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6'01 cVCIRGIDmDmst-pjs

 

ISOLATION OF TAB PIGJLLT OF RED CHERRIES

mo Id‘bo'rms RIFLE-twine 1T3 coLou

By

Mina Adeline Glidden

51' 9“};fi a? g er
In! luau .uz J.

Submitted to the School of Graduate Studies of Hichigen
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

HASTLR CF SCIENCE

Department of Foods and Nutrition
School of Home Economics

1955

Approved_~_

k” V V _. —A . .___.__
V—

 

 

Kine Adeline Glidden
l

The anthocyanin pigment or the skin or aontmorency
cherries was extracted with methanol. Isolation and
purification wemaacnieved Ly treatment with ion exchange
resins. The extract was passed through an anion exchange
resin to remove the anion of the pigment and any basic
constituents. The effluent was passed through a cation
exchange resin which absorbed the anthocyanin. The
neutral substances passed through in the effluent. The
anthocyanin was then eluted with 5% methanolic hydrochloric
acid. In concentration of the eluate hydrolysis apparently
occurred and a red powder presumed to be anthocyanidin
precipitated.

An attempt was made to obtain some indication as to
the structure of the anthocyanin. Tests indicated that
the sugar group might be a bioside or a pentoseglycoside.
The results of tests for the anthocyanidin were not
conclusive. However they indicated a structure closely
related to cyanidin, pelergonidin or peonidin.

Various factors which affect the color of the antho-
cyanin were studied. The anthocyanin was found to produce
a blue color in basic medium and to be decomposed in

very strongly basic solutions. In acid medium the pigment

 

"“1“”

nine Adelina Glidden

2

was red in color. In the pd range of 2 to 5.6 the intensity
of the color, but not the hue. was affected. Alcohol was
found to produce a mere blue color in tne pigment solutions.
Carbohydrates such as starch, pectin, and methyl cellulose
were found to stabilize the color of the anthocyanin in
aqueous solutions. Starch was particularly effective.

The effect of iron and tin ions on the color change of the
pigment in citrate buffered solutions held at 1000 F for

5 weeks was found to be very slight. The greatest factor

in the deterioration of color of the pigment appeared to

be the temperature of storage. In all cases the eluate of
the cation exchange resin (presumably quite pure anthocyaniri)
showed considerable fading. The effluent or neutral fracticnz
(probably containing tannins and flavones) darkened during
storage. The original methanol extract of the pigment also

showed a fading of red color.

 

Isommon or Ti-LE mourn or RED CHERRIES

AND icToab IuFLULNCIKG ITS COLOR

BY
Mina Adeline Glidden

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition
School of Home Economics

1955

I A- -IY (Kr-"Lt. f" '75? fV' grfn {-1
twink “U 3' “La- '“ingc—u“&u

The writer wishes to eXpress her sincere gratitude
to Dr. Elizabeth Osman, for her invaluable guidance and
counsel throughout this study; and to Dr. C. L. Bedford.
Horticulture. for his advice in planning the problem and
assistance in obtaining and processing the cherries used

in this study.

.1‘7 w. 1"‘-:‘3r-‘

. "1. .'"‘.‘ff '..
[wrath UP VUHI Lal‘xu

II‘éTfiCDJCTIoN . . o . . . . . . . . o . . . . . .
LI’Ith-SIL-ii; I‘iiVlLL‘e’ . . . . . . . . . . . . . . .
Chemical otructure . . . . . . . . . . . . . .
Factors Effecting Color in llents . . . . . .
Color Tests for flnthocyenins . . . . . . . . .
Isolation and Purification . . . . . . . . .
Eehevior of nnthocyanins in Food Products . .
fiik’lfilifilisl‘i-L PROCLDULEE. . . . . . . . . . . . . .
Treliminary Isolation Ltudies . . . . . . . .
Isolation Eethod . . . . . . . . . . . . . . .
Test for Identification of Pigment . . . . . .
Factors Influencing Color Changes . . . . . .
REUULIC n30 DISCUSS UN . . . . . . . . . . . . .
Preliminary Isolation Ltudies . . . . . . . .
Isolation of the Lutdocyenin . . . . . . . . .
Test for Identification of Pigment . . . . . .
Effects of Colloidal hystems . . . . . . . . .
Factors Affecting the Color . . . . . . . . .
The Iffect of Iron and Tin Ions on the Pigment

v 791*

f“ v v .v‘" 'IT'f' “'-"fi7- “v.7 -.q_--c.‘

'." F'v‘r f‘_

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Liar Ul' Alig'l-l‘.-:l¢v;...u‘ o o o o o o o o o o o o o o 0

fi'
Cu

0301!“

003

F3883

INTHUDJGT ION

The procuction of Eontmorency Cherries is one of the
important fruit industries in nichigen. The average
annual production for the decode 1940 to 1950 was about
43,0CG tons (Bureau of Agricultural Economics, 1352).

This represented approximately fifty per cent of the total
United States production. nbout half of the cherries
procuced were canned.

The problem of color deterioration in conned cherries
hes lOng teen recognized. It has commonly been thought
that the enthocysnin pigment, which is reopensible for
the red color, reacts with the metal of the cans, to
bring about a fading or color. however, the erect nature
of the reactions involved in color deterioration of
enthocyenin pigments still leaves many unsolved mysteries.
The early workers usually dealt with unyurified extracts
which UJQOUthGIY contained tannins, flevones and other
water soluble constituents. These impurities may have
led to inaccurate results and conclusions. however in
recent years investigators have develOped methods of
isolation and purification for the pigments. Thus the
study of pure pigments has been made possible. Although

some color reactions between the pure pigments and various

metal ions have been found to agree rather closely with
those observed by earlier workers with crude extracts.
certain discrepancies exist which can be exoleined only
by studies of the pure pigments.

The purpose of tnis study wee to isolate and purify
tne red pigment of cherries, to obtain some indications
as to its structure, and to determine the effect of iron
and tin ions, sugar, and verious colloidal systems on the
changes in the oigment color. The color change of the
pigment in relation to pd has also been observed. The
amount of color loss and the change in color were measured
by light absorption. using s spectrOpnotometer. Prom the
results of these exocriments it was heped thot information
would be gained concerning the nature of color deterio-

ration in canned red cherries.

'af rad-1‘

REVIEJ CF Llfifinl'gu
Chemical Ltructure

In the last fifty years the pigaent comgounds of
plants have aroused much interest. Among these compounds
are the antnocyanins, which impart an orange red to blue
color in many flowers. fruits, berries, vegetables and
leaves. The anthocyanins are water soluble and usually
are found in solution in the cell sap of glants (Hawzonek
1951). These pigments belong to a family of polyhydroxy~
flavylium salts, which can be hydrolyzed into two com-
poneuts, a sugar and a ben20pyrylium comoooud known as
on antnocyunidin. The anthocyanidins consist of three
general types, shown by formulas I. II, and III. The
numerous variations have been found to be methyl others
of those (Robinson 1333a, Robinson and Robinson 1332b,

Robinson, at L1. 1354)-

 
   

CI) _ (III) ”
+C._. ,, :9], 0R
OkilRO
H OR’
OF!
pelargonidin Stud cyanioin 33H. delyhinidin R'fi'afi"=d
apigeuidin R=J peouidin R8345 petuaidin R8035 fi'.aw=d
0P malvidin R=R"Cd5 R"'J
gesnerioin or 7
syringidin

hirsutidin 3=R'~R"=c§5

4
The anthocyenins end the enthocysnidins are emphoteric
in nature end yield salts of both acids end bases (Robinson
1935a). The acid salts are usually red in color while the
basic salts ere blue. There are two theories concerning
the formation of the basic salts.

The existence of a ring containing a quadrivelent

oxygen was postulated as early as 1905 (Dyson 1950).

+C| -

bel’—veu to be

no
(:5
i.)
C;

9 OH This structure

rBSponsible for the peculiarities of color in the antho-

  

cyonin pigments. some workers thought that the addition
of alkali to an anthocysnin resulted in a shift to e

quinone structure.

tsa:
HO OH_____ 0 =0 ‘
R R
OH ‘ 0H

Shrinor and fioffett (1333) honover, s owed that such

 

a quinoid structure foiled to exist for some relsted
benzooyrylium salts. They concluued further (Luriner
and Loifett 1540) thet both oxonium structures

+CH-

   

5
are untenable. They concluded (Shriner and hoffett 1941)
that the enthocyenidin chlorides, es yell as other
flsvylium salts, contain an ellylic system in the

heterocyclic ring

    

    

H *0"

end that these salts may tsutonerize end resonate between
the two structures represented. with the yositive charge
shifting between carbon 2 and carbon 4. They further
suggested toutomerizetion to o quinoid structure.

Tue behavior of the enthocyenins is dependent not
only on the structure of the enthocysnidin, but L150 on
the sugar groups present. Robinson (1952a. 13663) has

subdivided the unthocysnins into five g'oups depenuing

on the number, position, and tyoes of sugar molecules
attached to the snthocysnidin nucleus. His classifi-
cation is as follows: (a) monosides, which are mainly
5-monoglucosines, end 5-monognlsctosides, (b) o-rhamno-
glucosides one other 3-pentoseglycosides. (c) 3-biosides,
(d) 3,5-digluoosides and other dimonosides. (e) the
conglex anthocysnins or ucyleted derivatives of the
other four classes; in this lest group, organic acids
such as melonic, p-hydroxycinnemic end p-hydroxytcnzoic

are component Harts. The ecylated snthocyenins occur in

all the enthocyunidin series (dotinson lsddb). In the

above classification of enthocycnins, Robinson (l952a)
defined the sugar portion of the molecule as follows:

(a) in s glycoside, e single unit of e henose. (b) in a
pentose glycoside. e disnccheriue composed of a pentose
and s hexose. (c) in e Lioside, a diseccheride composed

of two hexose units. and (d) in a diglycoside, or dimonoside
we hexose units linked to two different hydroxyl groups

of the enthocyenidin nucleus.

a majority of the entnocyenins that have teen isolated

have been found to Le 3-monosioes or 3,5-diglycosides.
The color of most flowers and fruits is apparently dependent
on the presence of only one enthocysnin. noeever, when a
mixture of such pigments hes been identified, the components
have been found to be methylated end unmethyleted deriva-

tives of the Some enthocysnidin (Lawrence, e e .. 1959).

Factors Affecting Color in Plants

'7‘. a
$1“:

Vurietions in the color of plants containing
enthocysnins are influenced both by the structure of the
anthocyenin and by the presence of certain other comoounds
(jotinson 1935c, G. iohinson 1959). The effect of structure

on color varies with the number and potition of the

(’5

hydroxyl gr ups. Increasing the number of these groups

7
results in e Shift of color from orange red to blue red
in an acid medium. It is believed that the hydroxyl
groups on the phenyl ring attached to carbon 2 of the
pyrene nucleus have the greatest influence in determining
the color of the nnthocynnidin (fiewzonok 1951).

The presence of conigments such es tannins, end
flevone glycosides was fOUJd to deepen the color of
enthocyenins. The extent of deepening depends on the
concentration of cepigments in relation to the concen~
tretion of enthocyenins (hobinson 1959).

Some inorganic substances, neinly ferric salts, were
found to effect the color. The presence of iron salts
in a plant usually resulted in the formation of a blue
color. This was illustreted by growing hydrangea in
soil containing iron filings. Blue flowers usually were
produced. This reaction is not fully understood but
Robinson and Robinson (1959) proposed the theory that
iron functions in the initiation of physiolOgicel
disturbances in the plant whereby the enthocyenin
concentration is diminished. This theory was based
on the fact that solutions of enthocynnins were observed
to form a blue color on filter peter. if the pigment and

acid concentrations were low.

