Determination of HBC/steroid stability constant

The stability constants (K_S) of the inclusion complex between HBC and steroids were determined according to the modified protocol described by Ma et al.[31]. The mixtures of various concentrations of HBC (from 0 to 98 mM), 2% (v/v) EGME and the double molar excess of AD or cholest-4-en-3-one were incubated in the thermoblock at 30 °C, 1000 rpm for 48 h. Afterward, the suspensions were filtered through 0.45 μm membrane filters, diluted 0–200 times with ACN, centrifuged at 15 000 g for 15 min, and concentration of soluble steroid was quantified by HPLC–DAD using calibration on external standards [8]. The linear function was fitted to the data obtained for AD. The stability constant for AD:HBC 1:1 complexes (K_1:1) was calculated from Eq. (1), where S₀ is an y-intercept [32, 33].

The quadratic function ([S]=a[HBC]2+bHBC+S0) was fitted to the data attained for cholest-4-en-3-one. The stability constants for cholest-4-en-3-one, HBC 1:1 (K_1:1) and 1:2 (K_1:2) complexes, were calculated from the Eq. (2) and (3), respectively [33].

S₀ refers to the solubility of steroids in 2% (v/v) EGME/water solution (144.2 ± 7.1 μM for AD and 4.8 ± 0.5 μM for cholest-4-en-3-one).