Purified gastrodia elata polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses purified gastrodia elata polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of extraction of active ingredients of gastrodia elata. The method comprises the following steps: (1) adding an alcoholic solution into gastrodia elata powder for reflux extraction, and collecting filter residues; (2) dissolving the filter residues in pure water, and performing ultrasonic extraction to obtain filtrate; (3) adding absolute ethyl alcohol into the filtrate for low-temperature alcohol precipitation to obtain crude gastrodia elata polysaccharide; (4) performing deproteinization treatment on the gastrodia elata crude polysaccharide; (5) purifying by using macroporous resin; (6) dialyzing the crude polysaccharide purified liquid in pure water; (7) purifying by using a first chromatographic column, wherein a filler is cellulose-based anion exchange resin; (8) purifying by using a second chromatographic column to obtain purified gastrodia elata polysaccharide; and the filler is sephadex. According to the present invention, the gastrodia elata crude polysaccharide is purified by using the five purification means, such that the high-purity gastrodia elata purified polysaccharide can be rapidly, stably and efficiently obtained.

Description

Technical Field

[0001] The invention relates to the technical field of extracting active ingredients of gastrodia elata, and in particular to a gastrodia elata purified polysaccharide and a preparation method and application thereof. Background Art

[0002] In recent years, research on how Chinese herbal medicines exert their efficacy has focused more on small molecule active compounds. However, more and more scientific evidence shows that macromolecular components such as polysaccharides and proteins also play an important role in the application of Chinese herbal medicines. However, the extraction and separation of macromolecules is relatively cumbersome, and different operation methods will produce macromolecular fragments with different molecular weights or structures. How to stably obtain a uniform component with good activity has become a barrier to the development of macromolecular drugs.

[0003] Gastrodia elata is the dried tuber of Gastrodia elata, an orchidaceous plant. It has the effects of calming wind and stopping spasms, calming liver yang, and dispelling wind and unblocking collaterals. It is mainly used clinically for neurasthenia, insomnia, dizziness, epilepsy, convulsions, neuralgic headaches, hypertension and other diseases. Among them, gastrodin, an active ingredient in Gastrodia elata, has been developed into a drug to treat related diseases, while Gastrodia elata polysaccharide, another high-content active ingredient in Gastrodia elata, has relatively few studies on its structure and activity. Since the polysaccharide structure in Gastrodia elata is complex, contains a variety of monosaccharide units, and has different branched structures, the yield of Gastrodia elata purified polysaccharide obtained by the existing purification and extraction process is low and the composition is not uniform, resulting in the inability to guarantee the effect of its drug activity. There is an urgent need for a preparation process for extracting Gastrodia elata purified polysaccharide with high activity and uniform composition that can be obtained stably and efficiently. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a purified polysaccharide of Gastrodia elata and a preparation method and application thereof, so as to overcome the problems of low yield and non-uniform composition of purified polysaccharide of Gastrodia elata in the prior art.

[0005] In a first aspect, the present invention provides a method for preparing purified polysaccharides from Gastrodia elata, comprising the following steps:

[0006] (1) placing the Gastrodia elata powder in a reaction vessel, adding an alcohol solution with a volume fraction of 70% to 80%, refluxing for extraction, filtering, and collecting the residue;

[0007] (2) dissolving the filter residue in pure water for ultrasonic extraction, filtering, and collecting the filtrate;

[0008] (3) concentrating the filtrate under reduced pressure, adding anhydrous ethanol to the obtained concentrated solution to make the volume fraction of ethanol in the mixed solution be 70% to 80%, and then subjecting the mixed solution to low-temperature alcohol precipitation; after the alcohol precipitation is completed, collecting the precipitate and freeze-drying it to obtain crude Gastrodia elata polysaccharide;

[0009] (4) dissolving crude Gastrodia elata polysaccharide in pure water to obtain a crude Gastrodia elata polysaccharide aqueous solution, and deproteinizing the crude Gastrodia elata polysaccharide aqueous solution to obtain a deproteinized water extract;

[0010] (5) purifying the deproteinized water extract using a macroporous resin to obtain a crude polysaccharide purified solution;

[0011] (6) dialyzing the purified crude polysaccharide solution in pure water, and concentrating the crude polysaccharide solution obtained after the dialysis under reduced pressure, and freeze-drying the solution to obtain the dialyzed Gastrodia elata crude polysaccharide;

[0012] (7) dissolving the dialyzed crude Gastrodia elata polysaccharide in deionized water to obtain a crude Gastrodia elata polysaccharide solution, purifying the crude Gastrodia elata polysaccharide solution using a first chromatography column, gradient eluting with water, 0.1-0.15 mol / L NaCl solution, 0.3-0.35 mol / L NaCl solution, and 0.5-0.55 mol / L NaCl solution in sequence, combining the eluates obtained by eluting with the NaCl solution, dialyzing according to the method of step (6), concentrating under reduced pressure, and freeze-drying to obtain Gastrodia elata polysaccharide;

[0013] (8) Dissolving Gastrodia elata polysaccharide in deionized water to obtain a Gastrodia elata polysaccharide solution, purifying the Gastrodia elata polysaccharide solution using a second chromatography column, eluting with deionized water, concentrating the obtained eluate and then freeze-drying it to obtain purified Gastrodia elata polysaccharide.

[0014] Compared with the prior art, the present invention extracts the residue obtained after reflux extraction of Gastrodia elata powder with an alcohol solution by ultrasonic extraction in pure water to obtain a filtrate, and after decompression and concentration of the filtrate, adds anhydrous ethanol for low-temperature alcohol precipitation to obtain crude Gastrodia elata polysaccharide. The crude Gastrodia elata polysaccharide is subjected to deproteinization, adsorption of pigments and impurities by macroporous resin, dialysis, and two chromatography purifications to obtain purified Gastrodia elata polysaccharide. The present invention purifies the crude Gastrodia elata polysaccharide by using the above five purification means, and can quickly, stably and efficiently obtain a high-purity purified Gastrodia elata polysaccharide. Among them, the present invention performs gradient elution with water and NaCl solutions of different concentrations in turn when chromatographically purifying the crude Gastrodia elata polysaccharide for the first time, collects the eluate after elution with NaCl solutions of different concentrations, and can purify to obtain Gastrodia elata acidic polysaccharide, and the Gastrodia elata acidic polysaccharide is subjected to a second chromatography purification, and the two chromatography synergistic effects obtain a high-purity purified Gastrodia elata polysaccharide.

[0015] Furthermore, in step (1), the solid-liquid ratio of Gastrodia elata powder to 70% to 80% alcohol solution by volume is 1 g: (10 to 15) ml; the alcohol solution is ethanol; the reflux extraction temperature is 50 to 70° C., the extraction time for each extraction is 30 to 60 minutes, and the number of extractions is 3 to 5 times.

[0016] The above technical scheme further defines the condition parameters for reflux extraction of Gastrodia elata powder. Under these condition parameters, reflux extraction of Gastrodia elata powder using ethanol with a volume fraction of 70% to 80% can effectively remove non-polysaccharide components such as gastrodin that are soluble in ethanol solution in Gastrodia elata powder.

