journal of the science of food and agriculture volume 25 issue 10 1974 [doi...

8/18/2019 Journal of the Science of Food and Agriculture Volume 25 Issue 10 1974 [Doi 10.1002%2Fjsfa.2740251005] Ronald…

1/8

J . Sci. Fd Agvic. 1974,

25

1221-1228

Identification

of

Anthocyanins and Distribution

of

Flavonoids

in Tamarillo Fruit (Cyphomandra betaceae Cav.) Sendt.)

Ronald

E.

Wrolstad and David

A.

Heatherbell

Plant Diseases Division, Private Bag, Auckland, ew Zealand

Manuscript received 30 October I973 and accepted 26 May 1974)

The major tamarillo

Cyphomandra betaceae)

anthocyanin pigments were isolated

and identified as

pelargonidin-3-rutinoside

elargonidin-3-glucoside

yanidin-

3-rutinoside, cyanidin-3-glucoside, delphinidin-3-rutinoside and delphinidin-3-

glucoside. The intense purple-coloured jelly surrounding the seeds contained the

greatest concentrationof anthocyanins, delphinidin-3-rutinosidebeing the major

pigment. Flavones, flavonols and leucoanthocyanins were also present in this

material. The yellow-coloured flesh contained flavones and low concentration of

anthocyanins. The major anthocyanin

of

the skins is cyanidin-3-rutinoside;

flavones and leucoanthocyanins are also present.

It

is suggested that the presence

of leucoanthocyanins in pigment extracts induced degradation of anthocyanins

during isolation and purification.

1.

Introduction

The tamarillo or tree tomato ( C .

betaceae

Sendt), a native of Peru is grown in many

tropical-subtropical regions of the world, including South America, Ceylon, India,

Indonesia, Malaysia, the Philippines, the Mediterranean and New Zealand. 1-3 Apart

from in New Zealand, the tamarillo has been cultivated

on

a surprisingly limited scale

considering the appealing, unique flavour and attractive colour of the fruit. The red

strains, which are the predominant strains grown in New Zealand, have

a red skin,

yellow-orange flesh and an intense purple coloured jelly surrounding the seeds.

In New Zealand the crop is achieving considerable commercial importance, produc-

tion amounting to 2277 tons in 1972.4 The fruit is

a

popular fresh market fruit and in-

creasing amounts are being commercially processed into a range of products including

juice, jams, preserves and ch ~ t n e y s . ~number of colour-deterioration problems occur

when tamarillos are processed. Tamarillo juice is an attractive deep red-purple colour

but occasionally difficulties are encountered with browning of the product and develop-

ment of a red-coloured haze. Preserves become an unattractive reddish-brown with

storage. Glass containers with enamelled closures are used when canning the fruit as

detinning and a subsequent colour change to an undesirable purplish-black occurs

when cans with fruit enamels are used.6 These colour degradations prompted investiga-

tion of the anthocyanin pigments.

In

a preliminary study, Dawes6 reported that red

On leave from

Department

of

Food Science and Technology, Oregon State University, Corvallis,

Oregon

97331, USA .

1221


2/8

1222

R.

E Wrolstad and D. A.

Heatherbell

tamarillos contained at least eight anthocyanins, the major pigment being a delphinidin

derivative. Identification of the anthocyanins of red tamarillos and measurement of

their distribution within the fruit was the main objective of this investigation. The pre-

sence of leucoanthocyanins, flavones and flavonols is also reported.

2. Experimental

2.1.

Plant materials

Tamarillo juice was prepared from washed fruit which had been crushed and heated to

71

C.

The pulp was cooled to 37 Cand treated with pectolytic enzymes. The super-

natant was decanted and the residue pressed; the liquid phases were combined and

centrifuged. The juice (pH of 3.45) was stored at

-30

C .

