Total
phenolic contents and determination of Antioxidant activity by DPPH, FRAP, and
cyclic voltammetry of the fruit of Solanum nigrum (black nightshade)
growing in the south of Algeria
Imene Guediri1,4, Chérifa Boubekri2,4*,
Ouanissa Smara1,5 and Touhami Lanez3,4
1University Kasdi Merbah, Faculty of Material Science, Department of
Chemistry,
Ghardaia BP Road 511.
30,000, Ouargla, Algeria
2*University Mohamed
Khider, Faculty of Exact sciences, Natural and Lile Sciences,
Department of
Material sciences, BP145 RP Biskra 07000 Algeria.
3University of Echahid Hamma Lakhdar, Faculty of Exact sciences, PO Box 789,
39000, El Oued, Algeria
4University of El Oued, VTRS Laboratory, B.P.789, 39000, El Oued, Algeria
5Laboratory Valorisation et Promotion des Ressources Sahariennes (VPRS),
30000, Ouargla, Algeria
ABSTRACT:
In the present research, we studied the quantification
of the total phenolics, total flavonoids, total flavonols, and the evaluation
of the antioxidant activity of the fruits of Solanum nigrum L. by
chemical and electrochemical methods. The chloroform, ethyl acetate, and
n-butanol crude extracts are prepared from the hydroethanolic extract of the
fruits were tested. The results showed that ethyl acetate extract showed the
highest phenolic content (87.68mg GAE/g), chloroform extract showed the highest
flavonoid content (24.23mg QE/g) and highest flavonol content (71.60mg RE/g).
The evaluation of antioxidant activity by chemical methods revealed that the
highest DPPH radical scavenging activity was shown by ethyl acetate extract
(0.123mg/mL) while the highest ferric reducing capacity was shown by butanol
extract (610.46±0.0015mg FeSO4/g). The electrochemical study of the three
extracts by cyclic voltammetry method indicates that the highest antioxidant
activity was shown by ethyl acetate extract (17.75±0.238mg/g) equivalent
ascorbic acid. The superoxide scavenging assay of chloroform extract showed the
highest value (0.8 mg/mL).
KEYWORDS: Solanum nigrum L., fruit
extraction, phenolic profiles, antioxidant activity, cyclic voltammetry, superoxide anion radical.
INTRODUCTION:
Phenolic compounds are
commonly found in plants and have been reported to have several biological
activities including antioxidant properties and are useful indicator of
potential nutritional benefit [1]. Experimental studies revealed that cells and
organisms require defense against oxidants [2]. Free radicals such as hydroxyl
radical and superoxide anion are produced as normal products of cellular
metabolism [3].
Production
of free radicals have been implicated to cause several diseases [4], it is
usually necessary to evaluate the free radical-scavenging activity of herbal
extracts because they represent the common form of usage of the plant in
medicine as well as in food technology. Often, differential antioxidant
activities of a herbal extract are determined against different methods [5].
Several methods for evaluation of the antioxidant activity of compounds have
been developed. Cyclic voltammetry is a unique technique for the
electrochemical characterization of compounds [6]. Electrochemical methods of
analysis are preferable due to the advantages of high sensitivity, simplicity,
good stability, inexpensive and portable instrumentation, and less interference
from non-electroactive substances [7]. Oxidation or reduction peaks are
proportional to the concentration of a chemical species that oxidizes or
reduces the electrode. In CV, the potential sweep is cycled over time. The
potential cycling enables the visualization of the forward and backward
reactions. CV can be used to identify the potentials at which active redox
couples are oxidized and reduced and are an electrochemical technique [8]. Solanum
nigrum or «Black nightshade» belongs to the family Solanaceae is native to
Eurasia and was introduced in many parts of the world, it is widely used in
conventional medicine [9]. Pharmacological studies indicated that the plant
extract has a potential application for treating many types of cancer,
including liver cancer, cervical cancer, breast cancer, lung cancer, stomach
cancer, colon cancer, skin cancer, and bladder cancer [10,11]. It exhibited a
variety of biological activities including inflammation, infection, and
oxidative damage [12–14]. The present study aims to determine the content of
polyphenols, flavonoids, and flavonols in the fruit of Solanum nigrum
and to evaluate the antioxidant activity by the chemical method using DPPH,
FRAP, and electrochemical method. The interaction between different prepared
extracts of fruit and superoxide anion radical was evaluated. It was selected
because of its presence in the human body it has a longer half-life and is
capable of generating other harmful radicals such as hydroxyl radical [15].
