Assessment of cytotoxic and antimicrobial activity of Hymenaea courbaril L. stem barks extract

Avaliação da atividade citotóxica e antimicrobiana do extrato da casca do caule de Hymenaea courbaril L.

Abstract

Cerrado is Brazil's second largest biome, and its fauna and flora are rich in biodiversity. As a result, many plants are being studied to find new viable drugs. Among the species of interest is Hymenaea courbaril, commonly known as "jatobá". In this scenario, the extract's standardization and the execution of in vitro studies are crucial steps before investigating its applicability in human beings. This study aimed to obtain a standardized liquid extract of Hymenaea courbaril stem bark and analyze its phytochemical, antimicrobial, and cytotoxic profiles. The extract proved viable in RAW 264.7 cells and inhibited the bacterial growth of a methicillin-resistant Staphylococcus aureus (MRSA) clinical isolate. After High-Performance Liquid Chromatography (HPLC) screening, peaks suggestive of catechin and epicatechin were found. In silico analysis of these flavonoids indicated an LD₅₀ of 10000 mg/kg, fulfillment of Lipinski's criteria, and both were classified as druglike, with a safety level of 6. In conclusion, the standardized liquid extract of Hymenaea courbaril stem bark is non-toxic and rich in catechin and epicatechin. It has antimicrobial action against Staphylococcus aureus, opening up prospects for in vivo studies.

Keywords:
medicinal plants; cerrado; cytotoxic analysis; in silico; anti-infective agents

Resumo

O Cerrado é o segundo maior bioma do Brasil em extensão, cuja fauna e flora são ricas em biodiversidade. Isto posto, múltiplas plantas são estudadas à procura de novos fármacos viáveis. Entre as espécies de interesse, encontra-se a Hymenaea courbaril, comumente conhecida por "jatobá". Nesse cenário, a padronização do extrato e a execução de estudos in vitro são etapas primordiais para a investigação da aplicabilidade em seres humanos. O objetivo deste estudo foi obter o extrato líquido padronizado das cascas do caule da espécie Hymenaea courbaril e analisar seus perfis fitoquímico, antimicrobiano e citotóxico. O extrato mostrou-se viável em células RAW 264.7 e inibiu o crescimento bacteriano de um isolado clínico de Staphylococcus aureus resistente à meticilina (MRSA). Após o screening por Cromatografia Líquida de Alta Eficiência (CLAE), encontrou-se picos sugestivos para catequina e epicatequina. A análise in silico desses flavonoides indicou DL₅₀/mg/kg de 10000, ambos atenderam os critérios de Lipinski e foram classificados como druglike, com nível de segurança 6. Conclui-se que o extrato líquido padronizado das cascas do caule de Hymenaea courbaril é atóxico, rico em catequina e epicatequina e possui ação antimicrobiana contra Staphylococcus aureus, o que abre perspectivas para a execução de estudos in vivo.

Palavras-chave:
plantas medicinais; cerrado; análise citotóxica; in silico; ação antimicrobiana

1. Introduction

Cerrado is the second largest biome in Brazil, occupying 21% of the national territory. It has excellent biodiversity and contains many plants with medicinal properties corroborated by popular empirical practices and recent scientific evidence (Dourado et al., 2005). Medicinal plants are known to possess chemical compounds capable of promoting biological activities in the organisms of living beings, such as anti-inflammatory, antioxidant, anticancer, antimicrobial, and analgesic actions (Cruz et al., 2022; Nascimento et al., 2000). Consequently, numerous species have been the research subjects in pursuing new drugs.

In this scenario, there is Hymenaea courbaril L. or "jatobá", a Fabaceae (Leguminosae) family member and the Caesalpiniodeae subfamily. This species is popularly known for its curative effects on respiratory disorders such as asthma, bronchitis, colds, and flu, and its diuretic, healing, and antimicrobial effects. Apart from having its fruit used in cooking, the stem bark, unripe fruit, resin, flowers, and leaves of the jatobá are used to prepare infusions, decoctions, alcoholic macerates, homemade cough syrups, and poultices (Magalhães et al., 2019). Matos et al. (2024) suggested that H. courbaril possesses potential for antioxidant activity and for use in the pharmacological and food industries.

