University of the Pacific Scholarly Commons

University of the Pacific Theses and Dissertations Graduate School

1972

Studies on the pigments in Allium amplectens Torr

Chun-Mei Chu University of the Pacific

Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds

Part of the Biology Commons

Recommended Citation Chu, Chun-Mei. (1972). Studies on the anthocyanin pigments in Allium amplectens Torr. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/1787

This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. STUDIES ON THE ANTHOCYANIN" PIGHEHTS IN

Allium amplec~ ']~orr.

A Thesis Presented to The Faculty of the Department of Biological Sciences University of the Pacific

In Partial Fulfillment of the Requirements for the Degree r1aster of Science in Biological Sciences

by Chun-Nei Chu December 1972 This thesis, written and submitted by

is approved for recommendation to the Committee on Graduate Studies, University of the Pacific.

Department Chairman or Dean:

Thesis

Chairman -~·

ACKNOWLEDGEMENT

I am deeply grateful to Dr.o Dale W. ~1cNea.l of the University of the Pacific under whose guidance this study was made. I would also like to thank 11r. John P. IvlcGo'.'ran for his help.

. -~~- .. · .. ' .. :~ .. TABLE OF CONTENTS

PAGE INTRODUCTION ...... 1 LITERATURE REVIillv General review of ••••••••••••••••••••• 5

Isolation ~nd identification ••••••••••••••••••••••• 10 Biosynthesis of anthocyanins ...... 11

MATERIALS AND NETHODS .... ~ .. ~ ...... • ...... 15

RESULTS .••• 6 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ~ • • • • • • 19

DISCUSSION ...... ~ ~ . ~ . ~--~ 34

LITERATURE CITED •••••••••••••••• ·• 4 •••••••••• ~ ••••••••• 37 LIST OF TABLES

TABLE PAGE

1 • Naturally occurring • • • • • • • • • • • • • e • • 7 2. Glycoside structure·of naturally occurring anthocy·anins ...... ·...... ,. . . . . 9 3 •. Solvent systems for the chromatograms of cellulose

t~in. .. layer·····~·~································ 18

4. · . Rt values of anthocyanins of Allium &mplecj;en.~?_ on paper chromatograms •••••••••••••••••••••••••••.••• 22 5. Rf values of anthocyanidins of Allium §3-IDE}ectens

on cellulose thin layer chromatograms • 0 • • • • • • • • • • 0 32 ,. o. Rf values of anthocyanins of !Jjju~ ~~lectens on

cellulose thin layer chromatograms ·········~••e••• 33

?. The possible anthocyanins of Allium ~mplectens

from the known glycoside •••4••••••••••••• 36 LIST OJ.i1 FIGURES

FIGURE PAGE 1. Composite chromatogram of the anthocya.nin components of the Allium acuminatum alliance •••••••••••••••••• 2

2. Flavylium cation ••••••••••••••••• • ...... ~ 5 3. Biosynthetic path,Hays to • • • • • • • • • • • • • 14 4. Composite chromatogram of the anthocyanin components of the Allium a.mnlecte:ns • • • • • • • • . • • • • • • • • • • • . • • • • • • 21 .5. Chrome. to gram of unhydrolyzed anthocyanins, hydrolyzed anthocyanins and standard anthocyanidins

in n-BAW solvent system·············~····~········· 24 6. Chromatogram of u:nhydrolyzed anthocyanins, hydrolyzed anthocya.nins and standard anthocyanidJns in Forestal solvent system •••••••••••.••••••••••••• 26 7a Chromatogram of unhydrolyzed anthocyanins, hydrolyzed anthocyanins and standard anthocyanidins in HAc·HCl solvent system •••••••••.••••.••••••••••• 28 8. Chromatogram of unhydrolyzed anthocyanins, hydrolyzed anthocyanins and standard anthocyanidins in AA•H solvent system •••.••••••••••••••••••••••••• 30 9. Structure of cyanidin • . • • • • • • . • • . • • . • • • • • . • • • • • • • • • 31 INTRODUCTION

