Int J Curr Pharm Res, Vol 12, Issue 2, 78-84Original Article


ANTIMICROBIAL AND ANTIOXIDANT EFFECT OF NATURAL EXTRACTS FROM LEAVES, ROOT, STEM AND FLOWERS OF BACCHARIS LATIFOLIA FROM ECUADOR

FAVIAN BAYAS-MOREJON*, ANGELICA TIGRE, RIVELINO RAMON, DANILO YANEZ

Centro de Investigación y Desarrollo Biotecnologico, Facultad de Ciencias Agropecuarias, Universidad Estatal de Bolivar, CP: 020150, Guaranda Ecuador
Email: fbayas@ueb.edu.ec

Received: 22 Nov 2019, Revised and Accepted: 21 Jan 2020

ABSTRACT

Objective: The increase in chronic and degenerative diseases and the use of synthetic antioxidants such as (butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)) are being restricted because they can be considered carcinogenic. Therefore, there is a growing interest in the search for natural antioxidants, especially from plants, due to their content in different bioactive compounds, such as antioxidants and antimicrobials.

To evaluate the antibacterial and antioxidant activity of Baccharislatifolia extracts.

Methods: For the determination of the antimicrobial activity of extracts of leaves, root, stem and flowers of Baccharislatifolia (Bl), the disk plate diffusion method was used, the strains of Listeria, Salmonella and E. coli were studied; antibiotics Penicillin G and Ciprofloxacin were the controls. For the antioxidant activity, a solution of H2O2 (Abs at 230 nm) was prepared in Potassium Phosphate Monobasic-Sodium Hydroxide buffer.

Results: The antimicrobial activity against Listeria and Salmonella, showed that the extracts of leaves and flowers were more effective with inhibition zones>15 mm and>20 mm respectively. In front of E. coli, the extracts of flowers and stem were the best with zones>7.0 mm. Antibiotics studied inhibited the development of Listeria and Salmonella. However, E. coli isolates were resistant. In the antioxidant activity, the flower extract of Bl in 60 mg/ml presents a higher effect with 47.25%.

Conclusion: Bl extracts from leaves and flowers were more efficient both in their antimicrobial and antioxidant capacity.

Keywords: Baccharislatifolia, Natural extracts, Antimicrobial, Antioxidant

© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2020v12i2.37495. Journal homepage: https://innovareacademics.in/journals/index.php/ijcpr

INTRODUCTION

The continuous increase of microorganisms resistant to different antimicrobial agents has been a major problem for health and food safety, it is also known that with the appearance of antibiotics the lives of millions of human beings have been saved, however, is approaching a reality of dimensions not yet considered.

Among the most used antibiotics, are the antibiotics from the group of Fluoroquinolones [1]. On the other hand, Penicillin G, according to Flores et al. [2], is an antibiotic belonging to the group of Beta-lactams, they have a mechanism of action inhibiting the cell wall of the bacteria, especially Gram+such as Listeria, Salmonella, E. coli [3-5].

The world could be in a serious phase caused by multiple lethal bacteria resistant to antibiotics, this due as the lack of innovation and development of new antibiotics, especially of a natural origin [6]. The antibiotics derived especially from vegetables (medicinal plants), have been proven to be less toxic than synthetic agents [7].

The use of extraction techniques to obtain substances with bioactive principles is of great importance at the time of obtaining the component, even, when orthodox medicines are available, a large percentage of the population still uses herbal remedies together with conventional medicines [8, 9].

These compounds are obtained by contact with solvents through extraction techniques like maceration. The effectiveness of extraction generally depends on the polarity and nature of the solvent used [10].

Antioxidant activity of plants

Oxidative damage caused by free radicals is related to the development of various diseases such as atherosclerosis, cancer, arthritis and other inflammatory diseases [11]. The existence of synthetic substances (Butylated hydroxyanisole (BHA) or Butylated hydroxytoluene (BHT)) that are efficient scavengers of free radicals; but they are being restricted because they can be considered carcinogenic [12]. So, there is a growing interest in the search for antioxidants of natural origin, especially from plants. In most cases, the antioxidant activity of these plants is mainly due to the presence of phenolic compounds, which are powerful oxygen scavengers and are also capable of inhibiting enzymes that produce free radicals [13].