[0017] Furthermore, in step (2), the material-liquid ratio of the filter residue to pure water is 1 g: (50-60) ml; the temperature of ultrasonic extraction is 50-70° C., the extraction time for each extraction is 50-60 min, the ultrasonic power is 300-400 W, the ultrasonic frequency is 30-40 kHz, and the number of ultrasonic extractions is 3-5 times.

[0018] The above technical scheme further defines the condition parameters for ultrasonic extraction of the filter residue dissolved in pure water. Ultrasound under the condition parameters can break up cells and fully release the Gastrodia polysaccharides in the cells, so that the Gastrodia polysaccharides in the filter residue are further dissolved in pure water.

[0019] Furthermore, the pressure of the reduced pressure concentration in step (3) is -0.1 to -0.08 MPa, and the concentration is to 5% to 10% of the original volume; and the low-temperature alcohol precipitation is: alcohol precipitation at 4 to 8°C for 24 to 28 hours.

[0020] The above technical scheme further defines the condition parameters of reduced pressure concentration and low-temperature alcohol precipitation. Alcohol precipitation at 4-8°C for 24-28h can completely precipitate the ethanol-insoluble Gastrodia polysaccharide in the mixed solution, further remove impurities, and improve the purity of Gastrodia polysaccharide.

[0021] Furthermore, in step (4), the Gastrodia elata crude polysaccharide aqueous solution is subjected to a deproteinization treatment, comprising:

[0022] Add chloroform-n-butanol reagent to the aqueous solution of crude polysaccharide of Gastrodia elata, shake for 30-40 minutes and then centrifuge to remove the denatured protein in the middle layer, repeat the shaking, centrifugation and denatured protein removal operations until no flocculent denatured protein is produced in the middle layer after centrifugation, and collect the deproteinized water extract in the upper layer; the volume ratio of the aqueous solution of crude polysaccharide of Gastrodia elata to the chloroform-n-butanol reagent is (4-5):1; the volume ratio of chloroform to n-butanol in the chloroform-n-butanol reagent is (5-6):1.

[0023] Furthermore, the step (6) of dialyzing the crude polysaccharide purified solution in pure water comprises:

[0024] The crude polysaccharide purified liquid is placed in a dialysis bag, and the dialysis bag is placed in pure water for dialysis, with the water being changed every 2 to 3 hours for 2 to 3 days; the pore size of the dialysis bag is 8000 to 14000 Da.

[0025] The above technical solution further limits the dialysis time and the pore size of the dialysis bag when the crude polysaccharide purified liquid is dialyzed. Under the above conditions, small molecular impurities in the Gastrodia elata crude polysaccharide purified liquid can be further removed, thereby improving the purity of the Gastrodia elata crude polysaccharide.

[0026] Furthermore, during the gradient elution in step (7), the phenol-sulfuric acid method is used for color development tracking. When no color is developed, the next gradient eluent is used for elution. When no color is finally developed, the elution is stopped.

[0027] The filler in the first chromatography column is a cellulose-based anion exchange resin.

[0028] In the above technical solution, phenol-sulfuric acid method is used for color tracking during gradient elution, and cellulose-based anion exchange resin is selected as filler, so that the gastrodia acid polysaccharide in the gastrodia crude polysaccharide solution can be eluted more thoroughly.

[0029] Furthermore, during elution in step (8), the phenol-sulfuric acid method is used to draw an elution curve, and the eluate at the elution peak is collected according to the elution curve; and the filler in the second chromatography column is dextran gel.

[0030] The above technical solution defines collecting the eluate at the elution peak according to the elution curve drawn by the phenol-sulfuric acid method, and the collection method can obtain an eluate with more purified polysaccharides of Gastrodia elata. Selecting dextran gel as the filler of the second chromatography column can further purify the Gastrodia elata acidic polysaccharide obtained in the previous step to obtain purified polysaccharides of Gastrodia elata with higher purity.

[0031] In a second aspect, the present invention provides a purified polysaccharide of Gastrodia elata, which is prepared by any of the above methods. The purified polysaccharide of Gastrodia elata is composed of arabinose, galactose, glucose and galacturonic acid; the molar ratio of arabinose, galactose, glucose and galacturonic acid is (0.1-0.2):(0.18-0.25):(97-98):(1.8-2.5); the molecular weight of the purified polysaccharide of Gastrodia elata is 890-1085 kDa.

[0032] Compared with the prior art, the purified polysaccharide of Gastrodia elata of the present invention is a macromolecular substance with a novel structure, which has a specific composition and ratio, and further improves the preventive and therapeutic effects on Alzheimer's disease.

[0033] In a third aspect, the present invention provides an application of a purified polysaccharide of Gastrodia elata, and an application of the purified polysaccharide of Gastrodia elata in the preparation of a drug for preventing / treating Alzheimer's disease.

[0034] Compared with the prior art, the inventors studied the activity of the purified polysaccharide from Gastrodia elata and found that it has a specific and significant improvement effect on the pathological indicators of transgenic Alzheimer's disease Caenorhabditis elegans. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the elution curve in Example 1.

[0036] Figure 2 This is the NMR carbon spectrum of the purified Gastrodia elata polysaccharide in Test Example 2.

[0037] Figure 3 This is the NMR hydrogen spectrum of the purified Gastrodia elata polysaccharide in Test Example 2.

[0038] Figure 4 This is the high performance anion exchange chromatography in Test Example 2.

[0039] Figure 5 This is the UV spectrum scanning result diagram in Test Example 2.

[0040] Figure 6 This is the infrared spectrum scanning result diagram in Test Example 2.

[0041] Figure 7 This is a comparison chart of Aβ aggregation in nematodes in Test Example 3.

[0042] Figure 8 This is a comparison of the Aβ aggregation in vivo of CL4176 nematodes fed with common Gastrodia elata polysaccharide and purified Gastrodia elata polysaccharide in Example 1 after induction.

[0043] Fig. 9 This is the expression of the Aβ gene in the body of CL4176 nematodes after induction in Test Example 3. DETAILED DESCRIPTION

[0044] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] In a first aspect, an embodiment of the present invention provides a method for preparing purified polysaccharides from Gastrodia elata, comprising the following steps:

[0046] S1. Put the Gastrodia elata powder in a reaction container, add 70% to 80% alcohol solution by volume, reflux extraction, filter, and collect the filter residue.

[0047] The particle size of the Gastrodia elata powder can be 50-65 meshes, the alcohol solution can be ethanol, and the reaction container can be a round-bottom flask.

[0048] Furthermore, in the above steps, the solid-liquid ratio of Gastrodia elata powder to 70% to 80% alcohol solution by volume is 1g: (10 to 15) ml; the reflux extraction temperature is 50 to 70°C, the extraction time is 30 to 60 minutes each time, and the extraction times are 3 to 5 times.

[0049] For example, the volume fraction of the alcohol solution may be 70%, 75% or 80%, preferably 80%.

[0050] For example, the solid-liquid ratio of Gastrodia elata powder to alcohol solution can be 1g:10ml, 1g:11ml, 1g:12ml, 1g:13ml, 1g:14ml, or 1g:15ml, preferably 1g:(10-12)ml.

[0051] For example, the temperature of reflux extraction can be 50° C., 55° C., 60° C., 65° C. or 70° C. The extraction time can be 30 min, 40 min, 50 min or 60 min, preferably 50 to 60 min. The number of extractions can be 3, 4 or 5 times.

