Gastrodin and its preparation method and application

Abstract

The application discloses a purified polysaccharide of Gastrodia elata and a preparation method and application thereof, and belongs to the technical field of extraction of active components of Gastrodia elata. The method comprises the following steps: (1) adding an alcohol solution to Gastrodia elata powder for reflux extraction, and collecting filter residues; (2) dissolving the filter residues in pure water for ultrasonic extraction to obtain a filtrate; (3) adding anhydrous ethanol to the filtrate for low-temperature alcohol precipitation to obtain crude polysaccharide of Gastrodia elata; (4) performing deproteinization treatment on the crude polysaccharide of Gastrodia elata; (5) performing purification by using a macroporous resin; (6) dialyzing the crude polysaccharide purification liquid in pure water; (7) performing purification by using a first chromatographic column, and the filler is cellulose-based anion exchange resin; (8) performing purification by using a second chromatographic column to obtain the purified polysaccharide of Gastrodia elata; and the filler is dextran gel. The crude polysaccharide of Gastrodia elata is purified by using the above five purification means, and a high-purity purified polysaccharide of Gastrodia elata can be quickly, stably and efficiently obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active ingredient extraction of Gastrodia elata Blume, in particular to a purified polysaccharide of Gastrodia elata Blume, a preparation method thereof and an application thereof. BACKGROUND

[0002] In recent years, more attention has been paid to small-molecule active compounds in the research on how traditional Chinese medicinal materials play medicinal effects, and more and more scientific evidences show that macromolecular components such as polysaccharides and proteins also play important roles in the application of traditional Chinese medicinal materials. However, the extraction and separation of macromolecules are relatively complicated, and different operation methods can obtain macromolecular fragments with different molecular weights or structures, so how to stably obtain a uniform component with good activity has become a barrier to the development of macromolecular drugs.

[0003] Gastrodia elata Blume is the dried tuber of Gastrodia elata Blume of the Orchidaceae family, which has the effects of calming wind and convulsion, calming liver yang and dispelling wind to dredge collaterals, and is mainly used for treating neurasthenia, insomnia, dizziness, epilepsy, convulsion, nervous headache, hypertension and other diseases. The active ingredient gastrodin in Gastrodia elata Blume has been developed into a drug to treat related diseases, and as another high-content active ingredient in Gastrodia elata Blume, Gastrodia elata Blume polysaccharide has relatively less research on its structure and activity. Since the polysaccharide in Gastrodia elata Blume has a complex structure, contains various monosaccharide units, and has different branched structures, the yield of the purified polysaccharide of Gastrodia elata Blume obtained by using the existing purification and extraction process is low and the components are not uniform, so that the effect of the medicinal activity cannot be guaranteed, and a preparation process for stably and efficiently extracting the purified polysaccharide of Gastrodia elata Blume with high activity and uniform components is urgently needed. SUMMARY

[0004] Therefore, the present application aims to provide a purified polysaccharide of Gastrodia elata Blume, a preparation method thereof and an application thereof, so as to overcome the problem of low yield and non-uniform components of the purified polysaccharide of Gastrodia elata Blume in the prior art.

[0005] In a first aspect, the present application provides a preparation method of a purified polysaccharide of Gastrodia elata Blume, comprising the following steps:

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

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

[0008] (3) concentrating the filtrate under reduced pressure, adding anhydrous ethanol to the obtained concentrated solution, so that the volume fraction of ethanol in the mixed solution is 70% to 80%, and then low-temperature alcohol precipitation is performed on the mixed solution; after the alcohol precipitation is completed, the precipitate is collected and freeze-dried to obtain crude polysaccharide of Gastrodia elata Blume;

[0009] (4) dissolving the Gastrodia elata crude polysaccharide in pure water to obtain a Gastrodia elata crude polysaccharide aqueous solution, and performing a deproteinization treatment on the Gastrodia elata crude 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 liquid;

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

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

[0013] (8) dissolving the Gastrodia elata polysaccharide in deionized water to obtain a Gastrodia elata polysaccharide solution, and purifying the Gastrodia elata polysaccharide solution using a second chromatography column, and eluting using deionized water, and freeze-drying the eluate after concentration to obtain Gastrodia elata purified polysaccharide.

[0014] Compared with the prior art, the present application obtains a filtrate by ultrasonic extraction of the filter residue obtained by reflux extraction of Gastrodia elata powder in an alcohol solution in pure water, and obtains Gastrodia elata crude polysaccharide by adding anhydrous ethanol to the filtrate after concentration under reduced pressure. The Gastrodia elata crude polysaccharide is subjected to deproteinization treatment, macroporous resin adsorption of pigments and impurities, dialysis, and twice chromatography purification to obtain Gastrodia elata purified polysaccharide. The Gastrodia elata crude polysaccharide is purified by using the above five purification methods, and a high-purity Gastrodia elata purified polysaccharide can be quickly, stably and efficiently obtained. In the first chromatography purification of the Gastrodia elata crude polysaccharide, water and NaCl solutions with different concentrations are sequentially used for gradient elution, and the eluate obtained by elution using the NaCl solutions with different concentrations is collected, so that Gastrodia elata acidic polysaccharide can be purified. The Gastrodia elata acidic polysaccharide is subjected to second chromatography purification, and the two chromatography purifications synergistically obtain high-purity Gastrodia elata purified polysaccharide.

[0015] Further, in step (1), the ratio of Gastrodia elata powder to alcohol solution with a volume fraction of 70%-80% is 1 g:(10-15) 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 number of extraction times is 3-5.

