jsfa vol 010 is 01 jan 1959 pp 0063-0068 the phenolic constituents of prunus domestica. i.—the...

8/11/2019 JSFA Vol 010 Is 01 JAN 1959 pp 0063-0068 THE PHENOLIC CONSTITUENTS OF Prunus domestica. I.THE QUANTITA

1/6

SW AI N et a1.-PHENOLIC CONSTITUENTS

OF

P R U N U S DOMESTICA

63

is available for the commercial application of both of these techniquesz41

5

but the benefit to be

gained in canning of citrus juice must be regarded

as

doubtful.

Complete removal

of

oxygen from orange juice improves the retention of ascorbic acid

and flavour during processing but has little effect on the retention of these quality factors

during storage. In relation to pasteurised, canned citrus juices, the importance of the de-aera-

tion operation has been over-emphasised. Since oxygen disappears rapidly from the contents

and the atmosphere of the can during storage at ordinary temperatures, it is not profitable to

attempt to achieve highly efficient de-aeration. Removal of the bulk of the dissolved air,

particularly when the juice is supersaturated, is sufficient to confer the main benefits of better

initial flavour and ascorbic acid content, and decreased corrosion of the can.

With frozen juices, however, oxygen does not disappear rapidly from the container and

efficient de-aeration may be amply justifiable in terms of improved retention of flavour and

ascorbic acid.

Acknowledgments

The authors wish to express their sincere gratitude to Mr. G. G. Coote of the Division of

Mathematical Statistics, C.S.I.R.O., for his painstaking assistance with the statistical aspects

of this investigation, and

to

Mr. R. A . Edwards for analytical assistance.

Commonwealth Scientific and Jndustrial Kesearch Organisation

Division of Food Preservation and Transport

Homebush, N

S

., Australia

References

1 McDermott,

F.

A, Bull. F l u agvic. E,v@. S ta . , 1917,

2 Hunnikin, C., Tech. Conznzz~n. Bzir. Hovt. , E.

3

Lewis, V.

M.,

McKenzie,

H. .A Annalyt. Clzem.,

a

Boyd, J.

M.,

Peterson, G. T., I n d u s t v .

E ngng

C hem .

( f i z d u s t r . E d n ) , 1945. 37,

370

5 Hicks,

E. W.,

Kefford,

J.

F., McKee, H. S.

Report

on

Food Stores in New Guinea ', 1945,

p. 13 (Melbourne

:

Council for Scientific and

Industrial Research)

NO.

I35

Mulling,

1950, 21 68

'947. 19 643

6

Huelin,

F.

E.,

Stephens,

I.

Nyee, ilust

J .

ex@.

Biol. wed Sc i . , 1947,

25,

17

7 McKenzie, H.

A . ,

1 C o u n . scz. i ndus t r . IZcs. Aust.,

1945, 18, 1 8 r

* McKenzie,

H. A,, J .

T O Y . S o c . N.S.W., 1947, 1 147

B Evenden, W., Marsh,

G.

L.,

Food

R es . ,

1948, 13,

244

10 Huelin, F.

E.,

Food .Rcs., 1953, 18 633

11 Joslyn, M.

A.,

Miller,

J . ,

Food 12es.,

1949, 14

325

1 2 Freed,

M.,

Brenner,

S.

Wodicka, V. O.,

Food

13 Riester, D. W., Braun, 0 G., Pearce, W.

E.,

Tech. C ham f la ign , 1949, 3 148

Food

I n d . ,

1945, 17, 742

Received 28 May,

1958

l4 Feaster, J.

F.,

Tompkins,

M. D.,

Pearce, W.

E.,

l5 Huelin,

F. E.,

Stephens, I. Myee,

Aust. J .

sci.

l6 Huelin, F.

E.,

Stephens,

I.

Myee, A u s t . J .

sci.

l 7 Curl,

A.

L., Veldhuis, 31. K., F ru i t P rod .

J . , 1948,

Joslyn, M. X., Marsh, G.

L., I n d u s t r . Engng

C h e m . ( I n d u s t r . E d n ) ,

1934, 6,

295

Blair, J.

S.

Godar,

E.