[0052] After the reflux extraction is completed, the mixture is filtered using a filter with a pore size of 80 μm to 120 μm, and the residue is collected for the next step. Through the reflux extraction in the above steps, most of the alcohol-soluble impurities in the Gastrodia elata powder can be preliminarily removed, and the Gastrodia elata polysaccharide components can be concentrated in the residue.

[0053] S2, dissolving the filter residue obtained in the above step S1 in pure water, performing ultrasonic extraction, filtering, and collecting the filtrate.

[0054] Furthermore, in the above steps, the material-liquid ratio of the filter residue to pure water is 1g:(50-60)ml; the temperature of ultrasonic extraction is 50-70°C, the extraction time for each time is 50-60min, the ultrasonic power is 300-400W, the ultrasonic frequency is 30-40kHz, and the number of ultrasonic extractions is 3-5 times.

[0055] For example, the temperature of ultrasonic extraction can be 50°C, 60°C or 70°C, preferably 60-70°C; the ultrasonic power can be 300W, 330W, 350W, 370W or 400W, preferably 350-400W; the ultrasonic frequency can be 30kHz, 32kHz, 35kHz, 37kHz or 40kHz, preferably 35-40kHz; the number of ultrasonic extractions can be 3, 4 or 5 times.

[0056] In the above step, the filter residue is dissolved in pure water, so that the gastrodia polysaccharide in the filter residue can be dissolved in pure water, and the gastrodia polysaccharide in the cells can be further released by ultrasonic cell disruption, thereby fully extracting the gastrodia polysaccharide in the filter residue.

[0057] After the ultrasonic extraction is completed, filter using a filter with a pore size of 80 μm to 120 μm, discard the filter residue, and collect the filtrate.

[0058] S3. Concentrate the filtrate obtained in the above step S2 under reduced pressure, add anhydrous ethanol to the obtained concentrated solution to make the volume fraction of ethanol in the mixed solution be 70% to 80%, and then precipitate the mixed solution with alcohol at low temperature; after the alcohol precipitation is completed, collect the precipitate and perform freeze-drying to obtain crude Gastrodia elata polysaccharide.

[0059] The pressure of the reduced pressure concentration is -0.1 to -0.08 MPa, and the volume is concentrated to 5% to 10% of the original volume. By reducing the pressure and concentrating the filtrate, the volume of the solution is reduced, and the concentration of the Gastrodia elata polysaccharide in the solution is increased, which is convenient for subsequent operations.

[0060] Furthermore, low temperature alcohol precipitation is: alcohol precipitation at 4-8°C for 24-28h. For example, the temperature may be 4°C, 5°C, 6°C, 7°C or 8°C, preferably 4°C-6°C, and the alcohol precipitation may be for 24h, 26h or 28h.

[0061] Since the solubility of Gastrodia elata polysaccharide in ethanol is low, long-term alcohol precipitation at a low temperature of 4 to 8°C can effectively precipitate Gastrodia elata polysaccharide from the solution and improve the extraction rate of Gastrodia elata polysaccharide.

[0062] After the alcohol precipitation is completed, you can choose to use a vacuum freeze dryer to freeze-dry the precipitate, the condensation temperature is: -70~-80℃, vacuum degree: <10Pa. Among them, the model of the vacuum freeze dryer can be FD-2A, which is produced by Boyikang Instrument Co., Ltd.

[0063] S4, dissolving the crude Gastrodia elata polysaccharide obtained in the above step S3 in pure water to obtain a crude Gastrodia elata polysaccharide aqueous solution, and deproteinizing the crude Gastrodia elata polysaccharide aqueous solution to obtain a deproteinized water extract.

[0064] In the above steps, the ratio of crude Gastrodia elata polysaccharide dissolved in pure water is 1 g: (30-60) mL.

[0065] Furthermore, the deproteinization treatment of the crude polysaccharide aqueous solution of Gastrodia elata in the above step comprises the following steps:

[0066] Add chloroform-n-butanol reagent to the aqueous solution of crude polysaccharide of Gastrodia elata, shake for 30 to 40 minutes and then centrifuge to remove the denatured protein in the middle layer, repeat the shaking, centrifugation and denatured protein removal operations until no flocculent denatured protein is produced in the middle layer after centrifugation, and collect the deproteinized water extract in the upper layer. The centrifugation can be carried out at a speed of 3000 to 4000 r / min for 1 to 2 minutes.

[0067] The volume ratio of the aqueous solution of crude Gastrodia elata polysaccharide to the chloroform-n-butanol reagent is (4-5):1; the volume ratio of chloroform to n-butanol in the chloroform-n-butanol reagent is (5-6):1.

[0068] The above steps use the Sevage method to effectively remove the protein in the aqueous solution of crude Gastrodia elata polysaccharide, and further improve the purity of Gastrodia elata polysaccharide.

[0069] S5. Purify the deproteinized water extract using a macroporous resin to obtain a crude polysaccharide purified solution.

[0070] The macroporous resin in the above step can be D3520 macroporous resin, which is a non-polar adsorption resin. The deproteinized water extract is purified by using the resin, which can not only remove the pigment in the deproteinized water extract, but also further remove the residual protein therein.

[0071] For example, the purification process of the above deproteinized water extract can be referred to as follows:

[0072] Place D3520 macroporous resin in a chromatography column (30.0cm×1.5cm), balance the column with pure water, wait for the resin to settle naturally to 23-25cm, and slowly drip the deproteinized water extract along the column wall with a dropper. It should be understood that the dripping rate should be based on not destroying the surface flatness of the filler. After the sample solution has been balanced in the column for 1h, open the valve at the bottom of the column, let the sample solution pass through the column at a flow rate of 1-3cm / min, collect the sample solution, and obtain the crude polysaccharide purified solution.

[0073] S6. The crude polysaccharide purified solution obtained in the above step S5 is dialyzed in pure water, and the crude polysaccharide solution obtained after the dialysis is concentrated under reduced pressure and freeze-dried to obtain the dialyzed Gastrodia elata crude polysaccharide.

[0074] Further, the crude polysaccharide purified solution is dialyzed in pure water, comprising:

[0075] The crude polysaccharide purified solution was placed in a dialysis bag, and the dialysis bag was placed in pure water for dialyzing, with the water being changed every 2 to 3 hours for 2 to 3 days.

[0076] In the above steps, the pore size of the dialysis bag is 8000-14000Da, and the model of the dialysis bag can be MD34.

[0077] After the dialysis is completed, the dialyzed crude polysaccharide of Gastrodia elata is concentrated under reduced pressure at a pressure of -0.1 MPa to -0.08 MPa to 5% to 10% of the original volume, and freeze-dried to obtain the dialyzed crude polysaccharide of Gastrodia elata. The freeze-drying conditions refer to the above step S3.

[0078] By dialyzing the crude polysaccharide purified liquid, salts, small molecular organic matter and low molecular weight proteins in the crude polysaccharide purified liquid can be removed, thereby further improving the purity of the Gastrodia elata purified polysaccharide.