The circumboreal genus Allium (Amaryllidaceae) is composed of about: 500 species, 80 of which occur in the New World (Ownbey, personal .communication). The North American species form a distinct group with all but two species, !· schoenoprasum and A· tricoccum, being obviously closely related ·and showing no particular a,ffini ty to. any Old ivorld species •.... on the basis of morphology and cytotaxonomy, nine well-defined alliances are recognized within the New V/orld group (Ovrabey and Aase, 1955; Saghir tl al., 1966).- Preliminary chemotaxonomic surveys have ·oeen done in the two largest alliances, comprising a total of 43 taxa. These in:L tial surveys indicated that a large number of anthocyanin pigments are found j_n these species. Mingrone (1968) reported 14 anthocyan1ns in the 30 taxa of the Allium falcifolium alliance with 9 occurring in over half the taxa and 3 in all but one (Fig. 1, spots 1,2 and 3). I~cNeal (1970) reported 19 different anthocyanin spots in 13 taxa of the !_. acuminatum alliance. Based on comparative Rt values it appears that all 14 spots reported by Mingrone are found in one or more of the .f::...· acuminatum alliance taxa. Spots 1 and 3 occur in all of the A. acuminatum alliance taxa and ten of the remaining twelve reported by Mingrone occur in half or more of the taxa. The constancy of these spots across alliance

1 2

Fig. 1. Composite chromatogram of the anthocyanin

components of the Allium acuminat~ alliance. 3

1.0

0.8 t a0.6 "C (.) 0 ic" (.) --+- (}) (.) 0.4 0

0~ l()

0.2 8 ~~·=---~------~~ ~o 0.6 0.4 0.2 0

+- t- butanol: acetic acid: H 0 2 ( 3: I: I) 4 lines reinforces the theory of a close relationship among most of the New \'/orld species of Allium. · In this investigation, the basic pigment component, or anthocyanidin, of the ten most common spots was identified using paper and thin layer chromatographic techniques. LITERATUHE REVIE\v

General review of anthocyanin·:

An"thocyanins are water soluble pigments responsible for nearly all of the pink, scarlet, red, mauve, violet and blue coloes in plants. According to Hayashi (1962), the majority of these pigments occur in the dissolved state in the cell sap of flowers, fruits and other plant organs.

;~thocyanins belong to a group of glycosides and comprise one of the 12 classes of flavonoid compounds. Flavonoids are a group of water ~oJ..uble ppeno].ip_ pi,gm_e!lt_s that usually occur as glycosides with the aglycone (sugar-free portion) skeleton consisting of two aromatic rings linked by an aliphatic three­ carbon chain. The aromatic benzene rings are referred to as A and B (Harborne and Hall, 1964). Anthocyanidins, the aglycones of anthocyanins, occasionally associate with organic acids to form acylated or complex glycosides. All of the anthocyanidins have the same basic chemical structure (Fig. 2) : 2-phenylbenzopylium or flavylium (Pratt and Robinson, 1922).

Fig. 2. Flavylium cation.

5 6

There are 16 naturally occurring anthocyanidins : six common anthocyanidins, five rarer methylated compounds, four 3-deoxanthocyanidins ·and •. Anthocyanidins may be classified into ,four basic pigment groups .: , ...... ·t~ , cyanidin and (Table 1). Both anthocyanins and anthocyanidins are unstable ·in light, with the latter .fading more rapidly than the former. Hydroxyl groups, especially those attached to the B-ring, control colors in the anthocyanins. Generally, in acid solu­ tions, increasing the number of hydroxyl groups causes a varia­ tion in color from orange-red as in pelargonidin, to deep red as in cyanidin, to blue-red as in delphinidin. Methyl substi- tution also affects the color, resulting in a more or less dull bluish shade as in . Colors of the anthocyanins in acid solution also depend on the presence of various organic or inorganic substances. Fei'ric salts bring about an intense blue color which is characteristic for anthocyanins containing free vicinal hydroxyl groups, as in cyanidin and delphinidin (Hayashi, 1962). Glycosylation or changes in the pH value can also alter the color. Anthocyanins show red color in acid solutions and blue in alkali. The solubility and stability of anthocyanins depends on the presence of attached sugar residues. The positions at which sugars may be attached to hydroxyl groups in anthocyanins are restricted, and residues are normally found in the 3- position or in the 3- and 5- positions. Sugar residues found 7

Table 1. Naturally occurring anthocyanidins.

Apigeninidin(Gesneridin) : 5t7,4'-tri.hydroxyflavylium Luteolidin: 5,7,3',4'-tetrahydroxyflavylium : 5,7,3',5'-tetrahydroxyflavylium Columnidin : 5,6,7,3'-tetrahydroxyflavylium

Pelargonidin 3,5,7,4'-tetrahydrox.yflavylium Aurantinidin 3,5,6,7,4'-pentahydroxyflavylium

Cyanidin 3,5,7,3',4'-pentahydroxyflavylium

Peon.idin :3,5,7,4'-tetrahydroxy-3'-methoxyfla~lium : 3,5,4'-trihydroxy-7,3 1 -dimethoxyflavylium

