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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Shrishailappa Badami, Mahesh Gupta, Noble Mathew, Subramania Meyyanathan, Bhojraj Suresh, and David Bendell

Among the complex mixture of biologically active compounds in the bark of Grewia tiliaefolia , a plant used in folklore, lupeol, a constituent of the bark, has been used as an analytical marker indicative of the quality of the plant. A sensitive and reliable quantitative high-performance thin-layer chromatographic method has been developed for the determination of lupeol from Grewia tiliaefolia . Chloroform extracts of bark from five different sources were used for HPTLC on silica gel with benzene-ethyl acetate, 95 + 5, as mobile phase. Under these conditions the R F of lupeol was 0.40. The calibration plot was linear in the range 0.5 to 1.5 μg lupeol and the correlation coefficient, 0.999, was indicative of good linear dependence of peak area on concentration. The mean assay of lupeol was 2.902 ± 0.243 mg g −1 bark. The method enables reliable quantification of lupeol and good resolution and separation of lupeol from other constituents of Grewia tiliaefolia . To ascertain the purity of the peak from the test sample its in-situ reflectance spectrum was compared with that from standard lupeol; clear superimposibility indicated the purity of the peaks. Recovery values from 98.00 to 99.78% showed the reliability and reproducibility of the method were excellent. The HPTLC method proposed for quantitative monitoring of lupeol in Grewia tiliaefolia is rapid, simple, and accurate and can be used for routine quality testing.

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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.

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A simple and sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the evaluation of biomarker lupeol in the leaves of five different species of genus Ficus (Ficus carica, Ficus nitida, Ficus ingens, Ficus palmata, and Ficus vest) belonging to family Moraceae. Chromatography was performed on glass-backed silica gel 60 F254 precoated HPTLC plates with solvents toluene-methanol (9:1, v/v) as the mobile phase. After development, the HPTLC plate was derivatized with p-anisaldehyde reagent, scanned, and quantified at 540 nm. The system was found to give compact spot for lupeol at R F = 0.32 ± 0.01. The precisions and accuracy (n = 6) for lupeol were found to be 1.47–1.64% and 1.63–1.86%, and 99–100% and 99.4–99.7%, respectively, for inter-day and intra-day. Lupeol was found to be present in four species, i.e., F. carica (0.4%, w/w), F. nitida (1.4%, w/w), F. palmata (0.33%, w/w), and F. vest (0.59%, w/w), while it was absent in F. ingens. The statistical analysis proved that the developed method is reproducible and selective. The developed method can be used as an important tool to assure the therapeutic dose of active ingredients in herbal formulations as well as for standardization and quality control of bulk drugs and in-process formulations. This method can also be employed for the further study of degradation kinetics and determination of lupeol in plasma and other biological fluids.

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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.

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A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of anti-inflammatory compounds betulinic acid (BA, 1), 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO, 2), and lupeol (LU, 3) in the roots of Clerodendrum phlomidis. Extraction efficiency of marker compounds was studied using normal (cold and hot), ultrasonic, as well as microwave-assisted extraction techniques with various solvents. Well-resolved separation of marker compounds was achieved on silica gel 60F254 plates using the mobile phase consisting of chloroform-methanol (98:2, ½/½). Marker compounds were scanned using the densitometric reflection-absorption mode after post-chromatographic derivatization with vanillin-sulfuric acid reagent. Validation of method was performed as per the International Conference on Harmonization (ICH) guidelines. Report on the occurrence of betulinic acid for the first time in C. phlomidis is of chemotaxonomic importance. In addition, anti-inflammatory potential of the rare sterol ECTO (2) on lipopolysaccharide (LPS)-stimulated production of pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) was also evaluated as it was not reported earlier.

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Kalanchoe pinnata (Crassulaceae), Bombax ceiba (Bombaceae), and Morus alba (Moraceae) are important drugs in Indian and Chinese Systems of Medicine. Leaves of K. pinnata are reported to show anti-inflammatory, antifungal, and antibacterial activity and are reported to contain quercetin, kaempferol, α-amyrin, β-sitosterol, ferulic, and caffeic acid. Leaves of B. ceiba are reported for the treatment of diarrhea, dysentery, gonorrhea, and bladder ulceration and are reported to contain shamimin, lupeol, β-sitosterol, and α-amyrin. Leaves of M. alba are reported to show anti-inflammatory, emollient, hypolipidemic, and diuretic activity and are reported to contain quercetin, rutin, moracetin, and artocarpin. In the present work, a method for simultaneous quantification of three marker compounds, viz., α-amyrin, lupeol, and β-sitosterol from the leaves of K. pinnata, B. ceiba, and M. alba was developed and validated as per International Conference on Harmonization (ICH) guidelines. This is the first report of simultaneous quantification of these three compounds. α-Amyrin, lupeol, and β-sitosterol resolved at R F 0.71, 0.28, and 0.16, respectively, on thin-layer chromatography (TLC) when developed in the solvent system of toluene-ethyl acetate (9.5:0.5). The linearity range for α-amyrin, lupeol, and β-sitosterol was found to be 160–560, 150–900, and 80–480 ng spot−1, respectively, with correlation coefficients (r values) of 0.999, 0.997, and 0.995, respectively. The amount of α-amyrin, lupeol, and β-sitosterol were found to be in range of 0.013–0.047, 0.046–0.318, and 0.028–0.123 % w/w, respectively. The developed methods were found to be accurate, precise, and reproducible and can be used for routine quality control of herbal material and formulations containing K. pinnata, B. ceiba, and M. alba.