[0079] S7. Dissolve the dialyzed Gastrodia elata polysaccharide obtained in the above step S6 in deionized water to obtain a Gastrodia elata polysaccharide solution, and purify the Gastrodia elata polysaccharide solution using a first chromatography column, and gradient elute with water, 0.1-0.15 mol / L NaCl solution, 0.3-0.35 mol / L NaCl solution, and 0.5-0.55 mol / L NaCl solution in sequence, combine the eluates obtained by eluting with NaCl solution, dialyze according to the method of step S6, and then concentrate under reduced pressure and freeze-dry to obtain Gastrodia elata polysaccharide.

[0080] The filler in the first chromatography column is a cellulose-based anion exchange resin, for example, DEAE-52 can be selected. The ratio of crude Gastrodia elata polysaccharide to deionized water is 1 g: (5-10) mL.

[0081] In the above steps, when loading the sample, the crude polysaccharide solution of Gastrodia elata can be dripped along the inner wall of the chromatography column with a dropper to distribute it on the upper surface of the DEAE-52 filler as quickly as possible, and the dripping rate should be controlled to ensure that the flatness of the filler surface is not damaged during the dripping. During gradient elution, the linear velocity of the eluent is 50 to 100 cm / h.

[0082] In the above steps, the neutral polysaccharide of Gastrodia elata is eluted with water, which is not the target polysaccharide of the present invention and is discarded. The acidic polysaccharide of Gastrodia elata (target polysaccharide) is eluted with NaCl solution, and the acidic polysaccharide of Gastrodia elata in the solution can be fully eluted by gradient elution with 0.1-0.15 mol / L NaCl solution, 0.3-0.35 mol / L NaCl solution, and 0.5-0.55 mol / L NaCl solution.

[0083] Furthermore, during the gradient elution in the above step, the phenol-sulfuric acid method is used for color development and tracking. When no color is developed, the next gradient eluent is used for elution, and the elution is stopped when no color is finally developed.

[0084] S8. Dissolve the Gastrodia elata polysaccharide obtained in the above step S7 in deionized water to obtain a Gastrodia elata polysaccharide solution, use a second chromatography column to purify the Gastrodia elata polysaccharide solution, elute with deionized water, concentrate the obtained eluate and then freeze-dry it to obtain purified Gastrodia elata polysaccharide.

[0085] The filler in the second chromatography column is dextran gel, for example, the filler can be Sephadex G-100. The ratio of Gastrodia elata polysaccharide to deionized water is 1 g: (4-8) mL.

[0086] When deionized water is used for elution in the above step, the linear velocity of the deionized water is 0.5-0.8 ml / min.

[0087] Furthermore, during elution in the above step, the phenol-sulfuric acid method is used to draw an elution curve, and the eluate at the elution peak is collected according to the elution curve.

[0088] For example, the drawing of the elution curve can refer to the following steps:

[0089] Take 0.2mL of the eluent, add pure water to 1.0mL, then add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, leave at room temperature for 10 minutes, heat in a boiling water bath for 15 minutes, and measure its absorbance at 490nm after cooling. Draw the elution curve with the number of the elution tube as the horizontal axis and the absorbance at 490nm as the vertical axis, and merge the eluent at the elution peak according to the elution curve.

[0090] The eluate is concentrated under reduced pressure at a pressure of -0.1 MPa to -0.08 MPa to 5% to 10% of the original volume, and then freeze-dried to obtain purified Gastrodia elata polysaccharide. The freeze-drying conditions refer to the above step S3.

[0091] The above steps further purify the Gastrodia elata acidic polysaccharide obtained by the first chromatography column purification, and can obtain a high-purity Gastrodia elata purified polysaccharide with a novel structure.

[0092] In the second aspect, an embodiment of the present invention provides a purified polysaccharide of Gastrodia elata, which is prepared by the above method. The purified polysaccharide of Gastrodia elata is composed of arabinose, galactose, glucose and galacturonic acid; the molar ratio of arabinose, galactose, glucose and galacturonic acid is (0.1~0.2):(0.18~0.25):(97~98):(1.8~2.5); the molecular weight of the purified polysaccharide of Gastrodia elata is 890~1085kDa.

[0093] Among them, the molar ratio of arabinose, galactose, glucose and galacturonic acid is preferably (0.13-0.15):(0.21-0.22):(97.44-97.63):(2.01-2.22); the molecular weight of the purified Gastrodia elata polysaccharide is preferably 894.5-1082.8 kDa.

[0094] Compared with the prior art, the present invention prepares a purified polysaccharide of Gastrodia elata with a novel structure, which has a specific composition and ratio, and further improves the preventive and therapeutic effects on Alzheimer's disease.

[0095] In a third aspect, an embodiment of the present invention provides an application of a purified polysaccharide of Gastrodia elata, and an application of the purified polysaccharide of Gastrodia elata in the preparation of a drug for preventing / treating Alzheimer's disease.

[0096] Compared with the prior art, the purified Gastrodia elata polysaccharide of the present invention has a specific and significant improvement effect on the pathological indicators of transgenic Alzheimer's disease Caenorhabditis elegans.

[0097] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0098] Example 1

[0099] A method for preparing purified Gastrodia elata polysaccharide comprises the following steps:

[0100] S1. Place 50 mesh Tianma powder in a round bottom flask, add 80% ethanol by volume, reflux extract 3 times, 60 min each time, filter, and collect the residue. The reflux extraction temperature is 60°C, the solid-liquid ratio of Tianma powder to 80% ethanol is 1g:10ml, and the filter pore size is 100μm.

[0101] S2, dissolve the above filter residue in pure water and perform ultrasonic extraction for 3 times, each time for 50 minutes, the material-liquid ratio of the filter residue to pure water is 1g:50ml, filter, and collect the filtrate. The ultrasonic extraction temperature is 60°C, the ultrasonic frequency is 40kHz, the ultrasonic power is 400W, and the filter pore size is 100μm.

[0102] S3, the upper filtrate was concentrated under reduced pressure at a pressure of -0.09 MPa, concentrated to 7% of the original volume, anhydrous ethanol was added to the obtained concentrated solution, and the volume fraction of ethanol in the mixed solution was 80%, and the mixed solution was precipitated with alcohol at low temperature for 24 hours at 4°C. After the alcohol precipitation was completed, the precipitate was collected and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), the condensation temperature was -80°C, and the vacuum degree was <10Pa, and crude Gastrodia elata polysaccharide was obtained.

[0103] S4. Dissolve the above-mentioned crude Gastrodia elata polysaccharide in pure water to obtain a crude Gastrodia elata polysaccharide aqueous solution, the ratio of crude Gastrodia elata polysaccharide to pure water is 1g:40mL, and deproteinize the crude Gastrodia elata polysaccharide aqueous solution: add chloroform-n-butanol reagent to the crude Gastrodia elata polysaccharide aqueous solution, shake for 30 minutes, and then centrifuge at 4000r / min for 1 minute to remove the denatured protein in the middle layer, repeat the shaking, centrifugation and denatured protein removal operations until no flocculent denatured protein is produced in the middle layer after centrifugation, and collect the deproteinized water extract in the upper layer.

[0104] The volume ratio of the Gastrodia elata crude polysaccharide aqueous solution to the chloroform-n-butanol reagent is 4:1; the volume ratio of chloroform to n-butanol in the chloroform-n-butanol reagent is 5:1.

