include acid-pyridine, 35 acid amide 105-108 and hydroxylpyridine. 109-111 Figure 2.1 represents the acid-acid dimm er (homosynthon) and acid pyridine (heterosynthon). Figure 2.1 Examples of supramolecular ho mosynthon (left) and supr amolecular heterosynthon (right) Supramolecular homosynthons tend to be observed in single component crystals, but their existence can also be observed in mu ltiple component crystals in which each component has at least one identical f unctional group. Furthermore when multiple functional groups are present the formati on of a supramolecula r heterosynthon is possible. Supramolecular heterosynthon can ex ist between the func tional groups located on two different molecules with same chemical make up or on the two different molecules with different chemical make up. In terpretation of existi ng crystal structures with complete knowledge on in terplay between supramolecula r synthons would help in designing new multicomponent crystals. 2.4 CSD Statistics for Polyphenols The polyphenolic nature of the flavonoids pr ovoked an interest to conduct a survey on intermolecular interactions of phenols and polyphenols in the CSD. Phenols and polyphenols are evaluated by considering the following constraints, no ions, only 23

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organics, R factor: <=0.075, stru ctures containing 3D coordi nates. As EGCG contains ether, ester and alcohol groups, while the two known solvates of it contain acetonitrile and nitrobenzene, studies are conducted considering these functional groups. There are 6,032 phenols in CSD as of May 2007. Out of 6,032 phenols 1,005 are polyphenols. O O H H O O H H Figu re 2.2: Structure of polyphenol considered in the CSD search R O H O R O H O O R O H R O H I II III Figure 2.3: Synthon I; OH---OH, Synthon II OH---CO, Synthon IIIOH----O(ether) interactions, (R:C 6 H 5 OH) 24

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In CSD analysis, the frequency of occurrence of supramolecular synthons hydroxyl to hydroxyl (synthon I), hydroxyl to carbonyl of ester (synthon II) and hydroxyl to ether (synthon III) in phenols and polyphenols is evalua ted. In order to determine appropriate distance ranges, within which homo and hete rosynthons exist, distance histograms were generated. Based on visual inspection of the resulting interactions in the crystal structures included in the histograms, the lower and higher cut offs for hydrogen bond distances were determined. The histograms (Fig 0.2) revealed that hydroxyl to hydroxyl synthon (I) occurs within a distance range of 2.6-3.0 Supramolecular synthons II (hydroxyl to carbonyl of an ester) and III (hydroxyl to et her) exist within a range of 2.56-2.94 and 2.5-3.0 respectively. Figure 2.4:Histograms of contacts for supramol ecular synthons I (OH---OH), II (OH---CO) and (OH----O) III 25

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O N O R R O H R O H N C IV V Figure 2.5: Synthon IV represents OH---CN an d Synthon V represents OH---NO interaction Similarly hydroxyl to cyano group Synt hon (synthon IV as represented in figure 2.5) and hydroxyl to nitro group synthon (synthon V as represented in figure 2.5) were evaluated. Histograms revealed the distance ranges within wh ich these synthons occur are 2.73.2 and 2.78 3.06 respectively. Figure 2.6 : Histograms of contacts for supramolecular synthons IV 26

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27 Phenols Polyphenols Synthon I (OH---OH) 836 hits/6,032 14.8% 385 hits/1,005 38.3% Table 2.1: Frequency of occurrence of synthon I in phenols and polyphenols Phenol + Ester OH--CO (ester) 2.562.94 Phenol+ ether OH---O (ether) 2.5-3.0 Phenol + Cyano group OH---CN 2.7 -3.2 Phenol + Nitro group OH---NO 2.783.06 Phenols 799 hits/6,032 phenols 225 hits/799 2,074 hits/6,032 199 hits/ 2,074 187 hits/ 6,032 74 hits/ 187 370 hits/ 6,032 83 hits/ 370 Polyphenols 191 hits/ 1,005 polyphenols 81 hits/191 281 hits/1,005 50 hits/281 38 hits/ 1,005 21 hits/38 31 hits/ 1,005 11 hits/31 Table 2.2: Frequency of occurrence of synthons from II to V in phenols and polyphenols Table 2.2 represents th e frequency of occurrence for s ynthons II to V in the presence of the respective functional groups with phenols and polyphenols. Though the presence of ethers with phenols and polyphenols is high in comparison with other functional

