Antibacterial, cytotoxic, and schistosomicidal activities of the methanolic extract from Cassia grandis L.f. (Fabaceae) stem bark and its fractions

Antibacterial, cytotoxic, and schistosomicidal activities of the methanolic extract from Cassia grandis L.f. (Fabaceae) stem bark and its fractions Luciméri Paulino Machado Magalhães, Bárbara Azevedo Ramos, Márcia Vanusa da Silva, Maria Tereza dos Santos Correia, Kêsia Xisto Fonseca Ribeiro de Sena, Túlio Diego da Silva, André de Lima Aires, Mônica Camelo Pessôa de Azevedo Albuquerque, Gardênia Carmen Gadelha Militão, Teresinha Gonçalves da Silva, Renata Mendonça Araújo, Julianna Ferreira Cavalcanti de Albuquerque and Rafael Matos Ximenes


INTRODUCTION
The use of medicinal plants as a therapeutic resource in the cure and prevention of diseases is as old as the human culture. This ancient knowledge has been used in the research and development of modern medicines.
Currently, up to 35% of the commercially available medicines are originated from or inspired by natural products (Calixto, 2019). Plants are a limitless source of bioactive compounds potentially useful to treat many human or animal disorders (Guidoti et al., 2019). A broad investigation of their biological activities is the main step in development of effective alternative medications (Singh et al., 2017).
Fabaceae (Leguminosae) is one of the largest Angiosperm families with approximately 730 genera and almost 20,000 species from small herbs to high trees widespread around the world. In Brazil, Fabaceae is the largest plant family with more than 2,700 species sorted among 212 genera (Andrade et al., 2009;Forzza et al., 2010;Lima et al., 2015). Cassia species are distributed in tropical and subtropical regions of America, Africa, and Asia. Besides their uses in folk medicine as purgative, many other pharmacological activities have been reported, such as antibacterial, antifungal, antimalarial, anti-inflammatory, and hepatoprotective (Viegas Junior et al., 2006).
Cassia grandis L., commonly known as pink cassia, is native from the Brazilian Amazon forest and nowadays very common all-around Brazil due to its use in urban landscaping. This species is a large tree that can reach 12 m in height. It has small deciduous leaves, pinkishyellow flowers and pod fruits that can weigh up to a kilo (Joshi and Kapoor, 2003). C. grandis is considered a multipurpose plant due to its many uses. As a medicinal plant, it is used against worms and intestinal parasites, to treat stomach and respiratory problems, infected wounds, blood diseases, insulin resistance, among others (Parra and Sardiñas, 2000;Lodha et al., 2010;Meena et al., 2009). Phytochemical studies have shown the presence of alkaloids, phytosterols, saponins, flavonoids, tannins, and anthraquinones in different organs of the plant (Meena et al., 2010).
Based on its medicinal uses, this study aimed to evaluate in vitro the antioxidant, cytotoxic, antimicrobial, and schistosomicidal activities of C. grandis stem bark methanolic extract and to determine the phytochemical profile of its active fractions by GC-MS and UPLC-MS.

Plant material and extraction
The stem bark of C. grandis L.f. (Fabaceae) was collected inside the campus of the Federal University of Pernambuco, Recife, Brazil (8°02'47.4"S 34°57'03.2"W, sea level). Recife has tropical weather, with temperatures between 20-30ºC during all year, a rainfall season between April and August, and annual precipitation at about 2000 mm. A voucher specimen was identified by O. Cano at the IPA Herbarium and deposited under nº 89,340.
The plant material was dried at room temperature for seven days and powdered in a knife-mill (Willey type). The powder (2.6 kg) was extracted with methanol for 2 h under mechanical agitation. The solvent phase was filtered, and the procedure repeated twice. Thereafter, the organic phases were pulled together and evaporated to dryness at 40ºC under reduced pressure yielding 12.1% (CgME). The extract (50 g) was fractionated by solid-liquid partition with hexane and ethyl acetate to give the respective fractions: CgHF and CgEF, which yielded 1.5 and 18.5%, respectively. The remaining extract was called CgMF (79.9%).

