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Coreopsis tinctoria flowering tops (CTFs) is a popular health product as herbal tea or as a traditional medicinal herb that is rich in saponins and exerts substantial biological activity. In this study, an ultrasound-assisted aqueous two-phase system (ATPS) was utilized to extract total saponins from CTFs and optimize the extraction process by response surface methodology. Moreover, the nitric oxide and nitrite scavenging capability, and N-nitrosamine formation inhibitory activity of total saponins were evaluated. Results showed that the optimal conditions for total saponins were 37.76% (w/w) ammonium sulfate and 35.62% (w/w) ethanol in ATPS coupled with ultrasonic-assisted extraction. Under the optimal conditions, the maximum yield of total saponins of 33.4 g/kg can be obtained from the CTFs raw material. The nitric oxide radical scavenging, nitrite scavenging, and N-nitrosamine inhibitory activities (SC50) were 287.92 ± 7.42, 191.63 ± 7.69, and 1787.4 ± 51.26 μg/mL, respectively. The total saponins has a certain nitric oxide and nitrite scavenging capability, and N-nitrosamine formation inhibitory activity in vitro. Given these activities, research on saponins from CTFs provides profound and lasting implications for the novel applications of C. tinctoria. [ABSTRACT FROM AUTHOR]