Study On The Exploration Of Quality Marker-related Components Of Shen-zhi-ling Oral Liquid And Rapid Quality Evaluation Method

Alzheimer’s disease(AD)is a serious neurodegenerative disease.Shen-zhi-ling(SZL)oral liquid is the first new Chinese medicine for mild and moderate AD syndrome of insufficient heart and qi,including "monarch" Codonopsispilosul,Cinnamomum cassia Presl,"minister" Paeonia lactiflora,Glycyrrhiza uralensis Fisch,Wolfiporia cocos,and "adjuvant"-"courier" Rhizoma Zingiberis,Polygala tenuifolia Willd,Acorus tatarinowii,Os Draconis and Ostreidae,a total of ten drugs.At present,the research of SZL oral liquid mainly focuses on clinical study,pharmacological study,rapid analysis of near-infrared spectroscopy,and fingerprint quality evaluation method.The existing quality standards cannot fully reflect the quality,and the research on quality marker(Q-marker)is not enough.Near-infrared(NIR)spectroscopy is one of the important process analysis techniques,but its band overlap is serious,and chemical composition analysis is a bottleneck problem.Therefore,this study proposed a rapid quality evaluation method of SZL oral liquid based on quality markers.Firstly,HPLCMS/MS technology was used to study the chemical composition of SZL oral liquid,and the pharmacological substance basis was combined with network pharmacological research to explain the mechanism of action of SZL oral liquid,and the drug targets of SZL oral liquid were summarized.Secondly,the fingerprint effect relationship of SZL was studied,and the components related to the inhibition effect of Acetylcholinesterase(AChE)/Butoxylcholinesterase(BChE)/β-secretase(BACE1)were explored,which provided the reference for the determination of Q-marker.The conversion method of NIR spectrum to HPLC fingerprint was established to identify and characterize the chemical composition and Q-marker-asscociated components of the drug "visually".A rapid quality evaluation method for SZL oral liquid was developed by using the conversion model of NIR spectrum and chromatographic fingerprint.This main research includes the following three parts:1.Network pharmacology of SZL oral liquid based on the properties of medicinal substances.HPLC-MS/MS was used to identify the chemical components characterized by fingerprint of SZL oral liquid,and the chemical substances of the drug was explained.The medicinal properties and chemical composition information of 120 common Chinese herbal medicines were integrated,and the association analysis method was used to explore the relationship between medicinal properties and chemical substances.Based on the association analysis results,the medicinal properties information of components contained in SZL oral liquid was speculated,and the pharmacodynamic components and action mechanism of SZL oral liquid in the treatment of Alzheimer’s disease were analyzed in combination with the network pharmacological research.The relevant targets were summarized.The results of association analysis showed that the drug properties of sugar and glycosides,organic acids and steroids were classified as peaceful and sweet,the drug properties of phenylpropyl components were classified as mild and pungent,the drug properties of alkaloids were classified as hot and pungent,the drug properties of terpenoids were classified as mild and bitter,the drug properties of quinones were classified as cold and bitter,and the drug properties of flavonoids and tannins were classified as cold and acid.In addition,the eight herbal medicines in SZL oral liquid mainly contain eight types of substances and exert their effects through the eight medicinal properties of cold,warm,hot,flat,sweet,bitter,pungent and acid.Based on the literature,the known chemical components contained in SZL oral liquid were summarized,and 64 possible active components were screened out according to the analysis of pharmacokinetic parameters.According to the association results between drug properties and substances,the drug properties of active components were deduced.Sixty-four active components of SZL oral liquid acted on 95 target proteins,and 25 main targets were screened out including BACE1,AChE,PTGS2 and BChE through eight medicinal properties except cool and salty.At the same time,AAP,TNF,GAPDH,CTNNB1 and other target proteins were important nodes of PPI network.GO enrichment analysis and KEGG pathway analysis were carried out from the perspective of the whole and different drug properties to explain the biological processes,cell components,molecular functions and signaling pathways related to SZL oral liquid in the treatment of Alzheimer’s disease.Through the network pharmacological