In this sense, applying plant species in formal medicine requires verifying the safety and efficacy of their biological effects to rule out possible intoxications. Therefore, phytochemical analysis and preliminary pharmacological and toxicological tests are necessary (Colet et al., 2015; Ma et al., 2025). Analytical studies of the phytochemical profile of H. courbaril L. stem bark extracts have indicated the presence of flavonoids, saponins, tannins, anthocyanins, and terpenoids (Bezerra et al., 2013; Vencato et al., 2016). Moreover, Fernandes et al. (2005) and Gonçalves et al. (2005) observed in vitro antimicrobial activity of the jatobá stem bark extract against strains of Staphylococcus aureus, Proteus mirabilis, and Escherichia coli. There are some studies regarding the technological studies of this species regarding the leaf extracts. However, stem bark extracts have been little explored regarding the biological and technological features (Silva-Silva et al., 2023).

Given the medicinal potential of the species, this study aims to obtain a standardized liquid extract of Hymenaea courbaril L. stem bark and analyze its phytochemical, antimicrobial, and cytotoxic profiles.

2. Materials and Methods

2.1. Plant material

The stem barks of H. courbaril were collected in the municipality of Morrinhos, Goiás, Brazil (17º35´18.58´´S, 49º 2´18.15´´W, 694 m), in August 2021. At the State University of Goiás, Anápolis Campus, the collected stem barks were dehydrated in ovens with air circulation at 40 ºC until reaching constant weight.

2.2. Preparation of extract

After dehydration, the dried material was weighed and powdered in a knife mill. The extract was obtained by macerating 40 g of plant material powder with a 96% ethanol solution in a ratio of 1:5 for seven days. The solution was concentrated in a rotary evaporator (Buchi Vacuum Pump and Fisatom Glassware) at 37 °C, 625 mmHg, and 40 to 60 rpm. Subsequently, the solvent was eliminated with aeration, and the resulting extract was stored at -20 ºC until further analysis.

2.3. Physicochemical characterization

The stem bark extract of H. courbaril L. was subjected to physicochemical characterization studies to determine the moisture content, total ash content, acid-insoluble ash content, particle size distribution, and swelling index, according to the methodology of Costa (2001) and Matos (2009).

2.4. Qualitative analysis of secondary metabolites

A phytochemical screening was performed to identify the presence of saponins, tannins, flavonoids, alkaloids, coumarin, anthraquinone heterosides, and digitalis heterosides using standard methods for qualitative testing by Costa (2001) and Matos (2009).

2.5. Cell viability assay

The cytotoxic activity of the extract was evaluated using the MTT assay (Mosmann, 1983). In 96-well microplates, 5x10⁴ RAW 264.7 cells were added per well containing 200 μL of supplemented RPMI-1640 medium (Sigma Chemical Co., St Louis, MO, USA). After incubation of 24 hours in a CO₂ oven, the cells were treated with five solutions of different concentrations of H. courbaril L. stem bark extract (100%, 33.2%, 25%, 16.6%, and 12.5%). The samples received 100 μL of solution and were incubated for 48 h. The experiment was reproduced in triplicate, and in addition to the treatments, control wells were prepared to contain only cells and a supplemented medium. Cells containing only medium were considered blank.

After treatment, 100 μL of 10% MTT solution (0,5 mg/mL) in PBS was added, followed by a new incubation period of 2 h. Next, 100μL of dimethyl sulfoxide was added to the samples, including control and blank wells. Absorbance was measured on a plate spectrophotometer (Biolisa Reader, Bioclin, Belo Horizonte, MG) with a 550 nm filter. The cell viability was calculated according to Equation 1, where OD corresponds to the average optical density.

$Cellviability\left(\%\right)=\frac{\left(ODtreated-ODblank\right)}{\left(ODcontrols-ODblank\right)}x100$ (1)

2.6. Antimicrobial activity

The disk diffusion method was used to investigate the antimicrobial activity, which was performed in duplicate. The gram-negative clinical isolates Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis were isolated from uroculture, and the gram-positive species Staphylococcus aureus was obtained from the oropharyngeal secretion culture.

In Petri dishes containing MacConkey agar (NewProv) and Mannitol-salt agar (Biocen do Brasil), gram-negative and gram-positive microorganisms were inoculated, respectively, with subsequent incubation in a bacteriological oven at 36ºC ± 1ºC. After 24 h, the bacteria’s suspensions were prepared in sterile 0.85% NaCl saline solution until a turbidity equivalent to the 0,5 standard on the MacFarland scale was obtained (10⁸ CFU/mL).

In 1 mL of sterile 0.85% NaCl solution, 0.0224 µg of the crystallized stem bark extract was diluted. Using sterile swabs, Petri dishes containing Mueller-Hinton agar were inoculated. Next, three 9 mm diameter holes were created in each plate and filled with 100 µL, 200 µL, and 400 µL of H. courbaril L. extract. As positive controls, multidisc systems with commercial antibiotics were used. The plates were incubated at 36ºC ± 1ºC for 24 h. Afterward, the inhibition halos' diameters were read.

