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Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves

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ORAC method and a continuous flow injection method based on Folin-Ciocalteau reaction (FI-FC) were used for determining the antioxidant activity in extracts obtained by using focused microwaves. Analysis of the antioxidant capacity (AC) of the main compounds of vanilla (vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid and vanillic acid) was also carried out. Vanilla extracts obtained by using focused microwaves had a higher AC (between 72% and 117%) than the obtained by conventional methods. Vanillin had a linear correlation with the antioxidant capacity of the extracts and it is the most influential compound in the antioxidant power. The AC calculated by the ORAC method and the FI-FC method had a ratio 2:1 because of different kinetics and reaction mechanisms of the antioxidants with the reagents, so it is necessary more than one method to establish the antioxidant power in food. On base on the results of the present study microwaves energy can be used to obtain vanilla extracts to improve the AC of them.
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Food and Nutrition Sciences, 2013, 4, 244-253
http://dx.doi.org/10.4236/fns.2013.48A030 Published Online August 2013 (http://www.scirp.org/journal/fns)
Antioxidant Capacity in Vanilla Extracts Obtained by
Applying Focused Microwaves
Adalith Rojas-López, María P. Cañizares-Macías*
Departamento de Química Analítica, Facultad de Química, Universidad Nacional Autónoma de México, México D.F., México.
Email: *pilarm@unam.mx
Received April 6th, 2013; revised May 6th, 2013; accepted May 13th, 2013
Copyright © 2013 Adalith Rojas-López, María P. Cañizares-Macías. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
ABSTRACT
ORAC method and a continuous flow injection method based on Folin-Ciocalteau reaction (FI-FC) were used for de-
termining the antioxidant activity in extracts obtained by using focused microwaves. Analysis of the antioxidant capac-
ity (AC) of the main compounds of vanilla (vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid and vanillic acid)
was also carried out. Vanilla extracts obtained by using focused microwaves had a higher AC (between 72% and 117%)
than the obtained by conventional methods. Vanillin had a linear correlation with the antioxidant capacity of the ex-
tracts and it is the most influential compound in the antioxidant power. The AC calculated by the ORAC method and
the FI-FC method had a ratio 2:1 because of different kinetics and reaction mechanisms of the antioxidants with the
reagents, so it is necessary more than one method to establish the antioxidant power in food. On base on the results of
the present study microwaves energy can be used to obtain vanilla extracts to improve the AC of them.
Keywords: Focused Microwaves; Vanilla; Antioxidant Capacity; ORAC Method; Flow Injection; Folin-Ciocalteau
1. Introduction
The early news from vanilla are in the middle 15th cen-
tury, when Aztecs conquered the Totonaca Empire who
used vanilla to make a drink named “xocolatl” (choco-
late). Libellus de Medicinalibus Indorum Herbis, Cruz
Balbiano codex, were the first document where vanilla is
named with its Nahuatl name: “tlilxochitl” (black flower)
[1]. For three centuries Mexico was the only producer of
vanilla in the world. In 1841, Edmond Albins, a farmer
from Reunion Island had the idea of pollinating the flo-
wer with a bamboo stick. From then vanilla began to
spread through Africa and Asia [2].
Vanilla is the only orchid that produces an important
commercial fruit for its flavour and smell. There are more
than 110 species of vanilla, but the Vanilla planifolia, also
known as Vanilla fragans, is the most important source
of the natural commercial vanilla [1,3,4].
Vanilla has more than 250 compounds but only 26 have
concentrations higher than 1 mg·kg1. Among non-vola-
tiles compounds of vanilla are: tannins, polyphenols, free
amino acids and resins and among volatiles compounds
are: carbonyl aromatic and aliphatic alcohols, aromatic
acids, aromatic esters, phenols, lactones, aliphatic and aro-
matic hydrocarbons, terpenoids, etc. Vanillin (1% - 3%),
vanillic acid (0.1% - 0.2%), p-hydroxy-benzaldehyde
(0.1% - 0.2%) and p-hydroxybenzoic acid (0.01% -
0.02%) are the most important phenolics compounds pro-
duced during cured process [5-7].
