Industrial Crops & Products 133 (2019) 168–177

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Extracting phenolic compounds from Hibiscus sabdariffa L. calyx using T

microwave assisted extraction
⁎
Liliana Cassol, Eliseu Rodrigues, Caciano Pelayo Zapata Noreña
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, CEP 91501-970, Porto Alegre, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Hibiscus is an edible flower with great industrial potential due to the presence of bioactive compounds. The aim
Phenolic compounds of this study was to compare three extraction procedures of phenolic compounds from the calyx of hibiscus using
Hibiscus only microwave assisted extraction (MAE), using a solution of citric acid followed by MAE, and MAE followed by
Microwave extraction using a solution of citric acid. The results indicated that the best extraction condition was the com-
Extraction
bined extraction of 700 W for 8 min and subsequent extraction in acidic aqueous solution for 6 h, with the
contents of 1.63 mg g−1, 29.62 mg g−1, 133.25 μmol g−1 for anthocyanins, total phenolics, and antioxidant
activity, respectively. In extractions obtained by exhaustive extraction with methanol for 25 min and MAE at
700 W and 8 min, 13 compounds were found: six phenolic acids, two anthocyanins, and five flavonoids derived
from quercetin, kaempferol, and myricetin. The major phenolic compounds were 3-caffeoylquinic acid (2.58 and
1.32 mg g−1) and 5-caffeoylquinic acid (1.71 and 0.90 mg g−1) for exhaustive extraction and MAE, respectively.
The results suggest that MAE followed by extraction in acidic aqueous solution was the best alternative to
recovery of phenolic compounds from hibiscus.

1. Introduction
Hibiscus is a herbaceous plant used for medicinal purposes. Hibiscus
presents compounds with antioxidant activity, neutralizing reactive
oxygen and free radicals (Mohd-Esa et al., 2010) and conferring pro-
tection of the cells against the damage caused by lipid peroxidation
(Olalye and Rocha, 2007). The anthocyanins cyanidin-3-glucoside,
delphinidin-3-glucoside, cyanidin-3-sambubioside, and delphinidin-3-
sambubioside, were detected in high concentrations in the calyces of
hibiscus (Rodríguez-Medina et al., 2009).
The consumption of its calyx has antihypertensive activity, reducing
both systolic and diastolic pressures, heart rate, being considered va-
sodilator (Inuwa et al., 2012).
Extracts from different parts of the hibiscus have been widely stu-
died, being predominantly obtained by the solvent extraction method,
such as water, methanol, ethyl acetate, and hexane under different
conditions of time and temperature (Sindi et al., 2014; Formagio et al.,
2015; Zhen et al., 2016), where several phenolic compounds, phenolic
acids, flavonoids such as quercetin, luteolin or gossipetine, and its re-
spective glycosides have been identified (Ali et al., 2005). However, the
variety of extraction conditions (type of solvent, concentration, time,
and temperature) may potentially affect the polyphenolic profile of the
extracts (Segura-Carretero et al., 2008). In addition, conventional
methods such as maceration and Soxhlet extraction have many dis-
advantages, including the use of large amounts of organic solvents that
in some cases may be toxic, as well as high energy and time con-
sumption (Castro and Garcia-Ayuso, 1998) and also harmful to the
environment (Mandal et al., 2007).
Microwave assisted extraction (MAE) is a method that can be a
potential tool to reduce the amount of solvent to be used, time, tem-
perature, and energy consumption, resulting in a better separation and
recovery of compounds of interest (Rombaut et al., 2014). The principle
of microwave extraction is based on the direct effect of the microwaves
on the molecules either by ion conduction or dipole rotation, where the
two mechanisms can occur simultaneously. Polar molecules, such as
water, have positive and negative partial charges at opposite ends and
rotate as the incidence of the microwaves increases, in an attempt to
align with the formed electric field (Barba et al., 2008). The molecular
level movement and the friction between the molecules cause the en-
ergy of the electromagnetic waves to be transformed into thermal en-
ergy, increasing the temperature of the matrix, and this effect leads to
the cellular rupture that facilitates the extraction of the compounds
(Aspé and Fernández, 2011) and to the increase of the mass diffusion
constants of the solutes in the solvent, improving the extraction yield
(Mandal et al., 2007). These heating characteristics allow the MAE
method to be used in the extraction of bioactive compounds from

⁎
Corresponding author.
E-mail address: czapatan@ufrgs.br (C.P. Zapata Noreña).

https://doi.org/10.1016/j.indcrop.2019.03.023
Received 22 August 2018; Received in revised form 5 March 2019; Accepted 8 March 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
L. Cassol, et al.
natural products.
In this research, different methods of MAE were evaluated to find
the best extraction condition for total monomeric anthocyanins, total
phenolic compounds, and antioxidant activity and to compare the best
MAE condition, using an acidic aqueous solution, with the extract ob-
tained by exhaustive extraction using acidified methanol solution.
2. Materials and methods
2.1. Materials
Hibiscus flowers came from a community garden (GPS location
30°06′49.4″S and 51°06′30″W, Porto Alegre, RS, Brazil), in 2016. After,
selection and sanitization, the calyces were separated and then stored in
polyethylene bags at −18 °C.
