Silica gel 60h

Evaporated diethyl ether extracts were fractionated by adsorption preparative TLC on 20 cm × 20 cm glass plates coated manually with silica gel 60H (Merck, Darmstad, Germany). The solvent system, chloroform:methanol 97:3 (v/v), was applied for developing. The fractions of triterpenoid acids and free (non-esterified) steroids and neutral triterpenoids were localized on the plates by comparison to the appropriate standards, as described earlier [16 (link),24 (link)]. Fractions were eluted from the gel in diethyl ether. Subsequently, fractions containing free neutral triterpenes and sterols (R_F 0.3–0.9) were directly analyzed by GC-MS, whereas the fractions containing triterpene acids (R_F 0.2–0.3) were methylated with diazomethane. The average recovery of α-amyrin, uvaol, stigmasterol, and ursolic acid methyl ester from preparative TLC plates was 98.6, 97.2, 98.9, and 96.1%, respectively [25 (link)].

Methanol extracts from roots and n-butanol extracts from the culture medium were hydrolyzed by 11% HCl in 70% methanol for 2h on a heating mantle under reflux as described earlier [25 (link)]. Subsequently, the hydrolysates were diluted with distilled water, methanol was evaporated in a rotary evaporator, and the obtained aqueous remaining were extracted 3 times with 40 mL portions of diethyl ether in a separation funnel. The obtained extract was washed with distilled water 3 times and evaporated to dryness.
The dried extracts were fractionated by preparative TLC on 20 cm × 20 cm glass plates, manually coated with silica gel 60H (Merck, Darmstadt, Germany). A solvent system with a chloroform:methanol (95/5,v/v) mixture was used for developing the plates. The obtained OA was methylated with diazomethane [13 (link),14 (link),28 (link)].

Evaporated diethyl ether extracts obtained from the hairy roots, as well as roots and aerial parts of pot plants were fractionated by adsorption preparative TLC on 20 cm × 20 cm glass plates coated manually with silica gel 60H (Merck, Darmstadt, Germany). The solvent system chloroform: methanol 97:3 (v/v) was applied for developing. Four fractions were obtained as described by Sykłowska-Baranek et al. (2022): (i) esters; (ii) free steroids and neutral triterpenoids; (iii) free triterpenoid acids; and (iv) glycosides [51 (link)]. Fraction (ii) containing free steroids and neutral triterpenoids (alcohols) was directly analyzed using GC–MS, gas chromatography-mass spectrometer (Agilent Technologies 7890A); the triterpenoid acid fraction (iii) was methylated with diazomethane prior to GC–MS analysis as described previously [52 (link)]; the ester fraction (i) was subjected to alkaline hydrolysis to release the steroid core from ester forms; and the glycoside fraction (iv) to acidic hydrolysis to release the aglycones.

Evaporated diethyl ether extracts were fractionated by adsorption preparative TLC on 20 cm × 20 cm glass plates, manually coated with silica gel 60H (Merck, Darmstadt, Germany). The solvent system chloroform: methanol 97:3 (v/v) was used to develop the plates. Two fractions were obtained: (i) free steroids and neutral triterpenoids (Rf of 0.3–0.9), and (ii) free triterpenoid acids (Rf 0.2–0.3) as described earlier [37 (link)]. The individual fractions were localized on plated by comparison with standards of oleanolic acid, sitosterol and α-amyrin, visualized by spraying the relevant part of the plate with 50% H₂SO₄, followed by heating with a hot-air stream. Fractions were eluted from the gel in diethyl ether. The fraction of free steroids and neutral triterpenoids was directly analyzed without derivatization using GC–MS (Agilent Technologies 7890A, Santa Clara, CA, USA) while the fraction of triterpenoid acids was methylated with diazomethane [31 (link),32 (link)].

Method I Boc-Gly-Δ^ZPhe 0.160 g (0.5 mmol), Et₃N 0.196 mL (1.1 mmol) and (S)-glycidol 0.266 mL (2.0 mmol) were dissolved in 2.0 mL of acetonitrile, and TBTU (Abdelmoty et al., 1994 (link)) 0.208 g (0.65 mmol) was then added. Mixture was stirred at room temperature for 2.5 h, and solvent removed under reduced pressure. The residue was dissolved in 70 mL of ethyl acetate and washed subsequently with: 1 M HCl (3 × 5 mL), saturated KHCO₃ (3 × 5 mL) and brine. Organic phase was dried over MgSO₄ and filtered, and solvents were removed. We were unable to purify a mixture of products obtained using column chromatography with silica gel 60H (Merck) as stationary phase and various eluents. Thus, crude mixture was used in deprotection step. HRMS (ESI) indicated the presence of the desired product as a major one: m/z calcd for C₁₉H₂₄N₂O₆ (M + Na)⁺ 399.1526; found 399.1529.
Method II iso-butyl chloroformate 0.066 mL (0.5 mmol) was added to solution of Boc-Gly-Δ^ZPhe 0.160 g (0.5 mmol) and Et₃N 0.070 mL (0.5 mmol) in dichloromethane when cooling in ice bath to −15 °C. After 1.5 min, glycidol 0.133 mL (1.0 mmol) was added. The mixture was left to warm to room temperature, and stirring was continued for next 24 h. Further steps of synthesis were performed according to the methodology described for Method I and afforded similar mixture of products.

Evaporated chloroform extracts were fractionated by adsorption preparative TLC on 20 cm × 20 cm glass plates coated manually with silica gel 60H (Merck, Darmstad, Germany). The solvent system chloroform:methanol 97:3 (v/v) was applied for developing. Three fractions were obtained as described earlier: free (non-esterified) steroids and triterpenoids, triterpenoid acids, and steroid/triterpenoid esters [10 (link)]. Fractions were eluted from the gel in diethyl ether. Subsequently, fractions containing free neutral triterpenes and sterols (R_F 0.3–0.9) were directly analyzed by GC-MS, fractions containing triterpene acids (R_F 0.2–0.3) were methylated with diazomethane, and fractions containing triterpenoid (triterpene and sterol) esters (R_F 0.9–1) were subjected to alkaline hydrolysis. The average recovery of α-amyrin, uvaol, stigmasterol, and ursolic acid methyl ester from preparative TLC plates was 98.6, 97.2, 98.9 and 96.1%, respectively.