agronomy

Article
Optimization of Microwave-Assisted Extraction for
the Recovery of Bioactive Compounds from the
Chilean Superfruit (Aristotelia chilensis
(Mol.) Stuntz)
Mercedes Vázquez-Espinosa 1 , Estrella Espada-Bellido 1, * , Ana V. González de Peredo 1 ,
Marta Ferreiro-González 1 , Ceferino Carrera 1,2 , Miguel Palma 1 , Carmelo G. Barroso 1 and
Gerardo F. Barbero 1
1 Department of Analytical Chemistry, Faculty of Sciences, University of Cadiz, Agrifood Campus of
International Excellence (ceiA3), IVAGRO, 11510 Puerto Real, Cadiz, Spain;
mercedes.vazquez@uca.es (M.V.-E.); ana.velascogope@uca.es (A.V.G.d.P.); marta.ferreiro@uca.es (M.F.-G.);
ceferino.carrera@uca.es (C.C.); miguel.palma@uca.es (M.P.); carmelo.garcia@uca.es (C.G.B.);
gerardo.fernandez@uca.es (G.F.B.)
2 Andalusian Center for Wine Research, Agrifood Campus of International Excellence (ceiA3), IVAGRO,
University of Cadiz, P.O. Box 40, 11510 Puerto Real, Cadiz, Spain
* Correspondence: estrella.espada@uca.es; Tel.: +34-956-016355




Received: 15 October 2018; Accepted: 26 October 2018; Published: 28 October 2018


Abstract: Maqui berry (Aristotelia chilensis) is being explored in the food industry, and is considered
to be one of the healthiest berries due to its bioactive components and high commercial value.
Microwave-assisted extraction (MAE) was developed for the determination of total phenolic
compounds and anthocyanins from maqui. A Box–Behnken experimental design was employed in
conjunction with a response surface methodology to optimize the conditions based on 27 different
experiments. The extractions were carried out with four factors (i.e., methanol percentage, pH,
temperature, and solvent volume:sample mass ratio), and two responses—total phenolics and
anthocyanins. Temperature and methanol percentage were found to be the most influential
parameters for total phenolic compounds and anthocyanins, respectively. The optimum MAE
conditions were: 65% MeOH in water at pH 2, temperature of 100 ◦ C, and a ratio of 10:0.5 for
total phenolics; and 60% MeOH in water at pH 2, temperature of 50 ◦ C, and a ratio of 14:0.5 for
anthocyanins. Kinetics assays were carried out and an optimum time of only 2 min was identified
for the extractions. Repeatability and intermediate precision were also evaluated, and coefficients of
variation below 5% were obtained. The new methods were successfully applied to a foodstuff made
with maqui.

Keywords: anthocyanins; Aristotelia chilensis (Mol.) Stuntz; food analysis; maqui berry;
microwave-assisted extraction; phenolic compounds; superfruit

1. Introduction
There is growing interest within the population in the consumption of foods that are rich in
health-promoting compounds and in an increasing incorporation of vegetables and fruits into the diet
to improve quality of life [1]. As a consequence, research effort in food science has focused on the intake
of fruits such as berries, which are rich in nutrients and can prevent several diseases and disorders.
In fact, food and pharmaceutical industries have shown increasing attention in recent years to the
development of new formulations with integrated berry extracts as a source of bioactive compounds

Agronomy 2018, 8, 240; doi:10.3390/agronomy8110240 www.mdpi.com/journal/agronomy

Agronomy 2018, 8, 240 2 of 15

with high antioxidant capacity [2–4]. Maqui (Aristotelia chilensis (Mol.) Stuntz) is a small tree belonging
to the family Elaeocarpaceae and it is native to the center and south of Chile and southwestern
Argentina. The fruit of this tree contains one of the highest concentrations of antioxidants in the world,
far surpassing other highly recognized fruit species [5–7]. In December and January the tree produces
purple/black berries that are around 6 mm in diameter. The collection of this fruit is attributed to the
Mapuche Indians of South America, who have consumed and used it as a natural remedy for thousands
of years. In traditional herbal medicine, infusions of maqui berries have been used to treat sore throats,
kidney complaints, digestive ailments, ulcers, fevers, and scarred wounds [7]. At present, the fruit of
the maqui is highly appreciated for its characteristic flavor and aroma, in addition to its innumerable
beneficial properties such as the prevention of Alzheimer’s disease, cardiovascular diseases, and the
oxidation of low-density lipoproteins, analgesic, anti-inflammatory, anti-carcinogenic, anti-oxidant,
and anti-diabetic properties, and also for the control of obesity [8–10]. It is now known that the excellent
properties of this fruit are due to the fact that they are an extremely rich source of bioactive compounds
such as phenolic acids, tannins, stilbenes, flavonoids, and anthocyanins [6,10,11]. It is therefore not
surprising that maqui has recently been recognized as a “superfruit” [5,12]. Maqui berries are being
explored in the pharmaceutical, food, and cosmetics industries as a potential source of bioactive
compounds. Therefore, its consumption in both fresh and processed forms such as juice concentrates,
liqueurs, pills, capsules, lyophilized, beverages, jams, and jellies, is highly recommended [2].
The main phenolic compounds that have been identified in this fruit are gallic acid,
hexahydroxydiphenic acid, granatine B, punicacortein C, flavonols (myricetin, quercetin, kaempferol,
and its derivatives) and eight anthocyanins, which are delphinidin 3-O-sambubioside-5-O-glucoside,
delphinidin 3,5-O-diglycoside, cyanidin 3-O-sambubioside-5-O-glucoside, cyanidin 3,5-O-diglycoside,
delphinidin 3-O-sambubioside, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, and cyanidin
3-O-sambubioside [6,13], and these are responsible for its intense color and high antioxidant
potential [14].
Maqui is considered to be one of the healthiest fruits, but it is still an understudied and relatively
unknown berry. Although it has recently been commercialized, only a few references can be found in
the literature, so it is necessary to develop extraction and analysis methodologies for this fruit [13,15].
The development of such methodologies is essential for quality control, as food fraud is a
serious and increasing problem in the food industry [16,17]. Due to the high price of this berry,
the replacement of the fruit with cheaper similar berries or cheaper fruit juices as diluting agents
should not be ignored [18,19]. The extraction technique applied is the key step for the appropriate
recovery and to develop an efficient process. Thus, the availability of optimized extraction methods
for this fruit could help in the detection of possible food fraud. In recent years, there has been an
increase in the number of publications focused on developing new extraction methods for bioactives
from plants [20,21]. Among the extraction methodologies employed, ultrasound-assisted extraction
(UAE) and microwave-assisted extraction (MAE) are considered to be excellent green alternatives to
conventional methods for the extraction of bioactive compounds due to their low instrument set-up
costs on a laboratory scale [21–24].
Microwave-assisted extraction is considered to be an effective method for the extraction of
compounds from solid samples [25]. Microwaves are non-ionizing electromagnetic (EM) waves.
Microwaves consist of electric and magnetic fields which oscillate perpendicularly to each other in
high frequency range from 0.3 to 300 GHz. Microwave power provides localized heating in the
sample and acts as a driving force for the destruction of the plant matrix so the analyte of interest can
diffuse easily and dissolve in the solvent. The transfer of energy occurs through two mechanisms:
the inversion of dipoles and the displacement of charged ions [26]. One of the main differences from
other techniques is that instead of acting through conventional heating, microwave irradiation is a
“cold” technique in which the heating occurs at the core of the object and extends from the inside of
the body. In recent years, microwave extraction has been developed greatly due to the reduction in the
extraction time and the amount of solvents, the degree of automation, and its high performance [27].
Agronomy 2018, 8, 240 3 of 15