8

In agreement with the idea that the blue color wns
produced by some factor other than pH value, it was found
that the cell sop of blue flowers, though possessing a
higher pd value than that of red flowers, was nevertheless
acid.

Colloid association of snthocyenins, probably with
polysaccharides such as xylem and others not known,
appears to aid in the formation of blue colors. Robinson
(19533) studied a distilled water extract of blue corn-
floners, and found it to contain xylen and other poly-
saccharides. 0n eXnminntion of the extract with e slit
ultremicroscoye, ultremicrons were observed, which
possessed fairly rapid Brownian malement. Licroceta-
phoresis showed the particles to be negatively charged.
These particles did not precipitate in the presence of
two normal sodium chloride. Therefore it was concluded
that a protective colloid was present. Robinson explained
the phenomenon by the formation of a complex between the
colloid and onion of the pigment, which resulted in a
stable aggregate with a negative charge. imparting a
blue color to the solution. The colloid complexes are

'\

believed to form more readily at pH velues 01 5. or

(71

higher. The pH value of the cell sop of blue flowers

is usually between 5.0 and 5.6.

The theory of stabilization of the anionic charge
by the state of aggregation wns further tested by Robinson
(lQSBe). ne preoored two solutions of cysnin chloride.
One solution was prepared by adding a small amount of
pigment to toiling tap water, with the formation of a
violet color. The other solution was pregsred by tritu-
ration of the pigment in cold water for a minute, before
gradually heating to the boiling point. A blue solution
was formed. nobinson stated that he difference in
formation of the two colors could only be caused by the
State of aggregation whicn stabilized the anionic charge,
yielding a blue color. As fouad that the addition of s
colloid such as xylen, starch. and agsr~egsr to a pigment
solution greatly increased the ease of preparing e blue

solution.
Color Tests for Lnthocyunins

Based on the color Varietions of enthocyenins at
different pi Values Robertson end dobinson (lgfig) have
develoyed a series of color reactions which are useful
in characterization of the Specific types of enthocysnidins.
For best results these reactions should be carried out in
a system of buffer solutions within the desired pH range.

The color changes hsve been demonstrated to be of greater

10
value and reliability than use of the absorption spectrum
in characterization of anthocyonidins. Some or the
properties revealed by he color reactions were: ease
of pseudo-base formation whiCh caused e decrease in color
intensity; ease of formation of color buses. noting the
pH and length of time required for their precipitation;
and the ease of oxidation, again noting the pd and long h
of time required for the disappearance of the color.

Robinson and Robinson (1931. 1932:) also develoyed
other color tests to aid in the identification of the
anthocyeuidin portion of the molecule. in aqueous hydro-
chloric acid extract of the pigment may he need for these
tests. It may be tested with ferric onlOride for the
presence of tannins and flevonco. since they imoert a
green or brown color with iron colts (Choice 1925);
pure enthocynnina give a Line color or remain unchanged.
If cepigments are oreeent, it may be necessary to purify
the solution (Robinson and Robinson 1951, 1952a). To
obtain the enthocyenidin, the pigment is hydrolyzed and
extracted with emyl alcohol. Ease of transference of
the enthocynnioin from the n-emyl alcohol to eqneous
hydrochloric acid by addition of benzene gives an
indiCntion of the structure of the pigment. Three

volumes of 0.5 per cent hydrochloric ecid are adned

ll
to one volume of omyl alcohol extract. Toe cooroximate
volumes of benzene reguircd to cause complete transfer
of t: .c 1giggzrzcnt to the aqueo 1* layer are as follows:
dolphinidin, three to four; petunidin, f01r to five;

yanidin, iivc to six; m;lvi.oin, five to Six; peonidin,
ei;11t to nine; ono pelorgow1i1in te:1 to eleven. To obtain

a solution ior subsequent tests a one per cent hydrochloric
acid extzoct is used. '1osults of W1 i'ollox tug tests on
thisext1act are surmo1izcd in Table I:

(a) A soul p0: ion 0? the prepared extract
01 out uocya 11idin is extroc.od dwith amyl alcohol;
sodium acetate is added and the color observed.

A droo of ferric chloride solution is added and
the color again obs QPVed.

(b) A small p01tion oi the extract is shaken
with cyanidin reagent (8 mixture of one volume of
cyclohexane ond five volumes of toluene).

(c) A portion of extract is shaken with
delghinidin 1o"*PAt onsistiug of a mixanre of
five oar cont picric acid in one volume of amyl
ethyl other and four volumos of anisole.

(d) Oxidation test: A £m1ll gortion of

r?
)4
1‘

"act is shoéton with air and half its volume

Q

x
of ten yer cent sooi um hydroxidc is aoded.

 

AmeHv GomCHQom saw Comdfipoma
.coapooom mp6 :« caca>aoa on moafiaflm mo mane: coo maonmu whoooo afloauomnflm "ofiwauomnfiw
m“ .Hogooam :uas ooaosafio co
daoficowaoaom cusp mooao 1053 who mo>fiuo>ahoo :fioacoom .hoaoo ca nmammo
ma coaosaom owoo may pogo ca :«anomumao; Eon“ mauoano moommag :Hwfidoom unfluacoom

ozooooo
no soauauoo no
odflxouom: moan :oadoonw
flowcom oUHHomnmn andoom

moan :mH on oagapmdo ou magnum opmoonuoo

1coouw ucmfian moan was; sown noaoo uoaoap suave» Amy
coauoaom
caom
comaamfioam own uoaow> don ooaoa> con uoH0fi> won mo uoaoo on
womoypmon vomoapmoo oomomnozs mapoum hHaHoh domouumoc no: coaomoaxo nqv
vmpoonpxo cmuomhpxo douooamxm poomuoh
defipoouuxo on Cowpoonumo o: hamuoamcoo mfiopoMquo poo wamuoamfioo aficaoflmlaoc AOV
cmpowupxm unmwooa
cowponupxo on soapomnpxo oz :0fiuooapxo c: uoaoo omen maommma afloaaoho any
ooauoano
omdogo cc moan mono owzozo on moan woman» owzozo on cannom
vow
oumumoo
anacom dam
moan mzapuuoao«> uoaofiptmoan poaoa> moacmon can poaoa> Honooao Hmsm va

 

fificflocNdlllllmdedomumHoo

 

noomoom

 

momma Hoaoo Mo ano&55u

H mqmwa

13
immediately followed by the addition of concentrated
hydrochloric acid and omyl alcohol: the recovery of
the anthocyanidin is noted.

(e) Color in one per cent hydrochloric acid
at the boiling point is observed.

(f) One half volume of sodium carbonate is
added to a portion of the extract and the color
change observed.

The distribution of the anthocyanins between emyl
alcohol and one per cent aqueous hydrochloric acid was
found by thinoon and Robinson (19323) to be of value in
identifying the number and types of sugar groups. In this
system the monoglycosides are transferred mainly to the
alcOhol layer while the rhamnoglucosides and other
pentoseglycosides predominote in the aqueous layer.
RhamnOglucosides and the other pentoseglycosidea can be
distinguished from diglycosides by the addition of salt
to the system. The pentoseglycosides remain in the
aqueous solution. while the diglycosides become more
soluble in the alcohol layer.

Anthocyonidins can usually be distinguished from
onthocyenins by their distribution between a dilute acid
solution and isoamyl clcohol. If the isoemyl alcohol

extracts practically no pink color, the oigment is an

14
enthocyanin. If it extracts m at of the color the pigment
is an anthocyanidin. Lnthocyanidins are said to occur
only very rarely as such in plants (Eancroft and Rutzler

1958).
Isolation and Purification

Host isolation and purification methods are based on
the amphoteric properties of the anthocyanins.

In extraction of the anthocyanin pigment from plant
tissue methanolic hydrogen chloride or glacial acetic
acid usually has been used. Frequently the pigment~
containing materials. if flower petals, were dried in an
oven. With fruit the pigmented portion was freed from
the Juice by means or a hydraulic press. The fruit was
pressed until a fairly dry press cake was obtained. The
dried solid see then placed in a one to two per cent
methanolic hydrochloric acid solution and soaked for
several days (Price. g3_§;. 1939; Brown. 1940; Schindler.
1945). The pigment was precipitated from the methanolic
hydrochloric acid solution by the addition of a large
volume of anhydrous other. a sirupy precipitate usually
resulted. which was dissolved in one per cent methanolic
hydrochloric acid and reprecipitated with ether. in order

to obtain a more purified substance. After several

15
reprecipitations in this manner the pigment was further
purified by a conversion to a picrate or lead salt and
back to a chloride. The details or these procedures are
Specific for each pigment. however the picrete was ob-
tained essentially by dissolving the sirup in a warm
saturated aqueous solution of picric acid and allowing
it to stand several days (Grove. Robinson, 1931; Anderson,
1924). The picrate crystals were removed by filtration.
washed free of picric acid with ether. and allowed to dry.
The dried crystals were dissolved in three per cent
methanolic hydrochloric acid and the chloride was precipi-
tated by the addition of a large volume of ether. Often
several of these conversions were necessary to obtain a
pure compound. This method frequently resulted in the
loss of much pigment (Anderson 1993; fondheimer. Kertesz
1948b).

The lead salt usually was obtained by the addition
of-lead acetate to an aqueous solwtion. The lead precipi-
tate was removed by filtration and triturated with methanol
containing hydrochloric acid. The liquid was filtered
free of lead salts, the pigment precipitated and the
solution chilled. The chloride salt of the pigment was
removed by filtration, washed with ether and dried in a

vacuum desiccator. For further purification the process

16
was repeated (Pucher. et el. 1938). Schindler (1945)
used hydrogen sulfide gas to precipitate the lead Which
was removed as lead sulfide. Five to ten per cent sulfuric
acid has also been used for the removal of lead (Onslow
1325). The filtrate was evaporated to dryness under
reduced pressure. The residue was dissolved in ethyl
alcohol and the pigment precipitated by the addition of
ether.

Willstatter and Bollinger (1916) and Anderson (1923)
used glacial acetic acid for extracting the pigment from
the skins of sweet cherries, ngnug fixing. and grapes
respectively. The pigscnt was then precipitated and
purified in the some manner as when a methanolic hydro-
chloric acid solution was used for extraction.

fiillstatter and Zollinger (1916) found that several
of the snthocyenlns could be crystallized by dissolving
the crude pigment in methanol. Gentle warning on a water
bath was required to dissolve the pigment. in intensely
dark red solution was obtained which was allowed to stand
in a loosely covered dish at room teoperature for twenty-
four hours, after which a small amount of ethanol was
added and crystals appeared within a few hours.

In the last few years chromatographic methods have

been applied in the separation and isolation of anthocyanins.

l?
The choice of the most efficient absorbent end develooer
is rather difficult. since the various anthocyenins respond
differently to each system. A water solution of antho-
cyenins was passed over the absorbent column. and separation
of the various pigments was achieved (Kerrer, Strong 1936,
Ksrrer. water 1956). Spueth (1949) has used a mixture of
one volume of normal butenol and three volumes of ethyl
ether for the separation of mulvidin end petunidin. The
separation occurs since each substance has its Specific
rate of flow in'e given system. The ratio of the rate of
flow of the solute in relation to the rate of flow of the
solvent is known as the Rf value.

