Ginseng acidic polysaccharide, and preparation method and application thereof

Abstract

This invention provides a ginseng acidic polysaccharide, its preparation method, and its application, belonging to the field of biopharmaceutical manufacturing technology. The invention extracts polysaccharides from ginseng using water extraction and alcohol precipitation, then removes proteins using a combination of freeze-thaw and Sevag methods while retaining polysaccharide activity. Further purification is achieved using DEAE-cellulose ion exchange column chromatography and Sepharose CL-6B gel filtration column chromatography, yielding two homogeneous fractions. In vivo efficacy evaluation methods confirmed that the homogeneous acidic polysaccharide fraction GPA-0.2b possesses anti-AD activity, significantly reducing intracranial inflammatory responses and neurological damage caused by β-amyloid protein, and improving cognitive impairment and anxiety behavior. Therefore, GPA-0.2b provides a clear material basis for the precise development and utilization of ginseng polysaccharides.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical manufacturing technology, specifically relating to a ginseng acidic polysaccharide, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a central nervous system degenerative disease characterized by progressive cognitive impairment and behavioral disturbances. Its pathological features primarily include senile plaques formed by the abnormal deposition of β-amyloid (Aβ) protein and neurofibrillary tangles. Currently, commonly used AD treatments mainly include acetylcholinesterase inhibitors (such as donepezil and rivastigmine) and NMDA receptor antagonists (such as memantine). While these drugs can improve patients' cognitive symptoms to some extent, they cannot slow down or stop the pathological progression of the disease. Furthermore, they have significant side effects and are expensive, leading to poor patient adherence.

[0003] Ginseng polysaccharides (GP), one of the main bioactive components of ginseng, possess various pharmacological activities, including immunomodulation, antioxidation, antitumor activity, and neuroprotection. Studies have also shown that GP may exert potential anti-AD effects by inhibiting neuroinflammatory responses and reducing Aβ deposition. However, current ginseng polysaccharide extraction processes are relatively rudimentary, the anti-AD active components are unclear, and it is impossible to obtain single-component ginseng polysaccharides. Even when single ginseng polysaccharides are reported to inhibit Aβ aggregation, the available types are extremely limited, and whether they can achieve clinical efficacy requires further investigation. Summary of the Invention

[0004] The purpose of this invention is to provide a ginseng acidic polysaccharide GPA-0.2b with uniform molecular weight and a clear structure-activity relationship. It not only inhibits the deposition of Aβ plaques in the brain but also has anti-inflammatory effects, improves patients' learning and memory abilities, and reduces anxiety behaviors, thus achieving clinical therapeutic effects for Alzheimer's disease (AD).

[0005] Another objective of this invention is to provide a method for preparing ginseng acidic polysaccharide GPA-0.2b, which involves a combination of freeze-thaw and Sevag methods and fractional purification to obtain an acidic polysaccharide with high purity, uniform molecular weight, and good activity retention.

[0006] This invention provides a ginseng acidic polysaccharide GPA-0.2b with a molecular weight of 5011 Da, comprising the following molar percentages of residues: 2.60% terminal α-L-rhamnose pyranose residues, 3.04% terminal α-L-arabinose pyranose residues, 1.55% terminal α-L-arabinose pyranose residues, 1.81% terminal α-L-glucose pyranose residues, 6.7% terminal α-L-galacturonic acid pyranose residues, 4.31% terminal β-L-galactose pyranose residues, and 1.55% terminal α-L-arabinose pyranose residues. 57%, 3-β-L-galactopyranose residues 2.22%, 4-β-L-galactopyranose residues 4.43%, 4-α-L-galactopyranouronic acid residues 50.65%, 4-α-L-glucanopyranose residues 10.96%, 6-β-L-galactopyranose residues 1.61%, 2,4-α-L-glucuronic acid residues 1.62%, 4,6-β-L-galactopyranose residues 1.03%, 3,6-β-L-galactopyranose residues 1.56%; The ginseng acidic polysaccharide is composed of [→4)-α-GalA p -(1→4)-α-GalA p -(1→] n The main chain is composed of side chains including [α-Glc] p →4)-α-Glc p -(1→4)-α-Glc p -(1→]5、[β-Gal p →3)-β-Gal p -(1→]2 and [α-Ara f →4)-β-Gal p -(1→]3 structure.

[0007] This invention provides a method for preparing the ginseng acidic polysaccharide GPA-0.2b, comprising the following steps: extracting polysaccharide from ginseng using a water extraction and alcohol precipitation method to obtain ginseng extract; The ginseng extract was treated with Sevag reagent and subjected to repeated freeze-thaw cycles to remove impurities and proteins, yielding crude ginseng polysaccharide. The crude ginseng polysaccharide was purified using a DEAE-cellulose chromatography column. After eluting with water to remove neutral polysaccharides, it was eluted with 0.2 mol / L NaCl solution to obtain ginseng acidic polysaccharide GPA-0.2. The ginseng acidic polysaccharide GPA-0.2 was purified by Sepharose CL-6B gel column chromatography, eluted with 0.1~0.15 mol / L NaCl solution, and the fractions that elute later in the elution curve were collected to obtain ginseng acidic polysaccharide GPA-0.2b.

