A polysaccharide, cordyceps sinensis extract, and a preparation method and application thereof

Abstract

The present application relates to a kind of polysaccharide, cordyceps sinensis extract and its preparation method and application, relate to the field of traditional Chinese medicinal material extract.The polysaccharide has excellent immunomodulatory and / or antioxidant activity, and can be used to identify the authenticity and specification of cordyceps sinensis.

Description

[0001] Priority information

[0002] This invention claims priority and benefit to patent application 202410161554.3, filed with the China National Intellectual Property Administration on February 4, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of extracts from traditional Chinese medicinal materials, specifically to a polysaccharide and Cordyceps sinensis extract, its preparation method, and its application. Background Technology

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

[0005] Polysaccharides are a class of high-molecular-weight compounds widely found in living organisms. However, due to the influence of proteins, pigments, and other factors during the extraction process, and the inherent characteristics of polysaccharides such as poor stability, high polarity, difficulty in volatility, and high molecular weight, their extraction and purification face numerous challenges.

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

[0007] Therefore, we will continue to explore a Cordyceps sinensis polysaccharide with excellent pharmacological activity and its preparation method. Summary of the Invention

[0008] The purpose of this invention is to provide a polysaccharide, Cordyceps sinensis extract, and its preparation method and application, which have excellent immunomodulatory and / or antioxidant activities.

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

[0010] A polysaccharide with the following structure:

[0011]

[0012] Where n represents the number of repeating units, and the molecular weight of the polysaccharide is 1.0 × 10⁻⁶.6 Da~2.0×10 6 Da.

[0013] In some embodiments, the polysaccharide has a molecular weight of 1.0 × 10⁻⁶. 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 polysaccharide has a molecular weight of 1.3 × 10⁻⁶. 6 Da.

[0015] In some embodiments, n is 600-1200. 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 method for determining the molecular weight of the polysaccharide includes determination using high performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD).

[0017] In some embodiments, the molecular weight determination method for the polysaccharide uses a TSK gel Super Multipore PW-H chromatographic column.

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

[0019] In some embodiments, the method for determining the molecular weight 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 column temperature of the chromatographic column in the method for determining the molecular weight of the polysaccharide 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] Secondly, the present invention provides a Cordyceps sinensis extract.

[0023] A cordyceps extract comprising the polysaccharides described in the first aspect.

[0024] Thirdly, 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 described in the first aspect or the Cordyceps sinensis extract described in the second aspect includes the following steps:

[0026] (1) Take Cordyceps sinensis powder, mix it with ethanol solution, extract by reflux in water bath, cool, centrifuge, and obtain precipitate 1;

[0027] (2) Take precipitate 1, evaporate the ethanol, add water, mix, reflux in a water bath for extraction, cool and centrifuge to obtain supernatant and precipitate 2; take precipitate 2 and repeat the above operation of adding water, reflux in a water bath for extraction, cooling and centrifugation 0-5 times (e.g. 0, 1, 2, 3, 4 or 5 times), and combine the supernatant obtained from centrifugation in step (2); each 1g of precipitate 1 or precipitate 2 is mixed with 10ml-30ml or 20ml of water;

[0028] (3) The combined supernatant obtained in step (2) is concentrated to one-fifth to one-twentieth of the volume before concentration to obtain a concentrated solution. The concentrated solution is mixed with 2-6 times (e.g., 2, 3, 4, 5 or 6 times) the volume of the concentrated solution in anhydrous ethanol, placed at 2-8℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃ or 8℃) for alcohol precipitation, centrifuged, the precipitate is collected and the ethanol is evaporated to obtain precipitate 3;

[0029] (4) Evaporate the ethanol from precipitate 3 and dissolve it in water to remove protein. Dialyze and concentrate the solution. Take the precipitate obtained by dialysis and concentrate it under reduced pressure and freeze dry to obtain crude polysaccharide.

[0030] (5) Dissolve the crude polysaccharide obtained in step (4) in water, then mix it with anhydrous ethanol, place it at 2-8℃ (such as 2℃, 3℃, 4℃, 5℃, 6℃, 7℃ or 8℃) for alcohol precipitation, and then centrifuge to obtain the supernatant.

[0031] (6) The supernatant obtained in step (5) is mixed with an 80% vol-95% vol (e.g., 80% vol, 85% vol, 90% vol or 95% vol) ethanol aqueous solution, placed at 2-8℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃ or 8℃) for alcohol precipitation, centrifuged, the precipitate is collected, the ethanol is evaporated, and precipitate 4 is obtained, thus obtaining the polysaccharide or the Cordyceps sinensis extract.

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

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

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

[0035] In some embodiments, the ethanol solution in step (1) is a 95% vol-100% vol aqueous ethanol 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), each 1g of precipitate 1 or precipitate 2 is mixed with 10ml-30ml of water. In some embodiments, in step (2), each 1g of precipitate 1 or precipitate 2 is mixed with 10ml, 15ml, 20ml, 25ml, or 30ml of water.

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

[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, each 1g of precipitate 3 in step (4) is dissolved in 50ml-150ml of water. In some embodiments, each 1g of precipitate 3 in step (4) 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 performed using the Sevag method.

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

[0044] In some embodiments, the molecular weight cutoff for dialysis concentration in step (4) is 3.0 kDa-5.0 kDa. In some embodiments, the molecular weight cutoff for 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 vacuum concentration in step (4) is to concentrate to one-half to one-fifth of the original volume. In some embodiments, the vacuum concentration in step (4) is to concentrate to one-half, one-third, one-quarter, or one-fifth of the original volume.

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

[0047] In some embodiments, the volume ratio of water to anhydrous ethanol in step (5) for dissolving crude polysaccharide 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 aqueous ethanol 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 aqueous ethanol 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), each 1g of the precipitate 4 is mixed with 150ml-300ml of water. In some embodiments, in step (6), each 1g of the precipitate 4 is mixed with 150ml, 200ml, 250ml or 300ml of water.

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

[0053] (1) Take Cordyceps sinensis powder and mix it with ethanol solution. Each 1g of Cordyceps sinensis powder is mixed with 5ml of 95%vol ethanol solution. Reflux extraction in a water bath at 80℃ for 2h, cool, centrifuge, and obtain precipitate 1.

[0054] (2) Take precipitate 1, evaporate the ethanol, add water and mix, then reflux in a water bath at 100°C for 2 hours, then cool and centrifuge to obtain supernatant and precipitate 2; repeat the above operation of mixing with water and refluxing in a water bath at 100°C for 2 hours, then cooling and centrifuging, 0-5 times (e.g., 0, 1, 2, 3, 4 or 5 times), and combine the supernatant obtained from step (2) centrifugation; mix 1g of precipitate 1 or precipitate 2 with 20ml of water;

[0055] (3) The combined supernatant obtained in step (2) is concentrated to one-tenth of the volume before concentration to obtain a concentrated solution. The concentrated solution is mixed with anhydrous ethanol of 4 times the volume of the concentrated solution, placed at 2-8°C for alcohol precipitation, centrifuged, the precipitate is collected and the ethanol is evaporated to obtain precipitate 3.

