Chemical profile of Stachytarpheta schottiana by LC-HRMS/MS dereplication and molecular networking

Abstract

Restingas are extreme environments present in the Atlantic Rainforest biome. These ecosystems show peculiar characteristics, like sandy soil, high salinity, and high solar exposure, which brings scientific interest to their native species. Stachytarpheta schottiana is an endemic Brazilian species found in Jurubatiba Restinga, and just like other species of the genus Stachytarpheta, is used in folk medicine. In this paper, we describe, for the first time, 28 secondary metabolites from S. schottiana polar extract, among them iridoids, flavonoids, lignans and phenylethanoids, with the use of LC-HRMS/MS dereplication and molecular networking methodologies. Many of these compounds have not been described yet for the genus Stachytarpheta, like astragalin, taxifolin, lamiide and the lignans secondary metabolites class. Also, in this paper, High Speed Counter-Current Chromatography (HSCCC) isolation and Nuclear Magnetic Resonance (NMR) of two compounds were used to correct a misidentification in the dereplication procedure and to create seeds for molecular networking. Some of the suggested known compounds found in this work have had their biological activities described in the literature, such as the hepatoprotective activity of verbascoside, that matches those already related for the genus Stachytarpheta and for the folk use of Stachytarpheta schottiana itself.

Key words
dereplication; Jurubatiba restinga; LC-MS/MS; molecular networking; Stachytarpheta schottiana

Resumo

As restingas são ambientes com características extremas, que fazem parte do bioma da Mata Atlântica. Estes ambientes apresentam características peculiares, como solo arenoso e altas salinidade e exposição solar, o que torna as espécies presentes interessantes para a ciência. A espécie Stachytarpheta schottiana é endêmica do Brasil, presente em restingas e, assim como outras espécies do gênero, apresenta uso na medicina popular. Neste trabalho são descritos pela primeira vez 28 metabólitos secundários da espécie S. schottiana, como iridoides, lignanas, flavonoides e feniletanoides, através do uso de metodologias de desreplicação e molecular networking por LC-HRMS/MS. Destas substâncias, muitas sequer foram descritas para o gênero, como astragalina, taxifolina, lamiídeo e toda a classe de lignanas. Ainda neste trabalho, a Cromatografia Contracorrente de Alta Velocidade (High Speed Countercurrent Chromatography - HSCCC) e a Ressonância Magnética Nuclear (RMN) foram utilizadas para corrigir a identificação equivocada de duas substâncias, bem como para a criação de seeds que seriam posteriormente utilizadas nos molecular networkings. Algumas das substâncias conhecidas sugeridas neste trabalho apresentam atividades biológicas já descritas na literatura que coincidem com o uso popular descrito para a espécie e também para o gênero, como o efeito hepatoprotetor do verbascosídeo.

Palavras-chave
desreplicação; restinga de Jurubatiba; LC-HRMS; molecular networking; Stachytarpheta schottiana

Introduction

Restinga environments are part of the Atlantic Rainforest biome, characterized by large sandy plain areas near the sea (Scarano 2002). These characteristics make the restingas extreme environments and bring scientific interest to the species living there. However, these are extremely fragile ecosystems submitted to intense degradation processes and, despite being one of the most threatened ecosystems in Brazil, only a few areas are protected by the government, like Parque Nacional da Restinga de Jurubatiba (PARNA Jurubatiba) (Luz et al. 2011). The PARNA Jurubatiba is the first Brazilian national park composed exclusively by the restinga ecosystem, which makes it vital to the ecosystem preservation. Also, the ecosystem comprised of this park possesses plant species that are used in Brazilian folk medicine, which can be the starting point to researches of biological interest (Boscolo & Senna Valle 2008).

The genus Stachytarpheta (Verbenaceae) is consisted of about 40 species, native from America, and shows a wide array of biological activities, such as antioxidant, anti-inflammatory (Schapoval et al. 1998), gastroprotective (Penido et al. 2006), antibacterial (Awah et al. 2010) and anti-hypertensive (Okokon et al. 2008).

Stachytarpheta schottiana Schauer species, popularly known as “gervão-da-praia”, is a small shrub endemic in Brazil and can be found in the restingas of Espírito Santo and Rio de Janeiro states, like the PARNA Jurubatiba (BFG 2018). Stachytarpheta schottiana is used in folk medicine as a vermifuge and hepatoprotective plant, by the population living near PARNA Jurubatiba (Boscolo & Senna Valle 2008) but the phytochemical and pharmacological profiles of S. schottiana have not yet been addressed, and further studies are required to it.

