Method for identifying cordyceps sinensis powder

Abstract

The invention relates to an identification method of cordyceps sinensis powder, and belongs to the technical field of pharmaceutical analysis. The identification method comprises the following steps: respectively mixing test sample powder with a buffer solution or an adenosine-containing buffer solution, standing, carrying out high-temperature treatment, cooling to room temperature, filtering, and taking a subsequent filtrate to respectively obtain test sample solutions 2 (mixing the test sample powder with the buffer solution, and carrying out high-temperature treatment); mixing the test sample powder with the adenosine-containing buffer solution to obtain the test sample solution 1 (mixing the test sample powder with the adenosine-containing buffer solution, and calculating the inosine increment of the test sample solution 1 compared with the test sample solution 2), and calculating the content of the inosine in the test sample solution 2 according to the content of the inosine in the test sample solution 1. And judging whether the test sample powder is the cordyceps sinensis powder or not according to the inosine increment of the test sample solution 1 compared with the test sample solution 2. The identification method provided by the invention is simple in equipment, low in cost, small in difficulty, high in accuracy and high in speed, and can be used for rapidly, effectively, simply and conveniently identifying whether the sample powder to be detected is the cordyceps sinensis powder or not at low cost.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for identifying cordyceps sinensis powder. Background Art

[0002] Cordyceps sinensis powder is a powder of the Cordyceps sinensis (BerK.) Sacc. parasitized on the larvae of the bat moth family insects, which is a complex of the fruiting body and the larval corpse formed by the Cordyceps sinensis (BerK.) Sacc. of the ergot family. It is a traditional precious tonic Chinese herbal medicine in my country and has high application value in the field of medical care. The Cordyceps sinensis fungus has a specific parasitic property and requires a special living environment. Domestic researchers have obtained fermented Cordyceps sinensis powder by separating natural Cordyceps strains and performing deep fermentation. The types of its main compounds are basically the same as those of Cordyceps sinensis, but the content of each compound is different, which also leads to a certain difference in its medicinal value from Cordyceps sinensis. In addition, since Cordyceps sinensis is expensive, the production cost of fermented Cordyceps sinensis powder is low, the resources are abundant, the price is low, and the powder morphology is extremely similar to Cordyceps sinensis powder, which is not easy to distinguish with the naked eye. If identification is performed, there are problems such as the need for relatively expensive equipment and reagents, high cost, complex operation, high difficulty, long identification time, or the need for skilled operation and rich identification experience.

[0003] Therefore, in order to prevent unscrupulous merchants from using fermented Cordyceps powder to impersonate Cordyceps powder, it is necessary to establish a fast, effective, convenient and low-cost method to identify Cordyceps powder. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions.

[0005] The present invention provides a method for identifying Cordyceps sinensis powder, which comprises:

[0006] (1) Preparation of test solution 1: Mix the test sample powder with a buffer solution containing adenosine, leave it for a certain period of time, then treat it at high temperature, cool it to room temperature, filter it, and take the filtrate to obtain the test solution 1;

[0007] (2) Preparation of test solution 2: Mix the test sample powder with the buffer solution, let it stand for a certain period of time, then treat it at high temperature, cool it to room temperature, filter it, and take the filtrate to obtain the test solution 2;

[0008] (3) Preparation of reference solution: dissolving inosine reference substance in water to obtain a reference solution;

[0009] (4) Inosine detection: Detect the inosine content in the test solution 1, the test solution 2 and the reference solution respectively, and calculate the increase in inosine in the test solution 1 compared with the test solution 2;

[0010] (5) Determination: Determine whether the test powder is Cordyceps sinensis powder based on the increase in inosine in test solution 1 compared to test solution 2.

[0011] In some embodiments, the pH of the adenosine-containing buffer in step (1) is 4-10, and the pH of the buffer in step (2) is the same as the pH of the adenosine-containing buffer in step (1). In some embodiments, the pH of the adenosine-containing buffer in step (1) is 4, 5, 6, 7, 8, 9 or 10, and the pH of the buffer in step (2) is the same as the pH of the adenosine-containing buffer in step (1).

