A method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans

Abstract

The present invention relates to a method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans, comprising: (1) crushing and sieving the dried mycelia of Paecilomyces elegans to obtain mycelia powder of Paecilomyces elegans; (2) subjecting the mycelia powder of Paecilomyces elegans to a composite enzymatic hydrolysis treatment with cellulase and protease, filtering, and collecting the filtrate; (3) passing the filtrate through a macroporous resin column, eluting with water and an ethanol aqueous solution in sequence, and collecting the eluate; (4) purifying the eluate by high performance liquid chromatography to obtain the cordycepin. The method for separating and purifying cordycepin involved in the present invention does not involve the use of heat extraction and toxic solvents, and does not involve strong acid or strong base substances in the whole process. It has the advantages of short extraction time, mild reaction conditions, non-denaturation of the product, and high product purity, and is a low-carbon and green chemical preparation and separation process.

Description

Technical Field

[0001] The invention belongs to the technical field of cordycepin separation and relates to a method for separating and purifying cordycepin from mycelia of Paecilomyces avicularis. Background Art

[0002] Paecilomyces elegans is a Cordyceps fungus isolated from Cordyceps sinensis. It is widely found in the fungus. This strain can be fermented to produce a dried mycelium product. Its pharmacological effects and chemical composition are similar to those of Cordyceps sinensis, with benefits such as kidney protection, immune enhancement, and anti-tumor properties. Due to the high value and scarcity of wild Cordyceps sinensis, Paecilomyces elegans mycelium is becoming a growing alternative to Cordyceps sinensis. Rich in nutrients such as polysaccharides, nucleosides, various amino acids, important trace elements, and total flavonoids, Paecilomyces elegans mycelium has been approved by the State Food and Drug Administration for use in health foods.

[0003] Cordycepin is also known as cordycepin, cordycepin, alias 3'-deoxyadenosine, chemical formula is C 10 H 13 N₅O₃, the primary active ingredient in Paecilomyces elegans, is the first nucleoside antibiotic isolated from a fungus. Research results indicate that cordycepin possesses biological activities such as lung and kidney protection, anti-hypertension, anti-cancer, neuroprotection, anti-inflammatory, antioxidant, and immunomodulatory properties. Consequently, cordycepin has attracted considerable attention in fields such as anti-aging, healthcare, and new drug development, and holds promising clinical application prospects.

[0004] Currently, cordycepin is expensive, reaching tens of thousands of yuan per gram. Developing more efficient methods for its production is crucial. Wild Cordyceps sinensis has a relatively low cordycepin content, but fermentation with the strain Paecilomyces elegans can significantly increase this content. Combined with efficient separation and purification methods, this can yield high-purity cordycepin products, potentially providing a valuable addition to the Chinese Cordyceps market. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a method for separating and purifying cordycepin from the mycelium of Paecilomyces aviculare.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans, the method comprising:

[0008] (1) crushing and sieving the dried Paecilomyces elegans mycelium to obtain Paecilomyces elegans mycelium powder;

[0009] (2) treating the mycelium powder of Paecilomyces elegans with a combined enzymatic hydrolysis method using cellulase and protease, filtering, and collecting the filtrate;

[0010] (3) passing the filtrate through a macroporous resin column, eluting with water and ethanol aqueous solution in sequence, and collecting the eluate;

[0011] (4) The eluate is purified by high performance liquid chromatography to obtain the product.

[0012] The method for isolating and purifying cordycepin involves pretreatment, composite enzymatic hydrolysis, macroporous resin column purification, and high-performance liquid phase separation and purification. The entire process does not involve heat extraction or the use of toxic solvents, and no strong acids or bases are involved. Furthermore, the method has the advantages of short extraction time, mild reaction conditions, low product denaturation, and high product purity. It is a low-carbon, green chemical preparation and separation process. Therefore, the present invention has the dual significance of environmental protection and energy conservation, creating conditions for the effective utilization of cordycepin.

[0013] Preferably, the cellulase comprises β-glucosidase, endoglucanase and exoglucanase.

[0014] Preferably, the proteases include papain and trypsin.

[0015] The composite enzyme used in the separation and purification method of the present invention preferably uses the above-mentioned specific types of cellulase and protease to make the purity of the final product higher.

[0016] Preferably, the ratio of filter paper enzyme activity to β-glucosidase activity in the cellulase is (3-5):1, for example 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0017] Preferably, the ratio of papain activity to trypsin activity is 1:(1-3), for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, etc. Other specific values ​​within this numerical range can be selected and will not be described in detail here.

