Method for simultaneously detecting ergothioneine and adenosine and method for identifying cordyceps sinensis by using ergothioneine

Abstract

The invention relates to the technical field of traditional Chinese medicine detection and identification, in particular to a method for simultaneously detecting ergothioneine and adenosine and a method for identifying cordyceps sinensis by using ergothioneine. The method for identifying cordyceps sinensis provided by the invention comprises the following steps: identifying by taking the content of ergothioneine as an index, or identifying by taking the content of ergothioneine and the content of adenosine as indexes. The method for identifying the cordyceps sinensis can effectively identify whether the sample to be identified is the cordyceps sinensis or not, and is simple to operate, high in accuracy, strong in matrix interference resistance, good in repeatability, high in speed, safe, environment-friendly, low in cost and wide in counterfeit product distinguishing range. When the detection method provided by the invention is used for detecting the sample to be detected, the contents of the ergothioneine and the adenosine can be accurately detected at the same time, the component to be detected and an adjacent peak have excellent separation degree, and the method is high in sensitivity, simple to operate, high in accuracy, strong in matrix interference resistance, good in repeatability, high in speed, safe, environment-friendly and low in cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection and identification, and particularly relates to a method for simultaneously detecting ergothioneine and adenosine and a method for identifying Ophiocordyceps sinensis by using ergothioneine. BACKGROUND

[0002] Ophiocordyceps sinensis is a traditional Chinese medicinal material, and its medicinal value has been legally recorded in Chinese Pharmacopoeia. Modern pharmacological studies have shown that Ophiocordyceps sinensis contains bioactive components such as polysaccharides, adenosine, and gamma-aminobutyric acid, and has multiple pharmacological activities such as antioxidant stress, immune regulation, and neuroprotection, which has important application value in the field of dietary supplements, health foods, and drug development. With the rapid development of the health industry, the market demand for Ophiocordyceps sinensis continues to rise, but due to the scarcity of resources and the cost of collection, there is a phenomenon of using closely related species or artificial products such as Cordyceps militaris, Xinjiang Cordyceps, and fermented Cordyceps fungus powder to counterfeit the genuine product on the market, which seriously threatens the safety of consumers and the clinical efficacy.

[0003] The current quality control system of Ophiocordyceps sinensis mainly relies on the adenosine single indicator quantitative standard specified in Chinese Pharmacopoeia. However, adenosine, as a nucleoside basic metabolic product, is widely present in animals, plants, and microorganisms, and a single nucleoside indicator cannot reflect the unique quality attributes of Ophiocordyceps sinensis.

[0004] The existing detection technology of ergothioneine has significant limitations: the traditional spectroscopic method and thin-layer chromatography method have insufficient sensitivity for ergothioneine detection; the reported methods are only for edible fungi and fruiting bodies; and the reported analysis methods are mainly for detecting single components, and there is no analysis method suitable for simultaneous quantification of adenosine and ergothioneine in Cordyceps samples. It is particularly important to note that Ophiocordyceps sinensis and some of its counterfeit products are fungus-insect symbiotic organisms, and their matrix complexity is much higher than that of ordinary edible fungi. The existing technology cannot realize efficient co-extraction of two types of components with significant polarity differences, which makes it difficult to guarantee the accuracy and reproducibility of the detection results.

[0005] Therefore, there is still an urgent need for a rapid and accurate method for identifying Ophiocordyceps sinensis and a method for detecting ergothioneine content that is simple to operate, highly sensitive, accurate, and has high anti-interference ability. SUMMARY

[0006] To solve the above technical problems, the present application provides the following technical solutions.

[0007] In a first aspect, the present application provides a method for identifying Ophiocordyceps sinensis and its counterfeit products.

[0008] A method for identifying Cordyceps sinensis and its counterfeit products, comprising: identifying with the content of ergothioneine as an index, or identifying with the content of ergothioneine and the content of adenosine as indexes.

[0009] In some embodiments, the judgment method of the identification method comprises: calculating with the dry product of the sample to be identified, identifying as Cordyceps sinensis when the content of ergothioneine in the sample to be identified is ≥0.20 mg / g; identifying as a counterfeit product of Cordyceps sinensis when the content of ergothioneine in the sample to be identified is <0.20 mg / g.

[0010] In some embodiments, calculating with the dry product of the sample to be identified, identifying as a fermented fungus powder counterfeit product when the content of ergothioneine in the sample to be identified is <0.20 mg / g and the content of adenosine in the sample to be identified is >2.0 mg / g.

[0011] In some embodiments, calculating with the dry product of the sample to be identified, identifying as a non-fermented fungus powder counterfeit product when the content of ergothioneine in the sample to be identified is <0.20 mg / g and the content of adenosine in the sample to be identified is <2.0 mg / g.

[0012] In some embodiments, the fermented fungus powder counterfeit product comprises at least one of fermented Cordyceps sinensis fungus powder and Paecilomyces hepiali fungus powder.

[0013] In some embodiments, the non-fermented fungus powder counterfeit product comprises at least one of Cibotium barometz, Cordyceps militaris fruiting body, Cordyceps militaris whole plant, Ophiocordyceps xinhuijae, Ophiocordyceps daaii, White Caterpillar Fungus, and Herba Cordalines.

[0014] In some embodiments, the counterfeit product of Cordyceps sinensis comprises at least one of Cibotium barometz, Cordyceps militaris fruiting body, Cordyceps militaris whole plant, Ophiocordyceps xinhuijae, Ophiocordyceps daaii, White Caterpillar Fungus, Herba Cordalines, fermented Cordyceps sinensis fungus powder, or Paecilomyces hepiali fungus powder.

