L. _: {THESIS LIBRARY L Michigan State University This is to certify that the thesis entitled BIOSYNTHETIC PRECURSORS 0F PELARGONIDTN- 3-GLUCOSTDE IN THE STRAWBERRY. FRUIT presented by Henry Co has been accepted towards fulfillment of the requirements for Eh. I) . degree inland—Science 79% WW Major professor 4411mm W) #265 Date 0-169 ABSTRACT BIOSYNTHETIC PRECURSORS 0F PELARGONIDIN- 3-GLUCOSIDE IN THE STRNWBERRI FRUIT by Henry Co 1”Culabelled compounds were fed into maturing strawberries at the pinkawhite stage. The pelargonidin-B-glucoside was extracted and purified. The relative effectiveness of these compounds as precursors of the pigment was assessed by calculating the ¢ 140 converted. The order of decreasing effectiveness is as follows: cinnamic acid > shikimic acid > fructose > glucose > phenyl- alanine > quinic acid > sodium acetate > tyrosine. The pelargonidin-B-glucoside was degraded into sugar, phloro- glucinol and p-hydroxybenzoic acid by acid hydrolysis and alkaline degradation. Determination of these 3 degradation products showed that acetate contributed almost exclusively to ring A. Glucose and fructose contributed to both ring A and B. lac from shikimic acid, cinnamic acid-3-1%, quinic acid, phenylalanine (ring labelled), and tyrosine was incorporated into p-hydroxybenzoic acid. Phenylalanine, tyrosine, quinic acid, and cinnamic acid did not label the sugar moiety. Cinnamic acid-2-1% and phenylalanine-1-1% labelled the pigment but not any of the 3 degradation products, suggesting that the C6-C3 skeleton might be incorporated as a unit. The mode of attachment of glucose to the pelargonidin could be through sugar nucleotide, as shown by the labelling of the sugar in 14C) pelargonidin-B-glucoside when UDPG (glucose-UL- was fed. Glucose- Henry Co l-phosphate was almost as good a sugar donor as the UDPG. 14C-glucose was not incorporated into the pigment even after 3 hours of metabolism. The pelargonidin was not labelled in all cases. 'When 14C-pelargonidin-B-glucoside was fed to strawberries, the cyanidin-B-glucoside was not labelled. The reverse was also true. This indicates that there might not be any interconversion between the 2 pigments. The two-dimensional chromatogram of the ethyl acetate extract of the strawberry fruit, the reactions of the spots to various detection methods, and the UV absorption spectra of some of the compounds were presented. Leucoanthocyanins, catechin, kaempferol and quercetin were present in the ethyl acetate extract of the strawberry fruits. They were all labelled when cinnamic acid-3-1nC was fed to the detached strawberry fruit. BIOSYNTHETIC PRECURSORS OF PELARGONIDIN- 3-GLUCOSIDE IN THE STRAWBERRY FRUIT Henry Co A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1965 A CKN OWLE fill-{Eli T3 The author expresses his sincere thanks to Dr. Pericles Markakis for his guidance and helpful suggestions throughout this research. He eXpresses appreciation to the members of the guidance committee, Dr. C. L. Bedford, Dr. R. S. Bandurski, Dr. L. R. Dugan, Dr. S. H. Schanderl, and Dr. G. J. Karabatsos for their help. The author is most grateful to his wife Denise Yen-ching for typing the thesis and for her encourage- ment throughout this study. ii TABLE OF CONTENTS IDITRODUCTlorJO0.0.0.0000...0.0.0....OOOOOOOOOOOOOOOOOOOOOOO REVIEW OF LITERATURE A. B. C. Chemistry of anthocyanins........................ Biosynthesis of anthoeyanins and other flavonOid compounds...oooeooooooooooooooooooooeoo Methods of analyses of flavonoid compounds....... MATERIALS AND METHODS A. B. AnthocyaninSoooeeooooooooooe0.0000009000000000... PhenOlic compoundSeooeooeo00000000000000.0000...o RESULTS AND DISCUSSION A. B. C. D. E. Extraction and purification of the pigments...... Efficiency of various 1“C-compounds as precursors of pelargonidin-B-glucoside........... Distribution of 140 in the pigment............... Glucosylation of pelargonidin-B-glucoside........ Phenolic compounds in strawberry................. SUl‘fivIAM AND CONCLUSIONS...00.00....OOOOOOOOOOOOOOOOOOOOOOO BIBIIImRAPHY.0.0.0.....0.00000000000.00000000000000000.... APPEIJDIXOOOOOOOOOOOOOOOOOOOOOOOOOOO0.000000000000000000000 iii Page 16 22 26 27 28 38 55 FIGURE 10 11 12 13 14 LIST OF FIGURES The comon natural anthocyanidinSo o o o o e o o o o o o o o o o o o Hypothetical derivation of anthocyanins from related flavonOidSoooooooooooooooeoooooooooeooooooo Standard curve of pelargonidin-B-glucoside in 01% methanOliC HCleeoooooooooooooeoooooooooooooo Chemical degradation of pelargonidin-B-glucoside 0f StraWberry fruitooooooeooooooooeoooooooeoooooooo Incorporation of phenylalanine-l-luc into pelargonidinoocoooooeeoooooooo00.000000000000000... Incorporation of cinnamic acid-Z-lnC into pelargonidinoooeoooooooooooooooeooooooooooooooooeoe Incorporation of cinnamic acid-B-lac into pelargonidinoooooooooooooooooeooooooooooooooooeooee Incorporation of phenylalanine-14C (ring labelled) into pelargonidin...ooooeooeooeoooeoeooeooooooooooo Origin of the carbon atoms in anthocyanidin........ Two-dimensional paper chromatogram of the ethyl acetate extract of strawberry fruit.......... Absorption spectra of cyanidin in .1% ethanOliC H01..0.0......0.0...OOOOOOOOOOOOOOOOOOOOO UV absorption spectra of kaempferol in 95% ethanol. Absorption spectra of quercetin in 95% ethanol..... UV absorption spectrum of catechin in 95% ethanol.. iv PAGE 55 29 31 32 33 35 39 57 58 59 6O LIST OF TABLES TABLE PAGE 1 The Rf values of strawberry pigments............... 26 2 Efficiency of various compounds as precursors of pelargonidin-B-glucoside of strawberries........... 27 3 Distribution of 140 in pelargonidin-B-glucosi e of strawberries with administration of various 1 C- labelled compourldSeooooooooooooooooooooooooooeeoooo 28 a Efficiency of various sugar compounds as precursors of glucose in pelargonidin-B-glucoside of straw- berIV‘YIOOOOOOOO.O...O000......OOOOCCOOOOOOOOOOOOOOOIO 37 5 The radioactivity pg the 2 major strawberry pigments fed With c-pignlentSooooooooooooooooooooo 38 6 Reaction of spots on chromatogram to the detection methOdSoeoeooooooooeooooooooooooooooooooooooooooooo 1+0 7 R values of different sugar compounds and pelargonidin-B-glUCOSide.oe.e..e................... 56 INTRODUCTION Anthocyanins are one of the classes of flavonoid compounds which are characterized by a C6-CB-C6 skeleton (I). They are responsible (I) for most of the red, violet and blue coloration in plants (5A). The biosynthesis of anthocyanins has been of interest to geneticists for a long time. Earlier speculations on biosynthetic routes to flavo- noid substances were based on structure analyses (93). Geissman and Hinreiner (33) assumed that the common flavonoid precursor was a chalcone. The objective of this study was to test a number of compounds which may serve as precursors of pelargonidin-3-glucoside in an effort to elucidate the biosynthetic pathway of the pigment in the strawberry fruit. Strawberry fruits were chosen as the experimental material for the following reasons: 1. The anthocyanins of the fruit have not been studied biosynthe- tically. 