Psidium guajava L. hydroethanolic extract as endodontic irrigant: phytochemical analysis, antioxidant activity, antimicrobial action and biocompatibility

Preparation of the plant extract

The P. guajava L. hydroethanolic extract was prepared using young leaves, manually collected in March 2023 from shrubs in the southern region of São José dos Campos, São Paulo. After collection, the leaves were washed with distilled water, dried in the dark at temperatures between 20–27 °C for 5 days, and stored in a clean, dry environment. The dried plant material was ground using a blender. The solvent used for extraction was absolute ethanol (ethyl alcohol 99.5%; Merck, Darmstadt, Germany) and ultrapure water obtained from a Milli-Q® system (EtOH:H2O/50:50). The ratio was 30 g of plant material per 100 mL of solvent, with an extraction period of 48 h. The extracts were filtered in two stages: first, using a common article filter with micro-pores, the same used to filtrate coffee, made from bleached or unbleached cellulose fibers with pore size generally ranges from 15 to 25 µm, to remove solid residues and then sterilized using a 0.22 µm membrane filter (MilliporeSigma, Millex®) (Baldoni et al., 2023). The extract was stored for further analysis.

Soluble solid content of P. guajava L. hydroethanolic extract

Three empty 25 mL beakers were weighed, and their weights were recorded. Then, 5 mL of the extract were pipetted into each beaker (in triplicate) and dried at 80 °C for 24 h. After drying, the beakers were cooled in a desiccator and weighed again. The soluble solid content of the extracts was quantified using the formula (Meccatti et al., 2023; de Oliveira et al., 2024b):

$$\mathrm{\%}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}\mathrm{s}(\mathrm{m}/\mathrm{V})=(\mathrm{m}-\mathrm{b})\times 100/\mathrm{V}\mathrm{a}$$

$$\mathrm{\%}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}\mathrm{s}(\mathrm{m}/\mathrm{m})=\mathrm{\%}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}\mathrm{s}(\mathrm{m}/\mathrm{V})/\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}$$where: b = beaker mass; m = final mass of the extract after drying; Extract density = m/V (mass of the 5 mL aliquot weighed, and V is the volume of 5 mL).

The concentration of the extract was determined using the weight/volume (w/v) method. First, the soluble solid content was measured, and the corresponding values were converted from % v/v to w/v. This conversion was achieved by multiplying the percentage by 10, considering that 1% v/v is equivalent to 10 mg/mL. Consequently, the final concentrations are expressed in w/v in the results.

Total flavonoid content determination of P. guajava L. hydroethanolic extract

To determine total flavonoid content, a stock solution was prepared using 100 µL extract in a 10 mL volumetric flask filled with 9,900 µL of methanol (absolute methyl alcohol; Êxodo Científica, Sumaré, Brazil) to the meniscus in a proportion of (1:99). The procedure was performed in triplicate. A 200 µL aliquot from the stock solution was transferred to a 10 mL flask containing ~5 mL of methanol, followed by 200 µL of aluminum chloride (AlCl₃), and the volume was completed with methanol in a proportion of (2:2:98). The resulting solution was agitated and incubated in a water bath (Generalmed, São Paulo, Brazil) for 30 min at 20 °C. After adjusting the meniscus, absorbance was read using an ultraviolet-visible (UV-Vis) spectrophotometer at 425 nm (Micronal B-582; Micronal São Paulo, Brazil). Total flavonoid concentration (in mg/mL), expressed in quercetin, was calculated using a calibration curve (Cristina Marcucci et al., 2021; Meccatti et al., 2023; de Oliveira et al., 2024b).

Total phenol content determination of P. guajava L. hydroethanolic extract

In a volumetric flask of 100 mL, 1 mL of the extract was transferred and diluted in a 1 mL of ethanol (99.5% ethyl alcohol-Merck Darmstadt, Germany), then diluted to volume with distilled water (obtained using the Milli-Q® system) while stirring (stock solution) (10 μg/mL). From this point, the procedure was conducted in triplicate. A 0.2 mL (200 μL) aliquot was transferred to a 10 mL volumetric flask (1:50) containing 5 mL of distilled water, with the addition of 800 μL of Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA). The mixture was stirred for a few seconds, and between 1 and 8 min later, 1.2 mL of 20% sodium carbonate-tartrate buffer solution was added. The volume was completed with distilled water to the meniscus, and the solution was maintained in a water bath (Generalmed, São Paulo, Brazil) at 20 °C. After 2 h, the final volume was adjusted by adding distilled water up to the 10 mL mark at 20 °C with agitation for a few seconds, and the absorbance was measured at 760 nm using a UV-Vis spectrophotometer (Micronal B-582). The total phenol content was determined using the calibration curve equation. A stock solution of phenol (standard) was used to prepare the calibration curve for subsequent quantification (Cristina Marcucci et al., 2021; Meccatti et al., 2023; de Oliveira et al., 2024b).

