| Scutellariae Radix,the dried roots of Scutellaria baicalensis Georgi,is the most commonly used heat-clearing medicine in clinic.The 2015 edition of the "Pharmacopoeia of the People’s Republic of China",contains two processed products,including Scutellariae Radix and wine-processed Scutellariae Radix.Scutellariae Radix has strong detoxification power for clearing heat and purging fire due to the natures of bitter and cold.After wine-processed,the natures are weaken and Scutellariae Radix can be used for ascending lung heat and damp heat in the limbs.Given the natures of Scutellariae Radix will be changed after wine-processing,it is important to clarify the effects of processing on the natures,which is the premise of clinical application and modernization of traditional Chinese medicines(TCMs).However,according to the historical pharmacopoeia and the processing standards in several provinces and cities,the processing technology of Scutellariae Radix described usually referred to traditional experiences and lacked specific parameters,and the descriptions of the performance characteristics were different.Flavonoids,as the main active constituents of Scutellariae Radix,will be effected under high temperature.Furthermore,the performances in vivo will be different with the active components changed.In this paper,modern analytical instruments and multivariate statistical methods were combined to analyze the effects of wine-processing on Scutellariae Radix,providing references for its rational clinical applications.Research objectiveThis paper aims to optimize the processing parameters of Scutellariae Radix and discuss the effects of processing process on it in terms of colors,tastes,chemical profiles and metabolisms in vivo.Furthermore,the correlation between each index and the natures of TCMs will be analyzed in order to clarify how wine-processing changed TCMs natures and provide powerful evidence for the clinical applications of Scutellariae Radix.Research methods1.Taking surface characters,contents and alcohol-soluble extract as evaluation indexes,single-factor inspection combined with comprehensive scoring method were used to optimize the cutting process of Scutellariae Radix.Subsequently,orthogonal experiment design was carried out based on four factors,including the volume of rice wine,stuffiness time,roasting temperature and time,and the comprehensive scoring methods were used to optimize the process of wine-processed Scutellariae Radix.2.The colors and tastes of Scutellariae Radix and wine-processed Scutellariae Radix were quantified using colorimeter and electronic tongue technology respectively,and the differences were analyzed after multivariate statistical analysis,such as PCA and OPLS-DA.3.The characteristic maps of Scutellariae Radix and wine-processed Scutellariae Radix were established by HPLC and processed by the Chinese medicine chromatographic fingerprint similarity evaluation system(version 2012)to evaluate the overall quality and the differences between Scutellariae Radix and wine-processed Scutellariae Radix.Moreover,multi-components were qualitatively and quantitatively analyzed in Scutellariae Radix and wine-processed Scutellariae Radix using UPLC-QTOF-MS and UPLC-TQMS.The" volatile components of Scutellariae Radix and wine-processed Scutellariae Radix were compared using HS-SPME-GC-TQMS.4.A total of 30 mice were divided averagely into 3 groups,including Scutellariae Radix,wine-processed Scutellariae Radix and vehicle-treated groups.The metabolites in 3 groups were analyzed using 1H-NMR combined with multivariate statistical analysis to find the biomarkers and the differences.5.To clarify the changes of Scutellariae Radix caused by wine-processing,simple correlation analysis,canonical correlation analysis and PLS correlation model were used to analyze the potential factors of Scutellariae Radix pieces in terms of colors,tastes,constituents and metabolites.Research results1.Using single factor inspection and comprehensive scoring method to compare 3 softening methods of boiling,normal pressure steaming and pressure steaming,and the cutting process of Scutellariae Radix was finally determined as:the pure Scutellariae Radix was pressurized at 0.1 MPa for 10 minutes,then removed to cut into 1.5 mm slices,and blowing dried at 60℃ for 3 h.Orthogonal test and comprehensive scoring method were used to determine the preparation of wine-processed Scutellariae Radix:After mixed with rice wine,the Scutellariae Radix pieces were moisten for 15 minutes,and stir-fried at 140± 10℃ for 5 minutes.Method validation results indicated that the determined processing method had good repeatability and stability.2.Colorimeter test results showed that after wine-processed,pieces became darker,brownish red increased,and yellowish green weakened,but overall color changed mildly.Results of PCA and OPLS-DA showed that the colors of Scutellariae Radix before and after wine-processed were different,red and yellow were the diagnostic colors.Electronic tongue test results showed that bitterness of Scutellariae Radix was lower than that of wine-processed Scutellariae Radix,whereas salty was increased.Furthermore,salty and bitter tastes were the diagnostic tastes.3.The characteristic maps of Scutellariae Radix