Some Reactions of Anthocyanin Pigments

^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

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 considerable 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 corresponding 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 anthocyanin 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 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. It is important to know exactly what pigments are being studied since a lead can often be obtained to the type and extent of degradation which can be expected. For example evidence has been found^^^^ that more rapid oxidation of the delphinidin glycosides than the cyanidin glycosides occurs in blackcurrants, and so it might be anticipated that materials containing delphinidin derivatives would be more susceptible to colour loss on heat treatment in the presence of oxygen than those which contain cyanidin derivatives. This thesis is divided into two portions. Firstly the anthocyanins in the fruit of the blackcurrant (gibes nigrum) have been identified by the use of microchemical techniques involving chromatography and spectrophotometry. In the course of this work a number of reference conqpounds were isolated from other natural sources and one, cyanidin- 3-rutinoside, was prepared synthetically by the reduction of rutin. In the second part some of the properties of the anthocyanin molecule are examined. The polarographic "behaviour of an anthocyanin has "been investigated and a preliminary study has also "been made of the rate of degradation of blackcurrant anthocyanins under conditions which occur daring the processing of anthocyanin containing foods. PART I

THE IDMTIFICATIQN OF THE A3STH0CYANINS

OF BIACKCUHBAHT FRUITS INTBQDUOTION

The complete identification of an anthocyanin requires identification of the aglycone, the sugar group or groups present and their positions of attachment, and the identification of any acyl groups which may he attached to the anthocyanidin or the sugar. Early procedures involved extraction and purification of the pigment on a scale large enough to give quantities of material which could he studied hy the classical methods of organic chemistry. The purification consisted largely in repeated selective precipitation of the pigments from solution as picrates which were then suhgected to acid hydrolysis and alkaline degradation and the products identified "by comparative analyses. Such procedures established that the naturally occurring anthocyanins were hased upon the three anthocyanidins : pelargonidin, cyanidin and delphinidin.

Pelargonidin Cyanidin Delphinidin

Eohinson and co-workers^^^^ contributed greatly to anthocyanin chemistry by working out a general scheme for their syntlieBis which allowed the preparation of authentic reference ccxnpounds. This allowed the properties of all the anthocyanidins and a large nuniber of their glycosides to he tabulated and a series of coloar tests were devised by means of which many of the naturally occurring anthocyanins were identified. Although these colour tests made the identification of anthocyanins Emch easier there still remained several difficulties. Firstly, the interpretation of such tests as the colour given by the addition of soditim acetate to a solution of the anthocyanin was too subjective, since it depended upon differentiating between shades of colour, e.g. **blue-violet" and "violet-blue". Secondly, since the anthocyanins are structurally similar to each other, separation, when they occurred as a mixture in the plant extract, was very difficult by the methods then available and very often mixtures were described as a single pigment. This aspect is well illustrated in the present work on blackcurrants, in which only one pigment was originally identified, whereas a complex mixture of six anthocyanins is actually present. These difficulties were largely overcome by the application of paper partition chromatography to the field by Bate-Smith and Westall^^^^ a technique which allows an anthocyanin mixture to be readily separated into its component parts. In "brief, the present day method of anthooyanin identification is to chroraatograph an acid extract of the plant material on paper, in several solvent systems, and to observe the rnmber of spots and their positions on the paper. Some of the extract is then liydrolysed with 20 percent hydrochloric acid and an amyl alcohol 1 extract chromatographed to identify the aglucone/s present. The aqueous portion of the hydrolysate is likewise examined hy paper chromatography to identify the sugars split off by hydrolysis. The results of these examinations together with the Rf values should give a good indication of the identity of the anthocyanins present. The procedure is presented schematically "below. Conclusive identification is obtained "by comparative chpcsnatography, in a number of solvent systems, with an authentic reference anthocyanin with which identity is suspected. The reference anthocyanins are obtained either synthetically or from plant sources where they have "been positively identified. However, where such reference samples are not available or a mixture of anthocyanins is present, use must be made of a number of other techniques to resolve the mixture and obtain evidence for positive identification. The work to be described in the following pages, on the identification of the anthocyanins in blackcurrants, serves to illustrate this latter statement.

Blackcurrants were chosen for this study because they contain relatively large quantities of colouring matter and it was thought that they might provide a good source of material for future degradation studies.

PHEVIOUS BaVESTIQATIOHS The first attempt to identify the anthocyanins of (x^) blackcurrants was that of Robinson and Robinson^"^ using the colour test technique, in the course of a systematic survey of the plant kingdom. It was concluded that the skins of blackcurrants contained a cyanidin-3~bioside together with much co-pigment. Oyanidin was the only anthocyanidin identified "but it was

thought that delphinidin might also he present.

At the eommenc^ait of this work it had heen shown'(5 )

that the pigment from blackcurrants could he resolved

chromatographically into seven distinct components^

as follows :

BASD PEOPBRTIES IDMTIFICATIOK

1 Brown colour. Moved with solvent Degradation front product

II Ghromatographically identical Oyanidin with Oyanidin

III Ghromatographically identical Delphinidin with Delphinidin

IV Hydrolysed to Oyanidin and aiucose. Tentatively Partially hydrolysed to Oyanidin- Oyanidin- 3~monogluc0side diglucoside

V Hydrolysed to Oyanidin and Glucose. Oyanidin-3- Ohromatographically identical with monoglucoside Oyanidin-3-monoglucoside

VI Hydrolysed to Delphinidin and Tentatively Glucose. Partially laydrolysed Delphinidin- to VII- diglucoside

VII Hydrolysed to Delphinidin and Tentatively Glucose Delphinidin- monoglucoside

The subsequent discussion concerns the cc^plete characterization of hands IV, VI and VII as a result of which revised structures are presented. Using paper clircsmatography Fouassin^®^ carried oat a survey of the anthocyanins of a large immber of fruits of economic ii^ortance and also shoired the presence of two cyanidin glycosides and two delphinidin glycosides in "blackcurrant extracts. No attempt was made in this worls: to identify or compare the glycosides thoaselves hut their Ef values in a number of solvent systems were listed. (5) The previously published Rf values were compared with those of Pouassin and it was found that the Rf values published for cyanidin-3-iaonoglucoside (band V) were in agreement with those given by Pouassin for one of the cyanidin glycosides. The values for the second cyanidin glycoside (band IV) were also in agreement and corresponded with cyanidin glycosides present in Prunus arium, P. st>inosa and Sambucu^ nigra. The pigment frcan these fruits has previously been shown to be a cyanidin rhamnoglucoside (^0,30,39 ) ^ Cyanidin-rhamnoglucoside In view of the possibility that this second cyanidin glycoside from blackcurrants could he cyanidin-rhamnoglucoside, a search was made of the literature for a possible source of this anthocyanin for use as a reference material. Cyanidin-rhamnoglucosides have also been isolated from P. Serrulata^^^^. Canna generalis^^^\ Antirrhinum ma jus and Linaria vulgaris^^^\ Ribes sanguineum^^^^. Plea (2-^) (314.) euroDaea ^ and Syzygium caaiJal . However in no case lias the pigment been fully characterized as a di-monoside

or a "bioside. Although they are in most cases considered

to "be "biosides, "by reason of their colour reactions, there

is no chemical evidence to establish their correct

structure. Cyanidin-rhamnoglucoside has never been

reported to have been synthesised so that a direct

comparison of these pigments with one of proven constit-

ution has not been made. These pigments were therefore

considered to be unsuitable for comparison work, and it

was decided that synthesis and chemical evidence would

be necessary for complete characterization of the pigment.

Bauer, Birch and Hillis^^^ reduced rutin (quercetin-

3-rutinoside) to an anthocyanin which they considered

may be identical with antirrhinum, the pigment from

Antirrhinum ma jus, and keracyanin, the pigment from the

Prunus species mentioned previously. The flavonol,

present as the acetate, was reduced using lithium-

alTiminium-hydride in tetrahydofuran. The initial

reduction product required treatment with cold dilute

hydrochloric acid to produce the anthocyanin. The

reaction was considered to be a reduction of the flavonol

decaacetate (VII) to the l|.-hydroxy-compaund (VIII). a

form of the anthocyanin leucobase, which was converted by cold dilute acid to the anthocyanin (IX). fOhc.

i-Rutifloit. SA/ Acetate OAt. 0 OH H ^OM vri VIII

CI OH voO H -Riftinosc JX

The constitution of rutin has "been well established so that its reduction product is well suited for comparative work. Attree and Perkin^^^ methylated rutin with diazomethane and then r^oved the sugar "by- hydrolysis to give 5,7,3',i+'-tetramethoxyflavan-3-ol thereby establishing position-3 as the site for the sugar group. The nature of the sugar was established by Zemgl&D. and Gerecs^^^ by splitting off rutinose enzymically and converting it to a heptaacetyl derivative found to be identical with heptaacetyl rhamnoside-6-fl^-glucose. Eutin is therefore rhamno6ido-6-c:^-glucosido)-quercetin and its reduction product must be cyanidin-3-rutinoside. The only gap in the knowledge of the structure of this compound is whether the sugar is linked to the aglycone through an oC or /3 linkage. Identification of the Nature and Position of Sugar Suljstituents Acid hydrolysis is used to liberate the sugar from an anthocyanin for identification Toy paper chromatography. However> if a disaccharide is present it will he split into its component sugars hy this method and it will not he then known whether the sugars are present in the anthocyanin molecule as a hiose or individually on different carhon atoms, e.g. cyanidin-3»3~diglucoside. A number of methods have been used to overcome this problem, of which the most readily performed is the spectral method presented by Harborne^^^^ who showed that the absorption spectra of anthocyanins carrying unsubstituted hydroxyl groups at position 5 have a shoulder to the main peak at about 1^40 rap and that this shoulder is missing when glycosidation of this hydroxyl occurs. This difference may be eapressed by comparing the ratio of the optical density at with that at the wave-length of maximum absorption. With 5- substituted anthocyanins the ratio was found to be approximately half that of the iinsubstituted derivative. Thus the average ratio of cyanidin and its 3-glycosides was 0.23 while the 5-glycosides averaged 0.12. Methylation of the anthocyanin followed by acid hydrolysis has also been used to locate the positions of sugar attachment. For instance Nordstrom (26) used this method in the identification of 3-glucosido-5- arabinosidocyanidin in Dahlia variabilis, Methylation was effected "by dimethyl-sulphate in sodima hydroxide solution. In alkaline solutions the heterocyclic ring of the anthocyanin (X) is opened and the chalcone (XI) formed "but only when the 5-hydroxyl group is "blocked hy a sugar molecule can the anthocyanidin methyl ether (XII) be reformed during acid hydrolysis. Reformation of the

X" + .OH ,OCH, Ho Y^oh

^OH OCH, YU red anthocyanin colour after acid hydrolysis of the methylation product is therefore an indication that the anthocyanin is a 5-glycoside» In order to investigate further glycosidation in the aromatic nuclei the methylated anthocyanin was subjected to alkaline degradation (boiling sodium hydroxide solution for U hours) followed by acid hydrolysis and the phenolic and acid components identified by paper chromatography using synthetic standards for comparison. Phloroglucinol dimethyl ether (XIII) and veratric acid (XIV) were identified so that none of the other positions in the molecule could have been glycosylated. HOOtV^^VocHj

OH ML >oy

Although these methods reveal the positions of sugar attachment they do not assign the positions of the individual sugars when more than one is present. Partial hydrolysis has "been used to give information of this nature. Nordstrom^^^^ showed cyanidin-3-glurcoside to "be one of the products from the partial hydrolysis of the Dahlia anthocyanin discussed ahove thus locating the glucose molecule at position 3. Harborne^^^^ subjected one of the anthocyanins of the potato to partial hydrolysis, and obtained the 3-glucoside, 3,3-diglucoside and 5-glticoside of cyanidln plus scxne unchanged pigment. Hhamnose was the only other sugar fbund in the acid hydrolysate and its position of attachment was decided by finding cyanidin-3-rhamnoglucoside as a product of enzymic hydrolysis. The agljrfeone/glucose/rhamnose ratio was 1 :2:1 so that the pigment must have been cyanidin- 3-rhamnogluG o si de-5-monoglucosi de. The enzyme used in this investigation was an "anthocyanase" isolated from certain pectinase preparations and certain species of Asnergillus^'^^^ and had been found by Harbome and Sherratt^^^^ to hydrolyse monosides more rapidly than biosides. Thus with cyanldin-3~rhaninoglucoside-5- monoglucoside the 5-glucose was removed more rapidly than the 3-rhamnoglucose and the latter could "be detected in the partially hydrolysed material. Another enzyme that has found an application in investigations of this nature is "rhamnodiastase", an enzyme which is capable of hydrolysing rhamnoglucosides without further hydrolysis of the disaccharide. Charaux^^^ was the first to isolate this enzyme from the seeds of Rhamnus utilis and used it to hydrolyse the flavonol, rutin, into quercetin and rutinose. Similar enzyme activity was also noted in extracts of R. cathartica. R. frangula and infectoria L. Use was made of this enzyme "by Z^aplen and Qerecs^^^ to remove the sugar rutinose from rutin and establish its structure. This enzyme preparation has not previously "been applied to anthocyanin identification hut it will he shown in this work that it is a very useful aid in establishing the identity of sugar residues. A purely chemical method is also available for removing the sugar residue from position 3 of an anthocyanin without hydrolysing a di saccharide if present. The anthocyanin is oxidised with hydrogen peroxide which opens the C2-C3 bond of the heterocyclic ring to give a product of the "malvone" type (XV). Asimonolysis of this compeand splits off the sugar at position 3 without interfering with any other sugars which may he present in the molecnle. After removal of this sugar, as a phenylhydrazone, the residue may he subjected to acid hydrolysis for identification of

O-R

R and R c sugar5

Ho OH o fy 0 + c II / NH O-R'

sugars present at other sites in the molecule. This reaction was originally investigated by Karrer (18) and de Heuron and although it has been proposed as a useful method in anthocyanin identification (35) n has not been used in any recent investigations. In the present work it forms the basis for the identification of two of the blackcurrant anthocyanins. EXPERIMEIiTTAL

Materials and Apparatus Blackcurrants. The "blackcurrants used in these investigations were the variety "Whitebud", grown in Tasmania and harvested during the 1954-55 season. The fruit was held at -10°C in internally lacquered U-gallon cans until required for use. Cellulose Column. The cellulose column used for separation of the anthocyanins was packed from Whatman standard grade cellulose powder, which was first sieved to obtain the fraction larger than 200 mesh and finer than 100 mesh. A slurry, prepared "by "blending the powder with the organic phase from equal volumes of hutanol and 2N hydrochloric acid and then holding under vacuum for several minutes to remove air "bubbles, was packed into a glass column 50 cm x ii-.Ii. cm having at the bottom a sintered glass disc (porosity 3) upon which rested about half a centimetre of clean sand. As the slurry settled it was firmly packed in place, half a centimetre at a time, with a stainless steel tamper. After packing, the column was washed with a solution of 8-hydroxyquinoline in the solvent (2x10 ml). Polyamide Column. The polyamide columns were prepared from a sample of polyamide - "Celite" mixture* prepared from "Nylon" pellets by dissolving in formic

Prepared and donated by Dr. B. V. Chandler il acid and precipitating onto "Gelite" ty the slow addition of alcohol. The mixture was made into a slurry with a mixture of ethanol and hydrochloric acid (1:1) and packed into a column 20 cm. x 1 cm. "by allowing it to settle. Spectrophotometry. With the exception of the spectra shown in Fig. 5 which were recorded on a Beckman model "DU" spectrophotometer, all optical measurements were made on a Beckman model "B" spectrophotometer.

Methods Extraction of the PiiSgnent. Blackcurrants (500 g.) were thawed and macerated in a blendor with methanol containing hydrochloric acid (1 1.). After standing for about 30 minutes the deep red solution was filtered under light vacuxim. The residue was re-extracted up to six times "by which time most of the pigment had been removed. The bulk of the solvent was r^oved from the combined extracts by evaporation at ii-O" C in a rotary vacuum evaporator. The anthocyanin was then precipitated ai the blue lead salt by the addition of a saturated solution of lead acetate until no further precipitation occurred. The precipitate was removed by centrifuging and washed once with twice its volume of alcohol. The lead salt was decomposed by suspending, with vigorous stirring, in the minimum quantity of methanol and slowly adding hydrochloric acid until all the "blue complex had decomposed. The precipitate of lead chloride was filtered off and the filtrate evaporated, at less than W^C, under vacuum to a thick syrup which was dissolved in methanol (100 ml.)* The anthocyanins were then precipitated as a gummy residue "by adding slowly, with shaking, to anhydrous ether (500 ml.). After redissolving in methanol and reprecipitating in this way from four to six times a product was finally obtained as a light red powder which was filtered off, washed with ether and dried in an Abdorhalden drying pistol at 65^0. The final precipitate was very hygroscopic and rapidly turned to a gum if exposed to the atmosphere for more than a few minutes. The dry, crude anthocyanin was rapidly packed in tuhes, tightly stoppered, and stored in the refrigerator until required.

Separation of the Pigments. The crude anthocyanin was dissolved, with gentle warming, in lDutanol/2N hydrochloric acid so as to form a saturated solution. Some aqueous phase separated during this operation and was removed hy centrifuging. The saturated solution (30 ml.), representing about 0.5 g. pigment, was placed on the top of the cellulose column, allowed to soak in, and the chromatogram developed with the solvent. The eluate was collected in 10 ml. portions in a fraction collector. The optical density of each fraction was measured at 5k5 lEji. (diluting with solvent where necessary) and plotted against tu"be number to give the graph shown in Fig. The eluates representing each hand were concentrated "by two different methods. The slower running hands D, B, F, and Q (Fig, 1) were mixed with an equal volume of acetone and absorbed on a second cellulose column (IiOcm x 2 cm). The solvent was washed off with acetone ard the anthocyanin eluted with methanol containing 0.1% hydrochloric acid which could be readily concentrated at low temperature in the rotary vacuum evaporator. The faster running bands. A, B and C, were not retained sufficiently strongly by the cellulose column and were concentrated by precipitating them as the lead salt. Basic lead acetate (saturated solution) was added to the eluate until no further precipitation occurred. The precipitate was filtered off, washed with alcohol, dried, and then decomposed in methanol by the addition of hydrochloric acid. The solution was filtered to remove the lead chloride and evaporated under vacuum to dryness. In later work the procedure was simplified by the acquisition of a second type of rotary vacuum evaporator which allowed a much higher vacuum to be obtained through the use of a mechanical p\imp. With this apparatus the eluates, direct from the column, could be quickly evaporated to dryness at less than 23^0. The residues, in all cases, were further purified by solution in agueous hydrochloric acid and the addition CM

cij M Pt,

I20 TUBE of concentrated hydrochloric acid to "bring the final concentration up to 2095. After sereral days storage in the refrigerator precipitation of the anthocyanins was coH^ilete and the solid was filtered off "by means of a small filter paper (k mm.) on the end of a capil3ary tuhing connected to a vacmtim pfomp.

