Biological activity in hydroethanolic extracts from bark, stem, and leaves of the Stryphnodendron adstringens (Mart.) Coville

Atividade biológica em extratos hidroetanólicos da casca, caule e folhas de Stryphnodendron adstringens (Mart.) Coville

Abstract

Stryphnodendron adstringens (Mart.) Coville, commonly known as “barbatimão,” is native to the Cerrado biome in Brazil and belongs to the botanical family Fabaceae. The objective of this study was to evaluate the biological activity of crude hydroethanolic extracts formulated from the bark, leaves, and stems of S. adstringens. Soluble solids were determined using the incubation drying methodology. Colorimetric methods of complexation with ferric chloride were employed as a qualitative assay to identify the presence of tannins, while phenolics and flavonoids were quantified by the Folin-Ciocalteu method and aluminum chloride complexation, respectively. Antioxidant activity was assessed by the capture of DPPH free radicals. Antibacterial and antifungal analyses in vitro were conducted using the disk diffusion method against Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans var. grubii. The MTT assay was used to determine antiparasitic activity against Leishmania amazonensis and to assess cytotoxicity using differentiated THP-1 macrophages. The extracts demonstrated efficacy against yeasts, especially the stem extract against C. albicans (7.62 mm), and against bacteria, with emphasis on the stem and leaf extracts against M. tuberculosis (both 9 mm). All extracts exhibited high antioxidant capacity, particularly the leaf and stem extracts (both over 92%) and low cytotoxicity (Cytotoxic Concentration - CC50 > 300 µg/mL). No extract was effective against L. amazonensis (Inhibitory Concentration - IC50 > 100 µg/mL). In conclusion, S. adstringens is a potential source of compounds with antibacterial properties (particularly against Gram-positive bacteria) and antifungal activity, with low cytotoxicity and high antioxidant activity. This work emphasizes the use of this plant as a source of molecules for the development of drugs against bacterial and fungal infectious diseases, as well as for combating diseases, such as cancer and neurodegenerative disorders, that are linked to cellular and DNA damage due to oxidative stress.

Keywords:  antimicrobial activity; barbatimao; phenolic compounds; vegetative parts

Resumo

Stryphnodendron adstringens (Mart.) Coville, conhecido popularmente como “barbatimão”, é nativo do bioma Cerrado no Brasil e pertence à família botânica Fabaceae. O objetivo deste estudo foi avaliar a atividade biológica de extratos hidroetanólicos brutos formulados da casca, folha e caule de S. adstringens. Os sólidos solúveis foram determinados utilizando-se a metodologia de secagem em incubadora. Utilizou-se os métodos colorimétricos de complexação com cloreto férrico como ensaio qualitativo para identificar a presença de taninos, enquanto os fenólicos e flavonoides foram quantificados pelos métodos de Folin-Ciocalteu e complexação com cloreto de alumínio, respectivamente. A atividade antioxidante foi avaliada pela captura de radicais livres de DPPH. Análises antibacterianas e antifúngicas in vitro foram conduzidas utilizando o método de difusão em disco contra Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans e Cryptococcus neoformans var. grubii. Foi utilizado o método de viabilidade celular por MTT para determinação da atividade antiparasitária frente Leishmania amazonensis e para a citotoxicidade utilizando-se macrófagos diferenciados de células THP-1. Os extratos demonstraram eficácia contra leveduras, especialmente o do caule frente a C. albicans (7,62 mm) e bactérias, com ênfase nos extratos do caule e das folhas contra a M. tuberculosis (ambos 9 mm). Todos os extratos apresentaram capacidade antioxidante elevada, com destaque para os extratos da folha e caule (ambos superior a 92%) e baixa citotoxicidade (Concentração citotóxica - CC₅₀>300µg/mL). Nenhum extrato foi efetivo contra L. amazonensis (Concentração inibitória - IC₅₀>100µg/mL). Conclui-se que S. adstringens é uma potencial fonte de compostos com propriedades antibacterianas (particularmente contra bactérias gram-positivas) e antifúngicas, com baixa citotoxicidade e atividade antioxidante elevada. Este trabalho enfatiza o potencial uso desta planta como fonte de moléculas para o desenvolvimento de fármacos, frente doenças infecciosas bacterianas e fúngicas, assim como, para combater doenças como o câncer e distúrbios neurodegenerativos, que estão relacionados a danos celulares e ao DNA causados pelo estresse oxidativo

