Traceability Research on Geographic Erigeron breviscapus Based on High-Resolution Mass Spectrometry and Chemometric Analysis

A method was developed to identify and trace the geographic sources of Erigeron breviscapus using high-resolution mass spectrometry and chemometrics. The representative samples were collected from the geographic area of Honghe Dengzhanhua and other areas in Yunnan province and Guizhou province. The data points could be determined well using the PCA and PLS-DA diagram. A total of 46 characteristic compounds were identified from Honghe Dengzhanhua and within Guizhou province, but 37 compounds were different from Honghe Dengzhanhua and other counties in Yunnan province. Two biomarkers were found from three regions. Their structures were inferred as 8-amino-7-oxononanoic acid and 8-hydroxyquinoline, and they had the same molecular composition. This may suggest that a possible synthesis pathway can be proven in the future.


Introduction
Traditional medicines are cultivated in specific regions with a long history of plants.These traditional medicines in a characteristic region are called "orthodox" medicines.Erigeron breviscapus is an important traditional medicine.As one of the so-called "Yunyao" varieties, it has been listed as one of the "top ten Yunyao" and "five natural series" medicines in Yunnan province, China [1].It is known that the best-quality Erigeron breviscapus is planted in the Honghe area of Yunnan province, and due to its area of production, is named "Honghe Dengzhanhua" [2].Although this medicine is identified by its appearance, shape, and main chemical component (scutellarin) in the Chinese national standard, it is difficult to identify the geographic origin of Erigeron breviscapus in different plant regions.Since there are tens of major components, Li et al. [3] reported the structure of 64 volatile organic compounds from Erigeron breviscapus.However, no biomarker has been found to indicate its geographic origins, so it is urgent to develop an effective traceability technology to identify the geographic production of Honghe Dengzhanhua.
The technology used to determine geographic origin consists of stable-isotope mass spectrometry [4,5], mineral element analysis [6,7], and chromatographic fingerprinting [8][9][10].However, when an analyte consists of unknown compounds, it is difficult to qualitatively analyze.This problem can be solved by using high-resolution mass spectrometry with nontarget screening technologies [11,12].

Sample Preparation and Instrumental Method
Preparation of the Erigeron breviscapus sample: A sample of 1.0 g was precisely weighed and placed in a 50 mL centrifuge tube.After adding 25 mL of a 70% methanol solution, the sample was weighed again.After weighing, the samples were placed in an ice bath at 80 kHz and 25 • C and exposed to ultrasound for 40 min.After the sample was cooled to room temperature, the weight was replenished with a 70% methanol solution.After centrifugation at 5000 r/min for 5 min, the upper liquid was filtered by a 0.22 µm filter membrane, and a high-resolution mass analysis was performed.All of the Erigeron breviscapus samples were mixed equally to obtain QC samples.Then, the QC samples were prepared according to the preparation method used for the Erigeron breviscapus samples (n = 6).The preparation of blank samples only required placing 25 mL of 70% methanol in a 50 mL centrifuge tube, according to the method used for the Erigeron breviscapus samples.When the high-resolution injection was used again, the 6-pin blank sample was continuously injected first, and then the 20-pin QC sample was randomly injected and the breviscapine sample was randomly injected and detected.The injection rule is to add one QC sample to each 20-needle Erigeron breviscapus sample, and ensure that the last needle is a QC sample.