[0015] In some embodiments, the identification method further comprises: detecting the content of adenosine and the content of ergothioneine in the sample to be identified by high performance liquid chromatography, wherein the high performance liquid chromatography uses a hydrophilic interaction chromatographic column as a chromatographic column, and uses an aqueous solution and an acetonitrile solution as a mobile phase.

[0016] In some embodiments, the aqueous solution contains 0%vol-0.2%vol of formic acid. In some embodiments, the aqueous solution contains 0%vol, 0.05%vol, 0.1%vol, 0.15%vol, 0.2%vol of formic acid, or any value within any two value ranges thereof. In some preferred embodiments, the aqueous solution contains 0.1%vol of formic acid.

[0017] In some embodiments, the acetonitrile solution contains 0% vol to 0.2% vol or 0.1% vol of formic acid. In some embodiments, the acetonitrile solution contains 0% vol, 0.05% vol, 0.1% vol, 0.15% vol, 0.2% vol, or any value within a range between any two of the values. In some preferred embodiments, the acetonitrile solution contains 0.1% vol of formic acid.

[0018] In some embodiments, the hydrophilic interaction chromatography column comprises a Shimadzu Shim-pack Velox HILIC column.

[0019] In some embodiments, the hydrophilic interaction chromatography column has a column length of 50 mm to 250 mm. In some embodiments, the hydrophilic interaction chromatography column has a column length of 50 mm, 100 mm, 150 mm, 200 mm, 250 mm, or any value within a range between any two of the values. In some preferred embodiments, the hydrophilic interaction chromatography column has a column length of 50 mm to 100 mm.

[0020] In some embodiments, the hydrophilic interaction chromatography column has an inner diameter of 2 mm to 5 mm. In some embodiments, the hydrophilic interaction chromatography column has an inner diameter of 2 mm, 2.1 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 4.6 mm, 5 mm, or any value within a range between any two of the values. In some preferred embodiments, the hydrophilic interaction chromatography column has an inner diameter of 2.1 mm to 4.6 mm.

[0021] In some embodiments, the hydrophilic interaction chromatography column has a packing particle size of 1.8 pm to 5 pm. In some embodiments, the hydrophilic interaction chromatography column has a packing particle size of 1.8 pm, 2 pm, 2.5 pm, 2.7 pm, 3 pm, 3.5 pm, 4 pm, 4.5 pm, 5 pm, or any value within a range between any two of the values. In some preferred embodiments, the hydrophilic interaction chromatography column has a packing particle size of 2.7 pm.

[0022] In some embodiments, the hydrophilic interaction chromatography column is a Shimadzu Shim-pack Velox HILIC column having a column length of 50 mm, an inner diameter of 4.6 mm, and a packing particle size of 2.7 pm.

[0023] In some embodiments, the high performance liquid chromatography is performed using isocratic elution or gradient elution.

[0024] In some embodiments, isocratic elution includes isocratic elution of the aqueous solution and the acetonitrile solution at a volume ratio of 10:90 to 20:80. In some embodiments, isocratic elution includes isocratic elution of the aqueous solution and the acetonitrile solution at a volume ratio of 10:90, 15:85, 20:80, or any two of these values.

[0025] In some embodiments, the gradient elution includes eluting using the aqueous solution as mobile phase A and the acetonitrile solution as mobile phase B according to the following gradient procedure:

[0026] Time Mobile phase A Mobile phase B 0 min Any proportion in 90%~85% Any proportion in 10%~15% Any time point in 1.5 min~2.5 min Any proportion in 79%~82% Any proportion in 21%~18% Any time point in 4.5 min~10 min Any proportion in 79%~82% Any proportion in 21%~18%

[0027] or

[0028] Time Mobile phase A Mobile phase B 0 min 88％ 12％ Any time point in 1.5 min~2.5 min 80％ 20％ Any time point in 4.5 min~10 min 80％ 20％

[0029] or

[0030] Time Mobile phase A Mobile phase B 0 min 88％ 12％ 2 min 80％ 20％ 5 min 80％ 20％ .

[0031] In some embodiments, the identification method further includes the preparation of a test solution; the preparation of the test solution includes: mixing the powder of the sample to be identified with a dispersant, grinding to obtain a mixture, mixing the obtained mixture with a solvent, extracting, filtering, and obtaining the test solution.

[0032] In some embodiments, the dispersant includes at least one of diatomaceous earth and silica.

[0033] In some embodiments, the weight ratio of the powder to the dispersant of the sample to be identified is 1:0.5 to 1:2. In some embodiments, the weight ratio of the powder to the dispersant of the sample to be identified is 1:0.5, 1:1, 1:1.5, 1:2, or any value within any two of these ranges. In some preferred embodiments, the weight ratio of the powder to the dispersant of the sample to be identified is 1:1.

[0034] In some embodiments, the solvent comprises an aqueous methanol solution with a methanol concentration of 70% vol to 80% vol. In some embodiments, the solvent comprises an aqueous methanol solution with a methanol concentration of 70% vol, 71% vol, 72% vol, 73% vol, 74% vol, 75% vol, 76% vol, 77% vol, 78% vol, 79% vol, 80% vol, or any two of these values.

[0035] In some embodiments, 0.5 mL to 2 mL of the solvent is added for every 100 mg of the mixture. In some embodiments, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, or any two of the above values ​​of the solvent are added for every 100 mg of the mixture.

[0036] In some embodiments, the extraction includes vortex extraction.

[0037] In some embodiments, the vortex extraction time includes 10 seconds or more. In some embodiments, the vortex extraction time is 10 to 120 seconds. In some embodiments, the vortex extraction time is 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 120 seconds, or any value within any two of these ranges. In some preferred embodiments, the vortex extraction time includes 10 to 60 seconds. In some preferred embodiments, the vortex extraction time includes 20 to 40 seconds. In some more preferred embodiments, the vortex extraction time is 30 seconds.