Thin-layer chromatography analysis
A phytochemical screening was performed by TLC to determine the presence of the following classes of secondary metabolites: flavonoids, phenylpropanoids, terpenes and steroids, coumarins, quinones, alkaloids, and proanthocyanidins as described in Table  S1 (Supplementary material).

Determination of total phenolic content
The total phenolic content was determined using the Folin-Ciocalteu's reagent (McDonald et al., 2001). Briefly, aliquots of 0.5 mL of the extract/fractions (1 mg/mL) and 0.1 mL of Folin-Ciocalteu's reagent (0.5 N) were mixed and incubated at room temperature for 15 min. After this period, 2.5 mL of saturated sodium carbonate solution was added and further incubated for another 30 min at room temperature. Thereafter the absorbance was measured at 760 nm. The total phenolic content of the extract/fractions were expressed as gallic acid equivalent (mg GAE/g of extract/fraction) based on a standard curve of gallic acid (1.56 to 100 μg/mL).

Determination of flavonoid content
The amount of flavonoid in the extract/fraction was determined by the AlCl 3 colorimetric method (Woisky and Salatino, 1998). Shortly, 500 μL of the extract/fractions (1 mg/mL) and 500 μL of AlCl 3 (2%) were mixed and incubated at room temperature for 60 min. Then, the absorbance was measured at 420 nm. The flavonoid content of the extract/fractions was expressed in as quercetin equivalent (mg QE/g of extract/fraction) based on a standard curve of quercetin (1 to 100 μg/mL).

Gas chromatography-mass spectrometry (GC-MS) analysis
Gas chromatography and mass spectrometry (GC-MS) analysis was performed at the Technological Development Park of the Universidade Federal do Ceará using a GCMS QP 5050A (Shimadzu, Kyoto, Japan) equipped with a non-polar AW Scientific DB-1 MS capillary column (50 m × 0.25 mm × 0.25 μm). The oven temperature was set at 70°C with an increase of 4°C/min until 230°C and then maintained for 15 min. The carrier gas was helium, with a constant flow rate of 1.7 mL/min. The temperature of the ionization source was maintained at 280°C, ionization energy at 70 eV, and ionization current at 0.7 kV. Mass spectra were recorded from 30 to 450 m/z. Individual components were identified by matching their 70 eV mass spectra with those of the spectrometer database by using the Wiley L-Built library and by comparing their retention indices and fragmentation patterns with those of the NIST MS library (Stein et al., 1997) and those reported in the literature (Adams, 2001).

Ultra-performance liquid chromatography-mass spectrometry (UPLCMS) analysis
The chemical constituents of CgEF were analyzed on an Acquity H-Class UPLC (Waters®, MA, USA) equipped with a BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm x 100 mm. The mobile phases used consisted of aqueous solution containing 2% MeOH, 5 mM ammonium formate, and 0.1% formic acid (eluent A), and MeOH containing 0.1% formic acid (eluent B), at a flow rate of 0.3 mL/min. Elution was performed in gradient mode and the initial condition (98% A / 2% B) was maintained for 0.25 s. The proportion of B was linearly increased to 99% in 8.5 min and then maintained for 60 s, followed by the immediate decrease to 2% B, which was maintained for 90 s. Ten microliters of sample were injected. The column temperature was maintained at 40°C and the injector at 10°C. The UPLC system was coupled to a single quadrupole mass spectrometer SQ Detector 2 (Waters®). The optimized conditions were as follows: capillary voltage 3.5 Kv; cone voltage 30 V; desolvation temperature 450°C; and a desolvation gas flow rate of 650 L/h. The data acquisition was done in full scan mode, searching for masses between 100 and 1000 Da, in negative ionization mode. The chromatograms and mass spectra were acquired with MassLynx™ software (Waters®).