Palavras-chave:  atividade antimicrobiana; barbatimão; compostos fenólicos; partes vegetativas

1. Introduction

Secondary pathways in plant metabolism are significant sources for producing phytochemicals with diverse structures and broad medicinal applicability. Secondary plant compounds have a notable evolutionary adaptive value, functioning as biostimulants, insecticides, antimicrobials, and antioxidants, among others (Bourgou et al., 2008). These compounds are produced in all plant cells and tissues, and their production quantity is primarily linked to the biotic or abiotic environmental stimuli the plant is exposed to (Mrid, 2021).

Plant secondary metabolism involves the synthesis of terpenes, nitrogenous compounds, and phenolic substances. Phenolics (tannins, flavonoids, lignins, coumarins, phenylpropanoids, etc.) are characterized by having at least one aromatic ring covalently linked to a hydroxyl group and are often part of the phytochemical composition of numerous natural sources with biological properties. Interestingly, phenolic compounds represent the most structurally and biologically heterogeneous group of molecules derived from secondary metabolism (Tanase et al., 2019).

Flavonoids, derived from aromatic amino acids like phenylalanine and tyrosine (Khoddami et al., 2013), include chalcones, flavones, flavonones, flavonols, isoflavones, and anthocyanins (Marcucci et al., 2021). They are widely distributed in natural sources and are responsible for the colors and aromas of flowers, fruits, and seeds. Flavonoids regulate cell growth, attract pollinators and fruit dispersers, and help maintain plant integrity under stress, such as excessive radiation and attacks by phytopathogens (Dias et al., 2021). Tannins are also important phenolic molecules, complex and diverse, with high molecular weight, containing about 12 to 16 phenolic groups with many hydroxyls. They are widely distributed in nature and are primarily associated with plant stress, acting as powerful photoprotectors, antioxidants, and antimicrobials. They are currently classified as gallotannins, ellagitannins, condensed tannins, complex tannins, and phlorotannins (Fraga-Corral et al., 2021).

Empirical knowledge of plant use, particularly among traditional populations, forms the basis of ethnobotany (Hoffman and Gallaher, 2007; Höft et al., 1999). In developing countries, the World Health Organization estimates that 80% of people use traditional medicine. Historically, the prospecting of bioactives from natural sources, mainly botanical, has become a viable alternative for obtaining antioxidant, antimicrobial, anti-inflammatory, and wound-healing compounds. These compounds can serve as prototypes for developing more potent and less toxic molecules and for formulating pharmaceutical products for human health applications (Ozcan et al., 2014; Cosme et al., 2020). Focusing on health applications, flavonoids like anthocyanins exhibit a high capacity to scavenge oxidative free radicals and can reduce cellular and tissue degradation processes caused by reactive oxygen species (ROS) (Scalbert et al., 2005). Additionally, tannins act as potential antimicrobials by nonspecifically binding to various polymers, such as microbial cell wall peptides, sugars, and enzymes, denaturing them and leading to cell death (Daglia, 2012).

Numerous studies have evaluated the phenolic composition of natural ingredients derived from Stryphnodendron adstringens (Mart.) Coville and its potential as a source of biologically active molecules (Ribeiro and Santos, 2022; Cruz et al., 2022). The bark and leaves of this plant are widely used by conventional human populations (indigenous, quilombola, rural communities, etc.) (Oliveira et al., 2014). Commonly known as “Barbatimão,” S. adstringens is native to the cerrado, endemic to Brazil, and belongs to the Fabaceae family. Adult individuals average 5 meters in height, with trunks reaching up to 30 cm in diameter, and their leaves are bipinnate or recomposed (Lorenzi, 1992).

In conclusion, this work aimed to identify the presence of tannic compounds, determine soluble solids content, quantify phenols and flavonoids, and evaluate the antioxidant, antimicrobial, antiparasitic, and cytotoxic activities of hydroethanolic crude extracts from the bark, leaves, and stems of Stryphnodendron adstringens (Mart.) Coville. The results obtained not only aim to support future research but also emphasize the potential of this plant as a valuable source of biologically active molecules.

