Silymarin

Silymarin and paracetamol were acquired from Sigma-Aldrich, St. Louis, MO, USA. Based on a Certificate of Analysis by Sigma Aldrich, the Silymarin (Product Number: S0292, Batch No. BCCD8696) used in this study contains 46.5% of silibilin. From Sigma-Aldrich (Steinheim, Germany), reagents were supplied as follows: Trolox, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-s-triazine (≥99.0%) (TPTZ). 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammoniumsalt (98%) was purchased from J&K, Scientific Ltd. (Beijing, China). Ferro sulphate heptahydrate and ferric chloride hexahydrate were supplied from Centrohem (Stara Pazova, Serbia).

Enzymatic porcine placenta hydrolysate (EPPH) prepared by Ubio (Ubio, Seoul, Korea) was used. The porcine placenta enzymatic hydrolysate was washed several times with NaCl to remove the blood and crush the placenta from which foreign substances were removed. The crushed placenta was subjected to hydrolysis and heating after adding papain, a proteolytic enzyme, to inactivate the protease, followed by filtration and adsorption purification. The purified product was prepared by adsorption purification after leaving/concentrating by adding ethanol. In this experiment, a liquid porcine placenta enzyme hydrolysate was used. The porcine placenta enzyme hydrolysate was quantified/diluted based on the total nitrogen content, and the positive sample silymarin (silymarin, Sigma-Aldrich, St. Louis, MO, USA) was prepared according to the treatment concentration by weight and then used.

Transforming growth factor (TGF)-β1 was obtained from R&D Systems (#240-B; Minneapolis, MN, USA), and chloroquine (#C6628) and silymarin (#S0292) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile, methanol, ethanol, silymarin, glucose, and fructose were purchased from Sigma Aldrich (St. Louis, MO, USA), and 4–hydroxyquinoline, salicylic acid, vanillic acid, rutin, trans–4–hydroxycinnamic acid, ellagic acid, sinapinic acid, luteolin, trans–ferulic acid, hesperidin, naringin, 4–hydroxybenzoic acid, and 3,4–dimethoxycinnamic acid (purity > 98%) were purchased from Aladdin (Shanghai, China). Dihydroxybenzoic acid, methyl syringate, fisetin, abscisic acid, quercetin, trans–cinnamic acid, naringenin, apigenin, kaempferol, hesperetin, kaempferide, daidzein, and chrysin (purity > 98%) were obtained from J&K Scientific (Beijing, China). Formic acid was purchased from Merck (Darmstadt, Germany).

Analysis of Silymarin content in the in vitro raised plant samples was carried out according to the method of Khan et al. (2013 (link)). Finely ground and dried plant material (200 mg) of each sample was ultrasonicated in solvent mixture contained methanol (CH₄O) and 0.1 % phosphoric acid (H₃PO₄) in a ratio of 70 V:30 V, (1 mil each) for 30 min. We used Shimadzu Lc8A system for HPLC (High Performance Liquid Chromatography) set up with a binary pump, solvent vacuum degasser, a variable wavelength (λ) detector, and an auto sampler containing an injection loop (10 μl). The column used was C18 (ODS) with particle size (150 × 4.6 mm) and the chromatographic eluents consisted of ultrapure water containing 0.1 % H₃PO₄ (Pump A) and C₂H₃N; acetonitrile (pump B). The scheme for gradient elution of Silymarin was set as: 0–30 min, 10–20 % B; 30–110 min, 20–80 % B. The rate of elution flow was kept as 1.0 ml/min with the injection volume of 10 µl. Silymarin (Sigma; CA, USA) was used as a standard reference and samples were analyzed on the basis of comparison of peak areas and retention times of the samples with that of the standard. The content of Silymarin was quantified and expressed in mg g⁻¹ DW (dry weight).

Silymarin with a purity of 99% was obtained from Sigma (St Louis, MO, USA) and was dissolved in dimethylsulfoxide (DMSO) ( Sigma, St Louis, MO, USA) to make a stock solution. It was diluted with DMEM with the DMSO concentration kept below 0.1% in cell culture, which had no detectable effects on cells. U0126 and SB203580 were purchased from Cell Signaling Technology (Danvers, MA, USA) and Selleck Chemicals (Houston, TX, USA), respectively. Primary antibodies against LC3B, phospho-ERK, phospho-p38, β-actin and horseradish peroxidase-conjugated second antibodies were all pruchased from Cell Signaling Technology. The SuperSignal West Pico Chemiluminescent Substrate for horseradish peroxidase enzyme was obtained from Pierce (Rockford, IL, USA).