Method for identifying genuineness of radix codonopsis based on electronic tongue and GC-MS technology

Abstract

The application discloses a method for identifying the genuineness of radix codonopsis based on an electronic tongue and GC-MS technology, and discloses the following technical scheme: the electronic tongue technology is used to evaluate the tastes of radix codonopsis (Lu Dang, Bai Tiao Dang, Tai Dang, Wen Dang, Dao Dang and Tiao Dang) solutions of different commodities, principal component analysis and partial least square analysis methods are used to analyze the electronic tongue data of the radix codonopsis of different commodities, and it is found that the radix codonopsis of different commodities can be well distinguished through the taste identification of the electronic tongue. Through the primary metabolomics difference research of Lu Dang and other commodity radix codonopsis by GC-MS, 35 metabolites are identified and recognized, and PCA analysis and orthogonal partial least square analysis are conducted on the results, and 17 differential metabolites in the radix codonopsis are found. The Lu Dang and Bai Tiao Dang and Tai Dang of the same origin are analyzed, and 5 differential metabolites are found. The application can provide an efficient and objective analysis method for identifying the genuineness of radix codonopsis medicinal materials.

Description

Technical Field

[0001] This invention relates to a method for identifying Chinese medicinal materials, specifically a method for identifying the authenticity of Codonopsis pilosula based on electronic tongue and GC-MS technology. Background Technology

[0002] The 2020 edition of the Chinese Pharmacopoeia lists *Codonopsis pilosula* (Franch.) Nannf., *Codonopsis pilosula* Nannf. var. *modesta* (Nannf.) L.Shen, or *Codonopsis tangshen* Oliv. as medicinal materials. It possesses spleen-strengthening, lung-nourishing, blood-nourishing, and fluid-generating effects, and can be used to enhance immunity, fight tumors, and regulate blood lipids. Based on different origins and sources, common commercial *Codonopsis* varieties include "Wen Dang" from Wenxian County, Gansu; "Banqiao Dang" from Enshi, Hubei; "Lu Dang Shen" and "Tai Dang" from Shanxi; and "Bai Tiao Dang" and "Wen Dang" from Gansu. Among these, Lu Dang Shen (Franch.) Nannf. is a traditional medicinal material from Shanxi.

[0003] Authentic medicinal herbs are synonymous with high-quality medicinal materials characterized by their commercial value. They encompass geographical, quality, economic, and cultural concepts, arising from suitable environments, superior germplasm, or mature production techniques and processing methods within specific production areas. These herbs consistently and reliably influence market demand and are clinically or scientifically validated. Historically, the formation of authentic medicinal herb production areas has largely followed the principle of "survival of the fittest, selection of the best, and the formation of authentic herbs naturally." Therefore, the quality of authentic medicinal herbs is proven through practice. As an authentic medicinal herb from Shanxi, Lu Codonopsis pilosula is primarily cultivated, with its cultivation area expanding annually, resulting in good quality. However, the quality of Lu Codonopsis pilosula in the market currently varies, with some inferior products being sold as superior ones, leading to significant price differences. Traditional identification of Codonopsis pilosula focuses on "shape, color, aroma, and taste," reflecting its overall characteristics through physical features. This is an important means of identifying the commercial value and quality of Codonopsis pilosula, but it requires sufficient experience and is subject to subjective factors. The quality evaluation of modern Chinese medicine mainly includes four aspects: appearance evaluation, chemical evaluation, efficacy evaluation, and biological evaluation. However, this process is characterized by lengthy testing times, complex procedures, and the need for large amounts of chemical reagents. Therefore, it is necessary to establish a rapid and concise method for the identification and quality analysis of Codonopsis pilosula.

[0004] Electronic tongue, also known as taste fingerprint analyzer, is a detection technology developed in the 1980s for analyzing liquid samples. It simulates human taste and converts the "taste" of the sample into response values ​​of various sensors, thereby avoiding the uncertainty of results caused by subjective differences of tasters.