1 , Delphinidin :. 3, . 5, 7, 3',. 4 5 '-hexahydroxyi'lavylium : 3,5,7,4',5'-pentahydroxy-3'-methoxyi'lavylium (?) : 3,7,3',4',5'-pentahydroxy-5'-methoxyflavylium' . Malvidin : 3,5,7,4'-tetrahydroxy-3',5'-dimethoxyflavylium Eur.opinidin :. 3, 7, 4', 5 '-tetrahydroxy-3' , 5 '-dimethoxyflavylium (?) : 3,5,4'-trihydroxy-7,3',5'-trimethoxyflavylium : 3,7,4'-trihydroxy-5,3',5'-trimethoxyflavylium 8 in 3- and 7- positiqns are rare (Harborne, 1965). Surveys showed that most of the structural variation in anthocyanins resided not in the nature·of the aglycone but in the nature, number and position of attachment of sugars and other residues

(Forsyth and Simmonds, 1954; · La.wr·ence ~ g., 1939). The sugars vvhich have been found in naturally occurr­ ing anthocyanins involve : four monosaccharides, five di­ saccharides and three trisaccharides. The known glycoside structures are·shown in Table 2 (Harborne, 1967). Complex or acylated anthocyanins are acylated derivatives of biosides, in which organic acids are involved in ester combination with one of the anthocyanidin or-sugarhydroxyl··groups. Anthocyan.ins are difficult to characterize by means of the usual techniques of organic chemistry, since they are unstable in light and are difficult to isolate in pure state. After paper partition chromatography was first introduced by Bate-Smith in 1948, chromatography became especially important in characterizing anthocyanins. Rf values of anthocyanins following two dimensional paper chromatography are important in identification, since no two glycosides have exactly the same Rf values. Harbor·ne ( 1958), Abe and Hayashi ( 1956) have studied the Rf values of anthocyanins in a variety of solvents. The regular relation between the Rf values and structure of anthocyanins has been noted by other investigators as well (Bate-Smith and Wastall, 1950). Hydroxylation, methylation, glycosidation and acylation cause the following types of Rf I

9 !li ~~

Table 2. Glycoside structures of naturally occuring anthocyanins (Harborne, 1967).

3-monosides 3.;..arabionoside, 3-galactoside, 3-glucoside, 3-rhamnoside. 3-biosides 3-gentiobioside, 3-rhamnosylglucoside, ·3-sophoroside, 3-xylosylgalactoside, 3-xylosylglucoside. 3,5-dimonosides : 3-arabinoside-5-glucoside, 3,5-diglucoside, 3-galactoside-5-glucoside, 3-rhamnoside-5-glucoside. 3,7-dimonosides : 3,7-diglucoside. 3-triosides : 3-gentiotrioside, 3-·(2G-glucosylrutinoside), 3-(2G-xylosylrutinoside). 3-bioside-5-monosides : 3-r.harmoglucosid.e-5-glucoside, 3-sophoroside-5-glucoside, 3-xylosylglucoside-5-glucoside. 3-bioside--7-monosides · 3-·sophoroside-7-glucoside.

gentiobiose : diglucose (~, 1-6 linkage) rutinose : 6~-L-rhamnosido-D-glucose

sophorose : diglucose (~, 1-2 linkage) 10 value changes in different solvents. Hydroxylation : The greater the numbers of hydroxyl groups in the anthocyanidin molecule, 'the lower the Rf value in both alcoholic and aqueous solventso Hethylation : The reverse of the effect of hydroxylation with larger numbers of methoxyl groups resulting in higher Rf values in both types of solvent. The increase in Rf values brought about by methylation is somewhat less than the decreas.e caused by hydroxylation. Glycosidation : There is a direct relationship between Rf value and the number of sugar residues present on the antho­ cyanidin moleCule. Tb.e effect of glycosiaatiorl i.s -to inc~~ease ' Rf values in aqueous solvents, and decrease them in alcoholic solvents. Acylation : Reverses the effect shown by glycosidation, increasing the Rf value in alcoholic solvents and decreasing the Rt value in aqueous solvents. l§.Q).ation and identification :

The characterization of an unknown.pigment depends on identifying the anthocyanidin produced by hydrolysis of the pigment molecule and on determining the nature, position of attachment and number of sugars present. The first step in identifying an anthocyanin is isolating it in pure state. Paper chromatography has been a satisfactory technique for separating and identifying anthocyanins since its introduction 1 1

·by Bate-Smith (1948) •. Column chromatography is also used to separate the mixtures of· two or three anthocyanins (Karrer and Strong, 1936; Forsyth, 1952; Li and Wagenlmecht;· .1956). Paper partition chromatography, however, is useful in obtaining pure pigmrnts in small quantities (Harborne, 1959). Chromatographic procedures have also been used in identifying sugars, acyl components, anthocyanins and antho­ (Harborne, 1958). The most satisfactory method for identifying these compounds is by chromatographic comparison with standard markers. The procedure was described by Harborne and Sherratt (1957). The partial acid hydrolysis procedure of

Abe and Hayashi (1956) d.~termines t}le position and number of sugar residues attached to the anthocyanidin. Although chromatography can be used at each step in the identification of anthocyanins, the chromatographic methods should be combined with other techniques for complete structural determination. For example, spectral methods are important and can be used most profitably in conjunction with chromato­ graphic studies.

