Liquiritigenin

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with the broth dilution method as described by the Clinical & Laboratory Standards Institute 2017 (Wu et al., 2019 (link)). Liquiritigenin, glabrol, licoflavone A, licoflavone B, licoflavone C, licoisoflavone A, glabrene, glycyrrhisoflavone, and licochalcone A-E were purchased from Chengdu Biopurify Phytochemicals Ltd., with a purity of ≥95% (Chengdu, China). Briefly, the tested chemicals were diluted with Mueller Hinton Broth (MHB) in 96-well concave bottom plates. Bacteria were adjusted to obtain a bacterial suspension with approximately 1 × 10⁶ colony forming units (CFUs)/mL with MHB and then added into 96-well concave bottom plates (Pupo et al., 2017 (link)). To confirm the antibacterial activity of the tested chemicals, the antibacterial activity was also analyzed with Oxford cup plate methods (Aziz et al., 2016 (link)). Moreover, the constituents of the bacterial wall and membrane were used to screen for potential antibacterial targets.

Gallic acid (1), berberine chloride (8), and benzoic acid (12) were obtained from Sigma-Aldrich (St Louis, MO, USA). Neomangiferin (2), mangiferin (4), liquiritigenin (13), and benzoylpaeoniflorin (14) were obtained from Chengdu Biopurify Phytochemicals (Chengdu, China). Geniposide (5), albiflorin (6), paeoniflorin (7), liquiritin (10), and glycyrrhizin (15) were obtained from Wako (Osaka, Japan). liquiritin apioside (9) and atractylenolide III (16) were obtained from Shanghai Sunny Biotech (Shanghai, China). Chlorogenic acid (3, 99.6% purity) was obtained from Acros Organics (Pittsburgh, PA, USA), and nodakenin (11, 98.0% purity), decursin (17), and decursinol angelate (18) were obtained from NPC Bio-Technology (Yeongi, Korea). The chemical structures of each compound are shown in Figure 1, and the purity of these compounds was higher than 98%. The distilled water, acetonitrile, and methanol used for the mobile phase and formic acid were purchased from J.T. Baker (Phillipsburg, NJ, USA) and Merck KGaA (Darmastadt, Germany), respectively.

The nine standard compounds used as markers for quality assessment of GHT in this study are shown in Figure S1. These compounds were provided by commercial suppliers and used for LC–MS/MS analysis: liquiritin apioside (C₂₆H₃₀O₁₃, CAT No. DR10690, 99.6%), hesperidin (C₂₈H₃₄O₁₅, CAT No. DR10882, 98.7%), neohesperidin (C₂₈H₃₄O₁₅, CAT No. DR10883, 98.4%), and 6-shogaol (C₁₇H₂₄O₃, CAT No. DR10924, 99.2%) from Shanghai Sunny Biotech Co., Ltd. (Shanghai, China); neoeriocitrin (C₂₇H₃₂O₁₅, CAT No. TBW00746, 99.9%) from Wuhan ChemNorm Biotech Co., Ltd. (Wuhan, China); narirutin (C₂₇H₃₂O₁₄, CAT No. BP0985, 99.5%), liquiritigenin (C₁₅H₁₂O₄, CAT No. BP0873, 99.8%), and glycyrrhizin (C₄₂H₆₂O₁₆, CAT No. BP0682, 99.1%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China); naringin (C₂₇H₃₂O₁₄, CAT No. 71162, 95.0%) from KGaA (Darmstadt, Germany). Methanol and acetonitrile were LC–MS grade and supplied by ThermoFisher Scientific (San Jose, CA, USA). Purified water was used, specifically, produced through a Vivagen water purification system (EXL3 Analysis 16, Seongnam, Korea). Formic acid (≥99.5%) was supplied by Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan) and ammonium formate (99.0%) by Kanto Chemical Co., Inc. (Tokyo, Japan).