[0008] Preferably, the volume concentration of alcohol in the water extraction and alcohol precipitation method is 88%~92%.

[0009] Preferably, the water extraction and alcohol precipitation method includes water bath extraction; the number of water bath extractions includes 3 to 4 times; the mass of ginseng and the total volume of water are 1:36 to 40.

[0010] Preferably, the temperature for the repeated freeze-thaw treatment is -18 to -22 °C; The repeated freeze-thaw treatment is performed 3 to 4 times.

[0011] Preferably, the ginseng acidic polysaccharide GPA-0.2 or ginseng acidic polysaccharide GPA-0.2b further includes dialysis desalting; the molecular weight cutoff of the dialysis bag during dialysis desalting is 3500 Da.

[0012] This invention provides the application of the ginseng acidic polysaccharide GPA-0.2b or the ginseng acidic polysaccharide GPA-0.2b obtained by the preparation method in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease.

[0013] Preferably, the drug comprises at least one of the following: improving amyloid pathological damage, improving cognitive impairment and anxiety behavior, and inhibiting inflammatory responses in the brain.

[0014] Preferably, the dosage form of the drug includes at least one of the following: tablets, powders, capsules, granules, and oral preparations.

[0015] This invention provides a medicament for the prevention and / or treatment of Alzheimer's disease, comprising the ginseng acidic polysaccharide GPA-0.2b or the ginseng acidic polysaccharide GPA-0.2b obtained by the preparation method and pharmaceutically acceptable excipients.

[0016] This invention provides ginseng acidic polysaccharide GPA-0.2b. HPGPC detection yielded a single, narrow, symmetrical peak, confirming the high purity and uniform molecular weight of the tested polysaccharide component (5011 Da). FT-IR analysis of the monosaccharide composition showed that GPA-0.2b is an acidic polysaccharide composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), and fucose (Fuc). Methylation, hydrolysis, acetylation, and GC-MS analysis of GPA-0.2b revealed that GPA-0.2b includes t-Rha p、 t-Ara f、 T-Ara p、 2-Rha p、 T-Glc p、 T-Gal p -UA、T-Gal p、5-Ara f、 3-Gal p、 4-Gal p、 4-Gal p -UA, 4-Glc p、 6-Gal p、 2,4-Glc p -UA、4,6-Gal p、 3,6-Gal p residues ， Among them, 4-Gal p -UA accounted for as high as 50.65%. GPA-0.2b is likely an acidic heteropolysaccharide with an HG domain as the main chain and dextran, galactan, and AG-I domains as side chains. Furthermore, the efficacy of GPA-0.2b was evaluated using an APP / PS1 mouse model. GPA-0.2b showed a significant effect in reducing the levels of Aβ42 and Aβ40 in the brain compared to the ginseng acidic polysaccharide GPA-0.2a isolated from the same batch. Simultaneously, water maze, mine field, and new object recognition experiments showed that GPA-0.2b could also improve learning and memory abilities and reduce anxiety behavior in the model mice. Staining results indicated that GPN-0.2b could improve the pathological state of the model mice by reducing the formation of Aβ42 plaques or promoting their clearance. ELISA results showed that GPN-0.2b could also reduce brain inflammation and lower the levels of Aβ42 and Aβ40 in the brain. Therefore, the GPA-0.2b provided by this invention has good efficacy in preventing and treating AD. This invention provides a homogeneous ginseng acidic polysaccharide, clarifies its structure-activity relationship with Alzheimer's disease (AD), and provides a material basis and technical support for the development of highly efficient, low-toxicity, and targeted anti-AD natural polysaccharide drugs.

[0017] This invention provides a method for preparing the ginseng acidic polysaccharide GPA-0.2b. The method involves extracting the polysaccharide from ginseng using a water extraction and alcohol precipitation process. The obtained ginseng extract is treated with Sevag reagent to remove free proteins, followed by repeated freeze-thaw cycles to remove residual proteins and small molecule impurities. This combined technique ensures thorough protein removal while reducing the amount of organic solvent used, maximizing the preservation of the polysaccharide's natural structure and bioactivity. The extracted crude ginseng polysaccharide is purified using a DEAE-cellulose chromatography column, eluted with water and 0.2 mol / L NaCl solution, achieving effective separation of neutral sugars and polysaccharides with different acidity levels. The obtained ginseng acidic polysaccharide GPA-0.2b is purified by Sepharose CL-6B gel chromatography, eluted with 0.1–0.15 mol / L NaCl solution, yielding GPA-0.2b with a uniform molecular weight distribution. As can be seen, this invention uses a combination of freeze-thaw and Sevag methods to remove proteins, combined with gel purification, to achieve precise development and utilization of ginseng polysaccharides. The method is simple, reduces the use of organic solvents, and has good potential for widespread application. Attached Figure Description

