Authors:Prabha Shetty, Kiran Mangaonkar, and Ramesh Sane
Among the complex mixture of biologically active compounds in the leaves of
, a plant used in traditional medicine, ursolic acid, a constituent of the leaves, has been used as an analytical marker indicative of the quality of the plant. A sensitive thin-layer chromatographic method has been developed for determination of ursolic acid in the leaves of
. Chromatographic separation was performed on silica gel HPTLC plates with toluene-ethyl acetate-triethylamine-methanol, 7 + 2 + 1 + 1 (
), as mobile phase. The plates were scanned densitometrically at
= 366 nm. The method was validated for precision, repeatability, and accuracy. Intra-day and inter-day
were 0.43% and 1.54%, respectively. Instrumental precision and repeatability of the method were found to be 0.54 and 1.20 (% CV), respectively. The accuracy was checked by studying recovery at three levels; average recovery was 100.26%. The method proposed for quantitative monitoring of ursolic acid in
is rapid, simple, and accurate and can be used for routine quality testing.
A simple and rapid high-performance thin-layer chromatographic method has been established and validated for determination of ursolic acid in rabbit plasma after ingestion of an aqueous slurry of
R. Br. leaves. Chromatography was performed on silica gel HPTLC plates with toluene-ethyl acetate-triethyl-amine-methanol, 7 + 2 + 1 + 1 (
), as mobile phase. The plates were scanned densitometrically at
= 366 nm. The response to ursolic acid in spiked plasma was a linear function of concentration in the range 10 to 80 μg mL
. The detection and quantification limits were 3.0 and 10 μg mL
, respectively. The method has been successfully used to study the pharmacokinetics of
R. Br. using ursolic acid as a marker.
Authors:Kedar Rout, Bandita Dash, and Rajesh Singh
An important bioactive molecule, ursolic acid was isolated from the leaves of Diospyros melanoxylon and characterized with help of physical and spectroscopic data viz. m. p, IR, 1H, and 13C NMR. A high-performance thin-layer chromatography method has also been developed and validated for its quantification in D. melanoxylon leaves. The high-performance thin-layer chromatography analysis was performed on high performance thin-layer chromatography plates using chloroform-methanol (9.5:0.5, v/v) as mobile phase. The compound was quantified at 540 nm after derivatzation with sulphuric acid reagent. The sensitivity of the method with respect to limit of detection and limit of quantification were found to be 20 and 40 ng per spot. The response was obtained as a linear function of peak area and concentration in the range of 50 to 450 ng per spot with correlation coefficient of r2 = 0.9998. The method showed excellent accuracy greater than 97.54% with acceptable precession and was successfully validated according to International Conference of Harmonization protocols. Antimicrobial screenings of ursolic acid revealed potent activity against two Gram-positive bacteria viz. Staphylococcus aureus and Enterococcus faecalis and three fungal starins viz. Aspergillus niger, Candida tropicalis, and Candida albicans.
A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of ursolic acid and β-sitosterol in the methanolic fraction of Paederia foetida L. leaves was developed for the first time. For achieving good separation, a mobile phase of toluene‒ethyl acetate‒formic acid (8:2:0.1, v/v) was used. The densitometric determination was carried out at 550 and 522 nm in reflection/absorption mode for ursolic acid and β-sitosterol. The calibration curves were linear in the range of 100-600 ng per spot for ursolic acid and β-sitosterol. During the analysis, the methanolic fraction of P. foetida L. leaves showed the presence of ursolic acid (0.12 ± 0.05%) and β-sitosterol (0.08 ± 0.12%). The proposed method is simple, precise, specific, accurate, less time-consuming, and cost-effective. The statistical analysis of the data obtained proves that the method is reproducible and selective and can be used for the routine analysis of the reported phenolic compounds in crude drug and extracts. The simultaneous quantification of these compounds has not yet been reported in P. foetida L. leaves which may be utilized for the proper standardization of the plant.
Oldenlandia corymbosa Linn. (Rubiaceae) is an important herb traditionally used as a febrifuge and liver tonic. In this study, a high-performance thin-layer chromatography (HPTLC) method has been established for the quantification of four bioactive markers, oleanolic acid (OA), ursolic acid (UA), lupeol (LU), and stigmasterol (ST), in the whole plant of O. corymbosa. Separation was achieved on silica gel 60 F254 HPTLC plates using hexane-ethyl acetate-methanol (8.2:1.8:0.5, v/v) for oleanolic acid and ursolic acid; and toluene-methanol (9.4:0.6, v/v) for lupeol and stigmasterol as the mobile phases. The quantitation of the four markers was carried out using the densitometric scanning at 540 nm after derivatization using sulfuric acid reagent. The linear regression analysis data for the calibration plots showed a good linear relationship (r2 = 0.9831–0.9979) in the concentration range of 1200–4200 ng for oleanolic acid, 400–1400 ng for ursolic acid, 100–500 ng for lupeol, and 500–2500 ng per spot for stigmasterol with respect to area. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for oleanolic acid, ursolic acid, lupeol, and stigmasterol were 98.77 to 99.12%, indicating the good reproducibility. Stigmasterol 1.19 ± 0.04% w/w was present at high concentration, and oleanolic acid 0.012 ± 0.006% w/w was present at low concentration in the whole plant powder. The proposed HPTLC method was found to be simple, precise, sensitive, accurate, reproducible, and robust.
