Hesperidin

Hesperidin was purchased from Tokyo Chemical Industry (Tokyo, Japan). Trifluoroacetic acid (TCA) and methanol were purchased from Sigma (St. Louis, MO, USA). For the preparation of GME, unripe fruit of Citrus unshiu was obtained from the orchards of Jeju Island (Korea) and extracted with ethanol two times. Briefly, the dried unripe fruit was extracted with 4 L of 70% aqueous ethanol three times at 80 °C for 1 h, followed by further extraction with 95% aqueous ethanol to yield 90% ethanol-insoluble precipitate (GME). For analysis of the Hesperidin content in GME, an HPLC (LC-2000Plus, Jasco International, Tokyo, Japan) assay using a Luna^® C18 5 μm column (4.6 × 250 mm) was performed. A gradient elution was achieved with various ratios of solvent A (0.1% TCA in water) to solvent B (methanol), with a flow rate of 1 mL/min as follows: 0–25 min, 70:30, 26–30 min, 10:90, and 31 min onward, 70:30. The wavelength of acquisition was set to 280 nm. The average content of Hesperidin in the GME was 35% (Supplemental Fig. 1).

Baclofen, gabapentin (internal standard, IS), glycyrrhizin, hesperidin, and ferulic acid were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Naringin and 6-Gingerol were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Neohesperidin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Ecdysterone and R,S-Inokosterone were obtained from analytical chemistry laboratory in College of Pharmacy at Catholic University of Daegu. Acetonitrile, methanol, and distilled water (all HPLC grades) were purchased from Mallinkrodt Baker, Inc. (Phillipsburg, NJ, USA). Perchloric acid and formic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Oyaksungisan Extract Granule was obtained from Hankook Shinyak Corp. (Nonsan, Chungnam, Korea). Achyranthes bidentata radix was obtained from Daegu Haany University (Gyeongsan, Gyeongbuk, Korea).

Lime peel powder was obtained from Chiangmai Bioveggie Co., Ltd. (Chiang Mai, Thailand). The powder was produced by vacuum drying fresh lime peel, which is food waste from the GMP-certified lime juice factory. Limonin (≥95.0% purity) and hesperidin (≥90.0% purity) were purchased from the Tokyo chemical industry (Tokyo, Japan). Eagle minimum essential medium (EMEM), fetal bovine serum (FBS), Phosphate-buffered saline (PBS) pH 7.4, 0.25% trypsin + 0.03% EDTA solution, penicillin/streptomycin, and Geltrex LDEV-free reduced growth factor were purchased from Gibco by Invitrogen Life Science Technologies, Thermo Fisher Scientific Inc. (Waltham, MA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT dye), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit with propidium iodide (PI) from Immune tools (Friesoythe, Germany).