2. Material and Methods

2.1. Plant material collection

The botanical material was collected in the rural area of Santa Rita do Sapucaí (22°17'8.082” S, 45°48'18.174” W) in the state of Minas Gerais, Brazil (Figure 1). The collection took place during the winter, in August 2022. Samples of bark, leaves, and apical branches (20 cm, measured from the base of the cut to the apex) were collected. The plant material was transported to the Botany and Phytotherapy Laboratory of the University of Vale of Sapucaí, where it was identified and deposited as a type specimen under the responsibility of curator F. S. Braz at the UNIVAS Herbarium, with accession number UNIVAS-007.

Figure 1
Specimen of S. adstringens studied (UNIVAS-007).

2.2. Preparation of plant extracts

The plant samples were dried in an analog sterilization and drying oven (7LAB® model SSA - 30 L) for 72 hours at 55°C. Next, the samples were ground in a knife mill to obtain a fine and homogeneous powder. The extractive solutions were formulated at 5% (w/v) using 70% INPM ethyl alcohol by the dynamic maceration technique (Shaking table, Criemaq® model C-200) at 40 rpm for 48 hours. Subsequently, the extractive solutions were vacuum-filtered, and the extracts were stored in amber bottles, protected from light, and refrigerated.

The plant samples were dried in an analog sterilization and drying oven (7LAB® model SSA - 30 L) for a duration of 72 hours at 55°C. Subsequently, the samples were ground using a knife mill to achieve a fine and homogeneous powder. The extractive solutions were prepared at a concentration of 5% (w/v) utilizing 70% INPM ethyl alcohol through the dynamic maceration technique, employing a shaking table (Criemaq® model C-200) set at 40 rpm for 48 hours. Following this, the extractive solutions were vacuum-filtered, and the extracts were stored in amber bottles to protect them from light and were kept refrigerated.

2.3. Determination of total soluble solids content

The total soluble solids content was determined in accordance with the method established by Reis (2021). The quantification of total soluble solids was conducted in triplicate. Aliquots of the extracts were transferred to beakers with recorded initial mass (m¹) and placed in an oven at 55°C for a duration of 48 hours for drying. Following this period, the masses of the beakers containing the dried extract were recorded (m²), and the total soluble solids content was calculated using the following Equation 1:

$$Sst=m2-m1$$ (1)

Where:

Sst = total soluble solids content (mg/mL);

m¹ = initial mass of the beaker (g);

m² = final mass of the beaker (g).

2.4. Determination of total phenolic compounds content

The total phenolic compounds content was determined using the Folin-Ciocalteu colorimetric reaction method, as described by Furtado (2019). The extracts were evaluated at a concentration of 100 mg/mL. A reference standard curve was constructed using gallic acid (C₇H₆O₅) at concentrations of 1 µg/mL, 2 µg/mL, 3 µg/mL, 4 µg/mL, 5 µg/mL, 6 µg/mL, and 7 µg/mL in volumetric flasks. Subsequently, each flask received 8 mL of Folin-Ciocalteu reagent and 12 mL of a 20% sodium carbonate (Na₂CO₃) solution. The solutions were allowed to rest, protected from light, for 2 hours. The extract (test group) was evaluated using the same methodology employed for the construction of the standard curve, substituting aliquots of gallic acid with aliquots of the extracts. All solutions were prepared in triplicate, and absorbance readings were obtained using a spectrophotometer (BEL® UV-M51 model) at a wavelength of 760 nm. The absorbance values were analyzed using the standard curve equation via the least squares method. Results were expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g).

2.5. Determination of total flavonoid content

The total flavonoid content was determined using the aluminum chloride (AlCl₃) complexation method at a concentration of 2%, as described by Do et al. (2014), with adaptations. A methanolic quercetin standard curve was prepared at concentrations of 1.5 µg/mL, 3 µg/mL, 6 µg/mL, and 12 µg/mL by adding 4 mL aliquots of quercetin solutions and 4 mL of ethanolic aluminum chloride solution to each test tube. For the evaluation of the extracts (test group), the same procedure was followed, substituting the quercetin aliquots with aliquots of the extracts at a concentration of 100 mg/mL. The test tubes were allowed to rest for 30 minutes. Readings were performed in triplicate using a spectrophotometer (BEL® UV-M51 model) at a wavelength of 425 nm. The absorbance values obtained were utilized to calculate the standard curve equation through the least squares method. Results were expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g).

