The Research On The Chemical Analysis Of Guilingji And Its Improvement In Mild Cognitive Impairment

Background:Gui Ling Ji is a well-known and classical TCM formula that can be traced back hundreds of years ago to the Ming and Qing dynasties as elixir and has been used in clinical for more than 400 years.Gui Ling Ji is composed of over 20 herbs,has the effects of strengthening the body and nourishing the brain,strengthening the kidney and invigorating qi,and stimulating appetite.It can be used for kidney deficiency,yang weakness,memory impairment,nocturnal dream overflow,soreness of the waist and weakness of the legs,cough due to qi deficiency,diarrhea due to five diarrhea,and loss of appetite,according to the Pharmacopoeia of the People's Republic of China.However,Guilingji with various functions and indications,but few practical applications in clinic,and its clinical orientation is too broad.The market share of this classic famous prescription is not high,and the modern systematic and holistic research has not yet been carried out,which seriously limits the secondary development of Guilingji.First of all,Guilingji possessed multiple functions based on its multi-component,but the research on chemical components of Guilingji has not been reported.Secondly,Guilingji was the only the only existing compound herbal formulae made by the process of alchemy,and the scientific implications of"alchemy"was still unknow.Third,the compound formulae of Guilingji were huge and the production technique is complex,and the product quality consistency and production process stability have not been evaluated.Fourthly,Guilingji's instructions showed that it can be used for"memory decline".Our previous research also found that Guilingji can obviously improve the impairment of learning and memory functions caused by aging.Can Guilingji be be used as a routine treatment in mild cognitive impairment(MCI)?What is its pharmacological mechanism?The purpose of this topic is to study the above problems,and to inherit,develop and make good use of this classic compound formulae.Amis:(1)The chemical composition of Guilingji was preliminarily defined from organic compounds and inorganic elements.(2)The scientific connotation of alchemy of Guilingji was discussed from chemical composition.Establish the fingerprint of organic components and inorganic elements of Guilingji,and evaluate the consistency of existing products and the stability of production process,provide guarantee for clinical drug use.(3)Clarify the efficacy of Guilingji in improving MCI,and further explore its possible mechanism.Method:(1)The chemical composition of Guilingji was comprehensively identified and characterized by LC-MS and ~1H NMR combined with polar segmentation strategy.A series of methods,such as diagnostic ion filtering,mass defect filtering and database matching,was used to analyze the MS data.The concentrated inorganic elements of Guilingji were determined by ICP-MS.(2)LC-MS and ICP-MS technical combined with multivariate statistical analysis were used to compare the differences of organic components and inorganic elements before and after alchemy of Guilingji,and explain the significance of alchemy based on chemical substances.The fingerprints of organic components and inorganic elements of Guilingji were established by UPLC and ICP-MS respectively,and the consistency of Guilingji and the stability of production process were evaluated from both organic and inorganic aspects.(3)The MCI rat model was replication by D-galactose(D-gal)combined with semi-high fat diet.Rats were randomly divided into 6 groups:control group,model group,positive drug including donepezil group and Ginkgo biloba leaf tablet group,low-dose group(75 mg/kg)and high-dose group(150 mg/kg)of Guilingji for continuous administration for 30 days.The pharmacodynamic effects of Guilingji on MCI were evaluated by weight,appearance,behavior(Morris water maze),blood lipids(total cholesterol,triglyceride,high density lipoprotein,low density lipoprotein),visceral index,pepsin level,liver and kidney function(alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,urea nitrogen),pathological damage of hippocampus and other indexes.To explore the pathological mechanism of MCI and the possible pharmacological mechanism of Guilingji by the related indexes of oxidative stress,inflammatory factors,cholinergic,apoptosis and neurotrophic.LC-MS metabonomics was used to explore the metabolic profile of MCI and the possible mechanism of Guilingji in improving MCI.Result:(1)166 compounds in Guilingji were characterized by LC-MS and ~1H NMR techniques,including amino acids,organic acids,phenolic acids and saponins,coumarins,flavonoids,triterpenes and triterpene saponins,fatty acids,organic bases and saccharides.ICP-MS analysis showed that there are more than 70 kinds of inorganic elements in Guilingji.Almost all major group elements,transition metal elements,lanthanide and actinide elements were included in Guilingji,and 14 essential trace and major elements were screened out as an indicator of quality consistency evaluation according to the relative contents of elements and function of Guilingji.