Bioactive potential of plant extract of Abarema cochliacarpos Barneby & J.W. Grimes for oral health

Potencial bioativo do extrato vegetal de Abarema cochliacarpos Barneby & J.W. Grimes para saúde bucal

Abstract

Biotechnological advances have enabled the use of plant-derived secondary metabolites in innovative therapies, with growing applications in dentistry. Abarema cochliacarpos ("barbatimão"), a species with notable medicinal properties, is a promising candidate for antioxidant and antimicrobial formulations aimed at oral health. This study aimed to characterize the phytochemical profile of the ethanolic extract from A. cochliacarpos stem bark and its fractions (hexane, ethyl acetate, and hydromethanol) and to evaluate their antioxidant, antimicrobial, and antibiofilm activities against oral microorganisms. The phytochemical analysis of the ethanolic extract was carried out by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antioxidant capacity of the extract and its fractions was assessed by total phenolic content and DPPH•/ABTS•+ assays. Antimicrobial activities were evaluated against mixed oral cultures (MOC) through agar well diffusion, minimum inhibitory concentration (MIC) and anti-quorum sensing assays. Among all samples, the hydromethanol fraction, rich in 3,7-dimethylundecane and methyl benzoate, exhibited the most potent activity, showing significant antioxidant and antimicrobial effects (10 mm inhibition zone at 10 μg/mL, MIC = 5 μg/mL) while also inhibiting biofilm formation at a concentration of 12.5 μg/mL. The hydromethanol fraction of A. cochliacarpos demonstrated antimicrobial and antioxidant effects, supporting its potential for pharmaceutical development in oral care.

Keywords:
phytochemical profile; oral microorganisms; bacterial biofilm; dental care

Resumo

Avanços na biotecnologia possibilitaram terapias inovadoras utilizando metabólitos secundários derivados de plantas, com aplicações crescentes na odontologia para tratamentos orais. Abarema cochliacarpos ("barbatimão") apresenta notáveis propriedades medicinais, mostrando-se promissora para formulações antioxidantes e antimicrobianas voltadas à saúde bucal. Este estudo teve como objetivo caracterizar o perfil fitoquímico e avaliar os efeitos antioxidante, antimicrobiano e antibiofilme do extrato etanólico bruto da casca do tronco de A. cochliacarpos e suas frações hexânica, acetato de etila e hidrometanólica, frente a microrganismos orais. A análise fitoquímica do extrato etanólico foi realizada por espectrometria de massas por dessorção/ionização a laser assistida por matriz com analisador de tempo de voo (MALDI-TOF MS). Tanto o extrato quanto suas frações foram submetidos à avaliação da atividade antioxidante, que incluiu a determinação do teor de fenólicos totais e os ensaios de DPPH• e ABTS•+. Os efeitos antimicrobianos foram testados por difusão em ágar, concentração inibitória mínima (CIM) e ensaio de antiquorum sensing contra culturas orais mistas (MOC). A fração hidrometanólica (rica em 3,7-dimetilundecano e benzoato de metila) apresentou a maior atividade antioxidante e antimicrobiana (zona de inibição de 10 mm a 10 μg/mL; CIM = 5 μg/mL). Essa fração inibiu a formação de biofilme a 12,5 μg/mL. A fração hidrometanólica de A. cochliacarpos demonstrou efeitos antimicrobianos e antioxidantes, sustentando seu potencial para o desenvolvimento farmacêutico na área de cuidados orais.

Palavras-chave:
perfil fitoquímico; microrganismo oral; biofilme bacteriano; saúde bucal

1. Introduction

The increasing prevalence of oral diseases, associated with microbial biofilms and chronic inflammation, represents a significant challenge for modern dentistry. Conventional treatments, such as antibiotics and anti-inflammatory drugs, often present limitations, including microbial resistance, adverse effects, and low efficacy against established biofilms (Tewari et al., 2021; Farias et al., 2021). In Brazil, a country of remarkable biodiversity, medicinal plants have attracted growing scientific interest as potential alternatives for combating oral pathogens and inflammatory processes (Ellwanger et al., 2022).

The Caatinga biome stands out as one of Brazil’s most unique ecosystems, whose biodiversity has drawn increasing scientific attention, especially for its relevance in facing microbial resistance. A recent review highlighted the potential of natural products from the Caatinga in combating multidrug-resistant bacteria, including species such as Abarema cochliacarpos, reinforcing its importance as a promising source of bioactive compounds (Souza et al., 2024). Furthermore, studies demonstrate that secondary metabolites extracted from living species of the Caatinga from Caatinga species exhibit enhanced activity against pathogenic agents. (Silva et al., 2024).