[0038] In some embodiments, the identification method further includes injecting a reference solution, the reference solution comprising an ergothioneine reference solution, or a mixed reference solution of adenosine and ergothioneine; or the reference solution comprising an adenosine reference solution and an ergothioneine reference solution.

[0039] In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 3.88 μg / mL to 104 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 5.2 μg / mL to 103 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 3.88 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 25.9 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 104 μg / mL, or any value within any two of these ranges. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 20 μg / mL to 40 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 25 μg / mL to 40 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 20 μg / mL to 35 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 20 μg / mL to 30 μg / mL.

[0040] In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 1.49 μg / mL to 100 μg / mL. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 5.0 μg / mL to 99 μg / mL. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 1.49 μg / mL, 2 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 14.9 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, or any value within any two of these ranges. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 10 μg / mL to 30 μg / mL. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 10 μg / mL to 25 μg / mL.

[0041] In some embodiments, the method for preparing the reference solution includes: independently preparing each solution using an aqueous methanol solution with a methanol concentration selected from 75% vol to 85% vol as a diluent. In some embodiments, the method for preparing the reference solution includes: independently preparing each solution using an aqueous methanol solution with a methanol concentration selected from 75% vol, 76% vol, 77% vol, 78% vol, 79% vol, 80% vol, 81% vol, 82% vol, 83% vol, 84% vol, 85% vol, or any two of these values ​​as a diluent.

[0042] Secondly, the present invention provides a method for detecting the ergothionein content in Cordyceps sinensis.

[0043] A method for detecting ergothionein content in Cordyceps sinensis, the method comprising detecting Cordyceps sinensis using the high performance liquid chromatography method described in the first aspect of the identification method.

[0044] Thirdly, the present invention provides a method for detecting the content of ergothionein and adenosine in Cordyceps sinensis.

[0045] A method for detecting the content of ergothioneine and adenosine in Cordyceps sinensis, the method comprising detecting Cordyceps sinensis using the high performance liquid chromatography method described in the identification method of the first aspect.

[0046] In some embodiments, the detection method further includes injecting an ergothioneine reference solution and an adenosine reference solution, or the detection method further includes injecting a mixed reference solution of ergothioneine and adenosine.

[0047] In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 3.88 μg / mL to 104 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 5.2 μg / mL to 103 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 3.88 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 25.9 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 104 μg / mL, or any value within any two of these ranges. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 20 μg / mL to 40 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 25 μg / mL to 40 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 20 μg / mL to 35 μg / mL. In some embodiments, the concentration of ergothioneine reference standard in the ergothioneine reference solution or the mixed reference solution is independently selected from 20 μg / mL to 30 μg / mL.

[0048] In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 1.49 μg / mL to 100 μg / mL. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 5.0 μg / mL to 99 μg / mL. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 1.49 μg / mL, 2 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 14.9 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, or any value within any two of these ranges. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 10 μg / mL to 30 μg / mL. In some embodiments, the concentration of adenosine reference standard in the adenosine reference solution or the mixed reference solution is independently selected from 10 μg / mL to 25 μg / mL.

[0049] In some embodiments, the method for preparing the ergothioneine reference solution includes: preparing the ergothioneine reference solution using an aqueous methanol solution with a methanol concentration of 75% vol to 85% vol as a diluent. In some embodiments, the method for preparing the ergothioneine reference solution includes: preparing the ergothioneine reference solution using an aqueous methanol solution with a methanol concentration of 75% vol, 76% vol, 77% vol, 78% vol, 79% vol, 80% vol, 81% vol, 82% vol, 83% vol, 84% vol, 85% vol, or any two of these values ​​as a diluent.

[0050] In some embodiments, the method for preparing the adenosine reference solution includes: preparing the adenosine reference solution using an aqueous methanol solution with a methanol concentration of 75% vol to 85% vol as a diluent. In some embodiments, the method for preparing the adenosine reference solution includes: preparing the adenosine reference solution using an aqueous methanol solution with a methanol concentration of 75% vol, 76% vol, 77% vol, 78% vol, 79% vol, 80% vol, 81% vol, 82% vol, 83% vol, 84% vol, 85% vol, or any two of these values ​​as a diluent.

[0051] In some embodiments, the method for preparing the mixed reference solution includes: preparing the mixed reference solution using an aqueous methanol solution with a methanol concentration of 75% vol to 85% vol as a diluent. In some embodiments, the method for preparing the mixed reference solution includes: preparing the mixed reference solution using an aqueous methanol solution with a methanol concentration of 75% vol, 76% vol, 77% vol, 78% vol, 79% vol, 80% vol, 81% vol, 82% vol, 83% vol, 84% vol, 85% vol, or any two of these values ​​as a diluent.

[0052] Beneficial effects

[0053] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0054] (1) This invention creatively discovers that ergothionein can be used as a specific identification indicator to distinguish Cordyceps sinensis from its counterfeits, providing an original and novel technical concept for the identification of genuine and counterfeit Cordyceps sinensis.

[0055] (2) The identification method of Cordyceps sinensis and its counterfeits provided by the present invention can be used to identify the sample to be identified. It can effectively identify whether the sample to be identified is Cordyceps sinensis. It is simple to operate, highly accurate, strong against matrix interference, good repeatability, fast (short extraction time, short detection time for adenosine and ergothioneine content), safe and environmentally friendly, low cost, and can distinguish a wide range of counterfeits. It has excellent unexpected technical effects.

[0056] (3) Using the chromatographic conditions provided by the present invention to detect the sample, the contents of ergothioneine and adenosine can be detected simultaneously and accurately. Furthermore, ergothioneine and adenosine have excellent separation from their respective nearest neighbor peaks, resulting in excellent unexpected technical effects.

