Phytochemical study of Waltheria viscosissima and evaluation of its larvicidal activity against Aedes aegypti

Maria Denise Leite Ferreira Diégina A. Fernandes Fabíola C. Nunes Yanna C.F. Teles Yngred M. Rolim Camila Macaúbas da Silva Janderson B.L. de Albuquerque Maria de Fátima Agra Maria de F.V. de Souza About the authors

Abstract

The species Waltheria viscosissima A.St.–Hil, Malvaceae, which is known as ‘malva-branca', is traditionally used in the Brazilian northeast for the treatment of coughs. This research looks towards reporting the isolation of phytoconstituents of W. viscosissima, as well as the quantification of its phenolics, total flavonoid content, and free radical scavenging potential, along with an evaluation of its larvicidal activity against Aedes aegypti larvae. Chromatographic techniques were used to isolate the compounds and a structural elucidation was performed by 1D and 2D NMR. The quantification of total phenolics and flavonoids and the DPPH˙ radical scavenging activity was determined through spectrophotometric methods. Consequently, the phytochemical investigation led to the identification of fourteen compounds from the aerial parts of the W. viscosissima: steroids, triterpenes, alkaloids, and eight flavonoids previously reported in the literature. The quantification of compounds showed that the aerial parts extract possessed high concentration of flavonoids, while the roots extract were rich in other phenolic compounds. At the DPPH˙ free radical scavenging assay, the roots extract presented EC50 = 77.32 ± 4.37 µg/ml and the aerial parts extracts showed EC50 = 118.10 ± 1.21 µg/ml. W. viscosissima roots extract showed the most potent larvicidal activity against Ae. aegypti (LC50 = 4.78 mg/ml), with the potential of being used in effective and economically viable preparations that can be catered for domestic use towards controlling the vector insect of severe diseases, such as dengue and Zika.

Keywords
Sterculiaceae; Phytochemical study; Antioxidant activity; Larvicidal assay; Aedes aegypti

Introduction

Malvaceae sensu lato comprises the traditional families that can be found within the Malvales order: Bombacaceae, Sterculiaceae, Tiliaceae e Malvaceae sensu stricto (APG IV, 2016APG IV, 2016 , 2016. Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot. J. Linn. Soc. 181, 1-20.). The Waltheria genus, Malvaceae, occurs in both Asia and America, including Brazil. It possess around sixty species, many of which are used in human health treatments to treat respiratory and dental infections, along with inflammation and cases of malaria (Zongo et al., 2013Zongo et al., 2013 Zongo, F., Ribuot, C., Boumendjel, A., Guissou, I., 2013. Botany, traditional uses, phytochemistry and pharmacology of Waltheria indica L. (syn. Waltheria americana): a review. J. Ethnopharmacol. 148, 14-26.; Cretton et al., 2014Cretton et al., 2014 Cretton, S., Breant, L., Pourrez, L., Ambuehl, C., Marcourt, L., Ebrahimi, S.N., Hamburger, M., Perozzo, R., Karimou, S., Kaiser, M., Cuendet, M., Christen, P., 2014. Antitrypanosomal quinoline alkaloids from the roots of Waltheria indica. J. Nat. Prod. 77, 2304-2311., 2015Cretton et al., 2015 Cretton, S., Bréant, L., Pourrez, L., Ambuehl, C., Perozzo, R., Marcourt, L., Kaiser, M., Cuendet, M., Christen, P., 2015. Chemical constituents from Waltheria indica exert in vitro activity against Trypanosoma brucei and T. Cruz. Fitoterapia 105, 55-60.; Esteves, 2015Esteves, 2015 Esteves, G., 2015. Waltheria in Lista de Espécies da Flora do Brasil. Rio de Janeiro, Jardim Botânico do Rio de Janeiro, Available at: http://floradobrasil.jbrj.gov.br/2012/FB009270Accessed on 10 December 2018.
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; Yougbare-Ziebrou et al., 2016Yougbare-Ziebrou et al., 2016 Yougbare-Ziebrou, M.N., Lompo, M., Ouedraogo, N., Yaro, B., Guissoun, I.C., 2016. Antioxidant, analgesic and anti-inflammatory activities of the leafy stems of Waltheria indica L. (Sterculiaceae). Int. J. Appl. Pharm. Sci. Res. 6, 124-129.; Veeramani and Alagumanivasagam, 2016Veeramani and Alagumanivasagam, 2016 Veeramani, P., Alagumanivasagam, G., 2016. In-vitro antioxidant activities of ethanolic extract of whole plant of Waltheria indica (Linn.). Der. Pharm. Lett. 8, 299-302.; Silveira-Júnior et al., 2017Silveira-Júnior et al., 2017 Silveira-Júnior, C.E.A., Lima, L.C.L., Saba, M.D., 2017. Pollen morphology of Waltheria L. (Malvaceae-Byttnerioideae) from Bahia. Brazil. Acta. Bot. Bras. 31, 597-612.; Mundo et al., 2017Mundo et al., 2017 Mundo, J., Villeda-Hernandez, J., Herrera-Ruiz, M., Gutierrez, M.C., Arellano-Garcia, J., Leon-Rivera, I., Perea-Arango, I., 2017. Neuropharmacological and neuroprotective activities of some metabolites produced by 102 cell suspension culture of Waltheria americana Linn. Biomed. Pharmacother. 94, 129-139.).

Based on the Waltheria genus, previous studies have reported the occurrence of quinolone alkaloids, (Hoelzel et al., 2005Hoelzel et al., 2005 Hoelzel, S.C.S.M., Vieira, E.R., Giacomelli, S.R., Dalcol, I.I., Zanatta, N., Morel, A.F., 2005. An unusual quinolinone alkaloid from Waltheria douradinha. Phytochemistry. 66, 1163-1167.; Lima et al., 2009Lima et al., 2009 Lima, M.M.C., López, J.A., David, J.M., Silva, E.P., Giulietti, A.M., Queiroz, L.P., David, J.P., 2009. Acetylcholinesterase activity of alkaloids from the leaves of Waltheria brachypetala. Planta Med. 75, 335-337.; Cretton et al., 2016Cretton et al., 2016 Cretton, S., Dorsaz, S., Azzollini, A., Favre-Godal, Q., Marcourt, L., Ebrahimi, S.N., Voinesco, F., Michellod, M., Sanglard, D., Gindro, K., Wolfender, J., Cuendet, M., Christen, P., 2016. Antifungal quinoline alkaloids from Waltheria indica. J. Nat. Prod. 79, 300-307.), triterpenes, phenolics, and flavonoids (Monteillier et al., 2017Monteillier et al., 2017 Monteillier, A., Cretton, S., Ciclet, O., Marcourt, L., Ebrahimi, S.N., Christen, P., Cuendet, M., 2017. Cancer chemopreventive activity of compounds isolated from Waltheria indica. J. Ethnopharmacol. 203, 214-222.; Caridade et al., 2018Caridade et al., 2018 Caridade, T.N.S., Rusceli, D.A., Oliveira, A.N.A., Souza, T.S.A., Ferreira, N.C.F., Avelar, D.S., Teles, Y.C.F., Silveira, E.R., Araújo, R.M., 2018. Chemical composition of four different species of the Waltheria genus. Biochem. Syst. Ecol. 80, 81-83.).

Waltheria viscosissima A.St.-Hil, Malvaceae, popularly known as ‘malva-branca' and ‘malva-viscosa', is endemic of the Northeast region of Brazil, with its aerial parts having been traditionally used as antitussive and expectorant. However, the phytochemical investigations on the species are scarce (Corrêa, 1974Corrêa, 1974 Corrêa, M.P., 1974. Dicionario de Plantas uteis do Brasil e das Exoticas Cultivadas. Di Giorgio, Rio de Janeiro, pp. 44.; Vasques et al., 1999Vasques et al., 1999 Vasques, C.A.R., Côrtes, S.F., Silva, S.M., Medeiros, I.A., 1999. Muscarinic agonist properties of the hydrobutanol extract from aerial parts of Waltheria viscosissima St. Hil. (Sterculiaceae) in rats. Phytother. Res. 13, 312-317.). A preliminary phytochemical screening has detected the presence of triterpenes, steroids, phenolic compounds and saponins in the crude ethanol extract of W. viscosíssima (Vasques et al., 1999Vasques et al., 1999 Vasques, C.A.R., Côrtes, S.F., Silva, S.M., Medeiros, I.A., 1999. Muscarinic agonist properties of the hydrobutanol extract from aerial parts of Waltheria viscosissima St. Hil. (Sterculiaceae) in rats. Phytother. Res. 13, 312-317.), and one triterpene also has been identified (Soares et al., 1998Soares et al., 1998 Soares, F.P., Ronconi, C.A.V., Cunha, E.V.L., Barbosa-Filho, J.M., Silva, M.S., Braz-Filho, R., 1998. Four known triterpenoids isolated from three Brazilian plants: 1H and 13C chemical shift assignments. Magn. Reson. Chem. 36, 608-614.).

