Polysaccharide, cordyceps sinensis extract as well as preparation method and application of polysaccharide and cordyceps sinensis extract

Abstract

The invention relates to polysaccharide, a cordyceps sinensis extract as well as a preparation method and application thereof, and relates to the field of traditional Chinese medicinal material extracts. The polysaccharide has excellent immunoregulation and / or antioxidant activity, and can be used for identifying authenticity and specifications of cordyceps sinensis.

Description

[0001] Priority information

[0002] This application claims priority and benefits of patent application 202410161554.3 filed with the State Intellectual Property Office of China on February 4, 2024, and the entire text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of Chinese medicinal material extracts, and in particular to a polysaccharide, a cordyceps sinensis extract, and a preparation method and application thereof. Background Art

[0004] Cordyceps sinensis is a dried complex of the fruiting body and larval corpse of the Cordyceps sinensis (Berk.) Sacc., a fungus of the ergot family, which is parasitic on the larvae of insects of the bat moth family. It is mainly distributed in high-altitude areas such as Tibet, Qinghai, Gansu, Yunnan, and Sichuan in my country.

[0005] Polysaccharides are a class of high molecular weight compounds that are widely present in organisms. However, due to the influence of proteins, pigments, etc. during the extraction of polysaccharides, and the characteristics of polysaccharides themselves, such as poor stability, high polarity, difficulty in volatility, and high molecular weight, there are many problems in their extraction and purification.

[0006] The literature (Fang Qiuyue, Wang Junqiao, Chen Shuping, etc. Mechanism of action of different components of natural Cordyceps sinensis on intestinal damage repair in mice [J]. Journal of Nanchang University (Science Edition), 2021, 45(03): 251-256. DOI: 10.13764 / j.cnki.ncdl.2021.03.009) discloses a method for preparing different components of Cordyceps sinensis and the pharmacological activities of each component, including the preparation and activity of crude polysaccharides and pure polysaccharides of Cordyceps sinensis. However, the study found that although the crude polysaccharide and pure polysaccharide have certain immune activity, the activity is still not high.

[0007] Therefore, a Cordyceps sinensis polysaccharide having excellent pharmacological activity and a preparation method thereof are still desired. Summary of the invention

[0008] The purpose of the present invention is to provide a polysaccharide with excellent immunomodulatory and / or antioxidant activity, a cordyceps sinensis extract, and a preparation method and application thereof.

[0009] In a first aspect, the present invention provides a polysaccharide.

[0010] A polysaccharide having the structure:

[0011]

[0012] Where n represents the number of repeating units, the molecular weight of the polysaccharide is 1.0×106 Da~2.0×10 6 Da.

[0013] In some embodiments, the molecular weight of the polysaccharide is 1.0×10 6 Da, 1.1×10 6 Da, 1.2×10 6 Da, 1.3×10 6 Da, 1.4×10 6 Da, 1.5×10 6 Da, 1.6×10 6 Da, 1.7×10 6 Da, 1.8×10 6 Da, 1.9×10 6 Da or 2.0×10 6 Da.

[0014] In some embodiments, the molecular weight of the polysaccharide is 1.3×10 6 Da.

[0015] In some embodiments, n is 600-1200. In some embodiments, in some embodiments, n is 600, 650, 700, 750, 760, 770, 780, 785, 790, 800, 850, 900, 1000, 1100, or 1200.

[0016] In some embodiments, the molecular weight detection method of the polysaccharide comprises determination using high performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD).

[0017] In some embodiments, the molecular weight detection method of the polysaccharide uses a TSK gel Super MultiporePW-H chromatographic column.

[0018] In some embodiments, the specifications of the TSK gel Super Multipore PW-H chromatography column are 150 mm×6.0 mm, 8 μm.

[0019] In some embodiments, the molecular weight detection method of the polysaccharide uses 0.1 mol / L ammonium acetate as the mobile phase for isocratic elution at a flow rate of 0.4 mL / min.

[0020] In some embodiments, the chromatographic column temperature of the polysaccharide molecular weight detection method is 35°C.

[0021] In some embodiments, the carrier gas of the evaporative light scattering detector is nitrogen, the carrier gas pressure is 3.5 bar; the drift tube temperature is 60° C.; and the gain value is 6.

[0022] In a second aspect, the present invention provides a Cordyceps sinensis extract.

[0023] A Cordyceps sinensis extract, comprising the polysaccharide described in the first aspect.

[0024] In a third aspect, the present invention provides a method for preparing the polysaccharide described in the first aspect or the Cordyceps sinensis extract described in the second aspect.

[0025] The method for preparing the polysaccharide of the first aspect or the Cordyceps sinensis extract of the second aspect comprises the following steps:

[0026] (1) taking Cordyceps sinensis powder, mixing it with ethanol solution, extracting it under reflux in a water bath, cooling it, and centrifuging it to obtain precipitate 1;

[0027] (2) taking precipitate 1 and evaporating the ethanol, adding water to the mixture, extracting under reflux in a water bath, cooling and centrifuging, and obtaining a supernatant and precipitate 2; taking precipitate 2 and repeating the above steps of adding water to the mixture, extracting under reflux in a water bath, cooling and centrifuging 0-5 times (e.g., 0, 1, 2, 3, 4 or 5 times), and combining the supernatant obtained by centrifugation in step (2); mixing 10 ml-30 ml or 20 ml of water for every 1 g of precipitate 1 or precipitate 2;

[0028] (3) concentrating the combined supernatant obtained in step (2) to one fifth to one twentieth of the volume before concentration to obtain a concentrated solution, mixing the concentrated solution with 2 to 6 times (e.g., 2, 3, 4, 5 or 6 times) the volume of anhydrous ethanol of the concentrated solution, placing at 2 to 8° C. (e.g., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C. or 8° C.) for alcohol precipitation and centrifuging, collecting the precipitate and evaporating the ethanol to obtain precipitate 3;

[0029] (4) After the ethanol in the precipitate 3 is evaporated, it is dissolved in water to remove the protein, dialyzed and concentrated, and the retained solution obtained by the dialysis and concentration is concentrated under reduced pressure and freeze-dried to obtain crude polysaccharide;

[0030] (5) dissolving the crude polysaccharide obtained in step (4) in water, mixing with anhydrous ethanol, placing at 2 to 8° C. (e.g., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C. or 8° C.) for alcohol precipitation, and centrifuging to obtain a supernatant;

[0031] (6) mixing the supernatant obtained in step (5) with 80% vol-95% vol (e.g., 80% vol, 85% vol, 90% vol or 95% vol) ethanol aqueous solution, placing at 2-8° C. (e.g., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C. or 8° C.) for alcohol precipitation, and then centrifuging, collecting the precipitate, and evaporating the ethanol to obtain precipitate 4, thereby obtaining the polysaccharide or the Cordyceps sinensis extract.

[0032] In some embodiments, the step (6) further comprises dissolving the precipitate 4 in water and freeze-drying to obtain the polysaccharide or the Cordyceps sinensis extract.

[0033] In some embodiments, in step (1), every 1 g of the Cordyceps sinensis powder is mixed with 5 ml to 20 ml of ethanol solution. In some embodiments, in step (1), every 1 g of the Cordyceps sinensis powder is mixed with 5 ml, 10 ml, 15 ml or 20 ml of ethanol solution.

[0034] In some embodiments, the water bath reflux extraction in step (1) is water bath reflux extraction at 75° C.-85° C. In some embodiments, the water bath reflux extraction in step (1) is water bath reflux extraction at 75° C., 80° C. or 85° C.

[0035] In some embodiments, the ethanol solution in step (1) is a 95% vol-100% vol ethanol aqueous solution.

[0036] In some embodiments, the extraction time of the water bath reflux extraction in step (1) is 2h-6h. In some embodiments, the extraction time of the water bath reflux extraction in step (1) is 2h, 3h, 4h, 5h or 6h.

[0037] In some embodiments, in step (2), 10-30 ml of water is mixed with every 1 g of the precipitate 1 or precipitate 2. In some embodiments, in step (2), 10 ml, 15 ml, 20 ml, 25 ml or 30 ml of water is mixed with every 1 g of the precipitate 1 or precipitate 2.

[0038] In some embodiments, the water bath reflux extraction in step (2) is water bath reflux extraction at 95°C-100°C.

[0039] In some embodiments, the extraction time of the water bath reflux extraction in step (2) is 2h-6h. In some embodiments, the extraction time of the water bath reflux extraction in step (2) is 2h, 3h, 4h, 5h or 6h.

[0040] In some embodiments, in step (4), each 1g of the precipitate 3 is dissolved in 50ml-150ml of water. In some embodiments, in step (4), each 1g of the precipitate 3 is dissolved in 50ml, 60ml, 70ml, 80ml, 90ml, 100ml, 110ml, 120ml, 130ml, 140ml or 150ml of water.

[0041] In some embodiments, the alcohol precipitation time in step (3) is 8h-24h. In some embodiments, the alcohol precipitation time in step (3) is 8h, 9h, 10h, 11h, 12h, 15h, 16h, 18h, 20h, 22h or 24h.

[0042] In some embodiments, the protein removal in step (4) is carried out using the Sevag method.

[0043] In some embodiments, the protein removal in step (4) is performed by using the Sevag method until all proteins are removed. In some embodiments, the protein removal in step (4) is performed by using the Sevag method for 10-20 times. In some embodiments, the protein removal in step (4) is performed by using the Sevag method for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times.

[0044] In some embodiments, the molecular weight cutoff of the dialysis concentration in step (4) is 3.0 kDa-5.0 kDa. In some embodiments, the molecular weight cutoff of the dialysis concentration in step (4) is 3.0 kDa, 3.5 kDa, 4.0 kDa, 4.5 kDa or 5.0 kDa.