[0057] (4) The chromatographic conditions provided by this invention have high sensitivity, simple operation, high accuracy, strong resistance to matrix interference, good repeatability, fast speed (short elution time), safety and environmental protection, low cost, and excellent unexpected technical effects.

[0058] (5) In the preparation of the test solution, compared with neutral alumina, using diatomaceous earth and / or silica as dispersants is more conducive to improving the extraction rate and extraction efficiency of adenosine and ergothioneine, and has unexpected technical effects.

[0059] (6) Compared with other extraction solvents, using methanol aqueous solution as the extraction solvent is more conducive to improving the extraction rate and efficiency of adenosine and ergothioneine, and has unexpected technical effects.

[0060] (7) Compared with methanol solutions of other concentrations, using 70% vol to 80% vol methanol aqueous solution as the extraction solvent is more conducive to improving the extraction rate and efficiency of adenosine and ergothioneine, and has unexpected technical effects.

[0061] (8) Compared with methanol solutions of other concentrations, using 80% vol methanol aqueous solution as the extraction solvent is most beneficial to improving the extraction rate and efficiency of adenosine and ergothioneine, with unexpected technical effects.

[0062] (9) Compared with other chromatographic columns, the Shimadzu Shim-pack Velox HILIC column (50mm×4.6mm, 2.7μm) is more conducive to improving the separation degree of adenosine and the nearest neighbor peak and the separation degree of ergothioneine and the nearest neighbor peak, with unexpected technical effects.

[0063] (10) Compared with other mobile phase systems, the present invention preferably uses an aqueous solution containing formic acid and an acetonitrile solution containing formic acid as the mobile phase for separation, which is beneficial to improving the separation degree of adenosine and the most adjacent peak and the separation degree of ergothioneine and the most adjacent peak; more preferably, a mobile phase system of 0.1% vol formic acid aqueous solution and 0.1% vol formic acid acetonitrile solution is used as the mobile phase for separation, which is even more beneficial to improving the separation degree of adenosine and the most adjacent peak and the separation degree of ergothioneine and the most adjacent peak, and has unexpected technical effects.

[0064] Terminology Explanation

[0065] In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0066] The term “room temperature” refers to ambient temperature, which is between approximately 10°C and approximately 30°C, or approximately 20°C and approximately 30°C, or approximately 25°C.

[0067] The term "dried product" refers to a sample that has undergone appropriate drying treatment (such as natural drying, oven drying, or freeze drying), has a moisture content of no more than 15 wt%, which is significantly lower than that of fresh products, and is suitable for long-term preservation.

[0068] The term "Cordyceps militaris whole plant" refers to the complete complex of Cordyceps militaris, consisting of the pupa (insect body) and the stroma (grass part).

[0069] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0070] In the following content, all figures disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%, etc. Whenever a figure with a value of N is disclosed, any figure with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction. Attached Figure Description

[0071] Figure 1 The images show high-performance liquid chromatograms obtained from separation using different chromatographic columns in Example 2.

[0072] Figure 2 This is a high-performance liquid chromatogram of the separation performed using different mobile phases in Example 3.

[0073] Figure 3 The following are representative high-performance liquid chromatograms for the content determination in Example 4; wherein, Figure A is the high-performance liquid chromatogram of the blank solution, Figure B is the high-performance liquid chromatogram of the reference solution, and Figure C is the high-performance liquid chromatogram of the test solution of the cultivated Cordyceps sinensis sample. Detailed Implementation

[0074] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0075] The reagents or consumables used in this invention can be purchased from the market or prepared by the methods described in this invention.

[0076] Unless otherwise stated, the samples to be identified in the embodiments and comparative examples of this invention are dry products.

[0077] I. General Method of the Invention

[0078] Unless otherwise specified, the following methods shall be used:

[0079] 1. Solution preparation

[0080] (1) Preparation of mixed reference solution: Dilute adenosine reference standard and ergothioneine reference standard with 80% vol methanol aqueous solution to obtain a stock solution of adenosine reference standard containing approximately 1 mg / mL and a stock solution of ergothioneine reference standard containing approximately 1 mg / mL. Prepare two parallel solutions. After mixing the stock solutions of adenosine and ergothioneine, dilute with 80% vol methanol aqueous solution to prepare a mixed reference solution with the following concentrations: adenosine 15 μg / mL; ergothioneine 25 μg / mL.

[0081] (2) Preparation of test solution: Mix the powder of the sample to be identified with the dispersant (diatomaceous earth) at a weight ratio of 1:1, grind for 60 seconds to obtain mixed sample powder, weigh 100 mg of the obtained mixed sample powder, add 1 mL of extraction solvent (80% vol methanol aqueous solution) to obtain mixed solution A, vortex extract for 30 seconds, after standing, take the supernatant and filter it through a 0.22 μm organic filter membrane to obtain the test solution.

[0082] (3) Blank solution: 80% vol methanol aqueous solution.

[0083] 2. Chromatographic conditions

[0084] Instrument: High-performance liquid chromatograph;

[0085] Column: Shim-pack Velox HILIC (50mm × 4.6mm, 2.7μm);

[0086] Mobile phase: 0.1% vol formic acid aqueous solution (mobile phase A); acetonitrile solution containing 0.1% vol formic acid (mobile phase B).

[0087] Elution method: gradient elution, see Table 1 for specific elution procedures.

[0088] Table 1:

[0089]

[0090]

[0091] Flow rate: 1.2 mL / min;

[0092] Column temperature: 30℃;

[0093] Detection wavelength: 260nm;

[0094] Injection volume: 2 μL.

[0095] 3. Result Calculation

[0096] The calculation is performed using the external standard method, specifically as follows:

[0097] Content (mg / g) = (Sample A × Reference C × Sample V) / (Reference A × Sample M) × Conversion Factor

[0098] in,

[0099] Sample A: Peak area of ​​the analyte in the test solution.

