Acetohydroxamic acid aha

Urease inhibition assay was performed as described by Nagata et al. [11 (link)], where urease inhibition served as a proxy for H. pylori inhibition. A 10 μL of 6×10⁸ CFU/mL H. pylori suspension was challenged with 200 μL of a detection reagent composed of 50 mM PBS (pH 6.8) containing 500 mM urea and 0.02% phenol red. Color development was monitored by measuring the O.D. at 555 nm wavelength in a 5-min interval. Samples of bacteria with a detection reagent only or PPEE with a detection reagent only served as controls. The following equation calculates the percentage of inhibition:
% inhibition = ([activity without plant extract - activity with plant extract]/[activity without plant extract])×100.
A lack of potential urease inhibition was indicated if IC₅₀ was more than 12.5 mg/mL (inhibition of <60%). The IC₅₀ of PPEE was determined using ED₅₀ plus (v1.0) software (Vergas, 2000). The urease inhibitory activity of PPEE was compared to the reference urease inhibitor acetohydroxamic acid (AHA) from Sigma (USA).

Palmatine (Pal, CAS number: 3486-67-7, the structure shown in Fig 1) was purchased from Sichuan Pure Chemical Industries (Sichuan, China). Metronidazole (Met, CAS number: 443-48-1,) was purchased from TOKU-E Company (Tokyo, Japan). Acetohydroxamic acid (AHA, CAS number: 546-88-3, purity: 98%), urea (Molecular Biology Reagent), jack bean urease (JBU, type III with specific activity 40.3 U/mg solid), HEPES (Amresco > 99%), L-cysteine (L-cys), glutathione (GSH), dithiothreithol (DTT), boric acid (BA) and sodium fluoride (NaF) were all purchased from Sigma-Aldrich (St Louis, MO, USA). Bradford Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). HEPES buffer (20 mM, pH 7.5) was prepared by adjusting the pH with NaOH. Other chemicals and solvents were of analytical grade or HPLC grade and obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China).