Biosynthesis of a!lthocyani~ :

Anthocyanins are derived from two separate biosyn­ thetic pathvmys utilizing acetate and shikimic acid. The A­ ring arises by head-to-tail condensation of two malonyl CoA units and acetyl CoA. The B-ring and the C3 unit are derived from a C6 ring-C3 precursor, which may be shikimic acid, 12 cinnamic acid, or phenylalanine (Harborne, 1967). The pio- syn_th~~-ic .P~~hway for an tho cyanidins is s ho~ in Fig. 3 • Glycosylation is the last step in anthocyanin synthesis and requires a'specific group of enzymes, anthocyanases. Synthesis of mono-; di~, and triglycosides is a stepwise process involv­ ing separate additions of monosaccharides to approprlate precursors. The relative frequencies with which the various monosaccharides occur in bound form is glucose > galactose > rhamnose > xylose '> arabinose (Barbo me, 1963). Environmental factors can have profound influence on anthocyanin synthesis in plant tissues. Light is the most important external :fact'or and many experiments have been carried out on the light dependency of anthocyanin synthesis (Harborne, 1967). A two-step biosynthesis s scheme has been propooed : a light phase of activation and a dark phase of synthesis (Havelange and Schumaker, 1966). Anthocyanin synthe­ sis requires the presence of free sugar. The addition of carbohydrate (up to a certain concentration) to the system always stimulates anthocyanin synthesis, while conditions favoring rapid protein build~up inhibit it. All the enzyme systems important to anthocyanin synthesis occur in the buds of the flower blossom stage only. No intermediate stages of anthocyanin synthesis from the vege­ tative stage to the formation of buds are known (Hess, 1963). 13

Fig. 3. Biosynthetic pathways to anthocyan.idin. -(Harborne, 1965- andGrise-bach, 1965) 14

HOOC-CH-CH2-., \d~ NH2 phenylalanine shikimic acid 1-NH3 COOH · HOOC-CH:CH-0 2 CHz-CO-CoA cinnamic acid malonyl CoA + . ·~ CH3-CO-CoA '-¥ acetyl CoA HOI:?IOH_C-0. ~ ...... c..... 9' f\ OR C15-Intermediate J H H0~ 0)0o:H -~It . OH 0 .flavanone I coJ ~ H . HO'((~/Q-oH

bH'~ OR

cyanidin . !!

NATERIALS AHD :r.:IETHODS

Anthocyanins were studied in Allium a.mplectens to determine the identity of the anthocyanidin molecule in each.

Allium ~mplectens was chosen because 15 of the 19 anthocyanins reported in Allium (McNeal, 1970) occur in one or more of the 19 collections of the species under cultivation in the experimental garden at Stockton. The ten most common spots, quantitatively, were isolated by two dimensional paper chromatography, hydrolyzed and compared by thin-layer co­ chromatography, with known anthocyanidins to determine their identity. The anthocjatiins were extracted from dried, pressed plants with 1% hydrochloric acid in methanol (v/v). The

flowers, scapes, leaves and bulbs were cut into smal~ pieces and placed in a beaker. ·Enough extracting solvent was added to the beaker to just cover the plant material. Extraction was carried out in darkness for 24 hours at 4 c. The extract was drawn off with a pipette pulled from glass tubing and approximate 0.5 ml of extract applied to one corner of each it/hatman 3IVJN Chromatographic paper in a spot about 2.5 em in diameter. The chromatograms were de­ veloped by the descending method in two directions. The first direction was developed in the top layer of a n-BAW solvent system (normal butanol : acetic acid ·: water, 4:1:5 v/v, equilibrated for 8 hours before being separated) for