2.6. Identification of tannic compounds

The presence of tannic compounds was assessed using the ferric chloride (FeCl₃) complexation assay, in accordance with the protocol outlined by Tiago et al. (2020). In a test tube, 2 mL of extract was combined with 10 mL of distilled water, followed by the addition of 4 drops of a 1% methanolic solution of ferric chloride. This qualitative colorimetric test indicates the presence of tannins: a blue color signifies the predominant presence of hydrolysable or gallic tannins, whereas a green color indicates the presence of condensed or catechic tannins.

2.7. Determination of antioxidant activity

The antioxidant capacity was determined using the 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) radical scavenging method with a 0.06 mM ethanolic solution, as described by Bondet et al. (1997) and Molyneux (2004), with modifications. Aliquots of 100 µL of the extracts at a concentration of 100 µg/mL were added to test tubes containing 3.9 mL of ethanolic DPPH radical solution. A control was conducted using 100 µL of 70% INPM ethyl alcohol in place of the plant extract. The tubes were kept away from light for 30 minutes, after which the samples were analyzed using a spectrophotometer (BEL® UV-M51 model) at a wavelength of 517 nm. The analyses were performed in triplicate. The absorbance values obtained were converted to the percentage of inhibition (%I) of antioxidant activity using the following Equation 2:

$$\%I=\left[\left(Ac-At\right)/Ac\right]x100$$ (2)

Where:

%I = Percentage of DPPH radical capture;

Ac = absorbance reading of the control solution;

At = reading recorded for the test sample.

2.8. Evaluation of antibacterial and antifungal activity

The antibacterial and antifungal activities of the extracts were assessed using the disk diffusion method, also known as the Kirby-Bauer method, in accordance with documents Performance Standards for Antimicrobial Disk Susceptibility Tests (2003) and Method for antifungal disk diffusion susceptibility testing of yeast (2009) of Clinical and Laboratory Standards Institute (2003, 2009). The bark, stem, and leaf extracts were evaluated at a concentration of 100 µg/mL against strains from the American Type Culture Collection (ATCC). The reference microbial strains used included Escherichia coli ATCC 8739, Candida albicans ATCC 10231, Cryptococcus neoformans var. grubii ATCC 90113, Mycobacterium tuberculosis ATCC 25177, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923. Bacterial and yeast inoculum were previously reactivated on Nutrient Agar (NA) and Sabouraud Agar (SA) media for 24 and 48 hours, respectively. Subsequently, colony fragments were transferred to tubes containing a 0.9% saline solution, and the inoculum were standardized by spectrophotometry (BEL® UV-M51 model) at a wavelength of 625 nm to match the 0.5 McFarland standard.

Ethyl alcohol (ETOH) at 70% was utilized as the negative control, while the antibiotic amoxicillin combined with clavulanic acid (AMOX, 30 µg) and the antifungal fluconazole (FLUC, 25 µg) served as positive controls. For the disk diffusion assay, Mueller Hinton Agar was employed for bacterial strains, while the same medium supplemented with 2% glucose was used for yeast strains. Each plate received 200 µL of the standardized inoculum. Whatman discs with a diameter of 6 mm were impregnated with 10 µL of the extract at 100 µg/mL and 10 µL of 70% INPM ethyl alcohol. After complete drying, the discs were placed on the media containing the inoculum. The plates were incubated in a B.O.D. incubator (Solid Steel Model SS B.O.D - 120L) for 24 hours for bacteria and 72 hours for yeast. Following this incubation period, the inhibitory halos were measured, and the obtained values were subjected to analysis of variance (ANOVA). The mean halo measurements were compared using the Tukey test, both at a significance level of 5%, utilizing R Software version 2.5.1.

2.9. Determination of anti-protozoal activity

The anti-protozoal activity of the extracts was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability method, following the protocol established by Velásquez et al. (2017). Aliquots containing 1 × 10⁷ L. amazonensis promastigotes in both log and stationary phases were separately added to 96-well plates for interaction with extracts at increasing concentrations ranging from 1.56 to 100 µg/mL in 97 μL of LIT medium. The plates were subsequently incubated in a humid chamber (BOD) at 27°C for 72 hours. The assays were conducted in both experimental and biological triplicates. Medium with or without extracts, in the presence or absence of the parasite, served as control conditions. Amphotericin B was utilized as a positive control.