Antioxidants have beneficial effects on the conserva-
tion of the food, avoiding their oxidation, besides having
healthy effects; so industries of food, pharmaceutical and
cosmetic have been used antioxidants to give better ad-
vantage over yours products. The main synthetic anti-
oxidants used by the industry are: propyl gallate, buty-
lated hydroxy anisole, hydroxyl toluenehydroxyanisole,
tertiary butylhydroquinone, most of them are not very
healthy, therefore natural antioxidants instead of the syn-
thetic compounds have been more used [8-10]. Plants
and spices are the main target to look for natural antioxi-
dants. There are some studies to identify the compounds
that should act as antioxidants [11]. Among phytoche-
micals best characterized in vegetable food polyphenols
have been the most studied due to their high dairy ingest
[12]. Moreover, polyphenols are able to fix other reactive
compounds as HO•, NO2•, N2O3, ONOOH and HOCl and
also are able to fix metallic ions (specially iron and cop-
*Corresponding author.
Copyright © 2013 SciRes. FNS
Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves 245
per) to obtain poorly active compounds in the formation
of reactive species [13].
Inhibition of the chain reaction, oxygen captation, in-
hibition of singlet oxygen, metals quelation and inhibi-
tion of oxidative enzymes are mechanism that makes
very complex the antioxidant action in the food. There-
fore, several analytical methods are necessary to get a
precise evaluation of the antioxidant capacity (AC) [14].
Based on the chemical reaction involved, two categories
of the main antioxidant capacity tests can be named:
a) Tests based on the hydrogen atom transfer reaction:
these methods measure the competitive kinetic reaction
and the quantification comes from the kinetic curves.
Usually these are made from a free radicals synthetic ge-
nerator, an oxidative molecular probe and an antioxidant.
The Oxygen Radical Absorbance Capacity (ORAC) test
is one of the more used [15-17].
b) Tests based on the electron transfer reaction: in
these methods a redox reaction is carried out and the re-
duction of the oxidant marks the finish of the reaction.
The reaction with Folin-Ciocalteau reagent is one of these.
This method is principally used to determine total poly-
phenols [18,19].
In the last decade, there has been an increasing de-
mand for new extraction techniques enabling automation,
shortening extraction times and reduction of organic sol-
vents consumption [20,21]. Advances in preparation of
solid samples have brought a great number of new tech-
niques such as focused microwave-assisted energy [22-
24]. Microwaves have been used for different chemical
process accelerating digestion [25] and organic and inor-
ganic synthesis [26-28]. Focused microwaves have been
also used successfully to extract vanillin from vanilla
beans increasing the efficiency at 100%, so it has been
possible to accelerate extraction procedures of different
compounds without oxidation of them when the condi-
tions of extraction are controlled [29].
Vanilla is one of the species more consumed, mainly
in extracts, with a high antioxidant capacity (AC). Vanil-
lin is its main compound and the majority phenol. In this
paper the antioxidant capacity in ethanolic vanilla ex-
tracts obtained by applying of focused microwaves was
measured. A maceration procedure [30] was also carried
out and the results were compared. Vanillin concentra-
tion was determined and a study about the relation be-
tween vanillin and AC was carried out. AC was deter-
mined by two tests way: a) ORAC method, using fluo-
rescein as probeand 2,2’-azobis(2-methylpropionamidine)
dihydrochlorideas radicals’ generator and b) a flow in-
jection method using Folin-Ciocalteau reagent (FI-FC).
2. Material and Methods
2.1. Reagents and Solutions
All reagents were analytical grade and used water was
distilled.
Fluorescein (Sigma-Aldrich) and 2,2’-azobis(2-methyl-
propionamidine) dihydrochloride, 97% (AAPH) from
Sigma-Aldrich were used by the ORAC method. A fluo-
rescein stock solution (10 µg·mL1) was prepared in a
0.05 M pH 7 phosphates buffer. 0.03 µg·mL1 fluorescein
solutions to obtain the kinetics curves were prepared
from the stock solution using the same buffer. A 0.22 M
AAPH solution was also used. A 1:10 dilution from Fo-
lin-Ciocalteau reagent (Merck) and a 0.5 M NaOH (Ba-
ker) solution were used by the FI-FC method. Caffeic
acid (Sigma-Aldrich) was used as reference to determine
the AC by both methods.
