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<p><i>Cassia fistula</i> is a valuable medicinal tree which has various medicinal effects in conventional and modern pharmacological diagnosis systems. The present investigation dealt with the standardization of the extraction method as well as the development of a simple, effective, and validated thin-layer chromatography (TLC) method for the simultaneous determination of two biologically active constituents of this plant, <i>i.e.,</i> β-sitosterol and lupeol in flowers, fruits, root, stem bark and leaves of this species. Out of three different methods, <i>i.e.,</i> maceration, soxhlet, and reflux, used to extract the samples, reflux was found to be the most efficient method for the extraction of β-sitosterol and lupeol content. Moreover, the separation was achieved on TLC plates precoated with 0.2&#xa0;mm silica gel 60 F<sub>254</sub>, using a mobile phase toluene‒methanol‒chloroform (8:1:1, <i>V</i>/<i>V</i>). The plates were derivatized with freshly prepared reagent anisaldehyde‒sulphuric acid, followed by heating at 150&#xa0;°C for 5&#xa0;min on a CAMAG plate heater. The densitometric scanning of the plates was performed at <i>λ</i><sub>max</sub> = 525&#xa0;nm in a CAMAG TLC Scanner 4 equipped with winCATS software version 1.4.8, using a tungsten lamp. The linear regression data for the calibration curves showed a satisfactory linear relationship with <i>r</i><sup>2</sup> = 0.992 and 0.999 for β-sitosterol and lupeol. Accuracy of the method was checked by a recovery study conducted at three different levels, with average recoveries of 99.81% and 100.97% for β-sitosterol and lupeol, respectively. The method exhibited no interference from plant matrix. The results showed the maximum extraction of the targeted components in reflux conditions and the highest content of β-sitosterol (0.195 ± 0.01 mg/g ± SD) in fruit pulp extract and lupeol (0.092 ± 0.00 mg/g ± SD) in stem bark extract. The developed and validated TLC method may be utilized as an excellent technique for the quality control of different plant parts of <i>C. fistula</i>.</p>

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A validated thin-layer chromatography method for the concurrent determination of β-sitosterol and lupeol in Cassia fistula L.—an important species of Ayurveda

  • 期刊论文

  • 发表日期：

    2025年5月1日

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  • Samiksha Parihar,
  • Hari Om Saxena,
  • Ganesh Pawar,
  • Harish Singh Ginwal,
  • Neelu Singh

摘要

Cassia fistula is a valuable medicinal tree which has various medicinal effects in conventional and modern pharmacological diagnosis systems. The present investigation dealt with the standardization of the extraction method as well as the development of a simple, effective, and validated thin-layer chromatography (TLC) method for the simultaneous determination of two biologically active constituents of this plant, i.e., β-sitosterol and lupeol in flowers, fruits, root, stem bark and leaves of this species. Out of three different methods, i.e., maceration, soxhlet, and reflux, used to extract the samples, reflux was found to be the most efficient method for the extraction of β-sitosterol and lupeol content. Moreover, the separation was achieved on TLC plates precoated with 0.2 mm silica gel 60 F254, using a mobile phase toluene‒methanol‒chloroform (8:1:1, V/V). The plates were derivatized with freshly prepared reagent anisaldehyde‒sulphuric acid, followed by heating at 150 °C for 5 min on a CAMAG plate heater. The densitometric scanning of the plates was performed at λmax = 525 nm in a CAMAG TLC Scanner 4 equipped with winCATS software version 1.4.8, using a tungsten lamp. The linear regression data for the calibration curves showed a satisfactory linear relationship with r2 = 0.992 and 0.999 for β-sitosterol and lupeol. Accuracy of the method was checked by a recovery study conducted at three different levels, with average recoveries of 99.81% and 100.97% for β-sitosterol and lupeol, respectively. The method exhibited no interference from plant matrix. The results showed the maximum extraction of the targeted components in reflux conditions and the highest content of β-sitosterol (0.195 ± 0.01 mg/g ± SD) in fruit pulp extract and lupeol (0.092 ± 0.00 mg/g ± SD) in stem bark extract. The developed and validated TLC method may be utilized as an excellent technique for the quality control of different plant parts of C. fistula.