Study on the bioaccessibility and antioxidant activity of phenolics in mulberry leaf during simulated digestion in vitro

Fruit mulberry and leaf mulberry (2 years old) are harvested in September and August at the Medicinal Botanical Garden of Hexi University. Rinse the undamaged leaves of fruit mulberry and leaf mulberry with distilled water, dry in the shade, crush, pass through a 60 mesh sieve, and seal for storage. Determination of polyphenols: Quantitative analysis of phenolic substances in mulberry leaves was carried out in accordance with national standards. 1 mg/mL gallic acid was prepared, and a standard curve was drawn. The absorbance value was detected at 765nm based on the polyphenol standard curve; Determination of flavonoids: The flavonoid content in mulberry leaves was determined using the NaNO2-Al (NO3) 3-NaOH colorimetric method. A 0.209 mg/mL rutin standard solution was prepared, and the absorbance of the test solution was measured according to the flavonoid standard curve. Then, the in vitro mulberry leaf flavonoid content was calculated using the standard curve formula. HPLC analysis of phenolic substances: High performance liquid chromatography (HPLC) is used for qualitative and quantitative analysis of the content of phenolic substances. Prepare a mixed standard solution of chlorogenic acid (1.039 mg/mL), rutin (1.023 mg/mL), and quercetin (1.050 mg/mL), then dilute it by 2, 4, 8, 10, and 20 times and store it for future use. Determine the optimal detection wavelength as 345 nm. Column temperature at 30 ℃; The mobile phases A and B are 0.1% phosphoric acid aqueous solution and methanol, respectively; Flow rate 1 mL/min; Detection wavelength: 345 nm Injection volume: 10 μ L. Study on the antioxidant activity of phenolic substances: Determination of DDPH radical scavenging ability at 517 nm wavelength absorbance value, DPPH scavenging rate=A0-A/A0 * 100; The antioxidant value of ABTS free radicals was measured in the wavelength range of 734 nm using an equal volume of extraction solvent as a blank group. The absorbance value was determined using ABTA=A0-A/A0 * 100 and the nitrogen blue tetrazolium photoreduction method to evaluate the sample's ability to scavenge superoxide anion radicals (O2-). The absorbance value O2-=A0-A/A0 * 100 was measured at a wavelength of 560 nm. The experimental data were the average of three repeated experiments, and the final display format was mean ± standard deviation. Excel 2021 software was used to statistically analyze and organize the experimental data, and SPSS 26 software was used for significance analysis. The significance limit was represented by p<0.05, and the same letter indicated no significant difference. Different letters represented no significant difference. There is a significant difference. Using the graphic software Origin 2021 for mapping, conducting grey correlation analysis using SPSS AU, and conducting correlation analysis between active ingredients and antioxidants using Pearson correlation test