ORIGINAL RESEARCH published: 20 July 2021 doi: 10.3389/fphar.2021.703985 Antileishmanial Activity of Flavones- Rich Fraction From Arrabidaea chica Verlot (Bignoniaceae) João Victor Silva-Silva 1†, Carla Junqueira Moragas-Tellis 2†, Maria do Socorro dos Santos Chagas 2, Paulo Victor Ramos de Souza 2,3, Celeste da Silva Freitas de Souza 1, Daiana de Jesus Hardoim 1, Noemi Nosomi Taniwaki 4, Davyson de Lima Moreira 2, Maria Dutra Behrens 2†, Kátia da Silva Calabrese 1*† and Fernando Almeida-Souza 1,5† 1Laboratory of Immunomodulation and Protozoology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 2Laboratory of Natural Products for Public Health, Pharmaceutical Techonology Institute – Farmanguinhos, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 3Student on Postgraduate Program in Translational Research in Drugs and Medicines, Farmanguinhos, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 4Electron Microscopy Nucleus, Adolfo Lutz Institute, São Edited by: Paulo, Brazil, 5Postgraduate in Animal Science, State University of Maranhão, São Luís, Brazil Pinarosa Avato, University of Bari Aldo Moro, Italy Reviewed by: Acknowledging the need of identifying new compounds for the treatment of leishmaniasis, Edson Roberto Silva, this study aimed to evaluate, from in vitro trials, the activity of flavones from Arrabidaea University of São Paulo, Brazil chica against L. amazonensis. The chromatographic profiles of the hydroethanolic extract Miriam Rolon, Centro para el Desarrollo de la and a flavone-rich fraction (ACFF) from A. chica were determined by high-performance Investigacion Cientifica, Paraguay liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV) and *Correspondence: electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS). The flavones Kátia da Silva Calabrese fi calabrese@ioc.fiocruz.br luteolin (1) and apigenin (2), isolated from chromatographic techniques and identi ed 1 13 fi †These authors have contributed by Nuclear Magnetic Resonance of H and C, were also quanti ed in ACFF, showing equally to this work 190.7 mg/g and apigenin 12.4 mg/g, respectively. The other flavones were identified by comparing their spectroscopic data with those of the literature. The in vitro activity Specialty section: was assayed against promastigotes and intramacrophagic amastigote forms of This article was submitted to Ethnopharmacology, L. amazonensis. Cytotoxicity tests were performed with peritoneal macrophages of a section of the journal BALB/c mice. Nitrite quantification was performed with Griess reagent. Ultrastructural Frontiers in Pharmacology investigations were obtained by transmission electron microscopy. Anti-Leishmania Received: 01 May 2021 Accepted: 06 July 2021 assays indicated that the IC50 values for ACFF, apigenin, and luteolin were obtained at Published: 20 July 2021 40.42 ± 0.10 and 31.51 ± 1.13 μg/mL against promastigotes, respectively. ACFF and Citation: luteolin have concentration-dependent cytotoxicity. ACFF and luteolin also inhibited the Silva-Silva JV, Moragas-Tellis CJ, intra-macrophagic parasite (IC 3.575 ± 1.13 and 11.78 ± 1.24 μg/mL, respectively), with Chagas MSS, Souza PVR, Souza CSF, 50 Hardoim DJ, Taniwaki NN, Moreira DL, a selectivity index of 11.44 for ACFF. Promastigotes exposed to ACFF and luteolin Dutra Behrens M, Calabrese KS and exhibited ultrastructural changes, such as intense cytoplasm vacuolization and Almeida-Souza F (2021) fi Antileishmanial Activity of Flavones- mitochondrial swelling. These ndings data evidence the antileishmanial action of Rich Fraction From Arrabidaea chica flavone-rich fractions of A. chica against L. amazonensis, encouraging further studies. Verlot (Bignoniaceae). Front. Pharmacol. 12:703985. Keywords: L. amazonensis, flavonoids, Arrabidaea chica, macrophages, nitric oxide, transmission electron doi: 10.3389/fphar.2021.703985 microscopy Frontiers in Pharmacology | www.frontiersin.org 1 July 2021 | Volume 12 | Article 703985 Silva-Silva et al. Arrabidaea chica Fraction Against Leishmania INTRODUCTION Thus, the present study aimed to evaluate the antileishmanial activity in vitro of the hydroethanolic extract derived from A. Flavonoids are an important class of secondary metabolites with a chica, as well as of its flavone-rich fraction and the isolated low molecular weight polyphenolic structure, widely distributed flavones, luteolin and apigenin, against promastigotes, and in the plant kingdom among subgroups that include chalcones, intracellular amastigotes of Leishmania amazonensis. flavones, flavonols, and isoflavones (Panche et al., 2016). This metabolic class has its biological and therapeutic activity experimentally determined (Nijveldt et al., 2001), being able to MATERIALS AND METHODS affect enzymes and various cellular