# Preparation and Evaluation of the Biological Properties of Ethanolic Extract of Red Clover: An In Vitro Study

## TL;DR

This study evaluates the antimicrobial, antioxidant, and cytotoxic properties of an ethanolic extract from red clover, finding it to be a promising mouthwash candidate.

## Contribution

The study provides empirical evidence of red clover extract's biological properties, including its antioxidant and biocompatible characteristics.

## Key findings

- The extract showed increased antioxidant activity with higher concentrations in the DPPH assay.
- The extract exhibited minimal cytotoxicity at concentrations between 5-20 µL.
- Antibacterial activity showed similar inhibition zones compared to controls.

## Abstract

Introduction: Red clover, a perennial herbaceous plant, has been demonstrated to possess blood-purifying, expectorant, and calming properties. This research endeavors to create and evaluate the antimicrobial, antioxidant characteristics, and cytotoxic effects of the ethanolic extract derived from red clover.

Methods: A water-based solution of red clover was formulated and subjected to centrifugation. Various concentrations of the extract were applied to the wells of agar plates inoculated with E. coli, Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, and Candida albicans and then left to incubate. The inhibition zones for each concentration were subsequently measured. The antioxidant properties were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, while the cytotoxicity of the extract was assessed through the brine shrimp lethality assay.

Results: Initially, the extract was tested with a volume of 10 μL, which was subsequently incremented to 20 μL, 30 μL, 40 μL, and 50 μL. According to the DPPH assay, as the concentration of the extract solution increased incrementally by 10 μL, its antioxidant activity also exhibited a corresponding rise. The cytotoxicity assay indicated that the mouthwash formulated with red clover had minimal cytotoxic effects within the range of 5-20 µL. Antibacterial analysis revealed a similar zone of inhibition between the test and control groups.

Conclusion: The ethanolic extract obtained from red clover was identified as a powerful antioxidant, antibacterial, and biocompatible substance. Hence, it can be a potential candidate for application as a mouthwash.