MATERIALS AND
METHODS:
Plant
material:
The fruit of the black
nightshade was collected in October 2017 from the Debila, El Oued (south of
Algeria), and identified by the Botanist Halis Youssef (Biology Department,
Echahid Hamma Lakhdar University, El Oued, Algeria). The essential aim of this
work was to research the phenolic profile and the antioxidant capacity of three
extracts (chloroform, ethyl acetate, and n-butanol) of the fruit of Solanum
Nigrum.
Preparation
of sample:
The fruits of the plant
were washed with water and cleaned from insects, coarse parts, and gravel. They
were divided into small pieces and dried for several days, they were stirred
twice each day without exposure to the sun. the dried fruits were ground into
powder using an electric grinder.
Procedure
of extraction:
Air-dried
fruits (200g) of Solanum Nigrum were macerated at room temperature with
ethanol/water (80:20 v/v) for twenty-four h, 3 times. After filtration, the
filtrates were combined and concentrated up to 55°C under reduced pressure. the
obtained residue was diluted with distilled water and left to count overnight
in order to precipitate the maximum of chlorophyll. After filtration, the
resulting solution was successively extracted several times with n-hexane,
chloroform, ethyl acetate, and n-butanol. The organic phases were dried with Na2SO4,
filtered using filter paper, and concentrated until to get dry extracts.
Determination of total phenolic content:
The total phenolic
content of fruits of Solanum
Nigrum
was determined spectrophotometrically by the method
given by singleton et al. [16] involving Folin-Ciocalteu reagent and gallic
acid as standard with slight modifications. The reaction mixture was prepared
by mixing 100µL extract with 500µL 10% Folin-Ciocalteu’s reagent in water and
2mL 20% Na2CO3 aqueous. The mixture was incubated within the dark at temperature for
30 min and therefore the absorbance was read at wavelength 760nm using UV-Vis
spectrophotometer, analysis
was wiped out triplicate for every extract. Standard solutions of gallic
acid with different concentrations were
wont to obtain a typical curve. The total phenolic content was expressed
as milligrams of gallic acid equivalent (GAE) per gram of extract.
Determination of total flavonoid content:
The determination of total flavonoid content was
supported on the method described by Bahorun et al. [17] with slight
modifications using the aluminum chloride method. 1mL extract was mixed with
1mL 2% aluminum chloride (AlCl3). The mixture was incubated within
the dark at room temperature for 10 min and measured spectrophotometrically at
430nm against the blank. The analysis was wiped out triplicate for every extract.
The flavonoid content of different tested extracts was estimated by using the
quercetin standard calibration curve and therefore the obtained results of
flavonoids were expressed as milligrams of quercetin equivalent (QE) per gram
of extract.
Determination of total flavonol content:
The
total flavonol content was determined by using the method described by Kumaran
and Karunakaran [18] with slight modifications. Brief, 1mL extract was mixed
with 1mL 2% aluminum chloride (AlCl3), and three mL (50 g/L) sodium
acetate, the solution was incubated within the dark at room temperature for two
hours and therefore the absorbance was read by UV-Vis spectrophotometer at
wavelengths 440nm. The experiment was performed in triplicate. The total
flavonol content was calculated from the quality calibration curve constructed
with various concentrations of rutin and expressed as milligrams of rutin
equivalent (RE) per gram of extract.
Determination
of antioxidant activity:
The antioxidant
activity of phenolics is especially
due to their redox properties, which permit
them to act as reducing agents, hydrogen donators, and singlet oxygen
quenchers. Additionally, they
possess a metal chelation potential [19]. Therefore, it is important to search
for the safe and effective natural antioxidants by using various screening
methods [20].
