A method for identifying under-forest ginseng by mass spectrum based on marker components

Abstract

This invention provides a mass spectrometry identification method for forest-grown ginseng based on marker components, belonging to the field of traditional Chinese medicine identification technology. The marker component composition includes ginsenoside Jb isomer, notoginsenoside C isomer, ginsenoside L2, ginsenoside VI, and ginsenoside Rd. For the first time, it has been confirmed that the content of these five marker components in forest-grown ginseng is significantly higher than that in other non-forest-grown ginseng types, serving as specific markers to distinguish between forest-grown and non-forest-grown ginseng. Combining mass spectrometry detection and mass spectrometry data normalization, a precise quantitative method for trace components is established. This provides a highly specific, accurate, stable, reliable, and batch-applicable identification method for forest-grown ginseng, aiming to regulate the ginseng market order, curb the sale of inferior products and falsification of vintages, and effectively protect consumer rights and the quality of ginseng. It solves the technical problems of high misjudgment rates and difficulty in implementation due to reliance on common components, crude quantification, lack of specific markers, and inconsistent discrimination systems in existing technologies.

Description

Technical Field

[0001] This invention relates to the field of identification technology of Chinese medicinal materials, and in particular to a mass spectrometry identification method for forest ginseng based on marker components. Background Technology

[0002] Ginseng (Panax ginseng CA Mey.) is the dried root and rhizome of a plant in the Araliaceae family. It is listed in the 2025 edition of the *Pharmacopoeia of the People's Republic of my country* and is a traditional and precious Chinese medicinal herb. Based on different growing environments and cultivation methods, ginseng is divided into two categories: varieties cultivated intensively in artificial farmland, commonly known as "garden ginseng," have a short growth cycle and high yield; varieties sown in wild mountain forest environments and growing naturally are called "forest ginseng" (commonly known as "seed sea"), which have a longer growth cycle, superior quality, and significantly higher medicinal value and market price than garden ginseng. Modern research shows that the main active ingredient in ginseng is ginsenosides, and it also contains polysaccharides, volatile oils, amino acids, flavonoids, and other active ingredients. Modern pharmacological studies have confirmed that ginseng and its active ingredients have multi-target and multi-pathway pharmacological effects, covering aspects such as enhancing immunity, anti-fatigue, anti-oxidative stress, anti-tumor, improving cardiovascular function, regulating the central nervous system, protecting liver and kidney function, and delaying aging. It has extremely high application value in the fields of medicine, health products, and cosmetics.

[0003] Due to differences in growth cycle and habitat, the quality of ginseng varies greatly, making it extremely difficult to distinguish them based solely on traditional appearance. Since wild ginseng and cultivated ginseng share a high degree of homology in their origins and common chemical components, existing research, although attempting to identify them using techniques such as HPLC and mass spectrometry, primarily focuses on conventional saponin components. It lacks specific marker components that can accurately distinguish between the two, and a standardized discrimination model based on calculus-based quantification has not been established. Therefore, it is impossible to achieve rapid, accurate, and quantitative identification of wild ginseng and cultivated ginseng. Thus, there is an urgent need to develop a highly specific detection method to regulate the market and ensure the quality of medicinal materials.

[0004] Existing detection technologies mainly fall into four categories: First, HPLC fingerprinting combined with chemometric methods such as cluster analysis and principal component analysis is used to classify and identify common ginsenosides such as Rg1, Re, Rb1, Rc, Rb2, and Rd as indicators; second, high-resolution mass spectrometry such as UPLC-Q-TOF-MS is used to conduct non-targeted metabolomics analysis, and differentially expressed saponins such as Rb1, Rg1, Rb2, Ro, and Rg3 are screened as potential biomarkers using the OPLS-DA model; third, near-infrared spectroscopy and micro-infrared spectroscopy combined with data preprocessing and pattern recognition algorithms are used to achieve rapid and non-destructive identification of powder samples; and fourth, conventional physicochemical and physical indicators such as total saponin content, monomeric saponin content, ether extract, and ginseng powder density are used as auxiliary identification criteria. The above methods generally have significant drawbacks: they all rely on components, spectra, or physicochemical indicators common to both forest-grown and cultivated ginseng, lacking specific and highly characteristic marker components, leading to blurred discrimination boundaries and susceptibility to misjudgment; for trace difference components, they only use conventional peak area integration instead of calculus algorithms for precise quantification, resulting in insufficient detection sensitivity and repeatability; the discrimination results are easily affected by external factors such as origin, ginseng age, sample condition, and instrument model, resulting in poor stability and universality; various indicators have a large overlap between the two types of ginseng, and can only serve as auxiliary references, and a standardized system of "marker identification - precise quantification - threshold determination" has not been formed, making it difficult to apply on a large scale in actual supervision and quality inspection scenarios.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a substance for detecting marker components in the identification of wild ginseng or the preparation of products for identifying wild ginseng, so as to solve the technical problem in the prior art that samples cannot be accurately identified due to mixed origins, ambiguous age of ginseng, and processing interference.