2. The plants can be grown under green-house conditions. 'With light supplements, an almost continuous supply of strawberries is possible, with 2 major seasons, one in summer, and the other one in winter. 3- The major anthocyanins in strawberry fruits have been identified 1 as pelargonidin-3-glucoside and cyanidin-3-glucoside (72, 95, 105, 106). A. The production of anthocyanins in strawberry is very fast. The fruit changes from pinkawhite to fully red in 48 hours. This is 14 very suitable for feeding experiments with C-labelled compounds. 5. The pelargonidin-3-glucoside (II) was chosen as the anthocyanin ‘UH ’ (II) to be studied, because it constitutes more than 90% of the total anthocyanins in the strawberry. REVIEW OF LITERATURE The term anthocyanin was coined in 1835 by Marquart (78) to denote the blue pigment of the corn flower. Later, this term was used to in- clude the whole group of similar pigments soluble in the cell sap of flowers, fruits and other plant organs (118). ‘ Bate-Smith (8), Blank (16, 17), Geissman (3o), Harborne (45, 46), Hayashi (5h), Karrer (68) and Paech (85) have review articles on antho- cyanins. The two most recent books on this field are "Biochemistry of Phenolic Compounds", edited by J. B. Harborne (51), and "Chemistry and Biochemistry of Plant Pigments", edited by T. w. Goodwin (37). A. Chemistry of anthocyanins The chemical investigation of anthocyanins was pioneered by Willstgtter and his coaworkers during the period 1913-1916. The consitution common to all anthocyanins was first established by'Willstgtter (118), and was later confirmed by Robinson (87, 9#) in an extensive series of organic synthesis experiments to be the 2-phenylben20pyrylium, or flavylium structure: There are 6 basic anthocyanidins commonly found in nature (17): pelargonidin, cyanidin, peonidin, delphinidin, malvidin and petunidin, all of which are hydroxylated in the 3-, 5-, and 7- positions. Peonidin, malvidin and petunidin are the methylated derivatives of cyanidin and delphinidin. The 6 common natural anthocyanidins are shown in Fig l. The pigments generally occur as anthocyanidin glycosides, antho- cyanins, containing one or more sugar residues and occasionally various organic acid residues. The relative frequencies with which the various monosaccharides occur in bound form in anthocyanins are as follows: glucose > galactose > rhamnose > xylose ) arabinose (50). The attachment of sugars is found in the majority of cases to be at position 3 and less frequently at position 5 of the anthocyanidin. Only one pigment has sugar at position 7 (52). The sugar at position 5 is always glucose (50). Our present knowledge of the position of sugar attachment in the molecule is due to a series of investigations by Robinson and co- workers (11, 74, 91, 96, 97) and by Harborne (50). The organic acid residues commonly found are p-coumaric acid, p- hydroxybenzoic acid, malonic acid, sinapic acid and ferulic acid (50, 54). They could be attached to any of the hydroxyl groups of the anthocyanidin or the sugar residue. The hydroxyl groups attached to the B-ring affect the color of anthocyanins. A deepening of visual color is brought about by an in- crease in the number of hydroxyl groups attached to the B-ring, as illus- trated by the orange-red pelargonidin, deep-red cyanidin, and bluish- red delphinidin derivatives. Methyl substitution gives the anthocyanin a more or less dull bluish shade, as in peonidin and malvidin glycosides. The number, position and nature of sugar does not affect the shade of the anthocyanin color (511'). The colors of anthocyanins and anthocyanidins undergo conspicuous changes with alkali and inorganic salts. They are red in acid, and blue Peonidin Malvidin Fig. 1. The common natural anthocyanidins in alkaline solution. Their colors fade at pH near neutrality. Antho- cyanins containing free vicinal hydroxyl groups as in cyanidin, del- phinidin, petunidin and their glycosides give blue chlor with inorganic salts. Pelargonidin, peonidin, malvidin and their glycosides, which have only one free hydroxyl group in the B-rings, do not react with inorganic salts to give any noticeable color change. Blue anthocyanins have been isolated from natural sources (9, 10, 55). They are metal complexes of anthocyanins associated with high molecular weight compounds of unknown structures. B. Biosynthesis of anthocyanins and other flavonoid compounds The literature on the occurrence and biosynthesis of flavonoid compounds has been covered in several reviews and books (12, 19, 31, 33, 37. 51. 85. 99, 109). In 1899, Overton (84) prOposed the idea that anthocyanins originated from carbohydrates, and suggested that the red coloring in normal plants could be correlated with excessive sugar in those tissues. Onslow (83) suggested that the pigment was derived from flavone or flavonol pigments. a. Relationships of different flavonoid compounds The different classes of Cé-CB-Cé compounds are related by oxidation- reduction reactions of the 03-portion, but so far, inter-transformation has not been shown in any biological system (14). Birch (14) proposed 2 alternate pathways to the production of anthocyanins, Fig. 2. Route (i) through flavonol derivatives is a very early postulate (83). Birch (14) suggested that it is quite possible that the whole series of processes occur in one enzyme system and that no free intermediates are released. Fig. 2. Hypothetical derivation of anthocyanins from related flavonoids (14). Among polyphenolic compounds, leucoanthocyanins have been considered to be the precursors of the anthocyanins (3, 29, 103). However, there are indications from genetic studies that the biosynthetic pathway of these two classes of compounds diverge at a fairly early stage in syn- thesis (2, 63). Aurone (32, 63) and flavanone (49) have also been found by genetic studies to diverge from the main flavonol-anthocyanidin pathway fairly early in synthesis. Anthocyanidins, flavonols and flavones are reported to be closely related biosynthetically (49). Our present knowledge on the biosynthesis of flavonoid compounds is chiefly derived from feeding experiments with 1LAC-labelled compounds and from the genetic studies of flower colors. b. Biosynthesis of C6-CB-C6 skeleton Earlier speculations on biosynthetic routes to flavonoid substances were based on structural analyses. It was pointed out by Robinson (93) that the C6-CB-C6 skeleton (I) of flavonoid compounds could be related in part to the C6-C3 skeleton in substances such as phenylalanine, tyro- sine, and other natural products. The