A method for identifying artificial cordyceps sinensis and wild cordyceps sinensis

Abstract

This invention belongs to the field of traditional Chinese medicine (TCM) material authentication technology, specifically relating to a detection method for distinguishing between cultivated and wild Cordyceps sinensis. This invention, for the first time, uses lauric acid as a discriminant to differentiate between wild and cultivated Cordyceps sinensis, establishing a clear and intuitive standard for source identification. Specifically, "lauric acid ≥ 1.0%" is used to identify cultivated Cordyceps sinensis, and "lauric acid < 0.05%" is used to identify wild Cordyceps sinensis. For samples with lauric acid levels between 0.05% and 1.0%, the detection of caprylic acid and decanoic acid is further used for auxiliary determination: the detection of caprylic acid and / or decanoic acid indicates cultivated Cordyceps sinensis, while the absence of caprylic acid and / or decanoic acid indicates wild Cordyceps sinensis. This technical solution improves the ability to identify samples in the critical range through a "single-index initial screening + combined marker verification" approach.

Description

Technical Field

[0001] This invention belongs to the field of identification technology of genuine and counterfeit Chinese medicinal materials, specifically involving a detection method for distinguishing between artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis. Background Technology

[0002] Cordyceps sinensis typically refers to the complex formed when a fungus of the genus Cordyceps in the family Clavicipitaceae parasitizes the larvae of insects in the family Hepialidae. Due to its high nutritional, medicinal, and economic value, it has long been a focus of market attention. Wild Cordyceps sinensis is mainly distributed in the Qinghai-Tibet Plateau and surrounding high-altitude areas of my country. Its yield is relatively limited due to factors such as natural ecological conditions, host resources, harvesting season, and production environment. Furthermore, samples from different production areas and years exhibit certain differences in appearance and internal composition. With the continuous growth of market demand, various artificially cultivated related products and alternative Cordyceps sinensis products have emerged. These products often appear alongside wild Cordyceps sinensis during circulation, processing, and use.

[0003] In practical applications, wild and cultivated Cordyceps sinensis can be preliminarily distinguished based on appearance, stroma characteristics, insect morphology, and tissue structure when the samples are intact; however, this method is not accurate. Furthermore, when samples are crushed, cut, extracted, or processed into capsules, powders, medicinal slices, or other products, these morphological characteristics are often destroyed or difficult to preserve completely, increasing the difficulty of source identification. Especially in scenarios such as raw material acceptance, quality control, market supervision, and identification of high-value products, there is a real need to establish source identification methods suitable for crushed or processed samples.

[0004] In existing technologies, the identification and analysis of Cordyceps sinensis and related Cordyceps samples typically involve morphological observation, microscopic feature analysis, molecular biological detection, and chemical composition analysis. While morphological and microscopic identification methods are intuitive, their applicability to powdered, compound, or highly processed samples is limited. Molecular biological methods have high specificity, but they still have certain limitations in terms of sample DNA degradation, extraction efficiency, experimental condition control, and detection costs. Chemical composition analysis methods often use nucleosides, amino acids, polysaccharides, sterols, or other metabolites as detection targets for sample quality evaluation or auxiliary identification. However, due to the influence of culture conditions, production environment, harvesting, and processing methods on samples from different sources, some indicators may overlap or fluctuate between different samples, leaving room for further optimization when used alone for source identification.

[0005] Therefore, it is still necessary to develop a relatively simple and intuitive identification method to effectively distinguish between wild Cordyceps sinensis and artificially cultivated Cordyceps sinensis. Summary of the Invention

[0006] Firstly, a method for distinguishing between wild Cordyceps sinensis and artificially cultivated Cordyceps sinensis, characterized by comprising:

[0007] The lauric acid in the sample is tested. When the lauric acid content is greater than or equal to 1.0%, the sample is determined to be artificially cultivated Cordyceps sinensis; when the lauric acid content is less than 0.05%, the sample is determined to be wild Cordyceps sinensis.

[0008] Furthermore, when the lauric acid content in the sample to be tested is between 0.05% and 1.0%, octanoic acid and capric acid in the sample to be tested are detected, and a determination is made based on the detection results of octanoic acid and capric acid.

[0009] Furthermore, when caprylic acid and / or decanoic acid are detected in the sample to be tested, the sample to be tested is determined to be artificially cultivated Cordyceps sinensis; when caprylic acid and / or decanoic acid are not detected in the sample to be tested, the sample to be tested is determined to be wild Cordyceps sinensis.

