Preparation method and application of Cordyceps sinensis extract

Abstract

The invention discloses a preparation method of a Cordyceps sinensis extract and its application in drug preparation, the preparation method comprising the following steps: (1) subjecting Cordyceps sinensis raw materials to microwave water extraction, concentrating the extract and then precipitating with alcohol, separating solid and liquid, and obtaining an alcohol precipitate; (2) subjecting the alcohol precipitate obtained in step (1) to a first ultrafiltration, collecting the first ultrafiltration permeate for a second ultrafiltration, collecting the second ultrafiltration permeate for nanofiltration, and collecting a concentrated solution; (3) adjusting the pH of the concentrated solution obtained in step (2) to 6.5-7, obtaining a loading solution, loading the loading solution onto an adsorption resin column for adsorption, eluting with ethanol aqueous solution, and collecting the eluate; (4) concentrating the eluate obtained in step (3), heating, and recrystallizing to obtain. The present invention obtains high-purity cordycepin or N6-(2-hydroxyethyl)-adenosine by four-step separation techniques of alcohol precipitation, membrane separation, primary adsorption resin column chromatography, and recrystallization. The process is simple, highly operable, low cost, environmentally friendly, and easily applicable to industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and extraction of active substances in edible fungi, and specifically relates to a preparation method and application of a Cordyceps sinensis extract. Background Art

[0002] Cordyceps cicadae, also known as cicada antler, worm flower, cicada cordyceps or cicada antler fungus, formerly known as Penicillium cicadae, is a common insect pathogenic fungus in southern my country. It is a fungus-insect complex formed after the cicada nymph is parasitic on the cicada nymph. It is a traditional and precious Chinese medicinal material. Its active ingredients are similar to those of natural Cordyceps sinensis, and it has been proven in pharmacological and clinical experiments that it can be used as an important artificial substitute for Cordyceps sinensis. Cordycepin, also known as 3′-deoxyadenosine, is an important nucleoside active substance in Cordyceps cicadae, with a molecular formula of C 10 H 13 N5O3, molecular weight is 251, melting point is 230℃~231℃, can form needle-shaped or flake crystals in water, n-butanol and methanol. Cordycepin is soluble in water, methanol and ethanol, insoluble in benzene, ether and chloroform, anthrone reaction is positive, and the maximum absorption wavelength of ultraviolet light is 260nm. Cordycepin has pharmacological activities such as anti-HIV-Ⅰ virus, antibacterial, and tumor inhibition. N6-(2-hydroxyethyl)-adenosine (HEA), also known as cordycepin, has a molecular formula of C 12 H 17 N5O5, molecular weight 311.29, N6-(2-hydroxyethyl)-adenosine is a special nucleoside in Cordyceps sinensis. It is the first biological calcium antagonist. Its analgesic mechanism is different from the currently commonly used opioid analgesics. It is not addictive and relatively safe. It also has the effect of inhibiting tumor cell proliferation. HEA has strong anti-ultraviolet radiation, anti-platelet aggregation, analgesia, lowering blood pressure, increasing coronary and cerebral blood flow, reducing coronary resistance, protecting myocardium and killing insects. HEA combines the development potential of medicine, health care, cosmetics and biological control, and is an important bioactive substance indicator to measure the quality of some Cordyceps products.

[0003] At present, there are many reports on the preparation methods of single cordycepin samples, mainly including ion exchange resin method, silica gel column chromatography, supercritical fluid extraction method, reversed-phase high performance preparative liquid chromatography, etc., but they all have defects such as low recovery rate and complicated operation steps. There are few reports on the preparation methods of single N6-(2-hydroxyethyl)-adenosine samples. Zhu Bichun et al. prepared N6-(2-hydroxyethyl)-adenosine by macroporous resin chromatography and Sephadex LH-20 column chromatography (Zhu Bichun, Chai Yiqiu, Zhang Sisi, et al. Anticonvulsant effects of active ingredients from Cordyceps militaris [J]. Journal of Mycology, 2016, 35(05): 619-627.); Lv Ziming et al. separated and purified N6-(2-hydroxyethyl)-adenosine by macroporous resin chromatography, silica gel column chromatography, recrystallization and other steps (Lv Ziming, Jiang Yongtao, Wu Lijun, et al. Study on the chemical composition of artificial Cordyceps militaris fruiting body [J]. Chinese Journal of Traditional Chinese Medicine, 2008, 33(24): 2914-2917.); the separation and purification processes of N6-(2-hydroxyethyl)-adenosine in both methods are relatively complicated.