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28 groups, the occurrence of synthon III is relative ly lower compared to the other synthons. Supramolecular synthons D----A () Mean () OH----OH 2.6 -3.0 2.80 OH---CO (ester) 2.56-2.94 2.771 OH---O (ether) 2.5-3.0 2.805 OH----CN (cyano) 2.7-3.2 2.873 Table 2.2.3: Distance ranges within which different synthons exists This analysis has revealed that the fr equency of occurrence of hydroxyl to hydroxyl synthon (synthon I) is higher in phenols (14.8%) and polyphenols (38.3%), when compared to other synthons (II, III, IV and V). The prevalence of the specific supramolecular synthons in these crystal struct ures would provide a valuable insight for crystal engineering strategies in generati on of new multicomponent materials comprised of polyphenols.

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29 3. EXPERIMENTAL DATA Some solvents were obtained from Sigma Aldrich. Distillation of few solvents was performed in the lab itself, such as meth anol, ethanol, dichloro methane, chloroform. Distilled water is used. EGCG was provided by Dr. Roland Shytle. 3.1 Infrared Spectroscopy (IR) A Nicolet Avtar 320 Fourier transform In frared spectrometer was used for collecting the IR spectra of the samples (sample con centration 2mg). The spectra were measured over the range of 4000 400cm -1 Data were analyzed by using EZ Omnic software. 3.2 Differential Scanning Calorimetry (DSC) Thermal analysis of the samples was performed on TA instrument DSC 2920 equipped with liquid nitrogen cooling. The samp les of crystals (2mg-7mg) were placed in aluminum pans, and were scanned at 10 C/min in the range 25 350 C under a dry nitrogen atmosphere (flow rate 70ml/min). The data were managed by TA universal analysis. 3.3 Powder X-ray Diffraction (PXRD) PXRD patterns were collected on Br uker AXS D 8 powder diffractometer with a Cu K radiation (1.540 56). The tube voltage a nd amperage were set at 50kV and 40mA, respectively. Each sample was scanned between 3 and 40 in 2 with a step size of 0.02.

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30 The experimental PXRD patterns and calcu lated PXRD patterns from single crystal structures were compared to confirm the composition of materials. 3.4 Single Crystal X-Ray Crystallography Crystals of forms II, III and IV were examined under a microscope and suitable single crystals were selected for X-ray analysis Data were collected on a BrukerAXS SMART APEX CCD diffractometer with monochromatized Mo K radiation ( = 0.71073 ) connected to KRYO-FLEX low temperature device. Data were collected at 100 K. Lattice parameters were determined from leas t square analysis, and reflection data were integrated using the program SAINT. Lorent z and polarization corre ctions were applied for diffracted reflections. In addition, th e data was corrected for absorption using SADABS. Structures were solved by direct methods and refined by full matrix least squares based on F 2 using SHELXTL. All non-hydrogen atoms were refined with anisotropic displacement parameters. A ll H-atoms bonded to carbon atoms, except methyl groups, were placed geometrically a nd refined with an isotropic displacement parameter fixed at 1.2 times Uq of the atoms to which they were attached. N or O bonded protons, as well as H-atoms of methyl groups were located from Fourier difference map and refined isotropically based upon the corr esponding N, O or C atom (U(H)=1.5Uq(N, O)).

