Extraction method of dendrobium officinale polysaccharide

Abstract

The invention belongs to an extraction and separation technology of active ingredients of natural products in light industry and chemical engineering biotechnology in biology and new medicine, relates to the field of plant extracts, and in particular relates to an extraction method of dendrobium officinale polysaccharide. The method comprises the following steps: firstly, preparing dendrobium officinale stem micro powder by adopting freeze drying and superfine grinding technologies; then extracting polysaccharide in the dendrobium officinale stem micro powder by utilizing an ultrasonic wall breaking method and a hot water extraction method; and finally, purifying the dendrobium officinale polysaccharide by adopting high-speed counter-current chromatography, dialysis and alcohol precipitation methods. The dendrobium officinale polysaccharide prepared by the method is high in purity and can be directly used as a functional additive of food and health care products.

Description

Technical Field

[0001] The invention belongs to the separation and extraction technology of effective components of natural products of light industry and chemical industry biotechnology in biology and new medicine, relates to the field of plant extracts, and specifically is a method for extracting Dendrobium officinale polysaccharide. Background Art

[0002] Dendrobium candidum belongs to the orchid family and is a commonly used herb in traditional Chinese medicine. Dendrobium candidum is rich in polysaccharides, alkaloids, phenols, phenolic alkenes, acids, esters and amides. Therefore, it has unique functions and therapeutic effects and is often used as a drug, food additive, and cosmetic additive. Dendrobium candidum polysaccharide, as an important health-care ingredient of Dendrobium candidum, has pharmacological and physiological functions such as regulating immunity, lowering blood sugar, lowering blood pressure, anti-oxidation, anti-inflammatory and anti-cancer. At present, most of the Dendrobium candidum used in food is Dendrobium candidum powder or Dendrobium candidum stem segments. The purity and content of polysaccharides are low. It is difficult for consumers to fully absorb the active ingredients therein after consumption, and the health-care effect of the product is not obvious. Therefore, it is of great significance to develop a high-purity Dendrobium candidum polysaccharide extract to improve the health-care effect of Dendrobium candidum products.

[0003] At present, the methods for extracting polysaccharides from plants include immersion, reflux extraction, ultrasonic extraction, microwave extraction, enzymatic extraction and freeze-thaw extraction. However, a single extraction method has problems such as low extraction efficiency and high cost. And from a large number of previous studies, Dendrobium officinale polysaccharides are obtained through a series of complex separation processes, including graded precipitation, deproteinization, decolorization, anion exchange and Sephadex gel chromatography. In addition, the toxic reagents introduced in the traditional method will change the structural characteristics of natural polysaccharides, thereby affecting their biological activity. For example: in Chinese patent CN114848566A, polysaccharides are extracted by traditional methods, not only organic reagents are introduced, but also the final polysaccharide purity can only reach 75%. For example, in Chinese patent CN118745450A, Dendrobium officinale polysaccharides are extracted by microbial fermentation, but the fermentation time is long and the polysaccharide concentration after fermentation is reduced from the original 0.29 mg / mL to 0.17 mg / mL. Therefore, systematically developing a new method for separating and purifying Dendrobium officinale polysaccharides with good application value is the key to the refined separation and application of Dendrobium officinale polysaccharides.

[0004] High-speed countercurrent chromatography (HSCCC) is a continuous liquid-liquid distribution chromatography method that uses the difference in the distribution of solutes in a mutually immiscible two-phase system for separation, eliminating the irreversible adsorption loss of the solid matrix and the sample contamination problem, and can achieve efficient, rapid, and large-scale sample preparation without pretreatment. However, the solvent system of HSCCC is mostly organic reagents, and the solubility of polysaccharides in the solvent system is too small, resulting in limited solvent system screening and great difficulty in separation. The existing traditional separation methods use the sevage method and gel chromatography to purify Dendrobium officinale polysaccharides. The purity of the purified polysaccharides is 61.8%, which cannot be directly used in food additives. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a green and efficient method for purifying Dendrobium officinale polysaccharide. The method of the present invention can obtain Dendrobium officinale polysaccharide with high purity (polysaccharide content>84%, up to 92.7%).