After incubation, 10 μL of MTT/PMS solution (comprising MTT at 2.5 mg/mL and PMS at 0.2 mg/mL, where PMS refers to phenazine methosulfate) was added to all wells, followed by further incubation, protected from light, for 75 minutes at 28°C. Absorbance readings were taken using a UV/Visible spectrophotometer (Tecan®) at 570 nm after dissolving the formed crystals with the addition of 100 μL of ethanol and 100 μL of isopropanol mixed with PBS at a 1:1 ratio. The absorbance values were analyzed to determine the percentage of inhibition at different concentrations using the following Equation 3:

$$\%IC=100-\left[\left(Gc-Gp\right)/Gc\right]x100$$ (3)

Where:

%IC: the cytotoxicity of the substance.

Gc: refers to the quantity of viable parasites per milliliter in control wells that do not contain the substance. It is calculated using the formula: Gc = Ac - Am, where Ac represents the absorbance value of wells with Leishmania in the absence of the compound, and Am denotes the absorbance of the control without both the parasite and the compound.

Gp: corresponds to the number of viable parasites per milliliter at various concentrations of the substance, calculated as: Gp = Ap - Apm, where Ap is the absorbance of the well of interest, and Apm is the absorbance of the control well containing the same concentration of the substance.

Subsequently, the inhibition percentages corresponding to the evaluated concentrations were analyzed using BioEstat 5.3 software to determine the IC50 (inhibitory concentration for 50% of viable cells) through quadratic polynomial regression.

2.10. Evaluation of cytotoxicity

The activity of the extracts was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability method, following the protocol established by Velásquez et al. (2017), with modifications. A total of 1 × 10⁷ THP-1 cells were resuspended in RPMI 1640 medium containing 100 nM Phorbol-Myristate-Acetate (PMA) and seeded into 96-well plates for differentiation and adhesion. The plates were incubated in a humid chamber (BOD) for 96 hours at 37°C in an atmosphere containing 5% CO₂. After differentiation, the macrophages were incubated with the extracts at increasing concentrations ranging from 9.37 to 300 µg/mL in 97 μL of RPMI medium. The plates were incubated in a humid chamber (BOD) at 37°C for 24 hours. The assays were performed in both experimental and biological triplicates. Medium containing macrophages served as a negative control, while Amphotericin B (Cristália) was utilized as a positive control.

Following incubation, 10 μL of MTT/PMS solution (containing MTT at 2.5 mg/mL and PMS at 0.2 mg/mL) was added to all wells, followed by an additional incubation protected from light for 75 minutes at 37°C with 5% CO₂. Absorbance readings were taken using a UV/Visible spectrophotometer (Tecan®) at 570 nm after dissolving the formed crystals with the addition of 100 μL of ethanol and 100 μL of isopropanol mixed with PBS at a 1:1 ratio. The absorbance values were analyzed to determine the percentage of cytotoxicity at different concentrations using the following Equation 4:

$$\%CC=100-\left[\left(Gt/Gc\right)\right]x100$$ (4)

Where:

%CC: the cytotoxicity of the substance.

Gt: represents the absorbance value for the test group at the evaluated concentration.

Gc: represents the absorbance value for the control group at the evaluated concentration.

Subsequently, the percentages of cytotoxicity corresponding to the evaluated concentrations were analyzed using BioEstat 5.3 software to determine the CC₅₀ (cytotoxic concentration for 50% of viable cells) through quadratic polynomial regression.

3. Results

3.1. Levels of soluble solids, tannins, total phenolic compounds, and total flavonoids

The results pertaining to the levels of soluble solids, tannins, total phenolic compounds, and total flavonoids are presented in Table 1. Notably, the stem extract exhibited the highest concentration of polyphenols

Table 1
Tannins, soluble solids content, total phenols, total flavonoids for the extracts.

Parameters	Concentration
	HEB	HEL	HES
Total Soluble Solids (mg/mL)	9.9 ± 0.12	4.0 ± 0.34	2.3 ± 0.12
Total Phenols (mg AGE/g)	420.07 ± 0.26	542.74 ± 0.6	1243.19 ± 0.16
Total Flavonoids (mg QE/g)	843.69 ± 0.07	1285.43 ± 0.14	387.24± 0.33
Tannins	+/c	+/c	+/c

• HEB = Hydroethanolic extract from bark; HEL = Hydroethanolic extract from leaf; HES = Hydroethanolic extract from stem; +/c = positive ferric chloride complexation reaction for the presence of condensed tannins.

