Saccharine

Standards of artificial sweeteners and steviol glycosides were obtained from different sources: acesulfame-K from Nutrinova (Frankfurt am Main, Germany); saccharine, sucralose and neohesperidin DC from Sigma-Aldrich (St. Louis, USA); aspartame from Ajinomoto Foods Europe (Nesle, France); cyclamate from Merck KGaA (Darmstadt, Germany); alitame from Frapp’s Pharma (Hong Kong, China); neotame from CHEMOS (Regenstauf, Germany); and rebaudioside A, stevioside, rebaudioside C, dulcoside A, steviolbioside and steviol from LGC Standards (Łomianki, Poland). As the internal standard (IS), sodium N-(2-methylcyclohexyl)sulfamate was used [27 (link)]. Acetonitrile (ACN), methanol (MeOH) and acetone were purchased from Merck KGaA (Darmstadt, Germany). Acetic acid (AA) was obtained from POCH (Gliwice, Poland). Ultrapure water was prepared using the HLP5 system from Hydrolab (Wiślina, Poland).

Alexa Fluor-conjugated secondary antibodies were obtained from Life Technologies (Carlsbad, CA), IRDye-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE). Mouse anti-smMHC, anti-sm-α-actin, anti-calponin, rabbit anti-fibronectin were obtained from Sigma (St. Louis, MO) and anti-phospho Smad2 and anti-Smad2 antibodies were purchased from Cell Signaling (Danvers, MA). Sircol collagen assay was obtained from Biocolor Life Sciences, UK. Staining kits (PAS, Trichrome), chloroquine, quinine and saccharine were obtained from Sigma. H&E and Afog staining kits were obtained from Vector Labs. All other chemicals were of analytical grade.

The experimental stages involved preparing materials and equipment, making the GOx enzyme and glucose solutions, coating the GOx enzyme, and sensor testing. The materials used in this experiment were GOx enzyme ((Sigma Aldrich, Singapore), saccharine (Sigma Aldrich, Singapore), 70% alcohol (Sigma Aldrich, Singapore), and Aqua Dest (Merck, Indonesia). The equipment used included dropper drops (0.5 mL, 1 mL, and 5 mL), sample bottle, measuring cup, beaker glass, sample box, PVDF film, and I–V meter.

Sucrose, monosodium glutamate (MSG), NaCl, citric acid, PTC, quinine, sinigrin and saccharine were supplied by Sigma Aldrich (St. Louis, MO, USA). Distilled water was used as the solvent to prepare the corresponding solutions.

Formulation of the chewing gums was made by the Department of Pharmaceutical Technology. The Department has a licence to produce nutritional supplements. The sour cherry extract was prepared from the Hungarian sour cherry (Prunus cerasus L.) variety “VN1”, a selection of “Csengődi csokros”. The anthocyanin content was determined by Homoki and Nemes [24 (link)], where it contained mainly cyanidin 3-rutinoside, cyanidin 3-O-glucoside, delphinidin, malvidin, peonidin, and petunidin glycosides. The extract contained xylitol, chewing gum base, xylitol syrup, glycerine, citric acid, peppermint aroma, and sour cherry extract.
The anthocyanin-containing chewing gum was made as was written in our earlier study [16 (link)]. The main ingredients were Geminis T BHA gum (Cafosa) base, xylitol, citric acid, glycerol, saccharine (Sigma), peppermint volatile oil, and sour cherry extract. During the preparation, the flavourers (citric acid, glycerol, saccharine) were placed into purified water with 0.1 g anthocyanin-containing sour cherry extract, then at 60 °C, the water phase and melted gum base were mixed, and the peppermint volatile oil was added at 40 °C. Next, 2.5 g chewing tablets were formed from the mixture, and after 12 h of conditioning at room temperature, the tablets were put into plastic boxes and stored at 8–15 °C until consumption.