[0018] Figure 1 The elution curve of Sepharose CL-6B for ginseng acidic polysaccharide GPN-0.2; Figure 2 The results show the effects of ginseng acidic polysaccharides GPA-0.2a and GPA-0.2b on Aβ in the mouse brain, where A represents the Aβ40 measurement result; B represents the Aβ42 measurement result. Note: P <0.05, P <0.01; Figure 3 The HPGPC spectrum for GPA-0.2b is shown below. Figure 4 The infrared spectrum of GPA-0.2b; Figure 5 The results of monosaccharide composition analysis for GPA-0.2b are shown. Figure 6 The results show the effects of GPN-0.2b on the learning and memory abilities of mice, where A represents the results of the water maze test; B represents the results of the open field test; and C represents the results of the new object recognition test. Note: P <0.05, P <0.01; Figure 7 The results show the effect of GPN-0.2b on Aβ42 plaque deposition in the mouse brain; Figure 8 The results show the effects of GPN-0.2b on the levels of inflammatory factors and Aβ in the mouse brain. Note: P <0.05, P <0.01. Detailed Implementation

[0019] This invention provides a method for preparing the ginseng acidic polysaccharide GPA-0.2b, comprising the following steps: extracting polysaccharide from ginseng using a water extraction and alcohol precipitation method to obtain ginseng extract; The ginseng extract was treated with Sevag reagent and subjected to repeated freeze-thaw cycles to remove impurities and proteins, yielding crude ginseng polysaccharide. The crude ginseng polysaccharide was purified using a DEAE-cellulose chromatography column. After eluting with water to remove neutral polysaccharides, it was eluted with 0.2 mol / L NaCl solution to obtain ginseng acidic polysaccharide GPA-0.2. The ginseng acidic polysaccharide GPA-0.2 was purified by Sepharose CL-6B gel column chromatography, eluted with 0.1~0.15mol / L NaCl solution, and the fractions that elute later in the elution curve were collected to obtain ginseng acidic polysaccharide GPA-0.2b.

[0020] This invention uses a water extraction and alcohol precipitation method to extract polysaccharides from ginseng, thus obtaining ginseng extract.

[0021] This invention does not impose any special restrictions on the type and source of ginseng; any type and source of ginseng well-known in the art can be used. The ginseng preferably includes wild ginseng or artificially cultivated ginseng. The ginseng includes fresh ginseng or dried ginseng. In this embodiment of the invention, the ginseng is four-year-old ginseng. The ginseng is preferably pulverized before being soaked in water to improve the extraction efficiency of the active ingredients.

[0022] In this invention, the water extraction and alcohol precipitation method includes water bath extraction. The water bath extraction is performed 3-4 times. The water bath temperature is preferably a boiling water bath. The ratio of ginseng mass (g) to total water volume (mL) is preferably 1:36-40, and can also be 1:38. Each water bath extraction is preferably performed for 2-3 hours, preferably 3 hours for the first extraction and 2 hours for each subsequent extraction. After each water bath extraction, solid and liquid phases are separated, the liquid phases are collected and combined, concentrated, centrifuged to discard the precipitate, and the supernatant is collected. The centrifugation speed is preferably 4000-600 rpm, and can be 5000 rpm. The centrifugation time is 12-17 minutes, and can be 15 minutes. The supernatant is then subjected to alcohol precipitation to precipitate the polysaccharides, and the precipitate is collected. The alcohol is preferably ethanol. The volume concentration of the ethanol is preferably 88%-92%, and can be 90%. During the alcohol precipitation, anhydrous ethanol is slowly added to the supernatant. The preferred flow rate of the anhydrous ethanol is 5-10 mL / min, but it can also be 6-9 mL / min or 7-8 mL / min. The preferred precipitation time is 10-14 h, but it can be 12 h. The precipitation also includes washing twice each with 95% aqueous ethanol and anhydrous ethanol, and collecting the precipitate is the ginseng extract. The volume of washing solution used each time is 3-5 times the volume of the precipitate, but it can be 4 times. Multiple washings help remove pigments and alcohol-soluble impurities such as monosaccharides and oligosaccharides, thereby improving the purity of ginseng polysaccharides.

[0023] After obtaining the ginseng extract, the ginseng extract of the present invention is treated with Sevag reagent and subjected to repeated freeze-thaw treatment to remove impurities and proteins, thereby obtaining crude ginseng polysaccharide.

[0024] In this invention, the Sevag reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1. The preferred method for Sevag reagent treatment involves dissolving the ginseng extract in water and then thoroughly mixing it with the Sevag reagent to separate the proteins from the ginseng extract. The mixture is then centrifuged, and the supernatant is collected. The amount of Sevag reagent added is preferably 25% of the ginseng extract solution. The Sevag reagent treatment is preferably performed 4-5 times until the intermediate protein layer disappears.