[0056] (4) Evaporate the ethanol from precipitate 3 and dissolve it in water. Dissolve 1g of precipitate 3 in 100ml of water. Remove protein by Sevag method, and concentrate by dialysis. The molecular weight cutoff of the dialysis concentration is 3.0kDa. Take the dialysis concentration solution, concentrate it under reduced pressure to half the volume before reduced pressure concentration, freeze dry, and obtain crude polysaccharide.

[0057] (5) Dissolve the crude polysaccharide obtained in step (4) in water, with each 1g of crude polysaccharide dissolved in 200ml of water; then mix with anhydrous ethanol, with the volume ratio of the water to the anhydrous ethanol used to dissolve the crude polysaccharide being 4.0:1.0; place at 2-8℃ for alcohol precipitation and then centrifuge to obtain the supernatant.

[0058] (6) The supernatant obtained in step (5) is mixed with an 80% vol-95% vol ethanol aqueous solution, and the volume ratio of the supernatant to the 80% vol-95% vol ethanol aqueous solution is 2.5:1.0; after ethanol precipitation at 2-8℃, the mixture is centrifuged, the precipitate is collected, the ethanol is evaporated, and precipitate 4 is obtained, thus obtaining the polysaccharide or the Cordyceps sinensis extract.

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

[0060] Fourthly, 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] Fifthly, the present invention provides an application of the above-mentioned polysaccharide, Cordyceps sinensis extract, polysaccharide or Cordyceps sinensis extract obtained by the above preparation method, or the above-mentioned pharmaceutical composition.

[0063] The use of the polysaccharide described in the first aspect, the Cordyceps sinensis extract described in the second aspect, the polysaccharide or Cordyceps sinensis extract prepared by the preparation method described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of products for immunomodulation and / or antioxidation, or in the identification of the authenticity or specifications of Cordyceps sinensis.

[0064] In some embodiments, the immune modulation is to enhance immunity. In some embodiments, the immune modulation includes enhancing macrophage proliferation and / or phagocytic activity, 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 powder of the sample to be tested with water, centrifuge, take the supernatant, add ethanol for alcohol precipitation, centrifuge to obtain precipitate 5, evaporate the ethanol in the precipitate to obtain precipitate 6, add water to dissolve to obtain crude polysaccharide extract, remove protein from crude polysaccharide extract by Sevage method, filter to obtain test solution.

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

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

[0070] (4) Result judgment.

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

[0072] (ii) Criteria for determining the specifications of Cordyceps sinensis: The powder of the test sample in which the polysaccharide content in the test sample solution is greater than or equal to 0.03% and less than 2.24% is the powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis; and / or the powder of the test sample in which the polysaccharide content in the test sample solution is greater than or equal to 2.24% is the powder of large-sized Cordyceps sinensis, wherein the large-sized Cordyceps sinensis contains no more than 2,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

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

[0074] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.10% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0075] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.20% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0076] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.30% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0077] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.40% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0078] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.50% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0079] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.60% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0080] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.70% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0081] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.80% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0082] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.90% and less than 2.24% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0083] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.90% and less than 2.20% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0084] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.90% and less than 2.10% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0085] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.90% and less than 2.00% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0086] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.90% and less than 1.90% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0087] In some embodiments, the sample powder containing polysaccharides in the test solution at a concentration greater than or equal to 0.90% and less than 1.80% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0088] In some embodiments, the sample powder containing polysaccharides in the test solution of the first aspect at a content greater than or equal to 0.90% and less than or equal to 1.78% is powder of small-sized Cordyceps sinensis, wherein the small-sized Cordyceps sinensis contains no less than 5,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0089] In some embodiments, the polysaccharide content in the test sample solution of the first aspect is greater than or equal to 2.24%, and the powder of the test sample is powder of large-sized Cordyceps sinensis, wherein the large-sized Cordyceps sinensis is defined as having no more than 2,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0090] In some embodiments, the polysaccharide content in the test sample solution of the first aspect is greater than or equal to 2.30%, and the test sample powder is large-sized Cordyceps sinensis powder, wherein the large-sized Cordyceps sinensis is defined as having no more than 2000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0091] In some embodiments, the polysaccharide content in the test sample solution of the first aspect is greater than or equal to 2.40%, and the powder of the test sample is large-sized Cordyceps sinensis powder, wherein the large-sized Cordyceps sinensis is defined as having no more than 2,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0092] In some embodiments, the polysaccharide content in the test sample solution of the first aspect is greater than or equal to 2.50%, and the test sample powder is large-sized Cordyceps sinensis powder, wherein the large-sized Cordyceps sinensis is defined as having no more than 2000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0093] In some embodiments, the polysaccharide content in the test sample solution of the first aspect is greater than or equal to 2.60%, and the test sample powder is large-sized Cordyceps sinensis powder, wherein the large-sized Cordyceps sinensis is defined as having no more than 2000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0094] In some embodiments, the polysaccharide content in the test sample solution of the first aspect is greater than or equal to 2.69% and the powder of the test sample is powder of large-sized Cordyceps sinensis, wherein the large-sized Cordyceps sinensis is defined as having no more than 2,000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

[0095] In some embodiments, the sample powder containing polysaccharides in the test solution of the first aspect at a content greater than or equal to 2.69% and less than or equal to 5.04% is powder of large-sized Cordyceps sinensis, wherein the large-sized Cordyceps sinensis is defined as having no more than 2000 Cordyceps sinensis per 1 kg of dried Cordyceps sinensis.

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

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

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

[0099] In some embodiments, the volume ratio of ethanol to supernatant in step (1) is 2.0:1 to 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 to the water used to dissolve 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 to the water used to dissolve 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 removal of protein from the crude polysaccharide extract using the Sevag method in step (1) includes removing protein using the Sevag method until all protein is removed, or removing protein using the Sevag method 10-20 times (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some embodiments, the removal of protein from the crude polysaccharide extract using the Sevag method in step (1) includes removing protein using the Sevag method until all protein is removed, or removing protein using 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 to 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, step (3) is performed 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 packed with polymethyl methacrylate for separation.

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

[0106] In some embodiments, the column length of the chromatographic column is 100mm-250mm. In some embodiments, the column length of the chromatographic column is 100mm, 150mm, 200mm or 250mm.

[0107] In some embodiments, the inner diameter of the chromatographic column is 4.6 mm to 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 particle size of the chromatographic column packing material is 5 μm-12 μm. In some embodiments, the particle size of the chromatographic column packing material is 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, or 12 μm. In some embodiments, the particle size of the chromatographic column packing material is 8 μm.

[0109] In some embodiments, the chromatographic column has the following specifications: a column length of 150 mm, an inner diameter of 6.0 mm, and a packing particle size of 8 μm.

[0110] In some embodiments, the high-performance liquid chromatography method uses an aqueous solution of ammonium acetate as the mobile phase.

[0111] In some embodiments, the high-performance liquid chromatography (HPLC) uses a 0.05 mol / L to 0.2 mol / L ammonium acetate aqueous solution as the mobile phase. In some embodiments, the HPLC uses a 0.1 mol / L ammonium acetate aqueous solution as the mobile phase.

[0112] In some embodiments, the elution method of the high performance liquid chromatography is isocratic elution.

[0113] In some embodiments, the flow rate of the high-performance liquid chromatography (HPLC) is 0.3 ml / min to 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 high-performance liquid chromatography (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 3-4 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 gain value of the evaporative light scattering detector is 5-8. In some embodiments, the gain value of the evaporative light scattering detector is 5, 6, 7, or 8. In some embodiments, the gain value of the evaporative light scattering detector is 6.