Fresh ripe fruits

5

kg)

supplied by a local grower were washed and peeled; the seeds

and the surrounding intensely pigmented jelly were separated from the flesh. Yield

:

skins, 750 g; seeds and jelly, 1210 g; and flesh, 2990 g. The skins were liquid nitrogen

powdered and stored at -30 C.The flesh and seed-jelly fractions were frozen a t -30 C

and stored.

2.2. Isolation of anthocyanins from tamarillo juice

Anthocyanins from

1

litre of tamarillo juice (clarified by filtering through celite) were

isolated free from sugars and other non-phenolics by treatment with insoluble polyvinyl-

pyrrolidone (PVP).' The pigments were recovered in methanol containing

0.1

HCl

and further purified by precipitation with diethyl ether.

2.3. Extraction

of

flavonoids from skin, flesh and seed-jelly

An aqueous extract was obtained by macerating 500 g of material with boiling acetone,

filtering and partitioning with chloroform.26 Subsequent extraction with ethyl acetate

(six times) yielded an ethyl-acetate fraction; the anthocyanins in the residual aqueous

phase were purified with PVP as previously described. A procyanidin fraction was

obtained by treating the original filter cake residue with n-butanol containing

5

conc.

HCI .

2.4.

Chromatographic procedures

2.4.1.

Thin-layer t.l.c.), paper and column chromatography

One and two-dimensional t.1.c. was done with 20

x

20 cm cellulose plates, 0.25 mm

thick.

A PVP column was utilised for preparative separation of the anthocyanins into frac-

tions. The

2 x

30 cni column was filled with 60 to 80 mesh PVPs, and had a flow rate

of

11 ml/min when washed with water (gravity flow). The pigments were applied as an

aqueous solution and developed with methanol H 2 0

(60:40)

containing 0.1 conc.

HCI.

Descending chromatography on Whatman

No.

3 M M

paper was utilised for pigment

purification. Development time for PAW was 24 to 30 h and for AHW,

8

to 10 h.


3/8

Anthocyanins and flavonoids in tamarillo

1223

General solvent systems utilised in paper and thin-layer chromatography were :

AHW, acetic acid-conc. HCI-water

(25 :

3 72, v/v)

PAW, n-pentanol-acetic acid-water (2: 1 1, v/v)

FHW, formic acid-conc. HC1-water (5

:

2:

3, v/v)

Forestal, acetic acid-conc. HC1-water (30: 3: 10, v/v)

TBA, t-butanol-glacial acetic acid-water

(3 1

1, v/v)

HA, glacial acetic acid-water (15 85, v/v).

2.4.2. Visualisation

spray

reagents

Aluminum chloride: 5 AICl, in 95 ethanol.

Molybdate reagent : saturated aqueous solution of ammonium molybdate adjusted

pH

2 with 2

N-HCI.

Paulys reagent

:

(1) spray with 5 KOH in

80

ethanol

(2) spray with

0.5

g of sulphanilic acid and 0.5 g of KNOz in 100 ml of

1 N-HCI.

Vanillin-H,S04: 1 vanillin in conc.

H2S04.

Catechin reagent

l o (1)

spray with

1

2,4,6-trinitrophenol in 95 o/ ethanol

Flavan-3,Cdiols

:I1 (1)

spray with ethanol containing 5 vanillin and 5 p-toluene

(2) spray with 5 KOH in 80 ethanol

sulphonic acid

2.4.3.

Gas-liquid

chromatography (g.1.c.)

Sugars and acids were identified and quantitatively determined by g.1.c. of their tri-

methylsilyl (TMS) derivatives

as

described by Heatherbell.lz

2.5.

Hydrolysis and oxidation of pigments

Purified pigments (1.5 mg) and the total pigment isolate were subjected to total hydro-

lys i~ , '~lkaline hydr~lysis'~nd hydrogen peroxide oxidation.16 Aglycones were

examined by t.1.c. and sugars and acids by g.1.c. of their TMS derivatives.

2.6.