[0005] The quality of Codonopsis pilosula varies from place to place, and it is urgent to find a way to quickly and accurately identify authentic Codonopsis pilosula from Luzhou. Summary of the Invention

[0006] Objective of this invention: This invention aims to study the "taste fingerprint" information of different Codonopsis pilosula products using electronic tongue technology, and to extract sample feature values ​​through pattern recognition algorithms to characterize the differences in complex tastes, thereby establishing a method for rapidly identifying the authentic origin of Codonopsis pilosula from Luzhou. Furthermore, GC-MS is used to detect the differentially characteristic metabolites in different Codonopsis pilosula products, thus achieving the goal of identifying the authentic origin of Luzhou Codonopsis pilosula.

[0007] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0008] A method for identifying the geographical origin of Codonopsis pilosula based on electronic tongue and GC-MS technology includes the following steps:

[0009] (1) Preparation of electronic tongue sample solution

[0010] Collect Codonopsis pilosula from different products, crush them appropriately, sieve them, weigh them accurately, add ultrapure water, heat under reflux, filter while hot, take the filtrate, repeat the extraction several times, combine the filtrates, centrifuge, take the supernatant and make up to volume to obtain the sample solution.

[0011] (2) Take the electronic tongue sample solution from step (1) and use electronic tongue technology to determine the taste of different Codonopsis pilosula.

[0012] (3) The electronic tongue technology in step (2) will intuitively display the taste of Codonopsis pilosula of different products through the response value of the sensor, and concretize it with numerical values. Then, the electronic tongue data of Codonopsis pilosula of different products will be analyzed by principal component analysis (PCA) and partial least squares analysis (PLS-DA) to identify the taste of Codonopsis pilosula of different products.

[0013] (4) Preparation of GC-MS sample solutions

[0014] Take different types of Codonopsis pilosula, grind them appropriately, sieve them, weigh them accurately, add methanol solution containing formic acid, extract them by sonication, then centrifuge them, take the supernatant, add salicylic acid internal standard solution, blow dry with nitrogen, add methoxyamine pyridine solution, place in a metal bath, add MSTFA, place in a metal bath, centrifuge, and take the supernatant for later use.

[0015] (5) Take the GC-MS sample solution from step (4) and inject it into the GC-MS instrument to analyze the primary metabolites of Codonopsis pilosula from different products;

[0016] (6) The primary metabolite data of different Codonopsis pilosula products were analyzed by chemometric analysis using PCA, partial least squares analysis and orthogonal partial least squares analysis to screen out the differential metabolites of each Codonopsis pilosula as characteristic metabolites for identifying the authenticity of Codonopsis pilosula.

[0017] As a preferred option, the above-described method for identifying the authenticity of Codonopsis pilosula based on electronic tongue and GC-MS technology, step (1) preparation method of electronic tongue sample solution is as follows: collect Codonopsis pilosula from different products, crush appropriately, sieve, accurately weigh 25.00g, add 250mL of ultrapure water, heat under reflux for 1h, filter while hot, take the filtrate, repeat twice, combine the filtrates, centrifuge at 5000r / min for 10min, take the supernatant and dilute to 500mL volumetric flask to obtain the sample solution, each sample is in triplicate.

[0018] As a preferred option, the above-described method for identifying the authenticity of Codonopsis pilosula based on electronic tongue and GC-MS technology, the method for measuring the taste of different Codonopsis pilosula using electronic tongue technology in step (2) is as follows: using TS-5000Z electronic tongue measuring equipment, the data acquisition time for each Codonopsis pilosula sample is 120s, the samples are washed with ultrapure water for 10s, the samples are washed with a cup of ultrapure water between samples, each sample is repeated 6 times, the last 3 times are taken as the detection result, and the response value of the sensor is recorded.

[0019] As a preferred embodiment, in the method for identifying the authenticity of Codonopsis pilosula based on electronic tongue and GC-MS technology described above, step (4) involves the preparation of the GC-MS sample solution.

[0020] Take different types of Codonopsis pilosula, grind them appropriately, sieve them, weigh them accurately, add 1 mL of 70% methanol solution containing 2% formic acid, sonicate at 360W and 40kHz for 15 min, centrifuge at 13,000 r / min for 10 min, take 40 μL of the supernatant, add 20 μL of 1 mg / mL salicylic acid internal standard solution, blow dry with nitrogen, add 20 μL of 40 mg / mL methoxyamine pyridine solution, incubate in a metal bath at 30℃ for 90 min, then add 80 μL of MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide), incubate in a metal bath at 37℃ for 30 min, centrifuge at 13,000 r / min for 10 min, and take the supernatant for later use.

