Preliminary evaluation of the in vitro biological activities of crude wood extracts of Dinizia excelsa Ducke obtained using methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane solvents

Abstract

This study reports an exploratory in vitro screening of the biological potential of Dinizia excelsa Ducke, an Amazonian species and the only representative of its genus. Six wood extracts were prepared using solvents of different polarities and compared regarding their phytochemical profiles and biological activities. The methanolic extract presented the highest yield, followed by aqueous and acetonitrile extracts, which also contained the largest amounts of phenolics, flavonoids, flavonols, hydrolyzable tannins, saponins, and alkaloids. In antioxidant assays, polar extracts showed IC₅₀ values of 25.3–45.3 µg/mL (DPPH), 15.1–30.0 µg/mL (ABTS), 105–135 µg/mL (NO• and OH•), and 110.0–180.0 µg/mL (FRAP, CUPRAC, phosphomolybdenum, ferricyanide). Cytotoxicity tests revealed moderate effects against tumor cells (27.6–45.6 µg/mL), low toxicity in normal mammalian cells, and absence of hemolysis. Antimicrobial activity occurred against Gram-positive and Gram-negative bacteria and Candida spp. (16–512 µg/mL). Antiparasitic assays showed IC₅₀ values of 32.4–73.0 µg/mL against Trypanosoma cruzi, Leishmania amazonensis, Schistosoma mansoni, and Plasmodium falciparum. These findings indicate that D. excelsa wood residues contain bioactive compounds with measurable antioxidant, antimicrobial, and antiparasitic properties. The results provide a preliminary foundation for future studies focused on isolation, structural elucidation, and mechanistic evaluation of active metabolites.

Key words
Dinizia Excelsa; Phytochemical; Antioxidant; Antimicrobial; Antiparasitic

INTRODUCTION

The Amazon is the largest tropical biome on the planet and harbors one of the broadest known floristic diversities. This remarkable plant richness represents a natural source of chemical substances with biological potential, making the region strategically important for the prospection of new bioactive compounds. The combination of unique environmental conditions, high ecological heterogeneity, and evolutionary complexity enables Amazonian plants to produce a wide variety of secondary metabolites, molecules that play essential roles in defense, adaptation, and interorganismic communication. These compounds have attracted growing scientific and industrial interest because of their promising pharmacological activities and their role as scaffolds for the development of new drugs and bioactive prototypes.

Among the botanical families of greatest chemical and pharmacological importance in the Amazon, Fabaceae stands out as one of the most diverse in the plant kingdom. This family is widely recognized for its phytochemical richness, encompassing compounds such as flavonoids, alkaloids, terpenoids, coumarins, and tannins, which are often associated with antioxidant, antimicrobial, anti-inflammatory, antiparasitic, and cytotoxic properties. The wide spectrum of biological activities observed in Fabaceae species reinforces their relevance in bioprospecting programs and in the discovery of drugs from natural sources.

Within this family, the genus Dinizia is noteworthy, represented solely by the species Dinizia excelsa Ducke, popularly known as “angelim-pedra.” This large tree, typical of upland (terra firme) Amazonian forests, can exceed 60 meters in height and 4 meters in diameter (Ramos et al. 2025), making it one of the largest tree species in South America. Its prominent presence in the forest canopy contributes to the vertical structure of the forest, microclimatic regulation, and the maintenance of habitats for other species, playing a fundamental ecological role in the dynamics of Amazonian ecosystems.

In addition to its ecological importance, D. excelsa has high economic value due to the exceptional quality of its wood. Commercially known as angelim-pedra, it has a high apparent density (0.90–1.10 g/cm³), reddish-brown color, medium to coarse texture, and interlocked grain, characteristics that confer mechanical strength, durability, and dimensional stability. These properties ensure excellent performance under high humidity and temperature variation, justifying its use in outdoor structures such as bridges, railway sleepers, piles, industrial floors, beams, and decks. Moreover, its natural resistance to termites, fungi, and borers favors applications in naval construction, infrastructure, and high-end building projects.

In woodworking, angelim-pedra is appreciated for its aesthetic beauty and natural sheen after polishing, being widely used in the manufacture of furniture, window frames, and decorative panels. However, as a slow-growing species typical of primary forests, excessive exploitation raises environmental concerns. The use of wood residues generated by industrial processes emerges as a sustainable alternative, enabling the rational use of already harvested biomass and reducing the need for new tree extraction.

In this study, the wood samples were obtained from industrial leftovers (sawmill residues) originating from legally managed sources. This choice reinforces the commitment to sustainable research and was motivated by two main factors. First, wood is an important chemical compartment where structural and defense compounds such as lignans, simple phenols, condensed tannins, and polymeric flavonoids accumulate, contributing to the species’ natural resistance to microorganisms and degradation (Oliveira et al. 2024). Second, the use of industrial processing residues represents an environmentally responsible alternative, allowing the study of a matrix rich in secondary metabolites without causing additional impact on natural populations.

From a taxonomic and chemical perspective, D. excelsa is of particular relevance as the only valid species of the genus Dinizia, making it a monotypic genus within the Fabaceae family (Fassina Brocco et al. 2025, Santos et al. 2025). This condition suggests unique biosynthetic pathways that are potentially linked to the production of distinctive chemical compounds. Preliminary studies have reported the presence of flavonoids, terpenoids, phenols, and tannins, classes known for their roles in antioxidant, antimicrobial, antiparasitic, and anti-inflammatory activities.

Despite this potential, research on the biological properties of D. excelsa remains limited and has focused mainly on initial chemical analyses (Fassina Brocco et al. 2025, Oliveira et al. 2024). Therefore, it is essential to expand knowledge about the bioactive constituents of this species, particularly through initial bioactivity screenings that help identify promising extracts and guide further studies on the isolation and characterization of active compounds.

The use of solvents with different polarities represents a key step in this type of approach because it allows the selective extraction of various chemical classes, resulting in broader phytochemical profiles (Nawaz et al. 2020). Furthermore, several studies indicate that the biological effects of plant extracts generally arise from the combined action of multiple secondary metabolites, whose synergistic interaction may enhance pharmacological responses (Gunter et al. 2025). Among these compounds, phenolics stand out for their ability to modulate redox processes, cellular signaling, and gene expression, although their mechanisms of action are not yet fully elucidated (Zhang et al. 2022).

Therefore, the investigation of Dinizia excelsa extracts obtained using solvents of different polarities constitutes an essential initial step in assessing the chemical and biological potential of the species. Conducting in vitro screening assays enables the identification of antioxidant, antimicrobial, antiparasitic, cytotoxic, and immunomodulatory activities, providing a basis for future research on natural products. Thus, this study aimed to perform a preliminary bioactivity screening of D. excelsa extracts, contributing to the scientific and sustainable valorization of this important Amazonian species.

MATERIALS AND METHODS

Obtaining methanolic, aqueous, acetonitrile, dichloromethane, chloroformic and hexane extracts of Dinizia excelsa

The wood of Dinizia excelsa Ducke was provided by Mil Madeiras Preciosas Ltda (PRECIOUS WOODS), located in Itacoatiara, Amazonas, Brazil, and registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under number ACEE312. The plant material was oven-dried at 60 °C for 48 h, ground in a knife mill, sieved through a 0.11 mm mesh, and stored at 30 °C until use, following the protocol established by the Research Group in Chemistry and Therapeutic Innovation at UFPE (Oliveira et al. 2024, Santos et al. 2025).

Extracts were prepared according to the method described by Lezoul et al. (2020), with modifications. Briefly, 50 g of dried, ground wood were subjected to maceration with 500 mL of solvent (1:10 w/v) for 72 h at 25.0 ± 0.5 °C, without agitation. The solvents used separately were methanol, distilled water, acetonitrile, dichloromethane, chloroform, and hexane. After each extraction, the solutions were filtered through filter paper. Organic extracts (methanolic, acetonitrile, dichloromethane, chloroform, and hexane) were concentrated under reduced pressure at 40 °C using a rotary evaporator, while the aqueous extract was frozen at –80 °C and subsequently lyophilized in a freeze dryer (L101, Liotop®). All dried extracts were stored at –20 °C in amber vials until further use.

Phytochemical Characterization by UV/VIS Spectrophotometry

Phytochemical characterization was carried out by UV/Vis spectrophotometry according to the methodologies described by Tran et al. (2021) and Albuquerque Nerys et al. (2022), with minor modifications. Methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane extracts were solubilized in 1% (v/v) DMSO at 1 mg/mL.

Total phenolic content was determined using the Folin–Ciocalteu method. Aliquots of 200 µL of each extract (1 mg/mL) were mixed with 1 mL of 10% (v/v) Folin–Ciocalteu reagent. After 5 min, 800 µL of 7.5% sodium carbonate solution were added, and the mixture was incubated in the dark at 25 °C for 30 min. Absorbance was measured at 765 nm. Gallic acid (10–80 µg/mL) was used to construct the calibration curve, and results were expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g).

Total flavonoid content was quantified by aluminum chloride complexation. A mixture of 500 µL of extract, 500 µL of ethanol, 50 µL of 10% aluminum chloride, and 50 µL of 1 M sodium acetate was incubated at 25 °C for 30 min. Absorbance was read at 415 nm. Quantification was based on a quercetin calibration curve (10–80 µg/mL), and results were expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g).

Flavonol content was determined using the same extract solution. In test tubes, 500 µL of extract (1 mg/mL), 500 µL of ethanol, 50 µL of 10% aluminum chloride, and 50 µL of 1 M sodium acetate were mixed. The mixture was incubated at 25 °C for 2 h, and absorbance was measured at 440 nm. A quercetin calibration curve (10–80 µg/mL) under identical conditions was used. Results were expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g).