M., Reinke, H. G.,

Marshall, J. R.,

Food

Tech.

C ham paign , 195 11

61

2o

Blair, J.

S.

odar,

E.

M., Masters,

J. E.,

Riester,

D.

Mi. Food Res. ,

1952, 17, 235

21 Loeffler, H. J., Indus t r . E n g n g C h e m . ( I n d u s t r .

2 e

Joslyn, M. A., Marsh, G. L., Indus t r . Engng

23 Natarajan,

C.

P., Mackinney, G., Food T e c h , ,

2 4

Bayes,

A. L., F ood T ech . , C ham paign ,

1950,

4 Ijr

2 5

Lueck, R. H., Brighton, K. W.,

Int . Congr. on

Food Res. , 1949,

14

2 j

Res. , 1948,

I,

58

R e s . , 1948, 1 50

27, 342

Ed ),

1941, 33, 1308

C h em . ( I n d u s t r . E d n ) , 1935,

27,

186

C haw f la ign ,

1949, 3, 373

C anned

Fo o d s

(Paris), 19j1,

2,

XIV-8

THE PHENOLIC CONSTITUENTS

OF

P R U N U S

D O M E S T I C A

1.-The Quantitative Analysis of Phenolic Constituents

By T.

SWAIN

and

W. E.

HILLIS*

Methods for th e quanti tat ive ana lysis of anth ocyanins, leuco-anthocyanins, flavanols

and to tal phenols in plant tissue extrac ts are critically examined and suitable modifications

of existing methods are described.

*

Present address : Division of Forest Products, C.S.I.K.O., Melbourne, -1ustralia

J. Sci.

Food

Agric., 10, January,

1959


2/6

64 SWAiN

et a1.-PHENOLIC

COhTSTIlL7ENTS OF

PRUNUS DOMESTICA

Introduction

It

has recently been suggested tha t the formation of the troublesome defect of gum in the

flesh (mesocarp) of canned Victoria plums may be related to the t ransport of phenolic glycosides

from the kernel and hence to the degree and rapidi ty of lignification of the stone (endocarp).l

Lignification in plants has been shown t o be closely associated with the occurrence of leuco-

anthocyanins in leaves2 and it has also been suggested tha t these compounds are precursors of

heartwood extractives. An investigation of the variability in the amounts of leuco-anthocyanins

and other phenolic compounds in the leaves and other tissues of the Victoria plum (Prunus

domestica var. Victoria) during the course of

a

season was therefore undertaken to see what effect

these had on stone formation. Since the investigation involved the quant ita tive analysis of

over two hundred samples, methods involving preliminary separation of individual compounds

in each extract before analysis4 were out

of

the question. Methods in use in these labora-

tories for the analysis of individual groups of phenolic compounds were therefore modified and

improved and are reported here. The results of these analyscs will be given in Par t 11.

Experimental

Reagents

Methods of Analysis.5

with n-butanol to

500

ml.

concentrated sulphuric acid.

g

ml. of methanolic hydrochloric acid

( 5 :

v/v, 3 ~ ) .

Folin-Denis.-The reagents were prepared according to the procedure given in the A.O.A.C.

Leuco-anthocyanin reagent.--Concentrated hydrochloric acid (25 ml. of 3696 w i w ) is diluted

Vanilh reagent.-One

g. of

recrystallised vanillin is dissolved in

roo

ml. of 707; (VJ'V)

Anthocyanin reagent.-A freshly prepared solution of ml. of 30 hydrogen peroxide in

The reagent should be prepared freshly every

3

days.

Methods

Total

phenols.-A suitable aliquot

of

the solution under test, containing not more than

0 5 ml. of methanol or ethanol, is diluted with water to about 7 ml. in a 10-ml graduated test-

tube . The contents are well mixed,

0.50

ml. of th e Folin-Denis reagent is added, and the

tubes are thoroughly shaken again. Exactly

3

min. later,

1.0

ml. of saturated sodium carbonate

solution is added and the mixture made up to 10 ml. with good mixing. After

I

h., the

absorptivity is determined in I-cm. cells at 725 m,u using as a blank water and reagents only.

If the solution is cloudy or a precipitate forms,

it

should be filtered or centrifuged before readings

are taken.