Among the promising species is Abarema cochliacarpos (Gomes) Barneby & J.W. Grimes, a medium-sized tree of the Fabaceae family, popularly known as “barbatimão” or “bordão-de-velho” (Souza et al., 2024). Traditionally used for wound healing and inflammatory processes, its pharmacological potential has been attributed to phenolic compounds such as condensed tannins. (Aguiar et al., 2021).

Therefore, the present study aimed to characterize the phytochemical profile of A. cochliacarpos trunk bark extracts and evaluate their antioxidant, antimicrobial, and antibiofilm activities against microorganisms associated with oral health, in order to explore their potential for the development of innovative therapeutic formulations in dentistry.

2. Materials and Methods

2.1. Extract preparation - obtaining the crude extract, partitioning and standardization of A. cochliacarpos bark extracts

The bark of A. cochliacarpos (Gomes) Barneby & J.W. Grimes (Fabaceae) was provided by the Institute of Agronomy of Pernambuco (IPA), Pernambuco, Brazil (geographical coordinates: 8°03’57”S, 34°55’29”W; voucher number: 192889; identified by F. Gallindo). The material was oven-dried at 40 °C for 7 days, then grounded using a knife mill. The ethanolic extract was obtained by maceration of the bark in ethanol at a 1:10 (w/v) ratio, followed by solvent removal through rotary evaporation and dried in an oven at 45 °C for seven days. The crude extract was fractionated sequentially with solvents of increasing polarity (Oubannin et al., 2024): 200 mL of methanol-water (1:1, v/v), followed by 100 mL of hexane, and then 100 mL of ethyl acetate (1:1, v/v). Each resulting fraction was concentrated by rotary evaporation, dried in an oven at 45 °C for 7 days, and stored at -20 °C. Only the ethanolic extract was used for the phytochemical characterization, while the fractions were used in all subsequent assays.

2.2. Determination of total phenolic content

The quantification of total phenolic compounds was carried out using the Folin-Ciocalteau spectrophotometric method. (Wołosiak et al., 2021). For the assay, 100 μg/mL of the extract, 820 μL of distilled water and 20 μL of the Folin-Ciocalteu reagent were used. Next, 60 μL of 15% sodium carbonate were added and left to rest for 2h, protected from light. After the established time for the reaction, the ABS was read on a spectrophotometer (Asys Hitech UVM 340) at 760 nm. The content of total phenolic compounds in the extract was quantified using an external calibration curve equation (from the gallic acid standard), and the results were expressed as milligrams gallic acid equivalents per gram (mg GAE/g) dry weight of the sample, considering the standard deviation. The entire test determination procedure was carried out in triplicate.

2.3. DPPH• free radical scavenging activity

The antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay, following established protocols with adaptations (Wołosiak et al., 2021). The extracts were dissolved in ethanol to obtain final concentrations ranging from 1.0 to 10 μg/mL. The DPPH• solution was added, and mixtures were incubated under constant agitation for 30 min, in the absence of light and at room temperature. The solutions were transferred to 96-well microplates, and absorbance was measured at 517 ηm using a spectrophotometer. All assays were performed in triplicate. Negative control (DPPH solution + ethanol), blank (ethanol only), and positive control (DPPH solution + ascorbic acid) were included to ensure the reliability of the results and to account for any potential interference of the solvent in absorbance measurements. The percentage of scavenging activity (AS%) was calculated using the Equation 1:

$$AS\%=100x\left[\left(Abs\_control–Abs\_sample\right)/Abs\_control\right]$$ (1)

2.4. Assessment of the scavenging activity of the ABTS+ radical cation

The ABTS assay was performed following established protocols with minor modifications (Wołosiak et al., 2021). The ABTS+ radical cation was generated by reacting 7 mM ABTS with 140 mM potassium persulfate in distilled water. The mixture was kept in dark at room temperature for 12-16 h to ensure complete radical formation. The ABTS+ solution was diluted in ethanol (approximately 1:100, v/v) until an absorbance of 0.7 ± 0.05 was reached at 734 ηm. The extracts were tested at final concentrations ranging from 1.0 to 12 μg/mL by mixing the ABTS+ solution, adjusting to a final volume of 500 μL. After incubation for 6 min in an ultrasonic bath, protected from light and at room temperature, absorbance was measured at 734 ηm using a spectrophotometer. All assays were performed in triplicate. Trolox (6-hydroxy-2,5,7,8 tetramethylchrome-2-carboxylic acid) was used as positive control. Negative control (ABTS solution + ethanol), blank (ethanol only), and positive control (ABTS solution + Trolox) were included to ensure the reliability of the results and to account for any potential interference of the solvent in absorbance measurements. The percentage of scavenging activity (AS%) was calculated using the same equation described above.

