Authors:Katarzyna Szewczyk, Łukasz Komsta and Agnieszka Skalska-Kamińska
High performance thin-layer chromatography (HPTLC) combined with densitometry has been used for analysis of the triterpenoid content of acetone and ethyl acetate extracts of the leaves of
(Sims.) Opiz. After optimization of the extraction process, HPTLC separation was successfully standardized, using silica gel plates washed with methanol and dichloromethane, dichloromethane-ethyl acetate 18.5:1.5 as mobile phase, and 8% H
in ethanol (
) as spray reagent. Triterpenoids in the leaves of
were quantified by measurement of absorbance. Three spots were observed on chromatograms — the sum of α and β-amyrin, oleanolic acid, and an unidentified compound. All of the triterpenoid components were analyzed for the first time in this plant. The triterpenoid content was calculated statistically.
Authors:Nusrat Fayaz Bhatt, Raghbir Chand Gupta and Yogita Bansal
, R. Meyer , I.A. Dubery , Characterisation of two phenotypes of Centella asiatica in Southern Africa through the composition of four triterpenoids in callus, cell suspensions and leaves , Plant Cell Tiss. Organ Cult. 94 ( 2008
Authors:Alamgir A. Dar, Payare L. Sangwan, Nasseb Singh and Anil Kumar
Herein, the isolation, characterization, and method validation of 5 bioactive terpenoids, viz., taraxerol (1), 3ß-hydroxyoleanane-12-one (2), betulinic acid (3), ursolic acid (4), and oleanolic acid (5) from Codonopsis ovata has been reported. A novel, accurate, and cost-effective high-performance thin-layer chromatography method was developed for the simultaneous quantification of 5 natural products on silica-gel 60 F254 plate using a ternary solvent system, n-hexane-ethyl acetate-formic acid (8.0:2.0:0.4, V/V). Markers were quantified after post-chromatographic derivatization with ceric ammonium sulfate reagent. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. All calibration curves showed a good linear relationship (r > 0.995) within the test range. Precision was evaluated by intra- and inter-day tests with relative standard deviations <1.99%, and accuracy validation recovery 85.66–91.87 with relative standard deviations >2.00°%. Oleanolic acid (5) and ursolic acid (4) were found in major quantity which is advantageous because of their anticancer, anti-inflammatory, anti-mutagenic, and anti-obesity activities. Based on our results, the developed method features good quantification parameters and can serve as an effective quality control method for standardization of Codonopsis ovata.
Authors:V. K. Hridya, P. S. Godson and N. Chandrasekar
Mangroves are the source of several bioactive secondary metabolites and have proven activity against human, animal and plant pathogens. Rhizophora mucronata is a mangrove species belonging to the family Rhizophoraceae. Betulin and lupeol are two naturally occurring pentacyclic triterpenoids having excellent biological properties. Chloroform extract of R. mucronata, collected from Pitchavaram, Muthupett and Manakudy regions of Tamilnadu, was chromatographed on silica gel 60F254 plates with nhexane and ethyl acetate (80:20) (v/v) as the mobile phase. Derivatizations were done using anisaldehyde and sulfuric acid reagents. The triterpenoids such as betulin and lupeol were identified through HPTLC method. This is the first report for the thin layer chromatographic (TLC) identification of triterpenoids such as betulin and lupeol from R. mucronata, which will be the most valuable information to identify the antimalarial and antiviral activities.
A simple and rapid method for analysis of four major triterpenoids in Centella asiatica preparations is described. Thin-layer chromatography is widely used for saponin analysis; here it was used for identification and quantification of the pure compounds and the compounds in complex mixtures, by densitometric analysis after TLC. Ethanolic extracts of leaves from C. asiatica and authentic standards of asiatic acid, asiaticoside, madecassic acid, and madecassoside, were separated by normal-phase TLC with chloroform-methanol-acetic acid-water 60:32:12:8 (v/v) as mobile phase. These conditions enabled successful separation of the triterpenoid acids and their respective glycosides from other components of the crude extracts. The separated compounds were detected with anisaldehyde-sulfuric acid solution. The intensity of the resulting violet spots was proportional to the amount of saponin or sapogenin present. The correlation coefficients of calibration plots were between 0.9904 and 0.9982 and the plots were linear over the range 1.25–10 nmol standard, corresponding to approximately 0.6–5 μg for the acids and 1.2–10 μg for the glycosides. When the method was used for analysis of the specific saponins in C. asiatica leaves the results were consistent with literature reports.
