Agricultural and Biological Chemistry

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Stabilization Effect of Neutral Salts on

Anthocyanins: Flavylium Salts, Anhydrobases and
Genuine Anthocyanins

Toshio Goto, Tsutomu Hoshino & Masatoshi Ohba

To cite this article: Toshio Goto, Tsutomu Hoshino & Masatoshi Ohba (1976) Stabilization Effect
of Neutral Salts on Anthocyanins: Flavylium Salts, Anhydrobases and Genuine Anthocyanins,
Agricultural and Biological Chemistry, 40:8, 1593-1596, DOI: 10.1080/00021369.1976.10862247

To link to this article: https://doi.org/10.1080/00021369.1976.10862247

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 Agr. Bioi. Chern., 40 (8), 1593~ 1596, 1976

Stabilization Effect of Neutral Salts on Anthocyanins: Flavylium

Salts, Anhydrobases and Genuine Anthocyaninst

Toshio GOTO, Tsutomu HOSIDNO and Masatoshi OHBA

Department of Agricultural Chemistry, Nagoya University, Chikusa, Nagoya 464

Received March 3, 1976

Concentrated solutions of some neutral salts such as 4M MgCl z and 4 M NaCl strongly
stabilize anthocyanin anhydrobases as well as flavylium ions, and hence can effectively be used
for extraction and quantitative determination of genuine anthocyanins.

Generally anthocyanins are very unstable in
dilute neutral or weakly acidic aqueous solu-
tions; they decompose rapidly to form color-
1
less products. ) Extraction of anthocyanins
is, therefore, usually carried out using strongly
acidic solvents such as methanolic hydro-
2 pK = log [I]/[IV] + pH = 3.17
chloric acid. ) Willstiitter examined structure
change of anthocyanidins in aqueous solu-
tions with variation of pH.3) Recently, using
a simple model compound of anthocyanidin
Jurd 4 ) concluded that the anthocyanidin
exists in a flavylium cation (1) at pH below 2,
but when the solution is neutralized to pH 3
to 5 (the pH similar to that of cell sap in flower
petals) the pigment changes its structure to
the corresponding 2-hydroxychalcone (IV)
with hydration through the anhydrobase (II)
and colorless pseudobase (III). The flavylium
cation (I) and the chalcone (IV) are in equili-
brium as shown in the following equation!)
(I) + 2H2 0 ( > (IV) + HaO+
± 0.06
Recently many genuine anthocyanins (mostly
anhydrobase type) which are considered to
have the structure identical with that in the
original plants could be extracted using neutral
aqueous organic solvents. 5 ) Dilution with
water of the extracts causes hydration of the
pigments to form colorless products. Since
the amount of hydration is concentration de-
pendent, in more dilute solutions the pigments

OR
OH

HO., OMe
'/1
:::--....

f1avyJium ion (1) anhydrobase (II)

+H,O

o OR?' I
HO 7' OR . RO ~
.!.-I_
-OR OMe
~ I h-.
OMe
cis ·chalcone (IV) pseudobase (III)

. Studies· on Anthocyanin Complexes I.

t Partly presented at The 11 th Symposium on the Plant Chemistry,
Nagoya, Jan. 1975, Preprint p. 1 ~9.
1594 T. GOTO, T. HOSHINO and M. OHBA

4-
I
(\
\
1.0 ,,
t'
\ I
I \ I '
I \ t '",,"',
" ....
~ I
I
\ II> 0.8 if
It"
"'0-'\
"

~
I
~
3- \
" '\
II ..--... \ \
I I c !'~/
I /"

- \
\
\- '-...... " \
. ~ ,
0 I
I \ A'" 0.6 "
....0 .\
X I
\
\
~,
\ .
f \
I
ki
~
I \ A"'
I I
-< 0.4
<l>
u