[0021] As a preferred embodiment, the above-described method for identifying the authenticity of Codonopsis pilosula based on electronic tongue and GC-MS technology, wherein the GC-MS conditions in step (5) are as follows: using an Agilent HP-5MS flexible quartz capillary column with dimensions of 30m × 0.25mm × 0.25μm; splitless; high-purity helium as carrier gas; injection port temperature of 260℃; injection volume of 1μL; temperature program as follows: initial temperature of 60℃, increasing to 125℃ at 8℃ / min; increasing to 210℃ at 4℃ / min; increasing to 270℃ at 5℃ / min; increasing to 310℃ at 10℃ / min; using an EI ion source; electron energy of 70eV; interface temperature of 260℃; scan range of m / z 50~600; solvent delay time of 3min.

[0022] This invention describes a method for identifying the geographical origin of Codonopsis pilosula based on electronic tongue and GC-MS technology. A total of 17 differentially characteristic metabolites were identified among different types of Codonopsis pilosula: trimalic acid, xylanic acid, uridine, glucose, sorbitol, xylitol, myristic acid, sorbitol, proline, phenylalanine, glycerol, arabinose, erythritol, aspartic acid, butyric acid, succinic acid, and sucrose. The differentiating components between Codonopsis pilosula and Codonopsis pilosula var. baitiao and Codonopsis pilosula var. taiwanensis are fructose, galactose, sucrose, maltose, and allose.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] This invention first uses electronic tongue technology to evaluate the taste of Codonopsis pilosula solutions from different products. The results show that electronic tongue technology can intuitively display the taste of Codonopsis pilosula through sensor response values, concretizing it numerically. Principal component analysis (PCA), partial least squares analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the electronic tongue data of Codonopsis pilosula from Luzhou and other products. The results showed that the two groups of medicinal materials could be well separated through electronic tongue taste identification. GC-MS was used to study the differences in primary metabolomics between Codonopsis pilosula from Luzhou and other products, identifying 35 metabolites. PCA and orthogonal partial least squares analysis revealed 17 differential metabolites among the different Codonopsis pilosula varieties, and also identified 5 carbohydrate-related differential metabolites in Codonopsis pilosula from Luzhou, Codonopsis pilosula var. baitiao, and Codonopsis pilosula var. taiwanensis. Metabolic pathway enrichment results showed significant differences in C and N metabolism-related pathways between the two groups. The most significant differences were observed in the galactose metabolic pathway; the amino sugar and nucleotide sugar metabolic pathways; and the starch and sucrose metabolic pathways. In summary, the analytical methods based on electronic tongue technology and GC-MS technology to analyze the differences between *Codonopsis pilosula* and other *Codonopsis* species provide an efficient and objective analytical approach. This also offers a simple and easy method for identifying the authenticity of *Codonopsis pilosula* medicinal materials, revealing the causes of its authenticity. Furthermore, it provides a theoretical basis for studying the differences in secondary metabolite accumulation and pharmacological effects among different commercial *Codonopsis pilosula* products, and offers a theoretical foundation for the development of medicinal resources of *Codonopsis pilosula*. Attached Figure Description

[0025] Figure 1 The results of electronic tongue detection for Codonopsis pilosula extract samples.

[0026] Figure 2 The electronic tongue numerical radar image of the Codonopsis pilosula extract sample.

[0027] Figure 3 The images show the PCA (left) and PLS-DA (right) analysis results for samples of Codonopsis pilosula extract. (Note: Samples 1-3 are S1; samples 4-6 are S2; samples 7-9 are S3; samples 10-12 are S4; samples 13-15 are S5; samples 16-18 are S6)

[0028] Figure 4 Total ion current GC-MS chromatogram of Codonopsis pilosula sample

[0029] Figure 5 This is a PCA score chart for the Codonopsis pilosula samples. Note: Samples 1-3 are S1; samples 4-6 are S2; samples 7-9 are S3.