Total antioxidant activity
The total antioxidant activity of the extract/fractions was evaluated by the phosphomolybdenium method (Prieto et al., 1999). The assay is based on the reduction of Mo(VI) to Mo(V) by the extract/fractions and subsequent formation of a green phosphate/Mo(V) complex at acid pH. Each sample solution (0.3 mL) and ascorbic acid (100 mg/mL) was combined with 3 mL of reagent (0.6 M H 2 SO 4 , 28 mM sodium phosphate and 4 mM ammonium molybdate). The blank solution was made with 3 mL of reagent solution and 0.3 mL of the solvent used for sample dilution. All tubes were capped and incubated in a boiling water bath at 95°C for 90 min. After this period, the samples were cooled to room temperature, and the absorbance was measured at 695 nm. The experiment was performed in triplicate. Total antioxidant activity was expressed as equivalents of ascorbic acid.

DPPH free radical scavenging activity
The free radical scavenging activity was measured using DPPH (2,2-Diphenyl-1-picrylhydrazyl) according to the method described by Blois (Blois, 1958). The reaction mixture consisted of 1.0 mL of DPPH in MeOH (0.3 mM) and 1.0 mL of the extract/fractions (1.56 -50 μg/mL) diluted in MeOH. Thereafter, it was incubated in the dark for 10 min, after which the absorbance was measured at 517 nm. Gallic acid (1.56-50 μg/mL) was used as a positive control. The EC 50 was calculated by linear regression.

Cytotoxicity assay
MTT reduction assay was used to determine the cytotoxicity of the extract/fractions. Briefly, the cancer cells were plated in 96-well plates at 1 x 105 cells/mL for adherent cells (MCF-7 and NCI-H292) or 3 x 105 cells/mL for HL-60. The extract/fractions at 50 μg/mL) dissolved in DMEM or RPMI 1640 with 0.5% DMSO were added to each well. Then, cells were incubated for 72 h, after which 25 μL of MTT (5 mg/mL) was added. Three hours later, the supernatant was removed, and the formazan crystals were dissolved in 100 μL DMSO. The absorbance was measured at 570 nm in a microplate reader (Thermo, USA). For the extract/fractions that inhibited more than 70% of cell viability, the IC 50 was determined using different concentrations (50 -1.56 μg/mL). Doxorubicin (5-0.01 μg/mL) was used as a positive control (Mosmann, 1983).

Strains and growth conditions
The strains used in the disk diffusion test consisted of nine microorganisms: four Gram-positive bacteria Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, and Enterococcus faecalis; three Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, and Serratia marcescens; one alcohol-acid resistant bacteria Mycobacterium smegmatis; and one yeast Candida albicans. Three multidrug-resistant S. aureus clinical isolates from infected wounds were also used (Table S2 Supplementary material). All microorganisms were obtained from the Microorganism Collection of the Department of Antibiotics of the UFPE. Bacterial inocula were obtained from cultures with approximately 18-24 h of incubation at 35°C in Müller-Hinton agar for bacteria, and 24-48 h at 30°C in a Sabouraud agar medium. Microorganism suspensions were standardized according to the turbidity equivalent to 0.5 tube on the McFarland scale using a Genesys™ 10S spectrophotometer (Thermo Scientific, MA, USA), which corresponds to approximately 108 CFU/mL (CLSI, 2018).

Paper disc diffusion test
Whatman Nº 1 paper disks of 6 mm diameter were soaked with 20 μL of the extract/fractions (100 mg/mL) in DMSO, each disk having a final concentration of 0.4 mg of the extract/fractions. The disks were placed on the surface of solid medium previously seeded with the test microorganisms in Petri dishes. The dishes were incubated at 35°C for 24 h (bacteria) or 30°C for 48 h (yeast). The tests were performed in triplicate and the results were expressed in mm (Bauer et al., 1966). DMSO embedded disks were used as solvent control. As standard drugs, commercial kanamycin (30 μg) or ketoconazole (10 μg) disks were used.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
MIC was determined by broth microdilution in round-bottom 96-well plates according to the CLSI M100 guideline (CLSI, 2018). Serial dilution of the extract/active fractions in Mueller Hinton broth (2000 -3.91 μg/mL) was added to the wells containing an inoculum of 10 8 CFU/mL of the test microorganisms. The plates were incubated for 18 h at 35ºC, and then 20 µL resazurin (0.01%) was added to each well. MIC was determined as the concentration of the last well where there was no change from purple to pink. The content of the wells that remained purple was seed on Petri dishes containing Müller Hinton agar to determine the MBC, which is the concentration where there was no growth of colonies. All the analyses were performed in triplicate.

