Gallic acid

The total phenolic content (TPC) in the extracts was determined by a modified Folin-Ciocalteu method (19 ). Briefly, 100 μL of each diluted extract were mixed with 500 μL of Folin-Ciocalteu reagent (Sangon Biotech, Shanghai, PR China) and 6 mL of distilled water, and shaken for 1 min. Afterwards, 2 mL of a 15% (by mass per volume) Na₂CO₃ (Sinopharm Chemical Reagent Co., Ltd, Shanghai, PR China) solution were added to the mixture, shaken once again for 2 min, and the volume of the solution was adjusted to 10 mL with distilled water. Finally, the mixture was incubated in the dark for 2 h at room temperature. The absorbance at 750 nm was recorded on an InfiniteM200 PRO multifunctional microplate reader (Tecan, Männedorf, Switzerland) against a solution without sample as blank (100 μL of 70% ethanol instead of the test samples). A standard curve was prepared using gallic acid (Sigma-Aldrich, St. Louis, MO, USA): where A is the absorbance at 750 nm and c is the concentration of gallic acid (c=0.2–1 mg/mL, R²=0.9978).
Samples were independently analyzed in triplicate and the TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of extract.

For the extraction of polyphenols in GP, GE, diets and excreta, 0.50 g of sample was placed in a capped centrifuge tube, suspended in 20 mL of acidic methanol/water (50:50 v/v, pH = 2) and thoroughly shaken at room temperature for 1 h. The tube was centrifuged at 3500 rpm for 15 min and the supernatant was separated. Twenty millilitres of acetone/water (70:30 v/v) was added to the residue and shaking and centrifugation were repeated. The methanol and acetone extracts were combined and used for TEP quantification. The TEP content was determined by Folin–Ciocalteu procedure [36 (link)]. Briefly, a mixture of 0.5 mL of extract, 0.5 mL of Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) and 10 mL of 1 M Na₂CO₃ were introduced in a 25 mL volumetric flask. After reacting for 1 h, absorbance was measured at 750 nm using an ultraviolet-visible spectrophotometer (Hitachi U-2000; Hitachi, Ltd., Tokyo, Japan). Absorbance values were compared against a standard curve made with gallic acid (Sigma-Aldrich, St. Louis, MO, USA) ranging from 0.05 to 0.5 mg of gallic acid per mL. Results were expressed as grams of gallic acid equivalents (GAE) per 100 g of DM.

The methods described by Baba and Malik^{148 (link)} with slight modification were used to determine the total phenolic contents of the C. gigantea extracts. One milliliter of sample solution in methanol (1 mg/mL) was added to 10% Folin-Ciocalteu reagent in water (Merck, Germany, 1 mL) and then rigorously mixed for 5 min. Saturated sodium bicarbonate (60 g/L, 1 mL) was added, and the reaction mixture was kept in the dark at ambient temperature (27 ± 2 °C) for 90 min. The absorbance (725 nm) values of the mixtures were measured by using a UV/Vis spectrophotometer. The calibration curve of gallic acid (1.7–13.3 µg/mL, Sigma–Aldrich, China) was used to calculate the total phenolic contents of the extracts (Y = 0.144X − 0.066, R² = 0.9973, where Y represents the absorbance value of gallic acid at 725 nm, and X represents the gallic acid concentration (µg/mL). The average values of the total phenolic contents of the tested extracts ± SD (n = 3) are displayed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g extract).

The total phenolic content in the C. gigantea stem bark extracts was analyzed using a modification of the method described by Baba and Malik [28 ]. The sample solution (1 mL, 1 mg/mL) was mixed with 1 mL of 1:10 Folin-Ciocalteu reagent (Merck, Germany) and vortexed for 5 minutes. Then, 1 mL of saturated sodium bicarbonate (60 g/L) was added, and the mixture was allowed to stand for 90 min in the dark at ambient temperature for color development. The absorbance of the mixture was then determined at 725 nm using a UV/Vis spectrophotometer (Shimadzu UV-1800, Japan). Gallic acid (1.7−13.3 μg/mL, Sigma-Aldrich, China) was used as the standard (Y = 0.1441X − 0.0682, R² = 0.9974, where Y represents the absorbance of Gallic acid at 725 nm, X represents the concentration of Gallic acid (μg/mL), and R² represents the linear correlation coefficient). The total phenolic content of the extracts was expressed as milligram Gallic acid equivalents per gram of extract (mg GAE/g extract).

Total phenolic content (TPC) was measured using the Folin–Ciocalteu colorimetric assay [20 ,21 (link)]. Each replicate (10 g) was placed in a Warren blender with 90 g DI water and blended on the low setting for 3 min. Each replicate was analyzed in duplicate and DI water was the blank. Diluted replicates and blank (100 µL) were pipetted into a glass test tube. DI water (3.9 mL) was added to each test tube and vortexed for 5 s. Folin–Ciocalteu reagent (250 µL) (Fischer Scientific, Waltham, MA) was added to the test tube and vortexed for 5 s. Sodium carbonate solution (7.5% sodium carbonate anhydrous (Sigma-Aldrich, St. Louis, MO, USA) in DI water) (750 µL) was added to each test tube and vortexed for 5 s. The replicates were stored in the dark for 30 min and absorbance was read at 765 nm. Gallic acid (Sigma-Aldrich, St. Loius, MO, USA) was used to create a standard calibration curve where 0.5% Gallic acid solution was prepared and diluted to 0, 25, 50, 100, 150, and 200 mg/L of Gallic acid and the standard curve was made by plotting absorbance vs. concentration. TPC in the replicates were determined by using the Gallic acid calibration curve, dilution factor of the replicate, and the moisture content of the replicate to report TPC as mg/g Gallic acid equivalent (GAE).