2.5. Phytochemical analysis

The phytochemical characterization of the ethanolic extract from the trunk bark of A. cochliacarpos was carried out using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The analyses were performed on a Bruker UltrafleXtreme mass spectrometer (Bruker Daltonics). For sample preparation, an ethanolic extract rich in procyanidins was solubilized in distilled water and mixed with the matrix 2,5-dihydroxibenzoic acid (DHB) prepared in a 0.1M NaCl solution, using a 1:10:1 ratio (sample:matrix:salt). The resulting mixture was spotted onto the MALDI plate and allowed to dry at room temperature prior to analysis.

2.6. Isolation of microorganisms

Mixed oral cultures were obtained from the dental surface and back of the tongue of a healthy volunteer, previously selected within the eligibility criteria approved by the Human Research Ethics Committee (CAAE: 11397219.4.0000.5207). Mixed cultures from the oral cavity (MOC) were inoculated into Brain Heart Infusion (BHI) broth and placed in an incubator at 37 °C for 24h. After initial growth, the cultures were streaked in duplicate on Nutrient agar, Müeller- Hinton agar (MH), Blood agar, Mannitol agar and Eosin Methylene Blue agar (EMB) and incubated at 37 °C for 24h for isolation of pure colonies. Isolated colonies were then selected and preserved in BHI with 25% glycerol at −80 °C.

For molecular identification of microorganisms, bacterial genomic DNA was extracted and subjected to PCR amplification of the 16S rDNA gene using universal primers 8F and U1492R, following established molecular protocols for bacterial identification (Farias et al., 2021). The PCR products were sequenced on the LABCEN/CCB platform at the Federal University of Pernambuco using the ABI 3500 sequencer. The sequences obtained were submitted to the EzBioCloud database for identification of bacterial isolates.

2.7. Antimicrobial activity

The well diffusion assay was performed in accordance with Clinical and Laboratory Standards Institute (CLSI), document M100 (2019), to evaluate the antimicrobial activity of the plant extracts against the MOC described in the previous section. The powdered ethanolic extract was dissolved in sterile distilled water at concentrations of 50 mg/mL and 25 mg/mL, while the powdered hexane, ethyl acetate, and hydromethanol fractions were reconstituted at concentrations of 25 mg/mL and 10 mg/mL. The MOC inoculum was adjusted to the 0.5 McFarland scale (~1.5 x 10⁸ CFU/mL) in 1 mL of BHI broth and uniformly spread over the surface of Mueller-Hinton agar plates. Wells of 6 mm in diameter were aseptically punched into the agar, and 30 μL of each extract or fraction were dispensed into the wells. Chlorhexidine (0.12%) was used as a positive control. The plates were incubated at 37 °C for 24h, and the inhibition zones were measured using a caliper.

2.8. Minimum Inhibitory Concentration (MIC)

The MIC was determined using the microdilution method (CLSI, 2019), applied to the MOC. Extract and fractions were solubilized in Tryptone Soy Broth (TSB) at concentrations of 50, 25, 12.5, 6.25, 3.12, and 1.56 μg/mL. In 96-well microplates, each well received 100 μL of extract solution, 90 μL of BHI broth, and 10 μL of MOC (0.5 McFarland, ~1.5 x 10⁸ CFU/mL). Chlorhexidine (0.12%) was used as a positive control, and BHI with inoculum served as the negative control. Plates were incubated at 35 °C for 24h. After incubation, 30 μL of 0.02% resazurin solution was added to each well and plates were reincubated at 37 °C for 2 h. Color change indicated microbial viability. All tests were performed in triplicate.

2.9. Anti-quorum sensing activity

This assay was performed following the protocol described by Stepanović et al. (2007). The hydromethanol fraction was solubilized in TSB to obtain final concentrations of 50, 25, 12.5, 6.25, 3.12, and 1.56 μg/mL. The MOC was reactivated in TSB and adjusted to the 0.5 McFarland scale (~1.5 x 10⁸ CFU/mL). In 96-well microplates, each well received 90 μL of BHI broth, 100 μL of the plant extracts, and 10 μL of the MOC suspension. BHI broth with inoculum was used as the positive control. Plates were incubated at 37 °C for 24h, and optical density (OD) was measured at 600 ηm using a microplate reader. After incubation, the wells were washed with 0.9% NaCl to remove non-adherent cells and dried at 55 °C for 1h. Biofilms were then stained with 0.4% violet crystal for 15 min, followed by washing under running water. Subsequently, 200 μL of 99% ethanol was added to each well and incubated for 30 min to solubilize the dye. Final absorbance was measured at 570 ηm. All assays were performed in triplicate.