[0030] Samples 10-12 are S4; samples 13-15 are S5; samples 16-18 are S6.

[0031] Figure 6 PLS-DA score map and VIP values ​​of differential metabolites for Codonopsis pilosula samples.

[0032] Figure 7 OPLS-DA analysis chart and VIP values ​​of differential metabolites for the Lu Party's participation in the White Strip Party and the Taiwan Party.

[0033] Figure 8 A graph showing the differences in the composition of the Lu Party's participation in the White Strip Party and the Taiwan Party. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0035] Example 1

[0036] 1. Materials and Reagents

[0037] 1.1 Instruments and Reagents

[0038] Taste analysis system from INSENT Corporation (TS-5000Z, Beijing Yingsheng Hengtai Technology Co., Ltd.); electronic analytical balance of 0.01 g / L (ME104E, Mettler Toledo Instruments Ltd.); high-speed centrifuge (TGL-168, Shanghai Anting Scientific Instrument Factory); ultrasonic cleaner (KH-500B, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); circulating vacuum pump (SHZ-D(Ⅲ), Gongyi Yuhua Instrument Co., Ltd.); pure water purifier (EAC-10T, Nanjing Yipuyida Technology Development Co., Ltd.); Haisdike ceramic Buchner funnel (HKCL-256).

[0039] 1.2 Sample Information

[0040] The Codonopsis pilosula samples were collected during the optimal harvesting period of medicinal materials in 2020, and were collected from Gansu Province, Shanxi Province, Hubei Province and other places. They were identified by Professor Gu Wei of Nanjing University of Chinese Medicine as dried roots of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) L. Shen or Codonopsis tangshen Oliv., all belonging to the Campanulaceae family. The sample information is shown in Table 1-1.

[0041] Table 1-1 Information on Codonopsis pilosula samples

[0042]

[0043]

[0044] 2 Experimental Methods

[0045] 2.1 Electronic tongue detection

[0046] 2.1.1 Preparation of Codonopsis pilosula sample solution

[0047] The powdered herbs of Codonopsis pilosula, Codonopsis pilosula, Codonopsis pilosula, Codonopsis pilosula, Codonopsis pilosula, Codonopsis pilosula, and Codonopsis pilosula (passed through a 40-mesh sieve) were thoroughly mixed, and 25.00 g was accurately weighed. 250 mL of ultrapure water was added, and the mixture was heated under reflux for 1 hour. The mixture was then filtered while hot, and the filtrate was collected. This process was repeated twice. The filtrates were combined and centrifuged at 5000 rpm for 10 minutes. The supernatant was then diluted to a 500 mL volumetric flask to obtain the sample solution. Each sample was prepared in triplicate.

[0048] 2.1.2 Electronic tongue technology

[0049] This invention utilizes the TS-5000Z electronic tongue testing device, which employs artificial lipid membrane sensing technology similar to the working principle of taste bud cells. It simulates the taste perception mechanism of living organisms, evaluating five basic tastes (sour, sweet, bitter, salty, and umami) and astringency by detecting changes in membrane potential generated by electrostatic or hydrophobic interactions between various taste substances and the artificial lipid membrane, without requiring any statistical analysis or modeling. Six types of sensors are used, detailed in Tables 1-2. The electronic tongue can obtain initial taste (e.g., sour, sweet, salty) and aftertaste (e.g., bitter aftertaste, astringent aftertaste). For the pharmaceutical industry, initial taste detection is crucial, and aftertaste testing is even more essential.

[0050] After activation and calibration, the electronic tongue is used to test the sample. The data acquisition time for each sample is 120 seconds. The samples are rinsed with ultrapure water for 10 seconds, and the samples are rinsed with a cup of ultrapure water between each sample. Each sample is repeated 6 times, and the last 3 times are taken as the test result.

