J  Chromatogr Sci, 61(3), 225-233 (2023). Development of a method for analyzing rosmarinic acid (RA), caffeic acid (CA), ursolic acid (UA) and oleanolic acid (OA) in different organs of Salvia deserta Schang qualitatively and quantitatively by HPTLC (A) for RA and CA, on silica gel with chloroform – methanol - formic acid 20:4:1, quantitative absorption measurement at 330 nm; (B) for UA and OA, on silica gel F254 with cyclohexane - ethyl acetate - methanol 20:4:1, quantitative absorption measurement at 550 nm. The linearity ranges were 0.13-0.44, 0.01-0.09, 0.50-2.50 and 0.50-2.50 mg for RA, CA, UA and OA, respectively, with corresponding correlation coefficients of 0.9938, 0.9981, 0.9971 and 0.9969, respectively. The recovery rates were between 95 % and 105 %. LOD and LOQ were 50, 58, 25, 33 and 160, 191, 80, 106 ng for RA, CA, UA and OA, respectively.

J Chromatogr Sci, 61 (1), 66-73 (2023). Development of a rapid, easy and simple method for the isolation and purification of α-glucosidase inhibitors of the ethyl acetate extract of Thymelaea hirsuta (EaTh) by TLC-enzymatic test (TLC/EZ), after EaTh demonstrated previously a potent α-glucosidase inhibitory effect. Soxhlet extraction from Thymelaea hirsuta (T. hirsuta), separation on a silica gel column, TLC on silica gel. Detection of α-glucosidase inhibitors directly on the TLC plate using the glucose oxidase peroxidase method (GOD-POD), then characterization of active compounds using HPLC-MS analysis, resulting the main α-glucosidase inhibitors present in EaTh with a molecular ion [M + H]+ at m/z = 543. The proposed method is suitable for a reliable isolation and purification of α-glucosidase inhibitors present in EaTh and can be proposed as an interesting alternative to the classical method for the isolation and purification of α-glucosidase inhibitors in plant extracts.

J. Chinese Trad. Patent Med. 44 (6), 1770-1772 (2022). Ganqingqinglan (Dracocephalum tanguticum Maxim.) is a traditional Tibetan medicinal plant, containing flavonoids, terpenes, polysaccharides. It has antihypoxic, antibacterial, antiviral, and hypoglycemic effects and is used as the main component in the traditional Tibetan medicine for the treatment of hepatitis and gastritis. For quality control, TLC of the plant extracts on silica gel (1) for oleanic acid, with toluene - ethyl acetate - glacial acetic acid 28:8:1, detection by spraying with 10 % sulfuric acid in ethanol and heating at 105 °C until the zones are clearly visualized, evaluation under UV 366 nm; (2) for chlorogenic acid, with toluene - methyl acetate - propanone - methanol - methyl acid 30:5:5:20:1, evaluation under UV 366 nm. The method was applied for the analysis of 18 batches of real life samples and proved to be simple, specific, reproducible, robust and well suitable for the purpose.

J Pharmacogn Phytochem, 12(1), 159-167 (2023). TLC of methanolic Soxhlet extracts on silica gel with toluene – ethyl acetate – formic acid 16:4:1. Visualization under UV 254 nm and 366 nm. Two bands only (hRF values 10 and 45), observed with densitometric scanning at UV 366 nm, were present in Trianthema portulacastrum roots (Aizoaceae), whereas a different profile was observed with Boerhavia diffusa roots (Nyctaginaceae), which is sometimes substituted for T. portulacastrum.

J Pharm Bioallied Sci 15(Suppl.2), S948-S951 (2023). Sample was the ethyl acetate fraction of an ethanolic extract of Viola odorata aerial parts (Violaceae). Standards were coumarinic compounds: esculetin (1) and umbelliferone (2). TLC and HPTLC on silica gel with toluene – ethyl acetate – formic acid 5:4:1. Visualization under UV 254 nm and 366 nm; densitometric scanning at 366 nm. Both (1) and (2) were found in the extract (hRF values 30 and 53, respectively, in TLC). Alternative mobile phases were also tested (TLC only): toluene – ethyl acetate 1:1 (hRF values 47 and 68) and chloroform – methanol 97:3 (hRF values 20 and 41).