[0007] The second objective of this invention is to provide a mass spectrometry method for identifying forest ginseng.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the application of substances for detecting marker component compositions in the identification of wild ginseng or in the preparation of products for the identification of wild ginseng; The marker component composition includes ginsenoside Jb isomer, notoginsenoside C isomer, ginsenoside L2, ginsenoside VI, and ginsenoside Rd.

[0009] Furthermore, the substance used to detect the marker component includes a standard substance corresponding to the marker component, and / or an organic solvent used to dissolve the marker component in the standard substance, the test sample, and the non-forest-grown ginseng control sample. Preferably, the organic solvent includes methanol.

[0010] Furthermore, the product contains a substance for detecting marker components; Preferably, the product includes reagents and / or kits.

[0011] Secondly, the present invention provides a mass spectrometry identification method for forest-grown ginseng, comprising high performance liquid chromatography coupled with mass spectrometry to detect the aforementioned marker components in the test sample solution and the non-forest-grown ginseng control sample solution, respectively, to obtain the normalized peak area of ​​each marker component in each sample solution, and to calculate the determination ratio of each marker component in the test sample according to the following formula: ; R 标志性成分 S is the proportion of marker components for determination. 待测样品 S represents the normalized peak area of ​​the marker component in the sample. 非林下参对照样品 The normalized peak area of ​​the marker component in the non-forest-grown ginseng control sample; If the R of each iconic component 标志性成分 If all values ​​are ≥60%, the sample to be tested is forest-grown ginseng; otherwise, it is non-forest-grown ginseng.

[0012] Furthermore, obtaining the normalized peak area of ​​each marker component in each sample solution involves setting the sum of the peak areas of all detected mass spectrometry peaks in each sample solution to 100%, and converting the peak area of ​​the mass spectrometry peak corresponding to the marker component into a percentage value.

[0013] Furthermore, the non-forest-grown ginseng control sample includes at least one non-forest-grown ginseng sample; Preferably, when the non-forest-grown ginseng control sample includes two or more non-forest-grown ginseng samples, S 非林下参对照样品 This represents the arithmetic mean of the normalized peak areas of the marker components in all non-forest-grown ginseng samples.

[0014] Furthermore, the preparation method of the test sample solution and the non-forest-grown ginseng control sample solution includes dissolving the sample extract in an organic solvent and filtering it. Preferably, the organic solvent includes methanol; Preferably, the method for preparing the sample extract includes taking sample powder, extracting it with alcohol, centrifuging to obtain the supernatant, and freeze-drying it.

[0015] Furthermore, the chromatographic column is a silica matrix bonded with C18 alkyl chains, preferably a reversed-phase C18 chromatographic column; Preferably, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, gradient elution is used, and the injection volume is 2 μl.

[0016] Furthermore, gradient elution includes: 0~2min: 2%~25% mobile phase A, 98%~75% mobile phase B; 2~7min: 25% mobile phase A, 75% mobile phase B; 7~9min: 25%~30% mobile phase A, 75%~70% mobile phase B; 9~18min: 30~40% mobile phase A, 70%~60% mobile phase B; 18~21min: 40%~75% mobile phase A, 60%~25% mobile phase B; 21~21.5min: 75%~95% mobile phase A, 25%~5% mobile phase B; 21.5~24min: 95% mobile phase A, 5% mobile phase B; 24~24.5min: 95%~2% mobile phase A, 5%~98% mobile phase B; 24.5~30min: 2% mobile phase A, 98% mobile phase B; Preferably, the flow rate is 0.2~0.4 mL / min, more preferably 0.3 mL / min; Preferably, the column temperature is 25~35℃, and more preferably 30℃.