Some of the absorbents round to be efficient by horror
and otrong (1956) and by Kerrer and Weber (1936) were
gypsum for the delphinidin pigment of the black mellow.
and activated alumina for separation of a mixture of
peonin chloride and cyanin chloride. Usually the pigment
was absorbed from a water solution and eluted with a dilute
solution of hydrochloric acid. The eluete was concentrated
under diminished pressure. In the case of peonin chloride
the pigment Separated and was removed by filtration. It
was further purified by repeated absorption on activated
alumina and elution. followed by concentration. Finally

the precipitate was dissolved in hot 0.5 normel hydrochloric

18
acid, filtered and allowed to cool, whereupon crystals
separated out. Tee delphinidin pigment, after concentration.
was isolated as the picrete by addition of a saturated
solution of picric acid. This was converted to the chloride
by dissolving in two per cent methanolic hydrochloric acid
and precipitating with ether.

Paper corometogreyhy has been used for identification
purposes and es an indication of whether a single pigment
or a mixture of coloring msteriels is pr>sent. Bets-Smith
(1948) found s-solvent mixture of butanol—scetic acid-water
in the ratio of 481:5 to be effective in the separation of
pigments and the determination of the waxlues. The Rf
Values and color reactions obtained give some indication
as to the number of sugar groups. also the type and number
of entnocyenidins present. The results of these tests are
summarized in Table II. The rate of flow is influenced by
the temperature and the solvent system; therefore in
comyering results of Rf values it is necessary that the
conditions of eXperimentetion be identical.

The success of chrometogrephic methods has led to
the application of ion exchange resins in the isolation
and gurificetion of many natural products including amino
acids and tannins. However. to our knowledge, ion exchange
resins have not been used previously in the isolation of

entnocyenins.

19
TABLE II

 

 

 

 

Summary of Paper Chrometoaraphic Testsl
lcidity of

Luthocyonidin Anthocyanin finger ”soup

Doggxgglve Coluglon JiglKCU§;QUS‘LMQHO’lV 8 do
Cyanidin: __

color in air 1% £31 "lue mauve Mauve

color in ammonia ' Blue orey blue

color in air 5-55 J01 Lluo mauve mauve

color in ammonia Blue Grey blue
Peonidin:

color in air 13 ddl --- ---

color in ammonia --- -~-

color in air 5.5% HS Pink Pink

color in uamouiu clue dlue
Malvldln: ,

color in air 1% M31 --- -~-

color in ammonia --~ --~

C010? in air 5.5K n6 Eauve Kauve

color in ammonia blue green Blue
Hirsutidin:

color in air 1% HS Kauve -~-

color in ammonia mauve ---

color in air 5.5fi H31 Rosy mauve Rosy mauve

color in ammonia Blue mauve luisn
1

Bate-Lmith (1348)

Behavior of Anthocyanins in Food Products

The attractiveness and appeal of many fresh fruits
and vegetables is aided by the bright red and purple color
of the enthocyanin pigment. In general when these fruits
are canned, changes in color are observed. Culpepper and
Caldwell (1927) have studied several fruits in relation
to the color changes and have made the statement that.
"It is well established that enthocyanins generally.
regardless of the color of the free pigment, react with
many metels with an accompanying alteration of color
toward the violet end of the Spectrum.” They also
believed that reactions of snthocyenin with the metals
of the can were responsible for the corrosion and
perforation characteristic of many pigmented fruits.

The corrosion in the presence of higher enthocyanin
concentration was eXplained by the theory, that the

fruit acid reacts with the tin of the can forming salts
with only momentary existence. The salts are immediately
decoMposed with the transference of the tin to e combi-
nation with the enthocyenin. Thus there his a sustained
attack by the acid which could strip the tin completely
from the steel plate. Therefore the liriting factor in
corrosion is not the acid but the enthocyenin content.

the degree of attack on the can being determined by the

capacity of the anthocyanin to form a stable organic
complex with tin.

Culpeppor also specifically investigated the dis-
coloration of montmorency cherries, Prqngg Qggasgg, in
relation to use of tin cans in processing. The fruit
was processed in three types of co 8; glass. enameled
tin, and plain tin. After storing for various lengths
of time the cans were Opened and the fruit examined. The
cherries processed in glass were found to have lost in
brightness of color through diffusion of the pigment into
the water. however the color was clear red, proportional
in intensity to that of the fresh fruit. The enameled
cans yielded cherries more intense in color, but with a
purplish tint. The cherries canned in the plain tin
showed pronounced fading. In this instance the liquid
was a faint pinkish red with a decided milkiness. Rith
overnight eXposure of the fruit and Juice to air. they
became intensely purple.

A crude pigment. an aqueous extract of the cherries,
was made and the color changes observed after treatment
with stannous CJlOPidG, aluminum chloride. and ferric
chloride. The stannous chloride and aluminum chloride
caused a purpling. while ferric chloride caused a

blackening.t If the fruit acid in the extract was partially

22
neutralized. the discoloration was more intense. However.
the addition of sufficient acid to the discolored liquid
restored the original clear red color.

LathrOp's (1928) findings were in close agreement
with those of Culpepper. LathrOp found iron and tin to
be especially injurious to fruit pigments. He stated
that tin turns cherries a deep purple while iron produces
a dull brownish discoloration. 0n exposure to air the
metelnpigment complex becomes quickly oxidized. intensify-
ing the discoloration.

Morris (1946) also found that fruit Juices containing
snthocyanina frequently undergo color reactions with metals.
however since anthocyanins are usually accompanied by
impurities such as flavones and tannins. he believed it
was impossible to determine the amount of the color change
actually due to the anthocyanins and the amount due to the
impurities. Iron was found to turn many fruit Juices green
or greenish black, while tin salts resulted in a violet or
mauve color. Even two parts per million of iron were
found to darken strawberry pulp. The acidity was observed
to heve an inhibiting effect on discoloration caused by
metals. high acidity showed evidence or a lower degree
of discoloration. However after prolonged contact with

tin there was a tendency for the Juices to become turbid

25
or milky. Morris thought this was probably caused by the
formation of a hydrated stannous oxide. The concentration
of the anthocyanin was believed to influence the degree of
color change. When the cnthocyanin was present in abundance.
tin produced a more pronounced discoloration.

Cruess (1948) stated that prolonged heat as well as
metal ions injures the nnthocyanin color of canned fruits
and red Juices. The tin and iron salts dissolved from
the tin plate of the can either precipitates the antho-
cyanin or causes a blue discoloration of the canned fruit.
The pigments of prunes, red cherries. red plums. and berries
apparently not as catalysts in the corrosion of tin and
hasten the production of hydrogen swell and perforation.

Joslyn (1941) believed that the problem of color
retention in fruits was of a complex nature. Some of the
factors involved are. the nature and stability of the
pigments and of the minor constituents. such as tannins.
flavones, and enyzmes. The treatment of fruits in canning
and storage may also influence the color. however he
stated that the discoloration or changes in the tint of
canned berries and cherries was caused primarily by the
formation of stannous salts with the anthocyanin pigment.

Griswold (1944) found that the color of chtmorency

cherries was influenced by various factors. Cherries

24
became more orange in color with increased holding time
before canning. Soaking in cold water also caused a more
orange color. Processing time had little effect on color.
however, underprocessed fruit tended to be more orange,
while overprocessed fruit was more red. Increasing the
heed space or oxygen content of the can produced a more
orange and less intense color.

Glass Jars were used for canning most of the fruit.
In order to study the effect of metallic ions. ferrous
sulfate equivalent to 0.05 parts per million of iron was
added to some of the samples. Tin and iron suCh as might
be dissolved from a can were added in a concentration of
five part per million of tin as stannous chloride plus
one part per million of iron as ferrous sulfate. The tin
and high concentration of iron appeared to make the fruit
more orange than standard distilled water; however the
differences were not statistically significant. The
cherries canned in enameled tin cans were found to be
purplish and less intense in color than the standard
glass pack. After storage for eight months breaks were
noticed in the enamel and perforations were evident after
eleven months.

The deterioration of the color of cherries seemed

to occur gradually during storage; sugar was found to

25
preserve the brightness. Griswold proposed the theory
that sugar either depresses the decomoosition of antho-
cyenins to entnocyenidin plus monoseccherides or it protects
the pigment from oxidation. Cherries stored in the dark
and at ten degrees centigrede were found to be redder and
more intense in color than those stored in the light and
at high temgeretures.

Nebesky and co-workers (1949) studied the effect of
various factors on the stability of the color of several
fruit juices and the pigments isolated from strawberries
and currents. The factors studied were the effects of
time and temperature of storage, reletiozsnip of oxygen.
light, sugar. and pH on the deteriorative changes in the
color of the juices and pigment solutions. The color
changes were determined by measurement of light trans-
mission using a Colemon snectrOpnotometer. The absorption
maxime were determined for each Juice and subsequent
measurements made at the determined wave length.

Nebesky's finding were in agreement witn tnose of
Griswold. deet and oxygen were the most snecific
accelerating agents in the deterioration of color during
storage. Light, pH, and sugar were found to have very
little effect on the color. The solution of pigment

from currents resyonded to the treatments the same as

26
the current Juice. The strawber'y pigment and strawberry
Juice resoonded alike. with the exception that the pigment
was bleached by light, whereas the Juice was little
effected.

In their work with strawberries Sondheimer and Lee
(1949) found that on freezing oerries with D-glucose a
violet to blue coloration develOped. This color change
was reversed on thawing. The coloration was noted only
in semyles containing glucose in e solid fort. The
enthocyenin yigment of the strawberries was.found to play
a dominant role in the formation of the violet color. lhe
higher the concentration of pigment the more intense the
violet coloration. The pd of the pigment medium seemed
to have little effect on the color change.

This reaction was true only of the one sugar,glucose,
and it is not known wnether or not the resction is Specific
for pelargonidin 3-monoglucoside, the pigment of strawberries.

Sondheimer and Kertesz (1948a) have also studied the
color reactions of the enthocyenins in strawberries and
strawberry products during storage at stout zero degrees
ccntigrade. They found that a loss of red entnocysnin
pigment occurs and a brown pigment develOps. The change
in color was studied by means of ligat absorption of an

enthocysnin solution. A Beckmen quartz spectrOphotometer.

zooel 1%}. and 8 Solomon sooctrophotometo" were used.
The results of the two instruments ofirecd roughly within
ten per cent of each other.

is tne pl of the solution was lowered from 3.5 to
“.0 the absorption at 530 millimicrons exnrossod in ootical
density, was more than doubled. however within this pd
range the maximum absorption always occurred at the some
wave length. Thus at e pd between 2.0 and 3.4 the intensity
of color was altered. but not the hue. however,different
solvents were found to affect the point of maximum eb-
sorhtion. A shift from 500 to 520 millimicrons was found
when the snthocyenin of strawberries was dissolved in
so per cent ethanol instead of water.

enother esoect of the problem of color changes has
been recotnized Ly Kohmen (1932 . He observed that when
strained cranberry sauce was first canned. the liquid
that exuded was brilliantly red. however after several
months of storage the liquid was almost colorless. The
time required for this change was inversely related to
the storage temperature. If the sauce was then disoersed
in water and filtered, a colorless filtrate was obtained.
but if the sauce was dissersed in acetone a brilliantly
red filtrate resulted. Kohmen snagested that the water
insoluble portion of the pigment might be loosely bound

to the wax of the berry. with which it is closely associated.