[0045] In some embodiments, the reduced pressure concentration in step (4) is concentrated to one-half to one-fifth of the volume before reduced pressure concentration. In some embodiments, the reduced pressure concentration in step (4) is concentrated to one-half, one-third, one-quarter or one-fifth of the volume before reduced pressure concentration.

[0046] In some embodiments, in step (5), 1g of the crude polysaccharide is dissolved in 150ml-300ml of water. In some embodiments, in step (5), 1g of the crude polysaccharide is dissolved in 150ml, 200ml, 250ml or 300ml of water.

[0047] In some embodiments, the volume ratio of the water used to dissolve the crude polysaccharide to the anhydrous ethanol in step (5) is 3.0:1-4.0:1.

[0048] In some embodiments, the alcohol precipitation time in step (5) is 8h-24h. In some embodiments, the alcohol precipitation time in step (5) is 8h, 9h, 10h, 11h, 12h, 15h, 16h, 18h, 20h, 22h or 24h.

[0049] In some embodiments, the alcohol precipitation time in step (6) is 8h-24h. In some embodiments, the alcohol precipitation time in step (6) is 8h, 9h, 10h, 11h, 12h, 15h, 16h, 18h, 20h, 22h or 24h.

[0050] In some embodiments, the volume ratio of the supernatant to the 80% vol-95% vol ethanol aqueous solution in step (6) is 2.5:1.0-4.0:1.0. In some embodiments, the volume ratio of the supernatant to the 80% vol-95% vol ethanol aqueous solution in step (6) is 2.5:1.0, 3.0:1.0, 3.5:1.0 or 4.0:1.0.

[0051] In some embodiments, in step (6), 150-300 ml of water is mixed with every 1 g of the precipitate 4. In some embodiments, 150 ml, 200 ml, 250 ml or 300 ml of water is mixed with every 1 g of the precipitate 4.

[0052] In some embodiments, the method comprises the following steps:

[0053] (1) Take Cordyceps sinensis powder and mix it with ethanol solution. For every 1g of Cordyceps sinensis powder, mix it with 5ml of 95% vol ethanol solution; reflux extraction in a water bath at 80°C for 2h, cool it, and centrifuge it to obtain precipitate 1;

[0054] (2) taking precipitate 1 and evaporating the ethanol, adding water to the mixture, extracting under reflux at 100° C. in a water bath for 2 h, cooling and centrifuging to obtain a supernatant and precipitate 2; repeating the above-mentioned operation of mixing with water, extracting under reflux at 100° C. in a water bath for 2 h, cooling and centrifuging for 0 to 5 times (e.g., 0, 1, 2, 3, 4 or 5 times) for precipitate 2, and combining the supernatants obtained by centrifugation in step (2); mixing 1 g of precipitate 1 or precipitate 2 with 20 ml of water;

[0055] (3) Concentrating the combined supernatant obtained in step (2) to one tenth of its volume before concentration to obtain a concentrated solution, mixing the concentrated solution with anhydrous ethanol in an amount 4 times the volume of the concentrated solution, placing the solution at 2 to 8° C. for alcohol precipitation, and then centrifuging the solution, collecting the precipitate, and evaporating the ethanol to obtain precipitate 3;

[0056] (4) After the ethanol in the precipitate 3 is evaporated, the precipitate 3 is dissolved in water, and each 1 g of the precipitate 3 is dissolved in 100 ml of water; the protein is removed by Sevag method, and the dialysis concentration is performed, and the molecular weight cutoff of the dialysis concentration is 3.0 kDa; the retained solution obtained by the dialysis concentration is concentrated under reduced pressure to half the volume before the reduced pressure concentration, and freeze-dried to obtain crude polysaccharide;

[0057] (5) dissolving the crude polysaccharide obtained in step (4) in water, dissolving 1 g of the crude polysaccharide in 200 ml of water; then mixing with anhydrous ethanol, wherein the volume ratio of the water used to dissolve the crude polysaccharide to the anhydrous ethanol is 4.0:1.0; placing the mixture at 2 to 8° C. for alcohol precipitation and centrifuging to obtain a supernatant;

[0058] (6) mixing the supernatant obtained in step (5) with an 80% vol-95% vol ethanol aqueous solution in a volume ratio of 2.5:1.0; placing the mixture at 2-8° C. for alcohol precipitation and then centrifuging, collecting the precipitate, and evaporating the ethanol to obtain precipitate 4, thereby obtaining the polysaccharide or the Cordyceps sinensis extract.

[0059] In some embodiments, the step (6) further comprises dissolving the precipitate 4 in water and freeze-drying to obtain the polysaccharide or the Cordyceps sinensis extract.

[0060] In a fourth aspect, the present invention provides a pharmaceutical composition.

[0061] A pharmaceutical composition comprising the polysaccharide described in the first aspect, the cordyceps sinensis extract described in the second aspect, or the polysaccharide or cordyceps sinensis extract prepared by the preparation method described in the third aspect.

[0062] In a fifth aspect, the present invention provides a use of the above-mentioned polysaccharide, Cordyceps sinensis extract, the polysaccharide or Cordyceps sinensis extract obtained by the above-mentioned preparation method, or the above-mentioned pharmaceutical composition.

[0063] A use of the polysaccharide of the first aspect, the Cordyceps sinensis extract of the second aspect, the polysaccharide or Cordyceps sinensis extract prepared by the preparation method of the third aspect, or the pharmaceutical composition of the fourth aspect in the preparation of drugs for immunomodulation and / or anti-oxidation or in identifying the authenticity of Cordyceps sinensis or the specifications of Cordyceps sinensis.

[0064] In some embodiments, the immunomodulation is to enhance immunity. In some embodiments, the immunomodulation includes enhancing the proliferation and / or phagocytic activity of macrophages, and / or enhancing the secretion of cytokines (such as IL-6 and / or TNF-α).

[0065] In some embodiments, a method for identifying the authenticity or specifications of Cordyceps sinensis.

[0066] A method for identifying the authenticity or specifications of Cordyceps sinensis, comprising:

[0067] (1) Preparation of test solution: extract the sample powder with water, centrifuge, take the supernatant, add ethanol to precipitate, centrifuge to obtain precipitate 5, evaporate the ethanol in the precipitate to obtain precipitate 6, add water to dissolve to obtain a crude polysaccharide extract, remove protein in the crude polysaccharide extract by Sevage method, filter, and obtain the test solution;

[0068] (2) Preparation of reference solution: dissolving the polysaccharide described in the first aspect or the polysaccharide prepared by the preparation method described in the second aspect in water to obtain a reference solution;

[0069] (3) Detection: Detecting the content of the polysaccharide described in the first aspect in the sample powder to be tested in the test solution;

[0070] (4) Result judgment.

[0071] In some embodiments, the result is judged as follows: (i) Criteria for judging the authenticity of Cordyceps sinensis: if the content of the polysaccharide in the test solution of the first aspect is less than 0.03%, the powder of the test sample is not the powder of Cordyceps sinensis; and / or if the content of the polysaccharide in the test solution of the first aspect is greater than or equal to 0.03%, the powder of the test sample is the powder of Cordyceps sinensis; and / or

[0072] (ii) Criteria for judging the specifications of Cordyceps sinensis: the powder of the sample to be tested in which the content of the polysaccharide in the test solution of the first aspect is greater than or equal to 0.03% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5000; and / or the powder of the sample to be tested in which the content of the polysaccharide in the first aspect is greater than or equal to 2.24% is powder of large-sized Cordyceps sinensis, wherein the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not higher than 2000.

[0073] In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.10% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.20% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.30% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.40% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.50% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.60% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested in the first aspect with a content of greater than or equal to 0.70% of the polysaccharide in the test solution is a powder of Cordyceps sinensis. In some embodiments, the sample powder to be tested with a polysaccharide content greater than or equal to 0.80% in the test solution of the first aspect is Cordyceps sinensis powder. In some embodiments, the sample powder to be tested with a polysaccharide content greater than or equal to 0.90% in the test solution of the first aspect is Cordyceps sinensis powder.

[0074] In some embodiments, the sample powder to be tested in which the content of the polysaccharide in the test solution of the first aspect is greater than or equal to 0.10% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0075] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.20% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0076] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.30% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0077] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.40% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0078] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.50% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0079] In some embodiments, the sample powder to be tested in which the content of the polysaccharide in the test solution of the first aspect is greater than or equal to 0.60% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0080] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.70% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0081] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.80% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0082] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than 2.24% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0083] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than 2.20% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0084] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than 2.10% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0085] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than 2.00% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0086] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than 1.90% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0087] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than 1.80% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0088] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 0.90% and less than or equal to 1.78% is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000.

[0089] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 2.24% is a powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0090] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 2.30% is a powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0091] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution is greater than or equal to 2.40% of the first aspect is powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0092] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution is greater than or equal to 2.50% of the first aspect is powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0093] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 2.60% is powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0094] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 2.69% is powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0095] In some embodiments, the sample powder to be tested in which the polysaccharide content in the test solution of the first aspect is greater than or equal to 2.69% and less than or equal to 5.04% is powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not more than 2000.

[0096] In some embodiments, the extraction temperature in step (1) is 95° C.-100° C. In some embodiments, the extraction temperature in step (1) is 95° C., 96° C., 97° C., 98° C., 99° C. or 100° C.

[0097] In some embodiments, the extraction time of the extraction in step (1) is 2h-6h. In some embodiments, the extraction time of the extraction in step (1) is 2h, 3h, 4h, 5h or 6h.

[0098] In some embodiments, the water extraction in step (1) is performed with 20-60 ml of water per 1 g of the sample powder to be tested. In some embodiments, the water extraction in step (1) is performed with 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, 50 ml, 55 ml or 60 ml of water per 1 g of the sample powder to be tested.

[0099] In some embodiments, the volume ratio of ethanol to supernatant in step (1) is 2.0: 1-6.0: 1. In some embodiments, the volume ratio of ethanol to supernatant in step (1) is 4.0: 1.0.