[0017] Furthermore, the mass spectrometry conditions included: electrospray ionization (ESI) source, scanning in both positive and negative ion modes, with a scanning range of 100–1500 m / z; capillary voltage: 3.0 kV in positive ion mode and 2.5 kV in negative ion mode; ion source temperature: 150 °C; and desolvation gas temperature: 500 °C.

[0018] The application of the marker component composition provided by this invention in the identification of forest-grown ginseng or the preparation of products for identifying forest-grown ginseng has, for the first time, confirmed that the content of five marker components in forest-grown ginseng is significantly higher than that in other non-forest-grown ginseng species. These components can serve as exclusive markers to distinguish between forest-grown and non-forest-grown ginseng, improving accuracy and anti-interference capabilities, and avoiding cross-identification. This marker component composition breaks through the traditional common component analysis paradigm that relies on "content difference" as the criterion, instead relying on the specificity of metabolism, solving the technical problem in existing technologies where samples cannot be accurately identified due to mixed origins, ambiguous ginseng age, and processing interference. A precise quantitative method for trace components is established by combining mass spectrometry detection and mass spectrometry data normalization, forming an integrated standardized identification system of "marker qualitative analysis—mass spectrometry data normalization—judgment threshold." This provides a set of forest-grown ginseng identification methods that are highly specific, accurate, stable, reliable, and applicable in batches, thereby regulating the ginseng market order, curbing the sale of inferior products and falsification of age, and effectively protecting consumer rights and the quality of ginseng medicinal materials. It solves the technical problems of high misjudgment rate and difficulty in implementation of supervision caused by reliance on common components, rough quantification, lack of specific markers, and inconsistent discrimination system in existing technologies. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 A flowchart for establishing the screening and judgment criteria for the marker components of ginseng under forest cover provided in Embodiment 1 of the present invention; Figure 2 This is a comparison chart of the content of the marker component provided in Example 1 of the present invention between forest ginseng and cultivated ginseng; Figure 3 The mass spectrometry characteristic spectrum of the American ginsenoside Jb isomer provided in Example 1 of the present invention; Figure 4 The mass spectrometry characteristic spectrum of ginsenoside VI provided in Example 1 of the present invention; Figure 5 The mass spectrometry characteristic spectrum of the C isomer of notoginsenoside provided in Example 1 of the present invention; Figure 6 The mass spectrometry characteristic spectrum of ginsenoside Rd provided in Example 1 of the present invention; Figure 7 The mass spectrometry characteristic spectrum of ginsenoside L2 provided in Example 1 of this invention. Detailed Implementation

[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0022] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides, in one aspect, the application of a substance for detecting a marker component composition in the identification of forest-grown ginseng or in the preparation of products for identifying forest-grown ginseng; the marker component composition includes ginsenoside Jb isomer, notoginsenoside C isomer, ginsenoside L2, ginsenoside VI, and ginsenoside Rd.

[0025] This invention is the first to confirm that the content of five marker components in forest-grown ginseng is significantly higher than that in other non-forest-grown ginseng species. These components can serve as exclusive markers to distinguish between forest-grown and non-forest-grown ginseng, improving accuracy and anti-interference ability, and avoiding cross-judgment. This marker component composition breaks through the traditional common component analysis paradigm that uses "content difference" as the criterion, and instead relies on the specificity of metabolism, solving the technical problem in the prior art that samples cannot be accurately identified due to mixed origins, ambiguous ginseng age, and processing interference.

[0026] In some specific embodiments, the substance used to detect the marker component includes a standard substance corresponding to the marker component, and / or an organic solvent used to dissolve the marker component in the standard substance, the test sample, and the non-forest-grown reference control sample; in some specific embodiments, the organic solvent includes methanol.

[0027] In some specific embodiments, the product contains a substance for detecting marker components; in some specific embodiments, the product includes reagents and / or kits.