Parasites and hosts
Schistosoma mansoni strain BH cercariae (Belo Horizonte, Brazil) were obtained from Keizo Asami Immunopathology Laboratory (LIKA) at UFPE. Thirty-day old Swiss Webster mice (n = 10), weighing 28 ± 2 g, were infected percutaneously with approximately 120 cercariae and maintained under standard conditions, with water and food ad libitum, in LIKA's animal facility. After 60 days, mice were euthanized, and adult worms were harvested through perfusion of the hepatic portal and mesenteric systems. The experimental protocol was approved by the Ethics Committee on Animal Experimentation of UFPE, under the nº 0013/2019.

In vitro assay
The adult worms couples harvested from mice were washed four times with RPMI 1640 medium supplemented with 20 mM HEPES, 100 μg/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum. Then, intact couples were transferred to 24-well culture plates containing 2 mL of the same medium (two worm couples/well) and incubated at 37ºC in a humidified atmosphere with 5% CO 2 . After 2 h, the extract/fractions were added to each well from a stock solution of 1 mg/mL in 1.5% DMSO in RPMI 1640 to obtain concentrations of 50-400 μg/mL. Worm couples incubated with 1.5% DMSO in RPMI 1640 or praziquantel (PZQ) 3.12 μg/mL were used as negative and positive controls, respectively. All experiments were performed in triplicates and repeated twice. The worms were kept in culture for 3 days and monitored using an inverted optical microscope (Leica Microsystems, DM IL Wetzlar, Germany) every 24 h to evaluate the motor activity, oviposition, and mortality rate.

Motility and survival
The treatments were considered lethal when it was not possible to observe worms' movements for up to 2 min of observation. The motility of the worms was evaluated according to the viability criteria proposed by Horiuchi et al. (2005). Briefly, movements were scored in a 0-3 scale, being: Score 3-worms with typical movements, exhibiting peristalsis of the internal organs, suckers in movement, adhering to the bottom or sides of the culture plate; typical descriptions of worms of the negative control; Score 2-reduced movements throughout the body, peristalsis of internal organs and suckers; Score 1-movements only at the extremities or at only one of the extremities (anterior and/or posterior regions), with absence of peristalsis of the internal organs and not adhered suckers; Score 0-complete absence of motions and tegument with or without changes in coloration. The treatment was considered lethal when it was not possible to observe parasite movements for up to 2 min.

Cell viability assay of the S. mansoni couples
The cell viability of the S. mansoni couples after incubation with the extract/fractions or praziquantel was determined using MTT (Aires et al., 2014). Shortly, two worm couples were plated in each well using a 96-well culture plate containing 100 μL of MTT (5 mg/mL in PBS) and incubated at 37ºC for 30 min. Thereafter, MTT solution was replaced by 200 μL of DMSO to dissolve the formazan crystals. The absorbance was measured at 570 nm in a microplate reader (Bio-Rad Laboratories, USA).

Statistical analysis
The descriptive analysis (mean and standard deviation) was used to discuss the results, assuming the normal distribution of the studied variables. The t-test was used for the antimicrobial activity test. The value of p<0.05 was used to determine a significant difference. All calculations were performed using the Statistical program 8.0.

