Analysis of an Aqueous Extract from Turkish Galls Based on Multicomponent Qualitative and Quantitative Analysis Combined with Network Pharmacology and Chemometric Analysis

The current quality control method for Turkish gall (TG) is limited to assessing total tannin or gallic acid (GA), which offers a basic level of quality control but does not fully capture the true quality of TG. Therefore, it is essential to establish a comprehensive method that utilizes multiple indicators to assess the intrinsic quality of TG. This research utilized UPLC-Q-TOF-MS/MS technology to qualitatively analyze the chemical composition of TG. Subsequently, the potential main active ingredients, targets, and pathways of TG in treating recurrent aphthous ulcers (RAU) were explored and analyzed using network pharmacology technology. Quantitative analysis of multicomponents by single marker (QAMS) was then employed to quantify the primary pharmacodynamic components in TG. Finally, chemometrics analysis was utilized to interpret the measured results and identify the markers of scavenging quality. The study identified 36 chemical components in TG, highlighting ellagic acid (EA), GA, and so on as key components in treating RAU. A method for simultaneously determining GA, EA, 1,2,3,6-tetra-O-galloyl-β-D-glucose (TEGG) and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PEGG) in TG was established. Statistical analysis revealed significant differences in the content of these 4 components across 14 batches of TG, with GA and PEGG identified as the primary contributors to the variations. This study determined a quality index for TG, providing a reference for quality evaluation and introducing a cost-effective and efficient quality control method. Furthermore, it addressed the challenge of developing new Chinese medicine by overcoming the lack of reference substances.


Introduction
TG is the dry gall on the young branches of Quercus infectoria Oliv., a Fagaceae plant.It is formed by the larvae of Cynips gallae tinctoriae Oliv.TG, also known as Moshizi or Wushizi, is a commonly used medicinal herb in Uygur medicine due to its high medicinal value [1][2][3].It is recorded in many ancient Chinese medicine books such as "Haiyao defciency, diarrhea, Yin sweating, spermatorrhea, cough, toothache, slow-healing sores, hair loss, anal fssure, diabetes, and damp heat or hematogenous diseases [4,5].Te analysis reveals that TG has a higher concentration of polyphenols, polysaccharides, favonoids, cellulose, tannins, and other substances.Te tannin content is particularly high, ranging from 50 to 70%, and is mainly composed of GA tannin, which is a complex mixture of phenolic acid and glycoside extracted through water decoction.Gallo-tannins are mixtures of polygallic acyl groups, with two main types: one with TEGG as the core and the other with PEGG as the core [6,7].Pharmacological studies have demonstrated that TG exhibits a range of pharmacological activities, including antioxidant, anti-infammatory, antitumor, antibacterial, antiviral, local anesthetic, insecticidal, hypoglycaemic, and wound healing properties [8,9].
Although many studies have been conducted on TG's chemical composition and pharmacological activity, its chemical components are still unknown.In addition, there is a lack of in-depth research on its active ingredient, mechanism of action, and target of action.Currently, the quality control methods employed by TG are limited to determining the total tannin or GA content, resulting in a low overall quality control level that fails to refect its intrinsic quality.Additionally, the quality evaluation mode based on qualitative and quantitative analysis of only 1 or 2 active or index ingredients is no longer sufcient for evaluating the quality of traditional Chinese medicine (TCM).Te evaluation of TCM quality now demands a multi-index comprehensive model, which is a current research trend.
Tis study conducted qualitative and quantitative analyses of the chemical composition of TG.Network pharmacology and chemometrics analysis were used to confrm the quality markers of TG.Te aim was to effectively control the internal quality of TG, expand its application feld, and improve its application value.Tis study employed UPLC-Q-TOF-MS/MS technology to qualitatively analyze the chemical components of TG.Te main material basis of TG's role was identifed and, in combination with network pharmacology technology, the possible main active ingredients, targets, and pathways of TG in the treatment of RAU were discussed and analyzed.QAMS was then used to quantitatively analyze the main pharmacodynamic components in TG.Tis method can help solve problems such as difculty in obtaining reference materials and high costs associated with the simultaneous use of multiple standard substances.It is characterized by its economy, convenience, accuracy, and environmental friendliness and has good prospects for development and application [10][11][12][13][14]. Finally, multivariate statistical methods, such as cluster analysis, principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), VIP value, and T-test, were combined to analyze the determination results.Te quality markers of TG were screened, and the intrinsic quality of TG was characterized by changes in quality marker content [15,16].Te aim is to control the intrinsic quality of TG efectively and provide theoretical support for the development of new TG drugs.