Hydrolyzable tannins were quantified by a colorimetric method using vanillin and hydrochloric acid, with tannic acid as the standard. In test tubes, 500 µL of extract (1 mg/mL), 3 mL of 4% vanillin solution (in methanol), and 1.5 mL of 8% (v/v) HCl were combined. The mixture was incubated at 25 °C for 20 min, protected from light, and absorbance was measured at 500 nm. A calibration curve of tannic acid (10–80 µg/mL) was used, and results were expressed as milligrams of tannic acid equivalents per gram of dry extract (mg TAE/g).

Total saponins were determined by UV/Vis spectrophotometry at 544 nm using the colorimetric reaction with vanillin and perchloric acid. Extracts (1 mg/mL in methanol) were reacted with 250 µL of 8% vanillin solution (in glacial acetic acid) and 2.5 mL of 72% perchloric acid. The mixture (3.0 mL) was incubated at 60 °C for 15 min, cooled on ice for 5 min, and absorbance was measured. Oleanolic acid (10–80 µg/mL) was used as standard, and results were expressed as milligrams of oleanolic acid equivalents per gram of dry extract (mg OAE/g).

Total alkaloids were quantified at 470 nm using bromocresol green (BCG) in chloroform. Extracts were dissolved in 5 mL of 2 N HCl (1 mg/mL) and washed three times with 10 mL of chloroform. The aqueous phase was alkalinized with 1 mL of 0.1 N NaOH and reacted with 5 mL of 0.04% BCG and 5 mL of phosphate buffer (pH 4.7). After 15 min of stirring at 500 rpm, 5 mL of chloroform were added for extraction. The organic phase was evaporated under nitrogen, and the residue was redissolved in 10 mL of chloroform. Quinine (10–80 µg/mL) was used as standard, and results were expressed as milligrams of quinine equivalents per gram of dry extract (mg QE/g).

All assays were performed in quintuplicate.

Antioxidant Activity

Antioxidant activity was evaluated according to methodologies adapted from Albuquerque Nerys et al. (2022), Cavalcante et al. (2025), and Santos et al. (2025), using methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane extracts of D. excelsa wood. Extracts were diluted in 1% (v/v) DMSO at concentrations ranging from 3.9 to 1000 µg/mL. Four free radical scavenging assays (DPPH, ABTS, NO•, and OH•) and four redox-based assays (FRAP, ferricyanide, phosphomolybdenum, and CUPRAC) were performed.

For the DPPH assay, 0.32 mL of extract was mixed with 2.0 mL of DPPH solution (1.0 mM in methanol, Abs = 0.7 ± 0.01 at 517 nm), incubated for 25 min at 25 °C in the dark, and absorbance was measured at 517 nm.

In the ABTS assay, radicals were generated by reacting 5.0 mL of ABTS (7.0 mM) with 88 µL of potassium persulfate (140 mM) and incubating in the dark for 16 h. The solution was then diluted to an absorbance of 0.7 ± 0.01 at 734 nm. A volume of 30 µL of extract was added to 3.0 mL of ABTS solution, and absorbance was measured after 5 min.

For nitric oxide (NO•) scavenging, 1.0 mL of extract was incubated with 1.0 mL of sodium nitroprusside (5 mM) at 25 °C for 150 min in the dark. Then, 0.5 mL of Griess reagent (1% w/v sulfanilamide, 2% v/v H₃PO₄, and 0.1% w/v N-(1-naphthyl)ethylenediamine) was added, and absorbance was measured at 546 nm.

For hydroxyl radical (OH•) scavenging, 1.0 mL of extract was mixed with 0.5 mL of FeSO₄·7H₂O (1.5 mM), 0.35 mL of H₂O₂ (6.0 mM), and 0.15 mL of sodium salicylate (20 mM). The mixture was incubated at 37 °C for 1 h in the dark, and absorbance was measured at 515 nm.

In the phosphomolybdenum assay, 0.3 mL of extract was mixed with 1.5 mL of reagent (4.0 mM ammonium molybdate, 28.0 mM sodium phosphate, and 0.6 M H₂SO₄) and 1.5 mL of water, incubated at 95 °C for 90 min, and absorbance was measured at 695 nm.

For the FRAP assay, 0.5 mL of extract was added to 0.1 mL of water and 2.6 mL of FRAP reagent, incubated at 37 °C for 30 min, and absorbance was measured at 595 nm.

In the ferricyanide reducing power assay, 0.5 mL of extract was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% K₃[Fe(CN)₆], and incubated at 45 °C for 20 min. After addition of 2.5 mL of 10% trichloroacetic acid (TCA) and centrifugation (3000 rpm, 30 min), 2.5 mL of the supernatant was mixed with 2.5 mL of water and 0.5 mL of 0.1% FeCl₃, and absorbance was measured at 700 nm.

For the CUPRAC assay, 0.5 mL of extract was mixed with 0.5 mL of CuCl₂·2H₂O (0.01 M), 0.5 mL of ethanolic neocuproine (0.0075 M), and 0.5 mL of 1 M ammonium acetate buffer (pH 7.0). The mixture was incubated at 30 °C for 30 min, and absorbance was measured at 450 nm.

All assays were performed in quintuplicate using a UV/Vis spectrophotometer (Perkin Elmer, Lambda 650). Ascorbic acid and butylated hydroxytoluene (BHT) were used as positive controls, and distilled water served as the negative control. Results were expressed as percentage inhibition, and IC₅₀ values were determined by linear regression.

Cytotoxicity assessment in normal and tumor mammalian cells and hemolytic activity

The cytotoxicity of methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane extracts from Dinizia excelsa wood was evaluated using the MTT assay, following methodologies adapted from Albuquerque Nerys et al. (2022), Cavalcante et al. (2025), and Santos et al. (2025). Normal and tumor mammalian cell lines were used, all obtained from the American Type Culture Collection (ATCC®, USA). Normal cell lines included RAW 264.7 and J774-A1 (murine macrophages), Vero (African green monkey kidney cells), and V79 (Chinese hamster fibroblasts). Tumor cell lines included HepG2 (human hepatocellular carcinoma), Jurkat (human T-cell leukemia), HL-60 (human promyelocytic leukemia), K562 (human chronic myeloid leukemia), HeLa (human cervical carcinoma), HEp-2 (human laryngeal carcinoma), HCT-8, HT-29, and HCT116 (human colon adenocarcinomas), MCF-7 and T-47D (human breast adenocarcinomas), NCI-H292 (human lung carcinoma), and SF-295 (human glioblastoma).

Cells were cultured in RPMI 1640 medium (Sigma-Aldrich®, USA) supplemented with 5% fetal bovine serum (FBS, Gibco®, USA) and 1% penicillin-streptomycin (1000 IU/mL: 1000 µg/mL), maintained at 37 ± 0.5 °C in a humidified incubator with 5% CO₂ (Thermo Scientific®). Medium pH was maintained between 7.2–7.4. For the assays, cells were seeded in 96-well plates (Corning®, USA) at a density of 1 × 10⁴ cells/well in 100 µL of medium and incubated for 24 h to allow adhesion.

Extracts were first dissolved in 1% (v/v) DMSO (Sigma-Aldrich®) and then diluted in RPMI to final concentrations ranging from 3.9 to 1000 µg/mL. After 72 h of exposure, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well, followed by incubation for 3 h. The medium was removed, formazan crystals were solubilized in 100 µL of DMSO, and absorbance was measured at 570 nm using a microplate reader (Multiskan SkyHigh, Thermo Scientific®).

All experiments were performed in quintuplicate and repeated independently three times. IC₅₀ values were determined by nonlinear regression (GraphPad Prism® 5.0), and the selectivity index (SI) was calculated as the ratio of IC₅₀ values obtained for normal and tumor cells. Doxorubicin, amsacrine, and asulacrine (Sigma-Aldrich®, USA) were used as reference standards under the same experimental conditions. Statistical significance was assessed by one-way ANOVA followed by Tukey’s test, with p < 0.05 considered significant.

Hemolytic activity was assessed using a 1% suspension of murine erythrocytes in PBS, according to the same methodological references. A volume of 0.4 mL of each extract (3.9–1000 µg/mL) was incubated with 1.1 mL of the erythrocyte suspension for 60 min at 25 °C. PBS and Triton X-100 (1% v/v) were used as negative and positive controls, respectively. After incubation, samples were centrifuged at 1500 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm. All assays were performed in quintuplicate, in three independent replicates.

Antimicrobial Activity

The antimicrobial activity of Dinizia excelsa wood extracts was evaluated using the broth microdilution method, following the Clinical and Laboratory Standards Institute (CLSI) guidelines with adaptations from Albuquerque Nerys et al. (2022) and Santos et al. (2025). Six crude extracts obtained with solvents of different polarities (methanol, water, acetonitrile, dichloromethane, chloroform, and hexane) were tested. Extracts were dissolved in 1% (v/v) dimethyl sulfoxide (DMSO, Sigma-Aldrich®, USA) and serially diluted in culture medium to final concentrations ranging from 1024 to 2 µg/mL.

A total of ten bacterial strains were used: Acinetobacter baumannii UFPEDA-1024 and UFPEDA-1025, Enterococcus faecalis UFPEDA-69 and UFPEDA-138, Escherichia coli UFPEDA-224, Klebsiella pneumoniae UFPEDA-396, Pseudomonas aeruginosa UFPEDA-261 and UFPEDA-416, and Staphylococcus aureus UFPEDA-709 and UFPEDA-02. Additionally, sixteen Candida strains were evaluated: C. albicans HAM 11, HAM 13, HAM 15, UFPEDA-1007, UFPEDA-95, UFPEDA-4664, and URM49; C. parapsilosis HAM 14 and ATC22019; C. tropicalis HAM 33, HAM 43, and HAM 44; C. glabrata UFPEDA-6393; C. guilliermondii UFPEDA-6390 and URM62; and C. krusei ATC62.