[0100] A. Reference Standard: The peak area of ​​the analyte in the reference standard solution.

[0101] C. Reference Standard: The concentration of the reference standard corresponding to the analyte in the reference solution, in μg / mL.

[0102] V sample: The volume of solvent extracted from mixture A, in mL.

[0103] m sample: The actual mass of the sample to be identified, in mg, is the amount of 100 mg of the obtained mixed sample powder.

[0104] Conversion factor: The conversion factor for μg / mg to mg / g is 1.

[0105] Example 1: Investigation of Extraction Methods

[0106] This experiment investigated the effects of dispersant, mixing ratio, grinding time, extraction solvent concentration and volume, and vortex extraction time to establish an effective method for the simultaneous extraction of adenosine and ergothioneine. The results were evaluated using relative percentage content (%).

[0107] Relative percentage content (%) = (individual content / maximum content) × 100%.

[0108] Individual content: refers to the content detection value of each parameter when comparing different parameters of the same evaluation index.

[0109] Maximum content: refers to the highest detected content value when comparing different parameters of the same evaluation index.

[0110] The following extraction method was investigated using Cordyceps sinensis powder from the same batch as the sample to be identified.

[0111] 1. Investigation of dispersant types

[0112] The only difference from "I. General Method of the Invention" is the adjustment of the type of dispersant (see Table 2 for details). The detection results of adenosine and ergothioneine are shown in Table 2.

[0113] Table 2: Results of the investigation of dispersants

[0114] Dispersant Relative percentage content of adenosine (%) Relative percentage content of ergothioneine (%) Kieselguhr 95.4 100.0 Neutral alumina 100.0 35.4 Silica 90.2 98.8

[0115] Conclusion: As shown in Table 2, compared with neutral alumina, using diatomaceous earth and / or silica as dispersants is more conducive to improving the extraction rate and efficiency of adenosine and ergothioneine, with unexpected technical effects.

[0116] 2. Investigation of the mixing ratio of powder and dispersant in the sample to be identified

[0117] The only difference from "I. General Method of the Invention" is that the mixing ratio of the powder and dispersant of the sample to be identified is adjusted (see Table 3 for details). The detection results of adenosine and ergothioneine are shown in Table 3.

[0118] Table 3: Results of the investigation on the mixing ratio of powder and dispersant in the samples to be identified

[0119]

[0120] Conclusion: As shown in Table 3, the weight ratio of powder to dispersant of the sample to be identified is at least in the range of 1:0.5 (g:g) to 1:2 (g:g). The extraction rates and extraction efficiencies of adenosine and ergothioneine are relatively high. The preferred weight ratio of powder to dispersant of the sample to be identified is 1:1 (g:g).

[0121] 3. Examination of grinding time

[0122] The only difference from "I. General Method of the Invention" is that the grinding time of the powder of the sample to be identified after mixing with the dispersant is adjusted. The detection results of adenosine and ergothionein are shown in Table 4.

[0123] Table 4: Results of the study on grinding time

[0124] Grinding time Relative percentage content of adenosine (%) Relative percentage content of ergothioneine (%) 10 seconds 94.1 94.9 30 seconds 97.1 98.2 60 seconds 100.0 100.0 120 seconds 100.0 100.0

[0125] Conclusion: As shown in Table 4, the grinding time of the powder and dispersant of the sample to be identified is at least in the range of 10 seconds to 120 seconds, and the extraction rates and extraction efficiencies of adenosine and ergothioneine are relatively high.

[0126] 4. Investigation of extraction solvent concentration

[0127] The only difference from "I. General Method of the Invention" is the adjustment of the extraction solvent concentration. The detection results of adenosine and ergothionein are shown in Table 5.

[0128] Table 5: Results of the investigation on extraction solvents

[0129] Extraction solvent Relative percentage content of adenosine (%) Relative percentage content of ergothioneine (%) 70% vol methanol aqueous solution 96.9 96.6 80% vol methanol aqueous solution 100.0 100.0 90% vol methanol aqueous solution 90.3 79.5 100% vol methanol 65.1 46.5

[0130] in conclusion:

[0131] (1) Compared with methanol solutions of other concentrations, using 70% vol to 80% vol methanol aqueous solution as the extraction solvent is more conducive to improving the extraction rate and efficiency of adenosine and ergothioneine, and has unexpected technical effects.

[0132] (2) Compared with methanol solutions of other concentrations, using 80% vol methanol aqueous solution as the extraction solvent is most beneficial to improving the extraction rate and efficiency of adenosine and ergothioneine, with unexpected technical effects.

[0133] 5. Analysis of the volume of extraction solvent

[0134] The only difference from "I. General Method of the Invention" is the adjustment of the extraction solvent volume of the sample to be identified. The detection results of adenosine and ergothionein are shown in Table 6.

[0135] Table 6: Results of the investigation on the extraction solvent volume of the samples to be identified

[0136] Volume of extraction solvent of sample to be identified Relative percentage content of adenosine (%) Relative percentage content of ergothioneine (%) 0.5 mL 99.6 98.7 1 mL 100.0 100.0 2 mL 94.8 91.5

[0137] Conclusion: When the extraction solvent volume is in the range of 0.5 mL to 2 mL per 100 mg of the mixed powder of the sample to be identified, the extraction rate and extraction efficiency of adenosine and ergothioneine are relatively high. The preferred extraction solvent volume is 0.5 mL to 1 mL per 100 mg of the mixed powder of the sample to be identified, and the most preferred extraction solvent volume is 1 mL per 100 mg of the mixed powder of the sample to be identified.

[0138] 6. Vortex Time Examination

[0139] The only difference from "I. General Method of the Invention" is the adjustment of the vortex time of mixture A. The detection results of adenosine and ergothionein are shown in Table 7.

