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A simple normal-phase high-performance thin-layer chromatography (HPTLC) method has been developed for the quantification of β-sitosterol in hairy root cultures of Clitoria ternatea vis-à-vis natural plant parts. Chromatographic separation was achieved on normal-phase TLC plates by using optimized mobile phase of n-hexane. acetone (8:2, v/v). Densitometric scanning was performed after postchromatographic derivatization of the plate at 414 nm. The limits of detection and quantification were found to be 40 ng and 100 ng spot−1, respectively. A linear response of calibration line was observed over the range of 100 to 500 ng spot−1 with a correlation coefficient of r 2 = 0.999. Recovery values from 97.22 to 98.51% with small coefficient of variation showed excellent accuracy of the method. The method was validated according to the International Conference on Harmonization (ICH) protocol. The method enables excellent separation and accurate quantification of β-sitosterol in different plant parts of the two floral varieties of C. ternatea along with their cultured roots derived through genetic transformation. β-Sitosterol content was comparable between the blue-flowered and white-flowered plants; roots had a higher content than that in stem and leaf regardless of the two floral varieties. Transformed root cultures had a lower content of β-sitosterol compared to natural roots.

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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.

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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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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 R F 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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Extensive research on Ficus species has shown their excellent cytotoxic potential which motivated the authors for further evaluation of its other species. In this article, the β-sitosterol content in the chloroform extract of the leaves of five Ficus species (Ficus carica [FCCE], Ficus nitida [FNCE], Ficus ingens [FICE], Ficus palmata [FPCE], and Ficus vasta [FVCE]) was estimated by a validated high-performance thin-layer chromatography (HPTLC) method along with cytotoxic activity. The chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with hexane and ethyl acetate (8:2, v/v) as the mobile phase. The developed plate was derivatized with p-anisaldehyde reagent, scanned, and quantified at λ = 550 nm. It furnished a compact and intense peak of β-sitosterol at R F = 0.17 ± 0.001. The contents of β-sitosterol (μg mg−1 of the dried weight of the extract) in the selected Ficus species were found as: FCCE (1.047 μg mg−1) > FVCE (0.771 μg mg−1) > FNCE (0.372 μg mg−1) > FPCE (0.309 μg mg−1), while it was absent in F. ingens. Methylthiazol tetrazolium (MTT) assay was used to compare the cytotoxic potential of all Ficus species against HepG2 (liver), HEK-293 (kidney), MCF-7 (breast), and MDA-MB 231 (breast) cell lines. The FCCE exhibited good cytotoxic property against HepG2, HEK-293, and MDA-MB-231 cells (IC50: 32.5, 41.4, and 47.3 μg mL−1, respectively), while FICE showed against HepG2 and MDA-MB-231 cells (IC50: 31.4 and 41.2 μg mL−1, respectively). The remaining Ficus extracts were found to be very less effective or insignificant. The cytotoxic property of FCCE is also supported by the HPTLC estimation of β-sitosterol which is reported to exhibit anticancer properties by interfering with multiple cell signaling pathways, including cell cycle, apoptosis, and proliferation. Our data suggest that the developed HPTLC method can be further employed in the analysis of marketed herbal formulations, and the active Ficus species can be further subjected to isolation of cytotoxic phytoconstituents.

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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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A densitometric thin-layer chromatographic method has been established for quantitative determination of β-sitosterol in pumpkin seed oil ( Cucurbita pepo L.). Chromatography was performed on silica gel 60 F 254 TLC plates, with toluene-ethyl acetate-glacial acetic acid 15:4:1 ( v/v ) as mobile phase. Densitometric quantitation was performed at 525 nm. The method was validated by determination of linearity (15–750 μg mL −1 ), precision (RSD = 2.36%), and limits of detection (LOD = 0.65 μg per band) and quantification (LOQ = 1.99 μg per band). Average recovery from the pharmaceutical preparation was 96.15%. The method enables simple, rapid, and precise quantitative determination of β-sitosterol in pharmaceutical preparations and can be used for routine quality control.

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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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In this study, we have developed a validated high-performance thin-layer chromatography (HPTLC) method for the concurrent estimation of the biomarkers β-amyrin and β-sitosterol in dichloromethane and ethanol extracts of the aerial parts of Tinospora cordifolia (TCDC and TCET) and Calotropis gigantia (CGDC and CGET). Chromatographic estimations were carried out on HPTLC (glass-backed silica gel 60 F254) plates with solvents hexane and ethyl acetate in the ratio of 7.5:2.5, v/v (as the mobile phase). Post development, the plate was derivatized with p-anisaldehyde reagent to furnish compact spots of β-amyrin and β-sitosterol and scanned at λ max = 530 nm. Well-resolved, compact as well as intense peaks of β-sitosterol (R F = 0.26 ± 0.001) and β-amyrin (R F = 0.39 ± 0.001) were found. The linear regression equation and the correlation coefficient square (r 2) for β-amyrin (Y = 6.118x + 460.76 and 0.9959) and β-sitosterol (Y = 7.109x + 1069.1 and 0.9967) in the concentration range of 100–1400 ng spot−1 indicated good linear relationship. The low values of the percent relative standard deviation (% RSD) for intra-day and inter-day precisions for β-amyrin (1.003–1.148 and 0.993–1.105) and β-sitosterol (0.578– 0.969 and 0.513–0.813) suggested that the method is precise. The % recovery and % RSD values were found to be 98.42–99.29% and 1.103–2.103, respectively, for β-amyrin and 98.33–99.39% and 1.375–2.346, respectively, for β-sitosterol, which confirms the good accuracy of the proposed method. The quantity of β-amyrin in TCDC, CGDC, TCET, and CGET was found to be 70.14, 10.76, 4.85, and 0.87 μg mg−1, respectively, of the dried weight of the extracts, while the β-sitosterol content was found to be 19.4, 18.5, 1.18, and 0.27 μg mg−1, respectively. The highest quantity of β-amyrin and β-sitosterol in the dichloromethane fractions of T. cordifolia and C. gigantia validated the wide range of their proved activities including antioxidant, anticancer, and hepatoprotective features. The above developed method can be further employed in the analysis of these markers in marketed preparations.

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Reversed-phase thin-layer chromatography (RP-TLC) is a sensitive, reliable, and reproducible technique, which can analyze a number of components simultaneously. This technique was exploited in the present study to identify the adulteration of vegetable oils (groundnut, soybean, and sunflower oil) in ghee, using the fact that β-sitosterol, an unsaponifiable matter, is present in vegetable oils, while absent in pure ghee. For this, RP-TLC of reference standards, unsaponifiable matter of pure ghee (cow and buffalo), pure vegetable oils, and the ghee adulterated with these vegetable oils (≥1%) was carried out on plates with different stationary phases, viz., RP-18, RP-8, and RP-2 plates using solvent system comprising of petroleum ether, acetonitrile, and methanol. The results revealed that adulteration of vegetable oils in ghee can easily be detected at a level of as low as 1 percent on RP-18 and RP-8 plates, while RP-2 plate did not offer any help in detection of adulteration. Thus, with the help of RP-TLC, a robust technique, the purity of ghee can be screened with respect to the presence of vegetable oils.

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