The chilca (Baccharis latifolia) (fig. 1), is one of the 46 species of Baccharis genus that is widely distributed in Ecuador, in provinces such as Pichincha, Imbabura, Cañar, Cotopaxi, Chimborazo, Bolívar, Azuay, Loja, Napo, Sucumbíos and Zamora Chinchipe [14].

Fig. 1: Baccharis Latifolia plant

Baccharis latifoliait is commonly used in poultices to relieve external inflammations, fractures, dislocations and rheumatic pains; in infusions, it is used as an antidiarrheal, for asthma, menstrual pains, antidiabetic and insomnia [15]. Also, Baccharislatifolia, has been used in Latin America for medicinal purposes, such as antidiarrheal, anti-inflammatory, antidiabetic, antidepressant, analgesic and disinfectant of wounds, ulcers and antimicrobial [16].

Objective

The objective of the present study was; to determine the antimicrobial and antioxidant activity of the extracts of Baccharislatifolia.

MATERIALS AND METHODS

The leaves, stems and roots of Baccharislatifolia (Bl) were collected from young plants during the months of July 2017 to July 2018 on the grounds of the Faculty of Agricultural Sciences of the Universidad Estatal de Bolívar (Ecuador). The selected plants were clean and free of damage.

Preparation of extracts

The selected leaves, stems and flowers were exposed to maceration during 6 d in 96% ethyl alcohol in a ratio 50 gr of the vegetal matter: 100 ml of alcohol, after this time, the extracts were obtained by centrifugation (Sigma, 3-16C, United Kingdom) at 10,000 rpm and filtration using of cellulose filters pore size 2.5 μm (Whatman, 1001-110, USA). The preparation of the extracts was done in duplicate and the extracts with better performance were used to Antimicrobial analysis

Collection of bacterial inocula

Baccharislatifolia Extracts (Bl-E) were tested against three bacterial genera, Escherichia coli, Salmonellaspp and Listeriaspp, these bacterial strains were provided by the Molecular Biology laboratory of the Research Department of the Universidad Estatal de Bolívar. A number of 10 isolated for each bacterial genus isolated from meats were used in the study, as control were used the type strains of the Listeriaspp, Salmonellaspp and Escherichia, the bacterial strains used are described in table 1.

Table 1: Microorganisms used in the study

Type of meat (origin) Sample number and selected colony Code Identified microorganism
Beef Sample 1-Colony 2 B1C2 Listeria spp
Beef Sample3-Colony2 B3C2 Listeria spp
Chicken Sample3-Colony1 C3C1 Listeria spp
Chicken Sample4-Colony2 C4C2 Listeria spp
Chicken Sample5-Colony1 C5C1 Listeria spp
Chicken Sample6-Colony1 C6C1 Listeria spp
Chicken Sample8-Colony2 C8C2 Listeria spp
Chicken Sample14-Colony3 C14C3 Listeria spp
Chicken Sample18-Colony1 C18C1 Listeria spp
Pork Sample19-Colony1 P19C1 Listeria spp
ATCC 33090 Listeria innocua
Beef Sample3-Colony2 B3C3 Salmonella spp.
Beef Sample5-Colony1 B5C1 Salmonella spp.
Beef Sample15-Colony3 B15C3 Salmonella spp.
Beef Sample27-Colony1 B27C1 Salmonella spp.
Chicken Sample2-Colony1 C2C1 Salmonella spp.
Chicken Sample13-Colony2 C13C2 Salmonella spp.
Pork Sample1-Colony3 P1C3 Salmonella spp.
Pork Sample1-Colony5 P1C5 Salmonella spp.
Pork Sample5-Colony3 P5C3 Salmonella spp.
Pork Sample14-Colony3 P14C3 Salmonella spp.
ATCC 13314 Salmonella arizonae
Beef Sample2-Colony1 B2C1 Escherichia coli
Beef Sample3-Colony1 B3C1 Escherichia coli
Beef Sample3-Colony3 B3C3 Escherichia coli
Beef Sample5-Colony1 B5C1 Escherichia coli
Chicken Sample2-Colony3 C2C3 Escherichia coli
Chicken Sample3-Colony2 C3C2 Escherichia coli
Pork Sample2-Colony1 P2C1 Escherichia coli
Pork Sample4-Colony1 P4C1 Escherichia coli
Pork Sample5-Colony1 P5C1 Escherichia coli
Pork Sample14-Colony3 P14C3 Escherichia coli
ATCC 10536 Eschericha coli

The microorganisms used in this study were previously identified by biochemical and molecular methods.