Leuco-anthocyanins.-One ml. of the solution under test (which should not be more than

50%

with respect to either methanol or ethanol) is placed in each of two

6 x I

in. ground-glass

stoppered test-tubes of uniform bore and wall thickness and

10.0

nil. of leuco-anthocyanin reagent

added. The tubes are shaken until the solution is homogeneous and one

of

them is placed

unstoppered in a constant-level all-metal water-bath maintained at 97

IO.

After

3

min.

the s topper is placed firmly in position and the tube heated for

a

total of

40

min. After removal

of the stopper, t he tube is cooled in running ta p water for

j

nun. and the absorptiv it~of the

solution determined in

a

I-cm. cell

at 550

m,u and, if chlorophvll is present, a t

650

mp, using

as

a blank the contents of thc unheated tube.

Flavanols.--A suitable aliquot of solution, containing not more than 0.1ml. of methanol,

is placed in each of two 25-1111. conical flasks (A and B and diluted to 2-0 ml. with water.

Four ml. of the vanillin reagent are added from a burette during

10-15

sec. to flask A , and

4.0 ml. of 70

(v/v)

sulphuric acid to flask B, the flasks being shaken in

a

bath of cold water

to prevent the temperature from rising above

35 .

After shaking well the flasks are left for

exactly

15

min. at room temperature and the absorptivities of the contents of the two flasks

and that

of

a previously prepared blank

(C)

4.0 ml. of reagent and

2.0

ml. of water) are measured

in I-cm. cells at

500

m p against 47y0 sulphuric acid (D) (4.0 ml. of

70%

v/v H,SO, and 2.0 ml.

of water).

J. Sci. Food Agric., 10, January, 1959


3/6

SW AI N et a1.-PHENOLIC CONSTITUENTS OF P RUNUS DOMESTICA 65

The absorptivities of the reagent blank (C) and of the second flask (B) are subtracted from

the reading given by

A).

Alternatively the cells may be arranged in the holder

so

that A + D

are read against

B + C.

Anthocyanins.-Three ml. of hydrochloric acid in aqueous methanol (HC10.5~ CH,OH

80-85y0 v/v) are placed in each of two tubes. The solution in the first tube is

diluted with

1.0ml. of methanolic hydrochloric acid

(5

: v/v, 3N) , and 1-0 l. of the peroxide reagent is

added t o the second tube. The tubes are left for 15 min. in the dark and th e absorptivity of

the solution in the second tube measured in a I-cm. cell at

525

mp using the contents of the

first tube as a blank. In all cases, standard curves were prepared using known amounts of

suitable compounds.

Results and discussion

Total phenols

The quantitative estimation of total phenols in biological extracts can be accomplished

in a number of different ways. Most phenolic compounds are readily attacked by various

oxidising agents

;

many react with diazotised amines and, like electrophilic substances, form

coloured compounds ;

some form coloured complexes with certain metals ; all show definite

absorption peaks in the ultra-violet, and methods for their estimation have been devised based on

such properties. Since, however, individual compounds vary widely in their ability to react with

(a) oxidising agents such as phosphomolybdate or permanganate,G b ) coupling reagents such as

diazotised 9-nitroaniline' or Gibbs reagent,s (c) metals such as iron,s and (a) in their ultra-

violet or visible adsorption spectra

lo

all the procedures for the estimation of total phenols are

necessarily empirical.

In the course of these invesigations it was found th at methods based on the use of oxidising

agents are the most useful, since the variation between estimations of individual compounds

is much less than that obtained using methods based on the other properties listed above.

Joslyn and his co-workersG ave shown tha t there is little difference in the results obtained with

natural extracts using either the modified method of Folin Denis or th at of Lowenthal, but

in our experience the former has proved to be more convenient.

A number of variations of the method have been examined ( e g 11 but none seemed to

offer any particular advantage.

The addition of extra saturated sodium carbonate to the

solutions

(2.0

ml. instead of

1.0

ml.), although increasing the likelihood of precipitation, was

found to make no difference to the reading obtained, but t he use of other bases such as potassium

carbonate, sodium hydroxide or sodium phosphate buffers of various pH gave less reproducible

results The transmission curve of the blue complex showed a very broad maximum between

620

mp and 740 mp with a peak at 725 mp (cf. 12 and hence simple colorimeters can be used

with a suitable red filter without much loss of precision.