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3.5 Form I 90% pure EGCG provided by Dr.Roland D ouglas Shytle is considered as form I. Up to the date the reported form of egcg in li terature is form I. This is confirmed by PXRD. 88 3.5.1. Physical properties Melt-Temp: 220 C, Soluble in ethanol methanol, ethyl acetat e, acetonitrile, acetic acid, formic acid. It is insoluble in chloroform cyclohexane, toluene, and hexane. Egcg is sensitive to light, so it is stored at temp 28C. 3.5.2 IR of Form I The IR of form I matc h with the IR found in literature. 89 767.73 829.53 956.59 1014.94 1067.34 1145.47 1219.05 1291.37 1343.87 1446.30 1543.54 1615.14 1689.93 3354.16 82 84 86 88 90 92 94 96 98 100 %Transmittance 1000 2000 3000 4000 Wavenumbers (cm-1) Figure 3.1: IR of Form I 31

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3.5.3 DSC of Form I Phase transitions are observed at 126.43 C and 227.71C. DSC matches with the DSC in the literature. 88 Figure 3.2: DSC of Form I Figure 3.3: DSC of Form I from Literature 32

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3.5.4 Powder X-ray Diffraction of EGCG form I PXRD of 90% pure EGCG matches with PXRD of EGCG obtaine d in the literature. Major peaks; 90% egcg (literature) 5.32 (5.72), 8.63 (8.63), 10.48 (10.4), 12.24 (11.998), 17.14 (16.993), 20.86 (20.656), 24.541 (24.66), 29.68 (29.653). Figure 3.4: PXRD of Form I Figure 3.5: PXRD of Form I from literature 33

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3.6 Form II 3.6.1 Synthesis 65mg of 90% pure epigallocatechin gallate (0.141 moles) dissolved in 1ml of acetonitrile (99% pure) is laye red on 2.5 ml of dichloromethane and allowed to stand in refrigerator. After 72 hours colorless platel et crystals (Form II) were observed. An increase of 3-fold and 5-fold in contents has shown same results. Form II was also crystallized from acetonitrile and chloroform. 3.6.2 Physical properties MeltTemp: 235-238C. Solubility proper ties are observed to be similar to that of Form I. Crystals are stable up to four days at temperature 2 to 8 C. Upon heating at 120 C for twenty minutes, solvent is evaporated and Form II is converted to Form IV. 3.6.3 IR of Form II IR of form II shows O-H stretch 3323.8 cm-1, C=O stretch at 1605 cm-1, and aromatic carbon stretch at 742.4 cm-1. 742.49 824.22 987.29 1033.83 1097.22 1140.11 1208.03 1242.12 1315.48 1381.08 1449.94 1514.43 1535.38 1605.01 3323.81 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 %T 1000 2000 3000 4000 Wavenumbers (cm-1) Figure 3.6: IR of Form II 34

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3.6.4 DSC of Form II Phase transitions are observed at 101.43 C and 252.71C. Figure 3.7: DSC of Form II 3.6.5 TGA of Form II Below 100 C 16.68 % weight loss is observed. 20.20 % weight loss is observed between 230 C. Figure 3.8: TGA of Form II 35

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3.6.6 Powder X-ray Diffraction of form II The experimental PXRD matches with the calculated PXRD as shown in fig 3.11. Major peaks, experimental (calculated): 7.12 (7.12), 7.8 (7.92), 8.46 (8.56), 13.04 (13.3), 15.24 (15.28), 23.54 (23.86), 25.92 (25.44). Figure 3.9: Experimental PXRD of Form II Figure 3.10: Calculated PXD of Form II 36

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Figure 3.11: Calculated Vs Experimental PXRD of Form II 3.6.7 Picture of Single crystal Figure 3.12: Single crystal of Form II 37

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38 3.6.8. Single Crystal X-ray Crystallography Data Molecular formula: C 24 H 21 N O 11 ; Formula weight: 499.42; Crystal system: Monoclinic; Space group: C2; Unit cell dimensions: a = 22.876(3) b = 14.9054(17) ; c = 15.8886(18) =90 ; =102.688(2) ; = 90; Volume: 5285.4(10) 3 ; Z=8; Temperature:100(2) K; Dens ity (calculated): 1.433 Mg/m 3 ; (Mo-K ): 0.71073 ; Reflections measured: 12985; Independent reflections: 10165 [R(int) = 0.0277]; Final R indices [I>2sigma(I)]: R1 = 0.0831, wR2 = 0.1993; R indices (all data): R1 = 0.1013, wR2 = 0.2144 3.7 Form III 3.7.1 Synthesis In a typical reaction, a reaction tube with 6m l of dichloromethane is taken, and 1ml of nitrobenzene is layered on it. 200mgs (0.436m moles) of 90% pure EGCG dissolved in 2.5ml of acetonitrile is layered on nitrobenzene. Reaction test tube is left on refrigerator, after 24 hours yellow needle like crystals were observed 3.7.2 Physical properties Melt Temp: 235 -238; Solubility properties are similar to that of Form I. Form III crystal are more stable compared to Form II crystals at temp 28 C. Upon heating to 120 C for twenty five minutes, Form III converts to Form IV.