This technique has been used to extract antioxidant substances from a wide variety of matrices, such as
grapes [28], tomatoes [29], myrtle [30], or basil [31], amongst others [21].
The efficiency of the extraction process can be affected by various factors, such as the type
and volume of solvent, pH, and temperature. Solvent plays an important role in the solubility
of the phenolic compounds. In fact, hydroalcoholic mixtures with similar polarity of the phenolic
compounds are normally employed in the literature [32,33]. Regarding pH and temperature, both must
be controlled due to the degradation processes at certain conditions [34]. Therefore, it is necessary to
optimize the different variables of the extraction method to achieve maximum production yield [35].
In the study described here, a Box–Behnken design was carried out. This is a type of response surface
factorial design that allows the reduction of the number of experiments compared to other statistical
designs, while ensuring maximum possible information on the system response, in addition to an
adjustment with quadratic models [36]. The results were treated by the response surface methodology,
which generates a mathematical model that is adjusted to the experimental responses obtained and
this allows the effects of the independent variables to be analyzed. The optimum values of these
variables were obtained by solving the mathematical equation and the evaluation of the response
surface plots [37,38].
To the best of our knowledge, this is the first time that the microwave-assisted extraction of
anthocyanins and phenolic compounds from maqui has been developed. Thus, the results described
here offer efficient, green, and low instrument set-up methods to extract the bioactive compounds from
maqui berries, which is of great interest in the food industry due to their high commercial value and
the increasing demand related to the healthy properties of this fruit.

2. Materials and Methods

2.1. Biological Material

The extraction methods were developed using commercially available lyophilized maqui
as a powder from organic farming (SuperAlimentos, Mundo Arcoíris, Besalú, Girona, Spain).
The suitability of the final extraction method was evaluated by analyzing several real samples
containing maqui commercialized in different formats, including capsules, pills, and lyophilized
material. The lyophilized maqui sample and the commercial real samples were stored in a freezer at
−20 ◦ C prior to analysis.

2.2. Chemicals and Solvents

Methanol (Fisher Scientific, Loughborough, UK) and formic acid (Scharlab, S.L., Sentmenat,
Barcelona, Spain) were HPLC grade. Ultra-pure water was supplied by a Milli-Q water purification
system from EMD Millipore Corporation (Bedford, MA, USA). Hydrochloric acid (Panreac Química
S.A.U., Castellar del Vallés, Barcelona, Spain) and sodium hydroxide (Panreac Química S.A.U.,
Castellar del Vallés, Barcelona, Spain) used for the adjustment of pH were of analytical grade.
For the quantification of the total phenolic compounds, distilled water, Folin–Ciocalteu reagent
(Merck KGaA, EMD Millipore Corporation, Darmstadt, Germany), and anhydrous sodium carbonate
(Panreac Química S.A.U., Castellar del Vallés, Barcelona, Spain) were used. The phenolic compound
standard and the anthocyanin standard, gallic acid and cyanidin chloride, respectively, were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

2.3. Extraction Equipment and Procedure

The extraction of maqui by microwave-assisted extraction was carried out using a MARS 6
240/50 kit (One Touch Technology, CEM Corporation, Matthews, NC, USA). Approximately 0.5 g of
lyophilized sample was weighed and placed in an extraction vessel. Based on the experimental design,
a set volume and type of solvent was added, and the extraction was performed under controlled MAE
conditions. The variables to be controlled in the different experiments were: percentage of methanol
Agronomy 2018, 8, 240 4 of 15

(25–50–75%), pH (2–4.5–7), temperature (50–75–100 ◦ C) and solvent volume (mL): sample mass (g)
ratio 10:0.5–15:0.5–20:0.5. The program carried out consisted of a ramp of 3 min to reach the desired
temperature. The temperature was then maintained for 5 min for the extraction of the compounds,
and a time of 25 min was employed to allow the sample to cool to room temperature. In all experiments,
the power was set at 800 W, as this was sufficient to reach the appropriate temperatures. At the end of
the extraction, the extract was centrifuged twice for 5 min at 11,544 × g. In both cases, the supernatant
was transferred to a 25 mL volumetric flask and the volume was completed with the same solvent.
Finally, the extracts were stored in a freezer at −20 ◦ C prior to analysis.

