The Oral Wound Healing Potential of Thai Propolis Based on Its Antioxidant Activity and Stimulation of Oral Fibroblast Migration and Proliferation

Abstract

Introduction: Propolis has demonstrated wound healing effects. Propolis' effects vary based on its composition and geographical origin. However, there are few reports on the effects of propolis on oral wound healing. The aim of this study was to evaluate the antioxidant and in vitro gingival wound healing effects of the n-hexane extract of propolis (HEP), ethyl acetate extract of propolis (EEP), and aqueous extract of propolis (AEP) fractions of the ethanol extract of Thai propolis.

Materials and methods: The crude ethanol extract of propolis was obtained by maceration with 95% ethanol that was sequentially fractionated with hexane, ethyl acetate, and distilled water. The chemical profiles of the samples were assessed by thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). Antioxidant activity was determined using DPPH and FRAP assays. The effects of the propolis fractions on human gingival fibroblast (HGF) proliferation, migration, and in vitro wound healing were determined by MTT, modified Boyden chamber, and scratch assay, respectively.

Results: We found that solvent polarity greatly affected the extract yield and TLC profiles. The highest extract yield was found in HEP (38.88%), followed by EEP (19.8%) and AEP (1.42%). TLC revealed 7 spots in the crude ethanol extract (Rf 0.36-0.80), 6 spots in HEP (Rf 0.42-0.80) and EEP (Rf 0.36-0.72), and 4 spots in AEP (Rf 0.17-0.79). GC-MS analysis revealed a high amount of triterpenoids in HEP (82.97%) compared with EEP (28.96%). However, no triterpenoid was found in AEP. The highest antioxidant activity and stimulation of HGF proliferation were observed in HEP, followed by EEP and AEP. HEP and EEP, but not AEP, enhanced HGF migration. However, all propolis fractions induced wound closure.

Conclusions: HEP contained a large amount of triterpenoids. Antioxidant and in vitro wound closure effects were found in HEP, EEP, and AEP fractions.

Figure 1

Flowchart of the propolis extraction and solvent partitions. HEP, EEP, and AEP were obtained. The dry weight of each sample was indicated.

Figure 2

TLC analysis of the propolis samples. TLC profiling of the propolis extracts using (a) hexane/ethyl acetate/formic acid (30 : 70 : 1) and (b) ethyl acetate/methanol/formic acid (50 : 50 : 1) solvents. The TLC analysis was visualized under UV254 nm, UV366 nm, and an anisaldehyde-sulfuric acid agent compared with that of natural light. Samples no.1, 2, 3, and 4 represent the crude extract, hexane, ethyl acetate, and aqueous extracts, respectively.

Figure 3

GC-MS chromatograms of the standard compounds and propolis extracts. The chromatograms were plotted between total ion count and acquisition time of standards and the propolis fractions. (a) Chromatogram demonstrates the peaks of the 7 standard compounds: (1) m-coumaric acid, (2) ferulic acid, (3) caffeic acid, (4) galangin, (5) naringenin, (6) apigenin, and (7) quercetin. The chemical profiles of (b) HEP, (c) EEP, and (d) AEP are shown.

Figure 4

In vitro antioxidative effect of propolis extracts. (a) Free radical scavenging activity in each propolis extract (500 μg/ml) was evaluated by DPPH assay (n = 3). (b) Ferric radical scavenging activity in each propolis extract (500 μg/ml) was evaluated by FRAP assay (n = 3). Quercetin (10 μg/mL) was used as the positive control. Bars represent the mean ± SEM of the percentage of free radical scavenging. ^∗P < 0.05 versus the quercetin, ^#P < 0.05 versus HEP, ^$P < 0.05 versus EEP.

Figure 5

Proliferative effect of propolis extracts on HGFs. The cells were treated with 3 propolis extracts at various concentrations for 72 h. Cells in media without propolis extract were used as control. MTT assay was used to evaluate the cell proliferation effect of (a) HEP (15.62, 31.25, 62.5, 125, and 250 μg/mL), (b) EEP (20, 40, 80, 160, and 320 μg/mL), and (c) AEP (200, 400, 600, 800, and 1000 μg/mL) (n = 3). Bars represent the mean ± SEM of cell proliferation (% of control). ^∗P < 0.05 versus the control group.

Figure 6

Effect of propolis extracts on HGF migration. The effective doses of propolis extracts, HEP (15.62 μg/mL), EEP (40 μg/mL), and AEP (400 μg/mL), were used to treat the HGFs for 5 h in blind-well Boyden chambers. Cells in media without propolis extract were used as control. (a) The migrating cells were stained with crystal violet for 30 min. Scale bar = 100 μm. The migrated cells were counted under a light microscope in 5 random fields at 400x magnification. (b) The average migrated cell was calculated for each group (n = 3). Bars represent mean ± SEM of migrating cell no./field. ^∗P < 0.05 versus the control group.

Figure 7

Propolis extracts enhanced wound healing in vitro. The effective doses of propolis extracts, HEP (15.62 μg/mL), EEP (40 μg/mL), and AEP (400 μg/mL), were used to treat the HGFs for 24 h. The cells in media without propolis extract were used as control. (a) Scratch assay results. The wound closure pictures were taken at the initial time (T0) and after 24 h incubation (T24). Scale bar = 100 μm. (b) The wound distance in each group was measured at each time point and calculated as the percentage of wound closure using Image J software (n = 3). Bars represent the mean ± SEM of the percentage of wound closure. ^∗P < 0.05 versus the control group.