[0012] In some embodiments, the method for preparing the adenosine-containing buffer comprises: dissolving adenosine with the buffer to obtain the adenosine-containing buffer.

[0013] In some embodiments, the buffer of step (2) and the adenosine-containing buffer of step (1) are Tris-maleic acid buffer.

[0014] In some embodiments, the concentration of adenosine in the adenosine-containing buffer in step (1) is 0.5 μmol / L-20 μmol / L. In some embodiments, the concentration of adenosine in the adenosine-containing buffer in step (1) is 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 11 μmol / L, 12 μmol / L, 13 μmol / L, 14 μmol / L, 15 μmol / L, 16 μmol / L, 17 μmol / L, 18 μmol / L, 19 μmol / L or 20 μmol / L. In some embodiments, the concentration of adenosine in the adenosine-containing buffer in step (1) is 2 μmol / L.

[0015] In some embodiments, in step (1), the sample powder is mixed with the adenosine-containing buffer and placed at 20°C-65°C for a certain period of time, then treated at high temperature, cooled to room temperature, filtered, and the filtrate is taken to obtain the sample solution 1. In some embodiments, in step (1), the sample powder is mixed with the adenosine-containing buffer and placed at 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C for a certain period of time, then treated at high temperature, cooled to room temperature, filtered, and the filtrate is taken to obtain the sample solution 1.

[0016] In some embodiments, in step (2), after the sample powder is mixed with the buffer solution, it is placed at 20°C-65°C for a certain period of time, then treated at high temperature, cooled to room temperature, filtered, and the filtrate is taken to obtain the sample solution 2; wherein the temperature of the placement in step (2) is the same as the temperature of the placement in step (1). In some embodiments, in step (2), after the sample powder is mixed with the buffer solution, it is placed at 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C for a certain period of time, then treated at high temperature, cooled to room temperature, filtered, and the filtrate is taken to obtain the sample solution 2; wherein the temperature of the placement in step (2) is the same as the temperature of the placement in step (1).

[0017] In some embodiments, the certain time in step (1) is at least 10 minutes, and the certain time in step (2) is the same as the certain time in step (1). In some embodiments, the certain time in step (1) is 10 minutes to 120 minutes, and the certain time in step (2) is the same as the certain time in step (1). In some embodiments, the certain time in step (1) is 10 minutes, 20 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes or 120 minutes, and the certain time in step (2) is the same as the certain time in step (1).

[0018] In some embodiments, the ratio of the weight of the sample powder to the volume of the adenosine-containing buffer in step (1) is 5 mg / ml-200 mg / ml; the ratio of the weight of the sample powder to the volume of the buffer in step (2) is the same as the ratio of the weight of the sample powder to the volume of the adenosine-containing buffer in step (1). In some embodiments, the ratio of the weight of the sample powder to the volume of the adenosine-containing buffer in the step (1) is 5 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml, 160 mg / ml, 170 mg / ml, 180 mg / ml, 190 mg / ml or 200 mg / ml; the ratio of the weight of the sample powder to the volume of the buffer in the step (2) is the same as the ratio of the weight of the sample powder to the volume of the adenosine-containing buffer in the step (1).

[0019] In some embodiments, the concentration of inosine in the reference solution obtained in step (3) is 0.1 mol / ml-2.5 μmol / ml. In some embodiments, the concentration of inosine in the reference solution obtained in step (3) is 0.2 μmol / ml-1.0 μmol / ml. In some embodiments, the concentration of inosine in the reference solution obtained in step (3) is 0.1 μmol / ml, 0.2 μmol / ml, 0.3 μmol / ml, 0.4 μmol / ml, 0.5 μmol / ml, 1.0 μmol / ml, 1.5 μmol / ml, 2.0 μmol / ml or 2.5 μmol / ml.

[0020] In some embodiments, the high temperature treatment is at 90° C.-100° C. In some embodiments, the high temperature treatment is at 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C. or 100° C.