[0119] Beneficial effects

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

[0121] (1) The polysaccharide provided by this invention is a novel water-soluble polysaccharide, which has been discovered for the first time in natural extracts. The polysaccharide provided by this 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, and has unexpected technical effects.

[0123] (3) The method provided by the present invention for identifying the authenticity or specifications of Cordyceps sinensis is highly accurate, and there is a significant distinction between genuine and counterfeit Cordyceps sinensis, as well as between different sizes and specifications 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 Explanation

[0126] In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0127] The term “room temperature” refers to ambient temperature, which is between approximately 10°C and approximately 30°C, or approximately 20°C and approximately 30°C, or approximately 25°C.

[0128] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0129] The term "v / v" indicates a volume ratio. The term "%vol" indicates a volume percentage. ABTS represents 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid. DPPH represents 1,1-diphenyl-2-trinitrophenylhydrazine. FRAP refers to the ferric reduction / antioxidant capacity method. The term "solid-liquid ratio" indicates the ratio of the mass of the extract to the volume of the extraction solvent; for example, a solid-liquid ratio of 1g:100ml means that 1g of extract is extracted with 100ml of extraction solvent.

[0130] In the following content, all figures disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a figure with a value of N is disclosed, any figure with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0131] The "molecular weight" of the polysaccharide described in this invention refers to the weight-average molecular weight. Attached Figure Description

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

[0133] Figure 2 The chromatogram shows the monosaccharide composition analysis of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

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

[0135] Figure 4The infrared spectrum of CSWP-40, a polysaccharide from Cordyceps sinensis obtained in Example 1, after methylation.

[0136] Figure 5 The image shows the GC-MS chromatogram of the methylated product of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0137] Figure 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 The image shows the COSY diagram of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0140] Figure 6D The image shows the HSQC diagram of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

[0141] Figure 6E The image shows the HMBC diagram of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1.

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

[0143] Figure 8 The following diagrams show the results of the immunomodulatory activity of Cordyceps sinensis polysaccharide CSWP-40 obtained in Example 1. In the diagram, A shows the effect of CSWP-40 on the proliferation activity of RAW264.7 cells, B shows the effect of CSWP-40 on the phagocytic activity of RAW264.7 cells, C shows the effect of CSWP-40 on the secretion of IL-6 factor in RAW264.7 cells, and D shows the effect of CSWP-40 on the secretion of TNF-α factor in RAW264.7 cells.

[0144] Figure 9 The images show representative high-performance liquid chromatograms of large-sized Cordyceps sinensis (2000 pieces / kg, dried) and small-sized Cordyceps sinensis (5000 pieces / kg, dried) in Example 5.

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

[0146] Figure 11 The images show the high-performance liquid chromatograms of genuine and counterfeit Cordyceps sinensis in Example 6. Detailed Implementation

[0147] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0148] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0149] Materials and equipment for the preparation of 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 analytical grade and purchased from Chengdu Kelon Chemical Co., Ltd. n-Butanol was analytical grade and purchased from Guangdong Huaguang Science & Technology Co., Ltd. Dichloromethane, phosphoric acid, and sodium hydroxide were all analytical grade; glacial acetic acid was chromatographic grade and purchased from Xilong Scientific Co., Ltd. DPPH was analytical grade and purchased from Sigma-Aldrich, USA. 1-Phenylene-3-methyl-5-pyrazolone (PMP), iodomethane, and phenol were all analytical grade; trifluoroacetic acid and ammonium acetate were chromatographic grade; potassium bromide was spectroscopic grade; bovine serum albumin was molecular biology grade; and five monosaccharide standards (rhamnose, 99.0% purity; galactose, 99.6% purity; xylose, 99.4% purity; arabinose, 99.1% purity; fucose, 99.6% purity) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Five monosaccharide reference standards (glucose, 99.8% purity; mannose, 100% purity; glucosamine, 100% purity; glucuronic acid, 99.8% purity; galacturonic acid, 95.1% purity) were purchased from the China National Institutes for Food and Drug Control. Dimethyl sulfoxide (DMSO) was analytical grade. A series of dextran standards (weight-average molecular weights of 1, 12, 80, 150, 410, and 670 kDa) were purchased from Sigma-Aldrich, USA. Acetonitrile was chromatographic grade and purchased from KRUDE, USA. Coomassie Brilliant Blue G-250 was analytical grade and purchased from Bio-Rad, USA. Heavy water was analytical grade and purchased from Beijing Innocare 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 Research Institute, Japan; Neutrophil proliferation and cytotoxicity assay kit (batch number 092222230516) was purchased from Beyotime Biotechnology Co., Ltd., Shanghai; 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, 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 thermostatic water bath (Gongyi Yuhua Instrument Co., Ltd.); Sorvall ST40R general-purpose benchtop centrifuge (Thermo Fisher Scientific, USA); RV10 DS25 rotary evaporator, V3 S025 vortex mixer (IKA, Germany); Tissue / Grinder 2020 high-throughput tissue homogenizer (Creant (Beijing) Co., Ltd.); Synergy H1 multi-functional 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 UV-Vis spectrophotometer, 5977B gas chromatography-mass spectrometry system (Agilent Technologies, USA); SEDEX Evaporative light scattering detector (SEDERE, France); TENSOR II infrared spectrometer and Avance III HD 600 nuclear magnetic resonance spectrometer (Bruker, USA); ED115 electric thermostatic drying oven (BINDER, Germany); SBL-22DT thermostatic ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); TURBOVAP-LV nitrogen blower (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 Scientific, 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 polysaccharides or the Cordyceps sinensis extract

[0157] Prepare the polysaccharide or the Cordyceps sinensis extract according to the following steps:

[0158] (1) Take Cordyceps sinensis powder and mix it with ethanol solution. Each 1g of Cordyceps sinensis powder is mixed with 5ml of 95%vol ethanol solution. Reflux extraction in a water bath at 80℃ for 2h, cool, centrifuge, and obtain precipitate 1.

[0159] (2) Take precipitate 1, evaporate the ethanol, add water and mix (each 1g of precipitate 1 is mixed with 20ml of water), reflux in a water bath at 100℃ for 2h, then cool and centrifuge to obtain supernatant and precipitate 2; repeat the above operation of "mix with water and reflux in a water bath at 100℃ for 2h, then cool and centrifuge" twice (each operation is mixed with 20ml of water for each 1g of precipitate 2), and combine the supernatant obtained from the centrifugation in step (2) three times;

[0160] (3) The combined supernatant obtained in step (2) is concentrated to one-tenth of the volume before concentration to obtain a concentrated solution. The concentrated solution is mixed with anhydrous ethanol at 4 times the volume of the concentrated solution. After alcohol precipitation at 2-8℃, the mixture is centrifuged, the precipitate is collected and the ethanol is evaporated to obtain precipitate 3.