[0028] According to another aspect of the present invention, a mass spectrometry identification method for forest-grown ginseng is also provided, comprising using high performance liquid chromatography-mass spectrometry to detect the aforementioned marker components in the test sample solution and the non-forest-grown ginseng control sample solution, respectively, to obtain the normalized peak area of ​​each marker component in each sample solution, and calculating the determination ratio of each marker component in the test sample according to the following formula: ; R 标志性成分 S is the proportion of marker components for determination. 待测样品 S represents the normalized peak area of ​​the marker component in the sample. 非林下参对照样品 The normalized peak area of ​​the marker component in the non-forest-grown ginseng control sample; If the R of each iconic component 标志性成分 If all values ​​are ≥60%, the sample to be tested is forest-grown ginseng; otherwise, it is non-forest-grown ginseng.

[0029] This invention identifies highly specific marker components that can accurately distinguish between forest-grown and cultivated ginseng. It then combines mass spectrometry detection and data normalization to establish a precise quantitative method for trace components, forming an integrated standardized identification system encompassing "marker characterization—mass spectrometry data normalization—judgment threshold." Ultimately, this provides a highly specific, accurate, stable, reliable, and batch-applicable method for identifying forest-grown ginseng, aiming to regulate the ginseng market, curb the sale of inferior products and falsification of vintages, and effectively protect consumer rights and the quality of ginseng. This invention solves the technical problems of high misjudgment rates and difficulty in implementation due to reliance on common components, crude quantification, lack of specific markers, and inconsistent judgment systems in existing technologies.

[0030] In some specific implementations, obtaining the normalized peak area of ​​each marker component in each sample solution includes setting the sum of the peak areas of all detected mass spectrometry peaks in each sample solution to 100%, and converting the peak area of ​​the mass spectrometry peak corresponding to the marker component into a percentage value.

[0031] Normalization effectively offsets systematic errors such as differences in sample volume, instrument response fluctuations, and ionization efficiency between different samples. Utilizing normalized peak area data, no additional experimental design is required, allowing for seamless integration with conventional metabolomics analysis workflows. It is particularly suitable for research scenarios that have already implemented differential screening methods such as volcano plots and OPLS-DA but have not yet performed absolute quantification, thus revitalizing existing qualitative data and giving it patented application value.

[0032] In some specific embodiments, the non-forest-grown ginseng control sample comprises at least one non-forest-grown ginseng sample; in some specific embodiments, when the non-forest-grown ginseng control sample comprises two or more non-forest-grown ginseng samples, S 非林下参对照样品 This represents the arithmetic mean of the normalized peak areas of the marker components in all non-forest-grown ginseng samples.

[0033] In some specific embodiments, the preparation method of the test sample solution and the non-forest-grown ginseng control sample solution includes dissolving the sample extract in an organic solvent and filtering it.

[0034] Sample pretreatment is simple, requiring no complex equipment or absolute quantification, thus reducing detection costs and operational difficulty. Only qualitative or semi-quantitative mass spectrometry data are needed, and discrimination can be achieved by the inter-group distribution ratio of normalized peak areas, significantly reducing detection costs and simplifying the pretreatment and data analysis process.

[0035] In some specific embodiments, the organic solvent includes methanol.

[0036] In some specific embodiments, the method for preparing the sample extract includes taking sample powder, extracting it with alcohol, centrifuging to obtain the supernatant, and freeze-drying it.

[0037] In some specific embodiments, the chromatographic column is a silica matrix bonded with C18 alkyl chains, preferably a reversed-phase C18 chromatographic column; In some specific embodiments, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, gradient elution is performed, and the injection volume is 2 μl.

[0038] In some specific implementations, gradient elution includes: 0–2 min: 2%–25% mobile phase A, 98%–75% mobile phase B; 2–7 min: 25% mobile phase A, 75% mobile phase B; 7–9 min: 25%–30% mobile phase A, 75%–70% mobile phase B; 9–18 min: 30–40% mobile phase A, 70%–60% mobile phase B; 18–21 min: 40%–75% mobile phase A, 60%–25% mobile phase B; 21–21.5 min: 75%–95% mobile phase A, 25%–5% mobile phase B; 21.5–24 min: 95% mobile phase A, 5% mobile phase B; 24–24.5 min: 95%–2% mobile phase A, 5%–98% mobile phase B; 24.5–30 min: 2% mobile phase A, 98% mobile phase B.