2.10. Statistical analyses

Statistical analysis initially involved verifying the assumptions of normality, homoscedasticity, and independence using the Shapiro-Wilk, Levene, and Chi-square tests, respectively. Since these assumptions were met, Analysis of Variance (ANOVA) was performed to investigate the presence of significant differences among means and the influence of factors on the dependent variable. To identify significant differences between group means, Tukey's post-hoc test was applied. All analyses were conducted using R software (version 4.4.1).

3. Results and Discussion

3.1. Yield percentage of ethanolic extract and derived fractions

From the bark of A. cochliacarpos, 33.75 g of ethanolic extract was obtained. From the fractionation process of the ethanolic extract, 1.65 g of the hexanic fraction, 1.34 g of the ethyl acetate fraction, and 6.89 g of the hydromethanol fraction were obtained. The yield percentages were calculated based on the initial mass of the ethanolic extract (Table 1).

Table 1
Yield information for the ethanolic extract and fractions from the bark of Abarema cochliacarpos.

Extract/Fraction	Obtained Mass (g)	Yield (%) *	Relative Yield (%) **
Ethanolic extract	33.75	-	100
Hexanic fraction	1.65	4.89	4.89
Ethyl acetate fraction	1.34	3.97	3.97
Hydromethanol fraction	6.89	20.42	20.42

• ^*
  Yield calculated based on the initial mass of the ethanolic extract;
• ^**
  Relative yield represents the percentage of each fraction in relation to the total ethanolic extract.

The fractionation process resulted in a total recovery of 29.28% of the initial mass of the ethanolic extract, with the hydromethanol fraction representing the highest yield (20.42%), followed by the hexanic fraction (4.89%) and the ethyl acetate fraction (3.97%). It is noted that 70.72% was not recovered in the fractions, likely because these are compounds that remained in the aqueous phase or were lost during the partitioning procedures. The yield of the hydromethanol fraction suggests that A. cochliacarpos bark predominantly contains polar compounds, which is consistent with the high content of phenolic compounds and tannins identified in the phytochemical analysis.

The yield of the hydromethanol fraction indicates that A. cochliacarpos bark contains predominantly polar compounds, which is consistent with the phenolic and tannin content found in the phytochemical assay. These findings are in line with established solvent extraction principles, in which the amount of extracted phenolic compounds increases with increasing solvent polarity (Nawaz et al., 2020). Methanol and water solutions are effective for extracting tannins, as these are polar compounds that dissolve preferentially in polar solvents (Abda et al., 2025). The predominance of polar compounds in the hydromethanol fraction is further corroborated by studies demonstrating that hydroethanolic extracts primarily concentrate polar compounds, such as polyphenols (Dieng et al., 2020).

3.2. Antioxidant potential of ethanolic extract and fractions of A. cochliacarpos

The total phenolic compounds measured in the extracts showed values of 181.28 ± 1.27 mg GAE/g for the ethanolic extract. The result obtained with the hydromethanol fraction is noteworthy for presenting the highest phenolic content with 196.30 ± 1.51 mg GAE/g (Table 2). In the DPPH and ABTS assays, all fractions showed EC50 values below 50 μg/mL, which can be considered a relevant result.

Table 2
Total phenolic content and antioxidant activity of the crude ethanolic extract and derived fractions from Abarema cochliacarpos bark.

Samples	TP ± SD (mg of GAE/g)	DPPH• EC50 ± SD (μg/mL)	ABTS+ EC50 ± SD (μg/mL)
Ethanolic extract	181.28 ± 1.27	5.49 ± 0.20	0.09 ± 0.32
Hexane fraction	157.51 ± 0.92	2.63 ± 0.13	1.21 ± 0.35
Ethyl acetate fraction	186.58 ± 1.47	4.53 ± 0.26	1.26 ± 0.43
Hydromethanol fraction	196.30 ± 1.51	5.36 ± 0.16	0.03 ± 0.24
Ascorbic acid (control)	-	4.16 ± 0.22	-
Trolox (positive control)	-	-	3.065 ± 0.16

• TP = total phenolics; mg GAE/g = milligrams gallic acid equivalents per gram; DPPH• = 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); EC₅₀ = effective concentration 50%; SD = standard deviation.

Although the hydromethanol fraction exhibited the highest total phenolic content (196.30 ± 1.51 mg GAE/g), its DPPH radical scavenging activity (EC50 = 5.36 ± 0.16 μg/mL) was lower than that observed for the hexanic fraction (EC50 = 2.63 ± 0.13 μg/mL), which contained a smaller amount of total phenolics (157.51 ± 0.92 mg GAE/g). This apparent discrepancy is justified because the Folin-Ciocalteu method quantifies the total content of phenolic substances without distinguishing between classes of phenolic compounds (Wołosiak et al., 2021). Free flavonoids, condensed tannins, phenolic acids, and proanthocyanidins demonstrate variable electron-donating capacities due to their structural differences, particularly in the positioning of hydroxyl groups and conjugation patterns (Cosme et al., 2025).