2.4. Determination of Total Phenolic Compounds

The quantification of the total phenolic compounds was carried out using a Helios Gamma
(γ) Unicam UV–Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) by a
modified Folin–Ciocalteu (FC) procedure [39,40]. This method has been employed previously by
numerous researchers for the measurement of total phenolic compounds [41–43], and is based on the
reactivity of the phenolic compounds present in the sample with the reagent (a mixture of sodium
phosphomolybdate and sodium phosphotungstate). This reaction gives a complex with a blue color
and this absorbs at a maximum wavelength of 765 nm. This absorbance is directly proportional to the
concentration of polyphenols, as related by the Lambert–Beer law. The extracts were filtered through a
0.45 µm syringe filter (Nylon Syringe Filter, FILTER-LAB, Barcelona, Spain) prior to spectrophotometric
analysis. The FC assay was performed by transferring 250 µL of extract, 12.5 mL of distilled water,
1.25 mL of Folin–Ciocalteu reagent, and 5 mL of 20% anhydrous sodium carbonate to a volumetric
flask (25 mL). The solution was made up to the mark with water. Due to the kinetics of the reaction,
it was necessary to measure the absorbances in the spectrophotometer at 765 nm after a time of 30 min.
It was necessary to create a calibration curve under the same conditions and by the same procedure,
with standards of gallic acid of known concentrations between 100 and 2000 mg L−1 and measuring
the absorbance values (y = 0.0010x + 0.0065; R2 = 0.9998). Results are expressed as mg of gallic acid
equivalent per gram of lyophilized fruit.

2.5. Identification of Anthocyanins

The anthocyanins were identified by ultra-performance liquid chromatography (UHPLC), on a
chromatograph coupled to a quadrupole-time-of-flight mass spectrometer (QToF-MS) (Xevo G2 QToF,
Waters Corp., Milford, MA, USA). The chromatographic separation was performed on a reverse phase
C-18 analytical column (Acquity UHPLC BEH C18, Waters Corporation, Milford Massachusetts, MA,
USA) with dimensions of 100 mm × 2.1 mm and a particle size of 1.7 µm. The extracts were filtered
through a 0.22 µm syringe filter (Nylon Syringe Filter, Filtros Anoia, S.A., FILTER-LAB, Barcelona,
Spain) prior to chromatographic analysis. Milli-Q water acidified with 2% formic acid as solvent A
and pure methanol as solvent B, both degassed and filtered, and a flow rate of 0.4 mL min−1 were
used. The elution gradient employed was as follows: 0 min, 15% B; 3.30 min, 20% B; 3.86 min, 30% B;
5.05 min, 40% B; 5.35 min, 55% B; 5.64 min, 60% B, 5.94 min, 95% B; 7.50 min, 95% B. The total
run time was 12 min, including 4 min for re-equilibration. The determination of the analytes was
carried out using an electrospray source operating in positive ionization mode under the following
conditions: desolvation gas flow = 700 L h−1 , desolvation temperature = 500 ◦ C, cone gas flow =
10 L h−1 , source temperature = 150 ◦ C, capillary voltage = 700 V, cone voltage = 20 V, and trap collision
energy = 4 eV. Full-scan mode was used (m/z = 100–1200). Molecular ions [M]+ for the anthocyanins
identified in the maqui showed the following m/z ratios: delphinidin 3-O-sambubioside-5-O-glucoside,
759; delphinidin 3,5-O-diglucoside, 627; cyanidin 3-O-sambubioside-5-O-glucoside, 743; cyanidin
3,5-O-diglucoside, 611; delphinidin 3-O-sambubioside, 597; delphinidin 3-O-glucoside, 465; cyanidin
3-O-glucoside, 449 and cyanidin 3-O-sambubioside, 581. These anthocyanins are shown in Figure 1.
Agronomy 2018, 8, 240 5 of 15
Agronomy 2018, 8, x FOR PEER REVIEW 5 of 15

2.6. Separation and

2.6. Separation and Quantification
Quantification of
of Anthocyanins
Anthocyanins
The
The separation
separationofofthe anthocyanins
the anthocyanins waswascarried out by
carried outUHPLC
by UHPLC on anon Elite
anLaChrom Ultra System
Elite LaChrom Ultra
(VWR Hitachi, Tokyo, Japan) consisting of an L-2200 U autosampler,
System (VWR Hitachi, Tokyo, Japan) consisting of an L-2200 U autosampler, an L-2300 column an L-2300 column oven set
oven
at ◦
set50
at 50C,°C,
twotwo
L-2160
L-2160 U pumps,
U pumps, and a UV–Vis
and a UV–Vis L-2420
L-2420UUdetector.
detector.The
Thedetector
detector was
was setset at 520 nm,
at 520 nm,
as this corresponds to the maximum absorption of the anthocyanins. Anthocyanins
as this corresponds to the maximum absorption of the anthocyanins. Anthocyanins were analyzed were analyzed
on
on aa Fused-Core
Fused-Core C-18 C-18 column (Phenomenex Kinetex,
column (Phenomenex Kinetex, CoreShell
CoreShell Technology,
Technology, Torrance,
Torrance, CA, CA, USA),
USA),
with
with dimensions
dimensions of 100 ×
of 100 2.1 mm
× 2.1 mm andand aa particle
particle size
size of
of 2.6
2.6 μm. The extracts
µm. The extracts were
were filtered
filtered through
through aa
0.22
0.22 μm syringe filter (Nylon Syringe Filter, FILTER-LAB, Barcelona, Spain) and the injection volume
µm syringe filter (Nylon Syringe Filter, FILTER-LAB, Barcelona, Spain) and the injection volume
used
used was
was 15 μL. The
15 µL. The gradient
gradient method
method used
used 5%
5% acidified
acidified Milli-Q
Milli-Q water
water with
with formic
formic acid
acid as
as solvent
solvent A A
and
and pure
pure methanol
methanol as as solvent
solvent B, B, previously
previously filtered
filtered using
using aa 0.22
0.22 μm filter (Nylon
µm filter (Nylon Membrane
Membrane Filter,
Filter,
FILTER-LAB, Barcelona, Spain) and degassed in an ultrasonic bath (Elma
FILTER-LAB, Barcelona, Spain) and degassed in an ultrasonic bath (Elma S300 Elmasonic, Singen, S300 Elmasonic, Singen,
Germany). The flow −1 and the gradient was as follows: 0.0 min, 2% B; 2.0 min,
Germany). The flow rate
rate was
was 0.7
0.7 mL
mL min
min−1, ,and the gradient was as follows: 0.0 min, 2% B; 2.0 min,
2% B; 3.5 min, 15% B; 5.5 min, 25% B;
2% B; 3.5 min, 15% B; 5.5 min, 25% B; 6.5 min, 40%6.5 min, 40% B; B;
7.07.0
minmin 100%
100% B; 9.3
B; 9.3 min,min,
100% 100% B; 10.0
B; 10.0 min,min,
2%
2% B; 12.0 min, 2% B. The anthocyanins were quantified from a calibration curve,
B; 12.0 min, 2% B. The anthocyanins were quantified from a calibration curve, using cyanidin chloride using cyanidin
chloride as a reference
as a reference standard.standard.
The standardsThe standards of known concentrations
of known concentrations were prepared werebetween
prepared 0.05between
and 30
0.05 and 30 mg L −1 (y = 252,638.09x 2− 28,465.10; R2 = 0.9999). The limit of detection (0.179 mg L−1 )
mg L (y = 252,638.09x − 28,465.10; R = 0.9999). The limit of detection (0.179 mg L ) and quantitation
−1 −1