Standard solutions obtained from a 1000 µg·mL1 va-
nillin (Sigma-Aldrich) stock solution were used to build
the vanillin calibration curve. Vanillic acid, p-hydroxy-
benzaldehyde and p-hydroxybenzoic acid were also pur-
chased by Sigma-Aldrich.
A 70:30 ethanol (Baker): water mixture was also used
to obtain the vanilla extracts.
2.2. Instruments
A fluorimeter PMT-FL Fialab and a thermostated bath
Lab-line 1800 were used to determine the AC by the
ORAC method. A peristaltic pump (Gilson minipuls), an
injection valve (Rheodyne), Teflon and Tygon tubing, a
Hellma 18 µL internal volume flow cell and a Cary UV-
Vis spectrophotometer (Varian) as detector were used to
build the FI manifold. A FI manifold reported by Val-
déz and Cañizares [7] was used to determine vanillin.
A 300 W maximum power focused microwaves oven
(Prolabo) was used to obtain the vanilla extracts. An ul-
trasonic bath (Branson Model 2510R), a centrifuge
(HETTICH EBA 20) and a pHmeter (Oakton) were also
used. A controlled temperature oven (Rios Rocha) was
used for the moisture assay.
2.3. Samples
All vanilla samples were Mexican. Two different cured
vanilla beans quality were analyzed: a) gourmet quality,
from Papantla, Veracruz: 15 cm length, dark brown col-
our and glossy beans; b) second quality (named zacatillo)
from Ayotoxco, Puebla, used to make handicrafts: from
11 cm to 17 cm length, light brown colour with clear
stripes beans. Green beans from Papantla, Veracruz were
also used.
Gourmet vanilla was purchased in a local market from
Papantla. A Cooperative of Women from Ayotoxco, Pue-
bla, who cultivate, harvest and cure vanilla, gave the Za-
catillo vanilla beans.
For the analyses a stock of 500 g of each kind of va-
nilla beans was used. Beans were cut to a size of 2 mm ×
2 mm and homogenized. They were stored in refrigera-
Copyright © 2013 SciRes. FNS
Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves
246
tion until were used.
Vanilla green beans from Ayotoxco were also ana-
lyzed. The beans were collected and stored at 4˚C until
analysis.
2.4. Getting of the Extracts
Focused microwaves-assisted extraction (FMAE) [29]: 1
g of cut vanilla beans (2 × 2 mm) was poured into a 10
cm test tube of 3 cm of internal diameter, which was
placed into a water bath (test tube of 4.5 cm of internal
diameter). 25 ml of a 70% (v/v) ethanol-water solution
were added to the sample. A refrigerant was adapted to
the test tube to avoid loss of the extractant and a 150 W
microwaves irradiation power was applied to the sample.
Twenty cycles of 1 min irradiation each one with a delay
time between them of 3 min were carried out. The total
extraction time was 80 min. Once the extraction was over,
solution was filtered and water was added until reaching
a final volume of 100 mL.
Maceration extraction (ME) [29,30]: 1 g of cut vanilla
beans (2 × 2 mm) was poured into a 10 ml volumetric
flask covered with 2 ml of ethanol and 1 ml of water. The
solution was macerated for 12 hours. 2 ml of ethanol
were added mixing well all the content. Maceration con-
tinued for 3 days. The solution was drained funnel dry,
packing solids firmly and percolating slowly with a 50%
ethanol solution until reaching a final volume of 10 ml.
2.5. Determination of the Antioxidant Capacity
(AC) in Extracts of Vanilla Beans
2.5.1. ORAC Method
It was used, with some modifications, for the determina-
tion the AC in the vanilla extracts obtained from the va-
nilla beans. 2 mL of the 0.03 µg·mL1 fluorescein (FL)
solution, 2 mL of blank (phosphates buffer (0.5 M, pH 7)
and 1 mL of standard (caffeic acid) or sample (diluted
1:1000) were set into a 13 × 100 cm testing tube. During
15 min were incubated at 37˚C. Later 1 mL of AAPH
was added and then the fluorescence intensity was meas-
ured setting this moment as cero time (t0). After 5 min of
a new incubation step the signal was measured again.
This procedure was repeated until fluorescence intensity
decayed at 20% from initial value. The fluorescence in-
tensity was measured λex = 486 nm and λem = 500 nm.
The measurements were carried out in triplicate for each
sample and standard.
