Extraction method of cordyceps sinensis polysaccharide

Abstract

The application discloses a Cordyceps sinensis polysaccharide extraction method, through step-by-step pH adjustment and gradient ethanol precipitation, CS-F30 polysaccharide and OSWP-2 polysaccharide can be extracted in one Cordyceps sinensis raw material, not only the extraction efficiency is improved, but also the extraction amount and purity are increased, the defects of the reduction of the extraction amount and the extraction efficiency in the CS-F30 polysaccharide and the OSWP-2 polysaccharide extraction process in the prior art are solved; the sulfated groups of CS-F30 are ionized by adjusting the pH, the solubility is increased, and the CS-F30 is reserved in the supernatant, the OSWP-2 polysaccharide is precipitated due to the close neutral isoelectric point and is removed by centrifugation, and is reserved in the precipitate, the method for separating based on the differences of the polysaccharide molecular structure and the charge characteristics can accurately separate the two polysaccharides, the mutual doping of the polysaccharides in the traditional extraction method is avoided, and the extraction accuracy and efficiency are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a polysaccharide extraction method, in particular to a cordyceps sinensis polysaccharide extraction method. BACKGROUND

[0002] Cordyceps sinensis is a unique and precious traditional Chinese medicine, which is not a simple plant or animal, but a complex of cordyceps sinensis fungus and bat moth larvae. Its formation process is very peculiar: in the alpine area with an altitude of 3000-5000 meters, the larvae of bat moth insects hibernate in the soil, and are invaded by cordyceps sinensis fungus. The fungus absorbs the nutrients in the larvae and grows continuously, eventually causing the larvae to be petrified and die. In summer, the stroma of the fungus will grow out of the head or mouth of the petrified insect, and emerge from the ground, looking like a grass stem, hence the name "cordyceps sinensis".

[0003] Cordyceps sinensis contains CS-F30 polysaccharide and OSWP-2 polysaccharide. CS-F30 polysaccharide can activate immune cells such as macrophages and natural killer cells. Macrophages can phagocytose and eliminate pathogens, senescent cells and tumor cells, etc. CS-F30 polysaccharide can enhance the phagocytic ability of macrophages, making them more effective in immune defense function. NK cells can directly kill tumor cells and virus-infected cells without prior sensitization of antigens, and CS-F30 polysaccharide can promote the activation and proliferation of NK cells, and improve the body's anti-tumor and anti-viral ability.

[0004] OSWP-2 polysaccharide can promote the proliferation and differentiation of B lymphocytes, and enhance the humoral immune function. B lymphocytes will differentiate into plasma cells after being stimulated by antigens, and plasma cells can secrete specific antibodies to neutralize pathogens and play an immune protective role. OSWP-2 polysaccharide can increase the level of antibody production and enhance the specific immune response of the body to pathogens.

[0005] However, CS-F30 polysaccharide and OSWP-2 polysaccharide have different acid-base properties, and the extraction of one type of polysaccharide will increase the difficulty of extraction of the other type of polysaccharide and reduce the extraction amount, thereby affecting the extraction effect.

[0006] Therefore, it is necessary to improve the polysaccharide extraction method in the prior art to solve the above problems. SUMMARY

[0007] The present application overcomes the shortcomings of the prior art and provides a cordyceps sinensis polysaccharide extraction method, which aims to solve the problem of reduced extraction amount and extraction efficiency caused by separate extraction of CS-F30 polysaccharide and OSWP-2 polysaccharide in the prior art.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows: a cordyceps sinensis polysaccharide extraction method, comprising the following steps:

[0009] S1: After the pulverization of Cordyceps sinensis, the petroleum ether is used for Soxhlet extraction for 6-8 hours, and the extract in the petroleum ether is dried to obtain a first extract;

[0010] S2: The first extract in S1 is put into hot water and stirred for 2 hours, and the supernatant is extracted by centrifugation to obtain a first extraction liquid; sodium hydroxide solution is added to the first extraction liquid until the pH is 8.3-8.7, and the supernatant and the precipitate are taken by centrifugation to obtain a second extraction liquid and a first centrifugal precipitate, respectively; the temperature of the hot water is 80-90°C;

[0011] S3: The pH of the second extraction liquid in S2 is adjusted to 5.0, and an ethanol solution is added, and after centrifugation, the precipitate is dried to obtain CS-F30 primary product; the first centrifugal precipitate is dissolved in water, and an ethanol solution is added, and after centrifugation, the precipitate is dried to obtain OSWP-2 primary product;

[0012] S4: The CS-F30 primary product and the OSWP-2 primary product in S3 are respectively dissolved in water and added with Sevage reagent, shaken and centrifuged, and the supernatant is taken to obtain CS-F30 solution and OSWP-2 solution, respectively; the pH of the CS-F30 solution and the OSWP-2 solution is adjusted to 5.5 and 8.0, respectively, and hydrogen peroxide is added and left for 2 hours, and column chromatography is used for refining to obtain CS-F30 polysaccharide and OSWP-2 polysaccharide.

[0013] In a preferred embodiment of the present application, the pulverized product in S1 is passed through a 40-mesh sieve, and the temperature in the Soxhlet extraction process with petroleum ether is 60-90°C.

[0014] In a preferred embodiment of the present application, the mass ratio between the first extract and hot water in S2 is 1:20-25.