and quantitation
(0.597 mg L−1) were (0.597 mg L−1 )aswere
determined determined
the analyte as the analyte
concentration concentration
corresponding corresponding
to the standard deviationto the
standard deviation
of the signal of the of the signal
blank valuesof(nthe blank
= 10) values
plus 3 or 10(n = 10) plus
times, 3 or 10 times,
respectively, respectively,
divided divided
by the slope by
of the
the slope of the linear regression. Assuming that the different anthocyanins have
linear regression. Assuming that the different anthocyanins have similar absorbances, and taking into similar absorbances,
and taking
account theinto account the
molecular molecular
weights weights
of each of each anthocyanin,
anthocyanin, a calibrationa calibration
curve was curve preparedwas prepared
for each
for each anthocyanin present in maqui, which allowed the quantification
anthocyanin present in maqui, which allowed the quantification of the compounds of interest.of the compounds of interest.

Anthocyanins Present in the Maqui Berry R1 R2 R3 R4

Delphinidin 3-O-sambubioside-5-O-glucoside OH OH C11H19O9 C6H11O5
Delphinidin 3,5-O-diglucoside OH OH C6H11O5 C6H11O5
Cyanidin 3-O-sambubioside-5-O-glucoside OH H C11H19O9 C6H11O5
Cyanidin 3,5-O-diglucoside OH H C6H11O5 C6H11O5
Delphinidin 3-O-sambubioside OH OH C11H19O9 H
Delphinidin 3-O-glucoside OH OH C6H11O5 H
Cyanidin 3-O-glucoside OH H C6H11O5 H
Cyanidin 3-O-sambubioside OH H C11H19O9 H

Figure
Figure 1.
1. Radicals
Radicals of
of the
the different
different anthocyanins
anthocyanins present
present in the maqui
in the maqui berry.
berry.

2.7. Box–Behnken Design

2.7. Box–Behnken Design Experiments
Experiments (BBD)
(BBD)
BBD is a spherical response surface methodology in which all the limit points are at a distance
of the square root root of
of 22 from
from the
the center
center ofof the
the design.
design. This specific design avoids the need for
experiments under extreme conditionsconditions andand allows
allows optimal
optimal results
results to
to be
be obtained
obtained quickly
quickly [44,45].
[44,45].
During
During thetheoptimization
optimization procedure, twotwo
procedure, typestypes
of variables can be distinguished:
of variables independent
can be distinguished: variables
independent
or factorsor
variables (i.e., percentage
factors of methanol,
(i.e., percentage pH, temperature,
of methanol, and solvent
pH, temperature, volume:sample
and solvent mass ratio)
volume:sample mass
and
ratio)dependent
and dependent variables or responses
variables (i.e.,(i.e.,
or responses concentration
concentrationof of
total phenolic
total phenoliccompounds
compoundsand and total
anthocyanins). Each Eachindependent
independent variable
variable cancantake take three
three possible
possible values,
values, coded as −1,as0,−
coded 1, 0,
and 1, and
which1,
which are minimum, average, or maximum values, respectively. Therefore, the
are minimum, average, or maximum values, respectively. Therefore, the design indicates the need todesign indicates the
need
carry to
outcarry out 27 experiments,
27 experiments, which werewhich were performed
performed in duplicate,
in duplicate, to identifyto identify
the optimalthe
MAE optimal MAE
conditions
conditions to obtain the highest concentration of phenolic compounds
to obtain the highest concentration of phenolic compounds and anthocyanins. and anthocyanins.
The response of the total phenolic compounds and anthocyanins obtained in each of the
experiments was introduced into a second-order polynomial equation in order to correlate the
relationship between the independent variables and the response [38]:
Agronomy 2018, 8, 240 6 of 15

The response of the total phenolic compounds and anthocyanins obtained in each of the
experiments was introduced into a second-order polynomial equation in order to correlate the
relationship between the independent variables and the response [38]:

k k
y = β0 + ∑ βi ·xi + βii ·x2i + ∑ ∑ βij ·xi xj + r, (1)
i=1 i i=1

where y is the response; βi is the coefficient for each main effect; βij is the coefficient corresponding to
the interactions i, j; βii is the coefficient of the quadratic factors that represents the curvature of the
surface; x represents each factor; and r is the residual value.
The statistical significances of the model, lack of fit, and regression terms were evaluated from
the analysis of variance (ANOVA). The fitting quality of the polynomial model was evaluated by the
use of the determination coefficient (R2 ). The Design Expert software 11 (Trial Version, Stat-Ease Inc.
statistic made easy, Minneapolis, MN, USA) was employed for experimental design, data analysis,
and model building.

3. Results and Discussion

3.1. Development of the MAE Method

In order to identify the optimum extraction conditions for phenolic compounds and anthocyanins,
a three-level Box–Behnken design was used. The measured and predicted results for each trial for
both responses are shown in Table 1. The four independent variables and their corresponding levels
were: percentage of methanol in water (%): 25–50–75; pH: 2–4.5–7; temperature (◦ C): 50–75–100;
and solvent volume (mL): sample mass (g) ratio 10:0.5–15:0.5–20:0.5. The concentrations of total
phenolic compounds and anthocyanins extracted in each of the experiments were considered as the
response variables.

Table 1. Box–Behnken design matrix with coded variables and measured and predicted responses.