7'\r--r~ "r?“*"‘1 (‘1’ r - ..-‘:- 7")?“
le'3L¢KIéi,taziln.—I I}.KCV.LD‘J4\LL

The Lontmorency red cherries used in this study were
harvested in July at a suitable stage of maturity for eating
and brought to the laboratory. The cherries were washed.
pitted, and the pulp and Juice removed with a tomato Juicer.
The quite dry skins from about 150 pounds of cherries were
added to l2 liters of 90% methanol. A second Lottie was
prepared in a like manner. The alcohol solution rapidly
became very dark red in color. It was stored in the
refrigerator until the last of October. The cherry skins

were then removed by filtration with the aid of Ln eSpirstor.
Preliminary Isolation Studies
Ether grecipitction

Concentrated hydrochloric acid was added to a liter
of the stove methsnolic extract of the tigment to form a
1% solution in hydrochloric acid. The solution was concen-
trated in an atmosphere of nitrogen at a temperature below
30° C. and under diminished pressure to one fifth its
original volume. During the concentration a white precipitate
was obtained, which was removed Ly filtration. The material

burned in a flame, showing it to be an organic substance.

29
The solution remaining after concentration was probably
mainly aqueous with a hydrochloric acid concentration
greater then one per cent.

The distribution of the pignent between the aqueous
solution and Q-Lutenol. isotutyl alcohol. gysmyl alcohol.
and isonnyl alcohol was determined by mixing equal volumes
or the aqueous solution end the alcohol with shaking. In
all cases the pigment predominated in the aqueous layer.
The eddition of salt to the mixture had little effect on
the distribution of the pigment.

Two and one-half volumes of ethyl alcohol were added
to the remaining pigment concentrate, to give a solution
miscitle with ether. This solution had a total volume of
500 m1. SeVen volumes or 3.3 liter of ether were required
for complete precipitation or the pigment. nfter the
addition of ether the solution was placed in the refrigerator
for 24 hours. Thereafter the upper layer of liquid was
decanted from the hick sirup which had separated. The
siruy was dissolved in s minimum of alcohol and reprecipi-
tated with seven volumes of ether. Reprecipitntion of the
sirup was repeated six times end still a sirup was obtained.

In this method concentration was of no vslue since it
was necessary to add a large amount of alcohol to the

concentrate in order to obtain a solution miscible with ether.

50

A second portion of the methanolic extract or the
pigment was acidified with concentrated hydrocnloric acid
to form a solution containing % hydrochloric acid. Ether
was added and a thick sirup precipitated. Six to seven
volumes of ether were required for complete precipitation
of the pigment, regardless of whether fractional precipi-
tation in three steps was used or complete precipitation
in one. Repeated reprecipitations. as above. yielded only
a sirup. Therefore the ether precipitation method was
abandoned since the large amount of ether required was

not practical. and a pure product was not obtained.
Picrate method

An atteMpt was made to purify further the thick sirup
containing the pigment and also the methanolic extract of
pigment by the formation of a picrate. A sample of the aqueous
solution of thick sirup was made to a 1% concentration in
hydrochloric acid. The picrate was made by combining an equal
volume of the pigment solution with a saturated solution of
picric acid. The temperature of both solutions was 40° C. when
combined. The solution was placed in the refrigerator for
48 hours and bronze colored flakes precipitated. However,
much of the red pigment remained in solution. The crystals

were removed by filtration and placed in a desiccator to

51
dry. All attempts to purify the picrete or convert it
to a chloride failed. The unconcentreted methanolic
extract of the pigment gave no better results. Therefore

the method was abandoned.
girometmtrs 3211c SEflL‘cll'CtiOIl and p2_1_1;11.‘icstion

Absorption columns were filled with the absorbent to
be tested, eno w“shed with methanol and 1% methanolic
hydrochloric acid. The absorbents tested were calcium
sulfate, activated alumina. Decalso. Florisil, Permutit,
Zeolite, and Lloyd's reegent. The activated alumina and
Florisil columns absorbed the pigment. Aowever a solvent
was not found which would effectively elute the pigment
from the activated alumina. The Florisil column was not
used since its capacity was very limited. The others did

not absorb the pigment.

Isolation Hethod

 

Ion exchn_;e resin;

Anion and cation exchange resins were used and found
to form the most satisfactory method for the isolation
and purification of the cherry pigment. A column of the
strongly basic anion exchange resin, Amherlite XE-QS, was

prepared. The resin was soaked overnight in distilled water.

32
A column 52 mm. in diameter was filled to a depth of 14
inches with the resin, and backwashed with distilled water
until the overflow was clear. Cue liter of 4% sodium
hydroxide was passed over the column, witn a contact time
of 2 hours, to regenerate the resin. The column was washed
with distilled water until the WfiShiflgS produced a yellow
color with phenol red indicator, *nd a red color with methyl
red indicator, showing that the p3 of the washings was close
to neutral.

A column of the cation exchange resin Zeokerb d, a
sulfonuted coal, was prepared. The resin was soaked over-
night in distilled meter. A column 80 mm. in diameter was
filled to s depth of 6 inches with the resin, and eckweshed
with distilled water until the overflow was cleer.i Two
hundred milliliters or bi hydrochloric acid was passed over
the column, with a contact time of one hour, to regenerate
the resin. The column was washed with distilled meter
until the washings produced a yellow color with phenol red
indicator, and e red color with methyl red indicator.

The methanolic extract of pigment was filtered to remove
a white precipitate. The filtrate was passed through the
column of Amberlite XZ-QS resin at the rate of one liter
per hour. Jhen more than one liter of extract was added

to the column SOme of the pigment appeared to Le held by

35
the resin instead of passing through it. Therefore, the
capacity of the anion resin appeared to be one liter. insofar
as the desired reaction was concerned. The effluent, which
was deep violet to dark green in color, was collected and
immediately passed through the column of Zeokerb d resin.
at the rate of one liter per hour. If the effluent repre-
senting here than one liter of extract was passed through
the column of Zeokarb n resin, pert of the pigment was not
adsorbed; therefore the capacity of the resin appeared to
be one liter. The Amberlitc XS-QS resin was washed free
of pigment with distilled water and the washings were also
passed over the Zeokarb n resin. hhen the effluent from
the column of anion exchange resin was passed through the
cation exchange resin a yellowish brown to pinkish brown
effluent was obtained. The color of this effluent was
unchenged by the addition of hydrochloric acid. The
column of Zecharb d resin was washed with distilled water
until a clear effluent was obtained. then with 100 ml. of
5% aqueous hydrochloric acid. The pigment was not eluted.
The pigment was then eluted from the cation exchange resin
with L; methanolic hydrochloric acid. yielding a bright
red solution.

A lOC-ml. portion of eluete from the column of cation

exchange resin was concentrated to 20 ml. in en atmOSphere

34
of nitrogen under diminished pressure, obtained with a
water nepirstor. The temperature was maintained below
30° C. A second lCO-ml. portion ves concentrated in the
same manner except that an etuosbhere of air was used.
No difference could be observed between the two concen-
tretes. ”In both cases during concentration a dark red
precipitate was obtained which was insoluble in water,
but readily soluble in ethanol and methanol. The red
precipitate, probably the enthocyanidin, was removed by
centrifuging until a clear liquid was obtained.

Light absorption data for the various solutions were
obtained end compared to determine the effect of treatment
and solvent. A Beckmen quartz SpectrOphotometer Kodel LU
was emyloyed for the determination of the Spectra. Corex
cells with e 13 mm. light path were used. Distilled water
served as a blank. he Spectra for five of the solutions
were determined throughout the complete band of visible
light, 400 to 650 millimicrons in wave length. The readings
were taken at 10 millimicron intervals. The following
solutions were used (a) the original methanol extract of
pigment diluted 1:24 with water, having a finel pd of 3.64.
(b) mothenolic extract. 1% in hydrochloric acid. diluted
with water and having a final pd of 2.09. (c) eluete from
the column of cation exchange resin diluted with water

1:1.5. (d) eluete from the column of cation exchange resin diluted

55
with methanol 1:1.5. (e) a dilute methnnol solution of the
pigment precipitated in the concentration of the eluete
from the column of cetion exchange resin. light absorption
of three other solutions were 0 served at six different
wave length. (f) eluete from the column of.cetion excnnnge
resin diluted 1:24 with water. (g) supernatant liquor from
the concentration, in en atmosytere of sir, of the eluote
from the column of the cation exchange resin. diluted 1:49
with water. (h) some as (g) except the concentration was
Carried out in an atmosphere of nitrogen. The Spectre are
shown in Figure I. Since the wave lengths for maximum
absorption of solutions (f), (a). and (h) were all identical
the remaining eluate from the cntion exchange resin was
concentrated in an atmosphere of sir. The amorphous red
precipitate formed during concentreti n was removed by
filtrntion, washed with water and placed in s vacuum
desiccator to dry.

Various attempts to crystallize the substance were
tried without success. Willststter's method of crystalli-
zation from methanol as described by inderson (1926) was
used, Lut no crystals were obtained. The pigment_wes also
dissolved in alcohol and precipitated by the addition of
various organic reagents. such as ethyl acetate, ether,

petroleum ether and enisole. In ell cases on emorphorus

‘35 Transmittunoy

Effect of Oxygen,

 

 

 

 

Kethenol, and on o

 

 

 

* s '9"°‘:‘E1.
13.0 /s xcf”§37?::7:‘”*‘
/AA.T o—wi— -D
n/4‘/¥/ / [Ix/fl"
I \“
/./ / _’ --'d /'
i/fl‘ a—w'o D‘X-‘d
a", .7<// ./r o "‘ °
,/ /P/ f. ' <4" ' '
9;: ,- 9. ~_ ," /, 5'. ./
; 5“ / T'V'r/%
\.\ o /P/.'
’ X \ \ \ ’1 // I". .I
\ \ \ / f
\ \ x \ \ ,, .' /, / I
\ \ I, "'I A
I \ \ “‘-"l ' ' 'l
1 x , ' I /
A \ y .
bk; k \\ \o/ / I,
/O f -’
I \ . . [A
i ‘ -’
\
' \ / /
.f""'—\ \ 11’ .
\ I
/ \ 1
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7'3 / \ .
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F \ \ ‘
so :— - . \w/
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r ,4». ., 9.
{'0 '. \Q 1
a/ k \ .-..o gyuate from cation exchan7e.‘
/ ‘k‘ \c Vtsl'n 1:15 d'u’U'hon
-' '\\.' \~ n__.3 elqate concd In arm 0f
L‘ I \o \ n;t,o en ljj’adilut/on
UC A \\'_ 4 7 1
‘\ ‘(1 - .__x cm to Concd In atm ofasr ;
* \ \ \ a: o diluTnon—
x . 0 4
, \ .'
/ \t \ \v
- \x \ ...-'A\.
/ \ \
:_ - 3‘ ...p Mon extract a Muted
4O \ n45 pn.iby
‘ own ‘Unconcd eluate diluTcd :
\n I: 1-5 wt“! me‘fhanol ;
\ o o uncogducluate diluted 1
\ I'I'D wlfh wafer i
\ l
‘0‘ , 4-...0 PM, me»? oculae r, obtained
~ .- "0' from concantr‘afiop 07'
60 ante, (1.5501de In
methanol
——-x HcoH extract—I70 HC!
’l dnuted 1:25 gnaw?
*\
20 - \
\, / .
‘4 . l
n L 1 L «La 1.er
«00 450 500 sec boo bob 70o
Nave Length nuq

Figure I

57
precipitate was formed. quantitative analysis of the red
precipitate showed it to contain 63.2% carbon and 6.673

hydrogen.
Tests for Identification of Pigment
Faper Chromatography

Paper chromatographs were made of (e) the original
methanolic extract of the pigment, (b) the eluete from
the cation exonehge resin, (c) the effluent from the
column of cation exchange resin. and (d) a mixture of
(b) and (c). In making the chromatographs a strip of
filter paper was Spotted about one-half inch from the
tiy and allowed to dry. The paper was placed in a test
tube containing 5 ml. of the nonequeous layer from a
4:1:5 mixture of Qrbutenol. acetic acid, and tater,
respectively. The tip of the paper diprd Just below
the surface of the liquid in the tube. The colored
pigment moved up the pager with the solvent, forming e
cone shaped figure. The chrometogreph from the original
methanol extract consisted of two distinct Spots. The
upper one was yellow brown, and became bright yellow
when exyoeed to damp ammonia vapor. The lower portion
was red in color and turned blue on eXposure to damp

amyonie fumes. Tue chrometogreph of the eluete from

38
the column of Eeokarb d resin 1nd only a red Spot corres-
gonding to the lower snot on the chromstOgrenh of the
original methanol extract. The effluent from the column
of Zeoknrb h resin resulted in e Ciromet05rnph with a
yellow spot. correSponding to the yellow spot on the
chrometogrsph of the original methanol extract. then the
eluete and effluent from the column of Zeohnrb n resin
were combined and used. c chrometogrnph similar to the

one obtained with the original methanol extract reszlted.