All the phenolic compounds tested

showed an almost linear relationship between absorptivity and concentration, although the

slopes of the curves were different, the useful range using I-cm. cells being of th e order of

10-100

p g

of substance in the aliquot taken for examination.

Leuco-anthocyanins

All quantitative estimations of leuco-anthocyanins are based on the transformation of these

substances to anthocyanidins by heating in acid solution. The transformation is not quanti-

tative since

a

large amount of brown

'

phlobaphene-like

'

polymer

is

formed along with the

anthocyanidin.13 The proportion of anthocyanidin produced depends on the solvent used ;

in aqueous acid, for example, not more than 10 is obtained, whereas in alcoholic solvents

the yield is of t he order of 25%.

The first method devised for the estimation of leuco-anthocyanins involved purification

by multiple transfers between solvents of the anthocyanidin produced1* and is unsuitable for

routine analysis. A much simpler method using a direct measure of the colour produced on

heating the sample in isopropanol-HC1 a t

IOOO

was introduced by Pigman and his co-workers,l5

but this suffers from the disadvantage that precautions must be taken to prevent evaporation

of the low-boiling solvent.



4/6

66 SWAIN

et a1.-PHENOLIC

C0,VSTITUENT.S

OF

P R U N U S

DOMESTICA

A

number of other solvents with

a

boiling point over

100'

were thercfore investigated,

2-butanol being found to be the most satisfactory. The colour development was more or less

complete in 30-3j-min. (cf.

15)

and the Amax for leuco-cyanidins was found to be

at 550 m p

(Fig.

I .

In other solvents (water, acetic acid) the smaller shoulder at

450

mp developed into

a definite peak with

a

lowering of absorption

at 550

mp (Fig.

I)

and this is undoubtedly due to

the relatively greater production of phlobaphene-like polymers.

(+)

Catechin after heating

in m-butanol-HC1 gives

a

product having

a

similar peak at 450 mp (cf.

16 .

I3oilrd in n-butanol HCI

B Boiled in

acetic acid-HCl

C ( r ) ~ C a t ~ c h i n

oiled

in

n-butanol-HC1

Using the method described or that of Pigman et al. 15 good replicates can only be obtained

when careful atten tion is paid to obtaining uniform heating conditions. This necessitates

th e selection of tubes of uniform bore and wall thickness, maintenance of

a

constant level of

water in the heating bath and good control

of

temperature. The anthocyanidin produced

fades very slowly in the dark, bu t this process is accelerated by light1: and therefore a metal

bath constructed

so

that no direct light falls

on

the tubes, should be used for the analysis.

When methanolic leaf extracts were analysed for leuco-anthocyanins by the method

described, it was observed that chlorophyll contributes to the absorption at

550

mp. The

visible spectrum of chlorophyll-containing extracts free from Zeuco-anthocyanin (e.g. from

nettle leaves) was, however, found to be unchanged after heating in the butanol-HC1 reagent.

This observation was checked in routine determinations by measurement at 650 inp where

chlorophyll absorbs strongly and anthocyanidins do not intcrferc, and indicated that an un-

heated control can be used

as

a blank.

Similarly the contribution to the adsorption at

550

m p made by anthocyanins in extracts

from fruit skins

was

found to be unchanged after heating in the reagent, although in this case

hydrolysis

of

the glycosides undoubtedly occurs. It

is

apparent that the molecular extinction

coefficient of anthocyanidin glycosides is little different from tha t of aglycone and in this case

also

an

unheated control can be used as a blank. The useful range

of

the method using I-cm.

cells is 50-400 p g . but an absolute straight-line relationship between concentration and absorp-

tivity was not obtained.

Flavanols

The colour reaction for the detection of phloroglucinol using vanillin and concentrated

hydrochloric acidla has been used both q ~a li ta ti ve ly l~nd quantitativelyz0>

1

for compounds

other than phloroglucinol which contain the

: 3

: 5-trihydroxybenzene nucleus. The red

colour produced is due to the formation of

a

carbonium ionz2 3 which is unstable even in the

presence of 13 ) hydrochloric acid.