Antimicrobial analysis

The antibacterial activity of the four Baccharislatifolia extracts (Bl-E) of root, stem, leaves and flowers against Listeriaspp; Salmonellaspp and Escherichia coli strains were tested by the paper disc diffusion method applied by Shokeen et al. [17].

Colonies of fresh pure culture from each isolate and of the reference strains were suspended in the physiological saline solution until turbidity of 0.5 McFarland standard (equivalent to 1.5x108 UFC/m). Bacteria from each suspension were inoculated onto Muller Hilton Agar (MHA) (Neogen, 7101A, USA) using a sterile cotton-tipped swab and the plates were left standing for 10 min.

The sterile filter paper discs (Oxoid, CT0998B, United Kingdom) of 6 mm diameter were immersed in 10 ml of each extract for 7 min, then applied to the surface of the agar [18]. Sterile water was used as a negative control. The commercially available standard antibiotics, Penicilin G (Oxoid, CT0043B, UK) and Ciprofloxacin (Bioanalyse, 181129B, Turkey) were used as reference antibiotic controls. All assays were performed in duplicate. The sensitivity of microorganisms to natural extracts is related to the size of the microbial grown inhibition zone. According to the diameter of inhibition zone, microorganisms are classified in: resistant (d<8 mm), sensitive (9 mm.<d<14 mm), very sensitive (14 mm<d<19 mm) and extremely sensitive (d>20 mm) [19].

Antioxidant capacity of the Baccharislatifolia extracts

The ability of plant extracts to remove H2O2 can be estimated according to the method of Ruch et al. [20]. In this work a solution of H2O2 (40 mmol) was prepared in Potassium Phosphate Monobasic-Sodium Hydroxide buffer (50 mmol, pH 7.4) (SB108-1, Fisher Chemical, Belgium), the concentration of H2O2 was determined by absorption at 230 nm in a spectrophotometer Genesys 10 UV (Thermo Scientific 335902, USA). Subsequently, the extracts were added individually in concentrations of 20, 40 and 60 μg/ml on H2O2, the ABS was determined at 230 nm after 10 min against a blank solution containing phosphate buffer without H2O2. It should be noted that there are several methods to evaluate antioxidant capacity in vitro, although there is still no consensus on the most appropriate method, as well as, although there are markers of oxidative damage to biomolecules, the results in vivo remain contradictory [21].

The percentage of the sweep of hydrogen peroxide is calculated with the following formula:

Antioxidant capacity (% of H2O2 sequestered) = [(Ai-At)/Ai]x100.

Where: Ai= absorbance of the reference standard; At = Absorbance of the sample.

RESULTS AND DISCUSSION

Antimicrobial activity of Bl-E against Listeria isolates

Through the inhibition analysis, it was determined that the extract of leaves and flowers presented greater effectiveness against Listeria isolates, in fact, the size of the inhibition zone presented a mean greater than 15 mm. The third extract that showed effectiveness in the analysis was root, but with smaller zone size, as shown in fig. 2. The isolates: B1C2, B3C2 and CH5C1 showed resistance to the root extract of Bl; isolates: B3C2, CH5C1 and CH14C3 resisted the stem extract; the extracts of leaves and flowers did not show effectiveness against the isolated CH5C1 and CH14C3.

Fig. 2: Antimicrobial effect of Bl-E agaists Listeria, R-E: root extract; S-E: stem extract; L-E: leave extract; F-E: flower extract

On the other hand, in the control strain (Listeria innocua, ATCC 33090) presented 23 and 22 mm in diameter of the zone in the extracts of leaves and flowers respectively, however, these two extracts did not inhibit the development of the isolate, as shown in table 2. Resistant isolates were considered to those that presented a zone size ≤8 mm in diameter [19].