[0004] In summary, it is necessary to develop a method for preparing a Cordyceps sinensis extract, by which cordycepin and N6-(2-hydroxyethyl)-adenosine can be conveniently and efficiently prepared. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for conveniently and efficiently preparing cordycepin and N6-(2-hydroxyethyl)-adenosine from Cordyceps sinensis extract in view of the shortcomings of the existing cordycepin and N6-(2-hydroxyethyl)-adenosine extraction and separation technology, which has complicated technical processes.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for preparing a Cordyceps sinensis extract comprises the following steps:

[0008] (1) subjecting the dried Cordyceps sinensis raw material to microwave water extraction, concentrating the extract, precipitating with alcohol, and separating the solid from the liquid to obtain an alcohol precipitate;

[0009] (2) subjecting the alcohol precipitate obtained in step (1) to a first ultrafiltration, collecting the first ultrafiltration permeate to a second ultrafiltration, collecting the second ultrafiltration permeate to a nanofiltration, and collecting a concentrated solution;

[0010] (3) adjusting the pH of the concentrated solution obtained in step (2) to 6.5-7 to obtain a loading solution, loading the loading solution onto an adsorption resin column for adsorption, eluting with a first ethanol aqueous solution, and collecting the eluate;

[0011] (4) Concentrating the eluate obtained in step (3), heating, and recrystallizing to obtain the product.

[0012] The raw material of Cordyceps sinensis is any one of Cordyceps sinensis mycelium and Cordyceps sinensis fruiting body or a combination of two thereof.

[0013] Wherein, the Cordyceps sinensis extract is cordycepin or N6-(2-hydroxyethyl)-adenosine.

[0014] In step (1), when the microwave water extraction is performed, the mass ratio of the Cordyceps sinensis raw material to water is 1:10-15, the microwave power is 300W-400W, and the extraction time is 30min-45min; the number of microwave water extractions is 2-3 times.

[0015] In step (1), when the alcohol precipitation is performed, the precipitant used is a 95% to 99% v / v ethanol aqueous solution, the alcohol precipitation time is 6h to 18h; and the concentration of ethanol in the alcohol precipitation solution is 80% to 90% v / v.

[0016] In step (2), the first ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000-8000Da and a transmembrane pressure difference of 1-3Mpa; the second ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1000-5000Da and a transmembrane pressure difference of 1-3Mpa; the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 150-1000Da and a transmembrane pressure difference of 2-4Mpa.

[0017] In step (3), hydrochloric acid or sodium hydroxide is used to adjust the pH of the concentrated solution obtained in step (2) to 6.5-7. The reason for adjusting the pH of the concentrated solution to 6.5-7 before eluting with a macroporous resin column is that cordycepin and N6-(2-hydroxyethyl)-adenosine are both weakly alkaline substances, which will form salts in a slightly acidic environment, which is not conducive to the adsorption of the adsorption resin. In a weakly alkaline environment, cordycepin and N6-(2-hydroxyethyl)-adenosine will exist in molecular form, which is more conducive to their hydrogen bonding with the adsorption resin for adsorption.

[0018] When the Cordyceps sinensis extract is cordycepin, in step (3), the model of the adsorption resin is XAD-16, XAD-1600, KA-I, HD-01 or HD-06; the loading adsorption amount of the loading liquid is 1.5-3BV, and the loading flow rate is 2.5-4BV / h; the concentration of the first ethanol aqueous solution is 30%-50% v / v, the dosage is 1-3BV, and the elution flow rate is 2-3.5BV / h. Preferably, the concentration of the first ethanol aqueous solution is 50% v / v.