Instruments and Reagents.
Te Waters Xevo GZ-XS QTOF high-resolution mass spectrometer and Waters e2695 high-performance liquid chromatography were purchased from Waters Corporation, USA.Te Agilent 1260 high-performance liquid chromatograph was purchased from Agilent Corporation, USA.Te Shimadzu LC-20AT high-performance liquid chromatograph was purchased from Shimadzu Enterprise Management (China) Co., Ltd.ME204T and ME55 electronic balances were from Mettler Toledo Instrument (Shanghai) Co., Ltd.Te KH5200B ultrasonic cleaner was purchased from Kunshan Hechuang Ultrasonic Instrument Co., Ltd.Additionally, the XMTD-7000 electric constant temperature water bath pot was purchased from Beijing Yongguang Medical Instrument Co., Ltd.

Sample
Preparation.0.5 g TG medicinal powder was accurately weighed and placed in a 100 ml volumetric fask.An appropriate amount of purifed water was added, and the mixture was treated by ultrasonic for 30 min (100 W, 50 Hz).Te solution was then left to cool to room temperature, and Journal of Analytical Methods in Chemistry purifed water was added to the fask until it reached the scale.Te mixture was shaken well and passed through a 0.45 μm microporous flter membrane.2.5 ml of the continuous fltrate was accurately measured and placed in a 50 ml volumetric fask.Purifed water was added to the mark, and the mixture was shaken well to obtain the sample solution.Te keyword "current aphthous ulcer" was searched in the human genome database (GeneCards, https://www.genecards.org/),Online Mendelian Inheritance in Man (OMIM, https://omim.org/),and Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmaGKB, https:// www.pharmgkb.org/).Te targets from these sources were merged and treated as candidate targets for the disease after removing duplicates.

Protein-Protein Interaction (PPI) Network Analysis.
Te potential target for the treatment of RAU by TG was identifed by selecting the intersection of TG target and disease target.Te subsequent step involved the construction of an "intersection target fle" which was then imported into the STRING database platform (https://string-db.org/)."Multiple proteins" and "Homo sapiens" were selected for search.Te parameter was set to a minimum required score of 0.4, resulting in the acquisition of the PPI network diagram and network relationship.Te resulting "PPI network relationship fle" was then imported into Cytoscape 3.8.0.
For screening the core targets, the following centrality measures were selected: "Degree centrality (DC)," "Closeness centrality (CC)," "Betweenness centrality (BC)," "Eigenvector centrality (EC)," "Network centrality (NC)," and "Local average connectivity (LAC)."Te six targets with topological eigenvalues greater than the corresponding median were selected as the core targets for further analysis.Subsequently, a PPI topological analysis diagram was constructed to analyze the core targets in the treatment of RAU by TG.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes
and Genomes (KEGG) Analysis.Te David online analysis tool (https://DAVID.ncifcrf.gov/)was used to perform GO enrichment analysis of intersection targets, covering biological processes, molecular functions, and cellular components.Te bioinformatics data platform (https://www.bioinformatics.com.cn/) was used to draw the GO enrichment analysis results.Te David online analysis tool was used to conduct KEGG enrichment analysis for intersection targets, and the KEGG enrichment analysis results were plotted by bioinformatics data platform.According to the items obtained by the above enrichment analysis, the possible biological functions and involved biological pathways of the potential targets of TG were investigated, and then the main action pathways of TG in the treatment of RAU were obtained [19][20][21].