Classical methods of structural elucidation are usually costly and time-consuming. Therefore, dereplication methodologies using state-of-art techniques have been introduced to address this problem. This methodology uses separation techniques, as Liquid or Gas Chromatography coupled to detectors such as Mass Spectrometry (MS) or Nuclear Magnetic Resonance (NMR) to gather information of compounds in complex mixtures. The collected information is then compared with existing databases, such as Mass Bank (Horai et al. 2010) or PubChem (Kim et al. 2019), to allow the annotation of the compounds present in the sample (Gaudêncio & Pereira 2015).

The aim of this work was to investigate the chemical profile of the methanolic fraction of S. schottiana aerial parts by LC-HRMS/MS dereplication and molecular networking using the Global Natural Products Social Molecular Networking (GNPS) platform (Wang et al. 2016), in order to provide the first phytochemical report for the S. schottiana species.

Material and Methods

The Stachytarpheta schottiana collection was authorized under SISBIO/ICMBio No 62.455-11, and the work was authorized by SISGEN/MMA No: AAA989F. S. schottiana was collected in Parque Nacional da Restinga de Jurubatiba (PARNA Jurubatiba), in Carapebus, Rio de Janeiro state, Brazil, (coordinates: 22°16’13”S, 41°38’54’’W), in June, 2018. The botanical identification was carried out by Dr. Tatiana U.P. Konno, and a voucher specimen was deposited under RFA No 40922 in NUPEM, UFRJ Macaé.

The dried aerial parts powder was macerated three times with ethanol 96° GL (1:4 w/v) for 72 h (Casa Wolff, Rio de Janeiro, Brazil). About 50 mg of the dried crude extract was washed three times with 2 mL of n-hexane 95% to remove lipophyllic compounds (TEDIA, Fairfield, USA), and the supernatant was removed. The precipitate was weighed, dissolved in methanol and filtered with 0.45 μm PTFE Unichro syringe filters (Cobbeter, Hangzhou, China) to achieve a 0.5 mg/mL final solution. Then, a sample of 3 µL was injected and analyzed by LC-HRMS/MS.

The LC-HRMS/MS analyses were performed with a Thermo Scientific Dionex Ultimate 3000 liquid chromatography system chromatograph coupled to a Thermo Scientific^TM Q Exactive^TM Plus high-resolution mass spectrometer (Waltham, MA, USA). Liquid chromatography analyses were performed using an Ascentis C₁₈ Express (100 × 4.6 mm; 2.7 µm) column (with a guard column) (Supelco, Bellefonte, PA, USA) with ammonium formate (0.1% w/v) (mobile phase A): acetonitrile/formic acid (0.1% w/v) (mobile phase B) at a flow rate of 0.5 mL/min in gradient elution mode as follows: B -15% (0–1 min); B 15-95% (1–16 min); B 95% (16–21 min); B 95-15% (21–22 min) and B 15% (22–30 min). The oven temperature was kept at 40 °C. Source ionization parameters were: spray voltage 3.9 kV; capillary temperature 300 °C; S-Lens level 50, sheath gas 50, auxiliary gas 15. Samples were analyzed in the scan mass range of m/z 150 to 1000 at resolution of 35000 (positive and negative full scan) followed by data-dependent MS2 (ddMS2 Top3 experiments) using a resolution of 17500 and normalized collision energy (NCE) stepped 35-50%.

Files obtained by the LC-HRMS/MS analyses were processed in MzMine software, v 3.51 (Pluskal et al. 2010), to generate the peak lists that were to be used in the dereplication. Dereplication procedure was performed in the GNPS platform, using the following parameters: parent mass and MS/MS fragment tolerance of 0.02 Da and minimum cosine of 0.7 and 6 matched peaks to library match. Molecular Networking was performed using the same parameters described for dereplication, and tentative identification of compounds was performed using molecular masses, fragmentation patterns, network correlations and literature data. The molecular networking data was then analyzed in Cytoscape (Shannon et al. 2003).