A calibration curve was obtained by plotting the total
area under the curve (AUC) against caffeic acid concen-
trations in the 1 µg·mL1 - 6 µg·mL1 range. The area
under the kinetic curve (AUC) was calculated for each
standard and from these values the total area under the
kinetic curve (AUCTotal). In Figure 1 the integration
ranges and the fluorescence intensity decreasing curve
are shown. The trapezoid method according to Equation
(1) was used to calculate the AUC;
05
510 5
()
AUC 5
2
()
5... 5
22
nn
yy
yy y y






 


(1)
where y0 is the initial fluorescence signal and y5, y10,
yn-5, · , yn are the fluorescence intensity values at different
incubation time: 5, 10, n 5, ··· , n minutes.
AUCTotal was solved from Equation (2):
Total ANTIOXIDANT BLANK
AUC AUC AUC
(2)
2.5.2. FI-FC Method
This method has been also used to determine AC en dif-
ferent kind of food. FI methods are faster than batch
method and it has been proved that they are very precise.
Folin-Ciocalteau reagent has been used for phenolic
compounds analysis both by FI [31] and by batch meth-
ods obtained a very good precision [19,32]. In Figure 2
the used FI manifold is shown. 100 µL of sample or caf-
Blank
AUC
Sample
-AUC
Blank
Sample
(0, y
0
)
FluorescenceIntensity
Incubation time (min)
0 5 15
(5.y
5
)
(10, y
10
)
(n-5, y
n-5)
(n, y
n
)
n-5 n
Figure 1. Trapezoid method (area under the curve (AUC))
to determine the antioxidant capacity (AC) using the ORAC
method.
carrier
FC
NaOH
sample
IV
760 nm
reactor
PP
Figure 2. Flow injection manifold to determine the antioxi-
dant capacity (AC) using the Folin-Ciocalteau reagent (FI-
FC). PP: peristaltic pump; IV: injection valve; FC: Folin-
Ciocalteau reagent (10%); carrier: distilled water; NaOH:
0.5 M; reactor: 100 cm.
Copyright © 2013 SciRes. FNS
Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves 247
feic acid standard were injected into the carrier (distilled
water) to merge with Folin-Ciocalteau reagent and later
with a 0.5 M NaOH stream. The formed complex was
measured at 760 nm. Extracts were diluted 1:500 to mea-
sure the AC in the vanilla.
The precisions of the methods were calculated by de-
termination of the antioxidant capacity of six independ-
ent aliquots of the sample for each evaluated method.
2.5.3. Determination of Vanillin
A flow injection manifold was used to quantify vanillin
in accordance with Valdez and Cañizares [7]. 100 µl of
sample were injected into a carrier of 0.01 N NaOH,
which carries the sample to the reactor where the vanillin
was hydrolyzed. Finally, the hydrolyzed plug passed
through a flow cell located in the UV-VIS spectropho-
tometer, where the product was measured at 347 nm.
2.6. Determination of Moisture in Cured Vanilla
Beans
The procedure was carried out in accordance with the
established in the American Society for Testing and Ma-
terial [33]. From one and five grams of the sample were
weighed and placed into a previously dried container,
which was covered and then weighed again. Later, the
stopper was removed from the container and both were
placed in the oven. The sample was dried for 4 h at
105˚C ± 3˚C. At the end of the specified period, the con-
tainer was quickly covered and placed in a desiccator
during 1 h. After this time the sample was weighed. The
drying and weighing procedures were repeated until the
mass loss between two successive weightings was not
more than 0.005 g (or until the specimen showed a gain
in mass). The moisture was calculated by using the fol-
lowing equation:


Moisture content 100
MD
MT





where:
M = original mass of the specimen and weighing bottle
D = oven-dry mass of the specimen
T = mass of the empty weighing bottle
3. Results and Discussion
3.1. Characteristics of the Quantification
Methods
It has been widely accepted that the radical system used
for the antioxidant evaluation may influence the experi-
mental results [34]. There are many methods of analysis
to determine the antioxidant capacity and radical scav-
enging activity in food (iron(II) chelating activity, total
antioxidant capacity, DPPH assays and lipid peroxidation)
and a number of different reagents have been used. Each
one of these methods has different values of AC depend-
ing on the kind of reaction and the analytical measure-
ment (fluorescence, UV-Vis, electrochemical, etc.) [35].
