In vitro anti‑bacterial activity of diosgenin on Porphyromonas gingivalis and Prevotella intermedia

Abstract

Diosgenin (Dios), a natural steroidal sapogenin, is a bioactive compound extracted from dietary fenugreek seeds. It has a wide range of applications, exhibiting anti‑oxidant, anti‑inflammatory and anti‑cancer activities. However, whether the extracts have beneficial effects on periodontal pathogens has so far remained elusive. The aim of the present study was to investigate the anti‑bacterial effects of Dios on Porphyromonas gingivalis (P. gingivalis) and Prevotella intermedia (P. intermedia) in vitro. The anti‑microbial effect of Dios on P. gingivalis and P. intermedia was assessed by a direct contact test (DCT) and the Cell Counting Kit (CCK)‑8 assay at 60, 90 and 120 min. In addition, counting of colony‑forming units (CFU) and live/dead cell staining were used to evaluate the anti‑bacterial effects. The results of the DCT and CCK‑8 assays indicated that Dios had beneficial dose‑dependent inhibitory effects on P. gingivalis and P. intermedia. The CFU counting results also indicated that Dios had dose‑dependent anti‑bacterial effects on P. gingivalis and P. intermedia. Of note, Dios had significant anti‑bacterial effects on the biofilms of P. gingivalis and P. intermedia in vitro as visualized by the live/dead cell staining method. In conclusion, the present results demonstrated that Dios had a marked anti‑bacterial activity against P. gingivalis and P. intermedia in vitro, both in suspension and on biofilms. The present study highlighted the potential applications of Dios as a novel natural agent to prevent and treat periodontitis through its anti‑bacterial effects.

Figure 1.

Molecular structure of diosgenin.

Figure 2.

Effect of Dios on P. gingivalis and P. intermedia determined by a direct contact test. Effect of Dios against P. gingivalis over three periods: (A) 60 min, (B) 90 min, (C) 120 min; effect of Dios against P. intermedia over three periods: (D) 60 min, (E) 90 min, (F) 120 min. Bacterial suspensions in tryptic soy broth without Dios were used as a control. Data are presented as the mean ± SD (n=5). *P<0.05, **P<0.01 vs. control group. Dios, diosgenin; OD, optical density; CHX, chlorhexidine; P. gingivalis, Porphyromonas gingivalis; P. intermedia, Prevotella intermedia.

Figure 3.

Inhibitory effects of Dios on (A) P. gingivalis and (B) P. intermedia. The viability of P. gingivalis and P. intermedia was tested by a Cell-Counting-Kit 8 assay. Bacterial suspensions in tryptic soy broth without Dios were used as a control. Data are presented as the mean ± standard deviation (n=5). *P<0.05, **P<0.01 vs. control group. Dios, diosgenin; OD, optical density; CHX, chlorhexidine; P. gingivalis, Porphyromonas gingivalis; P. intermedia, Prevotella intermedia.

Figure 4.

To determine the concentration-dependent anti-bacterial activity of Dios, CFU counts at 24 h were determined. (A) Images of colonies formed by P. gingivalis in the presence/absence of Dios and (B) quantified numbers of colonies in the different groups. (C) Images of colonies formed by P. intermedia in the presence/absence of Dios and (D) quantified numbers of colonies in the different groups. Bacterial suspensions in tryptic soy broth without Dios were used as a control. Quantified values are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. control group. Dios, diosgenin; CFU, colony-forming units; CHX, chlorhexidine; P. gingivalis, Porphyromonas gingivalis; P. intermedia, Prevotella intermedia.

Figure 5.

Viability of P. gingivalis and P. intermedia biofilms, as assessed by CLSM. (A and B) CLSM images of (A) P. gingivalis and (B) P. intermedia biofilms (scale bar, 50 µm). (C and D) Percentage of live cells on (C) P. gingivalis and (D) P. intermedia biofilms. Bacterial suspensions in tryptic soy broth without Dios were used as a control. Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 vs. control group. Dios, diosgenin; CHX, chlorhexidine; CLSM, confocal laser scanning microscopy. P. gingivalis, Porphyromonas gingivalis; P. intermedia, Prevotella intermedia.