PariHcation on a poiyamii^ft nf>iTim-n. it was found necessary to purify farther the separated anthocyanins "by passage through a polyamide column. The solid material, obtained from the cellulose column, was dissolved in the minimum amount of solvent (ethanol- I9S hydrochloric acid (1:1) ) and applied to the top of the coltimn, and the chromatogram developed with more solvent. The coloured eluate was evaporated in vacuo to give a purplish-hrown solid which was used directly for identification of the sugar residues.

Paper Chromatography of the Aathocyanins. An aqueous or alcoholic solution of the material to -,"be chromatographed was spotted on a line drawn 3i in. from one end of strips of TOiatman No. 1 filter paper, 19 cm, long. The spots were dried in a current of warm air and the application continued until sufficient material was present to give a spot which was easily visible. The papers were hung from troughs in glass "battery ^ars, containing aome solvent in the bottom, and allowed to eguili'brate for several hours. Solvent was then added to the trough and irrigation continaed until the solvent had travelled a distance of 17 - 18 ins. The paper was then removed and air dried.

The following solvent systems were nsed:-

For anthocyanidins (the presence of hydrochloric acid in the solvent was necessary to prevent fading) :

1. The organic phase obtained hy mixing equal

voltuaes of n-hutanol and 2S hydrochloric acid.

2. Porrestal Solvent: acetic acid, water, hydro-

chloric acid in the proportions hy volume 30: 10: 3.

For anthocyanins. The ahove solvents together with the following :

3. The organic phase from the mixing of n-butanol,

acetic acid, water in the proportion by volume

if: 1: 5.

if. The organic phase from mixing m-cresol, acetic

acid, water in the proportion by volume hS: 2: 50.

5* Aqueous hydrochloric acid 1%.

Rt values were calculated using the distance from the origin to the leading edge of the spot.

All chromatography was carried out in a constant temperature room at 20^0.

The Rf values of the blackcurrant anthocyanins are presented in Table la and some values from the literature for cyanidin and delphinidin glycosides in 1b. 2Z

Paper Chpomatograp3ay of the Sggars. For separation of the sugars the solvent was regiiired to run off the paper and the ends were therefore given a serrated edge

"by trimming with pinking shears. Irrigation was continued for 2k hours. Glucose and rhamnose were run concurrently on each paper as reference sugars.

The solvents used were "butanol^ acetic acid, water

(i|.:1:5) and phenol, water (4:1).

The sugar spots were detected "by spraying with aniline phosphate reagent and heating at 110®G for 5 minutes.

The reagent was prepared according to the method of Ash and Reynolds ^^^ "by adding 9S% orthophosphoric acid (3 mlO to a solution of aniline (6 ml.) in "butanol saturated with water (600 ml.).

To test the sensitivity of this method, standard solutions of glucose and rhamnose were spotted onto paper in amounts varying "between 0.5 pg. and 5 pg. and it was found that the i^ot from 0.3 pg* glucose was ^st visible after development and spray treatment while 1 jig. rhaimose was required to give a spot of equal intensity. The difference was due to the fact that rhamnose travels farther on the paper than glucose ^d spreads out more thereby diminishing in intensity. TaTjle 1a Rf values, in various solvent systems, of blackcurrant anthocyanins and some cyanidin and delphinidin glycosides Rf. in Solvent Anthocyanin 1 2 3 k 5 B 0.73 0.14-5 Fades Fades C 0.1+3 0.31 Fades Fades D 0.30 0.76 0.38 0.37 0.22 £ 0.27 0.71 0.39 0.1+7 0.13 F 0. 22 0.69 0.25 0.15 0.16 G 0.18 0.63 0.19 0.17 0.05 Gyanidin-3- glucoside 0.26 0.72 0.36 0.U7 " -3,5-diglucoside 0.10 0.27 0.23 H "Z rutinoside 0.30 0.76 0.38 0.36 0.21+ Delphinidin 0.I|.2 0.31

Table 1b Gyanidin 0.69c 0.50"b » -3- glucoside 0.25^,0.27® 0.38^,0.33® 0.23^ 0.07^ " —3—i*hamno- glucoside 0.25^0.28® 0.37^,0.37® 0.25® 0.19^ " -3-diglucoside 0.22^,0.22® 0.33^,0.29® 0.18® 0.31+^ " -.3,5-ai- glucoside 0.06^,0.08® 0.28®,0.16® 0.19® 0.16®

"-3-rhamno-5- glucoside 0.08® (i 0.25® 0.36® Delphinidin 0.35® 0.30^ A "-3-glucoside 0.11+®,0.11® 0.16®,0.26® 0.11® 0.03

glue 0 side 0.06®, 0.03® 0.11®,0.15^ 0.03® 0.08® "-.3-Piiamno- glue0side 0.15^ 0.30® 0.11®

I. Organic phase of butanol/2N hydrochloric acid, 1:1. II, Acetic acid/water/hydrochloric acid, 3:10:3. III. Organic phase of butanol/acetic acid/water, :5. IV. Organic phase of m-cresol/acetic acid/water, 14.8:2:50. V. One per cent aqueous hydrochloric acid. a. Harborne, J.B., J.Chromatography 1, hl3 0958). b. Parkinson, T.L., J.Sci.Food Agr. 239 (195U). c. Bate-Smith, E.G., Biochem.Soc.Synrp. (Camb. )No.3>62(1930). d. Bate-Smith, E.G., Nature, 176. 10l6 (1955). Acid Hydrolysis, The anthoeyanin (generally aT3oat

0.1 mg.) was added to 20^ bydroehloric aeid (2 ml.) and heated in a "boiling water bath for 5 minutes. fhe aglycone was extracted "by washing with amyl aloohol

(2x1 ml.) and the extract spotted directly onto paper for chromatography.

The aqueous residue was passed through an ion exchange column (8 cm. x 1 cm. of "Amherlite IHliB") in the hydroxyl form. The washings were collected, evaporated to small volume (0.5 ml.) and spotted onto paper, together with known sugars, for chromatographic analysis of the sugars.

Only glucose could be detected by this method in all the blackcurrant anthocyanins as obtained from the cellulose column unless spotted heavily on the paper when a faint spot for rhamnose could be detected. After passage through a polyamide column bands E and Q gave only glucose while D and F gave rhamnose and glucose.

Partial Hydrolysis. The method of Nordstrom using boiling 1% hydrochloric acid for 30 minutes, was applied to the diglycoside anthocyanins from blackcurrants

(D and F) but only a small ammint of hydrolysis was found to occur. Better results were obtained by boiling the anthocyanin (about 0.1 mg.) for 30 minutes in aqueous hydrochloric acid (1 ml.). Althou^ some aglycone was produced nearly all the diglycoside was converted to the monoside and gave vastly superior results. io

The anthocyanins from "bands E and Qt gave only the aglycone "by this method, while B and F gave, as well as Bome aglycone, spots ehroraatographically identical with E and Q respectively, indicating that they were diglycosides.

Enzjaic Hydrolysis

Preparation of Ehamnodiastase. The enzyme from the seeds of PhAfflnna frangala» was prepared hy the procedure of Charaux^^\ The seeds (2.8 g.) were ground in a mortar and extracted fomr times with small quantities of ether and the extract discarded. The residue (2.2 g.), mixed with water (9 ml.) and a few small crystals of thymol as a preservative, was allowed to stand for two days, filtered, and the filtrate diluted with four volumes of ethanol. The precipitate was filtered off, washed with alcohol, and dried in a vacuum desiccator.

Test for Activity. A small spatula tip of cyanidin-

3~rutinoside, prepared as described below, was placed in a small test tube and several drops of an aqueous suspension of the enzyme added. A control was also set up consisting of an equal volume of enzyme suspension.

Each solution was covered with a layer of ether, the tubes shaken and left for kS hours. At the end of this time aliquots of the solutions were spotted onto paper, chromatographed, and examined for sugars (see

• Kindly donated by the Melbourne Botanic Gardens. il

A well defined spot conld "be olsserved having an in phenol/water and an Sg. (ratio of distance travelled to that of glueose) of 0.5^4- ia "bntanol/aeetie aeid/water, fhis spot was undoa'btedly due to ratinose.

Examination of the Aathoeyanins. The procedure msed for testing the enzyme was repeated on the anthocyanins obtained frc®i the eellalose colamn prior to their further purification on polyamide columns. Only glucose could he detected in samples P and Gr "but in D and 'B, besides an intense spot for glucose, a weak ^ot could he observed corresponding to rutinose.

Peroxide Oxidation A small spatula tip of the anthocyanin to he examined (approximately 0.1 mg.) was dissolved in purified methanol (0.1 ml.) and 30^W/V hydrogen peroxide solution (2 drops) added. After 2J4. hours a few grains of palladium catalyst were added to the solution to decompose the excess peroxide. After leaving for a farther 2k hours the mixture was then made strongly alkaline "by the addition of ammonium hydroxide solution (0.5 ml.) and heated in a boiling water bath for 3 minutes. The solution was then spotted onto paper, chromatographed, and examined for sugars. The blackcurrant anthocyanins, as obtained from the cellulose column, were examined by this technique and and only glucose could "be found in the reaction mixtures from E and G. The anthocyanins fr

Following further purification of these anthocyanins on a polyaiaide column glucose was still the only sugar detected in the anthocyanins E and "but rutinose was the ma^or sugar, acccaapanied hy small quantities of rhamnose and glucose, arising from D and P.

These results are illustrated in Tahle 2.

CCTnparison of Intensity of Spots obtained Before and After Polyamide Purification

Sugars detected Band from cellulose after polyamide column purification D glucose +++ + + rhamnose + +++ rutinose

E glucose + +

P glucose +++ + + rhamnose +++ rutinose +

G glucose + +

Quantitative estimation of the Sugars

The method used was that of Nordstrom^"'''(2§)' which depends upon the development of a green colour when a sugar is heated with anthrone in strong sulphuric acid. Preparation of the Standard Curve. Standard glucose soltitlons, containing UO, 100, 200 and 300 fig. per 5 ml. were prepared "by dilation of a stock solution containing pure dry "Analar" glucose (0.1 g.) in distilled water (500 ml.). Each of the standard solutions (5 ml.) and distilled water (5 ml.) to act as a blank, were pipetted into identical tnhes and anthrone solution (5 ml. of 0.2^^ anthrone in 95% sulphuric acid) carefully added from a "burette. The solutions were gently mixed and allowed to stand for 15 minutes for colour development. The optical densities were measured at 620 IE^ and plotted against sugar concentration to give a straight line (Fig- 2).

Estimation of the Sugars. A small quantity (approx, 1 mg.) of the anthocyanin to he analysed was dissolved in 0.05% hydrochloric acid (10 ml.) and 2 ml. pipetted into a standard test tube. Distilled water (3 ml.) was then added and the solutions thoroughly mixed with a stirring rod. After standing for 15 minutes the optical density was measured at 620 mp and by reference to the standard curve (Pig. 2) the sugar concentration was calculated. 2k Estimation of the Aglycone. One ml. of each of the anthocyanin solutions was placed in a small (5 ml.) flask containing hydrochloric acid (0.5 ml.) and the solution refluxed for 30 minutes in a hoiling water hath in total darlmess. The solutions were then cooled, made up to 10 ml. with ethanol, and the optical densities measured at 3U5 lEp. By using the extinction coefficient for cyanidin given "by Kordstrom^^^^ = 29,270 @ 5U5 nyi.) the concentration of the anthocyanin was calculated. The results obtained for the blackcurrant anthocyanins D and E are given in Table 3.

Exaiaination for Acyl Groups. The test used was that described by Robinson and Eohinson^-" ^ which depends upon the change in partition of an anthocyanin between amyl alcohol and water when the acyl group, if present, is split off by alkaline hydrolysis. The blackcurrant anthocyanins all reacted negatively to this test.

Preparation of Reference Materials Cyanidin-3~rutinoside. Rutin (10 g.) and anhydrous sodium acetate (5 g.) were dissolved in acetic anhydride (200 ml.) and refluxed for one hour. After cooling, the mixture was poured into ice water (l 1.) and the cream coloured precipitate recovered by filtration. The product was purified by redissolving in hot acetic acid and again pouring it into water when a white crystalline STANDARD CURVE FOR SUGAR DETERMINATION

> t

<-J 0'5 »O- a O

lOO 200 GLUCOSE Table 3

Results of Sugar ;Aglycone Ratio Analyses

Anthocyanin Opt. Density Anthocyanin Opt.Density Sugar Gone. Sugar/ at 3k5 n^ after 620 mji (as gluoose) Aglucone hydrolysis Cone, g.moles/L. Anthrone treated soln. )ig./ml. g.Moles/L.

Cyanldin-3- monogluc oslde 0.620 2.16x10*"^ 0.3i44 ko 2.2 xlO"^

Oyaiiid.ln-3- rutinoside 0.362 0.398 45 2.47x10"^ 1.99:1

D 0.340 1.16x10"^ 0.395 kh 2.44x10"^ 2.10:1

E 0.535 1.83x10"^ 0.347 ko 2.2 x10"^ 1.20:1 iZ solid g., 86%) was o"btained which was dried in

Tacuo over sulphuric acid. M.P. 110-i12°C (dried at

60® in vacuo). No melting point has previously "been recorded for this compound. Required for Oij.7H5o026:

C, H, U.85; 0, ko.k; acetyl,

Pound: C, 5h.26; H, 5.06; 0, M.O; acetyl, 39.k%.

Rutin decaacetate (2 g.) was dissolved in anhydrous tetrahydrofaran (kO ml,) and lithium aluminium hydride

(0.J+ g.) in tetrahydrofuran (10 ml.) slowly added.

The solution was then warmed on a water hath and finally refluxed for 10 minutes. The yellow-grey precipitate

(1.5 g.) was filtered off and dissolved in 3% methanolic hydrochloric acid (20 ml.) to give a red solution.

After filtration the solution was evaporated to dryness under vacuum, the residue dissolved in a minimum quantity of "butanol/2N hydrochloric acid and purified hy passage through a cellulose column as described for the "blackcurrant anthocyanins. A small yield {2%) was obtained of an anthocyanin which "behaved (partial hydrolysis, total hydrolysis, sugar/aglycone estimation, peroxide oxidation, paper chromatography) as eyanidin-3-rutinoside.

Cyanidin-3-monogluc oside (Chrvsanthemin). Cyanidin-3- monoglucoside was obtained from pink and mauve chrysanthemums where it had previously been identified as the major pigment by Willstatter and Bolton^^^^.

The petals (500 g.) were extracted several times by "blending with i% methanolie hydroehloric acid. The combined extracts were chromatographed on a cellulose colTonin as descrilsed at ore for the "blackctirrant ant ho- cyanins. Two ma^or "bands appeared which were collected separately. The first "behaved chromatographically as the aglycone, cyanidin, (Rf. "biitanol/2N hydrochloric acid, 0.7U: Porrestal solvent, 0.14-9: cf. Tahle 1"b, p.

The second "band was concentrated "by absorption on a second cellulose column, as descri"bed previously (p. 23) and recrystallized from 20^ hydrochloric acid. This material "behaved hcanogeneously upon paper chromatography

(Ta"ble 1a) and chemical tests (acid hydrolysis, partial hydrolysis, sugar-aglycone ratio) indicated that it was cyanidin-3-Hionoglucoside.

Cyanldin^3.5«-diglucoside. Cyanidin-3»5-diglucoside was previously identified as the pigment of red roses

"by Willstatter and Holan^^^^

Petals from red roses of mixed variety were macerated with methanolic hydrochloric acid, the extract filtered and evaporated to a'bout a quarter of its original volume. The pigment was then precipitated

"by the addition of excess saturated lead acetate solution, dried and then reconverted to the anthocyanin with methanolic hydrochloric acid as described above for the isolation of the "blackcurrant anthocyanins. The deep red solution was evaporated to a thin syrup and the aBthocyanins precipitated "by the addition of 5 volames of aahydroas ether. The precipitated gam was redissolved in methanol and precipitated again "by the addition of anhydrotus ether. Bleating this procedure six times finally gave a dry light red solid which was filtered off and dried under vacuum. The product was further purified "by dissolving in hydrochloric acid and mating the final concentration of hydrochloric acid up to 20^ when the anthocyanin precipitated as a dark red powder after several days storage in the refrigerator. The product was homogeneous upon paper chromatography in several solvent systems (Tahle 1a, page 28) and "behaved as cyanidin-3,5-diglucoside (Rf. values in solvents I and III, acid hydrolysis, partial hydrolysis).

