Study On The Inhibitory Effect Of Lycibarbarspermidines On Cholesterol-24-Hydroxylase

In recent years,with more and more knowledge of Chinese herbal medicine,Lycium barbarum has been widely used as a dietary supplement in people's life.Modern chromatographic analysis has proved that barbarum polysaccharide,carotenoids and polyphenols are its main active components.Modern pharmacological activity studies have found that Lycium barbarum plays an important role in neuroprotection,anti-aging,immune regulation,liver protection,anti-oxidation,eye care,anti-tumor and other aspects.Ji Nan University's research team found that the active compounds of Lycibarbarspermidines had a good effect on short-term memory,but the mechanism was not clear.Cholesterol-24-hydroxylase?CYP46A1?is a key enzyme in the metabolism of cholesterol in the brain.Cholesterol was metabolized by CYP46A1 to generate 24S-hydroxycholesterol and then expelled from the brain.Research evidence shows that the high level of24S-hydroxycholesterol may be related to some neurological diseases such as Alzheimer's Disease.Thus,it was speculated that Lycibarbarspermidines might play a neuroprotective role by inhibiting the activity of CYP46A1 and lowering the level of 24S-hydroxycholesterol.The purpose of this study was to investigate the inhibitory potential of CYP46A1 on Lycibarbarspermidines.The experiment used ultra-high performance liquid chromatography-tandem mass spectrometry?UPLC-APCI-MS/MS?technology based on atmospheric pressure chemical ionization to establish an analytical method for the determination of the CYP46A1 metabolite24S-hydroxycholesterol and study of species differences in the metabolic properties of CYP46A1 in rats,mice and humans.After methodological verification and analysis,the linear fit of 24-hydroxycholesterol was good R²=0.9983.In the low,medium and high concentration quality control samples,the precision,accuracy and stability of the day and day were in line with the biological samples.The analysis requires that the minimum limit of quantification at the ng/mL level.In the application of the method,the product24S-hydroxycholesterol formed by the recombinant human enzyme CYP46A1 conforms to the Michaelis-Menten equation,the constant K_m is 3.30±0.48?mol/L,and the V_max for CYP46A1 is 0.29±0.01 nmol/min/nmol CYP.Studies on species of rat,mice and human showed that the main metabolites of mice were 24,25 and 24,27-dihydroxycholesterol,while the main metabolites of rat and human CYP46A1 were 24S-hydroxycholesterol.Using the established CYP46A1 activity detection method and recombinant human CYP46A1,we evaluated in vitro the inhibitory ability of Lycibarbarspermidines compounds?DW-1 to 7?on CYP46A1.The research results showed that the compounds DW-1,DW-2and DW-3 can inhibit cholesterol-24-hydroxylation,the compounds DW-1 and DW-2 mixed inhibited CYP46A1 with K_i of 106.30±24.36 nmol/L and 258.40±171.70 nmol/L,respectively,while the DW-3 showed competitive inhibition of CYP46A1 hydroxylation,K_i was 12.89±1.64 nmol/L.The results suggest that even when low doses of DW-1,DW-2,and DW-3 are consumed,it is possible to reduce the content of 24S-hydroxycholesterol in human body by inhibiting the activity of CYP46A1,thereby reducing its toxic effect on nerve cells,especially neurons.This has significant significance for the study of late neurodegenerative diseases.