Method for extracting adenine phosphate in cordyceps militaris

Abstract

The invention relates to the technical field of natural biological extraction, and discloses a method for extracting adenine phosphate from cordyceps militaris, which comprises the following steps: raw material preparation: selecting fresh or dried cordyceps militaris as a raw material, cleaning, drying and crushing to obtain cordyceps militaris coarse powder; hydrolysis reaction: mixing the cordyceps militaris coarse powder with a proper amount of solvent and buffer solution, and carrying out hydrolysis reaction under proper conditions; separation and purification: after hydrolysis is finished, solid residues are removed through centrifugation or filtration, and hydrolysate containing adenine phosphate is obtained. And crystallizing and drying: concentrating the purified adenine phosphate solution, and crystallizing and separating out adenine phosphate by adjusting the pH value. The method for efficiently hydrolyzing and extracting adenine phosphate from cordyceps militaris has the advantages of high extraction efficiency, high purity, simplicity and convenience in operation and the like. The adenine phosphate extracted by the method disclosed by the invention can be widely applied to the fields of medicines, health care products, food additives and the like, and has important economic and social values.

Description

Technical Field

[0001] The invention relates to the technical field of natural biological extraction, in particular to a method for extracting adenine phosphate from Cordyceps militaris. Background Art

[0002] Cordyceps militaris, also known as Cordyceps militaris, is a precious medicinal fungus. Its chemical components mainly include nucleoside compounds, amino acids, sterols, and a variety of trace elements and vitamins. Among them, nucleoside compounds are one of the main active ingredients of Cordyceps militaris, such as cordycepin and adenosine. Adenosine is an important intermediate for the synthesis of biologically active substances such as adenosine triphosphate, adenine, and adenylic acid, and adenosine combined with phosphate can generate adenine phosphate, which is a key step in the biochemical process.

[0003] Adenine phosphate, chemically known as adenine-5'-phosphate, is an important nucleotide that is widely present in organisms, especially in the energy metabolism, signal transduction and genetic material synthesis of cells. Adenine phosphate has a chemical formula of C5H8N5O4P. It is soluble in boiling water and sodium hydroxide solution, slightly soluble in cold water and dilute hydrochloric acid, but insoluble in ethanol. This substance plays an important role in biochemical research. It is mainly used to promote leukocyte proliferation and is often used to prevent and treat leukopenia caused by various reasons. Adenine phosphate is extremely important in organisms and is an indispensable part of life activities. Therefore, the development of natural adenine phosphate provides an important basis for the treatment of related diseases and drug development.

[0004] Currently, there are few literature reports on whether Cordyceps militaris directly contains adenine phosphate, but based on the biochemical relationship between adenosine and adenine phosphate, we can reasonably speculate that Cordyceps militaris may contain adenine phosphate. In a specific biological environment, these precursors may be converted into adenine phosphate through hydrolysis biochemical reactions.

[0005] Cordyceps militaris, as a precious fungus that can be cultivated on a large scale, has attracted much attention due to its medicinal value and tonic effects similar to those of Cordyceps sinensis. Adenine phosphate, as an important bioactive substance, plays a key role in energy metabolism, signal transduction, etc., and has broad application prospects. However, there is currently no report on a method for efficiently extracting adenine phosphate from Cordyceps militaris, and an extraction process with simple operation and high extraction efficiency is urgently needed. Summary of the invention

[0006] 1. Technical issues to be resolved The invention relates to the field of biochemistry, and in particular to a method for discovering adenosine phosphate from Cordyceps militaris through a hydrolysis biochemical reaction and an extraction process thereof. The method comprises the steps of raw material pretreatment, solvent extraction, hydrolysis biochemical reaction, purification and drying, and can effectively improve the extraction rate and purity of adenosine phosphate.

[0007] (II) Technical solution To achieve the above object, the present invention provides the following technical solutions: 1. A method for extracting adenine phosphate from Cordyceps militaris, comprising the steps of: 1) Raw material pretreatment: Select fresh or dried Cordyceps militaris as raw material, remove impurities, rinse with pure water and cut into pieces or coarse powder, and dry the processed Cordyceps militaris raw material in a dark environment to constant weight for later use; 2) Solvent hydrolysis: the raw material and the solvent are mixed in a ratio of 1:5 and fully stirred for 15 minutes to 30 minutes, and soaked at room temperature of 25 to 40° C. for 2.0 to 3.0 hours; The solvent is a sodium hydroxide phosphate solution, and the pH value of the solvent is 7.0<PH value<7.8. The more preferred conditions are 40°C and 7.5<PH value<7.8 to ensure that Cordyceps militaris is fully hydrolyzed under weak alkaline conditions.