High-performance liquid chromatography analysis of P. guajava L. hydroethanolic extract

High-performance liquid chromatography (HPLC) was used to characterize the marker content and phytochemical profile of the extracts. The extract used in this analysis is not diluted, it is the same extract prepared inutility from the guava leaves. The analysis was performed on an HPLC system with a diode-array detector (HPLC-DAD) and an automatic injector (D-7000; Merck-Hitachi, Darmstadt, Germany). Chromatographic conditions included a mobile phase consisting of aqueous formic acid solution (95:5, solvent A) and chromatographic-grade methanol (Merck, Darmstadt, Germany, solvent B). The flow rate was set to 1 mL/min with a linear gradient starting at 0% B and ending at 70% B over 50 min. Detection wavelengths of 280 and 340 nm were used (Meccatti et al., 2023; de Oliveira et al., 2024b).

Determination of free radical suppressive activity of P. guajava L. hydroethanolic extract (Antioxidant Activity)

Samples were prepared at different dilutions based on previously determined soluble solid contents. The required extract volume was calculated to obtain 1% V/V and 0.01% V/V dilutions. For 1% V/V dilution, the appropriate aliquot of extract solution was added to a 10 mL flask, and the volume was adjusted with ethanol. The 0.01% V/V dilution was prepared using a serial dilution method. Eleven test tubes were labeled from 0 to 10, and the ethanol and extract solutions were added in sequence using 0, 40, 80, 120, 160, 200, 240, 280, 320, 360, 400 µL of the extract, and adjusted with ethanol to obtain 1,000 µL. In addition, tube 0 served as the control, containing only 100% DPPH (1,1-diphenyl-2-picrylhydrazyl) without plant extract. Also, 1,000 µL of DPPH was added to tube 1, and the reaction time was recorded for 1 min before sequentially adding DPPH with the same volume (1,000 µL) was added to the remaining tubes at 1-min intervals, with periodic shaking. After 30 min, spectrophotometric readings (Micronal B-582) at 517 nm were taken. Each measurement was performed in triplicate, and absorbance values were plotted as Absorbance (%) vs. extract concentration (µg/mL). The IC₅₀ value indicating the concentration required to eliminate 50% of free radicals, was determined using the least squares method (Alves et al., 2010; Veiga et al., 2017).

Determination of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) of P. guajava L. hydroethanolic extract

One standard (ATCC 4083) and two clinical strains (denominated 2 and 4) of E. faecalis and one standard (ATCC 18804) and two clinical strains (denominated 14 and 60) of C. albicans were used in this study. E. faecalis was cultured (37 °C/24 h) on Brain Heart Infusion agar (BHI ), and C. albicans strains were cultured (37 °C/24 h) on Sabouraud-dextrose agar (SD-Kasvi, São José dos Pinhais, Brazil). Microbial suspensions were prepared by diluting colonies of the respective strains in sterile saline solution (0.9% NaCl) and homogenized in a vortex mixer for 10 s to standardize the microbial solution according to each protocol.

The broth microdilution method, according to the Clinical and Laboratory Standards Institute (CLSI), standards M27-S4 and M7-A9, was used to determine the MIC and MMC of the extract for each microbial strain. The inoculum was standardized in a spectrophotometer (Micronal B-582). For E. faecalis, the wavelength of 760 nm with an optical density of 0.298 was used, resulting in a standard suspension of 1 × 10⁶ bacterial cells/mL. For C. albicans, the wavelength of 530 nm with an optical density of 0.284 was used, resulting in a standard suspension of 1 × 10⁶ yeast cells/mL.

In separate microplates (Kasvi K12-096; Kasvi, São José dos Pinhais, Brazil), a total of 10 serial dilutions (1:2) of the extract were performed in culture media: Mueller Hinton broth (HiMedia®, Mumbai, India) for E. faecalis, and RPMI 1,640 broth (with glutamine, without bicarbonate, and with phenol red indicator) (INLAB) for C. albicans. Aliquots of 100 µL of each microbial suspension were added to all wells. After 24 h incubation at 37 °C, the MIC was determined as the last well without turbidity indicating microbial growth.

To determine MMC, aliquots from all wells were plated on BHI agar and incubated at 37 °C for 48 h. The MMC was identified as having the lowest concentration without colony growth. A vehicle control group (EtOH:H₂O/50:50) was included to evaluate its potential interference with the extract’s antibacterial activity.

