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is the amount of free insulin in the supernatant. Partition Coefficient The partition coefficients of insulin, C1, and C2 were also determined. Briefly, 1 mg of insulin or the freeze-dried powder of C1 or C2 (equivalent to 1 mg insulin) was added to 1 mL of octanol/water (1:1) and incubated at 25°C for 24 h with vigorous stirring (600 rpm). After 24 h, the samples were centrifuged at 15,602 × g for 15 min at 4°C. Finally, the aqueous and oil phases were separated, collected, and analyzed using RP-HPLC as previously described. The partition coefficient was determined as follows: (2) denotes the concentration of insulin in the aqueous phase at equilibrium.

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