[0105] S5. Purify the deproteinized aqueous extract using D3520 macroporous resin:

[0106] D3520 macroporous resin was placed in a chromatography column (30.0cm×1.5cm). After the column was balanced with pure water, the resin was naturally settled to 24cm. The deproteinized water extract was slowly dripped along the column wall with a dropper. The dripping rate was controlled so as not to damage the surface flatness of the filler. After the sample solution was balanced in the column for 1h, the valve at the bottom of the column was opened to allow the sample solution to pass through the column at a flow rate of 2cm / min. The sample solution was collected to obtain a crude polysaccharide purified solution.

[0107] S6. Dialyze the above-mentioned crude polysaccharide purified solution in pure water: put the crude polysaccharide purified solution into a dialysis bag, put the dialysis bag into pure water for dialysis, change the water every 2 hours, and continue for 3 days; the pore size of the dialysis bag is 10000Da, and the model is MD34.

[0108] The crude polysaccharide solution obtained after dialysis was concentrated to 5% of the original volume under a pressure of -0.1 MPa, and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain the dialyzed Gastrodia elata crude polysaccharide.

[0109] S7. Dissolve the dialyzed crude Gastrodia polysaccharide in deionized water at a ratio of 1 g:10 mL to obtain a crude Gastrodia polysaccharide solution. Use a first chromatography column to purify the crude Gastrodia polysaccharide solution by gradient elution with water, 0.1 mol / L NaCl solution, 0.3 mol / L NaCl solution, and 0.5 mol / L NaCl solution in sequence. Combine the eluates obtained by eluting with NaCl solution, dialyze according to the method of step S6, and then concentrate under reduced pressure and freeze-dry to obtain Gastrodia polysaccharide. Wherein, the filler in the first chromatography column is DEAE-52.

[0110] During gradient elution, the linear velocity of the eluent is 80 cm / h, and the phenol-sulfuric acid method is used for color development and tracking. When no color is developed, the next gradient eluent is used for elution, and the elution is stopped when no color is finally developed.

[0111] S8, dissolving the above-mentioned Gastrodia elata polysaccharide in deionized water to obtain a Gastrodia elata polysaccharide solution, wherein the ratio of Gastrodia elata polysaccharide to deionized water is 1 g: 5 mL. Purifying the Gastrodia elata polysaccharide solution using a second chromatography column, eluting with deionized water, concentrating the obtained eluate and freeze-drying it to obtain purified Gastrodia elata polysaccharide; wherein the filler in the second chromatography column is Sephadex G-100.

[0112] During elution, the linear velocity of deionized water was 0.8 ml / min, and an elution curve was drawn using the phenol-sulfuric acid method. The eluate at the elution peak was collected according to the elution curve.

[0113] The process of drawing the elution curve is as follows:

[0114] Take 0.2mL of the eluent, add pure water to make up to 1.0mL, then add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, leave at room temperature for 10 minutes, heat in a boiling water bath for 15 minutes, and measure its absorbance at 490nm after cooling. Draw the elution curve with the number of the elution tube as the horizontal axis and the absorbance at 490nm as the vertical axis. The elution curve is shown in Figure 1. Figure 1 shown.

[0115] The collected eluate was concentrated under reduced pressure at -0.1 MPa to 5% of the original volume, and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain purified Gastrodia elata polysaccharide.

[0116] Example 2

[0117] A method for preparing purified Gastrodia elata polysaccharide comprises the following steps:

[0118] S1. Place 50 mesh Tianma powder in a round bottom flask, add 70% ethanol by volume, reflux extract 5 times, 30 minutes each time, filter, and collect the residue. The reflux extraction temperature is 50°C, the solid-liquid ratio of Tianma powder to 70% ethanol is 1g:10ml, and the filter pore size is 100μm.

[0119] S2, dissolve the above filter residue in pure water and perform ultrasonic extraction for 5 times, each time for 60 minutes, the material-liquid ratio of the filter residue to pure water is 1g:60ml, filter, and collect the filtrate. The ultrasonic extraction temperature is 50°C, the ultrasonic frequency is 30kHz, the ultrasonic power is 300W, and the filter pore size is 100μm.

[0120] S3, the upper filtrate is concentrated under reduced pressure at a pressure of -0.1 MPa, concentrated to 5% of the original volume, anhydrous ethanol is added to the obtained concentrated solution, and the volume fraction of ethanol in the mixed solution is 70%, and the mixed solution is precipitated with alcohol at low temperature, and precipitated with alcohol at 8°C for 28h. After the alcohol precipitation is completed, the precipitate is collected and freeze-dried using a vacuum freeze dryer (FD-2A, Bo Yi Kang Instrument Co., Ltd.), the condensation temperature is -80°C, and the vacuum degree is <10Pa, to obtain crude Gastrodia elata polysaccharide.

[0121] S4. Dissolve the above-mentioned crude Gastrodia elata polysaccharide in pure water to obtain a crude Gastrodia elata polysaccharide aqueous solution, the ratio of crude Gastrodia elata polysaccharide to pure water is 1g:60mL, and deproteinize the crude Gastrodia elata polysaccharide aqueous solution: add chloroform-n-butanol reagent to the crude Gastrodia elata polysaccharide aqueous solution, shake for 40 minutes, and then centrifuge at 4000r / min for 1 minute to remove the denatured protein in the middle layer, repeat the shaking, centrifugation and denatured protein removal operations until no flocculent denatured protein is produced in the middle layer after centrifugation, and collect the deproteinized water extract in the upper layer.

[0122] The volume ratio of the crude Gastrodia elata polysaccharide aqueous solution to the chloroform-n-butanol reagent is 5:1; the volume ratio of chloroform to n-butanol in the chloroform-n-butanol reagent is 6:1.

[0123] S5. Purify the deproteinized aqueous extract using D3520 macroporous resin:

[0124] D3520 macroporous resin was placed in a chromatography column (30.0cm×1.5cm). After the column was balanced with pure water, the resin was naturally settled to 24cm. The deproteinized water extract was slowly dripped along the column wall with a dropper. The dripping rate was controlled so as not to damage the surface flatness of the filler. After the sample solution was balanced in the column for 1h, the valve at the bottom of the column was opened to allow the sample solution to pass through the column at a flow rate of 1cm / min. The sample solution was collected to obtain a crude polysaccharide purified solution.

[0125] S6. Dialyze the above-mentioned crude polysaccharide purified solution in pure water: put the crude polysaccharide purified solution into a dialysis bag, put the dialysis bag into pure water for dialysis, change the water every 3 hours, and continue for 2 days; the pore size of the dialysis bag is 8000Da, and the model is MD34.

[0126] The crude polysaccharide solution obtained after dialysis was concentrated to 10% of the original volume under a pressure of -0.08 MPa, and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain the dialyzed Gastrodia elata crude polysaccharide.

[0127] S7. Dissolve the dialyzed crude Gastrodia polysaccharide in deionized water at a ratio of 1 g to 8 mL to obtain a crude Gastrodia polysaccharide solution. Use a first chromatography column to purify the crude Gastrodia polysaccharide solution by gradient elution with water, 0.15 mol / L NaCl solution, 0.35 mol / L NaCl solution, and 0.55 mol / L NaCl solution in sequence. Combine the eluates obtained by eluting with NaCl solution, dialyze according to the method of step S6, and then concentrate under reduced pressure and freeze-dry to obtain Gastrodia polysaccharide. Wherein, the filler in the first chromatography column is DEAE-52.