[0010] Furthermore, the lauric acid, caprylic acid, and decanoic acid are detected by gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry.

[0011] Furthermore, the sample to be tested can be any one of the following: raw Cordyceps sinensis, powder, freeze-dried product, extract, medicinal slices, capsules, tablets, granules, food, or health product.

[0012] In a second aspect, a product for identifying wild Cordyceps sinensis and artificially cultivated Cordyceps sinensis, characterized in that the product comprises a reagent, standard or detection module for detecting lauric acid, and is configured to perform the method described in the first aspect.

[0013] Thirdly, the product also includes reagents, standards or detection modules for detecting octanoic acid and capric acid.

[0014] Furthermore, the product is a test kit, a test device, a test system, or data analysis software.

[0015] Fourthly, the application of lauric acid in distinguishing between wild and artificially cultivated Cordyceps sinensis. Specifically, when the lauric acid content is greater than or equal to 1.0%, it is used to identify artificially cultivated Cordyceps sinensis, and when the lauric acid content is less than 0.05%, it is used to identify wild Cordyceps sinensis.

[0016] Fifthly, the application of lauric acid combined with caprylic and decanoic acid in identifying the source of Cordyceps sinensis samples. Specifically, when the lauric acid content is between 0.05% and 1.0%, the detection of caprylic and / or decanoic acid is used to identify artificially cultivated Cordyceps sinensis, while the absence of caprylic and / or decanoic acid is used to identify wild Cordyceps sinensis.

[0017] Beneficial effects

[0018] 1. This invention is the first to use lauric acid as a distinguishing indicator between wild and artificially cultivated Cordyceps sinensis, establishing a clear and intuitive standard for source identification. Specifically, artificially cultivated Cordyceps sinensis is identified by "lauric acid ≥ 1.0%", while wild Cordyceps sinensis is identified by "lauric acid < 0.05%". The criteria are clear, the results are easy to interpret, and they are convenient for practical application.

[0019] 2. The identification method provided by this invention has high specificity and practicality. For samples with lauric acid content between 0.05% and 1.0%, the detection of caprylic acid and decanoic acid is further combined for auxiliary determination: the detection of caprylic acid and / or decanoic acid indicates artificially cultivated Cordyceps sinensis, while the absence of caprylic acid and / or decanoic acid indicates wild Cordyceps sinensis. This technical solution improves the ability to identify samples in the critical range through a "single-index initial screening + combined marker verification" approach.

[0020] 3. The markers used in the method described in this invention are stable and easy to detect. They can be detected using conventional detection methods such as gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry. The detection process is mature, easy to standardize, and convenient for promotion and application in enterprise quality control, testing institutions, and market supervision.

[0021] 4. The identification method described in this invention is not only applicable to raw Cordyceps sinensis samples, but also to the source identification of processed samples such as powders, freeze-dried products, extracts and related preparations. It has a wide range of sample applicability and can meet the needs of multiple scenarios such as raw material acceptance, production process control, finished product release and market sampling inspection. Detailed Implementation

[0022] The present application is described in detail below with reference to embodiments, but this does not imply any adverse limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific implementations of the present application without departing from the spirit and scope thereof.

[0023] Example 1: Screening of characteristic indicators to distinguish between wild and artificially cultivated Cordyceps sinensis

[0024] 1. Sample Source

[0025] Cordyceps sinensis samples from different sources were collected, all of which underwent initial morphological screening and were classified in conjunction with the sample source records. The samples included 200 wild Cordyceps sinensis samples and 200 artificially cultivated Cordyceps sinensis samples.

[0026] All samples were stored in a cool, dry place after collection. Before testing, all samples were crushed and sieved to obtain powder samples with uniform particle size.

[0027] 2. Experimental Methods

[0028] (1) Sample pretreatment

[0029] Accurately weigh approximately 0.20 g of Cordyceps sinensis powder sample and place it in a stoppered centrifuge tube. Add 5 mL of n-hexane-methanol mixed solution for lipid extraction and sonicate for 30 min. Collect the supernatant after centrifugation. Repeat the extraction once and combine the extracts. Concentrate the extract to near dryness by nitrogen blowing or vacuum, then redissolve it with an appropriate amount of n-hexane for subsequent methyl esterification treatment.

[0030] (2) Fatty acid methyl esterification

[0031] The extracted lipid sample was added to a methyl esterification reagent and reacted at 50°C for 30 min to convert fatty acids into the corresponding methyl esters. After the reaction was completed, the mixture was cooled and extracted with pure water and n-hexane. The organic layer was collected, dried over anhydrous sodium sulfate, and filtered to obtain the test solution.