[0021] In some embodiments, the high temperature treatment time is 5 min-15 min. In some embodiments, the high temperature treatment time is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0022] In some embodiments, the determination of step (5) is:

[0023] The test sample whose increase in inosine in test solution 1 was greater than 0.05 μmol / 100 mg powder compared to test solution 2 was Cordyceps sinensis powder;

[0024] The test samples whose increased amount of inosine in the test solution 1 is less than or equal to 0.05 μmol / 100 mg powder compared with the test solution 2 are non-Cordyceps sinensis powders.

[0025] In some embodiments, the Cordyceps sinensis powder is Cordyceps sinensis powder containing Cordyceps sinensis bodies.

[0026] In some embodiments, the step (4) uses high performance liquid chromatography to detect inosine.

[0027] In some embodiments, the chromatographic column of the HPLC method is hydrophilic octadecylsilane bonded silica gel.

[0028] In some embodiments, the mobile phase of the HPLC method includes mobile phase A and mobile phase B, the mobile phase A is a buffered saline solution, and the mobile phase B is a mixture of mobile phase A and methanol in a volume ratio of 85:15-90:10 or 90:10; the elution method of the HPLC method is gradient elution.

[0029] In some embodiments, the HPLC column is an Agilent Polaris C18-A column or an equivalent column thereof.

[0030] In some embodiments, the gradient elution is:

[0031]

[0032]

[0033] In some embodiments, the pH of the buffered saline solution is 5.3-6.3. In some embodiments, the pH of the buffered saline solution is 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 or 6.3.

[0034] In some embodiments, the buffered saline solution is phosphate buffered saline.

[0035] In some embodiments, the buffer salt in the buffered saline solution is at least one of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0036] In some embodiments, the concentration of the buffer salt in the buffered saline solution is 0.01 mol / L-0.10 mol / L or 0.05 mol / L.

[0037] In some embodiments, the method for preparing the buffered saline solution comprises: adjusting 0.05 mol / L potassium dihydrogen phosphate aqueous solution to a pH of 5.3-6.3 using 0.05 mol / L dipotassium hydrogen phosphate aqueous solution.

[0038] In some embodiments, the detection wavelength of the HPLC is 255nm-265nm. In some embodiments, the detection wavelength of the HPLC is 255nm, 256nm, 257nm, 258nm, 259nm, 260nm, 261nm, 262nm, 263nm, 264nm or 265nm.

[0039] In some embodiments, the flow rate of the HPLC is 0.5 ml / min-0.7 ml / min. In some embodiments, the flow rate of the HPLC is 0.6 ml / min.

[0040] In some embodiments, the column temperature of the HPLC is 20°C-30°C. In some embodiments, the column temperature of the HPLC is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0041] In some embodiments, the mobile phase B is a mixture of mobile phase A and methanol in a volume ratio of 85:15, 86:14, 87:13, 88:12, 89:11 or 90:10. In some embodiments, the mobile phase B is a mixture of mobile phase A and methanol in a volume ratio of 90:10.

[0042] In some embodiments, the inner diameter of the chromatographic column is 2.0 mm to 4.6 mm. In some embodiments, the inner diameter of the chromatographic column is 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm or 4.6 mm. In some embodiments, the inner diameter of the chromatographic column is 4.6 mm.

[0043] In some embodiments, the column length of the chromatographic column is 100 mm-250 mm. In some embodiments, the column length of the chromatographic column is 100 mm, 150 mm, 200 mm or 250 mm. In some embodiments, the column length of the chromatographic column is 150 mm.

[0044] In some embodiments, the filler particle size of the chromatographic column is 1.7 μm-5 μm. In some embodiments, the filler particle size of the chromatographic column is 1.7 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm or 5 μm.

[0045] In some embodiments, the inner diameter of the chromatographic column is 4.6 mm, the column length is 150 mm, and the filler particle size is 5 μm.

[0046] Beneficial Effects

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The identification method provided by the present invention has simple equipment, low cost, low difficulty, high accuracy and high speed, and can quickly, effectively, simply and at low cost identify whether the test sample powder is Cordyceps sinensis powder.