A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the quantification of two bioactive lupane triterpenoids, namely, lupeol and betulin from Diospyros melanoxyon stem bark. Chromatographic separation was achieved on aluminium foil-backed HPTLC plates using ethyl acetate-hexane (1.8:8.2, v/v) as mobile phase. The compounds were quantified at their wave length of maximum absorbance in the range of 100–500 ng per spot. The instrumental precision was 0.82% and 1.07% (CV) and the repeatability of the method was 1.33% and 1.17% (CV), respectively, for lupeol and betulin. The minimum detectable amount was found to be 40 and 50 ng per spot for lupeol and betulin, respectively. The linear regression analysis data for the calibration plots showed a good linear relationship with r2 = 0.9996 for lupeol and 0.9997 for betulin. The method was validated for precision, recovery, and repeatability as per the International Conference on Harmonization guidelines. The developed HPTLC method is very accurate and precise, and has been successfully applied for the assay of these bioactive molecules in D. melanoxylon stem bark.
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.
Triterpenoid betulinic acid (BA) was detected, quantified, and reported for the first time from leaf extract of Achyranthes aspera along with much known oleanolic acid (OA). Extraction was achieved using ultrasonic exposure, and reversed-phase.ultra flow liquid chromatographic (RP.UFLC) technique was employed during investigation. RP.UFLC separation was achieved on a Hibar 250–4.6 mm, 5 μ, Lichrospher 100, C18e column using methanol and water (90:10) as mobile phase with pH adjusted to 5.0 using glacial acetic acid (GAA) in an isocratic mode. The content of BA (0.25 mg g−1 fresh weight [FW]) was ∼75% higher than OA (0.06 mg g−1 FW). These results suggest BA to be the major triterpenoid compared to OA in the leaf of A. aspera. High-performance thin-layer chromatography (HPTLC) separation of the two triterpenic acids (oleanolic and betulinic acid) was also achieved on silica gel G 60 F254, 50 ×~ 100 mm glass TLC plates, using benzene, ethyl acetate, and formic acid as solvent system in a ratio of 67.9:22.7:9.4. Anisaldehyde reagent was used for detection. The method was used for the screening of oleanolic and betulinic acids from A. aspera leaf extract. Similarly, Fourier transform-infrared (FT-IR) spectroscopic analysis was done in the mid IR region of 400–4000 cm−1 with 64 scan speed using OMNIC 8.1 (ver. 8.1.210) software. The results were also supported by HPTLC and FT-IR data.
This article enfolds a rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method for the estimation of four triterpenoids, namely, betulin (BU), betulinic acid (BA), lupeol (LU), and oleanolic acid (OA), from the bark, roots, and leaves of Betula utilis D. Don, an endangered Himalayan tree. All the four phytoconstituents have high therapeutic value. Separation was performed on thin-layer chromatography (TLC) aluminum plates precoated with silica 60 F254 (20 × 20 cm) followed by detection of betulin, lupeol, and oleanolic acid carried out by derivatizing the plate with ceric ammonium sulfate followed by heating at 110°C for 5 min. For betulinic acid, the plate was dried and visualized after spraying with Liebermann‒Burchard reagent. CAMAG TLC Scanner 4 equipped with winCATS software was used for densitometric scanning at 500–550 nm. The proposed technique was further validated in terms of linearity, precision, accuracy, and sensitivity as per the International Conference on Harmonisation (ICH) guidelines. A good linear relationship was obtained for the calibration plots with r2 = 0.9994, 0.9995, 0.9969, and 0.9998 for betulin, lupeol, oleanolic acid, and betulinic acid, respectively. Accuracy of the method was checked by recovery study conducted at three different levels with the average recovery between 98.9% and 99.3% for all the four markers.
Lupenone was isolated for the first time from the stem bark of Diospyros melanoxylon and characterized with the help of physical and spectroscopic data. A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the quantification of lupenone in D. melanoxylon stem bark. HPTLC analysis was performed on HPTLC plates by using a binary mobile phase of n-hexane–ethyl acetate (8.2:1.8, v/v). It was quantified at 395 nm after derivatization with methanol—sulfuric acid reagent. The limits of detection and quantification were found to be 40 ng and 100 ng per spot, respectively. The linear regression analysis data for the calibration plot in the range of 100–500 ng spot−1 showed a good linear relationship between peak area and concentration (r2 = 0.9997). The instrumental precision was 1.01% (coefficient of variation [CV]) and the repeatability of the method was 2.17% (CV). The method was validated for precision, recovery, and repeatability as per the International Conference on Harmonization guidelines. The developed HPTLC method is very accurate and precise and has been successfully used for the estimation of lupenone in D. melanoxylon stem bark.