[0019] When the Cordyceps sinensis extract is N6-(2-hydroxyethyl)-adenosine, in step (3), the model of the adsorption resin is AB-8, ADS-17, D-101 or DM130, the sample adsorption amount of the sample solution is 2-3BV, and the sample flow rate is 1-2.5BV / h; before eluting with the first ethanol aqueous solution, the impurities are washed with water and a 5%-30% v / v second ethanol aqueous solution in sequence, preferably, the impurities are washed with water and a 5% v / v second ethanol aqueous solution in sequence; when washing with the water, the amount of water is 3-4BV, and the washing flow rate is 2-3.5BV / h; when washing with the second ethanol aqueous solution, the amount of the second ethanol aqueous solution is 1-2BV, and the washing flow rate is 2-3.5BV / h.

[0020] When the Cordyceps sinensis extract is N6-(2-hydroxyethyl)-adenosine, in step (3), the concentration of the first ethanol aqueous solution is 5% to 30% v / v, the dosage is 3 to 4 BV, and the elution flow rate is 2 to 3.5 BV / h; preferably, the concentration of the first ethanol aqueous solution is 10% v / v.

[0021] In step (3), the adsorption resin is first soaked and washed 2 to 3 times with 6 times the volume of anhydrous ethanol before use. During the soaking process, it needs to be constantly stirred and the soaking time should exceed 4 hours so that impurities in the adsorption resin can be washed out. Then, the anhydrous ethanol in the adsorption resin is rinsed clean with pure water and set aside.

[0022] In step (4), the concentration is to concentrate the eluent to a solid-liquid ratio of 1:1 to 4 (mass ratio); the heating is to heat to above 90°C, and the main purpose of heating is to increase the solubility of cordycepin and N6-(2-hydroxyethyl)-adenosine to facilitate better crystallization and precipitation; the recrystallization is low-temperature recrystallization, the recrystallization temperature is 0°C to 10°C, and the time is 5h to 12h.

[0023] The adsorption resins AB-8, ADS-17, D-101, DM130, XAD-16 and XAD-1600 used in the present invention can be purchased through commercial channels. The adsorption resins XAD-16, XAD-1600, KA-I, HD-01 and HD-06 are provided by the National Biochemical Center of Kaohsiung University of Technology.

[0024] The application of the above Cordyceps sinensis extract in the preparation of medicines is within the protection scope of the present invention.

[0025] Among them, the application is the application in the preparation of antibacterial drugs, antiviral drugs, antitumor drugs, immune regulating drugs, lipid-lowering drugs, antiplatelet aggregation drugs, memory improving drugs or calcium ion antagonists.

[0026] Among them, calcium ion antagonists are widely used in the treatment of cardiovascular diseases, and their application has also expanded to reversing multidrug resistance, inhibiting ascorbic acid accumulation, inhibiting central nervous system inflammatory response, and delaying neuronal death.

[0027] Beneficial effects:

[0028] The invention obtains high-purity cordycepin or N6-(2-hydroxyethyl)-adenosine through four-step separation technology of alcohol precipitation, membrane separation, primary adsorption resin column chromatography and recrystallization. The process is simple, the process is highly operable, the cost is low, and it is easy to industrialize. The organic solvent used is only ethanol, and other toxic and harmful organic solvents are not used. The process is highly safe and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0030] Figure 1 This is a comparison chart of the adsorption-desorption performance of cordycepin on five resins in Example 2;

[0031] Figure 2 This is a comparison chart of the desorption effects of different concentrations of ethanol aqueous solution eluent on cordycepin adsorbed on KA-I resin in Example 3;

[0032] Figure 3 This is a comparison chart of the cordycepin adsorption performance of KA-I resin under different pH conditions in Example 4;

[0033] Figure 4 : is the penetration curve of cordycepin on the KA-I resin column at different loading flow rates in Example 5;

