Glycyrrhetinic acid

Chrysin was obtained from MP Biomedicals, LLC (Santa Ana, CA, USA). Glycyrrhetinic acid was obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals for biochemical and histological analysis were purchased from Sigma Chemical Co. and were of analytical grade or the highest grade available.

Glycyrrhetinic acid (GA), Poly(ethylene) glycol 2000 (HO-PEG₂₀₀₀–OH with 2 kDa of PEG chains), N,N′-Dicyclohexylcarbodiimide (DCC), Dichloromethane (DCM), 4-(Dimethylamino) pyridine (DMAP), anhydrous sodium sulfate (Na₂SO₄), succinic anhydride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Cholesterol (Chol) was obtained from Avanti® Polar Lipids (Alabaster, AL, USA). Murrayafoline A (MuA) was extracted and isolated from the roots of Glycosmis stenocarpa. All other reagents were of analytical grade.
Structural analysis: NMR spectra of Chol-suc, Chol-PEG, Acetyl Glycyrrhetinic acid, GA-PEG-Chol have been recorded on a Bruker Avance (500 MHz) spectrophotometer (Bruker Corporation, Massachusetts, USA) (CDCl₃ as a solvent).

Enzyme reactions were performed in 100 µl of buffer (50 mM Tris-HCl pH 8.0) containing 50 µg purified protein, 1 mM UDP-Glc (electronic supplementary material, figure S1, Yuanye Biotech, Shanghai, China) and 100 µM glycyrrhetinic acid (Sigma-Aldrich, Shanghai, China). After 6 h of incubation at 37°C, the reactions were stopped by the addition of 100 µl methanol. The conversion rates were calculated by HPLC peak area (A_product/A_substrate + A_product × 100%). The supernatants obtained by centrifugation (13 500 r.p.m. for 5 min) were analysed by Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA) and LC-QTOF-MS (Agilent Technologies, Santa Clara, CA, USA), and internal standards (GA-3-O-monoglucose and GA-30-O-monoglucose) were supplied by Prof. Ye Min's laboratory (State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University).

Flubendazole (FLU), the deuterated standard FLU-D₃, NADPH, glycyrrhetinic acid, mebendazole, menadione, naringenin and silybin were purchased from Sigma Aldrich (Prague, Czech Republic). Luteolin was obtained from TRC Canada (Toronto, Canada), and glyceraldehyde was purchased from VWR Chemicals BHD (England). Dimethyl sulfoxide (DMSO), formic acid (FA) (LC‒MS LiChropur™, 97.5–98.5%) and acetonitrile (ACN) (UHPLC‒MS grade) were obtained from VWR International s.r.o. (Prague, Czech Republic). Ultrapure water was prepared from deionized water using a Milli-Q ultrapure water purification system (Millipore, Bedford, MA, USA). Liquid sterile-filtered RPMI-1640 medium was purchased from Biosera (Biotech, Prague, Czech Republic). An ATP Bioluminescence Assay Kit CLS II was purchased from Roche (Mannheim, Germany).

The designation of antibacterial activity of 10 selected, plant-originated compounds (dioscin, aescin, glycyrrhetinic acid, ginsenoside Rg3, procyanidin A2 and C1, hypericin, robinin, amentoflavone, and zeaxanthin; all from Sigma-Aldrich, St. Louis, MO, USA) against H. pylori J99 was made by checking their minimum inhibitory concentrations (MICs) [93 (link),94 (link)]. To obtain this, bacteria in a density of 10⁷ CFU/mL were cultured in 12-well titration plates (Bionovo, Legnica, Poland) filled with 1 mL of Brain Heart Infusion broth (BHI; Oxoid, Dardilly, France) with 5% fetal calf serum (FCS; Gibco, Paisley, Scotland, UK) and a concentration gradient of the tested compounds (2–256 µg/mL). The titration plates were then followed for 3 days of microaerophilic incubation at 37 °C and 100 rpm shaking (MaxQ 6000, ThermoFisher, Waltham, MA, USA). Then, bacterial turbidity in each well of the plates was determined visually, and the lowest concentration with the lack of growth was considered as the MIC.

Streptozotocin, caffiene, pseudoephedrine, emodin, sennoside, naringenin, chrysin, hesperidin, isoquercitrin, kaempferol, luteolin, myricetin, prunetin, quercetin, caffeic acid, chlorogenic acid, vanillin, allyl anthranilate, carvone, caryophyllene oxide and glycyrrhetinic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Epicatechin, rutin, vitexin and cynarin were purchased from Bedoukian Research (Danbury, CT, USA). Khellin, umbelliferone were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Gallic acid, cuminaldehyde, isoeugenol and thymol were purchased from Shenggong Research (Shanghai, China). Mcl‐1 inhibitor S63845 was obtained from APExBIO (Houston, TX, USA).

GOLD Suite version 5.2.2 (license no. 4C72bqe56187) was purchased from Flexera software LLC (Belfast, UK). Croton oil and plant steroids, namely, glycyrrhetinic acid, guggulsterone, boswellic acid, withaferin A, and diosgenin, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fluticasone was obtained as a generous gift from Sun Pharmaceutical Industries Ltd. (Vadodara, Gujarat, India). Diagnostic commercial enzyme-linked immunosorbent assay (ELISA) kits for the proinflammatory markers, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were purchased from Krishgen Biosystems (Mumbai, India). All other reagents and chemicals used in this study were of analytical grade.