[0128] During gradient elution, the linear velocity of the eluent is 60 cm / h, and the phenol-sulfuric acid method is used for color development and tracking. When no color is developed, the next gradient eluent is used for elution, and the elution is stopped when no color is finally developed.

[0129] S8, dissolving the above-mentioned Gastrodia elata polysaccharide in deionized water to obtain a Gastrodia elata polysaccharide solution, wherein the ratio of Gastrodia elata polysaccharide to deionized water is 1 g: 8 mL. Purifying the Gastrodia elata polysaccharide solution using a second chromatography column, eluting with deionized water, concentrating the obtained eluate and freeze-drying it to obtain purified Gastrodia elata polysaccharide; wherein the filler in the second chromatography column is Sephadex G-100.

[0130] During elution, the linear velocity of deionized water was 0.5 ml / min, and an elution curve was drawn using the phenol-sulfuric acid method. The eluate at the elution peak was collected according to the elution curve.

[0131] The process of drawing the elution curve is as follows:

[0132] Take 0.2mL of the eluent, add pure water to 1.0mL, then add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, leave at room temperature for 10 minutes, heat in a boiling water bath for 15 minutes, and measure its absorbance at 490nm after cooling. Draw the elution curve with the number of the elution tube as the horizontal axis and the absorbance at 490nm as the vertical axis.

[0133] The collected eluate was concentrated under reduced pressure at -0.1 MPa to 5% of the original volume, and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain purified Gastrodia elata polysaccharide.

[0134] Example 3

[0135] A method for preparing purified Gastrodia elata polysaccharide comprises the following steps:

[0136] S1. Place 50 mesh Tianma powder in a round bottom flask, add 75% ethanol by volume, reflux extract 4 times, 50 min each time, filter, and collect the residue. The reflux extraction temperature is 60°C, the solid-liquid ratio of Tianma powder to 75% ethanol is 1g:15ml, and the filter pore size is 100μm.

[0137] S2, dissolve the above filter residue in pure water and perform ultrasonic extraction 4 times, each time for 55 minutes, the material-liquid ratio of the filter residue to pure water is 1g:55ml, filter, and collect the filtrate. The ultrasonic extraction temperature is 70°C, the ultrasonic frequency is 35kHz, the ultrasonic power is 350W, and the filter pore size is 100μm.

[0138] S3, the upper filtrate is concentrated under reduced pressure at a pressure of -0.08MPa, concentrated to 10% of the original volume, anhydrous ethanol is added to the obtained concentrated solution, and the volume fraction of ethanol in the mixed solution is 75%, and the mixed solution is precipitated with alcohol at low temperature, and precipitated with alcohol at 6°C for 26h. After the alcohol precipitation is completed, the precipitate is collected and freeze-dried using a vacuum freeze dryer (FD-2A, Bo Yi Kang Instrument Co., Ltd.), the condensation temperature is -80°C, and the vacuum degree is <10Pa, to obtain crude Gastrodia elata polysaccharide.

[0139] S4. Dissolve the above-mentioned crude Gastrodia elata polysaccharide in pure water to obtain a crude Gastrodia elata polysaccharide aqueous solution, the ratio of crude Gastrodia elata polysaccharide to pure water is 1g:30mL, and deproteinize the crude Gastrodia elata polysaccharide aqueous solution: add chloroform-n-butanol reagent to the crude Gastrodia elata polysaccharide aqueous solution, shake for 35 minutes, and then centrifuge at 4000r / min for 1 minute to remove the denatured protein in the middle layer, repeat the shaking, centrifugation and denatured protein removal operations until no flocculent denatured protein is produced in the middle layer after centrifugation, and collect the deproteinized water extract in the upper layer.

[0140] The volume ratio of the Gastrodia elata crude polysaccharide aqueous solution to the chloroform-n-butanol reagent is 4:1; the volume ratio of chloroform to n-butanol in the chloroform-n-butanol reagent is 5:1.

[0141] S5. Purify the deproteinized aqueous extract using D3520 macroporous resin:

[0142] D3520 macroporous resin was placed in a chromatography column (30.0cm×1.5cm). After the column was balanced with pure water, the resin was naturally settled to 24cm. The deproteinized water extract was slowly dripped along the column wall with a dropper. The dripping rate was controlled so as not to damage the surface flatness of the filler. After the sample solution was balanced in the column for 1h, the valve at the bottom of the column was opened to allow the sample solution to pass through the column at a flow rate of 3cm / min, and the sample solution was collected to obtain a crude polysaccharide purified solution.

[0143] S6. Dialyze the above-mentioned crude polysaccharide purified solution in pure water: put the crude polysaccharide purified solution into a dialysis bag, put the dialysis bag into pure water for dialysis, change the water every 2 hours, and continue for 3 days; the pore size of the dialysis bag is 14000Da, and the model is MD34.

[0144] The crude polysaccharide solution obtained after dialysis was concentrated to 8% of the original volume under a pressure of -0.09 MPa, and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain the dialyzed Gastrodia elata crude polysaccharide.

[0145] S7. Dissolve the dialyzed crude Gastrodia polysaccharide in deionized water at a ratio of 1 g:10 mL to obtain a crude Gastrodia polysaccharide solution. Use a first chromatography column to purify the crude Gastrodia polysaccharide solution by gradient elution with water, 0.12 mol / L NaCl solution, 0.32 mol / L NaCl solution, and 0.52 mol / L NaCl solution in sequence. Combine the eluates obtained by eluting with NaCl solution, dialyze according to the method of step S6, and then concentrate under reduced pressure and freeze-dry to obtain Gastrodia polysaccharide. Wherein, the filler in the first chromatography column is DEAE-52.

[0146] During gradient elution, the linear velocity of the eluent is 100 cm / h, and the phenol-sulfuric acid method is used for color development and tracking. When no color is developed, the next gradient eluent is used for elution, and the elution is stopped when no color is finally observed.

[0147] S8, dissolving the above-mentioned Gastrodia elata polysaccharide in deionized water to obtain a Gastrodia elata polysaccharide solution, wherein the ratio of Gastrodia elata polysaccharide to deionized water is 1 g: 6 mL. Purifying the Gastrodia elata polysaccharide solution using a second chromatography column, eluting with deionized water, concentrating the obtained eluate and freeze-drying it to obtain purified Gastrodia elata polysaccharide; wherein the filler in the second chromatography column is Sephadex G-100.

[0148] During elution, the linear velocity of deionized water was 0.6 ml / min, and the elution curve was drawn using the phenol-sulfuric acid method. The eluate at the elution peak was collected according to the elution curve.

[0149] The process of drawing the elution curve is as follows:

[0150] Take 0.2mL of the eluent, add pure water to 1.0mL, then add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, leave at room temperature for 10 minutes, heat in a boiling water bath for 15 minutes, and measure its absorbance at 490nm after cooling. Draw the elution curve with the number of the elution tube as the horizontal axis and the absorbance at 490nm as the vertical axis.

[0151] The collected eluate was concentrated under reduced pressure at -0.1 MPa to 5% of the original volume, and freeze-dried using a vacuum freeze dryer (FD-2A, Boyikang Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain purified Gastrodia elata polysaccharide.