[0016] The above technical solution 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 the ultrasonic extraction is 50-70° C., the extraction time is 50-60 min each time, 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 solution further defines the condition parameters for ultrasonic extraction of the filter residue dissolved in pure water. Ultrasound under these condition parameters can break up cells and fully release the Gastrodia elata polysaccharide in the cells, thereby further dissolving the Gastrodia elata polysaccharide in the filter residue 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 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 solution further defines the condition parameters of reduced pressure concentration and low-temperature alcohol precipitation. Alcohol precipitation at 4-8°C for 24-28 hours can completely precipitate the ethanol-insoluble Gastrodia elata polysaccharide in the mixed solution, further remove impurities, and improve the purity of Gastrodia elata polysaccharide.

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

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

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

[0024] The crude polysaccharide purified solution is placed in a dialysis bag, which is then 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 technical scheme is further limited to the dialysis time and the pore size of the dialysis bag when the crude polysaccharide purification liquid is dialyzed, and under the above conditions, small molecular impurities in the crude polysaccharide purification liquid of Gastrodia elata can be further removed, so that the purity of the crude polysaccharide of Gastrodia elata is improved.

[0026] Further, in the gradient elution in step (7), phenol-sulfuric acid colorimetry is used for tracking, when there is no color, the next gradient eluent is used for elution, and when there is no color at last, the elution is stopped.

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

[0028] In the technical scheme, phenol-sulfuric acid colorimetry is used for tracking when gradient elution is performed, and cellulose-based anion exchange resin is selected as the filler, so that the acidic polysaccharide of Gastrodia elata in the crude polysaccharide solution of Gastrodia elata can be completely eluted.

[0029] Further, in the elution in step (8), the elution curve is drawn by using phenol-sulfuric acid method, and the eluent at the elution peak is collected according to the elution curve; the filler in the second chromatographic column is sephadex.

[0030] In the technical scheme, the eluent at the elution peak is collected according to the elution curve drawn by using phenol-sulfuric acid method, and the collection method can obtain the eluent with more purified polysaccharide components of Gastrodia elata. The sephadex is selected as the filler of the second chromatographic column, so that the acidic polysaccharide obtained in the previous step can be further purified, and the purified polysaccharide of Gastrodia elata with higher purity can be obtained.

[0031] In a second aspect, the application provides a purified polysaccharide of Gastrodia elata, which is prepared by any of the above methods, and 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); and 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 is a macromolecular substance with a new structure, has a specific composition and ratio, and further improves the prevention and treatment of Alzheimer's disease.

[0033] In a third aspect, the application provides an application of the purified polysaccharide of Gastrodia elata, and the above purified polysaccharide of Gastrodia elata is applied to the preparation of a medicine for preventing / treating Alzheimer's disease.

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

[0035] Figure 1 Elution curve in Example 1.

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

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

[0038] Figure 4 High performance anion exchange chromatography in Test Example 2.

[0039] Figure 5 UV spectrum scanning result chart in Test Example 2.

[0040] Figure 6 Infrared spectrum scanning result chart in Test Example 2.

[0041] Figure 7 Comparison chart of Aβ aggregation in vivo of nematodes in Test Example 3.

[0042] Figure 8 Comparison chart of Aβ aggregation in vivo of CL4176 nematodes fed with common Gastrodia elata polysaccharide and purified polysaccharide of Gastrodia elata in Example 1 in Test Example 3.

[0043] Figure 9 Expression of Aβ gene in vivo of CL4176 nematodes in Test Example 3. DETAILED DESCRIPTION

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

[0045] In a first aspect, the present application provides a preparation method of purified polysaccharide of Gastrodia elata, comprising the following steps:

[0046] S1, placing Gastrodia elata powder in a reaction container, adding an alcohol solution with a volume fraction of 70% to 80%, refluxing and extracting, filtering, and collecting the filter residue.

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

[0048] Further, the ratio of the Gastrodin powder to the alcohol solution with a volume fraction of 70% to 80% in the above step is 1g:(10-15)ml; the temperature of the reflux extraction is 50-70°C, the extraction time is 30-60 minutes each time, and the extraction times is 3-5 times.

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

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

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

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

[0053] S2, the residue obtained in the above step S1 is dissolved in pure water and ultrasonically extracted, filtered, and the filtrate is collected.

[0054] Further, the ratio of the residue to the pure water in the above step is 1g:(50-60)ml; the temperature of the ultrasonic extraction is 50-70°C, the extraction time is 50-60 minutes each time, the ultrasonic power is 300-400W, the ultrasonic frequency is 30-40kHz, and the ultrasonic extraction times is 3-5 times.

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

[0056] The above step of dissolving the residue in pure water can dissolve the Gastrodin polysaccharide in the residue in the pure water, and the ultrasonic crushing of the cells can further release the Gastrodin polysaccharide of the cells, so as to fully extract the Gastrodin polysaccharide in the residue.

[0057] After the ultrasonic extraction is completed, a filter with a pore size of 80-120 μm is used to filter the solution, the residue is discarded, and the filtrate is collected.

[0058] S3, the filtrate obtained in the above step S2 is concentrated under reduced pressure, anhydrous ethanol is added to the concentrated solution, the volume fraction of ethanol in the mixed solution is 70-80%, and then the mixed solution is alcohol precipitated at low temperature; after alcohol precipitation is completed, the precipitate is collected and freeze-dried to obtain crude polysaccharide of Gastrodia elata.