[0049] Terminology

[0050] In the description of the present invention, “plurality” means two or more than two, unless otherwise clearly and specifically defined.

[0051] The term "room temperature" means ambient temperature, which refers to a temperature between about 10°C and about 30°C, or between about 20°C and 30°C, or about 25°C.

[0052] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0053] The term "v / v" means volume ratio.

[0054] The term "% vol" means volume percentage.

[0055] In the following, all numbers disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number with a value of N is disclosed, any number 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 "+ / -" means plus or minus. DETAILED DESCRIPTION

[0056] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

[0057] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0058] 1. General detection method:

[0059] Unless otherwise specified, the following examples or comparative examples were tested according to the following general test method.

[0060] 1. Solution preparation

[0061] (1) Preparation of test solution 1: 100 mg of test sample powder was mixed with 1 ml of Tris-maleic acid buffer having a pH of 7 and containing 2 μmol / L adenosine (preparation method of Tris-maleic acid buffer having a pH of 7 and containing 2 μmol / L adenosine: adenosine was dissolved in Tris-maleic acid buffer having a pH of 7 to a concentration of 2 μmol / L.) (the Tris-maleic acid buffer may also be replaced with other buffers, such as phosphate buffer or Teorell-Stenhagen buffer), placed at 35° C. for 20 min, then subjected to high temperature treatment at 100° C. for 5 min, cooled to room temperature, filtered, and the filtrate was collected to obtain test solution 1;

[0062] (2) Preparation of test solution 2: 100 mg of the test sample powder was mixed with 1 ml of Tris-maleic acid buffer (the Tris-maleic acid buffer may also be replaced with other buffers, such as phosphate buffer or Teorell-Stenhagen buffer) at pH 7, placed at 35° C. for 20 min, then treated at 100° C. for 5 min, cooled to room temperature, filtered, and the filtrate was collected to obtain test solution 2;

[0063] (3) Preparation of reference solution: Dissolve the inosine reference substance in water to obtain a reference solution with an inosine concentration of 0.5 μmol / ml;

[0064] (4) Inosine detection: Detect the inosine content in the test solution 1, the test solution 2 and the reference solution respectively, and calculate the increase in inosine in the test solution 1 compared with the test solution 2;

[0065] (5) Determination: Determine whether the test powder is Cordyceps sinensis powder based on the increase in inosine in test solution 1 compared with test solution 2;

[0066] The judgment criteria are: compared with the test solution 2, the test sample whose increased amount of inosine in the test solution 1 is greater than 0.05 μmol / 100 mg powder is Cordyceps sinensis powder; compared with the test solution 2, the test sample whose increased amount of inosine in the test solution 1 is less than or equal to 0.05 μmol / 100 mg powder is non-Cordyceps sinensis powder.

[0067] 2. Inosine detection method

[0068] The detection was performed by high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography were as follows:

[0069] Chromatographic column: Agilent Polaris C18-A column (4.6 mm × 150 mm, 5 μm);

[0070] Mobile phase: including mobile phase A and mobile phase B,

[0071] Mobile phase A: dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer aqueous solution with a pH of 5.8 (phosphate concentration is 0.05 mol / L) (0.05 mol / L potassium dihydrogen phosphate aqueous solution is used to adjust the pH of 0.05 mol / L potassium dihydrogen phosphate aqueous solution to 5.8 to obtain the mobile phase A);

[0072] Mobile phase B: a mixture of mobile phase A and methanol in a volume ratio of 90:10;

[0073] Elution mode: gradient elution, the elution procedure of the gradient elution is as follows:

[0074] time Mobile phase A (%) Mobile phase B (%) 0 100 0 14min 100 0 25min 75 25 35min 10 90 40min 0 100 55min 0 100 ;

[0075] Column temperature: 25°C;

[0076] Flow rate: 0.6ml / min;

[0077] Detection wavelength: 260nm;

[0078] Injection volume: 5 μL.

