In Vitro Study on Green Propolis as a Potential Ingredient of Oral Health Care Products

Abstract

Propolis is increasingly being discussed as an alternative to commonly used antiseptics. This in vitro study focused on the ethanolic extract of green Brazilian propolis (EEPg) as an additive in an oral health care product. We investigated (i) a potential inflammation-modulation activity of EEPg when a periodontal or Candida biofilm was exposed to monocytic (MONO-MAC-6) cells, (ii) the adhesion of oral pathogens to gingival keratinocytes and (iii) the antimicrobial and antibiofilm effect of different toothpaste formulations. EEPg decreased the levels of interleukin (IL)-1β and increased IL-10 in MONO-MAC cells challenged with a periodontal biofilm. In contact with TIGK cells, EEPg reduced the numbers of adherent Porphyromonas gingivalis to 0.5% but did not affect the adhesion of Candida albicans. The frequent brushing of a cariogenic biofilm with a toothpaste supplemented with EEPg reduced the surface microhardness loss of enamel specimens. Mixing an experimental erythritol toothpaste with 25 and 50 mg/mL of EEPg confirmed the antibacterial activity of EEPg against oral bacteria and particularly inhibited periodontal biofilm formation. The suggested toothpaste formulations seem to have potential in the prevention of caries, gingivitis and periodontitis and should be evaluated in further in vitro research and in clinical trials.

Keywords: Brazilian green propolis; biofilm; monocytic cells; toothpaste.

Figure 1

Released interleukin(IL)-1β (a,c,e) and IL-10 (b,d,f) from MONO-MAC-6 cells after being exposed to ethanolic extract of green propolis (EEPg) or ethanol without (a,b) and with a Candida biofilm (c,d) or with a periodontal biofilm (e,f) for 2, 4 and 8 h. Presented are mean and SD, */** p < 0.05/p < 0.01 vs. control of the respective time and assay. ^ΠΠ p < 0.01 vs. MONO-MAC-6 cells without biofilm at the respective time.

Figure 2

Monocytic (MONO-MAC-6-cells) mRNA expression of interleukin(IL)-1β (a) and IL-10 (b) after being exposed to 1.25 mg/mL ethanolic extract of green propolis (EEPg) and/or a periodontal biofilm for 30 min. Presented are mean and SD, ** p < 0.01 vs. control (The MONO-MAC-6 cells were tested for any cytotoxic reaction. Increased vitality of cells was found 2 h and 4 h after exposing them to 1.25 mg/mL or 2.5 mg/mL propolis; no difference existed after 8 h).

Figure 3

Adhering (incl. invaded) Candida albicans (colony forming units = cfu; mean and SD) to epithelial cells (a) in the presence of ethanolic extract of green propolis (EEPg) and formation of hyphae after being exposed to 2.5 mg/mL EEPg (b). Presented are mean and SD, */** p < 0.05/p < 0.01 vs. control.

Figure 4

Adhering (incl. invaded) Porphyromonas gingivalis (colony forming units = cfu; mean and SD) to epithelial cells in the presence of ethanolic extract of green propolis (EEPg). Presented are mean and SD, ** p < 0.01 vs. control.

Figure 5

Bacterial counts (colony forming units = cfu; mean and SD) present in the biofilms (a), enamel surface microhardness (SMH) loss (b) of the samples in percent (mean and SD) after 14 days of exchange of media twice daily and daily brushing of the enamel specimens with slurries of a commercially available toothpaste (TPcom) without and with added 50 mg/mL ethanolic extract of green propolis (EEPg). Presented are mean and SD, */** p < 0.05/p < 0.01 vs. control.

Figure 6

Activity of the ethanolic extracts of green propolis (EEPg, 25 mg/mL and 50 mg/mL), erythritol (Ery) and different toothpaste formulations (erythritol toothpaste (EryTP) without and with 25 mg/mL or 50 mg/mL EEPg, commercially available TP (TPcom)) on colony forming units (cfu); (a,b), and metabolic activity (c) in a formed two-species biofilm after 2 h (a) and 6 h (b,c). Presented are mean and SD, */** p < 0.05/p < 0.01 vs. control.

Figure 7

Activity of the ethanolic extracts of green propolis (EEPg, 25 mg/mL and 50 mg/mL), erythritol (Ery) and different toothpaste formulations (erythritol toothpaste (EryTP) without and with 25 mg/mL or 50 mg/mL EEPg, commercially available TP (TPcom)) on colony forming units (cfu); (a,b), and metabolic activity (c) in a formed cariogenic biofilm after 6 h (a) and 24 h (b,c). Presented are mean and SD, ** p < 0.01 vs. control.

Figure 8

Activity of the ethanolic extracts of green propolis (EEPg, 25 mg/mL and 50 mg/mL), erythritol (Ery) and different toothpaste formulations (erythritol toothpaste (EryTP) without and with 25 mg/mL or 50 mg/mL EEPg, commercially available TP (TPcom) on colony forming units (cfu); (a,b), and metabolic activity (c) in a periodontal biofilm after 6 h (a) and 24 h (b,c). Presented are mean and SD, */** p < 0.05/p < 0.01 vs. control.