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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.

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The present paper reports a validated high-performance thin-layer chromatography (HPTLC)‒densitometric method for the simultaneous quantification of phenolic (ferulic acid and caffeic acid) and terpenoid (β-sitosterol and lupeol) markers in Convolvulus pluricaulis Choisy. According to Ayurveda, it is commonly known as ‘Shankhpushpi’ due to its ‘Conch’ or ‘Shankh’-shape flower. The plant species, viz., Clitoria ternatea L., Evolvulus alsinoides (L.) L., and Tephrosia purpurea (L.) Pers., also having similar flowers are reported as its adulterants/substitutes. This creates a problem in its quality and efficacy in the commercial drug market of India. Therefore, a HPTLCmethod was performed on a pre-coated silica gel 60 F254 plate with the aforesaid markers. The solvent system toluene–ethyl acetate–formic acid (8.5:1.5:0.1) was determined to be the best system for the simultaneous separation of caffeic acid, ferulic acid, β-sitosterol, and lupeol at R F values of 0.14, 0.29, 0.48, and 0.63, respectively. A densitometric scanning profile of all the samples at 580 nm showed peaks for all the four markers of varying heights in the samples, except the absence of caffeic acid in Tephrosia purpurea. The developed method was standardized and validated for the quantification of active principal-based quality-control markers in terms of precision, accuracy, linearity, recovery, and repeatability. It will help to maintain batch-to-batch consistency and identification of adulterants/substitutes in raw materials during production of drug in the pharmaceutical units.

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Among the complex mixture of biologically active compounds in Leptadenia pyrotechnica, three compounds have been used as analytical markers. A sensitive high-performance thin-layer chromatographic (HPTLC) method has been developed for the estimation. Methanolic extracts of whole plants from three populations were used on aluminum pre-coated silica gel 60 F254 plates with different mobile phases to determine the amount of β-sitosterol, lupeol, and oleanolic acid with RF value of 0.64, 0.84, and 0.47, respectively. The calibration curve was linear in the range of 2–10 μg. The method is reliable for the quantification, separation, and good resolution of these compounds from other constituents of L. pyrotechnica. To ascertain the purity of the peak from the test sample, its in-situ reflectance spectrum was compared with that from standards; the clear superimposability indicated the purity of the peaks.

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Summary

A simple, specific, and quantitative high-performance thin-layer chromatographic (HPTLC) method has been developed for the quantitative determination of lupeol in 2 marketed formulations, namely, Manasamitra vatakam and Amree plus capsule. Chromatographic development was performed by using a pre-coated silica gel 60 F254 aluminum-backed plate, and the development was carried out using toluene-ethyl acetate (9.48:0.52, V/V) as the optimized mobile phase. The developed TLC plates were derivatized by using anisaldehyde-sulfuric acid reagent. The detection of lupeol was carried out at 600 nm. Box-Behnken design was applied for optimization of the chromatographic conditions, and combinations of factors, such as mobile phase composition (volume of ethyl acetate) (A), chamber saturation time (B), and migration distance (C) likely to affect R F were identified from preliminary trials and further optimized using a response surface design. Among 3 factors, the significant factor found was the volume of ethyl acetate that resulted in higher change in the R F value and can be considered as a critical method parameter. Full factorial design was applied for optimization of extraction efficiency. The factors selected for the optimization process were volume of methanol (A) and duration of extraction (B) with percentage yield of extract as response. The linear ranges were found to be 500–3000 ng per band. The accuracy and precision measured were less than 2% relative standard deviation for lupeol. The sensitivity of the method in terms of the limit of detection (LOD) and the limit of quantification (LOQ) was measured. The proposed method was found to be accurate, precise, reproducible, robust, and specific and can be applicable for the determination of lupeol in the quality-control testing of extract and polyherbal formulations.

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