22

Attempts to apply the Lindt reagent quantitatively

were found to give non-reproducible results, which was apparently due to the instability of

the carbonium ion form, since the difficulty could be overcome by using

7096

sulphuric acid

instead of

30

alcholic hydrochloric acidz1 in making up the reagent. Under these conditions

the adduct with both phloroglucinol and catechin had A

500

m,u (cf. 23 and the colour formed

only faded slowly (Fig.

2 ) .

Even

so

it can be seen that care must be taken t o measure the absorp-

tion a t a fixed time after addition of the reagent. It was found essential for best rcproducibility

that the initial mixing of the reagent with the solution under test should be done as rapidly

as

possible and tha t th e temperature of the solution should be kept below 40'.

The presence of excessive amounts

of

methanol, or more especially ethanol, materially

J, Sci. Food

Agric., 10,

January,

1959


5/6

SWAIN et a1.-PHE,I'OLIC CONSTITUENTS OF P R U N U S DOMESTICA 67

alters the colour obtained in the reaction to a bluer shade. This is probably due to the formation

of a coniferylaldehyde-like compound by the vanillin and alcohol in strong sulphuric acid which

then condenses with the phenol. Such addition products are known to have absorption maxima

at longer wavelengths than the original compound.z2

on

0 . 5

TIME, min

FIG.?-.-Dewease

of optical densi ty of vanillin-flavanol adduct with

tim

Examination of the method showed th at only compounds containing an undeactivated

phloroglucinol (or resorcinol) nucleus react when present in amounts in the useful range of the

method

(0-100

g. in the aliquot).

Compounds such as phloracetophenone, butein, hesperidin

or quercetin gave no colour with the reagent.

Measurements showed th at Beer's law was obeyed

for all reactive compounds tested.

A

thocyanins

The qualitative estimation of anthocyanins in aqueous buffer extracts of fruit tissues can

be satisfactorily carried out by measuring the difference in the red colour

of

the extract at

pH

2-0

and pH

3.4.24

As can be seen from Fig.

3,

however, the lower pH recommended by

the American workers is not low enough to ensure highest sensitivity or accuracy since a t this

pH the change in colour with a small change in pH is still large (cf. 2 5 .

It

was found better

to use a pH between 0 5 and 1.0 Fig. 3 ) . The change in absorptivity in the visible spectrum

of t he anthocyanin solution with pH is dependent on the change in the proportion of the com-

pound in the oxonium-carbonium ion form,26 and it is interesting to note tha t the absorptivity

due to the phenolic hydroxyl groups at 280 m,u is not changed greatly by changes in pH from

to 5 (Fig. 3 ) .

I

I

I

I

I

5

0

OO

I 2

3

4

PH

When methanolic hydrochloric acid was used to extract the tissue, however, the above

method was found to be unsatisfactory because of the large dilution required to ensure adequate

buffering. The possibility of bleaching the solution without change in

pH

was therefore

examined. A recommended methodz5using sodium sulphite was found to require far more

of the reagent than had been suggested, causing cloudiness, and hydrogen peroxidez7 was

therefore substituted. Under the conditions used, decolorisation of a standard anthocyanin

solution

was

found to be virtually complete in j min. and the reagent showed no serious influence

on the colour of other substances (such as chlorophyll) present in extracts from immature fruit

skins.



6/6

68

SOMERS-PREPARATION

OF

BORDEAUX

M I X T U R E

The method was found to be suitable over a range of

0.30 pg.

of anthocyanin and the curve

(Cyanin hydrochloride, 10 pg.,/ml., in methanolic HC1btained was linear with concentration.

gives optical density of 0.405 in a I-cm. cell at

525

mp.)

Acknowledgments

The work described in this paper was carried out as part of the programme

of

the Depart-

ment

of

Scientific and Industrial Research and of the Division of Forest Products, Commonwealth

Scientific and Research Organisation, Australia.

The authors wish to thank Dr. W. G. E. Forsyth for a gift of pure cacao Zeuco-anthocyanin.