[0051] Table 1-2 Introduction to the TS-5000Z Electronic Tongue Sensor

[0052]

[0053] 2.2 A Study on the Differences in Primary Metabolites among Different Commodities of Codonopsis pilosula

[0054] 2.2.1 Sample Pretreatment

[0055] Accurately weigh 0.1g of each type of Codonopsis pilosula powder (passed through a 40-mesh sieve), add 1mL of 70% methanol solution containing 2% formic acid, sonicate (360W, 40kHz) for 15min, centrifuge at 13000r / min for 10min, take 40μL of supernatant, add 20μL of 1mg / mL salicylic acid internal standard solution, blow dry with nitrogen, add 20μL of 40mg / mL methoxyamine pyridine solution, incubate in a metal bath at 30℃ for 90min, then add 80μL of MSTFA, incubate in a metal bath at 37℃ for 30min, centrifuge at 13000r / min for 10min, and take the supernatant for later use.

[0056] 2.2.2 GC-MS Conditions

[0057] An Agilent HP-5MS flexible quartz capillary column (30m × 0.25mm × 0.25μm) was used; splitless flow; high-purity helium as the carrier gas; injection port temperature was 260℃; injection volume was 1μL; temperature program was used (initial temperature 60℃, increased to 125℃ at 8℃ / min; increased to 210℃ at 4℃ / min; increased to 270℃ at 5℃ / min; increased to 310℃ at 10℃ / min); an EI ion source was used; electron energy was 70eV; interface temperature was 260℃; scan range was m / z 50–600; solvent delay time was 3 min.

[0058] 2.2.3 Data Processing and Multivariate Statistical Analysis

[0059] The characteristic peaks of the data were extracted and processed using the R software platform and the XCMS package. After normalization, the processed data matrix was imported into SIMCA14.1 software for PCA analysis and PLS-DA and other multivariate statistical analyses.

[19] The selection criteria for differentially metabolites were then based on a contribution score (VIP score) greater than 1 and a significant difference in inter-group changes (t-test) (P < 0.05). The differentially metabolites were then entered into the KEGG and MetaboAnalyst (http: / / www.metaboanalyst.ca / ) databases for enrichment and screening of metabolic pathways.

[20] .

[0060] 3 Results and Discussion

[0061] 3.1 Results of Electronic Tongue Measurement

[0062] Oral tasting results are uncertain due to subjective differences among tasters and cannot quantify individual flavors. Electronic tongue technology, however, can simulate human taste, converting the "taste" of a sample into response values ​​for various sensors, thus quantifying the "flavor." Therefore, this experiment uses electronic tongue technology to compare the differences in flavor among different types of Codonopsis pilosula and verifies the method through repeatability testing.

[0063] Samples of Codonopsis pilosula from different products were tested. Data acquisition for each sample was 120 seconds. Samples were rinsed with ultrapure water for 10 seconds between each sample, with a separate rinse of one cup of ultrapure water between samples. Each sample was repeated 6 times, and the last three values ​​were used as the test results. Sensor response values ​​were recorded. To characterize the taste differences between different Codonopsis pilosula products, a radar chart and bar chart of taste information for different Codonopsis pilosula products were established based on the sensor response values. Figure 1 and 2 The "Tasteless" value represents the taste value of the reference solution, which is composed of KCl and tartaric acid. Therefore, the tasteless value for sour taste is -13, and for salty taste, it is -6. Based on this, if the taste value of a sample is lower than the "Tasteless" value, it indicates that the sample lacks that taste; conversely, if it is higher, it does. The experimental data shows that the flavors in the extracts of various *Codonopsis pilosula* samples are consistent, containing sweet, umami, richness, astringency, saltiness, and bitterness. The bitterness and astringency have relatively low aftertaste values ​​and are not noticeably sour. However, significant differences exist in some indicators. *Codonopsis pilosula* from Luzhou has significantly higher sweetness, umami, and richness values ​​than other *Codonopsis pilosula* varieties. The identification of *Codonopsis pilosula* is based on "sweetness is superior," as sweetness is one of the standards for judging the quality of *Codonopsis pilosula*. As a traditional medicinal material from Shanxi Province, *Codonopsis pilosula* from Luzhou is of excellent quality and has significant therapeutic effects. The richness of the umami flavor indicates that it contains abundant amino acids, nucleic acids, and other nutrients, which may be the main reason for its good taste. This provides strong data support for the widespread clinical application of Codonopsis pilosula oral liquid.

