α solanine

In the work, the following enzymes were used for biosensor creation: urease (EC 3.5.1.5) from Canavalia ensiformis, activity 66.3 U/mg (Fluka, Switzerland) and activity 22 U/mg and 35 U/mg (Sigma, Switzerland) and butyrylcholinesterase (EC 3.1.1.8) from equine serum, activity 13 U/mg and activity 20 U/mg (Sigma, Germany). Glycerol, bovine serum albumin (BSA; fraction V), 50% aqueous solution of glutaraldehyde (GA), sodium and potassium chlorides, urea, and butyrylcholine chloride were from Sigma-Aldrich Chemie (Steinheim, Germany). Potassium-phosphate buffer (KH₂PO₄-K₂HPO₄) and NaOH were produced by Helicon (Moscow, Russia) and Sigma-Aldrich Chemie (Steinheim, Germany). Glycoalkaloids α-chaconine (purity 95%) and α-solanine (purity 95%) from potato sprouts were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and used as inhibitors for BuChE. Mercury nitrate (Hg(NO₃)₂) from Himlaborreaktiv (Kiev, Ukraine) was used as an activity inhibitor for urease. Other inorganic substances were of analytical grade (>98%).

Trans-chalcone and α-solanine were purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted in 10% aqueous DMSO. Terbinafine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted in 5% aqueous DMSO. The final concentration of DMSO used in the antifungal and coculture assays was fixed at a maximum of 0.5%. Terbinafine was used as positive control (commercial antifungal involved in ergosterol synthesis inhibition) [9 (link)]. Negative controls consisted of DMSO without the tested compounds at a final concentration of 0.5%.

Acetonitrile was of HPLC grade and was purchased from Sigma (Milano, Italy). Orthophosphoric acid (ACS ISO, for analysis, 85%) and ethanol (ACS-Reag. Ph.Eur.) were purchased from Carlo Erba Reagents S.r.l. (Milano, Italy). Water was distilled and filtered through a Milli-Q apparatus (Millipore, Milan, Italy). Standards of malvidin-3-O-glucoside, cyanidin, ascorbic acid, chlorogenic acid, α-solanine and α-chaconine were purchased from Sigma (Milano, Italy). C18 SPE columns (500 mg 3 mL, Supelco, Bellefonte, PA, USA) were used.

BafA1, α-solanine, CQ, doxorubicin,N-acetyl-L-cysteine (NAC), SRB sodium salt, AKT 1/2 kinase inhibitor, anti-LC3B and anti-β actin were purchased from Sigma-Aldrich. Rapamycin was procured from Millipore Corporation (Billerica, MA, USA). Anti-PARP, anti-Atg5, anti-phospho-Akt (Ser473), anti-4E-BP1, anti-phospho-mTOR (Ser2448), anti-phospho-4E-BP1 (Thr37/46), anti-caspase -3, anti-phospho p70 S6 kinase (Thr389), anti-phospho-Akt (Thr308), anti-Akt(pan), anti-mTOR, anti-phospho mTOR, anti-GRP78 (BiP), anti-IRE1α, anti-PERK, anti-CHOP and anti-LC3 were obtained from Cell Signaling Technology (Danvers, MA, USA). Paraformaldehyde, anti-ATF-6α, anti-XBP-1, anti-CREB-2, anti-GADD153/CHOP (B-3), anti-Beclin 1 and anti-LAMP-2 were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Imgenex (IMGENEX India Pvt. Ltd., Bhubaneswar, India). All fluorescence-conjugated and peroxidase-conjugated secondary antibodies were purchased from Invitrogen Corp., (Carlsbad, CA, USA) and Thermo Scientific (Rockford, IL, USA), respectively.

Methanol, acetonitrile, and formic acid (Merck, Darmstadt, Germany), and standards of α-Solanine and α-Chaconine (Sigma Aldrich, Milan, Italy), all of analytical grade, were used.
Starch and vitamin C contents were assessed by acid hydrolysis (AOAC 925.38) and indophenol (AOAC 967.21) methods, respectively [55 ]. The protein content was quantified through the Bradford protocol, using bovine serum albumin as the standard [56 (link)]. Total dietary fiber was determined by enzymatic-gravimetric assay using Megazyme Kit (K-TDFR) following the procedure reported in the instruction manual [57 ].

The methanolic fraction was evaporated to dryness under vacuum using rota-evaporator (Buchi Lab Equipments, USA) and stored at 4 °C for HPLC analysis. For preparation of standard, 10 mg of α-solanine (Sigma, USA) was weighed and added to a 10 mL volumetric flask, and made up to 10 mL with methanol (HPLC grade, Merck). 100 mg of the extract was weighed and made up to 100 mL, followed by filtration through a Millex syringe-driven filter unit (Millipore Corporation, Bedford, USA) before injection. The sample injection volume was 2.0 μl and the chromatogram was recorded at 210 nm. HPLC was performed in model LC-10AT vp (Shimadzu Corporation, Kyoto, Japan) using C18 column (3 μm size, 5 cm × 2.1 mm in length; Supelco, USA) with water-acetonitrile (HPLC grade; Merck, Darmstadt, Germany) as the mobile phase. Separation was carried out at a flow rate of 0.2 mL/min.