[0006] In order to solve the above technical problems, the present invention provides a method for extracting polysaccharides from Dendrobium officinale, comprising the following steps:

[0007] 1) Preparation of Dendrobium officinale stem powder:

[0008] The fresh stems of Dendrobium officinale are subjected to vacuum freeze drying, and then pulverized (ultrafine pulverization) to obtain freeze-dried stem powder of Dendrobium officinale;

[0009] 2) Obtaining crude extract of Dendrobium officinale:

[0010] The freeze-dried Dendrobium officinale stem powder obtained in step 1) is added to deionized water, and ultrasonically crushed (ultrasonic wall breaking) and then extracted; the obtained extract is filtered to remove the filter residue (i.e., separate the solid particles), and the obtained filtrate is concentrated to 0.02 to 0.04 times the volume to obtain a crude extract (Dendrobium officinale crude extract);

[0011] The mass ratio of freeze-dried Dendrobium officinale stem powder to deionized water is 1:30±2;

[0012] 3) Purification of Dendrobium officinale polysaccharide:

[0013] The crude extract is purified by high-speed countercurrent chromatography, dialysis and alcohol precipitation to obtain Dendrobium officinale polysaccharide.

[0014] As an improvement of the extraction method of Dendrobium officinale polysaccharide of the present invention, the step 3) comprises the following steps:

[0015] Step a: Preparation of a two-phase solution of a high-speed countercurrent chromatography two-phase aqueous system:

[0016] The two-phase aqueous system is composed of 10-20% by weight of polyethylene glycol, 10-20% by weight of inorganic salt and the balance of deionized water.

[0017] The two-phase aqueous system obtained by mixing polyethylene glycol, inorganic salt and deionized water is allowed to stand for separation; the upper layer is subjected to ultrasonic degassing and named as the stationary phase; the lower layer is subjected to ultrasonic degassing and named as the mobile phase;

[0018] After standing and stratifying, a separatory funnel can be used to separate the two phases;

[0019] Step b: Preparation of injection sample:

[0020] Add the crude extract (Dendrobium officinale crude extract) obtained in step 2) to the mobile phase, discard the insoluble matter (discard the insoluble matter by centrifugation), and obtain an injection sample;

[0021] Crude extract: mobile phase = 1:4 ± 0.5 volume ratio;

[0022] Step c, separation and purification of Dendrobium officinale polysaccharide by high-speed countercurrent chromatography:

[0023] Fill the spiral tube of the high-speed countercurrent chromatography with the stationary phase, and then pump the mobile phase from the head end at a flow rate of 1 to 2 mL / min, inject the sample after the system is stable (i.e., inject the sample after the two phases reach equilibrium), and then pump the mobile phase from the head end at a flow rate of 1 to 2 mL / min; collect the effluent containing polysaccharides (the effluent with peaks is detected by a differential detector);

[0024] Step d, dialysis and alcohol extraction:

[0025] The effluent containing the polysaccharide is dialyzed and concentrated, and then ethanol precipitation is performed, and the obtained precipitate (the precipitate can be collected by centrifugation) is freeze-dried to obtain the Dendrobium officinale polysaccharide extract.

[0026] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, in step a: the inorganic salt is Na2SO4, and the molecular weight of polyethylene glycol is 600-2000 (preferably 600-1000, more preferably 800).

[0027] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, in step d:

[0028] The dialysis bag used for dialysis is a dialysis bag with a molecular weight cutoff of 3500 to 15000 Da, and the dialysis time is 12 to 24 hours;

[0029] Concentrating the retentate obtained by dialysis to 6-10% (preferably 8-9%) of the original volume to obtain a concentrated solution;

[0030] According to the volume ratio of concentrated solution: ethanol = 1: (3 ± 0.5), ethanol (anhydrous ethanol) was added to the concentrated solution and precipitated at 3 to 5° C. for 16 to 20 hours.

[0031] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, in step 2):

[0032] The ultrasonic crushing power is 200-400W, the ultrasonic crushing frequency is 10-25kHz, and the time is 20-60min;

[0033] The extraction temperature is 60-100°C and the time is 1-5h (preferably 80°C, 2h).

[0034] The system obtained by adding water to 6-10 g of Dendrobium officinale stems can be operated in the above manner.

[0035] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, in step a: the standing and stratification time is 6 to 14 hours.

[0036] After standing and separating the two phases, a separatory funnel can be used to separate the two phases; thereby obtaining the upper phase and the lower phase.

[0037] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, in step 1):

[0038] The vacuum freeze drying temperature is -25℃~-35℃, and the time is 18~36 hours;

[0039] The mixture is crushed (ultrafinely crushed) until it passes through a 100-300 mesh sieve to obtain freeze-dried Dendrobium officinale stem powder.