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

[0060] Further, the low-temperature alcohol precipitation is: alcohol precipitation at 4-8℃ for 24-28h. For example, the temperature can be 4℃, 5℃, 6℃, 7℃ or 8℃, preferably 4-6℃, and the alcohol precipitation time is 24h, 26h or 28h.

[0061] Because the solubility of Gastrodia elata polysaccharide in ethanol is low, long-time alcohol precipitation at low temperature of 4-8℃ can effectively precipitate Gastrodia elata polysaccharide from the solution, thereby improving the extraction rate of Gastrodia elata polysaccharide.

[0062] After alcohol precipitation is completed, a vacuum freeze-drying machine can be selected to freeze-dry the precipitate, the condensation temperature is -70 to -80℃, and the vacuum degree is <10Pa. The model of the vacuum freeze-drying machine can be selected as FD-2A, which is produced by Bomedica Instrument Co., Ltd.

[0063] S4, the crude polysaccharide of Gastrodia elata obtained in the above step S3 is dissolved in pure water to obtain a crude polysaccharide of Gastrodia elata aqueous solution, and the crude polysaccharide of Gastrodia elata aqueous solution is subjected to deproteinization treatment to obtain a deproteinized water extract.

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

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

[0066] Chloroform-n-butanol reagent is added to the crude polysaccharide of Gastrodia elata aqueous solution, shaken for 30-40min, and then centrifuged to remove the denatured protein in the middle layer. The shaking, centrifugation and denatured protein removal operations are repeated until no flocculent denatured protein is produced in the middle layer after centrifugation, and the deproteinized water extract in the upper layer is collected. The centrifugation can be selected at a speed of 3000-4000r / min for 1-2min.

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

[0068] The above step can effectively remove the protein in the water extract of crude polysaccharide of Gastrodia elata Blume by using Sevage method, and further improve the purity of the polysaccharide of Gastrodia elata Blume.

[0069] S5, using macroporous resin to purify the deproteinized water extract 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. Using the resin to purify the deproteinized water extract can not only remove the pigment in the deproteinized water extract, but also further remove the residual protein.

[0071] For example, the purification process of the deproteinized water extract can refer to the following:

[0072] The D3520 macroporous resin is placed in a chromatography column (30.0 cm x 1.5 cm), and after the pure water balance column is used, the resin is naturally settled to 23-25 cm. The deproteinized water extract is slowly added along the column wall with a dropper. It should be understood that the addition rate is based on not damaging the flatness of the filler surface. After the sample solution is balanced in the column for 1 hour, the valve at the bottom of the column is opened, and the sample solution is allowed to pass through the column at a flow rate of 1-3 cm / min. The sample solution is collected to obtain a crude polysaccharide purified solution.

[0073] S6, the crude polysaccharide purified solution obtained in the above step S5 is dialyzed in pure water. After dialysis is completed, the crude polysaccharide solution is reduced pressure concentrated, frozen and dried to obtain the dialyzed crude polysaccharide of Gastrodia elata Blume.

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

[0075] The crude polysaccharide purified solution is loaded into a dialysis bag, and the dialysis bag is placed in pure water for dialysis. The water is changed every 2-3 hours, and the dialysis is continued for 2-3 days.

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

[0077] After dialysis is completed, the dialyzed crude polysaccharide of Gastrodia elata Blume is reduced pressure concentrated to 5%-10% of the original volume under a pressure of-0.1 MPa to-0.08 MPa, and then frozen and dried to obtain the dialyzed crude polysaccharide of Gastrodia elata Blume. The freezing and drying conditions are as described in the above step S3.

[0078] The purified polysaccharide solution is dialyzed to remove salts, small organic molecules and low molecular weight proteins, thereby further improving the purity of the purified polysaccharide of Gastrodia elata.

[0079] S7, the dialyzed Gastrodia elata crude polysaccharide obtained in step S6 is dissolved in deionized water to obtain a Gastrodia elata crude polysaccharide solution, and the Gastrodia elata crude polysaccharide solution is purified using a first chromatography column, and gradient elution is performed using 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, the eluate obtained by elution with the NaCl solution is combined and dialyzed according to the method of step S6, and then concentrated under reduced pressure and freeze-dried to obtain Gastrodia elata polysaccharide.

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

[0081] In the above step, when loading, the Gastrodia elata crude polysaccharide solution can be added along the inner wall of the chromatography column with a dropper to make it distribute on the surface of the DEAE-52 filler as soon as possible, and attention should be paid to control the dropping speed to ensure that the flatness of the filler surface is not damaged during dropping. During gradient elution, the linear velocity of the eluate is 50-100 cm / h.

[0082] In the above step, the water eluted is Gastrodia elata neutral polysaccharide, which is not the target polysaccharide of the present application and is discarded. The NaCl solution eluted is Gastrodia elata acidic polysaccharide (target polysaccharide), and gradient elution is performed using 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 to fully elute the Gastrodia elata acidic polysaccharide in the solution.

[0083] Further, in the above step, during gradient elution, phenol-sulfuric acid colorimetric tracking is used, when there is no color, the next gradient eluent is used for elution, and when there is no color at the end, the elution is stopped.

[0084] S8, the Gastrodia elata polysaccharide obtained in step S7 is dissolved in deionized water to obtain a Gastrodia elata polysaccharide solution, and the Gastrodia elata polysaccharide solution is purified using a second chromatography column, and eluted with deionized water, and the obtained eluate is concentrated and freeze-dried to obtain purified Gastrodia elata polysaccharide.

