Eleven compounds, including nine known flavonoid glycosides (1–4, 6–8, and 10–11), one isoflavone glycoside (5), and a glansreginic acid (9), were isolated from the 80% ethanol extract of commercial Astragali Complanati Semen (ACS). All chemical structures were determined by spectroscopic analyses, including 1D and 2D NMR. Compounds 2, 4, 5, 6, 9, and 10 were isolated and identified from the title plant for the first time. Biological evaluation revealed that all the isolates showed promising anti-NO production, and 1, 2, 3, and 8 were more potent in antioxidant activity than vitamin E. The major peaks in the UPLC and HPLC profiles identified their chemical structures by comparing their retention time and UV spectra with those of the reference substances. Furthermore, nine of the eleven samples collected from North, Middle, and South regions of Taiwan possessed similar HPLC fingerprints and were identified as Astragali Complanati Semen, whereas the other two samples from southern Taiwan would be the adulterants due to the different fingerprinting patterns. In addition, an HPLC-UV method was employed to determine the content of target compound complanatuside (11) with good linear regression (R2 = 0.9998) for ACS in the Taiwanese market. Of the isolates, flavonol glycosides 1 and 3 were the major peaks in HPLC/UPLC, and showed more potent antioxidant and anti-NO production activities than that of 11, revealing that these compounds can be the available agents for the quality control of ACS.

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