[0040] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, the temperature during high-speed countercurrent chromatography separation is 30-35°C.

[0041] As a further improvement of the extraction method of Dendrobium officinale polysaccharide of the present invention, the two-phase aqueous system consists of polyethylene glycol with a mass content of 16-18%, inorganic salt with a mass content of 10-15% and deionized water as the balance.

[0042] As a further improvement of the method for extracting Dendrobium officinale polysaccharides of the present invention, the two-phase aqueous system consists of PEG800 with a mass content of 18%, sodium sulfate with a mass content of 10% and deionized water as the balance.

[0043] In the invention process, the high speed countercurrent chromatography (HSCCC) method is optimized to more effectively separate different polysaccharide components, and the successful separation of HSCCC depends on the selection of a suitable two-phase solvent system. The details are as follows:

[0044] 1. The effect of inorganic salt set as sodium sulfate, PEG (polyethylene glycol) molecular weight 800, and inorganic salt mass concentration of 10% and 15% on the distribution coefficient of Dendrobium officinale polysaccharide. The calculation formula of the distribution coefficient K value is the distribution ratio of polysaccharide between two phases of solvent under equilibrium state. The formula is as follows:

[0045] K = CU / CL, where CU and CL represent the sugar content of Dendrobium officinale polysaccharides in the upper phase and lower phase, respectively; to achieve separation of substances, the K value must be between 0.5 and 2.

[0046] Finally, the distribution coefficients (K values) of 10% and 15% of the inorganic salt mass concentration were 0.96 and 0.73, respectively. The separation conditions were met, and the effect of 10% was good. Therefore, the specific method of setting the high-speed countercurrent chromatograph was as follows:

[0047] Sodium sulfate, PEG800 and water were mixed evenly in a mass ratio of 10:18:72 and allowed to stand for separation to obtain an upper phase solution and a lower phase solution; the upper phase solution was used as the mobile phase and the lower phase solution was used as the stationary phase.

[0048] 2. The effect of inorganic salt set to sodium sulfate, PEG (polyethylene glycol) molecular weight 600, and inorganic salt mass concentration of 10% and 15% on the distribution coefficient of Dendrobium officinale polysaccharide.

[0049] Finally, the distribution coefficients of 10% and 15% inorganic salt mass concentrations were 0.65 and 0.55 respectively. The separation conditions were met, and the effect of 10% was the best. Therefore, the specific method for setting the high-speed countercurrent chromatograph is:

[0050] Sodium sulfate, PE600 and water were mixed evenly in a mass ratio of 10:18:72 and then allowed to stand for separation to obtain an upper phase solution and a lower phase solution; the upper phase solution was used as a mobile phase and the lower phase solution was used as a stationary phase;

[0051] 3. The effect of inorganic salt set to sodium sulfate, PEG (polyethylene glycol) molecular weight 1000, and inorganic salt mass concentration of 10% and 15% on the distribution coefficient of Dendrobium officinale polysaccharide.

[0052] Finally, the distribution coefficients of 10% and 15% inorganic salt mass concentrations were 1.78 and 1.65, respectively. The separation conditions were met, and the 10% concentration had the best effect. Therefore, the specific method for setting the high-speed countercurrent chromatograph was as follows:

[0053] Sodium sulfate, PE1000 and water were mixed evenly in a mass ratio of 10:18:72 and allowed to stand for separation to obtain an upper phase solution and a lower phase solution; the upper phase solution was used as the mobile phase and the lower phase solution was used as the stationary phase.

[0054] The present invention combines high-speed countercurrent chromatography, dialysis and alcohol precipitation to purify Dendrobium officinale polysaccharide, so that the prepared polysaccharide has high purity; PEG (preferably PEG800)-Na2SO4-H2O is used to form a biphasic water phase according to a set mass ratio, and the upper phase is used as the stationary phase in the high-speed countercurrent chromatography, and the lower phase is used as the mobile phase in the high-speed countercurrent chromatography. Because the polysaccharide has high solubility in water, the system improves the purification efficiency of the polysaccharide. The present invention improves the process of countercurrent chromatography separation of polysaccharides and performs online detection of the effluent, which greatly reduces the workload of high-speed countercurrent chromatography separation. The purity of the Dendrobium officinale polysaccharide prepared by the present invention can be as high as 90% or more.