[0032] (3) Preparation of reference solution

[0033] Take methyl octanoate, methyl decanoate, methyl laurate and other fatty acid methyl ester standards, prepare them into single standard stock solutions respectively, and mix them to prepare a mixed reference solution of appropriate concentration for retention time comparison, mass spectrometry fragment verification and quantitative calibration.

[0034] (4) Chromatographic conditions

[0035] The following chromatographic conditions can be used for detection:

[0036] Chromatographic column: capillary column for fatty acid methyl esters; carrier gas: high-purity helium or nitrogen; split ratio: 10:1; injection volume: 1.0 μL. The temperature program can be set as follows: initial temperature 60℃, hold for 3 min, increase to 180℃ at 10℃ / min, then increase to 250℃ at 5℃ / min and hold for 5 min.

[0037] (5) Component identification and relative content calculation

[0038] The retention time was compared with the standard, and the fatty acids, including caprylic acid, capric acid, and lauric acid, were qualitatively identified using mass spectrometry information. The relative percentage of each fatty acid in the total fatty acids was calculated using the peak area normalization method. The relative content of lauric acid was calculated using the following formula:

[0039] Relative lauric acid content (%) = (Lauryl acid peak area / Sum of total fatty acid peak areas) × 100%

[0040] Caprylic acid and decanoic acid were determined using a combination of "detected / not detected" and peak signal-to-noise ratio, with S / N≥3 as the detection limit.

[0041] 3. Experimental Results

[0042] 3.1 Overview of Fatty Acid Differences in the Screening Groups

[0043] The test results showed significant differences in the fatty acid composition between the two groups of samples. In the 200 wild-type samples, the relative content of lauric acid in 190 samples ranged from 0.000% to 0.049%, with an average of 0.018 ± 0.012%, and another 10 samples fell within the range of 0.055% to 0.88%. In the 200 artificial-type samples, the relative content of lauric acid in 192 samples ranged from 1.02% to 3.86%, with an average of 2.21 ± 0.71%, and another 8 samples fell within the range of 0.06% to 0.95%. The overall difference in the distribution of lauric acid between the two groups was statistically significant. Caprylic acid and capric acid were detected at extremely high rates in the artificial-type samples, while they were not detected in the wild-type samples. Caprylic acid and / or capric acid were detectable in the artificial-type samples, while they were virtually undetectable in the wild-type samples.

[0044] Table 1. Detection of Lauric Acid, Caprylic Acid, and Decanoic Acid

[0045]

[0046] 3.2 Threshold Determination

[0047] Analysis of the lauric acid content distribution in the screening groups showed that when the lauric acid content was <0.05%, all samples were wild Cordyceps sinensis; when the lauric acid content was ≥1.0%, all samples were artificially cultivated Cordyceps sinensis. Therefore, samples with lauric acid ≥1.0% could be directly identified as artificial samples, and samples with lauric acid <0.05% could be directly identified as wild samples. However, the data showed that a small number of samples (10 wild and 8 artificial) had lauric acid contents between 0.05% and 1.0%, which overlapped, and relying on a single indicator could not achieve 100% accurate differentiation. Further observation revealed that all 8 artificial samples in this range tested positive for caprylic acid and / or capric acid, while none of the 10 wild samples tested positive. Therefore, the auxiliary judgment rule is determined as follows: For samples with lauric acid between 0.05% and 1.0%, the detection of caprylic acid and decanoic acid is further combined for auxiliary judgment: when caprylic acid and / or decanoic acid are detected, it is judged as artificially cultivated cordyceps, and when caprylic acid and / or decanoic acid are not detected, it is judged as wild cordyceps.

[0048] Example 2: Auxiliary determination verification of samples with lauric acid in the range of 0.05% to 1.0%.

[0049] 1. Sample Source

[0050] To verify the auxiliary determination capability of this invention in samples with lauric acid in the critical range of 0.05% to 1.0%, 60 Cordyceps sinensis samples with initial lauric acid content in the range of 0.05% to 1.0% were screened from an independent sample library. These included 30 wild Cordyceps sinensis samples and 30 artificially cultivated Cordyceps sinensis samples. All samples underwent initial morphological screening and confirmation based on provenance records. The samples included some that, after being pulverized, had a lauric acid content falling within the aforementioned range.

[0051] All samples were stored in a cool, dry place after collection. Before testing, all samples were crushed and sieved to obtain powder samples with uniform particle size.