oxygenation pattern of the A-ring of several flavonoid compounds such as anthocyanins and quercetin is typically that of phloroglucinol or resorcinol and the former in particular was considered as a possible intermediate. Geissman and Hinreiner (33) has published a review on the earlier work and ideas. The work of Underhill gt a; (111), Watkin 23515 (115), and Geissman and Swain (36) on the biosynthesis of quercetin (III) in buckwheat (FagOpyrum) showed that the ring A was formed with about equal efficiency (III) from either 1-140- or 2-1110- acetate, but very little 1“'0 from acetate was incorporated into ring B. The ring B was derived from a 03-06 com- pound such as phenylalanine, cinnamic acid or p-coumaric acid. These compounds were incorporated without loss or randomization of carbons to give ring B and the central C3 unit of the heterocyclio ring (111). Zaprometov (121) reported that acetate was incorporated mainly into ring A and shikimic acid was incorporated mainly into ring B of catechin in young tea shoots. Comte 9; g; (27), also showed that p-luC-cinnamic acid was incor- porated into the carbon atom at position 2 of the catechin nucleus. Hutchinson gt. 5; (58) reported that phenylalanine was incorporated into ring B and acetate into ring A of phloridzin (Phloritdn glucoside) in leaf disks of Malus. Avadhani and Towers (4) showed that ec-IuC- cinnamic acid was also readily incorporated into phloridzin. Although quercetin might arise from the acylation of phloroglucinol by phenylpropanoic acid, this path does not seem to operate in buckwheat, Since 11‘C-phloroglucinol was not incorporated into quercetin (79). Grisebach (38) studied the biosynthesis of cyanidin (IV) in red cabbage seedlings. Wanidin occurs here as a complex anthocyanin. Twa 01‘ the major pigments are the mono- and di-feruloyl esters of cyanidin- 10 (IV) 14 5-glucoside-3-sophoroside (50). Acetate-l- C was found to incorporate mainly into ring A. Degradation of the phloroglucinol obtained by al- 14C to be concentrated in the oxygen-bearing kaline fusion showed the carbon which showed that ring A was formed by head to tail condensation of acetate units. Grisebach (39) also found that phenylalanine-U-luc was incorporated into ring B and the attached carbons. So far, acetate was shown to be the best precursor of ring A (40, 116). Grisebach and Bopp (42) reported that both quercetin (III) and cyanidin (IV) were formed simultaneously from l-luC-phenylalanine in etiolated buckwheat seedling - there was no evidence that one was a precursor of the other. Both flavonoids reached their maximum specific activity at the same time and then declined. Grisebach and Patschke (43) have obtained evidence that a chalcone was a precursor of both cyanidin and quercetin. When 2', 4, 4', 6'-tetrabydr03qrchalcone-2-pJac- glucoside was administered to FagOpyrum, both quercetin and cyanidin were labelled. This chalcone was also converted to cyanidin in red cabbage, without randomization of the carbons. c. Glycosidation of flavonoid compounds Barber (5) has described the enzymic glycosylation of quercetin (III) to form rutin [quercetin-3-O- at -L-rhamnosyl-(l->6)- A -D-glucoside ] . 11 The enzyme was prepared by ammonium sulfate precipitation from a buffer extract of mung bean leaves. 'While the different preparations varied in their reSpective abilities, all but one preparation catalyzed the following two reactions involving different types of nucleoside diphosphate glucose (NDPG) or its rhamnose counterpart. NDPG + Quercetin —*- Quercetin-3-IB -D-glucoside + NDP NDP-L-rhamnose + Quercetin-glucoside -'- Rutin + NDP The reactions were followed by measuring the incorporation of radioactivity into the products from nucleoside diphosphate sugars labelled with 1" C in their sugar moieties. Some recent results have shown that UDP-rhamnose was a somewhat more effective donor than TDP- rhamnose for rutin synthesis (6). The work on rutin formation indicates that the pattern for enzymic glycosylation of phenols discovered by Yamaha and Cardini (119, 120) may be wideSpread, i. e., phenols are first converted to the le-monosides and then successively to di-, tri-, and tetra-saccharides. Mansell (75) reported on the glucosylation of anthocyanins in detached petals of Impatiens balsamina. Para-coumaryl-3,5-diglucoside was produced by the developing petals of a genetically white strain when pelargonidin-3-monoglucoside was fed. d. Biosynthetic pathway based on genetic evidence A pathway of flavonoid biosynthesis based on genetic studies was drawn up by Harborne (49). Glycosylation was considered to be the final steps in synthesis (33). Methylation and acylation of the anthocyanins were also considered to take place toward the end of the synthesis since 12 the co-occurring and related flavones are unaffected. The introduction of the 3-hydroxyl group was assumed to take place before ring closure of the central pyran ring since it is a common feature of both flavonols and anthocyanidins. Finally, hydroxylation of the B ring was assumed to take place at the C15 level rather than at the C9 level, because there is a closer correlation between the hydroxylation pattern of the different classes of flavonoids, than between flavonoids and cinnamic acids and other 09 compounds. C. Methods of analyses of flavonoid compounds a. General references Methods of identification of flavonoid compounds were reviewed by M. K. Seikel (102) in the book "Biochemistry of Phenolic Compounds", edited by Harborne. Seshadri (100), Seikel (101), Venkataraman (112) and Jurd (65) each has a chapter on the isolation and purification of flavonoid compounds in Geissman's book (31). Harborne (45, 46, 47, 48) and Geissman (30) both have review articles on the identification of flavonoid compounds. More recent books on flavonoid compounds include "The Chemistry of Flavonoid Compounds", edited by T. A. Geissman (31), "Biochemistry of Phenolic Compounds", edited by J. B. Harborne (51), "Biogenesis of Natural Compounds", edited by P. Bernfeld (12), "Chemistry of Natural Phenolic Compounds", edited by W. D. Ollis (82), "Methods in Polyphenol Chemistry" (90, "Enzyme Chemistry of Phenolic Compounds" (89) both edited by J. B. Pridham, and "The Chemistry and Biochemistry of Plant Pigments", edited by T. w. Goodwin (37). 