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3.7.3 IR of Form III 3272.3 cm -1 (O-H stretch), 1681.33 (N-O stretch) are observed. 816.22 848.86 1014.74 1031.06 1148.22 1193.97 1313.36 1513.08 1599.20 1681.33 3272.13 86 87 88 89 90 91 92 93 94 95 96 97 98 99 %T 1000 2000 3000 4000 Wavenumbers (cm-1) Figure 3.13: IR of form III 3.7.4 DSC of Form III Phase transitions are observed at 140.10C, 169.26 C, and 253.52C. Figure 3.14: DSC of Form III 39

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3.7.5 TGA of Form III 11.98 % of weight loss is observed at 131 C, 25.31 % of weight loss is observed at 211.36 C is observed. Figure 3.15: TGA of Form III 3.7.6 Powder X-ray Diffraction of form III The experimental PXRD matches w ith the calculated PXRD as shown in fig 3.18.Major peaks, Experimental (calculate d): 13.4 (13.4), 15.4 (15.4), 19.4 (19.44), 24.3 (24.4), 27.8 (27.65), 28.5 (28.5), 31.3 (31.3), and 37.6 (37.5) Figure 3.16: Experimental PXRD of Form III 40

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Figure 3.17: Calculated PXRD of Form III Figure 3.18: Calculated Vs Experimental PXRD of Form III 41

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3.7.7. Picture of single crystal Figure 3.19: Single crystal of Form III 3.7.8. Single Crystal X-ray Crystallography Data Molecular formula: C 28 H 25 N O 14 ; Formula weight: 599.49; Crystal system: Orthorhombic; Space group: P2(1)2(1)2(1); Unit cell dimensions: a = 13.2070(14) b = 13.2134(14) ; c = 14.5781(16) = = = 90; Volume: 2544.0(5) 3 ; Z=4;Temperature:100(2) K; De nsity (calculated): 1.565 Mg/m 3 ; (Mo-K ): 0.71073 ; Reflections measured: 11442; Independent reflections: 4964 [R(int) = 0.0608]; Final R indices [I>2sigma(I)]: R1 = 0.0605, wR2 = 0.1126; R indices (all data): R1 = 0.0787, wR2 = 0.120 42

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3.8. Form IV 3.8.1 Synthesis Form IV is obtained by heating form II and III at 120 for 20-25 minutes. Form IV crystals: EGCG (45mg, .098mmoles of form IV powder obtained by heating form III at 120 C for 25 minutes) is dissolved in 1ml of acetonitrile (99%pure, Aldrich). The solution is layered on 2.5ml dichloromethane (distilled and stored over molecular sieve) and seeded with form IV crystals obtained by heating form III (at 120 C for 25 minutes) and was allowed to stand in refrigerator. After one week colorless needle like crystals were observed. 3.8.2. Physical Properties Melt-Temp: 232-235. Solubility properties are similar to that of form I. 3.8.3. IR of Form IV 3466.81cm -1 (O-H stretch), 1606.19 (C =O) stretch are observed. Figure 3.20: IR of Form IV 43

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3.8.4 DSC form IV Phase transition is observed at 250.08 C Figure 3.21: DSC of Form IV 3.8.5 TGA form IV 30.02 % of weight loss is observed at 251.78 C. Figure 2.22: TGA of Form IV 44

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3.8.6 Powder X-ray Diff raction of form IV The experimental PXRD matches with the calculated PXRD as shown in fig 3.24. Major peaks (experimental): 13.9 (13 .84), 17.2 (17.12), 21.2 (21.16), 23.6 (23.64), 26.5 (26.6), 29.0 (29.16), and 31.5 (31.7). Figure 3.22: Experimental PXRD of Form IV Figure 3.23: Calculated PXRD of Form IV 45

