/ ^omc/ly BA1.0S k , HOOKBINniNG f- HiiRvicr I tfd HHONn HQAO Ok.BnNiii.juMcrios K Permission has been granted by the Head of the School in which this thesis was submitted for it to be consulted ^^^ and copied This permission is contained in the Administration file "AYailability of H.D. Theses" and applies only to those theses lodged with the University before the use of Disposition Declaration f orms, SOME REACTIONS OP ANTEOCYAKIlf PIGMENTS A Thesis submitted for the degree of Master of Science of the University of New South Wales hy Kevin Anthony Harper B.Sc. (Hons.) Submitted January 1961 UNIVERSITY OF N.SJ. 27878 26. FEB.75 LIBRARY DECLARATION The candidate, Kevin Anthony Harper, hereby declares that none of the work presented in this Thesis has "been submitted to any other University or Institution for a higher degree. AOKNOWLEDG-MENTS The author wishes to record this thanks to Dr. E. R. Cole of the UniTersity of New South Wales and to Mr. J. P. Kefford of the C.S.I,R»0, Bivision of Pood Preservation for their consider- able help and guidance in the work described in this Thesis. He is also deeply indebted to Dr. J. R. Vickery, Chief of the O.S.I.R.O. Division of Pood Preservation, for permission to carry out this work in the Division's laboratories. He also wishes to express his gratitude to Dr. B. V. Chandler for many helpful criticisms and discussions of the work and to Mr. D. J. Oasimir for his assistance in settin^g up the polarograph and instruction in the technique of polarography. SUMMARY Part 1 The pigment from blackcurrant fruit was separated into seven components on a cellulose column. They were identified as cyanidin, delphinidin, their -3-monoglucosides and -3- rutinosides and a reddish-brown decomposition product. Special techniques were used to remove the biose, rutinose, for identification by paper chromatography. Further purification of the pigments on a polyamide column was necessary prior to sugar identification to remove carbohydrate contaminants. Oyanidin- 3-rutinoside was prepared by the reduction of rutin. Part 2 (a) The polarographic behaviour of synthetic peonidin chloride was examined over a wide pH range. In strongly acid solutions the half- wave potential (-0.765 V.) was independent of pH and the reaction involved a single electron. At pH 3.05 the half-wave potential increases linearly with pH and an anodic wave appears. This inflection point corresponds to the pK of the flavylium ion - pseudo-hase transformation. The reaction still involved one electron. At pH 8.25 another inflection point occurred cor- responding to ionization of the phenolic groups. The half-wave potential increased more rapidly with pH since the negatively charged molecule resists addition of electron^. Concurrent spectral studies assisted in fonmilating the electrode reactions. ("b) Exploratory easperiments on anthocyanin degradation indicate that only of the antho- cyanin would be retained in "blackcurrant 3am manufacture. The presence of 150^ sugar decreased the anthocyanin retained after 20 minutes "boiling. In contrast with previous work on strawberry anthocyanins oxygen had no significant effect on degradation. Better colour retention in "blackcurrant pulp compared with ^uice was considered due to release of anthocyanin from the tissue during "boiling. TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 PART I THE IDENTIFICATION OF THE ANTHOCYANINS OF BLACKCURRANT FRUITS Introduction 7 Previous Investigations 10 Cyani din-rhamnoglucoside 12 Identification of the Nature and Position of Sugar Su"bstituents 15 Experimental Materials and Apparatus 20 Blackcurrants 20 Cellulose Column . 20 Polyamide Column , 20 Spectrophotometry 21 Methods Extraction of the Pigment 21 Separation of the Pigments 22 Purification on a Polyamide Column 25 Paper Chromatography of the Anthocyanins . 25 Paper Chromatography of the Sugars 27 Acid Hydrolysis 29 Partial Hydrolysis 29 Enzymic Hydrolysis 30 Peroxide Oxidation 31 Quantitative Estimation of the Sugars 32 Examination for Acyl Groups 3h Preparation of Reference Materials 3h Cyanidin-3-rutinosid.e 3k Cyanidin-3-monoglucoside 37 Gyanidin~3,5~diglucoside 38 Delplainidin 39 RESULTS AUD DISCUSSION kO Band A k^ B k^ C h^ D ki E ki P 33 G 30 CONCLUSIONS 5k ADDEICDA 57 BIBLIOGRAPHY 59 PART 2 (a) THE POLAROGRAPHIC BEHAVIOUR OF ANTHOCYANINS Introduction 63 Literature Survey 6k Reduction of the Anthocyanins Sk Experimental Apparatus 69 Supporting Electrotete 69 vii Experimental (continued) Page Anthocyanin Solution 70 Method 72 Results 79 Pi scussion 88 (a) pH less than 3.05 88 Calculation of "n" and the Diffusion Coefficient 89 Mechanism of the Reduction 92 (h) pH 3.05 to 8.25 93 Reaction Characteristics 93 Formation of the Pseudo-base 96 Evidence for Chalcone Formation 98 Reduction of the Chalcone 99 Oxidation of the Chalcone 10U Formation of the Anhydro Base 