DPPH
radical scavenging assay:
The DPPH
(1,1-diphenyl-2-picrylhydrazyl) radical-scavenging effect was estimated using
the method described by Villano et al. with some modifications [21]. Briefly,
1mL extract was mixed with 1mL DPPH solution prepared in methanol and incubated
within the dark at room temperature for 30min. the absorbance of the solution
obtained was measured at 517nm. Ascorbic acid was used as a standard which we
can compare its effect. The activity of the sample to scavenge DPPH radical was
determined from the equation below, where Ac and As are respectively control
absorbance and sample absorbance:
Ferric reducing antioxidant potential (FRAP):
The ferric reducing capacity of extracts was
determined by using the method described by Benzie and Strain [22]. Briefly,
100µL extract was added to 300µL distilled water and three mL of the FRAP
reagent. The FRAP reagent is freshly prepared just before the test by mixing
25mL of acetate buffer (0.3M) at pH = 3.6 and 2.5mL of 2, 4,
6-tripyridyl-s-triazine (TPTZ) (10mM) in HCl (40mM) and 2.5mL of ferric
chloride (20mM). The mixture was incubated for 30 min and the wavelengths were
measured at 593nm. FeSO4 was used as a standard with varying
concentrations. The results were expressed as mg Fe(II)/g of extract.
Cyclic
voltammetry assay:
Measurement of reducing capacity and electrochemical
behavior of compounds may provide useful information about the free radical
scavenging activity of naturallly occurring compounds [23]. The experimentation
consists of a PGP 301 potentiostat associated with an electrochemical cell with
a volumetric capacity of 25mL containing a glassy carbon working electrode 3 mm
diameter, polished before each measurement, a platinum wire counter electrode,
and a Hg/Hg2Cl2 reference electrode saturated with KCl
were used. Cyclic voltammogram was obtained by one single cycle performed at a
scan rate of 100 mV.S-1 and therefore the potential was swept in
direct scanning mode ranging from 00 to +1000mV. Ascorbic acid was used as a
standard in the calculation of the antioxidant capacity of the studied extracts
of the fruits of black nightshade. phosphate buffer at pH=7 was the supporting
solution (20mL, 0.2M).
Superoxide
scavenging assay:
The cyclic
voltammetry technique was used to generate the superoxide radical anion by the
one-electron reduction of oxygen molecular saturated in DMF media [24]. 10mL
of DMF solution containing the supporting electrolyte Bu4NBF4
was saturated by molecular oxygen. The cyclic voltammogram measurements of the
oxygen reduction were run from −1.6 to 0.0 V at a scan rate of 100 mV.S−1.
The capacity of the plant extract to quench 
was calculated using the equation :
The results were
expressed as percent inhibition (I %), where i0 and is
are the anodic peak current densities of the superoxide anion radical in the
absence and the presence of extract.
RESULTS AND
DISCUSSION:
Extraction
yield:
The mass of varied
polarities extracts from the fruit of the black nightshade is presented in
Table 1. The results revealed that the very best yield extracts were obtained
by the n-butanol (1.53%) and chloroform (0.15%) respectively, followed by ethyl
acetate (0.065%) and n-hexane (0.047%).
Table 1. Extract yield of the fruits of Solanum Nigrum.
|
Fruit extract |
Extract yield % (w/w) |
|
n-hexane |
0.047% |
|
Chloroform |
0.15% |
|
Ethyl acetate |
0.065% |
|
n-Butanol |
1.53% |
The difference in
the yields of extracts might be attributed to the difference in solvent
polarities used which also plays a key role in increasing the solubility of
phytochemical compounds [25]. This result justified by the polarity effect of
the solvents.
Determination of total phenolic content:
Phenolic compounds
contain hydroxyl groups which contribute to the free radical scavenging and act
as primary antioxidants [26]. Total phenolic content in different extracts of
the fruit of black nightshade was
calculated in terms of gallic acid equivalent (Figure 1) using the standard
curve equation y = 3.7143x - 0.0914, R˛ = 0.9951. Ethyl acetate extract
contains the highest phenolic content (87.68mg GAE/g) as compared to the
n-butanol (70.65 mg GAE/g) and chloroform (52.70mg GAE/g) extracts (Table 2,
Figure 4).
Figure 1. Calibration curve of standard gallic acid
for determination of total phenolic content.
Ethyl acetate
extract is expected to exhibit good results in antioxidant activity. The
various extraction solvents had an interesting effect on the extraction ability
of phenolics depending on their polarities, solubilities, and chemical
structures [27]. The total phenolic concentration could be used as a basis for
the rapid screening of antioxidant activity [28]. A number of scientific papers
indicate the phenolics as effective contributor to antioxidant activity and
inhibit oxidative mechanisms [29]. The antioxidative properties of phenolic
components have been attributed to their potential to chelating transition
metal ions, the inhibition of superoxide-driven Fenton reaction, hindering the
diffusion of free radicals, and modifying lipid peroxidation kinetics in
membranes by altering lipid pecking order [30]. A previous study demonstrated
that the fruits of Solanum Nigrum contain eight phenolic compounds [31].
