Plant Foods Hum Nutr (2013) 68:403–410
DOI 10.1007/s11130-013-0391-8
ORIGINAL PAPER
Physical, Chemical, and Antioxidant Activity
Characterization of Pitaya (Stenocereus pruinosus ) Fruits
Leticia García-Cruz & Salvador Valle-Guadarrama &
Yolanda Salinas-Moreno & Elvia Joaquín-Cruz
Published online: 20 October 2013
# Springer Science+Business Media New York 2013
Abstract Fruits with red and orange flesh of the colum-
nar cactus pitaya (Stenocereus pruinosus ) were studied
to evaluate physical characteristics, total soluble solids,
betalains and soluble phenols content, and antioxidant
activity. Fruits had, in average, weight of 179.0 g, 9.8
°Brix, 9.4 % carbohydrates, 1.25 % protein, 0.11 %
ethereal extract, 0.60 % crude fiber, and 0.62 % ash.
Also, fruits resulted rich in Fe (22.8–27.8 mg/kg). Hue
angle and contents of betacyanins, betaxanthins (μg/g
dry sample), and total soluble phenols (mg GAE/g fresh
sample) were 19.8°, 2860.0, 3210.0, and 1.6 in the red
material, and 28.9°, 470.0, 2670.0, and 1.2, respectively,
in the orange fruit. The antioxidant capacity was higher
in the red material, since the ABTS (2,2′-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid)) technique report-
ed 1090.6 and 735.4 μmol of Trolox equivalents/g fresh
flesh in red and orange fruits, respectively, while the
oxygen radical absorbance capacity (ORAC) assay re-
ported 7.84 and 5.16 μmol of Trolox equivalents/g fresh
flesh, respectively. The chromatographic profile showed
five betalains in red fruits, but only four of them were
observed in those orange fleshed.
Keywords Stenocereus pruinosus . Antioxidant activity .
ABTS . Betalains . HPLC . ORAC
L. García-Cruz : S. Valle-Guadarrama (*) : E. Joaquín-Cruz
Departamento de Ingeniería Agroindustrial, Universidad Autónoma
Chapingo, Carretera México-Texcoco, km 38.5,
Chapingo 56230, Mexico, Mexico
e-mail: svalleg@taurus.chapingo.mx
Y. Salinas-Moreno
Laboratory of Crops Quality, Instituto Nacional de Investigaciones
Forestales, Agrícolas y Pecuarias (INIFAP), P.O. Box 10,
Chapingo 56230, Mexico, Mexico
Abbreviations
AA Antioxidant activity
ABTS 2,2′-azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid)
GAE Gallic acid equivalents
HPLC High performance liquid chromatography
HSD Honest significant difference
ORAC Oxygen radical absorbance capacity
TE Trolox equivalents
Tsp Total soluble phenols
TSS Total soluble solids
Introduction
Pitaya (Stenocereus pruinosus) is a columnar cactus used for
its edible fruit occurring in natural forests, silviculturally
managed in agroforestry systems, and agriculturally managed
in home gardens [1] of arid and semi-arid regions, like those
located in the Balsas river basin and the Tehuacan Valley in
Central Mexico, where columnar cacti are used as food,
building material, fodder, living fences, and fuel [2]. In these
zones six variants, named Red, Yellow, Orange, Purple,
White, and Pink, have been identified, and their fruits have
been appreciated and consumed by people for centuries, tak-
ing into account their sweet flavor and pulp color [1]. Cur-
rently, the knowledge we have about pitaya fruits is limited to
morphological characteristics and studies about genetic vari-
ation [2]. However, the color in flesh suggests high betalains
content, and these, due to their antioxidant nature [3], could
impart nutraceutical properties that may have a beneficial
impact on human health.
Betalains are water soluble pigments that provide color in
flowers and fruits. They exhibit antioxidant and radical scaveng-
ing activity [3, 4] but, in contrast to anthocyanins, carotenoids,
404 Plant Foods Hum Nutr (2013) 68:403–410

and chlorophylls, have received little attention [5]. Recently they
have begun to be studied in fruits of cacti, mainly of the genera
Opuntia [6, 7], Hylocereus [4, 8], and just one species of the
Stenocereus genus [9]. On other hand, nowadays there is also
interest in identifying sources of natural pigments as betalains [3].
