| Curcuminoids(Cur)is an active ingredient extracted from the rhizomes of ginger,turmeric,and other plants of the family Zingiber officinale.Cur have the conjugated molecular structure consisting of?-diketone linked to two o-methylated phenols Polyphenols.Cur has a wide range of pharmacological effects,including hypolipidemic,anti-tumor,anti-inflammatory,anti-oxidant and protective effects on cardiovascular and nervous system.In recent years,studies have found that Cur has a high value in neuroprotective effects,for example,in neurodegenerative disease such as Alzheimer's disease(AD)and Parkinson's disease(PD).Curcumin is hardly soluble in water,its absorption rate after oral administration is low,and its metabolic excretion rate is fast,resulting in its low bioavailability.However,the extensive pharmacological effects of curcumin are not attenuated by its low bioavailability,and it can be inferred that turmeric The production of pharmacological activity of the hormone does not necessarily result directly from the action of its prodrug and may be related to the effect of curcumin metabolites or degradation products distributed in the tissue.The liver tissue contains various metabolic enzyme systems and is the main drug metabolism organ in the organism.It is an important means for drug metabolism research.In this study,curcumin solution and liver homogenate suspension were incubated together in a 37°C water bath.The product ofcurcumin liver metabolism in vitro was determined by HPLC.Studies have shown that one of the secondary metabolites of curcumin,tetrahydrocurcumin,also has a neuroprotective effect.Is curcumin metabolized into tetrahydrocurcumin and enters brain tissue,or does curcumin through the blood-brain barrier metabolize to produce tetrahydrocurcumin in brain,which lacks relevant research reports.Blood-cerebrospinal fluid barrier(blood-CSF barrier)is composed of brain microvascular endothelial cells(BMECs),astrocyte(As)foot processes,pericytes,A complex of cells,a dynamic regulatory interface that exists between the brain,spinal capillaries and nerve tissue.Methods to study drug delivery in the blood-CSF barrier are mainly divided into in vivo and in vitro experiments,which in vitro tests avoid the overall complexity of in vivo assays and are relatively suitable for mechanism studies.In this study,the same type of primary rat BMECs and As co-culture method to establish in vitro blood-CSF barrier model.Then transmembrance test of Cur and its liver metabolites across the in vitro blood-CSF barrier model.1.Establishment and evaluation of the in vitro blood-CSF barrier modelNewborn SD rats(within 24h)cortex tissue material,digestion,centrifugation of As,isolated and purified in As culture medium,showing typical As morphological features,GFAP positive expression.The cell purity was over 95%.The cerebral cortex gray matter in neonatal SD rats(within 7d)was used as material,and the isolated and purified brain microvascular segments were obtained by two-step enzymatic digestion and BSA centrifugation.To BMECs special medium,BMECs grew out from the brain microvessel section and purified.The typical characteristics "paving stone" of BMECs were observed.The positive expression of ? showed that the purity of BMECs was over 95%.BMECs and As at different times,inertial inoculation of Transwellsemipermeable membrane on the upper and lower surfaces of the intercellular communication through the semi-permeable membrane to Transwell chamber and cell culture plate-based in vitro blood cerebrospinal fluid barrier,clear BMECs and As clear morphology,adherent Fusion growth.The model group of high-resistance(378.97 ±11.38)?·cm2,4 hour liquid test leak test was positive by determining the trans-endothelial cell resistance(TEER)co-cultured model group,and the blood-cerebrospinal fluid barrier,And then the ?-Glutamyl transpeptidase(?-GT),Alkaline phosphatase(ALP)and sodium fluorescein permeation test Showed high enzymatic activity and strong ability to bypass restricted passage(P <0.01).It has been confirmed through experiments that an in vitro blood-CSF barrier model that is relatively simple,reliable,repeatable and nearly in-vivo is established and can be applied to subsequent drug transport experiments.2.Cur in vitro liver metabolites product determinationThe Cur solution and rabbit liver homogenate suspension were incubated in water bath at 37 ?.The content of curcumin was determined by HPLC,and the changes of chromatogram before and after the metabolism of curcumin were compared to detect the production of metabolites.After hepatic metabolism,the retention time was 3.7min appeared a peak,the peak area within a certain metabolic time as curcumin peak area decreased and increased,and with the curcumin concentration and liver homogenized Suspension concentration increased and increased,initially identified as curcumin liver metabolites.The optimal experimental conditions of liver metabolism were determined by single factor test.The results showed that under the conditions of curcumin concentration of 163?mol·L-1,suspension concentration of liver homogenate of 0.6g · m L-1 and incubation time of 120 min the best product yield.3.Transmembrance transport of Cur and Cur metabolites across thein vitro blood-CSF barrier modelBy Cur and Cur metabolites concentration screening experiments to determine the Cur of safe concentration is 652?mol·L-1 and Cur metabolites of safe concentration is 326?mol·L-1.The two solutions were added to the extracorporeal blood-CSF barrier.After 1 h transport experiment,the results showed that curcumin had a small amount of passage,but only a trace of the metabolite was detected after the metabolism of curcumin.The results showed that curcumin was a low permeability drug for the blood-cerebrospinal fluid barrier,and the permeability of its metabolites needed to be proved by experiments. |
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