[0055] Compared with the prior art, the method for extracting polysaccharides from Dendrobium officinale described in the present invention has the following beneficial effects: compared with the traditional extraction method, the present invention combines the ultrasonic wall breaking method with the leaching method to destroy the plant cell walls in Dendrobium officinale so that the polysaccharide compounds can be quickly leached, and has the characteristics of short operation time, low energy consumption and high extraction efficiency.

[0056] The present invention establishes an effective method for separating Dendrobium officinale polysaccharides based on HSCCC using sodium sulfate as an inorganic salt; the Dendrobium officinale polysaccharides with a purity of up to 92% are separated, solving the problems in the existing plant polysaccharide purification technology that the product concentration can generally only reach about 50%, the organic solvent consumption is large, and the product is difficult to purify. DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0058] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0059] The HSCCC instrument in the present invention is a TBE-300V HSCCC system (Shanghai Tongtian Biotechnology Co., Ltd.), equipped with a temperature and speed control system, a 360mL coil volume, a 20mL injection system, a BSZ-100 automatic fraction collector, and a differential detector (Labindex 2000). Each time the separation is performed, the high-speed countercurrent chromatograph is turned on, and the temperature of the chromatographic column is controlled at 30-35°C by a constant temperature circulation device. The upper phase is pumped into the chromatographic column at a flow rate of 30.0mL / min as the stationary phase, and the mobile phase is pumped into the chromatographic column at a flow rate of 0.5-2mL / min. The device rotates at speeds of 700, 800, and 900rpm. When the mobile phase flows out of the chromatographic column, 20mL of the sample is injected into the injection loop, 10mL of the effluent is collected in each tube, and according to the differential detector display signal, the test tubes of the polysaccharide components belonging to the same peak are combined and dialyzed (molecular weight cutoff: 3500Da) to remove PEG and salt. Finally, the purified polysaccharide component is obtained by alcohol precipitation and freeze drying. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0060] Example 1-1, a method for extracting polysaccharides from Dendrobium officinale, characterized by comprising the following steps:

[0061] 1) Preparation of Dendrobium officinale stem powder:

[0062] The fresh stems of Dendrobium officinale were placed in a vacuum freeze dryer for freeze drying (vacuum degree of 5 Pa), the freeze drying temperature was controlled to be -25°C, and the freeze drying time was 36 hours; then the freeze dried stems of Dendrobium officinale were ultra-finely crushed to exceed 300 meshes to obtain Dendrobium officinale stem powder;

[0063] 2) Obtaining crude extract of Dendrobium officinale:

[0064] 6g of Dendrobium officinale stem powder was added into 180g of deionized water, and then ultrasonically crushed and extracted; the details are as follows:

[0065] Dendrobium officinale stem powder: deionized water = mass ratio of 1:30;

[0066] The ultrasonic wall breaking power is 300W, the time is 20min, the ultrasonic crushing frequency is 10-25kHz; the extraction temperature is 80℃, and the time is 2h.

[0067] After the solid particles were separated by filtration, the filtrate was concentrated to 0.03 times of the original volume to obtain a crude extract (Dendrobium officinale crude extract);

[0068] 3) Purification of Dendrobium officinale polysaccharide:

[0069] The polysaccharide from Dendrobium officinale was purified by high-speed countercurrent chromatography, dialysis and alcohol precipitation. The specific steps are as follows:

[0070] Step a, preparation of a two-phase solution of a high-speed countercurrent chromatography two-phase aqueous system: sodium sulfate, PEG800, and deionized water are mixed in a mass ratio of 10:18:72 and then allowed to stand for stratification (stratification time is about 6 hours) to obtain an upper phase solution and a lower phase solution respectively; the upper phase solution is subjected to ultrasonic degassing treatment and is named as a stationary phase; the lower phase solution is subjected to ultrasonic degassing treatment and is named as a mobile phase;

[0071] The specific process parameters of the ultrasonic degassing treatment are 400W and time of 30 minutes.