3.2. Antioxidant activity

The hydroethanolic extracts evaluated demonstrated significant antioxidant activity, as indicated by their percentage reductions in DPPH oxidative radicals. The bark extract (HEB) exhibited a reduction of 86.48% (± 0.48), the leaf extract (HEL) showed a reduction of 92.18% (± 0.07), and the stem extract (HES) achieved a reduction of 92.4% (± 0.54). Notably, the hydroethanolic extract of the stem displayed the highest antioxidant activity, which correlates with its elevated phenolic concentration.

3.3. Antimicrobial activity

The results of the antimicrobial activity are detailed in Tables 2 and 3. Means that share the same letters in the columns do not differ significantly according to Tukey's test at a 5% probability level (P < 0.05). Measurements are expressed in millimeters (mm). The extracts demonstrated promising activity, particularly against M. tuberculosis, S. aureus, and P. aeruginosa.

Table 2
Antibacterial and antifungal activity of the extracts.

Microorganisms	Diameter (mm) of inhibitory halos and Tukey test
	HEB	HEL	HES
Mycobacterium tuberculosis	8.37a ± 0.1	9.0a ± 0.06	9.0a ± 0.1
Staphylococcus aureus	7.56ab ± 0.11	6.3c ± 0.03	6.5bc ± 0.05
Candida albicans	6.75b ± 0.05	6.3c ± 0.03	7.62b ± 0.13
Cryptococcus neoformans var. grubii	6.56b ± 0.08	7.18bc ± 0.09	6.43c ± 0.04
Pseudomonas aeruginosa	6.5b ± 0.03	7.51b ± 0.09	7.06bc ± 0.05
Escherichia coli	6.43b ± 0.04	0d	6.1c ± 0.02
p	3.47x10-5	2.2x10-16	2.59x10-8

• HEB = Hydroethanolic extract from the bark; HEL = Hydroethanolic extract from the leaf; HES = Hydroethanolic extract from the stem.

Table 3
Inhibition halos of the tested microorganisms against positive and negative controls (mm).

Microorganisms	AMOX	FLUC	ETAN
Candida albicans	Nt	27.33 ± 2.02	0
Cryptococcus neoformans var. grubii	Nt	26.5 ± 2.2	0
Escherichia coli	25.65 ± 1.4	Nt	0
Pseudomonas aeruginosa	33.95 ± 0.49	Nt	0
Mycobacterium tuberculosis	25.55 ±1.34	Nt	0
Staphylococcus aureus	29.86 ±1.0	Nt	0

• Nt = Not tested; AMOX = Amoxicillin-clavulanic acid combination; FLUC = Fluconazole; ETAN = 70% INPM ethanol.

3.4. Anti-Leishmania activity of the extracts

The extracts did not exhibit anti-Leishmania activity against L. amazonensis at increasing concentrations ranging from 1.56 to 100 µg/mL. In contrast, the positive control, amphotericin, demonstrated an IC₅₀ of 0.605 µg/mL (± 0.01).

3.5. Cytotoxicity of the extracts

Interestingly, the extracts did not exhibit cytotoxicity against macrophages differentiated from THP-1 cells. In contrast, amphotericin, used as a positive control, demonstrated a CC₅₀ of 150.15 µg/mL (± 7.15) when evaluated at increasing concentrations ranging from 9.37 to 300 µg/mL.

4. Discussion

The species S. adstringens (Mart.) Coville is an important representative of Brazilian flora regarding the use of medicinal plants. The success and benefits of utilizing this plant are related to its high concentration of phenolic compounds, particularly tannins and flavonoids, which are abundant in vegetative organs, especially documented for the bark and leaves (Santos et al., 2002). In addition to genotypic factors, environmental factors can also influence the chemical composition of a plant and, consequently, the biological activity of its derivatives, such as extracts and essential oils. Among these factors are rainfall, solar radiation, temperature, and the mineral composition of the soil. In plants, each cell possesses the metabolic machinery necessary for the synthesis of primary and secondary metabolites; however, not all secondary metabolites are produced in equal concentrations across different vegetative organs. The production and distribution of these compounds depend on the environmental conditions to which the organism is exposed, as well as the physiological needs of each organ. Two noteworthy examples are tannins and flavonoids: tannins are well-documented for the bark of various plants, while flavonoids are primarily associated with leaves, fruits, and flowers (Jacobson et al., 2005).