Factors Responses
Run Solvent pH Temp. Ratio Phenolic Compounds (mg g−1 ) Anthocyanins (mg g−1 )
X1 X2 X3 X4 Measured Predicted Measured Predicted
1 −1 −1 0 0 46.32 45.49 27.92 28.85
2 1 −1 0 0 49.95 50.81 37.70 39.48
3 −1 1 0 0 38.06 34.63 28.72 28.04
4 1 1 0 0 46.88 45.12 38.22 38.39
5 0 0 −1 −1 46.72 47.69 42.75 39.92
6 0 0 1 −1 62.03 60.15 20.20 26.20
7 0 0 −1 1 51.13 50.44 43.04 38.14
8 0 0 1 1 60.60 57.06 30.15 34.07
9 0 0 0 0 45.93 43.87 43.68 41.25
10 −1 0 0 −1 39.60 36.36 26.63 27.65
11 1 0 0 −1 57.18 49.82 37.57 37.61
12 −1 0 0 1 39.20 41.75 29.01 30.17
13 1 0 0 1 45.67 44.09 41.03 41.19
14 0 −1 −1 0 56.85 50.67 39.07 43.71
15 0 1 −1 0 49.43 49.47 25.63 33.43
16 0 −1 1 0 72.13 67.28 32.09 25.48
17 0 1 1 0 50.57 51.94 37.31 33.86
18 0 0 0 0 43.30 43.87 40.14 41.25
19 0 −1 0 −1 46.86 54.42 38.26 36.91
20 0 1 0 −1 43.48 47.43 38.86 35.98
21 0 −1 0 1 52.10 55.54 39.38 39.98
22 0 1 0 1 46.15 45.98 39.96 39.01
23 −1 0 −1 0 35.61 37.32 32.56 30.13
24 1 0 −1 0 43.00 47.15 41.67 39.38
25 −1 0 1 0 45.55 48.78 20.00 19.99
26 1 0 1 0 49.08 54.76 31.59 31.72
27 0 0 0 0 42.39 43.87 39.94 41.25
Agronomy 2018, 8, 240 7 of 15

Firstly, an analysis of variance (ANOVA) was performed to evaluate the effect of the variables,
to identify possible interactions between them, and to assess the statistical significance of the model
(Table 2). From the results obtained, it was determined that the analysis explained 80.44% of total
variability in the case of total phenolic compounds and 78.78% in the case of anthocyanins. These values
are consistent with a statistically significant agreement between the measured and predicted responses.
This analysis provides information on the mathematical model that is generated from the experimental
data. The extraction of both types of compounds is related to the experimental conditions by a
second-order polynomial equation.

Table 2. Analysis of variance (ANOVA) for the response surface quadratic model of the recovery.
(A). Total phenolic compounds; (B). Total anthocyanins.

(A)
Source Degrees of Freedom Sum of Squares Mean Square F-Value p-Value Coefficient
Model 14 1337.37 95.53 3.52 0.0174 43.87
A-Solvent 1 187.39 187.39 6.91 0.0219 3.95
B-pH 1 205.34 205.34 7.58 0.0175 −4.14
C-Temp. 1 272.84 272.84 10.07 0.0080 4.76
D-Ratio 1 0.0867 0.0867 0.0032 0.9584 −0.08
AB 1 6.73 6.73 0.2485 0.6274 1.29
AC 1 3.72 3.72 0.1374 0.7177 −0.96
AD 1 30.86 30.86 1.14 0.3065 −2.78
BC 1 49.98 49.98 1.84 0.1993 −3.53
BD 1 1.65 1.65 0.0609 0.8093 −0.64
CD 1 8.53 8.53 0.3146 0.5894 −1.44
A2 1 78.93 78.93 2.91 0.1135 −3.85
B2 1 84.82 84.82 3.13 0.1019 3.99
C2 1 259.69 259.69 9.58 0.0093 6.97
D2 1 47.38 47.38 1.75 0.2112 2.98
Residual 12 325.23 27.10
Lack of Fit 10 318.47 31.85 9.42 0.0997
Pure Error 2 6.76 3.38
Total 26 1662.60
(B)
Model 14 964.38 68.88 3.18 0.0256 41.23
A-Solvent 1 330.12 330.12 15.26 0.0021 5.24
B-pH 1 2.73 2.73 0.1260 0.7287 −0.47
C-Temp. 1 237.45 237.45 10.98 0.0062 −4.45
D-Ratio 1 27.91 27.91 1.29 0.2782 1.52
AB 1 0.0196 0.0196 0.0009 0.9765 −0.07
AC 1 1.54 1.54 0.0711 0.7943 0.62
AD 1 0.2916 0.2916 0.0135 0.9095 0.27
BC 1 87.05 87.05 4.02 0.0679 4.66
BD 1 0.0001 0.0001 4.623E−06 0.9983 −0.01
CD 1 23.33 23.33 1.08 0.3195 2.41
A2 1 172.52 172.52 7.98 0.0153 −5.69
B2 1 18.75 18.75 0.8668 0.3702 1.87
C2 1 147.42 147.42 6.81 0.0228 −5.26
D2 1 10.60 10.60 0.4902 0.4972 −1.41
Residual 12 259.59 21.63
Lack of Fit 10 250.73 25.07 5.66 0.1593
Pure Error 2 8.85
Total 26 1223.97

As far as total phenolic compounds are concerned, it can be seen that the variables that influenced
the process were the percentage of methanol in the extraction solvent, temperature, pH, and the
quadratic temperature interaction, as the p-values were less than 0.05 (95% confidence level). Once the
influence of the different factors was known, the second-order polynomial equation was obtained by
taking into account only the four significant variables mentioned above. Thus, the non-significant
terms were removed from the mathematical model and this gave similar results to those obtained with
the full equation. The reduced equation that could reliably predict the experimental results was: yTP =
43.87 + 3.95 Solvent − 4.14 pH + 4.76 Temperature + 6.97 Temperature2 .
In the case of anthocyanins, the factors that influenced the process in a statistically significant way
(i.e., a confidence level of 95%) were the percentage of methanol in the extraction solvent, temperature,
 significant terms were removed from the mathematical model and this gave similar results to those
obtained with the full equation. The reduced equation that could reliably predict the experimental
results was: yTP = 43.87 + 3.95 Solvent − 4.14 pH + 4.76 Temperature + 6.97 Temperature2.