Spectral methods

Visible and ultraviolet spectra of purified anthocyanins were measured in methanol

containing 0.01% HCl using

a

Unicam SP

800

spectrophotometer. Solutions had

absorbances

of 0.80

to 1.40 at the visible maximum. Spectral measurements were re-

ported after addition of a drop of aluminium chloride

(5 )

in methanol.

The total anthocyanin content was determined as cyanidin-3-rutinoside in tamarillo

skins and as delphinidin-3-rutinoside in tamarillo flesh and seed jelly. It was assumed

that the molar absorptivity of cyanidin-3-glucoside would be the same as cyanidin-3-

galactoside (4.48)13 and

delphinidin-3-rutinoside

would be the same as delphinidin-

3,5-diglucoside (4.37).

Spectra of flavonoid extracts were determined in methanol

;

spectral measurements

were repeated after adding 3 drops of sodium methoxideIg and

6

drops of AlCl,.


4/8

1224

R. E. Wrolstad and D. A. Heatherbell

3.

Results

3 1

Identification

of

tamarillo anthocyanins

The anthocyanins isolated from tamarillo juice could be resolved into seven spots

(Tam 1-7) by two-dimensional t.1.c. By utilising both PVP column chromatography

and preparative paper chromatography Tam 1 to 6 were isolated in pure form. The

spectral properties of the purified anthocyanins (Table

1

indicate that the five position

TABLE. Spectral and chromatographic properties of tamarillo anthocyanins

Tam 1 68

Tam 2 53

Tam 3 57

Tam 4 37

Tam 5 48

Tam 6 26

Tam

7

59

Tam 8 74

39 510,271

0

42

21 529 +42 25

18 528 +41 25

10

540,278 +39 19

9 541 +44 20

5

22

38 509,270 0 44

is free in Tam 1 ,2 ,3 ,4 ,5and 6. The RF values of the aglycones indicate Tam 1 and 2 are

pelargonidin glycosides, Tam 3 and 4 cyanidin glycosides and Tam 5 and

6

delphinidin

glycosides.

Partial hydrolysis of the purified pigments revealed Tam 2,4, and 6 to be monoglyco-

sides and Tam 1 ,3and to be diglycosides. The intermediate product formed on hydro-

lysis of Tam 1had the same R , in

A H W

as Tam 2. Similarly Tam

3’s

intermediate hydro-

lysis product has the same R F as Tam 6 .

G.1.c. identification of sugars in acid hydrolysates of the purified pigments revealed

that Tam 2,4 and 6 contained only glucose and Tam 1 , 3 and 5 contained glucose and

rhamnose. The sugar obtained by H,Oz oxidation of Tam

I ,

3, 5 co-chromatographed

with rutinose. Thus spectral and chromatographic data of the purified pigments along

with identification of hydrolysis products identify Tam

1

as

pelargonidin-3-rutinoside,

Tam 2 as

pelargonidin-3-glucoside,

Tam 3, cyanidin-3-rutinoside, Tam 4, cyanidin-3-

glucoside, Tam 5, delphinidin-3-rutinoside and Tam

6,

delphinidin-3-glucoside. Co

chromatography of tamarillo juice anthocyanins with black currant an tho cyan in^'^

by two-dimensional t.1.c. failed to resolve Tam 3,4, 5 and 6 from cyanidin-3-rutinoside,

cyanidin-3-glucoside, delphinidin-3-rutinoside and delphinidin-3-glucoside. Similarly

co-chromatography with pointsettia anthocyaninsZ0 onfirmed Tam 1 as pelargonidin-

3-rutinoside, Tam 2 as

pelargonidin-3-glucoside

am 3 as cyanidin-3-rutinoside and

Tam 4 as cyanidin-3-glucoside. Insufficient quantity of Tam 7 was isolated for spectral

analysis or identification of aglycones and sugars.

Its

chromatography behaviour

(Table I would suggest that it is a diglycoside and its colour change with molybdate

suggests a cyanidin or delphinidin derivative.