The hexanic fraction, due to its nonpolar nature, selectively concentrates lipophilic antioxidants such as tocopherols, methylated flavonoids, or other liposoluble phenolic compounds known for their high radical scavenging efficiency (Huang et al., 2024). In contrast, the hydromethanol fraction, which showed a considerable value of total phenolic compounds, predominantly extracted hydrophilic tannins and glycosylated flavonoids which, despite being abundant, may exhibit lower individual antioxidant efficiency per molecule (Angelini, 2024).

Previous studies demonstrate that antioxidant activity is not linearly related to the total phenolic content, being more influenced by the specific chemical structure of the present compounds (Cosme et al., 2025). The structural characteristics of phenolic compounds, including the position of hydroxyl groups, significantly influence their capacity for free radical stabilization and electron-donating properties (Dieng et al., 2020). It is also necessary to emphasize that the range of bioactive compounds in each fraction may exhibit synergistic or antagonistic interactions that influence the overall antioxidant capacity, a phenomenon well-documented in studies of complex plant extracts (Wołosiak et al., 2021).

3.3. Phytochemical analysis

The bioactive extract from A. cochliacarpos consists of two polymeric series (A, B, and C) rich in epicatechin/catechin monomer units as demonstrated by a distance of 288 Da (Table 3, Figure 1). This revealed that the main structural unit of A. cochliacarpos proanthocyanidins was epicatechin/catechin.

Table 3
Composition of peaks based on MALDI-TOF MS analysis of the ethanolic extract of Abarema cochliacarpos.

Ethanolic extract	[M+H]+	[M+Na]+	Composition a
Series A	697	721	(epi)afz-A-(epi)galloafz-A
	985	1009	(epi)afz-A-(epi)galloafz-A+(epi)cat
	1273	1297	(epi)afz-A-(epi)galloafz-A+(epi)cat+(epi)cat
	1561	1585	(epi)afz-A-(epi)galloafz-A+(epi)cat+(epi)cat+(epi)cat
	1849	1873	(epi)afz-A-(epi)galloafz-A+(epi)cat+(epi)cat+(epi)cat+(epi)cat
Series B	845	-	(epi)afz+(epi)cat+(epi)cat
	1133	-	(epi)afz+(epi)cat+(epi)cat+(epi)cat
	1421	-	(epi)afz+(epi)cat+(epi)cat+(epi)cat+(epi)cat
	1709	-	(epi)afz+(epi)cat+(epi)cat+(epi)cat+(epi)cat+(epi)cat
Series C	829	-	(epi)cat+(epi)afz+(epi)afz
	1117	-	(epi)cat+(epi)afz+(epi)afz+(epi)afz
	1405	-	(epi)cat+(epi)afz+(epi)afz+(epi)afz+(epi)afz
	1693	-	(epi)cat+(epi)afz+(epi)afz+(epi)afz+(epi)afz+(epi)afz

• ^a
  A = double (A-type) bond; (epi)cat = (epi)catechin; (epi)afz = (epi)afzelechin; (epi)galafz = (epi)galloafzelechin.

Figure 1
MALDI-TOF positive ion mass spectrum of condensed tannins from ethanolic extract of Abarema cochliacarpos.

In Figure 1, a series of ions corresponding to sodium adducts [M+Na]+ of procyanidin oligomers were observed, from the dimer (m/z 697) up to the hexamer (m/z 1847). A tentative structure of this hexamer (series A) is a dimer as starter unit (m/z 697) characterized as (epi)afzelechin-A-(epi)galloafzelechin-A, in which A represents an A-linkage. The spectrum is simple in appearance and the results obtained indicate a series of peaks with distances of 288 u corresponding to a mass difference of one catechin/ epicatechin between each polymer.

Series B consists of the A-type procyanidin hexamers with (m/z 1713) A-linkages were detected in the extract. The starter unit is identified as trimer (m/z 845) consisting of one (epi)afzelechin unit, two (epi)catechin units with A-linkages. This trimer is linked to repeat units of (epi)catechin drawn based on the product ions detected, indicating a series of peaks with distances of 288 u corresponding to a mass difference of one catechin/ epicatechin between each Polymer. These masses had been identified as heteropolymers of repetitive flavan-3-ol units, which indicated the presence also of epiafzelechin/afzelechin in other oligomers.

These findings demonstrated the coexistence of procyanidin and propelargonidin in A. cochliacarpos proanthocyanidins. These results demonstrated structural heterogeneity of proanthocyanidins in the A. cochliacarpos.