DelT?hinidin. Petals of the violet pansy, Viola tricolor, which has heen reported as containing the p-hydroxycinnamic acid ester of delph1.nl din rhamnoglucoside^^^'^®^, were macerated with 1% methanoUc hydrochloric acid and the extract evaporated under vacuum to a thin syrap. The concentrate (1 ml.) was added to kOf> aqueous hydrochloric acid (1 ml.) and heated in a "boiling water hath for 5 minutes. The solution was then twice extracted with amyl alcohol (1 ml. portions) and the amyl alcohol extract used for chromatographie canparison with suspected delphinidin samples. The Rf. values of this aglycone (solvent I, solvent II, 0.31) collared favourably with those reported for delphinidin (Tahle 1, page 28). RESULTS AMD DISCUSSION

The curve obtained by plotting the optical density at 3k5 against the tube nuinber for a typical separation of blackcurrant anthocyanins on a cellulose column is shown in Fig, 1 (page 28). Previous work showed the presence of seven distinct "bands whereas only six were ohvious here. Visual observation of the first band as it approached the end of the column showed that it was a mixture, since its leading edge was reddish-hrown in colour changing to a crimson-red at the trailing edge. Absorption spectra of the contents of tuhes 13 and 19 confirmed that two substances were present and fractions of the front and tail of this hand were kept separate as band A and band B respectively. It will he later shown that these fractions are identical with hands I and II of the earlier work. It will also "be d«aonstrated that hand B is due to cyanidin and hands D and S to cyanidin glycosides and that C is due to delphinidin and F and G to delphinidin glycosides. This corresponds to the conclusions reached in the previous work^*^' but the graph presented there shows that the aglycones are present at about half the concentration of the glycosides whereas Fig. 1 shows that delphinidin (hand C) is present to about the same extent as its glycosides (P and G). It is difficult to assess the quantity of cyanidin present because the M BEUAD (B) is combined with the first "band (A) but both these materials are present to greater extents than previously reported. It is obvious therefore that the extracts applied to the columns differed in the relative amounts of their component pigments and indicates the sensitivity of this technique in showing up quantitative differences in anthocyanin mixtures. In this work the blackcurrant pigments have been designated by the letters A, B, 0, D, E, P and G in contrast to the numbers previously used^^^. This has been done to avoid confusion with the Rcxaan numerals used to label formulae.

Band A The eluate containing band A had a brownish-red appearance and on paper chrcffljatograms ran as a brown diffuse spot with the solvent front. Its spectrum (?ig. 3) shows a relatively weak absorption peak at n^. contrasting with a large general absorption at shorter wavelengths, below 500 mji. This type of spectrum is generally associated with decomposed anthocyanins^^^^ and has been noted by Lakton et al. ^^^ K The behaviour of this material was identical with that reported for band which was previously regarded as a degradation product. It was not further considered in this work.

Band B - BAND

1-4

m s o i-a

0 lO w > t 0 zto CO Ui 0€ • D M W Q • s jU < p. • y 0-6 0 K a M O w a 0-4 o

o M

•ill, I 1 I I L 300 4cx:> 500 600 WAVE LENGTH mp. hi

Band B The material from "band B exhibited chromatographic "behaviour on paper consistent with its previous identification as cyanidin. The anthocyanidin was not isolated hut spotted directly onto paper from the coltmin eluate. The Rf. values in hutanol/aN hydrochloric acid and Forrestal solvent (Tahle 1a, page 28) approach closely those given "by Bate-Smith^-^' and Parkinson^^^^ (Tahle 1h) and were identical with those of cyanidin prepared hy the hydrolysis of cyanidin-3- monoglucoside isolated from chrysanthemums (page 37).

Band C The identification of the material from this "band as delphinidin was also confirmed, A comparison of the chromatographic behaviour of this pigment with the sample of delphinidin (page 39) showed that the two pigments were identical (Table 1a, page 28).

Band E The anthocyanin responsible for band E has previously (5) been identified as cyanidin-3-monoglucoside^-'^. It was isolated, as described above, as a dark reddish-brown solid, which gave cyanidin and glucose as the only sugar on acid hydrolysis. Partial hydrolysis gave only the unchanged compound plus some aglucone. Further evidence for the correctness of this identification is now provided by the sugar/aglycone Ml ratio of 1.20:1. The validity of this estimation was established by the concurrent determination of 1.07:1 for the sample of cyanidin-3-monoglucoside isolated from chrysantheimims (page 37). The spectra of anthocyanin E in butanol/2N hydrochloric acid and 0.01^ methanolic hydrochloric acid are shown in Pigs. 1+ and 5 respectively. In the latter solvent it shows an absorption maximum at 523 niyi. in agreement with the value quoted by Harborne^"^^^ for cyanidin-3-monoglucoside. However it does not show a shoulder at 1+40 mp.. which Harborne considers to be characteristic of anthocyanins having the 5-hydroxyl group unsubstituted. Nevertheless the ratio is O.2I4. which is close to the ratio of 0.22 given by that author for cyanidin-3-monoglucoside. A 5-substituted compound would give a ratio of approximately half this

On paper chromatography anthocyanin E gave Rf values, in three solvent syst^s, in approximate agreement with those reported in the literature (Tables la and lb, p.28) for cyanidin-3-monoglucoside. In m-cresol/acetic acid/ (27) water it gave an Rf of 0.1+7 while Parkinson^ reported an Rf of 0.23 for cyanidin-3-monoglucoBide. It is to be noted that none of the reference anthocyanins agree in Rf values, in this solvent system, with those reported in the literature. Of. Table 1, although care was taken to BAND 0 cn TJ e

F o

CO «t J m M Q

psi Q M

I> SI o

W O H 500 600 WAVE LENGTH — mjJ. CQ m Q

!0 r ANTHOCYANIN Ff^OM 9AND D

U •! E M C§Q

O • o M M p.

M 0 M03

600 2CO 400

WAVE LENGTH mp. la follow closely the techniques reccamnended "by Bate-Smith

When ran with the sample of cyanidiii-3-monoglTicoside, prepared from chrysanthemoms, the anthocyanin E gave identical Ef values in all solvent systems.

The anthocyanin from hand E is therefore considered to he cyanidin-3-nionoglucoside.

Band D

Earlier work^^^ had shown that the pigment from

"band D gave cyanidin on complete hydrolysis and cyanidin-

3-monoglticoside on partial hydrolysis. Since glucose was the only sugar detected "by chromatography following acid hydrolysis of the comhined anthocyanins from the blackcurrants, and since the test for acyl groups was negative it was considered that this pigment was prohahly C31) the 3-hioside, as proposed "by the Rohinsons^-^ \ or the

3,5-diglucoside, although its Rf values did not agree with those given hy Bate-Smith^^^ for either of these anthocyanins.

However the results presented hy Fouassin (page 11) indicated that this pigment was probably a cyanidin- rhamnoglucoside and hence the acid hydrolysate of the anthocyanin D was carefully examined by paper chromatography for traces of rhamnose. Initially only glucose could be detected but it was found that if a larger than usual amount of the acid hydrolysate was spotted onto the paper, so that an intense spot was obtedned corresponding to glucose, a faint spot for rhanmose coald be detected. Sensitivity measurenients of the method used for detecting the sugars (page 2?) revealed that it was only ah out half as sensitive to rhamnose than it was to glucose while the ratio of spot intensity was far greater than 1:2 in the acid hydrolysate of the anthocyanin. It was ohvious therefore that the method of detecting the sugar residues was not responsible for the large amounts of glucose observed, Cyanidin-3-rutinoside, prepared by the reduction of rutin (page 3^1-), was compared with anthocyanin D by paper chromatography and it was found that they behaved identically in all solvent systems (Table 1a, page 28). These two pigments also possessed identical spectra (Pig. 5, page U6) showing absorption maxima at 526 mji. and 280 mji. Harborne^^^^ gives X max. 525 lap. for cyanidin-3-nionoglucoside and 523 inp. for the rharano- glucoside, gentiobioside and xyloside. As was the case with cyanidin-3-monoglucoside no shoulder was observed at U^O n^. but the ratio f^li' pigment D, was 0,25* This compares with ratios of 0.22 for cyanidin-3-monoglucoside, 0.23 for -3-rhamnoglucoside, 0.25 for -3-gentiobioside and 0.13 for -3,5-diglucoside, given by Harborne.

Since there was now strong evidence indicating that pigment D was cyanidin-3-rhamnoglucoside attempts were made to remove the sugar residue intact and detect it "by paper chrcmatography. Enzymic hydrolysis and peroxide oxidation were used and "both methods revealed a sugar which was chromatographically identical, in two solvent systems, with the sugar from cyanidin-3-rutinoside and which must have "been rutinose. However, as with the acid hydrolysis, large amounts of glucose were also present in the hydrolysates frcm pigment It appeared probable therefore that the blackcurrant pigment D was contaminated with some glucose containing substance, possibly a flavonoid. Further purification was therefore carried oat by passing the anthocyanin through a polyamide column. This procedure has been recommended by Chandler and Swain^^^ to separate anthocyanins from flavonoids, the latter being more firmly retained by the adsorbent. Peroxide oxidation of the purified pigment D followed by ammonolysis liberated a sugar chromatographically identical with rutinose from authentic cyanidin-3- rutinoside. A weak spot could be observed for glucose in this material but it was accompanied by a similarly weak spot for rhamnose indicating that it had arisen from hydrolysis of the rutinose. Furthermore acid hydrolysis of the purified pigment now showed glucose and rhamnose to be present in approximately eguimolar amounts. It is evident then that separation on a cellulose column alone is not sufficient for complete separation of the pigments from other materials present and it is recommended that the eluates be further purified by passage through a polyamide column before identification is atteapted. A sugar aglycone determination was carried out on the polyamide purified pigment D (Table 3, page 36) and gave a ratio of 2.10:1. This result, together with the liberation of glucose and rhamnose on acid hydrolysis and rutinose on enzymic hydrolysis and peroxide oxidation plus its identity (spectra and chromatography) with authentic cyanidin-3-rutinoside, identifies the pigment from band D as cyanidin-3-rutinoside.

Band G On acid hydrolysis the pigment from band Gr gave delphinidin which was identified by comparison, on paper chromatograms, with an authentic sample obtained from the hydrolysis of the pigment of Viola tricolor (p. 39) The spectrum of pigment G in 0,01^ methanolic hydrochloric acid (Fig. 6) shows a maximum at 339 h^. ccmparing with 535 nfi. and 537 biji. given by Harborne^^®^ for the 3-glucoside and 3-rhamnoglucoside respectively of delphinidin. The ratio is 0.19 while Harborae gives 0^18 and 0.17 for these compounds and 0.11 for delphidinin-3,5-diglucoside, indicating that pigment Q is a 3-glycoside. Paper chromatography shows that the Rg values of pigment G do not compare with those of any of the CO tM a

o 0'5 BAND — F o band — G cn OA ^XJ > M H Q t/) pi Z ON UJ Q a 03 M ^ »O < y o H 0-2 CL O s (D fflO ^o o- H

J I I I L « i 1 < 1—J I I L t I 1 3od 400 500 600 WAVE LENGTH mjj. anthocyanins for which Rf. values are recorded in the literature (Tables 1a and Ih, page 28) hut approach more closely those given for the 3-glycoside than any of the others. That the comparison of Rf. values is not a reliable method for identifying anthocyanins is home out hy comparing the Efs. obtained for the anthocyanins D and E from the blackcurrants, which have been positively identified as the 3-rutinoside and 3-glucoside respectively, with those reported in the literature. Furthermore the low Rf. values given by the delphinidin glycosides make accurate Rf. measurement difficult and add to the uncertainty of this method. No reliable reference compound could be obtained which would have made the paper chromatographic identification more complete. The sugar-aglycone ratio determination could not be carried out on this material, or the anthocyanin from band P, since the extinction coefficient of delphinidin has never been recorded. Only glucose could be found in the acid hydrolysate and in the peroxide oxidized material following further purification on a polyamide column. Enzymic hydrolysis also gave only glucose. Pigment G is therefore a 3-glucoside. Partial hydrolysis yielded only the aglucone and some unchanged anthocyanin. This eliminates the possibility of a 3-bioside or a 3^5-diglucoside since in the former case 3 pigments and in the latter case k pigments would have been obsepved* The test for acyl groups (page was negative so that the anthocyanin from "band G must therefore "be delphinidin-3-monoglucoside.

Band P Acid hydrolysis of the material from "band P gave the anthocyanidin delphinidin which was identified "by comparison on paper chromatograms with an authentic sample of delphinidin. The spectrum of hand P is given in Pig. 6 and shows a maximum at 5kO doji. and a ratio of 0«19 which, as with the material frcxn "band G, is similar to the figures obtained by Harborne^^^^ for the 3-glucoside and 3-rhamnoglucoside of delphinidin and indicates that the pigment is not a 5-glycoside. The Ef. values of pigment P obtained on paper chromatography do not correspond with any of those in the literature (Tables 1a and 1b) and in the absence of reliable reference compounds no further information could be obtained by this technique. The presence of organic acids could not be detected in this pigment using the test for acyl groups (page 3^4-). Pollowing the difficulties experienced with the identification of the pigment from band D, pigment P was further purified on a polyamide column prior to the identification of the sugar moiety. Acid hydrolysis now liberated glucose and rhamnose and partial acid hydrolysis gave an anthocyanin which was chromatographically identical with pigment Q (delphinidin- 3-monogl\ic o side) • Peroxide oxidation followed "by ammonolysis of the polyamide purified pigtaent F li"berated a sugar which was identified as mtinose "by chroiaatographie comparison with the sugar liberated from cyanidin~3-rutinoside. Once again the polyamide purification had "been successful in removing some glucose containing mterial since enzymic hydrolysis carried out on the unpurified material had given an intense spot for glucose and only a faint spot corresponding to rutinose. These facts lead to the conclusion that the anthocyanin from "band F is delphinidin-3-rutinoside. Delphinidin-rhamnoglucoside has heen found in only (iA) two other natural products, Harhorne^ ' has recently identified delphinidin-3-rhamnoglucoside as one of ten anthocyanins present in the potato and Ahe and Gotoh have reported delphinidin-rhamnoglucoside as "being present in the fruit coat of the "Black Beauty" variety of the egg plant. In neither of these reports has the rhamnoglucoside residue "been reported as rutinose, COHOLUSIOHS The anthocyanins occurring in the fruit of the "Whitebud" variety of blackcurrants have now been completely identified. In previous work^"'' cyanidin, delphinidin and cyanidin-3-monoglucoside (chrysanthOBin) I'g were identified and in this work the presence of these three substances has "been verified and the remaining three glycosides characterized. They were cyanidin-3~ rutinoside, dclphinidin-3-monogl-acoside and delphinidin- 3-nitinoside. The term "extract" is used here "because it cannot be definitely stated that these six substances all exist in the growing fruit. It is possible that hydrolysis may have occurred during extraction leading to the formation of the aglycones and it is also possible that the monoglucosides may have arisen from partial hydrolysis of the rutinosides. However the chances of this occurring have been kept to a minimum by employing only weakly acidic solutions and performing all the extractions and concentrations at low temperature (less than i|.O^C). Harborne and Sherratt^ ^ examined the glycosidic pattern of a number of naturally occurring anthocyanin mixtures euad concluded that the occurrence of mono-, di- and tri-glycosides, in the one plant, indicates that glycosidation during the biosynthesis of anthocyanins, involves a nximber of steps. They consider that the sugar molecules are attached, one at a time, to the pigment molecule or its precursor rather than that a preformed di~ or tri-saccharide is attached directly in one step. Further evidence to support this theory has been presented very recently by Yamatia and Cardini^^^^ who isolated two enzymes from wheat germ which were able to catalyse glycosidation in phenols. The first enzyme gave a monoglucoside with hydrogainone as substrate while the second enzyme would act only on the monoglucoside to give the diglucoside. If this theory is correct then it would seem quite appropriate that aglycones, as well as glycosides, should exist in the living plant. The system of anthocyanins which has "been found to exist in the blackcurrants, that is the aglycones cyanidin and delphinidin together with the monoglucoside and rutinoside of each of them, is a further case of this systematic pattern of glycosydation.

« « « « 5Z

Addenda

After tills work was completed L. Eeichel and W. Beichwald (Natxirwiss. 1960. p.M) published an account of their investigations on the blackcurrant anthocyanins in which they identify one cyanidin glycoside and two delphinidin glycosides. These authors used two dimensional paper chromatography (solvents unstated) to show the presence of four pigments of which one was present in only very small quantities. Two fractions were obtained from a cellulose column, using 25% formic acid as solvent, of which the first was separated into two fractions, 1a and 1"b, "by paper chronatograpliy ("butanol/acetic acid/water, 1|.:1:5). Fraction la gave cyanidin, glucose and rhamnose on acid hydrolysis and was therefore concluded to be cyanidin-3-i^ -rutinoside. Similarly, fraction lb was identified as delphinidin-3-<9 -rutinoside. The second fraction from the cellulose column was found to be hcanogeneous on paper chromatography and on acid hydrolysis gave delphinidin and glucose. No farther mention is made of the fourth component. Althoagh agreeing with the identification presented in this thesis, the characterization of two of the pigments by Eeichel and Eeichwald as 3-p -rutinosides is "based only upon the identification of g la cose and phamnose in the acid hydrolysates. No evidence is presented to show that the pigments are -3-glycosides or that "both stxgar molecules are present as the disaccharide rutinose, or that they are joined to the aglyeone thrcmgh a ^ -linkage. It is to "be noted that no aglycones were found in the "blackcurrant extract "by these workers. This may "be due to the fact that the solvents used for chromatography did not contain hydroehloric acid which has "been foand^^®^ necessary to avoid fading of the aglycones during development. The authors do not state what solvent systQoas were used for the two dimensional chromatography where only one of the solvents would need to lack hydrochloric acid for fading of the aglycones to ocoir. The cellulose column was developed with 25% formic acid so that it is to "be expeeted that the aglycones would not have "been visilJle here had they "been present. BIBLIOGRAPHY 1. Ash, A.S.F. and Reynolds, T.M., Aust. J.Biol.Sci., 2, i^•35, (1951+). 2. Attree, G.P. and Perkin, A.G., J.Chem. Soe., 1222, 231+. 3a. Bate-Smith, E.G., Biochon.Soc.Symposia (CamT3ridge,Eng.) No.3, 62 (1950). "b. „ Bioch«n.J. (London), 58, 122, (195U). c. „ and Westal, R.G., Biochem.et Biophys.Acta, 14.27, (1950). k, Bauer, I., Birch, A.J. and Hillis, W.E., Chem. and Ind. 12^, h33. 5. Chandler, B.V. and Harper, K.A., Nature, 181. 13I (1958). 6. Chandler, B.V. and Swain, T., Ibid.. 185. 989, (1959).

7. CharauxIMd.,, M.C.8, 35, ,Bull.soe.ehim.'biol. (1926). , 6, G3k, (1921+): 8. Pouassin, A.. Revue Fermentations et Ind. Aliment., IJ., 173, (1956). 9. Geissman, T.A. and Hinreiner, E., Botan.Rev., 18, 77, (1952). 10. Harhorne, J.B., Biochem.J. (London), 20, 22, (1958). 11. Harhorne, J.B., rbid. 2k» 262, (196O).