5/8

Anthocyanins and flavonoids in tamarillo

225

While none of the identified pigments contained galactose, analysis of the total hydro-

lysate showed galactose to be present. The possibility of free sugars being in the total

anthocyanin isolate was checked by absorbing the pigment extract on PVP and washing

several times with distilled water. The anthocyanins were extracted with acidic

methanol he hydrolysate of this material still contained galactose. The percentage of

galactose, glucose and rutinose was estimated by triangulation of the peak areas of the

sugars obtained by

H,O,

oxidation of the total anthocyanins. The results were: galac-

tose-5 , glucose-26 and rutinose-70 .

3.2.

Distribution

of

flavonoids in the tamarillo

Table 2 gives the distribution of anthocyanins within tamarillo fruit. The flesh contained

very little anthocyanin. Delphinidin-3-rutinoside is the major pigment in seed jelly and

cyanidin-3-rutinoside is the major pigment in the skins. The pigments isolated from

TABLE . Distribution of anthocyanin pigments within tamarillo fruit

Quantity Relative concentration of

mmol/g individual pigments

Sample (fresh weight) 1max (decreasing order, visual estimation)

Skin 9.77 x 528,330,291,270 Tam 3$T am1 > T a m 4 >

Flesh 3.23 x 528,285

Jelly seed coat 6.42

x

536, 276 Tam 5 %Tam 1 >Ta m 3 >

Tam 5

Tam 2

>

Tam 6

>

Tam 7

Tam

4

> Tam

8

Tam 5 > Tam

1

>Tam

6

>

Tam 3> Tam 2 > Tam 7

Tamarillo juice

tamarillo juice contained a higher proportion of monoglucosides than either the seed

jelly or skin.

When seed jelly anthocyanins were co-chromatographed with tamarillo juice antho-

cyanins, an additional pigment (Tam 8) was detected. Its yellow-orange colour, bright

orange-fluorescent appearance under ultraviolet light, and chromatographic behaviour

(Table

1)

suggest it may be a pelargonidin 3,5-digly~oside.’~ he skin contained some

minor pigments in addition to Tam 1,3 ,4and 5. G.1.c. analysis of TMS derivitised skin

total anthocyanin hydrolysate, revealed trace peaks which co-chromatographed with

p-coumaric, caffeic and ferulic acid. I t is possible that some of the trace pigments may

contain acylated anthocyanins.

Treatment of two-dimensional chromatograms with visualisation spray reagents gave

evidence for the presence of anthocyanins, leucoanthocyanins, flavones and flavonols in

the ethyl acetate extracts. These colourless extracts became cherry-red during concen-

tration. The two orange-red spots could be further resolved into five different pigments

with AHW-PAW

21 1 ,

three of which co-chromatographed with Tam 1 ,3 and

5.

None

of the visible spots co-chromatographed with known anthocyanidins. Three colourless

areas on the chromatogram turned red with flavan-3,Cdiol spray. There were several


6/8

1226

R.

E.

Wrolstad

and D. A. Heatherbell

spots which appeared blue or blue-fluorescent under ultraviolet light and became yellow

when fumed with ammonia and bright yellow when sprayed with

KOH.

The ultraviolet

absorption maxima of the ethyl acetate extract (324 nm) and the spectral shifts with

sodium methoxide (346 nm) and AlCI, (343 nm)19 would substantiate the presence of

flavones. One region of the chromatogram which appeared purple under ultraviolet

light became fluorescent yellow after spraying with AIC13. An extract of a band from a

paper chromatogram containing this compound had an absorption maxima at 345 nm

which shifted to 400 nm with sodium methoxide and 396 nm with

AICIJ.

This would be

in accordance with the presence of a flavonol glycoside.22Similar evidence was obtained

for the presence of anthocyanins, flavan-3,4-diols and flavones in the skin ethyl acetate

extract and for flavones in the flesh ethyl acetate extracts.