In this context, the present study was conducted to evaluate
physical and chemical characteristics of S. pruinosus fruits with
red and orange flesh, including betalains analysis, phenolic com-
pounds content, and antioxidant activity, in order to assess their
nutraceutical properties.
Materials and Methods
Plant Material
Two batches of pitaya (Stenocereus pruinosus (Otto)
Buxbaum) fruits, developed in home gardens at Tepexi de
Rodríguez, Mexico (18° 35′ 46″ N; 97º 55′ 48″ W; 1,644
masl), were used. The stage of fruits corresponded to physio-
logical maturity, which is identified when spines are easily
detached and the skin begins to be shiny. The first batch
included two groups of six fruits with red and orange flesh,
respectively (Fig. 1b, c), collected in May of 2011, which were
used to carry out proximal and mineral content analyses, color
evaluation, and betalains identification. The second, collected
in May of 2013, contained two groups of 18 fruits, also with
red and orange flesh, respectively, which were used to perform
a morphological analysis and evaluations of total soluble
solids, acidity, betalains and phenolic compounds contents,
and antioxidant activity.
Physical Characterization
Weight and percentage of flesh and peel were determined with
an Ohaus® scale (USA). Equatorial and polar diameters were
evaluated with a Vernier gauge, taking the ratio of the former
to the latter as shape index. Also, color was expressed as
lightness (L*), hue angle (H*), and chroma (C*), and it was
evaluated with a Hunter Lab colorimeter (Mini Scan XE Plus
45/0-L, USA).
Proximate and Mineral Content Analyses
An Abbe refractometer (American Optical, USA) was used to
evaluate total soluble solids (TSS, °Brix) in juice of fruits.
Acidity was evaluated in the same juice by titration and was
expressed as malic acid percentage. A general proximate
analysis was applied to the lyophilized flesh of fruits using
AOAC [10] methods. Also, minerals content was evaluated
with digestion in acid mixture (HClO4-HNO3) [10], and flame
emission spectrophotometry (3000SC Perkin Elmer, USA).
Betalains Identification
Betalains Extraction
The method described by Wu et al. [4] was applied to pitaya
fruits and also to beetroot (Beta vulgaris) which was used as
reference. Two grams of flesh (including seeds) from each
fruit were macerated and mixed with 20 mL of an 80 % (v/v)
aqueous methanol solution. Samples were sonicated for
10 min in a Branson® bath (USA). Then, they were stirred
in dark for 20 min at room temperature and centrifuged at
2200×g for 10 min (Hettich Zentrifugen, Germany). Super-
natants were recovered and residues were subjected to a
similar second extraction. Supernatants were pooled, filtered
with Whatman paper no. 4, and concentrated to dryness at
40 °C, in a rotary evaporator (Laborata 4010, Germany). The
concentrated materials were re-suspended in 10 mL of 80 %
methanol solution, and stored in amber vials at -20 °C. In this
procedure, seeds were separated during centrifugation phases
without apparent damage.

Fig. 1 Stenocereus pruinosus

columnar cactus (a), red-fleshed
fruit (b), and orange-fleshed fruit
(c)
Plant Foods Hum Nutr (2013) 68:403–410
HPLC Analysis
A high performance liquid chromatograph (HPLC) (Perkin
Elmer, Series 200) was used with a Spherisorb ODS-2 column
(250×4.6 mm; 5 μm) and an UV–VIS photodiode array
detector. Methanol extracts were filtered through a nylon
syringe with membrane of 0.45 μm (Millipore Millex-HN),
and aliquots of 20 μL were injected to the equipment. The
method of Wu et al. [4] was adapted with trifluoroacetic acid
at 0.5 % (A) and acetonitrile (B) as solvents, at A:B ratio of
85:15, flow rate of 0.5 mL/min, run time of 35 min, and a
column temperature of 30 °C.
Antioxidant Compounds Characterization
Betalains Content
The method of Wu et al. [4] was again applied using two
grams of flesh from each fruit to obtain a methanolic extract,
and this was used to determine betacyanins and betaxanthins
concentrations with the method of Castellanos-Santiago and
Yahia [11], through spectrophotometry and the calculation
(B =(A ×DF ×W ×V)/(ε ×P ×L)), where B is betacyanins or
betaxanthins content (mg/g), A is absorbance (538 nm for
betacyanins and 483 nm for betaxanthins), DF is a dilution
factor at the moment of reading, W is the molecular weight
(550 g/mol for betanin and 308 g/mol for indicaxanthin); ε is
the molar extinction coefficient (60,000 L/mol·cm for betanin
and 48,000 L/mol·cm for indicaxanthin), and L is the length
of the cell (1 cm).