[0140] Table 7: Results of the investigation on vortex time

[0141] Vortex time Relative percentage content of adenosine (%) Relative percentage content of ergothioneine (%) 10 seconds 96.2 95.3 30 seconds 100.0 100.0 60 seconds 98.8 97.4

[0142] Conclusion: The extraction rates and efficiency of adenosine and ergothioneine are relatively high when the vortexing time of mixture A is in the range of 10 to 60 seconds, with 30 seconds being the optimal vortexing time.

[0143] Example 2: Column Chromatography

[0144] Take Cordyceps sinensis powder and prepare the test solution according to "I. General Method of this Invention" section "1. Solution Preparation". Then, examine the separation performance of different chromatographic columns (see Table 8 for column and elution conditions), and the results are shown in Table 8 and... Figure 1 .

[0145] Table 8: Chromatographic Column Investigation

[0146]

[0147] Conclusion: Compared with other chromatographic columns, the Shimadzu Shim-pack Velox HILIC column (50mm×4.6mm, 2.7μm) is more conducive to improving the separation of adenosine and its nearest neighbor peak, as well as the separation of ergothioneine and its nearest neighbor peak, with unexpected technical effects.

[0148] Example 3: Investigation of the mobile phase

[0149] Take Cordyceps sinensis powder and prepare the test solution according to "1. Solution Preparation" in "I. General Method of the Invention". Then, investigate the mobile phase under chromatographic conditions. The only difference between this and "2. Chromatographic Conditions" in "I. General Method of the Invention" is the different mobile phase (see Table 9). The results are shown in Table 9 and... Figure 2 .

[0150] Table 9: Investigation of the mobile phase

[0151]

[0152]

[0153] Conclusion: Mobile phase systems 1, 3, and 4 failed to effectively separate adenosine, with the resolution between adenosine and its adjacent peaks all <1.5. Mobile phase system 2 effectively separated adenosine and ergothioneine, with the resolution between both components and their adjacent peaks ≥4. Therefore, compared to other mobile phase systems, this invention preferentially uses mobile phase system 2 for separation, which is more conducive to improving the resolution between adenosine and its nearest peak, and the resolution between ergothioneine and its nearest peak, resulting in unexpected technical benefits.

[0154] Example 4: Content determination

[0155] 1. Procedure: Prepare the mixed reference solution according to the method described in "1. Solution Preparation" under "I. General Methods of the Invention". Take 8 batches of cultivated Cordyceps sinensis samples, 8 batches of wild Cordyceps sinensis samples, and 18 batches of non-Cordyceps sinensis samples, and prepare the test solution according to the method described in "1. Solution Preparation" under "I. General Methods of the Invention". Prepare blank solutions in parallel. Determine and calculate the content of adenosine and ergothioneine in the above-prepared test solutions according to the method described in "2. Chromatographic Conditions" under "I. General Methods of the Invention".

[0156] 2. Experimental Results:

[0157] See results Figure 3 See Table 10.

[0158] Table 10: Content detection results (n=2, mg / g dry product)

[0159]

[0160]

[0161] Note: "Not detected" means that the component to be detected did not show a peak or was below the detection limit.

[0162] in conclusion:

[0163] (1) The results show that the ergothioneine content in Cordyceps sinensis (breeded Cordyceps sinensis and / or wild Cordyceps sinensis) is >0.20 mg / g of dried product, while the ergothioneine content in non-Cordyceps sinensis is <0.20 mg / g of dried product. The ergothioneine content in the dried product of the sample to be identified can be detected to identify whether the sample to be identified is Cordyceps sinensis.

[0164] (2) The results show that the content of ergothioneine in fermented mycelium powder adulterants (such as fermented Cordyceps sinensis mycelium powder or at least one of Hepialus fasciatus mycelium powder) is <0.20 mg / g dry product, and the content of adenosine in them is >2.0 mg / g dry product. The presence of ergothioneine and adenosine in the dry product of the sample to be identified can be detected to determine whether the sample to be identified is a fermented mycelium powder adulterant (fermented Cordyceps sinensis mycelium powder or Hepialus fasciatus mycelium powder).

[0165] (3) The results show that the content of ergothioneine in non-fermented mycelium powder adulterants (such as: at least one of Cordyceps militaris, Cordyceps militaris fruiting body, Cordyceps militaris whole plant, Cordyceps sinensis, Cordyceps dauricum, Bombyx mori, and Stachys pilosa) is <0.20 mg / g dry product, and the content of adenosine in them is <2.0 mg / g dry product. The presence of ergothioneine and adenosine in the dry product of the sample to be identified can be detected to determine whether the sample to be identified is a non-fermented mycelium powder adulterant (such as: at least one of Cordyceps militaris, Cordyceps militaris fruiting body, Cordyceps militaris whole plant, Cordyceps sinensis, Cordyceps dauricum, Bombyx mori, and Stachys pilosa).

[0166] Example 5: Further Verification

[0167] To prove that ergothioneine is a marker for identifying Cordyceps sinensis and its adulterants, and to determine whether the content of ergothioneine in the dried sample can be used to identify whether the sample is Cordyceps sinensis, the experiment further tested 12 batches of blind samples.

[0168] Experimental Procedure: Twelve batches of blind samples were taken and named A to L. The test solution was prepared according to the steps in "Experimental Procedure" under "Example 4: Content Determination" and the ergothioneine content in the sample to be identified was determined.

[0169] Basis for judgment:

[0170] (1) When the ergothioneine content in the sample to be identified is >0.20 mg / g of dried product, the sample is determined to be Cordyceps sinensis; when the ergothioneine content in the sample to be identified is <0.20 mg / g of dried product, the sample is determined to be non-Cordyceps sinensis.