13 b. Isolation The flavonoid compounds are extracted from the plant materials with acetone, ethanol, methanol, or water (100, 102). The chlorOphylls, carotenoids, and other waxy materials are usually removed by extraction with petroleum ether, hexane or benzene. c. Purification Initial purification of flavonoid compounds can be carried out by extraction with ether, ethyl acetate, or butanol (102), or by precipita- tion with neutral or basic lead acetate (54). Column chromatography can be used for the separation of flavonoid compounds. Cation-exchange resins (60, 117), Magnesol (a synthetic hydrated magnesium acid silicate) (59), silicic acid (105), cellulose powder (113) and polyamide (24, 25, 80) were some of the absorbents used. Paper chromatography is the most important method for separating complex flavonoid mixture. The techniques of one-dimensional, two- dimensional, and preparative chromatography have been covered in several books on chromatography (18, 56, 104). The developing solvents (45, 47, 101, 102) and spraying reagents for detecting flavonoid compounds on paper chromatograms (101, 102) have been summarized in several publications. Thin-layer chromatography (13, 15, 57) and paper electrophoresis (77, 88) are some of the other methods for purification of flavonoid compounds. d. Identification Preliminary identification is carried out on the paper chromatogram. Anthocyanins, chalcones and aurones are visible under ordinary light. 14 The other flavonoid compounds, except flavanones, isoflavones, catechins and leucoanthocyanins (47), can be revealed with UV light, with or without the help of NH3 fumes. Catechins and leucoanthocyanins can be located with p-toluene sulfonic acid (101). Flavanones turn magenta with sodium borohydride spray (101). Micro methods are available for complete identification of flavonoid compounds. They are based on paper chromatographic study of the glycoside in several solvent systems and of the aglycone, the sugar; and the cinnamic acid derivatives (if present) formed on hydrolysis (45), followed by a spectral study of the glycoside and aglycone. The unknown glycoside is co-chromatographed with known compounds in at least 3 different solvent systems and the Rf values compared with the published data (8, 30, 45, 47, 101). The absorption spectra of the glycoside (46, 65), and the shift of absorption maximum upon addition of aluminum chloride (34, 44, 108), sodium ethylate (76), sodium acetate (66, 67), and boric acid-sodium acetate (64) are determined and compared with those in literatures. Infra-red spectroscOpy has been used to investigate the flavones, flavonols, flavanones and isoflavones (61, 71) and to identify different anthocyanidins (92). It is especially useful in confirming the presence or absence of methory group in the pigment. Several methods are available for the determination of the nature (1, 23, 52, 86) and the position of attachment (23, 24, 1+5, 1+6) of the sugar residues to the aglycone. The acyl group, if present, can be readily removed by cold basic hy- drolysis, isolated and identified (18, 101). 15 The aglycone, produced after complete acid hydrolysis, is identified by paper chromatography (45, 46, 54, 101) and spectral methods (46, 48, 98) as with the glycoside. The aglycone can also be degraded into smaller fragments by alkaline reagents (27) and the resulting phenolic compounds identified with paper chromatography and chromegenic reagents (21, 107). MATERIALS AND METHODS A. Anthocyanins a. Materials 1. Plant material Strawberries of the Ever-bearing type were grown in pots in the green-house. 2. 11‘Culabelled compounds D-glucose-UL-luC D-fructose-UL-laC Sodium acetate-l-luC DL-phenylalanine-l-luC DL-phenylalanine-luc (ring labelled) L-tyrosine-UL-lnC Shikimic acid-GL-luC Quinic acid-GL-luC Cinnamic acid-3-1“c Cinnamic acid—2-1“c Dbglucose-l-phosphate-UL-1uC Uridine-diphospho-glucose (glucose-UL-luC) 14C-cyanidin-3-glucoside 111’C--pelargonidin--3-glucoside Cinnamic acids were purchased from Merck, Sharp and Dohme of Canada, Ltd., Montreal, Canada. D-glucose-l-phosphate-UL- 11"C and uridine-diphospho-glucose (glucose-UL-lhc) were purchased from International Chemical and Nuclear Corporation, City of 16 17 Industry, California. 1I‘AC-cyanidin-Juglucoside and 11+0- pelargonidin-3-glucoside were isolated from strawberries fed with cinnamic acid-3-14C. The rest were purchased from New England Nuclear Corporation, Boston, Mass. Methods 1. Administration of 1"(C-labelled compound to strawberries At least 10 strawberries were used in each experiment. Maturing strawberries in the pinkawhite stage were removed from the plant by cutting the stems under water with a razor blade so that about one inch of stem remained attached to each berry. Individual berries was placed in an aqueous solution of the desired radioactive compound in a tube which was prepared from a 5 mm I.D. glass tubing by sealing off one end and cutting the tubing to make a one inch tube. The tube with the berry was placed in a beaker, and covered with a watch glass to minimize dehydration of the fruit. The cinnamic acid was administered as its sodium salt. About 1-34uc of the radioactive compound was administered to each strawberry depending on its size. The 1“‘0 solution (50-100,..1) was taken up within 3.4 hours. Water was added every time the tube was about to dry out so that the last trace of the 14C compound was washed into the plant and that a continuous supply of water was available to the strawberries thus preventing dessication. The strawberries were allowed to metabblize for 2 days at room temperature until they turned very red. The pigments were then extracted. 18 In the case of the experiments on glucosylation, the D- glucose-UL-luc, D-glucose-l~phosphate-UL-1uc, or uridine- diphospho-glucose (glucose—UL-luC) was injected into the straw- berries receptacle (70) with a 100 ‘41 syringe and #23 hypodermic needle. The fruits were allowed to metabolize for 1 or 3 hours before the extraction of the pigments. In the experiments on the interconversion of pelargonidin- 3-glucose and cyanidin-3-glucose, the 1“c pigments previously isolated and purified from strawberries fed with cinnamic—3-1% were injected into the receptacles of strawberries. The fruits were allowed to metabolize for 24 hours before the extraction of the pigments. 2. Isolation and purification of the pigments The fully ripened strawberries were extracted thoroughly with several portions of hot butanol containing 0.1% HCl. The anthocyanins were precipitated from the combined butanolic extracts by addition of about 10 volumes of petroleum ether (b.p. range 30-600). The pigments were then dissolved in a small volume of methanol and the solution was streaked on ‘Whatman #3MM paper and developed with BAW: n-butanol-acetic acid- water (4:1:1.25 v/v/v), in descending direction for 16 hours. The chromatograms were dried and the pigments, cyanidin-3- glucoside and pelargonidin-3-glucoside, were each cut from the paper and extracted several times with 0.01% methanolic HCl. The combined methanolic extracts were concentrated under reduced pressure in a flash evaporator at 38°C. The pelargonidin-3- 19 glucoside was again purified by descending paper chromatography using 2% acetic acid as develOping solvent. Further purification was made using paper electrOphoresis. The Durrum type paper electrophoresis cell was used, with a voltage of 480 volts in 0.5N acetic acid, on'Whatman #3MM paper. Another descending paper chromatography was carried out using n-butanol-acetic acid-water (4:1:l.25 v/v/v), as developing solvent. The purified pigment was dissolved in methanol containing 0.1% H01. 