[0152] Comparative Example 1

[0153] The purified polysaccharide of Gastrodia elata was prepared according to the method of Example 1, except that in step S5, an equal amount of D3520 macroporous resin was replaced by D101 macroporous resin.

[0154] Comparative Example 2

[0155] The purified polysaccharide of Gastrodia elata was prepared according to the method of Example 1, except that, during elution in step S7, gradient elution was performed using water, 0.1 mol / L NaOH solution, 0.3 mol / L NaOH solution, and 0.5 mol / L NaOH solution in sequence, and the eluates obtained by elution with NaOH solution were combined and then performed in step S8.

[0156] Comparative Example 3

[0157] The purified polysaccharide of Gastrodia elata was prepared according to the method of Example 1, except that step S8, ie, the second chromatography column purification, was not performed.

[0158] Comparative Example 4

[0159] The purified polysaccharide of Gastrodia elata was prepared according to the method of Example 1, except that, during elution in step S7, water and 0.1 mol / L NaCl solution were used in sequence, and the other conditions remained unchanged.

[0160] Test Example 1

[0161] The yield (relative to Gastrodia elata powder) and purity of the purified Gastrodia elata polysaccharides in Examples 1 to 3 and Comparative Examples 1 to 3 were tested, and the results are shown in Table 1:

[0162] Table 1

[0163] Yield of purified polysaccharides from Gastrodia elata Purity of Gastrodia elata purified polysaccharide Example 1 0.5％ 95％ Example 2 0.3％ 97％ Example 3 0.4％ 96％ Comparative Example 1 0.7％ 78％ Comparative Example 2 0.8％ 80％ Comparative Example 3 0.6％ 70％ Comparative Example 4 0.2％ 90％

[0164] As shown in Table 1, after the D3520 macroporous resin was replaced with the D101 macroporous resin in equal amounts in Comparative Example 1, the purity of the purified polysaccharide from Gastrodia elata was reduced, indicating that the use of the D3520 macroporous resin for purification in the present invention can improve the purity of the purified polysaccharide from Gastrodia elata. After eluting with water and NaOH solutions of different concentrations in step S7 of Comparative Example 2, the purity of the purified polysaccharide from Gastrodia elata was reduced, indicating that the use of a specific eluent in the present invention can further improve the purity of the purified polysaccharide from Gastrodia elata. After the second chromatography column purification in step S8 was not performed in Comparative Example 3, the purity of the purified polysaccharide from Gastrodia elata was reduced, indicating that the two chromatography purifications in the present invention can further improve the purity of the purified polysaccharide from Gastrodia elata. During the elution in step S7 of Comparative Example 4, water and 0.1 mol / L NaCl solution were used in sequence, and the other conditions remained unchanged, and the purity of the purified polysaccharide from Gastrodia elata was reduced, indicating that the use of NaCl solutions of different concentrations for elution in the present invention can improve the purity of the purified polysaccharide from Gastrodia elata while further improving the yield of the purified polysaccharide from Gastrodia elata.

[0165] Test Example 2

[0166] 1. Nuclear magnetic resonance spectroscopy detection:

[0167] The method of Example 1 was repeated 3 times to prepare 3 batches of purified Gastrodia elata polysaccharides, and the purified Gastrodia elata polysaccharides prepared in Examples 1 to 3 (5 batches in total) were subjected to nuclear magnetic resonance spectroscopy detection. The results are as follows: Figure 2 , Figure 3 As shown: the NMR carbon spectrum and hydrogen spectrum signals are consistent, indicating that the purified Gastrodia elata polysaccharides obtained in Examples 1 to 3 of the present invention are the same substance, and the preparation method of the purified Gastrodia elata polysaccharide of the present invention is repeatable and stable.

[0168] 2. Composition analysis of purified polysaccharides from Gastrodia elata:

[0169] The monosaccharide composition of the purified polysaccharide of Gastrodia elata in Example 1 was analyzed by high performance anion exchange chromatography amperometric detection. Figure 4 As shown: Gastrodia elata purified polysaccharide (GEP) is mainly composed of arabinose, galactose, glucose and galacturonic acid, with a molar ratio of 0.15:0.21:97.63:2.0. The molecular weight of GEP detected by gel exclusion chromatography is 1012.65 kDa.

[0170] 3. The purified polysaccharide from Gastrodia elata in Example 1 was scanned by ultraviolet spectrum. The results are as follows: Figure 5 As shown: There is no absorption at 260nm and 280nm, indicating that GEP does not contain nucleic acids and proteins.

[0171] 4. The purified polysaccharide from Gastrodia elata in Example 1 was scanned by infrared spectroscopy. Figure 6 Shown: 3000~3500cm -1 There is an obvious broad peak, which is the OH bond stretching vibration peak, 2931cm -1 is the CH stretching vibration peak, 848 cm -1 It is an α-pyranosidic bond, 1200~1400cm -1 CH variable angle vibration, 1025~1156cm -1 It is a pyranoside bond type. 1645cm -1 To bind water.

[0172] Test Example 3

[0173] The effects of the purified Gastrodia elata polysaccharides prepared in Examples 1 to 3 above on Alzheimer's disease Caenorhabditis elegans were detected.

[0174] 1. Thioflavin S staining was used to detect the aggregation of amyloid beta protein (Aβ) in CL4176 nematodes (Caenorhabditis elegans with Alzheimer's disease) after induction at 25°C:

[0175] After culturing CL4176 nematodes at 16°C for 48 hours, they were induced at 25°C. During induction at 25°C, the CL4176 nematodes in the control group were only fed with E. coli solution, the CL4176 nematodes in treatment group 1 were fed with E. coli solution containing 250 mg / L of purified Gastrodia elata polysaccharides prepared in Example 1, and the CL4176 nematodes in treatment group 2 were fed with E. coli solution containing 250 mg / L of common Gastrodia elata polysaccharides. The preparation method of the common Gastrodia elata polysaccharides is as follows: Zhang Shuangqi, Liu Lin, He Nianwu, et al. Study on the process optimization and antioxidant activity of ultrasound-assisted extraction of Gastrodia elata polysaccharides from Shaanxi Province [J]. Chinese Agricultural Science Bulletin, 2021, 37(09): 131-136.

[0176] The C. elegans from the control group, treatment group 1, and treatment group 2 were selected separately and stained with 1 mM Thioflavin S for 4 h. Then, the CL4176 nematodes were fixed in 4% paraformaldehyde / PBS, pH 7.4, at 4°C for 24 h. The fluorescence images were obtained with a 20× objective lens in an inverted fluorescence biological microscope. Figure 7 , Figure 8 shown.

[0177] Figure 7 Middle: A is a staining image of Aβ deposition in CL4176 nematodes in treatment group 1 after induction at 25°C; B is a staining image of Aβ deposition in CL4176 nematodes in the control group after induction at 25°C; C is a staining image of wild-type nematodes (normal non-Alzheimer's disease nematodes, fed only with Escherichia coli liquid) after induction at 25°C; D is a staining image of CL4176 nematodes (fed only with Escherichia coli liquid) that were cultured at 16°C without induction at 25°C.

[0178] Depend on Figure 7 It can be seen that when the CL4176 nematodes were induced from 16°C to 25°C, Aβ aggregation occurred. After being fed with the purified Gastrodia elata polysaccharide in Example 1, the Aβ aggregation of the CL4176 nematodes was significantly reduced.