2.7. High-performance liquid chromatography (HPLC)

Phenolic compounds were screened by high-performance liquid chromatography using a UHPLC 1290 Infinity II LC system (Agilent Technologies, USA). The column used was a 2.7 µm Agilent Infinity Lab Poroshell 120 EC-C18 (4.6x100 mm) with a flow rate of 1 ml/min at 30°C, injection volume of 5 µL, and wavelength scans of 280 nm, 306 nm, and 340 nm in 35 min. A solution of acetonitrile and 0.2% acetic acid was used as a solvent.

Furthermore, the presence of catechin was investigated at wavelengths of 200 nm, 210 nm, and 280 nm at 35°C, using acetonitrile and 0.05% orto-phosphoric acid with a solvent gradient ranging from 5:95 to 95:5 in 20 min. For comparison purposes with sample results, a mixture of phenolic compound standards (gallic acid, catechin, caffeic acid, epicatechin, rutin, ellagic acid, resveratrol, quercetin, apigenin, and kaempferol) was used in both procedures.

2.8. In silico analysis

The investigated molecules from H. courbaril L. species were selected through PubMed and ScienceDirect databases. Their molecular structures were obtained from PubChem (Kim et al., 2023). Pass Online (Filimonov et al., 2014) and Swiss ADME (Daina et al., 2017) were used to assess biological activity and pharmacokinetic characteristics. The toxicity screening was conducted using the Protox Prediction 3.0 web server (Banerjee et al., 2018, 2024) to identify the toxic profile in the selected molecules.

3. Results and Discussion

3.1. Physicochemical characterization

The stem bark analysis identified total and acid-insoluble ash contents of 4.7% and 4.2%. Most of the powder particles (75.85%) were retained between 2 mm and 850 µm. Around 12.10% were retained at 600 µm and 11.99% at 425 µm. Consequently, based on the Brazilian Pharmacopoeia classification, the plant material is coarse powder. Furthermore, the sample collected had a moisture content of 8.35% ± 0.11 and a swelling index of 0.43 mL ± 0.2.

Ash content measures the non-combustible components of a material after oxidation at high temperatures. The value indicates the total amount of minerals present in a sample. While investigating the ash content of Hymenaea courbaril L. fruit pulp and seeds, Dias et al. (2013) found values of 4.53% and 1.95%, respectively. Therefore, the total amount of minerals obtained in H. courbaril stem bark is similar to the pulp amount indicated in the literature.

3.2. Preliminary phytochemical screening and analysis by HPLC

Qualitative analysis of the sample revealed the presence of tannins, flavonoids, saponins, and anthraquinone heterosides (Table 1). The foam index observed in the saponin testing was 333,3. The data obtained are from previous phytochemical screenings of H. courbaril L. stem bark extracts performed by Bezerra et al. (2013) and Vencato et al. (2016).

Table 1
Phytochemical prospection of powdered plant material from Hymenaea courbaril L. stem bark.

Chemical class	Result
Alkaloids	-
Anthraquinone heterosides	+
Coumarin Heterosides	-
Digithalic Heterosides	-
Flavonoids	+
Saponins	+
Tannins	+

• (+) presence; (-) absence.

Studies carried out by Pettit et al. (2003) to analyze the chemical and biological properties of Hymenaea species found high concentrations of flavonoids in the bark of H. parvifolia and the leaves of H. palustris. Likewise, the chromatogram of the liquid extract evidenced the presence of flavonoids (Figure 1). It was observed that there were suggestive peaks for epicatechin at a retention time of 13.496 min at 280 nm (Figure 2) and for catechin at a retention time of 10.15 min at 200 nm (Figure 3). The presence of catechin was also detected in the H. courbaril sap extract by Costa et al. (2021).

Figure 1
Molecular structures of the chemical markers (A) catechin and (B) epicatechin found in the concentrated liquid extract of H. courbaril L. stem bark.

Figure 2
Chromatogram of H. courbaril L. stem bark concentrated liquid extract at 280 nm. Peak height (mAU), minutes (min), retention time (RT), coelution (CE).

Figure 3
Chromatogram of H. courbaril L. stem bark concentrated liquid extract at 200 nm. Peak height (mAU), minutes (min), retention time (RT).

Secondary metabolites are natural compounds that plants synthesize to protect against abiotic and biotic stresses. Among these, tannins and flavonoids are polyphenols known for their antidiarrheal, antimicrobial, antifungal, antioxidant, anti-inflammatory, and anticancer effects (Jesus et al., 2012; Shamsudin et al., 2022). Consequently, the ability to produce defensive substances against microorganisms makes plants potential alternative sources of antibacterial and antifungal agents for human pathogens.