[0171] (2) When the content of ergothioneine in the sample to be identified is <0.20 mg / g dry product and the content of adenosine in it is >2.0 mg / g dry product, the sample to be identified is judged to be a counterfeit product of fermentation powder.

[0172] (3) When the content of ergothioneine in the sample to be identified is <0.20mg / g dry product and the content of adenosine in it is <2.0mg / g dry product, the sample to be identified is judged to be a non-fermentation powder counterfeit product.

[0173] Experimental results: The results are shown in Table 11.

[0174] Table 11: Ergothioneine content in blind samples (n=2, mg / g dry product)

[0175]

[0176]

[0177] Experimental conclusion: The judgment criteria are valid, and the established method can effectively identify Cordyceps sinensis and its counterfeits, and can also distinguish whether the counterfeits are fermented mycelium powder counterfeits or non-fermented mycelium powder counterfeits.

[0178] Example 6: Methodological Validation

[0179] Unless otherwise stated, the methodological validation shall be performed in accordance with the preparation and detection methods described in Section I, "General Method of the Invention".

[0180] 1. Specificity: The specificity of the method was assessed by analyzing adenosine and ergothioneine in the blank (80% methanol), mixed reference solution, and test solution to eliminate potential interferences. Results are shown in Example 4. Figure 3 There were no interfering peaks near the chromatographic peaks of adenosine and ergothioneine in the test solution, indicating that this method has good specificity.

[0181] 2. Linearity, limit of quantitation, and limit of detection:

[0182] (1) Preparation of reference stock solutions: Dilute adenosine reference standard and ergothioneine reference standard with 80% vol methanol aqueous solution to obtain a reference stock solution containing approximately 1 mg / mL adenosine reference standard and a reference stock solution containing approximately 1 mg / mL ergothioneine reference standard.

[0183] (2) Preparation of mixed reference solutions of different concentrations: After mixing the stock solutions of adenosine and ergothioneine, the solutions were diluted with 80% vol methanol aqueous solution to prepare a series of mixed reference solutions, namely:

[0184] 1. A mixed reference solution containing 4.95 μg / mL adenosine and 5.18 μg / mL ergothioneine;

[0185] 2. A mixed reference solution containing 9.90 μg / mL adenosine and 10.35 μg / mL ergothioneine;

[0186] 3. A mixed reference solution containing 19.81 μg / mL adenosine and 20.71 μg / mL ergothioneine;

[0187] 4. A mixed reference solution containing 49.51 μg / mL adenosine and 51.77 μg / mL ergothioneine;

[0188] 5. A mixed reference solution containing 79.22 μg / mL adenosine and 82.83 μg / mL ergothioneine;

[0189] 6. A mixed reference solution containing 99.03 μg / mL adenosine and 103.54 μg / mL ergothioneine.

[0190] (3) Investigation of solutions for limit of quantitation and limit of detection

[0191] Reference solutions containing 0.74 μg / mL to 2.23 μg / mL adenosine and 1.29 μg / mL to 3.88 μg / mL ergothioneine were prepared as limit of quantitation (LOQ) and limit of detection (LOD) solutions for adenosine and ergothioneine, respectively, to investigate the LOD and LOD concentrations of the corresponding components.

[0192] The series of mixed reference solutions were determined according to the method described in "2. Chromatographic Conditions" under "I. General Methods of the Invention". A standard curve was plotted with peak area as the ordinate (Y) and standard concentration as the abscissa (X), and the linear regression equation was obtained.

[0193] By injecting solutions of different concentrations with limits of quantitation and limits of detection, the concentrations corresponding to signal-to-noise ratios (S / N) of 10 and 3 were recorded and used as limits of quantitation and limits of detection, respectively.

[0194] 3. Precision and Repeatability: Precision was assessed by performing six parallel determinations of the mixed reference solution on the same day; repeatability was assessed by performing six parallel determinations of the test solution. The relative standard deviation (RSD, %) was used as the evaluation index for precision and repeatability. If RSD ≤ 3.00%, the method was considered to have good precision and repeatability.

[0195] 4. Accuracy: Prepare the test solution according to the steps in "1. Solution Preparation" of "I. General Method of the Invention". Add known amounts of adenosine and ergothioneine mixed reference standards to the solution (each added in six parallel increments). Then, determine the solution according to "2. Chromatographic Conditions" of "I. General Method of the Invention". The accuracy is evaluated by the recovery rate.

[0196] 5. Stability: The test solution was placed at room temperature and analyzed at 0, 2, 4, 6, 8, 10, 12, 16, 20 and 24 hours. If the RSD ≤ 3.00%, the solution was considered to have good stability.

[0197] Table 12. Methodological Validation Results

[0198]

[0199]

[0200] Table 13 Results of adenosine and ergothioneine recovery rates

[0201]

[0202] Experimental conclusion: This method exhibits good specificity, with no interference from the peaks of the analyte. Furthermore, linearity, precision, repeatability, accuracy, and stability all meet the requirements and comply with the stipulations of the Chinese Pharmacopoeia.

[0203] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for identifying Cordyceps sinensis and its counterfeit, characterized in that, Comprising: Identified with the content of ergothioneine as an index, or identified with the content of ergothioneine and the content of adenosine as an index.