[0025] In this invention, the repeated freeze-thaw treatment method involves freezing the supernatant into ice, then thawing it at room temperature (20~28 ℃), centrifuging, and collecting the supernatant. The freezing temperature is preferably -18~-22 ℃, and can be -20 ℃. The centrifugation speed is preferably 7000~9000 rpm, and can be 8000 rpm. The centrifugation time is 15~25 min, and can be 20 min. The centrifugation temperature is preferably 4 ℃. The repeated freeze-thaw treatment is preferably performed 3~4 times. This repeated freeze-thaw treatment helps to further remove residual proteins and other small molecules, improving the purity and yield of the final active ingredient, while retaining high activity and reducing the amount of organic solvent used.

[0026] In this invention, the supernatant obtained after repeated freeze-thaw cycles preferably includes water removal. The method for water removal is preferably freeze-drying. The freeze-drying temperature is preferably -80°C. The freeze-drying time is preferably 3-4 days. The yield of the crude ginseng polysaccharide is 9.8%.

[0027] After obtaining the crude ginseng polysaccharide, the present invention purifies the crude ginseng polysaccharide using a DEAE-cellulose chromatography column. After eluting with water to remove neutral polysaccharides, it is eluted with 0.2 mol / L NaCl solution to obtain the ginseng acidic polysaccharide GPA-0.2.

[0028] In this invention, the ginseng crude polysaccharide is preferably dissolved into a solution before purification using a DEAE-cellulose chromatography column. The mass concentration of the solution is 13%~17%, and can be 15%. The DEAE-cellulose chromatography column is preferably prepared by fully swelling the DEAE-cellulose, treating it sequentially with alkali and acid, washing it until neutral, then soaking it in 2 mol / L NaCl solution for 1 h and washing it to remove gas. The column is then packed into a chromatography column (6.0 × 27 cm) and equilibrated with distilled water, NaCl solution, and distilled water, respectively. The alkali and acid treatment is preferably performed by soaking in 0.5 mol / L NaOH for 1 h, washing it until neutral, and then soaking it in 0.5 mol / L HCl for 1 h and washing it until neutral. During column equilibration, the flow volumes of distilled water, NaCl solution, and distilled water are 2, 1, and 3 times the column volume, respectively. The flow rate of the flow solution during equilibration is preferably 10 mL / min. The solvent in the NaCl solution is preferably water.

[0029] In this invention, during purification using the DEAE-cellulose chromatography column, water elution is first performed to remove neutral polysaccharides. The preferred flow rate for water elution is 10 mL / min. When eluting with 0.2 mol / L NaCl solution, the flow rate is also 10 mL / min. During DEAE-cellulose chromatography column purification, the eluent composition is preferably monitored simultaneously using both the phenol-sulfuric acid method and the m-hydroxybiphenyl method, with absorbance measured at 490 nm and 525 nm every 20 mL to monitor the elution progress of total sugars and uronic acids. Elution curves are plotted based on the detection results, and acidic sugar components are collected. After obtaining ginseng acidic polysaccharide GPA-0.2, dialysis desalting and freeze-drying are preferably further performed. The molecular weight cutoff of the dialysis bag during dialysis desalting is 3500 Da. During dialysis desalting, dialysis is preferably performed with running water and distilled water for 20–26 h each, or 24 h.

[0030] After obtaining ginseng acidic polysaccharide GPA-0.2, the present invention purified the ginseng acidic polysaccharide GPA-0.2 by Sepharose CL-6B gel column chromatography, eluted with 0.1~0.15 mol / L NaCl solution, and collected the fractions that eluted later in the elution curve to obtain ginseng acidic polysaccharide GPA-0.2b.

[0031] In this invention, the preferred concentration of the ginseng acidic polysaccharide GPA-0.2 for column loading is 50 mg / mL. The Sepharose CL-6B gel column is prepared by washing the Sepharose CL-6B gel with deionized water to remove impurities and air bubbles, then injecting the gel suspension into a chromatography column (2.5 cm × 100 cm). After complete sedimentation, the column is equilibrated for 24 h with a 0.1–0.15 mol / L NaCl solution as the mobile phase at a flow rate of 0.5 mL / min. The concentration of the NaCl solution can be 0.5–0.12 mol / L, or 0.8–0.10 mol / L. Water is preferred as the solvent in the NaCl solution. The preferred flow rate for elution with the NaCl aqueous solution is 0.5 mL / min. During purification using a Sepharose CL-6B gel column, the phenol-sulfuric acid method was employed for monitoring. Absorbance at 490 nm was measured every 10 mL, and an elution curve was plotted. The main peak components were collected, resulting in two peaks: the first eluting component was designated GPA-0.2a, and the second eluting component was designated GPA-0.2b. Preferably, the ginseng acidic polysaccharide GPA-0.2b also underwent dialysis desalting and freeze-drying. The molecular weight cutoff of the dialysis bag during dialysis desalting was 3500 Da. The yield of GPA-0.2b was 80 mg / kg.

