Single peak that eluted at about 5.67 min, with detection at 242 nm (Figure 9a,b). Ecdysterone was further subjected to liquid chromatography ass spectrometry (LC S) evaluation. Reversed-phase ultra-high-performance liquid chromatography separation was performed applying an UltiMate 3000 RSLC HPLC program (Thermo Fisher Scientific, Bremen, Germany) on a Kinetex C18 (100 2.1 mm, 2.6 , one hundred A particle size) column (Phenomenex, Torrance, CA, USA), coupled having a Q Exactive HF-X (Thermo Scientific, Bremen, Germany) mass spectrometer. Chromatographic evaluation was performed at a flow price of 200 /min with all the mobile phase β adrenergic receptor Activator web described above. Full scan mass spectra have been measured within a mass variety of m/z 100 to 1000 at a resolution of 240,000 (m/z 200), an automatic gain manage targetInt. J. Mol. Sci. 2021, 22,16 ofof three 106 , as well as a maximum injection time of 100 ms. High-resolution MS analysis in the sample revealed an precise mass of ecdysterone at m/z of 481.3152 [M+H]+ (Figure 9c).Figure 9. Chromatograms from the HPLC analysis of ecdysterone at a concentration of 150 /mL (a) and 750 /mL (b) in MeOH. (c) Representative high-resolution total ion current (TIC) chromatogram of ecdysterone.The basal diet program contained 19.four MJ gross energy/kg DM and offered the following crude nutrients as determined by official strategies [33] ( DM): crude protein, 21.2; crude fat, 5.6; crude ash, three.two; crude fibre, 3.8. The rats of all groups had totally free access to their diets which were fed for 4 weeks. Water was continuously readily available ad libitum from nipple drinkers. four.2. Sample Collection Rats had been decapitated beneath CO2 anaesthesia inside the non-fasted state. Blood was taken up in heparin-coated polyethylene tubes (AppliChem, Darmstadt, Germany) and the plasma was separated from the remaining blood components by centrifugation (1100g, 10 min) at four C. The liver was removed, washed in ice-cold NaCl option (0.9 ), weighed, and various tiny aliquots were placed in 2 mL reaction tubes and snap frozen in liquid nitrogen. Furthermore, several whole organs (heart, kidneys, M. rectus femoris, M. gastrocnemius, M. soleus, M. vastus intermedius, M. vastus medialis) had been excised and weighed. From kidneys, perirenal adipose tissue was removed prior to weighing. Plasma and liver samples were stored at -80 C until evaluation. four.3. Determination of TG and Cholesterol Concentrations in Liver and Plasma Liver samples had been ground within a mortar under liquid nitrogen, and lipids extracted from ground liver samples having a mixture of n-hexane and isopropanol (3:two, vol/vol) according to Hara and Radin [34]. The lipid extracts had been dried under nitrogen and lipids dissolved with chloroform and Triton X-100 (1:1, v/v), as described in [35]. Triglyceride and cholesterol concentrations of both, liver lipid extracts and plasma samples were determinedInt. J. Mol. Sci. 2021, 22,17 ofusing enzymatic reagent kits (OX1 Receptor Antagonist Species Fluitest CHOL, cat. no. 4241, Fluitest TG, cat. no. 5741, each from Analyticon Biotechnologies, Lichtenfels, Germany). 4.four. Determination from the Concentrations of Fatty Acids of Hepatic Total Lipids Concentrations of fatty acids of hepatic total lipids had been determined as fatty acid methyl esters by gas chromatography lame ionisation detection (GC ID) soon after transesterification of lipids from hepatic lipid extracts by trimethylsulfonium hydroxide, as described lately in detail [36]. four.5. RNA Extraction Total RNA from frozen liver aliquots (150 mg) was isolated making use of TRIzol reagent (Invitrogen, Kar.