The Basic Research Of Effective Substance In The Non-Saponins From Ginseng Which Showed The Effect Of Treatment Of Alzheimer's Disease

Purpose: China now has begun to enter an aging society and the increasing trend of prevalence rate of Alzheimer has brought a serious threat to the health of the aged and a huge pressure in mind and economy to the patients,their families and the society.Ginseng as rare herbal medicines in China has a medicinal history of over 4000 years and has good nootropic effects.In the prophase research work and the literature report we found that the non-saponins from ginseng have the function of improving learning and memory abilities and curing Alzheimer disease.This research is carried out for the purpose of determining preliminarily the structure of this active ingredient,which will lay good foundation for the future research.Method:(1)First,weigh the aqueous extracts of ginseng and dissolve it by adding water and stirring,and then get the ginseng ultrafiltrate Y-1,1K extra-dialysate Y-2 and 1K intra-dialysate Y-3 after centrifugation,ultrafiltration,dialysis,concentration and lyophilization.(2)The Morris water maze task: inject A?25-35 into the hippocampus of the rat to create a senile dementia model,and then measure the average escape latency(AEL)of the rat climbing up the underwater platform for rest by localization and navigation experiment,with its time length reflecting the learning and memory ability of the rat;then remove the underwater platform and measure the pass number of the rat through underwater platform by space exploration experiment;(3)Purification of active components: G-15 was used for further purification of the active component,and draw elution curve.(4)Research of physicochemical property: carry out study of physicochemical property on the active ingredient selected out via the Morris water maze task,apply the phenol-sulphoacid method to determine the absorbance of the reference substance under the wave length of490 nm,calculate the regression equation,and then bring this absorbance into the equation to calculate the content of neutral sugar;apply the m-hydroxydiphenyl method of 525 nm and neutral sugar content determination method to determine the content of uronic acid;apply Lowery method at the position of 660 nm to determine the content of protein,the same as above;(5)Determination of purity and molecular weight: apply the HPLC and take the BSA(66430 Da),VB12(1355.37 Da),L – Try(204.23 Da),protease inhibitor(6500 Da),cytochrome C(13000 Da)as reference substance to make standard curve,and apply thesoftware GPC to calculate the molecular weight distribution of the sample;(6)Sugar composition analysis: the screened active ingredient will be prepared into pre-column derivative by using PMP derivatization method,and determine its sugar composition via HPLC method;(7)Glycosidic bond connection mode analysis: apply the sodium hydroxide method to process the test sample,and prepare the processed test sample according into the sugar composition analysis method into the derivatives via hydrolysis,reduction,acetylation,and then determine its glycosides bond connection mode by GC-MS;(8)Amino-acid analysis:Prepared FDNB as a derivative after hydrolysis,and determine amino acid composition via HPLC method.Result:(1)Result of the Morris water maze task: the result of localization and navigation experiment showed that while comparing the model group,Y-1 group and Y-3group the rat AEL are all shortened significantly,where Y-3 group is much more obvious;the result of space exploration experiment showed that the pass number of rat through platform for Y-1 group and Y-3 group increases obviously comparing with model group,especially for Y-3 group;(2)After purification,Y-3A and Y-3B were obtained,Y-3A is the main component of Y-3,and according to the elution curve,Y-3A is ginseng glycoprotein.(3)Result of research of physicochemical property showed that the neutral sugar content of active ingredient Y-3A is 19.11%,and 6.79% and 51.60% respectively for uronic acid and protein;(4)Result of determination of purity and molecular weight showed that Y-3A is a mixture and the molecular weight distribution span is between 400Da-50000Da;(5)Result of sugar composition analysis showed that this substance mainly consists of 7 kinds of sugar including Man,Rha,Gal A,Glc,GlcNAc,GalNAc and Fuc,where the Glc content is 51.32%;(6)Glycosides bond connection mode is that the backbone is composed of(1?2)-linked Man,(1?3)-linked Glc and(1?3)-linked Gal NAc/GlcNAc.The branch point has existed on(1?4,6)-linkde Man.The non-reducing termination is composed of Glc,GalA,and the most are Glc with its content of 20.13 %;(7)Amino-acid analysis result: Y-3A consists of 17 kinds of amino acids where most of it is Arg,Gly and Glu,respectively for 39.45%,12.48% and10.58%.Conclusion: We identified by the Morris water maze task that the active ingredient for curing Alzheimer is 1K intra-dialysate(Y-3)and Y-3A is the main component of Y-3,and Y-3Ais a glycoprotein.The result of physicochemical property research and determination of purity and molecular weight showed that Y-3A is mixture.The preliminary structure analysis to Y-3A showed that this ingredient includes 7 kinds of sugar with content of Glc higher than others,the backbone is composed of(1?2)-linked Man,(1?3)-linked Glc and(1?3)-linked GalNAc/GlcNAc,the branch point has existed on(1?4,6)-linkde Man,and the non-reducing termination has more Glc,and ginseng glycoprotein contains 17 kinds of amino acids,in which the content of Arg,Gly and Glu are in higher level.