Studies of the phytochemical profile of extracts and essential oils from the exocarp, stem bark, and leaves of H. courbaril L. have mainly detected the presence of tannins, flavonoids, and terpenes, known in the literature for their antioxidant nature. In this study, no suggestive peaks were identified for rutin and ellagic acid, phenolic compounds found in the aqueous stem bark extract by Vencato et al. (2016). This contrast can be explained by interfering variables in phytochemical composition, such as climate, soil, altitude, water availability, stage of plant development, and the extract methodology (Martins et al., 2010).

3.3. Antimicrobial activity

The sensitivity evaluation criteria for the agar diffusion test were the formation of an inhibition halo. Among the clinical isolates evaluated, only methicillin-resistant Staphylococcus aureus proved sensitive to all extract volumes, with a maximum inhibition halo of 20 mm. No inhibition halos were observed in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis cultures (Table 2). Although S. aureus presented the highest resistance profile among the clinical isolates in the antibiogram (Table 3), it was the bacterial species with the greatest sensitivity to the plant extract, which supports the investigation of the antimicrobial potential of H. courbaril stem bark extract.

Table 2
Susceptibility of clinical bacterial isolates to the liquid extract of H. courbaril stem bark and inhibition halos (mm).

Clinical isolate	100 µL	200 µL	400 µL
Staphylococcus aureus	(S) 20	(S) 20	(S) 20
Escherichia coli	R	R	R
Klebsiella pneumoniae	R	R	R
Proteus mirabilis	R	R	R

• R=resistant; (S)=susceptible.

Table 3
Susceptibility of clinical isolates to commercial antibiotics.

Clinical isolate	Resistant	Susceptible
Staphylococcus aureus	AMP, AZI, ERI, OXA, PEN	CFO, CIP, CLI, CLO, GEN, LNZ, RIF, SUT, TET, VAN
Proteus mirabilis	TET(a)	AMC, AMI, AMP, ATM, CAZ, CFO, CFZ, CIP, CLO, CPM, CRO, GEN, MPM, SUT
Escherichia coli	AMC, AMP, CLO	AMI, ATM, CAZ, CFO, CFZ CIP, CPM, CRO, GEN, MPM, SUT, TET
Klebsiella pneumoniae	AMP(a)	AMC, AMI, ATM, CAZ, CFO, CFZ, CIP, CLO, CPM, CRO, GEN, MPM, SUT, TET

• AMC=amoxicillin, AMI=amikacin, AMP=ampicillin, ATM=aztreonam, AZI=azithromycin, CAZ=ceftazidime, CFO=cefoxitin, CFZ=cefazolin, CIP=ciprofloxacin, CLI=clindamycin, CLO=chloramphenicol, CPM=cefepime, CRO=ceftriaxone, ERI=erythromycin, GEN=gentamicin, LNZ=linezolid, MPM=meropenem, OXA=oxacillin, PEN=penicillin, RIF=rifampicin, SUT=sulfazotrim, TET=tetracycline, VAN=vancomycin.
• (^a) Intrinsic resistance.

Given the wide use of H. courbaril as a medicinal alternative, several studies on its biochemical, pharmacological, and biological characterization have revealed antifungal, antimicrobial, antioxidant, larvicidal, and molluscicidal activities (Suzuki et al., 2008). The antimicrobial activity observed may be associated with the abundant presence of tannins and flavonoids, capable of inhibiting certain microorganisms (Fernandes et al., 2005).

In this perspective, Gonçalves et al. (2005) verified the antibacterial activity of H. courbaril stem bark hydroethanolic extract on a clinical isolate of S. aureus using the agar diffusion method. The authors observed the development of a 23 mm inhibition zone in a culture of gram-positive bacteria and the extract's inhibitory action against the P. mirabilis species, a finding contrary to that observed in this experiment.

Likewise, Cruz et al. (2023) also reported the antimicrobial potential of jatobá stem bark and possible variations caused by different extraction techniques. The extracts prepared by ultrasound-assisted extraction, dynamic maceration, and static maceration at a concentration of 500 mg/mL produced inhibition halos of 16 ± 3.6, 13 ± 0.6, and 16 ± 0.6 mm in cultures of S. aureus ATCC 25923. Therefore, the inhibition halo values for Staphylococcus aureus found in this study coincide with data reported in scientific literature.