Table 2: Antibacterial activity of Bl-E against strains of Listeria spp

Antibiogram in Listeria Inhibition diameter in mm (24 h/incubation)
N ° Code R S L F P Cp
1 B1C2 8 12 12 18 21 24
2 B3C2 4 4 12 18 - 12
3 C3C1 10 11 14 11 22 -
4 C4C2 12 14 20 21 18 -
5 C5C1 8 2 - - 9 14
6 C6C1 15 12 22 24 - 24
7 C8C2 24 18 20 15 22 20
8 C14C3 10 8 2 2 21 14
9 C18C1 20 24 16 9 4 27
10 P19C1 12 11 20 18 18 18
Mean 12,3 11,6 15,3 15,1 16,9 19,1
Listeria innocua, ATCC 33090 20 18 23 22 21 14

R = root; S = stem; L = leaves; F = flowers; Cp = ciprofloxacin; P = penicillin

Anti-listerial effect of Bl-E and antibiotics for clinical use

For penicillin G (gr), an average of 16.9 mm in diameter was obtained, not so far from the average of our best Bl extracts. It should be noted that this antimicrobial is specific to fight infections caused by Listeria, taking as reference the parameters established by CLSI2012, [22], it can be said that of the 10 isolates studied, 4 were resistant to this agent (B3C2, C5C1, C6C1 and C18C1) with zone sizes ≤14 mm.

The control strain was susceptible to the antibacterial with 21 mm diameter. On the other hand, after applying ciprofloxacin discs, 5 isolates (B3C2, C3C1, C4C2, C5C1, C14C3), showed resistance with a zone size ≤15 mm, according to the CLSI2012 [22], the control strain proved susceptible to the quinolone (table 2).

Antimicrobial activity of Bl-E against Salmonella isolates

After having measured the inhibition diameters, it was determined that all the Salmonella isolates were susceptible to the Bl extracts studied, where the extracts of leaves and flowers presented greater effectiveness, in fact, the size of the zone had a mean greater than 20 mm fig. 3.

Fig. 3: Antimicrobial effect of Bl-E against Salmonella, R-E: root extract; S-E: stem extract; L-E: leave extract; F-E: flower extract

On the other hand, in the control strain, the root of Bl extract with a zone of 22 mm acted better, followed by the leaves extract with 21 mm of inhibition zone, as shown in table 3.

Table 3: Antibacterial activity of Bl-E against strains of Salmonella spp

Antibiogram in Salmonella Inhibition diameter in mm (24 h/incubation)
N ° Code R S L F P Cp
1 B5C1 20 20 18 14 - 28
2 B3C3 26 28 22 18 - 35
3 B15C3 16 24 25 25 22 28
4 B27C1 14 24 28 20 21 35
5 C2C1 20 16 14 16 - 31
6 C13C2 23 16 22 16 - 31
7 P1C3 20 20 24 31 - 35
8 P1C5 20 20 18 26 - 33
9 P5C3 24 26 30 28 - 31
10 P14C3 22 16 20 14 - 30
Mean 20,5 21 22,1 20,8 21,5 31,7
Salmonella arizonaeATCC 13314 22 18 21 18 14 32

R = root; S = stem; L = leaves; F = flowers; Cp = ciprofloxacina; P = penicilina

Anti-Salmonella effect of Bl extracts and antibiotics for clinical use

In the clinical antimicrobials, for penicillin G, a mean of 21 mm in diameter was obtained (in strains with inhibitory effect) value not so far from the average of the extracts of Bl, in fact, the extracts of Bl leaves acted better than this antibiotic. There were 8 strains of Salmonella that showed total resistance to Penicillin (B5C1, B3C3, C2C1, C13C2, P1C3, P1C5, P5C3, P14C3) zone size<14 mm as established by the CLSI 2012 [22], (table 2). However, it is important to consider that in the group of penicillins they are specific for Gram+microorganisms [23], so that it justifies the ineffectiveness of the antibiotic against Salmonella.

With ciprofloxacin, all isolates were shown to be susceptible to this antimicrobial agent with a zone diameter size>14 mm, according to the CLSI, 2012 [22]. The control strain was also susceptible to this quinolone.

