Astra 6 software

The weight-average molecular weight (Mw), number-average molecular weight (Mn), and the polydispersity index (Ip = Mw/Mn) of the samples were determined by high-performance size exclusion chromatography (HPSEC) coupled with a multiangle light scattering detector (MALS, Dawn Heleos-II, Wyatt Technology) and a differential refractive index (RI) detector (Hitachi L2490). HPSEC system was composed of an HPLC system Prominence Shimadzu, a PL aquagel-OH mixed, 8 μm (Varian) guard column (U 7.5 mm ×  L 50 mm), and a PL aquagel-OH mixed (Varian) separation column (U 7.5 × 300 mm, operating range 10²–10⁷ g/mol). GY785 DR and GY785 DRS were dissolved in distilled water at a concentration of 2 mg/mL and filtered through 0.45 μm cellulose acetate syringe filter before being injected. The elution was performed at 1 mL/min rate with 0.1 M ammonium acetate containing 0.03% NaN_3, filtered through 0.1 μm membrane (Durapore Membrane, PVDF, Hydrophilic type VVLP, Millipore). Data were computed with Astra software 6.1 (Wyatt Technology).

Astra software 6.1 (Wyatt Technology) was used for the data analysis. The molecular weight of lipase–control sample was obtained from MALS and dRI detectors, applying the Zimm model [24 (link)] with a first order fit using 12 scattering angles (from 44.8° to 149.0°). The refractive index increment (dn/dc) used was 0.185 mL/mg [25 ], a generic protein value based on BSA. The second virial coefficient was assumed to be negligible.
dRI fractograms were used for lipase quantification, where the peak maximum was used to calculate the relative concentrations, normalized in relation to the lipase-control sample.
The Stokes–Einstein equation was applied to determine the hydrodynamic radius (r_h),
${\mathrm{r}}_{\mathrm{h}}=\frac{\mathrm{k}\mathrm{T}}{6\mathrm{\pi }\mathrm{\eta }\mathrm{D}}$
where k is the Boltzmann constant, T is the absolute temperature, ƞ is dynamic viscosity of the solvent, and D is the translational diffusion coefficient, using the FFFhydRad 2.1 MATLAB App [26 (link)]. The channel thickness (w) was 286.4 µm, determined using BSA with a hydrodynamic diameter of 6.6 nm. The void time (t⁰) was calculated according to Wahlund and Nilsson, 2012.

The data analysis was performed using Astra software 6.1 (Wyatt Technology). The molecular weight of lipase in the positive control was determined by the Zimm model [24 ] with a 1^st order fit using 14 scattering angles, from 34.8° to 163.3°. The UV extinction coefficient used was 1.4 ml/(mg cm) and a refractive index increment (dn/dc) of 0.185 ml/mg [25 ]. As the dn/dc used is for a generic protein (BSA), the molecular weights reported for lipase are somewhat apparent. The second virial coefficient was considered negligible.
Once the lipase peak was identified, its retention time was used as reference for the rest of the samples, where only the UV detector was taken into consideration in order to calculate the free lipase in solution after its interaction with the respective flavonoid. For this, peak maximum was used for relative concentration that was normalized with respect to the control sample (containing only lipase).