Total Soluble Phenols
The Folin-Ciocalteu (FC) method [12] was used to determine
total soluble phenols content: 125 μL of FC reagent:water
(1:1) reacted in assay tubes with 100-μL aliquots of the
methanolic extract for 6 min. After, neutralization was applied
with 1.25 mL of 19 % Na2CO3, and volume was adjusted to
3 mL with distilled water. Mixtures were shaken on a vortex
and left in darkness for 90 min to achieve stabilization. Cen-
trifugation (Hermle Z200 equipment, Labortechnik, Germa-
ny) at 15,300 × g for 10 min removed turbidity and absor-
bance was determined (Perkin Elmer Lambda 25 UV/Vis) at
760 nm. A standard curve was prepared with gallic acid to
express total soluble phenols content in μg of gallic acid
equivalents (GAE)/g of fresh weight.
Antioxidant Activity
ABTS Method
An aqueous 7 mM solution of 2,2′-azino-bis(3-ethylbenzo-
thiazoline-6-sulphonic acid) (ABTS) was combined (2:1) with
405
a 2.45 mM solution of potassium persulfate (PP) and left in
darkness for 16 h to allow free radical generation. Mixture was
diluted with a pH 7.4-phosphates buffer until absorbance was
0.7 at 734 nm [4]. Aliquots of 200 μL of methanolic extracts
reacted during 7 min with 2.8 mL of ABTS-PP solution and
absorbance was measured every minute. The reduced ABTS
was calculated as [%ABTS =(A 0 −A n )100/A 0], where A 0 and
A n were blank and sample absorbances, respectively. Trolox
((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid) at different concentrations (50–300 μM) was used to
express antioxidant capacity in Trolox equivalents.
ORAC Assay
The oxygen radical absorbance capacity (ORAC) was deter-
mined by the method of Ou et al. [13] with minor modifica-
tions. Peroxyl radicals were generated using 2, 2′-azobis (2-
amidino-propane) dihydrochloride (AAPH) (Sigma, St. Lou-
is, MO). Fluorescein (3′,6′-dihydroxyspiro[isobenzofuran-
1[3H],9′[9H ]-xanthen]-3-one) (Sigma, St. Louis, MO) was
used as the fluorescent probe and Trolox was used as standard.
Extracts and reagents were diluted in 75 mM potassium phos-
phate buffer (pH 7.4). Assays were performed in a Synergy
HT microplate reader (Bio-Tek, Winooski, VT) at 37 °C in
flat-bottom black-opaque microplates (Nunc, Langenselbold,
Germany). Sample aliquots of 25 μL were incubated for
30 min with 150 μL of 0.1 μM fluorescein. Afterwards,
25 μL of a 153 mM AAPH solution was added and the
fluorescence decay was recorded for 1 h at 2 min intervals.
The excitation and emission wavelengths were 485 and
530 nm, respectively. The area under curve (AUC ) was cal-
culated for each kinetic fluorescence decay reaction and the
relative ORAC value (μmol Trolox equivalents/g fresh mass)
was calculated as [(AUC S −AUC B )/(AUC T −AUC B )]×(m T −
m S ), where subindex S, B , and T indicate sample, blank, and
Trolox, respectively, and m refers to molarity.
Data Analysis
A completely randomized design was considered to perform
analyses of variance and means comparison routines (Tukey,
0.05) to compare characteristics of fruits type.
