Development of a Supercritical Fluid Extraction Process for Preparation of Phytosteroids Rich Extract From <i>Tinospora cordifolia</i> Stem and Determination of Content of Three Phytosteroids Using a Validated Ultra‐High Performance Liquid Chromatogr

Tinospora cordifolia is an integral part of traditional medicine, rich in secondary metabolites of various chemical classes including phytosteroids. These phytosteroids possess cholesterol-lowering property; however, systematic studies are lacking regarding preparation of extract enriched with such bioactive compounds. In this study, supercritical fluid extraction (SFE) method was developed to obtain three phytosteroids (ecdysterone, stigmasterol, and β-sitosterol) enriched extract from T. cordifolia stem. The phytosteroid content was quantified using validated ultra-high performance liquid chromatography method. Separation of analytes was achieved using C-18 column (150 mm × 4.6 mm, 5.0 µm) as stationary phase and water-acetonitrile mixture (80:20) as mobile phase with detection at 242 nm (range of 190-350 nm; photodiode array detector). The limit of detection was 0.25, 0.23, and 0.39 µg/mL for ecdysterone, stigmasterol, and β-sitosterol respectively, whereas, limit of quantification was 0.85, 0.78, and 1.25 µg/mL for ecdysterone, stigmasterol, and β-sitosterol respectively. Accuracy of recovery was 80%-109%, with precision (RSD) of < 2 for all three targeted analytes. For SFE, under optimized condition i.e., automated back pressure = 100 bar, CO₂ flow = 30 g/min, modifier flow = 4 g/min, extraction and collection vessel temperature = 60°C and 45°C, respectively, and extraction time = 120 min; phytosteroid (ecdysterone + stigmasterol + β-sitosterol) content was obtained as 319.36 µg/g which was significantly higher than its content in extract (108.04 µg/g) obtained by refluxing with 50% aqueous ethanol as extraction solvent. Method performance and greenness of SFE was compared with ultrasound-assisted extraction. Result showed 2.06-fold increase in extract yield in case SFE than ultrasound-assisted extraction. Greenness score calculated for SFE and ultrasound-assisted extraction was 0.65 and 0.59, respectively. T. cordifolia SFE extract can be key component in developing herbal products to address obesity related problems. Thus, the developed extraction method can be recommended to herbal industries for preparation of effective medicines with diverse therapeutic applications.