[0008] 3) Hydrolysis extraction: ⑴ Primary extraction: Add the hydrolyzed Cordyceps sinensis and solvent in step 2) into a stainless steel extraction tank, add 10 times the amount of pure water, open the steam valve to raise the temperature to 70-80°C, the pressure in the tank is 0.9Mp-1.0Mp, and perform hydrolysis extraction for 2-3 hours. → When the pressure in the tank is close to 0.1Mp, open the tank pressure relief valve to release the pressure and release the extract; ⑵ Second extraction: For the second extraction, add 10 times the amount of pure water and raise the temperature to 80-90℃. The pressure in the tank is 0.9Mp-1.0Mp. Hydrolysis extraction is performed for 2 hours. → When the pressure in the tank is close to 0.1Mp, open the tank pressure relief valve to release the pressure and release the extract; Combine the extracts of the first extraction and the second extraction, and filter the extract by siphoning after settling for 2-3 hours; ⑶ The extract is added to the double-effect concentrator and heated to 60-65°C. The concentrated relative density is 1.15-1.2. When the pomei degree is 6-8 degrees, the material is led out of the concentrator into the transfer tank. (4) The concentrate is placed in a double-effect extraction tank, and 5-9 times the volume of the concentrate with a mass fraction of 95% ethanol is added, fully stirred and refluxed for 60-120 minutes, and then precipitated for 12-24 hours. After centrifugation, the crude adenine phosphate is weighed and the ethanol is recovered; ⑸ Weigh the crude adenine phosphate and put it into a precipitation tank, add 10-15 times the volume of 95% ethanol and stir for 5-10 minutes. After precipitation for 12-24 hours, centrifuge and weigh the refined adenine phosphate, and recover the ethanol; (6) Drying: Place the adenine phosphate product in a drying oven and dry it to constant weight at 60-80°C under circulating airflow to obtain the dry product of adenine phosphate. After cooling to room temperature in a dryer, weigh and package it to obtain the finished product.

[0009] (III) Beneficial effects The present invention firstly proposes to extract adenine phosphate from Cordyceps militaris through a hydrolysis biochemical reaction and an extraction method, which can improve the extraction efficiency and purity of adenine phosphate, has the advantages of high extraction efficiency, high purity, simple operation, etc., can meet the demand for high-quality raw materials in the fields of medicine, health products and cosmetics, and has important economic and social value. At the same time, it has the effect of simplifying the extraction process and reducing the production cost. It is environmentally friendly and energy-saving. No harmful solvents are used in the entire extraction process, which reduces environmental pollution, optimizes process conditions, and reduces energy consumption. Moreover, the process is simple, the operation steps are clear, and it is easy to realize industrial production, with good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a liquid chromatography analysis chart of the dried fine product of adenine phosphate obtained in this example. DETAILED DESCRIPTION

[0011] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0012] ⑴ Place 1000g of dried Cordyceps militaris raw material in a suitable container, add sodium phosphate (adjust pH <7.5--7.8) and pure water (5000ml) as a solvent in a ratio of 1:5 according to the amount of raw material, stir thoroughly for 15 to 30 minutes, and soak at room temperature of 25 to 40°C for 2.0 to 3.0 hours, preferably at 40°C to ensure that Cordyceps militaris is fully hydrolyzed under weak alkaline conditions.

[0013] ⑵ Add the hydrolyzed Cordyceps and solvent into the stainless steel extraction tank → add 10 times the amount of pure water (10000ml) → open the steam valve to raise the temperature to 70-80℃ and the tank pressure to 0.9Mp-1.0Mp → hydrolyze and extract for 2.5 hours, → when the tank pressure is close to 0.1Mp, open the tank pressure relief valve to reduce the pressure, → release the extract.

[0014] During the second extraction, add 10 times the amount of pure water (10000ml) and heat to 80-90℃. The pressure in the tank is 0.9Mp-1.0Mp → hydrolysis extraction for 2 hours. Combine the extracts (24000ml) and let them settle for 2 hours before siphoning and filtering.