Previous study report the presence of phenolic compounds, such as phenolic acids, flavonoids and, mainly, hydrolysable and condensed tannins (proanthocyanidins, prodelphinidins, prorobinetinidins) in “barbatimão” (Fabaceae) extracts (Keivani et al., 2024), corroborating the results obtained.

In a preliminary phytochemical analysis of A. cochliacarpos, the presence of tannins, phenols, anthraquinones, catechins, proanthocyanidins, leucoanthocyanidins and flavonoids was reported (Alves et al., 2022).

Due to its chemical composition, the species of A. cochliacarpos presents activities such as gastroprotective, healing, anti-inflammatory (Alves et al., 2022), antioxidant (Farias et al., 2021), antimicrobial (Santos et al., 2022).

3.4. Assessment of inhibition halos and Minimum Inhibitory Concentration (MIC) of A. cochliacarpos extract

In order to characterize the mixed oral cavity culture used in antimicrobial protocols, 16 samples were sent for genomic DNA investigation. The microorganisms were identified as Streptococcus mitis, Streptococcus parasanguinis, Staphylococcus argenteus, Streptococcus salivarius, Staphylococcus epidermidis, Streptococcus oralis subsp. Oralis, Streptococcus sp.

The evaluation of the inhibition halos of the MOC revealed antimicrobial activity for the 4 extracts from the trunk bark of A. cochliacarpos. The ethanolic extract and the hydromethanol fraction presented the most significant halos when compared to the other fractions (Table 4). The ethanolic extract from the trunk bark of A. cochliacarpos was able to inhibit the growth of the mixed culture tested, showing better results at a concentration of 50 mg/mL.

Table 4
Inhibition zone diameters of a mixed oral microbiota culture exposed to the ethanol extract and derived fractions of Abarema cochliacarpos.

Samples	Concentrations	Average (mm) ± SD	Lower CI (mm)	Upper CI (mm)
Ethanolic extract	50 mg/mL 25 mg/mL	12.67 ± 0.50 11.67 ± 0.50	11.43 10.43	13.91 12.91
Hexane fraction	25 mg/mL 10 mg/mL	10.16 ± 0.33 9.44 ± 0.53	9.34 8.12	10.98 10.76
Ethyl acetate fraction	25 mg/mL 10 mg/mL	9.89 ± 0.33 8.78 ± 0.67	9.07 7.12	10.71 10.44
Hydromethanol fraction	25 mg/mL 10 mg/mL	11.11 ± 0.78 9.89 ± 0.33	9.17 9.07	13.05 10.71
Chlorhexidine (positive control)	0.12%	20.89 ± 1.76	16.52	25.26

• SD = Standard deviation. The 95% CI was calculated using a critical t-value of 4.303 for n=3.

To further evaluate the differences in antimicrobial efficacy presented in Table 4, a statistical inference was performed by calculating the 95% confidence intervals (CI) for the mean inhibition zones. This analysis allows for a more robust comparison, where non-overlapping Cis between two groups suggest a statistically significant difference in their activity. The analysis reveals that the ethanolic extract at 50 mg/mL (CI: 11.43–13.91 mm) demonstrated a significantly higher antimicrobial activity compared to the hexane and ethyl acetate fractions at 10 mg/mL (Cis: 8.21–10.67 mm and 7.21–10.35 mm, respectively), as their confidence intervals do not overlap. In contrast, several groups showed comparable efficacy; for instance, the Cis for the hexane fraction at 25 mg/mL (9.36–10.96 mm) and the hydromethanol fraction at 10 mg/mL (9.09–10.69 mm) overlap, indicating that the observed difference in their mean inhibition zones is not statistically significant. As expected, the positive control, chlorhexidine (CI: 16.68–25.10 mm), was significantly more effective than all tested plant extracts, with its CI not overlapping with any other group. This detailed analysis provides clearer evidence of a dose-dependent effect for the ethanolic extract and highlights the superior performance of the hydromethanol fraction over the hexane and ethyl acetate fractions at equivalent concentrations.

The use of relatively high concentrations (10 to 50 mg/mL) in the agar diffusion assays was based on preliminary findings indicating limited antimicrobial activity at lower concentrations. It is important to emphasize that, given the crude nature of the plant extract and the chemically complex profile of its fractions, higher concentrations are often required during initial bioactivity screenings, as the bioactive constituents may be present at low relative abundance. However, elevated concentrations may induce nonspecific toxicity effects that could mask or overlap with genuine antimicrobial activity, and thus the results should be interpreted with caution, taking this methodological limitation into consideration (Pellenz et al., 2018).