All strains were stored at –80 °C in skim milk with 10% glycerol and reactivated prior to testing. Bacterial strains were cultured on Mueller-Hinton agar (24 h, 37 °C) and yeast strains on Sabouraud Dextrose agar (48 h, 28 °C). Colonies were suspended in sterile 0.85% NaCl solution, and turbidity was adjusted to 1.5 × 10⁵ CFU/mL for bacteria and 2.5 × 10³ CFU/mL for yeasts, confirmed by spectrophotometric absorbance at 600 nm (OD₆₀₀).

Assays were performed in sterile 96-well microplates (Corning®, USA), with 100 µL of extract solution and 100 µL of microbial suspension per well. Mueller-Hinton broth was used for bacterial assays, and RPMI 1640 medium buffered with MOPS (Sigma-Aldrich®, USA) was used for yeasts. Positive controls included ampicillin/sulbactam, amikacin, gentamicin, oxacillin, fluconazole, micafungin, and amphotericin B (20–0.4 µg/mL), while 1% DMSO and medium alone served as negative controls.

After incubation under appropriate conditions (24 h at 37 °C for bacteria and 48 h at 28 °C for yeasts), the minimum inhibitory concentration (MIC) was defined as the lowest extract concentration showing no visible microbial growth. To determine the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), 10 µL from each inhibited well was subcultured on Brain Heart Infusion (BHI) agar for bacteria or Sabouraud agar for yeasts. The lowest concentration showing no colony growth after incubation was recorded as the final MBC/MFC. All assays were carried out independently in quintuplicate.

Antiparasitic Activity

The antiparasitic activity of methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane extracts of Dinizia excelsa wood was evaluated against Leishmania amazonensis, Trypanosoma cruzi, Schistosoma mansoni, and Plasmodium falciparum, according to protocols adapted from Cruz Filho et al. (2023), Silva et al. (2023), and Santos et al. (2025). All assays were carried out in sterile, flat-bottom 96-well microplates, in quintuplicate and under controlled conditions. Reference drugs were included for each parasite as positive controls, and 1% DMSO and culture medium were used as negative controls. Extract concentrations ranged from 0.17 to 1000 µg/mL, prepared by serial dilutions in 1% (v/v) DMSO.

Leishmanicidal activity was assessed against promastigote and amastigote forms. Promastigotes were maintained in Schneider’s medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin–streptomycin (p.H 7.2), incubated at 26 °C until exponential growth (72 h). Parasites were washed three times with PBS, adjusted to 1 × 10⁶ parasites/mL, and incubated with extracts for 72 h at 37 °C in a 5% CO₂ atmosphere. Viability was assessed by direct counting using a Neubauer chamber, and IC₅₀ values were calculated by nonlinear regression. For amastigote assays, RAW 264.7 macrophages were seeded in 96-well plates (1 × 10⁵ cells/well), infected with promastigotes at a 1:10 (cell:parasite) ratio for 4 h, washed to remove free parasites, and subsequently treated with extracts for 72 h. Cells were fixed, stained with Giemsa, and analyzed by light microscopy to determine infection rates and parasite burden per cell.

Trypanocidal activity was tested against epimastigote, trypomastigote, and amastigote forms of the Y strain. Epimastigotes (3 × 10⁶/mL) were incubated in BHI medium supplemented with 10% FBS and treated with extracts for 96 h at 28 °C. Cell viability was determined by the MTT assay (5 mg/mL), with absorbance read at 540 nm. Trypomastigotes were obtained from the supernatant of infected Vero cells and incubated with extracts for 24 h at 37 °C; viability was assessed by direct counting in a Neubauer chamber. For amastigotes, RAW 264.7 macrophages were infected with trypomastigotes (2 × 10⁸/mL) for 4 h, washed, treated with extracts for 96 h, stained with Giemsa, and infection parameters (percentage of infected cells and mean parasite number per cell) were quantified by microscopy.

Schistosomicidal activity was evaluated against juvenile and adult worm pairs obtained from infected Biomphalaria glabrata. Parasites were transferred to 96-well plates containing RPMI 1640 medium supplemented with 10% FBS and treated with extracts for 24 h at 37 °C in 5% CO₂. After incubation, viability was assessed using the MTT reduction assay (5 mg/mL), with absorbance measured at 550 nm. Morphological changes (motility, tegument alterations, and mortality) were also recorded under an inverted light microscope.

Antimalarial activity was assessed using unsynchronized cultures of P. falciparum (3D7 strain), maintained at 0.6% hematocrit and 0.5% parasitemia in RPMI 1640 medium supplemented with 0.5% Albumax II, 25 mM HEPES, 25 mM NaHCO₃, and 50 µg/mL hypoxanthine. Parasite cultures were incubated with extracts for 72 h at 37 °C in a humidified 5% CO₂ atmosphere. After treatment, parasites were stained with SYBR Green I, and parasitemia was quantified by flow cytometry (Cytoflex) using the FL-1 channel (488 nm), counting 20,000–40,000 erythrocytes per well. IC₅₀ values were calculated from dose–response curves.

Evaluation of immunomodulatory activity

The immunomodulatory activity of methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane extracts of Dinizia excelsa wood was evaluated using splenocytes from BALB/c mice (6–8 weeks old), according to a methodology adapted from Cruz Filho et al. (2023). Splenocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL), and seeded into 96-well plates at a density of 1 × 10⁵ cells/well. Extracts were tested at final concentrations ranging from 3.9 to 1000 µg/mL, solubilized in 1% DMSO:distilled water. Vehicle controls (1% DMSO) and untreated cells were included as controls.

After 24 h of exposure, cytotoxicity was determined by annexin V-FITC and propidium iodide staining, followed by analysis on a flow cytometer (BD FACSCalibur). Cell proliferation was assessed by CFSE staining. Th1, Th2, and Th17 cytokines were quantified in culture supernatants using a Cytometric Bead Array (CBA) kit (BD Biosciences) and analyzed with FCAP Array™ software. Nitric oxide (NO) production was measured by the Griess reaction, with absorbance recorded at 540 nm using a microplate reader (SpectraMax).

Oxidative stress parameters were analyzed by flow cytometry using specific fluorescent probes: dihydroethidium (DHE) for cytosolic ROS, MitoSOX™ Red for mitochondrial ROS, Fluo-3AM for cytosolic calcium influx, and MitoStatus Red for mitochondrial membrane potential (ΔΨm). Immunophenotyping was performed with monoclonal antibodies conjugated for CD4, CD8, CD28, CTLA-4, CD14, CD80, CD86, and HLA-DR. All experiments were conducted independently in quintuplicate.

Ethical Considerations

All animal procedures followed the guidelines of the Brazilian Council for Animal Experimentation (COBEA) and were approved by the Animal Ethics Committee of the Federal University of Pernambuco (CEUA/UFPE, protocol no. 0087/2023).

Statistical Analysis

All experiments were conducted in quintuplicate, and results were expressed as mean ± standard deviation. Differences between extracts and reference standards were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. Differences were considered statistically significant at p < 0.05. Statistical analyses were performed using GraphPad Prism® version 5.0.

RESULTS AND DISCUSSION

Phytochemical Characterization

For the phytochemical characterization of Dinizia excelsa wood, crude extracts were obtained by maceration using solvents of varying polarities (hexane, dichloromethane, chloroform, acetonitrile, methanol, and water). These extracts were then subjected to quantification of total phenolic compounds, flavonoids, flavonols, hydrolyzable tannins, saponins, and alkaloids. The results were expressed in equivalents of specific standard compounds. Table I presents the percentage yields of the extracts and the concentrations of the main secondary metabolites, highlighting variations in phytochemical composition depending on the solvent used.

Table I
Yield and levels of phytochemical compounds (phenolics, flavonoids, flavonols, tannins, saponins and alkaloids) in Dinizia excelsa extracts.

Extracts	Yield (%)	Total Phenolics (mg GAE/g)	Total Flavonoids (mg QE/g)	Flavonols (mg QE/g)	Hydrolyzable Tannins (mg TAE/g)	Total Saponins (mg OAE/g)	Total Alkaloids (mg QE/g)
Methanolic	7.14 ± 0.05	452.30 ± 0.89	165.30 ± 0.65	47.80 ± 0.27	210.60 ± 0.91	38.60 ± 0.55	7.50 ± 0.26
Aqueous	5.78 ± 0.06	356.40 ± 0.91	138.20 ± 0.73	36.70 ± 0.32	239.70 ± 1.02	40.40 ± 0.57	8.20 ± 0.27
Acetonitrile	3.65 ± 0.04	224.60 ± 0.72	86.40 ± 0.51	31.50 ± 0.39	157.20 ± 0.83	31.50 ± 0.44	6.80 ± 0.31
Dichloromethane	2.40 ± 0.03	78.90 ± 0.54	28.30 ± 0.42	9.60 ± 0.23	38.70 ± 0.62	12.70 ± 0.34	3.20 ± 0.22
Chloroformic	1.27 ± 0.02	48.60 ± 0.33	15.40 ± 0.21	5.10 ± 0.19	12.30 ± 0.31	6.20 ± 0.23	1.40 ± 0.11
Hexanic	0.84 ± 0.01	15.20 ± 0.24	6.10 ± 0.14	1.80 ± 0.08	nd	2.80 ± 0.13	0.60 ± 0.08

• Values expressed as mean ± standard deviation; nd: not detected at the concentrations evaluated; milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g); milligrams of quercetin equivalents per gram of dry extract (mg QE/g); milligrams of tannic acid equivalents per gram of dry extract (mg TAE/g); milligrams of oleanolic acid equivalents per gram of dry extract (mg OAE/g).

The choice of solvent is a critical factor in the characterization and extraction of secondary metabolites, particularly when dealing with plant matrices rich in oxygenated compounds (Lapornik et al. 2005). The results presented in Table I demonstrate a correlation between solvent polarity and extraction efficiency, both in terms of yield and the chemical nature of the extracted constituents.