Indeed, phytochemical researches allied to biological assays have raised the potential uses of plant extracts or isolated phytoconstituents as insecticides and/or larvicides, with many studies confirming the efficacy of these natural products (Santiago et al., 2005Santiago et al., 2005 Santiago, G.M.P., Viana, A.F., Pessoa, O.D.L., Pouliquen, Y.B.M., Arriaga, A.M.C., Andrade-Neto, M., Braz-Filho, R., 2005. Avaliação da atividade larvicida de saponinas triterpênicas isoladas de Pentaclethra macroloba (Willd.) Kuntze (Fabaceae) e Cordia piauhiensis Fresen (Boraginaceae) sobre Aedes aegypti. Rev. Bras. Farmacogn. 15, 187-190.; Santos et al., 2012Santos, 2015 Santos, D.B., 2015. Atividade larvicida da Copaifera langsdorffii (Leguminosae), evidenciada pelas alterações morfohistológicas em Aedes aegypti (Diptera, Culicidae). Dissertação (Mestrado), Pós-graduação em Biologia da Relação Parasito-Hospedeiro, Universidade Federal de Goiás, Goiânia, Brasil, pp. 73.; Lakshmi et al., 2018Lakshmi et al., 2018 Lakshmi, K.V., Sudhikumar, A.V., Aneesh, E.M., 2018. Larvicidal activity of phytoextracts against dengue fever vector, Aedes aegypti-a review. Plant Sci. Today 5, 167-174.). These insecticides and/or larvicides are especially relevant in tropical countries, where mosquitoes contribute to the occurrence of severe viral diseases, such as yellow fever, dengue, and Zika (Fernandes et al., 2018Fernandes et al., 2018 Fernandes, D.A., Souza, M.S.R., Teles, Y.C.F., Oliveira, L.H.G., Lima, J.B., Conceição, A.S., Nunes, F.C., Silva, T.M.S., Souza, M.F.V., 2018. New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules 23, http://dx.doi.org/10.3390/molecules23112784..
http://dx.doi.org/10.3390/molecules23112...
).

Considering the popular use of W. viscosissima in Brazil and the lack of consistent information with regards to its phytoconstituents, this study seeks to report on the isolation of compounds from aerial parts of W. viscosissima, as well as the quantification of phenolics and flavonoids in the obtained extracts. The wide occurrence of flavonoids in Waltheria and the larvicidal activity of Sterculiaceae species have been recently reported, justifying the interest in evaluating the antiradical and larvicidal activity of W. viscosissima (Caridade et al., 2018Caridade et al., 2018 Caridade, T.N.S., Rusceli, D.A., Oliveira, A.N.A., Souza, T.S.A., Ferreira, N.C.F., Avelar, D.S., Teles, Y.C.F., Silveira, E.R., Araújo, R.M., 2018. Chemical composition of four different species of the Waltheria genus. Biochem. Syst. Ecol. 80, 81-83.; Fernandes et al., 2018Fernandes et al., 2018 Fernandes, D.A., Souza, M.S.R., Teles, Y.C.F., Oliveira, L.H.G., Lima, J.B., Conceição, A.S., Nunes, F.C., Silva, T.M.S., Souza, M.F.V., 2018. New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules 23, http://dx.doi.org/10.3390/molecules23112784..
http://dx.doi.org/10.3390/molecules23112...
).

Materials and methods

General procedures and chemicals

Chromatographic glass columns were used for the purification of the compounds, packed with Silica gel 60 (Merck), Sephadex LH-20 (Merck) or Amberlite XAD-2 as stationary phases, and hexane, EtOAc and MeOH in increasing polarity mixtures as the mobile phase (Fig. 1).

Fig. 1
Larvicidal activity of different concentrations of APCE and RCE of Waltheria viscosissima on Ae. aegypti larvae after 24 h. PC: Positive control; NC: Negative control. (*) statistically significant in relation to the NC. (**) statistically significant in relation to the PC (p-value < 0.05).

Thin layer cchromatography (TLC) was carried out using silica plates and the resulting spots were visualized either under UV light (254 and 366 nm) or with a p-anisaldehyde acid solution. The isolated compounds were identified by 1D and 2D NMR (1H - 500 MHz/13C -125 MHz - Varian and 1H - 400 MHz/13C -100 MHz Bruker. 1H - 300 MHz/13C -75 MHz – Bruker Avance DPX-300), using deuterated solvents. The chemical shifts were expressed as parts per million (ppm) and the coupling constants of (J) in Hz.

Plant material

The plant material (aerial parts and roots) of the Waltheria viscosissima A.St.-Hil, Malvaceae, was collected in Santa Rita City, during the month of August 2013. Plant identification was performed by Prof. Maria de Fátima Agra (PgPNSB/UFPB) and a voucher species was deposited at the Prof. Lauro Pires Xavier Herbarium (M. F. Agra 21709). This research has been registered in the National System of Genetic Resource Management and Associated Traditional Knowledge (SisGen - A568B8A).

Extraction and isolation of compounds

The plant material, aerial parts, and roots were oven dried at 40 °C for 72 h. The material was ground separately, and 2,000 g of powdered aerial parts and 350.3 g of powdered roots was obtained. Both materials were macerated with ethanol (95%) for 72 h. The obtained solutions were filtered and concentrated in a rotatory evaporator to obtain 550 g of aerial parts crude extract (APCE) and 33.5 g of roots crude extract (RCE).

The APCE was solubilized using EtOH:H2O (7:3) and the obtained solution was sequentially partitioned in a separation funnel using hexane (Hex), chloroform (CHCl3), ethyl acetate (EtOAc) and n-butanol, which yielded 125 g of hexane phase (HP), 25 g of CHCl3 phase (ChlP), 10 g of EtOAc phase (ACP), 40 g of n-butanol phase (BP), and 100 g of hydroalcoholic phase (HAP).

A sample of HP (50 g) was submitted to vacuum liquid chromatography (VLC) using silica gel and Hex, EtOAc, and methanol in increasing polarity mixtures. The obtained fractions were combined after TLC. The fractions eluted with Hex:EtOAc (7:3), Hex:EtOAc (1:1), Hex:EtOAc (4:6) and EtOAc were combined and named HX1. HX1 (12 g) was column chromatographed (CC) in silica flash using Hex, EtOAc and MeOH in increasing polarity mixtures, yielding 97 fractions. Those fractions were analyzed by TLC. Fractions 19–20 and 21 were found to be pure white solids, and were named as compounds 1 (210 mg) and 2 (140 mg). Fraction 22–23 was pure (colorless crystals), and named as compound 3 (110 mg).

The ChlP (12 g) was submitted to silica gel CC using Hex, EtOAc and MeOH to obtain 150 fractions which were combined after TLC. The combined fractions of 79–92 were submitted to Sephadex LH-20 CC eluted with MeOH to yield forty fractions. The fraction of 12–15 (yellow powder) was pure, and named as compound 4 (9 mg). The combined fractions of 110–150 were submitted to silica gel CC using Hex, EtOAc and MeOH, resulting in the purification of compound 5 (13 mg) as yellow powder.

The ACP (8 g) was chromatographed using Sephadex LH-20 eluted with MeOH and MeOH: CHCl3, resulting in 89 fractions. After TLC, the fractions were combined and selected for the new Sephadex CC. From successive Sephadex columns, the compounds 6 (25 mg), 7 (23 mg), 8 (15 mg) and 9 (9 mg) were purified.

A sample of HAP was chromatographed with the use of XAD-2 as stationary phase. The solvents utilized were H2O (100%), H2O:MeOH (7:3), H2O:MeOH (1:1), MeOH, Hex, acetone and EtOAc. The fraction H2O:MeOH (7:3) precipitated a pure solid, which was named as compound 10 (35 mg).

From XAD-2 column, the fraction eluted with MeOH was rechromatographed in the Sephadex LH-20 CC, resulting in 25 fractions. The fractions 13–18 was a pure yellow powder, which was named compound 11 (15 mg).

A sample of APCE (75 g) was submitted to acid-basic alkaloid purification in the separation funnel to obtain the total alkaloid fraction (TAF). The TAF was submitted to an aluminum oxide CC, using Hex, EtOAc and MeOH as eluents. From the column, 55 fractions were obtained and analyzed by TLC. The fraction 39–43 was chromatographed in Sephadex CC, and the resulting purification of compound 12 (9 mg) was a yellowish oil.

Quantification of total phenolic compounds

The quantification of total phenolic compounds was determined by the spectrophotometric through the use of the Folin–Ciocalteu reagent and gallic acid as a standard according to Gulcin et al. (2004)Gulcin et al., 2004 Gulcin, I., Sat, G., Beydemir, S., Elmastas, M., Kufrevioglu, O.I., 2004. Comparison of antioxidant activity of alove (Eugenia caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 87, 393-400.. The experiment was performed in triplicates.

Total flavonoids content

Total flavonoid content was determined by the spectrophotometric method as described by Maciel et al. (2016)Maciel et al., 2016 Maciel, J.K.S., Chaves, O.S., Filho, S.G.B., Teles, Y.C.F., Fernandes, M.G., Assis, T.S., Andrade, A.P., Felix, L.P., Silva, T.M.S., Ramos, N.S.M., Silva, G.R., Souza, M.F.V., 2016. New alcamide and antioxidant activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. Ex. Rowl. (Cactaceae). Molecules 21, http://dx.doi.org/10.3390/molecules21010011.
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, using quercetin as a standard. Total flavonoid content was determined by the spectrophotometric method described by Maciel et al. (2016)Maciel et al., 2016 Maciel, J.K.S., Chaves, O.S., Filho, S.G.B., Teles, Y.C.F., Fernandes, M.G., Assis, T.S., Andrade, A.P., Felix, L.P., Silva, T.M.S., Ramos, N.S.M., Silva, G.R., Souza, M.F.V., 2016. New alcamide and antioxidant activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. Ex. Rowl. (Cactaceae). Molecules 21, http://dx.doi.org/10.3390/molecules21010011.
http://dx.doi.org/10.3390/molecules21010...
, which used quercetin as a standard. The analysis was evaluated in triplicates and the total flavonoid content was determined from the calibration curve which was constructed with quercetin solutions (1.25 to 40 µg/ml). The result was expressed in milligrams of quercetin equivalents per gram of extract (mg QE/g of extract).

Free radical scavenging activity assay

The in vitro antiradical activity of APCE and RCE was evaluated by the DPPH˙ (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging method which was described by Maciel et al. (2016)Maciel et al., 2016 Maciel, J.K.S., Chaves, O.S., Filho, S.G.B., Teles, Y.C.F., Fernandes, M.G., Assis, T.S., Andrade, A.P., Felix, L.P., Silva, T.M.S., Ramos, N.S.M., Silva, G.R., Souza, M.F.V., 2016. New alcamide and antioxidant activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. Ex. Rowl. (Cactaceae). Molecules 21, http://dx.doi.org/10.3390/molecules21010011.
http://dx.doi.org/10.3390/molecules21010...
. The experiment was performed in a triplicate.