Antibiofilm activity of P. guajava L. hydroethanolic extract via MTT analysis

For E. faecalis, in 96-well microplates, a volume of 100 μL/well of BHI broth (Kasvi) was added. After preparation and standardization with a spectrophotometer (10⁷ cells/mL), the bacterial suspension was added to the microplates (100 μL/well), already containing culture medium, for a total of 200 μL/well. For C. albicans, the standardization was performed using a spectrophotometer to obtain 10⁷ cells/mL. Subsequently, 200 μL/well of the adjusted C. albicans suspension was added to the microplates and incubated at 37 °C for 90 min to allow initial cell adhesion to the wells. Afterward, the supernatant was discarded, and BHI broth was added. The plates were incubated at 37 °C for 48 h to allow biofilm formation, with the medium being replaced after 24 h (Marques Meccatti et al., 2022; Santos et al., 2023; de Lima et al., 2024).

After biofilm formation, they were exposed for 10 min to different concentrations of the plant extract (1.04. 2.09, 4.18 and 8.37 mg/mL), which were determined based on the results of the MIC and MMC tests. Culture medium was used as a negative control, while 2% CHX (Biofórmula Manipulação, São José dos Campos, SP, Brazil) and 2.5% NaOCl (Asfer, São Caetano do Sul, São Paulo, Brazil) were used as positive controls for all biofilm analyses. Each experimental group consisted of n = 10. Later, saline solution was added and discarded to wash the wells and remove non-adherent cells affected by the treatments. The microbial cell viability test was performed by adding 100 μL of MTT solution (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Sigma-Aldrich) to each well. The plate was incubated in the dark at 37 °C for 1 h. After incubation, the MTT solution was removed, followed by the addition of 100 μL of dimethylsulfoxide (DMSO). The plate was incubated again at 37 °C for 10 min and then placed on a shaker under constant agitation for another 10 min. Finally, optical densities (OD) were measured using a microplate reader at 570 nm, and the obtained OD values were converted into percentages of metabolic activity.

Cytotoxicity evaluation of P. guajava L. hydroethanolic extract

The cytotoxicity analysis of the P. guajava L. extract was performed on human keratinocyte cell lines (HaCaT) obtained from the Rio de Janeiro Cell Bank–Associação Técnico Científica Paul Ehrlich (APABCAM–RJ). The cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM–GC Biotecnologia, Cotia, Brazil) with high glucose concentration, supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA), and maintained in cell culture flasks (Kasvi, Brazil) at 37 °C in a humidified incubator with 5% CO₂. Once sufficient cell quantities were reached, the cells were subjected to the respective tests.

The cell monolayer was detached from the culture flask using trypsin. The cultures were transferred to 96-well microplates (Kasvi) at a concentration of 2 × 10⁴ viable cells per well and cultured in 200 µL of DMEM + 10% FBS. The plates were incubated at 37 °C with 5% CO₂ for 24 h to allow cell adhesion. Following the incubation period, the cells were exposed for 10 min to different concentrations of the extract (1.04. 2.09, 4.18 and 8.37 mg/mL) showing antimicrobial activity. The negative control group contained only DMEM medium in its liquid 1× form, while the positive control groups included 2% CHX and 2.5% NaOCl.

The cells were subjected to the Alamar Blue assay, using a stock solution of 5 g of Resazurin sodium salt (Sigma-Aldrich, Jurubatuba, Brazil) dissolved in 500 mL of PBS. The solution was added to the 96-well microplates at a volume of 100 µL/well, followed by light-protected incubation at 37 °C with 5% CO₂ for 4 h. The solution was then discarded, and 100 µL/well of dimethyl sulfoxide (DMSO–Sigma) was added. After 10 min of incubation and agitation on a shaker, the absorbance of the wells was measured with a microplate reader at a wavelength of 570 nm. The optical density (OD) values obtained were converted into percentages of cell viability.

Cytotoxicity was evaluated according to ISO 10993-5:2009 guidelines for in vitro cytotoxicity assessment, which classifies materials based on their impact on cell viability. A reduction in viability below 70% indicates a cytotoxic effect (ISO 10993-5, 2009).

Genotoxicity evaluation of P. guajava L. hydroethanolic extract

The micronucleus test was conducted in accordance with the OECD 2016 guideline (TG 487) for the HaCaT human keratinocyte cell line. For the test, 5 × 10⁵ cells/mL were cultured in 24-well plates for 24 h at 37 °C in a 5% CO₂ atmosphere. After this period, the cells were exposed to the extract at different concentrations (1.04. 2.09, 4.18 and 8.37 mg/mL), 2% CHX, and 2.5% NaOCl for 24 ho. Subsequently, the cells were incubated with cytochalasin B (Sigma-Aldrich) at a concentration of 6 µg/mL for 24 h at 37 °C in a 5% CO₂ atmosphere to inhibit cytokinesis and induce the accumulation of binucleated cells. The cells were subjected to hypotonic shock and fixed in a methanol and acetic acid solution (3:1) for 10 min, a procedure repeated three times. The wells were stained with the addition of one drop of DAPI for 5 min. The ethyl methanesulfonate (EMS) that induces the formation of micronuclei was used as a control group.