A two-step High-Speed Counter-Current Chromatography (HSCCC) separation of two of the major compounds was performed in an AECS QuikPrep Quattro HSCCC (Bridgend, UK), equipped with two bobbins containing two coils of 112 mL each. 1 g of crude ethanolic extract was dissolved in 10 mL of a biphasic solvent system (EbuWat, 1:0.05:1) and injected after hydrodynamic equilibrium was reached (Vm = 43 mL, S_F = 81%, 850 rpm, two-coils volume). 480 mL of the mobile phase was pumped at 2 mL/min under rotation, and 240 mL of stationary phase were used to extrusion without rotation.

In the second step, two samples containing higher concentrations of the desired compounds were injected in two separate analyses (Vm = 17 mL, S_F = 85%, 850 rpm, one-coil volume), using 240 mL of mobile phase under rotation and 120 mL of stationary phase for extrusion. Fractions obtained in both steps were first analyzed by thin-layer chromatography (TLC) (Silicycle, Québec, Canada) and HPLC-DAD (Shimadzu, Kyoto, Japan) were used to evaluate the separation. The chromatographic conditions were the same used in the LC-HRMS analysis.

After the isolation, the purified compounds were dissolved in CD₃OD (Cambridge Isotope Laboratories, Andover, MA, USA), and submitted to 1D (¹H and ¹³C) and 2D (COSY, HSQC, HMBC) NMR analysis using a 500 MHz Varian spectrometer (Palo Alto, CA, USA).

Results and Discussion

The MS/MS dereplication process returned a total of 38 library matches in both modes (28 positive and 10 negative), with 25 suggested compounds pointed as secondary metabolites, such as iridoids, flavonoids, lignans and phenylethanoids (Tab. 1; Fig. 1). The presence of some of these classes of metabolites, except for lignans, had already been described for the genus Stachytarpheta (Penido et al. 2006; Kumar et al. 2012). Since there are no previous descriptions of S. schottiana phytochemical composition, all suggested compounds are new to the species and many compounds are described for the first time for the genus.

Figure 1
Base Peak Chromatograms (BPC) of Stachytarpheta schottiana polar extract, in positive (top) and negative (bottom) modes. The numbered peaks are the suggested secondary metabolites (Tab. 1). The unmarked peaks are not secondary metabolites or are peaks without a library match.

Table 1
Secondary metabolites suggested by LC-HRMS/MS dereplication, molecular networking and NMR elucidation.