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

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

[0087] Further, in the above step, the elution curve is drawn by using the phenol-sulfuric acid method, and the eluent 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] 0.2 mL of the eluent is taken, and then 1.0 mL of pure water, 1.0 mL of a 5% phenol solution and 5.0 mL of concentrated sulfuric acid are added, and the mixture is shaken and placed at room temperature for 10 minutes, and then heated in a boiling water bath for 15 minutes, and then cooled and measured for the absorbance at 490 nm. The elution curve is drawn with the number of the elution tube as the abscissa and the absorbance at 490 nm as the ordinate, and the eluent at the elution peak is collected according to the elution curve.

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

[0091] The above step is used for further purification of the Gastrodia elata acidic polysaccharide purified by the first chromatographic column, and a high-purity purified Gastrodia elata polysaccharide with a novel structure can be obtained.

[0092] In the second aspect, the embodiments of the present application provide a purified Gastrodia elata polysaccharide, which is prepared by using the above method. 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.

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

[0094] Compared with the prior art, the purified Gastrodia elata polysaccharide with a novel structure prepared by using the method has specific composition and ratio, and the prevention and treatment effects on Alzheimer's disease are further improved.

[0095] In the third aspect, the embodiments of the present application provide an application of the purified Gastrodia elata polysaccharide. The above purified Gastrodia elata polysaccharide is used for preparing a medicine for preventing and treating Alzheimer's disease.

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

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

[0098] Example 1

[0099] A preparation method of a ginseng purified polysaccharide, comprising the following steps:

[0100] S1, 50 mesh ginseng powder is placed in a round-bottom flask, 80% ethanol by volume is added, and reflux extraction is performed for 3 times, each time for 60 min, filtration is performed, and the filter residue is collected. The temperature of the reflux extraction is 60℃, the solid-liquid ratio of the ginseng powder and the 80% ethanol is 1g:10ml, and the filter pore size is 100μm.

[0101] S2, the filter residue is dissolved in pure water and ultrasonic extraction is performed for 3 times, each time for 50 min, the solid-liquid ratio of the filter residue and the pure water is 1g:50ml, filtration is performed, and the filtrate is collected. The ultrasonic extraction temperature is 60℃, the ultrasonic frequency is 40kHz, the ultrasonic power is 400W, and the filter pore size is 100μm.

[0102] S3, the filtrate is concentrated under reduced pressure, the reduced pressure concentration pressure is-0.09MPa, the concentration is performed to 7% of the original volume, anhydrous ethanol is added to the obtained concentrated solution, the volume fraction of ethanol in the mixed solution is 80%, and then the mixed solution is alcohol precipitated at low temperature, alcohol precipitation is performed at 4℃ for 24h. After alcohol precipitation is completed, the precipitate is collected and freeze-dried by using a vacuum freeze dryer (FD-2A, Biomedica Instrument Co., Ltd.), the condensation temperature is-80℃, and the vacuum degree is <10Pa, to obtain ginseng crude polysaccharide.

[0103] S4, the ginseng crude polysaccharide is dissolved in pure water to obtain a ginseng crude polysaccharide aqueous solution, the ratio of the ginseng crude polysaccharide and the pure water is 1g:40ml, and the ginseng crude polysaccharide aqueous solution is subjected to deproteinization treatment: chloroform-n-butanol reagent is added to the ginseng crude polysaccharide aqueous solution, after shaking for 30 min, centrifugation is performed at 4000r / min for 1 min, denatured protein in the middle layer is removed, the operation of shaking, centrifugation and denatured protein removal is repeated until no flocculent denatured protein is generated in the middle layer after centrifugation, and the deproteinized water extract in the upper layer is collected.

[0104] The volume ratio of the ginseng crude polysaccharide aqueous solution and the chloroform-n-butanol reagent is 4:1, and the volume ratio of chloroform and n-butanol in the chloroform-n-butanol reagent is 5:1.

[0105] S5, the deproteinized water extract is purified by using D3520 macroporous resin:

[0106] D3520 macroporous resin is placed in a chromatographic column (30.0 cm x 1.5 cm), and after the pure level balance column, the resin is naturally settled to 24 cm. The deproteinized water extract is slowly added along the column wall with a dropper, and the dripping speed is controlled to not damage the flatness of the filler. After the sample solution is balanced in the column for 1 h, the valve at the bottom of the column is opened, and the sample solution passes through the column at a flow rate of 2 cm / min. The sample solution is collected to obtain a crude polysaccharide purification solution.

[0107] S6, the crude polysaccharide purification solution is dialyzed in pure water: the crude polysaccharide purification solution is placed in a dialysis bag, and the dialysis bag is placed in pure water for dialysis. The water is changed every 2 h, and the dialysis lasts for 3 d. The pore size of the dialysis bag is 10000 Da, and the model is MD34.

[0108] The crude polysaccharide solution obtained after dialysis is concentrated to 5% of the original volume under reduced pressure at a pressure of -0.1 MPa. Vacuum freeze-drying is performed using a vacuum freeze-drying machine (FD-2A, Bomedica Instrument Co., Ltd.). The condensation temperature is -80℃, and the vacuum degree is <10 Pa. The dialyzed Gastrodia elata crude polysaccharide is obtained.