Sample Preparation and Instrumental Method
Preparation of the Erigeron breviscapus sample: A sample of 1.0 g was precisely weighed and placed in a 50 mL centrifuge tube.After adding 25 mL of a 70% methanol solution, the sample was weighed again.After weighing, the samples were placed in an ice bath at 80 kHz and 25 °C and exposed to ultrasound for 40 min.After the sample was cooled to room temperature, the weight was replenished with a 70% methanol solution.After centrifugation at 5000 r/min for 5 min, the upper liquid was filtered by a 0.22 µm filter membrane, and a high-resolution mass analysis was performed.All of the Erigeron breviscapus samples were mixed equally to obtain QC samples.Then, the QC samples were prepared according to the preparation method used for the Erigeron breviscapus samples (n = 6).The preparation of blank samples only required placing 25 mL of 70% methanol in a 50 mL centrifuge tube, according to the method used for the Erigeron breviscapus samples.When the high-resolution injection was used again, the 6-pin blank sample was continuously injected first, and then the 20-pin QC sample was randomly injected and the breviscapine sample was randomly injected and detected.The injection rule is to add one QC sample to each 20-needle Erigeron breviscapus sample, and ensure that the last needle is a QC sample.
The instrument was tuned in the positive and negative ESI mode of 3.8 kV of spray voltage, 60 V of SLens, 325 °C of capillary temperature, and 350 °C of probe heater temperature.Positive calibration solutions and the FS/DIA mode were used.In FS, the scan range was from m/z 80 to 1200; mass resolution was 140,000 FWHM; and the AGC target and maximum IT were set at 1.0 × 10 6 and 100 ms, respectively.For DIA, the relevant parameters were set as follows: mass resolution, 140,000 FWHM; AGC target, 2 × 10 5 ; maximum IT, 30 ms; Loop count, 12; MSX count, 1; Isolation window, 50 Da; stepped The sample was analyzed by Ultra-Performance Liquid Chromatography, using a Q Exactive High-Resolution Mass Spectrometry System (Thermo Fisher Scientific, Rockford, IL, USA) and an HSS T3 C18 column (2.1 × 100 mm, 1.8 µm, Waters, Milford, MA, USA).Solvent A was 0.1% formic acid, and solvent B was acetonitrile.The flow rate was set at 0.3 mL/min with the following gradients programmed: 98% A (0~1.8 min), 98% A~60% A (1.8~10 min), 60% A~5% A (10~12.0 min), 5% A~98% A (12.0~12.5 min), constantly 98% A (12.5~18.0min) and with 1 µL injection.
The instrument was tuned in the positive and negative ESI mode of 3.8 kV of spray voltage, 60 V of SLens, 325 • C of capillary temperature, and 350 • C of probe heater temperature.Positive calibration solutions and the FS/DIA mode were used.In FS, the scan range was from m/z 80 to 1200; mass resolution was 140,000 FWHM; and the AGC target and maximum IT were set at 1.0 × 10 6 and 100 ms, respectively.For DIA, the relevant parameters were set as follows: mass resolution, 140,000 FWHM; AGC target, 2 × 10 5 ; maximum IT, 30 ms; Loop count, 12; MSX count, 1; Isolation window, 50 Da; stepped normalized collision energy (NCE), 20%, 40%, and 60%.The spray voltage in positive and negative modes was set as 3.5 kV and 3.0 kV, respectively.The flow rate of sheath gas and aux gas was 45 and 10 mL/min (in arbitrary units), respectively.The software was used with Trace Finder 4.1 EFS and Compound Discoverer 3.3.

Data Processing and Statistical Analysis
A response peak area of no more than 400 was ignored, and the ion pairs with a high response intensity were retained.Unsupervised methods (PCA) constituted a first step in the data where samples were clustered with the same compounds.The next steps were to classify by orthogonal partial least squares (OPLS)-DA, and identify more robust samples.The last step was Variable Information Processing analysis (VIP), including a volcano plot (VP) and heat map, in which compounds with a p-value < 0.01 and VIP > 1 (top 50) were selected as biomarkers.
The experimental data were analyzed by Compound Discoverer 3.3 software, including peak extraction, peak alignment, background subtraction, compound identification, and multivariate statistical analysis.The peaks extracted by the software were compared with the molecular ion peaks of MzCloud, MzVault, MassLists, and ChemSpider databases in the software through the information of primary and secondary mass spectrometry, and the structural identification of unknown metabolites were realized.Finally, the metabolites with a relative standard deviation of QC sample peak area less than 30% were retained for deduplication.First, internal deduplication was performed according to the priority order in the database: MzVault > MzCloud > MassLists > ChemSpider.When the level was the same, the higher score was selected as the identification result.For the deduplication of positive and negative ion modes, the higher-level results were preferentially retained.If the results were the same, the mode with the largest expression was selected as the reference standard.

