Vydac 218tp1022

The filtered extract was injected into a Vydac 218TP1022 (Grace, Deerfield, IL, USA) preparative stainless-steel column (C-18, 10 μm) (22 × 250 mm²) at a 5.0 mL/min flow rate with 0.12% (v/v) TFA/water. Acetonitrile concentration in the eluting solvent was mounted using linear gradients to 20% for 10 min and to 70% over a period of 40 min. Major peaks were tested for insulinotropic activity, as detailed above, and positive fractions were purified again at a flow rate of 1.0 mL/min following recent studies [2 (link),57 (link)].

Crude bark extract (HWHF) reconstituted in 0.12% (v/v; TFA/water) and analysed by RP-HPLC. Filtered extract was injected into a (22 x 250 mm) Vydac 218TP1022 preparative stainless steel 10 μm C-18 column (Grace, Deerfield, IL, USA), equilibrated with 0.12% (v/v; TFA/water) at the flow rate of 5 ml/min. Acetonitrile was used as eluent at linear gradients to 20% over 10min and to 70% over a period of 40 min. Peak fractions detected at 254 nm and 360 nm were collected and individual peak retention time noted [20 (link)]. Major peaks were tested for insulinotropic activity as per aforementioned methods, and positive peak fractions were analysed further using Vydac 208TP510 (10 x 250 mm) semi-preparative stainless steel 5μm C-18 column (Phenomenex, UK) at a flow rate of 1 ml/min.

RP-HPLC was used to analyse the EEAS crude extract diluted in 0.12% (v/v) TFA/water. The filtered extract was loaded on a Vydac 218TP1022 preparative stainless steel 10 μm C-18 column (22 × 250 mm) (Grace, Deerfield, IL, USA), equilibrated with 0.12% (v/v) TFA/water at a flow rate of 5 mL/min. Acetonitrile was used as an eluent using linear gradients of 20% over 10 min and 70% over 40 min. Individual peak retention times were recorded and peak fractions detected at 254 nm were collected [14 (link)]. Insulinotropic peak fractions were subsequently assessed using a semi-preparative Vydac 208TP510 (10 × 250 mm) 5 μm C-18 column (Phenomenex, UK) eluting with acetonitrile as indicated above, at flow rate of 1 mL/min.