Comparing with previously published works, regarding spectrophotometric assays, this study found that the phenolic concentration of the S. adstringens leaf extract was higher than that of the bark extract, whereas in the study by Da Cruz et al. (2022), the total phenolic concentration of the bark was greater than that of the leaf extract, with values of 970.6 mg AGE/g and 693.8 mg AGE/g, respectively. Interestingly, in the research by Santos et al. (2002), the bark extract of S. adstringens exhibited a phenolic concentration of 195.15 mg AGE/g and a flavonoid concentration of 2.87 mg QE/g, both of which were lower than the values found in this study for the same organ. Furthermore, to emphasize the variations in the quantitative content of phenolics and flavonoids expected for S. adstringens, we cite the study by Formagio et al. (2018), which reported an average phenolic concentration of 1034.9 mg AGE/g in leaf extracts, and in the same study, the flavonoid content was determined to be 846.17 mg QE/g. Due to its high phenolic content, the antioxidant properties of S. adstringens are commonly explored (Sabino et al., 2018), as reported in the study by Baldivia (2018), where the “barbatimão” stem extract demonstrated an antioxidant percentage of 82.92% using the DPPH radical capture method, a value close to that found in this study (92.4%). In the study by Reis et al. (2022), the bark extract of S. adstringens was assessed for its antioxidant capacity using the DPPH free radical scavenging methodology, showing an antioxidant capacity of 72.2%, a value lower but relatively close to that reported in this article.

Interestingly, the variations in composition documented in the spectrophotometric dosage assays using the same standards, such as gallic acid and quercetin, can also be explained by the different methodologies applied during extraction, as well as by the conditions and quality of the sample, such as granularity, temperature and pressure, factors that influence the yield of biologically active molecules like phenols and their flavonoids (De Freitas et al., 2018). An interesting example to cite is the work by Reis et al. (2024), where different solvents were used to obtain crude extracts from the bark of S. adstringens. Notably, the results showed different concentrations of flavonoids and antioxidant activity, with the highest flavonoid content derived from the hydroethanolic extract and the methanolic extract exhibiting the highest antioxidant percentage.

Extracts from different vegetative parts of S. adstringens have already been explored for antibacterial and antifungal activity. Here, we highlight some results from previous studies and compare them with the findings of this research. In the study by Trevisan et al. (2020), the aqueous fraction of the S. adstringens extract showed a minimum inhibitory concentration (MIC) of 250 µg/mL for most evaluated S. aureus strains, except for strain ATCC 25923, which had an MIC of 125 µg/mL. Almeida et al. (2017) evaluated the antimicrobial activity of the crude bark extract of S. adstringens against E. coli and S. aureus, and, like this article, the authors determined a higher inhibitory potential against S. aureus. Furtado (2019) assessed the antimicrobial potential of the leaf extract of S. adstringens against S. aureus, E. coli, P. aeruginosa, Klebsiella pneumoniae, C. albicans, Candida tropicalis, and C. parapsilosis, finding greater antibacterial capacity against S. aureus and P. aeruginosa strains and lower activity against E. coli. Regarding antifungal activity, the Candida spp. strains showed the same susceptibility, with no statistical differences. In the study by Gonçalves et al. (2005), the antimicrobial capacity of the crude bark extract of S. adstringens was determined using the disk diffusion technique against clinical infection isolates, with species such as Streptococcus pyogenes (average inhibitory halos of 28 mm), Proteus mirabilis (30 mm), Shigella sonnei (30 mm), and S. aureus (20 mm). Audi et al. (2004) evaluated the antimicrobial activity of the crude extract of S. adstringens against E. coli, S. aureus, and P. aeruginosa, and at a concentration of 50 µg/mL (half of the concentration used for evaluation in this study), the extract exhibited inhibitory action only against S. aureus and P. aeruginosa, with inhibitory halos of 11 mm and 8 mm, respectively. In the study by Bueno et al. (2022), the extract of S. adstringens was effective against the following S. aureus strains: clinical isolates of the methicillin-resistant S. aureus MRSA strain, BEC-S. aureus (Brazilian Epidemic Clone of MRSA), and S. aureus ATCC 29213. It was also effective against another important gram-positive pathogen, Streptococcus pneumoniae type 3 ATCC 6303. Notably, we observe that most of the studies described above demonstrate some activity against S. aureus. The effective inhibitory activity of the extract against S. aureus reported in this article and in most of the evaluated studies is justified by the synergy between the concentration of phenols present in this plant and the composition of the cell wall of gram-positive bacteria, which are rich in peptidoglycan. The peptidoglycan molecules present in the cell wall of S. aureus are rapidly complexed and precipitated by the phenols, leading to loss of plasticity and function of the cell wall, and consequently, bacterial cell death (Daglia, 2012). Crude extracts comprise a pool of molecules. An alternative for future studies on the evaluation of the biological activity of compounds derived from S. adstringens would be the fractionation and screening of isolated molecules, as done in the study by De Freitas et al. (2018), which identified polymeric tannins of proanthocyanidins effective against clinical isolates of Candida species: C. guilliermondii, C. glabrata, C. albicans, C. parapsilosis, C. krusei, and C. tropicalis.