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Figure 3.24: Calculated Vs experimental PXRD of Form IV 3.8.7 Picture of Single Crystal Figure 3.25: Single crystal of Form IV 46

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47 3.8.8 Single Crystal X-ray Crystallography Data Molecular formula: C 22 H 18 O 11 ; Formula weight:458.36; Crystal system: Monoclinic; Space group: P2(1); Unit cell dimensions: a = 13.006(10) b = 5.686(4); c = 13.089(10) = 90;=107.062(11) ; = 90; Volume: 925.4(12) 3 ; Z=2; Temperature: 100(2) K; Dens ity (calculated): 1.645 Mg/m 3 ; (Mo-K ): 0.71073 ; Reflections measured: 2623; Independent reflections: 2550 [R(int) = 0.0312]; Final R indices [I>2sigma(I)]: R1= 0.0641, wR2 == 0.1494; R indices (all data): R1 =0.0800, wR2 = 0.1674

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4. RESULTS AND DISCUSSION 4.1 Structure Description EGCG structure; EGCG consists of 4 rings (A, B, C, D) with 8 hydroxyl groups, as shown in Fig4.1. In all forms of EGCG the A and C ring lie in one plane while rings B and D lie in different planes with different dihedral angles. EGCG makes full use of all hydroxyl groups to form different synthons such as hydroxyl to hydroxyl, hydroxyl to ether, hydroxyl to carbonyl of ester, hydroxyl to cy ano and hydroxyl to nitro. A C B D Figure 4.1: Structure of EGCG 4.1.1 Form II The crystal structure of form II was obt ained from Single crysta l X-ray diffraction as described in the experimental section. Form II solvate of acetonitrile, crystallizes in the 48

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49 onoclinic C2 space group with two egcg and two acetonitrile molecules and some to n between, the two hydroxyl groups of the D two m disordered solvent molecules in asymmetric unit. Analysis of the structure of form II indicates the presence of four types s ynthons. They are hydroxyl to hydroxyl (OH--OH), hydroxyl to ether (OH---O), hydroxyl to cyano group (OH---CN), and hydroxyl carbonyl of ester (OH---CO) synthons. The OH---OH hydrogen bond exists i ring of molecule I and one hydroxyl group of B ring of molecule II. The OH---OH hydrogen bond is observed between hydroxyl gr oup on the B ring of molecule I and hydroxyl groups of D ring of molecule II and also in between the hydroxyl group on the A ring of molecule I and the hydroxyl gr oup of A ring on molecule III. The hydrogen bond distances in synthon OH----OH are 2.999, 2.733, 2.753, 2.838, and2.603 The distances are consistent with the CSD analysis (2.6 3.0). Figure 4.2: Representation of OH----OH synthon in Form II Molecule II Molecule I Molecule III

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50 he OH---O (ether) hydrogen bond exists between the oxygen of the C ring and the hydroxyl of the D ring. The hydrogen bond dist ance in OH---O synthon is 3.026 which is consistent with the CSD analysis (2.5-3.0). in form II The OH---CO (ester) hydrogen bo olecule I and the hydroxyl on the D ring of molecule II and also with th e two hydroxyl groups on the B ring of molecule II. The hydrogen bond distances in synthon OH---CO are 2.847, 2.924, and 2.680. The distances are consiste nt with the CSD analysis (2.56-2.94). T Figure 4.3: Representa tion of OH---O synthon nd exists between the ester carbonyl of m

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51 Molecule I Molecule II Molecule III Figure 4.4: Representation of OH---CO synthon in form II The OH---CN hydrogen bond exists between the hydroxyl group on the B ring and cyano group of acetonitrile molecule. Th e hydrogen bond distance in the synthon OH--CN is 2.898 which is consistent with the CSD analysis (2.7-3.2 ). Figure 4.5: Representation of OH---CN synthon in form II