Phytochemical and antioxidant analysis
The qualitative phytochemical screening of the methanolic extract (CgME) and its fractions by TLC revealed the presence of all tested secondary metabolites classes, except for alkaloids. The hexane fraction (CgHF) concentrated the terpenes and steroids, while the ethyl acetate fraction (CgEF) enriched all secondary metabolite classes, mainly flavonoids, and proanthocyanins. The methanolic fraction (CgMF) presents the same profile of the CgME, except for the absence of proanthocyanins (Table 1). The quantification of total phenolic compounds and flavonoids in the CgEM and its fractions confirmed the enrichment of the CgEF, which present higher levels of both secondary metabolite classes, as shown in Table 2. The antioxidant activity of the extract and its fractions were also evaluated through two assays: total antioxidant activity (phosphomolibdenium assay) and DPPH free radical scavenging assay. In the first test, CgEF presented the best results, followed by CgME and CgMF. In DPPH free radical scavenging assay, CgME and CgEF showed similar EC 50 values. In both assays, the CgHF showed only residual activity (Table 2).

Antimicrobial activity
A disk diffusion assay against a panel of micro-organisms was used to screen the antimicrobial activity of the extract and its fractions. The CgME presented inhibition zone diameters of 15.3 ± 0.6 mm for S. aureus, E. coli, and M. smegmatis, and of 13.3 ± 0.6 mm for B. subtilis. After the fractionation, the antimicrobial activity against E. coli and M. smegmatis was lost, while the CgEF showed an increase in the activity against S. aureus and B. subtilis, with inhibition zone diameters of 17.3 ± 0.6 and 16.3 ± 0.6 mm, respectively. CgHF was not active for any tested micro-organisms. Kanamycin antibiogram disks (30 μg/disk) were used as control, presenting inhibition zone diameters among 13.0 ± 0.5 and 40.0 ± 1.0 mm for the tested micro-organisms. As the next step, MIC and MBC values of the CgEM and the active fractions (CgEF and CgMF) were determined against S. aureus multidrug-resistant clinical isolates. MICs between 250 and 500 μg/mL were obtained for all extract/fractions and tested strains. On the other hand, MBC varied from 250 to 2,000 μg/mL, with the best results obtained for CgEF (Table 3). Table 4 shows the mortality kinetics of adult S. mansoni worm couples treated with C. grandis methanolic extract and its fractions incubated for 24, 48, and 72 h. During the 72 h incubation time, worm couples treated only with culture medium showed typical body movements, with peristalsis of the internal organs, suckers' movements, and adhering to the culture plate (score 3). CgME killed 18.75% of the worms at 400 μg/mL after 72 h of exposure. The fractionation of the extract enhanced its schistosomicidal activity, with at least 75% mortality for all fractions at 72 h. After 24 and 48 h of incubation, worm couples treated with PZQ were immobile (score 0) showing 87.5 and 100% mortality, respectively.

Schistosomicidal activity
The cell viability of S. mansoni worms after 72 h incubation time was determined by the mitochondrial reduction of MTT. The CgME (400 μg/mL) reduced the cell viability by 33.8%, while the CgEF, the most active fraction, at 400 and 300 μg/mL reduced the cell viability by 97.5 and 85.3%, respectively. PZQ reduced cell viability by 80.7% (Figure 1).

Chemical composition of the active fractions (CgHF and CgEF)
Quantitative chemical analysis of CgHF by GC-MS revealed the presence of 8 ubiquitous compounds (fatty acids, phytosterols, and triterpenes). The major compounds were clionasterol (23.9%), lupeol acetate Table 3. Antimicrobial activity of the methanolic extract from Cassia grandis stem bark (CgME) and its ethyl acetate (CgEF) and methanolic (CgMF) fractions against multidrug-resistant S. aureus clinical isolates.    1. In vitro effects of the methanolic extract from Cassia grandis stem bark (CgME) and its hexane (CgHF), ethyl acetate (CgEF) and methanolic (CgMF) fractions (400 -50 μg/mL) and praziquantel (PZQ, 3.12 μg/mL) on cell viability of adult Schistosoma mansoni couple worms after 72 h of incubation. The results were expressed as mean ± standard deviation (SD) and analyzed by ANOVA, followed by Tukey's test. *p < 0.05 compared to the control (-).  (Table 5). One can check the GC-MS chromatogram and mass spectra of all compounds in Figures S1-S11 (Supplementary Material). Qualitative chemical analysis of CgEF by UPLC-MS was able to identify 6 flavonoids (Clarke et al., 2014). A piperidine derivative was also proposed based on its occurrence in Cassia sp. (Pivatto et al., 2005) The compounds found in CgEF are shown in Table 6 and in Figure S12-S16 (Supplementary Material).