[0109] S7, the dialyzed Gastrodia elata crude polysaccharide is dissolved in deionized water, and the ratio of Gastrodia elata crude polysaccharide to deionized water is 1g:10mL. The Gastrodia elata crude polysaccharide solution is purified using a first chromatographic column. Water, 0.1 mol / L NaCl solution, 0.3 mol / L NaCl solution, and 0.5 mol / L NaCl solution are used for gradient elution in sequence. The eluate obtained by elution with NaCl solution is combined and dialyzed according to the method of step S6, then concentrated under reduced pressure and freeze-dried to obtain Gastrodia elata polysaccharide. The filler in the first chromatographic column is DEAE-52.

[0110] During gradient elution, the linear velocity of the eluate is 80 cm / h. The phenol-sulfuric acid method is used for color tracking. When there is no color, the next gradient eluent is used for elution. When there is no color at the end, the elution is stopped.

[0111] S8, the Gastrodia elata polysaccharide is dissolved in deionized water to obtain a Gastrodia elata polysaccharide solution, and the ratio of Gastrodia elata polysaccharide to deionized water is 1g:5mL. The Gastrodia elata polysaccharide solution is purified using a second chromatographic column, and deionized water is used for elution. The eluate is concentrated and freeze-dried to obtain Gastrodia elata purified polysaccharide. The filler in the second chromatographic column is Sephadex G-100.

[0112] The linear velocity of deionized water during elution is 0.8 ml / min. The phenol-sulfuric acid method is used to draw an elution curve. According to the elution curve, the eluate at the elution peak is collected.

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

[0114] Take 0.2 mL of the eluent, add pure water to 1.0 mL, then add 1.0 mL of a 5% phenol solution and 5.0 mL of concentrated sulfuric acid, shake well, and let stand at room temperature for 10 minutes. Then heat in a boiling water bath for 15 minutes. After cooling, measure the absorbance at 490 nm. Take the number of the elution tube as the abscissa and the absorbance at 490 nm as the ordinate to draw the elution curve. The elution curve is shown in Figure 1 .

[0115] The collected eluent is concentrated under reduced pressure to 5% of the original volume at a pressure of -0.1 MPa, and freeze-dried using a vacuum freeze dryer (FD-2A, Biomedica Instruments Co., Ltd.). The condensation temperature is -80°C, and the vacuum degree is <10 Pa. Gastrodine purified polysaccharide is obtained.

[0116] Example 2

[0117] A method for preparing gastrodine purified polysaccharide, comprising the following steps:

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

[0119] S2, dissolve the filter residue in pure water and ultrasonically extract 5 times, each for 60 minutes. The filter residue to pure water liquid ratio 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, concentrate the filtrate under reduced pressure to 5% of the original volume at a pressure of -0.1 MPa. Add anhydrous ethanol to the obtained concentrated solution to make the ethanol volume fraction in the mixed solution 70%, and then low-temperature alcohol precipitation is performed on the mixed solution at 8°C for 28 hours. After alcohol precipitation is completed, the precipitate is collected and freeze-dried using a vacuum freeze dryer (FD-2A, Biomedica Instruments Co., Ltd.). The condensation temperature is -80°C, and the vacuum degree is <10 Pa. Gastrodine crude polysaccharide is obtained.

[0121] S4, dissolve the Gastrodine crude polysaccharide in pure water to obtain a Gastrodine crude polysaccharide aqueous solution. The Gastrodine crude polysaccharide to pure water ratio is 1g:60ml. Perform deproteinization on the Gastrodine crude polysaccharide aqueous solution by adding chloroform-n-butanol reagent to the Gastrodine crude polysaccharide aqueous solution, shaking for 40 minutes, and then centrifuging at 4000r / min for 1 minute to remove denatured protein in the middle layer. Repeat the shaking, centrifuging, and removing denatured protein operation until no flocculent denatured protein is produced in the middle layer after centrifugation. Collect the deproteinized water extract in the upper layer.

[0122] The volume ratio of the Gastrodia elata crude 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 (30.0 cm × 1.5 cm). After equilibration with pure water, the column was allowed to settle naturally to 24 cm. The deproteinized aqueous extract was then slowly added dropwise along the column wall using a pipette, controlling the droplet rate to avoid damaging the surface smoothness of the filler. After the sample solution had equilibrated in the column for 1 hour, the valve at the bottom of the column was opened, and the sample solution was allowed to pass through the column at a flow rate of 1 cm / 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, place 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 under reduced pressure at a pressure of -0.08 MPa to 10% 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 the dialyzed Gastrodia elata crude polysaccharide.

[0127] S7. Dissolve the dialyzed crude Gastrodia elata polysaccharide in deionized water at a ratio of 1 g:8 mL to obtain a crude Gastrodia elata polysaccharide solution. Purify the crude Gastrodia elata polysaccharide solution using a first chromatography column, performing 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 elata polysaccharide. 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. Elution is stopped when no color is finally observed.

[0129] S8. Dissolve the 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. Purify the Gastrodia elata polysaccharide solution using a second chromatography column, elute with deionized water, concentrate the obtained eluate, and then freeze-dry to obtain purified Gastrodia elata polysaccharide; wherein the filler in the second chromatography column is Sephadex G-100.

[0130] The linear velocity of deionized water during elution is 0.5 ml / min, and the elution curve is drawn by using the phenol-sulfuric acid method, and the eluent at the elution peak is collected according to the elution curve.

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

[0132] 0.2 mL of the eluent is taken, and the volume is made up to 1.0 mL with pure water, then 1.0 mL of a 5% phenol solution and 5.0 mL of concentrated sulfuric acid are added, and the mixture is shaken and placed at room temperature for 10 minutes, and then heated in a boiling water bath for 15 minutes. After cooling, the absorbance value at 490 nm is measured. The elution curve is drawn with the number of the elution tube as the abscissa and the absorbance value at 490 nm as the ordinate.

