Preparation of glass-ionomer cement containing ethanolic Brazilian pepper extract (*Schinus terebinthifolius* Raddi) fruits: chemical and biological assays

The study addresses the need for alternative treatments to control subgingival infections in the oral cavity, minimizing the side effects of conventional antibiotics. The research focuses on developing a drug delivery system using glass-ionomer cement (GIC) as a carrier for ethanolic Brazilian pepper extract (BPE). GIC is chosen for its biocompatibility and suitability as a drug delivery system. The BPE, derived from *Schinus terebinthifolius* Raddi (Brazilian pepper), is known for its antimicrobial, anti-inflammatory, and antioxidant properties. However, the potential toxicity of plant extracts necessitates biocompatibility testing. This study aimed to investigate the antibacterial activity of BPE incorporated into GIC, focusing on the flavonoid release profile and the overall biocompatibility of the GIC-BPE composite for potential use in endodontic treatments. The existing literature demonstrates the efficacy of Brazilian pepper extract in various applications, but few studies have explored its incorporation into GIC and its flavonoid release profile within this context, highlighting the novelty of this research.

The literature review highlights the challenges of systemic antibiotic administration for treating subgingival infections, emphasizing the need for localized drug delivery systems with minimal side effects. The rising interest in phytotherapeutics, particularly the antimicrobial properties of *Schinus terebinthifolius*, is discussed, acknowledging the potential toxicity of some plant extracts and the importance of biocompatibility testing. Previous studies have reported the antimicrobial, anti-inflammatory, and antioxidant activities of BPE, but research on its integration into GIC and the release profile of its flavonoids is limited, justifying this investigation.

The study involved the preparation of ethanolic Brazilian pepper extract (BPE) from *Schinus terebinthifolius* fruits. The glass-ionomer cement (GIC) was prepared according to the manufacturer's instructions. GIC-BPE was prepared by incorporating BPE into the GIC powder, followed by mixing with the liquid phase. Characterization techniques included scanning electron microscopy (SEM) to analyze the morphology of the samples, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to identify functional groups and potential interactions, and thermal analysis (TG/DTG/DTA) to evaluate the thermal stability of the materials. The flavonoid release profile was assessed by immersing GIC-BPE pellets in simulated body fluid (SBF) over time, measuring flavonoid concentration using UV-Vis spectroscopy. Phytochemical analysis (UPLC-QTOF-MS/MS) determined BPE composition, and total phenolic and flavonoid contents were measured using colorimetric methods. Cytotoxicity was evaluated using the sulforhodamine B (SRB) assay on human fibroblast MRC-5 cells. Antimicrobial activity was tested using the agar diffusion method against *S. aureus*, *S. mutans*, *A. actinomycetemcomitans*, and *C. albicans*. Compressive strength was measured according to ISO standardization. Statistical analysis was performed using a student t-test.

SEM images showed significant differences in surface roughness before and after immersion in SBF. ATR-FTIR spectroscopy confirmed the incorporation of BPE into GIC and suggested an intermolecular interaction based on overlapping and disappearance of specific bands. Thermal analysis (TG/DTG/DTA) showed a difference in weight loss between GIC and GIC-BPE samples, attributable to the presence of the extract. The DTA curve for GIC-BPE suggested a chemical interaction, indicated by a shifted exothermic peak. The compressive strength of GIC-BPE (161.17 ± 5.01 MPa) was slightly lower than that of GIC (176.60 ± 6.46 MPa), but not significantly different. Phytochemical analysis identified 27 compounds in BPE, categorized as gallic acid derivatives, flavonoids, and catechins. The total phenolic content was 60.09 ± 7.65 mg of gallic acid/g, and the total flavonoid content was 1.88 ± 1.01 mg of quercetin/g. The flavonoid release profile showed a two-stage release with the majority of flavonoids released within 60 hours. The diffusion coefficient was calculated as 1.406 × 10⁻⁶ cm² s⁻¹. Cytotoxicity tests showed low cytotoxicity for MRC-5 cells at concentrations of GIC-BPE up to 250 µg/mL. The GIC-BPE composite exhibited antimicrobial activity against all tested bacterial species (*S. aureus*, *S. mutans*, and *A. actinomycetemcomitans*) but not against *C. albicans*. The BPE alone showed higher antimicrobial activity than the GIC-BPE composite.

The findings suggest that the Brazilian pepper extract was successfully incorporated into the GIC matrix, forming a composite with promising antimicrobial properties. The observed intermolecular interaction between BPE and GIC, supported by FTIR and thermal analysis, might explain the slight decrease in the mechanical strength of the composite. The two-stage release of flavonoids, with a significant portion released within 60 hours, points to a potential for sustained drug delivery. The antimicrobial activity demonstrated by GIC-BPE at non-cytotoxic concentrations highlights its potential as an alternative therapeutic agent for endodontic treatments. The identification of various bioactive compounds in BPE provides a molecular basis for the observed antimicrobial activity. The lower antimicrobial activity of GIC-BPE compared to BPE alone could be attributed to the controlled release nature of the composite. This study supports the use of plant-derived extracts in developing cost-effective dental treatments, particularly relevant for developing countries.

This study successfully developed a novel glass-ionomer cement composite incorporating ethanolic Brazilian pepper extract, demonstrating its potential for endodontic applications. The composite exhibited antimicrobial activity against several oral pathogens at non-cytotoxic concentrations, with a controlled release profile. Future studies could optimize the BPE concentration and explore other plant extracts for enhanced efficacy and explore the *in vivo* efficacy and long-term effects of this composite.

The study's limitations include the *in vitro* nature of the experiments. Further *in vivo* studies are necessary to validate the findings in a clinical setting. The sample size in some assays could be increased for improved statistical power. The study focused on specific oral pathogens; further investigation with a broader range of microorganisms is warranted.