Free Radical ScaVenging ActiVity of Betalains
Journal of Natural Products, 2009, Vol. 72, No. 6 1145
quaternary ammonium as an exchanger group, 90 µm particle size)
purchased from General Electric Healthcare was used. After sample
injection, the elution process was as follows: 0% B from 0.0 to 2.0
mL (10.0 mL for compound E); after washing, a linear gradient was
performed over 15 mL from 0% B to 26% B, with 1 mL fractions
being collected. Injection volume was 1 mL, and the flow rate was 1.0
to the catechol substructure of compound 5, which is shown to be
3 times more potent than Trolox. Taking into account that the FRAP
assay is performed at pH 3.6, these observations are related to the
results obtained with ABTS•+ at lower pH values (Figure 4).
Betaxanthins present high antioxidant and free radical scavenging
activities not linked to the presence of phenolic hydroxy groups.
This may be general to all betalains, which contain a similar
electronic resonance system. In addition to their intrinsic activity,
a clear enhancing effect on the scavenging of the free radical
ABTS•+ and on the antioxidant power of betaxanthins has been
demonstrated to be linked to the presence of one or two phenolic
hydroxy groups in their structure.
mL min-1
.
Free Radical Scavenging Activity. The antiradical capacity of
betaxanthins was evaluated by following their effect on stable free
radical ABTS•+ [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)].
Decolorizing activity on ABTS•+ solutions was monitored spectropho-
tometrically at λ ) 414 nm.10 ABTS•+ radical was prepared from 2
mM ABTS through peroxidase activity (88 units/L commercial
horseradish peroxidase type VI, obtained from Sigma) in the presence
of H2O2 (45 µM), in 12 mM NaOAc buffer, pH 5.0. The reactive was
then diluted by 2/3 with the addition of samples, carrying out the
reactions in 53 mM sodium phosphate buffer, pH 7.0. Other conditions
are specified in the text. Measurements of 96-well plates were performed
after 24 h incubations at 20 °C, in a Synergy HT plate reader (Bio-Tek
Instruments, Winooski, VT). All experiments were performed in
triplicate and mean values and standard deviations were plotted. Final
volume was 300 µL (calculated path length ) 0.87 cm). Detector
linearity under the assay conditions was confirmed (r ) 0.999). Data
analysis was carried out by using linear regression fitting using
SigmaPlot Scientific Graphing for Windows version 8.0 (2001; SPSS,
Chicago, IL). In each case, errors associated with the results provided
were calculated on the basis of the residual standard deviation around
the regression line. For continuous recordings, the above spectropho-
tometer was used with thermostats and tap-covered 1 cm path length
cuvettes.
Antioxidant Capacity. The antioxidant activity of betaxanthins was
characterized by means of the reduction of Fe(III) to Fe(II). The method
described by Benzie and Strain39 to evaluate the ferric reducing
antioxidant power (FRAP) was used. Briefly, FeCl3 solutions at a final
concentration of 1.48 mM, in 223 mM sodium acetate buffer, pH 3.6
were used. Fe(III) reduction to Fe(II) was observed through the addition
of the reagent 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) at a final concen-
tration of 741 µM, which is able to yield a colored complex with Fe(II).
The reduction reaction was monitored spectrophotometrically at λ )
593 nm. All measurements were performed in triplicate, and mean
values and standard deviations were plotted. Data analysis was carried
out by using linear regression fitting using SigmaPlot Scientific
Graphing for Windows version 8.0. In each case, errors associated with
the results provided were calculated on the basis of the residual standard
deviation around the regression line.
Experimental Section
General Experimental Procedures. A Shimadzu LC-10A apparatus
(Kyoto, Japan) equipped with a SPD-M10A photodiode array detector
(PDA) was used for analytical HPLC separations. Reversed-phase
chromatography was performed with a 250 × 4.6 mm Luna C-18(2)
column packed with 5 µm particles (Phenomenex, Torrance, CA).
