Application of bakuchiol compound
A bakuchiol and drug technology, which is applied in the directions of active ingredients of hydroxy compounds, medical preparations containing active ingredients, drug combinations, etc., can solve the problem of ineffectiveness, sexual dysfunction, and ineffectiveness of patients with psychoneurotic diseases, etc. problems, to achieve good market prospects and excellent efficacy
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Embodiment 1
[0052] The establishment of embodiment 1 in vitro screening model
[0053] The present invention relates to experimental methods of conventional molecular biology, which are widely known in the art and described in, for example, Molecular Cloning: a laboratory Manual 3rd edition, edited by Sambrook et al., Cold Spring Harbor laboratory Press, Clod Spring Harbor, N.Y. , 2001 and Current Protocols in Molecular Biology, method reference books edited by Ausubel etc. have detailed introductions. Establishment of activity screening cell lines targeting serotonin, norepinephrine, dopamine, and γ-aminobutyric acid four neurotransporters (5-HTT, NET, DAT, GAT-1). By cloning the full-length coding sequences of 5-HTT, NET, DAT, and GAT-1 of rats (the cDNA sequences are derived from GeneBank on the PnbMed website) into the pCDNA3 vector (Invitrogen, USA), transfection into Chinese hamsters by electroporation Ovary cell (CHO) cells were cultured in 1640 medium containing G418 after 48 hou...
Embodiment 2
[0054] Example 2 Determination of 13-hydroxybakuchiol selection specificity
[0055] S6, NET, D8, and GAT-1 cells were cultured in 48-well plates (Costar) until the plates were confluent (approximately 60,000 cells per well). Discard the culture medium, wash once with PBS, suck off the PBS solution, add 90ul HBS (10mM Hepes, 100mM NaCl, pH8.0) to each well, incubate at 25°C for 10 minutes, and add 10ul HBS reaction solution to each well. Add 80ul HBS, 10ul different concentrations of 13-hydroxybakuchiol (B310) and positive control drug bupropion (BPR) to the experimental group and the positive control drug, and 10ul 3 H-labeled substrate (S6 uses 3 H-5-HT; NET adopted 3 H-NE; D8 adopted3 H-DA), 100 μM vitamin C and 100 μM parjiline. Incubate at 25°C for 20 minutes, wash three times with ice-bathed PBS solution, lyse with lysate for 60 minutes, absorb the lysate from each well, add it to 1.2ml scintillation liquid, and put it into a liquid scintillation counter (Beckman LS50...
Embodiment 3
[0062] Example 3 Cytotoxicity test of 13-hydroxybakuchiol
[0063] CHO cells were cultured in 1640 medium (containing 10% calf serum). After the culture dish was overgrown, trypsinized, seeded in a 48-well cell plate, and continued to culture until the number of cells reached 10. 5 Left and right, respectively, were treated as follows: after the liquid was changed, 10 μl of control solution (HBS containing 1% DMSO) was added to the control group, and 10 μl of 13-hydroxybakuchiol with different concentrations were added to the drug groups with different doses, so that the final concentration was 1 mM respectively. , 0.1 mM and 0.01 mM. The culture was continued for 24 hours, and MTT was added at the 20th hour to make the final concentration 0.5 mg / ml. After the reaction, the culture solution was sucked off, 100% DMSO was added, and incubated at 37° C. for 10 minutes. Transfer to a 96-well plate, and measure the OD value of each well with an enzyme label detector at a waveleng...
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