[0161] (4) Evaporate the ethanol from precipitate 3 and dissolve it in water. Dissolve 1g of precipitate 3 in 100ml of water to obtain a crude polysaccharide solution. Remove protein by Sevag method until all protein is removed (repeatedly extract with one-quarter volume of Sevag solution of crude polysaccharide solution until the Sevag solution layer (lower layer) and water layer (upper layer) are clear (about 15 extractions)). Take the upper layer solution obtained after protein removal and dialyze to concentrate it. The molecular weight cutoff of the dialyzed concentration is 3.0kDa. Take the retentate obtained by dialyz concentration, concentrate it under reduced pressure to half the volume before reduced pressure concentration, freeze dry, and obtain crude polysaccharide.

[0162] (5) Dissolve the crude polysaccharide obtained in step (4) in water, with each 1g of crude polysaccharide dissolved in 200ml of water; then mix with anhydrous ethanol, with the volume ratio of the water to the anhydrous ethanol used to dissolve the crude polysaccharide being 4.0:1.0; place at 2-8℃ for alcohol precipitation and then centrifuge to obtain the supernatant.

[0163] (6) The supernatant obtained in step (5) is mixed with an 80% vol-95% vol ethanol aqueous solution at a volume ratio of 2.5:1.0; after ethanol precipitation at 2-8℃, the mixture is centrifuged, the precipitate is collected, and the ethanol is evaporated to obtain precipitate 4; precipitate 4 is dissolved in water and freeze-dried to obtain the polysaccharide CSWP-40, with a yield of 1.41%. Example 2: Structural characterization of Cordyceps sinensis polysaccharide CSWP-40

[0164] 1. Molecular weight distribution detection

[0165] A suitable amount of polysaccharide CSWP-40 was prepared into a 2 mg / mL solution with water. After filtration through a 0.22 μm microporous membrane, the molecular weight distribution of the polysaccharide was determined by high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD). Detection conditions: chromatographic column: TSK gel Super Multipore PW-H (150 mm × 6.0 mm, 8 μm); mobile phase: 0.1 mol / L ammonium acetate; isocratic elution for 20 min; flow rate: 0.4 mL / min; column temperature: 35℃; injection volume: 20 μL; ELSD detector carrier gas: nitrogen; carrier gas pressure: 3.5 bar; drift tube temperature: 60℃; gain: 6. The logarithm of the molecular weight of the dextran series standards (lg M) was plotted on the x-axis as retention time. w Using X as the ordinate, a standard curve was plotted: Y = -1.465X + 13.68, r = 0.9992. Based on the retention times of each chromatographic peak in the sample solution, the molecular weight M of the polysaccharide CSWP-40 was determined from the standard curve. w 1.3×10 6 Da, the results are as follows Figure 1 .

[0166] 2. Determination of total sugar content

[0167] Take an appropriate amount of polysaccharide CSWP-40 and prepare a sample solution of 0.1 mg / mL with water. Use glucose as a reference standard and determine the total sugar content using the sulfuric acid-phenol method. Pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose reference solution (0.1 mg / mL) and 1.0 mL of sample solution into 10 mL colorimetric tubes, respectively. If the reference solution is less than 1.0 mL, add water to bring the volume to 1.0 mL. Simultaneously, replace the sample solution with 1.0 mL of water as a blank control. Then add 1 mL of 5% (w / v) phenol solution and 5 mL of concentrated sulfuric acid to each tube, vortex to mix, and let stand for 30 min. Measure the absorbance at 490 nm. Plot the standard curve with glucose concentration on the x-axis and absorbance on the y-axis: Y = 9.6888X + 0.0106, r = 0.9997. Based on the absorbance readings of the samples, the total sugar concentration in the samples was determined using the standard curve. The total sugar content of polysaccharide CSWP-40 was calculated to be 98.25% using the following formula.

[0168]

[0169] In the formula: C is the total sugar concentration 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 and prepare a sample solution of 2.0 mg / mL with water. Use bovine serum albumin (BSA) as a reference standard and determine the protein content using the Bradford method. Pipette 0.01 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, and 0.10 mL of BSA reference solution (1.0 mg / mL) and 0.10 mL of sample solution into 10 mL colorimetric tubes, respectively. If the reference solution is less than 0.10 mL, add water to bring the volume to 0.10 mL. Simultaneously, replace the sample solution with 0.10 mL of water as a blank control. Add 5 mL of Coomassie brilliant blue staining solution to each tube, vortex to mix, and let stand for 5 min. Measure the absorbance at 595 nm. Plot the standard curve with BSA concentration as the x-axis and absorbance as the y-axis: Y = 0.7912X + 0.0842, r = 0.9984. Based on the absorbance reading of the sample, the mass concentration of protein in the sample is obtained from the standard curve. The protein content in polysaccharide CSWP-40 is calculated to be 0% according to the following formula.

[0172]

[0173] In the formula: 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 mass of polysaccharide CSWP-40 when preparing the sample solution, in mg.

[0174] The results of total sugar and protein content show 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 sample solution of 2 mg / mL 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 tightly, and hydrolyze at 120℃ for 2 h. Remove and cool to room temperature, blow dry with nitrogen, then add 1 mL of anhydrous methanol, mix well, blow dry with nitrogen, and repeat 3 times. Dissolve the residue in 1 mL of water, mix well, and the resulting polysaccharide hydrolysate is obtained. Accurately pipette 100 μL each of the polysaccharide hydrolysate and the mixed sugar reference solution (containing approximately 0.05 mg / mL each of mannose (Man), glucosamine (GlcN), rhamnose (Rha), glucuronic acid (GlcUA), galacturonic acid (GlaUA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc)) into 2 mL centrifuge tubes. Add 50 μL of 0.6 mol / L sodium hydroxide solution, followed by 0.6 mol / L sodium hydroxide solution. 100 μL of PMP-methanol solution was thoroughly mixed, sealed, and reacted in a 70℃ water bath for 60 min. After cooling to room temperature, 100 μL of 0.3 mol / L hydrochloric acid solution was added for neutralization, followed by 650 μL of ultrapure water. The mixture was thoroughly mixed, and 500 μL of the solution was transferred to a 2 mL centrifuge tube. 1 mL of chloroform was added, and the mixture was vortexed for 1 min. The tube was then centrifuged at 12000 rpm for 10 min. The supernatant was collected, and the monosaccharide composition was analyzed using high-performance liquid chromatography (HPLC). Detection conditions: Agilent Eclipse XDB-C column. 18 (250mm × 4.6mm, 5μm); Mobile phase A (0.1mol / L phosphate buffer (pH = 6.7)): Mobile phase B (acetonitrile) = 83:17, isocratic elution for 50 min; Injection volume 5μL; Detection wavelength 245nm; Flow rate 1.0mL / min; Column temperature 30℃. The results showed that the polysaccharide CSWP-40 is composed of glucose, as shown in the following figures. Figure 2 .

[0177] 5. Infrared spectroscopy analysis

[0178] Take an appropriate amount of potassium bromide powder and grind it finely in an agate mortar until no obvious reflective crystals remain. Weigh approximately 200 mg into another small agate mortar, add approximately 2 mg of polysaccharide CSWP-40 powder, and grind until thoroughly mixed to obtain the sample powder to be tested. Dry the sample powder under an infrared drying oven lamp for approximately 5 minutes. After cooling, transfer the sample powder to a tableting mold and press it under a pressure of 10 MPa for approximately 2 minutes. Remove the pressed sample tablet and place it in a sample holder for testing. Instrument parameters: Scanning spectral range: 4000-400 cm⁻¹ -1 Resolution: 4cm -1Scan count: 16. Data processing: Water vapor compensation was selected using water compensation and carbon dioxide compensation; Baseline correction was selected using concave rubberband correction (10 iterations; 64 baseline points; carbon dioxide band excluded); Smoothing points: 25.