Construction of "Component-Target-Pathway"
Network.Te fles containing the "intersection target list," "component list," "pathway list," "component-target-pathway" relationship, and "component-target-pathway" attributes were imported into Cytoscape 3.8.0software.Te nodes representing components, targets, and pathways were analyzed visually to refect their degree value.A visual analysis of the protein network structure was conducted, resulting in the construction of the "component-targetpathway" network for TG in the treatment of RAU.Trough comprehensive analysis, the core components, targets, and pathways of TG in the treatment of RAU were identifed and examined in detail.

Preparation of Standards and
Samples.GA, EA, TEGG, and PEGG were accurately weighed and added to a volumetric fask.Te control solution was prepared by diluting it with methanol.Te mass concentrations were 0.139 mg/ml, 0.0384 mg/ml, 0.320 mg/ml, and 0.145 mg/ml, respectively.0.5 g TG medicinal powder was accurately weighed and placed in a 100 ml volumetric fask.An appropriate amount of purifed water was added, and the mixture was treated with ultrasound for 30 minutes (100 W, 50 Hz).Te solution was then left to cool to room temperature, and purifed water was added to the fask until it reached the scale.After shaking well, the solution was passed through a 0.45 μm microporous flter membrane to obtain the sample solution.
2.5.Data Analysis.Firstly, the content determination data were imported into SPSS 23.0 software, and the peak areas of the 4 components to be measured in 14 batches of samples were used as variables.Systematic cluster analysis was performed on the 14 batches of samples using the square Journal of Analytical Methods in Chemistry Euclidean distance method and the number connection method between the groups.After standardizing the peak areas, the PCA was performed on the peak areas of the 4 components to be measured in 14 batches of samples.Secondly, the peak area of 14 batches of samples was imported into SIMCA-P14 software, and OPLS-DA was performed.Te components that caused the diferences between the groups were analyzed using the projection of the model variable value as an index.Finally, GraphPad Prism9.5.1 software was used to conduct an independent sample T-test analysis with the content of common components as the variable.

Analysis of Chemical Components in TG.
UPLC-Q-TOF-MS/MS was utilized to perform negative ion scanning on the tested products under the conditions specifed in Section 2.2.1.Te acquisition of a total ion fow diagram under the negative ion mode is shown in Figure 1.To confrm the composition and structure of the compounds, the primary and secondary fragment ions of the compounds were compared with the information available in various databases, including UNIFI's own database, a selfbuilt database, online databases, and existing literature reports.Based on this analysis, a total of 36 chemical components were identifed [17,18,22].Te retention time, peak mass charge ratio of excimer ions, fragment ions, and other relevant data for each chemical component are presented in Table 1.Tannins, phenolic acids, and their esters accounted for the majority of the identifed components.Tese compounds were likely to be the key components responsible for the efcacy of TG.