2.2. Extraction methods
Prior to extraction, the calyces were thawed and blanched (steam
water at 100 °C for 4 min) and then acidified water (2% citric acid, w/v)
was added, with pH ˜2.1, for the stability of the anthocyanins, in the
ratio of 1:5 (calyx: acidified water) (Piovesana and Noreña, 2018).
Three methods of separation were studied. The first consisted of
microwave assisted extraction. After preparation of the acidic aqueous
solution, it was immediately put in a conventional microwave oven
(Electrolux, MEF41) at 2450 MHz. Two factors were evaluated: power,
with levels of 200, 300, and 700 W, and time with levels of 2, 5, and
8 min. The response variables were total monomeric anthocyanins, total
phenolic compounds, and antioxidant activity. Statistical data analysis
was realized through of 3 × 3 two-way factorial design.
In the second method, aqueous extraction followed by microwaves
was performed. To this end, the blanched calyces were previously im-
mersed in the aforementioned acidic aqueous solution at times of 1, 2,
4, 6, 18, and 24 h at room temperature. After each time the mixture was
vacuum filtered using Whatman filter paper no. 01, and the retained
was again immersed in a new acidified solution and immediately placed
in the microwave oven, employing powers of 200, 300, and 700 W, and
times of 2, 5, and 8 min, respectively. Extraction only by acidic aqueous
solution was evaluated through one-way design, where the time is a
single factor. For extraction by microwave was used a one-way ran-
domized complete block design.
In the third method, the microwave assisted extraction was per-
formed, and after filtration, the retained was immersed in a new acid
solution at room temperature. The filtration and separation of the re-
tained, as well as factors and levels employed in each extraction, were
the same as those used in the second method. In this case, for statistical
analysis, for the first extraction was used one-way design, and for the
second extraction was used a one-way randomized complete block
design.
In order to compare the effect of MAE, of the best treatment ob-
tained in the first extraction method, an exhaustive extraction was also
carried out. In a Falcon tube, 2 g of hibiscus calyces were added to
10 mL of methanol:water (8:2, v/v) acidified (1% HCl, w/v). The tube
was then vortexed (Quimis, Q920-A2) for 5 min (Rodrigues et al., 2013)
and centrifuged (Sigma, 4K15) at 3000×g for 5 min and the super-
natant was reserved. The retained was subjected to the same procedure
mentioned five more times until no longer reacted with the Folin-Cio-
calteu reagent, totaling 25 min of reaction. The supernatants were
mixed and the solvent was evaporated in a rotary evaporator (40 °C) for
the total quantification of the compounds.
2.3. Spectrophotometric analyzes
Absorbance measurements were performed on a UV–vis spectro-
photometer (Genesys S10, Thermo Scientific).
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Industrial Crops & Products 133 (2019) 168–177
2.3.1. Total monomeric anthocyanins (TMA)
The extracts were mixed with potassium chloride (pH 1.0) and so-
dium acetate (pH 4.5), according to the method of Lee et al. (2005) and
absorbances at 520 and 700 nm were measured. The final concentration
was expressed in mg of delphinidin-3-sambubioside per g sample on a
dry basis (d.b), using the extinction molar coefficient Ɛ = 26,000 L
mol−1 cm-1.
2.3.2. Total phenolic compounds (TPC)
The analysis of total phenolic compounds was performed using the
Folin-Ciocalteu reagent at an absorbance of 765 nm, according to
Singleton and Rossi (1965) with modifications of Kuck and Noreña
(2016). The results were expressed as mg of gallic acid equivalent
(GAE) per g (d.b).
2.3.3. Evaluation of antioxidant activity using 2,2′-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS)
Performed according to the method described by Re et al. (1999),
where the ABTS radical was formed by the reaction between the 7 mM
ABTS•+ solution and the 140 mM potassium persulfate solution, at 25 °C
for 16 h. The radical was diluted with ethanol until it reached an ab-
sorption value of 0.700 ± 0.050 at 734 nm. An aliquot of 30 μL of
extract was added to 3 mL of the ABTS radical and the absorbance was
determined at 734 nm after 6 min of reaction. The results were ex-
pressed in μmol of Trolox equivalent per g (d.b).
2.4. Phenolic compounds by high performance liquid chromatography with
diode array detector coupled with electrospray ionization mass spectrometry
(HPLC-DAD-ESI-MS/MS)
The extracts from the exhaustive extraction and the best extraction
condition obtained in the first method were analyzed by HPLC-DAD-
ESI-MS/MS using a high performance liquid chromatography (HPLC,
Shimadzu) system with a rearrangement detector of diodes (DAD)
connected in series to a mass spectrometer (MS) containing electrospray
ionization source (ESI) (Bruker Daltonics micrOTOF-Q III) (Piovesana
et al., 2018).