[0015] ⑶ Take 24000ml of extract and add it into the double-effect concentrator and heat it to 60-65℃. The concentrated relative density is about 1.18. When the temperature is 6-8 degrees, the material can be exported from the concentrator into the transfer tank (4500ml-4800ml). When concentrating, be careful to prevent the material from being sucked out along the vacuum pipe.

[0016] (4) Place 4,500ml-4,800ml of the concentrated solution into a double-effect extraction tank, add 6 times the volume of 95% ethanol, stir thoroughly, reflux for 60 minutes, and then precipitate for 12 hours. After centrifugation, weigh and obtain 50g of crude adenine phosphate (ethanol recovery).

[0017] ⑸ Weigh 50g of crude adenine phosphate and put it into a precipitation tank, add 10-15 times the volume of 95% ethanol and stir thoroughly for 5 minutes. After precipitation for 12 hours, centrifuge and weigh 20g-25g of fine adenine phosphate (ethanol recovery).

[0018] ⑹ Drying: Place the adenine phosphate concentrate in a drying oven and dry it to constant weight at a suitable temperature (such as 80-90°C with circulating airflow) to obtain 10g ± 10% of the dry concentrate of adenine phosphate. After cooling to room temperature in a dryer, weigh and package to obtain the finished product.

[0019] Take 10g±10% of the dried fine product of adenine phosphate and purify and test it using chromatographic separation technology (such as liquid chromatograph WATERS2695e liquid chromatograph tandem mass spectrometer WATERS XEVO TQ-XS liquid chromatograph Agilent 1260). The test results are as follows: Figure 1 shown.

[0020] Test item: Adenine phosphate 954.2 mg / kg The results of liquid phase method (ultraviolet detector) showed that the content of adenine phosphate reached more than 95.40%.

Claims

1. A method for extracting adenine phosphate from Cordyceps militaris, characterized in that: Includes steps: 1) Raw material pretreatment: Select fresh or dried Cordyceps militaris as raw material, remove impurities, rinse with pure water and cut into pieces or coarse powder, and dry the processed Cordyceps militaris raw material in a dark environment to constant weight for later use; 2) Solvent hydrolysis: the raw material and the solvent are mixed in a ratio of 1:5 and fully stirred for 15 minutes to 30 minutes, and soaked at room temperature of 25 to 40° C. for 2.0 to 3.0 hours; The solvent is a sodium hydroxide phosphate solution, and the pH value of the solvent is 7.0<PH value<7.

8. 3) Hydrolysis extraction: ⑴ Primary extraction: Add the hydrolyzed Cordyceps sinensis and solvent in step 2) into a stainless steel extraction tank, add 10 times the amount of pure water, open the steam valve to raise the temperature to 70-80°C, the pressure in the tank is 0.9Mp-1.0Mp, and perform hydrolysis extraction for 2-3 hours. → When the pressure in the tank is close to 0.1Mp, open the tank pressure relief valve to release the pressure and release the extract; ⑵ Second extraction: For the second extraction, add 10 times the amount of pure water and raise the temperature to 80-90℃. The pressure in the tank is 0.9Mp-1.0Mp. Hydrolysis extraction is performed for 2 hours. → When the pressure in the tank is close to 0.1Mp, open the tank pressure relief valve to release the pressure and release the extract; The extracts of the first extraction and the second extraction were combined, and the extracts were filtered by siphon plate frame after settling for 2-3 hours; ⑶ The extract is added to the double-effect concentrator and heated to 60-65°C. The concentrated relative density is 1.15-1.

2. When the pomei degree is 6-8 degrees, the material is led out of the concentrator into the transfer tank. (4) The concentrate is placed in a double-effect extraction tank, and 5-9 times the volume of the concentrate with 95% ethanol by mass is added, fully stirred and refluxed for 60-120 minutes, and then precipitated for 12-24 hours. After centrifugation, the crude adenine phosphate is weighed and the ethanol is recovered; ⑸ Weigh the crude adenine phosphate and put it into a precipitation tank, add 10-15 times the volume of 95% ethanol and stir for 5-10 minutes. After precipitation for 12-24 hours, centrifuge and weigh the refined adenine phosphate, and recover the ethanol; (6) Drying: Place the adenine phosphate product in a drying oven and dry it to constant weight at 60-80°C under circulating airflow to obtain the dry product of adenine phosphate. After cooling to room temperature in a dryer, weigh and package it to obtain the finished product.

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