One of the reasons for evaluating the activity of the extracts against L. amazonensis was that previous works showed the extract of Stryphnodendron spp. exhibited antiparasitic activity against important trypanosomatids such as L. brasiliensis, L. infantum, and Trypanosoma cruzi (Ribeiro et al., 2015; Vandesmet et al., 2017). However, in this research, the extract evaluated at 100 µg/mL did not demonstrate inhibitory potential against L. amazonensis. The low efficacy of the S. adstringens extract against this protozoan may reinforce the potential and possible selectivity of the extract for prokaryotic organisms, which is well-documented in the literature. Interestingly, the extract also shows activity against yeasts (eukaryotes). However, considering the mechanism of action of phenolic molecules against the rigid cell walls of bacteria, we must consider that yeasts also possess this rigid structure, while protozoa do not, which may be associated with the extract's activity against the evaluated yeast strains and the lack of activity against L. amazonensis.

In summary, when assessed in macrophages derived from THP-1 cells, the extracts showed no significant cytotoxic effects, with the concentration capable of causing death of 50% of viable cells being 150.15 µg/mL. This concentration is higher than that employed in biological assays, indicating that the extracts exhibit activity against important infectious agents such as S. aureus, M. tuberculosis, and C. albicans at a concentration lower than the cytotoxic concentration. Moreover, at this same concentration, all extracts demonstrated antioxidant activity exceeding 80%. These results provide intriguing insights regarding the use of compounds derived from this plant for the treatment of infectious diseases, such as staphylococcal infections and tuberculosis. Additionally, their antioxidant potential opens avenues for combating the production of oxidizing radicals associated with degenerative diseases and cancer (Roleira et al., 2015, Sabino et al., 2018). Considering these findings, we emphasize the continuation of investigations into the biological potential of this plant. It is conceivable that the use of these extracts for screening and fractionation may lead to the discovery of new compounds with biological activity.

5. Conclusion

The results presented indicate that the vegetative organs of the specie S. adstringens have potential as a source of compounds with antimicrobial activity, particularly against prokaryotes, such as gram-positive bacteria. Furthermore, they are notable for their high antioxidant capacity and low cytotoxicity. We recommend further investigations into the biological activity of S. adstringens through the fractionation, screening, and isolation of the chemical constituents from its crude extracts. Given its low cytotoxicity, along with its antimicrobial and antioxidant activities, the biologically active molecules isolated from this plant may be explored in the development of drugs for the treatment of bacterial and fungal infectious diseases. Additionally, they may also prove useful in combating diseases such as cancer and neurodegenerative disorders, which are associated with cellular and DNA damage caused by oxidative stress.

Acknowledgments

We would like to acknowledge São Paulo State University “Júlio de Mesquita Filho” (UNESP), University of Vale of Sapucaí (UNIVAS), and BEEOTEC S/A for their support.

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Publication Dates

• Publication in this collection
  31 Jan 2025
• Date of issue
  2024

History

• Received
  21 May 2024
• Accepted
  04 Nov 2024