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

[0134] Example 3

[0135] A method for preparing a purified polysaccharide from Gastrodia elata, comprising the following steps:

[0136] S1, 50 mesh Gastrodia elata powder is placed in a round-bottom flask, 75% ethanol by volume is added, and reflux extraction is performed 4 times, each time for 50 min, and the filtrate is collected. The temperature of the reflux extraction is 60°C, the solid-liquid ratio of the Gastrodia elata powder to 75% ethanol is 1g:15ml, and the filter pore size is 100μm.

[0137] S2, the filtrate is ultrasonically extracted 4 times in pure water, each time for 55 min, the solid-liquid ratio of the filtrate to pure water is 1g:55ml, and the filtrate is collected. 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 filtrate is concentrated under reduced pressure, the pressure during the concentration under reduced pressure is -0.08 MPa, and the concentration is performed to 10% of the original volume. Anhydrous ethanol is added to the concentrated solution to make the volume fraction of ethanol in the mixed solution 75%, and then the mixed solution is alcohol precipitated at low temperature for 26h at 6°C. After alcohol precipitation, the precipitate is collected and freeze-dried using a vacuum freeze dryer (FD-2A, Bomedica Instrument Co., Ltd.), with a condensation temperature of -80°C and a vacuum degree of <10 Pa, to obtain the crude polysaccharide from Gastrodia elata.

[0139] S4, dissolve the above Gastrodia elata Blume crude polysaccharide in pure water to obtain a Gastrodia elata Blume crude polysaccharide aqueous solution, the ratio of Gastrodia elata Blume crude polysaccharide to pure water is 1 g:30 mL, and the Gastrodia elata Blume crude polysaccharide aqueous solution is subjected to deproteinization treatment: chloroform-n-butanol reagent is added to the Gastrodia elata Blume crude polysaccharide aqueous solution, after shaking for 35 min, centrifugation is performed at 4000 r / min for 1 min, denatured protein in the middle layer is removed, and the operation of shaking, centrifugation and denatured protein removal is repeated until no flocculent denatured protein is generated in the middle layer after centrifugation, and the deproteinized water extract in the upper layer is collected.

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

[0141] S5, the deproteinized water extract is purified by using D3520 macroporous resin:

[0142] The D3520 macroporous resin is placed in a chromatography column (30.0 cm*1.5 cm), and after the pure water balance column is used, the resin is naturally settled to 24 cm, the deproteinized water extract is slowly added along the column wall by using a dropper, and the dripping speed is controlled to not damage the flatness of the filler surface. When the sample solution is balanced in the column for 1 h, the valve at the bottom of the column is opened, the sample solution passes through the column at a flow rate of 3 cm / min, and the sample solution is collected to obtain a crude polysaccharide purified solution.

[0143] S6, the above crude polysaccharide purified solution is dialyzed in pure water: the crude polysaccharide purified solution is placed in a dialysis bag, the dialysis bag is placed in pure water for dialysis, the water is changed every 2 h, and the dialysis is continued for 3 d; the pore size of the dialysis bag is 14000 Da, and the model is MD34.

[0144] The crude polysaccharide solution obtained after dialysis is concentrated to 8% of the original volume under reduced pressure at a pressure of-0.09 MPa, and freeze-drying is performed by using a vacuum freeze-drying machine (FD-2A, Bomedica Instrument Co., Ltd.), the condensation temperature is-80℃, and the vacuum degree is <10 Pa, to obtain the dialyzed Gastrodia elata Blume crude polysaccharide.

[0145] S7, the above dialyzed Gastrodia elata Blume crude polysaccharide is dissolved in deionized water, the ratio of Gastrodia elata Blume crude polysaccharide to deionized water is 1 g:10 mL, to obtain a Gastrodia elata Blume crude polysaccharide solution, the Gastrodia elata Blume crude polysaccharide solution is purified by using a first chromatography column, and gradient elution is performed by using water, 0.12 mol / L NaCl solution, 0.32 mol / L NaCl solution and 0.52 mol / L NaCl solution in sequence, the eluate obtained by elution with the NaCl solution is combined, and dialysis is performed according to the method of step S6, then reduced pressure concentration and freeze-drying are performed to obtain Gastrodia elata Blume polysaccharide; wherein the filler in the first chromatography column is DEAE-52.

[0146] The linear velocity of the eluent is 100 cm / h during gradient elution. The elution is tracked by phenol-sulfuric acid method. When there is no color, the next gradient eluent is used to elute. When there is no color at last, the elution is stopped.

[0147] S8, dissolving the Gastrodia elata Blume polysaccharide in deionized water to obtain a Gastrodia elata Blume polysaccharide solution. The ratio of the Gastrodia elata Blume polysaccharide to the deionized water is 1 g:6 mL. The Gastrodia elata Blume polysaccharide solution is purified by using a second chromatographic column. The eluent is eluted by using deionized water. The eluent is concentrated and freeze-dried to obtain the purified Gastrodia elata Blume polysaccharide. The filler in the second chromatographic column is Sephadex G-100.

[0148] The linear velocity of the deionized water is 0.6 ml / min during elution. The elution curve is drawn by using the phenol-sulfuric acid method. The eluent at the elution peak is collected according to the elution curve.