[0034] Figure 5 The penetration curves of cordycepin on KA-I resin columns with different aspect ratios in Example 5;

[0035] Figure 6 These are the penetration curves of cordycepin in the KA-I resin column at different temperatures in Example 5. DETAILED DESCRIPTION

[0036] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0037] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0038] Example 1 Extraction of Cordyceps sinensis from Cordyceps sinensis by microwave water extraction, alcohol precipitation and membrane separation

[0039] (1) Microwave water extraction

[0040] Take 500g of Cordyceps sinensis fruiting body raw material, crush it to 60 mesh, add 7.5L of water, and extract it 3 times with 400W microwave assisted extraction, and the single extraction time is 45min. Mix the extracts and concentrate them under reduced pressure to a solid content of 20% (volume fraction), separate the solid and liquid, and collect the clear night.

[0041] (2) Alcohol precipitation

[0042] Add 95% by volume ethanol aqueous solution to the clear solution obtained in step (1) until the volume fraction of ethanol in the mixed solution is 80%, then perform alcohol precipitation for 10 hours, separate the solid and the liquid, and collect the alcohol precipitate and the alcohol precipitate.

[0043] (3) Membrane separation and purification

[0044] The alcohol precipitate obtained in step (2) is filtered using an ultrafiltration membrane with a molecular weight cutoff of 5000Da (the transmembrane pressure difference is 1Mpa) to remove most of the macromolecular substances such as proteins and pigments, and the permeate is collected. The permeate is then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000Da (the transmembrane pressure difference is 1Mpa), and the permeate is collected again. The permeate collected again is then separated and purified by nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 150 (the transmembrane pressure difference is 2Mpa), and the concentrated solution is collected to obtain the crude extract of Cordyceps sinensis.

[0045] The pH of the concentrated solution was adjusted to 7 to obtain a loading solution for subsequent resin separation and purification.

[0046] Example 2 Optimization of adsorption resin type for separation and purification of cordycepin

[0047] The present invention selects 5 different types of resins (XAD-16, XAD-1600, KA-I, HD-01 and HD-06, provided by the National Biochemical Center of Kaohsiung University of Technology), and investigates the adsorption capacity of different types of resins for cordycepin in the sample solution obtained in Example 1. Among them, XAD-16 and XAD-1600 are macroporous adsorption resins, KA-I is a mesoporous and microporous resin, HD-01 and HD-06 are ultra-high cross-linking resins, and the physical and chemical properties of the above 5 types of resins are shown in Table 1.

[0048] Table 1 Physical and chemical properties of five types of resins

[0049]

[0050] Static adsorption experiment:

[0051] The above five resins were pretreated and filtered, and then 2 g of each was placed in a 100 mL conical flask, 50 mL of the sample solution was added to the conical flask, and the conical flask was sealed with a sealing film, placed in a shaker, and shaken at 25°C and 120 r / min for 8 hours to allow the sample solution and resin to fully contact and cordycepin to be fully adsorbed on the resin, and then the concentration of cordycepin in the sample solution after adsorption was determined.

[0052] Another experimental group was set up. Except that the temperature of the shaker was set to 45°C, other experimental operations were the same as the above static adsorption experiment.

[0053] Adsorption capacity = (concentration of target compound in initial solution - concentration of target compound in solution at adsorption equilibrium) × volume of initial solution / mass of resin

[0054] Static desorption experiment:

[0055] The surface moisture of the saturated resin was absorbed with filter paper, and the resin was placed in a 100 mL conical flask. Then, 20 mL of eluent (50% v / v ethanol aqueous solution) was accurately added. The resin was placed in a shaker and shaken at 25°C and 120 r / min for 8 h to allow the eluent to fully contact the resin. The concentration of cordycepin in the eluent was then determined.