[0079] 3. Calculation method of inosine increase

[0080] The formula for calculating the increase in inosine in test solution 1 compared to test solution 2 is:

[0081]

[0082] Where:

[0083] G is the increase in inosine in test solution 1 compared to test solution 2, in μmol / 100 mg powder;

[0084] c x is the concentration of inosine in the test solution 1, in μmol / mL;

[0085] c0 is the concentration of inosine in test solution 2, in μmol / mL;

[0086] V is the volume of the extraction solution, in mL;

[0087] m is the sample weight, in mg;

[0088] 100 is the conversion factor.

[0089] Example 1: Investigation of extraction conditions

[0090] 1. Investigation of extraction solvent pH

[0091] With reference to the “Universal Detection Method”, the effects of different pH values ​​of Tris-maleic acid buffer and Tris-maleic acid buffer containing 2 μmol / L adenosine in the preparation of the test solutions (test solution 1 and test solution 2) on the results were investigated; the pH values ​​of the Tris-maleic acid buffer containing 2 μmol / L adenosine in test solution 1 and the Tris-maleic acid buffer in test solution 2 were adjusted according to Table 1, and the remaining operations were the same as those in the “Universal Detection Method”. The test powder was Cordyceps powder or the contents of Bailing Capsules (the contents of Bailing Capsules were Cordyceps mycelium, and the contents of Bailing Capsules described below were the same and will not be repeated). The results are shown in Table 1.

[0092] Table 1: Results of investigation of different pH values ​​of extraction solvents

[0093]

[0094] Result analysis:

[0095] Under the conditions of pH = 2 and pH = 11, the increase in inosine in both samples was < 0.05 μmol / 100 mg powder, and the two types of samples could not be distinguished.

[0096] Under the conditions of pH = 4, pH = 7 and pH = 10, the increase in inosine in Cordyceps sinensis powder was > 0.05 μmol / 100 mg powder, and the increase in inosine in the contents of Bailing capsules was 0, which can normally distinguish the two types of samples. Therefore, the pH range of Tris-maleic acid buffer in the test solution was determined to be 4-10.

[0097] 2. Placement temperature inspection

[0098] On the basis of referring to the “General Detection Method”, the influence of the storage temperature in the preparation of the test solution on the result was investigated. The storage temperature in the preparation of the test solution 1 and the test solution 2 was adjusted according to the temperature in Table 2. The rest of the operations were the same as the “General Detection Method”. The test powder was Cordyceps powder or the contents of Bailing Capsules. The results are shown in Table 2.

[0099] Table 2: Investigation results of different placement temperatures

[0100]

[0101] Result analysis:

[0102] Under the conditions of 5°C and 80°C, the increase in inosine in both samples was <0.05 μmol / 100 mg powder, and the two types of samples could not be distinguished.

[0103] Under the conditions of 20°C and 65°C, the increase in inosine in Cordyceps sinensis powder is greater than 0.05 μmol / 100 mg powder, and the increase in inosine in the contents of Bailing capsules is 0, which can normally distinguish the two types of samples. Therefore, the water bath temperature range of the technology of the present invention is determined to be 20°C-65°C (the results of the placement temperature of 35°C can be seen in the results of pH=7 in "Investigation of pH of the extraction solvent").

[0104] 3. Investigation of material-liquid ratio

[0105] Based on the reference of “General Detection Method”, the influence of the ratio of the weight of the test sample powder in the test solution 1 to the volume of Tris-maleic acid buffer with pH 7 and 2 μmol / L adenosine (i.e., the solid-liquid ratio) and the ratio of the weight of the test sample powder to the volume of Tris-maleic acid buffer with pH 7 (i.e., the solid-liquid ratio) in the preparation of the test solution 2 on the results were investigated. The solid-liquid ratio in the preparation of the test solution 1 and the test solution 2 was adjusted according to Table 3. The rest of the operations were the same as those in “General Detection Method”. The test sample powder was Cordyceps powder or the contents of Bailing Capsules. The results are shown in Table 3.