Color tests

 

The color tests of Robinson and Robinson (1951) as
described on page 12 used. A smell amount of the precipi-
tated red pigment obtained by concentration of the eluste
from the column of Zeokerb h resin was dissolve in 5 ml.
of ethenol and diluted to 103 ml. with aqueous 1%
hydrochloric acid. The solution was boiled 3 minutes.
cooled end extracted with aromyl alcohol. The upper layer
was sepsrsted end washed twice with enter and once with
1% aqueous hydrochloric acid. Three volumes of 1% aqueous
hydrochloric acid were edded, end the color was transferred
to the aqueous layer by addition of benzene. The solution
of pigment in la hydrochloric acid was separated and washed

twice with benzene to remove traces of flrsmyl alcohol.

59
The hydrochloric acid solution of pigment was used for the
following tests;

(a) A smell portion of the stove solution of pigment
was extracted with gremyl elcohol; to half of the solution
sooium acetate was added. A purpliSh blue color resulted.
On the addition of ferric chloride the solution turned a
clear blue.'

(b) Three volumes of O.oi hydrochloric acid was
added to the other half of the emyl alcohol extract
remaining from test (a). The addition of eight volumes
of benzene was required to transfer the pigment to the
aqueous layer.

(c) A small portion of the hydrochloric acid extract
was shaken with cyanidin reagent (a mixture of one volume
of cyclohexene and five volumes of toulene). 1 small
amount of the pigment was extracted by the cysnidin reagent.
This layer was bluish red in color.

(d) Oxidation test: A Small portion of the hydrochloric
acid extract wss shaken with air. One half its volume of
10% sodium hydroxide was added. A greenish blue color was
obtained. This solution was immediately acidified with
concentrsted hydrochloric acid and the red color was

recovered. There wee no apparent destruction of color.

40

(e) One-half volume of a saturated solution of sodium
carbonate was added to a small portion of aqueous pigment
solution. A bright blue color was obtained. On the
addition of acetone, two layers resulted. The lower layer
was blue and the acetone layer had a pale greenish cost.

(f) A smell portion of the aqueous soic extract was
heated to toiling and the color observed. The solution
was orange red in color.

(a) To e smell portion of the aqueous ecid extract
ferric chloride was added. No reaction occurred.

(h) A small portion of the hydrochloric acid extract
was diluted to three times its volume with methanol. he
solution became tluer in color.

The results of these color tests are summarized in

Table III.
Eiscelleneows Tests

(a) A few drops of ferric chloride were added to
the original methanol extract of pigment, and to the brown
effluent from the column of cation exchange resin. In
both instances 5 greenish black color was formed. dowever,
no color change occurred on the addition of a few drops
of ferric chloride to the eluste from the column of

cation ion excusnge resin.

TABLE III

Summary of Color Tests for IdentifiCetion of Structure

i E: rent
(a) nmyl alcohol plus sodium acetate
plus ferric chloride
(b) Three volumes of 0.5 per cent
hydrochloric acid and one

volume of nmyl alcohol plus
benzene

(c) Cysnidin reagent

(d) Oxidation test
(e) One-half volume of sodium

carbonate plus sddition of
acetone

(f) nydrocnloric acid extract
heated to boiling

(g) Ferric chloride

(h) Dilution with methanol

Results
purplish blue color
clear blue

eight volumes of
benzene were
required to transfer
the color to the
aqueous layer

some extraction.
bluish red

no loss of color
bright blue color
two layers--bottom
layer blue; acetone
layer pale green

orange red color

no change

becomes bluer in
color

48
(b) The solubility of the red pigment obtained by
concentration of the eluete from the column of cation

exchsnne resin was determined in ethanol, methanol, stmyl

’4.

alcohol, ester. and d lute hydrochloric acid. The pigment
was soluble in the alcohols and insoluble in ester end acid.

(0) An aliquot of the eluste from the column of cation
exchange resin. and an aliquot of the supernatant liquor
obtained by concentration of the eluete were treated with
Benedict's solution. A small amount of copper precipitated
in the supernetnnt liquor, but none in the eluste.

(d) A lolisch test wes performed on an aliquot of
the seme solutions used in test (c). A positive test was
obtained with the supernatant liquor.

(e) some of the thick sirupy precipitate of pigment
resulting from the addition of ether to the original methanol
extract wos dissolved in water. The method used by Robinson
(ldjze) for differentiation of the types of glycoside was
applied. Kernel smyl slcohol was added with shaking to the
solution. The color was found to predominate in the aqueous
layer. This distribution was not altered by the addition

of salt.

45

Factors Influencing Color Changes
leloid systemsAsnd5Tneir Lffect on the F fining

Tue effects of several colloids on e solution of
snthocyenin were studied. dulflgrcm senples of starch.
yectin, seer-eger, and gelatin were added to lOO~ml.
portions of water. The mixtures were heated to boiling
to dissolve the solids and cooled to room temperature.

A suSpension of ungeletinized starch of the some concen-
tration was prepared by adding starch to cold water. A
similar suspension of methyl cellulose was made by adding
the methyl cellulose to boiling water. Some of the eluete
from the column of cation exchange resin was adJ‘sted
with sodium bicarbonate to a pH of 2.25, filtered. and
used in tests with the above colloids. The reaction of
the pigment with tap water was used as a standard for
comparison. One milliliter of pigment solution was added
to 10 ml. of boiling tap water. The color faded rapidly.
The same result was also obtained by adding the pigment
to cold water and gently heating the solution to the
boiling point. The tests were repeated using each of

the colloidal systems in place of the tsp wster. The
solutions of gelatin behaved in the some manner as the

tap water. However, when solutions of starch, methylcellulose.

44
pectin, or agar-agar were used. the sink color 1 the pigment
remained stable. itn stc' rc‘.1, he color was retained even
when the solution was boiled for several minutes and allowed
to stend iorthiaawce ‘2 s. The color of the pigment was also
ststilized by the starch solution when it was diluted one

to ten.

Qplor Reoctions in Bugfer solution

Tne buffer solutions described by hobertson and Robinson
(19“3) wexe used in tnese reactions. 0n lite1 of an acid
solution containing 0.04 mole of phenylacctic acid, 0.04
mole of horic acid and 0.04 mole of potassium dihydrogen
phOSphate was preporcd. Lac h bufier solution .103 prejored

by soding tne pgescribed amount of 0.2006 fi_sodium hydroxide
to ?5 ml. of the acid solution and diluting to 50 ml. with

distilled water.

Lu11er ml. of Buffer ml. of

solution NaCH pH solution NaCH pH
(1) 0 3.60 (10) 11.1 0.56
(2) 1.20 4.06 (11) 12. 9.05
(a) 2.00 4.48 (12 15.7r .49
(4) 3.75 4.90 (is) is. 0 10.
(s) 0.00 0.00 (14) 16.20 10.90
(6) 6.2.6 6.36 (15) 17.50 11.20
(7) 7.00 6.66 (16) 20.00 11.6"
(8) b.7L 7.25 (17) 22.50 11.70
(9) 10.;0 7.88

45
Twenty-five milligrams of enthocyenidin. the red precipitate
obtained by concentretion of the eluete from the column of
cation exchange resin, was dissolved in 100 ml. of 00%
ethanol. One milliliter of the resulting solution was added
by pipette to 10 ml. of buffer solution. The light absorption
of the pigment in eech buffer sys em was determined by use
of o Beckmen quartz SpectrOpnotometer Looel 0U. These
results are shown in Figure II. The color chenges of the
solution were observed also at verious inter'els or time

up to 18 hours. These results are given in Tetle IV.

46

'v'.‘ i t h

did not restore the red

Acidification of the colOrless tubes

hydrochloric ecid

 

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mmoamoaoo
mmoanoaoo

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mmoanoaoo

 

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seems eoaams mo.aa Aoav
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Effect of pa on Color Changes
'- pr 333
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400 600 630

Have Length/on

Figure II

 

 

 

 

of the Pigment in Buffered Solutions
.01
l A—a P H 1/. 2 0 pegs"???
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400 300 600
'.'~."ave Length I724.

48

Effect of Iron end Tin Ions on the Pigment

The effect of sugar and metol ions on the color of
the Vsri us pigment solutions was studied in atmospheres
of both air and nitrOgen. A series of tubes containing
10 ml. of citric acid buffer. 5 ml. of pigment solution,
the desired amount or metal ions. and water to give a
final volume of 20 ml. were prepared. and stored in an
oven st 1000 F. for 5 weeks. The buffer was prepared by
adjusting he pH of one liter of l §_citric acid to 5.08
by the addition of S fi_sodium hydroxide. This solution
was diluted with distilled water to a final volume of
two liters. when sugar was added it was dissolved in
the buffer to give a solution containing 30% sugsr. Stock
solutions of ferric, ferrous. stsnnous and stonnic chloride
were preyured with a metal ion concentration of 100 parts
per million. A dilution of 1 ml. in 100 ml. was made and
used in the preyerstion of the color tubes. For a concen~
trstion of l5 parts per million, 3 ml. of solution were
used; for 10 parts per million, 2 ml.; for 5 parts per
million. 1 ml.; and for one part per million, 0.2 ml.

The eluote from the column of cation exchange resin
was adjusted with sodium ticsrbonnte to 3 p5 of 8.25.

The salt formed was removed by filtration before the

49
eluste was used. nhen an atmosghere of nitrogen was used,
nitrogen was bubbled through the solution for three minutes.
fill the tubes were sealed with Psrefilm. Seventy-six
variations, in duplicate, were included in the study.

They are described in Table V.

The color changes vcre studied with the aid of a
Coleman SpectrOyhotometer Kodel ll. The color absorption
st $20 will microns was observed the morning after the

v - u ,_ r--. I ‘r‘ r ~-‘ '0 "' ‘ ‘F " *-» - I J" " "in m' ' ‘ ‘
tutes were rleCJCd and nce a neon tneresitei. ine red

C)

tignent powder presumed to be an anthocy nidin soon
precipitated from solution. In the other tubes s white
precipitate formed. Ens otserved chtnnes in the color

of the solutions are summarized in Table V. At the end

of 5 weeks spectra were determined for 28 of the Variations.

The results are summarized in Figures III. IV, and V.