Biological assay

The larvicidal activity of W. viscosissima extracts (APCE and RCE) have been evaluated by the World Health Organization (1970). The larvae of Ae. aegypti in the 4th stage (L4) of the Rockefeller strain for this study were obtained from the Biotechnology Applied to Parasites and Vectors Laboratory of the Biotechnology Center (Universidade Federal da Paraíba).

Twenty larvae (L4) were transferred to Falcon tubes containing 10 ml of APCE and RCE solutions. The concentrations used to determine the LC50 of APCE and RCE ranged from 2 to 200 mg/ml and from 2.5 to 40 mg/ml, respectively. The positive control group consisted of twenty larvae which were exposed to 1 ml of a commercial insecticide (Imiprotrin 0.02%, Permethrin 0.05% and Esbiotrin 0.1%). The negative control group consisted of twenty larvae exposed to distilled water. The experiments were performed in triplicate.

The experiment tubes containing the larvae were incubated for 24 h at 28 ± 4 °C, under a photoperiod comprising of 12 h of light and darkness. After 24 h, the larvae mortality was visually verified. Statistical analysis and CL50 calculation were performed using the GraphPad Prism version 5.0 for Windows program (GraphPad Software, San Diego, CA). Significant differences between the groups were analyzed by ANOVA and Tukey post-test (p < 0.05)

Results

Identification of isolated compounds

From the chromatographic procedures, along with the 1D and 2D NMR spectroscopic methods, fourteen compounds were identified from the aerial parts of W. viscosissima, including steroids, triterpenes, alkaloids and flavonoids.

Compound 1: β-sitosterol e stigmasterol were identified based on spectral data and comparisons with the literature data (Maciel et al., 2016Maciel et al., 2016 Maciel, J.K.S., Chaves, O.S., Filho, S.G.B., Teles, Y.C.F., Fernandes, M.G., Assis, T.S., Andrade, A.P., Felix, L.P., Silva, T.M.S., Ramos, N.S.M., Silva, G.R., Souza, M.F.V., 2016. New alcamide and antioxidant activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. Ex. Rowl. (Cactaceae). Molecules 21, http://dx.doi.org/10.3390/molecules21010011.
http://dx.doi.org/10.3390/molecules21010...
).

Compound 2: 3-oxolup-20(29)-en-28-oico acid (betulonic acid): NMR 1H (δ, CDCl3, 400 MHz): 0.92 (s, 3H, H-23), 0.97 (s, 3H, H-24), 0.99 (s, 3H, H-25), 1.0 (s, 3H, H-26) 1.06, (s, 3H,H-27), 1.69 (s, 3H, H-28), 4.61 (bs, 1H, H-29), 4.74 (bs, 1H, H-29), 3.0 (1H, ddd, J1 = 10.5 Hz, J2 = 10.5 Hz and J3 = 4.5 Hz, H-19), NMR 13C (δ, CDCl3, 100 MHz): 37.0 (C-1), 34.2 (C-2), 218.4 (C-3), 47.4 (C-4), 55.05 (C-5), 19.7 (C-6), 33.7 (C-7), 40.8 (C-8), 49.9 (C-9), 37.1 (C-10), 21.5 (C-11), 25.6 (C-12), 38.6 (C-13), 42.6 (C-14), 30.7 (C-15), 32.2 (C-16), 56.5 (C-17), 49.3 (C-18), 47,03 (C-19), 150.5 (C-20), 29.8 (C-21), 37.05 (C-22), 26.7 (C-23), 21.1 (C-24), 16.0 (C-25), 15.9 (C-26), 14.7 (C-27), 182.4 (C-28), 109.9 (C-29), 19.5 (C-30) (Caridade et al., 2018Caridade et al., 2018 Caridade, T.N.S., Rusceli, D.A., Oliveira, A.N.A., Souza, T.S.A., Ferreira, N.C.F., Avelar, D.S., Teles, Y.C.F., Silveira, E.R., Araújo, R.M., 2018. Chemical composition of four different species of the Waltheria genus. Biochem. Syst. Ecol. 80, 81-83.).

Compound 3: 3-oxoolean-18-en-28-oic acid (3a) (Soares et al., 1998Soares et al., 1998 Soares, F.P., Ronconi, C.A.V., Cunha, E.V.L., Barbosa-Filho, J.M., Silva, M.S., Braz-Filho, R., 1998. Four known triterpenoids isolated from three Brazilian plants: 1H and 13C chemical shift assignments. Magn. Reson. Chem. 36, 608-614.). 3b: 3-oxoolean-12(13)-en-28-oic acid. NMR 1H (δ, CDCl3, 400 MHz): 0.89 (s, 3H, H-26), 0.92 (s, 3H, H-29), 0.96 (s, 3H, H-30), 1.03 (s, 3H, H-25) 1.07, (s, 3H, H-23), 1.13 (s, 3H, H-27), 2.37 (1H, dd, J = 6.8 and 3.6 Hz, H-2), 2.82 (1H, dd, J = 14.0 and 4.4 Hz, H-18), 5.28 (1H, t, J = 3.2 Hz, H-12), NMR 13C (δ, CDCl3, 100 MHz): 39.2 (C-1), 34.1 (C-2), 217.9 (C-3), 47.5 (C-4), 55.4 (C-5), 19.6 (C-6), 32.2 (C-7), 39.3 (C-8), 46.6 (C-9), 36.9 (C-10), 23.0 (C-11), 122.5 (C-12), 143.7 (C-13), 41.8 (C-14), 22.7 (C-15), 23.5 (C-16), 46.9 (C-17), 41.1 (C-18), 45.8 (C-19), 30.7 (C-20), 34.2 (C-21), 32.4 (C-22), 26.5 (C-23), 21.6 (C-24), 15.1 (C-25), 16.9 (C-26), 25.9 (C-27), 184.4 (C-28), 33.1 (C-29), 23.6 (C-30) (Kwon et al., 2011Kwon et al., 2011 Kwon, H.A., Cha, J.W., Park, J., Chun, Y.S., Moodley, N., Maharaj, V.J., Youn, S.H., Chung, S., Yang, H.O., 2011. Rapid identification of bioactive compounds reducing the production of amyloid β-peptide (Aβ) from South African plants using an automated HPLC/SPE/HPLC coupling system. Biomol. Ther. 19, 90-96.).

Compound 4: 5,7-dihydroxy-4′-methoxy-flavone (acacetin); Compound 6: 4′,5,7-trihydroxyflavon-3-ol (kaempferol); Compound 7: 3′,4′,5,7-tetrahydroxyflavon-3-ol (quercetin) were identified based on spectral data and comparisons with the literature data (Fernandes et al., 2018Fernandes et al., 2018 Fernandes, D.A., Souza, M.S.R., Teles, Y.C.F., Oliveira, L.H.G., Lima, J.B., Conceição, A.S., Nunes, F.C., Silva, T.M.S., Souza, M.F.V., 2018. New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules 23, http://dx.doi.org/10.3390/molecules23112784..
http://dx.doi.org/10.3390/molecules23112...
; Chaves et al., 2017Chaves et al., 2017 Chaves, O.S., Teles, Y.C.F., Monteiro, M.M.O., Mendes-Junior, L.G., Agra, M.F., Braga, V.A., Silva, T.M.S., Souza, M.F.V., 2017. Alkaloids and phenolic compounds from Sida rhombifolia L. (Malvaceae) and vasorelaxant activity of two indoquinoline alkaloids. Molecules 22, http://dx.doi.org/10.3390/molecules22010094.
http://dx.doi.org/10.3390/molecules22010...
; Teles et al., 2015aTeles et al., 2015a Teles, Y.C.F., Horta, C.C.R., Agra, M.F., Siheri, W., Boyd, M., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2015. New sulphated flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae). Molecules, http://dx.doi.org/10.3390/molecules201119685.
http://dx.doi.org/10.3390/molecules20111...
).

Compound 5: 7, 4′-di-O-metilisoscutellarein. NMR 1H (δ, DMSO-d 6, 200 MHz): 12.97 (1H, s, 5 - OH), 6.86 (s, 1H, H-3), 6.55 (1H, s, H-6), 8.11 (2H, d, J = 8,4 Hz, H-2′ and 6′), 7.13 (2H, d, J = 8.2 Hz, H-3′and 5′), 3.85 (s, OCH3-4′), 3.90 (s, OCH3-7). NMR 13C (δ, DMSO-d 6, 50 MHz): 163.5 (C-2), 103.0 (C-3), 182.4 (C-4), 153.1 (C-5), 95.7 (C-6), 154.3 (C-7), 126.2 (C-8), 144.4 (C-9), 103.9 (C-10), 123.0 (C-1′), 128.5 (C-2′), 114.5 (C-3′), 162.4 (C-4′), 114.5 (C-5′), 128.5 (C-6′), 55.30 (OCH3-4′), 56.50 (OCH3-7) (Teles et al., 2015aTeles et al., 2015a Teles, Y.C.F., Horta, C.C.R., Agra, M.F., Siheri, W., Boyd, M., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2015. New sulphated flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae). Molecules, http://dx.doi.org/10.3390/molecules201119685.
http://dx.doi.org/10.3390/molecules20111...
, 2015bTeles et al., 2015b Teles, Y.C.F., Ribeiro-Filho, J., Bozza, P.T., Agra, M.F., Siheri, W., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2015. Phenolic constituents from Wissadula periplocifolia (L.) C. Presl. and anti-inflammatory activity of 7,4′-di-O-methylisoscutellarein. Nat. Prod. Res. 7, 1-5.).