[0015] In a preferred embodiment of the present application, the molar concentration of the sodium hydroxide solution in S2 is 0.1-0.15M, the centrifugal speed after the addition of the sodium hydroxide solution is 4000-4500rpm, and the centrifugal time is 20-30min.

[0016] In a preferred embodiment of the present application, the volume concentration of the ethanol solution added to the second extraction liquid in S3 is 95%, and the volume ratio between the ethanol solution and the second extraction liquid is 3-3.5:1.

[0017] In a preferred embodiment of the present application, the mass ratio between the first centrifugal precipitate and water in S3 is 1:50-60, the volume concentration of the ethanol solution added is 95%, and the volume ratio between the ethanol solution and the solution after the dissolution of the first centrifugal precipitate is 4-4.5:1.

[0018] In a preferred embodiment of the present application, the temperature during the drying treatment of the secondary extraction liquid and the solution after the dissolution of the once centrifugal precipitation in S3 is 4-6 DEG C, and the drying time is 12-16h.

[0019] In a preferred embodiment of the present application, the mass concentration of the CS-F30 primary product and the OSWP-2 primary product after being dissolved in water in S4 is 2-4%, and the volume ratio of the solution of the CS-F30 primary product and the OSWP-2 primary product to the Sevage reagent is 1:4-5.

[0020] In a preferred embodiment of the present application, the Sevage reagent in S4 is configured by chloroform and n-butanol in a mass ratio of 5-6:1, the centrifugal speed after the addition of the Sevage reagent is 4000-4500 rpm, and the centrifugal time is 20-30 min.

[0021] In a preferred embodiment of the present application, the mass concentration of the hydrogen peroxide in S4 is 30-35%, and the volume fraction of the CS-F30 solution and the OSWP-2 solution is 2-5% and 3-6% respectively.

[0022] The present application solves the defects in the background art, and has the following beneficial effects:

[0023] (1) The present application provides a Cordyceps sinensis polysaccharide extraction method, which can extract CS-F30 polysaccharide and OSWP-2 polysaccharide from one Cordyceps sinensis raw material through stepwise pH adjustment and gradient ethanol precipitation, thereby improving the extraction efficiency, increasing the extraction amount and purity, and solving the defects of the reduction of the extraction amount and efficiency in the extraction process of CS-F30 polysaccharide and OSWP-2 polysaccharide in the prior art.

[0024] (2) In the present application, the pH is adjusted to ionize the sulfate groups of CS-F30, increase the solubility, and retain in the supernatant, while the OSWP-2 polysaccharide has no sulfation group and the isoelectric point is close to neutral, so that the precipitation is generated and removed by centrifugation, and retained in the precipitate. Compared with the prior art, the method based on the difference in polysaccharide molecular structure and charge characteristics can accurately separate the two kinds of polysaccharides, avoid the mutual doping of polysaccharides in the traditional extraction method, and improve the accuracy and efficiency of extraction.

[0025] (3) In the present application, the low-temperature drying environment of 4-6 DEG C can effectively avoid the thermal degradation or denaturation of polysaccharide molecules at high temperature, avoid the gradual loss of water of polysaccharide molecules under relatively mild conditions, and maintain the natural spatial structure and biological activity of polysaccharide molecules. Compared with the prior art, it is helpful to reduce the occurrence of oxidation reaction and improve the purity and quality of polysaccharide.

[0026] (4) In the present application, the molar concentration of the sodium hydroxide solution in S2 is 0.1-0.15M, the sodium hydroxide solution changes the charge distribution of the polysaccharide molecules, compared with the prior art, the repulsive force between the polysaccharide molecules is increased, the interaction between the polysaccharide and the impurities such as protein is reduced, and the subsequent further separation and extraction of the polysaccharide are facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 is a method step diagram of the preferred embodiment of the present application. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, therefore, the scope of protection of the present application is not limited by the specific embodiments disclosed below.

[0031] In the description of the present application, it should be understood that the terms "first", "second" and the like are only used for description purpose, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" and the like can explicitly or implicitly include one or more features. In the description of the present application, unless otherwise specified, the meaning of "multiple" is two or more.

[0032] SUMMARY

[0033] The present application is directed to the simultaneous extraction of CS-F30 polysaccharide and OSWP-2 polysaccharide in Cordyceps sinensis. In the traditional technology, stepwise extraction is generally used. When CS-F30 is extracted first, the acidic condition will change the structure of OSWP-2. Conversely, if OSWP-2 is extracted first, the residual reagent will affect the yield of CS-F30.

[0034] Applicants found that CS-F30 contains sulfate groups (acidic) while OSWP-2 is a neutral polysaccharide, and there is natural complementarity in their charge behaviors; the former dissolves in alkaline conditions, and the latter precipitates at the isoelectric point; it was unexpectedly found that by uniformly adjusting the water extract of Cordyceps sinensis to a weak alkaline environment, taking the narrow window of pH 8.3-8.7 as the balance point, the sulfate groups of CS-F30 are sufficiently ionized when pH>8.3, while the critical point pH<8.7 avoids excessive alkalinity triggering the dissolution of OSWP-2, realizing that the sulfate groups of CS-F30 polysaccharide are negatively charged and dissolved in the supernatant; while OSWP-2 polysaccharide lacks sulfate groups and has an isoelectric point close to neutral, it hydrophobically aggregates to form a precipitate under alkaline conditions. Through one-step pH regulation, the two polysaccharides are simultaneously separated, and the traditional step-by-step extraction is avoided, realizing single feeding, simultaneous purification.