[0179] The results showed that the polysaccharide CSWP-40 was at 3389.61 cm⁻¹. -1 The broad peak that appears is the absorption peak of the stretching vibration of OH; at 2926.15 cm⁻¹ -1 The peak that appears is the absorption peak of the CH stretching vibration; at 1643.74 cm⁻¹. -1 The peaks that appear are absorption peaks due to the small amount of water of crystallization in the sugar; in the range of 1100–1010 cm⁻¹ -1 There are three absorption peaks (located at 1153.88 cm⁻¹). -1 1081.93cm -1 1023.42cm -1 This indicates that the glycosidic bond is of the pyran type; 854.63cm -1 The characteristic absorption peak for α-glycosidic bonds is located at 1750–1700 cm⁻¹. -1 The absence of absorption peaks indicates that polysaccharide CSWP-40 does not contain uronic acid. See the infrared spectrum below. Figure 3 .

[0180] 6. Methylation analysis

[0181] Take approximately 5 mg of polysaccharide CSWP-40 into a screw-top test tube, add 1.5 mL of dimethyl sulfoxide, seal tightly, and sonicate for 30 min to dissolve the sample. Then add approximately 20 mg of dry sodium hydroxide powder, sonicate for 30 min, and cool in an ice bath until the sample solution solidifies. Under light-protected conditions, add 0.1 mL of iodomethane, sonicate for 30 min (controlling the temperature at 18-20℃), and repeat the operation 3 times. Finally, add 1 mL of water to terminate the methylation reaction. Add 0.5 mL of chloroform to the reaction solution, vortex to mix, and allow to stand for layering. Remove the chloroform layer, repeat the operation 4 times, combine the chloroform layers, wash 3 times with an equal volume of water, and dry the chloroform layer with nitrogen. Take an appropriate amount of solution, spread it on the prepared potassium bromide slide, dry it under an infrared lamp, and then detect its infrared spectrum at 3389.61 cm⁻¹. -1 Whether the hydroxyl peak disappears at a certain point indicates whether methylation is complete (see [reference]). Figure 5The methylated polysaccharide was added to 1 mL of 2 mol / L trifluoroacetic acid, sealed tightly, and hydrolyzed at 120 °C for 2 h. After cooling to room temperature, it was dried under nitrogen. Then, 0.5 mL of anhydrous methanol was added, and the mixture was dried under nitrogen. This process was repeated three times. 0.5 mL of freshly prepared 10 mg / mL sodium borodeuteride was added, mixed well, and allowed to react at room temperature for 12 h, shaking occasionally. 4 mol / L acetic acid was added dropwise to neutralize the solution, and the pH was checked with pH paper. 0.5 mL of 5% (v / v) acetic acid-methanol was added, and the mixture was dried under nitrogen. This process was repeated three times. Then, 0.5 mL of anhydrous methanol was added, and the mixture was dried under nitrogen. This process was repeated three times. Add 1 mL of acetic anhydride, mix well, seal tightly, react at 100 °C for 2.5 h, cool to room temperature, dry under nitrogen, add 0.5 mL of anhydrous methanol, dry under nitrogen, repeat 3 times, add 1 mL of dichloromethane, vortex, centrifuge at 5000 r / min for 5 min, take the supernatant, filter through a 0.2 μm filter membrane, and detect by gas chromatography-mass spectrometry (GC-MS). Detection conditions: chromatographic column: DB-5MS capillary column (30m×0.25mm×0.25μm); carrier gas: high-purity nitrogen; gas flow rate: 1mL / min; injection port temperature: 260℃; injection volume: 1μL; split injection, split ratio: 50:1; solvent delay: 2.2min; temperature program (from 100℃ to 165℃ at 15℃ / min, hold for 30min, then at 2℃ / min to 180℃, then at 15℃ / min to 300℃, hold for 5min); electron impact ion source (EI); ion source temperature: 230℃; quadrupole temperature: 150℃; electron energy: 70eV; transfer line temperature: 300℃; scan mode: full scan mode (SCAN), mass scan range (m / z): 30-600.

[0182] The detection results showed that the polysaccharide CSWP-40 had three structural derivatives: 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 approximately 12.4:77.5:10.0. The results are shown in Table 1. Figure 5 .

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

[0184]

[0185]

[0186] 7. Nuclear magnetic resonance spectroscopy analysis

[0187] Dissolve approximately 30 mg of polysaccharide CSWP-40 in 1 mL of heavy water, blow dry with nitrogen, repeat 3 times, then dissolve in 0.6 mL of heavy water. After complete dissolution, transfer to an NMR tube and detect using an NMR spectrometer. Sample detection conditions: 1 H-NMR temperature 98K, frequency 599M; 13 C-NMR temperature: 298K, frequency 150M. 1D ( 1 H and 13 The chemical structure of the polysaccharide of the present invention was characterized in detail by C) and 2D (COSY, HSQC, HMBC) NMR spectroscopy.

[0188] exist 1 In the 1H 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). These signals were analyzed by the cross-peaks in the anomeric region of the HSQC spectrum. 13 C10 NMR determined the anodic carbon signal of residue A to be 99.80 ppm, residue B to be 98.61 ppm, and residue C to be 98.44 ppm. These anodic proton and anodic carbon chemical shifts indicate that all three residues are in the α-configuration. Further analysis using COSY et al. on all... 1 H and 13 The C signal was assigned. Using COSY correlation spectroscopy, the proton chemical shifts of residue A from H-1 to H-5 were assigned (5.31 ppm, 3.52 ppm, 3.89 ppm, 3.58 ppm, and 3.89 ppm, respectively); using HSQC correlation spectroscopy, the proton chemical shifts of residue A from H-1 to H-5 were assigned. 13 Chemical shifts at C1 to C6 were assigned as follows: 99.80 ppm, 71.64 ppm, 73.21 ppm, 76.90 ppm, 71.62 ppm, and 60.54 ppm, respectively. Further HSQC and COSY correlation spectra assigned H-6a and H-6b signals of 3.78 ppm and 3.69 ppm, respectively, to residue A. The lower field shift of the C-4 position indicates substitution at C-4. Combined with methylation analysis, residue A is inferred to be: →4)-α-D-Glcp-(1→). COSY correlation spectra assigned proton chemical shifts from H-1 to H-5 to residue B (4.90 ppm, 3.49 ppm, 3.94 ppm, 3.65 ppm, and 3.76 ppm, respectively). HSQC correlation spectra were used to assign the proton chemical shifts of residue B from H-1 to H-5. 13Chemical shifts of residue C were assigned, with C1–C6 values ​​of 98.61 ppm, 72.30 ppm, 73.23 ppm, 69.35 ppm, 71.23 ppm, and 60.50 ppm, respectively. HSQC and COSY correlation spectra assigned H-6a and H-6b signals of 3.78 ppm and 3.69 ppm, respectively. Residue B was inferred to be α-D-Glcp-(1→). COSY correlation spectra assigned proton chemical shifts of residue C from H-1 to H-5 (4.89 ppm, 3.46 ppm, 3.84 ppm, 3.76 ppm, and 3.94 ppm, respectively); HSQC correlation spectra were used to assign proton chemical shifts of residue C. 13 Chemical shifts of C1 to C6 were assigned as follows: 98.44 ppm, 71.53 ppm, 72.85 ppm, 76.81 ppm, 72.30 ppm, and 69.38 ppm, respectively. The H-6 signal in residue C was assigned as 3.35 ppm using HSQC and COSY correlation spectra. The chemical shifts of C-4 and C-6 shifted to a lower field, and there was no coupled hydrogen signal at C-6, indicating substitution at C-4 and C-6. Residue C was inferred to be: →4,6)-α-D-Glcp-(1→). NMR data for the polysaccharide are shown in Table 2 and Figure 6.

