# Antimicrobial, Time–Kill Kinetics, and Biofilm Inhibition Properties of Diospyros lycioides Chewing Stick Used in Namibia Against Enterococcus faecalis

## TL;DR

This study shows that a chewing stick from the Diospyros lycioides plant in Namibia can effectively kill and inhibit biofilms of Enterococcus faecalis, a bacteria linked to oral infections.

## Contribution

This is the first study to quantify the antimicrobial and biofilm inhibition properties of D. lycioides extracts against E. faecalis.

## Key findings

- Methanol extracts of D. lycioides showed high levels of flavonoids, phenols, and antioxidant activity.
- The organic extract achieved a bactericidal effect on E. faecalis at 8× MIC within 4 hours.
- The extract inhibited E. faecalis biofilm formation by ≥50% at concentrations as low as 7.8 μg/mL.

## Abstract

Background: Medicinal plants are used in Namibia for oral hygiene and to treat oral diseases. Validating the content and efficacy of medicinal chewsticks used in communities helps to provide proof of concept of medicinal plants used as a complementary/alternative medicine for oral diseases.

Aim: This study presents the first report on quantified phytoconstituents, antimicrobial, time–kill kinetics, and biofilm inhibition properties of Diospyros lycioides organic and aqueous extracts against Enterococcus faecalis.

Methodology: Dry plant materials were ground into powder and macerated in methanol and distilled water. Different phytoconstituents were quantified by Folin–Ciocalteu colorimetric method, ferric reducing antioxidant power assay, and DPPH free radical scavenging. An antibacterial assay was performed using the agar well diffusion method and a resazurin 96-well-based assay. Kill–time assay was done at various concentrations over 4 h. Biofilm inhibition was done using the crystal violet method.

Results: Higher total flavonoid, total phenol contents, and free radical scavenging abilities were reported in methanol twig extracts. Inhibition zones of 28 ± 0.82 mm, with MICs of 15.6 ± 0.00 μg/mL, are reported against E. faecalis. The bactericidal endpoint of D. lycioides organic extracts for E. faecalis was reached after 4 h of incubation at 8 × MIC (124.8 μg/mL). These were comparable to the positive control, gentamicin. The organic extracts showed ≥ 50% biofilm inhibition against root canal-infecting E. faecalis at concentrations between 7.8 and 500 μg/mL, indicating strong biofilm inhibition.

Conclusion: The study demonstrated that D. lycioides crude extracts have promising antibacterial properties and can eradicate E. faecalis biofilms in root canal treatments.