[0052] 2. Experimental Methods

[0053] The sample pretreatment, fatty acid methyl esterification, preparation of reference solution, chromatographic detection conditions, and relative content calculation method described in Example 1 were performed.

[0054] The relative content of lauric acid is calculated using the following formula:

[0055] Relative lauric acid content (%) = (Lauryl acid peak area / Sum of total fatty acid peak areas) × 100%

[0056] When the relative content of lauric acid in the sample is between 0.05% and 1.0%, octanoic acid and capric acid are further tested, and the following rules are used for auxiliary determination:

[0057] When caprylic acid and / or decanoic acid are detected, it is determined to be artificially cultivated cordyceps;

[0058] When caprylic acid and capric acid are not detected, it is identified as wild Cordyceps sinensis.

[0059] Among them, "detection" is judged by the characteristic peak signal-to-noise ratio S / N≥3.

[0060] 3. Experimental Results

[0061] Table 2 lists the auxiliary identification results for samples with lauric acid content in the range of 0.05% to 1.0%, and Table 3 shows the specific lauric acid content and the detection status of octanoic acid and decanoic acid in some samples. As can be seen from Table 2, when the relative lauric acid content is in the range of 0.05% to 1.0%, relying solely on lauric acid as a single indicator is insufficient to directly determine the sample source; further combining this with the detection status of octanoic acid and decanoic acid can significantly improve the identification ability of samples in the critical range.

[0062] Of the 60 samples examined in this embodiment:

[0063] Although the relative content of lauric acid in the 30 wild Cordyceps sinensis samples was in the range of 0.05% to 1.0%, caprylic acid and decanoic acid were not detected in any of them. Therefore, they were all correctly identified as wild Cordyceps sinensis. The relative content of lauric acid in the 30 artificially cultivated Cordyceps sinensis samples was also in the range of 0.05% to 1.0%, and caprylic acid and / or decanoic acid were detected in all of them. Therefore, they were all correctly identified as artificially cultivated Cordyceps sinensis.

[0064] The above results indicate that for samples with lauric acid in the critical range of 0.05%–1.0%, octanoic acid and decanoic acid can be used as effective auxiliary markers. This invention, through a two-step determination method of "lauric acid initial screening + octanoic acid / decanoic acid verification," can accurately distinguish samples in the critical range, thereby further improving the accuracy and practicality of identifying the origin of wild Cordyceps sinensis and artificially cultivated Cordyceps sinensis.

[0065] Table 2. Auxiliary judgment results for samples with lauric acid in the range of 0.05% to 1.0%.

[0066]

[0067] Table 3. Specific content of lauric acid and detection of caprylic and capric acids in some samples.

[0068]

[0069] Example 3 Independent Blind Sample Validation

[0070] 1. Sample Source

[0071] Cordyceps sinensis samples from different sources were collected, all of which underwent initial morphological screening and were classified in conjunction with the sample source records. The samples included 200 wild Cordyceps sinensis samples and 200 artificially cultivated Cordyceps sinensis samples.

[0072] All samples were stored in a cool, dry place after collection. Before testing, all samples were crushed and sieved to obtain powder samples with uniform particle size.

[0073] 2. Experimental Methods

[0074] (1) Sample pretreatment

[0075] Accurately weigh approximately 0.20 g of Cordyceps sinensis powder sample and place it in a stoppered centrifuge tube. Add 5 mL of n-hexane-methanol mixed solution for lipid extraction and sonicate for 30 min. Collect the supernatant after centrifugation. Repeat the extraction once and combine the extracts. Concentrate the extract to near dryness by nitrogen blowing or vacuum, then redissolve it with an appropriate amount of n-hexane for subsequent methyl esterification treatment.

[0076] (2) Fatty acid methyl esterification

[0077] The extracted lipid sample was added to a methyl esterification reagent and reacted at 50°C for 30 min to convert fatty acids into the corresponding methyl esters. After the reaction was completed, the mixture was cooled and extracted with pure water and n-hexane. The organic layer was collected, dried over anhydrous sodium sulfate, and filtered to obtain the test solution.

[0078] (3) Preparation of reference solution

[0079] Take methyl octanoate, methyl decanoate, methyl laurate and other fatty acid methyl ester standards, prepare them into single standard stock solutions respectively, and mix them to prepare a mixed reference solution of appropriate concentration for retention time comparison, mass spectrometry fragment verification and quantitative calibration.