Figure 2.
Calibration curve of standard quercetin for determination of total flavonoid
content.
Table 2. Total
phenolic content of the fruit of Solanum Nigrum chloroform, ethyl acetate, and n-butanol extracts.
|
Fruit extract |
Total phenolic content (mg GAE/g) |
|
Chloroform |
52.70±0.0032 |
|
Ethyl acetate |
87.68±0.0005 |
|
n-Butanol |
70.65±0.0036 |
Chloroform extract had the very best flavonoid content
(24.23mg QE/g) followed by ethyl acetate extract (18.21mg QE/g) and therefore
the lowest (12.97mg QE/g) was given by n-butanol extract (Table 3, Figure 4).
This suggests that this plant contains more flavonoid aglycones than
heterosides. The importance of flavonoids in foods and herbal extracts appears
in their protective effects against a spread of diseases associated with ROS
through their capacity to transfer free radical electrons, chelate metal
catalysts, activate antioxidant enzymes, reduce α-tocopherol radicals and
inhibit oxidation [33].
Table 3. Total flavonoid content of the fruit of Solanum Nigrum chloroform, ethyl acetate and n-butanol extracts.
|
Fruit extract |
Total flavonoid content (mg QE/g) |
|
Chloroform |
24.23±0.008 |
|
Ethyl acetate |
18.21±0.0025 |
|
n-Butanol |
12.97±0.0103 |
Determination of total flavonol content:
The total flavonol content within the various extracts
were demonstrated in term of rutin equivalent (Figure 3) using the standard
curve equation y = 3.4583x + 0.0198, R˛ = 0.999. Chloroform extract had the
highest flavonol content (71.60mg RE/g) followed by ethyl acetate extract
(56.68mg RE/g) and therefore the lowest (22.97mg RE/g) was given by n-butanol
extract (Table 4, Figure 4). This suggests that this plant contains more
flavonol aglycones than heterosides.
Figure 3.
Calibration curve of standard gallic acid for determination of total flavonol
content.
However its wide
distribution in medicinal plants, the flavonol fraction represents relatively
minor concentrations within the
phenolic mixture. Epidemiological studies analyzing the dietary effect of one
class of flavonoids, the flavonols, suggested that a high dietary intake of
flavonols correlated with a reduced risk of coronary heart disease.
Table 4. Total flavonol content of the fruit of Solanum Nigrum chloroform, ethyl acetate, and n-butanol extracts.
|
Fruit extract |
Total flavonol content (mg RE/g) |
|
Chloroform |
71.60±0.0191 |
|
Ethyl acetate |
56.68±0.0026 |
|
n-Butanol |
22.97±0.0183 |
Figure 4. Total
phenolic, total flavonoid, and total flavonol contents of the fruit of Solanum Nigrum chloroform, ethyl acetate, and n-butanol extracts.
Evaluation of antioxidant activity:
DPPH radical scavenging activity:
DPPH assay is based on the measurement of the
scavenging ability of antioxidant towards the stable DPPH radical [34]. It is
free radical having a violet color in alcoholic solution, in the presence of an
antioxidant substance, the reduction reaction leads to a yellow color [35]. The
DPPH radical scavenging activity was evaluated by IC50, therefore
the results were presented in (Table 5, figure 5). The very best result was
obtained in ethyl acetate extract (0.123 mg/ml) followed by n-butanol extract
(0.380mg/ml) and therefore the lowest (1.001 mg/ml) was obtained in chloroform
extract. However, ascorbic acid (0.007mg/mL) exhibited higher antioxidant
activity than all Solanum Nigrum fruits extracts.
Table 5. DPPH scavenging activities of Solanum
Nigrum chloroform, ethyl acetate,
and n-butanol extracts.
|
Sample |
IC50 mg.mL-1 |
|
Chloroform |
1.001 |
|
Ethyl acetate |
0.123 |
|
n-Butanol |
0.380 |
|
Ascorbic acid |
0.007 |
Solvents used for polyphenol extraction had
significant effects on DPPH scavenging capacity determination for the fruit of Solanum
Nigrum. The concentration of an antioxidant needed to decrease the initial
DPPH concentration by 50% (IC50) is widely used to evaluate the
antioxidant activity [36].