[0100] In some embodiments, in the preparation of the crude polysaccharide extract in step (1), the volume ratio of the supernatant before alcohol precipitation used to prepare the precipitate 6 to the water dissolved in the precipitate 6 is 1.0:1.0-1.0:5.0. In some embodiments, in the preparation of the crude polysaccharide extract in step (1), the volume ratio of the supernatant before alcohol precipitation used to prepare the precipitate 6 to the water dissolved in the precipitate 6 is 1.0:1.0, 1.0:2.0, 1.0:3.0, 1.0:4.0 or 1.0:5.0.

[0101] In some embodiments, the step (1) of removing protein from the crude polysaccharide extract by the Sevage method comprises removing protein by the Sevag method until all the protein is removed, or removing protein by the Sevag method 10-20 times (e.g., 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times). In some embodiments, the step (1) of removing protein from the crude polysaccharide extract by the Sevage method comprises removing protein by the Sevag method until all the protein is removed, or removing protein by the Sevag method 15 times.

[0102] In some embodiments, the concentration of the polysaccharide described in the first aspect or the polysaccharide prepared by the preparation method described in the second aspect in the reference solution of step (2) is 2 mg / mL-5 mg / mL. In some embodiments, the concentration of the polysaccharide described in the first aspect or the polysaccharide prepared by the preparation method described in the second aspect in the reference solution of step (2) is 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0103] In some embodiments, the step (3) is detected using high performance liquid chromatography coupled with an evaporative light scattering detector.

[0104] In some embodiments, the high performance liquid chromatography method uses a chromatographic column filled with polymethyl methacrylate for separation.

[0105] In some embodiments, the HPLC method uses a TSK gel Super Multipore PW-H chromatography column for separation.

[0106] 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.

[0107] In some embodiments, the inner diameter of the chromatographic column is 4.6 mm-6.0 mm. In some embodiments, the inner diameter of the chromatographic column is 4.6 mm, 5.0 mm, 5.5 mm or 6.0 mm.

[0108] In some embodiments, the filler particle size of the chromatographic column is 5 μm-12 μm. In some embodiments, the filler particle size of the chromatographic column is 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm or 12 μm. In some embodiments, the filler particle size of the chromatographic column is 8 μm.

[0109] In some embodiments, the specifications of the chromatographic column are: column length 150 mm, inner diameter 6.0 mm, and filler particle size 8 μm.

[0110] In some embodiments, the HPLC method uses aqueous ammonium acetate as the mobile phase.

[0111] In some embodiments, the HPLC method uses 0.05 mol / L-0.2 mol / L ammonium acetate aqueous solution as the mobile phase. In some embodiments, the HPLC method uses 0.1 mol / L ammonium acetate aqueous solution as the mobile phase.

[0112] In some embodiments, the elution mode of the HPLC is isocratic elution.

[0113] In some embodiments, the flow rate of the HPLC is 0.3 ml / min-0.6 ml / min. In some embodiments, the flow rate of the HPLC is 0.3 ml / min, 0.4 ml / min, 0.5 ml / min or 0.6 ml / min.

[0114] In some embodiments, the column temperature of the HPLC is 25° C.-40° C. In some embodiments, the column temperature of the HPLC is 25° C., 30° C., 35° C. or 40° C. In some embodiments, the column temperature of the HPLC is 35° C.

[0115] In some embodiments, the carrier gas of the evaporative light scattering detector is nitrogen.

[0116] In some embodiments, the carrier gas pressure of the evaporative light scattering detector is 3 bar to 4 bar. In some embodiments, the carrier gas pressure of the evaporative light scattering detector is 3.5 bar.

[0117] In some embodiments, the drift tube temperature of the evaporative light scattering detector is 40° C.-60° C. In some embodiments, the drift tube temperature of the evaporative light scattering detector is 40° C., 45° C., 50° C., 55° C., or 60° C.

[0118] In some embodiments, the evaporative light scattering detector has a gain value of 5 to 8. In some embodiments, the evaporative light scattering detector has a gain value of 5, 6, 7, or 8. In some embodiments, the evaporative light scattering detector has a gain value of 6.

[0119] Beneficial Effects

[0120] Compared with the prior art, the present invention has the following beneficial effects:

[0121] (1) The polysaccharide provided by the present invention is a new type of water-soluble polysaccharide, which is firstly found in natural extracts. The polysaccharide provided by the present invention has excellent immunomodulatory and / or antioxidant activities.

[0122] (2) The polysaccharide provided by the present invention can be used to identify the authenticity or specifications of Cordyceps sinensis, which has unexpected technical effects.

[0123] (3) The method provided by the present invention for identifying the authenticity of Cordyceps sinensis or the specifications of Cordyceps sinensis has high accuracy, and can significantly distinguish between authentic and counterfeit Cordyceps sinensis, and between the sizes of Cordyceps sinensis.

[0124] (4) The polysaccharide obtained by the preparation method provided by the present invention has a high yield and high purity.

[0125] Terminology

[0126] In the description of the present invention, “plurality” means two or more than two, unless otherwise clearly and specifically defined.

[0127] The term "room temperature" means ambient temperature, referring to a temperature between about 10°C and about 30°C, or between about 20°C and 30°C, or about 25°C.

[0128] 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.

[0129] The term "v / v" means volume ratio. The term "%vol" means volume percentage. ABTS means 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid. DPPH means 1,1-diphenyl-2-trinitrophenylhydrazine. FRAP means "ferric ion reducing / antioxidant capacity method". The term "solid-liquid ratio" means the ratio of the mass of the material to be extracted to the volume of the extraction solvent. For example, a solid-liquid ratio of 1g:100ml means that 100ml of the extraction solvent is used for every 1g of the material to be extracted.

[0130] 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.

[0131] The "molecular weight" of the polysaccharide described in the present invention refers to the weight average molecular weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 This is the HPLC-ELSD chromatogram of the Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0133] Figure 2 This is a chromatogram of monosaccharide composition analysis of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0134] Figure 3 This is the infrared spectrum of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0135] Figure 4This is the infrared spectrum of the methylated Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0136] Figure 5 This is the GC-MS chromatogram of the methylation product of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0137] Fig. 6A The Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1 1 H-NMR spectrum.

[0138] Figure 6B The Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1 13 C-NMR spectrum.

[0139] Figure 6C This is the COSY graph of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0140] Fig.6D This is the HSQC chart of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0141] Fig. 6E This is the HMBC chart of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0142] Figure 7 Schematic diagram of the structure of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0143] Figure 8 These are the result graphs of the immunomodulatory activity of the Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1, wherein A is the result graph of the proliferation activity of CSWP-40 on RAW264.7 cells, B is the result graph of the phagocytic activity of CSWP-40 on RAW264.7 cells, C is the result graph of the effect of CSWP-40 on the secretion of IL-6 factor in RAW264.7 cells, and D is the result graph of the effect of CSWP-40 on the secretion of TNF-α factor in RAW264.7 cells.

[0144] Fig. 9 The representative HPLC chromatograms of large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) and small-sized Cordyceps sinensis (5000 pieces / kg, dry grass) in Example 5 are shown.

[0145] Fig.10 This is a statistical chart of the content range of Cordyceps sinensis polysaccharide CSWP-40 in large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) and small-sized Cordyceps sinensis (5000 pieces / kg, dry grass) in Example 5.

[0146] Fig.11 The HPLC chromatograms of the authentic and counterfeit Cordyceps sinensis in Example 6 are shown. DETAILED DESCRIPTION

[0147] 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.

[0148] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0149] Materials and equipment for preparing polysaccharides or the cordyceps sinensis extract

[0150] 1. Materials and reagents

[0151] 95% ethanol, anhydrous ethanol, anhydrous methanol, chloroform, acetic anhydride, hydrochloric acid and sulfuric acid were all of analytical grade and purchased from Chengdu Kelong Chemical Co., Ltd. n-Butanol was of analytical grade and purchased from Guangdong Huaguang Technology Co., Ltd. Dichloromethane, phosphoric acid and sodium hydroxide were all of analytical grade, and glacial acetic acid was of chromatography grade and purchased from Xilong Science Co., Ltd. DPPH was of analytical grade and purchased from Sigma-Aldrich, USA. 1-phenyl-3-methyl-5-pyrazolone (PMP), iodomethane and phenol were all of analytical grade, trifluoroacetic acid and ammonium acetate were of chromatography grade, potassium bromide was of spectral grade, bovine serum albumin was of molecular biology grade, and 5 monosaccharide reference substances (rhamnose, purity 99.0%; galactose, purity 99.6%; xylose, purity 99.4%; arabinose, purity 99.1%; fucose, purity 99.6%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Five monosaccharide reference substances (glucose, purity 99.8%; mannose, purity 100%; glucosamine, purity 100%; glucuronic acid, purity 99.8%; galacturonic acid, purity 95.1%) were purchased from the China Food and Drug Inspection Institute. Dimethyl sulfoxide was of analytical grade, and a series of dextran standards (weight average molecular weights of 1, 12, 80, 150, 410, and 670 Kda, respectively) were purchased from Sigma-Aldrich, USA. Acetonitrile was of chromatographic grade and purchased from KRUDE, USA. Coomassie Brilliant Blue G-250 was of analytical grade and purchased from Bio-Rad, USA. Deuterium hydroxide was of analytical grade and purchased from Beijing Inokai Technology Co., Ltd. DMEM high-glucose medium (batch number 2508886) was purchased from GIBCO, USA; CCK-8 kit (batch number PG679) was purchased from DOJINDO Institute, Japan; neutrophil proliferation and cytotoxicity detection kit (batch number 092222230516) was purchased from Shanghai Biotech Biotechnology Co., Ltd.; IL-6 ELISA kit (batch number 343160-005) was purchased from Invitrogen, USA; TNF-α ELISA kit (batch number P362560) was purchased from R&D Company, USA.

[0152] Sevag solution: a mixture of chloroform and n-butanol in a volume ratio of 4:1.