Using gram-positive and gram-negative strains Tenório et al. (2016) performed antimicrobial activities using extract and fractions of A. cochliacarpos, demonstrating that the strains were sensitive to the extract as much as the present study. Santos et al. (2022) observed in vitro antibacterial activity of cyclohexane, acetonic and ethanolic extracts from the bark of A. cochliacarpos against Staphylococcus intermedius, at all concentrations tested (100, 50, 25, 12.5 and 6.25 mg/mL), except for the cyclohexane extract, which did not demonstrate inhibition at concentrations of 12.5 and 6.25 mg/mL.

The MIC against MOC was 12.5 μg/mL for ethanol extract (OD 1.536 ± 0.078) and hydromethanol fraction (OD 1.818 ± 0.064). The fractions of hexane and ethyl acetate obtained a MIC of 25 μg/mL with OD 1.709 ± 0.091 and OD 2.581 ± 0.055, respectively. Tenório et al. (2016), using cyclohexane, acetone and ethanolic extracts from this same species, observed that the extracts were not inhibitory at concentrations lower than 10 μg/mL (MIC > 10 μg/mL).

Such results may be due to the chemical structure of flavonoids, which can interact with cell membranes in different ways, such as penetrating the nonpolar nucleus of the bacterial cell membrane or promoting hydrogen bonds between hydrophilic flavonoids and polar groups of lipids or even reducing the fluidity of the outer and inner layers of the bacterial cell membrane (Zhang et al., 2025).

Previous studies have shown the relationship of phenolic compounds and the antimicrobial potential in different strains (Kauffmann and Castro, 2023). Nisa et al. (2024) in a study with ethyl acetate fraction of Anacardium occidentale presents an important correlation between the presence of phenolic compounds and flavonoids and antimicrobial activity and states that phenolic compounds, due to their versatile structure, allow a wide range of chemical additions that inhibit microbial activity, pointing out that one of the causes is interference in the synthesis of the bacterial cell wall, DNA replication and enzyme production. Regarding the presence of catechins in the extract, he states that studies have explored the antimicrobial potential of catechins from green tea, pointing out the action of epicatechins on both gram positive and negative bacteria. This antimicrobial effect can be attributed to the different chemical interactions between epicatechins and cellular components such as genes, cell membrane and enzymes (Zhang et al., 2025).

3.5. Anti-quorum sensing potential

A biofilm can be defined as a group of associated microorganisms compressed into a matrix composed of different polysaccharides, proteins and DNA, with quorum sensing being an important communication channel in the development of the biofilm and its surrounding extracellular matrix (Shamim et al., 2023).

For all mixed culture samples from the oral cavity and isolates therefrom, the concentration capable of completely inhibiting biofilm formation was 12.5 μg/mL (Table 5).

Table 5
Anti-quorum sensing activity of Abarema cochliacarpos against mixed oral cavity culture.

	Concentration (µg/mL)
	50	25	12.5	6.25	3.12	1.56
Ethanol extract	PBP 0.157	PBP 0.121	PBP 0.225	MBP 0.449	MBP 0.455	SBP 0.525
Hexane fraction	NBP 0.102	NBP 0.087	PBP 0.175	MBP 0.405	MBP 0.420	MBP 0.440
Ethyl acetate fraction	NBP 0.111	NBP 0.110	PBP 0.149	MBP 0.458	MBP 0.453	SBP 0.716
Hydromethanol fraction	PBP 0.142	PBP 0.173	PBP 0.146	MBP 0.279	MBP 0.375	MBP 0.350

• NBP = non-biofilm producer; PBP = poor biofilm producer; MBP = moderate biofilm producer; SBP = strong biofilm producer.

Although the extracts demonstrated inhibitory activity on biofilm formation, particularly at higher concentrations, this study did not include specific cytotoxicity assays to determine whether such inhibition is due to bactericidal effects or targeted interference in quorum sensing mechanisms. Therefore, the possibility that the anti-quorum sensing effect is partially masked by general toxicity cannot be excluded. This limitation underscores the importance of complementary assays in future studies to distinguish nonspecific antimicrobial activity from true quorum sensing inhibition.

Nonetheless, literature reports validate the antibiofilm action of various plant-derived secondary metabolites, such as phloretin (phenolic compound) acting on efflux protein genes (Grooters et al., 2024) and vitexin (flavonoid) (Carević et al., 2024), although this study did not isolate or quantify specific compounds such as phoretin and vitexin, the literature validates the anti-biofilm action of secondary metabolites belonging to flavonoid classes, reinforcing the relevance of findings.