3. Measurement of the yield of pelargonidin-B-glucoside The optical density of the pigment in 0.1% methanolic HCl was measured with a Beckman DU spectrophotometer at 506 mm. (46). The concentration was obtained from a standard curve prepared with crystalline pigment, Fig. 3. 4. Measurement of radioactivity A measured amount of the pigment or sugar solution was transferred into a planchet and the radioactivity of the pigment or sugar was determined with a Nuclear Chicago Corporation's Decade Scaler (model 181B) with automatic sample changer (model C-110-B) and printing timer (model C-111-B). A gas mixture of 98.7% helium and 1.3% butane was used as the carrier gas. 5. Acid hydrolysis of the pigment Six normal HCl was added to the aqueous solution of a known amount of the pigment to make a 10% HCl solution, and the mixture was placed in a boiling water bath for 30 minutes. The mixture was cooled quickly. The pelargonidin was separated from the sugar 20 by extraction with a few drops of isoamyl alcohol (69). 6. Sugar moiety of the anthocyanin After removal of the isoamyl alcohol layer, a portion of the hydrolysate was spotted on a Whatman #1 paper and developed descendingly for 48 hours in n-butanol-acetic acidawater (4:1:1.25 v/v/v) solvent. Authentic glucose was run side by side with the sugar. The sugar was further purified with a second descending paper chromatography using acetone-butanol- water (7:2:1 v/v/v) (56). After the paper was thoroughly dry, the portion containing the sugar was cut as a strip and passed through an automatic chro- matogram scanner (model 880) by vanguard Instrument Company, La Grange, 111., to determine if the sugar was labelled or not. The helium-butane mixture mentioned above was also used as the carrier gas. 7. Alkaline degradation of the aglycone The pelargonidin was precipitated from the isoamyl alcohol extract by addition of about 10 volumes of benzene. The resulting pelargonidin was subjected to alkaline degradation by the procedure of Comte gt 2.; (27): The pigment was placed in a test tube and heated with KOH pellets under NZ to 180-190°F for 3 minutes, then the temperature was raised rapidly to 245- 250°F and maintained at this temperature for 3%; minutes. The solution was cooled down as quickly as possible by first blowing air on the outside of the tube by means of a hair dryer, then by placing the tube in ice water when the temperature was low 21 enough not to cause breakage of the tube. The solidified mixture was dissolved in distilled water and acidified with 6N HCl to a pH of 5. The resulting solution was extracted 4 times with 3 ml portions of diethyl ether. The combined ether extracts were evaporated at room temperature almost to dryness by blowing a stream of N2 over the solution. The resulting ether solution was transferred quantitatively to a spot on Whatman #1 paper and develOped in a descending direction for 4 hours with a n-butanol-benzene-acetic acidawater (1:5:1:0.5 v/v/v/v, upper phase) solvent mixture (27). A mixture of authentic phloroglucinol and p—hydroxybenzoic acid was run next to the mixture. The 2 phenolic compounds were further purified first by thin-layer chromatography on silica gel G as adsorbent and absolute methanol as the developing solvent, and then by another descending paper chromatography using benzene-acetic acidawater (2:2:1 v/v/v, upper phase) as the solvent (21). The portion of the chromatogram containing the 2 phenolic compounds was cut as a strip and run under the chromatogram autoscanner to locate the radioactivity. The phloroglucinol and p-hydroxybenzoic acid were visualized by spraying with diazotized sulfanilic acid (28, 35) (25 ml of freshly prepared 5% NaNOZ + 5 ml of sulfanilic acid solution [0.9 gm sulfanilic acid + 9 ml concentrated HCl, diluted to 100 ml] ), followed by 15% NaZCOB. Phloroglucinol gives a reddish brown spot and the p-hydroxybenzoic acid gives an orange spot. B. 22 Phenolic compounds a. Materials 1. Plant materials Strawberries as in section A. 2. 14C-labelled compounds Cinnamic acid-3-1%. Methods 1. Administration of 1u’C-compounds The cinnamic acid-3-1QC solution was injected into both green and pinkawhite strawberries. After 24 hours of metabolism, both pinkawhite and red strawberries were obtained. 2. Extraction of phenolic compounds The strawberries were dr0pped into boiling 80% ethanol and kept there for 3 minutes (90) to inactivate the enzymes. After filtering, the residue was extracted twice with methanol. The combined alcoholic extracts were washed with petroleum ether (b.p. range 30-600) to remove chlorophylls, carotenoids and waxy materials. The washed extract was then concentrated under vacuum at 38°C in a flash evaporator until all alcohol was evaporated. The aqueous solution was extracted 10 times with ethyl acetate. The combined ethyl acetate extracts were dried with anhydrous sodium sulfate, filtered and concentrated to a small volume. 3. Paper chromatography The ethyl acetate extract was chromatographed descendingly in two dimensions on Whatman #1 paper, using n-butanol-acetic 23 acid-water (6:1:2 v/v/v) (81) as the first solvent for 15 hours and then 2% acetic acid as second solvent for 4 hours. 4. Examination of the paper chromatograms and location of the phenolic compounds The air-dried chromatograms were observed in the visible, and under UV radiation, before and during fuming with NHB. The papers were examined in both 254 and 336 mm. of UV radiation, and after spraying with 5% AlCl3 in ethanol. Another chromatogram was sprayed with 0.5% FeCl3 + 0.5% K3Fe(CN)6 in aqueous solution. All phenolic compounds give blue spots. A permanent record could be obtained by dipping the chromatogram in 2N HCl solution followed by thorough wash- ing in distilled water (7). Another spray used was 15% NaZCO followed by diazotized 3. sulfanilic acid. Three percent p-toluene sulfonic acid in ethanol was used to locate the leucoanthocyanins and catechins. Leucoantho- cyanins give orange to red spots, catechins give brown spots. 5. Purification of the compounds The spots containing the compounds were cut from the chro- matograms, eluted, and concentrated. The compounds were puri- fied by paper chromatography using different solvent systems. 6. Identification of the compounds i. Leucocyanidins The portions of the paper (spots L1 to L5) containing leucoanthocyanins were each cut and eluted with 80% methanol. 