DISCUSSION
Cassia species play an important role in traditional medicine around the world, with reports of use in Chinese and ayurvedic medicines to treat several diseases, mainly due to their purgative effects (Prada et al, 2014). Many secondary metabolite classes have been found in different Cassia species, such as flavonoids, tannins, terpenes and sterols, saponins, alkaloids, and specially anthraquinones (Dave and Ledwani, 2014). The same chemical profile here described for the methanolic extract of the C. grandis stem bark was previously reported for C. grandis leaves and fruits (Meena, 2010;Prada et al., 2018). A study with the leaves methanolic extracts of seven Cassia spp. native from Egypt highlights C. grandis as the species with higher content of phenolic compounds (169.73 ± 2.9 mg GAE/g extract) and flavonoids (39.03 ± 1.0 mg QE/g extract). Here, higher values were found for phenolic compounds in the CgME and two of its fractions, CgEF and CgMF. On the other hand, only CgEF showed similar levels of flavonoids.
Oxidative stress is one of the main causes of human diseases and many phytochemicals, such as flavonoids and tannins, have been reported to prevent oxidative damage (Amaral et al., 2019). In vitro antioxidant activity of CgME and its fractions was carried out through phosphomolibdium and DPPH assays, which are methods frequently used to determine the radicalscavenging activity of natural products. In this study, CgME and its fraction CgEF showed EC 50 of approximately 4.3 μg/mL, agreeing with the values published by Prada et al. (2018) for the extracts of C. grandis fruits. The study performed by El-Hashash et al. (2010) also reports total antioxidant activity (TAA) for the same seven Cassia spp., where Cassia glauca presented the highest TAA with 53.4% followed by C. grandis with 24% in relation to the activity of ascorbic acid alone. In our study, we reported values greater than 70% for all extract/fractions, except for CgHF which presented only residual activity of about 23%.
The poor cytotoxicity of CgME and its fractions, CgEF and CgMF, is probably due to the high levels of phenolic compounds and flavonoids, which are cytoprotective. Hegazi and Hashim (2016) reported the low cytotoxicity of the polar fractions of C. grandis leaves against three different cancer cell lines, hepatocellular (HepG-2), breast (MCF-7), and prostate (PC3) by the neutral red uptake assay. The nonpolar fraction, CgHF, instead, showed relevant cytotoxicity against two cancer cell lines, NCI-H292, and HL-60. The major compounds found in the CgHF were clionasterol (γ-sitosterol) and lupeol acetate, in addition to the fatty acids, linoleic and palmitic acids. Several phytosterols have been reported as cytotoxic to cancer cell lines: γ-sitosterol induces G2/M cell cycle arrest and apoptosis through c-Myc suppression in MCF-7 and A549 cells (Sundarraj et al., 2012), while fucosterol, the main metabolite of clionasterol, exhibits selective antitumor anticancer activity against HeLa cell line by inducing mitochondrial-mediated apoptosis, cell cycle migration inhibition and downregulation of mTOR/ PI3K/Akt signalling pathway (Jiang et al., 2018). Lupeol and its esters have also been reported as cytotoxic against tumor cells through the inhibition of key enzymes, such as topoisomerase II, DNA polymerase β, and farnesyltransferase (Gallo et al., 2009). Lupeol acetate is the active principle of Himatanthus drasticus latex, known in Brazil as janaguba, which is used in folk medicine as an anti-inflammatory and to treat cancer (Lucetti et al., 2010).