Figure 5. DPPH scavenging capacity determination for
the fruit of Solanum Nigrum chloroform, ethyl acetate and n-butanol
extracts.
Ferric reducing antioxidant potential:
The FRAP assay
evaluates the metal reduction by antioxidants using electron donation [37]. The
results of the antioxidant capacity assessed by the FRAP assay are presented in
(Table 6, Figure 6).
Table 6. Ferric reducing antioxidant activities of Solanum
Nigrum chloroform, ethyl acetate and
n-butanol extracts.
|
Extract |
FRAP (mg FeSO4/g) |
|
Chloroform |
134.36± 0.001 |
|
Ethyl acetate |
299.52±0.002 |
|
n-Butanol |
610.46±0.0015 |
They revealed that
the very best ferric reducing capacity was shown by n-BuOH extract
(610.46±0.0015 mg FeSO4/g), while ethyl acetate extract exhibited a
significantly lower antioxidant capacity (approximately 50% lower). Chloroform
extract, on the other hand, led to the lowest antioxidant capacity (134.36±
0.001 mg FeSO4/g). Ferric reducing antioxidant power values showed that the
n-Butanol extract might be classified as extract with a strong ability to scale
back the ferric complex.
Figure
6. Ferric reducing antioxidant activities for the fruit of Solanum Nigrum chloroform,
ethyl acetate and n-butanol extracts.
Cyclic
voltammetry assay:
There
was no previous study that dealt with the antioxidant activity with cyclic
voltammetry assay of the fruit of Solanum Nigrum. The equation obtained
from the linear calibration of various standard concentrations of ascorbic acid
(Figure 7), Y= 75.95 X + 3.332, R˛ = 0.988. y represents the worth of the
anodic current density and x, the worth of standard concentrations. The
concentration of electro active species present in a solution also plays a
major role in determining the response observed in a voltammetric experiment
[38]. The diminution in peak current density was proportional to the diminution
in ascorbic acid concentrations. The antioxidant capacity of different extracts
of fruit of Solanum Nigrum was expressed in equivalent terms of ascorbic
acid equivalent (mg/g). All the experiments were performed in triplicates.
Cyclic voltammetry is often wont to identify the potentials at which active
redox couples are oxidized and reduced and are an electrochemical technique
capable of monitoring redox reactions [39]. Figure 8, represent cyclic
voltammograms of the three extract; chloroform, ethyl acetate, and n-butanol,
they were recorded employing a scan rate of 100 mV.s-1.
Figure
7. (a) Cyclic voltammograms referring to different ascorbic acid concentrations.
(b) calibration curve obtained by the cyclic voltammetry method expressed as
ascorbic acid equivalent.
|
|
|
|
Figure
8. Cyclic voltammograms of the fruit of Solanum Nigrum chloroform, ethyl acetate, and n-butanol extracts
Electrochemical
data obtained from voltammograms of the fruit of Solanum Nigrum chloroform, ethyl acetate, and n-butanol extracts are
summarized in table 7.
Table 7. Electrochemical data of the fruit of Solanum
Nigrum chloroform, ethyl acetate, and
n-butanol extracts.
|
Extract |
Epa (V) |
ipa (µA/cm2) |
|
Chloroform |
0.636 |
4.02 |
|
Ethyl acetate |
0.272 |
5.49 |
|
n-Butanol |
0.265 |
3.843 |
Only one anodic peak appeared for them (Figure 8). No
cathodic peak was observed on inverting the scan direction indicating the
irreversibility of oxidation of the three studied extracts. The peak anodic
current (ipa ) was the highest for ethyl acetate extract (5.49μA) while it
was minimum for n-Butanol extract (3.843μA). Since all the extracts were
used at the same concentration, higher ipa might be a sign of the upper antioxidant content of those three
extracts.