Te Main Active Components, Targets, and Pathways of TG in the Treatment of RAU.
Te research focused on the chemical components identifed in TG.A total of 36 chemical components were identifed, of which dibutyl phthalate was excluded due to it is regarded to be a contaminated compound from experimental environment.Te remaining 35 chemical components were retrieved in the TCMSP database, resulting in the identifcation of 114 corresponding targets.After removing duplicates, 57 unique targets were obtained.For RAU, a total of 610 targets were retrieved from databases such as GeneCards, OMIM, and PharmGKB.After removing duplicates, 574 targets were identifed.By comparing the targets of TG and RAU, 8 common targets were found, namely, NOS3, GOT2, LCT, FASLG, RELA, VEGFA, MMP2, and MMP9, as shown in Figure 2. Tese targets were used to construct a PPI network consisting of 8 nodes and 10 edges.Te average degree of the nodes was 2.5, and the PPI enrichment P value was less than 0.000121.Tis network relationship was imported into Cytoscape 3.8.0software for topological analysis.Te fltering threshold obtained from the frst round of topology analysis was BC ≥ 0, CC ≥ 0.714285714, DC ≥ 3, EC ≥ 0.392358303, LAC ≥ 2, and NC ≥ 3. Consequently, six central nodes were obtained, namely, NOS3, FASLG, RELA, VEGFA, MMP2, and MMP9, as shown in Figure 3.
In order to further clarify the potential mechanism of TG in the treatment of RAU, frstly, GO enrichment analysis was performed on 8 intersecting core targets, which were annotated from three levels: biological process (BP), cellular component (CC), and molecular function (MF).Te results revealed that the core targets were mainly enriched in 548 BP, 8 CC, and 47 MF.Te top 10 analysis results were visualized, as shown in Figure 4(a).According to the GO analysis results in the fgure, BP enrichment results showed that the targets of TG in the treatment of RAU were mainly involved in the regulation of the response of cells to reactive oxygen species, the negative regulation of apoptosis signaling pathways, the regulation of responses to reactive oxygen species, and other processes.Te results of MF enrichment indicated that TG afects RAU through amino acid binding, metal endopeptidase activity, and serine endopeptidase activity.Te CC enrichment results suggested that TG targets the regulatory caveola, plasma membrane raft, membrane raft, membrane microregion, and membrane region in the treatment of RAU.Secondly, KEGG enrichment analysis was performed on 8 intersecting core targets resulting in 25 pathways.Te analysis results were visualized, as shown in Figure 4(b).Te top 10 important pathways were relaxin signaling pathway, fuid shear stress and atherosclerosis, AGE-RAGE signaling pathway in diabetes complications, bladder cancer, diabetic cardiomyopathy, proteoglycan, lipids and atherosclerosis in cancer, cancer pathway, PI3K-Akt signaling pathway, and HIF-1 signaling pathway.It can be seen that the enriched signaling pathways under the target genes corresponding to drug components were mainly concentrated in the aspects of diabetes and infammatory signaling pathways, etc. Tese signals were closely related to RAU and had a high degree of agreement with the treating diseases of TG.
After mapping the 8 intersection targets and the chemical components of TG, fve key components against RAU were fnally obtained.Tey were EA MOL001002, GA MOL000513, 3-methoxy-4-hydroxybenzoic acid MOL000114, aspartic acid MOL000065, and vitamin C MOL001691.Subsequently, the 5 key components, 8 intersection targets, and 25 signaling pathways were imported into Cytoscape 3.8.0software to construct the "component-target-pathway" network, as shown in Figure 5.

Quantitative Analysis of 4 Components in TG.
Te network pharmacology analysis revealed that TG contains fve key components with anti-RAU properties; they were EA, GA, 3-methoxy-4-hydroxybenzoic acid, aspartic acid, and vitamin C. Additionally, the UPLC-Q-TOF-MS/MS analysis indicated that TG is rich in tannins, specifcally two types: one with TEGG as the core and the other with PEGG as the core.Based on these fndings, the contents of GA, EA, TEGG, and PEGG in TG were determined and are presented in Figure 6.