Compound 8: 5,7-dihydroxy-4′-methoxyisoflavone. NMR 1H (δ, (CD3)2CO, 500 MHz): 12.97 (1H, s, 5 - OH), 8.17 (s, 1H, H-2), 6.28 (1H, d, J = 1.9 Hz, H-6), 6.41 (1H, d, J = 1,9 Hz, H-8), 7.55 (2H,d, J = 8.75 Hz, H-2′ and 6′), 7.00 (2H, d, J = 8.75 Hz, H-3′ and 5′), 3.83 (s, OCH3-4′). NMR 13C (δ, (CD3)2CO, 125 MHz): 154.6 (C-2), 123.8 (C-3), 181.5 (C-4), 163.9 (C-5), 99.80 (C-6), 165.1 (C-7), 94.5 (C-8), 159.0 (C-9), 106.1 (C-10), 124.2 (C-1′), 131.0 (C-2′), 114.5 (C-3′), 160.7 (C-4′), 114.5 (C-5′), 131.0 (C-6′), 55.50 (OCH3-4′) (Almeida et al., 2008Almeida et al., 2008 Almeida, J.G.L., Silveira, E.R., Pessoa, O.D.L., 2008. NMR spectral assignments of a new [C - O - C] isoflavone dimer from Andira surinamensis. Magn. Reson. Chem. 46, 103-106.).

Compound 9: tiliroside, 1H RMN (500 MHz, DMSO-d 6) and 13C (125 MHz, DMSO-d 6) in accordance with the literature data (Teles et al., 2015aTeles et al., 2015a Teles, Y.C.F., Horta, C.C.R., Agra, M.F., Siheri, W., Boyd, M., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2015. New sulphated flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae). Molecules, http://dx.doi.org/10.3390/molecules201119685.
http://dx.doi.org/10.3390/molecules20111...
).

Compound 10: 5,7,4′-trihydroxyflavone-8-C-β-glucopyranoside (vitexin). 1H NMR (400 MHz, DMSO-d 6): 13.16 (1H, s, 5−OH), 6.78 (1H, s, H-3), 6.26 (1H, s, H-6), 8.02 (2H, d, J = 8.8 Hz, H-2′/6′), 6.89 (2H, d, J = 8.8 Hz, H-3′/5′), 4.69 (1H, d, J 10.0 Hz, H-1″), 3.83 (dd, J = 9.2 and 9.6 Hz, H-2″), 3.51 (m, H-3″), 3.34 (m, H-4″), 3.24 (m, H-5″), 3.52 (1H, dd, J = 6.0 and 11.8, H-6″), 3.76 (1H, d, J = 11.0 Hz, H-6″). NMR 13C (100 MHz, DMSO-d 6): 182.2 (C-4), 163.9 (C-2), 162.6 (C-7), 160.4 (C-5), 161.4 (C-4′), 156.0 (C-9), 128.9 (CH- 2′/CH-6′), 121.6 (C-1′), 115.8 (CH-3′/5′), 104.6 (C-8), 104.3 (C-10), 102.4 (C-3), 98.1 (C-6), 73.4 (CH-1″), 70.8 (CH-2″), 77.9 (CH-3″), 70.8 (CH-4″), 81.6 (CH-5″), 61.3 (CH2-6″) (He et al., 2016He et al., 2016 He, M., Min, J.-W., Kong, W.-L., He, X.-H., Li, J.-X., Peng, B.-W., 2016. A review on the pharmacological effects of vitexin and isovitexin. Fitoterapia 115, 74-85.).

Compound 11: luteolin 7-O-β-d-glucopyranoside. 1H NMR (400 MHz, DMSO-d 6): 12.95 (1H, s, 5−OH), 7.45 (d, J = 2.4 Hz, H-6′), 7.43 (d, J = 2 Hz, H-2′), 6.90 (d, J = 8.0 Hz, H-5′), 6.78 (d, J = 2.0 Hz, H-8), 6.74 (s, H-3), 6.43 (d, J = 2.0 Hz, H-6), 5.08 (d, J = 7.2 Hz, H-1″), 3.70 (m, H-6″), 3.45 (m, H-3″, H-6″), 3.28 (m, H-2″), 3.18 (m, H-5″). NMR 13C (100 MHz, DMSO-d6): 181.8 (C-4), 164.5 (C-2), 162.9 (C-7), 161.1 (C-5), 156.9 (C-9), 150.01 (C-4′), 145.8 (C-3′), 121.3 (C-1′), 119.2 (CH-6′), 115.9 (CH-5′), 113.5 (CH-2′), 105.3 (C-10), 103.1 (CH-3), 99.9 (CH-1″), 99.5 (CH-6), 94.7 (CH-8), 77.1 (CH-3″), 76.3 (CH-2″), 73.1 (CH-5″), 69.5 (CH-4″), 60.5 (CH2-6″) (Silva et al., 2006aSilva et al., 2006a Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S.C., Matias, W.N.M., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes químicos e atividade antioxidante de Sida galheirensis Ulbr. (Malvaceae). Quim. Nova 26, 1250-1254.).

Compound 12a: waltherione a 1H NMR (300 MHz, CD3OD): 7.57 (1H, d, J = 8.8 Hz, H-7), 7.42 (1H, d, J = 8.8 Hz, H-8), 4.74 (m, H-10), 1.99-2,06 (2H, m, H-11), 2.26-2,41 (2H, m, H-12), 6.68 (1H, dl, J = 6.19 Hz, H-13), 7.10 (dl, J = 8.1 Hz, H-3′), 7.25 (ddd, J = 8.10, 7.81 and 1.57 Hz, H-4′), 6.73 (ddd, J = 7.65, 7.81 and 1.35 Hz, H-5′), 6.31 (dd, J = 7.65 and 1.35 Hz, H-6′), 3.81 (s, OCH3-3), 4.00 (s, OCH3-2′), 2.47 (s, 2−CH3). NMR 13C (75 MHz, CD3OD): 14.3 (2−CH3), 23.09 (C-11), 35.2 (C-12), 56.0 (OCH3-3), 60.3 (OCH3-2′), 76.9 (C-13), 79.2 (C-9), 81.4 (C-10), 112.4 (C-3′), 118.02 (C-8), 119.9 (C-4a), 121.2 (C-5′), 128.9 (C-4′), 132.7 (C-7), 132.4 (C-6), 133.6 (C-6′), 81.3 (C-10), 143.1 (C-3), 143.5 (C-2), 140.5 (C-5), 142.6 (C-8a), 158.2 (C-2′ ), 176.01 (C-4) (Gressler et al., 2008Gressler et al., 2008 Gressler, V., Stuker, C.Z., Dias, G.O.C., 2008. Quinolone alkaloids from Waltheria douradinha. Phytochemistry 69, 994-999.).

Compound 12b: waltherione b. 1H NMR (300 MHz, CD3OD): 7.58 (1H, d, J = 8.8 Hz, H-7), 7.42 (1H, d, J = 8.8 Hz, H-8), 4.76 (m, H-10), 1.99-2,06 (2H, m, H-11), 2.26-2,41 (2H, m, H-12), 6.68 (1H, dl, J = 6.19 Hz, H-13), 7.02 (d, J = 8.98 Hz, H-3′), 7.25 (ddd, J = 8.10, 7.81 and 1.57 Hz, H-4′), 6.73 (ddd, J = 7.65, 7.81 and 0.7 Hz, H-5′), 6.81 (dd, J = 8.93 and 3.07 Hz, H-6′), 3.95 (s, OCH3-3), 3.52 (s, OCH3-2′), 2.47 (s, 2−CH3). NMR 13C (75 MHz, CD3OD): 14.4 (2−CH3), 23.3 (C-11), 32.0 (C-12), 55.5 (OCH3-3), 59.7 (OCH3-2′), 76.9 (C-13), 79.1 (C-9), 81.44 (C-10), 112.4 (C-3′), 118.02 (C-8), 119.9 (C-4a), 121.2 (C-5′), 128.93 (C-4′), 132.7 (C-7), 132.4 (C-6), 133.6 (C-6′), 81.44 (C-10 ), 152.3 (C-3), 143.5 (C-2), 140.5 (C-5), 142.6 (C-8a), 154.5 (C-2′), 176.01 (C-4) (Gressler et al., 2008Gressler et al., 2008 Gressler, V., Stuker, C.Z., Dias, G.O.C., 2008. Quinolone alkaloids from Waltheria douradinha. Phytochemistry 69, 994-999.).

Quantification of phenolics, flavonoids, and free radical scavenging activity of Waltheria. viscosissima

Spectrophotometric methods were used to quantify the total phenolic compounds and total flavonoids in the extracts of W. viscosissima.

A calibration curve was constructed with gallic acid to quantify the phenolic compounds. The value of the coefficient of linearity found was R² = 0.9963, and the total phenolic compounds was calculated from the obtained equation of the line (y = 0.000993696x − 0.00218). To quantify the flavonoid content, a calibration curve was built using quercetin, which obtained the coefficient of linearity R2 = 0.9997 and equation of the line: y = 0.00215x − 0.00243.

To calculate the concentration of extracts that were able to induce the reduction of 50% in DPPH˙ concentration (EC50), crescent concentrations of APCE and RCE were used according to the described method. A calibration curve was built for each extract that was in mixture with DPPH: Y = −0.001921.x + 0.4705 and Y = −0.0008214.x + 0.4725 for APCE and RCE, respectively (Table 1).

Table 1
Total phenolic, total flavonoids contents and DPPH free radical scavenging activity.

Biological assay

The mortality of L4 larvae in different extract concentrations of W. viscosissima is shown in Tables 2 and 3. The concentration of 150.0 mg/ml of APCE killed 100% of larvae. Concentrations of 75, 50, 20, and 200 mg/ml killed 88%, 65%, 21.6% and 0%, respectively. The tested concentrations of 200 and 150 mg/ml were not found to differ statistically (p < 0.05). The calculated LC50 of APCE was 38.37 mg/ml (Figure 2).

Table 2
Mean of mortality of Ae. aegypti larvae (L4) exposed to different concentrations of APCE of Waltheria viscosissima.
Table 3
Mean of mortality of Ae. aegypti larvae (L4) exposed to different concentrations of RCE of Waltheria viscosíssima.