The seed jelly acetone filter cake residue turned crimson when treated with butanolic

HCI. The pigments in this fraction co-chromatographed with Tam 1,2,3,4, $ 6 and

7;

three additional pigments were present in trace quantities. There was one colourless

region on the chromatogram which became pink with the flavan-3,4-diol spray, but

none

of

the other visualisation sprays gave positive reactions. The skin filter cake

residue gave a low yield of red-coloured compounds with butanolic HCI; acid hydro-

lysis of this fraction gave a trace of cyanidin. No flavan-3,4-diols or other flavonoids

could be detected in the extract itself. The flesh filter cake residue did not produce a

red colour when treated with butanolic HCI.

4. Discussion

The cyanidin and delphinidin anthocyanins identified in tamarillo juice contain the

ortho phenolic groups required for complexing with metal ions. Recommendations for

processing tamarillos have stressed the avoidance of tin, iron and copper equipment in

order to maintain desirable colour.6 Van Teeling, Cansfield and Gallopz3 pointed out

that the anthocyanin moiety of the various soluble and insoluble blue-coloured antho-

cyanin complexes described in the literature are most frequently glycosides of cyanidin

and delphinidin, the other constituents including cations, protein, polysaccharides and

polyphenols. Chlorogenic acid is present as a trace constituent in tamarillo flesh and is

the major acid in tamarillo skins; under certain conditions chlorogenic acid will form

insoluble anthocyanin-metal complexes.25 Consideration should be given to avoiding

extraction of chlorogenic acid from the tamarillo skins in juice production as its pre-

sence could be deleterious to colour quality.

The presence of larger quantities of anthocyanin monoglucosides in tamarillo juice

could be attributed to partial chemical hydrolysis of the rutinosides during juice pro-

duction.

The presence of galactose in the tamarillo juice anthocyanin isolate has not been

accounted for. No free sugars, cinnamic acids or flavonoids other than anthocyanins

were detected by the various visualisation reagents. Any galactose-containing antho-

cyanins might have been inadvertently separated from the six purified anthocyanins

during preparative

PVP

column and paper chromatography. The presence of galactose-

containing anthocyanins in tamarillos is a possibility which cannot be discounted and


7/8

Anthocya nins and flavonoids in tamarillo

1227

could easily have been overlooked if the sugars present in the total anthocyanin hydro-

lysate had not been determined. This has important implications where anthocyanin

identification is applied to adulteration and chemical taxonomy investigations.

The anthocyanin glycosides which were present in the ethyl acetate extracts seemed

to be formed during concentration. Possibly these compounds were loosely associated

with some other materials which rendered them colourless and ethyl acetate soluble, the

concentration conditions being severe enough to break the complex.

It

was anticipated that the intense red pigment formed when the seed filter-cake

residue was treated with butanolic HC1 was caused by anthocyanidins which were

formed by acid-catalysed dehydration of procyanidin material. The anthocyanin glyco-

sides which were actually found to be present might have been in association with

polymeric material, the complex being broken with mineral acid. Timberlake reportedz6

the possibility of anthocyanins being loosely associated with procyanidin material in

extracts of apple peel.

Considerable difficulty was encountered in the isolation of anthocyanins from tama-

rillo fruit. While the anthocyanins from blackcurrant fruit and pointsettia brachts could

be isolated with comparative ease, tamarillo anthocyanins often degraded during

concentration step to yield soluble and insoluble yellow-brown products. At this stage

of the purification process the isolate would have been free of sugars and other neutral

compounds as well as metal ions and enzymes. Several alternative methods for isolating

tamarillo anthocyanins were carried

out

including extracting with SO,-saturated ace-

tone,z1 cold methanolic HCl and water containing

1

/, acetic acid. Precipitating the

anthocyanins with neutral lead acetate19was an alternative means of purification tried.