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

[0150] 0.2 mL of the eluent is taken. The volume is supplemented to 1.0 mL by using pure water. Then, 1.0 mL of a phenol solution with a mass fraction of 5% and 5.0 mL of concentrated sulfuric acid are added. After being shaken uniformly, the solution is placed at room temperature for 10 minutes and then heated in a boiling water bath for 15 minutes. After being cooled, the absorbance value at 490 nm is measured. The elution curve is drawn by taking the number of the elution tube as the abscissa and the absorbance value at 490 nm as the ordinate.

[0151] The collected eluent is concentrated to 5% of the original volume under reduced pressure at a pressure of-0.1 MPa. The vacuum freeze-drying machine (FD-2A, Bomedica Instrument Co., Ltd.) is used for freeze-drying. The condensation temperature is-80℃, and the vacuum degree is <10 Pa. The purified Gastrodia elata Blume polysaccharide is obtained.

[0152] Comparative Example 1

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

[0154] Comparative Example 2

[0155] The purified Gastrodia elata Blume polysaccharide is prepared according to the method of Example 1. The difference is that, in step S7, the gradient elution is performed by using water, a 0.1 mol / L NaOH solution, a 0.3 mol / L NaOH solution, and a 0.5 mol / L NaOH solution in sequence. The eluent obtained by using the NaOH solution is combined and then subjected to step S8.

[0156] Comparative Example 3

[0157] The purified Gastrodia elata Blume polysaccharide is prepared according to the method of Example 1. The difference is that step S8 is not performed, i.e., the second chromatographic column purification is not performed.

[0158] Comparative Example 4

[0159] The ginseng purified polysaccharide was prepared according to the method of Example 1, except that in step S7, water and 0.1 mol / L NaCl solution were used for elution in sequence, and the other conditions were not changed.

[0160] Test Example 1

[0161] The yield (relative to ginseng powder) and purity of the ginseng purified polysaccharide in Examples 1-3 and Comparative Examples 1-3 above were detected, and the results are shown in Table 1:

[0162] Table 1

[0163] Yield of purified polysaccharide from Gastrodia elata Purity of purified polysaccharide from Gastrodia elata 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] From Table 1, it can be seen that after the D3520 macroporous resin in Comparative Example 1 was replaced with an equal amount of D101 macroporous resin, the purity of the ginseng purified polysaccharide decreased, indicating that the use of D3520 macroporous resin in the present application can improve the purity of the ginseng purified polysaccharide. After water and different concentrations of NaOH solution were used for elution in sequence in step S7 of Comparative Example 2, the purity of the ginseng purified polysaccharide decreased, indicating that the use of specific eluent in the present application can further improve the purity of the ginseng purified polysaccharide. After the second chromatographic column purification of step S8 was not performed in Comparative Example 3, the purity of the ginseng purified polysaccharide decreased, indicating that the two chromatographic purifications in the present application can further improve the purity of the ginseng purified polysaccharide. In Comparative Example 4, water and 0.1 mol / L NaCl solution were used for elution in step S7, and the purity of the ginseng purified polysaccharide decreased, indicating that the use of different concentrations of NaCl solution for elution in the present application can improve the yield of the ginseng purified polysaccharide while improving the purity of the ginseng purified polysaccharide.

[0165] Test Example 2

[0166] 1. Nuclear magnetic resonance spectroscopy detection:

[0167] Three batches of ginseng purified polysaccharide were prepared by repeating the method of Example 1 three times, and the ginseng purified polysaccharide prepared in Examples 1-3 (a total of 5 batches) was subjected to nuclear magnetic resonance spectroscopy detection, and the results are shown in Table 2: Figure 2 Figure 3 The NMR carbon spectrum and hydrogen spectrum signals were consistent, indicating that the ginseng purified polysaccharide obtained in Examples 1-3 of the present application was the same substance, and the preparation method of the ginseng purified polysaccharide of the present application had repeatability and stability.

[0168] 2. Composition analysis of ginseng purified polysaccharide:

[0169] The monosaccharide composition of the ginseng purified polysaccharide in Example 1 above was analyzed by high-performance anion exchange chromatography amperometric detection, and the results are shown in Table 3:​Figure 4 As shown in the figure, the purified polysaccharide of GEP is mainly composed of arabinose, galactose, glucose and galacturonic acid, and the molar ratio is 0.15:0.21:97.63:2.0. The molecular weight of GEP is 1012.65 kDa detected by gel exclusion chromatography.

[0170] 3, The purified polysaccharide of GEP in Example 1 above was scanned by ultraviolet spectrum, and the results are shown in the figure Figure 5 As shown in the figure, there is no absorption at 260 nm and 280 nm, indicating that GEP does not contain nucleic acid and protein.

[0171] 4, The purified polysaccharide of GEP in Example 1 above was scanned by infrared spectrum, and the results are shown in the figure Figure 6 As shown in the figure, there is a broad peak at 3000-3500 cm -1 , which is the O-H bond stretching vibration peak, 2931 cm -1 is the C-H stretching vibration peak, 848 cm -1 is the α-pyranose bond, 1200-1400 cm -1 is the C-H variable angle vibration, 1025-1156 cm -1 is the pyranose bond type. 1645 cm -1 is the bound water.

[0172] Test Example 3

[0173] The effects of the purified polysaccharide of GEP prepared in Examples 1-3 above on Alzheimer's disease C. elegans were detected.