Results and Discussion
Physical Characteristics
Fruits averaged 179.0 g, and polar and equatorial diameters
were 8.2 and 6.6 cm, respectively, without difference (P >
0.05) between variants, although red-fleshed fruits were
rounder (P ≤0.05). The mesocarp, which is the edible fraction,
was the largest component (72.5 %; Table 1), and the flesh to
406 Plant Foods Hum Nutr (2013) 68:403–410

peel ratio was significantly higher than proportion found in part of the betalains structure [3]. Results showed that pitaya is
other cactaceae fruits, as those of Opuntia spp. [14], which a fruit rich in Fe (Table 1) and, due to consumption of this
pointed out that Stenocereus pruinosus fruits exhibit a high element may be important in prevention of diseases like
industrial yield potential. Color attributes were different (P ≤ anemia [15], its inclusion in human diet could be recommend-
0.05) between fruit types: a magenta hue was found in red ed, mainly in rural zones where this material is produced.
fruits, which was similar to that observed by Ochoa-Velasco Besides, mineral composition was found different to that of
and Guerrero-Beltrán [9] in fruits of Stenocereus griseus . other cacti. In two species of prickly pear (Opuntia ficus
Also, these fruits had low lightness, and moderate chroma, indica and Opuntia dillenii) the content for K, Ca, and Mg
while orange material had orange-red hue, higher lightness, has been reported higher than values of Table 1, although Fe,
and also higher chroma values (Table 1; Fig. 1b, c). Cu, Zn, and Mn in prickly pear resulted lesser than pitaya [16].
Guzmán-Maldonado et al. [17] found that the content of K
Proximate Analysis and Mineral Content and Ca of gooseberry (Myrtillocactus ) fruits was higher,
although Mg resulted similar, and P, Fe, Cu, Zn, and Mn
The proximate and mineral analyses found slight differences were lesser than quantities found in pitaya.
in humidity, carbohydrates, ethereal extract, K, and Fe (P ≤
0.05) between fruit types (Table 1). The humidity was higher Betalains Identification
than 85 %, carbohydrates represented the second most impor-
tant component, and proteins were the third one, although this The HPLC technique showed peaks of maximum absorption
could be more a measure of nitrogen content, because this is at 483 and 538 nm for betaxanthins and betacyanins,
Table 1 Physical and chemical
characterization of pitaya fruits Variable Red fruits Orange fruits HSD
with red (RP) and orange (OP)
flesh Morphological characteristics
Fruit weight (g) 180.9±72.6 a 177.0±46.4 a 41.2
Mesocarp weight (g) 130.6±55.0 a 128.6±37.0 a 31.7
Epicarp weight (g) 50.3±29.0 a 48.4±23.7 a 17.9
Equatorial diameter (cm) 6.9±0.8 a 6.3±0.7 b 0.5
Polar diameter (cm) 7.9±0.9 a 8.5±0.9 a 0.6
Shape index 0.87±0.06 a 0.75±0.09 b 0.05
Color
Lightness (L*) 19.4±2.7 b 25.7±5.2 a 2.8
Hue angle (H*) 19.8°±4.0° b 28.9°±7.3° a 4.0
Chroma (C*) 24.7±7.2 b 35.0±8.7 a 5.4
Chemical characterization
Total soluble solids (ºBrix) 9.3±1.1 b 10.3±0.9 a 1.0
Titratable acidity (% malic acid) 0.17±0.09 a 0.13±0.05 a 0.07
General proximate analysis
Moisture (%) 87.1±1.4 b 89.0±1.05 a 1.6
Carbohydrates (%) 10.2±0.24 a 8.5±0.16 b 0.6
Protein (%) 1.3±0.05 a 1.2±0.013 a 0.3
Ethereal extract (%) 0.12±0.001 a 0.10±0.001 b 0.01
Crude fiber (%) 0.67±0.09 a 0.53±0.02 a 0.3
Ash (%) 0.63±0.11 a 0.61±0.16 a 0.2
Mineral content
P (mg/kg) 3.6±0.49 a 3.4±0.57 a 0.4
K (mg/kg) 1.1±0.01 b 1.2±0.01 a 0.04
Ca (mg/kg) 0.90±0.14 a 0.95±0.07 a 0.2
Mg (mg/kg) 2.35±0.21 a 2.15±0.07 a 0.3
Fe (mg/kg) 22.8±1.2 b 27.9±1.4 a 2.63
Means with the same letters indi- Cu (mg/kg) 9.67±1.0 a 14.31±1.22 a 8.43
cate non-significant difference Zn (mg/kg) 7.04±5.82 a 8.77±0.28 a 2.86
(Tukey, 0.05)
Mn (mg/kg) 4.61±5.0 a 4.34±0.42 a 0.74
HSD honest significant difference
Plant Foods Hum Nutr (2013) 68:403–410 407

respectively, which was consistent with the report of Stintzing the solvent front, which were numbered from 1–6 at 483 nm,
and Carle [5]. Subsequently, when chromatograms were and from 1′–4′ at 538 nm, as they were sequentially appearing
performed at those wavelengths to analyze samples from (Fig. 2). In the case of red fruits the retention time of peaks 2,
beetroot and pitaya fruits, several peaks were observed after 3, and 5 at 483 nm coincided with that of peaks 1′, 2′, and 4′ at

Fig. 2 Chromatograms of
betalains found in fruits of pitaya
(Stenocereus pruinosus) with red
(a and b) and orange (c and d)
flesh, and also in beetroot (e and
f), analyzed by HPLC. Analyses
of (a, c, and e) were carried out at
483 nm, while in (b, d, and f) a
wavelength of 538 nm was used.