4
Journal of Analytical Methods in Chemistry

Method Validation
(1) Linearity.Following Section 2.4.2, the mixed standard solution was accurately absorbed and diluted with methanol to prepare six standard solutions with diferent mass concentrations.Te samples were then injected and analyzed using the chromatographic conditions specifed in Section 2.4.1.Regression analysis was performed using the mass concentration of the standard solution as the X-coordinate and the peak area as the Y-coordinate.Te results of the regression analysis are presented in Table 2, indicating a satisfactory linear relationship among all components within their respective concentration ranges.
(2) Precision, Repeatability and Stability.Te mixed standard solution under Section 2.4.2 was precisely absorbed and injected for 6 times according to the chromatographic conditions under Section 2.4.1.Te relative standard deviation (RSD) of the peak area for GA, TEGG, EA, and PEGG were 0.93%, 1.70%, 0.86%, and 1.00%, respectively.It showed that the precision of the instrument was good.Te powder of TG was accurately weighed, and 6 sample solutions were prepared in parallel following the method described in Section 2.4.2.Te samples were then injected and analyzed using the chromatographic conditions specifed in Section 2.4.1.Te RSD of the peak area for GA, TEGG, EA, and PEGG was determined to be 2.89%, 1.86%, 2.91%, and 1.90%, respectively.Tese results indicated that the analysis method employed exhibited good repeatability.
Te powder of TG was accurately weighed, and the sample solution was prepared following the method described in Section 2.4.2.Te sample was then injected and analyzed at 0, 2, 4, 8, and 12 hours using the chromatographic conditions specifed in Section 2.4.1.Te RSD of the peak area for GA, TEGG, EA, and PEGG was determined to be 2.90%, 1.71%, 1.34%, and 1.25%, respectively.Tese results indicated that the sample solution exhibited good stability within the 12-hour time frame.
(3) Recovery.Te TG medicinal powder, weighing 0.5 g, was accurately measured.Each standard was added at 80%, 100%, and 120% of the known content of each ingredient, respectively.Te sample solution was prepared following the method described in Section 2.4.2, and three aliquots of each concentration level were prepared in parallel.Te sample was then injected and measured using the chromatographic conditions specifed in Section 2.4.1.Te recovery and RSD of the sample were calculated.Te average recoveries of GA, TEGG, EA, and PEGG were 101.53%, 100.76%, 98.01%, and 99.80% and those of RSD were 2.89%, 2.55%, 2.89%, and 2.88%, respectively, as shown in Table 3. Te results showed that the method was accurate.

Calculation of Relative Correction Factors (RCF).
Te common methods for calculating RCF include the multipoint correction method, slope correction method, and quantitative factor correction method [23][24][25].In this study, the linear standard solution in 3.3.1.1 was determined according to the chromatographic conditions in 2.4.1.Te slope correction method was used to calculate the RCF according to the calculation formula f a/k � F s /F k , where F k was the slope of the standard curve of the component to be measured and F s was the slope of the standard curve of the reference.GA was chosen as a reference due to its availability and cost-efectiveness.Te results indicated that the RCF of TEGG, EA, and PEGG was 1.2714, 0.2021, and 1.5321, respectively.

Durability Test.
Te mixed standard solution under Section 2.4.2 was taken and measured according to chromatographic conditions under Section 2.4.1.Te efects of various factors, including diferent HPLC chromatographs, chromatographic columns, fow rates, column temperatures, and detection wavelengths, on the RCF were investigated and are summarized in Table 4.It was evident that these factors did not have any signifcant impact on the RCF, indicating that the proposed method exhibited good robustness and durability.

Location of Chromatographic Peaks.
Using GA as the reference, the corresponding chromatographic peak was located by the relative retention time method (the ratio of the retention time of the component to be measured and the reference), and the determination by diferent HPLC chromatographs and chromatographic columns was investigated,  With GA as reference, the QAMS method was used to calculate the contents of TEGG, EA, and PEGG.Te results were compared with those measured by the external standard method (ESM) and evaluated by the relative error (RE), as shown in Table 5.It can be seen that the results obtained by the two methods are close (RE < 5.0%), indicating that the established QAMS method is highly accurate and can be used to determine the content of TG.