and wine-processed Scutellariae Radix were established,and contents of 5 flavonoids were quantified,14 common peaks were calibrated,similarity range of 10 batches of Scutellariae Radix was between 0.875～1,and the similarity range of wine-processed of Scutellariae Radix pieces was between 0.986～1,indicating that the overall quality of wine-processed Scutellariae Radix was more stable.After PCA analysis,there were no significant differences occurred between 2 pieces with the common peak areas as variables,which indicated that the overall quality of the Scutellariae Radix before and after wine-processing was similar.The qualitative analysis of Scutellariae Radix and wine-processed Scutellariae Radix were conducted by UPLC-QTOF-MS,and 44 components were identified.The results showed that Scutellariae Radix had not caused flavonoids increased or disappeared before and after wine-processing.UPLC-TQMS was used to quantitatively compare contents of 11 flavonoids before and after wine-processing.The quantitative results showed that contents of flavonoid glycosides such as Baicalin,Wogonoside decreased slightly,while the contents of flavonoid aglycones such as Baicalein and Wogonin increased slightly.PCA analysis results showed that 2 decoction pieces were different in content profiles.The contents of Baicalin,Wogonoside,Oroxyloside were the major factors that caused differences in the components before and after wine-processing.The volatile components in 2 pieces were qualitatively and semi-quantitatively compared using HS-SPME-GC-TQMS.A total of 33 chemical components were identified in Scutellariae Radix.A total of 32 chemical components were identified in wine-processed Scutellariae Radix.There were 17 common components in 2 pieces.Relative quantitative results showed that the intensity of the main chromatographic peaks of Scutellariae Radix tended to decreased,which may be related to the wine-processed4.With the increase of the administration time,the growth rate of mice’s body weight became slower.The weight growth rate of Scutellariae Radix group was slower than that of wine-processed Scutellariae Radix group.At the same time,both 2 decoction pieces could reduce the anus temperature of the mice,and anal temperature in Scutellariae Radix group decreased more.Metabolites in the serum and liver were analyzed by 1H-NMR.As a result,32 compounds were identified in serum and 44 compounds were identified in liver.After multivariate statistical analysis,11 and 19 biomarkers were identified in serum and liver respectively.Moreover,the results of metabolic pathways showed that the effects of Scutellariae Radix and wine-processed Scutellariae Radix in mice were mainly related to biosynthesis of Phenylalanine,Tyrosine,and Tryptophan,metabolism of D-glutamine and D-glutamic acid,and energy metabolism.Phenylalanine metabolism,Alanine,Aspartic acid,and Glutamic acid metabolism,Tyrosine metabolism,Citric acid cycle(Tricarboxylic acid cycle),Pyruvate metabolism,Arginine biosynthesis.Amino acid metabolism,Carbohydrate metabolism,Lipid metabolism,Ketone body decomposition and synthesis affect metabolism in mice.Among them,the contents of Glucose and Choline in mice of wine-processed Scutellariae.Radix group were higher than that of Scutellariae Radix group.The ability of energy metabolism was enhanced,the energy consumption was increased,and the coldness of Scutellariae Radix was reduced after wine-processed,which directly reflected the way and mechanisms of change of natures of Scutellariae Radix caused by wine-processing5.The results of simple correlation analysis indicated that a*value(red)had a significant negative correlation with Baicalin and a significant positive correlation with Baicalein However,the rules in b*value(yellow)were oppositely different.The CTS sensor(salty)has no correlation with Baicalin,Wogonoside,Baicalein,Oroxyloside,whereas the SCS sensor(bitter)had a significant positive correlation with Baicalin.The results of classical correlation analysis indicated that the Baicalin,Wogonoside,Oroxyloside in serum were positively correlated with Lipid,3-hydroxybutyric acid,Lactic acid,Acetic acid,Citric acid,Choline,Glycerophosphocholine and Inositol.It was negatively correlated with α-glucose and β-glucose,while Baicalein had the opposite rule.Baicalin,Wogonoside,Oroxyloside and Leucine,Isoleucine,Valine,Alanine,Glutamic acid,Choline,Hepatic glycogen,Fumaric acid,Tyrosine Phenylalanine and Adenosine were positively correlated,and were negatively correlated with Lactic acid,Acetic acid,Glutathione,Succinic acid,Glycerophosphocholine,Betaine,Taurine and Xanthine,whereas the patterns in Baicalein were opposite.Research conclusionWith the introduction of rice wine and high temperature,the surface characters and material basis of Scutellariae Radix changed,which eventually led to changes in the natures of Scutellariae Radix.Herein,the process technologies of cutting and softening of Scutellariae Radix were determined,and the chemical profiles of Scutellariae Radix were qualitatively and quantitatively analyzed.Moreover,the scientific connotations of the changes of the nature of Scutellariae Radix caused by wine-processing were preliminarily clarified. |
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