[0064] The "taste" of traditional Chinese medicine (TCM) is closely related to its chemical components. Sweetness is primarily attributed to carbohydrates, sourness mainly to organic acids and phenols, saltiness to inorganic salts and minerals, and umami to amino acids, nucleotides, and umami peptides. However, there are currently no studies determining the content of these components in different types of Codonopsis pilosula. Taste attributes arise from the interaction between various tastes. For example, sourness can enhance saltiness, while saltiness and sweetness can suppress bitterness; sweetness can suppress sourness, and sourness can enhance bitterness. The key to these interactions lies in concentration; different concentrations result in different taste interaction responses. The complexity of taste mirrors the complexity of its chemical components. Taste interactions can be considered as interactions between these chemical components, and these complex chemical components endow drugs with good biological activity. Therefore, detecting the taste attributes can, to some extent, reflect the quality of a drug.

[0065] 3.1.3 Analysis of Electronic Tongue Measurement Results

[0066] Principal component analysis (PCA) is widely used for dimensionality reduction of data. It is a technique for simplifying and analyzing datasets and is one of the most commonly used unsupervised dimensionality reduction methods in multivariate statistical analysis. It extracts a few "variables" from multiple variables that can better reflect the information of the sample without human interference. This invention uses SIMCA14.1 software to perform principal component analysis on different types of Codonopsis pilosula, with Ctr (centering) selected as the scaling method. Figure 3 The results showed that the two-dimensional graph composed of PC1 and PC2 could objectively reflect the overall quality differences among the Codonopsis pilosula samples. Codonopsis pilosula samples S1, S2, S4, and S6 could be well distinguished from other Codonopsis pilosula samples, but S3 and S5 had similar tastes and could not be distinguished. The VIP score graph showed that tastes with VIP > 1 included sweetness, astringency, aftertaste, and saltiness, with the most significant difference in sweetness among the different Codonopsis pilosula samples. Since Codonopsis pilosula from Luzhou was well distinguishable from other Codonopsis pilosula samples, it indicates that electronic tongue technology can effectively differentiate Codonopsis pilosula from other commercial Codonopsis pilosula samples, and the primary method for identification is the difference in sweetness.

[0067] 3.2 Results of a study on the differences in primary metabolites among different commercial Codonopsis pilosula products

[0068] 3.2.1 Analysis and Processing of Raw GC-MS Data

[0069] Depend on Figure 4It can be seen that the baselines of the GC-MS chromatographic peaks of different products of Codonopsis pilosula after derivatization were relatively stable, and the chromatographic peaks were effectively separated, indicating that the method is stable and reliable, and the research data can be used for subsequent statistical analysis and processing. After preprocessing the raw GC-MS data, the detected chromatographic peaks were qualitatively annotated in the NIST database and the Wiley Registry metabolomics database based on information such as the retention time of the chromatographic peaks. A total of 35 metabolites were identified, as shown in Tables 1-3.

[0070] Table 1-3 Analysis of primary metabolites in QC samples

[0071]

[0072]

[0073]

[0074] 3.2 Differential analysis of metabolomics in Codonopsis pilosula samples

[0075] (1) PCA Analysis

[0076] To analyze the metabolomics differences among different Codonopsis pilosula samples, principal component analysis (PCA) was first performed. A PCA model fitted with two principal components was obtained, with a cumulative R²X = 1 and Q² = 0.999. The parameters of both models were greater than 0.5 and showed little difference, indicating that the established PCA model was stable and suitable for metabolomics difference analysis. Figure 5 It can be seen that different products of Codonopsis pilosula are classified according to their origin, and the sample S1 of Codonopsis pilosula from Luzhou is also clearly separated from the sample from the same origin, indicating that there are obvious differences in the origin of Codonopsis pilosula from Luzhou in terms of metabolite composition. However, the disadvantage of PCA analysis is that it cannot ignore within-group errors and eliminate random errors that are irrelevant to the research objective; therefore, it is an unsupervised analytical method.

