Cytochalasin b

Neutrophils (1 × 10⁶ cells·mL⁻¹) were cultured for 1 h in a serum‐free condition to induce the formation of NETs. Cytochalasin B (5 μg·mL⁻¹; Wako) was then added and the cells were incubated further for 15 min. Subsequently, E. coli was added into the well at an MOI of 50 with or without DNase I (100 U·mL⁻¹), and the cells were cultured for 1 h. Appropriate dilutions of the culture supernatants were spread onto LB agar plates and the plates were incubated at 37 °C overnight. Colonies were counted to determine the colony‐forming units (CFU) of the surviving bacteria.

Mouse diploid parthenogenic embryos were generated as previously described with slight modifications [21,22]. Eight–12-week-old female B6D2F1 (C57BL/6 × DBA/2) (Japan SLC, Inc.) were injected with 5 IU pregnant mare serum gonadotropin (PMSG, ASKA Animal Health) followed by injection with 5 IU human chorionic gonadotropin (hCG, ASKA Pharmaceutical) 46–48 h later, and matured oocytes were obtained from oviducts 16 h later. Oocytes were treated with 0.1% hyaluronidase (Sigma-Aldrich) in M2 medium (Sigma-Aldrich) for 1 min to remove cumulus cells, washed with M2 medium and M16 medium (Sigma-Aldrich) three times each, and incubated in M16 medium supplemented with 2 mM EGTA (EGTA-M16) for 20 min. Diploid parthenogenetic embryos were produced by treating the oocytes with 2 mM SrCl₂ in EGTA-M16 in the presence of 5 µg/mL cytochalasin B (Wako) for 2.5 h, and then incubating them in KSOM medium (MTI-GlobalStem) in the presence of the same concentration of cytochalasin B for 3.5 h. Activated diploid parthenogenetic embryos were then washed with KSOM three times and cultured in the same medium at 37°C with 5% CO₂. In case diploid embryos were converted to tetraploid after parthenogenesis, E1.5 embryos were treated with 5 µg/mL cytochalasin B for 12 h to block the second cleavage, and washed three times with KSOM.

The leaves of E. globulus, used as the test material, were obtained from K. Kobayashi & Co. (Kobe, Japan). The extract of 100 g of dried leaves was obtained using 1 kg of ethanol–water (1:2, v/v) under reflux for 2 h. The obtained extract was filtered, evaporated to dryness in vacuo, and used as ELE throughout the experiments. The yield was approximately 20% of the leaves. HPLC- and LC/MS-grade solvents were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Compounds (Figure 2; oenothein B (1), quercetin 3-O-β-d-glucuronide (4), and kaempferol 3-O-β-d-glucuronide (5)) used as test samples in the sugar absorption assays were isolated from ELE [23 (link)]; cytochalasin B and gallic acid (2) were purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan) and ellagic acid (3) from Sigma-Aldrich Japan (Tokyo, Japan). Other reagents mentioned elsewhere in the text with the manufacturer’s name not specified were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) or Fujifilm Wako Pure Chemical Co. (Osaka, Japan).