[0056] Desorption amount = concentration of target compound in desorption liquid at desorption equilibrium × volume of desorption liquid / resin mass

[0057] Desorption rate = (concentration of target compound in desorption solution at desorption equilibrium × volume of desorption solution) / (mass of resin × adsorption capacity) × 100%

[0058] The static adsorption capacity of cordycepin on different types of resins at 25℃ and 40℃ and their desorption at 25℃ are shown in Figure 2. Figure 1 As shown. Figure 1 It can be seen that at 25℃ and 40℃, the adsorption capacity of cordycepin on various types of resins has the same trend. Among them, HD-01 and HD-06 resins have the highest adsorption capacity for cordycepin (20.2mg / g), followed by KA-I resin, while XAD-16 and XAD-1600 resins have lower adsorption capacity for cordycepin. This may be because the ultra-high cross-linked resin has a unique microporous structure and a large specific surface area, so the adsorption capacity for cordycepin is also large. Figure 1 It can also be seen that, except for HD-01 resin, the cordycepin adsorption capacity of the other four types of resins at 25°C is higher than that at 40°C.

[0059] In addition to the adsorption capacity, the desorption difficulty of cordycepin on different types of resins also needs to be considered. In order to ensure that most of the cordycepin is desorbed and to compare the desorption performance between different types of resins, 50% v / v ethanol is used as the eluent. The results show that the desorption rate of KA-I reached 72.6%, followed by HD-06, which reached 70.8%. Although the desorption performance of the two resins is similar, when adsorbing cordycepin in the actual sample solution, HD-06 has a stronger adsorption capacity for pigments. Therefore, in the subsequent elution process, the pigment adsorbed on the resin will also enter the cordycepin eluent, resulting in a darker color, affecting the color of the final product. Considering the resin's adsorption capacity for cordycepin, the difficulty of desorption, and the adsorption selectivity of the pigment, KA-I resin was selected as the adsorbent for separating cordycepin in the sample solution.

[0060] Example 3 Optimization of Cordycepin Eluent

[0061] Since cordycepin has a strong adsorption affinity on the resin and is difficult to desorb with water, ethanol aqueous solutions of different volume concentrations are considered as desorbents. At room temperature (25°C), a static desorption experiment (the experimental steps are the same as those in Example 2) was conducted to investigate the effect of ethanol concentration on the desorption rate of cordycepin adsorbed on KA-I resin. The results are as follows: Figure 2 As shown. Figure 2 It can be known that, along with the increase of ethanol volume concentration, its desorption capacity to the cordycepin adsorbed on the resin is enhanced, but when the volume fraction of ethanol exceeds 70%, the desorption rate decreases instead. This may be due to the low solubility of cordycepin in ethanol at room temperature, when the ethanol concentration in the ethanol aqueous solution exceeds a certain value, the desorption capacity to cordycepin decreases instead. In addition, the desorption rate of ethanol with a volume fraction of 50% is 79.9%, which is equivalent to the desorption rate of ethanol with a volume fraction of 70% (the desorption rate is 81.5%). Considering the consumption and recycling of ethanol in the elution process, therefore, the ethanol aqueous solution with a volume fraction of 50% is finally adopted as the desorbent.

[0062] Example 4 Effect of pH on Adsorption Capacity of Cordycepin

[0063] There is a nitrogen-containing base (adenine) in the cordycepin molecule, so the dissociation state of cordycepin under different pH conditions is different. Therefore, at room temperature (25°C), a static adsorption experiment was conducted to investigate the adsorption capacity of cordycepin on KA-I resin under different pH conditions. The pH of the concentrated solution obtained in Example 1 was adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, to obtain the sample solution, and the subsequent operation steps were the same as the static adsorption experiment in Example 2. The experimental results are shown in Figure 2. Figure 3As shown in the figure, the results show that with the increase of the pH of the sample solution, the adsorption capacity of cordycepin on the resin gradually increases; when the pH of the sample solution is greater than 7.0, its adsorption capacity reaches a stable value (about 21.5 mg / g). Therefore, under the condition of pH = 7.0, KA-I resin has a better effect on the adsorption of cordycepin.