12. Harbornei4.86, (1957), J.B. and Sherratt, H.S.A., Ezperientia, IJ., 13. Harhorne, J.B. and Sherratt, H.S.A., Biochem. J. (London), 65, 2ltP, (1957). lif. Hayashi, K., Acta Phytochim. (Japan), ik, 39, (19144): C.A., W6, (1951). 15. Harper, K.A., B.Sc. Thesis, University of New South Wales, January 1957. 16. Hayashi, K., Noguehi, T. and Abe, Y., Pharm.Bull. (Japan), 2, M (195U) : O.A., 10983, (1955).

17. Huang, H.T., J.Agric.Pood Chem., 3, IM, (1955): J.Am.Chem.Soc., JQ, 2390, (1956}. Go

18. Karrer, P. and de Eeuron, G., Helv.Chem.Acta, 15, 507, 1212, (1932). 19. Karrer, P. and de Meuron, Q., i"bid. 16, 272, (1932). 20. Karrer, P. and Widmer, R., iMd. 10, 67, (1927). 21. Lukton, A., Chichester, 0.0., and Mackinney, a.. Food Technol., 10, U27, (1956). 22. Mayer, F., "Chemistry of Natural Coloring Compounds" Trans, hy A.H. Cook, Reinhold, N.Y. p.226, (19U-3). 23. Musago, L., Boll, soc.ital.hiol. sper., lit, 620, (1939) C.A., iit, 1663, (19^1-0). 2k. Nolan, T.J. and Brady, T.a., Pr oc. Soy. Irish Acad., 1, (1936). 25. Nolan, T.J. and Casey, H.M.T., ihid.. i4-0B. 56, (1931). 26. Nordstrom, C.G., Acta Chem. Scand., 10, 1U91, (1956). 27. Parkinson, T.L., J.Sci.Pood Agr., 239, (1954). 28. Pratt, D.D. and Rohinson, R., J.Chon.Soc. Igl, 577, (1922): and suhsequent papers. 29. Reichel, I., Stroh, H.H. and Reichwald, W., Naturwiss., 12, U68, (1957). 30. RoMnson, G.M. and Rohinson, R., J.Chem.Soc., 1927, 2196. 31. RoMnson, G.M. and RoMnson, R., Biochem.J. (London), 25, 1687, (1931). 32. Shriner, R.L., Records Chem.Progress, H, 121, (1950). 33. Saodheimer, E., J.Am.Chean.Soc., 25, 1507, (1953). 3k. Venkateswarlu, G., J. Indian Chem. Soc., 22, k3h, (1952). 35. Wawzonek, S. in Blderfield "Heterocyclic Compounds" Vol.2, Wiley & Sons, N.Y., p.307 (1951). 36. Willstatter, R. and Bolton, E., Ann, Chem., M2, 136, (1916) 37. Willstatter, R. and Nolan, T.J., iMd., 1j28, 1, (1915). from Perkin, A.G. and Everest, A.E., "The Natural Organic Colouring Matters", Longmans, London, p.288, (1918). 38. Willstatter, E. and Weil, P.J., Aim.Chem. , ij.12. 178, (1917). 39. Willstatter, R. and Zollinger, E.H., ibid., p.l64. kO. Yamalia Tsutoma and Cardini, C.E., Archives Biochem. and Biophys. ^ 127, (i960): iMd., p.123. 1+1. YukeMde Abe and Kanje Gotoh, Ann.Rqpt.Nat. Inst. Genetics (Japan), No.7, i+9, (1937). Zemplen, G. and Gerecs, A., Chem.Ber., 68B. 1318, (1935).

m* PAET 2a

THE POLAHOaBAPHIG BEHAVIOUR

AIJTHOCYANINS INTRODUCTION

A study of the polarography of the anthocyanins was conmenced in order to obtain further data on the oxidation-reduction "behaviour of anthocyanins. The only previous work on this suh;)ect is that of Zuman ^^^^ who examined a large number of anthocyanins "but did not carry out wave analyses nor were any mechanisms proposed for the electrode reactions. It was thought that valuable information might he gained from a closer study of this type in particular from the relationship of half-wave potential to pH.

Consequently an anthocyanidin, peonidin, was subjected to polarographic reduction at various pH levels, the resulting current/voltage curves analysed and reaction mechanisms proposed. Concurrent spectral studies have been carried out to assist in identification of the molecular species present at the various pH levels. « « « LITERATURE SURVEY

The polarograpMc iDehaviaur of the anthoeyanins was first investigated "by Zuman^^^^ who studied pelargonidin, cyanidin and delphinidin and some of their glycosides in aqueous solutions of Britton- Eobinson "buffer using a saturated mercury sulphate electrode. Half-wave potentials of -0.14.05 volts for pelargonidin, -0.l|.00 v. for cyanidin and -0.U25 v. for delphinidin were obtained at pH 3.0 and it was found that the height of the polarographic step rose linearly with concentration between the range 1x10""^. to 8x10" The effect of hydrogen ion concentration on the polarography of pelargonidin was examined and multiple waves were observed as the pH was raised. A single wave, at negative potentials, was evident in acid solutions up to pH 3 but further increase in pH shifted this wave to more negative potentials. At the same time a second wave separated which was smaller and more negative than the other. This second wave continued to grow at the expense of the first until at pH 5.5 to 6 both waves were of about equal height. At higher pH values the second wave continued to increase until it reached the height of the first, as observed in the acid region. Above pH 11 a third wave appeared which was about double this height. No attempt was made in this paper to analyse the waves or to formulate the electrode reactions. No other work has "been published on the polarography of the anthocyanins although Eihereau-Gayon^^^^ attempted to repeat Zunian*s work "but was unable to obtain reproduc- ible results.

Reduction of the Anthocyanins In order to appreciate electrochemical reduction processes it is pertinent to discuss results of purely chemical reductions. It is to be expected that mild reduction would produce the flavene II while stronger conditions would give flavans III.

f^ T" + ZH + EH SAJ n lU Gompiete reduction to flavans has been found to occur on uncontrolled catalytic hydrogenation. For instance 2',3,i|.',5,7-pentamethoxyflavylium chloride was reduced over palladium on barium sulphate to a compound which analysed for the equivalent flavan^^^. Blstow and Piatt ^^^ successfully reduced 6-ethyl-7~hydroxy-3 * ,-dime thoxyf la vylium chloride using lithium aluminium hydride followed by hydrogenation over Eaney nickel catalyst to give the f la van in almost quantitative yield. The identification of the product as the flavan was confirmed "by synthesis, Flavans have also been obtained from the corresponding flavene by hydrogenation over (2) platinum on carbon^ Catalytic hydrogenation has also been used to reduce the flavylium salt to the flavene II- Thus Fosenka^^^^ using Willstatters platinum catalyst reduced di'benzoyl-3,7-dihydroxyflavylium chloride, with the absorption of 2.25 H, to yield a product which analysed for the sesquihydrate of the flavene. This material was soluble in ether and on treatment with sodixum hydroxide solution gave a deep blue colour. A product giving a similar colour reaction was obtained by Charlesworth et al. ^^^ from the hydrogenation (i mole H2 absorbed) of 2',3',U,7-tetrahydroxyflavylium chloride (morinidin chloride) over platinum black. Other forms of reduction yield different products depending upon the flavylium salt and the reducing agent. Morinidin chloride is reduced by zinc dust in dilute hydrochloric acid^-^-'^'^ to a coloarless solution which, on shaking with air, regenerates some of the original red colour. Prolonged treatment with zinc dust takes the reduction beyond this stage and an ether extractable substance is formed which gives the colour reaction (blue with alkali) associated with the flavene. Cyanidin chloride, upon treatment with zinc dust in hydrochloric acid^^^ or zinc dust in pyridine containing acetic acid^^^^, is decolourized but the colour is regenerated on shaking with air. Both groups of workers were satisfied that the regenerated colour was due to cyanidin (colour tests and distribution ratio) and not due to an oxidatively coupled bis derivative as was thou^t possible. The identity of this reoxidizable material (5) has never been solved but Gharlesworth et al. suggest that it could be dihydro-bis-cyanidin IV which would,

on oxidation, undergo a reversal of the ready reductive coupling which led to its formation. Support is lent to this theory by the isolation of free radicals of flavylium salts, chromenyls, Ziegler et al. ^^^^ reduced 2,I|.-diphenyl-3,6-dimethylbenzopyrylium perchlorate with chromous chloride to give the free (22) radical YJ and Lowenbein and Hosenbaum^ ^ used phenyl magnesium bromide to reduce 2,3,U-triphenylbenzopyrylium SQ perchlorate. The product, TDis-(2,3,l4--triphenylchromeny3), m.p. 130-163°, was partly dissociated to give de^ green solutions which were decolourized through formation of the peroxide on shaking with air. Oxidation in acid solution however gave the flavylium salt.

A free radical formed "by mild reduction of the simpler naturally occurring anthocyanin molecules would not he as well stabilized by resonance as the 2,l4.-diphenyl or the 2,3,U-triphenyl derivatives and, being less sterically hindered, would have only a transient existence and would dimerize completely. If no dissociation were to take place then the solution would be colourless since the strongly resonating forms are destroyed. Hence the proposal of Gharlesworth et al.^^^ that the reoxidizable material is a bis derivative formed by the dimerization of free radicals seems a most probable explanation. These considerations are of major importance in the present work where free radical formation followed by dimerization is postulated to account for the behaviour of peonidin at the dropping mercury electrode. EXPERIMEITTAL APPARATUS T3ae polarograph used was of the three electrode type similar to that of Lingane^^^ Details of the circuit, the polarographic cell, the dropping mercury electrode and purification of the mercury are presented in the appendix.

SuDPorting Electrolyte To obtain the wide pH range used in these experiments two "buffer solutions were used. The range from pH 2.2 to 8.0 was obtained with the Mcllvaine system^^^ which was prepared in the following way to permit the use of 50^ methanolic solutions. Stock solutions of 0.2 M citric acid (A.E.) and O.k M disodioim monohydrogen phosphate (Analar) were prepared and mixed in the proportions indicated "by Britton^^^ to give a combined volume of 25 ml. This solution was then diluted with 25 ml of purified methanol^^®^ except for the more alkaline "buffers of this system, when sane precipitation of sodium phosphate occurred, and the strength of methanol had to be reduced from to i+O^S. To obtain the solutions at pH 1.6 and 2.0 it was necessary to add nitric acid to the citric acid solution. Although this did not give a good buffer system, it produced a large concentration of hydrogen ions compared to which any change in hydrogen ion concentration at the electrode would "be negligible. Furthermore, as will he seen later, the actual pH is not of great importance because the half-wave potential is independent of pH in this range. For the solutions more alkaline than pH 9, mixtures of potassium dihydrogen phosphate and sodium hydroxide were used. Stock solutions, O.i^, were prepared and the buffer solutions obtained as previously, using the proportions given by Britton Anthocyanin Solution The anthocyanin used in these experiments, peonidin chloride, was prepared synthetically. Details of the preparation are given in the appendix. The material was purified before use by recrystalliz- ing several times from 209^ hydrochloric acid as follows. The anthocyanin was dissolved in 1% hydrochloric acid, by refluxing in the dark, to give a concentrated solution which was filtered and then made up to 20^ with respect to hydrochloric acid. After several days storage in the refrigerator precipitation was complete and the anthocyanin was filtered off and dried in vacuo over sulphori^j acid. Stock solutions of peonidin chloride were prepared, approximately 0.08M in purified methanol, and their purity checked by examination of the spectra from 320 to 600 inp. A typical curve is given in Pig. 1. > CO 2'Or o w TJ M O M CO ^ t>^d O o t 1-3 o G53 M • o (D H wS •XJ td o o^ M M

a t-« o w M

300 400 500 600 WAVE LENGTH — The exact concentration of peonidin chloride in the stock solution was determined by diluting (1 in iOOO) an aliquot with 0.01% ethanolic hydrochloric acid and measuring the optical density at 5^0 nyi. The extinction coefficient is not directly available from the literature hut Nordstrom^^^^ gives € 29,270 for cyanidin chloride in 0.01% eifehanolic hydrochloric acid. Eihereau-Gayon^^^^ states that methylation of the hydroxyl groups in the anthocyanins does not alter the position of the visible maximum or its intensity so that the extinction coefficient for peonidin may be taken as the same as that for cyanidin in this media: namely 29,270. On this basis the solution whose spectrum is depicted in Fig. 1 is 0.06M. Method The buffer solution was prepared, the pH checked, and UO ml. placed in the polarographic cell. Mercury was allowed to flow through the capillary which was lowered into the cell and the stopper secured. Nitrogen, purified by bubbling through two alkaline pyrogallol scrubbers and then through a water-methanol mixture (1:1), was bubbled through the cell for two hours to ensure the complete removal of oxygen from the buffer solution. This unusually long time was found necessary to obtain a reasonably flat curve for the supporting electrolyte, probably because of the very high sensitivity used in these e25)erinients. At the end of this period the electrolyte was sucked up into the tube "F" (Fig. 9) so as to form the connection "between the cell and the reference electrode. The nitrogen was now turned off and the average galvanometer deflection recorded (mean of maximiam and minimam) at 0.05 volt intervals over the range desired. The nitrogen was now turned on again and 0.1 ml of stock peonidin chloride solution, measared with a haemoglobin pipette (capacity 0,1 ml) admitted through the tube "C" (Fig. 9). The rubber stopper was immediately replaced in the tube and the nitrogen bubbling continued for another minute. At the end of this time the polarographic run was made from the lowest voltage at which a reading could be taken to the voltage where hydrogen discharge caused a sharp increase in current. The current-voltage curves were plotted for the supporting electrolyte and the anthocyanin solution. The difference between these two gave the true current- voltage curves for the anthocyanin, some of which are presented in Fig. 2. The experimental results of a number of others, representative of the pH range covered, are given in Tables 1 to k^ In a second series of experiments the spectra of the polarograph cell solutions were read from 330 to 600 nji. immediately after completion of each experiment. CQ O M Q • K o I\J ^ • o M ^

t-sl o JXJ M o t?d

APPLCD EJv1.F _ voLrs Ta'ble 1 Experimental Results pH 1*6 Applied E,M,P. Mean Galvanometer Heading (cms,) (volts) Buffer Soln. Anthocyanin Soln. Difference

0.2 7.0

0.3 8.1; 8.8 o.k

O.h 9.2 9.5 0.3 0.5 10.1 10.3 0.2 0.55 10.14- 10.7 0.3 0.6 10.8 11.1 0.3 0.65 11.0 11.6 0.6 0.7 11.3 12.5 0.8 0.75 11.5 13.8 2.3 0.8 11.7 15.6 3.9 0.85 11.8 17.0 5.2 0.9 12.0 17.6 5.6 0.95 12.1 18.0 5.9 1.0 12.2 18.2 6.0 1.1 12.U 18.6 6.2 1.2 12.8 19.2

1.3 1i4..7 21.2 6.7

Uh 17.5 25.6 8.1 Table 2

Experimental Results. pH i+.8

Applied E.M.P. Mean Galvanometer Reading (cms.) (volts) Buffer Soln. Anthocyanin Soln. Difference

o.U 10.0 8.9 - 1.0

0.5 10.95 9.9 - 1.05

0.6 11.7 10.7 - 1.0

0.7 12.3 11.14- - 0.9

0.75 12.55 11.7 - 0.85

0.8 12.7 12.1 - 0.6 0.85 12.9 13.3 o.h 0.9 12.95 114-.8 1.85

0.95 13.05 15.55 2.5

1.0 13.15 16.0 2.85

1.05 13.25 16.3 3.05

1.1 13.14. 16.6 3.2

1.2 13.7 16.9 3.2

1.3 13.95 17.25 3.3 l.h 1i|-.3 18.0 3.7 1.5 11^.6 19.6 5.0 Table 5

Experimental Results. pH 7*4

Applied E.M.F. Mean Galvanometer Reading (cms.) (volts) Buffer Soln. Anthocyanin Soln. Difference 0.6 12.3 9.6 - 2.9 0.7 13.1 lO.U - 2.7 0.8 13.33 10.83 - 2.7 0.83 13.7 11.0 - 2.7 0.9 13.83 11.20 - 2.63 0.95 11.3 - 2.3 1.0 1U.1 12.3 - 1.6 1.03 1i4..2 1U.1 - 0.1 1.1 114.. il 13.i4.6 1.03 1.13 1i|..63 16.03 1.2 1U.93 16.I|. 1.U3 1.23 13.23 16.33 1.3 1.3 13.3 16.7 1.2 1.U 16.1 17.13 1.03 1.3 16.73 17.83 1.1 1.6 17.U 18.6 1.2 Table k Experimental Results. pH 10.3 '— ' - 1 Applied E.M.F. Mean Galvanometer Reading (cms.) (volts) Buffer Soln. Anthocyanin Soln. Difference 0.6 10.2 8.2 - 2.0

0.7 11.0 9.1+ - 1.6 0.8 11.5 10.5 - 1.0 0.9 11.85 11.0 - 0.85 1.0 12. 11.55 - 0.85 1.05 12.6 11.75 - 0.85 1.1 12.8 11.95 - 0.85 1.15 13.05 12.5 - 0.55 1.2 13.3 13.3 0 1.25 13.55 1U..2 0.65 1.3 13.8 15.2 I.I4. 1.35 II4..I 16.5 2.I4. I.I4. ^k.k 18.3 3.9 1.14-5 IU.75 18.9 1^-.15 1.5 15.1 19.9 U.8 1.55 15.5 20.5 5.0 1.6 15.9 21.05 5.15 1.7 16.95 22.35 1.8 18.8 25.0 6.2

1.9 20.0 28.5 8.5 2.0 21.0 31.7 10.7 Where necessary, dilutions were made with "buffer solution.

These spectra are presented in Pigs. 3 and k^

Results

Some of the polar ©graphic curves o"btained are presented in Fig. 2. A summary of the results of all of them are given in Table 5 (page 83).

Of special interest is the shift of the polarographic wave helow the base line as the pH is increased followed by a return above the line at still higher pH values.

This division of a polarographic wave into a cathodic and an anodic wave is similar to that of the quinone-hydroquinone system^^^^ where the anodic wave (below the base line) represents oxidation of the hydroquinone while the cathodic wave indicates reduction of the quinone.