systems, having beneficial effects on the body (Silva et al., 2010). Furthermore, it is largely Plant Material known that flavonoids have a wide spectrum of antileishmanial Leaves of A. chica Verlot (Bignoniaceae) (morphotype IV) were activity (Fotie, 2008; Wong et al., 2012; Rocha et al., 2018). cultivated and collected in March 2016 at Fiocruz Atlantic Forest Leishmaniasis is a neglected tropical disease that seriously Campus, municipality of Rio de Janeiro, Rio de Janeiro State, affects humans and can lead to death if left untreated (Reithinger Brazil (S22.9406 W43.4046). Plant material was identified by Dr. and Dujardin, 2007). This protozoosis represents a global health Marcus Felipe Oliveira da Silva at the Botanical Collection of challenge, since it has a worldwide distribution, with an estimate Medicinal Plants of Farmanguinhos/FIOCRUZ, where a voucher of more than one billion people living in endemic areas and at risk specimen was deposited and registered under the number of Leishmania infection (World Health Organization, 2020). In CBPM666. addition to these circumstances, the anti-leishmanial drugs currently in use exhibit drug resistance, toxicity, and Extraction and Isolation high cost, which may explain the low adherence to treatment Arrabidaea chica leaves were dried in a forced circulation oven at (Sundar et al., 2019). The lack of new therapeutic alternatives to a temperature of 45°C for 4 days. After drying, the plant material leishmaniasis highlights the need to seek new compounds with was powered using a knife-mill affording 0.85 mm particles that leishmanicidal activities. In this context, the use of natural were stored in an amber flask. The hydroethanolic extract was products in traditional medicine has contributed to the prepared by exhaustive maceration of dried and powdered leaves identification of candidate compounds for the development (1.5 kg) in 70% ethanol solution (v/v) with three changes of of new drugs. Therefore, medicinal plants represent a solvent, once every 48 h for 7 days at room temperature. After repository of bioactive compounds potentially useful for the filtration, the solvent was evaporated under reduced pressure to development of new therapeutic alternatives for leishmaniasis yield 226.19 g of a red extract (ACCE) corresponding to yielding (da Silva et al., 2018). of 15.07%. The crude hydroethanolic extract (60 g) was then Arrabidaea chica (Humb. & Bonpl.) B. Verlot, syn. Bignonia submitted to liquid-liquid partition with n-hexane (3 × 200 mL), chica, belongs to the family Bignoniaceae. It comprises about 120 dichloromethane (3 × 200 mL), ethyl acetate (3 × 200 mL) and genera and 860 species (Fischer et al., 2004; Sampaio et al., 2016). n-butanol (3 × 200 mL). The final aqueous residue was discarded. A. chica occurs in tropical America, being a very common species Dichloromethane fraction was successively chromatographed by in the Amazon region (Takemura et al., 1995; dos Santos et al., column chromatography on Sephadex LH-20 (Sigma, St Louis, 2013) and is popularly known as crajiru or pariri (Behrens et al., MO, United States), using methanol as eluent to produce a 2012). It is traditionally used as a medicinal plant in the Amazon purified flavone-rich fraction determined by thin layer region (Takemura et al., 1995), with the use of tea made from chromatoghapy (TLC) analysis, and named ACFF. Successive leaves as an anti-inflammatory (Evangelista et al., 2013), to treat chromatographic fractionation steps of ACFF on Sephadex LH- skin inflammation and mycoses (Correa,ˆ 1984), and has 20 led to the isolation of two flavonoids (F1 24 mg; F2 4 mg). astringent properties (Lima de Medeiros et al., 2011). Isolated compounds and a flavone-rich fraction were analyzed by Amazonian Indians use the decoction of leaves to clean TLC (silica gel F254, Merck, Darmstadt, Germany) using acetone: wounds and ulcers to aid in healing, in addition to the use to chloroform: formic acid (75:16:0.8 v/v/v) as eluent and, treat fungal infections and herpes (Lorenzi and Matos, 2002), as subsequently, sprayed with 1% NP/PEG reagent well as for other skin conditions (Barbosa et al., 2008). (diphenylboriloxyethilamine/polyetileneglicol, Sigma, St Louis, Furthermore, the infusion (oral use) is used to heal wounds MO, United States), as well as 1H and 13C NMR spectrometry. and cleanse the blood (Bieski et al., 2012). Previous studies Comparison with literature records allowed the identification of have demonstrated its antioxidant (do Amaral et al., 2012; dos (1) luteolin and (2) apigenin (Ersöz et al., 2002; Özgen et al., 2011; Santos et al., 2013), wound healing (Aro et al., 2013; Cortez de Sá Siraichi et al., 2013; Grabsk et al., 2017). et al., 2015), trypanocidal (Barbosa et al., 2008; Miranda et