Ascorbic acid

Fermented milk was produced from Łaciate milk (2% fat) from SM Mlekpol (Grajewo, Poland). Organic powder of rosehips (Rosa canina L.) (400 mg of vitamin C × 100 g⁻¹) came from RADZIOWI SP. z.o.o. (Częstochowa, Poland). Acerola in powder form (25,000 mg vitamin C × 100 g⁻¹) was purchased from Aliness Health’n’beauty (Karczew, Poland) and L(+) ascorbic acid from Chempur (Piekary Śląskie, Poland). The starter culture of probiotic bacteria (Lactobacillus rhamnosus) was purchased from Chr. Hansen (Hoersholm, Denmark). MRS agars and peptone water came from Biocorp (Warszawa, Poland). Sodium hydroxide, phenolphthalein, ascorbic acid, sodium bicarbonate, oxalic acid, 2,6-dichlorophenolindophenol, were purchased from Chempur (Piekary Śląskie, Poland).
All of the reagents used were of analytical reagent grade.

Human serum albumin (HSA) was purchased from MP Biomedicals. 1-methyl-3-allylthio-4-(4′-methylphenylamino)quinolinium bromide (Qui1, Figure 3a), 1-methyl-3-allylthio-4-(3′-hydroxyphenylamino)quinolinium bromide (Qui2, Figure 3b) as well as 1-methyl-3-allylthio-4-(4′-hydroxyphenylamino)quinolinium bromide (Qui3, Figure 3c) were obtained by alkylating the corresponding 1-methyl-4-(phenylamino)quinolinio-3-thiolates (1-methyl-4-(4′-methylphenylamino) quinolinium-3-thiolate, 1-methyl-4-(3′-hydroksyphenylamino)quinolinium-3-thiolate, 1-methyl-4-(4′-hydroksyphenylamino)quinolinium-3-thiolate) with allyl bromide using the previously described method [2 (link),56 (link),57 (link)]. Spectroscopic parameters of the obtained compounds are given in Supplementary Materials. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TPTZ) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Sigma Aldrich. Potassium persulfate (PPS), hydrochloric acid (HCl) as well as ascorbic acid (AA) were purchased from Chempur. All chemicals were of analytical grade and used without further purification.

Acetone, calcium carbonate, ethanol, potassium persulphate, sodium acetate, and sodium carbonate were purchased from IDALIA (Radom, Poland); azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP), Folin–Ciocalteu's phenol reagent, gallic acid, tripyridyl-S-triazine (TPTZ), and Trolox were purchased from Archem (Łany, Poland); acetic acid, activated carbon, ammonium metavanadate, ammonium molybdate tetrahydrate, ascorbic acid, barium chloride dihydrate, cyclohexane, magnesium nitrate, 65% nitric acid, oxalic acid, sodium bicarbonate, and sodium sulfate were purchased from CHEMPUR (Piekary Śląskie, Poland); 2,6-dichlorophenolindophenol sodium salt hydrate was purchased from ACROS ORGANICS (ARGENTA; Poznań, Poland); chloroform, hydrochloric acid (38%), and methanol were purchased from STANLAB (Lublin, Poland); standard solutions and Tween TM 80 were purchased from Merck (Darmnstadt, Germany); 2-undecanone and BF₃/MeOH were purchased from Sigma-Aldrich (Saint Louis, MO, USA); hexane and sodium bicarbonate were purchased from UQF (Wrocław, Poland); n-hexane (99%) was purchased from POCH Basic (Gliwice, Poland); helium was purchased from Air Products (Warsaw, Poland); and potassium hydroxide was purchased from Avantor (Gliwice, Poland).

Valve interstitial cells (VICs) were isolated and cultured as previously described [26 (link)]. All experiments were performed on VICs between their third and fifth passages. To initiate calcification, VICs were cultured in a calcification medium containing β-glycerophosphate disodium hydrate salt (10 mmol/l; Sigma-Aldrich, St Louis, MO, USA), CaCl₂ (1.5 mmol/l; Chempur, Piekary Slaskie, Poland) and ascorbic acid (50 μg/ml; Chempur) and stimulated or not (a negative control) with TNF-α (50 ng/ml). In parallel, to investigate the influence of glucose, VICs were cultured in the calcification medium supplemented with the d-(+)-glucose (11 mmol/l; Sigma-Aldrich). BMP-2 was used as a marker of calcification and was quantified using immunofluorescence as described above. To inhibit oxidative stress generated by high concentration of glucose, the inhibitor of ROS was added (1 mmol/l N-acetyl-l-cysteine [NAC]; Sigma-Aldrich) to the calcification medium 1 h before glucose stimulation. Similarly, to inhibit the transcription pathway of NF-κB, an inhibitor (BAY 11-7082; Sigma-Aldrich) was added to the calcification medium at a concentration of 10⁻⁶ mol/l 30 min before glucose stimulation. VICs were cultured for 72 h. Each experiment was repeated three times using VICs isolated from another valve.