Stability and Repeatability of Instrument
The quality control samples (QC) were mixed with a total of 120 samples.The sensitivity and stability of the instruments were monitored by QC and corrected for the subsequent data analysis.Each biological replicate of the sample was clustered together and clearly separated from the quality control (QC) sample, indicating that the variation between the samples in the group was the smallest, thus ensuring the repeatability and reliability of the laboratory, as shown in Figure 2. In addition, after consulting the Pharmacopoeia of the People's Republic of China, the main marker compound in the Erigeron breviscapus was breviscapine.The RSD is 5.00~6.00%.Through the joint monitoring results for the breviscapine and QC samples, it can be seen that the performance of the instrument is very stable, the experimental repeatability is good, and the obtained data are acceptable and reliable.
classify by orthogonal partial least squares (OPLS)-DA, and identify more robust samples.The last step was Variable Information Processing analysis (VIP), including a volcano plot (VP) and heat map, in which compounds with a p-value < 0.01 and VIP > 1 (top 50) were selected as biomarkers.
The experimental data were analyzed by Compound Discoverer 3.3 software, including peak extraction, peak alignment, background subtraction, compound identification, and multivariate statistical analysis.The peaks extracted by the software were compared with the molecular ion peaks of MzCloud, MzVault, MassLists, and ChemSpider databases in the software through the information of primary and secondary mass spectrometry, and the structural identification of unknown metabolites were realized.Finally, the metabolites with a relative standard deviation of QC sample peak area less than 30% were retained for deduplication.First, internal deduplication was performed according to the priority order in the database: MzVault > MzCloud > MassLists > ChemSpider.When the level was the same, the higher score was selected as the identification result.For the deduplication of positive and negative ion modes, the higher-level results were preferentially retained.If the results were the same, the mode with the largest expression was selected as the reference standard.

Stability and Repeatability of Instrument
The quality control samples (QC) were mixed with a total of 120 samples.The sensitivity and stability of the instruments were monitored by QC and corrected for the subsequent data analysis.Each biological replicate of the sample was clustered together and clearly separated from the quality control (QC) sample, indicating that the variation between the samples in the group was the smallest, thus ensuring the repeatability and reliability of the laboratory, as shown in Figure 2. In addition, after consulting the Pharmacopoeia of the People's Republic of China, the main marker compound in the Erigeron breviscapus was breviscapine.The RSD is 5.00~6.00%.Through the joint monitoring results for the breviscapine and QC samples, it can be seen that the performance of the instrument is very stable, the experimental repeatability is good, and the obtained data are acceptable and reliable.

Unsupervised Classification by Principal Component Analysis (PCA)
In this analysis, the compounds detected in the sample and the chromatographic peaks corresponding to RSD ≤ 30% in the QC sample were considered to be a variable, and their integrated relative area was the response variable.The compounds selected in this way can be identified as key compounds in Erigeron breviscapus, which indicates that the subsequent statistical analysis of the data is credible.The obtained data set from the chemical components retained the maximum possible variability within the conditions.In the study, thirty samples from six areas divided by three classes were drawn in the PCA.

Unsupervised Classification by Principal Component Analysis (PCA)
In this analysis, the compounds detected in the sample and the chromatographic peaks corresponding to RSD ≤ 30% in the QC sample were considered to be a variable, and their integrated relative area was the response variable.The compounds selected in this way can be identified as key compounds in Erigeron breviscapus, which indicates that the subsequent statistical analysis of the data is credible.The obtained data set from the chemical components retained the maximum possible variability within the conditions.In the study, thirty samples from six areas divided by three classes were drawn in the PCA.The score plot of the PCA (Figure 2) showed that the clear difference in the comparisons was mainly reflected in the difference in the peak areas of the common components from the Honghe region and other areas.
From the scatter plot, the sample could be classified into four groups, including Honghe region, Kunming of Yunnan province, Dali of Yunnan province, and Xingyi of Guizhou province (Figure 2).It can be seen that the Honghe regions of Luxi and Mile can be separated from other areas, whether in Yunnan province or in Guizhou province, but the data were disparate from other areas of Yunnan province.The results show that the chemical composition relates to the plant environment and the climatic character creating biological diversity in Yunnan province.However, the clusters were not obviously distinct between Xingyi county in Guizhou province and Qujing county in Yunnan province.Because the two areas were neighboring, the environmental conditions were similar and associated with the same chemical properties/components.The two principal components, PC1 and PC2, accounted for 44.0% and 19.8% of the total difference, respectively, indicating that these clusters can effectively be distinguished from Honghe Dengzhan and other areas by the differences in the chemical components.

Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)
An appropriate model was established to analyze the collected data, and the OPLS-DA model was obtained.The OPLS-DA was performed to confirm the above four-classes data model.It can discriminate functions with linear combinations of the selected descriptors, and was proportional to the between-classes sum of squares and the within-classes sum of squares.The results were consistent with the PCA, which can be distinguished by the difference in the chemical components from Honghe Dengzhanhua and other areas.The prediction and discrimination ability of the model is greater than 99% (Figure 3).Because OPLS-DA is a supervised discriminant statistical analysis method, a permutation test is used to test the model (Figure 4).In order to avoid over-fitting in the process of model establishment, the intercept of R2 in the permutation test is generally not greater than 0.3~0.4.When the intercept between the Q2 regression line and Y axis (Q2 intercept) is < 0.05, it shows that there is no over-fitting phenomenon in the model.The prediction Because OPLS-DA is a supervised discriminant statistical analysis method, a permutation test is used to test the model (Figure 4).In order to avoid over-fitting in the process of model establishment, the intercept of R2 in the permutation test is generally not greater than 0.3~0.4.When the intercept between the Q2 regression line and Y axis (Q2 intercept) is <0.05, it shows that there is no over-fitting phenomenon in the model.The prediction abilities were 100% for all classes.An R2 of 0.99 is the correlation coefficient of cross validation, which indicates that the fitting degree was good.A Q2 of 0.985 represents the performance of the OPLS-DA model that can successfully predict the geographic origin in this experiment (Figure 4).Because OPLS-DA is a supervised discriminant statistical analysis method, a permutation test is used to test the model (Figure 4).In order to avoid over-fitting in the process of model establishment, the intercept of R2 in the permutation test is generally not greater than 0.3~0.4.When the intercept between the Q2 regression line and Y axis (Q2 intercept) is < 0.05, it shows that there is no over-fitting phenomenon in the model.The prediction abilities were 100% for all classes.An R2 of 0.99 is the correlation coefficient of cross validation, which indicates that the fitting degree was good.A Q2 of 0.985 represents the performance of the OPLS-DA model that can successfully predict the geographic origin in this experiment (Figure 4).A 3D score plot was generated to visualize the results from applying OPLS-LDA, as seen in Figure 5.The plot shows the distribution of the samples in the 3D scatter plot.Each point represents a particular class of sample, and is automatically color-coded according to its geographic origin.As can be observed, a complete separation of the four considered classes was achieved (Figure 5).A 3D score plot was generated to visualize the results from applying OPLS-LDA, as seen in Figure 5.The plot shows the distribution of the samples in the 3D scatter plot.Each point represents a particular class of sample, and is automatically color-coded according to its geographic origin.As can be observed, a complete separation of the four considered classes was achieved (Figure 5).

Variable Information Processing Analysis (VIP), Volcano Plot (VP), and Heat Map
In positive and negative ion toll scan modes, 4852 and 3178 peaks were detected, respectively.The collected mass data were searched using MarkerView 1.3 software (https://cloud.metware.cn).Differential marker compounds were analyzed by t-test, when p < 0.01 and the fold change (FC) was ≥2 or ≤0.5.The Variable Information Processing analysis (VIP), volcano plot (VP), and heat map were obtained in both positive ion mode and negative ion mode, and the ion pairs with a p-value < 0.01 and VIP > 1 were selected to draw plots.
A 3D score plot was generated to visualize the results from applying OPLS-LDA, as seen in Figure 5.The plot shows the distribution of the samples in the 3D scatter plot.Each point represents a particular class of sample, and is automatically color-coded according to its geographic origin.As can be observed, a complete separation of the four considered classes was achieved (Figure 5).

Variable Information Processing Analysis (VIP), Volcano Plot (VP), and Heat Map
In positive and negative ion toll scan modes, 4852 and 3178 peaks were detected, respectively.The collected mass data were searched using MarkerView 1.3 software The VIP scores, volcano map, and heat map (see Supplementary Materials, Figures S1-S3) showed the 46 compounds with large differences from Honghe Dengzhanhua and Guizhou provinces.As shown in Table 1, the content of 31 characteristic compounds was larger for Honghe Dengzhanhua than that of other areas, and the content of 15 characteristic compounds of Honghe Dengzhanhua was smaller than that of other areas.The 37 compounds that were differentiated from Honghe Dengzhanhua with other areas in Yunnan province are shown in Figures S1-S3, and the information for these compounds is provided in Table 2.There were 16 characteristic compounds, and their content in Honghe Dengzhanhua was larger than in other areas in Yunnan province, and the content of 21 characteristic compounds in Honghe Dengzhanhua was smaller than the content from other areas in Yunnan province.