For RCE, the concentration of 40 mg/ml was able to kill 100% of larvae. Concentrations of 30, 20, 10, 5, 2.5 and 1 mg/ml showed a mortality of 96.6%, 91.6%, 85%, 56.6%, 38.3% and 0%, respectively. The concentrations of 5 and 2.5 mg/ml were statistically different (p <0.05), and the calculated LC50 for RCE was 4.78 mg/ml (Figure 2).

Discussion

Identification of compounds

Compound 1 was identified as a mixture of the phytosteroids: β-sitosterol and stigmasterol, which is widely isolated from plants and known by their biological role as vegetal cell membrane constituents (Maciel et al., 2016Maciel et al., 2016 Maciel, J.K.S., Chaves, O.S., Filho, S.G.B., Teles, Y.C.F., Fernandes, M.G., Assis, T.S., Andrade, A.P., Felix, L.P., Silva, T.M.S., Ramos, N.S.M., Silva, G.R., Souza, M.F.V., 2016. New alcamide and antioxidant activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. Ex. Rowl. (Cactaceae). Molecules 21, http://dx.doi.org/10.3390/molecules21010011.
http://dx.doi.org/10.3390/molecules21010...
).

Compound 2 was identified as the lupane-type triterpene 3-oxolup-20(29)-en-28-oic acid (betulonic acid) that was previously isolated from Waltheria cinerencens (Caridade et al., 2018Caridade et al., 2018 Caridade, T.N.S., Rusceli, D.A., Oliveira, A.N.A., Souza, T.S.A., Ferreira, N.C.F., Avelar, D.S., Teles, Y.C.F., Silveira, E.R., Araújo, R.M., 2018. Chemical composition of four different species of the Waltheria genus. Biochem. Syst. Ecol. 80, 81-83.). The betulonic acid was demonstrated to possess leishmanicidal activity and anti-proliferative activity against cell cancer lineages (Alakurtti et al., 2010Alakurtti et al., 2010 Alakurtti, S., Bergström, P., Sacerdoti-Sierra, N., Jaffe, C.L., Yli-Kauhaluoma, J., 2010. Anti-leishmanial activity of betulin derivatives. J. Antibiot. 63, 123-126.; Yang et al., 2015Yang et al., 2015 Yang, S.J., Liu, M.C., Zhao, Q., Hu, D.Y., Xue, W., Yang, S., 2015. Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents. Eur. J. Med. Chem. 96, 58-65.).

The 1H NMR of compound 3, showed a triterpene aspect, as shown in compound 2. Absorptions for two hydrogen, at δH 5.28 (1H, t, J = 3.2 Hz) and δH 5.15 (1H, bs), indicate the presence of double bonds, suggested that the sample could be a mixture of two triterpenes. The 13C NMR spectra presented signals for sixty carbons, corroborating the claim regarding the presence of two triterpenes in the sample. The olefinic carbons at δC 122.5 (C-12) and δC 143.7 (C-13), characteristic of oleanan-type triterpenes, allowed an identification of the compounds in the mixture as 3-oxo-olean-12(13)-en-28-oic acid and the 3-oxo-olean-18-en-28-oic acid (moronic acid). The moronic acid was previously isolated from W. viscosíssima (Soares et al., 1998Soares et al., 1998 Soares, F.P., Ronconi, C.A.V., Cunha, E.V.L., Barbosa-Filho, J.M., Silva, M.S., Braz-Filho, R., 1998. Four known triterpenoids isolated from three Brazilian plants: 1H and 13C chemical shift assignments. Magn. Reson. Chem. 36, 608-614.; Kwon et al., 2011Kwon et al., 2011 Kwon, H.A., Cha, J.W., Park, J., Chun, Y.S., Moodley, N., Maharaj, V.J., Youn, S.H., Chung, S., Yang, H.O., 2011. Rapid identification of bioactive compounds reducing the production of amyloid β-peptide (Aβ) from South African plants using an automated HPLC/SPE/HPLC coupling system. Biomol. Ther. 19, 90-96.).

The NMR spectra of compounds 4, 5, 6 and 7, isolated as yellow powder, showed signals in the aromatic region that was compatible with flavonoids. By analyzing the 1D and 2D NMR of them, it was possible to identify the compounds as acacetin (4), 7,4′-di-O-methyl-isoscutellarein (5), kaempferol (6) and quercetin (7). The presence of these compounds in Malvaceae sensu lato species is in agreement with the literature (Silva et al., 2006aSilva et al., 2006a Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S.C., Matias, W.N.M., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes químicos e atividade antioxidante de Sida galheirensis Ulbr. (Malvaceae). Quim. Nova 26, 1250-1254.; Costa et al., 2008Costa et al., 2008 Costa, F.J., Bandeira, P.N., Albuquerque, M.R.J.R., Pessoa, O.D.L., Silveira, E.R., Braz-Filho, R., 2008. Constituintes químicos de Vernonia chalybaea mart. Quim. Nova 31, 1691-1695.; Chaves et al., 2017Chaves et al., 2017 Chaves, O.S., Teles, Y.C.F., Monteiro, M.M.O., Mendes-Junior, L.G., Agra, M.F., Braga, V.A., Silva, T.M.S., Souza, M.F.V., 2017. Alkaloids and phenolic compounds from Sida rhombifolia L. (Malvaceae) and vasorelaxant activity of two indoquinoline alkaloids. Molecules 22, http://dx.doi.org/10.3390/molecules22010094.
http://dx.doi.org/10.3390/molecules22010...
; Gomes et al., 2011Gomes et al., 2011 Gomes, R.A., Ramirez, R.R.A., Maciel, J.K.S., Agra, M.F., Souza, M.F.V., Falcão-Silva, V.S., Siqueira-Junior, J.P., 2011. Phenolic compounds from Sidastrum micranthum (A. St.-Hil.) Fryxell and evaluation of acacetin and 7,4′-Di-O-methylisoscutellarein as motulator of bacterial drug resistence. Quim. Nova 34, 1385-1388.; Dixit et al., 2011Dixit et al., 2011 Dixit, P., Khan, M.P., Swarnkar, G., Chattopadhyay, N., Maurya, R., 2011. Osteogenic constituents from Pterospermum acerifolium Willd. Flowers.. Bioorg. Med. Chem. Lett. 21, 4617-4621.; Fernandes et al., 2018Fernandes et al., 2018 Fernandes, D.A., Souza, M.S.R., Teles, Y.C.F., Oliveira, L.H.G., Lima, J.B., Conceição, A.S., Nunes, F.C., Silva, T.M.S., Souza, M.F.V., 2018. New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules 23, http://dx.doi.org/10.3390/molecules23112784..
http://dx.doi.org/10.3390/molecules23112...
). Flavonoids have been shown to be the major compounds in several species from the Sterculiaceae family, including species from the Waltheria genus (Muqarrabun and Ahmat, 2015Niraimathi et al., 2010 Niraimathi, S., Balaji, N., Venkataramanan, N., Govindarajan, M., 2010. Larvicidal activity of alkaloid from Sida acuta against Anopheles subpictus and Culex tritaeniorhynchus. Int. Curr. Res. 11, 034-038.; Cretton et al, 2015Cretton et al., 2015 Cretton, S., Bréant, L., Pourrez, L., Ambuehl, C., Perozzo, R., Marcourt, L., Kaiser, M., Cuendet, M., Christen, P., 2015. Chemical constituents from Waltheria indica exert in vitro activity against Trypanosoma brucei and T. Cruz. Fitoterapia 105, 55-60., 2016Cretton et al., 2016 Cretton, S., Dorsaz, S., Azzollini, A., Favre-Godal, Q., Marcourt, L., Ebrahimi, S.N., Voinesco, F., Michellod, M., Sanglard, D., Gindro, K., Wolfender, J., Cuendet, M., Christen, P., 2016. Antifungal quinoline alkaloids from Waltheria indica. J. Nat. Prod. 79, 300-307.; Teles et al., 2015b; Caridade et al., 2018Teles et al., 2015b Teles, Y.C.F., Ribeiro-Filho, J., Bozza, P.T., Agra, M.F., Siheri, W., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2015. Phenolic constituents from Wissadula periplocifolia (L.) C. Presl. and anti-inflammatory activity of 7,4′-di-O-methylisoscutellarein. Nat. Prod. Res. 7, 1-5.).

Compound 8 was isolated as orange powder. The ¹H NMR showed a couple of doublets coupling meta, which were characteristic of 5, 7-di-substituted flavonoids. The hydroxyl at position 5 was found at em δH 12.97 (s). A singlet was detected at δH 8.17, suggesting an isoflavonoid structure for compound 8. The 13C NMR showed fourteen signals, including one methoxyl. Among the signals, the highlights include the carbons at δC 154.4 and 123.8 assigned to the presence of a double bond between C-2 and C-3; the carbonyl, found at δC 181.5; and the high intensity carbons of a para substituted B-ring at 114.5 (C-3′ and 5′), 131.0 (C-2′ and 6′). The 2D spectral data and comparison with the literature led to the identification of compound 8 as the isoflavan: 5,7-dihydroxy-4′-methoxyisoflavone (biochanin A) (Almeida et al., 2008Almeida et al., 2008 Almeida, J.G.L., Silveira, E.R., Pessoa, O.D.L., 2008. NMR spectral assignments of a new [C - O - C] isoflavone dimer from Andira surinamensis. Magn. Reson. Chem. 46, 103-106.). The compound is being reported for the first time in the Sterculiaceae family. In previous studies, it has demonstrated anticancer and anti-hyperlipidemic activities (Harini et al., 2012Harini et al., 2012 Harini, R., Sundaresan, A., Pugalendi, K.V., 2012. Antihyperlipidemic effect of biochanin A on streptozotocin induced diabetic rats. J. Pharm. Res. 5, 707-710.; Muqarrabun and Ahmat, 2015Muqarrabun and Ahmat, 2015 Muqarrabun, L.M.R.A., Ahmat, N., 2015. Medicinal uses, phytochemistry and pharmacology of Family Sterculiaceae: a review. Eur. J. Med. Chem. 92, 514-530.; Xiao et al., 2017Xiao et al., 2017 Xiao, P., Zheng, B., Sun, J., Yang, J., 2017. Biochanin A induces anticancer effects in SK Mel 28 human malignant melanoma cells via induction of apoptosis, inhibition of cell invasion and modulation of NF κB and MAPK signaling pathways. Oncol. Lett. 14, 5989-5993.).