Blanching to inactive enzymes, shielding extracts from light, and carrying out operations

in the cold were some of the precautions taken. Eventually a procedure (described in

Experimental section 2) was found which yielded anthocyanins without degradation.

Extraction with ethyl acetate seemed to be critical to prevention of anthocyanin degra-

dation. This removed flavones, flavonols and most importantly, leucoanthocyanins.

Proanthocyanidins and flavan-3,4-diols would be expected to degrade to anthocyani-

dins and plobaphene material under the concentration conditions used (presence of

HC1,45 C). Such reactions could have initiated anthocyanin degradation. It is very

possible that leucoanthocyanins may play a major role in some of the colour degrada-

tion problems encountered during processing of tamarillos.

References

1. Dadlani, S. A.; Chandal, K P. S Indian Hort. 1970, Jan-Mar, 13.

2. Fletcher, W. A. Orchard N . Z . 1970,43, 69.

3. van der Mey, J. A. M.; van Hasselt, G. A. M .; Elsevier, D. E. M.

Cenetica

1969, 40 413.

4. Fletcher, W. A. Ministry

of

Agriculture and Fisheries, Auckland, New Zealand. Personal

communication.

5. Strachan, G. Fd. Technol.N . Z . 1970, 5 304.

6. Dawes, S. N.

Fd. Technol.

N . Z . 1972,7, 22.

7 . Wrolstad, R. E. ; Putnam,

T. P. J .

Fd

Sci.

1969,34 154.

8. Hrazdina, G. J . ugric. Fd Chem.

1970,

18,

243.

9.

Wrolstad, R. E.; Struthers,

B. J.

J . Chromat. 1971, 55 405.

10. Tirimanna, A.

S L . ;

Perera,

K.

P. W.

C .

J.

Chromat.

1971,

58

302.

11. Markham, K. R.; Porter,

L.

M. D.S.I.R. Chemistry Division, Petone, New Zealand. Personal

communication.


8/8

1228

12. Heatherbell,

D.

A. J . Sci. Fd Agric. 1974, 25, 1095.

13.

Anderson, D. W.; Julian, E. A.; Kcpner, R.

E.;

Webb, A. D.

Phytoc hemidtry 1970,9, 1569.

14. Lyn,

D. Y .

C.;

L u h , B. S

J .

Fd

Sri. 1964, 29, 735.

15. Albach,

R. F.;

Kepner,

R.

E.; Webb, A. D. J . Fd

Sci .

1965,30, 69.

16. Chandler, B. V.; Harper, K. A.

Aust.

J .

Chem.

1962, 15 114.

17.

Swain,

T.;

Hillis,

W.

E. J . Sc i. Fd Agric . 1959, 10, 63.

18. Swain,

T.

Analytical methods for flavonoids, In

Chemistry and Biochemistry

of

Plant Pigments

T.

W. Goodwin, ed.) Academic Press, New York. 1965, p. 541.

19. Mabry, T. J.; Markham, K.

R . ;

Thomas, M. B. The Systematic Identification of Flavonoids

Springer-Verlag, New York.

1970,

p.

35, 41.

20. Asen,

S.

PI.

Physiol.

1958, 33,

14

21. Harborne, J. B. J .

Chromat.

1958, 1, 473.

22. Duggan, M. B. J . Ass. 08 gric. Chem . 1969, 52, 1038.

23.

Van Teeling, C.

G.;

Cansfield,

P.

E.; Gallop,

R.

A.

J. Fd Sci. 1971, 36, 1061.

24. Heatherbell, D. A.;Surawski,

J.

R.; Withy,

L.

M.

J . Sci . Fd Agric.

Submitted for publication.

25. Jurd,

L.;

Asen, S .

Phytochemistry

1966,

5

1263.

26. Timberlake, C. F.; Bridle,

P.

J . Sci. Fd Agric. 1971, 22, 509.

R .

E Wrolstad and D. A. Heatherbell

journal of the science of food and agriculture volume 25 issue 10 1974 [doi...

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