Structural Identification of Characteristic Compounds
As an important herb in China, Erigeron breviscapus can treat cardiovascular and cerebral vessel diseases with the presence of a number of flavonoids and caffeic acid [55].Yang reported that caffeic acid was the main constituent in Dengzhan xixin injections [33].The characteristic components of these compounds may be different for Honghe Dengzhanhua, which mainly includes amino acid, caffeic acid, polyphenols, alkaloids, flavonoids, and their derivatives, as seen in Figure S4.

The Caffeic Acid of Compounds
Caffeic acid and its esters (CAEs) were widely distributed in the herbs as a class of biological activities, including anti-inflammatory, antiviral, anti-atherosclerotic, vasorelaxant, antioxidant, radical-scavenging, immunomodulatory, and hepatoprotective activities [55].Liao et al. reported [33] that di-O-caffeoylquinic acids had characteristic ions at m/z 353 in Erigeron breviscapus.Through a characteristic ion checker of m/z 353.0876, three peaks were found in Honghe Dengzhanhua (see Supplementary Materials Figure S4).The peak of 6. ).However, the reported di-O-caffeoylquinic acids [33] were found, and the content of three compounds were all lower in Honghe Dengzhanhua, which suggests that the three biomarkers were not the main biological activity in Honghe Dengzhanhua.

The Flavonoid of Compounds
Li reported [46] that the three flavonoids of scutellarin, scutellarein, and apigenin were the main compounds in Erigeron breviscapus and its extract injection.The results showed that only apigenin was found in Honghe Dengzhanhua, and the contents were higher, which suggests that the content of apigenin was high and bioactive in Honghe Dengzhanhua.Furthermore, a new polyphenol was found with the same molecular makeup of scutellarein, but it had a different retention time.The new polyphenols were identified at 7.38 min, which was earlier than for scutellarein at 8.91 min.The new polyphenols belonged to ellagic acid, which gave molecular ions at m/z 463.0868 [M + H] + and characteristic product ions at m/z 289.0341 ([M − arabinose-2CH 3 + H] + ) and 243.02905 ([quinic acid + H] + ), corresponding to the formula of C 21 H 18 O 12 and presumed as 2-hydroxy-3,8-dimethoxy-7-((3,4,5-trihydroxytetrahydro-2H-pyran-2yl)oxy)chromeno [5,4,3-cde]chromene-5,10-dione (Supplementary Materials Figure S4), which shows potent inhibition without cytotoxicity [44].
The other flavonoid was found at the peak of 8. 48  The relative content of two biomarkers from Honghe Dengzhanhua was higher than that from Guizhou province and other areas in Yunnan province, which may be mainly attributed to the geographic resources of Honghe Dengzhanhua.The peak of 6.In particular, 8-hydroxyquinolines have been widely used in therapeutics, demonstrating an array of medicinal applications (e.g., antibacterial, anti-cancer, anti-neurodegenerative, antiviral) [21].Except for 2-Acetyl-quinoline-8-ol, other quinolines were found, as presented in Tables 1 and 2 ).It corresponded to the formula of C 9 H 17 NO 3 and was inferred to be 8-amino-7-oxononanoic acid, as seen in Supplementary Materials Figure S4.Amino ketones are important structural moieties that are widely found in many pharmaceuticals and bioactive compounds.They are also valuable intermediates for the synthesis of complex compounds, including 2-amino alcohols and various N-heterocycles [18].The 8-amino-7-oxononanoic acid and 8-hydroxyquinoline had the same molecular composition, suggesting that a possible synthesis pathway may exist, as seen in Figure 6.It further proves the possible synthesis of the two biomarkers with the relationship to geographic resources and their bioactivity in the future.).It corresponded to the formula of C10H8O4, and was inferred to be Scopoletin [43].

