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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
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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).
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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.
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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
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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.
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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
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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
top related