[0035] As shown in Figure 1 A Cordyceps sinensis polysaccharide extraction method, comprising the following steps:

[0036] S1: After crushing Cordyceps sinensis, Soxhlet extraction with petroleum ether for 6-8h, drying the extract in petroleum ether to obtain a first extract; petroleum ether is a non-polar organic solvent that can effectively dissolve fat-soluble impurities such as oil, pigment, etc. in Cordyceps sinensis, while polysaccharides are polar substances with low solubility in petroleum ether. By Soxhlet extraction, the fat-soluble impurities in Cordyceps sinensis are removed, thereby enriching the polysaccharide components in Cordyceps sinensis, creating favorable conditions for subsequent polysaccharide extraction and purification, reducing the interference of impurities in subsequent steps, and improving the extraction efficiency and purity of polysaccharides.

[0037] S2: Put the first extract in S1 into hot water and stir for 2h, centrifuge to extract the supernatant to obtain a first extract liquid; add sodium hydroxide solution to the first extract liquid until the pH is 8.3-8.7, centrifuge to obtain the supernatant and precipitate, respectively, to obtain a second extract liquid and a first centrifugal precipitate; hot water can dissolve polysaccharides and dissolve part of the polar substances such as proteins and polysaccharides. By stirring, the contact and collision opportunities between the extract and hot water are increased, promoting the dissolution of polysaccharides and improving the extraction rate of polysaccharides. Under alkaline conditions, the charge distribution of polysaccharide molecules changes, increasing the repulsive force between polysaccharide molecules and reducing the interaction between polysaccharides and impurities such as proteins, which is beneficial to further separation and extraction of polysaccharides. At the same time, alkaline conditions can promote the denaturation and precipitation of part of the impurities such as proteins, making it easier for polysaccharides to separate from the mixture.

[0038] The pH adjustment ionizes the sulfate groups of CS-F30, increasing the solubility, and the CS-F30 is retained in the supernatant, while the OSWP-2 polysaccharide is precipitated and removed by centrifugation because it lacks sulfate groups and has an isoelectric point close to neutral. The method of separation based on the differences in the molecular structure and charge characteristics of the two polysaccharides can accurately separate the two polysaccharides, avoiding the mutual contamination of polysaccharides in traditional extraction methods, and improving the accuracy and efficiency of extraction.

[0039] The two polysaccharides can be maximally enriched in the supernatant and the precipitate, respectively, reducing the loss of polysaccharides during separation, avoiding the reduction of yield caused by mutual wrapping and adsorption of polysaccharides, and improving the extraction rate of polysaccharides, so that the polysaccharide resources in Cordyceps sinensis can be more fully utilized.

[0040] The pH range (8.3-8.7) is the key to accurately separating the two polysaccharides: under this weak alkaline condition, the sulfate groups of CS-F30 polysaccharide are fully ionized and negatively charged, the intermolecular electrostatic repulsion increases and the polysaccharide is dissolved in the supernatant; at the same time, the OSWP-2 polysaccharide lacks sulfate groups and has an isoelectric point close to neutral (pI≈7), and in an alkaline environment, the hydrophobic interaction is enhanced and the polysaccharide is precipitated. If the pH is lower than 8.3, the ionization of CS-F30 is not sufficient, resulting in partial residual precipitation; if the pH is higher than 8.7, the structure of the sulfate groups of CS-F30 is destroyed or the OSWP-2 is abnormally dissolved, resulting in incomplete separation of the two polysaccharides or loss of activity.

[0041] S3: Adjust the pH of the secondary extraction liquid in S2 to 5.0, add an ethanol solution, and after centrifugation, dry the precipitate to obtain the CS-F30 primary product; dissolve the primary centrifugation precipitate in water, add an ethanol solution, and after centrifugation, dry the precipitate to obtain the OSWP-2 primary product; the method of adjusting the pH of the secondary extraction liquid is to add 0.1M hydrochloric acid.

[0042] S4: Dissolve the CS-F30 primary product and the OSWP-2 primary product in S3 into water respectively and add Sevage reagent, shake and centrifuge, and take the supernatant to obtain CS-F30 solution and OSWP-2 solution respectively; adjust the pH of the CS-F30 solution and the OSWP-2 solution to 5.5 and 8.0 respectively, and add hydrogen peroxide to the CS-F30 solution and the OSWP-2 solution respectively and stand for 2h, and use column chromatography to refine to obtain CS-F30 polysaccharide and OSWP-2 polysaccharide. The method of adjusting the pH of the CS-F30 solution and the OSWP-2 solution is to add hydrochloric acid and sodium hydroxide solution respectively, and the concentration of the hydrochloric acid and the sodium hydroxide solution is 0.1M and 0.1M respectively.

[0043] By stepwise pH adjustment and gradient ethanol precipitation, CS-F30 polysaccharide and OSWP-2 polysaccharide can be extracted from one batch of Cordyceps sinensis raw materials, which not only improves the extraction efficiency, but also increases the extraction amount and purity, solving the defects of reduced extraction amount and efficiency in the extraction process of CS-F30 polysaccharide and OSWP-2 polysaccharide in the prior art.

