Leismanicidal Activity of Propolis Collected in the Semiarid Region of Brazil

Objective: The aim of the current study is to investigate the chemical composition, cytotoxic effect, and leishmanicidal activity of propolis collected in the semi-arid region of Bahia, Brazil. Methods: EtOH extract, hexane, EtOAc and MeOH fractions from propolis were analyzed by ultra-performance liquid chromatography coupled with diode array detector and quadrupole time-of-flight mass spectrometry. The identification was based on the exact mass, general fragmentation behaviors and UV absorption of the flavonoids. The in vitro cytotoxic effect and leishmanicidal activity of ethanolic extract, hexane, ethyl acetate, and methanolic fractions of propolis were evaluated. Results: Five triterpenes and twenty-four flavonoids were identified. The propolis did not present toxicity to the host cell up to the maximum concentration tested. In addition, all tested samples showed statistically significant activity against promastigotes of Leishmania chagasi and Leishmania amazonensis. Regarding the activity against amastigote forms of L. amazonensis, the hexane fraction, presented statistically significant activity with IC50 of 1.3 ± 0.1 μg/ml. Conclusion: The results support the idea that propolis can be used for future antileishmania studies.


INTRODUCTION
Leishmaniasis is one of the six major parasitic diseases targeted by the World Health Organization. It is endemic in 98 countries and more than 350 million people are at risk of infection. The disease causes 20,000-40,000 deaths per year globally (Sasidharan and Saudagar, 2021).
In humans, the clinical forms of leishmaniasis are broadly categorized into cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), and clinical manifestations depend on the pathogenicity of the parasite, which differs among species, and the genetically determined cellmediated immune response of the human host (Singh et al., 2012). Current leishmaniasis treatment strategy is based on chemotherapy with some attempts at immunotherapy, but the combination of factors including the development of parasite resistance to drug therapies, the absence of vaccines, problems with vector control, among other factors, has rendered the treatment options for leishmaniasis limited (Taslimi et al., 2018).
Pentavalent antimonials are the standard first line treatment, but the emergence of resistance has limited their usefulness. Alternative chemotherapeutic treatments with amphotericin B and its lipid formulation, miltefosine, and paromomycin are available but their use is limited either due to toxicity or the high cost of treatment (Braga, 2019).
Consequently, it is of the utmost importance to discover effective drugs and new drug targets for the treatment of leishmaniasis. Propolis has long been the subject of pharmaceutical interest because of its potent biological functions, such as antimicrobial, anti-inflammatory, antiprotozoan, and antitumoral activities (Popova et al., 2005;Dantas et al., 2006;Jin et al., 2008;Szliszka et al., 2013). The propolis resin is complex, and its common constituents include polyphenols (flavonoids, phenolic acids, and their esters, terpenoids, and steroids (Tran et al., 2012). Its chemical composition depends on the plant or plants from which the resin is collected, the geographical location, and the associated flora (Popova et al., 2005;Ishida et al., 2011).
Therefore, there has been an interest in the composition and biological properties of this natural product. Extensive investigations have been conducted with propolis from different countries in temperate and tropical areas (Sforcin, 2016); however, a limited number of investigations have been conducted to study the palynological analysis (Matos et al., 2014), chemical composition (Valcic et al., 1998;Hernandez et al., 2007) and biological activities of propolis from semiarid regions (Russo et al., 2004).
In our continuing studies of the chemical and biological activity of Apis and Melipona bee products (Silva et al., 2006;Silva et al., 2009;Freire et al., 2012;Silva et al., 2013;Souza et al., 2013;Souza et al., 2014;Sousa-Junior et al., 2019;Sousa-Fontoura et al., 2020), we investigated the chemical composition of propolis by ultra-performance liquid chromatography coupled with diode array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS/MS), and we also analyzed the cytotoxic effect and leishmanicidal activity of the ethanolic extract, hexane, ethyl acetate, methanolic fractions of propolis from semi-arid region of Bahia, Brazil.

Propolis Sample and Extraction
Propolis was collected in Brazil, State of Bahia, municipality of Casa Nova, which is an area of the Caatinga, a Brazilian biome. The propolis (79.4 g) was successively extracted with ethanol (EtOH) in an ultrasonic bath and evaporation was performed using a rotary evaporator in a vacuum at 40°C. 20 g dry extract was dissolved in methanol:water (1:1, MeOH) and successively fractionated with hexane (hexane, 7.4 g) and ethyl acetate (EtOAc,9.1 g). The amount of MeOH fraction was 1.1 g. The EtOH extract, hexane, EtOAc, and MeOH fractions, were subjected to UPLC-PDA-QTOF-MS analysis.