15 16 about 16 hours. The second direction was developed in 15% acetic acid {acetic acid : water, 15:85 v/v) for about 6 hours. After drying, the chromatograms were marked with a pencil and the anthocyanin spots were numbered in sequence following the system set up by Hingrone (1968). The Rf values were calcu­ lated for each spot in both solvent systems. Each spot was then cut from the paper and eluted in 1% methanolic hydro­ chloric acid. Because some of the spots (1,2 and 15; 3 and 14) were too close to be separated in a two dimensional system, they were cut out as a group and eluted together. The solutions were concentrated by evaporation under vacuum. The elutes of spots 1 ' 2, 15 and spots 3, 14 were applied at one end of a piece of half size 3:r-Tivr" whatman Chromatographic paper4 These chromatograms were dev·eloped in one direction by the descend- ing method in the n-BA\'1 solvent system for 16 hours. The chromatograms showed three distinct bands for spots 1, 2, 15 and two bands for spots 3, 14. Since the solvent system in the first direction of the two-dimensional and in the one-dim.ensional paper chromatography for spots 1, 2, 15 .and 3, 14 is n-BAW, we may compare the Rf values of the spots on both chromato­ grams. It was concluded that band 1 from spots 1, 2, 15 '\'ras probably spot 15, band 2 was spot 1 and band 3 was spot 2. For spots 3, 14, band 1 was 1)robably spot 3 and band 2 was spot 14. After drying, the separate bands were cut and eluted in 1~~ methanolic acid. The concentrated elutes were then sub- jected to mild hydrolysis by heating for 10 minutes in a boiling 17

water bath~ The hydrolyzed elute was applied to plastic thin layer sheets, commercially precoated with cellulose (Eastman Chromatogram 6064). Each chromatogram was spotted with a sample of the hydrolyzed material, an unhydrolyzed sample of the anthocyanin from the same solution, and four standard antho­ cyanidins : cyanidin, delphinidin, malvidin and pelargonidin. The standard anthocyanidins were also dissolved in 1% methanolic hydrochloric acid before spotting. These samples were applied to one end of the chromatogram with a micropipet in a spot

approximately 0.5 em in diameter. Th~ unhydrolyzed sample was · used- because after mild hydrolysis, only a portion of the antho­ cyanin breaks down to sugars and anthocyanidin. The cr...:::omato-· grams, therefore shew at least two separate spots for each hydrolyzed sample, representing the anthocyanin and anthocyanidin. By running an m1hydrolyzed sample, it is possible to distin­ guish between the unhydrolyzed anthocyanin and the anthocyanidin. The chromatograms were then developed in an Eastman sandwich developing apparatus. Four different solvents (Table 3) were used for separate chroma to grams bearing each of the unknO\vn anthocyanidins. After drying, the Rf values were calculated for each spot and the unknown was compared with each of the standard anthocyanidins and the undrolyzed anthocyanin. Using four different solvent systems allowed for comparison of the unknown and standard anthocyanidins under different conditions of mobility. 18

Table 3. Solvent systems for the chromatograms of cellulose thin layer.

abbreviation composition tim(3 of run

AA•H acetic acid . cone • HCl water 2 hrs. (3:1 :8, v/v) Forestal acetic acid . cone • HCl water 3-i hrs. (30:3:10, v/v) HAc•HCl acetic acid . cone • HCl water 1! hrs. (15:3:82, v/v) n-BA\v* n-butanol . acetic acid water 4 hrs. (4-:1:5, v/v)

*use top layer. after being equilibrated for 2 hours. RESULTS

According to l\1cNeal ( 1970 ), chromatograms of Allium

~mplec,:tens reveal 15 different spots in two-dimensional paper ch't'omatography in the solvent systems, t~BAv/ ( t-butanol acetic acid :water, 3:1:1, v/v) and 15% acetic acid. These spots are 1 , 2, 3, 4, 5, 6, 7, 8, 9, 11 ' 13, 14, 15' 16, 18 using the numbering system set up by Hingrone ( 1968), (Fig. 1 ) • In that investigation, ten of the spots (4, 6, 7, 8 9, ' 11 ' 13, 14, 15' 18) were not detected in all collections of the taxon. ·In this investigation using th.e n-BAW solvent system (4:1:5, v/v), ·only ten of' the anthocyanins were detected. These spots are 1, 2, 3, 4, 8, 9, 12, 13, 14, 15 (Fig. 4), and all of the spots occurred in every chromatogram except spot 13. These ten spots are the most common anthocya:u.ins that have been detected in the Allium accuminatum allianceo The Rt values of these spots in both solvent systems are shown in Table 4. Thin layer chromatograms of the unlmown spots, after mild hydrolysis, showed·two or more separate spots in each of the solvents tested (Fig. 5, 6, 7, and 8). Comparing these to the standard anthocya,nid.ins and the unhydrolyzed antho­ cyanins, it was possible to distinguish one of thespots as the unhydrolyzed portion of the anthocyanin and the others as the intermediates from anthocyanin to anthocyanidin. The Rf values of all the standard and unknown

19 20

Fig. 4. Composite chromatogram of the anthocya!l_~!l co_rnp_Q:n.ents· of the Allium .§lli]]:ecten..§_alliance., 21