[0044] In S1, the crushed powder is passed through a 40-mesh sieve, and the temperature during the Soxhlet extraction process with petroleum ether is 60-90℃. Crushing Cordyceps sinensis to pass through a 40-mesh sieve can significantly increase its contact area with petroleum ether. During the subsequent Soxhlet extraction process, petroleum ether can more fully penetrate into the internal structure of Cordyceps sinensis and contact the internal components more closely, thereby improving the extraction efficiency and making it easier for lipid-soluble impurities to be dissolved out by petroleum ether, reducing interference for the subsequent polysaccharide extraction.

[0045] A smaller particle size is beneficial to the destruction of the structure of Cordyceps sinensis cells, making it easier for the substances inside the cells to be released. During Soxhlet extraction, it helps the lipid-soluble components to be dissolved into petroleum ether more quickly, and also prepares for the subsequent polysaccharide extraction, because part of the cell structure related to polysaccharide is preliminarily destroyed, making it more convenient to contact polysaccharide during subsequent polysaccharide extraction.

[0046] Uniform particle size after crushing is beneficial to the uniformity of the extraction process. In a Soxhlet extraction device, uniform particle distribution can make the circulating extraction of petroleum ether more effective, avoiding problems such as insufficient or excessive extraction in local areas due to uneven particle size, making the entire extraction process more stable and controllable, and improving the consistency of extraction quality.

[0047] Within this temperature range, the solubility of petroleum ether will increase. Lipid-soluble impurities in Cordyceps sinensis, such as oils, waxes, and part of pigments, are more easily dissolved into petroleum ether at higher temperatures, so they can be extracted more quickly, reducing extraction time and improving extraction efficiency.

[0048] As the temperature increases, the flowability of petroleum ether increases, and the molecular motion becomes more vigorous, with an increased diffusion speed. This facilitates the deeper penetration of petroleum ether into the interior of Cordyceps sinensis, and also enables the faster diffusion of dissolved lipid-soluble impurities from the interior of Cordyceps sinensis to petroleum ether, further improving the thoroughness and efficiency of extraction.

[0049] The mass ratio between the primary extract and hot water in S2 is 1:20-25, and the temperature of the hot water is 80-90°C. An appropriate amount of hot water can provide a sufficient solvent environment to make the polysaccharide components in the primary extract fully dissolved. If the amount of hot water is too small, the polysaccharide cannot be completely dissolved, resulting in incomplete extraction; and if it is too much, the solution is diluted, reducing the efficiency of subsequent processing. A ratio of 1:20-25 can effectively balance the dissolution effect and solution concentration, ensuring that the polysaccharide is fully dissolved while facilitating subsequent centrifugation and other operations for solid-liquid separation. An appropriate solvent ratio can make the primary extract form a good dispersion and dissolution state in hot water, accelerate the diffusion speed of polysaccharide molecules from solid particles to the liquid phase, shorten the extraction time, and improve the overall extraction efficiency.

[0050] High temperature accelerates the movement speed of water molecules, increases their collision frequency and energy with polysaccharide molecules, and makes polysaccharide molecules more easily detach from the surface of solid particles and dissolve into water, significantly improving the extraction rate of polysaccharide. There are often hydrogen bonds and other forces between polysaccharide molecules and between polysaccharide and impurities. At a high temperature of 80-90°C, these hydrogen bonds are partially broken, reducing the interaction between polysaccharide and other substances, which is beneficial to the separation of polysaccharide from complex mixtures and more completely dissolving into hot water.

[0051] The molar concentration of the sodium hydroxide solution in S2 is 0.1-0.15M, and the centrifugal speed after adding the sodium hydroxide solution is 4000-4500rpm, and the centrifugation time is 20-30min. An appropriate concentration of sodium hydroxide solution can cause some impurities in the polysaccharide solution to react chemically or change their physical properties, making them more easily separated from polysaccharide.

[0052] A 0.1-0.15M sodium hydroxide solution can adjust the pH of the solution to about 8.5, causing the charge distribution of the polysaccharide molecules to change, increasing the repulsive force between the polysaccharide molecules and reducing the interaction between the polysaccharide and impurities such as proteins, which is beneficial to the further separation and extraction of polysaccharide.

[0053] Within this range of centrifugal speed and time, the precipitate in the solution can be fully settled at the bottom to form a precipitate, while ensuring that the polysaccharide in the supernatant will not be lost due to excessive centrifugal force, achieving effective separation of the polysaccharide solution from the precipitated impurities and improving the purity of the polysaccharide extraction.

[0054] Suitable centrifugation parameters can ensure that the solid-liquid separation is completed in a relatively short time, improving the extraction efficiency, reducing the time cost and energy consumption in the extraction process, and also facilitating the smooth progress of subsequent extraction steps.

[0055] The volume concentration of the ethanol solution added in S3 is 95%, and the volume ratio between the ethanol solution and the secondary extraction solution is 3-3.5:1. The 95% volume concentration of ethanol belongs to high concentration ethanol, which can significantly reduce the solubility of polysaccharide in the solution. When ethanol is added to the secondary extraction solution, the polarity and solvent properties of the solution change, causing polysaccharide molecules to gradually aggregate and precipitate. The use of this high concentration ethanol can effectively promote the precipitation of polysaccharide from the solution, realizing the separation of polysaccharide from other impurities in the solution.

