| Objective:Alzheimer’s disease(AD)is a common disease of the central nervous system,and its global prevalence is increasing every year,posing a serious threat to human health and quality of life.The existence of the blood-brain barrier limits the access of drugs to the brain for efficacy and poses a great challenge for drug development.Citri Reticulatae Pericarpium(CRP)has attracted much attention because of its multiple biological activities,and it has shown some effects in neuroprotection and treatment of AD.However,the therapeutic material basis and mechanism of action of CRP in improving AD symptoms are still unclear and need to be investigated in depth.Therefore,in this study,we combined the identification of the blood components of CRP with the cerebrospinal fluid components based on a sequential metabolic approach,analyzed the absorption and metabolism of CRP and the migrating components in blood,targeted the identification of brain components,and further investigated the potential of CRP for the treatment of AD based on acetylcholinesterase(AChE),a recognized target for AD therapy.Methods:(1)High performance liquid chromatography(HPLC)was employed to guide the selection of extraction solvent and extraction method of tangerine peel,and to screen the preparation method of CRP extracts.Meanwhile,ultra-high-performance liquid chromatography-high resolution mass spectrometry(UPLC-HRMS)was used to detect and analyze the components in the CRP ethanol extracts.By comparing the reference substances and references,and analyzing the retention time,accurate relative molecular mass,secondary fragment ions and fragmentation patterns of each component,the accurate identification of compounds was realized.(2)SD male rats were used as experimental subjects.The ethanol extracts of CRP was given by in situ intestinal perfusion,and blood samples were collected from mesenteric vein and femoral vein to obtain intestinal metabolism and hepatic metabolism blood samples.Comprehensive metabolic blood samples were collected via the abdominal aorta after intragastric administration.Different drug-containing plasma samples were analyzed using UPLC-HRMS,and their compositions were compared under different metabolic conditions to obtain a comprehensive list of blood components and their metabolic sites.Based on this analysis,we utilized the parallel reaction monitoring(PRM)technique to identify the components of CRP in cerebrospinal fluid.(3)The preparation of the effective parts was guided by the large class of components of CRP into cerebrospinal fluid,and the resins that could be used for the preparation of the effective parts were screened by selecting four different polar macroporous adsorption resins(MAR)of AB-8,D101,LSA-10 and NKA-9 as the study materials,and the saturation ratio,the absorbtion ratio and the eluation ratio as the investigated parameters.The best method for preparing and purifying the effective parts was determined using a single-factor test that examined the effects of the sample concentration,the loading amount,and the type and amount of desorption solvent on the purification of the effective parts.The blood and cerebrospinal fluid components of the CRP extracts’s affinity and binding sites with AChE were predicted using the molecular docking.Based on Ellman ’s colorimetric method,an in vitro enzymatic reaction system was established to study the inhibitory effect of CRP extracts,effective parts and monomer components on AChE activity.Results:(1)The ethanol extract of CRP was prepared by 60%ethanol and ultrasonic extraction,and 44 compounds were identified from it,including 35 flavonoids such as lucenin2,vicenin-2,stellarin-2,narirutin,hesperidin,nobiletin,tangeretin,sinensetin,naringenin,2 coumarins such as meranzin and isomeranzin,5 amino acids and peptides such as phenylalanine,proline and citrusin Ⅲ.Also included are nomilin as well as adenosine.(2)22 prototype blood components of CRP were detected in the intestinal metabolism samples,and 18 prototype blood components were detected in the hepatic metabolism samples.9 identical prototype blood components(narirutin,hesperidin,meranzin,isosinensetin,5,7,8,3’,4’,5’-hexamethoxyflavone,sinensetin,nobiletin,3,5,6,7,8,3’,4’-heptamethoxyflavone and tangeretin)were detected in the three metabolic samples of comprehensive metabolism,intestinal metabolism and liver metabolism,and nine prototype blood components were also detected in cerebrospinal fluid.Based on the high sensitivity and selectivity of PRM,it was found that 5,7,8,3’,4’-pentamethoxyflavavone could also enter the cerebrospinal fluid.(3)Among the four resins,NKA-9 type MAR showed the best adsorption and resolution of the five index components(narirutin,hesperidin,sinensetin,tangeretin and nobiletin)in the cerebrospinal fluid of CRP;The optimum purification conditions were as follows:the sample concentration was 0.5 g/mL,the maximum loading amount was 7.5 g/mL,impurities were removed using 5 BV deionized water and 20%ethanol,and effective parts Ⅰ and Ⅱ were prepared gradually using 5 BV 40%ethanol and 6 BV 95%ethanol.It was found by HPLC analysis in this process that the content of narirutin and hesperidin in effective parts Ⅰ were 20.84%and 10.51%,respectively,while the content of sinensetin,nobiletin and tangeretin in effective parts Ⅱ were 1.18%,8.13%,and 2.55%,respectively.The results of molecular docking showed that the components of CRP in cerebrospinal fluid had strong binding ability with AChE.The benzene ring and phenolic hydroxyl group may be the main binding sites,and they mainly interacted with phe330,tyr334,trp279 and trp84 residues through hydrogen bonding,hydrophobic interaction and π-π stacking.The in vitro enzyme activity inhibition assay showed that the inhibition of AChE was significantly enhanced after purification of CRP extracts,with the best inhibition effect of effective parts Ⅱ.At a concentration of 78.8 μg/mL,the inhibition rate was 55.23±0.40%.It was speculated that the inhibitory effect of the effective fraction Ⅱmight be due to the synergistic effect of polymethoxyflavones such as sinensetin,nobiletin and tangeretin.Conclusion and significance:This study comprehensively investigated the metabolism of the chemical components in CRP using sequential metabolism methods,clarifying its metabolic profile in vivo and further characterizing its chemical components in cerebrospinal fluid.This provides a more precise and comprehensive material basis for the treatment of AD and other brain diseases with CRP.In addition,this study examined the binding ability and action sites of CRP in vivo components to AChE as well as the inhibitory activity of CRP extract,effective parts,and monomers on AChE,with AChE as the target.These results reveal the potential of CRP in treating AD and provide a new research direction for screening AChE inhibitors from traditional Chinese medicine sources. |
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