[0153] 2. Instruments and equipment

[0154] XPE 205DR electronic balance (METTLER TOLEDO, Switzerland); HH-ZK8 electric constant temperature water bath (Yuhua Instrument Co., Ltd., Gongyi City); Sorvall ST40R general-purpose desktop centrifuge (Thermo Fisher, USA); RV10 DS25 rotary evaporator, V3 S025 vortexer (IKA, Germany); Tissue / Grinder 2020 high-throughput tissue grinder (Corient (Beijing) Co., Ltd.); Synergy H1 multifunctional microplate reader (BioTek, USA); Alpha 1-4LSC Basic freeze dryer (Christ, Germany); Milli-Q ultrapure water system (Merck, USA); 1260 high-performance liquid chromatograph, Cary 60 ultraviolet-visible spectrophotometer, 5977B gas chromatography-mass spectrometer (Agilent, USA); SEDEX Model 90 evaporative light scattering detector (SEDERE, France); TENSOR II infrared spectrometer, Avance III HD 600 nuclear magnetic resonance instrument (Bruker, USA); ED115 electric constant temperature drying oven (BINDER, Germany); SBL-22DT constant temperature ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); TURBOVAP-LV nitrogen blowdown apparatus (Orgnomation, USA); WS70-1 infrared drying oven (Changzhou Guowang Instrument Manufacturing Co., Ltd.); Atlas 15T tablet press (Beijing Youhua Venture Technology Co., Ltd.); HERAcell 150i carbon dioxide incubator (Thermo Fisher, USA); CKX31 biological inverted microscope (Olympus, Japan); TSK gelSuper Multipore PW-H (150mm×6.0mm, 8μm; TOSOH, Japan).

[0155] 3. Experimental cells: RAW264.7 cell line, purchased from the Cell Bank of the Chinese Academy of Sciences.

[0156] Example 1: Preparation of polysaccharide or the Cordyceps sinensis extract

[0157] The polysaccharide or the Cordyceps sinensis extract is prepared according to the following steps:

[0158] (1) Take Cordyceps sinensis powder and mix it with ethanol solution. For every 1g of Cordyceps sinensis powder, mix it with 5ml of 95% vol ethanol solution; reflux extraction in a water bath at 80°C for 2h, cool it, and centrifuge it to obtain precipitate 1;

[0159] (2) Take the precipitate 1 and evaporate the ethanol, add water and mix (every 1g of the precipitate 1 is mixed with 20ml of water), then reflux extract in a water bath at 100°C for 2h, then cool and centrifuge to obtain a supernatant and precipitate 2; the precipitate 2 obtained by centrifugation is repeatedly extracted twice according to the above-mentioned "mixing with water, reflux extracting in a water bath at 100°C for 2h, then cooling and centrifuging" operation (in each operation, every 1g of the precipitate 2 is mixed with 20ml of water), and the supernatants obtained by the three centrifugations in step (2) are combined;

[0160] (3) Concentrating the combined supernatant obtained in step (2) to one tenth of its volume before concentration to obtain a concentrated solution, mixing the concentrated solution with anhydrous ethanol in an amount 4 times the volume of the concentrated solution, placing the solution at 2-8° C. for alcohol precipitation, and then centrifuging the solution, collecting the precipitate, and evaporating the ethanol to obtain precipitate 3;

[0161] (4) After the ethanol in the precipitate 3 is evaporated, the precipitate 3 is dissolved in water, and each 1g of the precipitate 3 is dissolved in 100ml of water to obtain a crude polysaccharide solution; the protein is removed by the Sevag method until all the protein is removed (a quarter of the volume of the crude polysaccharide solution is repeatedly extracted with a Sevag solution until the clarification is between the Sevag solution layer (lower layer) and the water layer (upper layer) (extraction is performed about 15 times)), and the upper layer solution obtained after the protein is completely removed is dialyzed and concentrated, and the molecular weight cutoff of the dialysis and concentration is 3.0kDa; the retained solution obtained by the dialysis and concentration is concentrated under reduced pressure to half the volume before the reduced pressure concentration, and freeze-dried to obtain a crude polysaccharide;

[0162] (5) dissolving the crude polysaccharide obtained in step (4) in water, dissolving 1 g of the crude polysaccharide in 200 ml of water; then mixing with anhydrous ethanol, wherein the volume ratio of the water used to dissolve the crude polysaccharide to the anhydrous ethanol is 4.0:1.0; placing the mixture at 2-8° C. for alcohol precipitation and centrifuging to obtain a supernatant;

[0163] (6) The supernatant obtained in step (5) was mixed with 80% vol-95% vol ethanol aqueous solution, and the volume ratio of the supernatant and 80% vol-95% vol ethanol aqueous solution was 2.5:1.0; after being placed at 2-8°C for alcohol precipitation, centrifuged, the precipitate was collected, and the ethanol was evaporated to obtain precipitate 4; the precipitate 4 was dissolved in water and freeze-dried to obtain the polysaccharide CSWP-40, with a yield of 1.41%. Example 2: Structural Characterization of Cordyceps Polysaccharide CSWP-40

[0164] 1. Molecular weight distribution detection

[0165] Take an appropriate amount of polysaccharide CSWP-40, prepare a 2 mg / mL solution with water, filter through a 0.22 μm microporous filter membrane, and use high performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) to determine the molecular weight distribution of the polysaccharide. Detection conditions: chromatographic column is TSK gel Super Multipore PW-H (150 mm × 6.0 mm, 8 μm); mobile phase is 0.1 mol / L ammonium acetate; isocratic elution for 20 min; flow rate is 0.4 mL / min; column temperature is 35 ° C; injection volume is 20 μL; ELSD detector carrier gas is nitrogen; carrier gas pressure is 3.5 bar; drift tube temperature is 60 ° C; gain value is 6. With retention time (Time) as the horizontal axis, the logarithm of the molecular weight of the dextran series standard (lg M w ) as the ordinate, and the standard curve Y = -1.465X + 13.68, r = 0.9992 was drawn. According to the retention time of each chromatographic peak in the sample solution, the molecular weight M of polysaccharide CSWP-40 was calculated from the standard curve. w 1.3×10 6 Da, see the result Figure 1 .

[0166] 2. Determination of total sugar content

[0167] Take an appropriate amount of polysaccharide CSWP-40, prepare a sample solution of 0.1 mg / mL with water, use glucose as the reference substance, and determine the total sugar content by sulfuric acid-phenol method. Pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of glucose reference solution (0.1 mg / mL) and 1.0 mL of sample solution into a 10 mL colorimetric tube, and fill the reference solution less than 1.0 mL with water to 1.0 mL. At the same time, replace the sample solution with 1.0 mL of water as a blank control, and then add 1 mL of 5% (W / V) phenol solution and 5 mL of concentrated sulfuric acid, vortex mix, and measure the absorbance at 490 nm after standing for 30 minutes. With glucose concentration as the horizontal coordinate and absorbance value as the vertical coordinate, draw a standard curve Y=9.6888X+0.0106, r=0.9997. According to the sample absorbance reading, the mass concentration of total sugar in the sample was calculated from the standard curve, and the total sugar content of the polysaccharide CSWP-40 was calculated to be 98.25% according to the following formula.

[0168]

[0169] Wherein: C is the mass concentration of total sugar in the sample obtained from the standard curve, in mg / mL; V is the volume of water added when preparing the sample solution, in mL; m is the sample mass of polysaccharide CSWP-40 when preparing the sample solution, in mg.

[0170] 3. Protein content determination

[0171] Take an appropriate amount of polysaccharide CSWP-40, prepare a sample solution of 2.0 mg / mL with water, and use bovine serum albumin as a reference substance to determine the protein content by Bradford method. Pipette 0.01mL, 0.02mL, 0.04mL, 0.06mL, 0.08mL, 0.10mL of bovine serum albumin reference substance solution (1.0 mg / mL) and 0.10mL of sample solution into a 10mL colorimetric tube respectively, and make up the reference substance solution less than 0.10mL with water to 0.10mL, and replace the sample solution with 0.10mL of water as a blank control, and then add 5mL of Coomassie brilliant blue dye solution respectively, vortex mix, and measure the absorbance value at 595nm after standing for 5min. With bovine serum albumin concentration as the horizontal coordinate and absorbance value as the vertical coordinate, draw the calibration curve Y=0.7912X+0.0842, r=0.9984. According to the sample absorbance reading, the mass concentration of protein in the sample was calculated from the standard curve, and the protein content in the polysaccharide CSWP-40 was calculated to be 0% according to the following formula.

[0172]

[0173] Wherein: C is the mass concentration of protein in the sample obtained from the standard curve, in mg / mL; V is the volume of water added when preparing the sample solution, in mL; m is the sample mass of polysaccharide CSWP-40 when preparing the sample solution, in mg.

[0174] From the results of total sugar content and protein content, it can be seen that the main component of the obtained Cordyceps sinensis extract is polysaccharide and does not contain protein.