The x-axis is expressed in min
and the y-axis in absorbance units
408
538 nm (Fig. 2a, b), but due to the percentage of relative areas
was higher at 538 nm, it was accepted that compounds iden-
tified by such peaks corresponded to betacyanins. Therefore,
five different types of betalains were found in red material,
among which two corresponded to betaxanthins and three to
betacyanins. Peak 5 was identified as betaxanthin and peak 2
as betacyanin. In the case of orange fruits, four peaks were
observed at 483 nm and two at 538 nm, where peaks 2 and 5
coincided in time with peaks 1′ and 4′ (Fig. 2c, d). However,
due to at 538 nm areas were higher, the corresponding com-
pounds at that wavelength were identified as betacyanins.
Also, based on the retention time, the betacyanin identified
by peak 2′ was only present in the red material. On other hand,
beetroot showed four peaks (1–4) at 483 nm and three (1′–3′)
at 538 nm (Fig. 2e, f). Azeredo [3] pointed out that the most
abundant betalains in red beetroot are betanin, isobetanin, and
betanidin, which may correspond to compounds of peaks 1′,
2′, and 3′ in the present work.
Chemical Characterization
TSS and Acidity
Total soluble solids (TSS) resulted higher (P ≤0.05) in the
orange material (10.3±0.9 °Brix) than in the red one (9.3±
1.1 °Brix), and these values were slightly higher than those
found by Ochoa-Velasco and Guerrero-Beltrán [9] in S.
griseus fruits. On other hand, the acidity was around
0.15 %, without difference between fruits (P >0.05), and
the value from 54.9–79.5 in the ºBrix/acidity ratio sug-
gested that these fruits may provide a sense of sweetness
when consumed.
Fig. 3 Betacyanins (Bc), betaxanthins (Bx), total betalains (Bt), and total
soluble phenolic (Tsp ) content in flesh of beetroot and fruits of
Stenocereus pruinosus. RP red pitaya; OP orange pitaya; AA: Antioxi-
dant activity; TE Trolox equivalents; ds dry sample; fs fresh sample.
Plant Foods Hum Nutr (2013) 68:403–410
Betalains Quantification
Betacyanins (Bc) content (μg/g dry sample) was different (P ≤
0.05) between fruits, with 2,860.0 (±380.0) and 470.0
(±222.0) in red and orange fruits, respectively. Also, there
was difference (P ≤0.05) in betaxanthins (Bx) concentration,
which was equal to 3,210.0 (±560.0) μg/g in the red material
and 2,670.0 (±270.0) μg/g in the orange one (Fig. 3), thus the
total betalains content resulted higher in those red-fleshed
fruits (P ≤0.05). Ochoa-Velasco and Guerrero-Beltrán [9] de-
termined a content of 110 mg/L of betalains in red fruits of
Stenocereus griseus, although it seems higher than the content
of Stenocereus pruinosus, the basis was different, thus the
comparison may not be adequate. On other hand, beetroot
(Beta vulgaris) was used as reference in the present work, and
its Bc and Bx contents were 7,889.0 (±723.0) and 4,381.8
(±394) μg/g, respectively, which resulted greater (P ≤0.05)
than values of red and orange pitaya up to 2.0 and 3.9 times
higher, respectively. In this regard, Georgiev et al. [18]
showed that betalains content in beetroot can be even higher
and concentrations of 39,760.0 μg/g can be found. However,
the pitaya fruits can exhibit greater concentrations of betalains
than other cacti fruits. Therefore, the Bx content of red fruits
was several times higher than concentrations found by
Castellanos-Santiago and Yahia [11] in Rojo Pelota and
Cardona Opuntia spp. fruits, although betacyanins concentra-
tion was similar.