[0106] Table 3: Investigation results of different material-liquid ratios

[0107]

[0108] Result analysis:

[0109] Under the four different material-liquid ratio conditions, the amount of inosine added in Cordyceps sinensis powder was all greater than 0.05 μmol / 100 mg powder, and the amount of inosine added in Bailing capsules was all 0, which can distinguish the two types of samples. It can be seen that accurate identification can be performed at least within the material-liquid ratio range of (0.1 g: 0.5 ml)-(0.1 g: 20.0 ml).

[0110] 4. Adenosine concentration study

[0111] On the basis of referring to the “Universal Detection Method”, the influence of different adenosine concentrations in the preparation of the test solution 1 on the results was investigated. The adenosine concentration of the test solution 1 was adjusted according to Table 4. The rest of the operations were the same as the “Universal Detection Method”. The test powder was Cordyceps powder or the contents of Bailing Capsules. The results are shown in Table 4.

[0112] Table 4: Results of investigation on adenosine concentration

[0113]

[0114] Result analysis:

[0115] When the adenosine concentration in the adenosine solution was 0.1 μmol / mL, the increase in inosine in the two samples was less than 0.05 μmol / 100 mg powder, and the two types of samples could not be distinguished normally.

[0116] When the adenosine concentration in the adenosine solution reaches above 0.5 μmol / mL, the increase in inosine in the Cordyceps powder is greater than 0.05 μmol / 100 mg powder, and the increase in inosine in the contents of Bailing capsules is 0, which can normally distinguish the two types of samples. Therefore, the adenosine concentration range in the adenosine concentration of the technology of the present invention is determined to be ≥0.5 μmol / mL.

[0117] 5. Placement time inspection

[0118] Based on the reference of “General Detection Method”, the influence of the standing time in the preparation of test solutions (test solution 1 and test solution 2) on the results was investigated. The standing time in the preparation of test solutions was adjusted from 20 min to the time in Table 5. The rest of the operations were the same as those in “General Detection Method”. The test powder was Cordyceps powder or the contents of Bailing Capsules. The results are shown in Table 5.

[0119] Table 5: Results of the investigation on placement time

[0120]

[0121] Result analysis:

[0122] When the water bath time was 5 min, the increase in inosine in the two samples was ≤0.05 μmol / 100 mg powder, and the two types of samples could not be distinguished normally.

[0123] When the water bath time reaches more than 10 minutes, the increase in inosine in Cordyceps powder is greater than 0.05 μmol / 100 mg powder, and the increase in inosine in the contents of Bailing capsules is less than 0.05 μmol / 100 mg powder, which can normally distinguish the two types of samples. Therefore, the water bath time range of the technology of the present invention is determined to be ≥10 minutes.

[0124] Example 2: Identification of Cordyceps powder products and non-Cordyceps powder products

[0125] Cordyceps sinensis powder products (wild products from Tibet, Qinghai, Sichuan, and Hubei) and Cordyceps mycelium products (contents of Jinshuibao capsules, Bailing capsules, and Ningxinbao capsules) were taken as test powders respectively, and the step (1) of "mixing 100 mg of the test powder with 1 ml of Tris-maleic acid buffer having a pH of 7 and containing 2 μmol / L adenosine, and leaving it at a temperature of 35°C for 20 min" was modified to "mixing 100 mg of the test powder with 1.5 ml of Tris-maleic acid buffer having a pH of 7 and containing 2 μmol / L adenosine, and leaving it at a temperature of 50°C for 90 min", and the step (2) of "mixing 100 mg of the test powder with 1 ml of Tris-maleic acid buffer having a pH of 7 and leaving it at a temperature of 35°C for 20 min" was modified to "mixing 100 mg of the test powder with 1.5 ml of The samples were mixed with Tris-maleic acid buffer at pH 7 and placed at 50°C for 90 minutes. The remaining operations were the same as those in the “General Detection Method”. The results are shown in Tables 6 and 7.