Te Result of Chemometrics Analysis. Te cluster analysis results
indicate that the 14 batches of TG can be divided into two groups, with the exception of S4, S12, and S14.S2, S5, S8, S9, and S13 were clustered into Group 1, while S1, S3, S6, S7, S10, and S11 were clustered into Group 2, and there was a better similarity between the batches clustered into one group, as shown in Figure 7(a).Te score plot (refer to Figure 7(b)) indicates that the TG was divided into two groups, with the exception of S4, S12, and S14, which is consistent with the cluster analysis.After standardizing the area of each common peak, the peak areas of the four components tested in the 14 batches of TG underwent PCA.Te related PCA eigenvalues and variance contributions were then obtained.Table 6 shows the results, indicating that the frst 2 factors contributed to 85.418% of the cumulative variance and had eigenroots >1, meaning that they represented most of the information of the common peak.Table 7 shows the principal component loading matrix, which indicates the degree of contribution of each variable to the principal components.Principal component 1 mainly refects variables 1 (GA) and 3 (EA), while principal component 2 mainly refects variable 4 (PEGG).To evaluate the overall quality of the diferent batches, the composite score can be calculated using the matrix of component score coefcients, as presented in Table 8.OPLS-DA was performed on the peak areas of 14 batches of TG.Te established model had R 2 X � 0.906, R 2 Y � 0.798, and Q 2 � 0.611 > 0.5, indicating its validity.Te score plots (refer to Figure 7(c)) showed that the samples could be well clustered into 2 classes, which agreed with the results of the cluster analysis and PCA.Te permutation test was conducted by randomizing the model 200 times (refer to Figure 7(d)).Te intercepts of the replacement test parameters R 2 and Q 2 were 0.263 and −0.457, respectively.Te original R 2 and Q 2 of the OPLS-DA model (located in the upper right of Figure 7(d)) were larger than those of the randomly arranged R 2 and Q 2 on the left side.Tis indicates that there was no overftting phenomenon in the established OPLS-DA model, and it could be used for the pattern recognition of the herbs of TG.Te VIP plot (refer to Figure 7(e)) refects the degree of contribution of each peak.
Peaks with VIP values greater than 1 were GA and PEGG, respectively.Tese components were the main signature components causing the diferences between the TG batches and were critical in distinguishing the samples and classifying them.Te content of shared components was used as variables for the independent samples T-test analyses performed with GraphPad Prism 9.5.1 software.Te results showed that there were no statistically signifcant diferences for GA (P � 0.195) and PEGG (P � 0.144) at the test level of

Discussion
Tis study identifed 36 chemical components in TG, including 20 tannins, 7 phenolic acids and their esters, 3 amino acids, 2 favonoids, and 4 other components.It can be seen that the main compounds in TG were phenolic acids and gallic tannins composed of 1∼7 galloyl and glucose as the center.Although no new compounds were discovered in TG during this experiment, the identifed compounds were more abundant in TG and these compounds may be the key components of TG to exert a curative efect.RAU is a common oral mucosal disease characterized by solitary, round, or oval ulcers that occur on the lip, tongue, cheek, and soft palate.It is often accompanied by spontaneous pain and is prone to recurring and self-limiting.Tis disease is the most common ulcer disease in oral mucosal diseases, with a prevalence rate of up to 20%, ranking frst in oral mucosal diseases.Modern medicine primarily utilizes chemical drugs for treatment, such as vitamins, immunomodulators, and local anti-infammatory, hormone, and analgesic drugs, but there is still a lack of efective radical treatment [26][27][28].Previous literature studies have shown 10 Journal of Analytical Methods in Chemistry that TG has a good therapeutic efect on RAU, but its mechanism of action remains unclear.In order to clarify the mechanism of TG in the treatment of RAU, network pharmacological analysis was conducted on the basis of UPLC-Q-TOF-MS/MS.Te network pharmacological results showed that EA, GA, 3-methoxy-4-hydroxybenzoic acid, aspartic acid, vitamin C, and other active ingredients may act on 8 targets closely related to RAU, such as NOS3, GOT2, and LCT.Te study focused on 25 pathways, such as the relaxin signaling pathway, fuid shear stress and atherosclerosis, and the AGE-RAGE signaling pathway, a complication of diabetes, which have been implicated in the treatment of RAU.Based on the above results, the contents of GA, EA, TEGG, and PEGG in TG were determined.Currently, the current standard of TG does not contain the content measurement items, so it is urgent to establish its content determination methods and improve its quality standards.Meanwhile, previous studies on the quality control of TG mainly focus on the content determination of total tannin and total polyphenols or the analysis of one or two index components.As a result, the overall quality control level is low, and it is difcult to refect its intrinsic quality.Terefore, it is imperative to establish a method of multi-index synchronous control of the intrinsic quality of TG.In this study, QAMS was used to determine the content of four components in TG.Tis method can realize the simultaneous determination of multiple components by determining one easy to obtain, cheap, and efective component, which can reduce the detection cost and solve the problem of insufcient reference substances, and may become a new model for the quality evaluation of TCM in the future.Te results of this part of the experiment showed that the method can be used to determine the content of 4 components in TG, and the results were not signifcantly diferent from the ESM.Finally, this study conducted a stoichiometric analysis of the experimental data of content determination and fnally determined that GA and PEGG are important bases for classifcation and evaluation of TG and have the potential to become its quality markers.

