C (Eppendorf), sonication for 15 min, and centrifugation. For acid hydrolysis, the
C (Eppendorf), sonication for 15 min, and centrifugation. For acid hydrolysis, the methanolic extract (1 mL) was transferred to a 5.0 mL tube and reacted with 20 (w/w) hydrochloric acidMolecules 2021, 26,14 of(1 mL) for 1 h at 1000 rpm and 60 C utilizing a ThermoMixer C. For the handle (devoid of acid hydrolysis), the extract (1 mL) was reacted with water (1 mL) in place of hydrochloric acid. The reaction mixture was extracted with chloroform (two mL three) and concentrated. The crude extract was dissolved in methanol (1 mL) and evaporated beneath reduced pressure to remove the chloroform. The resulting residue containing sapogenin was dissolved in 1 tetrahydrofuran in methanol (1 mL), transferred to a 5-mL volumetric flask, and diluted with methanol to a total volume of 5 mL. The remedy was filtered by way of a 0.22- nylon syringe filter (Shimadzu GLC Ltd.) to prepare samples for LC-MS. A hederagenin resolution in methanol (0.96 /mL; 1 mL) was subjected towards the same acid hydrolysis treatment and chloroform extraction to estimate the recovery rate. Hederagenin was obtained from TCI Chemicals (Tokyo, Japan). Hydrochloric acid and tetrahydrofuran (HPLC grade) were purchased from FUJIFILM Wako (Japan). 4.three.3. HGS Content material Hederagenin concentrations with the acid hydrolyzed extracts of matoa peel and salak peel (Section 4.3.two) were measured by LC-MS in accordance with a previously described method [44] with modifications. HPLC was performed on a LC-20A Prominence program equipped with an SIL-20AC autosampler (Shimadzu, Japan). An LCMS-2020 mass spectrometer equipped with an electrospray Trisodium citrate dihydrate Cancer ionization supply operating in unfavorable mode was made use of to identify and quantify the target analytes using chromatographic data processed using LabSolutions computer software (Shimadzu). Sample solutions (1 ) had been injected into an XBridge BEH C18 column (three.5 , two.1 150 mm; Waters, Milford, MA, USA). The separation was achieved by applying a gradient elution of solvent A (10 mM ammonium bicarbonate) and B (methanol) as follows: 0 min, linear gradient 405 A; 38 min, 25 A; 180 min, 40 A. The flow rate was 0.2 mL/min, along with the column temperature was 40 C. The eluent was passed through an electrospray source. A capillary voltage of three.five kV was utilized within the damaging ion mode. Nitrogen was made use of because the drying gas at a flow rate of 15 L/min and nebulizing gas at a flow rate of 1.5 L/min. The desolvation line temperature was set at 250 C. The ion trap was operated in full scan mode from m/z 50 to 1000 and selected ion monitoring mode with m/z 471 for any molecular ion [M – H]of hederagenin. Identification and quantification were achieved by an external strategy using hederagenin standard solutions. Hederagenin (98 ; TCI Chemicals) was dissolved in a small level of tetrahydrofuran and diluted with methanol to prepare the standard options. Quantification with the integrated peak locations of the samples permitted comparison using the calibration curves of the o-Phenanthroline Autophagy normal options. Samples from the acid hydrolyzed matoa solutions were subjected to further dilution just before LC-MS injection. A single milliliter of your remedy was transferred to a 10-mL volumetric flask and diluted with methanol to a total volume of ten mL to adjust its concentration within the array of the calibration curves utilized for quantification (ten ppm conc. 10 ppb). The HGS content in the samples was estimated by multiplying the molar concentration of hederagenin within the samples by the molecular weight (MW) of saponin (1). The MW for 1 was calculat.