Although Trolox is the most used reagent by ORAC me-
thod due to its high sensibility, caffeic acid has been also
used because it has a high antioxidant power [36]. So, in
this paper all results have been expressed as mg caffeic
acid/g of vanilla bean.
3.1.1. ORAC Method
The calibration curve was built from the caffeic acid ki-
netics graphics. In Figure 3 is showed some signals of
three different concentrations of caffeic acid; some sig-
nals of vanilla extracts obtained by both extraction me-
thods are also showed. For blank, initial fluorescence in-
tensity falls down quickly because there is not an antioxi-
dant and the peroxiles radicals generated by thermic de-
composition of AAPH reacts with fluorescein obtaining a
non-fluorescent product. If the reaction is carried out
with an antioxidant the fall is not so quick therefore when
the caffeic acid concentration increases the lag phase is
larger, because caffeic acid fixes the peroxil radicals.
The linear range was from 1 µg·mL1 to 6 µg·mL1
with a lineal equation AUCTotal = 3589.7 (±179.70) [caf-
feic acid] + 1809.8 (±727.74) and a regression coefficient
of 0.9962. At concentration higher than 6 µg·mL1 the
decay of the fluorescence intensity in 20% was too long.
The detection limit (DL) was evaluated on the stan-
dard deviation of the response of the blank and the slope
using the ratio 3.3 s/m, where s is the standard deviation
of the response of the blank and m is the slope of the
calibration curve of the analyte. The detection limit was
0.81 µg·mL1 and the quantification limit using the ratio
10 s/m was 1.65 µg·mL1.
290
310
330
350
370
390
410
430
450
470
490
0 102030405060
Fluorescence Intensity
Time ( min)
Caffeic acid 1mg L-1 Caffeic acid 4mg L-1 Caffeic acid 5mg L-1
Blank FMAE1 FMAE2
ME1 ME2
Figure 3. Plot of fluorescence intensity signal vs time for
three caffeic acid standard solutions and for two vanilla
extracts obtained by focused microwaves assisted extraction
(FMAE1 and FMAE2) and by maceration extraction (ME1
and ME2).
Copyright © 2013 SciRes. FNS
Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves
248
3.1.2. FI-FC Method
The linear equation Abs = 0.011 (±0.0001) [caffeic acid]
+ 0.0085 (±0.0058) was obtained for a caffeic acid con-
centration range between 5 µg·mL1 and 100 µg·mL1.
The regression coefficient was 0.9998, the detection limit
of 3.55 µg·mL1 and the quantification limit of 6.35
µg·mL1.
3.2. Determination of Vanillin in Extracts
Extracts obtained by both methods were between light
and medium brown color (depending of the kind of bean),
with a vanilla characteristic odor. When focused micro-
waves were used the vanillin amount in the extracts ob-
tained from both kinds of beans increased. Once again it
was demonstrated that when focused microwaves are ap-
plied the vanillin extraction from vanilla beans increases:
for zacatillo beans between 75% and 87% and for gour-
met beans between 81% and 93%, comparing with the
obtained results by the official method (ME). For green
beans the vanillin concentration was too low therefore
this was only determined when microwaves were used.
The results are showed in Table 1. In this same table mo-
isture of the beans is also showed. Moisture is a quality
control parameter for vanilla beans and it has to be be-
tween 25% and 30% for gourmet vanilla and between 20
and 24 % for vanilla of second quality [30]. The evalu-
ated beans were in these classifications. Although the va-
nilla concentration was higher when moisture in the va-
nilla beans was higher, it was not possible to conclude
that moisture higher increases the vanillin concentration
in the vanilla extracts. On the other hand, in a previous
paper was demonstrated that when cured vanilla beans
were dried at 135˚C for 4 h, the vanilla concentration de-
creased between 30% and 40% [29]. The waves of mi-
crowave are non-ionizant radiations that cause the mobil-
ity of the molecules due to the migration of ions and the
bipolar rotation. When the electric camp decreases the
thermic disorder that produce energy as heat is restored.
