Antioxidant and antimicrobial characteristics of ethyl acetate polar fractions from walnut green husk

Abstract

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 nbutanol 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 (C 16 H 18 O 9 ), gallic acid (C 7 H 6 O 5 ), vanillic acid (C 8 H 8 O 4 ), ferulic acid (C 10 H 10 O 4 ), epicatechin (C 15 H 14 O 6 ), catechin (C 15 H 14 O 6 ), hesperetin (C 16 H 14 O 6 ), naringenin (C 15 H 12 O 5 ), hyperin (C 21 H 20 O 12 ), luteolin (C1 5 H 10 O 6 ), and so on. Therefore, WGH had the potential to be developed as a natural antioxidant and antibacterial material.K E Y W O R D S antimicrobial mechanism, antioxidant activity, chemical compounds, walnut green husk Meihua Xi and Yujie Hou contributed equally to this work.

Antimicrobial Activity and Mechanisms of Walnut Green Husk Extract

“…Additionally, variations in bacterial size were observed, with some demonstrating dissolution. Comparable findings have been previously reported in antibacterial assessments involving other WGH extracts [11]. Vanillic acid, chlorogenic acid, and cinnamaldehyde, which were found in WGHa, are responsible for these morphological and structural alterations in the cell membranes of the selected bacteria.…”

“…No antimicrobial activity was observed for WGHb and WGHc at concentrations of 100 mg/mL or lower. A previous study found that the MIC for the ethyl acetate fraction of a WGH extract against both E. coli and B. cereus was 31.25 mg/mL [11]. However, a combination of an n-hexane extract and a 80% hydroethanol extract of WGH exhibited MIC values of 2, 1, 0.5, and 4 mg/mL against S. aureus, B. subtilis, E. coli, and P. aeruginosa, respectively [10].…”

“…Previous studies have demonstrated that quercetin, the primary active component in WGHa, effectively disrupts the permeability of the bacterial cell membranes of E. coli and S. aureus, resulting in the leakage of intracellular proteins into the culture medium and elevating the concentration of soluble proteins within the medium [22]. This evidence indicates that the treatment of bacteria with WGHa not only caused damage to the cell wall, loss of cytoderm integrity, and leakage of AKP but also potentially led to the entry of other cellular proteins into the culture medium, consequently impairing cellular functions [11]. While the blank medium (containing the same amount of WGHa without bacterial inoculation) exhibited consistent AKP activity throughout the incubation period, the experimental groups displayed a sharp increase in extracellular AKP activity within 0 to 2 h of exposure to WGHa.…”

“…attributed to the efflux of intracellular ions such as Na + and K + as it has been demonstrated that K + efflux values of B. cereus and E. coli treated with the ethyl acetate polar fraction of a WGH extract were significantly higher compared to that of untreated cells [11,23]. The findings of the present study may be attributed to the interaction between active ingredients such as flavonoids and polyphenols in WGHa and the cell membrane, ultimately leading to an increased cell membrane permeability and the efflux of intracellular substances from the bacterial cell [24,25].…”

A Review on Extracts, Chemical Composition and Product Development of Walnut Diaphragma Juglandis Fructus

“…Numerous studies have shown that compounds extracted from plants, animals, and microorganisms have antibacterial activity and are expected to be novel antibacterial agents. Extensive experiments have demonstrated the antibacterial effects of walnut extract [ 69 , 70 , 71 , 72 , 73 ]. In fact, EeDJF has promising antibacterial activity.…”