114
Notes
Chem. Pharm. Bull. 49(1) 114—117 (2001)
Vol. 49, No. 1
Acid Mediated Hydrolysis of Blueberry Anthocyanins
c
Takashi ICHIYANAGI, ,a Kikuo OIKAWA,a Chigusa TATEYAMA,b and Tetsuya KONISHI
*
Department of Hygiene Chemistrya and Department of Radiochemistry-Biophysics,c Niigata College of Pharmacy, Niigata
950–2081, Japan and Niigata Woman’s College,b Niigata 950–0806, Japan.
Received July 10, 2000; accepted October 5, 2000
Acid mediated hydrolysis of anthocyanins was studied using capillary zone electrophoresis (CZE). A com-
mercially available wild blueberry (Bilberry) extract was dissolved in different concentrations of TFA (0.1, 1, 3,
9%), then was subjected to thermodecomposition reaction at 95 °C. After the reaction, the samples were ana-
lyzed by CZE. The hydrolysis rate of each anthocyanin and the formation of the aglycon were determined by the
change in the peak pattern of the anthocyanins in the electropherogram. Each anthocyanin peak decreased time
dependently in a first order kinetic fashion. It was revealed that the hydrolysis rate of each anthocyanin was de-
termined primarily by the type of conjugated sugar and not by the aglycon structure. The rate constant of antho-
cyanin hydrolysis was in the following order, arabinosideϾgalactosideϾglucoside without regard to the aglycon
structure. The kinetic behavior of this anthocyanin hydrolysis together with the CZE mobility allowed us to iden-
tify an unknown CZE peak as delphinidin 3-O-b-arabinoside. At low TFA concentration, significant decomposi-
tion of the anthocyanidin nucleus occurred, but the glycoside hydrolysis predominated at high TFA concentra-
tion. It was further revealed that the aglycon released reacted successively to form polymeric products at higher
TFA conditions.
Key words blueberry anthocyanin; capillary electrophoresis; anthocyanin decomposition; glycoside hydrolysis
lution was diluted 10 times with 1% TFA, then 100 ml of the solution was
taken in a sample tube, dried up in vacuo and finally dissolved in 1 ml of 1%
TFA. After passing through a 0.2 mmf membrane filter, the sample solutions
were subjected to CZE. Triplicate determinations were run for each sample.
The CZE conditions were as reported previously.9)
Anthocyanins are the reddish pigments widely distributed
in colored fruits and vegetables, such as eggplant, grapes and
blueberry. They are usually present as the glycosides having
arabinose, glucose or galactose attached to the anthocyanidin
nucleus. The phytoceutical significance of anthocyanins has
been discussed in relation to a wide range of physiological Results
functions such as vision improvement,1) antioxidant,2—5) anti-
Hydrolysis of each anthocyanin in the Bilberry extract was
cancer6) and so on. Few studies have been done on the struc- determined in 1% TFA using the CZE method. Twelve antho-
ture–radical scavenging activity relationship among antho- cyanin peaks were identified as shown in Fig. 2 from the
cyanin aglycons and the glycosides.7)
comparison of the mobility of standard anthocyanins as de-
It is known that the flavylium cation form of the antho- scribed in our previous report.9) When the TFA solution of
cyanidin nucleus is unstable under conditions such as neutral the Bilberry extract was heated in a sealed ampule filled with
and alkaline pH, high temperature and light illumination.8) Argon at 95 °C, the CZE peaks of each anthocyanin de-
From the point of view of using anthocyanins as a functional creased with reaction time, concomitantly with an increase of
food material, it is important to know the stability of antho- aglycon peak (Fig. 2). The time course changes of antho-
cyanins as a mixture.
cyanin and the aglycon peak are shown in Fig. 3 together
Since there are few studies on the degradation property of with the 525 nm absorbance (A525 nm) change of the reaction
anthocyanins, we precisely examined here the acid mediated solution that was determined as the indication of anthocyani-
reaction of anthocyanins by capillary zone electrophoresis din nucleus. It was revealed that the aglycon formation
(CZE) using commercially available wild berry (Bilberry)
extract as the sample of anthocyanin mixture. The results re-
vealed that the hydrolytic rate of anthocyanin glycoside was
not dependent on the aglycon structure, but dependent on the
type of conjugated sugar.
Materials and Methods
Reagents All the reagents including Na-borate (NaBO4), trans-1,2-di-
aminocyclohexane N,N,NЈ,NЈ-tetra acetic acid monohydrate (CyDTA) and
trifluoroacetic acid (TFA) were obtained from Wako Pure Chemical Indus-
tries, Co. Ltd., Japan. CLEAN 99 K200 was from Clean Chemical Co. Ltd.,
Japan.
Bilberon 25, the concentrated extract of Bilberry (Vaccinium myrtillus L.,
BILBERRY), was a kind gift from Tokiwa Phytochemicals Co. Ltd.
Methods. Acid Mediated Hydrolysis of Anthocyanin Bilberon 25
was dissolved in different concentrations of TFA (0.1, 1, 3, 9%) to make the
concentration to 6.47ϫ10Ϫ3 mol delphinidin eq/l (calculated using eϭ30906
at 525 nm absorbance). An aliquot was taken into an ampule, sealed with
Argon, then was subjected to hydrolytic reaction at 95 °C for a defined pe-
riod. After the reaction, each sample was analyzed by CZE to determine the
hydrolytic degradation rate of each anthocyanin.
Analysis of Anthocyanins by CZE An aliquot of the above reaction so- Fig. 1. Structure of Anthocyanins
To whom correspondence should be addressed. e-mail: kouji@niigata-pharm.ac.jp
© 2001 Pharmaceutical Society of Japan