106 Oxidation and Reduction of the Anhydro-hase 107 (c) pH greater than 8.25 107 CONCLUSIONS 113 APPENDIX I 115 Polarographic Cell 115 Circuit 115 Sensitivity 115 Calibration 116 Mercury 116 APPENDIX 2 121 Synthesis of Peonidin Chloride 121 BIBLIOGRAPHY 123 PART 2 (b) EXPLORATORY INVESTIGATIONS OF THE DESTRUCTION OP ANTHOCYANINS DURING PROCESSING Introduction 127 Literature Survey 128 viii Literature Survey (continued) Page Effect of Tengperature 128 Oxygen and Oxidizing Agents 131 Sugars 13^1. Other Factors 136 The Relative Stability of Anthocyanins 136 Quantitative Estimation of Anthocyanins 138 Experimental Blackcur raits 1M Pulp IM Serum 1U1 Buffer 114.1 Optical Measurements 11+1 Methods Estimation of the Anthocyanins 114.2 (a) Sulphite Procedure 1k2 (h) Peroxide Procedure 11+2 (c) Comparison of Procedures 1k2 Anthocyanin Degradation in Blackcurrant Pulp Blackcurrant Pulp 1U5 Pulp plus Added Sugar 1U6 Degradation in Serum 1i+7 Serum plus Added Sugar 11+7 Results and Discussion Effect of Heat Treatment 151 Effect of Added Sugar 152 Effect of Oxygen 156 CONCLUSIONS 159 BIBLIOGEAPHY 160 HtHnH imm. OP FIGURES PART I Page Figure 1 2k " 2 35 " 3 h2 h k3 5 kS " 6 51 part 2 (a) Figure 1 71 2 7k 3 80 k 81 5 82 6 86 7 87 8 9 118 10 119 11 120 PART 2 (ID) Figure "1 ^k3 " 2 3 15U k 157 TABLES PART I Page Table 1a • • 28 tf 1b • • 28 ti 2 • • 32 II 3 * • 36 PAHT 2 (a) Table 1 • • 75 2 • • 76 3 • • 77 h • • 78 5 • • 83 6 • • 100 PART 2 (b) Table 1 • • 11+8 II 2 • • 1i+9 II 3 • • 150 GENERAL INTRODUCTION Arithocyanins are a grcmp of naturally occurring glycosides of 2-phenylbenzopyrylium salts (f) whose parent aglycones are Imown as anthocyanidins. Only the six listed are widely distributed in nature differing from each other in the degree of hydroxylation and subsequent methozylation of the 2-phenyl- or "B" ring. Hydroxylation of the phenyl "A" ring almost always conforms to the pattern shown. The only exceptions so far found to this rule are 7-methoxymalvidin (hirsutidin), i4.',5,7-trihydroxy"benzopyrylium (gesneridin) and 6-hydroxy~ cyanidin. H=:E' =H Pelargonidin r t , R=:OH:R'=H Cyanidin OH R=OMe:R«=H PeoAidin R=R'=OH Delphinidin J R=:OMe:R»=OH Petunidin Rs^R'sOMe Malvidin In~^the anthocyanins glycosylation always occurs at position 3 and sometimes at position 5 as well hut never at 5 alone. Gesneridin derivatives are primarily glycoslated at the 5 position since this anthocyanidin lacks an hydroxy 1 at position 3. Sugars found have "been glucose, rhamnose, xylose and galactose, either alone, or combined as disaccharides as in pelargonidin-3'- rhaBmoglucoside-5-monoglticoside recently identified in Solamun i&^hereja. Orgaiiic acids may also "be present in the naturally occurring anthocyanins attached to the sugars or to the free phenolic groups as esters; malonic acid and sereral substituted cinnamic acids hare been found combined in this Although the structure of the anthocyanidin(T) is generally written as the oxonium salt with the positive charge located on the oxygen atom it is now recognized that this form exists in resonance with two other forms in which the charge is associated with carbon atoms 2 (II) and U (III) respectively^-(32^ )\ The positively charged ions are stable only in strongly acid solutions to which they impart a bright red colour. rfto nr'io 1 ^ I The pyrylium salt is in equilibrium, in aqueous solutions. with the pseudo-base(IV) and dilution of an acid solution causes a marked loss of colour as equilibrium is shifted in favour of pseudo-base formation; a pK of 2.98 has been recorded for tliis reaction with pelargonidin-3- mozLoglucoside. By raising the pH of the solution hoth these forms can be conrerte^o the anhydro-hase modification(V) which imparts a violet colour to the approximately neutral solution. In alkaline solution the deep "blue phenates are formed (VI). yc DM H»0 "frfO^m HX OH IV OH OH OH V The anthocyanins form part of a mach wider group of compounds, the flaTonoids, all of which possess the same "basic structure hut differ in the degree of oxidation of the heterocyclic ring. It has heen found that inter- conyersion may occur "between different types of flavonoids but their purpose in the plant organism is still a matter of con;)ecture. The complete mode of biosynthesis of flaronoids is still unknown although some plausible theories have been advanced^"^'. Anthocyanins are of particular interest to the food k technologist, since they are respcnsi'ble for the attractive colours of many edilsle fruits and berries. Tlie instalDility of these compounds during the processing and su"bsequent storage of such foods makes it desirable to learn more about tbeir chemical behaviour, so that the effects of processing conditions can be better understood. In any investigation of anthocyanin degradation, identification of the individual pigments present is always the first step.