Ten male Sprague–Dawley rats were randomly divided into three groups (n = 8) as control, treated with calycosin, and treated with calycosin-loaded nanoliposomes (dissolved in 1% ethanol). The doses of calycoin groups were 30 mg/kg for oral administration. After the rats were intragastrically administered with free or Nano-formulated calycosin, the 0.5 mL blood was taken from the fundus venous plexus of each rat at different time intervals. The blood samples were then centrifuged (4000 rpm, 10 min), and the plasma was removed and to stored − 80 °C. The content of the drug in the plasma sample was then calculated by the established LC-MS/MS method based on the previous report [35].

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