Among the tested solvents, methanol produced the highest extraction yield (7.14%) likely due to its ability to penetrate the lignocellulosic matrix and solubilize compounds with a wide range of polarities. Although more polar, water showed a lower yield (5.78%) probably due to its high cohesiveness and surface tension, which limit diffusion into denser tissues. Acetonitrile (3.64%) with moderate polarity and aprotic character was efficient in extracting moderately polar compounds. Nonpolar solvents such as dichloromethane (2.40%), chloroform (1.27%), and hexane (0.84%) produced lower yields, reflecting their limited affinity for polar constituents present in the wood.

Regarding total phenolic content, the highest concentrations were found in the methanolic extract (452.30 mg GAE/g), followed by water (356.40 mg GAE/g) and acetonitrile (224.60 mg GAE/g). Plant phenolics are highly polar compounds containing multiple hydroxyl groups that engage in extensive hydrogen bonding networks (Tran et al. 2021, Albuquerque Nerys et al. 2022). Methanol’s affinity for these structures is attributed to its ability to solvate both aglycones and glycosylated forms, whereas water, despite interacting with these groups, suffers from physical limitations in matrix penetration (Meira et al. 2020). Nonpolar solvents, in turn, yielded significantly lower extraction efficiencies (dichloromethane: 78.90 mg GAE/g; chloroform: 48.60 mg GAE/g; hexane: 15.20 mg GAE/g) as expected due to their inability to interact with highly oxygenated groups.

A similar trend was observed for total flavonoids. Methanol (165.30 mg QE/g), water (138.20 mg QE/g), and acetonitrile (86.40 mg QE/g) showed the highest levels. Flavonoids are a class of polyphenols often found in glycosylated forms, which confer high polarity to the molecules. The interaction between solvents and these compounds depends mainly on the ability to stabilize hydrogen bonds with hydroxyl groups on the aglycone and sugar units. Therefore, protic solvents such as methanol and water are the most suitable (Meira et al. 2020). The low extraction efficiency observed with dichloromethane (28.30 mg QE/g), chloroform (15.40 mg QE/g), and hexane (6.10 mg QE/g) reinforces the limitations of nonpolar solvents in solubilizing these species.

Flavonols, which are less glycosylated than flavonoids, were extracted in higher concentrations with methanol (47.80 mg QE/g), followed by water (36.70 mg QE/g) and acetonitrile (31.50 mg QE/g). Their lower water solubility compared to glycosylated flavonoids explains the intermediate performance of acetonitrile, while the lower efficiency of less polar solvents results from their weak interaction with the oxygenated groups of the aglycone.

Hydrolyzable tannins, which are polymers of highly hydrophilic phenolic acids, were best extracted with water (239.70 mg TAE/g), followed by methanol (210.60 mg TAE/g) and acetonitrile (157.20 mg TAE/g). These compounds have a high density of hydroxyl groups, requiring extensive hydrogen bonding for effective solubilization. Poor performance with dichloromethane (38.70 mg TAE/g), chloroform (12.30 mg TAE/g), and hexane (not detected) was expected due to their inability to solvate such macromolecules.

Saponins, amphiphilic compounds composed of triterpenoid or steroid aglycones linked to sugar chains, were extracted more efficiently with water (40.40 mg OAE/g) and methanol (38.60 mg OAE/g), followed by acetonitrile (31.50 mg OAE/g). The glycosidic moiety interacts strongly with polar solvents, while the apolar region can be accommodated by solvents such as methanol or acetonitrile. Dichloromethane (12.70 mg OAE/g), chloroform (6.20 mg OAE/g), and hexane (2.80 mg OAE/g) showed lower efficiency, consistent with the poor solubility of saponins in nonpolar media.

Finally, total alkaloids, nitrogen-containing compounds that may exist in various protonation states, were more abundant in aqueous (8.20 mg QE/g), methanolic (7.50 mg QE/g), and acetonitrile (6.80 mg QE/g) extracts. The presence of amine groups enables ionic and hydrogen bonding interactions with polar solvents, favoring extraction in protic media (Meira et al. 2020). In contrast, nonpolar solvents (dichloromethane: 3.20 mg QE/g; chloroform: 1.40 mg QE/g; hexane: 0.60 mg QE/g) were less effective, being limited to the extraction of free-base, lipophilic alkaloids.

Overall, the phytochemical analysis revealed that the methanolic extract presented the most comprehensive profile, with the highest concentrations of total phenolics, flavonoids, flavonols, hydrolyzable tannins, saponins, and alkaloids. The aqueous extract also stood out, particularly for its ability to extract tannins, saponins, and alkaloids, highlighting its effectiveness in solubilizing polar compounds. The acetonitrile extract showed intermediate concentrations of the analyzed metabolites, whereas dichloromethane, chloroform, and hexane extracts exhibited lower levels, with hexane being the least efficient. These findings confirm that polar solvents are the most suitable for extracting the bioactive compounds present in Dinizia excelsa wood.

Antioxidant Activity

The antioxidant activity of Dinizia excelsa extracts was evaluated using eight assays covering two main mechanisms: free radical scavenging (DPPH, ABTS, nitric oxide, and hydroxyl radical) and ion-reducing capacity (FRAP, ferricyanide, phosphomolybdenum, and CUPRAC). The combined use of these methods was essential to identify different biochemical response profiles, considering the chemical nature of the compounds present in each extract and the specificity of each reactive system (Gulcin 2020).

Antioxidant activity was classified based on IC₅₀ values (µg/mL) as follows: IC₅₀ < 50 µg/mL indicates high antioxidant activity; 50 ≤ IC₅₀ ≤ 100 µg/mL indicates moderate activity; and IC₅₀ > 100 µg/mL indicates low activity (Badarinath et al. 2010, Gulcin 2020). The results are presented in Table II, showing that antioxidant activity varied according to the polarity of the extraction solvents and the mechanisms evaluated.

In the DPPH and ABTS assays, radical neutralization occurs via electron or hydrogen donation, with phenolic compounds being the primary contributors due to their hydroxyl groups that stabilize free radicals. DPPH, being soluble in organic medium, is more sensitive to antioxidants of intermediate polarity, whereas ABTS allows detection of both polar and nonpolar compounds (Gulcin 2020).

The methanolic extract exhibited high activity (IC₅₀ of 25.3 µg/mL for DPPH and 15.1 µg/mL for ABTS), as did the aqueous (32.1 and 20.4 µg/mL) and acetonitrile extracts (45.3 and 30.0 µg/mL). Dichloromethane (70.4 and 55.2 µg/mL) and chloroform extracts (90.1 and 75.0 µg/mL) demonstrated moderate activity, while the hexane extract showed low activity (120.3 and 100.2 µg/mL). These variations are associated with the presence of phenolic compounds, which act as electron donors and radical stabilizers (Badarinath et al. 2010). Although all extracts had significantly higher IC₅₀ values than the standards ascorbic acid (7.79 and 13.94 µg/mL) and BHT (18.99 and 5.31 µg/mL), with p < 0.05, the results obtained for the methanolic, aqueous, and acetonitrile extracts are considered promising, given the complexity and unpurified nature of the matrices.

In the assays involving physiologically relevant radicals such as nitric oxide (NO•) and hydroxyl radical (HO•), IC₅₀ values for all extracts were above 100 µg/mL, indicating low activity. These assays simulate cellular oxidative stress conditions and require highly reactive compounds (Gulcin 2020). The methanolic extract showed IC₅₀ values of 105.0 µg/mL for NO• and 110.0 µg/mL for HO•, with no statistically significant differences compared to the aqueous (120.0 and 130.0 µg/mL), acetonitrile (130.5 and 135.0 µg/mL), dichloromethane (140.0 and 145.0 µg/mL), chloroform (165.0 and 155.0 µg/mL), and hexane (180.0 and 175.0 µg/mL) extracts (p > 0.05). All extracts showed significantly lower activity than the standards ascorbic acid (50.9 µg/mL for NO• and 1.58 µg/mL for HO•) and BHT (67.93 and 2.54 µg/mL), with p < 0.05. The low activity may be related to a lower concentration of highly reactive nucleophilic groups such as catechols and conjugated systems (Badarinath et al. 2010).

The ion-reducing capacity assays showed variation among the extracts, reflecting their ability to neutralize metal ions such as iron, molybdenum, and copper. Reducing these ions is crucial, as they can participate in redox reactions that generate reactive oxygen species (ROS), such as hydroxyl radicals, which have high cytotoxic potential (Gulcin 2020). The excessive presence of these species can damage lipids, proteins, and DNA, compromising cellular and tissue integrity. Therefore, the extracts’ capacity to reduce these ions contributes to their protective effect against oxidative stress (Badarinath et al. 2010).

In the FRAP assay, IC₅₀ values were as follows: methanol (160.2 µg/mL), water (138.3 µg/mL), acetonitrile (120.5 µg/mL), dichloromethane (110.0 µg/mL), chloroform (120.6 µg/mL), and hexane (130.5 µg/mL). All were classified as having low activity, except dichloromethane, which approached the moderate range. Compared to standards ascorbic acid (85.32 µg/mL) and BHT (7.12 µg/mL), all extracts showed significantly lower activity (p < 0.05).

In the ferricyanide reducing assay, the IC₅₀ values were: water (145.7 µg/mL), dichloromethane (150.0 µg/mL), chloroform (160.0 µg/mL), methanol (170.5 µg/mL), and acetonitrile and hexane (180.0 µg/mL), all indicating low activity. Again, the extracts were statistically less active than ascorbic acid (26.52 µg/mL) and BHT (9.30 µg/mL) (p < 0.05).

In the phosphomolybdenum assay, chloroform (95.3 µg/mL) and dichloromethane (100.0 µg/mL) extracts were classified as having moderate activity. The other extracts showed IC₅₀ values of 110.7 µg/mL (acetonitrile), 125.4 µg/mL (water), 150.1 µg/mL (methanol), and 90.0 µg/mL (hexane), all considered to have low activity. All extracts were statistically less potent than ascorbic acid (5.45 µg/mL) and BHT (8.90 µg/mL) (p < 0.05).