Table 5 shows the ratio of anodic to cathodic current, i (a)

It is also to be noted that there is a large change in total wave height (id)* which is least in the very neutral solutions and greatest in the very alkaline solutions. FIG^e

SPBCTRA OF CELL SOLUTIONS pH 1.6 TO h.6

600 500 400 300 WAVE LENGTH — mp FIG, h.

SPSCTRA OF CELL SOLUTIONS pH 6.5 TO 12.^

t/i z

y h- a O

SOO 400 WAVE LENGTH - mp. 1-8

1-6

\A k I he] -E M i o Q 12 M> tH w CJ

o« />K 3 0S

06i 8 lO 12 14 Table 5

PolarograpMc Data for Peonidin Solutions

-E i id ±U) t sees. n pH volts i(3uA id(a) id(c) M^ . t'/6 i(c)

1.6 0.77 0.166 - - - 3.83 1.7

2.0 0.76 0.272 0.126 - - 3.82 2.0

2.4 0.77 0.30 0.139 - - - 3.81 1.6

2.9 0.76 0.29U 0.137 - - - 3.81 1.9

3.U 0.78 0.266 0.12k 0.01 0.2144 0.04 3.81 1.5

3.9 0.83 0.1+ 0.186 0.0 o.k - 3.81 1.6

k.e 0,86 0.382 0.179 0. 07 0.31 0.23 3.82 1.3

0.87 0.25 0.116 0.05 0.20 0.25 3.81 1.3

6.5 0.97 0.125 0.058 0.053 0.072 0.74 3.80 0.9

6.8 0.98 0.110+ 0.067 0.062 0.78 0.'08 3.75 1.0

7.k 1.02 0.226 0.105 0.169 0.056 3.0 3.68 1.0

7.8 1.05 0.262 0.123 o.m 0.119 1.2 3.65 1.2

8.6 1.1i|. 0.319 0.15 0.075 0.2104- 0.31 3.60 1.4

9.1 1.22 0.1v68 0.219 0.137 0.3¥i. 0.41 3.56 1.6

9.3 1.27 0.531 0.251 0.014. 0.49 0.08 3.52 2.3

10.3 1.35 0.M5 0.196 0.05 0.362 0.14 3.49 1.6

11.1+ 1.78 0.381 0.183 0.02 0.36 0.05 3.09 1.5

12.3 1.85 0.56 0.27i+ - - 0.56 - 2.88 1.5 A small pre-wave is to "be noted in the wave at pH 2.14.,

This is probably an adsorption wave similar to that found by Kaye and Stonehill^''^^ in the polarography of acridine and is believed to be due to the easier reduction of a compound in the adsorbed state. It was also found in

some of the other waves which have not been shown in

Fig. 2.

Small maxima sometimes made their appearance and are

to be seen at the plateaus of the waves at pH and

12.3. No maximum suppressor was used as they only rarely appeared and generally were not troublesome.

A plot of the haIf-waee potential versus pH is given

in Pig. 5. The points fall on three intersecting

strai^t lines; good agreement being obtained up to pH 9.3 after which the points do not fit the line so closely. None the less it is evident that there is a 1 marked change in direction of the pB/'B^ curve and a

straight line has been drawn through these points which intersects the remainder of the curvi e at pH 8,23. The significance of this type of pH/E^ relationship (11) is discussed by Gardner and Lyons ^ ' who state that

each line represents the reduction of a different molecular species and the pK values are given by the

points of intersection.

In table 5 are listed the half-wave potentials

obtained together with some calculated results. Values for the diffusion current constant, which is independent of capillary characteristics, have "been calculated from the expression I = /"d where ia. is the diffusion current, m is the rate of fall of mercury from the electrode and t is the drop time in seconds. The number of electrons, n, participating in the electrode reaction is included and was calculated from the slope of the straight line formed "by plotting the values of E against log ( i (Pigs. 6 11-1 and 7) where ii is the limiting current and i is the current at E volts over the range of the polarographic step. The reciprocal of the slope divided hy 0.059 gives the value of n. The method applies only if the reaction is known to "be reversible^(11 ) FIG. 6 Log -H-7- V. Electrode Potential 'I" +0-7

r /bH i-e / 2-4 / 2 0 /

' fi-- 1 75 / -nsi-e / ft« a-o . /

. -di / - OS -0-7 /-0-8 - 0-7 /o-f

/ • -1-0 +0-7

' ybH 2-9 / /)H 3-4 / /DH3 9 I

; U^ 195 / TIHS / mri-e /

1 . 1 / o ! ' W 1 ! - -0-7 /O-^ - 0-7 /-0-8 -0 7 -0-8 /

-10 + 0 7

^ /jH 4-6 / /^H 4-8 / 6-5 J

-n = 1 3 J 71-0-8 /

-o'g / -0-9 -08 AO-9 -o'9 / -J^

/ «

-JO

Electrode. Pot&ntiai - ^oLts. FIG. 7 Log V. Elcctrodc Potential ii- i

+07f . ' # ' ! ^H 6-8 / M / ^ T8 / ' H s 1-0 / n -1-0 / /

. _ 1 y j 1 1 / f— 1 1' /# 1 Jt ' -0-9 / -10 -to/ M -l-a/ -13

\ ^H B'€ J ^H 91 / 11=1-6 J 11= 1-4 / 0 ' / ' \ J r- - f-• - 1— / fc 1 t -Mt J / -i-e1 . / -i.3 • /

-1-0 -•-10 ; /iHjO-3 / /)H 11-4 / I^H \Z'5 / - 1-6 y ^a^liT / » 1-5 • //

/ • — 1 / 1 I 1 ' • r —1 ' 1• 1 11^ 11 • y -J 4 -17 / -18 / -19

-0-7

EUttroeit PtttntiouL - volts piSOU^SljON

For the purposes of discussion it is convenient to consider separately each of the three pH zones corresponding to the strai^t lines obtained hy plotting half-wave potential against pH (Pig. 5, P- 82).

(a) pH Less than 3.05 Up to pH 3.05 the half-wave potential has an average value of -0.765 volts with respect to the saturated mercury sulphate electrode and is independent of pH. Since the ease of reduction is not affected hy hydrogen ion concentration hydrogen ions do not take part in the electrode reaction. Zuman^^^^, in his study of the polarography of the anthocyanins, found a single wave in acid media up to pH 3.0 and gives half-wave potentials of -O.l4.05 v., -0.i|.0 V. and -0.U25 v. for pelargonidin, cyanidin and delphinidin respectively, approximately 0.35 volts more positive than that obtained here for peonidin. Peonidin differs from cyanidin only by methylation of one of the hydroxyls of the B ring, a difference, which is most unlikely to be responsible for such a large shift in half-wave potential, especially in view of the fact that the presence of one, two, and three hydroxyls on the B ring, as in pelargonidin, cyanidin, and delphinidin cause so small a shift. It is possible that the yalaes obtained experimentally by Zuman using the saturated mercury sulphate electrode were calculated and reported in reference to the more common saturated calomel electrode. Since the latter is 0,14. volts more negative at 23°C this adjustment would "bring Zuman's values to ah out -0.8 volts and could explain the discrepancy with the values found in the present work. However no reference was made in the paper to any such adjustment nor any direct mention made of the electrode to which the results are referred (apart from the e^jperimental details), and it lias not "been possi"ble, as yet, to clarify this point. Zuman considered that this wave corresponded to reduction of the "benzopyrylium ion hut gave no information regarding the mechanism or end products of the reaction. Prom the present results it is now possible to offer a definite interpretation of this reaction.

Calculation of ^N" and the Diffusion Coefficient Two methods are available for determining the number of electrons participating in a polarographic reduction:- (a) The plot of log ( i ) against applied potential. il-i This method is described on page 85 and the results for each wave tabulated in Table 5 (page 83). The average value for the waves "below pH 3.05 is suggesting that two electrons are involved in the reaction. However it should he remembered that this method applies only if the reaction is known to be reversible.

(b) Another method of obtaining n is from the Ilkovic eqiaation, which is n = i^/ 605. D^. C. M^ . t where i^ is the diffusion current in micro-amps, D the diffusion coefficient in em /sec. for the substance being reduced, C its concentration in milli-moles per litre, m the rate of fall of mercury from the capillary in mg./sec. and t the drop time in seconds. The diffusion coefficient for an anthocyanin has never been recorded so that it is necessary to assume, as a first approximation, a value based upon that of a similar molecule. Quinaldinic acid has a diffusion coefficient of 0.08 x cmVsec^^^^ in aqueous

0 sA^ QUINAIiDINIC AOII) solution and its molecular size should approximate to that of an anthocyanin molecule. This value applies to an aqueous solution whereas the present determinations were carried out in methanol. The diffusion 2a coefficient is inversely proportional to the viscosity of the medium^^^^ and the coefficient of viscosity is 0.8cp. for water and 1.8cp. for hOf> methanol at

Hence, assuming that viscosity is the main factor to be considered, the diffusion coefficient should "be approximately half that in aqueous solution, i.e. 0.14- X 10 ^ cm /sec.

The stock solution used to obtain the polaro^rams at pH 1.6 and 2.i<. was 0.6M with respect to peonidin chloride (calculated from optical density) so that the concentration in the polarography solution was 0.15 milli-molar. Substituting this value for 0 in the —R o Ilkovic equation together with D = O.U x lO^^cm /sec. and J^d = 0.l6)iA/iQg t for the polarogram at pH 1.6 mt .t^ a value of n = 0.88 electrons is obtained. Similarly the polarogram at pH 2.h ( /d = 0.139 pi/mg^ t^ ) ml .t^ gives n = 0»77 electrons. It is apparent then, if the assumptions with regard to the diffusion coefficient are correct, that it is a one electron reaction.

A more accurate value for the diffusion coefficient can now be calculated by assuming that n is exactly equal to one. An average value of 0.2? x cm /sec. is obtained. It is to be expected that the anthocyanin molecule, because of the phenyl substituent, would be

larger and hence have a smaller diffusion coefficient

than qainaldinic acid so that the figure obtained for for peonidin chloride is of the correct magnitude.

If n were assumed to be two, as indicated by the log plot method, then the diffusion coefficient would be 6 2 0.68 X 10 cm /sec. which is much lower than is to be

e3Epected of such a molecule.

Mechanism of the Reduction

Under acid conditions, below pH 3, it is reasonable

to assume that the anthocyanin is present largely in the flavylium salt form, VII. This is verified by

the spectra (Pig. 3, page 80) which show a strong

absorption peak at 525 b^i. corresponding to the pres-

ence of the fully con^jugated double bond system. It

is to be expected that an electron would attach itself

readily at the positive centre, neutralizing the charge

and forming a free radical, VIII. Like the flavylium

ion from which it was fomed this free radical could

exist in a number of tautcaaeric forms, two of which

are shown.

+ ^CCH. ,oOCHc ) OH

vnr Y^"

vV There are several poesiMlities for the fate of this free radical. Further reduction can occur to form the flavene, K,, or it can dimerize or decon^^ose. It is not possible to decide from the ezperimental evidence which of these courses is taken "but it is evident that, if further reduction does occur, it must he at potentials beyond -1.14. volts where it is masked by the hydrogen discharge. It is more reasonable to assume that either dimerization takes place immediately upon formation of the radicals, while they are still in the electrode environment, or they decompose as diffusion into the body of the solution occurs.

(b) pH 5.Q5 to 8.25

Reaction Characteristics. All the waves in the region from pH 3«05 to 8.25 show an anodic diffusion current which increases up to pH 7 aud decreases again

(Table 5, page 83). This lowering of the wave to below the zero galvanometer deflection was also noted by

Zuman^^^^ who also observed multiple waves which were not obtained in this work.

As mentioned previously the presence of an anodic wave indicates that oxidation is taking place and the wave resembles that of an oxidation-redmction system (17) typical of which is the (iuinone-hydroq.uinone system^ '' shown in Pig. 8. Curve 1 was obtained with 0.001M. guinone, curve 2 with a mixture of 5 x each of E^ Volts vs. S.CE. ae.

Pig. 8

Polarograms of the Quinone-Hydroq.uinone System

(After Kolthoff and Llngane^^^^ p.21+8) quinone and liydroguinon© and curre 3 wit^ 0.001M. hydroqiiinone. TB/hen only the reduced form is present, curve 3, the wave is cosipletely anodic. Conversely the wave is eathodic when only the oxidised form is present. An equimolecular mixture shows a continuous wave with approximately equal cathodic and anodic diffusion currents. This reaction is laiown to iDe reversible at the dropping mercury electrode. It is prohahle therefore that a similar oxidation-reduction system exists in the anthocyanin solution in this pH range in which the oxidation reaction reaches a maximam at a"feout pH 7.

In this pH range the reaction is dependent upon hydrogen ion concentration as indicated "by the increase in half-wave potential with increase in pH. The plot of half-wave potential against pH (Pig. 5, page 82) gives a straight line with a slope of 0.0585 volts/pH unit which is in good agreement with the theoretical slope as indicated by the equation for a reversible (M) electrode reaction^ '':-

E^ = E® - 0.0591 log IsR - 0.0591 pH. n kRHji

The resemblance of the polarographic waves to the reversible quinone-hydroquinone system and the agreement of the slope of the E^ vs. pH graph with the theoretical slope for a reversible reaction strongly suggest that the reaction of the anthocyanins at the dropping mercury electrode is itself reTersi'ble over tMs pH range.

The plot of applied potential against log ( i_) may il-i therefore he used to calculate the numher of electrons involved in the electrode reaction. The points all fall on straight lines (Figs. 6 and 7, pp. 86,87) and the values of n, obtained from their slopes (Tahle 5), approximate to 1 with the exception of those at pH 3*k and 3.9 which give the values 1.5 and 1.6 respectively.

It will he shown later that the latter two values represent a transition stage between the irreversible reduction of the henzopyrylium ion in acid solution and the reversible electrode reaction predominating in this pH range.

Formation of the Pseudo-base. The spectra (Pigs.

3 and kf PP. 80,81) show that changes are occurring in the molecular species present as the pH is increased.

At pH 3.U there is only a small absorption in the visible region which is obviously due to the presence of some henzopyrylium ion and it has almost entirely disappeared in the solution at pH i|..6. It has been proposed^®'^'^^'^^ ^ and is generally accepted that the henzopyrylium ion, is in equilibrium with a hydrated (leuco-) modification

XI. which is colourless owing to the conjugation having been destroyed. Sondheimer^^^^ using optical measurements, obtained a pK value of 2.98 + 0.06 for pelargonidin-

3-monoglucoside for this equilibrium. The pK value OCH3

OH OH + X OH XT for peonidin indicated "by the b4 vb, pH graph (Fig. 3, p. 82) is 3#05, whieh c

OH fO-""

OH XT ai

This carbonyl group would behave essentially as an alicyclic ketone and is not in conjugation with either of the benzene rings. Such ketones are extremely difficult to reduce polarographically, a half-wave potential of -2.i|.5 volts (S.C.E.) in tetramethylammonium iodide having "been recorded for cyelohexanone, so that the 3-keto group is virtually eliminated as a possihl® site for the reduction.

Evidence fbr Chalcone Formation. Opening of the hetero ring in the pseudo-hase is generally considered to occur and some evidence for this has "been given "by Rihereau-aayon^^^^. Addition of sodium "bisulphite to an anthocyanin causes decolorization which is reversible in the case of a 3-glycoside upon the addition of acid or another carhonyl compound (formaldehyde, acetaldehyde). This would only he possible if ring opening occurs to form the open chain compound, XIII. with the fceto group at 02. In the case of the aglycone, where R s H (XIII), the

O /OCM^

M reaction is irreversible and Ribereau-Gayon attributes this behaviour to the formation of a second ketone graip through keto-enol tautomerism. Reaction with a second molecule of bisulphite can then occur and this step is considered to be irreversible although no reasons were given. RedXLction of the Clialeone, If ring opening does oecur, as indeed seems very proljable, then it allows an e^qplanation of the polarographic hehaviour of the pseudo- base since the chalcone structure is knoim to he reducible at the dropping mercury electrode. Geissman and Friess^''^^ examined the polarographic behaviour of a number of chalcones and found that they were reduced in three stages, each involving one electron. The following series of steps were suggested to aceount for the electrode reactions:-

CH H% CH II k Q, CH 1 OH

hV 5te.|> i A .CH I OH

f\ CH R-odacts. ff ^-O

OH Korslraiiov and Vodzinsfcii ^^^^ also examined the

reduction of unsnabstituted chalcone at the dropping mercury electrode in lithimn chloride solution "but,

contrary to Geissman and Friess, found only one wave

representing a single electrode reduction.

The half-waTe potentials, at pH 6.1, for some of the compounds examined by Geissman and Friess are

given in Table 6. The values have been related to

the saturated mercury sulphate electrode to bring th^

into line with the present work. These values show

that there is a marked increase in resistance to

reduction as hydroxylation is increased so that the

chalcone formed from peonidin, Till (E ss H), should

have a half-wave potential considerably more negative

Table 6

Half-Wave Potentials of Some Ohalconel^^^

Compound 1st Wave 2nd Wave 3rd Wave

Unsubstituted chalcone -1.29 V. -1.52 V. -1.8U V. 2 *-hydroxy- chalcone -1.33 -1.52 -1.87 2', i|.-dihydroxy- chalcone -1.59 -1.8i|. 2',U',i|.-trihydroxy- chalcone -1.50 -1.65 -1.95

than -1.50 volts. Geissman and Friess recorded their values at pH 6.1 in a 50^ isopropyl alcohol "buffer solution. The present detei»niinations were carried out in 50^ methanol "buffer mixtures and at pH 6.1 a half-wave potential of -0.9^5 volts is indicated "by the vs. pH graph (Pig. 5). It is unlikely that the effect of the different solvents in the supporting electrolyte wouH lead to a difference in half-wave potential of 0.5 volts, although this cannot he stated for certain^^^\ An inrportant difference however in the structure of the chalcone formed from peonidin ana those investigated "by Geissman and Friess is the presence of the hydroxyl group on the

I H * II II OH 0 0 O XIV XV

V^CH,- 3-c-cO % g m\ Some polarographie data is aTaila"ble which indicates that the presence of the second carlDonyl groap in a position a, to the first has a marked effect in lowering the half-waye potential. (*) -phenylacetophenone, XVI, has a half-wave potential of -1.38 v. in ©.1M. asmonlvm. chloride^"* ^ while that of henzil, SH, at pH k.9 is only -0.5^4- v., "both values "being related to the saturated calomel electrodeThus it is quite possi'ble that the of-hydroxyl group in the chalcone derived from peonidin is responsible for the very large difference in the half-wave potentials found in the present work and those given "by Geissman and Priess for the chalcone.