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Acetonitrile molecules are embedded in between egcg molecules and sustained by hydrogen bonding. Figure 4.6: Solvent en trapment in form II Unit cell packing of form II includes 8 egcg molecules, 8 acetonitrile molecules, and disordered solvent molecules. Figure 4.7: Unit cell packing of form II 52

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53 4.1.2 Form III Form III solvate of nitrobenzene crysta llizes in orthorhombic P2 (1) space group with one egcg molecule, one nitrobenzene and one water molecule in asymmetric unit. Analysis of form III crystal indicated the pr esence of four types of synthons. They are hydroxyl to hydroxyl (OH---OH), hydroxyl to carbonyl of ester (OH---CO), hydroxyl to nitro (OH---NO) and hydroxyl to oxygen in water (OH---O). Figure 4.7 represents the OH----OH interactions of molecule I and molecules II, III, IV and V. One of the hydroxyl group on the A ring of molecule I a nd hydroxyl groups on the B and D ring of molecule V form hydrogen bond. The other hydroxyl group of A ring in molecule I forms hydrogen bond with the hyd roxyl group on the B ring of molecule II. The hydroxyl group on the B ring (of molecu le I) forms hydrogen bond with hydroxyl group on the B ring of molecule III. The two hydroxyl groups on D ring (molecule I) form hydrogen bond with hydroxyl group on the A ring of molecule IV and also with hydroxyl group on the B ring. The hydrogen bond distances in the synthon OH----OH are 2.910, 2.827, 2.831, 2.827, 2.910, and 2.982. The distances are consistent with the CSD analysis (2.6-3.0).

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54 Molecule I Molecule II Molecule III Molecule IV Molecule V Figure 4.8: Representation of OH----OH synthon in Form III The OH---CO (ester) hydrogen bond exists betw een the carbonyl of the EGCG molecule and the hydroxyl group on the A ring of a nother EGCG molecule. The hydrogen bond distances in OHCO synthon is 2.671 which is consistent with the CSD analysis (2.56-2.94). Figure 4.9: Representation of OH---CO synthon in Form III

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The OH---NO hydrogen bond is observed between the hydroxyl group of B ring and nitro group of nitrobenzene. Hydrogen bond distance in the synthon OH---NO is 2.773 which is consistent with the CSD analysis (2.73.2). Figure 4.10: Representation of Synthon OH---NO in Form III The OHO hydrogen bond is observed between the hydroxyl groups on B ring and oxygen of water molecule. The hydrogen bond distances in the synthon OH---O (water) are 2.837, 2.957, and 2.610. Figure 4.11: Representation of Synthon OH---O in Form III 55

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Nitrobenzene molecule is sandwiched betw een two egcg molecules and sustained by hydrogen bond. Figure 4.12: Solvent entrapment in form III Unit cell of form III contain 4 egcg molecules, 4 nitrobenzene molecules and 4 water molecules Figure 4.13: Unit cell packing of Form III 56

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4.1.3 Form IV Form IV of egcg crystallizes in monoclinic P2 (1) space group with one egcg molecule in asymmetric unit. Form IV exhi bits three types of synthons. They are OH--OH, OH---CO (ester), and OH----O (ether). Figure 4.14: Representation of Form IV structure The OH---OH hydrogen bond in form IV exists between the hydroxyl groups on A ring of molecule I and hydroxyl group on B and D rings of molecules VI, and IV respectively. The hydroxyl groups on the D ring of molecule I exhibits OH---OH hydrogen bond with hydroxyl group on the A and B rings of molecules IV and III respectively. The hydroxyl group on the B ri ng of molecule I exhibits the OH---OH hydrogen bond with hydroxyl group on the A ring of molecule II. The hydrogen bond distances in the synthon OH---OH are 2.801 2.697, 2.696, and 2.960. The distances are consistent with the CSD analysis (2.6 -3.0 ). 57

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Molecule I Molecule II Molecule III Molecule IV Molecule V Molecule VI Figure 4.15: Representation of OH---OH synthon in Form IV The OHO (ether) hydrogen bond exists between the hydroxyl group of the B ring and the oxygen in the C ring of another mo lecule. The hydrogen bond distance of synthon OH---O is 2.875 which is consistent with the CSD analysis (2.5-3.0). Figure 4.16: Representation of OHO synthon in Form IV 58