[0175] 4. Monosaccharide composition analysis

[0176] Take an appropriate amount of polysaccharide CSWP-40 and prepare a 2 mg / mL sample solution with water. Take 1 mL of the sample solution and place it in a 15 mL centrifuge tube, add 1 mL of 4 mol / L trifluoroacetic acid, mix well, seal, hydrolyze at 120°C for 2 hours, take it out and cool it to room temperature, blow it dry with nitrogen, then add 1 mL of anhydrous methanol, mix well, blow it dry with nitrogen, repeat 3 times, dissolve the residue with 1 mL of water, mix well, and get the polysaccharide hydrolyzate. 100 μL of polysaccharide hydrolyzate and mixed sugar reference solution (containing mannose (Man), glucosamine (GlcN), rhamnose (Rha), glucuronic acid (GlcUA), galacturonic acid (GlaUA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc), about 0.05 mg / mL each) were accurately pipetted into 2 mL centrifuge tubes, 50 μL of 0.6 mol / L sodium hydroxide solution was added, and 0.6 mol / L 100 μL of PMP-methanol solution was mixed thoroughly, sealed, placed in a 70°C water bath for 60 minutes, removed and cooled to room temperature, 100 μL of 0.3 mol / L hydrochloric acid solution was added for neutralization, 650 μL of ultrapure water was added, mixed thoroughly, 500 μL of the above solution was pipetted into a 2 mL centrifuge tube, 1 mL of chloroform was added, vortexed for 1 minute, centrifuged at 12000 r / min for 10 minutes, the supernatant was taken, and the monosaccharide composition was detected by high performance liquid chromatography (HPLC). Detection conditions: The chromatographic column was Agilent Eclipse XDB-C 18 (250mm×4.6mm,5μm); mobile phase A (0.1mol / L phosphate buffer (pH=6.7)): mobile phase B (acetonitrile)=83:17, isocratic elution 50min; injection volume 5μL; detection wavelength 245nm; flow rate 1.0mL / min; column temperature 30℃. The results showed that polysaccharide CSWP-40 was composed of glucose, the results are shown in Figure 2 .

[0177] 5. Infrared spectroscopy analysis

[0178] Take an appropriate amount of potassium bromide powder, put it in an agate mortar and grind it until there are no obvious reflective crystals; weigh about 200 mg into another small agate mortar, and then add about 2 mg of polysaccharide CSWP-40 powder, grind it until it is fully mixed to obtain the sample powder to be tested. Dry the sample powder under the infrared drying oven light for about 5 minutes. After cooling, transfer the sample powder to a tablet pressing mold and press it at a pressure of 10MPa for about 2 minutes. Take out the pressed sample tablet and put it into the sample rack for testing. Instrument parameters: Scanning spectrum range: 4000-400cm -1 ; Resolution: 4cm -1; Number of scans: 16. Data processing: Water vapor compensation selected water compensation and carbon dioxide compensation; Baseline correction selected concave rubberband correction (iterations 10; baseline points 64; exclude carbon dioxide spectrum); Smoothing points 25.

[0179] The results showed that polysaccharide CSWP-40 was at 3389.61 cm -1 The broad peak is the stretching vibration absorption peak of OH; at 2926.15 cm -1 The peak that appears is the CH stretching vibration absorption peak; at 1643.74cm -1 The peak that appears is the absorption peak of a small amount of crystal water in sugar; at 1100~1010cm -1 There are three absorption peaks (located at 1153.88 cm -1 、1081.93cm -1 、1023.42cm -1 ), indicating that the glycosidic bond is pyranose; 854.63cm -1 The characteristic absorption peak of α-glycosidic bond is at 1750~1700cm -1 There is no absorption peak between the two, indicating that the polysaccharide CSWP-40 does not contain uronic acid. Figure 3 .

[0180] 6. Methylation analysis

[0181] Take about 5 mg of polysaccharide CSWP-40 in a screw-top test tube, add 1.5 mL of dimethyl sulfoxide, seal it, and sonicate for 30 minutes to dissolve the sample. Then add about 20 mg of dry sodium hydroxide powder, sonicate for 30 minutes, cool in an ice bath until the sample solidifies, add 0.1 mL of iodomethane under light-proof conditions, sonicate for 30 minutes (control the temperature at 18-20°C), repeat the operation 3 times, and then add 1 mL of water to terminate the methylation reaction. Add 0.5 mL of chloroform to the reaction solution, vortex to mix, let it stand for stratification, aspirate the chloroform layer, repeat the operation 4 times, combine the chloroform layers, wash with an equal volume of water 3 times, take the chloroform layer, and blow dry with nitrogen. Take an appropriate amount of solution, apply it on the prepared potassium bromide sheet, place it under an infrared lamp for drying, and detect the infrared spectrum at 3389.61 cm -1 The methylation is complete when the hydroxyl peak at Figure 5). Add 1 mL of 2 mol / L trifluoroacetic acid to the methylated polysaccharide, seal it, hydrolyze it at 120°C for 2 h, cool it to room temperature, blow it dry with nitrogen, then add 0.5 mL of anhydrous methanol, blow it dry with nitrogen, and repeat 3 times. Add 0.5 mL of freshly prepared 10 mg / mL sodium borodeuteride, mix well, and let it react at room temperature for 12 h, shaking it from time to time. Add 4 mol / L acetic acid dropwise to neutralize, check with pH test paper, add 0.5 mL of 5% (V / V) acetic acid-methanol, blow it dry with nitrogen, repeat 3 times, then add 0.5 mL of anhydrous methanol, blow it dry with nitrogen, and repeat 3 times. Add 1 mL of acetic anhydride, mix well, seal, react at 100 ° C for 2.5 h, cool to room temperature, blow dry with nitrogen, add 0.5 mL of anhydrous methanol, blow dry with nitrogen, repeat 3 times, add 1 mL of dichloromethane, vortex and shake, centrifuge at 5000 r / min for 5 min, take the supernatant and filter it through a 0.2 μm filter membrane, and detect it by gas chromatography-mass spectrometry (GC-MS). Detection conditions: the chromatographic column is a DB-5MS capillary column (30m×0.25mm×0.25μm); the carrier gas is high-purity nitrogen; the gas flow rate is 1mL / min; the temperature of the injection port is 260℃; the injection volume is 1μL; split injection, split ratio is 50:1; the solvent is extended for 2.2min; programmed temperature (from 100℃ to 165℃ at 15℃ / min, maintained for 30min, programmed to 180℃ at 2℃ / min, programmed to 300℃ at 15℃ / min, maintained for 5min); electron bombardment ion source (EI); ion source temperature is 230℃; quadrupole temperature is 150℃; electron energy is 70eV; transmission line temperature: 300℃; the scanning mode is full scan mode (SCAN), and the mass scanning range (m / z): 30-600.

[0182] The test results showed that the polysaccharide CSWP-40 had three structural fragment derivatives, namely α-D-Glcp-(1→), →4)-α-D-Glcp-(1→) and →4,6)-α-D-Glcp-(1→). The peak area ratio of the three main peaks was about 12.4:77.5:10.0. The results are shown in Table 1. Figure 5 .

[0183] Table 1 Ion peaks and fragment ions of methylated derivatives of polysaccharide CSWP-40

[0184]

[0185]

[0186] 7. Nuclear Magnetic Resonance Spectroscopy Analysis

[0187] Take about 30 mg of polysaccharide CSWP-40, dissolve it in 1 mL of heavy water, blow it to dryness with nitrogen, repeat 3 times, then dissolve it in 0.6 mL of heavy water, and after it is fully dissolved, transfer it to a nuclear magnetic resonance tube and detect it with a nuclear magnetic resonance instrument. Sample detection conditions: 1 H-NMR temperature 98K, frequency 599M; 13 C-NMR temperature: 298K, frequency 150M. Using 1D ( 1 H and 13 The chemical structure of the polysaccharide of the present invention was characterized in detail by 2D (COSY, HSQC, HMBC) and 2D (COSY, HSQC, HMBC) NMR spectroscopy.

[0188] exist 1 In the H NMR spectrum, three anomeric proton coupling signals were found in the anomeric region, with chemical shifts of 5.31 ppm (A), 4.90 ppm (B), and 4.89 ppm (C). 13 C NMR determined that the anomeric carbon signal of residue A was 99.80ppm, the anomeric carbon signal of residue B was 98.61ppm, and the anomeric carbon signal of residue C was 98.44ppm. The chemical shifts of the above anomeric protons and anomeric carbons showed that the three residues were all α-configured. Then, COSY and other methods were used to analyze all the residues. 1 H and 13 The COSY correlation spectrum assigned the proton chemical shifts of residue A from H-1 to H-5 (5.31ppm, 3.52ppm, 3.89ppm, 3.58ppm and 3.89ppm respectively); the HSQC correlation spectrum assigned the proton chemical shifts of residue A from H-1 to H-5 (5.31ppm, 3.52ppm, 3.89ppm, 3.58ppm and 3.89ppm respectively); 13 The chemical shifts of C were assigned to C1~C6 as 99.80ppm, 71.64ppm, 73.21ppm, 76.90ppm, 71.62ppm, and 60.54ppm, respectively; further, the signals of H-6a and H-6b in residue A were assigned to 3.78ppm and 3.69ppm, respectively, through HSQC and COSY correlation spectra. Among them, the chemical shift of C-4 shifted to the low field, indicating that substitution occurred at the C-4 position. Combined with the results of methylation analysis, it was inferred that residue A was: →4)-α-D-Glcp-(1→). The proton chemical shifts of residue B from H-1 to H-5 were assigned through COSY correlation spectra (4.90ppm, 3.49ppm, 3.94ppm, 3.65ppm, and 3.76ppm, respectively); the HSQC correlation spectrum was used to identify the chemical shifts of residue B. 13The chemical shifts of C were assigned to C1~C6, which were 98.61ppm, 72.30ppm, 73.23ppm, 69.35ppm, 71.23ppm, and 60.50ppm, respectively. The signals of H-6a and H-6b in residue B were assigned to 3.78ppm and 3.69ppm, respectively, through HSQC and COSY correlation spectra. Residue B was inferred to be: α-D-Glcp-(1→). The proton chemical shifts of residue C from H-1 to H-5 were assigned to 4.89ppm, 3.46ppm, 3.84ppm, 3.76ppm, and 3.94ppm, respectively, through COSY correlation spectra. The HSQC correlation spectra can be used to identify the residue C. 13 C chemical shifts were assigned, C1 to C6 were 98.44ppm, 71.53ppm, 72.85ppm, 76.81ppm, 72.30ppm, 69.38ppm respectively; the signal of H-6 in residue C was assigned to 3.35ppm by HSQC and COSY correlation spectra. Among them, the chemical shifts of C-4 and C-6 shifted to the low field, and there was no coupled hydrogen signal at C-6, indicating that substitution occurred at C-4 and C-6. It was inferred that residue C was: →4,6)-α-D-Glcp-(1→). The NMR data of the polysaccharide are shown in Table 2 and Figure 6.

