Study On Anti-AD Active Components Of Ginseng Based On Profile-effect

Purpose:The AD is persistent neurological degenerative disease associated with age,leading to the decline of memory and mental disorders,cognitive dysfunction,and a serious threat to the lives and health of the elderly,which is one of the most destructive diseases among the old.There are currently 35.6 million people estimated to be living with dementia worldwide,and this number is expected to double between the years 2030 and 2050.The purpose of this paper is to select the active components in ginseng to inhibit Acetylcholinesterase?AChE?based on one-dimensional chromatography,and the active components of AChE inhibition in ginseng were screened by LC-MS based on two-dimensional chromatography mass spectrometry.The active cellular protection constituents of human neuroblastoma cells?SH-SY5Y?were screened by high resolution liquid chromatography-mass spectrometry?RRLC-MS²?and multivariate statistical analysis.With molecular docking and cell metabolomics,the material foundation of medicinal effectiveness and action mechanism of anti-AD in ginseng were preliminarily determined to provide a scientific basis for the development of new drugs for the treatment of AD.Grey correlation analysis?GRA?and partial least squares analysis?PLS?combined with ginseng different medicinal parts common components of the HPLC fingerprint of 10 AChE inhibition screening of active componentsMethods:First of all,with AChE inhibition rate as active index,the method of Ellman improvement was used to screen the active AChE inhibition parts from the ginseng rhizome,main root,fibrous roots,total ginseng,xylem and phloem.Set up the first dimension of high performance liquid chromatography?HPLC?fingerprint of six ginseng different medicinal parts.The application of principal component analysis?PCA?and orthogonal partial least-squares discriminant analysis?OPLS-DA?were used to distinguish different medicinal parts from ginseng.Grey correlation analysis?GRA?and partial least squares?PLS?combined with 10 common fractions of HPLC fingerprint in different medicinal parts of ginseng root,to screen AChE inhibitory activity components.With the second dimension ultrafiltration liquid chromatography/mass spectrometry?UF-LC-MS?technology,screening AChE inhibition active component from ginseng rapidly,and studying on the mechanism of AChEIs and AChE receptors based on molecular docking technology in ginseng.Finally,we used GRA and PLS to screen the active protective components of SH-SY5Y cells in 19 common peaks in the RRLC fingerprints of 6 ginseng parts.Annexin V/PI double-dyeing method was used to detect the apoptosis rate of Rb₁ for glutamine-induced SH-SY5Y cells.Based on¹H NMR cell metabolomics,Rb₁ was used to investigate the changes in the metabolism profile of SH-SY5Y cells induced by glutamate,and to reveal the mechanism of neuroprotective action of Rb₁.Results:Different medicinal parts from ginseng have a certain AChE resisted ability,and fibrous root showed much higher AChE inhibition ability than phloem,total ginseng,taproot,xylem and rhizome,and the fibrous root's IC₅₀0 is 15.63?g/mL.PCA analysis showed that rhizome,fibrous root and total ginseng get good distinguish,but the difference of taproot,xylem and phloem is not obvious,thus the taproot,xylem and phloem have PCA and OPLS analysis alone.The result showed taproot,xylem and phloem can get distinguish,and the difference between the two is even greater.In the screening,the grey correlation coefficient was greater than 0.9 and the largest group of PLS positive correlation coefficient was divided into fraction 5?Fr.5?,which is the active AChE inhibition component of ginseng.In this way,5 potential active monomers with affinity degrees greater than 15%in Fr.5 were selected by affinity ultrafiltration?UF-LC-MS?technique.Ginsenoside Ri,Rd,Rb₃,Rb₁ and Rc were identified by LC-MS,and the activity verification results indicate that their IC₅₀0 is at4.27-20.00?g/mL.The molecular docking results show that the order of binding energy is parallel monomer AChE inhibition activity.In the screening of the protective active components of SH-SY5Y cells,Based on GRA analysis,Comprehensive multivariate statistical analysis results showed a total of 4 potential protective components of SH-SY5Y cells were screened,which correlation coefficient is greater than 0.8,and the VIP value obtained by OPLS-DA analysis is greater than 0.5 and PLS analysis coefficient is positively correlated.The LC-MS results indicated that ginsenoside Rb₁,Rb₃,Rb₂ and Rc were the active components,and the activity verification results indicate that ginsenoside Rb₁ was the most active component.It was found that ginsenoside Rb₁ could protect the SH-SY5Y cell by reducing apoptosis induced by glutamate and 18 metabolites of taurine,arginine,proline in ¹H NMR cell metabolomics analysis.Metaboanalyst showed taurine and hypotaurine metabolism,arginine and proline metabolism,alanine,aspartate and glutamate metabolism,D-Glutamine and D-glutamate metabolism were mainly involved.Ginsenoside Rb₁ has SH-SY5Y cell protection by up-regulating 15 metabolites of taurine,arginine,proline,alanine,glycine and threonine in the damage model and down-regulating glutamic acid,beta hydroxy butyric acid and scyllitol.Conclusions:In this paper,based on the spectral efficiency study on AD active components of ginseng are studied,ginsenoside Rb₃,Rb₁ and Rc were showed the both AChE inhibitory activity and SH-SY5Y cells protective activity.The active components of anti-AD in ginseng were preliminarily clarified.Based on molecular docking and NMR cell metabonomics technology to further clarify the molecular mechanism of ginseng of AD,molecular docking analysis showed that the specific amino acid residues in the complex of AChE and the specific amino acid residues of AChE activity center played an important role in their activity.¹H NMR based cell metabonomics research reveals the AD may be caused by excessive glutamate stimulation amino acid metabolic disorder caused by pathological changes,and a variety of energy Rb₁ mainly by influencing ginsenosides and amino acid metabolism of SH SY5Y cells protection.The results provide a reliable scientific basis for the development and application of anti-AD products,and provide references for the rapid screening and mechanism research of anti-AD active components in other traditional Chinese medicines.