TABLE V

Effect of Storage at 100° F. on the
Red Color of Pigment Solutions

 

% Transmittancy
gt :1 520rn/(
cs , '7

K'Trsnsmittancy
g; %. SSOInA

 

 

 

 

p§torage
Izeetment Q 4_weeks .Ireatment 0 week
Eluate of cation Eluate of cation
exchange resin exchange resin
Nitrogen atmosphere Eitrogen atmosphere
control-stored in control+l5fi dextrose 76.5 85.5
refrigerator 76.5 84.0 Stannous 5 ppm
control 77.0 86.5 +15% dextrose 75.0 82.5
Stannic 5 ppm
Stannous ion 1 ppm 76.5 86.5 +15% dextrose 74.0 82.0
5 ppm 75.5 86.1 Ferrous 5 ppm
10 ppm 74.5 85.5 +15% dextrose 75.0 81.5
15 ppm 76.6 86.8 Ferric 5 ppm _
+15% dextrose 75.5 85.0
Stannic ion 1 ppm 76.5 85.0 Ferrous-Stannic
5 ppm 74.5 85.5 5 ppm each +15% -
10 ppm 75.8 86.0 dextrose 5.0 85.4
15 ppm 74.5 84.0 Ferric-Stannous
5 ppm each4—15%
Ferrous ion 1 ppm 77.0 85.5 dextrose 74.5 85.0
5 ppm 76.5 87.0
10 ppm 76.5 86.8 Red pigment powder
15 ppm 75.5 86.5 from concentration
of eluate from
Ferric ion 1 ppm 77.0 85.0* column of cation
5 ppm 76.8 86.0 exchange resin
10 ppm 78.0 86.7 nitrogen atmosphere
15 ppm 78.5 86.0 control 84.5 89.0
Stannous 5 ppm 86.0 92.5
Ferrous-Stannic Stannic 5 ppm 84.0 2.5
5 ppm each 75.5 85.5 Ferrous 5 ppm 80.7 88.7
Ferric-Stannous Ferric 5 ppm 82.0 90.7
5 ppm each 78.0 85.5 Ferrous-Stannic
5 ppm each 79.5 86.0
controltl5% sucrose 77.5 84.6 Ferric-Stannous
Stannous 5 ppm 5 ppm each 79.0 85.5
r15% sucrose 77.2 85.5
Stannic 5 ppm Original pigment
+15% sucrose 77.5 85.5 extract
Ferrous 5 ppm Nitrogen atmosphere
+15% sucrose 76.7 84.7 control 7.0 10.5
Ferric 5 ppm Stannous 5 ppm 6.0 9.5
+15% sucrose 72.5 85.5 Stannic 5 ppm 7.0 9.0
Ferrous-Stannic Ferrous 5 ppm 6.5 8.1
5 ppm each {'1ng Ferric 5 ppm 6.5 7.7
sucrose 74.0 85.7 Ferrous-Stannio
Ferric-Stannousd F 1 5 ppm BBCh 5-5 7-7
c: . +- L'.., -0
” PP“ eafiBcr5§§ 74.7 85.1 err °5“p:$n2§2h 5.5 7,0

 

 

51

TABLE V (Continued)

 

 

 

 

 

 

 

 

flfiTransmittancy % Transmittancy
g; A 520 m4 at A EEO’WLL
Stoyage Steppes
ggeatment Q ¥_4fweeks Treatment 0 4 weeks_
Effluent from Eluate of cation
cation exchange exchange resin
resin Atmosphere of sir
Eitrogen atmosghere control+15£fsucrose 71.0 85.0
control 78.7 78.5 Stannous 5 ppm
Stannous 5 ppm 78.5 77.0 +152 sucrose 71.5 86.0
Stannic 5 ppm 77.5 78.0 Stunnic 5 ppm
Ferrous 5 ppm 78.0 67.0 +15% sucrose 72. 85.5
Ferric 5 ppm 78.2 70.0 Ferrous 5 ppm
Ferrous-Stannic +15% sucrose 70.0 86.2
5 ppm each 79.5“ 74.2 Ferric 5 ppm
Ferric-Stannous +15% sucrose 72.0 88.5
5 ppm each 77.7 75.5 Ferrous-Stannic
5 ppm +15% sucrose 71.5 87.2
Eluate of cation Ferric-Stannous
exchange resin 5 ppm-+15% sucrose 71.0 87.5
AtmOSphgge of Air
control 75.5 86.2 control+15% dextrose 69.7 84.0
Stannous 5 ppm 74.0 85.5 Stannous 5 ppm
Stannic 5 ppm 75.5 84.0 +15% dextrose 68.7 85.0
Ferrous 5 ppm 74.5 85.0 Stannic 5 ppm
Ferric 5 ppm 75.0 85.7 +15% dextrose 67.0 85.0
Ferrous-Stannic Ferrous 5 ppm
5 ppm each 75.2 84.5 +15% dextrose 67.8 85.5
Ferric-Stannous Ferric 5 ppm
5 ppm each 75.2 85.5 +15% dextrose 70.0 85.7
Ferrous-Stannic
5 ppm +15% dextrose 70.0 85.5
Ferric-Stannous
5 ppm t15% dextrose 68.0 85.7

 

 

lThe transmittancy was measured at a wave length of 52 millimicrons
with a Coleman SpectrOphotometer Model 11.

% Transmittaucy

% Transmittancy

 

 

 

 

 

 

lOC

 

Effect of Storage at 1000 F
EXCLlLinge
ico—- yes
0.. 3_ 1-)“ -K‘!
0:* 'M
030 ./
IIGI
.l‘ I
Q/'/ I
.4/
o".
a/ :a
I A; .'/,' , C)
a IL/f— ."li'ffi 1 C:
i ,5] U)
F .OII‘. J»)
l fipfi/ i.)
. ..o' .
I -o~-°"°.t'0/ :1
o-‘OI X-&::".’ ‘I T:
‘ O‘X’A/l.” ' U)
Q ‘2 I" f i ('4
-°/ /". ! t3
.Oi:+ * a
9. La/ '
p .‘/. -—-0 Ferrous : B
o ’ .
f/ a” m . as
I
1 x—x Fer-IC- r
f 5FF07 I
3 0 .. o Firrav 5 ‘ E
Stonnzc
5 m Q66 51
.. 1.... ___.. -J ’f
A, n _ I“
450 500 650
yr V ‘ T V ‘7‘
' 6J6: beflbuflr‘cu
4r———- --

 

 

.5)
C)
n_~ d ‘53
9V fi/ :5
.p ; p
9.? 9’. p
.‘ kftlQK: .{n 0 a?" i, 9/; .2-{
x.‘1’”wfl‘ ’3
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WEI-$0- p334,“ ./ EU
\°’ ’—“' ConYr-ol SZOPQJ H
an m r63~7¢r6£°5 E"
x—q COHf'O' stared $33
D M oven
n-—g Conn“ *670
dexTrosc
o...o control H5“)
SUC'OSL
l J_
400 500 600

Nave

Figure III

Lengthr./

 

 

 

 

 

 

 

 

 

on the Eluate from the tion
Resin
1C0 [ ('4‘ $115; -
. '2‘“ 4.3
I {€13-3’3‘o. 0.8
' {26.60
1 [3‘0
f 5,77
: ".14".
QOr— f:
5
1’}:
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‘ if? 0 6" ’7“; O a ‘r
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#7. ”ga— ~—- Control
b o
.‘p r—r Stennous
soefl‘ 5PPm
L/ OM42 ,FfirrOUS
u .me
O .-0 Ferrous
- 55¢ [70' ‘-
flopm "5&0“
_I_- -_l - ~L
i: \ I", w
400 VOL 000
nave Length,”
100 _ .1 ”1333' '
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6:
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.. 1p,» m
p
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’f’F ’7’
0.. o Ferr I (— "
5r 3 on a US
5ppm 64‘"
--~— - l ‘ ' TL .
nave Len t1rn~

% Transmittancy

Effect of Storage at 1000 F on the Effluent from the Cation
Exchange Resin

 

lOO

 

 

40'}

 

 

 

 

 

 

0’. CantrO’
no storage
(—1} Control
1 weeks Slat-69¢.
0’0 FerrOUS" Sterlnm
~5me of each
{ O..-O F rnc- SCh'InOUS
. PP”) of each
L 1 J
500 600

400

Wave Length mu

Figure IV

% Transmittancy

100

90

m.————.

 

 

 

-_.....- -... ' fi-

Sfdnnaus Slalom
5,0,om
{ppm

58””
1

Stennic.
Farrel-(3

Ftrric

_, cam-“up...” n...-

- - _.._.H.; _--o

 

 

400

500 600

Have Lengthrwm<

Transmittancy

c7
/0

1

1 O 1 fl 7 «\ . -' . ‘.- ‘ _ 1 9‘ -
53 b on the uriginel methanol Extract
of Pigment

Fr
4»
“D
(D
O
( f
O
H)
(”I
('9'
G
}.
C0
«I:
f.)
( 1’
1 .J
C

 

 

 

 
   
 
    
   
  
 
 

7o "‘**—“"* 'I 70
04—- COntro/ .'
(n. star373)

o» o SIannoqs Spp‘rh

1 : .— "" 533?:an 5/010»;
' x——x Control , ,
i. 5w¢QH3 S£0r57e :

A: ran-cu: 5/0707?

,3... Ferric. 5PF’77

E so *-

J—U Ferrous - Srinnlc
’fflm of COS-h

60 -O-“O FarrIC‘Sténnqqs
5,0,0”) of Edcn

~....._ -.___

.7....4

50

C".
O
I

40

.--._.T___..--___,_..__r___-.

% TranSmittancy

 

 

(‘1
(J

C)

 

 

 

l-i__{

-___-.Mi.-Lii_.iI _,
600

500‘ 600

Have Lengthnwa Na

 

11:811ch m A

Figure V

'\ . 9.3 a.

5'2“" or- . . :"- .— of .“r- .»-- 7-x
HuuUuiU bud dIebJuVIvh

Preliminary Isolation Studies

methods previously employed for isolation of anthocyanin
pigments failed to produce desi'etle results. The addition
of ether to the methanolic extract of pigment was impractical,
since large amounts of other were required, and only an impure
sirupy precipitate was formed. The sirup did not appear to
be improved in purity with repeated dilution with alcohol
and reprecipitation with ether.

Concentration of the methenolic extract of the pigment
seemed to be of no value. The concentrate was immiscible
with other tccnise of the high content of water. Kiccitility
could be obtained only by the addition of large ~mounts of
alcohol. n small amount of waits precipitate formed during
concentration and was removed. However the material precipi-
tated Ly the addition of ether was eflein sirupy in nature
and appeared to be nearly as impure as that obtained from
the direct addition of ether to the methanolic extract.

Other solvents used either failed to precipitate the pigment
or proouced a change in color.

attempts to precipitate the pigment as a picrate from

either the sirupy concentrate or the original mothanolic

55
eAtrect were not promising. Cnly e smell portion of the
pigment was precigitoted from the solution. ifforts none
to purify the precipitate were unsuccessful.

Separation of the constituents of the methonolic
extract by chrorneto; jrnphic eLsorption Appeared to be of
more value. however. suitetle systems of absorbents end
solvents were not easily discovered. Thus search for a
better system was abandoned when it appeared thet the use
of ion excnonge resins would produce tne unsired results.

Isolation of the Anthocycnin by Use of
Ion Exchange hesins

A stronr'lv basic anion exchente resin Amberlite

‘\J U

er.»

A 38, was used . It not only adsorhe.d the acid constituents
of the mixture. tut also split the salts present. adsorbing
the unions. The cetiox‘as of the withocyanin a 1d en"! other
salts piesent passed throu.; h the resin in tie iorsa 01‘ has es,
together with any neutr'i end besic constituents of the
mixture. «QGH the methanolic extract of the cherry skins
was passed through a column of anion exchange resin. the
effluent was a very dork violet color. The color wes
characteristic of the nnthocv'nin i.n e slightly basic
solution. Audition of hydrochloric acid to a few orOps

of the effluent restored the red color. showing that the

enthocynnin hed not been destroyed.