1.0

0.8 t 0.6 "0 () 0 ()

0.2

0.6~ 'F"'~· 0.2 _&.,0 0.4 ~-- 0 Rf n-BAW (4:1:5) 22

Table 4. Rf values of·anthocyanins of Allium amplectens on paper chromatograms.

solvent systems spot number n-:SAiv 15% acetic acid

1 0.28 0.36 2 0.30 0.39 3 0.35 0.50 4 0.43 0.48 8 0.20 0.72 9 0.24 0.74 12 0.14 0.55 13 0.37 0.61 14 0.36 0.40 15 0.25 0.34 23

n• i! ~g. 5 • Chromatogram of unhydrolyzed anthocye.nins., hydrolyzed anthocyanins and. standard anthocya:nidins. in n-BAW solvent system.

p pelargonidin M mal vi din D del:phinidin ·C cyanidin

1 (1 1 2,15) band 2 2 (1,2,15) band 3 3 (3,14) ban'd 1 14 ( 3, ·14 ) band 2 15 (1,2,15) band 1

* Parenthesis indicate hydrolyzed anthocyanins. • Broken line means spot in very low concentration. 24

q CQ ~ <;t: C\1 0 0 0 d 0 ~Mc~am-~.R-m~,~~Mm~~ma~eam.~~~ 0 .. 0 '·-· . ,.. ~-. C> ' ..... •'

., ... -...... It) ·~ 0 ~ ... ---. = ,. , ' tt) I.. I ._ ...-- ~. @ 0 ' ...... _., . .:::;.

~ 0 § -- 0) .... Q) ) Q".) ..0 0 E :::1 !II (l) c ' --- I 0 \._ ...... 0 : .. -~, 0. -- ~ Ul 0 ---~ .. - .I ·-· .' 'It 0 .•''..' E1 tt)

0 ~

C\1

......

0 u D 0 0 c__ :o) a. 25

Fig. 6. Chromatogram of unhydrolyzed a.rithocyanins, hydrolyzed.anthocyanins a.nd·standardanthocyanidins in Forestal solvent system.

* For the abbreviations and explanation see ~ig. 5. 26

...... 0::

(\J d 0

iO' ~

l()

.,.. - __-- ., .... %

·::t

=~

--.J 1'1) 0 -· @' c.:> =

Q:;. ~ -

!o. ~ Q) .n E ,. (j) ::J c .,_ .-.. 0 ~ a. (J) co

~ ·, ...

---_~ 0---~ \ ......

1'1)

0 (.) 0 0

a.. 27

Fig. 7. Chromatogram of unhydrolyzed anthocyanins, hydrolyzed anthocyaninsand standard anthocyani

* For the abbreviations and explanation see Fig. 5.

\ -...... --.. • C> " .. ··, -~-~,_I m

',.,...... C> § .... - ~' .... ~: +- 0 c...... _. __ -...... (/) c.:> "'" D g D t()

# .. - .... g .'·-,. ,..• 0 D C\1 .. .-----..... ' D ...... D 0 () 0 0 0 :E 0 a.

l

H!

ir 29

Fig. 8. Chromatogram of unhydrolyzed anthocyan.ins, hydrolyzedanthocyanins and standard anthocya!lidihs in AA·H solvent system.

~ For the abbreviations and explanation see Fig. 5.

\ 30

..... 0::

0

0 D !.!2 'it 0 ~ 0 ' !:2 ...... _, @' ..... __ .. c:::> = , ,.--, .. ~ •' __ , •

.... (\) ,... ----, _ , ..0 . -· E ::J c , -- ...... , .... '--...... - 0 0.. C> (/) C> 0 0 0 ,...,, ·-· __ , . 0 0

0 0 0... 31

anthocyanidins on cellulose thin layer chromatograms are shown in Table 5, and the Rf values of the unhydrolyzed anthocyanins in Table 6. Comparing the Rf values of the anthocyanidin and anthocyanin from the same spot,. we f.ind that all the Rf values of the anthocyanidins are less than the original unhydrolyzed anthocyanins in aqueous solvents (all of the solvent systems except n-BAW in this investigation) and are greater than antho­ cyanins in the alcoholic solvent (n-BAW). This can only be explained if sugar moieties are being removed from the hydro­ lyzed anthocyanins; removal of acyl groups, for example, would have the opposite effect. (For detailed explanation, see -introduction-on page 10). A comparison·of the anthocyanidins with the four standard makers shows the.t the Rf values of the anthocyanidins of all spots investigated are the same as, or very close to, the Rf values of cyanidin in all solvent systems while the other three standard anthoc_yanins have significantly different Rf values. This indicates that all of the ten anthocyanins of Allium amplectens possess the same aglycone, cyanidin (Fig. 9).

