Phosphoric acid

The purification of HO from supercritical fluid extraction involves four stages:
degumming, acid removal, washing and fractionation, bleaching and deodorization.
In the degumming stage, 1% phosphoric acid (85%, Samchun Pure Chemical, Seoul,
Korea) was added to the supercritical fluid extracted HO to remove the
fat-soluble gums. Subsequently, 1% of 3N NaOH was added to remove free fatty
acids, trace metals, and pigments during the acid removal process. The sample
underwent two cycles of washing using distilled water, followed by stirring in a
50°C water bath with an automatic stirrer for 20 min to eliminate
moisture. Separation was accomplished using a separation funnel during the
washing and fractionation phases. In the final stage of bleaching and
deodorization, various deodorants, including AC, PAC, and zeolite, were added at
a concentration of 2% to remove the off-odor and undesirable color from the HO.
The treatment groups were control group with no deodorant treatment (CON),
AC-purified HO (ACHO), PAC-purified HO (PACHO), and zeolite-purified HO (ZHO).
The deodorization process transpired in a shaking incubator for 30 min at a
temperature of 50°C, and the ultimate purified HO from supercritical
fluid extraction was obtained through vacuum filtration. Stringent safety
protocols were adopted throughout each phase of the study.

Aβ_25-35 and CA were purchased from Sigma Aldrich Co. (St. Louis, Missouri, USA). Aβ_25-35 was dissolved in saline solution (0.9% NaCl) and administered via intracerebroventricular injection (i.c.v.) to mice. CA was dissolved in water orally at doses of 10 or 50 mg/kg/day using a zonde. Butanol was obtained from Duksan Co. (Gyeonggi-do, Korea), N-(1-naphthyl) ethylenediamide was purchased Sigma Aldrich Co. (St. Louis, Missouri, USA), phosphoric acid was purchased from Samchun Pure Chemical Co. Ltd (Gyeonggi-do, Korea), and thiobarbituric acid (TBA) was purchased Acros Organics (New Jersey, USA).

Aβ_25-35 and Griess reagent were obtained from Sigma Aldrich (St. Louis, MO, USA). Phosphoric acid was procured from Samchun Pure Chemical (Pyeongtaek, Korea), thiobarbituric acid (TBA) from Acros Organics (Geel, Belgium), and 1-Butanol from Yakuri Pure Chemical (Kyoto, Japan). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits were purchased from Asan Pharmaceutical Co. (Eumseong, Korea).

Aβ_25-35, donepezil (DO), Griess reagent, malondialdehyde (MDA), and 2’,7’-dichloro-fluorescein diacetate (DCF-DA) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Sodium chloride (NaCl) was purchased from LPS Solution Co. (Daejeon, Korea). Trichloroacetic acid (TCA) and thiobarbituric acid (TBA) were obtained from Biosesang Inc. (Seongnam, Korea) and Acros Organics Inc. (New Jersey, USA), respectively. n-Butanol and acetic acid were acquired from Duksan (Ansan, Korea). Pyridine was purchased from Wako Pure Chemical Co. (Osaka, Japan). Dimethyl sulfoxide (DMSO) was purchased from Biopure Reagent Co. (Daejeon, Korea). Phosphoric acid was acquired from Samchun Pure Chemical Co., Ltd. (Seoul, Korea).