24 The eluates were concentrated under vacuum. Each con- centrate was transferred to a test tube, 0.6N butanolic HCl (110) added, and the tube was placed in a boiling water bath for 40 minutes. The converted pigments were purified twice with descending paper chromatography using Forestal solvent (22): acetic acid-concentrated HClawater (30:3:10 v/v/v) and n-butanol-ZN HCl (1:1 v/v) (45) as the develOping solvents. The purified pigments were used for spectra determinations. ii. Flavonols The spots (Q, K,) which fluoresced.yellow under UV in NH3 fumes, were each cut and eluted with 80% methanol. The eluates were concentrated and hydrolyzed in 2N HCl for 20 minutes in a boiling water bath. The aglycone was extracted with ethyl acetate, and chromatographed twice using Forestal solvent and n-butanol-acetic acidawater (6:1:2 v/v/v) as developing solvents. iii. Catechin Spot C was cut from the chromatograms, eluted, and purified by paper chromatography using three different solvents: water, 2% acetic acid, and n-butanol-acetic acid- water (6:1:2 v/v/v). 7. Absorption spectra The UV and visible absorption spectra of the purified com- pounds in 95% ethanol were obtained with a Bausch and Lomb Spectronic 505 recording spectrophotometer. The spectra were 25 also obtained after addition of AlCl3 (108), NaOAc, and NaOAc + H3803 (6a, 67). 8. Measurements of radioactivity The radioactivities of the purified spots were measured as described in the section on anthocyanins. RESULTS AND DISCUSSION A. Extraction and purification of the pigments Several solvents could be used to extract the anthocyanins from fruits (54). One percent HCl in methanol was found to be the best solvent for extracting anthocyanins from Bing cherries (73). But in these experiments, hot 0.5% butanolic HCl was used to avoid the ex- traction of excess sugars which will interfer in the separation of the pigments on paper. In order to ensure that the percent recoveries of the pigments from each experiment are as close to each other as possible, the iso- lation and purification procedures were standardized. It was found that 2 paper chromatographic runs, one paper electrophoresis and another paper chromatography were sufficient to give a pigment with constant specific activity. The paper should not be over or underloaded because overloading will result in inefficient separation and underloading will result in poor recovery of the pigments. The RT values of the 2 straw- berry pigments are presented in Table 1. Table 1. The Rf values of strawberry pigments Paper chromatography Paper electr0phoresis BAW (4:1:1.25) 2% HAc (0.5N HAc, 480 volts) Pelargonindin- Moved 3.3cm in 3-glucoside .36 .34 5 hrs to cathode Cyanidin- Moved 1.1cm in 3-glucoside ~29 .27 5 hrs to cathode 26 27 B. Efficiency of various 14C-compounds as precursors of pelargonidin- 3-glucoside. About 1-3,uc of the 11+C-labelled compound was administered into each strawberry depending on its size. Since the 140 compounds administer- ed had a sufficiently high specific activity (1-10 mc/m moles), there was no danger of overloading to a point which would affect the meta- bolism of the strawberries. The efficiency of the various 1LlC-labelled compounds as precursors of pelargonidin-3-glucoside was expressed as % 14C converted. It was suggested (114) that the percentage conversion value is the most useful way for recording the efficiency of a precursor in a tracer experiment in those cases where reliable analyses of the amount of end product are feasible. The efficiency of the various 14C-labelled compounds as precursors of the pigment is shown in Table 2. Table 2. Efficiency of various compounds as precursors of pelargonidin-3-glucoside of strawberries Compound fed % 11"C converted Sodium acetate-l-luc .29 L-tyrosine-UL-luC .15 DL—phenylalanine-1-1“c .85 D-glucose-UL-luC 1.59 D-fructose-UL-lnc 1.66 Quinic acid-GL-luC .37 Cinnamic acid-3-140 3.32 Shikimic acid-GL-luC 2.19 28 Shikimic acid and cinnamic acid were the best precursors among the compounds tested as shown by higher % 14C converted into pelargonidin- 3-glucoside. Glucose and fructose came next, followed by phenylalanine. Tyrosine was the poorest of them all. Sodium acetate and quinic acid were a little better than tyrosine. 0. Distribution of 1"c: in the pigment In order to locate the position of the 11+C, the pigment was degraded into glucose, phloroglucinol, and p-hydroxybenzoic acid by acid hydrolysis and alkaline degradation, Fig. 4, as described in the experimental section. After purification, the chromatograms were scanned for radioac- tivities. The results are shown in Table 3. Table 3. Distribution of 140 in pelargonidin-3-glucoside of strawberries with administration of various 1LAC-labelled compounds Compound fed Phloro- p-hydroxy- glucose glucinol benzoic acid Sodium acetate-1-140 + trace + L-tyrosine-UL-luc - + - 14 DL-phenylalanine- c - + - (ring labelled) D-glucose-UL-luC + + + D-fructose-UL-luC + + + Quinic acid-GL-luC - + - Shikimic acid-GL-iuC trace + + Cinnamic acid-3-1“c - + - DL-pheny1a1anine-1-1“c - - - 14 Cinnamic acid-Z- C - - - .._fi 29 acid Ho hydrolysis + glucose Pelargonidin- Pelargonidin 3-glucoside 0H , HO OH alkaline "“ , + HOOC 0H + other degradation products 0H Pelargonidin Phloro- p-hydroxy- glucinol benzoic acid Fig. 4. Chemical degradations of pelargonidin- 3-glucoside of strawberry fruit 3o Phenylalanine, tyrosine, cinnamic acid and quinic acid did not label the sugar moiety of the pigment. Acetate contributed almost exclusively to ring A. Glucose and fructose contributed to both ring A and B. 140 from Shikimic acid, quinic acid, cinnamic acid-3-1uC, phenylalanine-lac (ring labelled) and tyrosine was incorporated into p-hydroxybenzoic acid. Cinnamic acid-2-14C and phenylalanine-l-iuC labelled the pigment but not any of the three degradation products. Therefore the 1“ C must be in C3 or C4 of the pigment. This means that the cinnamic acid and phenyl- alanine should be incorporated as a unit. Figs. 5, 6, 7, and 8 show the incorporation of various 11+C compounds into pelargonidin. From the results of these experiments, and those of Grisebach (38), a scheme of the synthesis of the C6-C "C6 skeleton of anthocyanin can 3 be drawn. Fig. 9 shows such a scheme. Ring B and the 3 carbon atoms (2,3,4) of the heterocyclic ring are derived from a phenylpropane compound such as cinnamic acid, phenylalanine or tyrosine. These compounds are incorporated as a unit. Ring A originates from acetate. Grisebach (38) found that when acetate-1-14C was fed to red cabbage seedlings, the 14C was con- centrated in the oxygen-bearing carbons of phloroglucinol, and when he fed acetate-Z-luC, the 1“C was found in the non-oxygen-bearing carbons. This led him to assume a head-to-tail condensation of the acetyl units. Phenylalanine or tyrosine was considered not to take part directly in the condensation to the C6-C3-C6 compound (41). Since Shikimic acid and quinic acid were not as good precursors of the pigment as cinnamic acid, cinnamic acid or one of its derivatives 31 *1 NHZ HOOC Fig. 5. Incorporation of phenylalanine-1- 14C into pelargonidin 32 HOOC Fig. 6. Incorporation of cinniamic acid-2- 140 into pelargonidin 33 HOOC Fig. 7. Incorporation of cinnamic acid-3- 14C into pelargonidin 34 Fig. 8 Incorporation of phenylalanine-luC (ring labelled) into pelargonidin 35 (SH EODH "O “I .