Different varieties of fresh waxy corn cultivars of various colors, namely, ‘Jingkenuo 2000’ (white corn, WC), ‘Xiameinuo’ (yellow corn, YC), ‘Caizhen 100’ (red corn, RC), ‘Minuo 4’ (yellow and white corn, YWC), ‘Huanuo 1’ (multicolor corn, MC), and ‘Yuheinuo 600’ (black corn, BC) were planted on an experimental farm at the Shandong Academy of Agricultural Sciences on June 8, 2018. Ears of corn that were 16-18 cm in length and 4.5-5 cm in circumference at their upper ends were harvested at 22-25 d of milk stage post-pollination. The ears were then husked (Fig.1) and five ears were used for various assays. The kernels from five ears were removed from the middle of the ear with a sharp knife. The kernels of each sample were mixed equally, frozen in liquid nitrogen, and stored at -80 °C.
2,2-Diphenyl-1-picrylhydrazyl (DPPH), taurodeoxycholic acid, taurocholic acid, and glycodeoxycholic acid were purchased from Aladdin (Shanghai, China). α-Amylase and α-glucosidase were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). All other reagents used were of analytical grade.
Extraction of free and bound phenolics
Free and bound phenolics were extracted according to previous method . The samples (5 g) were ground to flour and mixed with 100 mL chilled acidified methanol (95% methanol and 1M HCl 85:15, v/v). Extraction was performed at room temperature for 2 h in a water bath. The supernatants were obtained by centrifugation at 2,500 ´g for 10 min. The residue was re-extracted as mentioned above. The mixed supernatants were concentrated undervacuum at 45 °C and reconstituted to a final volume of 25 mL. The product was stored at -20 °C and considered free phenolics.
The residue from the above free phenolic extraction was hydrolyzed with 200 mL 2M NaOH at room temperature for 1 h with continuous shaking in the presence of nitrogen gas. The mixture was defatted with hexane and then neutralized with concentrated HCl. The remaining mixture was then extracted five times with ethyl acetate. The supernatants were combined and evaporated at 45 °C, and then reconstituted to a final volume 50 mL with chilled acidified methanol. The product was considered bound phenolics and stored at -20 °C until further analysis.
Determination of phenolic acid content
The phenolic acid content was analyzed using the Folin Ciocalteu (FC) colorimetric method described previously . Gallic acid was used as a standard, and the total phenolic content was expressed as μg of gallic acid equivalents (GAE) per gram of sample dry weight (DW).
Determination of flavonoid content
The flavonoid content was determined according to previous report . The total flavonoid content was expressed as μg of (+)-catechin equivalents (CE) per gram of sample DW.
Determination of anthocyanin content
The anthocyanin content was determined according to previous report  with some modifications. The absorbance of the methanol extracts was measured in triplicate at 535 nm in a 96-well microplate against an acidified methanol blank. Anthocyanin concentration was quantified by comparing the sample’s average absorbance to a calibration curve of cyanidin 3-O-glucoside chloride in acidified methanol. The final values were expressed in anthocyanin equivalents (AE) in μg/g DW.
Determination of antioxidant activity
DPPH radical scavenging activity of phenolics was assessed by measuring the ability to bleach a black colored methanol solution of DPPH radicals as described by previous method [6, 16]. One milliliter phenolic extract was mixed with 5 mL 60 μM DPPH dissolved in methanol. The absorbance was measured at 517 nm against a solvent blank. The scavenging rate of DPPH radicals was expressed as the scavenging rate of one gram of dried samples and was calculated using equation (1).
(see Equation 1 in the Supplementary Files)
where Acontrol is the absorbance of the control solution, Asample is the absorbance in the presence of phenolic extracts in DPPH solution, and Aerror correcting, which is the absorbance of the extract solution without DPPH used for error correction.Vfinal represents the final volume of methanolic extract, Vtest represents the volume used for activity test, and Wsample represents the total weight of sample DW.