Number	t R	Theoretical mass	Fragments	Cosine Score	m/zerror (ppm)	Molecular Formula	Ionization Mode	Compound Name	Identification method
1	2.08	462.174	315, 135, 113, 67	0.81	2.2	C20H30O12	Negative	Decaffeoyl verbascoside isomer	Dereplication
2	2.19	422.142	447, 247,	0.81	0.0	C17H26O12	Positive	Lamiide †	Dereplication
3	2.94	432.163	325, 163, 145, 91, 85	0.74	0.0	C19H28O11	Positive	Benzyl gentiobioside	Dereplication
4	4.63	464.095	303, 85	0.90	0.0	C21H20O12	Positive	Isoquercitrin	Dereplication
5	5.12	596.138	465, 303	0.92	0.0	C26H28O16	Positive	Peltatoside	Dereplication
6	5.26	582.231	581, 419, 401, 89	0.75	1.6	C28H38O13	Negative	(+)/(−)-Lyoniresinol 9’-O-glucoside	Dereplication
7	6.03	448.100	287	0.91	0.0	C21H20O11	Positive	Astragalin	Dereplication
8	6.07	756.248	593, 161	0.90	1.4	C34H44O19	Negative	Phlinoside B isomer	Dereplication
9	6.08	624.205	461, 161	0.87	1.6	C29H36O15	Negative	Verbascoside †	NMR
10	6.11	610.153	477, 315, 299	0.72	3.3	C27H30O16	Negative	Rhamnetin 3-O-hexosyl-pentoside	Dereplication
11	6.15	580.143	449, 287	0.91	0.0	C26H28O15	Positive	6’’-O-L-arabinopyranosylastragalin	Dereplication
12	6.22	756.248	325, 163	0.87	1.3	C34H44O19	Positive	Hebeoside isomer	Dereplication
13	6.42	624.205	325, 163	0.92	0.0	C29H36O15	Positive	Isoverbascoside †	NMR
14	6.57	304.058	177, 125	0.87	0.0	C15H12O7	Negative	Taxifolin	Dereplication
15	6.58	208.183	121, 107, 95	0.98	0.0	C14H24O	Positive	2-Butanone, 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)	Dereplication
16	6.60	374.230	213, 195, 177	0.82	0.0	C19H34O7	Positive	Linarionoside B	Dereplication
17	6.74	580.216	401, 265, 235, 205	0.85	1.6	C28H36O13	Positive	Acanthoside B	Dereplication
18	6.82	520.194	311, 175, 163, 137	0.78	1.8	C26H32O11	Positive	Dehydrodiconiferyl alcohol 4-β-D-glucoside	Dereplication
19	6.89	638.221	461, 175, 161	0.84	1.6	C30H38O15	Negative	Leucosceptoside A isomer †	Dereplication
20	7.07	638.221	339, 177	0.90	0.0	C30H38O15	Positive	Leucosceptoside A †	Dereplication
21	7.15	476.096	299, 284, 113	0.92	2.1	C22H20O12	Negative	6-O-methylscutellarin	Dereplication
22	7.26	506.106	329, 314, 175, 113	0.80	2.0	C23H22O13	Negative	3,3’-dimethyl-quercetin-7-O-glucuronide	Dereplication
23	7.74	652.237	339, 177	0.86	0.0	C31H40O15	Positive	Martynoside †	Dereplication
24	7.91	652.237	475, 193, 175	0.87	1.6	C31H40O15	Negative	Martynoside isomer †	Dereplication
25	12.86	392.220	119	0.84	4.8	C22H32O6	Positive	(1S,2R,4aR,8aR)-1-Acetoxy-7-isopropylidene-1,4a-dimethyl-6-oxodecahydro-2-naphthalenyl 2,3-dimethyl-2-oxiranecarboxylate	Dereplication
26	-	592.215	325, 163	-	-	C29H36O13	Positive	Jionoside C	Molecular Networking a
27	-	470.142	325, 163	-	-	C21H26O12	Positive	Tangshenoside V	Molecular Networking a
28	-	610.153	465, 303	-	-	C27H30 O16	Positive	Rutin	Molecular Networking b

• t_R = retention time in minutes; Theoretical mass = calculated mass based in compound formula; Cosine score = score of similarity used to match a compound with GNPS library; Compound name = in dereplication identified compounds, the name relates to the identification of the GNPS library match; in NMR identified compounds, it relates to the name of the compound; and in the Molecular Networking it relates to the suggestion based in the literature. † = compounds already described in the genus Stachytarpheta. ^a = Shen et al. 2016; ^b = Fu et al. 2020.

The iridoid lamiide (compound 2 in Tab. 1) can be identified by the sodium adduct formed in ionization at [M + Na]⁺ (m/z 445.132), which is recognized by the platform. The identified lignans (compounds 6, 17 and 18 in Tab. 1) showed fragments representing the removal of their sugar moieties (loss of 162 Da) and the ionization of the remaining structure in the case of (7’R)-(+)-lyoniresinol 9’-glucoside (m/z 419) and acanthoside B (m/z 401).

Seven flavonoids were identified by dereplication and are highlighted in sequence: isoquercitrin, a flavonol constituted of quercetin, that is seen in the fragmentation as the m/z 303 signal, and a 3-glucosyl moiety; peltatoside, an isoquercetin with a xylanopyranosyl moiety (fragments m/z 465 and 303); astragalin, kaempferol, a flavonol with a 3-glucosyl moiety attached (fragment m/z 287 denotes the protonated aglycone); rhamnetin 3-O-neohesperidoside, a rhamnetin aglycone attached with a xylopyranosyl-glucopyranoside moiety; 6’’-O-L-arabinopyranosylastragalin is an astragalin flavonol with an arabinopyranosyl moiety (132 Da mass difference); taxifolin, a di-hydroflavonol aglycone that is a reduced form of quercetin (which explains the 2 Da mass increase); 6-O-methylscutellarin, a flavone derived from scutellarin and 3,3’-dimethyl-7-O-glucuronic acid quercetin, a dimethylated-glucuronyl derivative of quercetin (m/z 329 denotes the aglycone).