In a C18 column (4.0 μm, 150 × 4.6 mm, Phenomenex, USA) the
phenolic compounds were separated at flow rate at 0.7 mL min−1 and
column temperature at 29 °C with solvent A: water/formic acid
(99.5:0.5, v/v) and solvent B: acetonitrile/formic acid (99.5:0.5, v/v),
in elution gradients from A/B 99:1 (v/v) to 50:50 (v/v) in 50 min, then
50:50 (v/v) to 1:99 (v/v) in 5 min, the ratio (1:99, v/v) was maintained
for another 5 min (Rodrigues et al., 2013). Ultraviolet-visible spectra
were obtained between 200 and 800 nm and the chromatograms pro-
cessed at 260, 320, 360, and 520 nm. The column eluate was divided
into only 0.35 mL min−1 to enter the ESI interface. The mass spectro-
meter was operated under the following conditions: mass range in the
ratio m/z from 100 to 700, with source in the positive and negative
ionization modes with capillary voltage of 3000 V, temperature and
flow of nitrogen drying gas of 310 °C and 5 L min−1, MS/MS frag-
mentation energy of 34 eV, and nitrogen gas pressure nebulizer of
30 psi. The extracts were diluted with solvent A and filtered (Millipore,
0.22 μm) prior to analysis.
The identification of the phenolic compounds was performed based
on the order of elution and the retention time of the peaks on the re-
versed phase column against the standards, comparison of these UV–vis
and mass spectra with the standards, and analyzed under the same
conditions, with data from the literature (Rodríguez-Medina et al.,
2009; Ramírez-Rodrigues et al., 2012; Da-Costa-Rocha et al., 2014).
The quantification was made by external standardization using analy-
tical curves with limits of detection and quantification of 5-caffeine
(0.59 and 1.79 ppm), cyanidin (0.69 and 2.10 ppm), quercetin- 3-O-
glycoside (0.38 and 1.14 ppm), and kaempferol (0.17 and 0.52 ppm),
respectively.
L. Cassol, et al.
Fig. 1. Total monomeric anthocyanins (TMA) (A), total phenolic compounds
(TPC) (B) and antioxidant activity (ABTS) (C) obtained in microwave assisted
extraction. (T1: 200 W, 2 min, T2: 300 W, 2 min, T3: 700 W, 2 min, T4: 200 W,
5 min, T5: 300 W, 5 min, T6: 700 W, 5 min, T7: 200 W, 8 min, T8: 300 W, 8 min,
T9: 700 W, 8 min).
Different letters indicate significant differences (p < 0.05).
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Industrial Crops & Products 133 (2019) 168–177
2.5. Statistical analysis
The data were submitted to analysis of variance (ANOVA), and the
comparisons between the means of the treatments were performed
using the Tukey’s test. Hierarchical cluster analysis was used to classify
and identify similarities among extraction treatments. The calculations
were performed using SAS 9.3 statistical software.
3. Results and discussion
3.1. First method of extraction: microwave assisted extraction
Acid extraction condition used in the experiments was really im-
portant because, at low pH, anthocyanin is as flavylium cation that
shows high stability (Hubbermann et al., 2006).
The contents of total monomeric anthocyanins, total phenolic
compounds, and antioxidant activity obtained by the first extraction
method are presented in Fig. 1.
The contents of anthocyanins obtained (Fig. 1A) ranged from 0.1 to
1.2 mg g−1 (d.b) of delphinidine-3-sambubioside. In the 200 and 300 W
power, in the first five min, there was an increase in the contents.
However, when the extraction time was 8 min, there was a decrease in
anthocyanin contents, which may have been due to low power used,
were not able of provocating complete break of the cell structure, and
for longer contact time, at low power, begin to cause the degradation of
the compounds.
For the extracts obtained at 700 W, the concentrations increased
significantly (p < 0.05) with time (from 0.92 to 1.15 mg g−1 (d.b) of
delphinidin-3-sambubioside). Extractions obtained at 700 W were also
higher than treatments at 200 and 300 W. This shows that the use of
higher powers and times provides higher extractions of compounds,
which can compensate for the possible losses due to thermal degrada-
tion. Further studies would be needed to verify the actual effect of time
and power on membranes and cell walls.
Kappe et al. (2009) mention that the mechanism of interaction be-
tween the electric field component and the matrix is the dipole rotation,
that whether it does not have enough time to realign (high frequency
irradiation) or quickly reorient (low frequency irradiation) the heating
does not occur. Latha (2007) reported that higher temperatures
achieved during MAE increase kinetic diffusion coefficients and reduce
convective resistance to mass transfer, helping the compounds to dif-
fuse more rapidly through solvent and cells.
The behavior presented for extractions of total phenolic compounds
(Fig. 1B) and antioxidant activity (Fig. 1C) were similar to the behavior
showed for anthocyanins (Fig. 1A). The extractions were significantly
larger with both time and power (p < 0.05). The highest extraction
obtained with the T9 treatment (700 W and 8 min) corresponded to
23.54 mg g−1 (d.b) of gallic acid equivalent (GAE) and 93.01 μmol g−1
(d.b) of Trolox equivalent (TE), for total phenolics and antioxidant
activity, respectively. In the case of MAE, the main factors that may
affect the extraction process are the power, time, temperature, and
increase in internal cellular pressure.
