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This study aimed to identify Pheretima aspergillum (Guang‐Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high‐performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura‐2‐Parameter model was used to analyze genetic distance, and phylogenetic neighbor‐joining tree was constructed for species identification of 20 labeled Guang‐Pheretima samples. A high‐performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang‐Pheretima (P. aspergillum), and the others as the adulterants‐Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor‐joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant‐M. magna. The incidence of adulterants for Guang‐Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant‐ or animal‐derived products for safety concerns to avoid misidentification.

Ultrasonic-assisted extraction (UAE) was optimized using response surface methodology (RSM) to maintain the cyto-protective activity of M.toringoides against oxidative stress. The optimal conditions for UAE were a 58 mL/g liquid-solid ratio, a 38 °C extraction temperature, an 85% solvent concentration, and a 19-min extraction time, which resulted in a protection rate of 54.57% against hydrogen peroxide-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). These results were comparable to the predicted value of 53.75%. The extracts showed excellent antioxidant activity, and phlorizin was detected in the dried leaves of Malus.toringoides. The highest yield of phlorizin (101.239 mg/g) was also obtained using these conditions. Taken together, these results showed that the method successfully integrated RSM and partial least squares regression methods to optimize M.toringoides extraction to yield the highest cyto-protective activity and effectively increase the yield of phlorizin from M.toringoides.