Total Soluble Phenols Quantification
Total soluble phenols (Tsp) content of pitaya fruits resulted
higher (P ≤0.05) in the red material (1.6±0.2 mg GAE/g fresh
Error bars correspond to standard deviation. Means with the same letters
inside each group of bars indicate no statistical difference (Tukey, 0.05).
HSD: honest significant difference
Plant Foods Hum Nutr (2013) 68:403–410
sample) than in the orange one (1.2±0.2 mg GAE/g fresh
sample) (Fig. 3). Ochoa-Velasco and Guerrero-Beltrán [9]
found Tsp content of 0.405 mg GAE/mL in S. griseus, which
is similar to the content of S. pruinosus, but the comparison
may not be adequate since the mass basis was different. Also,
Wu et al. [4] found Tsp values of 0.42 mg of GAE/g of fresh
sample of Hylocereus polyrhizus fruits, which, on dry basis,
were similar to the content found in pitaya fruits. Besides, the
content was similar to values between 0.54 and 1.2 mg GAE/g
found by Coria et al. [19] in Opuntia spp. fruits. However,
Guzmán-Maldonado et al. [17] found values from 9.1–
10.5 mg GAE/g fresh sample of gooseberry (Myrtillocactus),
which points out that phenolic content in cacti fruits can be
variable. In addition, the soluble phenols content of pitaya
fruits was comparable with phenolic content of other fruits,
which are recognized as good source of these compounds, as
blackberry (1–4 mg/g) and blueberry (2.1 mg/g) [20].
Antioxidant Activity
ABTS Method
Antioxidant activities (AA) values of 1,090.6 (±152.8) and
735.4 (±111.6) μmol TE/g fresh sample were found in red and
orange pitaya, respectively (Fig. 3), and the difference was
significant (P ≤0.05), which showed that red fruits had higher
AA than orange fruits. However, the AA of red fruits was
lower than activity of S. griseus, since Ochoa-Velasco and
Guerrero-Beltrán [9] reported a value of 92 mg TE/100 mL of
juice (3,550.0 μmol TE/mL), and the difference is due to the
fact that these authors used juice instead of flesh in assess-
ments. In addition, these results indicated that the pitaya fruits
exhibit higher antioxidant potential than fruits of Opuntia
spp., since Zafra-Rojas et al. [21] reported values around
4368.5 μmol TE/L and Coria et al. [19] found values between
0.13 and 0.33 mg/g (33.7 and 85.6 μmol/g).
ORAC Assay
The relative oxygen radical absorbance capacity (ORAC)
value was 7.84 (±1.98) and 5.16 (±1.26) μmol of Trolox
equivalents/g fresh flesh in the red and orange fruits, the
difference being significant (HSD=1.77 μmol of Trolox/g),
which confirmed that the red material possesses higher anti-
oxidant activity than the orange-fleshed one. Wolfe et al. [22]
evaluated ORAC values for 25 common fruits in the range
from 5.65 to 96.21 μmol of Trolox equivalents/g fresh flesh,
where berries had the highest antioxidant activity and fruits
like mango, pineapple, banana, and cantaloupe the lowest.
According to this, the pitaya fruits had an antioxidant activity
comparable to the latter fruits.
409
Conclusion
The fruit of pitaya (Stenocereus pruinosus) possesses high
°Brix/acidity ratio, is rich in carbohydrates, iron, betalains,
and soluble phenols compounds. Since the mesocarp, the
edible fraction, is present in high proportion, this material
constitutes a good source of nutritional elements. Five types
of betalains are present in the red material, two correspond-
ing to betaxanthins and three to betacyanins. However, in
the orange material only two betacyanins and two
betaxanthins are present, which causes differences in color
between variants. The ABTS and ORAC techniques
showed a moderate to high antioxidant activity, and the
red material had the highest potential to be considered a
nutraceutical product.
Conflict of Interest All authors confirmed that they have no conflict of
interest.
Studies with Humans or Animals This article does not contain any
studies with human or animal subjects.
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