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The OH---CO (ester) interaction is observed between the carbonyl of molecule I and the hydroxyl groups on the B and D rings of molecules II and III respectively. The hydrogen bond distances in the synthon OH----CO are 2.813, 2.839, and 2.879. The distances are consistent with the CSD analysis (2.56-2.94). Molecule I Molecule II Molecule III Figure 4.17: Representation of OHCO synthon in FormIV 59

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Unit cell of form IV cont ain two egcg molecules Figure 4.18: Unit cell packing of Form IV 4.2 Conformational analysis Conformational flexibility in the EG CG molecule was previously analyzed by proton NMR. 115 It includes the orientation of the linkage between B, D and C ring owing to the puckering of C ring. The conformational change s in C ring with respect to the B and D rings, gives rise to 4 conf ormers (A, B, C, and D). Conformer A Conformer B Conformer C Conformer D R 1 = Pseudoequitorial R 1 = Pseudo axial R 1 = Pseudoequitorial R 1 = Pseudo axial R 2 = Pseudo axial R 2 = Pseudoequitorial R 2 = Pseudoequitorial R 2 = Pseudo axial 60

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Figure 4.19: Representation of different conformations of EGCG (116) The pseudoequitorial and pseudoaxial po sitions of the B and D rings with respect to the C ring in forms II, III, and IV suggest th at they exist as conf ormer A. The puckering of the C ring generates the pseudoequitorial a nd pseudoaxial positions of rings B and D. 61 Form III Form IV 1 and C2) on C ring exists in S, S, for form II and R, R for C1 C2 C1C2 C1 Form II The two chiral centers (C C2 forms III, IV respectively.

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624.3 Discussion Although flavonoids are abundant and co mmercially available they are difficult to purify and crystallize. In this respect developing new crystal forms of EGCG was challenging. Different crystallization techniqu es were explored in crystallizing EGCG, ch as solvent evaporation, vapor diffusion, slow cooling, and solv ent layering. Solvents hniques were ethanol, me thanol, toluene, cyclohexane, hexane, 1, 4nd to sults Figure 4.20: LC-MS analysis of 90% pure EGCG su used in these tec dioxane, dimethylsulfoxide, dimethylform amide, chloroform, dichloromethane, acetonitrile, nitrobenzene, benzene, and wate r. Solvent layering technique was fou be the most successful technique in crystall izing EGCG. As menti oned in chapter 3, two solvates and a new form of EGCG were obtained by solvent layering technique. Liquid chromatographyMass spectrometry (LC-MS) was used to evaluate the purity of crystals of the two solvates a nd the new form of EGCG. The LC-MS re have revealed the presence of impurities. An impurity with mass/charge ratio 443 (retention time 13 minutes), was observed in all three forms.

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Figure 4.21: LC-MS analysis of Form II 63 Figure 4.22: LC-MS analysis of Form III

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64 Figure 4.23: LC-MS analysis of of Form IV is of all four forms of EGCG.LC-MS e; EGCG: 7.609 (Mol.wt of EGCG: 458.4), Impurity: 13.039 Figures 4.20-4.23 represents the LC-MS analys Data: Retention tim minutes. Retention time Bulk Form II Form III Form IV 7.609 459 (M+1) 481 (M+23) 459 (M+1) 459 (M+1) 459 (M+1) 13.039 443(M+1) 443 (M+1) 443 (M+1) 443 (M 481 (M+38) 506 (M+63) 481 (M+38) +1) 523 (M+80) 506 (M+63) 506 (M+63) Table 4.1 : Representi ng the components eluted at different retention times

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65 Here in ble 4.1, bul represents th e 90% pure EGCG. LC-MS results have revealed forms, EGCG was elut inutespurities after es. Identities of impurities were not determined. gh cou analysis of the existing supramolecular synthons in the new crystal forms can provide an gscule men ents when designing c C hap lete knowledge on interplay between the supramolecula r OH, OH---CO, OH---O, OH--, OH---O) would help to develop a strategy in choosing the co-crystal formers. ta k that from a ll four ed after 7m and the im 13minut Thou crystallizatio n techniques ld not produ ce 100% pure EGCG, a careful educated uess to choo e other mole with comple tary compon o-crystals. Co nsider ing the SD analysis (c ter 2), a comp synthons (OH CN