[0039] Single sample detection takes less than 30 minutes, and batch applications are possible, making it suitable for large-scale screening. Data processing relies on open-source mass spectrometry data processing software, and percentage calculation and threshold determination can be automatically completed through simple scripts or Excel formulas.

[0040] In some specific embodiments, the flow rate is 0.2~0.4 mL / min, preferably 0.3 mL / min; In some specific implementations, the column temperature is 25~35℃, preferably 30℃.

[0041] In some specific implementations, the mass spectrometry conditions include: an electrospray ionization (ESI) source, scanning in both positive and negative ion modes, with a scanning range of 100~1500 m / z; capillary voltage: 3.0 kV in positive ion mode and 2.5 kV in negative ion mode; ion source temperature: 150 °C; and desolvation gas temperature: 500 °C.

[0042] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] Forest-grown ginseng samples: a total of 20 batches of samples, as shown in Table 1.

[0044] Table 1

[0045] Garden ginseng samples: a total of 10 batches of samples, as shown in Table 2.

[0046] Table 2

[0047] Red ginseng samples: a total of 38 batches of samples, as shown in Table 3.

[0048] Table 3

[0049] Example 1 Combination Figure 1 This document explains the establishment of criteria for screening and determining the marker components of ginseng grown in forests.

[0050] 1. Preparation of reference solution: Accurately weigh an appropriate amount of the reference standard, add 70% methanol, and dissolve to obtain the reference standard solution.

[0051] The reference standards included ginsenoside Rb1 43.687 μg / ml, ginsenoside Rb2 41.390 μg / ml, ginsenoside Rb3 44.843 μg / ml, ginsenoside Rc 787.910 μg / ml, ginsenoside Rg1 55.773 μg / ml, ginsenoside Rg2 2.831 μg / ml, 20(R)-ginsenoside Rg3 31.399 μg / ml, 20(S)-ginsenoside Rg3 3.973 μg / ml, ginsenoside Rg4 3.468 μg / ml, ginsenoside Rg5 41.003 μg / ml, ginsenoside Rg6 0.855 μg / ml, ginsenoside Rf5 8.324 μg / ml, and ginsenoside Rd 27.360 μg / ml. Ginsenoside Rh1 32.548 μg / ml, 20(S)-Ginsenoside Rh1 1.830 μg / ml, 20(R)-Ginsenoside Rh2 54.195 μg / ml, Ginsenoside Re 41.220 μg / ml, Ginsenoside Rk1 37.629 μg / ml, Ginsenoside Rk3 42.822 μg / ml, Ginsenoside F2 13.984 μg / ml, Ginsenoside Rh4 29.179 μg / ml, and Ginsenoside CK 38.361 μg / ml.

[0052] 2. Preparation of the test solution Whole samples of forest-grown ginseng, cultivated ginseng, and red ginseng were pulverized and passed through an 80-mesh sieve. 1g of powder was placed in a 50ml centrifuge tube, and 10ml of 70% methanol was added. The mixture was extracted twice by sonication for 40min. The supernatants were combined by centrifugation, and the methanol was evaporated. The lyophilized powder was then freeze-dried to a powder state. An appropriate amount of the freeze-dried powder was weighed, and a solution of approximately 1mg / ml was prepared by adding methanol to a mass spectrometer. The solution was then filtered through a 0.22μm filter membrane.

[0053] 3. Chromatographic conditions and system suitability test Chromatographic column: Morphling® AQ-C18 column (100 mm × 2.1 mm, 3 μm); acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, gradient elution as specified in Table 4; flow rate: 0.3 ml / min; column temperature: 30 °C; Mass spectrometry conditions: Electrospray ionization source was used, with scanning in both positive and negative ion modes.

[0054] Scan range: m / z 100-1500.

[0055] Capillary voltage: 3.0 kV (positive), 2.5 kV (negative); Ion source temperature: 150℃; Desolvation gas temperature: 500℃.