Mandal et al. (2007) reported that the fast increase in temperature
and internal pressure by the microwaves, it can cause cell rupture,
occasioning rapid solubilization of the solutes that were contained
within the cell, by the solvent. The temperature is directly related to the
power and time spent in the microwaves, due microwave energy is
converted into heat in the dielectric material, and at higher tempera-
tures, increase extraction efficiency and reduce residence times (Yan
et al., 2010). In addition, the increase in the temperature of the solvent
with the power and the time, solvation power increases due to the
decrease of the viscosity and surface tension (Mandal et al., 2007),
rising the mass diffusion coefficients.
At 700 W and times of 2, 5, and 8 min, the contents obtained were of
0.92, 1.00, and 1.15 mg g−1 (d.b) of delphinidin-3-sambubioside for
anthocyanins, of 6.90, 19.77, and 23.54 mg g−1 (d.b) of gallic acid
L. Cassol, et al.
Fig. 2. Total monomeric anthocyanin content (TMA) at power 200 (A), 300 (B)
and 700 W (C), respectively. (empty bar: acidic aqueous extraction; striped bar:
microwave assisted extraction).
Different upper and lower case letters indicate significant differences between
the aqueous and microwave extractions, respectively (p < 0.05).
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Industrial Crops & Products 133 (2019) 168–177
equivalent for phenolic compounds, and 70.57, 81.91, and
93.01 μmol g−1 (d.b) of Trolox equivalent for antioxidant capacity,
respectively; being significantly higher (p < 0.05) the contents in the
time of 8 min, being this condition selected for the later comparison
with the exhaustive extraction. In addition, the best results of each
power (200 W and 5 min, 300 W and 5 min, 700 W and 8 min) were
selected for using in the second and third extraction methods.
3.2. Second extraction method: acidic aqueous extraction followed by MAE
In the second extraction method, observed in Figs. 2A, 2B, and 2C,
the total monomeric anthocyanins contents during the first stage were
higher in the three powers for the times between 6 and 18 h of ex-
traction (1.04–1.12 mg g−1 (d.b) of delphinidin-3-sambubioside), de-
creasing significantly when the 24 h time was used, in the 200 and
300 W power (p < 0.05). In the three powers, the highest contents
(1.06, 1.10, and 1.11 mg g−1 (d.b) of delphinidin-3-sambubioside, re-
spectively) were obtained with six h of extraction, respectively, which
were not significantly different than with 18 h (p < 0.05). In the ex-
traction obtained during the second stage, the highest contents were
obtained at 700 W in the first two h and were significant (p < 0.05),
when compared with the other powers used. However, when the ex-
tractions performed by the two stages were compared together, the
highest extraction was obtained in 6 h of acidic aqueous extraction,
followed by 700 W of power, with 1.58 mg g−1 (d.b) of delphinidin-3-
sambubioside.
For the total polyphenol content (Fig. 3A, B, and C) for the acidic
aqueous extraction also the highest yields for the three powers were
from 6 h. The subsequent MAE at 700 W and times of 1 and 2 h were
significantly higher (p < 0.05) than those obtained in the other
treatments (12.18 and 9.41 mg g−1 (d.b) of gallic acid equivalent, re-
spectively). However, when the two steps were evaluated together, the
extraction obtained with six h in acidic aqueous extraction followed by
700 W, was the one with the highest concentration of phenols, with
26.95 mg g−1 (d.b) of gallic acid equivalent.
As for the antioxidant activity, shown in Fig. 4A, B, and C, similarly,
in the first step from the six h of aqueous extraction, the highest yields
were obtained. However, for the MAE, the highest significant yields
(p < 0.05) were obtained with 1 and 6 h in the power of 700 W, with
contents of 64.91 and 54.10 μmol g−1 (d.b) of Trolox equivalent, re-
spectively. When the total extraction was evaluated, the highest yields
corresponded to the time of six h of extraction followed by 700 W
(142.16 μmol g−1 (d.b) of Trolox equivalent).
The antioxidant capacity is directly related to the content of phe-
nolic compounds. Garofulić et al. (2013) verified higher extractions of
phenolic compounds and anthocyanins of the cherry Marasca (Prunus
cerasus) with the use of the highest power tested (500 W), due to the
greater rotation of the water molecules in the attempt to realign in the
electric field, generate higher heat with the consequent increase in
temperature.
The advantage of prior acidic aqueous extraction is that the solvent
molecules, at room temperature, penetrate the plant matrix progres-
sively (Crossley and Aguilera, 2001), facilitating the dissolution of ac-
tive compounds.
The best condition obtained in this method was of 6 h in acidic
aqueous extraction, followed by 700 W and 8 min, with values of
1.58 mg g−1 (d.b) of delphinidin-3-sambubioside, 26.95 mg g−1 (d.b) of
gallic acid equivalent, and 142.16 μmol g−1 (d.b) of Trolox equivalent
for anthocyanins, phenolics, and antioxidant activity, respectively.
