Walnut green husk (WGH) is rich in natural compounds and is valued as a potential source of antioxidant and antimicrobial properties. In this study, the antioxidant and antimicrobial activities of petroleum ether polar fraction, dichloromethane polar fraction, ethyl acetate polar fraction (EAPF), and n-butanol polar fraction from WGH were analyzed. The results showed that EAPF exhibited the highest total flavonoid content (65.74 ± 1.01 mg rutin equivalents [RE]/g dry weight [DW]) and total phenol content (48.73 ± 1.09 mg gallic acid equivalent [GAE]/g DW), with the highest 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical (•OH), and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonate scavenging activity compared with other fractions. EAPF also showed good antibacterial activity against Escherichia coli and Bacillus cereus vegetative cells, with a diameter of inhibition zones of 33.5 and 37.6 mm, respectively, a minimum inhibitory concentration of 31.25 mg/ml and a minimum bactericidal concentration of 62.5 mg/ml, which inhibited the growth of both bacteria. Analysis of the antibacterial mechanism demonstrated that EAPF damaged the integrity of the cell membrane, increased the membrane permeability, and triggered the leakage of intracellular material. In addition, ultrahigh performance liquid chromatography-tandem with mass spectrometry analysis revealed that 8 polyphenols and 14 flavonoids were mainly present in EAPF, such as chlorogenic acid (C₁₆ H₁₈ O₉ ), gallic acid (C₇ H₆ O₅ ), vanillic acid (C₈ H₈ O₄ ), ferulic acid (C₁₀ H₁₀ O₄ ), epicatechin (C₁₅ H₁₄ O₆ ), catechin (C₁₅ H₁₄ O₆ ), hesperetin (C₁₆ H₁₄ O₆ ), naringenin (C₁₅ H₁₂ O₅ ), hyperin (C₂₁ H₂₀ O₁₂ ), luteolin (C1₅ H₁₀ O₆ ), and so on. Therefore, WGH had the potential to be developed as a natural antioxidant and antibacterial material. PRACTICAL APPLICATION: Our work indicates that WGH contains abundant flavonoids and polyphenols compounds. Therefore, the plant byproducts like WGH may have a promising application as a source of antimicrobial and antioxidant additives.

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