The objective of this study was to determine the ultrasonication-assisted extraction conditions that maximize the DPPH radical scavenging activity of extracts obtained from the stems of Lespedeza bicolor Turcz through the application of the Response Surface Methodology (RSM). Before delving into the analysis of extraction conditions using the RSM model, we conducted efficiency validation of ultrasonication-assisted extraction and executed single-factor experiments for ethanol concentration, extraction time, and extraction temperature. The data obtained from these single-factor experiments were employed to construct the Box-Behnken Design (BBD). In these results, in the single-factor experiments, it was evident that the parameters for ethanol concentration, extraction time, and extraction temperature exhibited quadratic trends. The single-factor experiments allowed us to discern the trends for each parameter leading to the maximum antioxidant capacity, and this data was subsequently applied to the BBD. Following the completion of initial experiments, a Response Surface Methodology (RSM) model was constructed based on Box-Behnken Design (BBD). According to the predictive model developed in this study, it was anticipated that performing ultrasonic-assisted extraction for 85.0412 minutes at an ethanol concentration of 32.573% and an extraction temperature of 51.5608°C will result in a DPPH radical scavenging activity of 79.7146%. The predictive results were statistically verified through a comparative analysis with actual measurements and ANOVA analysis, confirming the statistical significance of the model. The finding of this study underscore the significance of optimizing extraction conditions in the precise quantification of the antioxidant potential for economic advantages in both experimental and industrial contexts.

Introduction
Materials and Methods
    1. Preparation of extract
    2. DPPH radical scavenging activity
    3. Experimental design for the development of theresponse surface methodology model
    4. Statistical analysis
Results and Discussion
    1. The impact of ultrasonication-assisted extraction onthe DPPH radical scavenging activity of L.bicolor
    2. Single-Factor Experiments
    3. Optimization of ultrasonication-assisted operationalparameters for DPPH radical scavenging activity
Acknowledgements
References

• Woo Seok Lim(Undergraduate student, Department of Environmental Forest Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Korea)
• Si Young Ha(Research Professor, Department of Environment Materials Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Korea)
• Ji Young Jung(Research Professor, Department of Environment Materials Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Korea)
• Hyeon Cheol Kim(Master course, Department of Environment Materials Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Korea)
• Jae-Kyung Yang(Professor, Department of Environment Materials Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Korea) Corresponding author