3.3. Third extraction method: MAE followed by acidic aqueous extraction
In the third extraction method, the effects of both extraction stages
can be seen in Fig. 5A, B, and C: the first, by microwaves and the
second, by acidic aqueous extraction. The extractions performed at the
lowest power (200 and 300 W) showed low concentrations of
L. Cassol, et al.
Fig. 3. Total phenolic compounds (TPC) (A, B, C) at power 200, 300 and 700 W,
respectively. (empty bar: acidic aqueous extraction; striped bar: microwave
assisted extraction).
Different upper and lower case letters indicate significant differences between
aqueous and microwave extractions, respectively (p < 0.05).
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Industrial Crops & Products 133 (2019) 168–177
Fig. 4. Antioxidant activity (ABTS) (A, B, C) at power 200, 300 and 700 W,
respectively. (empty bar: acidic aqueous extraction; striped bar: microwave
assisted extraction).
Different upper and lower case letters indicate significant differences between
aqueous and microwave extractions, respectively (p < 0.05).
L. Cassol, et al.
Fig. 5. Total monomeric anthocyanins (TMA) (A, B, C) at power 200, 300 and
700 W, respectively. (empty bar: acidic aqueous extraction; striped bar: mi-
crowave assisted extraction).
Different upper and lower case letters indicate significant differences between
aqueous and microwave extractions, respectively (p < 0.05).
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Industrial Crops & Products 133 (2019) 168–177
Fig. 6. Total phenolic compounds (TPC) (A, B, C) at power 200, 300 and 700 W,
respectively. (empty bar: acidic aqueous extraction, striped bar: microwave
assisted extraction).
Different upper and lower case letters indicate significant differences between
aqueous and microwave extractions, respectively (p < 0.05).
anthocyanins, varying from 0.24 to 0.34 mg g−1 (d.b) of delphinidin-3-
sambubioside. The highest extractions were performed at 700 W, with
contents around 1.3 mg g−1 (d.b) of delphinidin-3-sambubioside, in-
dicating an approximate increase of 4.6 times of extracted compounds
L. Cassol, et al.
Fig. 7. Antioxidant activity (ABTS) (A, B, C) at power 200, 300 and 700 W,
respectively. (empty bar: acidic aqueous extraction, striped bar: microwave
assisted extraction).
Different upper and lower case letters indicate significant differences between
aqueous and microwave extractions, respectively (p < 0.05).
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Industrial Crops & Products 133 (2019) 168–177
in relation to the lower power.
As for acidic aqueous extraction, the highest extractions were per-
formed at times greater than 6 h, in the extracts from the 200 and
300 W, representing an increase of 31.8 and 36.2 times in relation to
the MAE, with contents of 0.90 and 0.78 mg g−1 (d.b) of delphinidin-3-
sambubioside for the powers of 200 and 300 W, respectively. In the
extracts from 700 W, the extractions were significantly smaller
(p < 0.05), due to the fact that most of the anthocyanins were already
removed in the first stage, presenting in 6 h the highest content, being
0.35 mg g−1 (d.b) of delphinidin-3-sambubioside.
In Fig. 6A, B, and C indicate that the highest extractions of phenolic
compounds in the first step corresponded to 700 W and were sig-
nificantly higher than in the 200 and 300 W power (p < 0.05). In the
second stage, the highest concentrations were obtained in the extracts
from 200 and 300 W, using aqueous extraction times from 6 h, with
concentrations of 14.1 and 13.5 mg g−1 (d.b) of gallic acid equivalent,
respectively. In total extraction, however, significantly the highest yield
(p < 0.05) was at 700 W followed by 6 h of aqueous extraction (29.6
mg g−1 (d.b) of gallic acid equivalent).
As for the antioxidant activity (Fig. 7A, B, and C), in the MAE at
700 W, the extractions were also significantly higher (p < 0.05) (93.18
μmol g−1 (d.b) of Trolox equivalent) when compared with the powers
of 200 and 300 W (26.08 and 30.10 μmol g−1 (d.b) of Trolox equiva-
lent, respectively). For the three powers, the time did not significantly
influence the extractions in this stage (p > 0.05). The microwaves
generate ionic conduction and dipole rotation, resulting in dissipated
energy within the material with molecular motion and heating (Aspé
and Fernández, 2011). The extraction at 700 W caused the final tem-
perature of the extract to increase to 96 °C, and when the aqueous acid
extraction was applied, most of the compounds had already been re-
moved.
For the second stage, extraction at 300 W, followed by 18 h, was
significantly higher (p > 0.05) with 73.48 μmol g−1 (d.b) of Trolox
equivalent. In the comparison of total extraction, the highest contents
were obtained at 700 W, followed by times of 1 and 6 h, totaling 127.27
and 133.25 μmol g−1 (d.b) of Trolox equivalent.
For acid extraction, the mechanism of separation is by mass diffu-
sivity, meanwhile, for microwave, the mechanism of polarization of the
water propitiates high heat transfer rates due to friction among water
molecules. This way, the temperatures of samples rapidly increase, that
result in a rise of mass diffusivity.
