Ginsenoside F1

Kinase experiment:

Glycosylation ability is assayed with overexpressed BSGT1 enzyme and F1. The reaction mixtures contain 100 μL of 0.5 mM F1 and 100 μL of 2.5 mM UDP-glucose and 800 μL of purified enzyme (final concentration at 0.1 mg/mL) (pH 7.0). The mixtures are incubated at 30°C for 24 h. Moreover, three groups of controls are incubated under the same conditions: (1) control 1 (C1) consists of Ginsenoside F1 with BSGT1; (2) control 2 (C2) consists of BSGT1 with UDP-glucose; and (3) control 3 (C3) consists of Ginsenoside F1 with UDP-glucose[1].

Cell experiment:

B16BL6 cells are cultured in Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin at 37°C in a humidified 95% air/5% CO2 atmosphere. Cell viability is determined for Ginsenoside F1 and metabolite 1 using MTT conversion to formazan. Cells are seeded at a density of 1×105 cells/well in a 96-well plate, cultured for 24 h, and treated with various concentrations from 1 μM to 200 μM of Ginsenoside F1 and metabolite 1 for 5 d. Finally, 10 μL of MTT (5 mg/mL in PBS) is added to each well. Cells are incubated at 37°C for 3 h, and then DMSO (100 μL) is added to dissolve the formazan crystals. The absorbance is measured at 570 nm with the reference wavelength of 630 nm using an ELISA reader[1].

Animal experiment:

Mice[2]Six-week-old (17±1 g) male C57BL/6 mice and ApoE-/- mice with a C57BL/6 background are maintained in a temperature-controlled facility (temperature: 22±1°C; humidity: 60%) with a 14 h light/10 h dark cycle in conventional cages. Forty mice are randomly divided into four experimental groups (n=10/group): (I) C57BL/6 N mice, the control group; (II) ApoE-/- mice group; (III) ApoE-/- mice+ Ginsenoside F1 group; (IV) ApoE-/- mice+Probucol group. All mice are fed with a high fat diet (HFD, 0.3% cholesterol and 20% pork fat) for 8 weeks. Ginsenoside F1 (50 mg/kg/day, i.g.) and Probucol (2 g/kg, i.g.) are dissolved in carboxymethyl cellulose sodium (CMC-Na). Oral administration is given to mice every day for 8 weeks. The control and model groups receive the aseptic 0.5% CMC-Na treatment every day (i.g., 0.1 mL/10g) [2].

References:

[1]. Wang DD, et al. Rare ginsenoside Ia synthesized from F1 by cloning and overexpression of the UDP-glycosyltransferase gene from Bacillus subtilis: synthesis, characterization, and in vitromelanogenesis inhibition activity in BL6B16 cells. J Ginseng Res. 2018 Jan;42(1):42-49.
[2]. Qin M, et al. Ginsenoside F1 Ameliorates Endothelial Cell Inflammatory Injury and Prevents Atherosclerosis in Mice through A20-Mediated Suppression of NF-kB Signaling. Front Pharmacol. 2017 Dec 22;8:953.