Applications of pinoresinol in preparing products for preventing and treating diseases induced by oxidative stress
A technology of oxidative stress and pinoresinol, which is applied in the field of medicine to achieve the effect of increasing GSH levels and inhibiting cell damage and apoptosis
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[0044] The preparation and structural confirmation of embodiment pinoresinol
[0045] The obtaining method of pinoresinol is as follows: the cinnamon medicinal material is extracted with 95% ethanol to obtain an ethanol extract, and then sequentially extracted with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate part was eluted with a petroleum ether-ethyl acetate system gradient, and the eluent concentrations were: petroleum ether-ethyl acetate=100:0, petroleum ether-ethyl acetate=95:5, petroleum ether-ethyl acetate Ester=90:10, petroleum ether-ethyl acetate=85:15, petroleum ether-ethyl acetate=80:20, petroleum ether-ethyl acetate=70:20, petroleum ether-ethyl acetate=60:40, Petroleum ether-ethyl acetate=50:50, after combining, 8 fractions (Fr.1-8) were obtained. Fr.4 (petroleum ether-ethyl acetate=85:15 eluted fraction) was eluted through a silica gel column with petroleum ether / ethyl acetate (90:10→50:50) as the mobile phase gradient, and a total of 10 fract...
Embodiment 2
[0048] Example 2: Evaluation of NQO1-inducing activity of pinoresinol
[0049] (1) Culture of mouse hepatoma cell line Hepa 1c1c
[0050] The mouse hepatoma cell line Hepa 1c1c was purchased from the American Type Culture Collection (ATCC), using MEM medium containing 10% fetal bovine serum (FBS), placed at 37 °C, 5% CO 2 cultured in an incubator.
[0051] (2) NQO-inducing activity test
[0052] Hepa 1c1c cells were inoculated on a 96-well plate, and different concentrations of pinoresinol (confirmed in Example 1) were added after the cells adhered to the wall for 24 hours. The cells were lysed with 0.8% digitonin solution, and the detection solution (1.0 mL 0.5 M Tris Hydroxymethylaminomethane-hydrochloric acid (Tris-HCl), 15 mg bovine serum albumin, 6 mg MTT, 150 μL Tween-20, 150 μL 150 mM D-glucose-6-phosphate, 15 μL 7.5 mM flavin adenine dinucleotide, 27 μL 50mM nicotinamide adenine dinucleotide phosphate, 20 μL 50mM menadione), let stand for 3min, and measure the lumin...
Embodiment 3
[0054] Example 3: pinoresinol can up-regulate the protein levels of Nrf2, γGCS and NQO1
[0055] Method: Western blot analysis (Western blot) to detect changes in protein levels in cells
[0056] Beas-2B cells were inoculated in a 35 mm diameter petri dish, cultured until the density reached 70%-80%, and then treated with different concentrations of compounds to be tested for 16 h, washed twice with PBS, added cell lysate (50 μg / ml aprotinin , 0.5mM phenylmethylsulfonyl fluoride, 1mM sodium orthovanadate, 10mM sodium fluoride, 10mMβ-glycerol phosphate), the protein was collected and the protein concentration was determined by Bradford method. Each sample protein (100 μg) was loaded on the sample, and the protein components were separated by SDS-PAGE, and the protein bands were transferred to the nitrocellulose membrane by electrotransfer method. After the film was sealed with 5% skimmed milk powder solution prepared in TBS at room temperature for 1 hour, it was incubated with...
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