[0018] The complex enzyme used in the separation and purification method of the present invention preferably uses the above-mentioned specific enzyme activity ratio relationship to make the purity of the final product higher.

[0019] Preferably, the added amount of the cellulase is 0.1-0.8% of the mycelium powder of Paecilomyces avicularis, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, etc.

[0020] Preferably, the added amount of the protease is 0.5-1.5% of the mycelium powder of Paecilomyces avicularis, such as 0.5%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, etc.

[0021] Preferably, the composite enzymatic hydrolysis treatment is carried out at pH = 5.0-5.5 (for example, pH = 5.0, pH = 5.1, pH = 5.2, pH = 5.3, pH = 5.4, pH = 5.5, etc.) and a temperature of 45-55°C (for example, 45°C, 47°C, 48°C, 50°C, 52°C, 54°C, 55°C, etc.) for 30-70 min (for example, 30 min, 40 min, 50 min, 60 min, 70 min, etc.).

[0022] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0023] Preferably, the macroporous resin column is a D101 macroporous resin column.

[0024] Preferably, the filtrate is loaded onto a macroporous resin column and adsorbed at a flow rate of 0.5-1.5 BV / hr (e.g., 0.5 BV / hr, 0.7 BV / hr, 0.8 BV / hr, 0.9 BV / hr, 1.0 BV / hr, 1.2 BV / hr, 1.5 BV / hr, etc.).

[0025] Preferably, the elution volume of water is 1-4 times (eg, 1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, etc.) column volume.

[0026] The purpose of the first elution with water is to remove impurities.

[0027] Preferably, the ethanol aqueous solution is eluted at a flow rate of 2-6 BV / hr (eg, 2 BV / hr, 3 BV / hr, 4 BV / hr, 5 BV / hr, 6 BV / hr, etc.).

[0028] Preferably, the elution time of the ethanol aqueous solution is 1.5-2.5 h, for example, 1.5 h, 1.7 h, 1.8 h, 2.0 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, etc.

[0029] Preferably, the concentration of the ethanol aqueous solution is 50-98%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 98%, etc., more preferably 70-90%.

[0030] The macroporous resin column eluent used in the separation and purification method of the present invention is preferably an ethanol aqueous solution with a concentration of 50-98%, which can elute cordycepin more fully without affecting its stability.

[0031] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0032] Preferably, the stationary phase used in the high performance liquid chromatography method is a C18 column.

[0033] Preferably, the mobile phase used in the high performance liquid chromatography method is a methanol-water solution.

[0034] Preferably, the concentration of the methanol aqueous solution is 10-40%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, etc.

[0035] The HPLC mobile phase used in the separation and purification method of the present invention is preferably a methanol aqueous solution with a concentration of 10-40%, which can enable cordycepin to be more fully separated and purified without affecting its stability, thereby ensuring the purity of the final product.

[0036] Preferably, the detection wavelength in the high performance liquid chromatography method is 258-262 nm, for example, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, etc.

[0037] Preferably, the flow rate of the mobile phase in the high performance liquid chromatography method is 0.8-1.2 mL / min, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, etc.

[0038] Preferably, the detection temperature in the high performance liquid chromatography method is 28-32°C, such as 28°C, 29°C, 30°C, 31°C, 32°C, etc.

[0039] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0040] Preferably, the preparation method of the mycelial powder of Paecilomyces avicularis comprises:

[0041] The Paecilomyces elegans strain is inoculated into a liquid fermentation medium for fermentation, and then the fermentation liquid is filtered, the dried mycelium is collected, and then crushed and sieved to obtain the product.

[0042] Preferably, the liquid fermentation medium comprises sucrose, soybean cake powder, dipotassium hydrogen sulfate and magnesium sulfate, and has a pH of 6.0-7.0 (e.g., 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 6.9, 7.0, etc.).

[0043] Preferably, the fermentation conditions are: a rotation speed of 100-250 rpm (for example, 100 rpm, 120 rpm, 150 rpm, 180 rpm, 200 rpm, 220 rpm, 250 rpm, etc.), a temperature of 22-28°C (for example, 22°C, 23°C, 24°C, 25°C, 26°C, 28°C, etc.), and a time of 100-140h (for example, 100h, 110h, 120h, 130h, 140h, etc.).

[0044] Preferably, the drying is carried out at a temperature of 75-85°C (e.g., 75°C, 77°C, 78°C, 80°C, 82°C, 84°C, 85°C, etc.).

[0045] Preferably, the mesh size of the sieve is 60-100 mesh, such as 60 mesh, 70 mesh, 80 mesh, 90 mesh, 100 mesh, etc.