Crown

C o y i g h t Reserved

Low Temperature Kesearch Station

Downing St.

Cambridge

References

1 Bate-Smith,

E.

C., k Pridham, J . B., Personal

Date-Smith, E.

C.,

Biochoin .

J . , 1954, 56, 2

Hillis, W.

E.,

Aust.

J .

biol Sci., 1956, 9, 263

'ridham, J. B.,

A n a l y t . C h e m . ,

1957,

9,

116j

' Official and Tentative JIethods of -1nalysis

',

Ass. Off. hgric. Chem., Igjj, 8th Edn, p. 144

(Washington : A .O .A .C . )

Smit, C. J . B., Joslyn, 31. h. Lukton, A

A n a l y t . Chem., 1 9 j j . 27, 11j9

' Bray, H. G., Humphris,

3.

G., Thorpe,

W.

V.,

White, K., and Wood, I? B., Bi o c h a m .

J. ,

19.52,

King, I.. E., King, T.

J.,

c Nanniiig, L. C . , J .

c h e m .

communication

52, 416

l o

-4ulin-Erdtrnai1, G., Sz,r7is/z.

Pappe~s t id i z i7 zg ,19j3,

56, 287

I1 Englis, I>.

T.,

Miles, J . \Ir., Wollerman, 1,. -I.,

J .

A s s

off

agvic .

Ch e i u . , W a s h . , 1955, 38, 51s

l 9 Pro, 31. J.. J . Ass. 04..

p i r

Cheiiz., W a s h . , 1952, 35,

'5.5

XI

Bate-Smith, E. C., S: Slrain,

T.,

Cizeix G.

I i zd . ,

7953 .

P 377

Received

4

March. 1958

amcndfrl

iiianuscript

25

July,

r y j S

l 4 I-Iallas, C.

A., Keseavch Hecovd

h'o.

70,

1939, Brit.

Ass.

Res. for the Cacao, Chocolate, Sugar Con-

fectionery and Ja m Trades (quoted by Forsyth,

W. G. C., Biochem. J . , 1952, 51, 516)

l 5 Pigman, W., Anderson,

E.,

Fischer, R., Buchanan,

M . A , , Browning, B. L.,

TAPPI ,

1953, 36, 4

l 6

Hathway,

D. E.,

Seakins,

J.

W. T., J . chem.

Soc.,

1957. P. 1562

l 7 Nordstrom, C.

G.,

A c t a c h em . scand., 1956, 10,

1491

l9 Bradfield, A. E.,

Penney, IT. , J . chem. Soc.,

1948,

Lindt, O., Z .

anal.

Chem., 1887, 26, 260

P. 2249

Kursanov, A. L.,

B i o k h i m i y a ,

1941, 6, 128

21

W-atkin, J. E., Underhill,

E.

W., Neish, A. C.,

2 2

Pew, J. C., J . Amev.

chenz.

So r I q - j I , 73, 1678

2 3 Giniliano, R., Ann. c h i m . apfi l . , Kortza, 1939, 29, 86

2 4 Sondheimer.

E..

Iiertesz. Z . 1..

Aimlvt. Chent . .

C n n a d . J . Bi o c h e m . , 19.57, 35, 229

Soc

, 1940, 62, 2711

27

Karrer, P.,

de Xleuron,

G ,H e l v chwn A c t a , 1932,

15,

507

THE PREPARATION OF BORDEAUX MIXTURE

By

E. SOMERS

Using seven different methods of preparation, the sedimentation, tenacity, n

vitro

fungitoxicity, and rat e of crystallisat ion of

8 :

10

:

1 Bordeaux mixture have been in-

vestigated. It is suggested that the most effective Bordeaux mixture is that prepared

by

adding a concentrated lime

paste

to diluted copper wlphate.

Introduction

Although long-established as a fungicide, Bordeaux mixture is still widely used in plant

protection.

It has

been demonstratedl t ha t the chemical properties of the gelatinous precipitate,

formed by copper sulphate and calcium hydroxide, are not dependent on the manner in which

J.

Sci. Food Agric.,

10 January, 1959

jsfa vol 010 is 01 jan 1959 pp 0063-0068 the phenolic constituents of prunus domestica. i.—the...

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