''""
2-
I
I
I \
I
\ \'
. /\"--~
,.Q
h
0
Vl
I
I
I I
I
\
0.2 ,
•

' .
--~' . ~
'('." ,\,----
_--
....
~
\

-~ ... ~
on
-< 1-
I
I \
\ --
I
I I
\
250 300 350 400 500 600 700
I \
I \ Wavelength (nm)
/
I
I
\
,,
, --- ---"'" FIG. 3. Uv-vis Spectra of Commelinin (concn. 31.3
0
" ,
--
350 400 450 500 mg/liter; measured at 20°C).
Wavelength (nm) - - , in 4 M NaCl (stable for a few hours); - , in
water (2 min after dissolution); --, in water
FIG. 1. Visible Spectra of 4',7-Dihydroxyantho-
(1 hr after dissolution); ------, in water (3 hr after
cyanidin (1.83 x 10- 6 mole/liter) (both solutions are dissolution).
stable for a few hours).
------, fiavylium ion (in 4 M MgCl 2 containing 5 x 1.5
10- 4 N HCI); , anhydrobase (the above solution
was neutralized by addition of 0.05 ml conc. NH4 0H).

525
/1-, 560 1.0
I \
(B)
, I
I
, ~ 1.0
\
, I
I
\
(A) 0.8 dark red o
0'>
, I
I
\
\ '"
~
\ \
\ I
\ 0.6 '"
\ ,, /
I
\
\ "§
u
/
\ '- 515
\ 0.4
\
, ,1, 550
\
\ 'fo
' ,, 0.2 reddish
0.61 \
\
\ /
I
I
I
\
'
\
\
\
,,
------ 0
II> violet <"' 0.5
~ (A)
t
0.4 \
\
,, , - /
I
/ \
\
(B)
'f:'o"
\

0.2
, \
,,
,,
,
0,, 540
~, 580
-- -- -- '- A"'
-<
II>
\
, I
I \
\
u
C
\ , I
I l. 0 (D) o
'"
-""
....
, I
I
\
\
(C) light o 25 50 75
0 \
\
I
\
I
I
,
\
0.8 blue Concentration of commelinin, mg/liter
If! I \
\ \ I \
-""
-0:: 1.0 \ ,
\
I
I \
, 0.6
, I,
\ \
,,
I
\ FIG. 4. Plots of the Absorbance at 590 nm vs. Con-
\
0.8 \
\
\ ,, , /
I \
\
0.4 centration of Commelinin
\
I
\
, c
0-0, in 4 M MgCl 2 (0.5 hr after dissolution at 20 C);
0.6
\
\
\ ,, 0.2 bluish
\ ,
" --, violet e-., in water (0.5 hr after dissolution at 20°C).
0.4
\
\
\
o (C)
\ (D)
0.2 \
_-1-
555 600 Pansy petals are more hydrated. Therefore, it is very dif-
0
\
'-
-- -- -- - - , ,
------ used
ficult to measure the uv spectra of the pure
400 500 600 700
genuine pigments in such a dilute solution as
Wavelength (nm)
used for the usual uv measurements (ca. IO-5 M).
FIG. 2. Visible Spectra of Genuine Anthocyanins We found that anhydrobases as well as
of Pansy Petals in 4 M MgCl z Solution.
flavylium ions are strongly stabilized and do
A disc (12 mm diameter) of petal was extracted with
not form colorless pseudo bases by hydration
4 M MgCI z a few times and the combined extracts
were diluted with 4 M MgCh to 10 ml, whose visible when dissolved in concentrated aqueous solu-
spectrum was measured in a cuvette of path length tions of some neutral salts such as MgCl 2
10mm. (4 M) and NaCI (4 M). For example, in 4 M
- - , in 4 M MgCI 2 ; ------, in 4 M MgCl 2 (3 ml) con-
taining 0.05 ml of conc. HCI. MgCl 2 solution either flavyIium ion or anhy-
 Stabilization Effect of Neutral Salts on Anthocyanins 1595

Solvent measure the vis spectra of pure commelinin.

MNaCI
S
l: 1 However, in 4 M NaCI solution commelinin is so
<:>
0>
In
4MMgCh stable that we could obtain the uv-vis spec-
~
IMNaCI trum of pure commelinin; i. e. the ration of
.,'" 2MMgClz
..
"c
...0
.0
1 MLiCI
absorption intensity at 590 and 316 nm in the
NaCI solution (1.5) is larger than the value
., 0.5 hI MgCIz (1.0) measured in water as reported by Hayashi
.0
.,'" et aU) A linear relationship is observed
>
..-
.~