[0072] Step b: Preparation of injection sample:

[0073] Add the mobile phase to the crude extract obtained in step 2) and stir evenly, centrifuge and discard the insoluble matter to obtain an injection sample;

[0074] The volume ratio of the crude extract:mobile phase=1:4;

[0075] Step c, separation and purification of Dendrobium officinale polysaccharide by high-speed countercurrent chromatography:

[0076] Turn on the pump, inject about 400ml of the stationary phase into the spiral tube of the high-speed countercurrent chromatography (i.e., fill the spiral tube of the high-speed countercurrent chromatography with the stationary phase), and set the speed to a predetermined value (i.e., 30.0mL / min). Start the high-speed countercurrent chromatography, rotate clockwise, adjust the speed to 800-900r / min, and pump the mobile phase from the head end at a flow rate of 2mL / min. After the system stabilizes (i.e., after the two phases reach equilibrium, when the mobile phase flows out of the chromatographic column), use the injection loop to inject the sample (i.e., pump 20ml of the sample obtained in step b from the head end), and then pump the mobile phase from the head end at a flow rate of 2mL / min; use an automatic collector to collect the effluent containing polysaccharides (the effluent with peaks detected by the differential detector);

[0077] 10 mL of the effluent was collected from each tube, and the effluents from tubes 12 to 23 were collected and combined.

[0078] Step d, dialysis and alcohol extraction:

[0079] The combined solution obtained in step c was dialyzed (molecular weight cutoff: 3500Da) to remove PEG and salt. The dialysis process was as follows: 120 ml of the combined solution was placed in a dialysis bag with a molecular weight cutoff of 3500Da, and placed in deionized water (about 5000 ml) for dialysis. The deionized water was replaced every 8 hours during the dialysis process, and the dialysis time was 72 hours; the retentate in the dialysis bag was concentrated to 10 ml in a rotary evaporator at 55°C to obtain a concentrated solution.

[0080] According to the volume ratio of concentrate: ethanol = 1:3, ethanol was added to the concentrate for alcohol precipitation at 4°C for 20 hours, and then centrifuged (speed was 8000r / min), and the precipitate obtained by centrifugation was freeze-dried at -40°C for 48 hours to obtain the Dendrobium officinale polysaccharide extract.

[0081] According to the anthrone sulfate method, the polysaccharide concentration of the Dendrobium officinale polysaccharide extract was 92.7%.

[0082] Example 1-2: The following changes are made relative to Example 1-1:

[0083] In step 3), in "step a, preparation of a two-phase solution for a high-speed countercurrent chromatography two-phase aqueous system", the mass ratio of sodium sulfate, PEG800, and water is changed from "10:18:72" to "15:18:67", and the rest is the same as in Example 1-1.

[0084] The results show that the polysaccharide concentration of the Dendrobium officinale polysaccharide extract is 84.7%.

[0085] Example 1-3: The following changes are made relative to Example 1-1:

[0086] In step 3), in "step a, preparation of a two-phase solution for a high-speed countercurrent chromatography two-phase aqueous system", the mass ratio of sodium sulfate, PEG800, and water is changed from "10:18:72" to "14:16:70", and the rest is the same as in Example 1-1.

[0087] The results show that the polysaccharide concentration of the Dendrobium officinale polysaccharide extract is 89.8%.

[0088] Example 2: The following changes are made relative to Example 1-1:

[0089] In step 3), "step a, preparation of a two-phase solution of a high-speed countercurrent chromatography two-phase aqueous system" is changed to: sodium sulfate, PE600, and water are evenly mixed in a mass ratio of 10:18:72; the rest is the same as Example 1-1.

[0090] The polysaccharide concentration of the Dendrobium officinale polysaccharide extract is 86.7%.

[0091] Example 3: With respect to Example 1-1, the following changes are made:

[0092] In step 3), "step a, preparation of two-phase solution of high-speed countercurrent chromatography two-phase aqueous system" is changed to: sodium sulfate, PE1000 and water are mixed in a mass ratio of 10:18:72; the rest is the same as Example 1-1.

[0093] The polysaccharide concentration of the Dendrobium officinale polysaccharide extract is 84.6%.

[0094] Comparative Examples 1 to 3: Compared with the two-phase solution of the high-speed countercurrent chromatography two-phase aqueous system of Example 1-1, the type and mass concentration of the inorganic salt are changed, and PEG 800 is still used, as shown in Table 1 below:

[0095] Table 1

[0096]

[0097] The separation effect corresponding to the two-phase solvent system in the above comparative example is poor, resulting in the polysaccharide concentration of the Dendrobium officinale polysaccharide extract being far lower than that of the present invention.