Micronuclei were analyzed using fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) at 400× magnification, evaluating 2,000 cells per well in at least two independent experiments. Micronuclei were identified as DNA-containing structures in the cytoplasm, clearly separated from the main nucleus, surrounded by a nuclear membrane, and occupying an area smaller than one-third of the main nucleus. Cells with fewer than five micronuclei were counted (Abu Hasna et al., 2022).

Statistical analysis

Data normality was assessed with the Shapiro-Wilk test. Normally distributed data were analyzed using ANOVA and Tukey’s test; non-parametric data were analyzed using Kruskal-Wallis and Dunn’s test. GraphPad Prism 5.0 software was used, with a significance level of 5%.

Soluble solid, total flavonoid total phenol content determination of P. guajava L. hydroethanolic extract

The soluble solids content in Psidium guajava L. hydroethanolic extract was 3.35%. Using the quercetin standard curve, the total flavonoid concentration was 0.130 mg/mL (0.013%), with a standard deviation of 0.110 mg/mL (0.011%). In addition, using standard curve for phenolic acids, the total phenolic concentration was 1.770 mg/mL (0.177%), with a standard deviation of 1.540 mg/mL (0.154%).

High-performance liquid chromatography analysis of P. guajava L. hydroethanolic extract

The chromatographic analysis by HPLC revealed peaks of rutin at retention time of 10.33 min, quercetin at retention time of ~17.83 min, and kaempferol at retention time of ~24.40 min in the extract of P. guajava L., as shown in Fig. 1.

Determination of free radical suppressive activity of P. guajava L. hydroethanolic extract (antioxidant activity)

The concentration required to eliminate 50% of free radicals (IC₅₀) was 10.39 µg/mL for the P. guajava L. extract, with a standard deviation of 2.90 µg/mL.

Determination of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) of P. guajava L. hydroethanolic extract

The extract demonstrated bactericidal and fungicidal activity against all the tested strains of E. faecalis and C. albicans. The MMC values ranged 1.04–8.37 mg/mL as shown in Table 1, respectively. It was not possible to identify the minimum inhibitory concentration (MIC) values due to turbidity caused by the color of the extract, which hindered visual reading. Therefore, in this study, the MBC values were considered for biofilm evaluation.

Antibiofilm activity of P. guajava L. hydroethanolic extract via MTT analysis

All the tested concentrations were effective against E. faecalis ATCC after 10 min except of 1.04 mg/mL which had no statistically significant difference (P < 0.0001) with the control group. However, the extract at 8.37 and 2.09 mg/mL reduced the biofilm formation by 51.62% and 46.58%, respectively, being as effective as NaOCl and CHX without a statistically significant difference (P < 0.0001). Similarly, all the tested concentrations were effective against E. faecalis clinical strain 2 after 10 min except of 1.04 mg/mL which had no statistically significant difference (P < 0.0001) with the control group. However, the extract at 8.37, 4.18 and 2.09 mg/mL reduced the biofilm formation by 53.44, 44.41 and 33.74 %, respectively, with a statistically significant difference with the control group (P < 0.0001). Even more, all the tested concentrations were effective against E. faecalis clinical strain 4 after 10 min. The extract at 8.37 and 2.09 mg/mL reduced the biofilm formation by 69.27, 61.24%, respectively, being as effective as NaOCl and CHX without a statistically significant difference (P < 0.0001) as shown in Fig. 2.

The extract at 8.37 mg/mL was effective against C. albicans ATCC strain after 10 min, in which it reduced the biofilm formation by 60.90%, being as effective as NaOCl and CHX without a statistically significant difference (P < 0.0001). Furthermore, the extract at 8.37 and 4.18 mg/mL was effective against C. albicans clinical strain 14 after 10 min, in which it reduced the biofilm formation by 63.27 and 55.39%, being as effective as NaOCl and CHX without a statistically significant difference (P < 0.0001). In addition, the extract at 8.37 and 4.18 mg/mL was effective against C. albicans clinical strain 60 after 10 min, in which it reduced the biofilm formation by 50.14 and 38.68% without a statistically significant difference (P < 0.0001) as shown in Fig. 2.

Cytotoxicity evaluation of P. guajava L. hydroethanolic extract

All the tested concentrations of the extract were less cytotoxic than NaOCl and CHX with a statistically significant difference (P < 0.0001). However, they had a statistically significant difference (P < 0.0001) as well with the control group (Fig. 3).

Genotoxicity evaluation of P. guajava L. hydroethanolic extract

All the tested concentrations of the extract presented a relatively low quantity of micronuclei in comparison to the EMS (the micronuclei former), with a statistically significant difference (P < 0.0001), still all the tested concentrations of the extract were less genotoxic than NaOCl and CHX with a statistically significant difference (P < 0.0001) as shown in Fig. 4.