In 2018,
Agronomy the case
8, 240 ofanthocyanins, the factors that influenced the process in a statistically significant 8 of 15
way (i.e., a confidence level of 95%) were the percentage of methanol in the extraction solvent,
temperature, the quadratic interaction of temperature, and the quadratic interaction of the percentage
the quadratic interaction
of methanol. As mentionedof temperature, and thepolynomial
above, the reduced quadratic interaction
equation only of the percentage
contained of methanol.
the variables and
As
interactions between them that had a significant effect on the response, and it was: yTA =interactions
mentioned above, the reduced polynomial equation only contained the variables and 41.23 + 5.24
between
Solvent them
− 4.45 that had a significant
Temperature effect2on
− 5.69 Solvent the Temperature
− 5.26 response, and 2. it was: yTA = 41.23 + 5.24 Solvent −

4.45 Temperature − 5.69 Solvent 2 − 5.26 Temperature2 .

In both cases, phenolic compounds and total anthocyanins, lack of fit test showed a p-value
In both
higher thancases, phenolic
0.05 (not compounds
significant), whichand total anthocyanins,
means that the modellack of fit test showed a p-value higher
fit well.
than 0.05 (not
These significant),
results can be which means
graphically that the model
represented fit well.
in standardized Pareto charts (Figure 2), where the
significance of each of the variables analyzed in decreasing orderPareto
These results can be graphically represented in standardized can becharts
easily (Figure 2),sign
seen. The where the
of each
significance
effect is alsoofobserved
each of the
duevariables analyzed
to the two in decreasing
different colors of theorder
bars.can be easily
Positive seen.
signs The sign
indicate of
a direct
each effect is also observed due to the two different colors of the bars. Positive
relationship between the effect and the response variable, while negative signs refer to an inverse signs indicate a
direct relationship
relationship. between the effect and the response variable, while negative signs refer to an
inverse relationship.

Figure Pareto charts

Figure2.2. Pareto charts for
forthe
thestandardized
standardizedeffects:
effects:(A)(A) total
total phenolic
phenolic compounds
compounds and and (B)
(B) total
total anthocyanins.
anthocyanins.

ForForphenolic
phenoliccompounds,
compounds,ititwas wasobserved
observedthat,
that,ofofthe
thefour
fourvariables
variablesanalyzed,
analyzed,temperature
temperaturehad had
the most marked effect on the response variable. However, pH and percentage
the most marked effect on the response variable. However, pH and percentage of methanol of methanol alsoalso
had had
an
effect, thus confirming the information discussed above. The variable that had the smallest
an effect, thus confirming the information discussed above. The variable that had the smallest effect effect on the
response variable was
on the response the solvent
variable was the volume:sample mass ratio.mass
solvent volume:sample It wasratio.
also It
observed
was also that the percentage
observed that the
ofpercentage
methanol in of methanol in the extraction solvent and the temperature had a positiveindicates
the extraction solvent and the temperature had a positive effect, which that
effect, which
the use of higher temperatures and higher percentages of methanol led to higher
indicates that the use of higher temperatures and higher percentages of methanol led to higher levels levels of these
compounds in the extract.
of these compounds in theIn extract.
contrast,InpH had a negative
contrast, pH had effect on theeffect
a negative extraction.
on theOn the other On
extraction. hand,the
for anthocyanins it was clearly observed that, of the four variables analyzed, the
other hand, for anthocyanins it was clearly observed that, of the four variables analyzed, the onesones that had the
greatest
that hadinfluence wereinfluence
the greatest the temperature
were theand the percentage
temperature and the of methanol,
percentagewhile the pH and
of methanol, whilethethe
ratio
pH
hardly had any influence. In this case, the percentage of methanol had a positive effect, which means
that an increase in this variable favored the recovery of anthocyanins in the extract. Temperature had
a negative influence on the extraction, and an increase in this factor would decrease the recovery of
anthocyanins. The results mentioned above were recorded in three-dimensional (3D) response surface
plots obtained using the fitted model. These surface plots help to improve the understanding of the
 Agronomy 2018, 8, x FOR PEER REVIEW 9 of 15

and the ratio hardly had any influence. In this case, the percentage of methanol had a positive effect,
which means that an increase in this variable favored the recovery of anthocyanins in the extract.
Agronomy 2018, 8, had
Temperature 240 a negative influence on the extraction, and an increase in this factor would decrease
9 of 15

the recovery of anthocyanins. The results mentioned above were recorded in three-dimensional (3D)
response surface plots obtained using the fitted model. These surface plots help to improve the
main and interactive effects of the most influential parameters [46,47]. 3D plots are represented in
understanding of the main and interactive effects of the most influential parameters [46,47]. 3D plots
Figure 3 which show the influences of two selected factors on each response (anthocyanins and total
are represented in Figure 3 which show the influences of two selected factors on each response
phenolic compounds) keeping the rest of the variables constant. Methanol and temperature were
(anthocyanins and total phenolic compounds) keeping the rest of the variables constant. Methanol
selected as the most significant factors for anthocyanins. These variables as well as the effect of pH
and temperature were selected as the most significant factors for anthocyanins. These variables as
were selected for phenolic compounds. The combined effects of methanol–pH, methanol–temperature,
well as the effect of pH were selected for phenolic compounds. The combined effects of methanol–
and pH–temperature on the total phenolic compounds recovery are represented in Figure 3A–C,
pH, methanol–temperature, and pH–temperature on the total phenolic compounds recovery are
respectively. The interactive effect of methanol and temperature on the anthocyanins extraction is
represented in Figure 3A–C, respectively. The interactive effect of methanol and temperature on the
represented in Figure 3D. Thus, temperature and solvent composition were found to be the most
anthocyanins extraction is represented in Figure 3D. Thus, temperature and solvent composition
influential factors for both anthocyanins and total phenolics, and these had a marked effect on the
were found to be the most influential factors for both anthocyanins and total phenolics, and these
extraction. Similar findings were reported by other authors [48,49]. The largest difference in behavior
had a marked effect on the extraction. Similar findings were reported by other authors [48,49]. The
between total phenolics and anthocyanins was observed on considering temperature. For total
largest difference in behavior between total phenolics and anthocyanins was observed on considering
phenolics, the use of higher extraction temperatures led to a more efficient extraction [50], but high
temperature. For total phenolics, the use of higher extraction temperatures led to a more efficient
temperatures canbut
extraction [50], lead to the
high degradation
temperatures canoflead
individual anthocyanins
to the degradation [51].
of individual anthocyanins [51].