study,the relevant targets of SZL oral liquid were summarized,providing reference for the subsequent study on the fingerprint effect relationship.2.Study on Q-marker-related components of SZL oral liquid inhibiting AChE/BChE/BACE1 based on fingerprint effect relationship.Based on network pharmacology,AChE/BChE/BACE 1 were selected as the key targets to explore the fingerprint effect relationship.The fingerprints of 15 batches of SZL oral liquid were determined,and the inhibitory effect of each batch of SZL oral liquid on AChE/BChE/BACE1 was determined by in vitro experiment.The statistical difference in pharmacodynamic activity between the unexpired and the expired samples was evaluated by t-test.Grey correlation degree analysis,orthogonal partial least square method,and correlation coefficient method were all used to analyze the co-relationship between the fingerprint of SZL and the enzyme inhibition of each target.The fingerprint effect relationship model was established.The common peaks explored by the three methods was taken as the key components of SZL oral liquid’s inhibition of each target.The results showed that the related components of peak 1,16(Codonopyrrolidium A)and 31(Licoricesaponin H2)were considered to be the main components of SZL oral liquid to inhibit AChE.The related components of peak 1,16,19(Tenuifoliside A)and 31 were the main components related to the inhibition of BChE;the peak 31-related component was the key component associated with the inhibition of BACE1.The comprehensive analysis showed that these ingredients related to pharmacodynamic activities conformed to the "five principles" of Q-marker through the study of fingerprint effect relationship.At the same time,it was verified that the changes in the peak areas of the key characteristic peaks screened out have a certain correlation with the pharmacodynamic activity.3.Rapid quality evaluation method of SZL oral liquid based on NIR spectrum to HPLC fingerprint conversionTo establish a rapid quality evaluation method of SZL oral liquid,we studied the spectrum-chromatogram conversion method,which could convert the NIR spectrum to the corresponding HPLC fingerprint,so that the fingerprinting peaks and the Q-marker characteristic peaks could be both reflected in the transformed fingerprints,which could be then used for the rapid quality evaluation of SZL oral liquid.The theoretical explanation of spectrum-chromatogram conversion method and related modeling algorithm were both studied.The spectrum-chromatogram conversion model was established by associating the NIR spectra of samples with the corresponding HPLC fingerprints,and the theoretical basis of the conversion method was deduced.Inspired by the spectral space transform(SST)method,a spectrum fingerprint transform(SFT)method was then proposed and compared with the improved principal component analysis(iPCA)method in our previous studies.The spectrum-chromatogram conversion studies of the simulation system,the mixed reference-compound system,and the actual sample system showed that the two methods could both be used to convert the NIR spectrum to the HPLC fingerprint,and the chemical composition information contained in the near-infrared spectrum could be "visualized" identification and analysis through the converted fingerprint.For the simulated data system,the retention time and peak area of chromatographic peak can be predicted 100%accurately by two conversion methods.The spectrum-chromatogram transform model specificity results showed that when the abnormal sample with a missing component,it could be well identified,and the abnormal sample with an added components,it was not able to be accurately predicted,but could be solved by model updating strategy.In the mixed reference-compound system,the chromatographic peak could be predicted more accurately,and the conversion effect was improved after the selection of spectral variables by iPLS method.The fingerprinting similarity before and after conversion was greater than 0.999,and each component content and its corresponding peak area could maintain a good linear relationship before and after conversion.For the actual sample system of SZL oral liquid,the common fingerprinting peaks and the key characteristic peaks corresponding to Q-markers could be predicted and displayed by the SFT and iPC A methods,and the conversion hardly changed the similarity evaluation results and original properties between samples.For the actual sample system,the normal batch conversion model could accurately identify the abnormal batch samples,which had a good warning and guiding function,and could quickly and accurately screen out the problem samples.