[0080] (4) Chromatographic conditions

[0081] The following chromatographic conditions can be used for detection:

[0082] Chromatographic column: capillary column for fatty acid methyl esters; carrier gas: high-purity helium or nitrogen; split ratio: 10:1; injection volume: 1.0 μL. The temperature program can be set as follows: initial temperature 60℃, hold for 3 min, increase to 180℃ at 10℃ / min, then increase to 250℃ at 5℃ / min and hold for 5 min.

[0083] (5) Component identification and relative content calculation

[0084] The retention time was compared with the standard, and the fatty acids, including caprylic acid, capric acid, and lauric acid, were qualitatively identified using mass spectrometry information. The relative percentage of each fatty acid in the total fatty acids was calculated using the peak area normalization method. The relative content of lauric acid was calculated using the following formula:

[0085] Relative lauric acid content (%) = (Lauryl acid peak area / Sum of total fatty acid peak areas) × 100%

[0086] Caprylic acid and decanoic acid were determined using a combination of "detected / not detected" and peak signal-to-noise ratio, with S / N≥3 as the detection limit.

[0087] 3. Experimental Results

[0088] The validation group included 200 wild-type samples and 200 artificial samples from independent sources. The blind sample validation results are as follows:

[0089] Table 4 Blind Sample Validation Results

[0090]

[0091] Based on the results of the screening and validation groups, it was found that the relative content of lauric acid in the total fatty acids and the detection of characteristic chromatographic peaks of caprylic acid and decanoic acid in Cordyceps sinensis powder samples are of significant importance in distinguishing between wild and artificially cultivated Cordyceps sinensis. Using the method of this invention, artificial samples are identified as having "lauric acid ≥ 1.0%"; wild samples are identified as having "lauric acid < 0.05%". For samples with lauric acid content between 0.05% and 1.0%, the detection of caprylic acid and decanoic acid is further used for auxiliary determination: the detection of caprylic acid and / or decanoic acid indicates artificially cultivated Cordyceps sinensis, while the absence of caprylic acid and / or decanoic acid indicates wild Cordyceps sinensis.

[0092] It should be understood that the present invention is not limited to the specific embodiments described above, as variations can be made to the specific embodiments and they still fall within the scope of the appended claims.

Claims

1. A method for distinguishing between artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis, characterized in that, include: The lauric acid in the sample is tested. When the lauric acid content is greater than or equal to 1.0%, the sample is determined to be artificially cultivated Cordyceps sinensis; when the lauric acid content is less than 0.05%, the sample is determined to be wild Cordyceps sinensis.

2. The method according to claim 1, characterized in that, When the lauric acid content in the sample to be tested is between 0.05% and 1.0%, octanoic acid and capric acid in the sample to be tested are further detected, and the determination is made based on the detection results of octanoic acid and capric acid.

3. The method according to claim 2, characterized in that, When caprylic acid and / or decanoic acid are detected in the sample to be tested, the sample is determined to be artificially cultivated Cordyceps sinensis; when caprylic acid and / or decanoic acid are not detected in the sample to be tested, the sample is determined to be wild Cordyceps sinensis.

4. The method according to any one of claims 1 to 3, characterized in that, Lauric acid, caprylic acid, and decanoic acid were detected by gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry.

5. The method according to any one of claims 1 to 4, characterized in that, The sample to be tested is any one of the following: Cordyceps sinensis, raw herb, powder, freeze-dried product, extract, medicinal slices, capsules, tablets, granules, food, or health product.

6. A product for distinguishing between artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis, characterized in that, The product includes reagents, standards, or detection modules for detecting lauric acid and is configured to perform the method of claim 1.

7. The product according to claim 6, characterized in that, The product also includes reagents, standards, or detection modules for detecting octanoic acid and capric acid, and is configured to perform the method of claim 2 or 3.

8. The product according to claim 6 or 7, characterized in that, The product is a test kit, test device, test system, or data analysis software.

9. The application of lauric acid in differentiating wild and cultivated Cordyceps sinensis, among which, When the lauric acid content is greater than or equal to 1.0%, it is used to identify artificially cultivated cordyceps; when the lauric acid content is less than 0.05%, it is used to identify wild cordyceps.

10. The application of lauric acid combined with caprylic and capric acid in identifying the source of Cordyceps sinensis samples, among which, When the lauric acid content is between 0.05% and 1.0%, the presence of caprylic acid and / or decanoic acid indicates artificially cultivated Cordyceps sinensis, while the absence of caprylic acid and / or decanoic acid indicates wild Cordyceps sinensis.

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