[0046] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

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

[0048] The method for isolating and purifying cordycepin involves pretreatment, composite enzymatic hydrolysis, macroporous resin column purification, and high-performance liquid phase separation and purification. The entire process does not involve heat extraction or the use of toxic solvents, and no strong acids or bases are involved. Furthermore, the method has the advantages of short extraction time, mild reaction conditions, low product denaturation, and high product purity. It is a low-carbon, green chemical preparation and separation process. Therefore, the present invention has the dual significance of environmental protection and energy conservation, creating conditions for the effective utilization of cordycepin. DETAILED DESCRIPTION

[0049] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0050] The Paecilomyces hepiali strain involved in the following examples was purchased from Ningbo Testo Biotechnology Co., Ltd. as a Paecilomyces hepiali product with the strain number cgmcc 3.7845.

[0051] Example 1

[0052] This example provides a method for separating and purifying cordycepin from the mycelium of Paecilomyces avicularis, as follows:

[0053] (1) Prepare 200 mL of fermentation medium according to the culture medium formula. The culture medium composition is: 4% sucrose, 4% bean cake powder, 0.2% dipotassium hydrogen sulfate, 0.1% magnesium sulfate, and pH 6.5. Place the culture medium in a 500 mL shaking flask and sterilize it with high pressure wet heat. After sterilization, inoculate the bat moth with a 5% inoculation amount and culture it for 120 hours at 180 r / min and 25°C. Harvest the fermentation culture, filter the fermentation liquid through filter paper, and then place the filter paper with mycelium in an oven at 80°C to dry. Gently peel the dried mycelium from the filter paper, and then grind it in a grinder and pass it through an 80-mesh sieve to obtain mycelium powder.

[0054] (2) Take 2 g of mycelium powder and place it in a 100 mL Erlenmeyer flask, add 20 mL of deionized water, then add 0.5% of cellulase (β-glucosidase, endoglucanase and exoglucanase, the ratio of filter paper enzyme activity to β-glucosidase activity is 4:1), add 1.0% of protease (papain and trypsin, the enzyme activity ratio is 1:2), adjust the pH to 5.5, extract at 50 ° C for 40 min, dilute to 100 mL, filter with filter paper, and harvest the filtrate for use;

[0055] (3) The filtrate was passed through a D101 macroporous resin column at a flow rate of 1 BV / hr for adsorption. Impurities were eluted with 4 column volumes of distilled water, and then eluted with 90% ethanol aqueous solution at a flow rate of 4 BV / hr for 2 h. The eluate was collected;

[0056] (4) The resulting eluate was purified using a high-performance liquid chromatography (HPLC) Polaris C18 column with a detection wavelength of 260 nm, a mobile phase consisting of 25% methanol in water, a flow rate of 1 mL / min, and a detection temperature of 30°C to obtain cordycepin. Testing revealed that the purity of the cordycepin reached 98.5%, and the cordycepin content reached 0.732 mg / g (mycelial dry weight).

[0057] Example 2

[0058] This example provides a method for separating and purifying cordycepin from the mycelium of Paecilomyces avicularis, as follows:

[0059] (1) Prepare 200 mL of fermentation medium according to the culture medium formula. The culture medium composition is: 4% sucrose, 4% bean cake powder, 0.2% dipotassium hydrogen sulfate, 0.1% magnesium sulfate, and pH 6.5. Place the culture medium in a 500 mL shaking flask and sterilize it with high pressure wet heat. After sterilization, inoculate the bat moth with a 5% inoculation amount and culture it for 120 hours at 180 r / min and 25°C. Harvest the fermentation culture, filter the fermentation liquid through filter paper, and then place the filter paper with mycelium in an oven at 80°C to dry. Gently peel the dried mycelium from the filter paper, and then grind it in a grinder and pass it through an 80-mesh sieve to obtain mycelium powder.

[0060] (2) Take 2 g of mycelium powder and place it in a 100 mL Erlenmeyer flask, add 20 mL of deionized water, then add 0.3% of cellulase (β-glucosidase, endoglucanase and exoglucanase, the ratio of filter paper enzyme activity to β-glucosidase activity is 5:1), add 0.7% of protease (papain and trypsin, the enzyme activity ratio is 1:1), adjust the pH to 5.0, extract at 55 ° C for 30 min, dilute to 100 mL, filter with filter paper, and harvest the filtrate for use;

[0061] (3) The filtrate was passed through a D101 macroporous resin column at a flow rate of 1.5 BV / hr for adsorption. Impurities were eluted with 4 column volumes of distilled water, and then eluted with 80% ethanol aqueous solution at a flow rate of 6 BV / hr for 1.5 h. The eluate was collected;

[0062] (4) The resulting eluate was purified using a high-performance liquid chromatography (HPLC) Polaris C18 column with a detection wavelength of 260 nm, a mobile phase consisting of 35% methanol in water, a flow rate of 1 mL / min, and a detection temperature of 30°C to obtain cordycepin. Testing revealed that the purity of the cordycepin reached 98.2%, and the cordycepin content reached 0.703 mg / g (mycelium dry weight).