Figure 3. Three-dimensional response surface plots for the graphical representation of the influence of
Figure 3. Three-dimensional response surface plots for the graphical representation of the influence
(A) methanol–pH, (B) methanol–temperature, and (C) pH–temperature on total phenolic compounds,
of (A) methanol–pH, (B) methanol–temperature, and (C) pH–temperature on total phenolic
and the influence of (D) methanol–temperature on total anthocyanins extraction.
compounds, and the influence of (D) methanol–temperature on total anthocyanins extraction.
3.2. Optimized Conditions
3.2. Optimized Conditions
Having carried out the detailed experimental design for both total phenolic compounds and
Having carried out the detailed experimental design for both total phenolic compounds and
anthocyanins and having performed the statistical treatment of the data, it was concluded that the
anthocyanins and having performed the statistical treatment of the data, it was concluded that the
optimum MAE conditions for total phenolic compounds were as follows: extraction solvent with
optimum MAE conditions for total phenolic compounds were as follows: extraction solvent with 65%
65% of MeOH in water at pH 2, extraction temperature of 100 ◦ C and a solvent volume:sample mass
of MeOH in water at pH 2, extraction temperature of 100 °C and a solvent volume:sample mass ratio
ratio of 10:0.5. The optimum MAE conditions for anthocyanins were as follows: extraction solvent
of 10:0.5. The optimum MAE conditions for anthocyanins were as follows: extraction solvent with
with 60% of MeOH in water at pH 2, extraction temperature of 50 ◦ C, and a solvent volume:sample
60% of MeOH in water at pH 2, extraction temperature of 50 °C, and a solvent volume:sample mass
mass ratio of 14:0.5.
ratio of 14:0.5.
It was observed in both cases that the optimum percentage of methanol was an intermediate
value, which was determined by the polarity of both the solvent and the set of phenolic compounds
present in maqui.
Regarding temperature, although it appears to play a positive role by increasing the extraction
kinetics, it must be borne in mind that degradation and fragmentation of the phenolic compounds
 It was observed in both cases that the optimum percentage of methanol was an intermediate
value, which was determined by the polarity of both the solvent and the set of phenolic compounds
present in maqui.
Regarding temperature, although it appears to play a positive role by increasing the extraction
Agronomy 2018, 8, 240 10 of 15
kinetics, it must be borne in mind that degradation and fragmentation of the phenolic compounds
can occur at high temperatures [51,52]. As a consequence, other phenolic compounds can be
can occur at
produced, andhigh
theirtemperatures
hydroxyl groups [51,52]. As areact
will also consequence, other phenolic
with the Folin–Ciocalteu compounds
reagent, can bea
thus causing
produced,
significantand their in
increase hydroxyl groups will
the absorbance. Foralso
thisreact with
reason, thethe Folin–Ciocalteu
use of temperatures reagent,
above 100thus°C causing
was not a
considered.
significant As far as
increase in anthocyanins
the absorbance. areFor
concerned,
this reason,the opposite
the use ofbehavior was observed
temperatures above 100 ◦
for C this factor.
was not
considered.
High temperaturesAs far aslead
anthocyanins are concerned,
to fragmentation the opposite
and degradation of behavior
anthocyaninswas observed
and, as a for this factor.
consequence,
the optimum
High temperaturestemperature
lead to was the minimum
fragmentation andvalue used. Lower
degradation values were and,
of anthocyanins not tested, because this
as a consequence,
the
wasoptimum
the minimum temperature was the
temperature thatminimum
could be used valueinused. Lower values
the equipment. werepH
Finally, notvalues
tested, because
less than 2
werewas
this nottheconsidered
minimum because this may
temperature thatcause
could acid
be hydrolysis
used in theofequipment.
these compounds Finally,[53,54].
pH values The total
less
average
than 2 wereconcentrations
not considered in maqui werethis
because found
maytocause
be 54.27acidand 34.94 mgofg these
hydrolysis −1 for phenolic
compounds compounds
[53,54].
and total
The anthocyanins, respectively. in
average concentrations Similar
maquiresults were reported
were found to be 54.27 byandother
34.94 mg g−[2,4,13,55].
authors 1 for phenolic The
outstandingly
compounds andhigh total phenolic
anthocyanins, levels inSimilar
respectively. this fruit are due
results weretoreported
the remarkably
by other high
authors anthocyanins
[2,4,13,55].
content,
The which contribute
outstandingly high total the most tolevels
phenolic the antioxidant
in this fruitcapacity
are due[56].
to the remarkably high anthocyanins
content, which contribute the most to the antioxidant capacity [56].
3.3. Extraction Time
3.3. Extraction Time
An evaluation of the extraction kinetics was performed under the optimum conditions with
timesAnbetween
evaluation of the
2 and 25 extraction kinetics for
min (extractions waseachperformed
time wereunder the optimum
carried conditions in
out in triplicate) with times
order to
between 2 and 25 min (extractions for each time were carried out in triplicate)
identify the optimal extraction time (see Figure 4). For the extraction of both phenolic compounds in order to identify the
optimal extraction time
and anthocyanins, (see Figure
an optimal 4). For the
extraction timeextraction
of only of both was
2 min phenolic compounds
identified. and anthocyanins,
Regarding the phenolic
an optimal extraction
compounds, almost the timesame
of only 2 min was
amount was identified. Regarding of
extracted regardless thethe
phenolic compounds,
extraction almost
time employed
the same amount was extracted regardless of the extraction time employed
(statistically significant differences were not observed). In relation to anthocyanins, it was observed (statistically significant
differences
that there was wereanot observed).
slight decreaseInin relation to anthocyanins,
the amount of extracted it was observed that
anthocyanins there
as the was a slight
extraction time
decrease in the amount of extracted anthocyanins as the extraction time
increased. This finding may be due to the long exposure time to microwave radiation, which could increased. This finding may
be due
lead totoproduct
the longdegradation.
exposure time Anto microwave
optimum timeradiation,
of 2 minwhichwas used couldforlead
both tocompounds
product degradation.
in further
An optimum to
experiments timesaveof both
2 mintimewasandused for both
money. A compounds
reasonably rapid in further
method experiments
was proposed to savesinceboth time
most of
and money. A reasonably rapid method was proposed since most of the compounds
the compounds of high biological interest present in the maqui samples could be extracted in such a of high biological
interest present
short period of in the maqui samples could be extracted in such a short period of time.
time.

