Tlc f 254 plates

For IAA identification assay, 10 µl of extract and 5 µl of IAA standard solution (50 µg.ml⁻¹ in water/methanol) were spotted on a glass capillary plate coated with a thin layer of silica gel 60 (TLC F 254 plates; Merck, Germany). The solvent system used was N-butanol:ammonia:water (10:1:10) (Sethia et al., 2015 (link)). The plates were developed by spraying Salkowski reagent (0.01 M of FeCl3 in 35% perchloric acid) and heating at 40°C for 10 min. Spots were visualized under light and UV light (254 nm) and identified by comparing Retention factor (Rf) value of the IAA produced by RmBm31 with the standard.

Hexane, diethyl ether, petroleum ether, acetone, ethyl acetate, methanol, dimethyl sulfoxide, oleanolic acid (≥97%) and ursolic acid (UR) (≥97%) were purchased from Sigma‐Aldrich, Singapore. TLC F254 plates, aspirin, and indomethacin were purchased from Merck, Singapore. All chemicals and solvents were of analytical grade.

The TLC procedure described by Gambhava et al. (2013) was used to determine the number of chemical components in the crude CE, the petroleum ether bark extract fraction (PEF), and the hexane: ethyl acetate bark extract fraction (HEF) of S. buxifolia, with some minor modifications. Merck TLC F254 plates were loaded with 30 µl of the extracts, and the prepared plates were then developed in hexane: ethyl acetate: methanol (8.2:1.8:0.5; v/v/v). Oleanolic acid and ursolic acid were considered as commercial standards and prepared by dissolving in absolute methanol (100 µg/ml). The chromatograms were dried at 105°C for 5 min to remove solvents. The chemical components of the extracts were identified by either using UV light (254 and 365 nm) or by spraying with Liebermann reagent (10% H₂SO₄ in ethanol and 10% acetic anhydride (Sigma‐Aldrich, Singapore)) solution and then dried at 105°C. Identification of the components of S. buxifolia bark extracts was carried out by comparison of the retention factor (Rf) of the various spots. The Rf was obtained by using a meter rule to measure the distance moved by the solvent and the distance moved by the spot; based on this, the retention factor (Rf values) of the various spots was calculated using the following equation: $\text{Rf}=S_1/S_2,$ where S₁ is the distance travelled by the solute and S₂ is the distance travelled by solvent front on the TLC plates.