Table 8. Antioxidant capacities of the fruit of Solanum
Nigrum chloroform, ethyl acetate, and
n-butanol extracts.
|
Extract |
Ascorbic acid antioxidant capacity (mg/g) |
|
Chloroform |
4.53±0.010 |
|
Ethyl acetate |
17.75±0.238 |
|
n-Butanol |
3.37±0.010 |
Cyclic voltammetry
is a method used in electrochemistry for analysis, it provides important
results. Table
8, represents the ascorbic acid equivalent antioxidant capacity of the fruit of
Solanum Nigrum chloroform, ethyl
acetate, and n-butanol extracts calculated from the calibration graph. The
results indicate that ethyl acetate extract was the foremost effective with the
highest capacity
(17.75±0.238mg/g) followed by chloroform extract (4.53±0.010mg/g). n-butanol extract presents the lowest one (3.37±0.010mg/g) (Figure
12).
Superoxide
scavenging activity:
The superoxide anion radical was generated by
one-electron reduction of the atmospheric molecular oxygen (O2)
dissolved in DMF, at room temperature and the resultant cyclic voltammogram
response is presented in Figure 9.
Figure 9. Cyclic
voltammograms of oxygen in saturated DMF with supporting electrolyte Bu4NBF4
on GC as a working electrode at a scan of rate 100 mV/s.
The extract
volumes were added incrementally (0.1 ml to 1ml) to research their effect on
superoxide anion radical. Extract injection causes a proportional decrease in
the anodic current while the effect on the cathodic current was negligible.
Linear graphs of % changes in terms of concentrations permit to calculate the
IC50 values in (mg/mL); the concentration (mg/mL) of extract that inhibits the
formation of radicals by 50%. (figure 10, 11 and 12). The obtained results
indicate that the chloroform extract of the fruit of Solanum Nigrum
showed the highest scavenging activity (0.8 mg/mL) followed by butanol extract
(0.97mg/mL). The ethyl acetate extract was the lowest one (1.82mg/mL), Table 9,
Figure 13.
Table 9. 
radical scavenging activities of the fruit of Solanum
Nigrum chloroform, ethyl acetate, and
n-butanol extracts.
|
Fruit extract |
Equation |
R2 |
IC50 (mg/mL) |
|
CHCl3 extract |
y = 53.14 x + 7.618 |
0.971 |
0.8 |
|
ETOAc extract |
y = 25.02 x + 4.566 |
0.991 |
1.82 |
|
n-BuOH extract |
y = 43.53 x + 7.586 |
0.995 |
0.97 |
Figure 10. (a)
Cyclic voltammograms of chloroform extract at a scan rate of 100 mV/s. (b) Graphs
of % changes in terms of concentrations of chloroform extract, of the fruit of Solanum
nigrum.
Figure 11. (a) Cyclic voltammograms of ethyl acetate
extract at a scan rate 100 mV/s. (b) Graphs of % changes in terms
of concentrations of ethyl acetate extract, of the fruit of Solanum nigrum.
Figure 12. (a) Cyclic voltammograms of n-butanol extract
at a scan rate 100 mV/s. (b) Graphs of % changes in terms
of concentrations of n-butanol extract, of fruit of Solanum nigrum.
Figure 13. Antioxidant capacities of the fruit of Solanum
Nigrum chloroform, ethyl acetate, and
n-butanol extracts. (a) % radical scavenging activity (mg/mL). (b) Ascorbic acid antioxidant capacity (mg/g).
CONCLUSION:
In this study, we
report for the first time the evaluation of the effects of solvent polarities
on the content of phenolics, flavonoids, flavonols, and antioxidant activity
from the fruits of Solanum nigrum. The results showed that the solvent
used in extraction affects the yield, total contents, and antioxidant capacity.
Chemical and electrochemical methods were used for evaluation of the
antioxidant activity of the three crude extracts, different methods could give
different results. Cyclic voltammetry is expected to be a simple method for screening
antioxidants and estimating the antioxidant activity of medicinal plants. The
interaction of all extracts with superoxide anion radical was evaluated and all
results were electroactive.
ACKNOWLEDGMENT:
The authors were
grateful to Mr. Ali Tliba from the Laboratory of Valorisation and Technology of
Saharan Resources (VTRS), University of El Oued, staff for his assistance.
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Received
on 05.09.2020
Modified on 29.09.2020
Accepted
on 14.10.2020
©AJRC All right reserved
Asian
J. Research Chem. 2021; 14(1):47-55.
DOI:
10.5958/0974-4150.2021.00008.0