Antimicrobial activity of Bl-E against Escherichia coli isolates

After finishing each of the zones of inhibition, it was determined that the extract of flowers and stem showed greater effectiveness against isolates of Escherichia coli with a zone size of 7.2 and 7.1 mm respectively. However, according to Ponce et al. [19], it should be considered that the microorganism is susceptible to the natural extract or oil, if the zone size is greater than 9 mm, so that the root extract alone inhibited the P2C1 strain; stem extract, the strains: B3C3 and P4C1; leaves extract, the strains: B2C1 and C3C2 and flowers extract only strain B3C3, as shown in fig. 4.

Fig. 4: Antimicrobial effect of Bl-E against Escherichia coli, R-E: root extract; S-E: stem extract; L-E: leave extract; F-E: flower extract

On the other hand, in the control strain, the root of Blextract with a zone size of 12 mm acted better, definitively all the extracts acted better than penicillin against E. coli isolates, see table 4.

Table 4: Antibacterial activity of Bl-E against strains of Escherichia coli

Antibiogram in Escherichia coli Inhibition diameter in mm (24 h/incubation)
N ° Code R S L F P Cp
1 B2C1 8 4 10 8 6 30
2 B3C1 4 6 8 8 4 15
3 B3C3 2 14 4 20 8 26
4 B5C1 4 6 8 4 4 24
5 C2C3 4 8 4 6 6 22
6 C3C2 4 4 10 6 4 30
7 P2C1 20 8 6 4 6 38
8 P4C1 8 9 4 8 4 16
9 P5C1 4 8 4 4 4 24
10 P14C3 8 4 8 4 2 22
Mean 6,6 7,1 6,6 7,2 4,8 24,7
Eschericha coli ATCC 10536 12 5 7 4 4 21

R = root; S = stem; L = leaves; F = flowers; Cp = ciprofloxacina; P = penicilina

Anti-E. coli effect of Bl extracts and antibiotics for clinical use

For penicillin G, an average of 4,0 mm in diameter was obtained. Considering the parameters established by the CLSI 2012 [22], all the isolates showed resistance to this antibiotic, the size zone<12 mm. However, it is important to consider that in a group of penicillins they are specific for Gram+microorganisms [23], so that they justify the ineffectiveness against these isolates. After applying the ciprofloxacin discs, all the isolates sensitivity to this antibiotic, with inhibition zone sizes>13 mm.

Antioxidant capacity of Bl extracts

After this analysis it was possible to determine that the flower Bl extract in 60 mg/ml presents a higher percentage of sequestration of H2O2 with 47.25%; followed by the concentration of 20 mg/ml with 46.36%. In relation to the extract obtained from the stem of Bl, the concentration of 20 mg/ml was enough to be able to sequester H2O2 by 42.08%; followed by the 40 mg/ml concentration with 19.23%.

The leaves of Bl extract with the greatest effect on the retention of H2O2 was that of 20 mg/ml concentration. While the root extract did not show any positive effect for this analysis, as shown in table 5.

Table 5: Antioxidant capacity test of Baccharislatifolia extracts

Extract Concentration (mg/ml) % of kidnapped peroxide
Flower 20 46.36
40 41.04
60 47.25
Stem 20 42.08
40 19.23
60 18.72
Leaves 20 5.62
40 3.92
60 0.84
Root 20 0.00
40 0.00
60 0.00

DISCUSSION

The extracts of medicinal herbs showed inhibitory activity, as determined by a work developed by Yoon and Choi [24], where the extracts of Bogolji and Gosam showed antibacterial capacity with zone diameter>10 mm; also, Eruteya and Badón [25], obtained antilisteria activity of ethanol extracts of Moringa oleifera, with zone of inhibition>11 mm from extract concentrations of 200 mg/ml. Similar results were obtained by Ruilova et al. [18], obtained antilisteria effect of ethanolic extracts of Physalis peruviana fruits, but with zone sizes<7 mm. Odedina et al. [26], used Rhodomyrtustomentosa ethanolic leaf extract as biocontrol against Listeria monocytogenes. Also, in the work carried out by Carrizo et al. [27], reported that the essential oil of B. salicifolia inhibits the growth of Listeria monocytogenes CLIP 74904, but was inactive against the Gram-negative organisms analyzed. It should also be noted that there are no studies on the antilisterial activity of Baccharislatifolia, which shows that our research group is the first to work with extracts of this plant against Listeriaspp isolates.