Compound 9 was identified as kaempferol-3-O-β-d-(6″-E-p-coumaroyl) glucopyranoside (tiliroside). Tiliroside is widely produced by Malvaceae sensu lato and has demonstrated many pharmacological properties, such as vasorelaxant, antioxidant, hypolipidemic, antinociceptive and anti-inflammatory activities (Barbosa et al., 2007Barbosa et al., 2007 Barbosa, E., Calzada, F., Campos, R., 2007. In vivo antigiardial activity of three flavonoids isolated of some medicinal plants used in Mexican traditional medicine for the treatment of diarrhea. J. Ethnopharmacol. 109, 552-554.; Orhan et al., 2009Orhan et al., 2009 Orhan, N., Aslan, M., Hosbas, S., Deliorman, O.D., 2009. Antidiabetic effect and antioxidant potential of Rosa canina fruits. Phcog. Mag. 5, 309-315.; Zhang et al., 2015Zhang et al., 2015 Zhang, Z., Li, G., Szeto, S.S., Chong, C., Quan, Q., Huang, C., Cui, W., Guo, B., Wang, Y., Han, Y., Michael Siu, K.W., Lee, S.M., Chu, I.K., 2015. Flavonoid glycosides from Rubus chingii Hu fruits display anti-inflammatory activity through suppressing MAPKs activation in macrophages. Free Rad. Biol. Med. 84, 331-343.).

The NMR data obtained for compounds 10 and 11, along with comparisons with literature, led to them being identified as two glucosyl flavones: vitexin (10) and luteolin 7-O-d-glucopyranoside (11). Both of them have pharmacological properties and have been isolated from the species Pterospermum acerifolium and Theobroma cacao, Sterculiaceae (Silva et al., 2006aSilva et al., 2006a Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S.C., Matias, W.N.M., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes químicos e atividade antioxidante de Sida galheirensis Ulbr. (Malvaceae). Quim. Nova 26, 1250-1254.; Dixit et al., 2011Dixit et al., 2011 Dixit, P., Khan, M.P., Swarnkar, G., Chattopadhyay, N., Maurya, R., 2011. Osteogenic constituents from Pterospermum acerifolium Willd. Flowers.. Bioorg. Med. Chem. Lett. 21, 4617-4621.; Cuong et al., 2015Cuong et al., 2015 Cuong, L.C., Trang, T., Cuc, N.T., Nhiem, N.X., Yen, P.H., Anh, H.L.T., Uong, L.T., Minh, C.V., Kiem, P.V., 2015. Flavonoid glycosides from Antidesma ghaesembilla. Vietnam J. Chem. 53, 94-97.; He et al., 2016He et al., 2016 He, M., Min, J.-W., Kong, W.-L., He, X.-H., Li, J.-X., Peng, B.-W., 2016. A review on the pharmacological effects of vitexin and isovitexin. Fitoterapia 115, 74-85.).

Compound 12, which was isolated as yellowish oil, showed signals at NMR spectra that were compatible with quinoline alkaloids. The 1H NMR spectra presented a set of signals δH 6.31–7.59 which was compatible with tetra substituted ring of quinoline alkaloids. The chemical shifts in addition to integrals values led to suggest that this would be a mixture of two substances in the proportion of 65:35. The nucleus of 10,13-oxo-bicyclo heptane was suggested by the absorptions for oxymethine protons at δH 4.74 (m) and 6.68 (d, J = 6.19 Hz), besides the multiplets at 1.99–2.06 (m) and 2.26–2.41 (m). The 13C NMR spectra confirmed the proposal of a mixture of two compounds by showing carbons with very different intensities. Relevant signals for elucidating the structure of the compounds were α,β-unsaturated ketone carbonyl at δC 176.6; oxymethine carbons δc 82.44 (C-10, 12b); 81.39 (C-10, 12a) and 76.10 (C-13, 12a and 12b).

The analysis of the 2D spectra, compilation of chemical shifts, and comparisons with literature data, allowed an identification of compound 12 as a mixture of the alkaloids waltherione a (12a) and waltherione b (12b) in the proportion (65:35), respectively, along with antimicrobial compounds previously reported from Waltheria douradinha (Hoelzel et al., 2005Hoelzel et al., 2005 Hoelzel, S.C.S.M., Vieira, E.R., Giacomelli, S.R., Dalcol, I.I., Zanatta, N., Morel, A.F., 2005. An unusual quinolinone alkaloid from Waltheria douradinha. Phytochemistry. 66, 1163-1167.; Gressler et al., 2008Gressler et al., 2008 Gressler, V., Stuker, C.Z., Dias, G.O.C., 2008. Quinolone alkaloids from Waltheria douradinha. Phytochemistry 69, 994-999.).

Quantification of phenolics, flavonoids and free radical scavenging activity of Waltheria viscosíssima

It has been well reported that plants with a high production of phenolic compounds, such as phenolic acids derivatives, tannins, flavonoids, etc., have been established to have great antioxidant activity (Singh et al., 2016Singh et al., 2016 Singh, P.A., Brindavanam, N.B., Kimothi, G.P., Aeri, V., 2016. Evaluation of in vivo anti-inflammatory and analgesic activity of Dillenia indica f. elongata (Miq.) and Shorea robusta stem bark extracts. Asian Pac. J. Trop. Dis. 6, 75-81.). The quantification of W. viscosíssima phenolic compounds demonstrated 131.01 ± 0.04 mg de EAG/g of APCE and 137.04 ± 0.01 mg de EAG of RCE. The total phenolic content of other species of Malvaceae sensu lato, such as Sterculia striata, Sidastrum micranthum and Sida rhombifolia, have been determined by the same method. The W. viscosíssima extracts (both APCE and RCE) were shown to possess a concentration of phenolic compounds that were at least twice as great as the other investigated species (Costa et al., 2009Costa et al., 2009 Costa, D.A., Matias, W.N., Lima, I.O., Xavier, A.L., Costa, V.B.M., Melo, M.F.F., Agra, M.F., Batista, L.M., Souza, M.F.V., 2009. First secondary metabolites from Herissantia crispa L (Brizicky) and the toxicity activity against Artemia salina Leach. Quim. Nova 32, 48-50.; Oliveira et al., 2012Oliveira et al., 2012 Oliveira, A.M.F., Pinheiro, L.S., Pereira, C.K.S., Matias, W.N., Gomes, R.A., Chaves, O.S., Souza, M.F.V., Almeida, R.N., Assis, T.S., 2012. Total phenolic content and antioxidant activity of some Malvaceae family species. Antioxidants 1, 33-43.).

Flavonoids are compounds that have garnered the interest of researchers in biological field due their huge pharmacological potential and their common occurrence in medicinal plants and in functional foods (Teles et al., 2015aTeles et al., 2015a Teles, Y.C.F., Horta, C.C.R., Agra, M.F., Siheri, W., Boyd, M., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2015. New sulphated flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae). Molecules, http://dx.doi.org/10.3390/molecules201119685.
http://dx.doi.org/10.3390/molecules20111...
). In this research, eight flavonoid structures have been identified, justifying our interest in quantifying the total content of flavonoids in W. viscosíssima extracts. The obtained results showed a greater concentration of flavonoids in the APCE than the RCE. The result is in agreement with literature that reports the accumulation of these compounds in surface cells of vegetal organs that have a high exposure to sun light (Agati et al., 2012Agati et al., 2012 Agati, G., Azzarello, E., Pollastri, S., Tattini, M., 2012. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 196, 67-76.). The flavonoids have been shown to act as protective agents against light-induced oxidative damage, since they absorb UV and visible light. Indeed, studies have demonstrated that sun light up-regulates the flavonoids biosynthesis. Thus, the accumulation of flavonoids in external vegetal structures is consistent with UV and visible light protection functions (Havaux and Kloppstech, 2001Havaux and Kloppstech, 2001 Havaux, M., Kloppstech, K., 2001. The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants. Planta 213, 953-966.; Agati et al., 2012Agati et al., 2012 Agati, G., Azzarello, E., Pollastri, S., Tattini, M., 2012. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 196, 67-76.).

Due the ability of phenolic and flavonoid compounds to prevent oxidative stress and inflammation (Tao et al., 2016Tao et al., 2016 Tao, X., Sun, X., Xu, L., Yin, L., Han, X., Qi, Y., Xu, Y., Zhao, Y., Wang, C., Peng, J., 2016. Total flavonoids from Rosa laevigata Michx fruit ameliorates hepatic ischemia/reperfusion injury through inhibition of oxidative stress and inflammation in rats. Nutrients 8, http://dx.doi.org/10.3390/nu8070418.
http://dx.doi.org/10.3390/nu8070418...
; Herrera-Calderon et al., 2016Herrera-Calderon et al., 2016 Herrera-Calderon, O., Enciso-Roca, E., Pari-Olarte, B., Arroyo-Acevedo, J., 2016. Phytochemical screening, antioxidant activity and analgesic effect of Waltheria ovata Cav. Roots in mice. Asian Pac. J. Trop. Dis. 6, 1000-1003.; Hou et al., 2017Hou et al., 2017 Hou, Z., Hub, Y., Yangc, X., Chen, W., 2017. Antihypertensive effects of tartary buckwheat flavonoids by improvement of vascular insulin sensitivity in spontaneously hypertensive rats. Food Funct. 7, 01-32.), the wide occurrence of flavonoids that were previously reported in other Waltheria (Caridade et al., 2018Caridade et al., 2018 Caridade, T.N.S., Rusceli, D.A., Oliveira, A.N.A., Souza, T.S.A., Ferreira, N.C.F., Avelar, D.S., Teles, Y.C.F., Silveira, E.R., Araújo, R.M., 2018. Chemical composition of four different species of the Waltheria genus. Biochem. Syst. Ecol. 80, 81-83.) and reported here in the aerial parts of W. viscosissima might be related to the popular use of the Waltheria species as an anti-inflammatory (Yougbare-Ziebrou et al., 2016Yougbare-Ziebrou et al., 2016 Yougbare-Ziebrou, M.N., Lompo, M., Ouedraogo, N., Yaro, B., Guissoun, I.C., 2016. Antioxidant, analgesic and anti-inflammatory activities of the leafy stems of Waltheria indica L. (Sterculiaceae). Int. J. Appl. Pharm. Sci. Res. 6, 124-129.).