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

[0190] sugar residues 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] Through analysis and summarization of the above chemical, spectroscopic, and wave-spectral data, it was finally determined that the Cordyceps sinensis polysaccharide obtained in this invention has a novel structure, with its main chain being →4)-α-D-Glcp-(1→), and the branch point being a glucan at the C-6 position. A schematic diagram of the structure is shown below. Figure 7 That is, the structure is as follows:

[0192]

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

[0194] 1. Cell culture and drug preparation

[0195] RAW264.7 cells were cultured in DMEM complete medium at 37°C in a 5% CO2 incubator. They were passaged daily, the old medium was discarded, the cells were gently washed once with PBS, 1 mL of fresh medium was added, the culture flask was gently tapped, and microscopic observation was performed until 80% cell detachment was observed. The detached cells were collected and counted at a rate of 1×10⁻⁶ cells / mL. 7 Cell / bottle passage.

[0196] Sample solution preparation: Accurately weigh the polysaccharide CSWP-40 sample prepared according to the method described in Example 1, dissolve it in DMEM complete medium to prepare a stock solution of 2 mg / mL, filter it through a 0.22 μm sterile microporous 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 the logarithmic growth phase, count them, resuspend the cells in DMEM complete medium, and adjust the cell concentration to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL per well in a 96-well plate. After incubation at 37°C and 5% CO2 for 24 h, the culture medium was discarded. 100 μL of different concentrations of sample solution (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) were added to each well, with three replicates for each concentration, forming the CSWP-40 group. Negative control wells (cells + 100 μL DMEM complete medium) and blank wells (100 μL DMEM complete medium) were also included. After completing the above steps, the 96-well plate was incubated at 37°C and 5% CO2 for another 24 h. Subsequent procedures were then performed according to the CCK-8 kit instructions. The absorbance was measured at 450 nm using an ELISA reader, and the cell proliferation rate induced by the drug was calculated using the following formula:

[0200]

[0201] In the formula: 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 within the concentration range of 10–1000 μg / mL, indicating that CSWP-40 promotes the proliferation of RAW264.7 cells within this concentration range. (See attached results.) Figure 8 Figure A in the middle.

[0203] 2.2 Detection of phagocytic activity

[0204] Collect cells in the logarithmic growth phase, count them, resuspend the cells in DMEM complete medium, and adjust the cell concentration to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL per well in 96-well plates. After incubation at 37°C and 5% CO2 for 24 h, the culture medium was discarded. 100 μL of different concentrations of sample solution (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) were added to each well, with three replicates for each concentration, forming the CSWP-40 group. Negative control wells (cells + 100 μL DMEM complete medium) and blank wells (100 μL DMEM complete medium) were also included. After completing the above steps, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. Subsequent operations were performed according to the instructions of the Neutral Erythrocyte Proliferation and Cytotoxicity Assay Kit. The absorbance 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] In the formula: A s : Absorbance of the sample (cells + sample); A c Absorbance of the 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 significantly enhanced the phagocytic activity of RAW264.7 cells at concentrations ranging from 31.3 μg / mL to 500 μg / mL, but this effect was not concentration-dependent, with a peak concentration of 62.5 μg / mL. This indicates that CSWP-40 has a certain enhancing effect on the phagocytic activity of RAW264.7 cells at concentrations ranging from 31.3 μg / mL to 500 μg / mL. (See attached figures.) Figure 8 Figure B in the middle.

[0208] 2.3 Detection of IL-6 cytokine levels

[0209] Supernatant from RAW 264.7 cells treated for 24 h 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, and 1000 μg / mL) was collected, with each concentration in triplicate, serving as the CSWP-40 group. Negative control wells (cells + 100 μL DMEM complete medium) and blank wells (100 μL DMEM complete medium) were also included. 50 μL of each solution was added to a 96-well plate, and subsequent operations were performed according to the IL-6 kit instructions. The absorbance was measured at 450 nm using a microplate reader. A standard curve for IL-6 was plotted, and the IL-6 concentration was calculated.

[0210] Results: Compared with the negative control group, the CSWP-40 group significantly increased IL-6 secretion from RAW264.7 cells within the concentration range of 500 μg / mL to 1000 μg / mL, indicating that CSWP-40 promoted IL-6 secretion from RAW264.7 cells within this concentration range; its half-maximal effect concentration (EC50) was also significantly higher than that of the negative control group. 50 The concentration was 533.5 μg / mL. See the results below. Figure 8 Figure C in the middle.

[0211] 2.4 Detection of TNF-α cytokine levels

[0212] Supernatant from RAW 264.7 cells treated for 24 h 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, and 1000 μg / mL) was collected. Each concentration was processed in triplicate, forming the CSWP-40 group. Negative control wells (cells + 100 μL DMEM complete medium) and blank wells (100 μL DMEM complete medium) were also included. 50 μL of each solution was added to a 96-well plate, and subsequent operations were performed according to the TNF-α kit instructions. The absorbance was measured at 450 nm using a microplate reader. A standard curve for TNF-α was plotted, and the TNF-α concentration was calculated.

[0213] Results: Compared with the negative control group, the CSWP-40 group significantly increased TNF-α secretion from RAW264.7 cells within the concentration range of 62.5 μg / mL to 1000 μg / mL in a concentration-dependent manner, indicating that CSWP-40 promoted TNF-α secretion from RAW264.7 cells within the concentration range of 62.5 μg / mL to 1000 μg / mL; its half-maximal effective concentration (EC50) was also significantly increased. 50 The concentration was 649.2 μg / mL. See the results below. Figure 8 Diagram D in the middle.

[0214] Example 4: DPPH antioxidant activity test of Cordyceps sinensis 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, add 5 mL of ultrapure water to dissolve, vortex to mix, and obtain a sample solution with a concentration of 10 mg / mL. Then, take appropriate amounts of the 10 mg / mL sample solution and dilute with ultrapure water to obtain sample solutions with concentrations of 7.5 mg / mL, 5 mg / mL, 2.5 mg / mL, and 1 mg / mL, respectively.

[0217] 2. Activity Assay

[0218] Pipette 100 μL of sample solution and add 50 μL of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution (80 μg / mL). Mix thoroughly and incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm. Calculate the clearance rate using the following formula and convert it to IC50. 50 Value (mg / mL).