[0126] Table 6: Increased amount of inosine in Cordyceps sinensis powder products (μmol / 100 mg powder)

[0127]

[0128] Table 7: Increased amount of inosine in Cordyceps mycelium products (μmol / 100 mg powder)

[0129]

[0130] in conclusion:

[0131] The increased amount of inosine in Cordyceps sinensis powder products was greater than 0.05 μmol / 100 mg powder, and the increased amount of inosine in Cordyceps mycelium products was less than 0.05 μmol / 100 mg powder. The results showed that the patented method can effectively distinguish Cordyceps sinensis powder products from Cordyceps mycelium products.

[0132] Example 3: Identification of Cordyceps Sinensis Powder Products and Non-Cordyceps Sinensis Powder Products

[0133] Take appropriate amounts of Cordyceps sinensis (wild products from Tibet, Qinghai, Sichuan, and Hubei) and other Cordyceps medicinal materials (Cordyceps gunnii and Cordyceps cicadae), separate the insect body and the stroma, and make insect body powder products and stroma powder products as test sample powders, respectively. Two batches of each type of sample are tested, and the "Tris-maleic acid buffer with a pH of 7 and containing 2 μmol / L adenosine" in step (1) is modified to "Tris-maleic acid buffer with a pH of 9 and containing 20 μmol / L adenosine", and the "Tris-maleic acid buffer with a pH of 7" in step (2) is modified to "Tris-maleic acid buffer with a pH of 9". The remaining operations are the same as those in the "General Detection Method", and then the test is carried out. The results are shown in Tables 8 and 9.

[0134] Table 8: Increased amount of inosine in Cordyceps sinensis powder products and stroma powder products (μmol / 100mg powder)

[0135]

[0136]

[0137] Table 9: Increasing amount of inosine in other Cordyceps powder products and stroma powder products (μmol / 100mg powder)

[0138]

[0139] in conclusion:

[0140] (1) The increased amount of inosine in Cordyceps sinensis body powder products is greater than 0.05 μmol / 100 mg powder, and the increased amount of inosine in Cordyceps sinensis stroma powder products is less than 0.05 μmol / 100 mg powder. The results show that the identification method of the present invention can effectively distinguish Cordyceps sinensis body powder products from stroma powder products.

[0141] (2) The increased amounts of inosine in the Cordyceps sinensis powder and stroma powder were both less than 0.05 μmol / 100 mg powder. The results showed that the identification method of the present invention can effectively distinguish Cordyceps sinensis powder from non-Cordyceps sinensis powder.

[0142] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for identifying Cordyceps sinensis powder, It is characterized in that include: (1) Preparation of test solution 1: Mix the test sample powder with a buffer solution containing adenosine, leave it for a certain period of time, then treat it at high temperature, cool it to room temperature, filter it, and take the filtrate to obtain the test solution 1; (2) Preparation of test solution 2: Mix the test sample powder with the buffer solution, let it stand for a certain period of time, then treat it at high temperature, cool it to room temperature, filter it, and take the filtrate to obtain the test solution 2; (3) Preparation of reference solution: dissolving inosine reference substance in water to obtain a reference solution; (4) Inosine detection: Detect the inosine content in the test solution 1, the test solution 2 and the reference solution respectively, and calculate the increase in inosine in the test solution 1 compared with the test solution 2; (5) Determination: Determine whether the test powder is Cordyceps sinensis powder based on the increase in inosine in test solution 1 compared to test solution 2.

2. The method according to claim 1, wherein the pH of the adenosine-containing buffer in step (1) is 4-10, and the pH of the buffer in step (2) is the same as the pH of the adenosine-containing buffer in step (1). ； and / or Optionally, the method for preparing the adenosine-containing buffer comprises: dissolving adenosine with the buffer to obtain the adenosine-containing buffer; and / or Optionally, the buffer of the buffer in step (2) and the adenosine-containing buffer in step (1) are at least one of Tris-maleic acid buffer, phosphate buffer, and Teorell-Stenhagen buffer; and / or Optionally, the concentration of adenosine in the adenosine-containing buffer in step (1) is 0.5 μmol / L-20 μmol / L or 2 μmol / L.