60
mg compounds / g maqui

50
Total Anthocyanins
40

30

20

10

0
2 5 10 15 20 25
Time (min)

Figure4.4. Recovery
Figure Recoveryofoftotal
totalphenolic
phenoliccompounds (mgg−g1−1))and
compounds(mg andtotal
totalanthocyanins (mgg−g1−1)) using
anthocyanins(mg using
different extraction times (n = 3).
different extraction times (n = 3).

3.4. Analytical Precision of the MAE Method

In order to study the precision of the MAE of maqui samples on the same day and on different days,
the repeatability and intermediate precision were studied under the optimum conditions described
above. A total of 36 extractions were performed. For repeatability, 12 extractions were carried
out on the same day, and for intermediate precision, 12 extractions were performed every day on
three different consecutive days. Repeatability results were 3.91% for phenolic compounds and
3.62% for anthocyanins. In the case of intermediate precision, the values were 4.24% for phenolic
compounds and 3.78% for anthocyanins. In both cases, the coefficients of variation were less than
5%, which is usually considered to be the limit to confirm that a method is accurate in this type of
Agronomy 2018, 8, 240 11 of 15

test [57]. Therefore, the extraction methods had good precision. Similar repeatability and intermediate
precision were obtained for phenolic compounds in other matrices, such as mulberries [48], grapes [28],
and peppers [58].

3.5. Application to Real Samples

The developed methods were applied to the extraction of total phenolic compounds and total
anthocyanins from seven real samples made from maqui (capsules, pills, and lyophilized material)
using the optimum conditions. The samples were analyzed in triplicate, and the results obtained are
shown in Table 3. Firstly, the large difference between the two types of capsules analyzed (M-1 and M-2)
is remarkable, with a far higher concentration of compounds of biological interest found in the second
sample. It can also be seen that the amounts extracted from the various lyophilized samples (M-4–7)
were very similar, despite the fact that they were obtained from different lots or even from different
brands. In addition, relatively high concentrations of active compounds were obtained because
lyophilization allows the preservation of beneficial properties and characteristics during transport [59].
Finally, it should be noted that in the case of M-3, the extraction of the eight specific anthocyanins from
maqui was not observed. In contrast, only two anthocyanins (cyanidin 3-O-glucoside and delphinidin
3-O-glucoside) were detected in this sample. Therefore, it can be concluded that the composition of the
pill is not maqui, since the eight characteristic anthocyanins were not detected. These results confirm
the great importance of having appropriate extraction methods to guarantee the quality of products
consumed and to avoid possible food frauds. Such fraud mostly occurs when the fruit is squeezed and
processed to obtain a concentrate or a dried food [19].

Table 3. Concentrations of total phenolic compounds (mg g−1 ) and total anthocyanins (mg g−1 ) in
different real samples made with maqui (n = 3).

Foodstuff made Type of Sample Total Phenolic Total Anthocyanins

Sample Preparation
with Maqui Analyzed Compounds (mg g−1 ) (mg g−1 )
M-1 Opening each of the capsules and 8.22 ± 0.34 1.73 ± 0.16
Capsules
M-2 using the powder inside 103.30 ± 0.30 74.55 ± 3.80
Crushing with a conventional electric
M-3 Pills 11.45 ± 0.45 –*
grinder to get a fine powder
M-4 53.06 ± 1.53 30.35 ± 3.25
M-5 Does not need any previous 49.29 ± 2.17 27.66 ± 1.02
Lyophilized
M-6 preparation 59.57 ± 0.70 34.51 ± 1.40
M-7 52.13 ± 2.53 19.89 ± 1.44
* The extraction of the eight specific anthocyanins from maqui was not observed.

4. Conclusions
Based on the results obtained in this work, it can be stated that microwave-assisted extraction
(MAE) was proven to be a suitable and rapid method for the extraction of total phenolic compounds
and total anthocyanins from maqui. The most influential variable for total phenolic compounds was
the temperature, whereas for anthocyanins the most significant parameter was the percentage of
methanol in the extraction solvent. The extraction methods developed had a high repeatability and
intermediate precision, with a coefficient of variation of less than 5%. A short period of time (2 min)
was sufficient for the extraction of the bioactive compounds. In addition, the applicability of these
methods has been demonstrated by the successful extraction of both total phenolic compounds and
anthocyanins in real samples. Moreover, the developed methods proved to be useful for the detection
of possible fraud in food made from maqui. It can be concluded from all the results that MAE under
the optimum conditions can be considered as an easy, rapid, and economical tool for the extraction of
both total phenolic compounds and anthocyanins in maqui, and that this approach is an excellent an
affordable alternative for industry [21].

Author Contributions: Conceptualization, E.E.-B. and G.F.B.; methodology, M.V.-E. and A.V.G.d.P.; software,
M.F.-G.; formal analysis, C.C., M.V.-E., and A.V.G.d.P.; investigation, M.V.-E. and A.V.G.d.P.; resources, C.G.B.
and M.P.; data curation, E.E.-B., M.F.-G., and G.F.B.; writing—original draft preparation, M.V.-E. and A.V.G.d.P.;
Agronomy 2018, 8, 240 12 of 15

writing—review and editing, G.F.B. and E.E.-B.; supervision, E.E.-B. and G.F.B.; project administration, G.F.B. and
E.E.-B.
Funding: This research received no external funding.
Acknowledgments: The authors acknowledge V. la Andaluza and University of Cadiz for the funding provided
through the project OT2016/046.
Conflicts of Interest: The authors declare no conflict of interest.

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