A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of lupeol and ursolic acid in the methanolic fraction of four different species of Bauhinia leaves was developed for the first time. For achieving good separation, a mobile phase of toluene—ethyl acetate—formic acid (8:2:0.1, v/v) was used. The densitometric determination was carried out at 550 nm and 520 nm in reflection—absorption mode for lupeol and ursolic acid, respectively, which were linear in the range of 100–600 ng per band. During the analysis, lupeol (0.15%) and ursolic acid (0.11%) were found to be the highest in the leaves of B. acuminata. The proposed method is simple, precise, specific, and accurate. The statistical analysis of the obtained data proves that the method is reproducible and selective and can be used for the routine analysis of the reported terpenoids in crude drug and extracts. The simultaneous quantification of these compounds has not yet been reported in the leaves of the studied Bauhinia species which may be utilized for the proper standardization of these species.
A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination and validation of ursolic acid, β-sitosterol, lupeol, and quercetin in the methanolic fraction of Ichnocarpus frutescens L. was developed for the first time. For achieving good separation, a mobile phase of toluene‒ethyl acetate‒formic acid (8:2:0.1, v/v) was used. Densitometric determination was carried out at 500 nm for ursolic acid, 550 nm for β-sitosterol, 650 nm for lupeol, and 310 nm for quercetin in reflection–absorption mode, and the calibration curves were linear in the range of 100–600 ng per spot. During the analysis, the methanolic fraction of I. frutescens L. showed the presence of ursolic acid (0.34%), β-sitosterol (0.27%), lupeol (0.27%), and quercetin (0.26%). The proposed method is simple, precise, specific, and accurate. The obtained data can be used for routine analysis of reported biomarkers in crude drug and extracts. The simultaneous quantification and method validation of these biomarkers have not yet been reported in I. frutescens L., which may be utilized for the proper standardization of the plant.
A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for simultaneous analysis of three triterpene acids (corosolic, oleanolic, and ursolic acids) in extracts from inflorescences, leaves, and fruits of Prunus serotina Ehrh. (American black cherry). Separation of the acids was accomplished on a C18 column (5 μm, 250 mm × 4.6 mm i.d.) and recorded at 210 nm. The greatest resolution was achieved with 90:10 (υ/υ) methanol-1% aqueous orthophosphoric acid as mobile phase at a flow rate of 0.6 mL min−1. The correlation coefficients for all the calibration plots (r > 0.998) showed linearity good over the range tested. The relative standard deviation of the method was less than 3.3% for intra and inter-day assays, and average recovery was between 95.9 and 100.9%. Sensitivity was high; detection limits were between 0.034 and 0.067 μg mL−1. Total amounts of triterpene acids were 0.451–0.928, 0.031, and 0.911–1.455% in the inflorescences, fruits, and leaves of P. serotina, depending on the time of harvesting.
A new, simple, reliable, and validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of four bioactive markers, ursolic acid (1), betulinic acid (2), β-sitosterol (3), and lupeol (4) in the stem and root barks of Alstonia scholaris. Extraction efficiency of the targeted markers from the bark matrixes with organic solvents using cold percolation, hot extraction, and ultrasonic extraction were studied, which showed that ultrasonic extraction was best for sample preparation. The separation was achieved on silica gel 60F254 HPTLC plates using chloroform-methanol (99:1 v/v) as mobile phase. The quantitation of four markers was carried out using the densitometric reflection/absorption mode at 680 nm after post chromatographic derivatization using vanillin-sulphuric acid reagent. The linear regression analysis data for the calibration plots showed a good linear relationship (r2 ≥ 0.998) in the concentration range 2–10 μg per spot for the ursolic acid and lupeol and 4–20 μg per spot for the betulinic acid and β-sitosterol with respect to area. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD), and quantitation (LOQ). Method specificity was confirmed using retention factor (RF) and visible spectral (post chromatographic scan) correlation of marker compounds in the samples and standard tracks.
Authors:Katerina Naumoska, Breda Simonovska, Alen Albreht, and Irena Vovk
Separation of three triterpenic acids (ursolic, oleanolic and betulinic acid) was achieved on different thin-layer chromatography (TLC) (silica gel 60) and high-performance thin-layer chromatography (HPTLC) sorbents (silica gel 60, C2 RP and C18 RP) using several developing solvents, based on the non-polar diluent n-hexane, and ester (methyl acetate, ethyl acetate, ethyl propionate) as selector. Anisaldehyde and molybdophosphoric acid detection reagents were used. Finally, a simple method on a C18 RP HPTLC plate was developed using n-hexane-ethyl acetate (5:1 v/v) as a developing solvent in a horizontal developing chamber. The method was used for the screening of ursolic, oleanolic and betulinic acids in different vegetable extracts. Other plant triterpenoids (lupeol, α-amyrin, β-amyrin, cycloartenol, lupenone, friedelin, lupeol acetate, cycloartenol acetate) and phytosterols (β-sitosterol, stigmasterol) did not interfere. TLC-MS was used as a tool for the additional confirmation of the presence of ursolic, oleanolic, and betulinic acids in some of the studied vegetable extracts. Ursolic and oleanolic acids were found in radicchio Leonardo and white-colored radicchio di Castelfranco extracts for the first time, while betulinic acid was not detected in the eggplant extract by MS, although it was suggested at first by TLC analysis. Pre-chromatographic bromination on the HPTLC silica gel 60 plates and subsequent development in toluene-chloroform-diethyl ether-formic acid (20:16:4:0.1, v/v) provided a superior resolution of these compounds.