Gradients were formed with two He degassed solvents. Solvent A was
H2O with 0.05% TFA, and solvent B was composed of MeCN with
0.05% TFA. A linear gradient was performed over 25 min from 0% B
to 35% B. The flow rate was 1 mL min-1, operated at 25 °C. Injection
volume was 20 µL. An Agilent VL 1100 apparatus with a LC/MSD
trap (Agilent Technologies, Palo Alto, CA) was used for ESIMS
analyses. Elution conditions were as described above using a Zorbax
SB-C18 (30 × 2.1 mm, 3.5 µm) column (Agilent Technologies) with
a flow rate of 0.3 mL/min. Vaporizer temperature was 350 °C, and
voltage was maintained at 3.5 kV. The sheath gas was nitrogen, operated
at a pressure of 45 psi. Samples were ionized in positive mode. Ion
monitoring mode was full scan in the range m/z 50-600. For detection
the electron multiplier voltage was 1350 V. A Jasco V-630 spectro-
photometer (Jasco Corporation, Tokyo, Japan) attached to a Tectron
thermostatic bath (JP Selecta, Barcelona, Spain) was used for absor-
bance spectroscopy. For the quantification of betalains, pigment
concentration was evaluated taking a molar extinction coefficient of ε
) 48 000 M-1 cm-1 at 480 nm for betaxanthins5,31 and ε ) 65 000
M-1 cm-1 at 536 nm for betanin.41 Measurements were made in water
at 25 °C. 3-Hydroxyphenethylamine was purchased from Fluorochem
Ltd. (Old Glossop, England). Other chemicals and reagents were
obtained from Sigma (St. Louis, MO). Solvents were from Merck
Chemicals Ltd. (Dorset, England). HPLC-grade acetonitrile was
purchased from Labscan Ltd. (Dublin, Ireland). Distilled water was
purified using a Milli-Q system (Millipore, Bedford, MA).
Acknowledgment. This work was supported by MEC (Spain) and
FEDER (Project AGL2007-65907) and by Programa de Ayudas a
Grupos de Excelencia de la Regio´n de Murcia, de la Fundacio´n Se´neca,
Agencia de Ciencia y Tecnolog´ıa de la Regio´n de Murcia (Plan Regional
de Ciencia y Tecnolog´ıa 2007/2010).
Synthesis of Betaxanthins. Betaxanthins were obtained as immo-
nium condensation products of betalamic acid with the following
amines: 1-aminopropane, 2-phenylethylamine, 3-hydroxyphenethy-
lamine, 4-hydroxyphenethylamine (tyramine), and 3,4-dihydroxyphen-
ethylamine (dopamine). Syntheses were carried out following a method
described previously by Gand´ıa-Herrero et al.32 and by Wyler et al.40
In short, betanin purified from red beet was used as starting material.
Basic hydrolysis (pH 11.4) of 0.2 mM betanin released betalamic acid,
which was then condensed with the appropriate amine after reaching
pH 5.0. The corresponding betaxanthin was obtained, revealed by a
characteristic deep yellow color (betaxanthin maximum wavelength is
around λm ) 480 nm). The whole process was carried out under nitrogen
atmosphere to avoid the oxidation of substrates and products. Once
synthesis was achieved, a C-18 solid-phase extraction step was
performed, and an automated system was used for pigment purification.
One milliliter C-18 cartridges (Waters, Milford, MA) were conditioned
with 5 mL of MeOH followed by 10 mL of purified water. Salts and
buffers from the samples were removed by rinsing the column with
H2O. Betaxanthins were eluted with acetone and then concentrated to
dryness under vacuum. The residue was redissolved in water for further
use or stored at -80 °C.
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Purification of Betaxanthins. Anionic exchange chromatography
¨
of synthetic betaxanthins was performed in an Akta purifier apparatus
(General Electric Healthcare, Milwaukee, WI). The equipment was
operated via a PC using Unikorn software version 3.00. Elutions were
monitored at 280, 480, and 536 nm. The solvents used were 10 mM
sodium phosphate buffer, pH 6.0 (solvent A), and 10 mM sodium
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