[0179] Depend on Figure 8 It can be seen that after induction at 25°C, compared with feeding ordinary Gastrodia elata polysaccharides, feeding the purified Gastrodia elata polysaccharide in Example 1 of the present application can further reduce the Aβ aggregation of CL4176 nematodes.

[0180] 2. QRT-PCR detection of Aβ gene expression:

[0181] After CL4176 nematodes were cultured at 16°C for 48 hours, they were induced at 25°C. During the induction at 25°C, the CL4176 nematodes in the control group were fed only with Escherichia coli culture, and the CL4176 nematodes in the treatment group were fed with Escherichia coli culture containing 250 mg / L of purified polysaccharides of Gastrodia elata prepared in Example 1. After 48 hours of induction, the total mRNA of CL4176 nematodes was extracted with TRNzol Universal reagent, and cDNA was synthesized by reverse transcription. The expression levels of related genes were detected by qRT-PCR using gene-specific forward and reverse primers. The amplification conditions were: ① pre-denaturation 95°C-5min, ② cycle reaction 95°C: 10s-60°C: 30s, 40 cycles, ③ melting curve 95°C: 15s-60°C: 60s-95°C: 15s, and the relative gene expression values ​​of three biological replicates were determined by the 2-ΔΔCt method.

[0182] The results are as follows Fig. 9As shown: Compared with the control group, the expression level of AβmRNA in CL4176 nematodes treated with Gastrodia elata purified polysaccharide was significantly reduced by 39.75%, which may be because GEP increased the expression levels of sod3, hsp16.1, hsp16.2, gst4, gshpx, and gcs1 in CL4176 nematodes by 30.26%, 37.72%, 43.62%, 37%, 47.64%, and 66.71%, respectively.

[0183] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing purified polysaccharide from Gastrodia elata, characterized in that: The following steps are involved: (1) placing the Gastrodia elata powder in a reaction vessel, adding an alcohol solution with a volume fraction of 70% to 80%, refluxing for extraction, filtering, and collecting the residue; (2) dissolving the filter residue in pure water for ultrasonic extraction, filtering, and collecting the filtrate; (3) concentrating the filtrate under reduced pressure, adding anhydrous ethanol to the obtained concentrated solution to make the volume fraction of ethanol in the mixed solution be 70% to 80%, and then subjecting the mixed solution to low-temperature alcohol precipitation; after the alcohol precipitation is completed, collecting the precipitate and freeze-drying it to obtain crude Gastrodia elata polysaccharide; (4) dissolving the crude Gastrodia elata polysaccharide in pure water to obtain a crude Gastrodia elata polysaccharide aqueous solution, and deproteinizing the crude Gastrodia elata polysaccharide aqueous solution to obtain a deproteinized water extract; (5) purifying the deproteinized water extract using a macroporous resin to obtain a crude polysaccharide purified solution; (6) dialyzing the crude polysaccharide purified solution in pure water, and concentrating the crude polysaccharide solution obtained after the dialysis under reduced pressure, and freeze-drying the solution to obtain dialyzed Gastrodia elata crude polysaccharide; (7) dissolving the dialyzed crude Gastrodia elata polysaccharide in deionized water to obtain a crude Gastrodia elata polysaccharide solution, purifying the crude Gastrodia elata polysaccharide solution using a first chromatography column, gradient eluting with water, 0.1-0.15 mol / L NaCl solution, 0.3-0.35 mol / L NaCl solution, and 0.5-0.55 mol / L NaCl solution in sequence, combining the eluates obtained by eluting with the NaCl solution, dialyzing according to the method of step (6), concentrating under reduced pressure, and freeze-drying to obtain Gastrodia elata polysaccharide; (8) Dissolving the Gastrodia elata polysaccharide in deionized water to obtain a Gastrodia elata polysaccharide solution, purifying the Gastrodia elata polysaccharide solution using a second chromatography column, eluting with deionized water, concentrating the obtained eluate and then freeze-drying it to obtain purified Gastrodia elata polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step (1), the solid-liquid ratio of the Gastrodia elata powder to the alcohol solution with a volume fraction of 70% to 80% is 1 g: (10 to 15) ml; the alcohol solution is ethanol; The temperature of the reflux extraction is 50-70° C., the extraction time is 30-60 min each time, and the extraction times are 3-5 times.

3. The preparation method according to claim 1, characterized in that: In step (2), the material-liquid ratio of the filter residue to pure water is 1 g: (50-60) ml; The temperature of ultrasonic extraction is 50-70°C, the extraction time is 50-60min each time, the ultrasonic power is 300-400W, the ultrasonic frequency is 30-40kHz, and the number of ultrasonic extractions is 3-5 times.

4. The preparation method according to claim 1, characterized in that: The pressure of reduced pressure concentration in step (3) is -0.1 to -0.08 MPa, and the concentration is 5% to 10% of the original volume; The low-temperature alcohol precipitation is: alcohol precipitation at 4-8°C for 24-28h.

5. The preparation method according to claim 1, characterized in that: In step (4), the Gastrodia elata crude polysaccharide aqueous solution is subjected to a deproteinization treatment, comprising: Adding chloroform-n-butanol reagent to the aqueous solution of crude polysaccharide of Gastrodia elata, shaking for 30 to 40 minutes and then centrifuging to remove the denatured protein in the middle layer, repeating the shaking, centrifuging and removing the denatured protein operations until no flocculent denatured protein is produced in the middle layer after centrifugation, and collecting the deproteinized water extract in the upper layer; The volume ratio of the crude Gastrodia elata polysaccharide aqueous solution to the chloroform-n-butanol reagent is (4-5):1; The volume ratio of chloroform to n-butanol in the chloroform-n-butanol reagent is (5-6):

1.

6. The preparation method according to claim 1, characterized in that: The step (6) of dialyzing the crude polysaccharide purified solution in pure water comprises: The crude polysaccharide purified solution is placed in a dialysis bag, and the dialysis bag is placed in pure water for dialysis, with the water being changed every 2 to 3 hours for 2 to 3 days; the pore size of the dialysis bag is 8000 to 14000 Da.

7. The preparation method according to claim 1, characterized in that: During the gradient elution in step (7), the phenol-sulfuric acid method is used for color development tracking. When no color is developed, the next gradient eluent is used for elution. When no color is finally developed, the elution is stopped. The filler in the first chromatography column is a cellulose-based anion exchange resin.

8. The preparation method according to claim 1, characterized in that: During elution in step (8), an elution curve is drawn using the phenol-sulfuric acid method, and the eluate at the elution peak is collected according to the elution curve; The filler in the second chromatography column is dextran gel.

9. A purified polysaccharide from Gastrodia elata, prepared by the method according to any one of claims 1 to 8, characterized in that: The purified Gastrodia elata polysaccharide is composed of arabinose, galactose, glucose and galacturonic acid; the molar ratio of arabinose, galactose, glucose and galacturonic acid is (0.1-0.2): (0.18-0.25): (97-98): (1.8-2.5); the molecular weight of the purified Gastrodia elata polysaccharide is 890-1085 kDa.

10. An application of purified polysaccharide from Gastrodia elata, characterized in that: Use of the purified Gastrodia elata polysaccharide described in claim 9 in the preparation of drugs for preventing / treating Alzheimer's disease.

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