[0056] Table 4 Gradient elution conditions

[0057] Assay: Accurately pipette 2 μl of each test solution and inject it into the liquid chromatograph for determination.

[0058] 4. Analysis Process The original mass spectrometry data file was imported into MS-DIAL.v5.5.251021 software. Samples from forest-grown ginseng (batches R01-R07, R13-R20), cultivated ginseng (batches R21-R25, R29-R30), and red ginseng (batches S01-S38) were grouped. Peak areas were normalized, and the results were exported to obtain a matrix containing the normalized peak areas of all mass spectrometry peaks. Mass spectrometry components were identified, and 152 saponins were identified. The normalized peak areas of each saponin in all samples were summed to calculate the average normalized peak area of ​​each saponin in the three groups of forest-grown, cultivated, and red ginseng samples. The percentage of each group in the total was then calculated. One-to-one volcano plot analysis revealed that the content of five saponins in forest-grown ginseng was significantly higher than in other ginseng types (e.g., ...). Figure 2 As shown): Quinquenoside Jb isomer, Notoginsenoside C isomer, Quinquenoside L2, Chikusetsusaponin VI, and Ginsenoside Rd can be used as target ginsenosides (i.e., marker components) to distinguish between forest-grown ginseng and cultivated ginseng.

[0059] The mass spectrometry characteristics of each target ginsenoside are as follows: Figures 3-7 As shown.

[0060] 5. Judgment Criteria The determination includes the following steps: (1) Set up the sample group to be tested and set up at least one non-forest ginseng control sample group, and normalize the mass spectrometry peak area of ​​the target ginsenosides; (2) Obtain the normalized peak area S of each target ginsenoside in the sample group to be tested. 待测样品 and the normalized peak area S in the control sample group 对照样品 When the control sample group contains two or more non-forest-grown ginseng samples, S 对照样品 The arithmetic mean of the normalized peak areas of the target ginsenosides in all non-forest-grown ginseng samples.

[0061] Calculate the determination ratio of the target ginsenoside in the sample to be tested, denoted as R. 目标人参皂苷 The calculation formula is as follows: ; If the R of each target ginsenoside 目标人参皂苷 If all values ​​are ≥60%, the sample is determined to be forest-grown ginseng; otherwise, it is not forest-grown ginseng.

[0062] Example 2 Verification Group Identification Forest ginseng samples numbered R08~R12 and cultivated ginseng samples numbered R26~R28 were selected as test samples and designated as the verification group to verify the above judgment criteria. The remaining 7 batches of cultivated ginseng samples were used as control samples and designated as the cultivated ginseng modeling group.

[0063] The normalized peak area S of each target ginsenoside in the validation group was obtained according to the method provided in Example 1. 验证组 The normalized peak area S in the ginseng modeling group 园参建模 Since the number of control samples in the ginseng modeling group was 7, the arithmetic mean of the normalized peak areas of the target ginsenoside in all control samples was taken.

[0064] Calculate the determination ratio of the corresponding target ginsenoside in the test samples of each validation group, R. 目标人参皂苷 The calculation formula is as follows: .

[0065] The judgment ratios for each sample in the validation group are shown in Table 5.

[0066] Table 5

[0067] The results showed that the detection rates of all five target ginsenosides in forest-grown ginseng were >60%, while in eight batches of cultivated ginseng, the detection rate of Notoginsenoside C isomer was close to 60%, and in ten batches of cultivated ginseng, the detection rate of Chikusetsusaponin VI was >60%. This demonstrates that using a single target ginsenoside may produce false positives, and the content of a single saponin marker component is easily affected by individual sample differences. A single indicator is not robust enough for identification, and there is a risk that the value of this indicator in some cultivated ginseng samples may fall within the discrimination range of forest-grown ginseng samples. Therefore, the identification method of forest-grown ginseng using a combination of multiple ginsenoside marker components, utilizing the complementary discriminative information of each saponin among different samples, can effectively avoid the risk of misjudgment caused by individual differences due to a single indicator. The probability of multiple saponins simultaneously showing abnormal values ​​is much lower than that of a single saponin; therefore, the group discrimination stability of the combined indicator is significantly better than that of a single indicator.