[0056] The volume ratio of ethanol solution to secondary extraction solution is controlled at 3-3.5:1, which can ensure that enough ethanol is added to make the polysaccharide precipitate completely, while avoiding excessive use of ethanol that can cause the solution to have too high a concentration of ethanol. Excessive ethanol can cause some impurities to also precipitate, increasing the difficulty of separation and wasting ethanol reagent. The appropriate volume ratio can ensure complete precipitation of polysaccharide while minimizing the co-precipitation of impurities, improving the purity of polysaccharide.

[0057] The polysaccharide precipitate obtained after ethanol precipitation has relatively high purity and low impurity content. This provides a good initial material for subsequent purification steps, reducing the impurity load in the subsequent purification process, improving the purification efficiency and effect, and helping to obtain high-purity polysaccharide products.

[0058] The mass ratio of the first centrifugal precipitate to water in S3 is 1:50-60, and the volume concentration of the ethanol solution added is 95%, and the volume ratio between the ethanol solution and the dissolved solution of the first centrifugal precipitate is 4-4.5:1.

[0059] The appropriate amount of water can fully dissolve the polysaccharide components in the first centrifugal precipitate. If the amount of water added is too small, the precipitate cannot be completely dispersed and dissolved, causing some polysaccharide to be not extracted; while too much water will dilute the solution, increasing the difficulty of subsequent processing. The ratio of 1:50-60 can ensure that the precipitate is fully dissolved and the solution concentration is controlled within a suitable range, creating favorable conditions for subsequent ethanol precipitation operations. This ratio helps to ensure that the impurities in the precipitate are fully contacted with water during the dissolution process, and some soluble impurities are dissolved in water, which can be removed by subsequent ethanol precipitation operations, improving the purity of the polysaccharide extraction.

[0060] The 95% volume concentration of ethanol can significantly reduce the solubility of polysaccharide, promoting its precipitation from the solution. The addition of ethanol at a volume ratio of 4-4.5:1 can ensure that the ethanol concentration in the solution reaches the critical point for polysaccharide precipitation, achieving efficient precipitation of polysaccharide and improving the extraction rate. Excessive ethanol can cause impurities to precipitate together, while the appropriate ratio can minimize the co-precipitation of impurities while ensuring complete precipitation of polysaccharide, improving the purity of polysaccharide. The appropriate amount of ethanol allows the polysaccharide to form larger particles that precipitate, which is easy to separate by centrifugation, shortens the centrifugation time, improves the operation efficiency, and is also beneficial to subsequent drying treatment, ensuring the quality of the polysaccharide.

[0061] By controlling the ratio during the dissolution and precipitation process, polysaccharides can be effectively separated from other impurities, resulting in a higher purity polysaccharide product, laying the foundation for subsequent application. Reasonable ratio setting makes each step closely connected, smooth operation, avoids repeated adjustment of reagent dosage, saves time and cost, and improves the overall efficiency of the extraction process.

[0062] The temperature during the drying process of the secondary extraction solution and the solution after the dissolution of the primary centrifugal precipitate in S3 is 4-6℃, and the drying time is 12-16h. Polysaccharide molecules will undergo thermal degradation or denaturation at higher temperatures, leading to structural damage and reduced biological activity. A low-temperature drying environment of 4-6℃ can effectively prevent this from happening, allowing polysaccharide molecules to gradually lose water under relatively mild conditions, maintaining their natural spatial structure and biological activity. Under low-temperature conditions, the oxidation reaction rate of polysaccharides is greatly reduced. Oxidation reactions cause changes in the chemical structure of polysaccharides, producing harmful oxidation products that affect the quality and stability of polysaccharides. Low-temperature drying helps reduce the occurrence of oxidation reactions, improving the purity and quality of polysaccharides.

[0063] 12-16 hours of drying time is sufficient to remove water from the secondary extraction solution and the solution after the dissolution of the primary centrifugal precipitate. If the drying time is too short, the polysaccharides cannot be completely dried, and the residual water will affect the purity and stability of the polysaccharides, and also cause the polysaccharides to mold or degrade during storage. Excessive drying time will cause the polysaccharides to lose water excessively, making the polysaccharide molecules too brittle and hard, increasing the difficulty of subsequent processing, such as dissolution difficulty. A drying time of 12-16 hours can ensure that the polysaccharides are fully dried while avoiding the adverse effects of excessive drying, allowing the polysaccharides to maintain good physical properties, making it easier for subsequent processing and application.

[0064] The mass concentration of CS-F30 crude product and OSWP-2 crude product after dissolving in water in S4 is 2-4%, and the volume of Sevage reagent added to the solution after the CS-F30 crude product and OSWP-2 crude product are dissolved in water is 1:4-5.

[0065] A mass concentration of 2-4% can ensure that the CS-F30 crude product and OSWP-2 crude product are fully dissolved in water. Too low a concentration cannot fully utilize polysaccharides, while too high a concentration leads to incomplete dissolution, excessive solution viscosity, and other problems. This concentration range allows polysaccharides to be uniformly dispersed in the solution, providing a uniform reaction system for subsequent processing. The appropriate concentration facilitates the subsequent addition of Sevage reagent for thorough mixing and reaction, ensuring that the polysaccharide solution and Sevage reagent are in full contact, improving the efficiency of impurity removal.