 J. Pharmacogn. Phytochem. 12(1), 6-14 (2023). TLC silica gel layers were used to monitor the purification through column chromatography (CC) of a chloroform fraction of the methanolic root bark extract of Rauvolfia vomitoria (Apocynaceae). Mobile phases were petroleum ether – ethyl acetate 4:1 (MP1), dichloromethane – methanol 20:1 (MP2), and dichloromethane – methanol 15:1 (MP3). Visualization under UV 254 nm. Preparative TLC on thicker silica gel was performed on two subfractions: (A) with dichloromethane – methanol 100:7 for the isolation of the methyl esters of eudesmic acid and of trimethoxycinnamic acid (hRF values 35 and 28, respectively, in MP1); (B) with MP2 for the isolation of an indole alkaloid: kumujan B (= 1-carbomethoxy-β-carboline, hRF value 40 in MP2). Other indole alkaloids were isolated through CC: ajmaline, mauensine and reserpine (hRF values 35, 13 and 47, respectively, in MP3).

J. Chromatogr. A. 1683, 463522 (2022). HPTLC of powdered root sample of Saussurea costus on silica gel with n-hexane - toluene - tetrahydrofuran 10:1:2. Planar bioluminiscent cytotoxicity assay by dipping into concentrated PBS, followed by removal of excess liquid and adding of human embryonic kidney (HEK) 293T cells expressing ELuc, which uses D-luciferin as the substrate for light emission (cell suspension of 5000 cells/μL). To avoid drying out of the plate, two stripes of paper were added to the side of the chamber, which were wetted with 1.5 mL of bidistilled water. After incubation for 6 h, the HPTLC plate was completely dried under cold air, followed by dipping twice into the respective bioluminescent substrate solution for each cell type. Dose-dependent cytotoxicity activity was calculated for the bioluminescence signal reduction. 

J Chromatogr A 1653, 462442 (2021). Samples were peptides obtained through tryptic hydrolysis of the 5 most abundant milk proteins: α-lactalbumin (α-LA), β-lactoglobulin (β-LG), α-, β- and κ-casein (CA). As standards, synthetic whey and pea (Pisum sativum, Fabaceae) peptides (selected based on the in silico tryptic digest of α-LA, β-LG, legumin A, and vicilin with one or zero miscleavages) were only used in the last assay for prediction of the RF values of peptides with known amino-acid (AA) sequences. Two-dimensional HPTLC on silica gel (pre-washed with methanol and activated 10 min at 100°), first with basic mobile phase sec-butanol – pyridine – ammonia – water 39:34:10:26, and (after 12h drying) in the orthogonal direction with acidic mobile phase sec-butanol – pyridine – acetic acid – water 11:8:2:5. Derivatization for peptides and proteins by immersion into fluorescamine (0.05 % in acetone); visualization under UV 254 nm and 365 nm. Computer-assisted determination of the x- and y-coordinates of the derivatized zones. Repeatability (n=8) of the 2D-HPTLC was statistically tested with the Kolmogorov-Smirnov test for normal distribution and with Dixon’s Q test for outliers. Relative standard deviation (RSD) for the RF values was 12.9 % for the first dimension (y-coordinates) and 16.5 % for the second dimension (x-coordinates). According to their higher intensity and sharpness, 15 – 20 detected zones from each protein hydrolyzate were selected, manually scraped from the derivatized layer, dissolved in formic acid solution (0.1 % in acetonitrile – water 3:2), mixed with an equal volume of matrix (dihydroxybenzoic acid 2 % in acetonitrile – water 3:7), crystallized on air on a ground steel target, before being desorbed by the laser beam of the MALDI-TOF-MS/MS (matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry). Direct hyphenation of HPTLC to MS was not performed, to avoid zone diffusion during plate coating with the matrix and to circumvent the stronger binding of polar peptides on the layer.  The MS spectra were acquired in positive reflector mode in m/z range 340 – 4000 (10 – 2500 for fragments), using an external peptide as calibration standard. Identification of 51 from the 85 selected peptides according to AA sequences was performed, using software programs allowing m/z calculation of protein fragments and estimation of cleavage sites. Correlation of the retention behaviour of the peptides with their properties (molecular weight MW, isoelectric point IEP, charges, polarity) was tested with Student’s two-sided t-test after calculation of Pearson’s correlation coefficients. The correlation was significant with IEP, percentages of anionic AA and of non-polar AA; but not with the following properties: MW, percentages of cationic AA and of uncharged polar AA. Finally, based on the correlation results, regression formulas were found to calculate the x- and y-coordinates of any known peptide from the percentage of non-polar AA (or vice-versa). The prediction power of these formulas was verified by repeating the complete 2D-HPTLC-MS experiment with the standard peptides of whey and of peas, and measuring the absolute and relative deviations between the actual x- and y-coordinates and the predicted values. The absolute deviations were higher in the lower RF zones. The average, relative RF value deviations (range 22.1 – 25.7 %) were not different between whey and pea peptides.