Urease

To test the efficiency of the inhibitors, in vitro activity assays were performed using Jackbean (Canavalia ensiformis) urease (EC 3.5.1.5, Sigma Aldrich, Taufkirchen, Germany) dissolved in 0.1 M K-phosphate buffer, pH 7.3. To quantify urease activity, liberation of ammonium from urea was determined spectrophotometrically at λ620 nm, using the Berthelot method in a two-step reaction with hypochlorite and salicylate [22 (link)]. The reaction mixture contained 50 mM urea; 0.1 M K-phosphate buffer, pH 7.3; and 30 U of Jackbean urease in a total volume of 1.0 mL. After incubation for 30 min at 37 °C, 0.5 mL of sodium hypochlorite (0.5% (v/v) free chlorine) in 0.5 M NaOH and 0.1 M salicylic acid in 1.0 M NaOH were added, and color development was measured after 15 min of incubation at room temperature. The urease inhibitors were used at concentrations of 1, 5, or 10 mM.

Proteinase-K, non-specific esterase, phospholipase, phosphatase, urease, or α-mannosidase, (each at 0.1 IU, Sigma-Aldrich, Saint Louis, MO) was added to 150 μl of LAM-spiked urine, and lactase and caseinase (both at 0.1 IU, Sigma-Aldrich) were added to 150 μl of LAM-spiked milk at the concentrations indicated in the figures and figure legends, hand mixed and further incubated for 15 minutes at room temperature. Enzymatically treated LAM-spiked urine or milk was then directly used to perform LAM-tests following manufacturer’s instructions.

Protease inhibitor cocktail tablet was purchased from Roche (Mannheim, Germany). Sodium azide, urease, methoxyamine, pyridine, N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS), and methanol were purchased from Sigma-Aldrich (St. Louis, MO). methoxyamine was prepared in pyridine at a concentration of 15 mg/ml.

Gynura segetum (purchased from Bozhou, Anhui, China) was identified as the root of Gynura segetum (Lour.) Merr. by Professor Yajun Cui (School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China.). The plant was stored in the laboratory of the Shanghai University of Traditional Chinese Medicine. Gynura segetum roots were chopped and suspended in distilled water for 2 h. The mixture was then boiled for 1.5 h and filtered. The entire extraction procedure was repeated one time, and the extracts were merged and equilibrated to 1.5 g/mL.
Urease (Lot: SLBB0100V) was purchased from Sigma-Aldrich; methanol (AR) was purchased from Sinopharm Chemical Reagent Co., Ltd; nonadecanoic acid (Lot: E0810030) was purchased from ANPEL Instrument Co. Ltd; 2-chloro-phenylalanine was purchased from Aladdin; methoxyamine hydrochloride (Lot: BCBP2843V) was purchased from Sigma-Aldrich; pyridine (AR, 10018118) was purchased from Sinopharm Chemical Reagent Co., Ltd; BSTFA with 1% TMCS (Lot: 62894) was purchased from REGIS Technologies, Inc. The instruments were: Gas chromatography-mass spectrometry (GC-MS) (Agilent 7890A, 7000B QQQ-MS detector); 1730R centrifuge (Gene Company Limited); Tissue Lyser II homogenizer (Qiagen).