Analysis of Propolis by Ultra-Performance Liquid Chromatography Coupled With Diode Array Detector and Quadrupole Time-of-Flight Mass Spectrometry
The analysis was performed using a XEVO-G2XSQTOF mass spectrometer (Waters, Manchester, United Kingdom) connected to an ACQUITY UPLC system (Waters, Milford, MA, United States ) via an electrospray ionization (ESI) interface. Chromatographic separation of the compounds was performed on the ACQUITY UPLC with a conditioned autosampler at 4 C, using an Acquity BEH C18 column (150 mm × 2.1 mm i.d., 1.7 μm particle size). The column temperature was maintained at 40°C. The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile (solvent B), and it was pumped at a flow rate of 0.4 ml min −1 . The gradient elution program was as follows: 0-5 min, 5-10% B; 5-9 min, 10-95% B. The injection volume was 10 μl. MS analysis was performed on a Xevo G2 QTOF (Waters MS Technologies, Manchester, United Kingdom), a quadrupole time-of-flight tandem mass spectrometer coupled with an electrospray ionization source in the negative ion mode. The scan range was from 50 to 1,200 m/z for data acquisition. In addition, MS E experiments were carried out which allow precursor and product ion data to be acquired in one injection. The source conditions were as follows: capillary voltage, 2.0 kV; sample cone, source temperature, 100 C; desolvation temperature 250°C; cone gas flow rate 20 Lh −1 ; desolvation gas (N 2 ) flow rate 600 Lh −1 . All analysis were performed using the lockspray, which ensured accuracy and reproducibility. Leucine-encephalin (5°ngml −1 ) was used as a standard or reference compound to calibrate the mass spectrometers during analysis. All the acquisition and analysis of data were controlled using Waters MassLynx v 4.1 software.

Leishmanicidal Assay
Cells (1 × 10 5 /well) of promastigotes forms of L. amazonensis and L. chagasi were cultured in Schneider's medium supplemented with 10% FBS and 2% human urine, in the presence of various concentrations of EtOH extract, hexane, EtOAc and MeOH fractions (0.1, 1, 10 and 100 μg/ml), and pentamidine (0.1, 1, 10, and 100 µM) in triplicate for 48 h at 26°C. Cells were also cultured in a medium free of substances, a vehicle (basal growth control) or with 0.1% DMSO (vehicle control). After 48 h, the viability of the promastigotes forms was analyzed using the MTT assay. MTT (20 μL) was added to each well and incubated at 37°C for 2 h with 95% humidity and 5% CO 2. Formazan extraction was performed using 120 μL of isopropanol and left at room temperature for 2 h. The absorbance was measured using an ELISA reader at 550 nm (Moraes et al., 2014). The intracellular amastigote assay was performed in 24-well microplates with rounded coverslips on the bottom. J774.A1 macrophages were seeded at a density of 3 × 10 5 cells/well and maintained for 1 h in 5% CO2 at 37°C for adhesion in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. Afterward, the cells were infected in vitro with promastigote forms of L. amazonensis and L. chagasi at a ratio of 1:10 for 6 h in a humidified atmosphere of 95% air and 5% CO 2 at 37°C.
The parasites in the supernatant were removed by washing, and various concentrations of EtOH extract, hexane, EtOAc, and MeOH fractions (0.1, 1, 10, and 100 μg/ml) and pentamidine (0.1, 1, 10, and 100 µM) were added and maintained at 37 C in 5% CO 2 for 48 h. The glass coverslips were fixed with methanol, stained with May-Grünwald-Giemsa, and intracellular amastigotes were counted (one hundred macrophages were evaluated per glass coverslip). Data were expressed as infection index (percentage of infected macrophages multiplied by the average number of amastigotes per macrophage) (Hussain et al., 1993).

Statistical Analysis
Results were expressed as the mean ± SEM of an experiment in triplicate. The tests were performed with controls: media free from compounds, a vehicle (basal growth control), or media with 0.1% DMSO (vehicle control). Significant differences between the treated and control groups were evaluated using ANOVA and Dunnett post-hoc tests by Graph Pad Prism 5.0 software, and 95% confidence intervals were included.