[0032] In one embodiment of this invention, to screen and clarify the structure-activity relationship of ginseng acidic polysaccharides for treating Alzheimer's disease (AD), in vivo pharmacodynamic evaluation was conducted using an APP / PS1 double transgenic AD mouse model. The results showed that, compared to the model group, GPA-0.2b had a significant advantage in reducing Aβ42 and Aβ40 levels in the brains of model mice; while ginseng acidic polysaccharide GPA-0.2a did not exhibit the same activity as GPA-0.2b, and compared to the model group, it did not show a significant reduction in Aβ42 and Aβ40 levels in the brains of model mice. Therefore, the ginseng acidic polysaccharide with anti-AD properties is GPA-0.2b.

[0033] This invention also characterized the structure of ginseng acidic polysaccharide GPA-0.2b. The molecular weight of ginseng acidic polysaccharide GPA-0.2b is 5011 Da. The ginseng acidic polysaccharide GPA-0.2b contains monosaccharides: rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), and fucose (Fuc). The molar ratio of Rha, Ara, Gal, Glc, Gal-UA, Glc-UA, and Fuc is 1:8.93:6.65:7.09:14.62:54.69:2.28.

[0034] In this invention, methylation, hydrolysis, acetylation, and GC-MS analysis of GPA-0.2b revealed that the ginseng acidic polysaccharide GPA-0.2b comprises the following molar percentages of residues: terminal α-L-rhamnopyranose residues 2.60%, terminal α-L-arabinose furanose residues 3.04%, terminal α-L-arabinose pyranose residues 1.55%, 2-α-L-rhamnopyranose residues 1.81%, terminal α-L-glucose pyranose residues 6.7%, terminal α-L-galacturonic acid pyranose residues 4.31%, and terminal β-L-galacturonic acid pyranose residues 4.31%. The composition includes 4.34% lactose residues, 1.57% 5-α-L-arabinose furanoate residues, 2.22% 3-β-L-galactopyranose residues, 4.43% 4-β-L-galactopyranose residues, 50.65% 4-α-L-galactopyranoside residues, 10.96% 4-α-L-glucose pyranose residues, 1.61% 6-β-L-galactopyranose residues, 1.62% 2,4-α-L-glucuronic acid pyranose residues, 1.03% 4,6-β-L-galactopyranose residues, and 1.56% 3,6-β-L-galactopyranose residues. The ginseng acidic polysaccharide has an HG domain as its main chain and also contains dextran, galactan, and AG-I domains. Specifically, GPA-0.2b is mainly composed of [→4)-α-GalA p -(1→4)-α-GalA p -(1→] nThe main chain is composed of [α-Glc], and the side chains are composed of [α-Glc], [α-G p →4)-α-Glc p -(1→4)-α-Glc p -(1→]5、[β-Gal p →3)-β-Gal p -(1→]2 and [α-Ara f →4)-β-Gal p Acidic heteropolysaccharides composed of structural units such as -(1→]3.

[0035] This invention provides the application of the ginseng acidic polysaccharide GPA-0.2b or the ginseng acidic polysaccharide GPA-0.2b obtained by the preparation method in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease.

[0036] In this invention, the drug preferably comprises at least one of the following: improving amyloid pathological damage, improving cognitive impairment and anxiety behavior, and inhibiting intracranial inflammatory responses. Improving amyloid pathological damage includes reducing the formation of Aβ42 plaques or promoting their clearance, and inhibiting the expression levels of Aβ42 and Aβ40 in the brain. Improving cognitive impairment preferably includes enhancing the patient's learning and memory abilities. Inhibiting intracranial inflammatory responses preferably includes inhibiting the levels of IL-1β, IL-6, and TNF-α in the brain. Therefore, GPA-0.2b achieves the prevention and treatment of AD by exerting neuroprotective effects and improving brain function.

[0037] This invention provides a medicament for the prevention and / or treatment of Alzheimer's disease, comprising the ginseng acidic polysaccharide GPA-0.2b or the ginseng acidic polysaccharide GPA-0.2b obtained by the preparation method and pharmaceutically acceptable excipients.

[0038] In this invention, the dosage form of the drug preferably includes at least one of the following: tablets, powders, capsules, granules, and oral preparations. The drug preferably also includes pharmaceutically acceptable excipients. The types of excipients are conventionally selected according to the dosage form of the drug. This invention does not impose any particular limitation on the preparation method of the crop; any drug preparation method well-known in the art can be used. The preferred mass percentage of ginseng acidic polysaccharide GPA-0.2b in the drug is 5% to 95%, but can also be 10% to 80%, 20% to 70%, and more preferably 30% to 50%.

[0039] The following detailed description, in conjunction with embodiments, illustrates a ginseng acidic polysaccharide, its preparation method, and its applications, but these should not be construed as limiting the scope of protection of this invention.