Table 6 presents the results of the Analysis of Variance (ANOVA) for biofilm inhibition, considering ethanolic, hexane, ethyl acetate, and hydromethanol solutions. In all analyses, concentration proved to be a highly significant factor in biofilm inhibition (p < 0.001). These results indicate that, regardless of the type of solution used, the concentration of the substances is a determining factor in their ability to inhibit biofilm formation, with all p-values significantly lower than 0.001 (indicated by ***), corroborating the strong influence of concentration on the inhibitory effect.

Table 6
Analysis of Variance (ANOVA) results of biofilm inhibition considering the different fractions of Abarema cochliacarpos extract.

Sample	Source of Variation	Df	Sum SQ	Mean SQ	Fvalue	p-value
Ethanolic extract	Concentration	5	0.46789	0.093577	164.01	1.295 e-10***
	Residuals	12	0.00685	0.000571
Hexane fraction	Concentration	5	0.42131	0.084263	304.93	3.292 e-12***
	Residuals	12	0.00332	0.000276
Ethyl Acetate fraction	Concentration	5	0.94810	0.189619	185.32	6.3 e-11***
	Residuals	12	0.01228	0.001023
Hydromethanol fraction	Concentration	5	0.164063	0.032813	49.059	1.419 e-7***
	Residuals	12	0.008026	0.000669

• ^***
  p-value < 0.001.

Studies investigating the action of catechins and epicatechins have demonstrated their effectiveness against biofilm-forming bacteria, through the reduction of virulence factors in quorum sensing systems of Pseudomonas aeruginosa (Carević et al., 2024). These compounds have shown promising results against multi-resistant bacteria, with catechins demonstrating the ability to interfere with bacterial resistance mechanisms. Research indicates that catechins can inhibit bacterial toxins by binding to the plasma membrane, causing decreased cell permeability and blocking signal transduction processes, which contributes to their antimicrobial efficacy (Zhang et al., 2025).

Tukey’s test was applied to identify significant differences between the means of biofilm inhibition at different concentrations of the ethanolic, hexane, ethyl acetate, and hydromethanol solutions, considering a significance level of 5% (α=0.05). The results are presented in terms of p-values, where values less than 0.05 indicate a statistically significant difference.

For the Ethanol solution, most comparisons resulted in p-values of 0.0000, indicating highly significant differences. This suggests that the biofilm inhibition capacity varies considerably among the different tested concentrations of ethanol. Notable exceptions include the comparison between 3.12 vs. 1.56 (p=0.0339) and 6.25 vs. 1.56 (p=0.0214), which are still significant but with a slightly higher p-value. The results between 50 vs. 25 (p=0.2442) and 6.25 vs. 3.12 (p=0.9997) showed no significant difference, suggesting that in these concentration ranges, the inhibitory effect is similar.

In the Hexane solution, a similar pattern was observed, with p-values of 0.0000, indicating significant differences between most concentrations. However, some comparisons, such as 3.12 vs. 1.56 (p=0.6718), 6.25 vs. 1.56 (p=0.1763), 50 vs. 25 (p=0.8600), and 6.25 vs. 3.12 (p=0.8800), showed p-values above 0.05, indicating no statistically significant differences in these concentrations for biofilm inhibition with Hexane.

For the Ethyl Acetate solution, most comparisons resulted in low p-values (0.00000 to 0.00004), highlighting significant differences between concentrations in biofilm inhibition. However, for some results no significant difference was observed: 25 vs. 12.5 (p=0.6746), 50 vs. 12.5 (p=0.6962), 50 vs. 25 (p=1.0000), and 6.25 vs. 3.12 (p=0.8463). This suggests that for these pairs of concentrations, Ethyl Acetate exerts a comparable biofilm inhibitory effect.

The results for the Hydromethanol solution also showed significant differences in many concentration comparisons, with p-values ranging from very low (0.0000061) to higher values. However, the comparisons between 3.12 vs. 1.56 (p=0.8289), 25 vs. 12.5 (p=0.7827), 50 vs. 12.5 (p=0.9999), and 50 vs. 25 (p=0.6799) indicate that in these ranges, the concentrations of Hydromethanol do not differ statistically in their biofilm inhibition capacity.

4. Conclusions

The flavonoid-rich bark extracts of A. cochliacarpos, particularly the hydromethanol fraction, exhibit significant antioxidant and antimicrobial activities against a mixed oral microbiota. Additionally, all fractions effectively inhibited biofilm formation at this same concentration, suggesting anti-quorum sensing potential. These findings support the applicability of A. cochliacarpos as a promising natural source of bioactive compounds for oral health.

Acknowledgements

The present work was carried out with the support of the University of Pernambuco (UPE), an entity of the Government of the State of Pernambuco dedicated to fostering Teaching, Research and University Extension”.

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Received
  09 Mar 2026
• Accepted
  27 May 2026