The antimicrobial activity of Cassia species against Gram-positive bacteria is well documented and corroborates our findings Chanda et al. (2012) evaluated the antimicrobial activity of the polar extracts from Cassia auriculata, Cassia fistula, Cassia siamea, and Cassia tora, which were active mainly against S. aureus. Regasini et al. (2010) proposed a CMI-based classification for plant extracts, being considered those with CMI < 100 μg/mL as highly active; between 100-500 μg/mL as moderately active; from 500 to 1000 μg/mL as poorly active; and above 1000 μg/mL as inactive. Based on this classification, CgEM and its polar fractions, CgEF and CgMF, were considered moderately active for both standard and clinical isolate strains. Proestos et al. (2005) studied 27 Greek plants in which they identified several flavonoids, including quercetin, apigenin, luteolin, rutin, catechin, epicatechin, and naringenin. These compounds presented antimicrobial activity against E. coli, S. aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus. The chemical analysis of CgEF, which was the most active fraction, revealed the presence of gallocatechin/epigallocatechin, catechin/epicatechin, quercetin, and luteolin, corroborating the previously published results (Proestos et al., 2005).
Despite being used in folk medicine against worms and intestinal parasites, there is no report in the literature of the anti-S. mansoni activity of any Cassia species. Here, Magalhães et al. 273 it was possible to correlate the enrichment in phenolic compounds and flavonoid levels with the increase in schistosomicidal activity of the CgEF when compared to the CgME. Many flavonoids and phenolic acids, including kaempferol, gallic acid, and protocatechuic acid, have been reported in literature for their ability to impair reproduction and even kill adult S. mansoni worms, causing tegument damage and reducing egg production (Braguine et al., 2009). The anthelmintic effect of the flavonoid luteolin, isolated from Ajania nubigena (Wall.) C.Shih (Asteraceae) and also present in the CgEF, against S. mansoni and Trichuris muris has been reported by Wangchuk et al. (2016). In addition to killing adult worms, luteolin was effective against schistosomula (IC 50 13.3 μg/mL), the juvenile stage of S. mansoni which is refractory to praziquantel. Another class of secondary metabolites with strong and diverse biological properties are the alkaloids, which can cross biological membranes and accumulate in specific sites due to deprotonation/protonation of their nitrogen atoms. Piperidine alkaloids are very common in Cassia and Senna species, and for that reason we proposed piperidine derivative structure to the peak [M-H]-384.09. Castro et al. (2016) demonstrated the in vitro schistosomicidal activity of (−)-cassine/(−)-spectaline, the two major alkaloids found in Senna spectabilis flowers, against adult worms and cercariae.

Conclusion
The methanolic extract of C. grandis stem bark and its fractions showed in vitro antioxidant activity due to their high phenolic and flavonoid contents. The non-polar fraction CgHF was cytotoxic to two cancer cell lines and this effect was correlated to the presence of clionasterol and lupeol acetate, a phytosterol and a triterpene ester known for their antitumor effects. The ethyl acetate fraction (CgEF) exhibited both antimicrobial and schistosomicidal activities against multidrug-resistant S. aureus and S. mansoni, respectively, which have been attributed to the presence of flavonoids, such as catechin derivatives, quercetin, and luteolin in the CgEF. These results agree with the popular uses of C. grandis and should stimulate future research on this species.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors, except for the Ph.D. scholarship from Capes/Brazil.

SUPPLEMENTARY MATERIAL
Antibacterial, cytotoxic, and schistosomicidal activities of the methanolic extract from Cassia grandis L.f. (Fabaceae) stem bark and its fractions Table S1. Thin-layer chromatography conditions, standard compounds, and chemical developers used for the phytochemical screening of the methanolic extract from Cassia grandis stem bark and its fractions.