[0066] Sevage reagent is composed of chloroform and n-butanol, which can effectively remove impurities such as proteins in polysaccharide solution. The volume ratio of 1:4-5 can make Sevage reagent and polysaccharide solution mix well, providing sufficient impurity removal capacity to remove protein impurities in polysaccharide. Excessive Sevage reagent introduces new impurities or increases the difficulty of subsequent processing. The volume ratio of 1:4-5 can ensure the effect of impurity removal and avoid excessive Sevage reagent, reducing the burden of subsequent processing steps.

[0067] In S4, Sevage reagent is configured by chloroform and n-butanol at a mass ratio of 5-6:1, and the centrifugal speed after adding Sevage reagent is 4000-4500 rpm, and the centrifugal time is 20-30 min. Mixing chloroform and n-butanol at a mass ratio of 5-6:1 can fully exert the synergistic effect of the two, forming an efficient impurity removal system. This proportion of Sevage reagent has strong extraction capacity for protein impurities, which can effectively remove impurities in polysaccharide solution and improve the purity of polysaccharide.

[0068] The mass concentration of hydrogen peroxide in S4 is 30-35%, and the volume fraction added to CS-F30 solution and OSWP-2 solution is 2-5% and 3-6% respectively. The hydrogen peroxide concentration of 30-35% is moderate, which can produce a mild oxidation effect after being added to CS-F30 and OSWP-2 solutions. This oxidation can effectively remove residual proteins, pigments and other impurities in the solution, further improving the purity of polysaccharide. Excessive hydrogen peroxide concentration will cause excessive oxidation of polysaccharide molecules, damaging their structure and biological activity. While the concentration of 30-35% is relatively mild, it reduces the risk of damage to polysaccharide molecules while ensuring the effect of impurity removal.

[0069] The volume fraction of hydrogen peroxide added to CS-F30 solution is 2-5%, which can meet the needs of removing impurities and avoid the potential adverse effects of excessive hydrogen peroxide on CS-F30 polysaccharide. CS-F30 polysaccharide is sensitive to oxidation, and lower hydrogen peroxide dosage can more accurately remove impurities while maximizing the activity and integrity of polysaccharide.

[0070] The volume fraction of hydrogen peroxide added to OSWP-2 solution is 3-6%, which is relatively high and can more effectively deal with the more complex impurities in OSWP-2 solution. The structure and properties of OSWP-2 polysaccharide enable it to withstand slightly higher concentrations of hydrogen peroxide, thereby achieving better impurity removal effect.

[0071] In the refining process of OSWP-2, DEAE-Sepharose Fast Flow anion exchange column chromatography was used, the inner diameter of the column was 2.6 cm, the length was 30 cm, the filler was expanded with distilled water and then filled to a column bed height of 20 cm, and then eluted with a linear gradient of 0.3M NaCl after equilibration with 20mM Tris-HCl (pH8.0), and eluted at 0.6M NaCl, and then collected the main peak and eluted with a constant flow of distilled water through a Sephadex G-100 gel column (inner diameter 1.6 cm, length 50 cm) to remove small molecular impurities, and finally dialyzed, desalted and freeze-dried to obtain purified CS-F30.

[0072] In the refining process of CS-F30, DEAE-Sepharose Fast Flow anion exchange column (pH7.0) was used first, and then eluted with distilled water, and then purified by Sephadex G-100 gel column, and the flow process was consistent with the gel step of CS-F30, and finally dialyzed and dried to obtain OSWP-2 polysaccharide.

[0073] Example 1: The present embodiment provides a method for extracting Cordyceps sinensis polysaccharide, and the specific steps are as follows:

[0074] S1: The Cordyceps sinensis was crushed and passed through a 40-mesh sieve, and then Soxhlet extracted with petroleum ether for 6-8h, and the temperature during the Soxhlet extraction was 90℃, and then the extract in the petroleum ether was dried to obtain a first extract;

[0075] S2: The first extract in S1 was placed in hot water and stirred for 2h, the mass ratio between the first extract and the hot water was 1:20, the temperature of the hot water was 80℃, and the supernatant was obtained by centrifugal extraction, and then sodium hydroxide solution was added to the first extract, the molar concentration of the sodium hydroxide solution was 0.1M, until the pH was 8.1, and then the supernatant and the precipitate were obtained by centrifugation at a speed of 4000rpm for 30min, and the second extract and the first centrifugal precipitate were obtained respectively;

[0076] S3: The pH of the second extract in S2 was adjusted to 5.0, and then ethanol solution was added, the volume concentration of the ethanol solution was 95%, and the volume ratio between the added ethanol solution and the second extract was 3:1, and then the precipitate was dried after centrifugation to obtain the CS-F30 primary product, the drying temperature was 4℃, and the drying time was 12h; the first centrifugal precipitate was dissolved in water, the mass ratio between the first centrifugal precipitate and the water was 1:50, and then ethanol solution was added, the volume concentration of the ethanol solution was 95%, and the volume ratio between the added ethanol solution and the solution after the first centrifugal precipitate was dissolved was 4:1, and then the precipitate was dried after centrifugation to obtain the OSWP-2 primary product, the drying temperature was 4℃, and the drying time was 12h;

