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4 protocols using folin ciocalteu reagent

1

Quantifying Phenolic Compounds in Samples

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Extraction and assays were performed based on Anisworth and Gillespie [141 (link)]. Then, 100 mg sample and 1.9 mL ice-cold methanol were added to the microtubes and incubated in the dark at 25 °C for 24 h. Mixing was performed with a vortex mixer and incubated for another 24 h. After the samples were centrifuged at 13,000× g for 5 min, 100 μL supernatant was transferred to fresh microtubes. Gallic acid (standard) and 95% (v/v) methanol blank were added to other microtubes. Then, 200 μL of 10% (v/v) Folin-Ciocalteu reagent (20-fold dilution of Folin-Ciocalteu reagent from Wako Pure Chemical Corporation) were added to the tubes and vortexed thoroughly, and 800 μL of 700 mM Na2CO3 were added to each tube and incubated at 25 °C for 2 h. Then, a 200 μL sample, standard, or blank was transferred from the assay tubes to a clear 96-well microplate, and the absorbance of each well at 765 nm was determined using a microplate reader (SH-9000, Corona Electric, Hitachinaka, Japan).
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2

Ginseng Phytochemical Extraction Protocol

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Organic solvents (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany). Folin–Ciocalteu reagent was purchased from Wako Pure Chemicals (Osaka, Japan). The standard compounds of panasenoside and kaempferol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pure ginsenoside standards (98%) were purchased from ChromaDex (Santa Ana, CA, USA) and Ambo Institute (Daejeon, Korea).
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3

Quantifying Polyphenols and Antioxidant Activity in Perilla Pomace

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The levels of total polyphenols in perilla pomace were determined using the Folin-Ciocalteu method with a slight modification 21) . In brief, 100 μL of the sample was added to 500 μL of Folin-Ciocalteu reagent (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and incubated for 30 min at room temperature. The mixture was then added 400 μL of 7.5% sodium carbonate solution. After incubation for 1 h, 200 μL of the reaction mixture was transferred to a 96-well plate, and absorbance was measured at 765 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) . Gallic acid was used as the standard compound, and the total polyphenol content was expressed as gallic acid equivalents (mg GE/g dry weight) . To measure the radical oxygen species scavenging activity of perilla pomace polyphenols, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity was measured according to the method described by Oki et al. 22) . Briefly, the sample solution (360 μL) , 200 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0, 180 μL pH 6.0) , and 400 μM DPPH in ethanol (180 μL) were mixed in a microtube. After incubation for 20 min at room temperature, 200 μL of the reaction mixture was transferred to a 96-well plate, and the absorbance was measured at 520 nm using a spectrophotometer.
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4

Antioxidant Properties of Krill Extract

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Isada krill frozen and packed in a 1-kg plastic shrink wrap was purchased from Hamaichi (Wakayama, Japan) and stored in a freezer at -30°C until use. The average mass and length of the krill were 0.07±0.01 g and 18.00±0.65 mm, respectively. Diethyl ether, Folin-Ciocalteu reagent, trisodium citrate dihydrate, copper (II) sulfate pentahydrate, bovine serum albumin (BSA), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothaiazoline-6-sulfuric acid) (ABTS), 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), and potassium persulfate, and uracil were purchased from Wako Pure Chemical Industries (Osaka, Japan). Myoglobin, tri -tyrosine, di-L-phenylalanine, and L-phenylalanyl-L-alanine dihydrate were purchased from Sigma-Aldrich (Tokyo, Japan). Glucose oxidase was purchased from Nacalai Tesque (Kyoto, Japan). Other reagents were of analytical grade.
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