Hydroxyl radical scavenging activity (HRSA) was assessed according to Mäkynen et al.  with some modifications. The reaction mixture was generated by adding 30 μL 2-deoxy-2-ribose (17 mM), 30 μL extract, 30 μL 1.2 mM EDTA, 60 μL 0.3 mM FeCl3, 30 μL 34 mM hydrogen peroxide (H2O2), and 60 μL 0.6 mM ascorbic acid. The reaction was performed at 37 °C for 1 h. Thereafter, 150 μL 1% (w/v) thiobarbituric acid (TBA) and 300 μL 2.8% (w/v) trichloroacetic acid (TCA) were added to the mixture, which was subsequently incubated at 100 °C for 15 min. The absorbance was measured at 532 nm against a blank containing deoxyribose and buffer. The HRSA values were expressed as scavenging rate per gram of dried samples and were calculated using equation (2).
(see Equation 2 in the Supplementary Files)
where Abscontol is the absorbance of the control solution (30 μL distilled water instead of extract) and Abssample is the absorbance in the presence of phenolic extracts. Vfinal is the final volume of the extract, Vtest is the volume used for activity test, and Wsample is the total weight of sample DW.
Ferric reducing antioxidant power (FRAP) assay was performed according to previous report [16, 17]. Briefly, a FRAP solution was mixed with 10 mL 0.3 M sodium acetate buffer solution (pH3.6), 1mL 10mM 2,4,6-tripyridyl-S-triazine (TPTZ) in 40 mM HCl, and 1 mL 20 mM FeCl3. The FRAP reagent was warmed to 37°C in a water bath. Next, 0.1 mL phenolic extract was mixed with 1.8 mL FRAP reagent and 3.1 mL ultrapure water. The absorption of the reaction mixture was measured at 593 nm after incubation for 30 min at room temperature. FRAP values were calculated from a standard curve prepared using FeSO4. FRAP values were expressed as μmol FeSO4 per gram of dried samples.
Inhibition assays for a-amylase and α -glucosidase activities
The inhibition assays for α-amylase and α-glucosidase activities were performed using a method described previously . Solution without the extract was used as a control. Deionized water was used as the blank. The results were expressed as inhibition (%) per gram of sample DW. The inhibition of α-amylase and α-glucosidase activities was calculated using equation (3).
(see Equation 3 in the Supplementary Files)
where Abssample is the absorbance of the experimental sample, Absblank is the absorbance of the blank, Abscontrol is the absorbance of the control, Vfinal is the final volume of methanolic extract, Vtest is the volume used for α-amylase activity test, and Wsample is the total weight of sample DW.
Determination of bile acid binding activity
The bile acid binding assay was performed according to previous method [17, 18] with some modifications. Sodium glycocholate, sodium cholate, and sodium taurocholate were used as bile acids in this experiment. Briefly, 1 mL of the extract was incubated with 1 mL 0.01M hydrochloric acid solution with shaking at 37 °C for 30 min. The pH was adjusted to 6.24 and 4 mL of 10 mg/mL L-trypsin (prepared with 0.1 M phosphate buffer, pH 6.24) was added and incubated at 37 °C for 30 min at constant temperature. Four milliliters 1 mM cholate solutions (prepared with 0.1 M phosphate buffer, pH 6.24) was added to each sample. After shaking at 37 °C for 1 h, the mixture was transferred to a centrifuge tube and centrifuged at 3,000 ´g for 20 min. The cholate content in the supernatant was analyzed. Bile acid binding activities were calculated from a standard curve using cholate.
All the statistical analyses were performed using one-way analysis of variance (ANOVA) of the SAS 9.2 statistical software (SAS Institute Inc., USA). The data presented were the means of three experiments, along with the standard error of the mean. The means were compared using Fisher's least significant difference (LSD) test, and differences at P < 0.05 were considered significant.