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4 protocols using calcium chloride

1

In Vitro Digestion Protocol

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Pepsin from
porcine gastric mucosa, bile extract porcine, and pancreatin from
porcine pancreas were purchased from Sigma-Aldrich; rabbit gastric
extract was acquired from Lipolytech (Marseille, France). Calcium
chloride (CaCl2(H2O)2, 96%) and hydrochloric
acid (HCl, 37%) were provided by Scharlab. Ammonium carbonate ((NH4)2CO3, 30–34% in NH3), potassium chloride (KCl, 99%) sodium bicarbonate (NaHCO3, 99–101%), and sodium chloride (NaCl, 99.5%) were purchased
from Panreac (Barcelona, Spain). Monopotassium phosphate (KH2PO4, ≥99.0%) was provided by Sigma-Aldrich, and
magnesium chloride hexahydrate (MgCl2(H20)6, 99.0–101.0%) was obtained from Merck (Darmstadt,
Germany). Dialysis membrane tubing (12 000–14 000
Da) MWCO was supplied by Spectrum Laboratories, Inc. (Piscataway,
NJ).
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2

Alginate Extraction and Precipitation

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The alginate fraction solubilised in the liquid phase obtained after hydrothermal extraction was precipitated by adding 1% of calcium chloride (w/v) (Scharlau, Barcelona, Spain) and stirring gently at least 12 h at 300 rpm (MMH90E, OVAN, Barcelona, Spain). A centrifugation step, necessary to separate the alginate fraction from the alginate-free liquid fraction, was carried out for 20 min at 4500 rpm (Rotixa 50RS, Hettich Zentrifugen, Tuttlingen, Germany).
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3

Preparation of Probucol-UDCA Formulation

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Probucol, metformin, low viscosity sodium alginate, ursodeoxycholic acid and sodium alginate were purchased from Sigma Chemical Co, USA, while calcium chloride from Scharlab S.L, Australia. The reagents were purchased from Merck (Australia) and were used without modifications. Stock of PB (20 mg/mL) and UDCA (4 mg/mL) were prepared by vortexing the powders with 10% gel3 ,14 ,27 –29 (link). Preparations were mixed for 7 hours, and used within two day of preparation.
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4

Acetylcholinesterase Assay with Inhibitors

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Acetylcholinesterase (AChE) from electric eels, acetylthiocholine chloride (ATCh), potassium ferricyanide (K3Fe(CN6), 99%) and chlorpyrifos were purchased from Sigma-Aldrich Co, Ltd, China. Hydrochloric acid (HCl, 37%), sodium chloride (NaCl, 99%), potassium chloride (KCl, <100%), acetone (CH3COOH3, ≥99.8%), and ethanol (C2H5OH, ≥99.7) were purchased from RCI Labscan, Thailand. Magnesium nitrate (Mg(NO3)2, ≥99.4%) and cobalt(ii) nitrate hexahydrate (Co(NO3)2·6H2O, ≥98%) were purchased from Univar, Australia. Calcium chloride (CaCl2, ≥95%) was purchased from Scharlau, Spain. Mercury(ii) chloride (HgCl2, ≥99.5%) was purchased from QRec™, New Zealand. Chitosan ((C6H11O4)n, 100 000–300 000) was purchased from ACROS, China. Phosphate buffer saline (PBS, 1× concentration, osmolarity 280–315 mOsm kg−1) was purchased from Cepham Life Science, USA. Hydrogen peroxide (H2O2, 30%, ISO grade) was purchased from Merck Millipore, USA. Deionized water (DI) with specific resistivity of 18.2 MΩ cm was obtained from a RiOs™ Type I Simplicity 185 Millipore water purification system. Alkaline hydrogen peroxide (AHP) solution was prepared by adding 1 N NaOH to a 3% H2O2 solution, adjusted to a pH of 11.5. Water and juices were bought from a convenience store in Khon Kaen, Thailand. Sugarcane bagasse was bought from a local market in Khon Kaen, Thailand.
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