[0077] S4: The CS-F30 crude product and the OSWP-2 crude product in S3 were respectively dissolved in water with a mass concentration of 2%, and a Sevage reagent was added, the Sevage reagent was configured by chloroform and n-butanol with a mass ratio of 5:1, the volume ratio of the solution of the CS-F30 crude product and the OSWP-2 crude product dissolved in water to the Sevage reagent was 1:4, and the solution was shaken and centrifuged at a centrifugal speed of 4500 rpm for 20 min, and the supernatant was taken to obtain a CS-F30 solution and an OSWP-2 solution; the pH values of the CS-F30 solution and the OSWP-2 solution were respectively adjusted to 5.5 and 8.0, hydrogen peroxide with a mass concentration of 30% was added and left for 2 h, and the volume fraction of the hydrogen peroxide added to the CS-F30 solution and the OSWP-2 solution was 3% and 5% respectively, and then the OSWP-2 polysaccharide and the CS-F30 were respectively refined, in the refining process of the OSWP-2 polysaccharide, DEAE-Sepharose Fast Flow anion exchange column chromatography was adopted, the column had an inner diameter of 2.6 cm and a length of 30 cm, the filler was expanded with distilled water and then filled into the column bed with a height of 20 cm, 20 mM Tris-HCl (pH 8.0) was used for balancing, after loading, linear gradient elution was performed through 0.3 M NaCl, elution was performed at 0.6 M NaCl, the main peak was collected, and then Sephadex G-100 gel column (inner diameter: 1.6 cm, length: 50 cm) was used for constant-flow elution with distilled water to remove small molecular impurities, and finally dialysis desalination and freeze-drying were performed to obtain purified CS-F30. In the refining process of the CS-F30, DEAE-Sepharose Fast Flow anion exchange column (pH 7.0) was first used for elution with distilled water, and then Sephadex G-100 gel column was used for purification, the process was consistent with the gel step of the CS-F30, and finally dialysis and drying were performed to obtain the OSWP-2 polysaccharide.

[0078] Example Two: The difference between this example and Example One is that in S2, sodium hydroxide is added until the pH is 8.3, and the rest is consistent.

[0079] Example Three: The difference between this example and Example One is that in S2, sodium hydroxide is added until the pH is 8.5, and the rest is consistent.

[0080] Example Four: The difference between this example and Example One is that in S2, sodium hydroxide is added until the pH is 8.7, and the rest is consistent.

[0081] Example Five: The difference between this example and Example One is that in S2, sodium hydroxide is added until the pH is 8.9, and the rest is consistent.

[0082] Example Six: The difference between this example and Example Three is that in S4, the mass concentration after being dissolved in water is 1%, and the rest is consistent.

[0083] Example Seven: The difference between this example and Example Three is that the mass concentration after dissolving in water in S4 is 3%, and the rest is the same.

[0084] Example Eight: The difference between this example and Example Three is that the mass concentration after dissolving in water in S4 is 4%, and the rest is the same.

[0085] Example Nine: The difference between this example and Example Three is that the mass concentration after dissolving in water in S4 is 5%, and the rest is the same.

[0086] Comparative Example One: The polysaccharide extraction steps are as follows:

[0087] S1: After the Cordyceps sinensis is crushed, it is dissolved, and after dissolution, it is filtered to obtain supernatant;

[0088] S2: The supernatant in S1 is adjusted to pH 5.0 and 8.0, respectively, to obtain two precipitates;

[0089] S3: The two precipitates are dissolved in water, and then ethanol solution is added, the volume concentration of the ethanol solution is 95%, and the volume ratio between the added ethanol solution and the solution of the precipitate dissolved in water is 3:1, after centrifugation, the precipitate is dried;

[0090] S4: CS-F30 polysaccharide and OSWP-2 polysaccharide are obtained by column chromatography.

[0091] The mass of the Cordyceps sinensis before extraction in Examples One to Nine and Comparative Example One is measured, and the mass of the obtained CS-F30 polysaccharide and OSWP-2 polysaccharide is recorded, and the extraction rate is calculated, and the extraction rate of CS-F30 polysaccharide and OSWP-2 polysaccharide is shown in Table One.

[0092] Table One: Extraction rate of CS-F30 polysaccharide and OSWP-2 polysaccharide in Examples One to Nine and Comparative Example One

[0093]

[0094] As can be seen from Table One, the extraction rate of CS-F30 polysaccharide and OSWP-2 polysaccharide in Examples One to Nine is greater than that of CS-F30 polysaccharide and OSWP-2 polysaccharide in Comparative Example One, and the present application has advantages.

[0095] In Examples 1 to 5, with the gradual increase of pH, the extraction rates of CS-F30 polysaccharide and OSWP-2 polysaccharide both first increase and then decrease, because with the increase of pH, the electrostatic repulsion between polysaccharide and impurities increases, which is beneficial to the separation of polysaccharide from impurities. When the pH of the solution is higher than the isoelectric point of polysaccharide, the polysaccharide molecules are negatively charged, while the proteins are positively charged or electrically neutral. The electrostatic repulsion between polysaccharide and impurities helps to reduce the interaction between polysaccharide and impurities, and improve the extraction rate of polysaccharide. However, too high pH makes the electrostatic repulsion between polysaccharide molecules too large, resulting in the aggregation of polysaccharide molecules to form larger particles, thereby reducing the solubility and extraction rate of polysaccharide. At the same time, too high pH changes the charge state of proteins, which enhances the interaction between proteins and polysaccharide, resulting in the decrease of the extraction rate of polysaccharide. The preferred embodiment is Example 3.

