Quantification and optimization of ethanolic extract containing the bioactive flavonoids from Millettia pulchra radix

Millettia pulchra is traditionally used for treating diseases, including joint pain, fever, ane-mia, and allergies. It is also a potential resource of natural flavonoid derivatives, which represents major constituents of this plant. This study aimed to isolate the major compounds from M. pul-chra radix, develop and validate the HPLC-PDA method to determine their contents, and optimize its extraction. Four major flavonoid derivatives (karanjin, lanceolatin B, 2^′′,2^′′-dimethylpyrano-[5^′′,6^′′:7,8]-flavone, and pongamol) were isolated using silica gel column chromatography, crystalliza-tion techniques in large amounts with high purities (>95%). A simple, accurate high-performance liquid chromatography–photodiode array (HPLC–PDA) detection method has been developed and validated with significantly statistical impacts according to International Conference on Harmonization (ICH) guidelines. The Response Surface Methodology (RSM), Artificial Neural Network (ANN) models were employed to predictive performance and optimization of the extraction pro-cess. The optimized conditions for the extraction of major flavonoids were: extraction time (twice), solvent/material ratio (9.5), and ethanol concentration (72.5%). Our research suggests an effective method, which will be helpful for quality control in the pharmaceutical development of this species.