\\' 3 ll °r \.=,/ /p ‘ NHZ HOOC HOOC \9 1 / .— H0 r”.\*’o \\ ..// )\. OH v 1 n + J. OH Fig. 9. Origin of the carbon atoms in anthocyanidin 36 is probably the phenylpropane intermediate which condenses with acetate to form the C6-C3-C6 compound. In the experiment to be shown later, when cinnamic acid-3—1uC was fed to green strawberry fruit and the phenolic compounds isolated, no cinnamic acid-3-1uC was found in the extract. Most of the radioactivities ended up in a phenolic compound which was extractable with ethyl acetate. This indicates that cinnamic acid is metabolized very readily by strawberry fruit. Since it disappears before the anthocyanin is formed, an inter- mediary phenolic compound, such as the highly radioactive one detected here, would be an intermediate in the anthocyanin formation. It seems that anthocyanins are synthesized in a similar manner as the other flavonoid compounds such as quercetin in buckwheat and catechin in tea leaf. Various 03-06 compounds were found to be incorporated into ring B, and acetate was found to be incorporated into ring A of quercetin (111, 115) and catechin (121). The C3-C6 compounds were also found to be incorporated into the flavonoid compounds as a unit (111). D. Glucosylation of pelargonidin-3-glucoside The mode of glucosylation of pelargonidin-3-glucoside was investigated by injecting solution of ll(Culabelled D-glucose, D-glucose-l-phosphate or uridine diphospho glucose into maturing strawberries. Table 4 shows the results of the glucosylation eXperiments. 1“Cmglucose did not label the sugar in pelargonidin-3-glucoside even in 3 hours. Glucose-1-phosphate was shown to be a better glucose donor to the pigment. UDPG was only a little better as glucose donor. The pelargonidin was not labelled by any of the sugars fed, even up to 3 hours of metabolism. 37 Table 4. Efficiency of various sugar compounds as precursors of glucose in pelargonidin-3-glucoside of strawberry Compound fed Feeding time %1uC converted (hour) D-glucose-UL-luC 1 0 3 0 D-glucose-1-phosphate-UL-140 1 trace 3 .12 Uridine-diphospho-glucose 1 trace (glucose-UL-luC) 3 .18 It should be noted that there was no possibility of contamination of the isolated anthocyanin by the sugars fed as shown by the differences in R1. values of the various sugars and pigment in the different puri- fication steps, Table 7. E. Interconversion of the 2 pigments in strawberry Experiments were carried out to see whether there is interconversion taking place between the pelargonidin-3-glucoside and cyanidin-3- glucoside. 11+C-pelargonidin-3-glucoside and 1uC-cyanidin-3-glucoside isolated from strawberries fed with cinnamic acid-3-1UC were used. The cinnamic acid-fed pigments were used because they had the highest specific ac- tivities and because the sugar moieties were not labelled, Table 3. Labelled sugar moiety in the compound fed is undesirable because the sugar might be detached from the aglycone and be used for the synthesis of new aglycone. The radioactivity of the purified pigments was measured. Table 5 shows the results of the interconversion experiments. Table 5. Fed 11+C-pelargonidin-3-g1ucoside 1L"C--cyanidin-3--glucoside 38 The radioactivity of the 2 major strawberry pigments fed with 1 ”C-pigments Pigment isolated Total cpm Pelargonidin-3-glucoside 1110 Cyanidin-B-glucoside 0 Pelargonidin-3-glucoside 0 Cyanidin-B-glucoside 730 There was no apparent interconversion of the 2 major pigments of strawberry. Maybe the specific activities of the compounds fed were not high enough to show any interconversion. E. Phenolic compounds in strawberry In an attempt to find the other precursors of the pigment, the phenolic compounds in the fruit were investigated. a. Paper chromatogram A drawing of the two-dimensional chromatogram of the ethyl acetate extract of the strawberry fruit fed with cinnamic acid-3- 140 is shown in Fig. 10. All 89 9, 10 and 12. are phenolic compounds except spots The latter spots are useful in locating phenolic compounds in comparative paper chromatography. The reactions of the spots to the detection methods are given in Table 6. in Figs. 11, 12, 13 and 14. b. Identification of some of 1. Leucocyanidins Spots L-i to L-5 were The absorption spectra of some of the spots are shown the phenolic compounds each eluted from two-dimensional paper chromatogram, purified, and their UV absorption spectra deter- 2) n-but-nol-acetl‘c acid—water (6: I Fig. 100 39 2% Acetic acid Two-dimensional paper chromatogram of the ethyl acetate extract of strawberry fruit 40 Table 6. Reaction of spots on chromatogram to the detection methods Detection method visible light UV (long wavelength) UV (short wavelength) NH in UV (ldng wavelength) NH in visible light 15% Na2003 15% NaZCO + diazotized sulfanilic acid 3% p-toluene sulfonic acid 5% 11ch3 0.5% FeCl + 0.5% K3F6(CN)6 Appearance Spots reacting yellow green purple light.yellow yellow blue yellow green dark dark light.yellow yellow yellow green blue lightyyellow yellow orange red violet red purple grey black yellow brown yellow light blue 2. 3 7. 8. Q 9. K 10, c U\O\£?P* 2, Q, K 2’ 39 5: L‘1 to L-S. Co Q C\h* 2, L-1 to L-5, C 0H4 29 5’ 6 Q. K 19 2: 39 as 59 6s 11: L-1 to L-5, Q, K, C 41 mined in 95% ethanol (one peak at 280 ma.) and.with added AlCl . No shift was detected upon addition of AlCl This 3 3' indicated that they might be related to one of the flavans (119). 'When spots L-1 to L-5 were each heated in 0.6N butanolic HCl in a boiling water bath, red pigment was produced. This placed the compound in the general classification of leuco- anthocyanins. The red pigments were then chromatographed with the Forestal solvent. A red spot was detected in all fractions at Rf=.47. The spot corresponded to cyanidin as determined by co-chromato- graphy in the same solvent system. They were also chromatogra- phed in n-butanol-ZN HCl (1:1 v/v) solvent. Their Rf=.73 also corresponded to that of cyanidin by co-chromatography. The visible absorption spectra of all the red pigments, Fig. 11, showed a peak in 95% ethanol at 545 mp1, and shifted to a longer wavelength upon addition of AlClB, indicating the presence of a O-dihydroxyl group (34). The red pigments had the same spectral characteristics as cyanidin (46). Therefore, Spots L-1 to L-5 are leucocyanidins. They might be the same leucocyanidin with various degree of polymerization. 