[0096] In Examples 3 and 6 to 9, with the gradual increase of the mass concentration of S4 after dissolving in water, the extraction rates of CS-F30 polysaccharide and OSWP-2 polysaccharide both first increase and then decrease, because when the mass concentration is low, with the gradual increase of the concentration, the distance between polysaccharide molecules decreases relatively. This makes the interaction between polysaccharide molecules and water molecules relatively weak, while the interaction between polysaccharide molecules gradually increases. This change is beneficial to the aggregation of polysaccharide molecules from the solution to form larger particles, which are more easily extracted; too high mass concentration will cause the polysaccharide molecules to be too dense, and the interaction between molecules to be too strong, which is easy to form larger aggregates or even precipitate. These aggregates or precipitates are difficult to be effectively separated and collected by subsequent extraction operation, and part of the polysaccharide will remain in the precipitate or unextracted phase, thereby reducing the extraction rate. The preferred embodiment is Example 7.

[0097] The above is based on the ideal embodiment of the present application, through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the contents of the specification, and the technical scope must be determined according to the scope of claims.

Claims

1. A method for extracting polysaccharides from Cordyceps sinensis, characterized in that, Includes the following steps: S1: After pulverizing Cordyceps sinensis, extract it with petroleum ether using Soxhlet extraction for 6-8 hours. Dry the extract in petroleum ether to obtain a primary extract. S2: Place the primary extract from S1 in hot water and stir for 2 hours. Centrifuge to extract the supernatant to obtain the primary extract. Add sodium hydroxide solution to the primary extract until the pH is 8.3-8.

7. Centrifuge to collect the supernatant and precipitate to obtain the secondary extract and the primary centrifugation precipitate, respectively. The temperature of the hot water is 80-90℃, the molar concentration of the sodium hydroxide solution is 0.1-0.15M, the centrifugation speed after adding the sodium hydroxide solution is 4000-4500 rpm, and the centrifugation time is 20-30 min. S3: Adjust the pH of the secondary extract in S2 to 5.0 using hydrochloric acid, add ethanol solution, centrifuge, and dry the precipitate to obtain CS-F30 primary product; dissolve the precipitate from the first centrifugation in water, add ethanol solution, centrifuge, and dry the precipitate to obtain OSWP-2 primary product. The volume concentration of the ethanol solution added to the secondary extract is 95%, and the volume ratio between the added ethanol solution and the secondary extract is 3-3.5:

1. S4: Dissolve the CS-F30 and OSWP-2 primary products from S3 in water and add Sevage reagent. The mass concentration of the CS-F30 and OSWP-2 primary products after dissolving in water is 2-4%. The volume ratio of the solution of the CS-F30 and OSWP-2 primary products to the volume of Sevage reagent is 1:4-5. Shake and centrifuge, and take the supernatant to obtain CS-F30 solution and OSWP-2 solution respectively. Adjust the pH of the CS-F30 solution to 5.5 with hydrochloric acid and adjust the pH of the OSWP-2 solution to 8.0 with sodium hydroxide solution. Add hydrogen peroxide to the CS-F30 solution and OSWP-2 solution respectively and let stand for 2 hours. Purify by column chromatography to obtain CS-F30 polysaccharide and OSWP-2 polysaccharide.

2. The method for extracting Cordyceps sinensis polysaccharides according to claim 1, characterized in that: In S1, the material is pulverized and passed through a 40-mesh sieve, and the temperature during the Soxhlet extraction of petroleum ether is 60-90℃.

3. The method for extracting Cordyceps sinensis polysaccharides according to claim 1, characterized in that: The mass ratio of the primary extract to hot water in S2 is 1:20-25.

4. The method for extracting Cordyceps sinensis polysaccharides according to claim 1, characterized in that: In step S3, the mass ratio between the first centrifugal precipitate and water is 1:50-60, the volume concentration of the added ethanol solution is 95%, and the volume ratio between the added ethanol solution and the solution after the first centrifugal precipitate is dissolved is 4-4.5:

1.

5. The method for extracting Cordyceps sinensis polysaccharides according to claim 1, characterized in that: The temperature for drying the secondary extract and the solution after the first centrifugation precipitation in step S3 is 4-6℃, and the drying time is 12-16h.

6. The method for extracting Cordyceps sinensis polysaccharides according to claim 1, characterized in that: The Sevage reagent in S4 is prepared by mixing chloroform and n-butanol in a mass ratio of 5-6:

1. After adding the Sevage reagent, the centrifugation speed is 4000-4500 rpm and the centrifugation time is 20-30 min.

7. The method for extracting Cordyceps sinensis polysaccharides according to claim 1, characterized in that: The hydrogen peroxide in S4 has a mass concentration of 30-35%, and the volume fractions added to the CS-F30 solution and OSWP-2 solution are 2-5% and 3-6%, respectively.

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