Design of Experiments-Based Optimization of Flavonoids Extraction from <i>Daphne genkwa</i> Flower Buds and Flavonoids Contents at Different Blooming Stages

The flower buds of Daphne genkwa have been reported as a potent resource associated with anti-angiogenic, anti-tumor, anti-rheumatoid arthritis activities, as well as immunoregulation. This paper aimed to establish an optimal extraction method for flavonoids, as active phytochemicals, and to conduct a comparative analysis by profiling the different blooming stages. Optimized shaking extraction conditions from the design of experiments (DoE), such as minutely mixture design, 23 full factorial design, and polynomial regression analysis, involved an agitation speed of 150 rpm and temperature of 65 °C for 12 h in 56% (v/v) acetone solvent. After, a comparative analysis was performed on three blooming stages, juvenile bud, mature purple bud, and complete flowering, by ultra-high-performance liquid chromatography-photodiode array-mass spectrometry (UHPLC-PDA-MS). Most flavonoids increased during bud growth and then decreased when the bud opened for blooming. In particular, apigenin 7-O-glucuronide, genkwanin 5-O-primeveroside, and genkwanin strikingly showcased this pattern. Furthermore, the raw spectrometric dataset was subjected to orthogonal projection to latent structures discriminant analysis (OPLS-DA) to find significant differences in the flavonoids from the juvenile bud, mature purple bud, and complete flowering. In conclusion, the present study facilitates an understanding of flavonoid change at different blooming stages and provides a momentous reference in the research of D. genkwa