# Proteolytic Activity Inhibition in Gingival Fluid by Cysteine Protease Inhibitors Obtained from Egg White and Fallopia japonica Extract: An In Vitro Study

## TL;DR

This study explores natural inhibitors from egg white and Fallopia japonica to block harmful enzymes in gum disease, showing they are effective and less toxic than common treatments.

## Contribution

The study introduces natural cysteine protease inhibitors from egg white and knotweed as a less toxic alternative to chlorhexidine for periodontitis treatment.

## Key findings

- Natural inhibitors from egg white and knotweed significantly inhibited cysteine protease activity in gingival fluid.
- The toxicity of knotweed cystatin was much lower than chlorhexidine and E-64d.
- Inhibition rates were higher in periodontitis patients compared to healthy individuals.

## Abstract

Background/Objectives: Gingipains produced by P. gingivalis have been shown to be directly related to periodontal tissue degradation and are significant molecular targets in therapy of periodontitis. Blocking the activity of these enzymes should reduce survival of this pathogen and mitigate the effects of inflammation in periodontitis. Therefore, gingipains inhibitors and specific antibodies could be recommended in the treatment of periodontitis. Cysteine peptidase inhibitors can be obtained by chemical synthesis, or isolated from natural raw materials. This research has the following aims: 1. to analyze in vitro the inhibition of cysteine protease activity in the gingival crevicular fluid (GCF) and 2. to compare the toxicity of natural raw inhibitors (obtained from Fallopia japonica plant and egg white) with chlorhexidine (CHX) using an MTS viability test. Methods: Samples of GCF were collected from healthy (N = 17) individuals and (N = 65) periodontal patients. Cysteine peptidase activity was inhibited by adding a solution of cystatin from egg white (with 20% glycerol), or cystatin from knotweed, or low molecular weight inhibitors (MW < 3 kDa) from egg white and knotweed against Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride. Results: There was a statistically significant difference between the inhibition means of cysteine protease activity for the five groups (p < 0.001). Means for the four groups of patients with periodontitis were not statistically significant different from each other (p = 0.320). The inhibition rates were higher in periodontitis patients. The toxicity of knotweed cystatin inhibitor was several times lower than the toxicity of E-64d, and of CHX. Conclusion: Cysteine protease inhibitors isolated from egg or plants were non-toxic, effectively inhibited the activity of cysteine proteases in GCF, and may be a promising alternative to more toxic standard antimicrobials (CHX) in preventing periodontal tissue breakdown.