Polyvinyl polypyrrolidone pvp

Glycogen phosphorylase α from rabbit muscle, glycogen from rabbit liver (type III), α-D glucose-1-phosphate, HEPES [4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)], magnesium chloride (MgCl2), EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), ammonium molybdate, malachite green, caffeine and potassium chloride, butylated hydroxyanisole (BHA), 2,2 diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), gallic acid monohydrate, ferrozine, sodium nitroferricyanide (III) dehydrate, sodium nitrite, Griess reagent, curcumin, polyvinyl polypyrrolidone (PVP), sodium acetate trihydrate, sodium phosphate dibase, sodium phosphate monobasic, and quercetin dihydrate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ascorbic acid, acetic acid glacial, sodium chloride, ferrous sulfate (FeSO₄), ferric chloride hexahydrate (FeCl₃·6H₂O), ethylenediaminetetraacetic acid disodium dehydrate (EDTA-Na₂·2H₂O), Folin-Ciocalteu phenol reagent, and sodium carbonate were purchased from Merck Chemical Co. (Malaysia). HPLC grade water was purchased from Fisher Scientific (Malaysia). All chemicals used are of analytical grade and were used without further purifications.

Frozen fresh leaves of each cultivar were pulverized in liquid nitrogen. Each sample (100 mg) was homogenized in sodium borate buffer (0.3 mM, pH 8.8), 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich, St. Louis, MO, USA), 1 mM dithiothreitol (DTT) (Sigma Aldrich, St. Louis, MO, USA), and 5% polyvinylpolypyrrolidone (PVP) (Sigma Aldrich, St. Louis, MO, USA), totalizing 1 mL of extraction buffer. The samples were centrifuged at 12,000× g (15 min at 4 °C), and then an aliquot of supernatant (0.5 mL) was mixed with 1.0 mL of reaction buffer composed of sodium borate buffer (0.3 mM, pH 8.8), and 0.03 mM L-phenylalanine (Sigma Aldrich, St. Louis, MO, USA). After 15 min at room temperature, the absorbance was measured at 290 nm using a UV-visible spectrophotometer (Amersham Biosciences, Little Chalfont, UK). The specific molar extinction coefficient of (E)-cinnamic acid (9630 mol·L⁻¹·cm⁻¹) was used to determine the PAL activity based on its formation from the substrate, L-phenylalanine [110 (link),111 (link)].