[0219]

[0220] In the formula:

[0221] A2 is the absorbance of the sample group, which includes 50 μL of DPPH solution and 100 μL of sample;

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

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

[0224] 3. Conclusion:

[0225] The IC50 of the polysaccharide CSWP-40 provided by this invention in the DPPH antioxidant assay 50 The result was 4.09 mg / mL, demonstrating good antioxidant activity.

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

[0227] 5.1 Experimental Samples

[0228] Twelve batches of large-sized Cordyceps sinensis (2000 pieces / kg, dried) were labeled as SD-1 to SD-12; twelve batches of small-sized Cordyceps sinensis (5000 pieces / kg, dried) were labeled as SX-1 to SX-12.

[0229] 5.2 Detection Method

[0230] 5.2.1 Preparation of test solution

[0231] Weigh approximately 0.5g of Cordyceps sinensis powder accurately, add 20mL of ultrapure water and a few boiling stones, weigh, and reflux in a boiling water bath for 4 hours. Remove, cool, weigh, and replenish the lost weight with ultrapure water. Centrifuge. Accurately measure 5mL of the supernatant, add 20mL of anhydrous ethanol, seal, mix well, and place in a 4℃ refrigerator for ethanol precipitation for 12 hours. Centrifuge, discard the supernatant, and obtain precipitate 5. Evaporate precipitate 5 until no alcohol odor remains, obtaining precipitate 6. Accurately add 10mL of water to dissolve the precipitate, thus obtaining the crude polysaccharide extract. Remove protein using the Sevag method until all protein is removed (repeatedly extract with one-quarter volume of Sevag solution from the crude polysaccharide solution until the Sevag solution layer (lower layer) and the water layer (upper layer) are clear (approximately 15 extractions)). After complete protein removal, filter the resulting upper layer solution through a 0.2μm filter membrane into a sample vial for analysis.

[0232] 5.2.2 Preparation of reference solution

[0233] Weigh an appropriate amount of the polysaccharide CSWP-40 prepared by the method described in Example 1, and prepare a 2 mg / mL stock solution with water for later use.

[0234] 5.2.3 Testing Conditions

[0235] The separation was performed using high performance liquid chromatography, and the detection was performed using an ELSD detector (evaporative light scattering detector).

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

[0237] 5.2.4 Calculation Formula

[0238] A standard curve was plotted with the logarithm of the injected mass of the reference solution as the abscissa and the logarithm of the peak area as the ordinate, yielding a regression equation. The logarithm of the area of ​​the target peak in the test solution (i.e., the chromatographic peak with the same position as the main peak in the reference solution, which, after structural confirmation, is the component in the test solution with the same position as the main peak in the reference solution as CSWP-40 polysaccharide) was substituted into the regression equation to obtain the logarithm of the injected mass of the target substance in the test solution. The antilogarithm was then calculated to obtain the injected mass of the target substance in the test solution. This mass was then divided by the injected volume of the test solution 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] In the formula:

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

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

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

[0245] m — The weight of Cordyceps sinensis powder, expressed in milligrams (mg).

[0246] 5.3 Test Results

[0247] As shown in Table 5, the average content of CSWP-40 polysaccharide in large-sized Cordyceps sinensis (2000 pieces / kg) was 3.51±0.78% (n=12), while the average content of CSWP-40 polysaccharide in small-sized Cordyceps sinensis (5000 pieces / kg) was 1.27±0.26% (n=12). The content of the two was significantly different and can be used as a distinguishing point between large-sized and small-sized Cordyceps sinensis.

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

[0249]

[0250] 5.4 Experimental Conclusions

[0251] The results showed that the CSWP-40 polysaccharide content of large-sized Cordyceps sinensis (2000 pieces / kg, dried) was higher, ranging from approximately 2.69% to 5.04%; while the CSWP-40 polysaccharide content of small-sized Cordyceps sinensis (5000 pieces / kg, dried) was lower, ranging from approximately 0.90% to 1.78%. There was a significant difference in CSWP-40 polysaccharide content between large-sized and small-sized Cordyceps sinensis (5000 pieces / kg, dried). Therefore, the CSWP-40 polysaccharide content in the Cordyceps sinensis powder can be used to determine whether the Cordyceps sinensis powder was made from large-sized (2000 pieces / kg, dried) or small-sized (5000 pieces / kg, dried) Cordyceps sinensis before pulverization.

[0252] Therefore, the identification standard for whether Cordyceps sinensis powder belongs to large-sized Cordyceps sinensis (2000 pieces / kg, dried) or small-sized Cordyceps sinensis (5000 pieces / kg, dried) before pulverization 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 median value between the lowest CSWP-40 polysaccharide content of large-sized Cordyceps sinensis (2.69%) and the highest CSWP-40 polysaccharide content of small-sized Cordyceps sinensis (1.78%) in Table 5), it can be determined to be large-sized Cordyceps sinensis (2000 pieces / kg) powder; when the CSWP-40 polysaccharide content in the Cordyceps sinensis powder is less than 2.24% but greater than or equal to 0.03% (0.03% is the detection limit of this detection method), it can be determined to be small-sized Cordyceps sinensis (5000 pieces / kg) powder.

[0253] Example 6: Identifying genuine Cordyceps sinensis using Cordyceps sinensis polysaccharide CSWP-40

[0254] 6.1 Experimental Samples

[0255] Twelve batches of Cordyceps sinensis were collected (three batches of Cordyceps sinensis bred by Yichang Shancheng Shuidu Cordyceps sinensis Co., Ltd., marked as S1-S3; three batches of wild Cordyceps sinensis from Tibet, marked as S4-S6; three batches of wild Cordyceps sinensis from Sichuan, marked as S7-S9; three batches of wild Cordyceps sinensis from Qinghai, marked as S10-S12); three batches of Cordyceps sinensis from Liangshan, marked as S13-S15; three batches of Cordyceps militaris, marked as S16-S18; three batches of Cordyceps kuningii, marked as S19-S21; and three batches of Cordyceps daisy, marked as S22-S24.

[0256] 6.2 Sample Testing and Results

[0257] The test solution was prepared according to the "5.2 Detection Method" under Example 5, and the content of CSWP-40 polysaccharide was determined. The results are shown in Table 6. The results show that CSWP-40 polysaccharide was detected in all Cordyceps sinensis, while it was not detected in Cordyceps militaris, Cordyceps truncatula, Cordyceps guilloché, and Cordyceps daisy.

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

[0259]

[0260] 6.3 Experimental Conclusions

[0261] The results show that the Cordyceps sinensis polysaccharide CSWP-40 prepared by the technology of this invention can distinguish Cordyceps sinensis from its common adulterants (Cordyceps liangshanensis, Cordyceps militaris, Cordyceps guinea, and Cordyceps daisy). That is, the powder of the test sample that is not detected in the test solution (not detected indicates a content <0.03% (detection limit)) is not Cordyceps sinensis powder, while the powder of the test sample that has a content greater than or equal to 0.03% in the test solution (0.03% is the detection limit of this detection method) is Cordyceps sinensis powder.

[0262] The method of the invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A polysaccharide, characterized in that, The structure of the polysaccharide is as follows: ， Where n represents the number of repeating units, and the molecular weight of the polysaccharide is 1.3 × 10⁻⁶. 6 Da.