In the CUPRAC assay, IC₅₀ values were 160.0 µg/mL (methanol), 145.3 µg/mL (water), 180.0 µg/mL (acetonitrile), 170.0 µg/mL (dichloromethane), 160.0 µg/mL (chloroform), and 175.0 µg/mL (hexane), all classified as low activity. Ascorbic acid (12.97 µg/mL) and BHT (9.03 µg/mL) showed significantly higher activity (p < 0.05).

The use of multiple assays allowed for the quantification of antioxidant activity and a better understanding of the predominant mechanisms in each extract. The high activity observed in the DPPH and ABTS assays for extracts with higher levels of phenolic compounds suggests that these functional groups efficiently neutralize stable radicals through electron donation (Badarinath et al. 2010). On the other hand, the low activity in the other assays highlights limitations in metal-reducing capacity and neutralization of more reactive radicals (Gulcin 2020). Nevertheless, the results obtained for the methanolic and aqueous extracts are promising, particularly given the complex and unpurified nature of the matrices.

The methanolic extract stood out for its versatility in extracting compounds of different polarities, reflected in its consistent performance in the DPPH, ABTS, and some redox assays. The aqueous extract also showed noteworthy results, especially in radical scavenging assays, due to its efficiency in extracting highly water-soluble substances such as tannins, saponins, and ionizable alkaloids. The acetonitrile extract demonstrated intermediate and selective activity, with efficacy in stable radical neutralization assays, suggesting affinity for moderately polar molecules.

The dichloromethane and chloroform extracts showed moderate performance in the DPPH, ABTS, and phosphomolybdenum assays, indicating the presence of less polar compounds with some antioxidant activity. The hexane extract, the most nonpolar, was the least active in all assays, demonstrating its limitation in extracting biologically relevant antioxidants.

These findings demonstrate that the choice of solvent directly influences the chemical profile of the extracts and, consequently, their antioxidant bioactivity, making it a key factor in the strategic utilization of plant species with therapeutic potential.

Cytotoxicity in Normal and Tumor Mammalian Cells

The evaluation of the cytotoxicity of Dinizia excelsa wood crude extracts in normal and tumor cell lines revealed significant variations associated with the polarity of the extraction solvents and the biological characteristics of the analyzed cells. IC₅₀ values are presented in Table III, while selectivity indices (SI), calculated as the ratio between cytotoxicity in normal and tumor cells, are shown in Tables SI to SIV - Supplementary Material, enabling comparative analysis among extracts and across different cell lines.

Table II
In vitro antioxidant activity of six Dinizia excelsa extracts evaluated by radical scavenging assays (DPPH, ABTS, NO•, OH•) and redox-based methods (FRAP, CUPRAC, phosphomolybdenum, ferricyanide), with results expressed as IC₅₀ values (µg/mL) and compared with the reference standards ascorbic acid and butylated hydroxytoluene (BHT), respectively.

Antioxidant Assays	Methanolic	Aqueous	Acetonitrile	Dichloromethane	Chloroform	Hexanic	Ascorbic Acid	BHT
DPPH	25.3 ± 2.4	32.1 ± 2.6	45.3 ± 3.1	70.4 ± 4.3	90.1 ± 5.0	120.3 ± 6.4	7.79 ± 0.01	18.99 ± 0.1
ABTS	15.1 ± 1.3	20.4 ± 1.9	30.0 ± 2.4	55.2 ± 3.8	75.0 ± 4.3	100.2 ± 5.2	13.94 ± 0.8	5.31 ± 0.9
Nitric Oxide (NO•)	105.0 ± 12.0	120.0 ± 0.1	130.5 ± 16.5	140.0 ± 18.0	165.0 ± 10.0	180.0 ± 22.0	50.9 ± 1.3	67.93 ± 1.2
Hydroxyl Radical (HO•)	110.0 ± 13.0	130.0 ± 0.2	135.0 ± 17.0	145.0 ± 19.0	155.0 ± 20.0	175.0 ± 21.0	1.58 ± 0.01	2.54 ± 0.1
FRAP	160.2 ± 5.6	138.3 ± 4.9	120.5 ± 3.8	110.0 ± 3.0	120.6 ± 4.5	130.5 ± 5.2	85.32 ± 0.0	7.12 ± 0.01
Ferric Ion Reducer	170.5 ± 4.2	145.7 ± 5.1	180.0 ± 6.5	150.0 ± 4.8	160.0 ± 5.0	180.0 ± 6.5	26.52 ± 0.9	9.30 ± 0.1
Phosphomolybdenum	150.1 ± 3.8	125.4 ± 4.2	110.7 ± 4.1	100.0 ± 3.0	95.3 ± 2.7	90.0 ± 2.5	5.45 ± 0.1	8.90 ± 0.01
CUPRAC	160.0 ± 6.1	145.3 ± 5.4	180.0 ± 6.2	170.0 ± 5.5	160.0 ± 5.0	175.0 ± 5.9	12.97 ± 0.2	9.03 ± 0.02

• IC₅₀: concentration required to inhibit 50% of radical or reducing activity. Mean ± standard deviation. DPPH: scavenging of the 2,2-diphenyl-1-picrylhydrazyl free radical; ABTS: scavenging of the ABTS•+ cation radical [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]; FRAP: ferric reducing antioxidant power; CUPRAC: cupric ion reducing antioxidant capacity; BHT: Butylated hydroxytoluene.

Table III
In vitro cytotoxic activity of six Dinizia excelsa extracts, evaluated in mammalian normal and tumor cell lines, with results expressed as IC₅₀ values (µg/mL) and compared with the reference standards asulacrine (Asu), amsacrine (Ams), and doxorubicin (Dox).

Non-tumorigenic cell lines	Methanolic	Aqueous	Acetonitrile	Dichloromethane	Chloroform	Hexanic	Dox	Ams	Asu
RAW 264.7	112.4 ± 3.5	125.6 ± 4.0	136.2 ± 2.9	148.3 ± 3.8	160.1 ± 4.2	172.8 ± 5.1	<0.10	<0.10	<0.10
J774-A1	110.5 ± 4.1	123.0 ± 3.6	134.0 ± 3.3	146.7 ± 4.5	155.9 ± 4.7	170.2 ± 5.2	<0.10	<0.10	<0.10
Vero	105.6 ± 2.9	117.3 ± 3.1	128.5 ± 2.8	140.6 ± 4.1	150.2 ± 4.4	162.4 ± 5.0	<0.10	<0.10	<0.10
V79	108.7 ± 3.3	119.2 ± 3.5	131.6 ± 3.0	143.8 ± 3.9	152.8 ± 4.1	165.0 ± 4.9	<0.10	<0.10	<0.10
Cancer cell lines	Methanolic	Aqueous	Acetonitrile	Dichloromethane	Chloroform	Hexanic	Dox	Ams	Asu
HepG2	38.5 ± 1.2	41.2 ± 1.5	45.6 ± 1.6	52.3 ± 1.7	58.0 ± 1.9	64.7 ± 2.2	<0.10	<0.10	<0.10
Jurkat	29.4 ± 1.3	34.8 ± 1.4	39.5 ± 1.5	44.1 ± 1.6	49.9 ± 1.8	55.7 ± 2.1	<0.10	<0.10	<0.10
HL-60	27.6 ± 1.1	31.5 ± 1.3	36.0 ± 1.4	41.7 ± 1.5	48.4 ± 1.7	53.2 ± 1.9	<0.10	<0.10	<0.10
K562	33.2 ± 1.2	38.4 ± 1.4	42.7 ± 1.6	47.0 ± 1.6	54.8 ± 1.9	61.5 ± 2.1	<0.10	<0.10	<0.10
HeLa	36.7 ± 1.4	40.5 ± 1.5	45.2 ± 1.7	50.8 ± 1.9	57.1 ± 2.0	63.0 ± 2.3	<0.10	<0.10	<0.10
HEp-2	35.1 ± 1.3	39.8 ± 1.5	44.4 ± 1.6	49.5 ± 1.8	55.4 ± 1.9	61.3 ± 2.2	<0.10	<0.10	<0.10
HCT-8	31.5 ± 1.2	35.7 ± 1.3	40.9 ± 1.5	46.6 ± 1.7	53.2 ± 1.8	58.6 ± 2.0	<0.10	<0.10	<0.10
HT-29	32.9 ± 1.3	37.2 ± 1.4	41.8 ± 1.5	48.0 ± 1.7	54.6 ± 1.9	60.9 ± 2.2	<0.10	<0.10	<0.10
HCT116	30.7 ± 1.1	34.4 ± 1.3	39.7 ± 1.4	45.2 ± 1.6	51.8 ± 1.8	57.4 ± 2.0	<0.10	<0.10	<0.10
MCF-7	34.5 ± 1.2	38.6 ± 1.4	43.3 ± 1.5	49.0 ± 1.7	55.7 ± 1.9	61.6 ± 2.1	<0.10	<0.10	<0.10
T-47D	33.0 ± 1.2	36.9 ± 1.4	41.2 ± 1.5	47.6 ± 1.6	53.5 ± 1.8	59.4 ± 2.1	<0.10	<0.10	<0.10
NCI-H292	35.9 ± 1.3	40.0 ± 1.5	44.5 ± 1.6	50.3 ± 1.8	56.8 ± 2.0	62.5 ± 2.3	<0.10	<0.10	<0.10
SF-295	29.8 ± 1.1	33.6 ± 1.3	37.9 ± 1.4	43.8 ± 1.6	50.1 ± 1.8	55.9 ± 2.0	<0.10	<0.10	<0.10

• IC₅₀: inhibitory concentration required to reduce 50% of cell viability. Mean ± Standard deviation.