Fig. 9. Structure of cyanidin. 32

Table 5~ Rt values of ·anthocyanidins of Allium amplectens on cellulose thin layer chromatograms.

solvent systems spot number n-BAW Forestal HAc•HCl AA•H

Pelargonidin 0.76 0.70 0.14 0.22 l-lalvidin 0.39 0.76 0.46 0.59 Delphinidin 0.46 0.29 0.03 0.05 Cyanidin 0.58 0.50 0.06 0.11 (1 '2, 15.) .band 1 0.56 .... 0.-51 0.06 o.-11 (1,2,15) band 2 0.56 0.07 0 .. 11 (1,2,15) band 3 0.57 0.07 (3, 14) band 1 0.55 0.49 0.06 0.09 (3,-14) band 2 0.55 ··:':·0•48· 0.07 0.11 4 0.56 0.52 0.05 0.12 8 0.56 . 0.50 o.os 0.11

9 0.58 0~51 0.05 0.11 12 0.54 0.48 0.05 0.11 13 0.56 0.47 0.06 0.09 33

Table 6. Rf values of anthocyanins of Allium amplectens on cellulose thin layer chromatograms.

solvent systems spot number n-BA\'1 Forestal HAc•HCl AA•H

(1,2,15) band 1 0.65 0.23 0.30 (1,2,15) band 2 0.37 0.63 0.23 0.33 (1,2,15) band 3 0.37 --- 0.22 (3,14) band 1 0.35 0.63 0.21 0.31 (3,14) band 2 0.36 0.64 0.21 0.32 4 0."31 6.68 0.21 0.26 8 0.25' 0 .. 65 0.39 0.56

9 0.23 0.65 0~37 0.53 12 0.22 0.62 0.34 0.45 13 0.40 0.63 Oo21 0.30 DISCUSSION

Comparison chromatography definitely indicates that the anthocyanidin molecule in all ten anthocyanins investigated is cyanidin. The number of breakdown products. from hydrolysis, as indicated by the number of spots on each chromatogram, gives some indication of the number and :position of attach­ ment of sugar moieties. The chromatograms were not entirely satisfactory in this regard as there was some tailing of spots which could have masked some intermediate products. Likewise some of the intermediate spots were present in such low con- . cehtration a:s to make their detection uncertain• Some of the unhydrolyzed anthoc:vanins showed more than one· spot on. some chromatograms and since these were stored in acid soluti.ons it is assumed that some hydrolysis took place naturally in these solutions. As pointed out earlier only a limited number of sugars are known to occur in anthocyanins and the first sugar is always attached to the hydroxyl at the 3- position on the anthocyanidin molecule except in the case of 3-deoxyanthocyanins where the sugar is usually in the 5- position. This means tnat those anthocyanins v1hich showed only two separate spots on a chromatogram must have hydrolyzed to cyanidin in a single step, indicating that only one sugar is attached to the molecule. This means that these anthocyanins must be cyanidin-3-mono­ glycosides. Anthocyanins showing three separate spots must

34 ·.·-•• ,,.· • .!. 35

have hydrolyzed ·to cyanidin in two steps and have two sugar moieties attached to the molecule. Moreover, both sugars must

be attached in tandem at the 3- position forming a cyanidin~ 3-diglycoside.-· If. the suga:rs.were attached to hydroxyls at . :/," ~, .. : ,, .-:···~·· ··~:; the 3- and 5- positions we would 'expect to find four spots rather than three : cyanidin-3,5-diglycoside, cyanidin-3-mono­ glycoside, cyanidin-5-monoglycoside and cyanidin. The same condition would prevail if the sugars were attached to hydroxyls in the 3- and 7- positions. Anthocyanins containing three sugar moieties would show four spots if they were attached in

tandem a~ the 3- position or six spots if attached at both the

. 3.,;.. and 5~ (or 7:..;.) positions: All the dio.. and trisaccharides have at·lease one glucoae (or·in one instance galactose) unit in the pigments that are known at present.

~lith the chromatographic data available we can en- numerate the combinations of sugars and cyanidin which are possible in Allium amulectens from known cyanidin-glycosides (Table 7). However, in order to definitely identify the antho­ cyanin molecules it will be necessary to identify the sugar moieties involved in each. In addition, with some of the more complex molecules, it will be necessary to compare the Rf values of the unknown .in several solvent systems with Hf values

I of well characterized glycosides. In this investigation, due to the lack of standard glycosides and the lack of data on the

identi~y of the sugars the anthocyanins could not be positively identified. 36

Table 7. The possible anthocyanins of Allium amplectens from the known cyanidin glycosides.

number of .the spots separated on chroma­ cyanidin glycoside tograms

.2 3-arabinoside, 3-galatosidep

3-glucoside, 3-rhamnoside~o

3 3-gentiobioside, 3-rhamnosylglucoside, ·3-sophoro side, 3-xylosylglucoside,

3~xylosylglucoside.