The use of MAE is able of decreasing the extraction time due to the
raising of temperatures, which decrease the surface tension and solvent
viscosity, accelerating the solubilization of the analytes in this phase,
with the increase of extraction efficiency (Taamalli et al., 2012).
The subsequent aqueous acid extraction presented higher significant
contents (p < 0.05) in the lower power used, due to the lower ex-
tractions obtained in the first stage. An important factor of this meth-
odology was the cooling done in the extract from the MAE, when was
added aqueous acid solvent at room temperature, because extract after
microwave extraction was still hot, thus reducing the risks of thermal
degradation and oxidation (Chen et al., 2007).
Chumsri et al. (2008) reported extractions of phenolic compounds
from hibiscus calyces using water as a solvent, in the proportions of 1:2,
1:5, and 1:10, obtaining contents of 22.39, 22.12, and 22.08 mg g−1
(d.b) respectively, however, found that with the increase in tempera-
ture, from 50 to 60 °C, there was a reduction in phenolic and antho-
cyanin content. These values were similar to those found in this study in
the power of 700 W and 8 min of 23.54 mg g-1 (d.b).
The best condition obtained in this method was with 700 W and
8 min followed by 6 h of acidic aqueous extraction, with anthocyanins,
total phenolics, and antioxidant activity of 1.63 mg g−1 (d.b) of del-
phinidin-3-sambubioside, 29.62 mg g−1 (d.b) of gallic acid equivalent,
and 133.25 μmol g−1 (d.b) of Trolox equivalent, respectively.
When comparing the best condition obtained in each of the methods
studied: in the first method, using 700 W and 8 min (1.15B mg g−1 (d.b)
L. Cassol, et al. Industrial Crops & Products 133 (2019) 168–177

of delphinidin-3-sambubioside, 23.54C mg g-1 (d.b) of gallic acid solute due some compounds have higher solubility in water than in
equivalent, 93.01B μmol g−1 (d.b) of Trolox equivalent), in the second organic solvent (Haminiuk et al., 2011). However, in this study, it was
method performed at 6 h of acidic aqueous extraction, followed by MAE possible to verify good results obtained with the use of the acidified
at 700 W for 8 min (1.58A mg g−1 (d.b) of delphinidin-3-sambubioside, aqueous solvent, due to this affinity, besides the stabilization of the
26.9B mg g−1 (d.b) of gallic acid equivalent, 142.15A μmol g−1 (d.b) of anthocyanins in this pH.
Trolox equivalent) and in the third method, MAE at 700 W for 8 min Chumsri et al. (2008) using water as solvent to extract the bioactive
followed by 6 h of acidic aqueous extraction (1.63A mg g-1 (d.b) of compounds from the calyx of hibiscus at temperatures of 50 and 60 °C,
delphinidin-3-sambubioside, 29,62A mg g−1 (d.b) of gallic acid reported that the antioxidant activity had no direct correlation with the
equivalent, 133,25A μmol g-1 of Trolox equivalent) for anthocyanins, amount of total anthocyanin and total phenolics, indicating that other
total phenolics, and antioxidant activity, respectively, it is evident that compounds present, such as protocatechuic acid and L-ascorbic acid,
the latter presented the best extraction conditions. The use of MAE is contribute to this antioxidant activity (Hirunpanich et al., 2006).
suitable to extract polar active compounds such as phenolic compounds However, the antioxidant capacity is more related to the composition of
(Karami et al., 2015). the phenolic compounds of the matrix than to its content (Prenesti
et al., 2007).
3.4. Exhaustive extraction with acidified methanol Wu et al. (2012) mention that in conventional extractions, such as
Soxhlet or maceration, heat transfer occurs from the outside to the in-
In the total exhaustive extraction using acidified methanol, total side of the matrix. In the MAE, microwave oscillations provocate col-
monomeric anthocyanins, total phenolic compounds, and antioxidant lisions between polar molecules that result in a rapid heat transfer
activity were 1.12A ± 0.005 mg g−1 (d.b) of delphinidin-3-sambubio- through of entire matrix, causing a fast rise of temperature, and the
side, 42.95A ± 0.837 mg g−1 (d.b) of gallic acid equivalent, and increase of the mass diffusion coefficient.
63.59B ± 0.372 μmol g−1 (d.b) of Trolox equivalent, respectively.
When compared with the best extraction defined in the first method, 3.5. Cluster analysis
700 W and 8 min, whose contents were 1.15A ± 0.002 mg g−1 (d.b) of
delphinidin-3-sambubioside, 23.54B ± 0.007 mg g−1 (d.b) of gallic Dendrogram defines four main clusters (Fig. 8). The first cluster (I)
acid equivalent, and 93.01A ± 0.347 μmol g−1 (d.b) of Trolox contains ten treatments; in this group is the best treatment, extraction
equivalent, for anthocyanins, phenolic compounds, and antioxidant by microwaves in the condition of 700W × 8 min, followed by acidic
activity, respectively, it can be observed that the activity the anti- aqueous extraction for 6 min (3T9 × 6). Acid extraction for 6 min fol-
oxidant activity was significantly higher in the latter, being lower for lowed by microwaves 700W × 8 min (2T9 × 6) showed high similarity
the phenol content (p < 0.05), already for the anthocyanin content with 3T9 × 6, because smaller distances indicate greater similarities.
there were no significant differences (p > 0.05). In the MAE at 8 min, Except for treatments at 700 W for 2, 5, and 8 min (1T3, 1T6, and 1T9),
the temperature reached around 96°C, compared with the exhaustive extractions only using the microwave are in the first cluster (IV), and
extraction performed at room temperature for 25 min. In the exhaustive the dendrogram indicates low similarity of this group with the best
extraction, however, a subsequent solvent extraction was carried out in extraction treatment.
a rotary evaporator at 40°C.