Results for normal cell lines (Table III) revealed differences in cytotoxic response to the extracts depending on the cell type and solvent polarity. For RAW 264.7 cells (murine macrophages), the methanolic extract showed the highest toxicity (IC₅₀ = 112.4 µg/mL), followed by aqueous (125.6 µg/mL), acetonitrile (136.2 µg/mL), dichloromethane (148.3 µg/mL), chloroform (160.1 µg/mL), and hexane (172.8 µg/mL) extracts, with a statistically significant difference between the extremes (p < 0.001). This response may be related to the higher concentration of phenolics, flavonoids, tannins, and saponins in the more polar extracts. The phagocytic activity of this cell line may also favor the internalization of these compounds, enhancing their cytotoxic effects (Taciak et al. 2018).

A similar profile was observed for the J774-A1 cell line, also derived from macrophages, with IC₅₀ values of 110.5 µg/mL (methanolic), 123 µg/mL (aqueous), 134 µg/mL (acetonitrile), 146.7 µg/mL (dichloromethane), 155.9 µg/mL (chloroform), and 170.2 µg/mL (hexane), with significant differences between the most and least toxic extracts (p < 0.001). The susceptibility of these cells may be associated with a higher intracellular uptake of bioactive metabolites, especially phenolics and saponins (Castaño & Gómez-Lechón 2005).

Vero cells (African green monkey kidney epithelial cells) showed greater sensitivity to the extracts. The methanolic extract had an IC₅₀ of 105.6 µg/mL, followed by aqueous (117.3 µg/mL), acetonitrile (128.5 µg/mL), dichloromethane (140.6 µg/mL), chloroform (150.2 µg/mL), and hexane (162.4 µg/mL) extracts, with a significant difference between extremes (p < 0.001). This response may be attributed to higher membrane permeability, facilitating the intracellular action of compounds such as alkaloids, tannins, and saponins (Taciak et al. 2018).

Fibroblast V79 cells were the most resistant, with IC₅₀ values of 108.7 µg/mL (methanolic), 119.2 µg/mL (aqueous), 131.6 µg/mL (acetonitrile), 143.8 µg/mL (dichloromethane), 152.8 µg/mL (chloroform), and 165.0 µg/mL (hexane), with statistically significant differences between the extremes (p < 0.001). This resistance may be linked to lower cell permeability, reduced metabolite uptake, and the presence of efficient antioxidant systems capable of attenuating cytotoxic impacts (Castaño & Gómez-Lechón 2005, Taciak et al. 2018).

All extracts showed significantly higher IC₅₀ values compared to standard drugs (p < 0.0001), indicating a more favorable safety profile in normal cells. According to the literature, IC₅₀ values above 100 µg/mL are indicative of low in vitro cytotoxicity, which was observed for all tested extracts.

Extracts obtained with more polar solvents (methanolic, aqueous, and acetonitrile) showed greater toxicity due to the higher concentration of phenolics, flavonoids, tannins, and saponins. In contrast, apolar extracts (dichloromethane, chloroform, and hexane) showed lower toxicity, possibly due to the predominance of lipophilic metabolites. Cellular responses varied: epithelial cells (Vero) were more susceptible, macrophages (RAW 264.7 and J774-A1) showed intermediate response, and fibroblasts (V79) were more tolerant. None of the extracts caused hemolysis, indicating biocompatibility. These results demonstrate that the different D. excelsa extracts can be safely used under the tested conditions.

The extracts also exhibited cytotoxic activity against various human tumor cell lines, with IC₅₀ values varying according to extract polarity and cell type, as shown in Table III.

In the HepG2 cell line (hepatocellular carcinoma), polar extracts were more active, with IC₅₀ values of 38.5 µg/mL (methanolic), 41.2 µg/mL (aqueous), and 45.6 µg/mL (acetonitrile), followed by less polar extracts: dichloromethane (52.3 µg/mL), chloroform (58.0 µg/mL), and hexane (64.7 µg/mL). This effect may be associated with phenolic compounds that interact with hepatic transporters and, after metabolism, generate reactive oxygen species (ROS), leading to mitochondrial dysfunction and apoptosis. Moreover, the ability of this cell line to metabolize xenobiotics enhances the formation of toxic metabolites (Liu et al. 2023).

Among leukemic lines, HL-60 (promyelocytic) was the most sensitive, followed by Jurkat (T lymphoblastic) and K562 (chronic myeloid). For HL-60, IC₅₀ ranged from 27.6 µg/mL (methanolic) to 53.2 µg/mL (hexane); for Jurkat, from 29.4 µg/mL to 55.7 µg/mL; and for K562, from 33.2 µg/mL to 61.5 µg/mL. These differences relate to the degree of cell differentiation and susceptibility to oxidative stress. HL-60, being less differentiated, has more vulnerable mitochondria and reduced antioxidant capacity, making it more susceptible to phenolic-induced apoptosis (Esmeeta et al. 2022).

In cervical cancer lines, both HeLa (adenocarcinoma) and HEp-2 (epidermoid carcinoma) responded similarly. HeLa showed IC₅₀ values from 36.7 to 63.0 µg/mL and HEp-2 from 35.1 to 61.3 µg/mL. Sensitivity may be linked to alterations in the p53 pathway induced by HPV, which favor apoptosis induction by phenolic compounds that also affect cell cycle progression (Esmeeta et al. 2022, Liu et al. 2023).

Among colorectal cancer lines, HCT116 (deficient in mismatch repair) showed the lowest IC₅₀ values: 30.7 to 57.4 µg/mL, followed by HCT-8 (31.5 to 58.6 µg/mL) and HT-29 (32.9 to 60.9 µg/mL). HCT116’s greater sensitivity may relate to impaired DNA repair against ROS-induced damage, whereas HT-29 and HCT-8 have partially functional repair mechanisms (Liu et al. 2023).

In breast cancer lines, MCF-7 (luminal A) and T-47D (luminal B) had IC₅₀ values ranging from 34.5 to 61.6 µg/mL and from 33.0 to 59.4 µg/mL, respectively. Phenolic compounds may modulate hormone pathways and trigger apoptosis in cells with active oxidative metabolism, regardless of hormone receptor status (Esmeeta et al. 2022).

The lung cancer line NCI-H292, derived from epithelial carcinoma, exhibited IC₅₀ values from 35.9 to 62.5 µg/mL. The high basal ROS levels and intense mitochondrial activity in these cells may favor the pro-oxidant action of phenolic-rich extracts.

In the glioblastoma cell line SF-295, IC₅₀ values ranged from 29.8 to 55.9 µg/mL. This line has mitochondria with low functional stability and deficient antioxidant defenses, making it more vulnerable to ROS-induced dysfunction and apoptosis promoted by extract compounds (Esmeeta et al. 2022, Liu et al. 2023).

Methanolic (IC₅₀: 27.6–38.5 µg/mL), aqueous (31.5–41.2 µg/mL), and acetonitrile (36.0–45.6 µg/mL) extracts showed the highest cytotoxic activity, while dichloromethane (41.7–52.3 µg/mL), chloroform (48.4–58.0 µg/mL), and hexane (53.2–64.7 µg/mL) extracts were less active. This difference is attributed to the higher concentration of phenolic compounds in polar extracts, which synergistically induce oxidative stress, DNA damage, mitochondrial dysfunction, and apoptosis in tumor cells. All extracts had higher IC₅₀ values than standard drugs (doxorubicin, amsacrine, and asulacrine), which showed IC₅₀ values below 0.10 µg/mL in all tested cell lines (p < 0.0001).

Selectivity index (SI) analysis (Tables SI-SIV - Supplementary material), calculated as the ratio of IC₅₀ values in normal to tumor cells, showed that methanolic, aqueous, and acetonitrile extracts had greater selectivity toward tumor cells. The highest SI values were observed in HL-60, SF-295, Jurkat, HCT116, HCT-8, and HepG2 cell lines. For HL-60, maximum SI values were 4.07 (methanolic), 3.99 (aqueous), and 3.78 (acetonitrile). In SF-295, SI values reached 3.77, 3.74, and 3.59, respectively. In Jurkat cells, SI values were up to 3.82 (methanolic), 3.61 (aqueous), and 3.45 (acetonitrile). For HCT116, the highest SI values were 3.66, 3.65, and 3.43, respectively, while in HCT-8, values were 3.57, 3.52, and 3.33. In HepG2, SI values reached 2.92, 3.05, and 2.99. Other lines, including K562, HeLa, HEp-2, NCI-H292, MCF-7, and T-47D, also showed SI values above 2.8 for polar extracts.

Dichloromethane, chloroform, and hexane extracts had lower selectivity, with SI values generally between 2.5 and 2.9. In contrast, doxorubicin, amsacrine, and asulacrine exhibited SI values close to 1.0 in all cell lines, indicating low selectivity between tumor and normal cells.

All extracts were more cytotoxic to tumor cells than to normal cells. This profile is associated with the increased sensitivity of tumor cells to oxidative stress due to accelerated metabolism, accumulation of reactive oxygen species (ROS), and mitochondrial dysfunction. With impaired antioxidant defenses, these cells are unable to counteract the effects of phenolic compounds, leading to apoptosis induction (Gavanji et al. 2023). In contrast, normal cells possess greater redox stability and lower proliferation rates, reducing their sensitivity to the extracts (Setiawati et al. 2022, Gavanji et al. 2023).

Methanolic, aqueous, and acetonitrile extracts showed the highest IC₅₀ and selectivity values, with the methanolic extract being the most effective. The HL-60 cell line was the most sensitive, followed by SF-295, Jurkat, HCT116, and HepG2. This sensitivity may be associated with high proliferation rates, genomic instability, and low antioxidant capacity.

These findings reinforce the potential of polar extracts as selective antitumor candidates with low toxicity to healthy cells.

