Silymarin powder

To follow a physiological administration, silymarin extract (Sigma Aldrich, St. Luois, MO, USA) containing 210 mg/g of silybin was added into the HFHC diet and administrated orally. Briefly, the composition of the animal food provided by Research Diets Inc. (Research Diets, New Brunswick, NJ, USA) and designed for a high fat high carbohydrates diet (HFHCD) was modified opportunely to include silymarin in the product (D12331, 23% protein, 35.5% carbohydrate, 35.8% fat in form of soybean oil and hydrogenated coconut oil). In particular, silymarin powder (0.01 g/g) and Tween 80 (Sigma Aldrich, St. Luois, MO, USA) (0.1 g/g) were added to coconut oil (Unigrà, Conselice, Italy) previously heated at 40 °C. The system was maintained at 40 °C until silymarin complete solubilization. The oil enriched with silymarin was manually incorporated at a concentration of 14% by weight in paste obtained by grounding the animal feed pellet with a mixer (Kenwood, Prospero KM260, Havant, UK). Subsequently, silymarin-enriched paste was subdivided in aliquots of 4.5 g cylinders. The effective concentration of the bioactive principle (silybin) contained in each food cylinder was (270 mg/kg) determined by HPLC analysis.

Silymarin powder (SLM, ≥30% silybin (silybin A + silybin B) was obtained from Sigma Aldrich (https://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&symbol=S0292). The other compounds identified were the flavonolignans silychristin, silydianin, isosilybin A and isosilybin B and the flavanonol taxifolin. The extract title was 54.3% according to our previous work [24 (link)]), d-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), silibinin (≥98%, HPLC), fluorescein 5(6)-isothiocyanate (FITC, ≥90%, HPLC), pepsin, bile salts, pancreatic lipase, pancreatin, phosphate buffered saline BioPerformance Certified pH 7.4 (PBS), Tween^®80, lecithin (≥99%, TLC) lyophilized powder, cholesterol BioReagent (≥99%), 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), human serum albumin (≥96%, HSA) lyophilized powder and all analytical grade and HPLC grade solvents were obtained from Sigma Aldrich (Saint Louis, MO, USA) with the support of Sigma Aldrich Italia (Milan, Italy). Soluplus^® was a gift from BASF (Ludwigshafen, Germany) with the support of BASF Italia, BTC Chemical Distribution Unit (Cesano Maderno, Monza e Brianza, Italy). Distilled water was obtained from a Simplicity^®UV Water Purification System, Merck Millipore (Darmstadt, Germany).