2. The authentication method according to claim 1, wherein the judging method of the authentication method comprises: The content of ergothioneine in the sample to be identified is ≥ 0.20 mg / g when calculated based on the dry sample to be identified, and the sample to be identified is identified as Cordyceps sinensis; The content of ergothioneine in the sample to be identified is < 0.20 mg / g when calculated based on the dry sample to be identified, and the sample to be identified is identified as a counterfeit of Cordyceps sinensis; and / or The content of ergothioneine in the sample to be identified is < 0.20 mg / g, and the content of adenosine in the sample to be identified is > 2.0 mg / g when calculated based on the dry sample to be identified, and the sample to be identified is identified as a fermented fungus powder counterfeit; the content of ergothioneine in the sample to be identified is < 0.20 mg / g, and the content of adenosine in the sample to be identified is < 2.0 mg / g when calculated based on the dry sample to be identified, and the sample to be identified is identified as a non-fermented fungus powder counterfeit; Optionally, the fermented fungus powder counterfeit includes at least one of fermented Cordyceps sinensis fungus powder and Paecilomyces hepiali fungus powder. Optionally, the non-fermented fungus powder counterfeit includes at least one of Cordyceps sobria, Cordyceps militaris fruiting body, Cordyceps militaris whole plant, Cordyceps Xinjiangensis, Cordyceps Daii, White Caterpillar Fungus, and Herba Cossyepti.

3. The identification method according to any one of claims 1-2, wherein the counterfeit of Cordyceps sinensis includes at least one of Cordyceps sobria, Cordyceps militaris fruiting body, Cordyceps militaris whole plant, Cordyceps Xinjiangensis, Cordyceps Daii, White Caterpillar Fungus, Herba Cossyepti, fermented Cordyceps sinensis fungus powder, or Paecilomyces hepiali fungus powder.

4. The method of any one of claims 1 to 3, further comprising: The content of adenosine and the content of ergothioneine in the sample to be identified are detected by high performance liquid chromatography, and the high performance liquid chromatography uses a hydrophilic interaction chromatographic column as a chromatographic column and water solution and acetonitrile solution as mobile phases.

5. The identification method according to claim 4, wherein the water solution contains 0%vol-0.2%vol or 0.1%vol of formic acid; and / or The acetonitrile solution contains 0%vol-0.2%vol or 0.1%vol of formic acid; and / or The hydrophilic interaction chromatographic column includes a Shimadzu Shim-pack Velox HILIC chromatographic column; and / or The column length of the hydrophilic interaction chromatographic column is 50 mm-250 mm, preferably 50 mm-100 mm; and / or The inner diameter of the hydrophilic interaction chromatographic column is 2 mm-5 mm or 2.1 mm-4.6 mm; and / or The particle size of the filler of the hydrophilic interaction chromatographic column is 1.8 μm-5 μm or 2.7 μm.

6. The identification method according to any one of claims 4-5, wherein the high performance liquid chromatography is performed by isocratic elution or gradient elution; and / or The isocratic elution comprises: The water solution and the acetonitrile solution are isocratically eluted at a volume ratio of 10:90-20:80; And / or The gradient elution includes: using the water solution as mobile phase A and the acetonitrile solution as mobile phase B, and performing elution according to the following gradient program: Or Or 。 7. The method of discrimination according to any one of claims 4 to 6, further comprising the preparation of a test solution; the preparation of the test solution comprising: The powder of the sample to be identified is mixed with a dispersant, ground to obtain a mixture, mixed with a solvent, extracted, filtered to obtain the test solution. Optionally, the dispersing agent comprises at least one of diatomite and silicon dioxide; Optionally, the weight ratio of the powder of the sample to be identified to the dispersing agent is 1:0.5-1:2; Optionally, the solvent comprises a methanol aqueous solution with a methanol concentration of 70%vol-80%vol; Optionally, 0.5 mL-2 mL of the solvent is added per 100 mg of the mixture; Optionally, the extraction comprises vortex extraction; Optionally, the vortex extraction time comprises 10 seconds or more.

8. The identification method according to any one of claims 4-7, further comprising injecting a reference solution, wherein the reference solution comprises an ergothioneine reference solution, or a mixed reference solution of adenosine and ergothioneine, or the reference solution comprises an adenosine reference solution and an ergothioneine reference solution; Optionally, the concentration of ergothioneine in the ergothioneine reference solution or the mixed reference solution is independently selected from 3.88 μg / mL-104 μg / mL or 5.2 μg / mL-103 μg / mL, respectively; Optionally, the concentration of adenosine in the adenosine reference solution or the mixed reference solution is independently selected from 1.49 μg / mL-100 μg / mL or 5.0 μg / mL-99 μg / mL, respectively; Optionally, the method of preparing the control solution comprises: The dilution solvent is independently selected from a methanol aqueous solution with a methanol concentration of 75%vol-85%vol, respectively.

9. A method for detecting the ergothioneine content in Cordyceps sinensis, characterized in that, The detection method comprises detecting Cordyceps sinensis by the high performance liquid chromatography in the identification method according to any one of claims 4-8.

10. A method for detecting the content of ergothioneine and adenosine in Cordyceps sinensis, characterized in that, The detection method comprises detecting Cordyceps sinensis by the high performance liquid chromatography in the identification method according to any one of claims 4-8, The detection method further comprises injecting an ergothioneine reference solution and an adenosine reference solution, or the detection method further comprises injecting a mixed reference solution of ergothioneine and adenosine; Optionally, the concentration of ergothioneine in the ergothioneine reference solution or the mixed reference solution is independently selected from 3.88 μg / mL-104 μg / mL or 5.2 μg / mL-103 μg / mL, respectively; Optionally, the concentration of adenosine in the adenosine reference solution or the mixed reference solution is independently selected from 1.49 μg / mL-100 μg / mL or 5.0 μg / mL-99 μg / mL, respectively; Optionally, the preparation method of the ergothioneine reference solution comprises: preparing the ergothioneine reference solution by using a methanol aqueous solution with a methanol concentration of 75%vol-85%vol as a dilution solvent; Optionally, the preparation method of the adenosine reference solution comprises: preparing the adenosine reference solution by using a methanol aqueous solution with a methanol concentration of 75%vol-85%vol as a dilution solvent; Optionally, the preparation method of the mixed reference solution comprises: preparing the mixed reference solution by using a methanol aqueous solution with a methanol concentration of 75%vol-85%vol as a dilution solvent.

Images