This law makes that the interaction between the analytes
in a matrix and the extractant is better and the extraction
is more efficient [22]. There are some examples of fo-
cused microwave extraction that improve the extraction
of polar and non-polar compounds in different matrixes
as the extraction of ketoprofen in pharmaceutical cream
formulation [37], the extraction of heterocyclic amines in
meat [38] or in order to clarify the possible hazard of aci-
dic pharmaceuticals (ibuprofen, naproxen, ketoprofen,
and diclofenac) in river water and sediments [39]. Using
microwave in solid samples allows more efficient and
faster sample treatment, so it is being more used in ana-
lysis laboratories. Microwaves are non-destructive ener-
gy, so can be applied in food under controlled radiation
which has allowed to be used, for example to accelerate
the oxidation process in olive oil [40].
On the other hand, several studies have demonstrated
that vanillin helps to the stability in the beans avoiding
oxidation, but it is necessary good conservation condi-
tions.
3.3. Antioxidant Capacity (AC) in Vanilla Beans
ORAC method is the most used method to measure the
AC in food because of its high sensitivity [15,41]. Nev-
ertheless there are a number of methods to determine the
AC where the found values are very different between
methods. For this reason, usually more than one method
is used to evaluate the AC in food [42,43].
Vanilla, specie, is mainly used as flavoring and it is
used in a very low amount in our daily diet but it could
help to get the daily recommended antioxidants. So, it is
important to develop extraction methods of improving of
the antioxidant power in the extracts, such as the focused
microwaves assisted extraction method evaluated in this
research. In Figure 3 is showed the signals obtained by
the ORAC method of different vanilla extracts obtained
by the maceration method (ME) and by the FMAE me-
thod. The signals of vanilla extracts show a large lag
phase which is indicative of a high AC but they are lower
when the extracts are obtained by the ME. The extracts
analyzed by the FI-FC method had values of AC lower
than by the ORAC method although by both methods the
AC increased when microwaves were applied. The re-
sults of the AC obtained by both methods are showed in
Table 2. The AC for gourmet beans was higher than for
zacatillo beans between a 55% and a 57%, using both
Table 1. Concentration of vanillin in extracts obtained by
focused microwaves (FMAE) and by maceration (ME). It is
also shown the moisture for each kind of bean.
Vanillin concentration (%, w/w) Moisture (%)
Sample
ME FMAE
Zacatillo 24.70 ± 0.20
1 0.62 ± 0.012 1.16 ± 0.03
2 0.57 ± 0.025 1.08 ± 0.02
3 0.65 ± 0.015 1.14 ± 0.03
Gourmet 27.80 ± 0.24
1 1.07 ± 0.020 2.02 ± 0.02
2 0.95 ± 0.015 1.81 ± 0.02
3 1.05 ± 0.031 1.96 ± 0.03
Green beans nd 0.27 ± 0.01 -
nd: no detected.
Copyright © 2013 SciRes. FNS
Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves 249
Table 2. Values of the antioxidant capacity (AC) by the
ORAC method and by the Flow Injection-Folin Ciocalteau
(FI-FC) method for vanilla extracts obtained by focused
microwave (FMAE) and by maceration (ME).
AC (expressed as mg caffeic acid g1 bean)
ORAC FI-FC
Sample
FMAE ME FMAE ME
Gourmet
1 77.22 ± 1.03 37.86 ± 1.65 35.74 ± 0.30 20.32 ± 0.13
2 69.93 ± 1.10 33.69 ± 1.42 32.33 ± 0.95 18.37 ± 0.36
3 75.13 ± 0.85 37.17 ± 2.05 34.77 ± 0.65 19.99 ± 0.44
Zacatillo
1 45.09 ± 0.87 24.74 ± 0.74 24.03 ± 0.16 12.18 ± 0.22
2 44.58 ± 0.78 20.52 ± 0.84 20.48 ± 0.56 12.20 ± 0.42
3 46.66 ± 1.00 23.27 ± 1.25 21.61 ± 0.34 12.52 ± 0.16
Green 6.70 ± 0.32 nd nd nd
nd. no detected.
analysis methods. Also, when the vanillin concentration
increased the AC does, so the highest AC was for ex-
tracts obtained using focused microwaves because they
had an amount of vanillin higher than when the macera-
tion method was used. The AC:vanillin ratio by the
ORAC method was between 35:1 and 41:1 and by the
FI-FC method was between 17:1 and 21:1, with an ex-
cellent correlation coefficient between vanillin concen-
tration and AC: of 0.994 for the ORAC method and of
0.996 for the FI-FC method (Figure 4). The AC by the
ORAC method increased between 82% and 117% in the
extracts obtained by focused microwaves in comparison
with the obtained by the maceration method; by the FI-
FC method the increased was between 72% and 97%.