£6
The effluent from the anion exonengo reein was passed
immediately through a column of cation ex‘henge rcs’n.
Zeokerb d. The basic constituents, chiefly the enthocyunin.
were adeorteo. A yellow brown effluent was obtained. when
a few drape of ferric CJlOPiCe was added to the yellow
brown effluent a greenish black color resulted. This was
a reaction characteristic of tannins and flavones with
ferric chloride. deition of hydrochloric acid to the
effluent did not produce a red color. It therefore appeared
thct the ontcocyenin had been adsorbed by the resin, but
met the contaminating tannins. as well as other neutral
meterinl5,hcd been removed.

rvn'

Ln egucons oi Solution of hydrochloric a id failed to

4'
"9

remove the pigment from the cation resin. A 5' solution
of mothcnolic hydrochloric acid was used for the elation
of the pigment. a bright red solution was obtained which
did not become cork on the acdition of ferric Caloridc.
It therefore appeared free of tannins or similar imourities
which caused considerable carhening who? the test was
coplicd to the unpurified extract of the cherry skins.

n further indication of the effectiveness of ion
exchange resins in the separation of the enthocyanin from
the contaminating substances was obtained by use of pager

chromatograpns. ChronotoEchhs were mode of the original

57
pigment extract. the effluent, and the eluote of the cetion
exchange resin. The original extract produced two spots
on the deve10ped Chromatogregh. One was red and the other
yellow. The effluent from the cation exchange resin yroduced

F"!
L:

the yellow sect without the red soot. 1e eluete from the
cation exchange resin produced a red syot uitdout the yellow
Scot.

Concentration of the eluete of the cation exchange
resin was carried out under reouced pressure at a tempera-
ture below 500 C in an effort to prevent hydrolysis of the
ehtdocyenin. Jewever, such hydrolysis appareutly occurred.
since the red powder which precipitated was insoluble in
water and dilute aqueous acid, but soluble in alcohol.

Such solubility is character stic of enthocyenidins, the
products remaining after hydrolysis of the glycosidic
linkage of the unthocyenius. a further indication of
hydrolys.s was obtained by positive tests for carbohydrates
in the supernatant liquor with Benedict's solution and

holisch reagent. Tests on the unconcentrated eluete were

negative.

58

Tests for Identification of Pigment

Sugerggrouu_

The uistritution between enyl alcohol and water of
the entnocyanin of the thick sirup preci; tsted by the
addition of ether to the original methanolic ext'ect was
used as an indication of the type of sugar present. The
color was founu to predominate in the sgueous leyer, end
to be unaltered by the hdoition of salt. This is in agree-
ment with the reacticns observed by hot nscn and Robinson

(13525) for pentoseglycosices and hiosidss.

1:.11tho egg-.znidi;

 

an inuication of the tyre of anthocynnidin present
was guinea by eXposing the payer Chromatogreph of the
eluete from the column of cation ion exchange res n to
ammonia vapor. The pink shot on the chromatogrsph
immediately turned blue when excoseu to ammonia fumes.
This reaction was similar to the one found by Letes-tnith
(1948) to ‘e characteristic of peonidin.

The color tests of notinson and Jcbinson (lsol) were
periormeu in order to gain auditions; information concerning
the structure of the enthocyenidin. In the comparison of

the results or these tests LS shown in Table III, page 41,

59
1.-itn t1e re eeults in Table I, page 12, the enthocyanidin
were observed to react in eome inotonces as cyenidin and
in others as pelergonicin.

The color of the pigment in ‘Myl cleo1ol, which

ntei men a few ciur tale of sooium ncetotc, was believed
to be similar to cyanioin. Jewever. Hoxe uiiilculcy "'
cncoqntered in the interpretetioh of the IBSAltS, since
synthesized or pure cnthocyenidins were not uveiletle for
use as standards of comparison. The Lidition of ferric
chloride to he cmyl alcohol solution caused a c11engge in
color iron lmlli21 purple to a clear blue. This is character-
istic of cynnidin. when cyenidin reagent was added with
shnking to an aqueous 1X hydrochloric acid solution of the
pigment. two layers of liquid re sul ted. A 5115 1t Lluish
red color v:as impo: ted to the cyanidin reagent. This may
also indicate the presence of cyanioin. In the oxioetion
test the Pigment wes 1011d to turn Llu 1 on the audition of
sodium hydrolide; howeVer. the color was feirly etchle
since t1e red color could be 1ecove1cd Ly the immediate
eooition or concentrated hydrochloric ccio. This test
might inoicete either pelargonidin or cyanidin since both
are stable under theéc conditions. The case of transfer
of the entlocyenidin from amyl alcohol to 0.5% aqueous

h; drochlorlc Ly tE1e ecoition oi ttnzene > so is oi Value

60

in the identification of structure. The ont‘Locycniuin

required the addition of 6L:“t vollm?s of “oozene for

tire tronof‘cr. 331183sz the 5:: no :35. tin-2 amount found by

1:01: ins on and Elobinaon (1343-1. '3 to be JJL’iLi‘LLiL'SC‘: for the

transfer of poonidin. -Thc color of tho pi‘:ont in the

aqueous acid soLLtion was observed to to oraxjo red, wgich

mac-y to pnluv'miuil j_";_;70111tiifi.11 or cyanidin. AOL .‘egier the
addition of alcohol to the aqueous solution produced a

blue red, which is stated by Lobiuscn to to more character-
istic of poonidin. The coiitton of sodium carbonate to

the aqueous solution of pigment produced a bright blue
color, which is indicative of ova udin. On the addition

-of acetone two layers separated. The acetono or upper

layer became pole groan in color and the lower layer

rer aLuoo bright blit. Too . notién a th acetone does

not corrcsyood to the one given tv hooiuolo i‘or yelaigoniuin.
If the cathocyonidin was poonidin, this might cccouot for
orieus indications 0L-LLLci with the color cs 3. Hobinson

and nobinson (19 >1) found pwonidin to react in many instances

as pclarxoni din although it is a derivative of cyanidin.

The dificré3nce 11 structure of the three compounoe lies

in the group attached to carbon 5 of the aryl ruoicol.

Peonidin has a methoxyl groug attached to the 5 position,

cyanidin a hydroxyl group, and wexcr :Lid;Ln a hyurogcn atom.

61

TrLerei‘ore in color reactions that mic. ht involve the flee
hyoroxyl SPOUyS peonidin could react similarly to pelargonidin,
and in Other instances, peonidin might behave similarly to
cyanidin.

Color res CtiCJHS in curler solutions as desc ibed by
Robertson and iotinson (1329) were used also to gain
information concerniLJ the structure. From the buffer
solution, information can be obtained conce mi in: the case
of pseudo-base and color base formation, and ease of oxidation.
These reactions are Specific for a particular snthocysnidin.
“saint no lock 01 s synthesized sntnocysnidin for use as a
standard of comparison mode interpretation of the results
difficult. The Luiiei solltions 01' pigment snovied a marked
decrease in intensity of color, even at a pH of 6.50.
Between a pd of 4.Q6-4.9 the fading was very rapid. Tnis
sgysrently indicates tiat & pseudo-base is easily iorrned.
After the tubes become colorless acidification with
hydrochloric acid did not restore the color. This indicates
toot the pseudo-bsse of the pigment was unstable and only
an intermediate step in the reaction involved in the loss
of color. After 18 hours color bases were formed in the
buffer solutions with a pH oi 5.6 or less. Apparently
color base i‘ormttion is slow and only takes place in acid

solutions.

62

The Ligment did not appear to be oxidized very readily,
since a pd of 13.98 or etovc was reouired for the reoid
formation of e brownish yellow color. The results obtained
with the unknown enthocynninin did not agree vitn those
fOUud Ly notertson and HoLinson (1939) for pelergonidin,

peonidin, or cynnidin chloride.

1
"'2

feet 01 V'C‘lle-(ittl LL'Ett,.;S

 

 

The effect of gelatin, storcn, pectin, agar-agar, end
methylcellulosc on the color of e dilute solution of the
cnthocyt nin was studied. Too ICE nlts were con Jared with
the obserVntions of notinson (19 boa). fie found that,
althougn audition of e few crystals of cytnin chloride to
boiling water pronuced n reddisn violet color, addition
to a solution of starch or xylen brought about formation
of a Line color. It was found to be impossible to regroduce
the results of Robinson when the enthocycnin from the red
cherries was used. The oigment ens found to fade when
added to boiling wnter, or when added to cold water and
then slowly txented to boiling. bolutioas of gelatin were
found to react as water solutions. doweVer, when solutions
containing the ca LL-L ohydrz. too were used the red color comm-ed
to be stabilized. Starch Seemed to possess the greatest

capacity for stabilization of the red color. oven when

65
the starch solution was boiled for several m nutes and
allowed to stnnd at room temperature for three weeks-e
pink color wee-still reteined. Tue colloidal solutions
of tne antnocyenin from the red cherries wemanot found to
form blue colors, Lut tne certohydretes eypeered to aid

in the retention of the Lrignt red color.

{gctgrs Affectin;_dolor as Icngyred by Light Absorption

.‘n—o

The effects or oxygen. methanol and pH were studied
Ly measurements of light absorption. The Spectre given
in Figure I indie tes that the oxygen had no effect on
the light absorption. nowever the methanol and p1 appeared
to alter the spectra.

The presence of oxygen during the concentration of tne

eldete from tne column of cetion exchenge resin bgpuflde
to have no effect on the pigment. The unconcentrtted eluete.
the eluate concentrated in en atmOSphere of nitronen, and
the eluete concentrated in en etmosyhere of oxygen all showed
a yoint of maximum absorption between 5 5 end 320 millimicrons.
The difference noted in intensity of the color wee ceased by
a difference in the concentration of the ioxiégxrzent.

Ectnnnol egyeared to influence the color. is the amount
of alcohol pres nt in the solution increased, the point of

mexi um etsorption was :nifted from £15 millimicrons in en

64

f. r) ~13
‘1’ .'.l I;

aqueous solution to 540 nillimicrons in a methanol

solution. These results were in agreement tith observation
of Sondheiaer (1943s) on etrenterry pigment. The pH of the
Solition between 2.09 and 6.64 tiiecttd tzt intansity of the
color, but not the point or moximum etoorption. is the pd
of tne soluti.n was decreased from $.64 to 2.09, the per
cent oI absorytion increase ed iroz: cl to 84. Sondheiner
(lfiioe) also obs ervcd similar results, in his study of
stre rbeIry p13ment.

In the Ltndy or Spectra of the buffered Solutioney
firnre II, tne on In .5 found to produce a {PFUJL enii't in

the nave length for maximum ocaoIfltiOI. An increase in pH
from o.tO~l0.0E showed a gradual increase in blue color.

also e step-wise sniit was noted in the wave loin tn for
meninum absorption from £20 millinicrons to £30 nillimi cron:.
Above a pd of 10.90 a greenish blue color was formed which
re pidly cnenged to a brownion yellow, probably due to
oxidation. hithin the pd range of 3.5 to 5.60, the oer

cent or lignt transmission in relatively higll, which
coincides with the rnyid fading 01' Color Cexu ed posssiblgl

by pseudo-base formation.