[0063] Example 3

[0064] This example provides a method for separating and purifying cordycepin from the mycelium of Paecilomyces avicularis, as follows:

[0065] (1) Prepare 200 mL of fermentation medium according to the culture medium formula. The culture medium composition is: 4% sucrose, 4% bean cake powder, 0.2% dipotassium hydrogen sulfate, 0.1% magnesium sulfate, and pH 6.5. Place the culture medium in a 500 mL shaking flask and sterilize it with high pressure wet heat. After sterilization, inoculate the bat moth with a 5% inoculation amount and culture it for 120 hours at 180 r / min and 25°C. Harvest the fermentation culture, filter the fermentation liquid through filter paper, and then place the filter paper with mycelium in an oven at 80°C to dry. Gently peel the dried mycelium from the filter paper, and then grind it in a grinder and pass it through an 80-mesh sieve to obtain mycelium powder.

[0066] (2) Take 2 g of mycelium powder and place it in a 100 mL Erlenmeyer flask, add 20 mL of deionized water, then add 0.5% of cellulase (β-glucosidase, endoglucanase and exoglucanase, the ratio of filter paper enzyme activity to β-glucosidase activity is 3:1), add 1.0% of protease (papain and trypsin, the enzyme activity ratio is 1:3), adjust the pH to 5.5, extract at 45 ° C for 60 min, dilute to 100 mL, filter with filter paper, and harvest the filtrate for use;

[0067] (3) The filtrate was passed through a D101 macroporous resin column at a flow rate of 1 BV / hr for adsorption. Impurities were eluted with 2 column volumes of distilled water, and then eluted with 70% ethanol aqueous solution at a flow rate of 3 BV / hr for 2.5 h. The eluate was collected;

[0068] (4) The resulting eluate was purified using a high-performance liquid chromatography (HPLC) Polaris C18 column with a detection wavelength of 260 nm, a mobile phase consisting of 20% methanol in water, a flow rate of 1 mL / min, and a detection temperature of 30°C to obtain cordycepin. Testing revealed that the purity of the cordycepin reached 98.1%, and the cordycepin content reached 0.689 mg / g (mycelium dry weight).

[0069] Example 4

[0070] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. The method differs from Example 1 only in step (2): 2 g of mycelial powder is placed in a 100 mL conical flask, 20 mL of deionized water is added, and then cellulase (β-glucosidase, endoglucanase, and exoglucanase, with a filter paper enzyme activity to β-glucosidase activity ratio of 4:1) is added at a ratio of 1.5%. The pH is adjusted to 5.5, and the mixture is extracted at 50°C for 40 minutes. The volume is then fixed to 100 mL, filtered with filter paper, and the filtrate is harvested for later use. The other steps remain unchanged. After testing, the purity of cordycepin reached 98.5%, and the cordycepin content reached 0.543 mg / g (mycelial dry weight).

[0071] Example 5

[0072] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. The method differs from Example 1 only in step (2): 2 g of mycelial powder is placed in a 100 mL Erlenmeyer flask, 20 mL of deionized water is added, and then a 1.5% ratio of protease (papain and trypsin, with an enzyme activity ratio of 1:2) is added. The pH is adjusted to 5.5, and the mixture is extracted at 50°C for 40 min. The volume is then fixed to 100 mL, filtered with filter paper, and the filtrate is harvested for later use. All other steps remain unchanged. Testing shows that the purity of cordycepin reaches 98.1%, and the cordycepin content reaches 0.619 mg / g (mycelial dry weight).

[0073] Example 6

[0074] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. This method differs from Example 1 only in that in step (2), the proteases (papain and trypsin, with an enzyme activity ratio of 1:2) are replaced with papain alone, while the total enzyme activity remains unchanged. All other steps remain unchanged. Testing revealed a cordycepin purity of 98.0% and a cordycepin content of 0.598 mg / g (mycelial dry weight).