[0040] Example 1 Extraction, separation, purification and activity screening of ginseng polysaccharides 1. Extraction with 90% ethanol aqueous solution Take 1 kg of dried four-year-old ginseng segments, add distilled water at a material-to-liquid ratio of 1:20 (g / mL), and extract three times in a boiling water bath for 3 h, 2 h, and 2 h respectively. After the first extraction, filter. Add distilled water to the residue at a material-to-liquid ratio of 1:10 (g / mL) for the second and third extractions. Combine the three filtrates, concentrate, centrifuge at 5000 rpm for 15 min to remove the precipitate, and slowly add anhydrous ethanol (flow rate of about 5-10 mL / min) to the supernatant on a magnetic stirrer to achieve a final ethanol concentration of 90% (v / v). Let stand overnight, centrifuge at 5000 rpm for 15 min, and collect the precipitate. Wash the obtained precipitate twice each with 95% ethanol and anhydrous ethanol (each time the volume of precipitate is about 3-5 times).

[0041] 2. A combination of freeze-thaw and Sevag methods was used to remove proteins and small molecule impurities. The washed precipitate was dissolved in an appropriate amount of distilled water (approximately 1500 mL) to fully dissolve the polysaccharide. 1 / 4 volume of Sevag reagent (chloroform:n-butanol = 4:1, v / v) was added, and the mixture was shaken for 1 h. After centrifugation at 4000 rpm for 20 min, the supernatant was collected. This process was repeated 4-5 times until the intermediate protein layer disappeared. The supernatant was then frozen at -20 ℃ for 24 h. After thawing completely at room temperature, the supernatant was collected by centrifugation at 8000 rpm and 4 ℃ for 20 min. This freeze-thaw cycle was repeated 3 times. The collected supernatant was aliquoted into lyophilization containers, pre-frozen at -80 ℃ for 24 h, and then freeze-dried for 3 days to obtain 98 g of crude ginseng polysaccharide GP.

[0042] 3. Obtaining ginseng acidic polysaccharides Take 500 g of DEAE-cellulose and swell it fully in sufficient distilled water for 2-3 days. After removing the water, soak it in 0.5 mol / L NaOH and 0.5 mol / L HCl for 1 h each time. After each alkali and acid treatment, wash it with distilled water until neutral. Then soak it in 2 mol / L NaCl solution for 1 h and wash it. Finally, soak it in distilled water for later use. After removing air bubbles by filtration, pack the treated DEAE-cellulose evenly into a chromatography column (6.0 × 27 cm). Equilibrate the column by passing it through 2 column volumes of distilled water, 1 column volume of NaCl solution, and 3 column volumes of distilled water in sequence, controlling the flow rate at 10 mL / min.

[0043] 6 g of GP sample was dissolved in 40 mL of distilled water and slowly loaded onto a pre-equilibrated DEAE-cellulose chromatography column. Neutral polysaccharides were eluted with distilled water (flow rate 10 mL / min). After complete elution of neutral sugars, 0.2 mol / L NaCl solution was used for elution (flow rate 10 mL / min). The absorbance was simultaneously monitored using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, measuring the absorbance at 490 nm and 525 nm every 20 mL, until all acidic sugars were eluted. Elution curves were plotted, and the acidic sugar fraction was collected and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The sample was dialyzed with running water and distilled water for 24 h each, then freeze-dried and weighed to obtain ginseng acidic polysaccharide GPA-0.2.

[0044] 4. Purification of ginseng acidic polysaccharides GPA-0.2 was purified using Sepharose CL-6B gel column chromatography. 400 mL of Sepharose CL-6B gel was taken, and deionized water was added for gentle stirring and washing to remove impurities. This process was repeated several times. After removing air bubbles, the gel suspension was slowly injected into the chromatography column (2.5 cm × 100 cm) using a glass guide rod. After complete sedimentation, the column was equilibrated for 24 h with 0.1 mol / L NaCl solution as the mobile phase at a flow rate of 0.5 mL / min.

[0045] Weigh 500 mg of GPA-0.2 sample and prepare a 50 mg / mL solution with distilled water. Centrifuge at 3000 rpm for 10 min, and carefully add the supernatant to the surface of a gel column. Elute with 0.1 mol / L NaCl solution at a flow rate of 0.5 mL / min. Detect using the phenol-sulfuric acid method, measuring the absorbance at 490 nm every 10 mL, plotting the elution curve, and collecting the main peak components. Dialyze the samples in a 3500 Da dialysis bag with running water and distilled water for 24 h to remove salts. After freeze-drying, weigh to obtain purified acidic polysaccharides GPA-0.2a (30 mg) and GPA-0.2b (80 mg). Figure 1 ).

[0046] 5. Activity verification Six-month-old wild-type C57BL / 6 mice and APP / PS1 mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. to evaluate the ameliorative effects of GPA-0.2a and GPA-0.2b on APP / PS1 mice. The experimental groups were: control group, model group, GPA-0.2a group, and GPA-0.2b group, with six mice in each group. The dosage was 50 mg / kg body weight, administered by gavage for 60 consecutive days. Mice were euthanized by decapitation, and their brains were harvested. The levels of Aβ42 (MM-44819M2) and Aβ40 (MM-44560M1) were measured by ELISA (Jiangsu Enzyme Immunoassay Co., Ltd.).