In the polarographic reduction of the o(-diketone,

"benzil SIII> Pasternak (29) found a single wave involving two electrons and postulated a reduction to benzoin, XIX. which was confirmed "by controlled potential electrolyses.

In the peonidin chalcone, the cx-diketone, although having the effect of lowering the half-wave potential

of the adjacent carhonyl group, would not itself be readily reduced since it is not in conjugation with a benzene ring and is essentially, as mentioned previously.

+ ae. SAc- B II 0 0 JW an aliphatic ketone group. The mechanigan for the polarogpaphic reduction of the pseudo-hase of peonidin in the ehalcone form, can now he postalated. One electron and a hydrogen ion are added at the dropping mercury electrode to fom a free radical, XXI.

0CH3 OH OCH, H0,>^0H I OH OH

Vc/n -it XX / zs OH OCH^ OH OCH) ho^^^oh .OH I OH t LA Tu Hi VOH H» Xxii

Through resonance this free electron can shift to the oi-carhon atom and then to the oxygen atom attached here, XXII. A second shift is possible into the henzene ring to form a semiquinone, XXIII. The stability of semi- ,(17CM) ) quinones is favoured in alkaline medium^ '' hut in acid medium, such as is now under consideration, the radicals may not have sufficient stability and may not remain at the electrode long enough to undergo a second reduction. Instead dimerization or decomposition would take place thus accounting for the appearance of a single wave involving only one electron. 10U

Oxidation of the Ohalcone, The proposed meelianism also satisfies the need for reTersihility since these wares also show an anodic diffusion cxarrent. Oxidation can take place "by the removal of one electron and a hydrogen ion from the hydroxy 1 group at giving a free radical. The free electron can he transferred, via the

HO OH 11 OH

Hi OH YXJV

> pcPCH , OCHs HO^ .OH 'O 0 0 V OH Hjl XXV

"benzene ring to the conjugated carhonyl group, XXV. so that, in effect, the ketone group, which was reduced previously, has now been oxidized.

Essentially, the mechanisms proposed ahove for the

oxidation and reduction of peonidin pseudo-^hase are merely a modification of the qninhydrone system

discussed earlier and e3q)lain the similarity of the polarographic waves to the latter system. If the pseudo-base of peonidin is present as the chalcone then evidence of its existence should he available from the spectra of the polarography cell solutions.

Unsuhstitated chalcone has reported absorption maxima at 309.3 and 228 mfi. and both maxima are shifted to longer wave lengths by hydroxylation in the benzene rings. Thus

2,3S^»U'-tetrahydroxychalcone (butein), XXVJ, shows maxima at 382 and 263 However, when an hydroxyl

OH HO^ o V

Butein, rgvi group is present on the a-carbon atom, as in the chalcone from peonidin, (S), keto-enol tautomerism can take place and conjugation of the original carbonyl group with the second benzene ring is destroyed. It is to be expected therefore that both bands would be shifted to shorter wave-lengths ani the spectrum would resemble more closely that of benzil, XVIII ^ or acetophenone which show peaks at 319 and 2kk ni|i., and 325 and 250 mp. respectively

The absorption spectra of the cell solutions at pH 3»h and luS (Fig. 3, page 80) show a rise to a maximum where the spectra cut out at 320 mpi. which is in agreement with the above considerations. A shoulder is to be observed at alxmt 370 ffl|i. in the speetram of the solation at pH 3,i+ which may he due to some of the chalcone "being in the enol form. However, if this is so, it is difficult to eacplain the disappearance of this shc«lder in the solution at pH Hone the less the evidence points strongly to the presence of the chalcone form of peonidin in these solutions and supports the interpretation of the polar ©graphic "behaviour outlined a'bove. Formation of the Anhydro-Base. At higher pH values the spectra (Fig. U, p. 81) show evidence of a different molecular species. Thus at pH 7.B peaks at U^O mp.^ and 580 mp. are observed which increase in size and shift to slightly longer wave lengths in more alkaline solutions. These solutions exhi"bited the "blue-purple colour of the anhydro-"base, XXVII. which is in egailihrium with the pseudo-hase, the reaction "being shifted to the right "by the addition of hydroxyl ions.

XWTT At the intermediate pH, "both species appear to "be present together with deccxoposition products since the solution gave only a very weak red colour upon reacidification. This would also aecoant for the imieh

diminished diffusion current at this pH (Table 5, p.83.

Pig.2, v.lk).

Oxidation and Seduction of the Anhydro-Base. The anhydro-hase is a qninone and reduction at the dropping mercury electrode follows the normal pattern of such molecules^^^^ inTolving the gain of one electron and a hydrogen ion to form a free radical of the semiquinone type, XXVIII. The fate of this free radical would he

similar to that fomed from the chalcone, as discussed previously, since the solutions under consideration are only moderately alkaline and the plot of E against log ( ) (Figs. 6 and 7, pp.86,8?) still point to il-i

a one electron reaction.

.OCHj 0CH3

OH The oxidation reaction ©an "be explained in mck the

same way as previcmsly. Oxidation of the phenolic hydroxyl groups, or the hydroxyl at position 3> eaii take place leading to the formation of a free radical, XUXf

following the loss of one electron and a hydrogen ion.

Half-ware potential® of the order obtained in these investigations conld reasonably be expected of such a complex molecmle since the half-ware potential of gainone at pH 6»2h in 75% ethanol, related to the saturated caloEiel electrode^^^^, is lowered by substitmtion with hydroxyl or methoxyl groups. fhas tolmquinone, JJX. has a half-wave potential of +0,05 v., fumigatin, XXXI^ -0,16v.

and spinmlosin, XXXII. -0.30 v. If the half-wave potential

of peonidin at pH 6.2k (Fig.5, p.82) is related to the

saturated calomel electrode (-0.i|. v.) a value of -0.55 v.

is obtained which is comparable with that of the less

coi^licated quinones when the effect of substituents is

taken into account.

^on V HaCo'V o 0 m jwr\ The pK value for the transforaiation of the pseudo-T^ase

to the arOiydro-hase would not reveal itself on the Ej vs. pH graph (Pig. 5) as it did with the flavylium salt - pseudo-base transformation "because the electrode reactions

are essentially the same. Each involves the oxidation of

hydroxyl groups or the reduction of carhonyl groups and is

dependent to the same extent upon hydrogen ion concentration.

Henco no point of inflection is to he expected on the pH vs.

Ei graph.

(c) -DH Greater than 8>25

The plot of half-wave potential against pH (Fig, 5)

shows a further point of inflection at pH 8.25 followed

"by a sharp increase in slope indicating an increase in

hydrogen ion dependency.

A similar effect was noted "by Qeissman et al.

in the polarographic reduction of the flavonoids hut only

with those compounds having free hydroxyl groups in the

1+', 5 or 7 positions, yxxill. They concluded that the

effect was due to resonance of the ionized phenolic

group with the carhonyl group at position i4.. The negative

charge on the molecule is transferred to the carhonyl

xmn group giving it a negative charge BO that a more negative potential is required to add further electrons at this site. This hypothesis can he applied to the anthoeyaain anhydro-hase molecule where the hydroxyl groups at 5 and 7, XXXIV a and h, are ionizahle and can resonate with the guinone group at U' thereby retarding reduction. The increased resonance of such a molecule in the ionized state woald also contribute towards its stability.

y ^ J

OH a b XXXIV

The point of inflection, pH 8.25, on the pH vs. E^ curve (Pig. 5) would therefore represent the pK^ (25®C.) for the ionization of the phenol groups on the A ring. As is to be expected this value lies between that of resorcinol, pKa (20-25®C) = and phloroglucinol, pK^ (20-25®a) =

However this theory does not account for the increased diffusion ciirrent in the strongly alkaline solutions. Reference to Table 5 (p.83) shows that the diffusion current constants, /d increase in alkaline solution mFTF" to more than that required for a one electron reaction. It is peasonalDle to assxane that the diffusion coeffic-

ient of the anhydro-base would "be close to that of the

flavylium ion so that the diffusion current constant

in acid solution (0.16 at pH 1.6) would still represent

a one electron change in the alkaline solutions. It

is ohTious therefore that, in the latter solutions,

reactions involving more than one electron are taking

place. This fact is confirmed by the plots of B vs.

log i which show values for n increasing in alkaline il-i solution up to 2.3 at pH 9.3 and then decreasing to 1.5

at pH 12.3.

The addition of a second electron csin be accounted

for by assuming that the semiquinone formed in the

first step is reduced farther to the phenol. This

reaction would be possible in alkaline solutions because

of the increased stability of the semiquinones under

alkaline conditions^^^^ which would allow them to persist

at the electrode and undergo the second reduction.

Although the diffusion cvirrents show an increase beyond tliat required for a one electron step they do not increase to the extent of a two electron addition.

Two reasons can be advanced to accoiint for this.

Firstly the semiquinone is probably sufficiently stable

to undergo the second reaction only when it is formed from an ionized molecule because of the increased resonance energy of such molecules. Hence not all the molecules undergoing the first reduction would "be capable of participating in the second reaction. Secondly the concentration of the anhydro-hase, at the time of reduction, would be less than the actual amount of peonidin added because of decomposition under the alkaline conditions. Evidence for this latter theory is to be seen in the fluctuations in the diffusion current of the polarograms in this range. Although they all show a general increase in diffasion current there are large variations which can only be accounted for by differences in concentration of the reactive species. It is to be noted that the anodic wave disappears as the pH is increased above pH 9 and is entirely absent at pH 12.3 (Table 5) indicating that some change takes place which alters the anhydro-base so that it can no longer undergo the oxidation reaction. The only change that can be envisaged is the ionization of the phenolic groups as originally described but no reason can be advanced to suggest why electrons cannot be removed from a negatively charged group such as a phenoxy ion. No report of this effect having been experienced by other workers has been foiind in the literature relating to the polarography of the quinones and no explanation can be offered at pressit. m

CONCLUSIONS

The polarogpapMc behaviour of an anthocyanidin, peonidin chloride, has "been examined over the pH range 1.6 to 12.3 and meehanisms have "been proposed for the electrode reactions. These mechanisms may- he summarized as follows:- In strongly acid solutions the flavylium ion is present and is reduced in a one electron reaction which does not involve hydrogen ions. Reduction of the heterocyclic ring occurs to form a free radical which is not further reduced at the electrode. The flavylium salt is in equilibrium with the pseudo-base (pK, 3.05) and in slightly acid solution (pH 4-5) this form predominates. The pseudo-base is not itself reduced at the dropping mercury electrode but exists in solution as the chalcone which is able to undergo an oxidation- reduction reaction similar to the guinone-hydroguinone system. Once again free radicals are formed by the loss or gain of one electron and a hydrogen ion and are not further oxidissed or reduced at the electrode. The pseudo-base is, in turn, in equilibritim with the anhydro-base which possesses a quinone structure and undergoes a similar oxidation-reduction reaction. The anhydro-base predominates in alkaline solution (greater than pH 7) "btit as the pH rises the hydroxyl llii groups "become ionized conferring a negative charge to tlie molecule so tliat addition of electrons 'becomes more difficult. This is ohserred as a sharp rise in the pH vs. E^ graph at pH 8.25 corresponding to the pK for the ionization of the phenolic groups. The increased resonance of the ionized molecule confers greater stability to the semiquinone formed on reduction and it remains at the electrode to undergo a second addition of an electron and a hydrogen ion to form a phenol. At the same time the oxidation reaction ceases hut no explanation can he offered to account for this hehaviour. Of particular interest is the mechanism proposed for the redaction of the pseudo-base since it is necessary to postulate the formation of the chalcone for a satisfactory explanation. The peonidin chalcone differs from those which have previously "been investigated by the presence of an ot-hydroxyl group which, through formation of a tautomeric ketone form, alters both the polarographic and spectral characteristics of the molecule. The chalcone formed from the 3-glycoside would not be capable of forming the keto tautomer and it would be of considerable interest to investigate such an anthocyanin to prove this point. APPENDIX I

PolarograT3Mc Cell

Details of the polarographic cell used in these

experiments are shown in Pig. 9.

The cell and reference electrode were inaaersed in a

water hath thermostatically controlled to 25® +

The dropping mercury electrode, "D", consisted of

a six inch length of capillary tuhing connected hy a

length of "Tygon" tuhing to a 100 ml. reservoir of

mercury. The height of mercury ahove the tip of the

electrode was ad^sted to 30 cm. so that 2.25 mg,

mercury per sec. flowed from the capillary. A plat-

inum wire dipping into the mercury in the reservoir

connected the electrode to the rest of the circuit.

Circuit

The circuit of the polarograph and details of

component parts are presented in Pig. 10.

Sensitivity

The sensitivity of Ihe instrument was adjusted "by

means of the Ayr ton shunt "A" and the rheostat "E2"

and was set at the "beginning of each run so that a

potential of 0.5 volts gave a galvancsaeter deflection

of ifO cm. With a 200,000 ohm resistance, "R5", in

the circuit the current was 2.5 micro-amps, which

represents O.O625 micro-amps per cm. galvanometer deflection. Calibration In order to check the potential of the mercury sulphate reference electrode and to detect aiay stray potentials in the circuit a polarographic curre was made, at intervals throughout these investigations, using a substance whose half-wave potential was known. An 0.001 molar solution of zinc acetate was used in a supporting electrolyte of 0.1 molar potassium chloride. This has an observed half-wave potential of-1.02 volts with respect to the saturated calomel electrode at 25^0. so that the half-wave potential for the zinc ions, with reference to the mercury sulphate electrode, should be -1.U2 volts. A typical curve obtained using zinc acetate in 0.1 M, potassium chloride at 25^0. is presented in Pig. 11. This shows a half-wave potential of -1.39 volts which is in good agreement with the literature.

Mercury The mercury used in these investigations was purified before use in the following mannerAir was bubbled for several hours through the mercury under a weak solution of mercuric nitrate in distilled water. The mercury was then dropped into distilled water by means of a filter funnel which had its stem drawn out to a fine point. This operation was carried out several times until, following vigorous shaking with water, the mercury formed a foam wMch lasted for at least 5 seconds. It was then dried "by dropping into a dust-free "bottle from a folded filter paper with a small pinhole punctured in its apex. Fia. 9 Polarographic Cell

H c m

-il'-J

A = Nitrogen exit seal. B = Nitrogen inlet. C = Sample inlet. D = Dropping merciary electrode. E = Mercury pool connection. F = Connecting "bridge containing cell solution., G = Agar plug saturated with potassium sulphate. H = Saturated potassium sulphate "bridge. I = Saturated mercury sulphate electrode, M = Mercury pool anode. Circuit and Details of the Polarograph & L

J L

R.I

—IAAAAAAAAAAA. R2 T

R3 W J R 5 -3V. rr

MERCURY POOL MERCURY SULPHATE DROPPING MERCURY ANODE ELECTRODE CATHODE

A = Pye Universal Ayrton Shunt, Resistance 10,000 ohms, Sensitivity range 1/1000 to full. L = Lamp and curved scale, situated at 1 metre from the Galvancxueter, Q = Carahridge D'Arsonval 50 ohm Galvanometer. P = Doran P.O. Potentiometer. W = Standard Weston cell. 50 ohm resistance. R2= 3,000 ohm wire wound radio potentiometer- R3= 25 ohm wire wound radio potentiometer. RU= 170 ohm wire wound radio potentiometer. R5= 200,000 ohm + 1% precision resistance. 20 - — "I > z O I- § o u. f a > o oc. o tiJ UJI 5 ~ ! 39 V § M o Q • N H M H 3 O > O M >

10 -

± ± -0-5 -HO -1*5 ~20 E.M.F, - VOLTS APPLIED ro o APPiaiDIX 2 Synthesis of Peonidin Chloride The peonidin chloride used in these experiments was synthetically prepared commencing with phi or o- glucinol and veratrole from which was prepared O-henzoylphloroglticinaldehyde and »,i|.-dlacetoxy-3-" methoxyacetophenone respectively. Condensation of these two compounds was then effected according to the method of Murakami and Eohinson^^^^ to yield 3-0-benzoylpeonidin chloride which was then dehenzoylated to peonidin chloride. PhlorQglucinaldehyde. The method used was a modification of the preparation of m-xylorcylaldehyde "by Robertson and Eobins Yield .. 30 g., 2-0-benzoylphloroglucinaldehyde ^^^) Yield .. 10.5 g., 37. M.P. .. 172-173^0. Chloracetyl Chloride(^Q) Yield .. ka g., B.P. .. 108-110OC. O) -chloracetovanillone^^'') Yield .. i|1 g., 66%

CJ -ac et oxy"3-methoxy-ij.-hydroxyacet ophenone(26 ) Yield .. II4..3 g., kS^ M.P. .. 103~10l4.°C. 6J. U-dia eet oi.y-3-me thoxyae et ophenone Yield .. 15.2 g., 88^. :.p. 70-72°0. ^O-benzoylpeonidln chloride^(25) Yield .. 0.28 g., 5%. Peonidin Chloride^^^^ Yield .. O.II4. g., 70%. BIBLIOGRAPHY

1. Adkins, H. and Cox, P.W., J.Am.Ohem.Soc., 60, 1151 (1938). 2. Bergel, P., Jacob, A., Todd, A.R. and Work, T.S. J.Chem.Soc., 1958. 1375. 3. Bhalla, A.L. and Ray, J.N., iMd., 1955. 288. I4.. Britton, H.T., "Hydrogen Ions", Vol.1, p.30it. Chapman and Hall, London, 19^2. 5. Cliarlesworth, E.H., Chavan, J.J. and Robinson, R., J.Chem.Soc., 1955. 370. 6. Elstow, W.E. and Piatt, B.C., Chem. and Ind., 1950. 82U. 7. Sngelkemeir, D.W., Geissman, T.A., Crowell, W.R. and Priess, S.L., J.Am.Ohem.Soc., ^,155 (19^+7). 8. Pieser and Pieser, "Organic Chemistry", Heath and Co., Boston, 1950, p.605» 9. ibid. p.66i^. 10. Posenka, E.L., J.Chem.Soc., 19U7. 1683.