2. Flavonols Spots K and Q fluoresced yellow under UV with NH3 fumes, indicating that they might be flavonols. The spots were cut from chromatograms, eluted, concentrated and hydrolyzed with acid. The aglycones were extracted with ethyl acetate and chromatographed with the Forestal solvent. Spot Q, R =.42, was 42 quercetin, and spot K, Rf=.58, was kaempferol as shown by co-chromatography with kaempferol from instant tea extract (26) and quercetin from a commercial source. Their Rf values in n-butanol-acetic acidawater (6:1:2 v/v/v) were also the same as the known kaempferol, Rf=.83, and quercetin, Rf=.64. They also had the same UV absorption spectra, Figs. 12 and 13, as the known compounds. 3. Catechin Spot C is catechin. It appeared dark under short wave- length UV, showed blue fluorescence under long wavelength UV after exposure to NH3 fumes, and had the same R as known f catechin in several solvent systems. It had R of .39 in dis- f tilled water, .48 in 2% acetic acid, and .62 in n-butanol- acetic acidawater (6:1:2 v/v/v). It also had the same ab- sorption spectrum as catechin, Fig. 14. c. Determination of radioactivity The spots (L-1 to L-5, Q, K, C, and 11) were each cut from the chromatogram, eluted and the eluates concentrated. They were purified once more with descending paper chromatography using 20% acetic acid as solvent. The radioactivity of each compound was counted. Catechin, kaempferol, quercetin, and leucocyanidin were all radioactive, with a leucocyanidin (L—4) and catechin having the second highest radioactivity. Spot 11 was the most radioactive spot. d. Comparison of the chromatograms of red and green strawberry 43 extracts The spots corresponded to leucocyanidin (L-4 and L-5) and catechin (C) were higher in color intensity in the green than in red strawberry fruits. Since they were radioactive, they might be anthocyanin precursors. However, catechin has been considered as a metabolic end pro- duct, because radioactive d-catechin which was taken up by Chamaecyparis pisifera cuttings was not transformed into other flavonoids such as quercetin or taxifolin (53). Leucoanthocyanins have also been considered as metabolic end products (Ln). Bopp and Matthiss (20) attempted to show that direct transformation of leucoanthocyanins to anthocyanins can take place. ‘With the feeding of carboxyl-inc-phenylalanine to Impatiens balsamina, and plotting the specific activities of anthocyanin and leucoanthocyanin against time, they showed that the decreasing portion of the leucoanthocyanin curve intersected the anthocyanin curve approximately at its maximum. However, these authors offered no proof that the leucoanthocyanins in Impatiens were identical with flavan-3,4-diols. There are numerous substances (pro-anthocyanidins) (62) in plants which are transformed into anthocyanidin by the action of acids. It can be stated that there is no conclusive evidence that catechin and leucoanthocyanins are not precursors of anthocyanins. 2. 30 SUMMARY AND CONCLUSIONS 14C-labelled compounds were fed into maturing strawberries at the pink-white stage. The pelargonidin-3-glucoside was extracted and purified. The relative effectiveness of these compounds as pre- cursors of the pigment was assessed by calculating the % 1L‘C con- verted. The order of decreasing effectiveness is as follows: cinnamic acid ) Shikimic acid ) fructose ) glucose ) phenyl- alanine ) quinic acid ) sodium acetate ) tyrosine. The pelargonidin-3-glucoside was degraded into sugar, phloroglu- cinol and p-hydroxybenzoic acid by acid hydrolysis and alkaline degradation. Determination of these 3 degradation products showed that acetate contributed almost exclusively to ring A. Glucose and fructose contributed to both ring A and B. th from Shikimic acid, cinnamic acid-3-1“c, quinic acid, phenylalanine (ring labelled), and tyrosine was incorporated into p-hydroxybenzoic acid. Phenyl- alanine, tyrosine, quinic acid, and cinnamic acid did not label the sugar moiety. Cinnamic acid-Z-luc and phenylalanine-1-1”c labelled the pigment but not any of the 3 degradation products, suggesting that the C6'03 skeleton might be incorporated as a unit. The mode of attachment of glucose to the pelargonidin could be through sugar nucleotide, as shown by the labelling of the sugar in pelargonidin-3-glucoside when UDPG (glucose-UL- was fed. Glucose-i-phosphate was almost as good a sugar donor as the UDPG. h 1 C-glucose was not incorporated into the pigment even after 3 hours of metabolism. The pelargonidin was not labelled in all an 45 cases. When 1L‘C-pelargonidin--3-glucoside was fed to strawberries, the qyanidin-B-glucoside was not labelled. The reverse was also true. This indicaunsthat there might not be any interconversion between the 2 pigments. The two-dimensional chromatogram of the ethyl acetate extract of the strawberry fruit, the reactions of the spots to various detection methods, and the UV absorption spectra of some of the compounds were presented. Leucoanthocyanins, catechin, kaempferol and quercetin were present in the ethyl acetate extract of the strawberry fruits. They were all labelled when cinnamic-3-14C was fed to the detached strawberry fruit. On the basis of the information obtained in this work, it appears that a) acetic acid is a very likely precursor of ring A of pelargonidin-3-glucoside of strawberries, b) cinnamic acid, Shikimic acid or phenylalanine may serve as precursor of ring B, c) cinnamic acid and phenylalanine are incorporated intact, d) UDPG and glucose- 1-phosphate are mediating in the glycosidation of anthocyanidin. 1. 10. 11. 12. 13. 14. BIBLIOGRAPHY Abe, Y. and Hayashi, K. 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Standard curve of pelargonidin-3-glucoside in .1% methanolic HCl. 55 56 Table 7. values of different sugar compounds and pelargonidin-3-glucoside Compound Papegxfihromatography Paper electrophore51s (4:1:1.25) 2% HAc (0.5N HAc, 480 volts) Glucose .18 .34 O Glucose-l-phosphate .04 .94 moved 10.1 cm in 3 hours to anode Uridine-diphospho- .00 1.00 moved 16.8 cm in glucose 5 hours to anode Pelargonidin-3- .36 .34 moved 3.3 cm in glucoside 5 hours to cathode 10°F Optical density 02" 300 400 500 600 700 Wavelength , mu Fig. 11. Absorption spectra of cyanidin in .176 ethanolic HCl. 100 r w Opt} cal dens it)! 02" o - - - . 250 300 350 400 Wavelength , mu. Fig. 12. UV absorption spectra of kaempferol in 95% ethanol. 58 1.0 ' 08 " 257 a - 3N 31 (With AlCl ) ’5, .6 _ ,22‘0 371 .+. 3 S ’ \‘ 0 \ '0 \. .§ on b \\ 43 ‘ ‘\ 0... \ o .\ .2 r o I 1 1 n l 250 300 350 400 450 500 Wavelength , mu. Fig. 13. Absorption spectra of quercetin in 95% ethanol. 59 1.0 A 08 " >\ 280 1;. .6 . § 0 .0 "3 -._ ,L; . 4) 0— O 02 " 0 ‘ 1 J 200 260 300 340 Wavelength , mu Fig. 14. UV absorption spectrum of catechin in 95772 ethanol. 60 ..w 5:26»..- GI .61... .-.\ er kufir “‘7111171111111111111111111“ 6 5698