3.4. Cell viability assay

It investigated the percentage of cell viability in RAW 264.7 cell cultures when exposed to different Hymenaea courbaril L. stem bark liquid extract dilutions. After 48 hours of incubation, similar cell viability percentages were observed for different dilutions. In proportions of 1:2, 1:4, and 1:8 of water solvent, respectively, RAW 264.7 cell cultures showed 111%, 104%, and 82% of viable cells post-treatment. The experiment found 100% cell viability in vitro and, therefore, no cytotoxic effect in the analyzed dilutions of the extract (Figure 4).

Figure 4
Cell viability (%) of RAW 264.7 cells after treatment of 48 h with H. courbaril L. stem bark concentrated liquid extract diluted 2, 4 and 8 times in water.

Costa et al. (2014) tested the cytotoxic effect of fisetin-rich precipitate, a flavonoid compound, and the crude sap of H. courbaril L. The IC₅₀ of fisetin and crude sap were 158 μg/mL and 109 μg/mL, respectively. Therefore, the fisetin-rich residue proved to be less cytotoxic against 3T3-A31 fibroblasts. Cecílio et al. (2012) identified cytotoxic activity of the hydroethanolic leaf extract in MA-104 cells after 48 hours of incubation at a concentration of 5000 μg/mL, which was not observed at concentrations of 500 and 50 μg/mL.

In contrast, Spera et al. (2019) reported cytotoxicity of the H. courbaril L. seeds hydroethanolic extract in mouse epithelial cells with B16F10 melanoma, indicating antitumoral potential. An opposite effect was observed in healthy bone marrow cells from Swiss mice, in which the extract exhibited antigenotoxic and chemoprotective capacity against cell damage induced by cyclophosphamide.

3.5. In silico analysis

Based on the data obtained in the chromatographic study, the pharmacokinetic profiles and degree of toxicity of catechin and epicatechin were analyzed. The molecular structures of the identified flavonoids were selected from PubChem (2023).

Using the Protox Prediction software, a median lethal dose (LD₅₀) of 10,000 mg/kg of ingestion was obtained for both compounds (Table 4). Consequently, epicatechin and catechin are classified as class 6 compounds (LD₅₀ > 5,000) or non-toxic, which supports the results of the MTT assay (Banerjee et al., 2018). Regarding the prediction of biological activities, it was possible to detect 43 possible pharmacological activities, including antioxidant, anti-carcinogenic, and anti-seborrheic actions, and inhibition of histamine, kinase, and peroxidase release. In addition, the investigated flavonoids favor Lipinski's rule and, therefore, have good theoretical oral bioavailability, classified as druglike.

Table 4
In silico analysis of the pharmacokinetic and toxicological potential of catechin and epicatechin.

Compound	Druglikeness	LD50	Class	Toxicity
Catechin	Yes	10000 mg/kg	6	Non-toxic
Epicatechin	Yes	10000 mg/kg	6	Non-toxic

Lipinski et al. (2001) studied the physicochemical characteristics of drugs and observed that oral availability depended on good water solubility and intestinal permeability. Thus, parameters were established for the drug-likeness classification, namely: calculated log P > 5, molecular weight > 500 Da, more than 5 hydrogen donor bonds, more than 10 hydrogen bond acceptors, and points of interactions with the primary pharmacological targets (Daina et al., 2019).

Similarly, Souza et al. (2021) conducted an in silico study of the toxicity and physicochemical properties of the primary secondary metabolites present in H. courbaril L. Most analyzed compounds were in class 4 and fulfilled Lipinski’s rules, with fisetinidol being the only non-toxic metabolite. Understanding the level of toxicity of a drug is necessary to ensure therapeutic safety, as lower toxicities allow for larger therapeutic windows. Therefore, in silico analyses produce preliminary results that are fundamental to screening candidate molecules for future drugs.

4. Conclusion

The phytochemical profile of H. courbaril L. stem bark liquid extract comprises tannins, flavonoids, saponins, and anthraquinone heterosides. The HPLC analysis identified the presence of epicatechin and catechin, flavonoids with broad medicinal potential under investigation. The extract exhibited cell viability in RAW 264.7 macrophages and in vitro antibacterial activity against a methicillin-resistant Staphylococcus aureus clinical isolate. In silico, the study of epicatechin and catechin proved the absence of toxicity and good theoretical oral bioavailability. Therefore, the results of this study open up prospects for future in vivo investigations of H. courbaril stem bark extract biological potential.

Data Availability Statement

The dataset analyzed or produced in this study can be requested from the corresponding author.

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Edited by

• Editor:
  Marcelo A.M. Esquisatto

Publication Dates

• Publication in this collection
  15 Dec 2025
• Date of issue
  2025

History

• Received
  05 Sept 2025
• Accepted
  14 Oct 2025