Diosgenin

Ethanol, sulfuric acid, acetic acid, ammonium hydroxide, methanol, sodium carbonate, sodium nitrite, aluminum nitrate, aluminum chloride, ammonia, sodium hydroxide, sodium chloride, n-butanol, diethyl ether, petroleum ether, p-anisaldehyde, hydrochloric acid, potassium bismuth iodide, ethyl acetate, etc. were purchased from Biochem, Egypt. Advanced Dulbecco’s modified Eagle’s medium (DMEM), bovine serum, trypsin-EDTA, L-glutamine, fetal bovine serum, penicillin, streptomycin, tris-(hydroxymethyl) aminomethane (TRIS), trichloroacetic acid (TCA), dimethylsulfoxide (DMSO), sulforhodamine B (SRB), gallic acid, Folin and Ciocalteu′s phenol reagent, quercetin, diosgenin, were supplied from Sigma Aldrich (Steinheim, Germany), doxorubicin hydrochloride (Ebewe Pharma, Unterach, Austria).

All reagents and chemicals used in this study were of analytical grade. As major extraction reagents, n-butanol, ethanol, methanol, and ethyl acetate were obtained from Merck Co. (Darmstadt, Germany). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), (2,2′-azino-(3-ethybenzo thiazoline-6-sulphonic acid) (ABTS), ascorbic acid, gallic acid, quercetin, diosgenin, Na₂CO₃, Al(NO₃)₃, catechin hydrate, Folin–Ciocalteu reagents, all were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Poly-ε-caprolactone (PCL) (C6H10O2)n (Mw: 45,000 g/mol; density: 1.14 g/cm³) and Diosgenin (Mw: 414.62 g/mol;density: 1.1 g/cm³) were purchased from Sigma Aldrich (3050 Spruce Street, Saint Louis, MO, USA).Pluronic F-68 (Mw: 8400 g/mol)was purchased from Himedia (Mumbai, India), and Acetone (Merck, GmBH Germany) and deionized water were procured from the laboratory for experiments. The purity of chemicals was 98.9%. Pluronic F-68 was used as a surfactant.
The glioblastoma (U87- MG) cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. These cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing NaHCO₃ (3.7 g/L) and D-glucose (4.5 g/L), supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO₂.

Diosgenin (≥ 93%, Cas No: 512-04-9), poly lactic-co-glycolic acid 50/50 (PLGA), LMW chitosan (1526.5 g/mol), polyvinyl alcohol (PVA), folic acid (FA), 1-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Dichloromethane (DCM), 3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethylsulfoxide (DMSO) were purchased from Merck company. The 1.1-diphenyl-2-picrylhydrazyl (DPPH), Butylated hydroxyanisole (BHA) and acetonitrile HPLC grade were obtained from Fisher Scientific, UK. All requirements of cell culture were purchased from Invitrogen Company. Cell lines were purchased from the Cell Bank of Ferdowsi University of Mashhad, Iran.

Nthy-ori 3-1 cells (obtain from ATCC, Manassas, VA, United States) were cultured with RPMI (HyClone, United States) supplemented with 10% FBS (Gibco, United States) and 1% penicillin/streptomycin at 37°C and 5% CO2. When the thyrocytes were 70–80% confluent, they were starved in serum-free medium for 2 h and then preincubated with or without 100 ng/ml IGF-1 (cat. no. I1271, Sigma, United States; purity 95%) for 24 h. Then, cells were added with 10 μM diosgenin (cat. no. D1634, Sigma, United States; purity 93%) for another 24 h.

All reagents were purchased from Merk Millipore and Panreac (Barcelona, Spain). The solvents, with technical quality American Chemical Society (ACS)-certified, were dried and distilled before use. The diosgenin was purchased from Sigma-Aldrich (Oakville, ON, Canada). Column chromatography was performed on Merk silica gel 60 (70–230 mesh ASTM, Merck, Barcelona, Spain). Analytical thin-layer chromatography (TLC) was carried out on silica pre-coated (0.25 mm) Merck silica gel GF254 (50 mm × 100 mm, Merck). The samples were developed with a vanillin solution of 1% in 50% perchloric acid. Melting points (mp) were recorded using an oven with the Electrothermal 9100 capillary apparatus, and the temperatures were uncorrected. The FTIR spectra were recorded in a Philips Analytical PU 9600 FTIR spectrophotometer in the 450–4500 cm⁻¹ interval in KBr pellets at room temperature. NMR spectra were recorded on a Bruker DRX-400 spectrometer (Rheinstetten, Germany) operating at 400 MHz for ¹H and 100 MHz for ¹³C. The samples were dissolved in deuterated chloroform, and the chemical shifts are expressed in ppm. The separation and purification process of the synthesized compounds by column chromatography (CC) was developed by using as the solvent a mixture of n-hexane/ethyl acetate (v/v) according to the derivatives.

Commercial seeds of fenugreek (Trigonella foenum-graecum L.) were purchased from Murciana de Herboristeria (Murcia, Spain) and seeds of red quinoa (Chenopodium quinoa Willd.) were purchased from Hijo de Macario Marcos (Salamanca, Spain). Trizma base, maleic acid, sodium chloride, calcium chloride, HCl, Amano lipase A from Aspergillus niger, pepsin, pancreatin from porcine pancreas, bile salts, phosphatidyl choline from egg yolk, β-sitosterol (≥70%), oleic acid, lysine, 1,3-diolein, 1-oleoyl-rac-glycerol, myo-inositol, d-glucose, quercetin, diosgenin, hederagenin, protocatechuic acid, squalane, and N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Sucrose was from Panreac (Barcelona, Spain). Dioscin, protodioscin, 5-pentadecylresorcinol and oleanolic acid were from Cymit Quimica S.L (Barcelona, Spain). Methanol, hexane, 1-butanol, chloroform were from Macron (Gliwice, Poland).