Design of Experiments-Based Optimization of Flavonoids Extraction from Daphne genkwa 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 shak-ing 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 signifi-cant differences in the flavonoids from the juvenile bud, mature purple bud, and complete flower-ing. 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.