[0064] Example 5 Optimization of dynamic adsorption conditions of cordycepin

[0065] A glass chromatography column with a jacket was used, and the temperature was controlled by a water bath. The dimensions of the column were: an inner diameter of 1.6 cm and a height of 40 cm. The adsorption resin was loaded into the chromatographic column by a wet method, and the bed was evenly rinsed with pure water. The loading solution prepared in Example 1 was loaded onto a KA-I resin column, and the optimal loading conditions were selected by examining the loading flow rate, the height-to-diameter ratio of the chromatography column, and the temperature on the adsorption effect of cordycepin on the resin.

[0066] (1) Penetration curves of cordycepin on KA-I resin at different loading flow rates

[0067] At room temperature (25°C), the sample solution prepared in Example 1 was loaded onto the KA-I resin column at different flow rates (0.5, 1.0 and 1.5 mL / min). The penetration curve of cordycepin on the column was as follows: Figure 4 As shown. Figure 4 It can be seen that under the condition of constant column height-to-diameter ratio, temperature and injection concentration, as the injection flow rate increases, the penetration time of cordycepin is advanced. The penetration curve of cordycepin is relatively gentle, from the beginning of penetration (ρ e / ρ0>0.05,ρ 0、 ρ e represents the mass concentration of cordycepin in the resin column inlet and outlet solutions respectively) to complete adsorption saturation (ρ e / ρ0 is close to 1) for a long time. This is because the cordycepin mass concentration in the sample solution is low (2.06 g / L), the concentration difference between the cordycepin concentration in the solution and the resin is small, and the driving force for mass transfer is small, so it takes a long time for cordycepin to reach adsorption equilibrium on KA-I resin. Figure 4 It can also be seen that as the loading flow rate increases, cordycepin begins to flow out before it reaches adsorption equilibrium on the resin, so the penetration time is advanced. At a lower flow rate, it is beneficial for the adsorption of cordycepin on the KA-I resin and can reduce the length of the mass transfer zone of the chromatography column, but at a lower flow rate, the time required for loading is also correspondingly longer. Therefore, considering the mass transfer effect and production efficiency, it is more appropriate to choose a loading flow rate of 1.0mL / min.

[0068] (2) Penetration curves of cordycepin on KA-I resin columns with different aspect ratios

[0069] If the height-to-diameter ratio of the chromatographic column is increased, the number of theoretical plates will increase, and the separation degree between the substances in the multi-component system will also increase accordingly; but as the height-to-diameter ratio increases, the separation time will be prolonged, the column pressure will also increase accordingly, the corresponding energy consumption will increase, and the production efficiency will decrease. Therefore, the penetration curve of cordycepin in the KA-I column under different height-to-diameter ratios (10:1, 15:1 and 25:1) was investigated, and the results are shown in Figure 5 .Depend on Figure 5 It can be seen that as the height-to-diameter ratio increases, the penetration time of cordycepin is delayed, the time to reach adsorption equilibrium is longer, and the required injection volume is also larger. Under different height-to-diameter ratio conditions, the adsorption capacity of cordycepin on KA-I resin is similar (about 29.2 mg / g), which may be because in addition to cordycepin, there are also substances such as adenosine and cordycepic acid in the sample solution, especially the structure of adenosine is very similar to cordycepin, thus affecting the separation of cordycepin. In order to achieve efficient separation between these components, it is possible to consider operating under a larger height-to-diameter ratio condition.

[0070] (3) Penetration curves of cordycepin on KA-I resin at different temperatures

[0071] The penetration curves of cordycepin in KA-I resin column were investigated under different temperature conditions. Figure 6 .Depend on Figure 6 It can be seen that with the increase of temperature, the breakthrough curve is advanced, which is consistent with the adsorption isotherm results of cordycepin on KA-I resin. It can be seen that high temperature is more conducive to the desorption of cordycepin on KA-I resin; with the increase of temperature, the breakthrough curve of cordycepin is steeper, which is consistent with the expected results. This is because the Brownian motion of cordycepin molecules in aqueous solution intensifies with the increase of temperature, and the molecular diffusion coefficient increases, which increases the mass transfer rate on the resin and can reach adsorption equilibrium faster. Considering the adsorption amount of cordycepin on the resin and the time required to reach adsorption equilibrium, the temperature for loading is selected as 25°C.