The phenylethanoids suggested by dereplication showed similar fragmentation patterns, which are related to the bond break between the moieties and the central sugar. In decaffeoyl-verbascoside, for example, m/z 135 and 315 represent the aglycone fragment and the remaining molecular fragment, respectively. Similarly, benzyl gentibioside showed the m/z 163 and 325 fragments, related to the caffeoyl and the remaining structural fragments.

Dereplication showed two phenylethanoid isomers as two of the major compounds (Fig. 1 - negative mode) in the methanolic extract of S. schottiana, which have a molecular mass of 624.205 Da. They were identified in GNPS as isoverbascoside (t _R = 6.08 min) and forsythoside A (t _R = 6.42 min), composed by an aglycone (phenethyl alcohol derivatives), a phenylpropanoid (caffeoyl), and two sugar moieties (glucosyl and rhamnosyl). These moieties can be observed in the MS/MS fragments (m/z 463 and 163 fragments in the positive ionization and m/z 461 and 161 in negative). The observed fragmentations are related to the ones described in the literature (Marchetti et al. 2019) and the fact that these compounds have the same m/z and different retention (t _R) times (Fig. 1) indicates that they are structural or geometric isomers.

In order to isolate these compounds, for usage as seeds to the molecular networking procedure and, in addition, for elucidation by NMR, the HSCCC technique was employed. After NMR analyses the compound previously identified as isoverbascoside by dereplication consisted, in fact, of verbascoside, shown in Figure S1 and Table S1 (Supplemental File 1, available on supplementary material <https://doi.org/10.6084/m9.figshare.16688791.v1>). The other compound had been identified as forsythoside A by dereplication, but NMR analysis identified it as isoverbascoside (Fig. S2 and Tab. S2, Supplemental File 1, available on supplementary material <https://doi.org/10.6084/m9.figshare.16688791.v1>).

Verbascoside and isoverbascoside present different chemical shifts in ¹H NMR to the hydrogen H-4’ and H-6’, respectively. Verbascoside shows δ_H 4.94 ppm to H-4’, δ_H 3.56 ppm to H-6’a and δ_H 3.63ppm to H-6’b (Fig. S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.16688791.v1>), while isoverbascoside shows δ_H 3.36 ppm to H-4’, δ_H 4.48 ppm to H-6’a and δ_H 4.38 ppm to H-6’b (Fig. S2, available on supplementary material <https://doi.org/10.6084/m9.figshare.16688791.v1>). The deshield shifts of H-4’ to verbascoside and H-6’ to isoverbascoside indicated that the caffeoyl moieties were attached to C-4’ and C-6’ from glucose, respectively. All chemical shifts and scalar couplings values matches those described in the literature for both compounds (Kawada et al. 2002; Caufin et al. 2014).

Other observed phenylethanoids showed similar fragmentation patterns, but with variations. Phlinoside C and cassifolioside isomers showed mass increments that suggests the addition of a sugar moiety (132 Da). Leucosceptoside A and martynoside showed very similar fragmentation patterns to verbascoside and isoverbascoside, but with a fragment indicating the presence of a ferulic moiety instead of a caffeoyl one (m/z 175) in both cases, and a fragment indicating a methoxylated aglycone in martynoside (m/z 475). GNPS dereplication suggested identifying the compounds 19 and 20 as plantainoside C, but the isomer leucosceptoside A was already described for the genus Stachytarpheta (Froelich et al. 2008).

It is worth of note that dereplication has not allowed the identification of some of the compounds present in the methanolic extract. So, Molecular Networking in positive ionization mode was used for the tentative dereplication of some compounds, using their chemical similarity with suggested compounds used as seeds, fragmentation patterns and the available literature information. Three compounds were then suggested by this method. The first was a phenylethanoid which mass [M + H]⁺ (m/z 610.249) was directly connected to verbascoside (m/z 625.212, Fig. 2) and which showed the same fragments (m/z 163 and 325). Therefore, based on this information, a search was performed in the literature and available databases, leading to the suggestion that it is a jionoside C similar compound (Shen et al. 2016). The second compound was another phenylethanoid, with a precursor mass [M + H]⁺ m/z 471.149, which was also connected to verbascoside in the molecular network (Fig. 2) and had the fragments m/z 163 and 325. This information led the search to the suggestion that the compound is similar to tangshenoside V, a smaller phenylethanoid (Shen et al. 2016). The third compound has a precursor mass [M + H]⁺ (m/z 611.139) and was connected in the molecular network to peltatoside [M + H]⁺ m/z 597.145 and isoquercitrin [M + H]⁺ m/z 465.102 (Fig. 2), suggesting that the compound is a flavonoid. It had the fragments m/z 303 and 465, and this information led to the suggestion of rutin, a quercetin-3-O-rutinoside (Fu et al. 2020).