In this study, we demonstrated that the RCE presented greater free radical scavenging activity against DPPH˙ radical than the APCE. The ability of free radicals reduction is one of several cell mechanisms that prevent oxidative damage (Li et al., 2012Li et al., 2012 Li, T., Hou, Y., Cao, W., Yan, C.X., Chen, T., Li, S.B., 2012. Role of dopamine D3 receptors in basal nociception regulation and in morphine-induced tolerance and withdrawal. Brain Res. 1433, 80-84.). According to our results, greater reductive activity was carried out by the RCE. Studies have demonstrated that the free radical scavenging activity is more intense for phenolic compounds with ortho-dihydroxy aromatic rings (Rice-Evans et al., 1996Rice-Evans et al., 1996 Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933-956.). In fact, just two out of the eight flavonoids identified in APCE possess ortho-dihydroxy rings in their structures. Thus, we can attribute the greater scavenging activity to the presence of other phenolic compounds in the RCE, rather than the flavonoids that have been shown to be in very low concentration in the RCE. When taken into consideration with other Malvaceae sensu lato species that were previously investigated, W. viscosissima extracts have shown greater free radical scavenging activity (Oliveira et al., 2012Oliveira et al., 2012 Oliveira, A.M.F., Pinheiro, L.S., Pereira, C.K.S., Matias, W.N., Gomes, R.A., Chaves, O.S., Souza, M.F.V., Almeida, R.N., Assis, T.S., 2012. Total phenolic content and antioxidant activity of some Malvaceae family species. Antioxidants 1, 33-43.). Indeed, when compared to the other Waltheria species previously investigated, Waltheria ovata has presented the best result, with EC50 of 3.50 µg/ml (Herrera-Calderon et al., 2016Herrera-Calderon et al., 2016 Herrera-Calderon, O., Enciso-Roca, E., Pari-Olarte, B., Arroyo-Acevedo, J., 2016. Phytochemical screening, antioxidant activity and analgesic effect of Waltheria ovata Cav. Roots in mice. Asian Pac. J. Trop. Dis. 6, 1000-1003.).

Larvicidal activity

Concentrations of 2 mg/ml APCE and 1 mg/ml of RCE were used to perform the bioassays. Using the cited concentrations, no activity was observed. As such, the concentrations were gradually increased to get a mortality that was statistically different from the negative control. The mortality is observed when the larvae presented compromised mobility, lethargy and complete paralysis (Ravikumar et al., 2011Ravikumar et al., 2011 Ravikumar, S.M., Ali, S., Beula, J.M., 2011. Mosquito larvicidal efficacy of seaweed extracts against dengue vector of Aedes aegypti. Asian Pac. J. Trop. Biomed. 1, 143-146.; Oliveira et al., 2013Oliveira et al., 2013 Oliveira, G.L., Cardoso, S.C., Júnior, C.R.L., Vieira, T.M., Guimarães, E.F., Figueiredo, L.S., Martins, E.R., Moreira, D.L., Kaplan, M.A.C., 2013. Chemical study and larvicidal activity against Aedes aegypti of essential oil of Piper aduncum L. (Piperaceae). An. Acad. Bras. Cienc. 85, 1227-1234.; Santos, 2015Santos, 2015 Santos, D.B., 2015. Atividade larvicida da Copaifera langsdorffii (Leguminosae), evidenciada pelas alterações morfohistológicas em Aedes aegypti (Diptera, Culicidae). Dissertação (Mestrado), Pós-graduação em Biologia da Relação Parasito-Hospedeiro, Universidade Federal de Goiás, Goiânia, Brasil, pp. 73.).

After a statistical analysis, it was observed that in the W. viscosissima APCE, the utilized concentrations statistically differed, with the exception of the 200 mg/ml and 150 mg/ml, as observed in Table 2. For the RCE sample, concentrations of 10 mg/ml and 20 mg/ml did not differ statistically, and this was similar with the concentrations of 20 mg/ml, 30 mg/ml, and 40 mg/ml (Table 3). After 24 h of exposure, the mortality reached 100% for both extracts. The concentrations of 150 mg/ml for APCE and 40 mg/ml for RCE were significantly more effective when compared against other concentrations and the negative control.

The two extracts exhibited larvicidal activities with different LC50, which were calculated based on the statistical results obtained. For APCE, the LC50 was 38.7 mg/ml and the closest concentration tested was 50 mg/ml, which killed 13 larvae (mean), the equivalent of 65%. For the RCE, the LC50 was 4.78 mg/ml and the closest concentration was 5 mg/ml, which killed 11.3 larvae (mean), and this was equivalent to 56.6% (Table 3).

The RCE possess more significant larvicidal activity than the APCE. Indeed, different parts of plants contain a complex mixture of compounds with certain biological activities (Veni et al., 2016Veni et al., 2016 Veni, T., Pushpanathan, T., Mohanraj, J., 2016. Larvicidal and ovicidal activity of Terminalia chebula Retz. (Family: Combretaceae) medicinal plant extracts against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. J. Parasit. Dis. 41, 693-702.; Govindarajan et al., 2008aGovindarajan et al., 2008a Govindarajan, M., Jebanesan, A., Pushpanathan, T., Samidurai, K., 2008. Studies on effect of Acalypha indica L. (Euphorbiaceae) leaf extracts on the malarial vector, Anopheles stephensi Liston (Diptera: culicidae). Parasitol. Res. 103, 691-695., bGovindarajan et al., 2008b Govindarajan, M., Jebanesan, A., Pushpanathan, T., 2008. Larvicidal and ovicidal activity of Cassia fistula Linn. Leaf extract against filarial and malarial vector mosquitoes. Parasitol. Res. 102, 289-292.,cGovindarajan et al., 2008c Govindarajan, M., Jebanesan, A., Reetha, D., Amsath, R., Pushpanathan, T., Samidurai, K., 2008. Antibacterial activity of Acalypha indica L. Eur. Riv. Eur. Sci. Med. Farmacol. 2, 299-302.). The activities are usually related to the presence of toxins and secondary metabolites that act as larvicides (Niraimathi et al., 2010Niraimathi et al., 2010 Niraimathi, S., Balaji, N., Venkataramanan, N., Govindarajan, M., 2010. Larvicidal activity of alkaloid from Sida acuta against Anopheles subpictus and Culex tritaeniorhynchus. Int. Curr. Res. 11, 034-038.). The difference display by the LC50 when comparing different parts of a vegetal has been reported by Satana (2012), and has also been shown to affect the leaves and roots of the Murraya koenigii (Tennyson et al., 2012Tennyson et al., 2012 Tennyson, S., Ravindran, K.J., Arivoli, S., 2012. Bioefficacy of botanical insecticides against the dengue and chikungunya vector Aedes aegypti (L.) (Diptera:Culicidae). Asian Pac. J. Trop. Med. 3, 1842-1844.), seeds and leaves of Calophyllum inophyllum (Pushpalatha and Muthukrishnan, 1999Pushpalatha and Muthukrishnan, 1999 Pushpalatha, E., Muthukrishnan, J., 1999. Efficacy of two tropical plant extracts for the control of mosquitoes. J. Appl. Ent. 123, 258-262.), and stem and leaves of Guettarda grazielae (Oliveira et al., 2010Oliveira et al., 2010 Oliveira, P.V., Ferreira-Jr, J.C., Moura, F.S., Lima, G.S., Oliveira, F.M., Oliveira, P.E.S., Conserva, L.M., Giulietti, A.M., Lemos, R.P.L., 2010. Larvicidal activity of 94 extracts from ten plant species of northeastern of Brazil against Aedes aegypti L. (Diptera: Culicidae). Parasitol. Res. 107, 403-407.).

The concentration of RCE (LC50 = 4.78 mg/ml) was lower than other larvicidal activities previously reported. The LC50 found for the species Croton linearifolius, Euphorbiaceae, and Albizzia amara, Fabaceae, were 17.42 and 7.10 mg/m, respectively (Murugan et al., 2007Murugan et al., 2007 Murugan, K., Murugan, P., Noortheen, A., 2007. Larvicidal and repellent potential of Albizzia amara Boivin and Ocimum basilicum Linn against dengue vector, Aedes aegypti (Insecta: Diptera: Culicidae). Bioresour. Technol. 98, 198-201.; Silva et al., 2014Silva et al., 2014 Silva, S.L.C., Gualberto, S.A., Carvalho, K.S., Fries, D.D., 2014. Avaliação da atividade larvicida de extratos obtidos do caule de Croton linearifoliusMull. Arg. (Euphorbiaceae) sobre larvas de Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae). Biotemas 27, 79-85.).