65

The Lffect of Iron and Tin Ions on the Figment

he iron and tin ions acre found to have little effect
on the color changes of the pigment. A control solution of
,igment stored in the refrige~oto retained the pink color.
nonever all buffered solutions of elunto from the cation
excnunge resin (presumably quite pure enthocyonin) which
were stored in the oven showed a marked decrease or fading
of the red color. Neither sue: se nor dextrose oopenred
to prevent the loss of red colOr. also no difference could
be noted between the tubes with e hendSpece of sir and those
in whicn the air had been replaced with nitrOgen. The tubes
containing iron appeared to be a little more yellow and
had a greater absorotion between 410 and 450 millimicrons
than the others stored in the oven. moweVer the absorption
in this range was approximately equal to that swown by the
control stored in the refrigerator.

The tubes containing the effluent from the cation
exchange resin (a solution of non-nnthooyenin substances.
possibly including tannins) all showed increased absorption
with storage. The tubes contnining the ferrous ions had a
tendency to exhibit the greatest absorption over the conglete
spectrum. The tubes containing the stennous ions had a

tendency to snow the least absorption.

66

The original methanolic extract also showed a fading
of red color on storage in the oven. Lomever a yellowing
effect similar to that noticed in the effluent epyeered
to have occurred. Tue results in these tubes were as
might be exgected since they contained the substances
pFGSLHt in boti the eluete and the —ffluent.

In all cases the most noticeable color change seemed
to be caused by storage at elevated temperatures rather
than by the presence of metal ions, since the centrol tubes
resyonded in a like KLHHBP to the solution containing

metal ions.

("'7' t""’,_' v“‘ up... a‘!’~§j,-'\T try! (-91‘_;
kJo-AJ-hthKI I‘d“) UUI‘.U-43Ju.-Iu.ou

The enthocyenin pigment of the skin of montmorency
cherries was extracted with methtnol. Isolation and
purificetirm were achieved by treatment with ion exchange
resins. The extract was passed through on anion exchange
reein to remove the anion of the pigment end any bueic
constituents. The effluent was passed through a cation
exchange resin which eteorbed tne entnocynnin. The
neutral substances passed through in the effluent. The
entnocyenin was then eluted with 5% methanolic hydrochloric
acid. In concentration of the eluete hydrolysis angerently
occurred and 8 red powder presumed to te enthocyenidin
precipitated.

in attempt was made to obtain some indication as to
the structure of the enthocyenin. Tests inoiceted that
the sugar group might be e bioside or a pentoseglycoside.
The results of tests for the enthocyenidin were not
conclusive. hoxever they indicated e structure closely
related to cyenidin. pclergonidin or pconidin.

Various factors which effect the color of the nntho~
cyanin were Studied. The onthocgenin was found to produce
a blue color in basic medium and to be decompoeed in

very strongly basic solutions. In ecid medium the element

6

C)

was red in.color. In the pH range 01 2 to 6.6 the intensity
of the color, Lut not the hue, was effected. slcohol was
fOJnd to produce a nor: Lise color in the pigment solutions.
carbohydrates such as stsrci, pectin, and methyl cellulose
were found to stabilize the color of the enthocyenin in
aqueous solitions. Starch was we rticulo.rly c‘fective.

The effect of iron end tin ions on the color change of the
pigment in cit"ste Luffered solutions held at 180° F for

a weeks was found to be very slight. The greatest factor

in tEle ceteriora tion of color of the pigm:nt 8:391-2 ered to

be the tense reture of storsre. In all cases the eluete of
the etion {venom e res‘a in (pres 1:111.ny quite pure cuitnocyenin)
showed COd‘luefuLll 1in _ The effluent or quutrel fraction
(i‘Jrotale contoinir b tannins end fie voncs %) d1 rkencd during-
he original methanol extract of the 'iii .ent also

Showed U. 115111115: 01‘ i’Cd CO.LOlo

69

1' {‘T --1 P~u---‘ ‘..~‘.--.(—
DIHL Lip 5‘54? 1-11;.1SUL1U

Anderson, J. Concerning the anthocyans in Norton and
3T5 Concord Grayes, A Contribution to the
Chemistry of Grape iiggments. J. Biol.
Chem. g1; 793-813.

.—
I

__ . A Contleution to the Chemistry of Grape
19:4 iL nents. J. 1101. Chem. 6;: 085-694.

.- .

Anderson, P. J. and Nabcnhuuer, F. P. A Contri b ztion to
131‘4 the Chemifitry Of Grape iig;,..i€;]1t50 Jo 1.31010
‘hem. Q3 97-1070

Lancroft, T. D. and fiutzler. J. H. Colloid Chemistry of
1938 Leaf and hloner F gments. J. a”. Chem.
Soc. 60: $945-$947.

Bate -cm1tn, L. 0. Say er uxrowctoprayuy of L'nthocyanins
1343 12.nd {eluted bub: tunces in ietul £1trccts.
bidtul'c it’ll but— &)8.

Lroau A. L. The Lutnoc”cnin Pigment of tho Aunt
I J
1340 Kuscadine Grape. J. Am. Chum. Loo. 62:
Eureuu of buricnlturul Economics. Crap Report. Dec.
1951:»: U. (I. O 13(1pt O 1:1)1‘0
Cruess, n. V. Commercial Fruit mud Vegetable Products.

1943 3rd ed. N. Y. HcGraw-fiill Eook 00., Inc.
1.3 p . 3:37- C1,) «3.

C1111.)Gfi)1.)€3£.’ Co {3:13. 831‘} ”€11. II. {:0 The Li;:1\1v1’)l‘ 01‘ tt‘le
13:-7 I? JltuOCYI‘J‘l 11" ngIL’ntS 1“ (36111211110. Jo brII..
uCou..1r‘ 1 3L: 3.07-1'UF’.

Dyson, G. fialcolm. a :ana1 01' C-gonic Chemistry. Vol. I.
1950 NOY. LOZLgmM-JAS ”4.86.: 811 L116. 30. 1.)"). ét'iw-SL'IO

Griswold, hutn 1. Factors Inll:zc.c1n: the Quality 01' Lowe-
1944 Canned 2.):1t11015 eucy Cheem.i<-.:snic21. L-t.
tit-21‘. EAL.)- w'tcao bull. 1'00 1J4.

Grove. K. and hobiuson, H. An Anthocyunin of Oxycoccus
:“

1951 fluorocarous Pars. Liochem. J. £2:
1 7 06‘1711 o

 

7O

Joslyn, E. A. Color Retention in Fruit Products.
1941 Ind. inn. Chem. gg; 508-314.
Knrrer, P. and Strong, F. n. Heindnrstellumg von
19cc Anthocyenen durcn caromntograghiscne
Analyse. nelv. Chim. nets 19: 236-2T8.
Karrer, P. and neber, d. n. Zerlegung Natfirlicher
1956 untno*"enhemiscne cure}; uniomnto rsghisc.'1e
Absorptionsnnelyse II. Uber thneein.
delv. Shim. beta 19: 10£‘-1 3'.
Konmnn, L. F. Txe dolor in Crunberr.tes. r‘ood lec linol.
19‘2 §,:160.

1959

Lathroy, C. P. Chemistry and the Preserve, or Jam and
1928 Jelly Industry. Ind. Ln”. Chem. g9;
.Lewrence, 3. J. C., Erice, J. R., Robinson, G. $., nobinson, h.
1959 The Distribution of gntnocyenins in flowers.
Fruits, and Leaves. Trans. Roy. Soc.
London 2303: 149-177.
Link, K. P. The Anthocynnins and the Flavones. Gilmen.
1‘4 d. Organic Chemistry An Advanced Treatise.
2nd ed. Vol II. N.Y. Jonn Ailey and Sons.
Inc. pp. 1518-1331.
Lorris, T. N. Principles of Fruit Preservation. 2nd ed.
1946 London. Chapman and Neil Ltd. Part 4
pg). 13.1“187.
Nebesky, E. A. Esselen, W. 3., XcConnell, J. 3., and
1949 Fellers. C. R. Stability of Color in Fruit
Juices. food neseercn‘iéz $61-$74.
Onslow, Huriel nheldele. The .ntnocyanin Pigments or fients.
19“‘ nd ed. Cambridge, University rress.
Price, J. n. and nobinson, R. nitrogenous Antnocyenins.
19:)? J. Chem. {’0‘}. 449-4‘u30
Price, J. 3., Robinson, 3.. and Scott-Eoncxieff, R. The

Yellow Pigment of Pa DCV:- r hudicaule.
J. Chem. ooc. 1466-14E8.

Fuellel‘? Go {to .

1m

Hoter son, A.

1989

; otimon. :33
1 Jéba

 

1935b

3051118011, G.

lgél

 

 

1 JUL} 8 w
1 3.321:

 

lDdB
1101;111:2011, G.
1959

Schindler, S.

194:);

Sariner. R. L.

19J9

 

1940

 

1941

“ ondixe izner. L.

1943e

{.2110

Qurtis, L.. and Vickery,

71

a. 13. The Red
Pigment of the fleet of the ecet. J. Biol.
Chem. 125: 61-70.

.nouinson, R. .Note on the 31n1“*terlzetion.
of the nntnocyenins end untnocyenleins by
means of lucir Colour nee etion in nlkeline
toluticns. liocuem. J. 2g; 53-40.

Natural Colouring letters and Ideir analogues.

unemi str" enu Industry 11: 7&7-749.

Natural Colouring matters and Their Analogues.
Nature 132: ego-cue.

end Robinson, 3. A Siu‘vcy of Jn'zthocyanins

I. biocncm. J. 2;; 1687-1705.

A Survey of nnthocyunius II. Liochem. J.
EC: 1647-1064.

Develo;;ments in the Clie.mistry of the
xntnocynnins. nature 1J0: 21.

T10 Colloid Snenistry of Leaf end Blower
.igmants an-! the Plecursors of the :ntllo-
cynuins. J. Am. Chem. Loc. 61: 160‘5-1606.

Kotes on Variable Colors of 210\2:tr .etels.
J. .1371. Chem. LOCO CL: 103(3-lw7.

Utes den Gelben Verbstoff der Forsytuin-
Bluten. nelv. calm. Acta ge; 11:7-1139.

and Joffett, R. 3. Halogen Lutstituted

LenZOpyl'ylium belts. J. Am. Snem. LOO.
Q1} 1474~1477.

benzepyrylium Salts II CzoniZntion.

J. uh. Chem. Lac. 62: 2711-2714.
“enzojyxylium belts III {yntheses from
LWul tituted Coumerins end Cnromones.
J. [21“. C2161”. L400. Gs): 16‘34-16380

and liertesz, .2. I. Lntllccyenin Pigments

Colorimetric Determination in Ltreaberries
‘nd .th'mvberry g-‘l'oduct-S. 2.11241. Shem.

( n. r2 c r“ '3.-
4-.\.J' 5-4u-é4k-l.
m

r‘ "., ' . ‘ .
gonads-1.1015
lvdgb

Sondaeimexy
1943

um

..
d.

H
I
in.

Llpéiet'l’ E). Co

1949

hawzonek. b.

ldfll

1916

it.

72

and Kertes z, E. I. The Lnthocyanins of

atxunbcxries. J. Am. Chem. 300- 193
5476-3479.

and Lee, F. 3. Color Change of atr awterry

Authocyauins with D-Ulucoae. Lcieuce

and uOELCHL11tt U. A. lhltib.)fl ullblatOFlLth

"L
“th.

K‘,

of lutnoc"'nldi1..z. Lola: ' ca ‘13;

Chromeuols. Chromenes. and benzoyyrylium
belts, the unthocyunins. gluerficld,

R. C. neterocyclic Compounds. Vol. 2

N.I. John alley and tons, inc. pp. Sfi9-363.

and Lollinper, L. A. Uber oio larbstoffe
der 1 :cxe und der :chlehe. “an. 419:
164-175.

 

 

 

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Glidden, Nina Adeline

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