[0077] (2) Analysis results of PLS-DA and OPLS-DA models

[0078] The advantage of PLS-DA model analysis is that it can filter out orthogonal variables in metabolites that are not related to categorical variables, and analyze non-orthogonal and orthogonal variables separately, thus more accurately identifying differences in metabolites across different samples. For example... Figure 6 As shown, the distribution of the three types of Codonopsis pilosula samples exhibits a clustering pattern based on their origin, indicating that the model can effectively distinguish the Codonopsis pilosula samples. OPLS-DA model analysis was performed on the three commercial Codonopsis pilosula samples from the Codonopsis pilosula origin. Figure 7 It can be seen that Codonopsis pilosula S1 can be effectively distinguished from samples S2 and S3 of the same origin.

[0079] (3) Differences in metabolites between Codonopsis pilosula and other commercial Codonopsis pilosula samples

[0080] The VIP scores (VIP > 1) of the PLS-DA and OPLS-DA models, combined with the p-values ​​(P < 0.05) of the t-test, were used to identify the marker-differential metabolites between different primary samples and between *Codonopsis pilosula* and samples from the same primary sample. Figure 6 It can be seen that 17 differential metabolites were screened from the various Codonopsis pilosula samples, namely trimalic acid, xylanic acid, uridine, glucose, sorbitol, xylitol, myristic acid, sorbitol, proline, phenylalanine, glycerol, arabinose, erythritol, aspartic acid, butyric acid, succinic acid, and sucrose. Figure 7 It can be seen that the components that differentiate the Lu Party from the White Strip Party and the Taiwan Party are fructose, galactose, sucrose, maltose, and alloose.

[0081] (4) Pathway analysis

[0082] Since electronic tongue analysis revealed that sweetness is the most significant taste for identifying Codonopsis pilosula, the KEGG database was further utilized to find the KEGG IDs of all differentially metabolites. These IDs were then input into the MetaboAnalyst database for metabolic enrichment, identifying potential differential metabolic pathways. These pathways were then screened to ultimately determine the pathways with the highest correlation to metabolite differences. Enrichment analysis identified three important carbohydrate metabolism pathways: galactose metabolism, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism. Figure 8 To further analyze the compositional differences among the Lu Party, White Strip Party, and Taiwan Party, which share a common origin, and in combination with Figure 8 The differences in content revealed that the content of fructose, galactose, and alloose in Codonopsis pilosula was higher than that in the other two groups, indicating that the metabolic pathways for identifying the authenticity of Codonopsis pilosula are galactose metabolism, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism.