4 3,5-diglucoside, 3,7-diglucosidet 3-arabinoside-5-glucoside, 3-galactoside-5-glucoside, 3-rhamnoside-5-glucoside. I .I

III " LITERATURE CITED

Abe, Y. and Hayashi, K. 1956. Future studies on paper chro- matography of anthocyanins involving an examination of gly­ coside types by partial hydrolysis. Bot. Hag •. (Tokyo), 69: 577-585. Bate-Smith, E.C. 1948. Paper chromatography of anthocyanins and related substances in petal extracts. Nature. 161: 835 -838. ------and Westall, R.G. 1950. Chromatographic behavior and chemical structure. (I). Some naturally occurr­ Tng phenolic substances. Biochim. et. Biophs. Acta. 4: 427 -440 •. Forsyth, \v. G. C. 1952. Cacao polyphenolic substances. I. :F'ractionation of the fresh bean. Biochem. J. 51: 511-516. ----and Simmonds, N.W. 1954. A survey of the anthocyanins of some tropical plants. Proc. Roy. Soc. B142: 549-564. Grisebach, H. 1965. Biosynthesis of flavonoids. In "Chemis­ try and biochemistry of plants". (Goodwin, T.W. ed), Acad. Press. London. New York. Harborne, J.B. 1958. The chromatographic identification of anthocya.nin pJ.gments. J. Chromatogr. 1: 473-488. 1959. The chromatography of the flavonoid pigment. J. Chromatogr. 2: 581 1963. Plant polyphenols. IX. The glycosidic pattern of anthocyanin pigments. Phytochem. 2: 85-97.

37 I 38 I ~ ! a 'I It H 1965. · Flavonoid pigments. In "Plant Biochemis­ ]; .,i try". (Bonner, J. and Varner, J. eds.) pp. 618-640. Acad. r:. Press, Land, Hew Yorlc. -1967. Comparative biochemistry of the flavo- noids. Acad. Press, Londo:n, New York. and Hall, E. 1964. Plant polyphenols. XIII. The systematic distribution-and origin of anthocyanins containing branded· trisaccharides. :Phytochem. 3: 453 and Sherratt, H.S.A. 1959. Identification of the sugars of anthocyanins. Biochem. J. 65: 23p-24p. Havelange, A. and Schumaker, R. 1966. Metabolism of antho-· Hcyanins in Siila:psis a1ba. plants. I. The role of light.

Bull. Soc. Roy. Sci~ Liege. 35(1-2): 125-41. Hayashi, K. 1962. The anthocyanins. In "Chemistry of flavo­ noid compounds'!. (Geissman, T.A. ed.), pp. 248-285. Perga. Press, Oxford. Hess, D. 1963. Blossoms as the region of anthocyanin synthe­ sis. z. Botan. 51(2): 142-155. Karrer, P. and Strong, P.M. 1936. Preparation of pure antho­ cyanins by chromatographic analysis. Helv. Chim. Acta. 19: 25-28. Lawerence, W.J.C., Price, J.R., Robinson, G.M. and Robinson, R. 1939. The distribution of anthocyanins in flowers, fruits and leaves. Philos. Toans. Roy. Soc. London, 230B: 149

Li, ICC. ~nd 1t'lagenknecht, A.C. 1956. Anthocyanin pigments of sour cherries. J. Am. Chern. Soc. 78: 979-980. I I 39

MeN eal, D. W. 1970. Comparative studies of Alli urr: acumina tum alliance. Ph.D. thesis, Washington State Univ.ersity, l'ullman. Mingrone, L.V. 1968. A comparative study of the Allium falcifolium alliance. Ph.D. thesis, Washington State Univer­ sity, Pullman. University Nicrofilms. Ownbey, l-'1. and .A.ase, B.C. 1955. Cytotaxonomic studies in .Allium. I. Allium canadense alliance. Res. Stud. State Coll. Wash.

23(Suppl.): 1-106~ Pratt, D.D. and Robinson, R. 1922. Synthesis of pyrylium salts of the anthocyanidin type. J. Chem. Soc. 121: 1577-1585. Saghir, A. R. B., Jviann, L .K., Ownbey, IV1. and Berg, R. Y. 1966. Composition of volaties in relation to taxonomy of American alliums. Amer. J. Bet. 53: 477-484.