3. The method according to any one of claims 1 to 2, wherein in step (1), the sample powder is mixed with a buffer containing adenosine and placed at 20°C-65°C for a certain period of time, then subjected to high temperature treatment, cooled to room temperature, filtered, and a filtrate is obtained to obtain a sample solution 1; and / or In the step (2), the sample powder is mixed with the buffer solution and placed at 20°C-65°C for a certain period of time, then treated at high temperature, cooled to room temperature, filtered, and the filtrate is taken to obtain a sample solution 2; wherein the placement temperature in the step (2) is the same as the placement temperature in the step (1).

4. The method according to any one of claims 1 to 3, wherein the certain time in step (1) is at least 10 minutes or 10 minutes to 120 minutes, and the certain time in step (2) is the same as the certain time in step (1).

5. The method according to any one of claims 1 to 4, wherein the ratio of the weight of the sample powder to the volume of the adenosine-containing buffer in step (1) is 5 mg / ml-200 mg / ml or 100 mg / ml; the ratio of the weight of the sample powder to the volume of the buffer in step (2) is the same as the ratio of the weight of the sample powder to the volume of the adenosine-containing buffer in step (1); and / or The inosine concentration in the reference solution obtained in step (3) is 0.1 μmol / ml-2.5 μmol / ml or 0.5 μmol / ml.

6. The method according to any one of claims 1 to 4, wherein the high temperature treatment is a treatment at 90°C-100°C; and / or The high temperature treatment time is 5 min-15 min.

7. According to the method according to any one of claims 1 to 6, the determination of step (5) is: The test sample whose increase in inosine in test solution 1 was greater than 0.05 μmol / 100 mg powder compared to test solution 2 was Cordyceps sinensis powder; Test samples with an increase in inosine in test solution 1 of less than or equal to 0.05 μmol / 100 mg powder compared to test solution 2 are non-cordyceps powders; Optionally, the cordyceps sinensis powder is cordyceps sinensis powder containing cordyceps sinensis bodies.

8. The method according to any one of claims 1 to 7, wherein the step (4) uses high performance liquid chromatography to detect inosine; Optionally, the chromatographic column of the high performance liquid chromatography is a hydrophilic octadecylsilane bonded silica gel; Optionally, the mobile phase of the HPLC method includes mobile phase A and mobile phase B, the mobile phase A is a buffered saline solution, and the mobile phase B is a mixture of mobile phase A and methanol in a volume ratio of 85:15-90:10; the elution mode of the HPLC method is gradient elution.

9. The method according to claim 8, wherein the chromatographic column of the high performance liquid chromatography is an Agilent Polaris C18-A chromatographic column or an equivalent chromatographic column; Optionally, the gradient elution is: ； Optionally, the pH of the buffered saline solution is 5.3-6.3 or is 5.8; Optionally, the buffered saline solution is a phosphate buffer; Optionally, the buffer salt in the buffered saline solution is at least one of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate; Optionally, the concentration of the buffer salt in the buffered saline solution is 0.01 mol / L-0.10 mol / L or 0.05 mol / L; Optionally, the detection wavelength of the high performance liquid chromatography is 255nm-265nm or 260nm; Optionally, the flow rate of the high performance liquid chromatography is 0.5 ml / min-0.7 ml / min or 0.6 ml / min; Optionally, the column temperature of the HPLC is 20°C-30°C or 25°C; Optionally, the mobile phase B is a mixture of mobile phase A and methanol in a volume ratio of 85:15-90:10 or 90:

10.

10. The method according to any one of claims 8 to 9, wherein the inner diameter of the chromatographic column is 2.0 mm to 4.6 mm or is 4.6 mm; and / or The column length of the chromatographic column is 100 mm-250 mm or 150 mm; and / or The filler particle size of the chromatographic column is 1.7 μm-5 μm; and / or The inner diameter of the chromatographic column is 4.6 mm, the column length is 150 mm, and the filler particle size is 5 μm.

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