[0083] 3.3 Electronic tongue and GC-MS component analysis

[0084] Analysis using electronic tongue technology revealed that the main flavors of Codonopsis pilosula are sweet, umami, richness, saltiness, astringency, and bitterness. To analyze the material basis of these flavors, this invention analyzed the resource-based chemical components, revealing numerous sugars and amino acids in Codonopsis pilosula. GC-MS identification showed that 12 sugar components in Codonopsis pilosula exhibited sweetness, and 6 amino acid components were associated with umami and richness. This demonstrates that combining electronic tongue and GC-MS technologies is more effective in identifying authentic Codonopsis pilosula.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for identifying the geographical origin of Codonopsis pilosula based on electronic tongue and GC-MS technology, characterized in that, Includes the following steps: (1) Preparation of electronic tongue sample solution Collect Codonopsis pilosula from different products, crush them appropriately, sieve them, weigh them accurately, add ultrapure water, heat under reflux, filter while hot, take the filtrate, repeat the extraction several times, combine the filtrates, centrifuge, take the supernatant and make up to volume to obtain the sample solution. (2) Take the electronic tongue sample solution from step (1) and use electronic tongue technology to determine the taste of different Codonopsis pilosula. The method of using electronic tongue technology to determine the taste of different Codonopsis pilosula is as follows: use TS-5000Z electronic tongue measuring device, the data acquisition time for each Codonopsis pilosula sample is 120 s, the samples are washed with ultrapure water for 10 s, and the samples are washed with a cup of ultrapure water between samples. Each sample is repeated 6 times, and the last 3 times are taken as the detection result. Record the response value of the sensor. (3) The electronic tongue technology in step (2) will intuitively display the taste of Codonopsis pilosula of different products through the response value of the sensor, and concretize it with numerical values. Then, the electronic tongue data of Codonopsis pilosula of different products will be analyzed by principal component analysis (PCA) and partial least squares analysis (PLS-DA) to identify the taste of Codonopsis pilosula of different products. (4) Preparation of GC-MS sample solutions Take different types of Codonopsis pilosula, grind them appropriately, sieve them, weigh them accurately, add 1 mL of 70% methanol solution containing 2% formic acid, sonicate at 360W and 40 kHz for 15 min, centrifuge at 13,000 r / min for 10 min, take 40 μL of supernatant, add 20 μL of 1 mg / mL salicylic acid internal standard solution, blow dry with nitrogen, add 20 μL of 40 mg / mL methoxyamine pyridine solution, incubate in a metal bath at 30 ℃ for 90 min, then add 80 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide, incubate in a metal bath at 37 ℃ for 30 min, centrifuge at 13,000 r / min for 10 min, and take the supernatant for later use; (5) Take the GC-MS sample solution from step (4) and inject it into the GC-MS instrument to analyze the primary metabolites of different products of Codonopsis pilosula. The GC-MS conditions are as follows: Agilent HP-5MS flexible quartz capillary column with specifications of 30 m × 0.25 mm × 0.25 μm is used; splitless; high-purity helium is used as the carrier gas; the injection port temperature is 260 ℃; the injection volume is 1 μL; the temperature program is as follows: the initial temperature is 60 ℃, and it is increased to 125 ℃ at 8 ℃ / min; it is increased to 210 ℃ at 4 ℃ / min; it is increased to 270 ℃ at 5 ℃ / min; and it is increased to 310 ℃ at 10 ℃ / min; an EI ion source with an electron energy of 70 eV is used; the interface temperature is 260 ℃; the scan range is m / z 50 ~ 600; and the solvent delay time is 3 min. (6) Principal component analysis (PCA), partial least squares analysis, and orthogonal partial least squares discriminant analysis were used to perform chemometric analysis on the primary metabolite data of Codonopsis pilosula from different products, and the differential metabolites of Codonopsis pilosula from different products were screened out as characteristic metabolites for identifying Codonopsis pilosula from different products.

2. The method for identifying the geographical origin of Codonopsis pilosula based on electronic tongue and GC-MS technology according to claim 1, characterized in that, Step (1) The preparation method of electronic tongue sample solution is as follows: collect Codonopsis pilosula from different products, crush appropriately, sieve, accurately weigh 25.00 g, add 250 mL of pure water, heat under reflux for 1 h, filter while hot, take the filtrate, repeat twice, combine the filtrates, centrifuge at 5000 r / min for 10 min, take the supernatant and dilute to 500 mL in a volumetric flask to obtain the sample solution, and each sample is divided into 3 parallel portions.

3. The method for identifying the geographical origin of Codonopsis pilosula based on electronic tongue and GC-MS technology according to claim 1, characterized in that, The different components among the various Codonopsis pilosula species are 17, namely trimalic acid, xylanic acid, uridine, glucose, sorbitol, xylitol, myristic acid, sorbitol, proline, phenylalanine, glycerol, arabinose, erythritol, aspartic acid, butyric acid, succinic acid, and sucrose; the different components among Codonopsis pilosula var. lumborum and Codonopsis pilosula var. lumborum are 5: fructose, galactose, sucrose, maltose, and alloose.

4. The method for identifying the geographical origin of Codonopsis pilosula based on electronic tongue and GC-MS technology according to claim 1, characterized in that, Using the KEGG database to identify the KEGG IDs of differentially metabolized metabolites from different products of Codonopsis pilosula, the IDs of these differentially metabolites were input into the MetaboAnalyst database. Through metabolic enrichment, possible differential metabolic pathways between the two groups were identified. These metabolic pathways were then screened, and the galactose metabolic pathway, amino sugar and nucleotide sugar metabolic pathway, and starch and sucrose metabolic pathway, which had the highest correlation with the differences in metabolites, were finally obtained.

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