In Vitro Evaluation of Substantivity, Staining Potential, and Biofilm Reduction of Guava Leaf Extract Mouth Rinse in Combination with its Anti-Inflammatory Effect on Human Gingival Epithelial Keratinocytes

Abstract

This study aimed to assess the biofilm reduction, staining potential, and cytotoxicity of guava extract mouth rinse compared to chlorhexidine (CHX). Substantivity, staining, and antibiofilm potential were investigated by spectrophotometry, colony-forming units, and luminosity color meter, respectively. The cell viability assay was conducted using a colorimetric assay to determine nontoxic levels of guava (0.15%) and CHX in human gingival epithelial keratinocytes (HGEK-16). Cells were treated with lipopolysaccharides (LPS, 1μg/mL) and guava to assess inflammatory gene expression levels of interleukin-β1, tumor necrosis factor-α, and Prostaglandin E2. A scratch wound healing assay investigated the effects of guava on cell migration. The teeth coated in guava mouth rinse displayed 19.4% higher substantivity compared to CHX (0.2%), and the anti-biofilm reduction was observed with both guava and CHX mouth rinses (P < 0.05). The overall discoloration changes were higher with CHX and distilled water compared to guava. Also, guava significantly enhanced HGEK-16 cell viability (P < 0.05), and IL-β1, TNFα and PGE2 expression presented a 0.6-fold decrease when exposed to guava and LPS (P < 0.05). The present study showed that guava mouth rinse fulfilled the requirement for an effective and useful oral care product with desirable substantivity and anti-biofilm action. In addition, guava reduced the inflammation response in HGEK-16 and may be a potential oral rinse for oral anti-inflammatory therapies.

Keywords: biofilm; guava; human gingival keratinocyte; inflammation; staining; substantivity.

Figure 1

Evaluation of the cellular viability using cell viability assay of human gingival epithelial keratinocytes. A significant increase in cellular viability was detected for guava treatment (0.15%), whereas, CHX at concentrations higher than 0.00005%, were found to be cytotoxic. X-axis = points of measurement, Y-axis = optical density, * P < 0.05. Mean ± S.D.

Figure 2

Upregulation of inflammatory genes in HGEK-16. Increased expression of (A) ILβ1, (B) TNFα, and (C) PGE2 genes was found in LPS (1 μg/mL) compared to control (white bars). A reduction of expression was found on HGEK-16 upon treatment with guava 0.15% mouth rinse and LPS (1 μg/mL). x-Axis: LPS and guava treatments; y-Axis: relative gene expression. * P < 0.05. Mean ± S.D.

Figure 3

Effects of guava exposure on wound healing of confluent HGEK-16. During the 12–24 h after wounding, a significant wound closure rate was found to be similar for guava (0.15%) and the untreated control. No significant migration difference was found after guava (0.15%) and LPS (1 μg/mL) treatment. Each bar indicates the mean ± S.D. Wound closure rates are expressed as the difference between wound width at 0 h, 12 h, and 24 h (n = 3). * P < 0.05.