11. GardnerAppl.Chem, H.J. . (Australia)and Lyons. , L.E.,13I4 Rev.Pur. (1953)e . and 12. Geissman, T.A. in "Modern Methods of Plant Analyses", Paech and Tracey, VIII. Springer- Veriag, Berlin, 1955. 13. Geissman, T.A. and Priess, S.L., J.Am.Chem.Soc., 21, 3893, (19U9). Ilj.. Gillam, A.E. and Stem, E.S., "Electronic Absorption Spectroscopy", Arnold, London, (195^1-). 15. Karrer, P., Trugenberger, C. and Hamdi, G., Helv.Chem.Acta, 26, 2116 (19U3)- 16. Kaye, R.C. and Stonehill, H.I., J.Chem.Soc., 12S1, 27. 17. Kolthoff, I.M. and Lingane, J.J., "Polarography", Interscience, N.Y., 19U1. 12ij.

18. Korshunov, I.A. and Vodzinskii, Y., Zhup.Fiz.KMm., 22, 1152, (1953): C.A. ii8, 56?^+ (195^1-). 19. Kahn, R. and Winterstein, A., Chem.Ber., 65, 17^12, (1932): C.A. 2Z, 7214. (1933). 20. Leon, A., EolDertson, A., EoMnson, R. and SeshadPi, T.A., J.Chem.Soc., 1951. 2672. 21. Lingane, J.J., J.Am.Chem.Soc., £1, 2099 (1939). 22. Lowenbein, A. and RosenTDaiam, B., Ann.Chem., Iti^^. 223 0926): C.A. 20, 3167 (1926). 23. Maureau, H., Cc^pt.rend., 188» 50i+ (1929): C.A. 23, 2705 (1929). 2I4.. Mueller, O.H., in Weiss"bepgep, "Pibysical Methods in Organic Chemistry", Interscience, N.Y. (1914^), p. 1190. 25. Murakami, S. and Robinson, R., J.Chem.Soc., 1928« 1537. 26. Nolan, T.J., Pratt, D.D. and Robinson, R., ibid. 1926. 1968. 27. Nordstrom, C.G., Acta ChQa.Scand., 10, 1i4.91 (1956). 28. Page, J.B. and Robinson, F.A., J.Ch«3i.Soc., 19U3. 133. 29. Pasternak, R., Helv.Chem.Acta, H, 753 (l9i+8). 30. Perrypp. , 37J.H.2 an, dChem 1686. .Eng.Handbook , McGraw Hill, (1950) 31. Pratt, D.D. and Robinson, R. J.Chem.Soc., IgJ, 7^5 (1923). 32. Pratt, D.D. and Robinson, R., ibid. 12^, 188 (I92if). 33. Pratt, D.D. and Robinson, R., ibid. 122, 1128 (1925). 3U. Ribereau-Gayon, P., "Researches sur les Anthocyannes des Vegetaux", Librairie Generale de I'Enseignment, Paris, 1959. 35. EoTDertson, A, and Robins on, E., J.Chm.Soc., 1222, 2196. 36. Sondheimer, E., J.Am.Chem.Soc., "^507 (1953). 37. Stock, J., J.Ohon.Soc., 19ti.9. 586. 38. Vogel, A.I., "Practical Organic Chemistry", Longmans, London, (i9i<.8), 168. 39. iMd. p. 311. kO. iMd. p. 1361+. M. Willstatter, R. and Mallison, H., Ann.Chem.. 1+08. 15, (1915). ^ l|.2. Zeigler, K.. Friess, F.A. and Salzer, F., i"bid. 1^18, ^ 21^9 (1926): C.A. 20, 3167 (1926). 1^3. Znman, P., Collection Czech. Chem. GosBnan., 18, 36, (1953). PART 2b

EO>LOBATOSY IFySSTIQATIOHS

THE DESTHUGTIOH OF AmEOCHmiSQ

DURIHQ PROOESSINQ IMTRODUGTIOlf

This section reports some exploratory results on the "behavioar of anthocyanins during the processing and storage of foods.

Most of the previous work in this field has "been carried out on strawberries and the strawberry anthocyanin, pelargonidin-3-iiioiioglTieoside. This situation has arisen because of the economic importance of strawberries, particularly in the U.S.A., and the sensitivity of this fruit to discoloration. Very little work has been carried out on other fruits and the tendency has been to use the results of the investigations on strawberries to predict the behaviour of other pigments - a procedure which could lead to errors since it has been observed^ that the strawberry pigment is far more stable to heat treatment than the anthocyanins derived from a number of other fruits.

Experimental work in this section has been aimed at investigating the extent of anthocyanin losses during the processing of blackcurrants under various conditions. LI^EEATUBE SURVSy

It is convenient to group previous results ®n anthocyanin stalDility under a number of headings, viz.- effects of temperature, oxygen and oxidizing agents, sugars, and other miscellaneous factors. In the present discussion the effect of pH has "been considered in relation to the various other factors under the appropriate headings. A discussion is also included on the relative stability of a number of different anthocyanins. Finally, reference is made to various methods available for the quantitative estimation of anthocyanins in foods.

Effect of Temperature It has been found by Nebensky et al. ^^^^ and by Decareau et al.^^^ that high temperature (greater than ) is one of the most specific agents responsible for colour deterioration during the storage of anthocyanin containing foods. Mackinney, Lukton and Chichester^^^^ investigated commercial processes for the manufacture of strawberry preserves and found that from 50 to of the pigment may be lost during cooking. These workers devised a vacuum pan concentrator which, operating at a vacuum of 29.6 in. and a pan temperature of 93resulted in oiay 10^ loss of pigment.

Using model systems consisting of the strawberry anthocyanin, pelargonidin-3-ffionoglucoside, in "buffer (iS) solutions at pH 2.0 and 3.^ Markakis et al. ^ ^ olDserved tlaat "between and 110°C., in the a"bsence of oxygen, the destruction of the pigment was a first order reaction. A red-brown precipitate was formed and when G^^ labeled anthocyanin was degraded S5% of the radio-activity was located in this precipitate.

These workers consider that the degradation might proceed through opening of the heterocyclic ring to form the chalcone X which is then further degraded to the brown precipitate. As evidence for this theory the work of Berson^^^ is cited in which it

iO" 0-Glucose XOH Pelargonidln-3- monoglucoside was shown that crystalline triphenylpyrylium pseudo- bases, obtained from the corresponding oxonium salts,

II, occur in the open diketone form. III, only, and

on standing or warming fade with the formation of m

"benzoic acid, IV, and a neutral oil, V. The diketone, Ill, was found to have an absorption maximum at 21+7 xap. and Markakis et al. ^^^^ observed the appearance of an absorption maxiisuffi at 230 mp during the degradation of pelargonidin-3-nionoglueoside. o II 0 o OH 0 R-C-OH + U I R-C c-R R-C W r R V V CH 1 Jl H Y

A Similar red-brown precipitate was found by Lukton, Chichester and Mackinney^^^^ to arise from the oxygenation of this pigment while only minute amounts occur in the absence of oxygen. The precipitate was isolated and was considered to be a polymer of the glycoside on the basis of its high melting point, chronatographic behaviour and the appearance of glucose on acid hydrolysis. Hydrolysis of the glycoside to the aglycone may also occur during processing of anthocyanin containing foods, especially when highly acid foods are encount- ered. Kohman^^^^ considers that the rate of hydrolysis is probably doubled for every 10*^0. rise in t^aperature. In investigations on the enzymic degradation of cyanidin- 3-glucoside Huangfound that hydrolysis occurred to m the aglycone which decomposed more rapidly than its glucoside. This finding contrasts with the work of Lukton et al. ^^^^ previously reported where the degradation, under different conditions, occurred without previous hydrolysis. It is also to "be expected that the aglycone would "be less soluble than its glycoside and may precipitate from solution. Colquhoon^^^ states that as a result of hydrolysis the colour "base is thrown out of solution as a "brown amorphous mass "but it would appear more probable that such a precipitate is an anthocyanin decomposition product, of the type discussed previously, rather than the aglycone or its colour base.

Oxygen and Oxidizing Agents The detrimental effect of oxygen upon anthocyanin stability during processing and subsequent storage has been noted by a number of workers Lukton, Chichester and Mackinney^^^^ studied the rate of anthocyanin breakdown in buffer and in strawberry juice and found that, in both cases, it was much faster and pH dependent in oxygen whereas in nitrogen the pH had little effect. A red-brown precipitate was formed, the nature of which has been discussed above. Tinsley and Bockian^^^^, investigating the effect of a number of factors upon the destruction of pelargonidin-3-Bionoglucoside in "buffer solution, noted that the presence of oxygen (as air) enlianced the harmful effects of 5-hydroxylmethylfurfural and various sugars. In discussing this "behaviour the authors refer to the work of Wizinger and Luthiger^^^^ and Black"burn et al« ^^^ where it was shown that flavylium salts condense with dime thy lani line, diarylethylenes and malonic acid to give the products

VII and VIII respectively. The anthocyanin

kA/' CH CH R-C-R

+ MlCH,)^ VII Vl wr was found to "be active as the pseudo-hase and oxygen was required for the reaction, conditions which are similar to those found "by Tinsley and Bockian to increase the rate of anthocyanin destruction in model systems, so that these authors ccnsider that similar oxidative couplings might "be involved.

It has "been noted^^'^^^ that the addition of ascorbic acid to an anthocyanin solution caused an increased loss of colour and this change was further accelerated "by high temperature and the presence of air. This olJserTation was csirried further by

Sondhelmer and Kertesz^^^^ who related colour loss with ascorbic acid oxidation. No loss of colour occurred when ascorbic acid was added to anthocyanin solutions under anaerobic conditions proving that oxidation products and not ascorbic acid itself were necessary for anthocyanin destruction.

That a mutual destruction of ascorbic acid and (25) anthocyanin occurs was shown by Timber lake ^ who found that addition of anthocyanins, isolated from blackcurrants, to ascorbic acid solutions greatly increased the oxygen uptake and the loss of ascorbic acid. At the same time a considerable loss of anthocyanin was noted.

This behaviour has been explained^^^ by postulating the formation of hydrogen peroxide as a by-product of the oxidation of ascorbic acid which then oxidises the anthocyanin to form insoluble decomposition products.

Some evidence for the formation of hydrogen peroxide, under special circumstances, is found in the work of

Decker and Dickinson^^^ and Silverblatt et al. who showed that it arose from the oxidation of ascorbic acid to dehydroascorbic acid in the presence of oxygen and cupric ions.

The action of hydrogen peroxide upon anthocyanins has been investigated by Karrer et al. ) who olDtained malvone, from the oxidation of malvin (malvidiii-3,5~diglticoside). The kinetics of this

. ^ ^OtHj • O -Glucosft 0-GlucoAft 00 ci«osI * R = H or OH S reaction, for pelargonidin-3-Kionoglucoside, were (22) investigated "by Sondheimer and Eertes25^ ' and mechanisms were proposed for the decomposition. Suigars Nehensky et al. working with model systems of strawberry and currant anthocyanins and with normal packs of •blueberries, cherries, currants, grapes, raspberries and strawberries found that sugar had only a slight effect upon anthocyanin decomposition during storage. Likewise Decareau et al. stated that changes in sugar composition did not greatly influence the half life at 100^P. of the anthocyanin in stored jellies. However a number of workers have found that the presence of sugars and their decomposition products adversely effect the stability of anthocyanins.

Meschter^^^^, working on the storage of strawberry syrups, found that anthoeyanin degradation increases

"below pH 1.8. This was attributed to decomposition of the stigar to aldehydes whichfreact with the anthoeyanin to give colourless products, The addition of furfural or hydroxymethylfurfural was found to mrkedly increase the rate of decon^osition of the anthoeyanin.

This latter finding was confirmed by Markalsls et al. ^^^^ using pelargonidin-3-monogl'ucoside in model systems.

Tinsley and BoclLian^^^^ using the same anthoeyanin in buffer solutions at pH 3»k studied the effects of various carbohydrates at 90°C. The most rapid decomposition occurred in the presence of glucuronic acid and the ketose fructose (first order rate constant, k, 7*91 X lO^min."'^ in air and 2,80 x lO^min.""^ in nitrogen). Increase in concaitration of glucose, fructose or sucrose increased the rate of pigment degradation as also did the addition of furfural or

5-hydroxymethylfurfural. The decomposition products of the latter compound in acid solution, levulinic and formic acids, although increasing the rate of pigment destruction did not have as marked an effect as the parent compound.

It is evident therefore that sugars or their decoBg>o8ition products can be important factors in anthocyanin destruction in foods although the findings of Decareau et al>^^^ and Nebensky et al,^^^^ suggest that protective systems may soiaetiHies operate so that the effect is not of ma^or significance*

Other Factors Tinsley and Bockian^^^^ studied the effects of a number of amino acids, alone and in the presence of sugars, upon the breakdown of pelargonidin-3-monoglucoside in buffer at pH at 90^0. and found that their effect was only a minor one, A composite system consisting of pigment, sucrose amino acids and ascorbic acid at concentrations similar to those observed in strawberry ^uice was examined by these authors and it was found that the rate of degradation was almost equal to natural strawberry ^uice. It was concluded therefore that these materials are responsible for most of the degradation occurring in this medium.

The Relative Stability of Anthocyanins A number of naturally occurring anthocyanins were isolated from various fruits, using paper chromatography, and their behaviour at 57.2^0. in buffer at pH 2.0 examined by Lamort^ The degradation of all the anthocyanins were first order reactions and considerable variations were observed in their coefficients. By far the most rapidly reduced pigment was a cyanidin derivative fcnand in "blueberries, "blackcurrants, raspberries and cherries which had a reaction coefficient of

2,900 X 10"^ours"^. Other pigments, derivatives of cyanidin, peonidin and delphinidin, showed coefficients varying between 200 x to 650 x 10"^aars""^ with the exception of the pelargonidin derivative of strawberries (pelargonidin-3-monoglucoside) which was much more resistant to degradation, having a coefficient of

72.3 X 10"^ours"^,

In a previous experiment on the degradation of blackcurrant pigments ^^^ it was noted that on bubbling air through solutions of blackcurrant pigments in buffer at pH 3.5 and at 100^0. the delphinidin glycosides suffered slightly greater degradation than the cyanidin glycosides. Although this result would appear to contradict the results of Iiamort, it should be noted that oxMation reactions would be more prominent in the former since air was bubbled through the solutions whereas Lamort carried out his experiments in sealed tubes. The latter worker does not give details of how much air was trapped in the tubes with the anthocyanin so that it is difficult to draw comparisons between the two sets of results.

E^eriments to be reported in this thesis have been m aimed at evaluating the extent of degradation to "be expected in the processing of "blackcurrant products.

The use of model systems has been avoided in order to approach more closely processing conditions.

Quantitative Estimation of Anthocyanins

The property of anthocyanin solutions to a'bsor'b light strongly "between 500 and 550 mfi. provides a very useful means for their estimation. However a complic- ation arises "because decomposition of the anthocyanins gives rise to secondary red-"brown materials which absorb in this region of the spectrum. In food products in particular brown discolorations appear, inter alia from the enzymic and non-enzymic browning reactions, which are responsible for interference at these wave lengths. It is obvious therefore that the absorption at the X max. of the anthocyanin would be quantitatively related to the amount present. This is well illustrated in Pig. k where it can be seen that the peak at 520 mp., due to the anthocyanins, disappears but the absorption at this wave length actually increases due to the formation of brown products.

Sondheimer and Kertesz^^^^ developed a method for the estimation of anthocyanins in strawberries which depends upon the decrease in intensity of absorption of anthocyanins with increase in pH. The method devised "by these workers was to macerate the strawberries with citrate-hydrochloric acid "buffer at pH 3.4 and then determine the optical density of an aliquot at 500 mp. Another aliquot was adjusted to pH 2.0 with hydrochloric acid and the optical density also measured at 500 mfi. The difference in absorh- ance was then related to the anthocyanin content of the strawberry extract. Measurement of the absorption of an anthocyanin solution followed by bleaching of the pigment with sodium sulphite and a second measuroaent to obtain the background absorption was used by Dickinson and Gawler^^^ in an investigation of the roles of anthocyanins in the corrosion of tinplate. It was shown that the addition of sodium sulphite had no effect upon the brown pigments in an old sample of plum juice from which the anthocyanin had completely disappeared so that the difference between the two readings (before and after sulphite addition) was directly related to the amount of anthocyanin present. This was confirmed by obtaining satisfactory recoveries of anthocyanin added to plum juice.

Swain and Hillis^^^^ modified this method for use with methanolic extracts from Prunus domestica. It was found that methanolic solutions became cloudy JM upon the addition of sodium sulphite and this reagent was replaced by hydrogen peroxide, Decolorization of the anthocyanins was found to he virtually complete in 5 minutes and the reagent showed "no serious influence" on the colour of other substances present in the extracts. It appeared that either of the latter two methods would he the most convenient for following changes in anthocyanin content during the cooking of blackcurrants and some preliminary e^eriments were therefore carried out to determine which was the more suitable.

««« 1M

BXPBRIMMTAL

Materials

Blackcurrants. The blackcurrants were the "Whitehud" variety, grown in Tasmania and picked during the 195i4--55 season. The fruit was held at -1G®C. in internally lacquered l4.-gallon cans until required for use.

Pulp. After thawing, the "blackcurrants were pulped in a comminuting machine using a 3 inm. screen. The pulp was thoroughly mixed and packed in kOO g. lots in plastic hags which were then heat-sealed and frozen at

-10^0. until required.

Serum. Several pounds of thawed "blackcurrants were drained and macerated in a hlendor. The pulp was then filtered through a double layer of muslin under vacuum.

Several preparations of serum had to he made during the course of this work because fermentation occurred even though the material was stored under refrigeration.

Buffer. The buffer was prepared by dissolving crystalline sodium acetate g.) in water, adding hydrochloric acid (UI4-8 ml.) and diluting to 2 litres with distilled water. The pH, checked with a glass electrode, was foond to be 1.8.

