Erythromycin

MRC5 cells, a human embryonic lung fibroblast cell line (Riken BioResource Center, Tsukuba, Japan), were cultured with Dulbecco modified Eagle medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum, 100 μg/mL streptomycin, and 100 U/mL penicillin G. MRC5 cells (7 × 10⁴ cells per well) were placed in 24-well plates (Nunc, Roskilde, Denmark) and cultured in 5% CO₂ humidified atmosphere at 37 °C with or without clarithromycin, erythromycin (Wako Pure Chemical Industries, Osaka, Japan), josamycin (Sigma-Aldrich), ampicillin (Sigma-Aldrich), or dexamethasone (Wako Pure Chemical Industries). clarithromycin was kindly supplied by Taisho Toyama Co., Ltd. (Tokyo, Japan). clarithromycin, erythromycin, josamycin, and ampicillin were dissolved in ethanol (EtOH, Wako) to therapeutic concentrations [19 (link), 20 (link)]. dexamethasone was dissolved in EtOH to 100 nM [21 (link)]. The final concentration of EtOH added to cells was 0.5%. After 24 h of culture, cells were stimulated by 50 ng/mL human recombinant IL-13 (Peprotech, Rocky Hill, NJ, USA) for 24 h. Cell viability was evaluated using WST-8 assay (Cell Count Reagent SF, Nacalai Tesque, Kyoto, Japan).

Primaquine and erythromycin were obtained from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). Aflatoxin B1 and dimethyl fumarate were obtained from Sigma–Aldrich (St. Louis, MO, USA). Terbinafine was obtained from Tokyo Chemical Industry (Tokyo, Japan). These compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma–Aldrich). The final concentration of DMSO in culture medium was less than 0.1%. SDS and D-mannitol were purchased from Nacalai Tesque (Kyoto, Japan) and dissolved in distilled water.

Tsumura rikkunshito extract granules for prescription (product code TJ-43, Tsumura & Co. lot numbers E24652 and H05142, Tokyo, Japan) were used for the investigational product. It was manufactured according to GMP, and adapted to factory release test. The sample of the investigational drug used in this study is retained in Tsumura & Co. 7.5 g of this herbal preparation contains 4.0 g of dried extract obtained by spray drying of a hot water extract of a mixture of eight crude drugs: 4.0 g of Atractylodis lanceae rhizoma (Compositae; atractylodes lancea rhizome), 4.0 g of Ginseng radix (Araliaceae; ginseng), 4.0 g of Pinelliae tuber (Araceae; pinellia tuber), 4.0 g of Poria (Polyporaceae; poria sclerotium), 2.0 g of Zizyphi fructus (Rhamnaceae; jujube), 2.0 g of C. unshiu pericarpium (Rutaceae; citrus unshiu peel), 1.0 g of G. radix (Leguminosae; glycyrrhiza), and 0.5 g of Zingiberis rhizoma (Zingiberaceae; ginger). The standard components contained in rikkunshito and digoxin were supplied by Tsumura & Co. Atractylodin and atractylenolide III were supplied by Tsumura & Co. and Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Erythromycin was purchased from Wako Pure Chemical Industries, Ltd. (±)-Warfarin-d5 was purchased from C/D/N ISOTOPES INC. (Pointe-Claire, Quebec, Canada). Other chemicals were purchased from commercial sources.