The fact that MAE at 700 W and 8 min was selected, although pre- 3.6. High-performance liquid chromatography -DAD-ESI-MS/MS analysis
senting values statistically lower than those obtained in the best ex-
tractions of the second and third methods, were due to the fact that it Thirteen phenolic compounds of extracts from exhaustive extraction
wanted to evaluate only the effect of the MAE in obtaining the bioactive and MAE at 700 W and 8 min were identified or tentatively identified
compounds of the calyces of hibiscus.
In this study, in the exhaustive extraction with methanol, the con-
tent of polyphenols was significantly higher (45.2%) (p < 0.05), but
with a reducing capacity (ABTS) 46.3% lower; this result may be due to
the phenolic compounds extracted in greater quantity could present
lower antioxidant power than those compounds obtained in the MAE.
According to Sun-Waterhouse and Waterhouse (2015), variations in the
contents of phenolic compounds are due to the ability of these com-
pounds to react between them, hydrolyzing into smaller molecules, or
polymerizing to form larger molecules, forming larger or smaller phe-
nolic compounds with antioxidant activity (Kuck et al., 2017). It is also
worth noting that during heating, phenolic degradation, including an-
thocyanins, can form new compounds, such as anthocyanins, and due to
the anthocyanins analysis method, quantify only the total monomers
present in the samples (Lee et al., 2005), there may be differences in the
content of these compounds.
Microwave assisted extraction is advantageous because it provides
extraction yields comparable with exhaustive extraction, and requires
less extraction time because the microwaves rupture the plant structure
to increase extraction power (Mandal et al., 2007). In addition to the
MAE, the extraction method used, the use of different solvents may also
contribute to the differences in the contents of the extracts. Mohd-Esa
et al. (2010), in the study of the extraction of phenolic compounds in
hibiscus using water and methanol (80%) as solvents, during 2 h at Fig. 8. Dendrogram of distance cluster centroids among extractions.
room temperature, obtained higher phenolic compounds content with (The first digit represents the first, second or third method of extraction; sym-
methanol (2.91 mg g−1 (d.b) of gallic acid equivalent) than with water bols T1, T2, T3, T4, T5,T6, T7, T8, T9 are defined in Fig. 1; 1, 2, 4, 6, 18, 24
(1.85 mg g−1 (d.b) of gallic acid equivalent), demonstrating that the represent the time in hours of acidic aqueous extraction). EE: exhaustive ex-
difference in solvent polarity has an influence on the solubility of the traction; I, II, III, IV: clusters.

175
L. Cassol, et al. Industrial Crops & Products 133 (2019) 168–177

Table 1 temperatures. Furthermore, the same compounds are found in the two
Quantification of the phenolic compounds of the hibiscus in extracts from the extraction methods.
exhaustive extraction and the MAE at 700 W and 8 min obtained by HPLC-DAD- Prolonged extraction time may cause undesirable reactions, such as
ESI-MS/MS. enzymatic degradation and oxidation, and may result in lower extrac-
Phenolic compound Concentration (mg g−1) tion yields of phenolic compounds (Khoddami et al., 2013). In this
study, the exhaustive extraction was performed at room temperature,
Exhaustive (25 min) Microwave (8 min) but with methanol, whose results indicated that the high contents of
3-caffeoylquinic acida
2.58 ± 0.45 1.32 ± 0.04
phenolic components obtained are due to these components contained
delphinidin 3-sambubiosideb 1.20 ± 1.76 0.59 ± 0.21 in the calyx of the hibiscus being very soluble in this solvent.
3-p-coumaroylquinic acida 0.09 ± 0.02 0.05 ± 0.05 The selection of solvent is based in the solubility and polarity of the
cyanidin 3-sambubiosideb 0.36 ± 0.10 0.16 ± 0.04 material to be extracted, in the interaction with the sample, and on its
5-caffeoylquinic acida 1.71 ± 0.36 0.90 ± 0.12
dielectric constant (Cass et al., 2000). Among the solvents selected for
4-caffeoylquinic acida 0.05 ± 0.05 0.03 ± 0.01
myricetin 3-sambubiosidec 0.40 ± 0.17 0.04 ± 0.01 extraction, those with high dielectric constant absorb more strongly the
quercetin 3-sambubiosidec 0.25 ± 0.08 0.10 ± 0.11 energy of microwaves (Oufnac et al., 2007), thus, the water presents
5-o-caffeoylshikimic acida 0.19 ± 0.19 0.08 ± 0.10 good behavior, due to its 80 dielectric constant. It is also worth noting
5-p-coumaroylquinic acida 0.07 ± 0.07 0.06 ± 0.14
that the solvents used in each of the methods are different, methanol in
quercetin 3-rutinosidec 0.31 ± 0.41 0.16 ± 0.47
quercetin 3-glucosidec 0.04 ± 0.05 0.02 ± 0.07
the exhaustive extraction and water in the MAE, having different
kaempferol 3-o -rutinosided 0.03 ± 0.02 0.01 ± 0.02 characteristics, such as polarity, density (methanol: 0.79 g cm−3 and
water: 1 g cm−3).