The results show that the AC value is higher when mi-
crowaves are applied independently of the analysis me-
thod used.
F test was calculated to compare the standard devia-
tion from the two methods of extraction using the two
analysis methods, ORAC and FI-FC. The F test showed
that there was no difference in the variances of the me-
thods because experimental value (1.56) was lower than
critical value (4.026). So the precision was similar by
both methods. So t test was calculated based on equal va-
riance. The null hypothesis was: the AC is the same by
both methods, FMAE and ME. The results showed that
the values of the media were statistically different, so the
AC is higher when FMAE was used to obtain vanilla
extracts, t value was higher than the critical value (t9 =
2.26).
0
20
40
60
80
100
00.511.522.5
ORAC FI-FC
% vanillin
Antioxidant capacity
Figure 4. Correlation between the vanilla concentration and
the ORAC method () and the FI-FC method (). Antioxi-
dant capacity expressed as mg caffeic acid g bean1.
The vanillin concentration in green beans was very
low and therefore the AC, so it is shown the importance
of the vanillin in the in antioxidant capacity in cured va-
nilla beans. In green beans, vanillin is fixed to glucose as
glucovanillin and its antioxidant power is not strong
enough so green vanilla beans have a very poor AC.
On the other hand, the AC value is different between
an analysis method and another one for a same com-
pound, so when caffeic acid was used as standard, which
is a strong antioxidant, the vanilla AC values by the
ORAC method were approximately two times higher
than by the FI-FC method (Table 2) but by both methods
of analysis the AC increased or decreased in the same
proportion for the same extracts. This difference is due to
that vanillin is majority compound of vanilla and the
analytical response is not the same for the two methods.
The linear correlation between the ORAC and FI-FC me-
thods was 0.99. Different electrochemical methods for
the determination of antioxidant power in food have been
compared with DPPH and ABTS radical cation assays by
Buratti et al. [42,44]. The antioxidant power values are
different but the linear correlation among them have been
higher than 0.92.
So, a calibration curve using vanillin as standard to
react with the Folin-Ciocalteau was evaluated. The cali-
bration curve had a slope of 0.0081 mg·mL1 (1.36 times
lower than the slope for caffeic acid). For at same con-
centration (expressed as mmol·L1) the analytical signal
of vanillin was lower than for caffeic acid.
The AC of vanillin was also measure using the ORAC
method and was compared with others compounds of the
vanilla beans.
3.4. Antioxidant Capacity of Vanillin,
p-Hydroxybenzaldehyde (PHB), Vanillic
Acid and p-Hydroxibenzoic Acid (HBA) by
ORAC Method
Besides vanillin the vanilla beans have others compounds
which are extracted at the same time of vanillin. The
Copyright © 2013 SciRes. FNS
Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves
250
most important are p-HBA, vanillic acid and HBA. Va-
nillin is between 10 and 30 times more concentrated than
PHB and vanillic acid and the ratio increases for HBA
between 100 and 140 times.