Conclusions
In this study, the main components of were characterized based on UPLC-Q-TOF-MS/MS combined with network pharmacology, QAMS, and chemometrics analysis.A total of 36 chemical components were identifed, including tannins, phenolic acids, and their esters.Te results of network pharmacological experiments showed that EA, GA, 3-methoxy-4-hydroxybenzoic acid, aspartic acid, and vitamin C were the key components of TG in the treatment of RAU.Combined with the results of UPLC-Q-TOF-MS/MS and network pharmacology, the QAMS method was established for the quantitative analysis of GA, TEGG, EA, and PEGG in TG.Te linearity, accuracy, precision, repeatability, and recovery of the method were all qualifed, and the results were compared with those obtained by ESM, and they were found to be similar.Terefore, this method can be used to determine the content of TG.Te study found signifcant diferences in the contents of 4 components across 14 batches of TG.Finally, it was determined that GA and PEGG were the main signature ingredients that caused the diference between batches of TG and were used as the quality markers of TG.Tis study can evaluate the overall quality of TG scientifcally and efectively.On the one hand, it can quickly identify the complex chemical composition of TCM and screen the key pharmacodynamic components; on the other hand, it can conduct multicomponent quantitative analysis by the QAMS method, saving the testing cost and time and avoiding the limitation of quality control by a single index.Terefore, it has a promising application prospect in the quality evaluation model of TCM.

Figure 2 :
Figure 2: Te Venn diagram of the intersection of component targets and disease targets.

Figure 4 :
Figure 4: GO functional analysis of key action targets (a) and KEGG pathway analysis of key action targets (b).

Figure 7 :
Figure 7: Te cluster analysis tree diagram of 14 batches of samples (a), the PCA scores of 14 samples (b), the OPLS-DA score diagram (c), the permutation plot of OPLS-DA analysis (d), the VIP results of 4 components (e), the heat map of 11 batches of samples (f ), and the content analysis results diagram (g and h).

Table 1 :
UPLC-Q-TOF-MS/MS analysis of the characteristic information of compounds in TG.
respectively.Te relative retention time (RRT) of TEGG, EA, and PEGG is shown in Table4.Te RSD of RRT of each component was less than 3.0%, indicating that each chromatographic peak could be located by the RRT method.

Table 2 :
Regression equation, linear range and correlation coefcient of each component.

Table 3 :
Te result of recovery.

Table 4 :
Te result of the durability test and chromatographic peak location.

Table 5 :
Te results of content determination of each component (w/w, %).

Table 6 :
Eigenvalues, variance contribution rates, and cumulative variance contribution rates.

Table 7 :
Principal component loading matrix.