Quantitative Analysis of Chlorogenic Acid, Rutin, and Isoquercitrin in Extracts of Cudrania tricuspidata Leaves Using HPLC-DAD

A high-performance liquid chromatography (HPLC) method using a diode array detector (DAD) was developed and validated for the simultaneous quantification of chlorogenic acid, rutin, and isoquercitrin, which are key bioactive compounds in Cudrania tricuspidata leaves. The method demonstrated excellent specificity, precision, and accuracy in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). Calibration curves showed outstanding linearity (r² > 0.99), with recovery rates of 101.63%, 101.81%, and 102.18% for chlorogenic acid, rutin, and isoquercitrin, respectively. The limits of detection (LOD) were 0.286, 0.411, and 0.201 μg/mL, and the limits of quantification (LOQ) were 1.246, 0.866, and 0.608 μg/mL for chlorogenic acid, rutin, and isoquercitrin, respectively. Additionally, response surface methodology (RSM) based on a Box–Behnken design was employed to optimize the extraction conditions of the three marker compounds. The second-order regression models showed high coefficients of determination (r²) and significant ANOVA results (p < 0.05). RSM analysis revealed that extraction temperature and ethanol concentration exerted the most significant effects on the extraction yields, while extraction time played a supportive role. The optimal conditions (70 °C, 40% ethanol, 120 min) significantly enhanced compound recovery while reducing solvent and energy consumption, thereby contributing to the development of efficient and sustainable extraction processes. Collectively, the validated HPLC-DAD method and the optimized extraction strategy developed in this study provide a reliable framework for the quality standardization and industrial application of C. tricuspidata leaf extracts in functional food, cosmetic, and pharmaceutical products.