[0047] The results showed that ( Figure 2 In the AB group, compared with the AD group, GPA-0.2b significantly reduced the levels of Aβ42 and Aβ40 in the brain. P <0.01; P <0.05), while GPA-0.2a had no significant effect on Aβ42 or Aβ40 levels ( P > 0.05). Further investigation was conducted into the ameliorative effect of GPA-0.2b on AD.

[0048] Example 2 Structural characterization of ginseng acidic polysaccharide GPA-0.2b 1. The homogeneity and weight-average molecular weight of GPA-0.2b were determined using HPGPC. Mw For evaluation, please refer to existing technologies (Zhou S, Liu J, Zhao R, et al. Structural integrity of PGPW-a2, apolysaccharide from Panax ginseng stem-leaf, contributes to Nrf2-mediated cytoprotection against oxidative stress-induced apoptosis in KGN cells[J]. International Journal of Biological Macromolecules, 2026: 151771.).

[0049] The results are as follows Figure 3 As shown, the narrow and single symmetrical peak on the HPGPC spectrum indicates the homogeneity of GPA-0.2b. Substituting its retention time of 16.1 min into the dextran standard curve y = -0.3812x + 9.8474, the molecular weight of GPA-0.2b is found to be 5011 Da.

[0050] 2. The characteristic absorption band of GPA-0.2b was detected by FT-IR.

[0051] The results are as follows Figure 4 As shown, 3382.5 cm -1 The broad and strong absorption peak at 2942.8 cm⁻¹ is due to the stretching vibration of OH groups. -1 The weak signal is due to the stretching vibration of CH; 1732.2 cm -1 The absorption peak at 1615.1 cm⁻¹ is caused by the stretching vibration of the C=O group of the carboxyl group. -1 and 1422.2 cm -1 The absorption peak at 1099.7 cm⁻¹ corresponds to the COO-deprotonated carboxyl group. -1 The vibrational absorption peak of COC indicates that the polysaccharide contains a pyranose ring.

[0052] The monosaccharide composition results of GPA-0.2b are as follows: Figure 5 As shown in Table 1, the results indicate that GPA-0.2b is an acidic polysaccharide composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), and fucose (Fuc), with a molar ratio of 1:8.93:6.65:7.09:14.62:54.69:2.28.

[0053] Table 1. Monosaccharide composition analysis (mol%) of GPA-0.2b

[0054] 3. GPA-0.2b was subjected to methylation, hydrolysis, acetylation and GC-MS analysis.

[0055] Sixteen partially methylated sugar alcohol acetates (PMAAs) were observed in GC-MS total ion chromatography.

[0056] The results are shown in Table 2. Based on retention time and characteristic ion fragments, the residues were identified as t-Rha. p t-Ara f T-Ara p 2-Rha p T-Glc p T-Gal p -UA、T-Gal p 5-Ara f 3-Gal p 4-Gal p 4-Gal p -UA, 4-Glc p 6-Galp 2,4-Glc p -UA、4,6-Gal p 3,6-Gal p Among them, 4-Gal p -UA accounts for as much as 50.65%, suggesting that GPA-0.2b may be an acidic heteropolysaccharide with HG domain as the main chain and dextran, galactan and AG-I domain as side chains.

[0057] Table 2. Methylation analysis of GPA-0.2b (mol%)

[0058] Example 3 The effect of GPA-0.2b on AD To further investigate the ameliorative effect of GPN-0.2b on Alzheimer's disease (AD), an AD model was established using APP / PS1 double transgenic mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., Production License SCXK (Zhejiang) 2024-0004). Mice were randomly divided into a control group, a model group, and three polysaccharide dosage groups (25 mg / kg, 50 mg / kg, and 100 mg / kg), with six mice in each group. Except for the control and AD groups, which received saline daily, the three GPN-0.2b dosage groups received the corresponding dose of GPN-0.2b daily via gavage for 60 consecutive days. The effects of GPN-0.2b on learning and memory abilities and anxiety behavior in APP / PS1 mice were assessed using water maze, open field, and novel object recognition tests. After the behavioral experiments, mice were euthanized by decapitation, and Congo red staining was used to assess the effect of GPN-0.2b on Aβ42 plaque deposition in the mouse brain. Further ELISA was used to measure the levels of inflammatory factors in the brain (Jiangsu Enzyme Immunoassay Co., Ltd.) (IL-1β (MM-0181H1), IL-6 (MM-0163M1), TNF-α (MM-0132M1), and Aβ).

[0059] Behavioral results indicate that in the water maze experiment ( Figure 6 In mice, after administration of GPN-0.2b (A), the time spent in the target quadrant and the number of times the platform was crossed were significantly increased. P <0.01 indicates a significant improvement in learning and memory abilities. In the open field experiment ( Figure 6 GPN-0.2b can effectively reduce the total distance traveled, average speed, and number of times the center is crossed in mice. P <0.01), thus effectively reducing anxiety behavior in mice. In the new object recognition experiment ( Figure 6GPN-0.2b can effectively increase the recognition coefficient and novelty index in mice, further confirming its effectiveness in improving the learning and memory abilities of APP / PS1 mice.