<Ii H2O between concentration of the pigment and

~
intensity of the absorption maximum (Fig. 4).
o
o 1 2
When water is used for dilution of the pigment
3
Time (hr) solution, such a linear relationship cannot
be obtained. The effects of neutral salts on
FIG. 5. Stability of Commelinin in Aqueous Salt
Solutions (concentration: 31.3 mg/Iiter; path length the stability of commelinin are shown in Fig.
10 mm; 20°C) 5. The similar observation was also obtained
in the case of other complex anthocyanins
drobase of 4',7-dihydroxyanthocyanidin ex- such as cornflower pigment. One of the rea-
ists in their own structure and does not tend sons of this stabilization effect of salts may be
to decompose (Fig. 1). Incidentally, when attributable to the decrease of free water in the
dissolved in water they decompose rapidly to solution.
form colorless products.
Genuine anthocyanins in plants can be EXPERIMENTAL
extracted quantitatively with the salt solutions.
For example, genuine anthocyanins in pansy Uv and vis spectra were measured with a Hitachi
EPS-3T Spectrometer.
petals having different colors can be extracted
4',5-Dihydroxyflavylium chloride was synthesized
as follows: a small disc (1.2 cm diameter) according to Pratt et af.B) and Hayashi') and crystallized
obtained from each petal by cutting with a from cone. HCI. MS m/e: 238 (M+-HCl). Found:
corkborer is extracted with 4 M MgCl 2 a few C, 63.14; H, 4.42. Calcd. for C ,5 H l1 0 3Cl'1/2 H 20:
times. The combined extracts are diluted with C, 63.50; H, 4.26 %. Commelinin was prepared ac-
cording to Hayashi et al. e,7)
4 M MgCb to 10 ml and their vis spectra are
measured (Fig. 2). In this case, when water
is used instead of the salt solution the extract REFERENCES
becomes almost colorless. When each of the
1) L. Jurd, "Some Advances in the Chemistry of
MgCl 2 extracts is acidified with conc. hydro-
Anthocyanin Type Plant Pigments," in "Chemistry
chloric acid, its color is changed to that of of Plant Pigments," ed. by C. O. Chichester,
flavylium ion having the absorption maximum Academic Press Inc., 1972, p. 123 ~ 142.
at a wavelength 35,' -40 nm shorter than that of 2) J. B. Harborne, "Comparative Biochemistry of
the neutral solution. The ratio of absorption the Flavonoids," Academic Press, 1967, p. 1 ~ 36.
3) F. M. Dean, "Naturally Occurring Oxygen Ring
intensities between the anhydrobase and the
Compounds," Batterworth Co., London, 1963, p.
corresponding flavylium ion is almost constant
388.
in each case, indicating that the anhydrobase is 4) L. Jurd, J. Org. Chem., 28, 987 (1963); L. Jurd and
extracted with little, if any, decomposition. T. A. Geissman, ibid., 28, 2394 (1963); L. Jurd,
This method of extraction is applicable to Tetrahedron, 25, 2367 (1969).
complex anthocyanin pigments. For example, 5) N. Saito, K. Hirata, R. Hotta and K. Hayashi,
Proc. Japan Acad., 40,516 (1964); K. Takeda and
commelinin,6) an anthocyanin complexed
K. Hayashi, ibid., 41, 449 (1965); K. Takeda,
with a flavone, is rapidly decomposed when N. Saito and K. Hayashi, ibid., 44, 352 (1968); A.
its aqueous solution is diluted to the concentra- B. Durkee and J. D. Jones, Phytochem., 8, 909
tion about 10- 6 M, and hence it was difficult to (1969); N. Saito, Y. Osawa and K. Hayashi,
1596 T. GOTO, T. HOSHINO and M. OHBA

Bot. Mag. Tokyo, 85, 105 (1972); K. Yoshitama
and K. Hayashi, ibid., 87, 33 (1974).
6) K. Hayashi and K. Takeda, Proc. Japan Acad.,
46, 535 (1970).
7) S. Mitsui, K. Hayashi and S. Hattori, Proc.
Japan A cad. , 35, 169 (1959).
8) D. D. Pratt, R. Robinson and P. N. Williams, J.
Chern. Soc., 199 (1924).
9) K. Hayashi, Acta Phytochirn. (Japan), 8, 179
(1935).