The concentration of APCE (LC50 = 38.70 mg/ml) were also lower than Cymbopogon citratus (LC50 = 63.89 mg/ml) and Ocimum gratissimum (LC50 = 71.27 mg/ml). The Sterculiaceae Helicteres velutina have been evaluated and showed an LC50 of 138.89 mg/ml (stem) and 171.68 mg/ml (roots) (Santos et al., 2012Santos et al., 2012 Santos, E.A., Carvalho, C.M., Costa, A.L.S., Conceição, A.S., Moura, F.B.P., Santana, A.E.G., 2012. Bioactivity evaluation of plants extracts used in indigenous medicine against the snail, Biomphalaria glabrata, and the larvae of Aedes aegypti. Evid. Based. Complement. Alternat. Med., http://dx.doi.org/10.1155/2012/846583.
http://dx.doi.org/10.1155/2012/846583...
). However those concentrations were higher than the ones shown by aerial parts of Helicteres velutina (LC50 = 2.98 mg/ml) and Sida acuta (LC50 = 4.28 × 10−2 mg/ml) (Govindarajan, 2010Govindarajan, 2010 Govindarajan, M., 2010. Larvicidal and repellent activities of Sida acutaBurm. F. (Family: Malvaceae) against three important vector mosquitões. Asian Pac. J. Trop. Med. 3, 691-695.; Fernandes et al., 2018Fernandes et al., 2018 Fernandes, D.A., Souza, M.S.R., Teles, Y.C.F., Oliveira, L.H.G., Lima, J.B., Conceição, A.S., Nunes, F.C., Silva, T.M.S., Souza, M.F.V., 2018. New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules 23, http://dx.doi.org/10.3390/molecules23112784..
http://dx.doi.org/10.3390/molecules23112...
).

According to Guarda et al. (2016)Guarda et al., 2016 Guarda, C., Lutinski, J.A., Roman-Junior, W.A., Busato, M.A., 2016. Atividade larvicida de produtos naturais e avaliação da susceptibilidade ao inseticida temefós no controle do Aedes aegypti (Diptera: culicidae). Interciência 41, 243-247. e Simões et al. (2010)Simões et al., 2010 Simões, C.M.O., Schenkel, E.P., Gosmann, G., Mello, J.C.P., Mentz, L.A., Petrovick, P.R., 2010. Farmacognosia da Planta ao Medicamento, 6a ed. UFRGS/UFSC, Porto Alegre/Florianópolis, Brasil, pp. 1104., the presence of high levels of polyphenols and flavonoids in the extracts may be related to larvicidal activity. Those compounds are known to have toxicity that affects insects and larvae (Santiago et al., 2005Santiago et al., 2005 Santiago, G.M.P., Viana, A.F., Pessoa, O.D.L., Pouliquen, Y.B.M., Arriaga, A.M.C., Andrade-Neto, M., Braz-Filho, R., 2005. Avaliação da atividade larvicida de saponinas triterpênicas isoladas de Pentaclethra macroloba (Willd.) Kuntze (Fabaceae) e Cordia piauhiensis Fresen (Boraginaceae) sobre Aedes aegypti. Rev. Bras. Farmacogn. 15, 187-190.; Simões et al., 2010Simões et al., 2010 Simões, C.M.O., Schenkel, E.P., Gosmann, G., Mello, J.C.P., Mentz, L.A., Petrovick, P.R., 2010. Farmacognosia da Planta ao Medicamento, 6a ed. UFRGS/UFSC, Porto Alegre/Florianópolis, Brasil, pp. 1104.).

Based on the obtained larvicidal results of tested plant extracts, it can be speculated that the compounds can thus act as a potent phyto-complex with synergic effect, and may show greater bioactivity as a mixture than in its isolated constituents (Sumroiphon et al., 2006Sumroiphon et al., 2006 Sumroiphon, S., Yuwaree, C., Arunlertaree, C., Komalamisra, N., Rongsriyam, Y., 2006. Bioactivity of Citrus seed for mosquitoborne diseases larval control. Southeast. Asian. J. Trop. Med. Public. Health. 37, 123-127.). It is through this notion that researchers have supported the use of the plant crude extracts, instead of isolated compounds, as insecticides and larvicides (Veni et al., 2016Veni et al., 2016 Veni, T., Pushpanathan, T., Mohanraj, J., 2016. Larvicidal and ovicidal activity of Terminalia chebula Retz. (Family: Combretaceae) medicinal plant extracts against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. J. Parasit. Dis. 41, 693-702.; Guarda et al., 2016Guarda et al., 2016 Guarda, C., Lutinski, J.A., Roman-Junior, W.A., Busato, M.A., 2016. Atividade larvicida de produtos naturais e avaliação da susceptibilidade ao inseticida temefós no controle do Aedes aegypti (Diptera: culicidae). Interciência 41, 243-247.).

RCE showed the greater larvicidal activity. Due the low yield of the root extract it was not possible to perform columns for isolation of compounds. Thus, we analyzed its chemical composition by H1 NMR and TLC, in order to know better the chemical profile of compounds in RCE. In the NMR spectra (Figure 5, supplementary material), it has been verified the presence of typical signals for terpenes, steroids and phenolic compounds, compatible with the spots found on TLC dyed with p-anisaldehyde acid or acetic anhydride solutions. The total phenolic compounds and flavonoids from RCE were quantified by spectrophotometry.

The different results showed for separated parts of plants extracts are often showed in literature. The ethanolic extract of the leaves and roots of Piper alatabaccum, Piperaceae, and Azadirachta indica, Meliaceae, showed greater larvicidal potential for leaves than for the roots after 48 h of exposure, demonstrating that different parts of the plant may show particular larvicidal response because of their chemical composition (Nour et al., 2012Nour et al., 2012 Nour, A.H., Sandanasamy, J.D., Nour, A.H., 2012. Larvicidal activity of extracts from diferent parts of neem (Azadirachta indica) against Aedes aegypti mosquitoes larvae. Sci. Res. Essays. 7, 2810-2815.; Guarda et al., 2016Guarda et al., 2016 Guarda, C., Lutinski, J.A., Roman-Junior, W.A., Busato, M.A., 2016. Atividade larvicida de produtos naturais e avaliação da susceptibilidade ao inseticida temefós no controle do Aedes aegypti (Diptera: culicidae). Interciência 41, 243-247.; Castillo et al., 2017Castillo et al., 2017 Castillo, R.M., Stasehnko, E., Duque, J.E., 2017. Insecticidal and repellent activity of several plant-derived essential oils against Aedes aegypti. J. Am. Mosq. Control Assoc. 33, 25-35.).

This is the first study that has reported the larvicidal activity of plants from the Waltheria genus against Aedes aegypti. Based on the better activity demonstrated by the roots extract, we suggest that the extract can be used in effective and economically viable preparations for domestic use to control the vector insect of severe diseases, such as dengue and Zika (Caridade et al., 2018Caridade et al., 2018 Caridade, T.N.S., Rusceli, D.A., Oliveira, A.N.A., Souza, T.S.A., Ferreira, N.C.F., Avelar, D.S., Teles, Y.C.F., Silveira, E.R., Araújo, R.M., 2018. Chemical composition of four different species of the Waltheria genus. Biochem. Syst. Ecol. 80, 81-83.; Fernandes et al., 2018Fernandes et al., 2018 Fernandes, D.A., Souza, M.S.R., Teles, Y.C.F., Oliveira, L.H.G., Lima, J.B., Conceição, A.S., Nunes, F.C., Silva, T.M.S., Souza, M.F.V., 2018. New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules 23, http://dx.doi.org/10.3390/molecules23112784..
http://dx.doi.org/10.3390/molecules23112...
; Lakshmi et al., 2018Lakshmi et al., 2018 Lakshmi, K.V., Sudhikumar, A.V., Aneesh, E.M., 2018. Larvicidal activity of phytoextracts against dengue fever vector, Aedes aegypti-a review. Plant Sci. Today 5, 167-174.).

Considering the larvicidal potential of W. viscosissima extracts, the sub-fractions and isolated compounds of W. viscosissima are being tested in a bio-monitoring study against the Ae. aegypti in order to identify the bioactive compounds responsible for this activity (Fernandes et al., 2018bFernandes et al., 2018b Fernandes, D.A., Souza, M.S.R., Oliveira, L.H.G., Ferreira, M.D.L., Barros, R.P.C., Oliveira, M.S., Lima, J.B., Nunes, F.C., Scotti, M.T., Conceição, A.S., Souza, M.F.V., 2018b. Phytochemical study of Helicteres velutina K. Schum (Sterculiaceae) biomonitoring by the tests against Aedes aegypti L. (Diptera: culicidae). In: Anais eletrônicos, DDNDIC, Available at: http://ddndic.com/DDNDIC_2018_Book_of_abstracts.pdf. Accessed on 23 May 2019.
http://ddndic.com/DDNDIC_2018_Book_of_ab...
). Previous studies have shown that the substances may perform their larvae toxic effects in several ways, such as suppression of reproduction, fertility, and the inhibition of growth (Silva et al., 2015).

Conclusions

The present study contributed to the phytochemical knowledge of the species W. viscosissima. Fourteen compounds have been identified, including a mixture of steroids, two triterpenes, two alkaloids and eight flavonoids.

The spectrophotometric quantification of compounds demonstrated that the aerial parts extract possesses a high concentration of flavonoids and the roots extract is rich in other phenolic compounds. The roots extract also showed larvicidal activity against Ae. aegypti, with the potential of it being used in effective and economically viable preparations that cater towards domestic uses in the controlling of the vector insect of severe diseases, such as dengue and Zika.

  • Ethical disclosures
    Protection of human and animal subjects
    The authors declare that no experiments were performed on humans or animals for this study.
    Confidentiality of data
    The authors declare that no patient data appear in this article.
    Right to privacy and informed consent
    The authors declare that no patient data appear in this article.

Acknowledgment

The authors would like to thank the CNPq for financial support, CAPES - Finance Code 001 and the Multiuser Analytical Central Laboratory (LMCA-UFPB) for obtaining the spectra, and the Center of Biotechnology (BIOTEC-UFPB) for assistance in the biological assay.

Appendix A   Supplementary data

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.bjp.2019.05.008.

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Publication Dates

  • Publication in this collection
    09 Dec 2019
  • Date of issue
    Sep-Oct 2019

History

  • Received
    17 Apr 2019
  • Accepted
    29 May 2019
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