2. A Cordyceps sinensis extract, characterized in that, Includes the polysaccharide described in claim 1.

3. A method for preparing the polysaccharide of claim 1 or the Cordyceps sinensis extract of claim 2, characterized in that, Includes the following steps: (1) Take Cordyceps sinensis powder, mix it with ethanol solution, extract by reflux in water bath, cool, centrifuge, and obtain precipitate 1; (2) Take precipitate 1, evaporate the ethanol, add water, mix, reflux in a water bath for extraction, cool and centrifuge to obtain supernatant and precipitate 2; take precipitate 2 and repeat the above operation of adding water, reflux in a water bath for extraction, cooling and centrifugation 0-5 times, and combine the supernatant obtained from centrifugation in step (2); in step (2), each 1 g of precipitate 1 or precipitate 2 is mixed with 10 ml - 30 ml of water; (3) The combined supernatant obtained in step (2) is concentrated to one-fifth to one-twentieth of the volume before concentration to obtain a concentrated solution. The concentrated solution is mixed with 2-6 times the volume of anhydrous ethanol, placed at 2-8°C for alcohol precipitation, centrifuged, the precipitate is collected and the ethanol is evaporated to obtain precipitate 3. (4) After evaporating the ethanol from precipitate 3, dissolve it in water to remove protein, dialyze and concentrate, take the retentate obtained by dialysis and concentrate under reduced pressure, freeze dry, and obtain crude polysaccharide. (5) Dissolve the crude polysaccharide obtained in step (4) in water, then mix it with anhydrous ethanol, place it at 2~8℃ for alcohol precipitation, and centrifuge to obtain the supernatant; (6) The supernatant obtained in step (5) is mixed with an 80% vol-95% vol ethanol aqueous solution, placed at 2~8℃ for alcohol precipitation, centrifuged, the precipitate is collected, the ethanol is evaporated, and precipitate 4 is obtained, thus obtaining the polysaccharide or the Cordyceps sinensis extract.

4. According to the preparation method of claim 3, in step (2), each 1 g of precipitate 1 or precipitate 2 is mixed with 20 ml of water.

5. The preparation method according to claim 3, wherein step (6) further includes dissolving precipitate 4 in water and freeze-drying it to obtain the polysaccharide or the Cordyceps sinensis extract.

6. The preparation method according to any one of claims 3-5, wherein in step (1), each 1 g of the Cordyceps sinensis powder is mixed with 5 ml-20 ml of ethanol solution; and / or The water bath reflux extraction in step (1) is performed by water bath reflux extraction at 75℃-85℃; and / or The ethanol solution in step (1) is a 95% vol - 100% vol aqueous ethanol solution; and / or The extraction time for the water bath reflux extraction in step (1) is 2 h - 6 h; The water bath reflux extraction in step (2) is performed by water bath reflux extraction at 95℃-100℃; and / or The extraction time for the water bath reflux extraction in step (2) is 2 h to 6 h.

7. The preparation method according to any one of claims 3-5, wherein in step (4), each 1 g of the precipitate 3 is dissolved in 50 ml-150 ml of water; and / or The alcohol precipitation time in step (3) is 8 h-24 h; and / or The protein removal in step (4) is performed using the Sevag method; and / or The molecular weight cutoff for dialysis concentration in step (4) is 3.0 kDa-5.0 kDa; and / or The vacuum concentration in step (4) is to concentrate to one-half to one-fifth of the volume before vacuum concentration; In step (5), each 1 g of the crude polysaccharide is dissolved in 150 ml-300 ml of water; and / or In step (5), the volume ratio of water to anhydrous ethanol used to dissolve the crude polysaccharide is 3:1-4:1; and / or The alcohol precipitation time in step (5) is 8 h-24 h; and / or The alcohol precipitation time in step (6) is 8 h-24 h; and / or In step (6), the volume ratio of the supernatant to the 80% vol-95% vol ethanol aqueous solution is 2.5:1.0-4.0:1.0; and / or In step (6), each 1 g of the precipitate 4 is mixed with 150 ml to 300 ml of water.

8. The preparation method according to any one of claims 3-5, wherein the protein removal in step (4) is performed by the Sevag method until all protein is removed.

9. The preparation method according to any one of claims 3-5, wherein the protein removal in step (4) is performed by removing protein 10-20 times or 15 times using the Sevag method.

10. The preparation method according to any one of claims 3-5, wherein the molecular weight cutoff of the dialysis concentration in step (4) is 3.0 kDa.

11. The preparation method according to claim 3, characterized in that, Includes the following steps: (1) Take Cordyceps sinensis powder and mix it with ethanol solution. Each 1 g of Cordyceps sinensis powder is mixed with 5 ml of 95% vol ethanol solution. Reflux extraction in a water bath at 80℃ for 2 h, cool, centrifuge, and obtain precipitate 1. (2) Take precipitate 1, evaporate the ethanol, add water and mix, then reflux in a water bath at 100°C for 2 h, then cool and centrifuge to obtain supernatant and precipitate 2; repeat the above operation of mixing with water and refluxing in a water bath at 100°C for 2 h, then cooling and centrifuging 0-5 times, and combine the supernatant obtained from centrifugation in step (2); mix each 1 g of precipitate 1 or precipitate 2 with 20 ml of water; (3) The combined supernatant obtained in step (2) is concentrated to one-tenth of the volume before concentration to obtain a concentrated solution. The concentrated solution is mixed with anhydrous ethanol of 4 times the volume of the concentrated solution, placed at 2~8℃ for alcohol precipitation, centrifuged, the precipitate is collected and the ethanol is evaporated to obtain precipitate 3. (4) Evaporate the ethanol from the precipitate 3 and dissolve it in water. Dissolve 1 g of the precipitate 3 in 100 ml of water. Remove the protein by Sevag method, and concentrate by dialysis. The molecular weight cutoff of the dialysis concentration is 3.0 kDa. Take the dialysis concentration solution, concentrate it under reduced pressure to half the volume before the reduced pressure concentration, freeze dry, and obtain crude polysaccharide. (5) Dissolve the crude polysaccharide obtained in step (4) in water, with each 1 g of the crude polysaccharide dissolved in 200 ml of water; Then mix with anhydrous ethanol, the volume ratio of water to anhydrous ethanol in dissolving the crude polysaccharide is 4:1; after alcohol precipitation at 2~8℃, centrifuge to obtain the supernatant; (6) The supernatant obtained in step (5) is mixed with an 80% vol-95% vol ethanol aqueous solution, and the volume ratio of the supernatant to the 80% vol-95% vol ethanol aqueous solution is 2.5:1.0; after ethanol precipitation at 2~8℃, the mixture is centrifuged, the precipitate is collected, the ethanol is evaporated, and precipitate 4 is obtained, thus obtaining the polysaccharide or the Cordyceps sinensis extract.

12. According to the preparation method of claim 11, step (6) further includes dissolving precipitate 4 in water and freeze-drying it to obtain the polysaccharide or the Cordyceps sinensis extract.

13. A pharmaceutical composition, characterized in that, The polysaccharide or cordyceps extract prepared by the method described in any one of claims 1, 2, or 3-12 is included.

14. The use of the polysaccharide of claim 1, the Cordyceps sinensis extract of claim 2, the polysaccharide or Cordyceps sinensis extract prepared by any one of claims 3-12, or the pharmaceutical composition of claim 13 in the preparation of products for immunomodulation and / or antioxidation.

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