[0072] Example 6 Optimization of macroporous adsorption resin type for separation and purification of HEA

[0073] At room temperature (25°C), the adsorption and desorption effects of four macroporous resins, AB-8, ADS-17, D-101 and DM-130, on HEA were investigated. The experimental method was the same as the static adsorption experiment and static desorption experiment in Example 2. The results showed that the adsorption amounts of AB-8 and ADS-17 were relatively small. At 0 to 4 h, the adsorption amount of D-101 was larger than that of DM-130. After 6 h, the adsorption amount of DM-130 reached the maximum, and the desorption rates of the two were similar. In order to ensure that the HEA in the crude extract can be fully absorbed, DM-130 was selected as the best resin for separating HEA, and the adsorption time was 6 h.

[0074] Since the polarity of HEA is relatively small, the highly polar eluent causes HEA to fall off the resin together with impurities quickly, and the separation effect is not achieved. However, the polarity of the eluent is too small and the elution time is too long. Under room temperature (25°C), a static desorption experiment was conducted to investigate the effect of ethanol concentration on the desorption rate of HEA adsorbed on DM-130 resin. The experimental method is the same as that of Example 3. The results show that 20% v / v ethanol can quickly elute HEA on DM-130 resin, and 5% v / v ethanol has a too long elution time. Therefore, in order to more effectively separate HEA, after DM-130 resin absorbs 2-3 BV of the sample solution, the resin bed is first rinsed with 3-4 BV of distilled water to remove impurities, then 1-2 BV of 5% v / v ethanol aqueous solution is used to wash the impurities, and finally 3-4 BV of 10% v / v ethanol aqueous solution is used to elute.

[0075] Example 7 Separation and purification of cordycepin and HEA

[0076] The sample solution prepared in Example 1 was loaded onto a KA-I adsorption resin column (height-to-diameter ratio 25:1, column height 25cm, resin weight 5.31g) to separate and purify cordycepin, with a sample load of 2.5BV and a sample flow rate of 1.0mL / min. After loading, cordycepin was eluted with an ethanol aqueous solution having a volume fraction of 50% as an eluent, with an eluent dosage of 2BV and an elution flow rate of 2.5BV / h, and the cordycepin eluate was collected. The mass concentration of cordycepin in the sample solution was 1.65g / L, and the maximum mass concentration of cordycepin in the eluent could reach 9.68g / L.

[0077] The sample solution prepared in Example 1 was loaded onto a DM-130 macroporous resin column (height-to-diameter ratio 25:1, column length 25 cm, resin weight 7.56 g) to separate and purify HEA, with a loading volume of 2 BV and a loading flow rate of 1.5 BV / h. After loading, the resin bed was first rinsed with 3 BV of distilled water at a flow rate of 2 BV / h, then washed with 1 BV of 5% v / v ethanol aqueous solution at a flow rate of 2 BV / h, and finally eluted with 3 BV of 10% v / v ethanol aqueous solution to collect the HEA eluate. The yield of HEA in the eluate can reach 87%.

[0078] The cordycepin eluate was concentrated under reduced pressure to a solid-liquid ratio of 1:2.5 (mass ratio), heated to 90°C, and refrigerated at 10°C for 8 hours for crystallization to obtain 6.25 g of cordycepin extract with a purity of 82% and a cordycepin yield of 97%.

[0079] The HEA eluate was concentrated under reduced pressure to a solid-liquid ratio of 1:2.5, heated to 90°C, and refrigerated at 10°C for 8 h to crystallize, to obtain 1.25 g of cordycepin extract with a purity of 86% and a HEA yield of 85%.

[0080] In summary, the preparation method of the present invention obtains high content of cordycepin and N6-(2-hydroxyethyl)-adenosine, has strong process operability, low cost, easy industrial production, does not use toxic and harmful organic solvents, is highly safe, and is environmentally friendly.