Antimicrobial Activity

Plant extracts represent complex and promising sources of bioactive molecules with antimicrobial potential, especially in the context of increasing resistance to conventional antibiotics. Their phytochemical diversity, particularly the presence of phenols, flavonoids, tannins, alkaloids, and saponins, enables interactions with multiple microbial cellular targets, affecting membranes, enzymes, and essential metabolic processes (Manso et al. 2021, Silva et al. 2023). In this context, the evaluation of extracts obtained from the wood of Dinizia excelsa constitutes a relevant initial step in biological screening aimed at the prospection of metabolites with pharmacological potential and possible biotechnological applications.

The results presented in Table IV indicated that Gram-positive bacteria were more susceptible than Gram-negative ones. For Staphylococcus aureus UFPEDA-02, the methanolic, aqueous, and acetonitrile extracts exhibited MIC values of 64 µg/mL, whereas the dichloromethane, chloroform, and hexane extracts showed 256, 512, and 1024 µg/mL, respectively, suggesting that higher polarity and phenolic content are associated with greater activity. For S. aureus UFPEDA-709, MICs were 512 µg/mL for the more polar extracts, equivalent to oxacillin (512 µg/mL), while the less polar ones reached 1024 µg/mL.

Table IV
In vitro antimicrobial activity of six Dinizia excelsa extracts, evaluated against Gram-positive and Gram-negative bacterial strains and yeasts of the genus Candida, with results expressed as minimum inhibitory concentration (MIC) values in µg/mL and compared with reference antimicrobial agents.

Microorganism	MIC Methanolic (µg/mL)	MIC Aqueous (µg/mL)	MIC Acetonitrile (µg/mL)	MIC Dichloromethane (µg/mL)	MIC Chloroform (µg/mL)	MIC Hexanic (µg/mL)	Control
Acinetobacter baumannii UFPEDA-1024	512	512	512	1024	1024	1024	Ampicillin + Sulbactam 16.0 (I)
Acinetobacter baumannii UFPEDA-1025	256	256	256	1024	1024	1024	Ampicillin + Sulbactam 4.0 (S)
Enterococcus faecalis UFPEDA-69	16	16	16	64	128	256	Amikacin 8.0 (S)
Enterococcus faecalis UFPEDA-138	32	32	32	128	256	512	Amikacin 8.0 (S)
Escherichia coli UFPEDA-224	128	128	128	512	1024	1024	Gentamicin 0.5 (S)
Klebsiella pneumoniae UFPEDA-396	128	128	128	512	1024	1024	Gentamicin 0.5 (S)
Pseudomonas aeruginosa UFPEDA-261	512	512	512	1024	1024	1024	Amikacin 32.0 (I)
Pseudomonas aeruginosa UFPEDA-416	256	256	256	1024	1024	1024	Amikacin 8.0 (S)
Staphylococcus aureus UFPEDA-709	512	512	512	1024	1024	1024	Oxacillin 512.0 (R)
Staphylococcus aureus UFPEDA-02	64	64	64	256	512	1024	Gentamicin 0.25 (S)
Candida albicans HAM 11	64	64	64	256	512	1024	Fluconazole 0.5 (S)
Candida albicans HAM 13	64	64	64	256	512	1024	Fluconazole 4.0 (S)
Candida parapsilosis HAM 14	32	32	32	128	256	512	Fluconazole 1.0 (S)
Candida albicans HAM 15	16	16	16	64	128	256	Fluconazole 1.0 (S)
Candida tropicalis HAM 33	64	64	64	256	512	1024	Fluconazole 16.0 (R)
Candida tropicalis HAM 43	32	32	32	128	256	512	Fluconazole 32.0 (R)
Candida tropicalis HAM 44	32	32	32	128	256	512	Fluconazole 128.0 (R)
Candida albicans UFPEDA-1007	64	64	64	256	512	1024	Micafungin 0.06 (S)
Candida albicans 95	16	16	16	64	128	256	Micafungin 0.03 (S)
Candida albicans 4664	32	32	32	128	256	512	Micafungin 0.01 (S)
Candida glabrata UFPEDA-6393	256	256	256	1024	1024	1024	Micafungin 0.01 (S)
Candida guilliermondii UFPEDA-6390	128	128	128	512	1024	1024	Micafungin 0.01 (S)
Candida parapsilosis ATC22019	32	32	32	128	256	512	Amphotericin B 0.5 (S)
Candida krusei ATC62	64	64	64	256	512	1024	Amphotericin B 0.5 (S)
Candida guilliermondii URM62	128	128	128	512	1024	1024	Amphotericin B 1.0 (S)
Candida albicans URM49	64	64	64	256	512	1024	Amphotericin B 0.25 (S)

• R – Resistant; S – Sensitive; I – Intermediate resistance; Ampicillin + Sulbactam – Ampicillin + Sulbactam.

Enterococcus faecalis strains also showed relevant sensitivity. For UFPEDA-69, the methanolic, aqueous, and acetonitrile extracts exhibited MICs of 16 µg/mL, while the others ranged from 64 to 256 µg/mL. For UFPEDA-138, MICs were 32 µg/mL for the most active extracts and 128–512 µg/mL for the less active ones. Although these values were higher than that of the antibiotic amikacin (8 µg/mL), a significant inhibitory effect was observed, characteristic of preliminary screenings using complex extracts (Hossain 2024).

Among Gram-negative bacteria, MICs were higher, reflecting the intrinsic resistance associated with the lipopolysaccharide outer membrane. For Acinetobacter baumannii UFPEDA-1025, the methanolic, aqueous, and acetonitrile extracts exhibited MICs of 256 µg/mL, while the others reached 1024 µg/mL. The UFPEDA-1024 strain presented an even more resistant profile (512–1024 µg/mL), far above that of the ampicillin plus sulbactam control (4–16 µg/mL). Escherichia coli UFPEDA-224 and Klebsiella pneumoniae UFPEDA-396 showed MICs of 128 µg/mL for the more polar extracts and 512–1024 µg/mL for the others, indicating moderate inhibitory effects.

For Pseudomonas aeruginosa, a notoriously resistant genus, MICs ranged from 256 to 512 µg/mL for the more polar extracts and up to 1024 µg/mL for the less polar ones. These values were higher than those observed for amikacin (8–32 µg/mL). These findings reinforce the need for subsequent studies using purified fractions and isolated compounds to identify the constituents responsible for the activity.

Yeasts showed greater sensitivity compared to Gram-negative bacteria. Candida albicans HAM 15 and HAM 95 exhibited MICs of 16 µg/mL for the methanolic, aqueous, and acetonitrile extracts, while the others ranged from 64 to 256 µg/mL. C. parapsilosis and C. tropicalis presented MICs of 32–64 µg/mL for the most active extracts and up to 512 µg/mL for the less polar ones. Although these activities were lower than those of fluconazole and amphotericin B (1.0–0.5 µg/mL), they demonstrated a promising initial inhibitory effect, particularly against clinically resistant strains (Akwongo et al. 2024).

The differences observed among microbial groups can be attributed to cell wall structure. Gram-positive bacteria, with their thick peptidoglycan layer, are more permeable to phenolic compounds, whereas Gram-negative bacteria, with a lipopolysaccharide-rich outer membrane, present a stronger diffusion barrier (Manso et al. 2021, Zhang et al. 2022). In yeasts, the cell wall composed of chitin and β-glucans may modulate the absorption and action of bioactive compounds.

Although none of the extracts exhibited measurable bactericidal (MBC) or fungicidal (MFC) concentrations, the microbial inhibition results allow characterization of a preliminary antimicrobial potential, particularly in the more polar extracts. The observed activity may be attributed to the presence of phenolic compounds, known to interact with microbial membranes, alter lipid fluidity, denature structural proteins, and interfere with essential enzymatic processes (Silva et al. 2023, Chen et al. 2024).

Therefore, the findings of this study should be interpreted as an initial screening that demonstrates the potential of D. excelsa extracts as a source of bioactive substances. Additional assays with purified fractions, isolated compounds, and mechanistic approaches such as MIC, MBC, FICI determination, and molecular modeling will be essential to confirm and elucidate the constituents responsible for the observed effects and their mechanisms of action.

Antiparasitic Activity

Natural products are valuable sources of compounds with antiparasitic potential, acting on multiple cellular targets of protozoa and helminths. Various classes, such as phenolics, flavonoids, alkaloids, and saponins, have shown efficacy against both extracellular and intracellular parasitic forms, including drug-resistant strains (Cruz Filho et al. 2023). Therefore, plant extracts such as those from Dinizia excelsa represent promising alternatives for the development of new antiparasitic therapies. The antiparasitic activity results of D. excelsa extracts, including IC₅₀ values and selectivity indices (SI), are presented in Table V.

Table V
In vitro antiparasitic activity of six Dinizia excelsa extracts, evaluated against Trypanosoma cruzi, Leishmania amazonensis, Schistosoma mansoni, and Plasmodium falciparum, with results expressed as IC₅₀ values (µg/mL) and selectivity index (SI), respectively.