Standards of PHB, HBA and vanillic acid to concen-
trations closed at the vanillin concentration in the diluted
extracts were tested. The vanillin concentration in the ex-
tracts obtained by focused microwaves from zacatillo
beans was of 467 mg·L1 so it was diluted 1000 times
and AC was determined, the vanillin concentration was
0.50 mg·L1 approximately. The others compounds were
analyzed at this same concentration. At 0.5 mg·L1 of
each compound the AC could be only measured for va-
nillin and for vanillic acid, obtaining 2.3 mg caffeic acid
L1 and 0.53 mg caffeic acid L1, respectability. The AC
of vanillin is 4.3 times higher than vanillic acid. PHB and
HBA did not show antioxidant capacity. This behavior is
because the antioxidant activity of phenolics acids and
their esters depends on the number of hydroxyl groups in
the molecule that would be strengthened by steric hin-
drance [45]. The electron withdrawing properties of the
carboxylate group in benzoic acids has a negative influ-
ence on the H-donating abilities of the hydroxyl benzo-
ates. The monohydroxy benzoic acids show no antioxi-
dant activity in the ortho and para positions in terms of
hydrogen-donating capacity against radical generated in
the aqueous phase but the m-hydroxy acid has antioxi-
dant activity. This is consistent with the electron with-
drawing potential of the single carboxyl functional group
on the phenol ring affecting the o- and p-positions. The
monohydroxy benzoates are, however, effective hydroxyl
radical scavengers, due to their propensity to hydroxyla-
tion and the high reactivity of the hydroxyl radical. With
a methylene group between the phenolic ring and the car-
boxylate group, as in the vanillin, the o- and m-hydroxy
derivatives have high antioxidant activities. Carried out a
study of AC of vanillin using different assays were car-
ried out by Akiro Tai et al. [46]. They showed that vanil-
lin had a stronger antioxidant capacity than ascorbic acid
and Trolox by the ORAC method but it showed no activ-
ity in the DPPH radical and galvinoxyl radical scaveng-
ing assays. By the FI-FC method a 5 mg·L1 concentra-
tion of each analyte was used. At this concentration only
vanillin and vanillic acid showed AC where the vanillin
concentration was 2.43 times higher than the vanillic acid
concentration (4.94 mg caffeic acid L1 vs 2.03 mg caf-
feic acid L1). The AC for 500 mg·L1 PHB and HBA
solutions was 4.53 mg caffeic acid L1 and 35.7 mg caf-
feic acid L1, respectively. So, the analysis of AC using
different method is suggested to assure the antioxidant
properties from a compound.
4. Conclusions
The AC value was different among analysis methods so
it is convenient to use more than one method to establish
the antioxidant power of the food. In this paper is shown
that the vanilla has a high AC independently of used me-
thod, but the obtained numeric values were different. So,
when caffeic acid is used as standard, which is a strong
antioxidant, the vanilla AC values by the ORAC method
were approximately two times higher than by the FI-FC
method but by both methods of analysis the AC increas-
ed or decreased in the same proportion for the same ex-
tracts. The linear correlation between the ORAC and FI-
FC methods was 0.99. Also the antioxidant power values
reported by Buratti et al. [42,44] were different but the
linear correlation among them was higher than 0.92.
When microwaves were applied the AC in the extracts
increased between 72% and 117% with a strong relation-
ship with the vanillin concentration. The vanillin concen-
tration in the obtained extracts by using microwaves in-
creased between 75% and 93%. So, it has been showed
that when microwaves are applied to obtain natural va-
nilla extracts the AC increased because the vanillin con-
centration is higher and it is the most influential com-
pound in the antioxidant power.
Vanilla, specie, is mainly used as flavoring and it is
used in a very low amount in our daily diet but it could
help to get the daily recommended antioxidants. So, it is
important to develop extraction methods of improving of
the antioxidant power in the extracts, such as the focused
microwaves assisted extraction method used. On the other
hand, several studies have demonstrated that vanillin helps
to the stability in the beans avoiding oxidation, but it is
necessary good conservation conditions.
The results showed that the vanilla AC is given, prin-
cipally, for the vanillin amount in the beans and in the
extracts. They also showed that green vanilla beans have
a very poor AC.
5. Acknowledgements
The Faculty of Chemistry and the Programa de Apoyo a
Proyectos de Investigación e Innovación Tecnológica,
PAPIIT (grant No. IT202812-3) of the Universidad Na-
cionalAutónoma de México are gratefully acknowledged
for financial support. The Ayotoxco vanilla beans were
supplied by the Ayotoxco farmers with the support of the
InstitutoPoblano de Desarrollo Rural A.C.
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Antioxidant Capacity in Vanilla Extracts Obtained by Applying Focused Microwaves 253
Abreviations
AC: Antioxidant Capacity
AAPH: 2,2’-azobis(2-methylpropionamidine) dihydro-
chloride
AUC: Area under Curve
FI-FC: Flow Injection-Folin Ciocalteau method
FMAE: Focused Microwaves-Assisted Extraction
HBA: p-hydroxybenzoic acid
ME: Maceration Extraction
ORAC: Oxygen Radical Absorbance Capacity
PHB: p-hydroxybenzaldehyde
Copyright © 2013 SciRes. FNS
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