The Simultaneous Biomarkers Distinguished in Honghe Dengzhanhua from Other Areas in Yunnan Province, Guizhou Province
The relative content of two biomarkers from Honghe Dengzhanhua was higher than that from Guizhou province and other areas in Yunnan province, which may be mainly attributed to the geographic resources of Honghe Dengzhanhua.The peak of 6.28 min gave molecular ions at m/z 188.07068 [M + H] + and characteristic product ions at m/z 146.0602 ([M − COCH3 + H] + ) and 118.0655 ([M − COCH3-CHOH + H] + ).It corresponded to the formula of C11H9NO2, and was presumed to be 2-Acetyl-quinoline-8-ol, as seen in Supplementary Materials Figure S4.This heterocyclic structure is an important bioactive molecule [58].In particular, 8-hydroxyquinolines have been widely used in therapeutics, demonstrating an array of medicinal applications (e.g., antibacterial, anti-cancer, anti-neurodegenerative, antiviral) [21].Except for 2-Acetyl-quinoline-8-ol, other quinolines were found, as presented in Tables 1 and 2. They were 8-Hydroxyquinoline and 2-Methyl-8quinolinamine, and all included 118.0655 ([M − COCH3-CHOH + H] + ), which means that these quinoline metabolites will be important bioactive compounds in Honghe Dengzhanhua.
The other peak of 3.00 min gave molecular ions at m/z 188.12839 [M + H] + and characteristic product ions at m/z 142.12280 ([M − COOH-H] + ) and 74.0608 ([M − COOH-C4H8-H] + ).It corresponded to the formula of C9H17NO3 and was inferred to be 8-amino-7-oxononanoic acid, as seen in Supplementary Materials Figure S4.Amino ketones are important structural moieties that are widely found in many pharmaceuticals and bioactive compounds.They are also valuable intermediates for the synthesis of complex compounds, including 2-amino alcohols and various N-heterocycles [18].The 8-amino-7-oxononanoic acid and 8-hydroxyquinoline had the same molecular composition, suggesting that a possible synthesis pathway may exist, as seen in Figure 6.It further proves the possible synthesis of the two biomarkers with the relationship to geographic resources and their bioactivity in the future.

Conclusions
Erigeron breviscapus is widely used as a traditional medicine material.Although the chemical and biological morphology is described by the Chinese national standard, it is difficult to distinguish the geographic origin with other areas in Yunnan province and Guizhou province.Thus, chemometrics should be developed to distinguish functions with linear combinations of the selected descriptors of the geographic regions.In this work, a total of 424 metabolites in whole quinoa grains were detected and annotated, which included 35 compounds in Honghe Dengzhan and Guizhou province, and 37

Conclusions
Erigeron breviscapus is widely used as a traditional medicine material.Although the chemical and biological morphology is described by the Chinese national standard, it is difficult to distinguish the geographic origin with other areas in Yunnan province and Guizhou province.Thus, chemometrics should be developed to distinguish functions with linear combinations of the selected descriptors of the geographic regions.In this work, a total of 424 metabolites in whole quinoa grains were detected and annotated, which included 35 compounds in Honghe Dengzhan and Guizhou province, and 37 compounds in Honghe Dengzhan and other areas in Yunnan province.The two candidate biomarker constituents were determined by grouping discriminant compounds, which suggests that the possible synthesis of the two candidate biomarkers can be proven in the future.However, the differences in the metabolites produced in different geographic regions

Figure 1 .
Figure 1.The collected samples from the geographic region of Luxi and Mile, Honghe, and other areas of Yunnan province (Dali, Fuming, Qujing), Xingyi, and Guizhou province.

17 Figure 3 .
Figure 3. OPLS-DA score chart and model diagram of Honghe and the other four areas.

Figure 3 .
Figure 3. OPLS-DA score chart and model diagram of Honghe and the other four areas.

Figure 3 .
Figure 3. OPLS-DA score chart and model diagram of Honghe and the other four areas.

Figure 4 .
Figure 4. OPLS-DA cross validation details of five areas: (a) positive ion mass and (b) negative ion mass.

Figure 4 .
Figure 4. OPLS-DA cross validation details of five areas: (a) positive ion mass and (b) negative ion mass.

Figure 5 .
Figure 5.The PLS-DA 3D score plot for the Honghe geographic region and other areas in Yunnan province and Guizhou province.

Figure 5 .
Figure 5.The PLS-DA 3D score plot for the Honghe geographic region and other areas in Yunnan province and Guizhou province.

Figure 6 .
Figure 6.The possible synthesis of the two biomarkers in Honghe Dengzhanhua.

Figure 6 .
Figure 6.The possible synthesis of the two biomarkers in Honghe Dengzhanhua.

Table 1 .
Difference biomarker in Honghe Dengzhan and Guizhou province analyzed by High resolution mass spectrum.

Table 2 .
Difference biomarker in Honghe Dengzhan and other area in Yunnan province analyzed by High resolution mass spectrum.