[0189] Table 2 NMR data of polysaccharide CSWP-40

[0190] Sugar residue H1 / C1 H2 / C2 H3 / C3 H4 / C4 H5 / C5 H6 / C6 A:→4)-α-D-Glcp-(1→) 5.31 / 99.8 3.52 / 71.6 3.89 / 73.2 3.58 / 76.9 3.89 / 71.6 3.78,3.69 / 60.5 B:α-D-Glcp-(1→) 4.90 / 98.6 3.49 / 72.3 3.94 / 73.2 3.65 / 69.4 3.76 / 71.2 3.78,3.69 / 60.5 C:→4,6)-α-D-Glcp-(1→) 4.89 / 98.4 3.46 / 71.5 3.84 / 72.8 3.76 / 76.8 3.94 / 72.3 3.35 / 6.94

[0191] By analyzing and summarizing the above chemical, spectroscopic and spectroscopic data, it is finally determined that the Cordyceps sinensis polysaccharide obtained by the present invention has a novel structure, and its main chain is →4)-α-D-Glcp-(1→), and the branch point is glucan at the C-6 position. The schematic diagram of the structure is shown in Figure 7 , that is, the following structure:

[0192]

[0193] Example 3: Immunoactivity test of Cordyceps sinensis polysaccharide CSWP-40

[0194] 1. Cell culture and drug preparation

[0195] RAW264.7 cells were cultured in a 37°C, 5% carbon dioxide incubator using DMEM complete medium. Subculture once a day, discard the old medium, gently rinse once with PBS, add 1 mL of new medium, tap the culture bottle, and observe under a microscope that 80% of the cells have fallen off. Collect the fallen cells and count them according to 1×10 7 cell / flask passaging.

[0196] Preparation of sample solution: Accurately weigh the polysaccharide CSWP-40 sample prepared according to the method described in Example 1, dissolve it in DMEM complete medium and prepare it into a 2 mg / mL mother solution, filter it with a 0.22 μm sterile microporous filter membrane, and then add an appropriate amount of DMEM complete medium to dilute it into different concentration gradients.

[0197] 2. Drug efficacy testing

[0198] 2.1 Cell proliferation activity

[0199] Collect cells in logarithmic growth phase, count them, resuspend the cells in DMEM complete medium, and adjust the cell concentration to 1×10 5 cell / mL, 100 μL of cells were inoculated in each well of a 96-well plate. After incubation in a 37°C, 5% carbon dioxide incubator for 24 hours, the culture medium was discarded. 100 μL of sample solution of different concentrations (0.1 μg / mL, 1 μg / mL, 10 μg / mL, 31.3 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) was added to each well, and 3 replicates were made for each concentration as the CSWP-40 group; negative control wells (negative control group) (cells + 100 μL DMEM complete medium) and blank wells (blank group) (100 μL DMEM complete medium) were set up. After completing the above steps, the 96-well plate was placed in a 37°C, 5% carbon dioxide incubator for further incubation for 24 hours, and subsequent operations were performed according to the instructions of the CCK-8 kit. The absorbance was measured at 450 nm using an enzyme marker, and the proliferation rate of the drug on cell growth was calculated according to the following formula:

[0200]

[0201] Where: A s : Absorbance of the sample (cells + CCK8 + sample to be tested); A c : absorbance of negative control (cells + CCK8); A b : Absorbance of blank control (culture medium + CCK8).

[0202] Results: Compared with the negative control group, the CSWP-40 group showed a significant concentration-dependent increase in the proliferation rate of RAW264.7 cells in the concentration range of 10-1000 μg / mL, indicating that CSWP-40 has a promoting effect on the proliferation of RAW264.7 cells in the concentration range of 10-1000 μg / mL. Figure 8 Figure A in the middle.

[0203] 2.2. Cell phagocytic activity detection

[0204] Collect cells in logarithmic growth phase, count them, resuspend the cells in DMEM complete medium, and adjust the cell concentration to 1×10 5 cell / mL, 100 μL cells were inoculated in each well of a 96-well plate. After incubation in a 37°C, 5% carbon dioxide incubator for 24 hours, the culture medium was discarded. 100 μL of sample solution of different concentrations (0.1 μg / mL, 1 μg / mL, 10 μg / mL, 31.3 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) was added to each well, and 3 replicates were made for each concentration as the CSWP-40 group; at the same time, negative control wells (negative control group) (cells + 100 μL DMEM complete medium) and blank wells (blank group) (100 μl DMEM complete medium) were set up. After completing the above steps, the 96-well plate was placed in a 37°C, 5% carbon dioxide incubator for further incubation for 24 h, and subsequent operations were performed according to the instructions of the neutrophil proliferation and cytotoxicity detection kit. The absorbance value was measured at 540 nm using an ELISA reader, and the effect of the drug on the cell phagocytosis rate was calculated according to the following formula.

[0205]

[0206] Where: A s : Absorbance of the sample (cells + sample to be tested); A c : absorbance of negative control (cells + DMEM complete medium); A b : Absorbance of blank control (DMEM complete medium).

[0207] Results: Compared with the negative control group, the CSWP-40 group was able to significantly improve the phagocytic activity of cells at a concentration of 31.3μg / mL-500μg / mL, but it did not show concentration dependence. The peak concentration was 62.5μg / mL, indicating that CSWP-40 had a certain effect on improving the phagocytic activity of RAW264.7 cells at a concentration of 31.3μg / mL-500μg / mL. The results are shown in Figure 8 Middle Figure B.

[0208] 2.3. Detection of cytokine IL-6 levels

[0209] The culture supernatant of RAW 264.7 cells treated with different concentrations of sample solutions (3.9μg / mL, 7.8μg / mL, 15.6μg / mL, 31.3μg / mL, 62.5μg / mL, 125μg / mL, 250μg / mL, 500μg / mL, 1000μg / mL) for 24h was collected, and 3 replicates were made for each concentration as the CSWP-40 group; at the same time, negative control wells (negative control group) (cells + 100μL DMEM complete medium) and blank wells (blank group) (100μL DMEM complete medium) were set up. 50μL of each well was added to a 96-well plate, and subsequent operations were performed according to the instructions of the IL-6 kit. The absorbance value was measured at 450nm using an ELISA reader. The standard curve of IL-6 was drawn and the IL-6 concentration was calculated.

[0210] Results: Compared with the negative control group, the CSWP-40 group could significantly increase the IL-6 secretion of RAW264.7 cells in the concentration range of 500 μg / mL-1000 μg / mL, indicating that CSWP-40 has a promoting effect on the secretion of IL-6 by RAW264.7 cells in the concentration range of 500 μg / mL-1000 μg / mL; its half effective concentration (EC 50 ) was 533.5 μg / mL. Figure 8 Figure C.

[0211] 2.4. Detection of cytokine TNF-α level

[0212] The culture supernatant of RAW 264.7 cells treated with different concentrations of sample solutions (3.9 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.3 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) for 24 h was collected, and 3 replicates were made for each concentration as the CSWP-40 group. At the same time, negative control wells (negative control group) (cells + 100 μL DMEM complete medium) and blank wells (blank group) (100 μL DMEM complete medium) were set up. 50 μL of each well was added to a 96-well plate, and subsequent operations were performed according to the instructions of the TNF-α kit. The absorbance value was measured at 450 nm using an ELISA reader. The standard curve of TNF-α was drawn and the TNF-α concentration was calculated.

[0213] Results: Compared with the negative control group, the CSWP-40 group could significantly increase the secretion of TNF-α in RAW264.7 cells in the concentration range of 62.5μg / mL-1000μg / mL, and the relationship was concentration-dependent, indicating that CSWP-40 promoted the secretion of TNF-α in RAW264.7 cells in the concentration range of 62.5μg / mL-1000μg / mL; its half effective concentration (EC 50 ) was 649.2 μg / mL. Figure 8 Figure D.

[0214] Example 4: DPPH Antioxidant Activity Test of Cordyceps Polysaccharide CSWP-40

[0215] 1. Solution preparation

[0216] Sample solution: Weigh 50 mg of the polysaccharide CSWP-40 sample prepared by the method described in Example 1, accurately weigh, add 5 mL of ultrapure water to dissolve, vortex mix, and the concentration is 10 mg / mL sample solution. Then take appropriate amounts of 10 mg / mL sample solution and dilute them with ultrapure water to 7.5 mg / mL, 5 mg / mL, 2.5 mg / mL, and 1 mg / mL sample solutions.

[0217] 2. Activity determination

[0218] Pipette 100 μL of sample solution, add 50 μL of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution (80 μg / mL) and mix thoroughly. Leave the mixture at room temperature and away from light for 30 min. Measure the absorbance at 517 nm and calculate the clearance rate according to the following formula, which is converted into IC 50 Value (mg / mL).

[0219]

[0220] Where:

[0221] A2 is the absorbance of the sample set, including 50 μL DPPH solution and 100 μL sample;

[0222] A1 is the absorbance of the control group, including 50 μL of anhydrous ethanol solution and 100 μL of sample.

[0223] A0 is the absorbance of the blank group, including 50 μL DPPH and 100 μL deionized water.

[0224] 3. Conclusion:

[0225] IC of the polysaccharide CSWP-40 provided by the present invention in the DPPH antioxidant assay 50 The result was 4.09 mg / mL, showing good antioxidant activity.

[0226] Example 5: Identification of Cordyceps sinensis of different specifications by Cordyceps sinensis polysaccharide CSWP-40

[0227] 5.1 Experimental samples

[0228] Take 12 batches of large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) and mark them as SD-1 to SD-12; take 12 batches of small-sized Cordyceps sinensis (5000 pieces / kg, dry grass) and mark them as SX-1 to SX-12.