Optical Keasurements. All spectrophotometrie measurements were carried out on a Beckman model B spec trophot ©meter. IM

Metbiods Estimation of the Aathocyanins. Blackcurrant jam was prepared W boiling blackcurrant pulp (100 g.) with commercial sucrose (150 g.) for 10 minutes in an open beaker. The resulting was then used for experiments on anthocyanin estimation. (a) Sulphite procedure. A stock solution of jam extract was made by mixing jam (10 g.) with buffer (pH 1.8) and making up to 100 ml. The mixture was centrifuged to provide a clear solution, an aliquot of which (25 ml.) was diluted to 50 ml. with buffer (Soln. A). Some of this solution was placed in a spectrophotometer cell, a small quantity (3-^4- mg. ) sodium sulphite added, and the contents mixed thoroughly (Soln. B). (b) Peroxide procedure. At the same time a second dilution of jam extract was prepared consisting of 5 ml. extract and 30^ hydrogen peroxide (0.3 ml.) made up to 10 ml, with buffer (Soln. C). leadings were made at 515 m|i. and the optical densities recorded. (c) Comparison of procedurea The curves of optical density against time are shown in Fig. 1 where it can be seen that the minimum reading at 515 was obtained with sodium sulphite within 5 minutes and thereafter there was no change (curve B). With hydrogen peroxide as bleaching agent however (curve C), ik3

F1G> I

CHANGE m ABSORBANCE OF ANTHOCYANH4 SOLUTtONS WITH TIME

» i I lllilMI ill I > .1 ^ 20 40 60 TIME — Minutes

FIG, 2 SPECTRA OF TREATED SOLUTIONS hO

A Anthocyanin extract

08 B.... „ „ + sulphfte

C « „ + peroxide

t 0-6 z UJ o 04 -I

8 0'2

11 t lii, irfii iiiii i ± I I I I .1 300 400 500 60C WAVE LEmXH - nw. JM the optical density showed a steady decrease for 20 minutes although after this time the decrease was much slower. The absorption of the straight line section of the peroxide "bleached solution is much lower than the limiting absorption for the sodium sulphite and to check that this was not due to insufficient sulphite an additional quantity was added when no change in optical density was recorded. The spectra of the three solutions, after 60 minutes, are shown in Pig. 2. The peak due to the anthocyanins is evident at 513 Bp. in curve A and has almost entirely disappeared in the sulphited solution, curve B. The peroxide bleached solution (C) differs from B by the complete elimination of this peak. If the peak at 500 nqi. in B was due to the presence of unchanged anthocyanins it would have been affected by additional sulphite. Since this did not occur it must be due to some other material (possibly anthocyanin decomposition product). Hydrogen peroxide also bleached this material and it is therefore unsuitable for anthocyanin analysis in these extracts. The method involving addition of sodium sulphite was chosen for the estimations in tMs work. IM

Anthocyanln Degradation In Blackcurrant Palp

For each experiment a Taag of frozen Tjlaekctirrant pulp was thawed and 100 g. set aside for a parallel e^eriment involving the addition of sugar. The remainder of the palp was placed in a 500 ml. round- hottomed flask fitted with a side am and a refl\xx condenser. The flask was heated on a mantle controlled hy a "Simmerstat", set to maintain the contents at a fast hoil. The flask was shaken at 5 minute intervals to ensure even distribution of the contents. Samples were taken "by inserting a 25 ml, pipette, from which the tip had heen roaoved, through the side arm of the flask and withdrawing sufficient material to provide a 10 g, sample. The sample was inmediately mixed with buffer at pH 1.8 and made up to 100 ml. as discussed previously. Samples were rmoved gust at the commencement of boiling and at 10, 20 and 30 minutes thereafter. The temperature measured in the centre of the boiling pulp was 100°G. and at the conclusion of 30 minutes the pulp had a solids content of 1i4.°Brix. After the sample solutions had settled sufficiently to allow the removal of clear solution (approximately 30 minutes) 1 ml. aliquots were diluted to 10 ml. with more buffer. The diluted sample was then transferred 1U6 to two spectpophotometer cells and to one was added a small spatula tip (3 - U mg.) of sodiTom sulphite. The a'bsorption of each solution was then read at 520 mji, using buffer solution as a blank. This experiment was repeated six times and the results are presented in Table 1(a). A further series of six experiments was carried oat in which oxygen at a rate of 80 ml./min. was introduced into the atmosphere above the boiling pulp by inserting a glass tube down the centre of the reflux condenser to a distance of 1 cm. above the surface of the pulp. The oxygen was saturated with water vapour before entering the flask. The results are presented in Table 1(c). Six experiments were also carried out in which oxygen, at the same rate of flow as above, was bubbled through the boiling pulp by lowering the tube to the bottom of the flask. The results are given in Table 2(a).

Ptilp plus Added Sugar The 100 g. of pulp removed frcaa the package prior to each of the previous experiments was mixed with commercial cane sugar (150 g.) and the mixture treated in the same manner as previously except that 25 g. samples were taken for pigment analysis. The temperature in the centre of the boiling ^am Ik! was 107^0, and at the end of 30 minutes the pulp had a soluble solids content of 72®Brix. These experiments were also carried out six times (results Table 1(h)) and repeated with oxygen "blown onto the surface (Table i(d)) and then through the pulp (Table 2(b)). Degradation in Serum Blackcurrant serum (10 ml.) was placed in a 6" x 1" test tube fitted with a reflux condenser and rapidly boiled over a Buns en burner. At the commencement of boiling and at 10-minute intervals thereafter for 30 minutes 1 ml. samples were withdrawn and diluted to 25 ml. with buffer. The anthocyanin content of each sample was then measured, without further dilution, in the same way as previously. Six experiments were conducted in this manner (results Table 2(c)) and six in which oxygen was blown onto the surface at the same rate as for the pulp (Table 3(a)) and six in which the oxygen was bubbled through the solution (Table 3(c)). Serum plus Added Sugar The serum (10 g.) was mixed with commercial cane sugar (15 g.) and the syrup treated as for the serum above except that the 1 ml. sauries were diluted to 10 ml. instead of 25 ml. The experiments were also carried out with oxygen being blown onto the surface and bubbled through the solution. The results are presented in Tables 2(d), 3(b) and 3(d) respectively. iU8 Table_1

Difference in Absorption "before and after addition of NapSO:^ % Antho- cyanin retained Experiment 0 10 20 30 after last Mean ^ mins. mins. mins. 20 mins. (a) 0.i^97 O.I1.22 0.31+8 0.287 68.0 Boiling O.itS? 0.1+28 0.31+9 0.298 69.7 "blackcurrant 0.i|.06 0.281 69.2 pulp 0.377 0.335 69.8 0.37k 0.t|.88 0.1+05 0.359 73.6'i' 0.k59 O.U85 0.1+07 0.338 69.8 0M9 O.Ul+1 0.371|. 0.318 72.2 M 0.1414.5 0.383 0.288 0.216 56.5 Boiling 0.369 0.285 60.3 "blackcurrant 0.390 0.1^.73 pulp + 0.311+ 0.21+3 56.4 0.i^26 0.1+31 57.7 150^ sugar 0.321+ 0.366 0.291 0.211+ 58.5 0.1+55 O.l+OO 0.315 0.236 59.1 0.319 0.332 0.266 0.181+ 55.5 (c) 0.373 0.352 0.306 0.262 7k. 5 Blackcurrant 0.1+01+ 0.357 0,328 0.280 78.5 pulp with 0.381+ 0.31+1 0.293 76.5 oxygen over 0.373 7U.5 surface 0.14.59 0.1+32 0.31+0 0.321 71+. 5 0.1+85 0.U23 0.338 0.302 71. O.MU O.I1.O6 0.31+9 0.292 71.9 0.291 0.370 0.272 0.211+ 57.9 Pulp + sugar with 0.350 0.366 0.280 0.222 60.8 oxygen over 0.U13 0.1+31 0.310 0.21+5 56.9 59.7 surface 0.509 O.U73 0.362 0.291+ 62.1 0.U80 0.i|.M 0.356 0.282 61+. 1+ 0.503 0.507 0.371 0.285 56.3

• Result neglected in calculation of mean. m

Table 2

Difference in Absorption "before and after addition of Na2S03

% Antho- cyanin retained Experiment 0 10 20 30 after last Mean % mins. mins. mins 20 mins.

(a) 0.380 0.331 0.290 0.235 71.0 Boiling pulp 0.k2h 0.361 0.309 0.261 72.3 with oxygen O.hSS 0.378 0.309 0.265 70.0 bubTsled 70.7 through 0.1+30 0.1+35 0.376 0.320 73.6 0.377 O.I1.O6 0.335 0.281 69.2 0.395 0.1+10 0.31+3 0.278 67.9

o.uoo 0.337 0.258 0.183 5I+.3 0.14.10 0.332 0.2i4J+ 0.200 60.3 Boiling pulp -Ir 150^ O.U76 0.1+33 0.3i+6 0.272 62.8 61.0 added sugar O.Ui+8 0.1+21 0.31+8 and with 0.270 61+. 2 oxygen 0.3M 0.31+5 0.21+9 0.212 61.5 huhhled 0.21+0 0.250 through 0.207 0.157 62.9

(c) 0.3i+1 0.307 0.258 0.203 66.3 Serum 0.348 0.328 0.256 0.230 66.2 0.330 0.260 0.217 65.9 0.339 66.3 0.177 0.200 0.11+5 0.131 65.6 0.181 0.202 0.11+8 0.121+ 61.5 • 0.200 0.191 O.li+it 0.129 67.6

(d) 0.309 0.231+ 0.160 0.100 1+2.7 Serum 0.290 0.231+ 0.138 0.107 U5.7 + added 0.269 0.259 0.198 0.117 1+5.1 1+5.7 sugar 0.375 0.291+ 0.188 0.150 51.1 • 0.373 0.270 0.192 0.125 1+6.0 0.1^60 0.361 0.273 0.177 1+9.0

* Result neglected in calculating mean. Ta-ble 3

Difference in Absorption before

% Antho- cyanin retained E3cperiment 0 10 20 30 after last Mean % mins. mins. mins. 20 mins.

(a) 0,176 0.180 0.11^3 0.126 70.0* Serum with 0.176 0.182 0.11+0 0.115 63.2 oxygen over 0.180 0.192 0.131 0.113 59.0 surface 61.1+ 0.323 0.299 0.21+0 0.188 63.0 0.337 0.305 0.21+2 0.178 58.5 0.31i|. 0.316 0.255 0.200 63.3 ib) 0.258 0.215 0.165 0.113 52.6 Serum + 130^ 0.268 0.188 0.245 0.078 sugar wi th oxygen over 0.290 0.262 0.195 0.11+0 53.5 51.8 surface O.kdk 0.379 0.317 0.208 5k. 9 0.U17 0.353 0.259 0.178 50.5 0.39k 0.339 0.232 0.161 1+7.5

(o) 0.153 0.119 0.099 0.085 71.5* Serum with 0.161 0.130 0.107 0.088 67.8 oxygen passed 0.1 i+2 0.119 0.076 63.9 through 0.103 62.7 0.289 0.214-5 0.169 0.157 61+. 0 0.283 0.21+0 0.181 0.139 57.9 0.255 0.212 0.132 0.127 60.0

(d) 0.207 0.162 0.126 0.085 52.5 Serum + 0.20i4. 0.178 0.121f 50.0 sugar with 0.089 oxygen passed 0.198 0.176 0.120 0.093 52.8 through 50.7 0.322 0.31U 0.205 0.168 53.5 o.hoi 0.338 0.227 0.16U U8.6 0.378 0.318 0.198 0.11+9 kl.o

» Result neglected in calculation of mean. RESULTS AND DISCUSSION

The results of the degradation experiments on blackcurrant pulp and serum are presented in Tables i, 2 and 3. The percent anthoeyanin retained has

"been calculated for the last 20 minutes of each

eagperiment rather than for the entire 30 minutes because of the very large variation in anthoeyanin content found in the initial sample. It is also to be noted that, in many cases, there is an increase in measureable anthoeyanin during the first ten minutes of boiling. This ancxaalous behaviour may be due to lysis of cells during boiling so that more anthoeyanin is liberated into the liquid phase.

Release of anthoeyanin adsorbed onto colloidal particles may also take place and could account for this effect during the initial boiling of the

serum.

The large variation in anthoeyanin content of the serum is due to several preparations having to be made during the course of this work.

Effect of Heat Treatment

It will be shown later that there is no significant effect from the introduction of oxygen into the boiling blackcurrant pulp and serum so that an average value may be taken for the effect of boiling from the three experimental series of each group. For the straight palp the average amount of anthocyanin retained after

20 minutes "boiling is 12% and for the serum 635^. A similar difference "between pulp and serum is to be noted in the presence of sugar where the pulp shows

retention of anthocyanin and the serum only 1+9^.

It is ohvious therefore that the cellular material in the pulp must exert some protective action upon the anthocyanin.

It was noted a"bove that the increase in anthocyanin which often occurred during the first few aiinutes of

"boiling may "be due to the release of anthocyanin from the cellular material. This provides a ready explanation of the protective action of the cellular tissue since the anthocyanin, while retained within the intact cells or adsor"bed onto the tissue, may not "be so readily, if at all, degraded. The gradual "breakdown of the tissues during "boiling releases fresh anthocyanin into the solution there"by increasing the pigment content in the pulp in comparison with the serum.

Effect of Added Sugar

The effect of sugar on the "boiling "blackcurrant pulp has "been to cause greater decomposition of the anthocyanin. The addition of 150^ sugar caused an increased loss of ^2% anthocyanin after 20 minutes "boiliiig (mean of the three experiments). TMs is in line with the work of Meschter^"^^^ and Tinsley and Bockian^^^^ who observed a similar increased degradation in the presence of sugar and attributed the result to the effect of sagar decomposition products, farfaral and hydroxymethyl furfural, as discussed previously (cf. pp.128 and 135). An indirect effect from the addition of sugar is that it raises the boiling point of the mixture. In these experiments 150^ sugar was used giving a final soluble solids content of 72®Brix and resulting in the elevation of the boiling point to 107^0. This effect alone would undoubtedly have caused some increased degradation of the pigment but it is unlikely to acco\mt for it all. It is probable therefore that most of the added degradation is more closely connected with the sugar itself or its decomposition products. The spectra of the blackcurrant pulp and pulp plus sugar, before and after boiling for 30 minutes are presented in Fig. 3. These show that in the pulp the major effect of the heat treatment has be^ a decrease in the height of the anthocyanin peak at 520 ra^. In the presence of added sugar there has also been a large increase in end absorption in the blue-violet region of the spectrum corresponding to increased browning in the mixtures. i2t

FIQ. 3

SPECTRA OF BLACKCURRANT PULP

Puip

Pulp after boiling 30 minutes

Pulp \50% sugar

Pulp + 150% sugar after boiling 30 mins.

c 0 0-5 a u ou O

aoo 400 500 600 Wave Length — my. The quantity of sugar used raised the solu'ble solids content from 1M.®Brix to 72®Brix wMle in a preliminary experiment, in an open "beaker, a similar mixture concentrated to 78°Brix in 15 minutes. The conditions used therefore closely simulate the factory production of "blackcurrant ^ams and these experiments indicate that, at "best, only about of the anthocyanin would be retained after processing in open kettles. In actual fact the figure would probably be considerably less because the time at higher temperature would be much greater owing to the delay between preparation of the ^am and final filling and cooling of the individual packs. The effect of addition of 150% added sugar to blackcurrant serum was to reduce further the quantity of anthocyanin retained after 20 minutes boiling by This is not significantly different from the 12^ reduction obtained for the pulp. The visible spectra of the serum showing the effects of sugar addition and boiling for 30 minutes are shown in Fig. i|.. The change on boiling is much more marked in these spectra than in those of the pulp (Fig. 3) so that the anthocyanin peak at 520 mp.. appears only as a shoulder on the greatly increased end absorption in the blue-violet region. The serum at this stage appeared very brown and lacked all natural anthocyanin colour in contrast with tlie pulp which, after similar treatment, still showed an over- all bright red appearance due to the presence of the red tissue.

Effect of Oxygen Contrary to the findings of other irorkers^^^'^^^ that the presence of oxygen greatly increased the rate of degradation of pelargonidin-3-monoglucoside in "buffer solution and in strawberry juice the results reported here do not reveal any significant effect from the introduction of oxygen into the boiling blackcurrant pulp or serum. In the experiments in which the pulp or serum was boiled under reflux any oxygen entrapped in the pulp or dissolved in the serum would have been rapidly expelled and the space immediately above the liquid occupied by steam so that anaerobic conditions would have predominated. Some change in the percentage of anthocyanin remaining after 20 minutes was observed when oxygen was blown over the surface (simulating open kettle concentration) and when oxygen was bubbled through the solution (Tables 1, 2 and 3) but no distinct trend is shown and when the variation due to experimental error is taken into account the differences are not significant. 15Z

FIG. 4

SPECTRA OF BLACKCURRANT SERUM

Serum

Serum after boilhiQ 30 minutes

Serum 4- IS07. sugar Serum + I507^ sugar after boUfng 30 mhs.

a 0-5 o &u O

« ' ' « i ' » ii t r i 1—I—1—I, t ,.i 300 400 500 600 Wave . Length — m^i. As stated above this finding is contrary to expect- ations "based upon the work on strawberries and suggests that some protective ssrstem imist exist in the "blackcurrants which prevents aerial oxidation of the anthocyanins* If this is so then an entirely different result is to he anticipated if the same experiments were to "be carried out on model systotas of anthocyanins dissolved in "buffer. 1S2

CONOLUSIOHS

The esqperiments discussed in the foregoing pages show that, at the most, only 609S of the anthocyanin will he retained during nianufacture of "blackcurrant jams. That the figure is even as high as this woald seem to "be due to the presence of the cellular material which, hy gradually releasing anthocyanin into the liquid phase, prevents the rapid discoloration of the material.

Previous work on the detrimental effect of sugar on anthocyanin decomposition has heen confirmed.

In contrast with previous work on strawberry anthocyanins, the presence of oxygen has "been shown to he of little practical importance from the point of view of colour retention.

These facts illustrate that e3q>eriments conducted on model systems, while giving valuable information on the isolated effects of individual substances, may not give a true indication of results to be expected from commercial practice. BIBLIOGRAPHY

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