[0174] 1, The aggregation of β-amyloid protein (Aβ) in CL4176 nematodes (Alzheimer's disease C. elegans) induced at 25℃ was detected by using thioflavin S staining:

[0175] After CL4176 nematodes were cultured at 16℃ for 48h, they were induced at 25℃. When induced at 25℃, the control group of CL4176 nematodes was only fed with E. coli liquid, the CL4176 nematodes in treatment group 1 were fed with E. coli liquid containing 250 mg / L of the purified polysaccharide of GEP prepared in Example 1, and the CL4176 nematodes in treatment group 2 were fed with E. coli liquid containing 250 mg / L of ordinary GEP, and the preparation method of the ordinary GEP is referred to: Zhang Shuangqi, Liu Lin, He Nianwu, et al. Optimization of ultrasonic-assisted extraction of polysaccharides from Shaanxi Gastrodia elata and its antioxidant activity [J]. Chinese Agricultural Bulletin, 2021, 37(09): 131-136.

[0176] The CL4176 nematodes of the control group, the treatment group 1 and the treatment group 2 were respectively selected alone, dyed with 1 mM of thioflavin S for 4 hours, and then fixed in 4% paraformaldehyde / PBS with pH value of 7.4 at 4℃ for 24 hours. The fluorescence image was obtained by a fluorescence inverted biological microscope with 20x objective lens. The results are shown in Figure 7 , Figure 8 .

[0177] Figure 7 A is the staining diagram of Aβ deposition of CL4176 nematodes in the treatment group 1 after induction at 25℃; B is the staining diagram of Aβ deposition of CL4176 nematodes in the control group after induction at 25℃; C is the staining diagram of wild type nematodes (normal non-Alzheimer type nematodes, only fed with E. coli liquid) after induction at 25℃; D is the staining diagram of CL4176 nematodes (only fed with E. coli liquid) which are cultured at 16℃ all the time without induction at 25℃.

[0178] It can be known from Figure 7 that Aβ aggregation occurs when the CL4176 nematodes are transferred from 16℃ to 25℃ for induction, and the Aβ aggregation of the CL4176 nematodes is obviously reduced after feeding with the purified polysaccharide of Gastrodia elata in Example 1.

[0179] It can be known from Figure 8 that compared with feeding with common Gastrodia elata polysaccharide, feeding with the purified polysaccharide of Gastrodia elata in the application Example 1 can further reduce the Aβ aggregation of the CL4176 nematodes after induction at 25℃.

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

[0181] The CL4176 nematodes were cultured at 16℃ for 48 hours and then induced at 25℃. The CL4176 nematodes of the control group were only fed with E. coli liquid, and the CL4176 nematodes of the treatment group were fed with E. coli liquid containing 250 mg / L of the purified polysaccharide of Gastrodia elata prepared in Example 1. After induction for 48 hours, the total mRNA of the CL4176 nematodes was extracted by using TRNzol Universal reagent, and cDNA was synthesized by reverse transcription. The expression level of the related gene was detected by using gene-specific forward and reverse primers through qRT-PCR. The amplification conditions were as follows: ① pre-denaturation at 95℃ for 5 min, ② cycle reaction at 95℃ for 10 s and at 60℃ for 30 s, 40 cycles, ③ melting curve at 95℃ for 15 s, at 60℃ for 60 s and at 95℃ for 15 s, and the relative gene expression value of three biological repeats was determined by using 2-ΔΔCt method.

[0182] The results are shown in Figure 9As shown: compared with the control group, the expression of Aβ mRNA in CL4176 nematodes in the Gastrodia elata purified polysaccharide treatment group was significantly reduced by 39.75%, which may be because the expression of sod3, hsp16.1, hsp16.2, gst4, gshpx and gcs1 in CL4176 nematodes was increased by 30.26%, 37.72%, 43.62%, 37%, 47.64% and 66.71% respectively.

[0183] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that modifications, equivalent replacements and improvements of the technical solutions described in the foregoing embodiments can still be made. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing purified polysaccharide from Gastrodia elata, characterized in that: The following steps are involved: (1) Place Gastrodia elata powder in a reaction vessel, add 70% to 80% alcohol solution by volume, reflux extract, filter, and collect the residue; (2) dissolving the filter residue in pure water, performing ultrasonic extraction, filtering, and collecting the filtrate; (3) Concentrating the filtrate under reduced pressure, adding anhydrous ethanol to the obtained concentrated solution so that the volume fraction of ethanol in the mixed solution is 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 aqueous extract using D3520 macroporous resin to obtain a crude polysaccharide purified solution; (6) dialyzing the purified crude polysaccharide solution in pure water, concentrating the crude polysaccharide solution obtained after the dialysis under reduced pressure, and freeze-drying the solution to obtain dialyzed crude Gastrodia elata 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, and 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), and then concentrating under reduced pressure and freeze-drying to obtain Gastrodia elata polysaccharide; Wherein, the filler in the first chromatography column is a cellulose-based anion exchange resin; (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 freeze-drying it to obtain purified Gastrodia elata polysaccharide; Wherein, the filler in the second chromatography column is dextran gel.

2. The preparation method according to claim 1, characterized in that In step (1), the material-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 number of extractions is 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℃, the extraction time is 50~60min, the ultrasonic power is 300~400W, the ultrasonic frequency is 30~40 kHz, and the number of ultrasonic extractions is 3~5 times.

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

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 Gastrodia elata polysaccharide, shaking for 30-40 minutes and then centrifuging to remove denatured protein in the middle layer, repeating the shaking, centrifugation, and denatured protein removal 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 and tracking. When no color is developed, the next gradient eluent is used for elution. When no color is finally developed, the elution is stopped.

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.

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); and 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 according to claim 9 in the preparation of a drug for preventing / treating Alzheimer's disease.

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