RESULTS
The compounds were tentatively identified by ultra-performance liquid chromatography coupled with diode array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS/MS), as flavonoids (flavonol/flavone, flavanone, and chalcones) based on their characteristic UV-Vis (flavonoids) spectra peaks and mass detection as well as the accurate mass measurement of the precursor and product ions. Terpenes were suggested by the absence of absorption in the UV spectra, in addition to the high-resolution mass spectra. Figure 1 shows the  Table 1.
The analysis allowed the identification of 24 flavonoids including 13 flavonols/flavones (1,2,3,5,6,7,8,9,10,11,15,16,17,18,20,21,22 (Figure 2). The compounds shown at the end of the chromatogram of EtOH extract (Figure 1) can be identified as triterpenes because they do not absorb in the UV region and had a molecular constitution typical of triterpenes derivatives (25-29). The main product ions observed in the mass spectra were due to the loss of CH 3 (15 Da) and H 2 O (18 Da). These compounds are isomers and were not completely identified. The in vitro leishmanicidal activity was evaluated with extract and fractions of propolis. The results presented in Table 2 show the effects of the ethanol extract (EtOH), hexane fraction (hexane), ethyl acetate fraction (EtOAc), methanolic fraction (MeOH), and pentamidine against J774.A1 macrophages using the MTT assay. After 48 h of incubation, pentamidine showed deleterious activity to the host cell with maximum cytotoxicity of 93.8 ± 0.7% and IC 50 of 44.1 ± 0.8 µM. However, the extract, and fractions not showed deleterious activity to the host cell, presenting as promising substances for the other assays. Orsi et al. (2005), observed that Brazilian propolis was not toxic to macrophages, and using cytotoxicity assays, Blonska et al. (2004),observed that the ethanolic extract of propolis was not toxic to these macrophage cells.  Mean ± standard error of the mean maximum cytotoxicity in triplicates of a representative experiment. The values of maximum effect were considered significant when **p < 0.01 compared to the 0.1% DMSO group. IC 50 is the concentration required to give 50% inhibition. ND: Not determined. Maximum cytotoxicity compared to the DMSO group.
Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 702032 5 The leishmanicidal activity of EtOH extract; hexane, EtOAc, MeOH fractions, and pentamidine against the promastigote forms of L. amazonensis and L. chagasi was assessed in vitro. The inhibitory effects and IC 50 values are shown in Table 3. Hexane, EtOAc, and MeOH fractions exhibited the most significant leishmanicidal activity to L. amazonensis. The EtOH extract, hexane and EtOAc fractions exhibited the most significant leishmanicidal activity to L. chagasi. Thus, it is observed that EtOH extract and EtOAc fraction showed the best inhibitory activity against the growth of promastigote forms of L. amazonensis and L. chagasi, respectively. Moreover, EtOAc and MeOH fractions showed the antiamastigote activity against L. amazonensis and L. chagasi, respectively.

DISCUSSION
The presence of flavonoids and terpenes were identified in the active EtOH extract, hexane and EtOAc fractions. In this study, the chemical profile of the flavonoids is similar to that found for the geopropolis samples of stingless bees collected in the semiarid region (Sousa-Junior et al., 2019;Sousa-Fontoura et al., 2020). However, additional studies need to be carried out to know the origin of the plant species from which bees collect the resin in the semi-arid region of Brazil.
Propolis is an important source of substances applied in medicine due to its pharmacological activities (Potin et al., 2008) and wide range of biological properties, including leishmanicidal activity (Duran et al., 2008;Potin et al., 2008;Nina et al., 2016). In another study, the ethanolic extract of propolis showed leishmanicidal activity against L. tropica at concentrations of 250, 500 and 750 μg/ml, and statistically significant differences in cell counts were observed compared to the control group (p < 0.05) (Duran et al., 2008), whereas the fractionated propolis-rich phenolic showed maximum efficacy against L. amazonenis (IC 50 12.1 μg/ml) and L. brasiliensis (IC 50 10.9 μg/ml) (Nina et al., 2016). Extracts of propolis were tested against protozoal pathogens, including Crithidia fasciculata a close relative of Crithidia mellificae, a parasite of bees. High levels of activity were obtained for all the samples of different extracts. In the case of C. fasciculata highest activity was associated with flavonoids methyl ethers of galangin and pinobanksin (Alotaibi et al., 2019). According to Ruiz-Gonzalez and Brown (2006), the spread of the protozoal infection occurs via feces, coating the surfaces in the hive with propolis that is active against trypanosomiasis could prevent transmission.
According to Ayres et al. (2007), the ethanolic extract of propolis presented a direct effect on the amastigote forms of L. amazonensis, up to 96 h, at a concentration of 25 μg/ml.
Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 702032 6 leishmaniasis is a current public health issue with a high global social impact. The currently available treatments for this disease are limited in terms of toxicity, and they have variable efficacy and high costs, which potentiates the results obtained in this study.

CONCLUSION
To the best of our knowledge, this is the first study which describes the chemical composition and biological activity of propolis collected from the semiarid region in Northeast Brazil. The extract, and fractions from propolis anti-leishmanial effects. The observed effects could be associated with the presence of flavonoids. The chemical and biological characterization of the semiarid region could be important for the development of alternative treatment strategies against Leishmania sp.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
GC contributed to chemical and biological study and analysis of the biological data. CC and TS designed the chemical study, supervised the laboratory work, identification of chemical compounds, and contributed to critical reading of the manuscript. ES contributed to collecting propolis samples and identification. MA designed the biological study, supervised the laboratory work, and contributed to critical reading of the manuscript. MS, AL, AQ, AS, and MA contributed to the biological study. All authors reviewed and approved the final manuscript.