[0075] Example 7

[0076] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. This method differs from Example 1 only in that in step (2), the proteases (papain and trypsin, with an enzyme activity ratio of 1:2) are replaced with trypsin alone, while the total enzyme activity remains unchanged. All other steps remain unchanged. Testing revealed a cordycepin purity of 98.4% and a cordycepin content of 0.612 mg / g (mycelial dry weight).

[0077] Example 8

[0078] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. This method differs from Example 1 only in that the 25% methanol-water solution in step (4) is replaced with a 50% methanol-water solution; all other steps remain unchanged. Testing revealed a cordycepin purity of 97.2% and a cordycepin content of 0.695 mg / g (mycelial dry weight).

[0079] Example 9

[0080] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. This method differs from Example 1 only in that the 25% methanol-water solution in step (4) is replaced with a 5% methanol-water solution; all other steps remain unchanged. Testing revealed a cordycepin purity of 96.9% and a cordycepin content of 0.512 mg / g (mycelial dry weight).

[0081] Example 10

[0082] This example provides a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans. This method differs from Example 1 only in that the 25% methanol aqueous solution in step (4) is replaced with a 25% ethanol aqueous solution; all other steps remain unchanged. Testing revealed a cordycepin purity of 97.9% and a cordycepin content of 0.681 mg / g (mycelial dry weight).

[0083] The applicant states that while the above-described embodiment illustrates a method for isolating and purifying cordycepin from the mycelia of Paecilomyces elegans, the present invention is not limited to the above-described embodiment, nor does it necessarily rely on the above-described embodiment for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for raw materials in the product, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0084] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0085] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans, characterized in that: The method comprises: (1) crushing and sieving the dried Paecilomyces elegans mycelium to obtain Paecilomyces elegans mycelium powder; (2) treating the mycelium powder of Paecilomyces elegans with a combined enzymatic hydrolysis method using cellulase and protease, filtering, and collecting the filtrate; The cellulase includes β-glucosidase, endoglucanase and exoglucanase; the protease includes papain and trypsin; The ratio of filter paper enzyme activity to β-glucosidase activity in the cellulase is (3-5):1; the ratio of papain enzyme activity to trypsin enzyme activity is 1:(1-3) (3) Passing the filtrate through a macroporous resin column, eluting with water and 70-90% ethanol aqueous solution in sequence, and collecting the eluate; (4) The eluate is purified by high performance liquid chromatography to obtain the product.

2. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The added amount of the cellulase is 0.1-0.8% of the mycelium powder of Paecilomyces avicularis.

3. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The added amount of the protease is 0.5-1.5% of the mycelium powder of Paecilomyces avicularis.

4. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The composite enzymatic hydrolysis treatment is carried out at pH=5.0-5.5 and temperature of 45-55° C. for 30-70 minutes.

5. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The macroporous resin column is a D101 macroporous resin column.

6. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The filtrate was loaded onto a macroporous resin column for adsorption at a flow rate of 0.5-1.5 BV / hr.

7. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The elution volume of water is 1-4 times the volume of the column.

8. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The ethanol aqueous solution is eluted at a flow rate of 2-6 BV / hr.

9. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The elution time of the ethanol aqueous solution is 1.5-2.5h.

10. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The stationary phase used in the high performance liquid chromatography method is a C18 column.

11. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The mobile phase used in the high performance liquid chromatography method is a methanol-water solution.

12. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 11, characterized in that: The concentration of the methanol aqueous solution is 10-40%.

13. The method for separating and purifying cordycepin from the mycelium of Paecilomyces elegans according to claim 1, characterized in that: The detection wavelength in the high performance liquid chromatography method is 258-262 nm.

14. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 1, wherein: The mobile phase flow rate in the high performance liquid chromatography method is 0.8-1.2 mL / min.

15. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 1, characterized in that: The detection temperature in the high performance liquid chromatography method is 28-32°C.

16. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 1, wherein: The preparation method of the mycelial powder of Paecilomyces avicularis comprises: The Paecilomyces elegans strain is inoculated into a liquid fermentation medium for fermentation, and then the fermentation liquid is filtered, the dried mycelium is collected, and then crushed and sieved to obtain the product.

17. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 16, characterized in that: The liquid fermentation culture medium comprises sucrose, soybean cake powder, dipotassium hydrogen sulfate and magnesium sulfate, and has a pH of 6.0-7.

0.

18. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 16, characterized in that: The fermentation conditions are: rotation speed 100-250 rpm, temperature 22-28° C., and time 100-140 h.

19. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 16, wherein: The drying is carried out at a temperature of 75-85°C.

20. The method for separating and purifying cordycepin from the mycelia of Paecilomyces elegans according to claim 16, wherein: The mesh number of the sieving is 60-100 meshes.