[0068] 7. The identification data of the five target ginsenosides are shown in Table 6.

[0069] Table 6

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of substances that detect marker component compositions in the identification of wild ginseng or in the preparation of products for the identification of wild ginseng; The marker component composition includes ginsenoside Jb isomer, notoginsenoside C isomer, ginsenoside L2, ginsenoside VI, and ginsenoside Rd.

2. The application according to claim 1, characterized in that, The substances used to detect the marker components include standard substances corresponding to the marker components, and / or organic solvents used to dissolve the marker components in the standard substances, the test samples, and the non-forest-grown ginseng control samples. Preferably, the organic solvent includes methanol.

3. The application according to claim 1 or 2, characterized in that, The product contains substances that detect marker components; Preferably, the product includes reagents and / or kits.

4. A mass spectrometry identification method for forest-grown ginseng, characterized in that, The method includes high-performance liquid chromatography and mass spectrometry to detect the marker components described in any one of claims 1 to 3 in the test sample solution and the non-forest-grown ginseng control sample solution, respectively, to obtain the normalized peak area of ​​each marker component in each sample solution, and to calculate the determination ratio of each marker component in the test sample according to the following formula: ； R 标志性成分 S is the proportion of marker components for determination. 待测样品 S represents the normalized peak area of ​​the marker component in the sample. 非林下参对照样品 The normalized peak area of ​​the marker component in the non-forest-grown ginseng control sample; If the R of each iconic component 标志性成分 If all values ​​are ≥60%, the sample to be tested is forest-grown ginseng; otherwise, it is non-forest-grown ginseng.

5. The method according to claim 4, characterized in that, To obtain the normalized peak area of ​​each marker component in each sample solution, the sum of the peak areas of all detected mass spectrometry peaks in each sample solution is set to 100%, and the peak areas of the mass spectrometry peaks corresponding to the marker components are converted into percentage values ​​according to the ratio.

6. The method according to claim 4, characterized in that, The non-forest-grown ginseng control sample includes at least one non-forest-grown ginseng sample; Preferably, when the non-forest-grown ginseng control sample includes two or more non-forest-grown ginseng samples, S 非林下参对照样品 This represents the arithmetic mean of the normalized peak areas of the marker components in all non-forest-grown ginseng samples.

7. The method according to claim 4, characterized in that, The preparation method of the test sample solution and the non-forest-grown ginseng control sample solution includes dissolving the sample extract in an organic solvent and filtering it. Preferably, the organic solvent includes methanol; Preferably, the method for preparing the sample extract includes taking sample powder, extracting it with alcohol, centrifuging to obtain the supernatant, and freeze-drying it.

8. The method according to claim 4, characterized in that, The chromatographic column is a silica matrix bonded with C18 alkyl chains, preferably a reversed-phase C18 column; Preferably, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, gradient elution is used, and the injection volume is 2 μl.

9. The method according to claim 8, characterized in that, Gradient elution includes: 0~2min: 2%~25% mobile phase A, 98%~75% mobile phase B; 2~7min: 25% mobile phase A, 75% mobile phase B; 7~9min: 25%~30% mobile phase A, 75%~70% mobile phase B; 9~18min: 30~40% mobile phase A, 70%~60% mobile phase B; 18~21min: 40%~75% mobile phase A, 60%~25% mobile phase B; 21~21.5min: 75%~95% mobile phase A, 25%~5% mobile phase B; 21.5~24min: 95% mobile phase A, 5% mobile phase B; 24~24.5min: 95%~2% mobile phase A, 5%~98% mobile phase B; 24.5~30min: 2% mobile phase A, 98% mobile phase B; Preferably, the flow rate is 0.2~0.4 mL / min, more preferably 0.3 mL / min; Preferably, the column temperature is 25~35℃, and more preferably 30℃.

10. The method according to claim 4, characterized in that, Mass spectrometry conditions included: electrospray ionization (ESI) source, scanning in both positive and negative ion modes, with a scanning range of 100–1500 m / z; capillary voltage: 3.0 kV in positive ion mode and 2.5 kV in negative ion mode; ion source temperature: 150 °C; and desolvation gas temperature: 500 °C.

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