MAE: microwave assisted extraction. A lower yield in aqueous extraction can be explained by the polarity
HPLC-DAD-ESI-MS/MS: high performance liquid chromatography with diode of the solvent, which yields a lower extraction yield of phenolic com-
array detector coupled with electrospray ionization mass spectrometry. pounds, unlike organic solvents such as ethanol and methanol (Veggi
a
quantified as equivalents of 5-caffeoylquinic acid.
b et al., 2013). Thus, in the case of extraction of 3-sambubioside myr-
quantified as cyanidin equivalents.
c icetin, this compound may have little solubility in water (Lovrić et al.,
quantified as quercetin-3-O-glycoside equivalents.
d
quantified as kaempferol equivalents. 2017) or be a poorly stable compound or indicate the highest extraction
obtained with methanol.
by HPLC-DAD-ESI-MS/MS (Table 1). The identification of these com- It should be emphasized that, due to the good recovery of the
pounds will not be discussed in this study, since it was reported, with compounds present in the calyx of hibiscus and preservation of bioac-
the same mass fragments, in previous studies (Piovesana et al., 2018; tive value in the extracts by the use of aqueous acidic solution and of
Ramírez-Rodrigues et al., 2012; Da-Costa-Rocha et al., 2014). microwaves, it can be considered a good alternative to minimize the use
High-performance liquid chromatography quantification was per- of organic solvents and the generation of toxic residues.
formed only for compounds identified in Table 1, i.e., unidentified
compounds and in smaller amounts were not quantified because of the 4. Conclusions
limits of detection and quantification of the reference compounds.
Among the phenolic compounds, the major components were 3-caf- It was verified the possibility of extraction of phenolic compounds
feoylquinic acid and 5-caffeoylquinic acid, representing in both cases, of the hibiscus calyx by microwave assisted extraction using an acid-
more than 50% of the total phenolic compounds of the extracts. ified aqueous solution. The highest recovery, obtained through of
The total phenolic acids were 4.86 and 2.48 mg g−1 (d.b) for ex- Tukey´s test and hierarchical cluster analysis, corresponded to the
haustive extraction and MAE, respectively. The main phenolic acid was combined extraction of 700 W for 8 min and subsequent extraction in
3-caffeoylquinic acid, with concentrations of 2.58 and 1.32 mg g−1 acid aqueous solution of 6 h, with contents of 1.63 mg g−1 (d.b) of
(d.b) for exhaustive extraction and MAE, respectively. Then, in de- delphinidin-3-sambubioside, 29.7 mg g−1 (d.b) of gallic acid equiva-
creasing order, 5-caffeoylquinic acid is obtained with 1.71 and lent, and 133.25 μmol g-1 (d.b) of Trolox equivalent, for total phenolic
0.90 mg g−1 (d.b), followed by 5-O-caffoylsikimic acid with 0.25 and anthocyanins, and antioxidant activity measured by ABTS, respectively.
0.10 mg g−1 (d.b), of 5-p-coumaroylquinic acid with 0.19 and Microwave assisted extraction used 1/3 of the time used in the ex-
0.08 mg g−1 (d.b) of 3-p-coumaroylquinic acid with 0.09 and haustive extraction, with a recovery of 50% of the obtained compounds.
0.05 mg g−1 (d.b), and 4-caffeoylquinic acid with 0.05 and 0.03 mg g−1 The major compounds found in the extract of the hibiscus obtained by
(d.b), for exhaustive extraction and MAE, respectively. Such com- microwave at 700 W and 8 min and exhaustive extraction with me-
pounds are according to Clifford et al. (2003) and Sinela et al. (2017). thanol were 3-caffeoylquinic acid and 5-caffeoylquinic acid, showing
Among the anthocyanins in the extract of the hibiscus quantified by that the hibiscus can be considered a crop rich in phenolic compounds.
HPLC, delphinidin-3-sambubioside was quantified with contents of 1.20
and 0.59 mg g−1 (d.b) and cyanidin-3-sambubioside with 0.36 and Acknowledgment
0.16 mg g−1 (d.b) for exhaustive extraction and MAE, respectively. The
identification and quantification of these two anthocyanins are ac- The authors thank FAPERGS – RS - Brazil, CAPES - Brazil, and CNPq
cording to Ramírez-Rodrigues et al. (2011) and Sinela et al. (2017) who – Brazil for financial support.
reported them as the majority hibiscus anthocyanins.
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