Parasite	Methanolic	Aqueous	Acetonitrile	Dichloromethane	Chloroform	Hexanic
L. amazonensis Promastigote	34.2 ± 1.8 / 3.26	37.5 ± 2.0 / 3.35	39.6 ± 2.1 / 3.44	43.2 ± 1.9 / 3.43	48.5 ± 2.2 / 3.30	52.3 ± 2.8 / 2.96
L. amazonensis Amastigote	42.6 ± 2.1 / 2.62	45.7 ± 2.3 / 2.69	48.2 ± 2.2 / 2.78	50.5 ± 2.4 / 2.93	58.1 ± 2.5 / 2.77	67.5 ± 2.7 / 2.29
T. cruzi – Epimastigotes	32.4 ± 1.6 / 3.43	36.0 ± 1.7 / 3.42	38.5 ± 1.8 / 3.53	42.0 ± 2.0 / 3.53	47.5 ± 2.3 / 3.38	50.8 ± 2.5 / 3.01
T. cruzi – Trypomastigotes	40.2 ± 1.9 / 2.77	43.6 ± 2.1 / 2.82	47.0 ± 2.0 / 2.85	50.4 ± 2.3 / 2.91	54.2 ± 2.4 / 2.87	59.0 ± 2.6 / 2.68
T. cruzi – Amastigotes	43.5 ± 2.2 / 2.58	46.9 ± 2.3 / 2.62	50.2 ± 2.5 / 2.68	53.8 ± 2.7 / 2.75	59.3 ± 2.6 / 2.71	65.2 ± 2.8 / 2.33
S. mansoni – Young Worms	68.0 ± 3.4 / 2.57	70.0 ± 3.5 / 2.54	72.0 ± 3.6 / 2.63	75.0 ± 3.8 / 2.62	77.0 ± 4.0 / 2.56	79.0 ± 4.1 / 2.33
S. mansoni – Adult Couples	52.0 ± 2.6 / 3.23	54.0 ± 2.7 / 3.20	56.0 ± 2.8 / 3.24	59.0 ± 2.9 / 3.23	61.0 ± 3.0 / 3.20	64.0 ± 3.2 / 2.66
P. falciparum	66.0 ± 3.3 / 2.70	67.5 ± 3.4 / 2.64	68.9 ± 3.5 / 2.61	70.2 ± 3.6 / 2.60	71.6 ± 3.7 / 2.55	73.0 ± 3.8 / 2.35

• IC₅₀: inhibitory concentration required to reduce 50% of cell viabili_ty; IC50 (µg/mL) and SI values of positive standards, w_ith IC50 for macrophages in parentheses: Amphotericin B (Macrophages: 34.75) – L. amazonensis – Promastigotes (0.14 / 248.21), L. amazonensis – Amastigotes (0.18 / 193.06); Benznidazole (Macrophages: 32.24) – T. cruzi – Epimastigotes (1.1 / 29.31), T. cruzi – Trypomastigotes (0.88 / 36.64), T. cruzi – Amastigotes (2.1 / 15.35); Praziquantel (Macrophages: 28.03) – S. mansoni – Adult Couples (0.01 / 2803.00) Chloroquine (Macrophages: 127.05) – P. falciparum (0.156 / 814.42).

As shown in Table V, for Leishmania amazonensis in the promastigote form, the methanolic extract was the most active (34.2 µg/mL; SI = 3.26), followed by the aqueous (37.5; 3.35), acetonitrile (39.6; 3.44), dichloromethane (43.2; 3.43), chloroform (48.5; 3.30), and hexane (52.3; 2.96) extracts. These values differed significantly from those of amphotericin B (0.14 µg/mL; SI = 248.21; p < 0.0001). In the amastigote form, a similar decreasing activity pattern was observed according to the extract polarity, with values ranging from 42.6 (2.62) to 67.5 µg/mL (2.29), again lower than the reference drug (Amphotericin B: 0.18 µg/mL; SI = 193.06; p < 0.0001). The increased resistance of the amastigote form may be due to its intracellular location, which limits the accessibility of active compounds.

Regarding Trypanosoma cruzi, activity also decreased with reduced extract polarity. For epimastigote forms, IC₅₀ values ranged from 32.4 (SI = 3.43) to 50.8 µg/mL (3.01), with significant differences compared to benznidazole (1.1 µg/mL; SI = 29.31; p < 0.0001). Trypomastigote forms exhibited IC₅₀ values between 40.2 (2.77) and 59.0 µg/mL (2.68), also lower than the standard drug (0.88 µg/mL; SI = 36.64; p < 0.0001). Amastigote forms were the most resistant, with IC₅₀ values from 43.5 (2.58) to 65.2 µg/mL (2.33), compared to benznidazole (2.1 µg/mL; SI = 15.35; p < 0.0001). The differences among the parasite forms reflect variations in cellular barriers, with intracellular forms exhibiting higher resistance to the tested compounds (Cruz Filho et al. 2023).

For Schistosoma mansoni, the extracts were more active against adult worm pairs (52.0 to 64.0 µg/mL; SI = 3.23 to 2.66) than against juvenile worms (68.0 to 79.0 µg/mL; SI = 2.57 to 2.33), with statistically significant differences (p < 0.05). This behavior may be attributed to physiological differences between life stages, including tegument thickness, metabolism, and immune evasion mechanisms. All extracts were less active than praziquantel against adult worms (0.01 µg/mL; SI = 2803.00; p < 0.0001). Notably, there is no effective pharmacological treatment with praziquantel for juvenile worms, highlighting the biological potential of the extracts against this stage (Souza Silva et al. 2023).

For Plasmodium falciparum, IC₅₀ values ranged from 66.0 (SI = 2.70) to 73.0 µg/mL (2.35), with greater activity associated with the more polar extracts. All extracts showed statistically significant differences compared to chloroquine (0.156 µg/mL; SI = 814.42; p < 0.0001). The lower efficacy against this protozoan may be related to its intracellular environment within red blood cells, which hinders the action of natural compounds, especially the less lipophilic ones (Cruz Filho et al. 2023).

Overall, the toxicity ranking of the extracts against the parasites was: Trypanosoma cruzi > Leishmania amazonensis > Schistosoma mansoni (adults) > Plasmodium falciparum > S. mansoni (juveniles). The methanolic extract was the most active across all tested models, a result likely attributable to its higher content of phenolic compounds, flavonoids, and alkaloids, reinforcing its broad biological activity.

Immunomodulatory Activity

The immunomodulatory evaluation of the methanolic, aqueous, acetonitrile, dichloromethane, chloroform, and hexane extracts of D. excelsa revealed distinct effects among the extracts. These differences were primarily associated with the content of phenolic compounds. The results were presented in Tables SV to SIX of the supplementary material. Cell viability, assessed using propidium iodide and Annexin V staining, remained above 95% at all tested concentrations (p > 0.05). This indicates a lack of significant toxicity and is consistent with previously obtained cytotoxicity data in normal cell lines, as well as the absence of notable hemolytic activity. These findings confirm the safety of the extracts, even at high concentrations.

Proliferative activity was evident in all extracts, showing a statistically significant increase in cell division with increasing concentrations (p < 0.05). The methanolic, aqueous, and acetonitrile extracts promoted higher proliferation rates. In contrast, the dichloromethane, chloroform, and hexane extracts showed a milder, yet still significant, effect. This pattern was accompanied by an expansion of CD4⁺ and CD8⁺ lymphocyte subpopulations (p < 0.05), indicating a coordinated activation of the adaptive immune response. CD4⁺ T cells play a central role in cytokine release, while CD8⁺ T cells are critical for the elimination of infected or transformed cells.

The cytokine profile analysis showed a progressive and significant increase in the levels of IL-2, IL-4, IL-10, IL-12, IL-17, IFN-γ, and TNF-α with rising extract concentrations (p < 0.05). These results indicate the simultaneous activation of Th1, Th2, and Th17 responses. Meanwhile, TGF-β levels were significantly reduced (p < 0.05), suggesting the suppression of regulatory pathways. These cytokine profiles support the control of various pathogens: Th1 is involved in the response to Trypanosoma cruzi, Leishmania spp., and Plasmodium spp.; Th17 contributes to bacterial and fungal clearance; and Th2 is linked to humoral immune modulation. Furthermore, the pro-inflammatory cytokine pattern favors the recruitment of antitumor effector cells, indicating a role in immune responses against cancer.

Oxidative stress parameters including cytoplasmic and mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨ), and calcium ion influx showed a gradual and statistically significant increase at all concentrations (p < 0.05), indicating cellular activation without compromising viability. The significant reduction in nitric oxide (NO) levels further suggests control over nitrosative stress.

The high antioxidant capacity of the extracts, demonstrated in radical scavenging assays, explains the observed cellular protection despite increased oxidative stress. The integration of lymphocyte proliferation, CD4⁺/CD8⁺ activation, a pro-inflammatory cytokine profile, and maintained cell viability explains the previously observed biological effects, including antimicrobial, leishmanicidal, trypanocidal, antiplasmodial, schistosomicidal, and antitumor activities.

The extracts of Dinizia excelsa exhibited an immunomodulatory profile. They promote controlled cellular activation, stimulate adaptive pathways of the immune response, and enhance protective inflammatory reactions, indicating therapeutic relevance against infectious and neoplastic diseases. Variations in activity directly reflected the phenolic compound content, with the methanolic, aqueous, and acetonitrile extracts showing the highest potency.

CONCLUSIONS

This preliminary study demonstrated that the wood extracts of Dinizia excelsa possess a diverse phytochemical composition and exhibit in vitro biological activities of pharmacological interest. The polar extracts (methanolic, aqueous, and acetonitrile) showed higher levels of phenolic compounds, which may synergistically contribute, together with other metabolites, to the observed antioxidant, cytotoxic, antimicrobial, and antiparasitic activities. Although these activities were lower compared to standard drugs, the results reveal a consistent biological profile and justify further investigations of D. excelsa as a potential natural source of bioactive metabolites. Future studies should focus on bioassay-guided fractionation, isolation of active compounds, and in vivo mechanistic evaluations to more accurately define the pharmacological potential and safety of the most promising extracts.

SUPPLEMENTARY MATERIAL

Tables SI-SIX.

Tables SI-SIX.

Acknowledgements

The authors acknowledge financial support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) (APQ-0498-4.03/19, BFP-0038-04.03/21, BFP-0087-4.03/23, and BIC-1383-4.03/25), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (306865/2020-3). The authors also thank the Technological Platforms Core of the Aggeu Magalhães Research Center (FIOCRUZ Pernambuco) for their support.

• Data availability
  The datasets produced in the course of this study can be obtained from the corresponding author upon reasonable request.

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Edited by

• Handling editor
  Vasco Azevedo

Data availability

The datasets produced in the course of this study can be obtained from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  20 July 2026
• Date of issue
  2026

History

• Received
  5 July 2025
• Accepted
  13 Nov 2025