D galactose

Hawthorn of the variety “Big Venus” was purchased in local supermarket (Baoding, China) at a fully mature ready-to-eat stage. Polyphenols including epicatechin (≥96%) and chlorogenic acid (98%), monosaccharides standards (D-galacturonic acid (98%), D-glucose (98%), D-mannose (98%), D-rhamnose (98%), D-galactose (98%), D-fucose (98%), D-arabinose (98%)), cellulase (400 U/mg), D3520 macroporous resin, phenol and 3-phenylphenol were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other chemicals and reagents were of analytical grade and obtained from Baoding Qianyue Trading Co., Ltd. (Baoding, China).

Eighty C57/BL6 mice with an age of eight weeks were randomly divided into eight groups. The mice from the model group were subcutaneously injected with D-galactose (B21893, Yuanye, Shanghai, China) (400 mg/kg) for 60 days, while an equivalent amount of physiological saline was given to those from the control group. For the RNP and BNP treatment groups, except for 60 days’ D-galactose (400 mg/kg) injection, different doses of RNP and BNP (high dose, 400 mg/kg/d; medium dose, 200 mg/kg/d; low dose, 100 mg/kg/d) were intragastrically supplied from day 30, and thus named RNP-L, RNP-M, RNP-H, BNP-L, BNP-M and BNP-H, respectively. Physiological saline was intragastrically supplied as a control for the other two groups. After the last injection, the mice were fasted but allowed to drink water overnight, then the blood was collected for further biochemistry assays.

An Ultimate 3000 UPLC system (Thermo Fisher Scientific, USA) coupled with an electrospray ionization triple-quadrupole mass spectrometer (TSQ Endura, Thermo Fisher Scientific, USA) was used in this study. Chromatographic separation was carried out with an XAmide column (150 mm × 2.1 mm i.d., 5 μm) using an UltiMate 3000 HPLC system (Dionex, Sunnyvale, CA, USA). The column was kept at 40°C with a mobile phase of 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.2 mL/min. Gradient elution began with 80% B and was then programmed as follows: 75% B from 0 min to 5 min, 60% B from 5 min to 10 min, and 85% B from 10 min to 20 min. MS detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ion source in positive and negative ionization modes.
The optimized mass spectrometric conditions were as follows: positive mode voltage, 3.5 kV; negative mode voltage, 2.5 kV; sheath gas flow, 35 arb; aux gas flow, 10 arb; ion transfer tube temperature, 325°C; vaporizer temperature, 275°C; and mass scan range, m/z 50–1000.
The sample spectra were compared with those of standards. Standards of maltotriose, α-D-lactose monohydrate, maltose, D-galactose, sucrose, D-(+)-xylose, D-glcNAc, rhamnose, D-fructose, D-(-)-arabinose, D-mannose, and D-(+)-glucose were purchased from YuanYe Biotechnology Company (Shanghai, China).

D-galactose was purchased from Shanghai Yuan Ye Bio-Technology Co., Ltd. (Shanghai, China), MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-zolium bromide] was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used in this study including antibodies against Bcl-2, Bax, Bak, p62, LC3 I/II were purchased from Abcam (Cambridge, MA, USA). Antibodies against Cleaved caspase 3, Mcl-1, Pink, Parkin, Beta-actin, HRP-linked secondary antibody were purchased from Proteintech (Chicago, IL, USA). Antibodies against AKT, p-AKT, 4EBP1, p-4EBP1, p70s6k, p-p70s6k were purchased from Cell Signaling Technology (Boston, MA, USA).