Figure 2
LC-HRMS positive mode molecular networking of the Stachytarpheta schottiana polar extract, where the compounds’ precursor masses are correlated by similarities between their MS/MS fragmentation patterns. Seed nodes = grey contour and white filling; other nodes = grey contour and dark grey filling.

The chemical profiling of S. schottiana showed some compounds already described in the literature for the genus Stachytarpheta, such as the iridoid lamiide (Viccini et al. 2008) and the phenylethanoids verbascoside, isoverbascoside, leucosceptoside A and martynoside (Leitão et al. 2005; Froelich et al. 2008). However, the flavonoids isoquercitrin, peltatoside and astragalin; and the phenylethanoids, phlinoside B and hebeoside were not described for the genus. In addition, the class of lignans is described for the first time for the Stachytarpheta genus, represented by acanthoside B, (+)/(−)-lyoniresinol 9’-O-glucoside, and dehydrodiconiferyl alcohol 4-β-D-glucoside.

The presence of several phenolic compounds of the shikimate pathway can be correlated with the harsh environmental conditions that the specimen was exposed. The specimen was collected in the restinga habitat (Zaluar & Scarano 2000; Scarano 2002) in winter, dry season. High solar exposure and drought stress may be responsible for the production of phenylethanoids and flavonoids in this species (Quan et al. 2016; Falahi et al. 2018; Yang et al. 2020).

Some of the compounds suggested in this work show a wide variety of biological activities described in the literature, such as the anti-inflammatory activity of lamiide (Delaporte et al. 2002) and anti-oxidant and anti-inflammatory activities of the flavonoids isoquercitrin, astragalin and taxifolin (Yang et al. 2016; Han et al. 2017; Hobbs et al. 2018). The phenylethanoids verbascoside and isoverbascoside have described anti-hepatotoxic, anti-inflammatory, anti-nociceptive and antioxidant activities (Jiménez & Riguera 1994; Jin et al. 2004). Also, the suggested lignans have biological activitied already described, such as the ability to alleviate hepatic steatosis and reduce High Glucose (HG)-induced Reactive Oxygen Species (ROS) in vitro of both isomers (+)/(−)-lyoniresinol 9’-O-glucoside (Shi et al. 2019), the neuroprotective activity of acanthoside B (Karthivashan et al. 2019) and in vitro anti-inflammatory and antioxidant potential of dehydrodiconiferyl alcohol 4-β-D-glucoside (Shan et al. 2018; Yang et al. 2019) . The activities described for the compounds pointed by this work matches the effects related to the folk use of S. schottiana as a hepatoprotective plant, as cited by Boscolo & Senna Valle (2008), suggesting this species as a potential candidate for studies focused in hepatic diseases.

The polar extract of S. schottiana was submitted to LC-HRMS/MS dereplication and molecular networking using the GNPS platform, which allowed the identification of 28 natural products, 25 by dereplication and 3 by molecular networking. Some of the dereplication results suggested compounds in the extract have described biological activities, that match the folk use of the plant as a hepatoprotective by the population that lives in the area near the PARNA Jurubatiba, as cited by Boscolo & Senna Valle (2008). This work provided research of an endemic plant species in a preserved environment, corroborating one basic objective seek in the preservation plan (ICMBio 2007). Dereplication allowed the fast identification of a series of known compounds in a never described species, which can allow the focus in unknown compounds if present.

Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors also thank to Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), for Project grant (E26/010.001617/2016); and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors would like to thanks Dr. Nelilma C. Romeiro, for constructive suggestions and grammar revision.

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Supplementary Material

See supplementary material at <https://doi.org/10.6084/m9.figshare.16688791.v1 >

Edited by

Area Editor: Dr. Leopoldo Baratto

Publication Dates

• Publication in this collection
  22 Oct 2021
• Date of issue
  2021

History

• Received
  14 May 2020
• Accepted
  28 July 2020