[0081] The present invention provides a method and idea for preparing a Cordyceps sinensis extract. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a Cordyceps sinensis extract, characterized in that: The following steps are involved: (1) subjecting the raw material of Cordyceps sinensis to microwave water extraction, concentrating the extract, precipitating with alcohol, and separating the solid and liquid to obtain an alcohol precipitate; (2) subjecting the alcohol precipitate obtained in step (1) to a first ultrafiltration, collecting the first ultrafiltration permeate to a second ultrafiltration, collecting the second ultrafiltration permeate to a nanofiltration, and collecting a concentrated solution; (3) adjusting the pH of the concentrated solution obtained in step (2) to 6.5-7 to obtain a loading solution, loading the loading solution onto an adsorption resin column for adsorption, eluting with a first ethanol aqueous solution, and collecting the eluate; (4) Concentrating the eluate obtained in step (3), heating, and recrystallizing to obtain the product.

2. The preparation method according to claim 1, characterized in that: The Cordyceps sinensis extract is cordycepin or N6-(2-hydroxyethyl)-adenosine.

3. The preparation method according to claim 1, characterized in that: In step (1), when the microwave water extraction is performed, the mass ratio of the Cordyceps sinensis raw material to water is 1:10-15, the microwave power is 300W-400W, and the extraction time is 30min-45min.

4. The preparation method according to claim 1, characterized in that: In step (1), when the alcohol precipitation is performed, the precipitant used is a 95% to 99% v / v ethanol aqueous solution, the alcohol precipitation time is 6h to 18h; and the concentration of ethanol in the alcohol precipitation solution is 80% to 90% v / v.

5. The preparation method according to claim 1, characterized in that: In step (2), the first ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000-8000Da and a transmembrane pressure difference of 1-3Mpa; the second ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1000-5000Da and a transmembrane pressure difference of 1-3Mpa; the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 150-1000Da and a transmembrane pressure difference of 2-4Mpa.

6. The preparation method according to claim 1, characterized in that: When the Cordyceps sinensis extract is cordycepin, in step (3), the model of the adsorption resin is XAD-16, XAD-1600, KA-I, HD-01 or HD-06; the loading adsorption amount of the loading liquid is 1.5 to 3 BV, and the loading flow rate is 2.5 to 4 BV / h; the concentration of the first ethanol aqueous solution is 30% to 50% v / v, the dosage is 1 to 3 BV, and the elution flow rate is 2 to 3.5 BV / h.

7. The preparation method according to claim 1, characterized in that: When the Cordyceps sinensis extract is N6-(2-hydroxyethyl)-adenosine, in step (3), the model of the adsorption resin is AB-8, ADS-17, D-101 or DM130, the sample adsorption amount of the sample solution is 2 to 3BV, and the sample flow rate is 1 to 2.5BV / h; before eluting with the first ethanol aqueous solution, the impurities are washed with water and a second ethanol aqueous solution of 5% to 30% v / v in sequence; when washing with the water, the amount of water is 3 to 4BV, and the washing flow rate is 2 to 3.5BV / h; when washing with the second ethanol aqueous solution, the amount of the second ethanol aqueous solution is 1 to 2BV, and the washing flow rate is 2 to 3.5BV / h.

8. The preparation method according to claim 1, characterized in that: When the Cordyceps sinensis extract is N6-(2-hydroxyethyl)-adenosine, in step (3), the concentration of the first ethanol aqueous solution is 5% to 30% v / v, the dosage is 3 to 4 BV, and the elution flow rate is 2 to 3.5 BV / h.

9. The preparation method according to claim 1, characterized in that: In step (4), the heating is heating to above 90° C.; the recrystallization is low-temperature recrystallization; the recrystallization temperature is 0° C. to 10° C., and the time is 5 h to 12 h.

10. Use of the Cordyceps sinensis extract prepared by the preparation method according to any one of claims 1 to 9 in the preparation of medicines.

Images