[0229] 5.2 Detection Method

[0230] 5.2.1 Preparation of test solution

[0231] Weigh about 0.5g of Cordyceps sinensis powder, weigh accurately, add 20mL of ultrapure water and a few zeolites accurately, weigh, place in a boiling water bath for reflux extraction for 4h, remove, cool, weigh, and make up the lost weight with ultrapure water. Centrifuge. Accurately measure 5mL of supernatant, add 20mL of anhydrous ethanol, seal, mix, place in a 4℃ refrigerator, precipitate for 12h, centrifuge, discard the supernatant, and obtain precipitate 5. Evaporate precipitate 5 until there is no alcohol taste to obtain precipitate 6. Accurately add 10mL of water to dissolve the precipitate to obtain crude polysaccharide extract. Remove protein by Sevag method until the protein is completely removed (repeatedly extract with Sevag solution of one-quarter volume of crude polysaccharide solution until it is clear between the Sevag solution layer (lower layer) and the water layer (upper layer) (about 15 times of extraction)) Take the upper solution obtained after the protein is completely removed and filter it through a 0.2μm filter membrane into a sample injection vial for testing.

[0232] 5.2.2 Preparation of reference solution

[0233] An appropriate amount of the polysaccharide CSWP-40 prepared by the method described in Example 1 was weighed and prepared with water to prepare a 2 mg / mL mother solution for later use.

[0234] 5.2.3 Test conditions

[0235] High performance liquid chromatography was used for separation and ELSD (evaporative light scattering detector) was used for detection;

[0236] The chromatographic column was TSK gel Super Multipore PW-H (150mm×6.0mm, 8μm); the mobile phase was 0.1mol / L ammonium acetate; the isocratic elution was 20min; the flow rate was 0.4mL / min; the column temperature was 35℃; the injection volume of the test sample was 20μL; the injection volume of the reference sample was 1μL, 2μL, 5μL, 10μL, and 20μL. The carrier gas of the ELSD detector (evaporative light scattering detector) was nitrogen; the carrier gas pressure was 3.5bar; the drift tube temperature was 60℃; and the gain value was 6.

[0237] 5.2.4 Calculation formula

[0238] The standard curve is drawn with the logarithm of the injection mass of the reference solution as the abscissa and the logarithm of the peak area as the ordinate to obtain the regression equation; the logarithm of the area of ​​the target peak of the test solution (i.e., the chromatographic peak at the same position as the main peak in the reference solution, and after structural confirmation, the component in the test solution with the same main peak position as the reference solution is CSWP-40 polysaccharide) is substituted into the regression equation to obtain the logarithm of the injection mass of the target substance in the test solution, and the antilogarithm thereof is taken to obtain the injection mass of the target substance in the test solution, and then divided by the injection volume of the test sample to obtain the concentration C (mg / mL) of the target substance in the test solution.

[0239] The CSWP-40 polysaccharide content X (%) in Cordyceps sinensis powder is calculated according to the following formula:

[0240]

[0241] Where:

[0242] C——the concentration of CSWP-40 polysaccharide in the test solution, in milligrams per milliliter (mg / mL);

[0243] V——the volume of the test solution, in milliliters (mL);

[0244] μ——the dilution factor of the test solution relative to the Cordyceps sinensis powder;

[0245] m——Weighing amount of Cordyceps sinensis powder, in milligrams (mg).

[0246] 5.3 Test results

[0247] From the results in Table 5, it can be seen that the average content of CSWP-40 polysaccharide in large-sized Cordyceps sinensis (2000 pieces / kg) is 3.51±0.78% (n=12), and the average content of CSWP-40 polysaccharide in small-sized Cordyceps sinensis (5000 pieces / kg) is 1.27±0.26% (n=12). There is a significant difference in the content between the two, which can be used as a distinguishing point for identifying large-sized and small-sized Cordyceps sinensis.

[0248] Table 5 Polysaccharide content of Cordyceps sinensis CSWP-40 of different specifications

[0249]

[0250] 5.4 Experimental Conclusion

[0251] The results showed that the CSWP-40 polysaccharide content of large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) was relatively high, about 2.69% to 5.04%; the CSWP-40 polysaccharide content of small-sized Cordyceps sinensis (5000 pieces / kg, dry grass) was relatively low, about 0.90% to 1.78%; there was an obvious difference in CSWP-40 polysaccharide content between large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) and small-sized Cordyceps sinensis (5000 pieces / kg, dry grass), and the CSWP-40 polysaccharide content in the Cordyceps sinensis powder could be used to determine whether the Cordyceps sinensis powder belonged to large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) or small-sized Cordyceps sinensis (5000 pieces / kg, dry grass) before crushing.

[0252] Therefore, the identification standard for whether the Cordyceps sinensis powder belongs to the large-sized Cordyceps sinensis (2000 pieces / kg, dry grass) or the small-sized Cordyceps sinensis (5000 pieces / kg, dry grass) before crushing is determined as follows: when the CSWP-40 polysaccharide content in the Cordyceps sinensis powder is not less than 2.24% (2.24% is the intermediate value between the minimum CSWP-40 polysaccharide content of 2.69% of the large-sized Cordyceps sinensis and the maximum CSWP-40 polysaccharide content of 1.78% of the small-sized Cordyceps sinensis in Table 5), it can be determined to be the powder of the large-sized Cordyceps sinensis (2000 pieces / kg); when the CSWP-40 polysaccharide content in the Cordyceps sinensis powder is less than 2.24% and greater than or equal to 0.03% (0.03% is the detection limit of this detection method), it can be determined to be the powder of the small-sized Cordyceps sinensis (5000 pieces / kg).

[0253] Example 6: Identification of the authenticity of Cordyceps sinensis by Cordyceps sinensis polysaccharide CSWP-40

[0254] 6.1 Experimental samples

[0255] Take 12 batches of Cordyceps sinensis (3 batches of Cordyceps sinensis breeding products from Yichang Shancheng Shuidu Cordyceps Co., Ltd., marked as S1-S3; 3 batches of wild Cordyceps sinensis in Tibet, marked as S4-S6; 3 batches of wild Cordyceps sinensis in Sichuan, marked as S7-S9; 3 batches of wild Cordyceps sinensis in Qinghai, marked as S10-S12); 3 batches of Liangshan Cordyceps, marked as S13-S15; 3 batches of Cordyceps subspinipes, marked as S16-S18; 3 batches of Cordyceps gunnii, marked as S19-S21; 3 batches of Cordyceps daisi, marked as S22-S24.

[0256] 6.2 Sample testing and results

[0257] According to "5.2 Detection Method" under Example 5, the test solution was prepared and the CSWP-40 polysaccharide content was determined. The results are shown in Table 6. The results showed that CSWP-40 polysaccharide was detected in Cordyceps sinensis, while CSWP-40 polysaccharide was not detected in Cordyceps liangshanensis, Cordyceps subspinipes, Cordyceps gunnii, and Cordyceps daisi.

[0258] Table 6: CSWP-40 polysaccharide content in Cordyceps sinensis and its counterfeit products (%)

[0259]

[0260] 6.3 Experimental Conclusion

[0261] The results show that the Cordyceps sinensis polysaccharide CSWP-40 prepared by the technology of the present invention can distinguish Cordyceps sinensis and its common counterfeits (Liangshan Cordyceps, Cordyceps sub-spicate, Cordyceps gunnii and Cordyceps dai); that is, the powder of the sample to be tested that is not detected in the test solution (not detected means the content is less than 0.03% (detection limit)) is not the powder of Cordyceps sinensis, and the powder of the sample to be tested that is greater than or equal to 0.03% (0.03% is the detection limit of this detection method) in the test solution is the powder of Cordyceps sinensis.

[0262] The method of the 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 invention to implement and apply the technology of the 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 polysaccharide, characterized in that The structure of the polysaccharide is: Where n represents the number of repeating units, the molecular weight of the polysaccharide is 1.0×10 6 Da~2.0×10 6 Da.

2. The polysaccharide according to claim 1, characterized in that The molecular weight of the polysaccharide is 1.3×10 6 Da.

3. A Cordyceps sinensis extract, characterized in that: The invention comprises the polysaccharide according to any one of claims 1 to 2.

4. A method for preparing the polysaccharide according to any one of claims 1 to 2 or the Cordyceps sinensis extract according to claim 3, characterized in that: The following steps are involved: (1) taking Cordyceps sinensis powder, mixing it with ethanol solution, extracting it under reflux in a water bath, cooling it, and centrifuging it to obtain precipitate 1; (2) taking precipitate 1 and evaporating the ethanol, adding water to the mixture, extracting by reflux in a water bath, cooling and centrifuging, and obtaining a supernatant and precipitate 2; taking precipitate 2 and repeating the above steps of adding water to the mixture, extracting by reflux in a water bath, cooling and centrifuging for 0 to 5 times, and combining the supernatant obtained by centrifugation in step (2); in step (2), mixing 10 ml to 30 ml or 20 ml of water for every 1 g of precipitate 1 or precipitate 2; (3) concentrating the combined supernatant obtained in step (2) to one fifth to one twentieth of the volume before concentration to obtain a concentrated solution, mixing the concentrated solution with 2 to 6 times the volume of anhydrous ethanol, placing at 2 to 8° C. for alcohol precipitation, and then centrifuging, collecting the precipitate and evaporating the ethanol to obtain precipitate 3; (4) After the ethanol in the precipitate 3 is evaporated, it is dissolved in water to remove the protein, dialyzed and concentrated, and the retained solution obtained by the dialysis and concentration is concentrated under reduced pressure and freeze-dried to obtain crude polysaccharide; (5) dissolving the crude polysaccharide obtained in step (4) in water, mixing with anhydrous ethanol, placing at 2 to 8° C. for alcohol precipitation, and centrifuging to obtain a supernatant; (6) mixing the supernatant obtained in step (5) with 80% vol-95% vol ethanol aqueous solution, placing at 2-8° C. for alcohol precipitation and then centrifuging, collecting the precipitate, and evaporating the ethanol to obtain precipitate 4, thereby obtaining the polysaccharide or the Cordyceps sinensis extract; Optionally, the step (6) further comprises dissolving the precipitate 4 in water and freeze-drying to obtain the polysaccharide or the Cordyceps sinensis extract.

