Hesperetin

The sterilized gelatin sponge scaffolds were prepared by cutting absorbable gelatin sponge (porosity 80%, Nanjing Jinling, China) into discs (diameter: 5 mm, thickness: 2 mm). Then, hesperetin (Sigma) was dissolved in enthanol sulotion, and a concentration of 100 μM was used for preparing hesperetin-gelatin sponge scaffolds constructs. 100μL hesperetin sulotion was uniformly pipetted onto the gelatin sponge scaffolds (100 μL hesperetin/scaffold). The scaffolds were frozen at –80°C for 12 h and freeze-dried for 24 h.
The scaffolds were placed in the wells of a 12-well culture plate (Corning, USA). The density of MSCs was regulated to 1 × 10⁶ cells/mL. MSC aliquots of 100 μL were seeded onto each scaffold and allowed to attach for 3 hours at 37°C before being immersed in complete medium. The constructs were incubated in vitro at 37°C in 5% CO₂. After 12 days, the scaffold MSCs were used in an in vivo study.

Cells were seeded at a density of 2 × 10⁶ cells per well in 6-well plates. Cultures were dissociated in 0.05% (w/v) trypsin (Sigma-Aldrich, St. Louis, MO, USA) in a phosphate-buffered saline (PBS), pH 7.4, and then passaged when the cell was confluent. For AGE-induction studies, cells were cultured in a fresh medium with 1% FBS for 2 h. Later, cells were pretreated with hesperetin (Sigma-Aldrich, St. Louis, MO, USA; Cat. # 69097-99-0, purity ≥ 95%) at different concentrations (10–40 μmol/L), or rosiglitazone (Sigma-Aldrich, St. Louis, MO, USA; Cat. # 557366, purity, ≥99%) at 10 μmol/L [25 (link)] for 1 h, followed by exposure to appropriate concentrations of AGEs in bovine serum albumin (AGEs-BSA) (Sigma-Aldrich, St. Louis, MO, USA; 50–250 μg/mL) for different time points (6–48 h) without medium change. The stock solution (100 μmol/L) of hesperetin or rosiglitazone was made by dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and diluted in culture medium to the appropriate concentrations for subsequent experiments. The final concentration of DMSO was ≤0.1%, a level generally harmless to most cells [26 (link)]. The following experiments were assessed after treatment.

The analysis employed an Agilent 1260 HPLC-DAD system (Agilent Technologies, Waldbronn, Germany) for high-performance liquid chromatography (HPLC). A separation process was conducted using an Eclipse C18 column (4.6 mm × 250 mm i.d., 5 μm), with 10 μL of each fungal extract sample injected. The mobile phase comprising water and 0.05% trifluoroacetic acid in acetonitrile (HPLC grade, Alpha Chemika, Mumbai, India), flowed at a rate of 1 mL/min, following a linear gradient. The column temperature was approximately 35 °C, and a multi-wavelength detector performed the monitoring at 280 nm [75 (link)]. Cinnamic, caffeic, ferulic, syringic, gallic, ellagic, p-coumaric, and chlorogenic acids, in addition to hesperetin, kaempferol, rutin, catechin, quercetin, apigenin, methyl gallate, vanillin, daidzein, and naringenin were purchased from Merck, Darmstadt, Germany, and utilized as standard phenolics.