(2)The changes in organic chemical compositions and inorganic elements before and after alchemy of Guilingji were detected by LC-MS and ICP-MS combined with multivariate statistical analysis.It was found that the content of ionone,chalcone,amide and fatty acid compounds were increased after alchemy.The content of flavonoids,isoflavones,dihydroflavones,flavonoid glycosides and coumarins were decreased after alchemy,the content of inorganic elements B and Si were decreased,and the content of Mg,K,Cr and Ni were increased.The UPLC fingerprint of Guilingji was established and 19 common peaks were identified,and the similarity of 15 batches of Guilingji were calculated.The results showed that the similarity of each batch of samples was good(>0.93).The content of 14inorganic elements(Na,Mg,K,Ca,Cr,Mn,Fe,Co,Ni,Cu,Zn,Se,Mo and Sn)in Guilingji was determined by ICP-MS,and the fingerprint of inorganic elements was established with element type as abscissa and relative content as ordinate,and the similarity of 10 batches of products were calculated.The results showed that the similarity of each batch of samples was good(>0.97).(3)Pharmacodynamic effects:The rats in the model group showed aging characteristics such as dark coat color and loose skin.Morris water maze results showed that the escape latency of rats in model group was significantly prolonged;the residence time of the target quadrant was significantly shortened;the times of crossing the platform was significantly reduced.Liver and spleen index were significantly decreased,and pepsin level was significantly decreased.Serum biochemical indexes showed that blood lipid indexes,liver function and kidney function indexes of model group rats were changed significantly.Guilingji significantly improved the learning and memory function.Guilingji could also regulated liver and spleen index,pepsin level,blood lipid level,liver functional disturbance,lessen pathological damage of hippocampus.The effect of Guilingji was better than that of Ginkgo leaf tablets and Donepezil.Molecular pharmacology mechanism:Guilingji could significantly reduce the serum MDA level,increase the activities of SOD and GSH-Px,and significantly reduce the levels of TNF-?,IL-6 and IL-1?in MCI rats.Guilingji can significantly increase Ach level and decrease Ach E activity in hippocampus of MCI rats,and increase the level of Bcl-2,BDNF and decrease the level of Bax.Guilingji can regulate MCI more comprehensively than the two positive drugs,and can improve MCI by regulating oxidative stress,inflammatory reaction,cholinergic,apoptosis and neurotrophic.Metabonomics:the results of LC-MS metabonomics in serum and hippocampus showed that the metabolic profile of MCI rats was changed compared with the blank control group.There were 25 different metabolites were found in serum,mainly including unsaturated fatty acids such as lysolecithin,arachidonic acid,linoleic acid and linolenic acid,bile acids and sheath lipids.The results of pathway enrichment analysis showed that the main metabolic pathways involved in differential metabolites were linoleic acid metabolism,linolenic acid metabolism,arachidonic acid metabolism,glycerol phospholipid metabolism and primary bile acid biosynthesis.Then,a total of 19differential metabolites were found in hippocampus,mainly including amino acids such as valine,aspartic acid,leucine,glutamic acid,pyroglutamic acid,tryptophan and methionine fatty acids such as oleic acid,linoleic acid,linolenic acid and stearic acid;organic acids such as aconitic acid and citric acid;nicotinamide,purine,adenosine and so on.The main metabolic pathways include linoleic acid metabolism,nicotinic acid and nicotinamide metabolism,tricarboxylic acid cycle,alanine,aspartic acid and glutamic acid metabolism.The above results suggest that MCI rats have disorder of energy metabolism and lipid metabolism,and Guilingji has a significant regulatory effect on the above metabolic pathways,and has a better regulatory effect on different metabolites and pathways than the two positive drugs.Conclusion:(1)Clarifying the material basis of GLJ is helpful for the pharmacological studies.The analysis strategy could be applied to other TCM formula for comprehensive characterization and identification of chemical components.The identification results also provided bases for further researches on the scientific connotation of alchemy of Guilingji.(2)On the one hand,the chemical changes in organic compositions and inorganic elements after alchemy changed the cold and hot properties of Guilingj,showing the decrease of dryness,and the hot property was changed to warm and neutral properties.On the other hand,the membrane permeability and absorption of the compound compositions were improved.The established UPLC fingerprint and inorganic element fingerprint are simple,stable and reliable,which can be used to evaluate the stability of production process and product consistency of Guilingji.It is beneficial to improve the quality control of Guilingji from both organic and inorganic aspects.(3)Guilingji can alleviate the symptoms of MCI by improving spleen and stomach disharmony,liver and kidney deficiency and filling the marrow-sea.The possible mechanism was regulating oxidative stress,inflammatory reaction,cholinergic,apoptosis and neurotrophic,influence fatty acids metabolism,amino acids metabolism and energy metabolism.