5. The preparation method according to claim 4, wherein in step (1), 1 g of the Cordyceps sinensis powder is mixed with 5 ml to 20 ml of ethanol solution; and / or The water bath reflux extraction in step (1) is water bath reflux extraction at 75° C.-85° C.; and / or The ethanol solution in step (1) is a 95% vol-100% vol ethanol aqueous solution; and / or The extraction time of the water bath reflux extraction in step (1) is 2h-6h; The water bath reflux extraction in step (2) is water bath reflux extraction at 95° C.-100° C.; and / or The extraction time of the water bath reflux extraction in step (2) is 2h-6h.

6. The preparation method according to any one of claims 4 to 5, wherein in step (4), each 1 g of the precipitate 3 is dissolved in 50 ml to 150 ml of water; and / or The alcohol precipitation time in step (3) is 8h-24h; and / or The protein removal in step (4) is carried out by the Sevag method; and / or In the step (4), the protein removal is performed by using the Sevag method until all the protein is removed, or the Sevag method is used to remove the protein 10-20 times or 15 times; and / or The molecular weight cut-off of the dialysis concentration in step (4) is 3.0 kDa-5.0 kDa or 3.0 kDa; and / or The reduced pressure concentration in step (4) is concentrated to one-half to one-fifth of the volume before reduced pressure concentration; In step (5), each 1g of the crude polysaccharide is dissolved in 150ml-300ml of water; and / or The volume ratio of the water to the anhydrous ethanol used to dissolve the crude polysaccharide in step (5) is 3:1-4:1; and / or The alcohol precipitation time in step (5) is 8h-24h; and / or The alcohol precipitation time in step (6) is 8h-24h; and / or The volume ratio of the supernatant to the 80% vol-95% vol ethanol aqueous solution in step (6) is 2.5:1.0-4.0:1.0; and / or In the step (6), every 1 g of the precipitate 4 is mixed with 150 ml to 300 ml of water.

7. The preparation method according to claim 4, characterized in that: The following steps are involved: (1) Take Cordyceps sinensis powder and mix it with ethanol solution. For every 1g of Cordyceps sinensis powder, mix it with 5ml of 95% vol ethanol solution. Reflux extraction in a water bath at 80°C for 2h, cool it, and centrifuge it to obtain precipitate 1. (2) taking precipitate 1 and evaporating the ethanol, adding water to the mixture, extracting under reflux at 100° C. in a water bath for 2 h, cooling and centrifuging to obtain a supernatant and precipitate 2; repeating the above-mentioned operation of mixing with water, extracting under reflux at 100° C. in a water bath for 2 h, cooling and centrifuging for 0 to 5 times for precipitate 2, and combining the supernatants obtained by centrifugation in step (2); mixing 1 g of precipitate 1 or precipitate 2 with 20 ml of water; (3) Concentrating the combined supernatant obtained in step (2) to one tenth of its volume before concentration to obtain a concentrated solution, mixing the concentrated solution with anhydrous ethanol in an amount 4 times the volume of the concentrated solution, placing the solution at 2 to 8° C. for alcohol precipitation, and then centrifuging the solution, collecting the precipitate, and evaporating the ethanol to obtain precipitate 3; (4) After the ethanol in the precipitate 3 is evaporated, the precipitate 3 is dissolved in water, and each 1 g of the precipitate 3 is dissolved in 100 ml of water; the protein is removed by Sevag method, and the dialysis concentration is performed, and the molecular weight cutoff of the dialysis concentration is 3.0 kDa; the retained solution obtained by the dialysis concentration is concentrated under reduced pressure to half the volume before the reduced pressure concentration, and freeze-dried to obtain crude polysaccharide; (5) dissolving the crude polysaccharide obtained in step (4) in water, dissolving 1 g of the crude polysaccharide in 200 ml of water; Then, the mixture is mixed with anhydrous ethanol, wherein the volume ratio of the water used to dissolve the crude polysaccharide to the anhydrous ethanol is 4:1; the mixture is placed at 2-8° C. for alcohol precipitation and then centrifuged to obtain a supernatant; (6) mixing the supernatant obtained in step (5) with an 80% vol-95% vol ethanol aqueous solution, the volume ratio of the supernatant and the 80% vol-95% vol ethanol aqueous solution being 2.5:1.0; placing at 2-8° C. for alcohol precipitation and then centrifuging, collecting the precipitate, and evaporating the ethanol to obtain precipitate 4, thereby obtaining the polysaccharide or the Cordyceps sinensis extract; Optionally, the step (6) further comprises dissolving the precipitate 4 in water and freeze-drying to obtain the polysaccharide or the Cordyceps sinensis extract.

8. A pharmaceutical composition, characterized in that The invention comprises the polysaccharide according to any one of claims 1 to 2, the cordyceps sinensis extract according to claim 3, or the polysaccharide or cordyceps sinensis extract prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the polysaccharide according to any one of claims 1 to 2, the Cordyceps sinensis extract according to claim 3, the polysaccharide or Cordyceps sinensis extract prepared by the preparation method according to any one of claims 4 to 7, or the pharmaceutical composition according to claim 8 in the preparation of drugs for immunomodulation and / or anti-oxidation or in identifying the authenticity of Cordyceps sinensis or the specifications of Cordyceps sinensis.

10. A method for identifying the authenticity or specifications of Cordyceps sinensis, characterized in that: include: (1) Preparation of test solution: extract the sample powder with water, centrifuge, take the supernatant, add ethanol to precipitate, centrifuge to obtain precipitate 5, evaporate the ethanol in the precipitate to obtain precipitate 6, add water to dissolve to obtain a crude polysaccharide extract, remove protein in the crude polysaccharide extract by Sevage method, filter, and obtain the test solution; (2) Preparation of reference solution: dissolving the polysaccharide according to any one of claims 1 to 2 or the polysaccharide prepared by the preparation method according to any one of claims 4 to 7 in water to obtain a reference solution; (3) Detection: Detecting the content of the polysaccharide according to any one of claims 1 to 2 in the sample powder to be tested in the test solution; (4) Result judgment.

11. The method according to claim 10, wherein the result is judged as: (i) Criteria for judging the authenticity of Cordyceps sinensis: the powder of the sample to be tested with a content of the polysaccharide described in any one of claims 1 to 2 in the test solution of less than 0.03% is not the powder of Cordyceps sinensis; and / or the powder of the sample to be tested with a content of the polysaccharide described in any one of claims 1 to 2 in the test solution of greater than or equal to 0.03% is the powder of Cordyceps sinensis; and / or (ii) Criteria for judging the specifications of Cordyceps sinensis: the powder of the sample to be tested, in which the content of the polysaccharide according to any one of claims 1 to 2 in the test solution is greater than or equal to 0.03% and less than 2.24%, is powder of small-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not less than 5,000; and / or the powder of the sample to be tested, in which the content of the polysaccharide according to any one of claims 1 to 2 in the test solution is greater than or equal to 2.24%, is powder of large-sized Cordyceps sinensis, and the number of Cordyceps sinensis in each kg of dry Cordyceps sinensis is not higher than 2,000.

12. The method according to any one of claims 10 to 11, wherein the extraction temperature in step (1) is 95°C-100°C; and / or The extraction time of the extraction in step (1) is 2h-6h; and / or In the water extraction in step (1), 20 ml to 60 ml of water is used for every 1 g of the sample powder to be tested; and / or The volume ratio of ethanol to supernatant in step (1) is 2:1-6:1 or 4:1 and / or In the preparation of the crude polysaccharide extract in step (1), the volume ratio of the supernatant before alcohol precipitation used to prepare the precipitate 6 to the water used to dissolve the precipitate 6 is 1:1-1:5; and / or The step (1) of removing protein from the crude polysaccharide extract by the Sevage method comprises removing protein by the Sevag method until all the protein is removed, or removing protein by the Sevag method 10-20 times or 15 times; and / or The concentration of the polysaccharide according to any one of claims 1 to 2 or the polysaccharide prepared by the preparation method according to any one of claims 4 to 7 in the reference solution of step (2) is 2 mg / mL to 5 mg / mL; and / or The step (3) is performed by high performance liquid chromatography coupled with an evaporative light scattering detector; and / or The high performance liquid chromatography method uses a chromatographic column filled with polymethacrylate for separation; and / or The high performance liquid chromatography method uses a TSK gel Super Multipore PW-H chromatographic column for separation; and / or The column length of the chromatographic column is 100-250 mm, or 150 mm; and / or The inner diameter of the chromatographic column is 4.6 mm-6.0 mm; and / or The filler particle size of the chromatographic column is 5 μm-12 μm, or 8 μm; and / or The specifications of the chromatographic column are: column length 150 mm, inner diameter 6.0 mm, filler particle size 8 μm; and / or The high performance liquid chromatography method uses an aqueous solution of ammonium acetate as a mobile phase; and / or The high performance liquid chromatography method uses 0.05mol / L-0.2mol / L or 0.1mol / L ammonium acetate aqueous solution as the mobile phase; and / or The elution mode of the high performance liquid chromatography is isocratic elution; and / or The flow rate of the high performance liquid chromatography is 0.3 ml / min-0.6 ml / min or 0.4 ml / min; and / or The column temperature of the high performance liquid chromatography is 25°C-40°C or 35°C; and / or The carrier gas of the evaporative light scattering detector is nitrogen; and / or The carrier gas pressure of the evaporative light scattering detector is 3 bar-4 bar or 3.5 bar; and / or The drift tube temperature of the evaporative light scattering detector is 40°C-60°C; and / or The gain value of the evaporative light scattering detector is 5-8, or 6.

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