In a study developed by Shan et al. [28], reported that the extracts of 26 medicinal herbs positively inhibited the development of Salmonella anatum (mean = 7.2 mm, 4.7-19.2 mm). On the other hand, the acid environment improved the antibacterial activity of the extract of Filipendulaulmaria when tested against S. enteritidis PT4, whose aqueous methanol extract contains a variety of phenolic compounds [29].

In a study conducted by N'guessan et al. [30], the aqueous extracts of Thonningiasanguinea showed an antimicrobial effect for all Salmonella strains of multiple drug resistance (S. typhi, S. typhimurium and S. hadar). In another study conducted in South Korea, by Lee et al. [31], the aqueous and methanolic extracts of Schizandraefructus showed antibacterial activity against the three Salmonella serotypes (S. typhi ATCC 19943, S. paratyphi A and S. gallinarum ATCC 9184). In addition, the root of Euphorbia balsamifera had shown high activity against S. typhimurium in comparison with the extracts of leaves and stems [32]. As well, in the study developed by our research group, the inhibitory effect of extracts of Physalis peruviana L against isolates of Salmonella spp. with inhibition zones between 8 and 10 mm [33].

In the research developed by BachirRaho and Benali [34] shows that the essential oil of Eucalyptus globulus is effective to inhibit the development of Escherichia coli with zone sizes ranging from 8 to 26 mm in diameter. According to Argote-Vera et al. [35], mentions that the essential oils of eucalyptus and mandarin inhibit in a 13.2 μl/ml and lemon 14.6 μl/ml, demonstrating that the essential oils of eucalyptus, lemon peel and mandarin have the inhibitory capacity to the bacteria Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Also, in the research developed by Bastos [36], oregano oil was more effective against Escherichia coli with 0.35% CBM (minimum bactericidal concentration), with an inhibition zone of 29.5±3.4 mm.

Similarly, Sequeda et al. [16], studied extracts of Baccharislatifolia obtained by percolation, maceration and soxhlet, but did not observe an inhibitory effect on E. coli, although it did act positively against other pathogens.

In general, the chemical compound of the plant extract has revealed the presence of several components, most of which have important antimicrobial properties [37]. The properties present in Baccharis species are constituted mainly in flavonoids, monoterpenes, diterpenes, triterpenes, tannins, quinones, saponins, as well as some phenolic compounds, where flavonoids are distinguished by confer protection/resistance against attack of microorganisms [38, 37, 16]. Coumarins and essential oils have also been obtained of Baccharis species [39].

In addition, there are studies that show that monoterpenes are the components that also act in the inhibition of microorganisms, [40]. Other researchers state that the phenolic compound in the plant contributes significantly to its antimicrobial and antioxidant properties [41]. This study is the first to analyze the antibacterial effect of extracts of Baccharislatifolia (root, stem, leaves and flowers) on isolated Listeria, Salmonella and E. coli.

In a study developed by Guerra [42], they analyzed the antioxidant activity of the essential oil of the Baccharislatifolia-β carotene test, with concentrations of 26 and 64 mg/ml of Bl oil obtained values of 40.56% and 46.20% respectively. In our study, the extracts of flowers were the only ones that approximate these results. Cucurbita pepo extracts inhibited the peroxidation of linoleic acid at 5.1-30.4% after incubation for 96 h [43].

In another study conducted by Hossain et al. [44], obtained a value of 48.6% with lipophilic extracts of mixed Cucurbita, in Peru a study by Doroteo et al. [45], determined the antioxidant effect of cat's claw (Uncariatomentosa) with an effect of 47%.

So also, Rodriguez et al. [46], determined the effect of an extract of Boccaniafrutencens L with a capture value of 40% with an extract concentration of 25 mg/l.

CONCLUSION

Extracts of leaves and flowers of Baccharislatifolia acted better against Listeria and Salmonella isolates, whereas in E. coli isolates; Flower and stem extracts were the best. In short, these extracts proved to be equal to or better than the antibiotics for clinical use, thus considering the extracts of Bl as an alternative natural product to inhibit the development of pathogens.

ACKNOWLEDGMENT

The authors express their gratitude to the Departamento de Investigación of the Universidad Estatal de Bolívar for allowing the experiments in their facilities, as well as to the debt Exchange program Ecuador–Spain, for the support received for carrying out the present work.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declare none

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