| Processing is an important part in the field of traditional Chinese medicine(TCM).It is one of the characteristics of TCM and is a vital part to ensure the quality of Chinese herbal medicine,which can improve the clinical efficacy and ensure the safety of medication.Therefore,the study of processing mechanism has been particularly important in the modernization of TCM.Guilingji,which started in Ming Dynasty,is a famous TCM prescription that composed of 28 kinds of Chinese herbal medicines and has a significant effect.From ancient times to the present,each herb in Guilingji has been processed by ancient processing method which has been listed in the national intangible cultural heritage list as early as 2009.However,there is no relevant research on mechanism of the ancient processing in this prescription,and the quality of each single TCM using ancient processing has not been reported yet.In addition,the material basis and metabolism rule of single drug before and after the ancient processing have not been fully clarified.It may limit the further research and industrialization development of ancient processing method in the quality and clinical efficacy of Chinese herbal medicine.Therefore,it is of great significance to apply modern research techniques and new methods to the study of the processing mechanism of Chinese medicine and the quality control of Chinese medicinal materials before and after processing.The present study takes two representative medicines named Aconiti Lateralis Radix Praeparata(Fuzi in Chinese)and Cistanche tubulosa(C.tubulosa,RCR in Chinese),which processed by the unique processing technology,as the research object,using ultrahigh performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer(UPLC-Q-TOF/MS)analysis platform,combined with a public data platform,related analysis software and metabolomics,based on the influence of ancient processing on the material basis of Chinese medicine,the chemical components of Fuzi and RCR were analyzed comprehensively and systematically.Finally,the whole and the different components before and after processing were found out,the quantitative study of the representative active components were also conducted.We also carry out the characterization of migrating components in the plasma of Fuzi and RCR before and after processing to rats.Combined with metabonomics,this paper reveals the influence of Fuzi and RCR before and after processing and its metabolites in vivo so as to lay a foundation for clarifying the processing mechanism.The pharmacodynamic material basis and metabonomics of Fuzi and RCR before and after processing were clarified,which provided scientific data support for the influence of ancient processing and the follow-up research on these two traditional Chinese medicines.The specific research contents are as follows:1.Study on chemical substances and chemical markers of Fuzi and RCR before and after processing.UPLC-Q-TOF/MS method was used for data collection,and MS/MS-based molecular networking(MN)was used to analyze the chemical components of Fuzi and RCR.A combination of MN and Progenesis QI software were also conducted to analyze the different chemical markers in the same batch before and after processing of Fuzi and RCR.First,by consulting the relevant literature and retrieving the relevant database,333 chemical composition database of Fuzi and 264 of RCR were established,and then the fast-DDA data of raw and processed products collected by mass spectrometry were uploaded to GNPS platform.With the comparison of retention time,fragmentation rule,reference substance and the relevant literature,145 chemical components of Fuzi in the positive ion mode and 85 chemical components of RCR in the negative ion mode were finally identified.Due to the raw and processed products are processed in the same way,the relative area of each node in molecular network is the change of the compound compared with raw products.We can intuitively find in the molecular network that the content of toxic components of Fuzi obviously decreases or even disappears,and some active ingredients of RCR increase after processing.In order to verify the accuracy of the MN method in searching for differential markers,we also collected MSE mass spectrometry data,and used Progenesis QI and Ezinfo software to perform multivariate statistical analysis methods to find the differential components,and the results showed that the two analysis results were consistent.In the end,77 differential components of Fuzi and 34 differential components of RCR were found between raw and processed products through these two methods.2.Quantitative study on characteristic components of Fuzi and RCR before and after processing.On the basis of the above research,12 representative chemical components of Fuzi and 5 of RCR were selected,and the multiple reaction monitoring(MRM)based on positive(Fuzi)and negative(RCR)scanning model was carried out by using high performance liquid chromatography tandem triple quadrupole mass spectrometry(HPLC-QQQ-MS)technology for gradient elution and quantitative analysis of selected target components.The method established in this study had high specificity and sensitivity.The results showed that the content of toxic components in Fuzi decreased,while the content of active components in RCR increased or unchanged.The quantitative analysis of characteristic components before and after processing of Fuzi and RCR provides data support for the study of quality control and processing mechanism of medicinal materials.3.Metabolite profiling of Fuzi and RCR before and after processing.On the basis of qualitative and quantitative analysis of chemical components of Fuzi and RCR before and after processing,this study used UPLC-Q-TOF/MSE combined with MetasenseTM prediction system and UNIFITM data processing platform to qualitatively analyze the prototype components of Fuzi and the prototype components and metabolites of RCR in rats before and after processing.There are 31 prototypes in plasma,65 prototypes in urine and 67 prototypes in feces in Fuzi,13 prototypes in plasma,23 prototypes in urine and 51 prototypes in feces in RCR.In addition,the metabolites of RCR before and after processing were identified,including 12 in plasma,31 in urine and 37 in feces.The main metabolic types of RCR are deglycosylation,sulfonation products,methylation,acetylation,glucuronization,hydroxylation,hydrolysis,oxidation and reduction.4.Metabonomics study of Fuzi and RCR before and after processingThe ethanol extracts of raw and processed Fuzi were administered to rats.Combined with the results of cardiac tissue section and biochemical indicators,the raw product group has obvious toxic effects on the heart tissue,while the processed product group has significantly reduced cardiotoxicity.Then based on UPLC-Q-TOF/MS technology,the urine samples of raw Fuzi group,processed Fuzi group and control group were studied by metabonomics.The results showed that there was an obvious classification trend between the urine samples of the three groups compared with the control group on the multivariate statistical score chart of the orthogonal partial least squares discriminant analysis(OPLS-DA).In the end,108 different metabolites of the urine samples related to the processing of Fuzi were screened.Metabo Analyst software was used to further analyze the metabolic pathways involved in these differential metabolites.The result showed that six metabolic pathways were disordered,while the processed products group attenuated the toxicity mainly by reversing these metabolic pathways.It is proved that the processed Fuzi has the function of reducing toxicity,which is conducive to the wide circulation of Fuzi in the pharmaceutical market and the safe application in clinic.RCR has always been a valuable traditional Chinese medicine for tonifying kidney yang.In this study,highdose hydrocortisone was injected into rats to simulate kidney yang deficiency model and raw and processed products of RCR were given.It was found that RCR could improve the pathological behavior of rats with kidney yang deficiency combined with rats’ body weight,behavior and biochemical indexes.Then based on UPLC-QTOF/MS technology,metabonomics of urine sample were studied.Combined with the univariate statistical analysis method,47 urine differential metabolites related to kidney yang deficiency were screened out,these differential metabolites could be recalled after RCR administration,and the effect of processed products is better than that of raw products. |
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