[0098] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for extracting polysaccharides from Dendrobium officinale, characterized in that The following steps are involved: 1) Preparation of Dendrobium officinale stem powder: The fresh stems of Dendrobium officinale are subjected to vacuum freeze drying, and then crushed to obtain freeze-dried stem powder of Dendrobium officinale; 2) Obtaining crude extract of Dendrobium officinale: The freeze-dried Dendrobium officinale stem powder obtained in step 1) is added to deionized water, and ultrasonically crushed and then extracted; the obtained extract is filtered to remove the filter residue, and the obtained filtrate is concentrated to 0.02 to 0.04 times the volume to obtain a crude extract; The mass ratio of freeze-dried Dendrobium officinale stem powder to deionized water is 1:30±2; 3) Purification of Dendrobium officinale polysaccharide: The crude extract is purified by high-speed countercurrent chromatography, dialysis and alcohol precipitation to obtain Dendrobium officinale polysaccharide.

2. The method for extracting polysaccharides from Dendrobium officinale according to claim 1, characterized in that The step 3) comprises the following steps: Step a: Preparation of a two-phase solution of a high-speed countercurrent chromatography two-phase aqueous system: The two-phase aqueous system is composed of 10-20% by weight of polyethylene glycol, 10-20% by weight of inorganic salt and the balance of deionized water. The two-phase aqueous system obtained by mixing polyethylene glycol, inorganic salt and deionized water is allowed to stand for separation; the upper layer is subjected to ultrasonic degassing and named as the stationary phase; the lower layer is subjected to ultrasonic degassing and named as the mobile phase; Step b, preparation of injection sample: Add the crude extract obtained in step 2) to the mobile phase, discard the insoluble matter, and obtain an injection sample; Crude extract: mobile phase = 1:4 ± 0.5 volume ratio; Step c, separation and purification of Dendrobium officinale polysaccharide by high-speed countercurrent chromatography: Fill the spiral tube of the high-speed countercurrent chromatography with the stationary phase, then pump the mobile phase from the head end at a flow rate of 1 to 2 mL / min, inject the sample after the system is stable, and then pump the mobile phase from the head end at a flow rate of 1 to 2 mL / min; collect the effluent containing polysaccharides; Step d, dialysis and alcohol extraction: The effluent containing the polysaccharide is dialyzed and concentrated, and then ethanol precipitation is performed, and the obtained precipitate is freeze-dried to obtain the Dendrobium officinale polysaccharide extract.

3. The method for extracting polysaccharides from Dendrobium officinale according to claim 2, characterized in that In step a: the inorganic salt is Na2SO4, and the molecular weight of polyethylene glycol is 600-2000.

4. The method for extracting polysaccharides from Dendrobium officinale according to claim 3, characterized in that In the step d: The dialysis bag used for dialysis is a dialysis bag with a molecular weight cutoff of 3500 to 15000 Da, and the dialysis time is 12 to 24 hours; The retentate obtained by dialysis is concentrated to 6-10% of the original volume to obtain a concentrated solution; According to the volume ratio of concentrated solution: ethanol = 1: (3 ± 0.5), ethanol was added to the concentrated solution for precipitation at 3-5°C for 16-20 hours.

5. The method for extracting polysaccharides from Dendrobium officinale according to claim 4, characterized in that In the step 2): The ultrasonic crushing power is 200-400W, the ultrasonic crushing frequency is 10-25kHz, and the time is 20-60min; The extraction temperature is 60-100°C and the time is 1-5h.

6. The method for extracting polysaccharides from Dendrobium officinale according to any one of claims 1 to 5, characterized in that: In step a: the standing and stratification time is 6 to 14 hours.

7. The method for extracting polysaccharides from Dendrobium officinale according to claim 6, characterized in that In the step 1): The vacuum freeze drying temperature is -25℃~-35℃, and the time is 18~36 hours; The mixture was crushed until it passed through a 100-300 mesh sieve to obtain freeze-dried Dendrobium officinale stem powder.

8. The method for extracting polysaccharides from Dendrobium officinale according to claim 7, characterized in that: The temperature during high speed countercurrent chromatography separation is 30-35°C.

9. The method for extracting polysaccharides from Dendrobium officinale according to claims 2 to 7, characterized in that: The two-phase water system consists of polyethylene glycol with a mass content of 16-18%, inorganic salt with a mass content of 10-15% and deionized water as the balance.

10. The method for extracting polysaccharides from Dendrobium officinale according to claim 9, characterized in that: The two-phase aqueous system consists of PEG800 with a mass content of 18%, sodium sulfate with a mass content of 10%, and deionized water as the balance.

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