[0060] The results of Aβ42 plaque assays indicate that ( Figure 7 Compared with the model group, GPN-0.2b intervention effectively reduced Aβ42 plaque deposition in the mouse brain, indicating that GPN-0.2b can effectively improve the pathological characteristics of APP / PS1 mice by reducing the formation of Aβ42 plaques or promoting their clearance.

[0061] Results of inflammatory factors and Aβ levels Figure 8 The results indicate that GPN-0.2b can effectively reduce the levels of IL-1β, IL-6, and TNF-α in mouse serum and brain, and also reduce the levels of Aβ42 and Aβ40 in the brain. Therefore, GPN-0.2b can effectively inhibit brain inflammation and reduce Aβ42 and Aβ40 levels, thereby achieving an anti-AD effect.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A ginseng acidic polysaccharide GPA-0.2b, characterized in that, The molecular weight is 5011 Da, comprising the following molar percentages of residues: terminal α-L-rhamnose residue 2.60%, terminal α-L-arabinose residue 3.04%, terminal α-L-arabinose residue 1.55%, 2-α-L-rhamnose residue 1.81%, terminal α-L-glucose pyranopyranose residue 6.7%, terminal α-L-galacturonic acid pyranopyranose residue 4.31%, terminal β-L-galactose pyranopyranose residue 4.34%, and 5-α-L-arabinose residue 1. 57%, 3-β-L-galactopyranose residues 2.22%, 4-β-L-galactopyranose residues 4.43%, 4-α-L-galactopyranouronic acid residues 50.65%, 4-α-L-glucanopyranose residues 10.96%, 6-β-L-galactopyranose residues 1.61%, 2,4-α-L-glucuronic acid residues 1.62%, 4,6-β-L-galactopyranose residues 1.03%, 3,6-β-L-galactopyranose residues 1.56%; The ginseng acidic polysaccharide is composed of [→4)-α-GalA p -(1→4)-α-GalA p -(1→] n The main chain is composed of side chains including [α-Glc] p →4)-α-Glc p -(1→4)-α-Glc p -(1→]5、[β-Gal p →3)-β-Gal p -(1→]2 and [α-Ara f →4)-β-Gal p -(1→]3 structure.

2. A method for preparing the ginseng acidic polysaccharide GPA-0.2b according to claim 1, characterized in that, Includes the following steps: Polysaccharides were extracted from ginseng using a water extraction and alcohol precipitation method to obtain ginseng extract; The ginseng extract was treated with Sevag reagent and subjected to repeated freeze-thaw cycles to remove impurities and proteins, yielding crude ginseng polysaccharide. The crude ginseng polysaccharide was purified using a DEAE-cellulose chromatography column. After eluting with water to remove neutral polysaccharides, it was eluted with 0.2 mol / L NaCl solution to obtain ginseng acidic polysaccharide GPA-0.

2. The ginseng acidic polysaccharide GPA-0.2 was purified by Sepharose CL-6B gel column chromatography, eluted with 0.1~0.15 mol / L NaCl solution, and the fractions that elute later in the elution curve were collected to obtain ginseng acidic polysaccharide GPA-0.2b.

3. The preparation method according to claim 2, characterized in that, The volume concentration of alcohol in the water extraction and alcohol precipitation method is 88%~92%.

4. The preparation method according to claim 2, characterized in that, The water extraction and alcohol precipitation method includes water bath extraction; the number of water bath extractions includes 3 to 4 times; the ratio of ginseng mass to total water volume is 1:36 to 40.

5. The preparation method according to claim 2, characterized in that, The temperature for the repeated freeze-thaw treatment is -18 to -22°C; The repeated freeze-thaw treatment is performed 3 to 4 times.

6. The preparation method according to any one of claims 2 to 5, characterized in that, The ginseng acidic polysaccharide GPA-0.2 or ginseng acidic polysaccharide GPA-0.2b also includes dialysis for desalting; the molecular weight cutoff of the dialysis bag during dialysis for desalting is 3500 Da.

7. The use of the ginseng acidic polysaccharide GPA-0.2b according to claim 1 or the ginseng acidic polysaccharide GPA-0.2b obtained by the preparation method according to any one of claims 2 to 6 in the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease.

8. The application according to claim 7, characterized in that, The drug includes at least one of the following: improving amyloid pathological damage, improving cognitive impairment and anxiety behavior, and inhibiting inflammatory responses in the brain.

9. The application according to claim 7 or 8, characterized in that, The dosage form of the drug includes at least one of the following: tablets, powders, capsules, granules, and oral preparations.

10. A medicament for the prevention and / or treatment of Alzheimer's disease, comprising ginseng acidic polysaccharide GPA-0.2b as described in claim 1 or ginseng acidic polysaccharide GPA-0.2b prepared by any one of claims 2 to 6 and pharmaceutically acceptable excipients.

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