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665. CONCLUSION In summary the study presented here in involves the synthesi s of different crystal rms of EGCG with limited CSD analysis on supramolecular synthons that exist in these rystal forms. In crystallizing EGCG the technique known as solvent layering was found be the most successful technique. Two solvates, with acetonitrile and nitrobenzene, nd one new form of EGCG is reported. A tota l of five types of synthons exist in the rystal forms of EGCG. Synthons I (OH---OH), and II (OH---CO (ester)) exist in all three rms (II, III and IV). The absence of synt hon III (OH---O (ether)) in form III suggests at the occurrence of an OH---O (water) synthon in th e presence of a competing cceptor, ether moiety, is relatively high. Synthons IV (OH---CN) and V (OH---NO) xists in form II and form III respectively. All the five synthons occur within the distance ranges which are consistent with the CSD analysis. e s bient fo c to a c fo th a e Though EGCG shows profound benefi ciary effects on health, it suffers from the problem of poor bioavailability. This study, on structural features of EGCG, could potentially provide valuable information for structural modifications, which may change the physiochemical properties of EGCG. One of the future as pects of this study could b the design of co-crystals of EGCG. A brief description for co-crystals is provided here A broad definition for co-crystal is given by Dunitz as Crystals containing two or more components together. 112 In a more specific way others 113,114 defined co-crystal a a multiple component crystal formed between compounds that are solid under am

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67 ith g materials, due to being new and s herefore compounds like caffeine and theobromine conditions: at least one component is molecular and forms a supramolecular synthon w the remaining components. Co-crystals ex hibit different physiochemical properties (such as bioavailability, solubility) from thei r startin distinct solid state structures. To design co-crystals of EGCG co-c rystal formers can be chosen based on CSD analysis. A small survey which represents the competitiveness between aromatic nitrogen and the carbonyl of an ester group to form a hydrogen bond with the hydroxyl moiety on phenols is conducted in the CSD. Results have revealed that 36 hits were present containing hydroxyl, ester, and aromatic nitrogen groups. Out of these 36 hits, 14 hits contain the OH---N (arom) synthon, 7 hits contain the OH---CO (ester) synthon, and 4 hits contain the OH---OH synthon. All three synthons occur within the distance range of 2.53.0 (OH----N (arom) ), 2.56-2.94 (OH---CO (ester) ) and 2.6-3.0 (OH---OH) respectively. The distances are estimated fr om histograms generated by the CSD. Thi study has revealed that th e occurrence of the OH----N (arom) synthon is relatively more favored than other synthons. T (which contain aromatic nitr ogen) could potentiall y act as good co-crystal formers.

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Crystallization studies of epigallocatechin gallate
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ABSTRACT: Flavonoids are a long and well known class of natural products. Their potential health benefits can be attributed to their antioxidant activity, and modulation of cell signaling pathways. Green tea one of the most widely consumed beverages, consist of flavonoids such as catechins and tannins. Epigallocatechin gallate (EGCG) the major catechin of green tea exhibits multiple health benefits due to its antioxidant nature. The radical scavenging activity of EGCG is attributed to its structure. Therefore, a study on molecular features of EGCG would provide valuable information on structural modifications, which may change the physiochemical properties such as bioavailability and solubility. Although flavonoids are abundant and commercially available they are difficult to purify and crystallize. In this respect, crystallizing EGCG was challenging. By exploring different techniques EGCG was crystallized. Here in this study one new form of EGCG and two solvates, acetonitrile and nitrobenzene, have been synthesized and structurally characterized by differential scanning calorimetry (DSC), infrared (IR) and powder X-ray diffraction (PXRD). The crystal structures were solved by single-crystal X-ray diffraction and a detailed description of synthesis and about the supramolecular synthons that exist in these crystal forms are given.
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EGCG.
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